key: cord- - is skpw authors: lesser, william title: whither the research anticommons? date: - - journal: from agriscience to agribusiness doi: . / - - - - _ sha: doc_id: cord_uid: is skpw fifteen years ago, the “tragedy of the anticommons” article warned that excessive patenting of biotech products and research methods could deter rather than stimulate invention, but little evidence was offered. here, subsequent changes in patent law, public research support, and surveys of researchers are summarized. results indicate the anticipated anticommons has not materialized significantly, and while ongoing monitoring is warranted, declining public research funding may necessitate more patenting to stimulate private investment. nearly years ago, heller and eisenberg ( ) published in science a highly influential article (more than citations) warning of the accelerated use of biotech patents stifling subsequent developments rather than incentivizing them as intended. their analysis focused particularly on biomedical research, but the issues are general to biotech research applications, including agricultural and veterinary medicinal. they used the term "tragedy of the anticommons" in contrast with the "tragedy of the commons," which popularized the observation that common property resources are overexploited because no one has a preservation incentive (hardin ). while the "commons" concept argues for privatization, heller and eisenberg ( , p. ) cautioned that overprivatization creates fragmented ownership and high transaction costs-an anticommons. "privatization can solve one tragedy but cause another." the anticommons potential is of ongoing significance because of the cumulative nature of scientific research. reductions in access to past developments diminish current and all future research productivity. economists explain those reductions in terms of transactions costs: more and potentially overlapping patents increase the costs of negotiating access, which causes further declines in output. that point is tively preempt anyone else from using that gene itself for any medical or scientific basis." myriad genetics had patented two isolated genes-referred to as brca and brca -associated with an elevated risk for developing breast and uterine cancer and utilized in diagnostic testing. the unanimous june decision bans patents on naturally occurring dna segments ("isolated dna") as products of nature while permitting them for complementary dna (cdna), which is not naturally occurring. as part of their ruling, the justices quoted a lower court decision that allowing patents for isolated dna would create a "considerable danger" that "patents would 'tie up' the use of such tools and thereby 'inhibit future innovation premised upon them ' (p. ) ." indeed, the patent validity case was brought by a researcher who used a different diagnostic lab to perform the genetic testing but ceased after receiving a warning letter from myriad genetics. note should be made though that the plaintiff was involved with commercial use of myriad's invention, not research access. indeed, in its court filings, myriad pledged to grant open research access to its then-patented genes, balking only at use by fee-charging labs. concerns over patents "preempting" subsequent research were also emphasized in mayo collaborative services v. prometheus laboratories ( , p. which related to a method for determining drug dosages. the supreme court noted that "… the grant of patents that tie up [a law of nature] will inhibit future innovation premised upon them […] or otherwise forecloses more future invention than the underlying discovery could reasonably justify." like heller and eisenberg ( ) , the justices apparently saw a potential anticommons in biomedical research and constrained it by invalidating the prometheus patents, narrowing the field of patentable inventions. however, while the supreme court was categorical in asserting an anticommons effect, heller and eisenberg ( ) were circumspect. they identified the potential for patents to create an anticommons, using the conditionals "may/can/likely/potential/might" more than times. the intent here + years later is to examine the evidence for or against any actual patent-based anticommons in biomedical research. evidence must be multifaceted, as the authors identify multiple components under their heading of a biomedical anticommons: • privatization of "upstream" research previously public • multiple patents incorporated in a single product/research program • patents on components, not just complete products • long delays in examining patents, allowing possible overestimates of patent scope • licensing issues, including stacking and reach through licenses • heterogeneous interests and conflicting agendas of multiple patent owners, compounding licensing issues we begin with changes/reforms to the patent system itself. in addition to the recent supreme court patent decisions noted above, other limitations on patenting had been applied to gene components, particularly expressed sequence tags (ests). in in re fisher ( ), the us court of appeals for the federal circuit ruled ests lacked "specific and substantial utility"-that the disclosed uses "were generally applicable to any est" and hence unpatentable. going back further in time to when living organisms became patentable, the us patent and trademark office (uspto) began to require deposits of the material if necessary to assure availability to the public to satisfy the disclosure requirement. under patent law: "every patent must contain a written description of the invention sufficient to enable a person skilled in the art to which the invention pertains to make and use the invention. where the invention involves a biological material and words alone cannot sufficiently describe how to make and use the invention in a reproducible manner, access to the biological material may be necessary for the satisfaction of the statutory requirements for patentability under u.s.c. section ." (uspto ). that is, the disclosure requirement of patent law mandates the invention be publicly available, including through access to a sample if a written disclosure is deemed inadequate. this is an oft-overlooked component of the concept of patents, providing an incentive not only to invest in an inventive activity but also to make the invention public rather than holding it in secrecy. prof. potrykus, coinventor of golden rice, recognized the importance of disclosure despite the frustrations caused by private ownership: "at that time [of commercialization] i was much tempted to join those who radically fought patenting. fortunately i did a bit further thinking and became aware that 'golden rice' development was only possible because there was patenting. much of the technology i had been using was publicly known because the inventors could protect their right. much of it would have remained secret if this had been the case." (potrykus ). so while patenting encumbers use of an invention during its pendency, it fosters public availability, as interested parties have access once the patent expires. this aspect of the patent system-the provision of an incentive to make an invention public-is often overlooked. currently, most us patent applications are published months following first application worldwide (america invents act ), which means that applications are no longer secret during the full multiyear examination period. concurrently, the united states joined the rest of the world under the "first-to-file" system, which recognizes the first filer as the inventor (america invents act ). gone is the ownership uncertainty under the previous "first-to-invent" system and its complex "interference" proceedings. sometimes time itself has a mitigating effect on patents; they lapse due to end of term or failure to pay the requisite maintenance fees. this factor is particularly relevant for key "upstream" inventions, which have a disproportionate effect on subsequent research. and so it is with the basic plant transformation technology patents-the "gene gun" (# ) and agrobacterium tumefaciens (# ). both are now in the public domain, although some improvements remain under patent. this kind of broad pioneering patent grant is unlikely to be repeated in the biotech field due to an attribute of the us patent system which treats a patent as the right of an inventor. it is thus the responsibility of the uspto (the assigned examiner) to document why a patent should be withheld, typically meaning the application is either nonnovel and/or nonobvious . what happens in new fields of endeavor-as biotechnology was in the s-is that most applications by definition are nonobvious. to document lack of novelty, the examiner must identify a publication or use or related patent, which destroys the novelty. again, in new fields of research, there are limited numbers of such documents so lack of novelty is difficult to establish. the consequence is broad patent grants. the situation, though, is self-correcting since time provides more evidence for examiners to reject or narrow patent grants. thus, over multiple years, the scope of patentable inventions applicable to biotech research has been curtailed and the process simplified and made more transparent. all limit the anticommons potential. many observers have decried-as do heller and eisenberg ( ) -the privatization of research, placing many important discoveries and tools in private hands. however the public domain had shown itself not to engender use of many publicly supported inventions. the major justification for the bayh-dole act (pub. l. - ), which allows publicly supported inventions to be owned by nonprofit (research) institutions and small businesses, was a recognition that few such inventions were ever commercialized (cook-deegan and heaney ). most institutional inventions are at an early stage and require significant additional investment for which the absence of patents is a disincentive (nelsen ). an example of the kinds of privatization issues raised is exemplified by the -year skirmish between the national institutes of health (nih) and burroughs wellcome to the rights for azt, the first effective treatment for aids. azt was initially developed as a cancer drug by the national cancer institute and, according to some accounts, identified as effective against aids by researchers at duke university. a partnership between the nih and burroughs wellcome, however, led to burroughs wellcome receiving six patents for the production and use of azt, initially pricing the drug at $ -$ , annually per patient. due to pushback by the government, two % price reductions were instituted in and , but lawsuits by firms seeking to overturn the patents for lack of inventorship by burroughs wellcome-asserting that the nih and duke researchers were the true inventors-were eventually unsuccessful. the drug subsequently reached $ billion in yearly sales (yarchoan ) . the bayh-dole act allows the federal agency providing the funding underlying the research leading to the patented invention to grant additional licenses if the initial licensee refuses a reasonable request. these so-called "march-in" rights, a form of compulsory license, may be utilized if the granting agency determines the "action is necessary to alleviate health or safety needs" and is "necessary to meet requirements for public use specified by federal regulations" ( usc ). while potentially very powerful, the practical effect of this authority has been scant. an example of the constraints to application was the technical difficulties experienced in - by genzyme in producing fabrazyme, a medication for suffers of fabry disease. fabry is a rare genetic disease with varied symptoms and is potentially fatal. the manufacturing (contamination) problems necessitated dose reductions of two thirds followed by a return of symptoms in patients and a petition to the nih, the research funding agency, to exercise its march-in authority to enhance the supply of the medication (johnson ) . the nih's decision though was negative, primarily because of the time delays for alternative supplier(s) to receive regulatory approval as well as marketing authority under the orphan drug act (cassedy and love ) . but the nih did require the mount shasta school of medicine-the patent holder-along with genzyme to provide monthly reports while committing itself to reconsider licensing if a third-party request was submitted. additionally, the us food and drug administration (fda) allowed shire plc to give replagal away for free pending us approval of the substitute drug. replagal had been approved in europe as a treatment for fabry disease for more than a decade. during the fabrazyme shortage, the fda encouraged shire to apply for regulatory approval in the united states, but eventually decided to require some additional clinical trials for replagal, which led to shire dropping its application (kelley ) . these two examples, azt and fabrazyme, hinge more on drug pricing and availability than research access, but of course the evident strength of the private patent rights would extend to controlling research access, were that the issue. what the examples do indicate is that access and use of biomedical inventions is controlled by other legislation in addition to patent rights. in particular, fda safety and efficacy testing and other laws like the orphan drug act effectively control access and use, along with patent rights. patents for sure limit access, but often if they evaporated, use rights would still be restricted by other legislation. fully rectifying the research access situation would require more than changing the patent statutes. while an issue not identified by heller and eisenberg ( ) , concerns have arisen regarding so-called defensive patenting. in the current context, this is an effort by public research entities to patent genes and other gene-related materials so as to prevent private-sector control. the consequence can be duplicative and wasteful of research spending, as has been identified in the quest for control of the sars virus. "the race to patent the sars virus seems to be an inefficient means of allocating resources….it will also be difficult to resolve the competing claims between the various parties…" (rimmer , p. ) . economists have studied the "winnertake-all" aspect of patenting for decades with no definitive conclusions. on the one hand, patent races can be wasteful of resources compared to cooperative research; on the other hand, they tend to hasten the identification of solutions (see a brief literature review in jensen ( )). and then there is the recent emergence of "patent assertion entities," better known colloquially as "patent trolls." the troll "business model focuses not on developing or commercializing patented inventions but on buying and asserting pat-ents…" (yeh , p. ii) . while the trolls' patent claims are typically weak-they lose % of infringement cases-they prevail in private settlements by setting royalty demands below litigation costs, thus making a settlement a clear business decision (yeh ) . to date, patent trolls have focused on the it sector, which has its own acute patent thicket issues, but biomedical patents could be a future target. a final consideration of the public vs. private research issue is the ongoing reduction in public research support. the nih budget for human genome research (national human genome research institute) has been flat for the past decade, while the total budget declined slightly, both in real terms (nih n.d.) . the sharp annual budget increases of the s are over; maintaining research support for the foreseeable future depends increasingly on private monies, which often require incentives like patent rights. actual evidence of an anticommons is the most telling factor; however, empirical studies are few. hall and harhoff ( ) cite a study of how the cetus corporation's use of intellectual property led to reductions in research and development. however, another quoted study found such practices "had little impact thus far due to the work-arounds adopted by university researchers: taking out licenses, inventing around, using informal research exemption, and developing available research tools" (hall and harhoff , p. ) . even the golden rice example cited above had a positive outcome. the inventors teamed up with the international rice research institute (irri), a public research organization, and syngenta, a private firm, to improve on the original golden rice product. syngenta then negotiated a free humanitarian use license with the owners of the intellectual and technical property, while commercial users are required to pay royalties (irri n.d.). one semi-documented example is that of chiron, the patent holder for the hepatitis c virus (hcv). gilead sciences is on record for dropping work on a hepatitis c drug after it was sued for infringement because it was unwilling to pay chiron's high initial licensing fees. gilead and other small-to medium-sized companies did license the patents following a reduction in upfront licensing fees (although the postcommercialization royalty rate was increased). the high initial rates had not deterred larger drug firms from licensing the patents, as had done (gillene ). project, which had data publicly released within hours, with those privately held by celera for the - period, after which the ownership entered the public domain. by comparing the celera and public research and commercialization outcomes over the entire sequencing period as well as the post-celera privatization period, and making adjustments for the possibility the celera-sequenced genes had inherently less scientific/commercial value, the author concludes celera's brief ownership "led to reductions in subsequent scientific research and product development on the order of - %" (williams , p. ). these are very strong empirical findings, all the more so because celera's ownership was brief, raising the possibility (not explored in the study) that longer-term ownership would have suppressed research to an even greater extent. while strong, the results have several caveats. one is that celera's ownership was based not on patents-which was the focus of heller and eisenberg's ( ) concerns-but instead was based on "contract law-based ip." second, the analysis is based during the biotech boom, when the demand for prompt access to data and ownership rights could justify the willingness of major private firms to pay $ -$ million annually for access to materials they knew would be entering the public domain in a few years. at a minimum, the uniqueness of the situation makes it more difficult to generalize the results. the author explains the outcome as a result of transaction costs, including the uncertainties over celera's attempts to patent the genes it had sequenced and the conditions of granting free access to academic researchers for "noncommercial" research. and because celera used a different sequencing technique from the public-sector researchers, celera's involvement is often credited with speeding up the entire sequencing process. these are limited examples; more useful evidence is surveys of researchers' experiences. cho et al. ( ) surveyed directors of clinical genetic testing services, concluding that "virtually all laboratory directors felt that patents have had a negative effect on all aspects of clinical testing, except on the quality of testing" (p. ). the ability to conduct research decreased modestly. however, it is important to recognize that the respondents (all but one) were involved in genetic testing for clinical (fee based) rather than research purposes. it is unsurprising that patent holders prevented that group from using patented technologies with no charge and thwarted the development of alternative tests. walsh et al. ( ) contacted attorneys, business managers, and scientists from universities and pharmaceutical and biotechnology firms for in-depth personal interviews. their focus was the more extreme forms of access restriction. they first addressed the sheer number of patents potentially burdening research, a factor identified by heller and eisenberg ( ) when citing patents termed "adrenergic receptor." respondents, however, saw matters differently. only a "small number" of licenses were found to be required- in the final analysis. generally complicated cases involved - key patents, but the "more typical number was zero" (p. ). jenson and murray ( ) though found that "some genes have up to patents asserting rights to various gene uses and manifestations" (p. ) suggesting additional fto issues for researchers. next assessed were research tools (upstream inventions). this too was a problem area identified by heller and eisenberg ( ) , who cited the cetus and oncomouse patents. walsh et al. ( ) , however, found "almost no evidence of such [negotiation] breakdowns that led to a project's cessation" (p. ). nor was royalty stacking found to be a practical barrier, and while the royalty burden could at times become "onerous," "the research always went forward" (p. ). reasons for this outcome include discounts for university and government researchers (walsh et al. ) as well as various negotiation strategies. those include establishing a "ceiling" (as well as a "floor" for individual components) for combined royalties along with the choice of a lump-sum payment or use of a patent pool or employing fieldof-use licenses (shotwell ) . the federal trade commission ( ) subsequently concluded that concerns about the patenting of research tools potentially obstructing commercialization of new products have yet to materialize. for universities and other nonprofits, there is the option of infringement. generally, if the work does not involve fees (such as for clinical tests), infringements are largely ignored; some may receive a cease-and-desist notification, but that is rare and frequently ignored as well. myriad, for example, allowed tests so long as fees were not charged (walsh et al. ) . walsh et al. ( ) subsequently interviewed academic biomedical researchers with similar results. that is, patents in the field do not regularly prevent researchers from access to the knowledge inputs for their research. none of the researchers interviewed abandoned a research project due to impediments from patents; few noted delays. however, nearly % indicated that requests for materials or data had been denied. the cause was not patents per se but rather scientific competition, a history of business activity, and the time and effort needed to fulfill requests, among others. also in , hansen et al. ( ) sought answers on the same topics from the membership rolls of the american association for the advancement of science. a total of responses were received from a random sample of us-based members, with an overall response rate of %. sample weights were varied according to the interest in a scientist member's area of research, with % of respondents in the biological sciences. of particular relevance here, the researchers explored access to research technologies protected by intellectual property rights. those technologies included research tools. for all respondents, including academic respondents, industry was the major source of new technologies. among the biological scientists, research tools constituted the majority of new acquisitions. for academics the dominant exchange mechanism used was a material transfer agreement (mta), while industry scientists relied largely on licensing. most transfers within academia were completed within month; industry required or more months for completion. two thirds of respondents reported no difficulties with technology acquisition, but when difficulties appeared, they were more likely to come from academia than industry. when problems occurred, they resulted most often in delays ( %) and project modification ( %), with only % of projects needing to be abandoned. for all respondents, there was no increase in the amount of licensing required post- compared to the prior period. in , the organization for the economic co-operation and development (oecd) conducted an international workshop on just these topics. conclusions included the following: • the transaction costs of negotiating arrangements within the complexity or overlapping patent claims are real and should not be ignored. • the available evidence does not suggest a systematic breakdown in the licensing of genetic inventions. • evidence of fragmented patent rights, blocking patents, uncertainty, and abuses of the patent monopoly positions appear anecdotal and are not supported by existing economic studies. • in specific areas, there is evidence of problems associated with the numbers and breadth of gene patents, although the exact cause of those problems has not been fully identified. • fto is not unduly impeded (oecd ). adelman and deangelis ( , p. ) examined , + biotech patents over the period of - for trends in numbers and ownership. they concluded that "the lack of concentrated control, the rising number of patent applications, and the continuous influx of new patent owners suggest that overall biotechnology innovation is not being impaired by the growth in patents issued each year." holman ( ) approached the issue from the perspective of human gene patents that had been litigated. the author carefully notes that infringement actions are not the sole measure of negative effects of patents-the payment of royalties would be an obvious one-but nonetheless provide an objective measure of the degree to which patent rights are asserted. four categories of human gene patents are identified: ( ) recombinant production of human therapeutic proteins, ( ) research tools, ( ) genetic testing products and services, and ( ) gene therapy. of the gene patents previously identified, only were found to have been involved in six infringement actions. this is a litigation rate of . %, far below the %- % for all patents. of the six actions, four were settled privately, one dismissed for lack of standing by the plaintiff, and one determined to be non-infringing. that is, not a single human gene patent had been determined by the courts to have been infringing. access to research tools is of particular relevance to researchers, and all but seven infringement actions were identified in this manner. citing a relationship between the level of litigation frequency and non-litigation impact, the author "find[s] that the impact of human gene patent litigation has been relatively modest [which] suggests that non-litigation impact is not as extensive as commonly perceived" (p. ). when considering gene patenting in particular, two additional anticommonsrelated issues arise: ( ) does the uniqueness of a gene prevent "patenting around" it, creating a "double monopoly," and ( ) do patents on genes prevent the sequencing of an individual's genome, a promising new field? huys et al. ( ) examined us patents selected using key words and classifications for genetic-based diseases. the analysis involved scrutinizing by knowledgeable researchers to establish the necessity of having access to the technology for carrying out a diagnosis. three levels of blockage were established-easily circumvented, circumvention requires a substantial investment, and nearly impossible to circumvent ("blocking claims"). only % of the gene patents were considered to be "blocking"-too few to constitute a patent thicket. conversely, % of the method claims were categorized as blocking, enough to constitute a thicket. overall, the authors concluded that "the present analysis and accompanying observations do not point to the existence of a wide patent thicket in genetic diagnostic testing. rather, they highlight a problem of lack of transparency and clarity, leading to legal uncertainty" (huys et al. , p. ) . the recent supreme court decision in myriad, which invalidated patents for isolated genes (see above), will largely obviate this issue going forward. in addition, existing gene patents may possibly be revoked. while the trend in the privatization of biotechnology research is far from ideal, the anticipated anticommons has not materialized significantly. simply stated, that emperor is scantily clad. contributing factors mitigating the anticommons potential are changes in patent-granting practices, use by firms of nonexclusive licenses for research tools, and the facility of simple material transfer agreements used by academic researchers. in many cases, industry and academics developed "working solutions" under which research access is facilitated for noncommercial purposes. "the fact of the matter is that academic researchers who are not engaged in research for commercial use are not affected by the existence of a patent. biotech companies do not sue researchers who are conducting research for purely academic purposes" (feldbaum , p. ) . certainly there are, and have been, holdups and disruptions over access to materials, just not to the extent initially feared. these observations, though, are about the past, and "though fears that gene patents could stifle research have not been borne out, for the most part, commentators are now raising questions about how the many existing gene patents might be used in the future" (cook-deegan , p. ) . so what can be done? two legislative remedies were attempted in the s. one-the genomic research and diagnostic accessibility act of -was a "limited exemption from liability for certain uses of patented genetic sequences and genetic sequence information in the context of basic research and genetic diagnostic information" (holman , p. ) . the bill was not acted on by congress and was not reintroduced when the introducing representative left office. the second would bar the patenting of any nucleotide sequence, or its functions or correlations, or the naturally occurring products it specifies (the genomic research and accessibility act of ). this language is very broad and ambiguous, potentially encompassing all inventions involving polynucleotides (holman ) . the bill never made it out of committee. congressional action to control patent trolls has met a similar fate. following a yearlong effort, the process was declared all but dead when patent reform was withdrawn from the senate judiciary committee's agenda in may (wyatt ) . these experiences strongly suggest any legislative remedy is off the table for the foreseeable future. another possible "remedy" is to be more specific about defining the issue, at least as it applies to patent numbers and ownership. from their assessment of , patents in the biotechnology complex, adelman and deangelis ( , p. ) were able to say that their analysis "also reveals the many pitfalls of seeking to resolve this question at a synoptic level using simple metrics. in this sense, both the advocates of the anticommons theory and enthusiasts of patent characteristics err by oversimplifying the multidimensional character of patent dynamics." further, commentators on the anticommons oftentimes mix the issues of the commercialization of products incorporating patented genes and testing methods with the effect of patents on research access. for example, the patent and related rights issues surrounding golden rice related to commercial use, not research access, did not restrict product development (see above). both topics are worthy of discussion, but they are not the same issue and should not be conflated. and the issue with celera's ip of certain human gene sequences (see above) was based on contract, not patent, law. the consequences of the two may be similar, but a policy remedy would require an entirely different approach. and then it is important not to raise the level of rhetoric, as michael crichton did in a new york times op-ed ( ): "you, or someone you love, may die because of a gene patent that should never have been granted in the first place. sound far-fetched? unfortunately, it's only too real. gene patents are now used to halt research, prevent medical testing, and keep vital information from you and your doctor. gene patents slow the pace of medical advance on deadly diseases." such words do not advance the level of debate on a complex subject. but the concerned have a passionate audience; why else would the citations to the heller and eisenberg ( ) article continue to grow when there is so little empirical support for their cautionary note? so where does that leave the state of affairs of the anticommons? basically it is where it was in ; there "may/can/likely/potentially/might" be a problem (heller and eisenberg ) . but critically, the likelihood has lessened due to time, changes in patent practice, and the largely successful efforts by industry and academia to reach workable solutions while ongoing declines in public research funding will accelerate the need for patent-focused private funding. nonetheless, the potential remains and must be monitored, which is best done by more systematic empirical studies. policy should not be based on anecdotal evidence, especially when that policy is made by the supreme court. patent metrics: the mismeasure of innovation in the biotech patent debate (arizona legal studies discussion paper no. - ) timeline for fabrazyme, replagal effects of patents and licenses on the provision of clinical genetic testing services patents in genomics and human genetics gene patents patenting life (op-ed). the new york times. federal trade commission letter to the honorable lynn rivers chiron relaxes patent licenses intellectual property experiences in the united states scientific community: a report by the project on science and intellectual property in the public interest the tragedy of the commons can patents deter innovation? the anticommons in biomedical research the impact of human gene patents on innovation and access: a survey of human gene patent litigation legal uncertainty in the area of genetic diagnostic testing international rice research institute (irri). n.d. are private companies involved in the golden rice project? patent races the intellectual property landscape of the human genome petition to use authority under the bayh-dole act to promote access to fabryzyme (agalsidase beta), an invention supported by and licensed by the national institutes of health under grant no. dk- shire drops after pulling u.s. application for replagal the intellectual and technical property components of pro-vitamin a rice (golden rice): a preliminary freedom-to-operate review (isaaa issue brief no. - ) evaluating inventions from research institutions centre for the management of intellectual property in health research and development (mihr) and public intellectual property resource for agriculture (pipra) genetic inventions, intellectual property rights and licensing practices: evidence and policies (workshop report the 'golden rice' tale the race to patent the sars virus: the trips agreement and access to essential medicines field-of-use licensing effects of research tool patents and licensing on biomedical innovation where excludability matters: material versus intellectual property in academic biomedical research intellectual property rights and innovation: evidence from human genome legislation to protect against 'patent trolls' is shelved (business day section). the new york times the history of zidovudine (azt): partnership and conflict an overview of the 'patent trolls' debate key: cord- -up vftj authors: brayton, cory; mähler, michael; nicklas, werner title: viral infections date: - - journal: the laboratory mouse doi: . /b - - / - sha: doc_id: cord_uid: up vftj nan in interpreting the microbiological status of laboratory animals, it must be understood that infection and disease are not synonymous. infection refers to the invasion and multiplication of microorganisms in body tissues and may occur with or without apparent disease. disease refers to interruption or deviation from normal structure and function of any tissue, organ, or system. many of the infections with which we are concerned may not cause discernable disease in many strains of mice. however, they may cause inapparent or subclinical changes that can interfere with research. such interference often remains undetected, and therefore modified results may be obtained and published. the types of interference of an agent with experimental results may be diverse. there is no doubt that research complications due to overt infectious disease are significant and that animals with clinical signs of disease should not be used for scientific experiments. but also clinically inapparent infections may have severe effects on animal experiments. there are numerous examples of influences of microorganisms on host physiology and hence of the interference of inapparent infections with the results of animal experiments. many microorganisms have the potential to induce activation or suppression of the immune system or both at the same time but on different parts of the immune system, regardless of the level of pathogenicity. all infections, apparent or inapparent, are likely to increase inter-individual variability and hence result in increased numbers of animals necessary to obtain reliable results. microorganisms, in particular viruses, present in an animal may contaminate biological materials such as sera, cells, or tumours (collins and parker, ; nicklas et al., ) . this may interfere with in vitro experiments conducted with such materials and may also lead to contamination of animals (lipman et al., polymerase chain reaction (pcr) testing of biologics to be inoculated into mice is an important component of a disease prevention programme. finally, latent infections may be activated by environmental factors, by experimental procedures, or by the combination and interaction between various microorganisms. for all these reasons, prevention of infection, not merely prevention of clinical disease, is essential. unfortunately, research complications due to infectious agents are usually considered artefacts and published only exceptionally. information on influences of microorganisms on experiments is scattered in diverse scientific journals, and many articles are difficult to detect. to address this problem, several congresses were held on viral complications on research. the knowledge available was summarized in conference proceedings (melby and balk, ; bhatt et al., b; hamm, ) and has later repeatedly been reviewed (lussier, ; national research council, ; baker, ; nicklas et al., ) . this chapter covers only viral infections of laboratory mice. viral infections of mice have been studied in detail, and comprehensive information on their pathogenic potential, their impact on research, and the influence of host factors such as age, genotype, and immune status on the response to infection is available. bacterial agents may be similarly important, but with few exceptions (e.g. helicobacter species) little is known about their potential to influence host physiology and experiments. even less is known about most parasites in this regard. among fungal agents, only pneumocystis carinii can be expected to play a significant role in contemporary mouse colonies. the nomenclature and taxonomy of viruses described are based on recent nomenclature rules by the international union of microbiological societies ( ) and the universal virus database of the international committee on the taxonomy of viruses (http://www.ictvdb.iacr.ac.uk). retroviruses are not covered in this chapter because they are not included in routine health surveillance programmes and cannot be eradicated with presently available methods. this is because most of them are incorporated in the mouse genome as proviruses and thus are transmitted via germline. the ability to accurately determine whether or not laboratory animals or animal populations have been infected with virus depends on the specificity and sensitivity of the detection methods used. most viral infections in immunocompetent mice are acute or short-term, and lesions are often subtle or subclinical. the absence of clinical disease and pathological changes has therefore only limited diagnostic value. however, clinical signs, altered behaviour, or lesions may be the first indicator of an infection and often provide clues for further investigations. serology is the primary means of testing mouse colonies for exposure to viruses, largely because serological tests are sensitive and specific, are relatively inexpensive, and allow screening for a multitude of agents with one serum sample. they are also employed to monitor biological materials for viral contamination using the map test. serological tests detect specific antibodies, usually immunoglobulin g (igg), produced by the host against the virus and do not actually test for the presence of virus. an animal may have been infected, mounted an effective antibody response, and cleared the virus, but remains seropositive for weeks or months or forever, even though it is no longer infected or shedding the agent. active infection can only be detected by using direct diagnostic methods such as virus isolation, electron microscopy, or pcr. meanwhile, pcr assays have been established for the detection of almost every agent of interest. they are highly sensitive and depending on the demands, they can be designed to broadly detect all members of a genus or only one species. however, good timing and selection of the appropriate specimen is critical for establishing the diagnosis. in practice, combinations of diagnostic tests are often necessary including the use of sentinel animals or immunosuppression to get clear aetiological results or to avoid consequences from false-positive results. reports on the prevalence of viral infections in laboratory mice throughout the world have been published frequently. in general, the microbiological quality of laboratory mice has constantly improved during the last decades, and several agents (e.g. herpes-and polyomaviruses) have been essentially eliminated from contemporary colonies due to advances in diagnostic methodologies and modern husbandry and rederivation practices (jacoby and lindsey, ; zenner and regnault, ; livingston and riley, ) . they may, however, reappear, since most have been retained or are still being used experimentally. furthermore, the general trend towards better microbiological quality is challenged by the increasing reliance of biomedical research on genetically modified and immunodeficient mice, whose responses to infection and disease can be unpredictable. increasing numbers of scientists are creating genetically modified mice, with minimal or no awareness of infectious disease issues. as a consequence, they are more frequently infected than 'standard' strains of mice coming from commercial breeders, and available information on their health status is often insufficient. frequently, they are exchanged between laboratories, which amplifies the risk of introducing infections from a range of animal facilities. breeding cessation strategies that have been reported to eliminate viruses from immunocompetent mouse colonies may prove to be costly and ineffective in genetically modified colonies of uncertain or incompetent immune status. it must also be expected that new agents will be detected, although only occasionally. infections therefore remain a threat to biomedical research, and users of laboratory mice must be cognizant of infectious agents and the complications they can cause. two members of the family herpesviridae can infect mice (mus musculus). mouse cytomegalovirus (mcmv- ) or murid herpesvirus (muhv- ) belongs to the subfamily betaherpesvirinae, genus muromegalovirus. murid herpesvirus (muhv- ) or mouse thymic virus (mtv) has not yet been assigned to a genus within the family herpesviridae. both viruses are enveloped, doublestranded dna viruses that are highly host-specific and relatively unstable to environmental conditions such as heat and acidic ph. both agents are antigenically distinct and do not cross-react in serological tests, but their epidemiology is similar (cross et al., ) . seropositivity to mcmv- was reported in less than % of specified pathogen-free (spf) mouse colonies in the usa in (jacoby and lindsey, ) , and some institutions reported to have mice 'on campus' that were positive for mtv. in a more recent study, a low rate ( . %) of samples was found to be positive for mcmv- whereas no sample tested positive for mtv (livingston and riley, ) . the data available suggest that the prevalence of both viruses in contemporary colonies and thus their importance for laboratory mice is negligible. however, both mcmv- and mtv are frequently found in wild mice, which may be coinfected with both viruses (national research council, ; singleton et al., ) . natural infection with mcmv- causes subclinical salivary gland infection in mice. the virus persists in the salivary glands (particularly in the submaxillary glands) and also in other organs (osborn, ; kercher and mitchell, ; lenzo et al., ) . most information concerning the pathogenesis of mcmv- infection is based on experimental infection studies. these results are very difficult to summarize because the outcome of experimental infection in laboratory mice depends on various factors such as mouse strain and age, virus strain and passage history, virus dose, and route of inoculation (osborn, ) . in general, newborn mice are most susceptible to clinical disease and to lethal infection. virus replication is observed in newborn mice in many tissues (for details, see osborn, ) and appears in the salivary glands towards the end of the first week of infection when virus concentrations in liver and spleen have already declined. resistance develops rapidly after weaning between days and of age. experimental infection of adult mice results in mortality only in susceptible strains and only if high doses are administered. not even intravenous or intraperitoneal injections of adult mice usually produce signs of illness in resistant strains (shanley et al., ) . mice of the h- b (e.g. c bl/ ) and h- d (e.g. balb/c) haplotype are more sensitive to experimental infection than are mice of the h- k haplotype (e.g. c h), which are approximately -fold more resistant to mortality than are those of the b or d haplotype (osborn, ) . subclinical or latent infections can be activated by immunosuppression (e.g. with cyclophosphamide or cortisone). reactivation of mcmv- occurs also after implantation of latently infected salivary glands into prkdc scid mice (schmader et al., ) . immunodeficient mice lacking functional t cells or natural killer (nk) cells, such as foxn nu and lyst bg mice, are more susceptible than are immunocompetent animals. experimental infection in prkdc scid mice causes severe disease or is lethal, with necrosis in spleen, liver, and other organs, and multinucleate syncytia with inclusion bodies in the liver (reynolds et al., ) . similar to aids patients infected with human cytomegalovirus, athymic foxn nu mice experimentally infected with mcmv also develop adrenal necrosis (shanley and pesanti, ) . the virus also replicates in the lungs leading to pneumonitis whereas in heterozygous (foxn nu / ϩ ) littermates replication and disease are not seen (shanley et al., ) . the most prominent histological finding of cytomegaloviruses is enlarged cells (cytomegaly) of salivary gland epithelium with eosinophilic nuclear and cytoplasmic inclusion bodies. the inclusion bodies contain viral material and occur in other organs such as liver, spleen, ovary, and pancreas (osborn, ) . depending on inoculation route, dose, strain, and age of mice, experimental infections may result in inflammation or cytomegaly with inclusion bodies in a variety of tissues, pneumonitis, myocarditis, meningoencephalitis, or splenic necrosis in susceptible strains (national research council, ; osborn, ; percy and barthold, ) . virus is transmitted oronasally by direct contact and is excreted in saliva, tears, and urine for several months. wild mice serve as a natural reservoir for infection. the virus is most frequently transmitted horizontally through mouse-to-mouse contact but does not easily spread between cages. it is generally assumed that mcmv- has a very low prevalence in contemporary colonies of laboratory mice. the risk of introduction into facilities housing laboratory mice is very low if wild mice are strictly excluded. monitoring is necessary if populations of laboratory mice may have been contaminated by contact with wild mice. as for other viruses, enzyme-linked immunosorbent assay (elisa) and indirect immunofluorescence assay (ifa) are the most appropriate tests for detecting antibodies. as the virus persists, direct demonstration of mcmv- in infected mice is possible by pcr (palmon et al., ) or by virus isolation using mouse embryo fibroblasts ( t cells). although mcmv- does not play a significant role as a natural pathogen of laboratory mice, it is frequently used as a model for human cytomegalovirus infection (bolger et al., ) . however, the virus is known to influence immune reactions in infected mice and may therefore have impact on immunological research (osborn, ; national research council, ; baker, ) . mouse thymic virus was detected during studies in which samples from mice were passaged in newborn mice. unlike other herpesviruses, the virus can not yet be cultured in vitro and is propagated by intraperitoneal infection of newborn mice. the thymus is removed - days later, and thymus suspensions serve as virus material for further studies. the prevalence of mtv is believed to be low in laboratory mice, and for this reason and also due to the difficulties in virus production for serological assays, it is not included in many standard diagnostic or surveillance testing protocols. limited data are available indicating that it is common in wild mice, and it is also found in laboratory mice (osborn, ; morse, ; national research council, ) . further, mtv obviously represents a significant source of contamination of mcmv- (and vice versa) if virus is prepared from salivary glands since both viruses cause chronic or persistent salivary gland infections and can coinfect the same host. all mouse strains are susceptible to infection, but natural or experimental infection of adult mice is subclinical. gross lesions appear only in the thymus and only if experimental infection occurs at an age of less than about days. virus is present in the thymus but may also be found in the blood and in salivary glands of surviving animals. salivary glands are the only site yielding positive virus isolations if animals are infected as adults. mouse thymic virus also establishes a persistent infection in athymic foxn nu mice, but virus shedding is reduced compared to euthymic mice and virus recovery is possible only in a lower percentage of mice (morse, ) . pathological changes caused by mtv occur in the thymus, and reduced thymus mass due to necrosis in suckling mice is the most characteristic gross lesion (percy and barthold, ) . lymphoid necrosis also may occur in lymph nodes and spleen (wood et al., ) , with necrosis and recovery similar to that in the thymus. in mice infected during the first days after birth, necrosis of thymus becomes evident within - days, and its size and weight are markedly reduced at day - . intranuclear inclusions may be present in thymocytes between days - post infection. the thymus and the affected peripheral tissues regenerate within weeks after infection. regardless of the age of mice at infection, a persistent infection is established in the salivary glands, and infected animals shed virus for life. several alterations of immune responses are associated with neonatal mtv infection. there is transient immunosuppression, attributable to lytic infection of t lymphocytes, but activity (e.g. response of spleen cells to t cell mitogens) returns to normal as the histological repair progresses (wood et al., ) . selective depletion of cd ϩ t cells by mtv results in autoimmune disease (morse and valinsky, ; morse et al., ) . information about additional influences on the immune system is given by osborn ( ), national research council ( ), and baker ( . in experimentally infected newborn mice, oral and intraperitoneal infections similarly result in thymus necrosis, seroconversion, and virus shedding suggesting that the oral-nasal route is likely to be involved in natural transmission (morse, ) . the virus spreads to cage mates after long periods of contact. it is transmitted between mice kept in close contact, and transmissibility from cage to cage seems to be low. mouse thymic virus is not transmitted to foetuses by the transplacental route, and intravenous infection of pregnant mice does not lead to congenital damage, impairment in size or development, or abortion (st-pierre et al., ) . mouse thymic virus and mcmv- do not crossreact serologically (cross et al., ) . serological monitoring of mouse populations for antibodies to mtv is possible by ifa testing, which is commercially available; elisa tests have also been established (morse, b) . elisa and complement fixation yield similar results . it must be noted that the immune response depends on the age at infection. antibody responses are not detectable in mice infected as newborns whereas adult mice develop high titres that are detectable by serological testing. if neonatal infection is suspected, homogenates of salivary glands or other materials can be inoculated into pathogen-free newborn mice followed by gross and histological examination of thymus, lymph nodes, and spleens for lymphoid necrosis (morse, ) . alternatives to the in vivo infectivity assay for detecting mtv in infected tissues include a competition elisa (prattis and morse, ) and map testing, although this is slightly less sensitive than infectivity assays (morse, a) . very little experience exists on eradication methods for mtv due to its low prevalence in contemporary mouse colonies. methods that eliminate other herpesviruses likely will eliminate mtv. procurement of animals of known negative mtv status is an appropriate strategy to prevent infection. strict separation of laboratory mice from wild rodents is essential to avoid introduction into laboratory animal facilities. mousepox (ectromelia) virus (ectv) is a member of the genus orthopoxvirus belonging to the family poxviridae. it is antigenically and morphologically very similar to vaccinia virus and other orthopoxviruses. poxviruses are the largest and most complex of all viruses with a diameter of nm and a length of - nm. mousepox (ectromelia) virus contains one molecule of double-stranded dna with a total genome length of , nucleotides. it is the causative agent of mousepox, a generalized disease in mice. experimental transmission to young rats (up to days of age) is possible (jandasek, ; buller et al., ) . the virus is resistant to desiccation, dry heat, and many disinfectants. it is not consistently inactivated in serum heated min at Њc (lipman et al., b) and persists for weeks when maintained at Њc in foetal bovine serum (bhatt and jacoby, a) . effective disinfectants include vapour-phase formaldehyde, sodium hypochlorite, and iodophores (small and new, ; national research council, ) . historically, ectv has been an extremely important natural pathogen of laboratory mice. the virus was widespread in mouse colonies worldwide and can still be found in several countries. between and almost individual ectromelia outbreaks were reported in the usa. the last major epizootic in the usa occurred in - and has been described in great detail (e.g. wagner and daynes, ) . severe outbreaks were also described in various european countries (deerberg et al., ; owen et al., ; osterhaus et al., ) . a more recent outbreak in the usa, which resulted in the eradication of almost mice in one institution, was described by dick et al. ( ) . the most recent well-documented case of mousepox was published by lipman et al. ( b) . few additional but unpublished cases of ectromelia have been observed thereafter. in a recent survey conducted in the usa, one population was reported to be seropositive for mousepox (jacoby and lindsey, ) . natural infections manifest differently depending on many factors. mousepox may occur as a rapidly spreading outbreak with acute disease and deaths, or may be inconspicuous with slow spreading and mild clinical signs. the mortality rate can be very low in populations in which the virus has been present for long periods. the infection usually takes one of three clinical courses: acute asymptomatic infection, acute lethal infection (systemic form), or subacute to chronic infection (cutaneous form ; fenner, fenner, , manning and frisk, ; national research council, ; dick et al., ) . the systemic or visceral form is characterized clinically by facial oedema, conjunctivitis, multisystemic necrosis, and usually high mortality. this form is less contagious than the cutaneous form because the animals die before there is virus shedding. the cutaneous form is characterized by typical dermal lesions and variable mortality. the outcome of infection depends on many factors including strain and dose of virus; route of viral entry; strain, age, and sex of mouse; husbandry methods; and duration of infection in the colony. while all mouse strains seem to be susceptible to infection with ectv, clinical signs and mortality are strain-dependent (fenner, ; wallace and buller, , ; brownstein et al., b) . acute lethal (systemic) infection occurs in highly susceptible inbred strains such as dba/ , dba/ , balb/c, a, and c h/hej. immunodeficient mice may also be very susceptible (allen et al., ) . outbreaks among susceptible mice can be explosive, with variable morbidity and high mortality (Ͼ %). clinical disease may not be evident in resistant strains such as c bl/ and akr, and the virus can be endemic in a population for long periods before being recognized. furthermore, females seem to be more resistant to disease than males, at least in certain strains of mice brownstein et al., b) . the mechanisms determining resistance versus susceptibility are not fully understood but appear to reflect the action of multiple genes. the genetic loci considered to be important include h- d b (termed rmp- , resistance to mousepox, on chromosome ; o'neill et al., ) , the c genes (rmp- , on chromosome ), rmp- , localized to a region on chromosome encoding the nk cell receptor nkr-p alloantigens (brownstein and gras, ) , the nitric oxide synthase locus on chromosome (karupiah et al., ) , and the signal transducer and activator of transcription locus on chromosome (mahalingam et al., ) . clearance of the virus by the immune system is absolutely dependent upon the effector functions of cd ϩ t cells while nk cells, cd ϩ t cells, and macrophages are necessary for the generation of an optimal response (niemialtowski et al., ; delano and brownstein, ; karupiah et al., ) . mousepox (ectromelia) virus usually enters the host through the skin with local replication and extension to regional lymph nodes (fenner, (fenner, , wallace and buller, ; national research council, ) . it escapes into the blood (primary viraemia) and infects splenic and hepatic macrophages resulting in necrosis of these organs and a massive secondary viraemia. this sequence takes approximately week. many animals die at the end of this stage without premonitory signs of illness; others develop varying clinical signs including ruffled fur, hunched posture, swelling of the face or extremities, conjunctivitis, and skin lesions (papules, erosions, or encrustations mainly on ears, feet, and tail; figure . ). necrotic amputation of limbs and tails can sometimes be seen in mice that survive the acute phase, hence the original name of the disease 'ectromelia' (meaning absent or short limbs; figure . ). common gross lesions of acute mousepox include enlarged lymph nodes, peyer's patches, spleen, and liver; multifocal to semiconfluent white foci of necrosis in the spleen and liver; and haemorrhage into the small intestinal lumen (allen et al., ; fenner, ; dick et al., ; percy and barthold, ) . in animals that survive, necrosis and scarring of the spleen can produce a mosaic pattern of white and red-brown areas that is a striking gross finding. the most consistent histological lesions of acute mousepox are necroses of the spleen (figure lymph nodes, peyer's patches, thymus, and liver (allen et al., ; fenner, ; dick et al., ; lipman et al., b; percy and barthold, ) . occasionally, necrosis may also be observed in other organs such as ovaries, uterus, vagina, intestine, and lungs. the primary skin lesion, which occurs about a week after exposure at the site of inoculation (frequently on the head), is a localized swelling that enlarges from inflammatory oedema. necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. secondary skin lesions (rash) develop - days later as the result of viraemia (figure . ). they are often multiple and widespread and can be associated with conjunctivitis. the skin lesions also can ulcerate and scab before scarring. mucosal and dermal epithelial cells may have characteristic intracytoplasmic eosinophilic (cowdry type a) inclusion bodies ( figure . ). basophilic (cowdry type b) inclusions may be found in the cytoplasm of all infected cells, especially in hepatocytes. natural transmission of ectv mainly occurs by direct contact and fomites (fenner, ; wallace and buller, ; national research council, ) . the primary route of infection is through skin abrasions. faecal-oral and aerosol routes may also be involved (werner, ) . in addition, the common practice of cannibalism by mice may contribute to the oral route of infection (bhatt and jacoby, b) . intrauterine transmission is possible at least under experimental conditions (schwanzer et al., ) . virus particles are shed from infected mice (mainly via scabs and/or faeces) for about - weeks, even though the virus can persist for months in the spleen of an occasional mouse (bhatt and jacoby, b; national research council, ) . cage-to-cage transmission of ectv and transmission between rooms or units is usually low and largely depends on husbandry practices (e.g. mixing mice from different cages). importantly, the virus may not be transmitted effectively to sentinel mice exposed to dirty bedding (lipman et al., b) . various tests have been applied for the diagnosis of ectromelia. previous epidemics were difficult to deal with because of limited published data and information on the biology of the virus and the lack of specific and sensitive assays (wallace, ) . in the s, diagnosis relied on clinical signs, histopathology, and animal passages of tissues from moribund and dead animals. culture of the virus on the chorioallantoic membrane of embryonated eggs was also applied. serology is currently the primary means of testing mouse colonies for exposure to ectv. the methods of choice are elisa and ifa; they are more sensitive and specific than the previously used haemagglutination inhibition (hi) assay (collins et al., ; buller et al., ; aclad, ) . both tests detect antibodies to orthopoxviruses and do not distinguish between ectv and vaccinia virus. vaccinia virus is commonly used as antigen for serological testing to avoid the risk of infection for mice. thus, false-positive serological reactions may be found after experimental administration of replication-competent vaccinia virus. it has been shown that even cage contact sentinels may develop antibodies, and vaccinia virus leading to seroconversion may even be transmitted by dirty bedding (gaertner et al., ) . confirmation of positive serological results is important before action is taken because vaccinia virus is increasingly prevalent in animal facilities as a research tool (e.g. for vaccination or gene therapy). as observed in different outbreaks, serological testing is of little value in the initial stages of the disease. for example, in the outbreak described by dick et al. ( ) depopulation was nearly completed before serological confirmation was possible. for this reason, negative serological results should be confirmed by direct detection methods (pcr, immunohistochemistry, virus isolation) or by histopathology, especially when clinical cases suggestive of mousepox are observed. polymerase chain reaction assays to detect different genes of poxviruses in infected tissues have been described by dick et al. ( ) , neubauer et al. ( ) , and lipman et al. ( b) . the key to prevention and control of mousepox is early detection of infected mice and contaminated biological materials. all institutions that must introduce mice from other than commercial barrier facilities should have a health surveillance programme and test incoming mice. perhaps even more important than living animals are samples from mice (tumours, sera, tissues). the virus replicates in lymphoma and hybridoma cell lines (buller et al., ) , and such cells or material derived from them may therefore be a vehicle for inadvertent transfer between laboratories. the last two published outbreaks of ectromelia were both introduced into the facilities by mouse serum (dick et al., ; lipman et al., b) . lipman et al. ( b) found that the contaminated serum originated from a pooled lot of l that had been imported from china. because mouse serum commonly is sold to the end user in small aliquots (few millilitres), it has to be expected that aliquots of the contaminated lot are still stored in numerous freezers. both cases provide excellent examples of why map or pcr testing should be performed on all biological materials to be inoculated into mice. eradication of mousepox usually has been accomplished by elimination of the affected colonies, disinfection of rooms and equipment, and disposal of all infected tissues and sera. while culling of entire mouse colonies is the safest method for eradication of mousepox, it is not a satisfactory method due to the uniqueness of numerous lines of genetically modified animals housed in many facilities. several studies indicate that mousepox is not highly contagious bhatt and jacoby a,b ) and that it may be self-limiting when adequate husbandry methods are applied. therefore, strict quarantine procedures along with cessation of breeding (to permit resolution of infection) and frequent monitoring with removal of clinically sick and seropositive animals are a potential alternative. the period from the last births before the break until the first matings after the break should be at least weeks (bhatt and jacoby, b) . sequential testing of immunocompetent contact sentinels for seroconversion should be employed with this option. in the past, immunization with live vaccinia virus was used to suppress clinical expression of mousepox. vaccination may substantially reduce the mortality rate, but it does not prevent virus transmission or eradicate the agent from a population bhatt and jacoby, c) . after vaccination, typical pocks develop at the vaccination site, and infectious vaccinia virus is detectable in spleen, liver, lungs, and thymus (jacoby et al., ) . vaccination also causes seroconversion so that serological tests are not applicable for health surveillance in vaccinated populations. it is therefore more prudent to control mousepox by quarantine and serological surveillance than by relying on vaccination. mortality and clinical disease are the major factors by which ectv interferes with research. severe disruption of research can also occur when drastic measures are taken to control the infection. the loss of time, animals, and financial resources can be substantial. murine adenoviruses (madv) are non-enveloped, double-stranded dna viruses of the family adenoviridae, genus mastadenovirus. two distinct strains have been isolated from mice. the fl strain (madv- ) was first isolated in the usa as a contaminant of a friend leukaemia (hartley and rowe, ) ; the k strain (madv- ) was first isolated in japan from the faeces of a healthy mouse (hashimoto et al., ) . both strains are now considered to represent different species (hamelin and lussier, ; jacques et al., a,b) . in laboratory mice, seropositivity to adenoviruses was reported in % of spf colonies and in % of non-spf colonies in the usa (jacoby and lindsey, ) . antibodies were also detected at a low prevalence rate in french colonies (zenner and regnault, ) , but the virus strain used as antigen is not mentioned. a similar range of positive samples was reported by livingston and riley ( ) . antibodies to madv were also found in wild mice and in rats (otten and tennant, ; smith et al., ) . both viruses are not known to cause clinical disease in naturally infected, immunocompetent mice. however, madv- can cause a fatal systemic disease in suckling mice after experimental inoculation (hartley and rowe, ; heck et al., ; wigand, ) . disease is characterized by scruffiness, lethargy, stunted growth, and often death within days. experimental infection of adult mice with madv- is most often subclinical and persistent (richter, ) but can cause fatal haemorrhagic encephalomyelitis with neurological symptoms, including tremors, seizures, ataxia, and paralysis, in susceptible c bl/ and dba/ j mice (guida et al., ) . balb/c mice are relatively resistant to this condition. athymic foxn nu mice experimentally infected with madv- develop a lethal wasting disease (winters and brown, ) . similarly, prkdc scid mice succumb to experimental infection with madv- (pirofski et al., ) . gross lesions in response to natural madv infections are not detectable. occasional lesions observed after experimental infection with madv- include small surface haemorrhages in the brain and spinal cord of c bl/ and dba/ j mice (guida et al., ) , duodenal haemorrhage in foxn nu mice (winters and brown, ) , and pale yellow livers in prkdc scid mice (pirofski et al., ) . histologically, experimental madv- infection of suckling mice is characterized by multifocal necrosis and large basophilic intranuclear inclusion bodies in liver, adrenal gland, heart, kidney, salivary glands, spleen, brain, pancreas, and brown fat (heck et al., ; margolis et al., ; national research council, ; percy and barthold, ) . in experimentally induced haemorrhagic encephalomyelitis, multifocal petechial haemorrhages occur throughout the brain and spinal cord, predominantly in the white matter, and are attributed to infection and damage to the vascular epithelium of the central nervous system (cns; guida et al., ) . histopathological manifestations in madv- -infected prkdc scid mice are marked by microvesicular fatty degeneration of hepatocytes (pirofski et al., ) . in contrast to madv- , the tissue tropism of madv- is limited to the intestinal epithelium. naturally or experimentally infected mice develop intranuclear inclusions in enterocytes, especially in the ileum and caecum (takeuchi and hashimoto, ; otten and tennant, ; national research council, ; percy and barthold, ) . transmission of madv primarily occurs by ingestion. madv- is excreted in the urine and may be shed for up to years (van der veen and mes, ). murine adenovirus- infects the intestinal tract and is shed in faeces for only a few weeks in immunocompetent mice (hashimoto et al., ) ; immunodeficient mice may shed the virus for longer periods (umehara et al., ) . murine adenovirus infections are routinely diagnosed by serological tests. however, there is a one-sided cross reactivity of madv- with madv- (wigand et al., ) . serum from mice experimentally infected with madv- yielded positive reactions in serological tests with both viruses while serum from mice infected with madv- reacted only with the homologous antigen . smith et al. ( ) reported that sera may react with madv- or madv- or both antigens. occasional reports of mice with lesions suggestive of adenovirus infections and negative serology (with madv- ) indicate that the infection may not be detected if only one virus is used as antigen (luethans and wagner, ) . it has therefore become standard practice to test sera for antibodies to both madv- and madv- . the common methods are ifa and elisa, and both are more sensitive than the previously used complement fixation test. the low prevalence in colonies of laboratory mice indicate that madv can easily be eliminated (e.g. by hysterectomy derivation or embryo transfer) and that barrier maintenance has been very effective in preventing infection. the low pathogenicity and the low prevalence in contemporary mouse populations are the main reasons why adenoviruses are considered to be of little importance. however, immunodeficient mice are increasingly used and candidates for natural infections and wasting disease (richter, ) , and the viruses might easily be spread by the exchange of genetically modified mice and therefore re-emerge. only few influences on research attributable to madv have been published. for example, it has been shown that madv- significantly aggravates the clinical course of scrapie disease in mice (ehresmann and hogan, ) . natural infections with madv could also interfere with studies using adenovirus as a gene vector. polyomaviridae are enveloped, double-stranded dna viruses. two different agents of this family exclusively infect mice (mus musculus), and both belong to the genus polyomavirus. murine pneumotropic virus (mptv) has formerly been known as 'newborn mouse pneumonitis virus' or 'k virus' (named after l. kilham who first described the virus). the second is murine polyomavirus (mpyv). both are related but antigenically distinct from each other (bond et al., ) . they are enzootic in many populations of wild mice but are very uncommon in laboratory mice. even older reports indicate that both have been eradicated from the vast majority of contemporary mouse colonies, and their importance is negligible (national research council, ) . seropositivity to these viruses was not reported in a survey conducted in the usa (jacoby and lindsey, ) . in a retrospective study in french facilities, antibodies to mpyv were found in of colonies, and all samples tested for mptv were negative (zenner and regnault, ) . comparable data were reported by livingston and riley ( ) . due to their low prevalence, both viruses are not included in the list of agents for which testing is recommended on a regular basis by felasa (nicklas et al., ) . although polyomavirus genes, especially those of sv are used widely in gene constructs for insertional mutagenesis, very few reports have been published on spontaneous or experimental disease due to mpyv or mptv in the last - years. the reader is therefore referred to previous review articles for details (eddy ; parker and richter, ; richter, ; shah and christian, ; national research council, ; orcutt, ; porterfield and richter, ) . natural infections with mptv are subclinical. the prevalence of infection is usually low in an infected population. the virus may persist in infected animals for months and perhaps for life depending on the age at infection and is reactivated under conditions of immunosuppression. virus replicates primarily in endothelial cells, but renal tubular epithelial cells are the major site of viral persistence (greenlee et al., (greenlee et al., , . clinical signs are observed only after infection of infant mice less than - days of age. infected pups suddenly develop respiratory symptoms after an incubation period of approximately week, and many die within a few hours of onset of symptoms with an interstitial pneumonia caused by productive infection of and damage to pulmonary endothelium. endothelial cells in other organs are involved in virus replication also (ikeda et al., ; greenlee et al., ) . in older suckling mice, mptv produces a more protracted infection, and the virus or viral antigen can be detected for as long as months. in adult animals, the virus produces a transient asymptomatic infection. even in immunodeficient foxn nu mice, experimental infection of adults is clinically asymptomatic although virus is detectable for a period of several months (greenlee, ) . in vitro cultivation of mptv is difficult. no susceptible permanent cell line is known to support growth. it can be cultured in primary mouse embryonic cells, but viral titres are not sufficient for use in serological assays (greenlee and dodd, ) . for this reason, the hi test using homogenates of livers and lungs of infected newborn mice is still frequently used, but ifa and elisa tests are also available (groen et al., ) . furthermore, a pcr test for demonstration of mptv in biological samples has been published (carty et al., ) . murine polyomavirus was first detected as a contaminant of murine leukaemia virus (mulv) when sarcomas developed in mice after experimental inoculation of contaminated samples. it has later been shown to be a frequent contaminant of transplantable tumours (collins and parker, ) . natural infection of mice is subclinical, and gross lesions including tumours are usually not found. tumour formation occurs if mice are experimentally infected at a young age or if they are inoculated with high virus doses. development of tumours may be preceded by multifocal necrosis and mortality during the viraemic stage (percy and barthold, ) . parotid, salivary gland, and mammary tumours are common, and sarcomas or carcinomas of kidney, subcutis, adrenal glands, bone, cartilage, teeth, blood vessels, and thyroid occur also. virus strains vary with regard to the tumour types or lesions that they induce, and mouse strains vary in their susceptibility to different tumour types. those of c bl and c br/cd lineage are considered to be the most resistant strains; athymic foxn nu mice are considered to be most susceptible; c h mice are particularly susceptible to adrenal tumours and a mice tend to develop bone tumours. immunosuppression or inoculation into immunodeficient strains (e.g. foxn nu ) also support the growth of tumours. on the other hand, experimental infection of adult immunocompetent mice does not result in tumour formation because the immune response suppresses tumour growth, and newborn immunocompetent mice develop runting only if inoculated with high virus doses (atencio et al., ) . after experimental intranasal infection, mpyv initially infects the respiratory tract followed by a systemic phase in which liver, spleen, kidney, and the colon become infected (dubensky et al., ) . the virus is shed in faeces and in all body fluids, and transmission occurs rapidly by direct contact between animals, but also between cages in a room. further, intrauterine transmission has been documented after experimental infection (mccance and mims, ) . murine polyomavirus persists in all organs in prkdc scid mice while viral dna is detectable in immunocompetent mice after experimental infection for only a limited period of about weeks (berke et al., ) . however, virus may persist and can be reactivated by prolonged immunosuppression (rubino and walker, ) or during pregnancy, at least in young mice (mccance and mims, ) . biological materials of mouse origin are likely to be the most common source of contamination of laboratory mice emphasizing the importance of map or pcr screening of biological materials to be inoculated into mice. the most frequently used tests for health surveillance of mouse colonies are elisa and ifa (aclad, ) ; in addition, the hi test is still used. latent infections can be detected by intracerebral inoculation of neonate mice or by map testing, but direct demonstration of virus in biological samples is also possible by pcr testing (porterfield and richter, ; carty et al., ) . parvoviruses are non-enveloped small viruses (approximately nm in diameter) with a single-stranded dna genome of approximately nucleotides. murine parvoviruses are members of the family parvoviridae, genus parvovirus. they are remarkably resistant to environmental conditions like heat, desiccation, acidic and basic ph-values. two distinct serotypes infect laboratory mice: the mice minute virus (mmv) and the mouse parvovirus (mpv). nonstructural proteins (ns- and ns- ) are highly conserved among both viruses whereas the capsid proteins (vp- , vp- , vp- ) are more divergent and determine the serogroup (ball-goodrich and johnson, ) . both viruses require mitotically active cells for replication. severe infections are therefore not found in mature animals due to the lack of a sufficient number of susceptible cells in tissues. general aspects of rodent parvovirus infections and their potential effects on research results have been reviewed (tattersall and cotmore, ; national research council, ; jacoby et al., ) . already in the mid- s mouse colonies were identified that gave positive reactions for mmv by ifa but not by hi tests. it was subsequently shown that these colonies were infected with a novel parvovirus, initially referred to as 'mouse orphan parvovirus'. the first isolate of mpv was detected as a contaminant of cultivated t-cell clones interfering with in vitro immune responses (mckisic et al., ) and was named 'mouse parvovirus'. it does not replicate well in currently available cell cultures, and sufficient quantities of virus for serological tests are difficult to generate. hitherto, only very few isolates of mpv have been cultured and characterized on a molecular basis (ball-goodrich and johnson, ; besselsen et al., ) . at present, mpv is among the most common viruses in colonies of laboratory mice. the prevalence of sera positive for parvoviruses was nearly % in a study from livingston et al. ( ) , with the majority of sera being positive for mpv. this is consistent with a recent survey conducted in the usa showing that almost % of non-spf colonies were seropositive (jacoby and lindsey, ) . similar results were obtained for genetically modified mice in japan (yamamoto et al., ) , in contrast to earlier studies indicating that the infection was rare in japan (ueno et al., ) . clinical disease and gross or histological lesions have not been reported for mice naturally or experimentally infected with mpv. infections are subclinical even in newborn and immunocompromised animals . in contrast to many other viruses infecting mice, viral replication and excretion is not terminated by the onset of host immunity. tissue necrosis has not been observed at any stage of infection in infected infant or adult mice . humoral immunity to mpv does not protect against mmv infections and vice versa (hansen et al., ) . serological surveys have indicated that mpv naturally infects only mice. differences in mouse strain susceptibility to clinical mpv infection do not exist. however, seroconversion seems to be strain-dependent. after experimental infection, seroconversion occurred in all c h/hen mice, fewer balb/c, dba/ , and icr mice, and seroconversion could not be detected in c bl/ mice (besselsen et al., ) . diagnosis of mpv infection by pcr testing of small intestine and mesenteric lymph nodes also depended on the mouse strain. mpv dna was detected in all mouse strains evaluated except dba/ even though seroconversion was detected in these mice. after oral infection, the intestine is the primary site of viral entry and replication. the virus spreads to the mesenteric lymph nodes and other lymphoid tissues, where it persists for more than months , and seems to be excreted via the intestinal and the urinary tract. after experimental inoculation of weanling mice, mpv is transmitted to cagemates by direct contact for - weeks , and transmission by dirty bedding is also possible. these results implicate a role for urinary, faecal, and perhaps respiratory excretion of virus. another study showed that naturally infected mice may not transmit the virus under similar experimental conditions (shek et al., ) . serology is a useful tool to identify mpv infections in immunocompetent hosts, but reaching a diagnosis based on serological assays may be difficult and requires a good knowledge of the available techniques. neither the virion elisa nor hi are practical screening tests for mpv because they require large quantities of purified mpv which is difficult to obtain. diagnosis of mpv infections has long been made on the basis of an mmv hi-negative result coupled with an mmv ifapositive result. a generic rodent parvovirus elisa using a recombinant ns- protein as antigen has been developed , but mpv ifa and mpv hi assays are more sensitive techniques than the ns- elisa and the mmv ifa (besselsen et al., ) . recently, elisa tests have been described that use recombinant vp- and provide sensitive and serogroupspecific assays for the diagnosis of mpv infections in mice (ball-goodrich et al., ; livingston et al., ) . in immunodeficient mice that do not generate a humoral immune response, pcr assays can be used to detect mpv (besselsen et al., ; redig and besselsen, ) and other parvoviruses. mpv has been shown to persist for at least weeks in the mesenteric lymph nodes . this tissue is considered the best suited for pcr analysis, but spleen and small intestine can also be used with good success (besselsen et al., ) . the virus persists sufficiently long in mesenteric lymph nodes so that pcr assays may also be used as a primary screening tool for laboratories that do not have access to specific mpv antigenbased serological assays. polymerase chain reaction is further a good confirmatory method for serological assays and has also been described for the detection of parvoviruses in cell lines and tumours (yagami et al., ) . in addition, the map test has been reported as a sensitive tool to detect mpv (shek et al., ) . given the high environmental stability of the virus and the potential fomite transmission together with the long virus persistence in infected animals, spontaneous disappearance from a mouse population (e.g. by cessation of breeding) is very unlikely. eradication of infection is possible by elimination of infected animals and subsequent replacement with uninfected mice, and the agent can be eliminated from breeding populations only by embryo transfer or by hysterectomy. although there are few published reports of confounding effects of mpv on research, it is lymphocytotropic and may perturb immune responses in vitro and in vivo. infections with mpv have been shown to influence rejection of skin and tumour grafts (mckisic et al., , . mice minute virus is the type species of the genus parvovirus. the virus was formerly called 'minute virus of mice' (mvm) and was renamed recently (international union of microbiological societies, ) . it was originally isolated by crawford ( ) from a stock of mouse adenovirus, and this prototype isolate was later designated mvmp. its allotropic variant was detected as a contaminant of a transplantable mouse lymphoma (bonnard et al., ) and designated mvmi because it exhibits immunosuppressive properties in vitro. both variants have distinct cell tropisms in vivo and in vitro. the mmvp infects fibroblast cell lines and does not cause clinical disease (kimsey et al., , brownstein et al., . the mmvi grows lytically in t cells and inhibits various functions mediated by these cells in vitro. both strains are apathogenic for adult mice, but the immunosuppressive variant is more pathogenic for neonatal mice than is mmvp. serological surveys show that the mouse is the primary natural host (parker et al., ; smith et al., b; singleton et al., ) , but the virus is also infective for rats, hamsters (garant et al., ; ward and tattersall, ) , and mastomys (haag et al., ) during foetal development or after parenteral inoculation. natural infections are usually asymptomatic in adults and infants, and the most common sign of infection is seroconversion. kilham and margolis ( ) observed mild growth retardation a few days after experimental infection of neonatal mice with mmvp. studies of transplacental infection yielded no pathological findings in mice (kilham and margolis, ). the immunosuppressive variant but not the prototype strain is able to produce a runting syndrome after experimental infection of newborn mice (kimsey et al., ) . depending on the host genotype, experimental infections of foetal and neonatal mice with mmvi produce various clinical presentations and lesions. infection in c bl/ mice is asymptomatic, but the virus causes lethal infections with intestinal haemorrhage in dba/ mice. infection of strains such as balb/c, cba, c h/he, and sjl is also lethal and mice have renal papillary haemorrhage (brownstein et al., ) . the mmvi also infects haematopoietic stem cells and mediates an acute myelosuppression (segovia et al., (segovia et al., , . due to their dependency on mitotically active tissues, the foetus is at particular risk for damage by parvoviruses. mice minute virus and other parvoviruses may have severe teratogenic effects and cause foetal and neonatal abnormalities by destroying rapidly dividing cell populations, often resulting in foetal death. adult prkdc scid mice develop an acute leukopenia month after experimental infection with mmvi and die within months. the virus persists lifelong in the bone marrow of these mice (segovia et al., ) . mice minute virus is shed in faeces and urine. contaminated food and bedding are important factors in viral transmission because the virus is very resistant to environmental conditions. direct contact is also important and the virus does not easily spread between cages. routine health surveillance is usually conducted by serological methods. unlike mpv, mmv can easily be cultured in cell lines so that antigen production for hi and elisa (using whole purified virions) is easy. haemagglutination inhibition is a highly specific diagnostic test whereas ifa always exhibits some degree of cross reactivity with mpv and other closely related parvoviruses. enzyme-linked immunosorbent assay is probably the most frequently used test, but depending on the purity of the antigen preparation, cross reactions with mpv may occur due to contamination with nonstructural proteins that are common to both viruses. this problem can be avoided by the use of recombinant vp- antigen (livingston et al., ) . viral detection is also possible by pcr in biological materials and in organs (intestines, kidney, spleen) from infected animals (yagami et al., ; chang et al., ; redig and besselsen, ) . in contrast to mpv, pcr is not appropriate as a confirmatory method for serology because mmv has not been shown to persist in immunocompetent animals for sufficiently long periods. the virus can be eliminated from infected breeding populations by caesarean derivation or by embryo transfer. in experimental colonies, elimination of infected animals and subsequent replacement with uninfected mice is practical if careful environmental sanitation is conducted by appropriate disinfection procedures. it is important that reintroduction is avoided by exclusion of wild mice and by strict separation from other infected populations and potentially contaminated materials in the same facility. admission of biological materials must be restricted to samples that have been tested and found free from viral contamination. both allotropic variants of mmv have been used as models for molecular virology, and their small size and simple structure have facilitated examination of their molecular biology and expedited understanding of cell tropism, viral genetics, and structure. the significance for laboratory mouse populations was considered low or uncertain because natural infections are inapparent. however, various effects on mouse-based research have been published (tattersall and cotmore, ; jacoby et al., ; baker, ; nicklas et al., ) . due to their predilection for replicating in mitotically active cells, they are frequently associated with tumour cells and have a marked oncosuppressive effect (rommelaere and cornelis, ) . special attention is also necessary for immunological research and other studies involving rapidly dividing cells (embryology, teratology). in addition, mmv is a common contaminant of transplantable tumours, murine leukaemias, and other cell lines (collins and parker, ; nicklas et al., ; garnick, ) . lactate dehydrogenase-elevating virus (ldv) is a single-stranded rna virus of the genus arterivirus belonging to the family arteriviridae. lactate dehydrogenase-elevating virus has repeatedly been detected in feral mice (mus musculus), which are considered to be a virus reservoir (rowson and mahy, ; li et al., ) . only mice and primary mouse cells are susceptible to infection with ldv. after infection, virus titres of - particles per ml serum are found within - h after infection. the virus titre drops to particles per ml within - weeks and remains constant at this level for life. lactate dehydrogenase-elevating virus replicates in a subpopulation of macrophages in almost all tissues and persists in lymph nodes, spleen, liver, and testes tissues (anderson et al., a) . the virus can be stored in undiluted mouse plasma at Ϫ Њc without loss of infectivity, but it is not stable at room temperature and is very sensitive to environmental conditions. lactate dehydrogenase-elevating virus was first detected during a study of methods that could be used in the early diagnosis of tumours (riley et al., ) . it produces a persistent infection with continuous virus production and a lifelong viraemia despite ldv-specific immune reactions of the host ( van den broek et al., ) . lactate dehydrogenase-elevating virus has been found in numerous biological materials that are serially passaged in mice such as transplantable tumours including human tumours (nicklas et al., ; ohnishi et al., ) , monoclonal antibodies or ascitic fluids (nicklas et al., ) , or infectious agents (e.g. haemoprotozoans, k virus, clostridium piliforme). these materials are contaminated after passage in an infected and viraemic animal. contamination with ldv leads to the infection of each sequential host and to transmission of the virus by the next passage and remains associated with the specimen. it is therefore the most frequently detected contaminant in biological materials (collins and parker, ; nicklas et al., ) . infection with ldv is usually asymptomatic, and there are no gross lesions in immunocompetent as well as in immunodeficient mice. the only exception is polyomyelitis with flaccid paralysis of hind limbs developing in c and akr mice when they are immunosuppressed either naturally with aging or experimentally (anderson et al., b; monteyne et al., ) . it has been shown that only mice harbouring cells in the cns that express a specific endogenous mulv are susceptible to poliomyelitis (anderson et al., c) . the characteristic feature of ldv infection is the increased activity of lactate dehydrogenase (ldh) and other plasma enzymes (brinton, ; national research council, ) , which is due to the continuous destruction of permissive macrophages that are responsible for the clearance of ldh from the circulation. as a consequence, the activity of plasma ldh begins to rise by only h after infection and peaks - days after infection at - -fold normal levels, or even be up to -fold in sjl/j mice. the enzyme activity declines during the next weeks but remains elevated throughout life. antigen-antibody complexes produced during infection circulate in the blood and are deposited in the glomeruli (brinton, ; national research council, ) . in contrast to other persistent virus infections (e.g. lymphocytic choriomeningitis virus lcmv), these complexes do not lead to immune complex disease and produce only a very mild glomerulopathy. the only gross finding associated with ldv infection is mild splenomegaly. microscopically, necrosis of lymphoid tissues is visible during the first days of infection. in mouse strains that are susceptible to poliomyelitis, ldv induces lesions in the grey matter of the spinal cord and the brain stem (brinton, ) . lactate dehydrogenase-elevating virus is not easily transmitted between mice, even in animals housed in the same cage. fighting and cannibalism increase transmission between cage mates most likely via blood and saliva. infected females transmit the virus to their foetuses if they have been infected few days prior to birth and before igg anti-ldv antibodies are produced, but developmental and immunological factors (e.g. gestational age, timing of maternal infection with ldv, placental barrier) are important in the regulation of transplacental ldv infection (haven et al., ; zitterkopf et al., ) . maternal immunity protects foetuses from intrauterine infection. immunodeficient prkdc scid mice transmit virus to their offspring also during chronic infection (broen et al., ). an important means of transmission is provided by experimental procedures such as mouse-to-mouse passage of contaminated biological materials or the use of the same needle for sequential inoculation of multiple mice. in principal, serological methods such as ifa may be used for detecting ldv infection (hayashi et al., ) but they are not of practical importance. circulating virus-antibody complexes interfere with serological tests (aclad, ) , and sufficient quantities of virus for serological tests are difficult to generate because ldv replicates only in specific subpopulations of primary cultures of murine macrophages and monocytes for one cell cycle (brinton, ) . therefore, diagnosis of ldv infection is primarily based on increased ldh activity in serum or plasma of mice. lactate dehydrogenase-elevating virus activity in serum or plasma can be measured directly, or samples (e.g. plasma, cell or organ homogenates) are inoculated into pathogen-free mice and the increase in ldh activity within - days is measured. an - -fold increase is indicative of ldv infection. detection of infectivity of a plasma sample by the induction of increased ldh activity in the recipient animal is the most reliable means of identifying an infected animal. however, it is important to use clear nonhaemolysed samples because haemolysis will (falsely) elevate activities of multiple serum or plasma enzymes, including ldh. while this assay may be included in a commercial 'map test', it does not involve antibody detection. persistent infection makes ldv an ideal candidate for pcr detection in plasma or in organ homogenates (van der logt et al., ; chen and plagemann, ) . however, reports exist that pcr may produce false-negative results and should be used cautiously (lipman et al., a) . similarly important as detecting ldv in animals is its detection in biological materials. this may be done by assay for increased ldh activity after inoculation of suspect material into pathogen-free mice (collins and parker, ; nicklas et al., ) or by pcr (goto et al., ; bootz and sieber, ) . lactate dehydrogenase-elevating virus spreads slowly in a population because direct contact is necessary. therefore ldv-negative breeding populations can easily be established by selecting animals with normal plasma ldh activity. embryo transfer and hysterectomy derivation are also efficient. the presence of ldv in experimental populations is indicative of contaminated biological materials. in such cases, it is essential that the virus is also eliminated from these samples. this is easily achieved by maintenance of cells by in vitro culture instead of by animal-to-animal passages (plagemann and swim, ) . due to the extreme host specificity of the virus, contaminated tumour samples can also be sanitized by passages in nude rats or other animal species. lactate dehydrogenase-elevating virus is a potential confounder of any research using biological materials that are passaged in mice. once present in an animal, the virus persists lifelong. the most obvious signs are increased levels of plasma ldh and several other enzymes. lactate dehydrogenase-elevating virus may also exhibit numerous effects on the immune system (thymus involution, depression of cellular immunity, enhanced or diminished humoral responses, nk cell activation, development of autoimmunity, and suppression of development of diabetes in nod mice; cafruny and hovinen, ; nicklas et al., ; takei et al., ; markine-goriaynoff et al., ; gomez et al., ) and enhance or suppress tumour growth (brinton, ; baker, ; nicklas et al., ) . lymphocytic choriomeningitis virus is an enveloped, segmented single-stranded rna virus of the genus arenavirus, family arenaviridae. its name refers to the condition that results from experimental intracerebral inoculation of the virus into adult mice and is not considered to be a feature of natural infections. mice (mus musculus) serve as the natural virus reservoir (salazar-bravo et al., ) , but syrian hamsters are also important hosts (ackermann, ) . additional species such as rabbits, guinea pigs, squirrels, monkeys, and humans are susceptible to natural or experimental infection. infection in hamsters is considered to be asymptomatic (national research council, ) . natural infection of callitrichid primates (marmosets and tamarins) leads to a progressive hepatic disease that is known as 'callitrichid hepatitis' (montali et al., ; asper et al., ; lukashevich et al., ) . antibodies to lcmv have been found in wild mice in europe (ackermann et al., ) , africa (el karamany and imam, ), asia (morita et al., (morita et al., , , australia , and america (childs et al., ) . thus, it is the only arenavirus with worldwide distribution. infection with lcmv is rarely found in laboratory mice (smith et al., ) . seropositivity to lcmv was reported in approximately % of non-spf mouse colonies in the usa in (jacoby and lindsey, ) and in % of french colonies in - (zenner and regnault, ) . recent studies confirm that only a small percentage of mice tested are positive for lcmv (livingston and riley, ) . in addition to laboratory mice and other vertebrate hosts, the virus has frequently been found in transplantable tumours and tissue culture cell lines from mice and hamsters (bhatt et al., a; nicklas et al., ) . despite the low prevalence in laboratory mice, seropositivity to this zoonotic agent should raise serious concern for human health. lymphocytic choriomeningitis virus is frequently transmitted to humans from wild mice (childs et al., ) and is also endemic to a varying degree in the human population (childs et al., ; marrie and saron, ; lledo et al., ) due to contact with wild mice. lymphocytic choriomeningitis virus is further transmitted to humans by domestic syrian hamsters rousseau et al., ) . in addition, infected laboratory mice (dykewicz et al., ) and contaminated biological materials are important sources of infections for humans, and several outbreaks of lcm among laboratory personnel have been traced to transplantable tumours biggar et al., ; mahy et al., ) . in mice, clinical signs of lcmv infection vary with strain and age of mouse, strain and dose of virus, and route of inoculation (lehmann-grube, ; national research council, ) . two forms of natural lcmv infection are generally recognized: a persistent tolerant and an (acute) nontolerant form. the persistent form results from infection of mice that are immunotolerant. this is the case if mice are infected in utero or during the first days after birth. this form is characterized by lifelong viraemia and shedding. mice may show growth retardation, especially during the first - weeks, but appear otherwise normal. infectious virus is bound to specific antibodies and complement, and these complexes accumulate in the renal glomeruli, the choroid plexus, and sometimes also in synovial membranes and blood vessel walls. at - months of age, immune complex nephritis develops with ruffled fur, hunched posture, ascites, and occasional deaths. this immunopathological phenomenon is called 'late onset disease' or 'chronic immune complex disease'. the incidence of this type of disease varies between mouse strains. gross lesions include enlarged spleen and lymph nodes due to lymphoid hyperplasia. kidneys affected with glomerulonephritis may be enlarged with a granular surface texture or may be shrunken in later stages of the disease process. microscopically, there is generalized lymphoid hyperplasia and immune complex deposition in glomeruli and vessel walls, resulting in glomerulonephritis and plasmacytic, lymphocytic perivascular cuffs in all visceral organs (percy and barthold, ) . the nontolerant acute form occurs when infection is acquired after the development of immunocompetence (in mice older than week). these animals become viraemic but do not shed virus and may die within a few days or weeks. natural infections of adults are usually asymptomatic. surviving mice are seropositive and in most cases clear the virus to below detection levels of conventional methods. however, virus may persist at low levels in tissues (particularly spleen, lung, and kidney) of mice for at least weeks after infection as determined by sensitive assays such as nested reverse transcriptasepolymerase chain reaction (rt-pcr) or immunohistochemisty (ciurea et al., ) . such nonlethal infection leads to protection against otherwise lethal intracerebral challenge. protection from lethal challenge is also achieved by maternally derived anti-lcmv antibodies through nursing or by the administration of anti-ldv monoclonal igg a antibodies (baldridge and buchmeier, ) . in experimentally infected animals, the route of inoculation (subcutaneous, intraperitoneal, intravenous, intracerebral) also influences the type and degree of disease (lehmann-grube, ; national research council, ) . intracerebral inoculation of adult immunocompetent mice typically results in tremors, convulsions, and death due to meningoencephalitis and hepatitis. neurological signs usually appear on day postinoculation, and animals die within - days after the onset of symptoms or recover within several days. the classic histological picture is of dense perivascular accumulations of lymphocytes and plasma cells in meninges and choroid plexus. while infection following subcutaneous inoculation usually remains inapparent, reaction of mice to intraperitoneal or intravenous inoculation depends on the virus strain and on the mouse strain. infection by these routes primarily causes multifocal hepatic necrosis and necrosis of lymphoid cells. athymic foxn nu mice and other immunodeficient mice do not develop disease but become persistently viraemic and shed virus. as a general rule, all pathological alterations following lcmv infection are immune-mediated; and mice can be protected from lcmv-induced disease by immunosuppression (gossmann et al., ) . lymphocytic choriomeningitis virus disease is a prototype for virus-induced t-lymphocyte-mediated immune injury and for immune complex disease. for detailed information on the pathogenesis of lcmv infection, the reader is referred to a recent review article by oldstone ( ) . extensive information on the clinical and pathological features of lcmv infection in mice has been assembled by lehmann-grube ( ) . in nature, carrier mice with persistent infection serve as the principal source of virus. intrauterine transmission is very efficient, and with few exceptions all pups born from carrier mice are infected. furthermore, persistently infected mice and hamsters can shed large numbers of infectious virions primarily in urine, but also in saliva and milk. the virus can replicate in the gastric mucosa after intragastric infection (rai et al., (rai et al., , . gastric inoculation elicits antibody responses of comparable magnitudes as intravenous inoculation and leads to active infection with lcmv indicating that oral infection is possible, e.g., by ingestion of contaminated food or cannibalism. a self-limiting infection frequently results from infection of adult mice. the virus does not spread rapidly after introduction in populations of adult mice, and the infectious chain usually ends. however, if the virus infects a pregnant dam or a newborn mouse, a lifelong infection results, and soon a whole breeding colony of mice may become infected if the mice live in close proximity (which is the case under laboratory conditions). lymphocytic choriomeningitis virus is most commonly diagnosed by serological methods. methods of choice are ifa and elisa, which have replaced the relatively insensitive complement fixation test. it is important that bleeding of mice is done carefully because of a potential risk due to viraemic animals. historically, direct viral detection was performed by inoculating body fluids or tissue homogenates into the brain of lcmv-free mice or by subcutaneous injection into mice and subsequent serological testing (map test). more recently, pcr assays have been developed for the direct detection of viral rna in clinical samples or animals (park et al., . both map test and pcr can also be used to detect contamination of biological materials (bootz and sieber, ) . vertical transmission of lcmv by transuterine infection is efficient so this virus cannot reliably be eliminated by caesarean rederivation. caesarean derivation may be effective if dams acquired infection after the development of immunocompetence (nontolerant acute infection) and subsequently eliminated the virus, but such a strategy is difficult to justify in light of lcmv's zoonotic potential. in breeding colonies of great value, virus elimination might be possible soon after introduction into the colony by selecting nonviraemic breeders. this procedure is expensive and time consuming and requires special safety precautions. fortunately, infections of laboratory mice with lcmv are very uncommon. however, once lcmv has been detected in animals or in biological materials, immediate destruction of all contaminated animals and materials is advisable to avoid risk of human infection. foxn nu and prkdc scid mice may pose a special risk because infections are silent and chronic (mahy et al., ) . cages and equipment should be autoclaved, and animal rooms should be fumigated with disinfectants such as formaldehyde, vaporized paraformaldehyde, and hydrogen peroxide. appropriate precautions are necessary for experiments involving lcmv, or lcmv-infected animals or materials. biological safety level (bsl) will be considered to be sufficient in most cases. biological safety level practices may be considered when working with infected animals owing to the increased risk of virus transmission by bite wounds, scratching, or aerosol formation from the bedding. animal biosafety level (absl) practices and facilities are generally recommended for work with infected hamsters. appropriate precautions have been defined for different bsls or animal biology safety levels by cdc ( ) . lymphocytic choriomeningitis virus is an important zoonotic agent. it has been transmitted to humans working with infected animals or with contaminated biological materials and can cause mild to serious or fatal disease in humans (dykewicz et al., ; barton et al., ; barton and hyndman, ) . congenital infection in humans may result in hydrocephalus, or foetal or neonatal death (barton et al., ) . lymphocytic choriomeningitis virus is also frequently utilized as a model organism to study virus-host interactions, immunological tolerance, virus-induced immune complex disease, and a number of immunological mechanisms in vivo and in vitro (slifka, ; zinkernagel, ) . accidental transmission may have a severe impact on various kinds of experiments (for details, see lehmann-grube, ; bhatt et al., b; national research council, ; baker, ; nicklas et al., ) . mammalian orthoreoviruses (mrv) are nonenveloped, segmented double-stranded rna viruses of the family reoviridae, genus orthoreovirus. they have a wide host range and are ubiquitous throughout the world. the designation reo stands for respiratory enteric orphan and reflects the original isolation of these viruses from human respiratory and intestinal tract without apparent disease. the term 'orphan' virus refers to a virus in search of a disease. mammalian orthoreovirus can be grouped into three serotypes ( , , ). mammalian orthoreovirus- (synonyms: hepatoencephalomyelitis virus; echo virus) infection remains prevalent in contemporary mouse colonies and has been reported in wild mice barthold, a) . seropositivity to mrv- was found in less than % of spf colonies and in approximately % of non-spf mouse colonies in the usa in (jacoby and lindsey, ) . a study in france reported antibodies to mrv- in % of mouse colonies examined (zenner and regnault, ) . more recently, a study in north america found a low rate ( . %) of mouse sera to be positive for antibodies against this virus (livingston and riley, ) . in addition, contamination of mouse origin tumours and cell lines by mrv- has been reported many times (national research council, ; nicklas et al., ; barthold, a) . experimentally, mrv- infection of infant mice has been used to model human hepatobiliary disease, pancreatitis, diabetes mellitus, and lymphoma (kraft, ; national research council, ; fenner et al., ) . the literature on mrv- infections in mice is dominated by studies on experimentally infected animals. the virus can cause severe pantropic infection in infant mice (kraft, ; tyler and fields, ; barthold, a) . after parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes, and blood vessels. following oral inoculation, reoviruses gain entry by infecting specialized epithelial cells (m cells) that overlie peyer's patches. the virus then becomes accessible to leukocytes and spreads to other organs by way of the lymphatic system and the bloodstream. neural spread to the cns has also been well documented (morrison et al., ) . the mechanisms of viral pathogenesis and their interactions with the host cell are reviewed in detail by and . natural infection by mrv- in a mouse colony usually is subclinical although diarrhoea or steatorrhoea and oily hair effect in suckling mice may be noted (kraft, ; tyler and fields, ; national research council, ; barthold, a; percy and barthold, ) . the latter term has been used to describe the matted, unkempt appearance of the hair coat that results from steatorrhoea due to pancreatitis, maldigestion, and biliary atresia. in addition, runting (attributed to immune-mediated destruction of cells in the pituitary gland that produce growth hormone), transient alopecia, jaundice (due to excessive bilirubin in the blood, which is attributed to the liver pathology, especially biliary atresia), and neurological signs such as incoordination, tremors, or paralysis may develop. when present in natural infections, clinical signs and lesions are similar to but milder than in experimental neonatal infections. early descriptions of naturally occurring disease may have been complicated by concurrent infections such as mhv or murine rotavirus a (murv-a)/epizootic diarrhoea of infant mice (edim) virus that contributed to the severity of the lesions especially in liver, pancreas, cns, and intestine. the outcome of mrv- infection depends on age and immunological status of mouse, dose of virus, and route of inoculation. adult immunocompetent mice typically show no clinical signs and have no discernible lesions even in experimental infections. mucosal and maternally conferred immunity are considered to be important in protection from or resolution of disease (cuff et al., ; barthold et al., b) . experimental infection of adult prkdc scid mice is lethal (george et al., ) . depending on the route of inoculation, experimental infection of adult foxn nu mice is subclinical or results in liver disease (carthew, ; george et al., ) . histological findings reported to occur after experimental mrv- infection of neonatal mice include inflammation and necrosis in liver, pancreas, heart, adrenal, brain, and spinal cord; lymphoid depletion in thymus, spleen, and lymph nodes; and hepatic fibrosis with biliary atresia (papadimitriou and robertson, ; tyler and fields, ; barthold et al., b; barthold, a; percy and barthold, ) . transmission of reoviruses probably involves the aerosol as well as the faecal-oral route (national research council, ) . fomites may play an important role as passive vectors because reoviruses resist environmental conditions moderately well. serological screening with elisa or ifa is in widespread use for detection of antibodies to mrv- in diagnostic and health surveillance programmes. both elisa and ifa detect cross-reacting antibodies to heterologous mrv serotypes that can infect mice (aclad, ) . the hi test does not detect such cross-reacting antibodies but is prone to give false positive results due to nonspecific inhibitors of haemagglutination (kraft and meyer, ; van der logt, ; aclad, ) . reverse transcriptase-polymerase chain reaction methods for the detection of mrv- rna (steele et al., ) or mrv rna (leary et al., ) are also available. reports on contamination of mouse origin tumours and cell lines by mrv- and its interference with transplantable tumour studies (bennette, ; nelson and tarnowski, ) emphasize the importance of screening of biological materials to be inoculated into mice by map test or pcr. natural seroconversion to mrv- without clinical disease is also observed in laboratory rats, hamsters, and guinea pigs (national research council, ; barthold, a) . caesarean derivation and barrier maintenance have proven effective in the control and prevention of mrv- infection (kraft, ; national research council, ) . the virus may interfere with research involving transplantable tumours and cell lines of mouse origin. it has the potential to alter intestinal studies and multiple immune response functions in mice. in enzootically infected colonies, protection of neonates by maternal antibody could complicate or prevent experimental infections with reoviruses. it could further complicate experiments that require evaluation of liver, pancreas, cns, heart, lymphoid organs, and other tissues affected by the virus. the term murine hepatitis virus (mhv; commonly referred to as 'mouse hepatitis virus') designates a large group of antigenically and genetically related, singlestranded rna viruses belonging to the family coronaviridae, genus coronavirus. they are surrounded by an envelope with a corona of surface projections (spikes). murine hepatitis virus is antigenically related to rat coronaviruses and other coronaviruses of pigs, cattle, and humans. numerous different strains or isolates of mhv have been described. they can be distinguished by neutralization tests that detect strain-specific spike (s) antigens. the best studied strains are the prototype strains mhv- , mhv- , mhv- , jhm (mhv- ), a , and s, of which mhv- is regarded as the most virulent. murine hepatitis virus, like other coronaviruses, mutates rapidly, and strains readily form recombinants, so that new (sub)strains are constantly evolving. strains vary in their virulence, organotropism, and cell tropism (homberger, ) . based on their primary organotropism, mhv strains can be grouped into two biotypes: respiratory (or polytropic) and enterotropic. however, intermediate forms (enterotropic strains with tropism to other organs) exist. murine hepatitis virus is relatively resistant to repeated freezing and thawing, heating ( Њc for min), and acid ph but is sensitive to drying and disinfectants, especially those with detergent activity (national research council, ) . mus musculus is the natural host of mhv. it can be found in wild and laboratory mice throughout the world and is one of the most common viral pathogens in contemporary mouse colonies. while polytropic strains have historically been considered more common, this situation is thought to have reversed. a survey conducted in the usa in reported antibodies to mhv in more than % of spf mouse colonies and more than % of non-spf colonies (jacoby and lindsey, ) , though very recent monitoring results for research institutions across north america indicate that the prevalence of mhv has decreased during the past few years (livingston and riley, ) . a retrospective study in france covering the period from to reported antibodies to mhv in % of mouse colonies examined (zenner and regnault, ) . suckling rats inoculated experimentally with mhv had transient virus replication in the nasal mucosa and seroconversion but no clinical disease . similarly, deer mice seroconverted but showed no clinical disease after experimental infection (silverman et al., ) . murine hepatitis virus is also a common contaminant of transplantable tumours (collins and parker, ; nicklas et al., ) and cell lines (sabesin, ; yoshikura and taguchi, ) . the pathogenesis and outcome of mhv infections depend on interactions among numerous factors related to the virus (e.g. virulence and organotropism) and the host (e.g. age, genotype, immune status, and microbiological status; kraft, ; barthold, ; national research council, ; compton et al., ; homberger, ; percy and barthold, ) . murine hepatitis virus strains appear to possess a primary tropism for the upper respiratory or enteric mucosa. those strains with respiratory tropism initiate infection in the nasal mucosa and then may disseminate via blood and lymphatics to a variety of other organs because of their polytropic nature. respiratory (polytropic) strains include mhv- , mhv- , mhv- , a , s, and jhm. infection of mice with virulent polytropic mhv strains, infection of mice less than weeks of age, infection of genetically susceptible strains of mice, or infection of immunocompromised mice favour virus dissemination. virus then secondarily replicates in vascular endothelium and parenchymal tissues, causing disease of brain, liver, lymphoid organs, bone marrow, and other sites. infection of the brain by viraemic dissemination occurs primarily in immunocompromised or neonatal mice. additionally, infection of adult mouse brain can occur by extension of virus along olfactory neural pathways, even in the absence of dissemination to other organs. in contrast, enterotropic mhv strains (e.g. livim, mhv-d, and mhv-y) tend to selectively infect intestinal mucosal epithelium, with no or minimal dissemination to other organs such as mesenteric lymph nodes or liver. all ages and strains are susceptible to active infection, but disease is largely age-related. infection of neonatal mice results in severe necrotizing enterocolitis with high mortality within h. mortality and lesion severity diminish rapidly with advancing age at infection. adult mice develop minimal lesions although replication of equal or higher titres of virus occurs compared with neonates. the age-dependent decrease in severity of enterotropic mhv disease is probably related to the higher mucosal epithelium turnover in older mice, allowing more rapid replacement of damaged mucosa. another factor that is of considerable importance to the outcome of mhv infections is host genotype. for example, balb/c mice are highly susceptible to enterotropic mhv disease while sjl mice, at the other end of the spectrum, are highly resistant (barthold et al., a) . unlike in polytropic mhv infection where resistance is correlated with reduced virus replication in target cells (barthold and smith, ) , enterotropic mhv grows to comparable titres in sjl and balb/c mice at all ages (barthold et al., a) . therefore, the resistance of the sjl mouse to disease caused by enterotropic mhv seems to be mediated through an entirely different mechanism than resistance to polytropic mhv. furthermore, mouse genotypes that are susceptible to disease caused by one mhv strain may be resistant to disease caused by another strain (barthold, ) . it is therefore not possible to strictly categorize mouse strains as susceptible or resistant. the genetic factors determining susceptibility versus resistance in mhv infections are as yet poorly understood. both polytropic and enterotropic mhv infections are selflimiting in immunocompetent mice. immune-mediated clearance of virus usually begins about a week after infection, and most mice eliminate the virus within - weeks (barthold, ; barthold and smith, ; barthold et al., a) . humoral and cellular immunity appear to participate in host defences to infection, and functional t cells are an absolute requirement (williamson and stohlman, ; kyuwa et al., ; lin et al., ; haring and perlman, ) . therefore, immunodeficient mice such as foxn nu and prkdc scid mice cannot clear the virus (barthold et al., ; compton et al., ) . similarly, some genetically modified strains of mice may have deficits in antiviral responses or other alterations that allow the development of persistent mhv infection (rehg et al., ) . recovered immune mice are resistant to reinfection with the same mhv strain but remain susceptible to repeated infections with different strains of mhv (barthold and smith, a,b; . similarly, maternal immunity protects suckling mice against homologous mhv strains but not necessarily against other strains . however, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine . therefore, the susceptibility of young mice to infection significantly increases at weaning. most mhv infections are subclinical and follow one of two epidemiological patterns in immunocompetent mice (national research council, ; homberger, ) . enzootic (subclinical) infection, commonly seen in breeding colonies, occurs when a population has been in contact with the virus for a longer period (e.g. several weeks). adults are immune (due to prior infection), sucklings are passively protected, and infection is perpetuated in weanlings. epizootic (clinical) infection occurs when the virus is introduced into a naive population (housed in open cages). the infection rapidly spreads through the entire colony. clinical signs depend upon the virus and mouse strains and are most evident in infant mice. typically, they include diarrhoea, poor growth, lassitude, and death. in infections due to virulent enterotropic strains, mortality can reach % in infant mice. some strains may also cause neurological signs such as flaccid paralysis of hind limbs, convulsions, and circling. adult infections are again usually asymptomatic. as the infection becomes established in the colony, the epizootic pattern is replaced by the enzootic pattern. in immunodeficient (e.g. foxn nu and prkdc scid ) mice, infection with virulent polytropic mhv strains often is rapidly fatal while less virulent strains cause chronic wasting disease (compton et al., ) . in contrast, adult immunodeficient mice can tolerate chronic infection by enterotropic mhv, with slow emaciation and diarrhoea, or minimal clinical disease (barthold et al., ; barthold, ) . subclinical mhv infections can be activated by a variety of experimental procedures (e.g. thymectomy, whole body irradiation, treatment with chemotherapeutic agents, halothane anaesthesia) or by co-infections with other pathogens (e.g. eperythrozoon coccoides, k virus; reviewed by kraft, ; national research council, ) . in most natural infections, gross lesions are not present or are transient and not observed. gross findings in neonates with clinical signs include dehydration, emaciation, and in contrast to edim, an empty stomach (ishida et al., ; barthold et al., ; kraft, ) . the intestine is distended and filled with watery to mucoid yellowish, sometimes gaseous contents. haemorrhage or rupture of the intestine can occur. depending on the virus strain, necrotic foci on the liver (ishida et al., ; kraft, ; percy and barthold, ) and thymus involution godfraind et al., ) may also be seen in susceptible mice. liver involvement may be accompanied by jaundice and haemorrhagic peritoneal exudate. splenomegaly may occur as a result of compensatory haematopoiesis (fox et al., ) . histopathological changes in susceptible mice infected with polytropic mhv strains include acute necrosis with syncytia in liver, spleen, lymph nodes, gut-associated lymphoid tissue, and bone marrow (kraft, ; barthold, ; national research council, ; percy and barthold, ) . neonatally infected mice can have vascular-oriented necrotizing (meningo)encephalitis with demyelination in the brain stem and peri-ependymal areas. lesions in peritoneum, bone marrow, thymus, and other tissues can be variably present. mice can develop nasoencephalitis due to extension of infection from the nasal mucosa along olfactory pathways to the brain, with meningoencephalitis and demyelination, the latter of which is thought to be largely t cell-mediated (haring and perlman, ) . this pattern of infection regularly occurs after intranasal inoculation of many mhv strains but is a relatively rare event after natural exposure. syncytia arising from endothelium, parenchyma, or leukocytes is a hallmark of infection in many tissues including intestine, lung, liver, lymph nodes, spleen, thymus, brain, and bone marrow. lesions are transient and seldom fully developed in adult immunocompetent mice, but they are manifest in immunocompromised mice. highly unusual presentations can occur in mice with specific gene defects. for example, granulomatous peritonitis and pleuritis were found in interferon-␥-deficient mice infected with mhv (france et al., ) . histopathological changes caused by enterotropic strains of mhv are mainly confined to the intestinal tract and associated lymphoid tissues (kraft, ; barthold, ; national research council, ; percy and barthold, ) . the most common sites are terminal ileum, caecum, and proximal colon. the severity of disease is primarily age-dependent, with neonatal mice being most severely affected. these mice show segmentally distributed areas of villus attenuation, enterocytic syncytia (balloon cells), and mucosal necrosis accompanied by leukocytic infiltration. intracytoplasmic inclusions are present in enterocytes. erosions, ulceration, and haemorrhage may be seen in more severe cases. lesions can be fully developed within - h, but are usually more severe at - days after infection. surviving mice may develop compensatory mucosal hyperplasia. mesenteric lymph nodes usually contain lymphocytic syncytia, and mesenteric vessels may contain endothelial syncytia. pathological changes in older mice are generally much more subtle and may only consist of transient syncytia. an occasional exception seems to occur in immunodeficient animals such as foxn nu mice, which can develop chronic hyperplastic typhlocolitis of varying severity (barthold et al., ) , but other agents such as helicobacter species may have been involved. in general, enterotropic mhv strains do not disseminate, but hepatitis and encephalitis can occur with some virus strains in certain mouse genotypes. murine hepatitis virus is highly contagious. it is shed in faeces and nasopharyngeal secretions and appears to be transmitted via direct contact, aerosol, and fomites (kraft, ; national research council, ) . vertical (in utero) transmission has been demonstrated in experimental infections (katami et al., ) but does not seem to be of practical importance under natural conditions. diagnosis during the acute stage of infection can be made by histological demonstration of characteristic lesions with syncytia in target tissues, but clinical signs and lesions can be highly variable and may not be prominent. suckling, genetically susceptible or immunocompromised mice are the best candidates for evaluation. active infection can be confirmed by immunohistochemistry (brownstein and barthold, ) or by virus isolation. virus recovery from infected tissues is difficult but can be accomplished using primary macrophage cultures or a number of established cell lines such as nctc or dbt (aclad, ) . these cells, however, may not be successful substrates for some enterotropic mhv strains. virus in suspect tissue can also be confirmed by bioassays such as map testing or infant or foxn nu mouse inoculation (de souza and smith, ; aclad, ) . amplification by passage in these mice increases the likelihood of detection of lesions and antigen, or virus recovery. other direct diagnostic methods that have been successfully utilized to detect mhv in faeces or tissue of infected mice include monoclonal antibody solution hybridization assay (casebolt and stephensen, ) and a number of rt-pcr assays (homberger et al., ; kunita et al., ; yamada et al., ; besselsen et al., ) . because of the transient nature of mhv infection in immunocompetent mice, serology is the most appropriate diagnostic tool for routine monitoring. enzyme-linked immunosorbent assay and ifa are well established and sensitive, and all known mhv strains cross-react in both tests (smith, ; aclad, ) . the magnitude of antibody response depends on mhv strain and mouse genotype (nakanaga et al., ; barthold and smith, ) . dba/ mice are poor antibody responders whereas c bl/ mice produce a high antibody titre and are therefore good sentinels. antibody titres remain high over a period of at least months (barthold and smith, b; . infected mice may not develop detectable antibodies for up to days after initial exposure (smith, ). in such cases, a direct diagnostic method as discussed above may be useful. another drawback of serology is that mice weaned from immune dams can have maternal antibodies until they are weeks of age (homberger, ) . this may impact serological monitoring because the possibility must be considered that low positive results are due to maternally-derived passive immunity. because the virus can be transmitted by transplantable tumours and other biological materials from mice, including hybridomas (holmes et al., ) and embryonic stem cells (okumura et al., ; kyuwa, ) , these materials should also be routinely screened for mhv contamination. mouse inoculation bioassay, map test, and rt-pcr can be used for this purpose. the best means of mhv control is to prevent its entry into a facility. this can be accomplished by purchase of mice from virus-free sources and maintenance under effective barrier conditions monitored by a welldesigned quality assurance programme. control of wild mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbour virus are also important measures to prevent infection. if infection occurs, the most effective elimination strategy is to cull the affected colony and obtain clean replacement stock. however, this is not always a feasible option when working with valuable mice (e.g. genetically modified lines, breeding stocks). caesarean derivation or embryo transfer can be used to produce virus-free offspring, and foster-nursing also has been reported to be effective (lipman et al., ) . quarantine of an affected colony with no breeding and no introduction of new animals for approximately months has been effective in immunocompetent mice (weir et al., ) . the infection is likely to be terminated because mhv requires a constant supply of susceptible animals. this method works best when working with small numbers of mice. large populations favour the development of new mhv strains that may result in repeated infections with slightly different strains (adami et al., ) . it may be practical to select a few future breeders from the infected population and quarantine them for approximately weeks (compton et al., ) . this can be achieved in isolators, or in individually ventilated cages if proper handling is guaranteed. after this interval, breeding can resume. the -week interval should permit recovery from active infection, and the additional -week gestation period effectively extends the total quarantine to weeks. it is advisible to select seropositive breeders because the possibility of active infection is lower in such animals. the breeding cessation strategy may not be successful if immunodeficient mice are used because they are susceptible to chronic infection and viral excretion (barthold et al., ) . genetically engineered mice of unclear, unknown or deficient immune status pose a special challenge because they may develop unusual manifestations of infection or may be unable to clear virus. rederivation likely is the most cost effective strategy in such situations. along with the measures described, proper sanitation and disinfection of caging and animal quarters as well as stringent personal sanitation are essential to eliminate infection. careful testing with sentinel mice should be applied to evaluate the effectiveness of rederivation. if transplantable tumours are contaminated with mhv, virus elimination can be achieved by passage of tumours in athymic whn rnu rats (rülicke et al., ) . murine hepatitis virus is one of the most important viral pathogens of laboratory mice and has been intensively studied from a number of research perspectives (e.g. as a model organism for studying coronavirus molecular biology or the pathogenesis of viral-induced demyelinating disease). numerous reports document the effects of natural and experimental infections with mhv on host physiology and research, especially in the fields of immunology and tumour biology (reviewed by barthold, ; national research council, ; compton et al., ; homberger, ; baker, ; nicklas et al., ) . murine pneumonia virus, commonly referred to as 'pneumonia virus of mice' (pvm), is an enveloped, singlestranded rna virus of the family paramyxoviridae, genus pneumovirus. it is closely related to human respiratory syncytial virus (hrsv). the virus name is officially abbreviated as 'mpv' according to the international union of microbiological societies ( ); however, the former designation 'pvm' will be used in this chapter to avoid confusion with the official abbreviation of mouse parvovirus (mpv). 'pneumonia virus of mice' infection is relatively common in colonies of mice and rats throughout the world. seropositivity to pvm was reported in less than % of spf mouse colonies and in approximately % of non-spf mouse colonies in the usa (jacoby and lindsey, ) . a serological survey in france demonstrated antibodies to pvm in % of mouse colonies examined (zenner and regnault, ) . in a more recent study in north america, such antibodies were found in only . % of mice monitored (livingston and riley, ) . antibodies to pvm have also been detected in hamsters, gerbils, cotton rats, guinea pigs, and rabbits (parker and richter, ; richter, ; national research council, ) . experimentally, pvm infection of mice is used as a model for hrsv infection (domachowske et al., ) . in immunocompetent mice, natural infection with pvm is transient and usually not associated with clinical disease or pathological findings (parker and richter, ; national research council, ; brownstein, b) . however, natural disease and persistent infection may occur in immunodeficient mice (carthew and sparrow, ; richter et al., ; weir et al., ) . in particular, athymic foxn nu mice seem to be susceptible to pvm infection, which can result in dyspnoea, cyanosis, emaciation, and death due to pneumonia (richter et al., ; weir et al., ) . similar clinical signs have been reported for experimentally infected, immunocompetent mice (cook et al., ) . necropsy findings in naturally infected foxn nu mice include cachexia and diffuse pulmonary oedema or lobar consolidation (weir et al., ) . pulmonary consolidation (dark red or grey in colour) also has been found after experimental infection of immunocompetent mice (brownstein, b) . histologically, natural infection of foxn nu mice with pvm presents as interstitial pneumonia (richter et al., ; weir et al., ) . experimental intranasal inoculation of immunocompetent mice can result in rhinitis, erosive bronchiolitis, and interstitial pneumonia with prominent early pulmonary eosinophilia and neutrophilia (brownstein, b; domachowske et al., ) . hydrocephalus may result from intracerebral inoculation of neonatal mice (lagace-simard et al., ) . susceptibility to infection is influenced by age of mouse, dose of virus, and a variety of local and systemic stressors (parker and richter, ; national research council, ) . pneumonia virus of mice is labile in the environment and rapidly inactivated at room temperature (parker and richter, ; national research council, ) . the virus is tropic for the respiratory epithelium (carthew and sparrow, ; cook et al., ) , and transmission is exclusively horizontal via the respiratory tract, mainly by direct contact and aerosol (parker and richter, ; national research council, ) . therefore, transmissibility in mouse colonies is low, and infections tend to be focal enzootics. serology (elisa, ifa, or hi) is the primary means of testing mouse colonies for exposure to pvm. immunohistochemistry has been applied to detect viral antigen in lung sections (carthew and sparrow, ; weir et al., ) , however, proper sampling (see chapter on health monitoring) is critical for establishing the diagnosis due to the focal nature of the infection. an rt-pcr assay to detect viral rna in respiratory tract tissues has also been reported . however, the use of direct methods requires good timing because the virus is present for only up to about days in immunocompetent mice (brownstein, b) . embryo transfer or caesarean derivation followed by barrier maintenance can be used to rear mice that are free of pvm. because active infection is present in the individual immunocompetent mouse for only a short period, strict isolation of a few (preferably seropositive) mice with the temporary cessation of breeding might also be successful in eliminating the virus (richter, ; national research council, ) . pneumonia virus of mice could interfere with studies involving the respiratory tract or immunological measurements in mice. in addition, pvm can have devastating effects on research using immunodeficient mice because they are particularly prone to develop fatal disease (richter et al., ; weir et al., ) or become more susceptible to the deleterious effects of other agents such as pneumocystis carinii (roths et al., ) . murine rotavirus-a/edim (commonly referred to as 'mouse rotavirus' or 'epizootic diarrhoea of infant mice virus') is a nonenveloped, segmented double-stranded rna virus of the family reoviridae, genus rotavirus. it is antigenically classified as a group a rotavirus, similar to rotaviruses of many other species that cause neonatal and infantile gastroenteritis (fenner et al., ) . murine rotavirus-a/edim infection remains prevalent in contemporary mouse colonies and appears to occur worldwide. seropositivity to murv-a/edim was reported in approximately % of spf colonies and in almost % of non-spf mouse colonies in the usa in (jacoby and lindsey, ) . more recently, livingston and riley ( ) found a low rate ( %) of mouse sera to be positive for antibodies against murv-a/edim. experimentally, murv-a/edim infection in mice is used as a model for human rotavirus infection, especially in investigations on the mechanisms of rotavirus immunity and in the development of vaccination strategies (ward and mcneal, ) . clinical symptoms following murv-a/edim infection range from inapparent or mild to severe, sometimes fatal, diarrhoea. 'epizootic diarrhoea of infant mice' describes the clinical syndrome associated with natural or experimental infection by murv-a/edim during the first weeks of life (kraft, ; sheridan and vonderfecht, ; national research council, ; barthold, b; percy and barthold, ) . diarrhoea usually begins around h after infection and persists for about week. affected suckling mice have soft, yellow faeces that wet and stain the perianal region. in severe instances, the mice may be stunted, have dry scaly skin, or are virtually covered with faecal material. morbidity is very high but mortality is usually low. gross lesions in affected mice are confined to the intestinal tract. the caecum and colon may be distended with gas and watery to paste-like contents that are frequently bright yellow. the stomach of diarrheic mice is almost always filled with milk, and this feature has been reported to be a reliable means to differentiate diarrhoea caused by rotavirus from the diarrhoea caused by mhv infection. histopathological changes may be subtle even in animals with significant diarrhoea. they are confined to the small intestine and are most prominent at the apices of villi, where rotaviruses infect and replicate within epithelial cells. hydropic change of villous epithelial cells is the hallmark finding of acute disease. the villi become shortened, and the cells that initially replace the damaged cells are less differentiated, typically cuboidal instead of columnar, and lack a full complement of enzymes for digestion and absorption, resulting in diarrhoea due to maldigestion and malabsorption. undigested milk in the small intestine promotes bacterial growth and exerts an osmotic effect, exacerbating damage to the villi. intestinal fluid and electrolyte secretion is further enhanced by activation of the enteric nervous system (lundgren et al., ) and through the effects of a viral enterotoxin called nsp (for nonstructural protein ; ball et al., ) . it is hypothesized that nsp is released from virusinfected cells and then triggers a signal transduction pathway that alters epithelial cell permeability and chloride secretion. susceptibility to edim depends on the age of the host and peaks between and days of age (kraft, ; sheridan and vonderfecht, ; national research council, ; barthold, b; percy and barthold, ) . mice older than about weeks can still be infected with murv-a/edim, but small numbers of enterocytes become infected, there is little replication of virus, and diarrhoea does not occur. the exact reason for this age-related resistance to disease is unknown. pups suckling immune dams are protected against edim during their period of disease susceptibility (rosé et al., ) . in general, the infection is selflimiting and resolves within days. successful viral clearance is promoted by an intact immune response (feng et al., ; mcneal et al., ; rosé et al., ) , and some immunodeficient mice (e.g. prkdc scid and rag tm fwa mice) may shed virus for extended periods or become persistently infected (riepenhoff-talty et al., ; franco and greenberg, ) . protection against murv-a/edim reinfection is primarily mediated by antibodies (feng et al., ; rosé et al., ) . murine rotavirus-a/edim is highly contagious and transmitted by the faecal-oral route (kraft, ; sheridan and vonderfecht, ; national research council, ) . dissemination of the virus occurs through direct contact or contaminated fomites and aerosols. murv-a/edim is stable at Ϫ Њc but otherwise tends to be susceptible to extreme environmental conditions, detergents, and disinfectants. enzyme-linked immunosorbent assay and ifa are in widespread use for detection of serum antibodies to murv-a/edim in diagnostic and health surveillance programmes; other assay systems such as those using latex agglutination are also utilized (ferner et al., ) . rotazyme ii is a commercially available elisa for detection of rotavirus antigen in faeces; however, great care must be used in interpreting the results because some feeds have been reported to cause false positive reactions (jure et al., ) . electron microscopy of faeces of diarrheic pups should reveal typical wheelshaped rotavirus particles, - nm in diameter. reverse transcriptase-polymerase chain reaction also can be used to detect rotavirus rna in faecal samples (wilde et al., ) . good timing is critical for establishing the diagnosis from faeces because virus is shed for only a few days in immunocompetent mice. embryo transfer or caesarean derivation followed by barrier maintenance is recommended for rederivation of breeding stocks (kraft, ; national research council, ) . in immunocompetent mice in which infection is effectively cleared, a breeding suspension strategy combined with excellent sanitation, filter tops, and conscientious serological testing of offspring may also be effective. murine rotavirus-a/edim has the potential to interfere with any research utilizing suckling mice. it may have a significant impact on studies where the intestinal tract of neonatal or infant mice is the target organ. the infection also poses a problem for infectious disease and immune response studies, particularly those involving enteropathogens in infant mice (newsome and coney, ) . in addition, runting could be interpreted erroneously as the effect of genetic manipulation or other experimental manipulation. sendai virus (sev) is an enveloped, single-stranded rna virus of the family paramyxoviridae, genus respirovirus. it is antigenically related to human parainfluenza virus . the virus was named for sendai, japan, where it was first isolated from mice. infections of mice and rats are relatively common and occur worldwide. in addition, there is evidence that hamsters, guinea pigs, and rabbits are susceptible to infection with sev (machii et al., ; aclad, ; national research council, ; percy and palmer, ) ; however, some apparently seropositive guinea pigs may in fact be seropositive to other parainfluenza viruses instead of sev. seropositivity to sev was reported to be absent from spf mouse colonies and to be approximately % in non-spf mouse colonies in the usa (jacoby and lindsey, ) . a study in france reported antibodies to sev in % of mouse colonies examined (zenner and regnault, ) . a low rate of seropositive mice ( . %) was found in a recent survey in north america (livingston and riley, ) . furthermore, sev can contaminate biological materials (collins and parker, ) . sendai virus is pneumotropic and the leading cause of viral respiratory disease in mice. the pneumotropism is partially a consequence of the action of respiratory serine proteases such as tryptase clara, which activate viral infectivity by specific cleavage of the viral fusion glycoprotein (tashiro et al., ) . in addition, the apical budding behaviour of sev may hinder the spread of virus into subepithelial tissues and subsequently to distant organs via the blood. two epidemiologic patterns of sev infection have been recognized, an enzootic (subclinical) and epizootic (clinically apparent) type (parker and richter, ; national research council, ; brownstein, a) . enzootic infections commonly occur in breeding or open colonies, where the constant supply of susceptible animals perpetuates the infection. in breeding colonies, mice are infected shortly after weaning as maternal antibody levels wane. normally, the infection is subclinical, with virus persisting for approximately weeks, accompanied by seroconversion that persists for a year or longer. epizootic infections occur upon first introduction of the virus to a colony and either die out (self-cure) after - months or become enzootic depending on colony conditions. the epizootic form is generally acute, and morbidity is very high resulting in nearly all susceptible animals becoming infected within a short time. clinical signs vary and include rough hair coat, hunched posture, chattering, respiratory distress, prolonged gestation, death of neonates and sucklings, and runting in young mice. breeding colonies may return to normal productivity in months and thereafter maintain the enzootic pattern of infection. factors such as strain susceptibility, age, husbandry, transport, and copathogens are important in precipitating overt disease. dba and /j strains of mice are very susceptible to sev pneumonia whereas sjl/j and c bl/ /j strains and several outbred stocks are relatively resistant. a/j, balb/c, and swr/j are among the strains that show intermediate susceptibility. there is no evidence for persistent infection in immunocompetent mice, but persistent or prolonged infection may occur in immunodeficient mice and can result in wasting and death due to progressive pneumonia (ward et al., ; iwai et al., ; percy et al., ) . clearance of a primary sev infection is mediated by cd ϩ and cd ϩ t cell mechanisms (kast et al., ; hou et al., ) . heavier than normal, consolidated, plum-coloured or grey lungs are a characteristic gross finding in severe sev pneumonia (parker and richter, ; national research council, ; brownstein, a; percy and barthold, ) . lymphadenopathy and splenomegaly reflect the vigorous immune response to infection. histologically, three phases of disease can be recognized in susceptible immunocompetent mice: acute, reparative, and resolution phases (brownstein, a; percy and barthold, ) . lesions of the acute phase, which lasts - days, are primarily attributed to the cell-mediated immune response that destroys infected respiratory epithelial cells and include necrotizing rhinitis, tracheitis, bronch(iol)itis, and alveolitis. epithelial syncytiae and cytoplasmic inclusion bodies in infected cells may be seen early in this phase. alveoli contain sloughed necrotic epithelium, fibrin, neutrophils, and mononuclear cells. atelectasis, bronchiectasis, and emphysema may occur as a result of damage and obstruction of airways. the reparative phase, which may overlap the acute phase but continues through about the third week post infection, is indicated by regeneration of airway lining epithelium. adenomatous hyperplasia and squamous metaplasia (with multilayered flat epithelial cells instead of normal columnar cells) in the terminal bronchioles and alveoli are considered to be a hallmark of sev pneumonia. mixed inflammatory cell infiltrates in this phase tend to be primarily interstitial rather than alveolar as they are in the acute phase. the resolution phase may be complete by the fourth week post infection and lesions may be difficult to identify subsequently. residual, persistent lesions that may occur include organizing alveolitis and bronchiolitis fibrosa obliterans. alveoli and bronchioles are replaced by collagen and fibroblasts, foamy macrophages, and lymphoid infiltrates, often with foci of emphysema, cholesterol crystals, and other debris, which represent attempts to organize and wall off residual necrotic debris and fibrin. lesions are more severe and variable when additional pathogens such as mycoplasma pulmonis are present (national research council, ) . otitis media has also been reported in natural infections with sev although some of these studies have been complicated by the presence of other pathogens (ward, ) . sendai virus has been detected in the inner ear after experimental intracerebral inoculation of neonatal mice (shimokata et al., ) . sendai virus is extremely contagious. infectious virus is shed during the first weeks of infection and appears to be transmitted by direct contact, contaminated fomites, and respiratory aerosol (parker and reynolds, ; parker and richter, ; national research council, ) . serology (elisa, ifa, or hi) is the approach of choice for routine monitoring because serum antibodies to sev are detectable soon after infection and persist at high levels for many months, although active infection lasts only - weeks in immunocompetent mice. the short period of active infection limits the utility of direct methods such as immunohistochemistry (carthew and sparrow, ) and rt-pcr (hayase et al., ; wagner et al., ) . although sev is considered to be highly contagious, studies have shown that dirty bedding sentinel systems do not reliably detect the infection and that outbred stocks may not seroconvert consistently (dillehay et al., ; artwohl et al., ) . mouse antibody production test and rt-pcr can be used to detect sev in contaminated biological materials. sendai virus infection in mouse colonies has proven to be one of the most difficult virus infections to control because the virus is highly infectious and easily disseminated. depopulation of infected colonies probably is the most appropriate means to eliminate the virus in most situations. embryo transfer followed by barrier maintenance has also been used successfully in eliminating the virus (national research council, ) . a less effective alternative is to place the infected animals under strict quarantine, remove all young and pregnant mice, suspend all breeding, and prevent addition of other susceptible animals for approximately months until the infection is extinguished and then breeding and other normal acitivities are resumed (parker and richter, ; national research council, ) . vaccines against the virus have been developed (brownstein, ; national research council, ) , but these probably do not represent a practical means to achieve or maintain the seronegative status of colonies that is in demand today. sendai virus has the potential to interfere with a wide variety of research involving mice. reported effects include interference with early embryonic development and foetal growth; alterations of macrophage, nk cell, and t and b cell function; altered responses to transplantable tumours and respiratory carcinogens; altered isograft rejection; and delayed wound healing (reviewed by national research council, ; baker, ; nicklas et al., ) . pulmonary changes during sev infection can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other agents. they could also affect the response to anaesthetics. in addition, natural sev infection would interfere with studies using sev as a gene vector. theiler's murine encephalomyelitis virus (tmev) or murine poliovirus is a member of the genus cardiovirus in the family picornaviridae. members of this genus are nonenveloped viruses with singlestranded rna. the virus is rapidly destroyed at temperatures above Њc. it is considered to be a primary pathogen of the cns of mice and can cause clinical disease resembling that due to poliomyelitis virus infections in humans. antibodies to tmev have been identified in mouse colonies and feral populations worldwide, and mus musculus is considered to be the natural host of tmev (lipton et al., ) . the most well-known and most frequently mentioned tmev strain is gdvii, which is virulent for mice. infant or young hamsters and laboratory rats are also susceptible to intracerebral infection. the original isolate is designated to (theiler's original) and represents a group of tmev strains with low virulence for mice. many additional virus strains have been isolated and studied, and they all fall in the broad grouping of to and gdvii. a similar virus strain has also been isolated from rats, but in contrast to mouse isolates this virus is not pathogenic for rats and mice after intracerebral inoculation (hemelt et al., ) . recently, another rat isolate has been characterized and shown to be most closely related to but quite distinct from other tmev viruses (ohsawa et al., ) . antibodies to tmev (strain gdvii) have been detected in guinea pigs and are considered to indicate infection with another closely related cardiovirus (hansen et al., ) . seropositivity to tmev was reported in approximately % of spf mouse colonies and approximately % of non-spf mouse colonies in the usa (jacoby and lindsey, ) . zenner and regnault ( ) reported a prevalence rate of % in french mouse colonies in a retrospective study, and it has been one of the most common virus infections in rodent colonies. in a recent study, antibodies were found in . % of mice monitored (livingston and riley, ) indicating that tmev, like most viruses, has meanwhile been eliminated from the majority of mouse colonies. theiler's murine encephalomyelitis virus is primarily an enteric pathogen, and virus strains are enterotropic. in natural infections, virus can be detected in intestinal mucosa and faecal matter, and in some cases it is also found in the mesenteric lymph nodes. however, histological lesions in the intestine are not discerned. virus may be shed via intestinal contents for up to weeks, sometimes intermittently (brownstein et al., a) , and transmission under natural conditions is via the faecal-oral route by direct contact between mice as well as by indirect contact (e.g. dirty bedding). the host immune response limits virus spread, but it does not immediately terminate virus replication in the intestines. virus is cleared from extraneural tissues, but it persists in the cns for at least a year. clinical disease due to natural tmev infection is rare, with a rate of only in - , infected immunocompetent animals (percy and barthold, ) . in immunodeficient mice, especially in weanlings, clinical signs may be more common and mortality may be higher (rozengurt and sanchez, ) . this group of viruses usually causes asymptomatic infections of the intestinal tract. they may spread to the cns as a rare event where they cause different neurological disease manifestations. the most typical clinical sign of tmev infection is flaccid paralysis of hind legs. the animals appear otherwise healthy, and there is no mortality. experimental infection in mice provides models of poliomyelitis-like infection and virus-induced demyelinating disease including multiple sclerosis (mcgavern et al., ) . after experimental infection, tmev causes a biphasic disease in susceptible strains of mice. the acute phase is characterized by early infection of neurons in the grey matter. encephalomyelitis may develop during this phase and may be fatal, but most animals survive and enter the second phase of the disease at - months after the acute phase. this phase is characterized by viral persistence in the spinal cord white matter, mainly in macrophages, and leads to white matter demyelination. persistence and demyelination occur only in genetically susceptible mouse strains while resistant strains clear the infection after early grey matter encephalomyelitis through a cytotoxic t lymphocyte response. for this reason, the nude mutation (foxn nu ) confers susceptibility on mice with an otherwise resistant background. the severity and nature of disease depend on virus strain, route of inoculation, host genotype and age (downs, ; lipton and rozhon, ; national research council, ; percy and barthold, ) . in general, virus isolates with low virulence produce persistent cns infection in mice whereas virulent strains are unable to cause persistent infection. intracerebral inoculation results in the most severe infections, but the intranasal route is effective also. experimental intracerebral infections with virulent fa and gdvii strains of tmev are more likely to cause acute encephalomyelitis and death in weanling mice - days after inoculation ('early disease'). death may be preceded by neurological manifestations of encephalitis such as hyperexcitability, convulsions, tremors, circling and rolling, and weakness. animals may develop typical flaccid paralysis of hind limbs, and locomotion is possible only by use of the forelimbs. interestingly, the tail is not paralysed. experimental infections with low virulence virus strains (e.g. to, da, ww) are more likely to cause persistent infection with development of mild encephalomyelitis followed by a chronic demyelinating disease after a few months ('late disease'). these virus strains infect neurons in the grey matter of the brain and spinal cord during the acute phase of viral growth, followed by virus persistence in macrophages and glial cells in the spinal cord white matter. sjl, swr, and dba/ strains are most susceptible to this chronic demyelinating disease. cba and c h/he are less susceptible strains, and strains a, c bl/ , c bl/ , and dba/ are relatively resistant (lipton and dal canto, ) . differences in humoral immune responses play a role in resistance to tmev infection (pena rossi et al., a) , but genetic factors are also important. several genetic loci implicated in susceptibility to virus persistence, demyelination, or clinical disease have been identified, including the h- d region of the major histocompatibility complex (brahic and bureau, ) . furthermore, the age at infection influences the severity of clinical disease. in infant mice, intracerebral infection with low virulence virus strains (e.g. to) is often lethal. young mice develop paralysis after an incubation period of - weeks while adult mice often show no clinical signs of infection (downs, ) . the only gross lesions are secondary to the posterior paralysis and may include urine scald or dermatitis due to incontinence of urine and trauma to paralysed limbs, or wasting or atrophy of the hind limbs in long term survivors. theiler's murine encephalomyelitis virus infects neurons and glial cells, and histological changes in the cns include nonsuppurative meningitis, perivasculitis, and poliomyelitis with neuronolysis, neuronophagia, and microgliosis in the brainstem and ventral horns of the spinal cord (percy and barthold, ) . demyelination in immunocompetent mice is considered to be immune-mediated. susceptible strains develop a specific delayed-type hypersensitivity response which is the basis for inflammation and demyelination. this reaction is mediated by cytotoxic t lymphocytes (lindsley et al., ; pena rossi et al., b) and by the activation of cytokines as a consequence of infection of macrophages and other cells of the cns (rubio and capa, ; sierra and rubio, ; palma et al., ) . protection from chronic demyelinating disease is possible by vaccination with live virus given previously by subcutaneous or intraperitoneal inoculation (crane et al., ; kurtz et al., ) . early immunosuppression at the time of infection, e.g. by treatment with cyclophosphamide or antithymocyte serum, inhibits or diminishes demyelination. immunosuppression in mice chronically infected with tmev leads to remyelination of oligodendrocytes (rodriguez and lindsley, ) . further details related to the pathogenesis of tmev infections and the role of immune mechanisms have been reviewed by yamada et al. ( ) . experimental infection of foxn nu mice results in acute encephalitis and demyelination. demyelination associated with minimal inflammation and neurological signs including the typical hind limb paresis develop weeks after inoculation, and most animals die within weeks. in foxn nu mice, demyelination is caused by a direct lytic effect of the virus on oligodendrocytes (rosenthal et al., ) . demyelination and lethality are reduced after administration of neutralizing antibodies (fujinami et al., ) . histopathological changes in prkdc scid mice are very similar to those in foxn nu mice (rozengurt and sanchez, ) . young mice born in infected populations usually acquire infection shortly after weaning and are almost all infected by days of age. intrauterine transmission to foetuses is possible during the early gestation period, but a placental barrier develops during gestation and later prevents intrauterine infection (miyamae, ; abzug et al., ) . all tmev isolates are closely related antigenically and form a single serogroup as determined by complement fixation and hi (lipton and rozhon, ) . hemelt et al. ( ) demonstrated cross reactions among four strains used in experimental infections, but differences were evident in homologous and heterologous titres. the viral strain most commonly used as antigen for serological testing is gdvii. this strain agglutinates human type erythrocytes at Њc, and hi has been the standard test for routine screening of mouse populations. meanwhile, hi has been replaced by elisa or ifa, both of which are more sensitive and specific. virus isolation is possible from brains or spinal cords of mice with clinical disease or from the intestinal contents of asymptomatic mice. pcr techniques also are available to test for virus-specific nucleotide sequences in biological samples (trottier et al., ) . mice that have been shown to be free from tmev by serological testing can be selected for breeding populations. if the virus is introduced into a mouse population, depopulation of infected colonies may be the most appropriate means to eliminate tmev. embryo transfer or caesarean derivation are the methods of choice for eliminating virus from valuable breeding populations. foster-nursing has been reported to be effective in generating virus-free offspring (lipman et al., ) although transplacental transmission has been demonstrated with experimental infection early in gestation. lesions of demyelination in cns of mice with clinically inapparent chronic infection may interfere with investigations that require evaluation of the cns (krinke and zurbriggen, ) . conceivably, such lesions also could affect neuromuscular responses or coordination, and affect neurological and behavioural evaluations. viral and mycoplasmal infections of laboratory rodents: effects on biomedical research monographs on pathology of laboratory animals: digestive system monographs on pathology of laboratory animals: digestive system viral and mycoplasmal infections of laboratory rodents: effects on biomedical research the mouse in biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research monographs on pathology of laboratory animals: respiratory system monographs on pathology of laboratory animals: respiratory system biosafety in microbiological and biomedical laboratories (bmbl), th edn. u.s. department of health and human services proc. natl. acad. sci. usa the mouse in biomedical research the mouse in biomedical research the mouse in biomedical research veterinary virology complications of viral and mycoplasmal infections in rodents to toxicology research and testing viral and mycoplasmal infections of laboratory rodents: effects on biomedical research virus taxonomy. seventh report of the international committee on taxonomy of viruses the mouse in biomedical research the mouse in biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research proc. natl. acad. sci. usa the importance of laboratory animal genetics, health, and the environment in biomedical research proc. natl. acad. sci. usa national research council, committee on infectious diseases of mice and rats manual of microbiologic monitoring of laboratory animals the mouse in biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research the mouse in biomedical research the mouse in biomedical research pathology of laboratory rodents and rabbits proc. natl. acad. sci. usa manual of microbiologic monitoring of laboratory animals viral and mycoplasmal infections of laboratory rodents: effects on biomedical research lactic dehydrogenase virus viral and mycoplasmal infections of laboratory rodents: effects on biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research viral and mycoplasma infections of laboratory rodents: effects on biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research viral and mycoplasmal infections of laboratory rodents: effects on biomedical research handbook of animal models of infection the mouse in biomedical research key: cord- -qfpfllay authors: tiddi, ilaria; balliet, daniel; ten teije, annette title: fostering scientific meta-analyses with knowledge graphs: a case-study date: - - journal: the semantic web doi: . / - - - - _ sha: doc_id: cord_uid: qfpfllay a meta-analysis is a science of science method widely used in the medical and social sciences to review, aggregate and quantitatively synthesise a body of studies that address the same research question. with the volume of research growing exponentially every year, conducting meta-analyses can be costly and inefficient, as a significant amount of time and human efforts needs to be spent in finding studies meeting research criteria, annotating them, and properly performing the statistical analyses to summarise the findings. in this work, we show these issues can be tackled with semantic representations and technologies, using a social science scenario as case-study. we show how the domain-specific content of research outputs can be represented and used to facilitate their search, analysis and synthesis. we present the very first representation of the domain of human cooperation, and the application we built on top of this to help experts in performing meta-analyses semi-automatically. using few application scenarios, we show how our approach supports the various phases meta-analyses, and more in general contributes towards research replication and automated hypotheses generation. systematic literature reviews and meta-analyses in particular are a scientific method used in a number disciplines ranging from (bio-)medical to social sciences to summarise vast amounts of research outputs on a specific topic [ ] . in a nutshell, a meta-analysis exploits statistical models to quantify, aggregate and compare evidence from a set of (dozens, sometimes hundreds) experimental studies addressing the same research question, in order to derive generalisable conclusions [ ] . in this way, meta-analyses offer a snapshot of a research topic, supporting research transparency, reproducibility and re-usability -a more and more urgent topic in various research disciplines [ ] . performing meta-analyses is a knowledge-intensive process that can take months, sometimes years, due to methodological and technical barriers. and with the volume of research outputs growing exponentially every year, this problem is becoming more and more difficult [ ] . a new meta-analysis can require authors to spend a significant amount of time and effort to find studies that meet their criteria, identify the evidence in them, annotate their contents and statistically aggregate their results, before reaching any significant conclusion. moreover, meta-analyses can be large in scope, and planning for time and human resource allocation can be a hard task. this calls for new methods to help researchers in summarising scientific evidence in a more automated way, and more in general to facilitate publication and sharing of large bodies of scientific findings. our motivation stems from the cooperation databank (coda or data-bank henceforth), a large data repository aiming at analysing the entire history of laboratory and field research on human cooperation using social dilemmas. the goal of the databank is to encourage and facilitate sharing experiments as well as null findings (that tend to be hardly ever published), and consequently reduce the publication bias that currently affects the area [ ] . over the last years, a small pool of domain experts manually annotated approx. , studies collecting years of research publications with experimental settings, measured/manipulated variables of observation, and quantitative results, with the goal of establishing an open access database that researchers worldwide could consult to identify studies to include in their systematic literature reviews, as well as to directly conduct their own statistical (meta-)analyses. in this work, we show how semantic technologies, which provide support for scaling, reuse, and interoperability, can be exploited to tackle the scalability, methodological and technical issues of conducting meta-analyses. using a social science scenario, we show how the content of research outputs can be represented using semantic descriptions, and how to leverage this structured, domain-specific knowledge to facilitate search, analysis and synthesis of research outputs. our main contributions are ( ) the first structured representation of the field of human cooperation, that researchers from the field can easily reuse and extend; and ( ) a science of science application to help experts in performing meta-analyses semi-automatically, supporting the correct evaluation and interpretation of research conclusions. we discuss on the multiple benefits of our approach using few use-cases that demonstrate how the various phases of the meta-analytic process can be facilitated and, more in general, how this can significantly contribute to research replication and automated hypotheses generation. we introduce here the basic notions of scientific meta-analyses, the best practices and current applications, and overview the semantic approaches supporting scientific research. a meta-analysis is a process used to synthesise knowledge from studies addressing the same research question by using comparable methods. each study may observe a relation between two (one independent, one dependent) variables, which can be quantified as an effect size. meta-analytic techniques are then used to estimate an overall effect size average through aggregating the effect sizes observed in single studies [ ] . effect sizes can represent the differences observed between the experimental variations (treatments) of the independent variable and the dependent variable (such as a standardised difference between means d), or could also be the relation between two measured variables (such as a correlation coefficient ρ). in order to derive the overall estimate, a researcher first frames a problem statement, defining the research question, inclusion criteria, independent and dependent variables of observation etc., and then collects relevant studies (both published and non-published material) across scientific sources. conducting a meta-analysis then consists in: ( ) coding, i.e. annotating the studies with the relevant characteristics, including independent and dependent variables and effect sizes; ( ) analysis, i.e. estimating the overall effects using fixed and random effects models, determining heterogeneity in the studies, assessing publication bias, conducting moderator analyses through meta regression, performing statistical power analysis; ( ) interpretation, i.e. the presentation of the obtained results along with conclusions and graphical support, often including graphs such as forests, funnel, violin/scatter-box plots. these steps make a meta-analysis significantly different from a literature review conducted, for instance, in computer science, as the researcher numerically "pools" results from the studies (i.e. the effects sizes) and arrives at a statistical summary that can be integrated into the narrative of a publication. while meta-analyses are now established in the field, they are still seen as a controversial tool, as even small methodological violations can lead to misleading conclusions [ ] . researchers have argued that significant conclusions can only be derived from meta-analyses with large number of studies, while smaller meta-analyses can only support framing new research hypotheses [ ] . in response to this, a number of methodologies across disciplines have been published to assist experts in deriving reliable conclusions, e.g. the cochrane handbook by the campbell organisation [ ] , the york centre for reviews and dissemination guidelines for health care [ ] , the evidence for policy and practice information and co-ordinating centre . a considerable amount of statistical expertise is also needed to avoid deriving incorrect conclusions. a number of statistical tools are now available to overcome the technical barriers, resulting in tools and libraries such as revman [ ] , comprehensive meta-analysis [ ] , stata, and r packages such as meta, rmeta and metafor. finally, with the volume of research outputs growing exponentially, identifying relevant studies and annotate the evidence can require significant efforts. as a result, a number of research projects emerged in the latest years with the goal of making large bodies of research findings openly available, offering summaries of scientific evidence and, more in general, automating meta-analyses [ , ] . supporting science with semantic technologies. semantic web technologies have been used to provide an interoperable and machine-interpretable infrastructure for scientific enquiry in the context of semantic e-science [ ] . ontology engineering techniques have widely been employed to formally organise domain-specific knowledge and provide interactive, semanticallyenhanced data querying, exploration, and visualisation in a number of crossdisciplinary settings [ , , ] . a large number of vocabularies to semantically describe research outputs have been proposed, e.g. document publication [ , ] ; provenance, versioning, attribution and credits through research objects or nanopublications [ ] ; description of research hypotheses [ ] and scientific datasets [ ] . the benefit of using controlled vocabularies to describe research outputs guarantees them to be findable, exchangeable and interpretable across different applications (interoperability). another strand of research has focused on capturing knowledge from scientific processes, in order to support design and management of workflows [ ] , identify common patterns and motifs in them [ , ] , or recommend activities to tackle the cold start problem of experimental design [ ] . these works demonstrated that research reproducibility and support to frame research hypotheses can be supported by semantically describing and mining workflow components. a semantic approach could be used support conducting meta-analyses. domain vocabularies and descriptions could express the scientific knowledge contained in research outputs to facilitate search, analysis and synthesis. at the same time, replication of published results and support to derive the correct conclusions could be offered by the relying on semantic technologies, that enable scalability, interoperability and reuse. in the following, we show how our hypothesis was tested to support research replication and automated hypotheses generation in a social science scenario. the cooperation databank ( - ) is a large-scale effort involving a trained team of international researchers working with the goal of representing and publishing an open-access repository of over years of research on human cooperation using social dilemmas. social dilemmas are social situations that involve a conflict of interests and people must choose between doing what is best for themselves or what is best for the collective, either a dyad or group [ ] . in these situations, there is always one choice that results in the best outcome for each individual, regardless of what others choose to do. however, if everyone decides to behave this way, then the entire group receives a worse outcome, relative to when everyone decides to do what is best for the group. common social dilemma paradigms used to study cooperation include the prisoner's dilemma, public goods dilemma, and the resource dilemma. cooperation in these situations is operationalised as deciding to do what it best for the collective. in the databank, around , studies from the social and behavioural sciences published in english, chinese, and japanese were annotated with more than cooperation-related features. these features can be grouped in three categories, i.e. (a) characteristics of the sample participating in the study (e.g. sample size, average age of sample, percentage of males, country of participants), (b) characteristics of the experimental paradigm (structure of the social dilemma, incentives, repeated trial data, etc.), and (c) quantitative results (e.g., mean levels of cooperation, variance in cooperation, and effect sizes with cooperation). in this scenario, the coda experts are required to annotate and include new data (i.e. findings gathered from researchers worldwide) to the dataset in a continuous way. this can be inconvenient, costly and time-consuming, especially as data are not always directly accessible [ ] . in the long-term, we aim at supporting coda's maintainers in capturing and sharing knowledge more efficiently. starting with the assumption that scholars that consult the repository online act as domain experts, the solution we target is to crowdsource the meta-analyses that users conduct online to automatically enrich, fix and update the dataset. the procedural nature of the meta-analyses allows in fact to model them as scientific workflows of sequential activities, that we wish to capture and use to update the dataset, in a way that data maintainers do not have to input new data themselves. besides relieving the workload of the dataset maintainers, collecting workflows could benefit data consumers, as the expertise of previous scholars could support new users when performing their own analyses. in order to achieve this, our first goal is to make the databank available to the field to allow exploring data and conducting meta-analyses with it. following similar approaches, we use an ontology engineering approach to represent the databank as a structured dataset, describing both the bibliographic information and the domain-specific knowledge from the collected studies, and then to build semantically-enhanced application to perform meta-analyses. our work has two contributions, namely (i) we provide a detailed semantic description of the domain of human cooperation, so far never published in a structured way, that researchers from the field can easily reuse and extend; and (ii) we build a tool to conduct meta-analyses semi-automatically, reducing the time researchers need to test their new hypotheses. more in general, our work shows how semantic technologies can be used to tackle the limitations of meta-analyses at different levels (search, analysis and synthesis), fostering research reproducibility while facilitating the framing and testing of research hypotheses. in order to allow conducting meta-analyses over a knowledge graph, we follow two simple steps: first, we deal with the generation of the databank, by describing the research studies and their content and generating a knowledge graph from this; second, we focus on building the application conduct meta-analyses on it. in the following, we will imagine the example of a meta-analysis performed to study the impact (effect) of using communication (independent variable) over cooperation (dependent variable) in a control group. the first step is gathering the raw data annotated by the coda team and build a structured knowledge graph from it. the dataset consists a series of csv tables, roughly divided by topic, where published studies are annotated according to the features described in sect. , including both generic information (study characteristics such as country or year of publication) and specific characteristics (information relevant to cooperation games, e.g. types of priming or incentives given to the study participants). we therefore divide this task in three steps, i.e. establishing a general schema for papers, dois, authors, experiments (domain-independent knowledge), providing a more fine-grained model to describe the cooperation situations (domain-specific knowledge), and populating the knowledge graph accordingly. modelling domain-independent knowledge. figure presents the domain-independent schema we used , where a publication consists of a cdo:paper that includes an arbitrary set of cdo:study, i.e. experiments performed in different settings and with different goals. in the example of listing . , for instance, resource :eng represents a paper written by h. varian in reporting his experimental study :eng where students from the us played a prisoner's dilemma game. additional metadata about the paper such as publication date, authors etc. are collected directly by dereferencing the paper's dois and by including the collected triples as properties of a cdo:doi class. we then define these properties as cdo:scholarly prop. in our example, the doi allows to gather the paper's scholar information, such as his author h. varian and its year of publication ( ). each cdo:study has also specific properties, which we divided in cdo:sample prop and cdo:quantitative prop depending if they represent information about the study sample settings or the experimental quantitative/statistical information. for example, cdo:country, cdo:sample size, cdo:country, cdo:studentsonly and cdo:game are subproperties of cdo:sample props, while cdo:overall coop (that measures the overall participants' cooperation rate across different tests in an experiment) is a quantitative property defined as subproperty of cdo:quant prop. as said, a cdo:study reports one or more tests, modelled as cdo:observation. the significance of the tests is estimated in terms of statistical cdo:quantitative prop of an observation, e.g. effect size values, standard errors, variance, etc. in our example, study :eng reports one observation called :eng . note that the current work only focuses on using a semantic-based approach as a mean to simplify meta-analyses. in other words, vocabulary and data model are not finalised, and while alignments at schema and instance level are already under way, they remain out of this paper's scope. modelling domain-specific knowledge. in order to allow experts to understand the factors affecting cooperation, the next step is describe the content of each study in a fine-grained way. we model observations as comparisons of one or two different cdo:treatment, consisting in the experimental settings that an experimenter modifies with the goal of assessing how and if the cooperation between participants of a game varies significantly. for example, observation :eng . . . . compares a treatment in which participants were not allowed to communicate (line - ) with a second treatment, in which participants were playing with real partners (line ) and could only exchange promises about future game behaviours (line ). these experimental settings, modified across different treatments of the same independent variable (iv), are fundamental to perform meta-analyses. an experimenter could be interested in observing the effects of allowing or denying communication within participants of a game, or on the impact of specific characteristics (called moderators) such as age, gender or personality of the participants, type of communication exchanged. we therefore take all rdf properties whose domain is the class cdo:treatment, and organise them in a domain-specific taxonomy of information relative to cooperation in social dilemmas. the resulting taxonomy, shown in fig. , was built in a bottom-up fashion, i.e. (i) an initial list of key variables and definitions was drafted by the coda's team given their extensive expertise in the domain; (ii) the list was used to perform an initial annotation of ∼ k studies across universities, to report potential problems and additions, and (iii) further revised by a scientific advisory board of senior domain experts; (iv) existing papers were revised and new ones were annotated accordingly. all properties are by definition subproperties of a generic rdf:property called cdo:iv props, and can be either cdo:measured iv or cdo:manipulated iv, depending if it consists in a discrete (e.g. type of communication) or continuous (e.g. amount of money incentive) variable. additionally, up to four categories of properties can describe the cooperative game in a treatment, namely: • participant variables (cdo:participant iv), i.e. all variables related to the people taking part of a cooperation, including personal background (age, ethnicity, education), stable personality traits (e.g. hexaco/social value orientation), dynamic psychological states (e.g., emotions, moods); • decision variables (cdo:decision iv), i.e. all variables related to the decisions that people take during the game, e.g. intrapersonal features (priming, time constraints), or interpersonal features (communication, gossip); • game structure variables (cdo:game structure iv), consisting in all variables related to the structural aspects of the game, e.g. payment of the participants, protocol for forming teams, composition of the team etc; • institution variables (cdo:institution iv), involving the rules and norms for participants, such as punishment, reward or taxation during the game. taking back the example of listing . , cdo:communicationbaseline, cdo:realcommunication, cdo:communicationcontent are subproperties of cdo:communication iv, indicating that in his study, the experimenter only manipulated communication as an independent variable. of course, this is a rather simplified example, and treatments describe on average multiple ivs. once defined the two parts of the schema, we create statements with the support of python's rdflib library, additionally dividing them across a number of named graphs (i.e. studies, papers, observations, treatments, vocabulary descriptions) for storage and querying convenience. the generated dataset is hosted as a triplydb instance , allowing to easily upload and update datasets and expose them through apis such as sparql, restful, and textual search. while the complete dataset is in the process of being iteratively published, its preliminary online version currently includes , statements, including approx. . k studies and specific independent variables (cfr. table ). in the second phase, we build a web-based interface allowing experts to conduct their meta-analysis over the generated knowledge graph. the application, shown in fig. , is accessible through the main website, and allows to (i) explore the databank; (ii) select relevant studies and (iii) performing meta-analyses online. data exploration. at first, users are presented a global overview of the data. studies and the effect sizes they report can be explored using a number of criteria, e.g. year of publication, sample size, country of publication (as in fig. ). at the bottom, a tabular condensed view of the data is also offered, and users are given the possibility to click on studies, papers and observations to explore their properties in-depth directly from the triplydb instance. an additional panel at the top offers the possibility of visualising the taxonomy of independent variables described in the previous section. search&selection. the left panel allows users to select the desired studies before starting their statistical computations. the selection can be performed based on independent variables that are manipulated during treatments of a study (cfr. fig. ). in the example of fig. , the user selected observations from studies manipulating some cdo:communication iv, and specifically studies manipulating the properties cdo:realcommunication and cdo:communicationcontent, resulting in a selection of observations from different studies. multiple selection is allowed, e.g. one could additionally include cdo:personality iv or cdo:emotion iv variables. studies can be additionally filtered based on sample and quantitative properties, e.g. one could choose to include in the metaanalysis only observations from studies published between and , or those with at least participants. in order to foster data sharing and reuse, the portion of desired data can be downloaded in tabular format. in this way, as authors are relieved from the coding step, as the provided data are already annotated, and the model can be extended according to the meta-analysis' purposes. once selected the desired data, the user can perform his meta-analysis using the tabs in the app. due to space restrictions, fig. shows a simplified view of the meta-analytic steps, but the reader is invited to consult the online version to replicate our example. typical meta-analysis steps include: . fitting a meta-analytic model (fig. a) . this operation consists in choosing the model type (fixed, random and mixed effects), the method (maximum or restricted likelihood estimators), and the variables (single, or aggregated) to obtain the estimate of the overall population effect size. models can also be fitted by specific moderators (e.g. mean age of the sample as in fig. b ), corresponding to the ivs and study characteristics in the kg schema. . exploring the heterogeneity of single studies. using forest plots (fig. c) , a meta-analysts can illustrate the effect sizes of the studies ordered by year of publication, using confidence intervals that reflect the precision with which effects were estimated (the narrower the confidence interval, the greater precision). effect sizes can also be plotted to check their distribution and density using violin plots (fig. d) , where relevant statistics such as median and its % confidence interval, the quartiles and outliers are also shown. . checking for publication bias. using funnel plots (fig. e) , the user can plot effects sizes against the sample sizes in a symmetrical inverted funnel centred on the average effect, in a way that asymmetries in the distribution of the data should be revealed. an additional data-augmentation method called "trim&fill" can also be selected in order to estimate the number of studies that are missing from the meta-analysis. . computing power analysis. this activity (fig. f) allows to derive the optimal sample size for a desired effect size (either obtained by the fitted model, or specified by the user) with a given power and p-value. determining the optimal effect size given a desired sample size and p-value is also possible. with this operation, researchers can calculate the required sample size necessary to obtain high statistical power in future studies. similarly to the data selection, all meta-analytic results can also be comfortably downloaded through the interface. this is particularly beneficial to less experienced meta-analysts, as they can be relieved from the often tedious and time consuming task of writing efficient code. additionally, all statistical computations and activities are presented sequentially in order to support methodological design -thoroughly crafted using meta-analytic experts. finally, by allowing to compute meta-analyses online, published and unpublished meta-analyses can also be easily reproduced, benefitting study reproducibility and transparency for the whole research field. implementation. the above web-app is implemented using r shiny , a package for dashboard development straight from r code. the advantage of using shiny lies mostly in the fact that we can exploit the large variety of statistical techniques (linear/nonlinear modelling, classical statistical tests etc.) and their graphical outputs (funnel, forests and violin plots) to manipulate, interact and visualise data from the databank knowledge graph. data are selected through sparql queries stored as apis on the triplydb instance, allowing to further decouple the application from the dataset. offloading data maintainers. here, we are interested in reducing the workload of the experts in maintaining and updating the dataset. we therefore asked the coda team to quantify the time it takes the editorial board to include a new study in the dataset. table shows the main activities they need to perform, i.e. searching studies, skimming publications to assess if they fall under the eligibility criteria, coding studies based on the chosen characteristics, and computing the aggregate effect sizes if a work reports separate splits (e.g. a paper reporting different cooperation rates, because rounds of a game were experimented). we report answers of three experts e n supervising the databank, as well as one annotator a of more limited expertise. these are then compared with the data provided by [ ] , an established reference that analysed the problem of performing meta-analyses in the medical domain. the table illustrates that data maintainers invest most of their efforts in searching, skimming and annotating papers, reported as the most time-expensive activities needed to be performed. a straightforward conclusion here is that the this workload could be significantly reduced by allowing users consulting the databank to upload their studies using the same annotation schema. this would allow maintainers to focus only on the light-weight refinement and validation of the uploaded data. finally, the disproportion in time between activities in the social science and medical domain suggests that a substantial difference lies in the type of studies that experts need to analyse (e.g. lengths, regression analyses). here, we look at estimating how our approach supports meta-analysts in searching data. we asked the experts to write a set of competency questions, which can indicate how well the knowledge graph support the search phase through requirement fulfilment. table shows that a number of requirement, particularly related to the selection of studies with multiple characteristics or using effect sizes, could not be answered with the original the dataset. this means that a user would mostly have to go through the process of extracting and re-coding the studies of interests, before performing a new meta-analysis. additionally, the databank now includes over independent variables (vs. the original dataset with no controlled vocabulary for independent variables), which can be used both for the study selection and the moderator analyses at finer or coarser grain (e.g. users can decide to simply consider all communication variables, or to some specific communication characteristics). this is a great opportunity for the behavioural science community, which can easily investigate different research questions in a more automated and assisted way. we are interested in understanding if our approach facilitates users performing the statistical computations needed to finalise and publish a meta-analysis. table provides an estimate of the resources necessary to compute meta-analytic models in a normal setting (i.e. when not supported by the coda application) based on the answers of two experts that recently run a meta-analysis (ma , ma ), as well as the information provided by [ ] . we used lines of code as a measure for data preparation, model fitting and result plotting to show that users might be relieved from writing a significant part of code when using a semantically-enriched system, as opposed to a database of meta-analyses. of course, resource allocation is highly dependent on the type meta-analysis performed (i.e. number of studies analysed, complexity of the question framed, number of moderator analyses...), and the same would lie when conducting metaanalyses with the support of our framework. yet, users would be relieved from the data preparation and programming tasks, offered by the coda app as interactive activities to be performed in a customised way. to give a baseline over the current application, a simple sequence of model fitting, heterogeneity analysis and moderator analysis takes on average to min. where specific var n are siblings of a parent variable (e.g. cdo:communication iv and cdo:priming iv are children of cdo:decision iv). additionally, by relying on sparql queries over the dataset, we can monitor the users' activities, and offer recommendations of new variables to meta-analyse based on popularity (i.e. suggesting the most popular moderators for specific variables) or anti-patterns (i.e. suggesting to choose less popular and unexplored queries for the meta-analysis). finally, by describing meta-analysis as scientific workflows that manipulate data with specific parameters, we can collect and offer them as recommended practices to users. in this sense, the expertise of previous users could be leveraged to offer inexperienced practitioners a more automated way of performing meta-analyses -tackling to the cold start problem of designing experiments. in this work, we have shown how to use semantic technologies to support researchers in conducting meta-analyses and summarise scientific evidence in a more automated way. meta-analysis are a science of science method for research synthesis, which tend to suffer from scalability, methodological and technical issues also due to the exponentially growing volume of research. using a social science scenario, we showed how the content of research outputs can be semantically described and used to build an application to help meta-analysts in searching, analysing and synthesising their results in a more automated way. the use-cases we discussed have shown that the approach is beneficial at several levels of the meta-analysis, and has the potential of fostering research replication and facilitating the framing and testing of research hypotheses in the field of behavioural science. future work will be focused on: (i) publishing the databank following the fair principles , which will require alignment to existing vocabularies (e.g. data cubes) and linking instances to available datasets (e.g. microsoft academics); (ii) improving the web application with more advanced analyses, e.g. dynamics of citations, multivariate analyses, integration of cross-societal moderators from the linked open datasets (e.g gdp or gini indices from eurostats); (iii) implementing the collection and documentation of meta-analytic workflows using prov ; (iv) evaluation through user-testing, quantifying the time they take to perform a meta-analytic tasks with and without the support of the knowledge-based recommendations, workflows and documentation. promoting replicability in developmental research through meta-analyses: insights from language acquisition research meta-analysis: neither quick nor easy analysis of the time and workers needed to conduct systematic reviews of medical interventions using data from the prospero registry introduction to meta-analysis metabus as a vehicle for facilitating metaanalysis google dataset search: building a search engine for datasets in an open web ecosystem ontology use for semantic e-science review manager (revman) workflow-centric research objects: a first class citizen in the scholarly discourse an incremental learning method to support the annotation of workflows with data-to-data relations meta-analysis in clinical trials goweb: a semantic search engine for the life science web ontology-based discovery of workflow activity patterns strengths and limitations of meta-analysis: larger studies may be more reliable publication bias in the social sciences: unlocking the file drawer the publishing workflow ontology (pwo) common motifs in scientific workflows: an empirical analysis the disk hypothesis ontology: capturing hypothesis evolution for automated discovery from peer-reviewed to peer-reproduced in scholarly publishing: the complementary roles of data models and workflows in bioinformatics meta-analysis: pitfalls and hints the anatomy of a nanopublication meta-analysis and the science of research synthesis cochrane handbook for systematic reviews of interventions the datalegend ecosystem for historical statistics biosearch: a semantic search engine for bio rdf sharing interoperable workflow provenance: a review of best practices and their practical application in cwlprov keynote addresses. meta-analysis: reconciling the results of independent studies comparison of effect estimates from a meta-analysis of summary data from published studies and from a meta-analysis using individual patient data for ovarian cancer studies systematic reviews: crd's guidance for undertaking reviews in health care social dilemmas: understanding human cooperation key: cord- -vf xbaug authors: dysko, robert c.; nemzek, jean a.; levin, stephen i.; demarco, george j.; moalli, maria r. title: biology and diseases of dogs date: - - journal: laboratory animal medicine doi: . /b - - / - sha: doc_id: cord_uid: vf xbaug nan status as a cooperative companion animal of reasonable size. dogs were used in the mid- s by william harvey to study cardiac movement, by marcello malpighi to understand basic lung anatomy and function, and by sir christopher wren to demonstrate the feasibility of intravenous delivery of medications (gay, ) . the use of dogs continued as biomedical research advanced, and they were featured in many noteworthy studies, including those by pavlov to observe and document the conditioned reflex response and by banting and best to identify the role of insulin in diabetes mellitus. for a comprehensive but concise review of the use of the dog as a research subject, the readers are directed to the manuscript by gay ( ) . the breed of dog most commonly bred for use in biomedical research is the beagle. some commercial facilities also breed foxhounds or other larger-breed dogs for use in surgical research studies. some specific breeds with congenital or spontaneous disorders are also maintained by research institutions (see specific examples below). random-source dogs used in research are most frequently mongrels or larger-breed dogs (e.g., german shepherd, doberman pinscher, labrador and golden retrievers) that are used for surgical research and/or training. according to a computerized literature search for beagle for the years - , approximately % of the biomedical scientific publications identified were in the fields of pharmacology or toxicology. especially common were studies focusing on pharmacokinetics, alternative drug delivery systems, and cardiovascular pharmacology. the next most common areas of research using beagles were dental and periodontal disease and surgery ( % of publications), orthopedic surgery and skeletal physiology ( %), and radiation oncology ( %). other research areas that utilized beagles included canine infectious disease, surgery, imaging, prostatic urology, and ophthalmology. most large-sized dogs (either purpose-bred or randomsource) are used in biomedical research because of their suitability for surgical procedures. anesthetic protocols and systems for dogs are well established, and the organs of larger-breed dogs are often an appropriate size for trials of potential pediatric surgical procedures. surgical canine models have been used extensively in cardiovascular, orthopedic, and transplantation research. there are also some unique spontaneous conditions for which dogs have proven to be valuable animal models. a colony of gray collies is maintained at the university of washington (seattle) for the study of cyclic hematopoiesis. this condition is manifested by periodic fluctuations of the cellular components of blood, most notably the neutrophil population. these dogs are used to study the basic regulatory mechanisms involved with hematopoiesis, as well as possible treatments for both the human and the canine conditions (brabb et al., ) . golden retrievers affected with muscular dystrophy have been used as models of duchenne muscular dystrophy in human children. duchenne muscular dystrophy is caused by an absence of the muscle protein dystrophin, inherited in an x-linked recessive manner. the dystrophy in golden retrievers is caused by absence of the same protein and is inherited in the same way. the clinical signs (such as debilitating limb contracture) are also similar between the canine and human conditions (kornegay et al., ) . bedlington terriers have been used to study copper storage diseases (such as wilson's disease), and the development of spontaneous diabetes mellitus and hypothyroidism in a variety of dogs has also been studied for comparisons with the human conditions. although historically the dog has been a common laboratory animal, the use of dogs in research has been waning over the past few years. according to the u.s. department of agriculture ( ) , the number of dogs used in research has declined from a high use of , in , in to only , in . this decrease was caused by a variety of factors, including (but not limited to) increased cost, decreased availability, local restrictive regulations, conversion to other animal models (such as livestock or rodents), and shift in scientific interest from pathophysiology to molecular biology and genetics. dogs used for research are generally segregated into two classes: purpose-bred and random-source. purpose-bred dogs are those produced specifically for use in biomedical research; they are intended for use in long-term research projects and/or pharmacologic studies in which illness or medication would require removal from the study. usually these dogs are either beagles or mongrel foxhounds, although other breeds may be available. purpose-bred dogs typically receive veterinary care throughout their stay at the breeding facility. they are usually vaccinated against canine distemper virus, parvovirus, adenovirus type , parainfluenza virus, leptospira serovars canicola and icterohemorrhagiae, and bordetella bronchiseptica. rabies virus vaccination may also be included. purpose-bred dogs are also usually treated prophylactically for helminths and ectoparasites, intestinal coccidia, and bacterial ear infections (r. scipioni and j. ball, personal communication, ) . random-source dogs are not bred specifically for use in research. they may be dogs bred for another purpose (e.g., hunting), retired racing dogs, or stray dogs collected at pounds or shelters. the health status of these dogs can be the same quality as purpose-bred dogs, or it can be an unknown entity. randomsource dogs that have been treated and vaccinated in preparation for use in research are termed conditioned dogs. these dogs are then suitable for long-term studies or terminal preparations that require unperturbed physiologic parameters. conditioned dogs are often tested for heartworm antigen because of the implications that infestations can have on cardiovascular status and surgical risk. nonconditioned random-source dogs are useful only in a limited number of research studies, such as nonsurvival surgical training preparations. options for procurement of dogs for biomedical research typically include purchase from a u.s. department of agriculturedesignated class a or class b licensed dealer or directly from a municipal pound. the requirements for usda licensure are detailed in code of federal regulations (cfr), title , chapter ( - - edition), subchapter a, animal welfare, . , definitions, and . , requirements and application. briefly, class a licensees are breeders who raise all animals on their premises from a closed colony (suppliers of purpose-bred dogs are typically class a dealers). class b licensees purchase the dogs from other individuals (including unadopted animals from municipal pounds) and then resell them to research facilities. there are additional regulations that apply to class b dealers (such as holding periods and recordkeeping documentation) because of the public concern that stolen pets could enter biomedical research facilities in this manner. regulations regarding the sale of pound dogs to research facilities or class b dealers vary from state to state and include some bans on this practice. the best resource for identification of possible vendors is the "buyer's guide" issue of the periodical lab animal. typically the last issue of each year, the "buyer's guide" lists sources for both purpose-bred and random-source dogs and denotes such features as pathogen-free status, documentation of health status, and availability of specific breeds and timed pregnant females. some suppliers also have separate advertisements within that issue of the journal. welfare act ( cfr . , . , and . [g] ) are described in cfr chapter ( - - edition), subchapter a, animal welfare. regulations pertaining specifically to the care of dogs used in research are found in subpart a, specifications for the humane handling, care, treatment, and transportation of dogs and cats, of part (standards) of subchapter a. particular attention should be paid to section . c (primary enclosures--additional requirements for dogs), because the space required for housing dogs is calculated using the length of the dog rather than the body weight (which is used for other species and also for dogs, according to national research council (nrc) guidelines). section . (exercise for dogs) describes the requirements that dealers, exhibitors, and facilities must follow in order to provide dogs with sufficient exercise. the institute of laboratory animal research (ilar) has written the "guide for the care and use of laboratory animals" (seventh edition, ) . the "guide" is the primary document used by institutional animal research programs to develop and design their programs, as well as by the association for assessment and accreditation of laboratory animal care international (aaalac international) and other animal care evaluation groups to facilitate site visits and inspections. the ilar committee on dogs has also written "dogs: laboratory animal management " ( ) . this publication describes "features of housing, management, and care that are related to the expanded use of dogs as models of human diseases" and includes "an interpretive summary of the animal welfare regulations and the requirements of the public health service policy on humane care and use of laboratory animals." the reader is encouraged to use these publications to obtain further information on care and husbandry of dogs in the biomedical research setting. growth data for beagles from a purpose-bred dog breeding facility are provided in table i . table ii features hematology data from beagles from the same commercial facility. table iii lists serum and urine chemical data for beagles. normal physiologic data for dogs (no breed specified) are provided in table iv . the information presented in the tables represents a range of normal values that can vary, depending on the analytical method and equipment used as well as the age, breed, gender, and reproductive status of the animal. federal regulations promulgated by the animal and plant health inspection service, usda, in response to the animal good nutrition and a sound, balanced diet are essential to the health, performance, and well-being of the animal. the basic nutrient requirements for dogs have been compiled by the nrc and represent the average amounts of nutrients that a group of animals should consume over time to maintain growth and prevent deficiencies (national research council, ) . the reader is referred to these guidelines for useful reference points for management of an animal's diet during various physiologic states (e.g., gestation, lactation, maturational age). most commercially available balanced dog diets are "closedformula" diets, in which the labeled specific minimum requirements for protein and fat, and the maximum values for ash and fiber, are met. these diets do not necessarily provide the identical composition of ingredients from batch to batch. ingredient composition varies, depending on the cost relationships of the various ingredients as the manufacturer attempts to achieve the label requirements at the lowest ingredient cost. an "openformula" (or "fixed-formula") diet provides more precise dietary control. in these diets the ingredients are specified, and the percentage of each ingredient is kept constant from batch to batch. "semipurified" diets provide for the strictest control of ingredients and are formulated from the purified components: amino acids, lipids, carbohydrates, vitamins, and minerals. the animal care provider should be aware of the manufacture date of the diet, which should be clearly visible on the bag. as a general rule, diets are generally safe for consumption up to months following the manufacture date when stored at room temperature. refrigeration may prolong the shelf life, but the best strategy is to use each lot based on the date of manufacture in order to prevent food from expiring and to ensure that only fresh diets are fed. specifications for feeding and watering of dogs are provided in the regulations of the animal welfare act. recommendations for feeding the appropriate amount of diet are determined by the dog's metabolic requirements. the basal metabolic rate, or basal energy requirement (ber), refers to the amount of energy expended following sleep, - hours after food consumption, and during thermoneutral conditions (kleiber, ; lewis et al., ) . the maintenance energy requirement (mer) is the amount of energy used by a moderately active adult animal in a thermoneutral environment, which in the dog is approximately twice the ber (lewis et al., ) . for dogs weighing greater than kg, the mer may be calculated using this simplified linear equation: mer (metabolizable kcal/day) = ( weightkg + ) (national research council, ; lewis et al., ) . the quantity of a correctly balanced diet to be fed to each dog can then be determined by dividing the mer by the energy density of the diet. fat provides three major dietary functions, including absorption of fat-soluble vitamins (a, d, e, and k), enhancement of palatability, and provision of essential (unsaturated) fatty acids. dietary fat is an excellent, highly digestible energy source, providing . times more energy on a per weight basis than either soluble carbohydrates or proteins (lewis et al., ) . however, fats are not needed for this purpose when adequate carbohydrate and protein are present. consumption of fat in excess of an animal's ability to metabolize it results in steatorrhea and has been related to the development of acute pancreatitis, whereas lack of dietary fat may lead to a fatty acid/energy deficiency. fatty acid deficiency is associated with poor growth, poor physical performance, reduced reproductive performance, and weight loss. dogs are considered to be "easy keepers," because they do not have as many absolute nutritional requirements as their domestic counterpart, the cat. however, they do possess a unique requirement for certain polyunsaturated fatty acids, a deficiency of which may predispose them to decreased growth rates and dermatologic abnormalities, such as "hot spots." dogs require linoleic (f - ) acid, an essential fatty acid (national research council, ) , and more recently it has been demonstrated that the f - fatty acids may play a role in maintaining healthy skin (logas and kunkle, ) . supplementation with a balanced essential fatty acid product (e.g., derm caps) may alleviate allergy-related dermatoses such as flea-bite dermatitis and pyoderma (logas and kunkle, ; miller, ) . essential fatty acid deficiency can occur in dogs receiving low-fat dry dog food that has been stored too long, particularly under warm, humid conditions (lewis et al., ) . there are a-amino acids, of which cannot be synthesized in sufficient quantity to meet a dog's normal metabolic demands for growth and maintenance. hence, as their name implies, these essential amino acids are required by all dogs and must be provided in the diet. the essential amino acids and the minimal requirements for growth are listed elsewhere (lewis et al., ) . chronic excessive protein intake may be detrimental to the kidney by contributing to accelerated renal aging and subsequent glomerulosclerosis (lewis et al., ) . conversely, inadequate protein intake results in retardation of growth and adata graciously provided by r. scipioni and j. ball of marshall farms usa, inc., north rose, new york. beagles tested for period / / - / / . b s.d., standard deviation; wbc, white blood cells; rbc, red blood cells; hgb, hemoglobin; hct, hematocrit; mcv, mean corpuscular volume; mch, mean corpuscular hemoglobin; mchc, mean corpuscular hemoglobin concentration; rdw, red cell distribution width; hdw, hemoglobin distribution width; plt, platelets; mpv, mean platelet volume; neut, neutrophils; lymp, lymphocytes; mono, monocytes; eos, eosinophils; baso, basophils; luc, large unstained cells; li, lobularity index; mpxi, mean peroxidase activity index reduction in production and/or performance. protein deficiency, a potential consequence of decreased food intake, results in decreased energy intake. as a compensatory mechanism for a lack of fat or carbohydrate, body protein catabolism ensues in order to meet energy demands, thus exacerbating the negative protein balance and contributing to the clinical signs of edema/ascites, unkempt appearance, lethargy, and weight loss. thus, caloric needs must be met before protein needs (lewis et al., ) , an important concept to bear in mind in the event of research experiments that may predispose to anorexia. in general, providing a good quality commercial diet that supplies the required amount of amino acids and caloric requirements of the animal, while avoiding excess protein, will ensure nutritional stability and promote longevity. appropriate mineral balance in the diet is very important. the best approach in the laboratory setting is to feed a commercial diet that has been formulated with the proper amount and balance of minerals for normal growth. the recommended amount of dietary minerals and the major causes and clinical signs of deficiencies are published elsewhere (lewis, ) . determining the specific mineral involved in an imbalance can be a diagnostic challenge, because the clinical signs for several excesses/ . (basal) . (anestrus) . c < . c - c - r (continues) deficiencies are similar and nonspecific. a definitive diagnosis is often made only after the diet has undergone analysis of the mineral components. once the imbalance has been identified, the safest resolution to the problem is to discard the entire lot of misformulated diet. attempting to correct the imbalance through oral supplementation is likely to be more harmful than beneficial, and it risks intensifying the problem by creating additional mineral imbalances. vitamins function as enzymes that regulate a wide variety of physiologic processes. they are divided into two groups based on their solubility. the fat-soluble vitamins include a, d, e, and k, whereas the rest are water-soluble. a list of the vitamins, their requirements, and clinical signs associated with deficiencies and toxicities is published elsewhere (lewis et al., ) . cases of dietary deficiency are rarely encountered in the research setting, because laboratory dog chows are fortified with vitamins. additional vitamin supplementation may occasionally be required during prolonged clinical illnesses, such as polyuria or diarrhea, which predispose to loss of water-soluble vitamins (b complex and c) (lewis et al., ) . however, as with minerals, routine supplementation of vitamins may induce inadvertent toxicity and exacerbation of an imbalance. management of a breeding colony requires broad knowledge of the dog's anatomy, reproductive physiology, and behavioral needs during breeding, gestation, and parturition. although a comprehensive discussion of the biology of canine reproduction is beyond the scope of this chapter, essential features of the broad topics noted above are presented. this section is largely based on information assimilated from texts such as "miller's anatomy of the dog" (evans and christensen, ) , "veterinary reproduction and obstetrics" (arthur et al., ) , and an issue of veterinary clinics of north america: small animal practice devoted to pediatrics of puppies and kittens (hoskins, ) . the ovaries of the bitch are attached to the dorsolateral walls of the abdominal cavity caudal to the kidneys by the broad ligaments and are not palpable abdominally. the uterus consists of the cervix, uterine body, and uterine horns. the cervix is an abdominal organ, located approximately halfway between the birchard and sherding ( ) . ovaries and the vulva. when the bitch is in proestrus and estrus, the cervix can be distinguished during abdominal palpation as an enlarged, turgid, walnut-shaped structure. catheterization of the cervix is usually not possible in the normal bitch at any stage of the reproductive cycle, except during or immediately following parturition. thus, semen is deposited at the external cervical os during natural or artificial insemination. the vagina is a long musculomembranous canal that extends from the uterus to the vulva. when the vagina is examined, the gloved finger or examination instrument should be introduced through the dorsal commissure of the vulva so as to avoid the deep ventral clitoral fossa. examination should proceed at an angle of approximately ~ until the instrument or fingertip has passed over the ischial arch, after which it can be directed further craniad toward the cervix. the bitch has a monoestrous cycle, with clinical estrus occurring predominantly in january or february and again in july or august (although it can occur at any time of year). the estrous cycle consists of four stages: proestrus, estrus, diestrus, and anestrus. the average duration of proestrus is days. during this stage the vulva is enlarged, turgid, and firm, and a sanguinous vaginal discharge is present. endocrinologically, proestrus is the follicular stage of the cycle, and estrogen levels peak at this time. estrus generally lasts days, and the vulva is softer and smaller than in proestrus. a vaginal discharge persists during estrus and may remain serosanguinous or become straw-colored. the endocrine feature of estrus is the luteinizing hormone (lh) surge, followed by ovulation within - hours. diestrus begins approximately days after the onset of standing heat. the end of this stage is days later, which would be coincident with whelping if the bitch had become pregnant. serum progesterone levels peak during diestrus. the duration of anestrus is approximately months. anestrus is the stage of reproductive quiescence, characterized by an absence of ovarian activity and serum progesterone levels of less than ng/ml. components of the canine spermatic cord include the ductus deferens, the testicular artery and vein, the lymphatics and nerves, and the cremaster muscle. the cremaster muscle and pampiniform plexus aid in thermoregulation of the testicles, which are maintained at ~ lower than basal body temperature. sweat glands in the scrotum assist in lowering the scrotal temperature through evaporation. the penis is a continuation of the muscular pelvic urethra and is attached to the ischiatic arch by two fibrous crura. it is composed of fibrous tissue and three cavernous sinuses: corpus cavernosum, corpus spongiosum penis, and corpus spongiosum glandis. the accessory sex glands of the dog consist of only a well-encapsulated prostate gland that surrounds the pelvic urethra, and ampullary glands at the termination of the vas deferens in the urethra. the dog does not have seminal vesicles or bulbourethral glands. the onset of puberty ranges from to months of age and is affected by breed, season, and nutritional and disease status. testicular growth is rapid at this time, and the seminiferous tubules begin to differentiate. the sertoli cells form the bloodtestis barrier, the tubules become hollow, and spermatogenesis commences. this process is initiated by the secretion of lh from the anterior pituitary, which stimulates the production of testosterone by the interstitial, or leydig's, cells. secretion of follicle-stimulating hormone (fsh) by the anterior pituitary stimulates the production of other key hormones by the sertoli cells, including inhibin, androgen binding protein, and estrogen. fsh stimulates spermatogenesis in the presence of testosterone, while inhibin and estrogen play a role in a feedback loop on the pituitary gland to decrease fsh production. spermatogenesis in the dog is completed in days, with subsequent maturation of sperm occurring in the epididymis for approximately days. thus, the entire process from initiation of spermatogonial mitosis to delivery of mature sperm to the ejaculate is days. a breeding soundness exam should be conducted to assess the probability of a male dog's successful production of offspring. factors affecting male fertility include libido, ability to copulate, testicular size, and quality and number of sperm produced. problems with libido may occur in dogs due to early weaning, isolation, or inherited abnormalities that suppress sexual behavior. animals with poor hindlimb conformation or with trauma to the back or hindlimbs may be unable to properly mount the female. there is a positive correlation with the size of the testicles as measured by scrotal circumference and the number of sperm produced. finally, parameters used to assess the quality of sperm include motility, morphology, volume, and concentration. an ejaculate ( ml) that contains approximately million progressively motile sperm without significant morphological abnormalities (such as a kinked tail) is a good indicator of normal male fertility. in general, erection, which involves muscular contractions and increased arterial blood flow to the penis, is controlled by the parasympathetic nervous system, whereas ejaculation is under sympathetic control. on mounting, the initial thrusting and ejaculation of semen last about minute. the bulbus glandis becomes enlarged, which lodges the penis in the female reproductive tract. the male then dismounts and brings one hindleg over the female, and the two continue to be joined "rear to rear," a position classically termed "the tie." ejaculation of the accessory gland fluid continues for - minutes. the continued expulsion of prostatic fluid during the "tie" may serve to propel the semen from the vagina through the cervix into the uterus. fertilization occurs in the oviduct and may occur as late as days after coitus, because of the long life span of sperm in the dog. however, once ovulated, oocytes generally remain viable for only - hours. therefore, the bitch should be bred prior to ovulation to ensure the presence of sperm for fertilization of live oocytes. cells of the vaginal epithelium mature to keratinized squamous epithelium under the influence of estrogen. because of the rise in estrogen throughout proestrus, with peak levels occurring just prior to the onset of standing heat, the vaginal smear can be used as an indicator of the bitch's readiness for breeding. the smear will not confirm the presence of ovulation, nor is it of prognostic value in normal bitches during anestrus. the percentage of vaginal epithelial cell cornification is an index of estrogen secretion by the ovarian follicles. as cornification of vaginal epithelial cells proceeds, the cells become larger, with more angular borders. the nuclear-cytoplasmic ratio decreases until the nuclei reach a point where they no longer take up stain (coincident with the onset of estrus). the cells appear "anuclear" and are classified as "cornified" or "anuclear squames." cornification occurs approximately days prior to the estrogen peak and days prior to standing heat. the percentage of cornified cells (of the total number of epithelial cells) decreases gradually to zero after the onset of diestrus. the vaginal cytology smear of the bitch changes from predominantly cornified to noncornified days after ovulation. the day of this change is the first day of diestrus. other epithelial cell types noted on vaginal cytology include superficial cells (large, angular cells with small nuclei); intermediate cells (round or oval cells with abundant cytoplasm and large, vesicular nuclei); and parabasal cells (small round or elongated cells with large, well-stained nuclei, and a high nuclear-cytoplasmic ratio). based on vaginal cytology, the estrous cycle is classified as follows: although vaginal cytology is a useful tool, it is not a substitute for observation of behavioral estrus, which is the best criterion to use in breeding management. during proestrus the male is attracted to the bitch and will investigate her hindquarters, but she will not accept breeding. the behavioral hallmark of estrus is standing receptivity toward the male. during this stage the bitch will exhibit "flagging," or elevation of her tail with muscular elevation of the vulva to facilitate penetration by the male. in order to maximize the conception rate, and the number of pups whelped per egg ovulated, it is recommended to breed the bitch on days , , and of the standing heat. fertilization is completed in the mid-to distal oviduct. implantation is evident by areas of local endometrial edema - days after breeding. there is no correlation between the number of corpora lutea and the number of fetuses in the corresponding uterine horn, suggesting transuterine migration of embryos. the dog has endotheliochorial placentation. the endothelium of uterine vessels lies adjacent to the fetal chorion, mesenchymal, and endothelial tissues, so that maternal and fetal blood are separated by four layers. the canine placenta is also classified as zonary and deciduate, indicating that the placental villi are arranged in a belt and that maternal decidual cells are shed with fetal placentas at parturition. the length of gestation is - days. luteal progesterone is responsible for maintaining pregnancy, and canine corpora lutea retain their structural development throughout gestation. serum progesterone rises from less than ng/ml in late proestrus to a peak of - ng/ml during gestation, then declines to - ng/ml just prior to parturition. progesterone is essential for endometrial gland growth, secretion of uterine milk, attachment of the placentas, and inhibition of uterine motility. pregnancy detection can be performed by abdominal palpation of the uterus days after breeding. the embryos and chorioallantoic vesicles form a series of ovoid swellings in the early gravid uterus. they are approximately inches in length at - days, the time at which pregnancy is most easily and accurately diagnosed. by day the uterus begins to enlarge diffusely, so that the vesicles (and, therefore, pregnancy) are difficult to identify by palpation. fetal skeletons become calcified and are radiographically evident by day . bitches in which a difficult whelping is anticipated should be radiographed in late pregnancy to determine the litter size and to evaluate the size of the fetal skulls in relation to the bony maternal birth canal. real-time ultrasound can be utilized for pregnancy detection of vesicles as early as - days. an abrupt drop in body temperature to less than ~ indicates impending parturition within - hours. the process of parturition has been divided into three stages, stage of labor lasts - hours and is characterized by uterine contractions and cervical dilation. during this stage, the bitch may appear restless, nervous, and anorexic. other common clinical signs include hard panting and increased pulse and respiration rates. fetal expulsion occurs during stage , which lasts approximately - hours. as the fetus engages the cervix, the neuroendocrine system induces the release of oxytocin; this is referred to as the ferguson reflex. oxytocin strengthens the uterine contractions and may elicit voluntary abdominal contractions as well. the bitch is usually recumbent during stage but is able to inhibit this stage if labor if disturbed. the chorioallantois ruptures either during passage of each neonate through the birth canal or by the bitch's teeth at birth. interestingly, posterior presentation is common in dogs but does not predispose to dystocia. the time interval between delivery of each pup is irregular, but the average time lapse is less than hour between pups until parturition is complete. veterinary assistance is necessary if the bitch remains in stage for more than hours without delivering the first pup, or for more than hours before delivering subsequent pups. the placentas are expelled during stage of labor, immediately following delivery of a pup, or up to minutes thereafter. if two pups are delivered from alternate uterine horns, then the birth of both puppies may precede expulsion of the respective placentas. the bitch will lick the newborn vigorously to remove the membranes from its head and to promote respiration. she will also sever the umbilical cord. the bitch may ingest the placentas, although they confer no known nutritional benefit and may induce a transient diarrhea. thermal support should be provided prior to parturition. dogs housed on grated flooring should be provided with mats, and those on solid floors would benefit from blankets placed in a corner of the primary enclosure. shavings are discouraged as they have the potential to coat the umbilical cord, which may predispose to ascending infections. heat lamps may be placed hours prior to parturition and remain until all neonates dem-onstrate vigorous and successful suckling behavior. however, the use of heat lamps necessitates strict supervision in order to prevent thermal burns. if possible, whelping bitches should be housed in a quiet corridor in order to decrease periparturient stress, especially in primiparous or young mothers. thus, monitoring of parturition is important, but human intervention should be minimal in order to prevent stress-induced cannibalism. weak or debilitated puppies may be cannibalized by the bitch before the research staff recognizes the need for veterinary attention. the postpartum use of oxytocin is required only in the event of uterine inertia, stillbirths, or agalactia. in these cases, - units of oxytocin may be administered intramuscularly. uterine involution occurs during anestrus within - weeks of parturition. during this time a greenish to red-brown vaginal discharge, or lochia, may be noted. although lochia is normal, the presence of an odiferous, purulent discharge, accompanied by systemic signs of illness, indicates metritis or pyometra. desquamation of the endometrium begins by the sixth postpartum week, with complete repair by months. newborn puppies are easily sexed by examination of the anogenital distance. in female puppies the vulva is evident a short distance from the anus, whereas the prepuce of male puppies is nearly adjacent to the umbilicus. eyes are open at approximately days, and ears are patent at approximately - days. solid food can be introduced between . and weeks of age, and puppies can be weaned at - weeks. artificial insemination (ai) is indicated when the male is physically incapable of mounting or penetrating the bitch, when there are vaginal abnormalities such as strictures, or when the bitch refuses to stand for breeding. semen for ai is collected using a plastic centrifuge tube and rubber latex artificial vagina. the male is introduced to the bitch's scent and manually stimulated. after collection of the first two fractions, a sufficient amount of the third fraction, which consists predominantly of prostatic fluid, is collected to bring the total semen volume to - ml. the semen is then drawn into a sterile or ml syringe attached to a sterile disposable insemination pipette. the bitch is inseminated either standing or with raised hindquarters. a gloved index finger is inserted into the dorsal commissure of the vulva and directed craniodorsally until it is over the ischial arch. the tip of the insemination pipette is introduced and guided by the gloved finger toward the external cervical os. the semen is injected, and - ml of air are then flushed through the syringe and pipette. the pipette is withdrawn, and the gloved finger is used to feather the ceiling of the vagina until contractions of the vaginal musculature are palpable. the bitch's hindquarters are subsequently elevated to promote pooling of semen around the external cervical os. as with natural breeding, ai should be performed on days , , and of standing heat, or on the days of maximal vaginal cornification. the bitch should be palpated for pregnancy approximately weeks after the first insemination. false pregnancy (pseudocyesis), a stage of mammary gland development and lactation associated with nesting or mothering behavior, is common in the bitch. the condition occurs after the decline in serum progesterone toward the end of diestrus. there is no age or breed predisposition. pseudopregnancy does not predispose the bitch to reproductive disease or infertility. however, in the event of extreme discomfort due to mammary gland enlargement, bitches may be treated with mibolerone (cheque drops) at an oral dose of ~tg/kg q hr for - days (brown, ) . reproductive performance in the bitch is optimal prior to years of age. although normal cycle lengths are reported to occur up to the ages of - years, the interestrous interval tends to increase by years of age. cycling does not completely cease; however, after years of age, bitches demonstrate significant decreases in conception rate and number of live pups whelped. by - years of age, pathologic conditions of the uterus, such as cysts, hyperplasia, atrophy, and neoplasia are extremely common. beagles have been a popular animal model because of their docile nature. they are easily handled and for the most part respond favorably to repetitive manipulations such as body weight measurements, physical examination, electrocardiogram (ecg) recordings, oral gavage, and venipuncture. dogs are sexually mature by - months of age, but they are not socially mature until - months of age. the socialization process should begin early during development, when puppies are receptive to conspecific and human contact. for example, from - weeks of age, puppies are most capable of learning about how to interact with other dogs. between weeks and , puppies are most capable of learning how to interact with people. by - weeks of age dogs voluntarily wander and explore new environments. thus, early handling and mild stress (such as vaccination) appear to be extremely beneficial components of a dog's social exposure. the extent to which breed affects behavior has been the subject of popular speculation but is difficult to prove. in general, breed-specific patterns do tend to emerge. for example, it appears that beagle pups are very motivated by food reward (overall, ). this is not surprising, because the breed was selected to work with its nose, and this may be a useful attribute for laboratory investigations that are predicated on food restriction. canid social systems use signals and displays that minimize the probability of outright aggression. these behavior patterns are most likely elicited during distressful situations, such as strange environments, being handled by strange people, or encountering new animals. an excellent, illustrated discussion of normal canine behavior patterns can be found in the third chapter of "clinical behavioral medicine for small animals" (overall, ). by virtue of the dog's status as a companion animal, there are many veterinary publications and reference texts on the diagnosis, medical management, pathology, and epidemiology of the disorders that can affect this species. the authors of this chapter have chosen to emphasize those diseases that are more frequently encountered in the research setting. especially noted in this chapter are infectious diseases associated with the use of random-source dogs that have unknown vaccination history and have had intensive contact with other similar animals at pounds and/or shelters, or conditions seen frequently in the beagle, the most common breed used in biomedical research. for more thorough and detailed discussion of these diseases, as well as those not discussed in this chapter, the reader should consult standard veterinary textbooks, such as the "current veterinary therapy" series (j. d. bonagura and r. w. kirk, eds.), "veterinary internal medicine" (s. j. ettinger and e. c. feldman, eds.), and "infectious diseases of the dog and cat" (c. e. greene, ed.) . full citations of some chapters from these texts are listed in the references (w. b. saunders co. of philadelphia publishes all three texts.) canine infectious tracheobronchitis (kennel cough complex) etiology. infectious tracheobronchitis (itb) is a highly contagious illness of the canine respiratory tract that usually manifests as an acute but self-limiting disease. several organisms have been incriminated as causative for this condition: bordetella bronchiseptica; canine parainfluenza virus (cpiv); canine adenovirus types and (cav- , cav- ); canine herpesvirus; canine reovirus types , , and ; and mycoplasms and ureaplasms. clinical signs. clinical infectious tracheobronchitis can be subdivided into mild or severe forms. the mild form is the more common presentation and is characterized by an acute onset of a loud, dry, hacking cough. increased formation of mucus sometimes results in a productive cough, followed by gagging or retching motions. cough is easily elicited by tracheal palpation and may be more frequent with excitement or exercise. otherwise the dog is typically asymptomatic, with normal body temperature, attitude, and appetite. mild tracheobronchitis usually lasts - days, even if left untreated. the severe form of tracheobronchitis generally results from mixed infections complicated by poor general health, immunosuppression, or lack of vaccination. secondary bronchopneumonia can occur and may be the determinant of severity (sherding, ) . animals are clinically ill and may be febrile, anorexic, and depressed. productive cough and mucopurulent naso-ocular discharge are more common than in the mild form. these cases require more aggressive treatment and may be fatal. bordetella bronchiseptica is considered to be the respiratory tract of infected animals (bemis, ) . this bacterium is very easily spread by aerosol and direct contact, and fomite transmission is also possible (bemis, ) . transmission is favored by confined housing of multiple animals. in experimental studies, b. bronchiseptica transmission to susceptible individuals was % (thompson et al., ; mccandlish et al., ) . the incubation period is - days. cpiv and cav- are also spread by aerosols. of these two viruses, cav- is the most persistent, lasting for up to several months in the environment, whereas cpiv is fairly labile (hoskins, a) . both viruses can be destroyed by quaternary ammonium disenfectants. pathogenesis. the most common clinical isolates are cpiv and bordetella bronchiseptica. however, b. bronchiseptica may be a commensal organism, and it is often recovered from asymptomatic animals. in cases of clinical infection, b. bronchiseptica attaches to the cilia on the mucosal surface of the upper airway epithelium, causing suppurative tracheobronchitis and bronchiolitis. infections with cpiv or cav- alone are usually subclinical; coinfections with b. bronchiseptica or other microbes may result in clinical itb (keil and fenwick, ; wagener et al., ) . the characteristic lesion from cpiv or cav- infection is necrotizing tracheobronchiolitis (dungworth, ) . pathogenic infection of the upper airways typically results in inflammation and ciliary dysfunction. diagnosis and differential diagnosis. diagnosis of infectious tracheobronchitis is often based on clinical signs. isolation of bordetella bronchiseptica or mycoplasma by nasal swabs allows only a presumptive diagnosis. viral isolation or paired serology can be done but is often impractical and expensive. if cough persists for more than days, other disease conditions should be considered. canine distemper virus infection, pneumonia, heartworm disease, tracheal collapse, and mycotic infections are differential diagnoses for dogs with similar signs. bronchial compression as a result of left atrial enlargement, hilar lymphadenopathy, or neoplasia may also elicit a nonproductive cough (johnson, ) and should be considered as a differential for itb. prevention. prevention is best achieved by avoiding exposure to infected animals, but this is oftentimes not practical. dogs should be vaccinated prior to, or at the time of, admission to the animal research facility. intranasal vaccine combinations for bordetella bronchiseptica and cpiv are preferred. intranasal vaccines protect against both infection and disease, can be given to dogs as young as weeks of age, and can produce immunity within days. control. sanitation and ventilation are critical for control. the animal care staff must practice proper hygiene to prevent fomite transmission. symptomatic animals should be isolated, and animal-to-animal contact avoided. kennels should be disinfected with agents such as bleach, chlorhexidine (nolvasan) or quaternary ammonium chloride (roccal-d). proper ventilation and humidity are important in controlling spread of these infectious agents; - air changes per hour at % relative humidity are recommended (sherding, ) . no specific treatment is available for viral infections. bordetella bronchiseptica is typically sensitive to potentiated sulfas, chloramphenicol, quinolones, tetracyclines, gentamicin, and kanamycin. use of antibiotics is indicated when severe or persistent clinical signs occur, and it should be continued for days. use of empirical antibiotic treatment in mild cases may hasten the resolution of clinical signs. for severe or unresponsive infection, treatment should be based on bacterial culture sensitivity patterns; nebulized gentamicin may be helpful. cough suppressants (e.g., dextromethorphan) should be avoided if the cough is bringing up mucus (productive); however, their use is indicated if coughing is causing discomfort or interfering with sleep. bronchodilators such as aminophylline, theophylline, or terbutaline can be helpful in reducing reflex bronchoconstriction and minimizing discomfort. tis results in altered respiratory tract histology and impaired mucociliary clearance, infected animals should not be used for pulmonary studies. animals with clinical disease would also be poor surgical candidates. etiology. [ -hemolytic lancefield's group c streptococcus (streptococcus zooepidemicus) is a gram-positive non-spore-forming coccus and an etiologic agent for pneumonia and septicemia in dogs. clinical signs. clinical signs vary based on the organ system affected. pneumonic disease is typically associated with coughing, weakness, fever, dyspnea, and hematemesis. peracute death without clinical signs has been reported in a previously healthy research dog (bergdall et al., ) , and conjunctivitis can also be caused by this organism (murphy et al., ) . epizootiology and transmission. lancefield's group c streptococci have been isolated as commensal flora in the upper respiratory tract and the vagina of clinically normal dogs (olson et al., ) . epizootics have been reported in both racing greyhounds and research colonies (sundberg et al., ; garnett et al., ) . in these epizootics, and in the reported case of peracute death (bergdall et al., ) , recent transportation (within days) was associated with the disease. as such, lancefield's group c streptococcus may be an opportunistic pathogen in dogs. pathologic findings. in the peracute case reported (bergdall et al., ) , hemorrhage from the mouth and nose and within the pleural cavity was the most striking lesion. ecchymotic and petechial hemorrhages were seen on other organ surfaces. the lungs were heavy and wet, and blood oozed from cut surfaces. "bull's-eye" lesions were observed on the pleural surface of affected lung lobes, similar to ischemic lesions seen with fungal infections (fig. ) . histologically, the lungs were characterized by areas of hemorrhage surrounding foci of degenerative neutrophils, blood, and necrotic debris. gram-positive cocci were seen in both the lung and the tonsils. pathogenesis. the pathogenesis for disease caused by lancefield's group c streptococcus is unclear. strain variation with respect to virulence and host immune factors is probably significant. diagnosis and differential diagnosis. definitive diagnosis is made based on bacterial culture and identification. any cause of pneumonia and/or peracute death in dogs needs to be considered as a differential diagnosis. bacterial pneumonias or septicemias can be caused by other pathogenic streptococcus spp., staphylococcus spp., escherichia coli, pasteurella multocida, pseudomonas spp., klebsiella pneumoniae, and bordetella bronchiseptica. nonbacterial causes include rodenticide intoxication, coagulopathies, heartworm disease, pulmonary thromboembolism, ruptured aneurysm, and left-sided congestive heart failure. prevention and control. too little is known about the pathogenesis of lancefield's group c streptococcus to make any recommendations about prevention and control. treatment. antibiotic therapy should be provided, based on culture and sensitivity. intravenous fluids are indicated for febrile or systemically ill patients. for dyspneic patients, oxygen therapy and strict activity restriction are required. research complications. clearly, dogs with severe hemorrhagic pneumonia or septicemia are not appropriate for any research study. the association between epizootics of this disease and transportation shipment supports the philosophy of providing acclimation periods to animals upon arrival at research facilities to evaluate health status and enable the animals to normalize physiologically. etiology. serovars of the spirochete leptospira interrogans sensu lato cause canine leptospirosis. disease in dogs is primarily due to serovars canicola, icterohemorrhagiae, grippotyphosa, pomona, and bratislava. clinical signs. leptospirosis may present as either an acute or a chronic problem. clinical signs are nonspecific and include lethargy, depression, abdominal discomfort, stiffness, anorexia, and vomiting. animals may be febrile and may be reluctant to move, because of muscle or renal pain or meningitis. icterus, congested mucous membranes, or signs referable to disseminated intravascular coagulation (petechial/ecchymotic hemorrhages, melena, epistaxis, or hematemesis) are also possible. animals with peracute leptospirosis are characterized by septicemia, shock, vascular collapse, andrapid death. uveitis, abortions, and stillbirths have also been associated with leptospirosis. epizootiology and transmission. vaccination and reduced exposure to reservoir hosts have markedly decreased the prevalence of leptospirosis over the past years. wild animals, cattle, and rodents are reservoirs for leptospira. the epidemiology of the disease is not static, and recent changes have been observed. serovars pomona, grippotyphosa, and bratislava are becoming more common causes of canine disease, with canicola and icterohemorrhagiae becoming less common. this may be due to vaccination practices and increased movement of wildlife reservoirs (raccoons, skunks, and opossums) into urban/suburban areas. rats have been implicated as important in the transmission of serovars canicola and icterohemorrhagiae (rentko et al., ; brown et al., ; kalin et al., ) . transmission occurs primarily by environmental contact, and not directly from animal to animal. infected hosts shed leptospires in urine, thereby contaminating the environment; naive animals are infected when the organisms contact mucous membranes or abraded skin. recovered animals may shed organisms in their urine for months to years. the organisms are actually labile in the environment; moisture, moderate temperatures, and alkaline soil favor survival and subsequent transmission. close contact, bites, ingestion of infected meat, and transplacental and venereal transmission are also possible. leptospirosis is a zoonotic disease. pathologic findings. the kidneys consistently have gross and microscopic lesions. in the acute phase of the infection, kidneys are swollen and have subcapsular and cortical ecchymotic hemorrhages. petechial or ecchymotic hemorrhages and swelling of the lungs and liver may also be noted. hepatic lesions during the acute phase consist of diffuse hemorrhage and focal areas of necrosis (searcy, ) . in chronic stages of leptospirosis the kidneys become small and fibrotic. endothelial cell degeneration and focal to diffuse lymphocytic-plasmacytic interstitial nephritis are the characteristic histopathological findings. pathogenesis. infection occurs after the leptospires penetrate a mucous membrane or abraded skin. the organisms then invade the vascular space and multiply rapidly. several days postinfection the renal tubular epithelium (and, to a variable extent, the liver) is colonized. the hematogenous phase lasts - days. acute renal failure or progressive renal failure leading to oliguria or anuria may occur. the most common clinical syndrome is chronic or subclinical infections after recovery from the acute phase (greene, ) . the nephritis may or may not be accompanied by hepatitis, uveitis, and meningitis. icterus, if it develops, is most common in the acute phase. the combination of azotemia and icterus should alert the clinician to the possibility of leptospirosis. disseminated intravascular coagulation is often a secondary complication. the severity and course of leptospirosis depend on the causative serovar and the age and immune status of the patient. diagnosis and differential diagnosis. zinc toxicity in dogs most closely mimics the clinical syndrome of leptospirosis. other causes of acute and chronic renal failure, icterus, and acute hepatic failure must also be considered. paired serology is the most reliable means of definitive diagnosis; however, seroconversion may not occur until after the first week of infection. prevention and control. vaccination for leptospirosis is standard veterinary practice. bivalent inactivated bacterins for serovars of l. interrogans canicola and serovars of l. interrogans icterohemorrhagiae are commercially available. however, immunization does not prevent development of the carrier state or protect against other serovars. for outdoor-housed dogs, an effective program to prevent contact with wildlife reservoirs is important. control requires identification and either treatment or elimination of carrier animals. treatment. penicillins are the drugs of choice for treating leptospiremia, and prompt use reduces fatal complications. aggressive fluid therapy and supportive care may also be needed. elimination of renal colonization and the carrier state can be accomplished with dihydrostreptomycin or doxycycline administration. should not be used in research studies because of the effects of the disease on renal and hepatic function. etiology. campylobacteriosis in dogs is caused by campylobacter jejuni, a thin, curved or spiral, microaerophilic, thermophilic motile gram-negative rod. clinical signs. most adult animals infected with c. jejuni are asymptomatic carriers; clinical signs are most commonly noted in dogs that are less than months of age (greene, ; burnens et al., ) . in cases of clinical illness, small volumes of mucoid or watery diarrhea, with or without frank blood, are most commonly noted. these signs are usually mild, may be intermittent, and typically last - days. tenesmus, inappetance, vomiting, and a mild fever may accompany the diarrhea. epizootiology and transmission. the role of c. jejuni as a primary pathogen has been questioned; it may require a coenteropathy to produce disease (sherding and johnson, ) . clinical signs of disease most often occur in dogs less than months of age, although any age may be affected. stress or immunosuppression may make animals more susceptible to clinical disease. pound and shelter populations have the highest rates of fecal excretion of c. jejuni (sherding and johnson, ) . transmission is via the fecal-oral route, mostly through fecally contaminated food or water. unpasteurized milk, poultry, and meat are other sources of infection. campylobacter jejuni can be zoonotic; children and immunocompromised individuals are at the greatest risk. pathologic findings. the actual lesions observed depend upon the mechanism of the enteropathy (van kruiningen, ) . enterotoxin production results in dilated fluid-filled bowel loops, with little or no histopathologic alteration. in cytotoxin-mediated disease, hyperemia and a friable, hemorrhagic mucosal surface are noted. on histopathology the mucosal surface is irregular and ulcerated, and a lymphocytic-plasmacytic ileitis or colitis may be seen. when translocation occurs, the lamina propria becomes edematous and congested, with focal accumulation of granulocytes in the crypts and lamina propria. focal areas of epithelial hyperplasia and decreased numbers of goblet cells are also noted. with warthin-starry silver staining, c. jejuni may be seen between enterocytes but only rarely inside them. pathogenesis. clinical disease may be produced by several different mechanisms after the campylobacter has populated the intestinal tract (van kruiningen, ) . after colonization of the enterocyte surface, c. jejuni can produce an enterotoxin that causes a secretory diarrhea. campylobacterjejuni can also cause an erosive enterocolitis by invasion of the ileal and colonic epithelium along with production of a cytotoxic agent; this may be the mechanism that causes hematochezia. in addition, c. jejuni can produce illness by translocation, i.e., multiplication in the lamina propria and transportation to regional lymph nodes by macrophages. this causes mesenteric lymphadenitis. diagnosis and differential diagnosis. fresh feces (per rectum) are best for ensuring an adequate diagnostic sample. presumptive diagnosis may be made by demonstration of highly motile curved or spiral organisms with dark-field or phase-contrast microscopy. gram-stained c. jejuni appear as gull-winged rods. definitive diagnosis requires isolation of the organism (sherding and . culture requires selective isolation media, and growth is favored by reduced oxygen tension and a temperature of ~ any disorder that can cause diarrhea in dogs should be considered as a differential diagnosis, including canine parvovirus, coronavirus, distemper virus, giardia, and salmonella infections; helminth infestations; and hemorrhagic gastroenteritis. clinical signs. based on experimental infections in dogs, three phases to the disease have been described: acute, subclinical, and chronic. clinical signs observed vary with the phase of the disease, and the acute and subclinical phases are often missed or misdiagnosed (c. g. couto, personal communication, ; waddle and littman, ; woody and mcdonald, ) . a history of tick exposure may be noted prior to onset of signs. in the acute phase, clinical signs range from mild to severe and may last - weeks. they include inappetance, lethargy, fever, generalized lymphadenopathy, hepatosplenomegaly, exercise intolerance or dyspnea, petechial or ecchymotic hemorrhages, and peripheral edema. central nervous system (cns) signs may also be present such as hyperaesthesia, myoclonus, and cranial nerve deficits. clinical laboratory abnormalities noted during the acute phase include thrombocytopenia, anemia, neutropenia or neutrophilia, and bicytopenia or pancytopenia. hyperplastic bone marrow, mild hyperglobulinemia, and elevated hepatic enzymes may be noted during this phase (kuehn and gaunt, ) . clinical signs are generally absent during the subclinical phase. mild thrombocytopenia, anemia, or leukopenia may be seen. the chronic phase develops - months after the initial infection, and signs may be subclinical to severe. an extremely varied clinical picture can emerge during this time and can mimic several other clinical syndromes. the following constellation of clinical signs may be observed: chronic lethargy, weight loss, inappetance or anorexia, fever, generalized lymphadenopathy, hepatosplenomegaly, petechial or ecchymotic hemorrhages, epistaxis, hematuria, melena, pallor, anterior or posterior uveitis, chorioretinitis, peripheral edema, ataxia, upper and lower motor neuron deficits, altered mentation, cranial nerve deficits, and seizures. persistent thrombocytopenia is the most consistent laboratory abnormality noted for all three stages. many other hematologic abnormalites may be found, such as regenerative or nonregenerative anemia (more frequently the latter), positive coombs' test, bicytopenia or pancytopenia, and splenic plasmacytosis or lymphocytosis. on bone marrow evaluation, plasmacytosis along with hypoplasia of erythroid, myeloid, and/or megakaryocyte lines may be seen. hyperglobulinemia as a result of polyclonal or occasionally monoclonal gammopathy has been noted in - % of e. canis seropositive or infected dogs (kuehn and gaunt, ; breitschwerdt et al., ; shimon et al., ) . proteinuria and/or hypoalbuminemia have also been seen. epizootiology and transmission. ehrlichia canis is an obligate intracellular parasite that infects mononuclear cells. the definitive hosts are arthropods; domestic and wild canids are parasitized secondarily. the primary vector and reservoir is the brown dog tick, rhipicephalus sanguineus. ehrlichia canis is found worldwide and follows the distribution of the vector. infection in dogs is most prevalent in tropical and subtropical areas (greene, ) . in the united states, cases are concentrated in the southeastern and southwestern states but have been reported in almost every state (breitschwerdt, ) . transmission is primarily by tick bites, but it can also occur via blood transfusions from dogs infected for as long as years. ticks become infected by feeding on an infected dog that is in the first - days of an acute infection (lewis et al., ) , and ticks can shed the organisms for up to months. within the tick population, e. canis is transmitted transstadially (within developmental stages) but not transovarially (from female to offspring) (groves et al., ) . pathogenesis. in experimental infections, the incubation period prior to the onset of the acute phase is - days. during the acute phase, which can last from - weeks, the bacteria replicate within circulating and tissue monocytes, resulting in lymphoreticular hyperplasia in affected tissues. infected monocytes then spread hematogenously to other organs in the body, in particular the lungs, kidney, and meninges. infected cells adhere to the vascular endothelium and induce vasculitis, which is the primary mechanism whereby the organism causes disease. the thrombocytopenia during the acute phase is due to both sequestration and destruction, and the development of anemia is a result of red blood cell destruction and suppression of erythrocyte production. the subclinical phase of the disease occurs - weeks after initial infection. during this stage, dogs that can mount an effective immune response clear the infection. those that cannot mount such a response progress to the chronic stage. infection does not confer protective immunity in dogs that recover. german shepherds and doberman pinschers seem to be more severely affected than other breeds. pathologic findings. gross lesions are varied and change, depending on the phase of the disease. the most common findings are petechial and ecchymotic hemorrhages and edema of dependent tissues (woody and hoskins, ) . the most common histologic abnormality noted is lymphocytic-plasmacytic inflammation of numerous organs. mononuclear phagocytic system hyperplasia, extramedullary hematopoiesis, and splenic erythrophagocytosis may also be seen. diagnosis and differential diagnosis. the most sensitive, specific, and commonly employed method for diagnosing e. canis infections is the indirect fluorescent antibody (ifa) test. antibodies can be detected as early as days postinfection, although some dogs may not seroconvert until days postinfection (buhles et al., ) . cross-reaction may occur between e. canis, e. chaffeensis, and e. ewingii. titers greater than : are considered positive and indicative of infection and may persist for up to year. effective treatment typically produces seronegative results in - months. in some cases, asymptomatic dogs may remain seropositive for years after treatment or may be seropositive with a persistent hematologic abnormality (bartsch and greene, ) . the exact mechanism for this finding has not been elucidated. ehrlichia canis morulae can be demonstrated in circulating monocytes of giemsa-stained blood smears. however, this method is labor-intensive and has low sensitivity, as morulae are present transiently and in low numbers. using buffy coat smears from capillary blood may increase the diagnostic yield. polymerase chain reaction (pcr) assays are also available to identify e. canis. differential diagnoses include immune-mediated hemolytic anemia/thrombocytopenia, multiple myeloma, chronic lymphocytic leukemia, and lymphoma. prevention. preventing laboratory animals from contacting ticks is the primary means to avoid monocytic ehrlichiosis in research dogs. avoid exercising dogs in areas infested with ticks. use topical acaricides to prevent tick infestations. keep kennel areas tick-free. dogs used as blood donors and dogs from unproven sources should be tested for e. canis. treatment. doxycycline is the drug of choice for treating monocytic ehrlichiosis. oral doses of either . - mg/kg q hr or mg/kg q hr for days are very effective at eliminating the organism. tetracycline, chloramphenicol, and enrofloxacin are also effective antibiotics; however, chloramphenicol should not be used in animals with cytopenias. in chronic cases, antibiotic treatment should be extended for an additional - weeks. research complications. the most significant research complication is the thrombocytopenia that persists for all stages of the disease. additionally, there is probable alteration in immune function and increased susceptibility to infectious agents. for these reasons, dogs positive for antibodies to e. canis should not be used in research. etiology. this disease, caused by ehrlichia platys, was first described as cyclic thrombocytopenia by harvey et al. in . clinical signs. in most cases, infection with e. platys results in subclinical disease. a generalized lymphadenopathy may be noted. epizootiology and transmission. the vector for e. platys is assumed to be a tick; however, this mode of transmission has not been established. experimental studies by simpson et al. ( ) failed to demonstrate rhipicephalus sanguineus as a vector for e. platys. coinfection with e. canis has been reported, which suggests a common vector for both organisms (french and harvey, ; kordick et al., ) . dogs have been experimentally infected by inoculation with infected blood or infected platelets from other dogs (harvey et al., ; gaunt et al., ) . the geographic distribution of thrombocytic ehrlichiosis is assumed to follow that of other ehrlichia organisms. the highest concentration of cases seems to be in southeastern states, but isolated cases have been reported as far north as michigan and as far west as oklahoma (wilson, ; mathew et al, ) . the prevalence of seropositive dogs can be high in some parts of the country. a study by bradfield et al. ( ) reported that % of the dogs entering a research institute's quarantine facility from sources in eastern north carolina were seropositive for e. platys. hoskins et al. ( ) reported a . % seropositive prevalence in healthy dogs from kennels in louisiana. pathologic findings. gross and histopathologic findings during experimental e. platys infection in dogs have been described by baker et al. ( ) . generalized lymphadenopathy was the only gross lesion noted. follicular hyperplasia and plasmacytosis were the predominate findings in lymphoreticular tissues. all dogs also had extramedullary hematopoiesis, erythrophagocytosis, and crescent-shaped hemorrhages in the spleen. multifocal kupffer's cell hyperplasia was noted in the liver, and mild multifocal lymphocytic-plasmacytic interstitial inflammation was seen in the kidneys. pathogenesis. the pathogenesis of e. platys in dogs has primarily been determined through experimental infection (harvey et al., ) . after inoculation the organism directly infects platelets. thrombocytopenia occurs by day - and fluctu-ates, along with parasitemia, at to day intervals. in some cases the rebound may be within the normal range for thrombocyte counts. the nadir can be lower than , platelets/~d. concurrent with low platelet counts is the development of megakaryocytic hyperplasia in the bone marrow. interestingly, despite extremely low platelet counts, spontaneous bleeding has not been reported in cases of e. platys infection. the mechanism responsible for the cyclic nature of the infection has not been elucidated. diagnosis and differential diagnosis. ehrlichia platys infection may be diagnosed on stained blood smears by visualization of the organisms within platelets. however, this method is very unreliable due to the cyclic nature of the parasitemia and the low numbers of infected thrombocytes. available ifa assays are much more sensitive and specific, and there is reportedly no serologic cross-reaction with other ehrlichia species. dogs usually develop detectable titers - weeks postinfection. pcr assays for e. platys have now been developed as well (chang and pan, ; mathew et al., ) . differential diagnoses for thrombocytic ehrlichiosis include e. canis infection, immunemediated thrombocytopenia, and disseminated intravascular coagulation (dic). platys is the same as described for e. canis, above. research complications. ehrlichia platys infection may increase the risk of bleeding during surgical or traumatic procedures. coinfection with e. platys may potentiate the pathogenicity of other infectious agents, in particular e. canis (breitschwerdt, ) . etiology. lyme disease is caused by borrelia burgdorferi sensu lato, a microaerophilic spirochete that is primarily an extracellular pathogen. clinical signs. clinical signs may be highly variable; lameness due to polyarthritis has been reported as the most common sign. the onset of lameness may be acute or chronic, shift from limb to limb, and be accompanied by swelling and joint pain. synovial fluid analysis from affected joints is consistent with a diagnosis of suppurative arthritis. other clinical signs include fever, anorexia, lethargy, lymphadenopathy, and weight loss. over the course of the disease, signs may wax and wane over a period of weeks to months. dogs rarely develop erythema chronicum migrans (the characteristic rash seen in infected people) and do not exhibit the severe arthritis and neurologic sequelae seen in human beings (greene, ; manley, ) . hematologic and biochemical profiles are generally unremarkable. lyme disease is thought to be the most common arthropod-borne disease of human beings (and possibly of dogs) in the united states. it affects humans and dogs worldwide. the geographic distribution of canine borreliosis is assumed to follow that of the human disease and is related to the range of the arthropod vectors. three major endemic foci that have been identified in the united states account for % of reported human cases (appel and jacobson, ). the distribution of these cases is as follows: northeast/mid-atlantic focus, %; midwestern focus (michigan, wisconsin, minnesota, iowa, illinois, and missouri), %; and california and oregon, %. for the most part, dogs in the remainder of the country are not at risk for contracting lyme disease. borrelia burgdorferi is transmitted exclusively by ixodes ticks. other arthropod hosts may carry the organism but have not as yet been implicated in the transmission of disease. ixodes scapularis, a three-host tick with a to year life cycle, is the prototypical vector for north america. the spirochetes are spread by tick bites from both nymphs and adults. ticks become infected by feeding on an infected mammal and by transstadial transmission (transovarial passage is rare). in endemic areas, - % of adult ticks may be infected (appel and jacobson, ) . the primary reservoir for the organism is the whitefooted deer mouse, peromyscus ieucopus, which can carry spirochetes for its life span without becoming ill. evidence also indicates that the eastern chipmunk, tamias striatus, is an important reservoir (slajchert et al., ) , and birds may also be a significant reservoir. deer, however, serve only as hosts for the tick vectors and not as a reservoir for the spirochete. pathogenesis. the pathogenesis of lyme disease is poorly understood, primarily because of a lack of good animal models and the chronic nature of the disease. infection can be induced experimentally by the bite of a single infected tick. clinical signs develop - days postinfection. some evidence points to the host's inflammatory response to the organism as etiologic for disease (pershing et al, ; greene, ) . seroconversion in dogs occurs - weeks after infection with b. burgdorferi. antibody titers may remain extremely elevated for at least months. igm titers also remain elevated for several months and are indicative of neither acute nor active infection (appel and jacobson, ) . because antibiotic treatment may not eliminate the organism, persistent infections in dogs (treated for days with antibiotics) can be reactivated by steroid treatment up to days postinfection (straubinger et al., ) . diagnosis and differential diagnosis. appel and jacobson ( ) recommend that three of the following four criteria be met to establish a diagnosis of lyme disease in dogs: ( ) history of exposure to ixodes ticks in an endemic area, ( ) characteristic clinical signs, ( ) positive serology, and ( ) rapid resolution of clinical signs with antibiotic therapy. ifa or elisa tests for borrelia antibodies are the assays of choice. it should be re-membered, however, that a positive titer in an endemic area indicates exposure and not necessarily disease and that vaccinated dogs will also have a positive titer. responses to vaccine versus infection may be distinguished by western blot. culture or identification of the organism provides a definitive diagnosis but is very difficult to perform. differential diagnoses include immune-mediated polyarthritis and septic arthritis from other etiologic agents. prevention and control. prevention and control are the same as for the other tick-borne diseases (see discussion of monocytic ehrlichiosis, section iii,a,l,e above). a vaccine against b. burgdorferi is available but should not be necessary in a research setting. treatment. doxycycline is the drug of choice for treating lyme borelliosis. a typical dosing regimen is mg/kg q hr for - weeks. amoxicillin, tetracycline, and the quinolones are also effective. of significant note is that antibiotic treatment results in resolution of clinical signs but may not result in elimination of the organism. (fox and lee, ) . "helicobacter heilmannii" and h. bizzozeronii are thought be the same species, with the latter being the updated nomenclature. this species, as well as h. rappini and h. canis, is considered to be zoonotic (fox and lee, ) . clinical infections may present with vomiting, diarrhea, fever, and anorexia, pica, or polyphagia. epizootiology and transmission. the epizootiology and transmission of helicobacter spp. in the dog remains to be elucidated. the prevalence of canine helicobacter infections in colony or shelter situations has been reported to range from % to almost % (fox, ; hermanns et al., ) . both oral-oral and fecal-oral routes for transmission have been suggested. pathologic findings. no gross lesions are noted; the primary lesion is that of histologic gastritis. this is typically characterized by reduced mucus content of the surface epithelium; vacu-olation, swelling, karyolysis, and karyorrhexis of parietal cells; and multifocal infiltrates of plasma cells and neutrophils into the subepithelium, primarily around blood vessels and between the gastric pits (hermanns et al., ) . focal areas of lymphocytic inflammation and lymphoid follicles may also be seen. pathogenesis. some helicobacter spp. colonize the gastric epithelium exclusively and other species colonize lower parts of the gastrointestinal tract. helicobacter felis and "h. heilmannii" infections have been linked to gastric lesions in laboratoryraised beagles (fox and lee, ) . the mechanism by which these organisms cause disease may be related to the host's inflammatory response to colonization and the helicobacter's ability to produce urease. urease splits urea into ammonia and bicarbonate; ammonia is toxic for the epithelial cells, and bicarbonate may help the organism survive the acidic environment (marshall et al., ; shimoyama and crabtree, ). diagnosis and differential diagnosis. any of the numerous causes of acute or chronic vomiting and diarrhea in the dog (including canine distemper, viral or bacterial gastroenteritis, and ingested toxicants) should be considered as differential diagnoses. definitive diagnosis for dogs requires either endoscopic or surgical biopsy. confirmation of infection with helicobacter spp. requires demonstration of the organism in biopsy samples by histopathology, culture, or recognition by pcr. a positive urease test on a biopsy sample may give a presumptive diagnosis, but only for those species that produce urease. the use of warthin-starry silver stain may increase the sensitivity for histopathologic diagnosis. prevention and control. until more is known about the epizootiology and transmission of helicobacter spp. in the dog, specific recommendations cannot be made about prevention and control in this species. treatment. combination therapy has proven to be the most effective method for treating helicobacter spp. infections in dogs. combination therapy of amoxicillin ( mg/kg q hr), metronidazole ( mg/kg q hr), and sucralfate ( . - . mg/kg q hr) for days has been suggested for dogs (hall and simpson, ) . replacing the sucralfate with famotidine ( . mg/kg q hr), omeprazole ( . mg/kg q hr), or bismuth subsalicylate ( . ml/kg q - hr) may also be effective (marks, ; jenkins and bassett, ; denovo and magne, ) . the benefits of antimicrobial therapy in dogs still need to be established by controlled therapeutic studies. research complications. helicobacter spp. infections could result in altered gastrointestinal responses to drugs and toxic or carcinogenic compounds. therefore, dogs used in gastric physiology or oral pharmacology studies should be free from helicobacteriosis. clinical signs. clinical signs of canine parvovirus usually appear days after inoculation by the fecal-oral route and are characterized by anorexia, fever, depression, and vomiting. profuse, intractable diarrhea ensues, which may become hemorrhagic. approximately % of affected dogs develop severe leukopenia, with a total granulocyte/lymphocyte count ranging from - wbc/~d or less. repeated hemograms may provide prognostic value, because rebounds in leukocyte counts are indicative of impending recovery. terminally ill dogs may develop hypothermia, icterus, or disseminated intravascular coagulation due to endotoxemia. parvovirus can infect dogs of any age, but puppies between and weeks of age appear to be particularly susceptible. puppies less than weeks of age are generally protected from infection by passive maternal antibody. adult dogs probably incur mild or inapparent infections that result in seroconversion. pathogenesis. canine parvovirus has an affinity for rapidly dividing cells of the intestine and causes an acute, highly contagious enteritis with intestinal crypt necrosis and villus atrophy. the virus also has tropism for the bone marrow and lymphoid tissues; thus leukopenia and lymphoid depletion accompany the intestinal destruction. diagnosis and differential diagnosis. parvovirus can be detected in fecal samples with a commercially available elisa from cite. at necropsy, diagnosis is based on gross and histopathologic evidence of necrosis and dilatation of intestinal crypt cells with secondary villous collapse. other lesions include myeloid degeneration and widespread lymphoid depletion. parvovirus can also be demonstrated in frozen sections by fluorescent antibody techniques. differential diagnoses should include other viral enteritides, salmonellosis, and small intestinal obstruction. prevention and control. prevention of transmission begins with isolation of affected animals and quarantine for week after full recovery. disinfection of potentially infected kennel and diagnostic areas with diluted bleach ( : ) or commercially prepared disinfectant (such as kennesol, available from alphatech, lexington, massachusetts) is essential for elimination of the virus. six-week-old puppies should be vaccinated every - weeks with a commercially available modified live vaccine until - weeks of age. young rottweilers and doberman pinschers appear to be predisposed to parvoviral enteritis and should be vaccinated every weeks ( times) from - weeks of age. treatment. treatment is largely supportive and is aimed primarily at restoring fluid and electrolyte balance. research complications. infection with parvovirus obviously precludes the use of a particular dog in an experimental protocol. given the potential for significant discomfort of the affected animal, and the cost of therapy, humane euthanasia is usually the option chosen in a research setting. canine coronavirus infection is usually inapparent or causes minimal illness. this epitheliotropic virus preferentially invades the enterocytes of the villous tips, resulting in destruction, atrophy, and fusion and subsequent diarrhea of varying severity. subclinical infections are most common, but abrupt gastrointestinal upset accompanied by soft to watery, yelloworange feces is possible. definitive diagnosis by virus isolation or paired sera is usually not made, because supportive therapy generally results in rapid resolution of the diarrhea. inactivated coronavirus is present in commercially available combination vaccines, which are administered immunoprophylactically at - , - , and - weeks of age and then annually thereafter. the role of these vaccines in protection from coronaviral infection is unknown, because the virus typically causes inapparent or mild illness (hoskins, ) . etiology. canine distemper virus (cdv) belongs to the family paramyxoviridae, within the genus morbillivirus, which includes human measles virus and rinderpest virus of ruminants. although there is only one serotype of cdv, there is a wide difference in strain virulence and tissue tropism. some strains produce mild clinical signs that are similar to tracheobronchitis, whereas other strains cause generalized infections of the gastrointestinal tract, integument, and central nervous system, resulting in enteritis, digital hyperkeratosis, and encephalitis, respectively. other factors contributing to the severity and progression of clinical signs include environmental conditions, immune status, and age of the host. a transient subclinical fever and leukopenia occur - days after exposure, with a subsequent fever spike - days later, accompanied by conjunctivitis and rhinitis. other clinical signs associated with acute distemper include coughing, diarrhea, vomiting, anorexia, dehydration, and weight loss. secondary bacterial infections can cause progression to mucopurulent oculonasal discharge and pneumonia. an immune-mediated pustular dermatitis may develop on the abdomen; this is usually a favorable prognostic sign (greene and appel, ) , because dogs that develop skin lesions often recover. neurologic complications of distemper infection may occur weeks to months after recovery from an acute infection. dogs that develop late-onset disease are usually immunocompetent hosts, suggesting that the virus may have escaped complete elimination by the immune system, possibly because of protective effects by the blood-brain barrier. classic neurologic signs that may occur in acute or chronic cdv infection include ataxia, incoordination, vocalization, "chewing gum" seizures, and myoclonus with or without paresis of the affected limb. canine distemper is the most common cause of seizures in dogs less than months of age. dogs with extensive neurologic involvement often have residual clinical deficits, including flexor spasm and olfactory dysfunction. cdv has also been associated with two forms of chronic encephalitis in mature dogs: multifocal encephalitis and "old dog encephalitis." epizootiology and transmission. the virus is highly prevalent and contagious to dogs and other carnivores, especially at the age of - months, coincident with the waning of maternal antibody. transmission is primarily by aerosolization of infective droplets from body secretions of infected animals. pathologic findings. the predominant histopathologic lesion in neurologic forms of distemper is demyelination, which may .. be accompanied by gliosis, necrosis, edema, and macrophage infiltration. acidophilic cytoplasmic inclusions can be found in epithelial cells of mucous membranes, reticulum cells, leukocytes, glia, and neurons, while intranuclear inclusions are often present in lining or glandular epithelium and ganglion cells. diagnosis and differential diagnosis. diagnosis of cdv is based on history of exposure and clinical signs. young dogs who have not received routine immunoprophylaxis (or similarly, mature dogs with a questionable vaccination history) and present with rhinitis, mucopurulent oculonasal discharge, plus or minus hyperkeratosis of the footpads and neurologic signs, are highly likely to have cdv. ophthalmologic examination may reveal chorioretinitis with acute disease or retinal atrophy in chronic cases. definitive diagnosis of acute infection can be made by fluorescent antibody testing of intact epithelial cells from conjunctival and mucous membranes. attenuated strains of cdv, found in modified live vaccines, are not disseminated from lymphoid tissue to epithelial cells and thus are not detected by the fluorescent antibody. serologic testing is usually not useful, because dogs frequently fail to mount a measurable immunologic response. because of the variety of clinical signs, there are many differential diagnoses for canine distemper. an important differential diagnosis for respiratory illness is infectious tracheobronchitis (kennel cough). bacterial, viral, and protozoal causes of gastroenteritis must be considered for cases presenting with vomiting and diarrhea, and rabies, pseudorabies, bacterial meningitis, and poisonings are differential diagnoses for dogs with central nervous system disorder. prevention and treatment. a series of three immunizations from to weeks of age, followed by yearly boosters, is a recommended preventative. treatment is largely supportive, but because of the profound immunologic effects and significant morbidity of cdv, humane euthanasia is usually undertaken in the research setting. etiology. canine herpesvirus (chv) infection causes a generalized hemorrhagic disease with a high mortality rate in newborn puppies less than weeks of age. in adult dogs, chv causes a persistent, latent infection of the reproductive tract with recrudescence and shedding during periods of physiologic stress. clinical signs. clinically affected puppies do not suckle, cry persistently, become depressed and weak, and fail to thrive. petechial hemorrhages of the mucous membranes and erythema of sparsely haired regions such as the caudal abdomen and inguinal area are evident. older puppies, aged - weeks, develop less severe clinical signs and are likely to survive with neurologic sequelae such as ataxia and blindness resulting from reactivation of latent infection. infection in adult dogs may result in stillbirths, abortions, and infertility. lesions in adult bitches include raised vesicular foci in the vaginal mucosa, accompanied by mild vaginitis. adult males have preputial discharge due to vesicular lesions at the base of the penis and on the preputial mucosa. passage of puppies through the birth canal or venereally in adult dogs. puppies can also be horizontally infected by littermates. entire primiparous litters may be lost, with subsequent litters protected by colostral antibody. pathologic findings. pathologic findings include multifocal ecchymotic hemorrhages of the kidneys, liver, lungs, and gastrointestinal tract. basophilic intranuclear inclusions in necrotic areas of parenchymal organs are characteristic findings. diagnosis and differential diagnosis. diagnosis of canine herpesvirus infection in adult dogs is based on a history of reproductive infertility and the presence of genital vesicular lesions. differential diagnoses for stillbirths, abortions, and infertility include canine brucellosis, canine distemper virus and parvovirus infections, and pyometra. the diagnosis in infected puppies is usually made based on clinical history and characteristic lesions (multifocal systemic hemorrhages) (carmichael and greene, ) . differential diagnoses for the disease in neonates would include canine ehrlichiosis and causes of disseminated intravascular coagulation, including bacterial endotoxemia. there is no effective curative treatment. supportive therapy is unrewarding, and death usually ensues within hours in in-fected neonates. in general, adult bitches that have multiple abortions, stillbirths, or persistent infertility should be culled from the breeding colony. examination of these animals may reveal raised vesicular lesions on the vaginal mucosa. adult male dogs that have vesicular lesions on the base of the penis and preputial mucosa should be similarly culled. adult dogs would obviously interfere with production operations, and affected animals should be culled based on the criteria noted above in the discussion of prevention and treatment. because of the severity of clinical illness in puppies, such animals should be humanely euthanatized. etiology. rabies virus is a member of the rhabdovirus family and is essentially contagious to all species of warm-blooded animals. clinical signs. clinical progression of neurologic disease occurs in three stages. the first, or prodromal, stage is characterized by a change in species-typical behavior. the loss of the instinctive fear of humans by a wild animal is a classic sign of impending rabies. in the second, or furious, stage animals are easily excited or hyperreactive to external stimuli and will readily snap at inanimate objects. the third, or paralytic, stage is characterized by incoordination and ascending ataxia of the hindlimbs due to viral-induced damage of motor neurons. death usually occurs within - days of the onset of clinical signs, due to respiratory failure. epizootiology and transmission. wild animals such as raccoons, skunks, and bats are common reservoirs of infection for domestic animals, which in turn are the principal source of infection for humans. transmission occurs primarily by contact of infected saliva from a rabid to a naive animal (or human), usually via bite wounds. pathogenesis. the incubation period for rabies is generally - weeks from the time of exposure to the onset of clinical signs but can range from week to year. bites of the head and neck typically result in shorter incubation periods because of the proximity to the brain. following infection, the virus migrates centripetally via peripheral nerve fibers to the central nervous system and eventually to neurons within the brain, resuiting in neurologic dysfunction. on reaching the brain, the virus migrates centrifugally to the salivary glands, thus enabling shedding and subsequent transmission. diagnosis and differential diagnosis. diagnosis of rabies is based on clinical signs; differential diagnoses include pseudorabies, canine distemper, bacterial meningitis, and toxicants that affect neurologic function. definitive diagnosis is based on fluorescent antibody demonstration of the virus in negri bodies of hippocampal cells. prevention and treatment. puppies should be vaccinated at - months of age, "boostered" in year, then vaccinated annually or triennially, depending on state and local laws and which vaccine product is used. treatment of rabies is not recommended, because of the risk of human exposure. research complications. in a research setting, dogs are often not vaccinated for rabies, because of the low incidence of exposure to wild-animal reservoirs. a healthy, purpose-bred dog that bites a human in a research facility should be quarantined for days and observed for signs of rabies. this quarantine interval is based on the knowledge that dogs do not shed rabies in the saliva for more than a few days before the onset of neurologic disease. a random-source dog with an unknown vaccination history that bites a human should be immediately euthanized. the brain should be examined for rabies virus to determine if the dog was infected, and if the test is positive, postexposure immunization should be initiated for the human patient. a rabies vaccine licensed for use in humans is available, and immunoprophylaxis is recommended for animal care and research personnel who may have high work-related risks of exposure. a. protozoa i. giardiasis etiology. giardiasis is a small-intestinal disease of the dog caused by giardia duodenalis (lamblia), a binucleate flagellate protozoan. clinical signs. most giardia infections are subclinical. when dogs are clinically affected, diarrhea is the most prominent sign. the diarrhea is a result of intestinal malabsorption and is often characterized as voluminous, light-colored, foul-smelling, and soft to watery. weight loss has also been associated with clinical infection. clinical illness is more often seen in young animals. epizootiology and transmission. giardia has a direct life cycle. dogs (and people) typically become infected when they consume water (or food) contaminated with giardia cysts. the ph change from the stomach (acid) to duodenum (neutral) causes excystation. trophozoites migrate to the distal duodenum and proximal jejunum and attach to the villus surface. eventually the trophozoites encyst and pass in the feces to perpetuate the life cycle. pathologic findings. giardiasis is rarely fatal. on histopathology of duodenal or jejunal specimens, giardia trophozoites can be seen attached to enterocytes. mucosal inflammation and ulceration, and villous atrophy, have been observed. pathogenesis. the exact pathogenesis of giardia-induced illness is unknown. it is thought that tissue invasion, although occasionally observed, is unimportant for pathogenesis. it is suspected that illness is caused by physical obstruction of enteric absorption, enterotoxicity, competition for nutrients, excess mucus production, and/or secondary bacterial overgrowth. diagnosis and differential diagnosis. definitive diagnosis requires observation of the organism in fecal or intestinal samples. direct fecal smears are considered best for observing trophozoites, and zinc sulfate flotation is preferred for detection of cysts. commercial elisa kits and direct immunofluorescent tests are available to detect fecal giardia antigens, but the diagnostic specificity and/or sensitivity of these tests may not be sufficient to warrant substitution for the less expensive direct fecal examination or zinc sulfate preparation (barr, ) . differential diagnoses for giardiasis include bacterial and protozoal enteritis, coccidiosis, and whipworm infestation. prevention. high-quality water sources will eliminate the possibility of infection developing within an animal research facility. use of dogs with a known husbandry and medical background will minimize the chances of giardiasis developing in a research colony. control. once giardiasis has been diagnosed in a canine population, segregation of infected animals will help to reduce further infection (provided other dogs were not preinfected at the same source location as the signal case). disinfection with quaternary ammonium compounds, bleach, or steam is usually successful in eradication of giardia cysts. treatment. the most common treatment for giardiasis is metronidazole (flagyl) at - mg/kg per os twice per day for - days. quinacrine hydrochloride (atabrine) at mg/kg per os once per day for days, furazolidone (furoxone) at mg/kg per os twice per day for - days, and the anthelmintics albendazole and fenbendazole have been proposed for use against metronidazole-resistant strains of giardia. a bendazole is recommended at mg/kg per os q hr for days, and fenbendazole at mg/kg per os q hr for days. fenbendazole was thought to be safer for both puppies and pregnant females (nonteratogenic) (barr, ) . research complications. typical asymptomatic infections probably have no consequence on research protocols, with the exception of intestinal physiology or immunology studies. clinical diarrhea would clearly need to be treated before a dog could be used as a research subject. ii. coccidiosis etiology. intestinal coccidia that have been associated with enteropathy in dogs include cystoisospora canis, c. ohioensis, c. burrowsi, and c. neorivolta. clinical signs. dogs are typically asymptomatic when infected with intestinal coccidia, and oocysts are an incidental finding on fecal flotation or direct smear. dogs that are clinically infected usually develop diarrhea, which can vary from soft to watery and may contain blood or mucus. vomiting, dehydration, lethargy, and weight loss can also be seen. epizootiology and transmission. cystoisospora oocysts are typically spread by fecal-oral transmission, usually by ingestion of fecal-contaminated food or other objects in the environment. an indirect form of transmission is also possible, whereby the dog consumes a rodent or other animal that is serving as a transport host. once inside the small intestine, the cyst releases sporozoites that infect enteric epithelium. several generations of asexual reproduction can occur in the enterocyte before sexual reproduction produces gamonts. the gamonts fuse to become a zygote, which encysts, ruptures the enterocyte, and passes in the feces. once in the environment the cyst sporulates and is now an infective stage for ingestion by another host. pathologic findings. dogs with coccidiosis may have hyperemia or fluid retention at affected intestinal segments. the mucosa may appear normal, raised, or ulcerated. histologically, there may be necrosis of enterocytes, hyperemia, and submucosal inflammation. the oocysts are usually readily apparent within the epithelial cells (van kruiningen, ) . pathogenesis. intestinal coccidia are opportunistic organisms; they do not typically cause illness unless other predisposing factors are present. such factors include immunodeficiency, malnutrition, and/or concurrent disease. overcrowding and unsanitary conditions can also promote clinical coccidiosis by providing a high population of infective oocysts to stressed animals. diagnosis and differential diagnosis. diagnosis is somewhat difficult, as coccidian oocysts (of both cystoisospora and non-cystoisospora spp.) can be seen on fecal examinations of clinically healthy dogs, as well as animals with diarrhea. other causes for diarrhea (e.g., parvovirus, roundworms, giardia spp., campylobacter jejuni, and inflammatory bowel disease) should be excluded before a coccidial etiology is implicated. prevention. clinical coccidiosis can be readily prevented by adhering to proper sanitation guidelines, reducing any over-crowding, and providing as stress-free an environment as possible. treatment. treatment for the presence of coccidial oocysts may often not be necessary, because cystoisospora infections are typically self-limiting and clinically insignificant. treatment may, however, help to limit the number of oocysts shed in a kennel housing situation and may be necessary in cases of protracted clinical illness. possible choices for treatment include daily administration of sulfadimethoxine ( - mg/lb per os for days), trimethoprim sulfa ( mg/lb per os for days), or quinacrine ( mg/lb per os for days). amprolium, which is not labeled for dogs, can also be used as a coccidiostat. it can be given in gelatin capsules for - days at a daily dose of mg for small-breed pups and mg for larger breeds. research complications. as with any enteric disease, the presence of clinical coccidiosis can cause aberrations in gastrointestinal physiological parameters. dogs used in intestinal pharmacokinetic studies should be confirmed to be free of cystoisospora infections. b. nematodes i. ascarids etiology. the most common ascarid of dogs is toxocara canis. toxascaris leonina can also infect both dogs and cats. clinical signs. ascarid infestations are most commonly subclinical. however, large worm burdens can cause diarrhea, vomiting, dehydration, and abdominal discomfort with vocalization. puppies may have a classical "potbellied" appearance and dull hair coat. heavy infestations can cause intussusception and/or intestinal obstruction, in which case the young dogs may be found dead. visceral larval migrans caused by toxocara canis can cause pneumonia. epizootiology and transmission. toxocara canis typically infects puppies. in fact, a unique characteristic of t. canis is its ability to infect prenatal puppies by transplacental migration, and neonatal puppies by transmammary migration. ingestion of infective eggs that have been shed in the feces is another common route of transmission, and infection by ingestion of a transport or intermediate host is also possible. pathologic findings. puppies that die from ascarid infestations typically have large worm populations in the lumen of the small intestine. such populations can cause intestinal obstruction and may also result in intussusception or intestinal perforation. puppies that experience lung migrations of large larval worm populations can have severe pulmonary parenchymal damage and develop fatal pneumonia. pathogenesis. the infective stage of t. canis is the third-stage larva (l ). infections initiated by ingestion of infective eggs have three possibilities for larval migration: liver-lung migration (which leads to intestinal infection), somatic tissue migration, and intestinal wall migration. older dogs that become infected typically have an age-related resistance to liver-lung migration and instead experience the other two migratory patterns. these larval migrations are often asymptomatic, and progression of the l larvae is arrested in the tissues. it is these larvae that become reactivated in a pregnant bitch, thus establishing the transplacental and transmammary routes of transmission. if the source of infection is transplacental, puppies may be born with l larvae in their lungs, because larval migration is already in progress (sherding, ). diagnosis and differential diagnosis. the characteristic large ( - ~tm in diameter) and relatively round ascarid eggs can be readily diagnosed by standard fecal flotation methods. prevention and control. monthly administration of milbemycin or ivermectin plus pyrantel pamoate (heartgard plus) is recommended for prevention and control of canine ascarid infestation (hall and simpson, ) . treatment. most anthelmintics are effective for treatment of ascariasis. pyrantel pamoate (nemex) and fenbendazole (panacur) are commonly used. treatment should be started early in puppies ( , , , and weeks) because of the possibility of prenatal or neonatal infection. pyrantel pamoate, dosed at mg/kg per os, is safe for puppies and is also effective in treatment of hookworms (see section iii,a, ,b,ii). in breeding colonies in which ascarid infestation is a known problem, treatment of the pregnant and nursing bitch may be advantageous. extended fenbendazole therapy ( mg/kg per os twice per day for days or once per day from day of gestation through day of lactation) has been shown to be experimentally safe and effective in decreasing ascarid burdens in puppies. research complications. puppies with large worm burdens make poor research subjects and should be treated aggressively before placement on an experimental study. ii. hookworms etiology. the most common and most pathogenic hookworm of dogs is ancylostoma caninum. other, less pathogenic canine hookworms found in north america are a. braziliense, which can be found in the american tropics and southern united states, and uncinaria stenocephala, which is distributed in the northern united states and canada. clinical signs. only a. caninum infestation typically results in clinical illness, because of the amount of blood that it con-sumes. puppies with a. caninum infestations are typically pale and weak (from anemia), with bloody diarrhea or melena. other clinical signs include lethargy, anorexia, dehydration, vomiting, and poor weight gain. epizootiology and transmission. infective larvae (l ) are typically ingested by puppies and develop directly in the intestinal tract. ingestion can be from the bitch's milk (transmammary migration occurs with a. caninum), from food or objects contaminated with infective larvae, or from ingestion of a paratenic host. transplacental migration does occur with a. caninum, but to a much lesser extent than is seen with toxocara canis. larvae can also penetrate intact skin, migrate to the lung via somatic or circulatory routes, and be coughed and swallowed to reach the intestine. the prepatent period is weeks. pathologic findings. infected puppies often have severe anemia and eosinophilia. the anemia can be from acute blood loss or can also be an iron-deficiency anemia caused by chronic blood loss coupled with limited iron reserves. on gross necropsy, the small-intestinal tract contains worms admixed with intestinal contents containing fresh or digested blood (fig. a) . ulcerative enteritis caused by hookworm attachment is evident on histopathologic examination, and worms with mouthparts embedded in the mucosa can be identified in some sections (fig. b) . pathogenesis. the severe pathogenicity of a. caninum is a direct result of its voracious consumption of blood and body fluids. each adult hookworm can consume . - . ml of blood; thus an extensive infection could deplete a puppy of ml of blood per day, which is approximately % of the blood volume of a . kg animal. in contrast, a. braziliense and u. stenocephala consume . and . ml per worm, respectively. diagnosis and differential diagnosis. diagnosis of ancylostomiasis is made by identification of eggs or larvae from fecal samples by either flotation or direct smear. parvovirus should be considered for puppies with bloody diarrhea, and autoimmune hemolytic anemia should be considered in the diagnosis of a young dog with anemia. prevention and control. purchase of purpose-bred animals will limit the exposure to hookworm larvae, and effective sanitation programs will easily eradicate the infective larvae. unlike ascarid eggs, hookworm eggs are readily killed by drying, sunlight, or cold; however, they do survive readily in warm, moist environments. monthly administration of milbemycin or ivermectin plus pyrantel pamoate (heartgard plus) is recommended for prevention and control of canine ascarid infestation (hall and simpson, ) . treatment. pyrantel pamoate (nemex) is the anthelmintic of choice because it is safest in young ill animals and is also effective against ascarids and other enteric helminths. because of the possibility of transplacental or milk-borne infection, puppies should be treated every weeks from weeks - . a follow-up treatment at weeks is recommended to kill any larvae that have migrated and matured since the initial therapy. severely ill puppies may require supportive fluid therapy and possibly whole blood transfusions and iron supplementation. research complications. anemic puppies with large worm burdens make poor research subjects and should be treated aggressively before placement on an experimental study. iii. strongyloides etiology. strongyloides stercoralis is a small strongyle that can cause hemorrhagic enteritis in puppies. it is found in warm, humid climates such as the southeastern united states. fects dogs and other animals by third-stage larval penetration of the skin or mucous membranes. larvae migrate via the circulatory system to the lung and then are coughed and swallowed to initiate the intestinal parasitism. the eggs of s. stercoralis hatch within the gut lumen, and so it is the first-stage larvae that pass in the feces and need to be identified by diagnostic examination. once passed, the larvae can either develop into the infectious third-stage larvae or mature into free-living, nonparasitic adults. diagnosis and differential diagnosis. the baermann procedure is usually performed on fresh feces in order to detect the motile first-stage larva ( - ~tm x - ~tm). the larvae must be distinguished from larva of filaroides hirthi and hatched ancylostoma caninum. treatment. the usual treatment for s. stercoralis is fenbendazole (panacur) at mg/kg per day for days. iv. whipworms etiology. trichuris vulpis, the canine whipworm, can cause acute or chronic large-intestinal diarrhea. the adult whipworm typically resides in the cecum or ascending colon. clinical signs. most whipworm infections are subclinical. in symptomatic cases, the typical clinical sign is diarrhea with blood and/or mucus. abdominal pain, anorexia, and weight loss are also seen. dogs may have eosinophilia, anemia, and/or hypoproteinemia on clinical hematology. severe dehydration with electrolyte imbalance has occurred occasionally as an acute crisis episode. life cycle. adult worms residing in the canine large intestine intermittently release eggs that pass in the feces. the eggs are very hardy and can persist for years. in optimal conditions, the eggs develop into an infective embryo within days. after ingestion by a dog, the larvae hatch in the small intestine, burrow into the small-intestinal mucosa, and then reemerge several days later to travel and burrow into the cecal and colonic mucosa. the prepatent period is typically - months long. pathologic findings. dogs do not typically die from whipworm infestations. lesions seen as incidental findings feature adult worms embedded into the colonic and cecal mucosae, causing local granulomatous inflammatory reactions and mucosal hyperplasia. pathogenesis. the penetration of the adult worm into the enteric mucosa, and the associated inflammation, can lead to the clinical development of diarrhea. factors that influence the possible.development of clinical symptoms are the number and location of adult whipworms; the severity of inflammation, anemia, or hypoproteinemia in the host; and the overall condition of the host. diagnosis and differential diagnosis. whipworm infestation is diagnosed by the presence of characteristic trichurid eggs on fecal flotation. these eggs are barrel-shaped, with thick walls and bipolar plugs. because of the intermittent release of eggs by the adult female worms, negative fecal flotation does not exclude the possibility of clinical whipworm infection. adult worms can be seen on colonoscopy (jergens and willard, ) . differential diagnoses for whipworm infestation include giardiasis, coccidiosis, and bacterial enteritis. prevention and control. trichuris eggs are resistant to disinfection, making control difficult. dessication or incineration is the only completely effective means to eradicate whipworm eggs from the environment. treatment. fenbendazole, oxibendazole, and milbemycin have all been recommended for treatment of whipworms. treatment for whipworm infestation should be at monthly intervals for months (jergens and willard, ) . treatment is also suggested in cases wherein whipworm infestation is suspected but not confirmed by multiple fecal flotation. rapid response to treatment would be indicative of a correct diagnosis; lack of response should prompt further diagnostic efforts. research complications. whipworm infestation has not been documented to interfere with research protocols, although one would anticpate that aberrations in local enteric immune function and absorptive functions of the large intestine could result from trichuriasis. etiology. heartworm disease of dogs is caused by the filarial worm, dirofilaria immitis. adult heartworms reside in the pulmonary artery; severe infestations can result in the presence of worms in the right ventricle and atrium. microfilariae, the immature worms produced by the adults, circulate in the bloodstream until a mosquito (intermediate host) ingests them. clinical signs. most heartworm infestations are asymptomatic. the most common clinical signs observed are coughing and dyspnea. clinical signs of exercise intolerance and rightsided heart failure can be seen in severe infestations. epizootiology and transmission. successful heartworm transmission requires the presence of mosquitoes. for this reason, random-source dogs or dogs housed in outdoor kennels are much more likely to have heartworm infestations than indoor, purpose-bred dogs. mosquitoes become infested with heartworm microfilariae when they take a blood meal from the dog. the microfilaria progress through several larval stages within the mosquito, eventually terminating at the third stage. this stage is then returned to the canine bloodstream during feeding. this stage matures within the dog's circulatory system, and the adults reside in the pulmonary artery. male and female heartworms will then sexually reproduce to create more microfilariae and propagate the parasitic life cycle. in the united states, transmission of heartworm by mosquitoes occurs over a month or shorter period, except for the southeastern and gulf coast states. here, climatic conditions enable longer survival of the mosquitoes (possibly year-round), thus resulting in the highest prevalence of heartworm infestation (knight, ) . pathologic findings. on necropsy, the small, slender worms can be seen in the pulmonary artery, right ventricle, and/or right atrium (fig. a ). there may be no histologic abnormalities associated with a minor worm burden, although typically the arterial endothelium in these areas is hyperplastic (fig. b) . endothelial cell hyperplasia, vascular smooth muscle hyperplasia, inflammation, and thrombosis of the pulmonary arteries and arterioles characterize more significant infestations. severe infestations can lead to right-sided heart failure and its pathologic sequelae of ascites, pleural effusion, hepatomegaly, and right heart and pulmonary artery enlargement. verminous pulmonary embolism can result from treatment of dogs with anthelmintics when a worm burden is present. immune responses to circulating microfilariae can cause pathologic lesions, most commonly glomerulonephritis. pathogenesis. the physical presence of the worms in the pulmonary artery is partially responsible for clinical signs observed in severe cases. however, the host immunologic response to this infestation, coupled with secretion by the heart-worms of physiomodulative factors, contributes significantly to the complications seen with this disease. endothelial cell proliferation, damage, and sloughing stimulates periarteritis and proliferation of the vascular media of pulmonary arteries and arterioles. these changes lead to thrombosis of these vessels and the arterial truncation that can be seen radiographically in severe infestations. the heartworms also release circulating factors that affect vascular tone and can promote bronchoconstriction (dillon, ) . these factors are discussed in more detail below, under "research complications." diagnosis and differential diagnosis. for dogs used in biomedical research, diagnosis of asymptomatic heartworm disease is important, especially if the dogs are used in cardiovascular, pulmonary, or long-term studies. a diagnosis of dirofilariasis is typically made by detection of adult heartworm antigens in a blood sample. use of adult heartworm antigen tests has virtually eliminated the historical status of "occult" heartworm disease, which was caused by infestation of adult worms without corresponding microfilarial circulation. commercial test kits that assay for the presence of adult heartworm antigens, and designed for use by veterinary practitioners, are readily available. false-negative results can occur during the prepatent period after initial infection (first - months), and when the adult worm burden is light or predominantly male. infections consisting of more than three mature female worms are usually detected by antigenic serology (knight, ) . a significant feature of these tests for circulating antigen is that they have a very high specificity (low rate of false-positive resuits). if a dog were negative on initial testing because of prepatency or small worm burden, it will more than likely be detected on a follow-up test months later. examination for circulating microfilariae could be used to confirm an antigenic diagnosis of dirofilariasis or to establish that microfilarial production had occurred. microfilarial detection can be done by microscopic examination of the buffy coat of a microhematocrit tube or by concentration techniques, such as the modified knott test and filter tests. tests that examine for microfilariae have the inherent problem of false positives caused by microfilariae of dipetalonema reconditum, a nonpathogenic filarial worm. other serologic diagnostic tests that were more common historically, and that may still be useful, include detection of antibodies to either adult heartworm antigens or microfilarial antigens. these same techniques can be used to diagnose clinical heartworm disease. additional diagnostic tests that can augment a diagnosis of clinical heartworm disease include thoracic radiography (pulmonary artery and right-heart enlargement), electrocardiography (right-heart enlargement), and hematology (eosinophilia). differential diagnoses for symptomatic heartworm disease (coughing, dyspnea, and exercise intolerance) include canine distemper, canine infectious tracheobronchitis (complicated), streptococcal or other bacterial pneumonia, nocardiosis, and congestive heart failure. prevention and control. for dogs used in biomedical research, prevention is primarily via insect control and housing of the dogs in a controlled, indoor environment. purpose-bred dogs reared in such an environment are usually free from dirofilariasis. however, any dog (random-source or purposebred) exposed to mosquitoes could become inoculated with infective larvae and, if untreated, could develop adult heartworm disease. there are many commercial anthelmintic preparations used to prevent heartworm infestation by killing the larval stages in the canine bloodstream before they become adult worms (e.g., ivermectin, milbemycin, and diethylcarbamazine). these could be used in a research setting in which heartwormnegative dogs are housed outdoors and thus could potentially be infected through mosquito bites. if a research facility is conditioning random-source dogs for long-term use, the presence of circulating adult heartworm antigen should disqualify an animal from the conditioning program. treatment. treatment for eradication of heartworms (adults, juveniles, and microfilaria) is a long process that can pose a significant risk to the patient with regard to both drug side effects (hoskins, ) and immunologic reactions to dead worms lodged in the pulmonary vasculature. for this reason, medical treatment of heartworm disease is not usually attempted in research dogs. in a rare instance when such treatment was in the best interest of a long-term canine experiment, thiacetarsamide (caparsolate) and ivermectin (ivomec) were used to eradicate adults and microfilariae, respectively (authors' personal experience). alternative choices include melarsomine (immiticide) as an adulticide and milbemycin (interceptor), levamisole (levasol), or fenthion (spotton) as microfilaricidal agents. dosing regimens for these agents are detailed in dillon ( ) . research complications. the physiomodulative properties of heartworm infection have been studied. such studies have looked at factors released by adult heartworms, as well as changes in the function of host tissues in response to the worm presence. probably the most consistent finding is that endothelial cell-dependent relaxation of pulmonary arterial smooth muscle is depressed in heartworm-infected dogs as compared with control dogs, indicative of alterations in local endothelial cell behavior (maksimowich et al., ; matsukura et al., ; mupanomunda et al., ) . the extension of this effect on peripheral arteries (in vivo and in vitro) has been supported in some studies (kaiser et al., ) but refuted in others (tithof et al., ) . it is thought that the endothelium is perturbed by a factor released from the adult dirofilaria, possibly a cyclooxygenase product such as prostaglandin d (kaiser et al., (kaiser et al., , . these products have also been demonstrated to cause constriction in in vitro rat tracheal ring preparations (collins et al., ) , suggesting that bronchoconstriction could be an aspect of the pathogenesis of the infestation. platelet reactivity was also been found to be enhanced in dogs naturally infected with dirofilaria, when compared with uninfected controis (boudreaux and dillon, ) . based on these data, dogs that are positive for adult heartworm antigen should be considered inappropriate for use as research subjects and, if used, should be restricted to nonsurvival preparations that do not require physiological measurements. etiology. several species of cestodes (tapeworms) parasitize the small intestine of dogs. the most common is dipylidium caninum. other species include taenia pisiformis and, more rarely, echinococcus granulosus, multiceps spp., mesocestoides spp., and spirometra spp. clinical signs. most cestode infestations are subclinical. severe infestations with dipylidium can be associated with diarrhea, weight loss, and poor growth. epizootiology and transmission. the cestode life cycle requires an intermediate host. for dipylidium caninum, the intermediate hosts are fleas and lice. thus this species of tapeworm can be readily transmitted by ingestion of arthropods that are canine parasites in and of themselves. taenia pisiformis requires small ruminants, rabbits, or rodents for intermediate hosts, so spread is less likely, especially in a research setting. echinococcus granulosus uses not only sheep as an intermediate host but also human beings, and thus the zoonotic potential of this cestode must be considered. pathologic findings. adult cestodes in the small intestine are usually an incidental finding at necropsy. diagnosis and differential diagnosis. definitive diagnosis is usually made by the identification of egg capsules or proglottids (tapeworm segments) on the surface of the feces or around the anus. dipylidium egg packets are large ( x bm) and contain - eggs per packet (hall and simpson, ) . prevention and control. the most significant means to limit cestode infestation is to control the population of fleas and/or lice infesting the colony. see the sections on these ectoparasites for effective means to treat infested dogs and kennels. treatment. praziquantel at - . mg/kg orally or subcutaneously is the standard treatment for cestodiasis, especially taenia or echinococcus species. fenbendazole, mebendazole, or oxfendazole may also be effective against dipylidium caninum (hall and simpson, ) . clinical signs. most lung fluke infestations are inapparent, but coughing can develop in cases that prompt a strong inflammatory response. pneumothorax has been a sequela of cyst rupture, in which case dyspnea with reduced lung sounds would be the typical presentation. epizootiology and transmission. the lung fluke life cycle requires two intermediate hosts: a snail and then a crayfish. dogs become infested after eating crayfish, which essentially limits this disease to random-source dogs. on ingestion, the immature flukes (metacercariae) migrate to the lungs and encyst in the pulmonary parenchyma. eggs produced by adult flukes are passed into the bronchioles, coughed up, swallowed, and passed in the feces to complete the life cycle. pathologic findings. grossly, the trematode cysts containing adult flukes can be seen in the lung parenchyma. areas of eosinophilic inflammation surround the cysts, and eosinophilic granulomas can also be seen encircling released eggs. pleural hemorrhages may also be caused by the migrating metacercariae (lopez, ) . pathogenesis. clinical illness is usually a result of a severe eosinophilic inflammatory response, pneumothorax caused by cyst rupture, or secondary bacterial pneumonia. diagnosis and differential diagnosis. definitive diagnosis of paragonimus infestation requires identification of the characteristic ovoid eggs ( - ~tm long) with a single operculum in either the feces or a transtracheal wash. identification from fecal samples requires sedimentation techniques. other causes of coughing in dogs (e.g., infectious tracheobronchitis, dirofilariasis, congestive heart failure) need to be considered. radiographically, the appearance of (multi)focal densities within the air-filled lung field needs to be differentiated from pulmonary neoplasia (primary or metastatic) or systemic fungal pneumonias. prevention. use of purpose-bred dogs virtually eliminates the chance of pulmonary trematodiasis in a research animal. treatment. praziquantel (at mg/kg q hr x days) or fenbendazole ( - mg/kg q hr x - days) are recommended for treatment of canine paragonimus infestation (hawkins, ) . effectiveness is monitored by fecal sedimentation tests for eggs and resolution of radiographic lesions (which may never resolve entirely). early diagnosis of pulmonary trematodiasis should warrant discontinuation of a dog from a long-term study because of the possibility of more serious clinical sequelae, such as pneumothorax. research complications. experimental studies involving the immune system, especially eosinophilic or local pulmonary responses, would be significantly affected by even minor infestations. clinical illness would complicate almost any research project and makes dogs poor anesthetic risks. radiographic lesions may confound diagnostic evaluation for pulmonary metastasis of tumors. e. mites i. demodicosis etiology. canine demodicosis is caused by demodex canis, a commensal mite that lives in the hair follicles. it is considered to be normal fauna of dog skin, but certain conditions (i.e., immunosuppression) cause development of clinical illness. clinical signs. demodex canis infestation is typically asymptomatic. clinical demodicosis presents with variable and nonspecific clinical signs, such as alopecia, erythema, pruritus, crusts, and hyperpigmentation. it can occur anywhere on the body but is often seen on the feet and the face and around the ears (demanuelle, a). secondary bacterial pyoderma is a common complication. epizootiology and transmission. demodex canis mites pass to nursing pups from the dam. they live their entire lives on one dog and are not considered contagious to other dogs or humans. certain breeds are predisposed to the generalized form of demodex dermatitis (see "pathogenesis," below). beagles are among the predisposed breeds, as are german shepherds, doberman pinschers, old english sheepdogs, collies, boxers, and shorthair brachycephalic breeds (muller et al., ) . pathologic findings. histologically, demodex infections are characterized by perifolliculitis and folliculitis with mites and keratin debris visible in the hair follicles. cases with generalized demodicosis (see "pathogenesis," below) may have a minimal cellular response with no eosinophils, indicative of severe immunosuppression . pathogenesis. when clinical demodicosis develops, it is classified into "localized" or "generalized" (e.g., more than one foot affected, or five or more small areas, or one large body area). localized demodicosis is typically seen in juvenile dogs (< months) and usually resolves without treatment as natural immunological control develops. generalized demodicosis can develop in juvenile or adult populations. juvenile-onset generalized demodicosis occurs in dogs with a genetic predisposition, thought to be an inherited t-lymphocyte dysfunction. adult-onset generalized demodicosis is usually indicative of an underlying endocrine (hyperadrenocorticism, diabetes mellitus, hypothyroidism) or neoplastic disorder or can develop as a result of immunosuppressive therapy (such as corticosteroid administration). diagnosis and differential diagnosis. demodex is readily identified from deep skin scrapings of lesioned areas (campbell, ; noli, ) . demodex canis has a characteristic "cigar shape," with short, stubby legs on a body - ~tm long. differential diagnoses for local demodicosis include dermatophytosis, allergic contact dermatitis, and seborrheic dermatitis. the primary differential diagnosis for generalized demodicosis is primary bacterial pyoderma; remember, however, that bacterial pyoderma is a common secondary complication of the generalized form of this parasitism. prevention and control. dogs with generalized demodicosis should not be maintained in a breeding colony. treatment: ivermectin (ivomec) at - ~tg/kg and oral milbemycin (interceptor) at - mg/kg/day have been found to be effective treatments. these parasiticides are probably the most practical to use in a research setting, although they are not labeled for treatment of demodex canis. amitraz (mitaban) dips ( ppm every days) can be used for more problematic cases. treatment duration can be extensive and must be accompanied by repeated skin scrapings. research complications. dogs with generalized demodicosis should not be used in research studies, because this disease is indicative of another underlying disorder (endocrine or immunological). dogs that receive immunosuppressive agents or paradigms could develop generalized demodicosis as an unexpected consequence of the experimentation. ii. sarcoptic mange etiology. canine sarcoptic mange is caused by sarcoptes scabiei var. canis. clinical signs. the most common clinical sign is an intense pruritus, usually beginning at sparsely furred areas such as the ear pinnae, elbows, and ventral thorax and abdomen. lesions are characterized by alopecia and yellowish dry crusts with a macular papular eruption. these lesions may be exacerbated by excoriation due to the pruritic nature of the condition. epizootiology and transmission. sarcoptes mites live their entire lives in the stratum corneum of the host animal; however, they can survive for - weeks away from the host, and it is this ability that enables them to spread from dog to dog. sarcoptes scabiei var. canis can also infect cats and humans. pathologic findings. histologic examination can be unrewarding because mites are rarely seen on tissue sections, and the associated dermatitis is nondiagnostic: perivascular and interstitial dermatitis with hyperkeratosis, with or without eosinophilic infiltration. suggestive histopathologic lesions are epidermal "nibbles," small foci of edema, exocytosis, degeneration, and necrosis . pathogenesis. lesions and illness are a result of the female mites burrowing through the epidermal layers to deposit eggs, and the larvae migrating back to the surface. the typical locations of mange lesions are a result of the mite's preference for relatively hairless areas. diagnosis and differential diagnosis. sarcoptic mange can be difficult to diagnose because multiple skin scrapings can yield negative results with this parasitic disorder. hopefully, adult mites, mite eggs, or mite feces can be observed on superficial skin scrapings. even if scrapings are negative, however, a therapeutic trial should be initiated if the clinical signs and history suggest a sarcoptes etiology. demonstration of anti-mite ige in either the serum or via an intradermal antigen test can be used as a diagnostic aid (campbell, ). an important differential diagnosis is flea allergy dermatitis; in contrast, mange is nonseasonal and contagious. prevention and control. use of purpose-bred dogs limits the possibility of having research animals with sarcoptic mange. for random-source dogs, an ectoparasite control program should be in place to limit possible infestations. many institutions use ivermectin as a means to control both endoparasites and ectoparasites. treatment. unless treatment would interfere with research objectives, all dogs with sarcoptic mange (no matter how minor the lesions) and their kennel mates should be treated because of the contagious nature of the disease and its zoonotic potential. in research colonies, the usual means of treatment is either ivermectin (ivomec) at - ~tg/kg q days or milbemycin (interceptor) at oral doses of mg/kg q days . neither of these agents is approved for treatment of sarcoptic mange, but they are considered to be effective. acaricidal dips (e.g., lime sulfur, organophosphates, amitraz) can also be used. research complications. the local skin inflammation and systemic immune response to sarcoptic mange probably make infected dogs poor subjects for dermatologic and immunologic studies. f lice and ticks i. lice etiology. dogs can be infested by one species of sucking louse (linognathus setosus) and two species of biting lice (trichodectes canis and heterodoxus spiniger). clinical signs. mild cases of pediculosis may be asymptomatic or may cause pruritic areas of dry skin. more severe infestations can cause significant pruritus and produce alopecia, papules, and crusts. these lesions lead to excoriation and secondary bacterial dermatitis. severe linognathus infestations could cause anemia, because this species feeds on blood. epizootiology and transmission. louse infestations are uncommon in both pet animal practice and the research setting. they would most likely be seen in random-source dogs that were obtained from a pound or shelter. transmission is usually by direct contact, for lice spend their entire lives on the host species. lice are host-specific and not zoonotic. pathogenesis. the biting lice usually cause more local irritation than the sucking louse and therefore are more apt to induce clinical dermatologic signs. trichodectes canis can serve as vector for the canine tapeworm dipylidium caninum. the most severe complication of infestations by the sucking louse is the potential anemia. diagnosis and differential diagnosis. pediculosis is diagnosed by direct observation of the lice or nits (eggs) on the dog's skin. cellophane tape can be used to pick up surface debris from skin lesions, which may include nits or immobilized lice (muller et al., ) . differential diagnoses include dermal acariasis, flea allergy dermatitis, and seborrhea. prevention. use of high-quality conditioned dogs for research should prevent pediculosis from ever being seen within a research facility. random-source dogs should be shampooed or treated prophylactically with topical insecticide before being permitted to enter the research colony. treatment. most commercially available insecticide shampoos and dips readily treat louse infestations. treatment should be repeated in - days, because any nits that were not killed would have hatched by that time (muller et al., ) . there is probably minimal interference with research, unless severe linognathus infestations cause anemia. ii. ticks etiology. ticks are obligate arachnid parasites that require vertebrate blood as their sole food source. except for the brown dog tick (rhipicephalus sanguineus), ticks have a wide host range and are not especially host-specific; so any number of tick genera and species can be found on dogs. genera that more commonly infest dogs in the united states include species of rhipicephalus, dermacentor, and ixodes. the primary significance of tick infestation is the tick's ability to be a vector for many other infectious diseases, including rocky mountain spotted fever (caused by rickettsia rickettsii), lyme disease (borrelia burgdorferi), and the canine forms of ehrlichiosis (ehrlichia canis and e. platys), babesiosis (babesia canis), haemobartonellosis (haemobartonella canis), and hepatozoonosis (hepatozoon canis). clinical signs. as an entity unto itself, tick infestation causes minimal clinical signs. most infestations are subclinical, although some dogs may lick and bite at the site, aggravating the local lesion. some dogs can develop a hypersensitivity reaction after several tick bites; these dogs develop a more granulomatous response at the location of the bite (merchant and taboada, ) . some species of ticks (primarily dermacentor andersoni and d. variabilis) produce a salivary neurotoxin that can cause an ascending flaccid paralysis (malik and farrow, ) . the paralysis develops within - days of tick attachment and can result from a single tick. this paralysis is fatal once the respiratory musculature is affected. epizootiology and transmission. in dogs used for biomedical research, tick infestation may occasionally be seen in randomsource dogs, because these dogs are more likely to have been in tick habitats than purpose-bred dogs. ticks commonly reside in wooded areas until they contact a suitable host for a blood meal. the brown dog tick may reside within kennels (attics, bedding, wall insulation) (garris, ) . pathologic findings. under most circumstances, tick infestation will be an incidental finding on necropsy (unless tick paralysis was the cause of death). pathogenesis. tick-bite paralysis is caused by the presence of a salivary neurotoxin released by female ticks of certain genera (e.g., dermacentor) while consuming a blood meal (malik and farrow, ) . interestingly, dogs seem to be most affected by this condition, whereas cats appear to be resistant. the primary dysfunction appears to be at the neuromuscular junction, as stimulation of the motor nerves fails to elicit a response, but direct stimulation of the muscle tissue results in contractions. tick bites can also transmit pathogen microorganisms to the dog, because ticks serve as vectors for several infectious diseases, including lyme borreliosis, ehrlichiosis, babesiosis, and rocky mountain spotted fever. diagnosis and differential diagnosis. for uncomplicated tick bites and tick-bite paralysis, definitive diagnosis is made by identification of the offending arachnid (and improvement of paralysis after removal). differential diagnoses for tick-bite paralysis include botulism, snakebite, polyradiculoneuritis, and idiopathic polyneuropathy (malik and farrow, ) . prevention. purpose-bred dogs should be free from all ectoparasites, but ticks can occasionally be seen on randomsource animals. research dogs should not be exercised in outdoor areas infested with ticks, and kennels must be cleaned properly and regularly so as to remain free of ticks and other parasites. treatment. removal of the offending tick is the primary treatment for both local inflammation as well as tick-bite paralysis. dogs with tick-bite paralysis usually show improvement within hr, with complete recovery within hr (malik and farrow, ) to remove an attached tick from a dog, forceps should be used to grasp the tick as close to the dog's skin as possible. the tick should not be grabbed by the body, as this may cause the parasite to either rupture or inject its body contents into the dog. the tick should be pulled away from the dog with steady pressure. many of the diseases transmitted by ticks are zoonotic so precautions, such as wearing gloves, should be taken. use of topical acaricide/insecticides on newly arrived random-source dogs should help to limit infestations. probably have minimal impact on research variables. the significant concern for tick infestation is the possible development of tick-bite paralysis or of any one of a number of systemic diseases spread by ticks (see sections iii,a,l,e-g). g. other i. flea infestation etiology. fleas are laterally flattened wingless insects that feed on animal blood. the most common flea to infest dogs is ctenocephalides felis, the cat flea. other fleas that can affect dogs are ctenocephalides canis, pulex irritans, and echidnophaga gallinacea. the fleas are speciated by the shape of their head and by the presence or absence of ctenidae (spiny combs on or behind the head) (campbell, ) . clinical signs. flea infestations usually cause foci of alopecia and pruritus. dogs that are hypersensitive to antigenic proteins in flea saliva develop the more severe "flea allergy dermatitis," which features papules and crusting. acute moist dermatitis ("hot spots") can also be seen in these cases, and secondary pyoderma or seborrhea can develop. lesions from flea allergy dermatitis generally appear in the dorsal lumbosacral region, as well as the flanks, thighs, and abdomen (muller et al., ) . the lesions are typically worse in the summer and autumn months and are progressively more severe as the dog ages. epizootiology and transmission. fleas are readily transmitted between animals and even between host species. they move readily between the host and the environment, making transmission easy and control difficult. because fleas require host blood for food, they can survive off of a host for only - months (muller et al., ) . pathologic findings. biopsy samples are usually nondiagnostic in cases of flea allergy dermatitis. lesions are typically characterized by perivascular eosinophilic inflammation and may feature pustules and folliculitis if secondary pyoderma develops (muller et al., ) . pathogenesis. fleas are parasites that require animal blood for their meals. when they bite host animals, they inject some saliva into the host's skin. if the host develops an allergic response to the flea saliva, it will develop the more pruritic flea allergy dermatitis. fleas can also transmit or serve as vectors for other pathogens (e.g., dipylidium tapeworms). flea allergy dermatitis are definitively diagnosed by observing the fleas on the host's skin. given that this may be difficult because of the mobility of the flea and the majority of the time it spends off of the host, diagnosis is often based on clinical signs, history, and lesion distribution. sometimes the presence of flea excrement ("flea dirt") on the dog's skin can support a presumptive diagnosis (demanuelle, b) . circulating eosinophilia is seen in some dogs with flea allergy dermatitis. differential diagnoses include mite and louse infestations, bacterial folliculitis, and allergic or atopic conditions that present with skin lesions in dogs (e.g., food, drug, or contact hypersensitivity). prevention. most dogs obtained from high-quality purposebred facilities should be free from flea infestations. dogs received from pounds, shelters, or licensed dealers would be more likely to be affected by fleas (or any ectoparasitism). thorough knowledge of prevention, control, and treatment measures at these facilities should be obtained, and dogs from sources where proper prevention and/or therapy are not practiced should be evaluated and/or empirically treated upon arrival at the facility. control. thorough cleaning of the dog's housing environment should remove the risk of perpetuating or transmitting flea infestation in the colony. treatment. treatment for fleas needs to address treatment of both the dog and the environment. many insecticide formulations such as shampoos, sprays, dips, powders, and oral systemics can be used for initial treatment of the individual dog. the active ingredients include pyrethrins, pyrethroids, carbamates, and organophosphates. flea control in the kennel may need to include outdoor areas in warm climates. typically combinations of adult insecticides and juvenile growth regulators are used for environmental treatment. directed sprays are the most effective means of treating housing areas, because flea "bombs" or foggers do not penetrate adequately into tight areas where fleas might hide (demanuelle, b) . in addition to insecticide therapy, dogs with flea allergy dermatitis may also require anti-inflammatory medication to relieve clinical signs. oral prednisone or prednisolone at . mg/kg q hr for - days has been proposed as a starting therapy (muller et al., ) . the use of hyposensitization with flea-bite antigens is controversial and not practical for the research setting. research complications. mild flea infestation probably has minimal impact on most research protocols, and treatment measures may in fact be more detrimental to the experimental objective than the actual ectoparasitism. in a research setting, the residual effects of insecticides may preclude their use in experimental animals. such treatments should be used judiciously to ensure that experimental results are not more seriously affected by the therapy rather than the infestation. dogs with flea-allergy dermatitis are more severely affected by the flea infestation and should be treated apigropriately; however, systemic corticosteroids may also interfere with experimental objectives, especially in studies involving functions of the immune system. the ability of fleas to transmit other parasitic diseases must also be considered. etiology. dermatophytoses ("ringworm") are fungal skin infections, which in dogs in the united states are usually caused by either microsporum canis, m. gypseum, or trichophyton mentagrophytes (muller et al., ) . clinical signs. uncomplicated superficial dermatophytoses are characterized by circumscribed circular areas of alopecia, usually with minimal to no inflammation. these skin lesions are usually seen around the face, neck, and forelimbs but can be found anywhere on the body. secondary bacterial infections can develop; these lesions are called kerions and are selflimiting, for the fungus cannot survive in inflamed skin (muller et al., ) . ep&ootiology and transmission. the fungi that cause skin infections are very contagious and readily transmissible between dogs and other species (including human beings), but they can also be obtained from the soil. pathologic findings. on close inspection of skin samples, broken hair shafts (and not complete hair loss) would be seen with uncomplicated dermatophytosis. histologically, fungal elements can be seen within the stratum corneum or in and around the hair and hair follicles (muller et al., ) . stains that facilitate visualization of fungal elements include periodic acid-schiff (pas) or gomori methenamine-silver. the pattern of inflammation in the affected foci is very variable and can feature folliculitis, perivascular dermatitis, hyperkeratosis, and/or vesicular dermatitis. pathogenesis. the dermatophytes typically infect the hair shaft itself, the hair follicle, and possibly the skin around the affected hair. the hair follicle is not destroyed (unless by secondary bacterial infection), but the hair itself becomes brittle and breaks. this causes short stubbly hair to be seen within the lesion. as the lesion progresses, the hairs in the center recover from the infection, thus leading to the classic "ringworm" appearance of the alopecic areas. it is postulated that the inflammatory process produces an environment that is unfavorable for dermatophyte survival, whereas the periphery of the lesion still enables continued fungal growth (muller et al., ) . diagnosis and differential diagnosis. diagnosis of dermal fungal infection is typically made by scraping the affected area to obtain hair and superficial epidermal cells. these scrapings are then digested with potassium hydroxide to facilitate observation of fungal elements. fungal elements can also be seen on skin biopsy samples. for speciation of a fungus, skin scrapings can also be inoculated onto agars that promote fungal growth, such as sabouraud's medium or dermatophyte test medium (dtm). incubation should be at ~ with % humidity for - days. lesions caused by m. canis may fluoresce when inspected using a wood's ( . nm ultraviolet) light. unfortunately, some strains of m. canis do not fluoresce, and neither does m. gypseum or t. mentagrophytes. differential diagnoses for dermatomycosis include seborrhea, localized demodecosis, folliculitis, histiocytoma, and acral lick dermatitis (muller et al., ) . prevention. purpose-bred dogs are typically free of infectious dermatophytes, but ringworm may be diagnosed on randomsource animals. control. in cases of dermatophytosis, isolation of the affected animal(s) is prudent, because the fungi are easily spread to other dogs, as well as to people. treatment, if acceptable, should be started immediately. treatment. topical antifungal therapy is most commonly used. shampoos, rinses, and creams containing miconazole, ketoconazole, enilconazole, or chlorhexidine are commercially available to treat ringworm (stannard et al., ) . severe cases may require systemic therapy with griseofulvin, ketoconazole, itraconazole, or fluconazole. however, these systemic antifungal agents may have considerable side effects (such as vomiting and teratogenicity with griseofulvin). many of the newer agents are also expensive and not labeled for use in dogs. impact on most research applications for dogs. unfortunately, the zoonotic implications of dermatophytoses force the issue of aggressive treatment, and many antifungal agents may not be compatible with biomedical research studies. systemic fungal infections disseminate to multiple organ systems from a single mode of entry (usually through the respiratory tract). dogs are susceptible to several fungi that characteristically cause systemic mycosis, including blastomyces dermatitidis, histoplasma capsulatum, coccidioides immitis, and cryptococcus neoformans var. neoformans. these diseases are not typically seen in the research setting, because of the low overall incidence and noncontagious nature of these disorders and because of the use of purpose-bred animals. these conditions could, however, present in the rare random-source dog that was subclinical at its point of origin, especially if the animal becomes immunosuppressed (either naturally or by virtue of experimental manipulation). typical clinical signs include weight loss, fever, lymphadenopathy, and cough and dyspnea (if the lungs are affected). the reader is advised to read veterinary medical text chapters (e.g., taboada, ) for more complete information on these disorders and their possible treatments. although the incidence of hypothyroidism in the canine population is not high (kemppainen and clark, ) , deficiency in thyroid hormone can significantly affect basal metabolism and immune function. because these factors are important in many biomedical research studies, it is imperative that laboratory animal veterinarians be able to recognize, diagnose, and treat this problem. etiology. the majority of cases of canine hypothyroidism are due to lymphocytic thyroiditis, an autoimmune disorder, or idiopathic atrophy of the thyroid gland. both of these causes result in a gradual loss of functional thyroid tissue (kemppainen and clark, ) . lymphocytic thyroiditis is the major cause of hypothyroidism in laboratory beagles and appears to be familial in that breed (tucker, ; beierwaltes and nishiyama, ; manning ) . rarely, congenital defects or nonfunctional tumors may cause hypothyroidism (peterson and ferguson, ; kemppainen and clark, ) . clinical signs. because it affects metabolism in general, hypothyroidism can produce a large number of clinical signs referable to many organ systems. an individual dog with hypothyroidism may have one or any combination of clinical signs. hypothyroidism reduces the dog's metabolic rate, which then produces such signs as obesity, lethargy, cold intolerance, and constipation. additionally, hypothyroidism can produce several dermatologic abnormalities, including alopecia, hyperpigmentation, seborrhea, and pyoderma (peterson and ferguson, ; panciera, ) . several clinicopathologic abnormalities have also been reported in a large percentage of hypothyroid dogs. these aberrations include increased serum cholesterol and triglycerides due to a decrease in lipolysis and decreased numbers of low-density lipopolysaccharide receptors (peterson and ferguson, ; panciera, ) . normocytic normochromic nonregenerative anemia and increased serum alkaline phosphatase and creatine kinase have also been reported in a significant number of hypothyroid dogs (peterson and ferguson, ; panciera ) . neurologic signs of hypothyroidism, which include lameness, foot dragging, and paresis, may be caused by several mechanisms such as segmental nerve demyelination or nerve entrapment secondary to myxedema (peterson and ferguson, ) . mental impairment and dullness have also been reported in hypothyroid dogs, secondary to atherosclerosis and cerebral myxedema (peterson and ferguson, ) . hypothyroidism has been implicated in other neurological abnormalities such as horner's syndrome, facial nerve paralysis, megaesophagus, and laryngeal paralysis; however, these conditions do not always resolve with treatment (bischel et al., ; panciera, ) , and so the relationship between hypothyroidism and these problems has not been completely defined (panciera, ) . myopathies associated with hypothyroidism are caused by metabolic dysfunction and atrophy of type ii muscle fibers and can present with signs similar to neurological disease (peterson and ferguson, ) . hypothyroidism can also cause bradycardia as a result of decreased myocardial conductivity. abnormalities that may be detected by ecg include a decrease in p and r wave amplitude (peterson and ferguson, ) and inverted t waves (panciera, ) . these electrocardiographic abnormalities are caused by lowered activity of atpases and calcium channel function. several reports have suggested that hypothyroidism is associated with von willebrand's disease and bleeding abnormalities. however, the relationship is probably one of shared breed predilection and not a true correlation. it has been demonstrated that dogs with hypothyroidism are not deficient in von willebrand's factor when compared with other dogs. in addition, the replacement of thyroid hormone in dogs did not increase the levels of vwf:ag in naturally occurring (panciera and johnson, ) or experimentally induced (panciera and johnson, ) hypothyroidism. epizootiology. the prevalence of hypothyroidism in the general canine population has been reported to be less than % (panciera, ) . the disorder occurs most often in large-breed dogs but has been reported in several other breeds as well as mongrels. doberman pinschers and golden retrievers appear to have a higher incidence of hypothyroidism when compared with other breeds (panciera, ; peterson and ferguson, ; scarlett, ) . there have been several reports about hypothyroidism in laboratory colonies of beagles (manning, ; tucker, ; beierwaltes and nishiyama, ) . in general, the problem is usually recognized in middle-aged animals, and some reports state that there is a higher incidence of hypothyroidism in spayed female dogs (panciera, ; peterson and ferguson, ). diagnosis and differential diagnosis. because of the large number of clinical manifestations in dogs, the recognition of hypothyroidism is not always straightforward. likewise, the diagnosis of hypothyroidism can be difficult because of the lack of definitive diagnostic tests available for the dog. the tests currently available and in popular use will be discussed further. however, a complete understanding of the diagnosis of hypothyroidism requires a familiarity with thyroid hormone metabolism and function that is beyond the scope of this writing. for additional information, the reader is referred to one of several manuscripts available (peterson and ferguson, ; ferguson, ) . currently, the ability to diagnose hypothyroidism relies heavily on the measurement of serum total t (thyroxine) and free t (peterson and ferguson, ; ferguson, ) . t serves primarily as a precursor for t in the body and is heavily proteinbound. free t represents the unbound fraction that is available to the tissues (peterson and ferguson, ) . using the measurement of serum total t and free t , hypothyroidism can usually be ruled out if the values are within the normal range or higher. if both hormone concentrations are low, it is highly likely that the patient has hypothyroidism, and a therapeutic trial is in order (peterson and ferguson, ) . however, it must be noted that nonthyroidal illnesses and some drugs (e.g., glucocorticoids, anticonvulsants, phenylbutazone, salicylates) can falsely lower these values (peterson and ferguson, ; ferguson, ) . therefore, low values do not always indicate that hypothyroidism is present, and animals should not be treated solely on the basis of serum hormone levels if clinical signs are absent. if the clinical signs are equivocal or if only total t or free t is decreased, further diagnostic testing is warranted (peterson and ferguson, ) . although t is the most biologically active form of thyroid hormone in the body, the measurement of serum t levels is an unreliable indicator of hypothy-roidism (peterson and ferguson, ; ferguson, ) . like t , serum t can be falsely lowered by many nonthyroidal illnesses and many drugs (see above). in addition, t may be preferentially released, and conversion of t to t may be enhanced in the hypothyroid dog (peterson and ferguson, ; ferguson, ) . t was within normal limits in % of the hypothyroid dogs in one study (panciera, ) . autoantibodies can be responsible for false elevations in the concentrations of t and t found in these respective assays. it has been recommended that free t , measured by equilibrium dialysis, be assayed in dogs that are suspected of hypothyroidism and have autoantibodies with normal or high t and t . autoantibodies have been found in less than % of the samples submitted to one laboratory (kemppainen and behrend, ) . other means of diagnosing hypothyroidism have been described. in humans, endogenous tsh (thyroid-stimulating hormone) levels provide reliable information on thyroid status, and an assay for endogenous tsh is now available in dogs. however, tsh levels can be normal in some dogs with hypothyroidism, and high tsh levels have been noted in normal dogs. therefore, it is recommended that tsh levels be considered along with other information (clinical signs, t ) prior to diagnosis and treatment (kemppainen and behrend, ) . tsh stimulation testing using exogenous bovine tsh provides a good and reliable method for establishing a diagnosis. unfortunately, the availability and expense of tsh limit the use of this diagnostic tool (peterson and ferguson, ; ferguson, ) . another drawback of tsh testing is that the test must be postponed for weeks if thyroid supplementation has been given (peterson and ferguson, ) . when tsh is available for testing, there are several recommendations for dosage, routes of administration, and sampling times. one recommendation is . u of tsh per pound of body weight (up to a maximum of u) to be administered iv. for this protocol, blood samples are taken prior to administration of tsh and hours after. a normal response to the administration of tsh should create an increase of t levels at least ktg/dl above the baseline levels or an absolute level that exceeds ~tg/dl (peterson and ferguson, ; wheeler et al., ) . treatment. the treatment of choice for hypothyroidism in the dog is l-thyroxine (sodium levothyroxine). a recommended dosing regimen is . mg/kg once a day or . mg/m (body surface area)/day for very small or very large dogs. if drugs that decrease thyroxine levels are being administered concurrently, it may be necessary to divide the thyroxine dose for twice daily administration. after the supplementation has begun, the thyroid hormone level should be rechecked in - weeks, and blood samples should be drawn - hours after the morning pill. a clinical response is usually seen in - weeks and would include weight loss, hair regrowth, and resolution of other signs (panciera, ) . ecg abnormalities also return to normal (peterson and ferguson, ) . for dogs with neurologic signs, the prognosis is guarded, because the signs do not always resolve with supplementation (panciera, ) . weight gain and eventual obesity are also frequent findings in dogs in the research environment. because obesity can adversely affect several body systems as well as general metabolism, the laboratory animal veterinarian must be aware of the development of obesity and the potential effect that it can have on research. etiology. obesity is defined as a body weight - % over the ideal. in general, obesity occurs when the intake of calories exceeds the expenditure of energy. excessive caloric intake resuits from overeating or eating an unbalanced diet. overeating is a common cause of obesity in pet dogs and may be triggered by boredom, nervousness, or conditioning (macewen, ) . in addition, pet animals are often subjected to unbalanced diets supplemented with high-fat treats. in the laboratory animal setting, overeating is less likely than in a household, because access to food is more restricted and diets are usually a commercially prepared balanced ration. however, obesity can still be a problem if specific guidelines for energy requirements are not followed. in addition, the necessary caging of dogs in the research environment and thus the limitation to exercise reduces energy expenditure and predisposes dogs to weight gain. it is also important to realize that other factors may predispose dogs to obesity, even when guidelines for caloric intake and energy expenditure are followed (butterwick and hawthorne,. ). as in humans, genetics plays an important role in the development of obesity in dogs. it has been established that certain breeds are more predisposed toward obesity. in a study of dogs visiting veterinary clinics in the united kingdom, labrador retrievers were most likely to be obese. other breeds affected included cairn terriers, dachshunds, basset hounds, golden retrievers, and cocker spaniels. the beagle was also listed as a breed predisposed to obesity in the household environment (edney and smith, ) . in addition to genetics, several metabolic or hormonal changes are associated with obesity. it has been well established that neutering promotes weight gain. in one study, spayed female dogs were twice as likely to be obese when compared with intact females (macewen, ) . the authors proposed that the absence of estrogen promotes an increase in food consumption. a similar trend toward obesity was found in castrated male dogs (edney and smith, ) . in addition, hypothyroidism and hyperadrenocorticism may present with obesity as one of the clinical signs (macewen, ) . epizootiology. ewen, ) . obesity affects up to % of pet dogs (mac-diagnosis and differential diagnosis. the diagnosis of obesity is somewhat subjective and relies on an estimate of ideal body weight. the ideal body condition for dogs is considered to be achieved when the ribs are barely visible but easily palpated beneath the skin surface. when the ribs are not easily palpated and/or the dog's normal function is impaired by its weight, the animal is considered obese. there are few objective, quantifiable methods for establishing this diagnosis. ultrasound has been evaluated for measurement of subcutaneous fat in dogs, and measurements taken from the lumbar area can be used to reliably predict total body fat (wilkinson and mcewan, ) . after a diagnosis of obesity has been made, additional diagnostic tests should be performed to determine if there is an underlying cause for the problem. a complete physical exam should be performed to look for signs of concurrent disease and to establish if obesity has adversely affected the individual. serum thyroid hormones should be evaluated (see section iii,b,l,a), and serum chemistry may reveal an increased alkaline phosphatase associated with hyperadrenocorticism. treatment. restricting food intake readily treats obesity, and this is easily done in the research setting. it has been suggested that a good weight loss program involves restriction of intake to % of the calculated energy requirement to maintain ideal body weight. it has been shown that restriction of calories down to % produces no adverse health effects. however, t levels will decrease in direct proportion with caloric intake. ideally, weight loss will occur at a rate of - % of body weight per week (laflamme et al., ) . with more severe calorie restriction and more rapid weight loss, the individual is more likely to rebound and gain weight after restrictions are relaxed. there has been agreat deal of attention in humans as to the correct diet to be fed to encourage weight loss. likewise, the type of diet fed to dogs has been examined. as mentioned above, the restriction of calories is most important, and feeding less of an existing diet can do this. alternatively, several diet dog foods are available, and there is some evidence that these diets are superior to simple volume restriction (macewen, ) . there has been much concern about the addition of fiber to the diet in both humans and animals as a method for reducing caloric intake while maintaining the volume fed. studies in dogs have examined the addition of both soluble and insoluble fibers to calorierestricted diets. these studies have shown that the addition of fiber does not have an effect on satiety in dogs and therefore does not have a beneficial effect in weight loss protocols (butterwick and thorne, ; butterwick and thorne, ) . it is important to control weight gain in research animals, because of the association of obesity and several metabolic changes. although an association between obesity and reproductive, dermatologic, and neoplastic problems has been reported (macewen, ) , this relationship is not consistently apparent (edney and smith, ) . obesity in dogs over years of age appears to be related to an increase in cardiovascular problems (edney and smith, ) , and obesity has been linked to hypertension. joint problems including osteoarthritis and hip dysplasia have also been related to obesity (macewen, ; kealy et al., ) . in addition, diabetes mellitus has been linked to obesity, and obesity induces hyperinsulinism in several experimental models (macewen, ) . in the laboratory setting, the majority of traumatic wounds will be small in size. in facilities with good husbandry practices and a diligent staff, traumatic wounds will generally be observed quickly and attended to promptly. under these conditions, proper initial treatment will lead to uncomplicated wound healing. complications such as infection and delayed healing arise when wounds are not noticed immediately or. when the basic principles of wound management are not followed. to aid in the description of wounds and in decision making about wound therapy, several classification systems have been developed for traumatic injuries. at one time, decisions about wound therapy were largely based upon the length of time since wounding, or the concept of a "golden period." it is now recognized that several factors must be considered prior to initiating wound care, including (but not limited to) the type and size of the wound, the degree of wound contamination, and the capability of the host's defense systems (swaim, ; waldron and trevor, ) . one of the most widely used classification systems is based upon wound contamination and categorizes wounds as either clean, clean-contaminated, contaminated, or dirty (see table v ). the vast majority of the wounds seen in the laboratory setting will fall into the clean and clean-contaminated categories. these wounds may be treated with the basic wound care described below and primary closure of the wound. contaminated and dirty wounds, which are seen infrequently in the laboratory setting, require more aggressive therapy. dirty wounds can occur as postsurgical infections or complications of initial wound therapy. when one is in doubt as to the classification of a wound, the worst category should be presumed in order to provide optimal therapy and reduce the chance for complications. the initial treatment of a wound is the same regardless of the wound's classification. when first recognized, the wound should be covered' with a sterile dressing until definitive treatment is rendered. bleeding should be controlled with direct waldron and trevor ( ) . pressure; tourniquets are discouraged because of the complications that may arise with inappropriate placement (swaim, ) . it is best to avoid using topical disinfectants in the wound until further wound treatment (culture, debridement, lavage) has been performed (swaim, ) . when the treatment of a wound begins, anesthesia or analgesia may be necessary, and the choice of anesthetic regimen will depend on the size and location of the wound as well as the preference of the clinician. if the wound is contaminated or dirty, bacterial cultures, both aerobic and anaerobic, should be performed at this time. then a water-soluble lubricant gel may be applied directly to the wound. a wide margin of hair should then be clipped from around the wound, using a # blade. after the clipping, a surgical scrub is performed around the edges of the wound. povidone-iodine alternating with alcohol or chlorhexidine gluconate scrub alternating with water is most often recommended for surgical preparation of the skin surface (osuna et al., a,b) . simple abrasions that involve only a partial thickness of the skin do not generally require further treatment. full-thickness wounds require further attention, including irrigation with large quantities of a solution delivered under pressure. two solutions, . % chlorhexidine diacetate in water (lozier et al., ) and % povidone-iodine in saline, are most often recommended for wound lavage (waldron and trevor, ) . the chlorhexidine solution may offer the advantage of greater bactericidal activity but does not significantly alter wound healing when compared with povidone-iodine (sanchez et al., ) . actually, the type of solution chosen may not be as important to wound care as the volume and pressure at which the solution is delivered. it has been suggested that psi is required to obtain adequate tissue irrigation, and this may be achieved by using a ml syringe and an -or gauge needle (waldron and trevor, ) . for wounds that are contaminated or dirty, debridement is an important part of initial therapy. debridement usually proceeds from superficial to deeper layers. skin that is obviously necrotic should be removed. although it is often recommended to remove skin back to the point at which it bleeds, this may not be feasible with large wounds on the limbs. in addition, other factors such as edema or hypovolemia may reduce bleeding in otherwise viable skin (waldron and trevor, ) . if one is unsure about tissue viability in areas that are devoid of extra skin, the tissue may be left (swaim, ; waldron and trevor, ) , and nonviable areas will demarcate within - days (waldron and trevor, ) . necrotic fat should be resected liberally, because it does not have a large blood supply and will provide an environment for infection. often, resection of subcutaneous fat is necessary to remove debris and hair that could not be removed during wound irrigation. damaged muscle should also be liberally resected (swaim, ) . the wound should be irrigated several times during debridement and again after completion. after initial wound treatment, the options concerning wound closure must be weighed. the principles of basic surgery are discussed in several good texts, and readers are encouraged to pursue additional information. primary wound closure is defined as closure of the wound at the time of initial wound therapy and is the treatment of choice for clean and clean-contaminated wounds. closure is performed in two or more layers, carefully apposing tissues and obliterating dead space. if dead space will remain in the wound, a drain should be place d. subcutaneous closure should be performed with absorbable suture such as polydioxanone (pds), polyglactin (vicryl), or polyglycolic acid (dexon). it is best to use interrupted sutures and avoid leaving excess suture material in the wound. it may be necessary to choose tension-relieving suture patterns, such as horizontal mattress. skin closure is generally performed with nylon ( - or - ). in situations where gross contamination cannot be completely removed, closure of the wound should be delayed or avoided. after debridement and irrigation, the wound should be bandaged. initially, the wound can be covered by gauze sponges soaked in saline or chlorhexidine to create a wet-to-dry bandage. when the sponges are later pulled from the wound, dried exudates will also be removed. when the wound appears clean, the layer in contact with the wound may be changed to a nonadherent dressing such as vaseline-impregnated gauze (swaim, ) . the contact layer is covered by cotton padding, and the entire bandage is covered by a supportive and protective layer. the bandages should be changed once or twice daily, depending upon the amount of discharge coming from the wound. wound closure within - days of wounding (prior to the formation of granulation tissue) is considered delayed primary closure. when the wound is closed after days, this is considered secondary closure (waldron and trevor, ) . secondintention healing involves allowing the wound to heal without surgical intervention. this type of healing is often used on limbs when there is an insufficient amount of skin to allow complete closure (swaim, ) . it is important to note that second-intention healing will take longer than surgical repair of a wound, and in the case of large wounds it will be more expensive because of the cost of bandaging materials. several factors must be weighed concerning the use of antibiotics in traumatic wounds, including the classification and site of the wound, host defenses, and concurrent research use of the animal. when wounds are clean or clean-contaminated, antibiotics are seldom necessary unless the individual is at high risk for infection. when wounds have been severely contaminated or are dirty, antibiotics are indicated, and the type of antibiotic will ultimately depend on culture and sensitivity results. until such results are available, the choice of antibiotic is based on the most likely organism to be encountered. in skin wounds, staphylococcus spp. are generally of concern, whereas pasteurella multocida should be considered in bite wounds. cephalosporins, amoxicillin-clavulanate, and trimethoprim sulfas are often recommended for initial antibiotic therapy (waldron and trevor, ) . etiology. pressure sores (decubital ulcers) can be a problem in long-term studies that require extended periods of recumbency. decubital ulcers usually develop due to continuous pressure from a hard surface contacting a bony prominence such as the elbow, the tuber ischii, tarsus, or carpus. the compression of the soft tissues between the hard surfaces results in vascular occlusion, ischemia, and ultimately tissue death (swaim and angarano, ) . several factors that increase pressure at the site and/or affect the integrity of the skin will predispose an individual to develop pressure sores. these factors include poor hygiene, self-trauma, low-protein diet, preexisting tissue damage, muscle wasting, inadequate bedding, and ill-fitting casts or bandages (swaim and angarano, ) . clinical signs. at first, the skin at the developing site will appear red and irritated. over time, constant trauma can result in full-thickness skin wounds and can progress to necrosis of underlying structures such as bone. the severity of the sores may be graded from i to iv, according to the depth of the wound and the tissues involved, from superficial skin irritation to bone necrosis. epizootiology. the problem usually occurs in large-breed dogs, but any type of dog can be affected. prevention and control. minimizing or eliminating those factors that can predispose to decubital ulcers is important to both the prevention and the control of this condition. if the dogs are going to experience long periods of recumbency, adequate bedding or padding must be provided. skin hygiene is of the utmost importance when trying to prevent or treat pressure sores. the skin should be kept clean and dry at all times. if urine scalding is a problem, the affected area should be clipped, bathed, and dried thoroughly at least once or twice daily. finally, an appropriate diet to maintain good flesh and adequate healing is also important (swaim and angarano, ) . treatment. the treatment of pressure sores must involve care of the wound and attention to the factors causing the wound. the extent of initial wound management will largely depend on the depth of the wound. for simple abrasions and small wounds involving the skin only, simple wound cleansing and openwound management provide adequate treatment. when wounds involve deeper tissues, including fat, fascia, or bone, more aggressive therapy must be performed. the affected area should be radiographed to assess bone involvement, and the wound should be cultured. all of the damaged tissue should be debrided, and wound management guidelines should be followed (see section iii,c, ). when a healthy granulation bed has formed over the entire wound, a delayed closure over a drain may be performed (swaim and angarano, ) . with extensive lesions, reconstruction with skin flaps may be necessary. bandaging should be performed on all full-thickness wounds; however, it is important to remember that ill-fitting or inadequately padded bandages or casts may worsen the problem. the area over the wound itself should not be heavily padded, because this will increase the pressure over the wound. the wounded area should be lightly covered and then a doughnut, created from rolled gauze or towel, should be fitted around the wound. this will displace the forces acting on the wound over a larger area and over healthier tissue. then the doughnut is incorporated into the bandage. if a cast has been applied to the area for treatment or for research purposes, a hole can be cut over the wound to reduce pressure in that area and allow treatment of the wound (swaim and angarano, ). bandages should be removed at least once or twice a day to allow wound care. after wound care has been initiated the causative factors for the pressure sore must be addressed (see "prevention and control," above). recumbent animals should be moved frequently to prevent continuous compression on the wound. if the dog tends to favor a position that aggravates the problem, splinting the body part to reduce contact with hard surfaces may be necessary. etiology. acral lick granuloma is a psychodermatosis, a skin lesion caused by self-trauma. in a few cases, self-trauma begins because of identifiable neurologic or orthopedic causes (tarvin and prata, ) . however, the majority of the cases begin because of repetitive licking by dogs that are confined and lack external stimuli (swaim and angarano, ) . it has been theorized that the self-trauma promotes the release of endogenous endorphins, which act as a reward for the abnormal behavior (dodman et al., ) . the laboratory setting is an environment that could promote this abnormal behavior and lead to acral lick granuloma. epizootiology. the lesions associated with acral lick granuloma are seen most often in large-breed dogs, but any type of dog can be affected (walton, ) . clinical signs. at first, lesions appear as irritated, hairless areas usually found on the distal extremities (swaim and angarano, ). the predilection for the limbs may be due to accessibility or possibly may be caused by a lower threshold for pruritus in these areas. as the lesions progress, the skin becomes ulcerated, and the wound has a hyperpigmented edge. the wounds may partially heal and then be aggravated again when licking resumes. diagnosis and differential diagnosis. acral lick granulomas must be differentiated from several other conditions, including bacterial or fungal infection, foreign bodies, and pressure sores. in addition, mast-cell tumors and other forms of neoplasia can mimic the appearance of acral lick granuloma. many of these problems can be ruled out by the history of the animal. when in doubt, a biopsy should be taken. an uncomplicated acral lick granuloma would feature hyperplasia, ulceration, and fibrosis without evidence of infection or neoplasia (walton, ) . prevention and control. behavior modification and relief of boredom are important aspects of preventing (and treating) acral lick granuloma. the environment of a dog with this problem can be enriched with exercise and the introduction of toys. in addition, the relief of boredom or anxiety can be attempted through the use of drugs such as phenobarbital, megestrol acetate, and progestins. these drugs may produce side effects, however (swaim and angarano, ) , and may interfere with experimental results. treatment. several treatments have been reported for acral lick granuloma, and none of them have been proven to be successful in ah cases. one of the most important aspects of treatment is to break the cycle of self-trauma. mechanical restraint with an elizabethan collar is one of the easiest methods to accomplish this goal. several direct treatments have been examined, including intralesional and topical steroids, perilesional cobra venom, acupuncture, radiation, and surgery (swaim and angarano, ; walton, ) . opioid antagonists have been used in an attempt to treat acral lick granuloma by blocking endogenous opioids. in one study, either naltrexone ( mg/kg sq) or nalmefene ( - mg/kg sq) successfully reduced the excessive licking behavior in of dogs; however, lesions returned after the drug was discontinued (dodman et al., ) . the use of a mixture of flunixin meglumine, steroid, and dimethyl sulfoxide ( ml of banamine [schering] mixed with ml of synotic [diamond laboratories]) applied topically twice daily has also been shown to be effective (walton, ) . the prognosis for acral lick granuloma should be considered guarded, because the lesions often recur or new lesions develop when treatment is discontinued. etiology. hygromas are fluid-filled sacs that develop as a result of repeated trauma over a bony prominence. the area over the olecranon is most frequently affected, but hygromas have been reported in association with the tuber calcis, greater trochanter, and stifle (newton et al., ) . epizootiology. elbow hygromas are most frequently reported in large and giant breeds of dogs around - months of age (johnston, ; bellah, ) . elbow hygromas are seen infrequently in the laboratory animal setting because the commonly affected breeds are seldom used in research. however, the housing environment for research dogs predisposes them to hygromas, because these animals spend a large amount of time on hard surfaces such as cage bottoms or cement runs. for this reason, laboratory animal veterinary and husbandry staff should be familiar with this condition. clinical signs. a dog with an elbow hygroma presents with a unilateral or bilateral, painless, fluctuant swelling over the point of the elbow. the animals are not usually lame. over a long period of time, elbow hygromas may become inflamed and ulcerated. if the hygroma is secondarily infected, the animal may exhibit pain and fever (johnston, ; bellah, ) . pathology. the fluid-filled cavity in the hygroma is lined by granulation and fibrous tissue. hygromas lack an epithelial lining and therefore are not true cysts. the fluid within the cavity is yellow or red and is a serous transudate. this fluid is less viscous than joint fluid, and elbow hygromas do not communicate with the joint (johnston, ) . treatment. the treatment of elbow hygromas should be conservative whenever possible, and surgical options should be reserved for complicated or refractory cases. conservative management of the elbow hygroma is aimed at relieving pressure at the point of the elbow by providing a padded cage surface and/or bandaging the elbow in a manner similar to that used to treat pressure sores (see section iii,c, ). more aggressive therapy, including needle drainage and the injection of corticosteroid into the hygroma, has been described but is not recommended, because infection is a serious complication of this treatment (johnston, ) . likewise, simple surgical excision of elbow hygromas can be associated with complications such as wound dehiscence and ulceration (johnston, ) . a technique that has been used successfully involves placement of multiple penrose drains. the drains are kept in place for - weeks, and the limb remains bandaged for weeks with this technique (bellah, ) . another technique has been described that involves the removal of a crescent-shaped piece of the skin and capsule. the remaining dead space is closed with mattress sutures over stents, and then the wound is closed in a routine fashion. the stents are removed in - days, and the wound is bandaged until suture removal in - days (newton et al., ) . regardless of the method used to treat an elbow hygroma, recurrence of the problem is likely unless the predisposing factors are identified and relieved. etiology. in the research environment, corneal ulcers are most often associated with either direct trauma, contact with irritating chemicals, or exposure to the drying effects of air during long periods of anesthesia. chronic or recurrent corneal ulcers may also be associated with infection or hereditary causes in some breeds of dogs; however, these cases would be rare in the laboratory setting. clinical signs. the signs of corneal ulceration are blepharospasm, epiphora, and photophobia. the eye may appear irritated and inflamed. in minor cases, the cornea may not appear abnormal; however, in cases of deeper ulceration, the cornea may appear roughened or may have an obvious defect. in addition, the periocular tissues may be swollen and inflamed because of self-inflicted trauma from rubbing at the eye. a tentative diagnosis of corneal ulcer or abrasion may be based on the clinical signs. a definitive diagnosis of corneal ulcers may be made by the green appearance of the cornea when stained with fluorescein dye. when a corneal ulcer has been diagnosed, the eye should be inspected for underlying causes such as foreign bodies or abnormal eyelids or cilia. treatment. the treatment of corneal ulcers will depend on the depth and size of the affected area. deep ulcers may require debridement and primary repair. in such cases, a third eyelid or conjunctival flap may be applied to the eye until experienced help can be obtained. superficial abrasions are generally treated with topical application of antibiotics. a triple antibiotic ointment that does not contain steroids given times a day for - days usually provides adequate treatment. ointments are preferred over drops, because use of the former requires less frequent. simple corneal ulcers are restained with fluorescein after days and should show complete healing at that time. if the ulcer is not healed, this may indicate that the ulcer has an undermined edge impeding proper healing. topical anesthetic should be applied to the eye, and a cotton-tipped applicator can be rolled over the surface of the ulcer toward its edge. this will remove the unattached edge of the cornea and healing should progress normally after debridement. in all cases, an elizabethan collar or other restraint may be necessary to prevent additional trauma to the eye. indwelling intravascular catheters, including intracaths and vascular access ports, often play a vital role in research protocols. the catheters are most often placed in a central vein or artery where they may be used for repeated blood sampling, administration of anesthetics and experimental compounds, or measurement of hemodynamic parameters. although catheters vary in composition, number of ports, and port placement, the basic principles of their implantation and maintenance are similar. it is important that the laboratory animal veterinarian be familiar with these principles and the potential complications of catheter use. when appropriately maintained, indwelling catheters may remain functional for months without serious complication. the actual incidence of complications associated with indwelling vascular catheters in dogs is unknown. this is due largely to the fact that many of the problems may be incidental findings or related to a particular research protocol. one study (hysell and abrams, ) examined the lesions found at necropsy in animals with chronic indwelling catheters (exact vascular locations not specified). the lesions found were categorized as traumatic cardiac lesions, visceral infarcts, and fatal hemorrhages. the traumatic cardiac lesions consisted primarily of masses of fibrin and inflammatory cells on the heart valves. the visceral infarcts were noted in the spleen, kidney (fig. ) , and brain and resulted from fibrin embolization from either the valvular lesions or the catheter tip. fatal hemorrhages were most often found in animals with experimentally induced hypertension. these animals developed clinical signs of sepsis and later ruptured a major vessel associated with mycotic infection and aneurysm. etiology. the leading complication associated with the use of indwelling vascular catheters is infection, either systemic or local at the point of entry through the skin. septicemia may develop from bacterial colonization of either the tract around the catheter or the catheter lumen. clinical signs. the signs and treatment of systemic infection are covered in section iii,d, . problems with the skin defect associated with the catheter port vary from mild skin irritation to obvious infection. the signs may include redness and swelling of the skin around the external port, discharge from the skin wound, or even abscess formation. prevention. because indwelling catheters play an important role in many research protocols, it is highly desirable to prevent catheter complications that may result in loss of the device. the catheter should be made of nonthrombogenic material. in addition, it is recommended that catheters be as simple as possible. a catheter with extra ports or multiple lumens requires addi- tional management and supplies more routes for infection. the use of vascular access ports that lie entirely under the skin eliminates many problems with infection. it has also been found that a long extension of tubing connected to the port may actually reduce the potential for infection of the catheter (ringler and peter, ) . the initial placement of an indwelling catheter must be done under aseptic conditions by individuals who are familiar with the procedure. the placement of the catheter should be verified by radiography. catheters that are used for delivery of drugs or blood sampling should be positioned in the vena cava and not in the right atrium, thereby minimizing trauma to the tricuspid valve. after catheter placement, the animals should be observed daily for signs of either local or systemic infection. the catheter entry site should be disinfected, coated with antibiotic ointment, and rebandaged every other day. once a month, the catheter line may be disinfected with chlorine dioxide, as described below (see "treatment"). throughout the life of the catheter, injections into and withdrawals from the catheter should be done in a sterile manner, and the number of breaks in the line should be kept to a minimum. treatment. the treatment of catheter infections almost invariably involves removal of the catheter, as demonstrated in both dogs and monkeys (ringler and peter, ; darif and rush, ) . superficial wound irritation or infection may be treated locally with antibiotic ointment, sterile dressing changes and efforts to minimize catheter movement; however, more extensive problems require aggressive therapy. systemic antibiotic therapy should be initiated for a -day period. the choice of drug will ultimately be based on previous experience and culture results. aerobic and anaerobic cultures of blood and locally infected sites should be performed (ringler and peter, ) . localized abscesses or sinus tracts may be managed by establishing drainage and flushing with chlorhexidine. again, the catheter should be removed. if retention of a catheter is important, the catheter lumen may be disinfected by filling with chlorine dioxide solution. it has been shown that there are no adverse effects from the use of chlorine dioxide in catheters (dennis et al., ) . the solution is removed after min and replaced with heparinized saline. all of the extension lines and fluids used in the catheter should be discarded. the blood cultures should be repeated days after the antibiotic therapy has ceased. if bacteria are still cultured, the catheter must be removed. intestinal access ports have been used to study the pharmacokinetics of drugs at various levels in the intestinal tract. these catheters are usually vascular access ports with several modifications to allow secure placement in bowel (meunier et al., ) . when placed and managed correctly, these ports may remain in place for months without complications. the most frequently reported complication associated with these catheters is infection around the port site (meunier et al., , kwei et al., . these infections lead to removal of the catheters despite treatment with local lavage and systemic antibiotics. there have also been reports of catheters dislodging from the intestinal tract and resulting in peritonitis. this complication has largely been eliminated with the improved security afforded by a synthetic cuff added to the end of the catheter (meunier et al., ) . the chapter authors have also seen migration of the catheter end within the lumen of the intestine (caused by peristaltic motion to egest the catheter), extensive intra-abdominal adhesions, and intestinal torsion (figs. a,b) as complications of intestinal access ports. the procedures for placement and maintenance of the catheters are similar to those outlined previously for indwelling vascular catheters. it is important that the catheters be firmly secured to the intestine to prevent migration or dislodgment. an omental patch placed over the site of entry may help form a firm adhesion. in addition, it is important to place the proper length of catheter within the peritoneal cavity; excess catheter length can promote adhesion formation, whereas insufficient catheter length to account for visceral organ motion can result in detachment. the placement and patency of the catheters can be verified periodically by contrast radiography using iodinated contrast material or by fecal occult blood testing after a small amount of blood has been injected through the catheter (meunier et al., ) . etiology. sepsis is defined as the systemic response to infection. most often, sepsis is a result of infection with gramnegative bacteria; however, sepsis may also be associated with gram-positive bacteria and fungi. in laboratory animals, sepsis is seen as a complication of surgical procedures or associated with chronic implants. sepsis may also be seen as a complication of infectious diseases such as parvovirus. clinical signs. the signs of sepsis can vary, depending on the source of the infection and the stage of the disease. early in the course of sepsis, dogs will present with signs of a hyperdynamic response, including an increased heart rate, increased respiratory rate, red mucous membranes, and a normal to increased capillary refill time. systemic blood pressure and cardiac output will be increased or within the normal range. the animals will often be febrile. later in the course of the syndrome, the animals will show the classic signs of septic shock, including decreased temperature, pale mucous membranes, and a prolonged capillary refill time. cardiac output and blood pressure are decreased as shock progresses. peripheral edema and mental confusion have also been reported (hauptman and chaudry, ) . pathogenesis. the pathophysiology of sepsis is complex and is mediated by immune responses involving mediators such ~ as cytokines, eicosinoids, complement, superoxide radicals, and nitric oxide. the body responds to overwhelming infection with an attempt to optimize metabolic processes and maximize oxygen delivery to tissues. however, if inflammation is left unchecked, the system may be unable to compensate, and the result is cardiovascular collapse. diagnosis. in general, a presumptive diagnosis of sepsis is made based on the occurrence of several in a group of signs, including altered body temperature, increased respiratory and/or heart rate, increased or decreased white blood cell count, increased number of immature neutrophils, decreased platelet count, decreased blood pressure, hypoxemia, and altered cardiac output. however, extreme inflammation without infection (e.g., pancreatitis, trauma) may create similar signs. one study examined the diagnosis of sepsis in canine patients at a veterinary hospital based on easily obtainable physical and laboratory findings. that study found that septic individuals had higher temperatures, wbc counts, and percentage of bands than nonseptic individuals, whereas platelet counts were lower in the septic dogs. there were no differences in respiratory rate or glucose levels between the groups. using these criteria, the results had a high sensitivity and a tendency to overdiagnose sepsis (hauptman et al., ) . ultimately, the presence of a septic focus simplifies diagnosis greatly; however, the focus may not be obvious. if the signs of sepsis are evident but the focus is not, several systems should be evaluated for infection, including urinary tract, reproductive tract, abdominal cavity, respiratory tract, teeth, and heart valves (kirby, ) . treatment. the treatment of sepsis has three aims. the first aim is to support the cardiovascular system. all septic animals should be treated with fluids to replace deficits and to maximize cardiac output. crystalloids are most frequently used to maintain vascular volume, primarily because of their low cost. colloids offer the advantage of maintaining volume without fluid overload and may have other positive effects on the cardiovascular system. acid-base and electrolyte imbalances should also be addressed. after the animal has stabilized, the treatment of sepsis should be aimed at removing the septic focus. obvious sources of infection should be drained or surgically removed. if an implant is associated with the source of infection, the implant should be removed. antibiotic therapy should also be instituted. the choice of antibiotic will ultimately depend upon the results of culture; however, the initial choice of antibiotics is based on previous experience, source of infection, and gram stains. the organisms associated with sepsis are often gram-negative bacteria of gastrointestinal origin or are previously encountered nosocomial infections. ideally, the antibiotic chosen for initial therapy should be a broad-spectrum, bactericidal drug that can be administered intravenously. second-or third-generation cephalosporins provide good coverage, as does combination therapy with enrofloxacin plus metronidazole or penicillin. finally, the treatment of sepsis is aimed at blocking the mediators of the systemic response. several studies have examined the effects of steroids, nonsteroidal anti-inflammatory drugs, and antibodies directed against endotoxin, cytokines, or other mediators of the inflammatory response; however, none of these treatments have proven greatly effective in clinical trials. consequently, there is no "magic bullet" for the treatment of sepsis at this time. successful therapy remains dependent on aggressive supportive care coupled with identification and elimination of the inciting infection. etiology. in research animals, aspiration into the lungs may occur accidentally during the oral administration of various substances or by the misplacement of gastric tubes. aspiration of gastric contents may also occur as a complication of anesthesia. in pet animals, aspiration is often seen as a result of metabolic and anatomical abnormalities; however, such occurrence would be rare in the research setting. clinical signs. the signs of aspiration lung injury may include cough, increased respiratory rate, pronounced respiratory effort, and fever. when respiration is severely affected, the oxygen saturation of blood will be decreased. the diagnosis of this problem is based on a history consistent with aspiration and the physical findings. classically, radiographs of the thorax demonstrate a bronchoalveolar pattern in the cranioventral lung fields. however, these lesions may not appear for several hours after the incident of aspiration. in addition, the location of the lesions may be variable, depending on the orientation of the animal at the time of aspiration. pathogenesis. aspiration of gastric contents or other compounds can create lung injury of variable severity, depending upon the ph, osmolality, and volume of the substance. the compounds aspirated can produce direct injury to lung tissue, but more importantly, the aspiration provokes an inflammatory response probably mediated by cytokines. the result is a rapid influx of neutrophils into the lung parenchyma and alveolar spaces. the inflammation leads to increased vascular permeability with leakage of fluid into the alveolar spaces and can eventually lead to alveolar collapse. if the condition is severe, it may result in adult respiratory distress syndrome and respiratory failure. it should be noted that infection is not present in the early stages of this condition but may complicate the problem after - hr. treatment. the treatment of aspiration lung injury is largely supportive and depends upon the severity of the inflammation and the clinical signs. in cases in which a small amount of a relatively innocuous substance (e.g., barium) has been aspirated, treatment may not be necessary. when severe inflammation is present, systemic fluid therapy should be instituted. support of the cardiovascular system should be performed judiciously; fluid overload could lead to an increase in pulmonary edema. the use of colloids is controversial because of the increase in vascular permeability that occurs in the lungs. oxygen therapy is also controversial, because it may increase lung injury if administered at high concentrations for long periods of time (nader-djahal et al., ) . several studies have addressed the use of anti-inflammatory agents to reduce lung injury associated with aspiration; however, none are used clinically in human or veterinary medicine at this time. in humans, antibiotics are reserved for use in cases with confirmed infection, in order to prevent the development of antibiotic-resistant pneumonia. it has been suggested that dogs should be treated with antibiotics immediately when the aspirated material is either not acidic or has potentially been contaminated by oral bacteria associated with severe dental disease. amoxicillin-clavulanate has been recommended as a first line of defense, reserving enrofloxacin for resistant cases (hawkins, ) . the presence of pneumonia should be verified by tracheal wash and cultures. etiology. in laboratory animals, accidental burns usually result from thermal injury (heating pads, water bottles) or harsh chemicals (strong alkalis, acids, disinfectants). the insult to the skin results in desiccation of the tissue and coagulation of proteins. in addition, the severely injured area is surrounded by a zone of vascular stasis, which promotes additional tissue damage. even small burns can result in significant inflammation that could affect the outcome of some research investigations and cause considerable discomfort to the animal. the proper and immediate treatment of burn wounds can reduce the effects of the injury on both the individual and the research. clinical signs. the clinical signs vary with the type and degree of burn injury. initially, the injury may not be noticed. the first signs may be oozing from the skin and matting of the overlying hair. within a couple of days, progressive hair and skin loss may be observed (johnston, ) . the wounds may vary in severity from very superficial (involving only the epidermis) to those in which the epidermis and dermis are completely destroyed. superficial wounds appear as red, inflamed skin similar to sunburn in humans. the pain associated with these injuries usually subsides in - days, and the wound reepithelializes without complications in - days. deeper burns develop a thick covering, or eschar, composed of the coagulated proteins and desiccated tissue fluid. the wound heals by granulation under the eschar, which eventually sloughs or is removed to allow further healing by contraction and reepithelialization. within - days of injury, the burn wound will be colonized by grampositive bacteria that rapidly cover the entire wound. several days later, gram-negative organisms can appear in the burn wound (johnston, ) . at this point, signs of wound infection and sepsis may occur (see section iii,d, ). treatment. appropriate and timely treatment of a burn wound will reduce the extent of the injury. thermal injuries should be immediately cooled to reduce edema and pain (demling and lalonde, ) . chemical burns should be thoroughly lavaged for min after wounding. the damaged tissues may be unable to mount appropriate responses to changes in temperature; therefore, the lavage should be performed with warm water to prevent hypothermia. after the initial treatment, all burn wounds should be gently cleansed - times a day (demling and lalonde, ) . burns involving the epidermis and part of the dermis can be extremely painful, and analgesia should be addressed throughout the treatment period. systemic antibiotics are unable to penetrate eschar and are not adequately distributed through the abnormal blood supply of burned tissues. therefore, topical wound dressings are recommended in the early stages of treatment. a thin film of a water-soluble broad-spectrum antibiotic ointment should be applied to the wound surface after each cleaning. silver sulfadiazine has a broad spectrum, penetrates eschar well, and is often the preparation of choice for burn wound therapy. povidone-iodine ointment will also penetrate thin eschar and provides a broad spectrum. mafenide has a good spectrum that covers gram-negative organisms well and is often used to treat infected wounds, although it is associated with pain upon application (demling and lalonde, ) . when signs of wound or systemic infection are present, systemic antibiotics should be employed, and their ultimate selection should be based on culture and sensitivity results. after the topical antibiotic has been applied, a nonadherent dressing should be placed on the wound. burn wounds covered in such a manner tend to epithelialize more rapidly and are less painful than uncovered wounds. when the eschar over a burn wound has formed and become fully defined, a small or moderately sized wound may be completely resected. prevention. obviously, prevention of burn wounds is preferable to a long course of treatment. care should be taken to prevent direct exposure to harsh chemicals. tables, floors, and other surfaces should be rinsed thoroughly after chemical use, prior to allowing any animal contact. electric heating pads should be avoided, and only heated water blankets or circulating warm-air devices should be used to provide warmth to the animals. in rare instances, heated water blankets have also caused burns; therefore these devices should be carefully monitored. as a precaution, a thin towel may be placed between the animal and the water blanket. etiology. research and/or anesthetic protocols may require the intravenous injection of various solutions. when these substances have a ph or osmolarity significantly different from that of the surrounding tissues, the accidental perivascular extravasation of the solutions may result in tissue damage. several drugs have been shown to cause problems when injected perivascularly, including pentobarbital, thiamylal, thiopental, thiacetarsemide, vincristine, vinblastine, and doxorubicin (swaim and angarano, ; waldron and trevor, ) . clinical signs. the immediate signs of perivascular injection are swelling at the injection site and withdrawal of the limb or other signs of discomfort. later, the area may appear red, swollen, and painful as inflammation progresses. often there will be eventual necrosis of the skin around the injection site. in cases of doxorubicin extravasation, signs may develop up to a week after the injection, and the affected area may progressively enlarge over a to month period. this is because the drug is released over time from the dying cells (swaim and angarano, ) . prevention. because the degree of injury and extensive treatment associated with perivascular extravasation of a drug can be detrimental to research protocols and can cause severe discomfort to the dog, prevention of these injuries is preferred. prior to the use of any substance, the investigator should be aware of its chemical composition and the potential for problems. if a potentially caustic compound is to be used in a fractious subject, sedation of the dog is warranted if this will not interfere with the research protocol. whenever possible, insertion of an indwelling catheter is extremely important. access to a central vessel such as the cranial or caudal vena cava is preferred over the use of peripheral vessels. when peripheral catheters are used, the injection should be followed by a vigorous amount of flushing with saline or other physiological solution and removal of the catheter. additional injections are best given through newly placed catheters in previously unused vessels. the repeated use of an indwelling peripheral catheter should be approached cautiously and done only out of necessity. prior to use, the catheter should be checked repeatedly for patency by withdrawal of blood and injection of saline. any swelling at the catheter site or discomfort by the subject indicates that the catheter should not be used. treatment. the treatment of perivascular injections will depend on the amount and type of substance injected. in most cases, dilution of the drug with subcutaneous injections of saline is recommended. in addition, steroids may be infiltrated locally to reduce inflammation. topical application of dimethyl sulfoxide (dmso) may also be helpful in reducing the immediate inflammation and avoiding the development of chronic lesions (swaim and angarano, ). the addition of lidocaine to subcutaneous injections of saline has been used in cases of thiacetarsemide injection (hoskins, ) , and local infiltration of hyaluronidase accompanied by warm compresses has been suggested for use in cases of vinblastine injection (waldron and trevor, ) . despite these treatments, necrosis of skin may be observed and would require serial debridement of tissues with secondary wound closure or skin grafting. in cases of doxorubicin extravasation, early excision of affected tissues is advocated to prevent the progressive sloughing caused by sustained release of the drug from dying tissues (swaim and angarano, ) . in all cases, the condition can be painful, and analgesia should be addressed. etiology. hepatic encephalopathy is the result of the derangements in metabolism associated with abnormal liver function. this condition may be seen in young dogs with congenital portosystemic shunting of blood flow. however, in the research setting, encephalopathy occurs more often in canine models of hepatic disease that lead to liver failure. a well-developed knowledge of the pathophysiology of liver disease is necessary for the initial treatment and long-term management of hepatic encephalopathy. pathogenesis. when the liver function is severely impaired because of either portosystemic shunting of blood flow or loss of metabolically active hepatic tissue, the result is an accumulation of ammonia, toxic amines, aromatic amino acids, and short-chain fatty acids (hardy, ; center, ) . these compounds have several toxic effects that result in a decrease in cerebral energy metabolism and a decrease in excitatory neurotransmitter synthesis. concurrently, there is an increase in the concentration of false neurotransmitters and the inhibitory substance -aminobutyric acid (gaba). clinical signs. the signs of hepatic encephalopathy include lethargy, depression, muscle tremors, and convulsions. diagnosis and differential diagnosis. a presumptive diagnosis of hepatic encephalopathy may be based on the appearance of clinical signs following experimental manipulation of the liver. additional diagnostic tests to verify the loss of liver function can be performed to confirm the diagnosis. serum glucose and protein levels may be low if hepatic function is severely impaired. a low serum urea nitrogen level suggests that the normal hepatic metabolism of ammonia into urea has been impaired. elevated levels of serum bile acids and blood ammonia also verify the loss of liver function (hardy, ) . measurement of serum hepatic leakage enzymes are nondiagnostic, because they can be low, high, or normal. treatment. because of the severity of hepatic encephalopathy, treatment may be initiated based on a presumptive diagnosis. during initial treatment, supportive care with fluids and electrolytes should be instituted, based on the results of serum chemistry and blood gas analysis. the majority of animals with hepatic dysfunction will be hypokalemic, alkalotic, and hypernatremic; therefore, either . % sodium chloride or . % sodium chloride with . % dextrose, supplemented with potassium chloride, is recommended (hardy, ) . the type of drug to be used for seizure control is controversial. the short halflife of diazepam makes it an attractive choice compared with barbiturates, which have prolonged metabolism when hepatic function is impaired (maddison, ) . however, endogenous benzodiazepines mediate some of the cns signs seen with hepatic encephalopathy. therefore, the use of diazepam has been discouraged in favor of phenobarbital (johnson, ) . the drug selected for seizure control should be titrated carefully, given the altered liver metabolism. most importantly, the treatment of dogs with hepatic encephalopathy must be aimed at reducing the levels of toxic metabolites in the bloodstream. because protein metabolism is a major source of ammonia, all oral food intake should cease until the signs of hepatic encephalopathy have abated. because gastrointestinal bleeding may occur in individuals with liver failure and this is also a source of protein, the use of h blockers such as cimetidine or ranitidine is suggested (swalec, ) . in addition, lactulose retention enemas should be performed ( - ml/lb of a % solution in water, retained for - min) (hardy, ) . lactulose is an indigestible semisynthetic sugar that is metabolized in the gut to lactic and other acids. the decrease in colonic ph reduces ammonia levels in the bloodstream by converting intestinal ammonia into less diffusible ammonium ions. lactulose will also cause an osmotic diarrhea. antibiotics such as neomycin ( mg/lb, - times/ day) or metronidazole ( mg/lb, times/day) should also be used to reduce the intestinal load of urease-producing bacteria responsible for splitting urea into ammonia (hardy, ) . when the signs of hepatic encephalopathy have resolved, the dog may be fed a low-protein diet. diets suitable for dogs with renal insufficiency are recommended initially. this type of diet is not suitable for long-term use, however, because it appears that individuals with some types of hepatic disease actually have increased protein requirements. these requirements may be met by slowly increasing protein in the diet as long as signs of hepatic encephalopathy do not recur. to maintain the appropriate balance of aromatic and branched-chain amino acids, the diet should be based on vegetable and dairy protein instead of meat or fish protein (center, ) . in addition, the antibiotics suggested above should be continued to reduce the effects of increasing dietary protein levels. the prevalence of cancer in the general canine population has increased over the years (dorn, ) . this can be attributed to the longer life spans resulting from improvements in nutrition, disease control, and therapeutic medicine. because of these changes, cancer has become a major cause of death in dogs (bronson, ) . in a lifetime cancer mortality study of intact beagles of both sexes, albert et al. ( ) found death rates similar to the death rate of the at-large dog population (bronson, ) . approximately % of the male beagles died of cancer. the majority of the tumors were lymphomas ( %) and sarcomas ( %), including hemangiosarcomas of the skin and fibrosarcomas. of the female beagles dying of cancer ( % of the population studied), three-quarters had either mammary cancer ( %), lymphomas ( %), or sarcomas ( %). of the sarcomas in females, one-third were mast cell tumors. in addition to these tumors that cause mortality, the beagle is also at risk for thyroid neoplasia (hayes and fraumeni, ; benjamin et al., ) . because of the popularity of the beagle as a laboratory animal, discussion of specific neoplasms will focus on the tumors for which this breed is at risk, as well as tumors that are common in the general canine population. fine-needle aspirates are generally the first diagnostic option for palpable masses, because they can easily be performed in awake, cooperative patients. this technique allows for rapid differentiation of benign and neoplastic processes. in cases where cytologic results from fine-needle aspirates are not definitive, more invasive techniques must be used. needle-punch or core biopsies can also be performed in awake patients but typically require local anesthesia. an instrument such as a tru-cut needle (travenol laboratories, inc., deerfield, illinois) is used to obtain a mm x to . cm biopsy of a solid mass. a definitive diagnosis may be limited by the size of the sample acquired using this technique. incisional and excisional biopsies are utilized when less invasive techniques fail to yield diagnostic results. excisional biopsies are the treatment of choice when surgery is necessary, because the entire mass is removed. surgical margins should extend at least cm around the tumor, and cm if mast cell tumors are suspected (morrison et al., ) . incisional biopsies are performed when large soft-tissue tumors are encountered and/or when complete excision would be surgically difficult or life-threatening. when performing an incisional biopsy, always select tissue from the margin of the lesion and include normal tissue in the submission. etiology. lymphomas are a diverse group of neoplasms that originate from lymphoreticular cells. whereas retroviral etiologies have been demonstrated in a number of species (e.g., cat, mouse, chicken), conclusive evidence of a viral etiology has not been established in the dog. in humans, data implicate the herbicide , -dichlorophenoxyacetic acid ( , -d) as a cause of non-hodgkin's lymphoma, but studies in dogs with similar conclusions have come under scrutiny (macewen and young, ) . clinical signs. multicentric and alimentary lymphomas account for most cases of canine lymphoma. in multicentric lymphoma, animals usually present with enlarged lymph nodes and nonspecific signs such as anorexia, weight loss, polyuria, polydypsia, and lethargy. when the liver and spleen are involved, generalized organomegaly may be felt on abdominal palpation. alimentary lymphoma is associated with vomiting and diarrhea, in addition to previous clinical signs. less commonly, dogs develop mediastinal, cutaneous, and extranodal lymphomas. dogs with mediastinal lymphoma often present with respiratory signs secondary to pleural effusion. hypercalcemia is most frequently associated with this form of lymphoma and may result in weakness. cutaneous lymphoma varies in presentation from solitary to generalized and may mimic any of a number of other skin disorders. the tumors may occur as nodules, plaques, ulcers, or dermatitis. approximately half of the cases are pruritic. a number of extranodal forms of lymphoma have been reported, including tumors affecting the eyes, central nervous system, kidneys, or nasal cavity. clinical presentation varies, depending on the site of involvement. epizootiology. the incidence of lymphoma is highest in dogs - years old, accounting for % of cases. although the neoplasm generally affects dogs older than year, cases in puppies as young as months have been reported (dorn et al., ) . pathologic findings. enlarged neoplastic lymph nodes vary in diameter from to cm and are moderately firm. some may have areas of central necrosis and are soft to partially liquefied. the demarcation between cortex and medulla is generally lost, and on cut section, the surface is homogenous. the spleen may have multiple small nodular masses or diffuse involvement with generalized enlargement. the enlarged liver may have disseminated pale foci or multiple large, pale nodules. in the gastrointestinal tract, both nodular and diffuse growths are observed. these masses may invade through the stomach and intestinal walls. histologically, the most common lymphomas are classified as intermediate to high grade and of large-cell (histiocytic) origin. the neoplastic lymphocytes typically obliterate the normal architecture of the lymph nodes and may involve the capsule and perinodal areas. pathogenesis. all lymphomas regardless of location should be considered malignant. a system for staging lymphoma has been established by the world health organization. the average survival time for dogs without treatment is - weeks. survival of animals undergoing chemotherapy is dependent on the treatment regimen as well as the form and stage of lymphoma (macewen and young, ) . hypercalcemia is a paraneoplastic syndrome frequently associated with lymphoma. the pathogenesis of this phenomenon is not fully understood but may be a result of a parathormone-like substance produced by the neoplastic lymphocytes. diagnosis and differential diagnosis. differential diagnoses for multicentric lymphoma include systemic mycosis; salmonpoisoning and other rickettsial infections; lymph node hyperplasia from viral, bacterial, and/or immunologic causes; and dermatopathic lymphadenopathy. alimentary lymphoma must be distinguished from other gastrointestinal tumors, foreign bodies, and lymphocytic-plasmacytic enteritis. in order to make a definitive diagnosis, whole lymph node biopsies and full-thickness intestinal sections are frequently needed. treatment. therapy for lymphoma typically consists of one or a combination of several chemotherapeutic agents. the treatment regimen is based on the staging of the disease, the presence of paraneoplastic syndromes, and the overall condition of the patient. macewen and young ( ) provide a thorough discussion of therapeutic options for the treatment of lymphomas in the dog. research complications. given the grave prognosis for lymphoma with or without treatment, euthanasia should be considered for research animals with signifcant clinical illness. etiology. the fibrosarcoma group of tumors encompasses not only malignant tumors of fibroblasts but also a number of indistinguishable tumors, all of which are capable of collagen production (pulley and stannard, ) . frequently classified in this group are undifferentiated leiomyosarcomas, liposarcomas, malignant melanomas, and malignant schwannomas. clinical signs. although these neoplasms can arise throughout the body, they are most commonly found in the skin, subcutaneous tissues, and oral cavity. fibrosarcomas are extremely variable in size and can grow to be quite large. in general, they are irregular and nodular, poorly demarcated, and nonencapsulated, and they frequently invade deeper tissues. epizootiology. most fibrosarcomas develop in adult and aged animals but can affect dogs as young as months or less. pathogenesis. fibrosarcomas exhibit rapid, invasive growth, recurring frequently after excision. metastasis occurs in only one-fourth of cases, usually by the bloodstream to the lungs. less frequently, spread to local lymph nodes is observed. diagnosis and differential diagnosis. differential diagnoses for fibrosarcomas vary with the location of the tumor. histopathologic exam should be used to distinguish these tumors from round cell tumors (mast cell tumors, histiocytomas, transmissible venereal tumors), papillomas, and other neoplasms. treatment. treatment of any soft-tissue sarcoma would begin with wide surgical excision. if the tissue margins indicate incomplete resection, radiotherapy could be used. for any highgrade tumors, adjuvant chemotherapy would be recommended (see macewen and withrow, a , for a complete discussion). research complications. because fibrosarcomas are locally invasive and often recur, dogs with these neoplasms should not be considered good subjects for long-term studies. etiology. neoplasms of lipocytes and lipoblasts are welldifferentiated tumors referred to as lipomas. clinical signs. these growths can be found as single or multiple round, ovoid, or discoid masses in the subcutaneous tissues of the lateral and ventral thorax, abdomen, and upper limbs. generally they are well circumscribed, encapsulated, and soft on palpation. further, the skin is freely movable over the tumor. epizootiology. lipomas occur principally in aged animals (average years), and the incidence increases with age (pulley and stannard, ) . the tumors are most commonly seen in overweight female dogs, but no breed predisposition is observed. pathologic findings. histologically, lipomas are indistinguishable from normal adipose tissue except when a fibrous capsule is present. pathogenesis: lipomas are typically slow-growing and do not recur after complete surgical excision. diagnosis and differential diagnosis. lipomas are not frequently confused with other tumors but can sometimes be difficult to distinguish from normal adipose tissue. generally, the distinction can be made from the clinical history. treatment. treatment for lipomas is not usually necessary unless the mass is causing problems with normal ambulation. in such cases, surgical excision is usually curative. research complications. lipomas usually do not complicate research studies unless they are interfering with other systemic functions or ambulation. etiology. histiocytomas are benign skin growths that arise from the monocyte-macrophage cells in the skin. some debate exists as to whether this growth is actually a neoplasm or a focal inflammatory lesion (pulley and stannard, ) . clinical signs. the most frequent sites for histiocytomas are the head (especially the pinna) and the skin of the distal forelegs and feet. the masses are usually domelike or buttonlike (often referred to as "button tumors") and usually measure - cm in diameter. epizootiology. histiocytomas are the most common tumors of young dogs, mostly occurring in dogs less than years of age. pathologic findings. histologically, these tumors contain round to ovoid cells with pale cytoplasm and large nuclei. the cells infiltrate the dermis and subcutis, displacing collagen fibers and skin adnexa. despite being benign lesions, histiocytomas characteristically have a high mitotic index. pathogenesis. this tumor typically exhibits rapid growth ( - weeks) but does not spread. most histiocytomas will spontaneously regress in less than months. diagnosis and differential diagnosis. histiocytomas must be distinguished from potentially metastatic mast cell tumors. this is accomplished by staining with toluidine blue, which would stain the cytoplasmic granules of mast cells red or purple. treatment. although most histiocytomas will spontaneously resolve, conservative surgery or cryosurgery will provide an expeditious resolution. research complications. histiocytomas should not interfere with most studies. etiology. neoplastic proliferations of mast cells are the most commonly observed skin tumor of the dog (bostock, ) . mast cells are normally found in the connective tissue beneath serous surfaces and mucous membranes, and within the skin. clinical signs. well-differentiated mast cell tumors are typically solitary, well-circumscribed, slow-growing, to cm nodules in the skin. alopecia may be observed, but ulceration is not usual. poorly differentiated tumors grow rapidly, may ulcerate, and may cause irritation, inflammation, and edema. mast cell tumors can be found on any portion of the dog's skin but frequently affect the hindquarters, especially the thigh and in-guinal and scrotal areas. mast cell tumors usually appear to be discrete masses, but they frequently extend deep into surrounding tissues. epizootiology. these tumors tend to affect middle-aged dogs but have been observed in dogs ranging from months to years (pulley and stannard, ). pathologic findings. because of the substantial variation in histologic appearance of mast cell tumors, a classification and grading system described by patnaik et al. ( ) has become widely accepted. in this system, grade i has the best prognosis, and grade iii the worst prognosis. grade i tumors are well differentiated, with round to ovoid uniform cells. the nuclei are regular, the cytoplasm is packed with large granules that stain deeply, and mitotic figures are rare to absent. grade ii (intermediately differentiated) mast cell tumors have indistinct cytoplasmic boundaries with higher nuclear-cytoplasmic ratios, fewer granules, and occasional mitotic figures. grade iii (anaplastic or undifferentiated) mast cell tumors have large, irregular nuclei with multiple prominent nucleoli. the cytoplasmic granules are few, but mitotic figures are much more frequent. in addition to skin lesions, mast cell tumors have been associated with gastric ulcers. these lesions are most likely secondary to tumor production of histamine. histamine stimulates the h receptors of the gastric parietal cells, causing increased acid secretion. gastric ulcers have been observed in large numbers (> %) of dogs with mast cell tumors (howard et al., ) . the ulcers can be found in the fundus, pylorus, and/or proximal duodenum. although all mast cell tumors should be considered potentially malignant, the outcome in individual cases can be correlated with the histologic grading of the tumor. grade iii tumors are most likely to disseminate internally. this spread is usually to regional lymph nodes, spleen, and liver and less frequently to the kidneys, lungs, and heart. diagnosis and differential diagnosis. mast cell tumors can be distinguished histologically from other round cell tumors (such as histiocytomas and cutaneous lymphomas) by using toluidine blue, which metachromatically stains the cytoplasmic granules of the mast cells red or purple. treatment. initial treatment for mast cell tumors is generally wide surgical excision ( to cm margins). even with wide surgical margins, approximately % of mast cell tumors may recur. if the site is not amenable to wide surgical excision, debulking surgery and radiation therapy may be used. other alternatives include amputation (if on a limb) or radiation therapy alone. as an adjunct to surgery, grier et al. ( grier et al. ( , found that deionized water injected into surgical margins reduced tumor recurrence by hypo-osmotically lysing any mast cells left behind. this technique has recently been refuted by jaffe et al. ( ) . for systemic mastocytosis, and nonresectable or incompletely excised mast cell tumors, chemotherapy can be used. treatment options would include oral prednisolone, intralesional triamcinalone, and the combination of cyclophosphamide, vincristine, and prednisolone (graham and o'keefe, ) . research complications. because of the possibility of systemic histamine release and tumor recurrence, dogs with mast cell tumors are not good candidates for research studies. grade i mast cell tumors may be excised, allowing dogs to continue on study; however, monitoring for local recurrence should be performed on a regular basis (monthly). grade ii tumors are variable; animals that undergo treatment should be monitored for recurrence monthly, and evaluation of the buffy coat should be performed every - months for detection of systemic mastocytosis. because of the poor prognosis for grade iii tumors, treatment is unwarranted in the research setting. etiology. hemangiosarcomas are malignant tumors that originate from endothelial cells. clinical signs. these tumors may arise in the subcutis but are more commonly found in the spleen and the right atrium. clinical signs are associated with the site of involvement. vascular collapse is frequently observed secondary to rupture and hemorrhage from splenic masses. heart failure can be observed secondary to tumor burden or hemopericardium. when found in the skin, hemangiosarcomas are poorly circumscribed, reddish black masses that range in size from to cm in diameter. the most common cutaneous sites are the ventral abdomen, the prepuce, and the scrotum. epizootiology: hemangiosarcomas occur most frequently in -to -year-old dogs. the german shepherd dog is most commonly affected. pathologic findings. grossly, splenic hemangiosarcomas resemble nodular hyperplasia or hematomas (fig. ) . the masses are spherical and reddish black and can range in size up to - cm in diameter. on cut section the masses may appear reddish gray or black and have cavernous areas of clotted blood. when the masses are found in the heart, the endocardium may be covered by a thrombus, giving the to cm tumors a reddish gray or yellow appearance. histologically, hemangiosarcomas are composed of immature endothelial cells that form vascular channels or clefts. these spaces may be filled with blood or thrombi. the neoplastic cells are elongated with round to ovoid, hyperchromatic nuclei and frequent mitotic figures. pathogenesis. hemangiosarcomas can be found in one or many sites. in cases where multiple sites are involved, it may be impossible to identify the primary tumor. this neoplasia is highly malignant and spreads easily. metastasis occurs most frequently to the lungs but can be found in any tissue. diagnosis and differential diagnosis. splenic hemangiosarcoma may resemble nodular hyperplasia or some manifestations of lymphoma. when the heart is affected, other causes of heart failure must be ruled out. echocardiography is a valuable tool for identifying the primary lesion. histopathology should be used to differentiate dermal hemangiosarcoma from hemangiomas and other well-vascularized tumors. treatment. surgery is generally the first choice of treatment for hemangiosarcoma. dermal tumors are treated with radical resection, splenic tumors by total splenectomy, and heart tumors by debulking and pericardiectomy. because of the high likelihood of metastasis, adjunct chemotherapy should always be considered. research complications. dogs with dermal hemangiosarcoma may be cured after complete resection with margins, but monitoring should be done regularly for recurrence. the other forms of hemangiosarcoma have a much poorer long-term prognosis, and treatment is typically unwarranted in the research setting. etiology. also known as infectious or venereal granuloma, sticker tumor, transmissible sarcoma, and contagious venereal tumor, the transmissible venereal tumor is transmitted to the genitals by coitus (nielsen and kennedy, ) . the origin of this tumor is still unknown but has been described as a tumor of lymphocytes, histiocytes, and reticuloendothelial cells. although this tumor has been reported in most parts of the world, it is most prevalent in temperate climates (macewen, ) . clinical signs. the tumors are usually cauliflower-like masses on the external genitalia, but they can also be pedunculated, nodular, papillary, or multilobulated. these friable masses vary in size up to cm, and hemorrhage is frequently observed. in male dogs, the lesions are found on the caudal part of the penis from the crura to the bulbus glandis or on the glans penis (fig. ) . less frequently, the tumor is found on the prepuce. females typically have lesions in the posterior vagina at the junction of the vestibule and vagina. when located around the urethral orifice, the mass may protrude from the vulva. these tumors have also been reported in the oral cavity, skin, and eyes. epizootiology and transmission. transmissible venereal tumors are most commonly observed in young, sexually active dogs. transmission takes place during coitus when injury to the genitalia allows for transplantation of the tumor. genital to oral to genital transmission has also been documented (nielsen and kennedy, ) . extragenital lesions are believed to be a result of trauma prior to exposure to the tumor. pathogenesis. tumor growth is rapid after implantation but later slows. metastasis is rare (< % of cases) but may involve the superficial inguinal and external iliac lymph nodes as well as distant sites. diagnosis and differential diagnosis. transmissible venereal tumors have been confused with lymphomas, histiocytomas, mast cell tumors, and amelanotic melanomas. cytology may be of benefit in making a definitive diagnosis, so impression smears should be made prior to processing for histopathology. prevention. thorough physical examinations prior to bringing new animals into a breeding program should prevent introduction of this tumor into a colony. control. removing affected individuals from a breeding program should stop further spread through the colony. treatment. surgery and radiation can be used for treatment, but chemotherapy is the most effective. vincristine ( . - . mg/m ) iv once weekly for - treatments will induce remission and cure in greater than % of the cases (macewen, ). research complications. experimental implantation of transmissible venereal tumors has been shown to elicit formation of tumor-specific igg (cohen, ) . this response may occur in natural infections and could possibly interfere with immunologic studies. etiology. dogs are susceptible to a wide variety of mammary gland neoplasms, most of which are influenced by circulating reproductive steroidal hormones. clinical signs. single nodules are found in approximately % of the cases of canine mammary tumors. the nodules can be found in the glandular tissue or associated with the nipple. masses in the two most caudal glands (fourth and fifth) account for a majority of the tumors. benign tumors tend to be small, well circumscribed, and firm, whereas malignant tumors are larger and invasive and coalesce with adjacent tissues. epizootiology. mammary tumors are uncommon in dogs under years of age with the incidence rising sharply after that. median age at diagnosis is - years. mammary tumors occur almost exclusively in female dogs, with most reports in male dogs being associated with endocrine abnormalities, such as estrogen-secreting sertoli cell tumors. pathologic findings. based on histologic classification of mammary gland tumors, approximately half of the reported tumors are benign (fibroadenomas, simple adenomas, and benign mesenchymal tumors), and half are malignant (solid carcinomas, tubular adenocarcinomas, papillary adenocarcinomas, anaplastic carcinomas, sarcomas, and carcinosarcomas) (bostock, ) . extensive discussions of classification, staging, and histopathologic correlations can be found in macewen and withrow ( lb) and moulton ( ) . pathogenesis. mammary tumors of the dog develop under the influence of hormones. receptors for both estrogen and progesterone can be found in - % of tumors. futher, schneider et al. ( ) showed that the risk of developing mammary tumors increased greatly after the first and second estrus cycles. dogs spayed prior to the first estrus had a risk of . %, whereas dogs spayed after the first and second estrus had risks of % and %, respectively. malignant mammary tumors typically spread through the lymphatic vessels. metastasis from the first, second, and third mammary glands is to the ipsilateral axillary or anterior sternal lymph nodes. the fourth and fifth mammary glands drain to the superficial inguinal lymph nodes where metastasis can be found. many mammary carcinomas will eventually metastasize to the lungs. diagnosis and differential diagnosis. both benign and malignant mammary tumors must be distinguished from mammary hyperplasia and mastitis. prevention. mammary tumors can effectively be prevented by spaying bitches prior to the first estrus. this is commonly done in the general pet population at months of age. recently, the topic of spaying sexually immature dogs ( - weeks of age) has received much attention for the control of the pet population. kustritz ( ) reviewed the techniques for anesthesia and surgery, as well as possible pros and cons of spaying at this young age. treatment. surgery is the treatment of choice for mammary tumors, because chemotherapy and radiation therapy have not been reported to be effective. the extent of the surgery is dependent on the area involved. lumpectomy or nodulectomy should be elected in the case of small discrete masses, while mammectomy and regional or total mastectomies are reserved for more aggressive tumors. at the time of surgery, axillary lymph nodes are removed only if enlarged or positive on cytology for metastasis. inguinal lymph nodes should be removed any time the fourth and fifth glands are excised (macewen and withrow, lb). research complications. because % of mammary tumors are benign, treatment may be rewarding, allowing dogs to con-tinue on study. if removed early enough, malignant masses could yield the same results. all dogs should be monitored regularly for recurrence and new mammary tumors. etiology. beagles are among the breeds with the highest prevalence of thyroid carcinomas. benjamin et al. ( ) reported a correlation between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in the beagle. clinical signs. thyroid carcinomas generally present as palpable cervical masses. affected animals may experience dysphagia, dyspnea, and vocalization changes. precaval syndrome resulting in facial edema is also observed in some cases. epizootiology and transmission. the mean age of dogs presented with thyroid carcinomas is years, with equal distribution of cases between the sexes. pathologic findings. grossly, thyroid carcinomas are multinodular masses, frequently with large areas of hemorrhage and necrosis. they tend to be poorly encapsulated and invade local structures such as the trachea, esophagus, larynx, nerves, and vessels. the masses are unilateral twice as often as bilateral (capen, ) . histologically, thyroid carcinomasare divided into follicular, papillary, and compact cellular (solid) types (see capen, , for complete discussion). pathogenesis. thyroid carcinomas tend to grow rapidly and invade local structures. early metastasis is common and occurs to the lungs by invasion of branches of the thyroid vein. diagnosis and differential diagnosis. nonpainful cervical swellings such as seen with thyroid tumors are also consistent with abscesses, granulomas, salivary mucoceles, and lymphomas. often a preliminary diagnosis can be made by fineneedle aspirate. treatment. surgery is the treatment of choice for thyroid carcinomas that have not metastasized. when the tumor is freely movable, surgery may be curative. surgical excision may be difficult for tumors that adhere to local structures, requiring excision of the jugular vein, carotid artery, and associated nerves. when bilateral tumors are observed, preservation of the parathyroid glands may not be possible. in such cases, treatment for hypoparathyroidism will be necessary. both chemotherapy and radiation therapy have been suggested for extensive bilateral tumors and/or after incomplete excision, but no controlled trials have been performed (ogilvie, ) . in the research setting, treatment of this tumor may not be rewarding. only freely movable tumors can be practically treated without seriously affecting research efforts. euthanasia is warranted in the more advanced cases when clinical illness is apparent. beagles are subject to many of the inherited and/or congenital disorders that affect dogs in general. in a reference table on the congenital defects of dogs (hoskins, b) , disorders for which beagles are specifically mentioned include brachyury (short tail), spina bifida, pulmonic stenosis, cleft palate-cleft lip complex, deafness, cataracts, glaucoma, microphthalmos, optic nerve hypoplasia, retinal dysplasia, tapetal hypoplasia, factor vii deficiency, pyruvate kinase deficiency, pancreatic hypoplasia, epilepsy, gm gangliosidosis, globoid cell leukodystrophy, xx sex reversal, and cutaneous asthenia (ehlers-danlos syndrome). in addition, there are defects that affect so many breeds that the author simply lists "many breeds" for the breeds affected by those disorders. thus these defects could also affect beagles and include pectus excavatum, polydactyly, radial and ulnar dysplasia, hypoadrenocorticism, entropion, lens coloboma, factor viii deficiency (von willebrand's disease), renal agenesis or ectopia, and developmental defects of the reproductive and lower urinary tracts. at a commercial breeder of purpose-bred beagles, the most common birth defects were umbilical hernia ( . % of births) and open fontanelle ( . % of births) (r. scipioni and j. ball, personal communication, ) . other defects observed include cleft palate and cleft lip, cryptorchidism, monorchidism, limb deformity, inguinal hernia, diaphragmatic hernia, hydrocephaly, and fetal anasarca. each of these other congenital defects occurred at less than . % incidence. etiology. cataract is an opacification of the lens or the lens capsule. it is the pathologic response of the lens to illness or injury, because the lens has no blood supply. cataracts can be caused by metabolic, inflammatory, infectious, or toxic causes and can be congenital, juvenile, or degenerative. nuclear sclerosis is an apparent opacification of the lens caused by the compression of older lens fibers in the center of the lens (nucleus) as a consequence of the production of new fibers. because the nucleus increases in size as the animal ages, the sclerosis is more apparent in older animals and may be mistaken as a senile cataract. the ability to see the fundus during ophthalmoscopy persists with nuclear sclerosis but is obstructed by a true cataract. clinical signs. the first clinical sign is typically the ability to visualize the opaque lens through the pupil of the dog's eye. dogs have an impressive ability to tolerate bilateral lens opacity (especially when development is gradual), and often visual impairment is detected late in the development of the condition (helper, ) . moderate vision loss may cause the dog to be hesitant in moving in new surroundings or unable to locate movable objects (such as a toy). rapid cataract development can result in a sudden vision loss, such as can occur with diabetic cataracts. epizootiology and transmission. certain dog breeds can be predisposed to the development of juvenile or senile cataracts or to metabolic disorders that result in cataract development, such as diabetes mellitus. dogs in studies for diabetes mellitus should be observed regularly for cataract development. toxicological studies may also induce formation of cataracts. pathogenesis. lens fibers respond to all biological or chemical insults by necrosis and liquefaction (render and carlton, ) , because they have no blood supply with which to recruit an inflammatory and repair process. disruption of these fibers by any means, therefore, leads to opacification. the exact processes by which the varieties of congenital and juvenile cataracts are produced have not been determined. in diabetic cataracts, the excess glucose is metabolized to sorbitol and fructose. as these alcohols and sugars accumulate in the lenticular cells, they produce an osmotic imbalance, which brings fluid into the cells, causing swelling and degeneration of lens fibers and resultant opacity (capen, ) . diagnosis and differential diagnosis. the ability to visualize the retina and fundus during ophthalmoscopy differentiates true cataracts from nuclear sclerosis. dogs with cataracts should be evaluated for possible causes, especially diabetes mellitus. diabetes mellitus will typically affect middle-aged dogs and feature rapid cataract formation, whereas juvenile and senile cataracts are slow to develop and affect younger and older dogs, respectively. progressive retinal atrophy can also cause secondary cataract formation; pupillary light response is maintained with primary cataracts (even if the lens is completely opaque), whereas this reflex is obtunded by retinopathy. prevention. most forms of cataracts cannot be prevented, for their exact etiologic pathogenesis is unknown. diabetic cataracts, however, can be prevented by proper regulation of blood glucose concentrations with insulin therapy and proper diet. treatment. because dogs do not need to focus visual images as accurately as human beings, proper lens clarity and function are not necessary for an adequate quality of life. many dogs adjust quite well to the visual impairment caused by persistent cataracts. lens removal can be performed for dogs seriously affected by cataracts, but this would not be anticipated for dogs in the research setting. information on surgical lens extraction procedures can be found in helper ( ) or other veterinary ophthalmology textbooks. research complications. research complications would be minimal with cataracts, unless the dogs were intended for use in ophthalmologic or visual acuity-based studies. etiology. hip dysplasia is a degenerative disease of the coxofemoral joint. a specific etiology is unknown, but the development of hip dysplasia has a strong genetic component (pedersen et al., ) , modified by age, weight, size, gender, conformation, rate of growth, muscle mass, and nutrition (smith et al., ) . clinical signs. the initial clinical abnormality caused by hip dysplasia is laxity of the coxofemoral joint. this may present as a gait abnormality without any indication of lameness or stiffness. eventually, affected dogs will have periods of lameness and, in protracted cases, will be rendered immobile by severe pain. epizootiology and transmission. hip dysplasia has been seen in most dog breeds, but it typically affects larger breeds of dogs. in the research setting, it is primarily a condition of randomsource large-breed dogs used for surgical research. diagnosis and differential diagnosis. hip dysplasia is classically diagnosed by radiography of the pelvis and hip joints. radiographic abnormalities consistent with hip dysplasia include shallow acetabula with remodeling of the acetabular rim, flattening of the femoral head, subchondral bone sclerosis (caused by erosion of articular cartilage and exposure of underlying bone), and osteophyte production around the joint (pedersen et al., ) . hip dysplasia needs to be differentiated from other musculoskeletal or neurological conditions that can cause unusual gaits and/or lameness. this may be somewhat difficult, because clinical signs of hip dysplasia may develop before radiographic abnormalities. radiographic calculation of the distraction index (di) to measure joint laxity has proven to be a good means to predict future hip dysplasia before other radiographic changes are evident (smith et al., ) . prevention. because of the genetic component, dogs with hip dysplasia should not be used in breeding colonies. dogs should be provided a good plane of nutrition but not be allowed to become overweight. dogs that were limit-fed at % of the food amount eaten by ad libitum-fed dogs had lower body weights and decreased severity of radiographic lesions of hip dysplasia (kealy et al, ) . treatment. in the stages when clinical signs are episodic, cage rest and analgesics for several days can be used to treat the symptoms. more advanced cases may require continuous analgesia. sectioning of the pectineus muscle or tendon may provide some pain relief but does not affect the progression of the disease (pedersen et al., ) . surgical treatments for hip dysplasia include femoral head ostectomy and total hip replacement. neither surgical treatment is likely in a research setting. research complications. long-term studies using large-breed dogs may be affected by the eventual development of hip dysplasia. in studies where hip dysplasia would be a serious complication or confounding variable (e.g., orthopedic research), dogs should be radiographed upon arrival to assess possibility of early coxofemoral joint degeneration and suitability for use in the study. etiology. benign prostatic hyperplasia (bph) is an agerelated condition in intact male dogs. the hyperplasia of prostatic glandular tissue is a response to the presence of both testosterone and estrogen. clinical signs. bph is often subclinical. straining to defecate (tenesmus) may be seen because the enlarged gland impinges on the rectum as it passes through the pelvic canal. urethral discharge (yellow to red) and hematuria can also be presenting clinical signs for bph. epizootiology and transmission. bph typically affects older dogs (> years), although it has been seen as early as years of age. pathologic findings. in its early stages, canine b ph is hyperplasia of the prostatic glandular tissue. this is in contrast to human bph, which is primarily stromal in origin. eventually, the hyperplasia tends to be cystic, with the cysts containing a clear to yellow fluid. the prostate becomes more vascular (resulting in hematuria or hemorrhagic urethral discharge), and bph may be accompanied by mild chronic inflammation. pathogenesis. bph occurs in older intact male dogs because increased production of estrogens (estrone and estradiol), combined with decreased secretion of androgens, sensitizes prostatic androgen receptors to dihydrotestosterone. the presence of estrogens may also increase the number of androgen receptors, and hyperplastic prostate glands also have an increased ability to metabolize testosterone to a-dihydrotestosterone (kustritz and klausner, ) . diagnosis and differential diagnosis. bph is diagnosed in cases of nonpainful symmetrical swelling of the prostate gland in intact male dogs, with normal hematologic profiles and urinalysis characterized by hemorrhage, at most. differential diagnoses include squamous metaplasia of the prostate, paraprostatic cysts, bacterial prostatitis, prostatic abscessation, and prostatic neoplasia (primarily adenocarcinoma). these differential diagnoses also increase in frequency with age and, except for squamous metaplasia, can also occur in castrated dogs. as such, these conditions do not necessarily abate or resolve when castration is used for treatment of prostatic enlargement. prevention. castration is the primary means for prevention of benign prostatic hyperplasia. treatment. the first and foremost treatment for b ph is castration. in pure cases of b ph, castration results in involution of the prostate gland detectable by rectal palpation within - days. for most dogs in research studies this is a viable option to rapidly improve the animal's condition. the alternative to castration is hormonal therapy, primarily with estrogens. this may be applicable in cases in which the dog is a valuable breeding male (e.g., genetic diseases), and semen collection is necessary. if the research study concerns steroidal hormone functions, then neither the condition nor the treatment is compatible. newer drugs marketed for human males have also shown promise in treating canine bph. finasteride (proscar) is a a-reductase inhibitor that limits metabolism of testosterone to a-dihydrotestosterone. treatment at daily doses of - mg/kg has been effective in causing prostatic atrophy without affecting testicular spermatogenesis (kustritz and klausner, ) . dogs given . mg/kg were proven to still be fertile. there are also indications that lower doses may be effective in relieving b ph. androgen receptor antagonists (flutamide and hydroxyflutamide) have also been studied in the dog and found to be effective for treatment of bph while maintaining libido and fertility (kustritz and klausner, ) . unfortunately, both the areductase inhibitors and the androgen receptor antagonists are not presently labeled for use in male dogs in the united states. research complications. bph can cause complications to steroidal hormone studies, in that the condition may be indicative of abnormal steroidal hormone metabolism, and neither castration nor estrogen therapy is compatible with study continuation. it is presently unknown whether the use of the newer antihyperplastic agents systemically alters physiologic parameters outside of the prostate itself. the development of tenesmus as a clinical sign may also affect studies of colorectal or anal function. etiology. juvenile polyarteritis syndrome (jps) is a painful disorder seen in young beagles (occasionally reported in other breeds). the lesion consistent with the syndrome is systemic necrotizing vasculitis. the cause of the vasculitis has not been established, but it appears to have an autoimmune-mediated component and may have a hereditary predisposition. clinical signs. clinical signs of jps include fever, anorexia, lethargy, and reluctance to move the head and neck. the dogs tend to extend the neck ventrally. most dogs seem to be in pain when touched, especially in the neck region. the syndrome typically has a course of remissions and relapses characterized by - days of illness and - weeks of remission (scott-moncrieff et al., ) . there may be a component of this condition that is subclinical, given that a vasculitis has been diagnosed postmortem in beagles that had no presenting signs. epizootiology and transmission. jps typically affects young beagles ( - months), with no sex predilection. pathologic findings. on gross necropsy, foci of hemorrhage can be seen in the coronary grooves of the heart, cranial mediastinum, and cervical spinal cord meninges (snyder et al., ) . local lymph nodes may be enlarged and hemorrhagic. histologically, necrotizing vasculitis and perivasculitis of small to medium-sized arteries are seen. these lesions are most noticeable in the three locations where gross lesions are observed, but they may be seen in other visceral locations. the perivasculitis often results in nodules of inflammatory cells that eccentrically surround the arteries (fig. a) . the cellular composition of these nodules is predominantly neutrophils, but it can also consist of lymphocytes, plasma cells, or macrophages (snyder et al., ) . fibrinous thrombosis of the affected arteries is also seen (fig. b) . a subclinical vasculitis has also been diagnosed in beagles postmortem; it is not known whether this subclinical condition is a different disorder or part of a jps continuum. this subclinical vasculitis often affects the coronary arteries (with or without other sites). pathogenesis. the initiating factors for jps are unknown. it was once presumed to be a reaction to test compounds by laboratory beagles, but this may have been coincident to the fact that the beagle is the breed most often affected with jps. immune mediation of jps is strongly suspected, because the clinical signs have a cyclical nature and respond to treatment with corticosteroids, and the affected dogs have elevated a -globulin fractions and abnormal immunologic responses. there may be hereditary predisposition, given that pedigree analysis of some affected dogs has indicated that the offspring of certain sires are more likely to be affected, and breeding of two affected dogs resuited in / affected pups (scott-moncrieff et al., ) . diagnosis and differential diagnosis. differential diagnoses include encephalitis, meningitis, injury or degeneration of the cervical vertebrae or disks, and arthritis. in the research facility, the disorder may be readily confused with complications secondary to the experimental procedure, or with postsurgical pain. beagles with jps that were in an orthopedic research study were evaluated for postsurgical complications and skeletal abnormalities prior to the postmortem diagnosis of systemic vasculitis (authors' personal experience). are known at this time. no prevention and control measures brane (third eyelid). this is not considered a congenital anomaly, but there is breed disposition for this condition, including beagles. a specific etiology is not known. clinical signs. the glandular tissue of the nictitating membrane protrudes beyond the membrane's edge and appears as a reddish mass in the ventromedial aspect of the orbit (fig. ) . excessive tearing to mucoid discharge can result, and severe cases can be associated with corneal erosion. treatment. clinical signs can be abated by administration of corticosteroids. prednisone administered orally at . mg/kg, q hr, was associated with rapid relief of clinical symptoms. maintenance of treatment at an alternate-day regimen of . - . mg/kg was shown to relieve symptoms for several months. however, withdrawal of corticosteroid therapy led to the return of clinical illness within weeks. pathologic findings. typically the glandular tissue is hyperplastic, possibly with inflammation. rarely is the tissue neoplastic. pathogenesis. prolapse of the gland may be a result of a congenital weakness of the connective tissue band between the gland and the cartilage of the third eyelid (helper, ) . research complications. because of the potentially severe clinical signs and the need for immunosuppressive treatment, jps is often incompatible with use of the animal as a research subject. it is unknown whether subclinical necrotizing vasculitis causes sufficient aberrations to measurably alter immunologic responses. etiology. "cherry eye" is a commonly used slang term for hyperplasia and/or prolapse of the gland of the nictitating mem-prevention. hyperplasia of the third eyelid cannot be prevented, but dogs that develop this condition unilaterally should have the other eye evaluated for potential glandular prolapse. preventative surgical measures might be warranted. treatment. corticosteroid treatment (topical or systemic) can be used to try to reduce the glandular swelling. however, surgical reduction or excision of the affected gland is typically required to resolve the condition. in the reduction procedure, the prolapsed gland is sutured to fibrous tissue deep to the fornix of the conjunctiva (helper, ). if reduction is not possible (as with deformed nictitating cartilage) or is unsuccessful, removal of the gland can be performed. such excision is fairly straightforward and can be done without removal of the nictitating membrane itself. the gland of the third eyelid is important in tear production; although the rest of the lacrimal glands should be sufficient for adequate tear production, keratoconjunctivitis sicca is a possible consequence after removal of the gland of the nictitating membrane. research complications. in most cases, research complications would be minimal, especially if treated adequately. either the presence of the hyperplastic gland, or its removal, might compromise ophthalmologic studies. etiology. interdigital cysts are chronic inflammatory lesions (not true cysts) that develop in the webbing between the toes (fig. ). the cause for most interdigital cysts is usually not identified unless a foreign body is present. bacteria may be isolated from the site, but the lesions may also be sterile (hence the synonym "sterile pyogranuloma complex"). clinical signs. dogs with interdigital cysts are usually lame on the affected foot, with licking and chewing at the interdigital space. exudation may be noticed at the site of the lesion. the lesion appears as a cutaneous ulcer, usually beneath matted hair, with possible development of sinus tracts and purulent exudate. epizootiology and transmission. interdigital cysts are common in a variety of canine breeds, including german shepherds. beagles have been affected in the research setting. interdigital cysts usually occur in the third and fourth interdigital spaces (bellah, ) . pathologic findings. histopathologically, interdigital cysts are sites of chronic inflammation, typically described as pyogranulomatous. pathogenesis. initial development of the cysts is unknown, except for those cases in which a foreign body can be identified. diagnosis and differential diagnosis. bacterial culture swabs and radiographs should be taken of the cysts to rule out bacterial infection, and radiopaque foreign bodies or bony lesions, respectively. a biopsy should be taken if neoplasia is suspected. treatment. if a foreign body is associated with the lesion, then removal is the first order of treatment. if biopsy of the site provides a diagnosis of sterile pyogranuloma complex, then systemic corticosteroid therapy (e.g., prednisolone at mg/kg ql h) can be initiated and then tapered once the lesion heals. interdigital cysts that are refractory to medical therapy require fig. . interdigital cyst between the third and fourth digits of the forelimb of a research beagle. surgical removal. excision includes removal of the lesion and the interdigital web, and a two-layer closure of the adjacent skin and soft tissues is recommended (bellah, ) . the foot should be put in a padded bandage and a tape hobble placed around the toes to reduce tension when the foot is weightbearing. the prognosis for idiopathic interdigital cysts is guarded, because the cysts tend to recur (bellah, ) . research complications from the cysts are minimal, unless the dogs need to be weight-bearing for biomechanic or orthopedic studies. treatment with systemic steroids could be contraindicated with some experimental designs. post-therapy antibody titers in dogs with ehrlichiosis: follow-up study on patients treated primarily with tetracycline and/or doxycycline dog thyroiditis: occurrence and similarity to hashimoto's struma surgical management of specific skin disorders bordetella and mycoplasma infections in dogs and cats associations between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in beagles diagnostic exercise: peracute death in a research dog saunders manual of small animal practice neurologic manifestations associated with hypothyroidism in four dogs neoplasms of the skin and subcutaneous tissues in dogs and cats neoplasia of the skin and mammary glands of dogs and cats platelet function, antithrombin-iii activity, and fibrinogen concentration in heartworm-infected and heartworm-negative dogs treated with thiacetarsamide pancreatic adenocarcinoma in two grey collie dogs with cyclic hematopoiesis ehrlichia platys infection in dogs the rickettsioses monoclonal gammopathy associated with naturally occurring canine ehrlichiosis variation in age at death of dogs of different sexes and breeds leptospira interrogans serovar grippotyphosa infection in dogs efficacy and dose titration study of mibolerone for treatment of pseudopregnancy in the bitch tropical canine pancytopenia: clinical, hematologic, and serologic response of dogs to ehrlichia canis infection, tetracycline therapy, and challenge inoculation comparison of campylobacter carriage rates in diarrheic and healthy pet animals. zentralbl advances in dietary management of obesity in dogs and cats effect of level and source of dietary fiber on food intake in the dog effect of amount and type of dietary fiber on food intake in energy-restricted dogs external parasites: identification and control tumors of the endocrine glands thomson's special veterinary pathology infectious diseases of the dog and cat nutritional support for dogs and cats with hepatobiliary disease specific amplification of ehrlichia platys dna from blood specimens by two step pcr detection of humoral antibody to the transmissible venereal tumor of the dog dirofilaria immitis: heartworm products contract rat trachea in vitro dogs: laboratory animal management management of septicemia in rhesus monkeys with chronic indwelling catheters client information series: canine demodicosis client information series: fleas and flea allergy dermatitis management of the burn wound chlorine dioxide sterilization of implanted right atrial catheters in rabbits current concepts in the management of helicobacter associated gastritis dirofilariasis in dogs and cats use of narcotic antagonists to modify stereotypic self-licking, self-chewing, and scratching behavior in dogs epidemiology of canine and feline tumors epizootiologic characteristics of canine and feline leukemia and lymphoma study of obesity in dogs visiting veterinary practices in the united kingdom miller's anatomy of the dog update on diagnosis of canine hypothyroidism helicobacter-associated gastric disease in ferrets, dogs, and cats the role of helicobacter species in newly recognized gastrointestinal tract disease of animals serologic diagnosis of infectious cyclic thrombocytopenia in dogs using an indirect fluorescent antibody test hemorrhagic streptococcal pneumonia in newly procured research dogs control of ticks platelet aggregation studies in dogs with acute ehrlichia platys infection health benefits of animal research: the dog as a research subject soft tissue sarcomas and mast cell tumors textbook of veterinary internal medicine infectious diseases of the dog and cat canine lyme borreliosis mast cell tumor destruction by deionized water mast cell tumour destruction in dogs by hypotonic solution transmission of ehrlichia canis to dogs by ticks (rhipicephalus sanguineus) textbook of veterinary internal medicine diseases of the liver and their treatment cyclic thrombocytopenia induced by a rickettsia-like agent in dogs shock evaluation of the sensitivity and specificity of diagnostic criteria for sepsis in dogs textbook of veterinary internal medicine canine thyroid neoplasms: epidemiologic features magrane's canine ophthalmology helicobacter-like organisms: histopathological examination of gastric biopsies from dogs and cats thiacetarsamide and its adverse effects infectious diseases of the dog and cat pediatrics: puppies and kittens canine viral diseases textbook of veterinary internal medicine antibodies to ehrlichia canis, ehrlichia platys, and spotted fever group rickettsia in louisiana dogs mastocytoma and gastroduodenal ulceration complications in the use of indwelling vascular catheters in laboratory animals deionised water as an adjunct to surgery for the treatment of canine cutaneous mast cell tumours helicobacter infection textbook of veterinary internal medicine textbook of veterinary internal medicine hygroma of the elbow in dogs thermal injuries dirofilaria immitis: do filarial cyclooxygenase products depress endothelium-dependent relaxation in the in vitro rat aorta? depression of endotheliumdependent relaxation by filarial parasite products three cases of canine leptospirosis in quebec cvt update: interpretation of endocrine diagnostic test results for adrenal and thyroid disease etiopathogenesis of canine hypothyroidism five-year longitudinal study on limited food consumption and development of osteoarthritis in coxofemoral joints of dogs role of bordetella bronchiseptica in infectious tracheobronchitis in dogs kirk's current veterinary therapy : small animal practice the fire of life kirk's current veterinary therapy : small animal practice coinfection with multiple tick-borne pathogens in a walker hound kennel in north carolina tarsal joint contracture in dogs with golden retriever muscular dystrophy clinical and hematological findings in canine ehrlichiosis early spay-neuter in the dog and cat textbook of veterinary internal medicine chronic catheterization of the intestines and portal vein for absorption experimentation in beagle dogs evaluation of weight loss protocols for dogs the brown dog tick rhipicephalus sanguineus and the dog as experimental hosts of ehrlicha canis the clinical chemistry of laboratory animals double-blinded crossover study with marine-oil supplementation containing high-dose eicosapentaenoic acid for the treatment of canine pruritic skin disease thomson's special veterinary pathology effects of four preparations of . % chlorhexidine diacetate on wound healing in dogs transmissible venereal tumors kirk's current veterinary therapy : small animal practice soft tissue sarcomas tumors of the mammary gland canine lymphoma and lymphoid leukemias kirk's current veterinary therapy : small animal practice effect of heartworm infection on in vitro contractile responses of canine pulmonary artery and vein tick paralysis in north america and australia saunders manual of small animal practice thyroid gland and arterial lesions of beagles with familial hypothyroidism and hypedipoproteinemia bacterial gastroenteritis in dogs and cats: more common than you think urea protects helicobacter (campylobacter) pylori from the bactericidal effect of acid characterization of a new isolate of ehrlichia platys using electron microscopy and polymerase chain reaction decreased pulmonary arterial endothelium-dependent relaxation in heartworm-infected dogs with pulmonary hypertension vaccination against canine bordetellosis: protection from contact challenge dermatologic aspects of tick bites and tick-transmitted diseases a chronic access port model for direct delivery of drugs into the intestine of conscious dogs clinical trial of dvm derm caps in the treatment of allergic diseases in dogs: a nonblinded study diagnosis of neoplasia tumors of the mammary gland dirofilaria immitis: heartworm infection alters pulmonary artery endothelial cell behavior survey of conjunctival flora in dogs with clinical signs of external eye disease hyperoxia exacerbates microvascular injury following acid aspiration nutrient requirements of dogs surgical closure of elbow hygroma in the dog tumors of the genital system practical laboratory methods for the diagnosis of dermatologic diseases walker's mammals of the world tumors of the endocrine system beta hemolytic streptococcus isolated from the canine vagina comparison of three skin preparation techniques in the dog comparison of three skin preparation techniques in the dog. part : clinical trial in dogs clinical behavioral medicine for small animals hypothyroidism in dogs: cases ( - ) plasma von willebrand factor antigen concentration in dogs with hypothyroidism plasma von willebrand factor antigen concentration and bleeding time in dogs with experimental hypothyroidism canine cutaneous mast cell tumor: morphologic grading and survival time in dogs joint diseases of dogs and cats target imbalance: disparity of borrelia burgdorferi genetic material in synovial fluid from lyme arthritis patients textbook of veterinary internal medicine tumors of the skin and soft tissue thomson's special veterinary pathology canine leptospirosis: a retrospective study of cases dogs and cats as laboratory animals effects of chlorhexidine diacetate and povidoneiodine on wound healing in dogs epidemiology of thyroid diseases of dogs and cats factors influencing canine mammary cancer development and postsurgical survival muller and kirk's small animal dermatology systemic necrotizing vasculitis in nine young beagles thomson's special veterinary pathology textbook of veterinary internal medicine canine infectious tracheobronchitis (kennel cough complex) saunders manual of small animal practice serum protein alterations in canine erhlichiosis bacterial factors and immune pathogenesis in helicobacter pylori evaluation of rhipicephalus sanguineus as a potential biologic vector of ehrlichia platys role of the eastern chipmunk (tamias striatus) in the epizootiology of lyme borreliosis in northwestern illinois evaluation of risk factors for degenerative joint disease associated with hip dysplasia in dogs pathologic features of naturally occurring juvenile polyarteritis in beagle dogs textbook of veterinary internal medicine clinical manifestations, pathogenesis, and effect of antibiotic treatment on lyme borreliosis in dogs streptococcus zooepidemicus as the cause of septicemia in racing greyhounds trauma to the skin and subcutaneous tissues of dogs and cats chronic problem wounds of dog limbs portosystemic shunts textbook of veterinary internal medicine lumbosacral stenosis in dogs experimental respiratory disease in dogs due to bordetella bronchiseptica dirofilaria immitis: depression of endothelium-dependent relaxation of canine femoral artery seen in vivo does not persist in vitro thyroiditis in a group of laboratory dogs: a study of beagles of agriculture, animal and plant health inspection service thomson's special veterinary pathology a retrospective study of cases of naturally occurring canine ehrlichiosis role of canine parainfluenza virus and bortedella bronchiseptica in kennel cough management of superficial skin wounds serum concentrations of thyroxine and , , '-triiodothyronine before and after intravenous or intramuscular thyrotropin administration in dogs use of ultrasound in the measurement of subcutaneous fat and prediction of total body fat in dogs ehrlichia platys in a michigan dog ehrlichial diseases of dogs canine ehrlichiosis. miss albert, r. e., benjamin, s. a., and shukla, r. ( ). life span and cancer mortality in the beagle dog and human. key: cord- -ea nbkh authors: mitzner, veera title: conclusion and further thoughts date: - - journal: european union research policy doi: . / - - - - _ sha: doc_id: cord_uid: ea nbkh the conclusion chapter not only summarizes the main results of the research conducted for this book but also connects the events and discussions between the s and s to later political developments. it shows striking ideational and institutional continuity and reveals a substantial character of european integration: by relying on powerful political framings and discourses, as well as on sturdy institutions, the european community/union was able to move into areas that were not sanctioned by the treaties. the chapter further argues that to stay relevant, the eu research policy must be capable of breaking with the past and dramatically expand its mission to embrace the social and environmental challenges of the twenty-first century. in fact, with the existential threat of climate change and other global challenges, and the urgent need for socio-technological transformation at scale, opportunities and imperatives for european level activity in research might be greater than ever. separation of europe from the technologically more advanced united states, the european political and economic elites appeared open to arguments for unifying european resources in science and innovation. with the postwar market liberalization, rapid technological change, and the gradual shift of production to the newly industrialized countries, the case for joint european research effort seemed plausible. maria nedeva and linda wedlin have divided the development of european union research policy in two main phases: "science in europe" and "european science." according to these two scholars, the first phase, "science in europe," which roughly covers the time period analyzed in this book, was characterized by the principle of subsidiarity-an idea that european-level action would only be appropriate if action at a national level was insufficient; focus on increasing collaboration amongst researchers from different european countries; and concerns of the technology gap, which nedeva and wedlin call the "european paradox," as europe as a world leader of science seemed to lag behind in the industrial and economic exploitation of scientific ideas. furthermore, "science in europe" witnessed three distinct waves: the establishment of large, transnational facilities, such as european organization for nuclear research (cern), euratom, european molecular biology organization (embo), and european molecular biology laboratory (embl); the creation of "diffuse" organizations, such as european cooperation in science and technology (cost) and the european science foundation (esf), including all fields of research and providing platforms for cross-national cooperation rather than supporting science at european level; and finally the creation of the ec/eu framework program for research, bringing the scattered research programs under one administrative umbrella. for nedeva and wedlin, "science in europe" was a period "during which a partial and fragmented science and research system was developed." it complemented rather than challenged national science, research, and innovation arrangement; it had a limited impact on universities, research institutes, and national funding agencies and landscapes; and it "didn't in a rule, include explicitly european-level research performing organizations." this book doesn't challenge nedeva's and wedlin's analysis. for a long time, the ec/eu research policy had a partial character-the first initiatives were not designed to directly challenge activity in national settings. the proponents of european level activities in the field, while often ambitious and visionary, were aware of the political realities. even the "european paradox" argument is solid, although the debate had its ebbs and flows and occurred in different variations depending on the context and advocates. fears of european technological retard did push initiatives forward, at different degrees of success, giving them rationale and a sense of urgency. also, european level research facilities were still few and far in between, and decisions in brussels barely influenced the daily operations of universities and research institutes in the ec member states. however, with a closer historical analysis, we also see an emerging policy that was constantly challenged, contested, and vetted by a number of groups, and in particular those who would mostly have been affected by it. and this is the vital "more to the story" that nedeva's and wedlin's and others' accounts do not fully tell. this book also unveils the bigger ambitions of policy-makers and dreamers by fletching out the protracted struggles for initiatives that either never quite made it to the fruition or were realized as truncated versions, to the disappointment of many. it sheds light to the already forgotten and fluid policy spaces where the feasible and unfeasible were defined, and where future pathways were sometimes painfully determined. crucially, these spaces were shaped by broader political, social, and economic circumstances within and beyond europe, and the prevalence and strength of specific ideational shifts and conditions, such as the fading of the naïve enthusiasm about science that had characterized the immediate postwar period; increasing uneasiness about growth policies and calls for better accountability of economic and scientific activity; new limitations imposed by economic austerity; persisting distrust in european level bureaucracy among academics and national policy-makers; and ongoing political battles in other policy areas within the ec context. yet one distinct thread runs through all these events and debates: a basic agreement on the beneficial role of research and research policy in achieving greater growth and competitiveness, and the need to create joint political mechanisms to that end. how this would eventually be achieved remained open for contestation. besides being a story of continuity and struggle, this book also is an account of the vulnerability of a political idea that survived, grew big, and eventually transformed european research. it was just as strong as its proponents, which at the beginning were not many. although the commission made sure that science policy experts would be involved in the elaboration of the various schemes from the start, these experts often represented governments and national science funding bodies rather than the broader science community. the lack of active participation of scientists and their advocates, as well as of industry and the wider public in the early discussion on the topic, is striking; until the s, most proposals were developed in technocratic milieus with little public attention. the backing of pro-european associations such as union des industries de la communauté européenne (unice), the european parliament, and some individuals from national research institutions and administrations remained too weak and fragmented to put significant pressure on key decision-makers. this is the tragedy of the early ec/eu research policy. politicians in the ec member states, whose main concern was often winning the support of the electorate, tended to focus on issues with more immediate political benefit. it was not the case that the citizens in the community member countries had opposed the idea of the europeanization of scientific and technological activities: in the community's surveys of public opinion, research has consistently been at the top of the list of policies that people think should not be managed exclusively at a national level. research was, nevertheless, not the kind of "high politics" that would be surrounded by glamour and drama and that would have reached the headlines. it remained distant to the lives of ordinary citizens who barely knew of the commissions initiatives. it is also exactly this technical, abstract, complex, and distant nature of this policy domain that at least in part explains the persisting gap between rhetoric and reality in european research policy from the mid- s to the mid- s. for national politicians, expressing catchy phrases and stirring rhetoric about science and its many promises for the future, could be politically advantageous but also relatively safe: sacrifices in this sector rarely had a direct impact on the lives of voters, and therefore, they seldom provoked significant public protests. the late s' protests at the ispra euratom joint research centre stand out as a notable exception, as multiple employees were threatened to lose their jobs as a result of decisions made in brussels. but in most cases, lip service in research policy came at a low political price. in the absence of a systematic backing of scientists or their representatives, the commission alone was too weak to induce the national governments to truly commit to developing a community research policy. moreover, commission's strategy was not always consistent: important tensions prevailed between the dgs, while each commissioner in charge of research pursued a slightly different policy. the existence of dg research, under various names since , however, became a central structure for guaranteeing institutional and ideational continuity. the dg constituted a center where bureaucrats and experts, sharing some important ideas and interests, could further elaborate schemes for new community activity. this finding supports katia seidel's research on dgs iv (agriculture) and vi (competition), which shows that the administrative cultures developed in those dgs and the subsequent socialization of officials into these cultures, "helped to bring about policies in relevant areas, and then to keep them on track." through the discussions initiated by dg research and others, the ec gradually formed an arena for "europeanization" in research where socialization of different experts and political actors could take place. intra-european connections were reinforced, and similarities increased, while "europe" was given new contents and contours. at the same time, it would be misleading to argue that all contacts within the realm of the research policy debate would automatically have led to greater cohesion. these exchanges also exposed to governments and experts the diverse character of european research systems and made them aware of the difficulty in creating a strongly integrated common policy. yet they prepared the ground for subsequent initiatives that would take off with a more favorable political momentum. in the early s, the various plans and scattered activities of the previous twenty years finally evolved into something that could be said to have an important scope and political and economic significance. the first step into this new direction was the launch of sizeable technological programs, the pioneer being european strategic program on research in information technology (esprit), an initiative that was intended to develop a european strategic scheme based on collaboration between major european companies, small-and medium-sized firms, universities, and research institutes. the project was successful: of the proposals received in response to the first call, less than percent could be funded. not surprisingly, after , esprit became the model for successive community programs, such as research in advanced communications in europe (race) and basic research in industrial technologies (brite). the expansion of these activities led to the creation of the first framework program for research ( ) ( ) ( ) ( ) in . the € . billion budget of the framework program, together with the budget for esprit of € million, signified a threefold increase in the community's research spending in . moreover, the program sparked off a steady and fast increase of the community's research budgets. peter tindemans has observed that after esprit, there were a growing number of scientists and industrialists involved in the consultations, projects, and evaluations, and thereby directly benefiting from the community's activity in the field. this created "an almost unstoppable dynamic of pressure [that] arose in national capitals and in the directorates-general responsible for the fp's [framework programs] to increase the budget." although the role of the ec as a focal point of common european research activities remained contested, as was demonstrated by the creation of eureka, an intergovernmental initiative intended to promote "near-market research," in , and by the difficult negotiations of the subsequent framework programs, the political context had changed. national governments were now more willing to see the ec take a central role in the field, and a horizontal and vertical growth of brussels-led activities ensued. all this was supported by the favorable conjuncture in european integration that ensued the single market program. the first framework programs were still focused on advancing applied, marketoriented research, but during the s, these programs became more comprehensive and versatile as they included new mechanisms and stretched into different disciplines and sectors. they also opened up for countries outside the ec/eu framework, developed collaboration with other european and international organizations, and finally became more closely integrated with the ec/eu core policies. an eu-centric european research space started to emerge. this is, indeed, where wedlin and nedeva see the shift to "european science," marked by a changing understanding of the "european added value." whereas the previous ec/eu activities had been primarily focused on fostering collaboration between researchers, now there was a widely shared desire to increase the level of integration in different aspects of european science and research. new tools were designed to strengthen the european knowledge base and to support basic research through competitive funding. a key concept here is the european research area (era). originally dreamed up by commissioner ralf dahrendorf in the s, the idea was revived by commissioner alberto ruberti in the s, and finally pushed to the center of the european political agenda by commissioner philippe busquin. endorsed as an ambitious effort to pool european scientific and technological resources, the initiative marked a clear break with the distributive approach that had characterized the eu activities in the preceding years. while the definition of what the era stands for has evolved over the years, it has brought new inclusiveness into the eu research policy and signaled a move toward a more europeanized research policy field, "where the era agenda provides justification for the adoption of new institutions and funding tools." era aimed to increase the commission's autonomy in initiating projects and programs with direct implications for national activities. in , the commission defined era as "a unified research area open to the world based on the internal market, in which researchers, scientific knowledge and technology circulate freely and through which the union and its member states strengthen their scientific and technological bases, their competitiveness and their capacity to collectively address grand challenges." era was launched in the political framework of the lisbon european council of march , where the eu set itself the new strategic goal of becoming "the most competitive and dynamic knowledge-based economy in the world capable of sustainable economic growth, with more and better jobs and greater social cohesion." in lisbon, research and development were drawn to the center of the eu's strategy for achieving its goal by . essentially, scientific research, technological development, and innovation were defined as key factors in growth, competitiveness, and employment in a knowledge-based european economy. this trend was continued in the eu's new growth strategy, europe , launched in . one of the seven flagship initiatives announced in the strategy was innovation union, aiming to "improve conditions and access to finance and innovation, to ensure that innovative ideas can be turned into products and services that create growth and jobs." era was considered a core element of the innovation union and the europe strategy. the eu's barcelona target to achieve r&d funding of percent of gdp-an objective largely motivated by the higher r&d investment levels in the united states and japan-gave era additional political support and visibility. according to wedlin and nedeva, "european science" has created "an organizational space for the support of research that is aligned, and in competition with national-level research spaces." a key invention in this space was the european research council (erc), created in as a part of the seventh framework program. by allowing european researchers to compete with all other researchers in the eu area on the basis of excellence, erc revolutionized the eu's approach to supporting scientific activity. with scientific excellence as the sole criterion for selection and sizeable pots of money to distribute (the first budget in the seventh framework program ( - ) was . billion euros, and it almost doubled for horizon , constituting percent of the overall program budget), erc soon became a powerful tool for boosting european knowledge space and shaping science in europe. the shifting agendas and the broadening scope of the ec/eu activities have been paralleled by the gradual strengthening of the community's legal status. after the still very restricted formulation of the single european act ( ), the treaty of maastricht ( ) included a new article , which required that "the community and the member states shall coordinate their research and technological development activities so as to ensure that national policies and community policy are mutually consistent." moreover, the commission was granted the explicit right to take any "useful initiative" to promote such consistency. the amsterdam treaty ( ) then abandoned the requirement for council unanimity for adoption of the co-decision on the framework program. from now on, the program could be adopted by a qualified majority vote. the union's competences in the domain were further widened by the article of lisbon treaty ( ), stating that: . the union shall have the objective of strengthening its scientific and technological bases by achieving a european research area in which researchers, scientific knowledge and technology circulate freely, and encouraging it to become more competitive, including in its industry, while promoting all the research activities deemed necessary by virtue of other chapters of the treaties. . for this purpose the union shall, throughout the union, encourage undertakings, including small and medium-sized undertakings, research centres and universities in their research and technological development activities of high quality; it shall support their efforts to cooperate with one another, aiming, notably, at permitting researchers to cooperate freely across borders and at enabling undertakings to exploit the internal market potential to the full, in particular through the opening-up of national public contracts, the definition of common standards and the removal of legal and fiscal obstacles to that cooperation. . all union activities under the treaties in the area of research and technological development, including demonstration projects, shall be decided on and implemented in accordance with the provisions of this title. within ten years, the ec/eu research policy activity had firmly become woven into the legal fabric of the union. while the ec's/eu's legal base strengthened and its activities broadened, the community/union gradually moved into territories traditionally occupied not only by european nation-states but also by various intergovernmental organizations promoting research. in this book we have seen how already in the s and s, the ec challenged more established organizations such as the oecd and the council of europe. in the subsequent years, this trend continued. indeed, as the eu is thus increasingly dominating the european research field, one can observe an incremental transformation of previously non-ec/eu projects and institutions into eu or quasi-eu projects and institutions, or alternatively, a launching of new eu operations practically identical to existing activities in other european organizations. a good example here is space research. the cooperation agreement signed between the eu and the european space agency in constituted a new framework for a comprehensive european space policy involving the coordination of the actions of the european commission and esa through a joint secretariat, a small team of eu administrators and esa executives, as well as ministerial-level meetings in the space council. in there was a further development when twenty-nine european countries expressed their support for a european space policy, prepared jointly by the european commission and the esa's director-general. moreover, the lisbon treaty gave the european union an explicit competence in space, calling for the development of a european space program. the implementation of this program has further increased cooperation between the eu and the esa. in the recent past, almost percent of the funds managed by esa have originated from the eu budget. the european science foundation, on the other hand, after almost nearing death in , has acknowledged the eu's growing role in promoting european science and wound down all its collaborative research instruments and networks. with a stated mission to serve the european research area for the sole benefit of science, it now focuses on running an expert division called science connect that provides services to the science community. this is a significant shift: since its creation in , esf has run over world-class science programs and networks, with support from eighty member organizations in thirty countries. through this work, it has made a solid contribution to the creation of the european research space. in the most recent years, its financial resources, however, have faced a dramatic downturn. between and , the esf member contributions diminished by . million euros. these budget cuts, together with the wishes of its members, led esf to radically reduce its activities and staff and to move policy-related work into newly formed science europe. cost, another intergovernmental institution supporting european research set up in the early s and analyzed in this book, has weathered the emergence of a comprehensive eu research policy somewhat better. it fills a specific niche in the european research funding landscape as an organization providing financial support for the creation of european-wide multi-stakeholder research networks, called cost actions. most of cost's funding, however, comes from the eu research framework program, which makes it highly dependent on the political developments in the union. the various impacts of the emergent eu research policy on the wider european research space still deserve more thorough scholarly attention. one interesting trend, however, is the emergence of stronger and more organized organizations and networks promoting the interests of researchers, research funders, and the higher education sector at the european level. these include euroscience, a grassroots non-profit organization of researchers in europe, created in to shape policies for science, technology, and innovation. with its individual members, euroscience aims to be "a direct and democratic way to influence the construction of the europe of science and technology, for the benefit of europe's international position in science, and to enhance science's contribution to society, opportunities and mobility for both young and experienced researchers." the european association of research and technology organizations (earto) was established two years later to promote research and technology organizations and represent their interest in europe. its current activities are geared toward influencing the eu policy. in , a merger between the association of european universities and the confederation of european union rectors' conferences led to the creation of the european university association (eua). eua represents over universities in forty-eight countries, and its activities are targeted at influencing the eu policies on higher education, research, and innovation. a year later, another belgium-based organization, the league of european research universities (leru), was formed with the objective to "advance the understanding and knowledge of decision makers, policy makers and opinion leaders about the role and activities of research-intensive universities." both institutions seek to promote the interests of higher education and research institutions in eu policymaking. in , twenty-three countries joined in the founding assembly of science europe, an organization representing research funders and other research organizations in the eu. science europe was built from two former advocacy groups, the esf and eurohorcs (an organization of the heads of the european research councils), aspiring to give its members a stronger and more united voice on european level policy. the list could be continued, in particular if one includes more informal networks and alliances. the rise of these various groups is largely a response to the gradual strengthening of the eu's research arm, which has made advocating for researchers' and research and higher education institutions' interests in brussels a worthwhile endeavor. undoubtedly, there is a new reality for research in europe. the current eu research funding program is exceptional in its size, long duration (seven years), budgetary framework stability, and scope: it encompasses research as well as innovation; grants as well as loans, equity, and procurement; it combines a broad top-down focus on grand societal challenges with bottom-up "frontier" research; it is crossborder and cross-sectoral, encouraging inter-disciplinary collaboration, mobility, and coordination. and it is attracting increasing attention and interest within research communities both within and beyond europe. the horizon mid-term evaluation accounted a percent increase in the annual number of applications compared to the program's predecessor, the seventh framework programme (fp ). with an . percent success rate, which was much lower than under fp ( . percent), the competition for horizon grants had become fierce. while . percent of funding went to participants from the eu member states, the program involved people from over countries. moreover, eu-funded projects are also producing top-notch research. between and alone, horizon , supporting approximately , researchers, produced more than peer-reviewed publications that were cited more than twice the world average. two-thirds of them were also openly accessible to the public. it is also noteworthy that within the european institutions, research has taken a prominent role. in , a total of of the commission's , officials worked for dg research, making this dg the fourth largest division. if this number were to include staff members in the eu joint research centre, research would occupy the first place. research also continues to be the third largest item in the eu budget. despite the prevailing uncertainties about the future of european integration and the different priorities of the member states, the trend of strengthening the role of the eu in supporting and shaping european research is likely to continue. in june , the commission published its proposal for the next eu funding program, horizon europe, which would cover the years of - , involve a budget of billion euros-an increase from horizon -and be "the most ambitious research and innovation funding programme ever." there was little modesty in the commission's statement that "'[b]eneficiaries' research capacities and scientific outputs would have significantly decreased had they received national funding instead. this decrease would have been especially large in terms of their ability to collaborate with industry and business, transfer of knowledge, the number of participations in scientific conferences and the knowledge in new areas." the covid- outbreak in , effectively highlighting the importance of research in tackling global crisis is likely to give a further boost to eu research and innovation activities. the eu acted relatively fast by providing emergency funding from horizon and processing proposals at a record speed. one could note emerging tensions between european and national research spaces, as the national funding bodies and the eu instruments might increasingly compete for resources, applicants, and legitimacy. the larger the eu research budget grows, and the more extensive the european level programs become, the more accentuated this competition will become. already now the eu research funding programs include almost all research themes and forms of research promotion that exist in national research and technology policies. linda wedlin and maria nedeva explain the shift from "science in europe" to "european science" by two important changes in policy rationales and framing. first, the understanding of "european added value" shifted from focusing on coordination to incorporating competition. epitomized in the creation of the european research area, which signaled a move away from collaboration toward integration, this new framing precipitated the implementation of policy instruments that stretched beyond simple support for collaboration between researchers and encouraged genuine competition between them. second, there was growing evidence and concern that europe was not doing so well in science compared with its main commercial competitors. this new version of the technology gap debate argued for a european retard that was more fundamental than failure in making the link between scientific research and commercial applications. "by ," wedlin and nedeva write, "the long-standing assumption of this 'european paradox' morphed into a full-blown 'gap' argument, that is, that europe was clearly lagging behind the usa and japan both in terms of science and its application." this concern led to a change of rationales and the target for policy intervention. it served as a justification for a powerful move to the realm of basic science and the creation of the european research council. tim flink, through an analysis on the creation of erc, supports this finding. a renewed concern of european global retard and a conviction of the need to strengthen the european knowledge base underpinned a political discussion that ushered the birth of erc. moreover, language played a critical role: with the concept "frontier research," the european commission avoided directly violating the principle of subsidiarity, which had curtailed its activity in basic research-an area traditionally belonging to the nation states. "frontier research," falling somewhere in between basic and applied research, aligned well with the eu's traditional mission of fostering european competitiveness and the geopolitical objective of the lisbon strategy to make europe the world's leading commercial power, drawing its strength from knowledge. "frontier research" projected an image of global competition, and it made a solid reference to vannevar bush's monumental report science, the endless frontier and the founding myth of the us national science foundation. "[i]n this policy process," concludes flink, "the narrative structure only seemed to be successful if it followed the chimera of a geostrategic security understanding (isolation by the european research area) and a prosperity identity (market imperative of the eu)." also, terttu luukkonen, while noting the critical role of stakeholder groups, such as the leading life sciences organizations, stresses the role of the gap debate in the formation of the erc. "there was a concern," she writes, "about funding being too low for basic research and about quality of science and its institutions in europe and, as in european research policy in general, usa provided a benchmark with which comparisons were made." furthermore, the focus on frontier research, provided "a strong argument that the erc is justified from the point of view of technological and economic competitiveness, not just of scientific competitiveness." this is confirmed by just a cursory reading of the commission's documents from that time period. the prominence of economic concerns and the gap narrative is striking, as well as the discourse that highlights the insufficiency of national initiatives. the commission's proposal for era, for example, argues that in europe, "the situation concerning research is worrying. without concerted action to rectify this the current trend could lead to a loss of growth and competitiveness in an increasingly global economy. the leeway to be made up on the other technological powers in the world will grow still further. and europe might not successfully achieve the transition to a knowledge-based economy." the discursive continuity is remarkable. throughout this book we have seen how european commission officials and other supporters of greater eu competences in the field of research used a specific language and political framing to advance their initiatives. this language presented scientific research as an engine for economic growth and material prosperity, which were widely accepted as favorable political goals by the european governing elites. combined with arguments about european technological delay vis-à-vis its main commercial competitors, it presented an increasingly convincing case for new european level activity. by the early s, these framings had become dominant truth claims in the european debates, and they started to essentially determine political agendas. at the same time, the scope and nature of the scientific enterprise changed, the pace of discovery and innovation accelerated, and as a result, not only did competition between research institutes, nations, and individual researchers accentuate, but science and technology became increasingly vital forces shaping societies and determining the geopolitical and economic success of countries. it could be argued that one reason for the continuous popularity of the instrumental-economic conception of research policy has been its malleability and extraordinary ability to adapt to different political conditions. as amenable as it seemed to the western european expansive welfare economies of the s and s, it has proved perfectly compatible with the intensifying drive for market liberalism since the early s. this is not only because of the prevailing appeal of growth. research policy has proved useful in facilitating and accelerating the shift to the world of open markets. in this study we have seen how research policy, especially at european level, was commonly pictured as a means to adapt to an exogenous and irreversible transformation that later became labeled as globalization. the eu research policy came into being with an explicit objective of enhancing european economic performance in an international framework in which competition was accelerating. this goal remains as one of the guiding principles of the current eu research policy. when research commissioner máire geoghegan-quinn in november said: "fundamentally, support through horizon for research, innovation and science is an economic policy," she reiterated the principle on which the union's activity had been based since the very beginning. the economic framing remains sturdily in place. the horizon mid-term evaluation report, for example, made a blatant calculation a gdp gain, concluding that "[e]very euro invested under horizon brings back to . euros." the explanatory memorandum included in the commission's proposal for establishing horizon europe included similar calculations. according to the memorandum, horizon europe was expected to generate positive effects on growth, with an increase of gdp on average by . percent to . percent over twenty-five years, "which means that each euro invested can potentially generate a return of up to euro of gdp over the same period"-better ratio than in horizon . the memorandum went on to calculate that discontinuing the union research and innovation program could result in a decline of competitiveness and growth up to billion euros of gdp loss over twenty-five years. to some degree, the eu is tied to this language. initially, the lack of a solid juridical basis for research policy forced the commission to frame research as a tool for achieving prosperity, which has traditionally been one of the union's core objectives. although the gradual strengthening of the juridical basis of research policy during the last three decades has granted the commission more room for maneuver, the eu's activity and ambitions are still limited by the principle of subsidiarity and the constant search for european added value. the eu is most likely to gain legitimacy by anchoring its initiatives and programs to the objectives of competitiveness and growth-a territory where eu activity is seldom questioned. but continuously liking research and growth and articulating science and innovation policy in economic terms, is more than a smart political strategy. it is a durable policy framing that was constructed in the early s and that has been broadly embraced and absorbed by national decisionmakers, european officials, and a number of other actors shaping research policy. it embodies a very specific understanding of the role of science and research in society and the objectives of public support for research and innovation communities. policy framings can and need to change if they become unsuited to responding to the society's most pressing needs and challenges. johan schot and w. edward steinmueller, for example, have argued for a new framing for innovation policy, "linked to contemporary social and environmental challenges such as the sustainable development goals and calling for transformative change." this framing should take priority over the two existing framings, which emerged in the s and s, and which, while still relevant, "offer little guidance for managing the substantial negative consequences of the socio-technical system of modern economic growth to which they have contributed and of which they are a part." the first framing, which dominated during the time period analyzed in this book and-as we have seen-still influences eu policy, emphasized the role of research and innovation for increasing economic growth, aimed at boosting the potential for science and technology for prosperity, and cultivated socio-technical systems supporting mass production and consumption. the second framing gained ground toward the late s and more explicitly aimed to address challenges related to intensifying international competition and globalization. with a focus on developing and analyzing national systems of innovation, enhancing capacities for learning, and supporting connectivity between different societal sectors within the system, it added a layer of sophistication and complexity to the first framing. however, it didn't depart from the premises of increasing growth through maintained and improved competitiveness. the third framing suggested by schot and steinmueller draws upon the recognition that a deep and rapid transformation of socio-technical systems is needed in the backbone systems of modern societies, including energy, mobility, food, water, healthcare, and communication, and that research and innovation policy has a vital role to play in supporting this transformation. that requires a dramatic change in thinking, broadening of perspectives and alliances, and setting entirely new goals and objectives. according to the two authors, this new framing "focuses on innovation as a search process on the system level, guided by social and environmental objectives, informed by experience and the learning that accompanies that experience, and a willingness to revisit existing arrangements to deroutinize them in order to address societal challenges." further, the innovation process in this framing "is likely to be effective in achieving these goals if it is inclusive, experimental and aimed at changing the direction of socio-technical systems in all its dimensions." essentially, this framing will propose building a new knowledge base, setting up mission-oriented policies with missions formulated in an open-ended way, encouraging new forms of engagement and networks between public, private, and third sector actors, and supporting intermediary actors to advocate competitive niches, as well as new visions and policies. it will mean a shift away from the old research and innovation policy proposition that social and economic goals can be achieved through economic growth if surpluses are redistributed and technocratic elites can regulate the externalities of growth. a more comprehensive and ambitious-if not radical-approach is needed to ensure the continued relevance of research and innovation policy in the contemporary world in rapid transition. schot and steinmueller are not alone with this proposition. in the last few years, there has been increasing scholarly interest in rethinking the premises of science and innovation policy and exploring ways of better aligning policy objectives with broader social and environmental challenges. a new policy paradigm is emerging, "layered upon but not fully replacing earlier paradigms of science and technology and innovation systems policy." according to gijs diercks, henrik laren, and fred steward, this "'normative turn' that is currently taking place insists that innovation policy must not only optimize the innovation system to improve economic competitiveness and growth, but also include strategic directionality and guide processes of transformative change towards desired societal objectives." further, this "emerging paradigm of transformative innovation policy is still a heavily contested discursive space" and "there is considerable uncertainty regarding the paths of this paradigm shift in the making." looking at the most recent eu language on research and innovation policy, one can discern elements of this shift. while competitiveness and growth retain a solid place in the framework programs, with the increasing focus on strengthening the european knowledge base and the objective of tackling grand societal challenges through research, the political framing of the eu research policy has widened. one of the earliest visible expressions of this ambition was the declaration, endorsed by over researchers, policy-makers, and representatives from industry and research funding institutions during the "new world -new solutions" conference in lund, sweden. the declaration stated that "european research must focus on the grand challenges of our time moving beyond current rigid thematic approaches. this calls for a new deal among european institutions and member states, in which european and national instruments are well aligned and cooperation builds on transparency and trust." the "grand challenges" listed in the declaration included global warming; tightening supplies of energy, water, and food; aging societies; public health; pandemics; and security. the lund declaration also states that the "european knowledge society must tackle the overarching challenge of turning europe into an eco-efficient economy." since then, broader social and global concerns have found a firmer footing in the eu's research policy agenda. between and , a total of percent of horizon funding went to reinforcing and extending the excellence of european science base and consolidating the era. the second biggest pot, percent, went to stimulating a critical mass of research and innovation efforts to help address grand societal challenges. promoting industrial leadership came only as third, with percent of funding. the remaining percent was dedicated to other priorities, such as widening participation, including society, euratom, and the pilot for fast-tracking innovation. the three pillars of horizon europe-excellent science, global challenges and european industrial competitiveness, and innovative europe-seem to continue this trend, although the commission notes that "[i]industrial leadership will be prominent in this [global challenges] pillar and throughout the program as a whole." indeed, the proposal for horizon europe was "framed by the premise that research and innovation (r&i) delivers on citizen's priorities, boosts the union's productivity and competitiveness, and is crucial for sustaining our socio-economic model and values, and enabling solutions that address challenges in a more systematic way." in other words, "horizon europe will strengthen the union's scientific and technological bases in order to help tackle major global challenges of our time and contribute to achieving the sustainable development goals (sdgs). at the same time, the programme will boost the union's competitiveness, including that of its industries." these quotes reveal a striking feature in the commission's blueprints for horizon europe: an effort to achieve the double objective of responding to global challenges and maintaining economic growth. overall, however, the program seems to be defined as serving the european economy. in march , carlos moedas, the research commissioner, noted that "ware now on track to launch the most ambitious ever european research and innovation programme in , shaping the future for a strong, sustainable and competitive european economy and benefiting all regions in europe." even the lund declaration underlines that "[m]eeting the grand challenges will be a prerequisite for continued economic growth." a vital task for the commission and the union more broadly in the future will be resolving the tension between its traditional policy goals of pursuing growth and competitiveness and its newer mission of tackling grand challenges and promoting sustainability transitions. can both objectives be successfully pursued in parallel, and how possible conflicts between growth and broader socio-environmental issues can be reconciliated, has not explicitly been addressed in any of the key documents or declarations-until very recently. ursula von der leyen's commission's proposal for a european green new deal contains explicit language, suggesting that the challenge of climate change can be addressed without compromising economic growth and competitiveness. it presents the european green new deal as "a new growth strategy that aims to transform the eu into a fair and prosperous society, with a modern, resource-efficient and competitive economy where there are no net emissions of greenhouse gases in and where economic growth is decoupled from resource use." moreover, "careful attention will have to be paid when there are potential trade-offs between economic, environmental and social objectives." the language stressing the eu's "collective ability to transform its economy and society to put it on a more sustainable path," outlines an ambitious and transformative vision that departs from narrow sectorial propositions. in the european new green deal, science and innovation also has a solid place. the document calls for "new technologies, sustainable solutions and disruptive innovation" and notes that "conventional approaches will not be sufficient." the eu's new research and innovation agenda will emphasize experimentation, and working across sectors and disciplines, thereby taking the "systemic approach needed to achieve the aims of the green deal." for now it seems that four of horizon europe's five research missions would be closely linked to the green deal objectives. also, how the covid- pandemic transforms the european research landscape, remains to be seen. in the last few decades, the eu has shown an important capacity of selfcriticism and learning. horizon europe is being built on lessons learned in horizon , such as the need for increasing support for breakthrough innovation; adopting mission-orientation and encouraging citizen involvement; strengthening international cooperation; reinforcing openness; rationalizing the funding landscape; and encouraging participation. new initiatives in horizon europe, such as european innovation council, address these objectives. ursula von der leyen's commission has set a new level of ambition for achieving a pressing transformative change that transcends several sectors of european society. the rapid response to covid- also shows proactivity and agility from an institution often seen as stiff and bureaucratic. these serve as demonstrations that the eu does not need to or even cannot be constrained by its past policy framings and discourses. and this is a real opportunity. in today's europe, torn by nationalism and distrust in governing elites, preserving peace and producing prosperity no longer suffice to provide the legitimacy that carried the process of european integration this far. it is time for a more powerful and compelling mission, aiming at a profound socio-technological transformation that a union-wide science and research effort is well positioned to shape and realize. despite its contested origins, research policy has become one of the eu's core instruments for advancing its policies, promoting european integration, and achieving change. it has won the support of researchers, both in europe and beyond, gained prestige, and found a solid place on the eu political agenda. with its extensive programs and significant funding, it has transformed the european research landscape for good. however, to remain relevant, the eu research policy needs to be tailored to serve the most compelling needs of the european societies. if designed right, it can be a powerful force, enabling critical and truly transformative effort for which none of the individual member states would be capable alone. from 'science in europe' to 'european science qualitative study on the image of science and the research policy of the european union european commission, qualitative study on the image of science and the research policy of the european union the process of politics in europe. the rise of european elites and supranational institutions europeanization in the twentieth century: historical approaches post-war research, education and innovation policy-making in europe history and memory of an institution towards european science. dynamics and policy of an evolving european research space (cheltenham and political dynamics of the era," in changing governance of research and technology policy − the european research area explaining changes and continuity in eu technology policy: the politics of ideas european research area: an evolving policy agenda political dynamics european commission communication: a reinforced european research area partnership for excellence and growth the 'european research area' idea in the history of community policy-making european research area european commission communication: europe flagship initiative innovation union european commission communication: a reinforced european research area partnership for excellence and growth broadening aims and building support in science, technology and innovation policy: the case of the european research area towards european science die entstehung des europäischen forschungsrates: marktimperative -geostrategie -frontier research (weiserswist: velbrück wissenschaft consolidated version of the treaty on the functioning of the european union-part three: union policies and internal actions, title xix: research and technological development and space-article (ex article tec) esa website the european science foundation; death or mid-life crisis? the science connect website the european science foundation from 'science in europe science europe lobby group hit by sudden exodus. brussels-based advocacy group aimed to provide single voice for scientists in the eu-but is losing members key findings from the horizon eu-forschungspolitik-von der industrieförderung zu einer pan-europäischen wissenschaftspolitik? proposal for a decision of the european parliament and of the council on establishing the specific programme implementing horizon europe-the framework programme for research and innovation eu funding for research and innovation key findings from the horizon from 'science in europe eu-forschungspolitik from 'science in europe vannevar bush, science, the endless frontier. a report to the president by vannevar bush, director of the office of scientific research and development - ; - . the author's translation from german european research area european commission: communication from the commission to the council, the european parliament, the economic and social committee, and the committee of the regions: towards a european research area innovation and science: remarks at press conference launching horizon press conference brussels key findings from the horizon interim evaluation european commission: proposal for a regulation of the european parliament and of the council establishing horizon europe-the framework programme for research and innovation, laying down its rules for participation and dissemination three frames for innovation policy: r&d, systems of innovation and transformative change goal conflicts and goal alignment in science, technology and innovation policy discourse combining insights from innovation systems and multi-level perspective in a comprehensive 'failures' framework next generation innovation policy and grand challenges in addition to scholars in sts and sustainability transitions studies, there have also been historians arguing for a change. for instance, andrew jamison wrote in that "as in the s, there is a need for fundamentally rethinking the relations between science, technology, and society, in europe as well as internationally. in particular, there needs to be much more coordination between policies for science and technology and all the other policies that national governments, as well as local authorities and intergovernmental bodies pursue. in order to meet the challenge of climate change and sustainable development, science and technology need to be reconfigured so that the 'solutions' they provide can be relevant for the problems that humanity faces. and in order to provide appropriate solutions, scientists and engineers will need to be better educated about the problems that need to be solved transformative innovation policy european research area," ; ulnicane swedish presidency: research must focus on grand challenges european commission: the lund declaration: europe must focus on the grand challenges of our time european research area key findings from the horizon interim evaluation european commission: proposal for a regulation of the european parliament and of the council establishing horizon europe-the framework programme for research and innovation, laying down its rules for participation and dissemination the next eu research and innovation investment programme european commission, the lund declaration european commission: communication from the commission to the european parliament, the european council, the european economic and social committee, and the committee of the regions, the european green deal the next eu research and innovation investment programme key: cord- -cqpj f k authors: wen, jun; wang, wei; kozak, metin title: small but mighty: a newfound respect for brief research communications date: - - journal: ann tour res doi: . /j.annals. . sha: doc_id: cord_uid: cqpj f k nan tourism remain to be seen (mahara, zhang, & wang, ) . under such circumstances, brief papers can bring new findings to the broader community. the authors of this viewpoint and their collaborators are interested in covid- 's influence on the industry and seek to contribute to rapid information sharing by publishing concise papers. in the authors' experience, their research notes or viewpoints were published in tourism journals after fast-tracked peer review. comparatively, most of their full papers on covid- are undergoing a typical review process and may not receive decisions for months. while the authors and their colleagues hope their longer papers will be published, the lag between submission and print (while understandable) may date their findings. fifth, readers can benefit comparatively quickly from research notes and short communications. in medical studies, including short-form research, time is of the essence: stakeholders cannot necessarily afford to wait for insight. the same urgency applies to the tourism industry, one of the greatest victims of covid- . brief communications can help stakeholders strategise for industry recovery. might other research formats, such as rapid communications, also prove useful in this scenario? it is time that tourism academics consider how their scholarly community can benefit from concise information updates. in closing, "brilliant ideas require a huge amount of thinking, but communicating them does not necessarily require many words" (dolnicar, , p. ). brevity should not compromise credibility. as full-length and brief research is subjected to the same peer review process, should universities and scholars necessarily distinguish them in quality? today's researchers should also ponder the crux of knowledge creation. put simply, what is most important in research: innovation? brilliant ideas? timely information dissemination that can benefit the greater public? or the length of a study? the answer will not always come easily, and this paper is not meant to discount the merits of full-length work. yet the authors would encourage academics to review diverse brief communications in journals across disciplines. the knowledge displayed in these works should be readily apparent despite their brevity. in many cases, if an idea cannot be expressed succinctly, then it is simply not brilliant enough (dolnicar, ) . ultimately, a study's length is not exactly commensurate with its value; tourism scholars could surely engage in richer, more active discourse if journals accepted abridged article formats. no funding received for this paper. none. publish or perish: the proportion of articles versus additional sections in tourism and hospitality journals jun wen is a lecturer in tourism and hospitality management in the school of business and law at edith cowan university. his current research interests lie in chinese outbound tourism marketing, behaviours wang is a professor in public health in the school of medical and health sciences at edith cowan university. his current research interests lie in molecular epidemiology his main research interests focus on consumer behavior, benchmarking and competitiveness. he has widely published in top-tier journals. he sits on the editorial board of several journals including annals of tourism research key: cord- - g w d authors: shek, daniel t. l.; wu, florence k. y. title: the social indicators movement: progress, paradigms, puzzles, promise and potential research directions date: - - journal: soc indic res doi: . /s - - - sha: doc_id: cord_uid: g w d this paper is a response to the article entitled “fifty years after the social indicators movement: has the promise been fulfilled?” by ken land and alex michalos ( ) which constitutes a careful review of the historical development of the social indicators movement, utility of social indicators in shaping the concept of quality of life and subjective well-being, and issues deserving social indicators research in future. in this response paper, we join in the discussion by highlighting five issues—progress, paradigms, puzzles, promise, and potential research directions of social indicators research. in terms of progress, while we have accomplished many tasks proposed by solomon et al. (the quality of life, sage, london ), some of them are yet to be achieved. regarding research paradigms surrounding social indicators, researchers have primarily used positivistic or post-positivistic orientation to conduct and interpret social indicators research, with relatively fewer studies using interpretive, constructionist or critical theory perspective. there are also several puzzles deserving consideration. these include (a) the use of “other types of evidence”, particularly qualitative data; (b) evaluation of social programs; (c) feasibility of assessing “social progress”; (d) choice of social indicators; (e) interpretation of findings; (f) methodological debates; and (g) explanations for social change. finally, the promise of social indicators research to promote quality of life and potential future research directions of social indicators research are discussed. it is indeed my honor to write this paper responding to the groundbreaking paper entitled ''fifty years after the social indicators movement: has the promise been fulfilled?'' by ken land and alex michalos ( ) . after reading the paper, my immediate reaction is that the paper is an outstanding contribution to the field of social indicators because it carefully charts the history of the social indicators movement and highlights some important future research directions. as it is a ''heavy weight'' paper written by two gurus in the field of social indicators, it is a ''must read'' paper by students and researchers in the field of social indicators research. the paper by land and michalos ( ) is a comprehensive paper that gives both a historical and critical perspective on the social indicators movement. the paper starts by describing the social indicators movement in the s and its evolution in the past half of a century such as the development of different social indicators systems and academic journals. then the authors describe the contemporary state of social indicators, quality of life and well-being studies and conclude that ''numerous composite indices exist at all levels of analysis-from international comparisons to national-level indices to subnational/ regional levels and for various sub-populations' ' (p. ) and five examples are presented to illustrate such indicators. finally, agenda for future with reference to possible research directions is included in the paper. in this response article, we will add my two cents on several issues, including progress, paradigms, puzzles, promise and potential research directions (i.e., ''ps''). i will discuss the progress of the social indicator movement with reference to the tasks that social indicators researchers should accomplish as described by solomon et al. ( ) . concerning paradigms of research on social progress, we argue that quality of life research in different paradigms generates different pictures on ''social progress''. regarding puzzles in social indicators research, we consider several issues, including the role of qualitative data, role of evaluation in social programs, feasibility of assessing social progress, choice of social indicators to be used, interpretations of findings, methodological debates and explanations for social change. we will then examine, how social indicators research findings can inform policy according to its original promise. finally, future research directions will be highlighted. solomon et al. ( ) suggested a list of tasks to be undertaken at the initial stage of the social indicators movement. it would be interesting to ask the extent to which such tasks are accomplished after roughly years. • task -''studying objective life conditions and subjective life quality experience and their inter-relationships'' as pointed out by land and michalos ( ) , many frameworks for measuring objective life conditions have been developed and numerous studies have been conducted. similarly, many studies on subjective life quality experience have been conducted. however, studies on the inter-relationships between these two domains seem not to have been the primary focus in the field. thus, it is important to find ways to align these two domains in future. • task -''beyond description of conditions and experiences and to explore relationships amongst interdependent factors'' while models of objective indicators and experiences of life quality have been constructed and tested, more work is needed to explore the ''interdependent'' factors. • task -''to examine life quality not just on the societal level but also group and individual levels, and investigate their inter-relationships'' while most of the existing studies have examined quality of life at the societal level, there are comparatively fewer studies examining group and individual life quality. there are even fewer studies investigating the inter-relationships amongst quality of life on the different levels. • task -''to understand how individuals and groups participate to create their life quality'' most of the related studies focus on quality of life outcomes (social indicators of outcomes) with few studies examining how people create their life quality and the related process. there is a need to focus more on the human agency. • task -''to treat quality of life as a dynamic process than a static state'' although objective social indicators are usually collected over time, researchers usually regard the construct as static state. explanations of the changes are commonly ad hoc in nature. moreover, longitudinal research on subjective life studies is not common. • task -''foster cross-cultural comparisons and encourage institutions and international organizations to collaborate'' there are emerging studies examining crosscultural comparisons but the development is still in its infancy (shek et al. a; shek shek , . collaboration amongst institutions and international organizations is growing but is yet to mature. • task -''to stimulate quality of life research in places without related research tradition'' we have seen some progress in non-western contexts, such as studies conducted in asia (chua et al. ; ip and shek ) . however, the five illustrative examples given by land and michalos are all projects in english speaking communities. • task -''reflection and recognition about cultural differences'' this is a neglected aspect in the movement. social indicators researchers have commonly assumed that the indicators are universally valid, and can be used in different cultures. even though some social indicators researchers recognize cultural differences, the related reflection is not substantial. • task -''promoting inter-disciplinary projects'' we witnessed inter-disciplinary collaboration amongst sociologists, political scientists, social workers, and social policy scholars who were more focused on the ''macro'' aspect of quality of life. however, the link between macro and micro disciplines (such as psychology) remains to be achieved. for example, epidemiological studies are commonly conducted in psychology, psychiatry or mental health. however, such findings are seldom mentioned in social indicators research. • task -''regard biophysical and social environment and their perception as basic elements of quality of life'' most studies have actually mostly done this. however, the spiritual dimension, despite its importance, is not commonly included in social indicators research. • task -''conduct projects that have relevance for decision-makers'' some studies have attempted to do this, although some social indicators research (particularly those involving ranking of different sites) simply promote understanding without much relevance to decision-makers. social science research is inevitably shaped by the research paradigm one adopts (kuhn ) . the choice of research paradigm does not only dictate the research methods but also the interpretations of findings (neuman and kreuger ) . hence, it is important to reflect about social indicators research conducted within different paradigms. to be simple, we can regard paradigm as a lens through which we can see and understand the reality, such as social progress. it addresses the issue of ontology (i.e., the nature of reality one assumes, including whether it exists or not), epistemology (i.e., the question of ''how do we know?'') and methodology (i.e., ways through which the reality can be known). looking back in history, social indicators research is guided primarily by statistical data and analyses. in terms of paradigm, it is a product within positivism or post-positivism lincoln , ) . positivistic or post-positivistic research on social progress demands the use of social science methods involving deductive logic and precise observations with the research objectives of discovering and testing generalizable nomothetic universal laws, which can predict human behavior. researchers adopting a positivistic or post-positivistic paradigm typically assume that social reality is stable over time and preexisting regularities can be scientifically studied. through precise instruments and objective research methods, social indicator researchers can know the reality just like pressing a button of the camera. to maintain neutrality in research, common sense and values do not enter into the picture of positivistic social indicators investigation. while social science has made much progress based on positivistic or post-positivistic paradigms, there is growing dissatisfaction regarding whether it can give a ''true snapshot'' of the reality lincoln , ) . first, while positivistic research assumes that we can understand social reality in a ''social vacuum'', there is growing criticism of this ''context stripping'' view and there is a call for contextual understanding of observed social phenomena. second, the dualist assumption of the separation between theories and observations of facts is questionable and many researchers argue that ''facts'' are in fact theory-laden (i.e., thinking shaped by some existing preoccupation). third, the thesis that it is possible to be totally objective may not be tenable because values and facts are value-laden and interdependent. finally, there is the criticism that it is impossible to have impartial observers because inquirers might influence the observation process lincoln , ) . while positivists put up theories and test the related hypotheses, interpretive and constructionist social scientists define research studies as attempts to investigate meaningful social action and create as well as maintain understandings and interpretations of people. instead of relying on hypothetical-deductive models and supposedly ''objective'' observations, the purpose of research is to understand social reality that cannot be fully understood in terms of causal laws. as it is assumed that social reality is fluid and there are no preexisting regularities, individual interpretations and social constructions are important. according to this paradigm, common sense is an important source of understanding and values are indispensable in the research process. in contrast to the positivistic practice of using precise measurement, interpretive and constructionist social scientists commonly use interviews, narrative research, natural observations, qualitative studies, and ethnography. hence, profiles based on social indicators are regarded as ''naive realism'' because social reality is always fluid and thus beyond the description by static statistical indicators. statistical models can generate good maps but such maps are without ''blood and flesh'' and ''lived experience'' that cannot help people understand the experience of individuals and social groups. while interpretive and constructionist social science provides an alternative paradigm, through which we can understand social progress, there are several problems associated with this position. primarily, adopting a relativistic epistemology, no objective indicators of social progress are possible, because there can be different interpretations and constructions of the term. second, while interpretive studies can generate unique pictures based on individual or group experiences, how to construct an aggregated and presumably consistent picture on the ''profile'' would be a problem. thirdly, interpretive and constructionist research studies have been criticized as subjective and unscientific. critical social science endorses a critical process of inquiry which attempts not just to look at the ''observables'' (which are superficial illusions) but to examine the underlying structures and processes, which shape the real world phenomena (neuman and kreuger ) . besides looking at the underlying power relations that cannot be seen on the surface, critical social scientists help people build a better world for themselves. for example, according to neo-marxian thoughts, growth in gdp does not necessarily reflect social progress because the working class is exploited by the capitalists. in fact, because of its dynamic nature, exploitation, inequalities and injustice cannot be fully reflected in statistical measures. as far as preferred research methods are concerned, although quantitative and qualitative research methods can be used, critical theorists argue against the use of ''dominant'' methods (which create illusory pictures) and favor action research and participative research. obviously, different paradigms have different views on how to use social indicators to perform social auditing. positivistic and post-positivistic researchers would use valid and reliable measures to collect data from large samples to generate information about social progress. using housing and quality of life as an example, indices and profiles of housing affordability have been developed which help to assess the quality of life of the residents. for interpretive and constructionist researchers, instead of primarily collecting figures and statistical profiles, they tend to collect lived stories and listen to the voices of the unheard who are neglected in the mainstream discourses so that the fluid social reality can be better illustrated. using housing quality of life as an example, instead of building up ''profiles'' of housing quality of life, interpretive and constructionist social scientists would attempt to illustrate the subjective experiences of the informants and socially constructed realities in different groups of people on housing experience. for example, they would understand the subjective experiences of those who have to pay high mortgage or live in places with very bad environment, such as sub-divided flats and ''coffin rooms'' (very small rooms in hong kong). finally, critical theorists argue that the profiles related to housing quality of life shown by social indicators may be illusory and misleading. while positivistic researchers chart changes in housing affordability, critical social scientists look at the underlining power relations leading to such fluctuation, which is not only shaped by the home owners and buyers, but also by the interests and power relations amongst the real estate developers (who build the houses), bankers (who lend money to the buyers for mortgage) and the government (who owns the land and makes related regulations). in the seminal work on social indicators, bauer ( ) stated that ''this volume as a whole is devoted to the topic of social indicators-statistics, statistical series, and all other forms of evidence-that enable us to assess where we stand and are going with respect to our values and goals, and to evaluate specific programs and determine their impact'' (p. , emphasis added). obviously, there has been an abundance of statistical studies and statistical series in this area in the past years. nevertheless, the use of ''other forms of evidence'' such as qualitative data and mixed-methods data to reflect our position and direction is comparatively less widespread. there are several fundamental features of quantitative research (leung and shek ; lincoln and guba ; patton a, b) . firstly, its philosophical orientation is positivistic or post-positivistic and it assumes that the nature of reality is independent of human consciousness and that it is governed by causal laws. secondly, it aims at discovering universal laws (i.e. nomothetic emphasis). thirdly, the knowledge is achieved through ''fact accumulation'' where the facts are presumed to be derived from sense-impressions, which are value-neutral. fourthly, the role of research is to explain and predict phenomena using measured variables with reliability and validity as quality criteria. fifthly, quantitative research attempts to confirm theories through testing hypothetical deductive models. lastly, to ensure the objectivity of a study, quantitative research favors artificial research arrangement where the variables can be controlled. concerning quantitative researchers' characteristics, they are expected to be neutral and value free. to maintain objectivity, quantitative researchers should serve as outsiders controlling bias through error elimination and maintaining a distant, detached and neutral relationship with the subjects. on the other hand, subjects are expected to be passive in quantitative research. quantitative researchers also have certain beliefs about the research process: literature review is regarded as fundamental and as the skeleton for theory and hypotheses; operationalization of concepts; structured, predetermined and strictly planned research design; preference for random and representative samples; use of standardized and validated instruments; use of complex statistical analyses; generalization of findings. on the other hand, the philosophical orientation of qualitative research is derived from constructionist and interpretive philosophies which assume that the nature of reality is individually or socially constructed. on the nature of science, qualitative research takes an idiographic stand focusing on individual or group uniqueness. it looks at the nature of knowledge as construction of reality and understanding of meanings and interpretations, which are fluid in nature. qualitative research primarily attempts to induce theories through naturalistic and context-sensitive methodologies. instead of asking researchers to be value free, qualitative research expects that researchers to be reflexive and serve as the research instrument. to ensure context-specific study, qualitative researchers favor naturalistic research arrangements in the real world and regard research as understanding the reality focusing on individual unique experiences and/or group characteristics. for the quality criteria, it highlights trustworthiness and authenticity, including credibility, transferability, dependability and confirmability. concerning qualitative researchers' characteristics, they are expected to be empathetic and sensitive to feelings of people. to gain an in-depth understanding of human behavior, qualitative researchers should serve as insiders with close and empathetic neutrality. at the same time, participants are expected to be active in qualitative research. researchers doing qualitative research believe that in the research process literature review is auxiliary and helps to gain a better understanding of human experiences, and that there is no need to operationalize concepts. the research design should be unstructured, flexible and evolving. there is no preference for random and representative samples, and analyses should be qualitative and thematic. researchers are instruments in the research process. lastly it is believed that in qualitative research the findings should be conceptually generalized. according to flick ( ) , the dominant positivist approach to doing research requires the researchers to be emotionally distanced, unbiased and absolutely objective. however, qualitative researchers argue that it is virtually impossible to be totally value-free or objective. in mainstream social sciences research, quantitative research methods and statistical analyses are the conventional strategies while qualitative methods are regarded as a less preferred option which is less rigorous (flick ; griffin ; marshall and rossman ) . patton ( c) proposed five sets of criteria for judging the quality and credibility of qualitative research. these include: (a) traditional scientific research criteria (e.g., objectivity of inquirer, correspondence of findings to reality, systematic rigor of research procedures), (b) constructivist criteria (e.g., authenticity, connects and moves the audience, reflexivity), (c) artistic and evocative criteria (e.g., aesthetic quality, creativity, interpretive vitality), (d) critical change criteria (e.g., increase of consciousness about injustice, voices of less powerful group, historical and value contexts); and (e) pragmatic criteria (e.g., utility, feasibility and balanced approach). patton ( c) pointed out that ''many researchers mix and match approaches'' (p. ) and he has ''worked with and mixed criteria from all five frameworks to match particular designs to the needs and interests of specific stakeholders and clients' ' (p. - ) . the advantages and limitations of using qualitative research have been discussed in the scientific literature (flick ; griffin ; marshall and rossman ; willig ) . one of the advantages of using qualitative research is that it can enable researchers to focus on the uniqueness and individuality of the informants (i.e., meaning-making process of the informants). qualitative research best addresses the complexity and multiplicity of individual's lived experiences. the meanings of these experiences are usually understood through in-depth qualitative investigation on informants' frames of reference. this allows the researchers to understand the inconsistencies and contradictions within themselves and across different people. in addition, the face-to-face interactions between the researchers and the informants help develop the relationship of trust and they help to understand sensitive issues. another advantage of qualitative research is its flexible research design. the iterative process of the qualitative research practice helps the researcher situate himself/herself in the field of study and develop more and more knowledge about the field and people in it. in addition, qualitative researchers can explore in detail the experiences, motives and opinions of others and learn to see the world from perspective other than his/her own by using in-depth interviewing. with this new perspective, the long-held assumptions may be challenged and thus recasting reflections (griffin ; marshall and rossman ) . the more the researchers find out, the wider the perspective and horizons would be formed in the specific field. the third advantage of administering qualitative research is providing an emic understanding of the personal experiences of the informants (lincoln and guba ; marshall and rossman ; rubin and rubin ). the qualitative researchers are able to investigate the complexity and multiplicity of the context offers. by situating the researchers in the field and going into the contexts, researchers can elicit multiple constructed realities and study holistically the phenomena and thus obtain the tacit emic knowledge and subjective understandings and interpretations (marshall and rossman ; patton a) . whilst qualitative research methods can inquire the processes in particular contexts in considerable depth, sample size in qualitative research is usually small (flick ) . the statements are often based on analyses of relations, conditions and process in certain contexts. this rootedness in contexts often allows qualitative researcher a specific expressiveness and thus the knowledge produced might not generalize to other contexts. as such, academics, practitioners or policy makers may not take the related findings seriously (griffin ) . recently, some eminent qualitative researchers (see maxwell ; flick ) set forth some possible ways of mapping out the path from the case to the theory that will allow the qualitative researchers to reach at least a certain level of generalization. another limitation of utilizing qualitative research is that the analysis procedures are time-consuming and therefore expensive (griffin ) . while crafting the analyses, qualitative researchers have to carefully examine the recordings, field notes, verbatim and other relevant documents in detail and the process takes quite much time in digging in the depth of the informants' inferences. hennink et al. ( ) alerted the qualitative researchers the significance of this crafting process to gain verstehen, which is to understand the life of the people from their own perspective in their own contexts, though the researchers might spend prolonged time in the field and the analysis process. besides quantitative and qualitative methods, there is also a growing tendency to mix quantitative and qualitative data (creswell (creswell , tashakkori and teddlie ) . according to datta ( ) , there are five forceful and pragmatic reasons for integrating these two methods: (a) researchers have used both methods for a long time; (b) there are arguments supporting the use of both methods; (c) both methods are supported by funding bodies; (d) both methods and related findings have been used in social policies; and (e) we have learned much from both paradigms. greene et al. ( ) also noted five reasons to combine quantitative and qualitative methods in a single study. first, mixing methods (especially triangulation) can help to seek convergence of findings. for example, it is possible to show by using statistical data and subjective interviews that ordinary people cannot afford to buy a flat. second, complementarity mixed methods study is able to bring out different opinions about a phenomenon. for example, while quantitative housing affordability index suggests that housing is not affordable, qualitative interview data can give some indication how paying the high mortgage affects negatively people's lives. third, researchers can combine qualitative and quantitative methods in a developmental manner where the first method helps to inform the second method. for example, based on the housing affordability index information which suggests that housing is a problem, researchers can then carry out field observations about the lived experiences of people. fourth, mixed methods research can help to deal with contradictions in findings with the emergence of fresh perspectives (i.e., initiation). for example, researchers can use qualitative methods to understand the contradiction observed in the quantitative data, such as increase in income but decrease in happiness. finally, mixed methods can be used to add more understanding to a study (i.e., expansion). for example, qualitative research can be carried out to understand the information provided by the housing affordability index. there are an increasing number of mixed methods studies investigating quality of life. (e.g., mitra et al. ) . roy et al. ( ) used quantitative survey, qualitative interviews and participatory approach to construct social indicators of well-being ''by exploring the limits of quantification and considering an alternative action theory'' (p. ). tonon ( ) also advocated qualitative methodology in quality of life research and proposed mixed methods as the ''third methodological approach'' in quality of life studies, which are guided by some conceptual and methodological criteria (shek et al. b ). besides describing and monitoring social progress, social indicators can also be used as outcome indicators to measure changes after implementation of social intervention. interestingly, two observations can be highlighted on the use of social indicators in the evaluation of social programs. first, comparing with description and monitoring functions of social indicators, the evaluation function of social indicators has been given relatively weaker weight. one possible explanation is that macro social indicators such as indicators at the national level may not be sensitive measures for social programs, which may be more individualistic and community-based. it also takes time for social intervention programs to create outcomes, which can impact on changes in social indicators. nevertheless, for specific areas such as welfare, security and health, social indicators have been frequently used to reflect the effect of social intervention-poverty rates have been used to reflect the effectiveness of poverty alleviation programs; substance abuse rates have been used to reflect community anti-drug programs; infection diseases rates have been used to reflect flu shot programs. second, often social indicators research and evaluation research have been regarded as two separate research areas in the field and the literature has not been seriously addressing the possible linkages between these two domains. program evaluation has been defined by rossi et al. ( ) as ''the use of social research methods to systematically investigate the effectiveness of social intervention programs in ways that are adapted to their political and organizational environments and are designed to inform social action in ways that improve social conditions'' (p. , emphasis added). according to patton ( ) , program evaluation is ''the systematic application of research to inform evaluative judgments. it involves the systematic collection of empirical information about the activities, characteristics, and outcomes of programs to make judgments about the program's merit or worth, improve program effectiveness, and/or inform decisions about future programming'' (p. , emphasis added). obviously, it is possible to use social indicators on different levels as outcome indicators in evaluation research. the standards and principles in different evaluation models (e.g., american evaluation association ; european commission ; joint committee on standards for educational evaluation ; united nations evaluation group a, b) enlighten researchers in the field of social indicators research on at least two aspects. first, it is important to engage different stakeholders in the process. instead of just involving the experts in the process, other relevant stakeholders should be engaged as well. second, multiple types of evaluation and multiple data should preferably be used to evaluate social programs. different paradigms define social progress in different ways. positivists and post-positivists see social progress as something that can be assessed by social indicators. for interpretivists and constructionists, social progress is either subjectively experienced or socially constructed. hence, static averaged social indicators cannot capture the fluid nature of social reality. for critical theorists, it is possible to chart social progress, but it must be understood in terms of the historical perspective that helps to reveal the underlying contradictions. moreover, the concept of social progress is a rather philosophical one with an evaluative component attached to it. for example, while we enjoy many benefits of industrialization and urbanization, such as systematic provision of public utilities and affluence of material life, we also witness many problems, such as environmental pollution. similarly technological advancement has brought us the internet and smart phones. these technologies have freed many from the traditional working hours, but they have also often brought along increased stress and decreased face-to-face interaction between people. thus, many skeptics would argue that the costs we have to pay for all these social progresses are much greater than the benefits. unfortunately, there is a clear lack of discussion on the value bases of social indicators of the different schemes in the field. assuming that social progress can be measured, the next question that should be asked is what indicators should be used to chart social change. for researchers with different orientations, they have different views on what indicators constitute good measures of social indicators. there are three other issues that should be taken into consideration. first, for many social indicators research frameworks, there is a lack of credible and critical theoretical model. second, while some of the social indicators are common to all paradigms, there are also variations. while it is reasonable to gradually refine the indicators and find out whether it would be possible to use same indicators, the existing variations may also simply suggest that different value judgments are attached to different indicators. third, experts are usually involved in deciding what indicators should be included in a system. according to social constructionists, this is a form of social construction as it would be likely that the selected social indicators would be biased to fit the world views of the experts or elite academics. the choice of indicators is particularly critical when selecting social indicators to measure non-western cultures. for example, in the report titled ''reconceptualizing social indicators in the caribbean: a review and discussion'' (economic commission for latin america and the caribbean ), it is noted that a list of ''new indicators proposed by the eclac latin american meeting has been adapted to the caribbean reality for the purpose of inclusion into the framework of existing indicators on the family'' (p. ). to illustrate this problem further, an example from the european union can be used. atkinson et al. ( ) found in their analysis several properties that should be used in constructing social indicators that help to monitor national performance of the countries in the eu: . ''an indicator should identify the essence of the problem and have a clear and accepted normative interpretation. . an indicator should be robust and statistically validated. . an indicator should be responsive to effective policy interventions but not subject to manipulation. . an indicator should be measurable in a sufficiently comparable way across member states, and comparable as far as practicable with the standards applied internationally by the un and the oecd. . an indicator should be timely and susceptible to revision. . the measurement of an indicator should not impose too large a burden on member states, on enterprises, or on the union's citizens. of these principles, three refer to the portfolio of indicators as a whole. . the portfolio of indicators should be balanced across different dimensions. . the indicators should be mutually consistent and the weight of single indicators in the portfolio should be proportionate. . the portfolio of indicators should be as transparent and accessible as possible to the citizens of the european union' ' (p. ) . there are at least four further implications that can be drawn from this. first, it is not clear why a ''clear and accepted normative interpretation'' is important and whether it is possible to achieve. second, it is difficult, if not impossible for a social indicator not to be subjected to manipulation because of political consideration and the rise of social media. third, the requirement that social indicators ''should not impose too large a burden on member states'' is problematic because the concept of large is very vague and leaves too much opportunity for interpretation. fourth, the requirement of ''mutually consistent'' is a conceptual ideal only. for example, is the inclusion of indicators of economic development and poverty mutually consistent? the requirement of proportionate weight of single indicators is also unclear because weighting is commonly done in research. as statistical profiles are commonly used in social indicators research, many statistical issues are involved. perhaps a critical evaluation of the human development index can illustrate the existing methodological and conceptual issues highlighted in puzzle . for example, chakraborty ( ) argued against aggregation of scores across different domains for several reasons. first, aggregation would lead to a loss of important information because a composite index can contain only the information that the individual indicators are providing. ''it only presents that information in a form that is more convenient, more clearly understandable, and more amenable to some forms of analysis. but in the process, much useful information may be lost. this loss should be weighed against the gain from avoidance of the trouble of handling a large set of data'' (p. ). chakraborty ( ) further pointed out that ranking in hdi is problematic because ''even for planning purposes, the usefulness of an ordinal ranking of districts is not very clear'' (p. ). as the assessment units in hdi are not measured at an individual level, it is impossible to tell how human development attributes are distributed amongst individuals. finally, chakraborty ( ) argued that hdi only gives a state rather than a process view on human development. according to kovacevic ( ) , there are many criticisms of the human development index, including inability to assess human development accurately, over-simplification based on low quality data, high correlation between different components of hdi and hdi, biases and measurement errors in international data, problems of using composite measures, focusing on averages with under-focus on the vulnerable groups. several measures were proposed to improve the quality of the hdi. klugman et al. ( ) further pointed out several limitations of the hdi. they noted that the hdi was criticized as ''too simplistic, while others who accepted its self-imposed limitations still questioned its choice of indicators and its computational methodology'' (p. ). there was also a criticism on its lack of conceptual or theoretical basis (p. ). regarding the choice of indicators, hdi was criticized as excluding other important aspects of well-being such as equity, social justice, political openness, and happiness. besides, the choice of indicators was questioned and there were queries on the choice of using equal weights. although additional measures have been developed paradigms, puzzles… adjusted hdi, the gender inequality index, and the multidimensional poverty index), such measures have not received enough attention from the field. klugman et al. ( ) further presented three reasons why it is not recommendable for societies to try to maximize the hdi. first, the indicators in hdi are not able to reflect various aspects of human development, and also their weighting is problematic. second, focus on capability should not be regarded as the overriding concern. finally, it is very likely that policymakers in any particular country possess much more superior information to help them in decision-making. in response to the criticisms on the human development index several researchers have suggested alternative indicators. for example, pereira and mota ( ) proposed an alternative approach using the electre tri-c multicriteria method. neri ( ) argued for the use of perceived income, health and education to construct the perceived human development index (phdi). one key issue in social science research is interpretation of findings. it is noteworthy that social indicators research (particularly those on social indicators profiles) is primarily descriptive in nature, leaving the question of explanation and interpretation a thorny one. noll ( ) argued that ''using objective indicators starts from the assumption that living conditions can be judged to be favourable or unfavourable by comparing real conditions with normative criteria like values, goals or objectives. an important precondition, however, is that there is a societal or even political consensus about three key issues: first, about the dimensions that are relevant for welfare considerations; second, about good and bad conditions; third, about the direction in which society should move' ' (p. ) . to illustrate the difficulties highlighted by noll ( ) , we can refer to an example based on divorce rates. can we interpret that an increase in divorce rates is an indication of ''negative'' social development? people with religious beliefs may think so. however, radical feminists and those who are committed to liberation of women might interpret it as a good sign for women to liberate themselves. similarly, can we say that science and technology development brings forth positive social development? with rapid industrialization and urbanization, we witness the global warming effect, which might adversely affect the climate of the earth. in fact, the progress of humans also means worse development or even extinction for non-human species. these examples clearly highlight one important issue surrounding the interpretation of findings of social indicators researchthat value base affects how the findings are interpreted. in future, it would be helpful to further re-think the value choices and priorities in social indicators research. it is difficult to explain changes in social indicators and there are several issues involved. first, for cross-sectional studies utilizing social indicators, it is difficult to locate the causal relationships between changes in policies and social indicators outcomes. because data are collected at a single time point, it would be impossible to ascertain the cause-effect relationship. second, for longitudinal studies using macro social indicators, explanations may become post hoc in nature, if no a priori models are proposed. third, as sample size based on social indicators at the national level is small (such as income per capita in different countries and places), multivariate statistical analyses may not necessarily meet the assumptions of multivariate statistics. fourth, for subjective quality of life studies, it would be easier to propose models because data are usually collected at the individual level. nevertheless, we also need good theoretical models on the determinants of subjective well-being, which are not commonly seen in the field. in short, we need more effort to formulate models that can account for changes in social indicators across time and place. the original idea of social indicators research is to utilize social reporting or auditing to identify gaps in social policies that will lead to social intervention. thus, the problem of whether it is possible to use these findings to make policy and services initiatives arises. there are two observations regarding the potential use of social indicators research. first, application of findings to social intervention appears to be not sufficient. brown and corbett ( ) pointed out that social indicators have five basic policy uses where the ''hierarchical typology of uses which incur progressively exacting demands: description, monitoring, setting goals, outcomes-based accountability and evaluation'' (p. iii). according to noll ( ) , ''while social indicators and social reports have successfully been used as descriptive monitoring tools, their application and use for purposes like setting goals and priorities, or the choice and evaluation of political programs still seems to be problematic and questionable'' (p. ). fortunately, they also pointed out that there was a rising trend of using social indicators for policy making particularly in european countries. second, while some overarching social indicators such as the ones used in the human development index may not be strongly linked to social policies, social indicators in specific areas have impact on social intervention. for example, in the area of substance abuse, rise in the substance abuse figures in the last quarter would alert the law enforcement agencies to see whether there are any changes in the supply of or demand for drugs and what additional initiatives should be stepped up. similarly, rise in suicide rates among the elderly would prompt the government to re-think about the elderly policies and whether adequate support is given for old people. in the area of health, statistics on infectious diseases would engender quick response from the government to curb the problem as early as possible. in short, the relevance of social indicators related to social problems may capture greater attention of the government than social indicators related to the general well-being of the place. obviously, how those general social indicators can lead to social policies is an important issue to be addressed. in their paper, land and michalos pointed out several limitations in existing social indicators research. these included negligence of human agency, lack of multi-level studies, few longitudinal studies and few initiatives in building theories. they further pointed out that the drastic changes in social and economic structures (e.g., post-industrialization, globalization, digitalization, and rise of social media) have generated interesting topics for future research, such as social stratification, issues related to income disparity and migration issues. obviously, these are important and interesting directions for future research. there are several other areas that researchers in the field of social indicators research may consider (shek ) . the first issue is quality of life in the notion of ''uncertain world'' (farber ; inglehart ; manski ) where uncertainty emerging from different domains constitutes excellent research opportunities. regarding economic uncertainties, the economic cycle has become short and economic turbulence such as the financial tsunami in has become more frequent. besides, vocational uncertainties caused by the use of automation and information technology have reduced the demand for labor and employment opportunities. concerning political uncertainties, with the lost promise of capitalism and socialism, people in different parts of the world are also losing their confidence in the government. in particular, people lose confidence in those governments where welfare expenses and related debts are huge (e.g., greece). for social uncertainties, the emergence of the ''m-shaped'' society (i.e., growing upper and lower class with shrinking middle class) and lack of upward mobility amongst young people are also important social conditions affecting quality of life. besides, growing relationship uncertainties such as rise of marital disruption rates (e.g., divorce rates) and inter-group conflict also have adverse impact on quality of life. culturally speaking, uncertainties surrounding changing values and world views are emerging. with growing materialism, consumption and moral diversity, it is theoretically and practically important to ask how such cultural uncertainties influence quality of life. finally, growing global uncertainties such as health hazards (e.g., sars and ebola virus disease), global warming resulting in more extreme weather conditions, and mass extinction of non-human species also constitute quality of life issues for human beings throughout the world. obviously, issues surrounding the uncertain world constitute excellent research opportunities for future studies on social indicators and quality of life. in fact, research in response to these issues clearly underscores the importance of social indicators research in monitoring the improving quality of life. the second research direction is to promote more inter-disciplinary collaboration. an examination of the current picture shows that social indicators research involves primarily scholars and researchers from the more ''macro'' disciplines, including economics, sociology and political science, although the concept of health-related quality of life is also embraced by psychologists, psychiatrists, nurses, occupational therapists, counselors and other allied health professionals on the more ''micro'' level. it is noteworthy that many studies conducted by such ''micro'' researchers have great relevance to social indicators, such as epidemiological studies on mental disorders and surveys on happiness in different patient populations. hence, how to promote the collaboration between the ''macro'' and ''micro'' researchers would be a challenge. as a start, it would be conceptually stimulating to examine the concept of quality of life and the role of social indicators in different disciplines so that some conceptual integration can be achieved. for example, how we can shape the discourse that epidemiological studies on mental health issues address quality of life issues and that the related findings are regarded as social indicators would be exciting topics for future studies. as social scientists, critical appraisal is important. for positivists and post-positivists, it is important to use logical reasoning and critical debates in understanding human societies. for constructivists and critical theorists, adopting a reflective stand is important. hence, it is indispensable to reflect on some of the fundamental methodological and interpretational issues in the social indicators movement. essentially, we believe that human beings are capable of taking a step backward to ask questions about the reality. in this response article, the progress, paradigms, puzzles, promise and possible future directions surrounding the social indicators movement are presented. through this modest effort, we earnestly hope that the field can thrive further. guiding principles for evaluators social indicators: the eu and social inclusion social indicators social indicators and public policy in the age of devolution issues in social indicators, composite indices and inequality social development in hong kong: development issues identified by social development index (sdi) research design: qualitative and quantitative approaches research design: qualitative, quantitative, and mixed methods approaches paradigm wars: a basis for peaceful coexistence and beyond the landscape of qualitative research reconceptualizing social indicators in the caribbean: a review and discussion. caribbean: eclac subregional headquarters for the caribbean evaluation standards and good practice eco-pragmatism: making sensible environmental decisions in an uncertain world an introduction to qualitative research toward a conceptual framework for mixed-method evaluation designs 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approach: an interactive approach implementing a multidimensional poverty measure using mixed methods and a participatory framework a perceived human development index social work research methods: qualitative and quantitative approaches social indicators and quality of life research: background, achievements and current trends qualitative research & evaluation methods designing qualitative studies. qualitative research and evaluation methods two decades of developments in qualitative inquiry program evaluation human development index based on electre tri-c multicriteria method: an application in the city of recife evaluation: a systematic approach the challenges of participatory construction of social indicators of well-being qualitative interviewing: the art of hearing data special issue: quality of life of chinese people in a changing world special issue: quality in life research: responses to emerging issues in a changing world applied research in quality of life (arqol): where are we and issues for consideration special issue: quality of life research in chinese, western and global contexts evaluation of evaluation studies using qualitative research methods in the social work literature ( - ): evidence that constitutes a wake-up call unesco's policy-relevant quality of life research program handbook of mixed methods in social and behavioral research qualitative studies in quality of life: methodology and practice standards for evaluation in the un system introducing qualitative research in psychology: adventures in theory and method acknowledgements the preparation for this paper is financially supported by the hong kong jockey club charities trust. key: cord- -q x xb authors: nan title: th icar abstracts: date: - - journal: antiviral res doi: . /j.antiviral. . . sha: doc_id: cord_uid: q x xb nan the society was organized in as a non-profit scientific organization for the purpose of advancing and disseminating knowledge in all areas of antiviral research. to achieve this objective, the society organizes an annual meeting. the society is now in its th year of existence, and has about members representing countries. for membership application forms or further information, please contact dr. amy patick, secretary, isar; pfizer global r&d, department of virology, science center drive, san diego, ca ; phone + ; fax + ; e-mail amy.patick@pfizer.com. membership application forms will also be available at the conference registration desk, or from our website www.isar-icar.com. enzymes of the pol gene of hiv have been identified as important viral targets for the discovery anti-hiv therapeutic agents. while the viral targets, hiv reverse transcriptase and hiv protease, have been successfully investigated for the development of clinically useful therapeutic agents, research efforts on drug discovery on the third enzyme of the pol gene, hiv integrase, have not resulted in a single fda-approved drug. nevertheless, as integrase is essential for hiv replication, it remains an attractive target for the discovery of new anti-hiv agents. in this presentation, we report the discovery of a conceptually new beta-diketo acid, constructed on a nucleobase scaffold, that is a potent inhibitor of both the -processing and strand transfer steps of recombinant hiv integrase. this inhibitor and the positive control compound, azt, were tested in a pbmc cellbased, microtiter anti-hiv assay against the clinical isolate, hiv- teki (nsi phenotype) and hiv- nl - (si phenotype), and in a magi-x assay against hiv- nl - with hela-cd -ltr-beta-gal cells. our integrase inhibitor was found to have highly potent in vitro anti-hiv activity and efficacy. the discovery of this remarkably active molecule, representative of a unique set of diketo acids bearing nucleobase scaffolds, has uncovered a new chapter in the chemistry and biology of integrase inhibitors and their potential therapeutic applications. berta bosch , imma clotet-codina , julia blanco , eduard pauls , gemma coma , samandhy cedeño , francesc mitjans , anuska llano , margarita bofill , bonaventura clotet , jaume piulats , jose este retrovirology laboratory, fundacio irsicaixa, badalona, spain; laboratorio de bioinvestigación, merck farma y química, barcelona, spain macrophages are key cells for hiv infection and spreading inside the organism. macrophages cultured in vitro can be successfully infected after differentiation with cytokines such as macrophage colony stimulating factor (m-csf). in the monocyte to macrophage differentiation process with m-csf, av-integrins are upregulated concomitantly to the capacity of hiv to generate a productive virus infection. in the present study we show that an anti-av antibody, e , inhibited hiv- infection of primary macrophages. the effect of e on r or x hiv- replication in acutely infected macrophages was dose-dependent, with a % effective concentration (ec ) of ± g/ml in the absence of cytotoxicity. similarly, a monoclonal antibody targeting the avb integrin ( d .f ) also inhibited hiv- infection in this cell type. e reduced the detection of hiv- bal proviral dna in acutely infected macrophages but was completely ineffective against hiv- bal production in chronically infected macrophages, suggesting that e inhibited hiv infection at an early stage of the virus cycle. finally, a small molecular weight antagonist of the avb integrin reduced hiv replication at subtoxic concentrations. therefore, our results suggest that av-containing integrins could play a role in hiv replication in macrophages and indicate that small molecular weight compounds may be developed to interfere with hiv replication in macrophages through the interaction with av integrins. andrew vaillant , hong lu , shuwen liu , carol lackman-smith , roger ptak , jean-marc juteau , shibo jiang replicor inc., laval, que., canada; f. lindsay kimball research institute, new york blood center, new york, ny, usa; southern research institute, frederick, md, usa the sequence independent antiviral activity of phosphorothioate oligonucleotides in inhibiting hiv- by blocking interactions between the v loop and cd has been previously described. this activity was attributed to their polyanionic activity. here we show that ps-ons (and their fully -o-methylated derivatives) are also potent inhibitors of hiv- -mediated membrane fusion and hiv- replication in a sequence-independent, sizedependent (optimal size ∼ - bases) and phosphorothioation dependent manner (independent of stabilization). ps-ons interact with the heptad repeat regions of gp and the hiv- fusion inhibitory activity of ps-ons is closely correlated with their ability to bind to these heptad repeats and block gp six-helix bundle formation, a critical step during the process of hiv- fusion with the target cell. the requirement for ps-on interaction was also found to be dependent on phosphorothioation, suggesting that the v loop/ps-on interaction may also have a hydrophobic component. the increased hydrophobicity of longer (≥ base) ps-ons may contribute to their inhibitory activity against hiv- fusion and entry because these longer ps-ons have a greater hydrophobicity and are more potent in blocking the hydrophobic interactions involved in the gp sixhelix bundle formation than shorter ps-ons (< bases). this novel antiviral mechanism of action of long ps-ons has important implications for therapy against infection by hiv- and other enveloped viruses with type i fusion proteins. chris meier , soenke jessel , bastian reichardt , olaf ludek , jan balzarini university of hamburg, institute of organic chemistry, hamburg, germany; rega institute for medical research, katholieke universiteit leuven, leuven, belgium carbocyclic nucleoside analogues like abacavir showed very interesting antiviral properties. therefore, we are interested in a convenient stereoselective access to this class of compounds as potential antiviral agents. by using a new convergent synthetic strategy, starting from a chiral cyclopentenol, enantiomerically pure carbocyclic thymidine (carba-dt) was obtained as a key intermediate for further variations at the -position. this pathway allows an entry to d-and l-configurated nucleoside analogues. however, using this approach a mixture of side products avoids the formation of the product in very high yields. however, we will present that the side products can be recovered by a stereoselective hydroboration leading to one intermediate only that can be use as well for the synthesis of carbocyclic nucleosides. the condensation of the carbocyclic moiety and different pyrimidine and purine nucleobase was achieved by a mitsunobu reaction. various analogues have been prepared via this strategy, e.g. d-and l-carba-bvdu, nucleoside analogues known to be antivirally active against hsv- . additionally, carbocyclic ␣nucleosides and carbocyclic iso-nucleosides are accessible by this reaction sequence. all new nucleoside analogues were tested for their antiviral activity. particularly carba-dt was found to be a potent anti-hiv active derivative showing no toxicity. however, it can not be excluded that a non-activity of a compound is related to a missing phosphorylation to the monophosphate. in order to prove that, all nucleosides were converted into their cyclosal-phosphate trimesters and transferred into the nucleotides. detailed chemistry, enzymatic and antiviral activity data will be presented. in some cases the nucleotide releasing system showed improved antiviral activity as compared to the parent nucleoside. michela pollicita , candace pert , maria-teresa polianova , alessandro ranazzi , michael ruff , carlo-federico perno , stefano aquaro university of rome tor vergata, italy; georgetown university, washington, dc, usa monocytes/macrophages (m/m) are a strategic reservoir of hiv- commonly infected by ccr -using (r ) strains of hiv- . ccr is an attractive target for inhibition of ccr mediated hiv- entry. thus, ccr antagonists are expected to be a power-ful new class of receptor-based therapeutic agents against hiv- infection. d-ala-peptide t-amide (dapta) is an octapeptide derived from the gp v region of hiv- , able to bind ccr . dapta acts as selective viral entry inhibitor, displacing the binding of gp with ccr . dapta anti-hiv- activity was evaluated in m/m infected with two different hiv- r strains, bal and a, in presence of several doses of the compound. dapta inhibited hiv- replication in m/m (> % compared to control), measured by the p gag ag released in the cell culture supernatants, at concentration of - nm. pcr analysis of integrated hiv- proviral dna on cultured m/m proved that dapta is able to block hiv entry and so, to prevent hiv infection in m/m. moreover, the capability of different hiv- r strains produced and released by infected m/m in affecting neuronal homeostasis was assessed in a neuroblastoma cell line, sk-n-sh, expressing ccr . in sk-n-sh were evaluated cell morphology, propidium iodide binding and fluorescenceactivated cell sorting (facs) analysis. dapta, at concentration of - and - nm, strongly inhibited apoptosis in sk-n-sh of and %, respectively, compared to control. unexpectedly, tak- (a nonpeptidic ccr antagonist with potent anti-hiv- activity) inhibited apoptosis only of % compared to control. our results suggest that the development of new anti-hiv- compounds, such as dapta, could be important in synergistic combination with other antiretroviral treatments in prevent both central nervous system hiv-infection and the consequent neural damage. the mechanisms of dapta inhibition may include both suppression of hiv- r strains in the brain as direct inhibition of hiv- replication in m/m and gp related damage by ccr binding. pradimicin a (prm-a) is an antifungal non-peptidic benzonaphtacenequinone antibiotic that specifically inhibits human immunodeficiency virus (hiv) in cell culture. it markedly suppresses a variety of different hiv- clades in pbmcs, in primary macrophages and several hiv- and siv strains in laboratory cell lines (range of % effective concentrations: . - g/ml; % cytostatic concentration: > g/ml). prm-a also inhibits syncytium formation between persistently hiv- -infected hut- cells and uninfected sup t cells. prm-a behaves as an artificial lectin that selectively binds mannose-containing glycans. consequently, biacore experiments revealed that it binds to gp of hiv- /mn in the presence of ca + . prm-a is endowed with a high genetic barrier with regard to drug resistance development against hiv- . a variety of multiple mutations at n-glycosylation sites in hiv- gp are required before the virus looses marked sensitivity to the drug. there is no clustered pattern of hiv- gp glycan deletions that occur under prm-a drug pressure. the resistance spectrum and mode of action is unique among any of the existing anti-hiv drugs and warrant further (pre)clinical investigations. acknowledgement: this research was supported by the flemish "fonds voor wetenschappelijk onderzoek," the centers of excellence of the k.u. leuven (no. ef/ / ), and the european commission (empro). jan muench , ludger ständker , knut adermann , axel schulz , michael schindler , raghavan chinnadurai , wolf-georg forssmann , frank kirchhoff department of virology university of ulm, albert-einstein allee , ulm, germany; ipf pharmaceuticals gmbh, feodor-lynen-strasse , hannover, germany a variety of components in human blood might influence hiv- replication in infected individuals. peptide libraries derived from hemofiltrate (hf), an aqueous blood solution, contain essentially all circulating blood peptides with a molecular mass below kda, including chemokines, defensins, and cytokines. to identify the most potent natural occurring factors inhibiting hiv- replication, we screened a hf-derived peptide library for antiviral activities. the most active fraction contained a -residue peptide corresponding to a c-terminal fragment of ␣ -antitrypsin (␣ -at), a highly abundant serine proteinase inhibitor. further analysis of the corresponding chemically synthesized peptide, termed virus inhibitory peptide (virip), demonstrated that it inhibits infection by all hiv- variants tested, independently of their subtype and coreceptor usage. notably, virip also blocked multi-resistant hiv- variants and primary isolates. virip specifically inhibited hiv- env function, and did not affect infection by virions containing hiv- , siv, mlv, hcv, ebola or vsv env proteins. the antiviral activity proved to be highly specific for the -residue virip sequence since structurally closely related peptides were inactive. we found that virip inhibits hemolysis of erythrocytes induced by the hiv- gp fusion-peptide (fp). nmr spectroscopy confirmed that virip interacts directly with synthetic gp fp. our observations are evidence that a naturally occurring human substance inhibits hiv- infection by a new mode of action, i.e. binding of the highly conserved fp. furthermore, we performed a structure-activity-relation study with more than virip analogs and found that specific amino acid changes enhanced the antiviral potency of virip by up to two orders of magnitude. experiments in cell culture and animal models further demonstrated that virip exerted no cytotoxic effects. thus, virip derivatives might become a new class of hiv- entry inhibitors. stefano aquaro , valentina svicher , roberta d'arrigo , ubaldo visco-comandini , andrea antinori , mario santoro , giovanni di perri , sergio lo caputo , pasquale narciso , carlo-federico perno , university of rome tor vergata, italy; inmi l. spallanzani, italy; university of turin, italy; sm annunziata hospital, florence, italy to investigate gp -variability and correlation with viroimmunological parameters in hiv-infected patients (pts) receiving t added as a single active drug to a failing regimen. two hundred and ten hiv-gp sequences and clinical follow-up from t -treated patients were analyzed from baseline up to weeks (weeks) of treatment. the association of mutations with viremia (vl)/cd count (c/ul) was assessed by mann-whitney test. the addition of t to the failing antiretroviral regimen induced at weeks a significant vl decrease from . log (stable in the last weeks prior t ) to . log (p = . ) and a significant cd increase from c/ul (decreasing in the last weeks prior t ) to c/ul (p = . ). while vl rebounded to . - log at - weeks, respectively, cd increased to c/ul at weeks. t resistance mutations, absent at bl, occurred shortly after treatment and usually alone. v a was the most common sign of t failure ( . % of pts). the viroimmunological outcome of t -treated pts varied according to gp -mutations. v a/e ( . % of pts) was associated with a cd increase from bl ( c/ul) of . -fold ( c/ul) at weeks and . -fold ( c/ul) at weeks (p = . and . compared without v a/e, respectively). no significant correlation with vl was observed (from . log at bl to . - . at - weeks). by contrast, q h + l m ( . % of pts) was associated with cd loss from c/ul at bl to c/ul at weeks (p = . ), without significant changes in vl (from log at bl to log at weeks). mutation n k (observed in pts, but never found at bl) abrogates the th gp -glycosylation site and correlated with . -fold cd increase at weeks. conformational changes induced by v a/e in the highly conserved giv motif of gp -hr , are tightly related with a loss of hiv-induced damage of immune system. this facilitates cd -recovery through mechanisms that can be virus-(loss of fusion efficiency) and immune-mediated (exposure of new epitopes) not applicable to protease/rt-inhibitors, and thus important for innovative therapeutic strategies. the spread of highly pathogenic h n influenza viruses in humans in asia, with high mortality rates among infected individuals is a major public health concern. in the absence of a vaccine antigenically matching the pandemic virus, antiviral drugs can play an important role. in the present study we reported the antiviral activity of neuraminidase inhibitor oseltamivir against lethal h n influenza virus infection in ferrets, an appropriate animal model that closely resembles clinical signs of human influenza. inoculation of young adult ferrets with a viral dose as low as eid of a/vietnam/ / (h n ) influenza virus caused high fever ( . - . • c), weight loss ( . % of initial), anorexia, extreme lethargy and death of animals on days - post-virus inoculation (p.i.). oral administration of oseltamivir at a dose of mg/kg/day for days twice daily initiated h p.i. inhibited the febrile response, reduced weight changes ( . % of initial) and, most importantly, completely protected ferrets from lethal h n infection. in the treatment groups, virus replication in the upper respiratory tract of ferrets was prevented, whereas untreated animals shed virus at titers of . - . log eid /ml on days , and p.i. systemic spread of the h n virus was observed in untreated ferrets: virus was detected in multiple internal organs, including the brain. treatment with oseltamivir resulted in complete inhibition of virus replication in the lungs and small intestine on day p.i. in the brains of treated animals virus was detected in one of the two animals tested with > -fold reduction of titer. sequence analysis showed no amino acid substitutions at conserved residues in na or ha subunit in viruses isolated from ferret's internal organs after treatment. these results suggest that oseltamivir earlier treatment can prevent h n mortality in ferrets, however, further studies investigating optimal doses and treatment durations required to achieve protection against infection with highly pathogenic influenza viruses are much needed. natalia ilyushina, erich hoffmann, rachelle salomon, robert webster, elena govorkova st. jude children's research hospital, memphis, tn , usa in the present study we tested in the mouse model the hypothesis that combination chemotherapy with drugs targeting dif-ferent virus proteins may lead to more potent and beneficial effects. we applied plasmid-based reverse genetics technique to generate two recombinant a/vietnam/ / -like (h n ) viruses. one virus possessed asparagine at position of the m protein that was found in the naturally circulating virus (rgvn- ) and confers resistance to amantadine. the other recombinant virus possessed serine at that position and was sensitive to amantadine (rgvn- sens) . balb/c mice were administered oseltamivir ( or mg/kg/day) and amantadine ( . or mg/kg/day) twice daily for days by oral gavage; the first doses were given h before inoculation with mld of h n virus. combination treatment with mg/kg/day oseltamivir and mg/kg/day amantadine was given on the same schedule. single-drug oseltamivir produced a dose-dependent antiviral effect against both recombinant h n viruses (p < . ). treatment with oseltamivir at dosage of mg/kg/day significantly inhibited virus replication in the lungs, brain, spleen, and blood of mice at days , , and after inoculation (p < . ), but resulted in low survival rate ( %). single-drug amantadine showed dose-dependent effect only against rgvn- sens strain. notably, risk of death for mice that received mg/kg/day of amantadine or mg/kg/day of oseltamivir was similar (p < . ). in contrast, prophylactic treatment of mice with combinations of oseltamivir and amantadine completely inhibited virus replication in the animals infected with rgvn- sens (p < . ) compared to singledrug usage and protected % of animals. importantly combination chemotherapy completely protected h n virus spread to the brain of the mice: virus was not detected in brain of treated animals on days , , and after inoculation and neurological symptoms were not observed. our results suggest that combination chemotherapy provides an advantage over single-agent treatment. this strategy could be an option for the control of influenza virus infection, and combinations with other novel drugs should be explored. françois jean , reid asbury , meera raj , david lawrence , martin petric the university of british columbia, vancouver, bc, canada v t z ; ge healthcare bio-sciences, piscataway, nj , usa; british columbia center for disease control, vancouver, bc, canada v z r in late , severe acute respiratory syndrome (sars) became the first new severe and easily transmissible human disease to emerge in the st century. although it abated after six months, sars serves as a modern paradigm for human emerging infections with deaths reported from countries. the causative agent was found to be a new sars-associated coronavirus (sars-cov) . while the sequence of sars-cov genome was first reported by the bc genome sciences center, the full set of viral and cellular proteins that compose the sars-cov virion remains unknown. to approach this problem, we have utilized two-dimensional gel electrophoresis and liquid chromatography-tandem ms (lc-ms/ms) to identify the viral and cellular proteins in purified sars-cov virions obtained from human infected cells [huh : human liver] and primate (veroe : monkey kidney) infected cells. interestingly, analysis of the proteins from purified sars-cov preparations has revealed that the enveloped virions contain not only the predicted viral structural proteins (e.g. spike glycoprotein, nucleocapsid protein, and membrane glycoprotein) but also an important number of differentially incorporated host cellular proteins into or onto the newly formed viruses. we have unambiguously identified over host cellular proteins in sars-cov virions by lc-ms/ms. these proteins include members of the annexin superfamily, cytoskeletal proteins, chaperones, vesicular transport proteins, uracyl-dna glycosylases, and aldehyde oxidoreductases. this study provides the first comprehensive and comparative analysis of the viral and cellular proteins that compose infectious particles of sars-cov obtained from human and primate infected cells. the functions of these newly identified hostspecific proteins are currently being investigated using rna interfering systems; their contributions to structure, viral productive replication, and pathogenicity will be discussed. acknowledgement: supported by an early career ubc operating grant (f. jean) and cihr (m. petric) . dale barnard , craig day , robert montgomery , kevin bailey , matt heiner , larry lauridsen , robert sidwell , kurt berg institute for antiviral research, utah state university, logan, ut, usa; panum inst., immi, the ifn-lab, copenhagen, denmark severe acute respiratory syndrome (sars) is a life-threatening respiratory illness caused by sars-cov. there are no approved therapies for sars. some drugs inhibit sars-cov replication in vitro including human interferons and selected antiinflammatory agents (chihrin and loufty, . , - ) . interferons are very promising because of their potent in vitro inhibition of sars-cov. although anti-inflammatory agents are not very active in vitro, it is thought that they might be efficacious in reducing any deleterious inflammatory response associated with virus infections such as sars infections in humans. for example, troxerutin, a flavenoid with anti-inflammatory properties, is in clinical trials for treating rhinovirus (rv) infections, ameliorating rv-induced inflammation (turner et al., . apmis , - ) . therefore, troxerutin was tested for inhibition of sars-cov replication in the lungs of infected mice using a mix of four hydroxyethylrutosides that included troxuretin. in addition, mouse interferon-alpha, used as a model compound for human interferon-alpha, was evaluated for inhi-bition of virus lung titers. both mouse interferon-alpha administered i.p. daily beginning h pre virus exposure at doses of , and , iu and the hydroxyethylrutoside mix ( and mg/kg) administered i.p using the same schedule reduced virus replication in the lungs of mice to below detectable limits. when a hydroxyethylrutoside mix was given to mice in the drinking water at . mg/ml (likely equivalent to an i.p. dose of mg/kg, assuming that the mice drank freely), virus lung replication was also completely inhibited. all treatments appeared to be well tolerated, since all groups of mice gained weight. we also report on the efficacy of various combinations of two doses of these drugs administered i.p., using the same dosing regimen as described. these data support the supposition that interferon might be a useful therapy for treating human sars infections and that hydroxyethylrutosides should be investigated further as a potential therapy. acknowledgement: supported by contract no. n -ai- from the virology branch, niaid, nih. treatment options for human respiratory syncytial virus (rsv) are limited. an effective vaccine is not yet available. neutralizing polyclonal antibody (respigam tm , medimmune) and a humanized monoclonal antibody (synagis tm , medimmune), are licensed for prophylactic use. ribavirin is the only approved antiviral against rsv, but its efficacy is controversial and its use is limited to treatment of high-risk patients. there is a clear need for new anti-rsv therapeutics with improved efficacy and ease-of-use. many early efforts to identify anti-rsv compounds focused on blocking the process of fusion. we have developed a cell-based screening platform to identify antivirals that inhibit rsv transcription and replication. the assay does not require infection with wild-type virus. it is based on an rsv subgenomic replication system in baby hamster kidney (bhk- ) cells that express the essential viral replication proteins (n, p, l and m - ). the readout is expression of the reporter gene lacz from a subgenomic rna. screening of the apath small molecule library yielded compounds (hit rate = . %) with ec values ≤ m and with selectivity index (si) values ≥ . seventy-two compounds demonstrated antiviral activity against wild-type rsv (strain a ) in a cytopathic effects inhibition assay (ec < m; si > ). these anti-rsv compounds represent nine different chemical classes. two compounds, a -aminoquinoline (ec = . m) and a thienopyrimidine (ec = . m), were shown to have desirable pharmacokinetic profiles and were chosen for efficacy testing in the cotton-tail rat model of infection. sar studies to identify the pharmacophore of the compounds have been initiated. preliminary studies to characterize the mechanism-ofaction in virological assays will be discussed. acknowledgement: supported by nih r ai - . we have previously reported bicyclic furano pyrimidine nucleoside analogues (bcnas) as exquisitely potent and selective inhibitors of varicella zoster virus (vzv) , with subnanomolar activity for p-alkylphenyl substituted analogues such as lead compound cf (cf- ) ( ) . these compounds have entered preclinical development with fermavir pharmaceuticals. we now report the first chromatography-free synthesis of these agents, their scale up to multi-gramme amounts, and their pre-clinical characterisation. in addition, we were keen to address potential solubility and bioavailability issues of these highly lipophilic agents by the synthesis of more polar analogues in two categories; side-chain ethers ( ) as new analogues in their own right, and -phosphates ( ) as potential more soluble prodrugs. we report data on both of these new families at this meeting. finally, we note the application of our phosphoramidate pro-tide approach to this family, with a series of bcna protides ( ) designed as intracellular phosphate delivery forms to bypass the essential vzv thymidine kinase-mediated first phosphorylation step. graciela andrei , joos van den oord , pierre fiten , ghislain opdenakker , erik de clercq , robert snoeck rega institute for medical research, katholieke universiteit leuven, leuven, belgium; pathology department, u.z. leuven, leuven, belgium varicella (chickenpox) , the primary infection caused by vzv, is characterized by viremia and skin lesions. reactivation of the latent virus results in skin lesions characteristic of herpes zoster (shingles). as keratinocytes are one of the main target cells for productive infection in vivo for vzv, human epithelial cells represent a relevant model for the study of vzv pathogenesis and evaluation of antiviral compounds. organotypic epithelial raft cultures permit full differentiation of keratinocytes via culturing of the cells on collagen matrix at the air-liquid interface. we have previously shown that the susceptibility of cultures to infection with vzv depends on the stage of differentiation of the rafts. we have now quantified the activity of reference anti-vzv compounds by measuring viral dna load by realtime pcr. quantitative pcr for vzv dna was performed by using specific primers and a mgb-probe for the orf gene (single-stranded dna binding protein) by the taqman method. two series of raft cultures were infected with the wild-type oka strain after days of differentiation and treated with serial dilutions of the test compounds. at days post-differentiation one series of the cultures was processed for histology and the other one for viral dna quantification. acyclovir (acv), penciclovir (pcv) and brivudin (bvdu) at and . g/ml, foscarnet (pfa) at g/ml and cidofovir (cdv) at , . and . g/ml inhibited viral dna content by more than %. these results were in agreement with histological examination of the rafts, no cytopathic effect being observed at these concentrations. as expected, only cdv and pfa inhibited the replication of the thymidine-kinase deficient (tk-) - strain. a correlation between the degree of protection as determined by histological examination and viral quantification could also be demonstrated for cdv and pfa against the tk- - strain. since no animal model is available for the in vivo evaluation of antiviral agents against vzv, the organotypic cultures may be considered as a valuable ex vivo model to evaluate the efficacy of new anti-vzv antivirals. jae-seon hwang , oliver kregler , john c. drach , , leroy b. townsend , elke bogner institut für klinische und molekulare virologie, erlangen, germany; department of biologic and materials sciences, school of dentistry; interdepartmental graduate program in medicinal chemistry, college of pharmacy, university of michigan, ann arbor, mi, usa dna packaging is the key step in viral maturation and involves binding and cleavage of viral dna containing specific dnapackaging motifs. this process is mediated by a group of specific enzymes called terminases. we have previously demonstrated that the hcmv terminase is composed of two subunits, the large one encoding pul and the small pul , where each protein has a different function. while the large subunit mediates sequence specific dna binding and atp hydrolysis, pul is only required for duplex nicking. inhibitors targeting pul and/or pul are attractive alternatives as hcmv antivirals since mammalian cell dna replication does not involve cleavage of concatameric dna. we now have screened several members of the benzimidazole ribonucleoside class of replication inhibitors in order to determine if a compound has the capacity to block the atpase activity of the large terminase subunit pul . analysis by bioluminometric atpase activity assays identified bdcrb and one more compound [ -bromo- , , -trichloro- -( , , -tri-o-acetyl-␤-dribofuranosyl)benzimidazole (btcrb)] with inhibitory effects. although only btcrb and bdcrb were inhibitors of the atpase activity, two other compounds, dbdcrb and cl rb, inhibited virus replication in a plaque-reduction assay, thus indicating that those have a different mode of action. in addition, by electron microscopy of thin sections we observed that in the presence of btcrb only b-capsids and dense bodies were formed. furthermore, spherical capsids accumulated in the perinuclear cisternae indicating a block in nuclear egress thereby providing additional evidence that closely-related benzimidazole d-ribonucleosides may have differences in their antiviral modes of action. human cytomegalovirus (hcmv) is the cause of significant morbidity and mortality in a variety of immunocompromised patients. currently available anti-hcmv drugs interfere with dna replication; however, these drugs are highly toxic, pre-cluding their long-term use in humans. interrupting hcmv viral entry is largely unexplored as an antiviral drug development strategy and is potentially an ideal and tractable goal. hcmv is believed to rely upon formation of ␣-helical coiled coils in the viral glycoproteins gb and gh to promote virus-host membrane fusion; peptides encompassing heptad repeat sequences in these two proteins inhibit viral infection. we have explored nonnatural oligomeric molecules ("foldamers") that are designed to mimic elements of the putative ␣-helical segment of gb. this effort has led to the discovery of oligomers of ␤-amino acids ("␤-peptides") that block hcmv infection. the ␤-peptide scaffold offers several advantages for the design of protein-protein interaction inhibitors, as ␤-peptides are amenable to modular synthesis, resist proteolytic degradation, and can display large and tailored molecular surfaces. the most potent ␤-peptide inhibitor blocks hcmv infection with a micromolar ic in a cell-based assay. these compounds show specificity for hcmv relative to closely related viruses. mechanistic studies suggest that these inhibitors interfere with membrane fusion between hcmv particles and host cells. current efforts are focused on understanding in greater detail the origin of the observed biological activity, exploring other foldamer scaffolds as bases for inhibitor design, and developing specific fusion inhibitors for other herpesviruses. previous reports have indicated that herpes simplex virus (hsv) activates nuclear factor-kappab (nf-kb) during productive infections. nonsteroidal anti-inflammatory drugs (nsaids) have significant inhibitory effects on nf-kb. therefore, two nsaids, indomethacin and aspirin, were assayed for antiherpetic effects and utilized as tools to further study the role of nf-kb in hsv- infection. we report that indomethacin and aspirin inhibited hsv- replication at non-cytotoxic doses. in vero cells, um indomethacin and mm aspirin reduced hsv- titers . and . %, respectively. electromobility shift assays revealed that hsv- activation of nf-kb is inhibited by the nsaids at doses that coincide with reduction of hsv- titers. to investigate a pathway for nf-kb inactivation, protein levels of ikb-alpha, a cytoplasmic nf-kb inhibitor, were examined. ikb-alpha protein was present in uninfected samples, but decreased over time in all hsv samples, regardless of chemical treatment, suggesting localization of nf-kb to the nucleus. immunohistochemistry studies verified that p , a component of the dimeric nf-kb complex, translocated to the nucleus of hsv- infected cells in the presence or absence of the nsaids. finally, direct effects on viral gene activity were assayed by real-time rt-pcr analysis. indomethacin and aspirin reduced mrna for icp , an essential hsv immediate-early gene, . and . -fold, respectively, resulting in significant decreases of icp protein. but transcriptional analysis revealed that synthesis of mrna for thymidine kinase, an hsv early gene, was unaffected by chemical treatment. however, mrna for glycoprotein c, an hsv late gene was undetectable in indomethacin and aspirin treated samples. cumulatively, these data indicate that: (i) indomethacin and aspirin block hsv- replication and (ii) the in vitro anti-herpetic effects of nsaids may reside in their ability to block nf-kb activity within the nucleus, impairing activation of essentials hsv genes. increasing species-specificity constraints preclude study of human cytomegalovirus (hcmv) in animals, necessitating the use of rodent cmvs to model human disease. however, the susceptibility of animal cmvs to clinically useful antivirals is unpredictable. for example, the guinea pig cmv (gpcmv), a uniquely valuable virus for modeling congenital cmv infection, is highly resistant to ganciclovir (gcv) at medically relevant doses. we used a molecular virological approach to test the hypothesis that gcv susceptibility could be conferred on gpcmv by insertion of the human ul phosphotransferase gene into the gpcmv genome. the gpcmv genome, cloned as a bacterial artificial chromosome in e. coli, was modified by site-specific recombination, using a shuttle plasmid targeting the gp locus, and carrying the ul gene from hcmv strain towne. the resultant chimeric virus was replication competent, and was found to contain the hcmv ul by southern-blot and sequence analyses. northern-blot revealed that a hcmv ul -specific transcript was expressed with late gene kinetics. western-blot, using a hcmv ul -specific polyclonal antibody, detected protein in virus-infected cells. the chimeric virus was gcv-susceptible, compared to wild-type gpcmv, with an ic of m. chimeric virus also exhibited increased sensitivity to maribavir (mbv), exhibiting a -log reduction (compared to wild-type virus) in the presence of m mbv, and an ic of m. to study the in vivo pathogenesis of chimeric virus, cyclophosphamide-immunocompromised strain two guinea pigs were challenged intraperitoneally, resulting in evidence of disseminated infection, and mortality. ganciclovir treatment ( mg/kg/day) resulted in reduced weight loss, and mortality, compared to placebo. these studies confirm the key role of ul in cmv antiviral therapy, and demonstrate that a 'humanized' gpcmv can be generated with altered antiviral susceptibilities. genital herpes infections are a global health problem and impact hiv/aids epidemic. strategies to prevent transmission include treatment of infected subjects to suppress shedding and prophylaxis with vaginally-applied microbicides. we examined the in vitro and vivo activity of rep , a fully degenerate mer phosphorothioated oligonucleotide against hsv- infection of human cervical cells and in a vaginal murine model. rep has broad-spectrum anti-herpetic activity with potent in vitro activity against hsv- , hsv- , hcmv, vzv, ebv, and hsv- (vaillant et al., submitted for publication). at a concentration of m, rep inhibited -logs of hsv- infection if present during the entire experiment. synchronized infectivity assays demonstrate that, unlike sulfonated polyanions in clinical trials, which primarily block hsv attachment, rep acts at multiple steps and inhibits binding, entry and post-entry gene expression. in our in vivo studies, mice were treated once intravaginally with rep or pbs control at various times prior to vaginal challenge with a lethal dose of hsv- strain ( log pfu). rep prophylaxis provided protection to mice from hsv- infection and disease. protection was significant when challenged min after treatment (p < . ). additionally, treatment with an analog of rep , which cannot activate tlr- mediated immune stimulation, was at least as active as rep , suggesting that direct antiviral activity and not stimulation of innate immunity is the mechanism of action in vivo. utilizing this analog, protection was significant when challenged min after treatment (p < . ) with a trend toward protection when administered min prior to challenge (p = . ). in summary, treatment with the rep analog which has superior resistance to low ph and nuclease degradation was more effective than rep , in some experiments protecting % of mice from viral infection and disease. the testing of this ph resistant rep analog in a gel formulation is currently underway. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. a phosphorodiamidate morpholino oligomer (pmo) designed to hybridize to a highly conserved region including the aug translation start site of hcv, called avi- , has been evaluated for efficacy, toxicity, and pharmacokinetic properties. avi- inhibits translation initiated at the aug start site with ec of nm ( . ug/ml) and shows positive cooperativity. this pmo retains most of the activity in the presence of point mutations in the hcv genome. huh- cells were incubated with normal human serum (nhs) or hcv infected human serum (is) and hcv replication observed by rt-pcr. avi- produced robust inhibition of hcv in a dose and sequence-specific manner. studies conducted in vivo with avi- in the hcv infected trimera mouse (xtl) show reduction in viral titer which is dose dependent with approximately % of mice with undetectable viral titer and the remaining mice show log reduction in viral titer with . mg/mouse/day for consecutive days. the fractional bioavailability of avi- from a sq dose is approximately . the apparent elimination half life in rat, nonhuman primate and humans was . , . and . h, respectively. the volume of distribution ranged from . to . l/kg and the cmax is linearly related to the dose in mg/m . a phase i study in healthy volunteers in which daily sq doses of and mg has been completed. no serious adverse events have been observed. treatment of infected patients is currently planned. inhibition of hcv polyprotein synthesis is anticipated to contain therapeutic benefis of both protease inhibitors and polymerase inhibition. hcv infection can progress to fibrosis, reduced liver function, hepatocellular carcinoma, and death. currently, the standard treatment for hcv infection involves treatment with pegylated interferon in combination with the nucleoside analogue ribavirin. this treatment regimen effects a cure in approximately - % of the genotype- (gt- ) population; therefore a significant unmet clinical need exists in hcv therapy. virus-encoded polymerases have proven to be excellent molecular targets for chemotherapeutic intervention in numerous viral mediated diseases. in the case of hiv, hbv and herpes virus infections, deoxy-nucleoside analogues, which act as chain terminating agents, have been shown to have invaluable clinical utility. by analogy, appropriate ribonucleoside analogues might be expected to inhibit the essential rna polymerase (ns b) encoded by hcv. here we describe the preparation of nucleoside analogues as inhibitors of the hcv polymerase. in our design of nucleoside analogs as potential anti-hcv agents, we chose to investigate the effect of -substituted ribonucleoside derivatives. we reasoned that after incorporation of a ribonucleoside containing a -substituent, a disruption in elongation of the growing rna could be effected through either steric hindrance or via a conformational change of the carbohydrate moiety. our investigations on several such analogues will be presented. of particular interest is -azido-cytidine, which shows good activity in the genotype b sub-genomic replicon (ic = . m) with no measurable cytotoxic or cytostatic behavior. in addition, we have shown that the triphosphate of -azido-cytidine is a potent and highly selective inhibitor of ns b (ic = . m). joanna e. boerner, sue ma, choilai tiongyip, michael p. cooreman, teresa compton, kai lin novartis institutes for biomedical research, technology square, cambridge, ma , usa current drug discovery efforts for hepatitis c virus (hcv) focus on developing specific inhibitors of two viral enzymes, ns b polymerase and ns - a protease. however, resistant viral mutants are likely to emerge during therapy, compromising the effectiveness of these inhibitors. an alternative and complementary strategy is to target host factors that are also essential for viral replication. cyclophilins, a family of peptidyl-prolyl isomerases and the cellular targets of cyclosporin a (csa), present such an opportunity. it was reported recently that cyclophilin b bound to hcv ns b polymerase and stimulated its rnabinding activity, and that these functions were blocked in the presence of csa (watashi k. et al., molecular cell ) . nim , a csa derivative, is a more suitable candidate for hcv therapy because it binds to cyclophilins with higher affinity than csa while lacking the immunosuppressive activity associated with csa. using the hcv replicon system we demonstrated that nim exhibited potent anti-hcv activities in vitro. moreover, the combination of nim with a specific non-nucleoside inhibitor of hcv polymerase led to synergistic antiviral effects with no significant increase of cytotoxicity. resistant clones against both inhibitors were obtained in vitro, however, it was much more difficult to generate resistance against nim than the polymerase inhibitor. also, there was no cross-resistance between the two inhibitors. finally, addition of nim to the hcv polymerase inhibitor drastically reduced the emergence of resistance compared to polymerase inhibitor alone. taken together, nim , with a novel mechanism of action and a favorable pharmacokinetics and safety profile, represents a promising clinical candidate for treating hepatitis c and provides a rationale for specific combination therapy. the nucleoside analog r was identified as a specific inhibitor of hcv replication in subgenomic hcv replicon cells. r -tp is a competitive inhibitor of cmp incorporation by hcv polymerase ns b. in a transient replicon system r inhibited hcv rna replication driven by genotype b polymerase with similar potency as compared to that driven by genotype a polymerase. r -tp inhibited native hcv replicase and recombinant ns b from genotype a and b with similar potency. in contrast, r -tp did not inhibit human dna polymerases alpha, beta or gamma, including reverse transcriptase activities of dna polymerases beta and gamma, which were highly sensitive to inhibition by azt-tp and tc-tp. no significant inhibition was observed with human rna polymerases i, ii and iii derived from hela cells. in addition, the functionally related native influenza virus rna dependent rna polymerase (rdrp) activity in vitro was not inhibited by r -tp at concentrations up to mm, suggesting high selectivity for the hcv rdrp. thus, r was identified as a potent and highly selective inhibitor of hcv polymerase mediated rna synthesis. guangxiang luo, zhaohui cai, chen zhang, kyung-soo chang, jieyun jiang microbiology, immunology, and molecular genetics, university of kentucky college of medicine, lexington, ky, usa the study of hepatitis c virus (hcv) replication and the search for specific antiviral agents against hcv infection have been hampered by the lack of an efficient stable cell culture system of hcv infection and propagation. we have successfully constructed stable human hepatoma cell lines that contain a chromosomally integrated-genotype a hcv cdna and constitutively produce and secrete high titers of infectious virus into the culture media. transcriptional expression of the full-length hcv rna genome is under the control of a cellular pol ii polymerase promoter at the end and a hepatitis delta virus ribozyme at the end. the resulting hcv rna was expressed and replicated efficiently, as shown by the presence of high levels of hcv proteins as well as hcv rna in the stable huh cell lines. hcv secreted from the stable cell lines was infectious, as determined by antibody neutralization, blockage of putative hcv receptors, and inhibition of hcv replication by interferon. our findings demonstrate the establishment of a stable cell culture system of infectious hcv production and propagation, which allows the study of the entire hcv infectious cycle. the stable hcv-secreting cell lines are now being pursued to develop high throughput screens for effective hcv inhibitors. additionally, we established a novel and powerful hcv replication system in the mouse hepatocyte and mouse embryo fibroblasts (mef). hcv rna was found to replicate efficiently in both pkr +/+ and pkr −/− mef cells, demonstrating that hcv rna replication in mef cells is a powerful system to study host-virus interaction by using diverse gene-knockout animals. interestingly, hcv rna replicates more efficiently in the pkr −/− cell than in the pkr +/+ cell, suggesting a role of pkr in the control of hcv rna replication. however, ifn inhibited hcv rna replication in the pkr −/− cell with an efficacy similar to that in the pkr +/+ cell, suggesting a pkr-independent antiviral mechanism. clearly both pkr-dependent and pkrindependent antiviral mechanisms are important for the control of hcv replication and the mediation of the ifn-induced anti-hcv response. our studies set a stage for the development of transgenic mouse models of hcv replication and open up new avenues to study hcv and host interactions in mefs derived from diverse gene-knockout animals. andrea cuconati , haitao guo , gael westby , anand mehta , timothy block , institute for hepatitis and virus research; drexel institute for biotechnology and virology research, doylestown, pa, usa the high levels of hepatitis b surface antigen (hbsag)-bearing non-infectious particles in the serum of infected individuals is thought to play a role in suppressing hepatitis b virus (hbv)specific immune response by titering out hbv-specific antibodies and lymphocytes. current hbv therapeutics do not directly reduce this viral antigenemia. our group has focused on the enhancement of the immune response through the inhibition of viral antigen secretion in the infected hepatocytes, with the therapeutic goal being the use of hbv vaccination for the treatment of acute and chronic infection. the high-throughput screening of a small molecule library of , drug-like compounds was undertaken to discover novel inhibitors of hbsag secretion. using the stably hbv-transfected, human hepatoma cell line hepg . . , we developed an hts-compatible elisa protocol for the detection of hbsag secreted in the culture media. the screen resulted in initially positive hits, a hit rate of . %. subsequent retesting for activity and toxicity by mtt assay has narrowed the number of confirmed, non-toxic hits to , currently categorized in twelve chemical series. we have previously reported on a trio of related pyrazolo-pyridines with ec measurements below . m and cc measurements > . m. nascent structure-activity relationship (sar) suggests that a central moiety of the molecules is essential to activity, with an aromatic side group contributing to potency. among recently confirmed inhibitors, two currently under investigation include: ( ) an isobutyl-acetamide with an ec of . nanomolar, and a cc of > m, and ( ) a carbothiamide with an ec of . micromolar and a cc of > m. measurement of secreted hbv l and m antigens and cellular markers indicated that the pyrazolo-pyridines are not specific inhibitors of viral antigens, while the isobutyl-acetamide and the carbothiamide are indeed specific. measurement of intracellular viral dna indicated that none of these molecules are inhibitors of replication. we will be reporting on our studies of the potency, specificity, and potential mechanisms of action of these novel anti-hbv compounds. background: entecavir (etv) is a potent competitive inhibitor of hepatitis b virus (hbv) polymerase with activity versus all three enzymatic functions including priming, minus, and plus strand dna synthesis. virologic rebound due to etv resistance (etvr) has only been observed in lamivudine resistant (lvdr) hbv (m v/i ± l m), and requires at least one additional change in the reverse transcriptase domain (rt) at residues t , s , or m . these substitutions surround the dntp binding site or primer grip of rt. the objectives of this work were to further characterize etvr and its mechanism(s) using cell culture, in vitro enzyme, and molecular modeling studies. methods: hbv cell culture assays used transfected hepg cells and quantitation of released, immunocaptured hbv nucleocapsids. gradient-purified intracellular nucleocapsids were used for in vitro rt assays. a d homology model based on the hiv- rt structure was used to model resistance changes in hbv. results: reduced etv susceptibility of etvr hbv was observed both in culture and enzymatically in vitro. kinetic studies showed various etvr substitutions in lvdr hbv selectively reduced etv-triphosphate (etv-tp) binding (k i ) to rt without markedly changing the affinity for dgtp (k m ) or inhibition by ddgtp. etvr rts also displayed reduced enzymatic activity (k cat ) relative to wildtype and etvr hbv appeared growth impaired. modeling studies suggested a novel etv-tp binding pocket in hbv rt that became constrained with etvr changes. m changes in the primer grip region of rt were unique in that resistance was primarily seen during synthesis of minus strand dna. etvr changes in the absence of lvdr substitutions had greatly reduced impacts on etv susceptibility, confirming models suggesting etvr is imparted through lvdr changes. summary: etv provides a high genetic barrier to resistance, requiring additional changes at residues t , s or m along with pre-existing lvdr substitutions m v/i ± l m. kinetic parameters and molecular modeling indicated that etvr substitutions selectively affected etv-tp binding and reduced the replication capacity of hbv. a nonhuman primate (nhp) model of classical, lesional smallpox has been used to test the efficacy of intravenous (iv) cidofovir treatment. cynomolgus macaques were infected with a high dose ( pfu iv) of variola to produce an artificial primary viremia, and then treated with cidofovir at , , or h postinfection (pi). later treatment times were not evaluated. treatment at or h pi halted increases in peak blood viral genome titers measured by quantitative taqman-mgb real-time pcr, which were more than -fold less in cdv-treated animals compared to placebo. historically, the number of pox lesions provided the best correlation with human smallpox clinical severity, and cdv treatment in our model significantly reduced maximum pox lesion counts by > %; the number and size of skin lesions, and in untreated animals contributed significantly to the total viral burden with lesions containing - genomes/g. to better understand the role of viral burden and disease progression in major organ systems, a serial sample study was undertaken. in untreated animals at h pi, viral replication in spleen exceeded genomes/g while liver and bone marrow yielded genomes/ml. in comparison, titers in other tissues ranged between and genomes/g and blood yielded genomes/ml at h, suggesting that the liver, spleen, and marrow may be initial sites of replication. levels of virus in the bone marrow reached a peak of approximately genomes/g at day , then decreased to quantities consistent with those in blood. viral load in the blood increased with time, peaking around days - at genomes/ml. virus was also detected in intestine, skeletal muscle, and late in infection, testes. the ability to successfully treat with cdv h pi despite early extensive organ infection in the accelerated nhp variola model suggests that this treatment could be effective in reducing viremia and mortality after onset of symptoms in human smallpox, which demonstrates a more protracted disease course. work involving variola virus conducted in who-sanctioned cdc, atlanta bsl- laboratory. earl kern , kathy keith , robert jordan , dennis hruby , debra quenelle department of pediatrics, university of alabama school of medicine, birmingham, al, usa; siga technologies, inc., corvallis, or, usa although cidofovir (cdv) has been approved as an investigational new drug for emergency treatment of smallpox, its lack of oral activity and dose limiting toxicity dictates a need for continued development of better therapeutic agents for this potential bioterror disease. it has been reported previously that st- , a low-molecular weight compound, inhibits replication of all the orthopoxviruses in vitro and protects mice infected with vaccinia or ectomelia virus. in the present study, we have utilized cowpox virus (cv) and vaccinia virus (vv) infections in vitro and in vivo to evaluate the efficacy of st- for treatment of orthopoxvirus infections. in plaque reduction assays in human foreskin fibroblast cells, both cv and vv were inhibited by about . - . um of st- . for in vivo studies, st- was administered once daily by oral gavage to mice using mg/kg for , , , or days beginning or h after intranasal inoculation with vv or cv. st- was highly effective (p < . ) in preventing mortality due to vv or cv even when treatment was delayed up to h post-infection. a dosing duration of days was adequate for vv infected mice, but duration of days or longer was required for efficacy in cv infected mice. when st- was given once daily for days at , , or mg/kg daily at , , or h post-cv inoculation, mortality was significantly altered at all dosage levels and time points. to determine the effect of treatment on virus replication in target tissues, mice were inoculated with cv or vv and treated once daily with mg/kg of st- . on various days post-infection tissues were harvested and assayed for virus. in cv or vv-infected mice, st- treatment successfully reduced virus titers from to logs in liver, spleen, and kidney. little effect was noted in lung tissue. these results indicate that st- has significant activity against vv and cv infections in vitro and in vivo and may be a potential chemotherapeutic agent for treatment of human orthopoxvirus infections. cidofovir (hpmpc) is a broad-spectrum anti-viral agent that is used (vistide ® ) to treat aids-related cmv retinitis. currently, cidofovir is of particular interest as a potential therapy for orthopox virus infections, including smallpox. an important limitation of cidofovir and analogous nucleotide drugs in a therapeutic role is their low oral bioavailability and poor transport into cells. in principle, bioavailability of a drug can be improved by structural modification targeting transporters expressed in human intestine. to be effective, the transported prodrug must be cleaved by endogenous enzymes to its parent compound. we will present synthetic studies of novel cidofovir and cyclic cidofovir (chpmpc) prodrugs incorporating amino acids or small peptides, comparing different drug-amino acid linkage strategies. the compounds were evaluated for transporter-mediated uptake and cellular and plasma hydrolysis. the results will be compared with similar studies carried out on a series of peptidomimetic conjugates of foscarnet, the trisodium salt of phosphonoformic acid (pfa), an anti-viral agent that also has very low oral bioavailability and poor cell penetration. the question addressed in this study is if wnv-reactive antibody can improve disease signs in a hamster model after the virus is demonstrated to be in the brain. the hypothesis is based on the high activity of a humanized monoclonal antibody, he , in a mouse model when administered later in infection (oliphant et al., . nat. med. , ) . in this study, virus was demonstrated to be in the brains of hamsters at days post-viral injection (dpi) by cell culture assay, quantitative rt-pcr, and immunohistochemical staining of wnv in neurons. eighty percent of hamsters treated i.p. dpi with mg/kg of humanized monoclonal antibody, he , survived wnv disease, whereas, % of placebo-treated hamsters survived ( *** p < . ). if administered at dpi, % survived. we tested the hypothesis that he is effective if delivered directly into the brain instead of by peripheral administration. the antibody was delivered into the brain dpi using convectionenhanced delivery through a cannula implanted into the brain. the he was detected in the cns, but none was detected in the kidney. the survival of he -treated hamsters was % as compared to % of placebo-treated animals ( *** p < . ). for additional proof, the majority of hamsters having wnv in their cerebrospinal fluid, a marker for cns infection, were protected with he administered i.p. at dpi. this humanized monoclonal antibody, therefore, is a possible treatment for the post-exposure, wnv-infected humans that develop signs of neuroinvasive disease. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih, and grant -u ai - from the rocky mountain regional centers of excellence, nih. hemorrhagic fever viruses are of serious worldwide health concern as well as potential biological weapons. lassa fever virus in particular annually infects several hundred thousand individuals in west africa, and the export of this pathogen outside of this region, either intentionally or unintentionally, presents a serious risk to the developed countries of the world. the cdc and niaid have identified lassa fever virus as a category a priority pathogen, indicating the highest degree of threat to public health. no arenavirus-specific antiviral drugs are currently approved for use in humans. the purpose of siga's biodefense program is to develop safe and effective drugs for preventing and treating diseases caused by category a viruses. to that end, a large and diverse library of small molecule compounds was screened using a viral pseudotype assay to identify inhibitors that target the essential lassa surface glycoprotein (gp) and thus block viral entry into the host cell. twenty-six compounds were identified as quality hits, as defined by potency, selectivity, and chemical tractability. antiviral activity against authentic lassa fever virus was assessed in cell culture through a collaboration with colleagues at usamriid. a number of these potent antiviral compounds and their related analogs have exhibited informative chemical structure-biological activity relationships (sar). two potential lead compound series have emerged from these studies, each with % effective concentrations (ec s) of less than nm against lassa fever virus and with ec s of less than nm against lassa gp-pseudotyped virus. characterization of the in vivo properties of these compounds is underway. the in vitro antiviral potency and selectivity, animal pharmacokinetics, and the development process will be presented. these inhibitors represent an important step toward the development of a small molecule antiviral drug for lassa fever virus. sven enterlein , pramila walpita , allison groseth , heinz feldmann , ramon flick university of texas medical branch, department of pathology, galveston, tx, usa; national microbiology laboratory, public health agency of canada, winnipeg, man., canada nipah (niv) virus (family paramyxoviridae) is a recently emerged human and animal pathogen that can cause severe encephalitis with fatality rates of up to %. since no treatment or vaccination is available, and cross-species spread was observed, the virus has been classified as biosafety level (bsl- ) agent. to avoid bsl- containment for the study of cis-acting signals as target for antiviral strategies, we used an optimized plasmid-driven t minigenome rescue system (without the need for recombinant vaccinia virus mva-t ) as well as an newly established rna polymerase i-based approach. minigenome rescue is based on transfection of the minigenome niv-cat and the plasmids encoding for the three nucleocapsid proteins n (nucleoprotein), l (polymerase), and p (phosphoprotein) and measured by enzymatic cat assays. we used the established plasmid-based minigenome rescue systems to screen for potential antiviral compounds. in a first step we tried to determine the optimal strategy for the delivery of small hairpin (sh) interfering rna molecules. for this we compared three shrna delivery systems against another bsl agent-reston ebolavirus (family filoviridae); (i) plasmid-mediated pol i and (ii) pol iii-driven shrnas, and (iii) exogenously (t ) produced shrna, for their ability to induce gene silencing. interestingly, beside the in vitro-generated or pol iii-driven shrnas, pol i transcripts showed very efficient inhibition of minigenome rescue. however, the most efficient delivery method was transfection of in vitro transcribed shrnas. we will present the results of this comparison and, based on the most efficient approach, also first results of shrnas targeted either to niv n, p, and l genes or to the leader/trailer noncoding regions to interfere with minigenome replication. conformative data with live virus experiments under bsl conditions will be included. filoviruses, which include ebola virus and marburg virus, are among the most notorious human pathogens because they cause sporadic outbreaks of severe hemorrhagic fever. unfortunately, very few therapeutic agents are available to treat infections with these viruses. antiviral screening methods that determine the effect of compounds on viral replication involve working with infectious virus, which is obviously not practical for these biosafety level (bsl- ) agents. we developed an antiviral screening method based on a cell-based, infection-independent, ebola subgenomic replication system in which the expression of an easily measurable enzyme is dependent on the rna replication and transcription factors of ebola virus. using this system we screened a synthetic compound library for antiviral activity against ebola virus and have identified a number of inhibitors. we also used it to identify a peptide inhibitor directed against vp . anti-ebola virus activity for many of the inhibitors was confirmed in a viral replication assay using a gfp-expressing zaire ' strain of ebola virus. fifty-two small molecule inhibitors from at least six classes of compounds had ec values in the low micromolar range and good selectivity. several of these compounds have promising chemical, biological, and pharmacological profiles to pursue as potential anti-filovirus drugs. we are currently preparing to test these compounds in a mouse model of ebola virus. we have also begun a lead optimization program to improve antiviral potency and selectivity of aryl sulfonamide and -aminoquinoline compounds. acknowledgement: supported by nih r ai - and r ai - . human papillomavirus (hpv) has been a difficult virus to target by traditional antiviral methods due to its small size, its small number of obvious therapeutic targets, and its resistance to propagation in vitro. nevertheless, antiviral compounds that reduce hpv dna load have the potential to prevent carcino-genic progression in infected patients. to that end, we developed an approach that dramatically reduces the hpv episomal dna load of keratinocytes in vitro by targeting viral dna sequences. pyrrole-imidazole polyamides, with some containing fluorescent probes to aid in cell localization studies, were designed to recognize the hpv ori. all fluorescent compounds rapidly localized to the nucleus of cultured keratinocytes following addition to the culture media. the compounds were then tested for their ability to alter keratinocyte hpv episomal dna content. two of the compounds caused a dose-dependent reduction in hpv episomes as measured by taqman tm realtime pcr. while control and vehicle-treated cells maintained ∼ copies of hpv per cell, compounds -ta and -ta both reduced hpv dna levels to below copies per cell after h incubation with m compound. an alternative taqman tm amplicon within the hpv e gene produced identical results. a multiplexed taqman tm real-time pcr reaction that followed the ratio of hpv dna to the human apoe gene also demonstrated dramatic loss of hpv dna copies, further confirming our initial observations. finally, cells were treated with polyamides for h, polyamide-containing media was removed, and episome levels were followed for days. at day , days after removal of polyamide and days after sub-culturing of the cells, viral episome levels remained approximately % lower than control samples. by day , days after removal of polyamide, viral dna levels were beginning to recover but still remained significantly lower than control samples. together our results demonstrate that targeting the hpv origin of replication with dna-binding compounds dramatically reduces episomal dna levels. small interfering rnas (sirnas) are potent tools for gene down-regulation but are minimally stable in cells. to improve the efficacy of sirna, we replaced non-bridging oxygens in the phosphodiester linkages of natural rnas with bh groups. the resulting boranophosphates have unique properties, including enhanced nuclease resistance, altered hydrogen bonding of the phosphate, different interactions with metal ions, and increased thermal stability of rna:rna and rna:dna duplexes. anti-egfp sirnas containing boranophosphate modifications were prepared by in vitro transcription with t rna polymerase from ribonucleoside -(alpha-p-borano)triphosphates, as well as normal and phosphorothioate sirnas. after confirming the presence of the borane modifications with maldi-ms, several properties of borano-modified sirnas were investigated: ( ) the double stranded rna with borane modifications maintained the a-form conformation characteristics according to the circular dichroism (cd) spectra; ( ) the borane groups in the sirnas increased the thermal stability, with an enhancement of t m by . - . • c per modification; and ( ) sirnas with borano-modifications were shown to be at least -fold more resistant to rnase a digestion than normal ones. when these modified sirnas were used to down-regulate egfp expression in hela cell cultures, it was found that: ( ) borano-modified sirnas were consistently more effective than sirnas containing the corresponding phosphorothioate modifications; ( ) borano-sirnas were more effective than normal sirnas provided that the center of the antisense strand was not heavily modified; ( ) borano-sirnas were more potent than normal or phosphorothioate sirnas at lower concentrations; and ( ) finally, the silencing activity of boranophosphate singlestranded sirna (ss-rna) was comparable to that of unmodified ds-sirna. the borano ss-rna had excellent maximum silencing activity and was highly effective at low concentrations, and silencing activity was durable up to one week after transfection. results with anti-hpv sirnas will be discussed. boranophosphate modification is a potential new class of anti-viral therapeutic agents. this report describes the antiviral structure activity relationships that led to the discovery of phosphonomethoxy- -fluoro- , dideoxydidehydroadenosine (fd ap, gs ), a novel ntrti, with an excellent resistance profile toward hiv- variants containing major n(t)rti resistance mutations. methods: phosphonomethoxy analogs on purine and pyrimidine dideoxydidehydro (d ) and dideoxy (dd) ribose scaffolds were prepared. antiviral activity was measured against wildtype and n(t)rti-resistant recombinant viruses using cytopathic assay in mt- cells. mitochondrial toxicity was assessed in hepg cells by measuring mitochondrial dna content. results: the d scaffolds displayed superior antiviral activity compared to the dd scaffold and adenine was superior to other nucleobases. phosphonomethoxy- , dideoxydidehydroadenosine (d ap) inhibited hiv- replication with a mean ec of . m and an . -, . -, and . -fold change in potency against viruses containing m v, k r, and thymidine analog mutations (tams), respectively. further exploration of d ap was limited by its mitochondrial toxicity, which was then addressed in ways: (i) preparation of l-d ap or (ii) fluorine substitution. l-d ap exhibited an ec of . m but had substantially reduced potency ( -fold) toward m v mutant viruses. fd ap exhibited an ec of . m, with . -, . -, and . -fold change in potency against viruses containing m v, k r, and tams, respectively. no cytotoxic effects were measured up to mm in mt- cells and no effects on mitochondrial dna were detected up to m in hepg cells for both fd ap and l-d ap. conclusion: fd ap is a novel phosphonate ntrti with antiretroviral activity toward wild-type and resistant mutant hiv- strains. compared to d ap, the -fluorine atom significantly improved the in vitro toxicity profile while retaining the favorable resistance profile. in subsequent studies, the monoamidate prodrug strategy was applied to fd ap to achieve optimal in vivo pharmacokinetic properties. entry inhibitors, and ccr- antagonists in particular, have become one of the most actively pursued treatments for hiv within the pharmaceutical industry. recently, multiple groups have disclosed piperidine-based ccr- antagonists that -to the medicinal chemist's eye -might appear to share a common three-point pharmacophore comprised of a tertiary amine, a phenyl ring, and a carboxamide or sulfonamide group. in several of these cases, these pharmacophoric elements are tethered together by a flexible, aliphatic chain. we sought to improve the potency of and introduce structural novelty into this class of compounds by rigidifying this tether. herein, we describe stereoselective syntheses and sar of a series of ccr- antagonists wherein the tether has been replaced with four stereochemical isomers of a rigidified cyclopropyl scaffold. the regulation of hiv transcription is a complex, multistage process that requires the concerted action of viral and cellular proteins. we discovered the n-aminoimidazoles (naims) as a unique class of hiv inhibitors targeted at the viral transcription level. a prototype naim, nr- , prevents the reactivation of dormant virus by inhibiting both the hiv- p and viral mrna production from latently hiv- -infected cell lines upon stimulation with tnf-␣, pma, or tsa. extensive research revealed that nr- was unable to inhibit the nf-b activation pathway or chromatin remodeling at the viral promoter, both known to be crucial for viral transcriptional activation. focusing on the viral transcription process, chromatin immunoprecipitation (chip) experiments revealed that nr- was able to inhibit the ser phosphorylation of the c-terminal domain (ctd) of rna polymerase ii. this step is mediated by the cdk subunit of p-tefb, which is recruited to the viral promoter by the hiv- tat protein. since we did not find an inhibition at the level of cdk activity or tat-mediated transcription in tat-expressing cell lines transiently transfected with a ltr-gfp construct, we infer that nr- must interfere with the transcription process by a unique mode of action. evidence points towards a kinase, not belonging to the cdk family, to be the target of the naims, resulting in an antiviral action at the level of retroviral transcription. clara e. cases-gonzález , sandra franco , miguel a. martínez , luis menéndez-arias centro de biología molecular "severo ochoa", csic-uam, madrid, spain; fundació irsicaixa, hosp. university germans trias i pujol, badalona, spain a ser-ser insertion at codons - together with substitutions t s and t y in the reverse-transcriptase (rt)-coding region of hiv- are known to confer resistance to zidovudine (azt) and stavudine (d t). phenotypic resistance correlates with increased atp-dependent phosphorolytic activity on inhibitor-terminated primers. we have previously shown that an rt derived from a clinical isolate (ss rt) that contained the insertion and additional mutations related to drug resistance (including t y) showed > -fold increased unblocking activity on azt-and d t-terminated primers, when compared with an rt containing the insertion together with mutations t s and t y, in an otherwise wild-type bh sequence. these results suggested that other mutations associated with the complex t sss/t y in clinically relevant rts contributed to increase atp-mediated excision activity and conferred high-level resistance to azt and d t in phenotypic assays. to identify residues increasing the excision activity, we obtained recombinant enzymes bearing ss rt residues - and wild-type bh rt residues - (l rt), or residues - of the bh rt and - of the ss rt (l rt), as well as an l rt variant with the substitution t y (l rt) and an l rt derivative with t sss (l rt). additional rts containing mutations m l, a v, or k r together with the combination t sss/t y in the bh background were also obtained. atp-mediated excision activities on azt-and d tterminated primers were determined and the effects of mutations were tested in phenotypic assays using recombinant hiv- . the l rt containing mutations t sss/t y and additional changes in the n-terminal region showed the highest atp-dependent phosphorolytic activity on blocked primers, giving values similar to those reported for the ss rt. results were consistent with phenotypic data. in contrast, l , l , and l rts displayed low-level activity. further experiments revealed that three amino acid changes at the n-terminal region of the polymerase (m l, a v and k r) were responsible for the increased excision activity shown by rts bearing mutations t sss and t y. from a series of phenyl-substituted thiazolobenzimidazoles, several compounds were identified as selective inhibitors of coxsackie b virus replication in vero cells. a structure-activity relationship was established, from which the -trifluoromethyl substituted analogs emerged as the most potent congeners. the compounds were active against all six coxsackie b strains tested. the in vitro antiviral activity of one of the most selective compounds, i.e. chi- , was assessed by (i) mts-based cytopathic effect assays, (ii) virus yield reduction assays, (iii) real-time quantitative pcr (rt-qpcr) and (iv) by monitoring viral antigen expression. in all assays a clear concentration-response effect was obtained. the % effective concentration (ec ) was . ± . g/ml, while the cc ( % cytotoxic concentration) of chi- for vero cells was more than g/ml, thus resulting in a selectivity index of > . detailed single cycle time-of-drug-addition studies (in which viral replication was monitored by means of rt-qpcr) revealed that the compound interacts with viral replication at a time that coincides with the onset of intracellular viral rna synthesis. chi- resistant virus is being generated by culturing the virus in the presence of increasing drug concentrations. drug-resistant virus will be genotyped, which should allow us to identify the (putatively viral) molecular target of this class of compounds. retroviruses hiromichi tanaka , kazuhiro haraguchi , hiroki kumamoto , takao nitanda , masanori baba , ginger e. dutschman , yung-chi cheng school of pharmaceutical sciences, showa university, tokyo, japan; center for chronic viral diseases, kagoshima university, kagoshima, japan; school of medicine, yale university, new haven, ct, usa our recent research program on the development of synthetic methods for -carbon-substituted nucleosides has led to a new strategy, ring opening of , -epoxy-nucleosides with organoaluminum and organosilicon reagents. this enabled us to introduce alkyl, alkenyl, and alkynyl groups to the -position. as a result of this study, -ethynylstavudine ( -ed t) was found to be more anti-hiv active than the parent compound stavudine (d t). this compound ( -ed t) has several additional appeals as a promising anti-hiv agent: much less toxic to various cells and also to mitochondrial dna synthesis, better substrate for human thymidine kinase than d t, very much resistant to catabolism by thymidine phosphorylase, its activity enhances in the presence of a major mutation k n known for nnrti-resistant hiv. in this conference, we present the synthesis and sar studies of -ed t analogues modified mainly in the sugar portion. negatively charged polymers (np) possess a broad immunoadjuvant and antiviral activity topically useful for vaccine, drug, and microbicide development. but their efficiency is limited over a reversibility of electrostatic kind of interference with virusspecific nano-objects. to overcome this limitation the purposemade intra-molecular modifications of np were studied among non-toxic maleic acid co-polymers (npsa), dextran and chitin derivatives (npps) within varied alicyclic modifiers application. the configurationally flexible alkyls (i), as non-alicyclic control, are ineffective synergist for np antiviral potency. monocycles (ii) are moderate active too. on the contrary the hardconformation frame-structured spheroids (iii-vi) exhibit ability (at optimal macromolecular parameters) to be super-effective synergists for strength and diapason of np antiviral action. unlike small molecular iii/iv-containing prototypes (amantadin, rimantadin, deitiforin, etc.), narrowly-effective inhibitors mainly of influenza a viruses, the np-coupled modifications become effective also against many other viruses, including the drugs resistant strains [antivir. res. ( ), ]. in focus of the anti-hiv potency the ivs provide a - -fold elevation of np activity. the more available and less toxic iii species are similarly active, but iii* (with spatial-optimally contactable double bond due to the exo-configuration) turns out the best synergist - -fold amplifying the anti-hiv- selectivity up to is∼ . augmentation of the frame cycles from iii-iv toward v-vi results in no essential enhancement of antiviral activity, but stimulates toxicity. the recently involved in the investigation vii, cholesterol-like systems, as tools for novel raft-targeted strategy, demonstrate capacity for at least -fold amplification of anti-hiv- potency our earlier studies showed that esterification of cidofovir (hpmpc) with alkoxyalkanols increased antiviral activity by more that two logs and promoted oral bioavailability. to evaluate this approach with purine based nucleoside phosphonates, we synthesized several alkoxyalkyl esters of acyclic purine phosphonates such as , ,-diamino-( -[ -phosphonomethoxyethyl]purine (pme-dap) and -amino- -cyclopropylamino-( -[ phosphonomethoxyethyl]-purine (pme-cpr-dap) these purine phosphonates have been reported to be active against a wide range of viruses such as human immunodeficiency virus (hiv- ), other retroviruses, herpesviruses, poxviruses and hepatitis b virus. for this study several alkoxyalkyl analogs of acyclic , diaminopurine nucleoside phosphonates were synthesized and evaluated against hiv- . the alkoxyalkyl esters were more inhibitory than the unmodified compounds in p reduction assays in mt- cells infected with hiv- . for example, hexadecyloxypropyl (hdp) and oleyloxyethyl (ole) esters of pme-cpr-dap were > logs more active than unmodified pme-cpr-dap. in spite of increased cytotoxicity in mt- cells, the selectivity indexes are more than -fold higher then for unmodified compound. in conclusion, esterification of pme-dap and pme-cpr-dap with hexadecyloxypropyl-or oleyloxyethyl-residues greatly increased their antiviral activity and selectivity against hiv- in vitro. victor kuz'min , eugene muratov , anatoly artemenko , ludmila koroleva , vladimir silnikov , v. lozitsky , a. fedchuk a.v. bogatsky physical-chemical institute, odessa, ukraine; institute of chemical biology and fundamental medicine, novosibirsk, russian federation; ukrainian mechnikov research anti-plague institute, odessa, ukraine "chemical" ribonucleases hold promise as tools for studying the structures of rnas and rna-protein complexes, as reactive groups in conjugates intended for cleavage of particular rnas, as therapeuticals inactivating virus genome rnas or certain mrnas, and as a promising antiviral agents. drug design and development of new medicines directed against hiv are permanently actual tasks. the usage of modern quantitative structure-activity relationship (qsar) methods could allow us to solve these problems more effectively. the objective of the present work is qsar analysis of antiviral activity of various tetrapeptides-artifical ribonucleases and consequent molecular design of new antiviral agents. qsar approach based on simplex representation of molecular structure (sirms) has been used for the solution of the formulated problem. usage of sirms allows us to develop the molecular design of the new effective antiviral agents. thorough researches of relationship between antiviral activity (hiv- , % of rna p-o bond cleavage) and a structure of artifical ribonucleases have been carried out. statistic characteristics for pls (partial least squares model) are quite satisfactory (r = . , q = . ). on the base of these models the molecular fragments with positive or negative influence on the explored property have been determined. thus, for example, guanidine and triethylenediamine fragments promote antiviral action. it gives a possibility to realize based on elucidated rules molecular design of compounds with the high level of antiviral activity. the results of prognosis are verifying by the experimental investigations. thus, quite adequate simplex qsar model "anti-hiv activity-artifical ribonucleases structure" was obtained and used for drug design. the cyclotriazadisulfonamide (cada) compound specifically down-modulates the cd receptor expression on the surface of lymphocytes and monocytes/macrophages, the primary receptors utilized by hiv for infection of its target cells. cada thus inhibits the entry of hiv and hhv- (vermeire et al., . virology , - ) . cada chemotherapy may not be susceptible to the production of drug resistant strains of viruses, as its mechanism of action is completely different from those of any other anti-hiv drugs currently in clinical use. the cd down-modulating and antiviral potencies of more than cada analogs have been described (vermeire et al., . mol. pharmacol. , - ) . structural modifications of cada were made to increase potency, reduce cytotoxicity, and improve physical properties. several head group analogs were synthesized with polar groups and good leaving groups ( fig. ) . the anti-hiv and cd down modulation activities of these compounds are being studied. some of these head groups may regenerate the double bond of cada by elimination reactions, potentially producing water-soluble pro-drugs. isocada (sa ), an isomer of cada, was synthesized by cyclization of , , -triazabicyclo-[ . . ]dec- -ene (tbd) (fig. ). this structural modification may reveal a relationship between the symmetry of the molecule and its biological activity. two new fluorine-containing analogs were also synthesized by modifying the toluenesulfonamide side arms (fig. ) . the anti-hiv and cd down modulation activities of these new cada analogs are summarized. the center for drug discovery, university of georgia, athens, ga , usa drug discovery targeted at the elusive viral enzyme, hiv integrase, has not resulted in a single fda-approved drug. in this presentation we describe our molecular modeling studies with conceptually novel inhibitors of hiv integrase that also possess potent in vitro anti-hiv activity. docking was performed on the catalytic core of integrase represented by chain c of pdb structure code bl . building of molecules and primary modeling was done with sybyl . on a silicon graphics onyx (r ) workstation. the program gold . (genetic optimization for ligand docking) was used extensively in evaluating the docking poses of these compounds with the active site of hiv integrase and to give information on key residues involved in the recognition and binding of these ligands. the gold function consists of three basic components: protein-ligand h-bonding energy, protein-ligand van der waals energy, and ligand internal energy. post-processing gold output was done with the program silver . , a utility program supplied with gold for evaluating hydrogen-bonding interactions, metal coordination and van der waals factors. for comparison purposes, additional docking was performed using other docking protocols, notably the sybyl module flexx. data obtained from these and related studies including binding poses, binding affinities, functional and conformational considerations, and gold function scores will be presented and explained. the center for drug discovery, university of georgia, athens, ga , usa hiv integrase is essential for hiv replication and is an attractive target for drug discovery against aids. however, research efforts on drug discovery pertaining to hiv integrase have not resulted in a single fda-approved drug for which mechanism of action is inhibition of hiv integrase. recently, we have been exploring a novel class of diketo acids that are constructed on nucleobase scaffolds and that have a specific arrangement of the functional and hydrophobic group on the scaffold. these compounds are inhibitors both key steps of hiv integrase. one lead compound from this group has also been found to have remarkable in vitro anti-hiv activity. however, the syntheses of the inhibitors are quite challenging. this presentation will describe the synthetic methodologies specifically developed in our laboratory for the preparation of some representative examples of these integrase inhibitors. purification approaches to produce highly purified compounds for biological studies will be explained. structural, functional and conformational data obtained from extensive spectroscopic studies will be discussed. representative anti-hiv integrase data and in vitro anti-hiv screening results will be presented. we have recently reported the synthesis and antiviral activities of a ring-expanded ("fat") nucleoside analogue, called nz- , that inhibits both hcv and hiv in vitro with ec values ranging in micromolar concentrations or less, with little or low toxicity to the host cells. in this part i of the presentation on this subject, we report our preliminary findings on the mechanism of anti-hiv activity of this compound, along with the synthesis and antiviral activity of a few additional analogues. in view of the fact that a number of hiv patients also suffer from hcv as a major coinfection, and that a number of them ultimately die of end-stage hcv-related complications including liver cirrhosis and hepatocellular carcinoma, a drug with dual inhibitory characteristics against both viruses is highly desirable and timely. marina burshtein , alexander serbin , alissa bukrinskaya d.i. ivanovsky institute of virology, moscow, russia; health research and development found, moscow, russia introduction: amantadine is a well-known effective antiinfluenza drug. it was modified to enhance its antiviral activity by chemically linkage with the water-soluble polyanionic matrix via different spacer groups. the other group of used compounds was norbornene derivatives, as norbornene is an adamantane analogue on anti-influenza activity. methods: the absence of cytotoxic effect was shown by mtt test for estimating cytotoxic dose (ctd ). the antiviral effect of the compounds was analyzed in lymphoblastoid mt- cells and in hela cd +/b-galactosidase cells ("magi" cells). the effect of the compounds was registered by immunoblotting of cell lysates and by measuring of b-galactosidase activity. results: the strong inhibition of hiv- replication was observed when the compounds were added with the virus and was expressed even when the compounds added with the virus were removed h after infection. the anti hiv- effect of the compounds was gradually decreased if they were added and h after infection, no inhibition was observed when the compounds were added h after infection. the compounds did not impair the virion structure. adamantane and norbornene derivatives were shown also to inhibit azt resistant viral strains. conclusion: adamantane and norbornene were shown to be active hiv inhibitors with the high selectivity index. the compounds are promising candidates for further investigation including preclinical studies. less is known about the effect of their intracellular half-lives on the maintenance of antiviral activity. to investigate this question, we developed a novel in vitro antiviral persistence assay. measurement of the antiviral persistence of tenofovir (tfv) and abacavir (cbv) was coupled to measurement of the half-lives of their tfv-dp and cbv-tp anabolites. methods: mt- cells or stimulated primary cd + t-cells were incubated with graded concentrations of tfv or cbv for h (h); then extracellular drug was removed by washing. cells were further incubated without drug for - h and then infected with hiv- (iiib or bal). p was quantified on day ; inhibition of hiv- replication due to intracellular drug persistence (pc ) was determined relative to a standard ec . decay of intracellular dp/tps in cd + t-cells was measured using lc/ms/ms. results: in mt- cells, the pc value for tfv h after drug removal remained unchanged relative to the ec (< -fold shift) whereas the pc for cbv shifted > -fold, indicating less persistence of cbv. in cd + t-cells, the pc value for tfv also showed a minimal shift relative to the ec ( . -fold) h after drug removal. cbv showed a much larger relative shift (> -fold). quantification by lc/ms/ms of intracellular tfv-dp and cbv-tp in cd + t-cells in vitro demonstrated that tfv-dp had the longest intracellular half-life of the two drugs (tfv-dp, h versus cbv-tp, h). conclusions: a novel antiviral persistence assay was developed to study the relationship between intracellular nrti halflives and antiviral activity. in both mt- cells and primary activated cd + t-cells, tfv had the longest persistence of antiviral activity. in cd + t-cells, tfv-dp also had the longest half-life of the two nrtis. cbv-tp had a much shorter half-life than tfv-dp and showed less antiviral persistence. although both drugs are approved for qd dosing, the half-life of intracellular tfv-dp maintains antiviral suppression in vitro over a timeframe most consistent with qd dosing. karen m. watson, tracy l. hartman, lu yang, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa isis is a phosphorothioate oligonucleotide with a molecular structure of t g t . the g-quartet possessing molecule has been shown to be a potent inhibitor of hiv attachment and cell-cell fusion and acts by specifically interacting at the v loop of gp . mapping studies with monoclonal antibodies targeting epitopes in and around the v loop have been used to define the binding site of isis . in vitro, isis inhibits all laboratory and clinical strains of hiv- and hiv- tested, including representative subtype viruses, drug resistant viruses (including mdr viruses) and viruses that utilize the cxcr and ccr chemokine receptors. serial passage of virus in the presence of increasing concentrations of the oligonucleotide did not result in the selection of drug resistant virus strains and combination assays resulted in additive to synergistic interactions with other approved hiv inhibitors. the antiviral and toxicity profiles of isis resulted in the performance of human clinical trials for the therapeutic use of the oligonucleotide to treat hiv infection. the antiviral properties and mechanism of action of isis suggest that it may be an excellent anti-hiv topical microbicide. isis was found to be highly active in a cervical explant model of hiv infection with highly significant inhibition of ccr -tropic strains of virus. activity was also observed in cell-free and cell-associated virus transmission assays, as well as in cd -dependent and cd -independent acute infection inhibition assays. in microbicidal specific combination assays, significant efficacy has been observed with isis used in combination with other microbicidal compounds. the results of these studies suggest that isis may represent a new and novel anti-hiv topical microbicide. karen m. watson, tracy l. hartman, lu yang, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa though a variety of compounds are being developed as anti-hiv topical microbicides, such as polyanionic molecules, surfactants, natural products, peptides, proteins, heterocycles, and virucidal agents, clinical efficacy studies that demonstrate the ability of these agents to impact virus transmission are still in progress. it has been estimated that a microbicide that is only % effective would have the capacity to prevent millions of new infections each year. thus, one of the challenges in hiv drug development is the discovery of compounds that will inhibit the sexual transmission of infectious organisms between sexual partners. the rapid mutability of hiv and the known presence of drug resistant viruses in wild type virus populations suggests that microbicide development will suffer from the same problems that exist for all hiv therapies, namely the selection of resistant virus strains that will bypass the microbicide barrier and infect target cells in the vaginal or rectal environment even in the presence of the microbicide. thus, it is likely that haart-like combination drug therapies will become the most effective means of inhibiting the sexual transmission of hiv. we have evaluated a wide variety of anti-hiv and anti-sti compounds in vitro alone and in combination with one another and have demonstrated that certain patterns of inhibition (additivity, synergy, antagonism) occur between the various classes of compounds. recently, we have compared the combination anti-hiv activity of microbicide compounds in fresh human pbmcs infected with clinical isolates of hiv to the combination activity of the same test agents in cem-ss-based cultures. in general, these two assay systems yield similar combination assay results. to provide a rationale for the combination use of the compounds in a microbicide setting, the same combination of compounds was evaluated in a microbicide-like virus transmission assay. these combination results suggest that higher levels of synergy between virus attachment and reverse transcriptase inhibitors might be expected in the microbicide environment compared to levels predicted for the systemic therapeutic environment. the results of the combination assays with various microbicides will be presented. during the onset of the hiv disease, hiv rna is continually produced in the face of treatment with haart in circulating reservoirs and rt inhibitors are almost ineffective in the postintegration events. among the classes of anti hiv- drugs, protease inhibitors (pi) are the unique to inhibit the hiv- production in chronically infected macrophages. in the progression of hiv infection, the role of the monocytes-derived macrophages (m/m) is further confirmed as they represent chronologically the first cytotype where the viral replication restarts as a consequence of failure or interruption of antiviral therapy. aim of the work was to evaluate the rebound of hiv- production when pi have been removed in hiv- chronically infected m/m and, moreover, to verify the effect of this removal on virus maturation, infectivity and ability to trigger apoptosis in uninfected peripheral blood lymphocytes (pbl). a rebound of p gag protein was measured starting from h after drug removal yet virus infectivity remained log lower than control up to week. inhibition of hiv- replication was still % and % upon amprenavir and m, respectively. these data were confirmed by western blotting and electronic microscopy showing production and release of immature viral particles. moreover, pi (amprenavir and indinavir) treatment dramatically reduced apoptosis of pbl co-cultured with chronically infected m/m and kept cd /cd ratio above the levels of untreated controls until the th day of co-culture. taken altogether, these findings suggest a wide clinical importance for amprenavir and indinavir for their relevant long-lasting antiviral effect in persistently-infected reservoirs of hiv even in case of drug interruption and/or when hiv infection can restart in districts where drugs find not sufficient concentration. moreover, these results strengthen the evidence for an unique positive utilize of pi against ongoing and productive hiv infection. weili jin, salvatore santino, michael wang gilead sciences, foster city, ca, usa background: effective inhibition of hiv reverse transcriptase (rt) currently represents a crucial objective of antiretroviral therapy. capravirine is a second-generation non-nucleoside rt inhibitor (nnrti) that is capable of blocking the replication of certain nnrti-resistant strains of hiv and was recently in clinical development. in this study, we report on the in vitro selection and characterization of viral resistance to capravirine. methods: viral resistance selection experiments were performed in mt- cells with the hiv iiib isolate and increasing concentrations of capravirine. viruses were analyzed genotypically by population sequencing and by single genome sequencing (sgs). recombinant viruses with nnrti mutations were generated from proviral dna clones. phenotypic analyses were performed in mt- cells. results: capravirine resistance selections were initiated at nm (ec of . nm for capravirine). following nine passages in the presence of increasing concentrations of capravirine, the l i mutation emerged in rt and additional passaging led to v d and f c mutations at higher concentrations ( - nm). further increases in capravirine concentrations led to the emergence of a l i + v d + f c triple mutant, which confers > -fold resistance to capravirine. sgs of mixed viral populations from different passages showed that l i, v d and f c were present on the same genome, with l i as the primary mutation, and f c and v d were acquired sequentially at later passages. through sgs analysis, a l i + k r + v d + f c quadruple mutation on the same genome was also observed at higher capravirine concentrations (> nm). recombinant viruses carrying these mutations were produced to assess their susceptibilities to capravirine. conclusions: after extensive in vitro passaging of hivinfected cells in the presence of capravirine, neither k n nor y c mutations in rt were observed. instead, the l i mutation was initially acquired, followed by mutations f c and v d. addition of the k r mutation to the triple mutant genome, l i + v d + f c, appears to further enhance hiv resistance to capravirine. oluwafemi olawuyi , adeyemi falegan medical microbiology, university college hospital, ibadan, nigeria; dentistry, university college hospital, ibadan, nigeria issue: the percentage of aids/hiv is increasing every year in the third worlds, and this is reinforced by the factor that majority of youth in third worlds do not know his/her hiv status. description: a self developed validated and reliable questionnaire [r = . ] was used to collect the data and percentage was used to analyze the data. the population of the study was made up of youth [female and male] in higher institutions, working places, market places and community streets in nigeria, , -sample size, selected through simple random sampling technique. the mean age is . years old. relative risk [rr] calculated is . , i.e. rr > , indicating that the factor is the risk factor, and the confidential interval [ci] for rr at % significant level is . < . < . from the formula, ci lower limit < rr < ci upper limit. lessons learned: seventy percent of the sample population did know his/her hiv status and had had sexual intercourse in the past before, out which % had the unprotected intercourse once or more, % had protected sex while % were not sure of using protection means. while, % have knowledge about own hiv status and had had sexual intercourse before. ten percent have no knowledge about own hiv status and had no sexual intercourse before. conclusion: aids/hiv still remains a killer disease in the third world. however, the lack of knowledge of individual's hiv status remains the only highest risk factor for the spread of the disease in the third worlds. yuichiro habu , , jacob barnor , , norio yamamoto , kahoko hashimoto , , naoko miyano-kurosaki , , koichi ishikawa , naoki yamamoto , david ofori-adjei , hiroshi takaku , , department of life and environmental sciences, chiba institute of technology, chiba, japan; high technology research center, chiba institute of technology, chiba, japan; japan foundation of aids prevention; department of molecular virology, bio-response, tokyo medical and dental university, tokyo, japan; aids research center, national institute of infectious disease, tokyo, japan; department of virology, noguchi memorial institute for medical research accra-ghana, accra, ghana; bach tech corp. rna interference (rnai) is a potentially strong gene interference tool, which had been successfully used to silence many pathogenic viruses including hiv. however, many recent reports have shown that, in long-term assay cultures involving rna viruses such as hiv, escape mutants breakthrough the silencing effect. in the light of this conundrum, it had been proposed that, vector designed to target multiple genes in a synergistic manner, may address the problem. hence, we designed a chimeric rna expression vector which express vif shrna and decoy tar rna by combining vif shrna and decoy tar rna with linker to which dicer was able to recognize for cleavage, as a second generation rnai expression vector system. the synergistic effect of these molecules enhanced the inhibition of hiv- replication in a long-term transduced pbmcs, h , and jurkat cell culture assays ( weeks) and prevented virus breakthrough associated with sirna-mediated escape variants. notably, hiv- replication was similarly suppressed in the control cells expressing only vif shrna for about weeks, but an increase in virus replication was observed afterwards. hiv viral rna extracted and sequenced at this point indicated escape mutants in the cells expressing the vif target in hiv. we confirmed substitution of bases in the vif shrna target sequence. on the other hand, the incidence of mutation was not observed in a sequence of viral rna from the culture expressing the vif shrna-decoy tar rna at the fourth week. interestingly, virus production was inhibited for a long-term by an effect of decoy tar rna, through the rna-protein interaction. combining shrna with decoy tar rna as second-generation anti-hiv shrna may provide practical basis for applying sirna-based gene therapy to the treatment of hiv/aids. introduction: this is a designed efficient gene therapy against aids/hiv. the novelty of this aids vaccine design/concept is seen in the fact that the 'pol' gene encoding for nonstructural proteins (polyproteins that generate three enzymes: reverse transcriptase, integrase and protease) is cloned in a suitable retroviral vector and adult stem cells are transfected by this and reinfused into the circulation to effectively counter hiv replication and antigenic variation. method: the mrna are isolated from adult stem cells and transcribed into cdna with reverse transcriptase. the cdna are then cloned in a suitable retroviral vector (vacinia) carrying 'pol' gene that confers resistance to a strong reverse transcriptase inhibitor drug. the adult stem cells are transfected by the recombinant mixture, and reinfused into the circulation of hiv infected person. result: the transfected stem cells are reinfused to provide renewable source of more and better empowered normal blood cell types that would disrupt and half hiv replication in the circulation. there would be efficient induction of both humeral and cellular mediated immunity with prolonged expression of antigens and protective immunologic memory generation against hiv antigenic variation. conclusions: this aids vaccine design would lead to both efficient prophylactic and therapeutic therapy against aids in that it would effectively take care of the problematic factor of hiv antigenic variation which has long been the main obstacle to potent aids vaccine development. because of the real risk of interspecies transmission and/or reassortment between avian, swine and human influenza a strains, drug susceptibility monitoring of circulating avian and porzine virus strains appears to be warranted for effective application of antiviral drugs like amantadine. this study was designed to gain insight into amantadine susceptibility of avian and porcine influenza a viruses isolated in germany between and . virus strains were isolated in embryonated chicken eggs and passaged one time in mdck cells. plaque reduction assays were applied to examine virus susceptibility to amantadine. genotyping was used to confirm drug resistance. in the result of these antiviral studies, only of the porzine isolates but all avian isolates were shown to be amantadine-susceptible. interestingly, the three amantadinesensitive porzine strains were isolated between and . all porzine influenza a viruses isolated later on were drugresistant and contained the aa substitutions g e, s n, and r q in the matrix protein (m ). additionally, l a was detected in two h n strains. s n and/or l a are well known amino acid substitutions in m that confer amantadine resistance. the role of the pig as an intermediate host of avian and human influenza a viruses, the possible involvement of genetic reassortment, and the high incidence of naturally amantadineresistant porcine influenza a viruses suggest a real risk of emergence of amantadine resistant human viruses. therefore, further studies are ongoing now to evaluate the circulation of the resistant phenotype in pigs, birds and human. recently much attention has been devoted to searching for effective chemotherapeutic agents and vaccines for eradication of this notorious disease. at present only chemotherapy is available to combat avian flu, for instance, tamiflu, approved for the treatment by the us-fda. development of a simple, novel molecule with potential antiviral activity against is essential to treat avian flu viral infection. isatin ( , -dioxoindole), is a versatile lead molecule for designing of potential antiviral agents and its derivatives were reported to possess broad spectrum antiviral activity. methisazone (nmethylisatin- -thiosemicarbazone) was first clinically approved for treatment of pox viral infections, and its derivatives were documented to have anti-influenza activity. based upon this evidence, the present work was initiated to determine the antiviral activity of novel isatin derivatives against avian flu (h n ) in mdck cells. antiviral activity was studied by virus yield assay (ec ), and cytotoxicity by neutral red uptake assay by uninfected mdck cells. all five compounds of a series inhibited the replication of avian flu (h n ) virus replication in mdck cells and compounds spiii- h and spiii- cl were most active (ec . g/ml, cc > g/ml and si > ). details of these studies and results of treatment of influenza-infected mice are discussed. acknowledgement: supported in part by contract noi-ai- and noi-ai- from virology branch, niaid, nih]. arginine-rich peptide conjugated phophorodiamidate morpholino oligomers (arp-pmo) are nuclease resistant antisense compounds that hybridize to target rna in a sequence-specific manner resulting in disrupted rna function. eight arp-pmo were designed to base-pair with various regions of a/pr/ / (h n ) rna and were then evaluated by hemagglutination and plaque assays for their ability to inhibit fluav production in vero cell culture. arp-pmo targeting the aug translation start site of the np or pb segment mrnas, or the -terminus of their respective vrnas, were highly effective, reducing influenza virus titer by - orders of magnitude in a dose-dependent and sequence-specific manner over a period of days. two of the p-pmo, targeting the pb translation start site region (pb -aug) and the terminus of np vrna (np v ), were evaluated by endpoint dilution (tcid ) or elisa assays against another h n strain (a/wsn/ ), as well as a/memphis/ / (h n ) and a/thailand/ (kan- )/ (h n ). the pb -aug arp-pmo generated over % specific reduction of virus level, regardless of viral subtype or methodology, at concentrations in the range of - m. the np v p-pmo yielded similar results, with the exception of considerably lower efficacy against the h n strain, with which it has two base mispairings. studies are planned to further evaluate of at least two arp-pmos in animal models for h n and h n fluva subtypes. macroheterocyclic compounds containing crown fragments and nitrogen atoms show large-scale biological activity. we synthesized series of aza-crown ethers and their derivatives. we also studied anti-influenza and antiherpetic action of some of them. anti-hsv action of studied compounds was tested using cyto-morphological method. hep- cells were infected with hsv- strain us in dose ifu/cell. the cells were incubated in eagle's medium that contained compounds in a dose of − m in experimental samples, or without them in control samples. then cells were fixed with % ethanol and stained with . % acridine orange solution. the amount of infected cells with dna-containing virus inclusion bodies was counted by fluorescent microscopy. anti-hsv activity of compounds was calculated as the difference between of the percentage of infected cells in treated cell cultures to the percentage of infected cells in untreated cell cultures. anti-influenza activity was studied on the model of replication of a/hong kong/ / (h n ) strain in tissue culture of chorio-allantoic membranes of chicken embryos. compounds were used in a dose of − m during the study of their anti-influenza action. diaza- crown- and two of its derivatives have showmen neither anti-hsv nor anti-influenza activity. diaza- crown- derivatives that contain -oxyethyl-or ethoxycarbonyl-fragments decreased amount of cells infected by hsv- by and %, respectively. both of these compounds inhibited replication of influenza virus on . log tid aza- crown- did not show antiviral activity, but both its derivatives proved to be active inhibitors of hsv and influenza virus reproduction. aza- crown- derivatives that contain -amino- -phenyl-propanoyl-or -benzyloxy- -oxapentyl-fragments decreased amount of cells infected by hsv- with virus-specific intranuclear inclusions by and %, respectively. first compound inhibited replication of influenza virus on . log tid and the second one decreased virus amount on . log tid . the results of this study show that aza-crown ethers are the perspective class of compounds for search of new antiviral agents. acknowledgement: this work was partially supported by stcu (grant # ) . robert w. sidwell , kevin w. bailey , min-hui wong , donald f. smee , dale l. barnard , shanta bantia institute for antiviral research, utah state university, logan, ut, usa; biocryst pharmaceuticals, inc., birmingham, al, usa the cyclopentane neuraminidase inhibitor, peramivir (bcx- , rwj- ) has striking inhibitory effects on a spectrum of influenza viruses in vitro, and has also demonstrated significant effects against influenza a (h n , h n ) and b virus infections when administered orally to mice and ferrets. unfortunately, clinical trials with the drug administered orally were not successful, probably due to low blood levels obtained after oral administration. significant plasma drug levels of peramivir persist up to h after intramuscular (i.m.) injection; more importantly, however, is the observation that peramivir remains tightly bound to influenza virus n neuraminidase for over h, suggesting single i.m. or intravenous (i.v.) therapy with the drug may be highly effective against an influenza infection. experiments now in press have indicated that single i.m. peramivir therapy administered up to h after virus exposure was protective to mice infected with influenza a (h n ) virus. in the present study, peramivir was administered i.m. or i.v. in a single injection h pre-virus exposure in separate experiments to mice infected with an influenza a (h n ) virus; efficacy was compared to similar dosages of oseltamivir and oseltamivir carboxylate run in parallel. dosages of and mg/kg of peramivir administered by either route significantly prevented deaths, lessened arterial oxygen (sao ) decline, inhibited development of lung consolidation, and inhibited lung virus titers. the lung assays were performed at varying times after virus exposure. oseltamivir and oseltamivir carboxylate, which do not have the same neuraminidase binding abilities seen with peramivir, were less efficacious in these experiments. delaying the single i.v. therapy up to h after virus exposure also significantly inhibited the virus infection. peramivir appeared to be well tolerated in toxicity control animals run concomitantly with these studies. these data indicate parenterally administered peramivir may hold promise as a therapy for clinical influenza a (h n ) virus infections. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. hiroshi saitoh , naoko miyano-kurosaki , , hiroshi takaku , department of life and environmental sciences, faculty of engineering, chiba institute of technology, chiba, japan; department of life and environmental sciences, faculty of engineering and high technology research center, chiba institute of technology, chiba, japan background: influenza virus causes widespread infection in the human respiratory tract, but existing vaccines and drug therapy are of limited value. recently, small interfering rnas (sirnas) are a powerful tool for sequence-specific, post-transcriptional gene silencing and have a potential therapeutic and prophylactic application against cancer, as well as infectious diseases. here we show that short interfering rnas (sirnas) specific for conserved regions of the viral genome can potently inhibit influenza virus production in cell lines. the influenza virus np gene is a potential target for rnai technology. on the other hand, the baculovirus (acmnpv) can infect a variety of mammalian cells, facilitating its use as a virus vector for gene delivery in viral entry into cells. in this study, we describe the inhibition of influenza virus production by baculovirus-mediated shrna expression vectors. methods: the psv neo-u plasmid vectors and pvl based baculovirus vectors were used in this study. the influenza virus a and b np genes were made into the target and the shrna expression plasmid vectors were constructed under the control of the human u pol iii promoter. the shrna expression plasmids or shrna expression baculovirus vectors introduced into mdck cells, and h later the cells were infected with either a/pr or b/ibaraki virus at a moi of . . at h postinfection, culture supernatants were harvested and assayed to determine the virus titer by plaque assay. conclusion: the findings reveal that newly synthesized np proteins are required for influenza virus replication and provide a basis for the development of shrnas expression plasmids as prophylaxis and therapy for influenza infection in humans. julia serkedjieva, ekaterina krumova, tsvetanka stefanova, nadja nikolova, maria angelova institute of microbiology, bulgarian academy of sciences, sofia, bulgaria a semi-standardized polyphenol-rich extract (pre), obtained from geranium sanguineum l., inhibited the reproduction of influenza viruses types a and b in vitro and in ovo and protected mice from mortality in the experimental influenza virus infection (serkedjieva and manolova, ) . the selective in vitro virus-inhibitory activity of pre was fairly modest and this was in contrast with the significant protection in vivo. thus, the therapeutic effect of pre needed explanation. it was presumed that it might be attributed to a combination of more than one biological activities known for natural polyphenols. we have demonstrated previously that pre manifested strong antioxidant and radical-scavenging activities in model systems (sokmen et al., ) . the current study was undertaken to investigate the effect of the plant extract on the levels of the antioxidant enzymes superoxide dismutase (sod), catalase (kt) and peroxidase (po) in mice lungs during influenza virus infection as well as the effect of pre on the production of reactive oxygen species (ros) and reactive nitrogen intermediates (rni) by alveolar macrophages in influenza virus infected mice. mice were challenged intranasally (i.n.) with - ld of a/aichi/ / (h n ) influenza virus. pre was administered by i.n. instillation h before infection in the dose of mg/kg. it was established that influenza infection induced an increase in sod, kt and po production and on days and after infection their levels reached - % of placebo control. the application of pre brought enzymes values to control levels. influenza infection caused also a significant increase of h o , o •− and no production by alveolar macrophages; the generation of ros and rni peaked on day . pre-treatment before viral challenge reduced this excessive production. in conclusion, the obtained results outlined the antioxidant and radical scavenging properties of the plant extract; pre beneficially modulated the oxidative stress response in influenza virus-induced pneumonia. this alternative mechanism of action might contribute to the overall protective effect in the lethal murine experimental influenza infection. the antiviral activity of s , a natural herb extract, ji-sun kwon , hyun-jeong lee , chi-ung moon , jong-hwan kwak , youn-jeong lee , chang-seon song avian disease laboratory, college of veterinary medicine, konkuk university, seoul, korea; hanyang university, seoul, korea; sungkyunkwan university, seoul, korea; national veterinary research and quarantine services, seoul, korea the antiviral activity of s , one of the traditional korean medical herb extract, against influenza virus was investigated. the % effective concentration (ec ) using plaque reduction assay was . ug/ml and the mean % cytotoxic concentration (cc ) using wst- assay in the mdck cells was ug/ml. oral gavage treatment of the s to balb/c mice infected with a/pr/ / (h n ) influenza virus showed the therapeutic effects as delaying clinical signs, significant inhibition of death and reduction of lung virus titers. to identify the lead molecules, the s was subjected to further fractionation, purification, and isolation of active compounds. the antiviral activity of these natural herb compounds will be discussed. these results suggest that the s is a possible candidate for the development of new antiviral medicine for influenza therapy. hiroshi takaku , , , takayuki abe , hitoshi takahashi , naoko miyano-kurosaki , department life environ. sci., chiba inst. tech., chiba, japan; high tech. res. center, chiba inst. tech., chiba, japan; res. inst. microbial dis., osaka university, osaka, japan; bach tech corp background: the baculovirus autographa californica nuclear polyhedrosis virus (acnpv) has long been used as a biopesticide and as a tool for an efficient recombinant protein production in insect cells. in this study, we examined the immunization of a recombinant baculovirus expressing the influenza virus hemagglutinin (ha) against lethal influenza infection in mice. protection was observed in mice immunized intranasally with not only the recombinant baculovirus but also a wild-type baculovirus. baculovirus was also shown to induce secretion of inflammatory cytokines, such as tnf-␣ and il- , in murine raw . macrophage cell line. results: a varied route of immunization with a recombinant baculovirus expressing the influenza virus hemagglutinin protein of a/pr/ / (h n ) virus against lethal influenza infection was examined in mice. the recombinant baculovirus encoding the hemagglutinin gene under the control of chicken ␤ actin promoter was inoculated twice, weeks apart, at a dose of . × pfu per mouse by intramuscular, intradermal, intraperitoneal, and intranasal routes. mice intramuscularly and intraperitoneally immunized with the recombinant exhibited higher level of production of serum anti ha antibody than those immunized via the other routes, but protection was only achieved by the intranasal immunization. surprisingly, mice immunized with a wild-type baculovirus with intranasal route were also protected from the lethal influenza virus challenge. sufficient protection in mice was achieved by the intranasal immunizations with pfu of either the recombinant or wild-type baculovirus, as evaluated by the reduction of virus titer, production of inflammatory cytokines, and pulmonary consolidations in the lung. these results indicate that infection with a baculovirus induces a strong innate immune response and protection of mice from lethal influenza virus infection. conclusion: baculovirus (cpg motifs) induces a strong innate immune response and protection of mice from lethal influenza virus a and b infection. andrew vaillant , annie lebel , nathalie goyette , guy boivin , jean-marc juteau , phil wyde replicor inc., laval, que., canada; chuq-chul and laval university, st. foy, que., canada; baylor college of medicine, university of texas, houston, tx, usa potent antiviral activity of phosphorothioate oligonucleotides (ps-ons) was observed against influenza viral infections. antiviral activity was sequence-independent, size dependent (optimally active ps-ons were ≥ bases in length) and dependent on the presence of the phosphorothioate modification (hydrophobicity). binding studies showed that rep (a mer degenerate ps-on) interacts with both neuraminidase and hemagglutinin although the sialidase activity of neuraminidase was not affected, suggesting that the structural interactions of these proteins required for influenza activity are the target for this compound. the requirement for hydrophobicity further suggests that the alpha helical regions of hemagglutinin are one of the regions of interaction. the antiviral activity of rep was conserved in many influenza a and b strains suggesting potential therapeutic activity against avian flu and other newly emerging influenza strains. rep aerosol has excellent characteristics for lung deposition and aerosol treatment with rep was well tolerated and highly effective against infections with influenza a both in prophylaxis and h after infection. these results demonstrate the therapeutic potential of aerosolized ps-ons against influenza infection. acknowledgement: supported by nih contract no -ai- . irina v. alymova , y. sudhakara babu , allen portner virology division, department of infectious diseases, st. jude children's research hospital, memphis, tn , usa; biocryst pharmaceutical, inc., birmingham, al , usa bcx is a novel selective inhibitor of human parainfluenza virus infections, which design was based on the threedimensional structure of the hemagglutinin-neuraminidase (hn) protein of newcastle disease virus. compound exhibited striking activity against parainfluenza viruses in vitro and in vivo, and was efficacious in prophylaxis of lethal synergism between parainfluenza virus and streptococcus pneumoniae in a mouse model. present study was conducted to determine if bcx 's resistant variants of the recombinant sendai virus whose hn gene was replaced with that of human parainfluenza virus type (rsev(hpiv- hn) could be selected in tissue culture and animals. for this purpose virus was serially passaged in llc-mk cells at moi . in the presence of increasing (from to m) concentrations of compound; infected × /svj mice were treated with mg/kg/day of bcx twice for five days. treatment started h before infection. individual clones of viruses were analyzed for the presence of mutations. one mutation, e k, on the globular head region of the hn protein was selected in tissue culture after the fifth and eleventh passages of rsev (hpiv- hn) . several mutations in hn gene of rsev (hpiv- hn) were selected in an animal model after the second passage of virus from mice treated with bcx . two mutations, n s and p q, were located in the cytoplasmic domain of hn protein; mutations n s and t a were found on the globular head region of the glycoprotein. only nonconserved amino-acid residues of hn protein were involved in substitutions. all isolated mutant viruses were stable after the five passages in llc-mk cells without drug; did not develop other substitutions in the presence of drug and displayed no resistance to bcx both in vitro and in vivo. infectivity of all mutants was not altered to compare with the wild type of rsev (hpiv- hn) virus. taking together our results indicate that prophylaxis/treatment of human parainfluenza virus infections with bcx may not lead to appearance of clinically significant variant of viruses. kie-hoon jung , michelle mendenhall , lawrence m. blatt , robert w. sidwell , brian b. gowen institute for antiviral research, utah state university, logan, ut, usa; intermune, brisbane, ca, usa hantavirus pulmonary syndrome (hps) is an acute human respiratory disease with remarkably high case fatality rates ( - %) for which the etiological agents are members of the bunyaviridae family, genus hantavirus. maporal (map) virus is a recently identified hantavirus isolated in western venezuela, which is most similar phylogenetically to hantaviruses known to cause hps in southern regions of south america. despite the lack of evidence that map can productively infect humans and cause hps, infection of hamsters closely resembles disease manifestations associated with human hps. hantaviruses, in general, are known to produce little to no cytopathic effect (cpe) in cultured cell lines. unexpectedly, we found that map produces remarkable cpe in several vero cell lines facilitating the evaluation of known antiviral agents, ribavirin and interferon alfacon- . both drugs were highly effective at reducing cpe, as determined by visual examination and neutral red dye uptake, associated with map infection. since much of the observed cpe may be due to apoptosis of uninfected bystander cells, we also developed a quantitative (q)rt-pcr assay to detect copies of map genomic sequence to more directly assess the inhibition of viral replication. data obtained using the qrt-pcr-based assay were consistent with the visual cpe reduction and neutral red-uptake cytotoxicity findings. the development of in vitro antiviral testing methods for map are essential to the evolution of the in vivo hamster disease model of hps. the latter is of utmost importance considering the current need for effective antivirals for the treatment of hps and the lack of a suitable model that does not require biosafety level containment facilities. acknowledgement: supported by contract no -ai- from the virology branch, national institute of allergy and infectious diseases, national institutes of health. nucleoside analogues are widely used in antiviral and anticancer chemotherapy. for this class of drugs, intracellular conversion of the nucleoside analogue into the corresponding mono-, -di-, and -triphosphate after target cell penetration is a prerequisite for biological activity. because of the structural differences from natural nucleosides, this conversion is often inefficient and, as a consequence, therapeutic efficacy is sometimes limited. the free phosphates, or nucleotides, have limited utility in therapy on account of their poor membrane permeability and chemical stability. one approach to improve the therapeutic potential of nucleoside analogues is the delivery of the corresponding nucleotide entities via neutral, lipophilic prodrugs, or protides. the nucleoside aryl phosphoramidate approach, developed by mcguigan and co-workers ( ) has been successfully applied to a number of different nucleosides (azt, d t, dda, d a). the general structure of aryl phosphoramidates encompasses two masking groups, an amino acid ester and an aryl moiety bonded to the phosphate group. in order to apply this protide technology to nucleosides with the potential for anti-hepatitis c virus (hcv) activity, we have undertaken studies designed to probe the effect of varying the natural and unnatural amino acid esters and the aryl groups used as masking groups in the target phosphoramidates. these compounds have been synthesised and evaluated using genotipe b sub-genomic hcv replicon. we have prepared a variety of arylphosphoramidate derivatives from a range of -substituted nucleosides, including azido-cytidine, -azido-uridine, and , -protected variants. with certain nucleoside phophoramidates, we have observed dramatic enhancement (> -fold) of replicon activity relative to the parent nucleoside. the synthesis, biological activity and sar of these compounds will be presented. reference mcguigan, d. cahard, balzarini, j., . mini-review. med. chem. , - . we have identified a series of novel anthranilic acid derivatives that are potent, reversible inhibitors of hepatitis c virus (hcv) ns b polymerase, an essential enzyme for viral replication. the micromolar ns b polymerase inhibitors belong to the n-phenoxyacetylanthranilic acid chemotype. x-ray crystallography determined that the inhibitors bound to ns b between the thumb and palm regions adjacent to the active site. guided by crystallography, subsequent modifications to the hydrogen bonding and lipophilic regions of the inhibitors resulted in greatly improved activity against ns b. further sar studies revealed a second, more potent sub-series where the phenoxy group was replaced by an anilino group. analogs in both subseries showed antiviral activity in a cell-based replicon model of hcv. andrea brancale , dimitrios vlachakis , maria chiara barbera , romano silvestri , colin berry , johan neyts cardiff university, the welsh school of pharmacy, cardiff cf xf, uk; universita' degli studi "la sapienza", dipartimento di studi farmaceutici, roma, italy; cardiff university, cardiff school of biosciences, cardiff cf us, uk; rega institute for medical research, k.u. leuven, b leuven, belgium hepatitis c is a viral infection that affects million people worldwide, including million in the united states and million in europe. the virus establishes a chronic infection in - % of cases and % of affected individuals develop cirrhosis. at the moment there is neither a vaccine nor an effective antiviral therapy available and efforts to identify a specific anti-hcv inhibitor have dramatically intensified in the last few years. many research groups have focused their interest on the enzymes involved in the viral replication and, among these enzyme, the viral helicase/ntpase has proven to be a suitable target for developing novel anti-hcv compounds. compound is a potent inhibitor of the hcv helicase and, although its mode of action is still uncertain, it has been proposed that it acts as competitive inhibitor of rna binding. starting from this hypothesis, we have prepared a series of novel compounds based on the structure of where the benzimidazole moiety has been replaced by different chemical groups, including the negatively charged carboxylate moiety, which should mimic the phosphate backbone of the nucleic acid. the synthesis, the enzyme inhibition and the biological evaluation in replicon of these novel compounds will be presented and analyzed. dale r. cameron migenix inc., wesbrook mall, vancouver, bc, canada v s l hepatitis c virus (hcv), a leading cause of liver disease, continues to be an attractive target for new drug development. among the more favourable approaches to developing new hcv drugs is to target the rna-dependant rna (rdr) polymerase (ns b), which has been shown to be an essential enzyme for replication. there are several published non-nucleoside inhibitors of this polymerase (some in clinical development) and several published allosteric binding pockets on the protein they target. to be successful, traditional lead identification can be timeintensive, costly and have large infrastructure requirements. increasingly, a push towards effective computational-based screening has led to the development of virtual screening tools. such tools allow investigation of large quantities of compounds in silico for particular properties without the need for compound synthesis or high throughput screening. moreover, these techniques require only a modest infrastructure investment and are very efficient. we employed the openeye set of screening tools (omega, rocs and eon) in concert with publicly available hcv inhibitor information, and commercial databases to identify novel leads. the inhibitor coordinates from a protein-inhibitor complex crystal structure were utilized as the target. available compound databases (asinex and chembridge) were utilized as the testset of compounds. filtered compound conformers were generated using omega and compared with the template using rocs with post-analysis by eon. visual analysis to maximize particular desirable binding features while minimizing protein-inhibitor steric clash allowed the list of potential hits to be further narrowed. multiple classes of compound were identified from the above procedure and after sourcing a subset of the actual compounds or close analogs, they were tested for enzymatic inhibition activity and further characterized. iteration of the process resulted in the identification of a lead compound class containing multiple active compounds, one with reasonable replicon activity. in conclusion, readily available structural and database information and virtual screening tools can be successfully utilized to identify novel inhibitors of hcv rdr polymerase which, in turn, can serve as novel leads for developing new therapies for treating hcv. synthesis, antiviral activity, and cytotoxicity of some novel quinazolin- ( h)-one derivatives a series of novel -bromo/ , -dibromo- -( -oxo- -phenyl- h-quinazolin- y-l)-benzenesulphonamides were synthesized by condensation of -substituted benzo[ , ]oxazine- -ones and sulphonamide. their chemical structures were assigned by means of spectral analysis (ft-ir, pmr, ms). synthesized compounds were screened for in vitro antiviral activity against human pathogenic viruses (hiv, hcv, hsv, vv). -bromo- -( -oxo- -phenyl- h-quinazolin- y-l)benzenesulphonamide (sps-ii) and -( -oxo- -phenyl- hquinazolin- y-l)-benzenesulphonamide (sps-i) inhibits the replication of hiv- in acutely infected mt- cells at a concentration of approximately g/ml, while not being toxic to the host cell at a concentration of or > g/ml (selectivity index: and > ), respectively. in huh - cells sps-i inhibited hcv rna synthesis at ec of g/ml, while at cc for cell growth g/ml. sps-ii inhibited the virus-induced cytopathicity in human embryonic lung (hel) cell infection with hsv- , hsv- or vaccinia (vv) at a concentration of g/ml, while not being toxic to the cells up to a concentration of g/ml. further molecular modification in this series of compounds may help in optimising their antiviral activity. wengang yang, yongnian sun, avinash phadke, milind deshpande, mingjun huang achillion pharmaceuticals, new haven, ct , usa hcv nonstructural protein ns b is the catalytic subunit of the replication complexes, possessing a motif characteristic of rna-dependent rna polymerases. biochemical assays using recombinant ns b have been used to investigate ns b nonnucleoside inhibitors. however, the inhibitory effect of compounds often varies with the forms of recombinant ns b and the concentrations of the template and/or primer used in the assays. in addition, it does not always correlate to that obtained with replicon-containing cells. these observations have cast concerns about the validity of these cell-free assays. in the report, we explored replication complexes, isolated as crude membrane fractions from replicon-containing cells, for their competency to synthesize viral rna in vitro as well as their responsiveness to ns b inhibitors. after optimizing the experimental conditions, two species of nascent viral rna, one double-stranded and the other single-stranded, were readily detected. the addition of ns b nucleotide inhibitor blocked synthesis of both species. the presence of nonnucleoside inhibitors, however, inhibited mostly single-stranded rna (ssrna) synthesis. in addition, the replication complexes isolated from the cells containing a replicon that carried a resistant mutation in ns b to the nonnucleoside inhibitor were able to synthesize the same amount of ssrna in vitro regardless of the presence or absence of the inhibitor, demonstrating that the phenomenon is due to the specific inhibitory effect of the compound on ns b. combining with kinetic studies that ssrna synthesis was inhibited only when the nonnucleoside inhibitor was present during the pulse period, we conclude that ssrna synthesis catalyzed by the replication complexes in vitro is likely derived from the de novo initiation. we have recently reported the synthesis and antiviral activities of a ring-expanded ("fat") nucleoside analogue, called nz- , that inhibits both hcv and hiv in vitro with ec values ranging in micromolar concentrations or less, and little or low toxicity to the host cells. in this part ii of the presentation on this subject, we report our preliminary results on mechanistic studies of anti-hcv activity of this compound, along with the synthesis and antiviral activity of a few additional analogues in the series. in light of the fact that hcv is a major co-infection in patients infected with hiv, and that a number of them ultimately die of end-stage hcv-related complications including liver cirrhosis and hepatocellular carcinoma, a drug with dual inhibitory characteristics against both viruses is highly desirable and timely. nigel bourne, ronald veselenak, richard pyles, minkyung yi, stanley lemon the university of texas medical branch, galveston, tx, usa more than million people worldwide are estimated to be infected with hepatitis c virus (hcv). in the majority of these people a chronic infection is established which can result in serious long-term liver damage including progressive fibrosis, cirrhosis and hepatocellular carcinoma. in fact, hcv is believed to cause more than , cases of liver cancer annually worldwide and accounts for at least % of liver transplants in the us. current treatment options are limited and there is a high treatment failure rate. thus, there is a real need for new treatment options. amantadine has been evaluated as a treatment for chronic hcv infection in a number of clinical studies both as a monotherapy and in combination with other therapeutics. however, the results of these trials have been contradictory and at this time the clinical potential of amantadine as a therapy for chronic hcv infection remains unclear. recent studies have shown that the small hydrophobic hcv p protein forms an amantadine sensitive ion channel providing a possible basis for antiviral activity. we examined the ability of amantadine to reduce hcv replication in both subgenomic and full-length hcv replicons of genotypes a strain h c and genotype b strain n. in these studies amantadine failed to reduce viral rna replication in any of the replicons tested. further, in infectious virus assays using hcv genotype a strain jhf- um amantadine failed to reduce viral rna levels under any of the conditions tested. however, in these infectious virus studies, when the viral inoculum was treated with amantadine prior to infection of cell monolayers, or when the amantadine was added to cells h after virus adsorption there was a significant reduction in the number of infectious viral foci observed after h incubation (p < . and < . , respectively). these results suggest that even in the absence of a direct impact on rna replication amantadine has antiviral activity. we are currently evaluating amantadine for activity in infectious hcv genotype a assays to further define its antiviral spectrum of activity. studies to more fully define its mechanism of action in the virus life cycle are also underway. dominique dugourd, raymond siu, jeremy fenn migenix inc., vancouver, bc, canada celgosivir is an alpha glucosidase inhibitor that is being developed for the treatment of hepatitis c virus (hcv) infections in humans. the purpose of this study was to evaluate the in vitro antiviral activity of celgosivir and its primary active metabolite, castanospermine, when combined with current approved therapies (ribavirin, interferon ␣- b, or both) in a surrogate model of hcv (bovine viral diarrhea virus (bvdv)). compounds alone or in combination were tested against bvdv in infected madin-darby bovine kidney (mdbk) cells. synergies were analyzed using isobolograms and volume of synergy measurements (macsynergy ii tm software). the celgosivirinterferon ␣ b combination was significantly more synergistic than the celgosivir-ribavirin combination (∼ -fold), or the ribavirin-interferon ␣ b combination (∼ -fold). similarly, the castanospermine-interferon ␣ b double combination was more synergistic than the castanospermine-ribavirin combination (∼ -fold), or the ribavirin-interferon ␣ b combination (∼ . -fold). the combinations of celgosivir-interferon ␣ b or castanospermine-interferon ␣ b led to significant decreases in the ec s of celgosivir (up to > -fold) and castanospermine (up to > -fold). the effective ec s of celgosivir or castanospermine were further reduced by the addition of ribavirin. the cytotoxicity of the double and triple combinations was additive or less than additive, indicating that combinations of celgosivir or castanospermine with ribavirin and/or interferon ␣ b were generally less toxic than expected. these results indicate that the combination of celgosivir with interferon ␣ b or with interferon ␣ b and ribavirin may be effective in the treatment of hcv. pegylated interferon ␣ plus ribavirin is the current standard of care for the treatment of chronic hepatitis c virus (hcv) infections. this regimen results in sustained virologic response in only about % of patients and is associated with significant treatment-associated toxicities. a number of approaches are being used to identify novel therapeutic combinations with better tolerability and/or efficacy. inhibitors of endoplasmic reticulum (er) ␣-glucosidase have been shown to inhibit viral replication and secretion and may have utility as part of new multi-drug treatment cocktails. the ␣-glucosidase inhibitor celgosivir is currently being evaluated in combination with pegylated interferon ␣ and ribavirin in humans. the purpose of this study was to evaluate the antiviral effects of combinations of celgosivir and castanospermine, the primary active metabolite of celgosivir, with other antiviral agents having diverse mechanisms of action. the effect of the combination of celgosivir or castanospermine with the nucleoside analogue nm- , amantadine, and another iminosugar, n-butyl-deoxynojirimycin (nb-dnj) was determined in a cytopathic assay using the hcv surrogate virus bovine viral diarrhea virus in madin darby bovine kidney cells. synergies were analyzed using isobolograms and volume of synergy measurements (macsynergy ii tm software). volumes of synergy indicated that the castanospermine and nb-dnj combination was additive, while the celgosivir and nb-dnj combination was synergistic at high nb-dnj concentrations (> m). celgosivir and castanospermine were synergistic with both amantadine and nm- , with volumes of synergy between and m%. isobologram analysis confirmed these synergistic interactions. these results indicate that celgosivir could be considered in combination regimens containing drugs that directly target viral replication like nm- . mechanism(s) of synergy are under investigation. department of biotechnology, yonsei university, seoul - , korea hepatitis c virus (hcv) is an enveloped virus with positivestranded rna genome of approximately . kilobases and a major cause of non-a and non-b hepatitis, leading to liver cirrhosis and hepatocellular carcinoma. combination of interferon-␣ (ifn-␣) and ribavirin is the current standard therapy for the treatment of hcv infection, but there is no specific antiviral therapy available. the hcv viral genome encodes a single polyprotein of approximately amino acids, which is proteolytically processed by a combination of host and viral proteases into at least distinct structural and nonstructural proteins. the structural proteins include c, e , e , and p and the nonstructural (ns) proteins include ns , ns , ns a, ns b, ns a, and ns b. as new hcv specific therapies, small-molecule inhibitors against hcv enzymes including ns b protein, the viral rna-dependent rna polymerase (rdrp), and ns protease are in clinical tests. however, rapid emerging of drugresistant mutants has been hampering their practical clinical applications. recently, we have shown that phosphorylation of hcv rna polymerase by protein kinase c-like (prk ) regulates virus rna replication. hcv rna replication was inhibited when prk expression level was down-regulated by using a prk -specific sirna. in this study, we investigated the anti-hcv effect of prk inhibitors in an hcv subgenomic replicon system. treatment of the replicon cells with prk inhibitors suppressing the endogenous prk activity inhibited the phosphorylation of hcv rna polymerase and resulted in suppression of hcv rna replication in a dose-dependent manner. furthermore, the prk inhibitor in combination with ifn-␣ more effectively inhibited hcv rna replication than ifn-␣ alone. because the prk inhibitor did not show cytotoxicity in the cell-based drug inhibition studies and cellular proteins rarely get mutated, prk can serve as a cellular target for therapeutic intervention of hcv replication. specific inactivation of prk activity will provide an opportunity to interfere with hcv rna replication. haitao guo , tianlun zhou , ju-tao guo , andrea cuconati , anand mehta , timothy block drexel university college of medicine, doylestown, pa, usa; nucleonic inc., irvine, ca, usa; hepatitis b foundation, doylestown, pa, usa more than million people worldwide are chronically infected with hepatitis b virus (hbv). the major complication of chronic hepatitis b is the development of primary hepatocellular carcinoma (hcc), which causes an estimated , deaths annually. currently clinical treatments (␣-interferon and nucleoside analogs) of chronic hepatitis b rarely cure the virus infection. this is due, at least in part, to their failure to eliminate viral covalently closed circular (ccc) dna from the nuclei of infected hepatocytes. hbv cccdna is essential to the virus life cycle by serving as the template for the transcription of the pregenomic rna and of the subviral rna species. its elimination during chronic infection is considered critical to long-term therapy. however, cccdna has not previously been targeted in high throughput screens of small molecule libraries. to screen compound libraries for antiviral drugs targeting cccdna, we set out to develop a cell-based assay suitable for high throughput screening. since cccdna is time-consuming to assay, it was desirable to use a viral gene product that could serve as a reporter for intracellular cccdna level. we predicted that the secretion of hbv e antigen (hbeag) by hepad cells, a hepg -derived tetracycline inducible hbv expression cell line, would be cccdna-dependent. this is because a large portion of pre-core mrna leader sequence in the terminus of integrated viral genome was deleted, preventing hbeag expression from transgene, but could be restored from the terminal redundancy of pre-genomic rna during viral dna replication and subsequent cccdna formation. our experimental results showed that following induction, hepad produced and accumulated cccdna, which became detectable between and days. hbeag synthesis and secretion into culture fluid were dependent upon and proportional to the level of cccdna detected. therefore, the secretion of hbeag by hepad cells could potentially serve as a convenient reporter for the high throughput screening of novel antiviral drugs targeting hbv cccdna. kathy aldern, james beadle, karl hostetler university of california, san diego and the veterans medical research foundation, san diego, ca, usa (s)-hpmpa is a broad spectrum antiviral active against orthopoxviruses, hbv, cmv, hsv, and other herpes group viruses. we have shown that hdp-(s)-hpmpa has greatly enhanced antiviral activity against these viruses. in addition, while hpmpa itself is nearly inactive against hiv, we showed that hdp-(s)-hpmpa exhibited an ec > logs less than unmodified hpmpa in mt- cells by p reduction assay. to evaluate the metabolic basis for the increased antiviral activity, we studied and compared the cellular uptake of radiolabeled cdv, (s)-hpmpa and their hdp-esters and conversion to hpmpa-diphosphate (hpmpapp) and cdv-diphosphate (cdvpp) in mrc- human lung fibroblasts using hplc partisil sax ion exchange chromatography. cellular uptake of hdp-cdv and hdp-(s)-hpmpa was similar. however, when cells were exposed to the respective drugs for , and h, hpmpapp appeared much earlier than cdvpp and reached levels several fold greater than observed with hdp-cdv. drug wash out experiments were carried out in mrc- cells exposed to radiolabeled hdp-cdv and hdp-(s)-hpmpa. after h, the culture medium was removed and replaced with complete medium without drug and the levels of hpmpapp and cdvpp were measured by hplc every days for - days. levels of the diphosphates declined slowly with a t / of - days. in conclusion, hdp-(s)-hpmpa is converted to its diphosphate more rapidly than hdp-cdv and reaches higher intracellular levels. this may explain, in part, its greater antiviral activity. the antiviral activity and oral bioavailability of cidofovir (cdv) is enhanced when the phosphonate is esterified with various straight chain alkoxyalkyl groups. the length of this chain is an important determinant of antiviral activity and selectivity. however, in some cases, rapid metabolism to an inactive short chain metabolite was observed. to enhance the metabolic stability of these esters, we synthesized cidofovir alkoxyalkyl esters bearing methyl groups on the penultimate carbon of the alkyl chain. enzymatic stability of -me-hdp-cdv ( ) was tested in liver s fractions from various species. in mouse and human liver s fractions, compound was completely stable for min while - % of the straight chain hdp-cdv was metabolized. the branched alkoxyalkyl esters were then evaluated in cells infected with vaccinia, cowpox and ectromelia viruses. the branched methyl analogs were substantially more active than cdv and equal to or slightly more active than the straight chain analogs. compound retained full activity compared to hdp-cdv and compound showed greater activity against orthopoxviruses compared to its unbranched analog. we believe that the structural modification of the alkyl chain slows the formation of inactive metabolites, possibly by interfering with oxidation and may result in better pharmacokinetics and more potent antiviral activity against orthopoxvirus infection in vivo. cidofovir ([ -(s)- -hydroxy- -(phosphonomethoxy)propyl]cytosine, hpmpc) is a broad spectrum antiviral agent clinically used for treatment of aids-related cmv retinitis. cidofovir has limited oral bioavailability (< %), attributed to ionization of its phosphonic diacid moiety under physiological conditions. we have shown that masking of this group by conjugation of the cyclic form of the drug (chpmpc) via a ser side chain p-o ester linkage with x-ser dipeptides, where x = a hydrophobic amino acid, can result in prodrugs that afford significantly improved biological availability of parent drug in an animal model. here we describe the total synthesis of novel cyclic cidofovir prodrugs ab of l-val and l-phe using an alternative conjugation strategy, viz. via an ethylene glycol link utilizing p-o and c-o ester bonds. the preparation of the hpmpc synthon from r-glycidol used our modification of the literature procedure (brodfuehrer et al., ) , involving reaction of tritylated (r)-glycidol directly with unprotected cytosine to achieve regiospecific opening of the epoxide ring, followed by reaction with benzoic acid anhydride to obtain the desired n-benzoyl intermediate needed to continue the synthesis. pybop was used as condensing agent in a convenient, one-pot conversion of hpmpc to chpmpc and subsequent esterfication of the latter by the ethylene glycol-modified amino acids. the prodrugs were converted to drug by cellular (caco- , hff) and tissue (liver and intestinal) homogenates, but did not show enhanced oral bioavailability when evaluated in a rat model, suggesting that such compounds may be useful for understanding the effectiveness of in drug delivery. cidofovir (cdv) is a broad-spectrum anti-viral agent that is used to treat aids-related cytomegalovirus (cmv) retinitis and other cmv infections. cdv has good in vitro activity against orthopox viruses, including smallpox; however, its use is limited because of the drug's low oral bioavailability and poor transport into cells. in order to improve its oral bioavailability, our group has synthesized a series of dipeptide and amino acid prodrugs of the cyclic analog of cidofovir (ccdv). in the current project, we examined the cytotoxicity and antiviral activity of the prodrugs, showing that the compounds are not cytotoxic and have diverse activity against hcmv and orthopox viruses (vaccinia and cow pox) with % inhibitory concentrations ranging from . to . and m and greater, respectively for the two virus types. in vitro and in situ perfusion studies established that the permeability of the prodrugs is enhanced more than -fold and that the transport is mediated, at least in part, by the intestinal dipeptide transporter. we also have found that the bioavailability of the prodrugs is dependent upon the prodrug structure and that we can achieve up to an eight-fold increase in bioavailability over the parent compound in vivo. drug stability experiments showed that in gastrointestinal and liver homogenates, the ccdv prodrugs are enzymatically hydrolyzed to the parent compound. it is clear from this work that the biologically benign dipeptide moiety, strategically linked to the drug to mask its anionic properties, significantly enhances intestinal transport of ccdv, creating the possibility of an orally bioavailable form of ccdv with low toxicity. acknowledgement: supported by funds from tsrl inc, the university of michigan, and nih grants r ai and u ai . lawrence trost , bernhard lampert , lloyd frick , merrick almond , george painter chimerix, inc., durham, nc, usa; dmpk advisor, chimerix, inc., durham, nc, usa foscarnet, a pyrophosphate analog approved for the treatment of cmv retinitis and acyclovir-resistant herpes infections in immunocompromised patients, is active against highly drug resistant strains of hiv- . however, the clinical utility of foscarnet is limited because it requires controlled intravenous infusion and is associated with high risks of renal impairment and seizure caused by alterations in plasma minerals and electrolytes. lipid conjugation has been shown to increase the in vitro activity, improve oral bioavailability, and reduce the toxicity of several antiviral drugs requiring intravenous administration because of poor bioavailability. in the case of foscarnet, conjugation to methylbatyl alcohol (cmx ) decreases the apparent ec value against hiv- by up to -fold. cmx was esterified to produce cmx in order to increase solubility and to protect against decarboxylation of the foscarnet moiety during passage through the stomach. here we present the results of a preliminary toxicology and toxicokinetic study of the methylbatyl alcohol conjugate of foscarnet methyl ester (cmx ). rats were given oral doses of , and mg/kg cmx daily for days. there were no clinical signs of toxicity. body weight and food consumption were comparable to controls and serum biochemistry, hematology, coagulation parameters and urinalysis were normal. there were no gross findings at necropsy, no effects on organ weights and no findings by histopathological examination of a wide range of tissues. importantly, there were no changes in serum biochemistry parameters or histopathological examination that were indicative of the renal impairment or serum electrolyte changes that are associated with foscarnet. oral dosing resulted in significant plasma exposure to cmx (c max > g/ml), the biologically active deesterified form of cmx . in conclusion, cmx is absorbed after oral administration, converted to cmx , and has a good preliminary toxicity profile. these results support the development of cmx for the treatment of drug resistant hiv infection. zhiqian wu , julie breitenbach , ulrika erickson , john hilfinger , john drach , gordon amidon department of pharmaceutical sciences, college of pharmacy, the university of michigan, ann arbor, mi, usa; school of dentistry, the university of michigan, ann arbor, mi, usa; tsrl, inc., ann arbor, mi, usa vaccinia virus is a surrogate model system for study of pox virology and development of antiviral therapeutics. the potent anti vaccinia virus activity and various shortcomings of vidarabine make it a good candidate for improvement by utilizing prodrug strategy. vidarabine is a polar nucleoside drug with low membrane permeability and rapid degradation by adonesine deaminase. -monoester prodrugs of vidarabine with various amino acids promoieties (l-valine, l-isoleucine, l-phenylalanine. laspartic acid, l-proline) are synthesized and evaluated for their stability, permeability and activity against vaccinia virus. prodrugs exhibit different hydrolysis rate in caco- cell homogenate (t / : - min). -l-isoleucyl and -l-valyl monoester prodrugs exhibit comparable bio-conversion rate and hpept mediated uptake as well as caco- permeability with valacyclovir, a commercially marketed oral amino acid ester prodrug. both prodrugs have potent activity against vaccinia virus and are resistant to ada . preliminary animal study shows -lisoleucyl vidarabine results in > -fold increase in circulating vidarabine level. the results suggest that it may be possible to use amino acids prodrug strategy to improve vidarabine as anti vaccinia virus agent. vidarabine [ -␤-d-arabinofuranosyl)adenine or ara-a) was originally investigated as an anti-tumor agent and was later found to be active against herpes simplex virus (hsv) types and . it was the first fda-approved drug for treatment of systemic hsv infections. although replaced by acyclovir and analogs for most applications, vidarabine remains an alternative therapy for acyclovir-resistant hsv and varicella-zoster virus infections. despite its proven efficacy, vidarabine suffers some limitations including: (i) metabolism by adenosine deaminase (ada) to its inactive hypoxanthine homolog (ara-h); (ii) low lipophilicity and membrane permeability and (iii) poor aqueous solubility, thus limiting options for parenteral and peroral delivery. our recent interest in vidarabine was triggered by our discovery that it was ∼ -fold more active against vaccinia (vv) and cow pox (cpv) viruses than was cidofovir in plaque reduction assays. its activity was enhanced about -fold by combination with m -deoxycoformycin (pentostatin, a potent inhibitor of ada) thereby providing significant superiority to cidofovir. from these results and our earlier studies on -substituted vidarabine analogs (lipper et al., . mol. pharmacol. , - ), we determined that minimizing metabolism of vidarabine by synthesizing -amino acid substituted prodrugs gave a significantly more potent anti-pox virus agent. we found that amino acid ester prodrugs of vidarabine are active against vv at non-cytotoxic concentrations. further, using cell homogenates, purified enzyme and intact cell systems, we showed that the prodrugs are resistant to inactivation by ada. the prodrugs also had enhanced transport potential, most likely targeting the intestinal dipeptide transporter. finally, oral delivery of the prodrug to the small intestine resulted in a -fold increase in vidarabine plasma levels when compared to unsubstituted vidarabine. these properties make the prodrugs of vidarabine good candidates as orally bioavailable anti-pox virus agents that are stable in the presence of ada. acknowledgement: supported by funds from tsrl inc. and the university of michigan. ulf goerbig , anne baum , jan balzarini , chris meier university of hamburg, institute of organic chemistry, hamburg, germany; rega institute for medical research, katolieke universiteit leuven, leuven, belgium the cyclosal pronucleotide system has been designed for an intracellular delivery of therapeutically active nucleoside monophosphates. as part of recent work on the cyclosal approach, the interaction of cyclosal nucleotides with cholinesterases has been investigated. it is known that organo-phosphates may act as irreversible inhibitors of cholinesterases (suicide mechanism). in the case of cyclosal nucleotides, cholinesterase inhibition could lead to unwanted side effects in a possible therapeutic application. there are two types of cholinesterase found in humans, the highly specific, physiologically important acetylcholinesterase (ache) and the much more unspecific butyrylcholinesterase (bche) of unknown physiological importance. fortunately, no inhibition of ache was observed for a variety of different cyclosal nucleotides. in contrast, bche inhibition was found in some cases. the anti-hiv-active , -bis-tertbutyl- -fluoro-cyclosal-d t monophosphate is the first cyclosal derivative combining three desired properties: successful intracellular delivery of nucleotides, sufficient hydrolytic stability and strongly reduced inhibitor activity towards bche. because of the promising properties of this compound, we combined this mask developed for d t with the antiviral active nucleoside analogues like d a, dda, azt and acyclovir. in this contribution we present the synthesis, hydrolysis stability, inhibition behaviour towards bche and anti-hiv data of these new compounds. henning jessen , wolfgang fendrich , tilmann schulz , jan balzarini , chris meier university of hamburg, institute of organic chemistry, hamburg, germany, rega institute for medicinal research, katholieke universiteit leuven, leuven, belgium cyclosal-pronucleotides are used for the delivery of antivirally active nucleotides into cells via a ph triggered selective hydrolysis. to distinguish between intra-and extra-cellular environment enzyme-cleavable side chains were introduced in the aromatic moiety of the pronucleotide to enrich the compound inside cells. this behavior will further be described as "lock-in"effect. many other different cyclosal pronucleotides have been designed, all showing different hydrolysis properties and antiviral data, both originating from the nature of the cyclosal-moiety as well as of the nucleoside. to examine these differences, analytical tools of high accuracy and sensitivity were needed, being structurally as close as possible to the lead compounds. these requirements are met by intrinsically fluorescent nucleosides coupled to different cyclosal masking groups. for analysis of the purine-type nucleosides iso-da with high intrinsic fluorescence properties was chosen and converted into iso-a, iso-dda and iso-d a. for the pyrimidine-type series the fluorescent nucleoside m k was synthesized as well as the dideoxy-compound dm k. these nucleosides were transformed into different cyclosalpronucleotides and tested for their suitability for fluorescence analysis. in fact, an improvement of sensitivity by a factor of compared to uv-detection was found for some of the compounds (pmol detection). for all compounds fluorescence and absorbance spectra were recorded to determine the absorption and emission maxima. the new compounds lacked activity against hiv- and hiv- strains. however, the compounds showed low cytotoxicity, which is important for their usability as fluorescent probes in cells. due to the analytical sensitivity, a simple model uptake study could be carried out, employing an u-tube with two aqueous phases, which were separated by an unpolar organic solvent simulating a diffusion barrier. the properties of the aqueous phases were varied and an enzyme-driven enrichment of a "lock-in"-modified intrinsically fluorescent cyclosal-pronucleotide passing the diffusion barrier could be simulated. nicolas gisch , jan balzarini , chris meier university of hamburg, institute of organic chemistry, hamburg, germany; rega institute for medical research, katholieke universiteit leuven, leuven, belgium cyclosal-pronucleotides efficiently deliver therapeutically active nucleoside monophosphates in human cells. "lock-in"-cyclosal-pronucleotides -the so-called second generation of cyclosal-compounds -have been designed to trap the compound by intracellular cleavage of esterase-cleavable moiety. one disadvantage of the "lock-in"-compounds is their high chemical stability, which leads to a delayed drug delivery. therefore, conceptually different, enzymatically activated cyclosalpronucleotides have been developed. in this concept lipophilic donor substituents attached to the aromatic ring are converted into a polar acceptor substituent by intracellular enzymatic cleavage. as a consequence the liberated acceptor group leads to a strong decrease in hydrolysis stability and a rapid formation of a charged intermediate is the result. from the phosphodiester intermediate the nucleotide is released subsequently. the concept, synthesis, characterization and in vitro antiviral evaluation of the third generation of cyclosal-pronucleotides will be presented. tomas cihlar , richard mackman , adrian ray , dean boojamra , lijun zhang , deborah grant , hon hui , jennifer vela , neil parkin , yolanda lie , kirsten white , michael miller , gerry rhodes , manoj desai gilead sciences, foster city, ca, usa; monogram biosciences, so. san francisco, ca, usa background: n(t)rtis are currently used as a backbone of antiretroviral combination therapy. however, their long-term benefit can be limited by adverse effects, resistance development, drug-drug interactions, and sub-optimal efficacy in treatment-experienced patients. therefore, we searched for novel nucleotide analogs with improved pharmacological profiles. methods: phosphonomethoxy- -fluoro- , -dideoxydidehydroadenosine (gs ) was selected from a broad range of nucleoside phosphonate analogs. phosphoramidate prodrug technology previously explored with tenofovir was applied to gs , resulting in the identification of gs (ethylalaninyl phenyl ester of gs ). results: gs exhibits potent anti-hiv- activity in primary lymphocytes and t-cell lines (ec < nm). low cytotoxicity (cc > m) was observed in multiple cell types including renal cells. diphosphate metabolite of gs was shown to act as an obligatory dna chain terminator and a competitive inhibitor of hiv- reverse transcriptase (rt) (k i = . m). unlike ddi, d t, or d fc, neither gs nor its prodrugs inhibited mitochondrial dna replication in hepg cells. in a phenosense assay, gs retained its full activity against hiv- variants with k r, m v or l v mutations in rt. viruses with ≥ thymidine analog mutations showed ≤ fold reduced susceptibility to gs , a shift that was smaller than that of any other tested nrti. following an oral dose of mg/kg gs in dogs, the bioavailability of prodrug exceeded %, resulting in high intracellular levels ( . ± . m) and prolonged retention (t / > h) of gs diphosphate in blood lymphocytes. conclusions: both gs and its prodrug gs exhibit favorable in vitro pharmacological profiles including less resistance due to rt mutations than approved nrtis. gs possesses good in vivo pharmacokinetic properties and thus represents an attractive development candidate with potential for clinical efficacy in both treatment-naive and nrti-experienced patients. martin mcdermott, gabriel birkus, ruth wang, holly macarthur, xiaohong liu, nilima kutty, tomas cihlar, craig gibbs, swami swaminathan, arnold fridland, william lee gilead sciences, inc., foster city, ca, usa gs- and gs- are alkylalaninyl phenyl ester prodrugs of tenofovir (tfv; -[( -phosphonomethoxy)propyl]adenine) and a novel nucleotide analog fd ap (phosphonomethoxy- -fluoro- , -dideoxydidehydroadenosine), respectively. both gs- and gs- exhibit potent in vitro anti-hiv- activity, favorable resistance profile, and low cytotoxicity. compared to tenofovir disoproxil (viread), both prodrugs are significantly more stable in plasma and deliver > -fold greater levels of active diphosphate metabolites into pbmcs in vitro and in vivo. the initial step in the intracellular activation of gs- and gs- is the hydrolysis of the alanine carboxyester by an unknown hydrolytic enzyme. the isolation and identification of this enzyme from human pbmcs is reported here. results: a major enzyme capable of cleaving gs- and gs- was purified from human pbmcs and was separable from esterases able to cleave alpha napthyl acetate (ana). the increase in specific activity of prodrug hydrolase achieved was -fold. sds-page analysis showed the presence of a prominent protein band at kda, which was identified by ingel tryptic digestion and ms/ms sequencing of the resultant peptides as lysosomal carboxypeptidase a (cathepsin a, ec . . . , cata). the biochemical properties of purified prodrug hydrolase matched those of cata. recombinant cata and the isolated prodrug hydrolase displayed nearly identical susceptibility to hydrolase inhibitors and substrate preference against a panel of tenofovir phosphoramidate prodrugs. incubation of both enzymes with [ c]gs- and [ h]difluorophosphonate resulted in the labeling of an identical kda protein (catalytic subunit). both labeled bands reacted with polyclonal antibodies specific for human cathepsin a. finally, following incubation with gs- , approximately - -fold lower intracellular concentrations of tfv metabolites were detected in fibroblasts from patients expressing non-functional cat a (cat a-cells) compared to normal control fibroblasts (cat a+ cells). center for drug delivery and nanomedicine, university of nebraska medical center, omaha, ne, usa nucleoside -triphosphate (ntp) is the biologically active form of many antiviral nucleoside analogs capable of efficiently blocking the production of viral nucleic acids in infected cells. we describe novel microparticulate formulations for encapsulation of ntp, drug delivery and antiviral therapy of respiratory infections. polymer networks (poloxagels) consisted of crosslinked poloxamers and cationic polymer molecules were designed, synthesized and characterized by loading with ntp and interaction with cells. poloxagel-ntp formulations could be obtained by simple mixing of the aqueous solution of ntp with the aqueous dispersion of poloxagel and subsequent lyophilization. drug loading was equal up to % by weight. in this form, phosphates groups of ntp are complexed with amino groups of polycationic backbone of poloxagels, and the formulations could be stored at room temperature for many months without degradation of ntp. the particle size of aqueous poloxagel-ntp dispersions was low, with a hydrodynamic diameter of . - . m. the rate of passive drug release in physiological solution was from to % of loaded drug during the -h period. these formulations were effectively consumed by many types of cells. significant amounts of drug and poloxagels were detected in the cellular interior after only - h of incubation. in the presence of cellular membranes drug release from poloxagel-ntp formulations was dramatically increased. we attribute this effect to the triggered release of the bound ntp as a result of competitive interaction of polycationic backbone of poloxagels with phospholipids of cellular membranes. mucoadhesive properties of poloxamers may additionally enhance binding of poloxagels with airways/lung epithelium. -triphosphate of -␤-d-ribofuranosyl- h- , , -triazole -carboxamide (ribavirin) was synthesized using phosphorylation with a tris(imidazolyl) phosphate in a convenient one-pot approach. formulations of different poloxagels with the ribavirin -triphosphate were prepared and characterized as prospective antiviral formulations for prophylactic and therapeutic treatments of respiratory infections including influenza a virus. aerosolic route of application of these antiviral formulations and associated problems are discussed. edwin gong , ebrima gibbs , joel oger department of pharmacology and therapeutics, the university of british columbia, vancouver, bc, canada; neuroimmunology lab, ubc hospital, department of medicine, the university of british columbia, vancouver, bc, canada ifn-alpha and ifn-beta are currently employed in the treatment of many viral diseases, especially chronic hepatitis. ifn-beta is also employed for the treatment of multiple sclerosis (ms), a chronic and often debilitating disease of the central nervous system. however, as with other protein therapeutics, long-term ifn therapy can lead to the development of binding and neutralizing antibodies to ifns and thus lead to deceased clinical effect of ifns. in order to measure the bioavailability of ifns and the level of neutralizing antibody, we have developed a realtime rt-pcr (taqman) assay by quantitating the expression of mxa (an ifn-induced protein) mrna. the nucleotide sequences of mxa deposited in the genebank were aligned, and a pair of primers and the hybridization probe were designed based on the conserved regions. a house keeping gene, gapdh, was used as a calibrator for relative quantitation. the rna standards were generated by in vitro transcription from cloned mxa gene in a plasmid vector. the reaction parameters were optimized. the assay was validated using pbmcs of ms patients that were treated with ifn-beta. for evaluation of the ifn bioavailability, the total rna was extracted from pbmcs and quantitatively detected by one-step rt-pcr for both mxa and gapdh. the results calculated by the (− ct) method showed that the difference (signal-to-noise ratio) between samples with neutralizing antibodies and samples from untreated ms patients or healthy donors were approximately - -folds. this indicates that our assay is a reliable method for determination of ifn bioavailability. v. lozitsky , i. kravchenko , v. larionov research anti-plague institute, odessa, ukraine; national university, odessa, ukraine transdermal delivery (td) of drugs is a novel method for treatment of diseases. td is carried out by the help of transdermal therapeutic systems (tts), which are multilayer plasters that contain active ingredients. td have a number of advantages, such as: ( ) prolongation of the drug's action; ( ) drug's concentration is maintained in therapeutic range; ( ) there is no trauma to patient's skin while using td; ( ) removing tts from the skin immediately stops drug's entering to the organism; ( ) first-pass effect in the liver is reduced; and ( ) many highly active drugs are irritating the gastro-intestinal tract if administered orally, others have a short half-life time-these drugs do not have downsides mentioned above if used as tts. in our previous research we elaborated tts containing rimantadine (ttscr) and studied its efficacy during experimental influenza in mice. we had established that transdermal delivery of rimantadine is more effective than oral administration. the aim of this work was to increase the efficacy of ttscr. to solve this task we studied the influence of some permeability enhancers on anti-influenza efficacy of ttscr during experimental infection. applied tts had adhesion hydrogel matrix (polyvinyl alcohol and . -propylenglycol). they consisted of a base and a plastificator, which improves the administration of active substances through the skin and does not induce irritation. ttscr ( mg/mouse) were applied on shaven backs of experimental animals. tts for other groups additionally contained one of such permeability enhancers as: mg/mouse of dmso or mg/mouse of octanol or mg/mouse of papain. tts were applied on shaven backs of mice and stayed there from day before infection to th day after challenge. mice of all groups were infected intranasally with influenza virus a/pr/ / (h n ), which is highly pathogenic for them. challenge was carried out using four animals for each virus dilution within the range of − to − . deaths of animals were recorded for days. the results showed that proteolytic enzyme papain increased the anti-influenza efficacy of ttscr on log tid . dmso and octanol did not demonstrate such activity. mikhail dobrikov , serguei vinogradov , barbara ramsay shaw department of chemistry, duke university, durham, nc, usa; center of drug delivery and nanomedicine, and college of pharmacy, university of nebraska, omaha, ne, usa nucleoside reverse transcriptase inhibitors (nrti) are widely used in the antiviral chemotherapy. most nrtis require stepwise phosphorylation to the respective nucleoside triphosphates, which inhibit the viral dna synthesis. however, the emergence of hiv- reverse transcriptase-dependent drug resistance limits the effectiveness of treatment by nrtis. the ␣-p-borano-nucleotide analogues show several unique physico-chemical and biological properties: (i) enzymatic studies indicate that the rp-isomer of ␣-p-borano- , -ddndps is a better substrate for cellular ndp kinase than the parent ddndp; (ii) neither isomer of the ␣-p-borano-ddndps is a substrate for mammalian pyruvate kinase and shows very poor inhibitory properties to this enzyme; (iii) the rp-(␣-p-borano)-ddntp isomers are better inhibitors of drug-and multidrug-resistant viral reverse transcriptases and are poor substrates for dnadependent dna polymerises; and (iv) after incorporation into viral dna the borano-ddnmp residues are more resistant to atp-dependent removal from viral dna than parent ddntps. to by-pass inefficient phosphorylation of the nrtis, several prodrugs of ␣-p-borano-nucleotide analogues have been previously synthesized. a more efficient delivery system for ␣-p-boranonucleotide analogues based on nanosized cationic polymeric gel (nanogel) is proposed. selective inhibition of drug-and multi-drug resistant viral rts, poorer inhibition of intracellular kinases and dna polymerases by the ␣-p-borano-nucleotide analogues, and their specific delivery into infected cells in the complex with nanogel particles suggest a new approach to the design of more powerful antiviral drugs. acknowledgement: this work was supported by the nih grant r al to b.r.s. we have developed a high-throughput, cell-based assay to address the critical need for antiviral drugs for the treatment of influenza. in consideration of the demand to screen high volumes of compounds, we targeted the development of a microtiter plate format for the assay. in this assay, the inhibition of the influenza-induced cytopathic effect (cpe) in mdck cells was assessed using the celltiter-glo luminescent cell viability assay by promega. this reagent measures the amount of atp present in cells, which is directly proportional to the number of metabolically active cells. validation studies were executed to establish optimal cell density, viral concentration, dmso tolerance for compound dilution, incubation time for virus-induced cpe and effective control drug concentration. additional parameters, such as assay variability, reagent and read stability, edge effects, and ic stability were also investigated during validation. we are currently initiating use of the assay to screen chemical libraries and will report our findings from library screens in addition to the aforementioned validation. we believe the approach will also provide a mechanism for discovery of new antiviral leads for influenza as well as avian flu. fundacio irsicaixa, hospital universitari germans trias i pujol, badalona, spain we have developed bacteriophage lambda based genetic screen that can be used to isolate and characterize site-specific proteases. this genetic screen system is based on the bacteriophage lambda ci-cro regulatory circuit, in which the encoded repressor ci is specifically cleaved to initiate the lysogenic-to-lytic switch. we have adapted this simple, safe and rapid genetic screening system to predict the activities and phenotypes of human immunodeficiency virus type (hiv- ) proteases in the course of viral infection and antiretroviral therapy. a specific target for the hiv- protease, p -p , was inserted into the lambda phage ci repressor. the target specificity of the ci-hiv repressor was evaluated by coexpression of this repressor with an hiv- protease construct. upon infection of escherichia coli cells expressing the two constructs encoding the ci-hiv- repressor and hiv- protease, lambda phage replicated up to -fold more efficiently than in cells that did not express the hiv- protease. this assay responds appropriately to well-known hiv- proteases inhibitors and can be used to search for new proteases inhibitors. the high level of specificity of this system, in which modest differences in catalytic efficiency can be quantified, should be also useful for the characterization of different mutant viral proteases. we further demonstrated the broad applicability of this protease assay using other viral proteases and their cognate cleavage sites, including hepatitis c virus (hcv) ns protease and severe acute respiratory syndrome (sars) coronavirus (cov) (scov) c-like protease. compared with other protease assay methods, this assay has the following advantages: safe, highly sensitive, highly specific, easy quantification, and rapid generation of different protease cleavage substrates using molecular cloning and expression. this system may be useful for the development of a screening method to identify viral protease inhibitors and should be also useful to characterize cellular, viral, or other infectious agent proteases with different activities and specificities. karen m. watson, todd b. parsley, robert w. buckheit jr. imquest biosciences, inc., frederick, md, usa a virus transmission and rapid resistance selection assay has been developed in order to quickly evaluate the biological properties of anti-infective test compounds and rationally prioritize them for further development. the transmission assay specifically evaluates the ability of test agents to suppress and clear virus replication from cultures during the serial passage of virus in the presence of fixed concentrations of the test compounds alone or in combination. the growth and expansion of virus in the infected cultures has been shown to occur through the replication of originally infected cells in the absence of virus spread, through direct virus to cell infection, and through cell to cell transmission. in order to sterilize a culture a test compound must possess the ability to specifically and potently interfere with virus replication by each of these three methods and must be able to inhibit the replication of resistant viruses which pre-exist in the viral inoculum and which rapidly grow in the presence of the fixed concentrations of the test agent. twelve pyrimidinediones being evaluated for potential use as both anti-hiv therapeutic agents and topical microbicides were evaluated for their ability to inhibit virus transmission and to define their ability to rapidly select for resistant virus strains. these compounds were evaluated in parallel with known anti-hiv agents that inhibit virus entry (t ) and reverse transcription (sustiva, uc and azt). the results of the transmission assays suggest that significant biological differences exist between antiviral compounds and even between highly related congeners of the same class of pyrimidinediones, suggesting that the transmission and rapid resistant selection assay measures important antiviral properties of anti-hiv agents. biological studies that evaluate the mechanisms of virus growth in the presence of high concentrations of test compounds will be described. laure deflubé , kerstin angner , anna overby , david stein , patrick iversen , ramon flick utmb, department of pathology, galveston, tx, usa; avi biopharma, inc., corvallis, or, usa in the family bunyaviridae, several members on the genus phlebovirus have been reported to cause disease in humans or livestock. among these, rift valley fever (rvf) virus is an important human/animal pathogen. its widespread geographic distribution and its ability to produce severe human disease makes this virus a worldwide public health concern. the phosphorodiamidate morpholino-oligomers (pmo) are a class of dna-like antisense agents typically synthesized to a length of about subunits and contain purine or pyrimidine bases attached to a backbone composed of six member morpholine rings joined by phosphorodiamidate intersubunit linkages. they have been shown to be effective antiviral compounds for different virus families, e.g. coronaviridae and flaviviridae. pmo bind to rna preventing translation of the viral rnas. we used our recently developed plasmid-based minigenome rescue systems for uukuniemi (phlebovirus model virus) and rvf viruses to screen antiviral compounds based on the morpholino antisense oligonucleotide approach. for this the antiviral compounds were appraised on the basis of reporter gene activity (fig. b) . the inhibitory effects of the same compounds were also tested by measuring reduction in virus titer (fig. a) , by monitoring changes in viral antigen production using an indirect immunofluorescence procedure and facs analysis, and analysis of genome transcription/replication by rt-pcr. indeed several pmos could be identified with interfering effect at a low ic on bunyaviral minigenome rescue as well as virus proliferation. based on these results, we plan to confirm antiviral activity of the most promising compounds in suitable animal models. we have shown that the fractal approach to the problem of viruscell interaction gives the unique possibility to process the data through the sequence of the direct and inverse fourier transforms. the studies were carried on the herpes simplex virus us- interacting with the hep- sensitive cell culture. the object was imaged as system of bright peaks formed as a result of laser diffraction on the structural elements of the virus-cell system. the whole virus-cell interaction information is inserted into computer in a fastest parallel way. the laser intensity peaks, which form the speckle image of the system under consideration, could be transformed into the hierarchical system of the circles (or squares) according to the choice of the researcher, but conserving the same d value, which depends only on the true intermolecular interaction potential. this potential, being characteristic for every stage of virus-cell interaction, is responsible for the given structure of the dynamic virus-cell system and the unique, but the typical form of the fractal cluster corresponding both to the system itself and its image as well, was processed by computer techniques. the hierarchical fractal design of the virus-cell system, proposed here for the first time, gives the universality, needed for the quantitative description of any possible combination of the virus and corresponding sensitive cell. it should be noted, as well, that the fractal microscope use for viruscell dynamic system imaging have all the properties, required from all other experimental tools of monitoring, including the reliability, reproducibility and preciseness. this device could be used in drug design biological test stages with the scope of time and efforts economy during the compounds libraries screening. the fractal microscope combined with the qsar drug design technique makes the antiherpetic drug design more competitive as compared to the regular approaches. acknowledgement: the authors are indebted to the partial support of the stcu grant # . we have investigated experimentally the fractal properties of diffraction images obtained by laser irradiation of virus-cell system. it was shown that the diffraction process is mathematically equivalent to the direct fourier transform of the said system's components modeled with simple geometrical figures (e.g. circles). each viral family could be coded and described quantitatively with the average size of the free viral particle and the type of its symmetry. we propose here to use the inverse fourier transform of the virus-cell system in order to get the real enlarged image of the viruses attacking the sensitive cell as well as the cell's structural transformation caused by the sequent stages of virus reproduction process. the set of bright and dark spots, which forms the virus-cell system's diffraction image, could be coded into set of numbers (matrix form of correlation vector-function) using the quantification procedure. the correlation function was used as presented in polar coordinates because the system has the axial symmetry (laser beam taken as main physical axis). the full information included into the image peaks' diameters and color index is transformed using inverse fourier technique into set of intersecting bright and dark circles. the full in vitro dynamics of the structural changes of the virus-cell system are described by the changes of circles' diameters and the area of their intersection. it was shown, also, that the magnification of the fractal microscope could achieve , × to , ×, depending on the laser power used. proposed fractal microscope could be applied as well in vivo experiments until the required magnification will not make us to use projection laser with the output exceeding mw. we have shown that the fractal microscope based on the inverse fourier transform could be applied successfully in pharmaceutical antiviral drug design, laboratory and clinical trials of new antiviral preparations, especially effectively in hierarchic qsar research. acknowledgement: authors are grateful to the support under the stcu grant # . we have previously reported bicyclic furano pyrimidines as potent and selective inhibitors of varicella zoster virus (vzv) , with subnanomolar activity for palkylphenyl substituted analogues . these compounds however are highly lipophilic and poorly soluble in water. we then reported a series of p-alkyloxyphenyl compounds containing a phenolic ether aiming to enhance water solubility whilst retaining antiviral activity (mcguigan et al., ) . we will now report the synthesis, characterisation and antiviral evaluation of a novel series of p-alkyloxyphenyls where there is at least one methylene spacer between the phenyl and ether group to potentially boost the pharmacokinetic profile. the alkyl chain length was fixed to retain a high clogp value, a parameter that has previously been shown to correlate with high antiviral potency . the target structures were prepared by the pd-catalysed coupling of a series of para-substituted alkoxyphenyl acetylenes with -iodo- -deoxyuridine, to give intermediate -alkynyl nucleosides, which were subsequently cyclised in the presence of cui to give the desired bicyclic systems. the antiviral activity, cytotoxicity, and solubility of these compounds are to be reported. we have previously reported on some novel nucleoside analogues containing and unusual furano bicyclic pyrimidine base and long side chain , which were discovered to be both potent exquisitely and selective towards the varicella zoster virus. following this discovery, three main sites for modification were identified and explored: ( ) the side chain; ( ) the bicyclic base; and ( ) the sugar moiety. modification to the side chain by insertion of a phenyl ring, led to the most potent anti-vzv nucleoside to date (ec nm) . the investigation into modifications at the three sites stated above has continued and we herein report further adjustments to these analogues. replacement of the furo oxygen with sulfur on the parent nucleosides bearing an alkyl side chain has been reported to retain antiviral activity. however, those bearing a phenyl alkyl side chain are here shown to give a slight reduction in anti-vzv activity. modifications to the phenyl ring of the side chain have included halogen substitutions, and the fluorine in particular has produced some intriguing results in that, while the ortho and meta substitutions show some anti-vzv activity, the para analogue is completely devoid of antiviral activity. we now report further studies which include the di and tri substituted phenyl analogues. finally, we have also investigated sugar modification that has included substitutions of the hydroxyl group. previous modifications which have replacements of the hydroxyl groups, resulted in loss of activity against vzv . we now present some new substituted analogues which have provided interesting biological results. we have previously reported bicyclic furano pyrimidines as potent and selective inhibitors of varicella zoster virus (vzv) with subnanomolar activity for palkylphenyl substituted analogues srinivasan et al., ) . the sar is now further explored via the substitution of phenyl derivatives with electron withdrawing and electron donating groups. we now report the synthesis, characterisation, and biological evaluation of a novel series of mono substituted phenyl derivatives in order to probe the structure activity relationships in this region. the target compounds were synthesised under sonogashira conditions where a series of substituted phenyl acetylenes were coupled with -iodo- -deoxyuridine, to give intermediate alkynyl nucleosides that were subsequently cyclised in the presence of cui to give the desired bicyclic systems. diseases caused by herpes simplex virus (hsv) are widely distributed. prophylaxis and treatment of these infections are important health care tasks that require also the search, design and development of new antiherpetic drugs overcome drug resistance and toxic side effects of existing drugs. drug selection simply based on results of empirical screening is not very effective. computer-based technologies may help to optimize the structure of antiviral compounds as well as to design and develop new drugs. the objective of the present work is the quantitative structureactivity relationship (qsar) analysis of antiviral activity of various n,n -(bis- -nitropyrimidyl)dispirotripiperazines in connection with consequent drug design. the well-established simplex representation of molecular structure (sirms) qsar approach has been used to fulfill this objective. it allows the molecular design of new effective antiviral drugs. thorough investigation of the relationship between: (a) cytotoxic (hela cells and gmk cells, cc , g/ml); (b) antiherpetic activity (hsv- strain kupka, ic , g/ml); and (c) selectivity index (ratio of cc to ic ) and the structure of n,n -bis- -nitropyrimidyl derivatives of dispirotripiperazine have been conducted. statistic characteristics for pls (partial least squares) models are quite satisfactory (r = . - . , q = . - . ). the results are confirmed by experimental data. based on the obtained models, molecular fragments that promote and interfere with antiviral activity were defined. additionally, these models provide the possibility to predict molecular fragments that will enhance antiherpetic activity and to design new well tolerated highly virus-specific drugs. in summary, the developed simplex approach is an effective instrument for prediction and design of novel effective antiherpetic agents. several representatives of a series of -arylethynyl- deoxyuridines ( a) bearing bulky aryl groups were recently shown to possess unexpected activity towards hsv- . unlike common anti-hsv drugs, these compounds retain activity towards kinase-deficient acyclovir-resistant strains. therefore, an unusual mechanism of antiviral action is assumed. in order to investigate the mechanism and to discover more potent analogues we synthesized several novel -deoxy ( a) and -arabino ( b) uridine derivatives possessing different -arylethynyl substituents. dinucleosides containing two uridine moieties coupled to a single polycyclic aromatic hydrocarbon (e.g. pyrene) represent another type of structural variation of nucleosides a. these compounds as well as some of a and b possess bright fluorescence that can be used in biological evaluations. cytomegalovirus (cmv) is a wide spread opportunistic pathogen which belongs to the beta subfamily of the herpesviridae. primary infection is generally asymptomatic resulting in life long latency. however, morbidity and mortality rates post-transplantation are greatly increased following reactivation or recrudescence of cmv. ganciclovir (gcv) and cidofovir (cdv) have both been successful in suppressing cmv viral replication in immunocompromised patients. although sustained use of these drugs has resulted in the emergence of multi-drug resistant strains of virus. in this study we used plaque reduction assays to determine the antiviral efficacy of two ribonucleotide reductase inhibitors, didox (dx; , dihydroxybenzohydroxamic acid) and trimidox (tx; , , trihydroxybenzamidoxime) in inhibiting both wild type and drug-resistant strains of murine cmv (smith strain). the results presented here demonstrate that both dx and tx inhibit viral plaque formation in a dose dependent manner in both wild type and the resistant strain. a -and -fold increase in drug dose was required for cdv and gcv respectfully to inhibit plaque formation by % in the resistant strain (cdv wt: . m, r: . m/gcv wt: . m, r: . m). this compared to only a moderate increase in drug dose required for dx and tx to achieve % inhibition in the resistant strain (dx wt: . m, r: . m/tx wt: . m, r: . m), corresponding to a . -and . -fold increase respectfully. further work is currently underway to determine the possible mechanism of antiviral actions and toxicity profiles of these novel virostatics. in patients with human immunodeficiency virus (hiv) infection, coinfection with herpesviruses continues to be a problem for patients receiving antiviral hiv therapy. since treatment can be affected by the large number of drugs required for multiple infections, it would be useful to have antivirals that are active against both hiv and the herpesviruses. we reported previously that alkoxyalkyl ester prodrugs of cidofovir (cdv) are several logs more active against herpesvirus replication than unmodified cdv. to determine if this strategy would be effective for other acyclic nucleoside phosphonates which are active against hiv infections, hexadecyloxypropyl (hdp) esters were synthesized from -(phosphonomethoxyethyl)-cytosine (pme-c), -(phosphonomethoxyethyl)- -bromo-cytosine (pme- brc), -(phosphonomethoxyethyl)- -fluoro-cytosine (pme- fc), -(phosphonomethoxyethyl)- , -diaminopurine (pme-dap) and -(phosphonomethoxyethyl)- -amino- cyclopropylaminopurine (pme-cprdap) and assayed for activity against herpesvirus replication. overall, the hdp esters were more active than the unmodified acyclic nucleoside phosphonates, indicating that this is a useful strategy for increasing the antiviral activity of acyclic nucleoside phosphonates. one of the most active compounds was hdp-pme-cprdap which had ec values of . , . , and . m in hff cells infected with hsv- , hsv- or hcmv, representing a - -fold increase in efficacy over the parent pme-cprdap. another promising compound was hdp-pme-dap, which had ec values of . , . , and . um in hff cells infected with hsv- , hsv- , and hcmv, representing a - -fold increase over the parent pme-dap. the results presented here indicate that modified acyclic nucleotides with antiviral activity against hiv also inhibit the replication of some of the herpesviruses. further evaluation of their activity against other herpesviruses that are a problem in hiv-infected patients, such as human herpesviruses type and , is warranted and may provide new therapeutic options for patients with coinfections. julie m. breitenbach , katherine z. borysko , jiri zemlicka , john c. drach biologic & materials sciences, school of dentistry, university of michigan, ann arbor, mi, usa; karmanos cancer institute, wayne state university school of medicine, detroit, mi, usa we previously described first (qiu et al., . j. med. chem.) and second generation (zhou et al., . j. med. chem.) methylenecyclopropane purines that have potent and selective activity against hcmv. strains selected separately for resistance to first-generation analogs (synadenol, synguanol) were - -fold resistant to several first-generation purine analogs. similar resistance was observed to the second-generation guanine analog cyclopropavir [ic 's in plaque assays = . and m, respectively for wild-type (wt) and synguanol-resistant ( r) virus]. likewise a ul deletion mutant (prichard et al., . j. virol.) was resistant to both first and second-generation compounds (ic 's = . and . m in wt; and m in ul del , respectively for synguanol and cyclopropavir). ul from the hcmv strain selected for resistance to the synadenol was sequenced and two mutations were identified: m i and c y. because hcmv with either m i or the related c y mutation alone was sensitive to synadenol and synguanol (baldanti et al., . antiviral res.), we hypothesize that two mutations are required for resistance to first-and second-generation analogs. this hypothesis was tested by construction of three strains of hcmv from hcmv ad bac with one, the other, or both mutations in ul . as expected, the two strains with the single mutations were -to -fold resistant to ganciclovir but had little resistance to the first generation compounds synadenol and synguanol ( . -to -fold). both strains were somewhat more resistant to the second-generation compound cyclopropavir ( to -fold) but less so than observed in the r virus with two mutations. study of the resistance of the constructed virus with two mutations is underway. we conclude that a functional ul is required for activity against hcmv and that is likely that two mutations in ul are required for significant resistance. acknowledgement: supported by grants p -ai and r -ai from nih and funds from the university of michigan. svitlana zagorodnya , nadiya nesterova , inna alexeeva , larisa palchikovskaya , galina baranova , alexander kobko , anna golovan zabolotny institute of microbiology and virology of nas of ukraine, kiev, ukraine; institute of molecular biology and genetics of nas of ukraine, kiev, ukraine search of new effective preparations capable to inhibit herpesviruses reproduction is stipulated by their certain resistance to different groups of chemical preparations. new triazine bearing tricyclic bases and their n-glycosidic derivatives structures are widely used as potential antiviral agents. the objective of the present investigation was to study the activity triazine bearing tricyclic bases nos. and , as well as n-glycosidic derivatives no. against epstein-barr virus-lymphotropic and oncogenic virus from herpesviridae family. as a model of ebv-infection in vitro we used the line of lymphoblastoid b-cells raji, which infected by ebv. an inhibition of reproduction of ebv in a cell culture by no , no , and no was determined by reduction of a number of genome-equivalents of ebv dna on a cell, which were revealed by quantitative pcr with use of primers and reagents "amply-senc- r" (russia). the first stage of investigation of substances was the analysis of their cytotoxicity for cell line raji. they have been studied in concentrations of , , , , , , , , , . and . g/ml. the concentrations that inhibited the quantity of live cells on % (id ) were equal to substances no. - g/ml, no. - g/ml and no. - . the minimal inhibiting concentration (mic) of nos. , , and was equal to g/ml, because the amount of genome-equivalents of dna ebv on a cell was reduced with . up to . hence, the index of selectivity (is) was equal to and for triazine bearing tricyclic bases nos. and , for n-glycosidic derivatives- . in addition these compounds were also tested in transcription and replication model systems in vitro. our results indicate that bases and their n-glycoside derivatives effect rna and dna synthesis in different manner. r. sgarbanti , l. nencioni , g. macrì , c. nucci , u. benatti , m. magnani , e. garaci , a.t. palamara department public health sciences, university rome "la sapienza," rome, italy; department biopathology, physiopathological optics, university rome "tor vergata," rome, italy; department exp. medicine biochemistry section, university genova, genoa, italy; inst. biochemistry, university urbino, urbino, italy; department exp. med. biochem. sciences, university rome "tor vergata," rome, italy several studies have demonstrated that different viruses induce an imbalance in the intracellular redox state through a depletion of glutathione (gsh), the main intracellular antioxidant. the imbalance in the intracellular redox state represents a key event in the development of viral infection. indeed, our previous data showed that treatment with gsh prevents a decrease in intracellular gsh and inhibits replication of different rna and dna viruses in vitro and in vivo. our recent data demonstrated that a butanoyl derivative of gsh (gsh-c ), with increased hydrophobic properties, inhibited in vitro parainfluenza- and hsv- replication more efficiently than gsh. for this reason we evaluated the effectiveness of topical gsh-c administration in hsv- -induced keratitis in rabbits. for infection, the corneal epithelium, previously scratched, was inoculated with × pfu/ml of hsv- . gsh-c , dissolved in a saline solution ( mm, ul/eye), was administered as eyedrops four times daily for ten days. a saline solution was used for the control group. the clinical evaluation of conjunctival and corneal involvement, performed by using . % fluorescein sodium eyedrops and a slit lamp fitted with a cobalt blue filter, demonstrated that gsh-c treatment was effective in reducing the severity and progression of keratitis and conjunctivitis. moreover, in gsh-c treated animals, conjunctival hsv- titre, assayed by tcid on day post-infection, was significantly reduced as compared to that of control animals (mean = . × units/ml versus . × units/ml, n = for group). accordingly, similar results were obtained by measuring virus titre from the corneas of gsh-c -treated animals versus placebo animals (mean = . × units/ml versus . × units/ml, n = per group). such results highlight the antiviral activity of gsh-c in vivo and suggest that topical gsh-c treatment could be considered as complementary therapy of hsv- -induced keratitis. debra quenelle , deborah collins , latisha pettway , caroll hartline , james beadle , w. wan , karl hostetler , earl kern university of alabama school of medicine, birmingham, al, usa; department of medicine, university of california, san diego and veterans medical research foundation, san diego, ca, usa cytomegalovirus (cmv) can cause a wide variety of clinical manifestations in immunocompromised hosts or transplant recipients. we have utilized severe combined immunodeficient (scid) mice implanted with human fetal tissue and subsequently infected with hcmv or balb/c mice infected with mcmv to evaluate new antiviral therapies against cmv infection. in the current studies we used these two models to determine the efficacy of (s)- -[ -hydroxy- -(phosphonomethoxy)propyl]adenine ((s)-hpmpa), hexadecyloxypropyl-(s)-hpmpa (hdp-(s)-hpmpa), or octadecyloxyethyl-(s)-hpmpa (ode-(s)-hpmpa). in the hcmv model, human fetal thymus and liver (thy/liv) tissues were implanted under the kidney capsule of mice and inoculated - weeks later with pfu of hcmv. tissue samples were obtained at various time points for quantitation of hcmv titers by plaque assay. in general, replication of the toledo strain of hcmv in the implant tissue increased through - days and then gradually decreased to undetectable levels by weeks post-infection. to determine efficacy of these compounds, oral treatment with vehicle or mg/kg of (s)-hpmpa, hdp-(s)-hpmpa or ode-(s)-hpmpa was initiated h after infection and continued for days. cidofovir (cdv) at mg/kg was administered i.p. daily as a positive control. results indicated that (s)-hpmpa, hdp-(s)-hpmpa and ode-(s)-hpmpa were highly effective in significantly reducing replication when compared to the vehicle control. in mcmv infected mice, hdp-(s)-hpmpa was highly effective in preventing mortality when administered orally at or mg/kg beginning h post-viral inoculation and mg/kg when treatment was delayed until h postviral inoculation. these data indicate that these compounds were highly efficacious in two animal models of cmv infection and should be evaluated for use in hcmv infections in humans. cytomegalovirus (cmv) is a ubiquitous ␤-herpesvirus that asymptomatically infects immunocompetent individuals but leads to serious illness in immunocompromised individuals, such as transplant recipients, neonates and aids patients. thus, the need for well-tolerated and potent antiviral compounds with activity against cmv is well recognized. in our current studies, we have evaluated the in vivo activity of rep , a fully degenerate mer phosphorothioated oligonucleotide against murine cytomegalovirus infection (mcmv) in mice. rep has potent in vitro activity against hsv- , hsv- , hcmv, vzv, ebv, and hsv- (vaillant et al., submitted for publication). in our initial studies, infected mice were treated with rep and compared to saline-treated infected control mice. compound was administered intraperitoneally for consecutive days at mg/kg, starting at days prior to infection. mice were infected with × pfu mcmv on day , at h post-treatment. sera were collected at − h, at hpi, and at dpi for elisa analysis of ifn␥ production. spleens and livers were collected at dpi for determination of virus titers. at dpi, virus titers in the spleens and livers were significantly reduced by rep treatment as compared to control mice. splenomegaly was observed in infected mice treated with rep but not in saline treated, infected mice or in rep treated, uninfected mice. ifn␥ levels in mice treated with rep peaked at hpi compared to hpi for saline-treated control mice. these data suggests that immune stimulation might contribute to the antiviral activity of ps-ons, perhaps through ifn␥ levels. a second study comparing the in vivo activity of rep with two oligonucleotide analogs that do not activate tlr- mediated immune stimulation suggests that direct antiviral activity of rep and the analogs was the predominant therapeutic mechanism in vivo. moreover, one rep analog exhibited even greater antiviral activity than rep while causing no splenomegaly. additional experiments are underway to provide insights into the mechanism of action against mcmv infection. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. kathy keith , joseph maddry , namita bansal , kochurani jacob , secrist john , earl kern department of pediatrics, university of alabama school of medicine, birmingham, al, usa; southern research institute, birmingham, al, usa a series of novel antiviral agents was prepared based on lead compounds related to acyclic nucleoside phosphonates. these agents consist of a purine nucleus bearing a pendant phosphonic acid group. the design strategy was two-fold: ( ) following the approach of the hostetler group, to mask or partially mask the anionic phosphonate as a lipophilic ester and/or as an amino acid phosphonamidate prodrug that could enhance cellular uptake and be cleaved intracellularly; and ( ) to investigate new substituents at the purine -and -positions. for proof of concept, a phosphonomethoxyethyl adenine (pmea) scaffold was employed. over analogs of pmea substituted at the purine -or at the adenine n- site have been synthesized and evaluated for activity against orthopoxvirus infections. many n- substituents other than the previously recognized n-cyclopropyl have shown antiviral activity, and these structure-activity relationships are being investigated. an exciting finding has been that introduction of several novel moieties at the purine -position particularly the hydrazino, hydroxylamino, or the cyclopropylamino groups resulted in several compounds with excellent in vitro activity. for example, octadecyloxyethyl (ode) -amino-n( )-cyclopropyl pmea had ec values of . - . m and ode -hydroxylamino-n( )-cyclopropyl pmea had ec values of . - . m against cowpox and vaccinia viruses, respectively, using a plaque reduction assay in hff cells. under these conditions the parent molecule, pmea, was completely inactive. these two compounds had cc values of - m giving selective indices of - . these studies indicate that several modifications in the pmea scaffold can result in good antiviral activity against orthopoxvirus infections in vitro and the most active compounds are currently being scaled up for evaluation in animal models. isatin ( , -dioxoindole), a versatile lead molecule for designing of potential bioactive agents, and its derivatives have been reported to possess inhibitory activity against a variety of pathogenic viruses. methisazone(n-methylisatin- thiosemicarbazone) was one of the first synthetic antiviral agents used clinically for the treatment of orthopox virus infections. the presence of the thiosemicarbazone, however, can result in immunosuppresion and we have attempted to replace the thiosemicarbazone with a sulphonamide in order to modify the antiviral activity. the present work was performed to evaluate the antiviral activity and cytotoxicity of some novel -[( , dihydro- -oxo- h-indol- -ylidene)amino]-n-( , -dimethyl- pyrimidiny)-benzene sulphonamides against pox viruses such as vaccinia and cowpox virus in human fibroblast cells and the activity was compared with cidofovir(cdv). among the compounds tested, -[( -methyl- , -dihydro- -oxo- h-indol- -ylidene)amino]-n-( , -dimethyl- -pyrimidiny)-benzene sulphonamide(spiii- me), was the most active compound with an ec value of mol, compared with cdv, which had an ec of mol against vaccinia virus. all the compounds were non-toxic (> lm)using a neutral red uptake assay. substitution of a halogen atom in th position of isatin was found to abolish the antiviral activity. this compound should be evaluated in orthopox infections in animal to determine its potential for development as an effective agents for treatment of these infections. acknowledgement: supported in part by contract no -ai- from virology branch, niaid, nih, usa. evgeny belanov , svetlana kotovskaya , nikolay bormotov , sergey balakhnin , olga serova , nataliya perova , zina baskakova , galina dzhumbaeva , valerii charushin , oleg chupakhin state research center of virology and biotechnology "vector", koltsovo, novosibirsk reg., russia; ural state technical university, yekaterinburg, russia; institute of organic syntheses, yekaterinburg, russia during this study, we synthesized a series of , , -benzotriazine ( fig. ) derivatives in order to evaluate the structural features required for anti-orthopoxviruses activity. these derivatives were tested for cytotoxicity and activity against the vaccinia, cowpox, mousepox, monkeypox, and in some experiments with variola viruses in vero and mk- cells. the results from studies of structure-activity relationship revealed that only compounds containing phenyl group at c- and the alkoxy and fluoro substitutes in the benzene ring of benzotriazines showed anti-orthopoxviruses activity. the antiviral activity was reduced or lost after substitution with other substitutes. thus, we find a new class of heterocyclic compounds with antiviral activity. acknowledgment: this research was funded by istc project # . yali chen, guang yang, kady honeychurch, dennis hruby, robert jordan siga technologies, inc., sw research way-suite , corvallis, or , usa we have recently discovered a highly specific and potent antiorthopoxvirus compound (st- ) via high throughput screening (yang et al., . j. virol. , - ) . marker rescue of st- resistant variants localized compound resistance to the f l gene that encodes a major orthopoxvirus envelope protein (p ), which is required for extracellular viral particle formation. p participates in wrapping of intracellular mature virus (imv) in membranes derived from the trans golgi or late endosomal compartment to produce intracellular enveloped virus (iev) that are transported to the cell surface to form extracellular virus particles. to gain insight into the mechanism of action of st- , we examined the effects of st- on the production of the extracellular viral particles in bsc cells infected with recombinant vaccinia virus containing a gfp-tagged p protein. in the presence of st- , iev particle formation was dramatically reduced, plaque formation was almost completely inhibited, and imv particles appeared to be retained in intracellular vesicles as revealed by electron microscopy. furthermore, st- prevented the intracellular localization of p to the late endosome compartment as measured by confocal microscopy. in contrast, st- did not affect localization of p expressed from a st- resistant virus variant. more intriguingly, the compound did not affect the intracellular localization of p in transfected cells. these results suggest that st- inhibits an unknown virusspecific activity that requires f l. this work underscores the exquisite specificity of st- and supports continued development of st- as a potential anti-orthopoxvirus drug. guang yang, chris harver, dennis hruby, robert jordan siga technologies, inc. sw research way, suite corvallis, or , usa st- is a potent, orally bioavailable anti-orthopoxvirus compound that is active in vitro and in vivo. the frequency of naturally occurring st- resistant variants was measured by fluctuation analysis and found to be . × − . marker rescue of drug resistant variants localized changes associated with reduced compound susceptibility to the vaccinia virus f l gene. the spectrum of mutations that confer st- resistance was determined using an error-prone pcr procedure to increase the frequency of compound resistance by -fold relative to the frequency of naturally occurring resistance. using this procedure, random point mutations were introduced into the f l coding sequence by error-prone pcr and the mutated f l alleles were transferred into wild-type virus genome by marker rescue. sequence analysis of the input error-prone pcr products prior to marker rescue identified numerous nucleotide changes in the f l coding sequence, some of which created nonsense mutations. virus recombinants were selected that formed plaques in the presence of drug selection. this powerful selection procedure enriched for viruses that produced functional, st- resistant, f l proteins. sequence analysis of the compound resistant f l alleles identified numerous silent mutations scattered throughout the f l coding sequence and point mutations leading to amino acid changes that clustered around amino acid positions - within the f l gene. seven of these mutations resulted in single amino acid changes and could be correlated with reduced compound susceptibility. taken together, these results suggest that: ( ) mutations in at least positions within f l can confer resistance to st- and ( ) st- resistant mutations cluster to a amino acid domain in a region of the protein of unknown function. several -substituted pyrimidine analogs were identified as having antiviral activity against cowpox virus (cv) and vaccinia virus (vv) in primary human foreskin fibroblast cells. molecules containing benzopyran, cyanovinyl, and pyrazolone moieties at this position exhibited significant antiviral activity against both these viruses. three compounds in this series had ec values below m in a plaque reduction assay against both cv and vv. the antiviral activity of these compounds was also determined against herpes simplex virus (hsv) in a plaque assay. two compounds with cyanovinyl derivatives at the position had ec values below m against both hsv- and hsv- , whereas other substituents at this position exhibited weaker activity against one or both of these viruses. analogs containing the benzopyran substituents were the most effective against varicella zoster virus (vzv) and yielded ec values below m in a plaque reduction assay. none of the compounds were active against human cytomegalovirus. interestingly, all of the compounds were much less effective in a thymidine kinase (tk) negative strain of cv suggesting that the activation by this enzyme was important in their mechanism of action. tk deficient strains of hsv were also comparatively resistant to some of the compounds. the tk dependence of these compounds in cv and hsv taken together with the lack of activity against cytomegalovirus replication suggests that activation by a viral tk is important in their mechanism of action. these results indicate that pyrimidine analogs with large substituents at the position are substrates for the distinct tk homologs encoded by the herpesviruses and orthopoxviruses and suggest that they may be effective against infections with these viruses. synthesis and testing of additional analogs is warranted and should help identify the most potent analogs for in vivo testing. department of pediatrics, university of alabama school of medicine, birmingham, al , usa n-methanocarbathymidine ((n)-mct) is a conformationally locked nucleoside analog that is active against some herpesviruses and orthopoxviruses in vitro. this compound inhibits the replication of herpes simplex virus (hsv) with ec values below g/ml, and vaccinia virus (vv) and cowpox virus (cv) with ec values of . and . g/ml, respectively. assays using a thymidine kinase (tk) negative strain of cv yielded ec values -fold greater than a tk positive isolate. similarly, a tk negative strain of hsv- was -fold less sensitive to the drug than wild-type strains. thus, the antiviral activity of this molecule is dependent on the type i tk in hsv and the type ii tk expressed by vv and cv viruses, suggesting that it is a substrate for these divergent forms of the enzyme. the drug is also a good inhibitor of viral dna synthesis in both viruses and is consistent with inhibition of the viral dna polymerase once it is activated by the viral tk homologs. it is also possible that the phosphorylated forms of the drug may inhibit other enzymes such as thymidylate synthetase and inhibit viral dna synthesis indirectly. the interesting tk dependence of this molecule explains the rather unusual spectrum of activity that includes orthopoxviruses, alphaherpesviruses, epstein-barr virus (ebv), and human herpesvirus (hhv- ), since these viruses all express molecules with tk activity that could phosphorylate and thus activate the drug. conversely, n-mct is ineffective against the betaherpesviruses because they do not encode tk homologs. the compound is also highly effective in reducing the mortality of mice infected with cv, vv, and hsv when treatment is initiated h after infection and at doses as low as mg/kg. these results indicate that (n)-mct is active in vitro and in vivo and its mechanism of action suggests that the molecule may be an effective and selective therapeutic for orthopoxvirus and certain herpesvirus infections and that it warrants further development. we have previously reported the isolation and characterization of drug-resistant mutants obtained following repeated passages of the vaccinia virus (vv, lederle strain) in the presence of increasing concentrations of cidofovir (cdv). cdvr mutants encoded two mutations (a t and a v) not related to genetic polymorphism. we have now introduced these mutations in the pathogenic strain w western reserve and characterized the drug-susceptibility profile of the recombinant viruses and their pathogenicity in mice. both the a t and the a v recombinant viruses proved to be resistant to cdv and related compounds, such as cyclic cdv and -propoxy]pyrimidine}. the virus bearing both substitutions proved to be more resistant to cdv than the single mutants. interestingly, the a t and the a v mutants differed in their sensitivity to phosphonoacetic acid (paa); the a t and the a v mutants being, respectively, hypersensitive and resistant to paa. in contrast, the double mutant showed no change in sensitivity to paa as compared to the wild-type strain. unlike the a v mutant that showed only a two to three-fold decrease in susceptibility towards the -hydroxy- -phosphonomethoxypropyl (hpmp) purine derivatives, the a t mutant showed cross-resistance to the hpmp purine derivatives. it should be noted that in the process of selection of cdv-resistant mutants in the presence of increasing concentrations of the compound, the a t mutation appeared before the a v substitution, and the latter mutation only occurred in conjunction with a t. when tested for virulence in a lethal intranasal infection model in mice, all cdvr recombinant viruses proved to be attenuated, suggesting that cdvr mutations are associated with reduced pathogenicity. furthermore, we found that cdv at a dose of mg/kg/day for days was still able to protect mice (in terms of body weight loss) against an intranasal challenge with the a t + a v recombinant virus. evaluating the use of cpg dna as an antiviral therapy amanda phelps, linda eastaugh, amanda gates, david ulaeto, arthur krieg defence science and technology laboratories (dstl), salisbury, wiltshire, uk; coley pharmaceuticals ltd., ottawa, ont., canada at present there are no licensed antivirals against orthopoxvirus infections such as variola or vaccinia virus (vacv). although a stockpile of smallpox vaccine exists and has utility as a post-exposure treatment to infection, it is a live viral vaccine and as such cannot be administered to those with contraindications. bacterial dna contains unmethylated motifs that, together with their flanking regions, can stimulate an innate immune response. synthetic cpg dna mimics the immunostimulatory activity of bacterial dna and is recognised by intracellular toll-like receptor . there are four classes of cpg dna all of which have different properties, eliciting distinct initial immune responses. previous studies using an established lethal respiratory model of poxvirus infection demonstrated that a class b cpg dna ( ) could provide protection from lethality against vacv in balb/c mice when administered up to days prior to challenge. in order to evaluate efficacy balb/c mice were challenged intra-nasally with vacv and treated with doses of ranging between and ug/mouse. treatment was administered intra-nasally under light anaesthesia either on the day of challenge, , , , or days post-challenge. efficacy was determined by percentage body weight loss post-challenge. the optimum survival rate observed was % when treated with ug day post-challenge ( mld challenge). a survival rate of % was observed when treated with ug days post-challenge ( mld challenge). the delay of treatment to either or days post-challenge was ineffective, indicating that the window of opportunity for delivery of is within days. multiple doses of were used to attempt to extend this window of opportunity, delivering twice within a -day period. interestingly, this had a considerable detrimental effect, actually accelerating the onset of disease and ultimately death. further work is required to optimise the use of cpg dna as a potential antiviral therapy, and there is evidence to suggest that they may have immense utility as part of a co-administration therapy with other antiviral compounds, an area of work currently under investigation. © crown copyright dstl . department of virology, hebrew university, hadassah medical school, jerusalem, israel the pathogenicity and immunogenicity of the lister (elstree) strain of vaccinia virus, used for vaccination against smallpox, was studied in the mouse model. the virus did not reach the brain when inoculated intranasally, but when injected intracranially at a dose of × plaque forming units (pfu), was lethal for % of the mice. lower doses of virus caused the mice to initially loose some weight but they completely recovered thereafter. a significant level of protection against a lethal dose of the wr strain was achieved in mice following immunization with the lister strain, while higher doses and repeated vaccination procedure, were required with modified vaccinia virus ankara (mva). we found that the lister vaccine strain applied in israel is comprised of heterogeneous virus population. we isolated and plaque-purified three virus variants differing in their plaque size in bs-c- cell cultures. they were named: l-large plaque, m-medium plaque and s-small plaque variants. these isolates could be neutralized by rabbit antibodies prepared against the western reserve strain of vaccinia virus and their one-step growth curves in bs-c- cells were quite similar. however, they differ in their pathogenicity to mice following intranasal inoculation of pfu, or an intracranial injection of × pfu; the s variant being more virulent than the other two variants and resembles the pathogenicity of the lister strain. activity was also determined against a thymidine kinase (tk) deficient vaccinia virus in mouse and monkey cells. the potency of (n)-mct was similar to that seen with wild-type virus, suggesting that a cellular enzyme may be more important than viral tk to phosphorylate the compound. mice were intranasally infected with cowpox and vaccinia viruses followed h later by intraperitoneal treatment with (n)-mct ( x/day for days) or cidofovir ( x/day for days). (n)-mct treatment at and mg/kg/day resulted in and % survival from cowpox virus infection, respectively, compared to % survival (placebo). statistically significant reductions in lung virus titers on day occurred in , , and mg/kg/day treated mice. these doses did not spare mice from lethal vaccinia virus challenge, however, but the and mg/kg/day treatments significantly reduced day virus titers and lung weights, and the mg/kg/day treatment reduced lung consolidation. cidofovir ( mg/kg/day) protected all animals from death in both models. the evaluation of (n)-mct may be limited to mice based upon its greatly reduced efficacy in the cells of higher animals. acknowledgement: supported in part by contract no- -ai- from the virology branch, niaid, nih. chelsea byrd , elena sbrana , shu-yuan xiao , marina siirin , robert tesh , dennis hruby , robert jordan siga technologies, inc., corvallis, or, usa; university of texas medical branch, galveston, tx, usa st- is a potent small molecule inhibitor of orthopoxvirus replication that has been shown to protect mice from lethal challenge with vaccinia and ectromelia viruses. here we report the results of preliminary trials that show efficacy of st- against severe monkeypox virus infection in the ground squirrel model. ground squirrels infected with less than pfu of monkeypox virus develop a fulminant disease resembling human hemorrhagic smallpox: the most severe and lethal form of the disease. oral administration of st- at mg/kg once per day for days protected ground squirrels from a lethal challenge with and pfu of monkeypox virus. compound treated animals showed no weight loss or evidence of disease, and blood chemistry values were similar to uninfected animals. in contrast, placebo-treated animals showed elevated liver enzyme (alt and ast) levels and all animals died by day post-infection. when treatment with , , and h, % protection was observed in the , , and h groups, and % protection in the h group. severe pathologic changes were observed in the organs of the animals receiving placebo, especially in the lungs, liver, and spleen. in contrast, the organs of the animals receiving st- at , , , and h postinfection appeared grossly and microscopically normal. thus, st- appears to be a promising candidate for continued development as a therapeutic agent for severe orthopoxvirus infection. inge vliegen , guang yang , dennis hruby , erik de clercq , robert jordan , johan neyts rega institute for medical research, k.u. leuven, belgium; siga technologies, inc. corvallis, or, usa st- is a potent inhibitor of the replication of various orthopoxviruses. resistance of cowpoxvirus to st- maps to a mutation in v , which is homologous to vaccinia virus f l (yang et al., . j. virol.) . the latter encodes the envelope protein p required for production of extracellular virus. deleting f l resulted in a virus ( f l-vac) that is replicationcompetent in cell culture but that produces smaller plaques than the wild-type wr-vac. whereas intravenous (i.v.) inoculation of nmri mice with × pfu of wr-vac resulted in ± pox tail lesions per mouse, the same inoculum of f l-vac caused no lesions (p < . ). athymic nude (nu/nu) or scid mice inoculated iv with × pfu f l-vac did not develop tail lesions. the mean day of death in nu/nu mice inoculated with f l-vac was ± days as compared to ± days for wr-vac-infected mice (p < . ); scid mice survived the infection. we next studied whether f l-vac is able to protect mice against a subsequent infection with wr-vac. to mimic the human vaccination protocol, nmri mice were infected intracutaneously (i.c.) by means of scarification at the lumbosacral area with × pfu f l-vac or placebo. none of the infected mice developed lesions at the inoculation site. one week later, animals were infected ic with × pfu of wr-vac. all placebo animals, but none of the f l-vac animals developed poxvirus lesions. in a second set of experiments, mice were again inoculated ic with placebo or f l-vac and were infected one week later with × pfu of wr-vac by the iv route. placebo animals developed an average of ± pox tail lesions; no lesions developed in the f l-vac animals (p < . ). in a third set of experiments, nmri mice were inoculated iv with either × pfu of f l-vac or placebo, and none of the mice developed lesions. one week later, animals were inoculated iv with × pfu wr-vac. the placebo group developed an average of ± lesions as compared to . ± . lesions in the f l-vac mice (p < . ). f l-vac may thus be considered as a severely attenuated virus that may have potential for use as a smallpox vaccine. ji yuan , travis lim , shuan coughlin , dexin qiu , zhen liu , dave stein , decheng yang the james hogg icapture centre for cardiovascular and pulmonary research, university of british columbia, vancouver, bc, canada; avi biopharma, inc., corvallis, or, usa background: coxsackievirus b (cvb ) is the most common cause of viral myocarditis, but existing drug therapy is of limited value. antisense oligonucleotides (asons) designed to pair with viral rna could inhibit viral replication. however, the effectiveness of traditional asons is limited due to poor cellular uptake and degradation by nucleases. phosphorodiamidate morpholino oligomers (pmos) contain backbone modifications, which make pmos more resistant to nucleases. in addition, an arginine rich peptide (p ) is conjugated to the end of the oligomer to improve its delivery into cells. these features make p -conjugated pmos (p-pmos) promising candidates for the inhibition of cvb infection. methods: total p-pmos targeting distinct regions of viral genome and one scrambled sequence were designed and chemically synthesized. fitc labeled p-pmos were used to observe their distribution of by confocal microscopy. viral protein vp , viral titre, and cell viability were measured by western-blot, plaque assay, and mts assay, respectively. results: p-pmos showed increased cellular uptake compared to non-conjugated pmos. among the p-pmos, p-pmo- , targeting the internal ribosomal entry site in the utr, showed the most potent anti-cvb ability in a dose-dependent manner. both infected hela and cardiomyocytes hl- cells treated with p-pmo- showed drastically reduced vp production and . log decreases in viral titres as compared to the controls. cell viability assay revealed that and % of treated hela and hl- cells were still alive as compared to and % of control-treated cells and % antiviral activity still existed after days treatment. in addition, cells treated post-infection showed similar inhibition of viral replication. furthermore, the specificity of the p-pmos was demonstrated by their inability to inhibit rsv infection in hela cells. we have showed that p-pmos can effectively inhibit viral replication in vitro, providing a new possibility for antiviral intervention. picornaviruses are responsible for various human viral diseases including common cold, encephalitis, meningitis, myocarditis, etc. up to now, there is no specific antiviral therapy to treat or prevent such viral disease. the usage of modern computer technologies may help to solve this problem more effectively. the objective of the present study was the quantitative structure-activity relationship (qsar) analysis of antiviral activity of a set of [(biphenyloxy)propyl]isoxazole derivatives that inhibit cvb replication in hela cells. based on results from qsar, the structure of new potential antiviral agents should be predicted by using consequent molecular design. the qsar approach applied is based on simplex representation of molecular structure (sirms). the relationship between: (a) antiviral activity against the pleconaril-sensitive clinical cvb isolate - (ic , g/ml); (b) cytotoxicity in hela cells (cc , g/ml); and (c) selectivity index (si = ratio of cc to ic ), and structure of [(biphenyloxy)propyl]isoxazole derivatives has been studied systematically. quite adequate qsar models (r = . - . , q = . - . ) have been obtained using pls (partial least squares) method for all parameters studied. the models are in close correlation with experimental data. structural fragments with positive or negative influence on cytotoxicity as well as antiviral activity have been determined on the base of these models. for example, qsar analysis of antiviral activity of [(biphenyloxy)propyl]isoxazole derivatives revealed that the presence of m-nitrophenyl or p-trifluorophenyl fragment has distinctly negative influence on antiviral action. compounds with strong antiviral activity have to contain an oxadiazole fragment. moreover, our data allow the virtual screening and molecular design of new well-tolerated compounds with strong anti-cvb activity. ivanka nikolova , roumena petkova , boris atanassov , stoyan chakarov , angel s. galabov institute of microbiology, bulgarian academy of sciences, sofia, bulgaria; scientific technological service, ltd., sofia, bulgaria; institute of organic chemistry, bulgarian academy of sciences, sofia, bulgaria analysis of the rna sequence of the disoxaril-resistant mutants of the coxsackievirus b was carried out. the wild-type disoxaril-sensitive strain (connecticut ) and two disoxarilresistant mutants (one of them produced in fl cells and the other one isolated from brains of newborn mice) infected with coxsackievirus b and treated with disoxaril and a disoxarildependent mutant strain obtained from the resistant strain by passages in cell culture were included in the present study. a rt-pcr assay with primer sets selected from a region of the coxsackievirus b genome coding for the capsid protein vp was carried out. a parallel comparative analysis of the sequences of resulting fragments from the disoxaril mutants studied and the genbank sequence of origin of the vp gene of coxsackievirus b was performed with the blast alignment tool. distinct alterations in the vp locus of the disoxaril-resistant and the disoxaril-dependent mutants compared to the sequence of origin from the genbank (namely, a deletion of uug at ntt. - and an insertion of uuu at nt. ) were observed. high-degree similarity ( %) between the resistant mutant produced in cell cultures and the dependent strain was observed, while the similarity to the wild strain was only %. the resistant mutant obtained in mice was found to be very similar to the strain, developed in cell cultures. a putative -d model of the spatial folding of the target protein in disoxaril mutants is proposed. ralitsa vassileva-pencheva, angel s. galabov in previous study of ours we presented a new approach to combined application of antivirals-consecutive administration of the partners. this schedule could be considered especially suitable for treatment of enteroviral infections, in which the development of resistance is very rapid due to the extremely high viral mutation rate. this approach aims to restrict the resistance development in experiments in vivo, using antivirals with proved high efficiency in experiments in cell cultures. the screening of various double, triple, and quadruple combinations that we carried out showed that two of the triple combinations, namely disoxaril (win compound)/oxoglaucin (a new antiviral drug, developed in our laboratory)/ptu- (a classic enteroviral inhibitor) and disoxaril/oxoglaucin/guanidinehydrochloride (a classic enteroviral inhibitor) manifest significant effect of protection in newborn mice with neurotropic coxsackievirus b infection. in the current study the role of the chronology of arrangement of the antivirals included in the combinations was investigated. in the experiments carried out with the triple combination disoxaril/oxoglaucin/guanidine-hydrochloride, it was found that the optimal treatment course should start with disoxaril. the treatment course is quite successful when disoxaril is followed by guanidine-hydrochloride. the effect of the triple combination starting with oxoglaucine, followed by guanidine-hydrochloride was moderate. the course starting with guanidine-hydrochloride proved to be ineffective. furthermore, we studied the virus sensitivity to the inhibitorspartners (ic values) and some other phenotypic characteristics of the brain isolates, e.g. the size of the plaques and the pathogenicity for mice. recently our contribution to the development of new antipicornavirus agents has led to the discovery of methylthio- -aryl-isoxazole- -carbonitrile derivatives whose in vitro anti-coxsackievirus b activity were dependent on the nature of the substituents on the para position of the phenyl ring. particularly, compounds -methylthio- -[ -( -phenyl- -propoxy)phenyl]isoxazol- -carbonitrile (on- ) and -methylthio- -[ -( -phenoxy- -buthoxy)phenyl]isoxazol- -carbonitrile (on- ) exhibited an interesting antiviral activity with high selectivity indexes. in the present study, we investigated on the mechanism of action of these compounds. studies on time of addition experiments suggested that these compounds exert a different interference with an early step of the viral replicative cycle. in fact, compound on- was effective when added within h after the end of the adsorption period and no reduction was observed if it was added during the adsorption period. whereas a reduction of virus titer was observed for on- when was added during the adsorption period, while no reduction was observed if the compound was added after this period (time ). the influence of the compounds on virus adsorption step, studied by the infective center assays, indicated that on- primarily interferes with coxsackie b cellular attachment. at a concentration times the id , inhibition of adsorption of coxsackievirus by on- was complete, while similar concentration of on- had no effect. our experiments on neutralization of viral infectivity and on thermal stabilization demonstrated that the compounds were able to directly inactivate coxsackievirus, and the infectious titer was restored to the original value after extraction of the compound with chloroform. however, the compounds did not protect the viral infectivity against heat inactivation at the different concentrations used. the blood-brain barrier (bbb) fulfills a vital protective function by limiting entry of potential pathogens, toxins, and inflammatory cells into the central nervous system (cns). disruption of the bbb is a common component of many cns diseases, including viral diseases such as that caused by west nile virus (wnv). transforming growth factor-␤ (tgf-␤ ) has previously been shown to improve the function of an in vitro model of the bbb. we evaluated the role of the bbb in wnv infection in mice by determining the ability of intraperitoneally (i.p.) administered sodium fluorescein to move from the circulating blood to the central nervous system. to demonstrate bbb permeability a mean and normal range of permeability values was determined in non-infected c /bl mice. in subsequent experiments, any animal expressing a permeability value greater than sd above the mean was considered abnormally high. we determined that elevations in bbb permeability can be detected in mice days after subcutaneous inoculation with wnv. wnv inoculated animals were treated with doses of , , or ng/kg/day of tgf-␤ or with drug vehicle once daily via the i.p. route on and days post-virus inoculation (dpi), and then assayed for bbb permeability on dpi. sixty-two percent ( / ) of placebo-treated animals had abnormally high permeability values, while animals treated with and ng/kg/day of tgf-␤ had % ( / ) and % ( / ) of animals with abnormally high permeability values, respectively. in contrast, none of the animals treated with ng/kg of tgf-␤ ( / ) expressed abnormally high permeability values, which was significantly lower (p < . ) than placebo-treated animals. these results suggest that tgf-␤ may improve the function of the blood-brain barrier in wnv infected mice. acknowledgement: supported by grant -u ai - from the rocky mountain regional centers of excellence, nih. and contract no -ai- from the virology branch, niaid, nih. people infected with west nile virus (wnv) usually see their physicians after showing symptoms suggestive of neurological infection. wnv infects the central nervous system (cns) of rodents - days after s.c. viral challenge. yet, most published animal studies begin therapeutic treatments before or soon after viral challenge. the question addressed in this study is if neuroprotective agents can be efficacious when administered early before brain infection or later after the virus is demonstrated to be in the brain. the drugs evaluated in wnvinfected rodents were nmda and ampa receptor antagonists, modulators of nitric oxide synthase (nos) and nitric oxide production, and riluzole for reducing glutamate excitotoxicity. serial doses of diethyldithiocarbamate (ddtc) and n(g)monomethyl-l-arginine (l-nmma), an inducer or inhibitor of nos, respectively, administered i.p. daily for days beginning h before viral challenge slightly improved survival of mice, but the difference was not statistically significant. tolerated doses of two nmda-receptor antagonists, flupertine ( mg/kg) and mk- ( mg/kg), and one ampa-receptor antagonist, gyki ( mg/kg), were administered twice daily (b.i.d.) on though days post-virus inoculation (dpi). gyki slightly improved mouse survival and weight gain, but the difference was not statistically significant. talampanel, an ampa-receptor antagonist and a derivative of gyki , slightly improved hamster survival (p ≤ . ) when treatment began on dpi, but repeated experiments using different doses and slightly different protocols gave mixed results. riluzole, the only drug shown to improved survival of amyotrophic lateral sclerosis (als), presumably by reducing glutamate excitotoxicity, was not effective against wnv disease when administered b.i.d. beginning dpi. overall, neuroprotective agents did not consistently improve wnv disease, although slight improvements in animal survival might be relevant to improvement of neurological sequelae in wnv-patients. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. hamster and mouse models for west nile virus (wnv) disease were used in this study to identify infected cells of the central nervous system (cns) early in the course of infection. this information may be relevant to therapeutic strategies since most wnv-infected people visit their physicians after showing symptoms suggestive of neurological infection. we subcutaneously infected adult mice and hamsters using . tissue culture infectious doses of wnv. tissues of infected and control animals from to days post-viral injection (dpi) were fixed by cardiac perfusion using % paraformaldehyde. we localized wnv, neuronal and astroglial markers in the paraffin embedded tissue sections by immunofluorescence. the images were captured using the confocal microscope (bio-rad, mrc ). we observed the presence of wnv antigen in cns tissues of mice and hamsters as early as and dpi, respectively. a strong wnv-specific immunofluorescence staining was observed in the cytoplasm of neurons from the spinal cord, cerebellum, cerebral cortex, and midbrain of these rodents. the wnv-specific staining co-localized with neuron-specific markers; however, astroglial markers were not co-localized with wnv antigen in brain sections. the lack of tropism by wnv for astrocytes was also confirmed in primary murine astrocyte cultures. interestingly, infected neurons in the midbrain of -day infected hamsters co-localized with calbindin, which is a calcium-binding protein and mostly expressed in the interneurons of the cns. therapies were evaluated in hamsters or mice at a time-point when wnv-stained neurons were identified in the cns. acknowledgement: supported by grant -u ai - from the rocky mountain regional centers of excellence, nih. laboratory of virology, department of biological chemistry, school of sciences, university of buenos aires, argentina dengue virus (denv) is an arthropod-borne flavivirus that has re-emerged in recent years as an increasingly important public health threat with nearly million infections occurring each year. at present neither specific antiviral therapy nor vaccine exists for the treatment and prevention of denv infections. carrageenans are sulfated galactans that can be extracted from red seaweeds and comprise diverse structures with a wide range of biological properties useful in biomedicine. in a previous study we have demonstrated the antiviral activity of commercialand -carrageenans against denv type and in vero (monkey kidney cells) and hepg (human hepatoma cells), showing inhibitory concentration % (ic ) values in the range . - . g/ml and selectivity indexes (cc /ic ) in the range - . in the present work we studied the mode of action of these polysulfates against denv- in vero and hepg cells, first analyzing the influence of time of addition of compounds on anti-denv activity by an infectious centre assay. the highest inhibitory effect was observed when the compounds were added during adsorption or at h p.i., being ineffective at later times. then, the effect of compounds on virus adsorption and internalization was studied separately by a virus yield inhibition assay. significant antiviral efficacy was attained if compounds were present either only during denv- adsorption or internalization. the possible effect of carrageenans on viral protein synthesis, the subsequent stage of the virus cycle occurring during the first hour of infection, was analyzed by pulse-labeling with ( s)-methionine. no alterations in denv protein synthesis in carrageenan-treated cells were observed. when cells were transfected with purified denv- rna in the presence of -carrageenan no inhibition in fluorescent cell focus formation and virus production was detected. besides, no significant direct virucidal effect on denv- was shown by the compounds. these results indicate that both denv adsorption and internalization seem to be the main target for these compounds, lacking effect on the steps that occur once the viral genome is inside the cell during in vitro infection of human and monkey cells. multiple members of the flavivirus genus of the family flaviviridae cause lethal hemorrhagic fever or encephalitis. the public health significance of the hemorrhagic fever and encephalitis caused by such flaviviruses is enormous and global and there is a tremendous need for antivirals. imino sugar glucosidase inhibitors have been shown to have selective antiviral activity against viruses such as bovine viral diarrhea virus (bvdv) and west nile virus (wnv) that have common requirements for their glycoprotein processing during virus production. we are developing imino sugar deoxynojirycin (dnj) derivatives through chemical synthesis of compounds with various alkyl side chains and antiviral testing against bvdv and wnv as well as in wnv subgenomic replicon assays. briefly, using a single step growth (virus yield reduction) assay for bvdv and wnv, a series of dnj derivatives containing various conformational locking side chains were shown to have antiviral activity. pre-liminary structure-activity relationships (sar) were obtained for further modification of the alkyl side chain and improvement of these dnj derivatives. the activity and mechanisms of action of these compounds will be presented. several flaviviruses cause life-threatening diseases in man. currently, there is no therapy available for these infections. in recent years, several highly selective inhibitors of the replication of hepatitis c virus (hcv) were designed. most small molecule inhibitors of hcv that are in preclinical or clinical development are either protease or polymerase inhibitors. most of these compounds are highly selective for hcv and are unlikely to exhibit activity against flaviviruses. nucleoside polymerase inhibitors, however, may have the potential to inhibit the replication of flaviviruses as well. we evaluated in vitro whether the active component of the anti-hcv compound valopicitabine, i.e. -c-methylcytidine inhibits the replication of flaviviruses in cell culture. the compound was found to exhibit specific antiviral activity against yellow fever virus d (ec = . g/ml in cpe reduction assays and > % reduction at g/ml as assessed by qpcr) and dengue fever virus type (ec = . g/ml in cpe reduction assays). the compound also efficiently inhibited west nile virus replication (> % at g/ml as assessed by qpcr and > % by plaque reduction neutralization test at g/ml). in the absence of any drugs for the treatment of flavivirus infections, it may be envisaged that nucleoside polymerase inhibitors, when marketed for the treatment of hcv infections, could be used off-label for the treatment of lifethreatening flavivirus infections. even if such drug would not be able to completely inhibit flavivirus replication, a partial reduction of the viremia during the acute phase of the infection may be sufficient to prevent the development of a fulminate disease and thus protect against virus-induced mortality. yuri klimochkin , andrew shiryaev , igor moiseev , v sabynin , larisa rustamova , alexandr petkevich state technical university, samara, russia; institute of epidemiology and microbiology, minsk, belaruss arenaviruses are one of the most dangerous tools of bioterrorism in the view of pathogenicity and epidemiological threat. for the purpose of searching new remedies for treatment are-naviruses infections the synthesis of new derivatives of cage compounds has been carried out. the prepared compounds are bridgehead derivatives of cage compounds bearing different functional groups such as hydroxy, acylamino, alkoxycarbonylamino, alkylthiocarbonylamino groups as well as iminoalkyl adamantane derivatives, some adamantylated heterocycles and compounds containing two adamantane moieties in a molecule. the antiviral activity of the cage compounds has been studied in respect to arenaviruses lassa (sierra-leone strain) and pichinde (an- strain) on the vero cells culture. different level of antiviral activity was shown by compounds. the most active compounds are monosubstituted adamantane derivatives having sufur and nitrogen-containing substituent in the bridgehead position. the data on the activity confirm the availability of searching inhibitors of arenaviruses reproduction in the cage compounds series. brian gowen , donald smee , min-hui wong , anne pace , kie-hoon jung , kevin bailey , lawrence blatt , robert sidwell institute for antiviral research, utah state university, logan, ut, usa; intermune, brisbane, ca, usa several arenaviruses endemic to the south american (junin, machupo, and guanarito) and african (lassa) continents are known to cause frequently fatal hemorrhagic fever. with the exception of ribavirin, which has demonstrated efficacy in cases of lassa fever, there are no other effective therapeutics for the treatment of arenaviral hemorrhagic fever. the outcome of treatment is ultimately dependent upon early diagnosis and the tolerability of ribavirin by patients at the high doses required for effective antiviral activity. we have recently demonstrated that consensus interferon-alpha (ifn-alfacon ) can protect hamsters from lethal pichinde virus (pcv) infection (gowen et al., . antimicrob. agents chemother.), which serves as a model for acute arenaviral disease in humans. here we demonstrate highly effective therapy through the combined use of ribavirin and ifn alfacon- for the treatment of pcv infection in hamsters. ribavirin was given orally, twice per day for days, and ifn alfacon- was administered intraperitoneally, once per day for days. treatments were initiated - days post-infection with various dose combinations, many which were less than optimal when the drugs were given independently. combining suboptimal doses of ribavirin ( - mg/kg/day) with ifn alfacon- ( - mg/kg/day), we were able to show increased protection from mortality, reduced viral burden and liver disease, and greatly extended survival times as compared to treatments where drugs were administered alone. our data indicate that synergistic activity resulted from combination therapy and that this activity may slow down the progression of disease and decrease fatality rates seen with severe arenaviral infections. further, combination therapy reduces the effective dosage of ribavirin, which would serve to limit its toxicity. acknowledgement: supported by contract no -ai- from the virology branch, national institute of allergy and infectious diseases, national institutes of health. slobodan paessler , laure deflubé , andrew vaillant , jean-marc juteau , ramon flick department of pathology, university of texas medical branch, galveston, texas, usa; replicor inc., laval, que., canada rift valley fever virus (rvfv; genus phlebovirus, family bunyaviridae) is an arbovirus transmitted by many species of mosquitoes. this virus is a major public health concern in sub-saharan africa and egypt, which spread to yemen and saudi arabia. in this area, rvfv is responsible for dramatic epidemics/epizootics underlining the need for efficient antiviral/prophylactic measures. rep is a mer phosphorothioate oligonucleotide, which has previously been shown to have broad-spectrum activity in several viruses (vaillant et al., submitted for publication) . we used a vaccine strain (mp ) as well as the wild-type rvfv (zh ), to test the ability of rep to inhibit bunyavirus proliferation. in vitro data showed reduction of virus titer for both strains using rep at nanomolar concentrations. moreover, the absence of the phosphorothioate modification in a stabilized rep analog resulted in a loss of antiviral activity, suggesting that as in other viruses, the increased hydrophobicity of rep is essential for its antiviral activity. based on the inhibitory activity observed in vitro, we started with in vivo efficacy studies by utilizing a validated mouse model used in our laboratory. more animal experiments are ongoing to confirm the in vitro results and to evaluate the antiviral effect of the rep . adriana garozzo , rossella timpanaro , aldo stivala , gianna tempera , christian c.c. cutrì , angelo castro department of microbiological sciences, university of catania, via androne , catania, italy; department of pharmaceutical sciences, university of catania, viale a. doria , catania, italy our previous studies described the synthesis and the antiviral activity of , , -trisubstituted isothiazole derivatives that were found to be particularly effective against picornaviruses. compound -methylthio- -phenyl- -isothiazolecarbonitrile (is- ) exhibited an interesting anti-poliovirus activity with high selectivity index. in the present study, we investigated on the mechanism of action of this compound. studies on the time of is- addition to poliovirus type infected cells suggested that the compound may inhibit some early processes of viral replication. in order to determine its mechanism of action, we evaluated the rate of attachment and internalization of purified [ h]uridine-labeled poliovirus to hep- cells in the presence or absence of is- . no effect on poliovirus adsorption and internalization to host cells was detected. we also investigated the influence of the compound on virus uncoating using labeled poliovirus and measuring the radioactivity of oligoribonucleotides formed from viral rna susceptible to ribonuclease. these experiments demonstrated that poliovirus uncoating is influenced by is- action. justin julander , aaron olsen , john morrey , lawrence blatt , kristiina shafer , robert sidwell institute for antiviral research, utah state university, logan, ut, usa; intermune, brisbane, ca, usa alpha togaviruses are medically important arboviruses, with clinical cases occurring each year in north, south, and central america. the recent increase in the threat of the use of these viruses as bio-terrorism agents has led to increased efforts to develop therapeutic agents for treatment of these viruses. venezuelan (vee) and western equine encephalitis (wee) viruses have been listed as category b priority pathogens by the national institute of allergy and infectious disease (niaid). the goal of these studies was to characterize animal models for vee and wee for use in evaluation of antiviral therapies. c h/hen mice were infected through the intranasal (i.n.) route with a vaccine strain of vee, tc- . virus was detected in the brain days post-viral injection (dpi). brain titers increased to a peak titer of . % cell culture infectious doses per gram tissue (ccid /g) on dpi, maintained a titer of ccid /g through dpi, and dropped slightly to . ccid /g by dpi. virus was also detected in spleen, liver, and kidney. treatment of vee-infected mice with interferon alpha b/d, a human consensus interferon, resulted in % survival, whereas all placebotreated animals died by dpi. syrian golden hamsters were infected with ccid wee through intraperitoneal (i.p.) injection. morbidity, including hind limb paralysis, tremors, nasal bleeding, and hunching, and some mortality were seen as soon as dpi. the majority of deaths occurred on dpi. virus was detected in all organs assayed (brain, liver, and spleen) with peak titers occurring dpi. interferon alfacon (ifn alfacon), a human consensus interferon, active in hamsters, was effective in significantly reducing mortality (p < . as compared to placebo). there was a trend for reduction of brain titers in ifn alfacon-treated animals (mean titer . ccid /g) as compared with placebo (mean titer . ccid /g), although this difference was not statistically significant. these models will be useful in screening potential antiviral agents for efficacy against vee and wee. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. justin julander , kristiina shafer , john morrey , lawrence blatt , robert sidwell institute for antiviral research, utah state university, logan, ut, usa; intermune, brisbane, ca, usa yellow fever virus (yfv) has caused significant morbidity and mortality for centuries. primates were the only animal models for visceral yfv. recently, hamsters were found to have morbidity and mortality when injected with a hamster-adapted jimenez strain of yfv (tesh et al., j. infect. dis. , - . the objective of this study was to characterize this model of yfv viscerotropic disease for the study of effects of antiviral compounds and to test compounds with known efficacy for use as a positive control. animals were challenged with a − dilution (a dilution previously shown to cause high mortality) of a liver homogenate made from livers taken days post-viral injection (dpi) from hamsters challenged with the jimenez strain. there was % mortality in animals challenged with the virus up to dpi. virus titers in the liver peaked dpi as determined by qrt-pcr. a significant increase in serum levels of alt ( dpi), alkaline phosphotase ( dpi) and bilirubin ( dpi), and a significant decrease in amylase ( dpi), albumin ( dpi), and glucose ( dpi) were observed. hepatic icterus was observed in hamsters that exhibited disease signs at the time of necropsy. hamsters were treated with ribavirin or interferon (ifn) alfacon , a consensus interferon. ribavirin and ifn alfacon both significantly (p < . ) reduced mortality as compared with placebo-treatment. there was also significant reduction in weight loss with ribavirin (p < . ) and ifn alfacon (p < . ) treatment as compared with placebo. disease signs, such as lethargy and lying prostrate, were also reduced with treatment of ribavirin and ifn alfacon . viral liver titers from treated animals were not significantly different from titers in placebotreated animals. the hamster model of yfv disease will serve as a suitable model for the evaluation of antiviral compounds for efficacy against the virus. acknowledgement: supported by contract no -ai- from the virology branch, niaid, nih. polyomaviruses are small dna tumor viruses that depend on the host cellular dna polymerase for their replication. three polyomaviruses have been associated with tumor formation in humans: jc virus (jcv), bk virus (bkv) and simian vacuolating virus (sv ). in addition, some of them have been associated with viral diseases. jcv can cause progressive multifocal leukoencephalopathy in immunosuppressed patients, while bkv is considered to be the causative agent of polyomavirusassociated nephropathy, which leads to kidney transplant failure. sv has not been associated with a well-defined disease, but viral dna sequences and protein expression have been detected mostly in central nervous system (cns) tumors which strengthens the evidence for the association of this virus with human cancer. the activity of various acyclic nucleoside phosphonates (anps) such as cidofovir and adefovir against murine polyomavirus and primate sv in vitro has already been demonstrated (andrei et al., . antimicrob. agents chemother. , - ) . here, the activity of a new class of anp's, namely -[ -(phosphonomethoxy)alkoxy]- , diaminopyrimidines, against polyomaviruses was assessed. confluent uc -b cells were infected with either of the four murine polyomavirus strains mn/rde toronto, pta, pta or lid- , while bsc- cells were infected with either the primate sv strain a , the sv pml- strain ek or the sv pml- strain dar. after removal of the residual virus, serial dilutions of the test compounds were added. the viral cytopathic effect was recorded microscopically after - days (murine polyoma virus) or - days (sv ). hpmpo-dapy ( , -diamino- -(r)-[ -hydroxy- -(phosphonomethoxy)propoxy]pyrimidine) and pmeo-dapy ( , -diamino- -[ -(phosphonomethoxy)ethoxy]pyrimidine) were less active/selective than cidofovir and adefovir against the three sv strains tested. hpmpo-dapy and pmeo-dapy proved to be equally active as cidofovir and adefovir against the murine polyomaviruses. naresh sunkara , sylvester mosley , brian bakke , joshua sadler , katherine seley(radtke) , sunny zhou university of maryland-baltimore county, baltimore, md, usa; washington state university, pullman, wa, usa inhibition of biologically significant enzymes critical to nucleotide metabolism and viral replication is a well-established chemotherapeutic approach to the treatment of many diseases. transcriptional -capping of viral mrna has been implicated as an "elongation checkpoint" critical to the replication cycle of many viruses. this capping process is accomplished by various methyltransferases, therefore disruption of methylation becomes an attractive target for therapy. this can be accomplished in several ways; in particular, by direct inhibition of methyltransferases (metase) and/or indirect inhibition of s-adenosyl-l-homocysteine hydrolase (sahase), both established cellular targets for antiviral, antiparasitic and anticancer agents. modified nucleosides, in particular carbocyclic nucleosides, have exhibited potent inhibitory activity against both sahase and metase. inspection of the recent literature has revealed a close correlation between sahase inhibition and potent biological activity against negative stranded (−)-rna viruses (i.e. arenaviridae, paramyxoviridae, rhabdoviridae), double stranded (ą)-rna viruses (reoviridae), poxviridae, as well as hiv- , thus supporting the importance of sahase as a viable chemotherapeutic target. herein we report the design, synthesis, and preliminary biological activity of a new class of structurally novel carbocyclic nucleosides. phosphorylation of ␣-p-borano substituted nucleoside diphosphates charlotta wennefors, mikhail dobrikov, barbara ramsay shaw chemistry department, duke university, durham, nc - , usa most nucleoside antiviral agents require stepwise phosphorylation to their respective triphosphates in order to be activated in the cell. ␣-p-borano substituted nucleoside triphosphates are of interest because they have proven to be good substrates for hiv- reverse transcriptase (rt) and may therefore be useful antiviral agents. studies in our laboratory have indicated that the ␣-p-borano substitution of -dideoxycytidine triphosphate (ddctp) resulted in a -fold increase in efficiency of incorporation by mmlv rt compared to non-substituted ddctp. however, the potency of these ␣-p-borano substituted nucleoside analogs as anti-viral drugs highly depends on their ability to be activated to nucleoside triphosphate (ntp). the phosphorylation of nucleoside analog diphosphates to their respective triphosphates has remained largely unexplored. here, the roles of several phosphorylating enzymes are examined. in our laboratory, nucleoside diphosphate kinase, creatine kinase, and pyruvate kinase are being evaluated for their specificity towards nucleoside analog diphosphates. the effects of nucleobase, ribose, ␣-phosphate substitution and stereochemistry of the boranophosphate group are of interest. the binding affinities of the substrates for creatine kinase (ck) and pyruvate kinase (pk) were determined using a fluorescence-quenching assay, which allowed us to investigate the substrate affinity in the pre-steady state. rabbit muscle ck and pk were titrated with a wide range of ndps and ntps by monitoring a decrease in enzyme intrinsic fluorescence. the affinities of these substrates were determined to establish a structure-activity relationship for ck and pk and to evaluate the effect of a substrate ␣-p-borano modification. ck showed stereospecificity towards the sp isomer of adp␣b whereas pk showed stereospecificity towards the rp isomer of adp␣b. negative cooperativity was observed for all studied substrates. steady-state experiments are also being performed directly following the product formation using uv-visible spectroscopy and high performance liquid chromatography (hplc). these kinases were investigated because they may serve as a means for activation of antiviral ␣-p-borano substituted ndps. traditional antiviral targets encoded by the small human papillomavirus (hpv) genome are lacking. for this reason, we chose to target dna sequences within the hpv genome in an effort to identify compounds that would block viral dna replication in cells. we chose compounds known as polyamides, which are related to distamycin and other natural products, as our dna binding agents. unlike many literature studies where polyamides were designed to block formation of the transcription complex for a particular gene, we chose to target sequences within the origin of replication (ori). thus, pyrrole-imidazole polyamides, with some containing fluorescent probes to aid in cell localization studies, were designed to recognize the hpv ori. the principles used to design these compounds will be described. we used "traditional" hairpin polyamides and some more unusual structures related to very recent literature reports. from the focused library that we prepared, two highly active molecules were identified. the rest of the molecules had minimal or zero activity. no cellular toxicity was observed, either in this project or in a related program where polyamides were used to affect cox- transcription (and subsequent expression) in rheumatoid synovial fibroblasts. of particular interest is the difference between the active molecules and two closely related compounds that were inactive: the active species bind and recognize two more hpv dna base pairs than do the related but inactive structures. this presentation will provide detailed chemistry background and structural information to complement our cell work that is also being presented at the meeting. discovery of the chemokine receptor ccr as a co-receptor for hiv- infections revealed a novel approach to hiv- treatments and preventions. ccr , a member from the family of tm g-protein coupled receptors, thus became an attractive target pursued in the pharmaceutical industry. with the recent successful developments of several small molecules in clinic, these ccr antagonists hold great promise to be the next generation of anti-hiv medicines. this poster will describe our efforts at the n-terminal piperidine ring of template a to improve pharmacological properties of derived molecules. according to current models, proteolytic processing of hiv- gag precursor occurs within the virions which detach from infected cells. meanwhile, the viral protease is activated much earlier, and gag p cleavage initiates in infected cells. we followed the fate of matrix protein cleaved in infected cells (cma) in comparison with ma cleaved in the virions (vma) and showed that both forms differ in their localization in the infected cells and in the virions, both forms are involved into virus pathogenesis and represent the targets for antiviral compounds. mt- cells were labeled with [ h]-leucine or myristic acid, and h after labeling protease inhibitor was added to separate the cleavage of cma from vma. cma was found in the nuclear and membrane fractions of infected cells while cca resided in cytoplasm. - h after labeling cma was found in the virions localizing in the cores. vma was located under lipoprotein envelope of the virions. new membranotropic antiviral compounds based on adamantane-and norbornene-related derivatives not toxic for the host cells were added to mt- cells before infection or - h later and at concentration - ug/ml blocked reverse transcription, the transport of cma into the nuclei, and the production of infectious virus. the compounds inhibiting very early step of virus life cycle are optimal candidates for microbicides. to enhance their antiviral activity, we plan to associate polyanionic matrix with ma imitating peptides and cholesterol-like fragments. kurt vermeire , thomas bell , sreenivasa anugu , noah duffy , roger le grand , erik de clercq , dominique schols rega institute for medical research, katholieke universiteit leuven, leuven, belgium; department of chemistry, university of nevada, reno, usa; service de neurovirologie, fontenay-aux-roses, france the cyclotriazadisulfonamide (cada) compounds were shown to be potent inhibitors of hiv replication in human t-cell lines, pha-stimulated pbmcs, and monocytes/macrophages (ec : . - . m). the prototype compound, cada, had consistent activity against laboratory adapted and primary clinical isolates of hiv- , irrespective of chemokine receptor preference (r , x , r /x ). cada acted synergistically when evaluated in combination with various other hiv drugs, such as reverse transcriptase (rt), protease, and virus entry inhibitors. flow cytometric analysis revealed a significant decrease in the cell surface and intracellular expression of the cd receptor in human cells after cada-treatment. moreover, the anti-hiv activity of cada correlated with its ability to down-modulate the cd receptor in human t-cells. here, we report the consistent antiviral activity of cada against: (i) drug-resistant viruses (i.e. viruses resistant to rt inhibitors, protease inhibitors, and enfuvirtide); (ii) different hiv- subtypes (a, b, c, d, a/e, f, h, o); and (iii) various hiv- strains examined. in addition, cada potently inhibited sivmac infection of pbmcs isolated from macaques (ec : . m). comparable results were obtained in human cells infected with sivmac . flow cytometric analysis also demonstrated a significant and dose-dependent down-regulation of the cd receptor expression at the cell surface of simian pbmcs after treatment with cada. the combination of cada with cellulose acetate , benzenedicarboxylate (cap), an enteric coating polymer for capsules and tablets, resulted in a synergistic antiviral activity. in summary, our data indicate that cada may qualify as a potential anti-hiv microbicide drug candidate for the prevention of the sexual transmission of hiv. the preparation of gel formulations of cada (as single drug and in combination with cap) for vaginal administration in non-human primates is currently under investigation. department of micro & immuno, suny upstate medical university, syracuse, ny, usa varicella zoster virus (vzv, human herpesvirus ) infection causes chicken pox, latency is established in neurons, and reactivation leads to shingles. acyclovir and its derivatives are the treatment of choice for both manifestations of vzv. new therapeutics are needed because acyclovir-resistant strains exist, and treatment must begin within h. we have studied the anti-vzv properties of roscovitine, a cyclin dependent kinase (cdk) inhibitor. here, we tested more compounds that block the cell cycle and determined that vzv is acutely sensitive to them. their effects on vzv replication were tested in human foreskin fibroblasts (hffs) because these primary cultures should have a normal cell cycle (unlike tumor cell lines). the cytotoxicity of the drugs was determined by neutral red dye uptake assays. hffs were inoculated with a low moi ( . ) of vzvinfected cells, which remains entirely cell-associated, and then treated with drugs or diluent for h. vzv spread and replication were measured by infectious focus assay and quantitative real time pcr. all of the drugs tested (acyclovir [acv], phosphonoacetic acid [paa], aphidicolin, aloisine a, purvalanol a, roscovitine, r-roscovitine, s-roscovitine, indole- -carbanol [i c], l-mimosine, dichloro-␤-d-ribofurano-sylbenzimidazole [drb]) had some anti-vzv activity. the selective indices of aphidicolin ( ), purvalanol a ( ), and i c ( ) were greater than the positive controls acv ( ) and paa ( ). aphidicolin inhibits mammalian dna polymerase and is in clinical use for cancer; purvalanol a, a , , -trisubstituted purine, primarily inhibits cdk ; and i c is derived from cruciferous vegetables and inhibits cell proliferation. the concentrations of these compounds that inhibited vzv replication were much less than those needed to cause cell cycle arrest, suggesting that vzv depends on the enzyme activities targeted by these compounds and not on cell proliferation per se. these three drugs will be studied next in skin organ culture and in the scid-hu mouse model of vzv pathogenesis. the results presented here demonstrate that targeting cell functions can inhibit vzv replication and help us better understand virus-host interactions. the viruses could be identified as supra-biopolymeric nanoscale complexes, parasitic intervention in cells of which occurs on an inter-polymeric reactions level. so the antiviral safety can not be fully provided without adequate nano-responsible antivirals (nav). here we discuss a strategy and methodology for the multi-functional nav development by rational macromolecular sar-cooperation of: ( ) polyelectrolyte-specific interferon induction and immunomodulation; ( ) electrostatic-selective prevention of viruses absorption on plasma membranes; ( ) membrane-targeted blocking of post-absorption steps (fusion); ( ) macromolecular prevention of structure-specific interactions of viral and cellular receptors; as well as ( ) polymericassociated disruption of the latest stage of viral replication (virions assembly and maturation). a cooperation of the ( ) and ( ) functions was explored by synthesis and sar-optimization of succinate and carbohydrate polymeric derivatives modified with controllable combinations of anionic (a /a ) groups. the immune-mediated protectors against tick-born, rabies, and other viral infections in vivo, and hiv- absorption inhibitors in vitro, were developed. this pre-nav generation was used as a macromolecular platform to step-by-step targeted design and synthesis toward high effective multi-functional nav where virusresponsible nano-selectivity was achieved by rational intra-or inter-molecular cooperation of virus-specific membranotropic vectors (bi), raft-targeted anchors (ci), and peptide-kind mimickers of virus usable receptors of human cells (pi), particularly ccr /cxcr . as a result, the novel nano-sensitive systems possessed unique wide multi-synergistic antiviral potency on a high level selectivity up to si = , (against hiv- strains) were created and purposed for advancement of antiviral vaccines, drugs, and microbicides. marina kukhanova , alexander ivanov , , georgii galegov , valeria andronova , maxim jasko engelhardt institute of molecular biology, russian academy of sciences, moscow, russia; centre for medical studies, university of oslo, moscow, russia; ivanovsky institute of virology, russian academy of medical sciences, moscow, russia novel acyclic purine phosphonate derivatives bearing a double bond conjugated with the nucleic base, namely, (z)-and (e)- -[ -(phosphonomethoxy)prop- -en- -yl]purines, were synthesized, and their efficacies against hiv- and hsv- were evaluated in cell cultures. the activity of (z)isomer was higher against hiv than that of the reference -[ -(phosphonomethoxy)ethyl]adenine (adefovir) and comparable in respect to the activity of adefovir against hsv. the (e)-isomer showed low antiviral activity against both viruses. the compounds were less toxic towards cell cultures if compared to adefovir. the diphosphates (z)-and (e)- -[ -(phosphonomethoxy)prop- -en- -yl]purines were evaluated as substrates towards hiv- reverse transcriptase and hsv dna polymerase. (z)-isomer was shown to be a more efficient substrate for both enzymes than the (e)-isomer. human dna polymerase alpha could incorporate neither of the diphosphates into the -end of the growing dna chain. available to this virus. jev genome is an approximately -kb single-stranded positive-sense rna that has a cap structure at its terminus but lacks a poly(a) tail at its -terminus. the coding region of the genome is flanked by -and -untranslated region (utr). the -utrs on both plus-and minus-strand jev genome contain important cis-acting elements required for the replication of the viral rna genome. peptide nucleic acid (pna) is a synthetic oligonucleotide, in which the phosphodiester backbone of dna/rna is replaced with a polyamine-( -aminoethyl) glycine skeleton. pna offers a potentially powerful approach for recognition of rna and silencing of gene expression. in this study, we investigated the antiviral effect of the pnas targeted to the -utr region of jev genome. to evaluate the pnamediated inhibitory effect on rna synthesis in vitro, the rnadependent rna polymerase (rdrp) of jev, ns protein, which plays a major role in replication of the viral genomic rna, was expressed in escherichia coli and purified to near homogeneity by sequential column chromatographies. the recombinant jev ns protein exhibited a primer-dependent rdrp activity in vitro on a homopolymeric template, poly(a). in addition, it was able to accept both plus-and minus-strand -utrs as templates for rna synthesis in the absence of an exogenous primer. it could utilize the -end -nt of jev genome as a minimal template. in vitro rdrp assays using this functional recombinant jev rdrp in the presence of the pnas targeted to the jev -utr -nt showed a dose-dependent rna synthesis inhibition. delivery of the inhibitory pnas to the jev-infected cells suppressed jev replication, as determined by western-blot analyses and plaque assays. our results showed a sequence specific inhibition of jev replication by antisense pnas, suggesting the possible application of pna as a novel anti-jev agent. julia serkedjieva , reneta toshkova , milena nikolova , reneta tsvetkova , stefka antonova , ivana roeva , munnever sokmen , bektas tepe , medine gulluce , fikrettin sahin , atalay sokmen institute of microbiology; institute of experimental pathology and parasitology; institute of botany, bulgarian academy of sciences; faculty of biology, department of microbiology, sofia university, sofia, bulgaria; faculty of art and science, department of biology, cumhuriyet university, sivas, turkey; faculty of art and science, department of biology, atatürk university, erzurum, turkey natural products can be an important source of new pharmaceuticals. research on antivirals of natural origin is mainly focused on plants, since, among other reasons, they can be selected on the basis of their ethnobotanical use. plant extracts and natural plant products exhibit also a variety of biological activities with pharmacophoric utility. the population of the balkan peninsula, like people from all continents, has long applied poultices and imbibed infusions of hundreds of indigenous plants. the present report summarizes the antiviral screening study of plant products, obtained from bulgarian and turkish medicinal plants. they were tested for inhibitory effect on the reproduction of selected influenza virus (flu) strains in mdck cells and herpes simplex virus (hsv) strains in mdbk cells. the reduction of virus-induced cpe and infectious virus yields were used as measures of viral inhibition. fifteen samples ( . %) inhibited flu reproduction, and twelve samples ( . %) were active against hsv. the anti-flu activity was confirmed in vivo for all tested samples. the most effective products were tested further for their antiproteolytic, antioxidant and immunogenic capacities and for potential antibacterial and antifungal effects. the following correlations among the variety of biological and pharmacological activities of the plant products were observed: the anti-flu effect was associated with anti-hsv effect and vice-versa in . %; the antiviral effect was connected with antioxidant activity in %; the anti-flu effect was associated with immunogenic properties in %; there was found no correlation between the antiviral effect and the antiproteolytic capacity, the anti-viral properties and bacterial or fungal inhibition, the anti-viral activity and the polyphenol contents. our previous investigations have revealed antiviral activity of some proteolysis inhibitors such as e-aminocaproic acid (e-aca) and para-aminomethylbenzoic acid (pamba) in vitro, in vivo and in clinic. construction of qsar computer-assisted hierarchical system for the effective anti-herpetic (anti-hsv) and anti-influenza (anti-flu) agents' selection as well as the elaboration of new methods of their synthesis are permanently the object of keen interest of our team. the objective of this study was to investigate the efficacy , -di-substituted pyridines and their analogs combined with the fragments of proteolysis inhibitors in the framework of the qsar approach. molecules of new compounds consisted of "nucleus" (py or ar) and two symmetrical fragments: e-aca-carbonyl or pambacarbonyl taken from the inhibitors' molecules. anti-flu activity in dose - m was studied in vitro on the model of a/hong kong/ / (h n ) reproduction in tissue cultures of chorioallantoic membranes of days old chick embryos. anti-hsv activity in doses - m was studied on models of reproduction of hsv- on cell culture hep- . compounds with py-nucleus, contained pamba-carbonyl or e-aca-carbonyl fragments, demonstrated sufficient anti-hsv activity ( . and % of reduction of intra-nucleus virus-specific inclusions on infected cells account accordingly). , -dihydrazine-carbonyl- , -dimethylpyridine showed high anti-hsv ( %) activity. the efficacy of the designed antiherpetic compounds obtained with the combined efforts of qsar computer-assisted design, properties prediction, synthesis, and biological testing as well as the correction introduced after the iteration circle passsage have proven to be the efficient modern way of drug design. acknowledgement: this research was supported in part by stcu grant # and all the authors are indebted to stcu foundation courtesy. lubomira nikolaeva-glomb , angelina trifonova , stephan filipov , angel s. galabov * institute of microbiology, bulgarian academy of sciences, sofia, bulgaria; institute of organic chemistry, bulgarian academy of sciences, sofia, bulgaria a series of aporphinoid alkaloids isolated from glaucinum flavum l. or obtained synthetically, were tested in vitro for antiviral activity against viruses belonging to picorna-, orthomyxo-, paramyxo-and herpesviruses. one of them, oxoglaucine, manifested a well-pronounced inhibitory effect on poliovirus replication in fl cells measured by the semi-quantitative agardiffusion plaque-inhibition test. in virucidal activity testing the compound did not show direct virucidal effect on the extracellular virus. oxoglaucine's % inhibitory concentration for poliovirus (mahoney) was found to be . g/ml in the cpeinhibition test and . g/ml in the classical plaque-inhibition test. similar values were obtained for the vaccinal poliovirus type strain, lsc- ab. the antiviral effect of oxoglaucine on the replication of viruses belonging to another enterovirus species was tested, i.e. coxsackie and echoviruses (hev-b). cva- , the six coxsackie b viruses and echoviruses were tested for their sensitivity against the antiviral effect of oxoglaucine by the endpoint dilution method in the multi-cycle cpe inhibition set-up in fl cells. oxoglaucine revealed a marked inhibitory effect on all tested enteroviruses. the concentrations that reduced virus titer by lg ranged from . to . g/ml. selectivity index was greater than and even greater than for some of the viruses tested. time-of-addition study by the one-step virus growth cycle set-up showed strong virus inhibition during the early periods of virus replication. milka mileva , angel s. galabov department of medical physics and biophysics, medical university, sofia, bulgaria; institute of microbiology, bulgarian academy of sciences, sofia, bulgaria in this study an investigation and comparison of the effects of plant flavonoid polyphenols quercetin and its sugar-containing homologue (rutinoside) rutin on the "oxidative stress" in liver, isolated from influenza virus a/aichi/ / (h n ) ( . of ld ) inoculated mice, is carried out. it was found that experimental influenza virus infection is accompanied with graduated oxidative disturbances in the liver of mice, despite the absence of virus and inflammation in this tissue. it was found that experimental influenza virus infection is accompanied with a significant increase of lipid peroxidation products, a decrease of natural antioxidants (vitamin e, glutathione) and cyp, an inhibition of cytochrome c-reductase and liver monooxygenases (analgin-ndemethylase and amidopyrine-n-demethylase) as compared to control (non-infected) animals. the preliminary ( days) supplementation of mice with rutin, quercetin or its combination, and their subsequent virus inoculation influence significantly all analyzed parameters as compared to controls. the protective effect of rutin against influenza virus-induced lipid peroxidation and activities of cyp and liver monooxygenases in liver was better expressed than the effect of quercetin may be due to containing of rutinoside part or difference of its metabolism. hyun-jeong lee , ji-sun kwon , chi-ung moon , jong-hwan kwak , youn-jeong lee , chang-seon song avian disease laboratory, college of veterinary medicine, konkuk university, seoul, korea; hanyang university, seoul, korea; sungkyunkwan university, seoul, korea; national veterinary research and quarantine services, seoul, korea one of the traditional korean medical herb extract named s was investigated to determine the anti-influenza virus activity in vitro and in vivo. the s showed potent antiviral activities against a/pr/ / (h n ) influenza virus with the % effective concentration (ec ) values of . g/ml and the % cytotoxic concentration (cc ) values of . g/ml in mdck cells. treatment with the s appeared capable of significantly ameliorating the influenza virus infection in mice by oral gavage treatment. the s treated mice showed significantly higher survival rate and lower pathogenic index as well as lung virus titers than untreated control mice. further, the s was extracted with chcl , etoac and n-buoh for isolation of active compounds. the anti-influenza effects of these active compounds will be discussed. the antiviral effect of aqeous and ethanol extracts of ocimum gratissimum (og), terminalia catappa (tc), gynostemma pentaphyllum (gp), newbouldia laevis (nl), aspilia africana (aa) and phyllantus amarus (pa) leaves was examined by cultivation of virus and extracts in embryonated chicken eggs. extracts were inoculated immediately after virus (zero time) or h after virus inoculation. virus replication was compared with those of controls by haemagglutination assay. at zero time, aqeous extracts of og, tc, pa, and gp inhibited virus growth by , , , and %, respectively whereas those of nl and aa did not. ethanol extracts of og, tc, pa and gp at same time inhibited by , , , and %, respectively whereas nl and aa did not. at two h after virus inoculation aqeous extracts of og, tc, pa and gp inhibited virus growth by , , , and %, respectively whereas nl and aa had no effect. ethanol extracts of tc, pa and gp inhibited the virus by , , and %, respectively whereas those of og, nl, and aa did not. the herbs were studied because they were being used by some herbalists in the treatment of human infectious diseases. the th international conference on antiviral research will be held in the palm springs, california area. the conference will begin on sunday, april , and will end on thursday afternoon, may , . all scientific sessions will be held at the westin mission hills resort, rancho mirage, ca. the purpose of the international conference on antiviral research is to provide an interdisciplinary forum at which investigators involved in basic, applied, and clinical research worldwide can meet to review recent developments in all areas of antiviral research. specific topics to be covered in the program include synthesis and chemistry, biochemistry and mechanism of action, molecular biology and drug targeting, in vitro evaluation, animal models, pharmacokinetics, toxicology, and clinical trials. within these areas of interest, there will be invited overview speakers, oral presentations, and poster presentations. the famous el paseo shopping district of palm desert and downtown palm springs offer not only a large variety of galleries, boutiques and shops too numerous to mention, but there are restaurants for virtually every palate. whether your tastes run to burgers, sushi, pizza, escargot, steak, mexican or continental you will find it here with a california flourish in every price range. we hope you will take advantage of this opportunity to combine an important learning experience with a magnificent travel experience and join us in palm springs, california for the th international conference on antiviral research. isar conference committee future conferences acknowledgement: the study was supported by rfbs - - and russian ministry of sciences (lot ). key: cord- -rqo s g authors: kumar, sameer; markscheffel, bernd title: bonded-communities in hantavirus research: a research collaboration network (rcn) analysis date: - - journal: scientometrics doi: . /s - - - sha: doc_id: cord_uid: rqo s g hantavirus, one of the deadliest viruses known to humans, hospitalizes tens of thousands of people each year in asia, europe and the americas. transmitted by infected rodents and their excreta, hantavirus are identified as etiologic agents of two main types of diseases—hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome, the latter having a fatality rate of above %. although considerable research for over two decades has been going on in this area, bibliometric studies to gauge the state of research of this field have been rare. an analysis of articles, extracted from wos databases on hantavirus between and , indicated a progressive increase (r ( ) = . ) in the number of papers over the years, with the majority of papers being published in the usa and europe. about % papers were co-authored and the most common arrangement was – authors per paper. co-authorship has seen a steady increase (r ( ) = . ) over the years. we apply research collaboration network analysis to investigate the best-connected authors in the field. the author-based networks have components (connected clump of nodes) with vertices (authors) and , edges (co-author associations) between them. the giant component (the largest component) is healthy, occupying . % or vertices with , edges between them. by using edge-weight threshold, we drill down into the network to reveal bonded communities. we find three communities’ hotspots—one, led by researchers at university of helsinki, finland; a second, led by the centers of disease control and prevention, usa; and a third, led by hokkaido university, japan. significant correlation was found between author’s structural position in the network and research performance, thus further supporting a well-studied phenomenon that centrality effects research productivity. however, it was the pagerank centrality that out-performed degree and betweenness centrality in its strength of correlation with research performance. hantavirus, transmitted to humans through persistently infected rodents and their excreta, is a global public health threat hospitalizing tens of thousands of people every year throughout the world. since the isolation of the first hanta virus, htnv (or hantaan virus) in , several other hantaviruses have been identified, with at least being pathogenic to humans (bi et al. ) . one of the first major outbreaks of hantavirus was reported from to when close of american soldiers serving in korea became infected with the virus. in recent times, several cases have been reported in asia (zhang et al. ) , us, and europe. hantavirus genus belongs to bunyaviridae family and is identified as an etiologic agent of two different types of diseases-hemorrhagic fever with renal syndrome or hfrs and hantavirus pulmonary syndrome (hps). hfrs is also known by earlier names like korean hemorrhagic fever (khf), epidemic hemorrhagic fever (ehf), nephropathia epidemica (ne) (bi et al. ) . hprs affects close to , , - , , people throughout the world each year while hps infects just about . however, the fatalities caused by the latter are above % when compared to - % in the case of hprs depending on the severity of the virus (lednicky ; schmaljohn and hjelle ) . hfrs is more prevalent in the eurasian region and hps in the americas. china remains the most endemic nation accounting for close to - % hfrs cases in the world (zhang et al. ) . ne, the mild form of hfrs, is most dominant in western and central europe. a quick glance at the web of science databases reveals a progressive increase in research papers on hantavirus. the research in the field is paving the way to finding more pathogens, associated diseases, and vaccines. however, bibliometric studies to gauge hantavirus research are surprisingly rare. hence, we set out to mainly identify the prominent researchers in the research collaboration network and the bonded communities they were embedded in. research collaboration, a key mechanism that brings multiple talents together to accomplish a research task, could be effectively gauged through bibliometric records in research papers (heinze and kuhlmann ) . co-authorship in research papers has long remained the basis of investigating research collaborations (beaver and rosen ) . the co-authors of a research paper could reveal the exchange of knowledge among researchers in their effort to bring out a published paper. similarly, the affiliation details in the bibliometric records could be extrapolated to reveal collaboration happening at institutional and international levels. whether research collaboration could be gauged by just looking at the bibliometric records is a matter of academic debate (katz and martin ) . for example, a collaboration could take place (i.e. through research advise) even if the two researchers do not finally end up penning the research paper together. then there are some issues of honorary and ghost authorships (wislar et al. wislar et al. ) . while these concerns are serious, using bibliometric records is still the most concrete piece of evidence to establish a collaboration. given the fact that co-authorship associations could also help us in understanding the association at institutional, organizational, and international levels, their significance cannot be overlooked. the number of co-authored papers across disciplines has been growing over the years (sonnenwald ) . better communications facilities, faster commuting, and industrialization brought in significant changes in the way research was conducted. now there are researchers in large teams working on research projects and naturally these lead to published papers having significantly larger numbers of co-authors. price ( ) calls these large lab-based research projects, 'big science'. however, big science research projects aren't generally in the social domain, that is, researchers do not have much choice to decide on their co-authors. like the sciences, research conducted in the social sciences has also seen significant increase in the number of co-authors (moody ) . it remains imperative to note that collaboration is sharing of knowledge and may not always suggest improved quality of work. for example, in the humanities there are still a significant proportion of papers that are solo-written. research collaborations could also be seen from the perspective of networks (kumar and jan ) . in a network, two entities form a connection if there is some kind of association between them (newman ) . using bibliometric data, these associations could be constructed to understand knowledge flows at multiple levels. co-authorship in published papers is considered a reliable proxy to gauge research collaborations (sonnenwald ; melin and persson ; katz and martin ) . social network analysis, an established research method to analyse social networks, is a set of mathematical algorithms that quantitatively analyse these relationships between nodes (wasserman and faust ) . in a co-authorship network, for example, it could be applied to identify various patterns-i.e. the best connected nodes or key actors (taba et al. ) or the communities that the researchers form through their associations. specifically, these analyses reveal the pattern of network at both global and local levels. at the global level, the network pattern is seen from a whole network perspective, revealing, for example, the density, transitivity, scale-free pattern, small-world pattern, or the communities or clusters that the nodes form. at the local level, things are seen from the node perspective. centrality is an important concept when looking from the perspective of a node and its context in the entire network. centrality determines the relative importance (through centrality measures such as betweenness, closeness, and pagerank) and connectedness (through 'degree' metric) of nodes. hence, those with higher centrality scores are those who are the most prominent players in the network. another interesting aspect of social networks is that of the ties that the node is directly connected to. the strength of connection (depicted by a thicker line on a network graph) demonstrates a more frequent and stronger relationship than those that have an association of just a single or very few times. the idea of strength of relationship (coleman ) is challenged by the notion of structural holes (burt ) . structural holes theory postulates that the absence of ties in an ego network (network of ego-central node-and alters or immediate connections and those immediate connections connecting to one another) brings in more opportunities to the ego (the central node) as the ego then acts a bridge for the flow of resources between the 'alters'. yet another idea of ties is postulated by the concept of 'weak ties'. the theory argues that in contrast to strong ties, which bring in trust, weak ties bring in new knowledge in the network. growth and preferential attachment are the prime features of self-organising networks (barabasi and bonabeau ) . preferential attachment (kumar and jan ) is defined by the preference of nodes (due to affinity or similarity) to attach to another node. in the context of co-authorship network, it may be due to the fact that one author connects to another author because he or she is a well-known researcher or has the same nationality as others. preferential attachment causes some nodes to have much higher number of connections than most other nodes in the network. these hubs are kind of 'power houses' that tie together the network. this is the very reason why a self-organising network are small worlds (has a short path between any two random nodes) (watts and strogatz ) . a targeted attack or absence due to some other reason could break the network down into pieces, which could severely affect the flow of resources in the network. nonetheless, these self-organising networks are quite tolerant to random attacks (albert et al. ) . why do researchers collaborate? there are several benefits to collaboration (beaver ) . sharing of expertise and division of work are among the most prominent. collaboration also allows sharing of resources. for example, it is possible that certain equipment may not be available to certain researcher and collaborating with someone who has access to this equipment enables the conduct of research. collaboration, due to division of labour, technically reduces the duration for the completion of research project, enabling researchers to publish more papers. due to requirement for promotion and tenureships, which require papers to be published in high impact journals, collaboration does really help. our goals here are two pronged. first, we are interested in knowing the prominent and most connected authors in the field. a number of studies in recent times have found that the relative position significantly correlates with the research performance of researchers (abbasi et al. ; kumar and jan a) . we want to check if this stands true for our (hantavirus) dataset. however, another significant goal of this study is to detect the bonded communities of hantavirus research. with bonded communities we simply mean the cluster of researchers who interact more often with each other. a network of thousands of nodes otherwise only results in a hairball-like network that hardly provides much understanding or meaning. thus, in addition to common bibliometric analyses (i.e. annual paper production, average citations, top papers, number of papers per country, author research productivity, etc.), the present study has the following main objectives: a. investigate the prominent authors and the bonded-research communities clusters in hantavirus research. b. investigate if there is relationship between players or actors structural position in the network and research productivity. the study has significance as this would be perhaps one of the first studies to investigate research performance and bonded communities in hantavirus research from the perspective of research collaborations and networks. the idea of reaching out to bonded communities may be helpful to scientometricians wanting to get to the core of researchers who thickly interact with one another. they are the 'nucleus' or the real seat of knowledge of the network. gauging and mapping of research performance of a crucial area such as hantavirus is of immense relevance and importance to health and research policy makers. in addition, it attempts to understand if indeed the structural position in the network (i.e. the connectedness of actors in the network) has any significant correlation with research productivity. such results would add to the existing body of knowledge about whether or not structural connectedness in a network does affect academic performance. the rest of the paper is structured as follows: in the material and method section, we next discuss the data harvesting method and the keywords used to select the records. subsequently, we discuss the findings and finally we draw our conclusions. records were harvested from the web of sciences databases from to . important hantavirus related keywords such as ''hantavirus'', ''hantaan virus'', ''hemorrhagic fever with renal syndrome'', ''hantavirus pulmonary syndrome'', ''korean hemorrhagic fever'', ''epidemic hemorrhagic fever'' and ''nephropathia epidemica'' were used to refine the records selection. following search command was used: topic: (''hantavirus'' or ''hantaan virus'' or ''hemorrhagic fever with renal syndrome'' or ''hantavirus pulmonary syndrome'' or ''korean hemorrhagic fever'' or ''epidemic hemorrhagic fever'' or ''nephropathia epidemica''). refined by: document types: (article). timespan: - . indexes: sci-expanded, ssci, a&hci, cpci-s, cpci-ssh. the above keywords search and data cleaning resulted in the final availability of records for analysis. data cleaning is an arduous task in bibliometric studies. author name variations are among the most complicated as two or more authors may have same name and some even have the same institutional affiliation and hence their publications could be combined and shown as coming from a single author. on the other hand, an author may have different name variations and his or her publication may get split across these different name variations. at the institution and country levels, there is a need to make the names uniform. for example, in the present set of records, at the institution level, usa actually is an abbreviation of ''us army''. in older data some of the country names are not mentioned, hence, by manual checking, they were appended. by manual checking much of these issues were resolved and errors minimised. social network analysis (wasserman and faust ) is a main research method applied in this study. as mentioned earlier, a network could be constructed when two entities are related in some way. on the graph, nodes are represented by a 'dot' and the connection between nodes, as a line passing between them. hence, if two or more authors associate to co-write a research paper, the authors would be represented as nodes and the co-authored paper (the basis of relationship) is represented with a line passing between them. nodes are also referred to as 'vertices' and relationship between them as 'edges'. it is obvious that just one representation of co-authored with dots and lines on a graph does not reveal much but when hundreds and (at times thousands) of papers are represented in a graph, an interplay of association is revealed and how seemingly invisible associations become visible. data elements from the records are extracted and the co-authorship network constructed (see fig. ). scientometrics ( ) : - three centrality measures are calculated-the degree, betweenness centrality, and pagerank centrality. we also calculate the local clustering coefficient and the average geodesic distance of the network. we have not calculated closeness centrality as this centrality gives accurate results for one component (typically a giant component) at a time. it tends to give misleading results if the calculation is made for all the components in the network (for example, those in the dyadic network will have high closeness centrality that those nodes with high degree in the main component). since we are interested in all the authors in the network (and not just those in the giant component), we have chosen to leave out closeness centrality in our graph metrics calculations. degree, a popularity measure, is simply the number of direct connections a node has. betweenness centrality is path-based and checks how much 'in-between' a node is in the network. those with high betweenness centrality have positional advantage and work as bridges between communities. removal of these nodes could severely affect the flow of resources in the network. a pagerank measure is a prestige metric that not only checks the number of connection a node has but also the number of connection of alters. the mathematical formulae used to calculate are standard and are thus provided in ''appendix''. the centrality values are then correlated using ms-excel's correlation statistical function, with number of papers produced and citations accumulated, to check if there is any significant association between the two. nodexl (smith et al. ) was used to calculate graph metrics and visualize the network diagrams. the yearly paper production shows an upward trend. the worldwide alarm raised by the deadly virus has had researchers looking for the pathogens, its geographical reach, and its potential cure. from just four related papers published in , the number grew to in general bibiliometric record of a single paper author network: only those records with two or more authors are skimmed for analysis. (a dyad, the smallest building block of a network) author/s affiliaƟon addresses citaƟons … author n author paper code … author author fig. the extraction of data elements from bibliographic records and construction of co-authorship network . a linear trendline (r = . ) shows a good-fit, meaning that the growth in paper production on hantavirus has been steady over the years (fig. ) . however, a large proportion of paper production has been concentrated in certain regions of the world. majority of the research is going on in europe and the usa (see fig. ) when contrasted with the actual occurrence of hantavirus infection cases (see fig. ), we find that china (although a distant second in terms of number of paper produced) is probably doing comparatively much less research when compared to the number of hantavirus cases reported from the region. as mentioned earlier, china accounts for close to - % of all hanta virus cases in the world. the top ten countries in terms of research productivity are, usa ( papers the entire publication base of hantavirus's papers received a total of , citations or an average of . citations per paper. these are good averages and indicate sound 'health' of research in the field. the papers written have a downward trajectory in terms of average citations received-those papers that have been written earlier are cited significantly more than those that are published in later years (see fig. ). this is of course practical as papers that are written earlier have stayed in the knowledge base for a much longer time than the recent ones and thus have more opportunity to get more cited. some also get a chance (depending on its influence to the field) to enter the very 'seminal knowledge'. once these papers are in this select group, they are cited considerably more than the rest of the papers. papers published during - time frame were cited . times, compared to papers published older time period during - that were cited . times on average. % papers in the older time period had received at least one citations when compared to . % in the newer timeframe ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . of the authors, a large proportion or authors ( . %) have produced just one paper. authors ( . %) have produced two papers each, authors ( . %) have produced papers each, authors ( . %) papers each, and authors ( . %) papers and above. authors are highly productive and have produced papers and more. vaheri a ( papers), lundkvist a ( ), plyusnin a ( ), arikawa j ( ) and hjelle, b ( ) are the most productive authors in the dataset. in our dataset, authors have received no citations, authors had between and citations each, authors had between and citations each and the rest ( authors) have citations and more. authors had and more citations each with peters cj ( citations), ksiazak, tg ( ), vaheri a ( ), lundkist a ( ), rollin pe ( ) garnering the top five slots as the most cited authors. in both number of papers/author and citations/author, we notice few authors have been significantly more productive than the rest of the block, a common feature of research productivity in most disciplines. of , cumulative citations (if a paper has four coauthors and has received ten citations for the paper, cumulative citations for the authors would be ten for each author) by authors, , citations (or . %) are garnered by top % of authors, thus, almost fitting / rule or power law. as noted earlier, there is a whole host of research that has shown that the co-authorship in paper across disciplines has gone up especially in the last two decades. an analysis of coauthorship (or average number of co-authors on each paper) of our dataset shows that the same is true for publications in the field. however, there hasn't been a striking increase in the number of co-authors in the two time periods- - timeframe had an average of . authors per paper when compared to . in the time period between and . about % papers were co-authored (or had at least two authors on a paper). the most common arrangement was - authors per paper. there were -author papers, -author papers and -author papers. more authors per paper are symbolic of experimental research. two papers had and co-authors respectively. table shows the list of top ten most cited papers in hantavirus research. the most cited paper is the year paper by nichol et al. ( ) that was published after the outbreak of hantavirus in the four corners region of the united states. their study showed that the scientometrics ( ) : - comparison of the human and rodent sequences had a direct genetic link between the virus in infected rodents and infected human 'hantaviral ards' cases. on the heels of this study was another highly cited paper by duchin et al. ( ) or ne) and highly cited paper is that of brummerkorvenkontio et al. ( ) . their study concluded that the detection of ne antigen in rodents (bank voles) facilitates 'specific serologic diagnosis of ne'. the heat map drawn using vosviewer (van eck and waltman ) in fig. shows the scientific landscape based on co-citations. co-citation analysis looks at the relatedness of items based on the number of times they are cited together. we use author (first author only) co-citations for the analysis. visualization is automatically done by the software after a threshold is provided by the user. papers nichol st, (science) and schmalijohn c, (emerging infectious diseases) and lee hw, (journal of infectious diseases) are among the most influential papers. in this section, we investigate if the connectedness and relative position of authors have effect on the research performance and then analyze bonded communities embedded in coauthorship networks. the process of the construction of network is explained in the materials and methods section. the author-based networks have components (connected clump of nodes) with vertices and , edges between them (see fig. ). the giant component (the largest component) is healthy occupying . % or vertices and , edges between them. a healthy giant component may be an indication of frequent collaborative activity. the giant component is considered the seat of main activity in the research community (fatt et al. ) . knowledge flows in such networks are faster as they are not subject to disruptions which otherwise would have been the case had the giant component been small and the whole network having several small fragments of components. a recent study by liu and xia ( ) found that the development of an inter-disciplinary field is the giant components continue to grow as more components connect to it. after all, it takes just an edge from a disconnected component to connect to the giant component, thus, making the latter bigger in size. the average geodesic distance (shortest distance between any two random nodes in the network) between the nodes is just . meaning that, on average, two random authors in the hantavirus dataset are just about four hops away from one another. this is another indication that the authors are closely knit and resource flow and delivery would be faster in this network when compared to networks that are sparse and fragmented. this also confirms the small-world nature of this network (newman ) . small world networks typically have shorter geodesic distances. the centrality values (degree, betweenness and pagerank) of authors makes hjelle b. (brian hjelle) the most connected author in the hantavirus research community (see table ). dr. brian, a pathologist, is currently the md/ph.d program director at the university of new mexico (usa) and has several awards and recognitions to his credit. he has been conducting research on hantavirus since the s and was also a member of the hantavirus pulmonary syndrome clinical trial committee for the national institute of allergy and infectious diseases, national institutes of health (collaborative antiviral study group) from - (http://pathology.unm.edu/faculty/faculty/cvs/brian-hjelle. pdf). fig. the overall co-authorship networks of hantavirus dataset (drawn with fruchterman-reingold on repulsive force between vertices . and iterations per layout force directed layout). the darker clump at the center is symbolic of those nodes that are highly connected the local clustering coefficient provides an interesting picture-those with high degree have low clustering coefficient (correlation - . ). why is this the case? clustering coefficient or transitivity is a measure of prediction that if nodes b and c have common partner a, it is a likelihood that b would eventually connect with c. we surmise that this is due to the fact that a node or ego with many alters, will likely have alters that have less connections among them. this is true in many occasions as the ego with large connections would have these connections from several diverse set of nodes. several studies in the recent years have found that centrality measures indeed have significant effect on research performance (abbasi et al. ; uddin et al. ) . hence we set out to investigate if centrality measures have effects on research performance in the dataset of hantavirus research, too. our correlation test (see table ) confirms that indeed in hantavirus datasets there is a significant correlation (p \ . ) between centrality however, what stands out is the correlation of pagerank with research performance. its correlation coefficient strength with research performance demonstrates its efficacy that is even higher than the well-known measures such as degree and betweenness centrality. the very fact the pagerank is based not just on the connections an author has but the quality of these connections, provides it with a better predictability for research performance. here we also introduce an idea to detect 'bonded communities'. by increasing the threshold of edge-weight between nodes, a research community could be drilled down to a level where those nodes that frequently interact with one another are revealed. the importance of strength or 'bondedness' needs attention as this may provide new insights into the communities lying within. drilling down to the desired core (we call it as 'edgecore') is done by progressively increasing edge-weight, till the most bonded communities become visible-it could happen with just three or four in sparse communities and could be ten or more in dense communities. when our network is reduced and visualized with edge weight ten (edge-core-ten) network (the network only visualizes nodes that have an edge weight of ten and more between them), three distinct 'bonded' communities emerge. authors (or nodes) in these communities are involved in repeat associations with one another (see fig. ). quite interestingly, at a threshold of edge-weight ten and above, the community of hjelle b, the most connected author, becomes isolated. this probably goes to show that even best connected author/s may not be embedded in bonded-communities. community a, is led by vaheri a (vaheri antti) who has co-authored with vapalahti o (vapalahti olli) ( times), plyusnin a (plyusnin, alexander) ( times) and lundkvist a (lundkvist ake) ( times). antti vaheri works at dept of virology at university of helsinki, finland and has been active since the s. as a matter of fact, his papers, (brummerkorvenkontio et al. ; schmaljohn et al. ) are among the most cited hantavirus related papers. olli vapalahti and alexander plyusnin, too, are associated with university of helsinki while ake lundkvist is associated with karolinska institute, sweden. while community a has japanese authors, this community has european authors and is dominated by scholars from university of helsinki. within the edge-core-ten community, ake lundkvist is the author with the highest betweenness. he is a bridge node connecting to the sub-community of germany-based authors-ulrich r, meisel h, kruger dh and klempa b. community b that has prominent authors arikawa j (arikawa jiro), yoshimatsu k (yoshimatsu kumiko), takashima i (takashima ikuo) and kariwa h (kariwa hiroaki) are all from japan's hokkaido university. being from the same institution also provides the necessary geographical proximity to carry out joint research. community c has prominent authors ksiazek tg (ksiazek, thomas g); rollin pe (rollin, pierre e), nichol st (nichol, stuart t), peters cj (peters, clarance james), zaki sr and khan as, all associated with center for disease control & prevention, atlanta, usa. another prominent author mills, jn (mills, james n) is associated with emory university, atlanta, usa. clarence james peters is an accomplished physician who has a well-cited book (peters and olshaker ) , while zaki sr has, to his credit, papers (duchin et al. ; zaki et al. ) that are among the top ten most cited papers on hantavirus. however, peters cj, zaki sr and khan as have not published (in the dataset) after , and , respectively. as we see, the community is dominated by authors from the centers for disease control and prevention and all the prominent authors are stationed in atlanta, which again shows that geographical proximity is an important factor for deep-bonded association. university of helsinki, karolinska institute, and swedish inst of infectious disease control dominate the institutional collaborations in europe. at the same time, the centers for disease control and prevention and university of new mexico have a sustained and bonded relationship within usa. based on collaboration among institutions contributing at least ten or more research papers, university of new mexico has the maximum degree (collaborating with institutions), followed by university of helsinki ( ), centers for disease control and prevention ( ), and karolinska institute ( ). all the prominent authors as discussed also belong to these institutions. in the same stride, we thus see sweden and finland involved with extensive collaboration ( repeat associations) in europe while usa almost controls international collaboration with majority of countries including argentina ( repeat associations), south korea ( ), and peoples republic of china ( ). germany has a fair share of collaboration with sweden and slovakia. here we scientometrically analysed the research landscape of hantavirus research. by network reduction or by drilling down into the network based on the strength of ties (or edge weights), we revealed the communities that thickly interact with one other. we demonstrate that these bonded communities actually capture the most prominent authors, too. in our opinion, these bonded communities are the core or ''central brain'' of the network where central activity takes place. we also theorize that strength of relationship is an equally important criterion (apart from centrality measures) for sustainable research performance. pagerank stands out in its correlation with the research performance which further substantiate the idea that it is not only the number of other authors an author is connected to but the quality of these authors (how well those co-authors are connected) that ensures research visibility. where m jk is the number of geodesic paths from vertex j to vertex k(j, k = i) and m jik is the number of geodesic paths from vertex j to vertex k, passing through vertex i (otte and rousseau ; linton ) . pagerank is an importance measure that is calculated based on the premise that 'having links to page p from prominent pages, is a good indication that page p is important one too' (page et al. ) . identifying the effects of co-authorship networks on the performance of scholars: a correlation and regression analysis of performance measures and social network analysis measures error and attack tolerance of complex networks scale-free networks evolution of the social network of scientific collaborations reflections on scientific collaboration, (and its study): past, present, and future studies in scientific collaboration hantavirus infection: a review and global update nephropathia epidemica-detection of antigen in bank voles and serologic diagnosis of human infection the contingent value of social capital social capital in the creation of human capital hantavirus pulmonary syndrome-a clinical description of patients with a newly recognized disease the structure of collaboration in the across institutional boundaries? research collaboration in german public sector nanoscience a global perspective on hantavirus ecology, epidemiology, and disease what is research collaboration? research policy mapping research collaborations in the business and management field in malaysia on giant components in research collaboration networks: case of engineering disciplines in malaysia research collaboration networks of two oic nations: comparative study between turkey and malaysia in the field of 'energy fuels the assortativity of scholars at a research-intensive university in malaysia. the electronic library hantaviruses-a short review a set of measures of centrality based on betweenness structure and evolution of co-authorship network in an interdisciplinary research field studying research collaboration using co-authorships the structure of a social science collaboration network: disciplinary cohesion from to the structure of scientific collaboration networks coauthorship networks and patterns of scientific collaboration genetic identification of a hantavirus associated with an outbreak of acute respiratory illness social network analysis: a powerful strategy, also for the information sciences the pagerank citation ranking: bringing order to the web virus hunter: thirty years of battling hot viruses around the world big science, little science antigenic and genetic properties of viruses linked to hemorrhagic-fever with renal syndrome hantaviruses: a global disease problem analyzing (social media) networks with nodexl scientific collaboration. annual review of information science and technology towards understanding longitudinal collaboration networks: a case of mammography performance research trend and efficiency analysis of co-authorship network software survey: vosviewer, a computer program for bibliometric mapping social network analysis, methods and applications ( st edition, structural analysis in the social sciences) collective dynamics of 'small-world' networks honorary and ghost authorship in high impact biomedical journals: a cross sectional survey hantavirus pulmonary syndrome: pathogenesis of an emerging infectious-disease the epidemic characteristics and preventive measures of hemorrhagic fever with syndromes in china acknowledgments part of the analysis of this study was completed during s.k's research visit to tu-ilmenau, germany. the study is supported by high impact research, university of malaya, grant number um.c/ / /hir/mohe/sc/ / . sna measures (kumar and jan a, b) .a component is a set of nodes joined in such a way that any single random node in the network could reach out to any other random node by ''…traversing a suitable path of intermediate collaborators'' (newman ) .clustering coefficient, c, is also known as 'transitivity' and more accurately as the 'fraction of transitive triples' (wasserman and faust ) . mathematically, clustering coefficient is calculated as:where the number of triangles represents trios of nodes in which each node is connected to both others, and connected triples represent trios of nodes in which at least one node is connected to both others (barabasi et al. ; newman ) . degree is the most common and probably the most effective centrality measure to determine both the influence and importance of a node. a degree is simply the number of edges incident on the vertex. mathematically, degree k i of a vertex iswhere g ij = if there is a connection between vertices i and j and g ij = if there is no such connection. (otte and rousseau ) .betweenness centrality of a vertex i is the fraction of geodesic paths that pass through i, which could be mathematically represented as key: cord- -rn sbfm authors: guyonnet, sophie; rolland, y.; takeda, c.; ousset, p.-j.; ader, i.; davezac, n.; dray, c.; fazilleau, n.; gourdy, p.; liblau, r.; parini, a.; payoux, p.; pénicaud, l.; rampon, c.; valet, p.; vergnolle, n.; andrieu, s.; de souto barreto, p.; casteilla, l.; vellas, b. title: the inspire bio-resource research platform for healthy aging and geroscience: focus on the human translational research cohort (the inspire-t cohort) date: - - journal: j frailty aging doi: . /jfa. . sha: doc_id: cord_uid: rn sbfm background: the geroscience field focuses on the core biological mechanisms of aging, which are involved in the onset of age-related diseases, as well as declines in intrinsic capacity (ic) (body functions) leading to dependency. a better understanding on how to measure the true age of an individual or biological aging is an essential step that may lead to the definition of putative markers capable of predicting healthy aging. objectives: the main objective of the institute for prevention healthy aging and medicine rejuvenative (inspire) platform initiative is to build a program for geroscience and healthy aging research going from animal models to humans and the health care system. the specific aim of the inspire human translational cohort (inspire-t cohort) is to gather clinical, digital and imaging data, and perform relevant and extensive biobanking to allow basic and translational research on humans. methods: the inspire-t cohort consists in a population study comprising individuals in toulouse and surrounding areas (france) of different ages ( years or over — no upper limit for age) and functional capacity levels (from robustness to frailty, and even dependency) with follow-up over years. diversified data are collected annually in research facilities or at home according to standardized procedures. between two annual visits, ic domains are monitored every -month by using the icope monitor app developed in collaboration with who. once ic decline is confirmed, participants will have a clinical assessment and blood sampling to investigate markers of aging at the time ic declines are detected. biospecimens include blood, urine, saliva, and dental plaque that are collected from all subjects at baseline and then, annually. nasopharyngeal swabs and cutaneous surface samples are collected in a large subgroup of subjects every two years. feces, hair bulb and skin biopsy are collected optionally at the baseline visit and will be performed again during the longitudinal follow up. expected results: recruitment started on october and is expected to last for two years. bio-resources collected and explored in the inspire-t cohort will be available for academic and industry partners aiming to identify robust (set of) markers of aging, age-related diseases and ic evolution that could be pharmacologically or non-pharmacologically targetable. the inspire-t will also aim to develop an integrative approach to explore the use of innovative technologies and a new, function and person-centered health care pathway that will promote a healthy aging. electronic supplementary material: supplementary material is available for this article at . /jfa. . and is accessible for authorized users. aging is an important risk factor for several adverse health outcomes, particularly chronic and metabolic diseases and intrinsic capacity (ic) decline. since chronological age differs from biological aging, operationally defining biological aging is an essential aspect to understand the interplay between aging and health outcomes. individuals progress differently in the aging process ("normal" aging vs "accelerated" aging), which means, biological aging is a heterogeneous process. in this context, we need to develop researches to identify biomarkers of aging and healthy aging, and know how to measure biological aging. according to the geroscience field, understanding aging and the links with age-related diseases would contribute to prevent and/or delay the onset of various diseases and the decline in ic domains, in particular in the six operational ic domains crucial for independent living defined by the world health organization (who) (mobility, cognition, psychological, vitality, hearing and vision capacities) ( - ). the who has recently published the integrated care for older people, icope handbook guidance, to support healthy aging and to propose to health-care providers appropriate approaches to detect and manage declines in ic. with this integrated and individualized approach, who aims to reduce the number of dependent people by million worldwide by ( ) ( ) ( ) ( ) . studying concomitantly biomarkers of aging and the natural history of ic evolution in people of different ages and functional status is to date very challenging to understand the relation between biological aging and health outcomes. in this context, the inspire program was built to foster research in the field of geroscience and healthy aging. inspire is a research program dedicated to biological and healthy aging, aimed at constituting a bio-resource platform going from animals to humans, from cells to individuals, from research to clinical care. inspire will provide clinical, biological and technological resources for research and development on aging. the resources will be open to both academic and industry worlds in order to promote healthy aging and prevent dependency. it is a public-private initiative that brings together internationally recognized experts from basic and translational science (in particular, in the fields of immunology, metabolism, neurosciences and mesenchymal stem/stroma cells), clinical gerontology (i.e., researchers, but also physicians and nurses involved in clinical care), primary care and public health ( ) . one of the main challenges of inspire is to identify markers capable of determining biological aging with the implementation of human and animal cohorts. the inspire human translational cohort (inspire-t cohort) will recruit about individuals of several chronological ages (from years to +) and functional capacity levels (from robust to frail, and even disabled) with baseline and follow-up biological, clinical, imaging and digital data over years. these data should allow us to explore and identify a set of biomarkers of aging, age-related diseases and ic evolution. in addition, the inspire-t cohort aims: i/ to test the feasibility and acceptability of a new app for smartphone and tablet developed to monitor the six ic domains (locomotion, cognition, vision, hearing, vitality/nutrition, psychological status) according to the who recommendations; and ii/ to explore the development of digital markers of aging. this paper describes the study design of the inspire-t cohort. the inspire-t cohort, started in october , is a -year observational study. the study population will consist of subjects recruited in the city of toulouse and surrounding areas, south-western france, and covering the age range of years and over (no upper limit for age). several follow-up visits will be regularly scheduled during the -year period of this study. additional visits will be conditioned by the remote monitoring of ic and the onset of other major clinical conditions. at baseline, and then once a year, diversified data (clinical, digital, imaging) and biospecimens (blood, urine, saliva and dental plaques) are collected following standardized procedures. data collection is performed in the clinical research center (crc) of the gerontopole -chu toulouse. it can also be performed in participants' home (for more frail and disabled volunteers), or in selected gerontopole's collaborating centers by a mobile research team trained by the crc. participants are assessed by appropriately trained clinical research members. between yearly waves of data collection, participants are asked to record major clinical information, including adverse events (e.g. new diagnosis, sars cov- diagnosis, influenza, fracture…), medical consultations, hospitalizations, and changes in the drug prescription every -months. they also have the six ic domains monitored (with or without the help of a caregiver) (icope program step ) ( , ) in either the application developed in collaboration with who (icope monitor app) or a web platform; or through a phone call by a gerontopole's trained research nurse. when declines are detected in the icope step , a phone call is organized by the research nurse within one week to confirm this decline and to investigate the causes in collaboration with the medical research team. once an ic decline is confirmed, participants have a thorough clinical assessment following the recommendations of the icope step ( , ) and blood sampling (data are collected by research nurses in a home visit or at the research facility). such information will enable us to investigate the response of some markers of aging at the time declines are detected. the clinical assessments and biomarkers' exploration also allow us to propose a personalized prevention care plan to maintain function according to the recommendations from the who icope program for usual care (icope step ) ( , ). participants are trained to monitor their ic during the baseline visit by the gerontopole' research team. the remote monitoring of ic will last the whole length of this research study, i.e., up to ten years. the figure shows schematically the study procedures over one year. table describes the study flow chart with all data collected at each time point during the follow-up. to ensure quality of data collected, standard operating procedures are implemented covering subject's recruitment, biobanking, remote monitoring of ic, clinical assessments and digital data collection. all data are collected in the inspire-t database. preventive strategies to limit errors like miscoding, missing values, are applied before data entry to ensure the validity and quality of the performed data analysis. tools will be implemented for data exploration and data sharing between inspire consortium researches and later on with external scientists. the main objective of the inspire-t cohort is the appropriate data collection of key variables and biospecimens for at least people at baseline and people with at least four years of follow-up (i.e. four yearly post-baseline assessments) over the -year study. the key variables are clinical data on all six ic domains (locomotion, cognition, vo²max with blood sampling / maximal aerobic power b √ √ the cognitive composite score will be realized only in people lower than years; b. other examinations are proposed to a limited number of participants in a volunteer basis vision, hearing, vitality/nutrition, psychological status), and the collection of blood, urine, saliva and dental plaque samples. secondary objectives include: i/the identification of (a set of) biomarkers of aging through the constitution of a comprehensive biobank; ii/the assessment of the feasibility and acceptability of the icope monitor app used to measure and monitor intrinsic capacity; iii/ the study of the evolution of ic domains over time and its association with health outcomes; and finally, iv/ the study of the correlation between digital biomarkers to biological/imaging biomarkers and ic domains ( figure , online consultation). a mouse cohort in mirror of the human inspire-t cohort is being built in order to cross the results of translational research found in humans on aging animal models, and vice versa ( ) . the main objective of the inspire animal cohort is to define the relationship between the molecular mechanisms of cell premature senescence and frailty/accelerated aging ( ) . we will recruit about subjects, men and women, aged years-old or over (no upper limit for age), and affiliated to a social security scheme. people having a severe disease compromising life expectancy at years (or at year for subjects living in nursing homes) and people deprived of their liberty by administrative or judicial decision, or under guardianship, are excluded. recruitment is stratified per -year age groups, oversampling older people in order to be able to investigate major clinical events (e.g., declines on ic, onset of age-related diseases). due to the heterogeneity of biological aging, we opted for no too stringent eligibility criteria. by diversifying our recruitment sources and monitoring key risk factors for accelerated aging (e.g., age, obesity, frailty, and activities of daily living), we will be able to recruit participants with different trajectories of aging. sample size calculation was not relevant as many objectives of the inspire-t cohort are exploratory. we therefore considered an approach based more on the potential of the inspire-t cohort in terms of the ability to obtain parameter estimates with sufficient precision with a recruitment of subjects that corresponds to the maximum number of subjects that can be recruited and monitored with the funding provided. in case of evident underpowered population (for a particular subgroup of subjects), a reasoned additional recruitment of subjects may be considered in a second phase. to limit the attrition rate, subjects will be monitored by both active (visits, telephone calls) and passive ways (monitoring of several functions using new technologies via mobile phones or other connected devices). from all subjects enrolled, investigations include data collection at baseline and during follow-up visits (annual visits and additional visits planed in case of decline in ic). upon written informed consent, the following set of information is obtained by using a standardized questionnaire: inspire-t study procedures over one year. the remote monitoring of intrinsic capacity will last the whole length of this research study, i.e., up to ten years other examinations are proposed to a limited number of participants (all age ranges and functional status) in a volunteer basis: dual energy x-ray absorptiometry (dxa) for body composition assessment; whole body and brain magnetic resonance (mri); cardiorespiratory fitness (maximum oxygen consumption (v max) with blood sampling before and after the effort, and maximal aerobic power), and isokinetic muscle strength. these examinations are proposed annually for the dxa and, every two years for the other tests (mri, vo max, isokinetic muscle strength). participant-reported outcome for sarcopenia (sarqol) ( ) is completed for volunteers who perform cardiorespiratory fitness exploration. innovative digital assessments are also planned to be tested, such as home sensors (e.g., for measuring walking speed and its variability in daily environment), automated video analysis of mobility, and d facial images for the detection of digital markers of aging. a subgroup of patients monitored by ambient sensors at home or sensors worn on the wrist over the long term will allow to remote and continuous monitoring of the trajectories of the ic domains (especially mobility, sleep parameters and nutrition) (cart/smarthome research ancillary study, legal authorizations in process). this sub-study, developed in partnership with the cart research project team in united states (orcatech team, oregon health and science university, or, usa; pi, jeffrey kaye; www.ohsu. edu) will allow us to detect subtle changes that are not clinically perceptible, well before the appearance of signs and symptoms and therefore determine innovative digital biomarkers and decision thresholds. these digital biomarkers will be correlated with clinical data but also biological and imaging biomarkers. biospecimens are collected during the inspire-t cohort for the creation of a biobank. biospecimens, including blood, urine, saliva, dental biofilm, are collected from all subjects at baseline and then, annually (the genotyping sample will be collected only at the baseline visit). nasopharyngeal swabs and cutaneous surface samples are collected from all subjects every two years. feces, hair bulb, and skin biopsy, are collected optionally at baseline visit (see table ). aliquots of biological material are stored at - °c (dental biofilm, saliva, serum, plasma and urine) or at - ° c liquid nitrogen (pbmc) at the central lab (crb tbr, chu toulouse/ ifb purpan, toulouse, france). analysis will be either performed in toulouse by the local biological teams involved in the inspire project or by any third party not yet determined. the modality of laboratory data transfer from the central lab to other parties will be defined at a later stage. samples from the biobank may be moved to other us and european countries if required. the inspire-t biobank is supervised by the crb tbr where all measures are taken to ensure a quality service based on appropriate resources and adequate safety procedures: observance of good laboratory practice guidelines (the crb tbr is certified afnor since ), fully-equipped premises, appropriate, approved and safe equipment ( freezers - °c eppendorf cryocube, liquid nitrogen tanks with manual and documented filling, vigitemp probes provide metrological tracking), qualified personnel, safety test and system implementation, sample traceability (all of biological collections are tracked in a specific software (td biobank), and chu servers are daily backed up). all freezers are equipped with an alarm system. equipments are monitored three times per day. every failure is reported in the kalilab software as non-compliance statements. all the participants will be tested for sars cov- infection via serological tests from blood collection when these latter will be available. all biological samples are processed within min following a protocol elaborated for inspire purpose and split into smaller aliquots at the inspire-t biobank (figures & , for blood collection, all subjects are asked to donate blood ( ml) by venipuncture after overnight fasting. the blood sample is processed to obtain whole blood, plasma, red blood cells, serum and peripheral blood mononuclear cells (pbmc). for whole blood and serum, samples are immediately shipped after collection at room temperature to the crb tbr for the preparation of whole-blood and serum aliquots for freezing at - ° in the inspire-t biobank. for pbmc, blood samples are immediately transported to the crb tbr at room temperature and treated within hours from time of collection. pbmc are collected after density gradient-based separation, counted and frozen at - millions/ cells per vial. frozen vials will be stored in liquid nitrogen. for plasma and rbc (edta/lithium heparin/bdp blood), aliquots are immediately prepared after collection in the crc and stored at - °c until their shipment to the inspire-t biobank. when visits are organized by the mobile research team, some blood samples are not performed to limit quality procedures deviations (it concerns the lithium heparin tube, the whole blood edta tubes and the bd p blood tube). participants are asked to collect at least ml urine in a sterile screw-top container. the obtained volume is transferred into two vacutainer tubes of ml each and directly shipped at room temperature to the crb tbr where urine aliquots of ml are prepared and stored at - °c in the inspire-t biobank. participants are asked to collect ml saliva in ml falcon tube (at least min after a meal). the falcon tube is immediately shipped at room temperature to the crb tbr where saliva aliquots of ml are prepared and stored at - °c in the inspire-t. biofilm sampling consists in recovering the biofilm from the external surfaces of the teeth (from natural teeth in priority, from prosthetic teeth if not possible), at the juxta-gingival level by curettage at sites distributed over the dental arches: a sample from the upper anterior teeth, a sample from the upper posterior teeth, a sample from the lower anterior teeth, a sample from the lower posterior teeth. the product of each curettage is individualized in a sterile ml cryotube. the four cryotubes are frozen at - ° in the crc after the collection and regularly shipped to the crb tbr. nasopharyngeal swab will be addressed to the institute of biology of the chu toulouse within hours from time of collection for their analysis (detection and identification of multiple respiratory viral and bacterial nucleic acids). residuals samples will be stored at - ° to the crb tbr in the inspire-t biobank. swab samples are done on a skin exposed area (posterior face of the forearm) and a non-exposed area (lower back). specimens are stored immediately at - °c in the crc. frozen tubes are regularly shipped to the crb and stored at - °c in the inspire-t biobank. in addition, two mm diameter d-squames are applied successively on the exact same area of the posterior face of the forearm: the first one is discarded and the second one is stored in a ml tube. the same procedure is performed on the lower back. both tubes are regularly shipped to the crb and stored at - °c in the inspire-t biobank. feces are collected at the first visit and immediately stored at - °c. if it is not possible, the participant can return to the research facility within one week with its frozen sample in a coproculture pot, placed in a cool box. the frozen feces samples are regularly shipped to the crb tbr and stored at - °c in the inspire-t biobank. twenty hairs are taken with the bulb and immediately stored after the collection in a sterile ml cryotube in the crc until their shipment to the inspire-t biobank. a mm skin biopsy is obtained by using a punch. skin samples are prepared according two different procedures: half of the samples are immediately rinsed, dried and stored at - °c until its shipment to the crb tbr; the other are immediately placed in a cryotube with pbs for cells cultures to organize a biobank of skin fibroblasts. a biobank scientific committee will be set up, in the aim of determining the scientific directions and research priorities, of evaluating ongoing projects and their state of progress, and of resolving any methodological and ethical concerns raised by the studies. it shall i/examine the relevance, feasibility and conditions of implementation of the propositions concerning any analyses; ii/ ensure that national and international partnerships are made formal; iii/ control use of data, especially sample use, and iv/ ensure that participants rights are protected. the data disclosed will be made anonymous (coded, traceable data). since the primary outcome measure of the inspire-t cohort is related to reaching prespecified numbers for recruitment and retention, we will use numbers and percentages. hypothesis-testing statistics will be employed for some of the secondary outcome measures and the new hypothesis arising through the year duration of the inspire program. specific statistical analysis plan (sap) will be written to answer each research question. big data methods of analysis will be considered when examining the large and diversified amount of data that will be gathered from clinical and paraclinical evaluations, biospecimens, and digital assessments. significance will be set at p ≤ . . analyses will be performed using stata (v , statacorp), sas (v . , cary, nc, usa), and r (v . . ). statistical analyses will be done by researchers of the inspire program and professional statisticians. analyses by gender will be conducted. the inspire-t cohort is carried out in accordance with the declaration of helsinki, which is the accepted basis for clinical study ethics, and must be fully followed and respected by all engaged in research on human beings. the inspire-t cohort protocol has been approved by the french ethical committee located in rennes (cpp ouest v) in october . this research has been registered on the site http://clinicaltrials.gov (id nct ). the first participant was recruited on october . our objective is to recruit at least, people at baseline ( during the first year and during the second year of the project) from onwards, including robust, prefrail and frail older adults, as well as disabled people, to be able to better understand the biology of aging across age-ranges and functional status. all the recruiting work is currently carried out by the toulouse gerontopole research team on a single site; a mobile clinical research team is also currently active to recruit frailer population (e.g. people unable to come to research facilities) in toulouse and surrounding areas. current inclusion rates are participants per day. this rate will allow us to reach our objective of inclusions in years. two hundred and forty participants have been included by march ( women / men; mean age: . years), and new inclusions are planed until september . among the enrolled participants, are robust, prefrail and frail with fried criteria ( ). all participants gave their consent for the complete biobanking, participants have accepted the skin biopsy, hair bulb collection and feces collection. all subjects have accepted the dxa, and vo max and muscle strength assessment. the sub-study on mri is planned to start on september . however, recruitment has been temporarily suspended during the covid- pandemic. our first plans were to recruit a representative sample of users of primary care services, by inviting people to participate using patients' list of several family physicians in different areas (with different deprivation levels) of toulouse (all patients aged years or over being invited to be screened for participation). however, this recruitment approach proved to be unfeasible, mainly because many physicians have been very busy taking care of several viral pathologies during winter - (including covid- from february ) ( , ) . consequently, we decided to diversify the sources of recruitment. current recruitment relies mainly on the following strategies: flyers, community outreach strategies, media coverage, newspaper advertising, posters, online promotion, mass mailing, presentations at public events, conferences, study website, dissemination through institution newsletters, identification of participants from previous studies or existing registries, onsite recruitment /medical records review (by investigators/clinical research assistants), dissemination through health care providers : coordination with primary care, memory centers, hospital outpatient clinics, medical centers, physicians (site investigators, primary care physicians), specialists, hospital inpatient lists, private clinics, and finally, dissemination through residential homes, and nursing homes. the recruitment channels of the participant included (and planed over the next months) is detailed in table . applied strategies are constantly followed and adapted if necessary throughout the recruitment study period (weekly meeting with investigators and study staff). a mobile research team was implemented in january to recruit frailer population by collaborating with residential homes, long-term care facilities and post-acute and rehabilitation facilities. next collaborations are considered with the crct in oncopole-toulouse (an institution dedicated to cancer research and care) and a private clinic focused on the management of obese people. retention strategies are implemented in parallel. it consists of participant-centered values and strategies including (but not limited to) identify proxy contacts, minimize waiting time during study visits, facilitate transportation from and to research facilities, adapt comfortable waiting room facilities, build relationships with study participants; remind nonresponsive participants (contact via phone or email, make phone calls during optimal hours; offering regular feed-back during the follow-up (mailing study updates); offering regular gadgets during the follow up and postcards. the inspire-t cohort will gather clinical, biological (including imaging), and digital data for subjects of several chronological ages and functional capacity status regularly followed over up to years. one of the most innovative aspects of the inspire-t cohort is that, through a close monitoring of participants with the icope monitor app, we will obtain clinical and biological data at the moment declines in ic come up. the cohort will provide us the needed resources to improve our understanding of the biological mechanisms of aging and the natural history of loss of ic leading to dependency during aging. by following and monitoring the ic of participants over time, this study will provide information about a new, function-centered healthcare pathway, which would agree with who recommendations for an integrated care for older people. at the medium term, this data may inform the development of a pragmatically interventional study testing the effects of this new healthcare pathway on clinical outcomes in older people; this healthcare pathway may be integrated in daily practice in healthcare systems, becoming thus the usual care. innovative digital solutions (including sensors) proposed in the inspire-t cohort are a promising way to remotely collect and analyze real-life and continuous health related data and thus longitudinal trajectories over time. it makes it possible to detect subtle variation in the ic before a clinical event. the inspire-t cohort will also perform relevant and extensive bio banking to allow basic and translational research in humans in the field of geroscience and healthy aging. the inspire-t biobank might lead to improving our understanding about molecular and physiological mechanisms involved in healthy aging, interacting with changes linked to specific chronic diseases. this may contribute to establish a set of biomarkers, that could be pharmacologically or nonpharmacologically targetable, and that would characterize biological aging and, then, permit to identify an accelerated aging phenotype. in their recent paper, ahadi s et al ( ) have defined different types of aging patterns in different individuals, termed "ageotypes", on the basis of the types of molecular pathways that changed over time in a given individual. according to the authors, "ageotypes" may provide a molecular assessment of personal aging, reflective of personal lifestyle and medical history that may ultimately be useful in monitoring and intervening in the aging process". one of the inspire approach: an integrative view of biological aging the main objectives of the inspire-t cohort is to identify biological markers that could detect the inter-individual variability of biological processes before it becomes clinically perceptible ( ) . the identification of biomarkers of aging may help us to identify individuals who are with a high risk of developing age-associated diseases, decline in ic or disability, and to propose personalized strategies, including innovative therapeutics, to prevent or restore impaired functions. our clinical and biological data will give the opportunity to explore the interaction between changes with aging on inflammation, metabolism, gerosciences in general, and neurodegenerative process leading to alzheimer's disease ( , ) or physical frailty ( ), two major causes of loss of functions. the inspire-t cohort will benefit from the availability of plasma neurodegenerative biomarkers (plasma amyloid beta / , neurofilaments, plasma phospho tau) ( ) . the development of biological markers of frailty is also required to improve the treatment of frail individuals. the etiology of frailty is complex. proposed biomarkers of frailty include markers of inflammation. as recently proposed by the icsfr task force perspective on biomarkers for sarcopenia and frailty, «machine learning and information technology innovation could thus be used to develop risk scores that could be used in clinical and research settings. other technologies, such as induced pluripotent stem cells (ipscs) or skin fibroblasts, could be used to study markers of senescence and could also enable a move towards personalized medicine» ( ). interventions to promote healthy aging will be more effective in people with a risk of decline ( ) . hallmarks of aging are under scrutiny in particular dna alteration, epigenetics, unusual protein production, senescent cells secreting pro-inflammatory factors and others. new therapies aim to target senescent cells or their secretory proteins (the senolytic molecules) and therefore promote healthy ageing are presently under development ( ) ( ) ( ) ( ) . finally, the inspire-t cohort gives us the opportunity to federate clinical and biological research teams in toulouse and occitania region to build a research platform of gerosciences discovery to explore mechanisms of aging, and to implement comprehensive translational projects towards the goal of preventing the consequences of aging for a healthy, and long-lived society ( ) . the animal cohort, generated to "mirror" the human translational cohort, will facilitate the translation of results from basic research to humans and to the clinics. the identification of markers of aging will take advantage of three complimentary approaches to look for the best markers of aging: without a priori approaches (transcriptomics, proteomics, metabolomics); semi a priori approach (metabolism, inflammation, cell cycle, mitochondrial network…); and targeted approach (pre-identified targets such as (but not limited to) growth differentiating factor (gdf- ), apelin, senescent cells, amyloid protein in plasma) ( ) . from a biological viewpoint, the function-centred approach recommended by who represents a challenge due to the multidimensionality that characterizes ic' trajectories during aging. we will develop an integrative view of biological aging ( figure ). three classes of parameters transversal to the whole organism, present in all organs, strongly interrelated and crucial in tissue homeostasis have been selected: i) inflammation and immunity that represents both a warning signals and the house keeping guard of tissue integrity, ii) mesenchymal stem/ stroma cells (msc) allowing support for all function and their adaptation and iii) metabolism that controls any cell decision and the fate of most of them. for all these transversal components, senescence mechanisms will be carefully investigated. due to the covid , teleconsultation has been added for the pre-inclusion and some of the assessment, we will be able also to assess the" stay at home order" on the inspire-t cohort subjects ( ) . in conclusion, the inspire-t cohort, nested in the inspire platform, will contribute to healthy aging and dependency prevention. the inspire-t cohort will foster discoveries of human markers (i.e., biological, clinical, digital) of healthy aging capable of predicting functioning and resilience. challenges and opportunities for geriatric medicine measring biological aging in humans : a quest editorial: linking geroscience and integrated care to reinforce prevention who | who guidelines on integrated care for older people (icope) geneva: world health organization; . integrated care for older people: guidelines on community-level interventions to manage declines in intrinsic capacity editorial: who guidelines on community-level interventions to manage declines in intrinsic capacity: the road for preventing cognitive declines in older age? older people and the implementation in the inspire care cohort andrieu s et al for the inspire program group. the inspire research initiative: a program for geroscience and healthy aging research going from animal models to humans and the healthcare system frailty in older adults: evidence for a phenotype studies of illness in the aged. the index of adl : a standardized measure of biological and psychosocial function assessment of older people: self-maintaining and instrumental activities of daily living mini-mental state'. a practical method for grading the cognitive state of patients for the clinician screening for dementia by memory testing wechsler adult intelligence scale-revised formal and semantic lexical evocation in normal subjects. performance and dynamics of production as a function of sex assessing the nutritional status of the elderly: the mini nutritional assessment as part of the geriatric evaluation adherence to the french programme national nutrition santé guideline score is associated with better nutrient intake and nutritional status evidence-based protocol: oral hygiene care for functionally dependent and cognitively impaired older adults the phq- : validity of a brief depression severity measure a short physical performance battery assessing lower extremity function: association with self-reported disability and prediction of mortality and nursing home admission development and validation of criterion-referenced clinically relevant fitness standards for maintaining physical independence in later years. the gerontologist assessment of muscle function and physical performance in daily clinical practice : a position paper endorsed by the european society for clinical and economic aspects of osteoporosis, osteoarthritis and musculoskeletal diseases (esceo) the utility of the cognitive function instrument (cfi) to detect cognitive decline in non-demented older adults validation of the sarqol®, a specific health-related quality of life questionnaire for sarcopenia. j cachexia sarcopenia muscle editorial : covid- and older adults editorial : geriatric medicine in italy in the time of covid- personal aging markers and ageotypes revealed by deep longitudinal profiling revisiting the hallmarks of aging to identify markers of biological age editorial: geroscience and the role of aging in the etiology and management of alzheimer's disease plasma biomarkers of ad emerging as essential tools for drug development: an eu/us ctad task force report icfsr task force perspective on biomarkers for sarcopenia and frailty senescent cells: an emerging target for diseases of ageing targeting senescent cells in translational medicine the role of cellular senescence in ageing and endocrine disease creating the next generation of translational geroscientists a framework for selection of blood-based biomarkers for geroscience-guided clinical trials: report from the tame biomarkers workgroup. geroscience impact of the covid- outbreak on the clinical and research activities of memory clinics: an alzheimer's disease center facing the covid- the inspire program is supported by grants from the region occitanie/pyrénées-méditerranée (reference number: ), the european regional development fund (erdf) (project number: mp ), msd avenir and the inspire chairs of excellence funded by: alzheimer prevention in occitania and catalonia (apoc), edenis, korian, pfizer, pierre-fabre.conflict of interest: all authors of the paper "the inspire research initiative: a program for geroscience and healthy aging research going from animal models to humans and the healthcare system" declare no conflicts of interest related to this manuscript.open access: this article is distributed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits use, duplication, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license and indicate if changes were made. key: cord- -gct bmjl authors: enemark, christian title: influenza virus research and eu export regulations: publication, proliferation, and pandemic risks date: - - journal: med law rev doi: . /medlaw/fww sha: doc_id: cord_uid: gct bmjl an influenza pandemic would be a global health emergency, and laboratory-based research on influenza viruses is an important component of worldwide efforts to prevent and prepare for this. there are concerns, however, that publishing the findings of such research might sometimes increase the risk of a pandemic caused by a laboratory accident or the deliberate release of a deadly virus. this article addresses the challenge of governing scientific information sharing, with regard to public health benefits and risks, from an export-control perspective. the discussion focuses on research findings produced in by a team of influenza virologists in the netherlands, and on the dutch government’s unprecedented decision to regard the intended publication of these findings as being subject to european union regulations on the export of ‘dual-use’ items. i argue that, when a government is uncertain about whether the benefits of publishing particular research findings in a scientific journal outweigh the risks, a process of selectively disseminating those findings should be available as an alternative to official censorship. pandemic influenza is a public health risk of global concern. a variety of pharmaceutical and non-pharmaceutical measures can be used to mitigate that risk. however, the effectiveness of those measures, now and into the future, is in large measure dependent upon a strong scientific understanding of the changing properties and behaviour of influenza viruses. for this reason, laboratory-based influenza research, to the extent that it contributes to better treatments and disease-control methods, is important to all people everywhere. at the same time, such research can itself sometimes present risks to public health or national security. in the course of experimentation, a deadly virus might accidently escape from a laboratory and spark contagion, or a laboratory worker might decide to use the virus in a biological attack. just as there are benefits and risks associated with the conduct of laboratory-based influenza research, so too the written findings of such research have the potential to be put to good or bad uses. this expansion of the 'dual-use' problem in pathogen research is largely due to advances in genetic engineering and gene synthesis technologies. increasingly, gaining access to a virus with particular genetic properties is enabled by the sharing of a technique for producing it in a laboratory rather than by the shipping of physical specimens between scientists. from a security perspective, a shift from tangible to intangible technology transfer presents a challenge to export-control regimes aimed at preventing the international proliferation of 'weapons of mass destruction' (including biological weapons). and yet, from a public health perspective, the swift and extensive sharing of new discoveries about influenza viruses potentially improves global preparedness for a pandemic. this article focuses on the dilemma a government potentially faces when it attempts to prevent biological attacks by regulating the international transfer (export) of certain research findings. of central concern is the publication in scientific journals of experimental techniques for producing an influenza virus that is more dangerous (e.g. more transmissible or more virulent) to human health. on the one hand, it might be argued that the mass communication of information on exactly how a virus can be made more dangerous could further enable the use of that information for a harmful purpose. on the other hand, making such findings available to lots of other scientists could raise awareness and assist in the development of new and improved measures for preventing, containing, and treating influenza. the conundrum for policymakers is: what kind of regulation is necessary to reduce the risk of this intangible technology being harmfully misused, and what kind of regulation is likely to thwart the spread of knowledge applicable to health protection purposes? when it comes to the securing of populations against infectious disease risks-be they naturally occurring or deliberately caused-the required approach seems to be both to restrict and to facilitate the dissemination of certain research findings. in , the dutch government took the unprecedented decision to characterise research findings, produced by a team of virologists in the netherlands, as technology that was subject to european union (eu) regulations on the export of dual-use items. it was the first instance anywhere in europe of an export permit being required before a manuscript could be submitted to an international journal (science) for publication. the virologists, led by ron fouchier of the erasmus medical centre (emc) in rotterdam, intended to publish an account of how they produced an h n avian influenza virus that was airborne transmissible between ferrets. because a ferret's respiratory system is similar to a human's, this new virus was presumptively able also to spread easily between humans in a pandemic. although an export permit was eventually granted and publication went ahead, fouchier questioned whether the eu regulations were applicable in the first place, raising the issue in two dutch court cases (in and ). however, the broader public debate that ensued was about whether the dutch government did the right thing when it ultimately allowed the publication of these research findings, and more generally it was about how to govern transfers of intangible dual-use technology. to address these issues, the discussion here begins by exploring the tension between the governance imperatives of restrictiveness and permissiveness when it comes to the sharing of dual-use biotechnology. the article then considers legal and ethical questions surrounding fouchier's intended publication of his team's h n findings: was it lawful for the dutch government to require fouchier to apply for an export permit, and was it morally right to grant one? assuming that eu export control law was applicable, the problem in this case was that, despite deep uncertainty about the benefits and risks of publication, only two governance options were available: censorship (refusal of an export permit) or full publication. in the final section of the article, a third option is suggested: granting a permit for targeted export (pte). when a government is uncertain about whether the benefits of publishing particular research findings in a scientific journal outweigh the risks, it should be able to authorise the selective dissemination of those findings. such dissemination could be arranged so as to address security as well as public health concerns, although it would probably be less beneficial than publication to the careers of individual academic scientists. ii. dual-use biotechnology, export controls, and research publications biotechnology, along with virtually all other technologies, has the potential to be used for beneficial and malign purposes. in practice, dual-use potential rarely poses a governance challenge because the benefit of the widespread application of most technology usually far outweighs the risk of harmful misuse. occasionally, however, a particular technology can present a dual-use dilemma because the preponderance of benefits over risks (or vice versa) associated with its application is not obvious. in such circumstances, the challenge is to govern access to and use of a particular technology in a way that is likely to achieve more good than harm. here, 'good' includes both actual benefits and avoided harms, and 'harm' includes both actual harm and forgone benefits. when it comes to certain kinds of research on pathogenic microorganisms, the benefit to be gained is a better understanding of how to protect human health against infectious diseases. the countervailing risk is that the production of such knowledge might result in the accidental release from a laboratory of a diseasecausing microorganism or its use in a biological attack. dual-use biotechnology today includes gene modification and synthesis techniques that make it possible to produce pathogenic microorganisms with particular genetic properties in a laboratory setting. one use of such technology is to investigate the potential for a microorganism's genome to mutate naturally and so present a new kind of infectious disease risk. by anticipating that risk, the findings of research into the nature and behaviour of a genetically mutated microorganism can then inform the preparation of clinical and public health responses on a just-in-case basis. a scientist might, for example, set out to produce a bacterium resistant to a certain class of antibiotics to determine whether it could ever become resistant to that class through natural evolutionary processes. such information would be relevant to recommendations on how best to administer the antibiotic and it could help guide the medical management of infectious disease cases. alternatively, laboratory work of this kind might make that bacterium more useful for biological attack purposes precisely because it has been rendered capable of defeating human immune systems and pharmaceutical defences. in , a report by the us national research council on biotechnology research in an age of terrorism identified seven kinds of experiments as most likely to have biological weapons potential: (i) demonstrating how to render a vaccine ineffective; (ii) conferring resistance to therapeutically useful antibiotics; (iii) enhancing the virulence of a pathogen or rendering a non-pathogen virulent; (iv) increasing the transmissibility of a pathogen; (v) altering the host range of a pathogen; (vi) enabling the evasion of diagnosis and/or detection by established methods; and (vii) enabling the weaponisation of a biological agent. more recently, the us department of health and human services has referred to a similar list of experiment categories using the term 'dual use research of concern', defined as: life sciences research that, based on current understanding, can be reasonably anticipated to provide knowledge, information, products, or technologies that could be directly misapplied to pose a significant threat with broad potential consequences to public health and safety, agricultural crops and other plants, animals, the environment, material, or national security. from a governance perspective, when contemplating the benefits and risks of research on influenza viruses and other microorganisms, an important threshold question is whether it should be conducted at all. for present purposes, however, the key issue is whether, how and to whom the findings of research already conducted should then be communicated. to the extent that sharing those findings with other scientists would be beneficial to public health, there is a technology transfer imperative. but if there are risks associated with such sharing, there might also be a non-proliferation imperative to prevent this. a useful starting point for thinking about the tension between restrictive and permissive approaches to dual-use biotechnology transfers is the biological weapons convention (bwc). article i of the bwc binds member states never to national develop, produce, stockpile, or otherwise acquire or retain 'microbial or other biological agents, or toxins whatever their origin or method of production, of types and in quantities that have no justification for prophylactic, protective or other peaceful purposes'. article iv requires states to give effect to this rule through their domestic law. however, it can sometimes be difficult in practice to discern reliably the lack of a peaceful justification given the importance of biomedical research to human health and the scale of the scientific enterprise directed towards understanding pathogenic microorganisms. the task of preventing the use of microorganisms in biological attacks is also made difficult, from a legal standpoint, by two provisions of the bwc between which there is clear tension. on the one hand, article iii of the convention requires member states 'not to transfer to any recipient whatsoever, directly or indirectly, and not in any way to assist, encourage, or induce' the manufacture or acquisition of any '[biological] agents' for biological weapons purposes. on the other hand, article x requires states to 'facilitate . . . the fullest possible exchange' of technology and to 'cooperate in contributing . . . to the further development and application of scientific discoveries in the field of bacteriology (biology) for prevention of disease, or for other peaceful purposes'. the language of article x ('peaceful purposes') is such that the provision is subject to interpretations of intent, and it has long been a sign of international distrust that many developed countries are afraid to share certain kinds of biotechnology with some developing countries for fear that it will not be used peacefully. article iii of the bwc was reinforced in by united nations (un) security council resolution which requires all states to 'take and enforce effective measures to establish domestic controls to prevent the proliferation of nuclear, chemical, or biological weapons and their means of delivery, including by establishing appropriate controls over related materials'. to this end, states must establish, develop, review, and maintain 'effective national export and trans-shipment controls over such items . . .'. subsequently, many developed countries have continued to favour export controls (with more emphasis on the need to reduce proliferation risks) over the facilitating of biotechnology transfers. since , a group of states known as the australia group (ag) has cooperated to restrict exports of biological (and chemical) technology with a view to preventing the use thereof in weaponised form. the ag maintains lists that define dual-use biological agents and biological equipment, with participating states informally committed to ensure these items are subject to national export controls, and each state assesses export licence applications in accordance with an agreed set of guidelines. with respect to biotechnology, the list of controlled equipment includes, for example, fermenters, aerosol inhalation chambers, and spraying systems. in the decision to control 'the intangible transfer of information and knowledge' was made at an ag meeting in , and several participating states have since implemented enforceable legislation. for example, since and throughout the eu, council regulation / has provided a common legal basis for the eu member states individually to control exports of dual-use technology to non-eu states. for the purposes of the regulation, 'dual-use items' means 'items, including software and technology, which can be used for both civil and military purposes', and 'export' is defined to include 'transmission of software or technology by electronic media, including by fax, telephone, electronic mail or any other electronic means to a destination outside the european community' (article ). if an item of dual-use technology is listed in annex of the regulation, the government of an eu state must require the export of that item to be officially authorised (article ). in annex i, 'technology' means specific information, taking the form of 'technical data' or 'technical assistance', necessary for the development, production, or use of goods, and a general exception to technology transfer controls is made for 'basic scientific research' and information 'in the public domain'. in , regulation / was for the first time applied to the intended transmission (for publication purposes) of written research findings; specifically, the findings of particular research on the transmissibility of h n influenza virus that had been conducted in the netherlands. although this was an unprecedented application of export control law to this form of intangible technology transfer, it was not the first time that the risks of publishing the findings of virus research had generated public concern. over the preceding decade, a small number of cases had generated concern about the possible harmful application of published research methods. the controversies surrounding these cases in turn gradually intensified the political pressure on the editors of scientific journals to refrain from publishing (on security grounds) or else perhaps see governments one day take the decision out of their hands. one such controversy arose from an article that appeared in science in , less than a year after the anthrax envelope attacks in the usa. the article was authored by some us scientists who, sponsored by the us defense department, had spent years chemically synthesising the polio virus using sequences of genetic material purchased via the internet. the assembled material was then used to create live polio virus that paralysed and killed mice, thus showing that eradicating a virus in the wild might not mean it is gone forever. more broadly, the findings demonstrated how gene synthesis technology could obviate the need to source pathogens from natural reservoirs or from other laboratories. criticisms of the journal's decision to publish ranged from claims that the research findings were scientifically uninteresting to claims that the information could be maliciously and harmfully misused by its reader. one us congressman even tabled a congressional resolution accusing science of publishing 'a blueprint that could conceivably enable terrorists to inexpensively create human pathogens'. but the journal's editor at the time, donald kennedy, defended his decision by arguing: 'sticking one's head in the sand and hoping that unpleasant realities will go away has never been a fruitful approach to science or public policy'. he conceded, though, that 'there should continue to be serious conversations about the relationship between scientific research, publication, and security'. in , journal editors again came under political pressure in respect of two sets of written findings from research on the h n virus that caused the devastating spanish flu of - . one of the papers was eventually published in nature, and the other in science, but on this occasion, the us government (which had funded the research) first referred the papers to the newly formed national science advisory board for biosecurity (nsabb) for advice on whether the benefit to be derived from publishing this information outweighed the risk of harmful misuse. in combination, the findings revealed the genetic traits that made this influenza virus (which killed around million people worldwide) so deadly. the promise of sharing such information was that it could assist in further gene synthesis experiments aimed at better understanding the nature of influenza viruses and developing new vaccines or treatments. however, the intended publication of this information also gave rise to concerns that would-be bioterrorists could use it to reconstruct the spanish flu virus for the purpose of attacking a target population with no natural immunity. for example, an opinion article in the new york times described the published genome of this virus as 'essentially the design of a weapon of mass destruction'. members of the nsabb agreed that publication should proceed anyway, and science's donald kennedy explained that the risk of doing so was far outweighed by the benefit that it 'could help prevent another global flu pandemic'. six years later, when another influenza research controversy (involving the h n virus) began to unfold in the netherlands, the situation was quite different. the editor of science (a journal based in the usa) was less in control of whether the research findings should be published, because the dutch government decided that electronic transmission of the researchers' manuscript beyond the eu first required an export permit. as the next section will show, this decision raised legal and ethical questions to which the answers remain unclear: was this kind of research covered by regulation the continued circulation of h n viruses in poultry, and occasional infections of humans, has enabled ongoing viral evolution, and a key question for scientists has been whether this could eventually lead to the emergence of a virus with pandemic potential. such a virus would be able to spread through the air between humans via tiny droplets expelled during coughing and sneezing, and some influenza researchers have been interested to discover which genetic mutations might allow h n to do this. ron fouchier at the emc in rotterdam, the netherlands, was one of a number of scientists funded by the us national institutes of health (nih) to conduct experiments to investigate how h n might evolve to acquire the ability to spread from person to person. in september , at the fourth conference of the european scientific working group on influenza held in malta, fouchier announced a breakthrough: using a combination of genetic engineering and serial infection of ferrets, he and his team had succeeded in causing the mutation of h n into a form directly transmissible (through the air) between the animals. a ferret's respiratory system closely resembles that of a human, and ferret-to-ferret transmission of influenza virus is generally assumed to demonstrate human-to-human transmissibility. thus, it seemed, a new and presumably pandemic virus had emerged not through natural evolutionary processes but as a result of human experimentation at the molecular level of biology. a written account of this discovery, including a step-by-step description of how it was achieved, was made ready for submission to science. the journal's editorial board referred the manuscript to the nih (which had funded the research), and in october the nih in turn referred it to the nsabb to make recommendations regarding the responsible communication of fouchier's findings. two months later the board recommended against publication unless certain methodological details were omitted. the recommendation was unanimous, and nsabb member david franz explained: 'my concern is that we don't give amateurs -or terrorists -information that might let them do something that could really cause a lot of harm.' in february , however, participants in a meeting organised by the who recommended that the findings be published in full, on the grounds that 'the results . . . provide an important contribution to public health surveillance of h n viruses and to a better understanding of the properties of these viruses'. the following month, after considering a revised manuscript received from the dutch research team, the nsabb recommended that it should be published, although one-third of the board's members remained opposed to the publication of fouchier's findings. soon afterwards, with science poised to publish, the dutch government made an extraordinary intervention on the basis that highly pathogenic avian influenza viruses of subtype h are listed in annex i of eu council regulation / . under the terms of the strategic goods decree, which implements the regulation inside the netherlands, the ministry of economic affairs, agriculture and innovation announced in april that fouchier was required to apply for and be granted a permit to export his dual-use technology beyond the eu. a ministry spokeswoman explained that transferring technology without the required permit is 'an infringement of export-control legislation under the economic offences act' for which the 'maximum penalty, in case of premeditation or severe negligence, is six years' imprisonment or a e , fine'. prior to making a decision on whether to grant fouchier an export permit, the dutch government convened a closed meeting in the hague at which government officials discussed the risks and benefits of publication with an international group of scientists and security experts. after the meeting, fouchier applied for a permit despite earlier claiming he did not need one, and on april , the dutch minister for agriculture and foreign trade announced his decision to allow the export. the emc nevertheless filed a formal objection to the requirement for an export permit in this case, and the government disallowed the objection in december . thereafter, the matter of the applicability of regulation / to fouchier's h n research findings was pursued in court. was it lawful on this occasion for the dutch government to decide whether or not a scientific manuscript may be submitted for publication? fouchier claimed that it was not required for him to obtain an export permit, because the information to be published fell within two exceptions to the authorization rule in regulation / . with regard to items listed in annex i, one exception is that which applies to technology that is 'in the public domain', and another applies to 'basic scientific research'. in a case brought before the district court of noord-holland in haarlem, fouchier's lawyer argued that the methods used in the h n mutation experiment had been described before and were well-known (in the public domain), and that the virologists at emc had sought only to better understand mammalian transmissibility of an influenza strain (basic scientific research). on september , the district court rejected both arguments. in general, it considered that non-proliferation was a priority in the regulation and that exemptions should therefore be interpreted narrowly. regarding the first claimed exemption, the court found that fouchier's study went beyond known methods, by selecting and detailing the genetic changes needed to produce mammal-transmissible h n strains, and thus was not already in the public domain. the novelty of the research findings was also indicated by the willingness of science to publish them. the court found also that the research was not 'basic scientific research', defined in the regulation as 'experimental or theoretical work undertaken principally to acquire new knowledge of the fundamental principles of phenomena . . ., not primarily directed towards a specific practical aim or objective'. instead, the court characterised the development of an airborne h n virus as a practical goal, adding that it is not up to individual researchers to determine whether their research is basic. were the latter to occur, the court reasoned, it would compromise the obligation of states under un security council resolution to counter the proliferation of biological weapons. when fouchier appealed against this decision, the dutch court of appeal in amsterdam held (on june ) that it did not need to decide whether his research was basic or applied and thus whether the international transfer of the h n experiment data was subject to eu export regulations. rather, the court dismissed the appeal on procedural grounds alone. fouchier had applied to the dutch government for an export permit, he had then received one, and so he did not incur damage for which a remedy was necessary. that is, he no longer had a legal interest in pursuing the case. moreover, the court of appeal held that the government should not have taken fouchier's complaint into consideration, and that the district court b. benefits and risks of publication it is not clear exactly how the dutch government arrived at its decision to grant fouchier a permit to transfer his intangible h n technology to the editor of science. regulation / provides that, in deciding whether or not to grant an export authorisation, eu states 'shall take into account all relevant considerations including: (a) the obligations and commitments they have each accepted as members of the relevant international non-proliferation regimes and export control arrangements, or by ratification of relevant international treaties; . . .'. it is reasonable to assume, therefore, that the dutch government considered the requirements of the bwc, to which the netherlands is a party; and un security council resolution which is binding on all states. beyond that, taking into account 'all relevant considerations' appears to have involved assessing the perceived benefits and risks of allowing fouchier's h n findings to be published outside the eu. when export permission was announced on april , the dutch ministry for agriculture and foreign trade issued a statement : minister [henk] bleker has weighed all of the benefits and risks of publication of the avian influenza research, and has especially looked at the freedom of research and publication, health, and safety. he has also taken into consideration insights from national and international experts in the areas of security, health, and research; the positive advice of the u.s. national science advisory board on biosecurity to the u.s. government about publication of the research; and the u.s. government's decision to follow that advice. what, then, were the benefits and risks that the dutch government might have considered before deciding to grant fouchier an export permit? in march , when a majority of nsabb members recommended the communication in full of fouchier's data, methods, and conclusions, the benefit of publication was couched in the following terms: new evidence has emerged that underscores the fact that understanding specific [viral] mutations may improve international surveillance and public health and safety. global cooperation, critical for pandemic influenza preparedness efforts, is predicated upon the free sharing of information . . around this time, several of fouchier's colleagues publicly emphasised the importance of information sharing for disease surveillance and vaccine-testing purposes, and fouchier himself had previously argued in a televised interview: we have to be prepared for such viruses to emerge in the wild. if we would detect these viruses out in the field, then we could go out to outbreak areas and try to eradicate the virus and prevent a pandemic from happening. if that would fail, then we would still be in a good position to, ahead of that pandemic, evaluate our vaccines and anti-viral drugs and therefore gain months of time if a pandemic would hit and therefore we would be able to handle it better. however, the public health benefit of publishing research findings on influenza virus mutation is diminished if, in practice, systemic problems deny the theoretical opportunity to 'prevent a pandemic from happening'. on the issue of translating scientific discovery into actual benefit, a minority of the nsabb members who met in march concluded that 'the current [disease] surveillance infrastructure is ill-equipped to detect the emergence of highly transmissible influenza viruses in real time prior to their dissemination in nature'. nsabb chairman paul keim had earlier observed: i'm not confident at all that we have the surveillance capability to spot an emerging virus in time to stop it. and even if we did spot it early on, i don't think we have sufficient vaccines. the vaccines aren't good enough, and the drugs are not good enough to stop this emerging and being a pandemic. the message here seemed to be that the benefit of publication-to inform scientists worldwide of a discovery about h n influenza virus-is dependent upon the degree to which the information can then be exploited in a timely fashion by public health practitioners. if the prospect of beneficial application of published findings is slim, it is harder to claim that any risk associated with publication is outweighed. one such risk relates to laboratory biosafety. even if it was sufficiently safe for fouchier's team to conduct their h n experiment inside the emc in rotterdam, the same work done (using a published methodology) in less well-managed laboratories elsewhere in the world might carry a greater risk of an unintentional release of the mutated virus. according to marc lipsitch and alison galvani, the likelihood of a scientist somewhere being infected accidentally, for example, approaches % over a -year period. fouchier has insisted that 'scientific research has never triggered a virus pandemic', but accidental infections (such as with the sars virus) have gone undetected before. the view of a minority of nsabb members in march was that 'the revised fouchier manuscript provides information that would enable the near-term misuse of the research in ways that would endanger public health or national security'. as one nsabb member, michael osterholm, had earlier put it: 'we don't want to give bad guys a road map on how to make bad bugs really bad.' fouchier and two of his colleagues have dismissed this possibility, arguing that '[i]ndividuals with bad intentions do not need to read the details in our manuscript because the methods for creating similar viruses have already been published widely.' the implication here is that publication of their h n findings generated no additional risk. however, the problem with the 'it's already out there' argument is that the publication of a particular piece of writing is not simply an exercise in data dissemination; it also serves the purpose of assembling and packaging diffuse information for the reader. it can otherwise take much time and skill to bring together ideas and information from various sources and to craft them into an integrated, clearly explained message. thus, publication of fouchier's findings might have served qualitatively to increase accessibility to the necessary mutation technology. nevertheless, in respect of any 'recipe', there remains the possibility that it will not be followed correctly or that a key 'ingredient' will unknowingly be left out. regarding the problem of biological weapons proliferation, sonia ben ouagrham-gormley has found that 'such intangible factors as organizational makeup and management style greatly affect the use of acquired knowledge', and that these factors 'cannot be easily transferred among individuals or from one place to another'. if this is true of proliferation (in the form of publication of methodologies) in the realm of pathogen research more generally, it is a reason perhaps to afford less weight to the risk of information being harmfully misused. in any event, at the time the dutch government was attempting to weigh the benefits and risks of granting fouchier an export permit, the inherent dangerousness of the mutated virus itself (extant in fouchier's laboratory or reproduced elsewhere) remained uncertain. on the one hand, it is cause for concern that the global average case fatality rate for h n is high (around %). on the other hand, the who's figures reflect only confirmed cases of infection, and the proportion of deaths among all actual cases is probably much less. the government might have noted also that, in fouchier's experiment, '[n]one of the recipient ferrets died after airborne infection with the mutant a/h n viruses', and that this is consistent with the generally observed phenomenon that a virus's virulence decreases as its transmissibility increases. moreover, there might have been some doubt about the pandemic potential of fouchier's virus, given that virologists have disagreed on whether it would be as contagious and as pathogenic in humans as it is in ferrets. if, then, the dutch government judged that the mutant virus was probably less dangerous than was first thought, it could have downgraded the risk associated with publishing information on how to create it. for the same reason, though, the government might also have assessed that there was a lesser degree of public health benefit to be gained from allowing publication to proceed, because any pandemic caused by the virus would probably not be a severe one. assuming that the dutch government proceeded towards a decision in this wayassessing and weighing the benefits and risks of publishing fouchier's h n research findings, and authorising export on the grounds that the benefits outweigh the risksthe problem remains that this process is not a strong basis for decision-making. ideally, a government would indeed be able to conduct an integrated and exhaustive risk-benefit assessment of publication incorporating security, public health, and scientific perspectives. however, in the h n case, there were two fundamental problems attempting to do this under conditions of uncertainty. first, there was a lack of empirical, undisputed data upon which a probabilistic assessment of benefits and risks could be based. too little was known (or knowable) about the various claimed benefits and risks to allow measurement of the likelihood of their coming to pass and the consequences thereof. assessing the risk of bioterrorism, for example, is difficult given that an intelligent adversary can adapt to the presence of successful countermeasures. secondly, there was no commensurability among the different values potentially affected or advanced by the publication or censorship of fouchier's findings. it was not possible, for example, to balance concerns about public health benefits against concerns about national security risks in a precise, quantitative fashion. for these two reasons, judgments by the dutch government on the acceptability of risks and the desirability of benefits must have been based merely upon subjective assessments offered by numerous experts with different backgrounds, values, and opinions. one alternative approach to making a decision in this case would have been to apply the precautionary principle. originally conceived as a way to respond to the risk of irreversible environmental damage, this is a principle for 'making practical decisions under uncertainty when no reliable quantitative data is available'. because the prospect of such damage is the paramount consideration, other values (such as the financial interests of corporations) tend to be subordinated when decision makers invoke the precautionary principle. there has been some support for adopting this approach also in respect of dual-use dilemmas arising in pathogen research. for example, michael imperiale has argued that, under conditions of uncertainty, 'the burden should be placed upon those who wish to engage in research and publish their results to make a convincing argument as to why the risks of disseminating those results are insignificant'. in the h n case, applying the precautionary principle would probably have disposed the dutch government more towards deciding to refuse fouchier an export permit. however, it is important to note that the damage risked by publishing his findings is not, in contrast to some forms of environmental damage, irreversible. an influenza pandemic (whether caused naturally, accidently, or deliberately) eventually ends, and its effects are able to be mitigated. it seems excessively precautious, then, to constantly emphasise risk avoidance in the governance of pathogen research and for a government to withhold authorisation to transfer a particular intangible technology because it might in some way pose a risk. even in circumstances where benefits and risks are unquantified and the diverse values they affect are incommensurable, the achievement of some benefit remains important and indeed is what most influenza virus researchers probably have in mind when conducting laboratory experimentation. hence, with regard to fouchier's case and the issue of 'information security measures' generally, johannes rath has suggested that applying the proportionality principle is more appropriate for governance purposes. this is a principle that requires, first, that a particular governance measure is to some extent effective in protecting society from the harmful misuse of information. and it requires also that the intended measure is the least intrusive way of achieving that beneficial effect. unfortunately, however, there is presently little scope for pursuing a proportional response under regulation / . rather, a national government within the eu is afforded only two choices when it comes to the intended publication of dual-use research findings that are covered by the regulation: to refuse export authorisation (censorship) or to allow publication in full to proceed. this is highly unsatisfactory in circumstances where the merits of either choice are deeply contested; a government cannot be confident that full publication is not too risky or that censorship is not too heavy-handed. by contrast, having a third option available to deal with difficult cases (when the competing imperatives of non-proliferation and technologytransfer must both be addressed) would present an opportunity to apply the proportionality principle. accordingly, the next section proposes a governance measure that involves restriction short of censorship and communication short of publication. one suggested alternative to the censorship of fouchier's h n research findings was to allow the publication of a redacted article. according to david resnik, this would have been a proportional response to the security challenge posed by these findings: 'a compromise between promoting scientific research and preventing harm'. by omitting information on the precise methods used to mutate an influenza virus into a more transmissible form, 'terrorists' would have been denied 'a recipe for making a bioweapon' and other scientists would still have been able to learn more about h n . however, a number of objections were or could be raised against opting for redaction. with regard to states' obligations under the bwc, for example, it might be a bad idea for a government to conceal from other governments the methods used in an experiment that it deems to be of biological weapons concern. such concealment could give rise to international accusations that that government was developing (in a secret way) dangerous biological agents for a non-peaceful purpose in contravention of article i of the convention. and even if redaction of a dual-use technology publication were presented as consistent with the non-proliferation purpose of the bwc (article iii), it could also be perceived as a failure by a government to share with others (in accordance with article x) a scientific technique that could be applied for peaceful purposes. here, it is worth noting that, at a bwc meeting in december , there was general agreement on the value of ensuring that all states have 'access to the benefits of biotechnology' including 'developments of special relevance to disease surveillance . . .'. the omission of methodological data would instead inhibit access because the replication and testing of experiments requires this data. as such, a related objection is that redaction presents a barrier to researchers seeking to engage in further research, and that it thereby limits the scientific output essential to combat pandemic risk posed by influenza viruses. when nih director francis collins briefed the nsabb, in march , about the political problems with redaction in the h n case, one of her arguments was that limiting access to methodological data might jeopardise the pandemic influenza preparedness framework. this international agreement to share influenza virus samples and related information relies critically upon confidence that developing countries (from which h n samples are sourced) will benefit from influenza research carried out in the developed world. if confidence in the agreement were undermined by a perception that useful (albeit dual-use) technology was being deliberately withheld, the consequence might be a downgrading of international cooperation on disease surveillance and influenza vaccine development to the detriment of all. recent experience shows that redaction is also opposed by the editors of scientific journals. when resisting the idea of publishing anything less than a full account of research, they tend to refer to public health imperatives. for example, nature editor philip campbell argued, in december ,: 'it is essential for public health that the full details of any scientific analysis of flu viruses be available to researchers.' he was responding to the nsabb's original recommendation that a redacted version of fouchier's h n paper be published, and the response of science editor bruce alberts was similar: science has concerns about withholding potentially important public-health information from responsible influenza researchers. many scientists within the influenza community have a bona fide need to know the details of this research in order to protect the public, especially if they currently are working with related strains of the virus. alberts later attributed his journal's 'default position' (full publication) to 'the absence of any mechanism to get the information to those scientists and health officials who need to know and need to protect their populations and to design new treatments and vaccines'. consistent with these statements, and on the basis of a survey of journal editors in countries, daniel patrone and his colleagues have predicted: 'editors will be reluctant to withhold publication of dual-use research without at least having practical mechanisms for identifying responsible researchers and organizations with legitimate needs for the information and for distributing the information to them'. one practical mechanism worth considering, as an alternative to redaction, is the issuing of a permit for the targeted export of research findings that pose a dual-use dilemma. this would involve limiting the number of recipients rather than limiting the degree to which those findings were described. arguably, if findings like fouchier's could be disseminated directly to those 'scientists within the influenza community [who] have a bona fide need' for them, the public health concerns expressed by journal editors would be assuaged. as an additional mechanism for governing intangible technology transfers under regulation / , an eu system of ptes might operate as follows: . pte: the government issues a pte, covering all individual recipients named on the applicant's list, enabling intangible technology to be lawfully transferred accordingly. transmitted research findings could also be accompanied by a copy of the letter issued by a journal's editor indicating that its reviewers are satisfied with the scientific quality of the research. all the direct financial costs of technology transferal at this stage are borne by the authorising government. . bwc disclosure: immediately after the issuing of a pte, the government submits the applicant's research findings to the bwc implementation support unit (within the un office at geneva) to enable the transmission of the findings to the diplomatic representatives of all bwc member states. each member state is at the same time also provided with the list of the individuals authorised under the pte to receive this particular dual-use technology. in a case similar to that involving fouchier's h n findings, the availability of the option to issue a pte in this way would afford more scope for a government to respond to concerns about security risks and public health benefits in accordance with the proportionality principle. first, a pte is clearly more likely to satisfy the 'least intrusive means' test than is an outright refusal of export authorisation (censorship). and secondly, regarding the principle's 'effectiveness' test, the pte option has the advantage of facilitating the dissemination of information to individuals and governments who need to have it while also being effective in limiting the associated risk. at stages , , and of the process outlined above there would still be potential for the authorised recipients of data to transfer it to others. for example, there might be nothing preventing persons outside the eu from forwarding on the data. even so, preventing publication in major journals like science and nature, with their vast readerships, is what would prevent the greatest degree of information sharing. a pte would not prevent all dissemination of a given intangible dual-use technology, but it would prevent at least some dissemination and possibly most of what would have occurred as a consequence of publication. with every successful replication (done by reference to a written methodology) of fouchier's h n experiment, for example, there is a chance the mutated virus will escape its laboratory confinement and enter the general population. for this reason, as nicholas evans has argued, '[i]t is prudent . . . to limit not just who reproduces the studies, but how often these studies are reproduced.' in january , virologists michael osterholm and donald henderson argued that there was no need to share fouchier's h n mutation data 'outside of a small select group of established researchers already working within the who [influenza] network'. and fouchier himself earlier estimated that he could have shared his findings with 'well over organisations around the globe, and probably , experts'. however, this raises the question: why, then, did that intangible dual-use technology need to be accessible also to the hundreds of thousands of people worldwide who read science? part of the answer might indeed be that the benefit for pandemic preparedness purposes would thereby be maximised. but it is also worth acknowledging that, for academic scientists especially, a researcher's professional self-interest is well served by authoring an article that appears in a prestigious journal. this means there might be a strong temptation for a scientist to regard publication as something inherently good for one's career more (or rather) than as something instrumentally good for public health. moreover, as there is sometimes professional pressure also to be the first (among competing researchers) to publish, some scientists might sometimes feel disposed more towards haste than caution. as brendan maher has observed: 'it may be too much to expect scientists to coolly evaluate the risks of their own research against the benefits they gain personally from publication.' for these reasons, it might sometimes be healthy from a governance perspective that scientists alone do not decide whether or when their research findings should be submitted to a journal. if research findings were not published but rather disseminated through a ptebased process, the career advantage for the individual researchers involved would probably be less. already, there have been warnings that an inability to publish scientifically interesting results could have a chilling effect on certain kinds of pathogen research, driving ambitious scientists into other fields. however, one way to approach this problem would be to align more closely the imperatives of individual career advancement and socially responsible scientific practice. governments could afford more job security to influenza virus researchers in academia, for example, and thereby reduce the pressure to publish early and often. and public sponsors of research, who typically require grant recipients to publish their findings in full, could instead regard targeted dissemination as sufficient in circumstances where the benefits of publication do not appear clearly to outweigh the risks. research on pathogenic microorganisms has the potential to contribute indirectly to the saving of many lives in the event of an infectious disease outbreak caused naturally, accidently, or deliberately. national and international preparedness for an outbreak with global reach-an influenza pandemic-requires a strong scientific understanding of the changing properties and behaviour of influenza viruses. yet, the pursuit of that understanding brings risks as well as benefits. governments may therefore find it difficult sometimes to satisfy the competing governance imperatives of restrictiveness and permissiveness when it comes to the intended sharing of dual-use biotechnology. within the eu context, the fouchier case in the netherlands has demonstrated this, and it has also exposed at least two problems with the existing exportcontrol regime. ethical alternatives to experiments with novel potential pandemic pathogens virus experiments risk unleashing global pandemic, study warns' the guardian lab bungle china is scrambling to curb sars cases after a death reflections on the synthetic production of poliovirus' ( ) scientists brace for media storm around controversial flu studies' science the future of research and publication on altered h n viruses' ( ) journal of infectious diseases federal efforts to address the threat of bioterrorism: selected issues and options for congress rules of engagement the science of security versus the security of science' ( ) journal of infectious diseases a precautionary principle for dual use research in the life sciences meeting of the states parties to the convention on the prohibition of the development, production and stockpiling of bacteriological (biological) and toxin weapons and on their destruction risk assessment: laboratory-created a(h n ) viruses transmissible between ferrets' european centre for disease control and prevention research row as us raises terror fears' bbc news after receiving an application for permission to transmit dual-use research findings electronically to a journal editor located outside the eu, a government issues a preliminary permit (an 'individual export authorization' as defined in article of the regulation) for the sole purpose of allowing the scientific quality of the research to be scrutinised through the journal's normal peer-review processes scientific quality certification: following peer review, an applicant for an export permit submits to the government whatever letter he or she received from the relevant journal editor indicating that the research findings are fit to publish on scientific grounds distribution list: if the research findings are certified as scientifically sound, but the government determines (after consulting relevant experts) that the risks of regular publication are too great, the applicant is invited to submit a list of individuals located outside the eu (names and addresses) to whom the intangible technology should be exported for the sake of public health chief bruce alberts on publication of h n avian influenza research' american association for the advancement of science journal's concern over bird flu research' bbc news biosecurity and the review and publication of dual use research of concern public at last, h n study offers insight into virus's possible path to pandemic don't censor life-saving science four steps to rebuild trust in biology' the guardian the first problem relates to the exempting of intangible dual-use technology from the requirement for official authorisation under regulation / . following the dutch court of appeal decision in to overturn a district court decision in the dutch government's favour, it remains unclear whether research into the transmissibility of influenza viruses qualifies as basic scientific research. thus, it must be unclear to scientists inside the eu whether the publication of findings from such research requires an export permit. given that the penalty for unauthorised export of dual-use technology is severe, this is a highly unsatisfactory state of affairs. national governments within the eu should therefore engage in further discussion of the meaning of 'basic research' and do so with a view to issuing guidance for scientists and government decision makers. any such discussion should consider especially the practical difficulty of distinguishing basic from applied research. that is, in the everyday practice of science, there can be continuous interplay between the two categories: basic problems can inspire the development of new technologies, and the application of these can in turn open new avenues of basic research. for this reason, a scientist might get away with claiming that particular research is 'applied' in order to win public funding but 'basic' in order to circumvent export controls. such incoherence will need to be anticipated and addressed in an improved eu export-control regime.the second problem discernible from the fouchier case is that there are presently too few options available under regulation / for governing the intended communication of research findings that present a dual-use dilemma. assuming that the regulation was applicable at all in this case, the situation in which the dutch government found itself in was that, despite deep uncertainty about the balance of benefits and risks associated with sharing information about a mutated h n virus, it could only choose between two extreme alternatives: censorship or full publication. however, this need not be the case in the future. when faced with the difficult challenge of satisfying non-proliferation and technology transfer imperatives simultaneously, a third option could be tried: permits for the targeted export of research findings. such an arrangement for information sharing would address the dual need to reduce security risks and improve public health, although it would probably be less beneficial than publication to the careers of academic scientists. a further challenge, then, would be to maintain the professional attractiveness of pathogen research. although limiting transfers of intangible biotechnology might be protective of human health in the short term, it could be anti-protective over time if it adversely affects too many scientists' ability and willingness to discover more about global infectious disease risks. none declared. key: cord- -jsdpltji authors: wagner, david; bakker, arthur; meaney, tamsin; mesa, vilma; prediger, susanne; van dooren, wim title: what can we do against racism in mathematics education research? date: - - journal: educ stud math doi: . /s - - -w sha: doc_id: cord_uid: jsdpltji nan contribute to the oppression of black, immigrant, and indigenous children and youths (e.g., battey & leyva, ; khuzwayo, ; martin, gholson, & leonard, ; valoyes-chávez, ) . there is also some scholarship that identifies global disparities within mathematics education research communities (e.g., meaney, ; mesa, ) . we feel compelled to act, just like other journals (e.g., nature: https://www.nature.com/articles/d - - - ). we want to consider the role journals in general and esm in particular might play in sustaining racism and perpetuating inequities within the field of mathematics education within and across countries. while it is difficult to find instances of overt racism in the peer review process, it is easy to see that systemic issues in the research field favor scholars and research from some regions more than others. we see that many of these issues are outside our control, such as finance and other resources, but we need to examine ourselves to identify our role in the inequities. for example, the dominance of english as lingua franca in worldwide academic dialog tends to oppress other traditions (andrade-molina, montecino, & aguilar, ; chellougui, nguyen thi thu, & winsløw, ) , but we can begin to change that in our corner of the wider academic community. mesa and wagner ( ) reported on the countries of origin of contributors to esm and raised questions about the significant disparities among the countries represented. when we consider these disparities and similar imbalances in other journals (roberts, bareket-shavit, dollins, goldie, & mortenson, ) and conferences, we are compelled to ask whose lives are represented most and thus whose lives may appear to matter most in mathematics education research. we think of this question in two ways. first: to whose mathematics experiences does the research in esm pay attention? in other words, whose mathematics learning and teaching are considered important enough to study? whose mathematical practices are deemed interesting? second: which researchers matter? whose questions are recognized as important enough for the world to read about in esm? the disproportionate distribution of countries of origin of published papers might be taken as an indicator that readers and contributors to esm do value some persons' mathematics education more than others'. clearly, this needs to change. thus, in this editorial and the ongoing discussions among the editors, we examine our roles in this process and propose ways to begin to address the disparities. as noted in the article by mesa and wagner ( ) , we editors have committed ourselves to extra work to support contributors whose first language is not english and to inviting reviewers and editorial board members from a diversity of countries, but we see that more action is necessary. here are some of our current thoughts. we identify our different voices here to signal the importance of perspective in such considerations, presented in the order in which they were written. the timing of vacations influenced who was able to contribute among our editor team. racism has many different faces; in countries like germany (where the category "race" was banned from discourses after the nazi regime), it is disguised by distinctions between cultures or religions. in the international academic world, racism can be fueled by rankings of universities or countries. this is particularly subtle as there might be objective quality differences, but the global rankings can never do justice to an individual's achievements and can conceal the advantages of working in a privileged environment. in these contexts, it is hard to balance between the inclusion of all researchers notwithstanding their contextual conditions and the need for a journal such as esm to be selective due to the academic aim of high-quality standards. are we always sensitive enough to how knowledge about typical inequities (between countries, cultures, universities, …) can unconsciously turn into stereotypes and prejudices with an uncovered racist tendency? in the long run, the aim must be to reduce inequities, not only stereotypes. how can we be inclusive and adhere to quality demands at the same time? similarly to other contexts, in our editorial work, this tension means for us to keep asking ourselves: & how can we avoid labeling and prejudices on the basis of country or university when we consider (a) authors, (b) reviewers, and (c) people to be cited? & how can we contribute to more inclusion (a) by controlling our bias towards authors from underrepresented contexts in the journal and support the access to the international discourse for all authors, (b) by inviting reviewers from different contexts, including underrepresented countries, and (c) by making sure that authors from underrepresented countries are cited? & how can we contribute to developing quality (a) by working hard with all authors (also from less privileged contexts) so that they can express their ideas in the best way possible, (b) by working hard on the editorial letters to compensate if poor reviews provide too limited support for authors, and (c) by including suggestions for references from underrepresented contexts? & how can we continue becoming increasingly aware of the subtle mechanisms of labeling and stereotypes which we have internalized by socialization? as susanne writes, racism comes in many forms. yet, it has an impact wider than the journals that mathematics education research gets published in. for example, mathematics education is a combination of both mathematics and education. yet, in the articles that get published, "mathematics" is more often given precedence over "education." why is it the case that "what is it about the mathematics which makes it difficult to learn" is prioritized over "what is it about the contexts that produces under-achievement in the learning of mathematics"? when under-achievement is mentioned, it is most often done so as a way of labeling a group (see, for example, meaney, edmonds-wathen, mcmurchy-pilkington, & trinick, ) . generally, all the labeling does is to glue more strongly the under-achievement to these groups of students, so they or their families become responsible for the under-achievement. petra svensson described how teenagers in sweden whose parents were immigrants had learnt from public discourse, particularly politicians' statements, that failure in tests such as pisa meant that the only way to improve their chances of doing well in mathematics was by having parents who were swedish and by default who could help them with their homework (svensson, meaney, & norén, ) . ole skovsmose ( ) has written many times about how the majority of children are ignored in mathematics education research, often under the guise of "what can be learnt (by western countries) from this research?" on the other hand, it is presumed that children in these "other" countries can learn from what works in western countries. what is published and what is not has implications well beyond the readership of mathematics education journals such as esm. our choices about what we support for publication can be racist if we consistently deny the experiences of some groups as being important and value only those from a small group. consequently, as reviewers and editors, there is a constant need to reflect on how we review an article and what assumptions it draws upon about quality research, which may result in exclusion rather than inclusion. we rarely see blatantly racist statements made either in journal articles or by reviewers, but this does not mean that racism is not there. it may be easier to see this by turning the issue around and saying who is privileged in reviews and editorial work? david stinson ( ) writes about the need to consider white supremacy in mathematics education research as an alternative way of thinking about racism. this helps in the process of recognizing privilege. do native english speakers have an advantage in writing journal articles? it is not easy to see this as an issue of racism, but it is certainly easy to see it as an issue of privilege. when we recommend alternative literature, what is the underlying assumption about the literature which was being used? which researchers are we privileging by these comments as important mathematics education researchers? are these researchers reflective of mathematics education researchers from across the world or do they represent a group with very similar demographics? if it is the latter, who are reviewers and editors saying can be important researchers in our field and by default who are we saying cannot be? as reviewers and as editors, we need to not just look for offensive statements of racism but also for these more subtle forms. questions about race prompt me to think of my conversations with people who identify with groups that are marginalized: their descriptions of their experiences and our conversations about possibilities for challenging structural inequities. i rely on these conversations because i sit in the intersection of most privileged demographics (white, male, cis-gendered, canadian, etc.). the question of whose lives matter reminds me most vividly of an extended conversation with a friend when i was a teacher in eswatini. as we warmed our hands over the heater in the teacher work room one day, he asked why swazis are poor and canadians rich. he wanted to talk about stereotypes and how they are used to explain disparities. in particular, he said that north american and european attributions of africans being lazy and corrupt allowed us to feel comfortable with african poverty. for weeks, my swazi friend and i talked about the (in)accuracy of these stereotypes and also of stereotypes that valorize north american and european ingenuity. we problematized the stereotypes by discussing people who embodied the values of their cultures: we found flaws in the apparent virtues and wisdom in the apparent flaws. when is ambition also dominance and repression? what level of wealth can someone have before being implicated in the violence of disparity? when is sustainable living and efficiency seen as laziness? where is the line between corrupt corporate empires and corrupt government? i miss my friend. in mathematics education research communities, i have noticed that conversations about the underrepresentation of scholarship from certain regions eventually invoke a tension between diversity and quality. we all say that we wish for more research from the underrepresented places, but we wonder how this can be done without sacrificing expectations for quality. this tension suggests assumptions of superiority much like the ones discussed with my friend. it identifies, for example, the theoretical and conceptual richness of european research, the rigor of north american research methods, and the beauty of the body of research developed with these strengths and the associated consensuses. i think that narrow conceptions of quality are dangerous. they are incestuous, blinding us to potential flaws and thus allowing the flaws to grow into proclaimed virtues. there are different qualities of good research, different ways that research can give insight and challenge popular conceptions. i hope we can open discussion about different qualities of scholarship and how these qualities might represent and/or challenge conceptions in different contexts. in addition to talking about what can be good in research reporting, we need to talk about the problems with these apparent goods. i will point to one example of this kind of challenge to the status quo, but i mostly i want to leave the question open for discussion: lihua xu and david clarke ( ) drew attention to significantly different cultural norms in asian and english-speaking contexts to problematize assumptions and conceptualizations in research relating to what kind of communication is valued in mathematics classrooms. i suggest that the first priority should be to discuss different qualities that research can bring to the field, and that people from well-represented countries (people like me) need to make space for people from underrepresented countries to lead these conversations. for an example, i point to a symposium convened by aldo parra, arindam bose, jehad alshwaikh, monica gonzález, renato marcone and rossi d'souza ( ) that theorized crisis from the perspectives of scholars from "developing" countries. speaking in generalities (which is dangerous), i see researchers from underrepresented regions too often trying to fit the molds coming from western europe and the usa rather than building from their own perspectives on research qualities. of course, it is hard to do quality work in someone else's framework, but colonialist storylines and demands of peer reviewers push researchers to try. i want to challenge this push and encourage research that builds on the wisdom of local worldviews, not the co-opting or forced adoption of the worldviews of others. so far, my colleagues have used the word racism and the expression white supremacy, and their usage-without definition-has prompted me to query, what do i mean by these terms? we have mentioned power, structures, and stereotypes…. do we have a common understanding of these terms? this is my current understanding of racism: the belief that the inferiority of one group of people relative to another can be attributed to the color of their skin. adscriptions of inferiority can be made for many other categories, as the ones suggested by susanne. we know that genetically we are more alike across people from different skin colors than within people of the "same" color. yet, we do continue to use the marker of skin color to make split decisions about worth, ability, and competence. white supremacy, to me, is the belief that the way in which white people do things is the way in which things must be done; that the ideal to which all humanity needs to aspire or organize itself must follow what white people have established. white supremacy needs racism and any other form of making other groups inferior: religion, language, age, wealth, health, body ability, etc. ibram x. kendi ( ) beautifully traces the origin of racist ideas to the need to sustain economic advantage and the ways in which european and american thinkers (male and white, by the way) justified white supremacy and slavery. he describes how laws were amended time and again to maintain the status quo; how the laws (and white male lawmakers, thinkers, philosophers, and yes researchers) managed to, not only uphold whiteness as an ideal for beauty, intelligence, goodness, wisdom, etc. but also convince everybody else that this was true. power determines hierarchies among people; we construct these hierarchies with structures and symbols. how are these hierarchies expressed in our academic process of publishing in journals? esm has policies (e.g., length, style), procedures (one-way blind review, various experts read the same manuscript), and mechanisms to decide whether and when an article is ready for publication-based on a combination of the study reported in the manuscript, reviewers' comments, and editor's appraisal of both. but these have not been openly discussed nor historically scrutinized-and yet, we operate as if there is agreement and consensus about what they are. it is a struggle for me, to offer all the fair and equitable support that is needed to new researchers, especially to those from countries that are not well represented in the journal: i struggle to find reviewers who are willing to find merit in the work and put aside their own assumptions; i struggle with ways to present my suggestions so the researcher is not demoralized by the need of more revision; and i struggle with the demands on time that all of this requires. at the same time, i also wonder if i am participating in a colonialism project, another way of continuing imposing whiteness (relevancy, problems that matter, ways of writing, etc.) into the process of conducting and reporting research? but beyond my personal interactions with individual researchers, i think we need to look critically at how current structures supporting the journal make it easier to maintain the status quo. could our publisher ramp up their english language support? could we have a larger editorial board to include wider country representation? could we publish in more than one language? could the publisher make it enticing for the editors-in-chief and the associate editors to hold frequent meetings to discuss the workings of the journal, as dave suggested above? to what extent are the needs of the academics taking precedence over those of the publisher's shareholders? we need to critically assess how it is that the whole enterprise is being supported; otherwise, we will be seeking individual remedies to problems that are systemic. perhaps then we can provide an authentic answer to the question of whose lives matter in mathematics education research. as editors of one of the leading scientific journals in the field of mathematics education, we need to take issues of racism and any other inequities very seriously. as indicated in the introduction, it is very clear that papers that get published in esm are disproportionately distributed over countries of origin. as a consequence, certain mathematics experiences and practices remain in the blind spot of the research community. but a question that also needs to be addressed is whether this indicates that these mathematical practices are not deemed interesting, whether the questions raised by the non-published researchers are not considered important enough for the world to read about them. in other words, is the disproportionate distribution of published papers really an indicator that readers and contributors to esm do value some persons' mathematics education more than others'? in order to evaluate this, we first need to be aware that esm and other journals (and to some extent also conferences) are the "endpoint" of the research process, where results of research are published. a long and very complex process (from the education and training of academic researchers and mathematics teachers, over organization of research and the way it is funded and how its output is valued, to the reporting and submission in journals like esm) is involved. the range of manuscripts that we receive at esm-and certainly the subset of manuscripts that finally get published-reflects only a very small part of that complex process. so, we need to look critically at how the disproportionate distribution of published papers would indeed reflect that readers and contributors to esm do value some persons' mathematics education more than others'. looking at this complex process, from my experiences as an editor, i describe a few possible scenarios in which racism or other utterances of inequity could pop up, and lead to a disproportionate distribution in published papers. the elaboration of these scenarios is almost certainly incomplete, and i do not want to claim that all scenarios are equally real and/or relevant. some may even simply reflect my narrow perspective as a -year old white, male, european professor in instructional psychology being active as an esm associate editor (and probably having disproportionately many publications in journals). however, the scenarios do indicate that the disproportionate distribution of published papers does not necessarily reflect that some mathematics education practices and research practices are valued less. there are countries where a lot of mathematics education research happens, while in other countries, there may not even be research on mathematics education at all. this is an evident reason why some countries are underrepresented. but as an editor, i have seen that in some countries mathematics education research has started to grow only recently, while researchers are at the same time expected to show "scientific output" already after a very short time. this often seems to have led researchers from these countries to go for "quick gains": studies with limited novelty and no real contribution to the field, which again seriously hampers their chance of acceptance. in still other countries, a lot of novel research is done but there is no expectation at all that the research conducted is also published internationally. i take my own country (belgium) as an example, but various aspects can also be recognized elsewhere. most of our teachers (kindergarten, primary, and lower secondary) are not trained at university, but rather in institutions for higher education outside universities. until recently, these institutions were not expected to conduct research, and while this is now changing, the kind of research they do is typically aimed at direct use in our own educational context. there is no expectation that the research is reported in international scientific journals. this may be a reason for a disproportionate underrepresentation, simply related to how research is organized. related to this is that i have seen that the research being done in many countries is mostly educational in a general sense, rather than specific to mathematics, even though the profile of the researchers is a mathematical one, and these researchers are often training mathematics teachers. there are many good reasons, including the fact that educational challenges in the countries where these researchers work are overarching the specific domains. many of the submissions that i handle as editor are given a desk rejection for not fitting to the scope of the journal: while they in some way are about mathematics (for instance, the learning outcome is a mathematics test), they address a general educational theme that may be of interest to a wide readership, but they do not contribute specifically to a better understanding of the processes involved in teaching and learning mathematics. when a desk reject is given for such a reason, this is not a judgment of quality, but rather of the submission not being sent to the right journal. this would imply that countries underrepresented in esm could be more strongly represented in other journals. these scenarios describe reasons why certain countries may be disproportionately represented among the papers that are published-or even submitted-to esm. and these reasons can hardly be related to issues of racism or other inequity in the way submissions are handled within esm. but of course, many other scenarios do raise issues of racism or inequity that esm can work at. the most blatant case would be if submissions get a lower chance of acceptance simply because the editor and/or reviewers would let prejudices about the authors interfere in judging the value of the conducted research and the quality of the report about this research. but more subtly, it can happen when the conducted research is good on its own, but the way it is reported shows flaws in the language, the scientific style, the range of literature included, and/or formalities like references and the description of statistics. these are important aspects in a paper, and they need to meet certain standards before a paper can get published. but some authors may not get a fair chance and sufficient help to revise their paper and get it to meet this standard, and mechanisms of racism could play a role in that. it becomes even more subtle if one realizes that in certain countries, other kinds of mathematics education research are being conducted, with a focus on other practices, topics, theoretical frameworks, et cetera. these different kinds of research might not always be adequately appreciated by the handling editors and/or by the reviewers, and issues of racism may be at play here, too. the question, however, is whether a journal like esm can act on this on its own, or whether this is an issue of the broader mathematics education community that needs to take efforts to become more aware and more inclusive in light of such differences. as editors of esm, we can play a role in this, of course, but a broader initiative seems needed for it to take effect. the ideas raised by the others in this editorial are important, in that respect, and i genuinely feel compelled to take action when needed to eradicate any inequity, be it due to racism or other reasons. but we should be very well aware that the disproportionate representation of countries among the published papers has many more reasons, and that esm can only play its role within the broader mathematics education research community. what can i do against racism? to this big question, i only have humble, partial, temporary, and local answers that also depend on my positions as a human being, a white male, father, tourist, sports coach, consumer of coffee and chocolate, colleague, teacher, researcher, andmost relevant here-editor of esm. as background to the topics and questions that in my view require courageous conversations (singleton, ) , i use two anecdotes. the first dates back to when i traveled to the usa for the first time in my life. just before landing, we were asked to fill in a form, which included a question on race. in many european countries, it is forbidden by law to register race-one of the reactions to world war ii when it proved far too easy to identify jews and other groups in society. i was shocked: why would the us government need information about my race, and what would they do with such information? after a few minutes, i decided it was not the right moment to be anti-racist, but then i stumbled upon the next challenge: which box to tick? "white" was not on the list. it was suggested to me that "caucasian" came closest. given that the caucasus is just north of turkey, with very diverse ethnic groups, i found this hard to believe. it seems typical of white people that they are rarely confronted with the issue of race (cf. the story about teacher anne in horn, , pp. - ) , but this was one such moment for me: why would the usa government want to see me as caucasian whereas the dutch government puts me in their statistics as western immigrant, because my mother (a descendent of maori and british ancestors) was born in new zealand? the second anecdote is also about a confrontation between american and european perspectives on race, but this time between researchers. in , i participated in a -day workshop on engaging and diversifying stem education, organized by joe polman and melissa braaten (funded by the united states national science foundation, nsf grant no. , and the netherlands initiative for education research, nro). all the researchers from a dutch university would probably qualify as white, even though some of us prefer to stress the multiplicities of our identities (cf. akkerman & meijer, ; avraamidou, ) ; and none of us studied race or related themes. the us researchers were overtly more diverse, several with a background in critical race theory. tension was rising in the group until a us scholar started a courageous conversation, asking whether racism was really not a big issue in the netherlands or whether we were color blind. this confrontation ignited a much appreciated dialog that had an enormous impact on me (and i have been told on many others). for us, colorblindness was a virtue ("i only see people, not their race"), but we started to realize that there is a high price that we pay for not wanting to talk about race and not being allowed to register racial information. post-world war ii, the fear of racialization in europe (m'charek, schramm, & skinner, ) seems to have led to a situation where we do not know much about racism in our educational system. for example, i found hardly any studies related to racism or ethnicity in dutch mathematics education (what i found was mostly through a group of flemish authors: stevens, clycq, timmerman, & van houtte, ) . with the current protests in numerous countries and actions at universities, there seems to be more interest in finding ways to study racism but the european privacy law and guidelines of ethical review boards forbid collection of race information except under very special conditions. i stress this point because european reviewers may be surprised by reports on racial differences, whereas reviewers from the usa may wonder why their european colleagues did not collect that information. note for instance that roberts et al. ( ) advise authors to "detail the racial demographics of their samples" (p. )-a recommendation that makes sense in one context but is nearly impossible in another. note also that my examples from the usa versus europe show my ignorance about how these issues play out in other parts of the world (cf. kapuściński, ) . after the workshop, i have had many conversations with colleagues and students about the uncomfortable but helpful confrontation between us and dutch scholars. one recurring question was to what extent we could sensibly use the conceptual frameworks on race developed by researchers from the usa and south africa. the general opinion, in my own words, seems to be that much conceptual and empirical work needs to be done to answer this question. the usa and south africa have very specific histories in which the meanings of race, white supremacy (bonilla-silva, ; hooks, ) , and white innocence (wekker, ) are rooted. i also have the impression that other countries may need somewhat different languages to reach their local audiences. for example, i have noted that many dutch people prefer to talk about discrimination (ethnicity, gender, sexuality, etc.) rather than racism. furthermore, the meanings of many of these terms, in particular what counts as politically correct, change rather fast (m'charek et al., ) . although many mechanisms, in whichever formulation, may be rather universal (see examples mentioned by vilma), cultural-historical studies tend to show how local manifestations can be very different. as tamsin pointed out, stigmatizing and discrimination may manifest differently in different cultures, just like the experiences of maori, aboriginals, and other indigenous people may differ from turks in germany or the netherlands. de haan, keizer, and elbers ( ) discovered even subtler differences: "power relations in classrooms between dutch and immigrant students radically shift when students move from academic talk to more open, free talk" (p. ). their findings suggest that identity is much more contextually contingent than typical talk about black and white suggest (see also nasir & hand, ) . another recurring theme in our local discussions is that it is so hard to have transformative conversations such as we dutch had with the us visitors. along with other white colleagues, i have found it challenging to counter the anger expressed by antiracists (e.g., weiner, ) . i have felt guilt for being white and male as well as the colonial history of my country and its late abolishment of slavery (balkenhol, ) . in some discussions, arguments are swiped away as white innocence or white supremacy. the result is that we feel like elephants tiptoeing in a porcelain cabinet, as we say in dutch. and what is worse: we feel labeled white with many privileges but without any nuance or justice to individual identities. however, when it comes to anger and guilt, my view has changed after reading audre lorde's work, in particular her paper uses of anger: women responding to racism (lorde, ) . characterizing herself as black, lesbian, mother, warrior, and poet, she wrote: i cannot hide my anger to spare you guilt, nor hurt feelings, nor answering anger; for to do so insults and trivializes all our efforts. guilt is not a response to anger; it is a response to one's own actions or lack of action. if it leads to change then it can be useful, since it is then no longer guilt but the beginning of knowledge. (p. ) what do the aforementioned reflections imply for a journal such as esm? first of all, we need critical discussion about implicit norms and explicit guidelines in the review and publishing process. as these have grown historically, the problem is that some of these may privilege those who are already privileged. for example, it is quite common for reviewers to ask authors to join the international debate, but what if this debate is dominated by a particular group? in other words, it is possible that our norms and guidelines are defined in ways that are blind to some of the many colors of quality. the risk of monolithic views on research is criticized by biesta ( ) in an inspirational book titled educational research: an unorthodox introduction. the risk of the orthodox (greek for "the right opinion") is that guidelines become rigid and blind to context. his plea for unorthodox views on educational research appeals to me because if anything is clear from our discussions as editors, it is that current norms and procedures need to be scrutinized and rethought to ensure that inequitable shackles are broken. for example, it is an orthodox expectation in qualitative research to report interrater reliability with measures such as cohen's kappa or krippendorff's alpha. in some cases, such indicators may be useful, but there can be situations in which such reliability measures are not saying much about research quality (thomas, ) or where alternative quality procedures may be more suitable (e.g., akkerman et al., ) . another orthodoxy that biesta ( ) questions, with reference to dewey, is that of the desire for objectivity as opposed to subjectivity. in his view, educational research needs to go beyond the dichotomy between the two. under the banner of the ideal of objectivity, much harm can be done. when applied to the topic of avoiding racism in academia, i think we should avoid the orthodoxy of trying to make mathematics education research even more objective, with the risk of erasing the human from our work. rather, we need a more human science. second, although i have never seen reviewer comments that i would label as racist, we have heard stories about unfair comments or expectations, sometimes in disguise of a less controversial norm (e.g., the aforementioned quality of english). and there seems to be asymmetry in what authors are expected to explain about their country's educational system. for example, it would be unfair if researchers from the usa would not have to explain anything about their situation, whereas namibian researchers are asked to spell out the specificities of their educational context. third, does our (somewhat flexible) word limit privilege some types of research or researchers? what are the pros and cons of single and double blind and other review procedures (baldwin, ; mesa & wagner, ) ? can we require authors to engage conscientiously with the politics of citation (mott & cockayne, ) ? i think that no formal procedures will alleviate the racism problem (lorde, ) , but talking about procedures will help create awareness of the potential biases of each. the history of psychology shows that a focus on fear of bias may lead to rigor (literally "stiffness"), but not necessarily much theoretical progress (flis, ) . instead i would like to invite reviewers to articulate their own ethical ideals on reviewing. in my view, inspired by levinas (e.g., levinas, ; strhan, ) , we as editors and reviewers need to make an attempt to meet the other (which he mostly capitalizes), even if mediated through a manuscript about a scientific study. someone's face, levinas metaphorically argues, does an appeal to my ethical responsibility for this person. even though as an editor or reviewer, i only see someone's manuscript and not literally their face, i would rather focus on openness and vulnerability than fear and rigor. authors typically put their life and soul into their work, and devote scarce resources on their research. i know of no slower conversation than that between authors, editors, and reviewers, but it is a dialog. the key to me in realizing a fair, nonracist review process is recognizing that this dialog is not purely epistemological but primarily ethical. with such an ethical starting point, i am more optimistic that reviewers and editors continue to ask the big question: what can we do against racism? many wisdom traditions and literature on racism or oppression tell us to be reticent in trying to "fix" this. for example, in the tao te ching, we find: if a country is governed with tolerance, the people are comfortable and honest, if a country is governed with repression, the people are depressed and crafty. when the will to power is in charge, the higher the ideals, the lower the results. try to make people happy, and you lay the groundwork for misery. try to make people moral, and you lay the groundwork for vice. (mitchell, , chapter ) it is further clear that most of our editors are white and despite various origins, we all live in firstworld countries with resources to do our editorial work (even though most of us do not get time or "course relief" from our universities). the racism literature warns that white people trying to help others may only reinforce white supremacy (bonilla-silva, ; gillborn, ; wekker, ) . lorde ( ) notes that the master's tools will never dismantle the master's house (title of one of her papers). freire's ( ) work also shows how complex it is for the privileged to empower others. as a journal, we need to gain a better sense of institutional racism in the review process (assuming there is despite everyone's efforts) before implementing formal procedures. hence our call for dialog and action below. our reflection on our roles in privileging some lives over others in mathematics education research compels us to action as editors. we commit ourselves to increasing our awareness and understanding of challenges experienced by contributors, and to keep working on fair reviewing processes. we also see the need for action in our other roles as reviewers, as conference leaders, as university teachers educating future researchers, and in our many less formal interactions within mathematics education research communities. we hope to provoke others to reflection and action, too. we also invite mathematics educators to tell us stories of unfair treatment to improve our understanding. further, we invite suggestions for more equitable practices and of potential editorial board members from underrepresented communities. email any of us: arthur bakker at a.bakker @uu.nl, david wagner at dwagner@unb.ca, susanne prediger at prediger@math.tu-dortmund.de, tamsin meaney at tamsin.jillian.meaney@hvl.no, vilma mesa at vmesa@umich.edu, wim van dooren at wim.vandooren@kuleuven.be, using the subject "nonracist reviewing." another possibility is to submit a "letter to the editor" to esm (see e.g., proulx & maheux, ; simpson, ) . finally, we are pleased to announce an upcoming special issue of educational studies in mathematics on "race, racism, and racialization in mathematics education: global perspectives." this special issue will consider the concepts and practices much more deeply and broadly than we have in this editorial. it will be guest-edited by danny bernard martin, luz valoyes-chávez, and paola valero, who began organizing the special issue long before . see the journal's website under "updates" and the back matter of volume , issue , for details and the call for papers. auditing quality of research in social sciences a dialogical approach to conceptualizing teacher identity. teaching and teacher education beyond quality metrics: defying journal rankings as the philosopher's stone of mathematics education research i am a young immigrant woman doing physics and on top of that i am muslim": identities, intersections, and negotiations making "nature": the history of a scientific journal tracing slavery. an ethnography of diaspora, affect, and cultural heritage in amsterdam. (doctoral dissertation a framework for understanding whiteness in mathematics education educational research: an unorthodox introduction racism without racists: color-blind racism and the persistence of racial inequality in the united states language diversity in research on language diversity in mathematics education when the jokes aren't funny ethnicity and student identity in schools: an analysis of official and unofficial talk in multiethnic classrooms pedagogy of the oppressed discipline through method: recent history and philosophy of scientific psychology racism and education: coincidence or conspiracy? routledge teaching community: a pedagogy of hope motivated: designing math classrooms where students want to join in the other. verso books stamped from the beginning: the definitive history of racist ideas in america a history of mathematics education research in south africa: the apartheid years humanism of the other the master's tools will never dismantle the master's house. penguin technologies of belonging: the absent presence of race in europe mathematics as gatekeeper: power and privilege in the production of power the privileging of english in mathematics education research, just a necessary evil? keynote presentation distribution, recognition and representation: mathematics education and indigenous students jrme in the global village: parlez vous français? habla ud behind the door: a critical look at the process of publication in educational studies in mathematics tao te ching: a new english version citation matters: mobilizing the politics of citation toward a practice of 'conscientious engagement from the court to the classroom: opportunities for engagement, learning, and identity in basketball and classroom mathematics crisis" and interface with mathematics education research and practice: an everyday issue effect sizes, epistemological issues, and identity of mathematics education research: a commentary on editorial ( ) racial inequality in psychological research: trends of the past and recommendations for the future on the misinterpretation of effect size courageous conversations about race: a field guide for achieving equity in schools travelling through education: uncertainty, mathematics, responsibility researching race/ethnicity and educational inequality in the netherlands: a critical review of the research literature between researching race without researching white supremacy in mathematics education research: a strategic discursive practice levinas, subjectivity, education: towards an ethics of radical responsibility immigrant students' perceptions of their possibilities to learn: the case of homework how to do your research project: a guide for students colombian teachers' expectations of black and poor students' ability to learn algebra. (doctoral dissertation the ideologically colonized metropole: dutch racism and racist denial white innocence: paradoxes of colonialism and race speaking or not speaking as a cultural practice: analysis of mathematics classroom discourse in shanghai, seoul, and melbourne publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments we would like to thank colleagues who provided helpful input: lucy avraamidou, michiel and sofie doorman, na'ilah suad nasir, bill penuel, anne peters, joe polman, luz valoyes-chávez, and bjorn wansink. key: cord- - sx tso authors: perez, a; ball, g d c title: are we overlooking the qualitative ‘look' of obesity? date: - - journal: nutr diabetes doi: . /nutd. . sha: doc_id: cord_uid: sx tso nan during the opening ceremonies of the th canadian obesity summit held recently in toronto, along with the traditional speeches and awards, a woman who formerly had obesity shared her personal story. emotional, heart-felt, and humanizing, her experience of living with obesity as a child, a professional, a wife, and an artist provided a detailed and personal view of her ongoing personal struggles with her weight, which set the tone for the meeting over the next few days. her story and others like it can provide rich insight into individuals' perspectives of obesity and weight management. in our view, these perspectives have been under-represented in the field of obesity research where numbers from quantitative research often take precedence over meanings derived from qualitative inquiry. qualitative research has proved important in many areas of clinical and health research, including understanding patients' and clinicians' decision making and enhancing quality of health services delivery related to utilization, feasibility and appropriateness of care. , despite being on the rise, the publication of qualitative studies in medical journals is still low, especially in high-impact journals. this pattern is of concern given the role that high-impact journals have in disseminating new evidence to academic and clinical audiences as well as to the public through knowledge translation activities that follow publication, including both traditional (newspaper, television and radio) and social (twitter, blogs) media outlets. obesity research is not immune to this tendency. recently, we completed an online search of original manuscripts published from january to december in five obesity journals (childhood obesity, clinical obesity, international journal of obesity, obesity, and pediatric obesity). of the total number of papers published (n = ), qualitative reports comprised . % (n = ). we also reviewed the authorship guidelines for all five journals and found no explicit statements regarding the exclusion of qualitative research or specific preferences for quantitative research, although some details (for example, testing hypotheses; including controls) were applicable to quantitative study designs only. a search of bibliographic databases including pubmed and scopus with obesity and qualitative research as key words yields hundreds of publications per year over the past several years, so a low total volume of qualitative studies related to obesity may not be a primary factor for the under representation of qualitative reports in obesity journals. that said, the poor quality and novelty of qualitative manuscripts submitted to obesity journals may be an issue; however, the extent to which this factor has a role is difficult to determine given that details regarding editors' and reviewers' familiarity and expertise in qualitative research are required along with the criteria used to gauge manuscript quality and appropriateness. as health research has been predominantly quantitative, the low proportion of qualitative studies published in obesity journals may not relate to poor quality, but to a lack of understanding, making it difficult for editors and reviewers to judge the value and quality of qualitative reports. the tension between qualitative and quantitative research approaches and different underlying epistemologies have been documented. in our experience leading qualitative, obesity-related research with clinical and health services foci, we have gained some experience in addressing potential challenges with publication. for instance, the submission and resubmission processes provide opportunities for authors to include additional rationale for key methodological decisions, especially in relation to issues including sample size, hypothesis testing, reliability of coding, data saturation and generalizability of findings. reviewers of our manuscripts have, more often than not, appreciated our explanations. this experience highlights the value of describing differences and addressing potential misperceptions between quantitative and qualitative research during the peer-review process. in addition, the inclusion of quality assessment and reporting checklists (for example, , ) that accompany our manuscript submissions has allowed us to provide a better understanding of the design, implementation, analysis and implications of our research. using checklists to explain methodological and reporting details of qualitative studies may also benefit from a halo effect as it is consistent with many journal requirements for quantitative research. we have also been flexible in accommodating editors' and reviewers' recommendations to edit our manuscripts in circumstances when changes have not compromised the rigor of our qualitative research, but may not necessarily be consistent with the original intent of the research method (for example, including frequency data in thematic analyses). we also believe that several steps can be taken to enhance the presence of qualitative research in obesity research. first, consistent with the approaches taken by other organizations (for example canadian obesity network; obesity action coalition), the inclusion and participation of individuals living with obesity at academic meetings can enable conversations about obesity in a manner that is less stigmatizing and biased, encouraging research (qualitative and quantitative) that is relevant to people living with obesity and providing a forum for their perspectives to inform research. second, obesity journals can establish dedicated sections to highlight excellence in qualitative research, an approach that has been used to organize other research areas (for example, clinical trials and investigations; epidemiology/genetics). finally, the inclusion of explicit instructions within authorship guidelines for obesity journals can highlight the range of research considered for publication, which can include requiring applicable reporting checklists and be accompanied by the inclusion of scientists, clinicians, and administrators at all stages of the peer-review process who possess methodological expertise in both quantitative and qualitative research. the publication of qualitative research has been positively linked with journals' policies, authorship guidelines, and editorial content, so a proactive approach can be beneficial. collectively, these steps can encourage more inclusive discussions about obesity as well as provide academic venues for publishing and disseminating research of greater epistemological breadth and relevance. qualitative research: adding drive and dimension to clinical research how do you modernize a health service? a realist evaluation of wholescale transformation in london is qualitative research second class science? a quantitative longitudinal examination of qualitative research in medical journals paucity of qualitative research in general medical and health services and policy research journals: analysis of publication rates journal prestige, publication bias, and other characteristics associated with citation of published studies in peer-reviewed journals using quantitative and qualitative data in health services research -what happens when mixed method findings conflict? bmc the elephant in the living room: or extending the conversation about the politics of evidence critical appraisal skills programme: making sense of evidence consolidated criteria for reporting qualitative research (coreq): a -item checklist for interviews and focus groups this work is licensed under a creative commons attribution . international license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material key: cord- -j ruj authors: dreyfuss, didier title: is it better to consent to an rct or to care?: Μηδεν αγαν (“nothing in excess”) date: - - journal: intensive care med doi: . /s - - - sha: doc_id: cord_uid: j ruj nan twenty-five years ago the belmont report [ ] established a formal distinction between care and research in order to protect patients. the legitimate fear that research might be conducted under the pretence of medical care regardless of whether this increased the risk to patients drove this effort toward clarification. a sound ethical foundation to medical research was deemed essential after the heinous nazi experiments and the abhorrent -year-long tuskegee syphilis study [ ] . at the time that the belmont report was issued randomized clinical trials (rcts) were coming into vogue. an rct is not routine clinical care, even when the treatments in both study arms are consonant with standard practice. thus both the ability of rcts to produce high-quality medical knowledge and the distinction between care and research were considered of paramount importance at the time of the belmont report [ ] . whether research should be incorporated into medical care remains controversial, however, as reflected by several recent and conflicting statements of opinion [ , , ] . i argue here that things have changed since the belmont report, and that a formal distinction between care and research may no longer serve medical or ethical principles in many situations, most notably in the area of critical care. this seemingly provocative position stems from three arguments. first, a large part of the "research" conducted in icu patients consists in evaluating practices or comparing two widely used procedures or treatments [ ] . second, the scientific value and the historical role of rcts may have been overemphasized and their socioeconomic impact misinterpreted. third, analysis of the informed consent process, a mandatory preliminary intended to ensure the autonomy of patients included in rcts, suggests that the cure may be worse than the disease. although informed consent was a major stride towards protecting the rights and dignity of patients, the process may in some instances be perverted into protecting the physicians rather than the patients. the tragic jesse gellsinger case ("teen dies undergoing experimental gene therapy," washington post, september ) reminds us that "informed consent" is not sufficient to protect patients during research [ ] . the informed consent process has many flaws stemming from the frequent complexity of consent forms [ ] , poor comprehension of information by many families who must draw on their limited scientific culture to unravel complicated issues at a time when they are struggling with severe anxiety about their loved one [ ] , and dearth of communication skills among physicians. thus, informed consent to research may be stressful for families and reassuring for physicians. this was not its original purpose. to overcome these problems a reform allowing informed consent to be waived under specific circumstances has been suggested [ ] . although the opinions expressed by the advocates of this approach are worthy of respect, they conflict with the principle of patient autonomy [ ] . a similar wish is expressed by some european physicians who seem very anxious about the putative threat on medical research in emergency that a new european directive supposedly poses [ , , ] . indeed, this directive [ ] does not allow the waiving of consent by proxies. another contention of the present paper is this [ ] : critical care physicians may still believe that rcts remain the best tool for improving knowledge and care, and in this case they must accept to use the means needed to achieve the end and therefore to insist on mandatory informed consent from the patient or proxy; or they may realize that the game is not worth the candle and they must then turn to other forms of research that are ranked less highly in the pyramid of evidence-based medicine [ ] . in so doing they give priority to the well-being of families and must seek the help of innovative methodologists who accept to deal with the real world. before discussing the problem of informed consent to research a critical appraisal of the scientific and ethical validity of rcts in critical care medicine is in order. the role of rcts was overemphasized from the outset many critical care physicians believe in the superiority of rcts over other types of clinical studies. this belief may not be supported by the evidence. before discussing objective data on the value of rcts in critically ill patients it is important to recall a number of historical facts. rcts emerged in the s as a tool for overcoming the drawbacks of anecdotal experience. although rcts initially met with considerable resistance, they undoubtedly contributed to making clinicians aware of the need for rigorous methods. however, the data obtained proved only that rcts are feasible, not that they are superior over other forms of well-conducted studies. this is best illustrated by the trials of streptomycin in tuberculosis in the united kingdom and of the salk polio vaccine in the united states. streptomycin was discovered in in the united states. the british government could not afford to purchase streptomycin for all patients with tuberculosis, and the effectiveness of the drug was not yet established. the words of d'arcy hart [ ] , a member of the medical research council scientific staff that conducted the streptomycin trial, are worth quoting: "the trial proceeded from . the small amount of streptomycin available made it ethically permissible for the control subjects to be untreated by the drug-a statistician's dream." this gives the two reasons for the trial: fair allocation of a limited resource and the determination of a brilliant statistician, sir austin bradford hill, to prove the validity of rcts. d'arcy hart [ ] further stated: "secondly, the trial heralded the general conversion of clinical scientists to randomisation." shortly after this brilliant demonstration of the feasibility of rcts the salk polio vaccine trial was interpreted as a triumph of the new rct method in the united states. the unique circumstances surrounding the vaccine trial deserve mention [ ] : there was a raging scientific controversy between salk and sabin regarding the type of vaccine that should be developed (salk was developing a killed virus vaccine, whereas sabin thought that only an attenuated living strain could be a suitable vaccine), and salk was under considerable pressure to perform the trial, both to stop the polio epidemic that was costing so many lives among children and to win the race against sabin. for the trial children in a number of states were randomly allocated to receive the vaccine or a placebo. the results proved the vaccine dramatically effective. this both improved medical care and propelled the rct to prominence as the gold standard design for clinical research. however, an observational study was conducted in states that refused the placebo-controlled trial [ ] . the results were similar to those of the rct, but only the results from the rct were considered statistically valid [ ] . interestingly, salk described the placebo-controlled study of his vaccine as "a beautiful... experiment over which the epidemiologist could become quite ecstatic but (which) would make the humanitarian shudder" [ ] . it seems that this rct was driven by statisticians rather than by clinicians, in order to counter sabin's criticism and gain the support of the leaders of the medical community [ ] . in brief, rcts were performed because they could be performed, not because they had been proved superior over other study designs. the same may still hold true in critical care research today. what is the socioeconomic role of rcts? henry k. beecher, a professor in anesthesia research at the massachusetts general hospital commenting years ago on the huge increase in funds for research, said "there is reason to fear that... these resources may be greater than the supply of responsible investigators," and "every young man knows that he will never be promoted to a tenure post, to a professorship in a major medical school, unless he has proved himself as an investigator" [ ] . these two sentences acknowledge an uncomfortable reality: money and careers are at the center of clinical research. the importance of rcts for drug companies large rcts are vital for drug companies. these trials are mandatory not only for proving the efficacy of new products but also for obtaining regulatory approval and marketing licenses for drugs. thus drug companies have good reason to argue that rcts offer unequalled methodological purity. however, drug company sponsored trials of both hematology treatments and nonsteroidal anti-inflammatory drugs usually showed superiority of the new drug [ ] . this finding obviously violates the principle of equipoise, under which one would expect only one-half of the studies to find better outcomes with the new drug [ ] . a similar bias has been reported with other studies funded by the pharmaceutical industry and ascribed to the use of an inappropriate comparator or to publication bias [ ] . the importance of rcts for medical journals although the editors of major medical journals have recently warned against the threat to objectivity posed by some forms of industry-sponsored research [ ] and stated that "the use of clinical trials primarily for marketing makes a mockery of clinical investigation," the potential for disseminating pharmacological breakthroughs makes them likely to accept most of the industry-sponsored trials. the recent controversy on the efficacy and safety of activated protein c in sepsis highlights the difficulties faced by editors in this area [ , ] . the ties that link drug companies to investigators and to prestigious academic centers pose a worrisome threat to academic medicine [ ] . money from advertising may also weigh on editorial policies. methodologists and statisticians are consulted by drug companies and by independent investigators at the studydesign stage and by editors at the peer-review stage. they developed the rules of "methodological validity," and these rules are likely to be the same at each step of the design and publication of a clinical trial. the grading system for the quality of evidence from clinical research, with rcts at the top, is akin to a self-fulfilling prophecy: young researchers and renowned experts alike comply with these "golden" rules to ensure publication of their findings in a prestigious journal. this compliance with artificial rules is taken as firm evidence of validity, thus spinning the wheel endlessly. as stated by knottnerus and dinant [ ] , "finally, in using strict criteria in reviewing manuscripts for publication, we should worry about risk avoidance by clinical researchers. they might focus their energies on topics where the methodological criteria of reviewers and editors can be most easily met, rather than studying real life clinical problems which present substantial methodological problems." the adage "publish or perish" still applies. all these interests shared by drug companies, investigators, methodologists, and journals concur both to overproduction of rcts and to overestimation of their contribution to medical progress. rcts tend to become an uncontrolled activity driven by forces foreign to scientific goals. this results in an inextricable tangle of so-called evidence. then, meta-analyses are performed, supposedly to clarify an issue that has been artificially obscured. they may merely add to the confusion, as discussed below. how useful are rcts in critically ill patients? the validity of conclusions of earlier studies on hepatitis and cirrhosis has been evaluated by poynard and coworkers [ ] under the provocative title of "truth survival in clinical research: an evidence-based requiem." in this study the -year survival rate of conclusions derived from meta-analyses was lower ( %) than that from nonrandomized studies ( %) or rcts ( %). more importantly, the truth survival rate was similar for studies of high and low methodological quality. examination of three important areas of critical care further indicates that challenging the usefulness of rcts is not necessarily sacrilegious. the acrimony of the debate on these three topics in medical journals and at international meetings is a strong indicator that rcts fail to provide the "definitive" answer expected from them. these three topics are mechanical ventilation in adult respiratory distress syndrome (ards), selective digestive decontamination (sdd), and prevention of gastrointestinal bleeding. controversy erupted after publication of the findings of the ards network study on tidal volume reduction during ards. although this remarkable work showed that a low tidal volume of ml/kg predicted body weight resulted in better outcomes than a higher tidal volume of ml/kg [ ] , it did not tell us how to ventilate these patients. decreases in mortality [ ] and tidal volume [ ] over time occurred well before the study was initiated. patients are usually ventilated with tidal volumes that are intermediate between the two arms of the ards network trial (and probably closer to the lower volume). there is no rct telling us whether ml/kg is better than the - ml/kg generally reported in international surveys [ , ] . the results of this trial will encourage clinicians to use smaller tidal volumes, a practice that has not yet gained sufficient acceptance [ , , ] . in that sense this rct will prove useful, but not more useful than the earlier experimental, physiological, and nonrandomized clinical trials that resulted in the use of gradually decreasing tidal volumes over time [ ] . it is merely one more brick in the wall, and not a gold one. similarly, the results of the recent alveoli randomized study that compared two peep levels will probably not change current practice [ ] . in this study peep levels higher than the moderate values used in most surveys [ , ] did not improve patient mortality. finally, will the lack of effect of prone positioning on mortality in rcts [ ] discourage clinicians from using this very inexpensive and effective maneuver to improve patient oxygenation, and will these clinicians continue to await an rct providing "proof" of efficacy [ ] ? ards mortality rates have decreased substantially over the years [ ] and are probably declining further still, yet this improvement is ascribable not to rcts but to patient-oriented research based on sound physiological thinking [ , , , ] . selective digestive decontamination meta-analyses of rcts indicate a clear survival advantage with sdd [ , ] . however, sdd is seldom used because of the legitimate fear that this practice may promote the emergence of resistant bacteria [ , ] . how many studies will be needed to convince clinicians to use a method they do not want to use? or shall we wait until the "final" meta-analysis is "negative" and "proves" that clinicians were right when they refused to use sdd despite the accumulation of so-called evidence? few fields in critical care have generated as many rcts and meta-analyses. a recent meta-analysis concluded that sucralfate and ranitidine failed to prevent gastrointestinal bleeding in critically ill patients [ ] . the authors noted that a previous meta-analysis [ ] found reduced bleeding rates with h antagonists but included several positive trials of cimetidine, which has since then been superseded by drugs with better safety profiles. in addition, the use of proton-pump inhibitors seems to be increasing in critically ill patients, although there is no proof that this practice is beneficial. finally, no one knows whether prophylaxis should be given, and this uncertainty has recently been exacerbated by an observational study in , patients showing no difference in bleeding rates between a cohort of patients given prophylaxis and a subsequent cohort not given prophylaxis at the same institution [ ] . additional rcts may be needed if the obsessive goal is to discover the illusive "truth," but their drawbacks should be weighed against their utility. this paper does not claim that all rcts are useless in critically ill patients. rcts may be helpful for evaluating a single and simple intervention (even if this intervention is technologically sophisticated) in patients with a welldefined disease. this is obviously the case for acute coronary syndromes. however, many conditions seen in icu patients stem from extraordinarily complex pathophysiological mechanisms that preclude simple trial designs and interpretations [ ] . a typical example is the patient with ards and septic shock, multiple indwelling catheters, and a high risk of nosocomial respiratory and systemic infections. it is difficult to conceive of a single therapeutic intervention capable of improving such a complex situation. a further obstacle to studies on such an intervention is the highly heterogeneous nature of the icu population. these issues relate to the internal and external validity of a trial. methodologists seek to maximize the internal validity of rcts to decrease the effects of confounders. however, as internal validity increases, external validity (i.e., generalizability) decreases [ ] . this problem may exist for the ards network trial on tidal volume reduction [ ] . indeed, because extremely stringent inclusion criteria were used, only % of ards patients admitted to the participating centers were included in the trial [ ] . what ethical problems do rcts raise in critically ill patients? some rcts may conflict with currently accepted principles of medical ethics [ ] : beneficence, nonmaleficence, autonomy, and justice [ , ] . in addition, rcts may conflict with the principle that what is not scientific is not ethical. it is of course difficult if not impossible to determine a priori that a research protocol has a favorable risk-benefit ratio. however, rcts should rest on a foundation of strong experimental or clinical concepts. this may not have been the case in all instances, most notably in studies of new treatments for sepsis [ ] . without seeking to fuel the debate on the failure of antimediator agents in sepsis, one cannot but wonder whether the huge financial and academic stakes were in part responsible for the apparent haste with which some trials were conducted. in addition, the quality of research oversight in several trials with high mortality rates has been challenged [ ] . it is difficult to ensure that the prerequisites for beneficence and nonmaleficence are met when there is a major influence of financial incentives and academic competition. thus we still encounter problems similar to those met by the salk vaccine trial: the process of virus inactivation was not fully mastered at all the vaccine production sites, and therefore active virus was inoculated into a number of children in whom poliomyelitis developed (http:// www.pbs.org/wgbh/aso/databank/entries/dm sa.html, accessed july ; http://www.polio-vaccine.com/fr/ histoire/vaccins_experience.html, accessed july ). another aspect of beneficence and nonmaleficence that does not seem well addressed during the conduct of rcts concerns proxies and are discussed below. most critically ill patients are too ill to deal with issues of consent. consent is therefore sought from a surrogate in the united states and most european countries. consent by a surrogate is usually considered the best means of protecting patients during research [ , ] , although studies have shown that the decisions made by surrogates do not always reflect the wishes of patients [ , ] . however, some states in the united states either do not accept surrogate consent for research or authorized this form of consent only after the end of the ards network study [ ] . in addition, mistrust is gaining ground in the public at large, and organizations such as the alliance for human research protection (ahrp), whose stated goal is to ensure that human rights are protected during research (http://www.researchprotection.org, accessed july ) , are opposed to surrogate consent. the ahrp contributed to drive the inquiry of the office for human research protection on the ards network trials [ ] . surrendering the principle of autonomy to the principle of beneficence is ethically acceptable only when there is a reasonable certainty of nonmaleficence. this degree of certainty is not usually obtainable, as discussed above. tremendous amounts of money are invested in clinical research, in principle in the best interest of patients. the above words of beecher [ ] on the discrepancy between the abundance of funds and the scarcity of responsible investigators deserve careful consideration. financial resources for healthcare and for research are limited [ ] , and their fair allocation is both a political and an ethical imperative. because rcts are far more expensive than observational studies [ ] , they should provide answers that cannot be given by observational studies. although the impressive work conducted by the ards network investigators is worthy of respect, its failure to achieve this goal must be acknowledged. millions of dollars were spent [ ] , but, as discussed above, this study [ ] is merely one among several (including physiological and observational studies [ ] ) showing that patients should be ventilated with less than ml/kg body weight. in addition, it failed to determine whether volumes smaller than the - ml/kg noted in observational studies should be used [ , ] . similarly, the alveoli trial [ ] randomized a large number of patients but simply ruled out a need for peep levels higher than those in observational studies [ , ] . what have these multimilliondollar trials contributed? in the words of the belmont report [ ] , "the term 'research' designates an activity designed to test a hypothesis, permit conclusions to be drawn, and thereby to develop or contribute to generalizable knowledge." it is usually assumed that a nonscientific trial is unethical. but how generalizable are the data generated by rcts with excellent internal validity but limited external validity? as mentioned above, what makes the greatest contribution to clinical practice regarding the prevention of gastrointestinal bleeding in icu patients: "discordant" metaanalyses of multiple rcts [ , ] or a well conducted cohort study [ ] ? the informed consent dilemma this is a major ethical issue. as brilliantly discussed by several authors, informed consent by a proxy is a key safeguard for patients eligible for clinical trial inclusion [ , , ] . however, proxies may not consistently make the same choices as the patients or correctly interpret their best interests. in addition, the consent process can impose considerable suffering on the proxy. however, investigators are encouraged to disseminate the results of their work to the study participants [ ] , which is laudable. after the death of a loved one, a proxy might learn, for instance, that he or she consented to a clinical trial in which mortality was higher in the treatment arm. in that sense informed consent probably protects the icu patient and the physician in charge of the research project but not the proxy who is asked to provide consent. anxiety in patients asked to give their "full informed consent" to a study of a life-threatening disease has received attention [ ] . it has been rightly pointed out that this form of consent may be needlessly cruel [ ] . the same may hold true of "full informed consent" given by a proxy for a loved one. everyday practice teaches that proxies are highly vulnerable to distress and guilt when they are asked to provide consent to care for a critically ill patient (e.g., to a high-risk therapeutic procedure in a desperately ill patient). families of icu patients often exhibit major signs of anxiety and depression [ ] . the information that they receive in the name of the principle of autonomy may conflict with the principles of beneficence and nonmaleficence when proxies are asked to consent to research rather than to care. the primary focus of informed consent is risk disclosure [ ] , and the informed consent dilemma can be summarized as follows: if the consent form is to be reassuring for the family, it must fall short of providing honest information, and if the consent form is to be honest, as commendably proposed by the ards network investigators [ ] , it must supply information that is distressing to the family. surprisingly, most physicians fail to recognize that evaluation of the riskbenefit ratio, considered a requisite for ethical research [ ] , should not focus solely on the patient. the emotional risk to proxies should be balanced against the putative benefit (and risk) for the patient included in the rct [ ] . otherwise, proxies may be subjected to stress whose harmful effects exceed by far the objective benefits the patient may derive from participating in the study. does it make ethical sense to distress family members by asking them to consent to yet another study on sdd or on the prevention of gastrointestinal bleeding? informed consent documents and information sites for families of critically ill patients are sometimes so frightening that perhaps the door to the icu should bear the words inscribed on the gate to hell in dante alighieri's inferno (la divina commedia): "lasciate ogni speranza, voi ch'entrate!" ("abandon all hope, ye who enter here!"). two categories of solutions may be considered. some require no important qualitative changes in the system that governs clinical research but simply a tightening of research oversight procedures [ , , ] . other solutions would require a reshaping of many parts of the system, including the financial and academic incentives to publication, as well as a number of methodological dogmas. these two categories of solutions are not mutually exclusive. they both seek the best compromise between conflicting principles to protect the rights of patients and proxies and to improve scientific knowledge and quality of care, but not at the expense of scientific or ethical distortions. solutions that do not require significant changes in the system the protection of subjects who are unable to give or refuse consent must receive particularly close attention. there is little doubt that waiving of the requirement for consent should be reserved for highly unusual situations [ ] , which are described in detail in the united states code of federal regulations for the protection of human subjects [ ] . this text allows some forms of research in emergency situations without consent from the patient or surrogate provided certain conditions are met, including: the disorder is immediately life-threatening, available treatments are unsatisfactory, obtaining consent is not feasible, the research could not be carried out without the waiver, participation in the study holds the prospect of direct benefit, and the waiver of consent is given by an institutional review board. the situation is less clear in europe where research in emergency medicine can be performed without consent in some countries (including france) but not in others [ ] . however, the european parliament and council have issued a new directive that forbids research without consent, even in emergencies [ ] . this directive has been criticized as a serious potential threat to research in emergency situations [ , , , ] . before examining the magnitude of this threat, one must acknowledge the risk of overuse or abuse of waiving consent for emergency research in critically ill patients [ , ] . as recently argued by john luce, "few patients face true emergencies.... for example... studies of new modes of mechanical ventilation, novel therapies for sepsis... have a relatively long therapeutic window during which obtaining consent from patients or surrogates may be possible" [ ] . for research on true emergencies (e.g., treatment of cardiac arrest, acute brain injury) in the european union the directive and/or the research modalities will have to be modified, as discussed below. a strong argument that waiving consent may be appropriate for some rcts was put forward by truog and coworkers [ ] . they base their position on the frequently poor comprehension of the rct process and of informed consent documents by patients or surrogates. a waiver of consent could be obtained from an institutional review board provided the treatments offered in the trial are available outside the trial without the need for consent, the study carries only minimal additional risk, genuine equipoise exists among the studied treatments, and no reasonable person should have a preference for one treatment over any other. in the letters published in response to this thoughtful paper, the risk of jeopardizing patient autonomy was the principal argument against the contention by truog et al. [ ] . interestingly neither truog et al. nor their detractors questioned the validity of the opinion that governed their debate, namely, that rcts are useful under these special conditions. this is discussed below. solutions that require significant changes in the system from the above it clearly appears that the debate on informed consent to research stems primarily from two axioms that can be questioned: care and research are two separate activities, and rcts are superior to other forms of clinical research. investigators should acknowledge that they cannot have their cake and eat it too: they cannot both enjoy the putative methodological advantage of an rct and carry out their study without obtaining informed consent. consent is inherent in the rct process because it is needed to ensure compliance with basic ethical principles, most notably the principle of autonomy. except in the rare cases of true emergencies (see above), there is no obvious ethical justification of waiving consent to rcts. therefore if we want critical care to continue its amazing progress, we must rethink our research policies. first, the limited role (if any) of rcts in this progress must be acknowledged. second, the formal distinction between care and research must be reappraised. third, current methodological dogma must be challenged. as stated by miller and rosenstein [ ] in an article focusing mainly on rcts, "medical care is characterized by a convergence of the doctor's interests and the patient's interests.... by contrast, in clinical trials, the principal interests of the investigator and the participating patient may diverge." as mentioned above, the distinction between care and research was first made in the belmont report [ ] and should not be dismissed except in specific circumstances. critical care may be one of these circumstances, given the consensus that "there is instead a spectrum that extends from established, evidence-based interventions through unproved therapeutic innovations to formal rcts", as underlined by truog and colleagues [ ] in their response to the abundant correspondence generated by their publication [ ] . it is important to bear in mind that most if not all of the debate on the therapeutic misconception concerns the distinction between care and rcts [ , ] . this distinction is obviously valid. however, clinical research can also have an integral role in clinical care [ ] , most notably when the interventions are not allocated at random. this offers an opportunity for reconciling the interests of the patients and those of the physicians, provided adequate methodological changes are implemented. vandenbroucke and de craen [ ] wrote that "sometimes we accept the evidence from the randomized trial and overturn a theory-however beautiful it was-but at other times we stick with the theory and dismiss the evidence." there is no inviolable scientific reason to prefer rcts and their mandatory informed consent procedure to the well-being of patients and their proxies. there is, however, a moral obligation to improve the quality of critical care. therefore alternative methodological approaches that protect both the welfare and the autonomy of patients should be given preference. these approaches should "find ways of accommodating clinical reality, not ignoring it" [ ] , and should require acknowledgement that rcts can produce inconsistent results and can have limited external validity [ , ] . investigators will have to stop their obsessive quest for the so-called "absolute truth that can be given only by rcts" and acknowledge the subjective element in the evaluation of science [ ] . as underlined by jerome cornfield (inventor of both the odds ratio and logistic regression; cited in [ ] ), "good scientific practice... places the emphasis on reasonable scientific judgement and the accumulation of evidence and not on dogmatic insistence of the unique validity of a certain procedure." it is astonishing that physicians pressure institutional review boards (irbs) to accept waivers of consent and zealously lobby for changes in regulations that they feel may "impede research" [ , ] without questioning the validity of the diktats issued by a number of methodologists and journal editors. the real problem is not to obtain a waiver of consent to rcts from patients or proxies: the consent that we need is that of methodologists, from whom we seek creative study designs, and of medical journal editors, from whom we ask for greater openness to contributions that are less highly ranked in the pyramid of evidence-based medicine [ ] . trials with prerandomization. this design was introduced by zelen [ ] . patients are randomly preallocated to the conventional or new treatment before they are asked to consent to the study. in the patients allocated to the control arm no specific consent to research need be obtained. in contrast, informed consent is sought from the patients (or proxies) in the group allocated to the new treatment; when consent is refused, the patient receives the conventional treatment. this appealing design requires unblinded treatment administration, which is the case in many trials in icu patients (most notably on procedures). it has been used in pediatric trials of extracorporeal membrane oxygenation [ , ] . prerandomization may increase patient inclusion rates. studies have calculated the "price of autonomy," that is, the number of lives that may be lost if the inclusion rate is slower because prerandomization is not used, resulting in delayed implementation of a life-saving treatment [ , ] . however, ethical objections to prerandomization [ ] include denial of information, using people, denial of choice, and "overselling" of allocated treatments [ ] . observational studies. well-conducted observational cohort and case-control studies can provide the same level of internal validity as rcts [ , , ] . they are particularly well suited to research in the icu. indeed, as mentioned above, blinding is neither necessary nor feasible for most therapeutic interventions in the icu. in fact most of the major recent rcts were unblinded [ , , , , , , ] . why not evaluate new therapeutic interventions sequentially under conditions of genuine equipoise? in such conditions, rather than a detailed explanation of the randomization process, "such an elegant, reliable, sophisticated concept to the research clinician, but so brutal and harsh from the patient's view point" [ ] , only consent to general care [ ] and to the use of data obtained during usual patient care for research purposes [ ] would need to be obtained. renunciation of rcts in favor of observational studies does not imply that physicians have a free hand on their patients. irb approval and close monitoring of data quality and patient safety would still clearly be needed. in addition, cohort studies are probably the only option left for emergency research when legislation prohibits the waiving of consent, as may unfortunately become the case in the european union within the next few years [ , ] . interestingly, this approach was used in a recent study of thrombolytic therapy after initially unsuccessful cardiopulmonary resuscitation [ ] . the cohort design deserves to be considered before the alarm is sounded for the questionable reason that research in emergencies may stop if rcts are no longer feasible [ , ] . these are among the most dangerous studies, as recently shown by the results published for several new drugs in icu patients [ ] . furthermore, by definition there is a risk of major conflicts of financial interest. it follows that patient protection should receive particularly careful attention, and that the studies must be completely free of potential methodological weaknesses and ethical flaws. clearly such studies must be randomized (which is mandatory anyway for obtaining regulatory approval). in addition to a thorough review of the research protocol by a completely independent irb, unblinded monitoring of adverse effects should be conducted, as recently suggested by freeman and coworkers [ ] . finally, the informed consent document should provide detailed information and receive careful scrutiny by the irb. for instance, a fair informed consent document on new therapies for sepsis should explain what physicians know, namely, that the pathophysiology of sepsis is incom-pletely understood, and that the animal models on which the clinical trial is based are not fully valid [ ] . in addition, if another new treatment was associated with increased mortality rates in other studies [ ] , this fact should be disclosed. finally, the amount of money received by the physicians or their institution per patient included should be indicated, as well as any financial interests linking the physicians to the drug company (e.g., shares owned or position as paid consultants), as suggested by organizations such as the alliance for human research protection (http://www.researchprotection.org/informedconsent/informedconsent.html, accessed july ). patient autonomy should not stop where potential financial profit begins. as pointed out by luce [ ] , stronger research oversight may be as important as informed consent in protecting patient welfare. since we contend that, provided investigators desist from performing rcts, formal consent to research need not be sought and consent to care and to the use of data is the rule, it also holds that research oversight must be reinforced [ , , , ] . research projects that do not require specific informed consent should be examined thoroughly by irbs, which should obtain the opinions of independent consultants if needed. the primary goal is not to make research easier for physicians but to increase the safety of patients and proxies. if greater ease of research occurs as a side effect, this will be welcome. in conclusion, rcts were born under a shroud of original sin consisting of financial, political, and academic pressure. this was summarized by yoshioka [ ] in a publication about the medical research council trial on streptomycin: "the innovation of centrally controlled randomisation can be attributed to a combination of scientific logic and political and social pressures on medical bureaucracy." this sin may have remained unredeemed, as suggested by the extraordinary controversy about the ards network study [ , ] . before deciding which clinical study design is best suited to critically ill patients, consideration should be given to several points: -many rcts in critical care generated heated pro-con debate during medical conventions yet failed to improve patient care. -respect for patients and their families requires that investigators refrain from using a plethora of informed consent documents which constitute a perversion of ethical principles but rather wield this two-edged sword with discernment. -clinical trials should be conducted not to achieve methodological purity but to improve patient management. -in clinical trials the best possible compromise should be sought between the ethical principle of beneficence to the patient and that of nonmaleficence to the proxy, who is asked to give consent while struggling with overwhelming distress. -clinical trials should be appraised according to the ethical principle of distributive justice: what cost for what result? -society is changing and may no longer be ready to accept what many persons may consider, rightly or wrongly, to be a manifestation of medical power. the effects of the aids epidemic on patient mentalities and the growing influence of organizations such as ahrp should be pondered. similarly, owing to widespread public concern about the adequacy of protection for human research subjects, litigation against investigators, irbs, and academic institutions is becoming increasingly common [ ] . then, nolens volens, clinicians may have to forbear conducting rcts for ethical, scientific, sociological, legal, and financial reasons. one solution may consist in giving preference to forms of clinical research that are tightly linked to care. clinicians should work closely with innovative methodologists to find new designs that are acceptable to all. clinicians serve patients. and methodologists serve clinicians, not the opposite. national commission for the protection of human subjects of biomedical and behavioral research ( ) belmont report: ethical principles and guidelines for the protection of human subjects of research. united states government printing office unraveling the tuskegee study of untreated syphilis the therapeutic orientation to clinical trials the integral role of clinical research in clinical care is informed consent always necessary for randomized, controlled trials? what makes clinical research ethical readability standards for informed-consent forms as compared with actual readability symptoms of anxiety and depression in family members of intensive care unit patients: ethical hypothesis regarding decisionmaking capacity is informed consent always necessary for randomized, controlled trials? non-therapeutic research in the eu in adults incapable of giving consent? a european directive for clinical research implications of the eu directive on clinical trials for emergency medicine directive / /ec of the european parliament and of the council to consent or not to consent, that is (not) the (sole) question evidence-based medicine. a new approach to teaching the practice of medicine a change in scientific approach: from alternation to randomised allocation in clinical trials in the s a calculated risk": the salk polio vaccine field trials of essai clinique. in: lecourt d (ed) dictionnaire de la pen-sØe mØdicale ethics and clinical research alternative medicine: a "mirror image" for scientific reasoning in conventional medicine pharmaceutical industry sponsorship and research outcome and quality: systematic review sponsorship, authorship, and accountability risks and benefits of activated protein c treatment for severe sepsis assessing the use of activated protein c in the treatment of severe sepsis is academic medicine for sale? medicine based evidence, a prerequisite for evidence based medicine truth survival in clinical research: an evidence-based requiem? ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome improved survival of patients with acute respiratory distress syndrome evidence-based medicine or fuzzy logic. what is best for ards management? characteristics and outcomes in adult patients receiving mechanical ventilation: a -day international study epidemiology and outcome of acute lung injury in european intensive care units. results from the alive study impact of randomized trial results on acute lung injury ventilator therapy in teaching hospitals are we really reducing tidal volume-and should we? barriers to providing lung-protective ventilation to patients with acute lung injury higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome effect of prone positioning on the survival of patients with acute respiratory failure intermittent positive-pressure hyperventilation with high inflation pressures produces pulmonary microvascular injury in rats improved prognosis of acute respiratory distress syndrome years on searching for evidence: don't forget the foundations effectiveness of antibiotic prophylaxis in critically ill adult patients: systematic review of randomised controlled trials selective digestive decontamination: for everyone, everywhere? selective decontamination of the digestive tract and its effect on antimicrobial resistance prevention of hospital-associated pneumonia and ventilator-associated pneumonia bleeding and pneumonia in intensive care patients given ranitidine and sucralfate for prevention of stress ulcer: meta-analysis of randomised controlled trials stress ulcer prophylaxis in critically ill patients. resolving discordant meta-analyses clinically significant gastrointestinal bleeding in critically ill patients with and without stress-ulcer prophylaxis clinicians' approaches to mechanical ventilation in acute lung injury and ards the price of autonomy principles of medical ethics why immunomodulatory therapies have not worked in sepsis safeguarding patients in clinical trials with high mortality rates is the concept of informed consent applicable to clinical research involving critically ill patients? ethical considerations for research in critically ill patients do surrogate decision makers provide accurate consent for intensive care research? california's new law allowing surrogate consent for clinical research involving subjects with impaired decision-making capacity suspension of the nih ards network fluids and catheters treatment trial control group selection in critical care randomized controlled trials evaluating interventional strategies: an ethical assessment beyond randomised versus observational studies how best to ventilate? trial design and patient safety in studies of the acute respiratory distress syndrome informing clinical trial participants about study results bmj's present policy (sometimes approving research in which patients have not given fully informed consent) is wholly correct fully informed consent can be needlessly cruel influence of the law on risk and informed consent recommendations for informed consent forms for critical care trials. abstracts improving protection for research subjects protecting subjects with decisional impairment in research: the need for a multifaceted approach protection of human subjects: informed consent and waives of informed consent requirements in certain emergency research. final rules. title , code of federal regulations the european union directive and the protection of incapacitated subjects in research: an ethical analysis the effect of waiving consent on enrollment in a sepsis trial is informed consent always necessary for randomized controlled trials? a new design for randomized clinical trials extracorporeal circulation in neonatal respiratory failure: a prospective randomized study extracorporeal membrane oxygenation and conventional medical therapy in neonates with persistent pulmonary hypertension of the newborn: a prospective randomized study informed consent. numbers inform the debate zelen randomization: attitudes of parents participating in a neonatal clinical trial should zelen prerandomised consent designs be used in some neonatal trials a comparison of observational studies and randomized, controlled trials randomized, controlled trials, observational studies, and the hierarchy of research designs a multicenter, randomized, controlled clinical trial of transfusion requirements in critical care. transfusion requirements in critical care investigators, canadian critical care trials group a trial of goal-oriented hemodynamic therapy in critically ill patients. svo collaborative group early goal-directed therapy in the treatment of severe sepsis and septic shock intensive insulin therapy in the critically ill patients improving the process of informed consent in the critically ill patients' consent preferences for research uses of information in electronic medical records: interview and survey data efficacy and safety of thrombolytic therapy after initially unsuccessful cardiopulmonary resuscitation: a prospective clinical trial ethics, science, and oversight of critical care research: the office for human research protections use of randomisation in the medical research council's clinical trial of streptomycin in pulmonary tuberculosis in the s metaanalysis of acute lung injury and acute respiratory distress syndrome trials testing low tidal volumes the rise of litigation in human subjects research key: cord- - viikdbf authors: bucher, adrian; collins, andrew; heaven taylor, ben; pan, david; visman, emma; norris, james; gill, joel c.; rees, john; pelling, mark; tufet bayona, marta; cassidy, sonia; murray, virginia title: new partnerships for co-delivery of the agenda for sustainable development date: - - journal: int j disaster risk sci doi: . /s - - - sha: doc_id: cord_uid: viikdbf partnerships have become a corner stone of contemporary research that recognizes working across disciplines and co-production with intended users as essential to enabling sustainable resilience-building. furthermore, research that addresses sustainable development challenges brings an urgent need to reflect on the ways that partnerships are supported, and for the disaster risk management and resilience communities, efforts to support realization of the wider agenda for sustainable development bring particular pressures. in november , the uk disasters research group (drg) brought together a number of key stakeholders focused on disaster risk, resilience, and sustainability research relevant to official development assistance to consider how fit for purpose existing partnership models are for the pace of change required to deliver the priorities of the wider agenda. participants were invited to discuss how research partnerships across three levels (individual and project-based; national and institutional; and international) could be improved based on elements that facilitate robust partnerships and learning from aspects that hinder them. from the discussions, participants emphasized the importance of effective communication mechanisms in building partnerships, co-designing projects, and establishing shared objectives. enhanced approaches to addressing equitable partnerships and funding more substantive timelines will be key to responding to the challenges of the agenda. partnerships have become a cornerstone of the delivery of contemporary resilience-focused research. approaches that support multi-, trans-, or interdisciplinary research and coproduction with intended users are recognized as essential to research that can effectively strengthen resilience and support sustainable development. in disaster-affected states, particularly in low and middle income countries (lmics), partnerships can also ensure that research agendas are more relevant to local contexts, drive innovation, convene decision-making spaces, and support research infrastructure to generate evidence-based policy to build resilience and respond to disasters. partnerships among researchers are increasingly looked for by research funders, to bring together resource to confront challenges of sustainable development. these challenges are dynamic and bring a need to reflect on the ways that partnerships are supported and deployed in research. for the disaster risk management and resilience communities, ensuring address of the priorities articulated in the agenda for sustainable development (un ) , the sendai framework for disaster risk reduction - (unisdr , and the agenda for humanity, alongside meeting climate-related goals, brings both challenges to and opportunities for innovative partnerships. in november , the disasters research group (drg) brought together key uk stakeholders from across academia, government, civil society, think tanks, and the public sector focused on official development assistance (oda) relevant disaster risk, resilience, and sustainability research to explore new partnerships for delivery of the wider agenda. the drg is a forum convened by the uk collaborative on development research (ukcdr) comprising senior representation from organizations in the uk that fund and/or support disaster risk reduction research including the department for international development, uk research and innovation (ukri), and wellcome, as well as national representatives of several international bodies such as the united nations office for disaster risk reduction (undrr), the world meteorological organization, and the world health organization (ukcdr ). the aim of the drg is to enhance research and technology-based disaster risk reduction through improved coordination of research funders, providers, and users to deliver components of the uk's commitment to disaster risk reduction. the group also serves to guide the direction of disaster risk reduction funding. this is informed by reviewing trends in the emerging research landscape, which is largely achieved through active engagement with the uk alliance on disaster research (ukadr)-an independent network of uk research institutions, mainly universities active in oda-focused disaster research, associated with the global alliance of disasters research, gadri (ukadr ). since its establishment in , the drg has played an influential role in discussions surrounding disaster risk resilience at both the national and international levels and was credited by undrr as one of the most influential bodies in framing the role of science and technology within the sendai framework. there are four major demands placed on existing partnership models by the complexity and interconnectedness of global challenges inherent in the agenda. research should: • meet urgent needs; • respond to emergent phenomena; • enable equity in partnerships-both internal to project teams (partnering researchers and decision makers across levels) as well as with external stakeholders; and • be people-centered, not justified or led by innovation in technology alone. participants reflected on these demands by assessing three levels of partnerships (project-based, institutional and national, and international) to identify those factors that work well, those that work less well, and how future partnerships could be improved. while the discussions stemmed from a diversity of thought from across the disaster research space, it is acknowledged that the findings largely capture perspectives from those in the global north and that discussions on the development of future partnerships would greatly benefit from a range of perspectives of stakeholders in lmics. driven by the commitment of researchers, individual and project-based partnerships offer much in the form of flexibility, cost-effectiveness, and defined timelines which, in the context of disaster risk management and resilience research, has several advantages. for instance, rapid research responses can be mobilized quickly under this partnership model to fill knowledge gaps in an emergency situation and inform future resilience and potentially lifesaving strategies. when reflecting on the elements that work well in individual and project-based partnerships, participants highlighted the ease of relationship building, compared to the complex partnerships commonly associated with working in large consortia. in particular, participants discussed how healthy individual and project-based partnerships enable https://www.agendaforhumanity.org/. examples of such engagements include a combined drg and ukadr event in july on ''new points of departure in transitioning disaster reduction and sustainability challenges'' attended by people from across the disaster risk reduction research landscape. see https://tomorrowscities.org/tomorrows-cities-fit- -agenda. more effective and agile communication mechanisms that can help identify areas of cooperation from the outset of a research project, facilitate creative discussions, and more easily establish a shared vision. with the opportunity to take advantage of such communication mechanisms, project-based partnerships were especially valued for their ability to convene like-minded individuals with shared interests and foster an environment of trust and respect among researchers. in discussions among participants on those areas that work less well in project-based partnerships, many were quick to highlight the issue of partnerships being insufficiently equitable. from the way that much uk funding systems work, most grants are typically awarded to, and managed by, a designated lead institution, predominantly a uk university, which in turn disburses funds to their partners. this presents a number of challenges to overseas partner institutions, such as being paid in arrears and the requirement for funding to be channeled through the coordinating uk-based lead institution rather than via a project specific manner. this can have significant implications on research in terms of how far in advance research can be planned, the approaches used, and overall quality of outputs. these implications were said to be especially magnified for partnerships involving institutions based in lmics. further elaborating on the theme of insufficient equity, participants highlighted how project-based partnerships and associated funding tend to be concentrated around a small number of lmic-based institutions. as a result, research misses out on knowledge generated by lmic institutions with limited opportunities to partner with uk institutions, while those that are frequently engaged become overcommitted and overburdened on projects that often overlap with each other. this congestion for some lmic institutions, and failure of others to be recognized, was thought to be partially a result of a lack of understanding by uk institutions on the incentives that drive lmic-based partners to take on numerous projects despite lacking capacity, as well as lack of effective coordination among uk institutions, donors, and program management bodies. it also reflects the need to find mechanisms to broaden awareness among lmic researchers of research opportunities, and to expand networking to grow the pool of potential partnerships across lmic and uk institutions. project-based partnerships were also sometimes criticized for affording insufficient mechanisms for those people at risk and for whom resilience-building initiatives are intended to inform research prioritization, with agendas more reflective of external, preexisting funder and academic interests. there remains limited engagement with the existing knowledge of those agencies already engaging with at-risk populations. in the disaster risk and resilience space, future-focused approaches could be greatly improved if more opportunities would be afforded to those groups likely to be most directly affected by risks to inform the research agenda and inform monitoring and evaluation processes. this should be extended to vulnerable populations and those experiencing post-disaster recovery, as well as those agencies working to support these populations. in addressing issues around equity, participants suggested building the capacity of lmic partners to develop, lead, and manage research projects while also making proposal application processes for uk funding more accessible through language and call requirements. participants also welcomed recent initiatives by a selection of uk research funders to fund international partners directly. more broadly, participants underlined the significance of being explicit about the intended respective benefits of partnership. greater equity can be supported through partners from the outset of a project jointly developing a shared vision, an agreed set of deliverables, a common understanding of what each partner wants to gain from a specific collaboration and how the benefits will be shared beyond the end of funding. participants also highlighted the importance of developing more comprehensive impact frameworks that recognize social, environmental and economic, benefits, as well as the intangible benefits of new partnerships, altruism and individual motivation. communication around shared learning about the benefits and approaches was also said to play a crucial role in planning to bring forward research outputs from collaborative projects. specifically, it was emphasized that the knowledge built up by key individuals in project-based partnerships can be shared through establishing networks among colleagues and communities of practice. furthermore, early-career researchers from one project may be future research leaders and, through career progression, the interdisciplinary approaches from earlier projects may be adopted more widely. this will, in turn, require reviewing pathways for career progression, ensuring incentives that recognize the value of engaging in collaborative research that delivers tangible benefits for partnering at risk groups and those agencies that support them. research partnerships at the institutional and national level are characterized by their ability to facilitate collaborations that draw from multidisciplinary expertise across institutions and the experiences of different sectors to exchange ideas and address complex challenges. disaster risk reduction strategies have become increasingly informed by research generated from such partnership models, benefiting from the insights of a diverse range of partners such as from the financial sector, civil society, and government agencies. for partnerships at the institutional and national level, all groups underlined the importance of understanding and accepting the political, economic, and social situations where research takes place and the complexities of working in partnership with national institutions (which can be facilitated by diverse partnerships involving local policy intermediaries to help mediate the relationship between researchers and policymakers). this particularly applies to those instances involving government stakeholders as there may be challenges in using research and evidence to feed into policy debates in light of overlapping or competing mandates. in disaster-affected communities, there remains a need to consider how research is shared with participating populations, as research in these settings is at particular risk of being viewed as extractive. successful partnerships were therefore said to put a lot of emphasis on establishing clarity on the motivations and languages of different agencies, including between policymakers, researchers, and the media, and in identifying a common set of goals. in terms of what works less well, participants noted that institutional and national partnerships can be adversely impacted by a variety of exogenous influences. these include institutions viewing uk funding schemes as a means to pursue commercial interests rather than bring about long-term change, as well as pressure from research funders to build institutional partnerships in short funding cycles. as a result, participants noted that these can lead to the formation of partnerships based on questionable motivations that are unable to achieve sustainable outcomes. examples of these included top-down collaborations where inter-institutional partnerships are based on personal rather than professional relationships and, at a higher level, a duplication of efforts by groups of institutions to produce research using slight variation in approaches used. given the challenges in developing institutional and national partnerships, there were discussions on the role that research bodies and funders could play in facilitating matchmaking between different agencies. it was thought that the existence of such a platform could make partnership building easier as it could help, among other things, identify common approaches used by institutions and opportunities (and demand) to conduct interdisciplinary research. suggestions were also made on the possibility of having longer-term uk funding frameworks whereby small funding pools could be designated for longer-term and/or non-quantifiable components of disaster research (particularly institutional partnership building) where research outcomes are expected to be deferred outside of the standard - -year project timeline. extending the institutional partnership model to a global level, international partnerships are able to take advantage of an even larger community of knowledge and expertise to develop interdisciplinary research to address complex challenges. in addition to becoming more frequent in recent years (due to advances in air travel and communication technologies), international partnership models allow the disaster risk resilience community to benefit from evidence generated by lmic institutions, provide opportunities for research capacity strengthening, and can lead to the establishment of regional knowledge hubs. despite the amount of time required to build robust partnerships, participants praised the ability of the disaster risk and resilience research community to identify key people and organizations to work with when forming international partnerships. in particular, multi-sectoral partnerships that bring together the academic, public, private, and nongovernmental organization sectors were seen as effective tools to stimulate innovation. this was seen to largely be driven by the effective integration of stakeholder mapping into project design (rather than at later stages) to ensure that the right partners are involved, assumptions are challenged, and priorities are agreed. effective communications during the co-design and coproduction of research projects was especially highlighted by participants since this is crucial to the success of international and multi-sectoral partnerships as the careful selection of the project focus, including the phrasing of objectives, can help to bring together partners and helps to remove some of the politics from research. on those elements of international partnerships that work less well, participants noted that while there is a demand for (rapid) international partnerships, the resource investment needed to build such partnerships is often lacking. this was exemplified using the case of uk-led international partnerships involving lmic-based institutions when there is little to no follow-on funding available to grant holders to develop partnerships, resulting in scarcity of long-term capacity-building programs. similarly, participants lamented the lack of prioritization and support of uk funding schemes to support partnerships between lmic institutions. while participants noted that the research community is aware of cultural issues, an improved understanding of cultural norms and sensitivities would allow the community to be more effective. with international partnerships, it was said that there can be a tendency to homogenize groups (for example, academia, communities) and overlook the complexity of communities and the impacts of this on policy and implementation. this was said to be the case where organizations may be looking to create partnerships in new and emerging systems of governance. to improve international partnerships, participants spoke of the need to decolonize uk research and to continue the push for equitable partnerships by ensuring that partners in lmics have a greater input in shaping research priorities based on the needs of those countries and communities that research is seeking to help. this would involve giving those partners greater control over resource allocation, which in turn would also help address continued issues relating to capacity building. it was proposed that such shifts could be facilitated by broader application of processes of change in funding mechanisms. for instance, mentors could support lmic-based partners in identifying relevant funds and funding criteria and the designing of projects, including by ensuring that they have access to the relevant information in a format and language appropriate to their needs. to that end, participants highlighted the important role of trusted brokers to facilitate the research process as key in driving forward the research impact of increasingly complex international partnerships. while there has been a greater awareness of these issues as a result of the transparency commitments attached to increasing oda funding to support research in recent years, participants noted that this has resulted in a complex funding landscape with limited access to some institutions not considered to serve a core academic function. in response to this, it was suggested that there could be a better understanding and awareness of the different sources of funding available and funders could make it easier for a wider group of practitioners to access and help to influence the nature of this research funding. the urgent action required to address global challenges has only increased in recent years and progress towards the delivery of the wider agenda has become more reliant on rigorous research facilitated by effective partnerships. covid- and the economic consequences are likely to see reduced budgets for research and this places even greater importance on understanding how to manage partnerships to enable research that can be timely, adaptive, fair, and people-centered. while members of the uk disaster risk, resilience, and sustainability research community have developed important understanding of some of the components and mechanisms vital to enabling effective and agile partnership models, especially in terms of establishing shared objectives, the sustainability of partnerships and partnership processes are considered a cause for concern. a key way forward will be to develop research frameworks that support and value more equitable forms of partnership and resource more substantive timeframes that enable complex research partnership approaches to flourish. building partnerships of equals: the role of funders in equitable and effective international development collaborations kfpe (swiss commission for research partnerships with developing countries) the united kingdom alliance for disaster research transforming our world: the agenda for sustainable development united nations international strategy for disaster reduction) acknowledgements this paper draws from discussions convened by the disasters research group as part of its th anniversary celebration. john rees and joel c. gill publish with the permission of the executive director, british geological survey (ukri).open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons. org/licenses/by/ . /. key: cord- -pv doe authors: novossiolova, tatyana title: twenty-first century governance challenges in the life sciences date: - - journal: governance of biotechnology in post-soviet russia doi: . / - - - - _ sha: doc_id: cord_uid: pv doe the chapter explores the rapid advancement of biotechnology over the past few decades, outlining an array of factors that drive innovation and, at the same time, raise concerns about the extent to which the scope and pace of novel life science developments can be adequately governed. from ‘dual-use life science research of concern’ through the rise of amateur biology to the advent of personalised medicine, the chapter exposes the limitations of the existing governance mechanisms in accommodating the multifaceted ethical, social, security, and legal concerns arising from cutting-edge scientific and technological developments. so far as to suggest that the 'life sciences knowledge, materials and technologies are advancing worldwide with moore's law-like speed.' and whilst some commentators have questioned the extent to which the ongoing progress of biotechnology has translated into practical applications and novel products, there is some consensus that the biotechnology landscape has been fundamentally transformed over the recent decades with the possibilities now unlocked holding revolutionary potential. indeed, rapid advances in the field have produced a knowledge base and set of tools and techniques that enable biological processes to be understood, manipulated and controlled to an extent never possible before ; they have found various applications in numerous spheres of life, generating enormous benefits and offering bright prospects for human betterment; and they have come to be regarded as a key driver of economic development with potential to close the gap between resource-rich and resource-poor countries. the progress of biotechnology has been largely driven by three sets of forces, namely social, political and economic. the social dynamics at work in this context are understood as the efforts to improve public health and overall wellbeing of individuals both in the global north and global south, boost agricultural yields and encourage environment-friendly practices to mitigate the adverse effects of climate change. several factors account for the significant value attached to the life sciences in the context of intense globalisation and continuous change. surging population numbers and extended life expectancy are augmenting the demand for developing effective and affordable medications, novel approaches for the treatment of chronic diseases and additional cost-effective sources of energy and food production. at the same time, rising global trade and travel, coupled with increased urbanisation, and an uneven distribution of wealth are creating optimal conditions for disease outbreaks, pandemics and environmental degradation. against this backdrop, biotechnology appears full of promise and critical to tackling social and natural concerns; enhancing disease prevention, preparedness and surveillance; promoting development; and alleviating human suffering. economic dynamics include national expenditure on research and development, purchasing power, trends in consumerism and market pressures and fluctuations. besides public funding for r&d which remains a key factor in the growth and flourishing of bioindustry in developed and emerging economies alike, private investment from venture capital firms, start-up companies and transnational corporations (tncs) have also played an indispensable role in capturing new markets and further facilitating the extension of bioeconomy on a global scale. dupont's significant footprint in india is indicative in this regard, not least because of the depth and diversity of the activity that the company has undertaken via its offshore r&d centres ranging from crop science to biofuels. likewise, merck has outlined a . billion dollar commitment to expand r&d in china, as part of which it intends to establish an asia headquarters for innovative drug discovery in beijing. political dynamics are triggered by states' increasing commitment to support the progress of biotechnology as a way of maximising their power and boosting their status in the international arena. in the aftermath of / and the 'anthrax letters' attack of october , substantial effort has been given to harnessing life science research for the purposes of national security. biodefence and bioterrorism preparedness are thus considered high-priority areas for national investment by government agencies and the military alike. an illustrative example of this two-tiered approach is the funding policy in the usa, where biodefence research is financed by the nih, department of homeland security (dhs) and defense advanced research projects agency (darpa), to name a few. under the synergistic influence of these three sets of forcessocial, economic and politicalbiotechnology has been transformed into a truly global fast-evolving enterprise encompassing a multitude of stakeholders, delivering considerable benefits and holding out still greater promise, with profound and far-reaching implications for virtually every aspect of human well-being and social life. the pharmaceutical industry is a case in point, for its steady expansion would hardly be possible were it not for the vast array of techniques and methods enabled by the progress of the life sciences. worth roughly billion dollars, the global pharmaceutical market dominates the life sciences industry and arguably determines the trajectory of life sciencesrelated technological development and global spread. gene cloning, dna sequencing and recombinant construction of cell lines, to name a few, are all deemed indispensable for the development of novel medicines and therapeutics. it suffices to mention that more than half of the top selling commercially available drugs in the usa would not exist without those methods. agriculture, too, has been heavily influenced by the ongoing biotechnology revolution, as evidenced in the rapid growth and dispersion of commercialised transgenic crops (biotech crops) and the efforts to use gmos (both animals and plants) for the production of vaccine antigens and other biologically active proteins ('biopharming'). indeed, the increase in the area of farmland planted with transgenic crops rose dramatically from . hectares in to about million hectares in and is still growing. in addition, technological convergence between biotechnology, nanotechnology, information technologies and cognitive science has unlocked a broad scope of opportunities for maximising public (and private) welfare, offering substantial benefits in wideranging areas such as medicine, pharmacy, crime investigation and national security by ensuring precision and reliability, while at the same time, reducing the amount of time previously required for the performance of certain tasks. four key features of biotechnology make it so appealing to the majority of stakeholders involved. first, biotechnology innovation is characterised by duality, whereby research yields results that simultaneously lead to advances in basic knowledge and stimulate product development. second, the output that the life sciences generate in the form of new medicines, improved nutrition products, enhanced yields and novel materials, is 'strongly positive'. the increasing utility of tools and strategies for human enhancement, whether in professional sport, for cosmetic and aesthetic purposes, or on the battlefield, vividly reflects the firm conviction that the transformative capacity of biotechnology, even at the most fundamental level, is something to be welcomed and vigorously embraced. what is more, biotechnology possesses proven economic viability, as illustrated in the burgeoning industries and new markets it has spurred. against this backdrop, the high rate of biotechnology expansion is anything but surprising, since every increment in biological capability pays back the researcher and the researcher's sponsors in short order. payback comes in the esteem of peers, in promotions, and in increases in the academic or corporate salaries of the researchers whose work generates knowledge and new therapies. payback comes in the form of profits for the manufacturers of kits to perform the manipulations, royalties for the writers of the methods manuals profits for the drug industry. payback comes for the public in the form of new drugs and therapies. fourth, besides being cost-effective, many of the benefits that biotechnology offers are easy to obtain and disseminate. in other words, many of the various prospects for public (and private) betterment are not situated at some distant moment in the future but can be realised immediately, as a result of which pressing problems can be alleviated, if not fully resolved, and substantial revenue can be generated in the short term. last but not least, while there are some risks and concerns associated with the advancement of biotechnology, few of those are deemed urgent or significant enough to impact on the pace of innovation. as the actual manifestation of such risks is often contingent upon the interplay of a variety of factors, this renders the likelihood of a major crisis unfolding as a result of the progress of biotechnology low. moreover, there is a genuine belief that any challenges that may arise from the proliferation of novel technologies can either be foreseen or dealt with on a case-by-case basis. given the enormous potential of biotechnology for addressing societal, economic and environmental challenges, it is unsurprising that most states have readily endorsed scientific and technological innovation and embarked on largescale generously-funded r&d programmes in the life sciences. given the powerful multifaceted impetus for biotechnology advancement, it is possible to identify at least five key trends in the governance of biotechnology that are common for highly industrialised and developing countries alike. those include: high-level coordination, facilitation and funding; synergies within and between both the public and private sector; emphasis on strategic and competitive interests at the expense of precaution; regulations that seek to promote rather than restrict scientific and technological progress; and overreliance on technical solutions. at international level, the on-going expansion of biotechnology has been hailed not only as an inherently positive development but also as an essential prerequisite for enhancing human welfare and addressing various socio-economic, environmental and health concerns. in its world health report, the who called for: increased international and national investment and support in [life science] research aimed specifically at improving coverage of health services within and between countries. the who has also strived to promote research on specific diseases, such as hiv/aids, cancer, pandemic influenza, tuberculosis and malaria, with the goal to improve methods for prevention and diagnostics and facilitate the development of effective therapeutics and vaccines. in a similar fashion, the un food and agriculture organisation (fao) has highlighted the positive impact that biotechnology could have on the development of agriculture: . . . biotechnology could be a major tool in the fight against hunger and poverty, especially in developing countries. because it may deliver solutions where conventional breeding approaches have failed, it could greatly assist the development of crop varieties able to thrive in the difficult environments where many of the world's poor live and farm. it is not difficult to see how those assertions have been translated into national policies and practical steps across the globe. the us nih that provide the bulk financial support for medical and health-oriented r&d in the us spent over . billion dollars during the fiscal year , about a third of which was allocated for funding biotechnology and bioengineering projects. within its sixth framework programme for research and technological development spanning the period - the european union (eu) distributed more than . billion euro for projects under the theme 'life sciences, genomics and biotechnology for health'. developing countries, too, are increasingly investing in 'red' biotechnology as part of their efforts to address public health concerns. according to a recent who report, support for biotechnology and particularly, for cancer research, in cuba has soared over the past years, amounting to over one billion dollars. as a result, the cuban biotechnology industry is burgeoning, holding around international patents and exporting vaccines and pharmaceuticals to more than countries. the prospect of climate change coupled with rising population numbers has compelled governments in the global north and south alike to explore 'green' biotechnology as a means of ensuring food security. the usa remains by far the largest commercial producer of gm crops. several eu member states (france, germany, spain, poland, romania, czech republic, portugal and slovakia), canada and australia further feature in the list of industrialised nations that have embarked on growing gm plant breeds. more and more emerging economies are striving to expand their agrobiotechnology sector, most notably brazil, india, argentina, south africa, mexico, burkina faso, myanmar and chile. in , the chinese government launched a major r&d initiative worth billion dollars to develop new plant varieties by that will enhance yields, have improved nutritional value and be resistant to pests. public-private partnerships underpinned by access to early-stage risk capital and strong linkages between business, universities and entrepreneurial support networks constitute an important vehicle for promoting innovation and fostering technology transfer and product development. for instance, the chinese government has launched a major initiative mobilising . billion dollars in venture capital to support start-ups in the immense zhangjiang science park outside shanghai ; russia's rusnano has entered a million dollar partnership with the us venture capital firm domain associates to fund 'emerging life science technology companies and establish manufacturing facilities in russia for production of advanced therapeutic products'; and cleveland's university hospital has allocated million dollars for setting up a 'non-profit entity to fund and advise physician-scientists on transitional research and a related for-profit accelerator that will develop selected compounds to proof of concept.' the kauffman foundation in the usa, a wealthy philanthropic establishment dedicated exclusively to the goal of entrepreneurship has been particularly zealous in its quest for promoting university-based entrepreneurial activities nationwide. its kauffman campuses initiative launched in early enjoyed so much popularity among universities that following the initial round of grants totalling million dollars, the foundation announced its resolve to leverage a million dollar investment for the creation of new interdisciplinary education programmes. university-industry partnerships, while not a novel phenomenon in the area of biotechnology, have considerably intensified over the past several decades, thus facilitating the widespread commercialisation of life science research. indeed, per cent of the companies in the us surveyed by blumenthal et al. in had relationships with an academic institution in that year and in more than half of those cases industry provided financial support for research in such institutions. according to another study, the total industry investment in academic life science research in the usa tripled between and reaching almost billion dollars and has been growing ever since. against this backdrop, some commentators have put forward the 'triple helix' model, which serves both as a conceptual tool and a policy blueprint. in the former case, it is used to elucidate the academic-industry-government relationships that underpin the institutional arrangements and changing practices in the processes of production, transfer and application of knowledge in post-industrial societies; in the latter, it is promoted as a framework for economic development through state investment and knowledge sharing between academia and industry. others, however, have remained sceptical of the close integration of universities and the private sector voicing concerns about the possible deleterious effects arising therefrom: as in other activities, when big money flows fast, temptations and opportunities arise for risky behaviour and stealthy or even brazen wrongdoing in pursuit of personal or institutional advantage. the new world of academiccommercial dealings is characterised by some grey areas and evolving rules for permissible and impermissible conduct. the people who manage and conduct research in scientific organisations are not immune to the weaknesses and foibles so plentiful elsewhere, despite the accolades for probity that science bestows upon itself. with more and more universities joining the biotechnology 'gold rush' and corporate values and goals steadily penetrating the professional academic cultures, scholarship turns into a result-oriented activity subject to the priorities and interests of business partners and industrial sponsors. strategy and careful planning deemed essential to the pursuit of for-profit knowledge can have a restraining effect on the spontaneous vigour characteristic of academic research, limiting the range of problems that could be studied to those defined by the market. at the same time, scientists often find themselves under tremendous pressure striving to satisfy the demands of their industrial clients without utterly neglecting their academic duties ranging from mentorship through filing grant applications to publishing. the extensive workload coupled with the bright prospects for securing long-term research funding and achieving some individual gain and prominence provide a favourable environment in which instances of dubious, sometimes fraudulent, behaviour, conflicts of interest and lack of transparency, unless too severe, are unlikely to encounter widespread opprobrium and may even go unnoticed. in the race for patents and venture capital, the business mentality dulls scientific rigour and the ethics threshold appears not too difficult to cross. interests at the expense of precaution given the tremendous benefits that biotechnology is expected to generate in virtually any sphere of human activity, it is not difficult to understand why its progress is predominantly viewed through an explicitly positive lens by policy-makers. since the opportunities for achieving public betterment and enhancing state prestige and international standing are too tempting and too abundant, there is a powerful urge to dedicate both will and resources to promoting the large-scale expansion of the life sciences. for one thing, the prospect of conquering disease and maximising human wellbeing provides solid justification for a deliberate and sustained investment in fostering scientific and technological prowess. lack of commitment and reluctance to support r&d in the life sciences then becomes an unfavourable option in the political calculations of states regardless of their level of economic development and international status. within the context of political calculus pervaded by realist fears, competition and power, the perceived risks of inaction with regard to scientific and technological development justify vast expenditure, lower regulatory barriers to innovation and product development. political choices concerning biotechnology support are therefore frequently made at the expense of calls for caution and potential social, environmental and ethical concerns. the regulation of genetic engineering is a case in point. as discussed in the previous chapter, from the outset, the attempts of governments to impose strict controls on research involving rdna faced a severe backlash from academic scientists and business executives alike. by the s, the various legislative initiatives put forward in the usa were abandoned in favour of the regime established by the nih guidelines, which virtually exempted the biotechnology industry from formal regulation. while the leading us-based companies pledged to 'voluntarily comply' with the guidelines, behind the scenes they craftily continued to push for a system that would insulate them from governmental and public scrutiny. indeed, during the s when the states parties to the btwc strived to strengthen the treaty by negotiating a binding verification mechanism corporate interests proved too big and too important to be ignored. both the pharmaceutical research and manufacturers of america (phrma) which represented the country's major research-based pharmaceutical and biotechnology companies, and the biotechnology industry organisation (bio) which at that time represented some biotechnology firms, became vocal opponents of any measures designed to promote international arms control which seemed to hinder in any way the protection of proprietary information and intellectual property. in the period between and the associations invested considerable effort, time and ingenuity in lobbying the us government and influencing the diplomatic talks in geneva to secure an outcome that was in line with the demands of their constituencies. of course, it would be naive to ascribe the us resolve to reject in both the text of the protocol and its utility in general for providing adequate verification and enhancing confidence among states parties solely to the activity of the biotechnology industry; nevertheless, it would be equally naive to suppose that corporate interests played no significant role in the process. besides economic priorities, national security and military calculations can also provide a compelling rationale for downplaying the potential risks associated with biotechnology expansion. following the 'anthrax letters' attack in october , the us government embarked on a massive financial investment to boost its bioterrorism preparedness and enable the prevention, early detection, monitoring and emergency response to biological threats. as outlined in biodefense for the st century, a presidential directive that set out a comprehensive framework for national biodefence policy, between and the federal government provided roughly billion dollars 'to state and local health systems to bolster their ability to respond to bioterrorism and major public health crises'. along with the highly controversial vaccination programme that the government envisaged, another important development designed to enhance america's biodefence preparedness and capability was the drastic increase both in the number of high-containment labs (bsl- and bsl- ) and the number of researchers with access to some of the most dangerous pathogens known to mankind, including the causative agents of ebola, plague and q fever. some commentators have questioned the logic behind this policy highlighting the heightened risk of accidental or deliberate release of pathogens. far from being ill-founded or hypothetical, such fears stemmed from a range of high-profile cases that occurred after across the us in which the lack of proper training and professional negligence resulted in scientists being exposed to or infected with deadly microbes. real-life horror stories about vials of plague being transported in the hand-luggage of researchers on passenger aircraft without the required authorisation, and deadly cultures gone missing from what appeared to be secure laboratories further fuelled the criticism toward the us biodefence policy raising difficult questions about its appropriateness and actual goals even before the 'anthrax letter' investigation revealed that the attack was 'insider's business'. life science research, just as any other sphere of professional activity, is subject to a range of institutional, national and international regulations. along with the more general rules such as those related to occupational health and safety, fair pay and job competition, conflict of interests, labour rights, and professional liability, there are also specific ones addressing particular aspects of the research process including project clearing (e.g. review by local biosafety committees), safe laboratory practice and transport of pathogens (e.g. international health regulations), exchange of viral strains (e.g. pandemic influenza preparedness framework, ), handling of dangerous pathogens (e.g. us select agent programme) and ethical treatment of human subjects and samples obtained therefrom (e.g. the human tissue act in the uk). while hardly exhaustive, this list suffices to convey the idea that the regulatory regime governing the practice of life science research is dense and comprehensive. with more than international organisations overseeing biotechnology from various perspectives, there is a prima facie reason to assume that the regime in its current form is sufficiently flexible to accommodate novel advances and hold any potential risks, which they may pose, at bay. yet in reality over the past decade the opposite trend has prevailed, that is, the existing governance mechanisms have struggled to respond adequately to the proliferation of new scientific developments with multiple adaptive uses and the multiplicity of cutting-edge developments posing profound ethical quandaries. how to account for this discrepancy? part of the problem stems from the fact that since at least the late s the regulation of biotechnology has been streamlined so as to become compatible with and not a restriction on continued technological change and economic growth. as such, it rests upon the barely questioned assumption that the progress of biotechnology is inherently good and needs to be harnessed and vigorously promoted. needless to say, any measures that seem to slow down or restrain its advancement are deemed undesirable and even detrimental to socio-economic development. hence, when developing regulations, policy-makers have generally pursued a twofold objective: first, to promote the safe practice of life science research by reducing any risks arising therefrom both to scientists and the general public; and second, to ensure that any issues that may hinder the expansion of biotechnology are not subject to restrictive legislation. a vivid manifestation of this approach is the way in which the ongoing debate on 'dual use research of concern'benignly-intended research that seeks to maximise human welfare by responding to health, societal and environmental ills but could also facilitate to the development of more sophisticated and potent biological weapons and enable bioterrorism has been handled. for more than a decade, researchers, journal editors, security experts and policy-makers have strived to devise oversight mechanisms and governance initiatives that could adequately tackle the challenge of dual use without stifling innovation. unfortunately, to date their efforts have met with little success, as a result of which virtually each experiment of dual use concern is dealt with separately on a case-by-case basis. this is not to say that there are no similarities across the studies of this kind. on the contrary, a few of the most notable examples follow a similar paradigm, including the creation of a vaccine-resistant strain of the mousepox virus, the artificial synthesis of the polio virus, the recreation of the spanish influenza virus and, most recently, the production of a mammalian-transmissible h n avian influenza virus (see fig. . ). all four of them were performed in strict compliance with the rules and procedures in place for laboratory biosafety, biosecurity and biorisk management and under appropriate physical containment conditions; all had passed thorough review by the respective local biosafety and bioethics committees; and all of them were deemed essential in terms of public health benefits. above all, the ethical and security concerns that the studies have raised go far beyond the laboratory door, posing fundamental questions about how life science research is reviewed, conducted and communicated. yet none of the high-profile experiments of concern has proved critical enough to provoke a radical change in the way dual-use research is governed. three points merit consideration in this regard. the first pertains to the manner in which the dominant discourse on dual use is framed, that is, in purely ethical terms as a dilemma. while bioethics undoubtedly has a role to play in the discussions on dual use, the language in early , the journal of virology published a report of the creation of a highly virulent strain of the ectromelia virus, the causative agent of mousepox. the work described in the report was carried out by a group of australian scientists based in canberra. its original goal was the development of an infectious immunecontraceptive that could be used against wild mice for the purpose of pest control. to achieve this, the group drew upon previously published work. during the course of the experiment, the researchers unexpectedly discovered that the newly engineered strain of the mousepox virus, which they created, killed % more mice than the parent virus, including mice that had been vaccinated or that had natural immunity. when the research was published, concerns were raised that it could potentially be misapplied for hostile purposes, or even that the same technique could be utilised for creating a more virulent strain of the variola virus, which causes smallpox in humans. in , a team of scientists led by dr eckard wimmer from the university of new york at stony brook announced that they had successfully created a polio virus 'from scratch'. to carry out the research, the scientists 'followed a recipe they downloaded from the internet and used gene sequences from a mail-order supplier'. a once the virus created, it was tested on mice, as a result of which the infected animals became paralysed and died. the study spurred a wide-ranging debate, not least because it drew attention to the possibility of using synthetic biology for constructing de novo viruses for the purposes of bioterrorism. in , it was announced that cdc scientists, together with colleagues from several research institutions across the usa, had successfully recreated the influenza virus, that was responsible for the pandemic, which killed between and million people worldwide. using dna from a tissue of a flu victim buried in the permafrost in alaska, the researchers managed to reconstruct the influenza virus and thus study its pathogenesis and properties that contributed to its virulence. despite the scientific justification that was put forward, critics have argued that the study is 'a recipe for disaster', not least because the availability of the virus' full-genome sequence and detailed method for its reconstruction on the internet may facilitate its synthesis by a of 'dual-use dilemmas' is too abstract to offer appropriate analytical tools for dealing with the issues at play. as discussed above, the questions that dual-use research poses such as data sharing, research funding and project planning are far from hypothetical but they feature explicitly in everyday professional practice. however, the 'dilemma framework' automatically strips them of the complex socio-technical arenas in which they have actually presented themselves by laying an emphasis on what action should ideally be taken, rather than what is practically feasible given the circumstances. moreover, such issues are typically structural in nature for they constitute fundamental elements of the life sciences professional culture, and as such, could hardly be adequately addressed solely at the level of individual researchers. yet framing social, legal and security concerns in terms of moral dilemmas allows for structural issues to be omitted from the discussion, rendering life scientists the chief, if not the only, moral agents expected to reach what is deemed to be the 'right' answer. assigning abstract duties then comes to be regarded as an appropriate 'solution', even if those are virtually impossible to fulfil given the complexities of the working environment within which researchers operate. the second point is related to the reductionist view that dominates the discourse of what counts as a risk in life science research. perhaps one of the most significant legacies of the asilomar conference on rdna (see chapter ) is the emphasis on laboratory risk that could be effectively managed by dint of physical containment and rules and procedures for safe laboratory practice. it suffices to mention that the bulk of guidelines and formal regulations published by the who focus exclusively on promoting and refining measures that aim to maximise laboratory biosafety and prevent the accidental release of pathogens. hence, it is hardly surprising that the concept of dual use and the idea of risks beyond the laboratory door implicit in it seem alien to the majority of practising researchers. striking as it may appear, even though dual use research has been debated for more than a decade now, the level of awareness among life scientists of the broader social, security and legal implications of their work remains low. the third point deals with the way in which risks in life science research are assessed and mitigated. given the narrow definition of risk encompassing technical particulars, physical containment and biosafety, risk assessment is considered an appropriate and reliable tool for ensuring research safety. the heavy reliance upon risk assessing tools is underpinned by two underlying assumptions. one is that it is possible to foresee and calculate most, if not all, things that could potentially go wrong both during the development phase of the project and after its completion. the other is that it is possible then to use the produced data as a basis for devising measures and strategies for eradicating, or at least, mitigating the risks likely to occur. attractive as it may seem, this 'new alchemy where body counting replaces social and cultural values' presupposes a clear distinction between the risk assessment 'experts' and the general public, whereby the former are granted a licence to make decisions about the risks that the latter cannot do without. likewise, costbenefit analysis on the basis of which research proposals are screened for potentials risks and security concerns has attracted some serious criticism. in the view of some commentators, besides being sometimes deeply inaccurate, the cost-benefit analysis is 'ethically wrong' since 'applying narrow quantitative criteria to human health and human life' is unacceptable. but there are other problems, too. as pointed out by dickson, the cost-benefit analysis distorts political decision-making by omitting any factors that cannot be quantified, thus obscuring questions of equity, justice, power, and social welfare behind a technocratic haze of numbers. as a result, complex and politically charged decisions are reduced to a form that fits neatly into the technocratic ways of making regulatory decisions, whereby calculations and approximations made by the few substitute for the judgements of many. the wide-ranging controversy that unraveled in late when two teams of scientists working independently in the netherlands and the usa managed to produce an air-borne strain of the h n avian influenza virus, a highly pathogenic and lethal microbe with over per cent mortality rate in humans arguably constituted the pinnacle of the deliberation on dual use research. both studies set alarm bells ringing for the security community who almost immediately jumped in the debate voicing concerns over the possibility of biological proliferation and bioterrorism. some commentators even argued that the experiments ran counter to the spirit if not to the letter of the btwc. against this backdrop, the resultant controversy was deemed at least initially to offer a timely opportunity to evaluate the existing governance mechanisms, determine their gaps and weaknesses, and broaden the scope of deliberation inviting participation of a wide range of stakeholders. unfortunately, the outcome of the debate proved far more moderate, signalling preference for preserving the status quo without disrupting the established systems for governance and oversight. despite the extensive mass media coverage of the controversy, only few public consultations were held and none of those was designed as a platform for making policy proposals or developing action plans. moreover, the denselypacked agenda prepared duly in advance left very limited scope for posing 'tricky' questions which the participating 'experts' might have struggled to answer. needless to say, all consequential decisions were made behind closed doors away from public scrutiny and on some occasions the people with the greatest vested interest in the publication of the studies were also the ones with the greatest say in the process. there were no significant changes in terms of governance initiatives, either. far from being ground-breaking developments, the us government policy for oversight of life sciences dual use research of concern and the decision of the dutch government to invoke export control legislation before allowing the publication of the study conducted within its jurisdiction were little more than desperate moves that aimed to obscure the inadequacy and shortcomings of the measures already in place. overall, the manner in which the h n debate was handled could be treated as a missed opportunity, whereby those in charge of the decision-making process did little to address or even acknowledge the broader issues underpinning dual-use research of concern but simply 'kicked the can down the road to the next manuscript' waiting for the next controversy to erupt. technology seems to play a significant role in the governance of life science research. high-containment laboratories, well-equipped biosafety cabinets, sophisticated waste management systems, enhanced personal protective equipment and secure containers for the safe storage and transportation of biohazard materials are just a few of the tools and systems in place that allow the safe handling of dangerous pathogens and toxins and, at the same time, protect both laboratory personnel and the general public from exposure to deadly microbes. that said, the effectiveness of technical solutions should not be overstated if only for the fact that 'problems' of governance are barely technical matters per se but rather constitute complex issues of human relatedness. nevertheless, the attractiveness of technological fixes as offering reliable risk mitigation and reassurance in the safety of biotechnology is ever growing. it suffices to mention that the h n controversy discussed above was in part resolved after the lead researchers in the netherlands and the usa respectively agreed to add a detailed section on the technical specificities and laboratory biosafety and biosecurity measures taken during the experiments. the strategy has proven effective in diverting attention from the rather inconvenient questions regarding the utility and significant potential for hostile misuse of the so called 'gain-of-function' (gof) research and concentrating it on more mundane issues dealing with in-house precautions and safety procedures. once the latter were deemed adequately resolved, the former were effectively forgotten. still, the value of technical means in ensuring reliable risk management should not be taken for granted. for one thing, laboratory biosafety precautions, however sophisticated, are far from perfect and accidents do occur. such is the case with the pirbright site in the uk which was at the centre of a major outbreak of foot-and-mouth disease in , as a result of which over animals were slaughtered. in the bioterrorism bsl- laboratory at the us cdc in atlanta suffered repeated problems with airflow systems designed to help prevent the release of infectious agents. the faulty system could perhaps be regarded as an exception had it not been for the authoritative investigation report of the us government accountability office (gao) released in march . according to the report, the cost of building and maintaining high-containment laboratories, coupled with the absence of national standards for their design, construction, operation, and maintenance 'exposes the nation to risk'. far more critical is the situation in the developing world and emerging economies where lax regulations and technical failures have significantly heightened the risk of accidental release of pathogens, as demonstrated by the numerous 'escapes' of the severe acute respiratory syndrome (sars). but even if technology functions impeccably, this hardly reduces the likelihood for a human error or inappropriate behaviour. unlocked doors in high-containment facilities hosting deadly pathogens, eating and drinking in laboratories and poor waste disposal practices are just a small part of the otherwise long list of mundane mishaps that may result in severe consequences. it is worth mentioning that the us cdc came under the spotlight after internal e-mail correspondence revealed that doors in the bsl- block where experiments involving the causative agenets of anthrax, sars and influenza were performed were left unlocked on numerous occasions, thus increasing the risk of unauthorised access or theft. given the chance of technical flaw and the potential for human error, some life scientists have begun to question the reliability of existing laboratory precautions and demand thorough review and evaluation. in a recent letter to the european commission the foundation for vaccine research has asked for 'a rigorous, comprehensive risk-benefit assessment' of gof research that 'could help determine whether the unique risks posed by these sorts of experiments are balanced by unique public health benefits which could not be achieved by alternative, safe scientific approaches'. engines that drive biotechnology momentum by and large, the ongoing progress of biotechnology is largely viewed and assessed through an explicitly positive lens which allows focusing almost exclusively on the benefits likely to be accrued notwithstanding the risks, actual and potential. the resultant distorted image is problematic, not least because it precludes any comprehensive discussion on the potential side effects and negative implications of novel life science advances. above all, it sustains the barely questioned assumption that the existing governance mechanisms are adequate and sufficient to cope with the stresses and strains of the rapidly evolving biotechnology landscape. yet given the complex and multifaceted dynamics shaping the life science enterprise, the rapid pace of innovation, and the limits to predicting the synergistic and cumulative effects of the proliferation of new technologies, the uncritical acceptance of such assumptions is at best naïve and at worst dangerous. arguably the advancement of the life sciences has greatly benefited from the fascinating breakthroughs made in other areas of study, such as chemistry, engineering, computing, informatics, robotics, mathematics and physics. some commentators even talk about a third revolution in biotechnology underpinned by scientific and technological convergence: convergence does not simply involve a transfer of tools sets from one science to another; fundamentally different conceptual approaches from physical science and engineering are imported into biological research, while life science's understanding of complex evolutionary systems is reciprocally influencing physical science and engineering. convergence is the result of true intellectual cross-pollination. the resultant 'new biology' has opened up a range of marvellous possibilities enabling the manipulation of living matter at the full range of scales, as well as the application of biological systems principles for the development of novel materials, processes and devices. as such, it has been largely hailed as possessing the 'capacity to tackle a broad range of scientific and societal problems.' this is not an exaggeration. as noted by a recent report of the us nas, the precipitous decline in the cost of genome sequencing would not have been possible without a combination of engineering of equipment, robotics for automation, and chemistry and biochemistry to make the sequencing accurate. likewise, it is the combination of expertise from fields as diverse as evolutionary biology, computer science, mathematics, and statistics that has allowed both the analysis of raw genomic data and the subsequent use of these data to other fields. at the same time, advances in nanoscience and nanotechnology have considerably enhanced drug delivery making it more accurate by targeting specific parts of the body. yet the transformative potential of scientific and technological convergence comes at a price, not least because parallel to the benefits it offers, there are risks the effects of which could be truly devastating. take drug delivery, for instance. thanks to the technological breakthroughs over the past decade, doctors have gained unprecedented access to the human body which, in turn, has facilitated the treatment of previously incurable disease and conditions (e.g. some forms of cancer). nanoparticles and aerosols are now utilised for delivering a precise dose of therapeutics to tissues and cells via novel pathways circumventing body's natural defences and evading immune response. it is not difficult to imagine how such knowledge could be misapplied for malicious ends, including incapacitating and killing. research on bioregulators is a case in point. bioregulators are natural chemicals in the human body that play a vital role in the maintenance of the homeostasis but when administered in large quantities or in healthy individuals could be toxic and lead to serious disorders, even death. given their properties, bioregulators constitute the perfect bioweapon: efficient and virtually impossible to detect. and if in the past, security analysts discounted the risk of their weaponisation due to the instability of the compounds when released in the atmosphere, the emergence of novel drug delivery techniques has significantly altered the security calculus. this is just but one example of the challenges that the increasing convergence between biology and chemistry poses to the integrity of the international biological and chemical non-proliferation regimes. even though some effort has been made over the recent years to address those and other areas of concern and strengthen the international prohibition against biological and chemical warfare, in practical terms little has been achieved, as a result of which the risk of the hostile exploitation of novel scientific developments remains far from hypothetical. along with the risk of misuse of new knowledge, there is the risk posed by the lack of sufficient scientific knowledge. cross-disciplinary convergence opens a multitude of opportunities for manipulation and modification of living matter but, at the same time, it precludes almost any sensible assessment of the potential interactions likely to occur in the process. nano-based medicine is but one area that has attracted criticism in this regard. since some elements behave differently at nano-scale, it becomes extremely difficult to assess their level of toxicity or other negative side effects that they may exert. such is the case with long carbon nanotubes, which having been initially praised for their potential to improve implant development were later blamed for exhibiting asbestos-like behaviour that could lead to cancer. another area of converging science with far-reaching implications is synthetic biology, a cross-disciplinary field that draws upon strategies and techniques from molecular biology, chemistry, engineering, genomics, and nanotechnology and thus enables the design and modification of biological systems at a fundamental level. empowered by the tools of synthetic biology, in scientists managed to assemble a polio virus 'from scratch' in the absence of a natural template. and in craig venter and his team announced the construction of the first self-replicating synthetic cell which, in their view, was 'a proof of the principle that genomes can be designed in the computer, chemically made in the laboratory and transplanted into a recipient cell to produce a new self-replicating cell controlled only by the synthetic genome.' the controversial work has attracted criticism on several grounds, including the potential negative effects of the accidental or deliberate release of the novel organism in the environment and the arrogance of scientists to 'play god'. more broadly, both the polio and synthetic cell studies have exposed the obstacles to the regulation of synthetic biology. while some commentators dismiss the risk of bioterrorism, underscoring the key role of tacit skills and knowledge and the difficulties that the lack thereof poses to the replication of the experiments, other issues still merit attention. consider the question of access to commercially available genomic sequences. even though the oversight system for screening base pair orders has improved since the guardian report that exposed the lax regulations under which virtually anyone could order gene sequences, gaps still remain leaving scope for abuse by those with malign intent. for example, schmidt and giersch have outlined at least three areas of emerging challenges that the existing governance regimes would struggle to accommodate, including 'split orders', 'outsourcing', and the potential for non-natural biological systems. the human genome project completed in lasted over ten years and cost close to billion dollars; by contrast, about a decade later, wholegenome sequencing can be performed within hours at a price of roughly dollars or less. while still in its infancy, personalised medicine and individual genetic testing are steadily gaining popularity. indeed, 'up to people in england are expected to have their entire genetic makeup mapped in the first stage of an ambitious public health programme' launched by the national health service in that aims to 'revolutionise the treatment and prevention of cancer and other disease.' according to its proponents, genomic testing offers numerous advantages vis-à-vis traditional evidence-based medicine, including the possibility of early diagnostics of disease, of individually-tailored treatment and, perhaps most importantly, of disease prevention, as illustrated in the resolve of the hollywood actress angelina jolie to undergo double mastectomy after discovering she has an inherited genetic mutation that puts her at high risk of breast and ovarian cancer. but this is just the beginning. in scientists managed to sequence a foetus's entire genome using a blood sample from the mother and a saliva specimen from the father, a development that could potentially allow for a range of genetic disease conditions to be detected prenatally. and laboratory experiments have already demonstrated the efficacy of genetic therapy to cure mitochondrial disease by creating an embryo with genetic material from both parents and a third person acting as a donor. while truly breathtaking, the advances outlined above raise a host of thorny issues of ethical, social, and legal concern that merit public scrutiny and extensive deliberation before decisions regarding their widespread application are made. at a very basic level, there is the question of whether and to what extent we as individuals are capable of assimilating the information that our own genetic makeup may reveal. are we sufficiently resilient to cope with the emotional distress, anxiety, shame, stigma and guilt that the awareness of severe medical conditions that we or our closed ones are suffering or likely to develop? far from hypothetical, this question has prompted the establishment of a novel profession, that of the genetics counsellor whose task is to help patients overcome any negative effects, stress, or psychological trauma that the disclosure of their genomic map may create. this is just a partial solution though, for the crux of the matter lies in finding a way to deal effectively with risk and probabilities and we as humans are yet to demonstrate a capacity for understanding or relating them to our own lives. individual emotional turmoil, however significant, constitutes only the tip of the iceberg. according to daniel kevles, the torrent of new genetic information has already begun to fundamentally reconfigure social practices and inter-personal relations: it has been rightly emphasised that employers and medical or life insurers may seek to learn the genetic profiles of, respectively, prospective employees or clients. employers might wish to identify workers likely to contract disorders that allegedly affect job performance while both employers and insurers might wish to identify people likely to fall victim to diseases that result in costly medical or disability payouts. whatever the purpose, such genetic identification would brand people with what an american union official has called a life-long 'genetic scarlet letter' or what some europeans term a 'genetic passport'. linking genetic makeup with human identity would ultimately set the scene for the proliferation of technologies aimed at human enhancement: after all, if a gene therapy could allow one to stand a chance in a job competition, boosting one's capabilities would potentially make them a more desirable candidate. other issues of more immediate concern are also likely to arise. one is privacy. gene-sequencing companies usually hold the genetic data of their clients in digital format on online platforms, which automatically creates a risk that personal information may be leaked, hacked or stolen. further, there is the question of ownership. consider, for instance, the controversial issue of human gene patenting, whereby patented genes are treated as research tools and, as such, are controlled by the patent holder who may restrict and charge for their use. thus created, the system often operates to the detriment of patients by hindering research practice, elevating diagnostics prices and denying access to second and independent medical opinion. gene identification alone has a potential 'dark side' too, for it could enable the development of weapons targeted at groupspecific gene markers (e.g. ethnicity). pre-natal genetic testing is yet another significant bone of contention, not least because it evokes notions of state-mandated eugenic programmes and assaults on human rights and dignity. while a nazi-like campaign for a superior race seems improbable in the twentieth-first century, this is not to say that other forms of eugenics may not be encouraged. indeed, some commentators have highlighted the rise of 'homemade eugenics', whereby individual families can make decisions on the attributes of their progeny: the lure of biologically improving the human race, having tantalised brilliant scientists in the past, could equally seduce them in the future, even though the expression of the imperatives may differ in language and sophistication. objective, socially unprejudiced knowledge is not ipso facto inconsistent with eugenic goals of some type. such knowledge may, indeed, assist in seeking them, especially in the consumer-oriented, commercially driven enterprise of contemporary biomedicine. it is plausible to assume that when presented with the opportunity of having their future child tested for genetic disorders, many parents would barely hesitate to accept. such a resolve could have far-reaching implications though. for instance, some genetic therapies entail the use of donor dna different from that of the parents, whereby any genetic modifications in the embryo will pass down to future generations. despite the government support for the 'three-parent babies' in the uk, local religious organisations have protested vociferously against the legalisation of the technique. at the same time, there are certain genetic disorders that can be diagnosed at an early stage but, as of yet, cannot be cured, which inevitably poses the tough choice between raising an unhealthy child and abortion. to be sure, such questions constitute more than individual parents' dilemmas, for they touch upon established social and cultural values, something evident in the profound differences across national reproductive policies. more broadly, there are concerns that reproductive genomics may remain a prerogative of those affluent enough to afford it, thus further exacerbating the divide between the global rich and the global poor. the growth of life science capacity over the past few decades across the globe has been truly astonishing, leading to the emergence of a vibrant research community that brings together researchers from various parts of the world. indeed, a nas report highlights the extension of both north-south and south-south partnerships, which has played a key role in synergising strengths and maximising competitiveness by improving the quality and effectiveness of research and facilitating data sharing. at the same time, increasing collaboration in the realm of biotechnology industry has offered companies situated in emerging economies access to the global market, thus contributing to economic development and growth. recent advances in technology and laboratory and experimental equipment have further impacted on the practice of life science research in profound ways. improvements in dna sequencing technology have significantly shortened the time required for the preparation of nucleic base-lines, thus relieving scientists of the burden of completing the task themselves and allowing them to focus on their actual project instead. studies and experiments once performed by senior researchers with extensive experience are now carried out by masters students. aided by specially designed genetic engineering toolkits, children as young as the age of ten start exploring the realm of biology in an interactive and engaging manner. needless to say, their notion of science and the world in general would differ significantly from that of their parents whose primary sources of knowledge used to be textbooks and encyclopaedias. indeed, the increasing commercialisation of synthetic biology offers anyone curious enough to fiddle with biological systems the chance of doing so in the comfort of their own home. such modern gene hackers often lack formal background in biology and come from various walks of life. driven by an insatiable appetite for knowledge and the vision of a ground-breaking discovery that could be turned into a multimillion dollar profit, they take up the rather unusual hobby of biohacking which entails the redesign of existing and the creation of novel biological systems. for just few hundred dollars bio enthusiasts set up laboratories easily obtaining all essential requisites and equipment from online sales. and if to some biohacking equates to little more than an unusual hobby, others highlight its potential to generate substantial revenue and fuel economic development. contrary to popular expectation, biohackers are not just eccentric individuals who work in solitude away from public attention. rather, they are members of a wide global movement dedicated to the ideal of do-it-yourself biology (diy), which has branches in locations on four continents. the movement has been partially institutionalised through the establishment of the biobricks and international genetically engineered machine (igem) foundations, which seek to promote the open and ethical conduct of biological engineering and stimulate innovation and creativity. to this end, igem holds an annual competition open to high school students, university undergraduates and entrepreneurs from all over the world. with more than participating teams, the competition constitutes the premiere forum at which biohackers can showcase their skills through project presentation. exciting as it may seem, the ongoing diffusion of life science expertise poses an array of governance conundrums. at the level of professional practice, the proliferation of research facilities around the world has exposed the urgent need for laboratory biosafety and biosecurity training, especially in developing states where a tradition of handling dangerous pathogens is lacking. the issue is further complicated, for such countries often lack the required legal and institutional infrastructure to ensure that professional practice is in compliance with relevant international regulations. foreign aid has gone some way in helping overcome those deficiencies but it has given rise to new problems, too. for instance, it is far from unlikely for a donor state to provide material support for the construction of a state-of-theart laboratory eventually leaving its maintenance to the local government, which can hardly afford the subsequent costs. a similar trend is observed in the area of capacity building and human resource development. most projects that aim to promote biorisk management and a biological security culture tend to be severely constrained in terms of time and funding and overly ambitious in terms of agenda and expected outcomes. lack of adequate mechanisms for quality assessment hinders progress evaluation and sometimes leads to duplication of effort and resources. the emergence of the diy biologists in the life science arena has further added to the challenge of ensuring that novel scientific and technological developments are utilised in a safe and ethical manner. even at the level of everyday practice, difficulties still persist. for instance, many amateur scientists have complained of the lack of manuals and guidelines regarding the safe operation and maintenance of home laboratories. issues such as waste disposal, safe handling and storage of biological material and prevention of contamination pervade the work of biohackers who unlike professional researchers conduct experiments in a much more volatile environment. potential security concerns are also present. with more and more individuals gaining access to biological engineering technologies, ensuring appropriate oversight of what goes on in garage laboratories becomes increasingly difficult. the experience of the us fbi is a case in point. back in the fbi arrested steven kurtz, a professor at the university of buffalo under the suspicion of plotting a bioterrorist attack. the subsequent investigation revealed that all laboratory and dna extraction equipment found in kurtz's house was legitimately obtained and used in his artwork. in an attempt to avoid mistakes of this kind, the fbi has drastically changed its approach to dealing with the diy movement launching a series of outreach activities that seek to raise awareness of the potential security implications of biohacking. while undoubtedly necessary, such initiatives may well be seen as too little, too late in light of the wide spread of materials, tools and devices that could facilitate the malign misuse of the life sciences. indeed, it is worth noting that as early as the late s the us defence threat reduction agency (dtra) managed to build a research facility that simulated the manufacture of weaponised anthrax using only commercially available materials and equipment. the role of states: both a poacher and gamekeeper structural factors have an important bearing on the development and growth of biotechnology. economic considerations, power interests and realist fears generate potent dynamics that shape and influence and sometimes direct the life science trajectory. within this context, states assume a dual role. on the one hand, they are expected to act as gamekeepers and regulate, monitor and control the process of life science research and the dissemination of novel technologies. on the other hand, though, they also have powerful incentives to act as 'poachers', not least because of the fascinating opportunities for enhancing their prosperity, prestige and security that scientific and technological development open up. the following passage effectively outlines states' dual function: government has an important role in setting long-term priorities and in making sure a national environment exists in which beneficial innovations will be developed. there must be a free and rational debate about the ethical and social aspects of potential uses of technology, and government must provide an arena for these debates that is most conducive to results that benefit humans. at the same time, government must ensure economic conditions that facilitate the rapid invention and deployment of beneficial technologies, thereby encouraging entrepreneurs and venture capitalists to promote innovation. given that the agent (i.e. state governments) in charge of initiating ethical debates on the progress of biotechnology is also the one expected to provide the conditions that would allow this progress to generate outcomes likely to contribute to economic growth and political superiority, it is hardly surprising that any issues likely to slow down or otherwise hinder the enormous momentum of the life sciences are omitted from public discussion. this duality further informs how risks are perceived, framed and addressed. for instance, even though most of the developing countries lack capacity to manage dual use research of concern, they do not see this as an immediate priority and prefer to invest effort and resources in improving their laboratory biosafety and laboratory biosecurity infrastructure and capacity. in the view of their governments, the dangers of naturally occurring and circulating diseases constitute a far greater worry than the potential for misuse of cutting-edge research. by contrast, some developed countries, most notably the usa, have embarked on building their biological defence systems highlighting the grave threat posed by the potential use of bioweapons by non-state actors. their activities have encountered severe opprobrium as some analysts see them as a contravention of the norms embedded in the btwc. the evolution of the chemical and biological non-proliferation regime epitomises the attempts of states to avert the hostile exploitation of the life sciences whilst promoting their use for 'peaceful, prophylactic and protective purposes'. the entry into force of the btwc and the chemical weapons convention (cwc) in and , respectively, is indicative both of states' renunciation of chemical, biological, and toxin weapons and of their commitment to the goals of arms control and disarmament. that said, the imperfections and shortcomings of these treaties signify the influence of realist fears and political calculations that pervade international negotiations. in the case of the btwc, two points merit attention. the first pertains to the lack of verification mechanism when the treaty was first agreed back in the early s. subsequent revelations of secret state-led offensive biological programmes in the former soviet union, south africa and iraq up until the early s have significantly undermined the convention. second, the failure to negotiate a binding protocol in has further dimmed the prospects for strengthening the regime and thus ensuring universal compliance with its prescriptions. less acute but just as worrying is the situation regarding the cwc. even though the convention is exemplary in many respects, not least because of its verification system, almost universal membership and implementing body -organisation for the prohibition of chemical weapons (opcw)it still faces serious challenges that need to be considered. for instance, while the treaty bans the development, production, acquisition, and retention of chemical weapons, the definition of 'purposes not prohibited under th[e] convention' entails 'law enforcement including domestic riot control purposes' (article ii. d). some commentators have argued that given the lack of a universally agreed definition what kind of activities count as 'law enforcement', this text opens a major loophole in the convention. several states parties of the convention have voiced concerns in this regard. australia has noted that: the weaponisation of [central nervous system] acting chemicals for law enforcement purposes is of concern to australia due to the health and safety risks and the possibility of their deliberate misuse, both of which have the potential to undermine the global norm against the use of toxic chemicals for purposes prohibited by the convention. [ . . . ] australia's position is that it is not possible for a state party to disseminate anaesthetics, sedatives or analgesics by aerial dispersion in an effective and safe manner for law enforcement purposes. critics highlight the possibility for the deployment of novel chemical weapons for the purposes of countering terrorism, something evident in the moscow theatre siege (dubrovka) when the russian security forces used a fentanyl-derivative agent, as a result of which about a sixth of the hostages and all of the terrorists involved died. in the european court of human rights ruled with regard to the dubrovka operation that: there had been no violation of article (right to life) of the european convention on human rights concerning the decision to resolve the hostage crisis by force and use of gas. the court, nonetheless, noted that: even if the gas had not been a 'lethal force' but rather a 'non-lethal incapacitating weapon', it had been dangerous and even potentially fatal for a weakened person [ . . . ]. the court further confirmed some of the earlier criticisms that were levelled against the government, particularly in terms of preparedness and provision of medical assistance. according to the ruling, russia had to pay damages to all the applicantsrepresentatives of siege victims. to date, russian officials have withheld information concerning the exact formula of the gas, which was used during the dubrovka operation, on security grounds. given the lack of an internationally agreed definition of what constitutes 'terrorism' on the one hand, and the rise of irregular/asymmetric warfare and sporadic conflicts, on the other, some commentators have warned against the possibility of a 'grey area' which may enable states to utilise non-traditional methods of war to gain advantage. deliberative systems encompass a vast array of practices, processes and mechanisms, both formal and informal, whereby a polity considers the 'acceptability, appropriateness and control of novel developments in or impacting on, shared social and physical arenas'. by design, they reflect and are informed by the values, beliefs and standards shared among the group, or in other words, by the prevalent culture. as such, deliberative systems vary across societies with their intensity, inclusiveness and structure depending on the established political and social norms. yet their chief purpose and function remain virtually the same, namely to help societies adapt to the changing circumstance of their milieu in a way that ensures stability, sustainability and safety. public deliberation requires time; and wide-ranging life science advances, current and planned, offer profound challenges to shared ideas and ideals about the foundations of human relatedness and of social coherence, justice, human dignity and many other norms, both formal and informal. yet given the ruminative nature of deliberative processes, on the one hand, and the fast speed at which biotechnology innovation is evolving on the other, the danger of the former being steadily outpaced and overburdened by the latter is far from hypothetical. consider the following passage sketching the scale of social changes likely to arise from the increasing convergence between nanotechnology, biotechnology, cognitive neuroscience and information technology: in the foreseeable future, we will be inundated with new inventions, new discoveries, new start-ups, and new entrepreneurs. these will create new goods and new services. [ . . . ] as expectations change, the process of politics and government will change. people's lives will be more complex and inevitably overwhelming. keeping up with the changes that affect them and their loved ones exhausts most people. they focus most of their time and energy on tasks of everyday life. in the future, when they achieve success in their daily tasks, people will turn to the goods and services, the new job and investment opportunities, and the new ideas inherent in the entrepreneurial creativity of the age of transitions. no individual and no country will fully understand all of the changes as they occur or be able to adapt to them flawlessly during this time. this vision of a 'brave new world' merits attention on two important grounds. first, it implies that the changes likely to occur in the not too distant future as a result of the rapid progress of science and technology are imminent and unavoidable in the sense that their advent hardly depends on or even requires extensive public deliberation. second, given that our capacity for adaptation to and grasp of those changes will be considerably impaired, the age of transitions leaves little space for public deliberation. to add to this gloomy picture, there is already some evidence that the progress in the life sciences is overwhelming the existing deliberative mechanisms. for instance, kelle et al. argue that the rapidity of biotechnology advancement coupled with the immensity and complexity of the knowledge accumulated therefrom complicates efforts to deal with potential risks, something evident in the regulatory gap that the convergence of chemistry and biology has created in the area of arms control. this is problematic, for the reduced resilience of deliberative systems provides favourable conditions in which scientific and technological innovation can continue unabated. a vicious circle is thus created in which the inability of deliberative systems to cope with the strain exerted by biotechnology advancement fuels the latter turning it into a self-propelling force. the proliferation of contentious 'gain-of-function' research is a case in point. even though the h n controversy discussed in the preceding sections exposed the limitations of existing governance mechanisms for addressing the potential security, ethical, and legal implications arising from such studies, it hardly precluded scientists from conducting similar experiments. indeed, less than four months after the moratorium on research involving contagious h n virus was lifted, a team of chinese researchers announced the creation of a hybrid of the h n strain and the h n virus that caused the flu pandemic. and it was not long until the newly-emerged h n influenza virus became airborne, as well. if anything, those examples indicate that in light of the rapid pace of life science progress, addressing governance concerns on a caseby-case basis is not only self-defeating but given the number and variety of conundrums, it is likely to become unsustainable in the long run. given the significant potential of biotechnology to bring about multifaceted changes in different spheres of life and generate considerable benefits in the form of new products, enhancement of public and private capital and alleviation of social ills, there is a powerful urge to allow the ongoing expansion of the life sciences to proceed largely unfettered. risks are carefully calculated and, where possible, downplayed as hypothetical at the expense of comprehensive deliberation. and even when proposals for risk mitigation measures are entertained, preference is usually given to those unlikely to hinder the progress of life sciences. by and large, there is a genuine belief that the existing governance mechanisms in the area of biotechnology can accommodate and cope with the wide-ranging pressures exerted by scientific innovation and the rapid diffusion of technologies with multiple uses, by offering 'solutions' and handling concerns on a case-by-case basis. in particular, the technology of safety is still 'celebrated as an unadulterated improvement for society as a whole'. yet there are reasons for scepticism toward the adequacy and effectiveness of the governance approaches currently in place. much of the discussion in the preceding sections has focused on the ways in which the increasing pace, growth and global diffusion of biotechnology advances are beginning to expose the limits of the existing measures for control and risk management by challenging accepted values and beliefs and redefining established norms of practice. as the multifaceted dynamics driving the biotechnology momentum continue to intensify and multiply, it becomes more and more difficult to comprehend, let alone foresee, the various impacts that the large-scale deployment and proliferation of novel scientific and technological advances have both on our social systems and the environment. given the tight coupling between human-made and natural systems and their complex, often unanticipated interactions with catastrophic potential, the existing narrow definitions of risk are rendered inadequate. at the same time, the advent of new technologies with multiple adaptive applications opens up an array of possibilities for hostile exploitation thus compelling governments to make tough decisions in an attempt to reconcile the benefits of biotechnology with the potential security concerns arising therefrom. while the advancement of biotechnology promises tremendous public health benefits, it also holds a considerable catastrophic potential, as the case of 'gain-of-function' experiments illustrate. as scientific capabilities and work involving dangerous pathogens proliferate globally, so do risks and the prospects of failures, whether technical or arising from human error. indeed, assessing the rapidly evolving life science landscape some security commentators argue that 'current genetic engineering technology and the practices of the community that sustains it have definitively displaced the potential threat of biological warfare beyond the risks posed by naturally occurring epidemics' . laboratories, however well equipped, do not exist in isolation but are an integral part of a larger ecological system. as such, they constitute a 'buffer zone' between the activities carried out inside and the wider environment. and despite being technically advanced and designed to ensure safety, this 'buffer zone', just as other safety systems is far from infallible. for one thing, mechanical controls leave room for human error and personal judgement, both of which are factors that could be highly consequential but which could hardly be modelled or predicted with exact certainty. the speed at which the transformation of the life sciences is taking place is yet another factor that adds to the complexity of life science governance. stability is a fundamental condition for the development and preservation of human and natural systems alike. in social systems, culture is the primary source of stability, for it determines what values, beliefs, practices and modes of behaviour are deemed acceptable and, as such, lays the foundations of order. all forms of governance therefore are cultural artefacts and manifestations of culture. culture also provides the tacit standards whereby change is assessed and treated as acceptable or unacceptable. hence, any state of affairs in which the rate of change precludes regulation disrupts the ordinary functioning of the system and jeopardises its preservation: the breakdown of human regulation does not extinguish regulation of a simpler sort. [ . . . ] the system formed by men and the rest of the natural world will continue to regulate itself after a fashion, even if human regulation wholly fails at all levels above the primary group. but the resulting 'order' would exclude all those levels of human order which man-made stability makes possible. to be sure, a world characterised by a runaway biotechnology would be far different from the one we know. the main challenge to averting this prospect lies in ensuring that the systems of governance are in sync with the progress of life sciences. history has shown that even highly developed, long-standing systems of governance can fail for reasons as diverse as disasters; loss of authority/legitimacy of governing bodies; and pervasive corruption. one further source of failure includes the inability of a society to adapt to its changing milieu: men are adaptable; they can learn to live even in harsh and hostile environmentsso long as the environment remains constant enough to give them time to learn. [ . . . ] if they form the habit of adapting by constantly changing that to which they are trying to adapt, they build uncertainty into the very structure of their lives. they institutionalise cluelessness. the process of adaptation is closely connected to cultural patterns and any serious disruptions in the latter could have detrimental effects and impair it severely. the extent to which change is taking place within the framework of the prevalent culture defines the borderline between system evolution and system disintegration. the governance mechanisms currently in place, both formal and informal, are all a function of historical, cultural, and sociopolitical contingencies. as such, their capacity for adaptation largely depends on our ability to comprehend and assimilate the complex changes that the progress of biotechnology brings about. they can only evolve as fast as our shared standards, values, routines and perceptions allow them to. and that is why governance can hardly be reduced to a technocratic exercise; on the contrary, to be effective, it requires extensive deliberation and full appreciation of the far-reaching implications of novel life science advances. trends and drivers of change in the biomedical healthcare sector in europe: mapping report (dublin: european foundation for the improvement of living and working conditions chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template the test-tube synthesis of a chemical called poliovirus: the simple synthesis of a virus has far-reaching societal implications craig venter creates synthetic life form', the guardian moore's law pertains to the rapid rate of technological development and advances in the semiconductor industry, specifically the doubling of the number of transistors on integrated circuits that occurs approximately every months. although advances in the life sciences occur at more random intervals and are driven by new conceptual breakthroughs in understanding of biological processes, it is a useful metaphor for the exponential growth of knowledge related to biology. see committee on the advances in technology and the prevention of their application to next generation bioterrorism and biological warfare threats, an international perspective on advancing technologies and strategies for managing dual-use risks the myth of the biotech revolution: an assessment of technological, clinical and organisational change globalization, biosecurity and the future of the life sciences innovation in global industries: u. s. firms competing in a new world biotechnology and the un's millennium development goals top ten biotechnologies for improving health in developing countries rising to the challenge: u.s. innovation policy for global economy innovating in the new austerity, burrill & co's th annual report on the life sciences industry globalization and the future of the life sciences, op cit an international perspective on advancing strategies for managing dual-use risks the valley of the shadow of death', dspace@mit globalization and the future of the life sciences the biotechnology promise: capacity-building for participation of developing countries in the bioeconomy converging technologies for enhancing human performance: science and business perspectives converging technologies -shaping the future of european societies taking care of the symbolic order: how converging technologies challenge our concepts enhancement technologies and the modern self the ethics of nbic convergence bringing converging technologies closer to civil society: the role of precautionary principle', innovation: the innovation in global industries, op cit in the valley of the shadow of death see the 'infectious diseases' section of the who website for an overview of the nih budget for , see david malakoff and jeffrey mervis, 'first look: us spending deal a mixed bag for science within nih's flat budget, a few favourites', scienceinsider for information on the eu sixth framework programme and the activity area of life sciences cuba -battling cancer with biotechnology global status of commercialised biotech/gm crops plant genetic engineering: china hesitates on the brink', gmo safety rising to the challenge, op cit government academic, and venture firms come together in march to fund translational and early-stage development', fiercebiotech science for sale: the perils, rewards and delusions of campus capitalism relationships between academic institutions and industry in the life sciences -an industry survey the expanding role of university patenting in the life sciences: assessing the importance of experience and connectivity university-industry relationships and the design of biotechnology research entrepreneurial science in the academy: a case of the transformation of norms the triple helix of university-industry-government relations the triple helix: university-industry-government innovation in action the dynamics of innovation: from national systems and "mode " to a triple helix of university-industry-government relations commercialisation of the university and the problem choice by academic biological scientists see also dina biscotti et al. 'the "independent investigator": how academic researchers construct their professional identity in university-industry agricultural biotechnology research collaborations toward more secrecy in science: comments on some structural changes in science -and on their implications for an ethics of science varieties of secrets and secret varieties: the case of biotechnology for a detailed analysis on the us decision to reject the draft btwc protocol, see malcolm dando the white house the pitfalls of bioterrorism preparedness: the anthrax and smallpox experiences bioterrorism and smallpox planning: information and voluntary vaccination taking biodefence too far new labs, more terror a plague of researchers mixing bugs and bombs lab loses trio of plague mice', nature news anthrax letters' attack and the controversy of the us biodefence programme see jeanne guillemin army suspends germ research at maryland lab back to bioweapons? international health regulations (who, ), specifically section 'laboratory pandemic influenza preparedness framework for the sharing of influenza viruses and access to vaccines and other benefits (who for further discussion on the implementation of biotechnology regulations, see bo sundqvist et al. 'harmonisation of european laboratory response networks by implementing cwa : use of gap analysis and an "insider" exercise as tools on the development of health and safety regulations on the use of nanotechnology, see eileen kuempel et al. 'risk assessment and risk management of nanoparticles in the workplace: translating research into practice there is a debate on whether the 'american model' of science-policy making underpinned by neoliberal ideology is fully embraced in europe. see, for example, gabriele abels, 'the long and winding road from asilomar to brussels: science, policy and the public in biotechnology regulation further, a policy paper issued by a business taskforce appointed by the uk government issued a policy paper in late demanding the liberalization of the existing eu legislation which, in their view, 'places restrictions on products and technologies without adequate evidence of risk'. see department for business, innovation & skills and the prime minister's office, cut the eu red tape: report form the business task force proposed framework for the oversight of dual use life science research: strategies for minimising the potential misuse of research information biosecurity reconsidered: calibrating biological threats and responses some commentators have expressed scepticism toward the claim that scientific and technological advancement poses serious threats underscoring the importance of other factors, such as socio-economic and socio-technic contexts. see, for example, kathleen vogel, 'intelligent assessment: putting emerging biotechnology threats in context others, however, argue that advances in modern biology and medicine have implications for the evolution of biological weapon programmes. see malcolm dando, 'the impact of the development of modern biology and medicine on the evolution of offensive biological warfare programmes in the twentieth century expression of mouse interleukin- by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox the efficacy of cidofovir treatment of mice infected with ectromelia (mousepox) virus encoding interleukin- disaster in the making creation of killer poxvirus could have been predicted chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template a tale of two studies: ethics, bioterrorism and the censorship of science first synthetic virus created', bbc news university of bradford, available at www.brad.ac.uk/ acad/sbtwc (accessed / / ). see also german ethics council, biosecurity -freedom and responsbility of research on the framing of risk in biotechnology, see geert van calster, 'risk regulation, eu law and emerging technologies: smother or smooth? ethics of risk analysis and regulatory review: from bio-to nanotechnology european biotechnology regulation: framing the risk assessment of a herbicide tolerant crop framing the uncertainty of risk: models of governance for genetically modified crops denmark's regulation of agri-biotechnology: co-existence bypassing risk issues a distorted regulatory landscape: genetically modified wheat and the influence of non-safety issues in canada on the shortcomings of the existing models for public deliberation on the risks of biotechnology, see les levidow, 'european public participation as risk governance: enhancing democratic accountability for agrobiotech policy for critique of cost-benefit analysis, see also brian rappert the creation of contagious h n avian influenza virus: implications for the education of life scientists all meetings of the us national science advisory board for biosecurity (nsabb) convened to discuss the manuscripts were restricted to selected individuals and full proceedings were never published. moreover, the consequential consultation meeting organised by the world health organisation (who) in february which rejected the nsabb recommendation for a redacted publication of the manuscripts featured the lead scientists who conducted the experiments and representatives of the us national institutes of health flu experts -and one ethicist -debate controversial h n papers', scienceinsider fight over dutch h n paper enters endgame', scienceinsider fight over dutch h n paper enters endgame', scienceinsider dutch appeals court dodges decision on hotly debated h n papers', scienceinsider bias accusation rattles us biosecurity board experimental adaptation of an influenza h ha confers respiratory droplet transmission to a reassortant h ha/h n virus in ferrets airborne transmission of influenza a/h n virus between ferrets: materials/methods, supporting text, tables, figures, and/or refences safety incidents" at animal lab', bbc news notes airflow problems plague cdc bioterror lab high-containment laboratories: assessment of the nation's need is missing the scientist lab infection blamed for singapore sars case between and , several high-containment facilities in the us have experienced serious biosafety lapses. accidents involving dangerous pathogens such as the causative agents of anthrax and bird flu were reported biosafety in the balance' anthrax? that's not the real worry cdc closes anthrax and flu labs after accidents about the same time, there were also reports about smallpox vials being retrived after having been left unaccounted for over years. see ap, 'forgotten vials of smallpox found in storage room gain-of-function' influenza research and proposal to organise a scientific briefing for the european commission and conduct comprehensive risk-benefit assessment the third revolution: the convergence of the life sciences see committee on biomocular materials and processes, national research council, inspired by biology: from molecules to materials to machines a new biology for the st century nanotechnology: what it can do for drug delivery facing the truth about nanotechnology in drug delivery carbon nanotubes -bullets in the fight against cancer on the governance challenges brought about by the convergence between biology and other fields of science, see francis fukuyama and caroline wagner, information and biological revolutions: global governance challenges -summary of a study group report of the first workshop the body's own bioweapons innovation, dual use, and security: managing the risks of emerging biological and chemical technologies life sciences and related fields: trends relevant to the biological weapons convention building better implants some nanotubes could cause cancer and breeding: panacea or pandora's box?', news release on the security implications of synthetic biology, see international council for the life sciences synthetic biology and biosecurity awareness in europe on the social and ethical aspects of synthetic biology, see presidential commission for the study of bioethical issues, new directions: the ethics of synthetic biology and emerging technologies synthetic biology: social and ethical challenges framing biosecurity: an alternative to the biotech revolution model vogel's view are contested in jonathan tucker 'could terrorists exploit synthetic biology lax laws, virus dna and potential for terror on the issue of commercial order screenings, see stephen maurer et al. making commercial biology safer: what the gene synthesis industry has learned about screening customers and orders on the governance of synthetic biology, see catherine lyall dna synthesis and biological security industry self-governance: a new way to manage dangerous technologies unpacking synthetic biology: identification of oversight policy problems and options synthetic biology, security and governance'¸biosocieties dna synthesis and security science enters $ , genome era', bbc news happened when i had my genome sequenced', the observer for information about commercial companies offering full-genome sequencing genome-based therapeutics: targeted drug discovery and development: workshop summary dna of , people to be mapped for nhs', the guardian systems cancer medicine: towards realisation of predictive, preventive, personalised and participatory (p ) medicine' grateful and moved" by reaction to her mastectomy decision', the guardian dna blueprint for fetus built using tests of parents cure for illness raises ethical fear', the guardian what happened when i had my genome sequenced another point that cadwalladr raises is the danger of a negative placebo effect whereby doubts about certain genetic disorder may lead to psychosomatic symptoms from eugenics to patents: genetics, law, and human rights unhidden traits: genomic data privacy debates heat up from eugenics to patents see also harriet washington, deadly monopolies: the shocking corporate takeover of life itself -and the consequences for your health and our medical future taking life: private rights in public nature benefits and threats of developments in biotechnology and genetic engineering' in sipri yearbook, armaments ethnic-affiliation estimation by use of population-specific dna markers racial differences in the response to drugs -pointers to genetic differences the achilles' helix from eugenics to patents three-person" embryos to combat genetic disease', the guardian innovative genetic treatment to prevent mitochondrial disease, press release three-parent embryos for mitochondrial disease? twelve reasons for caution february , the uk passed legislation allowing the use of the technique. see james gallagher, 'uk approves three-person babies', bbc news building baby from the genes up one commentator distinguishes between 'big science' which was 'todown, hierarchical, vertical' and 'networked science' characterised by 'open systems, open software, open participation'. see diane rhoten biopunk: solving biotech's biggest problems in kitchens and garages life hackers biology is technology: the promise, peril, and new business of engineering life diffusion of synthetic biology: a challenge to biosafety do i understand what i can create: biosafety issues in synthetic biology charge dropped against artist in a terror case strategies to educate amateur biologists and scientists in non-life science disciplines about dual use research in the life sciences governing amateur biology: extending responsible research and innovation in synthetic biology to new actors, research report for the wellcome trust project 'building sustainable capacity in dual use bioethics secret project manufactured mock anthrax', the washington times biological weapons and america's secret war global governance and the twenty-first century technology converging technologies for improving human performance: nanotechnology, biotechnology, information technology and cognitive science report of the who informal consultation on dual use research of concern biological threat assessment: is the cure worse than the disease? dangerous ambiguities: regulation of riot control agents and incapacitants under the chemical weapons convention weaponisation of central nervous system acting chemicals for law enforcement purposes, xix session of the conference of the states parties moscow theatre siege: questions remain unanswered', bbc news press release: use of gas against terrorists during the moscow theatre siege was justified, but the rescue operation afterwards was poorly planned and implemented spasenie zalozhnikov ili unichtozhenie terroristov?', novaya gazeta the dubrovka and beslan hostage crises: a critique of russian counter-terrorism sekretov bol'she net', rossiskaya gazeta see also national research council, avoiding surprise in an era of global technology advances the challenge to deliberative systems of technological systems convergence converging technologies for improving human performance, op cit preventing a biochemical arms race, op cit bringing the bwc conventions closer together', the cbw conventions bulletin h n hybrid viruses bearing /h n virus genes transmit in guinea pigs by respiratory droplet infectivity, transmission, and pathology of human-isolated h n influenza virus in ferrets and pigs limited airborne transmission of h n influenza a virus between ferrets shifting the blame: literature, law, and the theory of accidents in nineteenth-century america normal accidents, op cit building a bio world shifting the blame, op cit freedom in a rocking boat: changing values in an unstable society some commentators have critiqued the work of diamond on the grounds of simplicity. for a summary of some of the criticisms levelled at his work, see eric powell key: cord- -jj fqcen authors: freudenberg, nicholas title: health research behind bars: a brief guide to research in jails and prisons date: journal: public health behind bars doi: . / - - - - _ sha: doc_id: cord_uid: jj fqcen while most people make staying out of jail and prison a priority, a growing number of researchers are eager to get into correctional facilities in order to study the criminal justice system, the causes and consequences of incarceration, and the role of corrections in our society. for health researchers and their collaborators, the audience for this chapter, correctional facilities offer several unique advantages: a population at high risk of many health problems including infectious and chronic diseases, substance abuse, and mental health problems; social and physical environments that can enhance or impede well-being; a setting that is a focal point for the class, racial/ethnic, and gender differences that divide the united states; a site where health and mental health services and prevention programs are offered and can be evaluated; a controlled environment for administration of treatments such as directly observed therapy for tuberculosis; and a stopping point in the cycle of incarceration and reentry that so profoundly affects community well-being. while most people make staying out of jail and prison a priority, a growing number of researchers are eager to get into correctional facilities in order to study the criminal justice system, the causes and consequences of incarceration, and the role of corrections in our society. for health researchers and their collaborators, the audience for this chapter, correctional facilities offer several unique advantages: a population at high risk of many health problems including infectious and chronic diseases, substance abuse, and mental health problems; social and physical environments that can enhance or impede well-being; a setting that is a focal point for the class, racial/ethnic, and gender differences that divide the united states; a site where health and mental health services and prevention programs are offered and can be evaluated; a controlled environment for administration of treatments such as directly observed therapy for tuberculosis; and a stopping point in the cycle of incarceration and reentry that so profoundly affects community well-being. in this chapter, i consider the benefits and perils of doing health research in jails and prisons. the chapter begins with a brief overview of the different types of health research conducted within correctional facilities and among those leaving jail or prison. i then describe some of the unique obstacles that correctional health researchers encounter and assess some of the methods they have used to overcome these obstacles. since researchers in correctional settings face significant ethical dilemmas, i next consider recent frameworks for making ethical decisions about this research. finally, i suggest an agenda for future health research in correctional settings. health research behind bars: a brief guide to research in jails and prisons in recent decades, researchers from a variety of disciplines including health services research, public health, medicine, criminal justice studies, sociology, psychology, anthropology, organizational studies, and others have initiated studies on health in the correctional system. a brief typology of the different categories of questions these investigators have asked will help to set the stage for our consideration of approaches to correctional research. . what are the health and social characteristics of people in jail and prison? numerous studies have examined the health and demographic profile of incarcerated populations. these vary from large studies based on national samples and using multiple health outcomes such as the reports of the national correctional health care commission on the health status of soonto-released inmates ( a, b) or of the health status of inmates in texas prisons (baillargeon, black, pulvino, & dunn, ) to studies of a single outcome such as hepatitis c among california inmates (fox et al., ) . the various reports of the bureau of justice statistics on mental health, substance use, and other health conditions (e.g., james & glaze, , karberg & james, summarize data across u.s. jurisdictions, providing an opportunity for correctional and health officials to identify incarcerated populations in higher need. other studies describe patterns of health care utilization among inmate populations (leukefeld et al., ) . investigators often compare the health status of different subpopulations, e.g., men to women (peters, strozier, murrin, & kearns, ) , or african-americans and latinos to whites (rounds-bryant, motivaus, & pelissier, ) . these descriptive studies are used to identify the needs of various segments of the incarcerated populations, to compare changing incidence or prevalence of conditions over time, or to serve as a baseline for the subsequent evaluation of interventions. research imperatives in these studies are consistent definitions of dependent and independent variables, uniformity in data collection methods in multisystem studies, and sampling strategies that enable generalizations to other settings. . how does the health of inmates differ from that of nonincarcerated populations? a second group of studies compare the health of incarcerated populations with the health of the general population or with samples of nonincarcerated people. for example, teplin and colleagues' studies of the prevalence of mental health conditions among women and juveniles in chicago jails found higher rates of some psychiatric conditions in incarcerated populations than in similar populations living in the same catchment area from which inmates had been arrested (teplin, ; teplin et al., ) . these studies set the stage for the next group of studies. methodological issues in this type of study include selecting an appropriate comparison group. . how does incarceration itself affect the health of incarcerated populastions? both correctional and public health authorities want to know whether observed differences between incarcerated and nonincarcerated populations are due to differences in the composition of the populations or to the experience of incarceration, a variant of the classic epidemiological task of distinguishing between compositional (i.e., characteristics of the population) and contextual effects (i.e., characteristics of an environment). for example, numerous investigators have sought to determine whether the higher prevalence of hiv infection among u.s. prison populations was due to intraprison transmission or to criminal justice policies that led to incarceration for people already hiv infected (hammett, ; krebs & simmons, ) . most studies suggest the latter route is more important, reassuring correctional authorities that within-prison transmission, while it does occur, is not a major factor in higher rates. on the other hand, studies in the early s established that tb transmission did occur within the facility, leading to substantial efforts to prevent such transmission (bellin fletcher, & safyer, ) . others have investigated whether incarceration is associated with homelessness and mental illness (mcneil, binder, & robinson, ) . the main analytic task in these studies is to distinguish between causal and noncausal associations between incarceration and selected health outcomes. . what are the health effects of criminal justice policies and practices on the health of inmates? criminal justice policies often have unintended effects on incarcerated populations. documenting the positive and negative impact of these policies can serve as a starting point for policy change. for example, a study in a large public hospital in new york city found that many admissions for diabetic ketoacidosis were related to the court practice of denying inmates access to insulin medications in court pens (keller et al., ) . health impact assessment, an analytic method developed to assess the health effects of both health and non-health-related policies, offers a promising approach to consider the health consequences of various prison and criminal justice policies (davenport, mathers, & parry, ; kemm, , veerman, barendregt, & mackenbach, . to date, however, this approach does not seem to have been used to assess the impact of u.s. correctional policies on inmate or community health. . what is the impact of interventions designed to care for or improve the health of incarcerated populations? a key practical question for correctional, public health, and correctional health officials is the effect of the programs they run on the well-being of the populations in custody. evaluation studies seek to document the utilization of health services (lindquist & lindquist, ) ; assess their impact on health or health care utilization (e.g., chan, vilke, smith, sparrow, & dunford, ; edens, peters, & hills, ) ; analyze the cost-benefits of an intervention (ncchc, a) ; or compare the cost-effectiveness of various approaches to a specified health problem, e.g., screening for hiv or other infectious diseases within correctional settings (resch, altice, & paltiel, ; kraut-becher, gift, haddix, irwin, & greifinger, ) . in these studies, methodological issues include the specification of clearly defined outcomes, the use of standard accepted measures for assessing costs and benefits of various interventions, and the design of evaluation studies that are both methodologically sound and operationally feasible. . how does reentry affect the health of incarcerated populations? in the last decade, correctional health researchers have begun to follow their research participants back into the community, examining their success in finding health services or drug treatment (jarrett, adeyemi, & huggins, ; lincoln et al., ) , maintaining control of a mental health condition (wilson & draine ) , or in improving hiv care or reducing hiv risk behavior (bauserman et al., ; rich et al., ; myers et al., ) . these studies can be part of an evaluation of a reentry program (e.g., needels, james-burdumy, & burghardt, ) or a descriptive study of the outcomes of the reentry process (e.g., freudenberg et al., ) . . what is the impact of incarceration rates on the well-being of communities and populations? finally, a growing number of researchers are studying the impact of incarceration and correctional policies on the health of families, communities, and populations. for example, some research looks at the impact of incarceration on children and other family members (murray & farrington, ; barreras, drucker, & rosenthal, ) . researchers have asked whether incarceration policies have contributed to the community transmission of hiv infection (leh, ; johnson & raphael, ) or other sexually transmitted infections (thomas & sampson, ) , community rates of violence (rose & clear, ) , or disparities in health between black and white u.s. populations (taxman, byrne, & pattav, ; johnson & raphael, ; iguchi, bell, ramchand, & fain, ) . these studies can help policy makers consider the impact of various incarceration policy choices. this brief summary of the types of questions that correctional health researchers have sought to answer illustrates the scope of the field. for neophyte investigators, becoming familiar with the findings and methodological challenges in the extant literature relevant to their question of interest can save years of trial and error in this difficult setting and avoid duplication of effort. for more experienced researchers, a familiarity with the scope of prior research can help them move from descriptive to analytic and intervention studies. several recent reviews provide a good starting place for becoming familiar with recent correctional health research (edens et al., ; freudenberg, ; magaletta, diamond, dietz, & jahnke, , morris, pollack, khoshnood, & altice, ) . successful health research in correctional settings requires familiarity with the existing literature described in the previous section, a knowledge of the research methods applicable to the correctional setting, discussed in the next section, and an understanding of the various stakeholders in correctional health, discussed here. without a map of this organizational landscape, even skilled researchers can lose their way. key participants in developing and implementing research studies in correctional settings include correctional officials, correctional health providers, public health authorities, other researchers and research institutions, elected officials, funders, prison and reentry advocacy groups and inmates and their families. each of these constituencies has the potential both to improve research and to stop studies before they get off the ground. thus, the practical researcher will want to understand how to enlist each of these groups in supporting the research process. correctional officials need to approve and at least not oppose any research study conducted in their facility. their main concerns are the extent to which research may pose a threat to safety and regular prison routines, fear of bad publicity, cost and liability concerns, or additional demands on their staff. researchers who can reassure correctional officials on these matters will have an easier time pursuing their studies. investigators who are unable (or un willing) to provide these assurances may need to consider other approaches to their research, such as interviewing participants after their release from jail or prison. in most situations, research studies will need the tacit support of at least three levels of correctional authorities: senior departmental managers (e.g., commissioners/directors or sheriffs); wardens of the facility(ies) where the study takes place; and frontline correctional staff. each level brings different concerns and requires different assurances in order to allow the research to proceed. for example, frontline correctional officers who may be required to bring participants to the researcher for interviews or medical examinations want to make sure these procedures do not interfere with their routines or increase staff workloads. wardens often need to be assured that no research procedure will jeopardize security. in another example, a jail security warden was concerned that a stylus for a handheld computer device used for interviews with inmates could be used as a weapon. it took several meetings between a warden and a research team to agree on a type of stylus and interview procedures. senior officials of corrections departments are sometimes ambivalent about studying illegal behavior such as drug use or voluntary or coercive sexual behavior. if they know that a problem exists, they may have an obligation to address it so that agreeing to research on these topics can have significant administrative, legal, and cost implications. researchers will need to be prepared to address these concerns. correctional health providers have a constitutional mandate to provide health care to people in custody, offering a theoretical rationale for research that helps to improve care or make it more efficient or economical. in practice, however, since the types and quality of these services are often the subject of litigation (nathan, ) , health providers often filter requests for participation in research projects through their potential impact on current or future litigation. in addition, similarly to corrections officials, correctional health authorities often believe that if they know about a problem they will be required to take action to address it. this has made some officials reluctant to support research on difficult-and expensive-conditions such as hepatitis c (spaulding et al., ) . researchers who want to study such topics will need to be able to address these concerns. correctional health providers operate under a variety of auspices, including public departments of corrections or health, universities, voluntary hospitals, or for-profit companies (mellow & greifinger, ) . these differing organizational sponsorships influence a unit's openness to research and their motivation to participate in research studies. as with other potential stakehold-ers, researchers need to initiate a straightforward discussion to identify areas of common interest and potential conflict before beginning a study. in some cases, correctional health providers have themselves initiated evaluation studies to guide practice. for example, the university of texas, which has a contract to provide health services for inmates in texas prisons, commissioned an independent evaluation of its services. the report generally lauded the texas program and made several suggestions for more systematic quality assessment (texas medical foundation, ) . public health authorities often have a legal mandate to provide oversight of correctional health services and always have responsibility for providing core public health services to people returning to their communities. these obligations provide an incentive for research that can identify unmet needs, improve the effectiveness or quality of care or reduce its costs, or demonstrate the impact of interventions. in practice, some state and municipal health departments have close and positive relationships with correctional health researchers and enlist their help in identifying and solving problems. others, either as a result of fears of litigation, new mandates for service, or unfavorable media attention, may be reluctant to establish partnerships with researchers. other researchers and research institutions can provide an important resource for both experienced and neophyte correctional health investigators. they can share their frontline experiences doing research in specific correctional systems or facilities, the study designs and instruments they have used, their solutions to issues of confidentiality and informed consent, or their findings from their previous research. in the last decade or so, a number of research centers focused on correctional health or reentry have been established, gaining valuable experience and producing a body of work that can inform future studies. some of these are listed in table . . since some federal funding agencies prefer multijurisdiction research projects in order to increase generalizability, establishing partnerships with experienced centers can help to design such studies and win funding for them. elected officials in both the executive and legislative branches are sometimes needed to approve funding for research studies (e.g., evaluation of publicly funded health or reentry interventions) or to pose questions that need study to correctional or health officials (e.g., how best to provide substance abuse treatment services to people in and returning from correctional facilities). in order to help these officials take on these roles, researchers can provide them with information documenting the problem, cost arguments on the potential savings from new approaches, and the public health benefits of correctional health services. many elected officials worry that supporting health services or even research on the health needs of people in jail or prison might lead to charges that they are "soft on crime" or coddling criminals. research evidence that can reframe the issues as public health, public safety, or economic concerns may help to provide a rationale for interest. funders provide the financial support for correctional and reentry health research and thus for this research to develop they must be willing to provide the level and continuity of funding needed to develop the field. given that both private and public funders always have more requests for support than resources, that prison health is always a less popular choice than, say, children's health or education, and that many funders change their priorities ( ) http://cira.med.yale.edu/ regularly, researchers face an uphill battle in winning the resources they need to pursue a comprehensive research agenda on correctional health. funders who have provided significant support to correctional health research include public agencies such as the national institutes of drug abuse, alcohol abuse and alcoholism, mental health, and allergy and infectious diseases, the centers for disease control and prevention, the national institute of justice, and some state and local governments. private funders include the robert wood johnson foundation, the kellogg foundation, the open society institute, and the jeht foundation, among others. to ensure long-term support, correctional health researchers will need to educate public and private funders about the connections between correctional health and public safety, public health, and social justice as well as to find ways to integrate correctional health issues into research on a variety of health and social problems. prison and reentry advocacy groups serve as important bridge between inmates and their families and the wider community. they also have the potential to influence policy makers, elected officials, and the media. their opposition to unsafe or unhealthy prison conditions, inadequate medical care, or violations of civil liberties have contributed to the development of standards for correctional health care and greater public attention to these issues (nathan, ) . the mission, scope, and activities of these groups vary widely, from national organizations such as the national prison project of the american civil liberties union, which brings legal action against correctional systems alleged to violate inmate rights, and critical resistance, an alliance of regional groups dedicated to radical reform of the criminal justice system, to local groups that seek to coordinate reentry programs or organize prison visiting programs. for researchers, these groups can provide detailed knowledge about prison conditions, inmate perceptions of problems, and the local political climate on correctional issues including health. establishing relationships of mutual trust and respect, even when the two parties may disagree on the causes or solution to a problem of interest, can deepen investigators' understanding of the context in which their research is carried out. finally, inmates and their families can provide the insider knowledge that can determine the success or failure of a research project. their understanding of the real-world intersection of policy and practice, the actual living conditions of inmates, and the problems that people leaving jail and prison face when they return home can help researchers to design their studies, develop their research instruments, and interpret their findings. many researchers have noted the benefits of participatory research-deeper knowledge of the problem under study, greater engagement of research participants in the process, and more meaningful interpretation of results (israel, schulz, parker, & becker, ; metzler et al., ) . in summary, correctional health researchers interact with a variety of stakeholders. at worst, these interactions can appear as a gauntlet of opponents, each with contradictory perceptions and demands that threaten the integrity of the research process and have the potential to disrupt or even halt any study. at best, however, each stakeholder can offer unique insights into the research problem, contribute distinct resources to the research process, and assist in making findings lead to improvements in practice, policy, and health. thus, developing skills in successfully negotiating these interactions is an essential prerequisite for the correctional health researcher. researchers in correctional facilities have used a wide variety of data sources to study inmate health. these include surveys of inmates or correctional authorities, clinical studies of inmate health, secondary analyses of national datasets, ethnographies, and reviews of existing prison health or criminal justice records. each of these sources of data has unique advantages and disadvantages. increasingly, researchers combine different types of data in order to gain deeper insights into the question of interest. for example, many correctional health studies will integrate survey data from participants, medical records from a correctional health service, and official criminal justice records in order to assess the impact of intervention programs. in general, the methodological questions in correctional health are similar to those in other settings: e.g., how to define variables of interest consistently, how to ensure that the data collected are reliable and valid, and how to select appropriate samples and comparison groups. a variety of standard research texts can help investigators to become familiar with these issues (e.g., boruch, ; datzker, ; noaks & wincup, ; patton, ) . research in correctional settings does pose some particular methodological challenges. for example, longitudinal studies that follow inmates into the postrelease period face the problem of locating participants after release. since people leaving jail or prison often lack residential stability and may not want further contact with those associated with the incarceration experience, achieving acceptable follow-up rates can be difficult. strategies that have been used to increase follow-up rates include collection of multiple contact names at study entry; frequent interim contacts in order to maintain updated locators, use of both service and financial incentives, and use of public records (e.g., "rap sheets" and criminal records) in lieu of face-to-face contacts. correctional health researchers, like other investigators, often struggle to design and implement multilevel studies that seek to understand the cumulative impact of more than one level of organization on inmate or community health. they may collect data on individuals, social networks such as family and peers, communities, correctional facilities, and jurisdictions, then seek to analyze the contribution each level makes to a specified outcome. for example, a study of women and male adolescents leaving new york city jails examined the impact of individual characteristics, the jail and reentry experience, conditions in the returning community, and changing municipal policies on crime, welfare, and housing on returning inmates' drug use, hiv risk behavior, and reincarceration (freudenberg et al., ) . multilevel analyses consider the contributions of variables at multiple levels to the variability in a particular individual-level dependent variable, e.g., drug use. in public health, multilevel research is increasingly used to assess the relative influence of neighborhood and individual-level variables on health (diez-roux, ) . by comparing these two influences within different jurisdictions, a third level of organization (i.e., city or state policies or services) can be studied. health research in correctional settings also faces organizational and logistical issues. these include finding space for confidential interviews (an extremely challenging task in overcrowded jails and prisons), negotiating use of technology such as computer-assisted interviewing devices with prison security officials, providing clearance and escorts for researchers, and gaining consistent and reliable access to research participants within the security confines of the facility. solving these logistical problems requires a close and collaborative relationship between researchers and correctional officials. defining common objectives at the inception of research, developing procedures for resolving conflicts before they emerge, and maintaining open communications with all levels of correctional authorities-from frontline correctional officers to wardens and commissioners-can help to reduce logistical problems. most importantly, researchers who choose to work in correctional settings must be willing to act as guests in someone else's house, rather than expect to develop their own rules of conduct. researchers who are unable or unwilling to accept this reality will face difficulty in working in prisons or jails. perhaps the most challenging aspect of health research in correctional settings is meeting the competing demands for ethical research practice as mandated by various bodies as well as the researcher's own ethical standards. prisoners pose ethical dilemmas for researchers not only because they lack the freedom to make the choices that most individuals in the free world take for granted, but also because so many prisoners experience other problems that make them vulnerable as research subjects: low levels of literacy, hiv infection, mental illness, victims or perpetrators of violence, as well as being adolescents. ethical questions correctional health researchers must address include: • what procedures ensure that all incarcerated people involved in studies have been given the opportunity to give informed and voluntary consent to participate in the research? • what research practices can guarantee that inmates have as much right to choose to participate in research as any other population? • how do correctional health researchers balance their ethical responsibilities to the correctional officials who commission their work or provide access to inmates with their responsibility to inmates? • what level of individual or population benefits in correctional health research balances potential risks? • how can researchers ensure that participation in correctional health research studies will not lead to harm through disclosure of confidential medical or criminal justice information to third parties? • what ethical responsibility do researchers have to bring the findings of their research in correctional settings to policy makers or others who can act on these finding? a brief review of the recent history of ethical issues in prison research helps to illustrate the competing forces and changing policy priorities. more in-depth discussion of this history can be found elsewhere (gostin, vanchieri, & pope, ; kalmbach & lyons, ; degroot, bick, thomas, & stubblefield, ; haney & zimbardo, ; hornblum, ) . in , hornblum observed that "from the early years of this century, the use of prison inmates as raw materials became an increasingly valuable component of american scientific research" (hornblum, ) . for example, in the s, major pharmaceutical companies, dow chemical, and the u.s. army tested experimental drugs at the holmesburg prison in pennsylvania (hornblum, ) . in , based in part on disclosures of research abuses in prisons, the national commission for the protection of human subjects of biomedical and behavioral research ( ) issued a report that set the framework for subsequent federal involvement in setting ethical standards for human experimentation. their report called for additional protection for certain "vulnerable" populations, including children, neonates, pregnant women, and prisoners. in , the commission issued a report titled "additional protections pertaining to biomedical and behavioral research involving prisoners as subjects" (u.s. dhhs, ) . the main goal of these early guidelines was to protect incarcerated individuals from serving as involuntary or coerced "guinea pigs" in research that offered no direct benefits and had the potential for harm. in the s and early s, the aids epidemic raised new ethical concerns for correctional health researchers. in some cases, prisoners with hiv infection or aids were not permitted to join clinical trials for new aids medications, based on various beliefs including their inability to give truly voluntary consent and their perceived unwillingness to comply with prescribed regimens. some health researchers and prisoners rights advocates argued that such a ban violated ethical principles and that prisoners should have the same access to experimental treatments and clinical trials as other sectors of the population. from this perspective, ethical guidelines should place a priority on ensuring access to potential beneficial treatments (dubler and sidel, ) -a priority that may conflict with the previous emphasis on protecting inmates from researchers. in , the institute of medicine commissioned another review of ethical issues involved in prisoner research (gostin, vanchieri, & pope, ) . based on several reviews of the more recent literature and testimony from dozens of witnesses including researchers, inmates, and correctional officials, the committee on ethical considerations for protection of prisoners involved in research made fourteen recommendations in five broad categories (table . ). these recommendations strive to find an appropriate and updated balance between the protection and access imperatives embodied in previous ethical standards. whether these institute of medicine recommendations lead to changes in federal guidelines for prison research or in practice remains to be seen. in practice, among the vexing problems correctional health researchers face are obtaining voluntary consent in jails or prisons, informing research participants about the benefits and risks of research, getting consent for randomized trials in which some participants receive no potential benefit, protecting the privacy of research participants, and negotiating with irbs that may lack expertise in the realities of prison research. defining "voluntary" consent in the coerced environment of a correctional facility is sometimes difficult. among the practices that can compromise free choice are promises of services not ordinarily available to inmates (e.g., certain types of health services), the presence of correctional officers in the area where consent is being solicited, the unavailability of the independent advice on participation that is normally available to research participants in the free world, or the implied offer to use participation in research in exchange for a shorter sentence or favorable consideration by a judge or parole board. since no set of rules can govern all the situations that can jeopardize voluntary consent, for any particular study the ethical researcher ought to consult experienced correctional researchers, correctional officials at the study site, prisoners rights advocates, and current and former inmates in order to obtain a variety of perspectives on the best procedures to insure voluntary consent. similarly, the process of informing research participants in correctional settings of the risks and benefits of a study can be challenging. many inmates have low levels of literacy; many distrust correctional and health authorities, sometimes based on their own past experiences; and, unlike most research in medical settings, an added risk is disclosure of information that can cause harm to participants from other inmates, correctional staff, legal authorities, or the wider public. research on stigmatized conditions such as hiv infection, mental illness, and substance use almost always poses such risks. methods that researchers have used to overcome these obstacles are to engage current and former inmates in the design of informed consent materials and as members of source: gostin et al. ( ) . irbs, to hire independent advisors who are not part of the research team to help inmates make decisions about participation, and to obtain federal certificates of confidentiality to minimize risk of disclosure of confidential information. while some inmates and ethicists express concerns about the coercion implicit in any research in the correctional setting, the recent iom report (gostin et al., ) also noted that other inmates strongly oppose restrictions on inmate participation in research. some are concerned about lack of access to cuttingedge treatments for hiv or cancer; others object to the loss of opportunities for compensation or enhanced living situations. a specific problem facing researchers involved in clinical trails in which some forms of treatment are withheld from some participants is convincing both staff and participants of the rationale for a randomized trial. from a researcher's point of view, the lack of definitive evidence of the benefits of an intervention is sufficient rationale for such a trial but for staff and participants, withholding services perceived to be beneficial may seem unethical. when staff are not convinced of the morality of a research study, they may intentionally or unintentionally undermine the study, either by providing services to the "control" group or by communicating their discomfort to research participants, thus discouraging enrollment in a study. for this reason, it is important for researchers to address this issue forthrightly. strategies to minimize this problem include offering all research participants some level of services above the standard care in the correctional facility, comparing different interventions to each other rather than to no special services, educating research staff about the ethics of offering unevaluated services to all participants, and, as the institute of medicine report on correctional research suggests (gostin et al., ) , joining advocacy efforts to improve the basic standard of care in all correctional facilities. in my experience, many correctional health researchers complain about the extensive and lengthy process required to get irb approval for their research study and suggest that it can discourage them from pursuing worthy projects. in some cases, several different irbs need to approve a single study and occasionally offer conflicting guidance on how to proceed. these complaints have a variety of sources: some investigators prefer the old way of business where researchers alone decided on the conduct of their studies. but even researchers who support the importance of protecting prisoners note that irb members often lack expertise in the day-to-day realities of correctional institutions and the nonresearch risks inmates encounter daily. they also report that irb committees often reflect the wider tension between protecting participants from research harm and ensuring access to beneficial services and in their effort to maximize both of these aims impose unreasonable demands on researchers. a possible solution is to assist irbs to find a member who is experienced in correctional settings and correctional research-not only to meet the dhhs regulatory requirement to include such a person but also to obtain practical advice on devising realistic and ethical resolution of problems. for example, one state prison system irb included an attorney who specialized in inmate litigation. another solution, as recommended by the iom report (gostin et al., ) , is to develop universal national standards for review of prison research so that all research is reviewed using uniform criteria. at present, correctional health researchers respond to a variety of heterogeneous influences -other criminal justice, medical, public health, and public policy researchers; local, state, and federal correctional and health officials; correctional health providers; a variety of professional organizations; elected policy makers; and various criminal justice and health advocacy organizations, among others. it is therefore not surprising that in this anarchic and complex environment correctional health researchers have yet to develop a coherent and comprehensive research agenda driven by existing scientific knowledge and public policy imperatives. however, the fact that it may be difficult to envision and articulate such an agenda should not stop the effort. in fact, as health and correctional officials and researchers request additional support for correctional health research, it is inevitable that they will be asked to set priorities. and if researchers themselves fail to take the lead in this process, others will impose an agenda on them. while the development of a comprehensive research agenda for correctional health is beyond the scope of this chapter, i conclude by suggesting some steps that might move the field in this direction. first, we need to begin a national dialogue on research needs that include researchers, correctional and health officials, policy makers, and advocates. questions to discuss include: what are the most promising avenues of research to lead to short-and middle-term improvements in the health of incarcerated populations? what are potential stable funding streams for this research? how best can we develop consistent frameworks for research so that clinical, practice, and policy decisions can be more evidence-based? who are the constituencies that will support a national research agenda on correctional health and how can these constituencies be organized into a coherent force? what correctional research might be particularly beneficial both to the health of the incarcerated and to the larger health of the public? organizations that can play a role in this national discussion include the national institute of justice, nih institutes and the centers for disease control and prevention, the national commission on correctional health care, various health professional organizations, and the reentry policy council. second, researchers need to synthesize the existing and disparate literature on correctional health to identify common findings, gaps in the literature, and future priorities. this literature is dispersed in several different disciplines and among the peer-reviewed and "gray" literatures, i.e., public and voluntary organization reports and studies. one possible sponsor for such a critical review would be the institute of medicine. third, as recommended by the recent iom report on correctional health research (gostin et al., ) , the united states should establish more consistent and uniform guidelines for ethical health research among incarcerated populations. such guidelines will protect researchers and inmates and help to resolve the continuing debate between protection from researchers and full access to the benefits of research. fourth, any agenda should consider the range of settings in which correctional health plays out, including courts, jails, prisons, parole and probation services, alternatives to incarceration, and reentry programs. too often, each setting has been its own silo with a cadre of researchers and officials. the evidence of the past decades suggests that in fact these settings constitute a single if sometimes disorganized system in which changes in one component affect all others. thus, health research needs to examine these systemic interactions in order to avoid shifting problems for one sector to another. finally, correctional health research has to be considered a branch of population health research and therefore address the broadest questions that affect the health of the public. in the past, some correctional health researchers have limited their attention to those individuals served in correctional health settings-the patients who walked through their clinic doors. while these concerns will continue to be important and warrant focused investigation, they are not sufficient to realize the full opportunity for correctional 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areas have not traditionally followed a systems approach towards mitigating food related health issues, and instead have relied upon specific issue interventions char deal with downstream indicators of illness and disease. in june of , the san francisco food alliance, a group of city agencies, community based organizations and residents, initiated a collahorarive indicator project called rhe san francisco food and agriculture assessment. in order to attend to root causes of food related illnesses and diseases, the purpose of the assessment is to provide a holistic, systemic view of san francisco\'s food system with a focus on three main areas that have a profound affect on urban public health: food assistance, urban agriculture, and food retailing. using participatory, consensus methods, the san francisco food alliance jointly developed a sec of indicators to assess the state of the local food system and co set benchmarks for future analysis. members collected data from various city and stare departments as well as community based organizations. through the use of geographic information systems software, a series of maps were created to illustrate the assets and limitations within the food system in different neighborhoods and throughout the city as a whole. this participatory assessment process illustrates how to more effectively attend to structural food systems issues in large urban areas by ( t) focusing on prevention rather than crisis management, ( ) emphasizing collaboration to ensure institutional and structural changes, and ( ) aptly translating data into meaningful community driven prevention activities. to ~xplore the strategies to overcome barriers to population sample, we examined the data from three rapid surveys conducted at los angeles county (lac). the surveys were community-based partic· patory surveys utilizing a modified two-stage cluster survey method. the field modifications of the method resulted in better design effect than conventional cluster sample survey (design effect dose to that if the survey was done as simple random sample survey of the same size). the surveys were con· ducte~ among parents of hispanic and african american children in lac. geographic area was selected and d .v ded int.o small c~usters. in the first stage, clusters were selected with probability proportionate to estimated size of children from the census data. these clusters were enumerated to identify and develop a list of households with eligible children from where a random sample was withdrawn. data collectmn for consented respondents involved - minutes in-home interview and abstraction of infor· ma~ion from vaccine record card. the survey staff had implemented community outreach activities designed to fost~r an~ maintain community trust and cooperation. the successful strategies included: developing re.lat on .w. th local community organizations; recruitment of community personnel and pro· vide them with training to conduct the enumeration and interview; teaming the trained community introduction: though much research has been done on the health and social benefits of pet ownership for many groups, there have been no explorations of what pet ownership can mean to adults who are marginalized, living on fixed incomes or on the street in canada. we are a community group of researchers from downtown toronto. made up of front line staff and community members, we believe that community research is important so that our concerns, visions, views and values are presented by us. we also believe that research can and should lead to social change. method: using qualitative and exploratory methods, we have investigated how pet ownership enriched and challenged the lives of homeless and transitionally housed people. our research team photographed and conducted one-on-one interviews with pet owners who have experienced home· lessncss and live on fixed incomes. we had community participation in the research through a partnership with the fred vicror centre camera club. many of the fred victor centre camera club members have experienced homelessness and being marginalized because of poverty. the members of the dub took the photos and assisted in developing the photos. they also participated in the presenta· tion of our project. results: we found that pet ownership brings important health and social benefits to our partici· pants. in one of the most poignant statements, one participant said that pet ownership " ... stops you from being invisible." another commented that "well, he taught me to slow down, cut down the heavy drugs .. " we also found that pet ownership brings challenges that can at times be difficult when one is liv· ing on a fixed income. we found that the most difficult thing for most of the pet owners was finding affordable vet care for their animals. conclusion: as a group, we decided that research should only be done if we try to make some cha.nges about what we have learned. we continue the project through exploring means of affecting social change--for example, ~eti.tions and informing others about the result of our project. we would like to present our ~mdmgs and experience with community-based participatory action resea.rch m an oral. presentarton at yo~r conference in october. our presentation will include com· mumty representation ~f. both front-hne staff and people with lived experience of marginalization and homdessness. if this is not accepted as an oral presentation, we are willing to present the project m poster format. introduction the concept of a healthy city was adopted by the world health organization some time ago and it includes strong support for local involvement in problem solving and implementation of solutions. while aimed at improving social, economic or environmental conditions in a given community, more significantly the process is considered to be a building block for poliq reform and larger scale 'hange, i.e. "acting locally while thinking globally." neighbourhood planning can he the entry point for citizens to hegin engaging with neighbours on issues of the greater common good. methods: this presentation will outline how two community driven projects have unfolded to address air pollution. the first was an uphill push to create bike lanes where car lanes previously existed and the second is an ongoing, multi-sectoral round table focused on pollution and planning. both dt•monstrate the importance of having support with the process and a health focus. borrowing from traditions of "technical aid"• and community development the health promoter /planner has incorporated a range of "determinants of health" into neighbourhood planning discussions. as in most urban conditions the physical environment is linked to a range of health stressors such as social isolation, crowding, noise, lack of open space /recreation, mobility and safety. however typical planning processes do not hring in a health perspective. health as a focus for neighbourhood planning is a powerful starti_ng point when discussing transportation planning or changing land-uses. by raising awareness on determmants of health, citizens can begin to better understand how to engage in a process and affect change. often local level politics are involved and citizens witness policy change in action. the environmental liaison committee and the dundas east hike lanes project resulted from local level initiatives aimed at finding solutions to air pollution -a priority identified hy the community. srchc supported the process with facilitation and technical aid. _the processs had tangible results that ultimately improve living conditions and health. •tn the united kmgdom plannm in the 's established "technical aid" offices much like our present day legal aid system to provide professional support and advocacy for communities undergoing change. p - (c) integrating community based research: the experience of street health, a community service agency i.aura cowan and jacqueline wood street health began offering services to homeless men and women in east downtown ~oronto in . nursing stations at drop-in centres and shelters were fo~lowed by hiv/aids prevent ~, harm reduction and mental health outreach, hepatitis c support, sleeping bag exchange, and personal tdennfication replacement and storage programs. as street health's progi;ams expanded, so to~ did the agency:s recognition that more nee~ed t~ be done to. address the underl~ing causes of, th~ soct~l and economic exclusion experienced by its clients. knowing t.h~t. a~voca~y ts. helped by . evtd~nce , street he.alt~ embarked on a community-based research (cbr) initiative to dent fy commumty-dnven research priorities within the homeless and underhoused population. methods: five focus groups were conducted with homeless people, asking participants to identify positive and negative forces in their lives, and which topics were important to take action on and learn more about. findings were validated through a validation meeting with participants. results: participants identified several important positive and negative forces in their lives. key positive forces included caring and respectful service delivery, hopefulness and peer networks. key negative forces included lack of access to adequate housing and income security, poor service delivery and negative perceptions of homeless people. five topics for future research emerged from the process, focusing on funding to address homelessness and housing; use of community services for homeless people; the daily survival needs of homeless people and barriers to transitioning out of homelessness; new approaches to service delivery that foster empowerment; and policy makers' understanding of poverty and homelessness. conclusions: although participants expressed numerous issues and provided much valuable insight, definitive research ideas and action areas were not clearly identified by participants. however, engagement in a cbr process led to some important lessons and benefits for street health. we learned that the community involvement of homeless people and front-line staff is critical to ensuring relevance and validity for a research project; that existing strong relationships with community parmers are essential to the successful implementation of a project involving marginalized groups; and that an action approach focusing on positive change can make research relevant to directly affected people and community agency staff. street health benefited from using a cbr approach, as the research process facilitated capacity building among staff and within the organization as a whole. p - (c) a collaborative process to achieve access to primary health care for black women and women of colour: a model of community based participartory research notisha massaquoi, charmaine williams, amoaba gooden, and tulika agerwal in the current healthcare environment, a significant number of black women and women of color face barriers to accessing effective, high quality services. research has identified several issues that contribute to decreased access to primary health care for this population however racism has emerged as an overarching determinant of health and healthcare access. this is further amplified by simultaneous membership in multiple groups that experience discrimination and barriers to healthcare for example those affected by sexism, homelessness, poverty, homophobia and heterosexism, disability and hiv infection. the collaborative process to achieve access to primary health care for black women and women of colour project was developed with the university of toronto faculty of social work and five community partners using a collaborative methodology to address a pressing need within the community ro increase access to primary health care for black women and women of colour. women's health in women's hands community health centre, sistering, parkdale community health centre, rexdale community health centre and planed parenthood of toronto developed this community-based participatory-action research project to collaboratively barriers affecting these women, and to develop a model of care that will increase their access to health services. this framework was developed using a process which ensured that community members from the target population and service providers working in multiculrural clinical settings, were a part of the research process. they were given the opportunity to shape the course of action, from the design of the project to the evaluation and dissemination phase. empowerment is a goal of the participatory action process, therefore, the research process has deliberately prioritized _ro enabling women to increase control over their health and well-being. in this session, the presenters will explore community-based participatory research and how such a model can be useful for understanding and contextualizing the experiences of black women and women of colour. they will address. the development and use of community parmerships, design and implementation of the research prorect, challenges encountered, lessons learnt and action outcomes. they will examine how the results from a collaborative community-based research project can be used as an action strategy to poster sessions v address che social determinants of women health. finally the session will provide tools for service providers and researchers to explore ways to increase partnerships and to integrate strategies to meet the needs of che target population who face multiple barriers to accessing services. lynn scruby and rachel rapaport beck the purpose of this project was ro bring traditionally disenfranchised winnipeg and surrounding area women into decision-making roles. the researchers have built upon the relationships and information gachered from a pilot project and enhanced the role of input from participants on their policy prioriries. the project is guided by an advisory committee consisting of program providers and community representatives, as well as the researchers. participants included program users at four family resource cencres, two in winnipeg and two located rurally, where they participated in focus groups. the participants answered a series of questions relating to their contact with government services and then provided inpuc as to their perceptions for needed changes within government policy. following data analysis, the researchers will return to the four centres to share the information and continue che discussion on methods for advocating for change. recommendations for program planning and policy development and implementation will be discussed and have relevance to all participants in the research program. women's health vera lefranc, louise hara, denise darrell, sonya boyce, and colleen reid women's experiences with paid and unpaid work, and with the formal and informal economies, have shifted over the last years. in british columbia, women's employability is affected by government legislation, federal and provincial policy changes, and local practices. two years ago we formed the coalition ior women's economic advancement to explore ways of dealing with women's worsening economic situations. since the formation of the coalition we have discussed the need for research into women's employabilicy and how women were coping and surviving. we also identified how the need to document the nature of women's employability and reliance on the informal economy bore significanc mechodological and ethical challenges. inherent in our approach is a social model of women's health that recognizes health as containing social, economic, and environmental determinants. we aim to examine the social contexc of women's healch by exploring and legitimizing women's own experiences, challenging medical dominance in understandings of health, and explaining women's health in terms of their subordination and marginalizacion. through using a feminist action research (far) methodology we will explore the relationship between women's employability and health in communities that represent bricish columbia's social, economic, cultural/ethnic, and geographic diversities: skidegate, fort st . .john, lumhy, and surrey. over the course of our year project, in each community we will establish and work with advisory committees, hire and train local researchers, conduct far (including a range of qualitative methods), and support action and advocacy. since the selected communities are diverse, the ways that the research unfolds will ·ary between communities. expected outcomes, such as the provision of a written report and resources, the establishment of a website for networking among the communities, and a video do.:umentary, are aimed at supporting the research participants, coalition members, and advisory conuniuces in their action efforts. p t (c) health & housing: assessing the impact of transitional housing for people living with hiv i aids currently, there is a dearth of available literature which examines supporrive housing for phas in the canadian context. using qualitative, one-on-one interviews we investigace the impact of transitional housing for phaswho have lived in the up to nine month long hastings program. our post<'r pr<·senta-t on will highlight research findings, as well as an examination of transitional housing and th<· imp;kt it has on the everyday lives of phas in canada. this research is one of two ground breaking undertakings within the province of ontario in which fife house is involved. p - (c) eating our way to justice: widening grassroots approaches to food security, the stop community food centre as a working model charles l.evkoe food hanks in north america have come co play a central role as the widespread response to growing rates of hunger. originally thought to be a short term-solution, over the last years, they have v poster sessions be · · · · d wi'thi'n society by filling the gaps in the social safety net while relieving govemcome mst tut ona ze . . . t f the ir responsibilities. dependent on corporate donations and sngmauzmg to users, food banks men so th' . · i i . are incapable of addressing the structural cause~ of ~u~ger. s pres~ntation w e~~ ore a ternanve approaches to addressing urban food security while bmldmg more sustamabl.e c~mmumt es. i:nrough the f t h st p community food centre, a toronto-based grassroots orgamzanon, a model is presented case e h'l k' b 'id · b that both responds to the emergency food needs of communities w e wor mg to. u ~ sustama le and just food system. termed, the community food centre model. (cfc), ~he s~op is worki?g to widen its approach to issues of food insecurity by combining respectful ~ rect service wit~ com~~mty ~evelop ment, social justice and environmental sustainability. through this approach, various critical discourses around hunger converge with different strategic and varied implications for a~ion. as a plac~-based organization, the stop is rooted within a geographical space and connected directly to a neighbourhood. through working to increase access to healthy food, it is active in maintaining people's dignity, building a strong and democratic community and educating for social change. connected to coalitions and alliances, the stop is also active in organizing across scales in connection with the global food justice movement. inner city shelter vicky stergiopoulos, carolyn dewa, katherine rouleau, shawn yoder, and lorne tugg introduction: in the city of toronto there are more than , hostel users each year, many with mental health and addiction issues. although shelters have responded in various ways to the health needs of their clients, evidence on the effectiveness of programs delivering mental health services to the home· less in canada has been scant. the objective of this community based research was to provide a forma· tive evaluation of a multi-agency collaborative care team providing comprehensive care for high needs clients at toronto's largest shelter for homeless men. methods: a logic model provided the framework for analysis. a chart review of clients referred over a nine month period was completed. demographic data were collected, and process and outcome indicators were identified for which data was obtained and analyzed. the two main outcome measures were mental status and housing status months after referral to the program. improvement or lack of improvement in mental status was established by chart review and team consensus. housing outcomes were determined by chart review and the hostel databases. results: of the clients referred % were single and % were unemployed. forty four percent had a psychiatric hospitalization within the previous two years. the prevalence of severe and persistent men· tal illness, alcohol and substance use disorders were %, % and % respectively. six months after referral to the program % of clients had improved mental status and % were housed. logistic regression controlling for the number of general practitioner and psychiatrist visits, presence of person· ality or substance use disorder and treatment non adherence identified two variables significantly associ· ated mental status improvement: the number of psychiatric visits (or, . ; % ci, . - . ) and treatment non adherence (or, . ; % ci, . - . ). the same two variables were associated with housing outcomes. history of forensic involvement, the presence of a personality or substance use disorder and the number of visits with a family physician were not significantly associated with either outcome. conclusions: despite the limitations in sample sire and study design, this study can yield useful informa· tion to program planners. our results suggest that strategies to improve treatment adherence and access to mental health specialists can improve outcomes for this population. although within primary care teams the appropriate collaborative care model for this population remains to be established, access to psychiatric follow up, in addition to psychiatric assessment services, may be an important component of a successful program. mount sinai hospital (msh) has become one of the pre-eminent hospitals in the world by contributing to the development of innovative approaches to effective health care and disease prevention. recently, the hospital has dedicated resources towards the development of a strategy aimed at enhancing the hospital's integration with its community partners. this approach will better serve the hospital in the current health care environment where local health integration networks have been struck to enhance and support local capacity to plan, coordinate and integrate service delivery. msh has had early success with developing partnerships. these alliances have been linked to programs serving key target populations with _estabhshe~. points of access to msh. recognizing the need to build upon these achievements to remain compe~mve, the hospital has developed a community integration strategy. at the forefront of this strategy is c.a.r.e (community advisory reference engine): the hospital's compendium of poster sessions v community partners. as a single point of access to community partner information, c.a.r.e. is more than a database. c.a.r.e. serves as the foundation for community-focused forecasting and a vehicle for inter and intra-organizational knowledge transfer. information gleaned from the catalog of community parmers can be used to prepare strategic, long-term partnership plans aimed at ensuring that a comprehensive array of services can be provided to the hospital community. c.a.r.e. also houses a permanent record of the hospital's alliances. this prevents administrative duplication and facilitates the formation of new alliances that best serve both the patient and the hospital. c.a.r.e. is not a stand-alone tool and is most powerful when combined with other aspects of the hospital's community integration strategy. it iscxpected that data from the hospital's community advisory committees and performance measurement department will also be stored alongside stakeholder details. this information can then be used to drive discussions at senior management and the board, ensuring congruence between stakeholder, patient and hospital objectives. the patient stands to benefit from this strategy. the unique, distinct point of reference to a wide array of community services provides case managers and discharge planners with the information they need to connect patients with appropriate community services. creating these linkages enhances the patient's capacity to convalesce in their homes or places of residence and fosters long-term connections to neighborhood supports. these connections can be used to assist with identifying patients' ongoing health care needs and potentially prevent readmission to hospital. introduction: recruiting high-risk drug users and sex workers for hiv-prevention research has often been hampered by limited access to hard to reach, socially stigmatized individuals. our recruitment effom have deployed ethnographic methodology to identify and target risk pockets. in particular, ethnographers have modeled their research on a street-outreach model, walking around with hiv-prevention materials and engaging in informal and structured conversations with local residents, and service providers, as well as self-identified drug users and sex workers. while such a methodology identifies people who feel comfortable engaging with outreach workers, it risks missing key connections with those who occupy the margins of even this marginal culture. methods: ethnographers formed a women's laundry group at a laundromat that had a central role as community switchboard and had previously functioned as a party location for the target population. the new manager helped the ethnographers invite women at high risk for hiv back into the space, this time as customers. during weekly laundry sessions, women initiated discussions about hiv-prevention, sexual health, and eventually, the vaccine research for which the center would be recruiting women. ra.its: the benefits of the group included reintroducing women to a familiar locale, this time as customers rather than unwelcome intruders; creating a span of time (wash and dry) to discuss issues important to me women and to gather data for future recruitment efforts; creating a location to meet women encountered during more traditional outreach research; establishing the site as a place for potential retention efforts; and supporting a local business. women who participated in the group completed a necessary household task while learning information that they could then bring back to the community, empowering them to be experts on hiv-prevention and vaccine research. some of these women now assist recruitment efforts. the challenges included keeping the group women-only, especially after lunch was provided, keeping the membership of the group focused on women at risk for hiv, and keeping the women in the group while they did their laundry. conclusion: public health educators and researchers can benefit from identifying alternate congregation sites within risk pockets to provide a comfortable space to discuss hiv prevention issues with high-risk community members. in our presentation, we will describe the context necessary for similar research, document the method's pitfalls and successes, and argue that the laundry group constitutes an ethical, respectful, community-based method for recruitment in an hiv-prevention vaccine trial. p - t (c) upgrading inner city infrastructure and services for improved environmental hygiene and health: a case of mirzapur in u.p. india madhusree mazumdar in urgency for agricultural and industrial progress to promote economic d.evelopment follo_wing independence, the government of india had neglected health promotion and given less emphasis on infrastructure to promote public health for enhanced human pro uct v_ity. ong wit r~p m astrucrure development, which has become essential if citie~ are to. act ~s harbmger.s of econ~nuc ~owth, especially after the adoption of the economic liberalization policy, importance _is a_lso ~emg g ve.n to foster environmental hygiene for preventive healthcare. the world health orga~ sat ~ is also trj:' ! g to help the government to build a lobby at the local level for the purpose by off~rmg to mrroduce_ its heal.thy city concept to improve public health conditions, so as to reduce th_e disease burden. this pape~ s a report of the efforts being made towards such a goal: the paper descr~bes ~ c~se study ?f ~ small city of india called mirzapur, located on the banks of the nver ganga, a ma or lifeline of india, m the eastern part of the state of uttar pradesh, where action for improvement began by building better sanitation and environmental infrastructure as per the ganga action plan, but continued with an effort to promote pre· ventive healthcare for overall social development through community participation in and around the city. asthma physician visits in toronto, canada tara burra, rahim moineddin, mohammad agha, and richard glazier introduction: air pollution and socio-economic status are both known to be associated with asthma in concentrated urban settings but little is known about the relationship between these factors. this study investigates socio-economic variation in ambulatory physician consultations for asthma and assesses possible effect modification of socio-economic status on the association between physician visits and ambient air pollution levels for children aged to and adults aged to in toronto, canada between and . methods: generalized additive models were used to estimate the adjusted relative risk of asthma physician visits associated with an interquartile range increase in sulphur dioxide, nitrogen dioxide, pm . , and ozone, respectively. results: a consistent socio-economic gradient in the number of physician visits was observed among children and adults and both sexes. positive associations between ambient concentrations of sul· phur dioxide, nitrogen dioxide and pm . and physician visits were observed across age and sex strata, whereas the associations with ozone were negative. the relative risk estimates for the low socio-«onomic group were not significantly greater than those for the high socio-economic group. conclusions: these findings suggest that increased ambulatory physician visits represent another component of the public health impact of exposure to urban air pollution. further, these results did not identify an age, sex, or socio-economic subgroup in which the association between physician visits and air pollution was significantly stronger than in any other population subgroup. eco-life-center (ela) in albania supports a holistic approach to justice, recognizing the environ· mental justice, social justice and economic justice depend upon and support each other. low income cit· izens and minorities suffer disproportionately from environmental hazards in the workplace, at home, and in their communities. inadequate laws, lax enforcement of existing environmental regulations, and ~ea.k penalties for infractions undermine environmental protection. in the last decade, the environmental ust ce m~ve~ent in tirana metropolis has provided a framework for identifying and exposing the links ~tween irrational development practices, disproportionate siting of toxic facilities, economic depres· s on, and a diminished quality of life in low-income communities and communities of color. the envi· ~onmental justice agenda has always been rooted in economic, racial, and social justice. tirana and the issues su.rroun~ing brow~fields redevelopment are crucial points of advocacy and activism for creating ~ubstantia~ social chan~~ m low-income communities and communities of color. we engaging intensively m prevcnnng co'.' mumnes, especially low income or minority communities, from being coerced by gov· ernmenta~ age_nc es or companies into siting hazardous materials, or accepting environmentally hazard· ous_ practices m order to create jobs. although environmental regulations do now exist to address the environmental, health, and social impacts of undesirable land uses, these regulations are difficult to poster sessions v enforce because many of these sites have been toxic-ridden for many years and investigation and cleanup of these sites can be expensive. removing health risks must be the main priority of all brown fields action plans. environmental health hazards are disproportionately concentrated in low-income communities of color. policy requirements and enforcement mechanisms to safeguard environmental health should be strengthened for all brownfields projects located in these communities. if sites are potentially endangering the health of the community, all efforts should be made for site remediation to be carried out to the highest cleanup standards possible towards the removal of this risk. the assurance of the health of the community should take precedence over any other benefits, economic or otherwise, expected to result from brownfields redevelopment. it's important to require from companies to observe a "good neighbor" policy that includes on-site visitations by a community watchdog committee, and the appointment of a neighborhood environmentalist to their board of directors in accordance with the environmental principles. vancouver - michael buzzelli, jason su, and nhu le this is the second paper of research programme concerned with the geographical patterning of environmental and population health at the urban neighbourhood scale. based on the vancouver metropolitan region, the aim is to better understand the role of neighbourhoods as epidemiological spaces where environmental and social characteristics combine as health processes and outcomes at the community and individual levels. this paper builds a cohort of commensurate neighbourhoods across all six censuses periods from to , assembles neighbourhood air pollution data (several criterion/health effects pollutants), and providing an analysis to demonstrate how air pollution systematically and consistently maps onto neighbourhood socioeconomic markers, in this case low education and lone-parent families. we conclude with a discussion of how the neighbourhood cohort can be further developed to address emergent priorities in the population and environmental health literatures, namely the need for temporally matched data, a lifecourse approach, and analyses that control for spatial scale effects. solid waste management and environment in mumbai (india) by uttam jakoji sonkamble and bairam paswan abstract: mumbi is the individual financial capital of india. the population of greater mumbai is , , and sq. km. area. the density of population , per sq. km. the dayto-day administration and rendering of public services within gr. mumbai is provided by the brihan mumbai mahanagar palika (mumbai corporation of gr. mumbai) that is a body of elected councilors on a -year team. mumcial corporation provides varies conservancy services such as street sweeping, collection of solid waste, removal and transportation, disposal of solid waste, disposal of dead bodies of animals, construction, maintance and cleaning of urinals and public sanitary conveniences. the solid waste becoming complicated due to increase in unplanned urbanization and industrialization, the environment has deteriorated significantly due to inter, intra and international migration stream to mumbai. the volume of inter state migration to mumbai is considerably high i.e. . lakh and international migrant . lakh have migrated to mumbai. present paper gives the view on solid waste management and its implications to environment and health. pollution from a wide varity of emission, such as from automobiles and industrial activities, has reached critical level in mumbai, causing respiratory, ocular, water born diseases and other health problems. sources of generation of waste are -household waste, commercial waste, institutional waste, street sweeping, silt removed from drain/nallah/cleanings. disposal of solid waste in gr. mumbai done under incineration . processing to produce organic manure. . vermi-composting . landfill the study shows that the quantity of waste disposal of through processing and conversion to organic ~anure is about - m.t. per day. the processing is done by a private agency m/s excel industries ltd. who had set up a plant at the chincholi dumping ground in western mumbai for this purpose. the corporation is also disposal a plant of its waste mainly market waste through the environment friendly, natural pro-ces~ known as vermi-composing about m.t. of market waste is disposed of in this manner at the various sites. there are four land fill sites are available and percent of the waste matter generated m mumbai is disposed of through landfill. continuous flow of migrant and increa~e in slum population is a complex barrier in the solid waste management whenever community pamc pat on work strongly than only we can achieved eco-friendly environment in mumbai. persons exposed to residential craffic have elevated races of respiratory morbidity an~ ~ortality. since poverty is an important determinant of ill-health, some h~ve argued that t~es~ assoc at ons may relate to che lower socioeconomic status of those living along ma or roads. our ob ect ve was to evaluate the association between traffic intensity at home and hospital admissions for respiratory diagnoses among montreal residents older than years. morning peak traffic estimates from the emmej montreal traffic model (motrem ) were used as an indicator of exposure to road traffic outside the homes of those hospitalised. the influence of socioeconomic status on the relationship between traffic intensity and hospital admissions for respiratory diagnoses was explored through assessment of confounding by lodging value, expressed as the dollar average over road segments. this indicator of socioeconomic status, as calculated from the montreal property assessment database, is available at a finer geographic scale than socioeconomic information accessible from the canadian census. there was an inverse relationship between traffic intensity estimates and lodging values for those hospitalised (rho - . , p vehicles during che hour morning peak), even after adjustment for lodging value (crude or . , cl % . - . ; adjusted or . , cl % . - . ). in montreal, elderly persons living along major roads are at higher risk of being hospitalised for respiratory illnesses, which appears not simply to reflect the fact that those living along major roads are at relative economic disadvantage. the paper argues that human beings ought to be at the centre of the concern for sustainable development. while acknowledging the importance of protecting natural resources and the ecosystem in order to secure long term global sustainability, the paper maintain that the proper starting point in the quest for urban sustainability in africa is the 'brown agenda' to improve che living and working environment of che people, especially che urban poor who face a more immediate environmental threat to their health and well-being. as the un-habitat has rightly observed, it is absolutely essential "to ensure that all people have a sufficient stake in the present to motivate them to take part in the struggle to secure the future for humanity.~ the human development approach calls for rethinking and broadening the narrow technical focus of conventional town planning and urban management in order to incorporate the emerging new ideas and principles of urban health and sustainability. i will examine how cities in sub-saharan africa have developed over the last fifty years; the extent to which government policies and programmes have facilitated or constrained urban growth, and the strategies needed to achieve better functioning, safer and more inclusive cities. in this regard i will explore insights from the united nations conferences of the s, especially local agenda of the rio summit, and the istanbul declaration/habitat agenda, paying particular attention to the principles of enablement, decentralization and partnership canvassed by these movements. also, i will consider the contributions of the various global initiatives especially the cities alliance for cities without slums sponsored by the world bank and other partners; che sustainable cities programme, the global campaigns for good governance and for secure tenure canvassed by unhabit at, the healthy cities programme promoted by who, and so on. the concluding section will reflect on the future of the african city; what form it will take, and how to bring about the changes needed to make the cities healthier, more productive and equitable, and better able to meet people's needs. heather jones-otazo, john clarke, donald cole, and miriam diamond urban areas, as centers of population and resource consumption, have elevated emissions and concentrations of a wide range of chemical contaminants. we have developed a modeling framework in which we first ~stimate the emissions and transport of contaminants in a city and second, use these estimates along with measured contaminant concentrations in food, to estimate the potential health risk posed by these che.micals. the latter is accomplished using risk assessment. we applied our modeling framework to consider two groups of chemical contaminants, polycyclic aromatic hydrocarbons (pah) a.nd the flame re~ardants polybrominated diphenyl ethers (pbde). pah originate from vehicles and stationary combustion sources. ~veral pah are potent carcinogens and some compounds also cause noncancer effects. pbdes are additive flame retardants used in polyurethane foams (e.g., car seats, furniture) fer sessions v and cl~ equipm~nt (e.g., compute~~· televisio~s). two out of three pbdes formulations are being voluntarily phased by mdustry due to rmng levels m human tissues and their world-wide distribution. pbdes have been .related to adv.erse neurological, developmental and reproductive effects in laboratory ijlimals. we apphed our modelmg framework to the city of toronto where we considered the southcattral area of by km that has a population of . million. for pah, local vehicle traffic and area sources contribute at least half of total pah in toronto. local contributions to pbdes range from - %, depending on the assumptions made. air concentrations of both compounds are about times higher downtown than km north of toronto. although measured pah concentrations in food date to the s, we estimate that the greatest exposure and contribution to lifetime cancer risk comes from ingestion of infant formula, which is consistent with toxicological evidence. the next greatest exposure and cancer risk are attributable to eating animal products (e.g. milk, eggs, fish). breathing downtown air contributes an additional percent to one's lifetime cancer risk. eating vegetables from a home garden localed downtown contributes negligibly to exposure and risk. for pbdes, the greatest lifetime exposure comes through breast milk (we did not have data for infant formula), followed by ingestion of dust by the toddler and infant. these results suggest strategies to mitigate exposure and health risk. p - (a) immigration and socioeconomic inequalities in cervical cancer screening in toronto, canada aisha lofters, rahim moineddin, maria creatore, mohammad agha, and richard glazier llltroduction: pap smears are recommended for cervical cancer screening from the onset of sexual activity to age . socioeconomic and ethnoracial gradients in self-reported cervical cancer screening have been documented in north america but there have been few direct measures of pap smear use among immigrants or other socially disadvantaged groups. our purpose was to investigate whether immigration and socioeconomic factors are related to cervical cancer screening in toronto, canada. methods: pap smears were identified using fee codes and laboratory codes in ontario physician service claims (ohip) for three years starting in for women age - and - . all women with any health system contact during the three years were used as the denominator. social and economic factors were derived from the canadian census for census tracts and divided into quintiles of roughly equal population. recent registrants, over % of whom are expected to be recent immigrants to canada, were identified as women who first registered for health coverage in ontario after january , . results: among , women age - and , women age - , . % and . %, rtspcctively, had pap smears within three years. low income, low education, recent immigration, visible minority and non-english language were all associated with lower rates (least advantaged quintile:most advantaged quintile rate ratios were . , . , . , . , . , respectively, p < . for all). similar gradients were found in both age groups. recent registrants comprised . % of women and had mm;h lower pap smear rates than non-recent registrants ( . % versus . % for women age - and . % versus . % for women age - ). conclnsions: pap smear rates in toronto fall well below those dictated by evidence-based practice. at the area level, immigration, visible minority, language and socioeconomic characteristics are associated with pap smear rates. recent registrants, representing a largely immigrant group, have particularly low rates. efforts to improve coverage of cervical cancer screening need to be directed to all ~omen, their providers and the health system but with special emphasis on women who recently arrived m ontario and those with social and economic disadvantage. challeges faced: a) most of the resources are now being ~pent in ~reventing the sprea.d of hiv/ aids and maintaining the lives of those already affected. b) skilled medical ~rs~nal are dymg under· mining the capacity to provide the required health care services. ~) th.e comphcat o~s of hiv/aids has complicated the treatment of other diseases e.g. tbs d) the ep dem c has led. to mcrease number of h n requiring care and support. this has further stretched the resources available for health care. orp a s d db . . i methods used on our research: . a simple community survey con ucte y our orgamzat on vo · unteers in three urban centres members of the community, workers and health care prov~ders were interviewed ... . meeting/discussions were organized in hospitals, commun.ity centre a~d with government officials ... . written questionnaires to health workers, doctors and pohcy makers m th.e health sectors. lessors learning: • the biggest-health bigger-go towards hiv/aids prevention • aids are spreading faster in those families which are poor and without education. •women are the most affected. •all health facilities are usually overcrowded with hiv/aids patients. actions needed:• community education oh how to prevent the spread of hiv/aids • hiv/aids testing need to be encouraged to detect early infections for proper medical cover. • people to eat healthy • people should avoid drugs. implications of our research: community members and civic society-introduction of home based care programs to take care of the sick who cannot get a space in the overcrowded public hospitals. prl-v a te sector private sector has established programs to support and care for the staff already affected. government provision of support to care-givers, in terms of resources and finances. training more health workers. introduction: australian prisons contain in excess of , prisoners. as in most other western countries, reliance on 'deprivation of liberty' is increasing. prisoner numbers are increasing at % per annum; incarceration of women has doubled in the last ten years. the impacts on the community are great - % of children have a parent in custody before their th birthday. for aboriginal communities, the harm is greater -aboriginal and/or torres strait islanders are incarcerated at a rate ten times higher than other australians. % of their children have a parent in custody before their th birthday. australian prisons operate under state and territory jurisdictions, there being no federal prison system. eight independent health systems, supporting the eight custodial systems, have evolved. this variability provides an unique opportunity to assess the capacity of these health providers in addressing the very high service needs of prisoners. results: five models of health service provision are identified -four of which operate in one form or another in australia: • provided by the custodial authority (queensland and western australia)• pro· vided by the health ministry through a secondary agent (south australia, the australian capital territory and tasmania) • provided through tendered contract by a private organization (victoria and northern territory) • provided by an independent health authority (new south wales) • (provided by medics as an integral component of the custodial enterprise) since the model of the independent health authority has developed in new south wales. the health needs of the prisoner population have been quantified, and attempts are being made to quantify specific health risks /benefits of incarceration. specific enquiry has been conducted into prisoner attitudes to their health care, including issues such as client information confidentiality and access to health services. specific reference will be made to: • two inmate health surveys • two inmate access surveys, and • two service demand studies. conclusions: the model of care provision, with legislative, ethical, funding and operational independence would seem provide the best opportunity to define and then respond to the health needs of prisoners. this model is being adopted in the united kingdom. better health outcomes in this high-risk group, could translate into healthier families and their communities. p - (a) lnregrated ethnic-specific health care systems: their development and role in increasing access to and quality of care for marginalized ethnic minorities joshua yang introduction: changing demographics in urban areas globally have resulted in urban health systems that are racially and ethnically homogenous relative to the patient populations they aim to serve. the resultant disparities in access to and quality of health care experienced by ethnic minority groups have been addressed by short-term, instirutional level strategies. noticeably absent, however, have been structural approaches to reducing culturally-rooted disparities in health care. the development of ethnic-specific h~alth car~ systems i~ a structural, long-term approach to reducing barriers to quality health care for eth· me mmonty populations. methods: this work is based on a qualitative study on the health care experiences of san francisco chinatown in the united states, an ethnic community with a model ethnic-specific health care infrastrucrure. using snowball sampling, interviews were conducted with key stakeholders and archival research was conducted to trace and model the developmental process that led to the current ethnic-specific health care system available to the chinese in san francisco. grounded theory was the methodology ijltd to analysis of qualitative data. the result of the study is four-stage developmental model of ethnic-specific health infrastrueture development that emerged from the data. the first stage of development is the creation of the human capital resources needed for an ethnic-specific health infrastructure, with emphasis on a bilingual and bicultural health care workforce. the second stage is the effective organization of health care resources for maximal access by constituents. the third is the strengthening and stability of those institutional forms through increased organizational capacity. integration of the ethnic-specific health care system into the mainstream health care infrastructure is the final stage of development for an ethnic-specific infrastructure. conclusion: integrated ethnic-specific health care systems are an effective, long-term strategy to address the linguistic and cultural barriers that are being faced by the spectrum of ethnic populations in urban areas, acting as culturally appropriate points of access to the mainstream health care system. the model presented is a roadmap to empower ethnic communities to act on the constraints of their health and political environments to improve their health care experiences. at a policy level, ethnic-specific health care organizations are an effective long-term strategy to increase access to care and improve qualiiy of care for marginalized ethnic groups. each stage of the model serves as a target area for policy interventions to address the access and care issues faced by culturally and linguistically diverse populations. users in baltimore md: - noya galai, gregory lucas, peter o'driscoll, david celentano, david vlahov, gregory kirk, and shruti mehta introduction: frequent use of emergency rooms (er) and hospitalizations among injection drug users (idus) has been reported and has often been attributed to lack of access to primary health care. however, there is little longitudinal data which examine health care utilization over individual drug use careers. we examined factors associated with hospitalizations, er and outpatient (op) visits among idus over years of follow-up. methods: idus were recruited through community outreach into the aids link to lntravenous experience (alive) study and followed semi-annually. , who had at least follow-up visits were included in this analysis. outcomes were self-reported episodes of hospitalizations and er/op visits in the prior six months. poisson regression was used accounting for intra-person correlation with generalized estimation equations. hits: at enrollment, % were male, % were african-american, % were hiv positive, median age was years, and median duration of drug use was years. over a total of , visits, mean individual rates of utilization were per person years (py) for hospitalizations and per py for er/op visits. adjusting for age and duration of drug use, factors significantly associated with higher rates of hospitalization included hiv infection (relative incidence [ri(, . ), female gender (ri, . ), homelessness (ri, . ), as well as not being employed, injecting at least daily, snorting heroin, havmg a regular source of health care, having health insurance and being in methadone mainte.nance treatment (mmt). similar associations were observed for er/op visits except for mmt which was not associated with er/op visits. additional factors associated with lower er/op visits were use of alcohol, crack, injecting at least daily and trading sex for drugs. % of the cohort accounted for % of total er/op visits, while % of the cohort never reported an op visit during follow-up. . . . lgbt) populations. we hypothesized that prov dmg .appomtments .for p~t ~nts w thm hours would ensure timely care, increase patient satisfaction, and improve practice eff c ency. further, we anticipated that the greatest change would occur amongst our homeless patients.. . methods: we tested an experimental introduction of advanced access scheduling (usmg a hour rule) in the primary care medical clinic. we tracked variables inclu~ing waiting ti~e fo~ next available appointment; number of patients seen; and no-show rates, for an eight week penod pnor to and post introduction of the new scheduling system. both patient and provider satisfaction were assessed using a brief survey ( questions rated on a -pt scale). results and conclusion: preliminary analyses demonstrated shorter waiting times for appointments across the clinic, decreased no-show rates, and increased clinic capacity. introduction of the advanced access scheduling also increased both patient and provider satisfaction. the new scheduling was initiated in july . quantitative analyses to measure initial and sustained changes, and to look at differential responses across populations within our clinic, are currently underway. introduction: there are three recognized approaches to linking socio-economic factors and health: use of census data, gis-based measures of accessibility/availability, and resident self-reported opinion on neighborhood conditions. this research project is primarily concerned with residents' views about their neighborhoods, identifying problems, and proposing policy changes to address them. the other two techniques will be used in future research to build a more comprehensive image of neighborhood depri· vation and health. methods: a telephone survey of london, ontario residents is currently being conducted to assess: a) community resource availability, quality, access and use, b) participation in neighborhood activities, c) perceived quality of neighborhood, d) neighborhood problems, and e) neighborhood cohesion. the survey instrument is composed of indices and scales previously validated and adapted to reflect london specifically. thirty city planning districts are used to define neighborhoods. the sample size for each neighborhood reflects the size of the planning district. responses will be compared within and across neighborhoods. data will be linked with census information to study variation across socio-eco· nomic and demographic groups. linear and gis-based methods will be used for analysis. preliminary results: the survey follows a qualitative study providing a first look at how experts involved in community resource planning and administration and city residents perceive the availability, accessibility, and quality of community resources linked to neighborhood health and wellbeing, and what are the most immediate needs that should be addressed. key-informant interviews and focus groups were used. the survey was pre-tested to ensure that the language and content reflects real experiences of city residents. the qualitative research confirmed our hypothesis that planning districts are an acceptable surrogate for neighborhood, and that the language and content of the survey is appropriate for imple· mentation in london. scales and indices showed good to excellent reliability and validity during the pre· test (cronbach's alpha from . - . ). preliminary results of the survey will be detailed at the conference. conclusions: this study will help assess where community resources are lacking or need improve· ment, thus contributing to a more effective allocation of public funds. it is also hypothesized that engaged neighborhoods with a well-developed sense of community are more likely to respond to health programs and interventions. it is hoped that this study will allow london residents to better understand the needs and problems of their neighborhoods and provide a research foundation to support local understandmg of community improvement with the goal of promoting healthy neighborhoods. p - (a) hiv positive in new york city and no outpatient care: who and why? hannah wolfe and victoria sharp introduction: there are approximately million hiv positive individuals living in the united sta!es. about. % of these know their hiy status and are enrolled in outpatient care. of the remaining yo, approx~mately half do not know their status; the other group frequently know their status but are not enrolled m any .sys~em of outpatient care. this group primarily accesses care through emergency departments. when md cated, they are admitted to hospitals, receive acute care services and then, upon poster sessions v di 'harge, disappear from the health care system until a new crisis occurs, when they return to the emergency department. as a large urban hiv center, caring for over individuals with hiv we have an active inpatient service ".'ith appr~xi~.ately discharges annually. we decided to survey our inpatients to better charactenze those md v duals who were not enrolled in any system of outpatient care. results: % of inpatients were not enrolled in regular outpatient care: % at roosevelt hospital and % at st.luke\'s hospital. substance abuse and homelessness were highly prevalent in the cohort of patients not enrolled in regular outpatient care. % of patients not in care (vs. % of those in care) were deemed in need of substance use treatment by the inpatient social worker. % of those not in care were homeless (vs. % of those in care.) patients not in care did not differ significantly from those in me in terms of age, race, or gender. patients not in care were asked "why not:" the two most frequent responses were: "i haven't really been sick before" and "i'd rather not think about my health. conclusions: this study suggests that there is an opportunity to engage these patients during their stay on the inpatient units and attempt to enroll them in outpatient care. simple referral to an hiv clinic is insufficient, particularly given the burden of homelessness and substance use in this population. efforts are currently underway to design an intervention to focus efforts on this group of patients. p .q (a) healthcare availability and accessibility in an urban area: the case of ibadan city, nigeria in oder to cater for the healthcare need of the populace, for many years after nigeria's politicl independence, empphasis was laid on the construction of teaching, general, and specialist hospital all of which were located in the urban centres. the realisation of the inadequacies of this approach in adequately meeting the healthcare needs of the people made the country to change and adopt the primary health care (phc) system in . the primary health care system which is in line with the alma ata declaration of of , wsa aimed at making health care available to as many people as possible on the basis of of equity and social justice. thus, close to two decades, nigeria has operated primary health care system as a strategy for providing health care for rural and urban dwellers. this study focusing on urban area, examimes the availabilty and accessibility of health care in one of nigeria's urban centre, ibadan city to be specific. this is done within the contest of the country's national heath policy of which pimary health care is the main thrust. the study also offers necessary suggestion for policy consideration. in spite of the accessibility to services provided by educated and trained midwifes in many parts of fars province (iran) there are still some deliveries conducted by untrained traditional birth attendants in rural parts of the province. as a result, a considerable proportion of deliveries are conducted under a higher risk due to unauthorised and uneducated attendants. this study has conducted to reveal the pro· portion of deliveries with un-authorized attendants and some spatial and social factors affecting the selection of delivery attendants. method: this study using a case control design compared some potentially effective parameters indud· ing: spatial, social and educational factors of mothers with deliveries attended by traditional midwifes (n= ) with those assisted by educated and trained midwifes (n= ). the mothers interviewed in our study were selected from rural areas using a cluster sampling method considering each village as a cluster. results: more than % of deliveries in the rural area were assisted by traditional midwifes. there are significant direct relationship between asking a traditional birth attendant for delivery and mother age, the number of previous deliveries and distance to a health facility provided for delivery. significant inverse relationships were found between mother's education and ability to use a vehicle to get to the facilities. conclusion: despite the accessibility of mothers to educated birth attendants and health facilities (according to the government health standards), some mothers still tend to ask traditional birth attendants for help. this is partly because of unrealistic definition of accessibility. the other considerable point is the preference of the traditional attendants for older and less educated mothers showing the necessity of changing theirs knowledge and attitude to understand the risks of deliveries attended by traditional and un-educated midwifes. p - (a) identification and optimization of service patterns provided by assertive community treatment teams in a major urban setting: preliminary findings &om toronto, canada jonathan weyman, peter gozdyra, margaret gehrs, daniela sota, and richard glazier objective: assertive community treatment (act) teams are financed by the ontario ministry of health and long-term care (mohltc) and are mandated to provide treatment, rehabilitation and support services in the community to people with severe and persistent mental illness. there are such teams located in various regions across the city of toronto conducting home visits - times per week to each of their approximately respective clients. each team consists of multidisciplinary health professionals who assist clients to identify their needs, establish goals and work toward them. due to complex referral patterns, the need for service continuity and the locations of supportive housing, clients of any one team are often found scattered across the city which increases home visit travel times and decreases efficiency of service provision. this project examines the locations of clients in relation to the home bases of all act teams and identifies options for overcoming the geographical challenges which arise in a large urban setting. methods: using geographic information systems (gis) we geocoded all client and act agency addresses and depicted them on location maps. at a later stage using spatial methods of network analysis we plan to calculate average travel rimes for each act team, propose optimization of catchment areas and assess potential travel time savings. resnlts: initial results show a substantial scattering of clients from several act teams and substan· rial overlap of visit travel routes for most teams. conclusions: reallocation of catchment areas and optimization of act teams' travel patterns should lead to substantial savings in travel times, increased service efficiency and better utilization of resourc_~· ~e l'<!tenri~i modifications to catchment areas must be conducted with appropriate attention to specific clients servtce needs, service continuity and applicable regulations by the mohl tc. venice lido is a popular island within and outside italy because it hosts the international cinema festival yearly. only few people remember the island for its hospital, which, in the fifties, was one of the most important hospitals in italy and in europe, being a modern center for wind-, sun-and thermal-therapy. since the sixties, its fame became to drop away, since its structure was no longer adequate for the evolving medicine science. at the end of the seventies all the wards were moved into a new big building in the same area. at present the hospital is made up by about forty 'pavilions' (mostly partially used or empty) and a big building, disseminated in a hectares area, on the sea front, on the m long beach. the aim of my research is to suggest a realistic solution, sustainable on the functional use of the area according to basic local needs and economically realizable. i used a philological and multi-disciplinary method, more in detail: -from historical documents, i found what was the very first call of the property and how it adapted to the needs of the demographic development and the sanitary supply over time; -by contacting some operators from different fields, i realized which functions were suitable to the buildings in existence, to the population's needs and to the present sanitary supply. moreover the planning idea considers: -the debate going on between local people who strongly want to preserve the property, and die ones who want to renew it by different new opportunities; and -the regional sanitary politics, aiming at rationalizing the regional hospitals dislocation, and the budget needs. this plan analyzes and proposes some new alternative solutions to satisfy different needs. the different activities will be dealt out and the environmental fall will be limited as much as possible. after having analyzed its basic call, considered its environmental peculiarities and its position, verified the emerging needs of the local population, the plan proposes different activities: the hospital; the social-rehabilitation center; the elderly house; the thermal center; the residential center; and other activities. such a plan wants to share in the solution of a basic problem by giving architectonic and functional dignity to an historical completely degraded area and by starting again a social-economical broken off process, by bringing in venice lido some fresh necessary elements for its so wished new throw. p - (c) dilemma of free health care in spokane, wa david bunting introduction: the paper reports on the activities of project access spokane [pas], a physician sponsored initiative to provide uncompensated health care to low income, uninsured residents of spokane county wa. program providers included all county hospitals, over physicians, other medical professionals, and specialized clinics. each prescription requires only a $ co-payment. sponsored and administered by the county medical society, pas is funded by local and national foundations, private corporations, municipalities, civic organizations and individuals. method: the paper is based on a first year assessment report funded by pas, using hcfa [health care financing administration] insurance claim data documenting utilization rates, disease categories and donated charge information and patient cares [centralized applications, referrals and enrollment status) data. results: while essentially free for enrollees, the program involved real costs. an administrative organization to refer over patients to any of over providers had to be developed and staffed, using both paid and unpaid personnel. methods to identify potential patients and veri.fy eligibility. had to ~devised. patient appointments and transportation had to be scheduled and monitored. pu~hc relanons, provider solicitations and fundraising activities were continuous. information requirements regarding program operation were also significant. data reporting the volume. ~nd value of dona~ed medical services were necessary to demonstrate accomplishments and attr~ct addmon~l support. providers required assurances their contributions were both significant and eqmtable. funding sources sought evidence that their support had important consequences. however, generating this ~~ta proved difficult. many providers failed to submit appropriate insurance claims forms because .of a~dmonal donated effort and administrative cost, thereby not only reducing their own apparent conmbut ons but also the overall significance of the program. conclusions: during its initial year, the estimated cost to deliver free health care was ~bout per enrollee. the lesson is that without an elaborate administrative structure and record keeping s.yst~~· efforts to provide free health care probably will fail. eligible enrollees c~n not be ~parated from in~hgi ble ones, care will not be accessed or delivered in an efficient manner, neither fundmg nor ~upport "". be offered without evidence of tangible benefits, and providers will withdraw if they perceive mequ table tteannent. v p - s (c) mobility in prostitution and the impact of health therese van der helm and henk sulman poster sessions introduction: many of the estimated . commer~ial ~ex wor~ers (c~w)in a'.' ~terdam ~~me from developing countries and eastern europe. to gain ins~ght mto their workmg and_ hvmg condinons the intermediary project (ip) at the municipal health serv ~e _contacts these women via ?utreach work on regular basis. since the new brothel law in october s ~troduc~d, ~sw ~rom outside the eu and who have no dutch residence permit are not allowed to work m prost tut n. smee then, many of these csw therefore have gone "underground." the consequences of the new law in the netherlands will be discussed with regard to the accessibility of csw and their risk for stl/hiv acquisition and unplanned pregnancies. methods: topics during outreach work are the interventions to increase the knowledge of safe sex and birth control for csw. written information is handed out in relevant languages and with addresses of health and social services. in conversations with the women, it appeared that many are not aware of these services. to provide easier access to health care for women, staff of the ip carries out free sti control csw working places, in windows brothels and sex houses. also, women are offered vaccination against hepatitis b (hbv). results: from january till july we approached csw for first contact. one third was dutch; others were from developing countries, other eu countries and eastern europe. sti consul· rations were done among non-iv drug using sex workers, of whom % was dutch. of these, % was diagnosed with syphilis, % with gonorrhea, and % with chlamydia. of women tested for hiv, none were infected. due to the high turnover of csw in .brothels, most wnmen were tested only once. among csw tested for hbv -of whom one third is dutch - % had antibodies for hbv, were carrier of hbv, most of whom came from hbv endemic areas. conclusions: sti control and hbv vaccination in brothels is an important support in health care. our findings suggest that csw do not play an important role in the transmission of sti. many csw are highly mobile and often not aware of the existing health and social services. continuous outreach is important to remain in contact with this mobile population. regulations in the new brothel law should not hamper contacts with (illegal) csw. heart disease (cho) is projected to become the leading cause of death. studies conducted in europe and the united states have proven a higher rate of cho in south asians who have migrated from south asia, most notably india, pakistan and bangladesh. approximately % of all heart attacks among south asian men occur under the age of ; % of them occur under the age of -unheard of in any other population. associated risk factors such as diabetes, high blood pressure and cholesterol are also com· mon in this group. the population of south asians residing in nyc has more than doubled over the past decade to over , , the majority being young, working-class and with limited health care access. many south asian men are employed as taxi drivers ( out of nyc taxi drivers are from south asial, working daily hour plus shifts. methods: acknowledging that this vulnerable, at-risk group was unavailable for outreach through conventional venues, three of the hospitals of the new york city health and hospital corporation, (elmhurst, queens and bellevue) conducted a one day outreach effort at nyc's jfk international air· ~rt's taxi holdin~ ar~a, ~here over one thousand taxis regularly assemble. bringing an outreach event directly to the taxi dnvers workplace, a tent was set up where cardiovascular-related health screenings, in~luding bl~ pressure, sugar, cholesterol, body mass index (bmi), were provided. results were shared with and explamed to the drivers and each participant received a south asian health education brochure. a total of taxi drivers who identified as south asian were involved. rau/ts: participants were all male, ranging from to years; mean age, . . screenings revealed % hypertensive; % had cholesterols over ; % blood glucose over ; % had a bmi greater than . conc~on: a unique outreach activity, adapting to logistical, occupational limitations, is pre· se~ted. on-site f~back and explanation of results, reiterating and reinforcing the concern that south asia~. men are at mcreased danger for early coronary heart disease, and the dissemination of a culturally sensmve and r~levant brochure, may heighten awareness of prevailing cardiac risk factors in this immi· grant community. p - (c) the community-hospital integration program framework: community-hospital panoenhips to improve the population's health john stevenson, richard blickstead, ann-marie marcolin, and sandi kendal v introduction: st. josephs health centre is a catholic community teaching hospital located in the heart of southwest toronto. st. joseph\'s was founded from a community-expressed need for hospital services, and since then has stayed committed to fostering a healthy community through collaboration and parmership. st. joseph's has developed an innovative model, the community-hospital integration program (chip), to strengthen st. joseph's partnerships with community stakeholders, and facilitates the building of links between community needs, service provision, and public policy outcomes to improve the health and wellness of the diverse neighbourhoods they together serve. methods: development of the chip framework st. joseph's health centre developed the chip framework through a multidisciplinary approach that included extensive international literature reviews, consultations, and key informant interviews with community service agencies and academics. to ensure active community voice, st. joseph's has hosted a number of community consultations including a community outreach forum attended by over ninety representatives from community partners. results: the chip framework the chip framework aims to improve the health and wellness of the urban communities served by st. josephs health centre through four intersecting pillars: • raising community voices provides an infrastructure and process that supports community stakeholder input into health care service planning, decision-making, and delivery by the hospital and across the continuum of care; • sharing reciprocal capacity promotes healthy communities through the sharing of our intellectual and physical capacity with our community partners; • cultivating integration initiatives facilitates vertical, horizontal, and intersectoral integration initiatives in support of community-identified needs and gaps; and • facilitating healthy exchange develops best practices in community integration through community-based research, and facilitates community voice in informing public policy. the chip framework drives the complex inter-relationships between community-hospital engagement, reciprocal capacity-building, integration initiatives, and community-based research and evaluation, to create an interconnected network of health care services. the fluidity and simplicity of this framework, and its dedication to balancing community needs and hospital mandate, posits the st. josephs health centre's chip model as an integral component in building healthy and thriving communities. p - (c) home based care promotion: improving acess to quality services and livelihood in the face of aids home based care is a service provided to persons affected/living with hiv/aids, a menu of care/support services at home/community. it is a prominent alternative approach. in uganda, hospital bed ocupacny is high, patient health care worker ratio deteriorating. empowering communities offers solutions to the problems; adressing cost, effectiveness of care. hiv/aids demands changes in attitudes, behaviour, approaches which is enhanced in home/community settings. for example art requires high levels of adherecnce, which medical workers have vety little opportunity to enforce. it also demands other support mechanisims in terms of nutrition, personal displine, etc which work best in stigma free environments. hence the relevance of home based care. this paper highlights the gaps that call for increased action in terms of home based care, examples of whre it has worked key challenges and recommendations for the future. p - (c) health care for one ... health care for alli katharina kovacs burns introduction: at the surface, it appears that every person in canada _has. ~ccess to a publicly funded health care system. those living in urban centers should have the best ava l~b hty, chmce, and access to a variety of health care services because of the distribution of health care services, fac lmes, and health professionals in concentrated in urban centers. this is not true for everyone -people with low income or who are homeless experience the challenges of social exclusion and being marginalized: there are many factors at play making it difficult for the latter group of people to access health care. service they need,.when they need them. health care is not as accessible or inclusive as intended. the quesuon to be explore.cl ~: if health care is available and accessible to one person, what makes it not available to all people? the ~ gmfic~nce of having marginalized people accessing health care and other services includes e~hanced quality of hfe and decreased risks associated with being homeless, and cost savings in ion~ ter~ w rh acute health care. methodology: community-based participatory research is applied m a case study on health care access and challenges experienced by low income and homeless people in one urban centre, edmonton, edmonton, and their challenges. the data is being gathered and ana_lyzed usmg basic m x~d methods. results: the results will focus on how services can be more mtegrated and accessible to all people with low income and who are homeless. there will also be discussion about specific perceptions of not only the service providers but also of people with low income an~ who are homeless~ and vario~s decision makers. the results will be compared to the literature regardmg health care services access mother urban centers in canada and elsewhere. conclusions: recommendations will need to address social exclusion and other factors which can make health care and other services more available to all people in the community. the implications for health care will also be discussed. additional information will be gathered in the next phases of the study to develop a community services access model. p - (c) availability and access exemplified: a case study beth hayhoe and ruth ewert introduction: access to health care in canada requires either the correct documentation or money. street youth have neither. in addition, health services in canada are designed by adults for adults. youth in general are often wary of having trust in adults with respect to their health concerns and frequently feel misunderstood. street youth are even more mistrustful of adults and public organizations, making their contact with health care professionals minimal. this combined with their risky behaviours and dangerous environment creates a population at risk for numerous health issues but with no place to have them investi· gated. at our non-government, not for profit health service designed specifically to provide a broad range of health care to street youth, professional services are provided almost entirely by volunteers. methods: using a retrospective analysis of the years of data gathered from this organization and reviewing the initial reasons gathered from youth about their needs, the success of the health centre was examined. results: all the things youth had listed as being important in a health centre have been implemented, and even more things have been added to improve the breadth and quality of services offered. the use of the health centre has increased by more than six times since its opening in . in addition, tens of thousands of visits have been paid to the health centre, costing the government and taxpayers nothing. as far as we know there is nothing else in canada that offers so many diverse and innovative services to youth in need. conclusion: it is clear from this case, that health care targeted at the needs of specific disadvan· taged populations can successfully provide them with appropriate health care. a model of accessible health care for marginalized populations can easily be developed for high impact and low cost from this successful case. toronto is one of the most ethno-racially diverse cities in the world of the estimated , , toronto residents, % ( , ) live in scarborough population growth in scarborough is faster than toronto. scarborough's population increased by % from to while toronto increased only by %. toronto's population is projected to increase by % ( ) in while scarbor· ough's will increase by %. (census canada ) low income, pove;ty, seniors, hidden homeless, new~omers to canada, the uninsured, are just a few of the issues concerning health care in our community. health care needs in scarborough are greatly impacted by diversity of ethnic and racial groups, poverty and settlement issues. this paper will share how the scarborough hospital (tsh) an urb~n communir_y hospital cares f~~ it~ community. tsh recognizes: •the changing community• b_arriers t~ accessing care • lnequalmes m health care the hospital in caring for its community pro· v ~es services that '!'e~e cu~turally, racially, and linguistically sensitive to our community. the paper will share the hospitals unique program on access and equity. the paper will share the needs in scar· borough and tsh's response to these needs: working in partnership with the local health committee, i:ne scarborough homeless co.mminee, the scarborough network of immigrant services organiza· nons a~d others. :' e paper will also share the many initiatives this urban community hospital has taken viz. community and home programs in service areas such as mental health, haemodialysis day care, t~~ home oxyg~n program, the palliative at-home program, and above all support for the volunteer clinic for the uninsured. j"'" tllu:lion: in canada's universal health care system it is generally assumed that everyone has equal access to health care. however, some immigrant groups in canada have historically been uninswed. these groups include failed refugee claimants, those overstaying their visas, and new immigrants in transition who are waiting to become eligible for health insurance. some estimates have suggested that at the present time there may be as many as one hundred thousand undocumented and thus uninsured immigrants in toronto alone. understandably, information on the characteristics of this marginalized population is very limited, since undocumented immigrants tend to have little contact with formal institutions. knowledge of the demographic characteristics of this population may aid in the development of health and social programs to better identify and meet the needs of undocumented immigrants in toronto. we conducted a review of all undocumented, uninsured patients, years of age or older at access alliance multicultural community health centre (aamchc) between and . demographic information such as age, gender, preferred language, length of time living in canada, selfreported education level and household income were recorded. rmdts: % of all adult patients at access alliance were undocumented and uninsured. % were under years of age (with a median age of years), % were women and only % spoke english. this group lived in canada for an average period of . years before they were first seen at aamchc. % of undocumented clients reported having completed at least a secondary or post-secondary education. % had self-reported household incomes of less than , $/year, while % of households reported earning less than $ , /year. the mean income per person in an average household was $/month. conchuions: uninsured individuals at aamchc were predominantly young females with limited knowledge of the english language. despite having attained a high level of education most were living well below the poverty line. recognition of these characteristics may assist in the development of health and social programs that are better adapted to meet the needs of undocumented immigrants. p - (c) sharing expertise: a role for the hospital lactation consultant in the community. badrgrmuul: st. michael's hospital is a tertiary care hospital that serves a large inner city population in downtown toronto. in order to provide care to this population, the hospital has developed a number of unique outreach roles. one such role was developed to work with pregnant and breastfeeding women living in regent park, a social housing complex located near the hospital. the population of regent park includes new immigrants, refugees and the socially disadvantaged. approach: the outreach lactation consultant provides care, as part of the canada prenatal nutrition program, in the community prenatally, in the hospital during their inpatient stay and postnatally when they return to the community. the continuity of care enhances the probability of long term successful breastfeeding. aside from the health benefits of breastmilk, breastfeeding is especially important for women who are financially needy and must depend on food banks for formula. . . lason learned: aher two years of partnership, the relationship between the an-hospital ~stpar tum experience for breastfeeding mothers and the community postpartum support, the breastfeeding rate ~thin this community is high. the professional support and visibility of the : ctation consultant, both m the hospital and in the community, contributes to the support of breastfee~m~.. . in light of ongoing funding constraints, this ha son between hospital and community could be at risk and discontinued. in light of the benefits to the mother/baby dyad, the lactation consultant as a community partner in the canada prenatal nutrition program should be safeguarded. . lldpodiu:tion: although the importance of adequate health care in pregna~cy is well recognised, ethnic disparities in utilization of prenatal care still exist in many western coun~~es. a fun~amental factor in these disparities may be language proficiency, as it influences women's ab hty. to ob~m and understand health information, to find the way in the health care system and to communicate with health care v poster sessions providers. we investigated the role of language proficiency in use and knowledge of folic acid as aspect of prenatal care in an urban multi-ethnic pregnancy c?hort. . . design: prospective cohort study. setting and parnc pants: amsterdam ~regnant women attending obstetric care providers for their first antenatal visit (n= ). country of birth defined ethnicity: the netherlands, surinam, antilles, turkey, morocco, ghana, other non-~est~m (nw) and other western (w) country. main outcome measures: knowledge about and use of.fohc a~d (fa) supplements in pregnancy, and determinants of these in diffe~~t ethnic ~~ups. deterrrunants mcl~ded age, ed~ tion, parity, pregnancy intention and dutch prof c ency. stansncs: ~ to co~p~re e~c groups, stranfied logistic regression (forward stepwise method) to explore determmants "'.'thm ethmc groups .. use of fa supplements was significantly lower among ghanaian, moroccan, turkish and other nw women ( %to %) than among dutch ( %) or other w women ( %). use amongsurinames and antillean women was intermediate ( %and %). ethnic differences in fa knowledge were similar. knowledge was the strongest determinant of use in all ethnic groups, with odds ratios (ors) ranging from . to . . language proficiency was the strongest determi~a~t of kno~ledge in ethnic groups with a mother tongue different from dutch (ors good vs. low proficiency ranging from . ro . ). educational level had a modifying role, as shown by an interaction effect between education and language proficiency in the nw group. here, the odds of having fa knowledge given a high education and good dutch proficiency was times the odds given a good proficiency but low education. conclusions: appropriate periconceptional use of folic acid supplements in non-western ethnic groups is low, reflecting an absence of knowledge that is largely determined by the inability to speak and understand the language of the habitual country. tailored interventions using communication channels most likely to address (pregnant) women from ethnic minority groups are necessary. apan from specific interventions, our results are in support of strong incentives on language education despite current political debate. introduction: recent research suggests living in an economically disadvantaged neighborhood is asso· ciated with decreased likelihood of undergoing mammography and increased risk of late-stage breast can· cer diagnosis. long distances and travel times to facilities offering low-or no-cost mammography may be imponant barriers to adherence to mammography screening recommendations for residents of economically disadvantaged neighborhoods. the purpose of this study was to examine whether facilities providing low-and no-cost screening mammography were less spatially accessible in low-income neighborhoods in chicago, and the extent to which the relationship between neighborhood income and the spatial accessibility of facilities varied by the proponion of african-american residents in the neighborhood. methods: the sample consisted of chicago neighborhoods. for each neighborhood, we con· structed three measures of the spatial accessibility of facilities: street network distance to the nearest facility, public transportation travel time to the nearest facility, and shortest automobile travel time to a facility. using decennial census data, we characterized the neighborhoods according to proportion of residents with incomes below the poverty line, proportion of residents in each of four raciavethnic groups (african-american, latino, white, and other), and population density. we used ordinary least squares (ols) and spatial exogenous lag regression to examine relationships. model estimated the relationship between neighborhood poverty and the spatial accessibility of facilities, adjusting for raciaveth· nic composition and population density. model added a multiplicative interaction term between neighborhood poverty and african-american. restllts: we identified deven facilities that provided low-or no-cost screening mammography to chicago residents in . we found that the distance and travel times via automobile and public trans· portation to facilities generally decreased as neighborhood poveny increased. however, we also found that the ~egative associations between neighborhood poverty and two of the spatial accessibility mea· sures -distance and public transportation travel time -were less strong in neighborhoods with the highest proponions of african-american residents. among neighborhoods in the highest tertile for poveny, th~ mean distance and mean public transportation travel time to facilities were over twice as long in neighborhoods in the highest tertile than in those in the lowest tenile of african-american residents. . ~ persistent socioeconomic and racial/ethnic disparities in breast cancer stage at diagno-s ~nd su~ val suggest that ensuring an equitable distribution of affordable mammography is a worth-w~e policy .goal. the ~~dy sugges~ that ~ture investigations should consider both neighborhood soc oecono d c charactensbes and rac avethmc composition when examining the spatial distribution of health resources. , ) . epidemiological data suggest tha~ hiv prevalence among msm is over % in some metropolitan cities, such .as beijing (ibid.). due to widespread homophobia in chinese society, however, this population remains invisible within current health/social services. lack of research on sexuality, specifically homosexuality, has impaired our understanding of health and health practices of chinese msm. focusing on the illness experiences of chinese msm with hiv/aids, this paper explores the socio-cultural impacts of hiv/ aids on this group. the data used for this paper were collected through semi-constructed in-depth face-toiace interviews with adult plwhas (including msm) in beijing, china. with the permission of the participants, the interviews were audio-taped or recorded in notes. the transcribed interviews and interview notes were analyzed by using n-vivo, a software program for qualitative data analysis. this phenomenological study aimed to understand the experiences chinese plwhas (including msm) from their own perspectives. results: it is found that the illness experiences of chinese msm with hiv/aids are profoundly shaped by the socio-cultural meanings of homosexuality, which are further complicated by the dominant discourses on hiv/aids in china. at a macro level, homophobia in the larger society has decreased the capability of this group to respond to this epidemic in a more efficient way. at a meso level, aids-phobia within the chinese msm circuits has prevented this group from openly confronting this disease and offering support to those who are already affected by it. at a micro level, however, these hiv-infected/ affected msm have tried their best to locate spaces to live and to construct hope for their future lives despite various barriers within family, community, and the larger society. this study suggests that facilitating chinese msm's response to the aids crisis urgently requires bridging awareness, commitment, knowledge and resources both within and without this group. it also illustrates the importance of understanding homosexuality in the local context of hiv i aids, which will be helpful for developing culturally sensitive hiv/aids programs for this population on the community, national and international levels. introduction: having a regular family doctor (rfd) is known to be positively correlated with a good medical follow up, including access to preventive and/or chronic care. inversely, a lack of primary care may lead to high rates of avoidable hospitalizations, especially among poor people and/or in underserved neighbourhoods. in such a matter, a recent comparative study showed that paris was better ranged than other cities, such as nyc, tokyo or london. nevertheless, despite a quasi universal health insurance and a high density of general practitioners, a noticeable fraction of the paris population does not have any regular physician and we aimed to understand who they are and for what reasons rhey don't have any rfd. mdbods: cross sectional population survey among a random sample of households in ~nderpriv ileged neighbourhoods in paris inner city, performed in , using a face-to-face quest nna re collecr- ng more than social and health characteristics. ruults: a quarter ( . %) of rhe study popularion did not have any rfd. this proporrion was iignificantly higher among male (or= . , %ci = [ . - . ), younger (e.g. or - /> .s l _= ._oo, " .ci = ( . - . )), and/or unemployed (or = . , %ci = ij . - . _ people. in multtvanate analysis, after a full adjustment on gender, age, health status, health insura~ce, income, occupat n and tducation level, we observed significant associations between having no rfd and: ~arrtal and_ pare~t hood status (e.g. or single no kids/in couple+kids = . , %ci = ( . - . ()~ quality of relattonsh ps with neighbours (or bad/good= . , %ci = [ . - . )), and length of residence m the neighbourhood (with a dose/effect statistical relationship). . co clusion: gender, age, employment status, mariral and parenthood stat~s as well as ~e gh bourhood anchorage seem to be major predictors of having a rfd, even when um.versa! health i~sur ance has reduced most of financial barriers. in urban contexts, where residential migrattons and single lift (or family ruptures) are frequent, specific information may be conducted to encourage people to ket rfd. :tu~y tries to assess the health effects and costs and also analyse the availability and accessibility to health care for poor. . methods: data for this study was collected by a survey on households of the local community living near the factories and households where radiation hazard w~s n?~ present. ~~art from mor· bidity status and health expenditure, data was collected ~n access, a~ail~b .hty and eff c ency of healrh care. a discriminant analysis was done to identify the vanables that d scnmmate between the study and control group households in terms of health care pattern. a contingent valuation survey was also undertaken among the study group to find out the factors affecting their willingness to pay for health insurance and was analysed using logit model. results: the health costs and indebtedness in families of the study group was high as compared to control group households and this was mainly due to high health expenditure. the discriminant analysis showed that expenditure incurred by private hospital inpatient and outpatient expenditure were significant variables, which discriminated between the two types of households. the logit analysis showed !hat variables like indebtedness of households, better health care and presence of radiation induced illnesses were significant factors influencing willingness to pay for health insurance. the study showed that study group households were dependent on private sector to get better health care and there were problems with access and availability at the public sector. conclusion: the study found out that the quality of life of the local community is poor due to health effects of radiation and the burden of radiation induced illnesses are so high for them. there is an urgent need for government intervention in this matter. there is also a need for the public sector to be efficient to cater to the needs of the poor. a health insurance or other forms of support to these households will improve the quality for health care services, better and fast access to health care facilities and reduces the financial burden of the local fishing community. the prevalence of substance abuse is an increasing problem among low-income urban women in puerto rico. latina access to treatment may play an important role in remission from substance abuse. little is known, however, about latinas' access to drug treatment. further, the role of social capital in substance abuse treatment utilization is unknown. this study examines the relative roles of social capital and other factors in obtaining substance abuse treatment, in a three-wave longitudinal study of women ages - living in high-risk urban areas of puerto rico, the inner city latina drug using study (icldus). social capital is measured at the individual level and includes variables from social support and networks, familism, physical environment, and religion instruments of the icdus. the study also elucidates the role of treatment received during the study in bringing about changes in social capital. the theoretical framework used in exploring the utilization of substance abuse treatment is the social support approach to social capital. the research addresses three main questions: ( t) does social capital predict parti~ipating in treatment programs? ( ) does participation in drug treatment programs increase social capital?, and ( ) is there a significant difference among treatment modalities in affecting change in ~ial capital? the findings revealed no significant association between levels of social capital and gettmg treatment. also, women who received drug treatment did not increase their levels of social capital. the findings, however, revealed a number of significant predictors of social capital and receiving drug ~buse treatment. predictors of social capital at wave iii include employment status, total monthly mcoi:rie, and baseline social capital. predictors of receiving drug abuse treatment include perception of physical health and total amount of money spent on drugs. other different variables were associated to treatment receipt prior to the icldus study. no significant difference in changes of social capital was found among users of different treatment modalities. this research represents an initial attempt to elucidate the two-way relationship between social capital and substance abuse treatment. more work is necessary to unden~nd. ~e role of political forces that promote social inequalities in creating drug abuse problems and ava lab hty of treatment; the relationship between the benefits provided by current treatment poster sessions v sctrings and treatment-seeking behaviors; the paths of recovery; and the efficacy and effectiveness of the trtaanent. and alejandro jadad health professionals in urban centres must meet the challenge of providing equitable care to a population with diverse needs and abilities to access and use available services. within the canadian health care system, providers are time-pressured and ill-equipped to deal with patients who face barriers of poverty, literacy, language, culture and social isolation. directing patients to needed supportive care services is even more difficult than providing them with appropriate technical care. a large proportion of the population do not have equitable access to services and face major problems navigating complex systems. new approaches are needed to bridge across diverse populations and reach out to underserved patients most in need. the objective of this project was to develop an innovative program to help underserved cancer patients access, understand and use needed health and social services. it implemented and evaluated, a pilot intervention employing trained 'personal health coaches' to assist underserved patients from a variety of ethno-linguistic, socio economic and educational backgrounds to meet their supportive cancer care needs. the intervention was tested with a group of underserved cancer patients at the princess margaret hospital, toronto. personal coaches helped patients identify needs, access information, and use supportive care services. triangulation was used to compare and contrast multiple sources of quantitative and qualitative evaluation data provided by patients, personal health coaches, and health care providers to assess needs, barriers and the effectiveness of the coach program. many patients faced multiple barriers and had complex unmet needs. barriers of poverty and language were the easiest to detect. a formal, systematic method to identify and meet supportive care needs was not in place at the hospital. however, when patients were referred to the program, an overwhelming majority of participants were highly satisfied with the intervention. the service also appeared to have important implications for improved technical health care by ensuring attendance at appointments, arranging transportation and translation services, encouraging adherence to therapy and mitigating financial hardship -using existing community services. this intervention identified a new approach that was effective in helping very needy patients navigate health and social services systems. such programs hold potential to improve both emotional and physical health out· comes. since assistance from a coach at the right time can prevent crises, it can create efficiencies in the health system. the successful use of individuals who were not licensed health professionals for this purpose has implications for health manpower planning. needle exchange programs (neps) have been distributing harm reduction materials in toronto since . counterfit harm reduction program is a small project operated out of a community health centre in south-east toronto. the project is operated by a single full-time coordinator, one pan-rime mobile outreach worker and two peers who work a few hours each week. all of counterfit's staff, peers, and volunteers identify themselves as active illicit drug users. yet the program dis~rib utes more needles and safer crack using kits and serves more illicit drug u~rs t~an the comb ~e~ number of all neps in toronto. this presentation will discuss the reasons behind this success, .s~ f cally the extended hours of operation, delivery models, and the inclusion of an. extremely marg ~ahzed community in all aspects of program design, implementation and eva.luat ?n. ~ounterfit was recently evaluated by drs. peggy milson and carol strike, two leading ep dem olog st and researchers in the hiv and nep fields in toronto and below are some of their findings: "the program has experienced considerable success in delivering a high quality, accessible and well-used program .... the pro· gram has allowed (service users) to become active participants in providing. services to others and has resulted in true community development in the best sense. " ... counterf t has ~~n verr succe~sful attracting and retaining clients, developing an effective peer-based model an.d assisting chen~s ~ th a vast range of issues .... the program has become a model for harm red~ctmn progr~ms withm the province of ontario and beyond." in june , the association of ?ntano co~mumty heal~~ <:en· ires recognized counterfit's acheivements with the excellence m community health initiatives award. in kenya, health outcomes and the performance of government health service~ have det~riorated since the late s, trends which coincide with a period of severe resource constramts necessitated by macro-economic stabilization measures after the extreme neo-liberalism of the s. when the govern· ment withdrew from direct service provision as reform trends and donor advocacy suggested, how does it perform its new indirect role of managing relations with new direct health services providers in terms of regulating, enabling, and managing relations with these health services providers? in this paper therefore, we seek to investigate how healthcare access and availability in the slums of nairobi has been impacted upon by the government's withdrawal from direct health care provision. the methodology involved col· leering primary data by conducting field visits to health institutions located in the slum areas of kibera and korogocho in nairobi. purposive random sampling was utilized in this study because this sampling technique allowed the researcher(s) to select those health care seekers and providers who had the required information with respect to the objectives of the study. in-depth interviews using a semi-structured ques· tionnaire were administered ro key informants in health care institutions. this sought to explore ways in which the government and the private sector had responded and addressed in practical terms to new demands of health care provision following the structural adjustment programmes of the s. this was complemented by secondary literature review of publications and records of key governmental, bilateral and multilateral development partners in nairobi. the study notes a number of weaknesses especially of kenya's ministry of health to perform its expected roles such as managing user fee revenue and financial sustainability of health insurance systems. this changing face of health services provision in kenya there· fore creates a complex situation, which demands greater understanding of the roles of competition and choice, regulatory structures and models of financing in shaving the evolution of health services. we rec· ommend that the introduction of user fees, decentralization of service provision and contracting-out of non-clinical to private and voluntary agencies require a new management culture, and new and clear insri· tutional relationships. experience with private sector involvement in health projects underlines the need not only for innovative financial structures to deal with a multitude of contractual, political, market and risks, but also building credible structures to ensure that health services projects are environmentally responsive, socially sensitive, economically viable, and politically feasible. purpose: the purpose of this study is to examine the status of mammography screening utilization and its predictors among muslim women living in southern california. methods: we conducted a cross-sectional study that included women aged ::!: years. we col· leered data using a questionnaire in the primary language of the subjects. the questionnaire included questions on demography; practices of breast self-examination (bse) and clinical breast examination (cbe); utilization of mammography; and family history of breast cancer. bivariate and multiple logistic regression analyses were performed to estimate the odds ratios of mammography use as a function of demographic and other predictor variables. . results: among the women, % were married, % were - years old, and % had family h story of breast cancer. thirty-two percent of the participating women never practiced bse and % had not undergone cbe during the past two years. the data indicated that % of the women did not have mammography in the last two years. logistic regression analysis showed that age ( r= . , % confi· dcnc~ interval (cl)=l. - . ), having clinical breast examination ( r= . , % cl= . - . ), and practtce of self-breast examination ( r= . , % cl= . - . ), were strong predictors of mammography use . . conclusions: the data point to the need for intervention targeting muslim women to inform and motivate th.cm a~ut practices for early detection of breast cancer and screening. further studies are needed to investigate the factors associated with low utilization of mammography among muslim women population in california. we conducted a review of the scientific literature and° government documents to describe ditnational health care program "barrio adentro" (inside the neighborhood). we also conducted qualiurivt interviews with members of the local health committees in urban settings to descrihe the comm unity participation component of the program. rtsmlts: until recently, the venezuelan public health system was characterized by a lack or limited access w health care ( % of the population) and long waiting lists that amounted to denial of service. moit than half of the mds worked in the five wealthiest metropolitan areas of the country. jn the spring oi , a pilot program hired cuban mds to live in the slums of caracas to provide health care to piople who had previously been marginalized from social programs. the program underwent a massive expansion and in only two years , cuban and , venezuelan health care providers were working acmss the country. they provide a daily average of - medical consultations and home visits, c lly out neighborhood rounds, and deliver health prevention initiatives, including immunization programs. they also provide generic medicines at no cost to patients, which treat % of presenting ill-ij!m, barrio adentro aims to build , clinics (primary care), , diagnostic and rehabilitation ctnrres (secondary care), and upgrade the current hospital infrastructure (tertiary care). local health committees survey the community to identify needs and organize a variety of lobby groups to improve dit material conditions of the community. last year, barrio adentro conducted . times the medical visits conducted by the ministry of health. the philosophy of care follows an integrated approach where btalrh is related to housing, education, employment, sports, environment, and food security. conclusions: barrio adentro is a unique collaboration between low-middle income countries to provide health care to people who have been traditionally excluded from social programs. this program shows that it is possible to develop an effective international collaboration based on participatory democracy. low-income americans are at the greatest risk of being uninsured and often face multiple health concerns. this evaluation of the neighborhood health initiative (nh!), an organization which uses multiple programmatic approaches to meet the multiple health needs of clients, reflected the program's many activities and the clients' many service needs. nh! serves low-income, underserved, and hard-to-reach residents in the des moines enterprise community. multiple approaches (fourth-generation evaluation, grounded theory, strengths-and needs-based) and methods (staff and client interviews, concept mapping, observations, qualitative and quantitative analysis) were used to achieve that reflection. results indicate good targeting of residents in the zip code and positive findings in the way of health insurance coverage and reported unmet health needs of clients. program activities were found to match client nttds, validating the organization\'s assessment of clients. important components of nhi were the staff composition and that the organization had become part of both the formal and informal networks. nhi positioned as a link between the target population and local health and social sc:rvice agencies, working to connect residents with services and information as well as aid local agencies in reaching this underserved population. p - (c) welfare: definition by new york city maribeth gregory for an individual who resides in new york city, to obtain health insurance under the medicaid policy one must fall under certain criteria .. (new york city's welfare programs ) if the individual _is on ssi or earns equal to or less than $ per month, he is entitled to receive no more than $ , m resources. a family the size of two would need to earn less than $ per month to qualify for no greater than ss, worth of medicaid benefits. a family of three would qualify for $ , is they earned less than $ per month and so on. introduction: the vancouver gay communiry has a significant number of asian descendan!l. because of their double minority status of being gay and asian, many asian men who have sex with men (msm) are struggling with unique issues. dealing with racism in both mainstream society and the gay communiry, cultural differences, traditional family relations, and language challenges can be some of their everyday srruggles. however, culturally, sexually, and linguistically specific services for asian msm are very limited. a lack of availability and accessibiliry of culturally appropriate sexual health services isolates asian msm from mainstream society, the gay community, and their own cultural communities, deprives them of self-esteem, and endangers their sexual well-being. this research focuses on the qualita· tive narrative voices of asian msm who express their issues related to their sexualiry and the challenges of asking for help. by listening to their voices, practitioners can get ideas of what we are missing and how we need to intervene in order to reach asian msm and ensure their sexual health. methods: since many asian msm are very discreet, it is crucial to build up trust relationships between the researcher and asian msm in order to collect qualitative data. for this reason, a community based participatory research model was adopted by forming a six week discussion group for asian msm. in each group session, the researcher tape recorded the discussion, observed interactions among the participants, and analyzed the data by focusing on participants' personal thoughts, experiences, and emotions for given discussion topics. ra lts: many asian msm share challenges such as coping with a language barrier, cultural differ· ences for interpreting issues and problems, and westerncentrism when they approach existing sexual health services. moreover, because of their fear of being disclosed in their small ethnic communities, a lot of asian msm feel insecure about seeking sexual health services when their issues are related to their sexual orientation. conclflsion: sexual health services should contain multilingual and multicultural capacities to meet minority clients' needs. for asian msm, outreach may be a more effective way to provide them with accessible sexual health services since many asian msm are closeted and are therefore reluctant to approach the services. building a communiry for asian msm is also a significant step toward including them in healthcare services. a communiry-based panicipatory approach can help to build a community for asian msm since it creates a rrust relationship between a worker and clients. p - (c) identifying key techniques to sustain interpretation services for assisting newcomers isolated by linguistic and cultural barriers from accessing health services s. gopi krishna lntrodaetion: the greater toronto area (gta) is home to many newcomer immigrants and other vulnerable groups who can't access health resources due to linguistic, cultural and systemic barriers. linguistic and cultural issues are of special concern to suburbs like scarborough, which is home to thousands of newcomer immigrants and refugees lacking fluency in english. multilingual community ~nterpreter. service~ (mcis) is a non-profit social service organization mandated to provide high quality mterpretanon services. to help newcomers access health services, mcis partnered with the scarborough network of immigrant serving organizations (sniso) to recruit and train volunteer interpreters to accompany clienrs lacking fluency in english and interpret for them to access health services at various locati?ns, incl~~ing communiry ~c:-lth centres/social service agencies and hospitals. the model envisioned agencies recruin~ and mcis ~.mm.g and creating an online database of pooled interpreter resources. this da.tabase, acces& bl~ to all pama~~g ?rganization is to be maintained through administrative/member · ship fees to. be ~ d by each parnapanng organization. this paper analyzes the results of the project, defines and identifies suc:cases before providing a detailed analysis for the reasons for the success . . methods:. this ~per~ q~ntitative (i.e. client numben) and qualitative analysis (i.e. results of key •~ormant m~rv ews with semce ~sers and interpreters) to analyze the project development, training and mplementanon phases of the project. it then identifies the successes and failures through the afore· mentioned analysis. poster sessions vss resljts: the results of the analysis can be summarized as: • the program saw modest success both ia l?lllls of numbers of clients served as well as sustainability at various locations, except in the hospital iririog. o the success of the program rests strongly on the commitment of not just the volunteer interprmr, but on service users acknowledgments through providing transponation allowance, small honororia, letter of reference etc. • the hospital sustained the program better at the hospital due to the iolume and nature of the need, as well as innate capacity for managing and acknowledging volunceers. collc/llsion: it is possible to facilitate and sustain vulnerable newcomer immigrants access to health !ul'ices through the training and commitment of an interpreter volunteer core. acknowledging volunteer commitment is key to the sustenance of the project. this finding is important to immigration and health policy given the significant numbers of newcomer immigrants arriving in canada's urban communities. nity program was established in to provide support to people dying at home, especially those who were waiting for admission to the resi<lential hospice. with the advent of haart and corresponding challenges for people living with hiv/aids in the community, the community program has developed a unique case management approach. this model features an interdisciplinary team providing a clientdrivcn service. the case manager provides continuity of care to clients in a variety of settings both within and outside of the health care system. a case study is used to demonstrate how formal and informal networks of support facilitate efficient and appropriate resource utilization to promote client health. this case study demonstrates the value of consistent coordination with a client who has complex physical, spiritual, substance use and mental health needs. the authors will show how they liaised with the housing authority, police, social services as well as the family physician, clinic staff and a hospital emergtncy room to ensure this client did not fall between the cracks. tarek hussain recognition of the importance of community involvement and sectoral cooperation in health and !ocial services, formalized in alma-ata declaration in , was reinforced at riga meeting held at the mid·point between alma-ata and the year . then at the th anniversary of alma-ata declaration, ir was declared that 'primary health care is everybody's business'. health does not exist in isolation. health cannot be defined only as an outcome of 'medical care' but also as one of 'social action'; the responsibility of disease prevention and health promotion rests not only on governments, but also with don·govemmentorgan izations, the community, and individuals. the major accomplishment to date may be that rhere is growing realization in the health sector that community involvement is more than a political right-it is absolute necessary. lessons have learned during the implementation of disease-specific l'cltical programmes, such as cdd, ari, and epi; first, integrated and holistic approaches to childcare art needed, and second, the need for community involvement has become evident. imc! is a broad inte-gra ed strategy with an overall objective contributing to reducing child morbidity and mortality in developing countries. and one of the imci components, 'community !mci', which is now broadening ~s an entry point for child-focused community development initiatives. community jmcl/c~ ld health is ~n mtcgrated approach to the promotion of key family and community practices that have impact on: child growth and development, disease prevention, home care for sick, malnourished child~en, a.nd care-seekmg behavior and compliance with advice and treatment. the presentation addres~es, m brief, the ~evel opinent of community !mci, operational aspect of community/im cl, key strategies and tools ava la~le for implementation of c/imci. the paper draws some successful programme examples on working logether for imc! with the community in various countries which had substantial benefns on programme outcomes. p - t (c) healthy child screening: an innovative service initiative ann-marie marcolin and joyce allen . introduction: with mounting attention for creative and integrated models of c~re .that are populallon and community focused, the healthy child screening initiative achiev.es t~ese pnncipl~s and more! . • parkdale high park rotary (sponsorship) the heal~h ~h ld scree? ?g is a ~opl~-centred model of care. agency and professional sector-specific silos are ehmmated, ~rov dmg f~~ hes wit~ one-sto~ access to primary prevention services in a local school gym! children at nsk are rece vmg early mtervent n and appropriate referrals to the right care provider, at the right time a~d in t~e right place. further, with a complimentary focus on prevention, the program serves to keep at nsk children healthy. healthy child screening results: the service model is developed around four distinct phases: ) planned outreach; ) coordinated intake and registration; ) interdisciplinary screening; and ) targeted referral. since the fall and through six collaborative healthy child screenings children ranging in age from to have benefited from this unique service model of care. conclusion: the presentation will provide practical information on the development and implementation of the model. there will also be a focus on lessons learned in building community service provider-hospital relationships. according to statistics canada ( ) % of the toronto population was born outside of canada and % were new immigrants. immigrants often arrive in canada in superior health compared to the canadian born population, but they lose this health advantage over time. changes in immigrant health status over time may be attributed to a variety of factors including barriers to accessing and receiving care as well as limitations in the mainstream health service organizations and their approaches to health care delivery (hyman, ) . for example, immigrant women are less likely to receive pap tests, which places them at a greater risk for developing cervical cancer. similarly, immigrant women receive less mammography screening than their canadian counterparts. the immigrant women's health centre mobile health unit (mhu) was created as an alternative care delivery model to address the need for increased accessibility as well as culturally and linguistically appropriate reproductive health care. toronto western hospital and the immigrant women's health centre have formed a collaborative partnership which incorporates a primary care nurse practitioner and lay ethnocultural counselors providing care on the mhu. currently, a qualitative ethnonursing study is underway to understand the unique experiences of women using the mhu for their reproductive health care as well as the perspectives of the mhu health care providers. based on a literature review and preliminary findings from provider and participant interviews, the proposed presentation will address the themes of health status for immigrant and refugee women, accessibility issues as well strategies to improve their reproductive health care. we will discuss benefits as well as limitations to health care provision using this alternative service delivery model. michael carden, brian edlin, andrew talal, elizabeth getter, and marla shu lntrod.ction: to date most active drug users have not had access to treatment for hepatitis c virus (hcv) infection and substantial barriers continue to exist that interfere with treatment availability for this population. methods: active illicit drug users interested in pursuing hcv care and treatment were recruited in partnership w~th co~munity-based organizations (cbo's) in new york city. baseline data were collected on quahty of hfe, substance use patterns, depression, health care utilization, hcv health beliefs and other relev.ant variables. medical evaluations were conducted, including liver biopsy when indicated, an~ treatment ts ~ffered when no contraindications are present. participants also receive psychiatric evaluahons to determme the appropriateness of interferon treatment. renlts: fifteen individuals have been recruited to date and received an initial medical evaluation. the mean age was years and _the average age of initial heroin or cocaine use was . . eight ( %) ha~ been homeless at least once m the last months and s ( %) were homeless for most or all of this penod. fourteen ( %) had a history of injection drug use with the mean age at first injection being s frsewons v ,an. elrven persc.!ns ( %) reported inje~~ng .heroin or cocaine within the last days while the .wing four ( l'o) reported regular non-m ect on use of cocaine and street-obtained benzodiazepines. sijiy seven percent of the sample (n= ) reported ever being referred to hcv treatment while ( t % ) ftponeddiattreann~nt had been offered by~ ~e.dical provider. none had ever received a liver biopsy or araanent. most believed that hcv was a s gmf cant threat to their health ( %) and that there was a pm)dchance they would eventually die if their hepatitis c was not treated ( %). though individuals % believed that there was no cure for hcv, ( %) reported that they would initiate treatment if i was recommended by their doctor. thirteen ( %) had a current psychiatric diagnosis including «pmsion, anxiety disorder, schizophrenia, schizoaffective disorder, and borderline and antisocial persooaliry disorders. among eight individuals who had the beck depression inventory administered, the mean score was . . attendance rates were % ( / ) at scheduled medical and psychiatric ppointments and % ( / ) at liver biopsy. <andtuions: active drug users, despite experiencing multiple psycho-social problems, can be mgaged in hcv care using a multidisciplinary approach, but require intensive follow-up support. hcv aunnent of active drug users should not be dismissed. n- (c) antiretroviral therapy in mv infected infants: when to initiate therapy an african experience kingsley okonkwo, ademola adeyemo, israel sokeye, and nwaife umeike /""°""ction: as technology for early diagnosis of human immunodeficiency virus [hiv] improves, m to commence highly active antiretroviral therapy [haarn is yet to be fully evaluated. this prospective study describes our initial experience with early haart in hiv infected nigerian infants and rqions the outcome. we also analyzed the socioeconomic implications of early haart therapy in infants in the african setting. method: hiv infected infants were started on haart before months and followed up at mkly intervals.we monitored baseline and monthly cd . conclusion: initial results suggest early haart before months resulted. in improv~d out~ome in african children. treatment appears to be as effective as in developed counmes. in. a~nca as. m most developing countries with limited resources it is possible and effective to use haart m ~~ants if p~oper llloniroring facilities are available. however the occurrence of long-term drug related tox c ty and mk of emergence of resistant viral strains create need for further evaluation of haart in infants. background: most risk factors for cardiovascular disease (cvd) can be mitigated through ~th clinical interventions and lifestyle change. we used data from a telephone survey of new york city (nyc) adults to characterize health care access and healthy behavior among those with three or more cvd risk factors. methods: the study population included respondents to the nyc community health survey (n= , ) self-reporting~ of the following: smoking, diabetes, high cholesterol level, high blood pres· sure, body mass index > , and age > (males) or > (females) (n= , ). results: based on self-report, an estimated . , ( %) of nyc adults have~ or more cvd risk factors. this population is % male, % white, % black, and % with s years of education. most report good access to health care, indicated by having health insurance ( %), regular doctor ( %), their blood pressure checked within last months ( %), and their choles· terol checked within the past year ( % ). only % reported getting at least minutes of exercise ~ times per week and only % eating ~ servings of fruits and vegetables the previous day. among current smokers, % attempted to quit in past months, but only % used medication or counseling. implications: these data suggest that most nyc adults known to be at high risk for cvd have access to regular health care, but most do not engage in healthy lifestyle or, if they smoke, attempt effective quit strategies. more clinic-based and population-level interventions are needed to support lifestyle change among those at high risk of cvd. introduction: recently, much interest has been directed at "obesogenic" (obesity-promoting) (swinburn, egger & raza, ) built environments, and at geographic information systems (gis) as a tool for their exploration. a major geographical concept is accessibility, or the ease of moving from an origin to a destination point, which has been recently explored in several health promotion-related stud· ies. there are several methods of calculating accessibility to an urban feature, each with its own strengths, drawbacks and level of precision that can be applied to various health promotion research issues. the purpose of this paper is to describe, compare and contrast four common methods of calculating accessibility to urban amenities in terms of their utility to obesity-related health promotion research. practical and conceptual issues surrounding these methods are introduced and discussed with the intent of providing health promotion researchers with information useful for selecting the most appropria e accessibility method for their research goal~ ~ethod: this paper describes methodological insights from two studies, both of which assessed the neighbourhood-level accessibility of fast-food establishments in edmonton, canada -one which used a relatively simple coverage method and one which used a more complex minimum cos method. res.its: both methods of calculating accessibility revealed similar patterns of high and low access to fast-food outlets. however, a major drawback of both methods is that they assume the characteristics of the a~e~ities and of the populations using them are all the same, and are static. the gravity potential method is introduced as an alternative, since it is ·capable of factoring in measures of quality and choice. a n~mber of conceptual and pr~ctical iss~es, illustrated by the example of situational influences on food choice, make the use of the gravity potential model unwieldy for health promotion research into sociallydetermined conditions such as obesity. co.nclusions: i~ ~ommended that geographical approaches be used in partnership with, or as a foun~ation for, ~admonal exploratory methodologies such as group interviews or other forms of commumty consultation that are more inclusive and representative of the populations of interest. qilhl in los angeles county ,,..ia shaheen, richard casey, fernando cardenas, holman arthurs, and richard baker ~the retinomax autorefractor has been used for vision screening of preschool age childien. ir bas been suggested to be used and test school age children but not been validated in this age poup. ob;taiw: to compare the results of retinomax autorefractor with findings from a comprehensive i!' examination using wet retinoscopy for refractive error. mllhods: children - years old recruited from elementary schools at los angeles county were iaml with snellen's chart and the retinomax autorefractor and bad comprehensive eye examination with dilation. the proportion of children with abnormal eye examination as well as diesensitiviry and specificity of the screening tools using retinomax autorefractor alone and in combinalion wirh snellen's chart. results of the children enrolled in the study (average age= . ± . years; age range, - years), ?% had abnormal eye examination using retinoscopy with dilation. for the lerinomax, the sensitivity was % ( % confidence interval [ci] %- %), and the specificity was % ( % ci, %- o/o). simultaneous testing using snellen's chart and retinomax resulted in gain in sitiviry ( %, % cl= , ), and loss in specificity ( %, % cl= %- %). the study showed that screening school age children with retinomax autorefractor could identify most cases with abnormal vision but would be associated with many false-positive results. simuhaneous resting using snellen's chart and retinomax maximize the case finding but with very low specificiry. mdhotjs: a language-stratified, random sample of members of the college of family physicians of canada received a confidential survey. the questionnaire collected data on socio-demographic characteristics, medical training, practice type, setting and hcv-related care practices. the self-adminisratd questionnaire was also made available to participants for completion on the internet. batdti: response proportion was %. median age was years ( % female) and the proporlionoffrench questionnaires was %. approximately % had completed family medicine residency lllining in canada; median year of training completion was . sixty-seven percent, % and % work in private offices/clinics, community hospitals and emergency departments, respectively. regarding ~practices, % had ever requested a hcv test and % of physicians had screened for hcv iafrction in rhe past months· median number of tests was . while % reported having no hcv-uaed patients in their practic~, % had - hcv-infected patients. regarding the level of hcv care provided, . % provide ongoing advanced hcv care including treatment and dose monitoring for ctmduions: in this sample of canadian family physicians, most had pro~ided hcv screening. to •least one patient in the past year. less than half had - hcv-infected patients and % provide ~:relared care the role of socio-demographic factors, medical training as wel_i as hcv ca~e percep-lldas rhe provision of appropriate hcv screening will be examined and described at the time of the canference. ' - (c) healthcare services: the context of nepal meen poudyal chhetri """ tl.ction healthcare service is related with the human rights and fundamental righ~ of the ci~ ciaaiuntry. however, the growing demand foi health care services, quality heal~care service, accessib b~ id die mass population and paucity of funds are the different but interrelated issues to .be ~ddressed. m nepat. n view of this context, public health sector in nepal is among other sectors, which is struggling -.i for scarce resources. . . . nepal, the problems in the field of healthcare servic~s do not bnut ~o the. paucity of faads and resources only, but there are other problems like: rural -urban imbalance, regional unbalance, poster sessio~ f the ll ·m ·ted resources poor healthcare services, inequity and inaccessibility of the poor management o , . poor people of the rural, remote and hilly areas for the healthcare services and so on.. . . . · . i f ct the best resource allocation is the one that max m zes t e sum o m ivi ua s u · ea t services. n a , · h d' ·b · · · h . ·t effi.ciency and efficient management are correlated. it might be t e re istn utmn of mes. ence, equi y, . . . . . income or redistribution of services. moreover, maximizanon of available resources, qua tty healthcare services and efficient management of them are the very important and necessary tools and techmques to meet the growing demand and quality healthcare services in nepal. p - (a) an jn-depth analysis of medical detox clients to assist in evidence based decision making xin li, huiying sun, ajay puri, david marsh, and aslam anis introduction: problematic substance use represents an ever-increasing public health challenge. in the vancouver coastal health (vch) region, there are more than , individuals having some probability of drug or alcohol dependence. to accommodate this potential demand for addiction related services, vch provides various services and treatment, including four levels of withdrawal management services (wms). clients seeking wms are screened and referred to appropriate services through a central telephone intake service (access i). the present study seeks to rigorously evaluate one of the services, vancouver detox, a medically monitored -bed residential detox facility, and its clients. doing so will allow decision makers to utilize evidence based decision-making in order to improve the accessibility and efficiency of wms, and therefore, the health of these clients. methods: we extract one-year data (october , -september , from an efficient and comprehensive database. the occupancy rate of the detox centre along with the clients' wait time for service and length of stay (los) are calculated. in addition, the effect of seasonality on these variables and the impact of the once per month welfare check issuance on the occupancy rate are also evaluated. results: among the clients (median age , % male) who were referred by access! to vancouver detox over the one-year period, were admitted. the majority ( %) of those who are not admitted are either lost to follow up (i.e., clients not having a fixed address or telephone) or declined service at time of callback. the median wait time was day [q -ql: - ], the median los was days iq -qt: - ], and the average bed occupancy rate was %. however, during the threeday welfare check issue period the occupancy rate was lower compared to the other days of the year % vs. %, p conclusion: our analysis indicates that there was a relatively short wait time at vancouver detox, however % of the potential clients were not served. in addition, the occupancy rate declined during the welfare check issuance period and during the summer. this suggests that accessibility and efficiency at vancouver detox could be improved by specifically addressing these factors. background: intimate partner violence (ipv) is associated with acute and chronic physical and men· tal health outcomes for women resulting in greater use of health services. yet, a vast literature attests to cultural variations in perceptions of health and help-seeking behaviour. fewer studies have examined differences in perceptions of ipv among women from ethnocultural communities. the recognition, definition, and understanding of ipv, as well as the language used to describe these experiences, may be different in these communities. as such, a woman's response, including whether or not to disclose or seek help, may vary according to her understanding of the problem. methods: this pilot study explores the influence of cultural factors on perceptions of and responses to ipv among canadian born and immigrant young women. in-depth focus group interviews were con· ducted with women, aged to years, living in toronto. open-ended and semi-structured interview questions were designed to elicit information regarding how young women socially construct jpv and where they would go to receive help. interviews were transcribed, then read and independently coded by the research team. codes were compared and disagreements resolved. qualitative software qsr n was used to assist with data management. . ruu~ts_: res~nses_abo~t what constitutes ipv were similar across the study groups. when considering specific ab.us ve ~ tuanons and types of relationships, participants held fairly relativistic views about ipv, especially with regard to help-seeking behaviour. cultural differences in beliefs about normaive m;ile/femal~ relations. familial.roles, and customs governing acceptable behaviours influenced partictpants perceptions about what n ght be helpful to abused women. interview data highlight the social l ter srnfons v suucrural _impact these factors ha:e on you?g women and provide details regarding the dynamics of cibnocultur~ m~uences on help-~eekmg behav ur: t~e ro~e of such factors such as gender inequality within rtlaoo?sh ps and t_he ~erce ved degree of ~oc al solat on and support nerworks are highlighted. collc~ the~ findmgs unde~score the _ mporta_nc_e of understanding cultural variations in percrprions of ipv ~ relanon to ~elp-seekmg beha~ ':'ur. th s_mformation is critical for health professionals iodiey may connnue developmg culturally sensmve practices, including screening guidelines and protorol s. ln addition, _this study demonstr~tes that focus group interviews are valuable for engaging young romen in discussions about ipv, helpmg them to 'name' their experiences, and consider sources of help when warranted. p -s (a) health problems and health care use of young drug users in amsterdam .wieke krol, evelien van geffen, angela buchholz, esther welp, erik van ameijden, and maria prins / trod ction: recent advances in health care and drug treatment have improved the health of populations with special social and health care needs, such as drug users. however, still a substantial number dots not have access to the type of services required to improve their health status. in the netherlands, tspccially young adult drug users (yad) whose primary drug is cocaine might have limited access to drugrreatment services. in this study we examined the history and current use of (drug associated) treatmmt services, the determinants for loss of contact, and the current health care needs in the young drug mm amsterdam study (yodam). methods: yodam started in and is embedded in the amsterdam cohort study among drug mm. data were derived from y ad aged < years who had used cocaine, heroin, ampheramines and i or methadone at least days a week during the months prior to enrolment. res lts:of yao, median age was years (range: - years), % was male and % had dutch nationality at enrolment. nearly all participants ( %) reported a history of contact with drug llt.lnnent services (methadone maintenance, rehabilitation clinics and judicial treatment), mental health car? (ambulant mental care and psychiatric hospital) or general treatment services (day-care, night-care, hdp for living arrangements, work and finance). however, only % reported contact in the past six l!xlllths. this figure was similar in the first and second follow-up visit. among y ad who reported no current contact with the health care system, % would like to have contact with general treatment serl' icts. among participants who have never had contact with drug treatment services, % used primarily cocaine compared with % and % among those who reported past or current contact, respectively. saied on the addiction severity index, % reported at least one mental health problem in the past days, but only % had current contact with mental health services. concl sion: results from this study among young adult drug users show that despite a high contact rm with health care providers, the health care system seems to lose contact with yao. since % indicatt the need of general treatment services, especially for arranging house and living conditions, health m services that effectively integrate general health care with drug treatment services and mental health care might be more successful to keep contact with young cocaine users. mtthods: respondents included adults aged and over who met dsm-iv diagn?snc criteria for an anxiety or depressive disorder in the past months. we performed two sets of logisnc regressmns. thtdichotomous dependent variables for each of the regressions indicated whether rhe respondenr_vis-ud a psychiatrist, psychologist, family physician or social worker in the _past_ months. no relationship for income. there was no significant interaction between educatmn an mco~:· r: ::or respondents with at least a high school education to seek help ~rom any of the four servic p were almost twice that for respondents who had not completed high school. th . d ec of analyses found che associacion becween educacion and use of md-provided care e secon s · · be d · · ·f· ly ·n che low income group for non-md care, the assoc anon cween e ucatlon and was s gm icant on -· . . . . use of social workers was significant in both income groups, but significant only for use of psychologists in che high-income group. . . . conclusion: we found differences in healch service use by education level. ind v duals who have nor compleced high school appeared co use less mental he~lt~ servi~es provided ~y psyc~iatrists, psycholo· gists, family physicians and social workers. we found limited e.v dence _suggesting the influence of educa· tion on service use varies according to income and type of service provider. results suggesc there may be a need to develop and evaluate progr~ms.designe~ to deliver targeted services to consumers who have noc completed high school. further quahtanve studies about the expen· ence of individuals with low education are needed to clarify whether education's relationship with ser· vice use is provider or consumer driven, and to disentangle the interrelated influences of income and education. system for homeless, hiv-infected patients in nyc? nancy sahler, chinazo cunningham, and kathryn anastos introduction: racial/ethnic disparities in access to health care have been consistently documented. one potential reason for disparities is that the cultural distance between minority patients and their providers discourage chese patients from seeking and continuing care. many institucions have incorporated cultural compecency craining and culturally sensicive models of health care delivery, hoping co encourage better relacionships becween patients and providers, more posicive views about the health care system, and, ulcimacely, improved health outcomes for minority patients. the current scudy tests whether cultural distance between physicians and patients, measured by racial discordance, predicts poorer patient attitudes about their providers and the health care system in a severely disadvantaged hiv-infected population in new york city that typically reports inconsistent patterns of health care. methods: we collected data from unscably housed black and latino/a people with hiv who reported having a regular health care provider. we asked them to report on their attitudes about their provider and the health care system using validated instruments. subjects were categorized as being racially "concordant" or "discordant" with their providers, and attitudes of these two groups were compared. results: the sample consisted of ( %) black and ( %) latino/a people, who reported having ( %) black physicians, ( %) latino/a physicians, ( %) white physicians, and ( %) physicians of another/unknown race/ethnicity. overall, ( %) subjects had physicians of a different race/ethnicity than their own. racial discordance did not predict negative attitudes about rela· tionship with providers: the mean rating of a i-item trust in provider scale (lo=high and o=low) was . for both concordant and discordant groups, and the mean score in -icem relationship with provider scale ( =high and !=low) was . for both groups. however discordance was significantly associated with distrust in che health care syscem: che mean score on a -icem scale ( =high discrust and l=low distrust) was . for discordant group and . for che concordant group (t= . , p= . ). we further explored these patterns separacely in black and lacino/a subgroups, and using different strategies ro conceptualize racial/ethic discordance. conclusions: in this sample of unscably housed black and latino/a people who receive hiv care in new york city, having a physician from the same racial/ethnic background may be less important for developing a positive doctor-patient relationship than for helping the patients to dispel fear and distrust about the health care system as a whole. we discuss the policy implications of these findings. ilene hyman and samuel noh . .abstract objectiw: this study examines patterns of mental healthcare utilization among ethiopian mm grants living in toronto. methods: a probability sample of ethiopian adults ( years and older) completed structured face-to-face interviews. variables ... define, especially who are non-health care providers. plan of analysis. results: approximately % of respondents received memal health services from mainstream healthcare providers and % consulted non-healthcare professionals. of those who sought mental health services from mainstream healthcare providers, . % saw family physicians, . % visited a psychiatrist. and . % consulted other healthcare providers. compared with males, a significantly higher proportion gsfer sessions v ri ftlnales consulted non-healthcare_ professionals for emotional or mental health problems (p< . ). tlbile ethiopian's overall use of mamstream healthcare services for emotional problems ( %) did not prlydiffer from the rate ( %) of the general population of ontario, only a small proportion ( . %) rjerhiopians with mental health needs used services from mainstream healthcare providers. of these, !oj% received family physicians' services, . % visited a psychiatrist, and . % consulted other healthll/c providers. our data also suggested that ethiopian immigrants were more likely to consult tradioooal healers than health professionals for emotional or mental health problems ( . % vs. . % ). our bivariate analyses found the number of somatic symptoms and stressful life events to be associated with an increased use of medical services and the presence of a mental disorder to be associated with a dfcreased use of medical services for emotional problems. however, using multivariate methods, only die number of somatic symptoms remained significantly associated with use of medical services for emooonal problems. diu#ssion: study findings suggest that there is a need for ethnic-specific and culturally-appropriate mrcrvention programs to help ethiopian immigrants and refugees with mental health needs. since there ~a strong association between somatic symptoms and the use family physicians' services, there appears robe a critical role for community-based family physicians to detect potential mental health problems among their ethiopian patients, and to provide appropriate treatment and/or referral. the authors acknowledge the centre of excellence for research in immigration and settlement (ceris) in toronto and canadian heritage who provided funding for the study. we also acknowledge linn clark whose editorial work has improved significantly the quality of this manuscript. we want to thank all the participants of the study, and the ethiopian community leaders without whose honest contributions the present study would have not been possible. this paper addresses the impact of the rationalization of health-care services on the clinical decision-making of emergency physicians in two urban hospital emergency departments in atlantic canada. using the combined strategies of observational analysis and in-depth interviewing, this study provides a qualitative understanding of how physicians and, by extension, patients are impacred by the increasing ancmpts to make health-care both more efficient and cost-effective. such attempts have resulted in significantly compromised access to primary care within the community. as a consequence, patients are, out of necessity, inappropriately relying upon emergency departments for primary care services as well as access to specialty services. within the hospital, rationalization has resulted in bed closures and severely rmricted access to in-patient services. emergency physicians and their patients are in a tenuous position having many needs but few resources. furthermore, in response to demands for greater accountability, physicians have also adopted rationality in the form of evidence-based medicine. ultimately, ho~ever, rationality whether imposed upon, or adopted by, the profession significantly undermines physu.: ans' ability to make decisions in the best interests of their patients. johnjasek, gretchen van wye, and bonnie kerker introduction: hispanics comprise an increasing proportion of th.e new york city (nyc) populanon !currently about %). like males in the general population, h spamc males (hm) have a lower prrval,nce of healthcare utilization than females. however, they face additional access barriers such as bnguage differences and high rates of uninsurance. they also bear a heavy burden of health problems lllehasobesity and hiv/aids. this paper examines patterns of healthcare access and ut hzat on by hm compared to other nyc adults and identifies key areas for intervention. . . . and older are significantly lower than the nhm popu anon . v. . , p<. ), though hi\' screening and immunizations are comparable between the two groups. conclusion: findings suggest that hm have less access t? healthcare than hf or nhm. hown r, hm ble to obtain certain discrete medical services as easily as other groups, perhapsdueto!rtor are a hm. i i . subsidized programs. for other services, utilization among s ower. mprovmg acc~tocareinthis group will help ensure routine, quality care, which can lead to a greater use of prevennve services iii! thus bener health outcomes. introduction: cancer registry is considered as one of the most important issues in cancer epidemiology and prevention. bias or under-reporting of cancer cases can affect the accuracy of the results of epidemiological studies and control programs. the aim of this study was to assess the reliability of the regional cancer report in a relatively small province (yasuj) with almost all facilities needed for c llcll diagnosis and treatment. methods: finding the total number of cancer cases we reviewed records of all patients diagnoicd with cancer (icd - ) and registered in any hospital or pathology centre from until i n yasuj and all ( ) surrounding provinces. results: of patients who were originally residents of yasui province, . % wereaccoulll!d for yasuj province. the proportion varies according to the type of cancer, for exarnplecancetsofdiglstive system, skin and breast were more frequently reported by yasuj's health facilities whereas cancmoi blood, brain and bone were mostly reported by neighbouring provinces. the remaining cases ( . % were diagnosed, treated and recorded by neighbouring provinces as their incident cases. this is partly because of the fact that patients seek medical services from other provinces as they believed that the facil. ities are offered by more experienced and higher quality stuffs and their relative's or temporary acooiii' modation addresses were reported as their place of residence. conclusion: measuring the spatial incidence of cancer according to the location of report ortht current address affected the spatial statistics of cancer. to correct this problem recording the permanm! address of diagnosed cases is important. p - (c). providing primary healthcare to a disadvantaged population at a university-run commumty healthcare facility tracey rickards the. c:ommuni~y .h~alth ~linic (chc) is a university sponsored nurse-managed primary bealthwt (p~c:l clime. the clm c is an innovative model of healthcare delivery in canada that has integrated tht principles of phc ser · · h' . vices wit ma community development framework. it serves to provide access to phc services for members of th · · illi · dru is ii be . . e community, particularly the poor and those who use or gs, we mg a service-learning facil'ty f d · · · · · · d rionll h . . .,m.:. · t · . meet c ient nee s. chmc nursing and social work staff and srudents r·--· ipa em various phc activities and h .l.hont" less i . f . outreac services in the local shelters and on the streels to'"" popu auon o fredericton as well th chc · model iii fosterin an on oi : . • e promotes and supports a harm reduction . · local d!or an~ h ng ~art:ersh p with aids new brunswick and their needle exchange program, w tha ing condoms and :xu:t h:~~~e e~aint~nance therapy clients, and with the clie~ts themselves ~_r; benefits of receiving health f ucation, a place to shower, and a small clothing and food oai~· care rom a nurse p · · d d · --""~'i"· are evidenced in th r research that involved needsaans mvo ves clients, staff, and students. to date the chc has unacn- · sessment/enviro i . d ; •• '"""" ll eva uanon. the clinic has also e . d nmenta scan, cost-benefit analysis, an on-go...,, "".'i'~ facility and compassionate lea x~mme the model of care delivery' focusing on nursing roles wi~ cj rmng among students. finally, the clinic strives to share the resu•p v . -arch with the community in which it provides service by distributing a bi-monthly newsletter, and plllicipating in in-services and educational sessions in a variety of situations. the plan for the future is coolinued research and the use of evidence-based practice in order to guide the staff in choosing how much n~ primary healthcare services to marginalized populations will be provided. n- (c) tuming up the volume: marginalized women's health concerns tckla hendrickson and betty jane richmond bdrotbu:tion: the marginalization of urban women due to socio-economic status and other determinants negatively affects their health and that of their families. this undermines the overall vitaliry of urban communities. for example, regarding access to primary health care, women of lower economic surus and education levels are less likely to be screened for breast and cervical cancer. what is not as widely reported is how marginalized urban women in ontario understand and articulate their lack of access to health care, how they attribute this, and the solutions that they offer. this paper reports on the rnults of the ontario women's health network (owhn) focus group project highlighting urban women's concerns and suggestions regarding access to health care. it also raises larger issues about urban health, dual-purpose focus group design, community-based research and health planning processes. mdhods: focus group methodology was used to facilitate a total of discussions with urban and rural women across ontario from to . the women were invited to participate by local women's and health agencies and represented a range of ages, incomes, and access issues. discussions focussed on women's current health concerns, access to health care, and information needs. results were analyzed using grounded theory. the focus groups departed from traditional focus group research goals and had two purposes: ) data collection and dissemination (representation of women's voices), and ) fostering closer social ties between women, local agencies, and owhn. the paper provides a discussion and rationale for a dual approach. rax/ts: the results confirm current research on women's health access in women's own voices: urban women report difficulty finding responsive doctors, accessing helpful information such as visual aids in doctors' offices, and prohibitive prescription costs, in contrast with rural women's key concern of finding a family doctor. the research suggests that women's health focus groups can address access issues by helping women to network and initiate collective solutions. the study shows that marginalized urban women are articulate about their health conctrns and those of their families, often understanding them in larger socio-economic frameworks; howtver, women need greater access to primary care and women-friendly information in more languages and in places that they go for other purposes. it is crucial that urban health planning processes consult directly with women as key health care managers, and turn up the volume on marginalized women's voices. women: an evaluation of awareness, attitudes and beliefs introduction: nigeria has one of the highest rates of human immunodeficiency virus ihivi seroprrvalence in the world. as in most developing countries vertical transmission from mother to child account for most hiv infection in nigerian children. the purpose of this study was ro. determine the awareness, attitudes and beliefs of pregnant nigerian women towards voluntary counseling and testing ivct! for hiv. mnbod: a pre-tested questionnaire was used to survey a cross section '.>f. pregnant women ~t (lrlleral antenatal clinics in awka, nigeria. data was reviewed based on willingness to ~c~ept or re ect vct and the reasons for disapproval. knowledge of hiv infection, routes of hiv transm ssmn and ant rnroviral therapy iart) was evaluated. hsults: % of the women had good knowledge of hiv, i % had fair knowledge while . % had poor knowledge of hiv infection. % of the women were not aware of the association of hreast milk feeding and transmission of hiv to their babies. majority of the women % approved v~t while % disapproved vct, % of those who approved said it was because vct could ~educe risk of rransmission of hiv to their babies. all respondents, % who accepted vc.i ~ere willing to be tnted if results are kept confidential only % accepted to be tested if vc.t results w. be s~ared w .th pinner and relatives % attributed their refusal to the effect it may have on their marriage whale '-gave the social 'and cultural stigmatization associated with hiv infection for their r~fusal.s % wall accept vct if they will be tested at the same time with their partners. ~ of ~omen wall pref~r to breast feed even if they tested positive to hiv. women with a higher education diploma were times v more likely to accept vct. knowledge of art for hiv infected pregnant women as a means of pre. vention of maternal to child transmission [pmtct) was generally poor, % of respondents wm aware of art in pregnancy. conclusion: the acceptance of vct by pregnant women seems to depend on their understanding that vct has proven benefits for their unborn child. socio-cultur al factors such as stigmatizationof hiv positive individuals appears to be the maj_or impedi~ent towards widespread acceptanee of ycr in nigeria. involvemen t of male partners may mpro~e attitudes t~wa~ds vct:the developmentofm novative health education strategies is essential to provide women with mformanon as regards the benefits of vct and other means of pmtct. p - (c) ethnic health care advisors in information centers on health care and welfare in four districts of amsterdam arlette hesselink, karien stronks, and arnoud verhoeff introduction : in amsterdam, migrants report a "worse actual health and a lower use of health care services than the native dutch population. this difference might be partly caused by problems migrants have with the dutch language and health care and welfare system. to support migrants finding their way through this system, in four districts in amsterdam information centers on health care and welfare were developed in which ethnic health care advisors were employed. their main task is to provide infor· mation to individuals or groups in order to bridge the gap between migrants and health care providers. methods: the implementat ion of the centers is evaluated using a process evaluation in order to give inside in the factors hampering and promoting the implementat ion. information is gathered using reports, attending meetings of local steering groups, and by semi-structu red interviews with persons (in)directly involved in the implementat ion of the centers. in addition, all individual and groupcontaets of the health care advisors are registered extensively. results: since four information centers, employing ethnic health care advisors, are implemented. the ethnicity of the health care advisors corresponds to the main migrant groups in the different districts (e.g. moroccan, turkeys, surinamese and african). depending on the local steering groups, the focus of the activities of the health care advisors in the centers varies. in total, around individual and group educational sessions have been registered since the start. most participants were positive about the individual and group sessions. the number of clients and type of questions asked depend highly on the location of the centers (e.g. as part of a welfare centre or as part of a housing corporation). in all districts implementa tion was hampered by lack of ongoing commitment of parties involved (e.g. health care providers, migrant organization s) and lack of integration with existing health care and welfare facilities. discussion: the migrant health advisors seem to have an important role in providing information on health and welfare to migrant clients, and therefore contribute in bridging the gap between migrants and professionals in health care and welfare. however, the lack of integration of the centers with the existing health care and welfare facilities in the different districts hampers further implementation . therefore, in most districts the information centres will be closed down as independent facilicities in the near future, and efforts are made to better connect the position of migrant health advisor in existing facilities. the who report ranks the philippines as ninth among countries with a high tb prevalence. about a fourth of the country's population is infected, with majority of cases coming from the lower socioeconomic segments of the community. metro manila is not only the economic and political capital of the philippines but also the site of major universities and educational institutions. initial interviews with the school's clinicians have established the need to come up with treatment guidelines and protocols for students and personnel when tb is diagnosed. these cases are often identified during annual physical examinations as part of the school's requirements. in many instances, students and personnel diagnosed with tb are referred to private physicians where they are often lost to follow-up and may have failure of treatment due to un monitored self-administered therapy. this practice ignores the school clinic's great potential as a tb treatment partner. through its single practice network (spn) initiative, the philippine tuberculosis initiatives for the private sector (philippine tips), has established a model wherein school clinics serve as satellite treatment partners of larger clinics in the delivery of the directly observed treatment, short course (dots) protocol. this "treatment at the source" allows school-based patients to get their free government-suppl ied tb medicines from the clinic each day. it also cancels out the difficulty in accessing medicines through the old model where the patient has to go to the larger clinic outside his/her school to get treatment. the model also enables the clinic to monitor the treatment progress of the student and assumes more responsibility over their health. this experience illustrates how social justice in health could be achieved from means other than fund generation. the harnessing of existing health service providers in urban communities through standardized models of treatment delivery increases the probability of treatment success, not only for tb but for other conditions as well. p - (c) voices for vulnerable populations: communalities across cbpr using qualitative methods martha ann carey, aja lesh, jo-ellen asbury, and mickey smith introduction: providing an opportunity to include, in all stages of health studies, the perspectives and experiences of vulnerable and marginalized populations is increasingly being recognized as a necessary component in uncovering new solutions to issues in health care. qualitative methods, especially focus groups, have been used to understand the perspectives and needs of community members and clinical staff in the development of program theory, process evaluation and refinement of interventions, and for understanding and interpreting results. however, little guidance is available for the optimal use of such information. methods: this presentation will draw on diverse experiences with children and their families in an asthma program in california, a preschool latino population in southern california, a small city afterschool prevention program for children in ohio, hiv/aids military personnel across all branches of the service in the united states, and methadone clinic clients in the south bronx in new york city. focus groups were used to elicit information from community members who would not usually have input into problem definitions and solutions. using a fairly common approach, thematic analysis as adapted from grounded theory, was used to identify concerns in each study. next we looked across these studies, in a meta-synthesis approach, to examine communalities in what was learned and in how information was used in program development and refinement. results: while the purposes and populations were diverse, and the type of concerns and the reporting of results varied, the conceptual framework that guided the planning and implementation of each study was similar, which led to a similar data analysis approach. we will briefly present the results of each study, and in more depth we will describe the communalities and how they were generated. conclusions: while some useful guidance for planning future studies of community based research was gained by looking across these diverse studies, it would be useful to pursue a broader examination of the range of populations and purposes to more fully develop guidance. background: the majority of studies examining the relationship between residential environments and cardiovascular disease have used census derived measures of neighborhood ses. there is a need to identify specific features of neighborhoods relevant to cardiovascular disease risk. we aim to ) develop methods· data on neighborhood conditions were collected from a telephone survey of s, fesi· dents in balth:.ore, md; forsyth county, nc; and new york, ny. a sample of of the i.ni~~l l'elpondents was re-interviewed - weeks after the initial interview t~ measure the tes~-~etest rebab ~ ty of ~e neighborhood scales. information was collected across seven ~e ghborho~ cond ~ons (aesth~~ ~uah~, walking environment, availability of healthy foods, safety, violence, social cohesion, and acnvmes with neighbors). neighborhoods were defined as census tracts or homogen~us census tra~ clusters. ~sycho metric properties.of the neighborhood scales were accessed by ca~cu~~.ng chronba~h s alpha~ (mtemal consistency) and intraclass correlation coefficients (test-r~test reliabilmes) .. pear~n s .corre~anons were calculated to test for associations between indicators of neighborhood ses (tncludmg d mens ons of race/ ethnic composition, family structure, housing, area crowding, residential stability, education, employment, occupation, and income/wealth) and our seven neighborhood scales. . chronbach's alphas ranged from . (walking environment) to . (violence). intraclass correlations ranged from . (waling environment) to . (safety) and wer~ high~~~ . ~ for ~urout of the seven neighborhood dimensions. our neighborhood scales (excluding achv hes with neighbors) were consistently correlated with commonly used census derived indicators of neighborhood ses. the results suggest that neighborhood attributes can be reliably measured. further development of such scales will improve our understanding of neighborhood conditions and their importance to health. childhood to young adulthood in a national u.s. sample jen jen chang lntrodfldion: prior studies indicate higher risk of substance use in children of depressed mothers, but no prior studies have followed up the offspring from childhood into adulthood to obtain more precise estimates of risk. this study aimed to examine the association between early exposure to maternal depl'elsive symptoms (mds) and offspring substance use across time in childhood, adolescence, and young adulthood. methods: data were obtained from the national longitudinal survey of youth. the study sample includes , mother-child/young adult dyads interviewed biennially between and with children aged to years old at baseline. data were gathered using a computer-assisted personal interview method. mds were measured in using the center for epidemiologic studies depression scale. offspring substance use was assessed biennially between and . logistic and passion regression models with generalized estimation equation approach was used for parameter estimates to account for possible correlations among repeated measures in a longitudinal study. rnlllta: most mothers in the study sample were whites ( %), urban residents ( %), had a mean age of years with at least a high school degree ( %). the mean child age at baseline was years old. offspring cigarette and alcohol use increased monotonically across childhood, adolescence, and young adulthood. differential risk of substance use by gender was observed. early exposure to mds was associated with increased risk of cigarette (adjusted odds ratio (aor) = . , % confidence interval ( ): . , . ) and marijuana use (aor = . , % ci: . , . ), but not with alcohol use across childhood, adolescence, and young adulthood, controlling for a child's characteristics, socioeconomic status, ~ligiosity, maternal drug use, and father's involvement. among the covariates, higher levels of father's mvolvement condluion: results from this study confirm previous suggestions that maternal depressive symptoms are associated with adverse child development. findings from the present study on early life experi-e~ce have the potential to inform valuable prevention programs for problem substance use before disturbances become severe and therefore, typically, much more difficult to ameliorate effectively. the ~act (~r-city men~ health study predicting filv/aids, club and other drug transi-b~) study a multi-level study aimed at determining the association between features of the urban enyjronment mental health, drug use, and risky sexual behaviors. the study is randomly sampling foster sessions v neighborhood residents and assessing the relations between characteristics of ethnographically defined urban neighborhoods and the health outcomes of interest. a limitation of existing systematic methods for evaluating the physical and social environments of urban neighborhoods is that they are expensive and time-consuming, therefore limiting the number of times such assessments can be conducted. this is particularly problematic for multi-year studies, where neighborhoods may change as a result of seasonality, gentrification, municipal projects, immigration and the like. therefore, we developed a simpler neighborhood assessment scale that systematically assessed the physical and social environment of urban neighborhoods. the impact neighborhood evaluation scale was developed based on existing and validated instruments, including the new york city housing and vacancy survey which is performed by the u.s. census bureau, and the nyc mayor's office of operations scorecard cleanliness program, and modified through pilot testing and cognitive testing with neighborhood residents. aspects of the physical environment assessed in the scale included physical decay, vacancy and construction, municipal investment and green space. aspects of the social environment measured include social disorder, social trust, affluence and formal and informal street economy. the scale assesses features of the neighborhood environment that are determined by personal (e.g., presence of dog feces), community (e.g., presence of a community garden), and municipal (e.g., street cleanliness) factors. the scale is administered systematically block-by-block in a neighborhood. trained research staff start at the northeast corner of an intersection and walk around the blocks in a clockwise direction. staff complete the scale for each street of the block, only evaluating the right side of the street. thus for each block, three or more assessments are completed. we are in the process of assessing psychometric properties of the instrument, including inter-rater reliability and internal consistency, and determining the minimum number of blocks or street segments that need to be assessed in order to provide an accurate estimate of the neighborhood environment. these data will be presented at the conference. obj«tive: to describe and analyze the perceptions of longterm injection drug users (idus) about their initiation into injecting. toronto. purposive sampling was used to seek out an ethnoculturally diverse sample of idus of both genders and from all areas of the city, through recruitment from harm reduction services and from referral by other study participants. interviews asked about drug use history including first use and first injecting, as well as questions about health issues, service utilization and needs. thematic analysis was used to examine initiation of drug use and of injection. results: two conditions appeared necessary for initiation of injection. one was a developed conception of drugs and their (desirable) effects, as suggested by the work of becker for marijuana. thus virtually all panicipants had used drugs by other routes prior to injecting, and had developed expectations about effects they considered pleasureable or beneficial. the second condition was a group and social context in which such use arose. no participants perceived their initiation to injecting as involving peer pressure. rather they suggested that they sought out peers with a similar social situation and interest in using drugs. observing injection by others often served as a means to initiate injection. injection served symbolic purposes for some participants, enhancing their status in their group and marking a transition to a different social world. concl ion: better understanding of social and contextual factors motivating drug users who initiate injection can assist in prevention efforts. ma!onty of them had higher educational level ( %-highschool or higher).about . yo adffiltted to have history of alcohol & another . % had history of smoking. only . % people were on hrt & . % were receiving steroid. majority of them ( . ) did not have history of osteoporosis. . % have difficulty in ambulating. only . % had family history of osteoporosis. bmd measurements as me~sured by dual xray absorptiometry (dexa) were used for the analysis. bmd results were compare~ w ~ rbc folate & serum vitamin b levels. no statistical significance found between bmd & serum v taffiln b level but high levels of folate level is associated with normal bmd in bivariate and multivariate analysis. conclusion: in the studied elderly population, there was no relationship between bmd and vitamin b ; but there was a significant association between folate levels & bmd. introduction: adolescence is a critical period for identity formation. western studies have investigated the relationship of identity to adolescent well-being. special emphasis has been placed on the influence of ethnic identity on health, especially among forced migrants in different foreign countries. methodology: this study asses by the means of an open ended question identity categorization among youth in three economically disadvantaged urban communities in beirut, the capital of lebanon. these three communities have different histories of displacement and different socio-demographic makeup. however, they share a history of displacement due to war. results and conclusion: the results indicated that nationality was the major category of identification in all three communities followed by origin and religion. however, the percentages that self-identify by particular identity categories were significantly different among youth in the three communities, perhaps reflecting different context in which they have grown up. mechanical heart valve replacement amanda hu, chi-ming chow, diem dao, lee errett, and mary keith introduction: patients with mechanical heart valves must follow lifelong warfarin therapy. war· farin, however, is a difficult drug to take because it has a narrow therapeutic window with potential seri· ous side effects. successful anticoagulation therapy is dependent upon the patient's knowledge of this drug; however, little is known regarding the determinants of such knowledge. the purpose of this study was to determine the influence of socioeconomic status on patients' knowledge of warfarin therapy. methods: a telephone survey was conducted among patients to months following mechan· ical heart valve replacement. a previously validated -item questionnaire was used to measure the patient's knowledge of warfarin, its side effects, and vitamin k food sources. demographic information, socioeconomic status data, and medical education information were also collected. results: sixty-one percent of participants had scores indicative of insufficient knowledge of warfarin therapy (score :s; %). age was negatively related to warfarin knowledge scores (r= . , p = . ). in univariate analysis, patients with family incomes greater than $ , , who had greater. than a grade education and who were employed or self employed had significantly higher warfarm knowledge scores (p= . , p= . and p= . respectively). gender, ethnicity, and warfar~n therapy prior to surgery were not related to warfarin knowledge scores. furthermore, none of t~e. m-hospital tea~hing practices significantly influenced warfarin knowledge scores. however, panic ~ants who _rece v~d post discharge co~unity counseling had significantly higher knowledge scores tn comp~r son with those who did not (p= . ). multivariate regression analysis revealed that und~r~tandmg the ~oncept of ?ternational normalized ratio (inr), knowing the acronym, age and receiving ~ommum !' counseling after discharge were the strongest predictors of warfarin kn~wledge. s~ oeconom c status was not an important predictor of knowledge scores on the multivanate analysis. poster sessions v ~the majority of patients at our institution have insufficient knowledge of warfarin therapy.post-discharge counseling, not socioeconomic status, was found to be an important predictor of warfarin knowledge. since improved knowledge has been associated with improved compliance and control, our findings support the need to develop a comprehensive post-discharge education program or, at least, to ensure that patients have access to a community counselor to compliment the in-hospital educatiop program. brenda stade, tony barozzino, lorna bartholomew, and michael sgro lnttotl#ction: due to the paucity of prospective studies conducted and the inconsistency of results, the effects of prenatal cocaine exposure on functional abilities during childhood remain unclear. unlike the diagnosis of fetal alcohol spectrum disorder, a presentation of prenatal cocaine exposure and developmental and cognitive disabilities does not meet the criteria for specialized services. implications for public policy and services are substantial. objective: to describe the characteristics of children exposed to cocaine during gestation who present to an inner city specialty clinic. mnbods: prospective cohort research design. sample and setting: children ages to years old, referred to an inner city prenatal substance exposure clinic since november, . data collection: data on consecutive children seen in the clinic were collected over an month period. instrument: a thirteen ( ) page intake and diagnostic form, and a detailed physical examination were used to collect data on prenatal substance history, school history, behavioral problems, neuro-psychological profile, growth and physical health of each of the participants. data analysis: content analysis of the data obtained was conducted. results: twenty children aged to years (mean= . years) participated in the study. all participants had a significant history of cocaine exposure and none had maternal history or laboratory (urine, meconium or hair) exposure to alcohol or other substances. none met the criteria of fetal alcohol spectrum disorder. all were greater than the tenth percentile on height, weight, and head circumference, and were physically healthy. twelve of the children had iqs at the th percentile or less. for all of the children, keeping up with age appropriate peers was an ongoing challenge because of problems in attention, motivation, motor control, sensory integration and expressive language. seventy-four percent of participants had significant behavioral and/or psychological problems including aggressiveness, hyperactivity, lying, poor peer relationships, extreme anxiety, phobias, and poor self-esteem. conclusion: pilot study results demonstrated that children prenatally exposed to cocaine have significant learning, behavioural, and social problems. further research focusing on the characteristics of children prenatally exposed to cocaine has the potential for changing policy and improving services for this population. methods: trained interviewers conducted anonymous quantitative surveys with a random sample (n= ) of female detainees upon providing informed consent. the survey focused on: sociodemographic background; health status; housing and neighborhood stability and social resource availability upon release. results: participants were % african-american, % white, % mixed race and % native american. participants' median age was , the reported median income was <s , year, and % reported receiving a high school diploma or equivalent. women reported a number of health conditions rypical of underserved populations, including asthma ( %), sexually transmitted infections ( %), high blood pressure ( %), hcv ( %), diabetes ( %) and hiv ( %). nearly half ( %) did not anticipate having a place to stay for at least days upon release. factors significantly associated housing stability upon release were: high family support score (adjusted odds ratio [aor) . ; % confidence interval ( % cl) . , . ), high neighborhood stability score (aor . ; % cl . , . ), wanting a commercial sex worker (csw) support group (aor . ; % cl . , . ); and identifying as bisexual (aor . ; % cl . , . ). women desired employment ( %), housing ( %), education ( %), drug treattnent ( %), help with custody of children ( %), childcare ( %), and domestic violence suppon ( %) services. conclusions: female detainees represent one of urban centers' most marginalized pcjpwatioos. short periods of detention represent a unique opportunity for _interven?~ns bri~ co~s and public health institutions. service providers should collaborate ~ th local ~ails to .p~de a con uwnof care for female detainees upon release that includes transportation , housing, residential drug treallllcnt, employment, education, family reunification, childcare and domestic viol~nce su~rt. ~pecial attenlion is warranted to lesbian and bisexual women and csws, who may be especially margmalized &om family and service-based support networks. introduction: "health care and ethnic minority immigrants" examines how health care policy dil· ferences between canada and the united states impact the health-related hardships, access and use of preventative care, and health outcomes of urban ethnic minority immigrants in both countries. methods: the findings of this paper build on the results of my qualitative comparative study of hotel workers in vancouver, bc and seattle, wa that revealed greater health related hardships for similarly matched employees in seattle compared to vancouver. in this paper, i examine the generalizability of these findings at the cross-national level for similar ethnic minority immigrant groups. i compare the impact of health care policy differences on specific groups that have emigrated to cities in both canada and the united states. these include immigrants from china, viemam, philippines, west indies, africa, sri lanka, pakistan, and latin america. comparative statistical analysis -utilizing rel· evant data from statistics canada and the u.s. census -reveal the extent to which health policy differ· ences help explain how current health policy in the united states disadvantages urban ethnic minority immigrants. results: many ethnic minority immigrants are a part of the working poor, a group which disproportionately lacks health insurance coverage in the united states. their position in the labour market makes them most vulnerable to exclusion from health care services. the data reveals how current u.s. health policy creates barriers for recently arrived ethnic minority immigrants to access health insurance and care. it remains difficult to ascribe health outcome differences directly to policy differences because of the challenge in disentangling the complex interacting interventing variables, including income inequality, that determine health outcomes. yer suggestive evidence suggests that health policy in the united states is harming the health of urban ethnic minority immigrants. the current health policy regime in the united states harms the health access, care, and outcomes of urban ethnic minority immigrants. comparing the fortunes of similar immigrants in cana· dian and u.s. cities reveals these patterns of disadvantage and some of their consequences. these barriers are an understudied factor in the sociological literature on "segmented assimilation" and social exclu· sion. the paper ends with policy recommendatio ns to improve access to health care among ethnic minor· ity immigrants in both countries, with the goal of reducing health related hardships, and improving health outcomes. mass index deyu pan, richard baker, and keith norris badtgrou~ over the past decades, there has been dramatic increase in prevalence of obesity in the us population. however, the relationship between obesity and the prevalence of cardiovascular dis· ease (cv~) risk factors in different race/ethnic groups over time is not well known. . ik~rgn: _we use cross-sectional , nationally representative surveys: national health and nutri· t nal examination survey (nhanes iii - (n= ) and nhanes nhanes - , including white, black, and hispa~ic races who were no~preg~ant, aged to years. res lt : the preval~nce of high cholesterol level (~ mg/dl), untreated high blood pressure ( : l / mm hg) an~ diabetes were calculated according to bmi group (lean, < ; overweight, - ; and obese, : ) for different race/ethnicity groups. over the approximately years period (from l ~ ~o [ ] [ ] [ ] [ ] , reducrion in cvd risk factors in non-hispanic white has been observed. for mmonty race/~hnicity groups, black and hispanic, there had been increases in prevalence in high blood fa pressure and diabetes. the lean group experienced larger increase in prevalence of those cvd risk ctors. . fo~~ the prevalence in cvd risk factors over time had reduced in most of the bmi ca egoes r t e w ~~e population. however, for black and hispanic, prevalence of of cvd risk factors ave generauy uncreased, especially in overweight and obese groups. methods: strategies identified to achieve these goals include: developing meaningful neighbourhood and district boundaries for comparing health information, providing free access to neighbourhood health profiles (including relevant data at the smallest level for which valid rates could be calculated); mapping relationships of inequality at a variety of nested levels; providing a range of formats (maps, tables) to meet the needs of users; providing technical support; seeking user input to advance and improve the site; and conducting workshops to foster access and use of data for decision-making , advocacy and collaboration. an evaluation strategy is being developed to assess the efforts and impact of these strategies. a preliminary assessment of the results of the first six months of activity will be completed in october . results: significant differences in health are shown at all the geographic levels indicating success in developing the boundaries. results tabulated for the first six months use of the site include: site visits; media use; reference/acknow ledgement of the site in other documents; number of contacts through presentations and workshops; feedback from users identifying strengths and limitations; and, response from health decision makers. members of the partnership are identifying additional tools and services to further support actions that reduce health inequalities. the assessment will be used to develop more specific goals, targets and monitoring mechanisms. conclusions: our experience with paper-based health profiles indicates that they have been used extensively for program planning and advocacy. the greater availability of information in a publiclyavailable web-based format is expected to translate into even greater usefulness and wider application. the web site has had considerable usage to date, extensive media coverage. site users and workshop participants have provided positive feedback and suggestions for next steps. the six month review will be available in october . in india, more than and a half people are hiv-positive and half a million have aids. india currently has the largest number of positive people in the world, oumumbering south africa and accounting for nearly one tenth of the global hiv/aids prevalence. the world bank predicts that by , there will be million hiv/aids cases in india. the central challenge facing hiv prevention efforts in south asia today is learning how to respond to the societal determinants of vulnerability to hiv. hiv/aids is an issue largely engulfed by social stigma. stigma and discrimination are often considered the foremost barriers to effective poster sess ns v prevention and care initiatives. stigma can make a person afraid of disclosing their status~ ~yone el se, and may make them feel depressed or alone. stigma can .also le~d to poor adherence to medicanon, ~ likelihood to access health and social services, and less desire to hve. hiv+ men and women may beafraidtotd! their co-workers that they have hiv for fear of their reactions or for fear of losing their jobs. in a study conducted of people living with hiv/aids in delhi, india ov~r. -~, many respo~ts shared their experiences of discrimination in their families, in their communmes, an~ m health ~e settmgs. these findings will be shared in the workshop, and will be compa~ed to m~ ex~nences workmg as an ~ co~l~ at the center for aids prevention studies in san francisco, cahforma and congreso de lannos unidos m philadelphia, pennsylvania. the workshop will include an overview of hiv/aids in urban contexts in tht united states (los angeles, san francisco, new york, philadelphia) and south asia (delhi, hyderabad, chennai). differences in methods for outreach, prevention and treatment efforts between devdoped world and developing world contexts will be discussed. a large portion of the workshop will be centered on discussion. participants will engage in an exercise to categorize their ideas of stigma. the workshop will also include a powerpoint presentation on the quantitative data gathered from the survey. the number of homeless and runaway youth is increasing in toronto, which is currently home to out of youth in the gt a who live alone, and it is the preferred locale for the street-involved and homeless youth. the city's youth population is expected to grow by nearly % by , the school dropout rate is rising, and there are is a paucity of empirical data on what works with these at-risk youth. over a two-year period, ( ) ( ) ( ) , youthlink-inner city, conducted an evaluation of the impact of a harm reduction program aimed at reducing street youth's risk of contracting/infecting others with the hep c virus, which has both short-term and long-term deleterious effects. demographic data from street-youth, along with resources/services used, employment methods, type and frequency of drug use, health status, police contacts, and their views of program impact were analyzed using a standardized sur· vey. additionally, focus groups with youth, agency staff and community key informants were conducted, as well as in-depth interviews with three youth, over a sixth-month period. this paper details study results, which both inform both practice and future study about what works, why it works, and how best to work with these vulnerable youth. sho~ed .that the wasteland in this area was contaminated by as cd zn pb cu simultaneously. the con-ta~matton of as ~d was quit significant. the contamination problems and environmental issues in this region are very. serious. the awareness on environmental and health issues was investigated by spot survey and analysis. overall awareness among the residents in the region is very poor. the measures must be taken to assu~e t~e .sustainable economy development by local and central gorvernments. keywords nandan guangx ; mmmg area; environmental awareness. concern like, epileptic fits, vision problems, earache, cough of more than weeks and urinary problems. ninety-five subjects did not take bath regularly and were exposed to sex. the health problems identified are only of a particular point of time and it may be difficult to interpret the depth of "iceberg" of the street children's world also draw the health-illness continuum. periodic interaction and follow-up is very difficult as they generally disappear from the research setting due to their frequent mobility for survival measures. as per the prediction of the iceberg theory, only some problems may have been identified, larger problems in respect to their health may not have been identified. in spite of various limitations, this attempt had proven that, it is possible to enter the street children world, explore the iceberg of illnesses, and establish data of health-illness continuum through an effective nursing agency. it is recommended to deworm these children, provide nutritional education, establish "condom corners" and "street children help line" in the city and urban slum areas and undertake action research on their health. malnutrition is a serious problem in developing countries like bangladesh. malnutrition causes a great deal of child suffering. malnutrition is one of the major causes of morbidity and mortality among the child. the aim of present study was to investigate the nutritional status and its relation to socioeconomic conditions of urban disadvantaged child of under five years age. nutritional status was determined by anthropoemetric measurement(heig ht, weight, mid-arm circumference etc.). determination of total protein and serum albumin were done for biochemical examination. haematological examination was done by blood haemoglobin profile estimation by cynmethhaemoglob in method and determination of haematocrit value or packed cell volume (pcv). stool examination for ova of hook worm, round worm, trichuris species and other species were also done. relevant information on socioeconomic characteristics was recorded by interviewing the house hold head using pretested questionnaire. our study demonstrated that nutritional status of the children are positively correlated with family income, parents level of formal education and negatively related to the family size. the effect of family income on the nutritional status, haematological and biochemical indices of urban disadvantaged child under five will be discussed in detail which will help us to determine proper way of utilization of health care facilities exists in developing countries. introduction: with the increase in morbidity and reduction in mortality and with the introduction of highly active antiretroviral therapy (haart), there is increasing focus on the determinants of healthrelated quality of life (hrqol) in hiv-infection. the focus on the present study is to examine the relationship between social support, depression, and hiv disease severity, and the extent to which these variables impact on hrqol in adult men with hiv-infection. methods: as part of a larger ongoing natural history study focusing on the neurobehavioural complications of hiv and aids, we administered questionnaires to assess depression (beck depression inventory), social support, and hrqol (mos-hiv) to adult men with hiv-infection. a structured interview was used to collect health information and data on hiv disease severity. physical and mental health summary scores (phs and mhs) were derived through principal components analysis. participants were grouped according to level of social support: "well supported" (n= ), "moderately supported" (n= ), "little or ineffective support" (n= ). analysis of variance was conducted to assess the effects of three levels of perceived social support, depression status (present vs absent), and hiv disease severity (aids vs non-aids) on mental and physical health dimensions of hrqol. potential interaction effects were also evaluated. results: social support was found to have a significant effect on both mhs and phs. presence of depression was found to have a significant effect on mhs but not phs. hiv disease severity (presence of aids diagnosis) was found to have a significant effect on phs but not mhs. manov a analyses revealed no significant interactions effects. conclusions and implications: level of social support, presence of depression and hiv disease severity were shown to have independent effects on hrqol. feeling "well supported" appears to have significant benefits for mental and physical health. biological indicators of hiv disease appear to have a selective impact on physical health while presence of depression is related to significant reductions in mental health. the development and evaluation of behavioural interventions to improve perceived social support network and depression will likely result in significant improvements in health outcomes of adults with hiv and aids. introduction: ongoing medical advances have greatly enhanced the longevity of penons living with aids (plwa). however, for certain subgroups living with hiv/aids, including, members of minority groups, women and the economically underprivilege d, aids outcomes have not changed so favorably. one obvious factor to consider when explaining disparities in aids outcomes is the environ· ment within which an individual resides. few studies of aids outcomes have focused on how commu· nity setting influences the effectiveness of care delivery. the principle research questions were: ) what is the relationship between the locations of areas with concentrations of pl wa and the distribution of both primary and ancillary service providers? ) how has the widespread availability of haart (after ) impacted aids outcomes at the community level? ) how do aids mortality and fatality rates vary when related community characteristics, such as household income, ethnic mix, and geographic access to primary and ancillary hiv/aids services are considered? we examine this issue across residential communities in the los angeles area. methods: los angeles county's comprehensive aids service providers database (compiled and maintained by aids project los angeles) was geo-referenced and distances between weighted mean population centers and the nearest hiv/aids service providers were calculated to provide a measure of access which was then merged with aids diagnosis and mortality data along with relevant socioeconomic and population indicators and for analysis using bivariate and multivariate statistics at the zip code level. results: there is a growing disparity in hiv/aids outcomes between low-income minority areas and affluent non-minority areas in the post-haart era (p = . ). ancillary aids services such as case management and counseling and testing are located near the places where low income hiv/aids service consumers are likely to live (p =. ). in spite of having more access to ancillary services low income areas continue to be associated with smaller decreases post-haart aids mortality rates (p= . ). conclusions: given the increasing disparities in aids outcomes between lower and upper income communities in the post -haart era, more specific research into the capacities of aids services providers and the efficiency of their interventions is required. although it is clear that ancillary services are operating in the areas of greatest need, their effectiveness remains limited. understanding the reasons for t~is, be they lack of adequate resources for inner city service providers, failure to reach target popula· tmns or other causes, requires additional research. in ? , y.out~ who were able to speak either french or english and had been absent from their parent's/ c~regivers ~es. dence for at least three consecutive nights took part in the survey which consisted of inter· viewer-adm ms tered question~aires. p.arti~i~an~s were recruited from drop in centres in cities across <?-nada.y~uth self-report as either using in ect on or non-injection drugs. statistical analyses were car· r ed out using sas version . the 'home' is a contested site for many women living in urban areas. women living in poverty and using illicit drugs are largely excluded from participation in dominant 'home-base' gender roles of wife and mother because of economic disadvantage. they thus 'make home' in ways that are specific to their everyday experiences. while research has established links between housing, health and drug use, this literature does not tend to consider the meanings that home may have within the lives of women who lack access to adequate tenured living spaces. this presentation reports the findings of my master's thesis work. using grounded theory methodology within a poststructuralist feminist conceptual framework, i conducted in-depth, open-ended interviews with women living in the toronto area. the women were recruited from within the community through posters at different social service agencies (e.g., drop-in centres, community health centres). they were asked to describe their housing experiences and what home meant to them over the course of their lives. through the data analysis process, a substantive theory of 'making home' for women living in poverty and using illicit drugs emerged. the central concept of making home is a continual process of learning about and interacting with the environment. the process of making home entails different subprocesses: finding home (knowing what is available and accessible, and accessing home), adaptation, and leaving home. the different meanings of home that women developed from their experiences and knowledge emerged as critical components for the process of making home. the women i interviewed were very mobile and experienced constant change with regards to home. home was not considered only in relation to physical living space. home could be made within a relationship or with regards to a possession. while dominant meanings of home relation to tenured living spaces are generally positive, my research indicated that home could also be a negative context. the presentation will describe my research process and findings as well as the theoretical and policy implications of my results. in the western democracies, social citizenship bundles the right to social provision (defined as a share of the social good, not necessarily proportionate to the market value of a citizen's contribution) with participation in the paid labour force. for this reason, social citizenship rights, which usually involve some kind of redistributive measures, are a key mechanism by which states ensure economic equality, or at least, economic equality of opportunity, but are also a mechanism of social exclusion. social rights are inscribed around a single kind of relationship, that which exists between a male family breadwinner and the paid labor market; those who make economic contributions that fall outside this relationship -such as unpaid care-work, or, work that occurs in shadow or underground economiesare to a greater or lesser degree barred from accessing citizen-based social provisions. furthermore, social provision that occurs outside of the breadwinner/market-eco nomy dyad is very often meanstested and is accompanied by regulatory state apparatuses (this includes a range of social services, from welfare payments, to child-care subsidies, to disability pensions). social citizenship is thus stratified; the legal relationship that is at the heart of social citizenship acts as a form of social closure against claims for a share of the social good made by those who are 'lesser' citizens than others. of particular interest in this paper is how citizenship inequalities translate into inequalities in the social provisions that emerge from economic participation: the right to safety on the job, protection from employer harassment, protection from the vagaries of the market as well the right to adequate, comprehensive, and appropriate health services. drawing on mixed methods, longitudinal data from adult sex workers (n= ) located in two research sites with different social welfare regimes -one in canada and one in the u.s. -we examine the consequences of working in a "shadow" or "underground" economy on various facets of health and access to health services, taking into consideration the mediating role that citizenship constructs and social welfare regimes play in accessing other key social and economic determinants of health. methods: the study population included methadone patients (re)admitted at the mhs in the period from / / to / / , born in the netherlands, morocco, surinam, dutch antilles or turkye, and officially registered in the population register of amsterdam. the population register was used to ascer· rain the vital status. causes of death were provided by the central bureau of statistics. mortality was categorised as hiv related, directly drug related (overdose) and other.mortality. results: the methadone patients had the following characteristics: years of age at entry; % male; % ethnic minority; % ever injected. in total, personyear (py) of observation time and deaths ( % hiv related, % overdose; % other) were observed. hence, the crude mortality rate was / py ( % ci - / py). the percentage of (ever) injectors was higher among the native dutch than among the ethnic minorities, % and % respectively. higher rates were observed among native dutch drug users compared to those belonging to an ethnic minority (age adjusted hazard ratio (hr) . ( % ci . - . ). the age adjusted hr of (ever) injecting drug users versus non injecting users was . ( % cl . - . ). after additional adjustment for route of administration a non significant difference between the dutch and ethnic minorities remained (hr . , % cl . - . , p-value . . conclusion: the mortality rate among the heroin users in amsterdam is strongly affected by the high prevalence of non-injecting drug use. moreover, this study confirms that differences in mortality between the native dutch and the ethnic minorities is related to differences in route of administration. the ongoing reduction of injecting drug use (due tot selctieve mortality, and switching route ofadministration and popularity of crack cocaine) may lead to a further reduction of mortality rates among heroin users in amsterdam. interventions preventing the initiation of injecting should be encouraged. . introduction: food insecurity is an inequality that is central in the lives of many low-income cana· d ans. people lack food security when regular access to nutritious food is limited or variable due to high prices'. low income, lack of transportation or inadequate food distribution, or they become disadvan· taged m other w~ys through the acquisition of food. a group comprising academics, health profession· als, and commumty workers who have experience in food insecurity, developed a pilot study in ottawa that sought to und~rstand the lived experience of food insecurity. the study also used the united states department of agriculture (usda) community food security module. methods: a series of group meetings determined the best research approaches; questionnaires and consent forms were developed by a working group. twenty-three self-identified food insecure respon· dents were interviewed. the data were analysed using frequency distributions· the food security module was coded according to the usda coding guide, and open-ended responses w~re coded using a grounded approach. renlts: preliminary results from our pilot study have given the research team cause for considerable concern. out of households without children, experienced food insecurity with hunger; ~ere at the severe l~vel. out of households with children, experienced food insecurity with hunger. ~•rst person reflecnons commonly featured feelings of depression low self-esteem and despair. participants' account tha d . an cu ty gettmg to the store, severely limited putting knowledge and skills mto practice. l'oster sessions v conclusion: based on these early findings, our research team is looking to challenge what appear to be assumptions about poverty and food insecurity and for best ways to use this information. some policy approaches and interventions that encourage people to eat better and exercise more, may be missing the mark for low-income individuals, and may serve to maintain food insecurity. future research will build on this pilot. with methods augmentation, and a comprehensive knowledge dissemination plan, we hope co contribute to research and policy frameworks at all levels. ps- (a) mental health and the corrections system: population-based analyses in urban, semi-urban, and rural settings julian somers, robert watts, and michelle patterson introduction: according to recent epidemiological research, rates of mental disorders vary considerably across countries and across regions within countries. nevertheless, rates of mental disorders (including substance use disorders) are consistently higher in populations involved in the corrections sys- em. courts have long recognized the heavy burden of mental illness within their purview, and have developed innovative programs to better manage the needs of such individuals. the majority of these innovations (e.g., specialized courts) exist in urban settings. however, few studies have examined the degree of variability in rates of different mental disorders between courts in urban and other settings (e.g., semi-urban, rural). variability between courts in different settings, if present, holds implications for needs-based resource planning. the present study was conducted as part of a long-term inter-ministerial collaboration in british columbia (bc). analyses were conducted on linked administrative data regarding population health and correctional services. a database was constructed including all individuals sentenced in bc in a single year (n= , ). corrections records were matched to records of health services utilization (hospital and outpatient services) for mental disorders and substance use disorders in the years preceding sentencing. services for mental health and substance-related problems were aggregated into three groups: severe mental illness (smi); less-severe mental illness (lsmi); alcohol/other drug (ad). courts were grouped, based on their annual volumes, inro three categories: "urban"; "semi-urban" and "rural." rates of service use for mental disorders were compared between groups for -year and -years preceding sentencing. results: there were significant differences in the rates of mental disorders in courts of different size (urban, semi-urban, rural) . however, differences between the rates of disorders within each of these groups exceeded the discrepancies between groups. comparatively high and low rates of disorders were observed in courts of each size. moreover, the courts with the highest rates of certain disorders (e.g., ad) did not have the highest rates of other types of mental disorders (e.g., smi). conclusions: rates of mental disorders differed significantly among the annual populations sentenced across courts in bc. urban courts (which process comparatively large numbers of people) have implemented services to address the needs of the mentally ill. the present analyses strongly suggest that program development should be closely linked to the demonstration of need. the results caution that a uniform approach to court programs for the mentally ill is likely to be inefficient, over-supplying services in some settings and underestimating need in others. results: public settings used for injection are characterized by highly unhygienic conditions, the presence of unsafely disposed syringes, limited availability of sterile syringes and a lack of sterile water for injecting. a number of unsafe injection practices observed were common among public injectors. the presence of police officers nearby and the potential of assault by street predators fostered rushed injecting among those consuming drugs in public spaces. the ecological features of these environments encourage unhygienic and unsafe injecting practices, heighten risk for overdose and the transmission of blood borne viruses. introduction: approximately % of women experience depression in the first weeks or months after giving birth. although it has been argued that postpartum depression (ppd) is a culture-bound p~ nomenon, experienced only in western, industrialized countries, recent international research studies have confirmed that the prevalence of ppd is similar around the world. however, little is known about variables that may influence the experience of ppd in women from diverse cultures and immigrant women. research is needed to identify the role culture may play in the presentation of ppd (i.e., types of symptoms most commonly reported), how women from various racial, ethnic and cultural backgrounds perceive and attribute their symptoms of ppd, and finally, how culture can be appropriately addressed in ppd treatment programs. method: to examine the role of culture in ppd, semi-structured interviews and self-report question· naires were administered to clients of the women's health centre of st. joseph's health centre, tor· onto. participants were identified as either first generation canadians (relative newcomen) (n= ) or second generation canadian (parents were immigrants to canada) (n= ). data were collected qd these two groups of women based on: severity of depression, symptom presentation, perceived role of social supports and culturally-specific traditions, and barriers and responses to treatment. the interview data were analyzed using qualitative methods (thematic content analysis), and the following themes were identified: ) stress about passing on their culture to their children, ) lack of social support and feelings of isolation, ) perceived importance or lack of importance of cultural based tra· ditions, ) conflicts with family members. about cultural traditions and beliefs, ) mental health stigma within ethnic communities and beyond, ) race-and sex-based discrimination, and ) language barriers. conclrllion: identifying the role culture plays in the presentation of ppd, and how women from diverse backgrounds perceive and attribute their symptoms of ppd, is critical in order to establishcultur· ally appropriate guidelines for treatment options. furthermore, information gathered from this study can be used to develop policies and resources for delivering culturally competent care for newcomer wo~ who are dealing with ppd as well as the issues around acculturation (i.e., language barriers, racism). llus is of particular importance for urban health centers which provide postpartum care to diverse groups of women, and particularly recent immigrants or newcomers. ps-~ (al. contaminated 'therapeutic landscape': perceptions of the aamjiwnaang fint nation keyln smith and isaac luginaah . in~: this. paper pres;ents the findings of an ongoing study among the aamjiwnaang f!rst nanon. aam wnaang is located m the heart of samia's 'chemical valley', surrounded by cherrucal ~(ants such as esso, imperial oil, shell, suncor energy, and canada's largest hazardous waste disposal sate. safety kleen. this fint nation community is located within the st. clair river •area of concem'. as destgna~ by health canada for the population of samia and the surrounding region. although this comm_umty, by way of. its proximity to the numerous chemical plants is already exposed to high levels pollu~on, recent che?ucal dumping in ~mjiwnaang, as well as a failed proposal for another ethanol plant m the community, only helped to mtensify community concerns about potential health impacts of exposure. research on the cultural beliefs and traditions of first nation communities has established that th~ ~isting rel~tionship between first nations people and 'mother earth' is not only physical, but also_ spmtual. in this study we explore how the complex relationship between the aamjiwnaang first naaon and 'mother earth' be ha · · · i .. may c ngmg a contammated 'therapeutic' landscape. the study a so ~lores h?w aam wnaang residents are coping with these changes and with the probable conramina· boa of their community. ra!jts: aamjiwnaang residents acknowledge that they are living in a contaminated environment and are being impacted by emissions from the surrounding 'chemical valley' especially for future generations, and have expressed concerns that members of the community and mother earth are 'sick' as a result of this exposure. while moving from that 'place' has been discussed, residents have strong personal and generational connections to the land and insist however, that aamjiwnaang is their 'home' and will remain that way despite growing community concerns about the impact from industry. the contribution of this study lies in the direct involvement of community leadership in designing and conducting this research and distributing the results to further local policy development regarding community concerns on the reserve. background: truck driver are at very high risk for drug abuse because the factor contributing to drug abuse among driver are multiple. it is important to gain an understanding of the key factors that contributing to drug abuse among truck drivers. methods: seventy-truck driver aged - years were conveniently selected from the population. all the participants were male. an interview schedule was developed in the light of literature data and data were collected by personal interview with informed consent in selected urban area of eastern part of nepal. results: a considerable percentage of truck driver have less knowledge about drug abuse, its effect on body and their complication. majority of the participants ( %) explained that lack of education, financial crisis were leading factors of drug abuse. the average numbers of respondents were believed that lack of meaning in life, marital disharmony. friendship with drug abuser, stress, tiredness, modernization of life pattern and freedom from the home pressure were the influencing risk factors to drug abuse. conchuion: this study reveals those truck drivers are at risk in urban area for developing drugs abuse, mostly influenced by social and environmental factors. hence, these groups should undergo certain educational programme to prevent not only drug abuse, but also prevent transmission of infectious disease among these groups. introduction: food insecurity is an inequality that is central in the lives of many low-income canadians. our group of university researchers, health professionals, and community developers conducted a study in ottawa with community workers who work with people identified as food insecure. the purpose of the study was to understand the nature of their work, and their perspectives, perceptions and experiences of the causes and consequences of food insecurity. (a parallel study was conducted with food insecure participants). methods: sampling was through local knowledge of organizations involved in the provision of food to community members. written permission for the researchers to contact workers in confidence was obtained from each organization. thirteen in-depth interviews were conducted. the interviews consisted of a number of open-ended questions. the data were collated, and analysed using a grounded approach. results: workers interviewed represented organizations that offered diverse programming and methods of food distribution intended to address the needs of the community. the provision of food was seen as a necessary response to address hunger that arose for the majority from poverty caused by low income, or levels of government income support and safety nets that were insufficient to support food requirements. workers reported that food requirements varied according to different. ethnic backgrounds, ages of recipients and family configurations, and with food preferences, and d'.etary (health) requirements. workers' observations of the effects of insufficient food on adults and children such as depression, low energy and non-participation or social disengagement matched those of food insecure respondents. food insecurity was cited as "another srressor among the mountain of problems." overall, workers presented a picture of a silent, stigmatized, poor population including children, and a lack of advocacy to address the issues. be tha th · ·ty the results suggest that workers and organizations, like the commuruty mem rs t ey msecun • · · · · f ilab'l' f food · t d ·n thei·r ab 'l 'ty to address food insecurity. l m tanons m terms o ava ty o , serve, are restnc e . . funding, and donations appeared to set the para? eters of pr~g:am r.espon~ to commuruty food msecurity. workers were concerned with program des g": and a~m mstranon that m general addr~d hunger on an emergency short-term basis, although food msecunty was long-ter_m for ~any. sustainable solutions that include knowledge translation strategies, and health and social pohcy responses were suggested and will be explored in future research that hopes to build on this pilot. background: socioeconomic deprivation as well as low levels of soc!al s.uppoi:r have .been associated with poor outcomes following cardiac surgery. pre-operative depression m ~anents with coron~ry artery disease awaiting cabg ranges from - % and is an independent predictor of post-operanve mortality. since st. michael's hospital has a large inner city population, this study was designed to determine the both the prevalence of depressive symptomatology (ds) in patients awaiting cabg as well as its relationship with socioeconomic status (ses) and social support. methods: consecutive patients referred for isolated cabg were invited to complete the centre for epidemiological studies depression scale (ces-d) as well as a social support scale and a life stressors inventory. a ces-d score of : represents mild ds and a score of : represents moderate to severe ds. participants also recorded information on functional status, perceived progression of heart failure, demographic and ses variables. all participants with ces-d scores : were considered to have ds. results: of the patients ( %) who returned the survey, ( . %) of participants had a ces-d score : with ( . %) of those having scores suggestive of severe depression. females and those with ongoing medical concerns tended to have more ds (p= . and p= . respectively). age was not related to ds. perceived worsening of symptoms and increased ccs angina class were significantly related to the presence of ds (p= . and p= . respectively) but not the number of diseased heart vessels. satisfaction with personal relationships including having someone to confide in (p= . ), feeling lonely (p= . ) and not having a partner (p= . ) were significantly related to ds. being very upset by a recent breakup in the immediate family was also significantly related to ds (p= . ). having greater than a grade education was the only ses variable related to ds (p= . ). multivariate logistic regression suggested that not being partnered and having low education were the most significant predictors of ds. conclnsion: pre-operative depression is present in at least one quarter of patients referred for cabg. since ds is related to poor outcomes following cabg, single patients with few social supports and low education should be considered at high risk for ds. these findings support the need for the development of supportive interventions in patients at risk in order to decrease post-operative morbidity. this study examines the relationship between childhood sexual abuse, sexual assault during adolescence, and hiv-risk-related behaviours in a sample of homeless youths. over the past decade, there has been increasing research devoted to the impact of childhood sexual abuse. some studies have suggested that the experience of childhood sexual abuse is associated with substance use and abuse, at· risk sexual behaviour and subsequent higher rates of hiv infection (ompad et al., ; cinq-mars et al., ; brown et al., ) . other studies have found that childhood abuse, especially sexual abuse, is c_o~n among stree~ youth (noell et al. ). this study compares sexually abused males and females livtng on the street ~th non-sexually abused street youth, with regards to unsafe sexual behaviours and e~ures to potmnal t:dv sources (e.g., tattooing, body piercing and injection drug use). the hypothe-sis oft~ ~nt study is that the prevalence of hn-risk-related behaviours will be higher among street youth vtctuns of sexual abuse than those who have not been sexually abused. the sample includes youths aged to who met specific criteria's for itinerancy ( male and female, with a mean ~of . y· and a standard deviation of . ). they were recruited through five organisations working ~th~ you~ and were~ of an ongoing cohort study. all youth completed a to minute ques· ~onnaue on their sexual behaviours, use of drugs and alcohol and other hiv risk behaviours. oral spec· unens for hiv testing were. also collected. a total of ( . %) youth reported at least one incident of ~i •~use/assault ( ~ girls ( . % of all the girls) and boys ( . % of all the boys]). preliminary descnpnve analyses pomt to sexually abused/assaulted youth showing the highest prevalence of sexual poster sessions v at-risk behaviours. for example, . % of sexual abuse/assault victims have engaged in prostitution in their lives compared with . % in the non-sexually abused/assaulted group. in addition, . % of sexual abuse/assault victims reported having injected drugs, compared with . % in the non-sexually abused/assaulted group. more statistical analyses will bring other significant factors to light, especially when we analyze separately those victim of childhood sexual abuse as compare to those victim of sexual assault after childhood. elucidating the risk factors for getting involved in hiv-risk-related behaviours is important for the development of comprehensive and appropriate prevention and treatment intervention strategies. introduction: new immigrants and refugees to canada frequently emigrate from regions of the world where intestinal parasitic infections with worms are endemic. of note, some of these parasites area capable of surviving for years to decades within a given host and can have life threatening consequences many years after initial infection. thus, early diagnosis and treatment of intestinal parasitic worms is considered important in high risk immigrant populations, however there remains a lack of consensus regarding the best method of accomplishing this. diagnosing worm infections through stool examinations is costly and has limited sensitivity, while presumptive treatment of all immigrants, although advocated by some, is expensive and results in healthy individuals being unnecessarily treated. herein, we examine the utility of a hematologic marker (i.e. peripheral blood eosinophilia) as a screening instrument to identify parasitic worm infections in new immigrants to toronto. we identified adults, years of age or older, who were screened for intestinal parasitic infections by stool examination as part of a recently developed screening protocol at a downtown toronto community health centre. we examined the prevalence of infections with worms and subsequently evaluated the impact of several demographic factors on the presence of worm infections. we also determined the sensitivity and specificity of peripheral blood eosinophilia (defined as greater than eosinophils per ml) as a marker for intestinal parasitic worm infections. results: overall, % of the immigrants tested had evidence of at least one worm infection on a single stool examination, while % of such individuals bad infections with multiple worms concurrently. age did not appear to be associated with the presence of worms, however % of males had evidence of worm infections compared with % of females (p= . ). immigrants from asia, sub-saharan africa, and central america had the highest burden of overall infection. the sensitivity and specificity of peripheral blood eosinophilia for worm infections was % and % respectively. conclusions: one in five recent immigrants presenting to a community health centre in downtown toronto had evidence of at least one intestinal parasitic worm infection. the highest burden of illness was in men and in those emigrating from less developed regions of the world. peripheral blood eosinophilia is a poor screening test for worm infections however its presence is highly suggestive of the existence of worms in new immigrants. more than a decade ago, the journal of american college health recommended that a national study be conducted to measure the health status of african american college students, 'both health disparities and [their] positive healthful behaviors' (fennell, ) . it was later decided that when exploring the level of health risk behaviors for african american students, gender is an important variable and should be included in the research (fennell, ) . at historically black college and universities (hbcus) college health researchers must tackle gender disparities through highly-effective health promotion and disease prevention programs. the purpose of this review is to examine the literature that addresses the overarching health behaviors (i.e. risky sexual behavior, poor diet, smoking, physical inactivity, etc.) of african american men who attend hbcus. despite the various social, cultural, and academic benefits of african american students who attend hbcus, the number of research studies that adequately address their health and health behaviors is limited. studies indicate that african american men who attend hbcus are actively engaged in poor health behaviors. compared to females at hbcus, african american men are more likely to behave in ways that could result in injuries and the use tobacco, alcohol, and ~ther dru~. the city of toronto has recently proposed to conduct a street count of the homeless. like proposals that have come before, this most recent proposal is controversial among many homeless advocates and service providers because it is perceived as intrusive, likely to undercount the target population, and unnecessary for addressing the problem. advocates of the count view it as necessary to gauge the scope of the problem and advise city leaders about how to most effectively direct resources ro.the various homeless services. this paper first reviews the history of efforts to count homeless populat ns m toronto, describing the motivations and arguments of people on both sides of the issue. second, the paper reviews the methodological complexities of counting homeless populations, describing approaches that use shelter-counts, administrative data and street observations. those interested in counts for toronto and elsewhere can benefit from this review of approaches that have been proposed and carried out in other municipalities. third, the paper proposes a new community-base d strategy that joins the skills of methodologists, local experts, and service providers to ensure a fair and accurate count. the method of systematic social observation avoids some of the problems of early counts by utilizing a repeated identification approach to minimize undercount bias. the method also has the advantage of involving and compensating homeless individuals for assisting with the street count estimates. the estimation approach can be implemented on a rolling basis throughout the year. drug transitions) is a multi-level study aimed at examining the associations between features of the urban environment and mental health, drug use, and risky sexual behaviors. research participants are being recruited in new york city (nyc) neighborhoods. an essential first step in the implementation of this study was to delineate boundaries for each target neighborhood that could then be used to establish sampling frames and as key units of analytic interest. although many neighborhood studies rely on pre-established definitions (e.g. those used for the census or administrative purposes), such definitions do not always reflect contemporary settlement patterns. we felt that street level observations were a neces· sary component of the definition process. methods: the procedures used to delineate neighborhoods were: ( ) development of census block maps of targeted areas; ( ) review of land use maps and census tract data to ensure, prior to going into the field, tha.t particular blocks are residential and likely to have sufficient population for recruitment purposes; and ( ) field vislts and observation in each of the targeted areas. field observations focused on: housing characteristics, including housing type and condition; characteristics of commercial areas, such as likely customer base (e.g. local or no~, socio-economic status and ethnicity of customers); pedestrian volume (including con· gregauons of people m parks and outside stores or residences); obstructions to pedestrian traffic (e.g. ma or thoroughfares, multi-block industry or institutions); and maintenance and use of open spaces. results: in making the final decisions regarding neighborhood boundaries and the inclusion or exclusion of particular. blocks, we considered a broad range of factors including evidence of homogeneity an~ heterogeneity (socmeconomic, ethnic, housing type, environmental) across blocks within and blocks ad acent to a nei.ghborhood, and across the sample neighborhoods; the potential for efficient recruit· ment of appropriate particip.ants (i.e. sufficient local pedestrian traffic); and boundaries used in reporting census data (s? that population data can be used in our analysis). . altho~gh utilization of field observations for neighborhood definitions is relatively time consuming (ap~~ox mately hours for a block area, with more diverse neighborhoods taking even longer), we annc p~t~ that it will substantially improve the quality and consistency of our data gath· ~rmg ~nd analyse~. spec f c e?'amples from neighborhoods defined through this process will be given dur· m~ this presentatmn. we will also discuss the merits and limitations of characterizing neighborhoods usmg street level observations. has been no consideration for how social networks are associated with sse. the objective of this study was to identify personal and social network characteristics associated with being a recipient of sse. in this cross-sectional study, idus who injected in the past months were recruited from syringe exchange programs in montreal, canada, between april and january . information on each participant and on the persons with whom contact had occurred in the past month were elicited using a structured, interviewer-administe red questionnaire. participants provided detailed demographic and drug use information on up to idu members with whom drugs or injecting materials were shared. using logistic regression, personal and network characteristics were examined in relation to receipt of sterile syringes. res.its: of idus, % reported receiving sterile syringes in the past month from another idu. the sample mean age was years (range - ), % male, % caucasian, and % cocaine injectors. in multivariate analyses adjusted for age and gender, recipients of sterile syringes were more likely than non-recipients to share drugs after preparation ( r= . ( . - . )), to require help or to have helped inject another idu (or= . ( . - . )), to have asked someone to get sterile syringes from a syringe exchange program (or= . [ . - . )), to lend syringes (or= . [ . - . ) ) and to use drug preparation water that was previously used by another iou ( r= . ( . - . ) ) during the past months. recipients also had a higher rate of change (p= . ) of idus in their drug injecting network during the past month. with respect to social networks, % ( / ) of idu network members were providers of sterile syringes and were more likely than non-providers to inject everyday (or= . ( . - . )). when stratified by their role as a sexual partner or as someone who provides support (e.g. friend, family member), further risk behaviours were observed in relation to sharing injecting materials with the participant. conclusion: recipients of sterile syringes and their idu network members had several high-risk behaviours for infection with bloodborne diseases. sse may afford an opportunity for the dissemination of other salutary behaviours such as information sharing on safe injecting practices between recipients and distributors. furthermore, drug injecting networks may be an avenue to reach idus who may not otherwise be exposed to preventive measures. there are numerous sources of stigma and labelling of mental and physical illness. first, the sociocultural dimension to stigma and labelling clearly influences patients, families, health care professionals and society's perception and treatment of illness. this creates multiple challenges related to illness identification and recovery, particularly in culturally diverse societies. second, the media is a major source of stigma and stereotyping with regard to a variety of mental and physical illnesses. despite increased public knowledge of many illnesses, studies have shown that stigma has intensified over the years in certain cases. a third element of stigma and labels appears with the use of diagnosis as a solution to human problems with the result that difficult to treat patients are repudiated and social issues are unaddressed. this presentation will serve to outline the development of stigma, various expressions of stigma, and consequences of stigma. it will provide some practical recommendations for the reduction of stigma related to mental and physical illness. purpose: to assess the knowledge, attitude, and practice about immunization for parent of - years old hispanic and african american children in los angeles county (lac). methods: we conducted two cluster surveys in lac. the sample was selected with probability proportionate to estimated size at the first stage and simple random sample of children at the second stage. data were collected through interview. we absuacted vaccine coverage data from immunization record card (irc) at home or from clinic records. the participation was % in hispanics and % in african americans populations. irc was not available at home for % of african american and % of hispanic children. results: all parents perceived their children got all necessary vaccine~. fa~orable attitudes to.w~rd immunization were reported by % of african american and % of h s?amc parents. '.he m~ ority of the parents ( % african american and % hispanic) agreed that vaccines prevent senous d se~ses among children. regarding vaccine safety, significantly more hispanic parents ( %) than african american parents ( %) mentioned that vaccines are safe. introduction: cervical cancer has been shown to be preventable by papanicolau tests in heterosexual women; however, the utility of papanicolau test in lesbians is controversial. cervical cancer is caused by the human papillomavirus (hpv) which is transmitted by sexual _intercourse; howev_er, its' transmission by homosexual intercourse is also controversial. the goal of this study was to review the evidence for papanicolau tests in lesbians. methods: a pub med, ovid ( ovid ( to , and cochrane library search was performed using the mesh headings "homosexuality , female," "vaginal smears," and "papillomavirus, human." in pub med, the clinical queries feature was used with "diagnosis" and "broad, sensitive" filters. a google advanced search of the internet was conducted with the terms "lesbian," "cervical cancer," and "pap rest." the url was limited to ".ca," ".edu," or ".gov." results: the search yielded original articles and reviews, but no cochrane reviews. none of the publications were canadian. website hits were found and selected websites were reviewed. studies showed that lesbians have fewer papanicolau tests than heterosexual women; however, many reported risk factors for cervical cancer, including multiple past or current sexual partners (male and female), early age of first coitus, history of sexually transmitted diseases, and cigarette smoking. hpv has been detected in - % of lesbians and - % of lesbians who have never had sex with men. case studies of abnormal papanicolau tests in lesbians who have never had sex with men have also been reponed. sexual transmission of hpv among lesbians can be explained by several factors: ) - % of lesbians have had sex with men and - % of lesbians continue to have sex with men. ) hpv may be transmitted by sexual behaviours among women, like digital-vaginal sex, digital-anal sex, oral sex, and the use of insertive sex toys. ) hpv transmission requires only skin-to-skin contact. genital hpv has been identified on human fingers. some professional organizations, like the society of obstetricians and gynecologists of canada and the american cancer society, recognize the need for papanicolau tests in lesbians. other organizations, like the canadian cancer society, do not have official policy statements. the current ontario cervical screening program does not specifically address lesbians. conclusion: although the data was limited, most studies recommended that lesbians should receive papanicolau tests. the frequency of these tests was controversial. policy makers and professional organizations should address the needs of this special population and make specific recommendations . monique chaaya, ahia sibai, hiam chemaitelly, and zana el roueiheb . literature concerning the mental and physical effect of work at an old age is contradictory. in addinon, urban environments are physically and socially harsh with reduced potential for income generation and employment and increased potential for depression. the present paper is an attempt to study how do work, or lack of work, link to the experience of depression among older adults in underprivileged lebanese urban communities. the data comes from a cross-sectional survey, where elderly residing in underprivileged communities in beirut, including a palestinian refugee camp, were successfully inter-v ewed through face-to-face interviews. logistic regression was performed to assess the effect of work on depression ad_justing for many other covariates. data showed that . % of people aged years and more were still workmg. there was a highly protective effect of work on depression in elderly males (or= . , % cl= . - . ). the current study is the first of its kind in the arab region and more work shou_ld be _don_e in this area. it is important for the lebanese government to consider elderly rights for social mclus n m terms of employment opportunities. amam nuru-jeter, nancy adler, and burton singer . l~u~on:_ the socioeconomic gradient is perhaps the most persistent and significant finding in social epidem ological research. the insistent nature of the ses effect is evidenced by its significance after acc?untmg for ~umerous confounds. the significance and magnitude of the effect, however, varies by ::•al group. evidence_ o_f the relative effect of race and ses is inconclusive. further, less attention has f ~ ptid to the specific interactions of race and ses than to examinations of which is a better predictor d ~ th. ~sues are further complicated by the explanatory complexity of the various measures of ses anl . owht ey relate to the health of various social groups. understanding the specific nature of these re a ons ips s necessary for guiding he ith d · i · · h a an soc a po mes and programs aimed at improving t e poster sessions v health of our most disadvantaged groups, and therefore population health, overall. this study examines the synergistic effects of ses with both race and gender in predicting group differences in mortality. methods: analyses use data from the national longitudinal mortality survey (n= , ) for the years , , and - ; and were linked to the national death index for deaths occurring through . results were confirmed using data from the national mortality followback survey for people who died in (n= , ) and the national health interview survey (denominator). we used correlation analysis and the standardized mortality ratio to examine ses (education and income), race, and gender as predictors of group differences in changes in mortality. results: for the total population, analyses support the income gradient showing significant declines in mortality ratios from low to high income along the income strata. education shows a significant drop in mortality with completion of high school and completion of college, exhibiting a threshold rather than a gradient effect. compared to whites, blacks receive diminishing health returns for each subsequent level of income and education; with whites closely mirroring the total population in ses thresholds. similarly, compared to men, women do not experience the same health gains at comparable ses levels. for education, black men and women only show significant mortality declines upon completion of high school. the ses gradient operates differently among race and gender subgroups. threshold effects suggest resource gains in life opportunities at particular milestones along the ses gradient. further, ses matters less for blacks and women compared to whites and men suggesting differing implications for health and social policy aimed at reducing disparities. objective: we assessed the impact of diabetes in a large puerto rican community of chicago by measuring the prevalence of diagnosed diabetes and calculating the diabetes mortality rate. methods: we analyzed data from a comprehensive health survey conducted in randomly selected households in community areas. questions on diagnosed diabetes and selected risk factors were asked. in addition, mortality data were analyzed in order to calculate the age-adjusted diabetes mortality rate. when possible, rates were compared to those found in other studies. results: the diabetes prevalence located in this community ( . %: % cl= . %- . %) is one of the highest ever reported for puerto ricans. for instance, it is more than three times higher than the prevalence for the us ( . %) and twice the prevalence for puerto ricans in new york ( . %) and puerto rico ( . %- . %). diagnosed diabetes was found to be significantly associated with obesity (p= . ). the prevalence was particularly high among older people, females, those horn in the us, and those with a family history. the diabetes mortality rate ( . per , population) was more than twice the rate for all of chicago ( . ) and the us ( . ). conclusions: understanding why the diabetes and mortality rates for puerto ricans in this commu· nity are so much higher than those of other communities is imperative. collaboration between researchers, service providers and community members can help address the issues of diabetes education, early screening and diagnosis and effective treatment needed in this community. introduction: sars is a respiratory illness that is spread from person to person through dose contact. this infectious disease outbreak was unusual due to the high rate of infection among health care workers. the aim of the study is to explore the demographic and attitudinal determinants of psychological distress due to sars among health care workers of a toronto hospital. methods: a total of adult participants completed questionnaires co assess psychological distress (impact of event scale -jes) and attitudes to sars. of these, participants completed the questionnaire during the sars i outbreak, and participants completed the questionnaire during the sars ii outbreak. participants also provided information on demographic variables, including occupation and exposure to sars. principal components analysis was used to derive eight attitudinal scales: fear: system, protection, prevention, job stress, stigma, instrumental coping, and psychological copm~. l ear regression analyses were applied to evaluate the associations of the demographic and amtudmal variables, as well as time, with psychological distress. . . regression analyses showed that time and higher scores on fear, ob stress, stigma'. and instrumental coping were associated with a higher total ies score: (r =. ). contrary to expectations, none of the demographic variables was associated with total ies score. conclusion: the presence of psychological distress in health care workers is indicative of intrusive or avoidant responses to thoughts, feelings, and memories associated with the sars outbreak. this study shows that ( ) fear for one's health and the he~lth of.others, ( ) ex~erien~in~ job stress, ( )_tbe per· ception of stigma, ( ) usage of instrumental copmg skills, ~n~ ( ) t m_e s grufi~an~ly c~nmb~te to increased psychological distress in health care workers. potential mterventmns during mfecttous diseaie outbreaks in health care settings should be targeted to minimize the impact of these factors. purpose: the purpose of this study was to expand knowledge regarding feeding practices, know!· edge and nutritional beliefs of mothers currently residing in the dominican republic (dr). the rising incidence of obesity in children is a major national health concern and obesity in first and second-generation immigrant children also continues to rise. we know that parents (particularly mothers) shape children's early diet patterns and that immigrant mothers experience additional challenges related to acculturation and assimilation into a new culture, however there is a paucity of literature concerning the effect of immigrant adaptation to american culture on the nutrition of children. in addition, new immigrant knowledge about the causes of obesity and the resulting health implications for childhood obesity has not been assessed and reported. this qualitative study used focus group methodology to discuss views and practices related to the introduction of food for infants and feeding practices for young children, along with a discussion on knowledge and beliefs related to the causes and health implications of child· hood obesity. ten dominican mothers' of young children (birth to years old) who reside in a small town in the western frontier area of the dr were participants in the focus group. results from this srudy will be compared to results from an identical study that will be conducted in fall with new immi· grant mothers from the dr to the boston area. we will compare feeding practices, beliefs and knowledge about nutrition for young children between these two groups. methods: using participatory principles, a focus group design was used to interview a group of mothers in the dr. the investigator along with a bilingual translator conducted the focus group. the session was tape recorded and is currently in the process of being transcribed and translated. transcribed data will be systematically analyzed themes will be identified. a qualitative data analysis software will be used to organize and code the data according to the specific aims of the study. supporting quotations will be selected to substrate each theme. lmpli~tions for urban health practice: an ultimate goal of this study is to lay a foundation for understanding the process of acculturation on feeding practices of mothers when they immigrate to the us. understanding beliefs, knowledge and feeding practices that mothers use with young children will help m ~he ~evelopment of culturally and linguistically appropriate educational strategies and materials for use m primary prevention of pediatric obesity. in canada more than seventy-five percent of immigrants choose to stay in three major urban centers of toronto, montreal and vancouver. approximately fifty percent of the immigrants eventually set· tie m ~~e greater tor<_>nto area. there is mounting evidence that the increasing immigrant population has a_ sigmfic~nt health disadvantage over canadian-born residents. this health disadvantage manifests particularly m the ma "ority of "mm "gr t h h d be · · h . . . . an s w o a en m canada for longer than ten years. this group as ~n associ~te~ with higher risk of chronic disease such as cardiovascular diseases. this disparity twccb n ma onty of the immigrant population and the canadian-born population is of great importance to ur an health providers d" · i i · b as isproporttonate y arge immigrant population has settled in the ma or ur an centers. generally the health stat f · · · · · · h h been . us most mm grants s dynamic. recent mm grants w o av_e ant •;ffca~ada _for less ~han ~en years are known to have a health advantage known as 'healthy imm • ~ants r::r · ~:s eff~ ~ defined by the observed superior health of both male and female recent immi- immigrant participation in canadian society particularly the labour market. a new explanation of the loss of 'healthy immigrant effect' is given with the help of additional factors. lt appears that the effects of social exclusion from the labour market leading to social inequalities first experienced by recent immigrant has been responsible for the loss of healthy immigrant effect. this loss results in the subsequent health disadvantage observed in the older immigrant population. a study on patients perspectives regarding tuberculosis treatment by s.j.chander, community health cell, bangalore, india. introduction: the national tuberculosis control programme was in place over three decades; still tuberculosis control remains a challenge unmet. every day about people die of tuberculosis in india. tuberculosis affects the poor more and the poor seek help from more than one place due to various reasons. this adversely affects the treatment outcome and the patient's pocket. many tuberculosis patients become non-adherence to treatment due to many reasons. the goal of the study was to understand the patient's perspective regarding tuberculois treatment provided by the bangalore city corporation. (bmc) under the rntcp (revised national tuberculosis control programme) using dots (directly observed treatment, short course) approach. bmc were identified. the information was collected using an in-depth interview technique. they were both male and female aged between - years suffering from pulmonary and extra pulmonary tuberculosis. all patients were from the poor socio economic background. results: most patients who first sought help from private practitioners were not diagnosed and treated correctly. they sought help form them as they were easily accessible and available but they. most patients sought help later than four weeks as they lacked awareness. a few of patients sought help from traditional healers and magicians, as it did not help they turned to allopathic practitioners. the patients interviewed were inadequately informed about various aspect of the disease due to fear of stigma. the patient's family members were generally supportive during the treatment period there was no report of negative attitude of neighbours who were aware of tuberculosis patients instead sympathetic attitude was reported. there exists many myth and misconception associated with marriage and sexual relationship while one suffers from tuberculosis. patients who visited referral hospitals reported that money was demanded for providing services. most patients had to borrow money for treatment. patients want health centres to be clean and be opened on time. they don't like the staff shouting at them to cover their mouth while coughing. conclusion: community education would lead to seek help early and to take preventive measures. adequate patient education would remove all myth and conception and help the patients adhere to treatment. since tb thrives among the poor, poverty eradiation measures need to be given more emphasis. mere treatment approach would not help control tuberculosis. lntrod#ction: the main causative factor in cervical cancer is the presence of oncogenic human papillomavitus (hpv). several factors have been identified in the acquisition of hpv infection and cervical cancer and include early coitarche, large number of lifetime sexual partners, tobacco smoking, poor diet, and concomitant sexually transmitted diseases. it is known that street youth are at much higher risk for these factors and are therefore at higher risk of acquiring hpv infection and cervical cancer. thus, we endeavoured to determine the prevalence of oncogenic hpv infection, and pap test abnormalities, in street youth. ~tbods: this quantitative study uses data collected from a non governmental, not for profit dropin centre for street youth in canada. over one hundred females between the ages of sixteen and twentyfour were enrolled in the study. of these females, all underwent pap testing about those with a previous history of an abnormal pap test, or an abnormal-appearing cervix on clinical examination, underwent hpv-deoxyribonucleic (dna) testing with the digene hybrid capture ii. results: data analysis is underway. the following results will be presented: ) number of positive hpv-dna results, ) pap test results in this group, ) recommended follow-up. . the results of this study will provide information about the prevalence of oncogemc hpv-dna infection and pap test abnormalities in a population of street youth. the practice implic~ tions related to our research include the potential for improved gynecologic care of street youth. in addition, our recommendations on the usefulness of hpv testing in this population will be addressed. methods: a health promotion and disease prevention tool was developed over a period of several years to meet the health needs of recent immigrants and refugees seen at access alliance multicultural community health centre (aamchc), an inner city community health centre in downtown toronto. this instrument was derived from the anecdotal experience of health care providers, a review of medical literature, and con· sultations with experts in migration health. herein we present the individual components of this instrument, aimed at promoting health and preventing disease in new immigrants and refugees to toronto. results: the health promotion and disease prevention tool for immigrants focuses on three primary health related areas: ) globally important infectious diseases including tuberculosis (tb), hiv/aids, syphilis, viral hepatitis, intestinal parasites, and vaccine preventable diseases (vpd), ) cancers caused by infectious diseases or those endemic to developing regions of the world, and ) mental illnesses includiog those developing among survivors of torture. the health needs of new immigrants and refugees are complex, heterogeneous, and ohen reflect conditions found in the immigrant's country of origin. ideally, the management of all new immigrants should be adapted to their experiences prior to migration, however the scale and complexity of this strategy prohibits its general use by healthcare providers in industrialized countries. an immigrant specific disease prevention instrument could help quickly identify and potentially prevent the spread of dangerous infectious diseases, detect cancers at earlier stages of development, and inform health care providers and decision makers about the most effective and efficient strategies to prevent serious illness in new immigrants and refugees. lntrodmction: as poverty continues to grip pakistan, the number of urban street children grows and has now reached alarming proportions, demanding far greater action than presently offered. urbanization, natural catastrophe, drought, disease, war and internal conflict, economic breakdown causing unemployment, and homelessness have forced families and children in search of a "better life," often putting children at risk of abuse and exploitation. objectives: to reduce drug use on the streets in particular injectable drug use and to prevent the transmission of stds/hiv/aids among vulnerable youth. methodology: baseline study and situation assessment of health problems particularly hiv and stds among street children of quetta, pakistan. the program launched a peer education program, including: awareness o_f self and body protection focusing on child sexual abuse, stds/hiv/aids , life skills, gender and sexual rights awareness, preventive health measures, and care at work. it also opened care and counseling center for these working and street children ar.d handed these centers over to local communities. relationships among aids-related knowledge and bt:liefs and sexual behavior of young adults were determined. rea.sons for unsafe sex included: misconception about disease etiology, conflicting cultural values, risk demal, partner pressur~, trust and partner significance, accusation of promiscuity, lack of community endorsement of protecnve measures, and barriers to condom access. in addition socio-economic pressure, physiological issues, poor community participation and anitudes and low ~ducation level limited the effectiveness of existing aids prevention education. according to 'the baseline study the male children are ex~ to ~owledge of safe sex through peers, hakims, and blue films. working children found sexual mfor~anon through older children and their teachers (ustad). recommendation s: it was found that working children are highly vulnerable to stds/hiv/aids, as they lack protective meas":res in sexual abuse and are unaware of safe sexual practices. conclusion: non-fatal overdose was a common occurrence for idu in vancouver, and was associated with several factors considered including crystal methamphetamine use. these findings indicate a need for structural interventions that seek to modify the social and contextual risks for overdose, increased access to treatment programs, and trials of novel interventions such as take-home naloxone programs. background: injection drug users (idus) are at elevated risk for involvement in the criminal justice system due to possession of illicit drugs and participation in drug sales or markets. the criminalization of drug use may produce significant social, economic and health consequences for urban poor drug users. injection-related risks have also been associated with criminal justice involvement or risk of such involvement. previous research has identified racial differences in drug-related arrests and incarceration in the general population. we assess whether criminal justice system involvement differs by race/ethnicity among a community sample of idus. we analyzed data collected from idus (n = , ) who were recruited in san francisco, and interviewed and tested for hiv. criminal justice system involvement was measured by arrest, incarceration, drug felony, and loss/denial of social services associated with the possession of a drug felony. multivariate analyses compared measures of criminal justice involvement and race/ethnicity after adjusting for socio-demographic and drug-use behaviors including drug preference, years of injection drug use, injection frequency, age, housing status, and gender. the six-month prevalence of arrest was highest for whites ( %), compared to african americans ( %) and latinos ( % ), in addition to the mean number of weeks spent in jail in the past months ( . vs. . and . weeks). these differences did not remain statistically significant in multivariate analyses. latinos reported the highest prevalence of a lifetime drug felony conviction ( %) and mean years of lifetime incarceration in prison ( . years), compared to african americans ( %, . years) and whites ( %, . years). being african american was independently associated with having a felony conviction and years of incarceration in prison as compared to whites. the history of involvement in the criminal justice system is widespread in this sample. when looking at racial/ethnic differences over a lifetime including total years of incarceration and drug felony conviction, the involvement of african americans in the criminal justice system is higher as compared to whites. more rigorous examination of these data and others on how criminal justice involvement varies by race, as well as the implications for the health and well-being of idus, is warranted. homelessness is a major social concern that has great im~act on th~se living.in urban commu?ities. metro manila, the capital of the philippines is a highly urbanized ar~ w. t~ the h gh~st concentration of urban poor population-an estimated , families or , , md v duals. this exploratory study v is the first definitive study done in manila that explores the needs and concerns of street dwdlent\omc. less. it aims to establish the demographic profile, lifestyle patterns and needs of the streetdwdlersindit six districts city of manila to establish a database for planning health and other related interventions. based on protocol-guid ed field interviews of street dwellers, the data is useful as a template for ref!!. ence in analyzing urban homelessness in asian developing country contexts. results of the study show that generally, the state of homelessness reflects a feeling of discontent, disenfranchisem ent and pow!!· lessness that contribute to their difficulty in getting out of the streets. the perceived problems andlar dangers in living on the streets are generally associated with their exposure to extreme weather condirioll! and their status of being vagrants making them prone to harassment by the police. the health needs of the street dweller respondents established in this study indicate that the existing health related servias for the homeless poor is ineffective. the street dweller respondents have little or no access to social and health services, if any. some respondents claimed that although they were able to get service from heallh centers or government hospitals, the medicines required for treatment are not usually free and are beyond their means. this group of homeless people needs well-planned interventions to hdp them improve their current situations and support their daily living. the expressed social needs of the sucet dweller respondents were significantly concentrated on the economic aspect, which is, having a perma· nent source of income to afford food, shelter, clothing and education. these reflect the street dweller' s need for personal upliftment and safety. in short, most of their expressed need is a combination of socioeconomic resources that would provide long-term options that are better than the choice of living on the streets. the suggested interventions based on the findings will be discussed. . methods: idu~ aged i and older who injected drugs within the prior month were recruited in usmg rds which relies on referral networks to generate unbiased prevalence estimates. a diverse and mon· vated g~o~p of idu "seeds." were given three uniquely coded coupons and encouraged to refer up to three other ehgibl~ idu~, for which they received $ usd per recruit. all subjects provided informed consent, an anonymous ~t erv ew and a venous blood sample for serologic testing of hiv, hcv and syphilis anti~!· results. a total of idus were recruited in tijuana and in juarez, of whom the maion!)' were .male < .l. % and . %) and median age was . melhotls: using the data from a multi-site survey on health and well being of a random sample of older chinese in seven canadian cities, this paper examined the effects of size of the chinese community and the health status of the aging chinese. the sample (n= , ) consisted of aging chinese aged years and older. physical and mental status of the participants was measured by a chinese version medical outcome study short form sf- . one-way analysis of variance and post-hoc scheffe test were used to test the differences in health status between the participants residing in cities representing three different sizes of the chinese community. regression analysis was also used to examine the contribution of size of the chinese community to physical and mental health status. rmdts: in general, aging chinese who resided in cities with a smaller chinese population were healthier than those who resided in cities with a larger chinese population. the size of the chinese community was significant in predicting both physical and mental health status of the participants. the findings also indicated the potential underlying effects of the variations in country of origin, access barriers, and socio-economic status of the aging chinese in communities with different chinese population size. the study concluded that size of an ethnic community affected the health status of the aging population from the same ethnic community. the intra-group diversity within the aging chinese identified in this study helped to demonstrate the different socio-cultural and structural challenges facing the aging population in different urban settings. urban health and demographic surveillance system, which is implemented by the african population & health research center (aphrc) in two slum settlements of nairobi city. this study focuses on common child illnesses including diarrhea, fever, cough, common cold and malaria, as well as on curative health care service utilization. measures of ses were created using information collected at the household level. other variables of interest included are maternal demographic and cultural factors, and child characteristics. statistical methods appropriate for clustered data were used to identify correlates of child morbidity. preliminary ratdts: morbidity was reported for , ( . %) out of , children accounting for a total of , illness episodes. cough, diarrhoea, runny nose/common cold, abdominal pains, malaria and fever made up the top six forms of morbidity. the only factors that had a significant associ· ation with morbidity were the child's age, ethnicity and type of toilet facility. however, all measures of socioeconomic status (mother's education, socioeconomic status, and mother's work status) had a significant effect on seeking outside care. age of child, severity of illness, type of illness and survival of father and mother were also significantly associated with seeking health care outside home. the results of this study have highlighted the need to address environmental conditions, basic amenities, and livelihood circumstances to improve child health in poor communities. the fact that socioeconomic indicators did not have a significant effect on prevalence of morbidity but were significant for health seeking behavior, indicate that while economic resources may have limited effect in preventing child illnesses when children are living in poor environmental conditions, being enlightened and having greater economic resources would mitigate the impact of the poor environmental conditions and reduce child mortality through better treatment of sick children. inequality in human life chances is about the most visible character of the third world urban space. f.conomic variability and social efficiency have often been fingered to justify such inequalities. within this separation households exist that share similar characteristics and are found to inhabit a given spatial unit of the 'city. the residential geography of cities in the third world is thus characterized by native areas whose core is made up of deteriorated slum property, poor living conditions and a decayed environment; features which personify deprivation in its unimaginable ma~t~de. there are .eviden~es that these conditions are manifested through disturbingly high levels of morbidity and mortality. ban · h h d-and a host of other factors (corrupt n, msens t ve leaders p, poor ur ty on t e one an , . · f · · · th t ) that suggest cracks in the levels and adherence to the prmc p es o socta usnce. ese governance, e c . . . . . ps £factors combine to reinforce the impacts of depnvat n and perpetuate these unpacts. by den· grou o . · "id . . bothh tifying health problems that are caused or driven by either matena _or soc a e~nvanon or , t e paper concludes that deprivation need not be accepted as a way. of hfe a~d a deliberate effon must be made to stem the tide of the on going levels of abject poverty m the third world. to the extent that income related poverty is about the most important of all ramifications of po~erty, efforts n_iu_st include fiscal empowerment of the poor in deprived areas like the inner c~ty. this will ~p~ove ~he willingness of such people to use facilities of care because they are able to effectively demand t, smce m real sense there is no such thing as free medical services. ). there were men with hiv-infection included in the present study (mean age and education of . (sd= . ) and . (sd= . ), respectively). a series of multiple regressions were used to examine the unique contributions of symptom burden (depression, cognitive, pain, fatigue), neuropsychologic al impairment (psychomotor efficiency), demographics (age and education) and hiv disease (cdc- staging) on iirs total score and jirs subscores: ( ) activities of daily living (work, recreation, diet, health, finances); ( ) psychosocial functioning (e.g., self-expression, community involvement); and ( ) intimacy (sex life and relationship with partner). resnlts: total iirs score (r " . ) was associated with aids diagnosis (ii= . , p < . ) and symptoms of pain (ii= - . , p < . ), fatigue (ji = - . , p < . ) and cognitive difficulties (p = . , p < . ). for the three dimensions of the iirs, multiple regression results revealed: ( ) activities of daily living (r = . ) were associated with aids diagnosis (ii = . , p < . ) and symptoms of pain <p =- j , p < . ), fatigue (ji = - . , p < . ) and cognitive difficulties (ji = . , p < . ); ( ) psychosocial functioning (r = . ) was associated with was associated with symptoms of fatigue (ii= - . , p < . ) and cognitive difficulties <ii = . , p < . ); and ( ) intimacy (r = . ) was related to was associated with symptoms of fatigue (ij = - . , p < . ) and cognitive difficulties (ii= . , p < . ). methods: the sif evaluation methodology involves a comprehensive database located at the sif, a randomly selected p~o.spective coh~rt of sif users, and two pre-existing external control cohorts. . . ~ts: in addmon to reporting process data and descriptions of the sif users, we report on data indicating that the establishment of the sif has been independently associated with reductions in public ~':°g ~ (p <?. ), and syringe sharing (aor = . [ %ci: . _ . ); p = . ) and other unsafe m ecnon pract _ces_ (p ~ . ), and increased uptake of detox services (log-rank p = . ). as well, we report on da~ md cat ng that the sif has not prompted adverse changes in community drug use patterns. the vancouver sif has been well accepted by the target population, and while adve~ events s~c~ as overdoses have occurred, these events have been successfully managed. externally compiled data indicate that the sif has been associated with substantial declines in public disorder !'oster sessions v associated with injection drug use, syringe sharing, and increased uptake of detox services. as well, the establishment of the sif has not exacerbated community drug use patterns. ongoing evaluation activities wiu involve assessing the impact of the sif on a variety of outcomes, including infectious disease transmission, fatal overdose, and utilization of health and social services. city, brazil, - maria cristina almeida, waleska caiaffa, renato assum;iio, and fernando proietti dengue cases were described according to time, space, intensity, age, gender, onset of symptoms, residence address and census tract. spatial distribution of two groups (children and women over years old and men aged - ) were compared, under assumption that they have distinct behaviors regarding their displacement around the city. analysis included local moran index, ripley\\'s k function and recurrence of census areas over time. about , cases were identified in seven epidemic waves. concentration of cases in small areas, followed by a spread either in space or time suggested endemic patterns. regarding age-gender groups, distinct patterns suggested that residence might not be a good proxy in determining the local of infection for men aged - . dengue is shifting to endemic pattern in this city and transmission may not be sole related to home environment, suggesting that additional spatial information must be couected aiming efficient control measures. murukan kandamuthan and subodh kandamuthan lnl uduction: illness or disablement of a child may affect the economic functioning of a family as it alters the employment pattern and earnings of parents this paper tries to assess the health status and the quality of life of disabled children, and how generally, severe disablement in a child affected their fami-lies\' finances, and to quantify any such effects the problems faced by children in their home and to provide information relevant to the formation of criteria for new or increased cash support. methods: a case-control study was done in the thiruvananthapuram district, capital city of kerala by selecting a random sample of families with severely disabled children below years and a random sample of normal children matched for age and occupation of the parents. comparative and subjective data were collected. money spent on children\'s medical care, loss of earnings of the parents, and other economic burden to the family due to the disability of the child in the family. a discriminant analysis along with univariate analysis was done to assess the factors that mostly differentiate the disabled and normal children. the mean expenditure of the families with a disabled child was rs. /-per month, which is significantly higher than the corresponding expenditure of rs. /-per month of families with normal child, (t= . , p <. ). of the disabled children, % were not getting any social security payments and % had no special concessions for medical and other educational purposes. the analysis of income and expenditure pattern indicated that financial impact of disablement in a child on the family is significant. the discriminant model results revealed that medical expenditure was a significant variable that differentiated the disabled and normal child. the study found that the health status and quality of life of the disabled children were far from satisfactory. this in fact affected the economic status of the disabled children. in addition to reduced earnings, there are extra costs for disabled children for travel, domestic help, medical care, and health care expenditures (hospital care, physician services, dentistry, drugs and others) for disabled individuals. a strong case can be made for their long-term care by improving the financial support to such families possibly through the introduction of an allowance specially to compensate for the earnings lost by parents due to the disability of their children. widows in india are considered disadvantage group of population. because they are characterized by pangs of separation from spouse, and consequently they go through mental agony, social ~s?lation, and economic dislocation. in addition to these, poverty, landlessness, homelessness, malnutr non etc. endure serious deterioration of their health. according to census , there are million women are widowed and one fourth live in urban areas. there is a noticeable increase of urban widows from the previous census (almost half). the paper examines the type of disability and chronic ailment borne among elderly widows ( years and above). fifty second round of national sample survey aske~ the individuals three sets of questions regarding their health: their status of health, whether they are physically poster sessions v immobile, and whether or not they have had any specific chronic illnesses. although health infrastruc- · ble ·n urban setting in india as compared to rural counterpart, prevalence of tures are arge y ava a . . . . . h · · · h h"gher "n urban areas analysis shows omt problems m old age qmte common c romc ness is muc • . . and nearly percent elderly widows are suffering from this ail'. ent in urban areas. one-fifth of widows are suffering from high/low bp. heart disease, diabetes and urinary problems are much ~ore pre~alent in urban areas though disabilities is comparatively lower in urban areas. however, the d~fference is not statistically significant. disability with respect to vision is more pronounced and one third_ of the total respondents have problems regarding eyesight. one fifths ofdderly widows are _ha.rd of hearing. though disability increases with age, similar trend is not observed with respect to chr~mc illness. the percepnon of self-health among elderly becomes poorer as their age increases. the perceived health status need not be always corrected to objective indicators of health. it is noted that around per~en~ of those perceived their health as "excellenr" or "very good" are found to be suffering from chrome disease of omts and percent have visual disability. to conclude an elderly widow living in urban areas have similar health problems as she jives in rural areas. this can be easily explained, as they were not made aware to seek treatment from health facility. in addition, the study shows that the economic conditions are deterrent factors for their illness. it is essential that intervention should be taken up improving their economic conditions so that wellbeing of this most vulnerable group of the society can be taken care of. heart failure is a disease that affects nearly million people world wide. the united states alone accounts for one third of this population. blacks are stricken with heart failure twice as often as whites. men are more likely to develop chf than women, however since the majority of the chf population are seniors, there are actually more women than men. congestive heart failure (chf) has become a pandemic. as the baby boomer generation ages, the number of chf cases will rise dramatically. (young, j. & mills, r., ). after the age of , each proceeding decade doubles the incidence level of chf. the average person has a in chance of developing heart failure within their lifespan according to the framingham study (nih.gov). individuals aging or older compose over half of the entire documented heart failure population. in this age group, chf is the leading cause of hospitalization. many patients are continuously readmitted. it is estimated that the chf population will increase by one million within the next years. mortality was at % for a two year period and % for five years (young, j. & mills, r., ). data from: aha as technology evolves, the understanding of the nature of heart failure has become clearer. in the beginning of the th century heart failure was diagnosed as a condition that presented with edema (swelling) in the extremities caused by water retention. treatment in the beginning of the past cenrury was limited to abdominal drainage and diuretics. the discovery of the heart's insufficient pumping ability in heart failure patients lead to new surgeries and devices such as heart transplants and ventricular assist devices (bridge until transplant is available). ventricular hypertrophy often occurs before the development of heart failure. hypertrophy is the term given for cardiac remodeling. cardiac remodeling also includes chamber dilation (young, j. & mills, r., ) . chf financial burden the us healthcare financing administration has ranked chf as the highest cost illness in the diagnose-related area. direct costs related to chf in the us for were nearly $ billion. these costs included hospital admissions, physician fees, home health aids and medications. indirect costs were over $ billion in . loss of jobs or death due to chf was the criteria for this category (young, j. & mills, r., ). . introduction: the injection drug user quality of life scale (iduqol) is a relatively new scale ~es gned to capture the unique and individual circumstances that determine quality of life among injection drug users (idus). the life areas comprising the instrument were based on the research literature an~ ~ocus group discussions with idus. the purpose of the present study was to evaluate the content validity of t.h~ iduqol using judgmental methods based on subject matter experts' (smes) ratings. content vah~ ty refers to the de~ee to which elements of an assessment tool are representative of the construct of interest and appropnate for a given population. methods: data were obtained from six smes, from the field of idu research and practice, who ~ere. asked t.o evaluate various aspects of the iduqol, including the title of the instrument, administraon instruchons, clarity of the names and descriptions of each life area, response format, scoring instruc-no~s and examples, record form and whether each life area should be included in the instrument. sme ratings were made using either or point scales. sm es were also given the opportunity to comment on poster sessions v each section and to suggest whether important life areas had been overlooked, were redundant, not relevant, or in need of revision. (cvi) and the ~v.erage ~eviation index (ad) were used to evaluate smes' agreement on ratings of the content vahd ty vanables. both measures provided support for the content validity of various aspects of the iduqol, although results from the stricter ad index noted some areas of disagreement, including the description of particular life areas (e.g., being useful, drugs, sex), the inclusion of some life areas (e.g., drug treatment, education, independence and free choice), and the ease of the scoring instructions. the findings from this study provide (a) content validity evidence to support the use of the iduqol as well as (b) recommendations to suengthen both the instrument (e.g., improved descriptions for some items) and the accompanying administration and scoring manual (e.g., an easier scoring template). changes made to the instrument and its manual make the iduqol even easier for researchers, practitioners, and program evaluators to use as a way of assessing, and tracking changes over time or as a result of interventions in, quality of life in injection drug users. introduction: strict adherence to prescribed regimens is required to derive maximal benefit from highly active antireuoviral therapy (haart) in persons living with hiv/aids (phas). adherence is a key determinant of the degree and durability of viral suppression. adherence is also essential in reducing the spread of hiv and ensuring that today's treatments remain effective for as long as possible. the objective of this study is to conduct a systematic review of the research literature on the effectiveness of education and patient support strategies for improving adherence to haart. methods: a systematic search of the core databases was performed from january until may . randomized controlled trials examining the effectiveness of education and patient support interventions to improve the adherence to haart were considered for inclusion. only those studies that measured adherence at a minimum of six weeks were included. study selection, quality assessments, and data abstraction were performed independently by two reviewers. results: study heterogeneity with respect to differing populations, interventions, outcomes, and length of follow-up did not allow for meta-analysis. we included studies involving , ph as. sample sizes ranged from to . study duration ranged from a single session to a variable number of sessions delivered over one year. many of the interventions involved the provision of an educational component to improve adherence and often addressed strategies to overcome barriers to adherence and the development of problem-solving skills. they ranged from simple interventions (e.g., the provision of reminder devices) to complex interventions (e.g., cognitive-behavioural therapy delivered by licensed psychologists). eleven of the studies demonstrated a statistically significant advantage associated with the described adherence intervention. the advantage associated with adherence outcomes does not seem to translate into the more clinically relevant outcome of viral suppression. only out of studies that reported virological or immunological results found a significant effect associated with the intervention. the studies had several methodological shortcomings. conclusions: there is a need for standardization and methodological rigour in the conduct of adherence trials. some interventions may have a significant impact on improving adherence. further research is required before any specific adherence improving strategy can confidently be incorporated into standard clinical practice. focus groups were used to collect data from a purposive sample of treatmentexperienced participants. focus group data were analyzed using a grou~ded t~eory appr~ach. i:ne themes identified from the interviews were used to identify the effects of ant rerrov rals on quality of hfe. the content of the mos-hiv was appraised against the themes identified from our analysis. participants also completed the mos-hiv survey and were asked whether the survey covered all important aspects of quality of life on antiretroviral medications. results: five key informant interviews and five focus groups with participants were used to identify effects on quality of life that are not directly ~aptu~ed by_ the mos-hiv health survey. our~~ showed that our participants described quality of hfe as mvol~mg_ a set of ~rsonal trad~ffs ~stay alive. in most cases, worsened quality of life was traded-off for gams ~ longevlty from ~~canon use. these eff~'ts or tradeoffs included: ( ) downstream consequences of side effects and toxlanes; ( ) loss of lrufe. pende~t decision-making privileges, ( ) having to choose drugs over ~areer; (~)burden of medication-taking responsibilities; and ( ) living life under a pretense and havmg to hide-the net effect of the tradeoffs, or "prices" to pay led to what was described as "longevity with hn without hope and future~. conclusion: our study highlights five major themes summarizing the impact of haart on quality on life, and suggests that currently used scales for measuring quality of life do not a~atd~ cap~e these findings and the associated consequences. the conceptual framework for measuring quality of bfe in hiv should be reconsidered in order to better capture the important effects of the medications on quality of life. this may involve widening the boundaries of the definition of health-related quality oflife to include aspects such as finances, employment, and housing. we should further consider the development of a haart adverse effect specific instrument, using a broad definition of adverse effect in order to capture all types of adverse impacts of hiv treatments on quality of life. introduction: measles (rubeola) is the most contagious vaccine preventable disease of humans. while cases of measles are uncommon in canada today, the disease continues to be a leading cause of morbidity and death in the developing world. as a result of human migration, measles cases still occur in canada, primari~ among immigrants, returning travelers, and other susceptible groups. canada's national advisory council on immunizations (naci) recommends that immigrants without records of measles immunization be considered for vac:cination since these individuals may not have been appropriately vaccinated in their native country. on rhe other hand, natural immunity to measles in immigrants is likely to be high given the high incidence of disease in developing parts of rhe world. thus it remains unclear if the most efficient strategy to achieve high levels of measles immunity in immigrants should entail initial serologic screening followed by vaccination of susceptible individuals or preemptive vaccination of all persons lacking immunization records. understanding the epidemiology of measles immunity in immigrant populations could help guide such decisions. . methods: we identified adults, years of age or older, who were screened for serologtc immunity to measles as part of a recently developed screening protocol at a downtown toronto community health centre. we subsequently determined if these individuals had any immunization records from their native country or from within canada. we then examined the relationship between several demographic factors and immunity to measles. results: % of immigrants had no prior immunization records. overall, % of the immigrants rested for immunity to measles were found to be immune; % of those under the age of , % of those between and years of age, % of those between and years of age, and % of those years of age or older. gender, education status, household income, and immigration status were nor ~ssoc ated wirh immunity to measles. immunity to measles was lowest among immigrants from eastern europe ( ~%), whil~ immigrants from other regions of the globe had greater than % immunity. c~lusrons: immigrants and refugees appear to have reasonably high levels of immunity to mea· sics, possibly related to natural infection wirh the measles virus. immigrants from eastern europe appear to have slightly. lower l~vel~ of immunity to the measles virus. universal screening for immunity to meales or preemptive vac:cmauon may both be inefficient public health strategies, however further research is needed to corroborate these findings. nancy r~ss, stephane tremblay, saeeda khan, daniel crouse, mark tremblay, and jean-marie berrhelor o~ve: the speed of the rise in obesity in places like canada suggests that rather than a shift in the gcncnc_ com~sirion of the population, the root of the obesity pandemic is an environment that suprrs ~besity. this paper examines the influence of neighbourhood and metropolitan characteristics. bour~lts:. while accounting. for individual socio-demographics and behaviours, residents of neighs with a large proportmn of poorly educated individuals had higher bmis than those living in poster sessions v neighbourhoods with more highly educated individuals (p <. ). residing in a neighbourhood with a high proportion of recent immigrants was associated with lower bmi for men (p<. ), but not women. neighbourhood dwelling density, a proxy for walkability, was not associated with bm! for either sex. metropolitan sprawl was associated with higher male bmi (p=. ) but the effect was negligible for women (p=. ). bmi was significantly lower for women (p<. ) living in a city in the province of quebec, even after accounting for the influence of individual and neighbourhood covariates. conclusions: bm! was strongly patterned by individual-level social position in urban canada. the incremental influence on bmi of neighbourhood related to the neighbourhood's social conditions and not to their physical form. metropolitan sprawl was associated with higher bm! for men, a group with longer car-dependent commutes than women. the findings suggest that both individuals and their environments (both social and physical) influence the distribution of bmi in urban populations. introduction: urban environments have been linked to a range of human health issues. in singapore, prevalence of end stage renal disease (esrd) is predicted to rise ( in to in , kidney international, vol. , . the clinical and economic burden of esrd has promoted development of strategies aimed at preventing the development and progression of chronic kidney disease. these include population based screening programs such as the national kidney foundation, sin-gapore\'s (nkfs) "partnership for prevention."the program, aims to reduce incidence of esrd through comprehensive intervention and screening for early detection of urinary abnormalities, blood pressure (bp), and random blood glucose (rbg). objective of this study is to examine gender, race and age wise prevalence of elevated bp, rbg and proteinuria. methodology: this current analysis includes , subjects, age to years, who underwent screening during september and december . participants were asked to give demographic information and the history of diseases. tests were given to get clinical information such as proteinuria, blood glucose, sbp, and dbp. blood pressure abnormalities were defined according to ]nc vi criteria. proteinuria was defined as the presence of plus or greater protein (equivalent to> mg/di) on dipstick analysis. results: there were , ( . %) males. racial distribution was chinese ( . % ), malay ( . % ), indians ( . %) and others ( . % ).among participants, who were apparently "healthy" (asymptomatic and without history of dm, ht, or kd), gender and race wise % prevalence of elevated (bp> / ), rbg (> mg/di) and positive urine dipstick for protein was as follows male: ( . ; . ; . ) female:( . ; . ; . ) chinese:( . ; . ; ) malay: ( . ; . ; . ) indian:( . ; . ; . ) others: ( . ; . ; . ) total:(l . , . , . ). percentage of participants with more than one abnormality were as follows. those with bp> / mmhg, % also had rbg> mg/dl and . % had proteinuria> i. those with rbg> mgldl, % also had proteinuria> and % had bp> / mmhg. those with proteinuria> , % also had rbg> mg/dl, and % had bp> / mmhg. conclusion: we conclude that sub clinical abnormalities in urinalysis, bp and rbg readings are prevalent across all genders and racial groups in the adult population. the overlap of abnormalities, point towards the high risk for esrd as well as cardiovascular disease. this indicates the urgent need for population based programs aimed at creating awareness, and initiatives to control and retard progression of disease. introduction: various theories have been proposed that link differential psychological vulnerability to health outcomes, including developmental theories about attachment, separation, and the formation of psychopathology. research in the area of psychosomatic medicine suggests an association between attachment style and physical illness, with stress as a mediator. there are two main hypotheses explored in the present study: ( t) that individuals living with hiv who were upsychologically vulne~able" at study entry would be more likely to experience symptoms of depression, anxiety and phys ca! illness over. the course of the -month study period; and ( ) life stressors and social support would mediate the relat nship between psychological vulnerability and the psychological ~nd physical outcomes. . (rsles), state-trait anxiety inventory (stai), beck depr~ssi~n lnvento~ (bdi), and~ _ -item pbys~i symptoms inventory. we characterized participants as havmg psychological vulnerability and low resilience" as scoring above on the raas (insecure attachment) or above on the das (negative expectations about oneself). . . . . . " . . ,, . results: at baseline, % of parnc pants were classified as havmg low resilience. focusmg on anxiety, the average cumulative stai score of the low-resilience group was significandy hi~e~ than that of the high-resilience group ( . sd= . versus . sd= . ; f(l, )= . , p <. ). similar results were obtained for bdi and physical symptoms (f( , )= . , p<. and f( , )= . , p<. , respec· tively). after controlling for resilience, the effects of variance in life stres".°rs averaged over time wa~ a_sig· nificant predictor of depressive and physical symptoms, but not of anxiety. ho~e_ver, these assooan~s became non-significant when four participants with high values were removed. s id larly, after controlling for resilience, the effects of variance in social support averaged over time became insignificant. conclusion: not only did "low resilience" predict poor psychological and physical outcomes, it was also predictive of life events and social support; that is, individuals who were low in resilience were more likely to experience more life events and poorer social support than individuals who were resilient. for individuals with vulnerability to physical, psychological, and social outcomes, there is need to develop and test interventions to improve health outcomes in this group. rajat kapoor, ruby gupta, and jugal kishore introduction: young people in india represent almost one-fourth of the total population. they face significant risks related to sexual and reproductive health. many lack the information and skills neces· sary to make informed sexual and reproductive health choices. objective: to study the level of awareness about contraceptives among youth residing in urban and rural areas of delhi. method: a sample of youths was selected from barwala (rural; n= ) and balmiki basti (urban slums; n= ) the field practice areas of the department of community medicine, maulana azad medical college, in delhi. a pre-tested questionnaire was used to collect the information. when/(calen· dar time), by , fisher exact and t were appliedxwhom (authors?). statistical tests such as as appropriate. result: nearly out of ( . %) youth had heard of at least one type of contraceptive and majority ( . %) had heard about condoms. however, awareness regarding usage of contraceptives was as low as . % for terminal methods to . % for condom. condom was the best technique before and after marriage and also after childbirth. the difference in rural and urban groups was statistically signif· icant (p=. , give confidence interval too, if you provide the exact p value). youth knew that contra· ceptives were easily available ( %), mainly at dispensary ( . %) and chemist shops ( . %). only . % knew about emergency contraception. only advantage of contraceptives cited was population con· trol ( . %); however, . % believed that they could also control hiv transmission. awareness of side effects was poor among both the groups but the differences were statistically significant for pills (p= . ). media was the main source of information ( %). majority of youth was willing to discuss a~ut contraceptive with their spouse ( . %), but not with others. . % youth believed that people in their age group use contraceptives. % of youth accepted that they had used contraceptives at least once. % felt children in family is appropriate, but only . % believed in year spacing. . conclusion: awareness about contraceptives is vital for youth to protect their sexual and reproduc· tive health .. knowledge about terminal methods, emergency contraception, and side effects of various contraceptives need to be strengthened in mass media and contraceptive awareness campaigns. mdbot:ls: elderly aged + were interviewed in poor communities in beirut the capital of f:ebanon, ~e of which is a palestinia~. refugee camp. depression was assessed using the i -item geriat· nc depressi~n score (~l?s- ). specific q~estions relating to the aspects of religiosity were asked as well as questions perta rung to demographic, psychosocial and health-related variables. results: depression was prevalent in % of the interviewed elderly with the highest proportion being in the palestinian refugee camp ( %). mosque attendance significantly reduced the odds of being depressed only for the palestinian respondents. depression was further associated, in particular communities, with low satisfaction with income, functional disability, and illness during last year. condiuion: religious practice, which was only related to depression among the refugee population, is discussed as more of an indicator of social cohesion, solidarity than an aspect of religiosity. furthermore, it has been suggested that minority groups rely on religious stratagems to cope with their pain more than other groups. implications of findings are discussed with particular relevance to the populations studied. nearly thirty percent of india's population lives in urban areas. the outcome of urbanization has resulted in rapid growth of urban slums. in a mega-city chennai, the slum populations ( . percent) face greater health hazards due to overcrowding, poor sanitation, lack of access to safe drinking water and environmental pollution. amongst the slum population the health of women and children are most neglected, resulting in burden of both communicable and non-communicable diseases. the focus of the paper is to present the epidemiology profile of children (below years) in slums of chennai, their health status, hygiene and nutritional factors, the social response to health, the trends in child health and urbanization over a decade, the health accessibility factors, the role of gender in health care and assessment impact of health education to children. the available data prove that child health in slums is worse than rural areas. though the slum population is decreasing there is a need to explore the program intervention and carry out surveys for collecting data on some specific health implications of the slum children. objective: during the summer of there was a heat wave in central europe, producing an excess number of deaths in many countries including spain. the city of barcelona was one of the places in spain where temperatures often surpassed the excess heat threshold related with an increase in mortality. the objective of the study was to determine whether the excess of mortality which occurred in barcelona was dependent on age, gender or educational level, important but often neglected dimensions of heat wave-related studies. methods: barcelona, the second largest city in spain ( , , inhabitants in ) , is located on the north eastern coast. we included all deaths of residents of barcelona older than years that occurred in the city during the months of june, july and august of and also during the same months during the preceding years. all the analyses were performed for each sex separately. the daily number of deaths in the year was compared with the mean daily number of deaths for the period - for each educational level. poisson regression models were fitted to obtain the rr of death in with respect to the period - for each educational level and age group. results: the excess of mortality during that summer was more important for women than for men and among older ages. although the increase was observed in all educational groups, in some age-groups the increase was larger for people with less than primary education. for example, for women in the group aged - , the rr of dying for compared to - for women with no education was . ( %ci: . - - ) and for women with primary education or higher was . ( %ci: . - . ). when we consider the number of excess deaths, for total mortality (>= years) the excess numbers were higher for those with no education ( . for women and . for men) and those with less than primary education ( . for women and - for men) than those with more than primary edm:ation ( . for women and - . for men). conclusion: age, gender and educational level were important in the barcelona heat wave. it is necessary to implement response plans to reduce heat morbidity and mortality. policies should he addressed to all population but also focusing particularly to the oldest population of low educational level. introduction: recently there has been much public discourse on homelessness and its imp~ct on health. measures have intensified to get people off the street into permanent housing. for maximum v poster sessions success it is important to first determine the needs of those to be housed. their views on housing and support requirements have to be considered, as th~y ar~ the ones affected. as few res.earch studies mclude the perspectives of homeless people themselves, httle is known on ho~ they e~penence the mpacrs on their health and what kinds of supports they believe they need to obtain housing and stay housed. the purpose of this study was to add the perspectives of homeless people to the discourse, based in the assumption that they are the experts on their own situations and needs. housing is seen as a major deter· minant of health. the research questions were: what are the effects of homelessness on health? what kind of supports are needed for homeless people to get off the street? both questions sought the views of homeless individuals on these issues. methods: this study is qualitative, descriptive, exploratory. semi-structured interviews were conducted with homeless persons on street corners, in parks and drop-ins. subsequently a thematic analysis was carried out on the data. results: the findings show that individuals' experiences of homelessness deeply affect their health. apart from physical impacts all talked about how their emotional health and self-esteem are affected. the system itself, rather than being useful, was often perceived as disabling and dehumanizing, resulting in hopelessness and resignation to life on the street. neither welfare nor minimum wage jobs are sufficient to live and pay rent. educational upgrading and job training, rather than enforced idleness, are desired by most initially. in general, the longer persons were homeless, the more they fell into patterned cycles of shelter /street life, temporary employment /unemployment, sometimes addictions and often unsuccessful housing episodes. conclusions: participants believe that resources should be put into training and education for acquisition of job skills and confidence to avoid homelessness or minimize its duration. to afford housing low-income people and welfare recipients need subsidies. early interventions, 'housing first', more humane and efficient processes for negotiating the welfare system, respectful treatment by service providers and some extra financial support in crisis initially, were suggested as helpful for avoiding homelessness altogether or helping most homeless people to leave the street. this study is a national homelessness initiative funded analysis examining the experiences and perceptions of street youth vis-a-vis their health/wellness status. through in-depth interviews with street youth in halifax, montreal, toronto, calgary, ottawa and vancouver, this paper explores healthy and not-so healthy practices of young people living on the street. qualitative interviews with health/ social service providers complement the analysis. more specifically, the investigation uncovers how street youth understand health and wellness; how they define good and bad health; and their experiences in accessing diverse health services. findings suggest that living on the street impacts physical, emotional and spiritual well·being, leading to cycles of despair, anger and helplessness. the majority of street youth services act as "brokers" for young people who desire health care services yet refuse to approach formal heal~h care organizational structures. as such, this study also provides case examples of promising youth services across canada who are emerging as critical spaces for street youth to heal from the ravages of ~treet cultur~. as young people increasingly make up a substantial proportion of the homeless population in canada, it becomes urgent to explore the multiple ways in which we can support them to regain a sense of wellbeing and "citizenship." p - (c) health and livelihood implications of marginalization of slum dwellers in provision of water and sanitation services in nairobi city elizabeth kimani, eliya zulu, and chi-chi undie . ~ntrodfldion: un-habitat estimates that % of urban residents in kenya live in slums; yet due to their illegal status, they are not provided with basic services such as water sanitation and health care. ~nseque~tly, the services are provided by vendors who typically provide' poor services at exorbitant prices .. this paper investigates how the inequality in provision of basic services affects health and livelihood circumstances of the poor residents of nairobi slums . . methods: this study uses qualitative and quantitative data collected through the ongoing longitudmal .health and demographic study conducted by the african population and health research center m slum communities in n ·rob" w d · · · · ai . e use escnpnve analytical and qualitative techmques to assess h~w concerns relating to water supply and environmental sanitation services rank among the c~mmumty's general and health needs/concerns, and how this context affect their health and livelihood circumstances. results: water ( %) and sanitation ( %) were the most commonly reported health needs and also key among general needs (after unemployment) among slum dwellers. water and sanitation services are mainly provided by exploitative vendors who operate without any regulatory mechanism and charge exorbitantly for their poor services. for instance slum residents pay about times more for water than non-slum households. water supply is irregular and residents often go for a week without water; prices are hiked and hygiene is compromised during such shortages. most houses do not have toilets and residents have to use commercial toilets or adopt unorthodox means such as disposing of their excreta in the nearby bushes or plastic bags that they throw in the open. as a direct result of the poor environmental conditions and inaccessible health services, slum residents are not only sicker, they are also less likely to utilise health services and consequently, more likely to die than non-slum residents. for instance, the prevalence of diarrhoea among children in the slums was % compared to % in nairobi as a whole and % in rural areas, while under-five mortality rates were / , / and / respectively. the results demonstrate the need for change in governments' policies that deprive the rapidly expanding urban poor population of basic services and regulatory mechanisms that would protect them from exploitation. the poor environmental sanitation and lack of basic services compound slum residents' poverty since they pay much more for the relatively poor services than their non-slum counterparts, and also increase their vulnerability to infectious diseases and mortality. since iepas've been working in harm reduction becoming the pioneer in latin america that brought this methodology for brazil. nowadays the main goal is to expand this strategy in the region and strive to change the drug policy in brazil. in this way harm reduction: health and citizenship program work in two areas to promote the citizenship of !du and for people living with hiv/aids offering law assistance for this population and outreach work for needle exchange to reduce damages and dissemination of hiv/aids/hepatit is. the methodology used in outreach work is peer education, needle exchange, condoms and folders distribution to reduce damages and the dissemination of diseases like hiv/aids/hepatitis besides counseling to search for basic health and rights are activities in this program. law attendance for the target population at iepas headquarters every week in order to provide law assistance that includes only supply people with correct law information or file a lawsuit. presentations in harm reduction and drug policy to expand these subjects for police chiefs and governmental in the last year attended !du and nidu reached and . needles and syringes exchanged. in law assistance ( people living with aids, drug users, inject drug users, were not in profile) people attended. lawsuits filed lawsuits in current activity. broadcasting of the harm reduction strategies by the press helps to move the public opinion, gather supporters and diminish controversies regarding such actions. a majority number of police officer doesn't know the existence of this policy. it's still polemic discuss this subject in this part of population. women remain one of the most under seviced segments of the nigerian populationand a focus on their health and other needs is of special importance.the singular focus of the nigerian family welfare program is mostly on demographic targets by seeking to increase contraceptive prevalence.this has meant the neglect of many areas of of women's reproductive health. reproductive health is affected by a variety of socio-cultural and biological factors on on e hand and the quality of the service delivery system and its responsiveness on the other.a woman's based approach is one which responds to the needs of the adult woman and adolescent girls in a culturally sensitive manner.women's unequal access to resources including health care is well known in nigeria in which stark gender disparities are a reality .maternal health activities are unbalanced,focusi ng on immunisation and provision of iron and folic acid,rather than on sustained care of women or on the detection and referral of high risk cases. a cross-sectional study of a municipal government -owned hospitalfrom each of the geo-political regions in igeria was carried out (atotal of ce~ters) .. as _part ~f t~e re.search, the h~spital records were uesd as a background in addition to a -week mtens ve mvesuganon m the obstemc and gynecology departments. . . . : little is known for example of the extent of gynecological morbtdtty among women; the little known suggest that teh majority suffer from one or more reproductive tr~ct infect~ons. although abortion is widespread, it continues to be performed under ilegal and unsafe condmons. with the growing v poster sessions hiv pandemic, while high riskgroups such ascomn;iercial sex workers and their clients have been studied, little has been accomplished in the large populat ns, and particularly among women, regardmgstd an hiv education. . . conclusions: programs of various governmentalor non-governmental agen,c es to mvolve strategies to broaden the narrow focus of services, and more importan~, to put wo~en s reproducnve health services and information needs in the forefront are urgently required. there is a n~d to reonent commuication and education activities to incorprate a wider interpretation of reproducnve health, to focus on the varying information needs of women, men, and youth and to the media most suitable to convey information to these diverse groups on reproductive health. introduction: it is estimated that there are - youths living on the streets, on their own with the assistance of social services or in poverty with a parent in ottawa. this population is under-serviced in many areas including health care. many of these adolescents are uncomfortable or unable to access the health care system through conventional methods and have been treated in walk-in clinics and emergency rooms without ongoing follow up. in march , the ontario government provided the ct lamont institute with a grant to open an interdisciplinary and teaching medical/dental clinic for street youth in a drop-in center in downtown ottawa. bringing community organizations together to provide primary medical care and dental hygiene to the streetyouths of ottawa ages - , it is staffed by a family physician, family medicine residents, a nurse practitioner, public health nurses, a dental hygienist, dental hygiene students and a chiropodist who link to social services already provided at the centre including housing, life skills programs and counselling. project objectives: . to improve the health of high risk youth by providing accessible, coordinated, comprehensive health and dental care to vulnerable adolescents. . to model and teach interdisciplinary adolescent care to undergraduate medical students, family medicine residents and dental hygiene students. methods: non-randomized, mixed method design involving a process and impact evaluation. data collection-qualitative:a) semi-structured interviews b) focus groups with youth quantitative:a) electronic medical records for months b) records (budget, photos, project information). results: in progress-results from first months available in august . early results suggest that locating the clinic in a safe and familiar environment is a key factor in attracting the over youths the clinic has seen to date. other findings include the prevalence of preventative interventions including vaccinations, std testing and prenatal care. the poster presentation will present these and other impacts that the clinic has had on the health of the youth in the first year of the study. conclusions: ) the clinic has improved the health of ottawa streetyouth and will continue beyond the initial pilot project phase. ) this project demonstrates that with strong community partnerships, it is possible meet make healthcare more accessible for urban youth. right to health care campaign by s.j.chander, community health cell, bangalore, india. introduction: the people's health movement in india launched a campaign known as 'right to health care' during the silver jubilee year of the alma ata declaration of 'health for all' by ad in collahoration with the national human rights commission (nhrc). the aim of the campaign was to establish the 'right to health care' as a basic human right and to address structural deficiencies in the pubic health care system and unregulated private sector . . methods: as part of the campaign a public hearing was organized in a slum in bangalore. former chairman of the nhrc chaired the hearing panel, consisting of a senior health official and other eminent people in the city. detailed documentation of individual case studies on 'denial of access to health care' in different parts of the city was carried out using a specific format. the focus was on cases where denial of health services has led to loss of life, physical damage or severe financial losses to the patient. results: _fourte_en people, except one who had accessed a private clinic, presented their testimonies of their experiences m accessing the public health care services in government health centres. all the people, e_xcept_ one person who spontaneously shared her testimony, were identified by the organizations worki_ng with the slum dwellers. corruption and ill treatment were the main issues of concern to the people. five of the fourteen testimonies presented resulted in death due to negligence. the public health cen· n:s not only demand money for the supposedly free services but also ill-treats them with verbal abuse. five of these fourteen case studies were presented before the national human right commission. the poster sessions v nhrc has asked the government health officials to look into the cases that were presented and to rectify the anomalies in the system. as a result of the public hearing held in the slum, the nhrc identified urban health as one of key areas for focus during the national public hearing. cond#sion: a campaign is necessary to check the corrupted public health care system and a covetous private health care system. it helps people to understand the structure and functioning of public health care system and to assert their right to assess heath care. the public hearings or people's tribunals held during the campaign are an instrument in making the public health system accountable. ps- (a) violence among women who inject drugs nadia fairbairn, jo-anne stoltz, evan wood, kathy li, julio montaner, and thomas kerr background/object ives: violence is a major cause of morbidity and mortality among women living in urban settings. though it is widely recognized that violence is endemic to inner-city illicit drug markets, little is known about violence experienced by women injection drug users (!du). therefore, the present analyses were conducted to evaluate the prevalence of, and characteristics associated with, experiencing violence among a cohort of female idu in vancouver. methods: we evaluated factors associated with violence among female participants enrolled in the vancouver injection drug user study (vidus) using univariate analyses. we also examined self-reported relationships with the perpetrator of the attack and the nature of the violent attack. results: of the active iou followed between december , and may , , ( . %) had experienced violence during the last six months. variables positively associated with experiencing violence included: homelessness (or= . , % ci: . - . , p < . ), public injecting (or= . , % ci: . - . , p < . ), frequent crack use (or= . , % ci: . - . , p < . ), recent incarceration (or = . , % cl: . - . , p < . ), receiving help injecting (or = . , % cl: . - . , p < . ), shooting gallery attendance (or = . , % ci: . - . , p < . ), sex trade work (or = . , % cl: . - . , p < . ), frequent heroin injection (or= . , % cl: . - . , p < . ), and residence in the downtown eastside (odds ratio [or] = . , % ci: . - . , p < . ). variables negatively associated with experiencing violence included: being married or common-law (or = . . % ci: . - . , p < . ) and being in methadone treatment (or = . , % ci: . - . , p < . ). the most common perpetrators of the attack were acquaintances ( . %), strangers ( . %), police ( . %), or dealers ( . %). attacks were most frequently in the form of beatings ( . %), robberies ( . %), and assault with a weapon ( . %). conclusion: violence was a common experience among women !du in this cohort. being the victim of violence was associated with various factors, including homelessness and public injecting. these findings indicate the need for targeted prevention and support services, such as supportive housing programs and safer injection facilities, for women iou. introduction: although research on determinants of tobacco use among arab youth has been carried out at several ecologic levels, such research has included conceptual models and has compared the two different types of tobacco that are most commonly used among the lebanese youth, namely cigarette and argileh. this study uses the ecological model to investigate differences between the genders as related to the determinants of both cigarette and argileh use among youth. methodology: quantitative data was collected from youth in economically disadvantaged urban communities in beirut, the capital of lebanon. results: the results indicated that there are differences by gender at a variety of ecological levels of influence on smoking behavior. for cigarettes, gender differences were found in knowledge, peer, family, and community influences. for argileh, gender differences were found at the peer, family, and community l.evels. the differential prevalence of cigarette and argileh smoking between boys and girls s therefore understandable and partially explained by the variation in the interpersonal and community envi.ronment which surrounds them. interventions therefore need to be tailored to the specific needs of boys and girls. introduction: the objective of this study was to assess the relationship between parents' employment status and children' health among professional immigrant families in vancouver. our target communmes v poster sessions included immigrants from five ethnicity groups: south korean, indian, chine~e, ~ussian, and irani~ with professional degrees (i.e., mds, lawyers, engineers, ma?~ger~, and uru~ers ty professors) w h no relevant job to their professions and those who had been hvmg m the studied area at least for months. methodology: the participants were recruited by collaboration from three local community agencies and were interviewed individually during the fall of . ra#lts: totally, complete interviews were analyzed: from south-east asia, from south asia, from russia and other eastern europe. overall, . % were employed, . % were underemployed, % indicated they were unemployed. overall, . % were not satisfied with their current job. russians and other eastern europeans were most likely satisfied with their current job, while south-east asians were most satisfied from their life in canada. about % indicated that their spouses were not satisfied with their life in canada, while % believed that their children are very satisfied from their life in canada. in addition, around % said they were not satisfied from their family relationship in canada. while most of the responders ranked their own and their spouses' health status as either poor or very poor, jut % indicated that their first child's health was very poor. in most cases they ranked their children's health as excellent or very good. the results of this pilot study show that there is a need to create culturally specific child health and behavioral scales when conducting research in immigrant communities. for instance, in many asian cultures, it is customary for a parent either to praise their children profusely, or to condemn them. this cultural practice, called "saving face," can affect research results, as it might have affected the present study. necessary steps, therefore, are needed to revise the current standard health and behavioral scales for further studies by developing a new scale that is more relevant and culturally sensitive to the targeted immigrant families. metboda: database: national health survey (ministry of health www.msc.es). two thousand interviews were performed among madrid population ( . % of the whole); corresponded to older adults ( . % of the . million aged years and over). study sample constitutes . % ( out of ) of those older adults, who live in urban areas. demographic structure (by age and gender) of this population in relation to health services use (medical consultations, dentist visits, emergence services, hospitalisation) was studied using general linear model univariate procedure. a p . ), while age was associated with emergence services use ( % of the population: %, % and % of each age group) and hos~italisation ( % .oft~~ population: %, % and %, of each age ~oup) (p . ) was fou~d with respect to dennst v s ts ( % vs %), medical consultations ( % vs %), and emergence services use ( % vs %), while an association (p= . ) was found according to hospitalisation ( % vs %). age. an~ g~der interaction effect on health services use was not found (p> . ), but a trend towards bosp tal sanon (p= . ) could be considered. concl.uions: demographic structure of urban older adults is associated with two of the four health se~ices use studi~. a relation.ship ber_ween age. and hospital services use (emergence units and hospitalisanon), but not with ~ut-hosp tal sei:vices (medical and dentist consultations), was found. in addition ro age, gender also contnbutes to explam hospitalisation. . sexua experiences. we exammed the prevalence expenences relation to ethnic origin and other sociodemographic variables as wc i as y j die relation between unwanted sexual experiences, depression and agreuion. we did so for boys and prts separately. mdhods: data on unwanted sexual expcric:nces, depressive symptoms (ce.s-d), aggrc:uion (bohi-di and sociodemographic facron were collected by self-report quescionnairc:s administettd to students in the: nd grade (aged - ) of secondary schools in amsterdam, the netherlands. data on the nature ol unwanted sexual experiences were collected during penonal interviews by trained schoolnursn. ltaijtj: overall prevalences of unwanted sexual experiences for boys and girls were . % and . % respectively. unwanted sexual experiences were more often ttported by turkish ( . %), moroc· an ( . %) and surinamese/anrillian boys ( . %) than by dutch boys ( . %). moroccan and turkish girls, however, reported fewer unwanted sexual experiences (respectively . and . %) than durch girls did ( . %). depressive symptoms(or= . , cl= . - . ) covert agression ( r• . , cl• . - . ) and cmrt aggression (or= . , cl• . - . ) were more common in girls with an unwanted sexual experi· met. boys with an unwanted sexual experience reported more depressive symptoms (or= . ; cl• i . .l· . ) and oven agression (or= . , cl= . - . ) . of the reported unwanted sexual experiences rnpec· timy . % and . % were confirmed by male and female adolescents during a personal interview. cond sion: we ..:an conclude that the prevalence of unwanted sexual experiences among turkish and moroccan boys is disturbing. it is possible that unwanted sexual experiences are more reported hy boys who belong to a religion or culture where the virginity of girls is a maner of family honour and talking about sexuality is taboo. more boys than girls did not confirm their initial disdosurc of an lllwalltc:d sexual experience. the low rates of disclosure among boys suggcsu a necd to educ.:atc hcahh care providen and others who work with migrant boys in the recognition and repomng of exu.il ... iction. viramin a aupplc:tmntation i at .h'yo, till far from tafl'eted %. feedinit pracn~:n panku· lerty for new born earn demand lot of educatton ernpha a• cxdu ve hrealt fecdtnit for dnared rcnoj of months was observtd in only .s% of childrrn thoulh colckturm w. givm n rn% of mwly horn ct.ildrm. the proportion of children hclow- waz (malnounshrdl .con" a• h!jh •• . % anj "rt'i· acimy tc.. compared to data. mother's ~alth: from all is womm in ttprod~uvr •ill' poup, % were married and among marned w~ .\ % only w\"rt' u mic wmr cnntr.-:cruve mt h· odl % were married bdorc thc •ar of yean and % had thnr ftnc prcicnancy hcftitt dlt' •icr nf yean. the lt'f'vicn are not uutfactory or they arc adequate but nae unh ed opumally. of thote' l'h mothen who had deliverrd in last one year, % had nailed ntmaral eum nat on ira" oncc, .~o-... bad matt rhan four ttmn and ma ortty had heir tetanus toxotd tnin,"t or"'" nlht "'"'"· ljn r ned rn· win ronductrd . % dchvcnn and % had home deh\'t'oc'i. ~md~: the tervtcn unbud or u led are !tu than dnarame. the wr· l'kft provided are inadequate and on dechm reprcwnttng a looun t ~p of h hnto good coytti\#' ol wr· ncn. l!.ckground chanpng pnoriry cannoc be ruled out u °"" of thc coatnbutory bc f. ps-ii ia) dcpn:wioa aad anuccy ia mip'mu ia awccr._ many de wn, witco tui~bmjer. jack dekker, aart·jan lttkman, wim gonmc:n. and amoud verhoeff ~ a dutch commumry-bucd icudy thawed -moarh•·prc:yalm«i al . ' . kw anx · ay daorden and . % foi' dqrasion m anmttdam. nm .. p tficantly hlllhn than dwwhrft .. dw ~thew diffamca m pttyalcnca att probably rdarcd to tlk' largr populanoa of napaan ..\mturdam. <turkish %, moroccan %, and surinam % . lndttd. ic'wt'll ltudla hatt ~ ~ high prevalena rata among migrant poupi in rbe ncdwrlands. howc'ytt au ttlluchn iuffenod from wry low raporne rara, or med screnung talel thac lack a ~y yabdarcd ~ in order to study the prevalence of depression and anxiety, and the (barriers to) use of mental health care among the different ethnic groups the following study was recently conducted: . the study consisted of a two-step approach. the first step was mcl~ded m the ~ h ith survey of (ahs), and consisted of three screening scales for depression and aruaety (klo, g::q- and mhl- ). in this survey all respondents were asked_ permission for~ second app~ :' t consisted of a structured interview containing the cidi for aruaety and depression, and questtollllalres on health care seeking, amongst others. all respondents who gave permission for a second approachwere invited by jetter for a suuctured interview with multilingual interviewers at home at a preset date and time in the following week. . in total, dutch, turkish, moroccan and surinam respondents took part in the ahs and gave permission for a second approach. in the second step, fo~ ~ tur~h, m~roc can surinamese and dutch respondents, information from the extensive interview was available (res~nse rates between and % ). since the data collection was completed only recently (june ), prevalence estimates can be presented at the conference for the first time. we have shown that with this approach it is feasible to achieve an acceptable response rate in a study on mental health among migrants. therefore we expect that this study will provide reli· able estimates of depression and anxiety in the turkish, moroccan and surinam migrants in amstw! ~· for the first time. in addition, insight into mechanisms underlying the differences between and within ethnic groups and into barriers to mental health care will provide pretexts for the improvement of pre· vention and mental health care for migrant groups. this study aimed to determine spatial patterns of mortality and morbidity of five major health problems in an urban environment: homicides, pregnancy among adolescents ((< years old), asthma hospitalization among young children(< years old) and two mosquito-borne diseasesdengue and visceral leishmaniasis, during - . all events were obtained through the city health database and, subsequently, geoproccsscd using the address of residence and the geographical and administrative division of the municipality, composed by unit of planning (up), which in tum were formed, each one, by census tract units. two research questions were investigated: are there spatial patterns to the events dis· tribution, and moreover, are these patterns overlapping across space? we use thematic maps, index of comparative mortality/morbidity by up and the overlapped rank of the th worse up rates for each event. a spatial pattern of high rates of homicides, proportion of young mothers and hospitalization of asthma were overlapping in areas social and economically disadvantaged. for mosquito-borne diseases, high rates with great dispersion were found in unprivileged areas in contrast with very low rares among privileged ones. these results pointed toward a coexistence of heavier burden of diseases for those living in areas of the city where misery, poverty, lack of political public health may be modulating social health problems. a possible environmental intervention in one mosquito•bome disease might be playing a role in the occurrence of other. although with limitations, this study may provide useful information for a joint urban planning under the public perspective, articulated for use in health impact assessment. jalil safaei bdrod#aion: the association of socioeconomic status (ses), usually measured by income, and health status is an established result in bcalth studies. the poor and low income individuals have lower health sta· tus then_ those with higher incomes. in general. such finding has been usually obtained from sample surveys on speafic populations. a problem with many sample survey$ is that their results are not comparable aaoss ~ndaries as they may use different sampling methods, ask different questions, categorize the answers differently, or define groups differently. moreover, the sample data need to be standardized using the demographic: katurea of a ~ population. this study, however, uses the national population health survey (nphs) cycle public use mictodata files which is based on a common survey template ·~~three~ area in canada, namely montreal, toronto, and vancouver. it also utili .es the built-m stanclatdizaaon of the nphs data which accounts for its complex survey design. . ~:the stu~ usc:s two well-known measures of health inequalitythe relative index of meqaality and die concentranon indexto capture the extent of income related health inequality among poster sessions v urban female and male populations in each of the three metropolitan areas. personal income is classified into ten groups by the nphs with "no income" as the first group and "more than $ , " as the last. health status is measured by three variables -having a chronic condition (chc), self assessed health (sah), and health utility index (hui) -using appropriate indices. alternative formulations are used to calculate the standard deviations of the estimated or calculated measures. the findings of the study suggest that the measured health inequality depends on the health measure used. for chc and hui the measured inequality indices do indicate poorer health for lower income individulas, however, they are not statistically insignificant. health inequalities are more pronounced when health is measured by sah. the results do not support any systematic ranking of the three metropolitan areas in terms of health inequalities. they do not reveal any systematic pattern of inequality between men and women, either. conclusions: despite the use of highly aggregated nphs data, income related health disparities are observed in the three metropolitan areas in canada. this is more the case with self perceived (sah) health. such disparities are preventable and call for broader health policies that address poverty and low income among other social determinants of health. introduction: the analysis of health indicators under the spatial perspective is configured as an important instrument in the detection of intra-urban differentials. this study aimed to examine the spatial distribution of the births occurred in belo horizonte, in , analyzing the presence of spatial clusters of health indicators for newborns (rn) and their mothers, using data from the information system on live birth database (sinasc). method: for each covered area of the basic units of health (ubs), we calculated the proportion of: adolescent mothers, mothers with less than years of schooling, first pregnancy, mothers with four or more pregnancies, dead babies born on previous pregnancies, stillbirths, cesarean section, less than four prenatal care attendance, moderate and severe hypoxemia in the st and th minutes of life, low or very low birth weight. we used empiric bayesian methods for smoothing the estimates. for spatial analysis, the indicators obtained from the global moran (i) index and local indicators of spatial association (lisa) were used. maps were built to allow the visualization of the spatial clusters. in all analysis, significance statistics was considered p~ . . results: a total of , births of residents in belo horizonte were registered in i. the estimated bayesian proportions for the entire municipality was close to the one observed for all variables. analysis using lisa showed the presence of relevant spatial clusters for adolescent and low educated mothers, dead babies form previous pregnancies, cesarean section, low attendance to the prenatal care especially in area with low socio-demographic characteristics. three areas presented consistent clusters, with important spatial auto-correlation for almost all studied indicators. conclusion: the used methodology was configured as a great instrument of detection risk areas where clustering occurs. it can easily be incorporated in any surveillance system as a mechanism for controlling events related to births in the municipality. moreover, it can be applied to discriminate target areas for prompt public health interventions, such as improving health services access and the consolidation of better obstetric practices. introduction: gentrification, the displacement of low income and non-majority people out of longtime neighborhoods and residences is a global phenomenon. it has been identified as occurring in both developed and less developed countries and as inequality persists or increases, many communities are vulnerable to disruption and loss. while there may be some benefits to gentrification, these benefits are less likely to impact those who have been forced to move out of a gentrifying community or those who remain but economically stressed. . . . . this paper lays out a theoretical framework for dent fymg and ~ddressm~. the_ health consequences of gentrification on residents living in a community prior .to o.r durmg gent~ ~ cat. n. by drawing on the literature of housing, sociology, health and urban plannmg, t places genmf catt~n and neighborhood change into the context of other forces affecting th~ .hea.lth of vulnerable populations. it then proposes solutions to prevent or mitigate the impacts of genmf cat on. results: four main categories of consequences potentiallr re~ult from ~entr~fica~~n: !'°°r housing· related issues, spatial effects, mental health impacts and contr bunons to racial ~spanttes m heal~. the housing issues include increased susceptibility to asthma, lead exposure, allergiesl~topy an~. acetdents/ injuries. spatial effects include reduced access to health ~are, reduce~ access to obslttadinonal food sources, decreased physical activity, and increased obesity. the __ pnmary mental health effects_ :ire increased stress increased risk of depression and disruption of trad t onal support networks. in addinon to the above he~lth issues, gentrification has the potential to increase racial disparities in health by reduc· ing access to long term and multi-culturally experienced health care provide~s. conclusions: public health practice provides a framework for addressmg the health consequences of gentrification. in this context primary prevention would involve preventing gentrification in the first place along with a renewed commitment to helping distressed communities a~~ im_proving the quality ~f life for long term residents. the next layer of meeting the challenges of gentrification would be to assjst long rime residents to stay in a community and thus potentially allowing them to participate in the bene~ts of gentrification (such as better public services). only if all these efforts fail -and these other prevennon strategies must be a priority, public policy should then work to help people and instirutions move to other communities through grants and the provision of alternative safe affordable housing and neighborhoods. sarah romans, marsha cohen, and tonia forte lnh'odlletion: there has been sustained interest in urban rural (ur) differences in mental health over the last years of epidemiological research, with most studies reporting higher urban rates of mor· bidity, particularly when psychotic disorders have been studied. most recently, in britain, urban rates of non-psychotic disorder were greater than rural and semi-rural rates, both before and after the more adverse circumstances of urban dwellers were considered (paykel et al ) . surprisingly, there were no ur differences in help seeking. in canada, wang ( ) found greater urban rates for major depression only after he controlled for the confounding effects of race, immigration, employment and marital status, a result reminiscent of work from the us (blazer et ) . rural participants were less likely to have sought help for their mental health problems. in this study, we sought to examine urban/rural differences in rates of depression and help seeking in canada. method; data came from the canadian community health survey . , a community survey conducted by statistics canada of people aged and older, with the total sample size of , respon· dents, % response. analyses used weighted data; differences were assessed by chi-squared. ra.its: bivariate cross-tabulations showed a modest increase in month depression rates for urban ( . %) over rural ( . %, p= . ) dwellers; there were no ur differences when the sample was examined separately by gender and age group ( - , - , - ) . in general, more urban ( . %) than rural people ( . %, p= . ) had sought mental health treatment in the past year. there was no ur difference in helping seeking amongst those with depression (u . %, r . % ns). amongst the whole population, helping seeking was gendered with more urban men accessing help ( . %) than rural men ( . %, p= . ); this did not apply for women ( . % vs . % p= . ). ~ons: the urban-rural demographic continues to generate intriguing findings, even recently when internal migration is common and people can seek out the environment most conducive to their health, ~th physical and mental. people with depression are a disadvantaged, frequently stigmatized group, with ~r quality of life a_nd often impaired function. unlike many other factors associated wi~h urban or rural hfe per se, depression can be treated. the low help seeking rates is a cause for concern, m both the ur~n and_ rural populations. it behooves researchers, policy makers and service planners to ensutt effective services are available for both humane and economic reasons. methods: using united states census income data, we assigned the manhattan community districts to two major socioeconomic groups, manhattan i and ii. manhattan i is composed of community districts with average household incomes above the median for new york city; manhattan ii is those below. with public new york city department of health death records and us census data, we calculated a "standard" new york city population and estimated mortality rate distributions for manhattan i and ii. we then compared these age-adjusted actual mortality distributions with a standard t-test. results: we explored premature deaths using several cut-off points (before the ages of , , and ), and with subcomparisons for males and females. every comparison showed the wealthier area, manhattan i, to have significantly lower premature mortality rates than the lower income manhattan ii. further, we compared age-adjusted mortality rates among the city's five boroughs, and found no significant differences based on local geography alone. conclusions: these findings suggest that in manhattan, characterized by its extremes of wealth and poverty, health status, as measured by premature mortality, does vary significantly with the local income level. in manhattan, the relative average income by community district varies as much as : . this ratio is greater than that found in the other cities we have studied: for example, the range among tokyo's kus is half that in manhattan, or about : . paris has shown the longest life expectancy and changing premature mortality rates. from our preliminary work, we expect to find less variation in premature mortality rates in comparable cities. this study will continue to explore the relationship of premature mortality rates and life expectancy to income levels and different health systems among wcp cities with a view to measuring health policy options. introduction: according to the world health organization (who), smoking-related illness is currently the world's second major cause of death, currently responsible for one out of ten adult deaths worldwide. the who's framework convention on tobacco control (fctc) aims to reduce tobaccorelated disease and deaths by changing national public policies. mexico, which, according to the who, had smoking prevalences of % among adults in and % among health care providers (hcps) in , ratified the fctc in . objective: to examine knowledge of and attitudes towards cigarette smoking and smoking cessation among hcps, and clinical practices regarding smoking cessation. methods: in june , a convenience sample of hcps from one public clinic in urban oaxaca, mexico, part of mexico's national network of public health care clinics, were interviewed in spanish using a verbally administered questionnaire that was standard among all participants. during primary care outpatient visits, hcps were observed treating patients to assess the relative importance of smoking cessation as a health care priority. results: of the hcps interviewed, including ten physicians, three nurses, and five medical students, % reported smoking regularly. all hcps reported assessing patients' history of smoking, knowledge of the health risks associated with smoking, and feeling qualified to discuss these risks with patients. all hcps reported counseling patients who currently smoked to quit. all hcps reported recommending nicotine replacement therapy (eg., patch and/or gum) and/or behavioral therapy (eg., psychologist, support groups) to patients who smoked. all hcps reported that there was no government funding for smoking cessation, and that all costs associated with smoking cessation were patients' out-of-pocket expenses. observation of hcp interaction with patients revealed that hcps always inquired about smoking history with new patients and rarely inquired about smoking history with returning patients. hcps were not observed counseling patients who reported current smoking on smoking cessation methods and its benefits. observation of the clinical environment suggested a focus on preventative child health (eg., immunization, water-borne illnesses), family planning, and chronic diseases (eg., diabetes, hypertension). conclusions: while the mexican government has made smoking cessation a public health priority and hcps report addressing smoking cessation, observation of hcps suggests: ) smoking status is assessed only in new patients; and ) smoking cessation methods are not addresse~. observ_atio~ of hcps and the clinical environment in a public clinic in oaxaca suggests that smoking cessation s of lower priority than other heath care issues. p - (a) urban agriculture and food and nutrition security in kampala, uganda fiona yeudall, renee sebastian, abdelrahman lubowa, selahadin ibrahim, and donald cole introduction: urban agriculture is an important livelihood strategy contributing to household food security by increasing access and availability to food in urban settings (koc et al., ) . the purpose of poster sessions v rhis study was to examine relationships between child nutritional securiry outcomes, household food security, and urban agriculture activiries in kampala, uga~da. . . questionnaires assessing socio-demographic, farmmg and household food secunry (hfsi characreristics were administered to households. food diversiry was ~lculate~ from ~e number of foods consumed over hours for one child ( - years) per household. heigh~ weight,. nud-upper·ann· circumference (muac) and tricep skinfolds (tsf) were measured for the mdex child. z-sc.ores for height-for·age (haz), weighr-for-age (waz) and body mass index (zbmi) were ~~ulated usmg cen· rres for disease control and prevenrion reference data. z-scores for body composition measures were calculated using nhanes i and ii data for african americans. the lms method was used to c~.t for skewed z·score indices. all dara was checked for normaliry and univariable and backward mulnvanablc linear regression analysis conducted. ruwlts: household food securiry was significantly associated with wealth (b= . , p< 'a acre were more dependenr on assets for hfs. although sex of head of household was ~ot related to j-:ifs, it modi· fied relationships in rhar female headed households had greater food securtry when fai:mmg less than , acre compared ro male headed households, and vice versa. hfs was significantly associated with food diversiry (s= . , p). urbanization, especially in developing countries, has substantially increased the vulnerability of rhe mass of low-income urban dwellers. the livelihoods and quality of life for many of the poor espe· cially in larin america, asia, and africa, have deteriorated significantly over the years. urban areas are increasingly unable to provide for their populations, resulting in poverry, massive unemploymenr, job cuts, poor housing, lack of or poor public services, and a compromised health status. botswana, a country rhat lies in the sourhern parr of africa, has had its share of urban problems such as rhe mush· rooming of squarters and low-income urban sertlements. despite efforts to address the substandard living conditions in low-income urban areas, these problems have continued to grow. these living con· diuons are porenrially stressful to rhe residenrs and likely affects their health. the primary aim of the udy wa to examine the complex relationship between communiry-level stressors and interveners and health-related quality of life among residents of low-income neighborhoods in francistown, botswana. u i"" a croa -icctional quantitative design (both descriptive and explanatory) and using primarily cloae-mded interview• with a random sample of residents, this study examined the role of chrome life trnson and environmental quality on overall health status qualiry of life) and the physical, psy· chological and level of independence domains of health. the major hypothesis of the study was that community-levrl stre sor would influence health-related quality of life and that social capital would moderate these relationships. findings indicate that neighborhood quality is a powerful predictor of healrh tatu rhan socioeconomic status and individual life stressors. social capital was also found to he a ificant positive predictor of healrh and also moderator of structural factors. social capital moderated the effects of low environmental quality on level of indrpendence and on physical health outcomn, but nor on psychological and global health outcomes. these findings suggest that as the environment get betttr. stresson are reduced, hence promoting berter health outcomes. the study ends with implication• for social justice, public health and social work practice, and research, focusing mostly on the ~ole of social capital and the environmental quality in predicting health outcomes. spt· cafically • anenhon should be focused on political and civic society's commitment for social justice and poveny alleviation, ~uction of the threat of insecuriry and violence; cultivating social capital and good governance; and improving the health and social environments, especially housing and environ· mental aiutanon to name a few. urban and other uprisings by non-elites that lead to transitions, can disrupt sexual and injection "risk" networks that transmit infectious diseases by changing mixing patterns. they can also weaken urban social networks, their associated protective norms and their informal social control mechanisms which can lead to increased sexual and drug risk behaviors and violence (fullilove ; wallace & wallace , aral , friedman & reid hankins et al ) . for example, the revolution and transition to theocratic rule, and subsequent urban and national conflicts, have been followed by many urban youth in iran engaging in clandestine high-risk sexual and drug behaviors. in palestine, conflicts and restrictions on movement have led to increased fatalities from chronic diseases due to limited access to hospitals and modern medical facilities (union of palestinian medical relief committees); and heroin use and violence-related blood exposure have also increased. social movements "from below" that are rooted both in urban social network dynamics and in underlying patterns of injustice can have both protective and risky effects. for example, the social movements that led to the collapse of the ussr and its dependent governments in other countries-with subsequent increases in sex trade, alcoholism, drug use, tuberculosis, hiv, stis, and mortality in many localities-were based originally on such city-based movements in eastern europe. their impacts on health were mediated by urban social dynamics and structures. some urban social movements have improved health by prevent· ing the spread of hiv, hepatitis and other diseases. examples include movements of (a) gays and lesbians and (b) drug users. these have been rooted in social networks that overlap with risk networks. theories of urban health should include social conflict as a core concept. mechanisms that generate conflicts and the pathways by which conflicts affect health should be a major part of urban health research agendas. p - (c) demographic characteristics of people seen with tuberculosis in lagos state university teaching hospital (lasuth) chest clinic john bako, adewale akeredolu, and wale alabi tuberculosis has become a resurgent public health problem in recent times in the world. because resources are limited, control programmes frequently must target population at greatest risk. the purpose of this study was to study the demographic characteristics of people seen with tuberculosis in lagos state university teaching hospital. a total of patients who have been both radiologically and bacteriologically confirmed tuberculosis patients were used for this study between january and march. ( %) were males, while ( %) were females. notable risk factors among patients with tuberculosis were overcrowding ( . % ), homelessness ( . %) cigarette smoking ( . % ), alcoholism ( . %), non vaccination ( %), secondary contact ( . %) and poor knowledge ( . l 'x,). man· agement history patterns among the patients were herbs ( . 'yo), orthodox medicine ( .h . .l'x.). intervention targeting early-identified groups may be an effective way to reduce the incidence of tuber· culosis. such intervention should focus on health education, modification of life style and improvement of standard of living. p - (c) profiles in urban health in cities of the americas in the countries of the americas, there is an increasing migration of national and international populations to urban centers. this presentation will focus on some of the issues created by this influx of populations, the ways that cities in countries are dealing with them, and the efforts to promote local participation and solutions in management and decision-making. the methodology used to collect this information has been to draw upon and analyze information produced by the mayor's and ministry of health and development offices in each of the cities under consideration. an analysis of the impact of urbanization on health and health determinants will be presented. the information covers issues such as health status by geographic areas within the cities, highlighting issues such as marginality, barriers and physical, economic and cultural constructs and inequities in the delivery of and access to essential services (such as health, education, water, and basic sanitation). issues of governance and citizen participation will be highlighted to provide insight in~o the balance of power and mechanisms for decision-making at the local level. part of the analysis will show the relationship between democratic processes and social participation. public ~olicies will be reviewed to indicate those that are most beneficial in promoting health and overcoming barriers to it, as well as ways of capitalizing on local assets and resources. final~y, evidence of effectiveness of various strategies will be indicated. the presentation will conclude wuh suggesuons for future strategic directions to improve the quality of life and the promotion of health in large urban centers of the americas. introduction: much of the illicit drug in mexico is concentrated in northern border areas. the mile border between the u.s. and mexico is also characterized by migration; the border crossing between tijuana, baja california, mexico and san diego, california, u.s.a. is rep~rt~dl~ the busiest land border crossing in the world. we attempt to describe the migration scene among m ect on drug users (idus) m tijuana and investigate service needs. methods: migration trends were investigated in a cross-sectional study conducted from february to june among idus in tijuana. enrollment criteria included informed consent, being years or older, and having injected drugs within the prior month. subjects were recruited by respondent-dnven sampling and were administered a quantitative survey and serology for hiv, hcv, and syphilis. logistic regression was used to compare idus who had migrated to the u.s. versus those who had not. results: of idus enrolled, % were male and median age and age at first injection were and , respectively. drug combinations injected most frequently in the past months were heroin ( % and crystal methamphetamine mixed with heroin ( %). of those enrolled, responded to quesnons on migration and drug treatment and were included in this analysis. although % had resided in tijuana for at least years, % of users were born outside of baja california. working outside of mexico was common, with % working abroad in the last years ( % in u.s.), and % in the past year. in the last months, % of idus had crossed the border to the u.s., with % crossing at least once per month. those working outside of mexico in the past year were less likely to have ever received substance abuse treatment, [ % vs. %, or . , ] and were marginally less likely to have received drug treatment in the past months [ % vs. %, or . , % ci ( . - . )]. drug use patterns, age and gender did not differ between migrants and non-migrants, although migrants were more likely to report being in need of substance abuse treatment ( % vs. %, respectively). conclusions: migration is common among idus in tijuana. maintaining drug treatment regimens and determining how to best target education and services to a highly mobile population pose challenges to officials on both sides of the border. these data underscore the need to develop coordinated binational prevention and treatment efforts. introduction: despite the expanding literature on the important role public policy plays in influencing the broader determinants of the public\'s health, profound differences exist among jurisdictions in rhe attention placed upon such activities. we take an international perspective on health by examining rhe d?minant public _health paradigms of canada, usa, uk, and sweden and exploring how these paradigms shape public health practice. _method: we carefully analyze governmental and public health agencies documents to discern ~he dommant para~igms driving public health approaches and practices. we also consider the unique paht · cal and economic contexts within each nation and consider how these contexts drive public health pahcy and approaches. . . ~u~: the canadian and usa public health communities -with some exceptions --focus upon m~ividuahzed approaches to risk management. in contrast, the uk and swedish public health scenes are oriented toward broader approaches to health determinants. we find that the extent to which govern· ments, public health agencies and public health workers concern themselves with public policy approaches £<_> ~ddress ~ro.ad~r .determinants of health depends upon the particular health parad'.~ adhered. ~o withm each urisd ctton. and whether a paradigm is adopted depends upon the ideologi~a and pol~ncal context of each nation. nations such as sweden that have a long tradition of public policies promonng social jus~ce an~ equity are naturally receptive to evolving population health concepts. '[he usa represen~ a ~bey en~ro~~t where such is~ues are clear!~ subordinate. ., our findings mdicate that there s a strong political component that influences pubh ~ealth a~proaches and practi~ within the jurisdictions examined. the implications are that those seek· m~ to raise the broader detennmants of the public's health should work in coalition to raise these issues with non-health organizations and age · ca d d th · - badrgrollnd: in developed countries, social inequalities in health have endured or even worsened comparatively throughout different social groups since the s. in france, a country where access to medical and surgical care is theoretically affordable for everyone, health inequalities are among the high· est in western europe. in developing countries, health and access to care have remained critical issues. in madagascar, poverty has even increased in recent years, since the country wenr through political crisis and structural adjustment policies. objectives. we aimed to estimate and compare the impact of socio· economic status but also psychosocial characteristics (social integration, health beliefs, expectations and representation, and psychological characteristics) on the risk of having forgone healthcare in these dif· fercnt contexts. methods: population surveys conducted among random samples of households in some under· served paris neighbourhoods (n= ) and in the whole antananarivo city (n= ) in , using a common individual questionnaire in french and malagasy. reslllts: as expected, the impact of socioeconomic status is stronger in antananarivo than in paris. but, after making adjustments for numerous individual socio-economic and health characteristics, we observed in both cities a higher (and statically significant) occurrence of reponed forgone healthcare among people who have experienced childhood and/or adulthood difficulties (with relative risks up to and .s respectively in paris and antananarivo) and who complained about unhealthy living conditions. in paris, it is also correlated with a lack of trust in health services. coneluions: aside from purely financial hurdles, other individual factors play a role in the non-use of healthcare services. health insurance or free healthcare seems to be necessary hut not sufficienr to achieve an equitable access to care. therefore, health policies must not only focus on the reduction of the financial barriers to healthcare, but also must be supplemented by programmes (e.g. outreach care ser· vices, health education, health promotion programmes) and discretionary local policies tailored to the needs of those with poor health concern .. acknowledgments. this project was supported by the mal>io project and the national institute of statistics (instat) in madagascar, and hy the development research institute (ird) and the avenir programme of the national institute of health and medical research (inserm) in france. for the cities of developing countries, poverty is often described in terms of the living standard~ of slum populations, and there is good reason to believe that the health risks facing these populations are even greater, in some instances, than those facing rural villagers. yet much remains to be learned ahour the connections between urban poverty and health. it is not known what percentage of all urban poor live in slums, that is, in communities of concentrated poverty; neither is it known what proportion of slum residents are, in fact, poor. funhermore, no quantitative accounting is yet available that would sep· arare the health risks of slum life into those due to a househoid•s own poverty and those stemminic from poveny in the surrounding neighborhood. if urban health interventions are to be effectively targeted in developing countries, substantial progress must be made in addressing these cenrral issues. this paper examines poverty and children's health and survival using two large surveys, one a demographic and health survey fielded in urban egypt (with an oversampling of slums) and the other a survey of the slums of allahabad, india. using multivariate statistical methods. we find, in both settings: ( substan· rial evidence of living standards heterogeneity within the slums; ( strong evidence indicating that household-level poverty is an imponant influence on health; and ( ) staristically significant (though less strong) evidence that with household living standards held constant, neighborhood levels of poverty adversely affect health. the paper doses with a discussion of the implications of these findings for the targeting of health and poverty program interventions. p - (a) urban environment and the changing epidemiological surfacr. the cardiovascular ~ &om dorin, nigeria the emergence of cardiovascular diseases had been explained through the concomitants o_f the demographic transition wherein the prevalent causes of morbidity and monality ~hangr pr~mmant infectious diseases to diseases of lifestyle or chronic disease (see deck, ) . a ma or frustration m the v poster sessions case of cvd is its multifactural nature. it is acknowledged that the environment, however defined is the d · f · t' b tween agents and hosts such that chronic disease pathogenesis also reqmre a me an o mterac ion e . spatio-temporal coincidence of these two parties. what is not clear is which among ~ever~( potennal fac· · h b pace exacerbate cvd risk more· and to what extent does the ep dem olog cal trans · tors m t e ur an s ' . . . . tion h othesis relevant in the explanation of urban disease outlook even the developmg cities like nigeri~: thesis paper explorer these within a traditional city in nigeria. . . . the data for the study were obtained from two tertiary level hospitals m the metropolis for years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the data contain reported cases of cvd in the two facilities for the period. adopting a series of parametric and non-parametric statistics, we draw inferences between the observed cases of cvds and various demographic and locational variables of the patients. findings: about % of rhe cases occurred in years ( ) ( ) ( ) coinciding with the last year of military rule with great instability. . % occurred among male. . % also occurred among people aged - years. these are groups who are also likely to engage in most stressful life patterns. ~e study also shows that % of all cases occurred in the frontier wards with minor city areas also havmg their •fair' share. our result conformed with many empirical observation on the elusive nature of causation of cvd. this multifactoral nature had precluded the production of a map of hypertension that would be consistent with ideas of spatial prediction. cvd -cardiovascular diseases. mumbai is the commercial capital of india. as the hub of a rapidly transiting economy, mumbai provides an interesting case study into the health of urban populations in a developing country. with high-rise multimillion-dollar construction projects and crowded slums next to each other, mumbai presents a con· trast in development. there are a host of hi-tech hospitals which provide high quality care to the many who can afford it (including many westerners eager to jump the queue in their healthcare systems-'medical tour· ism'), at the same time there is a overcrowded and strained public healthcare system for those who cannot afford to pay. voluntary organizations are engaged in service provision as well as advocacy. the paper will outline role of the voluntary sector in the context of the development of the healthcare system in mumbai. mumbai has distinct upper, middle and lower economic classes, and the health needs and problems of all three have similarities and differences. these will be showcased, and the response of the healthcare system to these will be documented. a rising hiv prevalence rate, among the highest in india, is a challenge to the mumbai public healthcare system. the role of the voluntary sector in service provision, advocacy, and empowerment of local populations with regards to urban health has been paramount. the emergence of the voluntary sector as a major player in the puzzle of urban mumbai health, and it being visualized as voices of civil society or communiry representatives has advantages as well as pitfalls. this paper will be a unique attempt at examining urban health in india as a complex web of players. the influence of everyday socio·polirical-cultural and economic reality of the urban mumbai population will be a cross cutting theme in the analysis. the paper will thus help in filling a critical void in this context. the paper will thus map out issues of social justice, gender, equiry, effect of environment, through the lens of the role of the voluntary sector to construct a quilt of the realiry of healthcare in mumbai. the successes and failures of a long tradi· tion of the active advocacy and participation of the voluntary sector in trying to achieve social justice in the urban mumbai community will be analyzed. this will help in a better understanding of global urban health, and m how the voluntary sector/ngos fir into the larger picture. ba~und: o~er. half _of n~irobi's . million inhabitants live in illegal informal settlements that compose yo of the city s res dent al land area. the majority of slum residents lack access to proper san· iranon and a clean and adequate water supply. this research was designed to gain a clearer understand· mg of what kappr · · · h f . . opnate samtanon means or the urban poor, to determine the linkages between gender, hvehhoods, and access to water and sanitation, and to assess the ability of community sanitation blocks to meet water and sanitation needs in urban areas. m~tbojs_: _a household survey, gender specific focus groups and key informant interviews were conducted m maih saba, a peri-urban informal settlement. qualitative and quantitative research tools were u~ to asses~ the impact and effectiveness of community sanitation blocks in two informal settlements. results ropna e samtarmn me u es not only safe and clean latrines, but also provision ° adequate drainage and access to water supply for cleaning of clothes and homes. safety and cleanliness poster sessions v were priorities for women in latrines. levels of poverty within the informal settlements were identified and access to water and sanitation services improved with increased income. environmental health problems related to inadequate water and sanitation remain a problem for all residents. community sanitation blocks have improved the overall local environment and usage is far greater than envisioned in the design phase. women and children use the blocks less than men. this is a result of financial, social, and safety constraints. the results highlight the importance a need to expand participatory approaches for the design of water and sanitation interventions for the urban poor. plans need to recognize "appropriate sanitation" goes beyond provision of latrines and gender and socioeconomic differences must be taken into account. lessons and resources from pilot projects must be learned from, shared and leveraged so that solutions can be scaled up. underlying all the challenges facing improving water and sanitation for the urban poor are issues of land tenure. p - (c) integrating tqm (total quality management), good governance and social mobilization principles in health promotion leadership training programmes for new urban settings in countries/ areas: the prolead experience susan mercado, faren abdelaziz, and dorjursen bayarsaikhan introduction: globalization and urbanization have resulted in "new urban settings" characterized by a radical process of change with positive and negative effects, increased inequities, greater environmental impacts, expanding metropolitan areas and fast-growing slums and vulnerable populations. the key role of municipal health governance in mitigating and modulating these processes cannot be overemphasized. new and more effective ways of working with a wide variety of stakeholders is an underpinning theme for good governance in new urban settings. in relation to this, organizing and sustaining infrastructure and financing to promote health in cities through better governance is of paramount importance. there is a wealth of information on how health promotion can be enhanced in cities. despite this, appropriate capacity building programmes to enable municipal players to effectively respond to the challenges and impacts on health of globalization, urbanization and increasing inequity in new urban settings are deficient. the who kobe centre, (funded by the kobe group( and in collaboration with regional offices (emro, searo, wpro) with initial support from the japan voluntary contribution, developed a health promotion leadership training programme called "prolead" that focuses on new and autonomous structures and sustainable financing for health promotion in the context of new urban settings. methodology: country and/or city-level teams from areas, (china, fiji, india, japan, lebanon, malaysia, mongolia, oman, philippines, republic of korea, tonga and viet nam) worked on projects to advance health promotion infrastructure and financing in their areas over a month period. tools were provided to integrate principles of total quality management, good governance and social mobili .ation. results: six countries/areas have commenced projects on earmarking of tobacco and alcohol taxes for health, moblization of sports and arts organizations, integration of health promotion and social health insurance, organizational reforms, training in advocacy and lobbying, private sector and corporate mobilization and community mobilization. results from the other six areas will be reported in ..;obcr. conclusions: total quality management, good governance and social mobilization principles and skills are useful and relevant for helping municipal teams focus on strategic interventions to address complex and overwhelming determinants of health at the municipal level. the prolead training programmes hopes to inform other processes for building health promotion leadership capacity for new urban settings. the impact of city living and urbanization on the health of citizens in developing countries has received increasing attention in recent years. urban areas contribute largely to national economies. however, rapid and unplanned urban growth is often associated with poverty, environmental degradation and population demands that outstrip service capacity which conditions place human health at risk. local and national governments as well as multi national organizations are all grappling with the challenges of urbanization. with limited data and information available, urban health characteristics, including the types, quantities, locations and sources in kampala, are largely unknown. moreover, there is n? basis for assessing the impact of the resultant initiatives to improve health ~onditions amo~g ~o": ": um ties settled in unplanned areas. since urban areas are more than the aggregation ?f ~?pie w~th md_ v dual risk factors and health care needs, this paper argues that factors beyond the md v dual, mcludmg the poster sessions v · i d h · i · ment and systems of health and social services are determinants of the health soc a an p ys ca environ . of urban populations. however, as part of an ongoing study? ~s pape~ .addresses the basic concerns of urban health in kampala city. while applying the "urban hvmg conditions and the urban heal~ pen· alty" frameworks, this paper use aggregated urban health d~ta t~ explore the role of place an~ st tu· tions in shaping health and well-being of the population m kampala by understanding how characteristics of the urban environment and specific features of the city are causally related to health of invisible and forgotten urban poor population: results i~dica~e that a .range o~ urb~n he~l~h hazards m the city of kampala include substandard housing, crowdmg, mdoor air poll.ut on, msuff c ent a~d con· taminated water, inadequate sanitation and solid waste management services, vector borne .diseases, industrial waste increased motor vehicle traffic among others. the impact of these on the envtronment and community.health are mutually reinforcing. arising out of the withdra"'.al of city pl~nning systems and service delivery systems or just planning failure, thousands of people part cularl~ low-mc~me groups have been pushed to the most undesirable sections of the city where they are faced with ~ va_r ety ~f envj· ronmental insults. the number of initiatives to improve urban health is, however, growing mvolvjng the interaction of many sectors (health, environment, housing, energy, transportation and urban planning) and stakeholders (local government, non governmental organizations, aid donors and local community groups). key words: urban health governance, health risks, kampala. introduction: the viability of urban communities is dependent upon reliable and affordable mass transit. in particular, subway systems play an especially important role in the mass transit network, since they provide service to vast numbers of ridersseven of the subway systems worldwide report over one billion passenger rides each year. surprisingly, given the large number of people potentially affected, very little is known about the health and safety hazards that could affect both passengers and transit workers; these include physical (e.g., noise, vibration, accidents, electrified sources, temperature extremes), biological (e.g., transmission of infectious diseases, either through person-to-person spread or vector-borne, for example, through rodents), chemical (e.g., exposure to toxic and irritant chemicals and metals, gas emissions, fumes), electro-magnetic radiation, and psychosocial (e.g., violence, workstress). more recently, we need to consider the threat of terrorism, which could take the form of a mass casualty event (e.g., resulting from conventional incendiary devices), radiological attack (e.g., "dirty bomb"), chemical terrorist attack (e.g., sarin gas), or bioterrorist attack (e.g., weapons grade anthrax). given the large number of riders and workers potentially at risk, the public health implications are considerable. methods: to assess the hazards associated with subways, a structured review of the (english) litera· ture was conducted. ruults: based on our review, non-violent crime, followed by accidents, and violent crimes are most prevalent. compared to all other forms of mass transit, subways present greater health and safety risks. however, the rate of subway associated fatalities is much lower than the fatality rate associated with automobile travel ( . vs. . per million passenger miles), and cities with high subway ridership rates have a % lower per capita rate of transportation related fatalities than low ridership cities ( . versus . annual deaths per , residents). available data also suggest that subway noise levels and levels of air pollutants may exceed recommended levels. . ~: there is a paucity of published research examining the health and safety hazards associated with subways. most of the available data came from government agencies, who rely on passively reported data. research is warranted on this topic for a number of reasons, not only to address important knowled~ gaps, but also because the population at potential risk is large. importantly, from an urban perspecnve, the benefits of mass transit are optimized by high ridership ratesand these could be adversely affu:ted by unsafe conditions and health and safety concerns. veena joshi, jeremy lim. and benjamin chua ~ ~rban health issues have moved beyond infectious diseases and now centre largely on chrome diseases. diabetes is one of the most prevalent non-communicable diseases globally. % of adult ¥ benefit in providing splash pads in more parks. given the high temperature and humidity of london summers, this is an important aspect and asset of parks. interviewed parents claimed to visit city parks anywhere between to days per week. corrduion: given that the vast majority of canadian children are insufficiently active to gain health benefits, identifying effective qualities of local parks, that may support and foster physical activity is essential. strategies to promote activity within children's environments are an important health initiative. the results from this study have implications for city planners and policy makers; parents' opinions of, and use of city parks provides feedback as to the state current local parks, and modifications that should be made for new ones being developed. this study may also provide important feedback for health promoters trying to advocate for physical activity among children. introdt clion: a rapidly increasing proportion of urban dwellers in africa live below the poverty line in overcrowded slums characterized by uncollected garbage, unsafe water, and deficient sanitation and overflowing sewers. this growth of urban poverty challenges the commonly held assumption that urban populations enjoy better health than their rural counterparts. the objectives of this study are (i) to compare the vaccination status, and morbidity and mortality outcomes among children in the slums of nairobi with rural kenya, and (ii) to examine the factors associated with poor child health in the slums. we use data from demographic and health survey representative of all slum settlements in nairobi city carried out in by the african population & health research center. a total of , women aged - from , households were interviewed. our sample consists of , children aged - months. the comparison data are from the kenya demographic and health survey. the outcomes of interest include child vaccination status, morbidity (diarrhea, fever and cough) and mortality, all dichotomized. socioeconomic, environmental, demographic, and behavioral factors, as well as child and mother characteristics, are included in the multivariate analyses. multilevel logistic regression models are used. l'nlimin ry rest lts: about % of children in the slums had diarrhea in the two weeks prior to the survey, compared to % of rural children. these disparities between the urban poor anj the rural residents are also observed for fever ( % against %), cough ( % versus %), infant mortality ( / against / ), and complete vaccination ( % against %). preliminary multivariate results indicate that health service utilization and maternal education have the strongest predictive power on child morbidity and mortality in the slums, and that household wealth has only minor, statistically insignificant effects. conclruion: the superiority of health of urban children, compared with their rural counterparts, masks significant disparities within urban areas. compared to rural residents, children of slum dwellers in nairobi are sicker, are less likely to utilize health services when sick, and stand greater risk to die. our results suggest policies and programs contributing to the attainment of the millennium development goal on child health should pay particular attention to the urban poor. the insignificance of socioeconomic status suggests that poor health outcomes in these communities are compounded by poor environmental sanitation and behavioral factors that could partly be improved through female education and behavior change communication. introduction: historic trade city surat with its industrial and political peace has remained a center of attraction for people from all the comers of india resulting in to pop.ulatio~ explosio~ a~d stressed social and service infrastructure. the topography,dimate and demographic profile of the city s threat to the healthy environment. aim of this analysis is to review the impact of managemt'nt reform on health indicators. method: this paper is an analysis of the changing profile of population, sanitary infr~s~rucrure, local self government management and public health service reform, secondary health stat st cs data, health indicator and process monitoring of years. . . health of entire city and challenge to the management system. plague outbr~ak ( ) was the turning point in the history of civic service management including p~blic ~e~lth service management. ~ocal self government management system was revitalized by reg~lar_ field v s ts o~ al~ cadre~, _decentraltzanon of power and responsibility, equity, regular vigilant momtormg, commumcanon facility, ream_approach and people participation. reform in public health service management was throu_gh stan~~rd zed intervention protocol, innovative intervention, public private partnership, community part c panon, academic and service institute collaboration and research. sanitation service coverage have reached nearer to universal. area covered by safe water supply reached to %( ) from % ( ) and underground drainage to % ( ) from % ( ) the overhauling of the system have reflected on health indicators of vector and water born disease. malaria spr declined to . ( ) from . 'yo(! ) and diarrhea case report declined to ( ) from ( ). except dengue fever in no major disease outbreaks are reported after . city is recipient of international/national awards/ranking for these achievements. the health department have developed an evidence and experience based intervention and monitoring system and protocol for routine as well as disaster situation. the health service and management structure of surat city have emerged as an urban health model for the country. introduction: the center for healthy communities (chc) in the department of family and com· munity medicine at the medical college of wisconsin developed a pilot project to: ) assess the know· ledge, attitudes, and behaviors of female milwaukee public housing residents related to breast cancer; develop culturally and literacy appropriate education and screening modules; ) implement the developed modules; ) evaluate the modules; and ) provide follow-up services. using a community-based participatory research model the chc worked collaboratively with on-site nurse case management to meet these objectives. methods: a "breast health kick off event" was held at four separate milwaukee public housing sites for elderly and disabled adults. female residents were invited to complete a -item breast health survey, designed to accommodate various literacy levels. responses were anonymous and voluntary. the survey asked women about their previous physical exams for breast health, and then presented a series of state· ments about breast cancer to determine any existing myths. the final part gathered information about personal risk for breast cancer, the highest level of education completed, and whether the respondents h;td ever used hormone replacement therapy and/or consumed alcohol. responses were collected for descriptive analysis. results: a total of surveys (representing % of the total female population in the four sites) were completed and analyzed. % reported that they had a physical exam in the previous rwo years. % of respondents indicated they never had been diagnosed with breast cancer. % reported having had a mammogram and % having had a clinical breast exam. those that never had a mammogram reported a fear of what the provider would discover or there were not any current breast problems ro warrant an exam. % agreed that finding breast cancer early could lower the chance of dying of cancer. over % reported that mammograms were helpful in finding cancer. however, % believed that hav· ing a mammogram actually prevents breast cancer. % indicated that mammograms actually cause cancer and % reported that a woman should get a mammogram only if there is breast cancer in her family. conclusion: this survey indicates that current information about the importance of mammograms and clinical breast exams is reaching traditionally underserved women. yet there are still critical oppor· tunities to provide valuable education on breast health. this pilot study can serve as a tool for shaping future studies of health education messages for underserved populations. located in a yourh serv· ~ng agency m downtow~ ottawa, the clinic brings together community partners to provide primary medical care. and dent~i hygiene t? the street youths of ottawa aged - . the primary goal of the project is to provide accessible, coordinated, comprehensive health and dental care to vulnerable adolescents. these efforts respond to the pre-existing body of evidence suggesting that the principle barrier in accessing such care for these youths are feelings of intimidation and vulnerability in the face of a complex healthcare system. the bruyere fhn satellite clinic is located in the basement of a downtown drop-in and brings together a family medicine physician and her residents, a dental hygienist and her nd year students, a nurse practitioner, a chiropodist and public health nurses to provide primary care. the clinic has been extremely busy and well received by the youth. this workshop will demonstrate how five community organizations have come together to meet the needs of high risk youths in ottawa. this presentation will showcase the development of the clinic from its inception through its first year including reaction of the youths, partnerships and lessons learned. it will also focus on its sustainability without continued funding. we hope to have developed a model of service delivery that could be reproduced and sustained in other large cities with faculties of medicine. methods: non-randomized, mixed method design involving a process and impact evaluation. data collection-qualitative-a) semi structured interviews with providers & partners b)focus groups with youth quantitative a)electronic medical records for months records (budget, photos, project information). results: ) successfully built and opened a medicaudental clinic which will celebrate its year anniversary in august. ) over youths have been seen, and we have had over visits. conclusion: ) the clinic will continue to operate beyond the month project funding. ) the health of high risk youth in ottawa will continue to improve due to increased access to medical services. p - (a) health services -for the citizens of bangalore -past, present and future savita sathyagala, girish rao, thandavamurthy shetty, and subhash chandra bangalore city, the capital of karnataka with . million is the th most populous city in india; supporting % of the urban population of karnataka, it is considered as one of the fastest growing cities in india. known as the 'silicon valley of india', bangalore is nearly years old. bangalore city corporation (bmp), is a local self government and has the statutory commitment to provide to the citizens of bangalore: good roads, sanitation, street lighting, safe drinking water apart from other social obligations, cultural development and poverty alleviation activities. providing preventive and promotive heahh services is also a specific component. the objective of this study was to review the planning process with respect to health care services in the period since india independence; the specific research questions being what has been the strategies adopted by the city planners to address to the growing needs of the population amidst the background of the different strategies adopted by the country as a whole. three broad rime ranges have been considered for analysis: the s, s and the s. the salient results are: major area of focus has been on the maternal and child care with activities ranging from day-care to in-patient-care; though the number of institutions have grown from to the current day , their distribution has been far from satisfactory; obtaining support from the india population projects and major upgradarions have been undertaken in terms of infrastructure; over the years, in addition to the dispensaries of modern system of medicine, local traditional systems have also been initiated; the city has partnered with the healthy cities campaign with mixed success; disease surveillance, addressing the problems related to the emerging non-communicable diseases including mental health and road traffic injuries are still in its infancy. isolated attempts have been made to address the risks groups of elderly care and adolescent care. what stands out remarkably amongst the cities achievements is its ability to elicit participation from ngos, cbos and neighbourhood groups. however, the harnessing of this ability into the health sector cannot be said totally successful. the moot question in all the above observed development are: has the city rationally addressed it planning needs? the progress made so far can be considered as stuttered. the analysis and its presentation would identify the key posirive elements in the growth of banglore city and spell a framework for the new public health. introduction: anaemia associated with pregnancy is a major public health problem all over the world. different studies in different parts of india shown prevalence of anaemia between - %. anaemia remains a serious health problem in pregnancy despite of strong action taken by the government of india through national programmes. in the present study we identified th~ social beha~iors, responsible for low compliance of if a tablets consumption in pregnancy at community level and intervention was given with new modified behaviors on trial bases. . in vadodara urban. anganwadies out of were selected from the list by random sampling for tips (trials of improved practices) study. . . participants: pregnant women ( , intervention group+ , control. group) registered m the above anganwadies. study was conducted in to three phases: phase: . formative research and baseline survey (frbs). data was collected from all pregnant women to identify behaviors that are responsible for low compliance of ifa tablets. both qualitative and quantitative data were collected. haemoglobin was estimated of all pregnant women by haemo-cue. phase: . phase of tips. behaviors were identified both social & clinical for low compliance of ifa tablets consumption in pregnancy from frbs and against those, modified behaviors were proposed to pregnant women in the intervention group on trial bases by health education. trial period of weeks was given for trial of new behaviors to pregnant women in the interven· tion group. phase: . in this phase, feedbacks on behaviors tried or not tried were taken from pregnant women in intervention group. haemoglobin estimation was carried out again in all pregnant women. at the end of the study, messages were formulated on the bases of feedbacks from the pregnant women. results: all pregnant women in the intervention group had given positive feedback on new modified behaviors after intervention. mean haemoglobin concentration was higher in intervention group ( . ± . gm%) than control group ( . ± . gm%). ifa tablets compliance was improved in intervention group ( . %) than control group ( . %). conclusion: all pregnant women got benefits after trial of new modified behaviors in the intervention group. messages were formulated from the new modified behaviors, which can be used for longterm strategies for anaemia control in the community. introduction: in order to develop a comprehensive mch handbook for pregnant women and to assess its effect among them, a pilot study was carried out at the maternal and child health training institute (mchti), in dhaka, bangladesh. methods: from mchti a sample of pregnant women was selected and all subjects were women who were attending the first visit of their current pregnancy by using a random sampling method. of the subjects, women were given the mch handbook as case and women were not given the handbook as control. data on pre and post intervention of the handbook from the cases and controls were taken from data recording forms between the st of november and st of october, and data was analysed by using a multilevel analysis approach. this was a hospital-based action (case-control) research, and was applied in order to measure the outcome of pre and post intervention following the introduction of the handbook. data was used to assess the effects of utilisation of the handbook on women's knowledge, practice and utilisation of mch services. results: this study showed that the change of knowledge about antenatal care visits was . % among case mothers. knowledge of danger signs improved . %, breast feeding results . %, vaccination . % and family planning results improved . % among case. results showed some positive changes in women's attitudes among case mothers and study showed the change of practice in antenatal care visits was .u. % in the case. other notable changes were: change of practice in case mother's tetanus toxoid (ti), . %; and family planning . %. in addition, handbook assessment study indicated that most women brought the handbook on subsequent visits ( . %), the handbook was highly utilised (i.e. it was read by . %, filled-in by . %, and was used as a health education tool by . %). most women kept the handbook ( . %) and found it highly useful ( . %) with a high client satisfaction rate of . %. conclusion: pregnant women in the case group had higher knowledge, better practices, and higher utilisation of mch services than mothers in the control groups who used alternative health cards. if the handbook is developed with a focus on utilising a problem-oriented approach and involving the recomendations .of end~users, it is anticipated that the mch handbook will contribute significantly to ensuring the quahry of hfe of women and their children in bangladesh. after several meetmgs to identify the needs of the community, a faso clinic was opened at ncfs. health care professionals from smh joined with developmental and social service workers from ncfs to implement the faso diagnostic process and to provide culturally appropriate after-care. the clinic is unique in that its focus is the high risk urban aboriginal population of toronto. it accepts referrals of not only children and youth, but also of adults. lessons learned: response to the faso clinic at native child and family services has been overwhelming. aboriginal children with f asd are receiving timely diagnosis and interventions. aboriginal youth and adults who have been struggling with poveny, substance abuse, and homelessness are more willing to enter the ncfs centre for diagnosis and treatment. aboriginal infants prenatally exposed to alcohol born at st. michael's hospital or referred by other centres have access to the developmental programs located in both of the partnering agencies. the presentation will describe the clinic's development, and will detail the outcomes described, including interventions unique to the aboriginal culture. p - (c) seeds, soil, and stories: an exploration of community gardening in southeast toronto carolin taran, sarah wakefield, jennifer reynolds, and fiona yeudall introduction: community gardens are increasingly seen as a mechanism for improving nutrition and increasing food security in urban neighbourhoods, but the evidence available to support these claims is limited. in order to begin to address this gap in a way that is respectful of community knowledge and needs, the urban gardening research opportunities workgroup (ugrow) project explored the benefits and potential risks of community gardening in southeast toronto. the project used a community-based research (cbr) model to assess community gardens as a means of improving local health. the research process included interviews, focus groups, and participant observation (documented in field notes). we also directly engaged the community in the research process, through co-learning activities and community events which allowed participants to express their views and comment on emerging results. most of the research was conducted by a community-based research associate, herself a community gardener. key results were derived from these various sources through line-by-line coding of interview transcripts and field note review, an interactive and iterative process which involved both academic and community partners. results: these various data sources all suggest that enhanced health and access to fresh produce are important components of the gardening experience. they also highlight the central importance of empowering and community-building aspects of gardening to gardeners. community gardens were thought to play a role in developing friendships and social support, sharing food and other resources, appreciating cultural diversity, learning together, enhancing local place attachment and stewardship, and mobilizing to solve local problems (both inside and outside the garden). potential challenges to community gardens as a mechanism for communiry development include bureaucratic resistance to gardens, insecure land tenure and access, concerns about soil contamination, and a lack of awareness and under· standing by community members and decision-makers of all kinds. conclusion: the results highlight many health and broader social benefits experienced by commu· nity gardeners. they also point to the need for greater support for community gardening programs, par· ticularly ongoing the ongoing provision of resources and education programs to support gardens in their many roles. this research project is supported by the wellesley central health corporation and the centre for urban health initiatives, a cihr funded centre for research development hased at the univer· sity of toronto. p - (c) developing resiliency in children living in disadvantaged neighbourhoods sarah farrell, lorna weigand, and wayne hammond the traditional idea of targeting risk reduction by focusing on the development of eff~ctive coping strategies and educational programs has merit in light of the research reportmg_ that_ ~ lupl.e forms of problem behaviour consistently appear to be predicted by increasing exposure to den_uf able risk factors. as a result, many of the disadvantaged child and youth studies have focused on trymg to better _unde.r· stand the multiple risk factors that increase the likelihood of the development of at nsk behaviour m ch ldren/youth and the potential implications for prevention. this in turn has led t_o. the conclus on that community and health programs need to focus on risk reduction by helpm~ md v duals develop more effective coping strategies and a better understanding of the limitations of cenam pathologies, problematic v poster sessions coping behaviours and risk factors potentially inheren~ in high needs co~unities. ~owever, another ai:ea of research has proposed that preventative interventions should cons de~ .~rotecnve fa~ors alo~~ with reducing risk factors. as opposed to just emphasizing problems, vulnerab ht es, and deficits, a res liencybased perspective holds the belief that children, youth and their families. have strengths, reso~ce.s and the ability to cope with significant adversity in ways that are not only effective, but tend to result m mcreased ability to constructively respond to future adversity. with this in mind, a participatory research project sponsored by the united way of greater toronto was initiated to evaluate and determine the resiliency profiles of children - years (n = ) of recent immigrant families living in significantly disadvantaged communities in the toronto area. the presentation will provide an overview of the identified protective factors (both intrinsic and extrinsic) and resiliency profiles in an aggregated format as well as a summary of how the children and their parents interpreted and explained these strength-based results. as part of the focus groups, current community programs and services were examined by the participants as to what might be best practices for supporting the development and maintaining of resiliency in children, families and communities. it was proposed that the community model of assessing resiliency and protective factors as well as proposed best strength-based practice could serve as a guide for all in the community sector who provide services and programs to those in disadvantaged neighbourhoods. p - (c) naloxone by prescription in san francisco, ca and new york, ny emalie huriaux the harm reduction coalition's overdose project works to reduce the number of fatal overdoses to zero. located in new york, ny and san francisco, ca, the overdose project provides overdose education for social service providers, single-room occupancy hotel (sro) residents, and syringe exchange participants. the project also conducts an innovative naloxone prescription program, providing naloxone, an opiate antagonist traditionally administered by paramedics to temporarily reverse the effects of opiate overdose, to injection drug users (idus). we will describe how naloxone distribution became a reality in new york and san francisco, how the project works, and our results. the naloxone prescription program utilizes multiple models to reach idus, including sro-and street-based trainings, and office-based trainings at syringe exchange sites. trainings include information on overdose prevention, recognition, and response. a clinician conducts a medical intake with participants and provides them with pre-filled units of naloxone. in new york, funding was initially provided by tides foundation. new york city council provides current funding. new york department of mental health and hygiene provides program oversight. while the new york project was initiated in june , over half the trainings have been since march . in san francisco, california endowment, tides foundation, and san francisco department of public health (sfdph) provide funding. in addition, sfdph purchases naloxone and provides clinicians who conduct medical intakes with participants. trainings have been conducted since november . to date, nearly individuals have been trained and provided with naloxone. approximately of them have returned for refills and reported that they used naloxone to reverse an opiate-related overdose. limited episodes of adverse effects have been reported, including vomiting, seizure, and "loss of friendship." in new york, individuals have been trained and provided with naloxone. over overdose reversals have been reported. over half of the participants in new york have been trained in the south bronx, the area of new york with the highest rate of overdose fatalities. in san francisco, individuals have been trained and provided with naloxone. over overdose reversals have been reported. the majority of the participants in san francisco have been trained in the tenderloin, th street corridor, and mission, areas with the highest rates of overdose fatalities. the experience of the overdose project in both cities indicates that providing idus low-threshold access to naloxone and overdose information is a cost-effective, efficient, and safe intervention to prevent accidental death in this population. p - (c) successful strategies to regulate nuisance liquor stores using community mobilization, law enforcement, city council, merchants and researchers tahra goraya presenta~ion _will discuss ~uccessful environmental and public policy strategies employed in one southen: cahf?rmna commumty to remedy problems associated with nuisance liquor stores. participants ~ be given tools to understand the importance of utilizing various substance abuse prevention str~tegi~ to change local policies and the importance of involving various sectors in the community to a~_ st with and advocate for community-wide policy changes. recent policy successes from the commultles of pa~ad~na and altad~na will highlight the collaborative process by which the community mobilized resulnng m several ordmances, how local law enforcement was given more authority to monitor poster sessions v nonconforming liquor stores, how collaborative efforts with liquor store owners helped to remove high alcohol content alcohol products from their establishments and how a community-based organiz,uion worked with local legislators to introduce statewide legislation regarding the regulation of nuisance liquor outlets. p - (c) "dialogue on sex and life": a reliable health promotion tool among street-involved youth beth hayhoe and tracey methven introduction: street involved youth are a marginalized population that participate in extremely risky behaviours and have multiple health issues. unfortunately, because of previous abuses and negative experiences, they also have an extreme distrust of the adults who could help them. in , toronto public health granted funding to a non governmental, nor for profit drop-in centre for street youth aged - , to educate them about how to decrease rhe risk of acquiring hiv. since then the funding has been renewed yearly and the program has evolved as needed in order to target the maximum number of youth and provide them with vital information in a candid and enjoyable atmosphere. methods: using a retrospective analysis of the six years of data gathered from the "dialogue on sex and life" program, the researchers examined the number of youth involved, the kinds of things discussed, and the number of youth trained as peer leaders. also reviewed, was written feedback from the weekly logs, and anecdotal outcomes noted by the facilitators and other staff in the organization. results: over the five year period of this program, many of youth have participated in one hour sessions of candid discussion regarding a wide range of topics including sexual health, drug use, harm reduction, relationship issues, parenting, street culture, safety and life skills. many were new youth who had not participated in the program before and were often new to the street. some of the youth were given specific training regarding facilitation skills, sexual anatomy and physiology, birth control, sexually transmitted infections, hiv, substance use/abuse, harm reduction, relationships and discussion of their next steps/future plans following completion of the training. feedback has been overwhelmingly positive and stories of life changing decisions have been reported. conclusion: clearly, this program is a successful tool to reach street involved youth who may otherwise be wary of adults and their beliefs. based on data from the evaluation, recommendations have been made to public health to expand the funding and the training for peer leaders in order ro target between - new youth per year, increase the total numbers of youth reached and to increase the level of knowledge among the peer leaders. p - (c) access to identification and services jane kali replacing identification has become increasingly more complex as rhe government identification issuing offices introduce new requirements rhar create significant barriers for homeless people to replace their id. new forms of identification have also been introduced that art' not accessible to homekss peoplt-(e.g. the permanent resident card). ar rhe same time, many service providers continue to require identifi· cation ro access supports such as income, housing, food, health care, employment and employmt·nt training programs. street health, as well as a number of other agencies and community health centres, h, , been assisting with identification replacement for homeless peoplt· for a number of years. the rnrrt·nr challenges inherent within new replacement requirements, as well as the introduction of new forn ' of identification, have resulted in further barriers homeless people encounter when rrring to access t:ssential services. street health has been highlighting these issues to government identification issuing offices, as well as policy makers, in an effort to ensure rhar people who are homeless and marginalized have ac'ess to needed essential services. bandar is a somali word for •·a safe place." the bandar research project is the product of the regent park community health centre. the research looks ar the increasing number of somali and afri· can men in the homeless and precariously house population in the inner city core of down~own toronto. in the first phase of the pilot project, a needs assessment was conducted to dennfy barners and issues faced by rhe somali and other african men who are homeless and have add cr ns issues. th_e second phase of rhe research project was to identify long rerm resources and service delivery mechamsms that v poster sessions would enhance the abiity of this population to better access detox, treatment, and post treatment ser· vices. the final phase of the project was to facilitate the development of a conceptual model of seamless continual services and supports from the streets to detox to treatment to long term rehabilitation to housing. "between the pestle and mortar" -safe place. p - (c) successful methods for studying transient populations while improving public health beth hayhoe, ruth ewert, eileen mcmahon, and dan jang introduction: street youth are a group that do not regularly access healthcare because of their mis· trust of adults. when they do access health care, it is usually for issues severe enough for hospitalization or for episodic care in community clinics. health promotion and illness prevention is rarely a part of their thinking. thus, standard public health measures implemented in a more stable population do not work in this group. for example, pap tests, which have dearly been shown to decrease prevalence of cer· vical cancer, are rarely done and when they are, rarely followed up. methods to meet the health care needs and increase the health of this population are frequently being sought. methods: a drop-in centre for street youth in canada has participated in several studies investigating sexual health in both men and women. we required the sponsoring agencies to pay the youth for their rime, even though the testing they were undergoing was necessary according to public health stan· dards. we surmised that this would increase both initial participation and return. results: many results requiring intervention have been detected. given the transient nature of this population, return rates have been encouraging so far. conclusion: it seems evident that even a small incentive for this population increases participation in needed health examinations and studies. it is possible that matching the initial and follow-up incentives would increase the return rate even further. the fact that the youth were recruited on site, and not from any external advertising, indicates that studies done where youth trust the staff, are more likely to be successful. the presentation will share the results of the "empowering stroke prevention project" which incor· porated self-help mutual aids strategies as a health promotion methodology. the presentation will include project's theoretical basis, methodology, outcomes and evaluation results. self-help methodology has proven successful in consumer involvement and behaviour modification in "at risk," "marginalized" settings. self-help is a process of learning with and from each other which provides participants oppor· tunities for support in dealing with a problem, issue, condition or need. self-help groups are mechanisms for the participants to investigate existing solutions and discover alternatives, empowering themselves in this process. learning dynamic in self-help groups is similar to that of cooperative learning and peertraining, has proven successful, effective and efficient (haller et al, ) . the mutual support provided by participation in these groups is documented as contributory factor in the improved health of those involved. cognizant of the above theoretical basis, in the self-help resource centre initiated the "empowering stroke prevention project." the project was implemented after the input from health organizations, a scan of more than resources and an in-depth analysis of risk-factor-specific stroke prevention materials indicated the need for such a program. the project objectives were:• to develop a holistic and empowering health promotion model for stroke prevention that incorporates selfhelp and peer support strategies. • to develop educational materials that place modifiable risk factors and lifestyle information in a relevant context that validates project participants' life experiences and perspectives.• to educate members of at-risk communities about the modifiable risk factors associated with stroke, and promote healthy living. to achieve the above, a diverse group of community members were engaged as "co-editors" in the development of stroke prevention education materials which reflected and validated their life experiences. these community members received training to become lay health promoters (trained volunteer peer facilitators). in collaboration with local health organizations, these trained lay health promoters were then supported in organizing their own community-based stroke prevention activities. in addition, an educational booklet written in plain language, entitled healthy ways to prevent stroke: a guide for you, and a companion guide called healthy ways to pre· vent stroke: a facilitator's guide were produced. the presentation will include the results of a tw<>tiered evaluation of the program methodology, educational materials and the use of the materials beyond the life of the project. this poster presentation will focus on the development and structure of an innovative street outreach service that assists individuals who struggle mental illness/addictions and are experiencing homelessness. the mental health/outreach team at public health and community services (phcs) of hamilton, ontario assists individuals in reconnecting with health and social services. each worker brings to the ream his or her own skills-set, rendering it extremely effective at addressing the multidimensional and complex needs of clients. using a capacity building framework, each ream member is employed under a service contract between public health and community services and a local grassroots agency. there are public health nurses (phn), two of whom run a street health centre and one of canada's oldest and most successful needle exchange programs, mental health workers, housing specialists, a harm reduction worker, youth workers, and a united church minister, to name a few. a community advisory board, composed of consumers and professionals, advises the program quarterly. the program is featured on raising the roors 'shared learnings on homelessness' website at www.sharedlearnings.ca. through our poster presentation participants will learn how to create effective partnerships between government and grassroots agencies using a capacity building model that builds on existing programs. this study aims to assess the effects of broadcasting a series of documentary and drama videos, intended to provide information about the bc healthguide program in farsi, on the awareness about and the patterns of the service usage among farsi-speaking communities in the greater vancouver area. the major goals of the present study were twofold; ( ) to compare two methods of communications (direct vs. indirect messages) on the attitudes and perceptions of the viewers regarding the credibility of messengers and the relevance of the information provided in the videos, and ( ) to compare and contrast the impact of providing health information (i.e., the produced videos) via local tvs with the same materials when presented in group sessions (using vcr) on participants' attitudes and perceptions cowards the bc healrhguide services. results: through a telephone survey, farsi-speaking adults were interviewed in november and december . the preliminary findings show that % of the participants had seen the aired videos, from which, % watched at least one of the 'drama' clips, % watched only 'documentary' clip, and % watched both types of video. in addition, % of the respondents claimed that they were aware about the program before watching the aired videos, while % said they leaned about the services only after watching the videos. from this group, % said they called the bchg for their own or their "hildren's health problems in the past month. % also indicated that they would use the services in the future whenever it would be needed. % considered the videos as "very good" and thought they rnuld deliver relevant messages and % expressed their wish to increase the variety of subjects (produ\:e more videos) and increase the frequency of video dips. conclusion: the results of this study will assist public health specialists in bc who want to choose the best medium for disseminating information and apply communication interventions in multi\:ultural communities. introduction: many theorists and practitioners in community-based research (cbr) and knowledge transfer (kt) strongly advocate for involvement of potential users of research in the development of research projects, yet few examples of such involvement exist for urban workplace health interventions. we describe the process of developing a collaborative research program. methods: four different sets of stakeholders were identified as potential contributors to and users of the research: workplace health policy makers, employers, trade unions, and health and safety associations. representatives of these stakeholders formed an advisory committee which met quarterly. over the month research development period, an additional meetings were held between resc:ar~h~rs and stakeholders. in keeping with participant observation approaches, field notes of group and md v ~ ual meetings were kept by the two co-authors. emails and telephone calls were also documented. qu~h tative approaches to textual analysis were used, with particular attention paid to collaborattve v poster sessions relationships established (as per cbr), indicators of stakeholders' knowledge utilization (as per kt), and transformations of the proposed research (as per cbr). results: despite initial strong differences of opinion both among stakeho~ders .an~ between stakeholders and researchers, goodwill was noted among all involved. acts of rec~proc ty included mu.rual sharing of assessment tools, guidance on data utilization to stakeho~der orga~ zat ns, and suggestions on workplace recruitment to researchers. stakeholders demonstrated mcreases m concep~ual. un~erstand ing of workplace health e.g. they more commonly discussed more complex,. psychosocial md cators of organizational health. stakeholders made instrumental use of shared materials based on research e.g. adapting their consulting model to more sophisticated dat~ analysis. sta~ehol?~rs recogni_zed the strategic use of their alliance with researchers e.g., transformational leadership trainmg as a~ inducement to improve health and safety among small service franchises. stakeholders helped re-define the research questions, dramatically changed the method of recruitment from researcher cold call to stakeholderbased recruitment, and strongly influenced pilot research designs. owing a great deal to the elaborate joint development process, the four collaboratively developed pilot project submissions which were all successfully funded. conclusion: the intensive process of collaborative development of a research program among stakeholders and researchers was not a smooth process and was time consuming. nevertheless, the result of the collaborative process was a set of projects that were more responsive to stakeholder needs, more feasible for implementation, and more broadly applicable to relevant workplace health problems. introduction: environmental groups, municipal public health authorities and, increasingly, the general public are advocating for reductions in pesticide use in urban areas, primarily because of concern around potential adverse health impacts in vulnerable populations. however, limited evidence of the relative merits of different intervention strategies in different contexts exists. in a pilot research project, we sought to explore the options for evaluating pesticide reduction interventions across ontario municipalities. methods: the project team and a multi-stakeholder project advisory committee (pac), generated a list of potential key informants (kl) and an open ended interview guide. thirteen ki from municipal government, industry, health care, and environmental organizations completed face to face or telephone interviews lasting - minutes. in a parallel process, a workshop involving similar representatives and health researchers was held to discuss the role of pesticide exposure monitoring. minutes from pac meetings, field notes taken during ki interviews, and workshop proceedings were synthesized to generate potential evaluation methods and indicators. results: current evaluation activities were limited but all kls supported greater evaluation effons beginning with fuller indicator monitoring. indicators of education and outreach services were imponant for industry representatives changing applicator practices as well as most public health units and environmental organizations. lndictors based on bylaw enforcement were only applicable in the two cities with bylaws, though changing attitudes toward legal approaches were being assessed in many communities. the public health rapid risk factor surveillance system could use historical baseline data to assess changes in community behaviour through reported pesticide uses and practices, though it had limited penetration in immigrant communities not comfortable in english. pesticide sales (economic) data were only available in regional aggregates not useful for city specific change documentation. testing for watercourse or environmental contamination might be helpful, but it is sporadic and expensive. human exposure monitoring was fraught with ethical issues, floor effects from low levels of exposure, and prohibitive costs. clinical episodes of pesticide exposure reported to the regional poison centre (all ages) or the mother risk program (pregnant or breastfeeding women) are likely substantial underestimates that would be need to be supplemented with sentinel practice surveillance. focus on special clinical populations e.g., multiple chemical sensitivity would require additional data collection efforts . . conc~ons: broad support for evaluation and multiple indicators were proposed, though con-s~raints associate~ with access, coverage, sensitivity and feasibility were all raised, demonstrating the difficulty of evaluating such urban primary prevention initiatives. interventionists. an important aim of the youth monitor is to learn more about the health development of children and adolescents and the factors that can influence this development. special attention is paid to emo· tional and behavioural problems. the youth monitor identifies high-risk groups and factors that are associated with health problems. at various stages, the youth monitor chancrs the course of life of a child. the sources of informa· tion and methods of research are different for each age group. the results arc used to generate various kinds of repons: for children and young persons, parents, schools, neighbourhoods, boroughs and the municipality of rotterdam and its environs. any problems can be spotted early, at borough and neigh· bourhood level, based on the type of school or among the young persons and children themselves. together with schools, parents, youngsters and various organisations in the area, the municipal health service aims to really address these problems. on request, an overview is offered of potentially suitable interventions. the authors will present the philosophy, working method, preliminary effects and future developments of this instrument, which serves as the backbone for the rotterdam local youth policy. social workers to be leaders in response to aging urban populations: the practicum partnership program sarah sisco, alissa yarkony, and patricia volland "'" tliu:tion: across the us, . % of those over live in urban areas. these aging urban popu· lations, including the baby boomers, have already begun encounter a range of heahh and mental hcahh conditions. to compound these effects, health and social service delivery fluciuates in cities, whit:h arc increasingly diverse both in their recipients and their systems. common to other disciplines (medicine, nursing, psychology, etc.) the social work profession faces a shortage of workers who are well-equipped to navigate the many systems, services, and requisite care that this vast population requires. in the next two decades, it is projected that nearly , social workers will be required to provide suppon to our older urban populations. social workers must be prepared to be aging-savvy leaders in their field, whether they specialize in gerontology or work across the life span. mllhotu: in , a study conducted at the new york academy of medicine d<> :umcntcd the need for improved synchroniciry in two aspects of social work education, classroom instruction and the field experience. with suppon from the john a. hanford foundation, our team created a pilot proj~"t entitled the practicum pannership program (ppp) in master's level schools of social work, to improvt" aginr exposure in field and classroom content through use of the following: i) community-university partnrr· ships, ) increased, diverse student field rotations, ll infusion of competcn ."}'·drivm coursework, enhancement of field instructors' roles, and ) concentrated student recruitment. we conductt"d a prr· and post-test survey into students' knowledge, skills. and satisfaction. icarlja: surveys of over graduates and field inltnk."tors rcflected increased numlk-n of . rrm:y· univmity panncrships, as well as in students placed in aging agencin for field placements. there wa marked increase in student commitments to an aging specialization. onr year por.t·gradu:nion rcvealrd that % of those surveyed were gainfully employed, with % employed in the field of aginic. by com· bining curricular enhancement with real-world experiences the ppp instilled a broad exposurr for llu· dents who worked with aging populations in multiple urban settings. coltdtuion: increased exposure to a range of levels of practicr, including clinical, policy/ajvocaq, and community-based can potentially improve service delivery for older adulh who live in elfin, and potentially improve national policy. the hanford foundation has now elected to uppon cxpantion of the ppp to schools nationwide (urban and rural) to complement other domntic initiatives to cnhalk"c" holistic services for older adults across the aging spectrum. bodrgnn.ntl: we arc a team of rcscarcbcn and community panncn working tcj c(her to develop an in"itepth understanding of the mental health needs of homeless youth ~ages to ) (using qualiutivc and quantitative methods ' panicipatory rncarch methods). it is readily apparmt that '-neless youth cxpcricnce a range of mental health problems. for youth living on the street, menul illnew may be either a major risk factor for homelessnal or may frequently emcsge in response to coping with rhe multitudinous stressors associated with homclcslllcsi including exposure to violence, prasutt to pamaplte in v poster sessions survival sex and/or drug use. the most frequent psychiatric diagnoses amongst the homeless gencrally include: depression, anxiety and psychosis. . . . the ultimate ob ective of the pr~am of rei:e~ is to ~evelop a plan for intervention to meet the mental health needs of street youth. prior t_o pl~nnmg mtervenbons, .it is necessary to undertake a comprehensive assessment ~f mental health needs m this ~lnerable populanon. thus, the immediate objective of this research study is to undertake a comprehensive assessment of men· tal health needs. . . melbotlology: a mixed methodology triangulating qualitative, participatory acnon and quantitative methods will capture the data related to mental health needs of homeless youth. a purposive sample of approximately - subjecrs. ages to , is currently being ~ted ~participate from the commu.nity agencies covenant house, evergreen centre fo~ srrc;et youth, turning p? ?t and street ~ serv~. youth living on the street or in short -term residennal programs for a mmimum of month pnor to their participation; ages to and able to give infonned consent will be invited to participate in the study. o..tcomes: the expected outcome of this initial survey will be an increased understanding of mental health needs of street youth that will be used to develop effective interventions. it is anticipated that results from this study will contribute to the development of mental health policy, as well as future programs that are relevant to the mental health needs of street youth. note: it is anticipated that preliminary quantitative data ( subjects) and qualitative data will be available for the conference. the authors intend to present the identification of the research focus, the formation of our community-based team, relevance for policy, as well as preliminary results. p - (a) the need for developing a firm health policy for urban informal worken: the case of despite their critical role in producing food for urban in kenya, urban farmers have largely been ignored by government planners and policymakers. their activity is at best dismissed as peripheral eveo, inappropriate retention of peasant culture in cities and at worst illegal and often some-times criminal· ized. urban agriculture is also condemned for its presumed negative health impact. a myth that contin· ues despite proof to the contrary is that malarial mosquitoes breed in maize grown in east african towns. however, potential health risks are insignificant compared with the benefits of urban food production. recent studies too rightly do point to the commercial value of food produced in the urban area while underscoring the importance of urban farming as a survival strategy among the urban poor, especially women-headed households. since the millennium declaration, health has emerged as one of the most serious casualties consequent on the poverty, social exclusion, marginalisation and lack of sustain· able development in africa. hiv/aids epidemic poses an unprecedented challenge, while malaria, tuber· culosis, communicable diseases of childhood all add to the untenable burden. malnutrition underpins much ill-health and is linked to more than per cent of all childhood deaths. kenya's urban poor people ~ace ~ h~ge burde~ of preventable and treatable health problems, measured by any social and bi~ medical md cator, which not only cause unnecessary death and suffering, but also undermine econonuc development and damage the country's social fabric. the burden is in spite of the availability of suitable tools and re:c=hnology for prevention and treatment and is largely rooted in poverty and in weak healah •rstems. this pa~ therefore challenges development planners who perceive a dichotomy instead of con· tmuum between informal and formal urban wage earners in so far as access to health services is con· cemed. it i~ this gap that calls for a need to developing and building sustainable health systems among the urban mformal ~wellers. we recommend a focus on an urban health policy that can build and strengthen the capacity of urban dwellers to access health services that is cost-effective and sustainable. such ~ health poli<=>: must strive for equity for the urban poor, displaced or marginalized; mobilise and effect ~ely use sufficient sustainable resources in order to build secure health systems and services. special anenti_on. should ~ afforded hiv/aids in view of the unprecedented challenge that this epidemic poses to africa s economic and social development and to health services on the continent. methods: a review of the literature led us to construct three simple models and a composite model of exposure to traffic. the data were collected with the help of a daily diary of travel activities using a sample of cyclists who went to or come back from work or study. to calculate the distance, the length of journey, and the number of intersections crossed by a cyclist different geographic information systems (gis) were operated. statistical analysis was used to determine the significance between a measure of exposure on the one hand, and the sociodemographic characteristics of the panicipants or their geographic location on the other hand. restlltj: our results indicate that cyclists were significantly exposed to road accidents, no matter of where they live or what are their sociodemographic characteristics. we also stress the point that the fact of having been involved in a road accident was significantly related to the helmet use, but did not reduce the propensity of the cyclists to expose themselves to the road hazards. condlllion: the efforts of the various authorities as regards road safety should not be directed towards the reduction of the exposure of the vulnerable users, but rather towards the reduction of the dangers to which they could face. keywords: cyclist, daily diary of activities, measures of exposure to traffic, island of montreal. p - (a) intra urban disparities and environmental health: some salient features of nigerian residential neighbourhoods olumuyiwa akinbamijo intra urban disparities and environmental health: some salient features of nigerian residential neighbourhoods abstract urbanization panicularly in nigerian cities, ponends unprecedented crises of grave dimensions. from physical and demographic viewpoints, city growth rates are staggering coupled with gross inabilities to cope with the consequences. environmental and social ills associated with unguarded rapid urbanization characterize nigerian cities and threaten urban existence. this paper repons the findings of a recent study of the relationship between environmental health across inrraurban residential communities of akure, south west nigeria. it discuses the typical urbanization process of nigerian cities and its dynamic spatial-temporal characteristics. physical and socio-demographic attributes as well as the levels and effectiveness of urban infrastructural services are examined across the core residential districts and the elite residential layouts in the town. the incidence rate of cenain environmentally induced tropical diseases across residential neighborhoods and communes is examined. salient environmental variables that are germane to health procurement in the residential districts, incidence of diseases and diseases parasitology, diseases prevention and control were studied. field data were subjected to analysis ranging from the univariate and bivariate analysis. inferential statistics using the chi-square test were done to establish the truthfulness of the guiding hypothesis. given the above, the study affirms that there is strong independence in the studied communities, between the environment and incidence of diseases hence health of residents of the town. this assertion, tested statistically at the district levels revealed that residents of the core districts have very strong independence between the environment and incidences of diseases. the strength of this relationship however thins out towards the city peripheral districts. the study therefore concludes that since most of the city dwellers live in urban deprivation, urban health sensitive policies must be evolved. this is to cater for the urban dwellers who occupy fringe peripheral sites where the extension of facilities often times are illegally done. urban infrastructural facilities and services need be provided as a matter of public good for which there is no exclusive consumption or access even for the poorest of the urban poor. many suffer from low-self esteem, shame and guilt about their drug use. in addition, they often lack suppon or encounter opposition from their panners, family and friends in seeking treatment. these personal barriers are compounded by fragmented addiction, prenatal and social care services, inflexible intake systems and poor communication among sectors. the experience of accessing adequate care between services can be overwhelming and too demanding. the toronto centre for substance use in pregnancy (t-cup) is a unique program developed to minimize barriers by providing kone-stop" comprehensive healthcare. t-cup is a primary care based program located in the department of family medicine at st. joseph\'s health centre, a community teaching hospital in toronto. the interdisciplinary staff provides prenatal and addiction services, case management, as well as care of newborns affected by substance use. regular care plan meetings are held between t-cup, labour and delivery nurses and social workers in the y poster sessions maternity and child care program. t-cup also connects "'.omen with. inpatient treatment programs and community agencies such as breaking the cycle, an on-site counselmg group for pregnant substance users. · f · d d h ith method: retrospective chart review, qualitative patient ~ans action stu ~· an ea care provider surveys are used to determine outcomes. primary outcomes mclude changes m maternal su~tance use, psychosocial status and obstetrical complications (e.g. pre-rupture of membrane, pre-eclampsia, placen· ral abruption and hemorrhage). neonatal measures ~~nsisted of .bir~h pa_rame~ers, length of h~spital st.ay and complications (e.g. feral distress, meconium stammg, resuscitation, aund ce, hypoglycemia, seventy of withdrawal and treatment length). chart review consisted of all t-cup patients who met clinical cri· reria for alcohol or drug dependence and received prenatal and intra-partum care at st. joseph's from october to june . participants in the qualitative study included former and current t-cup patients. provider surveys were distributed on-site and to a local community hospital. raulb: preliminary evaluation has demonstrated positive results. treatment retention and satisfaction rates were high, maternal substance use was markedly reduced and neonatal outcomes have shown to be above those reported in literature. conclusion: this comprehensive, primary care model has shown to be optimal in the management of substance use in pregnancy and for improving neonatal outcomes. future research will focus on how this inexpensive program can be replicated in other health care settings. t-cup may prove to be the optimal model for providing care to pregnant substance users in canada. lntrod ction: cigarette smoking is one of the most serious health problems in taiwan. the prevalence of smoking in is . % in males . % in females aged years and older. although the government of taiwan passed a tobacco hazards control act in , it has not been strongly enforced in many places. therefore, community residents have often reported exposure of second hand smoke. the purpose of the study was to establish a device to build up more smoke-free environments in the city of tainan. methods: unique from traditional intervention studies, the study used a healthy city approach to help build up smoke-free environments. the major concept of the approach is to build up a healthy city platform, including organizing a steering committee, setting up policies and indicators, creating intersectoral collaboration, and increasing community participation. first, more than enthusiastic researchers, experts, governmental officers, city counselors and community leaders in tainan were invited in the healthy city committee. second, smoke-free policies, indicators for smoke-free environments, and mechanisms for inter-departmen· tal inspections were set up. third, community volunteers were recruited and trained for persuading related stakeholders. lastly, both penalties and rewards were used for help build up the environments. raults: aher two-year ( aher two-year ( - execution of the project, the results qualitatively showed that smoke-free environments in tainan were widely accepted and established, including smoke-free schools, smoke-free workpla~es, smoke-free households, smoke-free internet shops, and smoke-free restaurants. smoke~s were. effectively educated not to smoke in public places. community residents including adults and children m the smoke-free communities clearly understand the adverse effects of environmental tobacco smoke and actively participated anti-smoking activities. conclruions: healthy city platform is effective to conquer the barrier of limited anti-smoking rc:sources. nor. only can it enlar:ge community actions for anti-smoking campaigns, but also it can provide par_merships for collaboratjon. by establishing related policies and indicators the effects of smoke· free environments can be susta ·ned a d th · · · ' · n e progression can be monitored m a commuruty. these issues are used ~· oi::c it~ goals, weuha identifies issues that put people's health at risk. presently, team com~u:c: ran ee~tion !earns. (iats) that design integrative solutions ~tesj'°~ g om six to fifteen members. methods in order to establish wo-poster sessions v projects for weuha, the following approach was undertaken: i. a project-polling template was created and sent to all members of the alliance for their input. each member was asked to identify thdr top two population groups, and to suggest a project on which to focus over a - month period for each identified population. . there was a % response to the poll and the top three population groups were identified. data from the toronto community health profile database were utilized to contextualize the information supplied for these populations. a presentation was made to the steering committee and three population-based projects were selected, leaders identified and iats formed. three population-based projects: the population-based projects and health care issues identified are: newcomer prenatal uninsured women; this project will address the challenges faced by providers to a growing number of non-insured prenatal women seeking care. a service model where the barrier of "catchments" is removed to allow enhanced access and improved and co-ordinated service delivery will be pilot-tested. children/obesity/diab etes: using a health promotion model this team will focus on screening, intervention, and promoting healthy lifestyles (physical activity and nutrition) for families as well as for overweight and obese children. seniors health promotion and circle of discharge: this team will develop an early intervention model to assist seniors/family unit/caregivers in accessing information and receiving treatment/care in the community. the circle of discharge initiative will address ways of utilizing community supports to keep seniors in the community and minimize readmissions to acute care facilities. results/expected outcomes: coordinated and enhanced service delivery to identified populations, leading to improved access, improved quality of life, and health care for these targeted populations. introduction: basic human rights are often denied to high-risk populations and people living with hiv/aids. their rights to work and social security, health, privacy, non discrimination, liberty and freedom of movement, marriage and having a family have been compromised due to their sero-positive status and risk of being positive. the spread of hiv/aids has been accelerating due to the lack of general human rights among vulnerable groups. to formulate and implement effective responses needs dialogue and to prevent the epidemic to go underground barriers like stigma need to be overcome. objective: how to reduce the situation of stigma, discrimination and human rights violations experienced by people living with hiv/aids and those who are vulnerable to hiv/aids. methodology and findings: consultation meetings were strm.-rured around presentations, field visits, community meetings and group work to formulate recommendations on how govt and ngos/cbos should move forward based on objective. pakistan being a low prevalence country, the whole sense of compl;u:enc.:y that individuals are not subject to situations of vulnerable to hiv is the major threat to an explosion in th•· epidemic, therefore urgent measures are needed to integrate human rights issues from the very start of the response. the protection and promotion of human rights in an integral component of ;tll responses to the hiv/aids epidemic. it has been recognized that the response to hiv/aios must he multi sectoral and multi faceted, with each group contributing its particular expertise. for this to occur along with other knowlcdg<" more information is required in human rights abuses related to hiv/ aids in a particular scenario. the ~·on sultarion meetings on hiv/aids and human rights were an exemplary effort to achieve the same ohj<..:tivc. recommendations: the need for a comprehensive, integrated and a multi-sectoral appro;u.:h in addressing the issue of hiv/aids was highlighted. the need social, cultural and religious asp•·ct' to he: prominently addressed were identified. it was thought imperative measures even in low prevalence countries. education has a key role to play, there is a need for a code of ethics for media people and h<"alth care providers and violations should be closely monitored and follow up action taken. p - (c) how can community-based funding programs contribute to building community capacity and how can we measure this elusive goal? mary frances maclellan-wright, brenda cantin, mary jane buchanan, and tammy simpson community capacity building is recognized by the public health agency of canada (phac) as an important strategy for improving the overall health of communities by enabling communities to addre~s priority issues such as social and economic determinants of health. in / phac.:, alberta/nwf region's population health fund (phf) supported community-based projects to build community capacity on or across the determinants of health. specifically, this included creating accessible and sup· portive social and physical environments as well as creating tools and processes necessary for healthy policy development and implementation. the objective of this presentation is to highlight how the community capacity building tool, developed by phac ab/nwf region, can demonstrate gains in v poster sessions · · the course of a pror· ect and be used as a reflective tool for project planning and community capacity over . . . . i · a art of their reporting requirements, pro ect sites completed the community caparny eva uanon. s p . . th t i ii i'd d . building tool at the beginning and end of their ~ne-year prorect. e oo ~o ects va an reliable data in the context of community-based health prorects. developed through a vigorous ~nd collabora ve research process, the tool uses plain languag~ to expl~re nine key f~atures o~ commuruty cap~city with 't ch with a section for contextual information, of which also mdude a four-pomt raong ems, ea f fu d · scale. results show an increase in community capacity over the course o the nde prorects. pre and post aggregate data from the one-year projects measure~ statistic.ally si~n~ficant changes for of the scaled items. projects identified key areas of commumty capacity bmldmg that needed strengthemng, such as increasing participation, particularly among people with low incomes; engaging community members in identifying root causes; and linking with community groups. in completing the tool, projects examined root causes of the social and economic determinants of health, thereby exploring social justice issues related to the health of their community. results of the tool also served as a reflec· cion on the process of community capacity building; that is, how the project outcomes were achieved. projects also reported that the tool helped identify gaps and future directions, and was useful as a project planning, needs assessment and evaluation tool. community capacity building is a strategy that can be measured. the community capacity building tool provides a practical means to demonstrate gains in community capacity building. strengthening the elements of community capacity building through community-based funding can serve as building blocks for addressing other community issues. needs of marginalized crack users lorraine barnaby, victoria okazawa, barb panter, alan simpson, and bo yee thom background: the safer crack use coalition of toronto (scuc) was formed in in response to the growing concern for the health and well-being of marginalized crack users. a central concern was the alarm· ing hepatitis c rate ( %) amongst crack smokers and the lack of connection to prevention and health ser· vices. scuc is an innovative grassroots coalition comprised of front-line workers, crack users, researcher! and advocates. despite opposition and without funding, scuc has grown into the largest crack specific harm reduction coalition in canada and developed a nationally recognized sarer crack kit distribution program (involving community-based agencies that provide outreach to users). the success of our coalition derives from our dedication to the issue and from the involvement of those directly affected by crack use. setting: scuc's primary service region is greater toronto, a diverse, large urban centre. much ofour work is done in areas where homeless people, sex trade workers and drug users tend to congregate. recently, scuc has reached out to regional and national stakeholders to provide leadership and education. mandate: our mandate is to advocate for marginalized crack users and support the devdopmentof a com.p.rehensive harm reduction model that addresses the health and social needs facing crack users; and to fac htare the exchange of information between crack users, service providers, researchers, and policy developers across canada. owrview: the proposed workshop will provide participants with an overview of the devdopment of scuc, our current projects (including research, education, direct intervention and consultation), our challenge~ and s~ccesses and the role of community development and advocacy within the coalition. pre-senter~ will consist of community members who have personal crack use experience and front-line work· ers-, sc.uc conducted a community-based research project (toronto crack users perspectives, ) , in w~ich s focus groups with marginalized crack users across toronto were conducted. participants iden· t f ed health and social issues affecti h b · · · d " red . . ng t em, arrsers to needed services, personal strategies, an oue recommendations for improved services. presenters will share the methodology, results and recommen· datmns resulting from the research project. conc/usio": research, field observations and consultations with stakeholders have shown that cradck shmoke~s are at an. increased risk for sexually transmitted infections hiv/aids hepatitis c, tb an ot er serious health issues health · ff, · ' ' · · . · issues a ectmg crack users are due to high risk behavmurs, socio· economic factors, such as homeless d. · · · · d · . . ness, scrsmmat on, unemployment, violence incarceraoons, an soc a so at on, and a lack of comprehe · h i h · ' ns ve ea t and social services targeting crack users. · · sinct · s, owever arge remains a gross underesurnaoon. poster sessions v these are hospital-based reports and many known cases go unreported. however teh case, young age at first intercourse, inconsistent condom use and multiple partnersplace adolescents at high risks for a diverse array of stls, including hiv. about % of female nigerian secondary school students report initiating sexual intercourse before age years. % of nigerian female secondary school students report not using a condom the last time they had sexual intercourse. more than % of urban nigerian teens report inconsistent condom use. methods: adolescents were studied, ages to , from benin city in edo state. the models used were mother-daughter( ), mother -son( ), father -son ( ), and father-daughter( ). the effect of parent-child sexual communicationat baseline on child\'s report of sexual behavior, to months later were studied. greater amounts of sexual risk communication were asociated with markedly fewer episodes of unprotected sexual intercourse, reduced number of sexual partners and fewer episodes of unprotected sexual intercourse. results: this study proved that parents can exert more influence on the sexual knowledge attitudes and practise of their adolescent children through desired practises or rolemodeling, reiterating their values and appropriate monitoring of the adolescents\' behavior. they also stand to provide information about sexuality and various sexual topics. parental-child sexual communication has been found to be particularly influential and has been associated with later onset of sexual initiation among adolescents, less sexual activity, more responsible sexual attitudes including greater condom use, self efficacy and lower self -reported incidence of stis. conclusions: parents need to be trained to relate more effectively with their children/wards about issues related to sex and sexuality. family -based programs to reduce sexual risk-taking need to be developed. there is also the need to carry out cross-ethnicaland cross-cultural studies to identify how parent-child influences on adolescent sexual risk behavior may vary in different regions or countries, especially inthis era of the hiv pandemic. introduction: public health interventions to identify and eliminate health disparities require evidence-based policy and adequate model specification, which includes individuals within a socioecological context, and requires the integration of biosociomedical information. multiple public and private data sources need to be linked to apportion variation in health disparities ro individual risk factors, the health delivery system, and the geosocial environment. multilevel mapping of health disparities furthers the development of evidence-based interventions through the growth of the public health information network (phin-cdc) by linking clinical and population health data. clinical encounter data, administrative hospital data, population socioenvironmental data, and local health policy were examined in a three-level geocoded multilevel model to establish a tracking system for health disparities. nj has a long established political tradition of "home rule" based in elected municipal governments, which are responsible for the well-being of their populations. municipalities are contained within counties as defined by the us census, and health data are linked mostly at the municipality level. marika schwandt community organizers from the ontario coaliti~n again~t pove~, .along ":ith ~edical practitioners who have endorsed the campaign and have been mvolved m prescnbmg special diet needs for ow and odsp recipients, will discuss the raise the rates campaign. the organizati~n has used a special diet needs supplement as a political tool, meeting the urgent needs o.f .poor ~ople m toront~ while raising the issues of poverty as a primary determinant of health and nutrtnous diet as a preventative health mea· sure. health professionals carry the responsibility to ensure that they use all means available to them to improve the health of the individuals that they serve, and to prevent future disease and health conditions. most health practitioners know that those on social assistance are not able to afford nutritious foods or even sufficient amounts of food, but many are not aware of the extra dietary funds that are available aher consideration by a health practitioner. responsible nurse practitioners and physicians cannot, in good conscience, ignore the special needs diet supplement that is available to all recipients of welfare and disabiliry (ow and odsp). a number of toronto physicians have taken the position that all clients can justifiably benefit from vitamins, organic foods and high fiber diets as a preventative health measure. we know that income is one of the greatest predictors of poor health. the special needs diet is a health promotion intervention which will prevent numerous future health conditions, including chronic conditions such as cardiovascular disease, cancer, diabetes and osteoporosis. many communiry health centres and other providers have chosen to hold clinics to allow many patients to get signed up for the supplement at one time. initiated by the ontario coalition against poverty, these clinics have brought together commu· niry organizers, community health centers, health practitioners, and individuals, who believe that poverty is the primary determinant of poor health. we believe that rates must be increased to address the health problems of all people on social assistance, kids, elders, people with hiv/aids -everyone. even in the context of understaffing, it could be considered a priority activity that has potentially important health promotion benefits. many clients can be processed in a two hour clinic. most providers find it a very interesting, rewarding undertaking. in the ontario coalition for social justice found that a toronto family with two adults and two kids receives $ , . this is $ , below the poverty line. p - (c) the health of street youth compared to similar aged youth beth hayhoe and ruth ewert . lntrod~on: street youth are at an age normally associated with good health, but due to their risky ~hav ours and th~ conditions in which they live, they experience health conditions unlike their peer~ an more stable env r~nments. in addition, the majority of street youth have experienced significant physical, sexual ~nd em.ot onal abuse as younger children, directly impacting many of the choices they make around their physical and emotional health. we examined how different their health really is. . , methodl: using a retrospective analysis of the years of data gathered from yonge street mis· ~ • evergreen health centre, the top conditions of youth were examined and compared with national tren~s for similar aged youth. based on knowledge of the risk factors present in the group, rea· sons for the difference were examined. d' ~its: street youth experience more illness than other youth their age and their illnesses can bt . irect t ·~kc~ to the. conditions in which they live. long-term impacts of abus~ contribute to such signif· ~~nt t e t d~slpl air that youth may voluntarily engage in behaviours or lack of self care in the hope at t cir ve~ w perhaps come to a quicker end. concl non: although it has ion b k h th' dy clearly shows d'fi . h g ee~ no~n t at poverty negatively affects health, ~siu be used to make ; erence m t .e health of this particular marginalized population. the infonnanon can relates to th . ecommendatio.ns around public policy that affects children and youth, especially as it e r access to appropriate health care and follow up. p - (cl why do urban children · b gt . tarek hussain an adesh die: how to save our children? the traditional belief that urban child alid. a recent study (dhs d fr r~n are better off than rural children might be no longer v urban migrants are highata th om h c~untn~s i demonstrates that the child survival prospects of rural· er an t ose m their r j · · ·grants. in bangladesh, currently million ~r~ ~ gm and lower than those of urban non-idi million. health of the urban ~ p~e are hvmg m urban area and by the year , it would be so the popu at on s a key a eals that urban poor have the worse h h . concern. recent study on the urban poor rev ea t situation than the nation as a whole. this study shows that infant poster sessions v mortality among the urban poor as per thousand, which are above the rural and national level estimates. the mortality levels of the dhaka poor are well above those of the rest of the city's population but much of the difference in death rates is explained by the experience of children, especially infants. analyzing demographic surveillance data from a large zone of the city containing all sectors of the population, research showed that the one-fifth of the households with the least possessions exhibited u child mortality almost three times as high as that recorded by the rest of the population. why children die in bangladesh? because their parents are too poor to provide them with enough food, clean water and other basic needs to help them avoid infection and recover from illness. researchers believed that girls are more at risk than boys, as mothers regularly feed boys first. this reflects the different value placed on girls and boys, as well as resources which may not stretch far enough to provide for everyone. many studies show that housing conditions such as household construction materials and access to safe drinking water and hygienic toilet facilities are the most critical determinants of child survival in urban areas of developing countries. the present situation stressed on the need for renewed emphasis on maternal and child healthcare and child nutrition programs. mapping path for progress to save our children would need be done strategically. we have the policies on hand, we have the means, to change the world so that every child will survive and has the opportunity to develop himself fully as a healthy human being. we need the political will--courage and determination to make that a reality. p - (c) sherbourne health centre: innovation in healthcare for the transgendered community james read introduction: sherbourne health centre (shc), a primary health care centre located in downtown toronto, was established to address health service gaps in the local community. its mission is to reduce barriers to health by working with the people of its diverse urban communities to promote wellness and provide innovative primary health services. in addition to the local communities there are three populations of focus: the lesbian, gay, bisexual, transgendered and transexual communities (lgbtt); people who are homeless or underhoused; and newcomers to canada. shc is dedicated to providing health services in an interdisciplinary manner and its health providers include nurses, a nurse practitioner, mental health counsellors, health promoters, client-resource workers, and physicians. in january shc began offering medical care. among the challenges faced was how to provide responsive, respectful services to the trans community. providers had considerable expertise in the area of counselling and community work, but little in the area of hormone therapy -a key health service for those who want to transition from one gender to another. method: in preparing to offer community-based health care to the trans community it was clear that shc was being welcomed but also being watched with a critical eye. trans people have traditionally experienced significant barriers in accessing medical care. to respond to this challenge a working group of members of the trans community and health providers was created to develop an overall approach to care and specific protocols for hormone therapy. the group met over a one year period and their work culminated in the development of medical protocols for the provision of hormone therapy to trans individuals. results: shc is currently providing health care to registered clients who identify as trans individuals (march ) through primary care and mental health programs. in an audit of shc medical charts (january to september ) female-to-male (ftm) and male-to-female (mtf) clients were identified. less than half of the ftm group and just over two-thirds of the mtf group presented specifically for the provision of hormones. based on this chart audit and ongoing experience shc continues to update and refine these protocols to ensure delivery of quality care. conclusion: this program is an example of innovative community-based health delivery to a population who have traditionally faced barriers. shc services also include counselling, health promotion, outreach and education. p - (c) healthy cities for canadian women: a national consultation sandra kerr, kimberly walker, and gail lush on march , the national network on environment and women's health held a pan-canadian consultation to identify opportunities for health research, policy change, and action. this consultation also worked to facilitate information sharing and networking between canadian women working as urban planners, policy makers, researchers, and service workers on issues pertaining to the health of women living in canadian cities. methods: for this research project, participants included front-line service workers, policy workers, researchers, and advocates from coast to coast, including francophone women, women with disabilities, racialized women, and other marginalized groups. the following key areas were selected as topics for du.bnes i alto kading .:auk of end·sugr ieaal clileue ia singapore, accounting for more than so% of new can singapore (nkfs) to embark on a prevention program (pp) empo~r d ahc j u f dieir condition bttter, emphasizing education and disease sdf·managemen lkilla a. essennal camponenn of good glycaemic control. we sought explore the effects of a pecialijed edu.:a on pro· pun od glycacmic conuol, as indicated by, serum hba ic values budine serum hba ic values were determined before un<k-rgmng the education progr ;am, which couisted of comprehensive dietary advice, ideal weight goals, and improving lipid profiln. serum hbaic values were obtained ar , , months later to determine impact of the program. analy • wu done uling paired !·tests significant reduction in hba le levels from ro month• was observed in females, chinese race, older age group(> so yean). ohew-ibmi ~ .nwm , wai hip ratio> l),up to primary and above secondary level education and those having om urine iclt showed that increasing hbalc levels ( ) had increasing urmary protein ( .± ; . ±i ih so± ) and crearinine (s .s ± s ± ; ioi± s) levels fbg rnults showed that the management nf d abetn m the nkfs preven· tion programme is effec;rive. results also indicated har hba le leve have a linnr trend wnh unnary protein and creatinine which are imponant determinants of renal diseate tal family-focused cinical palbway promoce politivc outcollln for ua inner city canu allicy ipmai jerrnjm care llctivirits in preparation for an infanr'' dilchargr honlr, and art m endnl lo improve effi.:k'fl.:tn of c.are. lere i paucity of tttran:h, and inconsi trncy of rnulta on ht-•m!*- of f m ly·fc"-'uw d nm a: to determinr whrthrr implrmentation of family.focuted c:pt n ntnn.tt.tl unit w"n mg an inner city ;ommunity drcl't'aki leftarh of lf•y (i.osi and rromclll'i family uo•fkllon and rt. j nest for dikhargr. md odt: family-focuk"d cpi data wm coll«ted for all infant• horn btrwttn and wft"k• t"lal mi atr who wrtt . dm ed to the ntonatal unit lmgdl of -.y . n. . day'o p c o.osi ind pma . d•mr., ho.nr . t . n. . ± i. i wb, p < o.os) wett n« fiamly f.lfrt n the pre.(]' poup. ~ .fxtmon icofn for famihn wrre high. and families noctd thc:y wnr mott prepued to ah thrar t..lby "'-· thett was .a cosi uving of s , (cdn) per patient d teharpd home n the pmi-cp poap c.-pated the p"''lfoup· cortclaion· lmplrmrnr.rion of family·foanrd c:p. in a nrona . i umt tc"fyidi an nnn an com· muniry decre.ned length of'"'" mft with a high dcgrft of family uujamon, and wrre coll~nt at least % percent of the kathmandu population lives in slum like conditions with poor access to basic health services. in these disadvantaged areas, a large proportion of children do not receive treatment due to inaccessibility to medical services. in these areas, diarrhea, pneumonia, and measles, are the key determinants of infant mortality. protein energy malnutrition and vitamin a deficiency persists and communicable diseases are compounded by the emergence of diseases like hiv/aids. while the health challenges for disadvantaged populations in kathmandu are substantial, the city has also experienced various forms of innovative and effective community development health programs. for example, there are community primary health centers established by the kathmandu municipality to deliver essential health services to targeted communities. these centers not only provide equal access to health services to the people through an effective management system but also educate them hy organizing health related awareness programs. this program is considered one of the most effective urban health programs. the paper/presentation this paper will review large, innovative, and effective urhan health programs that are operating in kathmandu. most of these programs are currently run by international and national ngos a) early detection of emerging diseases in urban settings through syndromic surveillance: data pilot study kate bassil of community resources, and without adequate follow-up. in november shelter pr.oviders ~et with hospital social workers and ccac to strike a working group to address some of th~ issues by mcre.asing knowledge among hospital staff of issues surrounding homelessness, and to build a stro?g workmg relationship between both systems in hamilton. to date the hswg has conducted four w~lkmg to~ of downtown shelters for hospital staff and local politicians. recently the hswg launched its ·~ool.k t for staff working with patients who are homeless', which contains community resources and gu dehnes to help with effective discharge plans. a scpi proposal has been submitted to incre~se the capacity of the hswg to address education gaps and opportunities with both shelters and hospitals around homelessness and healthcare. the purpose of this poster presentation is to share hamilton's experience and learnings with communities who are experiencing similar issues. it will provide for intera~tion around shared experiences and a chance to network with practitioners across canada re: best practices. introduction and objectives: canadians view health as the biggest priority for the federal government, where health policies are often based on models that rely on abstract definitions of health that provide little assistance in the policy and analytical arena. the main objectives of this paper are to provide a functional definition of health, to create a didactic model for devising policies and determining forms of intervention, to aid health professionals and analysts to strategize and prioritize policy objectives via cost benefit analysis, and to prompt readers to view health in terms of capacity measures as opposed to status measures. this paper provides a different perspective on health, which can be applied to various applications of health such as strategies of aid and poverty reduction, and measuring the health of an individual/ community/country. this paper aims to discuss theoretical, conceptual, methodological, and applied implications associated with different health policies and strategies, which can be extended to urban communities. essentially, our paper touches on the following two main themes of this conference: •health status of disadvantaged populations; and •interventions to improve the health of urban communities.methodology: we initially surveyed other models on this topic, and extrapolated key aspects into our conceptual framework. we then devised a theoretical framework that parallels simple theories of physkal energy, where health is viewed in terms of personal/societal health capacities and effort components.after establishing a theoretical model, we constructed a graphical representation of our model using selfrated health status and life expectancy measures. ultimately, we formulated a new definition of health, and a rudimentary method of conducting cost benefit analysis on policy initiatives. we end the paper with an application example discussing the issues surrounding the introduction of a seniors program.results: this paper provides both a conceptual and theoretical model that outlines how one can go about conducting a cost-benefit analysis when implementing a program. it also devises a new definition and model for health barred on our concept of individual and societal capacities. by devising a definition for health that links with a conceptual and theoretical framework, strategies can be more logically constructed where the repercussions on the general population are minimized. equally important, our model also sets itself up nicely for future microsimulation modeling and analysis.implications: this research enhances one's ability to conduct community-based cost-benefit analysis, and acts as a pedagogical tool when identifying which strategies provide the best outcome. p - (a) good playgrounds are hard to find: parents' perceptions of neighbourhood parks patricia tucker, martin holmes, jennifer irwin, and jason gilliland introduction: neighbourhood opportunities, including public parks and physical activity or sports fields hav~ been. iden.tified as correlates to physical activity among youth. increasingly, physical activity among children s bemg acknowledged as a vital component of children's lives as it is a modifiable determinant of childh~d obesity. children's use of parks is mainly under the influence of parents; therefore, the purpose of this study was to assess parents' perspectives of city parks, using london ontario as a case study.m~~: this qualitative study targeted a heterogeneous sample of parents of children using local parks w thm london. parents with children using the parks were asked for minutes of their time and if willing, a s.hort interview was conducted. the interview guide asked parents for their opinion 'of city parks, particularly the one they were currently using. a sample size of parents is expected by the end of the summer.results: preliminary findings are identifying parents concern with the current jack of shade in local parks. most parents have identified this as a limitation of existing parks, and when asked what would make the parks better, parents agree that shade is vital. additionally, some parents are recognizing the v poster sessions focused discussions during the consultation: . women in _poverty . women with disability . immi· grant and racialized women . the built and _physica_l environment. . . . . r its· participants voiced the need for integration of the following issues withm the research and policy :::na; t) the intersectional nature of urban women's health i~sues wh~ch reflects the reality of women's complex lives ) the multisectional aspect of urban wo_m~n s health, ss~es, which reflects the diversity within women's lives ) the interse~roral _dynamics within _womens hves and urban health issues. these concepts span multiple sectors -mdudmg health, educat n, and economics -when leveraging community, research, and policy support, and engaging all levels of government.policy jmplicatiom: jn order to work towards health equity for women, plans for gender equity must be incorporated nationally and internationally within urban development initiatives: • reintroduce "women" and "gender" as distinct sectors for research, analysis, advocacy, and action. •integrate the multisectional, intersectional, and intersecroral aspects of women's lives within the framework of research and policy development, as well as in the development of action strategies. • develop a strategic framework to house the consultation priorities for future health research and policy development (for example, advocacy, relationship building, evidence-based policy-relevant research, priority initiatives}.note: research conducted by nnewh has been made possible through a financial contribution from health canada. the views expressed herein do not necessarily represent the views of health canada.p - (c) drugs, culture and disadvantaged populations leticia folgar and cecilia rado lntroducci n: a partir de un proyecto de reducci n de daiios en una comunidad urbana en situ· aci n de extrema vulnerahilidad surge la reflexion sobre el lugar prioritario de los elementos sociocuhurales en el acceso a los servicios de salud de diferentes colectivos urbanos. las "formas de hacer, pensar y sentir" orientan las acciones y delimitan las posibilidades que tienen los individuos de definir que algo es o no problema, asf como tambien los mecanismos de pedido de ayuda. el analisis permanenre del campo de "las culturas cotidianas" de los llamados "usuarios de drogas" aporta a la comprension de la complejidad del tema en sus escenarios reales, y colabora en los diseiios contextualizados de politicas y propuestas socio-sanitarias de intervenci n, tornandolas mas efectivas.mitodos: esta experiencia de investigaci n-acci n que utiliza el merodo emografico identifica elementos socio estructurales, patrones de consumo y profundiza en los elementos socio-simb icos que estructuran los discursos de los usuarios, caracterizandolos y diferenciandolos en tanto constitutivos de identidades socia les que condicionan la implementaci n del programas de reduccion de daiios.resultados: los resultados que presentaremos dan cuenta de las caracteristicas diferenciadas v relaciones particulares ~ntre los consumidores de drogas en este contexto espedfico. a partir de este e~tudio de caso se mtentara co ? enzar a responder preguntas que entendemos significativas a la hora de pensar intcrvcnciones a la med da de poblaciones que comparten ciertas caracteristicas socio-culturales. (cuales serian las .motivaciones para el cambio en estas comunidades?, cque elementos comunitarios nos ayudan a i:nnstnur dema~~a? • cque tenemos para aprender de las "soluciones" que ellos mismos encuenrran a los usos problemat cos? methods: our study was conducted by a team of two researchers at three different sites. the mapping consisted of filling in a chart of observable neighbourhood features such as graffiti, litter, and boarded housing, and the presence or absence of each feature was noted for each city block. qualitative observations were also recorded throughout the process. researchers analyzed the compiled quantitative and qualitative neighbourhood data and then analyzed the process of data collection itself.results: this study reveals the need for further research into the effects of physical environments on individual health and sense of well-being, and perception of investment in neighbourhoods. the process reveals that perceptions of health and safety are not easily quantified. we make specific recommendations about the mapping methodology including the importance of considering how factors such as researcher social location may impact the experience of neighbourhoods and how similar neighbourhood characteristics are experienced differently in various spaces. further, we discuss some of the practical considerations around the mapping exercise such as recording of findings, time of day, temperature, and researcher safety.conclusion: this study revealed the importance of exploring conceptions of health and well-being beyond basic physical wellness. it suggests the importance of considering one's environment and one's own perception of health, safety, and well-being in determining health. this conclusion suggests that attention needs to be paid to the connection between the workplace and the external environment it is situated in. the individual's workday experience does not start and stop at the front door of their workplace, but rather extends into the neighbourhood and environment around them. our procedural observations and recommendations will allow other researchers interested in the effect of urban environments on health to consider using this innovative methodology. introduction: responding ro protests against poor medical attention for sexually assaulted women and deplorable conviction rates for sex offenders, in the late s, the ontario government established what would become over hospital-based sexual assault care and treatment centres (sactcs) across the province. these centres, staffed around the clock with specially trained heath care providers, have become the centralized locations for the simultaneous health care treatment of and forensic evidence collection from sexually assaulted women for the purpose of facilitating positive social and legal outcomes. since the introduction of these centres, very little evaluative research has been conducted to determine the impact of this intervention. the purpose of our study was to investigate it from the perspectives of sexually assaulted women who have undergone forensic medical examinations at these centres.method: women were referred to our study by sactc coordinators across ontario. we developed an interview schedule composed of both closed and open-ended questions. twenty-two women were interviewed, face-to-face. these interviews were approximately one-to-two hours in length, and were transcribed verbatim. to date, have been analyzed for key themes.results: preliminary findings indicate that most women interviewed were canadian born ( 'yo), and ranged in age from to years. a substantial proportion self-identified as a visible minority ( 'x.). approximately half were single or never married ( %) and living with a spouse or family of origin ( %). most were either students or not employed ( %). two-thirds ( %) had completed high school and onethird ( %) was from a lower socio-economic stratum. almost two-fifths ( %) of women perceived the medical forensic examination as revictimizing citing, for example, the internal examination and having blood drawn. the other two-thirds ( %) indicated that it was an empowering experience, as it gave them a sense of control at a time when they described feeling otherwise powerless. most ( %) women stated that they had presented to a centre due to health care concerns and were very satisfied ( % ) with their experiences and interactions with staff. almost all ( %) women felt supported and understood.conclusions: this research has important implications for clinical practice and for appropriately addressing the needs of sexually assaulted women. what is apparent is that continued high-quality medical attention administered in the milieu of specialized hospital-based services is essential. at the same time, we would suggest that some forensic evidence collection procedures warrant reevaluation. the study will take an experiential, approach by chroruclmg the impa~ of the transition f m the streets to stabilization in a managed alcohol program through the techruque of narrative i~:uiry. in keeping with the shift in thinking in the mental health fie!~ ~his stu~y is based on a paradigm of recovery rather than one of pathology. the "inner views of part c pants hves as they portray their worlds, experiences and observations" will be presented (charm~z, , ~· ~)-"i?e p~ of the study is to: identify barriers to recovery. it will explore the exj?cnence of ~n~t zanon pnor to entry into the program; and following entry will: explore the meanmg ~nd defirutto~s of r~overy ~~d the impact of the new environment and highlight what supports were instrumental m movmg pan apants along the recovery paradigm.p -st (a) treating the "untreatable": the politics of public health in vancouver's inner city introdudion: this paper explores the everyday practices of therapeutic programs in the treadnent of hiv in vancouver's inner city. as anthropologists have shown elsewhere, therapeutic programs do not siinply treat physical ailments but they shape, regulate and manage social lives. in vancouver's inner city, there are few therapeutic options available for the treatment of -ilv. public health initiatives in the inner city have instead largely focused on prevention and harm reduction strategies such as needle exchange programs, safe injection sites, and safer-sex education. epidemiological reports suggest that less than a quarter of those living with hiv in the downtown eastside (dtes) are taking antiretroviral therapies raising critical questions regarding the therapeutic economy of antiretrovirals and rights to health care for the urban poor.methods: this paper is drawn from ethnographic fieldwork in vancouver's otes neighborhood focusing on therapeutic programs for hiv treatment among "hard-to-reach" populations. the research includes participant-observation at inner city health clinics specializing in the treatment of hiv; semi· structured interviews with hiv positive participants, health care professionals providing hiv treatment, and administraton working in the field of inner city public health; and, lastly, observation at public meetings and conferences surrounding hiv treatment.r.awlts: hiv prevention and treatment is a central concern in the lives of many residents living in the inner city -although it is just one of many health priorities afflicting the community. concerns about drug resistance, cost of antiretrovirals, and illicit drug use means that hiv therapy for most is characterized by the daily observation of their medicine ingestion at health clinics or pharmacies. daily observed treatment (dot) is increasingly being adopted as a strategy in the therapeutic management of "untreatable" populations. dot programs demand a particular type of subject -one who is "compliant" to the rules and regimes of public health. over emphasis on "risky practices," "chaotic lives," and "~dh~rence" preve~ts the public health system from meaningful engagement with the health of the marginalized who continue to suffer from multiple and serious health conditions and who continue to experience considerable disparities in health.~ the ~ffec~s of hiv in the inner city are compounded by poverty, laclc of safe and affordable houamg, vanous llegal underground economies increased rates of violence and outbreaks of ~~~·~ly tr~nsmitted infections, hepatitis, and tuberculosi: but this research suggests 'that public health uunauves aimed at reducing health disparities may be failing the most vulnerable and marginal of citiztl s. margaret malone ~ vi~lence that occurs in families and in intimate relationships is a significant urban, ~unity, and pu~hc health problem. it has major consequences and far-reaching effects for women, ~~--renho, you~ sen on, and families. violence also has significant effects for those who provide and ukllc w receive health care violence · · i · · . all lasses, · is a soc a act mvolvmg a senous abuse of power. it crosses : ' : ' ~ s;nden, ag~ ~ti~, cultures, sexualities, abilities, and religions. societal responseshali ra y oc:used on identificatton, crisis intervention and services for families and individuajs.promoten are only "-"--:-g to add h · ' · i in intimate relationshi with"-~"'.". ress t e issues of violence against women and vjoence lenga to consider i~ m families. in thi_s p~per, i analyze issues, propose strategies, and note c~· cannot be full -...l'-~ whork towards erad canng violence, while arguing that social justice and equity y -. ucvcu w en thett are people wh mnhod: critical social theory, an analysis that addresses culturally and ethnically diverse communities, together with a population health promotion perspective frame this analysis. social determinants of health are used to highlight the extent of the problem of violence and the social and health care costs.the ottawa charter is integrated to focus on strategies for developing personal skills, strengthening community action, creating supportive environments, devdoping healthy public policies, and re-orientating health and social services. attention is directed to approaches for working with individuals, families, groups, communities, populations, and society.ratdts: this analysis demonstrates that a comprehensive interdisciplinary, multisectoral, and multifaceted approach within an overall health promoting perspective helps to focus on the relevant issues, aitical analysis, and strategies required for action. it also illuminates a number of interacting, intersecting, and interconnecting factors related to violence. attention, which is often focused on individuals who are blamed for the problem of violence, is redirected to the expertise of non-health professionals and to community-based solutions. the challenge for health promoters working in the area of violence in families and in intimate relationships is to work to empower ourselves and the communities with whom we work to create health-promoting urban environments. social justice, equiry, and emancipatory possibilities are positioned in relation to recommendations for future community-based participatory research, pedagogical practices for health care practitioners, and policy development in relation to violence and urban health. the mid-main community health center, located in vancouver british columbia (bc), has a diverse patient base reflecting various cultures, languages, abilities, and socio-economic statuses. due to these differences, some mid-main patients experience greater digital divide barriers in accessing computers and reputable, government produced consumer health information (chi) websites, such as the bc healthguide and canadian health network. inequitable access is problematic because patient empowerment is the basis of many government produced chi websites. an internet terminal was introduced at mid-main in the summer of , as part of an action research project to attempt to bridge the digital divide and make government produced chi resources useful to a broad array of patients. multi-level interventions in co-operation with patients, with the clinic and eventually government ministries were envisioned to meet this goal. the idea of implementing multi-level interventions was adopted to counter the tendency in interactive design to implement a universal solution for the 'ideal' end-user [ ), which discounts diversity. to design and execute the interventions, various action-oriented and ethnographic methods were employed before and during the implementation of the internet terminal. upon the introduction of the internet terminal, participant observation and interviews were conducted using a motion capture software program to record a digital video and audio track of patients' internet sessions. this research provided insight into the spectrum of patients' capacities to use technology to fulfil their health information needs and become empowered. at the mid-main clinic it is noteworthy that the most significant intervention to enhance the usefulness of chi websites for patients appeared to be a human rather than a technological presence. as demonstrated in other ethnographic research of community internet access, technical support and capacity building is a significant component of empowerment ( ). the mid-main wired waiting room project indicates that medical practitioners, medical administrators, and human intermediaries remain integral to making chi websites useful to patients and their potential empowerment. ( ) over the past years the environmental yo~th alliance has been of~ering a.youth as~t. mappin~ program which trains young people in community research and evaluation. wh ~st the positive expenenc~ and relationships that have developed over this time attest to the success of this program, no evaluations has yet been undertaken to find out what works for t.he youth, what ~ould be changed, and what long term outcomes this approach offers for the youth, their local community, and urban governance. these topics will be shared and discussed to help other community disorganizing and uncials governments build better, youth-driven structures in the places they live.p - (a) the world trade center health registry: a unique resource for urban health researchers deborah walker, lorna thorpe, mark farfel, erin gregg, and robert brackbill introduction: the world trade center health registry (wfchr) was developed as a public health response to document and evaluate the long-term physical and mental health effects of the / disaster on a large, diverse population. over , people completed a wfchr enrollment baseline survey, creating the largest u.s. health registry. while studies have begun to characterize / bealth impacts, questions on long-term impacts remain that require additional studies involving carefully selected populations, long-term follow-up and appropriate physical exams and laboratory tests. wtchr provides an exposed population directory valuable for such studies with features that make ita unique resource: (a) a large diverse population of residents, school children/staff, people in lower man· hattan on / including occupants of damaged/destroyed buildings, and rescue/recovery/cleanup work· ers; (b) consent by % of enrollees to receive information about / -related health studies; (c) represenration of many groups not well-studied by other researchers; (c) email addresses of % of enrollees; (d) % of enrollees recruited from lists with denominator estimates; and (e) available com· parison data for nyc residents. wfchr strives to maintain up-to-date contact information for all enrollees, an interested pool of potential study participants. follow-up surveys are planned.methods: to promote the wtchr as a public health resource, guidelines for external researcher.; were developed and posted on (www.wtcregistry.org) which include a short application form, a twopage proposal and documentation of irb approval. proposals are limited to medical, public health, or other scientific research. researchers can request de-identified baseline data or have dohmh send information about their studies to selected wfchr enrollees via mail or email. applications are scored by the wtchr review committee, comprised of representatives from dohmh, the agency for toxic subst~nces and disease registry, and wtchr's scientific, community and labor advisory committees. a data file users manual will be available in early fall .~suits: three external applications have been approved in , including one &om a non-u.s. ~esearcher, all requesting information to be sent to selected wtchr enrollees. the one completed mail· mg~~ wtchr enrollee~ (o , wfc tower evacuees) generated a positive survey response rate. three additional researchers mtend to submit applications in . wfchr encourages collaborations between researchers and labor and community leaders.conclusion: studies involving wtchr enrollees will provide vital information about the long· term health consequences of / . wtchr-related research can inform communities, researcher.;, policy makers, health care providers and public health officials examining and reacting to and other disasters. t .,. dp'"f'osed: thi is presentation will discuss the findings of attitudes toward the repeat male client iden· ie as su e a and substance us'n p · · · · i · 'd . . - g. articipants will learn about some identified effective strategies or service prov ers to assist this group of i · f men are oft · d bl men. n emergency care settings, studies show that this group en viewe as pro emaric patient d i r for mental health p bl h h an are more ikely to be discharged without an assessmen !) ea rofr ems t. an or er, more cooperative patients (forster and wu · hickey er al., · r y resu ts om this study suggest th · · ' ' l · d tel' mining how best to h . d at negative amtudes towards patients, difficu nes e · as well pathways l_e_ p patientsblan ~ck of conrinuity of care influence pathways to mental health care. • uc\:ome pro emat c when p ti k · che system. m a ems present repeatedly and become "get stuc id methods: semi-structured intervie d . · (n= ), ed nurses (n= ) other ed ;s were con ucted with male ed patients (n= ), ed phys oans ' sta (n= ) and family physicians (n= ). patients also completed a poster sessions v diagnostic interview. interviews were tape-recorded, transcribed verbatim and managed using n . transcripts were coded using an iterative process and memos prepared capture emergent themes. ethics approval was obtained and all participants signed a detailed informed consent form.introduction: urban settings are particularly susceptible to the emergence and rapid spread of nt•w or rare diseases. the emergence of infectious diseases such as sars and increasing concerns over the next influenza pandemic has heightened interest in developing and using a surveillance systt·m which detects emerging public health problems early. syndromic surveillance systems, which use data b, scd on symptoms rather than disease, offer substantial potential for this by providing near-real-rime data which are linked to an automated warning system. in toronto, we are piloting syndromic data from the · emergency medical services (ems) database to examine how this information can be used on an ongoing basis for the early detection of syndromes including heat-related illness (hri), and influenza-like-illness (ill). this presentation will provide an outline of the planned desi_gn of this system and proposed evaluation. for one year, call codes which reflect heat-related illness or influenza-like-illness will be selected and searched for daily using software with a multifactorial algorithm. calls will he stratified by call code, extracted from the -ems database and transferred electronically to toronto public health. the data will be analyzed for clusters and aberrations from the expected with the realtime outbreak and disease surveillance (rods) system, a computer-based public health tool for the early detection of disease outbreaks. this -ems surveillance system will be assessed in terms of its specificity and sensitivity through comparisons with the well-established tracking systems already in place for hr! and ill. others sources of data including paramedic ambulance call reports of signs and . this study will introduce complementary data sources t~ the ed ch e~ complamt an~ o~~rthe-counter pharmacy sales syndromic surveillance data currently bemg evaluated m ~ther ontar~o cltles. . syndromic surveillance is a unique approach to proactive(~ dete~tmg early c.hangesm the health status of urban communities. the proposed study aims to provide evidence of differential effectiveness through investigating the use of -ems call data as a source of syndromic surveillance information for hr! and ili in toronto. introduction: there is strong evidence that primary care interventions, including screening, brief advi<:e, treatment referral and pharmacotherapy are effective in reducing morbidity and mortality caused by substance abuse. yet physicians are poor at intervening with substance users, in part because of lack of time, training and support. this study examines the hypothesis that shared care in addictions will result in decreased substance use and improved health status of patients, as well as increased use of primary care interventions by primary care practitioners (pcps). methods: the addiction medicine service (ams) at st. joseph's health centre's family medicine department is in the process of being transformed from its current structure as a traditional consult service into a shared care model called addiction shared care (asc). the program will have three components: education, office systems and clinical shared care. as opposed to a traditional consult service, the patient will be booked with both a primary care liaison worker (pcl) and addictions physician. patients referred from community physicians, the emergency department and inpatient medical and psychiatric wards will be recruited for the study as well as pcps from the surrounding community. the target sample size is - physicians and a similar number of patients. after initial consult, patient will be recruited into the study with their consent. the shared-case model underlines the interaction and collaboration with the patient's main pcp. asc will provide them with telephone consults, advice, support and re-assessment for their patients, as well as educational sessions and materials such as newsletters and informational kits.results: the impact of this transition on our patient care and on pcp's satisfaction with the asc model is currently being evaluated through a grant provided by the ministry of health & long term care. a retrospective chart review will be conducted using information on the patient's substance use, er/clinic visits, and their health/mood status. pcp satisfaction with the program will be measured through surveys and focus groups. our cost-effectiveness analysis will calculate the overall cost of the program per patient..conclusion: this low-cost service holds promise to serve as an optimal model and strategy to improve outcomes and reduce health care utilization in addict patients. the inner city public health project introduction the inner city public health project (icphp) was desi.gned to explore new an~ innovative ways to reach marginalized inner city populations that par-t c pate m high health-nsk beha~ ors. much of this population struggles with poverty, addictions, mental illness and homelessness, creatmg barriers to accessing health services and receiving follow-up. this pro ect was de~igned to evaluat~ .~e success of offering clinics in the community for testing and followup of communicable diseases uuhzmg an aboriginal outreach worker to build relationships with individuals and agencies. v n(demographics~ history ~f testi~g ~nd immunization and participation in various health-risk behaviors), records of tesnng and mmumzat ons, and mterviews with partner agency and project staff after one year.. results: t~e chr ~as i~strumental in building relationships with individuals and partner agencies ' .° the c~mmun_ ~ re_sultmg m req~ests for on-site outreach clinics from many of the agencies. the increase m parnc pat n, the chr mvolvement in the community, and the positive feedback from the agen? staff de~onstrated that.the project was successfully creating partnerships and becoming increasingly integrated m the community. data collected from clients at the initial visit indicated that the project was reaching its target populations and highlighted the unique health needs of clients, the large unmet need for health services and the barriers that exist to accessing those services. ~usion: the outreach clinics were successful at providing services to target populations of high health-nsk groups and had great support from the community agencies. the role of the chr was critical to the success of the project and proved the value of this category of health care worker in an urban aboriginal population. the unmet health needs of this disadvantaged population support the need for more dedicated resources with an emphasis on reducing access barriers. building a caring community old strathcona's whyte avenue, a district in edmonton, brought concern about increases in the population of panhandlers, street people and homeless persons to the attention of all levels of government. the issue was not only the problems of homelessness and related issues, but feelings of insecurity and disempowerment by the neighbourhood residents and businesses. their concerns were acknowledged, and civic support was offered, but it was up to the community itself to solve the problem. within a year of those meetings, an adult outreach worker program was created. the outreach worker, meets people in their own environments, including river valley camps. she provides wrap-around services rooted in harm reduction and health promotion principles. her relationship-based practice establishes the trust for helping clients with appropriate housing, physical and/or mental health issues, who have little or no income and family support to transition from homelessness. the program is an excellent example of collaboration that has been established with the businesses, community residents, community associations, churches, municiple services, and inner-city agencies such as boyle street community services. statistics are tracked using the canadian outcomes research institute homes database, and feedback from participants, including people who are street involved. this includes an annual general meeting for community and people who are homeless. the program's holistic approach to serving the homeless population has been integral, both in creating community awareness and equipping residents and businesses to effectively interact with people who are homeless. through this community development work, the outreach worker engages old strathcona in meeting the financial and material needs of the marginalized community. the success of this program has been surprising -the fact is that homeless people's lives are being changed; one person at a time and the community has been changed in how they view and treat those without homes. over two years, the program has successfully connected with approximately seventy-five individuals who call old strathcona home, but are homeless. thirty-six individuals are now in homes, while numerous others have been assisted in obtaining a healthier and safer lifestyles by becoming connected with other social/health agencies. the program highlights the roots of homelessness, barriers to change and requirements for success. it has been a thriving program and a model that works by showing how a caring community has rallied together to achieve prosperous outcomes. the spn has created models of tb service delivery to be used m part~ers~ p with phannaceunca compa-. · · -. t' ns cooperatives and health maintenance orgamzanons (hmos). for example, the mes, c v c orgamza , . · b tb d' · spn has established a system with pharmaceutical companies that help patients to uy me cmes at a special discounted rate. this scheme also allows patients to get a free one-_month's worth of~ dru_g supply if they purchase the first months of their regimen. the sy_s~e~s were ~es gned to be cm~pattble with existing policies for recording and documentation of the ph hppme national tuberculosis program (ntp). aside from that, stakeholders were also encouraged to be dots-enabled through the use of m~nual~ and on-line training courses. the spn initiative offers an alternative in easing the burden of tb sc:rv ce delivery from rhe public sector through the harnessing of existing private-sector (dsos). the learnmgs from the spn experience would benefit groups from other locales that _work no~ only on ~ but other health concerns as well. the spn experience showcases how well-coordinated private sector involvements help promote social justice in health delivery in urban communities.p - (c) young people in control; doing it safe. the safe sex comedy juan walter and pepijn v. empelen introduction: high prevalence of chlamydia and gonorrhoea have been reported among migrants youth in amsterdam, originating from the dutch antilles, suriname and sub-sahara africa. in addition, these groups also have high rates of teenage-pregnancy (stuart, ) and abortions (rademakers ), indicating unsafe sexual behaviour of these young people. young people (aged - ) from the so· called urban scene (young trendsetters in r&b/hip hop music and lifestyle) in amsterdam have been approached by the municipal health service (mhs) to collaborate on a safe sex project. their input was to use comedy as vehicle to get the message a cross. for the mhs this collaboration was a valuable opportunity to reach a hard-to-reach group.mdhods: first we conducted a need assessment by means of a online survey to assess basic knowledge and to similtaneously examine issues of interest concerning sex, sexuality, safer sex and the opposite sex. second, a small literature study was conducted about elements and essential conditions for succesful entertainment & education (e&e) (bouman ), with as most important condition to ensure that the message is realistic (buckingham & bragg, ) . third a program plan was developed aiming at enhancing the stl/hiv and sexuality knowledge of the young people and addressing communication and educational skills, by means of drama. subsequently a safe sex comedy show was developed, with as main topics: being in love, sexuality, empowerment, stigma, sti, hivand safer sex. the messages where carried by a mix of video presentation, stand up comedy, spoken word, rap and dance.results: there have been two safe sex comedy shows. the attendance was good; the group was divers' with an age range between and year, with the majority being younger than year. more women than men attended the show. the story lines were considered realistic and most of the audients recognised the situations displayed. eighty percent of the audients found the show entertaining and % found it edm:arional. from this %, one third considers the information as new. almost all respondents pointed our that they would promote this show to their friends.con.clusion: the s.h<_>w reached the hard-to-reach group of young people out of the urban scene and was cons d_ered entert~mmg, educational and realistic. in addition, the program was able in addressing important issues, and impacted on the percieved personal risk of acquiring an sti when not using condoms, as well as on basic knowledge about stl's. introduction: modernity has contributed mightily to the marginality of adults who live with mental illnesses and the subsequent denial of opportunities to them. limited access to social, vocational, educational, and residential opportunities exacerbates their disenfranchisement, strengthening the stigma that has been associated with mental illness in western society, and resulting in the denial of their basic human rights and their exclusion from active participation in civil society. the clubhouse approach tn recovery has led to the reduction of both marginality and stigma in every locale in which it has been implemented judiciously. its elucidation via the prism of social justice principles will lead to a deeper appreciation of its efficacy and relevance to an array of settings. methods: a review of the literature on social justice and mental health was conducted to determine core principles and relationships between the concepts. in particular, fondacaro and weinberg's ( ) conceptualization of social justice in community psychology suggests the desirability of the clubhouse approach in community mental health practice. a review of clubhouse philosophy and practice has led to the inescapable conclusion that there is a strong connection between clubhouse philosophy-which represents a unique approach co recovery--and social justice principles. placing this highly effective model of community mental health practice within the context of these principles is long overdue. via textual analysis, we will glean the principles of social justice inherent in the rich trove of clubhouse literature, particularly the international standards of clubhouse development.results: fondacarao and weinberg highlight three primary social justice themes within their community psychology framework: prevention and health promotion; empowerment, and a critical pnsp<"<·tive. utilizing the prescriptive principles that inform every detail of clubhouse development and th<" movement toward recovery for individuals at a fully-realized clubhouse, this presentation asserts that both clubhouse philosophy and practice embody these social justice themes, promote human rights, and empower clubhouse members, individuals who live with mental illnesses, to achieve a level of wdl-heing and productivity previously unimagined.conclusion: a social justice framework is critical to and enhances an understanding of the clubhouse model. this model creates inclusive communities that lead to opportunities for full partic pil!ion civil society of a previously marginalized group. the implication is that clubhouses that an· based on the international standards for clubhouse programs offer an effective intervention strategy to guarantee the human rights of a sizable, worthy, and earnest group of citizens. to a drastic increase in school enrollment from . million in to . million in .s. however, while gross enrollment rates increased to °/., in the whole country after the introduction of fpe, it remained conspicuously low at % in the capital city, nairobi. nairobi city's enrollment rate is lower .than thatof all regions in the country except the nomadic north-eastern province. !h.e.d sadvantage of children bas_ed in the capital city was also noted in uganda after the introduction of fpe m the late s_-many_ education experts in kenya attribute the city's poor performance to the high propornon of children hvmg m slums, which are grossly underserved as far as social services are concerned. this paper ~xammes the impact of fpe and explores reasons for poor enrollment in informal settlements m na rob city. methods: the study utilizes quantitative and qualitative schooling data from the longitudinal health and demographic study being implemented by the african population and health research center in two informal settlements in nairobi. descriptive statistics are used to depict trends in enrollment rates for children aged - years in slum settlements for the period - . results: the results show that school enrollment has surprisingly steadily declined for children aged - while it increased marginally for those aged - . the number of new enrollments (among those aged years) did not change much between and while it declined consistently among those aged - since . these results show that the underlying reasons for poor school attendance in poverty-stricken populations go far beyond the lack of school fees. indeed, the results show that lack of finances (for uniform, transportation, and scholastic materials) has continued to be a key barrier to schooling for many children in slums. furthermore, slum children have not benefited from fpe because they mostly attend informal schools since they do not have access to government schools where the policy is being implemented.conclusion: the results show the need for equity considerations in the design and implementation of the fpe program in kenya. without paying particular attention to the schooling needs of the urban poor children, the millennium development goal aimed at achieving universal primary education will remain but a pipe dream for the rapidly increasing number of children living in poor urban neighborhoods.ps- (c) programing for hiv/aids in the urban workplace: issues and insights joseph kamoga hiv/aids has had a major effect on the workforce. according to !lo million persons who are engaged in some form of production are affectefd by hiv/aids. the working class mises out on programs that take place in communities, yet in a number of jobs, there are high risks to hiv infection. working persons sopend much of their active life time in workplaces and that is where they start getting involved in risky behaviour putting entire families at risk. and when they are infected with hiv, working people face high levels of seclusion, stigmatisation and some miss out on benefits especially in countries where there are no strong workplace programs. adressing hiv/aids in the workplace is key for sucessfull responses. this paper presents a case for workplace programing; the needs, issues and recommendations especially for urban places in developing countries where the private sector workers face major challenges. key: cord- -l xuu a authors: bergström, anna; ehrenberg, anna; eldh, ann catrine; graham, ian d.; gustafsson, kazuko; harvey, gillian; hunter, sarah; kitson, alison; rycroft-malone, jo; wallin, lars title: the use of the parihs framework in implementation research and practice—a citation analysis of the literature date: - - journal: implement sci doi: . /s - - - sha: doc_id: cord_uid: l xuu a background: the promoting action on research implementation in health services (parihs) framework was developed two decades ago and conceptualizes successful implementation (si) as a function (f) of the evidence (e) nature and type, context (c) quality, and the facilitation (f), [si = f (e,c,f)]. despite a growing number of citations of theoretical frameworks including parihs, details of how theoretical frameworks are used remains largely unknown. this review aimed to enhance the understanding of the breadth and depth of the use of the parihs framework. methods: this citation analysis commenced from four core articles representing the key stages of the framework’s development. the citation search was performed in web of science and scopus. after exclusion, we undertook an initial assessment aimed to identify articles using parihs and not only referencing any of the core articles. to assess this, all articles were read in full. further data extraction included capturing information about where (country/countries and setting/s) parihs had been used, as well as categorizing how the framework was applied. also, strengths and weaknesses, as well as efforts to validate the framework, were explored in detail. results: the citation search yielded articles. after applying exclusion criteria, articles were read in full, and the initial assessment yielded a total of articles reported to have used the parihs framework. these articles were included for data extraction. the framework had been used in a variety of settings and in both high-, middle-, and low-income countries. with regard to types of use, % used parihs in planning and delivering an intervention, % in data analysis, % in the evaluation of study findings, and/or % in any other way. further analysis showed that its actual application was frequently partial and generally not well elaborated. conclusions: in line with previous citation analysis of the use of theoretical frameworks in implementation science, we also found a rather superficial description of the use of parihs. thus, we propose the development and adoption of reporting guidelines on how framework(s) are used in implementation studies, with the expectation that this will enhance the maturity of implementation science. there has been an increased use of theoretical frameworks in the field of implementation science in the last decade, with most developed in the last two decades [ , ] . tabak et al. identified theoretical models used in dissemination and implementation science [ ] . however, while theoretical frameworks are increasingly being cited, more research is needed to understand how they are chosen and applied, and how their use relates to improved implementation outcomes [ , ] . variously described in the form of theories, frameworks, or models, all strive to provide conceptual clarity on different aspects of implementation practice and research. for consistency, we will refer to these as theoretical frameworks, or simply "frameworks." the promoting action on research implementation in health services (parihs) framework is a multidimensional framework which was developed to explicitly challenge the pipeline conceptualization of implementation [ ] . the parihs framework is a commonly used conceptual framework [ , ] that posits successful implementation (si) as a function (f) of the nature and type of evidence (e) (including research, clinical experience, patient experience, and local information), the qualities of the context (c) of implementation (including culture, leadership, and evaluation), and the way the implementation process is facilitated (f) (internal and/or external person acting as a facilitator to enable the process of implementation); si = f(e,c,f). the framework was informed by rogers' diffusion of innovations [ ] and various organizational theories and theories from social science [ ] and generated inductively by working with clinical staff to help them understand the practical nature of getting evidence into practice. the parihs framework was initially published in [ ] and updated based on a conceptual analysis in [ ] and further primary research [ ] . a further refinement was undertaken in [ ] , resulting in the integrated or i-parihs. articles using the revised version are not included in the citation analysis reported here. the parihs framework has been described as a determinant framework in that it specifies determinants that act as barriers and enablers influencing implementation outcomes [ ] . skolarus et al. [ ] identified kitson et al. [ ] as one of the two primary originating sources of influence in their citation analysis of dissemination and implementation frameworks. despite the growing number of citations of theoretical frameworks in scientific articles, the detail of how frameworks are used remains largely unknown. systematic reviews of the application of two other commonly used frameworks [ ] , the knowledge to action framework [ ] and the consolidated framework for implementation research [ ] , both reported that use of these frameworks, beyond simply citation, was uncommon. while parihs has been widely cited, it has also been scrutinized; in , helfrich et al. published a qualitative critical synthesis of studies that had used the par-ihs framework [ ] , finding six core concept articles and empirical articles. one of the reported findings was that parihs was generally used as an organizing framework for analysis. at the time, no studies used parihs prospectively to design implementation strategies [ ] . a systematic review applying citation analysis to map the use of parihs (similar to those undertaken for the knowledge to action framework (kta) [ ] and the consolidated framework for implementation research (cfir) [ ] ) has not yet been performed. systematic reviews can contribute to the development of existing theoretical frameworks by critically reviewing what authors state as their weaknesses and strengths; they can also direct future and current users of frameworks to examples of using the frameworks in different ways. to contribute to this development from the perspective of the parihs framework, we undertook a citation analysis of the published peer-reviewed literature that focused on the reported use of parihs (and its main elements), in what contexts the framework has been applied, and what scholars who have used the parihs framework (and its main elements) report as its strengths, limitations, and validity. the method used for this study is citation analysis, i.e., the examination of the frequency and patterns of citations in scientific articles, in this case articles citing the core parihs framework publications. a team of researchers with engagement in the development and/or use of the parihs framework was constituted. initially, the group decided on the core publications for the citation analysis. four articles were selected as they represented the key stages of the framework's development, namely the original paper that described parihs, plus • the findings underline that descriptions of the use of the framework generally were not that transparent and often partial. • findings also point at difficulties in using the framework, such as lack of guidance on key steps to overcome barriers and support implementation • identifies the need of common guidelines on how theories, models, and frameworks should be reported in research articles. citation searches were performed by an information specialist (kg) to retrieve published articles citing any of the four core articles. the searches were performed in two citation databases: web of science and scopus. the first searches were performed between march and april . later, september , additional searches were performed in respective databases. these searches were limited to citations that were published april - august to update the result from the first searches. all citations that were published september (i.e., when kitson et al was published)- august (i.e., prior to the search date) in respective databases were collected in endnote library. endnote was used for checking duplicates and retrieving full texts. to manage the scope of the citation analysis, we opted to only include articles in english published in peer-reviewed scientific journals. the searches in web of science were, because of the subscription, limited to web of science core collection without book citation index. the preferred reporting items for systematic reviews and meta-analyses (prisma) flow diagram [ ] for the data extraction is provided in fig. . initially, an assessment to identify the articles that used the parihs framework in any other way than merely referencing one or more of the core articles was performed (additional file ). for this initial assessment, all articles were read in full. after identifying articles where the parihs framework was used, data extraction was undertaken using a tailor-made data capture form (additional file ). the data capture form was developed and piloted in iterative cycles by the research team. apart from capturing information about where (country/countries and setting/s) and with whom (professional groups and roles) parihs had been applied, the form included questions on whether parihs was used in one or more of the following ways: ) in planning and delivering an intervention, ) in data analysis, ) in the evaluation of study findings, and/or ) in any other way. each of these questions was followed by an openended item for extracting information on how this was reported [ ] . to enhance reliability and data richness, each reviewer copy-pasted sections of the article corresponding to the open-ended reply into the data extraction form when appropriate and indicated page, column, and row. two additional items captured whether the parihs framework had been tested or validated, as well as any reported strengths and weaknesses of the framework. thus, we report on what the authors of the included articles claim to have done, rather than a judgment as to how and to what extent they actually used the parihs framework. for data extraction and validation, the research team was divided into four pairs, ensuring that each article was assessed separately by at least two research team members. the pairs received batches of articles at a time. variations in the assessments were discussed until consensus was reached within the pair(s). further, queries detected within the pairs were raised and discussed with the whole research team, until consensus was achieved. regular whole-team online meetings were held to consolidate findings between every new batch of articles and throughout the development and analysis process. in total, the group had > online meetings and four face-to-face meetings from the initial establishment of the group in january . categorical data were analyzed using descriptive statistics, whereas the open-ended items were analyzed qualitatively [ ] , including the collated extractions of data to illustrate each of the four types of use (i.e., how the parihs framework was depicted in terms of ( ) planning and delivering an intervention, ( ) analysis, ( ) evaluation of study findings, and/or ( ) in any other way). applying a content analysis approach [ ] , members of the research team worked separately with the texts extracted from the reviewed articles. the extracts for each open-ended item were read and reread, to get a sense of the whole. next, variations were identified and formed as categories. findings for each question were summarized in short textual descriptions, which were shared with the whole team. in a face-to-face meeting, the data relating to each question were critically discussed and comparisons were made between the findings for each question, to identify overlaps and relationships about how parihs has been used. after duplicate control, references remained. these were sorted by language and type of publication. in this phase, references categorized as books, book chapters, conference proceedings, and publications written in non-english language were excluded. also, three of the four core articles (i.e., the three citing kitson et al. [ ] which was the starting point for development of the parihs framework and therefore did not appear in the citation search) were excluded from the database [ , , ] , as were four articles expanding and refining par-ihs [ ] [ ] [ ] [ ] . accordingly, articles remained, and after the assessment excluding those merely citing par-ihs, a further articles were excluded, leaving articles that cited one or more of the core articles, and made explicit use of the parihs framework (see fig. and table ) . of these articles, cited kitson et al. [ ] , cited kitson et al. [ ] , cited rycroft-malone et al. [ ] , and cited rycroft-malone et al. [ ] . in total, the articles consisted of protocols [ , , , - , , , - , - , , ] . a further articles reported empirical studies: ▪ where parihs guided the development of the intervention [ - , - , , , - ] , ▪ intervention studies where parihs did not guide the development of an intervention [ , , , , , , , , , , , , , , - , , - , - , , , , , - , , - , - , - , , , - , , , , - , , , - , - , , , - , - ] , ▪ non-intervention studies [ , , , , , , - , , , - , , , , - , - , , , , , , , , - , - , - , , , , , , , , , , , - , , ] in addition, the database included empirical review studies [ , , , , ] and opinion/ theoretical articles [ , , . in terms of professional focus, about % of the included articles involved nursing. in the following sections, references have been added to the categorical items in the data extraction while we have opted only to provide examples of references to the findings from the qualitative exploration of how the parihs framework was operationalized in detail. of the articles reporting type of setting where the implementation project/research took place, a majority were undertaken in hospitals (n = ) [ , , , , , , , , , - , , - , , - , , , , , , - , , , , , - , - , , , , , , , , , , , - , , , , - , , , , - , , , - , , , , , , , , , , , , , , - , , , , , , , - , - , , , , - , , , , , , , , , , , , , , , , , , ] , followed by a combination of multiple healthcare settings (n = ) [ , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , - , , , , , , , , , , , , , , , , , , , , - , , , , , , , , , , , , , , , , , , , , ] , community/social care settings (n = ) [ , , , , , , , , , , , - , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ] , primary health care (n = ) [ , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ] , and home-based care (n = ) [ , , , , , , ] . five articles were derived from special settings such as construction [ ] , education [ ] , pharmacies [ ] , urban planning [ ] , and public health institutions [ ] . in articles [ , , , , , , , , , , , , , , , , , , , , , , - , - , , , , , , , , , , , , , , ] , the setting was not reported or not applicable (e.g., opinion/theoretical articles). for empirical studies and published protocols, about % were derived from research in the usa, % from canada, % from sweden, and % from the uk. the remaining articles mainly originated from other high-income countries in europe; in addition, there were a few articles reporting studies in low-and middle-income countries, including vietnam, tanzania, mozambique, and uganda [ , , , , , ] . the types of articles published using the parihs framework changed over time, with an increase in the number of empirical studies from onwards, as illustrated in fig. . as the search for articles for this review only included the first eight months of , the graph is limited to full years (i.e., through ). figure depicts how parihs was used by type of article. although authors frequently claimed that parihs was used in one or more ways, details as to how the framework was used were often lacking. in total, ( %) articles claimed to use the parihs framework to plan and deliver an intervention [ - , - , - , , , , , - ] . predominantly, these were empirical studies (n = ) [ - , - , , , - ] but also two opinion/theoretical articles [ , ] and protocols . of the articles, about half stated that the framework was used for theoretically informing, framing, or guiding an intervention (e.g., [ , , , , ] ). however, in these studies, parihs was referred to only in a general sense, in that the core elements of the framework were said to have informed the planning of the study. there was a lack of detail provided about what elements of the framework were used and how they were operationalized to plan and deliver an intervention. in the other half of the articles, it was described more specifically that one or more elements of the framework had been used. most commonly the facilitation element (e.g., [ , , , , ] ) was referred to as guiding an implementation strategy. the articles that provided explicit descriptions of interventions using facilitation employed strategies such as education, reminders, audit-andfeedback, action learning, and evidence-based quality improvement, and roles including internal and external facilitators and improvement teams to enable the uptake of evidence (e.g., [ , , , ] ). some articles drew on the parihs framework more specifically, to understand the role of organizational context in implementation (e.g., [ , , , ] ). there were ( %) articles where the parihs framework was reported to be used in the analysis [ , , - , - , - , - , - , , , - , , - , , ] . most of these involved empirical studies (n = ) [ - , - , - , ] where parihs often was described as guiding or framing the data collection, e.g., developing an interview guide, and/or analysis, but with no further details. in articles that provided more detailed information, parihs was used to guide or frame qualitative analyses in about studies (e.g., [ , , , , ] ). of these, around used a deductive approach in that they used the elements and sub-elements to structure the analytic process (e.g., [ , , , , ] ). about studies applied parihs for quantitative analysis, (e.g., [ , , , , ] ). in half of these, the alberta context tool (e.g., [ , , , , ] ) and the organizational readiness to change assessment tool (e.g., [ , , , ] ) were used; both these tools being derived from parihs. empirical studies using the parihs framework in the analysis encompassed primarily all three main elements of parihs (e.g., [ , , , ] ) and the context domain (e.g., [ , , , ] ), and in lesser extent the evidence (e.g., [ , , ] ) and the facilitation domain (e.g., [ , , , ] ). eleven review studies [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] ] used the framework for the analysis; findings were mapped to parihs elements in a few studies [ , , ] ; one described that their data had been "analysed through the lens of parihs" (p ) [ ] . a couple of the review studies had parihs as the object for analysis, comparing it with other frameworks [ , ] . this approach was also common in the opinion/theoretical articles [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , where the parihs framework itself was the focus of the analysis (e.g., [ , , ] ). in these articles, the analysis was performed in different ways, primarily through mapping and comparing parihs to other frameworks or models or even policies, but also for general discussions on implementation and evidence-based practice. among the articles that reported using the parihs framework in the analysis, there were also protocols where authors reported that the intention was to use the framework in the analysis [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the application of parihs in the evaluation of study findings a total of ( %) included articles provided information on how the parihs framework was used in the evaluation of study findings, in terms of contributing to the discussion and interpretation of results [ , , - , - , - , - , - , - , , , - , - , , - , - , , ] . the majority (n = ) of these were empirical studies [ , - , - , - , - , - , - , ] . we found two main approaches to how the parihs framework was used in the evaluation of study findings. first, parihs was used to organize the discussion of the findings (e.g., [ , , , , ] ), where the framework and/or its elements were used to provide a structure for reporting or generally discussing the findings, or both, for example in stating that the key elements of parihs were reflected in the study findings. second, the framework was used to consider the implications of the study's findings (e.g., [ , , , , ] ), where the framework or its elements (varying between one (e.g., [ , , ] ), two (e.g., [ , , ] ), and all the three main elements (e.g., [ , , ] )) enabled authors to elaborate on findings, or reflect on the implications of their study to evaluate the parihs framework itself. specifically, we found some empirical articles reported evaluating the parihs element "context" by means of context tools (e.g., [ ] ). in addition, an evaluation of the study findings using the framework was identified in opinion/theoretical articles [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and empirical review studies [ , , , - , - , ] . among the opinion/ theoretical articles, there were papers evaluating other theoretical constructions in relation to the parihs framework (e.g., [ ] ). a total of ( %) reported using parihs in some other way than directly informing the planning and delivery of an intervention or analyzing and evaluating findings [ , - , , , - , - , , - , - , , - , - , - , - , , , - , , - , - ] . a majority of these articles (n = ) were empirical studies [ - , , - , - , - , - , - , - ] , and about half of these described the use of par-ihs as an overall guide to frame the study (e.g., [ , , , , , ] ). a similar finding was apparent in the protocols [ - , , , - , , ] ; about half of these also referred to the use of parihs to guide and frame the study design (e.g., [ , , , ] ). an alternative use of parihs in empirical studies involved focusing on one of the three parihs elements (n = ) and investigating them in greater depth, most notably context (n = ) (e.g., [ , ] ) and facilitation (n = ) (e.g., [ , ] ). a total of opinion/theoretical articles [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] reported using the parihs framework in some other way, including a discussion about parihs as part of presenting a general overview of theories and frameworks to inform implementation (e.g., [ , , , ] ), using parihs to augment, develop, or evaluate other implementation models and frameworks (e.g., [ , , , , ] ), and informing education and learning and teaching initiatives [ , ] . empirical review articles (n = ) included reviews of implementation frameworks [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , including parihs, a review of the facilitation dimension of parihs and a discussion of the potential to combine implementation and improvement methodologies. testing and providing views on the validity of the framework a total of ( %) articles described testing or validating parihs, or provided comments on the validity of the framework [ , , , , , , , , , , , , , , , - , , , , , , , , , , , , , , , , , - , , , , , - , , , , , , , , , , - , , , , , , , , , , , , , , , , , , , , , , - , , , , , , , , , , , - , , , , , , ] . of these, were empirical studies [ , , , , , , , , , - , , , , , , , , , , , , , , , , , - , , , , , - , , , , , , , , , , - , , , , , , , , , , , , , , , , , , , , , ] , five were study protocols [ , , , , ] , opinion/theoretical articles [ , , - , , , , , ] , and empirical reviews [ , , , - , , , , , , , , ] . empirical studies either tested the whole or parts of the framework with a focus on: ▪ the validity of the whole framework (e.g., [ , , , , ] ) ▪ the validity of context (e.g., [ , , , , ] ) ▪ the validity of facilitation (e.g., [ , , , ] ) ▪ the validity of evidence (e.g., [ ] ) ▪ identifying gaps in the framework (e.g., [ , , ] ) over the review study period ( to ), among empirical studies, there was a shift from primarily studying the context element of the framework to more articles evaluating the whole framework. this was also evident in the pattern found in the protocols, which mostly focused on testing facilitation (e.g., [ , , ] ). opinion/theoretical articles tended to critique the whole framework (e.g., [ , , , , ] ). of the empirical reviews, the majority focused on the whole framework (e.g., [ , , ] ), then on context (e.g., [ , , ] ) and then on facilitation (e.g., [ ] ). of note is the lack of attention in the literature to the element of "evidence" in the parihs framework (examples of articles paying attention to evidence include [ , ] ). the articles varied in detail, depth, and quality in terms of descriptions of how they went about testing the validity of the parihs framework. approaches ranged from general observations of whether the research teams/users found the elements and sub-elements easy to use (e.g., [ , , ] ), to studies that used elements of context described in the parihs framework to validate new context measures across settings and groups (e.g., [ , , ] ). as one example, the alberta context tool started from the parihs conceptualization of context to include dimensions of culture, leadership, and evaluation. regarding the strength and limitations of the parihs framework, about one third of the included articles reported on its strengths and about % commented on perceived limitations. the identified strengths included: ▪ holistic implementation framework (e.g., [ , , , ] ) that is perceived as intuitive and accessible. ▪ both practical and theoretical and therefore feasible to use by both clinicians and researchers; also seen as intuitive to use and accessible (e.g., [ , , ] ). ▪ can be used as a tool: diagnostic/process/evaluative tool; predictive/explanatory tool or as a way to explain the interplay of factors (e.g., [ , , , , ] ). ▪ can accommodate a range of other theoretical perspectives (including approaches such as social network theory, participatory action research, coaching, change management and other knowledge translation frameworks) (e.g., [ , , , ] ). ▪ can be used successfully in a range of different contexts (low-and middle-income countries) [ ] and services and for various groups of patients (disability, aged care) (e.g., [ , , , ] ). limitations of the parihs framework included: ▪ poor operationalization of key terms leading to difficulties in understanding and an overlap of elements and sub-elements (e.g., [ , , ] ). ▪ lack of practical guidance on steps to operationalize the framework (e.g., [ , ] ) with a subsequent lack of tools. ▪ lack of information on the individual and their characteristics (e.g., [ , ] ) and their lack of understanding of evidence (e.g., [ , ] ). ▪ too structured and does not acknowledge the multidimensionality and uncertainty of implementation (e.g., [ , ] ). ▪ lack of acknowledgement of wider contextual issues such as the impact of professional, socio-political, and policy issues on implementation (e.g., [ , , , ] ). ▪ not providing support in how to overcome barriers to successful implementation (e.g., [ ] ). in a recent survey among implementation scientists, the parihs framework was found to be one of the sixth most commonly used theoretical frameworks [ ] . yet, in our review, about % (n = ) of the identified articles citing any of the four selected core parihs articles used the framework in any substantial way. similarly, a review of the cfir found that / ( %) of articles citing the framework were judged to use the framework in a meaningful way (i.e., used the cfir to guide data collection, measurement, coding, analysis, and/or reporting) [ ] . a citation analysis of the kta framework found that about % ( / ) of screened abstracts described using the kta to varying degrees, although only articles were judged to have applied the framework in a fully integrated way to inform the design, delivery, and evaluation of implementation activities [ ] . parihs has been used in a diverse range of settings but, similarly to other commonly used implementation frameworks, most often superficially or partially. the whole framework has seldom been used holistically to guide all aspects of implementation studies. implementation science scholars have repeatedly argued that the underuse, superficial use, and misuse of implementation frameworks might reduce the potential scientific advancements in the field, but also the capacity for changing healthcare practice and outcomes [ ] . the rationale for not using the whole parihs framework could be many, including the justified reason of only being interested in a particular element. as such, partial use cannot always be considered as inappropriate. simultaneously, many researchers entering the field might be overwhelmed with the many frameworks available and the lack of guidance about how to select and operationalize them and using their elements [ , , ] . the current citation analysis can thus help remedy a gap in the literature by revealing how the parihs framework has been used to date, in full or partially, and thus provides input to users of its potential use. the use of theoretical frameworks in implementation science serves the purpose of guiding researchers' and practitioners' implementation plans and informing their approaches to implementation and evaluation. this includes decisions about what data to gather to describe and explain implementation, their hypotheses about action steps needed, how to account for the critical role of context, and providing a foundation for analysis and discussion [ ] . the advancement of theoretically informed implementation science will, however, depend on much improved descriptions as to why and how a certain framework was used, and an enhanced and betterinformed critical reflection of the functionality of that framework. this review shows that the parihs framework has rarely been used as a whole; rather, certain elements tend to be applied, often retrospectively as indicated in fig. underlining the use of parihs in the evaluation of study findings, which resonates with the findings of reviews about the use of the kta [ ] and cfir [ ] frameworks. this could be as a result of a lack of theoretical coherence of some frameworks making them difficult to apply holistically, and/or a function of a general challenge that researchers face in operationalizing theory. however, this could also be a result of publishing constraints. while the parihs framework may have guided implementation or been implicitly used in the study design, it was rarely the focus of the publications. further, the aims and scopes of scientific health care journals have historically prioritized clinical outcomes over implementation outcomes where one could expect a more detailed description of the use of theoretical frameworks. this may have resulted in authors not fully reporting their use of, e.g., the parihs framework. the number of empirical studies using the parihs framework has steadily increased over the review period. there is also evidence to show that more research teams have contributed to critiquing the framework in terms of reporting on its strengths and limitations and its validity. the pattern of investigation is moving from studies on context, to more systematic explorations of facilitation, thus contributing to a more detailed understanding of the elements and sub-elements of the framework. the lack of focus on "evidence" identified in this review highlights the need for researchers and clinicians to focus on the multi-dimensionality of what is being implemented. common patterns emerging in this review support the changes made to the most recent refinement of the parihs framework [ ] . consistent with other reviews of the use of theoretical frameworks in implementation science, we found that parihs was often not used as intended. further, it was not always clear why the particular framework was chosen. frequently, authors merely cite a framework without providing any further information about how the framework was used. the lack of clear guidance on how to operationalize frameworks might be one of the underlying reasons for this. lastly, to enable a critical review of frameworks and further build collective understanding of implementation, we urge authors to be more explicit about how theory informs studies. development and adoption of reporting guidelines on how framework(s) are used in implementation studies might assist in sharpening the link between the used framework(s) and the individual study, but could potentially also enhance the opportunities for advancing the scientific understanding of implementation. to increase study reliability during the review process, more than one person identified, assessed, and interpreted the data. we had regular meetings to discuss potential difficulties in assessing included articles, and subsequently, all decisions were resolved by consensus to enhance rigor. we used a rigorous search strategy, which was undertaken by an information specialist. the standardization of our processes across the team was also enhanced by the creation of an online data extraction form via google. however, as the form was not linked to other software (e.g., endnote), this added timeconsuming processes. as we did not include articles that were not written in english, we may have limited the insights about the application of the parihs framework, particularly with relevance to different country contexts. additionally, we did not search the grey literature for practical reasons concerning the size of the literature, which may also have provided some additional insights not reflected in this publication. we also limited our search to two databases, which may mean we missed some relevant articles. however, we are confident that we found the majority of relevant published evidence to address the review questions because of a rigorous approach to retrieval. thus, we think the findings of our citation analysis on the use of parihs are generalizable for studies in english published in peer-reviewed journals. the importance of theoretically underpinned implementation science has been consistently highlighted. theory is important for maximizing the chances of study transferability, providing an explanation of implementation processes, developing and tailoring implementation interventions, evaluating implementation, and explaining outcomes. this review of the use of the parihs framework, one of the most cited implementation frameworks, shows that its actual use and application has been frequently partial and generally not well described. our ability to advance the science of implementation and ultimately affect outcomes will, in part, be dependent on better use of theory. therefore, it is incumbent on theory developers to generate accessible and applicable theories, frameworks, and models, and for theory users to operationalize these in a considered and transparent way. we propose that the development and adoption of reporting guidelines on how framework(s) are used in implementation studies might enhance the maturity of implementation science. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file : form for initial assessment and form for data extraction assessing citation networks for dissemination and implementation research frameworks making sense of implementation theories, models and frameworks bridging research and practice: models for dissemination and implementation research criteria for selecting implementation science theories and frameworks: results from an international survey enabling the implementation of evidence based practice: a conceptual framework diffusion of innovations models and frameworks for implementing evidence-based practice: linking evidence to action ingredients for change: revisiting a conceptual framework an exploration of the factors that influence the implementation of evidence into practice implementing evidence-based practice in healthcare: a facilitation guide: routledge using the knowledge to action framework in practice: a citation analysis and systematic review a systematic review of the use of the consolidated framework for implementation research a critical synthesis of literature on the promoting action on research implementation in health services (parihs) framework preferred reporting items for systematic reviews and meta-analyses: the prisma statement real qualitative researchers do not count: the use of numbers in qualitative research methodological challenges in qualitative content analysis: a discussion paper the qualitative content analysis process evaluating the successful implementation of evidence into practice using the parihs framework: theoretical and practical challenges getting evidence into practice: the role and function of facilitation the parihs framework--a framework for guiding the implementation of evidence-based practice what counts as evidence in evidence-based practice? getting evidence into practice: the meaning of 'context' provision of peer specialist services in va patient aligned care teams: protocol for testing a cluster randomized implementation trial a complex culturally targeted intervention to reduce hispanic disparities in living kidney donor transplantation: an effectiveness-implementation hybrid study protocol implementing a knowledge application program for anxiety and depression in community-based primary mental health care: a multiple case study research protocol implementation of a knowledge mobilization model to prevent peripheral venous catheter-related adverse events: prebacp study-a multicenter cluster-randomized trial protocol prioritising responses of nurses to deteriorating patient observations (pronto) protocol: testing the effectiveness of a facilitation intervention in a pragmatic, cluster-randomised trial with an embedded process evaluation and cost analysis case management and self-management support for frequent users with chronic disease in primary care: a pragmatic randomized controlled trial brief cognitive behavioral therapy in primary care: a hybrid type patient-randomized effectiveness-implementation design scaling up safer birth bundle through quality improvement in nepal (sustain)-a stepped wedge cluster randomized controlled trial in public hospitals monitoring and managing metabolic effects of antipsychotics: a cluster randomized trial of an intervention combining evidence-based quality improvement and external facilitation a study protocol for applying the co-creating knowledge translation framework to a population health study accessibility and implementation in uk services of an effective depression relapse prevention programme -mindfulness-based cognitive therapy (mbct): aspire study protocol study protocol for the translating research in elder care (trec): building context through case studies in long-term care project (project two) implementing health research through academic and clinical partnerships: a realistic evaluation of the collaborations for leadership in applied health research and care (clahrc) protocol: adaptive implementation of effective programs trial (adept): cluster randomized smart trial comparing a standard versus enhanced implementation strategy to improve outcomes of a mood disorders program an inpatient rehabilitation model of care targeting patients with cognitive impairment development of a management algorithm for post-operative pain (mapp) after total knee and total hip replacement: study rationale and design improving quality and outcomes of stroke care in hospitals: protocol and statistical analysis plan for the stroke implementation study comparison of high and low intensity contact between secondary and primary care to detect people at ultra-high risk for psychosis: study protocol for a theorybased, cluster randomized controlled trial implementing the i-decided clinical decision-making tool for peripheral intravenous catheter assessment and safe removal: protocol for an interrupted timeseries study translating health care-associated urinary tract infection prevention research into practice via the bladder bundle evidence-based intervention to reduce avoidable hospital admissions in care home residents (the better health in residents in care homes (bhirch) study): protocol for a pilot cluster randomised trial fire (facilitating implementation of research evidence): a study protocol developing family-centred care in a neonatal intensive care unit: an action research study protocol implementing knowledge into practice for improved neonatal survival; a cluster-randomised, community-based trial in quang ninh province knowledgeto-action processes in shrtn collaborative communities of practice: a study protocol study protocol for the translating research in elder care (trec): building contextan organizational monitoring program in long-term care project (project one) the prevention and reduction of weight loss in an acute tertiary care setting: protocol for a pragmatic stepped wedge randomised cluster trial (the prowl project) evidence into practice: evaluating a child-centred intervention for diabetes medicine management. the epic project adjunctive acupuncture for pain and symptom management in the inpatient setting: protocol for a pilot hybrid effectiveness-implementation study study protocol: addressing evidence and context to facilitate transfer and uptake of consultation recording use in oncology: a knowledge translation implementation study improving quality of care through routine, successful implementation of evidencebased practice at the bedside: an organizational case study protocol using the pettigrew and whipp model of strategic change exploring the interpersonal-, organization-, and system-level factors that influence the implementation and use of an innovation-synoptic reporting-in cancer care labouring together: collaborative alliances in maternity care in victoria, australia-protocol of a mixed-methods study determining factors in evidencebased clinical practice among hospital and primary care nursing staff sustaining transfers through affordable research translation (start): study protocol to assess knowledge translation interventions in continuing care settings process evaluation of a knowledge translation intervention using facilitation of local stakeholder groups to improve neonatal survival in the quang ninh province newborn care and knowledge translation -perceptions among primary healthcare staff in northern vietnam urban and suburban hospital system implementation of multipoint access targeted temperature management in postcardiac arrest patients. therapeutic hypothermia and temperature management south west community care access centre home care c. evolving the theory and praxis of knowledge translation through social interaction: a social phenomenological study implementation of evidence-based practice for a pediatric pain assessment instrument improving hospital environmental hygiene with the use of a targeted multi-modal bundle strategy to assess prerequisites before an implementation strategy in an orthopaedic department in sweden improving care for people after stroke: how change was actively facilitated implementation of a suicide nomenclature within two va healthcare settings exploring psychological safety as a component of facilitation within the promoting action on research implementation in health services framework facilitating successful implementation of a person-centred intervention to support family carers within palliative care: a qualitative study of the carer support needs assessment tool (csna t) intervention the influence of context and practitioner attitudes on implementation of person-centered assessment and support for family carers within palliative care why is it so hard to implement change? a qualitative examination of barriers and facilitators to distribution of naloxone for overdose prevention in a safety net environment from workshop to work practice: an exploration of context and facilitation in the development of evidencebased practice enhancing the quality of oral nutrition support for hospitalized patients: a mixed methods knowledge translation study (the eqons study) evaluations of implementation at early-adopting lung cancer screening programs: lessons learned changing practice to support self-management and recovery in mental illness: application of an implementation model designing and implementing two facilitation interventions within the 'facilitating implementation of research evidence (fire)' study: a qualitative analysis from an external facilitators' perspective a randomized controlled study of practice facilitation to improve the provision of medication management services in alberta community pharmacies improving patient participation in a challenging context: a -year evaluation study of an implementation project improving oral health for older people in the home care setting: an exploratory implementation study implementation of video telehealth to improve access to evidence-based psychotherapy for posttraumatic stress disorder the inter-play between facilitation and context in the promoting action on research implementation in health services framework: a qualitative exploratory implementation study embedded in a cluster randomized controlled trial to reduce restraint in nursing homes blending critical realist and emancipatory practice development methodologies: making critical realism work in nursing research effect of facilitation of local maternal-and-newborn stakeholder groups on neonatal mortality: cluster-randomized controlled trial protocol-based care: impact on roles and service delivery a pragmatic cluster randomised trial evaluating three implementation interventions the role of evidence, context, and facilitation in an implementation trial: implications for the development of the parihs framework a realist process evaluation within the facilitating implementation of research evidence (fire) cluster randomised controlled international trial: an exemplar optimizing the mobility of residents with dementia: a pilot study promoting healthcare aide uptake of a simple mobility innovation in diverse nursing home settings getting evidencebased pressure ulcer prevention into practice: a process evaluation of a multifaceted intervention in a hospital setting the development and implementation of a participatory and solution-focused framework for clinical research: a case example implementing brief cognitive behavioral therapy in primary care: a pilot study implications of translating research into practice: a medication management intervention cluster randomized adaptive implementation trial comparing a standard versus enhanced implementation intervention to improve uptake of an effective re-engagement program for patients with serious mental illness i had to somehow still be flexible": exploring adaptations during implementation of brief cognitive behavioral therapy in primary care action-oriented study circles facilitate efforts in nursing homes to "go from feeding to serving": conceptual perspectives on knowledge translation and workplace learning types of internal facilitation activities in hospitals implementing evidence-based interventions. health care manage rev implementing a fetal health surveillance guideline in clinical practice: a pragmatic randomized controlled trial of action learning staff experiences in implementing guidelines for kangaroo mother care--a qualitative study key components of external facilitation in an acute stroke quality improvement collaborative in the veterans health administration the role of organizational context and individual nurse characteristics in explaining variation in use of information technologies in evidence based practice implementation outcomes of evidence-based quality improvement for depression in va community based outpatient clinics a model (cmbp) for collaboration between university college and nursing practice to promote research utilization in students' clinical placements: a pilot study moving beyond resistance to restraint minimization: a case study of change management in aged care process evaluation of appreciative inquiry to translate pain management evidence into pediatric nursing practice the effect of using high facilitation when implementing the gold standards framework in care homes programme: a cluster randomised controlled trial can oral healthcare for older people be embedded into routine community aged care practice? a realist evaluation using normalisation process theory the effects of an interprofessional patient-centered communication intervention for patients with communication disorders development, implementation, and outcomes of post-stroke mood assessment pathways: implications for social workers factors affecting adherence to use of hip protectors amongst residents of nursing homes--a correlation study if really we are committed things can change, starting from us": healthcare providers' perceptions of postpartum care and its potential for improvement in lowincome suburbs in dar es salaam implementation of a facilitation intervention to improve postpartum care in a low-resource suburb of dar es salaam implementing the best available evidence in early delirium identification in elderly hip surgery patients collaborative action around implementation in collaborations for leadership in applied health research and care: towards a programme theory facilitating implementation of research evidence (fire): an international cluster randomised controlled trial to evaluate two models of facilitation informed by the promoting action on research implementation in health services (parihs) framework statewide dissemination of trauma-focused cognitive-behavioral therapy (tf-cbt) getting evidence-based pressure ulcer prevention into practice: a multi-faceted unit-tailored intervention in a hospital setting implementation of evidence into practice for cancer-related fatigue management of hospitalized adult patients using the parihs framework outcomes and challenges in implementing hourly rounds to reduce falls in orthopedic units making cognitive decision support work: facilitating adoption, knowledge and behavior change through qi formative evaluation of a multi-component, education-based intervention to improve processes of end-of-life care pilot study for evidence-based nursing management: improving the levels of job satisfaction, organizational commitment, and intent to leave among nurses in turkey effectiveness of structured hourly nurse rounding on patient satisfaction and clinical outcomes intervening with practitioners to improve the quality of prevention: oneyear findings from a randomized trial of assets-getting to outcomes knowledge brokering as an intervention in paediatric rehabilitation practice improving the identification and management of chronic kidney disease in primary care: lessons from a staged improvement collaborative a collaborative project to improve identification and management of patients with chronic kidney disease in a primary care setting in greater manchester employing external facilitation to implement cognitive behavioral therapy in va clinics: a pilot study implementation of pediatric early warning score; adherence to guidelines and influence of context pilot to policy: statewide dissemination and implementation of evidence-based treatment for traumatized youth improving pain care through implementation of the stepped care model at a multisite community health center intervention to improve care at life's end in inpatient settings: the beacon trial implementation of a rapid chest pain protocol in the emergency department: a quality improvement project health care redesign for responsive behaviours-the behavioural supports ontario experience: lessons learned and keys to success can we help care providers communicate more effectively with persons having dementia living in long-term care homes? a theory-informed approach to mental health care capacity building for pharmacists partners in caring: an innovative nursing model of care delivery stopdvts: development and testing of a clinical assessment tool to guide nursing assessment of postoperative patients for deep vein thrombosis pressure ulcer awareness and prevention program: a quality improvement program through the canadian association of wound care clinical expert facilitators of evidence-based practice: a community hospital program geriatrics, interprofessional practice, and interorganizational collaboration: a knowledge-to-practice intervention for primary care teams implementing point of care "e-referrals" in clinics to increase access to a quit smoking internet system: the quit-primo and national dental pbrn hi-quit studies change in patient outcomes after augmenting a low-level implementation strategy in community practices that are slow to adopt a collaborative chronic care model: a cluster randomized implementation trial pain in hospitalized children: effect of a multidimensional knowledge translation strategy on pain process and clinical outcomes promoting physical therapists' of research evidence to inform clinical practice: part --theoretical foundation, evidence, and description of the peak program developing leadership in managers to facilitate the implementation of national guideline recommendations: a process evaluation of feasibility and usefulness promoting nurses' knowledge in evidence-based practice: do educational methods matter? study circles improve the precision in nutritional care in special accommodations institutional interventions to prevent and treat undernutrition evaluating implementation of methicillin-resistant staphylococcus aureus (mrsa) prevention guidelines in spinal cord injury centers using the parihs framework: a mixed methods study knowledge translation in uganda: a qualitative study of ugandan midwives' and managers' perceived relevance of the sub-elements of the context cornerstone in the parihs framework using stake's qualitative case study approach to explore implementation of evidence-based practice the hidden complexity of long-term care: how context mediates knowledge translation and use of best practices influence of organizational characteristics and context on research utilization predicting research use in nursing organizations: a multilevel analysis development and assessment of the alberta context tool. bmc advancing the argument for validity of the alberta context tool with healthcare aides in residential long-term care how does context influence collaborative decision-making for health services planning, delivery and evaluation? bmc an environmental scan of an aged care workplace using the parihs model: assessing preparedness for change organizational readiness to change assessment (orca): development of an instrument based on the promoting action on research in health services (parihs) framework implementation of coherent, evidencebased pathways in danish rehabilitation practice survey of providers' attitudes toward integrating smoking cessation treatment into posttraumatic stress disorder care development and testing of the context assessment index (cai) the interplay of contextual elements in implementation: an ethnographic case study assessing feasibility and acceptability of study procedures: getting ready for implementation of national stroke guidelines in out-patient health care measuring the context of care in an australian acute care hospital: a nurse survey health information use in home care: brainstorming barriers, facilitators, and recommendations. home health factors related to the implementation and use of an innovation in cancer surgery implementation of evidence-based psychotherapies for posttraumatic stress disorder in va specialty clinics an exploration of context and the use of evidencebased nonpharmacological practices in emergency departments clinicians' perception of patient readiness for treatment: an emerging theme in implementation science? improving the implementation of evidence-based practice and information systems in healthcare developing an instrument for evaluating implementation of clinical practice guidelines: a test-retest study does the 'diffusion of innovations' model enrich understanding of research use? case studies of the implementation of thrombolysis services for stroke evidence-based practice and determinants of research use in elderly care in sweden a psychological intervention (conquerfear) for treating fear of cancer recurrence: views of study therapists regarding sustainability digging into construction: social networks and their potential impact on knowledge transfer specifying an implementation framework for veterans affairs antimicrobial stewardship programmes: using a factor analysis approach a model for evaluating knowledge exchange in a network context knowledge brokers in a knowledge network: the case of seniors health research transfer network knowledge brokers the relationship between characteristics of context and research utilization in a pediatric setting facilitators and barriers to applying a national quality registry for quality improvement in stroke care implementation of safe patient handling in the u.s. veterans health system: a qualitative study of internal facilitators' perceptions occupational therapists' perceptions of implementing a client-centered intervention in close collaboration with researchers: a mixed methods study effects of computer reminders on complications of peripheral venous catheters and nurses' adherence to a guideline in paediatric care--a cluster randomised study implementation of smoking cessation treatment in vha substance use disorder residential treatment programs promoting consultation recording practice in oncology: identification of critical implementation factors and determination of patient benefit use of the parihs framework for retrospective and prospective implementation evaluations factors associated with using research evidence in national sport organisations virtual knowledge brokering: describing the roles and strategies used by knowledge brokers in a pediatric physiotherapy virtual community of practice danish translation and adaptation of the context assessment index with implications for evidence-based practice the influence of context 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remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors wish to acknowledge sayna bahraini, heledd owen griffiths, and veronica costea for partaking in the initial assessment of retrieved articles. all authors made substantial contributions to the manuscript. kg and ab conducted the citation searches. ab led the initial assessment. ab, ae, ace, idg, gh, ak, jrm, and lw developed the data extraction form and undertook data extraction in pairs of two. ab coordinated the data extraction. ab analyzed the descriptive data and ae, ace, gh, sh, ak, and lw analyzed the qualitative data. ab prepared figures and tables and drafted the manuscript together with lw, ae, ace, idg, kg, gh, sh, ak, and jrm revised the manuscript. all authors have read and gave final approval of the version of the manuscript submitted for publication. idg is a recipient of a cihr foundation grant (fdn # ) and ab the recipient of a forte grant (cofas- , - ) . the funders had no role in designing the study, retrieving, or analyzing included articles, decision to publish, or preparation of the manuscript. open access funding provided by uppsala university. the datasets generated and analyzed during the current study can be obtained through contacting the first author.ethics approval and consent to participate not applicable. not applicable. we acknowledge that gh, ak, and jrm were all involved in the development of the parihs framework. further, idg, jrm, and lw are all members of the bmc implementation science editorial board. key: cord- - tf dxc authors: gomez-diaz, teresa; recio, tomas title: a policy and legal open science framework: a proposal date: - - journal: nan doi: nan sha: doc_id: cord_uid: tf dxc our proposal of an open science definition as a political and legal framework where research outputs are shared and disseminated in order to be rendered visible, accessible, reusable is developed, standing over the concepts enhanced by the budapest open science initiative (boai), and by the free/open source software (foss) and open data movements. we elaborate this proposal through a detailed analysis of some selected ec policies, laws and the role of research evaluation practices. even if nowadays the open access, free/open source software (foss), open data, etc. (that we will call open science movements from now on) find more and more followers, adepts, and even addicts among the different key actors in the research population, our experience provides everyday examples of scientists that do not know well these movements and their consequences. some have an idealistic or anecdotical point of view, and many are still not really aware of the deep changes that they do carry on for the research practice. the use of licenses is well acknowledged in many places, but again, our experience provides examples of scientists that do not even possess a basic understanding of the license issues. many use software licenses under the advice of collegues that have no further understanding on licenses, author's rights and their consequences. but the use of a wrong license can have devasting results, oppossed to what it was initially expected. it is also well known within software developer communities that code writters do pay much more attention to the quality of their outcome if it is openly and freely available, and, thus, easily exposed to other developers' examination (and criticisms), which may have real consequences, for example, upon their career. likewise, researchers that put preprints in places like arxiv do pay special attention to the quality of the initial version in order to attract collaborators, citations, and maybe comments, that will improve the content of the initial work. another example of potential consequences of open science movements in the research practice appears when noticing the current evolution of the scholarly publishing system. indeed, there are clear signals that show that the old model dominated by few big (predator) editors is slowly becoming out of date. yet, part of the research evaluation system is still favoring publications in some journal titles selected under impact factor and science citation index criteria, something that can also become outdated, see for example the analysis in [ , , ] . to illustrate here some of the current publishing evolutions we can mention the recent adoption of an open roadmap by the association for computing machinery (acm) council as well as the code ocean integrations in the acm digital library to make software and data more discoverable . despite the increasing presence of open science policies and its benefits for the scientific community and the research practices, open science can be still considered a young issue requiring, in particular, a deeper understanding of the different ingredients that conform this movement. one of the oldest references on the open science subject is [ ] , that approaches this concept through the definition of closed science: closed science is defined as research which, in its production, communication, or utilization, is inaccessible to potential consumers. the grounds for such closure are always political, in the sense that certain interests, fortified by legitimate power, can exercise democratic control. the information denied to interested parties becomes the focus of a dispute or controversy which includes the means of control and ways of opening it. paul a. david places in [ ] the historical formation of key elements in the ethos and organizational structure of publicly funded 'open science' in the late sixteenth and early seventeenth centuries, where ... the idea and practice of 'open science' [...] represented a break from the previously dominant ethos of secrecy in the pursuit of nature's secrets, to a new set of norms, incentives, and organizational structures that reinforced scientific researchers' commitments to rapid disclosure of new knowledge. addressing as well its positive caracteristics [ ] (p. ): ... its economic and social efficiency properties in the pursuit of knowledge, and the supportive role played by norms that tend to reinforce cooperative behaviors among scientists. despite these ancient origins, and although open science best practices are currently fastly evolving, real barriers remain for its universal adoption. in particular, many areas of scientific research are still far from being open, as remarked in [ ] . in our opinion, one important reason for this circumstance has to do with the lack of a clear definition of what open science is, which difficults the dissemination of a persuasive message to a wider community of scientists. this deficiency is a well known issue. as stated in [ ]: ... there is no single, accepted, unified definition or vision of 'open science'... this perception is shared by the oecd international organization as it confirms in [ ] the absence of such generally accepted definition, while, at the same time, establishes the key ingredients to be used in the oecd study: open science. there is no formal definition of open science. in this report, the term refers to efforts by researchers, governments, research funding agencies or the scientific community itself to make the primary outputs of publicly funded research results -publications and the research data -publicly accessible in digital format with no or minimal restriction as a means for accelerating research; these efforts are in the interest of enhancing transparency and collaboration, and fostering innovation. contrasting with this imprecise scenery, the open science benefits for science and for scientists are widely accepted, as mentioned in [ ] , referring to benefits of public investment in research, or as listed in [ ]: ... greater transparency throughout the entire research process, including peer review ... to combat the 'reproducibility crisis', to expose or prevent research misconduct, to introduce greater accountability for researchers, or to increase the verifiability of the research record in order to engender greater public trust for the scientific enterprise... in the same direction, the oecd report [ ] provides further insight about open science benefits, including emerging estimations of the economic value of increasing accessibility to public sector research outputs. from a larger, worldwide perspective, the recent codata coordinated report [ ] emphasizes the relevance of open science in the starting century: open science is best characterised as the necessary transformation of scientific practice to adapt to the changes, challenges and opportunities of the st century digital era to advance knowledge and to improve our world. this requires changes in scientific culture, methodologies, institutions and infrastructures. note that this report also studies in its section some of the negative impacts of open science and how to address them. getting back to our main issue here, that is, on the need for a clear and broad definition, let us observe that the authors of [ ] explicitly acknowledge this fact: ... there is a lack of awareness about what open science is, mainly due to the fact that there is no formal definition of open science... after performing a thorough review of publications of the period - , ending up by expressing the need for further research to clarify this concept. it is our aim, in the present work, to contribute towards the fulfillment of this purpose. rather than to concentrate here in the philosophical aspects involved in the definition of open science [ ] , we will focus in more practical endeavours, represented by the sharing and dissemination conditions of the research production, or by the norms and rules governing disclosure of these research outputs (in the words of paul a. david) , that is norms of full disclosure and cooperation in the search for knowledge [ ] (p. ). thus, in the following section we will present the three fundamental components supporting our proposal of a coherent framework that develops open science policy and legal issues. section emphasizes the relevant role of licenses in this context. section describes, through some examples, our proposal for an open science framework comprising policies, laws and research evaluation practices. this work ends by describing the advantages of our proposal, which are argued in the final section and exemplified via the analysis of a recent case covered by the media. there is an extended literature studying open science key issues and, although it is a pending task, it is not the goal of our present work to provide a bibliographic survey on the foundations of open science. rather, in this section, we summarily present what we consider the three pillars where our open science vision stands over. we place our first pillar on the boai, the budapest open access initiative ( ) , that defines open access to the scholarly journal literature as follows: by "open access" to this literature, we mean its free availability on the public internet, permitting any users to read, download, copy, distribute, print, search, or link to the full texts of these articles, crawl them for indexing, pass them as data to software, or use them for any other lawful purpose, without financial, legal, or technical barriers other than those inseparable from gaining access to the internet itself. the only constraint on reproduction and distribution, and the only role for copyright in this domain, should be to give authors control over the integrity of their work and the right to be properly acknowledged and cited. let us remark that a historic vision, initiated by peter suber, of the open access movement can be found at the open access directory (oad) , with a pioneer online library dated back to . the open access movement launched by the boai was partially inspired by the free/libre/open source software movements (foss or floss ), which constitute, for us, the second pillar of our open science vision. free software was defined by the free software foundation (fsf), started by r. m. stallman ( ), as follows: "free software" means software that respects users' freedom and community. [...] the four essential freedoms. a program is free software if the program's users have the four essential freedoms: -the freedom to run the program as you wish, for any purpose (freedom ). -the freedom to study how the program works, and change it so it does your computing as you wish (freedom ). access to the source code is a precondition for this. -the freedom to redistribute copies so you can help others (freedom ). -the freedom to distribute copies of your modified versions to others (freedom ). by doing this you can give the whole community a chance to benefit from your changes. access to the source code is a precondition for this. the third pillar is based in the open data movement. as we are not aware of older initiatives, we place its initial step in codata, the committee on data for sciences and technology launched by the international council of scientific unions (icsu) in [ ] (p. ): codata est un comité au niveau scientifique international le plusélevé [...] a cause de l'importance qui s'attacheà l'évaluation des données [...] c'est un comité de coordination et sa principale tâche est de prendre des initiatives et de sougliner l'importance des aspects communsà plusieurs domaines de la science et de la technologie, ce qui comprend les activités suivantes: a) l'évaluation des méthodes de contrôle de la qualité, b) la définition des besoins des utilisateurs, c) les standards divers, d) les techniques de l'information [...] as we can see in [ ] (p. ), data issues were already part, by that time, of the international scientific considerations: the compilation of evaluated numerical and other quantitative scientific data is an important part of the general problem of a science information system which encompasses abstracting, storage and retrieval of unevaluated scientific information as well as the evaluation of this information in the form of selected and critical sets of quantitative data, including critical review papers. moreover, in [ ] , we can find a guide to procedures for the publication of thermodynamic data, which shows how data dissemination issues were, even then, at the heart of the codata considerations. a modern version of this kind of international initiative to deal with scientific data issues is the research data alliance (rda) , launched as a community-driven initiative in by the european commission, the united states government's national science foundation and national institute of standards and technology, and the australian government's department of innovation. rda's goal is to build the social and technical infrastructure to enable open sharing and re-use of data. beside these three key points that we have swiftly described, there are other scientific initiatives and movements that do enrich our open science vision and that we would like to mention here. this is the case of the reproducible research initiative : an article about computational science in a scientific publication is not the scholarship itself, it is merely advertising of the scholarship. the actual scholarship is the complete software development environment and the complete set of instructions which generated the figures. (d. donoho) as well as the open peer review studies and the current evolutions in its practices [ , , ]. our three open science pillars set the dissemination conditions for research outputs, where licenses play an important role. a software is free if it is released complying with the four freedoms of the above mentioned definition given by the fsf; and this compliance should be stated in the license, a (legal) document that is included in the set of files that constitute the software, and that comprises the source code, the compiled code, documentation, etc. the running, loading, reproducing, translating or arranging of a computer program can only be done upon the corresponding (written) authorisation [ ] , and, if there is not license, then "all rights are reserved". in particular no one outside the circle of the authors and the rightholders can run the software (legally speaking). a license sets then the sharing conditions of the disseminated software. . https://www.rd-alliance.org/about-rda . http://reproducibleresearch.net/ thus, another important point of the free software foundation was to develop the gnu gpl license, a license according to its philosophy and that accompanies its software production . this is a curious difference between the open access movement and the foss movement, as it took some time in the open access movement to speak about the use of licenses in the dissemination of written works, mainly mentioning the creative common (cc) licenses . once you have access to a document, there is not legal barrier to its reading. so the important point for the open access movement was to have access to the scientific literature. on the other hand, even if you have access to a software, its use can encounter legal barriers. this is why the first freedom (freedom ) of the free software definition is about the use of the software. on the other hand, freedom and freedom are related to the access to the source code in order to be able to study it and to produce new software. these principles correspond to the production of (new) scientific knowledge, which should be eased by the open access to the existing scientific literature. nowadays, despite these initial differences, the important role of licences is now clear for the open acces movement, as we can see in recent publications such as [ , ] . licenses are in fact the tool to set aside the legal barriers referred to in the boai open access definition. other important issues related to licenses that we introduce here deal with: corresponding to item ii), we remark that cc licenses are not adapted to software dissemination . software licenses can be found, for example, in the free software foundation (fsf) web site , in the open source initiative (osi) web site or in the software package data exchange (spdx) web site . on the other hand, we observe that the version . of the creative commons (cc) licenses was published in and was developed to be more user-friendly and more internationally robust, and to cover more explicitly sui generis database rights [ , ] so they can also be used in the dissemination of databases. finally, let us remark that, regarding item iii), each license sets its own sharing (legal) framework giving rights but also conditions that should be respected. in the case that the planned use of the research output does not agree with the default conditions given by the license, it may be possible to contact the rightholders to set other agreements or collaboration contexts, that is, other sharing conditions. we consider that a proposal for a sound open science framework has to declare, at least, its position concerning two basic issues: the related policies and legal aspects. in this section we describe our perspective on these core points, highlighting as well the consequences of our choices on a practical context that involves research evaluation practices. our proposal for a sound open science framework begins with the adoption of open access policies such as the ones decided by the european commission (ec) as an answer to the open access movement and that has have deep consequences in the ec research founding program horizon (h ). in its communication "towards better access to scientific information: boosting the benefits of public investments in research" [ ], the commision: ...sets out the action that intends to improve access to scientific information and to boost the benefits of public investment in research. it also explains how open access policies will be implemented under 'horizon ', the eu's framework programme for research and innovation ( - ). [...] to improve access to scientific information, member states, research funding bodies, researchers, scientific publishers, universities and their libraries, innovative industries, and society at large need to work together [...] so that the 'fifth freedom' of the eu -the free circulation of knowledgecan become a reality. give open access to copies of their publications even if they have granted the copyright to a publisher. this law also sets the legal framework for open data and goes along with a décret [ ] to list the licenses that should be used for data and software . other licenses are possible, but there is an homologation process to be applied . this french law has been followed by a policy document, the plan national pour la science ouverte published by the ministère de l'enseignement supérieur, de la recherche et de l'innovation. this plan contains three main axes dedicated to publication's open access, research open data and a sustainable, european and international dynamic framework. each axis establishes three mesures, including: . rendre obligatoire la publication en accès ouvert des articles et livres issus de recherches financées par appel d'offres sur fonds publics. . rendre obligatoire la diffusion ouverte des données de recherche issues de programmes financés par appelsà projets sur fonds publics. these two components (policies, legal) of our proposal are direct consequences of the emphasis of the open science movements on these issues. but still there is a long path to be built in order to make open science practices to become part of the everyday practices in research. in our view, there are three keystones that have to be considered to pave this path: -the required evolution of policies of universities and research performing organizations, . https://www.openaire.eu/ . https://www.openaire.eu/mission-and-vision . https://www.openaire.eu/os-eu-countries . the second chaper entitledÉconomie du savoir refers toécrit scientifique issu d'une activité de recherche financée au moins pour moitié par des dotations de l'etat, and, as a consequence, also includes software. . note that in here, software and data refer to the french law concept that can be larger and include the research outputs like software and data. . please note that the list of licenses available at h t t p s : / / w w w . d a t a . g o u v . f r / f r / l i c e n c es do not include the creative common licenses nor the european union public licence (eupl) for software, available at https://joinup.ec.europa.eu/collection/eupl/eupl-text-eupl- . -the development of open science-oriented infrastructures and services, -the transformation of evaluation policies and practices. he have already mentioned in this section the ec evolutions in open science policies and the consequent evolutions in national laws and policies. another example of open science policy evolution at large scale, perhaps one of the most relevant at the time of writting this work, is the unesco global consultation initiative on open science , that aims to build a unesco recommendation on open science, following the commision of the member states at the th session of unesco's general conference in order to develop an international standard-setting instrument on open science in the form of a unesco recommendation on open science (see also [ ] ). as a consequence, universities, funders and research organizations are reviewing their policies in order to adapt to law and policy changes and to adopt and implement an answer to open science requirements. to illustrate more in detail this first keystone, we would like to mention some examples of such evolutions. the first example corresponds to the local environment of one of the authors of this work. the newly named université gustave eiffel is the result of the fusion of several academic organizations including the université de paris-est marne-la-vallée (that hosts the laboratoire d'informatique gaspard-monge (ligm)) and the institut français des sciences et technologies des transports, de l'aménagement et des réseaux (ifst-tar). this universty has been launched on january st , and one of the first decisions of the research vice-présidence has been to start a working group in order to elaborate the open science policies of the university, the groupe de travail "politiques recherche ouverteà la société" (gtpros). this group counts with several subgroups, with two of them dedicated to data and software, counting with the participation of one of the authors of this paper. other examples to show university open science evolutions correspond to the göttingen eresearch alliance and the central role of the vilnius university library. the göttingen university institutional data management policy was published in , and then the eresearch alliance was established to serve research projects and provide direct support to researchers, combining library and it services and expertise [ ] . the vilnius university senate approved the regulations of open access to the university scientific works and the results of scientific research prepared by the vilnius university library in . the vilnius university library has then the task to develop the scholarly communication tool dedicated to sustain open access to information and open science [ ] . both articles highlight the great value of the direct collaboration with researchers, like, for example, embedding research-data managers into research teams (göttingen), or their participation into determine roadmaps and priorities for the infrastructures and services under development (vilnius) . both examples show as well how the evolution in policies goes along with the development of infrastructures and services to accompany researchers with their output's dissemination, as there is still a lot of work to be done -the second keystone -in order to tackle the current imbalance between already imposed legal requirements for researchers, and the still ongoing and unfinished work to build the necessary infrastructures and services. the human ressources in charge of developing the needed infrastructures and services are also simultaneously and increasingly acquiring the necessary knowledge and expertise to deal with these challenges. the institution decision-makers are in a similar evolving situation. as a consequence, researchers have to adapt (and sometimes to improvise with) their working mechanisms to the new policies and funding requirements, while contributing to their implementation by the expression of their new, urgent research needs. in this context, we can also mention the ifsttar experience of an institutional data deposit developed to answer the researchers' needs. its development towards building the institutional data deposit for the gustave eiffel university is currently under consideration at the gtpros. at european level, the european open science cloud (eosc), is the open science infrastructure currently under construction, see for example [ , ] . but the keystone that we analyse here in more detail is the necessary evolution in research evaluation practices, as it has been pointed out in [ ] , with procedures like the ones proposed in [ , ] . research evaluation happens in recruitment and career progression, and the institution's evaluation policies will drive the selection of best candidates with open science good practices. it is also a general practice of research . https://en.unesco.org/science-sustainable-future/open-science/consultation . https://www.univ-gustave-eiffel.fr/ . https://research-data.ifsttar.fr/ institutions and funders to evaluate the laboratories, institutes, research units... regularly, and evaluation policies and practices should evolve from now on to include best open science practices. research evaluation also happens in project funding: the funder policies do establish under which conditions the projects are selected for funding, as well as to give the instructions for free/open access of the project outputs. the final evaluation of the project assesses the quality of the submited work and verifies if the outputs are open in compliance with the open access instructions. in the case in which the outputs are not open, the evaluators should consider the alleged reasons for the avoidance of public access, but, in any case, they need to have access to the outputs in order to realize a sound evaluation. other evaluation contexts appear, for example, when looking for collaborators for a project, or for a publication, or in the selection of a journal for a publication. this is what we call here the "research community evaluation", in which the perception of a colleague (or of a journal) reputation plays an important role. for example, authors or reviewers can choose or avoid a journal following its open peer review practices. they can also stand for more "openess" when they are contacted for their participation in a new project. this is the power of the research community and the challenge of the reputation system. funders and institution evaluations, research community evaluations are therefore a powerful tool to enhance effective open science evolutions, and constitute, in our view, the third cornerstone in the open science path. but, as the cat biting its own tail, the evaluation wave can only play fully its role if policies and laws are well into place. in summary, without the necessary evolution in policies and laws, whithout a sound research evaluation system to enhance full compliance with the resulting policies and laws, it is not possible to achieve a solid fulfillness of the open science movements. on january the world health organization (who) declared that the sars-cov- outbreak constitutes a public health emergency of international concern (pheic) [ ] . as a consequence, the european commission stressed the urgency to provide immediate open access to their related publications, data and any other output possible for researchers receiving h funding in sars-cov- related research, following the guidelines of [ ] . in particular, the openaire project centralized in its web site the information comming from the zenodo community, from the openaire gateway, the rda covid- fast track working group and from other ec ongoing efforts. moreover, researchers, funders and some publishers expressed the importance of open access to research outcomes in this context, as we can see for example in the references cited by [ ] . but the urgency of any situation like this one should remain, more than ever, incompatible with speedy publications of poor scientific content. in the current pandemic context, many papers have been delivered in preprint servers without proper peer review, and some high-profile journals have published papers that have been retracted [ ] . moreover, as remarked in [ ] , some of the open access publications available on the publishers platforms have not license at all, and thus the publisher can revoke this kind of access at any time, and then, further study can be restricted in the future. among these publications, possibily the most unhappily famous is the one of the the lancet journal, that has been fully retracted . this publication deals with the use of the hydroxychloroquine drug for the treatment of covid- disease, as presented for example in the french media . let us recall that all the lancet journals endorse the wellcome trust statement to ensure that relevant research findings and data are shared rapidly and openly. we will no enter here in the scientific details of this work, as they can be found for example in this entry blog of the barcelona institute for global health (isglobal) . rather we consider here some of the involved open science aspects. in general, data gathered and used to produce a publication should be available for co-authors and for reviewers before publication, but maybe the data has ethical or personal issues, and then it should be available for a restricted number of persons in a restriced environment for its study in order to allow the validation of the work. reviewers should be given the correct time to referee a scientific work. yet, the covid- urgency has restrained the reviewing period to four days. but in the case this interval is considered too short for doing a correct assessment, reviewers should ask for more time or retract from the referee process. and this has not happen in the the lancet case. this affair shows the importance of having open access to publications, which has allowed, in this case, the rapid post-publication evaluation and a fast reaction of the scientific community, yielding the detection of several irregularities of this work and, thus, its retraction. it also shows the way to go forward in order to react after suffering these kind of problems. indeed, authors who have been involved in such situations, are prone to experience further issues when seeking for new collaborations or for new project funding, unless their research practices and accountability methods evolve to adopt and follow clear and transparent open science rules. similar implications can be formulated concerning journals, as they can be also exposed to negative "research community evaluation", being prompted then to adopt more sound open science best practices, like open peer review. we have initiated this article reflecting on the lack of a clear definition of open science, and on the need to contribute towards its clarification. as supported by the ideas developed in this work, it is our vision that open science is the framework that renders research outputs visible, accessible, reusable. thus, it is most important to analyse what outputs are diseminated and by who, and when. next, once the dissemination conditions have been finally decided, it is crucial to establish -in order to render the outputs visible, accessible, reusable -how the outputs are disseminated, in which sharing conditions, and in which places researchers will do the dissemination. as we have introduced in section and placed into context in section , the sharing conditions of the research outputs do involve scientific (political) movements, policies, and laws and legal issues (author rights, licenses). it involves also the producer's choices [ ] (when and where outputs are made accessible, and in which conditions they can be reused), and the places where the dissemination is realized, that can include journals (scientific journals publishing articles, data papers and/or software papers, etc.) and other infrastructures and services for preprints, data, software and other research outputs deposit, output search and retrieval interfaces, and that do provide or facilitate the outputs' reuse. therefore, we conclude that open science should refer to the political and legal framework where research outputs are shared and disseminated in order to be rendered visible, accessible, reusable. landscaping is evolving quickly nowadays, as policies and laws evolve at many levels, and it is becoming more common to have open access to scientific publications as well as to other research outputs like software and data. as mentioned in section , research evaluation is the tool to reinforce the basis of the open science political and legal framework, enabling it to reach its final goals, and, thus, spreading its benefits everywhere. evaluation happens every day at every level, from the selection of collaborators to the recruitment and career evolutions, and include research funding decisions. research evaluation is not the only keystone to enhance open science implementation, there is also a needed to develop the infrastructures in which the outputs are accessible and reutilisable, as well as the supporting services to accompany the researchers. these items (research evaluation practices, infrastructures and services) are evolving to adapt to this, somehow new, open science context, both at supra-national level (e.g. eosc) and at national and local levels which include the evolutions of universities and other research performing institutions. would have been more established this open science framework, the the lancet retracted publication would have neither been co-authored , nor passed the peer-reviewed stage, or accepted by editors for publication. in this paper we have introduced some examples of the pros and cons of the current progression of the open science movement. reacting to the declared need to advance toward a more clear understanding of the open science concept, we have highlighted three relevant intiatives (boai, foss, open data movements) that conform our open science vision, emphasizing their role on the dissemination procedures for research outputs, and supporting our vision of open science as the political and legal framework where research outputs are shared and disseminated in order to be rendered visible, accessible, reusable. the important role of licenses in this context has been reported in section as a key ingredient to understand our proposal for a sound open science framework that includes ec promoted open access policies and legal provisions such as the french digital republic act. we have as well developed the needed elements to put in to practice the proposed framework, placing the accent over its implementation with research evaluation practices. we have concluded bringing up some suggestions for policies, laws and evaluation systems to evolve in order to achieve the goals of open science. open access of covid- -related publications in the first quarter of : a preliminary study based in pubmed caj södergøard, klaus tochtermann, and ross wilkinson architectures of knowledge: the european open science cloud open science for a global transformation: codata coordinated submission to the unesco open science consultation open science and closed science: tradeoffs in a democracy the historical origins of 'open science': an essay on patronage, reputation and common agency contracting in the scientific revolution décret n. - du avril relatif aux licences de réutilisationà titre gratuit des informations publiques et aux modalités de leur homologation european commission, directorate-general for research & innovation. guidelines to the rules on open access to scientific publications and open access to research datain horizon covid- ), the severe acute respiratory syndrome coronavirus (sars-cov- ), and related topics: guidelines for open access to publications, data and other research outputs. version . opening science. the evolving guide on how the internet is changing research, collaboration and scholarly publishing spanish version: software libre, software de código abierto, licencias. donde se propone un procedimiento de distribución de software y datos de investigación logiciel : questions juridiques et de politique scientifique dans la production de logiciels. -bulletin de la société informatique de france on the evaluation of research software: the cdur procedure oldenburg's long shadow future of scholarly publishing and scholarly communication opening science with institutional repository: a data intelligence winter-spring corporate author(s): directorate-general for research and innovation (european commission). evaluation of research careers fully acknowledging open science practices. rewards, incentives and/or recognition for researchers practicing the peer reviewers' openness initiative: incentivizing open research practices through peer review making open science a reality. oecd science, technology and industry policy papers open science now: a systematic literature review for an integrated definition what is open peer review? a systematic review acknowledgements this work benefits of the interesting and fruitful exchanges at the gtpros working group of the university gustave eiffel. it is also partially funded by the vice-présidence international of this university. key: cord- -ad i wgj authors: nan title: th international congress on amino acids and proteins : vienna, austria, august – , date: journal: amino acids doi: . /s sha: doc_id: cord_uid: ad i wgj nan the raised concentration of protein bound homocysteine in homocystinuric (hcu) patients displaces protein bound cysteine and increases the free/bound cysteine ratio in plasma. this ratio is independent of albumin concentration. results from hcu patients were compared to controls. free cystine concentrations in hcu were poorly discriminated from the control range but the total cysteine results were almost invariably lower than control data. this appears to result from an increased free/bound cysteine ratio in hcu [mean (range) for control . ( . - . ) and for hcu . ( . - . ); p ϭ . ]. ex vivo protein binding experiments in albumin solution revealed the free/bound cysteine ratio to be linearly related to the amount of homocysteine bound (r ϭ . , p Ͻ . ). we conclude that measurement of total cysteine is essential for assessment of the true cysteine status in hcu. however, any cysteine deficit, or alteration to free/bound cysteine ratios, does not obviously effect glutathione synthesis as assessed by measurement of plasma total glutathione. ( nmol/g; . %); sprague-dawley rat (rattus norvegicus, rodentia) (n ϭ ; - nmol/g; . - . %); rabbit ( nmol/ g; . %); pig (sus scrofa f. domestica, artiodactyla) ( nmol/ g; . %); bovine (bos primigenius f. taurus, artiodactyla) ( nmol/g; . %); seal (phoca vitulina, carnivora) ( nmol/g; . %), and rob (halichoerus grypus, carnivora) ( nmol/g; . %). from the date it is concluded that d-aas are common in body fluids and certain tissues of vertebrates. in order to determine the quantity of cyst(e)ine and methionine, the oxidation of cyst(e)ine and methionine (Ϫ) refers to not detected or not determinable; asx ϭ asp ϩ asn; glx ϭ glu ϩ gln; his, arg, trp, cys not determined; a) feed fortified with dl-met; b) nmol/g lyophilized serum. mentation and subsequent purification. the separation of the enantiomers of cysteic acid, methionine sulphone, aspartic acid and glutamic acid is displayed on the chromatogram. into cysteic acid and methionine sulphone with performic acid is often applied before hydrolysis of protein. the authors examined the applicability of this process in case of quantification of cyst(e)ine and methionine enantiomers. the rp-hplc analytical method was developed for the determination of the amount of cysteic acid and methionine sulphone enantiomers. the rate of conversion during oxidation from cyst(e)ine into cystic acid and from methionine into methionine sulphone was determined. the racemisation of l-cyst(e)ine and l-methionine was negligible during oxidation with performic acid, therefore this process can be applied before hydrolysis during quantification of cyst(e)ine and methionine enantiomers. after the performic acid oxidation and the m hcl hydrolysis of the protein, opa/tatg (o-phthaldialdehyde/tetra-o-acetyl- -thio--d-glucopiranoside) precolumn derivatisation method was used, and the enantiomers of sulphur containing amino acids were separated by rp-hplc (lichrosphere rp- e, ϫ mm, µm column, merck-hitachi lachrom hplc). the resolution of the peak of cysteic acid and methionine sulphone enantiomers was better than , . the method was used to determine the amount of l-and d-cyst(e)ine and land d-methionine containing preparations prepared by fer- d/l rate of aspartic acid and the individual age of specimens. a method for age determination based on d-aspartic acid content and on the racemisation of l-aspartic acid of teeth was developed. d-glutamic acid, beside d-aspartic acid, was found to be eminently suitable for the estimation of individual age, as it showed a sufficiently high sensitivity. calibration curves based on these investigations were used for the age estimation of adults ( males and females) of unknown individual age from the avar period series of kereki-homokbánya (hungary). the age distribution of the sample was the following: individuals ( %) belonged to the adult age group, persons ( %) to the mature and ( %) to the senile one. the correlation between our results and those obtained using standard paleoanthropological methods was over . . quantitative determination of free and bound -nitrotyrosine in rat plasma and tissues using isotope dilution liquid chromatography-electrospray tandem mass spectrometry t. delatour, p. a. guy, j. richoz, j. vuichoud, and nestlé research centre, nestec ltd, vers-chez-les-blanc, lausanne, switzerland since -nitrotyrosine was reported to be readily formed in proteins by reactions with nitrite or nitrogen dioxide, it has been postulated to be a possible marker for investigating peroxynitrite-mediated nitration of proteins. thus, several methods were developed to assess nitration of tyrosine in proteins and determine -nitrotyrosine in physiological fluids. methods based on hplc or gc/ms techniques were described to quantify -nitrotyrosine within tissues or biological fluids. unfortunately, it has been demonstrated that an artifactual nitration of tyrosine occurs with gc/ms assays leading to an overestimation of the response. in the present work, lc-esi-ms/ms methods for quantification of free -nitrotyrosine in rat plasma as well as bound -nitrotyrosine in tissue samples are reported. plasma samples were spiked with , , -d - -nitrotyrosine and the following steps were applied prior to injection into the lc-esi-ms/ms system used in selected reaction monitoring (srm) mode (m/z ae for the analyte and m/z ae for the internal standard): protein precipitation, solid phase extraction on aminopropyl cartridge and derivatization in nbutanol in hcl n. -nitrotyrosine butyl ester has lead to a dramatic increase of the sensitivity (ca. -fold) by comparison with -nitrotyrosine. under such conditions, calibration curves exhibited excellent linearity (r Ͼ . ) within concentration range . to . nm (equivalent to . - , fmol on column) and recoveries above %. inter-and intra-assay precision was determined below % over the concentration range . to . nm. no artifactual nitration of tyrosine occurring during sample clean-up was observed. this was unambiguously established by plotting experimental ratio of analyte response/ internal standard response versus expected within the range . - . nm. this curve strongly correlated with a linear model (r Ͼ . ) and slope was . Ϯ . (mean Ϯ sd). basal level of -nitrotyrosine in rat plasma was measured to be within concentration range Ͻ lod to . nm. -nitrotyrosine basal level in rat plasma, kidney and liver proteins was established by performing enzymatic hydrolysis in order to avoid artifactual nitration of tyrosine which may occur under strong acidic conditions (hcl n at °c). resulting hydrolysates were analysed by lc-esi-ms/ms and nitrotyrosine was monitored in srm mode (m/z ae for the analyte and m/z ae for the internal standard). t. guszczynski , r. b. kapust , d. s. waugh , and t. d. copeland basic research laboratory, and macromolecular crystallography laboratory, national cancer institute at frederick, maryland, u.s.a. the set-can fusion gene was first detected as associated with acute undifferentiated leukemia. set (also called phap ii) is a nuclear phosphoprotein with a long acidic tail. set has been shown to inhibit phosphatase pp a and is a substrate of human granzyme a. in order to determine any zn(ii) binding properties of set, we utilized affinity capillary electrophoresis (ace) to detect shifts in mobility as zn(ii) ions bind to the protein. we have earlier employed ace to measure the binding constants of zn(ii) to the nucleocapsid protein of hiv- . with a constant concentration of recombinant set as a receptor and varying concentrations of zn(ii) as ligand in the sample buffer, we observed changes in electrophoretic mobilities of set when complexes were formed with zn(ii). scatchard analysis of the mobility provided the stoichiometry and binding constant of zn(ii) to set. interdisciplinary research center, institute of nutritional science, department of food sciences, university of giessen, germany peptaibols are defined as fungal polypeptides containing a high proportion of aib (α-aminoisobutyric acid) and a cterminal bound amino acohol. the mold trichoderma aureoviride (strain imi ; commonwealth mycological institute, kew, uk) was cultured in complex medium consisting of casein peptone, g; soy peptone, g; yeast extract, g; dglucose, . g; nacl, g; dipotassium hydrogen phosphate, . g in l demineralized water adjusted to ph . . fermentation was conducted in nineteen -l shake flasks, each containing ml medium, for d at °c. mycelia were obtained by filtration and extracted with meoh and meoh/chloroform. extracts were evaporated to dryness and subjected to sephadex and silica gel chromatography (eluent chloroform/meoh/acoh/water : : : ) yielding . g and . g, respectively, crude peptaibol mixture named trichoaureocins. the peptide mixture was uniform on tlc but could be separated by analytical (fig. ) and semipreparative hplc (nucleosil c- ; ϫ mm id; µm). six peptides could be isolated each of which was subjected to sequencing using on-line hplc (fluorocarbon stationary phase) esi-ms/ ms (lcq, thermoquest, finnigan mat) as described for peptaibols trichovirins and antiamoebins. sequences are presented in fig. . the -residue peptaibols represent a natural peptide library and cause hemolysis of sheep erythrocytes and exert antibiotic activity against bacillus subtilis and staphylococcus aureus. national institute of chemistry, ljubljana, slovenia wine consists of several hundred components present at different concentrations. the dominant ones are water, ethanol, glycerol, sugars, organic acids, and various ions, while amino acids are present at much lower concentration. the composition of amino acids is of great importance in wine production. they act as a source of nitrogen for yeast during fermentation, they influence the aromatic composition of wine and their composition can be used to differentiate wines according to vine variety, geographical origin, and year of production. among already established analytical methods high-field nmr has been shown to be a promising method for the nondestructive analysis of low-molecular mass compounds in complex mixtures like wine due to its selectivity and capability of simultaneously detecting a great number of compounds. h and c one-dimensional nmr spectra of wine are very crowded and many signals are overlapped. due to a great difference in concentration levels the signal intensities of particular compounds may vary for the factor of . the tails of the dominant frequencies of water, ethanol and glycerol obscure weak signals of minor compounds like amino acids in the near surroundings. the use of d homo-and heteronuclear experiments and the suppression of strong signals are a prerequisite for a successful h and c signal assignment. a complete assignment of h and c nmr resonances of seventeen amino acids commonly present in wine and of γ-aminobutyric acid at ph was accomplished using gradient-selected cosy, tocsy, gradientselected hsqc and hmqc experiments with incorporated wet pulse sequence for the supression of large signals. unambiguous assignment of h and c nmr resonances of amino acids is necessary for the selection of appropriate signals in fast and simple one-dimensional nmr that can serve as parameters in the chemometric classification of wines according to the provenance, vine variety, and year of production. institute of medical biochemistry, jagiellonian university collegium medicum, kraków, poland highly sensitive colorimetric method for determination of aldehydes in the reaction with n-methyl benzothiazolone hydrazone (mbth) turned out to be not very specific for such carbonyl compounds. namely, it has been found that tryptophan and to higher degree its n-derivatives (n-acetyl-trp, ala-trp, gly-trp) and also tripeptides (gly-trp-gly and leu-trp-leu) in the reaction with mbth and fe ϩ are converted to coloured products, with maximum wavelength at nm. the properties of the products and the kinetics of the reaction under defined conditions are described in the spectrophotometric procedure. proteins containing tryptophan are also substrates in the reaction with mbth. comparison of molar extinction coefficients of mbth-fe ϩ -treated various proteins with those of simple n-derivatives of tryptophan shows, that not all molecules of tryptophan in proteins are accessible to the reagents, and in order to determine all tryptophan moieties partial unfolding of protein has to be performed. it should be emphasized that aldehydes cannot be detected and accurately determined in the presence of tryptophan derivatives and protein, and also aldehydes interfere with determination of tryptophan derivatives. natural product laboratory, department of chemistry, the university of burdwan, w. bengal, india detection of protein amino is of utmost importance for the evaluation of protein structure and also their presence in numerous natural products. several specific and non-specific reagents have been used for their detection using thin-layer chromatography, an important tool for such purpose. of the reagents in general use, ninhydrin is the most popular for its high sensibility, however, nihydrin produces same purple color with most of the amino acids (only proline and hydroxproline produce yellow color). an endeavour has been made to resolve this color problem with a reagent which is capable of developing various distinguishable colors with many of the protein amino acids and also shows its high sensitivity comparable to ninhydrin. a probable mechanism for such color formation has also been proposed. measuring enrichments below the sensitivity range of conventional gc-ms. the gc-c-irms technique combines the resolution capabilities of gc with the accuracy and precision of irms. at low abundance gc-c-irms analysis it is superior in terms of time, labor, and sample requirement as compared to the conventional off-line analysis. we discuss some latest advancements and applications of gc-c-irms amino acid analysis related to nutrition research. plasma amino acids in omnivorous human subjects show a characteristic n-isotopic pattern with phenylalanine and threonine showing the lowest abundance, whereas e.g. alanine and leucine are higher by ‰ δ n. in rats fed diets containing intrinsically labeled c casein or the corresponding amino acid mixture labeled with c leucine and n lysine whole-body protein homeostasis is better supported by casein-bound than free amino acids. there is no adaptation to a low lysine diet by an enhanced bioavailability of intestinal microbial lysine to extra-splanchnic tissues in minipigs. highly selective hplc determination of tyrosine, tryptophan and their related compounds based on precolumn derivatization followed by intramolecular fluorescence resonance energy transfer detection h. nohta , m. yoshitake , h. yoshida , t. yoshitake , and m. yamaguchi faculty of pharmaceutical sciences, fukuoka university, nanakuma, johnan-ku, fukuoka, and chemical evaluation and research institute, ishii machi, hita, oita, japan we have developed highly selective hplc method for the determination of tyrosine, tryptophan and their related compounds (l-dopa, catecholamines, -hydroxytryptamine, etc.). the compounds were precolumn-derivatized with a commercially available fluorogenic reagent for amines by usual manner. each derivative afforded intramolecular fluorescence resonance energy transfer (fret) from the tyrosyl or tryptophoryl moiety (donor) to the labeled fluorophore (acceptor); the acceptor fluorescence was observed with the excitation of the donor at nm. the derivatives were separated on a reversedphase column and then effectively detected by monitoring their fret. through the screening study of fluorogenic reagents, o-phthalaldehyde (with -mercaptoethanol) and dansyl chloride gave the best results for the purpose. the fret detection method was highly selective and sensitive by comparison with the previous methods detecting native fluorescence of the compounds or typical fluorescence of the acceptor. the presented study was devoted to determination of the energetic effect of interactions in aqueous solutions between urea and neutral amino acid derivatives. the principal reason for studying of interactions of peptides with urea is the hope that such investigations will give insight into the factors affecting protein denaturation in aqueous solutions. the enthalpies of solution of n-acetylglycinamide, n-acetyl-l-alaninamide and n-acetyl-l-leucinamide were measured in water and in aqueous solutions of urea of molality . to . mol·kg Ϫ using the "isoperibol" type calorimeter at . k. from the obtained standard dissolution enthalpies ∆ sol h ϱ m the enthalpic pair interaction coefficients h xy for urea-nacetylamino acid amide pairs in water were calculated. these parameters derived from mcmillan-mayer theory are regarded as a measure of effect of interactions between solute molecules in solution. the h xy values for the systems investigated suggest that the interactions between urea and amide molecules dominate the effects of dehydration of nonelectrolyte and of peptides. the replacement of the hydrogen atom in the hydrocarbon chain with a methyl group causes a positive change in the value of the enthalpic pair interaction coefficient. the obtained results were compared with those of earlier studies of interactions between electrolytes, namely sodium chloride, potassium chloride and sodium iodide and the same n-acetylamino acid amides. the effect of the solute type on the magnitude of the interaction parameter was also analysed. the side chains of amino acids in solution react in various ways with the water molecules which surround them as well as with other components of solution depending on the fact whether they possess non-polar, polar or ionic groups. many research laboratories carry out studies intended to describe precisely the intermolecular interactions with the participation of amino acid side chains. such a description may allow one to describe better the spatial structures of protein and the mechanisms of folding its surface area. the present work reports the results of calorimetric measurements of the dilution enthalpies of l-α-amino acids in water. using modified mcmillan-mayer's theory, these results served to calculate the enthalpic homogeneous interaction coefficients which characterise interactions between the amino acid zwitterions with the competitive participation of water molecules. thus, these coefficients illustrate the differences in amino acid molecules interactions both with the homogeneous amino acid molecules and water molecules around them, and consequently they may play the part of a parameter which differentiates the hydrophobic/hydrophilic properties of amino acid side chains. the enthalpic interaction coefficients of the homogeneous pairs of l-α-amino acids were compared also with the hydrophobicity parameters obtained by fauchere et al., which describe the side substituents of natural amino acids as well as aminobutiric acid (aba). based on the above statement, one may conclude that the obtained enthalpic homogeneous pair interaction coefficients of l-α-amino acids in water make it possible to systematise amino acid side chains according to their affinity to water or their hydrophobic-hydrophilic properties. thus the enthalpic homogeneous pair interaction coefficients may play the role of parameter describing the lipophilicity (hydrophobicity) of amino acid side chains. compounds (iii) with amino acid ligands. in this work we present results of x-ray investigation of fourth amino acid complexes of rhenium (iii), which have different coordination of amino acids around binuclear complexforming center -re ϩ . substances (glyh) . h o -in inner, but gaba has cisposition according to re -re bond. influences of fatty radical length in the amino acid ligand on week interaction between binuclear anion [re cl ] Ϫ and protonized amino acid are discussed. role of hydrogen bonds in formation of crystal unit cell of investigated substances is shown. these two factors are the reason of formation of staggered conformation of an anion [re cl ] Ϫ in the substance (glyh) [re cl ]cl together with existence of quadruple re -re bond that is described first. in the substance [re (gaba) cl (h o)]cl . h o axial position of re ϩ fragment are substituted by ligands of different kind: h o and cl Ϫ -that says about possibilities to coordinate a substrate of biological nature exactly to these position. a precise, sensitive and reliable rp-hplc/uv method was developed to enable determination of α, and k caseins in cow's milk. the optimised method using a chrompack p- -rp column allowed separation of caseins in min. this column differs from conventional alkyl-bonded silica rp matrices in that it is an underivatised polystyrene-divinylbenzene matrix, a material which proved excellent chemical and ph stability. gradient elution was carried out at a flow rate of ml/min and a temperature of °c, using a mixture of two solvents. solvent a . % trifluoroacetic acid in water and solvent b was % acetonitrile- % water- . % trifluoroacetic acid. the effluent was monitored by a uv detector at nm. the determinations were performed in the linear range of . - . mg/ml for k-casein, . - . mg/ml for α-casein and . - . mg/ml for -casein. the detection limits were . , . and . mg/ml, respectively. the validity of the method was verified. the recoveries ranged from to % for cow's milk. the precision of the method was also evaluated, the % cv being less then . %. the developed methodology was also applied with success to the separation of caseins in ewe and goat milks. different chromatographic profiles were obtained for the three kinds of milk. department of aquatic biosciences, the university of tokyo, bunkyo-ku, tokyo, japan several aquatic crustaceans and bivalve molluscs accumulate a large amount of free d-alanine ( - µmol/g wet wt.) in their muscle tissues. during seawater acclimation from freshwater to % seawater, red swamp crayfish procambarus clarkii largely accumulated d-and l-alanine by . -and . fold, respectively, together with l-glutamine, l-proline, and glycine. the percentage of d-alanine to total alanine increased from % in freshwater to % in % seawater. these data indicate that d-and l-alanine are the major compatible osmolytes responsible for the intracellular isosmotic regulation of this species as well as other crustaceans. under anoxia stress for h in freshwater, and % seawater, crayfish increased d-and l-alanine in muscle and hepatopancreas in addition to the increase of lactate. the increase was much higher in seawater than in freshwater. thus, d-and l-alanine may be anaerobic end products during prolonged anoxia of this species. alanine racemase [ec . . . ] has been proved to catalyze the interconversion of d-and l-alanine in crustaceans and bivalve molluscs. this enzyme was isolated to homogeneity from the muscle of black tiger prawn penaeus monodon. the purification was , -fold with % yield. the molecular weight of the enzyme was estimated to be kda on sds-page and kda on gel filtration, suggesting the dimeric nature of this enzyme. the amino acid sequences of the peptide fragments obtained from the isolated enzyme showed low homology below % with those of microbial enzymes. syntheses and immunological effect of thymic humoral factor-γ analogues research laboratory, global shinwa pharmaceutical co. ltd., yoriki, matsuomura, iwate-gun, iwate-ken, japan nine analogues of thymic humoral factor (thf)-γ , were prepared by the solid-phase method and their in vitro restoring effect on the impaired blastogenic response of phytohemagglutinin (pha)-stimulated t-lymphocytes of uremic patients with infectious diseases were examined. the results were as follows: [arg ]-thf-γ exhibited higher restoring activity than that of our synthetic thf-γ . phylos has developed a powerful combinatorial biology platform for peptide and protein selections. phylos' proprietary profusion tm technology enables the selection of peptides and proteins with desired properties. the fundamental advance represented in this unique platform is the in vitro covalent linkage of a peptide or protein (phenotype) to the encoding messenger rna (genotype). this linkage permits the selection of a protein based on its characteristics and allows the recovery and amplification of that protein through pcr, an efficient means of bring the desired proteins to easily detectable levels. profusion tm technology has routinely selected peptide and protein binders with affinity constants in the nanomolar to picomolar range. the starting library size of randomized peptide or protein profusion tm constructs is typically . linear and constrained loop peptide libraries, for ligand generation, enzyme: substrate interaction, peptidomimetic design, and epitope mapping have been successfully used. randomized constrained loops have also been incorporated in a betasandwich scaffold, resulting in the successful selection of binders against targets of therapeutic interest. antigenic properties of three biological active de novo proteins were investigated by peptide scanning approach, using noncleavable multipin technology. a de novo protein albebetin (pid caa ) was engineered to attain a pre-designed d structure and later modified by grafting short peptide fragments from human α interferon (aag ), and insulin molecules (aag ). such protein constructs carrying important biological activities may be used in future as potential protein pharmaceuticals. despite artificial proteins are investigated for more than years, immunological properties of these substances are not known. in our experiments we applied an innovative approach of raising antibodies in yolks of egg-laying hens. three continuous antigenic determinants with different immunogenic potentials have been revealed in two proteins with partially overlapping sequences. it was shown that the octapeptide interferon fragment is the immunodominant site in albeferon and albeferon-insulin molecules. on the contrary, the hexapeptides, corresponding to the insulin fragment displayed low immunogenic activity. thus we recognise that the fragments attached to the de novo frame could essentially govern immunological properties of resulting construct. no preference of any type of secondary structure was observed in antigenic determinants. nevertheless, all of them are located at the boundaries of the secondary structure elements and on the predicted surface-located sites of albebetin molecule. peptide fragments from human α -interferon and insulin corresponding to the functionally important sites of their molecules were grafted into de novo protein albebetin (pid caa ) engineered to attain a pre-designed tertiary structure with a unique topology that has not been observed in natural proteins. by means of genetic engineering the dna fragments corresponding to these peptides were inserted into the albebetin gene to obtain two variants of albebetin with antiviral fragment of human α -interferon and two variants of albebetin with insulin-like peptide. the chimerical genes were expressed in escherichia coli in a fusion expression system with thioredoxin based on the plasmid pet- (novagen). the fusion proteins were digested by highly specific protease factor xa and the target chimerical proteins were purified and tested for their structure and biological activity. according to the cd spectroscopy study the chimerical proteins maintained the pre-designed structural properties of albebetin. toxicological testing of the proteins in the mtt-test did not reveal their cytotoxicity. antiviral activity of de novo proteins with human α -interferon fragments was studied in vitro using human fibroblasts cell line l- and simian cell line vero. treatment of these cell lines with the proteins revealed the dose-dependent stimulated antiviral activity on fibroblasts and direct dose-dependent antiviral activity on the vero cells. one of two de novo proteins including insulin-like fragment (pid aag ) acquired ability to stimulate glucose uptake by l- cells although the efficacy of stimulation was lower than that for the synthetic peptide and insulin. these results demonstrated that albebetin can be used as a scaffold for constructing of the functionally active de novo proteins possessing the pre-designed tertiary fold of albebetin and various biological activities. the identification of genes encoding unique tumor associated antigens (taas) has facilitated the development of novel immunotherapeutic strategies in cancer patients. clinical investigations have focused on targeting these cancer antigens for the generation of anti-tumor t-cell responses. taa epitopes come from differentiation antigens, from embryonal reexpressed or overexpressed proteins, from mutated proteins and from viral proteins in viraly associated tumors. we have recently developed a novel screening system for identification of immunogenic and antigenic ctl peptide epitopes using d b-/-x m -/double knockout mice, transgenic for a single-chain hla-a - m molecule (hhd mice). specific ctl were derived by immunization of hhd mice with tumor peptide extracts loaded on antigen presenting cells and with hhd transfected human tumor cell lines ctl induced against peptides from various tumors recognized tumor peptides more effectively than peptides extracted from normal tissues and also reacted with a serie of peptides derived from overexpressed candidate proteins, identified by differential display methods (sage, microarrays) comparison of ctl derived from hhd mice to ctl induced from patient's pbmc showed overlapping recognition of many candidate peptides. using these hhd mouse derived ctl we identified novel peptide sequences from prostate, bladder, breast and colon carcinomas, antigens pap and steap, from breast carcinoma antigens muc and ba - . analysis of tumor differentially expressed genes by the sage method in colon, followed by screening for hla-a binding peptides resulted in candidate peptides for immunogenicity screening. we have identified antigenic peptides of which peptides were found to be immunogenic in hhd mice. interestingly of these peptides are derived from the same protein. differential expression studies, using "dna chips" were performed on prostate and bladder tumors versus normal tissues. ten new candidate genes from tcc were analysed for expression and potential immunogenic peptides. novel peptides from uroplakins and from mage- were identified. surface plasmon resonance biosensing in the study of viral antigenic sites mimicked by synthetic peptides p. gomes , e. giralt , and d. andreu centro de investigação em química da universidade do porto, portugal department de química orgànica, universitat de barcelona, spain antigen-antibody binding has been regarded as one of the most representative examples of specific molecular recognition in nature. the simplistic view of antigenic recognition in terms of a lock-and-key mechanism is superseded, since it is now evident that both antigens and antibodies are flexible and can undergo substantial mutual adaptation. this flexibility is the source of complexities such as degeneracy and non-additivity in antigenic recognition. we have used surface plasmon resonance to study the effects of combining multiple amino acid replacements within the sequence of the antigenic gh loop of foot and mouth disease virus. our aim was two-fold: to explore to what extent can antigenic degeneracy be extended in this particular case, and to search for potential non-additive effects in introducing multiple amino acid replacements. combined analysis of one such multiply substituted peptide by spr, solution nmr and x-ray diffraction shows that antigenic degeneracy can be expected as long as residues directly interacting with the paratope are conserved and the peptide bioactive folding is unaltered. structural properties of creatine kinase from amphioxus, branchiostoma belcheri gray f. inoue, s. obase, t. suzuki, and t. imai department of physiological chemistry, faculty of sciences, toho university, funabashi, japan to further our knowledge of creatine kinase (ck) in the fields of molecular evolution and comparative enzymology, we analyzed the ck gene of the protochordate amphioxus. amphioxus is thought to be the phylogenetic predecessor of vertebrates and thus possesses characteristics, such as enzymological properties, that are associated with ancestral vertebrates. the results clarified the sequence of bases including the active site. the homology of the active site and the surrounding bases for the amphioxus ck gene to that of the human and electric ray ck-m gene was . % and . %, respectively. the amino acid sequence of this region of amphioxus ck was also identical to that of human and electric ray ck-m. in addition, the estimated secondary structure of amphioxus ck was compared to that of human and electric ray. there were no marked differences in the relative ratio of the α helix, sheet and turn structures for the peptide structure of ck consisting of amino acid residues. there was a high degree of homology in the sequence of amino acid residues (met ϳhis ) near the active site of ck between amphioxus and other organisms, suggesting that this region of ck is functionally essential for transphosphorylation. gelsolin is a ca ϩ -activated and phosphoinsitide-regulated cytoskeletal actin-binding-and-severing protein, its fragments - : ksglkykk (g - ) and - khvvpnev vvqrlfqvkgrr (g - ), are responsible for the binding of this protein to actin and the cellular messenger phosphatidylinositol , -bisphosphate (pip ). the binding of peptides g - and g - to a cluster of four pip molecules in a dimyristoyl-phosphatidylcholine lipid was in vestigated by means of molecular-dynamics (md) simulations of , ps. the binding of the pip molecules to the peptides g - , g - showed both electrostatic and hydrophobic nature: lysine residues of the peptides formed salt bridges with the phosphate groups of the pip molecules, while hydrophobic interactions occurred between the nonpolar residues of the peptides and the fatty-acid tails of pip . during the binding some of the pip molecules were dragged out of the lipid, thus disrupting the bilayer. after the binding dissociated a draggen-out pip molecule tend to incooporate back to the lipid. division of applied physiology, institute of veterinary physiology, university of zürich, switzerland chemical modification of the proteins: bovine serum albumin, α-lactalbumin, -lactoglobulin and chicken egg white lysozyme by -hydroxyphthalic anhydride ( hp) yielded compounds which exerted antiviral activity in vitro as compared with the native unmodified proteins. of the three enveloped viruses tested: human herpes simplex virus (hsv- ), bovine parainfluenza virus (pi- ) and porcine respiratory corona virus (prcv), the hp proteins were shown to be active against human herpes simplex virus only indicating that a perturbation of the viral envelope is unlikely. pre-incubation of vero cells with hp-albumin, hp--lactoglobulin and hplysozyme resulted in protection against hsv- infection whereas pre-incubation with hp-α-lactalbumin had no antiviral effect. however, all hp modified proteins showed a more significant inhibition when present during or after the viral infection step. thus multiple mechanisms appear to be involved in the inhibition of hsv- infection. the blocking of cell receptors may contribute to the antiviral activity as shown by the preincubation data. however, a direct interaction between the modified proteins and the hsv- glycoproteins responsible for viral entry and spread, seems to play a more important role, as indicated by the smaller ec values obtained during and after the infection. a dummy or a protagonist on the stage of inflammation? r&d department, zambon group, bresso, milan, italy amino acids are usually present in large excess in healthy and the excess is used as source of calories. however, metabolic alterations are observed in ill patients and preferential retention of sulphur amino acids (saa) occurs during the inflammatory response. the metabolism of cysteine is modified during the acute phase of sepsis in rats. sulphate production is lower, whereas the higher liver production of taurine seems to play a protective role; glutathione concentration is greater in liver, kidney and other organs and cysteine incorporation into proteins was higher in spleen, lung and plasma (acute phase proteins) while albumin level decreases. another important phenomenon is the impairment of methionine conversion to cysteine during stressed condition. premature infants or hiv patients synthesise cysteine from methionine at a much lower rate. thus, the metabolic flow through the trans-sulphuration pathway may be insufficient to meet the glutathione and cysteine requirement in critical conditions. the pro-inflammatory cytokines, interleukin- , interleukin- and tnf-α are the main initiates that alter protein and amino acid metabolism. in this complex picture, saa supply may contribute to the immune system regulation. our previous investigations showed some biological activity of newly synthesized cluster rhenium compoundtetrachlorodi-µ-(γ-aminobutirato)dirhenium(iii) chloride -i such as antitumour activity, cell-stabilizing activity against osmotic hemolysis, changing of morphology of cells, and other. there exists some information about stabilizing effects of some metal-organic substances with antitumour properties on the isolated ishaemic-reperfused rat heart (leperre a. ) throughout decrease of malonaldehyde (mda) production. some new investigations showed the influence of metal-organic substances on apoptotic processes (winter b. , syrkin a. , that are considered now as the main mechanism of such tissue damages as ishaemia, myocardial infarct, etc. thus we tried to analyze such activity of i. two models of hemolytic anemia was used: a -on rabbits by introducing of pbac -solutions; this model permits to investigate dynamics of anemia in one experimental animal; bon rats by introducing of phenylhydrazine chloride. i was administrated as in solution as in lyposomic (lyp) forms. all measurements and models were accomplished according to described procedures. administration of i led to: increase of hemoglobin and resistance of erythrocytes and to prolonging of life for hemolytic animals; significant decrease in quantities of mda and increase in quantities of reduced glutathion (gsh), glutathionreductase (gsr) and glutathionperoxidase (gsp) in myocardium, blood, brain, liver, splenic and entherocites of anemic animals. the most effective was i in lyposomic form. mechanism of antioxidant action of rhenium cluster compound is speculated and experiments with some well-known antioxidants to compare with i are working out. at present problem of finding remedies against the mostly dangerous human disease -aids is one of higher interest. the aim of this work was the investigation of inhibiting effect of high-pure l-lysine-α-oxidase (lo) e.c. . . . , extracted from trichoderma sp., on hiv-virus reproduction, comparatively to azidotymidin (azt), being now in use for treatment of aids-patients. for studying of inhibiting effect of lo, the mt- cells, sensitive to citopathical action of virus, were used. the experimental studying has shown, that the enzyme at concentration - ng/ml suppresses hiv reproduction and synthesis of virus' proteins, not exerting toxical effect on mt- cells. toxical dose of lo has been determined preliminary. a comparison with standard preparation -azidotymidin, which causes suppression of virus reproduction at concentration mkm ( , mg/ml) not exerting toxical effect on mt- cells. the same effect is attained having used lo in doses - ng/ml. using lower concentrations of enzyme leads to partial increasing of virus' titre comparatively to control cultures. obtained data allow to conclude that lo from trichoderma sp. is more high specific agent than azidotymidin, because it needs times lower concentration for the same action. comparison of azt and lo action on synthesis of virus' antigens presenting in cultural media of mt- cells infected with virus, leads to conclusion, that lo has inhibitory action both on virus' reproduction and virus' protein synthesis. department of microbiology, dokkyo university school of medicine, mibu, tochigi, japan our previous studies showed that the cellular amino acid composition obtained by amino acid analysis of whole cells, differs in various organisms. these results suggest that the difference in the cellular amino acid composition reflects biological evolution. however, the basic pattern of cellular amino acid composition is relatively constant in all organisms, and the cellular amino acid compositions of the archaeobacteria are quite similar to those determined from codon usage data, based on the complete genomes. in the present study, the free amino acid compositions in archaeobacteria, eubacteria, protozoa, blue-green alga, green alga, slime mold, plants and mammalian cells were analyzed, to investigate whether changes in their free amino acid compositions reflected biological evolution. cell homogenates were treated with - % ethanol to separate cellular proteins and free amino acids contained in the cells. rat hepatoma cells (r-y b) were cultured in eagle's minimum essential medium (mem) containing % serum or in a modified mem lacking arginine, tyrosine and glutamine. no significant difference in the free amino acid composition was observed between the two cell groups cultured under two different conditions. the patterns of the free amino acid compositions differed completely from those of the cellular amino acid compositions, and from each other in various organisms. characteristic differences were observed between plant and mammalian cells, and between archaeobacteria and eubacteria. the patterns of the free amino acid composition in blue-green alga, green alga, protozoa and slime mold differed from each other and from those of eubacteria and archaea cells. it has been suggested that the free amino acid composition reflects apparent biological changes as the result of evolution. ) catalyzes the hydrolysis of gamma-glutamyl compounds such as glutathione, and the transfer of their gamma-glutamyl moieties to amino acids and peptides. we previously developed enzymatic methods for the synthesis of various gammaglutamylamino acids using the transfer reaction of ggt from e. coli k- as a catalyst. it has been reported that gamma-lglutamyltaurine has a potent and long-lasting antiepileptic action, and its chemical synthesis has also been reported, but it required protecting and deblocking of reactive groups. thus, the purpose of this study was to develop an enzymatic method for the synthesis of gamma-l-glutamyltaurine using ggt. the optimum reaction condition was mm l-glutamine, mm taurine and . unit/ml ggt, ph , and -hr incubation of °c. forty-three mm gamma-glutamyltaurine was obtained and the yield was .%. gamma-glutamyltaurine was purified by dowex ϫ column and c column, and then identified with gamma-l-glutamyltaurine by nmr and polarimeter. in this study the yield of gamma-l-glutamyltaurine was comparatively low because synthesized gamma-lglutamyltaurine was promptly converted into the by-product, gamma-l-glutamyl-gamma-l-glutamyltaurine. the production of antimicrobial peptides is an important aspect of host defense in animals ranging from insects to mammals. they do not target specific molecular receptors on the microbial surface, but rather assume amphipathic structures that allow them to interact directly with microbial membranes, which they can rapidly permeabilize. they are thus perceived to be one promising solution to the growing problem of microbial resistance to conventional antibiotics. insects express a battery of potent antimicrobial proteins in response to injury and infec-tion. until now, approximately immune peptides have been characterized from insects and other invertebrates. an antimicrobial gene (md-cecropin) belonging to cecropin family was cloned from the bacteria-charged adult house fly, musca domestica. expressed in the vector pgex- t . mrna was isolated and degenerated primers were designed according to the conserved sequences of cecropins. the full-length cdna encoding md-cecropin was cloned by rt-pcr and Ј, Ј-race and sequenced. the deduced amino acid sequence indicated that a prepeptide with amino acid residues is first translated and then processed to a mature peptide with amino acids. the dna encoding the mature peptide was subcloned into expression vector pgex- t , and expressed efficiently in e. coli bl as a fusion protein. the fusion protein was purified and specifically digested and the md-cecropin was further purified to homogeneity and the activity spectrum was investigated. escherichia coli with metabolic engineering methods l. yun, x. zhang, s. wang, q. xu, and l. ma biotechnology laboratory, institute of beijing radiation medicine, beijing, p.r. china a bioengineering escherichia coli strain was obtained by metabolic engineering method. three genes related to the biosynthesis of phenylalanine, arog, phea, and tyrb encoded key enzymes: -deoxy-d-arabino-heptulonate- -phosphate synthetase (ds), a bifunctional protein-chorismate mutase (cm)/prephenate dehydratase (pd) and aminotransferase (at), respectively. in this work, the feedback inhibition of ds and cm/pd were relieved by site-directed mutagenesis on bases of homology comparison of related sequences of the key enzyme. the feedback inhibition resistant genes encoding ratelimiting enzymes in the main and terminal pathways were amplified by co-expressed in order of arog-phea-tyrb on the plasmid by their own operator plpr, pl, and pr. in the recombinant strain showed great resistant to the l-phenylalanine analogues, the specific activities of ds, cm, pd and at were increased by . , . , . and . folds, respectively. as the result, the amount of phenyalalnine biosynthesis of the bioengineered strain was increased greatly compared with that of the host strain. an enzymatic approach for the mapping of phosphoproteins resolved on two-dimensional polyacrylamide gels hiroshima proteome laboratory, regional science promoter program, kagamiyama higashihiroshima, japan an enzymatic approach for high-throughput mapping of phosphorylated proteins resolved on two-dimensional ( -d) polyacrylamide gels is presented. proteins of cultured rat skin fibroblasts were divided into two aliquots, one of which was dephosphorylated using recombinant protein phosphatase. the two aliquots were then subjected to -d electrophoresis. the phosphoproteins could be mapped on the -d gel by com-paring the gels of the phosphatase-and non-treated samples, because the dephosphorylated proteins shifted to more basic positions on the gel. this technique revealed that approximately % of the detectable proteins were phosphorylated. fifteen phosphoproteins were identified by mass spectrometry, including proteasome component c and small glutamine-rich tetratricopeptide repeat-containing protein. furthermore, the extent of phosphorylation of two actin-modulating proteins, destrin and cofilin, was found to be significantly reduced when the cells were chemically or enzymatically detached from the culture dishes. the presented technique can be applied to all biological materials because it requires no protein-labeling step, and is therefore useful for high-throughput mapping of phosphoproteins in proteome research. with the completion of the human genome sequence maldi-tof-ms is increasingly becoming an established method for identification of proteins separated by d gel electrophoresis. mono-isotopic peptide mass fingerprinting (pmf) has been previously shown to be amenable to full automation encompassing the process of acquisition, data processing and databank searching under full software control. until now the throughput of maldi-tof-ms for proteomics has been limited to several hundred samples in a working day and this represents approximately - % of the total proteins resolved by a large format d gel. to reduce the number of proteins to be identified the d gels are imaged and analysed to determine differences in expression levels within a set of gels. although much of the image processing is semiautomated the comparison is labour intensive as manual pattern matching has a role in the gel alignments (land marking). increased ms sample throughput allows the possibility of identifying every protein spot in a d gel within a day. this could eliminate the potentially erroneous step of human gel image alignment, whereby land marking could be achieved using the ms data. increased sample throughput requires greater capacity and robust unattended instrument operation. in this poster we describe an integrated robotic multiple plate loader that allows overnight unattended ms operation. other improvements include an increased laser repetition rate that allows the data capture rate to increase four fold. sample tracking, data archiving and data reporting are essential attributes of this new technology and these aspects are outlined in the presentation. the proteinchip tm biology system for ciphergen biosystems: a novel proteomics platform for rapid biomarker discovery, validation and identification ciphergen biosystems ltd., surrey technology centre, the surrey research park, guildford, surrey, u.k. the proteinchip system uses seldi (surface enhanced laser desorption/ionization) proteinchip technology to perform the separation, mass detection and analysis of proteins at the femtomole level directly from biological samples. surfaces are based on either chromatographic based chemistry (ion exchange, reverse phase, imac etc.) that bind large classes of proteins or biologically defined surfaces (antibodies, dna, receptors, etc.) that are used to investigate specific proteininteraction events. as with conventional elution chromatography each type of surface is designed to bind a different subset of proteins from a crude mixture. sample complexity is reduced on the surface by washing with standard biological buffers compatible with the chosen proteinchip array. unlike elution chromatography, proteins are detected directly from the stationary phase using laser based mass spectrometry greatly increasing throughput whilst reducing sample loss and improving reproducibility. multiple proteinchip surface and wash conditions are explored with a small sample set to resolve hundreds of proteins and establish assay conditions that reveal candidate biomarkers or diagnostic protein profiles. the resulting custom built assay is then used to monitor disease processes or drug toxicity profiles by screening large banks of samples such as tissue extracts or physiological fluids (serum, urine, csf, etc.). pharmaceutical research, genomics technologies, f. hoffmann-la roche ltd., basel, switzerland to the present, samples representing the total protein mixture have been usually analyzed by proteomics technologies mainly only the abundant, hydrophilic components have been visualized. these proteins could be solubilized with reagents compatible with isoelectric focusing, for example urea and chaps. such an analysis provides us with a limited image of the proteome, which is insufficient for the detection of the majority of the proteins. in a -d gel, where about mg of protein amount has been resolved, , - , protein spots can be detected, using coomassie blue staining. the spots represent the products of only - different genes. other gene products, not visualized, are most likely expressed at too low levels for detection or they can not be identified because of limitations of the current technology, they are too small, too large, basic or hydrophobic. here we will discuss protein enrichment approaches prior to the analysis, which we have applied for the enrichment of bacterial and eukaryotic proteins. proteomic analysis of the rat liver mitochondrial proteins m. fountoulakis , j.-f. juranville , and l. suter genomics technologies, and drug safety, f. hoffmann-la roche ltd., pharmaceutical research, basel, switzerland subcellular fractionation increases the probability of detection of low-abundance proteins. we prepared mitochondrial, microsomal and cytosolic protein fractions from total liver of male rats. the proteins of the three fractions were analyzed by two-dimensional electrophoresis using broad and narrow ph range immobilized ph gradient strips. the proteins were identified by martix-assisted laser desorption ionization mass spectrometry. in the mitochondrial fraction, different gene products were detected. approximately % of the identified mitochondrial proteins are enzymes with a broad spectrum of catalytic activities. most of the identified proteins had been detected before in other samples as well, analyzed in our laboratory. eight gene products were detected for the first time. these were represented by one spot each, whereas most of the frequently detected proteins were represented by multiple spots. in average, approximately spots corresponded to one gene product. centre for molecular medicine, university college london, u.k. three kinds of experiments have been carried out successfully in our labs. ( ) identification of post-translational modifications of the endothelin a and b receptors (etar and etbr) including both phosphorylation and acylation. we have developed new, very efficient methods for single step isolation of highly pure etar and etbr from cells. this has allowed us to obtain evidence that the post-translational modifications are very complex and result in multiple phenotypes showing different forms of modification for receptor. as with other systems, e.g. insulin-like growth factors, it is probable that these multiple phenotypes of the et receptors correspond to different forms of signalling dependent on cellular state, e.g. the cell cycle. it is, for example, already clear from the phosphorylation of the receptor that a series of different kinases must be involved. ( ) following stimulation of fibroblasts with endothelin, phosphorylation/dephosphorylation signalling cascades involving several hundred proteins have been observed by use of high resolution d electrophoresis and detection of phosphorylated proteins labeled with p by autoradiography or immunological methods. the large number of proteins involved are being identified by mass spectrometric methods such as mass fingerprinting or sequencing by mass spectropmetry. ( ) differential gene expression has been followed by using s met pulse chase labelling concurrently with endothelin stimulation. at least proteins showed significant changes in expression of d gels and these proteins are also being identified. these experiments demonstrate that it is now possible to use proteomics methods to investigate the integration of response to an extracellular signal at the levels of the receptor itself, the subsequent signalling cascades and the ensuing gene expression. the proteomics technology permits concurrent monitoring of large numbers of protein phenotypes (the forms and amounts of individual proteins and is therefore able to provide a global overview of signalling processes which greatly augments more traditional investigations of individual proteins or pathways. furthermore, these new methods will allow quantitative determination of the changes in protein phenotypes, which is very important in view of the highly non-linear amplification properties of such signalling processes. an integrated approach to automated high throughput protein identification by d gel electrophoresis and mass spectrometry d. gostick , s. cohen , p. young , b. karol , j. langridge , j. randell , t. slyker , and a. jacobson micromass, manchester, u.k. waters corporation, milford, massachusetts, and bio-rad laboratories, hercules, california, u.s.a. establishing the function of gene products is the major challenge of the post genomic era. the rate-limiting step in this endeavour is the speed with which proteins can be isolated and identified. separation of proteins from cell lysates or sub-cellular domains by d gel electrophoresis is an established method of visualising these complex systems. recently mass spectrometry has proved to be a powerful method of further characterising these proteins. from the mass spectrum of the enzyme digest of a d gel spot, the resulting digest map is compared with the theoretical maps from the databases and the protein identified when these correlate. maldi-tof is of great benefit in these studies since it requires a minimal amount of sample, is relatively tolerant to salts and other contaminants arising from the gel and may be configured for automated sample analysis. high sample throughput with automated analyses including data processing and client-server database searching are already available. our system automatically acquires the data and processes the maldi mass spectrum into a monoisotopic peak list. this peak list is then automatically sent to a networked database for protein identification. when proteins are not identified from the maldi analysis or an ambiguous result is obtained, then further analysis of the sample by electrospray caplc-ms-ms is required. the development of a hybrid quadrupole orthogonal acceleration timeof-flight mass spectrometer (micromass, q-tof) has facilitated the generation of unambiguous amino acid sequences from the ms-ms analyses of tryptic peptides. these ms-ms spectra can be automatically searched against protein, nucleotide or est databases. thus enabling protein identification from gel spots, despite non-specific enzymatic cleavage, protein co-migration and post transitional modifications. for organisms who's genome sequences are poorly represented in the data bases de novo amino acid sequencing may be required. inferring de novo peptide sequences from ms-ms data is complex and is often the rate-determining step in this method. however, it is now possible to interpret the ms-ms spectrum automatically. in our approach the raw ms-ms spectrum is reduced to the plausible single-charge, monoisotopic mass spectrum. sequence interpretation is achieved by generating "trial sequences" consistent with the experimentally determined molecular weight. a probabilistic fragmentation model is used to transform the trial sequences to predicted spectra for comparison to the single-charge, monoisotopic spectrum and to calculate the likelihood that the trial sequence would account for the observed data. the possible number of trial sequences for any peptide is large, for example there are possible sequences for a peptide containing any of the naturally occurring amino acids and having residues. to reduce the scale of the problem a terminated markov chain monte carlo algorithm is used to produce sequences. this bayesian method simulates an exhaustive search of all sequences having the correct mass. the huge increase in genomic sequence information available, combined with the increased sensitivity and selectivity provided by mass spectrometry, has allowed large-scale protein identification. however the analysis of the post translational modifications present on the identified proteins is a more challenging problem. currently the approach that offers the most expedient and specific solution, to determine modified peptides, is precursor ion scanning. this approach has primarily been performed on a triple quadrupole mass spectrometer where the rear quadrupole, (ms ) is set to transmit only the fragment ion of interest. the ms quadrupole is then scanned across the appropriate mass to charge range. in this paper we describe a method that allows specific post translationally modified peptides to be identified and sequenced during the course of an hplc experiment on the q-tof mass spectrometer. during the hplc run the instrument is switched alternately at one-second intervals between low and high collision energy with argon in the collision cell. the quadrupole, ms is not mass selective, operating in the rf only mode. the first data set at low energy ( ev) shows only the normal pseudo molecular ions. the second at higher energy shows their fragments. wherever a product ion of interest occurs in the high-energy data all its possible precursors are revealed by the corresponding ev data. since the two data sets contain the entire set of precursor and product ions that can be formed it is clearly possible to generate the equivalent of a constant neutral loss scan. this is invaluable in the case of phosphorylated peptides where the neutral loss of da (h po ) occurs via -elimination from the phosphoserine and phosphothreonine residues. this allows the q-tof mass spectrometer to switch from the ms mode to the ms/ms mode of operation when a potential pseudo molecular ion exhibits a neutral loss of da between the high energy and low energy data sets. the product ion ms/ ms spectrum can then be acquired on the phosphorylated precursor ion. in the case of phosphotyrosine, neutral loss of the h po moiety is not observed, however a low mass immonium ion at m/z can be detected. this characteristic ion (from the high energy data) is used to direct the mass spectrometer to fragment potential phosphopeptide precursor ions, which are selected from the low energy data. in this case several precursor ions may require ms/ms interrogation at one decision making time-point. with the first draft of the human genome completed largescale protein identification by mass spectrometry, even for samples originating from higher organisms has become relatively straightforward. this requires a high throughput facility to identify proteins that have usually been separated by d page. the approach providing the highest level of automated sample throughput, in terms of samples per hour, is currently maldi-tof-ms. this technique provides a peptide mass fingerprint of the protein digests and allows the rapid and accurate identification of the parent protein by comparison to a databank. however, under some circumstances, for example if the number of peptides detected is small or if the sequence coverage is poor, it is advantageous to be able to include even a short piece of sequence information to provide added specificity. in a conventional maldi-tof-ms instrument post source decay (psd) can be used to try and generate sequence information, however this approach is notoriously unreliable in producing good quality ms/ms data. one reason for this is that the peptide ions do not undergo fragmentation in a controlled environment such as a gas cell with selected collision gas and collision energy. an alternative approach is to use the predictable fragmentation obtained from a hybrid quadrupole ortho maldi source has been fitted to a hybrid quadrupole orthogonal acceleration time-of-flight (q-tof) mass spectrometer. in contrast to a conventional maldi-tof-ms instrument the resolution and mass measurement accuracy of the data is comparable between the ms and ms/ms modes. this allows superior data acquisition in the ms-ms mode compared to conventional maldi-tof-ms. a number of modifications have been made to optimise the system for high throughput proteomics. the maldi source has been configured with a high-density target plate, compatible with a well microtiter plate. the acquisition software has been modified for automated data acquisition in both the ms mode and the ms to ms/ms switching mode. dedicated processing software has been developed to fully automate the post acquisition and databank searching. this software has been optimised to consider the unique nature of the data acquired from this configuration of instrument. in this paper we demonstrate the how an maldi-q-tof instrument can be used for high throughput proteomics. we also compare and contrast is functionality in comparison with alternative strategies for high throughput proteomics, namely conventional maldi-tof-ms and electrospray lc-ms/ms. pseudomonas putida is an ubiquitous, metabolically and physiologically extremely variable soil bacterium. it is kown to be a good colonizer of plant roots and a plant growth promoter. now, after the sequencing of the total genomic dna has been finished we have focused on the functional analysis of this strain. plant growth promotion is achieved in different ways. one is the inhibition of fungal and bacterial phytopathogens, which is known to be a multifactorial mechanism. an important factor of this mechanism is the production of siderophores (iron-transport-agents), small linear or cyclic peptides, which are synthesized in a ribosomal-independent manner by special synthetases. the siderophore production is induced by iron limitation. the regulation of this process was investigated by pulselabelling with [ p] inorganic phosphate. d-protein patterns generated from cells grown with and without fesupplementation were compared. proteins which were phosphorylated under iron limitating conditions were analysed by maldi-tof peptide mass fingerprint. for the identification of the proteins we used an in-house peptide mass database which has been built based on the genomic sequence data. bio-rad laboratories, inc., hercules, california, u.s.a. worksbase software for proteomics is a platform independent information management system encompassing laboratory experimental workflow and bioinformatics for protein and biochemical research. the worksbase system is designed to allow direct internal integration between laboratory experimental data and background biological knowledge found in reference and in-house data, such as gene, protein and functional annotation databases. worksbase provides a crossdisciplinary research infrastructure for drawing together multiple lines of evidence for characterization of proteins, and integration of this data with domains such as gene expression, pharmacological screening, structure and related areas. while the focus is on the biology underpinning the experimental work, the system is also designed with the capability of providing a sample and workflow tracking system for use in the wet lab, effectively a proteome lims (laboratory information management system). as experimentation proceeds in the laboratory, worksbase software can be used for development of hypotheses on protein, biochemical pathway, and post-translational processing involvement in biological systems and disease processes. as such, identifications that are derived from lab work and user observation can be used to augment the reference data repository. however, unlike databases ands systems where the methods and reasoning for assignment of annotations are obscure, by maintaining the link between the source data and the biological roles derived from them, the accuracy and integrity of any information stored in the worksbase system can be directly ascertained. changes in the brain protein levels following administration of kainic acid k. krapfenbauer , , m. berger , g. lubec , and m. fountoulakis f. hoffmann-la roche ltd., pharmaceutical research, genomics technologies, basel, switzerland institute of cancer research, and department of pediatrics, university of vienna, austria kainic acid (ka), a potent neurotoxin and excitatory amino acid, leads to derangements and modulation of brain proteins. no global brain protein expression pattern induced by ka-treatment has been reported yet. we studied the effect of systemic ka administration on the levels of brain proteins. rats were injected placebo or ka intraperitoneally and brain was taken after one week. the mitochondrial and cytosolic fractions of the brain proteins were analyzed by proteomics technologies. heat shock protein hsp was exclusively detected in brains of animals treated with ka. the levels of neurofilaments and alpha-internexin were significantly decreased and a fragment of tubulin alpha- chain was manifold increased in ka-brains. the mitochondrial enzymes dihydrolipoamide dehydrogenase, atp synthase beta chain and isocitrate dehydrogenase were reduced and pyruvate kinase m was increased following ka treatment. the results indicate altered regulation of heat shock proteins, neuronal death, cytoskeletal disruption and mitochondrial derangement by systemic ka administration. this report confirms and extends previous studies on the effect of ka on the expression of brain proteins and suggests that our analytical system can serve as a model for neurotoxicological, neurobiological and neuropathological proteome studies. the rat brain mitochondrial proteins genomics technologies, f. hoffmann-la roche ltd., pharmaceutical research, basel, switzerland we constructed a two-dimensional database for rat brain mitochondrial proteins. rat is a useful model of human diseases of the central nervous system. in order to detect alterations in the levels of the low abundance brain proteins, the mitochondrial, microsomal and cytosolic fractions were prepared. the proteins of each fraction were analyzed by two-dimensional electrophoresis, followed by martix-assisted laser desorption ionization mass spectrometry. approximately proteins were identified in the mitochondrial fraction, which were the products of different genes. about % of the identified proteins were detected in the mitochondrial fraction only and the rest were detected in the cytosolic and about % were found in the microsomal fraction as well. of the proteins had not been detected before in our laboratory. the identified proteins were in the majority enzymes or enzyme subunits with a broad spectrum of catalytic activities and heat shock proteins. whilst lc-ms/ms has been utilised for the identification of proteins from complexes and cell lysates (qualitative proteomics), the quantitative study of gene expression using differential display has until recently been the preserve of a d gel based proteomic experiment. however, recently a great deal of interest has been generated on the use of isotope coded affinity tags (icat) for the quantitative study of gene expression at the proteome level. the technique is based upon chemically modifying the cysteine residues of proteins isolated from cells in two different states with light and heavy isotopically labeled reagents. the two cell states are then combined, digested with trypsin and the cysteine containing peptides preferentially selected by binding to an avidin column, prior to analysis by mass spectrometry. the eluent from this column is then analysed by capillary lc esi-ms/ms. interrogation of the eluting peptides by tandem mass spectrometry and databank searching results in the identification of the associated protein. we describe how icat data analysis has been automated within a software environment. the ms and msms data acquired using the qtof instrument are processed and analysed using a new algorithm which recognises related isotope clusters and quantifies their relative intensities. based on a user defined ratio threshold the software will automatically carry out an lc-ms/ms experiment and databank search in a client-server mode and provide a report of the identified proteins and their expression ratio in the two cell states. deterioration of the transcriptional, splicing and elongation machinery in brain of fetal down syndrome b. lubec and m. fountoulakis department of neonatology, university of vienna, austria gene technologies, cns research, f. hoffmann la roche, basle, switzerland perturbation of brain development i.e. regulation of gene expression, differentiation, growth and migration in down syndrom (ds) has been reported to occur early in life pointing to impairment of the complex system of transcription and or translation and indeed, altered expression of transcription factors has been reported in adult ds brain. we therefore decided to compare the transcriptional and translational machinery in cortex of brains of controls and fetuses with down syndrome in the second trimenon of gestation. we determined a series of transcription/translation factors by d-electrophoresis followed by maldi -identification and quantification with specific software. the protooncogene c-crk, crk-like protein, elongation factor -alpha , elongation factor , elongation factor tu and two out of four spots representing ptb-associated splicing factor psf were significantly downregulated in brain of fetal ds fetuses as compared to controls. the finding of reduced transcription and translation factors may indicate deranged protein synthesis. the underlying cause for individual reduced transcription, splicing and translation factors may be explained by chromosomal imbalance or by posttranslational modifications as e.g. phosphorylation, known to be aberrant in ds. reduced expression of transcription factors in fetal ds during early life may be responsible or reflecting impaired brain development and deficient wiring of the brain in ds. r. mazzoli , m. g. giuffrida , e. pessione , g. dellavalle , c. barello , e. griva , and c. giunta dipartimento di biologia animale e dell'uomo, università di torino, and csaapz-cnr. c/o bioindustry park canavese colleretto giacosa (to), italy a fast phenol degrading acinetobacter radioresistens strain was isolated in our laboratories and selected for bioremediation applications. this bacterium is also able to grow on benzoate and catechol as sole carbon-energy sources, metabolizing them via the ortho route. in previous researches we detected, by means of proteome analysis, some marker enzymes of the phenol and benzoate degradative pathways. in the present work we extend the identification of the proteins involved in the aromatic-ring opening (the different components of the phenol hydroxylase and benzoate dioxygenase, the catechol dioxygenase isozymes) together with other satellite proteins specifically induced by the aromatic growth substrate. of these last proteins some are probably related to the cellular uptake of benzoate and phenol while others are ascribed to the groel family of heat-shock chaperonines, involved in proteins processing and folding. aromatic substrates may thus act as stress-agents like heat or cold. proteomic studies on rat body fluids i. miller , r. wait , l. sironi , i. eberini , m. gemeiner , e. tremoli , and e. gianazza veterinärmedizinische universität, wien, austria imperial college school of medicine, hammersmith, london, u.k. universita' degli studi, milano, italy previously, we have characterized rat serum proteins, both under "normal conditions" and during experimental inflammation, using two-dimensional electrophoretic separation, densitometric quantitation and identification by mass spectrometry and immunological procedures (http://linux.farma.unimi.it/ homeframed.html). we have now extended these studies to the protein composition of cerebrospinal fluid (csf) and urine, and have identified several proteins specific to these fluids, including major urinary protein, uromodulin, and prostaglandin d synthase. these baseline data provide a useful comparison to the biological fluids of stroke-prone spontaneously hypertensive rats, an inbred strain, which develops cerebrovascular abnormalities following high blood pressure. our studies have detected signs of an inflammatory condition several weeks prior to stroke. we have confirmed the sharp rise in proteinuria preceding stoke onset, and have identified the excreted proteins. following stroke we observe a massive increase in csf protein concentration as serum proteins, even those of large molecular size, cross an impaired blood-brain barrier. as a first step to discover useful disease markers from the urinary proteome, we have developed a unique and systematic approach for detection of low molecular weight urinary proteins by using high resolution two-dimensional ( d) electrophoresis and mass spectrometric methods. unlike previous studies on urinary proteins, and most importantly as observed in present study, our results show that a large number of low molecular weight protein spots can be visualized in the d electrophoresis pattern. it was observed that protein concentration and fractionation methods were critical for our ability to detect many proteins in the gel pattern. therefore, several approaches were carefully considered to concentrate and fractionate proteins in urine samples. initially, urine specimens from normal individuals were concentrated by using centrifugation and ultrafiltration methods. the concentrated samples of urine proteins were then fractionated by size exclusion and immunoaffinity chromatography. the size exclusion method was used to generate two fractions of proteins based on their native molecular weights. further, this method allowed us to enrich concentrations of less abundant proteins for each fraction. the immunoaffinity method was used to specifically remove well-known abundant urinary proteins (such as albumin) from the above mentioned two fractions. that the d pattern includes many native low molecular weight proteins was confirmed by analyzing both protein fractions from size exclusion chromatography. a detailed mass spectrometric analysis of the protein spots is carried out to identify the proteins observed in d pattern. since urine is an ultrafiltrate of plasma, many factors in urine are present in proportion to their rate of synthesis in the body. these factors include many low molecular weight proteins that remain undiscovered due to their low abundance. therefore, the present analysis of urinary proteins would serve as the most useful guide for the discovery of novel diagnostic markers in urinary proteins. i. pucci minafra , , s. fontana , p. cancemi , g. alaimo , and s. minafra , centre of experimental oncobiology, department of cell biology and development, and institute of histology and embryology university of palermo, italy breast cancer is one of the leading causes of death for cancer among women. there are different types of breast cancers, grouped as invasive and non-invasive types. among the invasive types "infiltrating ductal carcinoma" (idc) accounts for about % of all breast cancers. in order to study some biological properties related to this type of cancer, we have developed and well characterized an "in vitro" system, consisting of an idc-derived cell line, -bc (minafra et al., br. j. cancer, , - , ) and some of its cloned cell lines, selected for their high and low invasive activity in matrigel. using this model we are producing proteomic maps to compare with that of non-tumoral breast epithelial cells and with breast tissue fragments, existing in our collection or available at the expasy proteomics server. protein identification is currently done by means of gel matching, edman-microsequencing and immuno-detection. to rationalize data we grouped proteins into functional categories: a) cytoskeletal proteins, b) metabolic enzymes, c) chaperonins and other functionally related proteins, d) peptides and enzymes with regulatory functions. a fifth group consists of peptides with unknown identity. among these sets of proteins we found that glycolitic enzymes and some chaperonins are overexpressed in cancer cells. in addition, new isoforms of potential interest as biomarkers for breast cancer, were identified by means of microsequencing. a. santucci , l. trabalzini , d. soldateschi , e. ferro , a. paffetti , and p. martelli dipartimento di biologia molecolare, sezione di chimica biologica, universita' degli studi di siena, and diesse diagnostica senese srl, siena, italy human cytomegalovirus (hcmv) is an ubiquitous virus, belonging to the herpesviridae family, betaherpesvirinae subfamily, able to induce morbidity in immunocompromised patients and congenitally infected new-borns. hcmv has the largest genome among the herpes-viruses ( kbp): ad strain genome was completely sequenced, containing about open reading frames encoding polypeptides, most of which are not characterized. the viral genes are activated in a cascade fashion: ) alpha, immediate-early genes, coding for regulatory proteins necessary for the activation of ) beta, early genes, needed for dna replication, and, finally ) gamma, late genes, coding for structural proteins of the mature virions. this latter category includes the virus surface antigenic proteins responsible for the main immune response during hcmv infections. although the sequencing of hcmv genome has been completed, very little is known about the actual nature of the viral proteins. the most appropriate approach to characterize hcmv phenotype is to study its protein expression as it is carried out within the host cell. for this purpose, we analyzed by two-dimensional electrophoresis ( d-page) the protein phenotipic repertoire of human fibroblasts and compared it with that of the same cell type following infection with hcmv strain ad . the phenotypic d map of human fibroblasts dramatically changes following infection with hcmv. a relevant amount of newly appeared spots is attributable to hcmv proteins, mainly of the structural category, since we analyzed host cells at the - th day of infection, when the late, gamma genes are supposed to be the only to be activated. on the other hand, a marked decrease of protein synthesis can be easily evidentiated in the infected fibroblasts respecting to uninfected cells. a temptative mapping of the main structural viral proteins (those against which patients sera are directed) was carried out by immunoblotting, microsequencing and mass spectrometry. comparative proteomics of cultured cells: identification of genetic defects and molecular mechanism of apoptosis regulation v. seyrantepe , k. landry , s. taurin , s. n. orlov , and a. v. pshezhetsky sainte-justine hospital research centre, and research centre, chum, university of montreal, montreal, pq, canada we employed a comparative proteomics of cultured cells to study mechanism of genetic disorders and for identification of key proteins involved in cell proliferation, differentiation, and death. in particular, this technology proved to be very useful to understand molecular basis of severe inherited diseases resulting from deficiency of lysosomal membrane transporters, and a role of programmed cell death (pcd) of vascular smooth muscle cells (vsmc) in cardiovascular disorders. to increase sensitivity of the identification of cellular proteins we have either have isolated cellular organelles such as lysosomal membranes or performed the differential extraction of soluble, membrane and cytoskeletal proteins. by comparison of pro-teomic cell maps from normal controls and individuals affected with lysosomal transport disorders we have selected and identified several candidate disease-causing proteins, which have to be further studied by mutation analysis and functional expression. for the second group of disorders we identified proteins, which de-novo synthesis could result in survival of vsmc including a two members of hsp family, a molecular chaperone grp , and so-called mortalin (grp ) highly expressed in non proliferative tissues and associated with mortal cell phenotype. two-dimensional polyacrylamide gel electrophoresis ( d-page) is the established technology employed for the separation of proteins from a cell lysate, sub-cellular organelle or tissue sample prior to identification of the excised protein spots by mass spectrometry. in the order of several hundred to several thousand proteins, can be separated and visualised on a d gel by conventional staining or utilising fluorescent labelling techniques. the advantage of performing a two dimensional gel based separation is the ability to obtain quantitative information by comparing and contrasting two samples in a differential display experiment, for example, between a healthy and diseased state. the last stage however stipulates that the gels are reproducible which can be both difficult and time consuming to achieve. the relativity poor dynamic range that the gels exhibit also limits quantification. other restrictions include the under representation of certain classes of proteins, such as membrane proteins, large or small proteins and very acidic/basic proteins. for these reasons, amongst others, alternatives to d-page are being investigated. advances in both lc and mass spectrometry instrumentation have allowed the analysis of protein complexes, which have not been separated on a d gel. in this case protein identification is achieved via database searching of esi-ms/ms data. this provides qualitative information on the proteins that are present and has recently been coupled with isotope dilution experiments to provide relative quantiative information. these experiments normally involve separation of the complex digest mixture by microcapillary liquid chromatography connected to an instrument capable of data dependant switching between the ms and ms/ms modes. using this approach it has been demonstrated that hundreds of ms/ms spectra can be acquired in a fully automated fashion, resulting in the identification of significant numbers of proteins, including low copy number proteins, from a single lc-ms/ms experiment. if, however, a complex protein mixture is to be investigated then a fractionation step prior to separation of the peptides on the basis of their hydrophobicity would be advantageous. we have, therefore, adopted a d lc-ms/ms approach using a capillary lc system (caplc) operating at nanoliter per min flow rates coupled to a q-tof mass spectrometer. by replacing the standard sample loop within this system with a strong cation exchange (scx) cartridge followed by a c trap cartridge it is possible to pre-fractionate the peptides before separation on a c column. after loading the sample, discreet fractions are sequentially eluted from the cation exchange cartridge using a salt step gradient; the eluted peptides are then retained on the trapping c cartridge whilst they are desalted. finally the peptides are eluted from the c pre-column, at nl/min, onto a um id ϫ cm waters symmetry analytical column for separation and elution into the mass spectrometer. this analytical approach will be discussed with examples where this methodology has been used for the analysis of standard protein mixtures and also for the analysis of cell lysates and sub-cellular fractions. monoclonal igg are commonly observed in various b cell disorders, the most clinically relevant being multiple myeloma. in a series of serum samples, immunofixation identified igg , igg , igg , and igg in , , , and cases, respectively. their light-chains were k in cases and λ in cases. these monoclonal igg were further characterized by high resolution two-dimensional polyacrylamide gel electrophoresis ( -de) with various isoelectric focusing conditions as well as by -de ( -de of the proteins extracted from agarose after serum protein agarose electrophoresis). after -de or -de, the monoclonal γ-chains were not visualized in out cases, whatever the isoelectric focusing conditions that were tested. in cases, γ-chains were only detected using alkaline ph - gradients. monoclonal γ-chains and light chains were highly heterogeneous in terms of pi and mr. however, a good correlation (p Ͻ . ) was observed between the index of migration of the monoclonal igg in agarose gels and the pi of their γand of their light-chains (r ϭ . , multiple linear regression). because of the extreme diversity of the different γ-chains as well as of the k-and γ-chains, it appears that a classification of monoclonal igg based only on their electrophoretic properties is not possible. alzheimer's disease (ad) is one of disorders caused by protein conformational changes and recent studies have shown that several chaperone proteins are involved in this process. as information of chaperone expression in ad brain is limited, we aimed to study the expressional pattern of chaperones in several brain regions as this may be essential to understand how folding defects can lead to disease. we studied the concomitant expressional patterns of molecular chaperones in seven brain regions of adults with ad using two-dimensional polyacrylamide gel electrophoresis ( -de) and matrixassociated laser desorption ionization mass spectroscopy (maldi-ms). we unambiguously identified and quantified nine different chaperone proteins. six chaperone proteins, heat shock protein (hsp ), hsp ry, heat shock cognate (hsc) , alpha crystallin b chain, glucose regulated protein (grp) and grp showed aberrant expressional patterns depending on brain region. hsp . , grp and t-complex (tcp- ) epsilon subunit did not show any significant expressional change. these findings are compatible with neuropathological and biochemical abnormalities in ad brain and this report presents the first approach to quantify nine different chaperones simultaneously at the protein level in individual ad brain regions providing evidence for the relevance of aberrant chaperone expression to ad neuropathology. the mainstream approach to protein separation, visualisation and identification has been to use two-dimensional gel electrophoresis coupled to mass spectrometry for the identification of the separated proteins. however this approach is limited with the level of protein that may be loaded onto the d gel and the nature of the proteins that may be incorporated onto the first dimension (ipg strip). an alternative approach for the qualitative analysis of complex protein mixtures is the use of tryptic digestion followed by electrospray lc-ms/ms. this approach is dependent on a high degree of chromatographic separation prior to the mass spectrometer, such that ideally individual peptides are eluted into the source. if this is the case then the dynamic range of protein identification can be increased and low copy number proteins can be identified. often, however there is a large degree of redundant sequence information acquired, as in theory one peptide ms/ms spectrum is sufficient to identify a protein from a sequence databank. if a protein identification is obtained from a databank search of an ms/ms spectrum, it is potentially valuable to exclude the rest of the theoretical tryptic peptides to "mine" deeper into the protein complex being studied. we have introduced a new protein databank search engine capable of matching a tryptic peptide from the swissprot/ trembl databank to an ms/ms spectrum in one second. using this search engine we are able to generate dynamic tryptic peptide exclude and include lists, based upon the theoretical tryptic peptides from the identified protein, which can be passed to the acquisition software of our q-tof mass spectrometer in real time. thus, we are able to automatically steer the q-tof, during acquisition, to select and switch to the ms/ms mode only on those peaks that meet the modified selection criteria. experiments can be designed in which peaks that belong to a protein already identified during acquisition can be avoided. this exclusion list is based upon m/z, charge state and a user definable mass tolerance. the mass measurement of the data from the q-tof mass spectrometer is typically better than ppm and as a consequence of this a tight mass tolerance can be selected, thus making the exclude list extremely specific. alternatively, in the case of samples derived from d gel spots, the mass spectrometer may abandon the current sample, re-equilibrate the lc column and move on to the next sample. to illustrate this methodology we show examples, both on standard samples and complex protein mixtures where q-tof data acquisition has been directed based upon the results from a databank search. this data will be compared and contrasted to data acquired in the normal automated lc-ms/ms mode. the specific anti-cancer activity of green tea (؊)-epigallocatechin- -gallate (egcg) department of molecular biology, hebrew university-hadassah medical school, jerusalem, israel the effect of the green tea polyphenol-(Ϫ)epigallocatechin- -gallate (egcg) was tested in cultures of normal and transformed nih-patmras fibroblasts. in this system transformation can be induced at will by the addition of dexamethasone, which induces the expression of h-ras by activating the mammary tumor virus long terminal repeat (mmtv-ltr) promoter. this facilitates a reliable comparison of the susceptibility of normal and transformed cells to egcg. it has been shown that egcg inhibited the growth of transformed but not of the normal fibroblasts. in an attempt to elucidate the mode of the preferential inhibitory activity of egcg, its effect on growth promoting factors has been examined. the level of ornithine decarboxylase (odc, ec . . . ), which is a signal for cellular proliferation, was reduced by egcg in the transformed but not in the normal cells. egcg also showed strong inhibition of tyrosine kinase and mitogen-activated protein kinase (mapk) activities, without affecting the kinases in the normal cells. similarly, egcg also preferentially decreased the levels of the oncogenes ras and jun in transformed cell. egcg preferentially induced apoptosis in the transformed fibroblasts. in vitro chemosensitivity tests demonstrated that egcg inhibited the proliferation of leukemic cells. these findings suggest that egcg has a therapeutic potential in the combat against cancer. objectives: to develop a safo, affordable immune supportive therapy for hivϩ patients. design: a randomised, double blind, placebo-controlled study, testing an internationally patented l-methionine combination (lmc), in approximately hivϩ patients; not yet on anti-viral treatment (cd count to ). methods: parameters measured included: cd count, total lymphocyte court, viral load, several clinical, as well as mechanistic parameters. the difference in the change from the baseline (active -placebo) was determined for each parameter. the study is ongoing. results: within months, significant trends are noted. the cd count of the patients on the active therapy, presented with a slower rate of decrease, compared to the placebo group, mean difference (md) in this change from baseline; . /cmm and % confidence interval (c ), this was confirmed by the total lymphocyte court values. after months the placebo group was placed on active, causing the difference to disappear. conclusions: although further trials are needed, these results already indicate t-methionine as an important role player in the immune system of patients with impaired immune function. c. chiarla , i. giovannini , j. h. siegel , g. boldrini , and m. castagneto centro di studio per la fisiopatologia dello shock cnr, catholic university, rome, italy department of surgery, umdnj, newark, new jersey, u.s.a. in critical illness and sepsis, changes in amino acid plasma levels (aapl) have been assessed extensively, while little is known about the relationship with changes in other plasma components, such as those involved in fluid-electrolyte and osmotic balance; their investigation is also limited, in large clinical samples, by inter-patient variability. we analyzed the relationships between plasma sodium (na ϩ pl, meq/l) and aapl (µm/ l) in eighty consecutive measurements performed in one single patient with post-traumatic sepsis and severe, prolonged illness. unique feature of plasma taurine (tau) was maintenance of a highly significant inverse correlation with na ϩ pl (r ϭ . , p Ͻ . ). all other aapl were correlated directly, or unrelated, to na ϩ pl, the only exception being a weak inverse correlation between tryptophan and na ϩ pl. tau was correlated, strongly and directly, also to phosphoethanolamine (pea), glutamate (glu) and aspartate (asp): tau ϭ . ϩ . (pea) Ϫ . (na ϩ pl) r ϭ . , p Ͻ . tau ϭ . ϩ . (asp) Ϫ . (na ϩ pl) r ϭ . , p Ͻ . tau ϭ . ϩ . (logglu) Ϫ . (na ϩ pl) r ϭ . , p Ͻ . and unrelated, or weakly and inversely related, to other aapl (measurements of beta-alanine were not included). co-variation of na ϩ pl and these aapl (particularly tau and pea) was influenced by severity of illness, and more complex regressions were needed to quantify this effect. these results provide useful information on interdependency of tau, na ϩ pl and other aapl in critical illness. the central nervous system (cns) shows an exceptionally high degree of vulnerability to reactive oxygen species. considerable evidence suggests that free radical formation and oxidative stress might play an important role in the pathogenesis of parkinson's disease (pd). moreover, it has been reported that the levels of glutathione and vitamin e increase in the brain of patients with pd as a compensatory mechanism to deal with oxidative stress. since vitamin e is an effective free radical scavenger in the brain, its neuroprotective function is the issue of new therapeutic approaches in neurodegenerative diseases. to elucidate the possible role of vitamin e in the pathogenesis of pd, we assessed the plasma levels of vitamin e, measured by high-performance liquid chromatography, in patients with pd. vitamin e concentrations were also assessed in age and sex matched normal individuals. the mean plasma levels of vitamin e did not differ significantly between these two groups ( . Ϯ . mmoli/l for pd patients and . Ϯ . mmoli/l for controls). the results of our study suggest that plasma vitamin e concentrations do not play a major role in the pathogenesis of pd. vitamin e and cardiovascular disease: nutritional and intervention approaches f. galli , institute of biological chemistry, university of urbino, italy department of cardiovascular research, st thomas' hospital, london, u.k. vitamin e is represented by a family of eight natural vitamers ( tocopherols and tocotrienols) of which αtocopherol (α-t) form has the highest biological activity. this vitamin accounts for most of the lipid-soluble, chain-breaking antioxidant activity in mammalian tissues and plasma. in addition, it shows nonantioxidant properties through which it modulates cell signaling and the expression of specific enzyme in cell models playing a role in atherogenesis (e.g. endothelial and inflammatory cells). the preventive effect of vitamin e on acdv is still a matter of debate. the largest epidemiological investigations and out of main intervention studies at yet available have suggested a correlation between levels of vitamin e and incidence of atherosclerotic cardiovascular disease (acvd) and related mortality. an overall conclusion rising from these studies is that the major effect (if any) of vitamin e is to be found with intakes higher than iu ( mg all-rac α-tocopheryl acetate) per day. however, other investigations have failed to demonstrate a beneficial effect of vitamin e against acvd, suggesting the need for more studies on its metabolism and function. recently a family of tocopherol binding and transport proteins has been identified. they play a key role in the selective uptake and delivery of tocopherols to lipoproteins and tissues. genetic abnormalities of these proteins have been demonstrated to be responsible for conditions of vitamin e deficiency in humans. their tissue distribution and regulation are now under investigation. the information available on vitamin e metabolism and its response to supplements or diet changes are at yet poorly characterized. the synthesis of stable isotopes and the characterization of major metabolites of main vitamers provide important advances in this research. in the last years, both plasma levels and urinary excretion of relevant metabolites of α-t have been characterized. little information is available on metabolites formed by other vitamers. the emerging role of γ-t and its main catabolite , , -trimethyl- -(b-carboxyethyl)- hydroxychroman (γ-cehc) in the defense against nitrogen oxide species formed during the activation of inflammatory cells is now well established and suggests the need for further studies on the bioavailability and transformation of this homologue of vitamin e in humans. at the same time, an oxidation byproduct of α-t found in human plasma, namely α-tocopherylquinone, has been proposed to be also de novo synthesized from phenylalanine with a role in the genesis of a defective polyunsaturated fatty acid metabolism observed in phenylketonuric patients. this suggests a possible, and at yet unexplored relationship between vitamin e and phenylalanine/fatty acid metabolism which might have also a role in atherosclerotic process. r. gaspari , s. mensi , g. mercurio , c. callà , l. colacicco , e. sacco , and s. lippa department of anaesthesiology and intensive care medicine, and department of biochemistry and clinical biochemistry, catholic university of rome, italy four patients ( females, male; aged from to years) affected by severe liver failure, were treated by a new blood purification method, namely molecular adsorbent recycling system (mars). mars removes albumin-bound toxins using a specific membrane with a dialysate solution containing albumin. in the patients the plasma levels of methionine (meth), branched chain and aromatic amino-acids and liposoluble antioxidants were measured. the fischer's index did not show any significant variation, whereas the plasma levels of meth were well correlated with the levels of liposoluble antioxidants (vitamin e and coq ). in fact, in the patients receiving just branched chain amino-acids, the plasma levels of both meth and antioxidants progressively decreased. on the contrary, if meth and branched chain amino-acids were administered, the plasma levels of coq and vitamin e showed a positive correlation with the plasma meth levels (p Ͻ . ; r ϭ . and p Ͻ . ; r ϭ . , respectively). since vitamin e and coq are mutually dependent-molecules, the administration of meth, essential substance for coq synthesis, may be effective to maintain a good antioxidant status in patients with severe liver failure undergoing mars treatment. we obtained new synthetic peptide preparation epitalon to be widely applied as a pharmaceutical due to its properties important in medical care. epitalon was found to stimulate repair processes in retinal diseases via restoring the retinal functions, in particular its photoreceptors. this promising peptide drug is a linear tetrapeptide of formula h-ala-glu-asp-gly-oh (alanyl-glutamyl-aspartyl-glycine). the substance was obtained by classic peptide synthesis in a solution (scheme: ( ϩ ) ϩ ) with n-oxysuccinimide activated esters. coohgroups of lateral radicals of glutamic and aspartic acids were defended as benzyl esters, benzyloxycarbonyl (ala) and tert.butyloxycarbonyl (glu) n-defending groups were employed, deblockade conducted by trifluoroacetic acid and catalytic hydrogenolysis. preparative hplc on a reverse phase was applied for purification. the product was fully characterised by the data of analytical hplc (substance content - %), amino acid analysis, ir-and hmr-spectra. the ready drug form is ampoules containing µg of the substance in ml of isotonic solution. epitalon application in patients with pigmented retinal degeneration stopped the pathology development in % and increased visual functions in % of the cases. in % of the patients visual acuity raised by . - . . electroretinography confirmed the retinal functional activity increase. an increasing number of proteins are implicated in apoptosis and several of them have been shown to be altered in alzheimer's disease (ad) brain. because of this apoptosis is thought to be the underlying mechanism of neuronal cell loss in ad. to further substantiate this hypothesis we investigated the expression of a recently identified apoptosis related proteins and other apoptosis regulators in frontal cortex and cerebellum of ad by western blot and elisa techniques. quantitative analysis revealed unaltered levels of bax and raidd (receptor interacting protein associated ich- (caspase- )/ ced- (caenorhabditis elegans death protease- )-homologous protein with death domain) in both regions. zip (zipper interacting protein) kinase, bim/bod (bcl- interacting mediator of cell death/bcl- related ovarian death gene) and p were significantly increased only in ad frontal cortex (p Ͻ . , in all cases). cerebellar bcl- levels were significantly increased in ad (p Ͻ . ) while in ad frontal cortex, although the levels tended to increase did not reach significance level. the results indicate that apoptosis indeed account for the neuronal loss in ad. however, it does not seem to involve bax and raidd. a. magyar , m. brózik , r. tóbi , t. szabó , j. szakonyi , b. rojkovich , p. gergely , and f. hudecz research group of peptide chemistry hungarian academy of science, budapest, central laboratory of immunology, semmelweis university, budapest, and national institute of rheumatology, budapest, hungary rheumatoid arthritis (ra) is a systemic autoimmune disease of unknown etiology. it is the most common of the inflammatory joint diseases, affecting - % of the world population. anti-filaggrin antibodies (afa) directed against the epidermal protein, filaggrin, belongs to the most specific markers of ra. epitopes, containing citrulline within the sequence of filaggrin, have been recently identified as major antigenic sites recognised by afa. the aim of our study was to identify these epitopes of filaggrin derived-peptides targeted by ra specific antibodies to provide further information about the nature of the initial autoantigenic substance. the most immunogenic six sequences of filaggrin and further, on the n-and c-terminal, shortened version of the original peptide ( shqestrgrsrgrsgrsgs ) were synthesized. we used conventional solid-phase peptide synthesis (fmoc strategy) carried out on "multipin ncp" noncleavable kit. in elisa experiments the presence of afa was deter-mined using serum samples of ra patients and healthy blood donors. in conclusion our results provide further evidence that not simply the presence of citrulline but also the nature of its surrounding amino acids have important role in the creation of autoantigenic epitope reactive with anti-filaggrin antibodies. the autoimmune nature of multiple sclerosis (ms) has introduced cytokine genes as logical candidates for the loci determining susceptibility to the disease and/or influencing disease progression. interleukin (il)- alpha and beta are major proinflammatory cytokines that have been related with several chronic inflammatory diseases such as ms. the il -receptor antagonist (il- ra) is a protein structurally related to il- beta that effectively inhibits the proinflammatory effects of il- . a polymorphism in the Ј-flanking regulatory region at Ϫ of the il- alpha gene, which may cause an overexpression of il- alpha and a variable number tandem repeats (vntr) polymorphism in the il- ra gene have been also associated with several inflammatory diseases. two biallelic base change polymorphisms in the il- beta gene have been reported to influence the protein production: one is located in the promoter region at position Ϫ and the other is in exon at position ϩ . to analyze the contribution of il- alpha, il- beta and il- ra genes in the genetics predisposition to ms, we have examined four polymorphic genetic markers in italian patients with clinically definite ms and healthy controls. in summary, no significant differences in genotypes and allele frequencies were found between ms patients and healthy controls. fibronectin -the extracellular matrix protein is oxidatively modified with oxygen reactive species (ros) in inflammation site. activated neutrophiles release the hypochlorite acid (hocl) and chloramines as products of myeloperoxidase/ h o /cl Ϫ system. these reactive chlorine species chlorinate in turn matrix proteins. the resulting changes of tertiary protein structure could be evaluated by monitoring the antigen/antibody complex formation. the formation of the complexes between native/chlorinated fibronectin and igg class antibodies were examined by means of elisa with luminol chemiluminescence detection. the degree of fibronectin modification was monitored with spectroscopic methods. since the oxidation leads to the fibronectin aggregation -the tryptophane contents in resulting aggregates were evaluated with stern-volmer approach (acrylamide quenching). moreover, the aldehydes influence on the ag/ab complex formation was examined -since aldehydes are known products of amino acids n-chloramines deamination. also the native and modified fibronectin adherence to the matrix proteins was monitored with use of hrp labeled antifibronectin antibodies. the preliminary results suggest that chlorination impairs the ab/ag complex recognition but also prove that igg bounded chlorinated fibronectin promotes igg clusters formation. it was found also that mm concentration of the serine derived glycoaldehyde decreases the fibronectin/igg recognition and the effect could be attributed to the igg aggregates formation. we demonstrate also that hrp-labeled iggs detect the collagen and fibrynogen adherent fibronectin in a dose dependent manner-details of the elisa method are discussed. in subjects with rheumatoid arthritis (ra) oxidized low density lipoproteins (ldl) are supposed to serve as mediators for joint damage, further exacerbating the inflammatory process. to better understand mechanisms of ldl oxidation in ra a specific marker of oxidative modification of apolipoprotein (apo) b- proline and arginine residues, hydroxy- -aminovaleric acid (hava), had been measured in plasma and synovial fluid ldl subfractions (ldl , svedberg units (s f ) - and ldl , s f - ) by gc-ms. paired knee synovial fluid and plasma samples were collected from subjects with ra. additionally, plasma samples were collected from healthy controls. the ldl hava content in plasma was not different between the groups (ra, . Ϯ . vs controls, . Ϯ . mol/mol apob- , p ϭ . ). the ldl hava content in plasma was significantly higher in ra ( . Ϯ . vs . Ϯ . mol/mol apob- , p ϭ . ). furthermore, synovial fluid ldl and ldl in ra contained elevated hava levels when compared with plasma concentrations (ldl syn , . Ϯ . mol/mol apob- (p Ͻ . ) and ldl syn , . Ϯ . mol/mol apob- (p Ͻ . )). results suggest that proline and arginine residues of apob- are highly reactive toward oxygen radicals in both plasma and synovial fluid in ra. furthermore, susceptibility of apob- to oxidative modification increases along the lipoprotein metabolic cascade. particularly small dense ldl were prone to direct oxidation of apob- . correlation between hava content in plasma and synovial fluid ldl and ldl in ra may allow the use of hava as a clinical marker of antioxidant barrier impairment in ra. vascular collagen accumulation contributes to development of hypoxic pulmonary hypertension (ph). we have shown that injections of a polymer of the proline analogue cis- -hydroxy-l-proline (chyp) in liposomes attenuated acute ph in rats (ajrccm ; : ) . we now treated rats with established ph with a new polymer containing an increased "payload" of chyp. chyp was conjugated to a low mw poly(ethylene glycol)-lysine carrier {poly (peg )-lys-chyp} to increase the % by wt of the analogue. rats were exposed to % o for da to induce ph. on da , and after da of hypoxia, animals were injected iv with chyp polymer in liposomes (hc) or bioinactive trans-hyp polymer in liposomes (ht). air controls received thyp polymer in liposomes (at). at and da, we measured mean right ventricular pressure (rvp) and hydroxyproline (hyp) content in main pulmonary arteries. on da , rvp (mmhg) was Ϯ and hyp (µg/vessel) was Ϯ in at. rvp and hyp increased to Ϯ * and Ϯ *, respectively, in hypoxic animals (n ϭ ; *p Ͻ . vs. at). on da , rvps were at Ϯ , ht Ϯ *, hc Ϯ * †; hyps were at Ϯ , ht Ϯ *, hc Ϯ * † (n ϭ ; *p Ͻ . vs. at; †p Ͻ . vs. ht). from da to , rvp did not increase and hyp decreased in the hc group vs. ht. we conclude that weekly injections of polymeric chyp prevented progression of established hypoxic ph and reversed hyp accumulation. targeted delivery of antifibrotic polymers may prevent and reverse the progression of ph. (support: phs, barbara cornwall foundation). glucosinolates are amino acid-derived natural plant products found throughout the capparales order, which includes agriculturally important crops such as oilseed rape, brassica vegetables and the model plant arabidopsis. glucosinolates and their degradation products have a wide range of biological activities, e.g. in plant defense as deterrents against insect and fungi and as attractants to insects that are specialized feeders in brassicaceae. the conversion of amino acids to oximes is a key step in glucosinolate biosynthesis. we have recently shown that cytochromes p belonging to the cyp family catalyze the conversion of aliphatic, aromatic as well as indole amino acids to the corresponding oximes. cyp e catalyzes the oxime-metabolizing step in the biosynthesis of the cyanogenic glucoside dhurrin. we have recently shown that the oxime-metabolizing enzyme in the glucosinolate biosynthetic pathway is a cytochrome p homologous to cyp e . the post-oxime enzymes in the glucosinolate pathway have high substrate-specificity for the functional groups, and low substrate-specificity for the side chain. therefore, we have been able to metabolically engineer new glucosinolate profiles into arabidopsis by altering the level of endogenous cyp s and by introducing new cyp s. the approach has great potential for design of "biotech crops" with improved pest resistance and increased nutritional value. hypercalcemia as a potential threat in the dietary treatment of maternal phenylketonuria f. eyskens and s. beernaert pediatrician, metabolic diseases and dietitian, azm-koningin paola childrens hospital, metabolic lab pcma, antwerp, belgium over % of infants born to mothers with blood phenylalanine (phe) concentrations above µmol/l exhibit evi-dence of foetal dammage, low birth weight, microcephaly, dysmorphic facies, slow postnatal growth and development and long-term intellectual impairment. keeping maternal phe concentrations below µmol/l before conception and throughout pregnancy reduces significantly the risk of abnormalities in the offspring of women with phenylketonuria (pku). we describe a woman, years old, who showed phe blood levels of - µmol/l under a strict diet (total protein content of . g/kg body weight/day with . g/kg natural proteins and . g/kg proteins provided by the aminoacid mixture pku (milupa, germany); , cal/day) at the beginning of her first pregnancy. the first weeks she developed vomiting which gradually increased in severity. at weeks of pregnancy, she had diarrhea, severe bouts of vomiting and manifested a deficient nutritional status with intake of . g/kg bw proteins and , cal/day. she was hospitalized to start refeeding using continue drip feeding administered by nasogastric tube. after days on this regimen she developed vomiting, heart palpitations and mental confusion. her serum calcium level, that was normal at admission in the hospital, showed an elevation to . - meq/l (ref. value . - . meq/l). the feeding was stopped immediately and under an intravenous infusion and gradually introducing a feeding composed of pku , carbohydrates and mct fats the serum calcium and the blood phe levels dropped to normal values. and volatile components of caramel obtained by heating commercial maltose solution for different time intervals. one sample containing maltose only was used as control, the caramelization was conducted at c° for total time period minutes and subjected to sensory analysis and isolation of volatile components. the odour and colour sensory tests were evaluated according to the international standard methods (iso). the results showed that addition of lysine as a catalyst gave rise to a significant (p Ͻ . ) increase in intensity of the whole flavour in comparison with the control sample. the sweet and caramel notes, the most characteristic attributes of caramel, showed remarkable increase. on the other hand the increase in heating time in presence of lysine as a catalyst resulted in high significant increase in browning rate of caramel solution. the volatile components of each sample were isolated by using the new technique, solid phase microextraction (spme) and subjected to gc and gc-ms analysis. over volatile components were separated, however only the most important component for caramel flavour were reported. maltol and hydroxymethyl- -furfural (hmf) and . h-pyran- -one, , dihydro- , -dihydroxy- -methyl (dihydro dihydroxy maltol), the main characteristic caramelization products were present in high concentration in samples contaning lysine heated for minutes. in addition one pyrazine was only identified in the samples contaning lysine. a comparative study between the present results and those of our previous study concerning addition of alanine as a catalyst was carried out. short-term exposure of human umbilical vein endothelial cells (huvecs) to hyperglycemia increases l-arginine transport (system y ϩ /cats) and nitric oxide (no) production (via enos). it has been reported that enos could also be activated by a ca ϩ -independent mechanism involving phosphorylation of ser by a phosphatidylinositol -kinase (pi -kinase) dependent pathway. we investigated the involvement of pi kinase on the stimulatory effect of acute hyperglycemia on enos and l-arginine transport in huvecs. l-arginine transport, no synthesis and phosphorylation of ser in enos were increased by d-glucose ( mm, min). similar results were obtained in huvecs exposed to insulin. incubation of cells with wortmannin (pi -kinase inhibitor) prevented the effects of d-glucose and insulin. no changes in the intracellular ca ϩ and enos protein levels were detected. thus, acute hyperglycemia increases l-arginine transport and enos activity through a pi -kinase dependent, ca ϩ independent mechanism in huvecs. [ the hypercalcemia in this patient was due to a very high content in calcium of the feeding administered ( - times the adh value) associated with a high vitamine d concentration (see table) and a clinical state of dehydratation. the further pregnancy was uncomplicated and a healthy girl was born who developed normal. • the aminoacid mixtures used in the treatment of pku contain a high level of calcium, phosphate, magnesium and iron. they also contain a high concentration of vitamine d. • nutritional monitoring of pregnant pku patients should include the calcium, phosphate, iron, zinc and vitamins status. • vitamins a and d suppletion is contraindicated in these patients based on the high concentrations of these vitamins in the aminoacid mixtures used in the dietary treatment. flavour and aroma chemistry department, national research centre, dokki, cairo, egypt caramelization of various carbohydrates leads to product with a high tinctorial strength provided by different additives catalyzing the process. the present study was conducted to evaluate the catalytic effect of lysine on the sensory attributes administered pku ( g ϭ adh , g/kg/bw) lipoic acid is a prosthetic group of h-protein of the glycine cleavage system and e components of the pyruvate, oxoglutarate and branched-chain -oxoacid dehydrogenase complexes. in mammals, attachment of lipoic acid to these proteins requires two enzymes. lipoate-activating enzyme (lae) catalyzes the activation of lipoate to lipoyl-nucleoside monophosphate. then, lipoyltransferase transfers the lipoyl moiety to the specific lysine residue of the proteins. we purified lae from bovine liver mitochondria. lae activated lipoate with gtp at a -fold higher rate than with atp. the reaction absolutely required lipoate and mggtp, and the reaction product was lipoyl-gmp. lae activated both r-and senantiomers of lipoate to the respective lipoyl-gmp although preference for r-lipoate was observed. lipoyltransferase equally transferred both r-and s-lipoyl moiety from respective activated lipoate to apoh-protein. however, only h-protein carrying r-lipoate was active in the glycine cleavage reaction. cdna clones encoding a precursor lae with a mitochondrial presequence were isolated. amino acid sequence of lae was identical with that of xenobiotic-metabolizing/medium-chain fatty acid : coa ligase-iii, but an amino acid substitution due to snp was found. these results indicate that the medium-chain acyl-coa synthetase in mitochondria plays a novel function with gtp, the activation of lipoate. instituto di chimica biologica "g. fornaini", università di urbino, italy nitric oxide (no) can modulate red blood cells (rbc) glycolysis by translocation of the enzyme glyceraldehyde- phosphate dehydrogenase (gapd) [e.c. . . . ] from the cytosolic domain of the membrane protein band (cdb ) in the cytosol. in this study we have investigated which no-reactive thiols might be involved influencing gapd translocation, and which is the role of glutathione (gsh) in this context. two highly reactive cys residues (k ϭ . m Ϫ s Ϫ and . m Ϫ s Ϫ , respectively) were identified by transnitrosylation with nitrosoglutathione (gsno) of cdb and gapd. the cys in the catalytic site of gapd is exclusively involved in this gsno-induced nitrosylation. reassociation experiments carried out at equilibrium with preparations of rbc membranes and gapd revealed that different no-donors may form Ϫsno on, and decrease the affinity between, gapd and cdb . in intact rbc, both the no-donors -morpholino-sydnonimine (sin- ) and peroxynitrite (onoo Ϫ ) significantly increased gapd activity in the cytosol and glycolysis measured as lactate production and energy charge levels. however, we obtained data suggesting that onoo Ϫ is the main no-derivative able to cross the rbc membrane leading to gapd translocation and Ϫsno formation. both in cell-free experiments and intact rbc, diamide (a thiol oxidant able to inhibit gapd activity) was observed to reverse the effect of sin- on gapd translocation. the results demonstrate that cdb and gapd contain reactive thiols that can be transnitrosylation mainly by means of gsno, these can ultimately influence gapd translocation/ activity and the glycolytic flux. abteilung für allgemein-viszeral-und gefässchirurgie, kliniken dr. erler gmbh, nürnberg, germany new surgical procedures like minimal-invasive-surgery brought many advantages for the surgical patient: less pain and shorter hospitalization. regarding nutrition, patients gets normal food on the ward still on the operation-day and need only saline-infusions overnight for fluid and electrolyte substitution but no hypocaloric parenteral nutrition. hypocaloric parenteral nutrition had been developped as a peripheral intravenous nutritional concept for patients with a normal body mass index over a period not longer than - days. multiple clinical studies showed that bowel movements increase earlier after an early postoperative enteral feeding which allows an earlier discharge of the patient. the result is a remarkable decrease of costs and an increase in patient benefit. still some years before surgeons preferred in visceral surgery parenteral nutrition over a period of - days under the opinion not to stress an anastomosis. this opinion changed in the last years under the aspect that about , - , ml of bile fluid, , - , ml pancreatic juice and , - , ml gastric juice per day are passing a small intestine anastomosis without any complications. concerning colon-anastomoses, the colon is preoperatively washed out, so it lasts until days until defecation. multiple studies also showed a benefit for the patient regarding immunostimulation by early postoperative enteral feeding. conclusion: in our hospital with surgical patients we recommend postoperatively either early normal enteral feeding or a high caloric parenteral nutrition if parenteral nutrition is needed for longer than days. if artificial nutrition is necessary for more than days we recommend enteral nutrition given by a tube or peg (percutaneous endoscopic gastrotomy). department of food technology, national research centre, dokki, cairo, egypt in the near east, "frekeh" has been known for many centuries as a stable food made from wheat. it is generally claimed that "frekeh" is better than wheat regarding its storage stability. the protein quality of parched immature durum wheat (frekeh) produced from variety was evaluated. frekeh from four maturing levels during the dough stage of the seed development, were analyzed for approximate analysis. results showed that "frekeh" produced at the beginning of the dough stage was of better nutritional value than that produced at the following maturity levels, since the former was higher in protein, fat, minerals and crude fiber as well as in reducing sugar content. in addition, it was shown that these results confirm well with the sensory quality evaluation of the cooked product. further more, it was found that the cooking time was suitable to produce a "freqkeh" meal with high levels of acceptability. the observed decrease in protein content with increasing maturity level raised the question of how the protein quality of "frekeh" versus that of nature wheat grains varied. in this investigation, the amino acid of "frekeh" was determined. dietary treatment and carnitine supplementation has greatly improved long-term outcome of patients with ppa and (vitamin b unresponsive) mma. however, metabolic decompensation may be frequent and final outcome in most patients show various handicaps. to investigate the usefulness of measuring free carnitine and acylcarnitines in dried blood by tandem mass spectrometry, we investigated patients with ppa and with mma in a period of months by weekly capillary blood punctures performed by the parents. age of the patients were from . until years. clinical status at the time of blood drawing was evaluated by regular phone calls. free carnitine in all patients substituted by oral carnitine treatment ( - mg/kg/day bw) was normal. the parameter best reflecting clinical status was the c /c -acylcarnitine quotient. mean value in mma and ppa patients showed a range of . - . (normal . ϩ/Ϫ . , n ϭ ), there was no difference between ppa and mma patients. individual mean values of the patients significantly increased when the patient was ranked higher in the clinical score system or during decompensation. since measurement of acylcarnitines in dried blood by tandem mass spectrometry is easy to perform, this method may be used for home monitoring of patients with mma and ppa. influence of acute treatment with , , , tetrahydroisoquinoline on the levels of glutathione and reactive oxygen species, and on the enzymatic activity of γ-glutamyl transpeptidase in dopaminergic structures of rat brain e. lorenc-koci , m. sokoĺowska , m. zapaĺa , and l. wĺodek institute of pharmacology, polish academy of sciences, kraków, and institute of medical biochemistry, collegium medicum, jagiellonian university, kraków, poland , , , -tetrahydroisoquinoline (tiq) and its derivatives generated considerable interest as molecular species that may be implicated in the pathogenesis of parkinson's disease (pd). in pd, apart from the lack of dopamine in the striatum, a decreased concentration of glutathione (gsh) is found in the substantia nigra (sn). it is also known that gsh depletion potentiates the toxicity of mptp and -hydroxydopamine. however, there are no data available on the tiq influence on gsh metabolism. the aim of the present study was to exemine the effect of acute tiq administration on the levels of gsh and reactive oxygen species (ros), and on the enzymatic activity of γ-glutamyl transpeptidase (γ-gt) in dopaminergic structures of rat brain. the investigation was carried out h after a single dose of tiq ( mg/kg i.p.). at that time, a marked increase in the tissue gsh level and simultaneous significant inhibition of γ-gt were found in the structures studied. in tiq-treated rats, the production of ros was reduced in the sn, but it was markedly enhanced in the striatum. our results suggest that the increase in gsh level in dopaminergic structures stems from inhibition of γ-gt and refers to the extracellular pool of this peptide. apparently, the tiq-mediated alterations in the levels of gsh and ros may have some implications for the etiology of pd. tetrahydrobiopterin-responsive phenylalanine-hydroxylase deficiency with mutations distant from the tetrahydrobiopterin binding site z. lukacs , r. steinfeld , a. kohlschütter , j. zschocke , and k. ullrich department of pediatrics, university of hamburg, and university-children's hospital, heidelberg, germany phenylalanine hydroxylase (e.c. . . . ) catalyzes the hydroxylation of phenylalanine to tyrosine in the presence of oxygen and the cofactor ( r)-l-erythro- , , , tetrahydrobiopterin (bh ). mutations in the phenylalanine hydroxylase gene may cause phenylketonuria or hyperphenylalaninemia. alternatively, disorders in bh metabolism also result in an increase in phenylalanine concentrations but simultaneously affect other bh -dependent enzymes, consequently, causing a severe neurological disorder. recently, several patients with a phenylalanine-hydroxylase deficiency but with normal bh -metabolism were reported who showed a significant decrease in blood phenylalanine concentrations upon treatment with bh . indeed, two such patients in our hospital were also sensitive to daily oral doses of - mg bh /kg. the subsequent molecular genetic analysis revealed that patient was homozygous for the widespread mutation y c and patient was compound heterozygous for the mutations a d and k n. it is striking, that all mutations are located distant from the known bh -binding site and thus, should not be associated with bh -sensitivity. additionally, further patients who share the same genotype are not sensitive to bh . therefore, it must be concluded that factors independent of the phenylalanine hydroxylase gene, like e.g. individual chaperone proteins, influence the three-dimensional structure of the enzyme and thereby, enhance enzymatic activity in the presence of elevated concentrations of bh . retrotransposons are structurally similar to retroviral gag and pol which are required for their replication via reverse transcription, and seem to be an ancestral form of specialized retroviruses. reverse transcription of retrotransposons was assumed to occur in virus-like particles as well as in retroviruses. rna-packaging in this particles suggests a possibility of infection. presumably, the formation of functional virus-like particles requires the interaction of gypsy rna with a protein encoded by gypsy first open reading frame (orf ) or a product of its processing. the objective of this work was to study whether the protein by this frame can bind with nucleic acids similarly to retroviral gag-protein and how phosphorilation of that protein may influence to this interaction. then gypsy orf was cloned and expressed in escherichia coli, and its protein product was purified by ion-exchange chromatography on deae-cellulose and affinity chromatography on heparin-sepharose and tested electrophoretically. it was shown that recombinant protein bound with its own mrna and with dna. the affinity for ssdna bing higher than for dsdna. the binding constant was estimated with rna. the method utilizes the ability of nitrocellulose to bind proteins but not nucleic acids. binding of % gypsy rna was achieved with about ng of the protein in ml of the reaction mixture. the binding constant was &times; m- , which is consistent. the structure of the putative nucleic acid-binding domain suggests that the protein is more similar to the core proteins of spumaviruses of the family retroviridae that to those of other retroviruses. phosphorilation of gag-like protein encoded by first open reading frame of retrotrasposon gypsy (mdg ) affects to interaction with nucleic acid. tryptophan (trp) in humans is catabolized by several pathways leading to various metabolites of kynurenine and indolic compounds formation. a number of diseases are connected with abnormalities in its excretion, but relationship of cause and effect is usually unclear. we introduced a two-step procedure for the detection of defects in metabolism of trp: ) tlc is employed when starting the investigation, ) two hplc methods were proposed and used at the next step, when pathological findings are to be proved and the individual metabolites quantified. the first hplc procedure enables the assessment of tryptophan, indolylacry-loylglycine (iag) and other five indolic compounds. the second method is intended to the monitoring of kynurenine and seven of its catabolites. the same sep-pack pre-treated sample of plasma and urine is used for all methods. the reference values and the excretion pattern in some groups of patients ( in total) were assessed. hepathopathy, gastrointestinal defects, myopathy and seizures with other neurological symptoms were the conditions connected with changes in the excretion of some metabolites of trp. significant decrease of iag excretion was found in burn patients early after the injury. urine analyses were performed at patient with hartnup disease and benign xanthurenic aciduria, inherited metabolic defects of trp. in other experiments, trp effect on the decarboxylation of other aromatic amino acids in the liver was investigated; only weak inhibition under physiological conditions was recognised. ( two hypothetical proteins of escherichia coli, ybbq and yhae, show high sequence similarity to d-threonine dehydrogenase from pseudomonas cruciviae ifo . we cloned each gene encoding ybbq and yhae into e. coli jm . both ybbq and yhae showed no d-threonine dehydrogenase activity and showed significant activities for d-serine in the presence of nad. ybbq and yhae were purified to homogeneity from the e. coli clones. ybbq consisted of two identical subunits with a molecular mass of kda, whereas yhae was a tetramer (native molecular mass, kda). ybbq showed the maximum activity at ph . for the oxidation of d-serine. whereas optimum ph of yhae was ph . . they catalyzed oxidation of glycerate and -hydroxyisobutyrate. d-glycerate was the best substrate for both enzymes. both enzymes also catalyzed reduction of tartronate semialdehyde in the presence of nadh. at physiological ph, the rate of tartronate semialdehyde reduction was much higher than that of d-glycerate oxidation. the ybbq gene is in the operon of glyoxylate utilization and the yhae gene is in the operon for d-glucarate/galactarate utilization. these results suggest that both ybbq and yhae are dglycerate -dehydrogenases and function physiologically in conversion of tartronate semialdehyde into d-glycerate. a serine protease inhibitor model: synthesis and biology z. mucsi , Á. bódi , l. gráf , a. perczel , a. patthy , and g. orosz department of organic chemistry, biochemistry, eötvös university, budapest, agricultural biotechnology centre, gödölló´, and research group of peptide chemistry, hungarian academy of sciences, budapest, hungary sgci is structurally related to the pmpd- family of canonical serine protease inhibitors. in these peptides, there is a p -p Ј position which is responsible for reversible binding to chymotrypsin. their structure is characterized by structural compactness: the molecule contains three -sheets and three disulfide bonds. in the sgci molecule the p -p Ј corresponds to lys-leu bond, which is cleaved by chymotrypsin extremely slowly. the question arises why an excellent substrate behaves at the same time as inhibitor. it was assumed that the threedimensional structure of the molecule is responsible for the inhibitory activity. a model was designed to include all the known features of the inhibitor: the structurally necessary -sheet structure and the fragment containing the p -p Ј environment. three model peptide were synthesized. two model peptides had no inhibiting effect and were cleaved by chymotrypsin. one of the cleavage points is the expected p -p Ј position, while the other positions found to be chymotrypsin preferred positions after the first cleavage. the three-dimensional structures of the model peptides were mapped by nmr. on the basis of nmr structures obtained it has been shown that the cyclopeptide part is more flexible in the models than in sgci. the initial process in the reaction mechanism of a bisubstrate enzyme, rat mercaptopyruvate sulfurttransferase: inactivation study by using chloropyruvate n. nagahara , t. nakagawa , and m. minami department of hygiene and public health, nippon medical school, sendagi bunkyo-ku, tokyo, japan institute for organic chemistry, darmstadt university of technology, darmstadt, germany to investigate the reaction mechanism of a bisubstrate enzyme, rat mercaptopyruvate sulfurtransferase (ec . . . , mst), inactivation kinetics with -chloropyruvate (chloropyruvate) was studied; each inactivation reaction was completed in a preincubation procedure. chloropyruvate is an analog of -mercaptopyruvate (mercaptopyruvate) and irreversibly inhibits mst. the inactivation depended on incubation time and the concentration of chloropyruvate and showed saturation kinetics. the plot for the logarithm of % activity remaining versus preincubation time showed pseudo-first-order. the kinact is . ϫ Ϫ min Ϫ and k is . mm. these suggest that chloropyruvate serves as a mechanism-based inactivator. mercaptoethanol , so that chloropyruvate can approach cys via the donor substrate route and acceptor substrate one, and a ternary complex may be formed prior to the inactivation. these findings suggest that a donor substrate enters the catalytic cavity prior to an acceptor one in the initial process of the mst reaction: mst follows an ordered sequential mechanism. polyketides are natural products of bacteria, fungi, marine organisms and higher plants, many of which have clinical usage. actinorhodin ( ) is an antibiotic produced by streptomyces coelicolor via an iterative type ii polyketide synthase (pks) system. this consists of a multi-enzyme complex with a single catalytic function for each enzyme. departments of chemistry and pediatric surgery, school of medicine, fujita health university, toyoake, japan it has been reported that, in rats with a single intoxication of α-naphthylisothiocyanate (anit), acute liver injury develops with enhanced lipid peroxidation and neutrophil infiltration in the liver tissue. melatonin functions as an antioxidant. melatonin is known to inhibit neutrophil infiltration into damaged liver tissues. therefore, we examined whether melatonin exerts a protective or preventive effect on anit-induced acute liver injury. male wistar rats received a single i.p. injection of anit ( mg/kg) and oral administraton of melatonin ( mg/kg) at or h after anit injection. animals administered with melatonin at and h after anit injection were sacrificed and h, respectively, after the injection. liver injury appeared h after anit injection and developed at h. melatonin administered at h after anit injection prevented liver injury formation with attenuation of increases in hepatic lipid peroxide level and myeloperoxidase activity, an index of neutrophil infiltration. melatonin administered at h after anit injection prevented liver injury development with attenuation of further increase in hepatic lipid peroxide level. thus, melatonin protects against and prevents anit-induced acute liver injury in rats possibly through its antioxidant action and/or its inhibitory action against neutrophil infiltration in the liver tissue. k. okamura-ikeda, s. katayama, k. fujiwara, and y. motokawa t-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. mutation in human t-protein (ht) gene results in clinical nonketotic hyperglycinemia (nkh). eight point mutations have been identified so far in nkh patients with t-protein deficiency. to understand the structure and function of ht, the wild-type (wtht) and three mutant t-proteins (g r, g d and r h) were expressed in escherichia coli with chaperons groel and groes which facilitated the recovery of the expressed proteins as a soluble form. levels of expression of these proteins were similar but the recovered soluble forms of mutants were about one-third of wtht. g r showed comparable specific activity to wtht, whereas g d and r h mutants exhibited remarkable reduction in specific activity. since homoallelism for g d mutation and heteroallelism for g r and r h mutation were identified in typical and atypical nkh, respectively, these results suggest that g and r h mutations are highly deleterious in the aspects of not only protein folding and/or stability but also catalytsis. on the other hand, g r mutation might affect mainly on the protein stability. detailed characterization of these mutants is now in progress. laboratory of animal nutrition and biochemistry, miyazaki university, miyazaki-shi, japan the minimal actinorhodin pks, shown in black below, consists of the ketosynthase (ks), chain length factor (clf) and acyl carrier protein (acp) and is the minimum set of enzymes required for polyketide production. we have investigated the stoichiometry of the ks-clf complex and the ks-clf:acp minimal system using three methods: . native gel electrophoresis. . cross-linking of proteins using dibromoacetone. . radical cross-linking of proteins. this new method has also been used with wild type s. coelicolor cell free extract with % ks-clf in order to elucidate which proteins are in close proximity to ks-clf during in vitro actinorhodin production. in ruminant animals, essential amino acids have never been completely established, because of the difficulty of its estimation due to the presence of microorganisms such as bacteria and protozoa in the first stomach called rumen. in our previous paper, histidine was shown to be the first limiting amino acid in the rumen contents when evaluated by chemical score. recently we have also reported that rumen microorganisms cannot synthesize histidine from histidinol. on the other hand, there have been some reports which showed that nitrogen balance of ruminants was not improved by supplementation of histidine to rumen microbial protein together with methionine, lysine and threonine which had been known to improve. based on these facts, we have a hypothesis that histidine may not be an essential amino acid for ruminants. in the present paper, we will report about the abilities of cattle liver and kidney to synthesize histidine from histidinol comparing with those of swine liver and kidney. the ability was demonstrated by examining the activities of histidinol dehydrogenase (crude enzyme) by means of direct measurement of an increase in histidine and decrease in histidinol. the amount of histidine produced from histidinol by the enzyme seemed sufficient for meeting the histidine requirement of cattle. the browning reaction is the sequence of events which begins with the reaction of amino group in amino acids, peptides or proteins with glycosidic hydroxyl group of sugars; the sequence terminates with the formation of brown nitrogenous compounds or melanoidines. this reaction gives rise to tremendous number of components such as volatile alcohols, ketones, aldehydes, esters, ethers and sulfur and nitrogen containing heterocycles in addition to nonvolatile amadori compounds and complex brown pigments of medium to high molecular weights. the present study was designed to choose a currently occurring system (aspartic acid -fructose) as a model system, since aspartic acid was found to be one of the most important amino acids in many kinds of food varieties. the reaction was done under controlled conditions of reactants ratios, temperature and time. the reaction mixtures were subjected to successive extractions with suitable solvents where the obtained corresponding flavour concentrates were thoroughly investigated. the results indicated different classes of compounds such as aldehydes, furans, alcohols and alkylated pyrazines varying in quantities depending on the reaction conditions. these products were also investigated concerning their toxicological effects. so, such products of nonenzymatic reactions showed different chemical and biological properties. purification and characterization p. piyarat , s. nagata , h. misono , and k. packdibamrung department of biochemistry, faculty of science, chulalongkorn university, bankok, thailand department of bioresources science, faculty of agriculture, kochi university, nankoku, kochi, japan nad ϩ dependent alanine dehydrogenase was purified fold to homogeneity from aeromonas hydrophila. molecular mass of , daltons was estimated for alanine dehydrogenase by sephadex g- chromatography. sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified en-zyme showed polypeptide band with molecular mass of , daltons, indicating that the enzyme is hexamer. the enzyme is highly specific for alanine and nad ϩ . sulfhydryl group of the enzyme plays an important role in the catalysis. the enzyme retained its activity on heating at °c for h. optimum ph for reductive amination and oxidative deamination were . and . , respectively. the steady state kinetic studies including product inhibition on the enzyme reaction indicated that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism in which nad ϩ binds first to the enzyme followed by l-alanine and products are released in the order of pyruvate, ammonia and nadh, respectively. the k m values for nad ϩ , l-alanine, pyruvate, ammonia and nadh were . , , . , and . mm, respectively. an elevation of apolipoprotein (apo) b- concentrations is a particular feature of several metabolic disorders, such as type diabetes (t d), impaired glucose tolerance (igt), and familial combined hyperlipidemia (fchl). to further understand the in vivo turnover of apolipoprotein b- of very low density lipoprotein subfractions (vldl , svedberg units (s f ) - and vldl , s f - ) kinetic studies were performed in subjects with t d, igt, fchl, and healthy controls using a tracer of either l-[ring- c ]-phenylalanine or l-[ , , - h ]leucine. these studies showed direct hepatic vldl apob- secretion to be increased in patients with t d and igt when compared with controls. in contrast, patients with fchl showed a discrete increase in hepatic vldl apob- secretion. in all patients vldl catabolism is not essentially impaired. vldl apob- secretion is associated with plasma insulin and free fatty acid (ffa) concentrations, resp., whereas vldl apob- secretion is correlated with plasma mevalonate and lathosterol levels. in conclusion, vldl overproduction is supposed to be completely responsible for higher triglyceride (tg) levels found in patients with t d, igt, and fchl. vldl overproduction seems to be regulated by tg and ffa substrate and appears to be an indicator of decreased insulin sensitivity. in contrast, vldl overproduction is more likely to be regulated by the availability of cholesterol substrate. these data give further in vivo evidence that vldl and vldl secretion is regulated independently. arabidopsis resulted in enhanced production of cysteine and glutathione graduate school of pharmaceutical sciences, chiba university, chiba, japan serine acetyltransferase (satase) catalyzes the formation of o-acetyl-l-serine (oas) which is the key intermediate of cysteine biosynthesis. oas is not only a dominant limiting factor but recently suggested as a possible signal molecule for gene expression in cysteine biosynthesis. in has been shown that the activity of cytosolic satase from watermelon was feedback inhibited by l-cysteine. to enhance the ability of cysteine biosynthesis in plants and to reveal the role of oas in the regulation of sulfur assimilation, we made the point-mutated watermelon satase gene (satg c) whose product was not inhibited by cysteine, and introduced satg c into arabidopsis. the contents of oas, cysteine, and glutathione in transgenic arabidopsis were increased significantly as compared to the wild-type arabidopsis. we are currently dealing with the expression analysis of sulfur-related genes in transgenic arabidopsis accumulating oas due to the overexpression of satase. certain amino acids as source of specific branched chain fatty acids in fish sauce manufacture n. g. sanceda , e. suzuki , and t. kurata institute of environmental science for human life, and department of human biological studies, ochanomizu university, tokyo, japan the source of some branched volatile fatty acids (vfa) during the fermentation process in the manufacture of fish sauce was investigated. we previously reported that straight chain volatile acids seemed to have been derived from fish fats but unlikely for branched fatty acids which was believed to be derived from other sources. to clarify the source of branched volatile acids, specific amino acids, alanine, leucine, iso-leucine and valine were used in this study. these amino acids were first mixed with salt and added to fish. the fish mixtures were then aerobically and anaerobically incubated for one and a half months. results showed that addition of valine significantly increased the production of iso-butyric and iso-hexanoic acids and leucine increased that of iso-valeric in the aerobically fermented fish mixtures. a similar tendency was observed in the anaerobically fermented fish mixture except that an increase in the amount of iso-hexanoic acid was observed in the leucine added mixture, which was not observed in the aerobically fermented one. it seemed that specific branched volatile fatty acids were derived from certain amino acids. glutathione (gsh) is an important component of the cellular defense mechanisms that protect cells from oxidative injury. in the retina, the glial (müller) cells have been shown to synthesize and transport gsh, and thus are likely to be involved in regulating gsh levels. in the present study, we have characterized gsh transport system in a müller cell line using s-gsh uptake. the results showed that gsh was taken up in a na ϩ -and concentration-dependent manner with a k m of . mm. moreover, cellular gsh had no effect on the rate of gsh uptake. in related studies, we found that oxidative stress induced the expression of γ-glutamylcysteine synthetase (gcs) subunits, and that gcs mrna levels were correlated with the degree of gsh depletion. because organic anion transporters (oatps) have been implicated in glutathione cotransport, we examined expression of oatp members using rt-pcr. we found that the müller cell line expressed transcripts for oatp , oatp and oatp . these studies indicate that the müller cell plays important role in gsh homeostasis in the retina. in the active site of human porphobilinogen synthase (ec . . . , pbgs), two zinc ions are coordinated by cys , cys and cys , and his and cys , respectively. the fomer zinc ion, closer to catalytic site lys , plays an important role in catalysis. on the other hand, a role of the latter (distal) one has not been clarified. interestingly, in human hep b cell, his was replaced with arg (h r). to elucidate the role of his in catalysis, the kinetic properties of wild type and h r mutant enzymes were studied. these cdnas were cloned by rt-pcr with total rna from human peripheral lymphocyte and hep b cell, respectively. each cdna encoding pbgs with Ј non-coding region was inserted into pet- b(ϩ) vector and then the constract was transformed into e. coli strain bl (de ). the cells were cultured in lb medium containing mg/ml ampicillin and µm zn ion for h at °c. after addition of mm isopropyl--d(Ϫ)-thiogalactopyranoside, cells were further cultured for h at °c. the highly purified pbgss were obtained by ultora centrifugion, fractionation with ammonium sulfate and column chromatographies with deaecellulose, hydroxylapatite and superdex , serially. we are now investigating molecular properties of these pbgss. agriculture and agri-food canada, lacombe research centre, lacombe, alberta, canada handling and management procedures such as capture and restraint can be significant stressors for recently domesticated animals such as elk (cervidae elaphus). the objective of the current study was to investigate the use of pre capture nutritional therapy in attenuating hpa response and improving animal welfare. fourty eight adult male elk stags ranging in age from - years and raised on pasture were used in the study with as control and as nutritionally treated. twenty four hours prior to capture the elk were offered either kg of a cereal grain based dietary supplement or kg of a cereal grain based nutritional therapy product containing specified amino acids (usa patent # ). the amino acid content of the nutritional therapy product was minimally . g per kg animal weight of ala, lys, phen, meth, thre, isoleu, val and tryp plus g per kg weight of leu and g/ kg weight of glut. the animals were subsequently captured and held in appropriate facilities designed to handle elk. saliva samples were collected on all animals immediately following capture and salivary cortisol was monitored by ria. animals offered the nutritional therapy product containing the amino acid mixture displayed lower cortisol levels ( . nmol/l) compared to the untreated controls ( . nmol/l; p Ͻ . ). the data suggest that amino acid therapy can be used to attenuate hpa response to a stressor in captured elk. department of bioengeneering and technology, delhi, new delhi, india resistance to analogues of methionine by corynebacterium lilium results in the partial de-repression of methionine biosynthetic enzymes. the levels of enzymes involved in methionine biosynthesis also increased step-wise by successive endowing the resistant markers, resulting in the overproduction of methionine. moreover, the repressibilities of the enzymes were also reduced by the addition of methionine analogue resistance. analogue resistant mutants were developed by uv induced mutagenesis of corynebacterium lilium (wild type) strain. the single analogue (norleucine) resistant mutant c. lilium nl- produced µg/ml methionine in shake flasks with methionine yield at . g methionine/g glucose and specific methionine production at . mg/g dcw, while double analogue (norleucine and triazole) resistant mutant c. lilium nt- produced µg/ml methionine. a triple analogue (norleucine, triazole and ethionine) resistant mutant c. lilium nte- produced . g/l methionine. the methionine yield was . g methionine/g glucose and its specific productivity was . g methionine/g dcw. clinical biochemistry, laboratory , luxemburg, grand duchy of luxemburg blood plasma glucose level was compared on fast and minutes after oral administration of mg of acetylcysteine. in the group of healthy persons the plasma glucose level feel by . % over the minute period. in the diabetics on the contrary, the plasma glucose level observed minutes after administration of acetylcysteine was . % higher than in blood plasma taken on fast. similar tests were carried out "in vitro" to interpret these different results. the control group consisted of ml of distilled water ϩ . ml % glucose ϩ . ml god pad (boringer mannheim gmbh). in the acetylcysteine group the distilled water was replaced by ml . % solution of acetylcysteine. in the glucagon group the distilled water was replaced by . % solution of glucagen hypokit novo nordisk. spectrometric determination was carried out after minutes of incubation. a % diminution of glucose was observed in the acetylcysteine group in comparison with the control group. a . % increase in glucose was observed in the glucagon group in relation to the controls. the results with healthy persons and the tests "in vitro" indicate that acetylcysteine lowers the level of glucose. but it elevates the level of glucose in the blood plasma of diabetics. it may be presumed that acetylcysteine modifies the insulinglucagon balance in favour of glucagon. the objective of this study was to fortify yogurt with three oilseed protein hydrolysates prepared from soybean (glycine max), sesame (sesanum indicum) and rice bran (oryza sativa) flours. hydrolysis was carried with two enzymes one of plant origin (papain) and the other of microbial origin (alcalase). a yogurt fortification experiment was then carried using the previous hydrolysates. the hydrolysates were added to yoghurt at , and % levels of fortification and the fortified yoghurt was analyzed fresh, and after and days of consuming period. fortified yogurt was chemically examined for fermentation activity (ph values, acidity and proteolysis) as well as its organoleptic properties. results of this experiment indicate that the addition of soybean hydrolysates with papain ( . units/g) for minutes (tb) and rice bran hydrolysates with alcalase ( . units/g) for minutes (te) to yoghurt can ex-ceed - %, while fortification with sesame hydrolysed with papain ( . units/g) for minutes (td) and soybean hydrolysed with papain ( . units/g) for minutes (tc) can not reach up to %. it is well known that dna is fragile to reactive oxygen intermediates (rois) damage. evidences that dna fragmentation and apoptosis occur in cardiomyopathies, in the failing heart and in cultured cells under hyperbaric oxygen (hbo) stress, demonstrated that oxygen free radicals also play a critical role in heart failure. as a consequence, myocardial cell survival depends on response to oxidative stress. experimental data obtained in vitro suggested that polyamines, by acting as rois scavengers, play a role in prevention of endonucleasemediated dna fragmentation and inhibition of alkylating agents-mediated damage, potentially exerting a protective role against rois damage. thus we studied polyamine metabolism and superoxide dismutase (sod) expression in an in vivo model of heart oxidative stress, such as rats subjected to hbo. four experimental groups were used: ) controls; ) rats subjected to hbo for min once and immediately sacrificed; ) rats treated as group but for consecutive days and immediately sacrificed; ) rats treated as group but sacrificed h later (recovery). northern blot analyses showed that odc mrna accumulation increased immediately (paralleled by activity) in groups - , while ssat mrna decreased remarkably, thus leading to higher polyamine concentration in rois-stressed hearts. contrariwise, sod mrna level decreased rapidly in groups - . this suggests that hbo-induced compensatory mechanism in rat heart is based on specific and rapid boosting of polyamine concentration, caused by coordinate induction of biosynthesis and inhibition of catabolism, and not of enzymes known to metabolise rois such as sod. amino acids oxidation was greater in tumor-bearing rats muscle. leucine is an important ketogenic amino acid that proves energy to the skeletal muscle. leucine supplemented diet was used to analyze the effects produced by walker growing in pregnant rats which were distributed into six groups. three groups received normal diet ( % protein): control (c), tumor-bearing (w), pair-fed rats (cp). three groups were fed with diet supplemented with % leucine ( % protein plus % leucine): pregnant fed with leucine (l), tumor-bearing with leucine (wl) and pair-fed with leucine (lp). after days, the animals were submitted to intestinal perfusion to measure leucine, methionine and glucose absorption. leucine absorption increased in w and wl groups. glucose absorption reduced in tumor-bearing. in pregnancy with cancer, metabolic changes provided both reduced fetal and tumor development. tumor-bearing rats showed increase in methionine and leucine absorption, probably diverting this nutrients to tumor cells. glucose absorption reduced in w and wl. leucine supplemented diet group promoted high leucine absorption which could be used by neoplasic cells, and mainly by fetus and host. probably, the transamination of the branch long chain amino acid provided energy substract for the skeletal muscle, keeping the nitrogen offered to host carcass. ( undernutrition cause several changes as body weight loss, in biochemical parameters, even microscopic alteration in absorptive epithelium. this means the nutrients absorption process has been harmfully and consequently increase the damages caused by malnourished. knowing leucine is used as a ketonic and oxidative amino acid our main propose was to recovery the malnourished young rats with normal (rc) and leucine supplemented diet (rl, % of leucine) for days. it was measured body, liver, and muscle weight, intestinal absorption of glucose, methionine and leucine, and body chemical composition. the body weight gain in rc and rl was higher than control group, suggesting that nutritional replacement for these groups could provided nutrients to support the body weight recovery, reaching as the same weight as the control. methionine and glucose absorption was reduced in malnourished group, but it was recovered (glucose, methionine and leucine) after nutritional replacement. leucine supplemented diet promoted a good recovery of carcass collagen nitrogen, keeping the carcass structural nitrogen. further studies are necessary to investigate this mechanism. [financial support: fapesp ( we diagnosed the very rare autosomal recessive disorder hyperprolinaemia type ii (deficiency of ∆ -pyrroline- carboxylate dehydrogenase, ec . . . ) in a girl aged months presenting with seizures and encephalopathy. l-∆ pyrroline- -carboxylic acid accumulates in this disorder and there is a - -fold increase in plasma proline. surprisingly, she also had vitamin b deficiency. this was an unrecognised association, which was not explained by her diet or medications. we hypothesised that pyridoxal phosphate (vitamin b coenzyme) was de-activated by l-∆ -pyrroline- -carboxylic acid. with high resolution h nuclear magnetic resonance spectroscopy and mass spectrometry, we have shown that these two compounds react at ph . and °c in vitro to form three novel adducts, which we characterised. they are products of a claisen condensation (or knoevenagel type of reaction) of the activated c carbon of the pyrroline ring with the aldehyde carbon of pyridoxal phosphate. if this previously unreported interaction occurs in vivo, pyrroline- -carboxylic acid is a unique endogenous vitamin antagonist. preliminary observations show that pyrroline- -carboxylic acid also condenses with other biologically important aldehydes and ketones. some of these reactions may contribute to the brain disturbances in hyperprolinaemia type ii. we have already identified adducts with acetoacetic acid in urine from our child, which is evidence that condensation can occur in vivo. the kidneys are characterized by a high activity of γglutamyl transpeptidase (γ-gt), as well as by a high cysteine level. the present paper was aimed to obtain information on how the activity of γ-gt and the levels of non-protein sulfhydryl compounds (npsh) changed with age in rat kidneys. simultaneously, protein-bound cysteine (pb-cys) and sulfane sulfur compounds were estimated. the kidneys were from following rats groups: young ( -month-old), middle-aged ( month-old) and old ( -month-old). the obtained results showed that the activity of γgt and npsh levels in the kidneys fell with age. at the same time, a significant increase in the level of protein-bound cysteine was observed. on the other hand, the content of sulfane sulfur compounds was elevated in the group of the oldest animals. these findings indicate that -due to disturbances in the γ-glutamyl cycle -the capacity for extracellular glutathione degradation and, in consequence, the availability of cysteine for intracellular gsh biosynthesis may be impaired. the increased pb-cys level indicates potentiation of the thiolation reaction, i.e. development of protein-mixed disulphides, cysteine, sulfane sulfur compounds, oxygen reactive species. national research centre, dokki, cairo, egypt in the past few years, many attempts have been made to prepare a synthetic insulin. the biological activity of insulin is known to be closely related to the c-terminal octapeptide fragment of its b-chain. this does not necessarily mean, however, that each of the amino acid residues of the octapeptide fragment is essential for its activity. it was found that b gly and b phe were present in all insulins so far obtained from various animal species indicating the significance of these two residues. it would therefore seem desirable to study the effect of each of these two amino acid residues or both on biological activity of the octapeptiede fragment of the b-chain. weitzel et al. found that the substitution of arginine b with another amino acid resulted in a very large decrease in biological activity, which indicates that it participates in the action of insulin. also it was found that the aromatic amino acid residues (b -b ) participate in the action of insulin. a heptapeptide arg-phe-tyr-thr-pro-lys-ala-och , corresponding to (b -b ) insulin des gly -phe , and an octapeptide arg-phe-phe-tyr-thr-pro-lys-ala-och , des gly were synthesized using the solid phase method. the c-termenal ends of both peptide were converted to methyl ester by transesterification cleavage from the resin. the side chain protecting groups were removed by hf. manual counter current distribution method was used for purification of the free peptides. the way to solve the evaluation of tyrosine containing peptide was studied. the free methyl ester peptides were administrated for insulin-like activity test by glucose metabolism in the rat fat cells technique in vitro. nitric oxide synthase inhibitors influence dynorphin immunoreactivity in the rat brain following hyperthermia p. alm and h. s. sharma department of pathology, university hospital, lund university, lund, and laboratory of neuroanatomy, department of medical cell biology, biomedical centre, uppsala university, uppsala, sweden nitric oxide (no) is a free radical gas that influences neuronal communication in the central nervous system (cns). recent reports suggest that no can influence dynorphin neurotransmission in the normal brain as well as in several pathological states. previous reports from our laboratory show that the enzyme nitric oxide synthase (nos) responsible for no formation is upregulated in several brain regions following hyperthermia. the present investigation was carried out to find, whether hyperthermia can influence dynorphin immunoreactivity in the brain, and if so, whether inhibition of nitric oxide synthesis will alter its distribution in heat stressed rats. rats subjected to hyperthermia at °c for h in a biological oxygen demand incubator (bod) resulted in marked redistribution of dynorphin immunoreactivity in several brain regions e.g., cerebral cortex, hippocampus, cerebellum and brain stem. pretreatment with two potent nos inhibitors, l-name ( mg/kg, i.p.) and l-nmma ( mg/kg, i.p.) min before heat stress significantly altered the dynorphin immunoreactivity in the brain. these drugs alone however, did not influence the peptide expression in normal rats. the results suggest that (i) hyperthermia has the capacity to influence dynorphin immunoreactivity in the brain, and (ii) inhibition of nitric oxide synthase considerably influences the dynorphin immunoreaction in hyperthermia, not reported earlier. the functional changes induced by uncompetitive and competitive nmda antagonists, memantine, amantadine and mk- , and cgp , respectively, were studied in both saline-pretreated and mptp-pretreated c bl/ mice. the nmda antagonists were administered acutely by themselves or in combinations of either: nmda antagonist plus subthreshold l-dopa dose or nmda antagonist plus suprathreshold l-dopa dose, to either the mptp-pretreated or the salinetreated mice. activity-enhancing or functional restorative effects of the nmda antagonists were variable with memantine and mk- distinguished from amantadine and cgp . in the study of long-term effect of nmda antagonists mk- was administered postnatally and spontaneous motor behaviour and motor activity in response to several pharmacological interventions was assessed. marked alterations associated possible with apoptotic penchance are discussed. t. archer and a. fredriksson department of psychology, university of göteborg, and department of psychiatry, university of uppsala, ulleråkers hospital, uppsala, sweden synergistic antiparkinsonian actions of different classes of putative therapeutic agents co-administered with a subthreshold dose of l-dopa ( mg/kg) in drug-naive mptp-treated mice as well as the restorative actions of those compounds in suprathreshold l-dopa-tolerant mptp-treated mice subjected to "wearing-off" of l-dopa efficacy were assessed in a series of experiments. the classes of compounds studied included the noncompetitive nmda antagonists, memantine, amantadine and mk- , the anticonvulsive and putative anticonvulsive agents, lamotrigine, fce , phenytoin, the monoamine oxidase inhibitors, l-deprenyl, amiflamine, α-ethyltryptamine, clorgyline and phenelzine, and the α -adrenoceptor agonists, clonidine and guanfacine. in this final case, the restorative effects of clonidine and guanfacine were antagonised by the α -adrenoceptor antagonist, yohimbine, but not the α adrenoceptor antagonist, prazosin. within each class of potentially therapeutic agents a differential restorative efficacy was obtained, but the combination of different doses of apomorphine with clondine failed to restore motor activity. in vivo proton mr-spectroscopy of the human brain: assessment of n-acetylaspartate (naa) reduction as a marker for neurodegeneration w. block , f. träber , s. flacke , f. jessen , ch. pohl , and h. h. schild department of radiology, department of psychiatry, and department of neurology, university of bonn, germany proton magnetic resonance spectroscopy ( h-mrs) is a well accepted non-invasive method to investigate changes in brain metabolite composition in different types of cerebral disease. we performed proton spectroscopy in patients with dementia of the alzheimer's type (ad) and in patients with motor neuron disease (mnd) with the aim to detect a specific metabolic pattern for each of these two neurodegenerative disorders. overall, more than spectroscopic data sets of patients with mnd and more than data sets of ad patients were acquired within the last years. in the mnd group we found a significant reduction of naa/tcr metabolite ratios in the central region, which correlates with the disease severity and the clinical lateralisation of neurological symptoms and increases in the time course of the disease. in ad patients a similar reduction in relative naa contents was observed in the medial temporal lobe. the observed regional metabolic alterations correlate well with the characteristic neurological symptoms in ad (dementia) and mnd (muscular palsy) and seem to follow the disease process over time. since naa is exclusively expressed in neurons as shown by immunohistochemical studies, reduced naa levels suggest neuronal loss or dysfunction in the observed regions. center for molecular imaging research, massachusetts general hospital, boston, massachusetts, u.s.a. non-invasive measurement of hemodynamic parameters and imaging neovasculature architecture during angiogenesis is highly important in determining tumor prognosis and in assessing treatment efficacy. we suggested a technique to map the tumor vascular (vvf) and interstitial volume fraction (ivf) noninvasively in vivo. a poly-l-lysine based macromolecular probe (mpeg-pl-gddtpa) with extended circulation in the bloodstream designed to shield chelated paramagnetic lantanide with poly(ethylene glycol) chains. we hypothesized that a magnetic resonance signal after intravenous administration of a vascular paramagnetic probe can be maximized so the signal change after administration of a second comound (gddtpa) reflects the ivf but not the vvf. the method and its assumptions were verified in animal models of cancer. tumoral vvf and ivf values were consistent with histology data and literature values. imaging showed heterogeneity of both parameters at submillimeter pixel resolution. this technique was used for characterizing differential angiogenesis in human mammary adenocarcinoma lines as well as for imaging anti-angiogenic drug effects. anti-angiogenesis was induced using synthetic d-reverse peptides derived from thrombospondin- . this study showed that peptide treatment results in slower brain tumor growth due to inhibition of de novo blood vessel formation and synergistic anti-proliferative effect on tumor cells. in conclusion, in vivo mr imaging can be used for non-invasive treatment assessment of novel antiangiogenic drugs. wallenberg neuroscience centre, lund university, lund, sweden we have recently found that -hydroxydopamine lesioned rats gradually develop dyskinetic-and dystonic-like movements upon repeated administration of a therapeutic dose of l-dopa. such movements simulate the time course of peak-dose dyskinesia in parkinson's disease. in this rat model, the severity of l-dopa-induced dyskinesia is strongly correlated with an upregulated expression of the prodynorphin gene in striatal neurons. using antisense technology and gel-shift assay analyses, we have addressed the role of transcription factors which may mediate this response. we have found that the camp response-element binding protein (creb) is essential in maintaining a basal expression of prodynorphin mrna in the intact striatum, but it is not required for l-dopa to induce the prodynorphin gene in dopamine-denervated striatal neurons. we have thus addressed the role of fos-and jun family tran-scription factors, and found very high levels of fosb-and jundlike proteins in the striata of dyskinetic animals. these proteins could bind to both ap and cre sites in the prodynorphin promoter. moreover, intrastriatal fosb knockdown could inhibit both the upregulation of prodynorphin gene expression and the development of dyskinesias under chronic l-dopa treatment. we propose that dimers of fosb-and jund-like proteins mediate abnormal changes in striatal gene expression which are linked to the development of l-dopa-induced dyskinesia. department of pharmacology, grünenthal gmbh r&d, aachen, germany glutamate plays important roles in both normal and pathophysiological nocieception. upon physiological conditions, glutamate release from primary afferents in the spinal cord activates largely ampa receptors. as those are ubiquitously involved in fast transmission in the cns, ampa antagonists have a broad side-effect profile. prolonged activation of nociceptors by tissue damage, inflammation or nerve injury evokes a long-lasting release of glutamate and neuropeptides, activating nmda receptors in the spinal cord. this mechanism appears to play a key role in pain chronification. the nmda receptor is, therefore, an important target for chronic pain treatment. both animal and human studies confirm the efficacy of nmda antagonists in chronic pain, however, clinically available compounds are weak or have unacceptable side-effects. glycine b antagonists and compounds selectively blocking nr b-containing receptors appear to be safer, the reasons for this remain unclear. central side-effects could potentially be avoided by using nmda antagonists with restricted central access. peripheral nmda receptors (as well as some other subtypes of glurs) could be activated by glutamate released from the site of injury, thus contributing to peripheral hyperexcitability. some other subtypes of glurs can also contribute to peripheral sensitisation. of ionotropic glurs, kainate receptors appear important in inflammatory and neuropathic pain. they can be activated by high intensity stimulation of nociceptive afferents, and may act as autoreceptors controlling release of glutamate. group i metabotropic glurs are also present on primary afferents and on second order neurones in the spinal cord, and may play a similar role. antagonists of these subtypes of glurs are active in some models of chronic pain. specific upregulation of group ii metabotropic glurs in some pain-relevant structures could reflect a possible adaptive role of these inhibitory receptors under chronic pain conditions; their selective agonists also have a potential for the treatment of chronic pain. we have performed a series of studies of the distribution and function of mglur subtypes in the basal ganglia that suggest that members of this receptor family could serve as targets for novel therapeutic agents that would be effective in treatment of pd. for instance, two group ii mglurs (mglur and mglur ) are localized on presynaptic terminals of striatal neurons in the globus pallidus where they could reduce gaba release. furthermore, activation of group i mglurs results in a depolarization and increased cell firing of neurons in the subthalamic nucleus (stn) and projection neurons of the substantia nigra pars reticulata (snpr). interestingly, this effect is mediated by mglur in snpr projection neurons and mglur in stn neurons. finally, activation of group ii mglurs results in inhibition of glutamate release from stn terminals in the snpr. furthermore, selective agonists of group ii mglurs inhibit haloperidol-induced catalepsy in rats, suggesting an antiparkinsonian effect of these compounds. the rich distribution and diverse physiological roles of mglurs in basal ganglia raises the possibility that these receptors may provide targets for novel therapeutic agents that could be used for treatment of pd and related disorders. a. cupello , m. parodi , and m. balestrino centro di neurofisiologia cerebrale, cnr, genova and dipartimento di scienze neurologiche, university of genova, italy in vitro rat hippocampal slices are commonly used to study the effects of hypoxia in the central nervous system, because they allow to differentiate the effects of hypoxia in the brain from that of systemic (e.g., respiratory and cardiac) failure that may accompany hypoxia. we used electrophysiology to monitor and evaluate the damage caused by transient hypoxia to the nervous tissue. a few minutes after oxygen deprivation brain tissue suddenly depolarizes. this event, which is termed ìanoxic depolarizationî is accompanied by dramatic metabolic changes: transmembrane ionic gradients disappear (na ϩ enters, k ϩ exits the neurons), neurons swell, there is intra-and extra-cellular acidosis. this event is caused by functional inactivation of (na ϩ / k ϩ )atpase caused by decreased atp content, as it is suggested by the fact that it is mimicked by ouabain treatment. one of us has contributed to show that if this event is not promptly reversed by reoxygenation it causes irreversible damage, mainly by determining a massive entry of ca ϩ into neurons. pretreatment of tissue with creatine ( mm or more) both increases neuronal energy store by increasing neuronal phosphocreatine and protects brain tissue from irreversible damage. in vivo increase in phosphocreatine has been shown using lower ( . mm) creatine concentration, injected directly into the lateral ventricle. a different type of hypoxia-induced damage is observed when hypoxia is of shorter duration. in this case upon reoxygenation one does not observe disappearance of evoked potentials but their increase. this phenomenon, originally described as ìpost-hypoxic hyperexcitabilityî has been later called ìanoxic long-term potentiation (ltp)î. we showed that this event can be prevented by inactivating the nuclear protein s- . while this damage is milder than that induced by anoxic depolarization, it may explain stroke-induced epileptic fits. we are currently investigating what role, if any, pretreatment with creatine may have in preventing also this type of damage. cytoskeleton is subject to continuous modification to yield changes in cell shape and function of plasmamembrane proteins linked to the cytoskeleton. gelsolin (gsn) depolymerizes filamentous actin and thus causes dynamic uncoupling of membrane ion channels. we have studied alteration of neuronal ca ϩ influx by the absence of gsn and its pathophysiological consequences during cerebral ischemia. cytosolic ca ϩ concentrations were determined ratiometrically in synaptosomes preloaded with fura- am. glutamate release from synaptosomes superfused with krebs' buffer was measured by hplc. transient focal cerebral ischemia was induced by h occlusion of the middle cerebral artery (mca). in gsn deficient mouse brain cortical synaptosomes [ca ϩ ] i increase in response to k ϩ ( mm) depolarization was % higher than in wild-type. ω-agatoxin iva . µm decreased ca ϩ -influx in neocortical wild-type synaptosomes by %, and abolished differences between gsnϩ/ϩ and Ϫ/Ϫ genotype. k ϩinduced release of glutamate in neocortical synaptosomes was % higher and lesion size after mca occlusion was % higher than in wild-type. it is concluded that presynaptic ca ϩ influx is increased in gsn deficient nerve terminals which, together with subsequently increased glutamate release, increases neuronal vulnerability. in vivo assessment of tissue alteration in cerebrovascular and neurodegenerative diseases s. flacke, w. block, f. träber, p. mürtz, h. urbach, and h. schild the combined used of perfusion imaging (pi) and molecular diffusion imaging (dwi) are opening a new window into the processes that occur during the first hours of ischemia. dwi detects early changes of proton diffusion associated with cytotoxic edema. pi has the potential to characterize the degree of regional hypoperfusion. mismatches between dwi and pi, i.e. hypoperfused areas with normal adc are considered potentially salvageable. we present results of patients with an angiographically defined thrombembolus in the middle cerebral artery and a spontaneous stroke evolution. whereas the infarct core was clearly visible on both dwi and pi, tissue at risk of infarction could only be detected by an increased blood volume and transit time. however only in a subgroup of patients (n ϭ ) these areas were incorporated into the final infarct. in these patients perfusion parameter of tissue at risk of infarction were more pronouncedly altered than in those where the tissue at risk was spared from infarction (ratios of tissue at risk vs normal (rcbv . Ϯ . , mtt . Ϯ . , ttp . Ϯ . , p Ͻ . ). these human data show that a detailed analysis of diffusion/ perfusion mismatches allow the identification of tissue at risk of damage. glucose deprivation enhances the sensitivity of cerebellar granule cells to die by excitotoxicity. we have previously reported that neither min of glucose deprivation, a treatment that depletes cell energy resources, nor exposure to µm glutamate (glu) for min, induce significant cell death in cerebellar granule cell cultures, h after treatment. in contrast, the combined treatment with µm glu and glucose deprivation induces both cell death and choline (cho) release. we investigated whether the neurotoxic effect of this treatment is related with inhibition of phosphatidylcholine (pc) synthesis. we found that exposure to µm glu alone for min, glucose deprivation for min, and the combination of both treatments inhibited pc synthesis when measured at the end of treatment by %, % and %, respectively. furthermore, we found that exposure to either µm glu, glucose deprivation or µm glu ϩ glucose deprivation decreased incorporation of [ h]cho into phosphocholine and increased the intracellular content of free [ h]cho, indicating that all these treatments inhibit the synthesis of pc by inhibiting choline kinase activity. since only the combined treatment with µm glu plus glucose deprivation evoked cho release and excitotoxic cell death, the present results indicate that other factors in addition to inhibition of pc synthesis are required to induce cho release and excitotoxic cell death in cerebellar granule cells. (supported by cicyt, saf - .) the role of striatal metabotropic glutamate receptors in degeneration of dopamine neurons k. goĺembiowska, j. konieczny, k. ossowska, and institute of pharmacology, polish academy of sciences, kraków, poland the present study was undertaken to characterize the effect of blockade of the mglu receptor subtype by -methyl- -phenylethynylpyridine (mpep), as well as the effect of stimulation of the mglu / receptor by (Ϫ)- -oxa- aminobicyclo[ . . ]hexane- , -dicarboxylic acid (ly ) on spontaneous and stimulated dopamine (da) release in rat striatum using an in vivo microdialysis. mpep ( - µm), perfused through a microdialysis probe affected neither the basal nor the veratridine ( µm)-stimulated striatal da release. however, mpep given intraperitoneally ( mg/kg) diminished either the basal or the veratridine-evoked da release. ly ( - µm) administered locally also inhibited the veratridine-evoked da release in rat striatum. antagonists of mglur-i and agonists of mglur-ii have been shown to have neuroprotective properties in several models of neurotoxicity in animals. we have approached this issue using a selective mglu antagonist in an animal model of neurotoxicity induced by methamphetamine. in our preliminary experiments, methamphetamine ( ϫ mg/kg sc every two hours) decreased the tissue content of striatal da and its metabolites dopac and hva.mpep ( ϫ mg/kg ip) given before every methamphetamine injection reversed its action. the effect exerted by the mglu antagonist mpep seem to be mediated by sites located outside the striatum due to relieving da neurons of the facilitatory influence of glutamate. in turn, the attenuation of da release from nigrostriatal terminals by ly may be a consequence of activation of striatal mglu / receptors. reversal of the methampetamine-induced da depletion suggests a potential for neuroprotective activity of mpep. o. golubnitschaja , h. h. schild , and j. flammer department of radiology, university of bonn, germany university eye clinic, basel, switzerland glaucoma remains a major eye illness with unknown etiology. although elevated intraocular pressure has been shown to be the major risk factor, there is a cohort of relatively young patients developing normal-tension glaucoma (ntg). assymptomatic ischemic events in brain have been shown to be often attributable to galucoma. perfusion of the retina and optic nerve head suffering from observed vasospastic dysfunction may be further reduced by changes in the intraocular pressure. ocular ischemia developed due to these blood flow deficits may play a major role in initiation of glaucoma. possibly secondary to ischemia the autoimmunogenic capacity is activated by ntg patients having an increased prevalence of systemic autoimmune diseases. therefore, the determination of potential molecular markers in blood lymphocytes could be useful for early diagnostics of ntg. our recent study using "gent hunting"-techniques showed indeed altered gene expression in lymphocytes of ntg patients. the demonstrated downregulation of xpgc gene expression which subsequently leads to the accumulation of damaged dna and an elevated p expression, together with the upregulation of a new abc-transporter seem to be specific for the pathogenesis of ntg. molecular imaging of ntg provides insights in mechanisms of disease initiation and allows the early diagnostics and preventive treatment. (supported by "bio-rad" and "amersham pharmacia aggregate cell cultures prepared from fetal rat telencephalon were used to study neuronal amino acid consumption during glucose restriction. to that end, both mixed (neuronglia) and neuron-enriched cultures were grown in chemically defined medium and tested at an advanced maturational stage. it was found that h of exposure to reduced glucose ( . mm instead of mm) caused significant increases in the consumption of several amino acids and the accumulation of ammonia. it also greatly changed the intracellular level of several amino acids in neurons, particularly of aspartate and glutamate. irreversible neuron-specific damage was observed one week after the insult. elevated glutamine media concentrations ( mm instead of . mm) during glucose restriction further increased ammonia production and neuronal damage, although the overall rate of glutamine metabolism remained practically unchanged. taken together, our findings suggest that glucose deficiency caused (i) the dysfunction of crucial transamination pathways; (ii) a shift towards the oxidative deamination of glutamine and several other amino acids used by neurons as alternative energy substrates; and (iii) the accumulation of neurotoxic ammonia levels. institute for brain research, university of vienna, austria the racemic (d,l) mixture of the naturally occurring neutral aromatic amino acid , -dihydroxy-l-phenylalanine (l-dopa) was first synthetized in . in , the natural levorotatory isomer was isolated from vicia faba beans and declared to be biologically inactive. however, in l-dopa was observed to lower the blood pressure in the rabbit, an effect opposite to the vasopressor effect of adrenaline. following the discovery, in , of the enzyme l-dopa decarboxylase, ldopa's conversion in tissues (by decarboxylation) to dopamine (da), the first biologically active substance in the biosynthetic pathway of catecholamines, was demonstrated. subsequent pharmacological studies, done between and , showed that the biological actions of l-dopa were, in principle, due to da formed from it in the body. in , the central antireserpine effect of d,l-dopa was described in mice and confirmed in with l-dopa in humans. following the demonstration of da's occurrence in the brain in the years / , d,l-dopa was found (in rabbits) to restore brain da levels, reduced by reserpine. in , the severe brain da deficit, confined to patients with parkinson's disease (pd) was reported and a year later l-dopa's superior anti-akinesia effect in patients with pd demonstrated. finally, in the high-dose oral l-dopa regimen was successfully introduced into clinical practise. in contrast to these supreme achievements, two related early studies remained, for different reasons, without consequence. despite some initial doubts about its mechanism of action, there is now convincing evidence for l-dopa therapy being a classic example of a central neurotransmitter replacement therapy, with the severe brain da deficit furnishing a rational basis for the amino acid's clinical use and high efficacy in patients suffering from pd. b. jakobsen , a. tasker , and j. zimmer anatomy and neurobiology, sdu-odense university, odense, denmark department of anatomy and physiology, university of prince edward island, canada the toxicity of domoic acid (dom) was studied in rat hippocampal slice cultures, prepared from -days old rats and grown on semipermeable membranes for - weeks before exposure. dom ( . - µm) was added to the medium, alone or together with the glutamate receptor antagonists ns- , nbqx or mk- , for hrs followed by hrs in normal medium. neuronal degeneration was monitored and ec values estimated by densitometric measurements of the cellular uptake of the propidium iodide (pi) at , , and hrs. the lowest ec values, obtained at hrs, were: ca ( µm), dentate granule cells (dg) and ca ab ( µm),ca c ( µm). protective effects of µm nbqx at hrs were seen against µm dom in dg, ca and ca c and against µm dom in ca and ca c. µm ns and mk only displayed protective effects together with nbqx. mk thus significantly increased the protective effect of µm nbqx in ca against µm dom in combination with µm nbqx and µm ns . we can confirm that dom neurotoxicity primarily involves ampa/kainate receptors, but also nmda receptors at high concentrations (glutamate release). department of physiology/neuroscience, medical university of south carolina, charleston, south carolina, u.s.a. although dopamine has been most clearly tied to the development of addiction to drugs of abuse, recent studies indicate that once addiction has been established the expression of addictive behaviors, such as drug craving, is mediated more by long-term neuroadaptaions in glutamate transmission. data will be presented which supports and extends this hypothesis. repeated cocaine injections were given for one week and three weeks after the last drug injection a number of molecular, neurochemical and behavioral neuroadaptations were measured. it was found that in the nucleus accumbens there is a increase in the expression of genes encoding mglur / and glur , and a decrease in the expression of mglur and its accompanying scaffolding proteins homer bc. this was accompanied by an increase in the capacity of mglur / receptors to regulate presynaptic glutamate release and a blunting in the effects of stimulating mglur / receptors. in addition, there is reduced activity in the cystine/glutamate exchanger wks after repeated cocaine. as a result of these changes there is a decrease in the basal release of glutamate, and a relative increase in the releasibility of glutamate upon stimulation. by using the reinstatement model of drug seeking behavior, it was shown that glutamate transmission in the projection from the prelimbic cortex to the core of the nucleus accumbens was particularly affected by the cocaine-induced changes in gene expression. taken together, these findings support the use of glutamate autoreceptor agonists as possible therapeutic adjuvants in treating the cravings associated with addiction. dopamine (da), a catechol that autoxidizes to an oquinone, is implicated as an endogenous pro-toxin, however, the following studies suggest that da has dual neurodegenerative and neuroprotective roles. in rats treated as neonates with -hydroxydopamine ( -ohda; :g icv), there was a % reduction in striatal tissue da content in adulthood, and a to fold increase in spontaneous hydroxyl radical (ho*) formation (indirect salicylate trapping method: dihydroxybenzoic acid analysis). additionally, systemic l-dopa ( mg/kg i.p.) suppressed ho* formation. however, when glutamate ( mm) was added to an in vivo microdialysate, ho* formation was increased substantially more in the microdialysate from dainnervated striatum. these findings indicate that da innervation is inherently neuroprotective, but in the presence of a high level of an excitatory amino acid, da innervation predisposes to formation of reactive oxygen species. ongoing neuronal activity is likely to interact with and to determine the role of da as a neurotoxic or neuroprotective substance. (supported by ns .) the glutamate hypothesis of schizophrenia along with the dopamine hypothesis was intensively discussed in the past. the last years however suggest more and more that neither a hypofunction of the glutamatergic system alone nor a hypofunction of the dopaminergic system alone is responsible for symptoms found in schizophrenia. the basal ganglia (bg) as the critical structures mediating symptoms of schizophrenia are innervated by dopaminergic fibers from the mesencephalon as well as by glutamatergic fibers from limbic structures; like prefrontal cortex, hippocampus, entorhinal cortex and amygdala. thus, limbic input is able to modulate information processing in each structure of the bg and by this way control dopaminergic functions through feedback mechanisms. dysfunction in limbic structrues may result in an imbalance of information processing via the bg and terminates in behavioral symptoms of schizophrenia. we showed in recent neurochemical studies in combination with behavioral analysis that a simple, generalized hypofunction of limbic glutamatergic input on bg nuclei is not the key mechanism inducing schizophrenic behavior. a dysfunction of a particular limbic structure or pathway seems to be responsible for an imbalanced information processing via the bg and imbalanced behavioral adaptation terminating in schizophrenic symptoms. [ there is a need to identify subtype-specific ligands for mglu receptors to elucidate the potential of these receptors for the treatment of nervous system disorders. to date, most mglur antagonists are amino acid-like compounds acting as competitive antagonists at the glutamate binding site located in the large extracellular n-terminal domain. we have investigated novel subtype-selective mglur antagonists which are structurally unrelated to competitive mglur ligands. using a series of chimeric receptors and point mutations we demonstrate that these antagonists interact with novel allosteric binding sites in the tm domain via a noncompetitive mechanism of action. recent studies in animal models implicate mglu receptors as a potentially important therapeutic target particularly for the treatment of pain and anxiety. vascular endothelial growth factor (vegf) is a major mediator in angiogenesis and vascular permeability. in central nervous system (cns) vegf plays pivotal roles such e.g., inductor of endothelial cell proliferation, migration and inhibition of apoptosis, as well as mediator of blood brain barrier (bbb) breakdown and subsequently of brain edema formation. these ubiquitous epiphenomena are major complications in several cns pathologies, including head trauma and stroke. reduced tissue oxygen tension (hypoxia) and hypoglycaemia triggers vegf expression that occurs in ischemic regions around postraumatic or postinfarct necrosis. after brain injury, the expression of vegf is increased contributing to disruption of the bbb. vegf increases the permeability of bbb via the synthesis/release of nitric oxide and subsequent activation of soluble guanylate cyclase. the immunohistochemistry shows an increase of stained astrocytes around a cortical micronecrosis. vegf participates in the response of the cns to injury in a dose dependent way. immunostaining correlates with infarct volume and clinical disability. vegf-antagonists reduce ischemic brain edema and injury, involving vegf in pathogenesis and eventually in treatment of stroke and related disorders. this cytokine also exerts a neuroprotective effect mediated by its receptor flk- . functions related to the inflammatory response, co-expression with proteins of the ecm and interaction with the two main receptors, flk- and flt- , will be discussed. n-methyl-d-aspartate (nmda) receptors can mediate excitotoxic or neuroprotective responses. one of the molecular mechanisms responsible for nmda neuroprotection involves the release of brain-derived neurotrophic factor (bdnf) which in turn binds to and activates its cognate receptor trkb. bdnf levels in the neuronal culture medium increased -fold when cells were preincubated for three hours with nmda. at three hours, the increase in bdnf protein levels in the medium was accompanied by a concomitant increase in bdnf mrna. thus, nmda elicited two temporally distinct responses: an early release of bdnf protein followed by a later transcriptional activation of dbnf mrna and protein release. these results suggest that nmda activates the trkb receptor via a bdnf autocrine loop resulting in neuronal survival. in addition, extracellular regulated kinases (erk / ) were rapidly activated, which peaked within six hours of nmda treatment. erk / activation is completely blocked by mk- and partially blocked by k a, suggesting the nmda and trkb receptors act in a coordinated fashion to activate erk / . as an extension of this work, we discovered a single nucleotide polymorphism in the human nr gene that, when transfected into hek cells, alters the electrophysiological properties of the nmda receptor complex. possible consequences of this nmda receptor variant in signaling will be discussed. this overview summarizes our recent knowledge of the role that tyrosyl radical (tyro • ) can play in neurochemical systems of brain and thereby lead to neural disorders (pd, ad, als). these could involve the interactions of tyrosine and tyro • with reactive oxygen species (ros) and reactive nitrogen species (rns), via radical mechanisms and chain processes in the presence of o and endogenous brain antioxidants. concentrations of tyro • , ros and rns can increase dramatically under conditions of generalized stress: oxidative, nitrative or reductive. this in turn can directly damage (by lipid peroxidation) or indirectly damage (by protein oxidation and/or nitration) cellular substructures which ultimately can lead to apoptotic neuronal cell death or autoschizis. enzymatically (classical peroxidase mechanisms) or non-enzymatically formed tyro • can react with no • and this reversible and intrinsic "combination" acts to "buffer' tyro • concentrations. the reaction of tyro • with superoxide (o •Ϫ ) is a scavenging reaction which proceeds rather by addition, not by electron transfer; and major resultant products are tyrosine hydroperoxides (tyrooh). however, the decay of tyro • can be also terminated by self-termination (dimerization) resulting in dityrosine (dt) formation. tyro • can catalyze ldl oxidation, although the precise mechanisms of this reaction in vivo remain unknown. nitration of tyrosine to -nitrotyrosine ( -nt) requires a one-electron oxidation as a primary step, with formation of tyro • , followed by addition of the nitrogen dioxide radical (no • ). the promoting effect of carbon dioxide on peroxynitrite-mediated tyrosine nitration (via radical mechanisms) (tyro • /no • /o •Ϫ /no • system) is due to the selective reactivity of the putative carbonate radical anion, as compared to that of the oxidizing hydroxyl radicals ( • oh). moreover, once formed, -nt may act to promote repetitive redox cycling; it may be reduced to the corresponding nitroanion radical, which is then oxidized by molecular o to o •Ϫ and parent -nt. one-electron oxidation of -nt can result in catalytically active imminoxyl radical. dt formation can outcompete tyrosine nitration at lowsteady state concentrations of peroxynitrite. it is unquestionable that very high fluxes of no • and o •Ϫ are requisite intermediates of peroxynitrite, a tyrosine nitration agent formed via tyro • . evidence for the existence of generalized stress within neurons includes the presence of protein peroxides (tyrooh), dt, and -nt. the nitration/denitration processes can be pathologic, but these also may play: ) a signal transduction role; ) a role of "blocker" for radical-radical reactions (scavenging of no • , no • and co •Ϫ by tyro • ); or ) a role of delimiting factors for peroxynitrite formation. it is still unknown whether oxidation/nitration of tyrosine (as dopamine precursor or protein residue) via tyro • formation, is a footprint of generalized stress and neuronal disorders, an important part of o •Ϫ and no • metabolism, or just a part of integral processes for maintaining neuronal homeostasis. the complete answer of these questions should be the first priority task of our recent search, wherein the problem of increased free radical formation in the brain and/or the imbalance of ratios: ros/rns/tyro • may be all important in determining neural cell and tissue injuries under pathological conditions resulted from generalized stress. [acknowledgements. this work was supported in part by kbn ( more than % of patients with type diabetes have coronary heart disease, related to silent ischemia, caused by an autonomic denervation of the heart in diabetic patients. oxidative damage to dna has been well documented in cardiac cells isolated from diabetic patients and rats with streptozotocin-induced diabetes mellitus (dm) . this dmmodel shows already seven days after onset of disease structural changes in vascular tissue typical for the development of atherosclerosis. this study evaluates possible molecular mechanisms for early events in the development of dm-induced cardiomyopathy. methods: using "expression array" we examined the activation of cardiac cell death in heart of dm-rats. ms-pcr was used to examine a differential dna methylation. results: an increased expression of genes encoding renin, angiotensinogen and p was detected in heart of dm-rats. substantial changes in the methylation status of the p dependent p waf /cip -gene and the cyclin d -gene were detected in dm-rats. conclusions: the renin-angiotensin system is upregulated with diabetes, and this may contribute to the development of cardiomyopathy via oxidative damage and p -dependent activation of cardiac cell death. this pathway includes de novo methylation of the p -inducible p waf /cip -gene encoding a protein which binds to and inhibits a broad range of cyclincyclin-dependent kinase complexes. (supported by "bio-rad" and "amersham pharmacia biotech") department of pharmaceutical biosciences, uppsala university, uppsala, sweden during the past decade studies have indicated that growth hormone (gh) may exert effects on the central nervous system (cns). for instance, gh replacement therapy was found to improve the psychological capabilities in adult gh deficient (ghd) patients. furthermore, beneficial effects of the hormone on certain functions, including memory, mental alertness, motivation and working capacity have been reported. likewise gh treatment of ghd children has been observed to produce significant improvement in many behavioural problems seen in these individuals. studies also indicated that gh therapy affects the cerebrospinal fluid (csf) levels of various hormones and neurotransmitters. further support that the cns is a target for gh emerges from observations indicating that the hormone may cross the blood-brain-barrier (bbb) and from studies confirming the presence of gh receptors in the brain. it was previously shown that specific binding sites for gh are present in discrete areas in the cns of both humans and rats. in peripheral tissues gh is shown to elicit its effects through a second mediator insulin-like growth factor (igf- ). igf- is well recognized as a protective agent against neural injury in the cns. the neuroprotective effect of this peptide has a broad spectrum affecting many brain regions and acts through its antiapoptopic effect. the production of igf- is upregulated in areas of brain damage and the igf- system may be an important part of an endogenous neuroprotective system. in spinal cord injuries, however, the content of igf- is reduced. we recently observed a neuroprotective effect of topical application of igf- in animals subjected to spinal cord trauma. the observed effect may be mediated via a mechanism involving nitric oxide. in the same animal model we have very recently observed a neuroprotective effect of gh. recent reports suggest that the level of gh is drastically reduced in patients with spinal cord injury. in victims of spinal cord injury the secretion of gh and igf- , as well, is known to be decreased. therefore, exogenous substitution of gh and igf- might be a promising approach in the future therapy of spinal cord injury victims. in fact, there is one report indicating that prolonged treatment with synthetic gh of spinal cord injured rats attenuates some of the neurological motor dysfunction seen in these animals weeks following trauma. in our animal model we observed that topical application of rgh significantly reduced traumainduced disturbances in the fluid micro-environment. we also noted that gh was capable of attenuating the trauma-induced depression of spinal cord evoked potentials. the mechanism by which gh exerts it neuroprotective effects will be discussed. chronically administered levodopa in parkinson's disease (pd) treatment is ultimately associated with alterations in motor response. in -hydroxydopamine lesioned hemiparkinsonian rats, chronic twice-daily administration of levodopa progressively shortens duration of contralateral turning and augments the period of turning at or below % of peak turning rate. the pathogenesis of the response alterations involves in part sensitization of the corticostriatal glutamatergic synaptic activity. characteristic changes involving interactions between striatal kinase and phosphatase signaling now appear to contribute to sensitization of spiny-neuron glutamatergic receptors. glutamate-mediated striatal dysregulation, subsequently, modifies basal ganglia output system in ways that favor the appearance of parkinsonian motor response complications. at a molecular level, transcriptional activation of striatal creb contributes to the persistent expression of the levodopa-induced motor response alterations. conceivably, a safer and more effective therapy for all stages of pd can be provided by drugs that target intracellularly on striatal kinases or phosphotases, or by agents that interact extracellularly on non-dopaminergic striatal receptors such as ampa and nmda, adenosine a , adrenergic a , opiod, and serotonergic b. the primary cause of parkinson's disease is a loss of dopamine in the corpus striatum. it has been postulated that this effect leads to disinhibition of the striopallidal pathway and, secondarily, to a functional shift towards glutamatergic stimulation. the aim of the present study was to find out whether inhibition of glutamatergic transmission at a level of metabotropic glutamate receptors (mglurs) in the striatum may alleviate parkinsonian-like symptoms in rats. the non-competitive antagonist of receptor subtype (mglur ), mpep ( . - mg/kg ip), or the agonist of group ii mglurs, ly ( - mg/kg ip), reduced the haloperidolinduced muscle rigidity and catalepsy. intrastriatal injections of the antagonist of mglur , (rs) aida ( . - µg/ . µl), but not of the agonist of group ii mglurs, r, r-apdc ( . - µg/ . µl), inhibited the muscle rigidity induced by haloperidol. in order to search for an influence of mglurs on the striopallidal pathway, the effect of mpep or of the agonist of group ii mglurs, dcg-iv, on the preproenkephalin mrna expression in the striatum was examined. the obtained results suggest that blockade of group i mglurs, or stimulation of group ii mglurs may be important to the amelioration of parkinsonian symptoms. striatal mglurs may contribute to at least some of these effects. several lines of evidence suggest an important role of glutamate in depression. the involvement of group i mglurs in depression has also been proposed. thus, we decided to evaluate whether group i mglurs antagonists have antidepressantlike effects. we also investigated if antidepressant treatment influences group i mglu receptors in the brain. the experiments were performed on male wistar rats ( - g) and male c bl/ mice ( - g). aida (group i mglurs antagonist) given i.v. in the dose of µg, decreased the immobility time in the despair test in rats. mpep (noncompetitive, systemically active mglur antagonist) given i.p., was not effective in the despair test in rats. however, in doses of . , and mg/kg, it significantly decreased the immobility time of mice in the tail suspension test. moreover, the deficit in passive-avoidance learning, which was observed in bulbectomized rats, was reversed by chronic, but not acute mpep ( mg/kg) treatment. prolonged imipramine treatment resulted in significant increase of the level of expression of mglu receptors in the ca field of the hippocampus, while prolonged electroconvulsive shock treatment (ect) enhanced significantly the chemiluminescence of mglu receptors in the ca field. the results indicate that group i mglu receptors are modified by chronic antidepressant treatment and that group i metabotropic glutamate receptors antagonists may play a role in the therapy of depression. (this study was supported by kbn grant no. .po a. . ) institute of pharmacology, polish academy of sciences, krakow, poland chronic exposure to nicotine, alcohol, opioids, sedatives, and cannabis results in development of drug dependence that becomes evident upon a cessation of drug administration and expresses itself as a withdrawal syndrome (with its physiological and motivational manifestations). adaptations at the nmethyl-d-aspartate receptor (nmda-r) complex have been observed in different brain areas during chronic exposure to, and upon withdrawal from, opioids, ethanol, benzodiazepines and barbiturates. behavioral studies employ the assessment of the effects of nmda-r antagonists on: a) the development of dependence (nmda-r antagonists are co-administered with the drug), b) the maintenance of dependence (nmda-r antagonists are administered to animals with pre-established dependence, and -most relevant to the clinical situation -c) on the expression of drug dependence (assessment of the withdrawal severity in subjects with nmda-r antagonists administered just before the expected emergence of withdrawal). the development of dependence to opioids and benzodiazepines is significantly retarded by nmda-r antagonists. studies from this laboratory demonstrate similar inhibition by nmda-r antagonists of the maintenance of opioid dependence. both in rodents and humans, the expression of opioid antagonist-precipitated as well as spontaneous (natural) withdrawal is inhibited by nmda-r antagonists, and animal data demonstrate similar inhibition of the expression of dependence produced by ethanol, barbiturates and benzodiazepines. the involvement of the excitatory amino acid, glutamate and the inhibitiory amino acid, gamma-amino butyric acid (gaba) in the pathophysiology of spinal cord trauma is not known in details. this investigation is focused on the involve-ment of glutamate and gaba in a rat model of spinal cord injury using immunohistochemistry. spinal cord injury induced by an incision into the right dorsal horn of the t - segments resulted in profound edema formation and cell damage in the adjacent t and t segments at h. pretreatment with h- / ( mg/kg, p.o.), a potent antioxidant compound, effectively reduced the edema formation and cell injury following trauma. at this time period, untreated traumatised rats exhibited a marked increase in glutamate immunoreactivity and a distinct decrease in gaba immunostaining in the t and t segments compared to the control group. the changes in glutamate and gaba immunoreactivity in traumatised rats were considerably attenuated by pretreatment with h- / . these results suggest that (i) oxidative stress contributes to alterations in glutamate and gaba in spinal cord injury, (ii) glutamate and gaba are contributing to edema formation and cell damage and (iii) the antioxidant compound h- / has a potential therapeutic value in the treatment of spinal cord injuries. dov pharmaceutical, inc., hackensack, new jersey, u.s.a. both preclinical (i.e., behavioral despair models) and clinical studies indicate that compounds reducing transmission at nmda receptors are antidepressant. conventional antidepressants may be viewed as "monoamine-based", increasing the synaptic availability of serotonin, norepinephrine, and/or dopamine. however, chronic administration of of conventional antidepressants alters both mrna levels encoding nmda receptor subunits and radioligand binding to this family of ligand-gated ion channels in circumscribed areas of the cns indicating that nmda receptors may be a downstream target of these monoamine-based agents. we have recently reported (li, et al., neuropharmacology, in press ) that a class of ampa receptor potentiators also exhibits antidepressant-like actions in preclinical models. in this presentation, i will describe how these two distinct, and (at a cellular level) seemingly diametric approaches employing glutamatergic mechanisms converge on intracellular targets that are also impacted by chronic treatment with biogenic amine-based agents. kainic acid is an essential pharmacological tool for many forms of neurobiological research. until several years ago, all commercially available kainic acid was derived from a single biological source (digenia simplex). commercial isolation of kainic acid in japan ceased in , creating a void in the marketplace. recently several different companies have become providers of kainic acid, but each uses a different source of the compound ( biological and synthetic) and different isolation procedures. our objective was to use three common assay systems to evaluate the comparative pharmacological and neurotoxicological properties of these three sources of kainic acid. dose response curves, both alone and in the presence of receptor selective antagonists, were constructed for each kainate formulation using (a) cerebellar granule neurons in culture, (b) isolated hippocampal slice preparations, and (c) whole animal behavioural toxicity studies. preliminary results reveal many similarities, but also distinct differences between the three formulations, especially when challenged with antagonists for different eaa receptors. full results will be presented and discussed with respect to their implications for both extending the known kainite literature and for future studies employing kainic acid as a ligand in both mechanistic investigations and in animal models of neurodegenerative disease. our results from in vitro studies further elucidate the role of cell-cycle related proteins in neuronal apoptosis induced by excitotoxins. exposure of primary cerebellar neurons to toxic concentrations of glutamate was found to produce a significant, short lasting increase in the expression of p and cdc . transcriptional activity of p was shown by increased p dna binding activity and by the concomitant induction of the cdk inhibitor p , the cell cycle regulator gadd and the apoptotic induced bax. cell-cycle proteins are also expressed concomitantly to dna damage in neurons undergoing excitotoxic degeneration. we found that excessive activation of glutamate receptor by nmda results in the formation of -oh-deoxyguanosine, which is a marker of oxidative dna damage. in addition, the expression of the dna repair factor msh increases in cultured cerebellar neurons or in ca pyramidal cells that have been challenged with excitotoxins. excitotoxicity may thus provide a further example of how re-expression of cell-cycle proteins might be tightly connected to dna damage and repair in neurons. rush-presbyterian-st. luke's medical center, chicago, u.s.a. patients with parkinson's disease by definition benefit from levodopa therapy. however, after years of therapy % of patients experience motor response complications (mrc's): the benefit from each dose becomes shorter (wearing-off), more unpredictable (on-off) and associated with involuntary movements (dyskinesias). when dyskinesias first arise, they are associated with high levodopa levels and may be prevented or minimized by lowering levodopa intake. later on, the therapeutic window of levodopa narrows progressively and dyskinesias occur at doses equal to those needed to induce an antiparkinson effect. while the pathogenesis of motor complications remains incompletely understood, recent clinical studies implicate mechanisms downstream from the degenerating nigrostriatal dopamine system, possibly involving glutamatergic projections to the basal ganglia. in a rat model of pd, blockade of striatal glutamate receptors of the n-methyl-d-aspartate (nmda) subtype reverses levodopa-induced motor fluctuations. similarly, in -methyl- -phenyl- , , , tetrahydropyridine (mptp) lesioned primates, several nmda-antagonists reduce levodopa-associated dyskinesias. in parkinsonian patients the nmda-antagonists dextromethorphan, dextrorphan and amantadine improve dyskinesias as well. these findings have lead to the suggestion that hyperfunction of nmda receptors on striatal efferent neurons, as a consequence of chronic non-physiologic dopaminergic stimulation, contributes to the pathogenesis of motor response complications. protein misfolding and aberrant polymerization are salient features of virtually all central neurodegenerative disorders, including alzheimer's disease (ad), parkinson's disease, polyglutamine diseases, tauopathies, and prion diseases. in many instances, a single amino acid change can predispose to disease by increasing the production and/or changing the biophysical properties of a specific protein. possible pathogenic similarities among the cerebral proteopathies suggest that therapeutic agents interfering with the proteopathic cascade might be effective against a wide spectrum of diseases. however, testing compounds preclinically will require diseaserelevant animal models. numerous transgenic mouse models of ad-like pathology have now been produced. our studies have found that tg mice overexpressing human -amyloid precursor protein (huapp k n/m l) produce copious deposits of diffuse and compact -amyloid as they age, and that females are more susceptible than are males (callahan et al., am. j. pathol. , - , . recently, we also found that the overexpression of p protein, an activator of the kinase cdk , results in tau hyperphosphorylation, axonopathy and severe motor deficits in transgenic mice, in the absence of neurofibrillary tangles. none of the existing transgenic models of -amyloidosis or tauopathy fully recapitulates the pathology of ad. in an attempt to more authentically model the human disease, we infused dilute ad-brain extracts into tg mice at -months of age (i.e. - months prior to the usual onset ofamyloid deposition). we found that infusion of ad brain extracts results in: ) earlier and more abundant deposition of -amyloid in app-transgenic mice (kane et al., j. neurosci. , - ); ) evidence for the spread of pathology to other brain areas, possibly by neuronal transport mechanisms; and ) tau hyperphosphorylation (but not neurofibrillary pathology) in axons passing through the injection site. the seeding ofamyloid by ad brain extracts suggests pathogenic similarities between -amyloidoses such as ad and other cerebral proteopathies, and could provide a new model for studying the proteopathic cascade and its neuronal consequences in neurodegenerative diseases. supported by warner-lambert/pfizer. purpose: the effects of essential amino acid deficiencies on function of cornea and lens were investigated. methods: dietary deficiencies of tryptophane and methionine were studied in young rats over months. transparency of cornea and lens were evaluated using slitlamp microscope and scheimpflug camera. after sacrifice, lens fresh weight and crystallin patterns were determined to evaluate effects on lens growth and protein synthesis. results: methionine deficiency had no effect on the parameters investigated. tryptophane deficiency caused severe loss of body weight in both rat strains (brown-norway, bn; sprague-dawley, sd), sd rats also lost their hair. they developed corneal neovascularisations and cortical cataracts. bn rats developed faint neovascularisations and a discontinuity zone in the lens. diet intermission arrested pathological processes restarting when feeding diet again. this observation is supported by lens fresh weight data. dna staining evidenced that tryptophane deficiency arrested lens fiber maturation. conclusion: a difference has been found for essential amino acids in their effects on transparency of cornea and lens. tryptophane deficiency stimulated corneal neovasculariseration, but arrested lens fiber cell maturation. the difference in reaction of cornea and lens to tryptophane deficiency between bn and sd rat eyes remains to be elucidated. dynorphin is a neuropeptide that is present in the dorsal horn of the spinal cord. the peptide is actively involved in pain processing pathways. however, its involvement in spinal cord injury is not well known. alteration in dynorphin immunoreactivity occurs following a focal trauma to the rat spinal cord. infusion of dynorphin into the intrathecal space of the cord results in ischemia, cell damage and abnormal motor function. antibodies to dynorphin when injected into the intrathecal space of the spinal cord following trauma improves motor recovery and reduces edema and cell changes. however, influence of dynorphin on trauma induced alteration in spinal cord bioelectrical activity is still not known. spinal cord evoked potentials (scep) are good indicator of spinal cord conduction that are altered following trauma. therefore, in present investigation, influence of dynorphin antibodies on trauma induced changes in scep was examined in our rat model. in addition, spinal cord edema formation and microvascular permeability disturbances were also investigated. our results show that intrathecal administration of dynorphin antiserum prior to injury has a beneficial effect on trauma induced electrical activity, microvascular permeability disturbances, and edema formation. these observations indicate that dynorphin is somehow involved in the altered bioelectrical activity of the spinal cord and participates in the pathophysiological processes leading to cell injury. fatty-acid binding proteins (fabps) are involved in the intracellular binding, targeting and transport of long-chain fatty acids (fas) to modulate cell growth and/or differentiation. fabp form a family of proteins displaying tissue-specific expression. the expression of brain type fabp (b-fabp) is spatially and temporally correlated with neuronal differentiation during brain development. heart type fabp (h-fabp) is widely distributed and present in skeletal muscles, kidney, lung, brain and endothelial cells. it is neuron-specific in postnatal brain and participates in neurite formation and synapse maturation. epidermal type fabp (e-fabp) is expressed at high levels during neurogenesis, neuronal migration, and terminal differentiation. although all three fabps could be involved in normal brain function in prenatal and postnatal life, a neurobiological role of fabps in neurodegenerative diseases has not been reported yet. these made us evaluate the protein levels of fabps in brains from patients with down syndrome (ds) and alzheimer's disease (ad) and fetal cerebral cortex with ds using two-dimensional ( -d) gel electrophoresis with subsequent matrix-assisted laser desorption ionization mass spectroscopy (maldi-ms) identification and specific software for quantification of proteins. in fetal brain, b-fabp and e-fabp levels were comparable between control and ds. in adult brain, b-fabp was significantly increased in occipital cortex of ds, and h-fabp was significantly decreased in ds (frontal, occipital, parietal cortices) and ad (frontal, temporal, occipital and parietal cortices). we conclude that aberrant expression of fabps, especially h-fabp in neurodegenerative diseases could be involved in impaired neurite outgrowth and synapse maturation. in our previous paper, it was shown that gaba-a receptor antagonist picrotoxin suppressed etoh (ethanol) selfadministration. recently, several authors indicated that systemic injection of dopamine or serotonine agonists reduced ethanol drinking in rats. therefore, in the present study we investigated the effects of thip ( , , , -tetrahydroizokasazolo, , -c pyridin- -ol) gaba-a receptor agonist in naive and long-term ethanol-experienced rats on etoh selfadministration and on cardiovascular system. adult - week-old male, normotensive wistar-kyoto (wky) and spontaneously hypertensive rats (shr) were used. naive rats were examined according to smith method. long-term ethanolexperienced rats were studied according to boyle method. thip was injected in naive rats at a dose of and mg/kg i.p. metabotropic glutamate receptors are coupled to phospholipase c stimulation and adenylyl cyclase inhibition through g-proteins. c glioma cells, that endogenously express the phospholipase c coupled metabotropic glutamate receptor type, were treated with different specific agonists of these receptors and the effect of these treatments on different components of metabotropic glutamate receptor pathway was studied by radioligand binding, phospholipase c activity and rt-pcr assays. agonists treatment caused a decrease in l-[ h]glutamate binding to intact cells and membranes in a time dependent manner being maximum at - hours and recovered at - hours. this decrease was associated with a significant increase in the mrna level coding mglurs. no changes on g q/ mrna level were detected in any case. however, a significant decrease in l-glutamate stimulated phospholipase c activity was detected after agonist treatments in both membranes and intact cells. this decrease was not associated to significant variations in mrna level coding phospholipase c isoform. all these results suggest that agonist exposure causes a desensitisation of glial metabotropic glutamate receptor decreasing not only receptors number but its functionality. in this study the interaction between these two nuclei were investigated by means of microinjection and microdialysis techniques in sprague-dawley rats. steroetaxic surgery was performed by placing intracerebral parenchymal microinjection cannula into the right dmh and microdialysis probe into the left pvn. iliac artery was also cannulated to monitor the pulsatile blood pressure and heart rate by means of pressure transducer connected to a polygraph microinjection of pmol nmda into the dmh was performed and microdialysis perfusates were collected simultaneously from the pvn in conscious rat model. γ-aminobutyric acid (gaba) and l-glutamic acid levels were analyzed by an isocratic hplc (high pressure liquid chromatography) method with the aid of a fluorescent detector. microinjection of pmol nmda into dmh produced significant increases in mean arterial pressure and heart rate. nmda microinjection into the dmh produced significant increase in l-glutamic acid release in the pvn, but no significant change in gaba release was observed. these results suggest that stimulation of dmh by nmda results in subsequent stimulation of the pvn. [this study was sponsored by marmara university research foundation (project no: /sag/ ).] and in long-term ethanol-experienced rats only at a dose mg/ kg i.p. control group (cg) received saline ml/kg i.p. as can be seen in fig. and table the lower consumption of ethanol in shr in comparison to wky rats was observed. systemic injection of thip decreased dosedependently etoh intake in naive rats of both strains. this effect was more pronounced in shr (fig. ) . similar phenomenon was observed after thip injection in long-term ethanolexperienced rats. there were no effect on systolic blood pressure and heart rate after thip treatment. born-bunge foundation, university of antwerp, and university of ghent, belgium increased neuronal excitability may underlie some of the neurological complications in uremic patients. in an effort to identify candidate neuroexcitatory compounds, different uremic retention solutes, including several amino acids and amino acid derivatives, were applied to mouse spinal cord neurons in primary dissociated cell cultures. using the tight-seal whole-cell technique, a few of the candidate toxins were shown to evoke whole-cell currents in cells clamped at Ϫ mv. in a first survey, each of the solutes was briefly applied in a concentration of mm. significant inward whole-cell currents were evoked by guanidinosuccinate, spermine, and -indoxyl sulfate, whereas phenol evoked an outward current. further experiments indicated that guanidinosuccinate-evoked whole-cell currents were due to activation of nmda-type glutamate receptors in concentrations similar to those found in uremic patients. high (mm) concentrations of spermine activated voltage-gated calcium channels, whereas low (µm) concentrations were found to potentiate guanidinosuccinate-evoked currents through its action on the nmda receptor-associated polyamine binding site. whole-cell currents evoked by indoxyl sulfate or phenol seemed to be due to complex interaction with several different ion channels. we conclude that guanidinosuccinate-evoked nmda receptor activation, possi-bly potentiated by the neuroexcitatory effects of polyamines and other putative uremic neurotoxins, could be an important mechanism underlying the increased neuroexcitability in uremic brain. glutamine (gln) is one of the key metabolites in the cns (energy metabolite, precursor of neurotransmitter amino acids, end product of ammonia detoxication, osmolyte), and as such is a routine supplement of cns cell culture media. c glioma cells relatively easily adapt to culturing in a gln-deprived medium. the present study investigated the effects of gln deprivation on the characteristics of the different systems that mediate gln cell membrane transport in the cells. in contrast to a variety of cns and non-cns cells, the absence of gln did not derepress the methyl-amino-isobutyric acid (meaib)-sensitive ("system adependent") uptake. system asc became relatively more-, and system n less active than in cells grown in the presence of gln, but the ion -and substrate specificity of the uptake remained unaltered. system asc in c cells grown in a glnsupplemented medium shows two features distinct from most other cell types: a) strong ph sensitivity and b) partial tolerance of lithium substitution, pointing to domination of system asct -an asc variant strongly expressed in cultured astrocytes. cells grown in gln-deprived medium lost lithium tolerance, but not ph-dependence of the uptake, their properties thus resembling system glnt (sat ), a neuron-specific variant of system a. by contrast, transport of threonine, a standard asc system substrate, was not affected by gln deprivation and showed neither ph dependence nor lithium tolerance, which is typical of an asc in all the non-cns tissues. (supported by scsr grant no. p a .) the classical the hypothalamic-neurohypophysial system (hns) is comprised of neurons originating within the supraoptic nucleus (son) which project to the neurohypophysis to release the nonapeptides oxytocin (oxt) and vasopressin into the blood after appropriate stimulation. previous experiments have shown that a single social defeat experience triggers the release of oxt from somata and dendrites into the extracellular fluid of the son, but not from axon terminals in the neurohypophysis. to further investigate the regulatory mechanisms underlying this dissociated release, we exposed male wistar rats to a -min social defeat experience and monitored the release of the inhibitory amino acids gamma amino butyric acid (gaba) and taurine into the son using microdialysis. social defeat caused a significant increase of the intra-son pre-stress basal release). to reveal the physiological significance of the intrahypothalamically released gababicuculline, a specific gaba a -receptor antagonist -was administered into the son by retrodialysis. this treatment increased significantly the release of oxt both within the son ( %; p Ͻ . vs. pre-stress basal release) and -as measured via chronically implanted jugular venous catheters -into blood under basal and stress conditions (up to %; p Ͻ . vs. prestress basal release). however, bicuculline did not affect plasma vasopressin. these data demonstrate that gaba is released within the son during social defeat to act as an inhibitor of both, central and peripheral oxt secretion during emotional stress. the mechanism described here contributes to the regulatory capacity of the hns to ensure the appropriate involvement of oxt in the stress response of the animal (supported by dfg, en - ). down syndrome (ds) is the most common human chromosomal abnormality caused by an extra copy of chromosome and characterized clinically by somatic anomalies, mental retardation and precocious dementia. the phenotype of ds is thought to result from overexpression of a gene or genes located on the triplicated chromosome or chromosome region. reports that challenge this notion, however, have been published. to add to this body of evidence, the expression ofamyloid precursor protein (app), ets- and collagen α (vi) chain precursor, encoded on chromosome , was investigated in fetal brain by western blot and two-dimensional electrophoresis ( -de). western blot detected app and ets- that migrated at ϳ and kda, respectively. subsequent densitometric analysis of app and ets- immunoreactivity did not produce any significant change between controls and ds. since the metabolic fate of app determines the propensity of amyloid production, the expression of the secreted forms of app (sapp) had been examined. neither the expression of sappα nor sapp showed any detectable changes among the two groups. collagen α (vi) chain precursor, a protein resolved as a single spot on d gel was identified by matrix associated laser desorption ionization mass spectroscopy. quantitative analysis of this spot using the d image master software revealed a significant decrease in fetal ds (p Ͻ . ) compared to controls. linear regression analysis did not show any correlation between protein levels and age. the current data suggest that overexpression per se can not fully explain the ds phenotype. apoptosis is the mechanism by which cells are programmed to die under a wide range of physiological and developmental stimuli. accumulating evidence indicates that enhanced apoptosis (programmed cell death) in down syndrome (ds) may play a role in mental retardation and precocious neurodegeneration of the alzheimer-type. in this regard, alteration of several apoptosis related proteins have been reported in adult ds brain. fetal ds neurons exhibited increased reactive oxygen species leading to early apoptosis, however, expression of apoptosis related proteins in fetal ds, has never been considered. to address this issue, we investigated the expression of proteins involved in apoptosis including fas (cd , apo- ), caspase- , bcl- and annexins in the cerebral cortex of control and ds fetal brain by western blot and two dimensional electrophoresis. here, we report that no detectable changes were obtained in fetal ds brain in the expression of fas, caspase- , bcl- and annexins (i, ii, v, and vi) compared to controls. in parallel experiment, we also examined the expression of neuron specific enolase (nse), a neuronal marker found to be decreased in adult ds brain, to see if there is any neuronal loss and no difference was observed between the two groups. protein expression did not correlate with age. the unchanged levels of fas, bcl- and annexins together with unaltered caspase- expression, a predominant caspase that executes apoptosis in the developing nervous system, suggest that enhanced apoptosis may not be apparent in fetal ds brain as demonstrated for adult ds brain. introduction. among the various metabolites indicating neuronal damage, amino acids are regarded particularly important. detection of amino acids by microdialysis is currently introduced as a neuromonitoring tool in patient care. here, we present changes in the extracellular concentrations of various amino acids in stroke patients and in experimental stroke in cats. method. cat focal ischemia was produced by occlusion of the middle cerebral artery (mca) for h followed by h reperfusion. glutamate, aspartate, gaba, taurine, glycine, serine, glutamine, methionine, threonine, tyrosine, asparagine, valine, phenylanaine, isoleucine and leucine were sampled by microdialysis in the ischemic core and subsequently analyzed by hplc. human microdialysis was performed in patients with large mca infarction. the microdialysis probes were inserted into primarily non-infarcted tissue in the border zone of the ischemic territory. results. transmitter amino acids rose immediately after occlusion in the cat model. correspondingly, these substances increased sharply in the human brain, when the tissue around the probes became infarcted, as shown by positron emission tomography (pet) and ct scan. in contrast, structural amino acids did not show marked increases or even decreased during severe ischemia in both, experimental ischemia and stroke patients. these substances did increase, however, when the brain tissue was only slightly ischemic, i.e. after reperfusion of the cat brain, when brain swelling occurred, or in human brain, when tissue did not show any infarction in the ct scan but hypoperfusion in the pet image. conclusion. extracellular amino acids detected by microdialysis can serve as markers for secondary ischemia. severe ischemia is reflected by rapid increases of transmitter amino acids, due to various mechanisms including synaptic release and reversal of reuptake systems. oligemia seems to be reflected by slow increases of structural amino acids, possibly due to a reduction in cerebral protein synthesis. apoptosis has been implicated in the selective neuronal loss of down syndrome (ds). apoptosis activates a family of cysteine proteases with specificity for aspartic acid residues referred to as, caspases that play a key role in dismantling a cell committed to die. caspases activity is regulated by a variety of proteins that possess a domain resembling the prodomains of caspases. little is known, however, about the changes of caspases and their regulatory proteins in ds. here, we investigated levels of nine such different proteins by western blot technique in frontal cortex and cerebellum of control and ds subjects. the protein levels of dff (dna fragmentation factor ), and flip (fadd like interleukin- -converting enzyme inhibitory proteins) were significantly decreased whereas that of rick (rip-like interacting clarp kinase) increased in both regions of ds. in contrast, cytochrome c, apaf- (apoptosis protease activating factor- ), procaspase- and arc (apoptosis repressor with caspase recruitment domain) were unchanged. procaspase- and - were significantly decreased in frontal cortex but no significant change was observed in cerebellum. regression analysis revealed no correlation between postmortem interval and levels of the investigated proteins. however, inconsistent correlation was found between age and levels of proteins as well as amongst the density of individual proteins. these findings demonstrate that dysregulation of apoptotic proteins does exist in ds brain and may underlie the neuropathology of ds. the study further suggests that apoptosis in ds may occur via the death receptor pathway independent of cytochrome c. hence, therapeutic strategies that target caspase activation may prove useful in combating neuronal loss in this disorder. in order to examine the differential roles of nitric oxide (no) induced by either endothelial no synthase (enos) or neuronal no synthase (nnos) after transient cerebral ischemia, we investigated the effects of the relatively selective cnos inhibitor, l-n -( -iminoethyl)ornithine (l-nio), the relatively selective nnos inhibitor, -nitroindazole ( -ni) and the no scavenger, -( -carboxyphenyl)- , , , tetramethylimidazole- -oxyl -oxide (ptio) on hippocampal dysfunction caused by cerebral ischemia. we measured no concentration, mean arterial blood pressure (mabp), hippocampal blood flow, direct current potential, ca population spike (ps) and release of amino acids from rat hippocampus after transient forebrain ischemia, which was induced by vessel occlusion for min. l-nio ( mg/kg), -ni ( mg/kg) and ptio ( mg/kg) were administered intraperitoncally min before ischemia. ptio, -ni and l-nio reduced ischemiainduced no production in the hippocampus during the early period of reperfusion. the rank order of inhibitory potency was ptio Ͼ -ni Ͼ l-nio. l-nio, but not -ni, reduced hippocampal blood flow during ischemia and increased mabp before, during and after ischemia, compared with the vehicle group. ptio increased mabp during and after ischemia. ptio and -ni, but not l-nio, reduced amplitude of anoxic depolarization induced by ischemia. -ni recovered in part ps amplitude min after ischemia. -ni, but not l-nio, reduced ischemiainduced release of aspartate and glutamate, but not taurine. the present study provides further evidence for the idea that in the early stages of transient forebrain ischemia, enos-derived no has a neuroprotective effect in the hippocampus, while nnos-derived no has a neurotoxic effect. the estrogen affects brain protein synthesis in ovariectomized female rats k. hayase , m. tanaka , k. tujioka , e. hirano , and h. yokogoshi department of home economics, aichi university of education, kariya, aichi, and laboratory of nutritional biochemistry, school of food and nutritional sciences, the university of shizuoka, japan the purpose of this study was to determine whether -estradiol affected the rate of brain protein synthesis in ovariectomized female rats. experiments were conducted on three groups of wk old female rats: group . ovariectomized to reduce the level of plasma estradiol; group . ovariectomized and treated with estradiol; and group . sham-operated control. the fractional rates of protein synthesis in brain of ovariectomized rats treated with estradiol were significantly greater than in ovariectomized rats without estradiol treatment. in brain, the rna activity [g protein synthesized/(g rnaᮀd)] significantly correlated with the fractional rate of protein synthesis. the rna concentration (mg rna/g protein) was not related to the fractional rate of protein synthesis in any organ. the results suggest that estrogen treatment of ovariectomized female rats is likely to increase the rate of protein synthesis in the brain, and that rna activity is at least partly related to the fractional rate of brain protein synthesis. we have synthesized a series of new peptides that have demonstrated potent antidepressant activity in animal models for depression and in phase iia and iib clinical trials. mif- (prolyl-leucyl-glycinamide) an endogenous brain peptide has been reported to have some clinical activity in patients with unipolar depression with few apparent side effects. we have undertaken a study to determine the effect of molecular structural changes on the antidepressant activity of this peptide. we evaluated our new derivatives in a stress-induced animal model for depression, i.e. porsolt test, we have found that -f-phe- -oh-pro-arg-gly-trp-nh (inn ) is superior in all the statistical parameters used. in comparative testing inn was more active than prozac (fluoxetine) and zoloft (sertraline) in our antidepressant model. a u.s. patent has been granted on these compounds. the clinical results of inn show that it is effective in over % of depressed subjects when blood levels exceeded therapeutic threshold with no significant side effects. inn has a rapid onset of action, - days (vs. - weeks for currently marketed products) with sustained effects for months following to doses over - weeks. h. iwama , , a. umino , a. hashimoto , k. takahashi , n. yamamoto , and t. nishikawa , section of psychiatry and behavioral science, tokyo medical and dental university graduate school, and department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan using an in vivo dialysis technique, we have studied the extracellular contents of endogenous free d-serine in comparison with that of l-serine, glycine and l-glutamate in the brain of the freely moving rat. a high amount of d-serine was detected in the dialysate obtained from the medial prefrontal cortex and striatum, whereas the cerebellar dialysate contained only a trace concentration of the d-amino acid. intra-medial prefrontal cortex perfusion of a sodium channel activator, veratrine, augmented the extracellular release of glycine and l-glutamate but a slight decrease in that of d-serine in a tetrodotoxin-sensitive manner in the prefrontal area. moreover, selective destruction of neuronal cell bodies in the medial frontal region by means of local infusion of an excitotoxin quinolinate resulted in a marked reduction of extracellular and tissue levels of d-serine in the infused prefrontal region. these findings suggest that endogenous d-serine might be liberated into the extracellular space from non-neuronal cells or certain exceptional neuronal cells probably by a carrier-mediated process in the mammalian prefrontal cortex. also, the endogenous d-amino acid has been indicated to be accumulated or synthesized, at least in part, in the neuronal cells. nucleoside diphosphate kinase (ndpk) catalyzes a transfer of the terminal phosphate from nucleoside triphosphates ((d)ntps) to nucleoside diphosphates ((d)ndps) and has been suggested to be involved in the regulation of wide variety of cellular functions. in addition, ndpk isoforms (a and b) are encoded by nm genes (h and h ) , which are related with the metastatic potential of some tumors. although ndpk/ nm has been also implicated to modulate neuronal cell proliferation, differentiation and neurite outgrowth, a neurobiological role of ndpk/nm in neurodegenerative diseases has not been reported yet. here we evaluated the protein levels of ndpk-a/nm -h in brains from patients with down syndrome (ds) and alzheimer's disease (ad) using twodimensional ( -d) gel electrophoresis with subsequent matrixassisted laser desorption ionization mass spectroscopy (maldi-ms) identification and specific software for quantification of proteins. ndpk-a/nm -h was significantly decreased in brain regions (frontal, occipital, parietal cortices) of both ds and ad compared to controls. we conclude that the down-regulated ndpk-a/nm -h upon neurodegeneration could play a pivotal role in a wide range of neurobiological functions such as neurite outgrowth and consequently these could result in functional disturbance of the nervous system in ds and ad. brain α-endosulfine is manifold decreased in brains from patients with alzheimer's disease: a tentative marker? and drug target? α-endosulfine has the sulfonylurea-like ability to block atp-sensitive potassium (k atp ) channels and is expressed in a wide range of tissues. although the blockade of k atp channels has been reported to be involved in the release of neurotransmitters, the neurobiological role of α-endosulfine has not been studied yet. we examined the levels of αendosulfine protein in frontal cortex and cerebellum from patients with alzheimer's disease (ad). α-endosulfine was extremely decreased in both regions of ad compared to controls. this could result in the continuous opening of k atp channels with subsequent decrease of neurotransmitters release and change of potassium fluxes. this study is of great significance for providing a neurobiological function of α-endosulfine in brain and furthermore, α-endosulfine could serve as a useful marker for the diagnosis of ad and a target for drug treatment. children's hospital heidelberg, and department of pharmacology and toxicology, university of marburg, germany d- -hydroxyglutaric aciduria is an inherited neurometabolic disorder of unknown etiology characterized by progressive neurodegeneration of vulnerable brain regions during infancy and early childhood, resulting in psychomotor retardation, hypotonia, seizures, macrocephaly, enlarged lateral ventricles, delayed cerebral maturation as frequent neurological presentation in affected children. the disease is biochemically characterized by the accumulation of d- hydroxyglutarate (d- ), which is structurally similar to lglutamate (ϭ -amino-glutarate). we therefore investigated in primary neuronal cultures from chick and rat, whether d- induces excitotoxic neuronal damage. here we report that d- decreased cell viability in a concentration-and time-dependent fashion by disturbance of intracellular calcium homeostasis as determined by fura- measurement. furthermore, fluorescence microscopy using dihydrorhodamine- revealed an increased generation of reactive oxygen species (ros) elicited by expo-sure to d- . n-methyl-d-aspartate (nmda) receptor blockade specifically prevented excitotoxic neuronal damage as well as increased calcium influx and ros production, suggesting that d- is an agonist at nmda receptors. patch-clamp investigation confirmed that d- activated recombinant nmda receptors in hek cells. furthermore, activity measurement of single respiratory chain complexes revealed a specific inhibition of complex v activity by d- . we conclude that excitoxic mechanisms contribute to the neuropathology of d- hydroxyglutaric aciduria, highlighting new neuroprotective strategies for this neurometabolic diseases. these studies were designed to determine the effects of aging and an aging intervention on nmda subunit expression. in situ hybridization and receptor autoradiography were performed on naïve or behaviorally-tested c bl/ mice of different ages ( , - , and - months old) and diet groups (ad lib-fed and diet restricted). there were age-related decreases in both e and z mrna density in naïve, ad lib-fed mice. correlations were found between changes in e subunit mrna and agonist binding and z mrna expression and antagonist binding. diet restriction significantly improved learning ability, slightly spared glutamate binding to nmda sites and improved z mrna expression in older mice. significant correlations were found between agonist binding and both learning ability and e and e mrna density. learning ability in the old mice also correlated with the ratios of mrna expression for e and e and/or z subunits. these results suggest that changes in nmda receptor binding and the relationship between subunit expression levels are important for maintaining memory functions in older animals. extracts of st john's wort (hypericum perforatum l.) are widely prescribed for the treatment of mild to moderate depression and the putative antidepressant constituent is probably hyperforin. in this study the effect of hyperforin was investigated on the release of neurotransmitter amino acids. coronal cortical slices ( mm) were cut and perfused with gassed ( % o , % co ) acsf at °c. two-minute samples of perfusate were collected and aspartate and glutamate were assayed by hplc. potassium-and veratridine-stimulated release was elicited by administering pulses of kϩ ( mm) or veratridine ( mm) minutes apart. in control experiments the second kϩ pulse elicited glutamate release which was % of the first pulse. hyperforin ( mm) perfused for minutes prior to, and during, the second kϩ pulse significantly increased glutamate release to % (p Ͻ . , n ϭ - ). release elicited by the second veratridine pulse was % of the first pulse for both glutamate and aspartate. hyperforin ( mm) increased this release to the second pulse to % and % respectively (p Ͻ . , n ϭ - ). when perfused on its own for minutes, hyperforin ( mm) increased the basal release of glutamate (p Ͻ . , n ϭ - ). in conclusion, the increase in the release of neurotransmitter amino acids observed following hyperforin is possibly mediated through a facilitatory action on voltage-operated ca ϩ or naϩ channels. glaxosmithkline group, glaxo wellcome s.p.a., medicines research centre, verona, italy n-methyl-d-aspartate (nmda) receptors are ligand gated ion channels widely distributed in mammalian brain, which play a crucial role in important physiological mechanisms, such as excitatory transmission, neuronal migration and memory formation. a peculiar feature of nmda receptors is the absolute requirement of l-glutamic acid and glycine for the opening of the channel. noteworthy, these two aminoacids reciprocally modulate binding at their respective recognition sites. aim of this work was to study nmda receptor glycine/glutamate interactions in rat and human brain. binding of the nmda antagonist [ h]cgp to rat cerebral cortical membranes was inhibited by glycine. the overall effect of glycine consisted in a decrease of [ h]cgp affinity, with a parallel increase of the receptor affinity for glutamate. the glycine antagonist gv a competitively reversed glycine inhibition, proving that the modulation was via the glycine binding site. [ h]cgp binding to rat brain sections revealed the existence of regionally distinct nmda receptor subtypes with difference glycine/glutamate interactions. in most regions of the human brain nmda receptors presented the same allosteric modulation of [ h]cgp binding, as revealed by large section autoradiography technique. nevertheless, detection of any regional variation was not possible, probably due to the high intersubject variability. the effect of long-term high k ϩ -treatment on neuronal survival, cellular maturation, nmda receptor (nr) splice variant expression, and receptor function was investigated in primary cultures of rat cortical neurones. long-term incubation (up to days) with mm k ϩ significantly increased neuronal survival and induced multiple morphological changes associated with promoted cellular maturation. cultures grown in medium containing mm k ϩ also exhibited multiple changes in nr splice variant expression according to rt-pcr studies performed with primer pairs flanking the alternatively spliced regions, in order to estimate the ratios of the corresponding Ј and Ј splice variants. nr - and nr - (each containing exon ) were decreased, whereas nr - and nr - (each lacking exon ) were increased, accordingly. the predominant expression of nr -b was further increased. after administration of ttx, each of the k ϩ -induced changes on mrna expression was virtually abolished. in voltage-clamp recordings (holding potential: Ϫ mv), nmda induced inward currents in a concentration-dependent manner with a maximum effect of Ϫ pa under control conditions. neurones treated with mm k ϩ showed a significantly diminished response to nmda (max. response: Ϫ pa). in conclusion, the present data indicate that a sustained increase in neuronal activity induces adaptive changes in nr splice variant expression and a decrease in receptor function. thus, alternative splicing associated with a diminished receptorcytoskeletal linkage may be important compensatory mechanism in preventing cellular damage due to long-term activation of excitatory nr. it seems conceivable that this mechanism contributes to the promoting effects of mm k ϩ on neuronal survival and maturation. (supported by bmbf grant gg / ) there is accumulating reports that kainate-induced seizures elicit expression of various heat-shock proteins (hsps) in the brain, such as hsp , hsp , and hsp . however, no investigation has been carried out on changes in level of apg- , a member of hsp family, after excitatory amino acid-induced seizures. by means of an immunoblot assay, we determined the levels of hsp and apg- in discrete brain structures of mice after a single intraperitoneal injection of kainate or nmda. apg- level was significantly decreased in the frontal cortex, hippocampus, and striatum days after kainate administration, while hsp level was increased in these regions following the administration. decreased level of apg- returned to the control levels in the three regions days after kainate administration. no significant changes were observed in levels of both hsp and apg- in hypothalamus, midbrain, medulla-pons, and cerebellum of kainate-treated mice. by contrast, nmda administration did not significantly affect both levels in any of the regions examined. these results suggest that, unlike the case of hsp , apg- expression could be temporarily down regulated by signals peculiar to kainate, but not by those peculiar to nmda, in murine telencephalon. high concentrations of glucagon-like peptide- ( - ) amide (glp- ) and its specific receptor (glp- r) have been found in the rat hypothalamus. in this study the actions of glp- and its related peptides, exendin- (glp- r agonist), exendin ( - ) (glp- r antagonist) and glp- ( - )amide (major glp- metabolite) on levels of amino acids (glu, asp, gln, gly, tyr, trp, gaba) in the hypothalamus were investigated. i-glp- binding in rat hypothalamic membranes was competed by the peptides in the following order of potency; glp- Ͼ exending- Ͼ exendin ( - ) Ͼ glp- ( - )amide. intracerebroventricular (icv) glp- ( nmoles) produced a statistically significant reduction in levels of all measured amino acids compared with saline injected controls, whereas nmoles of exendin ( - ) was ineffective. exendin- produced a statistically significant reduction in the levels of trp, glu, and tyr. glp- ( - )amide showed a statistically significant increase in the level all the amino acids tested in this study. prior administration of exendin ( - ) or glp- ( - )amide blocked the effects of glp- on the levels of the amino acids. these data are consistent with exendin- being a glp- r agonist and exendin ( - ) being a specific glp- r antagonist. glp- ( - )amide, a primary metabolite of glp- , appears to act as an endogenous antagonist at the glp- r. department of biophysics, instituto de fisiología celular, universidad nacional autónoma de méxico, méxico city, méxico cholecystokinin (cck), a family of neuropeptides, seems to be involved in anxiety. evidence from several laboratories indicates that the ansiogenic effects of cck are mediated by cck b receptors. however it has been reported that cck a receptors have been found in brain and cck a receptor antagonists have ansiolytic properties. the aim of this work was to study whether or not cck a receptors are also involved in the modulation of anxiety. male rats were cannulated in the lateral ventricle and cck ( fmol) and/or cck antagonists ( fmol) were injected days after surgery. anxiety was evaluated in the elevated plus-maze test and locomotion in an open-field test. ansiogenic effects were observed when cck b receptor agonists (cck ns; cck ) or a mixed cck b and cck a receptor agonist (cck s) were injected. in contrast, cck , a cck a receptor agonist or cck - and cck - were uneffective. furthermore, the ansiogenic effects of cck s were prevented by the previous ( min) administration of l , (cck b receptor antagonist) but not by devazepide (cck a receptor antagonist). no effects on locomotion were observed in any condition. these results indicate that cck a receptors are not involved in anxiety, as measured by the elevated plus-maze test. congenital conditions (i.e. neural tube defects: ntg) have a multifactorial aetiology. deficiencies in the folate and transsulfuration pathways have, in recent years, been positively linked to ntd and other dysmorhogenic syndromes. efficient one-carbon metabolism is crucial for the synthesis of dna precursors, the remethylation of homocysteine and biomethylation of dna. more than % of the one-carbon units that flow through the metabolic system in mammals and birds are derived from l-serine and glycine, the natural substrates for shmt. the mitochondrial glycine cleavage enzyme system (gces) can potentially compete with shmt for tetrahydrofolate (thf) in the generation of the methylenetetrahydrofolate pool. valproate (depakene, epilim), an anti-epileptic agent, appears to be strongly associated with hyperhomocysteinemia, several other induced metabolic conditions, the inhibition of the gces and an increased incidence of ntd in epileptic women of child-bearing age. the exact mechanisms of valproate-induced ntd are not yet clear. we investigated the association of the teratogenic properties of valproate with the inhibition of shmt and/or the gces in developing embryos. chicken embryos were treated with sodium valproate (vpa) and pregnant female mice (c bl) received intraperitoneal injections of vpa, during the critical period of embryonic neural tube development. control embryos were treated with sterilised saline solution. harvested embryos were subsequently investigated for congenital abnormalities and hepatic shmt and gces activities quantified with radiometric assays. the effect of vpa on hepatic dna synthesis was monitored ( h-thymidine incorporation into embryonic dna) and the dna-methylation status determined (dna n -methylcytosine levels). dose-responsive incidences of ntd were observed in vpa treated embryos. very few defects occurred in control embryos. shmt and gces appeared to be inhibited in liver extracts of vpa-treated embryos. hepatic dna synthesis was significantly compromised and -mc levels were altered in vpa-treated embryos. the inhibition of either shmt and/or gces activities appeared to be associated with valproateinduced ntd in the chicken and mouse embryo models. the primary mechanism of this effect can probably be ascribed to a restriction in the flow of one-carbon units through the metabolic system, decreased synthesis of dna precursors and alterations in the methylation status of dna. department of neuroscience, university of cagliari, italy ethanol is long known to cause dose-related biphasic effects and we recently found that ethanol bidirectionally affects also working memory. the euphoriant and excitatory effects produced at low doses are associated with the rewarding action of ethanol and are thought to be mediated by the activation of the mesolimbic dopamine (da) system. however, ethanol monophasically stimulates mesolimbic da release in the nucleus accumbens, even at doses that cause hypnosis and coma. in contrast, ethanol biphasically modulates mesocortical da release in the prefrontal cortex (pfc). the changes in da release induced by ethanol are time locked with corresponding changes in extracellular glutamate levels. these biphasic effects of ethanol on pfc da and glutamate are matched by biphasic changes in the performance in a spatial delayed alternation task -a working memory test that is sensitive to proper function of the pfc -suggesting a link between da and glutamate transmission in the cognitive effects of ethanol. focal application in the pfc of the competitive ampa/kainate receptor antagonist cnqx suppresses both da release and the improvement of working memory induced by low doses of ethanol. these results suggest that ethanol may increase da transmission in the pfc and enhance working memory functions by increasing the release of glutamate, thereby stimulating non-nmda glutamate receptors. the enhancing effect on working memory by low, excitatory doses of ethanol may be perceived as rewarding and could constitute an important neurobiological mechanism for excessive ethanol drinking. physiology department, faculty of medicine, al-quds university, jerusalem, palestine glutamate and asparate are considered as the main excitatory neurotransmitters in brain and spinal cord, in addition to their role in energy metabolism, synthesis of proteins and detoxification of ammonia. glutamate and aspartate are centrally involved in basic mechanisms generating epileptic seizures and in epileptogenesis. stimulated release of glutamate and aspartate was detected in vivo and in vitro following neuronal depolarization. photic stimuli has increased glutamate release from visual cortex, and afferent brachial stimulation has increased the endogeneous release of glutamate from contra-lateral sensorimotor cortex compared to ipsi-lateral side. similar results were achieved after local application of tityustoxin or veratridine to the sensorimotor cortex. implantation of cobalt powder over the right sensorimotor cortex of rats produced an epileptogenic lesions characterized by contra-lateral fore and hind limb jerks and an increase in the frequency of eeg spikes. the jerks started after days with maximum myoclonic activity ( jerks/min). the concentration of glutamate in the epileptogenic focus was decreased significantly by % (p Ͻ . ) compared to the non-epileptogenic area on the left sensorimotor cortex, which was dissected but not treated with cobalt. part of the decrease in glutamate could be related to the enhancement of in-vivo release from the epileptogenic lesion to the extra-cellular fluid. kindling is the best model for studying the development of the epileptic focus (epileptogenesis), it could be achieved by repeated intra-cerebral micro-injection of glutamate ( . µ mol), aspartate ( . µ mol) or nmda ( - n mol), or repeated electrical stimulations of specific brain regions. in addition, glutamate antagonists particularly those specifically acting on the nmda receptor type e.g. -amino- -phosphonovaleric acid (ap ) and -amino- -phosphonopheptanoic acid (ap ) have been found to inhibit seizures in epileptic animals and inhibit the development of electrically kindled epilepsy. pre-synaptic glutamate receptor agonists like ( s, s)-acpd the agonist of group ii, and l-ap the agonist of group iii receptors has reduced ca ϩϩ uptake and glutamate release, thus it has inhibited epileptogenesis by preventing the increase in both seizure score and after-discharge duration. injection into fully kindled animals has produced an anti-epileptic effect by reducing the mean seizure score and by increasing the mean generalized seizure thresholds. this results suggest the mechanism by which pre-synaptically active glutamate receptor agonists block the development of the chronically epileptic state induced by electrical kinding, and indicate that their anticonvulsive activity is due to inhibition of pre-synaptic glutamate and/or asparate release following blockade of pre-synaptic ca ϩϩ entry. testing the changes in glutamate release from hyperactive brain tissues, and the effect of different glutamate agonists and antagonists, supports the role of glutamate in initiating the process of epileptogenesis, and contributes in developing new anti-epileptic agents. (this project was supported by a grant from alexo) the functional roles of cl(Ϫ) and divalent cations in the na(ϩ)/cl(Ϫ)/gaba cotransport were examined in xenopus oocytes expressing the human gat- (hgat- ) gaba transporter cdna. our results showed that cl(Ϫ) was not absolutely required for na(ϩ)/gaba transport via the hgat- (loo et al., j biol. chem. : - , ) . the cl(Ϫ) interacted with the transporter to modulate the binding of external na(ϩ). although hgat- transported cl(Ϫ) across the membrane with a stoichiometry of na(ϩ) : cl(Ϫ) : gaba, the transported cl(Ϫ) did not contribute to the net charge translocated across the membrane, suggesting a cl(Ϫ)/cl(Ϫ) exchange mechanism during the gaba transport cycle. the gaba transport via the hgat- is also modulated by divalent cations. the uptake of [ h]-gaba was inhibited significantly when both ca( ϩ) and mg( ϩ) were removed from the uptake buffer. several divalent cations tested were individually able to sustain the gaba uptake. in contrast to uptake, the gaba efflux was enhanced significantly upon removal of both ca( ϩ) and mg( ϩ) from the efflux buffer. the gaba transporter inhibitor skf a blocked the enhanced efflux, suggesting that the hgat- operated faster in the reverse mode in the absence of external divalent cations. these results suggest a regulatory role for the divalent cations in gaba transport. merck sharp & dohme, neuroscience research centre, terlings park, harlow, essex, u.k. the role of alpha containing gaba a receptors in hippocampal synaptic function has been investigated using pharmacological and electrophysiological techniques, as well as following disruption of the alpha subunit gene in knockout mice (ko). in the ca region of the hippocampus the induction of long-term potentiation (ltp) is powerfully regulated by gaba mediated synaptic currents (ipscs). agents that inhibit gaba-mediated transmission potentiate ltp induction, whereas allosteric agonists such as benzodiazepine-site agonists which slow the decay kinetics of ipscs suppress ltp induction. in alpha ko mice paired pulse facilitation of the amplitude of excitatory synaptic potentials is selectively enhanced in the ca region but not dentate gyrus. likewise, the frequency and rise time of spontaneous ipscs were similar in wt and ko slices. however their amplitude was significantly smaller in ko mice. furthermore, a significantly greater proportion of ipscs were best fitted to a mono exponential function in ko mice compared to wt animals. thus alpha containing gaba a receptors contribute to functional postsynaptic receptors on ca pyramidal cells in the hippocampus and modulate a postsynaptic component of synaptic facilitation. pharmacological research institute, volgograd medical academy, volgograd, russia the purpose of the study is to investigate effect of phenil (karphedon, mephebut, gammoxin) and circle (pyracetam) gaba derivatives on reproductive function of stressed male rats. the adult male rats were stressed by immobilization exposure ( hours) twice in week during weeks. four from five groups of stressed males were given substances (daily) at doses: karphedon - mg/kg, mephebut - mg/kg, gammoxin - mg/kg, pyracetam - mg/kg. the treated males were mated with intact females during days. after the mate the treated males and more in days all the mated females were sacrificed and investigated. analysis of our data indicates that the time of spermatozoa motion and epididymal sperm counts were decreased . % (p Յ . ) and . % (p Յ . ) respectively when compared with their intact controls. gaba derivatives have a softening effect on functional parameters of spermatozoa stressed males. karphedon and pyracetam increased the time of motion spermatozoa . % (p Յ . ), karphedon and mephebut drew near sperm counts to intact control level. the result of mate show that pregnancy rate was increased (p Յ . ) by stress exposure and pregnancy rate of females mated with gaba stressed males was some more (p Ն . ) than that of intact controls. the general embryonic morality was increased twice by stress and so the number of embryos was reduced . %. the gaba derivatives exposure to stressed male rats reduced the embryonic mortality of their posterity and increased the number of embryos to intact control level. our findings demonstrate that gaba derivatives administration has a protective effect on reproductive function of stressed male. transmission on the brain. the realization that glutamatergic pathways are involved in such diverse processes in epilepsy, ischemic brain damage and parkinsons' disease, is of a great practical interest. there are at least three functional classes of ionotropic glutamate receptors: n-methyl-d-aspartate (nmda), α-amino- -hydroxy- methyl- -isoxazolepropionic acid (ampa) and kainate (ka). other central neurotransmitter systems are under nmda influence. some data point on neuroprotective action of nmda antagonist on nigrostriatal pathway. in the present study female wistar rats were exposed during pregnancy with daily injected mk- (dizocilpine) . mg/kg sc. control rats received tap water only. behaviour of month old male offsprings was investigated by several psychopharmacological methods. oral activity, yawning, locomotor activity, stereotypy and catalepsy were recorded following respective central dopamine receptors agonists and antagonists administration (skf , quinpirole, apomorphine, haloperidol). our results indicate that mk- applied during pregnancy modulate reactivity of the central dopamine receptors in adult offspring rats. [ the development of mammalian ingestive behavior is characterized by a transition from suckling to chewing, two distinct motor behaviors. we hypothesize that this transition is accompanied by changes in brainstem circuitry underlying these movements. since glutamatergic neurotransmission is critical for the proper functioning of brainstem circuitry responsible for mastication, we investigated the development of glutamate receptors in trigeminal motoneurons (mo ) and mesencephalic trigeminal neurons (me ); neurons comprising the circuitry responsible for jaw movements. we conducted a series of receptor immunohistochemistry experiments that characterized the expression of iontotropic and metabotropic glutamate receptors (mglurs) during early postnatal development. the functional roles of nmda, ampa and mglurs in neonatal mo were investigated using in vitro electrophysiological experiments. results demonstrated that the spatial and temporal expression of ampa, nmda and group i and ii mglurs are developmentally regulated within and between mo and me during early development. electrophysiological data demonstrate that mglurs function pre-and postsynaptically to modulate synaptic transmission between trigeminal premotoneurons and mo . furthermore, nmda induced bursting is developmentally regulated and coincident with the transition from suckling to chewing behaviors. our studies suggest that the transition from suckling to chewing is accompanied by changes in the composition and function of glutamate receptors. fetal life in down syndrome starts with normal neuronal density but impaired dendritic spines and synaptosomal structure r. weitzdoerfer , m. dierssen , m. fountoulakis , and g. lubec department of pediatrics, university of vienna, austria information on fetal brain in down syndrome (ds) is limited and there are only few histological, mainly anecdotal reports and no systematic study on the wiring of the brain in early prenatal life exist. histological methods are also hampered by inherent problems of morphometry of neuronal structures. it was therefore the aim of the study to evaluate neuronal loss, synaptic structures and dendritic spines in the fetus with down syndrome as compared to controls by biochemical measurements. dimensional electrophoresis with subsequent mass spectroscopical identification of spots and their quantification with specific software was selected. this technique identifies proteins unambiguously and concomitantly on the same gel. fetal cortex samples were taken at autopsy with low postmortem time, homogenized and neuron specific enolase (nse) determined as a marker for neuronal density, the synaptosomal associated proteins alpha snap [soluble n-ethylmaleimidesensitive fusion (nsf) attachment protein], beta snap, snap and the channel associated protein of synapse (chapsyn ) as markers for synaptosomal structures and drebrin (drb) as marker for dendritic spines. nse, chapsyn and beta snap were comparable in the control fetus panel and in down syndrome fetuses. drebrin was significantly and remarkably reduced and not even detectable in several down syndrome brain samples. quantification of snap revealed significantly reduced values in ds cortex and alpha snap was only present in half of the ds individuals. we conclude that at the time point of about weeks of gestation (early second trimester) no neuronal loss can be detected but drebrin, a marker for dendritic spines and synaptosomal associated proteins alpha snap and snap were significantly reduced indicating impaired synaptogenesis. early dendritic deterioration maybe leading to the degeneration of the dendritic tree and arborization, which is a hallmark of down syndrome from infancy. pathfinding of growing axons to reach their target during brain development is a subtle process needed to build up contacts between neurons. abnormalities in brain development in down syndrome (ds) are described in a couple of morphological reports but the molecular mechanisms underlying abnormal wiring in fetal ds brain are not yet elucidated. we therefore performed a study using the proteomic approach to show differences in protein levels involved in the guidance of axons between control and ds brain in early prenatal life. proteins obtained from autopsy of human fetal abortus were applied on -dimensional gel, identified and quantified. we quantified members of the semaphorin/collapsin family, the dihydropyrimidinase related proteins - and the collapsin response mediator protein- (crmp- ) in ds and control cortex samples. drp- and crmp- levels were comparable in the control and ds samples. evaluation of drp- , drp- and drp- revealed significantly decreased levels of of the spots assigned to drp- and increased levels of one spot assigned to drp- and increased drp- in ds brain. we conclude that as early as from the th week of gestation pathfinding cues of the outgrowing axons are impaired in ds. these findings may help to elucidate mechanisms leading to abnormalities in neural migration of ds brain. inflammatory processes play an important role in the degeneration of basal forebrain cholinergic cells alzheimer's disease. the proinflammagen lipopolysaccharide (lps) was infused chronically into the basal forebrain of young rats. we then determined whether the administration of two novel nonsteroidal anti-inflammatory drugs or a pancaspase synthesis inhibitor, zvad, could provide neuroprotection from the cytotoxic effects of the neuroinflammation. we also determined whether the administration of the non-competitive n-methyl-d-aspartate (nmda) receptor antagonist, memantine, could provide neuroprotection from the cytotoxic effects of the neuroinflammation. chronic lps infusions decreased choline acetyltransferase activity and increased the number of activated microglia within the basal forebrain. caspases , and activity was increased in ventral caudate/putamen. non-steroidal antiinflammatory drug therapy attenuated the toxicity of the inflammation upon cholinergic cells and reduced caspases , , and activity in the caudate/putamen. zvad significantly decreased the levels of caspases , and but did not provide neuroprotection for cholinergic neurons. memantine significantly attenuated the cytotoxic effects of chronic inflammation upon cholinergic cells. these results suggest that prostaglandins contribute to the degeneration of forebrain cholinergic neurons in alzheimer's disease and that the cytotoxic effects of prostaglandins occur upstream to nmda receptor activation. intracranial administration of n-methyl-d-aspartate (nmda) receptor antagonists block learning of classical and avoidance conditioning in goldfish. studies with goldfish have shown that nmda receptors are mostly dense in the telencephalon and telencephalon ablation impairs avoidance learning. the present study investigated amnestic effects of microinjection of nmda receptor antagonist ap to the goldfish telencephalon in avoidance conditioning. in experiment , fish received no injection or microinjections of saline or various doses of ap to their telencephalon minutes before three semiweekly training sessions. fish were tested without injec-tions in session . a one-way anova with multiple comparisons on the test scores showed that ap produced anterograde amnesia in a dose-dependent manner. in experiment , fish received several training sessions and a microinjection of various doses of ap minutes before testing. the test scores showed that ap did not decrease avoidance responses, suggesting that microinjection of ap did not impair performance processes. in experiment , fish received microinjections of ap or saline to their telencephalon immediately following three semiweekly training sessions and were tested without injections in session . a one-way anova on the test scores showed that ap did not produce retrograde amnesia. (supported by gvsu grant-in-aid.) tryptophan modulates striatal serotonergic activity relative to fatigue t. yamamoto and e. a. newsholme health science laboratory, tezukayama university, nara, japan department of biochemistry, university of oxford, u.k. we have been reported that mechanism of fatigue in the brain relates to enhanced extracellular tryptophan and serotonergic function. brain concentration of tryptophan is not only dependent on the change of tryptophan which originates from the centarl nervous system, but also enhance tryptophan entering the brain from the blood-brain barrier and peripheral circulating tryptophan which is a trigger. supplementation of ltryptophan ( um) into the incubation medium with the synaptosomal striatum causes tryptophan to the extrasynaptosomal release by high kϩ stimulation. injecting l-tryptophan ( mm/ min) into the left striatum by microdialysis method can induce early fatigue for running time of rats. on the other hand, tryptophan deficiency rats (body weight average g) were made by tryptophan free feeding for weeks, and the rat's running time increased (Ͼ min difference). these results suggests that tryptophan is a potent active substance for fatigue in the brain. the active zone may be presynaptic terminal and the tryptophan itself may be releasing neuromodulators. (we appreciate that tryptophan free diet was provided by ajinomoto co., inc., japan.) our recent studies on the distribution of free d-serine, together with the d-serine action on the glycine site of the nmda type glutamate receptor, suggest that the d-serine can be an endogenous modulator of the nmda receptor. to explore the possible removal systems for brain d-serine signaling, we have evaluated the uptake of [ h]d-serine into the synaptosomal p fraction from the rat cerebral cortex. the cortical p fraction was able to accumulate [ h]d-serine in a temperatureand ph-dependent and saturable manner. the kinetic analysis indicates that cortical d-serine transport occurs by an apparent single-component system with km value of µm and a vmax value of pmol/mg protein/min. depletion of na ϩ and cl Ϫ ions remarkably decreased d-serine uptake into the cortical p fraction. the pharmacological profile of the inhibition of dserine uptake by various amino acids was different from those of glycine uptake system and other amino acid transporters reported. d-serine uptake activity was preferentially observed in the brain tissues such as cerebral cortex and cerebellum to the peripheral tissues. the present data support the view that the endogenous d-serine is taken up mainly through a carriermediated transport system to regulate the extracellular concentration in the mammalian brain. a. bocheva et al. the mammalian brain contains all the urea cycle intermediates, whereas enzymes participating in the conversion of lornithine (l-orn) into l-citrulline (l-cit) are absent, resulting in an incomplete urea cycle. the discovery of nitric oxide (no) synthase that catalyses the formation of no and l-citrulline as a co-product from l-arginine (l-arg) in the brain has indicated an additional pathway for l-arg metabolism. l-canavanine (l-cav), is a potent antimetabolite and structural analog of larginine, produced by legumes such as the jack bean, canavalia ensiformis. l-canaline (l-can) is a potent inhibitor of ornithine aminotransferase. our previous results indicated that l-cav, l-cit, l-arg, and l-orn exerted an antinociceptive effect, whereas l-canaline induced hyperalgesia in rat. l-canavanine exert stronger antinociceptive effect than l-arginine, l-ornithine and l-citrulline. the aim of the present study was to investigate are d-arg, l-cav and naloxone reversed the analdesic effects of l-ornithine, l-citrulline and l-arginine. the experiments were carried out on male wistar rats. the changes in the mechanical nociceptive threshold of the rats were measured by the radall-selitto paw pressure test using and analgesimeter (ugo basile). the amino acids were applied intracerebroventricularly (i.c.v.) at a dose µg/rat. the present results shown that d-arg, l-cav and naloxone reversed antinociception. the regulation of lysine metabolism in cereal crops r. a. azevedo , p. j. lea , s. a. gaziola , a. p. pellegrino , and s. m. g. molina departamento de genética, escola superior de agricultura luiz de queiroz, universidade de são paulo, brazil department of biological sciences, university of lancaster, u.k. a major nutritional drawback of cereal seeds is a deficiency in some amino acids, in particular lysine. biochemical, molecular and genetic studies have considerably increased our knowledge concerning the regulation of the aspartate pathway, by which lysine is synthesized. among the enzymes involved in lysine metabolism, aspartate kinase (ak) and dihydrodipicolinate synthase (dhdps) control the regulation of lysine biosynthesis, whereas lysine: -oxoglutarate reductase (lor) and saccharopine dehydrogenase (sdh), have been shown to play a key role in the breakdown of lysine. in general, lysine overproduction can be obtained by altering the sensitivity of dhdps to lysine, but accumulation of this amino acid in cereal seeds requires further manipulation of lor and/or sdh. this suggestion is strongly supported by five main points: ( ) cereal mutant or transgenic plants do not exhibit any significant accumulation of lysine in seeds, but only in other tissues. ( ) the enzymes of lysine degradation, lor and sdh, are endosperm specific in cereals only. ( ) the opaque- mutant, which exhibits higher concentration of soluble lysine and protein lysine in the seed, contains several-fold lower lor and -fold lower sdh activity when compared to the wild-type maize. this reduction in activity in the opaque- mutant is due to a reduced protein lor-sdh concentration by reduction of the zlkrsdh gene transcript. furthermore, the opaque- maize gene has been shown to regulate ak and lor activity. ( ) intermediates of lysine catabolism accumulated in the seeds of soybean and canola lysine overproducing plants, suggesting the presence of reduced lor and/or sdh activities. ( ) among cereals and although still below the recommend values by fao, rice exhibits the higher concentration of lysine, but lor and sdh are present in much lower activities. also, in phaseolus vulgaris, lor and sdh activities were shown to be around fold lower then in maize endosperm. the regulation of the lor activity is complex and involves a calcium dependent phosphorylation/dephosphorylation mechanism. it remains to be seen whether this latter mechanism can be controlled, so as to allow the production of more crop plants that contain elevated concentrations of lysine in the seed. the genetic progress for nue can be accelerated with the use of secondary traits that possess high inheritance and correlation with productivity. several traits have been studied such as chlorophyll concentration, plant height, leaf senescence, anthesis-silking interval, kernel number, activities of enzymes of n assimilation and loci of quantitative traits for assisted selection. (we are grateful for financial support from fapesp, brazil and the british council.) (termed hyperaccumulators) that grow on metalliferous soils, are able to translocate cd from the roots and accumulate it in high concentrations in the shoots. cd may be detoxified in plants by combination with a family of sulphur rich peptides termed phytochelatins. cd has the capacity to inhibit a range of enzyme activities in plants, in particular those of the calvin cycle and chlorophyll biosynthesis. evidence that cd causes the production of reactive oxygen species (ros) has also been obtained. we have investigated the antioxidant responses of radish, soybean and sugarcane to cd treatment. seedlings were grown in increasing concentrations of cdcl , ranging from . - mm, for up to h in a hydroponic system. analysis of cd uptake indicated that most of the cd accumulated in the roots, but some was also translocated and accumulated in the leaves. roots and leaves were analysed for catalase (cat), glutathione reductase (gr) and superoxide dismutase (sod) activities. gr activity increased considerably in the roots of all plant species tested after exposure to the metal, indicating a direct correlation with cd accumulation. cat activity also increased in roots but to a much lesser extent when compared to gr and also varied depending upon the plant species. the analysis of native page enzyme activity staining, revealed several sod isoenzymes in leaves of all plant species, however, only in radish was a clear increase in activity observed. the results suggest that in these plants, the activity of antioxidant enzymes responds to cd treatment. the main response may be via the activation of the ascorbate-glutathione cycle for the removal of hydrogen peroxide, or to ensure the availability of glutathione for the synthesis of cd-binding proteins. (we are grateful for financial support from fapesp, brazil and the british council.) all plant cells, tissues and organs provide the biosynthetic machinery and capacity to synthesise aliphatic polyamines. however, in physiological conditions only some organs and tissues synthesise polyamines, such as apical buds and sprouts, root apex, lateral buds of branches and secondary roots, as well as superficial layers of young stems and leaves, like epidermis, subepidermis and parenchyma cells. apical roots can also synthesise polyamines, but these activities in physiological conditions are lower than that of the shoots. this patterns recalls the one of auxins. polyamines are accumulated in high concentrations in storage organs, such as seeds, but not in tubers like helianthus tuberosus, potato or tuberised roots such as the carrot. also some fruit, e.g. oranges, contain high level of free polyamines, putrescine in particular. all other organs obtain polyamines through translocation via phloem tubes and xylem vessels. in plants, in addition to free polyamines, many polyamines are conjugated to hydroxycinnamic acids, the hydroxycinnamic amines, that only rarely represented outside the plant kingdom. this compounds are paticularly abundant in solanaceae family, where they can represent as much as % of the total polyamine pool, but they can be detected in different concentrations in many other families. the role of free and conjugated polyamines and their importance in food is discussed. drought, salinity or other environmental stressors promote the accumulation of free amino acids, amines and other organic n-metabolites with low molecular weight. in this contribution the influence of drought on the accumulation of amino acids, polyamines and trigonelline in growing barley plants and barley grains was examined. in comparison to non-stressed plants we obtained in stressed plants, exposed to drought before flowering, a higher concentration of proline (increase: -fold), n-trimethylglycine ( -fold), histidine ( -fold), tryptophane ( , -fold), putrescine ( , -fold), spermine ( , -fold) and trigonelline ( -fold) in the dry matter of barley sprouts. in addition to this, drought caused an increase of the n-content in the plant biomass ( %) as a result of growth inhibition ( %). six weeks later the content of soluble n-metabolites and protein was analyzed in non-stressed and pre-stressed barley plants again. during this reproductive period of plant development all the test groups were cultivated under the same moisture conditions. the analysis of n-metabolites in the ripening grains showed, surprisingly an after-effect of the drought stress. for example, in grains of pre-stressed barley the concentrations of free proline, histidine, tryptophane and asxϩglx were threefold to fivefold higher than in grains of non-stressed barley. depending on the resistance of barley cultivars to drought the biochemical response was different: in plants with low resistance the increase of amino acids and amines was higher than in resistant cultivars. however, resistant cultivars have already high genuine concentrations of n-metabolities in non-stressed plants. by treatments with choline or -aminoethanol the stresspromoted accumulation of amino acids and trigonelline was diminished. consequently, different biochemical responses of cereals to drought result in changes of product quality and nitrogen use. our goal is to increase the lysine content in corn. we have used genetic engineering to increase lysine synthesis and to prevent metabolic breakdown of lysine. to increase synthesis we circumvented the normal feedback control of a key enzyme in the lysine biosynthetic pathway, dihydrodipicolinic acid synthase (dhdps). lysine-feedback-insensitive dhdps, encoded by the corynebacterium dapa gene, was expressed from seed-specific promoters in transformed corn seeds. expression of dhdps in the corn embryo, but not in the corn endosperm, resulted in a to -fold increase in the accumulation of free lysine in the seeds and the total seed lysine content nearly doubled. lysine breakdown products have been observed in transgenic seeds that accumulate high levels of free lysine. we isolated a corn gene for the bifunctional enzyme lysine ketoglutarate reductase (lkr)/saccharopine dehydrogenase (sdh), which catalyzes the first two steps in lysine breakdown. knockout of lkr/sdh in corn by either mutation or genetic engineering results in a -fold increase in seed free lysine. combination of feedback-insensitive dhdps with knockout of lkr/sdh results in to -fold higher levels of free lysine than dhdps alone. no adverse effects on seed or plant agronomic performance are associated with the high lysine trait. biotechnology center for agricultural and the environment and the plant science department, rutgers university, new brunswick, new jersey, u.s.a. Ј-adenylylsulfate (aps) reductase catalyzes a key reaction in the plant sulfate assimilation pathway leasing to the synthesis of cysteine and the antioxidant glutathione. in arabidopsis thaliana aps reductase is encoded by a family of genes. in vitro studies revealed that the enzyme product derived from one of the aps reductase genes (apr ) is activated by oxidation, probably through the formation of a disulfide bond. redox titrations show that the regulation site has a midpoint potential of Ϫ mv at ph . and involves a -electron redox reaction. exposure of a variety of plants to ozone induces a rapid increase in aps reductase activity that correlates with the oxidation of the glutathione pool and is followed by an increase in free cysteine and total glutathione. during the response to ozone the level of immuno-detectable aps reductase enzyme does not increase. treatment of a. thaliana seedlings with oxidized glutathione or paraquat induces aps reductase activity even when transcription or translation is blocked with inhibitors. the results suggest that a post-translational mechanism controls aps reductase. a model is proposed whereby redox regulation of aps reductase provides a rapidly responding, self-regulating mechanism to control the glutathione synthesis necessary to combat oxidative stress. in aspergillus nidulans the structural genes coding for nitrate reductase (niad) and nitrite reductase (niia), share a common promotor region of , bp. we have previously characterized in vitro and in vivo the physiologically relevant cisacting elements for the two synergistically acting transcriptional activators, nira and area. we have further shown that area is constitutively bound to a central cluster of four gata sites and is directly involved in opening the chromatin structure over the promoter region and thus making additional cis-acting binding sites accesible. here we show that the asymmetric mode of nira-dna interaction determined in vitro is also found in vivo. binding of the nira transactivator is not constitutive as in other binuclear c -zn ϩϩ -cluster proteins but depends on nitrate induction and additionally, on the presence of a wild type area allele. dissecting the role of area further, we found that it is required for intracellular nitrate accumulation and therefore could indirectly excert its effect on nira via inducer exclusion. but in a strain accumulating nitrate independently of area nira binding and chromatin rearrangement is not triggered by nitrate in the absence of area. v. nikiforova , m. zeh , o. kreft , s. maimann , h. hesse , and r. höfgen max-planck-institut für molekulare pflanzenphysiologie, potsdam, and institut für biologie, angewandte genetik, freie universität berlin, germany higher plants, being a source of reduced sulfur for animal nutrition, assimilate inorganic sulfate into cysteine which is subsequently converted to methionine, another sulfur-containing amino acid. in order to investigate the possible regulatory points of the cysteine and methionine biosynthesis pathway a series of transgenic potato plants was engineered using clones encoding enzymes of the branched pathway from serine to cysteine as a pathway intermediate and from aspartate further on to methionine. increased cysteine levels were obtained in the leaves of serine acetyltransferase (sat) sense and cystathionine -lyase (cbl) antisense transformants. furthermore, glutathione levels were elevated in sat plants while downregulation of cbl was desastrous for plant growth, eventually. increased methionine levels were successfully obtained in potato by antisense inhibition of threonine synthase (ts). accumulation of free methionine was not only observed in source leaf tissues but as well in tubers. this enzyme competes with cystathionine gamma-synthase for the common substrate o-phosphohomoserine at the branchpoint between threonine and methionine synthesis, respectively. important control points of the biosynthesis of cysteine and methionine in potato, thus, turned out to be sat and ts, while further studies on overexpression of cystathionine gamma-synthase, cbl and ms did not reveal any substantial effect on potato methionine biosynthesis. dniepropetrovsk national university, board of biophysics and biochemistry, ukraine amino acids in root exudates of plants may be chelate agents as an alpha-amino acid can act like a bidentate ligand, forming a five-membered heterocyclic ring with suitable metal cations thus increasing mobility of metals. recently we have showed that application of growth regulators led to sharp increase of root exudative activity of some cultural (zea maize l.) and wild cereals (festuca rubra l., lollium perenne l.) during first days of germination. in this work we present results obtained in experiments with lollium perenne l., grown on sterile sand and on soils contaminated with great quantities of zn. detailed analysis of amino acid content of root exudates of several types of maize (hybrid, several lines, an opaque- mutant line) showed that the specie had more certain amino acids (cysteine, aspartic and glutamic acids and their amides, serine) in root exudates than cultural ones. these amino acids has more possibility for chelation due to existance of one more polar or ionogenic functional groop. seeds of lollium perenne l. were treated with growth regulater and planted on soils contaminated with salts of zink. it was shown that during days of germination quantity of zn in primary leaves increased from , to , % and decreased in soil: in upper layer from , to , , midde layer from , to , , lower layer from , to , mkg/kg correspondingly. thus, it was shown that stimulation of root exudative activity by pretreatment with a growyh regulator may be succesful in cleaning of soils and basicly this is a good method for phytoremediation. erenol exerted the strongest effect. exercise completely abolished the levels of cysteine in the atrial heart muscle. propranolol, isoproterenol, caffeine and pentylenetetrazol increased the ratio of cysteine to the total free amino acids in the atrial muscle, while physical stress and all cardioactive drugs tested increased this ratio in the ventricle muscle. disappearance of cysteine from the heart's atrial muscle after intensive exercise may be attributed to its utilization for atrial natriuretic factor and/or for endothelin synthesis, during stress. on the other hand it seems that hypoxia and isoproterenol are strong stimulants of no production, and consequently decrease the tissue levels of l-arginine, which is the major endogenous donor of no acting as the endothelin antagonist. measurement of serum levels of vitamin b is a screening test for detection of deficiency of this vitamin but low levels do not always indicate a deficiency of the vitamin. measurements of serum homocysteine and methylmalonic acid (mma) are used to confirm this deficiency because two enzymes involved in their metabolism have been shown to require vitamin b , but these results can also be inaccurate. vitamin b deficient white cells exhibit ultrascopic nuclear appenages which have been shown to contain dna; this finding could possibly be used as another confirmatory test of vitamin b deficiency. twenty-seven patients (mean age - . years) with low serum b were studied by electron microscopic determination of the percent of neutrophils exhibiting these appendages and routine clinical parameters. only one patient did not have nuclear appendages; the others had a range of . %- . % of neutrophils examined. there was a significant correlation of homocysteine (r ϭ . , p Ͻ . ) and mma (r ϭ . , p Ͻ . ) with serum b levels but no correlation of appendage number (r ϭ . ) with serum b . there was no correlation of appendage number with homocysteine (r ϭ . ) or mma (r ϭ . ). these results suggest that b -deficient white cell nuclear appendages do not measure the same metabolic pathways as homocysteine and methylmalonic acid and may be useful in confirmation of vitamin b deficiency. further extensive clinical evalution would be necessary to explore this possibility. the hypothesis: "l-theanine has relaxing effects of central nervous system of human beings", was verified by electroencephalographical methods. methods: male, healthy sport-students, free of drugs or stimulants, participated weekly in a cross-over study. after exhaustive bicycle-ergometer test as an individual, reliable, stress model, the subjects recovered by lying in a segregated shaded room. three testdrinks with different l-theanine content (d ϭ placebo, d ϭ mg, d ϭ mg) were given in a randomised, double-blind order. all test-conditions were standardized strictly. eeg-recordings (closed eyes) were carried out (m ϭ min. after stress/before testdrink, m ϭ min.-, m ϭ min.-, m ϭ min.-, m ϭ min. after testdrink) with the cateem ® system. absolute and relative eeg-spectralpower were examined. results: significant reductions in all frequencies (exception theta-power) were found in early recovery, being not significant influenced by testdrinks. qualitative different behavior trends were found in frontal-, central-, occipital-regions with increased alpha , theta (frontal) and decreasing beta relative-power earlier in recovery with d . these findings were related to relaxing effects. after ingestion of l-theanine alpha -, beta -power at occipital regions decreased faster (m ) to placebo recovery levels (m /m ). thus it may be concluded that l-theanine has no pharmaceutical effect on the down regulation system but supports the physiological mechanisms during recovery after physical stress in human brain. arginine and cysteine in muscle cytosol of rats' heart after exercise, hypoxia or challenge with six selected cardioactive drugs r. brus , j. gabryś , j. konecki , and j. shani department of pharmacology and department of histology and embriology, medical university of silesia, zabrze, poland department of pharmacology, the hebrew university school of pharmacy, jerusalem, israel levels of the amino acid l-arginine (a major endogenous donor of nitric oxide-no), cysteine (sulfur-containing amino acid, important for atriopeptins and endothelins synthesis), and of total free amino acids, were assayed by gas-liquid chromatography in cytosols of rats' atrial and ventricular muscle cardiomyocytes. the tissues were assayed after the rats had been exposed to either exercise (swimming), hypoxia or one of six cardioactive drugs such as propranolol, digoxin, pentylenetetrazol, reserpine, isoproterenol and caffeine. physical stress and the examined drugs significantly reduced the total amount of cytosolic free amino acids in both cardiac muscles. in the cytosol of the heart atrial muscle, reserpine, propranolol and pentylenetetrazol increased the relative content of l-arginine, while hypoxia and digoxin decreased it. in the cytosol of the ventricular heart muscle, hypoxia and all six drugs used, decreased the relative levels of l-arginine. hypoxia and isoprot-addition of somatostatin- or of some of its analogs was found to cause a selective inhibition, up to %, of the uptake of large neutral amino acids by isolated brain microvessels. although the luminal and abluminal sides of brain endothelial cells are both capable of taking up large neutral amino acids, only the uptake from the abluminal side was apparently inhibited by somatostatin. the involvement of a type- somatostatin receptor was suggested by assays with a series of receptorspecific somatostatin agonists, and was confirmed by the inhibition release caused by a specific type- receptor antagonist. a type- specific mrna was indeed shown to be present both in bovine brain microvessels ex vivo and in primary cultures of endothelial cells from rat brain microvessels. hemorphins represent a bioactive peptide class which contents between and amino acids and generated from the proteolysis of an hemoglobin "strategic zone". many activities have been related to hemorphins such as in vitro anti tumour effect, analgesia effects in vivo, and a potential role in the renin angiotensin system (ras). as far as their activity towards the ras is concerned, it was demonstrated that they could inhibit angiotensin converting enzyme (ace) and aminopeptidase n activity. so they could reduce angiotensin ii formation and angiotensin iv degradation. moreover some hemorphins, lvv-hemorphin- and vvhemorphin- , could behave like angiotensin iv receptor binding competitor. further it could be interesting to study the angiotensin iv potentiality to interact with ace. inhibition studies showed that it was possible that angiotensin iv could behave like a competitive inhibitor of ace. so some hemorphins could interact at different ras steps to inhibit ace. additionally to their inhibition of angiotensin i conversion, they could inhibit angiotensin iv degradation and consequently cause ace feedback inhibition. inhibition studies have been checked with ras natural substrate (angiotensin i) and confirmed that angiotensin iv, vv-hemorphin- and mainly lvv-hemorphin- could be natural ace inhibitors. so the hemorphins regulatory role in the ras appears to be more and more probable. the role of administration of each of methionine and finastride on the testicular function of both normal and prostate precancerous old male rats was investigated. for normal animals, neither methionine nor finastride has exerted any significant change in the hormonal profile after teatment for days. however methionine alone could exert a significant change in both testosterone and prostatic specific antigen {psa} levels after treatment for days. on the other hand, both methionine and finasteride significantly increased the levels of testosterone and androstenedione, whileas markedly reduced the levels of dihydrotestosterone and prostatic specific antigen {psa} after treatment of prostate precancerous old male rats for a period of days. noteworthy, continuation of treatment for aperiod of days realized marked improvement of hormonal profile of the prostate precancerous old male rats. several observations in our department point to some role of glycine in fatigue and exercise. ) in the framework of a study on the involvement of one-carbon matabolism in patients suffering from a polymorphic episodic psychosis, amino acid loading tests with serine, glycine and alanine were performed. a few hours after the administration of glycine, approximately % of the patients reported overwhelming feelings of fatigue and/or showed vegetative symptoms. ) in patients suffering from chronic fatigue syndrome, we found increased plasma levels of glycine in % of the female patients. moreover, - % of these patients omplained about a distorted sensory perception of objects. ) young soccer players were observed during a period of months, while in the course of this period eight blood samples were taken for amino acid analysis. based on the number and severity of injuries this population was divided into injury-prone and not injury-prone soccer players. it was found that in injury-prone soccer players plasma glycine levels during the whole observation period were significantly lower than in subjects who were not injury-prone. the consequences of the above mentioned observations will be discussed. institute of sportsmedicine, university of paderborn, germany percent of amino acids in green tea leaves are represented by l-theanine ( -n-ethylglutamine). previous rat experiments demonstrated effects of l-theanine to act on metabolism of neurotransmitters. it was therefore suggested that is causes the relaxing effects of green tea. to examine its influence as a component of a drink on the sympathetic nervous system after maximal physical exercise skin resistance measurements through electrosympathicography (esg) were used. after individual maximal exercise on a bicycle-ergometer testdrinks with different amounts of l-theanine ( , and mg) were administered to healthy volunteers in a randomised cross-over double-blind distribution on a weekly base. esg was monitored before and immediately after exercise as well as , , , , and minutes after end of exercise. all testconditions were standardized strictly. a characteristic esgcourse with subsequent qualities could be shown: . decreasing skin resistances after exercise could be established in each volunteer. . esg-activation levels before exercise could not even be reached again after a period of regeneration of / hours. . maximal electrodermal activity did not appear immediately after exercise, but after minutes. however, l-theanine could not significantly influence peripheral sympathetic electrodermal activity during the regeneration after maximal physical exercise. a. mero and h. pitkänen , neuromuscular research center, department of biology of physical activity, university of jyväskylä, and rehabilitation center of kankaanpää, finland essential amino acid leucine has many important roles in the body. therefore the purpose of the present study was to investigate if leucine supplementation has effects on serum amino acid profile and performance following training period or following single training sessions. all experiments were carried out in a randomized double blind cross-over procedure during a training season. thirty six adult male track and field power athletes served as subjects. in experiment ten of them were given leucine ( mg/kg body weight per day) as tablets. the concentration of leucine decreased significantly ( %) in the placebo group (p; n ϭ ) during weeks but not when leucine was taken. also total amino acids (taas) decreased strongly ( %) during weeks when dally protein intake was . g/kg body weight. in experiment the subjects (n ϭ ) carried out a single strength training seasion (sts) and consumed a drink containing leucine mg/kg body weight. following sts leucine in serum increased by % (ns) when leucine was taken but decreased strongly ( %) in p, in experiment the subjects (n ϭ ) underwent at days interval two maximal anaerobic running exercise (mare) tests on treadmill (n ϫ s with a recovery of s between the runs) until exhaustion. the subjects consumed drinks containing leucine ( mg/kg body weight) or placebo min before the test runs. following mare the concentration of leucine strongly increased by % whereas isoleucine ( %) and valine ( %) strongly decreased with the supplementation but no changes occurred in p. there were no improvements in physical performance either in mare or in explosive strength (experiment ) with leucine supplementation. the date suggest that leucine supplementation during a training period and before single training sessions prevents decreases in serum concentration of leucine and may have also effects on some other single amino acids. this may be beneficial during intensive training although improvements in performance were not observed in this study. since there are only limited data regarding effects of training period or training sessions on serum amino acid profile, the purpose of this study was to investigate serum amino acid changes following training period and following three different training sessions. the subjects consisted of track and field adult male power athletes. in experiment eleven of them performed a -week training period including a training sessions per week, which included sprint work, speed endurance work, endurance work, weight training, and jumps. significant decreases in the fasting concentrations of total amino acids (taas) ( %), branched chain amino acids (bcaas) ( %), essential amino acids (eaas) ( %) and leucine ( %) were observed following training with the daily protein intake of . g/kg body weight. in experiment eleven subjects performed a short run session (srs) of ϫ ϫ m with recoveries of s and s, and a long run session (lrs) of s runs with recoveries of s until exhaustion. there were no significant changes in taas following the sessions but bcaas decreased by % in srs and by % in lrs. leucine decreased by % following srs but only by % (ns) following lrs. the peak blood lactate concentrations after srs and lrs were . Ϯ . mmol/l and . Ϯ . mmol/l, respectively. in experiment sixteen subjects carried out a strength training session (sts), which consisted of jumps and heavy resistance exercises (speed and maximum strength) during minutes. the taas decreased significantly by %, bcaas by % and leucine by % following sts, while the peak blood lactate concentration was . Ϯ . mmol/l. these data indicate that remarkable decreases occur in the concentration of amino acids during a training period with the daily protein intake of . g/kg body weight. the decreases in serum amino acids are more pronounced following a strength training session than following lactic anaerobic running sessions. glutamine acts as a multipurpose regulator of amino acid and peptide transport across the blood-brain barrier departments of cellular biotechnology and haematology and of biochemical sciences, university "la sapienza", rome, italy isolated brain microvessels, the in vitro equivalent of the blood-brain barrier, have distinct na ϩ -independent uptake systems for the uptake of large hydrophobic amino acids, of enkephalins and of deltorphins, as shown by the absence of reciprocal inhibition. both d-and l-glutamine were capable, if added to the extracellular buffer, of exerting a competitive inhibition on the uptake of all these substrates. a trans-stimulatory effect was instead induced, in all cases, by l-glutamine preloading of the microvessels -the d-stereoisomer being instead ineffective, probably because of only l-glutamine could be taken up, in a concentrative manner, by some na ϩ -dependent concentrative system(s). all the na ϩ -independent systems present in brain microvessels seem therefore to share some structural feature responsible for their common susceptibility to interference by l-glutamine. this amino acid, whose synthesis can take place in the astrocytes, in the pericytes and also in the endothelial cells of the microvessels, plays a critical role in regulating the movements of several different substrates across the blood-brain barrier. department of applied bioorganic chemistry, division of life science, graduate school of agricultural science, tohoku university, aoba-ku, sendai, japan isolation and structure analysis of two amino acids from bovine ligamentum nuchae elastin hydrolysates revealed the presence of pyridine cross-links in elastin. the structures of these amino acids were determined to have , , -and , , -trisubstituted pyridine skeletons both with three carboxylic acids and a mass of (c h n o ), identified as -( -amino- -carboxybutyl)- , -di-( -amino- -carboxypropyl)pyridine and -( -amino- -carboxybutyl)- , -di-( -amino- carboxypropyl)-pyridine. we have named these pyridine cross-links, desmopyridine (desp) and isodesmopyridine (idp), respectively. structure analysis of these pyridine crosslinks implied that the formation of these cross-links involved the condensation reaction between ammonia and allysine. the elastin incubated with ammonium chloride showed desp and idp levels increased as the allysine content decreased. desp and idp were measured by hplc with uv detection and were found in a variety of bovine tissues. the desp/desmosine and idp/isodesmosine ratios in aorta elastin were higher than in other tissues. desp and idp contents in human aorta elastin were found to be gradually increased with age. the concentration of idp was significantly elevated in aorta elastin of rat with chronic liver cirrhosis induced by carbon tetrachloride when compared with normal rats. the provision of glutamine to marathon runners has resulted in a decreased, self-reported incidence of illness. increasing evidence -in vitro; and in vivo suggests that neutrophils in humans may benefit from exogenous glutamine. the provision of glutamine in vivo should replete the marked decrease in the blood concentration observed after stress such as clinical trauma or prolonged, strenuous exercise. beneficial effects of glutamine supplementation include increased phagocytic activity and reactive oxygen intermediate production in vitro; decreased neutrophilia and il- production (a chemoattractant for neutrophils) in vivo and ex vivo. the aim of the present study was to establish whether glutamine supplementation in vitro and in vivo affects neutrophil function at rest and after exhaustive exercise. in addition, it was planned to establish the presence of glutaminase in human neutrophils, which has not yet been achieved, although glutaminase is present in rat neutrophils. methods: blood samples were taken from marathon runners receiving either glutamine or placebo, immediately after and one hour after a race. measurements included the plasma concentration of glutamine (enzymatic assay), il- production (elisa), and neutrophil activity. the latter was measured with two different techniques for measuring oxidative burst in whole blood, one of which was a novel chemiluminescence assay (knight scientific ltd, u.k.) with the fluorescent label, pholasin, and two different stimuli, f-met-leu-phe (fmlp) and phorbol-myristate-acetate (pma). in addition, isolated whole cells and subcellular neutrophil fractions were assayed for the presence of glutaminase. results: the plasma glutamine concentration was reduced overall by % hr after the race (p Ͻ . ). there was an apparent decrease (only close to significance, p Ͻ . ) in il- production in the glutamine group compared with the placebo group. neutrophil function did not change between groups at any stage. the incidence of illness was % higher in the placebo group than the glutamine group in the week after the race. neutrophils from four out of six subjects gave an increased response ( . %) to fmlp when incubated with glutamine compared with no glutamine, and four out of four gave an increased response to pma ( . %). in the fmlp experiments there were two individuals who did not respond to the addition of glutamine. however, the response was not diminished whether or not glutamine was present. in separate studies, the effect of glutamine on lipopolysaccharide-induced il- production was also monitored. conclusions: the provision of glutamine after prolonged, exhaustive exercise appears to modify exercise-induced neutrophilia via a reduction in il- production and to reduce the incidence of illness in the following week. in vitro data suggest a role for glutamine in neutrophil metabolism. disappointingly, little or no evidence of the presence of glutaminase was found in human neutrophils. the three different methods used, freeze-thaw, homogenisation, nebulisation were apparently not sufficient to break open the granules. current studies are addressing this problem. r. j. ward and l. m. castell departments of biochemistry, university catholique de louvain, belgium oxford university, oxford, u.k. it is essential that the developing muscle has adequate amino acids for the synthesis of actin and myosin as well as those required for a multitude of enzymes involved in muscle metabolism. with carbohydrates and lipids, the body is able to store a reserve as glycogen and triglycerides respectively; however this is not the case with amino acids creatine supplementation in increasingly being used as a dietary supplement by athletes during high intensity, short term exercise to improve physical performance since it is converted in the muscle to phosphocreatine. transporters which permit creatine to cross the muscle membrane namely crt and crt (a na ϩ and clЈ dependent mechanism) have now been identified. creatine uptake is enhanced by the ingestion of carbohydrate at the same time as supplementary creatine. this may be due to increased circulating levels of insulin or insulin-like growth factor . more recently attention has been focussed upon the various transporters for amino acids across the muscle membrane. certain criteria are needed for the amino acids to enter the blood which include the presence of specific carriers for its transport across cells of the gastrointestinal tract, such as enterocytes, as well as minimal metabolism within these cells. a wide number of different transporters has been identified, which include neutral amino acids and cationic amino acids. despite the evidence which suggests that supplementation with some amino acids can influence metabolism, and therefore athletic performance, much more experimental work is still required in this area. m. weiss , t. barthel , r. schnittker , k. e. geiss , w. falke , and l. r. juneja university of paderborn, germany taiyo kagaku co., yokkaichi, japan, isme gmbh mörfelden, germany in animal studies l-theanine was shown to influence neurotransmitter systems. thus it may be helpful in managing stress regulation. so we observed the down regulation after physical stress in the brain (measured by eeg-mapping) and in peripheral hormonal systems (plasma levels of catecholamines, cortisole, prolactine, serotonine, measured by hplc). n ϭ healthy students consumed drinks containing either , or mg l-theanine in randomized double-blind trials in the min - after a near maximal bicycle step test. measurements were done directly after exercise (m ) and (m ), (m ), (m ), (m ) min after the drink. l-theanine seemed to accelerate the normalization of eeg spectral power in high frequency waves (barthel in this congress). the physiological return of increased hormon levels to basal levels / the circadianic rhythm up to m (catecholamines) or m (cortisole, serotonine, prolactine) was not influenced by the drinks. but in the l-theanine trials correlations between eeg spectral power and some hormones were altered (slow wave power/some catecholamines except norepinephrine/delta disappeared and new correlations with prolactine appeared). thus we conclude that l-theanine acts at the switch from the brain to the peripheral stress regulation and thereby supports physiological relaxing after severe exercise. polyamines the development from callus to plantlets, both activities increased, reaching the maximum at this latter stage. also sadenosylmethionine decarboxylase activity displayed a similar trend. all the activities were present in supernatant and in particulate fraction. higher activity of enzymes assayed in the small embryos rather than in the embryo with higher shape, was consistent with following polyamine accumulation. department of biology, laboratory of cell biochemistry, university of rome "tor vergata", rome, italy intracellular transglutaminase (tg, ec . . . ), which catalyzes the formation of ε-(γ-glutamyl)lysine isopeptide cross-links between polypeptides, has been related to a variety of important biological processes and in the development of senile cataract. the majority of the dry weight of the eye lens is composed of protein called crystallins. in the mammalian lens, these proteins are divided into three major classes: α-, -and γcrystallins. native -crystallins are oligomers, which elute in two or more size classes during gel filtration, ranging from - kda. they contain different types of subunits, named b , b , b , a , a , a , ba , ranging from - kda. in the rabbit eye lens two -crystallin subunits ( b and b ), among the water soluble proteins, have been shown to act selectively as acyl donors substrates for lens tg. calpains are cytoplamic ca ϩϩ -dependent cystine proteinases. the cleavage of αand -crystallins, the main substrates of lens calpain ii, has been associated to the increase of lens turbidity, due to insolubilization of peptides. we observed that tg-induced post-translational modification of b -and b -crystallins with polyamines, enhances their cleavage by calpain ii. this finding suggests that the enhancement of calpain ii activity, after conjugation of polyamines into -crystallins, could represent an important regulatory mechanism which may contribute to the opacification process of the eye lens, conducting to cataract formation. transglutaminases represent a family of enzymes, widely diffused in nature, from bacteria to plants and higher animals. the present discussion will focus on isoenzymes in mammals, which have been well characterized from the structural and functional point of view. they act on tissular proteins catalyzing crosslinkage through isopeptide bonds at peptidyl glutamine and lysine residues or incorporation of small molecular weight primary amines, usually polyamines, in an irreversible, calcium dependent reaction. in several instances the expression of transglutaminases is regulated at the transcriptional level. these enzymes help in maintaining structural integrity of tissues intervening in wound repair and in cellular homeostasis at the levels of cell activation, receptor signaling, cell proliferation, differentiation and death. these general roles involve bis(guanylhydrazones) are a class of compounds known to interfere with the metabolism of polyamines by virtue of their ability to inhibit s-adenosylmethionine decarboxylase (samdc), a key enzyme of polyamine biosynthesis. this property has made them useful tools to study the biological functions of these compounds. a curious feature of bis(guanylhydrazones) is their structural relationship with two molecules involved in polyamine biosynthesis, namely spermidine and s-adenosylmethionine. the methylglyoxal derivative of bis(guanylhydrazones), mgbg, has been actively studied both in animal and plant systems. in the present work the male pollen from actinidia deliciosa has been utilized to investigate the role of polyamines on the pollen tube growth. the effect of several bis(guanylhydrazones) was tested on pollen germination, length of pollen tube, levels of free and conjugated polyamines and samdc activity. all bis(guanylhydrazones) tested (glyoxal-bis-guanylhydrazone, gbg, methylglyoxal-mgbg, methylpropylglyoxal-mpgbg, ethylmethylglyoxal-emgbg) inhibit pollen germination and their effect is dose-dependent. a clear reduction of spermidine, both in free and conjugated form, was observed, as well as a pronounced decrease in samdc activity. these results suggest that the mechanism by which bis(guanylhydrazones) reduce the germination of kiwi pollen is related to their effect on spermidine biosynthesis. molecules structurally related to polyamines ( , diaminooctane, , -diaminononane, , -diaminodecane) and other inhibitors of their metabolism (cyclohexylamine, cha) are also tested on kiwi pollen germination. n. bagni , d. bertoldi , , e. candioli , l. martinelli , and a. tassoni istituto agrario, san michele a/adige, and dipartimento di biologia, università di bologna, italy in the frame of the study aiming to enlighten developmental programs during regeneration in grapes, polyamine content (free and conjugated to hydroxycinnamic acids) and biosynthetic enzyme activities were assayed during somatic embryogenesis. aliphatic polyamines are growth regulators affecting plant growth and development both in vivo and during in vitro cultures, being involved in several morphogenic processes related ti their action in cell division. the study was conducted on samples of callus, embryogenic callus, embryo at different stages and plantlets of vitis vinifera brachetto and chardonnay cultivars induced from anthers and ovaries. polyamine content (putrescine, spermidine and spermine) free plus conjugated to percloric acid soluble fraction, referred to unit, was higher in the cv. brachetto than in the cv. chardonnay, and reached the higher levels in the fullydeveloped embryo stage. besides, ornithine decarboxylase activity resulted higher than arginine decarboxylase and during multiple catalytic processes: the receptor signaling activity (demonstrated only for isoenzyme ) is related to an intrinsic gtp-ase activity of type transglutaminase; the processes leading to control of cell proliferation, differentiation and death are mainly related to the protein crosslinking activity, while the cell activation is tentatively considered dependent on the polyamidation of endogenous proteins at glutamine residues. the knowledge on this last aspect lies far back in comparison to the other roles of transglutaminases and requires further accurate investigation, which must further extend to the role of the enzyme in human pathology. the examination of polyamine metabolism at the present time suggests that vitamin b is implicated in polyamines metabolism. literature data speak that spermine and spermidine stimulate activity of cobalamin-dependant methionine synthase, the enzyme that catalyses the recycling of homocysteine to methionine; polyamines inhibit methionine adenosyltransferase. beside the wellknow significance of vitamin b , in transmethylation reaction, the significance of ,-deoxyadenozyl cobalamin, except the conversion of methylmalonyl-coa to succinyl coa, is not well elucidates. methionine as s-adenosylmethionine (sam) is essential amino acid for polyamine biosynthesis. sam has frequently usage in treatment of liver diseases. according the mentioned facts the aim of our experiments is to exanimate the significance of application of vitamin b alone and altogether with methionine to rats without and with experimentally induced cholestasis. our preliminary results speak about the disturbance of polyamine metabolism in hepatic tissue of rats with cholestasis. application of methionine alone increases the amount of polyamine in rat liver tissue, in-group without cholestasis and with bile duct obstruction. the animal treatment with cobalamin has higher amount of polyamines and lower activity of polyamine oxidase in liver tissues in both groups. the effects of vitamin b may be in direct relation with the formation of ,-methylthio deoxyadenosine (mta), the by-product of spermidine and spermine biosynthesis. the explanation the exact roles of vitamin b in polyamine metabolism of liver tissue need the futher investigations. department of molecular genetics, the weizmann institute of science, rehovot, israel exposure of mouse myeloma cells that massively overproduces ornithine decarboxylase (odc) but not of parental cells to ornithine results in a massive increase in the intracellular concentration of putrescine, followed by rapid cell death. the treated odc overproducing cells display fragmented nuclei, chromatin condensation and an oligonucleosome-size dna "ladder"; consequently, their death can be described as apoptosis. the apoptotic process induced by the accumulated putrescine involves the release of cytochrome c from the mito-chondria, activation of caspases cascades demonstrated by the cleavage of caspase- and parp, a substrate of caspase- . the general inhibitor of caspases, bd-fmk, effectively inhibited parp cleavage but failed to inhibit cell death. the intracellular ca ϩ chelator bapta/am and the antioxidant bha inhibit parp cleavage. however, only bapta/am inhibit the induction of cell death. it seems that bha subverted the death into caspase independent pathway. treatment with bapta/am did not interfere with the accumulation of putresine following ornithine treatment, suggesting that the accumulated putrescine induces the elevation in the concentration of intracellular ca ϩ which then activates the apoptotic process. the dominant anti-apoptotic effect of bapta/am over egta suggests that internal stores are the main source of the elevated ca ϩ , but that putrescine is also capable of inducing influx of extracellular ca ϩ . extensive small intestine resection results in the loss of absorptive surfaces, acceleration of intestinal transit and, as a consequence, in malnutrition, weight loss, diarrhoea and other complications of short bowel syndrome. the availability of human recombinant growth hormone rgh and its stimulatory effects on gut growth suggested its use in the treatment of short bowel syndrome. the trophic response of gi tract epithelium to hormones such as growth hormone is mediated by polyamines, which are vital in cell proliferation. this study was undertaken in rats to: / evaluate the effects of rgh by monitoring polyamine and amine metabolism parameters in the adapting short bowel and / determine whether erythrocyte (rbc) polyamine concentrations reliably reflect the proliferative activity of the remaining bowel. seventy per cent resection of the small intestine of wistar rats was performed under ether anesthesia leaving equidistant lengths of bowel from pylorus and ileocecal valve. recombinant human gh ( . iu, s.c., saizen, serono, switzerland) was administered once daily for or days, to randomly selected rats on the second postoperative day. animals were sacrificed , and days after the operation. enzyme activities were measured with radioassays or fluorimetry. polymines were determined as dansyl derivatives by hplc/fluorimetry. gh treated animals had significantly higher intestinal odc and sat and lowel dao activities; higher (non-significant) mucosal growth index and polyamine concentrations than in untreated counterparts on th postoperative day. thereafter the two groups did not differ in the investigated parameters. rbc polyamine concentrations were higher in operated verses control rats; rgh treatment had no significant effect. however, rgh treatment significantly reduced hepatic mao a and b activities. our results suggest gh accelerated the adaptive growth of the bowel remnant. they justify use of erythrocyte polyamine concentration measurement as the marker of small bowel proliferative activity. however, side-effects of this treatment must be considered. tissue transglutaminase (ttg) activity has been evaluated in different neural tissues, such as brain, spinal cord and peripheral ganglia, and appears to be expressed in cerebellar granule cells (cgn) as well as in astrocytes. the role of ttg in neuronal functioning is likely to be quite complex. other than the role during development, significant changes of enzyme activity have been evaluated in different neurodegenerative conditions. it is well known that nmda receptor activation may be able to trigger excitotoxicity. the nmda-induced injury is mainly associated to ion influx and subsequent calcium overload. the effects of nmda application to both, cerebellar granule cells and glial cell cultures, have been assessed. in cgn, ttg activity increased rapidly after a brief stimulation with µm nmda, whereas in glial cell cultures, high levels of enzyme activity was obtained after incubation of h in presence of the same concentration of nmda. such results rule out the possibility that excitotoxicity can modify numerous proteins making them better substrates of ttg, and this could contribute to enhanced ttg-modifications of proteins in response to excitotoxicity. the pote protein can catalyze both uptake and excretion of putrescine. the km values of putrescine for uptake and excretion (putrescine-ornithine antiport) are . µm and µm, respectively. amino acid residues, cys , trp , glu , trp and tyr are strongly involved in both activities, and that glu , tyr , cys , cys , cys and glu are moderately involved in the activities. mutations of tyr , trp and trp mainly affected uptake activity, indicating that these amino acids are involved in the high affinity uptake of putrescine by pote. mutations of lys and tyr mainly affected excretion activity, indicating that these amino acids are involved in the recognition of ornithine. the putrescine and ornithine recognition site on pote was found to be located at the cytoplasmic surface and the vestibule of the pore consisting of twelve transmembrane segments. the cadb protein has % sequence homology with pote protein. cadb can catalyze both uptake and excretion of cadaverine. the km values of cadaverine for uptake and excretion (cadaverine-lysine antiport) are µm and µm, respectively. it was found that two glutamate residues (glu and glu ) and four tyrosine residues (tyr , tyr , tyr and tyr ) are involved in the both activities. the difference of the substrate recognition site on pote and cadb is discussed. a. lentini, b. provenzano, and s. beninati department of biology, laboratory of cell biochemistry, university of rome "tor vergata", rome, italy tissue transglutaminase (ttg, e.c. . . . ) is a protein cross-linking enzyme which catalyzes an acyl transfer reaction where the carboxamide group of a peptide-bound glutamine is the acyl donor, and a lysine residue the acyl acceptor. polyamines may act as acyl acceptors, leading to the formation of mono-and bis-(γ-glutamyl)derivatives. we provided evidence that ttg activity is directly associated to differentiation markers, and inversely related to cell proliferation and invasion. we have shown the in vivo reduction of experimental melanoma metastasis by i.v. injection of a plasmid (psg ) carrying the ttg gene sequence to c bl /n mice. tumor cell metastatization requires specific interactions with subendothelial basement membrane (bm) and migration through the endothelial wall, allowing the colonization of the target tissue. therefore, the investigation on the possible mechanisms responsible for ttg effects is focused on the posttranslational modification of bm proteins. we detected that "matrigel", a tumor-derived complex of bm proteins, modified with polyamines after ttg catalysis, reduces both melanoma cell adhesion and invasion in an in vitro metastatic assay. similar results were obtained using polyamines conjugated to laminin, one of the major bm components, as unique substrate. our findings suggest that the increase of bm proteins conjugated to polyamines may be responsible for impairments of the invasive properties of melanoma cells. we demonstrated that interferon-α (ifnα) induces apoptosis in human epidermoid cancer cells. tissue transglutaminase (ttgase) is an enzyme involved in the regulation of apoptosis through the inactivation of some cell components. among these eukaryotic initiation factor- a (eif a) is peculiar because its activity is modulated by the formation of the amino acid hypusine. recently, we found that growth inhibition induced by ttgase is paralleled by reduced hypusine levels. here we report the effects of ifnα on the apoptosis, ttgase modulation and eif a activity in human epidermoid lung h cancer cells. we have found that h exposure to , iu/ml ifnα induces % growth inhibition and % apoptosis in h cells. moreover, ifnα induced a -fold increase of ttgase activity and expression that already occurred after h of exposure to the cytokine. this effect was paralleled by a . -fold enhance of ttgase mrnas. ifnα induced also a % increased eif a expression while an about % decrease of hypusine levels was observed. increased ttgase activity was paralleled by a decrease of hypusine content and of eif a activity. therefore, ifnαinduced apoptosis could occur through an increase of ttgase activity and the mechanism by which ttgase regulates biological functions can be the reduction of eif a activity. adometdc deficient mice k. nishimura , f. nakatsu , , k. kashiwagi , h. ohno , t. saito , and k. igarashi graduate school of pharmaceutical sciences, chiba university, chiba, graduate school of medicine, chiba university, chiba, and cancer research institute, kanazawa university, kanazawa, japan the amd gene encodes s-adenosylmethionine decarboxylase (adometdc) that is one of the key enzymes of polyamine biosynthesis. to examine the physiological role of polyamines, we performed the targeted disruption of the gene in mice to generate spermidine-and spermine-free mice. although the level of adometdc mrna decreased by % in amd ϩ/Ϫ mice, adometdc activity reduced only by % and spermidine and spermine contents did not change significantly. they showed normal phenotype and life span. to obtain amd Ϫ/Ϫ mice, we intercrossed amd ϩ/Ϫ mice and determined the genotype of the resulting offspring. however, we could not obtain any amd Ϫ/Ϫ mice from heterozygous intercrosses over. amd Ϫ/Ϫ embryos died early in development, between e . and e . days post coitum. in culture of blastocysts at e . , the shapes of all cell lines were normal, but amd Ϫ/Ϫ cells appeared to arrest the cell proliferation at day after the onset of cell culture. the arrest of amd Ϫ/Ϫ cell proliferation was rescued by addition of spermidine. these data indicated that the lethal phenotype of amd Ϫ/Ϫ mice was caused by growth retardation by polyamine depletion at early developmental stage. the formation of active species such as h o and aldehydes during the oxidative deamination of biogenic amines by amine oxidases (ao) suggests for these enzymes a key role in cellular processes. the ability of bovine serum amine oxidase (bsao) to oxidase free amino groups of lysozyme and ribonuclease a has been observed indicating a possible ao involvement in the post-translational protein modification. furthermore, bsao inhibition by h o formed during substrate oxidation under limited turnover conditions was demonstrated, which may be relevant to cellular physiopathology. we have also observed that some inhibitors of mitochondrial amine oxidases (mao) protected human melanoma cell line (m ) against apoptosis. the protection by catalase of mao-substrates induced membrane permeability transition was also obtained in isolated rat liver mitochondria, thus confirming a role of mao-derived h o in apoptosis. enrichment in ao activity by treatment with vegetal ao has been obtained in a erythroleukemia cell line (k ), substaining the possibility to modulate the intracellular ao activity. an antiarrhythmic and cardioprotective effect of bsao has been also observed on isolated rat heart in reperfusion; a protective effect during anaphylaptic crisis has been shown "in vivo", thus suggesting aos as a possible therapeutic agents. tetrakis( -aminopropyl)ammonium, a unique polyamine produced by an extreme thermophile, stabilizes nucleic acids at high temperature t. oshima and y. terui department of molecular biology, tokyo university of pharmacy and life science, hachioji, tokyo, japan an extreme thermophile, thermus thermophilus, produces tetrakis( -aminopropyl)ammonium; a novel polyamine containing a quaternary ammonium nitrogen. to clarify the roles of the unique polyamine in thermophily, the effects of tetrakis( aminopropyl)ammonium on biochemical reactions related to nucleic acids have been investigated. the unique polyamine stabilized both double and single stranded dnas and rnas. tm of a double stranded dna was raised by °c by the addition of . mm of tetrakis( -aminopropyl)ammonium. at around the boiling temperature of water, depurination of dna takes place. other long polyamines produced by the thermophile such as caldopentaamine also stabilized dnas and rnas. we found that tetrakis( -aminopropyl)ammonium prevents depurination most effectively. tetrakis( aminopropyl)ammonium activated the protein biosynthesis catalyzed by a cell-free extract of the thermophile at high temperature. the effects of this unique polyamine on dna and rna polymerases are also being investigated and the results will be presented. tissue transglutaminase (ttg) catalyses the cross-link formation between glutamine (q) residues and nh -donor molecules present in the cells (polyamines, lysine-donor proteins). recently, it has been correlated to neurodegenerative disorders characterised by polyglutamine (q n ) expansion, like huntington's disease. studies carried out on cell extracts revealed that glyceraldehyde -phosphate dehydrogenase (gapdh) was found covalently linked to q n domains. however, to date no structural data are available to solve the issue of which residues of gapdh are substrates for ttg. by coupling classical protein chemistry procedures and mass spectrometric techniques we achieved this goal by using as ttg substrates the substance p, an -aa peptide bearing the simplest q n domain (q ), and polyamines of different size and shape as q-and nh -donor, respectively. in the present study we report that out of the lysines present in gapdh only three are sites of ttgasedependent cross-link formation in vitro. moreover, to characterize the ttg catalysed cross-link between gapdh and polyq protein we used a synthetic q -peptide as ttg substrate in the catalysed reaction with polyamines. we found that any q residue is a potential ttg substrate, no matter the specific position in the sequence or the steric hindrance of the specific amine under investigation. cjf inserm - , institut contre les cancers de l'apareil digestif (ircad), strasbourg, france as soon as the key role of odc in polyamine metabolism was recognised, it became the major target for selective inhibi-tion. s. harik presented in the first potent odc inhibitor, α-hydrazino ornithine. although efforts continued until today, with the aim to improve odc inactivation, -(difluoromethyl)ornithine (dfmo) remained the most important compound among all polyamine-directed drugs. a known anti-leukaemic drug, methylglyoxal-bis(guanylhdrazone), was recognised early on by g. williams-ashman and his collaborators as an inhibitor of adometdc, the other highly regulated biosynthetic decarboxylase, and served as matrix for more recent developments. in the course of the years selective inhibitors for all enzymes involved in polyamine biosynthesis and degradation were synthesised. moreover, a series of polyamineuptake inhibitors were reported. however, only some of these numerous compounds reached a stage above evaluation as growth inhibitors of cancer cells. owing to the sophisticated homeostatic regulation of the polyamines in cells and organs by de novo synthesis, degradation, uptake and release, and due to the fact that exogenous polyamines (i.e. gut polyamines) can be utilised by the vertebrate organism, the efficacy of selective enzyme and uptake inhibitors remained modest in cancer therapy. the fact the dfmo became the most important drug for the therapy of west and central african sleeping sickness relies on differences of vertebrate and parasite biochemistry. a novel approach, initiated by carl porter, involved the design and synthesis of structural analogues of spermidine and spermine, which do not share the growth-promoting effects of the natural polyamines. a very large variety of homologues, mostly of spermine, with different alkyl-substituents on the primary amino groups, have been studied systematically with regard to their ability to alter enzyme and polyamine patterns, and to inhibit cell growth. in addition polymine-like chains with interposed heteroatoms ( , s, si etc.), and analogues with rigid aliphatic chains (due to inbuilt double and triple bonds, or of small rings) have been explored. the structural analogues either mimic regulatory functions of the natural polyamines, and thus lead to the depletion of endogenous pools of putrescine, spermidine and of spermine, or they prevent growth effects of the natural polyamines by displacing them from functionally important binding sites. the later type may be considered as polyamine antagonists. the actual drugs usually exhibit to some extent polyamine mimetic and antagonist properties. at present several polyamine analogues are in clinical trial. however, after more than years of active research, a polyaminerelated anticancer drug is still not available. one may conclude from this fact that the polyamines are an inappropriate target for cancer treatment. however, it is more likely that polyamine metabolism is a difficult target, because the differences between normal and cancer cells are mainly of quantitative nature. moreover, numerous mechanisms have developed in the course of evolution, which enable the vertebrate organism to prevent lethal polyamine losses. nine novel chemically modified polyamine (pa) analogs were evaluated for their ability to inhibit the pa biosynthesis in rat hepatoma g- cell-free system as well as the growth of caov tumor cells. the final concentration of oxy-and aminoadenosine pa analogs or two uracils modified pa analogs were . mm in the reaction mixture. bis(uracilyl)-analogs and -( -oxyethyl)ami-no- --d-xylofuranosyladenine supressed pa and putrescine synthesis and in the same conditions were more effective than dl-α-difluoromethylornithine (dfmo) -strong specific inhibitor of ornithine decarboxylase (odc). the other adenosine modified compounds could act both as activators of odc and inhibitors both diamine and polyamine oxidase activities in regenerating liver test system. in contrast to those mentioned above two uracils modified agents as well as dfmo were able to inhibit odc and to increase the rate of oxidative deamination of pa in the same system. thus bis(uracilyl) pa analogs were the most active and may be useful for further investigation as substances having potential antitumor and antiproliferative properties. several studies concerning the periodontal status in adult and adolescent patients treated with fixed ni-ti archwires have been performed, but until now it is not yet available any information about the influence of patient age on gingival tissue responses to ni-ti alloy. recently, researches by us demonstrated that the prolonged use for over months of ni-ti appliances may contribute to local pathological proliferative processes early detectable only through salivary polyamine concentration increase. although other data from our laboratory showed that salivary polyamine amounts are age and sex-independent, nothing is known about the influence of the age on salivary polyamine content m subjects wearing ni-ti appliances. eighty patients, under orthodontic treatment for months, were divided into four groups: the pre-, the mid-, the late-and the post-pubertal. salivary polyamine concentrations were determined by hplc. only the late pubertal group revealed a significant increase in both the spermine and spermidine content, while the other groups showed no modification. the results suggest that gingival pathological responses to a long-term appliance's use may be related to the endocrine modifications that occur in the late-pubertal age. sexual hormones appear to be in synergy with ni-ti alloy in promoting proliferative activity of gingival cells. the effects of polyamines on the synthesis of various σ subunits of rna polymerase were studied to determine how polyamines influence the functional specificity of transcription using western blot analysis. synthesis of σ was stimulated . -fold and that of σ was stimulated . -fold by polyamines, whereas synthesis of other σ subunits was not influenced by polyamines. stimulation of σ synthesis by polyamines occurred at the level of transcription. since our hypothesis is that polyamines regulate macromolecular synthesis mainly at the translational level, we searched for a target protein, related to the polyamine stimulation of σ synthesis, whose translation is altered by polyamines. stimulation of σ synthesis was due to an increase in the level of camp, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. polyamines were found to increase the translation of adenylate cyclase mrna by facilitating the uug codon-dependent initiation. analysis of rna secondary structure suggests that exposure of the shine-dalgarno (sd) sequence of mrna is a prerequisite for polyamine stimulation of the uug codon-dependent initiation. antitumor quinones are approved for clinical use and others antitumor quinones are in different stages of clinical and preclinical development. the efficiency of the quinonic compounds in inhibiting cancer cells growth is believed to stem from their participation in key cellular redox mechanisms with consequent generation of highly reactive oxygen species (ros). the ros is turn modify and degrade nucleic acids and proteins within the cells. recently, quinonic drugs were attached to the neurodecapeptide lh-rh and evaluated as potential drugs in the treatment of different tumours. we have synthesized several series of n-quinonyl amino acids in which five ω-amino acids are attached to p-quinones with different values of redox potentials. the attachment was made via michael-like reductive addition of the amino acids to the quinonic ring or via substitution of a chlorinated atom. the n-ω-quinonyl amino acids were characterized as to their ability to form semiquinone anion radicals by epr and cyclic voltammetry technique. the preparative methods, the redox potentials as well as the physical and spectral data ( h-nmr, ir, uv-vis and hrms) of these n-ω-quinonyl amino acids will be presented. the de novo design of biologically active peptides and proteins, mostly has involved consideration and design of backbone conformations (secondary structures) such as α-helix, -sheets, -turns, etc. (η/ψ space). however, for many bioactive peptides and proteins, especially those critical for information transduction such as neurotransmitters, hormones, antigens, neurocrines, etc. molecular recognition via side chain moieties is of paramount importance. thus far, the specific three dimensional orientations of side chain groups ( angles; chi space) in terms of biological activity has received only modest attention. in part this may be due to the energetics of chi space compared to ramachandran space. in order to overcome the current limitations of evaluating the importance of chi space in critical biological functions related to disease and behavior, we have designed amino acids with novel structures and unique constraints in chi space ( , , etc.), with special attention to their ability to mimic the chi space of native proteins and peptides. we have developed novel and simple asymmetric synthetic methods for such amino acids, often with ees greater than %. incorporation of these novel amino acids into bioactive polypeptide neurotransmitters has provided ligands with unique biological activities that effect unique behaviors including feeding, sexual, pain, and addictive behaviors. (supported by grants from the usphs and nida.) protein technology, wallenberg laboratory ii, lund university, lund, sweden we describe a method for comparative quantitation and de novo peptide sequencing of proteins separated either by standard chromatographic methods or by one and two-dimensional polyacrylamide gel electrophoresis. the approach is based on the use of an isotopically labelled reagent to quantitate (by mass spectrometry) the ratio of peptides from digests of a protein being expressed under different conditions. the method allows quantitation of the changes occurring in spots or bands that contain more than one protein, and has a greater dynamic range than most staining methods. since the reagent carries a fixed positive charge under acidic conditions and labels only the n-terminal of peptides, the interpretation of tandem mass spectra to obtain sequence information is greatly simplified. the sequences can easily be extracted for homology searches instead of using indirect mass spectral based searches and are independent of post-translational modifications. dehydroamino acids and their derivatives play important roles as constituents of various natural products and as synthetic intermediates for the preparation of optically pure amino acids. a large number of amino acid derivatives containing a pyrazol- -yl, isoxazol- -yl and other heterocyclic moieties has been prepared as potential agonists or antagonists for central glutamate receptors in connection with (r,s)- -amino- -( hydroxy- -methylisoxazol- -yl)propanoic acid (ampa), a bioisostere of (s)-glutamic acid. -hetaryl-α, -didehydroalanines might be considered as conformationally constrained ampa analogs and might be potential candidates for the synthesis of novel types of ampa analogs, for example, via their hydrogenation. compounds containing h-pyran- -one ring are also very useful synthons in selective synthesis. recently we have shown their use for the preparation of (e)-α, -didehydroα-amino acid derivatives containing a pyrazolyl moiety (vraničar l, polanc s, kočevar m ( ) tetrahedron : ). as a continuation of our investigation in this field we report here a detailed study of the transformation of h-pyran- -one derivatives with hydroxylamine ( , x ϭ o) and various hydrazines ( , x ϭ nr ) towards novel types of (e)-and (z)α, -didehydroamino acid derivatives . in most cases, the reactions were performed under basic conditions in a mixture of ethanol and pyridine. depending on the substrate and the reagent used the reaction could be controlled to give either (e)-or (z)-isomers; in some cases decarboxylation to the corresponding enamines also occured during the reaction course. some attempts to hydrogenate compounds towards α-amino acid derivaties by homogeneous or heterogeneous catalysis were also performed. analogs of endomorphin and were prepared to investigate the effect of the positional and c-terminal amide replacements and modifications on the biological activity. modifications in position and were studied. in position several hydroxy-and serine related amino acids were incorporated, whereas in position the amide bond was replaced by hydroxymethyl and allyl group. protected peptide derivatives were synthesized on chlorotrityl resin and further transformed to the corresponding derivatives in solution phase. among the analogs tested, in in vitro tests the most effective compound found was d-ser -endomorphin Ϫ . quite surprisingly, the partial agonist/antagonist properties of the derivatives in receptor binding and g-protein stimulation tests have been shown behave differently. the differences in efficacy and receptor binding properties of the compounds may explain the discrepancies between the in vitro and receptor binding tests. we have been assessing the possible applications of substituted h-pyran- -ones containing α, -didehydroamino acid unit in their structure as dienes in [ ϩ ]-cycloaddition reactions. as dienophiles we have been using different acetylene derivatives as well as n-phenylmaleimide and maleic anhydride. as it is evident from the structure of h-pyran- -ones upon the cycloaddition of acetylene derivatives the first intermediate formed ( ) still contains the carbon dioxide bridge. in many cases easily expels co and substituted benzene derivative is produced. when the alkenes are used, the first part of cycloaddition is the same as when acteylene derivatives are used, but after the extrusion of co from the adduct there are two possible paths: so formed cyclohexa- , -diene ( ) is either aromatized into benzene derivative ( ) or it acts as another diene with favourably positioned double bonds and unusual double cycloadducts ( ) are formed. since co -containing adducts are thermally unstable it is advantageous to use high pressure techniques. with the acetylene derivatives we have not been able to isolate co -containing adducts ( ), while with alkenes we have isolated, depending on the structure pattern of the compound , all three types of products: aromatized , co containing and double adducts . especially the type is suitable for further transformations into other heterocycles containing amino acid moiety. research group of peptide chemistry, hungarian academy of sciences, budapest, hungary among the opioid receptors family, the cloning of µ, k and δ receptors was followed by another member, named lc or orl . searching for an endogenous ligand for this receptor resulted in successful identification of a peptide (fggft garksarklanq) called noc or ofq. in vitro and in vivo studies have demonstrated that noc mediates a variety of biological actions. results from structure-activity experiments suggest that the whole sequence of noc is not required for binding to the lc receptor and for full biological activities. noc( - )-oh seem to be the minimum and essential sequence for good interaction with the receptor. this neuropeptide, similarly other peptides, are unresisting for enzymatic degradation and the releasing metabolites are very weakly active or inactive. some previous experiments refer to that the c-terminal amidation may protect the peptide from degradation. we purposed to synthesize carbamoyl analogues of noc( - )-nh , hoping that these derivatives retain the ability to bind lc receptor and are resistant against biological degradation: phe-nh-co--ala-noc( - )-nh phe--ala-nh-co-phe-noc( - )-nh phe-gly-nh-co--hphe-noc( - )-nh the first step in the synthesis of the carbamoyl analogues was the preparation of the building block [r-co-nh-co-nh-hc(rЈ)-cooh] by the classical method and then it was incorporated into the peptide by solid phase peptide synthesis. [ nonproteinogenic amino acids and their derivatives are valuable compounds from their pharmacological and biochemical effects. they can be used also in synthesis of peptides, as biomarkers, as the ligands in catalitically active transition metal complexes and so on. it is possible to prepare such amino acids by asymmetric hydrogenation of their prochiral precursors. however high enantioselektivities was reached only in the case of chiral phosphine-rhodium catalysts. recently we showed that high diastereoselectivity in the hydrogenation of linear dehydrodipeptides may be achieved over achiral catalyst in the catalytic system substrate -salts of ca ore mg -pd/c due to formation of dehydrodipeptides complexes with ions Ñà ϩ or mg ϩ and hence increasing of the conformational rigidity of substrates. this phenomenon may as well happen in other dehydrodipeptides, containing nonproteinogenic amino acids. among unnatural amino acids those bearing heterocyclic rings have attracted considerable attention due to the possibility of the heteroatoms participation in coordination with ions of metals. we have received some n-acyldehydrodipeptides, containing in the prochiral unit of dipeptides nonproteinogenic dehydroamino acids. all this n-acyldehydrodipeptides form in alcohol solution complexes with cax and mgx where one metal ion binds together several (up to ) substrate molecules. this kind of complexation leads to the increase of conformational rigidity and to the diastereoface shielding of cϭc bond. moreover the combination of cations (ca ϩ or mg ϩ ) and anions (x) and the sequence of their mixing with a substrate determine the assembly inside complex particles and hence the sign and degree of asymmetric induction. indeed hydrogenation of these complexes formed in situ over achiral heterogeneous catalyst (pd/c) gives two diastereomers of corresponding n-acyldipeptides with the substantial increase of the reaction diastereoselectivity (up to %). in living cells, glutamine represents one of the main storage forms of nitrogen and is a major physiological source of ammonia for the biosynthesis of aminoacids, aminosugars, purine and pyrimidine nucleotides and coenzymes. glutamine-dependent amidotransferases perform nitrogen transfer from the amide group of glutamine to various electrophiles. when the latter is fructose- p, the product of the reaction catalysed by glucosamine- p synthase is d-glucosamine -phosphate, a structural building block of peptidoglycane (bacteria) and of chitin and mannoproteins (fungi). fluorinated analogues of glutamine are expected to interfere with this biological process due to the strong electron withdrawing effect of fluorine atom (without significant steric consequence), inducing modulation of binding and/or electronic properties. these compounds might therefore behave as reversible or irreversible active site-directed enzyme inhibitors. synthesis of optically active from d-serine will be described and first results in the biological evaluation on glucosamine -phosphate synthase will be included. o. melnyk , d. bonnet , e. loing , l. bourel , and h. gras-masse -umr , -sedac-therapeutics, biological institute of lille, france lipopeptides, owing to their ability to cross passively the cell membrane or biological barriers, are unique tools for the intracellular delivery of bioactive peptides. the structure of the lipophilic moiety is known to have a profound effect upon the interaction with the membrane and its alteration. the stepwise solid phase synthesis of lipopeptides is limited by the necessity to perform a complex rp-hplc purification following the cleavage and deprotection step. in addition, the harsh conditions used during the final acidolysis procedure does not allow the introduction of unsaturated or sensitive fatty acids. to speed up the access to large lipopeptides modified by various fatty acid moieties or cholesterol derivatives, we have designed novel synthetic methods which involve the chemoselective reaction of fully deprotected and purified hydrazinopeptides with fatty acid succinimidyl esters or glyoxylyl derivatives. application of these methodologies to the c-terminal - portion of interferon (ifn)-γ allowed the selection of the optimal lipopeptide ifn-γ agonist, as determined by its ability to induce the expression of surface mhc-ii molecules through interaction with the intracellular components of ifn-γ receptor. graduate school of science, osaka city university, osaka, japan glutamate receptors in mammalian cns are implicated in the construction of memory and early learning as well as in the pathogenesis of neuron damage to cause various neuronal diseases. in recent years, we have studied the conformational role of l-glutamate when it binds to the receptors through the synthesis of l- -(carboxycyclopropyl)glycines (ccgs) and their related analogs. the works have demonstrated that not only the receptors require a specific conformation of l-glutamate, but also these analogs can be used as important tools for the neuropharmacological research. among them, dcg-iv, a Јsubstituted analog of ccg-i, is used as a potent and selective agonist of mglur . as an extension of these works, next program was focused on the synthesis of α-substituted glutamate analogs which would enable to develop potent and subtype-selective ligands for mglurs and transporters. α-alkoxymethylglutamate and ly and its c epimer were chosen for the synthetic targets, since the former slightly restricts the glutamate conformation to an extended form and the latter rigidly fix to an extended or a folded form on its bicyclo[ , , ]hexane skeleton. the key to the synthesis was a stereoselective construction of the quarternary carbon center, which was efficiently performed based on an asymmetric version of the strecker synthesis. details of the synthesis and their neuropharmacological activities will be described. using a genetically modified organism a broad variety of linear unsaturated amino acids are now accessible in enantiomerically pure form via this methodology, which can be used as starting materials for the synthesis of highly functionalized pipecolic acid derivatives. these compounds can be used to restrict conformations in polypeptides or can serve as scaffolds in synthesizing libraries for drug discovery. the synthetic approach involved both a pd-catalyzed amidopalladation reaction of alkoxy-allenes, in which the nh is added across one allene double bond and a ruthenium catalyzed ring closing metathesis step, to form a benzyloxypipecolic acid. further reaction of this n-sulfonyl-iminium-ion precursor with a nucleophile results in the formation of cis-substituted pipecolic acids. due to the unique electronic properties of fluorine, incorporation of α-fluoroalkylated amino acids is a new approach to design biologically active peptides with increased metabolic stability and defined secondary structure and provides a powerful nmr label for spectroscopic investigations. the application of proteases especially for cn-ligations is an attractive alternative to chemical methods, because the enzymatic formation of peptide bonds is highly regio-and stereospecific and, therefore, does not require large efforts to protect side chains of trifunctional amino acids. recently, the enzyme-catalyzed incorporation of α-fluoromethyl amino acids into the p , p and p Ј-position (nomenclature according to schechter and berger) of peptide fragments has been successfully performed. carboxypeptidase y was now shown to be suitable to catalyze the incorporation of α-trifluoromethyl alanine into the p position of peptides. furthermore, the general applicability of the substrate mimetic concept in enzymatic peptide synthesis was expanded to the transfer of c-terminal α-fluoroalkyl substituted amino acids. generally, each trifluoromethyl-and difluoromethyl amino acid guanidinophenyl esters can be applied as acyl donor in trypsin and chymotrypsin catalyzed peptide bond formation independently of the acyl moiety and the natural enzyme specificity, respectively. via these two approaches, incorporation of αfluoroalkylated amino acids into the p position of peptides using enzymatic methods was successfully applied for the first time. this investigation was performed in search of new Јdeoxynucleoside analogues modified at Ј-and Ј-positions with amino acids and possessing antiviral activity. substrate mimetics strategy: an efficient approach to protease-catalyzed peptide ligation n. wehofsky and f. bordusa , max-planck-society, research unit "enzymology of protein folding", halle, and institute of biochemistry, university of leipzig, germany two main drawbacks seriously restrict the synthetic value of proteases as reagents in peptide fragment coupling: ( ) native proteolytic activity and, thus, risk of undesired peptide cleavage; (ii) limited enzyme specificities restricting the amino acid residues between which a peptide bond can be formed. the latter can be overcome by the use of substrate mimetics. contrary to common acyl donors, substrate mimetics bear a binding site specific ester leaving group instead of having a specific amino acid moiety at the c-terminus of the acyl residue. this replacement mediates the acylation of the protease by nonspecific acyl residues. deacylation of the artificial acyl enzyme intermediate by the amino component added results in peptide bond formation regardless of the primary specificity of proteases enabling nonspecific coded and noncoded amino acid derivatives and even non-amino acid-derived acyl moieties to be coupled. the successful application of these artificial substrates for model peptide ligations catalyzed by the argspecific trypsin, the glu-specific staphylococcus aureus strain v protease (v protease), and α-chymotrypsin, which is specific for aromatic amino acid moieties, will be demonstrated. new development in the tritium labelling of peptides and proteins using solid state catalytic isotopic exchange with spillover-tritium yu. a. zolotarev , a. k. dadayan , b. v. vaskovsky , and n. f. myasoedov institute of molecular genetics, russian academy of sciences, and shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia the reaction of high temperature solid state catalytic isotope exchange (hscie) of hydrogen in peptides and proteins with spillover-tritium was studied. the reaction ability of amino fragments in hscie was shown to depend both of their structure and on the availability and the mobility of the polypeptide chain. [ h] peptide analysis using h nmr spectroscopy was carried out, and the modified fragment [ h]actc - (met-glu-his-phe-gly-pro), with molar activity of ci/mmol and [ h] zervamicin iib (ac-trp-ile-gln-iva-ile-trh-aib-leu-aib-leu-hyp-gln-aib-hip-aib-pro-phl, where aib ϭ amino-isobutyric acid) with molar activity of ci/mmol was produced. the obtained preparations completely retained their biological activity. with the -galactosidase protein from termoanaerobacter ethanolicus as example, the interrelation between the protein's tertiary structure and the isotopic label distribution incorporated due to the hscie reaction was used. the labeled protein with the molecular mass of kda was brought to fragmentation by glu-proteinase. peptide fragments were separated by hplc and were identified by maldi mass spectrometry. a correlation between the position of the amino acid fragment in the protein tertiary structure and its reaction ability in the hscie reaction was obtained. data on the retention of thegalactosidase enzymatic activity in condition of tritium label introduction are supplied. taurine chloramine modulates cytokine production by peripheral blood mononuclear cells m. chorą z . y , e. kontny , j. marcinkiewicz , and w. maśliń ski institute of rheumatology, warsaw, and jagiellonian university, cracow, poland objective. proinflammatory cytokines are produced in a cascade fashion, where monocyte-derived tnfα and il- trigger production of il- and il- also in the other cell types. we reported recently that taurine chloramine (tau-cl) inhibits production of the latter cytokines in fibroblast-like synoviocytes. in present study the effect of taurine (tau) and tau-cl on tnfα, il- and il- production was examined. methods. peripheral blood mononuclear cells from healthy volunteers were stimulated with lps ( h) in the presence of tau or tau-cl ( - µm). cytokine production was measured in culture supernatants (secreted) and cell lysates (intracellular) using elisa. results. in lps-stimulated cells both secreted and intracellular il- and il- were inhibited by tau-cl with ic Ϸ µm and µm, respectively. however, tau-cl exerted dual effect on tnfα production, raising it slightly ( . times) at low ( - µm) while reducing it (ic Ϸ µm) at higher concentration. tau did not significantly affect cytokine production. tau-cl modulates proinflammatory cytokine cascade and eventually might down-regulate it when present at high (Ͼ µm) concentration. department of biology, division of general physiology, university of oslo, blindern, norway every living cell must deal with osmotic and hydrostatic pressure changes between its environment and its interior and counteract volume changes. swelling activated channels is one group of effectors in the cell membrane that is important in preventing excessive volume increases by releasing inorganic ions and organic solutes that include taurine. such channels are associated with several physiological processes, but little is known about their activation mechanisms. we have used a rat thyroid cell line (frtl- ) to investigate the activation of a swelling sensitive [ h]taurine efflux pathway. hypo-osmolality and thyrotropin (tsh, µm) increased transiently the rate coefficient for [ h]taurine efflux with a similar pattern of activation. the phosphodiesterase inhibitor -isobutyl- -methyl xanthine ( µm) increased the swelling activated efflux rate coefficient . times above the control level and the camp analogue dibutyryl-camp ( µm) activated the pathway. these results indicate that both swelling and tsh activation of the taurine efflux pathway are mediated by camp. other aspects of the signal transduction pathway will be discussed. based on the inclination of n-chloroamines to disproportionate, the endogenous bactericidal agent n-chlorotaurine (nct), mainly at ph Ͻ , is accompanied by n, ndichlorotaurine (ndct). since pure ndct could be synthesized as crystalline sodium salt, a first evaluation of its chemical and bactericidal properties was possible. ndct-na (melting point: - °c, decomp.) is very well soluble in water and poorly in ethanol where it can be recrystallized from. on storage the initial ph of the aqueous solution decreases which correlates with a decrease of oxidation capacity of . % per day, probably originated by the elimination reaction r-ch -ncl ae r-chϭncl ϩ h ϩ ϩ cl Ϫ as a first step. contrary to nct-na an immediate decomposition occurs when ndct-na comes into contact with undiluted dmso. in aqueous solution, however, ndct does not react with dmso. the bactericidal activity of mm ndct at ph and against the gram-positive bacteria s. epidermidiand two strains of s. aureus was the same as with equimolar nct though ndct bears twice the oxidation capacity. against the gramnegative bacteria e. coli, p. aeruginosa, and p. mirabilis, however, a significantly higher activity of ndct was observed at both ph. the mechanism of taurine chloramine inhibition of fibroblastlike synoviocytes growth e. kontny , m. kurowska , j. kowalczewski , i. janicka , j. marcinkiewicz , and w. maśliń ski institute of rheumatology, warsaw, and jagiellonian university, cracow, poland objective. in rheumatoid arthritis (ra) enhanced proliferation of fibroblast-like synoviocytes (fls) leads to hyperplasia of synovial membrane (sm). therapeutic approaches to inhibit an excessive growth of these cells are not satisfactory. thus, we investigated the effect of taurine (tau) or taurine chloramine (tau-cl) on ra fls growth. methods. fls isolated from sm of ra patients were stimulated for hours with rhpdgf or rhtnf-α. tau or tau-cl were added at - µm concentrations. cell proliferation was determined by incorporation of h-thymidine into dna. expression of proteins regulating cell-cycle progression or apoptosis, was estimated by western blotting. results. at µm concentration tau-cl inhibited (by Ϸ %) both pdgf-and tnf-α-triggered cell proliferation and similarly reduced expression of pcna (a cofactor for dna polimerase δ). however, tau-cl affected neither the expression of cell-cycle inhibitors (p , p ) nor anti-apoptotic bcl- protein. tau has no effect on tested responses. conclusion. we report that tau-cl inhibits proliferation of ra fls by affecting expression of pcna, that is critical for cell cycle progression. e. kontny , k. szczepań ska , j. kowalczewski , m. kurowska , i. janicka , j. marcinkiewicz , and w. maśliń ski institute of rheumatology, warsaw, and jagiellonian university, cracow, poland objective. proinflammatory cytokines play critical role in the pathogenesis of rheumatoid arthritis (ra). we reported recently that taurine chloramine (tau-cl), but not taurine (tau), inhibits production of il- and il- by fibroblast-like synoviocytes (fls). in present study the mechanism of tau-cl inhibitory action was investigated. methods. fls isolated from synovial membrane of ra patients were stimulated with rhil- . tau or tau-cl were added at - µm concentration. after . - h or h the dna binding activity of nfkb and ap- (emsa) and the expression of il- and il- mrnas (rt-pcr) was examined, respectively. results. il- raised nfkb and ap- activity, followed by the elevation of cytokine mrnas expression. tau-cl, but not tau, reduced both the expression of cytokine mrnas (il- Ͼ il- ) and the activity of transcription factors (nfkb Ͼ ap- ). conclusion. tau-cl inhibits transcription of il- and il- genes due to its ability to diminish the activity of key transcriptional factors, that regulate these proinflammatory cytokine expression. institut für organische chemie, universität bremen, germany the synthesis of taurine and hypotaurine from cysteine can be followed up in astroglia-rich primary cultures obtained from brain of neonatal wistar rats. using h and c nuclear magnetic resonance spectroscopy cell extracts of glia cells incubated with c labelled cystein show the label subsequently in hypotaurine, taurine, lactate and gluthathione. within h, % of the total intracellular hypotaurine and . % of taurine were newly synthesized from cysteine. both metabolites were also released to the medium. neurones are capable to take up both metabolites from glia media to recruit their organic osmolite. part of newly synthesized glutathione and lactate are also exported to the medium. by this means lactate may serve as an energy substrate for neurons. in-vivo mrs of lactate is obscured by line splitting and signal overlay. using various two dimensional pulse sequences as spin preparation sequences prior to localized single voxel, in-vivo mrs or spectroscopic imaging sequences will provide homonuclear non-coupled resonance signal of taurine. these singlet signals are detectable and quantified. diffusion weighted spectroscopy is used to characterize the mobility of taurine in living tissue. department of pharmacology and ophthalmology and visual sciences, texas tech university health sciences center, lubbock, texas, u.s.a. taurine depletion, whether by removing taurine from the diet or by using a taurine transport inhibitor, has demonstrated various pathologies in various animal models including man. the first reported pathology associated with dietary taurine depletion was in the retina of the cat. in this animal model, taurine deficiency resulted in disorganization of the tapetum (the light reflecting membrane), disruption of the outer segments, photoreceptor dysfunction, and cell loss. when allowed to proceed for a number of months the result was blindness. subsequent studies demonstrated that taurine deficiency also had a profound effect on cardiac physiology. echocardiograms of the left ventricle of the cat heart depleted in taurine showed a dilated cardiomyopathy reflected in an extended end-diastolic diameter and an extended end-systolic diameter. dietary taurine supplementation resulted in the above parameters returning to normal. the cat is a difficult animal model to use for a variety of reasons and thus the rat was chosen to further probe the consequences of taurine depletion. unfortunately, the tissue taurine levels in the rat do not respond to dietary taurine depletion, and thus other experimental means had to be designed. guanidinoethanesulfonic acid (ges), an analogue of taurine and a taurine transport inhibitor, has been utilized for the last years to deplete rat tissues of their taurine content (j. e. shaffer and j. j. kocsis, methods of reducing tissue taurine levels, and r. j. huxtable, h. e. laird, and s. lippincott, rapid depletion of tissue taurine content by guanidinoethyl sulfonate. in: the effects of taurine on excitable tissues, spectrum publications, new york, ) . ges, when administered to rats in their diet in the drinking water as a - . % solution, usually produces a significant decrease in the taurine content of all tissues within one week of treatment. within - weeks the levels of taurine reach their nadir ( - % of control) and continued feeding of ges does not further reduce the levels of taurine. unfortunately, ges replaces taurine and thus one must always consider the effects of ges on physiological events that occur within the tissues in question. again, as in the cat, taurine depletion manifested itself in retinal pathology: disruption of the photoreceptor structure, dissociation of the disc membranes, and abnormal electroretinograms (erg). other animals models such as the monkey have also demonstrated structural disorganization of the photoreceptors and abnormal ergs. finally, the ultimate test is whether taurine deficiency has an effect in man. in , koppel and associates (geggel et al., n. eng. j. med. : - , ) demonstrated that children on long term parenteral nutrition devoid of taurine had abnormal erg. supplementation of the parenteral nutrition with taurine restored the ergs to normal in the majority of the children. because of these definitive studies, all infant formulae in the united states, europe and japan now contain taurine. (supported in part by a grant from the taisho pharmaceutical co., ltd., tokyo, japan.) department of pharmacology and ophthalmology and visual sciences, texas tech university health sciences center, lubbock, texas, u.s.a. it has been demonstrated previously in our laboratory that taurine inhibits the phosphorylation of an ϳ kdalton protein present in the mitochondrial fraction of the rat heart (j. mol. cell. cardiol. : - , . upon administering . % guanidinoethanesulfonic acid (ges) in the drinking water of rats for weeks, the taurine levels in cardiac tissue decline by %. however, the phosphorylation of a ϳ kdalton protein in the mitochondrial fraction of the heart tissue increased by % (j. mol. cell. cardiol. : - , ) . reversal of these effects could be accomplished by feeding the rat . % taurine in the drinking water for weeks. the ϳ kdalton protein was isolated by -dimensional polyacrylamide gel electrophoresis (page) using traditional glycine buffers followed by re-electrophoresing the cut out portion of the gel, which corresponds to the ϳ kdalton protein, on a tricine-buffered gel resulting in sufficient pure protein for digestion and sequence analysis. it was determined that the ϳ kdalton was pyruvate dehydrogenase (amino acids : - , ) which indicates a significant regulatory role for taurine in energy metabolism in cardiac tissue. these data are of significant interest in that taurine may be an additional effector of this enzyme or of the enzyme complex. studies are in progress to determine if taurine has a direct effect on either the kinase (inhibition) or the phosphatase (stimulation) associated with the pyruvate dehydrogenase complex. it has also been demonstrated and now reported that taurine depletion utilizing ges in vivo in rats affects the phosphorylation of myelin basic protein (mbp). in these experiments the animals were given ges ( %) for weeks in their water and then killed; the hearts were removed and homogenized. the homogenate was then incubated with buffer containing mbp ( ì) and radioactive atp for minutes. animals were also treated with taurine ( %) in their drinking water for - weeks or treated with taurine following the ges treatment. page of the incubation mixture, autoradiography on the dried gel, and densitometry of the mbp band gave the following results: relative % activity Ϯ sem (normalized to mg protein) control Ϯ (n ϭ ) ges-treated Ϯ (n ϭ ) p Ͻ . (paired -test) control Ϯ (n ϭ ) taurine-treated Ϯ (n ϭ ) p Ͼ . control Ϯ (n ϭ ) ges followed by taurine Ϯ (n ϭ ) p Ͼ . these data confirm previous reports that it is easier to deplete animals of their cardiac taurine content than it is to raise the levels of taurine. these data on the effects of taurine depletion (increase in mapkinase activity) and taurine supplementation (no change in mapkinase activity) on mapkinase activity reflect these past observations. (supported in part by a grant from the taisho pharmaceutical co., ltd., tokyo, japan.) act additively, or in the case of mpo negated each other's effects. regarding our results there is significance to pharmacological regimens which enhance the supply of propofol or taurine in whole blood. these regimens influence considerably pmn intracellular amino acid concentrations and it is this pmn "labile free amino acid pool" which may be one of the determinants in cell nutrition positively or adversely affecting pmn immune functions. taurine supplementation to pmn seems to interfere independently from the effects of propofol on pmn free amino acids and on immune functions tested. institut für hygiene, universität innsbruck, austria n-chlorotaurine (nct) is a long-lived oxidant produced by activated human leukocytes during the oxidative burst. it has activity against a broad spectrum of pathogens including bacteria, fungi, viruses and helminths. as a special feature, the killing of microbes by nct can be increased significantly in the presence of ammonium and also of some amino acids (alanine, glycine). this is explained by transfer of the active chlorine ("transhalogenation") from nct to ammonium and amino acids to form the corresponding, stronger microbicidal n-chloro derivatives monochloramine and n-chloro amino acids, respectively. especially addition of ammonium to nct provokes rapid inactivation of fungi and even mycobacteria. because of its good tolerability, nct solution can be applied to human tissue to treat infections. in ammoniumcontaining body fluids like nasal mucus and urine, fungi and bacteria are killed within minutes. therefore, amino compounds of human secretions can be transformed to the above quoted endogenous and highly microbicidal chloramines by nct via transhalogenation -a unique property of an antimicrobial agent. successful treatment in cases of urinary tract and otorhinolaryngological infections and conjunctivitis in phase iia clinical trials provides strong support for this concept. the endogenous sulfonated amino acid taurine has numerous functions in the central nervous system, including positive modulation of gaba a receptor function. recently we found that mice lacking protein kinase c -epsilon (pkcε) are behaviorally and biochemically supersensitive to ethanol and other positive allosteric modulators of the gaba a receptor. in addition, these mice consume - % less ethanol and wildtype controls in two separate self-administration paradigms. microdialysis studies in pkcε-deficient mice revealed elevated extracellular levels of taurine, which may account for the supersensitivity of gaba a receptors in these mice and resulting decreases in ethanol intake. in light of the fact that the taurine derivative acamprosate (calcium acetylhornotaurinate) is moderately effective in reducing craving and relapse in detoxified alcoholics, we examined the effect of taurine-related compounds on acute ethanol consumption in a two-bottle choice paradigm in rats. taurine ( - mg/kg ip) was only slightly effective in reducing ethanol intake but not preference, while the highest dose of taurine ( mg/kg) also suppressed water intake. the taurine precursor hypotaurine ( - mg/kg ip) was also weakly effective in reducing ethanol intake but not preference or water intake. the most effective compound tested was homotaurine ( - mg/kg ip), which suppressed ethanol intake and preference by approximately % without altering water intake. these data indicate that endogenous taurine may regulate sensitivity to ethanol and subsequent ethanol self-administration, and that taurine-related compounds may be effective in reducing alcohol intake in humans. we are currently exploring whether taurine and related compounds are able to suppress ethanol-stimulated mesolimbic dopamine release, a primary neural substrate of ethanol reinforcement. (this work was supported by funds provided by the state of california for medical research on alcohol and substance abuse through the university of california at san francisco.) organic osmolytes, such as taurine, regulate a cell's osmotic balance without directly altering either the cell's ionic composition or the membrane potential. this property of the organic osmolyte often renders the cell resistant to damage during a pathological insult. indeed, ischemia is associated with a massive efflux of taurine from the cell, an event that minimizes the severity of the osmotic imbalance that develops from the accumulation of lactate, inorganic phosphate and sodium. however, taurine depletion also activates specific signaling pathways that provide further protection to the cell. among the signaling pathways activated by taurine depletion is a pi -kinase (phosphatidylinositol -kinase) linked pathway that catalyzes the phosphorylation and inactivation of the pro-apoptotic factor, bad. taurine depletion also activates protein kinase c, which in turn elevates the intracellular content of the antiapoptotic factor, bcl- . increases in the extracellular osmolality by either addition of mm taurine or mm mannitol to the incubation medium activates similar pathways. however, pi kinase assumes a more important role in the mannitol treated cell than the taurine depleted cell. moreover, p map kinase is activated by mannitol treatment but not by taurine depletion. despite these differences, both taurine depletion and mannitol treatment protect the cell against hypoxia-induced apoptosis. the data suggest that osmotic stress protects the cell against apoptosis by increasing cellular levels of bcl- and promoting the inactivation of bad. this work was supported by a grant from the american heart association. a dummy or a protagonist on the stage of inflammation? r&d department, zambon group, bresso, milan, italy amino acids are usually present in large excess in healthy and the excess is used as source of calories. however, metabolic alterations are observed in ill patients and preferential retention of sulphur amino acids (saa) occurs during the inflammatory response. the metabolism of cysteine is modified during the acute phase of sepsis in rats. sulphate production is lower, whereas the higher liver production of taurine seems to play a protective role; glutathione concentration is greater in liver, kidney and other organs and cysteine incorporation into proteins was higher in spleen, lung and plasma (acute phase proteins) while albumin level decreases. another important phenomenon is the impairment of methionine conversion to cysteine during stressed condition. premature infants or hiv patients synthesise cysteine from methionine at a much lower rate. thus, the metabolic flow through the trans-sulphuration pathway may be insufficient to meet the glutathione and cysteine requirement in critical conditions. the pro-inflammatory cytokines, interleukin- , interleukin- and tnf-α are the main initiates that alter protein and amino acid metabolism. in this complex picture, saa supply may contribute to the immune system regulation. department of applied biological chemistry, the university of tokyo, japan the intracellular level of taurine is maintained not only by the taurine transporter that transports extracellular taurine inside cells but also by endogenous synthesis from methionine and cysteine. we therefore investigated the regulation of both the taurine transporter and the cysteine dioxygenase, one of the main taurine biosynthetic enzymes, in hepg human liver cells. the intracellular taurine content of hepg cells was extremely increased by culturing in a hypertonic medium. the activity of taurine transport was increased by hypertonic conditions, which was due to the increased expression of the taurine transporter gene. the expression level of the cysteine dioxygenase gene was also increased, suggesting that the expression levels of both the taurine transporter gene and the cysteine dioxygenase gene were regulated in harmony by hypertonic conditions to accumulate taurine inside cells. on the other hand, the activity of taurine transport in hepg cells was down-regulated on culturing the cells in taurine-rich medium, the expression level of the taurine transporter gene being also markedly decreased. however, the expression level of the cysteine dioxygenase gene was not significantly altered under taurine-rich conditions, indicating that the gene expression of the taurine transporter and that of the cysteine dioxygenase was independently regulated by extracellular concentration of taurine. the amino acid, taurine, is found in very high concentration in the heart. although its most important putative function is osmoregulation, it also serves as a regulator of cell growth. isolated cardiomyocytes exposed to medium containing nm angiotensin ii undergo hypertrophy, a process blocked by mm taurine. the amino acid also inhibits angiotensin iiinduced activation of c-fos, upregulation of atrial natriuretic factor and induction of tgf-betal. central to virtually all of these actions of angiotensin ii is the translocation and activation of key protein kinase c (pkc) isoforms. therefore, we proposed that taurine inhibited the hypertrophic actions of angiotensin ii by interfering with the translocation of one or more of the pkc isoforms. indeed, taurine and angiotensin ii exhibited different effects on the translocation of several pkc isoforms. while taurine promoted the translocation of pkcalpha, pkcdelta and pkcepsilon from the particulate fraction to the cytosol, the levels of the three isoforms in the particulate fraction were elevated following treatment with angiotensin ii. by contrast, both taurine and angiotensin ii increased the pkczeta content of the particulate fraction and the pkcbeta content of the cytosol. when the isolated cardiomyocytes were incubated with medium containing both angiotensin ii and taurine, the effects on pkc distribution were largely additive. these data support the notion that taurine prevents the hypertrophic effects of angiotensin ii by interfering with the translocation of either pkcalpha, pkcdelta, pkcepsilon or a combination of more than one of the isoforms. (the study was supported by a grant from taisho pharmaceutical co.) main final metabolites of l-cysteine in mammals are sulfate and taurine, and they are excreted in the urine. our previous studies in rats have shown that the ratio of urinary sulfate and taurine in rats fed diet containing sufficient methionine and cysteine is : - . in the present study, we determined urinary sulfate and taurine in urine samples of healthy japanese women after h starvation following usual meal. free (inorganic) and total (free ϩ ester) sulfate were determined with ion chromatography, and taurine by reversed-phase hplc after dabsylation. average excretions (micromols per mg of creatinine) were: total sulfate, . Ϯ . ; free sulfate, . Ϯ . ; ester sulfate, . Ϯ . ; taurine, . Ϯ . ; urea, . Ϯ . . the ratio of total sulfate and taurine was : . . this suggests that sulfate formation in humans is more dominant than taurine formation as in rats and this tendency is more evident in humans than in rats, which is in accordance with low cysteinesulfinate decarboxylase activity in humans. sum of sulfate and taurine excretions was significantly correlated with that of urea: correlation coefficient, . . this indicates that sulfur metabolism in humans is in the state of sulfur equilibrium similar to that of nitrogen and reflects protein metabolism. h. yokogoshi and h. oda laboratory of nutritional biochemistry, school of food and nutritional sciences, the university of shizuoka, and department of applied biological sciences, nagoya university, nagoya, japan the effect of taurine on hypercholesterolemia induced by feeding a high-cholesterol (hc) diet to rats was examined. when various amounts of taurine ( . - g/kg) were supplemented to hc for wk, serum total cholesterol gradually and significantly decreased in a dose-dependent manner, compared with the control (cholesterol free) diet group. by contrast, serum hdl-cholesterol was elevated by taurine supplementation. in the hypercholesterolemic rats fed the hc diet, the excretion of fecal bile acids and hepatic cholesterol α-hydroxylase (cyp a ) activity and its mrna level increased significantly, and the supplementation of taurine further enhances these indexes, indicating an increase in cholesterol degradation. agarose gel electrophoresis revealed that, in hypercholesterolemic rats fed the hc diet, the serum level of the heavier vldl increased significantly, but taurine repressed this increase and normalized this pattern. significant correlations were observed between the time-and dose-dependent increases of cyp a gene expression and the decrease of blood cholesterol concentration in rats fed the hc diet supplemented with taurine. these results suggest that the hypocholesterolemic effects of taurine observed in the hypercholesterolemic rats fed the hc diet were mainly due to the enhancement of cholesterol degradation and the excretion of bile acid. in vitro studies have shown that ammonia, which is responsible for neurological symptoms associated with hyperamonemia, causes a massive release of taurine from cultured cns cells and brain slices. in this study, taurine (tau) release was measured in vivo in rat striatum following direct application to the microdialysis tube of mm ammonium chloride which renders the final ammonia concentration in the extracellular space of ϳ mm. various in vivo stimuli evoke taurine efflux either by opening osmosensitive anion channels and/or by a mechanism secondary to glu accumulation and its interaction with nmda or ampa receptors. the following compounds were coadministered with ammonia to distinguish between these mechanisms: anion/cation transport inhibitors -dids and furosemide, a glu transport inhibitor-pdc, and nmda and ampa/ka receptor antagonists dizocilpine and dnqx. ammonia stimulated tau accumulation in the microdialysates to ϳ % of basal value. dids and furosemide moderately inhibited the effect of ammonia (furosemide by ϳ %), albeit dids added alone induced massive accumulation of tau with a delayed onset as compared to ammonia. ammonia-dependent tau accumulation was increased by ϳ % in the presence of pdc and reduced in an equal degree (ϳ %) by dizocilpine and dnqx. none of the agents affected tau accumulation in the absence of ammonia. the results show that ammonia in vivo evokes tau accumulation both via anion channels, possibly secondary to cell volume changes, and in consequence of stimulation of both nmda and ampa/ka receptors. (supp. by a scsr grant no p a and cimo, the acad. of finland) biosciences department, university of hertfordshire, hatfield herts, u.k. the discovery in that endothelium-derived relaxing factor is nitric oxide (no) was followed a year later with reports that the cationic amino acid l-arginine is the physiological precursor for nitric oxide. it has since been established that the terminal guanidinium nitrogen of l-arginine is metabolised via a series of oxidation reactions resulting in no production, with citrulline being formed as a co-product. of interest was the parallel observation that uptake of l-arginine was enhanced in inos expressing cells and that this was due to de novo synthesis of carrier proteins. the precise signaling pathway that regulates the enhanced expression of these carriers has been the subject of intense studies in recent years. current literature suggests that activation of upstream signaling molecules such as protein kinase c may be critical. in addition, downstream kinases thought to be points of convergence for various signals originating from cell surface receptors have also been implicated. two of these downstream targets include the and kda forms of mitogen-activated protein kinase (mapk) and the stressactivated kda mapk. it is worth noting however that the involvement of these different transduction pathways in the regulation of the induction of l-arginine transporters is not universal, and likely to be different from system to system. as a result there has been conflicting data on the relevance of these signaling proteins in inducing l-arginine transport in different cell. these issues will be discussed and the individual signaling pathways assessed on a cell type and species basis. moreover, the role of downstream signaling molecules will be examined in more detail, looking in particular at the critical dependency on the p mapk. this kinase currently exists in four different isoforms which are p α, , γ and δ. the involvement of individual isoforms of p in enhancing the expression of carrier proteins for l-arginine will be discussed. gw is an acetamidine derivative of heterosubstituted lysine which has been shown to have a marked selectivity for the human inducible nitric oxide synthase isoform (young et al. . bioorg. med. chem. lett., : , - ) . the systems associated with transport of this compound have been investigated using the macrophage cell line j . prior to each study, j cells were seeded in -well culture plates and allowed to adhere for h in dulbecco's modified eagle's medium (dmem). transport studies were carried out using hepes buffered krebs solution ( µl; °c) containing l-[ c]gw ( µciml Ϫ ) in the presence of either . mm or . - mm unlabelled substrate. in parallel studies transport ( µciml Ϫ , . mm) was monitored in the presence of mm excess of various other amino acids known to be substrates for distinct transport systems. time course experiments revealed that transport of . mm of l-[ c]gw occurred in a time-dependent manner and was linear for up to min. in addition, uptake was only marginally dependent on extracellular na ϩ . kinetic studies revealed that transport was saturable, and michaelis-menten analysis revealed single affinity entry with an apparent k t of . mm and v max of . pmol·µg protein Ϫ min Ϫ . at mm, -methylaminoisobutyric acid (meaib), lalanine, l-valine and - -amino-bicyclo-( , , )-heptane- carboxylic acid (bch) caused little or no inhibition of l-[ c]gw ( . mm) uptake. in contrast, transport of l-[ c]gw was inhibited markedly by l-arginine, llysine, l-leucine, l-methionine, -diazo- -oxo-l-norleucine (don) and l-glutamine. with the exception of l-arginine and l-lysine, the inhibition caused by the other substrates was critically dependent on extracellular na ϩ and was completely reversed when extracellular na ϩ was replaced with choline. in parallel kinetic inhibition experiments, transport of . mm l-[ c]gw was inhibited in a concentration dependent manner by l-arginine (ki ϭ . mm), l-leucine (ki ϭ . ), don (ki ϭ . mm) and l-glutamine (ki ϭ . mm). taken together, these data suggest that gw may be transported, at least in part, by system y ϩ . however, the marked inhibition caused by l-leucine, l-glutamine and l-methionine, substrates for the relatively high affinity cationic amino acid transporter system y ϩ l, would suggest that this system may also contribute to the uptake of gw ; if so, the monophasic substrate kinetics imply that the two systems handle gw with similar affinity. other systems such as b ,ϩ could be ruled out on the grounds that this transporter is critically na ϩ -dependent while uptake of gw is largely (ϳ %) na ϩ -independent. similarly, b ,ϩ , another broadspectrum aminio acid transporter that may be capable of transporting gw does not interact with l-glutamine and thus unlikely to be involved in transport of gw , at least in j cells. although a large number of different amino acid transporters have been identified on a molecular basis, some of themfunctionally described in mammalian cells -are still missing. in search of mammalian est sequences, which contain the signature of the aaap (amino acid/auxin permease) family, we identified a murine full length cdna, which encodes a membrane protein with - putative transmembrane domains. the transporter mrna is expressed in various murine tissues, including lung, heart and kidney. for functional characterization we used the xenopus laevis oocyte expression system and employed flux studies and electrophysiological analysis. oocytes injected with the crna showed an increased uptake of h-l-alanine and h-l-proline. detailed electrophysiological analysis revealed an electrogenic transport mode, independent of sodium and chloride ions. lowering the extracellular ph increased significantly substrate induced currents in crna injected oocytes. out of the proteinogenic amino acids the transporter recognizes only small amino acids, such as gly, ala, pro and ser. distinct structural analogues of these amino acids also interact with the transporters substrate binding site. in conclusion, we describe the molecular and functional characteristics of the first electrogenic proton driven amino acid transporter of mammals. pharmacology department, dr. willmar schwabe gmbh, karlsruhe, germany it is now well established that transport of amino acid neurotransmitters (like glutamate, aspartate, gaba and glycine etc.) from and to the neurones is essential for their proper functioning. like in the case of other neurotransmitters, specific pre-and post-synaptic as well as vesicular transporters are involved in such processes. extensive efforts to clarify the mechanisms and processes involved in the control and/or proper functioning of the amino acid transporters are now, therefore, being made in numerous laboratories. such efforts have not only led to the identification of a few specific ligands and/or modulators of neuronal amino acid transporters, but also have started unravelling the complex and diverse processes regulating their functions. aim of this communication is to point out potential usefulness of some neuroactive constituents isolated from therapeutically used medicinal herbs for clarifying the mechanisms involved in neuronal amino acid transport. our interest in such studies was initially triggered by the observations made with hyperforin, i.e. quantitatively the major neuroactive component of hypericum perforatum extracts widely used for the treatment of mild to moderate depressive disorders. this acyl phloglucinol derivative not only modulate synaptic transports of biogenic amines but also of glutamate, aspartate and gaba. since it does not interact with any of the till now described transporters for these neurotransmitters, efforts were made to clarify the mechanisms involved in their observed effects (both in vitro and as well as in vivo). the results of the in vitro studies available to date strongly suggest that its effects on neuronal amino acid transport processes is mediated via some novel extracellular mechanism controlling the h ϩ (and/or other ionic) concentrations of neurones. these observations not only demonstrate that hyperforin represent a structurally and mechanistically novel class of therapeutically useful agent but also suggest that it could be useful tool for clarifying the complex mechanisms involved in the control of neuronal amino acid transport. these observations stimulated us to screen other putative psychoactive herbal extracts and their active constituents on neuronal amino acid transport and on the consequences of disturbances caused by malfunction of specific transporters. observations made with several such agents indicate that either modulation of mechanisms and/or processes involved in neuronal amino acid transport or reversal of pathologies caused by anomaly of transporter functions could be involved in their modes of actions. these observations reinforce our conviction that studies directed towards clarifying the effects of herbal constituents on neuronal amino acid transport might not only be a feasible way for identifying novel types of therapeutically interesting molecules but also could expedite our knowledge on these complex processes. glutamate-regulated sodium dynamics in cortical astrocytes: implications for cellular bioenergetics j.-y. chatton, p. marquet, and p. j. magistretti the mode of na ϩ entry and the dynamics of intracellular na ϩ concentration (na ϩ i ) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. an elevation of na ϩ i was evoked by glutamate, whose amplitude and initial rate were concentration-dependent. the glutamate-evoked na ϩ increase was primarily due to na ϩ -glutamate cotransport. the rate of na ϩ influx decreased during glutamate application, with kinetics that correlate well with the increase in na ϩ i and which depend on the extracellular concentration of glutamate. a tight coupling between na ϩ entry and na ϩ /k ϩ atpase activity was revealed by the massive na ϩ i increase evoked by glutamate when pump activity was inhibited by ouabain. during prolonged glutamate application, na ϩ i remains elevated at a new steady-state where na ϩ influx through the transporter matches na ϩ extrusion through the na ϩ /k ϩ atpase. a mathematical model of the dynamics of na ϩ i homeostasis will be presented which precisely defines the critical role of na ϩ influx kinetics on the establishment of the elevated steady-state and its consequences on the cellular bioenergetics. indeed, extracellular glutamate concentrations as low as µm approximately doubled the energetic demands of the astrocytes. department of biochemistry and molecular biology, faculty of biology, university of barcelona, spain in the last years a new family of amino acid transporters composed by two different subunits has been defined. two heavy subunits (rbat and f hc) and seven light subunits are known. rbat and the light subunits b ,ϩ at and y ϩ lat are responsible for the inherited aminoacidurias type i cystinuria, non-type i cystinuria and lysinuric protein intolerance, respectively. the heavy subunits are highly glycosylated type ii proteins, while light subunits are very hydrophobic unglycosylated membrane proteins, displaying a polytopic (generally transmembrane domains) predicted structure. the specificity of the amino acid transport activity depends on the light chain expressed. this, together with its topology, indicates that the transport function mainly relies on the light subunits. i will summarize some of our current studies directed to the understanding of structure-function relationships of these heteromeric carriers, specially concerning their oligomeric structure and initial attempts to reconstitute them. ongoing work on the isolation of new rbat-associated light subunits and new b ,ϩ at-associated heavy subunits, which could also play a role in cystinuria, will also be discussed. department of pharmacology, joh. gutenberg university, mainz, germany mammalian cationic amino acid transporters (cats) catalyze the transport of basic amino acids through the plasma membrane. the cat family comprises at least five related carrier proteins (cat- , - a, - b, - and - ) with cat- a and - b being splice variants. in humans, only the "old" members of the family have been characterized (hcat- , - a and - b). hcat- and - b exhibit high affinity for cationic amino acids and are sensitive to trans-stimulation, consistent with the classical system y ϩ . in contrast, hcat- a is a low affinity carrier relatively insensitive to trans-stimulation. interestingly, hcat- a and hcat- b differ only in a region of amino acids. cat- , so far only identified in rat and mouse, exhibits also system y ϩ activity. however, the substrate recognition and maximal transport activity seems to differ from other y ϩ transporters. cat- expression has been reported to be restricted to the brain in adult animals. a cdna encoding for human hcat- has recently been isolated, however, the transport activity of hcat- has not been characterized. when optimally aligned, the amino acid sequence of hcat- shows only about % identity with the other hcat isoforms. in contrast, the amino acid sequences of hcat- , - (a or b) and - are about % identical. to elucidate which amino acids are responsible for the difference in the transport properties of the hcat proteins, we constructed chimeric proteins between hcat- and hcat- a and performed site directed mutagenesis. using this approach, we identified two amino acid residues that are responsible for the different transport properties of hcat- a compared to the high affinity cat-isoforms. to characterize the human cat- , we cloned a cdna encoding hcat- . when expressed in xenopus laevis oocytes, hcat- had a similar transport activity and affinity for l-arginine as hcat- or - b. hcat- mediated l-arginine transport was trans-stimulated and independent of extracellular na ϩ ions. expression studies demonstrated that hcat- is not only expressed in different regions of the human brain, but also in peripheral tissues. to investigate if hcat- also functions as an amino acid transporter, we measured the transport of cationic, neutral and acidic amino acids in xenopus laevis oocytes expressing hcat- , but could not detect an transport activity for any substrate tested. a bright fluorescence could be detected in the plasma membrane of oocytes expressing hcat- with the green fluorescent protein attached to the c-terminus. therefore, hcat- might either need a complementary protein to function as an amino acid transporter or serve as a transporter for a yet unidentified substrate. renal amino acid reabsorption in immature and adult rats as a sensitive marker of heavy metal-induced nephrotoxicity (pt, cr, tl) institut für pharmakologie und toxikologie, klinikum der friedrich-schiller-universität jena, germany the effects of cis-platinum (cp; . mg/ g b. wt. i. p.), sodium dichromate (cr; mg/ g b. wt. s. c.) and tl so (tl, mg/ g b. wt. i. p.) on renal amino acid excretion and plasma amino acid composition were investigated in -(both sexes) and -day-old (female) anaesthetised wistar rats (han : wist). on the basis of diuresis experiments on conscious rats (determination of urinary volume and protein excretion) the mentioned doses and times ( st day after cr in both age groups and in -day-old rats after cp and rd day after cp in adult rats; nd [ -day-old rats] and th - th day [ -day-old rats] after tl) were found out to be optimal for the characterisation of amino acid transport after heavy metal poisoning. interestingly, in conscious -day-old rats cr nephrotoxicity is not detectable after mg/ g b. wt. whereas all of the other experimental groups showed nephrotoxic effects of cr, tl and cp in conscious rats. urine volumes were lower, but not significantly, in anaesthetised immature rats, independently of the administered nephrotoxin. glomerular filtration rate (gfr) is significantly lower in -day-old rats compared to adults. after cp, cr and tl gfr is significantly reduced only in adult rats and age differences disappeared nearly completely. in principle the renal fractional excretion (fe aa ) of amino acids was distinctly higher in immature rats as a sign of lower amino acid reabsorption capacity. nevertheless, the amino acid plasma concentrations were relatively high in immature control rats. however, both cr and cp did not distinctly influence molecular cloning and functional characterization of ata , a novel subtype of the amino acid transport system a medical college of georgia, augusta, georgia, u.s.a. recent molecular cloning studies have revealed that the amino acid transport system a consists of more than one subtype. two different system a subtypes, called ata and ata , have been cloned and functionally characterized. ata is expressed primarily in the brain and placenta whereas ata is expressed ubiquitously. heterologous expression studies have shown that these two subtypes cannot be distinguished functionally based on substrate affinity nor substrate specificity. we have now cloned a third subtype of system a, designated ata . it is expressed primarily in the liver. apart from the liver, detectable level of expression is noted only in the skeletal muscle. interestingly, ata can be easily differentiated from the other two subtypes of system a based on functional characteristics. we first isolated rat ata cdna from a skeletal muscle cdna library using rat ata cdna as the probe. rat ata consists of amino acids and exhibits a high degree of homology in amino acid sequence to rat ata ( % identity) and rat ata ( % identity). interestingly, this new transporter also has a comparable degree of homology to sn and sn , the two known subtypes of the amino acid transport system n. however, when expressed heterologously in xenopus laevis oocytes, rat ata transports α-(methylamino)isobutyric acid (meaib), a specific model substrate for system a, confirming that this transporter is definitely a subtype of system a. system n does not transport this system a model substrate. with two-microelectrode voltage-clamp technique, we have shown that exposure of rat ata -expressing oocytes to neutral, short-chain aliphatic amino acids induces inward currents. the amino acid-induced current is na ϩ -dependent and phdependent. analysis of the currents with alanine as the substrate has shown that k . for alanine (i.e., concentration of the amino acid yielding half-maximal current) is . Ϯ . mm and that the na ϩ : alanine stoichiometry is : . subsequently, we have cloned the human homolog of rat ata from a liver cell line (hepg ) cdna library. human ata also contains amino acids and shows % identity in amino acid sequence with rat ata . the sequence identity of human ata with human ata and human ata is % and %, respectively. the homology of human ata with human sn and sn is also similar ( % and % identity, respectively). the gene coding for human ata contains exons and is located on chromosome p . in the human, ata is expressed almost exclusively in the liver. when expressed in mammalian cells heterologously, human ata mediates the transport of neutral amino acids, including meaib, in a na ϩ -dependent manner. interestingly, while characterizing the function of this clone, we have uncovered a unique feature of this system a subtype. human ata is capable of mediating the transport of cationic amino acids. in fact, the affinity of human ata for cationic amino acids is higher than for neutral amino acids. however, the human ata -mediated cationic amino acid transport is na ϩ -independent. in this respect, ata is similar to transport system y ϩ l that also transports neutral amino acids in a na ϩ -coupled manner and cationic amino acids in a na ϩindependent manner. in contrast, ata and ata have not been shown to interact with cationic amino acids. in addition to this difference in substrate specificity, ata also differs from ata and ata in substrate affinity. ata and ata interact with meaib with a k t of ϳ . mm whereas the affinity of ata for this model substrate is comparatively at least -fold lower (k t , ϳ mm). but, ata interacts with arginine with a k t value of . mm. since liver does not express any of the previously known high affinity cationic amino acid transporters, amino acid plasma concentrations. but in both age groups the administration of cr and cp significantly decreased amino acid reabsorption capacity (increase in fe aa ) as a sign of nephrotoxicity, most pronounced in adult rats after cp. on the other hand, after tl, the fe of amino acids was distinctly higher only in adult rats as a sign of lower amino acid reabsorption capacity and, thus, as a sign of higher nephrotoxicity. in immature animals fe aa was increased only for few amino acids. however, in both age groups tl administration significantly decreased plasma amino acid concentrations, more pronounced in immature rats. the investigation of renal amino acid handling confirmed: ( ) cr, cp and tl were more nephrotoxic in -day-old animals compared to immature rats as could be demonstrated previously using other parameters for nephrotoxicity testing. ( ) the extent of toxic effects of heavy metals on the kidney is related to the maturity of renal functions involved in the enrichment of the respective metal in renal tissue and in its toxicity mechanism. ( ) changes in the fractional excretion of amino acids (reduction in renal amino acid reabsorption capacity, e.g. increase in fe aa ) and in amino acid plasma concentrations (especially decreases as a consequence of enhanced renal loss of amino acids) are early indicators of nephrotoxicity. ( ) therefore, the determination of renal amino acid handling is a highly sensitive marker for nephrotoxicity testing, both in immature and in adult rats. the mammalian h ϩ /peptide cotransporter pept was initially identified in the brush border membrane of renal proximal tubular cells as a high affinity type ptr -family member. here we describe the synthesis and functional analysis of novel high affinity inhibitors for pept that will be useful in further studies on structure and functions. starting from lys[z(no )]-pro a series of different lysine-containing dipeptide derivates were synthesized and studied for interaction with pept based on transport competition assays in pichia pastoris yeast cells and in epithelial skpt cells, both expressing pept . the twoelectrode-voltage-clamp technique in x. iaevis oocytes expressing pept was used to determine whether the compounds are transported electrogenically or block the uptake of dipeptides. synthesis and functional analysis of lys-lys derivates containing z(no ) side chain protections provided a set of inhibitors that reversibly inhibited the uptake of dipeptides by pept with k i values as low as nm. this is the highest affinity of a ligand of pept ever reported. moreover, based on the structure-function relationship we can conclude that the spatial location of the ε-amino protecting group in a lys containing dipeptide and its intramolecular distance from the alpha catom are key factors for the transformation of a substrate into an inhibitor of pept . ata is likely to provide the major route for the uptake of arginine in this tissue. institute of pharmacology and therapeutics, faculty of medicine, porto, portugal the present study examined the nature and regulation of the l-dopa transporter in two functionally different clonal subpopulations of opossum kidney (ok lc and ok hc ) cells. the inward transfer of l-dopa was largely promoted through an energy-dependent and sodium-insensitive transporter, though a minor component (ϳ %) was found to require extraceilular sodium. l-dopa uptake was insensitive to meaib, but competitively inhibited by bhc (ok lc , ic ϭ µm; ok hc , ic ϭ µm). l-and d-neutral amino acids and basic amino acids markedly inhibited l-dopa accumulation. l-dopa, lleucine, l-arginine, bhc or l-arginine plus bhc stimulated [ c]-l-dopa efflux. the accumulation of l-dopa was significantly higher at an acidic ph, and incubation of cells with l-dopa ( µm) resulted in marked intracellular acidification. modulators of pka, pkg, pkc and ptk failed to affect the accumulation of l-dopa. only the ca ϩ / calmodulin inhibitors inhibited l-dopa uptake. it is likely that system b ,ϩ might be responsible for the sodium-dependent uptake of l-dopa in ok cells, whereas sodium-independent uptake of l-dopa may include systems b ,ϩ and lat , the activation of which results in trans-stimulation of l-dopa outward transfer. the trans-stimulation of l-dopa inward transfer by an imposed h ϩ gradient suggest that ok cells are provided with an l-dopa-h ϩ cotransport system. amino acids are essential nutrients for cell growth and maintenance. the essential amino acids arginine and lysine, are mainly transported via the cationic amino acid transporter protein (cat ). the regulation of translation of the cat mrna during amino acid starvation was studied. an adaptive response to amino acid starvation and stress is a global decrease of protein synthesis, by phosphorylation of the translation initiation factor eif a. translation of the transporter mrna increases when eif a is phosphorylated, allowing synthesis of the essential for survival arginine/lysine transporter protein. the mechanism of increased translation of this mrna involves the induction of activity of a uorf-containing internal ribosomal entry sequence (ires). translation of the uorf and phosphorylation of eif a are required for increased activity. we propose that eif a phosphorylation triggers translational attenuation within the uorf, converting a relatively inactive, to a high activity ires. this study demonstrates that like yeast, mammalian cells have developed a sophisticated response to stress conditions: when expression of most genes decreases, synthesis of stress response proteins increases to support cell survival. amino acid transport, cell volume and the regulation of cell death f. lang, s. fillon, i. setiawan, p. lang, v. tanneur, d. häussinger, and s. bröer department for physiology, university of tübingen, germany cell volume regulatory mechanisms participate in a wide variety of cellular functions including regulation of epithelial transport, excitability, hormone and transmitter release, metabolism, migration, cell proliferation and apoptotic cell death. besides ion transport, polyols, betaine and glycerophosphorylcholine, cells utilize amino acids including taurine to balance extracellular osmolarity and regulate their volume. cells counteract shrinkage by uptake and swelling by release of amino acids including taurine. moreover, cell swelling stimulates synthesis and cell shrinkage favours breakdown of proteins which are osmotically less active than the sum of the amino acids thus generated. conversely, amino acid transport does influence cell volume. concentrative uptake of amino acids leads to cell swelling, amino acid release to cell shrinkage. through alterations of cell volume the amino acids participate in the regulation of protein metabolism. thus, concentrative amino acid transport inhibits and release of amino acids favours proteolysis. these mechanisms participate in the regulation of cell death. cd induced apoptotic death of jurkat t lymphocytes is paralleled by the release of taurine. the taurine release occurs with a delay of some min following cd receptor triggering but immediately preceedes apoptic cell shrinkage and dna fragmentation. the signaling leading to taurine release is in large part elusive but requires at some stage activation of caspases. moreover, taurine release and apoptotic dna fragmentation are strongly inhibited by lowering of temperature. preloading of the cells with taurine retards cd induced dna fragmentation pointing to an active role of taurine in the regulation of apoptosis. peptide transporters of the ptr-family are integral plasma membrane proteins, that mediate the electrogenic protoncoupled transport of di-and tripeptides and peptide-like drugs across cell membranes. the physiological role of pept , one member of this family in mammals, is mainly the uptake of small peptides into intestinal and renal tubular epithelial cells. in caenorhabditis elegans a homologue to mammalian pept is encoded by the pep- gene, which is expressed in the intestinal cells and a subset of sensory neurons in the head of the animal. to study the physiological role of the pep- transporter in vivo, a c. elegans pep- mutant was constructed. the animals deficient in pep- show a remarkable phenotype with pronounced signs of malnutrition, characterised by a delayed development, less eggs in the uterus, a smaller brood size and a prolonged mean life-span compared to wild-type animals. we rescued the phenotype by the expression of the wt pep- gene in the mutant. the observed starved phenotype in pep- mutants might be best explained by the reduced intestinal absorption of peptide bound amino acids that are required for protein synthesis and energy metabolism and provides the first direct evidence for the predominant role of the intestinal peptide transporter in amino acid absorption. adenosine is a potent vasodilator in many vascular beds and modulated tone via elevation of intracellular camp and/or release of nitric oxide (no). we have previously reported that adenosine (ado) stimulates l-arginine transport and no production in human cultured umbilical vein endothelial cells (sobrevia et al., j. physiol. , - , ) , and here further characterise the signalling cascades. rt-pcr demonstrated that fetal endothelial cell possess mrna levels for a a , a b and a -adenosine receptor subtype, whereas negligible levels were detected for the a -receptor. adenosine ( µm, min) induced increases in l-arginine transport and no production were ca ϩ and camp independent and stimulated transport was abolished in cells depolarised with mm k ϩ . whole-cell patch clamp experiments revealed that adenosine activated inward k ϩ currents, resulting in a membrane hyperpolarization and enhanced influx of the cation substrate l-arginine. adenosine induced l-arginine transport and no production were also abolished by inhibitors of tyrosine kinases (genistein), mek / (pd , u ) but unaffected by inhibitors of pkc (calphosin c) and pi- kinase (ly ). these data suggest that adenosine induces membrane hyperpolarization by activating inward k ϩ currents, increasing the driving force for cationic amino acid influx via system y ϩ . the discovery of nocardicine a by aoki et al. and aztreonam showed that monocyclic -lactams, collectively known as monobactams, can have antibiotic activity. this activity is poor but compensated by the unique effect they can induce on certain microbial cell membranes. our quest for new non-conventional surfactants for various biomedical applications led us to synthesize bioactive compounds with structural similarities to nocardicins. we present here the preparation and the study of original trimodular biosurfactants of type i: spermine and amine oxidase induce a cytotoxic effect on multidrug resistant chinese hamster ovary cells e. agostinelli , s. lord-fontaine , e. przybytkowski , and d. a. averill-bates department of biochemical sciences "a. rossi fanelli", university of rome "la sapienza" and cnr, centre of molecular biology, rome, italy department de chimie/biochimie and toxen (centre de recherche en toxicologie de l'environnement), université du québec à montréal, canada the occurrence of resistance to cytotoxic agents in tumor cells is a major obstacle to successful anticancer chemotherapy. multidrug resistance (mdr) is associated with several phenotypic alterations. cells with the mdr phenotype display decreased drug accumulation due to overexpression of pglycoprotein (p-gp), encoded by the mdr- gene, which acts as an energy-dependent pump involved in extrusion of drugs. we studied a new strategy to eliminate mdr cells using an enzyme, bovine serum amine oxidase, capable of forming cytotoxic products, h o and aldehyde(s), from polyamines (spermine). the involvement of both toxic products, formed by the bsao/spermine enzymatic system, in causing cytotoxicity was investigated in multidrug resistant chinese hamster ovary cells, ch r c , at and °c. we observed that hyperthermia, depletion of intracellular glutathione (by l-buthionine sulfoximine) and inhibition of glutathione s-transferase (by ethacrynic acid), sensitized ch r c cells to the cytotoxic effect of spermine enzymatic oxidation products. mdr cells showed no resistance to h o and aldehyde(s) relative to their drug-sensitive counterparts, auxb cells, in experimental conditions of: higher temperature, higher spermine concentration and longer incubation time. the inhibition of cellular detoxification systems led to increased cytotoxic effects of spermine enzymatic oxidation products on both mdr and sensitive cell lines. these results might be of great interest and suggest that toxic oxidation products formed from spermine and amine oxidase could be used in anticancer therapy, mainly against multidrug resistant tumor cells. [acknowledgements: this work was supported by cnr "target project on biotechnology", ministero della sanità tar these compounds present a hydrophobic part introduced by an ester or amide linkage with an aminoacid, a junction modulus which corresponds to -lactam, and a hydrophilic part which contains a triazole, well-known in pharmaceutical industry for its inhibitor effect against -lactamase. the compounds are synthesised from -hydroxymethyl- methyl propionic acid in five steps. selective activation of one of the primary hydroxyl groups was accomplished by the formation of alkoxy tris(dimethylamino)phosphonium (atdp) salts from the corresponding diol. treatment of with excess potassium carbonate in refluxing anhydrous acetone yields the monobactams . activation by atdp salts followed by treat-ment with sodium azide and reflux in toluene gives the azido compound. the reaction with acetylenic derivatives allows to obtain the surfactants. the compounds show classical surfactant behavior and the evaluation of their biological properties give evidence for their antibacterial and antiviral activity, which corresponds apparently to antiprotease activity. a prodrug approach to glutathione derivatives with in vitro antiparasitic activity department of chemistry and materials manchester, faculty of science and engineering, metropolitan university, manchester, u.k. the potential chemotherapeutic activity of peptides are lost in many cases in vitro, due to their inability to cross cell plasma membranes. the recent identification of a series of glutathione diesters with high antiparastic activities in vitro against t.b.brucei (african sleeping sickness) lead us to investigate the determinants associated with their activity. a qsar study on some twenty-five diester derivatives against t.b.brucei and t.b. rhodesiense lead us to conclude that the mechanism of action of these compounds is related to membrane penetration and hydrolysis, controlled by hydrophobicity and steric factors. a hplc and sensor study have confirmed the de-esterified diacid as the active agent of these prodrugs. dietary taurine prevents oxidative stress and morphological alterations in the retina of diabetic rat f. franconi , m. a. s. di leo , s. caputo , n. gentiloni silveri , and g. ghirlanda department of pharmacology, university of sassari, and department of internal and geriatric medicine, catholic university, rome, italy diabetes mellitus can cause various complications including retinopathy, which is the earliest and most common complications of diabetes mellitus, affecting % of diabetics and progressing to blindness in about %. considerable evidence implicates oxidative stress in the pathogenesis of diabetic retinopathy. in fact, hyperglycemia generates reactive oxygen species and free radical defense is reduced in diabetic patients. thus, the prevention of oxidative stress may have important implications for pharmacological attempts to prevent diabetic retinopathy. at this regard, it has been found that taurine, a semi essential amino acid with antioxidant activity, is decreased both in type and type diabetes mellitus. moreover, taurine seems to have a peculiar role of taurine in terms of cellular physiology and pathophysiology of the retina. among others, taurine is thought to produce important physiological effects through osmoregulation, calcium modulation and antioxidant effects. therefore, we examined the effect of dietary chronic ( months) taurine ( % and %) supplementation in diabetic rats in comparison with vitamin e ( and ui). dietary taurine supplementation, for months, does not influence conjugated dienes (cd), lipid peroxides (lp) and na/k atpase activity in the retina of non diabetic rats. using rats streptotozocin (stz) induced diabetes of -month duration, we found that cd, lp are significantly increased and they remained elevated for months. while, the na/k atpase is significantly decreased during the whole experimental time ( months). moreover, an inverse correlation has been found among the cd and lp and atpase activity. in the retina of stz rats, these biochemical alterations are accomplished with marked profound morphological changes. in stz rats, taurine enriched diets decrease the lipid peroxidation and preserve the atpase activity, being % taurine more effective than % diets. the morphological examination reveals that in rats feed with % taurine no proliferative changes are present. moreover, the beneficial effects of taurine are more marked than of those of vitamin e. these results and previous findings encourage new investigations to evaluate the efficacy of taurine as an adjunctive agent ch ch (ch ) n xco iran applicated be ( mg/kg - days) and the third -control. enzyme activities were determined spectrophotometrically in brain homogenate. results: polyamine oxidase activity decreased significantly lower dose of be didn't induce any significant change in diamine oxidase activity gaba-transaminase activity increased significantly (p Ͻ . ; p Ͻ . ) and dose dependently upon be treatment we have been examined the effects of propofol, taurine and propofol combined with taurine on free intracellular amino acid (aa) profiles, superoxide anion formation (o Ϫ ), hydrogen peroxide production (h o ) and released myeloperoxidase activity (mpo) in polymorphonuclear leucocytes (pmn). propofol led to significant changes in pmn free taurine, glutamine, glutamate, aspartate, methionine, basic, neutral (naa) and branched chain amino acid concentrations. exogenous taurine reduced pmn naa while increasing intracellular taurine. taurine supplemented to propofol significantly reversed the changes in taurine, naa and alanine only. regarding pmn immune functions propofol significantly decreased o Ϫ , h o formation and mpo. taurine decreased o Ϫ and h o production, while increasing released mpo. when propofol and taurine were combined they appeared to by reacting tyrosine with -nitroso- -naphthol in the presence of nitric acid - -benzyo- -(alanyl)- -phenoxazone (blp) an analog of actinomycin d is produced. the structural similarity of blp to actinomycin d prompted the national cancer institute (nci) to investigate its antitumor activities. the nci investigations revealed that blp exhibits growth inhibitory effects on various cancer cells and as a result blp has received the u.s. patent from the u.s. patent office. the purposed of this investigation was to synthesize similar benzo phenoxazone derivative by reacting -nitroso- -naphthol with -(α-hydroxy -methylaminopropyl)phenol in the presence of nitric acid. during the study, it was found out that , -benzo- phenoxazone derivative is not produced but a hydrogenated form of , -benzo- -phenoxazone which is probably , -benzo- -(α-hydroxy -methylaminopropyl)- -hydroxyphenoxazine (bhmhp) which has been suhhested from mass spectra obtained by electron ionization, ei, chemical ionization, ci and electro-spray ionization, esi, methods. bhmhp was screened against various cancer cell lines by nci and has shown promising effect against three ( ) breast cancer cell lines: mda-mb- , mda-n and hs- t. the % growth inhibitory (gi ) concentrations for these three cell lines were . ϫ Ϫ , . ϫ Ϫ and . ϫ Ϫ molar respectively. a. bocheva and t. pajpanova institute of molecular biology, bulgarian academy of sciences, and institute of physiology, bulgarian academy of sciences, sofia, bulgariathe histamine is an endogenous substance with neurotransmitter and neuromodulator functions in the organism. its antagonists are used in the therapy of allergic diseases and inflammatory reactions and as antiulcer drugs.the limited potentialities of the antihistamine therapy together with the increasing number of the people suffering from allergic diseases give rise to the design and synthesis of new histamine analogues as a perspective area in the chemistry of therapeutic drugs.additionally, compounds containing the guanidine, oxyamino and sufonamide moieties are known to elicit a variety of pharmacological responses and are present in several marketing drugs or drug candidates.on the other hand, similar compounds, being a part of bigger structures (for instance peptides), can imitate the molecules of already known at ii-receptor antagonists.having in mind these data we aimed to synthesize new analogs of histamine containing sulfo-and oxy-guanidino groups with common formula: a. bocheva , s. pancheva , and t. pajpanova institute of physiology, bulgarian academy of sciences, and institute of molecular biology, bulgarian academy of sciences, sofia, bulgariathe problem of the efficient therapy of pain is important not only from clinical but from social and economic point of view. the great achievements in medicine are connected with the research on the development of antinociceptive drugs.melanocyte-inhibiting factor (mif) is a tripeptide (pro-leu-gly-nh ) that was discovered in hypotalamus.the mif- exerted a weak analgesic effect. the synthesis of non-protein amino acids and their incorporation into biologically active peptides might become a powerful method for the design and development of modified analogues of natural peptides. having in mind these data we synthezied a number of new mif-analogues, containing unnatural amino acids such as cav, slys, sleu, slle and snie and in vivo experiments were performed to study their action on the nociception. the changes in nociceptive effects were examined in male wistar rats by the tail-flick (tf) and hot-plate (hp), as well as, the randall-seitto paw-pressure tests. the peptides were applied intaperitoneal (i.p) injection at a does mg/kg. the results show that the newly sinthesized analogues exert an antinociceptive effects in all tests used. naloxone at a dose mg/kg (i.p) antagonized the antinociceptive effects of mif-analogues. the interaction between platelets and fibrinogen is known to be mediated by the intergrin gp iib/iiia. the arg-gly-asp (rgd) sequence located on fibrinogen and other proteins of blood and extracellular matrix is the minimum requirement for cell attachment and adhesion. it has been found that peptides containing the rgd sequence can effectively inhibit the binding of fibrinogen to gp iib/iiia. in addition aspirin has been shown to be beneficial in the treatment of stable and unstable angina, acute myocardial infraction. aspirin acetylates and inhibits the enzyme cyclooxygenase, the first enzyme involved in thromboxane a (txa ) synthesis, an activator of platelet aggregation and adhesion.we have already reported that the combination in the same molecule of dipeptide amides, containing amino acid(s) of rgd sequence, with salicylic-residue -ro-c h -coϳ, {where rϭh or ch co} at their n-terminal amino group have shown inhibitory activity on human platelet aggregation. continuing this research project on salicyl-peptides we have synthesized a series of rgd analogs, incorporating salicylic acid derivatives, by conventional solution techniques and/or by solid phase. the synthesized rgd analogs were identified by ir, nmr and es-ms spectra and tested for inhibitory activity on human platelet aggregation in vitro, by adding common aggregation reagents (collagen, adp, thrombin) to citrated platelet rich plasma (prp). platelets were obtained from venous blood of healthy donors and the prp was isolated by centrifugation at g for min at °c. the aggregation was determined using a dual channel electronic aggregometer. malonyl dialdehyde (mda) production was measured using thiobarbituric acid reagent. in order to confirm these results, flow cytometry with monoclonal antibodies against gpib, gpiib/iiia, gpiiia and gmp was used. the ic values of the synthesized and tested compounds, as well as their mda production and flow cytometry results will be discussed. amino acids have a long tradition as building blocks, chiral auxiliaries and/or ligands in advanced organic synthesis and catalysis. at dsm an enzymatic kinetic resolution process has been developed, based on an aminopeptidase catalyzed stereoselective hydrolysis of racemic amino acid amides to form a mixture of l-amino acid and unchanged d-amino acid amide.several small peptides currently are under investigation as possible anti-tumor agents. neuropeptides such as substance p (sp) and neuropeptide y (npy), have been studied for their ability to prevent tumor growth or the proliferation of several cancer cell lines. these neuropeptides have been investigated for their effect to prostate cancer, small cell lung cancer (sclc) and breast cancer. the synthetic sp analog [d-arg , d-phe , d-trp , , leu ]sp (antagonist d) and the c-terminal analog [arg , d-trp , , mephe ]sp - (antagonist g) inhibit sclc cell proliferation in vitro and in vivo, while the analogs [glp , glu(bu t ) ]sp - and [glp , glu(bu t ) ]sp - showed significant inhibition in the proliferation of the cancer cell lines hela and t d.in the present study the c-terminal analogs of sp [glp , d-trp , glu(bu t ) ]sp [ ] [ ] [ ] [ ] [ ] [ ] ( ), [glp , d-trp , , glu(bu t ) ]sp [ ] [ ] [ ] [ ] [ ] [ ] ( ), [glp , d-trp , , mephe , glu(bu t ) ]sp - ( ), [glp , d-trp , mephe , glu(bu t ) ]sp - ( ), [glp , trp , mephe , glu(bu t ) ]sp - ( ), [glp , mephe , d-trp , glu(bu t ) ]sp - ( ), [glp , d-trp , mephe , glu(bu t ) -oh]sp - ( ), [glp , d-trp , cys(acm) -oh]sp - ( ), [glp , d-trp , mephe , cys(acm) -oh]sp [ ] [ ] [ ] [ ] [ ] [ ] ( ), [glp , d-trp , , mephe , cys(acm) -oh]sp - ( ) have been synthesized and tested for their antineoplastic properties in several cancer cell lines. they were also examined for their cytotoxicity to normal cells.the analogs - are peptide amides whereas the analogs - are peptide acids. they were performed using the stepwise synthesis either in solution, using the method of mixed anhydrides with carbonic acids or in spps using the fmoc/bu t methodology. the fragment condensation method in solution, using phosphonium reagents, such as pybop, was also applied. the analogs were purified (hplc) and identified (ft-ir, es-ms, h-nmr).the antineoplastic properties of the analogs were studied using sister chromatide exchange (sce) and proliferation rate index (pri). as it is known the sce method is an indicator of dna damages or its repair mechanism, while the method of pri is a sensitive marker of cytotoxicity. the experiments were carried out using cultured human lymphocytes from healthy donors and these results will be discussed.semiempirical quantum chemical investigation of some thymidine derivatives modified with amino acids and peptides at Ј, Ј-positions j. velkov , i. stankova , a. ivanova , and a. tadjer department of chemistry, south-west university "neophit rilski", blagoevgrad, and department of chemistry, sofia university "st. kl. ohridsky", sofia, bulgaria optimized geometry and electron charge distribution for some thymidine derivatives ( Ј, Ј-bis-o-n-α-benzyloxycarbonyl-alanyl-, Ј, Ј-bis-o-n-α-benzyloxycarbonyl-valyl , Ј, Ј-bis-o-n-α-benzyloxy-carbonyl-glycyl-glycyl-glycyl, Ј, Јbis-o-n-α-benzyloxycarbonyl-phenylalanyl, Ј, Ј-bis-o-n-αbenzyloxycarbonyl-glycyl) were calculated at the semiempirical (am ) level. the choice of method is limited by the molecular size. in addition, the differences between the ground state energy of the compounds and that of the hydrolysis reaction intermediates were compared to the experimentally found stability towards hydrolysis.with a few notable exceptions, attempts to crystallise integral membrane proteins have failed due to the difficulties in finding appropriate conditions for proteins that have both hydrophobic and hydrophilic domains. thus structural information is largely limited to predictions of secondary structure from the amino acid sequence and computer modelling, neither of which can as yet give high resolution detail. thus alternative approaches are required, and one that we have employed is to look at the substrate binding/transport characteristics of compounds and predict what features the binding site might have. the membrane transport protein that we are interested in is the proton-coupled di/tri-peptide transporter, which has a wide range of natural substrates and is known to transport therapeutically important non-peptides such as ᮀ-lactam antibiotics and angiotensin converting enzyme inhibitors.the initial question that interested us was what makes a di/ tri-peptide a substrate, but not an amino acid? while the obvious answer is the peptide bond, studies with 'space mimic' compounds (which have the space filling properties of a dipeptide but no peptide bond) gave the surprising result that the peptide bond was not essential for binding and translocation. although these space mimics had n and c termini, studies from our laboratory and others have shown that the presence of free amino or carboxyl groups are not a prerequisite for binding or translocation either. this leaves the question of what does distinguish a pept substrate from a non-substrate?computer modelling of a large number of pept substrates has allowed the development of a substrate template, whereby potential substrates can be scored according to their predicted binding affinity. from this it is clear that it is a sum of energies derived from a number of substrate-transporter interactions that determine binding affinity, including the n-and c-termini, the peptide bond components and the substrate side-chain groups. further studies aim to refine this model through the complimentary approaches of novel substrate design and sitedirected mutagenesis of the transporter protein.why are we interested in this? a large number of promising therapeutic compounds are found to have little or no bioavailability. compared with most membrane transporters pept has a wide range of potential substrates, and amongst its non-peptide substrates are a range of peptidomimetic therapeutic compounds. the recent finding that a peptide bond is not a prerequisite for transport opens up the possibility of designing prodrugs to be substrates for pept , and this has found to be an effective strategy for example with the antiviral drug valacyclovir.(we thank the wellcome trust for their generous support.) nuklearmedizinische klinik und poliklinik der technischen universität münchen, germanyaim: the high amino acid metabolism of tumor cells allows tumor imaging with radiolabeled amino acids as c-methionine (met) by positron-emission-tomography (pet). however in recent experimental and clinical studies met uptake was also found in inflammatory tissue thus leading to false positive results. the aim of the study was to compare [ f]fluoroethyltyrosine (fet), a new amino acid analogue, with met to assess their suitability for differentiating between tumor cells and inflammatory cells in vivo and in vitro.methods: popliteal lymph nodes of balb/c and dba/ mice were stimulated either by streptocotocin (stz), causing chronic lymphadenitis, or by concanavalin a (con a), causing in acute lymphadenitis. tumor infiltrated lymph nodes were induced by inoculating cells from a lacz transfected t-cell mouse lymphoma line into the footpads of syngenic dba/ mice. the uptake of met and fet was determined quantitatively in tumor infiltrated and inflammatory lymph nodes as well as in the lymph nodes of untreated mice. in vivo imaging of tracer uptake in mouse lymph nodes was performed using a high resolution ( . mm) small animal pet (madpet). in vitro the uptake of the amino acids met and fet was investigated in different cells, such as sw human colon carcinoma cells and c rat glioma cells, stimulated human lymphocytes and macrophages. about ϫ cells of each cell line were incubated in a buffered medium containing either different concentrations of unlabeled amino acids or con a (stimulation of lymphocytes) or the transport inhibitors amino-norbornane-carboxylic acid (bch, l-system), α-(methylamino)-isobutyric acid (meaib, a-system) or l-serin (asc-system). . mbq of each amino acid tracer were added and incubated. uptake was stopped by using ice-cold pbs, cells were washed three times and uptake was analyzed.results: in tumor infiltrated lymph nodes uptake of both tracers was higher than in control lymph nodes. met showed an increased uptake in both lymphadenitis models, whereas fet did not accumulate significantly. met and fet uptake in tumor infiltrated lymph nodes was also seen in madpet images, however inflammatory lymph nodes could only be detected in met images.the amount of tumor uptake was different in the various cell types investigated. c cells showed the highest uptake of all cells investigated and a slightly lower uptake was found in sw cells. in con a stimulated lymphocytes, the uptake of fet was negligible, while met uptake was significantly higher than in both tumor cell lines. since bch reduced the uptake of fet and met to approximately %, fet seems to be also predominantly transported into tumor cells by the l-system. the results indicate, that fet appears to differentiate between tumor and inflammatory tissue, as a result of the low uptake of fet in inflammatory cells. nuklearmedizinische klinik und poliklinik der technischen universität münchen, germanyover the past few years numerous studies have documented the high diagnostic accuracy of positron emission tomography (pet) using the glucose analogue f- -fluordeoxyglucose (fdg) for detection and staging of malignant tumors. a significant limitation of fdg-pet, however, is that increased uptake is not only observed in malignant tumors but also in activated inflammatory cells. due to the high glucose utilization of the normal brain and the lower protein synthesis in the normal gray matter the radiolabelled amino acid c- -methionine (met) gives higher contrast between brain tumors and normal tissue than fdg-pet. rapid uptake of met has been documented for several malignant tumors like gliomas, lung cancer, bladder cancer and malignant lymphomas since amino acid transport and protein synthesis are generally increased in malignancies. the application of met-pet however has been limited by the short half life of the radioactive label c- ( min) in contrast to f- ( min). amino acid analogous labeled with f- like f- -fluoro-α-methyltyrosine (fmt), f- -fluoro-ethyltyrosine (fet), f- -fluoro-phenylanaline, f- -fluore-proline will allow a more widespread application of amino acid pet in oncology. an other amino acid analogue i- -iodo-α-methyltyrosine (imt) is of clinical interest because the radionuclid i- allows it applicability for single-photoemission-computer-tomography (spect). the uptake of the amino acid analogues can only be regarded as a measure for the increased amino acid transport in the tumor cells because they are not incorporated into proteins. clinical data show that radiolabelled amino acids that are only transported into the cells are not inferior to those that enter protein synthesis. this tracers may also help to differentiate tumor lesions from inflammatory lesions when the expression of the transport systems for amino acids in tumor cells and inflammatory cells is different.lysinuric protein intolerance: understanding the pathophysiology of a multi-system disorder of dibasic amino acid transport m. p. sperandeo , , v. fiorito , a. pietrosanto , a. pepe , g. andria , and g. sebastio telethon foundation, rome, and department of pediatrics, federico ii university, naples, italy lysinuric protein intolerance (lpi; mim ) is an autosomal recessive disease, mainly found in finland and italy. clinical findings of lpi include: vomiting, diarrhea, failure to thrive, hepatosplenomegaly, osteoporosis, episodes of coma, and mental retardation. a life-threatening lung involvement (alveolar proteinosis) and renal insufficiency were also reported. metabolic derangement of lpi includes: reduced intestinal absorption of cationic amino acids (lysine, ornithine, arginine, caa), increased renal excretion of caa and dysfunction of the urea cycle leading to hyperammonemia and orotic aciduria. most of the clinical findings cannot be explained by a selective deficiency of amino acid transport, as indeed observed for cystinuria (mim ), a cognate disease of lpi. the molecular basis of lpi resides in an abnormal caa carrier functioning at the level of basolateral membrane of epithelial cells in the intestine and the kidney. caa transport is mediated by y ϩ l system, that is exerted by heterodimers consisting of the f heavy chain ( f hc) and a light chain represented by either the solute carrier family a, member (slc a ) or (slc a ). after excluding the f hc as the causative gene of lpi, we identified slc a as the lpi gene and characterized mutations in twenty-five patients from families ( italian, japanese, moroccan, greek, and pakistani; independent alleles) affected by lpi. thirty-two of the independent alleles ( . %) were characterized and fourteen mutations were identified. only five mutations (namely insatca, w x, delctct, ivs ϩ gaea, s r) were identified in more than one independent family. most mutations are located in the slc a coding region, except for two splicing mutations. the pathogenesis of some clinical findings of lpi, namely alveolar proteinosis and renal involvement, remains mostly unknown. we are currently investigating the role of slc a gene in lpi, which, in addition to slc a , is responsible of the y ϩ l activity. in fact, the regulation of the y ϩ l system, exerted by either f hc/ slc a or f hc/slc a , is still unknown. hypothetically, the activation of f hc/slc a in all tissues might be the "simple" way to a lpi gene-therapy.[acknowledgements: m. p. s. is supported by telethon-italy (grant n. cp) and is an assistant telethon scientist.] pre-eclampsia (pe) is a potentially life threatening complication of pregnancy and is one of the leading causes of maternal and fetal morbidity and mortality. pe is associated with endothelial cell dysfunction and inadequate placental perfusion. fetal plasma l-arginine levels are decreased in pe and there is controversy as to whether nitric oxide (no) production is altered. we have investigated whether the kinetics of l-arginine transport via system y ϩ and no production are altered in fetal umbilical vein endothelial cells (huvec) from pe pregnancies. kinetics of l-arginine transport were similar in huvec isolated from normal, preterm and pe pregnancies, however nethylmaleimide inhibited transport in normal but not pe huvec. basal and histamine-stimulated no production was similar in normal and preterm huvec, whereas pe increased basal ( Ϯ vs . ϫ pmol/ cell/ min) and histaminestimulated ( Ϯ vs Ϯ pmol/ / min) no production. whole-cell patch clamp measurements revealed similar inward rectifying k ϩ currents in normal and pe huvec, with resting membrane potentials of Ϫ Ϯ and Ϫ Ϯ mv in normal and pe huvec, respectively. increased enos activity in pe endothelial cells may serve as a compensatory mechanism to counteract the hypertension observed in pe, however, elevated no production is apparently not associated with enhanced larginine transport. department of pharmacology, university of cambridge, u.k.over the past years, concerns have heightened over the escalating numbers of pathogenic microorganisms that are resistant to multiple antibiotics. this phenomenon poses major problems in the treatment of patients with hospital or community-acquired infections caused by bacteria, yeast, fungi and parasitic organisms. particularly intriguing are the so-called multidrug transporters, which have specificity of compounds with very different chemical structures and cellular targets. this lecture will focus on the molecular properties of the atpbinding cassette multidrug transporter lmra in the lactic acid bacterium lactococcus lactis. lmra is a close homolog of the human multidrug resistance p-glycoprotein, overexpression of which is one of the major causes of resistance of human cancers to chemotherapy. surprisingly, lmra can even substitute for pglycoprotein in human lung fibroblast cells. recent biochemical and pharmacological studies on lmra suggest that the protein may operate by a two-cylinder engine mechanism to transport amphiphilic drugs from the inner leaflet of the plasma membrane. this mechanism will be discussed in more detail. bone and bone marrow are important sites of metastasis formation in breast cancer; so, we studied the level of bone sialoprotein (bsn) and fibronectin (fn), two key connective tissue antigens, in patients with metastatic breast carcinoma. our data reveled that bsn have a statistically significant association with bone metastases in that disease. fn level was also significantly changed in metastatic breast carcinoma when compared to the non metastatic cases. kharkov national university, radiophysical department, chair of molecular and applied biophysics, kharkov, ukraine * present address: institute of cell and molecular biology, university of edinburgh, edinburgh, scotland, u.k.in the work the temperature dependencies of dielectric parameters of human serum albumin (hsa) and fibrinogen solutions ( . m nacl, ph . ) were obtained in the temperature interval - c degrees. the measurements of the dielectric parameters were carried out at the frequency of . hhz, i.e. in the range of free water molecules dispersion. in contrast to dependencies for poor solvent, temperature dependencies of dielectric parameters for protein solutions are of nonmonotonous character; they have a number of peculiarities in the temperature ranges of - , - and - c degrees. this fact means that at these temperatures redistribution of free and bound water in protein-water system occurs due to structural changes in protein molecules. the dependencies of hydration of hsa and fibrinogen on temperature were obtained as well.in the work the mechanism of temperature changes of spatial organisation of protein molecules was proposed. perhaps, this mechanism is responsible for maintenance of thermal stability of the functionally active conformation of native proteins. as peculiarities on temperature dependencies of dielectric parameters of solutions of globular (hsa) and fibrillar (fibrinogen) proteins were in the same temperature regions, one may to assume that the mechanism of proteins thermal stabilisation in physiological temperatures interval has a general character. laboratory of cell pharmacology, university of leuven, medical school, campus gasthuisberg (o&n), leuven, belgium n-pomc was purified from conditioned medium of att cells using a sequence of concentration, fractionation by ion exchange, rp-hplc and gel-filtration. twenty isoforms of n-pomc, for both and kda, were identified by means of mass spectrometry and n-terminal sequencing. these isoforms are assumed to be pomc - or pomc - with heterogeneous glycosylation.the n-pomc isoforms were tested on prolactin (prl) gene expression and lactotroph mitosis in pituitary cell aggregate cultures. prl mrna content was quantified by means of real time rt-pcr. three kda n-pomc fractions enhanced prl mrna levels by - %, while all other isoforms were inactive. this effect was abolished by immunoneutralization with n-pomc monoclonal antibody. only one fraction stimulated lactotroph proliferation ( . Ϯ . %) as assessed by brdu incorporation in prl-immunoreactive cells. several (but not all) kda n-pomc fractions stimulated prl mrna level and lactotroph mitosis. on the other hand, all and kda isoforms activated the mc- and mc- receptor in cell lines in which these receptors were transfected. thus, att cells produce various n-pomc isoforms, only a part of which display an effect on prl mrna expression. even fewer isoforms affect lactotroph proliferation. since all isoforms activate the mc- and mc- receptor, it is suggested that the effect of the few isoforms on lactotrophs is mediated by (a) different receptor(s). are widely prescribed for the treatment of mild to moderate depression and the putative antidepressant constituent is probably hyperforin. in this study the effect of hyperforin was investigated on the release of neurotransmitter amino acids.coronal cortical slices ( µm) were cut and perfused with gassed ( % o , % co ) acsf at °c. two-minute samples of perfusate were collected and aspartate and glutamate were assayed by hplc. potassium-and veratridine-stimulated release was elicited by administering pulses of k ϩ ( mm) or veratridine ( µm) minutes apart.in control experiments the second k ϩ pulse elicited glutamate release which was % of the first pulse. hyperforin ( µm) perfused for minutes prior to, and during, the second k ϩ pulse significantly increased glutamate release to % (p Ͻ . , n ϭ - ). release elicited by the second veratridine pulse was % of the first pulse for both glutamate and aspartate. hyperforin ( µm) increased this release to the second pulse to % and % respectively (p Ͻ . , n ϭ - ). when perfused on its own for minutes, hyperforin ( µm) increased the basal release of glutamate (p Ͻ . , n ϭ - ).in conclusion, the increase in the release of neurotransmitter amino acids observed following hyperforin is possibly mediated through a facilitatory action on voltage-operated ca ϩ or na ϩ channels.glucagon-like peptide- ( - ) amide (glp ) is the main product of the glucagons gene expression in intestinal l cells into the cirulation in response to the ingestion of food and is the most potent stimulator of glucose-induced insulin secretion. glp receptors have also been detected in discrete areas of rat brain and intracerebroventricular glp has been shown to inhibit feeding in fasted rats. in this study hplc techniques were employed to evaluate the effects of glp on serotonin ( -ht) and γ aminobutyric acid (gaba) metabolism in rat brain. glp ( . µm) produced a significant decrease in levels of -ht by % after minutes of incubation with combined hypothalamus and brain sterr. synaptosomes. levels of hydroxyindolacetic acid ( -hiaa), the principal metabolite of -ht, and tryptophan the amino acid precursor of -ht, were also decreased significantly by % and % respectively. gaba and its amino acid precursor glutamic acid were both measured at the same conditions as above, but a precolumn derivatization hplc technique was used. the increase in levels of gaba ( %) and glu ( %) by glp was not significant.the results suggest that decreased synaptosomal levels of -ht and -hiaa caused by glp are due to diminished availability of typtophan by the peptide. in experimental model of iron overload we obtained the following results: the concentration of carbonyl groups tended to increase, while mda level significantly increased after feso treatment ( . Ϯ . vs control . Ϯ . µmol/mg prot.) and ( . Ϯ . vs control . Ϯ . nmol/mg protein p Ͻ . ) respectively. it was associated with significantly increased iron content ( . Ϯ . µg/mg prot. vs control . Ϯ . p Ͻ . ). it is clear that oxidative stress occurs in experimental iron overload, if sufficiently high levels of iron within hepatocytes are achieved. in group treated with feso and spermine, iron content was significantly decreased ( . Ϯ . p Ͻ . compared with fe treated only) and carbonyl group content tended to be lower in comparison to feso treated only ( . Ϯ . ), but mda level didn't change ( . Ϯ . ). in addition, treatment with spermine alone resulted in increase of mda level ( . Ϯ . vs control p Ͻ . ), iron content didn't change ( . Ϯ . ), but carbonyl groups were decreased ( . Ϯ . vs control p Ͻ . ). feso treatment increased gsh level ( . Ϯ . nmol/mg prot. vs control Ϯ . ; p Ͻ . ) while in combination with spermine this increase was more profound ( . Ϯ . ; p Ͻ . vs control, p Ͻ . vs feso ). spermine alone produced similar increase of gsh level ( . Ϯ . , p Ͻ . vs control; p Ͼ . vs feso ). the results emanating from the human genome programme have required a reappraisal of protein science and have led to the rapid upsurge in interest in the area of proteomics. this sudden re-emergence of protein science, in fact, was predictable and should not have been surprising.recent experience of protecting group design with respect to lysine and aspartic acid will be discussed together with aspects of chemical synthesis of small proteins of biological significance and in the context of chemical synthesis methodology making contributions to the general field of proteomics. using a cell line permanently expressing the mouse taurine transporter (mtaut) as a fusion protein, we investigated the underlying mechanism by which the immunosuppressive drug cyclosporin a (csa) inhibits taurine transport. csa inhibited the recombinantly expressed mtaut function both in dose and time dependent manner. the inhibitory effect of csa was reversible. thus, washing out the csa resulted in almost complete recovery of taurine uptake. to obtain further insight, we examined the surface abundance of the mtaut as a function of csa treatment using a surface-labeling assay. our results demonstrated that csa treatment altered the surface expression of the mtaut without significantly altering its total expression level, and the reduction in the cell surface expression paralleled the decrease in taurine uptake. upon removal of csa, the virtual recovery in taurine uptake was due to the concomitant increase in the number of taurine transporters on the cell surface. taken together, our results suggest that csa induced inhibition of taurine uptake was either due to the impaired targeting of the taurine transporters to the cell surface or due to the removal of the transporters from the cell surface. polyamines are neuromodulators in a number of physiological and pathological conditions in cns. since application of ethylene glycols causes hypoactivity and lethargy of experimental animals, depression of cns and various neurological symptoms, the aim of this study was to examine the effects of butoxyetanol on polyamine and gaba catabolism, taking in account an alternative pathway of gaba synthesis from putrescine. methods: male wister rats were allocated into three groups: first treated by be ( mg/kg - days), second key: cord- - jt ksha authors: taylor-cousar, jennifer l.; maier, lisa; downey, gregory p.; wechsler, michael e. title: how i do it: restarting respiratory clinical research in the era of the covid pandemic date: - - journal: nan doi: . /j.chest. . . sha: doc_id: cord_uid: jt ksha the clinical research we do to improve our understanding of disease and development of new therapies has temporarily been paused or delayed as the global healthcare enterprise has focused its attention on those impacted by covid- . while rates of sars-cov- infection are decreasing in many areas, many locations continue to have a high prevalence of infection. nonetheless, research must continue and institutions are considering approaches to re-starting non-covid related clinical investigation. those restarting respiratory research must navigate the added planning challenges that take into account outcome measures that require aerosol generating procedures. such procedures potentially increase risk of transmission of sars-cov- to research staff, utilize limited personal protective equipment, and require conduct in negative pressure rooms. one must also be prepared to address the potential for covid- resurgence. with research subject and staff safety and maintenance of clinical trial data integrity as the guiding principles, here we review key considerations and suggest a step-wise approach for resuming respiratory clinical research. clinical research is critical to our understanding of disease mechanisms and leads to life-saving therapies. during the time that worldwide efforts have been appropriately focused on describing and treating disease caused by the sars-cov- virus, most non-covid clinical research has paused and or pivoted to a covid- research focus. however, now that we have navigated the initial surge of sars-cov- cases, many are considering how to reintroduce non-covid- clinical research conduct while protecting participants, staff and ensuring data integrity. because of the many aerosol generating procedures (agp) that are necessary to generate critical outcomes in respiratory research (e.g. lung sampling with bronchoscopy, nasal brushing, spirometry, administration of nebulized medications, and sputum induction amongst others), the challenges to resuming clinical research in pulmonary disease are numerous, and potentially more complicated than in other areas. here we review key considerations and suggest a step-wise approach for resuming clinical research including observational research, registry trials, and interventional trials, as well as potential data confounding related to covid- infections that are important to consider as research studies restart and data are analyzed. in january , a -year-old woman enrolled in an week, phase randomized trial with planned open label rollover. based on the increasing number of cases of covid- in her state, clinical research conduct was put on hold with the exception of studies that were deemed to have potential benefits to subjects. because the subject was scheduled to roll-over to open label study drug at the end of march , her continued participation was agreed to have benefit. the subject's local government issued a shelter-in-place notice on march , . this ordinance, in combination with the potential risk of infection, decreased the subject's enthusiasm to travel to the research site. development of a new process was deemed necessary to ensure her access open label study drug. to resume clinical research in the era of covid- , several guiding principles should be followed. -research subjects must be kept safe -clinical research staff must be kept safe -the integrity of protocols, data, and outcomes must be maintained. -the process of resuming research should be flexible and dynamic, varying over time and by geography as dictated by the prevalence of covid- , the availability of testing and personal protective equipment (ppe), and by local institutional and governmental policies. clinical research inherently has certain risks, due to either procedures or investigational agents. in the spirit of "do no harm", it is critical that institutional policies and processes are in place to ensure that there is no significant additional risk of contracting viral respiratory or other infections in the normal course of participation in research studies; now during the covid- pandemic, these principles are even more critical. careful consideration should be given regarding screening, enrollment, and continued protocol participation for high-risk participants. equally important, it is imperative that clinical research staff are protected from potential risks of contracting respiratory tract infections including sars-cov- from research subjects or individuals accompanying them. maximizing safety of all can be addressed by adopting several strategies to reduce potential exposure to those infected. covid- screening/testing: the first line of protection is preventing subjects and staff at risk of infection from contracting and propagating disease. while there is some risk associated with asymptomatic individuals spreading disease, the vast majority of covid- transmission is from symptomatic people. in that vein, all staff must self-monitor and self-report any symptoms daily. if these symptoms occur, staff must remove themselves immediately from work and from serving as vectors of infection. similarly, research subjects should be screened for symptoms in the - days before each visit to evaluate whether the subject has symptoms of covid- , or is otherwise at high risk of being infected or infectious. this evaluation is particularly important for immunocompromised patients as they may shed virus longer. screening for symptoms should also be repeated upon arrival at the institution with both survey questions and temperature checks. symptomatic or febrile subjects must undergo covid- testing with research visits postponed until test results have returned and/or symptoms have resolved. whether all subjects, including asymptomatic subjects, should be tested prior to participation in research depends on several factors including local community standards of care, prevalence and testing availability including rapid turnaround time ( ) . testing of some individuals before j o u r n a l p r e -p r o o f they undergo a procedure that is likely to produce a high level of aerosol generation such as bronchoscopy, should be strongly considered and has been implemented at many academic institutions ( ) . at this time, sars-cov- antibody testing has significant limitations due to sensitivity and specificity depending on the platform, and is currently not recommended. however, as testing characteristics improve in the future, this may be helpful in evaluating the safety of research participation and potential for confounding due to covid- as noted below. reducing exposures with administrative and engineering controls and personal protective equipment (ppe). other measures should be taken to reduce potential exposure, including directing access of participants strictly to entrances where monitoring stations are present, limiting or avoiding visitors accompanying participants, minimizing the number or duration of participant visits, using remote monitoring such as telehealth if possible, and staggering participant visits to reduce exposures. additional administrative measures to reduce exposures include limiting non-essential/non-research staff interactions with participants and research areas, and limiting research staff from directly interacting with participants; the latter will also conserve ppe. these preventive measures can be done by managing access to research units, developing one way hallways for -minute walk testing and using plexiglass/plastic barriers and good ventilation in areas when staff and or participants are in close proximity. furthermore, staff and participants should be safely positioned in workplaces and research areas to optimize social distancing. adherence to centers for disease control (cdc), professional society and/or local and state guidelines and policies should be followed for allocation of ppe for research, with priority of allocation for use in clinical care. as outlined by the cdc, central purchase and storage of ppe may be considered to ensure adequate supplies as well as conservation of ppe, especially for face masks and protective eyewear (e.g. face shields, safety glasses and goggles). participants and research staff should be educated in the appropriate use of ppe, including donning and doffing and exposure reduction measures. all must be required to wear face masks or surgical masks as well as protective eyewear for study personnel if available to reduce the spread of infectious droplets and aerosols. aerosol generating procedures, including pulmonary function testing, exhaled nitric oxide measurement, sputum induction, nasopharyngeal sampling, laryngoscopy, and bronchoscopy require additional ppe, inclusive of at least an n filtering facepiece respirator (ffr) and a face shield or a controlled air purifying respirator (capr), gown and gloves. while the occupational safety and health administration (osha) has currently waived the yearly fit testing requirement for ffrs, this testing is necessary if it has not been done recently. the agps should be performed with appropriate engineering controls, including negative pressure and increased air exchanges and filtration, allowing appropriate time for air clearance between procedures, as well as donning and doffing of ppe. scheduling patients' visits must include consideration of ppe, as well as air clearance, required intensified sanitization of the room and or equipment; consideration should be given to limit the number of visits occurring at any given time and to space out participant visits. environmental sampling for sars-cov virus should be considered to evaluate exposures and cleaning and disinfection processes. as an example, surface sampling based on world health organization ( ) or other methods could be considered in potential areas of exposure, including rooms in which agps are performed as well as the equipment used for testing. viral sampling of air in examination and j o u r n a l p r e -p r o o f procedure rooms, and uv light decontamination of rooms and surfaces may be considered using routine industrial hygiene techniques depending on available resources and expertise. a key principle of clinical research is ensuring protocol and data integrity to maximize the generalizability of clinical trial/study results including the endpoints, efficacy, and safety of studied interventions. research goals include timely recruitment, proper adherence to protocolspecified procedures, high retention of participants, and proper statistical analyses to avoid undue loss of statistical power and increased risk of bias due to informative missing data. fleming et al recently recommended several strategies to protect scientific integrity ( ). these approaches include potentially delaying or pausing enrollment, pre-specifying analyses to address effects of the pandemic on trial integrity, and addressing analytical issues such as missing data with validated statistical approaches. considerations for remote study visits: another consideration is to modify protocols to accomplish study goals without interfering with the spirit of the study. these modifications may involve reconsideration of the necessity of all study visits and procedures. in that regard, one could consider implementing telehealth, home health visits and smart technology to facilitate clinical research. prior to the pandemic, the use of telehealth and home outcome assessment (e.g. spirometry, sputum collection and even patient reported outcomes) were limited. the rapid deployment of these clinical tools has allowed their application to clinical research studies, and in some cases ensuring the continued conduct of study consent, safety visits and assessments, and collection of outcome measurements. based on the positive reception from trial participants, it is anticipated that, as in clinical care, telehealth and home assessment options will continue to be incorporated into clinical trials subsequent to the pandemic. the standardization of acquisition and reproducibility of home measurements for cross-sectional and longitudinal studies will need to be established. for example, increased variability in home spirometry measurements may lead to the need to increase sample size to maintain adequate study power to detect differences in outcomes. use of research coordinator tele-coaching for the maneuver could improve reproducibility. as another example, collection of a sputum sample at home by the participant could be inadequate, or the sample could be lost in transit, thus increasing the work of study statisticians to account for missing data points. furthermore, assessment of physical exam findings is limited to those that can be ascertained by observation, and may limit recording of study-related adverse events. on the other hand, use of telehealth for clinical research may increase access to research for potential participants who have previously avoided participation because of time and/or distance from the research site. sponsors and investigators will need to find the balance between access to study participation and the current limitations of telehealth and accuracy of home outcome measures. ). the impact on other assays, such as genomic, epigenetic or immunologic and the duration of the impact is unknown, but is likely to occur with the potential for extensive impact on the immune system ( ) and needs to be considered in analyses. finally, for multi-center studies, the variance in regional allowance of on-site visits and study procedures will need to be considered and the resulting bias adjusted for in data analysis. in addition, consideration may be given to modifying the study procedures to minimize risk. for example, if repeated agps are required to obtain specimens from bronchoscopy, induced sputum or nasopharyngeal sampling, limiting the number of these procedures if required over multiple time points, or pairing with blood specimens to allow comparisons and or allowing studies to substitute collection of specimens with lower risk of viral transmission more frequently might be an option. ensuring use of leftover specimens from procedures being done for clinical purposes and limiting control participant procedures or number are other possibilities. the processing of the specimens needs to be considered as a risk of exposure to staff and some centers have used heat or uv light inactivation of biofluid samples or chemical inactivation (e.g. placement of blood, nasal or bronchial epithelial cells directly into acid-guanidinium-phenol based reagent such as trizol or other virus-inactivating medium) for genomic studies to minimize the possibility of viral exposure. propagation of respiratory epithelial cells collected from a sars-cov- infected individual in submerged or air-liquid interface culture may result in generation of high viral titers and should be done under biosafety level- (bsl- ) conditions. ideally participants and/or their cells should be tested for sars-cov- prior to propagation of respiratory cells in culture. j o u r n a l p r e -p r o o f as each clinical research unit contemplates the approach to re-starting non-covid clinical research at its institution, guidelines from the national, regional and local level should be considered well in advance to allow appropriate preparations to be completed ( figure ). as described above, center for disease control (cdc) ( ) recommendations should be considered in establishing return to clinical research approaches in the health care setting and or other setting. institutional and local mandates: the establishment of crisis standards of care in hospitals (e.g. temporary restrictions on elective procedures), as well as at-home isolation orders and travel restrictions for state citizens are impacted by regional variation in leadership and rates of sars-cov- infection. similarly, the rescinding of such mandates is not uniform across the country and must be considered to determine not only when participants may travel to the research site for visits, but also when research sponsors and monitors can begin study initiation and oversight. the interpretation of city/state mandates by each institution will also dictate the revision of pandemic policies that restrict clinical study activity, including the conduct of outcome measures that result from agps and require ppe. changes made to protocols must be passed by local and central irbs/ethics committees as well as granting agencies and or sponsors as appropriate. federal guidance: both the fda and national institutes of health (nih) have issued publicly available recommendations ( , ) regarding conduct of research in the setting of the covid- pandemic. in the midst of the pandemic, the nih suggested limitation of study visits to those needed for participant safety or those that would be coincident with clinical care, to consideration of the conduct of virtual visits, use of local laboratories for safety monitoring, limitation of unnecessary travel and the cancellation of large gatherings. in acknowledgement of the potential pandemic-related delays in research progress and the incurrence of unanticipated costs, the nih will allow for project extensions and requests for administrative supplements in some cases. similar to the approach of the nih, the fda guidance has used patient safety as the overarching principle to guide recommendations. adherence to good clinical practice (gcp) and minimizing risks to trial integrity are also required. fda guidance encourages sponsors to work closely with investigators and independent ethics committees to determine in which situations subjects' participation in a trial will continue or be paused. furthermore, sponsors and investigators are advised to work closely with institutional review boards to address urgent or emergent changes to the protocol or informed consent that resulted from the pandemic, and to prospectively define procedures to prioritize reporting of protocol deviations that may impact participant safety. necessary protocol modifications that will impact efficacy assessments, data management and statistical analysis plans, should be discussed with the applicable review division. as with all fda research conduct, documentation of changes and their rationale remains critical during the pandemic including changes that "impact on the informed consent process, study visits and procedures, data collection, study monitoring, adverse event reporting, and changes in investigator(s), site staff, and/or monitor(s) secondary to travel restrictions, quarantine measures, or covid- illness itself." with the restart of clinical research, reversal of temporary changes must occur and be documented, including updates to study status on clinicaltrials.gov. finally, when submitting clinical trial study reports to the fda, sponsors will need to address the impact of the covid- pandemic on the reported safety and efficacy results. in order to re-start clinical trials/studies put on hold as a result of the pandemic, and to prepare for the start of new trials, one should consider implementing a multi-stage, risk-based approach that continues to emphasize participant and staff safety ( table ). in each stage, one should consider the pre-pandemic status of the trial (active or in the study initiation phase), the location of the participant (local versus out of state requiring travel), and most importantly, the procedures that will be associated with the visit. unlike research in many other fields, respiratory clinical trials often include a large number of potential and known agps that are used as primary or secondary efficacy measures and outcomes, including spirometry, induced sputum, nasal potential difference, cardiopulmonary exercise tests, laryngoscopy and bronchoscopy amongst others. as outlined above, the availability of negative pressure rooms, ppe fit-tested staff, adequate well-ventilated space for social distancing of returning staff, safe specimen processing areas, disinfection protocols, and availability of ppe must be established before progression to subsequent re-opening stages. to insure that each of the guiding principles has been considered prior to re-starting clinical research, we created the following checklists: research participant and staff safety establish guidelines for location of specimen processing based on source (lowrisk versus known sars-cov- infected subjects and their bodily fluids) ensure laboratory processing space meets safety guidelines for processing of biological specimens re-design mwt area to ensure social distancing between subjects/staff re-evaluate study design and ability to use lower risk procedures and/or samples such as peripheral blood cells versus bronchoalveolar lavage cells or change in study timepoints protocol integrity discuss all potential protocol modifications with sponsor, whether federal and or industry, as well as the institutional review board report any pandemic-related unanticipated events and protocol deviations to the sponsor/irb establish sop for conducting research telehealth visits establish process for shipping of investigational product, home testing kits (e.g. urine, sputum) submit protocol modifications to institutional review board/data monitoring committee/fda/nih update clintrials.gov with protocol modifications/enrollment holds (if indicated) contact sponsor (nih, foundation, etc.) to establish impact on study funding flexibility weekly covid planning/update meetings with research managers and medical director to review local case data, changes in state/federal/institutional guidelines weekly review of environmental testing of clinical research areas by medical director adjust newly established sops as indicated by changing case data and state/federal/institutional guidelines clear plan for regular communication to investigators regarding the move forward through stages of re-opening (or cessation of research based on increased infection rates/lack of ppe and/or testing capabilities) this year has taught all of us that flexibility and adaptability are critical aspects of navigating the uncertainties caused by the pandemic. thus, timelines for progressing to the next stage must be adjusted for subject and staff safety based on successful transition through the previous stage. successful transition will include objective measurements, such as the absence of positive sars-cov- environmental testing on the clinical research unit, absence of covid related adverse outcomes in research subjects (e.g. positive testing for sars-cov- after contact with an asymptomatic but subsequently positive member of the research staff), or local outbreaks of disease in the community. changes in the availability of adequate ppe could also delay the transition from one stage to the next. other considerations moving forward will include the criteria that would lead to another clinical research pause if there is a substantial resurgence of sars-cov- infections. this may include rates of covid- positive infections, hospital and icu capacity, availability of ppe, participant willingness to continue to be involved in research, and movement to crisis standards of care to name a few. these measures should be discussed and outlined ahead of time so that reversion to a prior stage or staying at a current stage can be clear and transparent for staff and participants. this pandemic has taught us that we may need to consider not just sars-cov- infection, but also more closely scrutinize the impact of confounding by other respiratory viruses and infections in many of our vulnerable patient populations as the 'cold and flu season' comes upon us. in order for the subject to remain in the study and continue in the open label arm, the sponsor was contacted to discuss and approve the plan to obtain the minimum required safety tests (urine pregnancy test, complete metabolic panel and complete blood count) at the subject's home, to conduct consent by telephone, and to ship the investigational product to the subject's home. the sponsor reviewed federal drug administration (fda) covid- guidelines ( ), and submitted the necessary changes to the central institutional review board. at the time of the subsequent scheduled follow-up safety visit, telehealth was in place and the subject accepted the option of a visit via telehealth with a home nurse visit to draw safety laboratory values versus an on-site visit. after the sponsor was notified that research spirometry could not be performed per institutional policy, the subject was provided with a home spirometer from the sponsor. if aerosol generating procedures are able to be performed at the time of her next scheduled safety visit, the subject will be given the option of a remote visit, with blood draw and home spirometry, or an on-site visit with spirometry performed in office or at home prior to the visit. throughout the subject's participation in clinical research during the pandemic, she expressed her appreciation for the opportunity to continue in the study from which she believed she was benefiting, with minimal risk of exposure to infection from sars-cov- . in sum, while the world has appropriately focused on combating covid- for the last several months, we are all now shifting our focus to the resumption of clinical care and clinical research under new circumstances. the main priority in clinical research conduct should continue to be the safety of participants, while also considering the safety of staff and data integrity of trials. flexibility, creativity, vigilance and resilience will be critical aspects of restoring and reinventing clinical research participation, and of designing and conducting the trials of the future. public health emergency guidance for industry, investigators, and institutional review boards recommendation from european respiratory group . (respiratory function technologists/scientists): lung function testing during covid- pandemic and beyond the use of bronchoscopy during the covid- pandemic: chest/aabip guideline surface sampling of coronavirus disease (covid- ): a practical "how to" protocol for health care and public health professionals. version conducting clinical research during the covid- pandemic protecting scientific integrity pulmonary fibrosis secondary to covid- : a call to arms? immunology of covid- : current state of the science guidance for nih-funded clinical trials and human subjects studies affected by covid- clinical trials press on for conditions other than covid- . will the pandemic's effects sneak into their data? capr-controlled air purifying respirator cdc-centers for disease control fda -federal drug administration gcp-good clinical practice ffr-filtering facepiece respirator mbw-multiple breath washout nih-national institutes of health no-nitric oxide npd-nasal potential difference, osha-occupational safety and health administration key: cord- -ug ler e authors: ramos-rincón, josé m.; pinargote-celorio, héctor; belinchón-romero, isabel; gonzález-alcaide, gregorio title: a snapshot of pneumonia research activity and collaboration patterns ( – ): a global bibliometric analysis date: - - journal: bmc med res methodol doi: . /s - - - sha: doc_id: cord_uid: ug ler e background: this article describes a bibliometric review of the scientific production, geographical distribution, collaboration, impact, and subject area focus of pneumonia research indexed on the web of science over a -year period. methods: we searched the web of science database using the medical subject heading (mesh) of “pneumonia” from january , to december , . the only document types we studied were original articles and reviews, analyzing descriptive indicators by five-year periods and the scientific production by country, adjusting for population, economic, and research-related parameters. results: a total of , references were retrieved. the number of publications increased steadily over time, from publications in to in (r( ) = . ). the most productive country was the usa ( . %), followed by the uk ( . %) and japan ( . %). research production from china increased by more than %. by geographical area, north america ( . %) and europe ( . %) were most dominant. scientific production in low- and middle-income countries more than tripled, although their overall contribution to the field remained limited (< %). overall, . % of papers were the result of an international collaboration, although this proportion was much higher in sub-saharan africa ( . %) and south asia ( . %). according to the specific mesh terms used, articles focused mainly on “pneumonia, bacterial” ( . %), followed by “pneumonia, pneumococcal” ( . %) and “pneumonia, ventilator-associated” ( . %). conclusions: pneumonia research increased steadily over the -year study period, with europe and north america leading scientific production. about a fifth of all papers reflected international collaborations, and these were most evident in papers from sub-saharan africa and south asia. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. acquired pneumonia (cap) remains the primary cause of death from infectious disease globally, and its high impact on morbidity and mortality is especially concentrated in children under five and the elderly [ , [ ] [ ] [ ] . the world health organization (who) predicted that deaths from lower respiratory tract infections would remain among the top four causes of deaths up to at least [ ] . antibiotic-resistant strains have also been on the rise, although resistance does not appear to be related to mortality. however, pneumonia is associated with high rates of hospitalization and length of hospital stay. moreover, it has considerable long-term effects on quality of life, and long-term prognosis is worse in patients with pneumococcal pneumonia [ ] . despite the public health importance of the disease, few studies have evaluated research in the area using bibliometric methods. indeed, only head et al. ( ) have analyzed publications on pneumonia, and their work was limited in geographical scope to the uk [ , ] . in this study, by analyzing scientific papers on pneumonia published in the main international scientific journals, we aimed to identify the scientific contribution of different countries to the worldwide research effort, the most cited landmark articles, the degree and nature of scientific collaboration, and the topics addressed. this bibliometric description can provide relevant information for researchers in the field, particularly new scientists, giving a snapshot of strong research areas in pneumonia and global health as well as possible gaps requiring additional investments [ ] [ ] [ ] . the paper also provides clues for addressing the weaknesses observed, such as the need to promote north-south collaborations and other research initiatives with countries that have relatively little scientific development on the topic [ , ] . the aim of the present study is to assess the scientific literature on pneumonia that is indexed in the web of science (wos). specifically, we will analyze: ( ) the evolution of scientific production; ( ) its distribution by countries and regions; ( ) the impact of the research papers; and ( ) the degree of international collaboration. finally, we will present details on the subject area focus of different publications according to the medical subject headings (mesh). for the performance of the study, we opted to identify documents about pneumonia by means of the mesh thesaurus in the medline database because this is a detailed instrument for controlled terminology. the thesaurus employs both a human team of specialist indexers to analyze each article and assign medical subject headings to it, plus automated processes to improve indexing; the result is a highly consistent system of classification for research topics [ ] [ ] [ ] . the pneumonia descriptor was introduced in as a disease of the respiratory tract and the lung, and it was defined as "infection of the lung often accompanied by inflammation" [ ] . synonyms of this descriptor (and therefore also included in search results) are "lung inflammation" and "pulmonary inflammation". additional file : table s shows the mesh tree structure for "pneumonia". the next step was to identify the documents assigned with the medline descriptor of "pneumonia" indexed in the wos. this body of research constitutes the population of documents for the present study. conceived by eugene garfield but now maintained by clarivate analytics, wos is the top scientific citation search and analytical information platform worldwide, serving both as a multidisciplinary research tool supporting a variety of scientific tasks and as a dataset for large, dataintensive studies [ ] . the use of the wos databases enables the analysis of all institutional affiliations reported in the documents and the calculation of citation indicators. the wos brings together the most visible literature at a global level. these qualities justify its choice as the database platform used in this study despite some limitations related to covering non-english biomedical journals [ ] . although initially no limitations were imposed on our search, to calculate the bibliometric indicators we considered only two types of documents, articles and reviews, as these are the primary references for researchers. the study period was limited to - , as delays associated with assigning mesh descriptors to documents mean that information on the most recent articles on pneumonia is not updated. the searches took place on the clarivate analytics wos platform, which includes medline database, on march , . for each of the retrieved documents, data on the following bibliographic characteristics were extracted: year of publication, journal of publication and wos subject category, document type, authorship, citations, institutional affiliation(s), and mesh descriptors. data were then standardized: institutional affiliations corresponding to england, northern ireland, scotland and wales were grouped together under "united kingdom," while affiliations in overseas france, british overseas territories, and island dependencies were also assigned to their ruling countries (for example, the documents signed by authors from french polynesia, guadeloupe, martinique, new caledonia, and reunion were assigned to france), although regional designations correspond to geographical rather than political criteria. scientific production from taiwan, which in wos is considered independently from the democratic republic of china (china) but whose status is disputed at an international level, was analyzed separately. countries responsible for publications were categorized according to their world bank classification by income level: low-income (< usd ), lower-middleincome (usd to usd ), upper-middle-income (usd to usd , ) , and high-income (≥ usd , ) countries. each of the countries identified was assigned to a macro geographical (continental) region according to the groups established by the world bank based on geopolitical and economic criteria and reflected in the world bank country and lending groups (see additional file : tables s and s ) [ ] . two kinds of indicators were obtained: descriptive indicators for the evolution of scientific production production by country, adjusted for demographic and economic parameters as well as for human resources dedicated to research activities we determined standardized indicators for each country's productivity with respect to: -population: number of publications per million inhabitants (population index). data were obtained from world development indicators in the world bank online databases [ ] . we calculated a mean value for each indicator based on available data from the study period. the analysis was limited to countries participating in the top articles in the field of pneumonia in order to facilitate comparison between countries' scientific production, demographic indicators, and economic development. results for the top articles are shown in the main text, while those for the top are provided in additional file . we calculated the following citation indicators by journal, country, and geographic region: -citation of the publications. absolute number of citations received. -citation rate (cr). number of citations divided by number of publications. -hirsch index (h-index). the h-index is a semiqualitative proxy measure to assess the impact of an author's or country's research output on the scientific community [ ] . an h-index of indicates that out of published papers have been cited at least times. in order to assess the differences in the distributions of the publications according to the prestige of the journals, we performed a specific analysis of a sub-sample of publications in journals occupying the top % in the impact factor ranking in their respective subject categories in the journal citation reports ( edition). we analyzed participation in these "prestigious journals" according to geographical location (regions and countries), collaboration level and number of citations. we calculated the percentage of documents produced in international collaboration and the evolution by quinquennium in order to estimate the scope of cooperative practices at a global level, considering the whole population of documents analyzed (research field) by country and geographic region. to specifically analyze collaboration between countries, collaboration networks were generated for each of the three quinquenniums using pajek software. to specifically analyze collaboration between countries, collaboration networks were generated for each of the three quinquenniums using pajek software. the collaboration network is a graphic representation (graph), wherein the nodes represent authors' countries (as determined from their institutional affiliations) and links between the nodes represent coauthorships between countries, that is, an international collaboration in published research. the more intense the collaboration, the thicker the links between the nodes. the spatial distribution of the nodes responds to the execution of the kamada-kawai algorithm in pajek, which places the most prominent nodes (those with a greater number of documents and collaboration links) in the center of the map, and the nodes with a smaller number of publications and degree of collaboration towards the periphery. based on an analysis of mesh terms, we identified the main research focus of the studies in the area, generating density maps using the vosviewer program with a spatial description of the main mesh terms for each type of pneumonia [ ] : (a) "pneumonia, aspiration" (b) "pneumonia, bacterial," (c) "pneumonia, ventilator-associated," (d) "pneumonia, viral," and (e) "pneumonia, pneumocystis"). the process of generating and interpreting the maps proceeded as follows: -determination of the co-occurrence of the descriptors assigned to the documents and generation of a matrix of absolute values. the joint assignment of two descriptors in a single document implies a thematic affinity, as both aspects are addressed simultaneously in the same paper. this affinity will be more intense as it is repeated a greater number of times in the collection of documents analyzed. -elimination of generic descriptors. in order to facilitate the analysis, we eliminated some excessively generic descriptors (like "humans" or "animals"), along with geographical descriptors and those related to age groups. these descriptors showed very high-density relationships, complicating the analysis and the interpretation of the results, so we analyzed their frequency more specifically. -visual representation of the network. to establish the main topics that exist for each type of pneumonia and to represent them visually, we used a clustering algorithm in the vosviewer program, which helps to detect the communities (clusters) within a network, made up of groups of homogeneous items that are strongly related to each other. the different groupings, in the form of "islands" in red tones, represent the main clusters of the thematic networks, while the chromatic gradation illustrates the areas with a lower density of relations between the mesh in yellow and green tones. the spatial distribution of the mesh and their proximity to each other responds to the intensity of co-occurrence between them. all data used to perform the study, including the information downloaded from the database as well as that derived from the treatment of the bibliographic entries, are available in the dataverse project, an open access public repository [ ] (https://dataverse.harvard.edu/, doi: https://doi.org/ . /dvn/ bune). due to the nature of the study and dataset, it was not necessary to obtain informed consent or approval from an institutional ethics committee. the search yielded a total of , documents published between and and assigned with the descriptor "pneumonia" in the medline database. of these, , ( . %) were indexed in the wos core collection databases; , ( . %) of them were classified as articles and ( . %) as reviews. thus, the population of study documents was a dataset of , articles and reviews, which we used to calculate the indicators presented below. letters (n = ; . %), editorials (n = , ; . %), news (n = ; . %), proceedings (n = ; . %) and other document types (n = , . %) were excluded from the analysis. the number of publications rose from in to in .the evolution of scientific production by year was fitted to a linear growth model, showing an r value of . . overall, the study period saw a two-fold increase in scientific production (additional file : figure s ). the country with the greatest number of documents was the usa ( . %), followed at some distance by the uk ( . %), japan ( . %), germany ( . %) and france ( . %). table shows the number of documents and the evolution of scientific production in the most productive countries by quinquennium (see additional file : table s for results on the top countries). although the usa ranks first in all periods, its relative contributions have declined, from . % of all documents in - to . % in - . on the other hand, china's emergence is highly notable, with a . % share of total scientific production in the first period (rank = ), compared to a . % share in the third (rank = ). south korea has also seen considerable growth, contributing just . % to total research production in - (rank = ) but . % in - (rank = ). likewise, taiwan and brazil have increased their production from . and . %, respectively, to . and . %. scientific production in different countries and geographic regions, and its evolution by quinquennium, is concentrated in north america and europe & central asia; together these regions are responsible for . table ) (see additional file : figure s for a visual representation of density equalizing mapping projections). table ranks the production of the top countries, adjusted for demographic and economic indicators (see additional file : table s for results on the top countries). when normalized by population, the most productive countries were switzerland, the netherlands, iceland, and denmark. adjusted for the gdp index, the most productive lmics were the gambia, malawi, uganda, and guinea bissau. if we calculate the ratio of pneumonia publications to gni per capita index, the usa, china, india, malawi y brazil were the most productive. adjusting by r&d expenditure index, the usa ranked first, followed by spain, the uk, china, and italy. in relation to the researchers in r&d index, the usa also leads the ranking, followed by india, uganda, and china. (see additional file figure s and figure s for a visual representation of density equalizing mapping projections of the number of documents and world development indicators, by gni per capita index, gdp index and population index plus r&d expenditure index). figure shows the collaboration networks between different countries by quinquennium. the most prominent countries in all time periods, occupying central positions in the networks with multiple cooperative links, are the usa, canada, the uk, germany, france, and the netherlands. the presence of south american and african countries is scarce in all periods. only south africa has a notable presence in the third quinquennium (fig. a) . a few other countries also "emerge" with a high degree of collaborative links in the second period, like spain, greece, italy, australia, china, and japan, although the latter two countries are not fully integrated in global networks, showing collaborative ties only with the usa (fig. b) . finally, other european countries, while present throughout all three periods, stand out to a greater degree in the third period. this is the case of sweden, switzerland, belgium, and austria. at the same time, china and japan seem more implicated in the network in this third period, while india and south korea also gain relevance (fig. c) . the documents we analyzed were published in scientific journals. twelve journals accounted for . % of the pneumonia literature table table s for results on the top journals with highest absolute and relative citations). the comparative analysis of the scientific production and crs of different journals is noteworthy in that some journals (such as the american journal of respiratory and critical care, critical care medicine, and intensive care medicine) present a very high cr in relation to their total scientific production (additional file : figure s for the top journals producing the most research on pneumonia, plus citation rates). with regard to the subject categories to which the journals are assigned, the most prominent are "infectious diseases" ( . % of the documents), "respiratory system" ( . %), "immunology" ( . %), "microbiology" ( . %), and "critical care medicine" ( . %) table . many of the most productive journals in pneumonia also fall into these subject categories. moreover, over the course of the three study periods, nearly all of the subject categories saw a moderate decrease in their relative contribution, as research articles became more dispersed and made headway into different disciplines producing less research on pneumonia table . the analysis of the documents published in the top % of prestigious journals shows a higher participation from the usa ( . %, compared to . % in the overall body of documents) and from some other european countries like the uk or spain. in contrast, the weight of asian countries, particularly japan and china, is much lower (table ) . overall, international collaboration in these journals (n = , . %) was sensibly higher than in the overall body of documents ( . %), and the greater degree of collaboration was much more pronounced for countries like brazil, japan, china, and even european countries like italy and germany ( table ). the high degree of collaboration was also confirmed between regions in the publications appearing in these journals (table ). with regard to the degree of citation, we observed notable increases in the citation rate of the usa and the european countries; these were even more significant for countries in the middle east & north africa, and for sub-saharan africa when they participated in these journals ( table ). with regard to types of pneumonia studied, the mesh terms to appear most frequently were "pneumonia, bacterial" ( . %), followed by "pneumonia, pneumococcal" ( . %), and "pneumonia, ventilator-associated" ( . %). table shows the number of documents assigned to each term describing the different types of pneumonia (additional file : table s for the top general mesh). table ranks the top countries in crude numbers of retrieved articles, stratified by types of pneumonia (additional file : table s for information on the most productive countries). for "pneumonia, aspiration", the main countries were the usa, japan, and germany; for "pneumonia, bacterial", the usa, france, and spain; for "pneumonia, pneumocystis", the usa, france, and the uk; for "pneumonia, ventilator-associated", the usa, france, and spain; and for "pneumonia, viral", the usa, china, and japan. table shows the relationship between mesh terms referring to age groups with those corresponding to different types of pneumonia. the closest associations for "aged, and over" and "aged" were with "pneumonia, aspiration" ( . and . %, respectively), while "pneumonia, viral" was the most frequent topic for studies in pre-adults ("infant", "child", "child, preschool" and "adolescent"). the one exception to this was "infant, newborn", where the highest proportion of articles was about "pneumonia, pneumocystis." in "adult" and "middle aged" people, studies most frequently focused on "pneumonia, bacterial" and "pneumonia, ventilator-associated." figure shows the subject area maps with the main mesh terms in the documents on (a) "pneumonia, aspiration"; (b) "pneumonia, bacterial"; (c) "pneumonia, ventilator-associated"; (d) "pneumonia, viral"; and (e) "pneumonia, pneumocystis." the principal mesh term related to "pneumonia, aspiration" is "deglutition disorder", but research is linked to a broad array of topics, including epidemiological aspects ("incidence", "risk factor", "retrospective studies"), treatment approaches in intensive care, and surgical techniques procedures facilitating breathing, swallowing, and feeding (fig. a) . the two main mesh terms that appear most frequently with "pneumonia, bacterial" are "community-acquired infections" and "anti-bacterial agents", reflecting the central focus that research has taken to identify risk factors and test different therapeutic approaches. mesh terms related to specific bacteria and infections, such as streptococcus, chlamydia, acinetobacter, and haemophilus influenzae, are also prominent (fig. b) . for its part, research on "pneumonia, ventilatorassociated" seems more disperse, although three areas of interest can clearly be differentiated: (a) epidemiological studies, clinical protocols, and treatment in intensive care units (the term "intensive care unit" is the most prominent in this area); (b) treatment outcomes ("treatment outcome" and "anti-bacterial agents"); and (c) cross infections ("cross infection") (fig. c) . research on "pneumonia, viral" also shows a disperse nature, with different areas of interest. epidemiological aspects are covered under terms such as "communityacquired infections" and "hospitalization", while at a researcher level, interests reside in the virus "influenza, human" and "orthomyxoviridae infections" (fig. d) . with regard to "pneumonia, pneumocystis", one prominent subject focus is on "aids-related opportunistic infections" and another is on "pneumocystis jirovecii" (fig. e ). our analysis shows that the number of publications on pneumonia increased notably over the study period, with annual research outputs doubling from to . different factors may have contributed to this. the first of these is the growing research relevance of pneumonia as a clinical entity, as this disease is one of the community-acquired infections with the highest incidence and is an important cause of hospital admissions. it is also associated with a high global burden of morbidity and mortality in both children and adults [ ] [ ] [ ] ] . the second potential factor relates to advances in basic immunological and microbiological research along with deepening knowledge on the pathogenesis of the disease with regard to aspects like microbiological resistance and preventive interventions (e.g. vaccines) [ ] . thirdly, increased funding has been directed toward research and particularly "proactive investments for emerging infectious threats" [ , ] , and finally, the increase in scientific production could be related to scientific development and international dissemination of scientific research in the wos databases. this is particularly the case of china and other emerging economies like brazil, where the rates of growth were highest relative to their respective regions [ ] [ ] [ ] . we observed a substantial increase in research worldwide, but particularly in some geographical regions and countries of south asia, east asia & the pacific, latin america & and the caribbean, and sub-saharan africa. to a great extent, this increase is simply a reflection of the limited contribution to global research that these countries made in the first period analyzed ( ) ( ) ( ) ( ) ( ) . the bulk of scientific production continues to come from countries with more economic and scientific development in europe and north america (together, these countries participated in % of all publications). despite the striking increase in scientific production across lmics, the relative contribution to pneumonia research remains very modest, and the fact that some countries rank highest in demographic and economic indicators may not be a positive feature, but rather a reflection of the scant development in their scientific systems. furthermore, the increase in international collaboration could have played a role in these indicators, multiplying the assignment of articles to different countries and possibly inflating some values, masking the real contribution of countries with less scientific development in research activities [ ] . the usa is undoubtedly the main reference for pneumonia researchers in quantitative terms, as it produces by far the largest volume of publications-four times that of the next most productive country in the last period. other european countries with important scientific systems (e.g. the uk, germany, france, and spain), along with other countries like japan, canada, china, india, and brazil, also stand out in relation to some of the indicators of scientific production and economic development (gni per capita index, and r&d expenditure index). the other significant aspect in the analysis of how scientific production evolved over the study period is the emergence of china, which in the last period of study ( ) ( ) ( ) ( ) ( ) trailed only the usa in research output. this growth has come about in large part from the investments and scientific policies to foster openness that have been implemented over the past several decades to promote internationalization [ , ] . the level of international scientific collaboration that we have observed in the field of pneumonia ( %) is below that seen in other areas of knowledge [ , , , [ ] [ ] [ ] [ ] . thus, even though the trend is toward increased international cooperation, rising from to % over the study period, implementing new strategies that favor collaboration is still necessary [ ] . initiatives promoting research could include those launched by international organizations, such as the world health organization (who) and the bill & melinda gates foundation, which have both invested considerable resources to investigate the etiology of childhood pneumonia in low-income countries [ ] [ ] [ ] . however, these initiatives [ ] are also collaborating in different projects related to hiv, tuberculosis, and malaria, and these organizations are largely responsible for the important degree of collaboration between european and sub-saharan african countries [ ] . research for operational health services is necessary to improve the distribution and accessibility of pneumonia treatments, including antibiotics in primary healthcare centers and oxygen in hospitals. likewise, new vaccines still need to be developed for strains of pneumococcus that current multivalent conjugate vaccines do not protect against [ ] . in addition to programs focused on financing and implementing collaborative north-south and south-south projects, other efforts could be directed toward reducing obstacles associated with publication processes fig. subject area maps with the main mesh terms associated with different types of pneumonia-(a) "pneumonia, aspiration" (b) "pneumonia, bacterial, " (c) "pneumonia, ventilator-associated, " (d) "pneumonia, viral, " and (e) "pneumonia, pneumocystis" groupings in the form of "islands" in red tones represent the main clusters of the thematic networks, while the chromatic gradation in yellow and green tones illustrates the areas with a lower density of relations between the mesh. the spatial distribution of the mesh and their proximity to each other responds to the intensity of co-occurrence between them that limit the dissemination of lmics through the main international scientific journals. the literature has described obstacles related to linguistic skills and methodological deficiencies, which highlights the need to improve these areas in particular [ , ] . other authors have pointed to the costs associated with publishing in open access journals, so it is worth assessing whether the programs to support open access publishing implemented at an institutional level and by publishers such as plos, biomed central, or the lancet journals, are sufficient [ ] [ ] [ ] . with regard to the impact of research, although europe and north america are balanced in terms of the absolute number of citations, north america holds an advantage in terms of the citation rate. research from sub-saharan africa also has a very high citation rate, which almost reaches that achieved in europe. the fact that these african countries present a high degree of collaboration with researchers in the usa and europe, who represent the "mainstream" international research interests, could help explain the high citation rates seen in this region. on the other hand, latin america & caribbean, south asia, and east asia & pacific are all regions with generally lower citation rates, although this difference is not so pronounced in the case of papers produced in collaboration, as reported elsewhere [ ] . by country, the hegemony of the usa and several european countries in terms of the number of citations received was evident, as was the lower ranking of some asian countries, such as japan and china, in relation to their scientific production. the positioning of china as a reference for scientific production and participation in international research networks does not correspond to its ranking with regard to citation indicators, despite their improved standing over the past several years [ ] . on these indicators, china still lags behind the usa as well as the leading european countries, canada, australia and even nearby countries such as japan. for now at least, the countries that have traditionally occupied the "mainstream" of scientific research still maintain their hegemony [ ] . as with the relative indicators of scientific production adjusted for economic and demographic parameters, some countries surpass the major scientific systems with regard to the citation rate, which links the degree of citation with the volume of scientific production [ ] . these countries may have participated in certain highly relevant contributions, or they may be small countries with highly developed scientific systems, such as vietnam, switzerland, south africa, new zealand, and saudi arabia. these countries also stand out for their high levels of international collaboration, which is a factor associated with more citations. the high mean citations received by publications produced in sub-saharan africa, and the participation of different emerging countries like vietnam and south africa in some of the highest cited papers we identified, underlines the capacity of these countries to contribute to high-impact and excellent-quality scientific studies. this result is consistent with previous studies that have also demonstrated these countries' capacity to participate in emerging research topics [ ] . these specialists therefore represent an excellent asset, strengthening the human capital from high-income countries and enabling the advancement of research [ , ] . in general, the most prestigious journals show a greater concentration of research from the usa and europe, with greater collaboration and impact when countries from other geographical regions also participate [ ] . bacterial pneumonia is the main branch for the multidisciplinary and multipathological mesh of "pneumonia", with the main areas of interest ("community-acquired infections", "anti-bacterial agents" and "treatment outcome") reflecting the focus of research on identifying risk factors and assessing different treatments and their outcomes. in publications pertaining to the mesh "pneumonia, ventilator-associated," the main axes of the subject content according to the mesh terms were the group of epidemiological studies and clinical and treatment protocols in intensive care. "pneumonia pneumocystis," is closely related to infection due to hiv and immunodepression. the main areas of research interest for "pneumonia, viral," were the epidemiological aspects related to the setting for the infection ("community-acquired infections" and "hospitalization") along with the viruses responsible ("influenza, human" and "orthomyxoviridae infections"). finally, for the mesh "pneumonia, aspiration" the main research focus is "deglutition disorder". the main limitation of this present study is its analysis of only the documents included in the wos databases and medline ( % of the documents). thus, a number of papers were excluded from the study, particularly those written in languages other than english, as well as the proceedings included in wos, as our searches were based on the journals included in medline. on the other hand, our approach also allowed us to precisely characterize collaboration in the area, as only recently has medline begun to include all the institutional affiliations of the authors. we were also able to analyze the citations of the publications, with a focus on the journals with the highest impact and dissemination at an international level [ ] . in conclusion, pneumonia research increased steadily over the -year study period, with europe and north america leading scientific production. about a fifth of all papers reflected international collaborations, and these were most evident in papers from sub-saharan africa and south asia. additional file : table s . descriptors included under the mesh "pneumonia" in pubmed. table s . top countries in crude numbers of retrieved articles in "pneumonia, aspiration", "pneumonia, bacterial", "pneumonia pneumocystis", "pneumonia, ventilator-associated", and "pneumonia, viral" mesh. figure s . evolution of scientific production on pneumonia ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . clinical and economic burden of communityacquired pneumonia among adults in europe burden of community-acquired pneumonia in north american adults global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for causes of death, - : a systematic analysis for the global burden of disease study impact of age and comorbidity on cause and outcome in communityacquired pneumonia the burden of community-acquired pneumonia in the elderly: the spanish evan- study burden of pneumococcal community-acquired pneumonia in adults across europe: a literature review all cause mortality estimates for - . who mapping pneumonia research: a systematic analysis of uk investments and published outputs - investments in respiratory infectious disease research - : a systematic analysis of uk funding scientometrics analysis of research activity and collaboration patterns in chagas cardiomyopathy evolution of cooperation patterns in psoriasis research: co-authorship network analysis of papers in medline bibliometric analysis of leishmaniasis research in medline ( - ) investment in pneumonia and pneumococcal research medical subject headings used to search the biomedical literature mesh now: automatic mesh indexing at pubmed scale via learning to rank years on -is the nlm medical text indexer still useful and relevant? 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publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we gratefully acknowledge the assistance of meggan harris in translating our manuscript from spanish.authors' contributions jmrr: study conception, study, design, data analysis, manuscript writing and final manuscript approval; hpc: data collection, data analysis, manuscript writing and final manuscript approval; ibr: study conception, manuscript writing and final manuscript approval; gga: study conception, study design, data collection, data analysis, manuscript writing and final manuscript approval no funding was received for this work. all data used to perform the study, including the information downloaded from the database as well as that derived from the treatment of the bibliographic entries, are available in the dataverse project, an open access public repository [ ] (https://dataverse.harvard.edu/, doi: https://doi.org/ . /dvn/ bune). due to the nature of the study and dataset, it was not necessary to obtain informed consent or approval from an institutional ethics committee. the authors give consent to publish the manuscript. the authors declare that they have no competing interests. key: cord- -sj qdi q authors: matzke, lise a; watson, peter h title: biobanking for cancer biomarker research: issues and solutions date: - - journal: biomark insights doi: . / sha: doc_id: cord_uid: sj qdi q biomarkers are critical tools that underpin precision medicine. however there has been slow progress and frequent failure of biomarker development. the root causes are multifactorial. here, we focus on the need for fast, efficient, and reliable access to quality biospecimens as a critical area that impacts biomarker development. we discuss the past history of biobanking and the evolution of biobanking processes relevant to the specific area of cancer biomarker development as an example, and describe some solutions that can improve this area, thus potentially accelerating biomarker research. biomarkers have been defined broadly as "a defined characteristic that is measured as an indicator of normal or pathogenic biological processes, or the biological responses to an exposure or intervention." the importance of biomarkers lies in their central role in the implementation of "precision medicine" to guide decisions and management strategies. , there are many types of biomarkers and these can be delineated broadly as either molecular, histologic, physiologic or in some cases radiographic characteristics. nevertheless, access to human biospecimens and accompanying data is essential to enable the discovery, translation, validation and implementation of most gene based biomarkers. , cancer gene biomarkers can be classified into those that delineate risk of disease, define diagnosis, or provide prognostic, predictive, or monitoring information to guide decisions around therapy and management. but despite their importance and potential in cancer care, the actual numbers of biomarkers currently deployed routinely for the management of common cancers are still few and far between. for example, breast cancer management remains founded on mostly a mix of a few historical prognostic features (eg, tumor size and grade) and a very limited number of prognostic gene expression panel type biomarkers (eg, oncotype dx) and predictive gene biomarkers (eg, er and her ). the development of the now well established her biomarker is illustrative of the problem. even if you discount the decade or more of basic research that created knowledge of these genes and tools to interrogate them, a period of ~ years elapsed from initial human tumor studies to implementation of a robust assay and scoring approach. for the more recently adopted oncotype dx gene panel assay, one can also discount the decade or more required to compile the first cohorts of biospecimens and data used in establishing the prognostic value of individual component genes as candidates to be considered for the final panel. but a period of over years elapsed from then to the point of recognition and clinical approval to deploy the final assay. one could argue that several other genes that were once considered to be promising biomarkers and did not make it on their own are part of current prognostic gene panels (eg, ki , c-myc within the oncotype dx panel), but for the most part the progress in developing new gene biomarkers remains well short of the promise conveyed by the literature. there are many established factors and reasons for slow progress and frequent failure in the biomarker development pipeline that have been discussed by others. [ ] [ ] [ ] but there are also other issues that have not been widely considered. one issue that remains to be proven is the interesting recent suggestion that the potential or promise of early research findings is systematically overexaggerated. and that the explanation for the gap between high promise and actual outcomes in the scientific literature may be attributable to overrepresentation of male gender and imbalance in the main authors of papers. another issue that has not been widely discussed that we will focus on in this review is the challenges in obtaining fast, efficient, and reliable access to the right quality biospecimens and biospecimen cohorts. we will first discuss the current landscape of research biobanking, and then the most important factors around obtaining biospecimens for current research. and finally, we present some solutions that facilitate better access to biospecimens and data for research. biospecimens and annotating data are compiled for biomarker research through a process called biobanking that is coordinated by an entity called a biobank. there are many types of biobanks. some prefer to restrict the term "biobank" to mean only a dedicated entity that compiles a biospecimen collection with the intention of making this collection available for future research and for qualified researchers. but the term "biobank" is best considered to encompass all types of research collections irrespective of size, type or intended use because their origins and intended purposes are the same. in the early 's biobanks were rare as they developed as a research tool mostly associated with a small emerging sector known as "translational research." since then the use of human biospecimens and data has expanded dramatically to the point that biospecimens contribute to the data in ~ % of all cancer research papers and biobanks of all types have proliferated and expanded to serve research across all sectors from basic discovery to clinical validation research. the process of biobanking has also changed significantly over the past decades, driven largely by the need for increased scale and by increased emphasis on quality, as appreciation of the potential impact of preanalytical variables has slowly developed. , , this evolution has been accompanied by maturation of best practices that have served as the basis for the development of national and international standards and most recently, external quality assurance programs for biobanks. [ ] [ ] [ ] [ ] another aspect of changes in biobanking since the early 's was the gradual separation of research biobanking from clinical biobanking. this has served research fairly well until the 's when several new factors emerged that have begun to drive the consideration of reintegration of research and clinical biobanking. paramount amongst these new factors is the increased research appetite for access to the clinical pathology ffpe archives. , biobanks and challenges these changes in models and processes have contributed to some acute challenges that now limit biomarker research and threaten the realization of our current investment in developing precision medicine. one issue is that the research biobanking system has become inefficient and hard to sustain. further systemic issues exist: too many biobanks are hidden or hard to find; were created with inadequate quality considerations; or are comprised of biospecimens and data of an unknown standard; and difficult legacy decisions around what collections to continue to store and what to discard, are becoming commonplace. furthermore, information about the source of biospecimens and the quality standards associated with them is not systematically reported in published research and is rarely considered in peer review, making the research findings harder to reproduce. the publication of relevant reporting standards by the translational research (remark) and biobank (brisq) communities have attempted to address this. for individual researchers the challenge of obtaining the right biospecimens for a biomarker research study is very familiar. this challenge has in part been ameliorated by the increasing availability of high quality "digital" biospecimen derived datasets comprising research data generated from biospecimen cohorts that is often included as supplemental data in papers on individual studies, or is available from large national and international "omics" initiatives that continue to be improved and expanded (eg, tcga and human protein atlas , ). with the recent disruption to biobanking caused by the covid- pandemic these existing digital resources have become even more important. however, the utility of these datasets for cancer biomarker studies have limitations, , and new biospecimen cohorts continue to be needed. biospecimens can be obtained by locating an existing collection, by collecting the necessary biospecimens specifically for the study, or by contracting with another entity to provide some or all of the necessary biobanking services to collect them for the study. in any given research situation, the relevance and best choice between these options, varies depending on many factors. sometimes there is factor that is decisive or only option is feasible. but more often there are several factors that are relevant but have different degrees of importance and so each has a graded influence that needs to be weighed in order to make the best and informed decision as to the most appropriate option. we will discuss some of the most important factors in determining the best option relating to the ( ) study design; ( ) the biospecimen and data quality needed to address the research question; ( ) the representation required of the biospecimen; and ) the overall cost and effort that can be justified and supported. important features of research study design involving biospecimens have been summarized by simon et al. these include the observational versus intentional characteristics of the study, statistical design considerations, and biospecimen factors such as the degree to which the biospecimens and data are/were collected with the focus of a specific study research question in mind, and the degree to which control is/was exerted over the details of the biospecimen and data collection process. these biospecimen factors can essentially be distilled to the retrospective vs prospective nature of the collection process and this strongly influences the strength or "level of evidence" , that the results of a study are considered to be capable of generating and the extent of validation needed. for biomarker discovery research, the demand for sets of biospecimens is often based on tissue level criteria. these are selected on the basis of factors such as representative pathology in the tissue and on the basis of preservation formats or ability to obtain live cells. in the subsequent "translation" and "validation" phases, the research focus moves to disease and then participant levels and more specifically defined cohorts and then establishing potential clinical relevance in preclinical studies. these latter phases in particular, are dependent on biospecimen quality achieved through meticulous adherence to standards and documentation of all processes including the biobanking component. quality is usually considered in terms of the intrinsic features of a biospecimen and its annotating data that determines its fitness for research purpose. intrinsic qualities include the composition, condition, and preservation of the biospecimen, the data concerning the collection and processing, and the data concerning the source that can include patient demographic and clinical data at the time of collection. verification of these aspects of quality and standardization of the processes associated with collection and storage of biospecimens and data is increasingly recognized to be important. quality control (qc) approaches to determine the quality of extracted derivatives such as dna, rna, and proteins are well recognized in the laboratory. in addition several organizations have created internationally recognized biobanking standards, including the recent development of the iso standard, and associated external quality assurance (qa) programs for biobanks that provide assurance of a known standard and level of quality around the broader "biobanking processes." there are also other aspects or extrinsic quality features that determine the degree to which a biospecimen is valued for an intended research purpose. these extrinsic or "complex" qualities include; spatial and temporal relationships that link a biospecimen to other biospecimens from the same participant (eg, primary tumor resection and subsequent biopsy of a later metastasis), information and annotation generated by additional analysis of the biospecimen during handling by the biobank, and the degree to which the biospecimen is representative of the population under study. this last challenge, obtaining biospecimens that are truly representative of the population or acknowledging the extent to which a research cohort under study is representative of the population, may be a significant factor in failure of biomarker validation. however this is often not addressed in the cancer biomarker literature. many translational cancer research studies are based on retrospective case cohorts with to -year follow-up data that are provided by tumor biobanks. however even a biobank that accrues cases across a geographical population can experience significant changes over time in the proportion of incident cases that it is able to collect and therefore changes in the degree to which cases are representative. in the example biobank shown in figure , major factors in the decline in accrual were the diminishing tumor size and increased clinical requirements for sampling, precluding collection of fresh tumor tissue from earlier stage tumors for research. even if known factors such as tumor size are controlled for in the selection of research cohorts from a biobank, other important selection biases can persist. for example, the widespread use of tissue microarrays to study cancer biomarkers is subject to bias in the construction of the tmas. tmas are typically based on coring central regions of tumor blocks and so invasive margins are less well represented. therefore, biomarkers and other features that have a regional distribution within tumors, such as those associated with tertiary lymphoid structures, are underrepresented in most tmas. to illustrate further potential bias in cohorts used for biomarker studies, table shows how different cohort compositions can be observed with respect to breast cancer molecular subtypes between examples of translational studies , based on selected cohorts as compared to population based studies , based on population/registry based data from the same regions. the overall cost and the level of evidence that the research is intended to generate are often among the most important factors influencing the investigator's decision about acquiring biospecimens. acquiring biospecimens for research involves harnessing or deploying resources such as capital, operating costs, personnel time, and effort. these expenses may not be incurred directly by the researcher collecting the biospecimen graph showing biobank collection profile over time. the graph shows the decline in the proportion of all incident breast cancer cases from which a frozen sample for research was collected by a provincial tumor biobank in canada over a -year period. but they will be incurred by someone in the process of acquisition. these costs are typically highest if the researcher creates their own biobank, intermediate if an existing biobank can be contracted to obtain the necessary biospecimens, and lowest if biospecimens are already available in an existing biobank. however, the level of evidence generated when biospecimens are collected prospectively for a specific study (as in creating a new biobank directly or through a contract) is higher than when biospecimens are obtained from an existing biobank. cancer research is dynamic and constantly evolving. this means that the types and details of biospecimen requirements also evolves and that the main pathways for researchers to obtain annotated biospecimens should be expected to change. while the emphasis differs in different areas of cancer research, the dominant route for research focused on biomarkers for guiding management of disease has, until recently, driven the widespread adoption and use of the "classic" biobank operating model which is to collect biospecimens and annotating data in order to store them and generate a large stock collection from which specific biospecimen cohorts could be selected for a given study. other biospecimen pathways (direct from person or patient and indirect via a clinical archive) are also important in cancer gene biomarker research (see figure ). however in the mid to late 's new methodologies (eg, high throughput proteomics applied to formalin-fixed-paraffin-embedded "ffpe" materials), technologies (eg, detection of ctdna in blood plasma by sequencing), research concepts (eg, a new focus on the dynamic balance between tumor and immune system and appreciation of tumor heterogeneity and clonal evolution), and the expanding scale of biomarker research (eg, cancer genome atlas, gwas studies, etc.), have all driven a new appetite for biospecimens. in particular, in research focused on biomarkers for guiding management of disease there is a significant switch to seeking access to biospecimens obtained direct from patients (eg, serial blood samples) or indirectly via pathology (eg, ffpe blocks ) (see figure ). this means that the dominant biospecimen pathway supporting this area of research is now changing rapidly. key solutions that improve access to these changing pathways include ( ) transforming biobank services; ( ) utilizing biobank and biospecimen locators; ( ) implementing programs that facilitate the research clinical access pathway. these are discussed further in the next sections. tumor biobanks can best serve changes in research needs by refocusing their operating models and the investment in their expertise in biobanking. many disease focused biobanks such as tumor biobanks, operate principally on a "classic" or "stock" based operation model. but their existing stocks of preassembled frozen cases representing primary tumors are now less in demand. at the same time these biobanks have distinct expertise in assembling cases and often well-established interactions with pathology departments. instead of prioritizing and expanding their own preassembled biospecimen collections, biobanks should shift their operations to prioritize providing brokerage services to facilitate research access to clinical pathology archives and biobanking services to assist with consenting and collection of bespoke cohorts for individual research studies on a contract service basis. finding a biobank can be a challenge for researchers. many biobanks and networks of biobanks are represented through locators which are online displays of contact and requirements details, or simple lists of biobanks and categories of materials available (eg, fresh and or ffpe specimens ), or more complex displays of "aggregated" data and are often combined with query tools. the most extensive locator developed by the bbmri-eric is based on methodical data sharing terminology and augmented by the addition of a "negotiator" function to improve the ability to follow through a query to secure approval for access. however all locators have to overcome perceptions of barriers to specimen access and a preference for local known sample sources. , many of these locators can be found online by using relevant search terms, as shown in table . however, choosing the right terms can still be challenging and many locators are unable to provide sufficient biospecimen level detail on existing stocks or to identify biobanks that can provide specific bespoke services. nevertheless, use of locators that allow researchers to query across groups of dedicated biobanks can be a very efficient way for a researcher to obtain the necessary biospecimens. this is because dedicated biobanks will have (a) a governance structure and operational capacity that makes it possible to apply for biospecimens, (b) an open access policy to the materials determined only by research quality criteria and not restricted by the condition of establishing a research collaboration, (c) appropriate consent and research ethics approval already in place to allow rapid distribution of biospecimens and data, and (d) expertise and advice available to refine the best selection of cases to match the research question and assays proposed. researchers can access some important types of biospecimens (eg, blood samples) directly from normal individuals and patients. but, as noted above, to access biospecimens representing the full range of pathological processes in tissues, researchers increasingly seek access to ffpe biospecimens in clinical archives. these ffpe biospecimens have been obtained in the course of clinical diagnosis and treatment and are stored as part of the patient record and to support future care and testing. unlike many biobank biospecimens these materials hold several distinct advantages: they contain most categories of pathology (that in some cases are very rarely available in a research biobank), they are preserved and processed in the same clinical format used for clinical testing, and the scope of these biospecimens is population based. accessing archival ffpe materials is contingent on a complex process that the clinical laboratory communicates and operates for the purpose of retrieving materials for clinical purposes, as well as adapting to support research requests. while no one uniform standard for the research access process exists, it typically involves an application by the researcher, and evaluation of regulatory documentation (such as proof of ethics review, consent of the patient, and proof of study funding), an evaluation by the laboratory of whether materials can be provided without eroding the capability to support possible future assessments, and then a release of materials accordingly. in many cases the added task of determining the most suitable ffpe block for research from review of h&e stained clinical slides cut from the many sample blocks associated with each pathology case adds a significant extra step. finally, the process is keenly dependent on an ability for the block material to be recalled efficiently from short-or long-term storage that in many instances involves another service provider and competing workload priorities with clinical requests, and the entire process needs to be appropriately tracked and recorded to ensure integrity of the clinical archive. as a result, accessing clinical archives can be a lengthy processes and clinical priorities or lack of clear process can add further delays for researchers accessing the materials that are critical to the research pipeline. as listed in table there are many discrete steps, for the researcher and for pathology, involved in requesting and obtaining ffpe blocks. perhaps not surprisingly there are many interface issues that arise related to this process that have been identified by both pathology departments and researchers in the canadian landscape (see figure ). strategies to enhance this access process have been proposed in the netherlands, denmark, and canada including concepts that have been designed to create platforms that embed staff in pathology departments with the sole focus of receiving and processing research requests. these platforms also aim to provide researchers with better communication on how to apply, a mechanism to catalog and track research requests, retrieve and ship materials to researchers and monitor demand. a common goal of shortening turnaround times and ensuring sound process that meets the needs of clinical pathology and researchers are a hallmark of all these platforms. however, embedding an understanding of research needs and a standard process for response to researchers at the level of individual pathology laboratories is also important. to address this the canadian tissue repository network (ctrnet) has developed a program called the pathology research support certificate program to provide standards and education for personnel within clinical pathology laboratories who are involved in the decisions or the processes of providing support for research involving biobanking. the program is accessible online, self-paced, and provides information on the key issues for consideration by a diagnostic pathology department with respect to supporting research involving biobanking and a national standard that delineates a standardized approach and the practices to be implemented to provide research access and support (see figure ) . the education and standards integral to biomarker insights this program have undergone review and input from leaders in pathology and biobanking across canada and australia and the program is accredited by university of british columbia (continuing professional development accreditation for . maintenance of competence section self-assessment hours). the inability to efficiently access the right biospecimens, contributes to failures in biomarker development. in particular, the high demand in the face of multiple challenges in accessing clinical archival materials are specific factors that ought to be recognized as significant issues to address. improved mechanisms that enable appropriate and efficient access by researchers whilst maintaining integrity of the clinical archives and adhering to the requirements set out by the needs of clinicians, is critical. biobanks can play an important role in addressing these issues by redirecting their expertise to brokering access to clinical specimens as well as focusing on services that provide researchers with bespoke models of collecting and processing biospecimens that are right for their biomarker research. the current evolution of biobanks from the existing prevalent classic model to prospective and services-based models, coupled with development of tools and programs aimed at improving the ways researchers can find biobank resources and disseminating common standards for access to clinical archives, and will ultimately improve biomarker discovery. us food and drug administration. the best resource: harmonizing biomarker terminology what are biomarkers? implementation of biomarker-driven cancer therapy: existing tools and remaining gaps national institutes of health. best (biomarkers, endpoints, and other tools) resource. bethesda, md: national institutes of health use of archived specimens in evaluation of prognostic and predictive biomarkers the importance of human tissue bioresources in advancing biomedical research prognostic and predictive factors in early-stage breast cancer development of the -gene assay and its application in clinical practice and clinical trials waste, leaks, and failures in the biomarker pipeline breaking a vicious cycle biomarker validation and testing papers with male authors are more self-promoting. the economist research perspective on utilizing and valuing tumor biobanks biobanking . : evidence based and customer focused biobanking biospecimen use in cancer research over two decades in search of an evidencebased strategy for quality assessment of human tissue samples: report of the tissue biospecimen research working group of the spanish biobank network biobanking in health care: evolution and future directions certification for biobanks: the program developed by the canadian tumour repository network (ctrnet) the evolution of biobanking best practices the college of american pathologists biorepository accreditation program: results from the first years standardization and innovation in paving a path to a better future: an update of activities in iso/ tc /wg biobanks and bioresources precision medicine: driving the evolution of biobanking quality the factors that drive the increasing use of ffpe tissue in basic and translational cancer research building a 'repository of science': the importance of integrating biobanks within molecular pathology programmes a framework for biobank sustainability fundamental considerations for biobank legacy planning reporting recommendations for tumor marker prognostic studies (remark): explanation and elaboration biospecimen reporting for improved study quality (brisq ) overcoming the translational roadblocks: a cancer care and research model before and after: comparison of legacy and harmonized tcga genomic data commons' data the human protein atlas: a spatial map of the human proteome an integrated tcga pan-cancer clinical data resource to drive high-quality survival outcome analytics pan-cancer analysis of genomic sequencing among the elderly rules of evidence and clinical recommendations on the use of antithrombotic agents biospecimen complexity and the evolution of biobanks biobanks for life sciences and personalized medicine: importance of standardization, biosafety, biosecurity, and data management comparison and analysis of two internationally recognized biobanking standards biospecimen complexity -the next challenge for cancer research biobanks? biobanking in the twenty-first century: driving population metrics into biobanking quality the single-cell pathology landscape of breast cancer cd and intratumoral immune response in breast cancer risk of locoregional recurrence and distant metastases of patients with invasive breast cancer up to ten years after diagnosis -results from a registry-based study from germany us incidence of breast cancer subtypes defined by joint hormone receptor and her status commentary on improving biospecimen utilization by classic biobanks: identifying past and minimizing future mistakes factors that drive the increasing use of ffpe tissue in basic and translational cancer research a model to estimate frozen tissue collection targets in biobanks to support cancer research the utilization of biospecimens: impact of the choice of biobanking model the australian and new zealand children's haematology/oncology group biobanking network bbmri-eric directory: biobanks with over million biological samples. biopreserv biobank a minimum data set for sharing biobank samples, information, and data: miabis extending the minimum information about biobank data sharing terminology to describe samples, sample donors, and events a decentralized it architecture for locating and negotiating access to biobank samples the barriers and motivators to using human tissues for research: the views of uk-based biomedical researchers pathology databanking and biobanking in the netherlands, a central role for palga, the nationwide histopathology and cytopathology data network and archive evolutionary concepts in biobanking -the bc biolibrary pathology research support certificate program we gratefully acknowledge support for this work by the biobanking and biospecimen research program at bc cancer key: cord- -zap dta authors: bai, xiao; sun, huaping; lu, shibao; taghizadeh-hesary, farhad title: a review of micro-based systemic risk research from multiple perspectives date: - - journal: entropy (basel) doi: . /e sha: doc_id: cord_uid: zap dta the covid- pandemic has brought about a heavy impact on the world economy, which arouses growing concerns about potential systemic risk, taking place in countries and regions. at this critical moment, it makes sense to interpret the systemic risk from the perspective of the financial crisis framework. by combing the latest research on systemic risks, we may arrive at some precautions relating to the current events. this literature review verifies the origin of systemic risk research. by comparing the retrieved and screened systemic literature with the relevant research on the financial crisis, more focus on the micro-foundations of systemic risk has been discovered. besides, the measurement methods of systemic risks and the introduction of interdisciplinary methods have made the research in this field particularly active. this paper synthesizes the previous research conclusions to find the appropriate definition of systemic risk and combs the research literature of systemic risk from two lines: firstly, conducting the division according to the sub-branch fields within the financial discipline and the relevant interdisciplinary research methods, which is helpful for scholars within and outside the discipline to have a more systematic understanding of the research in this field. secondly predicting the research direction that can be expanded in this field. the study of systemic risk suddenly increased after the subprime crisis. compared with the previous financial crisis, although there is an overlap between the two, systemic risk includes not only the information asymmetry, liquidity, and crisis transmission pathways involved in the financial crisis, but also the research on the stability problems caused by the structure of the system itself, the quantification of systemic risks, and early warning issues. as a result, further research on the market microstructure and agent heterogeneity has also been triggered. meanwhile, cross-disciplinary research methods from other disciplines have been introduced, such as the introduction of complex network models when studying the structural stability of the system, linking the contagious effects of financial systemic risks to the transmission pathways of infectious diseases or bio-food chains [ ] [ ] [ ] [ ] [ ] [ ] , establishing new measures to measure systemic risk [ ] [ ] [ ] [ ] . different measurement methods in the financial sub-branch field are also applied to empirically verify and predict the tail loss of systemic risks [ , , ] . what does systemic risk research study? what methods are used to study systemic risks? these are the questions that this review will always revolve around. the former one refers to a framework the scope of systemic risk and systematic risk is not the same. the latter refers to the risk factors shared by the entire economy, the non-distributable risk, also known as the market risk; it is opposite to non-systematic risk. systemic risk refers to the risk of an overall collapse of the entire system due to the impact of individual member units, and systemic collapse is reflected in the impact of most or all individual members [ ] . according to the econlit economic literature database search results, the term "systemic risk" can be traced back to the speech of andrew f. brimmer, a fed official in , at a joint annual meeting of aea (american economic association) and the society of government economists in new york (this speech was published in the journal of economic perspective the following year. see: [ ] ), there are different views on the clear definition of systemic risk in academia. [ ] first proposed the systemic risk and defined it as the occurrence and spread of a dilemma that is likely to seriously affect a country's financial order. the dilemma itself may be derived from micro factors or macro factors. the bis describes systemic risk as to the possibility that a party to a contract cannot perform an agreement and causes other contractors to default, and thus the chain reaction leads to a wider range of financial difficulties. this definition focuses on the overall risk of the system caused by the breach from microbodies. besides, darryl hendricks [ ] defines it from the perspective of equilibrium theory: systemic risk refers to the risk of moving the system from one equilibrium to another worse equilibrium, and this risk is difficult to reverse due to the catalysis of many self-reinforcing mechanisms. following the systemic elaboration of systemic risks in , [ ] summarized the definitions of systemic risks and classified them into three categories: ( ) the huge negative impact that the entire financial system faces at the same time, that is, systemic risks have the characteristics of simultaneity [ ] ; ( ) all or a part of the financial institutions face the same risk exposure, which means systemic risks have the same cause [ ] ; ( ) the impact can trigger a chain reaction. in other words, systemic risk is contagious [ ] . [ ] believe that these definitions have the following points in common: ( ) systemic risk concerns are for all financial institutions; ( ) systemic risk focuses on tail loss characteristics; ( ) all need to consider the relevance and cross-cutting of the various institutions within the financial system. besides, there are differences and linkages between the concepts of systemic risk and financial crisis. [ ] believes that systematicity is the most noteworthy cause of crisis formation and crisis development. the former is the reason and the characterization of the latter, that is, the financial crisis necessarily presents systemic risks, but systemic risks do not necessarily lead to financial crises. moreover, current systemic risk research is mainly limited to financial systemic risk issues within an economy. the financial crisis has a wider scope and more diversified forms of expression. for example, depending on the scope of the spread, a financial crisis can be divided into bank crisis (the bank run), currency crisis, bubble crisis of capital market speculation, sovereign debt crisis, etc., and may trigger a comprehensive economic crisis. on the other hand, systemic risk is similar to financial risk in that it is contagious, and most or all financial institutions in the system will face the same impact at the same time. however, systemic risk emphasizes the impact of financial institutions on the overall risk of the system, that is, the endogenous nature of systemic risks, while the financial crisis may origin from endogenous or exogenous causes, for instance, for iceland in the recent financial crisis, the us-centric financial crisis was transmitted to iceland's entire banking system, which led to the overall collapse of the country's economy [ ] . besides, although systemic risks are not all triggered by the leverage (for example, on october , , due to the stock market crash, the us stock index futures prices plummeted, causing the margin account to not add in time, leading to the collapse of the broker), the impact on the system after it occurs must be amplified with leverage. therefore, the study of systemic risks is essentially related to the credit creation process and the degree of leverage. according to the different sectors created by credit, it can be divided into systemic risks of the banking system and the shadow banking system; according to the classification of financial markets and instruments that occur, it can be divided into systemic risks in the real estate market, banking system, bill market, securities market, bond market and derivatives market; according to the different debt departments, it can be divided into systemic risks of public debt (including systemic risks of sovereign debt), systemic risks of corporate debt, and systemic personal risks of households. on the other hand, because the financial crisis is highly correlated with the concept of systemic risk, the scope of the financial crisis and systemic risk research also has great overlap. by entering the keywords "financial crisis" and "risk", a total of ssci core documents and working papers published since were searched through web of science. by entering the keyword "systemic risk", a total of core documents and working papers of the same period were searched. through screening the titles and abstracts of all these papers by adding "financial markets", papers are selected out. with the help of the software citespace, further bibliometric analysis reveals that financial risk and systemic risk research have more crossovers, and the research concentrated areas are almost overlapping ( figure ). the sub-branches of financial crisis research are numbered according to the timeline of theory development. it demonstrates that early crisis research mainly focused on macroeconomic issues [ ] [ ] [ ] as time went by, there was a detailed exploration into specific financial markets and product markets [ , ] . from another perspective, financial intermediaries and investor behaviors were discussed in terms of pricing credit risks [ , ] . as financial intermediaries have similar research methods with corporate finance, more later attention was drawn to bank governance [ ] . as the subprime crisis erupted, more studies were oriented to the stability of the financial system, which overlaps with the systemic risk research [ , , [ ] [ ] [ ] . a new branch, the extreme value approach, is also adopted in systemic research. systemic risk research is not distinct from crisis research, as the former one also refers to such bank behaviors and liquidity problems. among the subdivisions, more co-citation clusters are formed in regional contagion, monetary policy, credit risk pricing and systemic risk. within the systemic risk field, the co-citation cluster of multidisciplinary research is the largest one, also with most focus on the financial network. thus, by combing the literature on financial crisis theory and systemic risk research, it can be found that the research on systemic risk is closer to the micro-level than the original financial crisis research, although systemic risk management involves macro-level management and supervision. there are more studies on the micro-market structure and participant behavior, and more emphasis on systemic risk transmission and contagious research, as well as the endogenous mechanism research that pays more attention to the occurrence of risks; it can be said that systemic risk research is the further deepening and refinement of the theoretical research on the financial crisis. therefore, although the academic community still has differences in the definition of systemic risks, by comparing the concepts of systemic risk and financial crisis, and summarizing the definition of systemic risk in the academic world, the concept of systemic risk can be defined from an economic perspective: triggered by macro or micro-events, the institutions in the system are subjected to the sub-branches of financial crisis research are numbered according to the timeline of theory development. it demonstrates that early crisis research mainly focused on macroeconomic issues [ ] [ ] [ ] as time went by, there was a detailed exploration into specific financial markets and product markets [ , ] . from another perspective, financial intermediaries and investor behaviors were discussed in terms of pricing credit risks [ , ] . as financial intermediaries have similar research methods with corporate finance, more later attention was drawn to bank governance [ ] . as the subprime crisis erupted, more studies were oriented to the stability of the financial system, which overlaps with the systemic risk research [ , , [ ] [ ] [ ] . a new branch, the extreme value approach, is also adopted in systemic research. systemic risk research is not distinct from crisis research, as the former one also refers to such bank behaviors and liquidity problems. among the subdivisions, more co-citation clusters are formed in regional contagion, monetary policy, credit risk pricing and systemic risk. within the systemic risk field, the co-citation cluster of multidisciplinary research is the largest one, also with most focus on the financial network. thus, by combing the literature on financial crisis theory and systemic risk research, it can be found that the research on systemic risk is closer to the micro-level than the original financial crisis research, although systemic risk management involves macro-level management and supervision. there are more studies on the micro-market structure and participant behavior, and more emphasis on systemic risk transmission and contagious research, as well as the endogenous mechanism research that pays more attention to the occurrence of risks; it can be said that systemic risk research is the further deepening and refinement of the theoretical research on the financial crisis. therefore, although the academic community still has differences in the definition of systemic risks, by comparing the concepts of systemic risk and financial crisis, and summarizing the definition of systemic risk in the academic world, the concept of systemic risk can be defined from an economic perspective: triggered by macro or micro-events, the institutions in the system are subjected to negative impacts, and more organizations are involved in risk diffusion and the existence of internal correlations strengthens the feedback mechanism, causing the system as a whole to face the risk of collapse. the definition here emphasizes ( ) events triggering harms to institutions and systems; ( ) systemic risks that spillover and are contagious; ( ) due to the psychological and behavioral characteristics of the micro-subjects, a self-reinforcing feedback mechanism is formed; ( ) the potentially serious consequence of systemic risk is the overall collapse. we found that in web of science, from the journal of economics, finance, accounting, behavioral psychology, and management, from to march , all the articles on systemic risk published in the ssci core journals totaled articles. from figure a , we can tell the research on systemic risk has ups and downs from year to year, echoing with worldwide risk events. besides, the search for working papers on systemic risk research on the nber website has exceeded publications in the past years. furthermore, the number of work papers published in this field newly released in is still considerable (figure b) . especially, under current circumstances, systemic risk research deserves further exploring, as the covid- pandemic struck the real economy by threatening human life first, then financial systems are impacted in terms of both assets and liabilities, which may further hurt real economies through systemic risk chains. another peak in this area can henceforth be expected. negative impacts, and more organizations are involved in risk diffusion and the existence of internal correlations strengthens the feedback mechanism, causing the system as a whole to face the risk of collapse. the definition here emphasizes ( ) events triggering harms to institutions and systems; ( ) systemic risks that spillover and are contagious; ( ) due to the psychological and behavioral characteristics of the micro-subjects, a self-reinforcing feedback mechanism is formed; ( ) the potentially serious consequence of systemic risk is the overall collapse. we found that in web of science, from the journal of economics, finance, accounting, behavioral psychology, and management, from to march , all the articles on systemic risk published in the ssci core journals totaled articles. from figure a , we can tell the research on systemic risk has ups and downs from year to year, echoing with worldwide risk events. besides, the search for working papers on systemic risk research on the nber website has exceeded publications in the past years. furthermore, the number of work papers published in this field newly released in is still considerable (figure b) . especially, under current circumstances, systemic risk research deserves further exploring, as the covid- pandemic struck the real economy by threatening human life first, then financial systems are impacted in terms of both assets and liabilities, which may further hurt real economies through systemic risk chains. another peak in this area can henceforth be expected. the research literature on systemic risk is sometimes the overlap of the fine branches of the section . above. for example, the study of tail risk identification and quantification will involve specific empirical methods and recommendations including risk management. therefore, the literature review in this chapter is mainly summarized and reviewed from the following four aspects: the concept of systemic risk appears late, so systemic research on it is still being explored. however, according to the previous literature comparison, it has many similarities with the research theme of the financial crisis. the study of systemic risk formation mechanisms also draws on the research results of the formation mechanism of the financial crisis. therefore, in the study of the formation mechanism of systemic risk, the research context of the formation mechanism of the financial crisis is first outlined. the study on the formation mechanism of the financial crisis is mainly divided into two categories, namely, caused by emergencies or related to the economic cycle [ ] . in the [ ] and [ ] , bank runs come from depositors' expected self-realization, which can explain some of the bank runs the research literature on systemic risk is sometimes the overlap of the fine branches of the section . above. for example, the study of tail risk identification and quantification will involve specific empirical methods and recommendations including risk management. therefore, the literature review in this chapter is mainly summarized and reviewed from the following four aspects: the concept of systemic risk appears late, so systemic research on it is still being explored. however, according to the previous literature comparison, it has many similarities with the research theme of the financial crisis. the study of systemic risk formation mechanisms also draws on the research results of the formation mechanism of the financial crisis. therefore, in the study of the formation mechanism of systemic risk, the research context of the formation mechanism of the financial crisis is first outlined. the study on the formation mechanism of the financial crisis is mainly divided into two categories, namely, caused by emergencies or related to the economic cycle [ ] . in the [ , ] , bank runs come from depositors' expected self-realization, which can explain some of the bank runs and the currency crisis, but it is difficult to use the model to make a prediction. [ ] proposed that there is information asymmetry between banks and depositors. by establishing a global game model, the bank-runs problem is well explained, and the theoretical boundary value of bank runs is put forward. [ ] extended this model to the mutual fund market and proposed an empirical approach to the global game model. the research of this branch is mainly related to the micro-market structure from the perspective of the micro-level and individual agent. another focus of research on the causes of the financial crisis is its procyclical nature. [ ] pointed out that when the economy goes down, it is difficult for banks to realize the possibility of redemption. if depositors anticipate the impact of the economic downturn on the banking industry, they will withdraw funds from their banks, which may cause a run on the banks. [ ] examined the economic volatility indicators of the united states from the end of the th century to the beginning of the th century and found that the leading indicators of the scale of debts of closed enterprises can accurately predict the occurrence of the banking crisis. [ ] , however, systematically studied the us comprehensive monetary policy from to , and found that the four financial crises that occurred during this period were all caused by panic, but not related to the real economy. this led to a series of subsequent empirical tests on the causes of the financial crisis [ , ] , which conducted a more detailed investigation of the four financial crises and collected more extensive data. it has been empirically found that the first three financial crises were triggered by the impact of the real economy, while the fourth financial crisis broke out due to panic. the research on the systemic risk formation mechanism is carried out in the context of the establishment of the credible monetary system, and the degree of regulation of financial markets and the links between financial institutions and product innovations that have surpassed the previous era. therefore, against this backdrop, the occurrence of systemic risk not only means that the liquidity of the bank or the banking industry itself causes a banking crisis but also means that the liquidity of the inter-bank market or the lending market in which it is involved in financing is exhausted. furthermore, the resulting chain reaction, and even spread to other financial segment markets, eventually led to the collapse of the entire system. therefore, the research on the formation mechanism of systemic risk is divided into two directions. the first one is the research on the formation mechanism of the financial crisis, which focuses on the micro-level; dynamic analysis is used to explore the effects of interactions within the system between individual behaviors, organizations, and financial markets. the other one is to focus on the institutional roots of systemic risk from the perspective of normative economics, or law and sociology. [ ] found that the frequency of financial crises in the world, such as the great depression, was, in recent decades, twice the sum of the number of financial crises in the bretton woods period and the gold standard period ( - ). this cannot but make people wonder whether the shortcomings of the credit monetary system itself lead to the risk of a financial crisis, because the marginal cost of credit currency issuance is almost zero compared to its face value. therefore, the central bank is more inclined to issue more than a limited amount of money to obtain money as a gain from credit capital investment or to achieve other policy objectives, especially when the central bank has a monopoly position and no other financial institution issues currency to compete with it. moreover, the central bank is not bound by a bankruptcy (the self-owned capital ratio can be extremely low), so this tendency is more obvious. besides, according to the guidelines proposed by the central bank theory founder walter bagehot that the central bank should follow (the loans provided by the central bank should set higher interest rates to prevent those who should not get loans, and the central bank should accept good bank collateral in the financial crisis to distinguish borrowers who are not solvent), this type of criterion, which can only rely on the central bank's self-discipline, can easily be broken before or even during the financial crisis, as the fed did in the subprime crisis. therefore, [ ] believe that the financial crisis is essentially a lack of restraint of central bank behavior, resulting in excessive credit funds, which is caused by an excess supply of high-quality assets. this view is consistent with the findings of [ , [ ] [ ] [ ] [ ] [ ] [ ] , which showed that credit booms and asset price bubbles prevailed before the systemic banking crisis. [ ] found that the financial crisis in many countries stemmed from the puncture of asset bubbles in the real estate market. as the financial crisis broke out, asset prices fell sharply and continued to have a major impact on real economic output and employment. further exploration of the deeper reasons, the premise of financial systemic risk accumulation as excessive debt, the root cause of excessive debt and what is the "degree" of liability, becomes an important part of the discussion of the formation mechanism of systemic risk. the discussion of this issue has even surpassed the scope of mainstream western economic theory, so it is difficult to see the whole picture in the previous classic systemic risk research literature. george bragues linked the act of capital interest-taking with the liberal democratic trend of thought, arguing that a large amount of lent funds had no tangible foundation beyond the credit creation ability of the banking system, and the fact that people exchange real goods and labor for this kind of currency without receiving any substantial return is unfair. it is the exploitation of the borrower by the lenders of the greeks proposed by philosopher aquinas. however, the idea of the rise of the liberal democratic notion that "everyone should be free to enter into a loan contract at an interest rate that is considered appropriate" justifies loan profitability. the function of credit creation in the banking system and the nature of profit-pursuing operation of capital will naturally lead to excessive debt of the overall economy, because "the main activity of finance is to create a huge debt chain and obtain more profits from it". that is, due to the existence of compound interest, even if there is no new debt, the scale itself will have endless compound growth. the growth rate of the real economy is relatively limited, and the duration of high-speed growth is short-lived [ ] , which will inevitably lead to a continuous increase in leverage. therefore, when debts accumulate to a certain scale, credit funds will inevitably spread more than high-quality assets, and due to the decentralized nature of globalization (financial centers and distribution centers in the west, product centers in the east, savings in the east, debt accumulation in the west, [ ] ), will become more dangerous, systemic risks and even the outbreak of financial crisis is inevitable. therefore, the free development of economy and capital may lead to disastrous consequences, requiring external supervision and constraints to restrict, and regulatory forbearance and legal loopholes are also important aspects of the study of systemic risk formation mechanisms. [ ] believe that the outbreak of the subprime mortgage crisis stemmed from the sec's policy of exempting investment banks from restricting the regulation of the balance sheet liabilities in , which was converted into an institutional voluntary supervision model, and the leverage ratio of investment banks generally rose sharply. the commodity futures modernization act, introduced in , prevented state governments from using their laws to prevent wall street from using derivative financial instruments. [ ] pointed out that relying on technocrats or over-reliance on technology to manage financial systemic risks will easily create an over-confidence illusion for regulators. as greenspan admitted at the october congressional hearing, he relied too much upon and believed in the "self-correction of the free market." jonathan c. lipson, a professor of law at temple university, believes that the design and trading of financial derivatives cds are divorced from the nature of their guarantees. unregulated over-the-counter trading has made this market far larger than the actual guarantee, which has created hidden dangers for the outbreak of the subprime mortgage crisis. compared with the research on the formation mechanism of the financial crisis, the research on systemic risk formation mechanisms is not entangled in whether it is related to the real economy, but whether it is at the macro level or the micro level, whether it is institutional research or event research. on the other hand, research is more dispersed, and the formation mechanism of systemic risk spans the research scope of financial disciplines, which deserves more in-depth and detailed discussion. [ ] [ ] [ ] studied several financial crisis events from high-income and low-income countries, and found that the crisis would cause the average price of real estate to fall by % in the next six years; in the next three and a half years, asset prices fell by an average of %. in two years, real output fell by an average of %. in four years, the unemployment rate rose by %, while the government deficit averaged % higher than the pre-crisis level. once the systemic risk evolves into a financial crisis, the impact on the real economy is obvious. as [ ] put it, this is because external factors that are not related to the fundamentals of the company will lead to market participation shocks caused by changes in capital participation. this will affect the price of securities in the capital market, and thus change the investment behavior of micro-enterprises. the investment of enterprises has externalities and ultimately affects the operation of the real economy. he believes that different financing models have different impacts on the real economy. the equity financing market is suitable for investment projects that use equity financing methods (such as high-tech enterprises). such enterprises have positive externalities to the real economy. therefore, although the bursting of the technology bubble hit the securities market in the early st century, investment in high-tech industries has accelerated technological progress and is conducive to the development of the real economy. the bond financing market is suitable for investment projects that use debt financing (such as fixed assets investment such as real estate). such investments are generally highly leveraged and have negative externalities to the real economy, so once the bubble bursts, the damage to the real economy is significant. the theoretical study of the impact of systemic risk on the real economy has been dissociated from the mainstream of macroeconomic research for a long time after keynes, because whether it considers from the real economic cycle model [ ] [ ] [ ] or the keynesian is-lm model, the market is a complete market due to the alienation of individual heterogeneity or information asymmetry. therefore, the financing method of enterprises does not affect the value of enterprises (mm theorem), so similar financial friction and systemic risk issues are difficult to match with the traditional theoretical framework, which makes the research on such problems lack micro-foundations. until the new dynamic keynesian theoretical framework (dnk) based on the dsge method was put forward ((cee) [ ] , (acel) [ ] ), it was compatible with the assumption of price stickiness, and incorporated the investment and financing behaviors and leverage changes under the information asymmetry into the model. the financial accelerator theory proposed by bernanke et al., the bgg model [ ] , has become a pioneer in this field. due to the limitation of the model's solution, dsge's utility function and production function setting are both in the form of linear functions or quasi-linear functions. however, linear functions cannot explain the nonlinear changes brought about by economic shocks, and leverage has a nonlinear relationship with the economic cycle. the former has the most direct connection with systemic risk occurrence [ ] . to explain the nonlinear relationship between leverage and economic cycles, bernanke proposed a nonlinear investment and financing decision model linked to corporate net assets. in addition, because information asymmetry includes the cost of external investigations (costly state verification, csv), there exists a risk premium between corporate internal financing and external financing (external financial premium). the existence of the premium will make the financing situation of the enterprise worsen when the economic shock occurs, which will accelerate the decrease in investment and bring about the "financial accelerator effect". to simplify the research, bernanke expressed the functional forms of other economic sectors as linear relationships, focusing only on the internal and external financing differences of enterprises and the resulting crisis transmission mechanism. subsequent economists further extended financial frictions to the household sector, the banking system, and the government sector, and further explained the real estate market [ ] , the monetary policy transmission mechanism of government departments [ ] , and the risk transmission mechanism of the banking system [ , ] through the dnk theoretical framework. the transmission path that affects the real economy through leverage is not limited to the ways depicted in figure . the empirical study of the impact of systemic risk on the real economy is relatively infrequently referred only two of the top works of literature in the annual average are related to this field. even empirical studies of the impact of the financial crisis on the real economy are few and far between, which may stem from the following reasons: . the number of observable systemic risk events is limited, and fewer samples are analyzed using time series or panel analysis; . in the quantification of systemic risks due to the existence of many differences, the results of using quantitative indicators to assess the impact of systemic risks on the real economy may vary. . because of the different ways of quantification, there will be large differences in the prediction of real economic fluctuations. if the results are not significant, the value of the research will be affected. [ ] collected all systemic risk event samples after world war ii in the united states and europe to circumvent or mitigate the effects of the above problems, using different methods to quantify and assess systemic risk. the quantile regression method was used to test the predictive power of systemic risk indicators on the deterioration of the real economy. it was found that only a small number of quantitative indicators can capture the risks of the macroeconomic downturn, and they can only be roughly predicted. another reason for the lack of theoretical research on the impact of systemic risk on the real economy is that traditional macroeconomic theory itself has a weak microfoundation, however, the systemic risk is mainly reflected in the systemic problems caused by the interaction of individual heterogeneous behaviours at the micro-level. by using traditional macroeconomic models, it is assumed that individual identity or limited heterogeneity has greater limitations. therefore, [ ] believe that the agent model (agent model) should be introduced from other disciplines to better simulate the micro-individual behaviour, and to obtain a more realistic macroeconomic model. the empirical study of the impact of systemic risk on the real economy is relatively infrequently referred only two of the top works of literature in the annual average are related to this field. even empirical studies of the impact of the financial crisis on the real economy are few and far between, which may stem from the following reasons: . the number of observable systemic risk events is limited, and fewer samples are analyzed using time series or panel analysis; . in the quantification of systemic risks due to the existence of many differences, the results of using quantitative indicators to assess the impact of systemic risks on the real economy may vary. . because of the different ways of quantification, there will be large differences in the prediction of real economic fluctuations. if the results are not significant, the value of the research will be affected. [ ] collected all systemic risk event samples after world war ii in the united states and europe to circumvent or mitigate the effects of the above problems, using different methods to quantify and assess systemic risk. the quantile regression method was used to test the predictive power of systemic risk indicators on the deterioration of the real economy. it was found that only a small number of quantitative indicators can capture the risks of the macroeconomic downturn, and they can only be roughly predicted. another reason for the lack of theoretical research on the impact of systemic risk on the real economy is that traditional macroeconomic theory itself has a weak micro-foundation, however, the systemic risk is mainly reflected in the systemic problems caused by the interaction of individual heterogeneous behaviours at the micro-level. by using traditional macroeconomic models, it is assumed that individual identity or limited heterogeneity has greater limitations. therefore, [ ] believe that the agent model (agent model) should be introduced from other disciplines to better simulate the micro-individual behaviour, and to obtain a more realistic macroeconomic model. besides, the dnk model focuses more on the interpretation of risk-transfer pathways, and the endogenous interpretation of systemic risks is difficult. it is generally assumed that shocks are external, so it is difficult to predict the occurrence of crises. as to the endogenesis, based on the general theory, [ ] proposed that capitalist economies had not shown to follow the neo-classical theory or in the "dis-equilibrium state" as keynesian economics stated, because of the functioning of sophisticated financial institutions. two sets of prices, for current outputs and capital assets, aligned within a capitalist economy are determined by different proximate variables, as well as consumption demand and investment demand having different horizons, the former upon shorter run expectations and the later on longer ones. both form the aggregate (effective) demand. consumption demand is a function of current factors, while investment demand is a function of the price of capital assets, supply price of investment goods, expected profits and external financing conditions. different from the micro-based general equilibrium methodology, minsky initiated his theory from a macroeconomic perspective, based on kalecki profit function, which always stands in a close or an open economy. by decomposition of the profit function, variables including monetary issues are demonstrated as follows: where π is the current profit of the whole economy, i is the current investment of the private department, df is the government deficit, sw is the savings of the private department, and c is the current consumption. thus, profits are not only determined by current productive factors, government activities, savings and consumption, but also by asset prices, which connect closely with financial markets. furthermore, profits, identified as cash flows, are the essential signals for investments, critically linking different payment commitments in the past, present and future. according to different cash flow commitments being availably financed, the financial system and debt structure are composed of a mixture of three types of financial postures (hedge finance, speculative finance and ponzi finance). as the natural growth of the economy, profits, in the form of cash flows, fluctuate due to the endogenous discrepancy in expectation horizons, which causes the ratios of the speculative finance and ponzi finance to increase in prosperous times, leaving the financial system to gradually become more sensitive to interest variations. when the current cash flows cannot sustain the current payment commitments, it initiates a deflationary break, and a financial crisis takes place. the implementation of mitigation policies can only alleviate the destruction yet not solve it, making the whole financial system more fragile to economic fluctuations. (financial instability hypothesis). since the complication of interactions between key factors, as well as variant vast, complicates markets in capitalist economies, minsky mainly demonstrated his theory in a narrative approach. thus, even though minsky's theory became the kernel of post-keynesian economics, it is difficult to further develop a comprehensive mathematical model ( [ ] once put minsky's fih hypothesis into the framework of is-lm model to explain and get the equilibrium result, but the conditional formula that equates the price of investment goods and the price of assets implies the assumption that the capital market and the commodity market will automatically maintain the equilibrium. even though there are endogenous factors that cause deviation from the equilibrium of financial markets and commodity markets, the economy will return to equilibrium because of the implicitly assumed equality condition. besides, the decision of asset price depends solely on investment demand, which is also contrary to minsky's view, who emphasizes that the determination of asset price depends on various factors.). it was also confronted with various criticism. [ ] proposed that, as in the upwards phases of an economy, investment increases cause a higher level of aggregate profits so that the leverage ratio does not necessarily increase. while, in the downside phases, investment decreases negatively affect the aggregate profits more than expected, which will increase the leverage ratio. the paradox of debt goes against minsky's theory. this contradiction should be directed to the preconditions of fih, which assume an extent of monopoly in commodity markets, so that producer can earn excess profits from market power. as the aggregate profits increase in the upward phases while the profit rates decrease, the over-increased investment would bring up leverage ratios, which could imperil future payment commitments. as in the downside phases, shrunk investments decrease aggregate profits while profit rates recover, which is good for bringing down leverage ratio. thus, in the framework of minsky' theory, the paradox of debt is explainable on the presumption of monopoly markets. further discussion can be referred to [ ] . some studies try to expand the kaleckian model [ ] [ ] [ ] by bringing a normal degree of capacity utilization into endogenous investment function. [ ] utilize the profit share instead of the profit rate, [ ] introduced external competition into an imperfect commodity market. all the adjustments provide a supplementary explanation of the demand regime. furthermore, monetary and financial variables are incorporated into the profit function [ , ] . the late research intends to contain fih and the paradox of debt through a neo-kaleckian model [ ] . moreover, by following a centre-periphery structure, the model explains how financial movements in the periphery associate with international financial markets' movements and external vulnerability. recent research in this field mainly focused on empirical research to support and verify fih in different economies [ ] [ ] [ ] . minsky's achievements are more than fih. instead, he focused on the whole system, aiming at constructing a general theory to explain the whole operational law of capitalist economies, including the upward and the downward phases of the economic cycle. the discrepancy in horizons and the amplification mechanism, which is realized through the interaction between profits and investment activities, as well as the positive (negative) feedbacks between asset prices and liabilities, not only explains the endogenesis of financial crises but also demonstrates his economic vision in a dialectical way, so whether the economy attains an equilibrium gives way to the depiction of economic operation processes, as the former is only a temporary stability status compared with the whole process. with regard to the difference in expectations on consumption demand and investment demand, it should not be criticized as a lack of rationality; it should be interpreted as generalized rationality, as in the "wall street view", which is intended to be rationally applied to the real economy and sophisticated financial systems. early research on the formation of the financial crisis from the perspective of micro-market structure mainly began from the following two aspects: . information asymmetry; . liquidity. besides, there are studies on the impact of the financial crisis from the perspective of currency and interest rates and expectations. compared with the study on the financial crisis in the field of market microstructure, the study on the micro-market structure of systemic risk has increased the study of the complexity of the overall system, and it will be reviewed from the following three aspects: before the outbreak of the subprime mortgage crisis in , the conclusions of the close relationship between institutions and the complex financial network's overall risk resilience were generally positive [ , , ] , and it was considered that sufficiently decentralized inter-bank liabilities could produce a more stable financial system. nevertheless, some scholars then began to link the increase in systemic risk to the complexity of financial networks. for instance, [ ] believes that as banks' counterparties increase, the probability of a systemic crash increase. [ , ] demonstrated that the financial system itself has an amplification effect on the increase in systemic risk by modelling the inter-bank infection behavior. the research idea of this kind of amplification draws on the research methods of the previous network infection model [ , ] , and the amplified transmission pathways are various. from assets and liabilities, studies by [ ] showed that financially interrelated companies were more prone to systemic risk in the face of market shocks due to cross-shareholdings. similarly, [ ] argued that the similarity of assets held between banks determines the extent to which relevant information was disseminated and the likelihood of a systemic crisis. by studying bank exposures in the us and europe, [ ] argued that systemic risk was affected by common risk factors, and the interdependence of financial institutions was mainly due to systemic factors. [ , ] used asset securitization as an example to explain the transmission path of a financial network to amplify systemic risk from the perspective of debt. it is believed that through the interconnected balance sheet network, asset securitization essentially magnifies the financial leverage of the entire financial system, thereby increasing the vulnerability of the system. [ ] showed that the higher the degree of financial integration, the more stable the interbank interest rate under normal conditions, and the higher the interest rate soars during the crisis. combining the positive and negative views, [ ] demonstrated through modelling that a closely linked financial system provides sufficient stability in the face of smaller market shocks. however, when the impact scale exceeds a certain threshold, the tightly connected financial network will amplify the risk and make the financial system more vulnerable, and the structure of the financial network has an important impact on the stability of the financial system. the research of complex financial networks draws on the game model and the research results of the previous network theory. the network theory originated from the seven bridges problem and then developed into the random graph theory, and then evolved into complex network theory. network theory and topological methods provided the basis for theoretical modelling of the structure of the research network itself [ ] and also provided empirical methods [ ] . because how individuals interacting with each other in the network and their importance can significantly affect the results of network structure and impact transmission, the current research in this field is still very popular. [ ] pioneered the use of an interbank debt matrix to describe the network structure of the banking system. this idea was followed by scholars who later studied systemic risk [ , ] . however, early network models used to be a non-direct model, which assumed that the connections between the participants are reciprocal and have the same interaction, even the same importance, such as if the inter-bank liability matrix is assumed to be positive, bank a's claims against bank b are the same as bank b's claims against bank a. this is because the inter-bank liability data is not available (therefore, in the empirical case, the inter-bank liability behavior will be further assumed, and the banking system liability matrix will be determined through technical processing such as the information entropy method). on the other hand, for the sake of simplifying model simulation, as [ ] described, the simplified model and the same subject are to study the relationship between the structure of the financial network itself and financial stability. the results of many empirical tests are contrary to the assumptions of the theoretical model. [ ] studied the italian interbank market structure from to and found that they are in line with the core-peripheral network structure, that is, a few banks continue to play a central role, maintaining lending with peripheral banks and providing liquidity to the market. while capital lending activities between core banks and non-core banks are rare, market liquidity at the time of the financial crisis is often caused by the reduction in borrowing by core banks, which means that the network distribution is not symmetrical. the core-peripheral network structure was first proposed by [ ] , other related works are contributed by [ ] , and [ , ] covered the most core-periphery network structures and a further supplementary version [ ] was published in . a series of empirical studies showed that in other financial markets, such as india, mexico, the netherlands and the uk, the banking system network also presented the characteristics of the core-peripheral structure [ ] [ ] [ ] . thus, more complicated networks with directional connections are introduced, which is more common in the financial contagion subdivision within the same e-n framework [ , ] . moreover, since there are various types of connections between financial institutions, the financial network should have multi-layers, including credit, insurance, derivatives, collateral obligations cross-holding assets. the research on financial multi-layer networks is surging. [ ] study the interaction of short-and long-term bilateral secured and unsecured lending. [ ] study the mexican banking system on all market layers and find that market-based systemic risk indicators systematically underestimate expected systemic losses. the research on the relationship between financial market participants' behavior and systemic risk is more dispersed. the division of financial research segments can be mainly classified into behavioral finance research, corporate finance research, and micro-market structure research. participants vary in different financial markets, the relationships between their behavior and systemic risk, and the transmission pathway, are also various. according to the type of participant, the most studied is the bank. despite the trend of bank disintermediation, in the financial crisis, the irreplaceable role of the banking system and its impact on the financial system determined that it is still at the core of the financial system. the idea that competition promotes the efficiency of financial allocation has promoted the wave of financial liberalization in developed countries in europe and america since the s (the second banking directive issued by the european union in allowed banks to engage in banking, insurance and other financial operations; in the us gramm-leach-bliley act also eased similar restrictions.). financial innovation products and means are constantly emerging, and competition among banks is becoming increasingly fierce, which brings about financial deepening [ ] and improved resource allocation efficiency [ ] , but it may also lead to instability in the banking system [ ] . the relationship between interbank competition and the stability of the banking system has always been controversial. on the one hand, competition is conducive to reducing the cost of capital, thereby increasing the return on investment of enterprises, and increasing profitability, which maintains the controllable credit risk of banks and increases the stability of the system [ ] . on the other hand, it will lead to bank rent-seeking behavior, especially the bank deposit insurance system will make banks tend to take more radical measures to pursue profits in a fiercely competitive environment [ ] . on this basis, without deliberately screening customers, the systemic risk will be increased [ , , ] . this is because it is difficult for perfect competition to exist in the competition between banks. the different scales and core-peripheral banking system structure will inevitably have a monopolistic effect on this market [ ] , and there are moral hazards and adverse selection due to information asymmetry [ ] . an extended study on the [ ] models modifies the absolute inverse relationship between the two proposed [ , ] , and presents that it should be u-shaped, that is, increased competition can improve the stability of the system and also make the banking system more vulnerable. the intensity of competition and other factors are the decisive factors that determine the negative or positive relationship between the two. [ ] considered the role of policy incentives and constraints in the interaction between the two, and empirically tested and compared the relationship between the fierce competition in the banking industry and the stability of the banking system under different policy environments in different countries. it was found that the more standardized the regulation, the more developed the securities market, the more perfect the bank insurance system and the more efficient the credit information sharing, and that increased competition will increase the vulnerability of the system. the method of balance sheet effect is not only adopted to analyze banking defaults, but also applied to study other market participants' behavior. the fire sale and funding correspond to the activities of the bank's assets and liabilities, respectively. when there is a run (trader's run, deposit run or collateral run), the bank has to hedge the risk by recovering the loan in advance or raising the collateral mortgage rate, but there may be a risk of payment due to coordination failure. [ ] believe that when the market lacks liquidity, banks have fewer opportunities to hedge their overall risk or deal with liquidity shocks. this will push banks to begin fire sales in response to future liquidity demands, which in turn will lead to excessive fluctuations in market capital prices. moreover, when monopolistic behavior exists in the interbank market, it will further reduce the effective supply of funds in the market [ ] , and even market freeze appears. [ ] classify liquidity risk into liquidity risk in the market and liquidity risk in trader financing. traders provide liquidity to the market through financing, but the trader's financing capabilities (such as equity and margin financing) rely on the liquidity of the asset market. however, market liquidity will suddenly dry up under certain circumstances and will affect many securities, resulting in large market fluctuations. it also leads to difficulties in the financing of traders and the realization of assets. the interaction between financing liquidity and market liquidity forms a liquidity spiral and triggers systemic risks. the liquidity spiral has a variety of transmission pathways, such as the loss spiral of erosion capital, and the guarantees spiral, which all lead to a fire sale. there are many different interpretations of the reasons for the formation of market liquidity shocks: ( ) information asymmetry some of the research on financial crises caused by the lack of market liquidity is associated with information asymmetry, which is often one of the causes of liquidity exhaustion [ ] . the financial crisis caused by information asymmetry is more typical in bank run events. the global game model proposed by [ ] is also based on the assumption of information asymmetry, that is, participants can only obtain noisy observations, and the lack of common knowledge leads participants to choose risk-dominant equilibrium as the only equilibrium. this global game model is extended to the currency crisis model [ ] and the bank crisis model [ ] . when information asymmetry exists in the interbank market, it will exacerbate the liquidity of the market due to counterparty default risk, leading to system collapse [ ] . moreover, the information asymmetry between the supervisory authority and the regulated institution will cause the central bank to misjudge. since regulators do not have a complete understanding of the asset quality of the entire banking system, therefore, even if a central bank participates in the interbank market to prevent the inefficient allocation of market resources due to adverse selection, moral hazard and monopolistic behaviour, the result of such hedging is difficult to be optimal [ ] . ( ) liquidity mismatch [ ] believe that the common feature of financial markets in the recent financial crisis is that financial institutions generally have more serious mismatches in liquidity. for example, the terms of interest rate fluctuations in the subprime borrower's loan contract will affect their ability to repay and refinance. commercial banks are burdened with over-proportionate contingent liabilities relative to capital under unregulated off-balance sheet business. the rise in the proportion of investment banks that rely mainly on repurchasing commercial paper financing means that the proportion of the entire financial system that relies on market financing is rising, so the excessive leverage ratio is the risk of mismatch in liquidity. in the event of a liquidity shock, market liquidity is rapidly depleted by the balance sheet effect, resulting in the interaction of collateral run, bank run, and counterparty run. the central bank has to ease the crisis by releasing a large amount of liquidity, but this has further increased the moral hazard of financial institutions to increase leverage, which has further worsened the problem of liquidity mismatch. ( ) actual demand shock [ ] argued that the demand for liquidity stems from the mismatch between the supply and demand of goods by consumers in different spaces. once they needed to spend at different times in different locations, they constituted cross-regional liquidity needs, and banks needed to provide liquidity through the crediting behavior of the payment system or the interbank market. [ ] agreed that random demand of consumers for liquidity will become a liquidity shock for a depository institution due to incomplete interbank market transmission. [ ] suggested that systemic risk is mainly transmitted through the payment system, the interbank market and the derivatives market and that the random withdrawal demand of consumers at different times and in different places will have a liquidity impact on the payment system and the interbank market. the existence of the interbank market reduced the incentives for banks to hold non-profit cash; banks had solvency under certain conditions, while the market was caught in coordination failures. the financial accelerator model proposed by [ ] explained the external financing premium problem faced by enterprises from the perspective of the incomplete credit market. the incentive mechanism of the company has prompted the agent to favour debt financing. when the negative impact causes the net value of the enterprise to decrease, the solvency declines, which in turn has an impact on the asset side of the loan bank. when the assets of the loan bank are impaired due to corporate default and further affect the scale of the risk reserves, the bank faces a liquidity shock, and the impact has a nonlinear amplification effect through the interbank market. other factors affecting market liquidity risk were institutional and systemic risk management systems, financial system structures, and the institution's own reputation risk [ ] . moreover, the phenomena associated with liquidity shocks include market freeze, asset sales, contagion effects, and institutional bankruptcy, which also possibly appear when systemic risks occur. financial contagion occurs as the distress of one or a small group of banks risks the stability of other financial institutions, even ultimately spreading to the real economy. the financial contagion can occur due to both local contractual obligation connections, and global market connections, e.g., through the prices of assets due to mark-to-market valuation [ ] . [ ] established a framework for contagion analysis, which studies the spread of obligation default within the financial system due to unpaid liabilities. since there are various types of connections between financial firms, the shocks can transmit in the network via different channels, and the channels also interact with each other in some historic events. according to [ ] taxonomy, there are mainly four types of channels: asset correlated contagion, default contagion, liquidity contagion, market illiquidity and asset fire sales. except for the default contagion, for the rest of the channels, the observed domino or spillover effects are demonstrated through the slump in mark-to-market prices. thus, contagion research can be classified into two main branches: default contagion and price-mediated contagion. ( ) default contagion [ ] explored how the probability and potential impact of contagion is influenced by aggregate and idiosyncratic shocks. they found that the robust-yet-fragile financial system has a low probability of such contagion. [ ] extend the basic default contagion model by introducing default costs into the system. [ ] assume minimal information about network structure, and through such key node-level quantities as asset size, leverage and the fraction of a financial institution's liabilities held by other financial firms, they derive explicit bounds on the potential magnitude of network effects on contagion and loss amplification. other models with extension in default costs include [ , , ] . ( ) price-mediated contagion these contagion models consider the mark-to-market asset price slumps due to the extreme tension in market liquidity and individual liquidity, as demonstrated in fire sales [ ] [ ] [ ] [ ] [ ] [ ] cross-holdings have also been studied [ , ] . [ , ] provide a framework for modelling asset prices during a fire sale. [ ] generalizes the model of [ ] by allowing for differing liquidation strategies. [ ] study the three extensions that are bankruptcy costs, multiple assets and fire sales within a single model. the survey of empirical work on contagion can refer to [ ] . empirical studies are revealing that financial contagion can not be well explained by the base model of obligation default [ , , , ] . system stress tests are usually adopted to study the exposure of the whole system towards indirect contagion [ , ] . in general, the network-based models are mainly rooted in the general equilibrium approach, which brings sufficient economic connotation to these models. it also means that systemic shocks are usually assumed from outside instead of endogeneity. thus, through limited amplification, contagions can suspend automatically and the whole system returns to a new equilibrium. even referring to the multiple assets models, the shock on the prices of assets and consequent price variations distribute independently with the network contagion, otherwise, the mathematical model shall be too complicated to be solved. however, the financial contagion research does offer great achievements in: ( ) explaining the contagion path via networks of all types; ( ) empirically revealing the impact of contagion on the whole financial system; ( ) providing supportive evidence for capital requirements, mark-to-market accounting and limitation of the scale of interbank business. systemic risk identification and measurement are one of the most important aspects of systemic risk research. in recent years, the literature in this field has also increased. the indicators of systemic risk are ses [ ] , mes [ ] , klr [ ] , covar [ ] and capital shortfall [ ] . some scholars have reversed the financial systemic risk through the market prices of derivatives (credit default swaps, cdss) issued by financial institutions [ , ] . a survey of measurements on systemic risk can be referred to [ ] , and they also offered codes for such measures. besides, [ ] adopted the herfindahl index to measure the concentration of credit portfolios of the emergency loans programs provided by the fed in the - crisis. they found that strongly connected institutions received the most of the funds, while small dispersed shocks could have triggered the systemic default event, which indicates that the critical banks are "too-central-to-fail". i [ ] constructed an insurance pricing model based on the put option framework of the merton model, including asset relevance, banking systemic risk and joint default rate, suggesting that systemic risk leads to low insurance pricing, which leads to insufficient liquidity in the event of a crisis. [ ] used a multivariate extremum theory to model the tail dependence between multiple assets and adopted a non-parametric estimation to estimate and then to measure systemic risk. [ ] studied the monthly yield data of hedge funds, banks, securities, insurance and other financial institutions based on principal component analysis combined with granger causality, and concluded that the internal correlation of these four types of finance has increased the systemic risk. [ ] viewed the banking system as a credit portfolio, analyzing the value of the portfolio and the unanticipated losses to calculate systemic risk. [ ] considered financial departments as a time-varying systemic risk of overall measurements, and defined systemic risk as to the conditional default probability, and analyzed the default probability through credit repayment. when the systemic risk is defined as a series of debt contract defaults, systemic risk is linked to credit risk. the biggest characteristic of credit risk is its tail effect. therefore, many researchers have introduced the copula model to simulate the tail dependence of financial institutions' extreme losses under systemic risk [ , ] . [ ] proposed a new systemic risk analysis framework, which incorporated marginal default probability, credit risk structure, consistent information multivariate density optimization (cimdo), generalized dynamic factor model and t-copula model into the framework. [ ] propose the endogenous risk index (eri) to measure the spillovers across portfolios, as well as the indirect contagion index (ici) to capture the importance of a bank via measuring the loss it inflicts on other financial firms. the two indicators provide a complementary measure of interconnectedness. [ , ] provide reviews on more recent measurement studies. therefore, it can be seen that, due to the tail characteristics and nonlinear characteristics of systemic risks, there are many methods for the quantitative assessment of systemic risks. all approaches and definitions should serve their purposes. due to the fast development of systemic risk research, this paper may not cover all the latest methods, while it can provide a series of frameworks for reference. according to the survey work, the approaches are classified into five categories: theoretical foundation, mathematical models, econometric methods, simulation and agent-based models. as to the simulation methods, more can be referred to [ ] , which will not be discussed in this section. systemic risk research rests its theoretic root in equilibrium approaches, which follow a micro-based research paradigm. to achieve the equilibrium, the route of deduction shall be clarified, and the micro-based equilibrium or activities need to be coordinated with the purpose of general equilibrium by a series of preset hypotheses. to explain the frictions on financial markets, the preset conditions are adjusted accordingly, based on more convincing and practical micro-foundations. there are at least three types of endogenous inconsistencies: inconsistency in preference or expectation [ , ] , heterogeneity [ , , ] , information asymmetry [ , ] , and bgg model [ ] . besides, through obligation contracts [ , ] or institutions' capitals [ ] or other generalized agreements [ , ] , connections are established within the financial system, which establishes the network analysis of contagions, domino effects and spillovers. as mentioned above, the study of the generation and transmission mechanism of systemic risk is in virtue of the dnk model framework. linking the activity entities and market structure at the micro-level with macroeconomic growth and volatility makes the performance of macroeconomic fluctuation of systemic risk possess a certain micro foundation. because systemic risk is accompanied by rising leverage, financial friction, large fluctuations before and after liquidity, and other derivative phenomena, it is closely related to individual heterogeneity and the characteristics of the market structure itself. to explain the impact of individual heterogeneity or heterogeneous expectations on financial market asset prices and the real economy, calvo and rotemberg's sticky-price model is used for reference. what's similar is the application of the two-sector model in the utility sector or the constraint function in the family sector, for example, [ ] distinguish the buyers from the superior and sub-optimal levels and separate their constraint functions, explaining the intrinsic relationship between the rapid rise of mortgage loans and the rapid rise in housing prices before the subprime mortgage crisis. the game model is also a more common method for explaining financial friction and systemic risk. [ ] proposed an analytical approach to coordinated games. by dividing the depositor's investment behaviour into two categories, patient and impatient, in the case of incomplete information, the investor will choose whether to withdraw the deposit to the bank in advance according to the expectation and then the bank run may occur due to the individual's rational behaviour. this model successfully explains the reasons for the bank's asset-liability maturity mismatch, and also explains the transmission mechanism that triggered the banking crisis. however, the deficiency is that there are multiple equilibriums in this model, and it is impossible to predict the occurrence of the bank run behaviour. the coordinated game model is then further optimized and expanded [ ] . [ ] proposed a method to ease public information hypotheses, allowing participants' post hoc observations to satisfy certain random distribution characteristics. the game based on this hypothesis is named as a global game. compared to the coordination model, the global game model eases the assumption of public knowledge and reduces the quantity of equilibrium. [ ] introduced the global game method into the study of financial crisis theory and studied the mechanism of currency attack activities; the sequential global game method is also widely used in the fields of macroeconomics and financial crises. chinese scholars also used this model to explain the liquidity crisis of banks and the government's rescue policy [ ] , and infectious mechanisms for the liquidity of informal financial institutions to formal financial institutions [ ] . as for the modelling of contagion and spillover risks among financial intermediaries, the covariance of assets [ ] or liabilities [ , , ] was introduced based on micro-level maximization, in which the agent is either risk-neutral or risk-averse. asymmetric information assumption is also brought in. thus, the general equilibrium approach and its loose conditions can explain these endogenous questions: how participants react to an unexpected shock; what expectation they would form and action they would take thereafter; how the systemic risk is transmitted or spillovers occur within the system. however, although frictions are invited into the equilibrium models, they have to always assume the exogeneity of the shocks. another series of theoretical approaches are based on an analysis of evolutionary history and institutions, like marxism and minsky's theory. the latter inherited the former's essence in explanation of profits in that they both agree that profits go down as investment increases in the phases of economic expansion, and a crisis takes place when profits cannot cover the current payable obligations. while minsky further developed his theory by establishing a profit function on an identical equation, it follows a macro-micro-macro non-equilibrium paradigm, which unravels the necessity of present assumptions. through decomposition, the profit function can embrace various endogenous factors from different departments of an economy, including technical improvements, public policies and the financial institutions, etc., while marxism mainly attributes the profits to the surplus-labour and capital structure. minsky's methodology can also be applied to analyze the household and government behaviours and its subsequent effects on the financial system. this methodology solves the problem of exogeneity, as the financial crisis phases are integrated into a whole theory to explain the operation of capitalist economies. to avoid mathematical formalism, he insisted on delivering his theory via a narrative approach, which leaves more space for his successors to explore [ ] [ ] [ ] , etc. network and contagion models are mainly established in the general equilibrium approach [ ] . after [ ] , the baseline of the network model is proposed by [ ] , which is expanded from explaining default contagions to price-mediated contagions. based on that, [ ] offered a model involving hard defaults, in which interbank debts of defaulted banks recover zero value. there are also other models with extensions on bankruptcy costs, multi assets and fire sales. [ ] provides a framework to prove the existence of clearing asset prices and liability payments for equilibrium in a network contagion. [ ] expand it to a dual-risky-asset equilibrium. no matter the eisenberg-noe model, or the gai-kapadia model or the amini framework, they all belong to static cascade models, which assume the structure and scale of the network itself be static. while networks can evolve due to the interactions of participants' inter-connections of assets and shocks from the outside, so random graph models are brought in [ , , , , [ ] [ ] [ ] . moreover, to capture the stochastic and dynamic structure of the network with a vast volume of nodes, the mean-field models are borrowed from the discipline of physics, which can be applied to explain the herding effect and endogenous contagion [ , ] . these models focus on quite simple interbank interactions that neglect defaults and contagion [ , , ] . the latest models involve credit risk to explain default contagion [ , , ] . since, in most economies, the interaction data of financial institutions in the system is difficult to derive, it is also impossible to acquire the specific counterparty-trading information from a bank's balance sheets. thus, alternatives are adopted to settle this problem, one of which is the maximum entropy approach. the debtrank approach is another measure to estimate the distress contagion without observing failing institutions [ ] . the main methods used are the copula model to simulate the joint distribution of risk factors, and the introduction of option pricing models and multivariate extreme value theory, or principal factor analysis to extract key factors using the dcc-garch model to model the correlation between multiple assets [ ] , and to establish multivariate quantitative indicators. furthermore, to verify the economic structure variation as systemic risk takes place, var is usually adopted [ ] [ ] [ ] tc. [ ] proposed the introduction of an agent-based model (abm) to solve individual heterogeneity problems, and monte carlo simulation can be used to predict the impact of micro-subject heterogeneity on macroeconomic variables. compared with the game model, abm is more compatible with more differences and can link the internal economic evolution logic with external data consistency. the key contributions by [ , ] are to incorporate the leverage accelerator into agent-based simulated network models. the limitation of such models is that it is difficult to calibrate and test their results. in addition to financial heterogeneity issues, systemic risk draws more attention to the stability of the system itself, and complex network models are borrowed to solve such problems. because of its similar structure to infectious diseases and bio-food chains, the financial system network links various financial institutions through financial flows, and triggers a domino effect due to risk events in individual institutions [ , , ] . however, the financial network system is more complicated than the natural food web or infectious disease transmission. this is because the activities of participating individuals are interactive, the interactions are not always symmetrical, the behavioral choices of the entity change with expectations or event shocks, and the results of behaviors are also characterized by uncertainty. this makes the financial system's structural stability research more complicated and has to restrict the behavioral characteristics of the system to individuals through many strict preconditions [ ] . the methods for identifying and quantifying systemic risks have been discussed previously. therefore, in general, the system of research methods for systemic risk is as in figure . in addition to financial heterogeneity issues, systemic risk draws more attention to the stability of the system itself, and complex network models are borrowed to solve such problems. because of its similar structure to infectious diseases and bio-food chains, the financial system network links various financial institutions through financial flows, and triggers a domino effect due to risk events in individual institutions [ , , ] . however, the financial network system is more complicated than the natural food web or infectious disease transmission. this is because the activities of participating individuals are interactive, the interactions are not always symmetrical, the behavioral choices of the entity change with expectations or event shocks, and the results of behaviors are also characterized by uncertainty. this makes the financial system's structural stability research more complicated and has to restrict the behavioral characteristics of the system to individuals through many strict preconditions [ ] . the methods for identifying and quantifying systemic risks have been discussed previously. therefore, in general, the system of research methods for systemic risk is as in figure . systemic risk is understood from the perspective of classical economics as the transformation between multiple economic equilibriums. from the perspective of risk management, it is tail risk management. from the perspective of behavioral finance, it is because the micro-subjects' psychological and behavioral characteristics form a self-reinforcing feedback mechanism, which belongs to the systemic overall collapse risk from the perspective of complex network theory. from systemic risk is understood from the perspective of classical economics as the transformation between multiple economic equilibriums. from the perspective of risk management, it is tail risk management. from the perspective of behavioral finance, it is because the micro-subjects' psychological and behavioral characteristics form a self-reinforcing feedback mechanism, which belongs to the systemic overall collapse risk from the perspective of complex network theory. from a broader perspective, it also covers the fields of sociology, psychology, and political science. there are a thousand hamlets in the eyes of a thousand people. different starting points focus on different problems, and the methods used are different. it is also the complexity and change of systemic risk that attracts researchers to constantly try to innovate. the systemic risk research branch is extensively involved, both in connection with traditional financial crisis research and expanding research on system structure and risk warning. financial systemic risk research can be said to be the most intersecting field between finance and even economics and other disciplines. the main research methods used are both tools of traditional economic theory, as well as social network models and natural science experimental simulations. for the generation of systemic risks, the endogenous problems of shocks are difficult to solve using traditional economic models. therefore, dnk's dsge model often assumes that shocks are exogenous; to predict systemic risks, it is urgent to introduce interdisciplinary approaches to innovate research on endogenous factors. on the other hand, the adoption of complex network models often requires many rigorous assumptions that are outside of reality, and these assumptions may exclude the potential risk factors of reality. after all, the financial system itself is much more complicated than the food chain, while other new interdisciplinary research methods also have insufficient internal economic explanatory power. it is, therefore, necessary to use appropriate analytical tools based on specific issues of the specific financial sector. to better sort out the hotspots, the latest years' ( - ) systemic risk research was analyzed and was connected with the development of crisis research, articles in total. it turns out that two divisions are mainly focused on ( figure ). intersecting field between finance and even economics and other disciplines. the main research methods used are both tools of traditional economic theory, as well as social network models and natural science experimental simulations. for the generation of systemic risks, the endogenous problems of shocks are difficult to solve using traditional economic models. therefore, dnk's dsge model often assumes that shocks are exogenous; to predict systemic risks, it is urgent to introduce interdisciplinary approaches to innovate research on endogenous factors. on the other hand, the adoption of complex network models often requires many rigorous assumptions that are outside of reality, and these assumptions may exclude the potential risk factors of reality. after all, the financial system itself is much more complicated than the food chain, while other new interdisciplinary research methods also have insufficient internal economic explanatory power. it is, therefore, necessary to use appropriate analytical tools based on specific issues of the specific financial sector. to better sort out the hotspots, the latest years' ( - ) systemic risk research was analyzed and was connected with the development of crisis research, articles in total. it turns out that two divisions are mainly focused on ( figure ). a total of articles are screened and analyzed according to their titles, keywords, abstracts and references. among the subdivisions, more co-citation clusters are formed in systemic risk measurement, financial market structure, and financial stability. this map reveals that more recent studies focus on network stabilization and systemic risk measurements. although the extreme value approach was brought into this area, it caught only a small amount of attention. furthermore, the high concentration on risk measurement may imply that the measuring methods gradually mature, which leaves limited room for subsequent researchers. considering the ongoing worldwide recession, the research directions that may be further expanded in the future are as follows: a total of articles are screened and analyzed according to their titles, keywords, abstracts and references. among the subdivisions, more co-citation clusters are formed in systemic risk measurement, financial market structure, and financial stability. this map reveals that more recent studies focus on network stabilization and systemic risk measurements. although the extreme value approach was brought into this area, it caught only a small amount of attention. furthermore, the high concentration on risk measurement may imply that the measuring methods gradually mature, which leaves limited room for subsequent researchers. considering the ongoing worldwide recession, the research directions that may be further expanded in the future are as follows: ( ) more work on models with multiple shocks and spillover effects be done. no matter endogenous or exogenous shocks, the contagion rotation shall not be limited to a specific market or intermediaries. more than one spillover risk is discussed within a model. moreover, at present, there are many types of research on systemic risks in the banking system, the real estate market and the foreign exchange market. there are few studies on the systemic risks of the shadow banking [ , ] and internet financial markets, which may be related to the lack of available data and difficulties in providing supporting empirical studies. ( ) dynamic multi-layer networks expect further exploration, especially when the prices of assets have a connection with the financial contagions. ( ) more empirical studies of the impact of systemic risk on the real economy are necessary. it is expected that recent advances in large-scale data and computing tools benefit systemic risk studies, acquiring more real-world and large-scale data for empirical analysis [ ] . ( ) event studies shall be focused on the modification of market structure, which is being affected by new technology or policies that help promote information disclosure, for example, the alternative index (like sofr) in place of libor. moreover, digital currency issued by central banks substituting banknotes means that the monetary multiplier is no longer applicable, which will profoundly alter the whole financial system. therefore, these innovative measures should also be paid attention to. besides, there are other areas worth exploring, such as more comprehensive risk warning indicators, more effective econometric means, and crossover with other fields of economics or sociology, psychology, etc. in short, this is an active research field and has a strong practical significance and policy reference value. the research of systemic risk will be more vibrant because of the participation of more researchers. economic networks: the new challenges systemic risk in a unifying framework for 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(ord), va investigators are involved in over , funded research projects and often focus on psychogeriatric patient-centered research due to the demographics of veterans and their family caregivers (ord, ) . hence, it is important to understand how the ord is handling clinical research during the current pandemic. the current pandemic is nothing like what the existing research infrastructure has previously witnessed. on march , , the world health organization characterized coronavirus disease (covid- ) as a pandemic. at that time, there were approximately , cases in countries, and , people had died worldwide due to covid- . there were , total covid- cases in the us; recommended coronavirus mitigation efforts included self-isolation and avoidance of healthcare centers where symptomatic patients congregate for medical care and where patient-centered research is typically conducted (cdc, ). since the pandemic declaration of covid- , there has been rapid spread of covid- in the world and clear recognition of the burden of illness and excess mortality among older adults, especially those with cognitive impairment, frailty, and residing in nursing homes (shahid et al., ) . as we experience ongoing rapid changes in clinical care related to the pandemic, views regarding conducting clinical research involving older adults are also evolving (nicol et al., ) . despite the benefits of patient-centered research, a pandemic brings huge disruptions in safely conducting clinical research (gobat et al., ) . therefore, va ord prioritized the health and safety of study participants and appropriately deemed clinical research visits as nonessential during the covid- pandemic, in line with the us centers of disease control and the us national institute of health (cdc, , nih, . however, at the same time there are calls for using the existing va clinical research infrastructure for covid- -related research. while researchers have discussed challenges to conducting clinical trials during covid- , including efficient accrual and randomization, intervention adherence and delivery, and outcome collection (mcdermott and newman, ) , less is known about the perspectives of older participants and their caregivers related to their involvement in patientcentered research during a pandemic. this commentary highlights implications for psychogeriatric research based on the perspectives of older adults and caregivers in the context of the early days of the covid- pandemic. in march of , recognizing the importance of rapidly engaging patients and caregivers during the covid- pandemic, we briefly surveyed participants who were enrolled in several ongoing psychogeriatric research studies pertaining to mild cognitive impairment, as well as major neurocognitive disorder with and without behavioral problems at the visn little rock geriatric research education and clinical center, little rock, arkansas, us (padala et al., ) . this was before the va ord released guidance to temporarily halt all new enrollments and in-person research visits unless such a halt would be detrimental to the participants. we spoke with participants either during their in-person visit or via phone if they had canceled/ postponed the visit. we asked open-ended questions to explore participants' potential concerns about handling the pandemic, the va healthcare system's covid- screening process, and general advice to improve the process of healthcare visits (e.g. what helped you decided to attend this appointment or skip it? do you have any concerns about how the va is handling the situation? what advice do you have to improve our processes? do you prefer an in-person visit or a telephone visit?) we also asked about their primary source of covid- -related information and the reasons they preferred that source over others. a total of participants were approached all of whom responded. of these, were research participants and were caregivers with a mean age of . (± . ) years and a mean education of . (± . ) years, % were males and % were caucasian. while some responses from these older adult psychogeriatric research participants were expected, other input was surprising. deciding whether to attend visits: influenced by trust in the healthcare system regarding the decision to come to the va healthcare facility for a scheduled research appointment, key positive factors were trust in the va system, extra screening at the facility entrance related to covid- , and the fact that the research visit was not in a group format. for example, participants noted, "i am in a safe spot with the va," "we have limited going around. we did stock up. we feel safe coming to the va," and "extra screening was great." the low number of cases in the community also seems to have played a role in the decision process, "low cases in arkansas, no need to panic." related to trusting the va, participants felt that the va was prepared, professional, and acting in the veterans' best interests. some older veterans voiced concerns about participating in research during the covid- pandemic that were expected, while other concerns suggested the participants were influenced by myths or misconceptions. multiple expected responses focused on risks for increased infection, underlying conditions or frailty of the participant making him/ her more vulnerable for infections and worry about exposing older parents. a concerning reason for deciding not to participate in the research visit was "i am concerned that researchers were trying to give me coronavirus as a guinea pig." some lack of concern related to the pandemic seemed to be based on myths or mis-information, "i have terrific immune system as i work in the schools," and "it does not affect black people." close family members and friends and traditional news media were the common resources that participants accessed for covid- information. three respondents each obtained information from the va social media/newsletter and the us centers for disease control newsletter. several participants expressed concern that social media may be contributing to spread of unauthenticated information and that public should turn to experts. when given a choice of conducting the research visit over the telephone, half of the participants preferred a telephone visit over in-person visit. several participants had variable levels of acceptance related to the choice of telephone visits for research purposes. one participant advised, "don't go out. glad that you are offering televisit," reflecting the decision some made to limit infection risk was by deferring in-person visits. however, many others were not comfortable with telephone visits due to hearing impairment, uncertainty about the quality of care, and concern about when they would get their next appointment if they canceled the appointment. one participant felt that he would be more wary of research if it was all done over the phone. the voices of older research participants and their caregivers in the design and conduct of patientcentered research are of utmost importance. similarly, the perspectives of these research participants are also important in public health emergencies, like pandemics. we hope to elevate the perspectives of research participants and caregivers enrolled in psychogeriatric studies during the early days of the covid- pandemic. these individuals offer real-world perspectives on decision-making related to research participation that has implications for research during highly disrupted times. while many veterans expressed high levels of trust in the va healthcare system, they also had tangible concerns related to risk of infection and logistics of how to continue participation in research during the pandemic. education about covid- infection, transmission, and universal precautions should be provided continually to both patients and caregivers (wang et al., ) . an important implication is that communication from research staff is key when enacting researchspecific protocol changes (e.g. telephone visits instead of in-person visits) or new facility-level processes (e.g. new health screenings). clear communication about current status and any changes from the research staff may help prevent dropout and/or nonadherence by reassuring the participants that their involvement in the study remains important, even during the pandemic (mcdermott and newman, ). one example noted in the study was the need for clear communication about extra screening at in-person visits. for example, a phone call to describe a new health screening process might prepare participants and could reinforce accurate information about the pandemic. improving the screening processes based on concerns such as not reusing fomites such as pens to sign-in names needs to be undertaken. one simple solution would be for the healthcare staff member to document the temperature and the needed details of those being screened. given the importance of outcome collection, researchers may plan to transition to collecting outcomes remotely such as by telephone or online (mcdermott and newman, ) . the department of va has invested significantly in telehealth technology, but the research enterprise may lag behind the clinical division due to regulatory burdens (young et al., ) . this study, however, recognizes that some older veterans and caregivers may have barriers to these modalities and emphasizes the importance of communicating the rationale and exercising as much flexibility as is safely possible based on participants' needs. using va social media to make user-friendly promotional videos about telehealth use might improve its reach. it also appears that some participants were concerned about getting the next appointment if they were to cancel the current one. reassuring participants about clinic availability (for research or clinical purposes) and process would be helpful. family members and caregivers were noted to be the primary source of information for most respondents. it is imperative to work on messaging the caregivers about the pandemic. education is needed because studies have shown that african americans are at higher risk of covid- -related mortality than other ethnicities (yancy, ) . we heard that some respondents were concerned that social media contributes to spread of unauthenticated information. thus, providing a clearing house of credible information sources would be helpful. for example, many va medical centers have started telephonebased hotlines to mitigate false information about covid- called, "myth busters." similar hotlines for research participants would be helpful. in conclusion, we seek to elevate the opportunity for psychogeriatric researchers to understand the needs of older adult research participants and adapt research studies based on input from patients and caregivers. even as participants may place a high level of trust in their healthcare system and its preparedness in combating the covid- pandemic, clear communication from the research staff may help prevent dropout or nonadherence. researchers need to reassure participants that their involvement in the study remains important, even during the pandemic. additionally, because family members and caregivers often serve as a primary source of information, it is imperative to work on broad and accurate information and messaging about the pandemic. none. dr. hdl was funded by va ord and national institute of health. dr. prp was supported by grants from the department of veterans affairs, and the national institute of health. dr. kpp was supported by grants from the department of va and the national institute of health. the sponsor has no role in the design, methods, subject recruitment, data collections, analysis, or preparation of paper. all authors have contributed significantly to the paper and approve the final version. detailed author contributions are as follows: hdl interpreting data analysis and manuscript preparation; kpp conceptualization, facilitating participant interviews, interpreting data analysis, and critical review of the manuscript; ktd facilitating participant interviews and critical review of the manuscript; prp conceptualization, recruiting, consenting and conducting the study, interpreting data analysis, and critical review of the manuscript. talking to the people that really matter about their participation in pandemic clinical research: a qualitative study in four european countries preserving clinical trial integrity during the coronavirus pandemic action at a distance: geriatric research during a pandemic covid guidance [online]. nih.gov: nih. available at about the office of research & development participant and caregiver perspectives on clinical research during covid- pandemic covid- and older adults: what we know immediate psychological responses and associated factors during the initial stage of the coronavirus disease (covid- ) epidemic among the general population in china covid- and african americans home telehealth: patient satisfaction, program functions, and challenges for the care coordinator we are indebted to all participants in the va clinical research. key: cord- -vlmoa dr authors: mcculloch, peter; sedrakyan, art title: covid- has no effect on gravity date: - - journal: bmj surg interv health technol doi: . /bmjsit- - sha: doc_id: cord_uid: vlmoa dr nan covid- has transformed our world. none of us alive have ever seen anything quite like it in scale, except the very few who still remember world war . like that conflict, the pandemic has elicited responses from governments unparalleled in scope and speed. massive restrictions on people's liberties have been accompanied by massive efforts to prevent the complete collapse of the economy. extraordinary things have happened as a result. right-wing governments have funded workers' wages while they are idle. indians in some cities have discovered that their grandparents were telling the truth when they said you used to be able to see the himalayas. people in atomized cosmopolitan neighborhoods are finding out who their neighbors are, and looking after them. of course medical research has been hugely affected, this being a medical crisis. governments have swept aside sedate procedures which normally add months or years to the life cycle of research projects, and offered huge sums to speed any projects which offer hope of a solution. many researchers too are galvanized by the urgent need to do something and eager to offer their talents to what all deem a worthwhile cause. some of the results have been truly dramatic. the recovery trial of drugs which might ameliorate the effects of the virus went from protocol to first patient enrolled in days, and recruited over patients in weeks, and the oxford vaccine trial launched last week are among the most impressive examples. these achievements required not just money, but the willing cooperation of many people who would not normally have countenanced such haste. but the crisis has, as crises do, brought out the worst as well as the best in us. the remarkable research achievements listed above have been paralleled by a tsunami of proposals and projects whose chance of improving the lives of patients, like their prospects of contributing to the sum of medical knowledge, is near to zero. it would be invidious (and legally risky) to name names, but many proposals we have seen are quite clearly doomed to fail because of obvious flaws in design or in basic logic, or because they are asking a question of absolutely no importance. do we really need detailed studies of the effect of the virus on practice in every conceivable specialty? qualitative research with affected medical personnel has a valuable place, and will help us to understand the responses we have seen to the epidemic, from the heroic to the disgraceful, but some of the proposed studies of reactions to it are not research-journalism, perhaps, history possibly, or in rare cases art, but not science. trials and observational studies whose focus is too poorly defined to make a valid conclusion possible, or which rely on data which clearly will not be possible to collect are exercises in futility. much of this is classic research waste, as was recently pointed out in the bmj. it is also critical that we don't make premature or exaggerated claims. the adaptive covid- treatment trial of remdesivir was highlighted before any details were available even on preprint servers, and inferences made publicly about mortality reduction, although the trial did not show this. on the same day another randomized clinical trial was published showing no trend towards improved survival with remdesivir, and meta-analysis of these two trials shows no difference in mortality outcomes (p value . ) (see figure ). why is this happening? and what should we do about it? while hope for cure is biasing many policy leaders, the lure of easy money is also hard to resist, and scientists are as susceptible as anyone else to a bonanza. this is undoubtedly one major underlying cause of the covid-associated flood of junk science. an opportunity to do something large and impressive is presenting itself to many researchers, because so much goodwill and effort is being put into the drive to do something constructive by researchers, patients, clinicians and even the muchmaligned bureaucrats who run our systems. but bad science will leave its footprint everywhere: in our memory, in public trust and in overcoming fear: it will not help us either in open access figure relative risk of mortality in the trials of remdesivir. the short or the long run. the laws of scientific inference and statistics have not been affected by the virus, and studies whose design guarantees they cannot produce a valid result still will not do so during the crisis. and of course, applying for funding in the full knowledge that it will not be used for the purposes stated, or that it cannot yield the knowledge ostensibly sought, is still unethical. the crisis has shown that the normal processes of peer review and prioritization, both in funding and in publication, can be radically accelerated, but should be robust to protect the conduct of meaningful clinical research. those responsible for managing these activities have a responsibility to ensure that funds are not wasted. many processes already have an executive filter for clearly inappropriate submissions before peer review is even activated, and this needs to be exercised whether or not the terms covid or coronavirus appear in the title or abstract. we hope the research world, like the rest of society, will keep some of the helpful adaptations it has made to cope with the crisis. however, it is also essential that the rigor of the scientific process is maintained if it is to continue to have value and meaning. this is not just the responsibility of the regulators, funders and publishers. the scientific community also needs to reflect and conduct itself according to the high standards of integrity it claims to espouse. twitter art sedrakyan @artsytwits funding the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. competing interests none declared. patient consent for publication not required. provenance and peer review commissioned; internally peer reviewed. open access this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by-nc . ) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by-nc/ . /. peter mcculloch http:// orcid. org/ - - - recovery trial: the uk covid- study resetting expectations for clinical trials covid- : what do we know so far about a vaccine? mike clarke: how can we avoid research waste during the covid- pandemic and plan for the future available: https:// clinicaltrials. gov/ ct / show/ nct fda will reportedly authorize use of remdesivir for covid- after trial shows 'positive effect' on recovery time remdesivir in adults with severe covid- : a randomised, doubleblind, placebo-controlled, multicentre trial key: cord- -iefkv g authors: leal filho, walter; azul, anabela marisa; wall, tony; vasconcelos, claudio r. p.; salvia, amanda lange; do paço, arminda; shulla, kalterina; levesque, vanessa; doni, federica; alvarez-castañón, lorena; mac-lean, claudia; avila, lucas veiga; damke, luana inês; castro, paula; azeiteiro, ulisses m.; fritzen, bárbara; ferreira, paula; frankenberger, fernanda title: covid- : the impact of a global crisis on sustainable development research date: - - journal: sustain sci doi: . /s - - -y sha: doc_id: cord_uid: iefkv g the crisis caused by covid- has affected research in a variety of ways. as far as research on sustainable development is concerned, the lockdown has significantly disrupted the usual communication channels and, among other things, has led to the cancellation of meetings and long-planned events. it has also led to delay in the delivery of research projects. there is a gap in the literature in regards to how a global crisis influences sustainability research. therefore, this ground-breaking paper undertakes an analysis of the extent to which covid- as a whole, and the lockdown in particular, has influenced sustainability research, and it outlines the solutions pursued by researchers around the world to overcome the many challenges they have experienced. this paper also outlines some measures that may be implemented in the future to take more advantage of existing technologies that support research on sustainable development. electronic supplementary material: the online version of this article ( . /s - - -y) contains supplementary material, which is available to authorized users. the global health crisis caused by covid- impacted research activities undertaken by higher education institutions, research centres and research groups in a variety of ways. despite significant efforts to decrease the effects of the pandemic crisis, some researchers around the world have been (or are) forced into social isolation, and they had to interrupt face to face meetings with their colleagues, which had an adverse effect on the work within research teams. furthermore, during the peak of the pandemic, several activities in research laboratories were interrupted. this has led to delays in the scheduling of many research projects. in many cases, researchers were forced to apply for an extension of the deadlines for their activities. research centres are redesigning and adjusting their research policies to try to respond to their partnerships and stakeholders while trying to maintain the strongest local impact (mckinsey & company ) . particularly, research on sustainable development (sd) encompasses more critical issues than other fields of research. from different approaches and disciplines, sustainability research has been characterised as facing the multidimensional challenges of the complex relationship between the environment, the economy and society's prosperity. thus, the spread of covid- is upsetting climate and biodiversity meetings around the world. 'coronavirus hits a critical year for nature, climate' (uwaegbulam ) disrupting 'un meeting plans around the world' (leone ) . several meetings planned for were postponed, rescheduled or virtualized, such as the sdg planning calendar, ipbes meetings, iucn, unfccc and the un ocean conference (un a, b; unfccc ) . this response to covid- negatively distresses the potential establishment of a treaty on a new global framework to limit warning and to protect marine and land biodiversity over the next decade. in spite of the coronavirus crisis, there are increasing research outputs regarding sars-cov- and covid- . the european commission plays a crucial role in supporting research and innovation by coordinating european and global research efforts, including preparedness for pandemics. the commission is launching several special actions in that address epidemiology, the development of diagnostics, vaccines, as well as the infrastructures needed for this research (eu ) . apart from research on the virus (yan et al. ; shang et al. a ; among others) and vaccines (amanat and krammer ; shang et al. b; among others) , the interfaces between nature, biodiversity, viruses and zoonotic diseases/zoonotic viruses (johnson et al. ; lam et al. ; salata et al. ; zhou et al. c; xu ) are highlighted, and reservoirs and interspecies transmission viruses (chan et al. ) should be further discussed in relation to sustainability. on the basis of this consideration, wwf italy published a document on pandemics and their related effects on the destruction of ecosystems (wwf ) . evidence demonstrated that most of the so-called emerging diseases, such as ebola, aids, sars, swine flu and the new coronavirus, are not random catastrophes but the consequence of our impact on natural ecosystems. human activity has significantly altered three quarters of the emerged land surface and two-thirds of the oceans, and it has determined the birth of a new era called the 'anthropocene' (wwf ) . wealth and the abundance of species, two important components of biodiversity, can counteract the spread of diseases in several ways. among these, the dilution effect and the coevolution effect should be analysed and discussed (wwf ) . another relevant aspect is the reduction of pollution and carbon emissions during this period of lockdown. some organisations, at the local and international levels, are checking the current benefits from the shutdown of factories and business activities. for instance, the european environment agency (eea) is assessing how coronavirus measures have influenced the concentrations of air pollution by developing a viewer that tracks the weekly average concentrations of nitrogen dioxide (no ) and particular matter (pm and pm . ). the drop-in air pollution should be investigated in view of forward-looking investments and ambitious policies to move towards a resilient and sustainable society (eea ). a common axiom in sustainability research is the intersection between complex networks of interactions and the cultural, territorial, organisational, institutional, and infrastructural aspects (leahey and barringer ; waas et al. ). this has motivated the transition from sustainability to participatory and transdisciplinary research approaches to accelerate their impacts and magnify them in the long term or to succeed in problem-focused research (williams and robinson ; rau et al. ) . transnational collaborations can support effective progress in sustainability research. caniglia et al. ( ) reported some successful factors such as 'combining local and global considerations; making effective use of digital technologies; capitalizing on cultural and national differences; and making the best of available resources' (caniglia et al. , p. ) . transdisciplinary research models have been proposed as routes that use science to solve complex socio-environmental problems, despite the implied disciplinary tensions (klenk and meehan ) . particularly, in view of the implementation of the sustainable development goals (sdgs), sustainable research requires integrative science and the active involvement of academic institutions and non-academic actors, such as innovative companies, municipalities and civil society, namely as a living lab (bergvall-kåreborn and ståhlbröst ; schneidewind ; wiek et al. ) . to ensure the effectiveness of such research and its societal consequences, a transition to participatory sustainability approaches may be a successful solution. based on this framework, the crisis caused by covid- has posed new challenges for sustainability research, and it is relevant to study how a global crisis influences such research. the socio-political measures taken under high uncertainty to contain its spread have been absolutely essential (fong et al. ; zhou et al. a) . beyond the significant interruptions of the usual communication channels or the delays in the delivery of research projects findings, these measures equally represent a high uncertainty for sustainability research. ivanov ( ) shows that new research tensions are surfacing in regards to the impact of covid- with variables that hinder them; the pandemic is a special disruption risk that begins with a limited scope on a large scale, and spreads across many geographic regions. according to angeloni ( ) , a large part of recent research has focused on the impact of technological progress and globalisation to achieve sustainability. however, there is a latent demand for new ways of doing sustainability research to achieve transdisciplinarity and blur the geographic and communication boundaries that inhibit research processes. the literature reports some alternatives with potential in sustainability research in times of epidemic outbreaks; digital technologies such as data analysis, artificial intelligence, machine learning or digital twins (ivanov ) , big data technologies in geographic information systems (zhou et al. a) , or drones, g and robotics (show et al. ) are some of them. beier et al. ( ) explain that these technologies have a significant influence on the alignment of the objectives of sustainable development. these alternatives for sustainability research open a relevant research area. according to zhou et al. ( a) , the main challenge is to determine the strategies that have the appropriate mixtures of traditional methods and new technologies to impact sustainability. besides, show et al. ( ) state that it is essential to consider that the extensive use of emerging technologies goes beyond a pandemic response; it is necessary to appropriately link technology to the socioeconomic and scientific context. therefore, this article analyses to what extent covid- as a whole and the blockade in particular have influenced sustainability research, and it describes the action paths that researchers around the world identify for overcoming the experienced challenges and the measures that can be implemented in the future based on existing technologies. the remainder of this paper is structured as follows: section presents covid- , related facts and figures and its impact on research, with a specific focus on sustainable development. section discusses the methodological aspects by describing the questionnaire delivered via a sophisticated on-line survey. the findings are discussed in sect. , and sect. makes some final remarks about further development and practical implications of this research. a new pathogen, identified as a different coronavirus (sars-cov- ), caused an unusual pneumonia (covid- ) outbreak in december , starting in wuhan city in hubei (a central province in china) (zhou et al. b ) and spreading rapidly in the first three months of to other countries and territories (la et al. ). this pathogen is a beta coronavirus and shares a genetic sequence and viral structure with severe respiratory syndrome coronavirus (zhou et al. b) . the alignment of the genome sequence of the covid- virus revealed that the closest relationship was with the sars-like bat coronavirus (who a). according to the world health organization (who a), covid- is transmitted mainly via droplets and fomites during close, unprotected contact with an infector. regarding symptoms, the disease presentation can range from no symptoms (asymptomatic) to severe pneumonia and death. the fast contagion and severity of this new disease has driven the who to change its statement from classifying the outbreak as a 'public health emergency of international concern' on the th of january, to a 'pandemic' on the th of march, (who b). from the first situation report of coronavirus disease (covid- ) on the st of january , there were confirmed cases from four countries, including china ( ), thailand ( ), japan ( ) and the republic of korea ( ). of the confirmed cases, cases were severely ill, were in critical conditions, and were death. the cases in thailand, japan and the republic of korea were exported from wuhan (who c). on the th of february, the scenario was completely different: the novel coronavirus ( -ncov) situation report mentioned a total case count of , confirmed ( new) with new deaths in china. outside of china, there were confirmed ( new) in countries and death. the who risk assessment in china was very high at the regional level and high at the global level (who d). the following month, the situation report of the th of march (who d) reported a total of , confirmed cases. in china, there were , confirmed cases and , deaths; outside of china there were , confirmed and deaths in countries. the who risk assessment both in china and globally was very high (who e). after a month, the situation report of the th of april presented a total of , , confirmed cases spread throughout the european, americas, western pacific, eastern mediterranean, and south-east asia regions. at this point, the risk assessment at the global level was very high (who f). given the urgency of this outbreak, the international academic community is mobilising ways to accelerate the development of disease detection and intervention (la et al. ) . schools, universities, and research institutions are temporarily closed around the world in order to slow the spread of the covid- pandemic, impacting over % of the world's students (unesco ). the closure of higher education institutions (hei) affects not only the students at these schools but also the faculty who normally conduct research . this paper focuses specifically on the effect of covid- closures on sustainable development research. the un secretary general had considered as a 'super year' for sustainability action, prior to the covid- outbreak (djalante et al. ) . sustainability efforts are supported by research that explores environmental, social and economic dimensions of complex problems. this research is often collaborative, involving university researchers and non-academic stakeholders, with the goal of advancing sustainable development goals in specific places (van kerkhoff and lebel ) . the lockdown that has occurred in many countries can influence current work of researchers through work place restrictions, movement constraints for accessing research sites, facilities constraints, interruption of social and political processes in which research is situated, access to physical libraries or to high-speed internet, lack of peer support, and work-family interface challenges, among others. furthermore, the increased uncertainty amongst the population, accompanied by stress, actual sickness, and mental health challenges, can influence researchers as well. while there are many potential impacts of the covid- closures on research, very little has been documented in published literature. it has been reported that the closures have influenced the biomedical scientists who rely on wet labs (nature medicine ). there are also news stories of impacts on sustainability research. for example, the smithsonian magazine has reported that the northern gulf of alaska long term ecological research project may halt the three research cruises planned for , interrupting the physical and biological data that has been collected for decades to understand oceanographic trends in this region (pope ) . similarly, they report that a professor of engineering at a us-based college has delayed a community-based collaborative research project in greenland for creating sustainable energy solutions, relying instead on internet-based communications to gather preliminary data (pope ) . lab experiments may also be impacted, which is especially troubling for researchers that must keep plants and animals alive during strict laboratory closures (kimbrough ) . data collection procedures and actual fieldwork for surveys for research projects had to be adapted, in particular if targeting the elderly population (scherpenzeel et al. ) . also, sastry et al. ( ) and gummer et al. ( ) showed that covid- had a negative effect on fieldwork. this included the reduction of survey response rates and the termination of home visits or face-to-face interviews that can represent an important component of studies, including the ones that address the social dimension of sustainable development. for sustainability research, the exchanging events, forums and open communication arenas are of particular importance. international conferences, due to their interdisciplinary character and diverse stakeholder's participation, are used by higher educational institutions as a means to promote sustainability (berchin et al. ) . around the world, many such events are cancelled or postponed, impacting the exchange of knowledge, ideas, approaches, and further international collaborations. an adaptation of sustainability research themes or change of the research's trajectories can also be expected. knowledge from social scientific research related to the contextual behaviour aspects of communities, for instance, can be aligned with biomedical insights of the epidemic (la et al. ) . similarly, the provision of knowledge and science in understanding disaster and health-related emergency risks, as part of the sustainability research on current strategies for disaster resilience as outlined in the sendai framework, can contribute to responses to covid- (djalante et al. ) . emergency research can be a response for the risk management of newly created situations at chinese universities . constraints in doing sustainability research can be exacerbated by institutional capacity to change (spoelstra ) . the degree to which covid- will have lasting impacts on sustainability science research is unclear; disasters tend to facilitate societal change (cohen ) . it is likely that the global health crises, with their associated environmental, social and economic aspects, will continue in the future, and it is important that scientific institutions develop methods for dealing with the associated uncertainties in order to continue to provide context-specific knowledge to aid in decision making (djalante et al. ). this study aimed at ascertaining the extent to which the lockdown and other societal aspects of covid- have impacted researchers and influenced their studies on sustainable development. to address the established goal, a crosssectional descriptive study was performed. the descriptive approach is frequently used when little research has been done in an area to understand new concepts or phenomena (tarzian and cohen ) . to assess the impacts of the crises caused by covid- on sustainable development research, a questionnaire was delivered via a sophisticated on-line survey. the instrument incorporated three parts, in which questions were grouped and composed as follows: seven open-ended questions, four dichotomous questions, seven closed-ended questions and eleven five-point likert scale questions. these questions gathered relevant information on aspects such as respondents' backgrounds, the impact of the lockdown on their work in terms of distance learning, workload and challenges faced, as well as future projections regarding the covid- crisis influence on research. the full questionnaire is presented in appendix a. a pre-test was carried out by a group of academics whose fields of expertise lie within the scope of sustainable development research (hair et al. ) . the final version of the survey was implemented through the google forms system and distributed by email, collecting responses for weeks ( th april to th may). using the snowball sampling strategy, the instrument was initially shared with the inter-university sustainable development research programme (iusdrp) and also within each co-author's institution. the analysis procedure involved: (a) the statistical package for the social sciences-spps for quantitative data analysis, in which the following tests were performed: frequency, mean and a correlation of factors, and (b) the qualitative analysis by nvivo software was performed, performing tests of frequency and word cloud, applied to the main terms. the qualitative approach adopted here followed the experiences documented by bardin ( ) . in total, responses were received from all continents and different countries: australia, austria, belgium, brazil, canada, china, colombia, ethiopia, finland, germany, greece, guatemala, india, indonesia, iran, ireland, israel, italy, kenya, malaysia, malta, mexico, netherlands, nigeria, norway, pakistan, poland, portugal, romania, scotland, serbia, south africa, spain, sweden, switzerland, turkey, the united arab emirates, the united kingdom and the united states of america (fig. ) . more than % of the sample are from europe, the continent that reported more than , , cases and more than , deaths months after who was informed of cases of pneumonia of unknown cause in china and a novel coronavirus was identified as the root of these disease cases (who f; ecdc ). among european respondents, % were from portugal, where over , cases were registered, and almost , deaths in the months of covid- (sns ). respondents from south and north america countries represent and % of the sample, respectively. regarding south america, brazil represents . % of the respondents and mexican participants perform more than half ( %) of the north american participants. since the beginning of the outbreak of the novel coronavirus, mexico is in the th position amongst countries with the most confirmed cases in the world, and brazil has the second-highest number, after the united states-all three of which are located in the american continent (jhu ). for the scope of the study, fig. presents the main sample features of the respondents: (a) type of institution; (b) academic position; (c) gender. the sample also reveals that % of the respondents hail from universities and % represent institutes of technology or other higher education institutions. an academic career in universities has different routes in each country; however, many academic jobs include teaching and conducting over half ( %) of the participants were academic staff (professors, teachers, lecturers) and % were researchers with knowledge about sustainable development research. sixty-two percent of the respondents identified themselves as female, highlighting the representativeness of women in the research field. engineering, manufacturing, and construction ( %), natural sciences, mathematics, and statistics ( %), and business, administration, and law ( %), as well as education ( %), were the research areas most cited by the sample. the first two are areas that require, in many cases, practical activities in laboratories or outdoor fields. the main limitation of the study relates to the geographic coverage of the study, with only % of the study from the asia region. there is a significant amount of sustainability research undertaken in this region, and it is expected that researchers in china (where the initial lockdown occurred) may well have a different experience of how the lockdown has affected their research work. the first part of the survey asked respondents about the conditions in which they performed their job during the shutdown. figure shows how long the research had been affected by the shutdown (a) and the level of the researchers' agreement regarding the adopted measures (b). most of the sample ( %) indicated that they were unable to carry out their research, with % of respondents indicating that this was for a period of between and months. this reflects the current understanding, which argues that to minimise the spread of covid- , it is necessary to mitigate the risk of exposure, not only to covid- cases but also to asymptomatic carriers who may harbor the virus and, therefore, pose a significant risk to the health of other people (kimbrough ) . it also reflects the timing of the mass closure of universities and research institutions (unesco ), when research centers were redesigning their research policies and implementing emergency operation plans (mckinsey and company ; wang et al. ; omary et al. ) . this was particularly the case in relation to the potential damage or complete loss of research, and the need to keep plants or animals alive during university and laboratory closures (kimbrough ) . almost a fifth of respondents ( %) stated that they were able to continue conducting their research normally. twenty five percent of them do research in engineering, manufacturing and construction (fig. ) , and % percent are doing research in more than one specific field. more than % are researching in fields that need less access to specialist scientific labs and resources, such as business, administration, law, social sciences, journalism, information and education. although the majority of the sample stated that they were unable to conduct their research normally at university, almost % either agreed or strongly agreed with the actions taken by their institution during this period, almost % either disagreed or strongly disagreed, and % neither agrees nor disagrees. such agreement might reflect the scale and significance of the issue which are encapsulated within the who guidelines to maintain social distance, or the fear that people have of being contaminated or contaminating their relatives who belong to some risk group (rajagopaian et al. ). the implication for research institutes suggests that acting in line with who or other agency advice is helpful to retain the support of their researchers. driven by the need for social distancing and individual executive orders from the state, many institutions have been severely reducing onsite research and maintaining there only the activities considered essential. research activities considered critical are allowed, with the possibility of the presence of a single 'critical laboratory member' on-site at a time, with a possible rotation system (omary et al. ) . during the crisis caused by covid- , most researchers ( . %) have worked only from the home office. the researchers considered the available infrastructure to perform the work on sustainability research from home 'acceptable' ( %) and 'good' ( %), and % of the sample considered the support for research given by their institute as 'good' or 'very good'. figure shows the research workplaces during the crisis (a) and how the researchers evaluate their infrastructure and institute support during the covid- crisis (b). this supports the earlier implication for research institutes to provide appropriate and relevant support, perhaps differentially across research fields. the access and consultation of journal articles ( %) and the use of on-line media reports ( %) were reported as the main means of information necessary for performing the research during the shutdown. sustainability-oriented research requires transdisciplinary models to solve socioenvironmental problems, and communication among the researchers is essential (klenk and meehan ) . therefore, apart from e-mail, the sample used videoconferencing tools to communicate with research groups or attend meetings, such as zoom ( %) and skype ( %). other responses mainly included microsoft teams and google tools (meeting, classroom, and hangout) ( %). figure represents the main information means and communication tools used during shutdown. the use of technologies in sustainability research is an important strategy for the shutdown period, and it can impact sustainability research beyond the pandemic (show et al. ; zhou et al. a, b) . the implication of this is to provide professional development and support in relation to the use of technologies to support ongoing research activity. this part of the survey aimed to understand the extent to which the lockdown triggered by covid- has influenced research. around % of the respondents suggested that the lockdown has had negative or very negative influences on their research (from 'a little bit' to 'a great extent'), % reported neutral effects, and % reported a 'positive' or 'very positive' influence on their research. the neutral effects might be explained by the % who are still able to perform their research under covid lockdown (fig. a) . nevertheless, . % of the sample reported that the main direction of the research has not been adapted or changed as a consequence of this pandemic. table shows the extent to which the shutdown has influenced research and the nature of the influence. impacts on research caused by closures have been reported in different scientific areas (nature medicine ), and the extant literature suggests that the most negative impacts are predominantly related to delays and adjustments in the projects' schedules and programs (pope ) . the sample suggested a more nuanced analysis of these impacts, such as substantial adjustments in the project's schedule ( %), delays ( . %), project meeting cancellations ( . %), and disrupted communication ( %) . impacts on research workload were reported by . % of the participants, with some experiencing a decrease in workload ( . %) and others a moderate or great increase in workload ( %). the respondents also reported difficulties in adapting to the home office regime and in following their research. the main challenges related to covid- shutdown that were experienced by sustainability researchers were the lack of personal interaction and dialogue with colleagues or research stakeholders ( %), the lack of materials or resources ( %), and the lack of interest or motivation ( %). the shutdown has increased the use of communication technologies; academic and scientific staff members have become more familiar with these tech-tools, and these digital technologies have a significant influence on the alignment with the objectives of sustainable development (beier et al. ). however, these results perhaps highlight the need for face-to-face interaction in sustainability research, which is often transdisciplinary and collaborative (heaphy and dutton ; nie and hillygus, ) . it might be the case that some researchers fear that an additional emphasis on technology-mediated perceptions of reality may amplify existing disconnections (ibid). figure illustrates the challenges researchers have overcome during the coronavirus shutdown. the lack of materials and resources might be related to the logistical changes and difficulties in delivering raw material and inputs for research or office materials for basic administrative tasks associated with research. around a fifth of participants reported a lack of support in relation to the administration ( %) and lack of expertise in the use of technologies (also %), which echoes perceptions of those researchers who rate the support given by research establishments as 'poor' or 'very poor' ( % of the sample- fig. b) . these challenges represent high uncertainty for sustainability research (ivanov ) . despite the negative impacts and challenges, some sustainability researchers used the open answers to point out other positive aspects within their regime of work, such as bond improvements with relatives, being able to spend extra time with family members, and learning about their personal and professional life. such sensations of social connection and belonging are fundamental to people's physical, mental, and professional well-being (omary et al. ) . again using the open answers, researchers reported that the physical and social isolation, associated with maintaining the research activities from the home office, has opened to them to a new universe (something that rarely used to occur before the novel coronavirus); this new experience has stimulated empathy and solidarity on a daily basis, as well as the ability to produce more, as they have more time to devote exclusively to research. figure thinking about new research topics; (l) creating new working methods at the university; and (j) actions for the community and partnerships. the pandemic has created a new scenario for science professionals and institutions, bringing with it a scenario for rapid adaptation to change, problems and also opportunities as new drivers for research agendas and projects for sustainability. these aspects also reflect the challenges of the lockdown and the home office. figure shows that several participants ( . %) strongly agree that there is an adaptation or change in the way research is done, adding more on-line meetings and using more on-line resources; they expect this to continue in some way after the crisis, since it reduces the need for people to travel and move around, providing an economy of time for these people and collaborating in the reduction of carbon emissions. almost % agree or strongly agree that the covid- crisis has generated environmental and social impacts. cicala et al. ( ) cite that during social distancing in los angeles, there was a reduction in carbon emissions of . million metric tons. authors also affirm that behavioural changes in other major cities also led to substantial reductions in co emissions. also in fig. , it is possible to observe that over half of the sample ( %) 'agree' that the impact of the covid- crisis has influenced the way researchers understand or interpret change in wider systems, and almost % of the sample reported changes in project planning and new proposals focused on issues such as a health crisis, climate change, resilience, and environment, among others. the covid- topic has been added as a topic in current research activities by % of the researchers, and % 'agree' or 'strongly agree' that a review of research methods to highlight the impact of this crisis on life was assumed. these results reinforce the role of sustainabilityoriented research in addressing transdisciplinary issues that may trigger social and environmental impacts (klenk and meehan enable researchers to adapt when they are required to. this would include both the practical and administrative flexibilities that are required to do their work, but also the capabilities to be able to flex within their own research practise. this does not only include the sites in which their work is conducted, but also the ways in which they conceptualise the methodological aspects of their research (and most probably the theoretical dimensions which are linked to their methodological commitments). however, it is likely that research establishments need to be sensitive to the differential impacts across disciplinary fields, and so will need to reflect this in their own strategies and policies. the last part of the survey aimed to offer an understanding of future perceptions of the respondents on how sustainability research will be performed after the covid- pandemic. the majority of the sample ( %) 'agree' that the covid- crisis will influence research in the long-term. figure presents the main ways the respondents believe this influence will occur. the most common, longer term influence of covid- reported by researchers ( %) was that more technological resources would be deployed for coping with working from home. this is reflected in other studies which argue that the next generation of researchers will retain the memory of living and working through the crisis with them throughout their careers and will be shaped by the environment created by the situation (omary et al. ) . for example, the more extensive use of virtual lab meetings, research seminars, conferences, events, and meetings with students and fellow researchers will cause an increase in the current frequency and speed at which current activity is being organised (omary et al. ) . therefore, it is necessary to adapt and shift to more it-based communication technologies and consider the possibility of extreme events when preparing new schedules for research development (djalante et al. ) . the degree to which covid- will have lasting impacts on sustainability science research is unclear (cohen ) . new research opportunities are emerging, such as the european commission's recent announcement of up to € million to research the covid- crisis (eu ). in this sense, many of the researchers responding to the study ( %) also reported an increase in creativity or new ideas this study aimed to foster a better understanding of the impacts of the covid- lockdown on sustainable development research; our survey coincided with the period immediately after the peak in several european countries, the confirmation of covid- in all north american territories, and the rapidly rising count in south america. the information gathered highlight important aspects of the impact of covid- in terms of daily-based research routines. it also illustrates the adaptation measures needed to allow a continuation of research-along with teaching and other university activities-as well as future planning and the capacity to resume sustainable development research. this study has some limitations. first, it focused on sustainability research only and not on other activities. the reason for this is that the project teams have also undertaken other studies that look, for instance, at the impacts of the covid- pandemic on teaching. second, the sample is limited to responses, and this can by no means be regarded as representative of the world academic community. but the fact that these responses have been provided by academics from countries in all continents who are active in sustainability research means that a rough profile of the opinions from researchers can be built. an advantage of the study is that it is one of the very few which are fully devoted to research on sustainable development. in terms of gender issues, almost two-thirds of the respondents were female, and % of them were students or researcher fellows. such results are consistent with the reflections of the impact of covid- : ( ) on academic mothers (staniscuaski et al. ; gibson et al. ) and how this pandemic is exacerbating the gender inequalities in science; and ( ) on students and research fellows and how the interruption of scientific activities will be reflected in their research work, which is typically based on insecure contracts (inouye et al. ; gibson et al. ) . the study did not explore the impact of covid- closures in terms of the individuals' contexts, but many scientific journals contain testimonies from researchers who are facing challenging responsibilities regarding family (staniscuaski et al. ) . flexible-work from home has been adopted in most institutions, but again the expectations and adjustments seen in the modalities of remote working have not been reported evenly in the sustainable development research field. about one-eighth of our sample was from the natural and agricultural sciences, onequarter from engineering and technology, and one-third from fields that need less access to specialist scientific labs and resources. despite the constraints on continuing current research activities, which were reported by the majority of our sample, about % stated that they agree with the lockdown measures and % believe that their research institutions are supporting them. over half of our sample admitted that the impact of the covid- crisis influenced new angles of interpreting changes in wider complex systems, and almost % reported the intention of considering the health crisis, climate change, resilience, and environment, among others, in new research proposals. after a period of meetings cancelled all over the world, scientists have discovered the upside of innovative and hightech solutions, such as video conferencing, that provide the opportunity for virtual meetings while also making an important contribution to the move forward towards a lowcarbon economy. despite this advantage, one of the main challenges experienced by sustainability researchers was a lack of personal interaction, such as in participatory research involving multi-stakeholder groups. scientific journals have also been facilitating free access to covid- papers, bringing forth new opportunities for integrating science and rethinking sustainable development research without borders. by the time this paper has been prepared, about one in five individuals worldwide could be at risk of being affected by the covid- pandemic (clark et al. ) . the challenges brought by covid- also opens venues for further research on data collection methods and procedures that require personal interactions and face-to-face interviews, as these methodological transitions require changes in traditional collection instruments and data analysis, as debated by burton et al. ( ) or . some of the measures that may be implemented in the future, towards a greater support of research on sustainable development, are: (a) better planning of sustainable development research in a way that it becomes more resilient to such pandemics; (b) a more systematic use of existing technologies in a way that they may support research, for instance, a wider use of international, multilingual scientific studies and a greater dissemination of their findings and knowledge; (c) greater reflection on the adequacy of some research methods, so that studies may be undertaken even in the case of lockdowns; (d) consideration of the carbon neutrality of research, coupled with social innovation. this includes, for instance, how institutions may adapt their research labs and campus facilities for a greater provision of technical training and online support, which may help to make research projects more efficient. finally, the covid- pandemic has shown that, with due care and planning, 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probable bat origin publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. walter leal filho , · anabela marisa azul · tony wall , · claudio r. p. vasconcelos , · amanda lange salvia · arminda do paço · kalterina shulla · vanessa levesque · federica doni · lorena alvarez-castañón · claudia mac-lean · lucas veiga avila · luana inês damke · paula castro · ulisses m. azeiteiro · bárbara fritzen · paula ferreira · fernanda frankenberger , key: cord- -nws ug authors: islam, muhammad nazrul; inan, toki tahmid; rafi, suzzana; akter, syeda sabrina; sarker, iqbal h.; islam, a. k. m. najmul title: a survey on the use of ai and ml for fighting the covid- pandemic date: - - journal: nan doi: nan sha: doc_id: cord_uid: nws ug artificial intelligence (ai) and machine learning (ml) have made a paradigm shift in health care which, eventually can be used for decision support and forecasting by exploring the medical data. recent studies showed that ai and ml can be used to fight against the covid- pandemic. therefore, the objective of this review study is to summarize the recent ai and ml based studies that have focused to fight against covid- pandemic. from an initial set of articles, a total of articles were finally selected through an extensive inclusion-exclusion process. in our review, we have explored the objectives/aims of the existing studies (i.e., the role of ai/ml in fighting covid- pandemic); context of the study (i.e., study focused to a specific country-context or with a global perspective); type and volume of dataset; methodology, algorithms or techniques adopted in the prediction or diagnosis processes; and mapping the algorithms/techniques with the data type highlighting their prediction/classification accuracy. we particularly focused on the uses of ai/ml in analyzing the pandemic data in order to depict the most recent progress of ai for fighting against covid- and pointed out the potential scope of further research. down the covid- outbreak [ ] . therefore, various bodies have committed themselves to conduct research focusing on covid- to support global response. with new discoveries being announced at a breathtaking pace, artificial intelligence (ai) has re-emerged into scientific consciousness. ai is a branch of computer science that can be used to build intelligent systems. it is often instantiated as a software program [ ] . recent application of ai in diagnosing disease s has broadened the frontier of ai which once was a humans expertise. medicine and the health care systems are among the most promising areas of application of ai which can be traced back to as early as mid-twentieth century [ ] . researchers proposed and successfully developed several decision support systems [ ] . the rule-based system gained success in the late s [ ] and had been useful in detecting disease [ ] , interpreting ecg images [ ] , choosing appropriate treatment and generate hypothesis by the physicians [ ] . unlike this first-generation knowledge-based ai system, which relies upon the prior medical knowledge of experts and the formulation based rules, modern ai leverages machine learning algorithms to find pattern and associations in data [ ] [ ] [ ] . the recent renaissance in ai can be attributed to the successful application of deep learning to a great extent-training an artificial neural network with a large number of labelled datasets. a modern deep learning network usually contains hundreds of hidden layers [ ] . the recent resurgence of ai has been fueling a question of whether aidoctors will replace human physicians shortly. yet to be confirmed, researchers believe that ai driven intelligent systems can significantly help human physicians in making better and quick decisions and even sometimes remove the necessity of human decision i.e. radiology [ ] . the increasing data in health care resulting from increased use of digital technology and the advancement of big data analytics can be attributed to the recent success of ai in healthcare [ ] . although ai research in healthcare is emerging, most of the research is concentrated on three diseases: cancer, neurology and cardiology. guided by evidence, a strong ai can reveal the insight of the medical data which eventually can be used for decision support and forecasting [ ] , [ ] , [ ] . as ai has been proved useful in healthcare, researchers suggest that it may also be helpful in fighting against covid- . from forecasting of a pandemic to designing anti-viral-replication molecules, ai has made a paradigm shift in health care. recent research on covid- using ai suggests that ai can be helpful in detecting covid- infection, detecting infected population, predicting the next outbreak, finding the attack pattern and even finding a cure [ ] , [ ] , [ ] . the objective of this review is to explore the existing aibased research that has been conducted to fight against covid- pandemic. the organization of the remaining section is as follows. the methodology to conduct this review study is discussed in section . the review data analysis and findings are discussed in detail in section . section presents the main findings and the potential scopes of future research to fight against the covid- . finally, the concluding remark, research limitation and future work are presented in section . in this research, a systematic literature review procedure [ ] was adopted to attain the research aim. for selecting the primary arxiv: . v [cs. lg] aug articles, the major databases such as ieee xplore, springer link, acm digital library, science direct, and google scholar were searched for related articles . the search strings used to find the literature were machine learning and covid- , machine learning and coronavirus, artificial intelligence and covid- , artificial intelligence and coronavirus, artificial intelligence and lockdown and pandemic and covid- , machine learning and lockdown and pandemic and covid- , and coronavirus prediction and outbreak prediction and machine learning and artificial intelligence. these strings were applied for all the above-mentioned databases as well as google search engine. the search returned more than articles. the exclusion criteria include (a) duplicate articles that are found through several scholarly databases; (b) articles that are not focused on our research objectives; (c) the article was written other than english; and (d) the earlier version of any article that has been published on the same set of data and explored the same objective. after applying this inclusionexclusion process, we finally selected articles that include original research, review articles, and short articles including perspective, commentary, and letter to the editor. the prisma flowchart in figure shows the article selection process in different phases following the exclusion-inclusion criteria. the selected articles were reviewed systematically to extract data primarily related to the article type, publication time, research objectives, study context, study outcomes/findings, methodology/algorithm/techniques used, dataset used, and study subject. finally, the extracted data were synthesized and analyzed to summarize the existing research on covid- pandemic using ai and to identify the potential scopes for future research. to date ( th june, ) out of articles, ( %) were published as original research. among others ( % articles), three articles were the review articles, two were editorial and the remaining three were published as research perspective (short conceptual article). again, ( . %) articles were published in academic journals, while ( %) articles were archived as pre-print. among the pre-print, . % ( ) were original research. all the selected articles were published or archived in the online databases during january-may . we synthesise the existing research in terms of their purposes and aims to explore the contribution of ai to fight against the covid- pandemic. a summary of the synthesized data is presented in table i to show the research scopes and purposes of the original research. most of the articles (n = , %) were published focusing to detect the covid- infected patients using different ai-based algorithms that include, for example, the convoluted neural network (cnn) model, support vector machine (svm), generative adversarial network (gan), and the transfer learning. the chest x-ray images, ct images, mobile sensors data, and covid- symptoms were used to predict/detect the covid- patients. these researches aimed to identify, screen and detect the covid- patients; and also to predict, differentiate, or classify the patients into covid- infection, no infection, and other viral or bacterial infection. for example, wang et al. [ ] proposed a cnn based prediction system named covid-net that identifies non-covid- infected, covid- infected, and no infected patients using chest x-ray images. the proposed model was pre-trained on imagenet (open source) dataset and then trained on the covidx (author-created) dataset that includes , chest x-ray images of , patients that includes images from covid- positive patients, images are of healthy patients and images are of non-covid patients. a total of seven ( %) articles focused on diagnosing covid- patients through ai ( figure ). in these articles, the ai was used to diagnose the identified covid- patients to classify in to patients categories (severe, mild) and tracking their progress [ ] , distinguish covid- from pneumonia [ ] , efficiently diagnose covid- using x-ray images [ ] , predict survival and death for severe covid- patients [ ] , identify patients who would develop more severe illness [ ] , and to estimate uncertainty in deep learning solutions to improve the diagnostic performance from posterior-anterior (pa) x-ray images of lungs with covid- cases [ ] . two ( %) articles aimed to forecast the covid- epidemic to estimate the progress of the epidemic in terms of its size, lengths, peaks, and ending time as well as predict the development trend of the epidemic for the next certain time period in a specific country or geographical region [ ] [ ] . we found only one study [ ] focused the sustainable development that analyzes confirmed cases of covid- through a binary classification using ai and regression analysis and explores the correlation among confirmed cases of covid- in four countries (china, italy, south korea, japan) and environmental factors (low, high & average temperature, humidity, wind flow). an article [ ] compared the prediction performance of the proposed algorithm with the existing vgg- , googlenet, and resnet method using two different subsets of data, while in [ ] , an ai-based model is developed based on the existing studies [ ] detect covid- with the help of ai and smartphone sensors [ ] use an anomaly model based on deep learning network to make the screening process faster for covid- detection from x-ray images [ ] detect covid- from x-ray images using transfer learning with cnn [ ] detect covid- from x-ray images using deep cnn model [ ] propose an algorithm to detect covid- from ct images using deep cnn model and svm classifier [ ] develop a deep learning model coronet using the xception cnn to detect covid- from xray images [ ] build a framework which uses smartphone sensors to detect covid- [ ] diseases detection classify patients in to non-covid infection, covid- infection, and no infection from x-ray images using deep cnn model [ ] compare the performance of seven dl model to find the best model for covid- detection [ ] develop and evaluate the performance of an ai model to detect covid- and also evaluate the performance of radiologist to detect the disease by using and without ai support [ ] detect the covid- by identifying the characteristics from chest x-ray using a deep learning model(cad covid-xray) [ ] detect covid- from x-ray images using generative adversarial network (gan) and deep learning transfer [ ] diagnosis the identified patients to classify (in to patients' categories) and tracking the progress covid- patients [ ] distinguish covid- from pneumonia using deep learning [ ] efficiently diagnosis covid- using x-ray images through deep cnn models [ ] developing a tool to predict survival and death for severe covid- patients [ ] diseases diagnosis diagnosis covid- positive case faster using both non-image and image clinical data [ ] develop a system to identify patients who would develop more severe illness among the patients with mild cases of covid- [ ] develop a system to improve the diagnostic performance from posterior-anterior (pa) x-ray images of lungs with covid- cases [ ] forecast of the covid- to estimate size, lengths and ending time of covid- across china [ ] epidemic forecasting predict the trend of the infection for the next days using deep learning [ ] to predict the progress of the epidemic (epidemic sizes and peaks) [ compare the prediction performance of the proposed algorithm with the existing methods [ ] compare seven different dl model to find out the best model for disease detection [ ] performance comparison the remaining ( %) articles include review articles, editorial, perception, commentary, and short communication. the review articles are summaries of the existing research with the aim to highlight the contributions and constraints of ai [ ] , and to identify a roadmap of ai applications to fight against covid- pandemic [ ] . another review analyzes the ai-based techniques used in the ct and x-ray based medical imaging to fight against covid- pandemic [ ] . one of the two editorials highlighted how ai-based solutions may assist to fight against the pandemic by forecasting the pandemic to design anti-viral replication molecules, but with the supervision of humans [ ] . in another editorial, a workflow is presented to highlight the processes and applications of ai to fight the covid- pandemic [ ] . the perspective articles highlight firstly the needs of ai and the ways of data sharing (via smart city networks) for better monitoring and management of urban health on the covid- outbreak [ ] ; secondly, the importance of active learning-based ai tools for coronavirus outbreak [ ] ; and finally, suggested how ai and blockchain can be used to help the community during the covid- pandemic with equipment and donations [ ] . by using a private blockchain network to make donations for the pandemic, there would be no alterations and the donations would go to their destinations. a summary of the synthesized data is briefly presented in table ii to present the research scopes and purposes of the other types of research. the editorial constitutes existing works, current efforts and potential work ideas to fight against covid- using ai, ml algorithms, deep learning, neural networks. [ ] highlighted the needs of ai and the ways of data sharing via smart city networks for a better monitoring and management of urban health on the covid- outbreak [ ] perspective discussed the importance of active learning based ai tools for coronavirus outbreak. tools that use cross population training/testing methods and multitudinal and multimodal data. [ ] introduced ai and blockchain and suggested how they can be used to effectively help the community with equipment and donations.introduced ai and blockchain and suggested how they can be used to effectively help the community with equipment and donations. [ ] some of the articles focused their research on specific countries while others conducted research with a global perspective. a total of articles ( %) focused on specific country as shown in table iii . one of these articles considered confirmed cases from provinces of china for their research on a forecasting system of those areas [ ] . another article focused on provinces in japan, china, south korea and italy for environmental parameters, weather trends and confirmed cases to measure correlations and also build a classification model [ ] . in one study ct scan of lungs from patients of both usa and china [ ] were used. in another study, ct scan of lungs only from china [ ] were used for training and testing automated aibased tools for diagnosis and tracking. epidemiological data of three provinces of china (hubei, guangdong and zhejiang), sars epidemic data of all over china were collected and the prediction was made for the whole china [ ] . ct images of patients of italy were used as well in another study [ ] . data was collected from only wuhan, china in one of the articles [ ] and wenzhou city of zhejiang province in another article [ ] . as we see most articles concentrated on data from china as it is the original epicentre of the pandemic. contextual articles focused mainly on epidemic forecasting and sustainable development. most of the disease detection related articles and all of the recommendation type articles used global perspectives as well as public datasets and are not context-sensitive ( figure ) . thus, it can be said that the disease detection techniques mentioned in the articles are mostly not context-dependent; and for the epidemic forecasting purpose researchers need contextual data. there were two cross-country studies -one of them [ ] focused on finding correlation among multiple countries covid- cases and the other study [ ] focused on two different country cases to enhance the performance of their disease detection tool. table iii briefly shows the details on the data used in these contextual studies. [ ] . two of the other studies collected data from various hospitals from china and the usa (table iv) . one study collected data from societa italiana di radiologia medica e interventistica [ ] , a hospital from italy. the other seven articles ( %) used non-image data, predominantly in the form of text and numbers with the purpose of disease detection, epidemic forecasting, sustainable development, introducing advanced concepts, and disease diagnosis and progression (table i) . thousands of data points were collected through a mobile survey for a study [ ] that included information related to location, age, gender, race, travel and close contact to any affected person. furthermore, the study collected health data related to covid- symptoms during a period of days. clinical data including information on baseline most of the research papers (n= , %) aim to use ai to do some kind of classification (covid- detection, differentiate covid- from other respiratory diseases) forecasting, and prediction ( table i) . the cost and time associated with the gold standard of testing covid- : pcr -takes up to two to three days to get the results, drive researchers to find an easier, cheaper and faster way to detect covid- using computational technique. therefore, our study found most of the research work ( %) aimed to detect and diagnose covid- . table iv briefly presents the objective, scope and the results of using different ai algorithms. covid- has put researchers, health professionals at a critical situation due to the lack of timely information and historical data. intelligent systems cannot work unless they are trained with reliable data. application of ai and other related techniques: machine learning, deep learning is done based on the previous experiences i.e data and models. given that very little information related to covid- are available, researchers mostly rely on x-ray images and ct scan images. although a very small amount of chest-x ray (cxr) of covid- is available, cxrs are prescribed as one of the first diagnostic tests by the physician. most of the earlier research works use cxr images to detect covid- ( . %) and others use chest ct images (figure ) . the recent development of deep neural networks has opened up a new frontier in image classification. we found most of the research papers ( %) use different architecture of deep neural networks ( figure ) to classify images, both cxr and ct scan (table v) . when it comes to image data, convoluted neural networks dominated over all other algorithms and techniques(table . using cnn as a base, several studies come up with their architecture [ ] . our observation finds out among several cnns, the res-net architecture as the most used one ( figure other than deep learning approaches, traditional machine learning algorithms have also been applied when it comes to non-image data (table iv) . using a combination of regression analysis and group method of data handling(gmdh), pirouz et al. [ ] tried to find a correlation and forecast based on demographic factors. moreover, fig. : literature frequency of different ai techniques yang et al.[ ] combined epidemiological models with an ml model to show the effectiveness of the disease containment in china and predict the epidemic. with an addition to this, loey et al. [ ] used neural networks (gan network) for detecting covid- , whereas jiang et al. [ ] used decision tree, svm based algorithm to detect covid- from xray images. the authors depend on the train-test split method for validation of their models as none of these models is used to test on real patients. as a validation metric, the studies have used accuracy, specificity, sensitivity, f- score and area under receiver operating characteristic(roc) curve (auc). among other evaluation matrics authors also used false positive rate (fpr), true positive rate (tpr), positive and negative predictive values (ppv and npv respectively). our finding suggests that deep learning algorithms achieve a higher score in most of the evaluation matrices (see table iv , and v). in this section, we have briefly presented the challenges and further research opportunities on ai/ml not only to fight against covid- pandemic but also for the future pandemic. we observed that only one-third of the research ( %) used contextual data while the rest ( %) of them conducted research cnn -biraja et al. [ ] using data from more than one country. the countries or regions that are affected more than others have more opportunities to conduct contextual research. as china was the primary hotspot for the pandemic, a comparatively greater number of contextual studies have been conducted in china owing to the availability of more data and increased time to observe the nature of the pandemic. as the pandemic progressed, data from other countries also became available. hence, there is considerably more scope for future contextual research that will aim to explore and predict the similarity of the pattern of the pandemic among chinese studies and other regional studies. the existing research has been conducted to detect and diagnose covid- , epidemic forecasting, sustainable development, and patient management. we observe that a relatively small number ( . %) of research has been conducted on epidemic forecasting, sustainable development and patient management. further research can be done focusing on these areas. we observed that studies on epidemic forecasting and sustainable development used contextual data. we think that epidemic forecasting based research should always be contextual. there are opportunities to collect different types of data (e.g., images, texts, videos, etc.) and making it available for the researchers to conduct different experiments. such efforts will be highly valuable for fighting the pandemic. most disease detection and half of disease diagnosis-based research were conducted using global data. however, we suggest more research in this direction could use diverse global data in the future for better performance. a few research studies ( %) on disease detection and the other half research on disease diagnosis also performed context-based analysis on specific regions. hence, future studies may consider other affected regions for disease pattern exploration as well. it can be insinuated from the studies considered in this review paper that a significant amount of data was not used for conducting machine learning and deep learning research. future research could investigate if bigger datasets could result in better structured, authenticated and generalized outcomes. additional work can be done to validate some of the original research studies ( [ ] , [ ] ) that did not use data and only proposed models. the claims found in the studies can be explored more with sufficient data in the future. the majority of the existing research that has developed an ai/mlbased tool with the purpose of disease detection and progression has employed training and testing methods. the methods, in general, require related datasets to train and validate the systems to correctly predict the outcome of a given problem, which in this case is detecting the disease accurately. several deep learning based research ( %) studies have used chest x-ray and ct scan images to determine only one cross-country research has been conducted with the purpose of sustainable development, where correlation among confirmed cases, environmental and demographic factors of four different countries were calculated and compared. other cross-country research studies, similar to the one stated above, could be pursued in the future to determine if the virus spread depends on the environmental factors. it has been reported that the hospitals decided to provide service only to the young people, leaving elder citizens who have less survival possibility as the hospitals were out of capacity. further research can be pursued to predict which patient has higher likelihood to be a critical case based on their medical history and symptoms. this would help the hospitals determine which patient can be cured at home and who will need icu support. research studies focusing on icu admission would be helpful on early releasing of some patients, and thereby, leaving space for the patients who need it most. at the same time, studies can also prevent early termination from icus. the lack of health professionals has been observed in highly affected areas. they had to work beyond their limit making them vulnerable to human errors. ai-assisted systems could be helpful here. a rule-based ai can monitor all the data in the icu and suggests the professionals to take necessary steps. an efficient ai can help allocate and control the flow of oxygen level: a crucial treatment given to the covid- patients. researchers all over the world currently have been working on developing drugs and vaccines for covid- . different organizations have already been using ai to find a vaccine for covid- . from data analysis to decoy generation of covid- , ai can be helpful. therefore, there are plenty of opportunities for improvement of these ai algorithms (rosetta [ ] and quark [ ] ). furthermore, ai can be used for simulation and analysis of different candidate vaccine. this paper presents a systematic review of exploring ai and ml techniques in fighting the covid- pandemic. a total of research articles were reviewed. these papers are analyzed and compared in various dimensions including the data types, input features, the ai techniques (the machine learning classification algorithms), as well as their objectives. overall, there are three main contributions of our work. first, we have provided a summary of the findings in terms of objectives, data sources, data type, and volumes. second, we have explored ai techniques that include various machine learning and deep neural network techniques in the field and compared their outcomes considering several popular evaluation metrics. finally, we have identified several research issues based on our analysis and introduced corresponding new directions for future research. our study has a number of limitations, but at the same time, it provides some avenues for future research. first, we have used some specific keywords for searching the relevant materials. although, our search keywords provided effective results to achieve the goal of our study, there might be a risk to miss some important materials that did not emerge from our search queries. second, we think timely and up-to-date materials related to coronavirus and ai techniques are the key things that we have identified, studied, and summarized in this paper. therefore, future works would be needed to collect more resources. third, in addition to exploring ai and ml techniques in fighting the covid- pandemic, future research can be conducted to analyze data privacy and security in the relevant areas. although we have several limitations mentioned above, our analysis and discussion can have significant implications for both the health practitioners and researchers. we believe that our review opens a promising path and can be used as a reference guide for future research in this area. who director-generals opening remarks at the media briefing on covid- - public health emergency of international concern (pheic) artificial 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artificial intelligence: protecting health-care workers and curbing the spread artificial intelligence and machine learning to fight covid- on the coronavirus (covid- ) outbreak and the smart city network: universal data sharing standards coupled with artificial intelligence (ai) to benefit urban health monitoring and management ai-driven tools for coronavirus outbreak: need of active learning and cross-population train/test models on multitudinal/multimodal data a viral warning for change. the wuhan coronavirus versus the red cross: better solutions via blockchain and artificial intelligence a deep learning algorithm using ct images to screen for corona virus disease (covid- )," medrxiv, covid- detection using artificial intelligence identification of covid- can be quicker through artificial intelligence framework using a mobile phonebased survey when cities and towns are under quarantine detection of coronavirus disease (covid- ) based on deep features rosetta : an object-oriented software suite for the simulation and design of macromolecules ab initio protein structure assembly using continuous structure fragments and optimized knowledge-based force field key: cord- - g qr e authors: bhattacharya, sujit; singh, shubham title: visible insights of the invisible pandemic: a scientometric, altmetric and topic trend analysis date: - - journal: nan doi: nan sha: doc_id: cord_uid: g qr e the recent sars-cov- virus outbreak has created an unprecedented global health crisis! the disease is showing alarming trends with the number of people getting infected with this disease, new cases and death rate are all highlighting the need to control this disease at the earliest. the strategy now for the governments around the globe is how to limit the spread of the virus until the research community develops treatment/drug or vaccination against the virus. the outbreak of this disease has unsurprisingly led to huge volume of research within a short period of time surrounding this disease. it has also led to aggressive social media activity on twitter, facebook, dedicated blogs, news reports and other online sites actively involved in discussing about the various aspects of and related to this disease. it becomes a useful and challenging exercise to draw from this huge volume of research, the key papers that form the research front, its influence in the research community, and other important research insights. similarly, it becomes important to discern the key issues that influence the society concerning this disease. the paper is motivated by this. it attempts to distinguish which are the most influential papers, the key knowledge base and major topics surrounding the research covered by covid- . further it attempts to capture the society's perception by discerning key topics that are trending online. the study concludes by highlighting the implications of this study. coronaviruses are viruses that circulate among animals and are named because of the crownlike spikes (protein spikes) that protrude from their surface resembling the sun's corona. the first transmission of this type of virus from animals to humans happened in in the guangdong province of china which resulted in sars (severe acute respiratory syndrome). bats were thought to be the potential source of this virus. a novel coronavirus (ncov) was identified in early january which was traced to the severe pneumonic outbreak of an undocumented cause in early december in the city of wuhan, china. similar to sars, bats are seen as the potential source from which this virus has spread to humans. due to rapid spread of the virus within a short time globally resulting in health emergencies in a number of countries, who declared this as a pandemic on th march . initially the virus was named -ncov but later was named sars-cov- due to its "genetic relationship to the sars-cov- virus"(medscape, ) the entire human population is at potential risk as being a new virus nobody has prior immunity to it. there is no vaccine and no specific treatment for the disease and is highly transmissible. epidemiological estimation at present is that on an average, one infected person will infect between two to three other people. the spread has been from respiratory droplets and from contaminated surfaces. the world is facing a common challenge; how to control the spread of and what can be the effective interventions to control mortality. the early examples from china suggested the most effective method to control the spread of the virus is through lockdowns and social distancing measures. the example of south korea suggested testing as the major component of the mitigation measures. some studies pointed out the importance of hand hygiene and face masks in controlling the virus. with new hotspots emerging, the number of new cases and those not able to recover are raising new concerns every day. risk and uncertainty behind this disease control has generated a global concern for health, economy, and for persons at large. the alarming spread of the virus has shocked people across the world pushing among others researchers to understand the virus-its structure, transmission, replication mechanism, latency, etc. and promising interventions that can effectively control it. extensive global efforts are undertaken to develop vaccine and drug. this is unsurprisingly leading to huge volume of research activity within a short period of time increasing at an exponential rate. as the recent editorial published in the lancet highlights "the whole-genome sequence of sars-cov- had been obtained and shared widely by mid-january, a feat not possible at such speed in previous infectious disease outbreaks" the editorial points out the importance of the need for development of effective diagnostics, therapeutics and vaccine for the virus. examining from the dimensions database as of april, it was found that clinical trials are being conducted on the virus and policy documents have been published so far. the number of research papers, clinical trials at different phases within such a short period is unprecedented and shows the intensive efforts of the global research community to understand the different aspects of this disease and address it. seven patents have also been granted. it is important to capture insights of influential research and innovation from this ongoing activity for policy makers and research scholars from cross-disciplinary areas to build up further on this valuable repository. societal impact and what aspects are of concern to the people at large are difficult to capture. one useful method would be from online trends surrounding this disease that would indicate to some extent the key issues that are influencing the society at large. the present study is motivated by this and applies tools and techniques of scientometrics to uncover insights from research papers. scientometrics applies various mathematical and statistical techniques to capture insights of research activity from research papers and patents and other published sources including online sources (altmetrics) by constructing various types of indicators. citation based analysis is a prominent method to capture academically significant and theoretically relevant material (see for example glanzel, ) . keeping in view the research activity in this area started primarily with the outbreak of this disease, impact captured through citations would not give a correct picture as citations takes time to accrue. this is true for research paper as well as patent citations. citations that influence current research activity would however be useful to construct the present knowledge base. one of the useful method that can do so is based on cocitation analysis which captures frequency with which two documents are cited together (small, ) . co-citation establishes an intellectual relationship with earlier literature in a field/subfield/area of research; strength of relationship based on frequency of co-citation pairs. the rise of social networking websites like twitter, facebook etc. provides researchers a wider scope to share their scholarly publications. altmetrics allows to track and capture online impact of scholarly research and thus broadly indicates papers that are influencing the research community. to put it in a proper perspective, one can borrow from william ( ), "altmetrics are measurements of how people interact with a given scholarly work". altmetrics or article level metrics according to das and mishra ( ) is a "new trendsetter" to measure "impact of scientific publication and their social outreach to intended audience". it reflects "a scholarly article's popularity, usage, acceptance and availability" by using an altmetric score. google trends which was launched in , primarily shows how frequently a particular search term is entered in comparison with all other search terms in different regions and languages (google, ). in google trends level of interest in a topic is approximated using search volume of google. sullivan ( ) estimated searches on google trends reached trillion in ! thus, this is one of the most significant source of data if it is properly analysed. one of the most influential study was by ginsberg et al. ( ) which showed that google trends traced and predicted the spread of influenza earlier than the centers for disease control and prevention. jun et al. ( ) provides a good assessment of research studies in the past decade which have utilized google trends. they highlight the diverse fields in which this has been used for, from merely describing and diagnosing research trends to forecasting changes. according to mavragani et al. ( ) "google trends shows the changes in online interest for time series in any selected term in any country or region over a selected time period, for example, a specific year, several years, weeks, months, days, days, hours, hour, or a specified time-frame." they argue that as the internet penetration is increasing web based search activity has become a valid indicator of public behaviour. the paper positions itself in this direction; applying various tools and techniques of scientometrics, altmetrics and google trends to draw meaning from the huge volume of research papers and online activity surrounding this pandemic. the study attempts to answer the following research questions:  what are the key papers that captures the most relevant research, areas and topics on covid- ?  what is the knowledge base that influences current research on this pandemic?  what are the key aspects of this pandemic that is influencing the society at large? the study has used various types of data sets and analytical techniques as highlighted below to capture the research trends and also assess this disease influence on the society. the dimensions database (www.dimensions.ai) was used for this study. this database has various unique features which makes it very useful to capture various aspects of research activity. it provides dynamic altmetrics score for each article. the database unlike source based classification (journal classification) used in indexing articles in sci and scopus database uses article level classification. only when an article cannot be classified individually due to lack of information, it uses the fields of research (for) classification system. the for has three hierarchical levels: divisions (represents a broad subject area or research discipline), with the next two levels groups and fields representing increasingly detailed subsets of these categories. in for there are divisions, groups and fields. dimensions has incorporated only the groups in its classification system. thus classification article level provides a more informed assessment of the topic covered by it then based on journal level classification which is a macro level classification. these features motivated us to use this database for this study. the articles on this virus were extracted using the search string "covid- " or "sars-cov- " or "sars-cov " or " -ncov" on april , from this database. the final search string was developed based on review of contemporary studies and deleting those search keywords that lead to noises. for example, it was found that ncov which some studies have used also identifies papers that cover mers (middle-east respiratory syndrome). this was first reported in , was initially called novel coronavirus or ncov as it was a species of coronavirus. many studies had applied search string without hyphen which also results in extracting papers not covering this disease. the search string applied on the publications database of dimensions resulted in papers, containing articles and pre-prints. this data set of papers were further used for analysis. influential papers were distinguished by using altmetric score which is a weighted count of all the online attention of a research paper. the altmetrics data was captured from dimensions database which draws data from altmetrics.com of capturing online activity of research papers on facebook, twitter, blogpost, news reports etc. the score changes as people mentioning the paper increases (only one mention per user is considered). each category of mention carries different base amount so a news article contributes more than a blog post which in turn contributes more than a tweet in the final score. country wise analysis showed that around percent of the total papers were contributed by ten countries. further analysis of research activity of the ten identified countries was done using altmetric and citation analysis. word cloud provides a high visual representation of concepts that a paper had frequently applied. it is based on burst algorithm that captures the sudden rise in the usage of a word. mane and borner ( ) highlighted the usefulness of burst words as according to them "it helps humans mentally organize and electronically access and manage large complex information spaces". using r programming tools, word cloud was constructed from keywords of the data set. the words with higher frequency in the overall corpus of papers (herein ) have a larger font size and acquires more space in a visualisation. word cloud was used to get visualisation of most frequent words; the number of words chosen was limited by the clarity of visualisation. co-citation analysis helps to capture papers that are co-cited together in a large number of papers. the highly co-cited papers is seen as the core knowledge base of research area at a particular period. this analysis was undertaken to identify the key knowledge base behind the identified papers. dimensions database was used to extract a bibliographic mapping file for the papers. two software's pajek and vosviewer were used for co-citation analysis. initially the bibliographic mapping file was run on the vosviewer software to identify the most co-cited papers. the co-cited papers were identified at four levels (trim levels) to have a deeper insight of the core knowledge base: level identified papers co-cited or more times; level identified papers co-cited or more times; level identified papers co-cited or more times, and level identified papers co-cited or more times. for each of the trim levels a network file was obtained from the vosviewer software. the network file was then run on pajek software to create a refined co-citation network map so as to avoid overlapping of nodes. the final visualisation was done for the refined map in the vosviewer software. data for the policy documents referencing these top ten co-cited papers was done by accessing altmetrics.com directly from the dimensions database. another question which the study explored is the impact of this virus on the society primarily what are the key aspects of and related to this disease that has influenced the society at large. google trend analysis of key topics have been undertaken to capture this aspect. google trends website (https://trends.google.com/trends/?geo=us) was first accessed on th april . the topics were chosen based on closely monitoring the news items, and also finally choosing from a large set of topics. choice for example of 'pandemic' was seen to have initial burst but declined quickly. vaccine was trending highly but we found lot of noise in this term. the final six topics chosen were "social distancing", "quarantine", "covid- ", "coronavirus", "face mask" and "hydroxychloroquine". data for each of the topics was finally taken on april, . for country specific comparison data for five countries having maximum cases of covid- namely usa, italy, spain, france, germany and two emerging economies india and brazil was also obtained. hydroxychloroquine was not used in country specific search as it was only visible trending for three countries among the chosen seven countries. google trend analysis was not done for china as there is much restricted access to google in that country. one of the first important observation is the intensity with which research on covid- and related aspects is going on globally. search conducted in two different time periods, th march and then on th april showed that and papers were published in these two periods; almost percent growth during such a short time. the insights that we draw from our analysis of the papers is presented in different sections below ten most popular research papers among the covid- papers were extracted on the basis of their altmertrics score on april , . table highlights these influential papers. ), the study most popular on social media platforms (number of tweets more than three times the next popular paper) commented that "sars-cov- " is the seventh coronavirus to infect humans". the study found that sars-cov- is not a product of purposeful manipulation and is most likely the result of natural selection of human or human-like ace receptor. the study also found that sars-cov- spike protein has high affinity to bind to human ace receptor. the study also estimated that the undocumented cases contagiousness or transmission rate was % of documented infections, yet % of documented infection cases were due to these undocumented infections. the suggestion of this study that undocumented infections "isolation and identification is necessary to fully control the virus" is very important and the spread of this virus may be seen as a consequence of this. this study also was cited in policy documents. leung et al. ( ) explored "the importance of respiratory droplet and aerosol route of transmission" by quantifying the "amount of respiratory virus in exhaled breath of participants" that have acute respiratory virus illness (ari). the participants were divided in two groups, one wearing surgical face mask and other not wearing face mask. the study found that surgical face masks can efficaciously reduce the respiratory droplet emission of influenza virus particles but not in aerosols. they also found that surgical face masks can be used by ill patients of covid- to reduce "onward transmission". face mask is getting increasing attention and now being incorporated as essential guideline in health policies of different countries. table points to some interesting aspects of research activity in this area. these ten countries account for almost percent of total papers with china and usa accounting for percent of the total. china, usa and uk are actively collaborating among themselves and also with other countries. this is a good indication as global collaborative efforts, pooling each other resources are required to meet the challenges posed by this disease. a few leading universities can be discerned which are actively involved in this research. popularity of a paper can also be seen influenced by journals; papers with high altmetrics score strongly correlate with journals that have high reputation in the field (high impact factor, leading journal of the community). table highlights the areas covered in the covid- papers. the table provides a broad indication of intensity of research happening in different fields. figure presents a word cloud of most frequently used terms in covid- papers. the word cloud shows key aspects that have been part of many studies. the word cloud maps the topics of research surrounding this disease. the two keywords, for example "pandemics" and "china" that have maximum occurrence in papers indicated by large font size which shows that these two aspects were discussed in many papers. it is known that china was the source of this infection and who declared this disease as a pandemics. thus increasing research mention of these two keywords is not surprising. coronavirus primarily affects animals, sars disease as a result of transmission of coronavirus from animal to humans, travel has contributed maximum to the spread of this disease, are all visible prominently in this word cloud. thus, examination of the word cloud is useful to have a broad view of key areas of research in the papers. figure shows co-citation networks at four trim levels. it can be observed from figure that trim level that contains top co-cited papers is a complete cluster. a complete cluster according to gmÜr ( ) is when "each reference is connected to other references and there is no dominant document within the cluster". table highlights the details of these co-cited papers at trim level which identifies top cocited papers. it also includes the top co-cited paper at trim level (refer methodology for details). pandemic's influence on the society figure provides global google trends of six topics (refer methodology for details) currently talked about extensively on social media, news reports or in general public discussion. it can be observed the otherwise flat line of covid- started seeing spikes in late february, . this is because the term came into existence when who on feb named the disease from the virus as covid- (coronavirus disease ). it can also be observed that the term reached maximum level of interest during the end of march as cases started showing significant increase in countries usa, india etc. measures like quarantine has strong societal influence and thus useful to look at trend in this topic. another topic "pandemic" saw maximum interest around march , as who announced covid- pandemic on that day. it can also be seen that the interest about this topic fell shortly thereafter. as discussed in the methodology, this topic hence was not chosen further in this study for google trend examination. hydroxychloroquine term has seen a great amount of interest from middle of march, . this can be traced to the study by reputed french physician and microbiologist didier raoult who highlighted the use of this antimalarial drug in the treatment of this disease. it led to french president and us president endorsing this line of treatment which created favourable public opinion in many countries towards this drug. liu et al. ( ) whose work has also attracted high altmetrics attention found the drug to be effective in "inhibiting sars-cov- in vitro". this line of treatment and the robustness of the methodology and findings have also generated critical comments, see for example grens ( ) . india has become a key source for this medicine and already exported it to number of countries. thus a high degree of activity in google as seen through google trend can be due to various factors, positive as well as adverse reactions. similar public opinion generated by studies and endorsement can be seen in youtube searches in face mask. source: google trends figure provides the comparison of google trends of "social distancing", "covid- ", "quarantine", and "lockdown" and "face mask" from feb to april . figure (a) presents a global picture of these topics and figure (b) shows data for five countries having maximum cases of covid- namely usa, italy, spain, france, germany and two emerging economies india and brazil. three of the five topics chosen restrict people's movement. social distancing which basically means keeping a safe distance of around feet from others and avoiding places where this kind of distance cannot be made like schools, workplaces, a sports game or a temple. the second one is quarantine which applies to a person who have been in exposed to coronavirus or patients having coronavirus. the person has to avoid contact with people till the specified incubation period of the virus to see if they develop symptoms. third lockdown, the term mainly used to describe the confinement of prisoners to their cells has now a changed definition during this outbreak. through lockdown people are not allowed to leave their local area, building and it is used as a control measure to prevent covid- disease transmission. it can be seen from figure (b) that "lockdown" is the most popular topic worldwide as well as at the country level. according to world economic forum . billion people i.e. one thirds of the world population are under some kind of lockdown. this figure alone constitutes india's . billion, the largest lockdown in the world. thus it is unsurprising to see the popularity of this topic over others in india. these control measure have major economic, psychological and social impacts and in turn affects lives of all the people involved. a worrying trend is the low comparative interest in social distancing in most of the countries and almost negligible comparative interest in countries like indian and brazil. the stand taken by brazil through her president of opening up and has been critical of measures like social distancing and lockdown may have contributed to this type of trend. proximal origins of sars-cov- a trial of lopinavir-ritonavir in adults hospitalized with severe covid- challenges of coronavirus disease epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study genesis of altmetrics or article-level metrics for measuring efficacy of scholarly communications: current perspectives the cognitive paradigm : cognitive science, a newly explored approach to the study of cognition applied in the psychology of scientific knowledge and of education in science . rug. faculteit psychologische en pedagogische wetenschappen hydroxychloroquine and azithromycin as a treatment of covid- : results of an openlabel non-randomized clinical trial detecting influenza epidemics using search engine query data co-citation analysis and the search for invisible colleges: a methodological evaluation bibliometrics as a research field: a course on theory and application of bibliometric indicators journal publisher concerned over hydroxychloroquine study clinical features of patients infected with novel coronavirus in wuhan ten years of research change using google trends: from the perspective of big data utilizations and applications hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro mapping topics and topic bursts in pnas google trends in infodemiology and infoveillance: methodology framework analysis of the capacity of google trends to measure interest in conservation topics and the role of online news coronavirus disease (covid- ): a global crisis co-citation in the scientific literature: a new measure of the relationship between two documents google now handles at least trillion searches per year the donut and altmetric attention score: an at-a-glance indicator of the volume and type of attention a research output has received document co-citation analysis to enhance transdisciplinary research altmetrics: an overview and evaluation characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china the authors thank dr vivek singh, professor, department of computer science, banaras hindu university for providing access to dimensions database which helped in framing the pilot study for this research. we are grateful to dimensions for providing us direct access. glad to see the initiates they have taken in promoting covid- research. note: comparative interest over time of social distancing for india and face mask and social distancing for brazil were negligible, so these terms have been removed for the graph for these countries. covid- disease took the world by surprise. the alarming spread of the disease, challenge to control its spread and its grave health consequences, lack of vaccine or effective drug among others has prompted researchers to actively work on various aspects of this disease. this has led to a huge volume of research output within a short period of time. the various aspects surrounding this disease has also effected society's perception of this disease. drawing insights from this huge volume of research output is a challenge as well as an exercise of significant importance for the policy makers and research community. scientometrics provides various tools and techniques to uncover insights from research papers. altmetrics and google trends are novel approaches to track online impact. these tools and techniques was used in this study to analyse papers that were discerned from the dimensions database covering covid- for the period upto th april, . content analysis of a set of ten key papers was also undertaken to draw qualitative insights of these influential papers. some of the insights from this study reveals the key areas in which research has progressed. their visible impact can be seen in their altmetrics score and more directed impact in their citation in key policy documents. key policy influence such as period of quarantine, treatment type, use of face masks, population more vulnerable to disease etc can be traced to the influential papers that were discerned from this study. it would be however fallacy of generalisation if we say that these papers were the only influential factors behind the policy decisions. it is also interesting to see how papers had attracted attention from different online sources like twitter, news, blog, and facebook. thus, the importance of these sources in influencing research impact calls for researchers to aggressively use these modes for dissemination of their study findings. another important insight comes from the active collaboration seen in research papers. china and usa drive this research globally and are also actively engaging with other countries in research. this finding is drawn from top ten active countries which constitute almost % of the total research output as visible from research papers. word cloud showed the influential topics of research surrounding covid- research. this type of visual maps provide a good indication of key topics that constitute research activity in a area at a period of time. co-citation analysis identified the knowledge base that influences current research on this pandemic. it was observed that all of the top co-cited papers were published in high impact factor journals. it was also observed that most of the studies were driven by epidemiology and clinical characteristics of the disease. google trends analysis showed how the disease shapes the public opinion on certain topics. global trending topics incorporated interest over time in web, news, google shopping and youtube searches. a sudden rise in a topic's interest could be traced to various exogenous factors. the trend, for example in hydroxychloroquine, use of this antimalarial drug in the treatment of this disease could be seen the interplay of various forces, the impact of research paper, political endorsement and critical questioning of the research community, doctors etc to the effectiveness of this line of treatment. the trends observed in measures like lockdown, social distancing and quarantine at global and country level showed the societal increasing concern with these aspects.the findings of this study suggests how the research and public interest has been shaped around this disease. with so much information surrounding this disease, the study provides a space for understanding its various aspects. the study however is limited as it has not examined patents, clinical studies and policy documents. this may provide indications of implementable aspects that draw from research. future research intends to examine this. key: cord- -ug xki authors: sweileh, waleed m. title: bibliometric analysis of scientific publications on “sustainable development goals” with emphasis on “good health and well-being” goal ( – ) date: - - journal: global health doi: . /s - - - sha: doc_id: cord_uid: ug xki background: global progress in the united nations’ sustainable development goals (sdgs) requires significant national and international research efforts and collaboration. the current study aimed to provide policymakers, academics, and researchers with a snapshot of global sdgs-related research activity. method: this was a cross-sectional descriptive bibliometric study. sciverse scopus was used to retrieve sdgs-related research publications for the period from to . results: in total, , documents were found. the sustainability journal ranked first (n = ; . %) in the number of sdgs-related publications. the world health organization was the most active institution in publishing sdgs-related documents (n = ; . %). most of the retrieved documents belonged to sdg (partnership) followed by sdg (climate action), and sdg (responsible consumption and production), while sdg (affordable and clean energy) had the least number of publications. the european region (n = ; . %) had the highest research contribution while the eastern mediterranean region (n = ; . %) had the least contribution. after exclusion of sdg , the sdg (good health and well-being) was the top researched sdg for the african region, the eastern mediterranean regions, and the south-eastern asian region. for the region of the americas, european region, and the western pacific region, the sdg (climate action) was the most researched. the sdg (affordable and clean energy) was the least researched in the african region, the region of the americas, the european region, and the south-east asian region. in the eastern mediterranean region, sdg (reduced inequality) was the least researched while in the western pacific region, sdg (gender inequality) was the least researched. the most researched targets of sdg were targets (sexual and reproductive health services) and (universal health coverage) while the least researched targets were (substance use disorders) and (death from hazardous materials). international research collaboration within sdg between high- and low-income countries was inadequate. conclusion: the analysis presented in the current study are useful for researchers, institutes, governments, funding agencies, and policy-makers. countries in africa, the middle east, and south-east asia need to increase their funding and research collaboration in the field of sdgs. the united nations' (un) sustainable development goals (sdgs), adopted on september , are a universal political agenda that call for a collective action to achieve peace, prosperity, and well-being for all by . the goals and targets of the sdgs were developed to succeed the eight millennium development goals (mdgs) which ended in [ ] . the transition from eight mdgs to sdgs marked a shift from vertical to horizontal approach in addressing global issues [ ] . achieving cross-sectional progress in the ambitious agenda requires the involvement of policymakers at all levels and in all sectors [ ] . the list of sdgs includes ending poverty, ending hunger, encouraging good health and well-being, providing quality education, promoting gender equality, providing clean water and sanitation, promoting affordable and clean energy, providing decent work and economic growth, addressing industry, innovation and infrastructure, reducing inequalities, developing sustainable cities and communities, encouraging responsible consumption and production, taking action on climate change, promoting life below water, promoting life on land, working towards peace, justice and strong institutions, and creating partnerships to achieve sdg goals [ ] . the third sdg of good health and well-being is currently of special interest given the global health threats imposed by serious pandemics including the recent covid- which might interrupt the national and international progress to achieve the goals [ , ] . global warming has also created a serious threat to human lives by accelerating the spread of certain infectious diseases, particularly mosquito-borne diseases which threaten millions of people around the world [ ] . furthermore, conflicts and fragility in various world regions including africa and the eastern mediterranean regions created massive waves of refugees and migrants with minimum or poor health services [ , ] . poverty, poor sanitation, lack of access to essential medicines and health services have augmented the disease burden on most vulnerable categories particularly women, children, and elderly [ ] . the global health challenges imposed by emerging pandemics, conflicts, climate change, and poor economic growth affect the plans to achieve the sdgs particularly the third one focusing on good health and well-being. researchers and academic staff in universities are key players in promoting sdgs [ , ] . contribution of scientists in different disciplines enlightens policy-makers and politicians on certain goals that need to be addressed in the future as well as on the extent of success or failure in achieving a certain goal. the sdgs are based on the presence of serious global problems that need to be addressed and solved through research and innovation [ ] . researchers are in a position to provide genuine solutions and inventions to various national and international problems facing the pathway to achieving the agenda [ ] . local and international authorities need to adopt research results and recommendations made by academic staff and put them into a practical agenda to strengthen partnership with all stakeholders and to accelerate the practical steps toward achieving sdgs. to shed light on the contribution of academics and researchers to sdgs-related literature, the current study was performed aiming to assess research volume and research trends of scientific publications on sdgs over the past years ( - ). the analysis was based on traditional bibliometric methodology [ ] , which has been extensively used to analyze and assess a wide range of medical and scientific topics [ ] [ ] [ ] . the current study will shed light on most and least-researched goals and targets. the third sdg will be given special consideration and analysis. the current study was a cross-sectional descriptive analysis of scientific literature on "sustainable development goals" retrieved from scopus database. scopus, an online database with approximately , available journals across all fields of research, was used [ ] . scopus is commonly used in bibliometric studies and is considered suitable for this purpose because it includes a larger number of indexed journals than web of science [ ] . the current study was carried out on july th, , and all data analysis, including citation analysis, was carried out on the same day. research that supports national or global implementation and development to achieve sdg targets can be classified into two types: ( ) those that do not mention the phrase "sustainable development goals" and ( ) and those that explicitly mention the phrase "sustainable development goals". the second type of research is directly related or linked to policies, implementation, barriers, and other aspects of the sdgs [ ] . in the current study, we focused on the second type of research in which the phrase "sustainable development goal" was mentioned in the paper (title, abstract, introduction, methodology or discussion). these fields can be searched in any article using the function "all" in scopus search engine. of course, this type of research does not represent all research on sdgs. however, it will relatively reflect the contribution of various countries, world regions, journals, research themes and shed light on other indicators. furthermore, the results of the analysis of this type of research can be used to compare the extent of research on various sdg goals among world regions with an acceptable level of accuracy. . the search strategy was explained in additional file . documents with the phrase "sustainable development goal*" mentioned in any place in the paper were retrieved from scopus for the study period from to . the documents were limited to journal articles but no language restriction was imposed. the author believed that using this approach will retrieve the maximum potential number of documents of the second type in which the author(s) tried to link their study with dsgs. a very recently published article on sdgs used the title-abstract-key methodology to retrieve sdgs-relevant articles and retrieved approximately documents [ ] . using the methodology adopted in the current study, the number of sdg-related documents was at least three times larger than the number obtained from the title-abstract-key methodology. the , scientific publications obtained in the current study using the "all" function were considered to be sdgs-related publications and used for further research. . the search strategy for the number of sdgs-related documents contributed by each world region was calculated based on the methodology stated above and the list of all countries located in each world region. the world regions considered in the current study were those adopted by the who: african region, the region of the americas, the european region, the south-east asian region, the eastern mediterranean region, and the western pacific region. . the search strategy for each sdg goal was entirely based on the search queries for each sdg goal posted by "aurora universities network" [ ] , which is an initiative started from the aurora universities network in . for each goal and each target a search query is available and can be directly inserted into scopus advance search system [ ] . the search queries for the sdg goals using the keywords listed in aurora universities network yielded more than million documents relevant to keywords in the fields of the sdgs. however, the author is interested in documents in which the phrase "sustainable development goal*" is mentioned in any place in the study. when the phrase "sustainable development goal*" was added to the overall search query of the million documents and the time was limited from to , the result was , publications in peer-reviewed journals. this number represents approximately . % of the total number of publications with keywords listed in aurora website and related to the sdgs. the information retrieved from the scopus database included: annual number of publications, active countries, active journals, active institutions, and citation information. data in scopus was exported to excel software for tabulation or mapping. the number of publications on each sdg goal was analyzed using the search query in the aurora universities networ for that goal after adding the phrase "sustainable development goal*" in all fields. the same method was applied to find out the research output for each target in sdg . data obtained for sdg was exported to vosviewer program for mapping purposes [ ] . mapping was made for ( ) the most frequently encountered terms in titles/ abstracts of the retrieved documents and ( ) for countries with a minimum contribution of documents to visualize international research collaboration in sdg . the search query found , documents. the majority of documents were research articles (n = , ; . %) followed by review articles (n = ; . %). the annual growth of publications showed steep growth (fig. ). the number of publications in was approximately . times the number of publications in . of the retrieved publications, , ( . %) declared receiving funding for the study. the most active funding sponsor was bill and melinda gates foundation bmgf (n = ; . %). the retrieved documents were analyzed for the sdgs ( table ). the most researched goal was sdg (partnership) followed by sdg (climate action), (responsible consumption and production), (life on land), sdg (good health and well-being), and sdg (no poverty). the least researched goal was sdg (affordable and clean energy) followed by sdg (reduced inequality), (gender equality), and (peace, justice, and strong institutions). documents on sdg , (good health and well-being), and received the highest number of citations per document while documents on sdg (quality education) and (industry, innovation, and infrastructure) received the least number of citations per document. for all sdg goals, the usa and the uk were among the top five active countries. china was among the top five active countries for sdgs , , , and . the annual growth of the five sdgs with the highest number of publications (excluding sdg , which represents partnership) is depicted in fig. . the climate action (sdg ) showed the steepest growth with time while the growth of the remaining sdgs (good health, responsible consumption and production, no poverty, and life on land) showed similar annual growth pattern. the retrieved documents were published in journals that belonged to different subject areas. approximately % of the retrieved documents were published in journals within the subject area of social sciences. subject areas of medicine and environmental sciences ranked second with % for each. agriculture/biological sciences and energy subject areas ranked third and fourth with and % respectively. geographic distribution of the retrieved documents table lists the contribution of each who region to the retrieved documents. the european region (n = ; . %) had the highest research contribution followed by the region of the americas (n = ; . %) and the western pacific region (n = ; . %). the african region (n = ; . %) made higher contribution than either the eastern mediterranean region (n = ; . %) or the south-eastern asian region (n = ; . %). figure shows the annual growth from different world regions. the annual growth in the six world regions showed a similar pattern with a leading role for the european region. top ten active journals the sustainability, a multidisciplinary journal ranked first (n = ; . %) in the number of sdg-linked publications followed by plos one (n = ; . %) and journal of cleaner production (n = ; . %). nine of the top active journals were based in europe and one was based in the usa (table ) . analysis of active institutions based on author affiliation indicated that the who was the most active institution in publishing sdg-linked documents (n = ; . %) followed by london school of hygiene & tropical medicine (n = ; . %), and harvard university (n = ; . %) ( table ). the top ten active institutions included nine academic institutions in addition to the who. the number of sdg -related publications was . the distribution of these publications on the health targets was shown in table . the most researched targets were targets (sexual and reproductive health services) and (universal health coverage) while the least researched targets were (substance use disorders) and (death from hazardous materials). network visualization of the terms in titles and abstracts of sdg related publications showed three distinct clusters representing three major research themes ( fig. ) : universal health coverage (red color), women/ maternal health (green color), and the global burden of diseases (blue color). the total was less than the overall number (n = , ) either because of the presence of limited number of false positive results or because the keywords used to retrieve the documents on each sdg were not % accurate. the difference, which is approximately . %, will not affect the overall analysis network visualization of countries with a minimum contribution of documents for sdg linked publications (fig. ) . the map was characterized by the presence of countries from poor regions such as africa, the eastern mediterranean, and south-east asian regions far away from the center and with limited connections with countries in the center of the map indicative of poor international research collaboration. countries in the center of the map with the largest node size (the usa and the uk) had the largest research connections with other countries while countries such as egypt, zambia, and nepal located at the margin of the map with small node sizes had the least international research collaborations in sdgs-related research field. the current study was undertaken to assess and analyze scientific publications in which the phrase "sustainable development goal*" was mentioned in any part of the article assuming that such publications represent research related to one or more aspects of sdgs. the current study showed a steep increase in the number of sdgs-related publications with time reflecting an increasing interest of researchers in topics that are related to sdgs. it also reflects national and international support and commitment to achieve the sdgs. research volume on sdgs of poverty (sdg ), health (sdg ), responsible consumption and production (sdg ), climate action (sdg ), sustainable cities and communities (sdg ), and life on land (sdg ) dominated the overall retrieved literature. a recent qualitative study using snowball approach for experts on sdgs research indicated that sdgs , , and were among the most researched sdgs while sdgs and were among the least researched ones which is in agreement of the findings presented in the current study [ ] . another recent study that mapped the australian research on sdgs found that sdgs number , , , and were among the most researched sdgs while sdgs and were the least researched [ ] . it seems that there are regional differences regarding research interest in various sdgs. for example, sdg experts in northern america and the european region pointed to sdgs , , , and as the most preferred ones while specialists in the african region indicated that sdgs , , , , , and were most important [ ] . the current study showed that most researched sdg goals were those related to health, environment, and science. however, when overall literature was considered, the bulk of the retrieved documents were in social sciences, followed by medicine and environmental science subject areas. this was not surprising given that at least half of the sdgs are related to social issues such as poverty, hunger, education, inequality, gender, and peace. in fact, social and health targets in the sdgs represent global problems that need tremendous research and solution [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . environmental issues such as climate change and global warming are also a major global challenge to life and economy for planet earth [ , ] . in the current study, most active journals were in the q rank suggesting that the sdgrelated papers are of interest to journal editors. this also suggests that research content and quality of sdgs-related publications are high and beneficial for policymakers. none of the active journals was based outside europe or north america. this might be one possible reason for the large contribution of european and north american countries to sdgs-related literature compared to other countries that lack scientific journals indexed in scopus. the current study indicated the sustainability journal ranked first followed distantly by the plos one journal. the finding regarding the list of top active journals is in agreement with the findings of a recent paper on sustainable development goals in which the author used the title-abstract-key to search for sdg-related documents [ ] . this finding confirms that the methodology adopted in the current study was valid and the difference between the two approaches is in the number of the retrieved documents while the findings were relatively close. in the study by christine meschede [ ] , a total of target achieve universal health coverage, including financial risk protection, access to quality essential health-care services and access to safe, effective, quality and affordable essential medicines and vaccines for all. universities and academics in various countries are in a position to interact and have a leading role through research collaboration on various sdgs. the finding that nine of the top active institutions were academic institutions reflects the growing role of academics in the implementation and research on sdgs. researchers and academic staff must teach and train today's students, tomorrow's decision, and policymakers, to think on genuine approaches to solve global serious and complex societal and economic problems [ ] . academic institutions need to integrate sdgs in research, teaching, and student training to become leaders of change at the national and global levels. the current study showed that countries in the african region, the eastern mediterranean region, and the south-east asian region had the least contribution to sdgsrelated research publications. the online database for sustainable development report , showed that the achievements of most countries in these regions were coded red or orange suggesting major or significant challenges in goal achievements across the sdgs. in contrast, most countries with green or yellow coding for the sdgs were based in the european region, which could be attributed, in part, to the outstanding research output from this region. the current study indicated that the us and the uk were in the frontline regarding research activity across the sdgs. however, the sustainable development report indicated that the us is facing significant and even major challenges in many of the sdgs. this might suggest that national research output in sdgs does not significantly correlate with the sdg index score or goal achievement. research activity is one important factor in creating knowledge and innovation that will lead to sustainable growth and development. however, politics is also a major key player in this regard and might facilitate or hinder goal achievement. the current study showed that sdg , which refers to partnership for the goals, was the most researched item as an overall and for each world region. the sdgs included targets and has the largest number of keywords in its search query as presented in the aurora universities network web site. the sdg demands partnerships between governments, the private sector and civil society. the type of research publications that have been analyzed in the current study is the one in which sdg phrase was mentioned and we expect that such research focuses on policies and agendas that require governmental and private sector partnership. if all research on sdgs was included, i.e. the million documents, we expect that sdg will be one of the top researched goals but not number one. similar findings were presented in a recently published article [ ] . the current study indicated that sdg , which was one of the top researched goal across different world regions, was characterized by inadequate international research collaboration. an important aspect of sdgs is the global approach and partnership. the current study showed that the extent and strength of research collaboration in sdgs-related research vary based on the geographical region. european and american researchers have relatively strong collaboration while research collaboration with researchers in asian or african countries was poor. policymakers in regions with low research output in sdgs need to encourage and support joint research with european and north american researchers. such research collaboration is believed to attract more funding and better chances of publishing higher impact documents [ , ] . analysis of sdg indicated that the prevention and treatment of substance use disorder was the least researched target. according to the who, some million persons have drug use disorders at the global level with almost million people injecting drugs. substance use disorders have been associated with increased risk of hiv, tuberculosis, hepatitis and may even accelerate the spread of serious pandemics such as covid- [ , [ ] [ ] [ ] . the social stigma attached to drug users acts as a strong barrier to improve support services [ ] . in several parts of the world, governments impose compulsory detention with beating and humiliation of people who use drugs in the name of treatment [ , ] . such human rights violations are not in accordance with sdg . and research on preventive and management policies of drug users need to be carried out in all parts of the world. the bmgf was the most active in funding sdgsrelated research. the bmgf is the largest private foundation in the world with the primary aims of enhancing healthcare and reducing extreme poverty in the world. at the usa level, the foundation aims to expand educational opportunities and access to information technology. funding is a major driving force for publications, growth of scientific collaboration, and enhanced impact of scientific articles [ ] [ ] [ ] [ ] [ ] . the present study has some limitations. first, our study did not include articles published in non-scopus databases. this negatively affected the number of publications retrieved from countries/regions with local or regional journals that are not indexed in scopus. second, the current study focused on peerreviewed literature and did not include grey literature such as governmental reports. sometimes, grey literature included important information and assessment of the national progress in implementing sdgs. third, all types of publications such as notes, letters, reviews, and editorials were included in the analysis. however, these types might not represent original research contribution. it is well known that the lancet has many editorials and such documents might overestimate the role of the journal in publishing documents on sdgs. fourth, the method adopted in the current study did not distinguish articles that focus on sdgs from those that only mention the term. the opposite is also true. a document might discuss a problem such as sanitation without mentioning the phrase sdgs. therefore, this limitation should be taken into consideration when reading the current study. despite all this, the current study provides a comprehensive picture of sdgsrelated research productivity and subject areas of interest to worldwide researchers. research, innovation, education, and funding are crucial elements to achieve the sdgs. these elements need to be investigated periodically to assess the involvement of academia and researchers in partnership toward achieving the global agendas. the results of the current study showed that sdgs-related literature published in peer-reviewed journals were growing rapidly with an obvious emphasis on sdg and sdg . the majority of publications came from the european regions while the eastern mediterranean region had the least contribution. good health and well-being (sdg ) was the focus of researchers in low-income regions such as africa, the eastern mediterranean, and the south-east asia. climate action (sdg ) was the focus of researchers in highincome regions such as the americas, europe, and western pacific regions. from millennium development goals to sustainable development goals integration: the key to implementing the sustainable development goals united nations:sustainable development goals accessed covid- and the un sustainable development goals: threat to solidarity or an opportunity? sustainability impact of covid- 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effect of public funding on research output: the new zealand marsden fund collaboration in orthodontic clinical trials: prevalence and association with sample size and funding research funding and academic output: evidence from the agricultural university of athens how collaboration type, publication place, funding and author's role affect citations received by publications from africa: a bibliometric study of lis research from to joint modeling of the association between nih funding and its three primary outcomes: patents, publications, and citation impact publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank an-najah national university for giving us the opportunity to access most recent information sources.author's contributions ws started the idea, designed the methodology; did the data analysis, graphics, and data interpretation; wrote and submitted the manuscript. the author read and approved the final manuscript. none. supplementary information accompanies this paper at https://doi.org/ . /s - - - .additional file . availability of data and materials all data presented in this manuscript are available on scopus database using the search query listed in the methodology section.ethics approval and consent to participate not applicable. irb at an-najah national university, palestine requires no approval for bibliometric studies. not applicable. the authors declare that they have no competing interests.received: march accepted: july key: cord- -q zhz authors: papamitsiou, zacharoula; mikalef, patrick title: mapping the intellectual progress in e-business, e-services and e-society from to date: - - journal: responsible design, implementation and use of information and communication technology doi: . / - - - - _ sha: doc_id: cord_uid: q zhz this study aims to identify the conceptual structure and the thematic progress in e-business, e-services and e-society and to elaborate on backbone/emerging topics in the field from to . to address this objective, this paper employs hierarchical clustering, strategic diagrams and network analysis to construct the intellectual map of the i e community and to visualize the thematic landscape in this field, using co-word analysis. overall, a total of papers from the proceedings of the e-business, e-services and e-society (i e) conference, and the respective author-assigned keywords, were included in the analyses. the results indicate that the community has significantly focused in areas like technology adoption models, social media, e-government and business models; sentiment analysis and m-payments are peripheral themes, yet topics like cloud computing and open data are emerging. the analysis highlights the shift of the research interest throughout the past decades, and the rise of new topics, comprising evidence that the field is expanding and evolving. limitations of the approach and future work plans conclude the paper. the subject area of e-business, e-services and e-society has delved into the topic of digitalization with regards to different facets of how we live, work, and conduct business [ ] . since the early s when the dot com period hit its peak, the research interest has centered on the opportunities, challenges and implications that novel digital technologies introduce. the proliferation of such technologies, coupled with the rapid adoption of computer-based systems and the investments in network infrastructures, created a new wave of opportunities on digitalized processes [ , ] . along this progression, research has focused on issues related to each transition, examining obstacles of adoption from the individual to the industry level, value generating mechanisms, and the anticipated and unanticipated effects of integrating these technologies in everyday life. now that the field counts years, based on the number of the e-business, e-services and e-society (i e) conferences being organized, it is a good time to analyze its past and current state. doing so will allow researchers to understand the evolution of the field over the past two decades and identify the challenges and opportunities that lie ahead. the most appropriate way to examine all publications presented in the conferences throughout these years is by applying a methodology that quantifies the core topics, the marginal contributions, the under-developed themes, and the forthcoming ideas that worth investing on, as well as how these topics are related and move between these states during the last years. the main objective of this work is to capture, interpret and understand the big picture in the i e domain from a quantified viewpoint. the research presented in this paper is a mapping study of the i e field; it is a review that seeks to identify, not results, but linkages, shows the internal dynamics and structure of the domain, and pinpoints the topics with impact in the given discipline. towards facilitating this objective, this paper employs co-word analysis, and examines the associations and networks among concepts, ideas, and issues that have contributed to the evolution of the field to date [ ] . co-word analysis allows for and supports the identification of key patterns and trends that point to particular changes in research topics (e.g., emerging or declining interests) or specific research directions (e.g., paradigm shifts), using a graph of key-terms [ ] , extracted directly from the metadata of the papers. considering this, the present study maps the intellectual progress of the e-business, e-services and e-society landscape, as reflected in the records of i e conferences. the proceedings of this event provide a solid foundation to the related work published to date. during the past years, considerable work has been published, allowing us to observe where the field currently stands, what are the challenges and opportunities the researchers are facing, and what are the potential driving forces in the near future. accordingly, this work mainly contributes as follows: • brings new insights on the intellectual mapping and progress of the area of i e; • raises awareness of the community on the mature, under-developed, emerging, or declining research themes; • highlights individual topics as popular, core or backbone topics within the discipline. the community of i e is inherently diverse at the intersection of digital technologies, business, social sciences, big data, artificial intelligence, and network infrastructures [ , ] . digital services can make a global impact and therefore, the understanding of the economic, technical, and social aspects of service development and innovation is a demand [ ] . the quantity and quality of the research activity within this community has been the topic to a variety of literature reviews, aiming to evaluate the research progress, impact and societal value, from different viewpoints (e.g., [ , , , ] ). for instance, persuasiveness of electronic word-of-mouth (ewom) communications has received much attention from scholars because it can affect consumers' purchase attitudes, intention, decisions, and hence sales. a dedicated review of existing literature on the topic offered an overview of the determinants of ewom persuasiveness, identified gaps in current research and provided directions for future research [ ] . another study reviewed inclusive egovernment strategies and socially-aware egovernment policies, and highlighted the need for a better understanding of the role of new intermediaries as actors that can impact the qualities of the citizens' and publicsector relationships [ ] . the results recognized the usefulness of placing the intermediaries in an institutional framework and the authors proposed an agenda for future research. more recently, the emergence and extended discussions about blockchain and what it can offer to businesses in relation to consumers (b c), businesses (b b) and governments (b g) were under the lens of a systematic literature review of articles from business, management and accounting peer review journals [ ] . the findings demonstrate the role of blockchain as a facilitator of instant payments, trusted interfaces and traceability of goods for the consumers. specifically, businesses can be benefited from blockchain in terms of machine-to-machine transactions, accounting, business process management and provenance traceability. new business opportunities also arise in government sector such as digital storage, authentication and maintenance of records, smart trust codification and new market for digital payment services and global commerce [ ] . in another systematic review of articles, social media applications over the marketing context were explored [ ] . the study synopsized the main themes and trends, including the role of social media on advertising, the ewom, customers' relationship management, and firms' brands and performance. apparently, despite the several literature reviews in the field, they are sparse and there is no previous attemptto the best of our knowledgethat maps the field as a whole. as i e is a highly diverse and continuously evolving field, it is important to (a) identify and understand its core foundations that might contribute to reinforcing the community's identity; (b) detect under-represented or under-developed themes that require attention for their inclusion and success; (c) highlight research gaps in bridging theory and practice; and (d) find challenges and opportunities that hold the promise for improving the digitization processes. as the digitalization process and its outcomes in the st century accelerate transformation and the creation of sustainable societies, offering tremendous opportunities for revising current business methods and practices [ ] , there is a critical need for understanding and evaluating the field as a whole. the data analyzed in this study were downloaded from the i e springer proceedings between and . overall, peer-reviewed papers were produced within the community of i e and published to-date. the keynote speeches, prefaces to the conference-, or track proceedings, as well as the papers that did not contain any keywords, were excluded from the analysis. from the collected papers, the author-assigned keywords were extracted from the metadata of each paper and were used as a unit of analysis. the keywords used for the description of the content of a publication can be seen as the basic building blocks of the structure of a research field; an article's keywords provide an adequate summary of its content, and thus can be utilized to reduce a large space of descriptors (i.e., article text) to a network graph of smaller related spaces (i.e., keywords) [ ] . although when authors choose keywords to describe their work those keywords can either be very generic or very specific, still, those keywords are human annotations on the content of the papers, reflecting human judgement and perspective. the idea is to understand the conceptual structure and evolution of a field directly from the interaction between keywords: if two keywords co-occur within a paper, then the two topics they represent are related; higher co-word frequency implies stronger correlation in keywords pairs, further suggesting that two keywords are related to a specific theme [ ] . the papers are distributed per year of publication as shown in fig. . from the published papers, the had author-assigned keywords. specifically, keywords (m = . per article) were identified in total. the retrieved authorassigned keywords were manually pre-processed and standardized through merging words that convey similar meaning (e.g., "electronic government" and "e-gov" were merged into "egovernment"), fixing misspelled keywords (e.g., "goverment"), following a common spelling for uk and us terms (e.g., "behaviour" and "behavior"), and filtering broadly used terms (e.g., "iot" and "internet of things"; "wom" and "word of mouth") -following the approach recommended in [ , ] , in a noninvasive manner. keywords appearing in singular and plural forms of nouns and gerunds were also merged. at the end of this pre-processing, keywords ( . % of the original dataset) were identified as unique and were subjected to further analysis. in order to be able to apply this method, papers from more than years of research are required. the kolmogorov smirnoff test shown that the frequency of keywords follows a power-law distribution with an alpha of . . due to this heavy-tailedness, the research landscape of i e is a scale-free network, with small number of popular terms acting as "hubs": they connect different topics, capture major research directions and influences in the field, and shape its intellectual structure [ ] . a scale-free network also suggests that major research themes can be detected with small subset of popular terms. a previous analysis in the hci research field demonstrated that less than keywords are enough to describe the intellectual progress of a field [ ] . thus, in the present study we decided to include only those keywords that appear more than four times (n ! ) in the period - . this decision was grounded on two facets: (a) the frequency of a term reflects its significance for a research community, i.e., the higher the frequency is, the more often the term attracts the researchers' attention/interest; and (b) the retained keywords (total frequency = , . % of the total unique keywords) cover ( . %) of the articles (with keywords) published. furthermore, for the given datasets of terms and papers, n = is the minimum term frequency that achieves the highest inclusion of papers in the datasets. for example, for authorassigned keywords with n ! , the retained keywords are n = and cover . % of the papers, whereas for keywords with n ! , n = keywords, covering . % of the papers. thus, with fewer yet highly frequent terms we could satisfactorily describe the i e network of terms. this study employs co-word analysis to shed light on the intellectual progress in the i e field. co-word analysis has been proposed as a content-analysis technique to map the strength of relations between terms in texts and to trace patterns and trends in term associated-ness [ ] . the idea behind co-word analysis rests on the assumption that keyterms identified within an article (e.g., keywords) can adequately describe and communicate the content of that article; the co-occurrence of at-least two keywords in the same article indicates a linkage between the topics, i.e., a "theme" [ ] . the main units of analysis are keywords, clusters (i.e., sets of keywords) and keyword networks [ ] . co-word analysis is applied to reduce the broad network of keywords into a smaller network of related topics using graph theory [ ] . graphs consist of nodes that represent the keywords, and links that represent the interactions between the nodes. given a network of keywords, a combination of clustering, network analysis and strategic diagrams is used to model the conceptual structure of a field and to characterize it [ ] . the graph theory concepts employed are centrality (i.e., the strength of the links from one research theme or cluster to others, indicating its significance in the development of the community [ ] ) and density (i.e., the coherence of a cluster and a measure of a theme's development [ ] ). combining centrality and density allows for the creation of two-dimensional strategic diagrams [ ] (fig. ) : the position of a cluster in the diagram corresponds to the importance of the cluster in the whole network (i.e., centrality x-axis) in relation to how well the theme of this cluster is developed (i.e., density -yaxis). as one can observe, quadrant i (q ) holds the motor themes (i.e., mainstream themes) that have strong centrality and high density. quadrant ii (q ) contains themes that are internally well-structured but have weak external ties. these research themes are more specialized and peripheral to the mainstream work that is central in the research field. quadrant iii (q ) includes the themes with low density and low centrality, that are either emerging, or disappearing. finally, quadrant iv (q ) covers basic and transversal themes of considerable significance to the entire research network, i.e., central to the community, with potential to become important to the field as a whole. to identify the major research themes in the i e domain, hierarchical clustering analysis on a correlation matrix with the retained terms was performed, using the ward's method with squared euclidean distance as the distance measurement [ ] . the supervised clustering method allows to maintain content validity and cluster fitness for the highest number of clusters [ , ] . each cluster represents a research theme or sub-field. the co-word network was further analyzed using the following measures: • keywords: set of terms that constitute a cluster; • size: number of keywords in the cluster; • frequency: how many times all keywords (in a cluster) appear in the dataset; • co-word frequency: how many times at-least two keywords (from a cluster) appear in the same paper. computing this results in a symmetrical co-occurrence matrix [ ] : values in the diagonal cells are term frequencies, and values in nondiagonal cells are co-word frequencies. high frequency of co-occurrence between terms indicates connection between the topics they represent; • transitivity: how tightly connected is the cluster (the clustering coefficient), i.e., how close the keywords are to being a "clique". transitivity is the frequency of loops of length three in the cluster; a loop of length three is a sequence of nodes x, y, z such that (x, y), (y, z) and (z, x) are edges of the graph [ ] . the value range is [ , ]; • centrality: the degree of interaction of a theme with other parts of the network, i.e., how many other clusters a cluster connects to [ ] ; centrality refers to a group of metrics that aim to quantify the "importance" of a particular node (or cluster) within a network (e.g., betweenness centrality, closeness centrality, eigenvector centrality, degree centrality) [ ] . here we used betweenness centrality (c), with c ; • density: how cohesive is the cluster of terms, i.e., the number of direct ties observed for the cluster divided by the maximum number of possible ones [ ] . density is graph-dependent and can be any positive real number [ ] . based on the clustering results, we plotted the strategic diagram for the years - to visualize the cohesion and maturity of the i e themes [ , ] . in addition, a keyword network graph was created from the keywords list. in this graph, each keyword is represented as a node, and the keywords that co-appear on a paper are linked together. by creating associations between keywords, multiple networks associated with different themes are also created. in this case, bridges are built between the nodes of keywords, to allow communication and information flow between isolated regions in the whole network. those nodes are known as structural holes [ ] . keywords acting as structural holes serve as a "backbone" of a network: if removed, the network will lose its cohesion and will disintegrate into separated and unconnected concepts. thus, the network's core-periphery structure needs to be computed, to determine which nodes are part of a densely connected core (i.e., with a higher number of bridges), or a sparsely connected periphery [ ] . core nodes are reasonably well-connected to peripheral nodes, while peripheral nodes are sparingly connected to a core node or to each other. a node belongs to a core only if it is well-connected to other core nodes and to peripheral nodes [ ] . a follow-up core-periphery analysis was performed to spot the core research topics from the perspective of the whole network. in this analysis, keywords were categorized according to their popularity, coreness (i.e., connectedness with other topics) and constraint (i.e., backbone). the whole approach is illustrated in fig. . the analysis on the retained author-assigned keywords led to clusters (labeled as c -c , in table ), with each cluster representing a research theme or a sub-field. in order (a) to better understand the relative "position'' of these clusters within the overall i e field (i.e., what is the distance from each other in terms of cohesion and maturity of research themes they correspond to); and (b) to create the conceptual structure of the i e discipline, we constructed strategic diagrams (plots) using the centrality and density of each cluster [ , ] . the overall results can be seen in reading fig. and table together. in the plots, both axes are centralized to the average centrality and average density respectively (i.e., . , . ). the overall network's density was . . as it can be observed from fig. , one motor theme (mainstream theme), represented by cluster c (i.e., literature review, tam, adoption) is detected using the human descriptors (keywords) of the papers. in other words, the field is in general fragmented, with only one theme having received substantial attention from the community, in terms of human annotations. furthermore, in fig. , the author-assigned keywords indicate that the community has few internally well-structured research themes, yet with weak external ties (ivory towers), acting as peripheral nodes to the global network (i.e., connect only to core nodes, yet not necessarily to mainstream topics only), and classified in clusters c , c and c (e.g., mpayments, digital payment, twitter, sentiment analysis, utaut , perceived risk). those topics appear to have high-density, i.e., the clustering coefficient is high and the topics within each of the cluster are very well connected to each other, but they lack strong ties with topics that are external to them. the following-up core-periphery analysis will provide insight on that issue. regarding the themes that are either emerging or disappearing (chaos/ unstructured), the author-assigned keywords revealed that researchers have developed a considerable number of topics within a sense -"marginal" interest in the i e network, classified in clusters c , c , c , c , c and c (e.g., ewom, online reviews, motivation, information seeking, eservices, cloud computing, saas, business intelligence, ebusiness models, open data, information technology), as illustrated in fig. . the term "marginal" here is used to describe both the cases of "close-to-disappearing" and "nearly rising" topics, i.e., topics that either tend to no-longer attract major interest, or they have recently started to attract attention, but have not yet been well-developed. finally, a substantial number of transversal themes (bandwagon) have been detected as well, i.e., themes that are strongly linked to specific research interests throughout the network yet are only weakly linked together. these are categorized in the clusters c (e.g., customer engagement, facebook, social media, use and gratification theory, social networks), c (e.g., social commerce, wom, trust, privacy) c (e.g., ecommerce, interoperability, interorganizational system, security, supply chain, ebusiness, web services, qos) and c (e.g., iot, smart cities, artificial intelligence, egovernment, b b). a network of keywords demonstrates the relationships among different themes; to better understand and visualize the interactions between the research themes in table , network analysis was used to create a granular map of the keywords. figure displays the results. each node in the graph represents a keyword that is linked to other keywords that appear on the same paper. the size of the nodes is proportional to the frequency of the keywords, the color of the node corresponds to the cluster the keyword has been classified in, and the thickness of the links between the nodes is proportional to the co-occurrence correlation for that pair of keywords. to reduce visual clutter, a centralized subset of the complete network is illustrated, omitting isolated nodes and keywords with less than strong ties, that would lead to a highly disconnected network. finally, core-periphery analysis was performed to identify the core research topics, from a whole-network perspective, as individual keywords, regardless of the cluster they belong to. the analysis yielded topics in each of the categories ( table ): • popularity: how frequently a keyword is used; • coreness: how connected is a keyword with other topics; value range: [ - ]; • constraint: how connected is a keyword with other otherwise distinct topics (i.e., if the topic creates a backbone of the field); constraint is measured on a [ - ] scale. high core value indicates a topic that is well connected to other topics. lower constraint suggests a keyword that brings together otherwise isolated topics ("bridges"). burts constraint (i.e., constraint) [ ] is commonly used for this purpose (accurately speaking, the lack of it, because the larger the constraint, the less structural opportunities a node may have for bridging structural holes). topics with high popularity and coreness and low constraint can be considered as driving forces for advancements in the field: without these topics, the field of i e would be completely fragmented. the i e conference on e-business, e-services and e-society has grown and evolved over the years. the mere fact that the conference has managed to keep and expand its active community of researchers for almost two decades, demonstrates that the field is of increased and growing relevance. the objective and contribution of this paper is twofold: (a) to have a methodological contribution in the field (i.e., the method presented in this paper has not been employed in this research domain before); and (b) to demonstrate and apply a quantified (objective) methodology that is beyond the qualitative (subjective) perspective of a systematic literature review, aiming to shed light to the maturation of the field throughout the past two decades and to map the research streams in the field, how they are connected, and detect those that would benefit for more focus or integration. the analysis conducted in this study highlighted the major themes that have dominated the researchers' interest, as well as those that are likely to be the core focus of future studies. the co-word analysis of the i e proceedings to date revealed some interesting findings regarding the progression of the field as a whole. in coherence with the title of the conference, the three main (i.e., motor) themes have been on e-business (i.e., e-commerce), e-services (i.e., e-government) and e-society (i.e., social media) as is depicted in fig. , and in line with previous systematic literature reviews [ , ] . the underlying topic that has linked these three focus areas has been adoption models, examining the factors concerning individuals, users and organizations intention to adopt such systems, also noted in previous studies [ , ] . this trend also indicates the maturity of these technologies, with substantial research still concerning why and how adoption of emerging digital technologies can be enhanced. an intriguing finding is the emergence of big data as a linkage between the main three pillars (figs. and ). research themes that fall within the "chaos/unstructured" quadrant will attract attention in the years to come: as more and more people are moving from the adoption of digital technologies to the routinization in everyday life, so is the amount of generated data increasing. this has led to a surge of research on the potential of big data analytics for extracting actionable insight confirming previous findings [ ] . as seen from the network map (fig. ) , big datain particular with relevance to egovernment and social mediais an area with much future interest, also highlighted in [ ] . the use of user generated data, and the integration with social media platforms offers an interesting perspective into the generated insight, the services that can be built based on this insight, as well as the implications and ethical issues that accompany such ventures. big data and business analytics ecosystems may pave the way towards digital transformation and sustainable societies [ ] . furthermore, the strong link between e-commerce and social media (table ) , denotes the increased prevalence of social commerce efforts over the last years (e.g., [ ] ). we witness an increased number of social media platforms featuring commercial aspects, with some notable examples being facebook and instagram. as more vendors utilize these platforms, trust emerges as a core aspect [ ] , both regarding the sales side and the products featured on these, as well as on the data exchanged and utilized for marketing and sales promotions [ ] . recent events (e.g. the cambridge analytica scandal) surfaced issues about privacy violation and use of data by third-parties which have come to the attention of the general population. as social media and e-commerce vendors become increasingly more integrated, data sharing and ownership issues will be brought in the spotlight of research/practice. while these are some of the major trends that can be detected throughout an analysis of the field covered by the i e conference, there are many more sub-fields that are likely to be fruitful areas for future research. our ambition was to illustrate some of these as well as to identify the core areas that have received the center light of attention by researchers over the past two decades. our analysis indicates that the three pillars of the i e conference are becoming increasingly more fussed, and that future research is likely to bridge all three domains to derive important research and practical knowledge. i e a systematic review of extant literature in social media in the marketing perspective structural holes and good ideas co-word analysis as a tool for describing the network of interactions between basic and technological research: the case of polymer chemsitry from translations to problematic networks: an introduction to co-word analysis historical scientometrics? mapping over years of biological safety research with coword analysis science mapping software tools: review, analysis, and cooperative study among tools i e . iaict blockchain for businesses: a systematic literature review knowledge discovery through co-word analysis a co-word analysis of library and information science in china persuasiveness of ewom communications: literature review and suggestions for future research i e investigating patterns of interaction in networked learning and computer-supported collaborative learning: arole for social network analysis co-occurrence matrices and their applications in information science: extending aca to the web environment i e . iaict chi - : mapping two decades of intellectual progress through co-word analysis determining consumer engagement in word-of-mouth: trust and network ties in a social commerce setting ward's hierarchical agglomerative clustering method: which algorithms implement ward's criterion? a measure of betweenness centrality based on random walks sustainable development: the role of network communication i e big data and business analytics ecosystems: paving the way towards digital transformation and sustainable societies core-periphery structure in networks (revisited) approximating clustering-coefficient and transitivity intermediaries in e-inclusion: a literature review the battle of brain vs. heart: a literature review and meta-analysis of hedonic motivation use in utaut key: cord- -ktrw u authors: gupta, abhishek; lanteigne, camylle; institute, victoria heath montreal ai ethics; microsoft,; lab, algora title: report prepared by the montreal ai ethics institute (maiei) on publication norms for responsible ai date: - - journal: nan doi: nan sha: doc_id: cord_uid: ktrw u the history of science and technology shows that seemingly innocuous developments in scientific theories and research have enabled real-world applications with significant negative consequences for humanity. in order to ensure that the science and technology of ai is developed in a humane manner, we must develop research publication norms that are informed by our growing understanding of ai's potential threats and use cases. unfortunately, it's difficult to create a set of publication norms for responsible ai because the field of ai is currently fragmented in terms of how this technology is researched, developed, funded, etc. to examine this challenge and find solutions, the montreal ai ethics institute (maiei) co-hosted two public consultations with the partnership on ai in may . these meetups examined potential publication norms for responsible ai, with the goal of creating a clear set of recommendations and ways forward for publishers. in its submission, maiei provides six initial recommendations, these include: ) create tools to navigate publication decisions, ) offer a page number extension, ) develop a network of peers, ) require broad impact statements, ) require the publication of expected results, and ) revamp the peer-review process. after considering potential concerns regarding these recommendations, including constraining innovation and creating a"black market"for ai research, maiei outlines three ways forward for publishers, these include: ) state clearly and consistently the need for established norms, ) coordinate and build trust as a community, and ) change the approach. • create tools to navigate publication decisions: the use of extrinsic measurements like benchmarks, or third-party expert panels could be a crucial step to navigating publication decisions in a fair way. such methods could set a certain standard in terms of the acceptable level of risk associated with a publication. in line with this suggestion, it would also be pertinent to keep a record of the papers that were rejected due to their inherent risk, as well as some metrics on these. • offer a page number extension: it may be beneficial to extend the page limit for published papers to allow researchers to include negative results (results that are insignificant or disprove researchers' hypotheses), which aren't traditionally printed. in addition to expanding the number of pages, there should also be a significant change in the culture surrounding the publication of negative results. • develop a network of peers: developing a network of peers to evaluate researchers' ai models in terms of potential risks and benefits may be an important tool towards better and safer publication norms. if such a mechanism were put in place, the evaluation could be fully or partly based on philosopher john rawls's "veil of ignorance." if this idea was applied to reviewing ai research, peer reviewers could be asked to consider the potential advantages and risks of a new ai research from different social perspectives. • require broad impact statements: the neurips conference requires that submitted papers include a statement of broader impact with respect to the research presented. this incentivizes researchers to think about potential risks and benefits by making reflection a requirement for one's work to be considered at neurips. similar measures at all conferences and publications would encourage researchers to critically assess their research in terms of its effects, positive and negative, on the world • require the publication of expected results: requiring that researchers write and publish the expected results of their research project (including but not limited to its broader social and ethical impacts) could help foster reflection around potential benefits and harms even before researchers undertake their project. • revamp the peer-review process: the well-established practice of peer review is a great opportunity for exchanges on the risks and benefits each reviewer sees in the paper they are revising. if a question or requirement were added to this effect when papers were reviewed, it may have a rapid and widespread impact in inciting researchers to consider what may follow from their research. an effective review process should promote limiting risks while also being clear, fair, and efficient. one way of doing this is to intensify the requirements for publication proportionally to how risky the research is deemed by peers reviewing the paper. the history of science and technology shows that seemingly innocuous developments in scientific theories and research have enabled real-world applications with significant negative consequences for humanity; from the eugenics movement in the late th and early th centuries to the cataclysmic nuclear destruction in japan in . this history reveals that the real-world impacts of scientific research cannot be separated from the research itself, and it's important to look at the social, cultural, political, and economic realities that shape the way science is used and the norms that regulate it. it's also important to consider the impacts these developments may have on people across the world. often, researchers do not consult with or consider individuals that may be negatively affected by scientific developments, reflecting existing power imbalances in which a small group of privileged individuals make decisions or take risks that impact millions, if not billions, of lives. we need to be aware of these power imbalances and how they're rooted in existing inequalities-this is especially true in the field of artificial intelligence (ai). the pace and scale of impact from ai far exceed other technologies. thus, critical examination is necessary, especially as this technology becomes increasingly deployed throughout society. the aforementioned power imbalances and inequalities in scientific research are apparent in the general disconnect between the research priorities of funders (e.g. grant-making bodies, companies, governments, etc.) and the broader societal interest. the academic paradigm of " publish or perish" and the undue pressure it creates, overshadows more fundamental questions that need to be asked. the most important of which is, "why is this research project being pursued in the first place?" this lack of critical reflection and external pressure has given rise to predatory journals with lax quality standards regarding what gets accepted for publication-this is especially an issue for researchers who are new to an academic field and are uncertain about publishing norms. for example, research in phrenology continues to be accepted in highly revered journals despite decades of precedent demonstrating that phrenology is pseudoscience. the egregious inclusion of this type of research resulted in severe backlash from scholars that led to a retraction and apology from the journal. this example highlights how fallacious research can slip through the cracks, especially without critical reflection. in order to ensure that the science and technology of ai are developed in a humane manner, we must develop research publication norms that are informed by our growing understanding of ai's potential threats and use cases. unfortunately, there haven't been many efforts to create a set of publication norms for responsible ai because the field of ai is currently fragmented in terms of how this technology is researched, developed, funded, etc. thus, the norms around ai publications and ethical standards are not only fragmented, but also contradictory in many cases. a standardized approach to publication norms in ai across a large number of jurisdictions is essential. many subfields in ai are experiencing a boom in interest, with growing demands to produce novel research. thus, there is an elevated risk of a lack of awareness on what adequate and rigorous publishing norms are. additionally, there is a high degree of susceptibility to predatory journals that lure budding researchers through a "pay-to-play" model. this amplifies the potential impacts of harmful research that must be critically reviewed before published for public consumption. most scientific and academic journals have particular guidelines for submissions, which are a form of publication norms. however, problematic research can still be published. for example, researchers exerted external pressure on springer to withdraw the publication of a paper in which the researchers claimed to "predict criminality" using neural networks for facial recognition. this demonstrates that the publication ecosystem requires norms with multiple filters. as well as editorial boards that have sufficient demographic diversity and a range of expertise to be able to flag problematic research and prevent its publication, especially in cases where there is potential to render harm on marginalized peoples. however, the prevalent practice in peer-reviewed journals is to evaluate a work purely on its scientific merit, which overlooks the inherent interaction between fundamental research and the social context in which it's conducted. publication norms have a strong role to play in ensuring interdisciplinarity in review processes so that too narrow a focus does not allow potentially harmful work to pass as seemingly innocuous. beyond the social implications of research, it's also important to consider the technical implications. for example, in cryptography, it's important to find vulnerabilities in a system to improve robustness. cryptographers do this by looking at a system from an adversarial perspective, as well as by sharing their systems openly so they can be evaluated for undue risks by as many people as possible. for example, through this process decades ago, unnecessary risks arose from the use of substitution boxes (s-box) in data encryption standard (des), which led to the subsequent discovery of differential cryptanalysis. this is also important type of vulnerability was seen in the jboss middleware, which shipped quietly with dozens of consumer grade softwares but often with an unpatched, or even unpatchable, version. similarly, software which incorporates ai that relies on an unpatchable library will be vulnerable. to expose such vulnerabilities, as well as better understand the social implications of ai research, journal editors must be better equipped to identify all actors who may engage with ai research and make well-informed decisions around whether research should be published, and how. we believe developing a standardized approach to publication norms in ai across a large number of jurisdictions is the first step towards ensuring that the science and technology of ai are developed in a humane manner. the use of extrinsic measurements like benchmarks, or third-party expert panels could be a crucial step to navigating publication decisions in a fair way. such methods could set a certain standard in terms of the acceptable level of risk associated with a publication. in line with this suggestion, it would also be pertinent to keep a record of the papers that were rejected due to their inherent risk, as well as relevant metrics. for example, what subfield of ai was the paper part of? what institution or corporation was the authors affiliated with (if any)? demographic and identity metrics may also be pertinent to help ensure the review process is not discriminatory. to this end, mechanisms like retraction watch that are invested in by the scientific community and held to the highest academic standards will be a cornerstone to strategies combating spurious research. awareness of such mechanisms and their integration into tools like google scholar, akin to covid- research warnings for non-peer-reviewed articles, is essential. it may be beneficial to extend the page limit for published papers to allow researchers to include negative results (results that are insignificant or disprove researchers' hypotheses), which aren't traditionally printed. there is a bias towards publishing papers with positive results. that is, results that confirm or partially confirm one's hypothesis. in addition to expanding the number of pages, there should also be a significant change in the culture surrounding the publication of negative results. while a higher maximum of pages may help, it should be accompanied by other measures to encourage researchers to share their negative results and push the broader scientific community to consider negative results in their analysis. publishers could require the inclusion of all negative results that led the researchers to the positive results published and/or submitted. these could also be indexed through a standardized mechanism for retrieval by engines like google scholar so that downstream researchers are aware of what areas have already been explored. improving citations and value of such negative results work will also incentivize the ecosystem to invest in making negative results more public and elevate the quality of scientific publishing in the space. developing a network of peers to evaluate researchers' ai models in terms of potential risks and benefits may be an important tool towards better and safer publication norms. if such a mechanism were put in place, the evaluation could be fully or partly based on philosopher john rawls's "veil of ignorance," which asks individuals to create a new society and choose its governing principles without knowing their individual characteristics (e.g. gender, race, social class, etc.). the idea is that they'll choose principles that will benefit everyone in society since they're "ignorant" of their personal circumstances and social standing. if this idea was applied to reviewing ai research, peer reviewers could be asked to consider the potential advantages and risks of a new ai research from different social perspectives. specific details about fictional but realistic situations and personas may be used to help reviewers think more accurately and relevantly about privileges and disadvantages they might not have themselves. of course, this shouldn't replace important efforts to make the field of ai more diverse and inclusive-but the exercise of imagining how another person may be affected, positively or negatively, by an ai model is relevant for everyone. this practice has precedent in design thinking approaches and has shown to create products and services that are ultimately more empathetic to the needs of the communities that they are meant to serve and encourage the construction of more inclusive work. an important method to incite researchers to reflect on the impacts of their work is to give them incentives to do so. for example, the neurips conference recently added a requirement for papers that are submitted: they must include a statement of broader impact with respect to the research presented. this incentivizes researchers to think about potential risks and benefits by making reflection a requirement for one's work to be considered at neurips, one of the most prestigious conferences in the field of ai. therefore, similar measures at all conferences and publications may be a good way of encouraging researchers to critically assess their research in terms of its effects, positive and negative, on the world. iclr has followed suit and has included a code of ethics for their edition which is an important step in the proliferation of these standards becoming mainstream, not just something that one gives a brief nod to. it's important to note that how the research community adopts the formulation of broader impact statements is of fundamental importance. if broader impact statements become yet another "ethics stamp" on poorly conceived research, then the entire endeavour becomes counterproductive. researchers need to critically evaluate the broader impacts of their research before starting a project and use these insights to guide a conscientious research methodology and not construct these perspectives after the fact. the point of writing a broader impact statement must be to introspect the ethics of one's work and not as a mechanism to avoid desk rejection. one way of doing so is to popularize the importance of these norms in the early stages of a researcher's journey into the publishing world. during the formative years in the field, early-career researchers might be naturally drawn to emulating the behaviour of the more experienced researchers in their labs and workplaces. an educational push for holding research ethics, norms, and standards as the paramount element of doing research and development in a consequential field like ai needs to be strongly encouraged. principal investigators (pis) and other senior researchers in both academic and industry labs should shepherd those who are still growing accustomed to the modes of operation of research in the domain. the widespread presence of such a requirement could also incite greater awareness among technical researchers in the field of ai regarding fields and areas like ethics, psychology, anthropology, sociology, and critical race theory. furthermore, making impact statements and other educational pushes necessary could spark interdisciplinary collaborations between researchers. requiring that researchers write and publish the expected results of their research project (including but not limited to its broader social and ethical impacts) could help foster reflection around potential benefits and harms even before researchers undertake their project. for example, a list of journalists who would be interested in hearing about the benefits of the author's thesis on the rest of society could be created, incentivizing the consideration of their project benefits (scientists will want their papers to be promoted in newspaper articles). this would not only encourage researchers to reflect on the impacts of their work, but perhaps guide them towards generally less risky and more beneficial research. this could be similar to how many psychologists now publish a preliminary report on how they will conduct their research, the variables they will be measuring, and their hypothesis, before they perform an experiment. this prevents data manipulation in attempts to find statistically significant results. in the language of software engineering, it could create something akin to test-driven development (tdd) whereby the researchers can preemptively highlight what it is that they are aiming to achieve with their research and view negative results in a positive light as a way of showing ways that don't lead to results that were stated as the goals of the research nonetheless led to an understanding of some aspects of the field helping to improve the knowledge base for future researchers in the domain. of course, having much greater diversity of researchers in the field of ai-in terms of race, gender identity, sexual orientation, geography, language, lived experiences, and socioeconomic status-could also make a significant difference in how and how much researchers think about the impacts of their work. it seems reasonable that, in at least some cases (if not many), the potential benefits and pitfalls of researchers' work may be more pronounced for a certain demographic, and people who are part of that demographic are more likely to be attuned to how it may impact them. thus, having more diverse actors in the field of ai could help foster more comprehensive reflection on the potential impacts of a research project. additionally, a framing whereby the researchers take on the onus to surface these impacts rather than placing the burden on the people who might be impacted (often they might not have adequate knowledge, resources, or abilities) to defend themselves creates a more pro-social way forward for conducting research. the well-established practice of peer review is a great opportunity for exchanges on the risks and benefits each reviewer sees in the paper they are revising. if a question or requirement were added to this effect when papers were reviewed, it may have a rapid and widespread impact in inciting researchers to consider what may follow from their research. this could be done in a way that is similar to how the world health organization (who) highlights dual use research of concern (durc): research in the realm of the life sciences "that is intended for benefit, but which might easily be misapplied to do harm." to have a similar category for ai research would be pertinent, and developing guidelines for this kind of research seems crucial, both to define it and to control it, especially given that ai squarely falls under the category of a dual-use and general-purpose technology. such guidelines might include needing special authorization to conduct this type of research, or it may be required that the research be reviewed more extensively before being published. further, it's obvious that an effective review process should promote limiting risks while also being clear, fair, and efficient. one way of doing this is to intensify the requirements for publication proportionally to how risky the research is deemed by peers reviewing the paper. one way to go about this could be to identify a list of specific risks that any ai model proposed in a research paper could have. these risks may have numerical weights assigned to them depending on how dangerous their consequences may become. each peer reviewer could identify the risks they deem relevant to the ai model they are examining, and assign a score between and to each risk in accordance with how likely it is that the risk will materialize. the risks' weights could then be multiplied by their corresponding likelihood score. each of these quotients can be compiled into a sum. the sums obtained by each reviewer for one paper can then be added up and made into an average. if this 'total risk average' is higher than a pre-established number, the paper could then be immediately rejected, require further reviewing and discussion by a third-party group of experts, or be published under much stricter requirements than other papers that are below the 'total risk average' threshold. such a threat modelling approach is already used extensively in the field of cybersecurity to prioritize risks and vulnerabilities to guide the efforts of researchers and practitioners in working to address them . this process, or one with a similar structure, has the advantage of remaining somewhat efficient by not requiring that papers that are considered non-risky be subjected to more time-consuming reviewing or more stringent requirements unnecessarily. of course, a procedure like the one presented above risks potentially wrongly identifying a model as low risk, whereas a model that held all papers to higher standard regarding risk avoids this. however, it's unlikely that applying more stringent standards to all papers is necessary, efficient, or realistic considering the sheer volume of papers published. a more targeted approach seems better suited to the reality of publishing in the domain of ai. a consideration that publishers can adopt is to think about the rate of false positives and track them over the period of reviews and adjust as they go along to judge the efficacy of this mechanism. to the risk of this becoming repetitive, the cornerstone of an effective review process, as previously mentioned, will be a diverse body of reviewers from the point of view of race, gender, disability, socioeconomic status, geography, and more. risk assessment is unlikely to be fair and comprehensive if all reviewers have similar backgrounds and experiences. this will result in direct harm to those not represented among reviewers, which are often individuals who are already marginalized and most at risk. this is something that needs to be created proactively and will not necessarily emerge organically since reviewers are often sourced from a tightly knit network of people one is already familiar with; breaking free from that requires constant and conscious effort. one important area where there may be some pitfalls is innovation. if publishing norms are more stringent, then cutting-edge research may be ignored or underfunded. in some cases this may be because the potential risks outweigh the benefits. however, in other cases it may be the result of a lack of awareness from the researcher's part on what those publication norms are. this could have a particularly negative impact on emerging scholars in ai from regions and countries where ai research and development is in nascent stages, especially those which have a less than proportionate representation in the scientific publications at major conferences and journals (which are mostly concentrated in the western hemisphere). this could harm scientific diversity in the field. one can undoubtedly see this as a missed opportunity, but it's better framed as an opportunity for better work and innovation. what if we could use this as an opportunity to build ai that did more good than harm? by erring on the side of caution, we may encourage researchers to better understand the consequences of developing and deploying a certain system or application. understanding the tail risks of innovation is critical. the insights from fields such as cryptography are that the risks are enormous from poor research, and given the nature of ai systems, a similar level of risk is to be expected. in light of this understanding of asymmetric returns, we need to raise our standards of what constitutes "innovation," and who gets to decide if a new technological application constitutes positive innovation; bringing more good than harm. from this perspective, constraining innovation is not a pitfall of changing publishing norms. instead, changing publishing norms may foster higher quality, more inclusive and positive innovation. it can be viewed as a mechanism to bend the field of research towards a prosocial direction, something that governments use frequently in the form of regulations to guide how innovation happens in the market. in a similar vein, like many other scientific disciplines, machine learning and artificial intelligence have been affected by their own reproducibility crisis, where a worrying number of algorithmic research results cannot be reproduced when other data scientists run the same experiments. this is of particular concern in fields such as digital biomedicine, where faulty models for disease diagnosis and monitoring can place human lives at great risk. one key component of the problem is the absence of information about the training and evaluation code, the number of training runs required, and datasets used. another part of the issue is the environment in which data scientists operate, where there is a pressure to publish quickly, a reluctance to report failed replications and a lack of computational and human resources to test every condition and fine-tune each hyperparameter. better publication norms will not only encourage more rigour in scientific methodology, but also limit the number of cases where research is modelled on "false starts." the danger of this can ensure that future innovation in the field is based on a set of verifiable and veritable discoveries. in an inherently stochastic domain like ai, the degree of variability that can occur in experiments where one might possibly find an experimental run through sheer luck to get the results that showcase a correlation that they want is all the more reason for advocating for higher rigour; a focus on causation over correlation is going to be essential in addition to the points mentioned above. with more stringent norms on what gets published in the field of ai, there is a possibility that research deemed too risky will be driven underground. meaning, researchers whose work is rejected may publicize their research in other ways, circumventing the measures put in place. due to the stricter publishing norms, the rejected research may become even more dangerous as it's excluded from mainstream critique and necessary scrutiny. put simply, stringent publishing norms may actually increase the likelihood of harmful ai by creating a "black market" for research that has subversive aims. of course, there are counterarguments against this scenario. first, the field of ai/ml is heavily dominated by individuals who are affiliated with universities and companies. in both cases, getting research published by a reputable journal or presented at a recognized conference is key to advancing their career. simply putting out research without it being published or affiliated with their institution (whether academic or corporate) because it is too risky according to publishing norms is of limited use. hence it seems unlikely that someone with the knowledge and qualifications necessary to build innovative yet risky ai would have an incentive to share it using an alternative method. there may be a greater incentive for scientists to conduct research that is more likely to be published in accordance with the new, more stringent standards. it is highly unlikely that the risks for underground research are big enough to warrant not moving towards more stringent publishing standards in terms of security and risk in the field of ai. it's also important to note that the field's current exclusivity to those with affiliations to large and wealthy institutions is not in itself positive. we believe the field of ai should be more accessible than it currently is in that regard. paradoxically, there are questions as to whether it's actually more dangerous if risky ai research goes "underground" than if it's publicized and sanctioned by highly regarded institutions and publications. if an ai research submission is rejected by a publication, then it's likely that it'll receive minimal attention. however, if risky ai research were to be published by these journals and publications then it's likely it may do significant harm because people (especially academics and fellow researchers) tend to refer to these sources for the latest relevant information about progress in the field; under the assumption that the information published has been peer-reviewed. it's this same research that often gets widespread media coverage as well. this follows from the principle of using "strategic silence" as a way of limiting the oxygen that is provided to mis-, dis-, and mal-information by not offering it a platform by way of discussion, time, and resources. furthermore, general audiences are less likely to scrutinize research sanctioned by a journal or an institution. in other words, when risky papers are published, we may not be able to count on public scrutiny to highlight its dangerous possibilities. there have been numerous instances of popular media coverage for dubious research stemming from low-quality journals, preprint servers, and other places that had a significant impact on the public's perception of ai. as an example, research that claimed to create a "gaydar" identifying a person's sexual orientation was thoroughly debunked by leading researchers in the field of ai, but the harmful and offensive work still received significant media attention. we posit that this might be because of the highly technical nature of the field (not that this isn't the case in other domains, but there is a disproportionate attention paid to the advances in this field while the ability of the general public to parse the advances for what they truly are might not be sufficient) which leads to an overestimation in the capabilities of the systems. the fact that such research was published-and thus, endorsed by the publication or institution behind it-created a false sense of security and legitimacy for many people. papers that are published are usually revised and reviewed, and this process, along with the metaphorical seal of approval from the publication/institution, can obfuscate the overly risky nature of the results being published. it essentially shields the paper from any scrutiny because, at first glance, the paper has all the characteristics of an acceptable or even outstanding paper. thus, getting widespread public scrutiny, especially from individuals outside the ai community, is likely to be quite difficult as nothing seems particularly questionable. with this in mind, there is a responsibility on the part of the journal's editorial board to have experts outside of the field review papers in order to identify errors or risks with the research. homogenous editorial boards (i.e. dominant avatars of society), don't have the required perspective to ask essential, critical questions like that of a diverse board. in this sense, a diverse board will be able to provide additional points of failure for controversial scrutiny, before even requiring any public intervention. hence, stricter publication norms that include such a board would be desirable. in addition, one of the mechanisms trialled with neurips and icml over the last few months is the concept of ml retrospectives (co-organized by abhishek gupta, one of the authors of this document) where researchers are asked to reflect on their prior work and identify shortcomings, improvements, changes, and any other comments that they have on those papers to highlight the need to revise understandings of discoveries over time as new evidence and knowledge comes to light. normalizing the "owning" of faults in prior publications can move accountability back to authors, no matter their prestige, leading to a healthier intellectual ecosystem. research progress may be slowed to allow for more thorough risk evaluation. this may affect the quick rate at which ai papers are currently published. nevertheless, pausing the influx of information on ai can provide more time to dissect, digest, and process research. furthermore, a stricter process to pass, and one that takes time, could discourage researchers against cutting corners, which could lead to having their paper rejected and making the process unnecessarily long. this can then help guard against unnecessary research being proposed. one of the trends of taking rejected submissions from one conference or journal and making quick, minor tweaks and pushing it out to the next conference is a particularly problematic scenario which encourages the behaviour whereby researchers attempt a "keep trying to till you win" mindset that has the potential to lower the quality of overall scholarship in the field. making explicit perhaps where this idea was submitted before and reasons for rejections, how those shortcomings were addressed can serve two purposes: improve allocation of scarce reviewing resources in the field and push higher standards of transparency in the field. cross-linking submissions on a platform like openreview (acknowledging though that there are many caveats on the platform) can help us move in that direction. some publishers may feel as if they have lost an element of control over what they publish if more stringent norms are put into place. they may also perceive these new norms as constricting what they traditionally viewed as "worthy" of scientific regard. whether true or not, many publications perceive themselves as being apolitical; taking an explicitly agnostic stance towards scientific developments. therefore, norms that have an arguably social and/or political bent could present a fundamental challenge and potentially give rise to an increase in the bifurcation of journals split on social, political, or other lines. there are a host of concerns that must be accounted for in the case that an external body-such as a journal, a norm-enforcement committee, an ethics board, etc.-oversees the release of research findings; one being the importance of equal opportunity and diverse representation in the technological sphere. this standard not only ensures that the domain of technological research remains democratic, but also prevents it from becoming an echo chamber or a site of informational homogeneity. in order to frame best publication practices for high-stake ai-related research, we must ensure that this set of practices is predicated upon a firm anti-oppression mandate, whether this oppression is related to socioeconomic status, race, gender, ethnicity, mental/physical disability, or any other characteristic that incites unjust discrimination. responsible publication norms, by their very nature, must not only strive to mitigate harms related to the exposure of certain research findings, but also to inhibit the epistemic threat of information suppression under the guise of best practices. in order for a novel set of publication practices to be truly responsible in nature, it must be acknowledged that while a regulatory framework for ai research has the potential to reduce threats to public safety, it may also inadvertently serve as a site of gatekeeping, suppression and other forms of exclusionary practice. such a framework therefore risks consolidating epistemic injustice within the academic, technological and political spheres of influence, the prevention of which will require a defined set of safeguarding measures. injustice can be mitigated through the use of radical transparency where the publisher maintains an open list of papers that were rejected and the reasons for rejection in a public repository that is subject to public scrutiny and analysis preventing biases against any groups. to ensure that reactive measures for harm mitigation (i.e. flagging papers post-publication) are not discriminatory in nature or grounded in any form of identity-based prejudice, a more robust set of flagging requirements must be established. in addition to allowing the user to select a general reason for flagging certain content, the process could be made less enticing and/or gameable to those with unfounded reasoning and discriminatory motives by requiring a more exhaustive and substantiated explanation as to why the content should be removed. to this end, making the process involved enough to prevent trolling behaviour can be one proposed approach. in the case of suspected discriminatory practice throughout the publication process, there ought to be an appeal protocol in place whereby subjects of discrimination can formalize a complaint against the relevant publication or platform and negotiate reconsideration of their research by a designated secondary screening committee. such a protocol may include an instrument of a similar nature to that of the checklist or dread-like (damage, reproducibility, exploitability, affected users, discoverability) scoring principles . rather than measuring security threats, this instrument could assess and systematize the long-term personal impacts of discriminatory publication practices whether they be social, professional, or economic in nature. within a novel regulatory framework for responsible release norms, this impact checklist could be appealed to and taken up with a third party entity in instances of publication prejudice; this entity having authority over the reconsideration of initially rejected research, along with the pursuit of certain disciplinary measures if necessary. such an organizational framework could serve to encourage publisher accountability, caution against future discriminatory practices, and ultimately preserve epistemic justice in the domain of ai research. more staged or closed publication practices pose a threat to democratized exposure under the guise of responsible release norms, which may result in the omission of crucial information or gatekeeping. for instance, if research unveils potential risk of a given ai/ml model and there exists a conflict of interest between the publication platform and stakeholders invested in the model, the pretence of "alternative release strategies" could serve as something of a loophole for these stakeholders to suppress certain information and preserve their interests (whether financial, social, etc.) without being held liable for censorship. the malicious application of release norms not only entails self-interested nondisclosure, but also the suppression of projects carried out by actors who occupy marginalized identities, which could theoretically be falsely attributed to the overstated "harms" that their findings may yield, should they be disclosed publicly. such scenarios lend themselves to the risk of epistemic discrimination against marginalized groups. they can also have a chilling effect on the field of research and promote underground attempts at recreating the systems without proper controls in place because of this obfuscation of the real impacts of the system, thus potentially leading to more harm. it's clear that additional restrictions and/or guidelines in the field of ai are required because there are risks that need to be addressed without exception. unfortunately, however, there is no consensus on this in the field. therefore, more individual researchers, institutions, governments, etc. need to clearly and publicly communicate the potential societal impacts of ai research in order to convey the need for third party regulation. in addition, more researchers and institutions supporting and replicating efforts like the neurips impact statement requirement would make such considerations more standardized across the field. forming alliances, making connections, and building relationships are the hallmarks of effective community collaboration. one way this can be enacted is through connecting with a publication standard-setting body, whereby concerned researchers and institutions would be able to lobby for greater action on the topic and witness first-hand how the publication norms are created. for example, a potential partnership with cope could prove fruitful. cope is an organization committed to educating and supporting editors, publishers and those involved in publication ethics with the aim of moving the culture of publishing towards one where ethical practices become a normal part of the publishing culture. so far, it seems they haven't done any work related to the field of ai, but perhaps they would be interested in expanding their work to account for new ethical issues arising in the technology and computer science space. on a larger scale, a solid foundation for this collaboration within the community is the potential for an institution or process that is recognized worldwide and respected in terms of trust around technology. maiei's work on secure and green lighting ml are potential options. these initiatives could provide a standardized comparative measure to evaluate the trust in ai systems across the broad range of topics and themes contained within the community, providing a much-needed lingua franca . as previously mentioned with cope, the field of ai must work towards a new publication culture that prioritizes ethical practices and de-prioritizes progress for the sake of progress. to create safe and responsible ai, the core values and culture of the field must be informed by ethical practices, principles, and guidelines. additionally, it may be important to place a greater emphasis on the application of the technology after it has been deployed, rather than simply the publishing of the information itself. for example, canada has developed national ai standards, which regulate the application of the technology rather than the published content about said technology. this isn't to say that ai publication norms shouldn't be created and followed, but physiognomy's new clothes gaydar" study and the real dangers of big data the invention of ai 'gaydar' could be the start of something much worse . the verge that they should be done in parallel with other efforts necessary to ensure that the science and technology of ai are developed in a more humane manner committee on publication ethics (cope) secure: a social and environmental certificate for ai systems green lighting ml: confidentiality, integrity, and availability of machine learning systems in deployment canada's cio strategy council publishes national ai standards key: cord- -tbrdis authors: norton, alice; bucher, adrian; antonio, emilia; advani, nicole; grund, henrike; mburu, sheila; clegg, emma; boily-larouche, genevieve; lay, a. morgan; carson, gail; tufet bayona, marta title: baseline results of a living systematic review for covid- funded research projects date: - - journal: wellcome open res doi: . /wellcomeopenres. . sha: doc_id: cord_uid: tbrdis background: the coronavirus disease (covid- ) has resulted in an unprecedented research response, demonstrating exceptional examples of rapid research and collaboration. there is however a need for greater coordination, with limited resources and the shifting global nature of the pandemic resulting in a proliferation of research projects underpowered and unable to achieve their aims. methods: the uk collaborative on development research (ukcdr) and global research collaboration for infectious disease preparedness (glopid-r), two funder coordination groups have collaborated to develop a live database of funded research projects across the world relating to covid- . drawing data continually from their members and further global funding bodies, as of (th) july the database contains , projects, funded by funders, taking place across countries. to our knowledge it is one of the most comprehensive databases, covering a wide breadth of research disciplines. the database is aligned to the world health organisation (who) global research roadmap: novel coronavirus. it is being used by the who, governments and multi-lateral policy makers, research funders and researchers. this living systematic review aims to supplement the database by providing an open accessible and frequently updated resource summarising the characteristics of the covid- funded research portfolio. both descriptive and thematic analysis will be presented and updated frequently to aid interpretation of the global covid- funded research portfolio. results: in this baseline analysis we provide the first detailed descriptive analysis of the database and focus our thematic analysis on research gaps, study populations and research locations (with a focus on resource-limited countries). conclusions: this living systematic review will help both funders and researchers to prioritise resources to underfunded areas where there is greatest research need and facilitate further strategic collaboration. researchers and research funders in global health have been preparing for a pandemic such as that caused by severe acute respiratory syndrome coronavirus (sars-cov- ) for decades; however, the urgency and global scale of the research needs and response has been difficult to respond to and coordinate. research funders have rapidly supported repurposing of existing studies and launched rapid funding calls to support research into the most pressing needs. lessons in expediting research have been learnt from undertaking research in the recent north kivu ebola outbreak and west africa ebola, zika and sars epidemics, however the truly global nature of the coronavirus disease (covid- ) pandemic has led to unprecedented needs and challenges for coordination. the world health organisation (who) triggered a rapid response, building on the r&d blueprint , and co-organised the global research and innovation forum: towards a research roadmap for the novel coronavirus meeting with the global research collaboration for infectious disease preparedness (glopid-r) on february - , at which over global experts identified research priorities for covid- . in march , the who released the who coordinated global research roadmap: novel coronavirus (who roadmap) to coordinate and accelerate the global research response against the identified priorities. the who roadmap is an unprecedented galvanizing document for global research collaboration. this project builds on this to help shepherd the global response. in a joint effort to further coordinate and synergise the funding of research to address the who roadmap identified priority areas, the uk collaborative on development research (ukcdr) partnered with glopid-r to launch the covid- research project tracker (the tracker) on april , . the tracker is a live database of funded research projects across the world related to the current covid- pandemic. it includes both newly funded research projects and repurposed research projects across all disciplines and categorises them against the mid-to longterm research and development priorities and sub-priorities identified in the who roadmap. mapping of projects as soon as funding is announced allows visibility of the funded research portfolio well in advance of any outputs such as publications. the ukcdr epidemics preparedness and response funders group and the glopid-r key funders group have each been meeting at least fortnightly during the pandemic to strengthen uk and global covid- research funding coordination activities respectively, informed by the data and analysis from the tracker. several members of both organisations have recently launched calls for research on covid- in low and middle-income country (lmic) settings. there is a particular concern that due to the resource limitations in lmics an uncoordinated approach could potentially lead to unaddressed local research needs, failure of research to inform policy or unsustainable research capacity to respond to future outbreaks. the ukcdr and glopid-r funders groups have agreed to align to further strengthen their response by agreeing to a set of funder principles for supporting high-quality research for the most pressing global needs in epidemics and pandemics and with the formation of a new jointly hosted initiative for covid- research coordination and learning (covid circle), encompassing the tracker and with a particular focus on resource-limited settings . as part of the covid circle initiative, this living systematic review has been established to regularly update and incorporate newly funded research projects as they become available and review their alignment to the who roadmap priorities. a living systematic review (lsr) is needed due to the rapidly expanding number of funded research projects and the importance of the review to inform funding decision making. here we present the results of the baseline review of all research projects within the tracker as of th july and a descriptive and thematic analysis to aid interpretation of the global covid- funded research portfolio. the lsr protocol outlined herein was prospectively designed. due to the rapid need for this project to be conducted to inform research responses during the pandemic, data extraction commenced before the protocol could be formally registered with prospero. the protocol is outlined in this paper. funding bodies have responded rapidly to the covid- pandemic through repurposing existing grants and rapidly funding projects with both rolling and one-off funding calls. this has resulted in new research projects being funded at short intervals necessitating a living systematic review for this work. the regular update of this review will help coordinate ongoing researcher and funder responses. eligibility criteria all research projects funded by any research funder around the world (including regional funding organisations, national research funders and non-profit/ philanthropic organisations), with a focus on covid- were eligible for inclusion in this analysis. this includes data from all types of research activities and was not limited to just biomedical and health research. furthermore, this analysis includes grants identified by funders as having been repurposed to address covid- research priorities. the database and subsequent analysis make use of data from publicly announced covid- research grants and were obtained using one of two methods. the more common of the two methods to obtain data was through direct communication with research funders by requesting the completion of a template spreadsheet (extended data ). these requests were made to ukcdr and glopid-r funder groups members , on a fortnightly basis (as part of funder coordination meetings) and to wider funder contacts beyond these groups. alternatively, data were also obtained from online databases belonging to research funders using "covid" and/or "coronavirus" as search terms (see extended data ). the tracker remains open to the submission of new funding data relating to covid- from any global funder at any time. screening of submitted data occurs on a weekly basis. though the set of data fields varied between funders, the data fields presented in table were considered a priority for the purposes of the tracker and subsequent analyses: update schedule all figures will be updated on a three-monthly basis, when the discussion will also be revised to reflect any changes and trends over time. this living systematic review will continue to be updated for the duration of the covid circle initiative funding. the frequency of screening will not be reduced for the duration of covid circle, although updates will only continue where new grants are included. manually coded data fields data entry of additional manually classified variables was completed by one reviewer with each variable cross-checked by a second reviewer. abstracts not in english (french and german) were coded by project team members fluent in those languages. projects were coded to the following classifications: projects were assigned to one or more who priority areas of primary focus (extended data ). an assignment of 'n/a' was made where: information provided was insufficient for classification; funds were allocated for research administration; or where projects clearly fell outside the who broad priority areas. subsequently, projects were assigned to appropriate who sub-priority area(s). the assignment of 'n/a' was made if insufficient information limited further sub-categorisation or the projects fell outside the who sub-priority areas. in addition, suitable secondary priority area(s) with corresponding subpriority(ies) were determined for those projects that significantly addressed other priority areas. hence, projects were assigned with multiple primary and/or secondary who priority ad subpriority areas of research focus. the priority list will be updated if future iterations of the who roadmap are released. priority classification for those projects that were not considered as addressing any of the who research priorities, they were assigned 'n/a' and new sub-priorities were developed and assigned on an initial data set of projects. an inductive approach was used to develop new codes that emerged from the funded research and themes were confirmed through an iterative process through the projects in this baseline assessment. six new sub-priority codes were defined under the social science priority (mental health; digital health; policy and economics; education; logistics and food security). a new priority focusing on the environmental impacts of covid- , was developed as well. all newly identified categories were validated using the full baseline dataset. research projects involving lmics were additionally assessed for their alignment with their research priorities identified in table . priority data fields for the uk collaborative on development research (ukcdr) and global research collaboration for infectious disease preparedness (glopid-r) tracker and analysis. the latest and previous versions of this table are available as extended data . abstract scientific summary of the project total amount awarded by the responsible funder for the duration of the project (with currency stated) all countries where research is being conducted the names of all funding organisations (including co-funding) the name of the organisation that holds the grant and is leading the research the name of any partner institutions located in the country(-ies) where the study is being conducted name of the awarded project's lead investigator based at the lead institution (primarily used for project de-duplication) project id/reference number any unique reference number / project id assigned by the funder organisation to this project (primarily used for project de-duplication) during the data coding process, a number of cross-cutting themes identified by the project team for analysis. for this baseline assessment, the following additional variables were identified: repurposed projects; capacity strengthening and innovation. to capture information on the type of research taking place in relation to covid- with a stable classification system that is comparable to research on other health topics, the projects included in the tracker were also assessed against the research activity codes outlined by the health research classification system (hrcs) -a classification system developed by uk clinical research collaboration and used by health research funders around the world to classify "the full spectrum of biomedical and health research -from basic to applied -across all areas of health and disease." . while not all of the data in the tracker have been classified against the hrcs research activity codes yet ( of , coded), it is anticipated that all research projects will be classified appropriately with future updates to this analysis. for those projects that have already been coded against the hrcs, each project was assigned with an activity code or 'n/a' either when the research activity fell outside of those listed under the hrcs codes or where there was not enough project information to make an assessment. a study population categorisation structure was proposed using an inductive approach on an initial data set of projects and validated using the full data set, allowing the categories to be specific to the populations represented in the funded research. for the purposes of this analysis, a hierarchical categorisation system was produced to examine the study populations of the research projects included in the tracker. at the highest levels, research projects are assessed on whether they involve animal populations, human populations, literature reviews, policy analysis or only focus on the virus itself. research projects focused on human populations, were classified against three additional sub-categories. table outlines the categories, sub-categories and levels in full. in light of the stated aim of the tracker (and subsequent analyses) to provide an overview of trends in funded covid- research, descriptive and comparative analyses are used in this analysis to present the information included in the version of the tracker updated july th . the data used for this analysis can be obtained from the covid- research project tracker page on ukcdr's website, as mentioned in the data availability statement. data on the tracker (and subsequent analyses) will continue to be updated as more data becomes available and are obtained by the project team. the charts and figures produced in this analysis were produced using microsoft office (office versions of excel and powerpoint) and tableau (version . ) limitations of the data among the main challenges of the analysis is the varying degrees of completeness of data across funders which hindered assignment of projects to broad priority and sub-priority areas where the qualitative details of projects provided were insufficient. therefore, the assigned priority areas may have failed to completely capture all aspects of the projects relevant to the who roadmap. the same can be said for any value that was assigned to a given research project by the project team, including the study population and type of research activity. the data validation process by reviewers with expertise in global health research, policy, and funding outlined in the project selection section was used to address this and ensure that any assigned value was as accurate as possible, given the information provided. as far as funding amounts are concerned, this analysis is limited in providing a holistic picture of trends in covid- research funding data was available from of funders ( . % of all projects). however, as the analysis makes use of all possible information that is publicly available, it can still be considered as being as comprehensive as is possible. at a higher level, the comprehensiveness of the tracker is limited to the funders that have either provided data for the tracker or had their data extracted from online sources (if available). in this respect, there were challenges in engaging with (and obtaining data from) health research funders beyond existing networks either due to a lack of contacts or capacity from funders to contribute to the project (especially for funders whose data is not in english). few funders have yet identified or made available details on grants repurposed towards covid- to date. this lsr of funded covid- research projects uses descriptive and thematic analysis to summarise the scope of funded covid- research projects. no attempts are made to assess the quality of individual studies or whether the studies meet their objectives. the potential sources of bias with project selection, quality of data reviewed, and data extraction and classification are addressed by robust fortnightly searches, template completion by funders and independent assessment and review during project classification respectively, as mentioned in the information sources and search strategy. while the intention of the tracker and subsequent analyses are to provide as comprehensive a picture as possible of the covid- research landscape, the data obtained for the tracker is more likely to be derived from funders of research that are members of ukcdr (all uk and broad disciplinary focus) and/or glopid-r (global membership spanning hics to lics with a biomedical focus). this would likely skew the results to show that more research being funded from these organisations and reflect trends in their respective portfolios (in terms of location, research focus and research activity type) than may necessarily be the case of the landscape more generally. the funders whose data are included in the tracker currently are based in just (high-income) countries. we are anticipating funding data from several ongoing calls by funders based in lmics. in total, , projects were assessed against the eligibility criteria outlined in the methods and were excluded for being duplicate projects or failing to meet the eligibility criteria as they were not related to covid- (prisma flow diagram provided in figure ). the remaining , projects were assigned to the manually coded data fields by nine project team members before being validated by an independent reviewer not involved with the initial screening and assigning process. all reviewers had broad expertise in global health research, policy, and funding. summaries of the characteristics of the , projects included in the latest version of the tracker are provided in the discussion of the results (below) which breaks down the projects by: • funder; • priority and sub-priority areas; • location; • activity type; • study population. a full list of the projects is provided as underlying data . project funder. the , projects included in the latest version of the tracker comprises of data compiled from research funders based in different countries (figure ). of these funders, nearly half of the total number of research projects on the tracker ( . %) were awarded by funders based in the uk, with uk research and innovation (ukri) ranking first among all funders included in the analysis -more than the combined total of the next four highest-ranked funders. such an occurrence can, at least partially, be attributed to the stated data limitations of the tracker (see limitations and challenges) whereby ukri data were relatively more convenient to access and that ukri is one of the first funders to make data available on repurposed grants. of the funders that provided data on known funding amounts, while ukri invested the most funds according to the latest version of the tracker ($ . m without taking co-funding into consideration), average grant amounts were largest under the coalition for epidemic preparedness innovations (cepi) at $ . m. the medical research future fund ($ . m) and projects co-funded by the uk's department for international development (dfid) and wellcome ($ . m) were the only other research funders with average awards exceeding $ m. all projects were categorised against the priorities and sub-priorities identified by the who in their coordinated global research roadmap, with several research projects being assigned multiple priority and/or sub-priority areas. who priority areas. figure displays both the number of projects listed under each priority area and the known funding amounts (as not all funders provided financial information for their awarded research projects). with the novelty of covid- , it is not surprising that the priority area under 'virus: natural history, transmission and diagnostics' ranks first among all nine priority areas in terms of known funding amounts ($ . m ) and second in terms of the number of research projects ( ) as researchers seek to improve their basic biological understanding of this new disease and apply that to diagnosis. while the ranking of most of the priority areas in terms of the number of research projects are relatively consistent with the ranking in terms of known funding amounts, there are three instances where the respective rankings significantly differ: 'infection prevention and control'; 'candidate vaccines r&d'; and 'social sciences in the outbreak response'. these discrepancies are a reflection of the different grant values for different types of research funded in response to the covid- pandemic. to illustrate this point the average value of 'candidate vaccines r&d' projects (a research area typically associated with high financial costs) was $ . m -the largest of any priority area. this amount is more than ten-times the average grant amount awarded under the priority area with the smallest average 'infection prevention and control' ($ k). the two priority areas with the fewest number of research projects and lowest known funding amounts, 'animal and environmental research on the virus origin, and management measures at the human-animal interface' ( projects totalling $ . m) and 'ethics considerations for research' ( projects totalling $ . m) are also the areas with the fewest number of research funders ( each). this contrasts with the rest of the priority areas that each have between and funders investing in such research. looking in greater detail, figure shows how the , covid- research projects included in the latest version of the tracker have been categorised against abbreviated versions of the sub-priorities mentioned in the who roadmap. the names of the sub-priorities are listed in full as extended data . the funding patterns at the who priority-level are reflected at the sub-priority level. notably, of the seven sub-priorities with at least awarded research projects, three are from the 'virus: natural history, transmission and diagnostics' priority and two are from 'social sciences in the outbreak response'. the sub-priority area with the greatest total investment was "identification of candidates for clinical evaluation in relation to 'candidate vaccines r&d'". this priority area also features three of the top four sub-priority areas with the largest average funding amounts. the contrasts, however, in the findings between the priority and sub-priority areas highlight important issues in the distribution of funding for covid- research within priorities. for instance, within the 'clinical characterization and management' priority area, it can also be seen that the overall priority-level investments concentrated on research addressing only half of the six sub-priority areas. in fact, two of the sub-priority areas ('optimal endpoints for clinical trials' and 'develop core clinical outcomes to maximize usability of data across range of trials') rank among the bottom three of all sub-priorities in terms of both number of projects and known funding amounts, but 'optimal endpoints for clinical trials' is the only sub-priority area not addressed by any of the , covid- research projects in the latest version of the tracker. more specific research investment gaps emerge within the two priority areas with the lowest levels of investment. within the priority area 'animal and environmental research on the virus origin, and management measures at the human-animal interface' just three projects (total value $ k) focused on 'socioeconomic and behavioural risk factors for spill-over' and only two projects (total value $ k) address 'risk reduction strategies at the human-animal environment interface'. for the 'ethics considerations for research', a single project (no financial information available) has been funded to address the sub-priority on 'sustained education, access, and capacity building'. while the sub-priority area to 'promote adequate supply of therapeutics showing efficacy' includes just three projects (total value $ k), this is likely to be a sub-priority that will receive further investment once candidate therapeutics are identified. the majority of research projects which did not categorise against the who roadmap are social sciences research that does not align with the sub-priorities in the who roadmap. these are shown by the newly created categories in figure . these highlight important themes for covid- research, which both researchers and funders are prioritising. 'mental health' and 'digital health' are the two most prominent emergent categories which do clearly fall within a health remit and it is notable both that these are receiving research attention, but also that currently this is limited to research from a social science perspective, rather than a clinical perspective. the further newly emergent social sciences related sub-priorities of 'policy and economy', 'education', 'logistics' and 'food security' and newly emergent priority of 'environmental impacts' are all focussed on the broader social and economic impacts of the covid- recovery and reflect the broader covid- research focus of the tracker and remits of the funders whose data are currently incorporated. location of projects. figure summarises the location where research projects are taking place. research is being conducted in countries with the greatest number of projects taking place in the uk ( projects) followed by the united states ( projects) and then canada ( projects). it should also be noted that, of the , research projects, ( . %) take place across multiple countries, with research partnerships between canada and china being the most common ( projects). co-operation and development's (oecd) development assistance committee (dac) list, figure also shows that nearly all research projects ( . %) are taking place, at least in part, in high-income countries. projects are taking place in at least one of the official development assistance (oda) recipient countries identified-of which are taking place in the least developed and low-income countries, in lower-middle-income countries and in upper-middle-income countries. just projects are taking place in only low-or middleincome countries. of these, projects are underway in uganda, followed by burkina faso and south africa . in total, projects are taking place in china, though we are aware there is much more nationally funded research occurring for which we have not yet been able to obtain data. among the projects taking place exclusively in low-and middle-income countries, just under a third ( . %) are being conducted across multiple countries. all research projects in resource-limited settings could be categorised against one or more who research priorities. in addition, several were also categorised against the context-specific research priorities outlined identified by the ukcdr, african academy of sciences (aas) and the global health network (tghn) and are shown in figure and figure . figure shows that some projects mapped to the context specific sub-priorities under the following who priorities: social sciences in the outbreak response; epidemiological studies; virus' natural history, transmission and diagnostics; and candidate vaccines r&d. the predominant theme was research to understand covid- in conflict zones and among refugees and migrant populations whilst research focussed on co-infections and comorbidities such as hiv and tuberculosis and capacity strengthening ranked second. similarly, a few projects mapped to the new broad priority areas with the highest category being the cross-cutting theme involving the use of technology in various aspects of the pandemic response. figure shows those projects mapping to existing who priorities 'requiring greater research emphasis in lmics' here the most projects did map to 'understanding zoonotic leap between humans and animals' showing perhaps unsurprisingly that the few projects in the entire tracker focussing on this were taking place in lmics. in contrast the highlighted priorities of 'health systems research strengthening to mitigate impact of covid- on capacity' and research on 'adherence to trust in public health interventions such as quarantine and social distancing' were lacking. and uk research and innovation (ukri) fund the most projects involving lmics as shown in figure (bubble diagram). further, anrs, cihr and edctp-funded projects are well distributed across the sub-groups of oda-recipients. notably, ukri projects involve comparatively fewer countries since most of their funded projects are concentrated in uganda and gambia (the location of two ukri mrc centres). the national science foundation (nsf) has funded two projects involving china, and the national institutes of health (nih) funded projects span least developed countries. the majority of projects funded by coalition for epidemic preparedness innovations (cepi) are in hics with only in china and india. this likely speaks to the availability of the requisite research capacity in hics for carrying out preclinical and early stages of vaccine research which these projects are primarily concerned with. it is expected that future updates to the tracker will feature more research being conducted in lmics as the outcome of funding calls become known following the increased number of lmic-specific calls launched by research funders. cross-cutting themes. during the review and classification process three additional characteristics of the grants were identified: innovation, repurposed grants and capacity strengthening. figure shows the case of the two cross-cutting themes with the greatest number of projects, namely 'innovation' and 'repurposed grants', these figures were mainly driven by the large presence of ukri data -constituting a significant proportion of the number projects ( . % and . %, respectively). the nih was the second major source of repurposed projects, with many projects on other coronaviruses having been repurposed early in the pandemic. the number of repurposed grants in the tracker is expected to increase as funders start to make this data available. when restricting the analysis to just those coded against cross-cutting themes, the distribution of projects against the who priority areas reveal some notable findings in figure . in the case of 'capacity strengthening', much of these projects fell under the priority area of 'virus: natural history, transmission and diagnostics'( %) -nearly double the priority area with the next highest number of projects ('epidemiological studies' at %). in particular, of these projects, all but two of them have been taking place in, at least, one of african lmics. interestingly, among those repurposed grants, there were two priority areas with significant numbers of projects -namely 'social sciences in the outbreak response' ( . %) and 'virus: natural history, transmission and diagnostics' ( . %) with all other priority areas being assigned with less than % of repurposed projects. the priority areas most common for those coded against 'innovation' were 'infection prevention and control' ( . %) and 'social sciences in the outbreak response' ( . %). to improve our understanding of the type covid- research funded and provide a categorisation that would not change during the course of the pandemic and was comparable to other disease research portfolios, the projects included in the tracker were coded against the research activity codes outlined by the hrcs (figure ). due to covid- being identified relatively recently, most of the funded research projects included in the latest version of the tracker address the more elementary stages of biomedical and health research, specifically underpinning research ( . %), aetiology ( . %) and the prevention of disease and conditions, and promotion of well-being ( . %). while only of , projects have been classified against the hrcs research activity codes, it is anticipated that all research projects will be classified with future updates to this analysis. most research projects included in the latest version of the tracker deal with human populations ( . %) with a significant emphasis on populations that have tested positive for covid- ( . % of research projects studying human populations). more than one in seven projects ( . %) studying humans is focused on vulnerable population groups. figure summarises how the research projects are classified across all levels of the study population categorisation system outlined above. this baseline review of the ukcdr and glopid-r covid- research project tracker has described the huge investment and wide range of research projects repurposed or newly funded related to covid- between january and july , . we are keen for researchers, funders and policy makers to engage with these data directly for their areas of specialism and interest, through extracting the relevant data from the tracker and undertaking their own analyses to aid decision making. given the time demands on all parties in the pandemic, we hope that the regular provision of these descriptive and thematic analyses provide broad insights to help inform the research community and improve the efficiency and effectiveness of the research response going forwards. importantly, here we have aligned the funded research to the mid-long term research and innovation priorities of the who roadmap, and disaggregated the data by locations and population to give a detailed picture of how the research landscape aligns to these global research priorities. there are gaps in the global research funding portfolio, specifically related to the priority areas of 'ethics considerations for research' and 'animals and environmental. we believe that these do represent important and real research gaps, towards which the research community should be shifting its attention. however, it is also important to note the intention and detail of these and all priority areas within the who roadmap, where there is variability in who is best positioned to address the research sub-priorities with some clearly needing external research activity and others indicating research activity which the who planned to undertake directly themselves. the lack of alignment of funded research projects to the 'ethics considerations for research' priority may be one such example, as it misses the direct activity that the who has undertaken in to address this priority through direct research and provision of important guidance on ethical matters relating to covid- which align to the sub-priorities as well as the clear strength of ethical consideration across the majority of research projects (which don't have a core focus on ethics). despite these considerations, both researchers and research funders need to pay greater attention to the prominence placed on ethical considerations for research by the who and ensure that further research is undertaken on those aspects outlined under the roadmap priority area explicitly. for 'animals and environmental research', again the who is currently undertaking direct activity in this regard (through their developing mission with china to identify the specific source and intermediate pathways of transmission for sars cov- into humans ). however, except for a few notable projects in lmics, the instigation of necessary broader research activity in this area, particularly looking towards gaining broader understanding of how such viruses emerge in the human population and proactive surveillance is certainly limited and needs to be expanded and longer term in nature. this research needs to be undertaken in locations where diseases are most likely to emerge, due to the nature of interactions between humans and animals , many of which are lmics and this could therefore play an increasingly important role in the research portfolios in these locations going forward. it is also important that this research activity for this priority expands beyond the remit of the who and through collaborations with the un food and agriculture organisation (fao) and the world organisation for animal health (oie) through a one health framework . beyond the clear gaps at the priority level, it is inherently difficult to conclude when a particular priority area has received sufficient research funding or research projects at the grant award stage, as this is only apparent when the outcomes are achieved and it is clear that the research question has been sufficiently answered. we do however further note, that within some of the better funded who research priority areas, there are still certain sub-priorities which again are clear gaps. this is the case for 'optimal endpoints for clinical trials' and 'develop core clinical outcomes to maximize usability of data across range of trials' within the 'clinical characterisation and management' priority area. this may result from the fact that these activities will be implicit but not explicit in clinical research projects; however, this in itself may indicate a clear issue where both these sub-priorities are essential for collation of results across studies and should therefore be explicit, pointing to the generally observed lack of coordination beyond a few pre-established clinical trial networks as highlighted in discussions at the recent glopid-r synergies meetings . in contrast, the variability of research activity indicated within the 'candidate therapeutics r&d' priority area appears to reflect the inter-dependencies of these sub-priorities rather than necessarily a gap needing immediate funding, with research into 'supply of therapeutics' depending to some extent on the identification of particular safe and effective therapeutics. for those sub-priorities where research investments have been focused there will be benefits to enhanced coordination. we have already highlighted the wide range of social science research projects addressing 'uptake of public health measures' and 'media & communication' sub-priorities to the who covid- social sciences working group. the basic virus research on 'diagnostic products', 'virus compartments shedding and history' and 'charactering immunity' are further areas where coordination should be explored globally due to large funded research portfolios. most of the funded research projects in the tracker are located in hics, reflecting national funding by some of the wealthiest research funders during the first phase of this pandemic, with the truly global nature of the pandemic meaning that virus was circulating in these countries to enable relevant clinical research. a large amount of research has also been funded within china, although as explained in the limitations we have not managed to incorporate this. the global distribution of funding is now expected to start to shift, with a review of the ukcdr and glopid-r funders revealing at least eight open or recently closed funding calls specifically related to lmics. there appears to be a growing recognition that context specific research is needed in lmics , although the results presented here show little funding dedicated to context specific research priorities relating to health systems, trust in public health interventions, optimal personal protective equipment use, health care worker support and community engagement. we will continue to expand our analyses in this area as this is an area of focus for covid circle. the disaggregation of research projects by populations is particularly insightful with regards to the 'social sciences' who roadmap priority, but also for the 'clinical management' and 'epidemiological studies' priority areas. a range of vulnerable populations appear to be well represented for the social sciences including 'minority populations' with recent funding calls in the uk (by ukri and national institute for health research (nihr)) having focussed on researching black, asian and minority ethnic (bame) populations due to the emerging evidence that they are at higher risk from covid- than white people. a range of health care worker populations and other frontline workers are also included in research funded which again is important due to the clear evidence on greater risks of exposure to individuals in certain occupations in this pandemic. children are well represented in the epidemiological studies in accordance with the prioritisation of understanding their role in transmission. given the funded research projects within the tracker relate to disciplines beyond health (with relevance to it is unsurprising that several important emergent research themes identified relating to broader social sciences disciplines (policy and economy; education; logistics and food security) and also environmental research, extend beyond the priorities included in the who roadmap priorities. these all represent important areas for covid- research which funders and researchers are already prioritising with research projects. the two emergent themes of mental health and digital health are however directly relevant to the health research remit and appear to have not been sufficiently covered in the who roadmap document, although projects on these are being funded. we may also be observing the evolution of research priorities from response to recovery and expect to see further examples of this. the expansion of covid- research beyond the original who roadmap document illustrates the wide-reaching social, economic and cultural impacts of the pandemic. a key strength of this tracker is its breadth and we have therefore undertaken some initial cross-cutting thematic analyses across it here to highlight additional variables that cross-cut disciplines with the inclusion of capacity strengthening, innovation and repurposed grants for this baseline review. the analyses on these themes will be given greater focus in future iterations as greater data becomes available on repurposed grants, which we view as crucial in allowing researchers to respond rapidly within an outbreak. in conclusion, we have here provided a detailed baseline review and thematic analysis across the covid- funded research available and we now encourage the research community to use this and the tracker tool to support informed decision making on further research prioritisation going forwards, based on the knowledge of what research is already initiated. we encourage research funders to continue to submit their data to the tracker to ensure it can be as effective as possible. we have focussed our analysis in this baseline paper on highlighting the clear gaps in the portfolio; however, it is also important to note that for those sub-priorities receiving the most funded projects enhanced coordination would also be beneficial and we will expand on our consideration of these areas in future iterations of this review as the numbers of research projects expand. we have also shown here the power of tracking research funding at source in real-time, which is particularly important in the fast-moving research environment created by a pandemic, but may have benefits for other global collaborative research efforts going forward. the emerging issue within this pandemic of nationally funded projects resulting in underpowered studies not achieving their aims, means that researchers and funders need to be much more strategic going forwards to efficiently and effectively advance knowledge within epidemics and pandemics. to the best of our knowledge we have compiled the most comprehensive database of funded covid- research. we are however very mindful of its inherent limitations and the difficulties in gaining a fully comprehensive picture in what is a truly global research effort to a global pandemic. one main limitation is the absence of commercial research data making inferences on gaps in the vaccine and therapeutics portfolios difficult (this is lacking due to associated intellectual property restrictions). this tracker however has rich data on the early stage development research for those same priorities which is valuable for public funder coordination efforts and enables thematic analyses across disciplines. another limitation is the fact that few funders to date have shared data on repurposed grants or grants for institutional funding which may have been used for covid- related research. we are also aware of several funders across wider geographies and disciplines, from whom we have not yet been able to incorporate data. we call here for further research funders (especially within lmics) to submit their data to make this tracker and associated analyses more accurate to improve the ongoing coordination and help focus limited resources. the alignment of research in this tracker to the priorities outlined in the who roadmap also has its challenges, given the roadmap was produced at speed by drawing together findings from different working groups operating in different ways. the resulting priorities are unsurprisingly imbalanced with some covering much broader research areas than others and with not all sub-priorities intended to be addressed by newly funded research. we have tried to account for this in the discussion of the results here. another limitation of these priorities and indeed any priorities in a pandemic is their limited temporal nature. the who roadmap priorities that we have mapped here, although named mid-to long-term priorities, were identified by world experts in february , at a time when the majority of cases of covid- were still in china and a pandemic had not yet been declared. we will therefore be updating our mapping as soon as an anticipated new version of the who roadmap is released as research priorities have evolved with increasing knowledge. sustainability and future work this living systematic review will be updated on a quarterly basis for the duration of the covid circle initiative. future planned work includes incorporation of any new priorities or sub-priorities from the anticipated revision of the who roadmap (following the st - nd july meeting). given the tracker contains a broad range of research relating to covid- (beyond health research) and the evolution towards longer term thinking around research priorities, we are also discussing incorporating coding to the un covid recovery research roadmap and are in discussions with the team developing this. future iterations will focus in more depth on highlighting areas for potential collaboration in those parts of the portfolio with many funded projects and will identify any trends in funding over time. this project contains the following extended data: © smith p. this is an open access peer review report distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. - this assembly of the database on which this "systematic review" is based clearly represents a major effort. the database is likely to be most useful to funders, who will wish to avoid supporting research that is unnecessarily duplicative and it will also be of value to researchers seeking to identify others who are working in a similar area or to identify research areas that seem to have been relatively neglected. the authors have gathered data on research supported by funders, mostly based in high income countries. how complete this is, even for these funders is difficult to judge, but based on the projects supported by each funder, there seems to be a strong bias towards uk funders. it is surprising, for example, to see the us nih having supported less than % of the number supported by ukri. a notable deficiency in the database, acknowledged by the authors, is the lack of data from china, where there has been substantial research on sars-cov- , and lmics in general (e.g. brazil, india). however, it is understandable that there are challenges in accessing comprehensive information from these sources. another challenge is that most, but not all, of the information presented relates to the number of projects supported rather than the magnitude of the support. the data base is also missing information from private companies, including pharma companies, where much research on vaccines, therapeutics and diagnostics is likely to be supported. it is also unclear whether the databases of registered clinical trials have been trawled to identify ongoing research. for these reasons, i am not convinced that it is appropriate to label the paper as a systematic review as it seems to be a review only of what has been accumulated in the database to date. i recognize that the database is early in its life, but as an ongoing check on completeness it may be useful, in the future, to link published papers on sars-cov- to the database to see what is not being picked up in the database, acknowledging that it is not infrequently difficult to link a particular paper to a particular research grant. in the review, projects in the database are linked to who blueprint list of research priorities defined in february . this helps identify areas where there are possible funding gaps. however, there are some limitation in using the data base for this purpose. for example, the authors note that there are clear gaps in support for 'optimal endpoints for clinical trials and core clinical outcomes' and on 'ethical considerations for research'. both of these areas have had considerable attention, the former in the context of treatment and vaccine trials (the authors acknowledge the problems in identifying research that may be embedded in other research undertakings) and much of the ethical work that has been done has not required specific funding. despite some of the deficiencies identified above, the generation of the database has been a valuable undertaking, and as more funders come on board it will become even more useful. similarly, the ongoing systematic review will give a useful summary of what is in the database and will help identifying trends in funding and areas where support has been lacking. are sufficient details of the methods and analysis provided to allow replication by others? yes are the conclusions drawn adequately supported by the results presented in the review? yes world health organization: a research and development blueprint for action to prevent epidemics -plan of action reference source covid- research project tracker reference source united kingdom collaborative on development research (ukcdr): epidemics preparedness and response group ukcdr reference source pubmed abstract | publisher full text | free full text african academy of sciences: research and development goals for covid- in africa the african academy of sciences priority setting exercise the remaining unknowns: a mixed methods study of the current and global health research priorities for covid- reference source . ethics and covid- . world health organisation reference source . world health organisation media briefing on covid- . th reference source pubmed abstract . operational framework for strengthening human, animal and environmental public health systems at their interface glopid-r covid- research synergies meetings: meeting summaries and recordings. reference source uk office for national statistics: which occupations have the highest potential exposure to the coronavirus (covid- )? reference source baseline results of a living systematic review for covid- clinical trial registrations publisher full text replication data for baseline results of a living systematic review for covid- funded research projects. harvard dataverse extended data for baseline results of a living systematic review for covid- funded research projects. figshare. figure we would like to thank all funders who have provided data to the tracker to date. we thank laura scott and alice cross from ukcdr for their support to the authors in coding on the tracker. we also thank the infectious diseases data observatory (iddo) for inspiring us to undertake this analysis as a living systematic review, through their living systematic review of covid- clinical trial registrations . no competing interests were disclosed. reviewer report september https://doi.org/ . /wellcomeopenres. .r © vaughn d. this is an open access peer review report distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. bill and melinda gates foundation, seattle, wa, usa this is a well-written report by the ukcdr and glopid-r on their living systematic review (project tracker) of covid- r&d including projects supported by funders across countries as of july aligning said r&d efforts with the who global research roadmap for covid- r&d published in march . the tracker also identifies research objectives not included in the who roadmap (seven new areas) that may influence planning for future pandemics (including future who roadmaps). a spin-off of the effort (covid circle) focuses on r&d in resource-limited settings.the tracker allows the global health community to assess the covid- research portfolio to identify gaps (animal and environmental research? clinical trial endpoints and outcomes?) and to reduce duplication (more than vaccines being advanced?); reduction of excessive duplication is important given limited global r&d resources.the authors are transparent concerning the limitations of the effort. the data is skewed to glopid-r members. it is heavily ukri biased. the us nih, in particular, seems under-represented. repurposed funded research may be missed. there is limited data from a number of governments (e.g., china as noted by authors) and private sources (e.g., companies, private foundations such as the bmgf, and ethics think tanks). this publication may encourage more lmics to participate in the accounting.is it possible to comment on measured or perceived impacts? who uses? is there documentation of times accessed? is it timely information if the figures are updated only every three months? the group should conduct a survey at the end of the calendar year to see if funders changed direction based upon this effort. has there been a reduction of duplication? or, will this be a retrospective of what happened with little real-time impact on what was happening? shifting of funding to lmics is one positive example of impact. again, we will need a later assessment to build upon this baseline review focusing on impact on the pandemic response. has the scope been too broad? are the conclusions drawn adequately supported by the results presented in the review? yes competing interests: no competing interests were disclosed. i confirm that i have read this submission and believe that i have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. key: cord- -xcpv k authors: bresalier, michael title: uses of a pandemic: forging the identities of influenza and virus research in interwar britain date: - - journal: soc hist med doi: . /shm/hkr sha: doc_id: cord_uid: xcpv k this paper counters the tendency to retrospectively viralise the – pandemic and to gloss the important historiographical point that, in britain, such knowledge was in-the-making between and . it traces the genesis of influenza's virus identity to british efforts in – to specify the cause of the pandemic and it examines how, in the s, the british medical research council used the connection between a virus and the pandemic to justify the development of virus research and to make influenza a core problem around which it was organised. it shows that the organisation of medical virus research was inextricably linked to the pandemic before the actual discovery of flu virus in . recognising that the relationship between the virus and the disease itself has a history demands we rethink the pandemic's medical scientific legacy and the crucial role of virus research in shaping its history. in may , walter morley fletcher, secretary of the medical research council (mrc), organized a secret meeting of pathologists at the war office to outline a new scheme of research on 'diseases probably caused by filter-passing organisms.' created in , the mrc had used the war to apply laboratory science to military medicine. in peace time it was seeking new challenges and the still relatively unexplored filter-passing organisms offered just such opportunities, for medical science and the health of the nation. an immediate reason for the mrc's interest was the - influenza pandemic. in his annual report for - , fletcher stressed that investigations during the pandemic of the purported connection between a filter-passer and influenza were a key motivation for the new scheme. there could hardly be a set of problems whose solution has more potential importance for the community than this. influenza kills regularly, though its slaughter is chiefly effected during epidemics. in a few months in - it killed more persons in india than died from the plague there during the previous twenty years …. the pandemic had ignited interest in the nature of filterable viruses, however, the way forward was unclear, as fletcher observed: 'the chief problem which the investigator of [filterable viruses] meets is the difficulty of proceeding by sound experimental methods.' the purported influenza agent was one of a group of pathogens that could not be seen with light microscopes or studied by the culture methods that had been so successful with bacteria. only their pathogenic effects were evident in animal models and could be traced by serological tests. the mrc's new scheme was to build on these methods to make viruses into workable laboratory objects and to develop new methods for their control. this paper traces how, in the decade after , the pandemic and virus research became inextricably linked in the work supported by the mrc. fletcher and his colleagues became convinced that if questions about influenza's virus identity-and more general questions about virus diseases-were to be successfully investigated new researchers and institutions would have to be created. the plan charged the council's flagship laboratory, the national institute for medical research (nimr), with, among its primary goals, developing research on human and animal virus diseases and linking virus researchers to medical and veterinary institutions. work on the influenza filter-passer was pivotal. british medical science played an important role in the battle against the pandemic, but it was also shaped by it. donald fisher has argued that the pandemic represented a 'central turning point' in efforts to modernize british medicine: 'the death of millions of persons as a result of influenza made the advancement of medical knowledge and practice urgent.' according to fisher, the pandemic prompted the british state to take a new role in medical scientific administration and education, primarily through the mrc. the mrc's collaboration with the rockefeller foundation to reform british medical education was an important legacy. but the pandemic reached into the very organization of medical scientific research. the mrc's virus research scheme clearly demonstrates this point. fletcher regularly invoked the idea that a filterable virus might have caused the greatest pandemic since the black death to bolster support for the scheme. more than a symbolic resource, the identities of the pandemic and virus research were forged together as part of broader mrc plans to scientifically modernise pathology. experience during the war and the pandemic convinced fletcher and his colleagues that pathology needed to be founded on experimental principles, and located above all in university science departments and research institutions. the nimr emerged as one of the key institutional supports for this new pathology, and virus research became closely allied to its development. my argument takes up roger cooter's recent challenge to historicise the relationship between war and epidemics. cooter claims, rightly i believe, that medical historians have taken this relationship as self-evident. this is true of much work on the - pandemic, which has retrospectively linked influenza's emergence and virulence to specific war conditions. such approaches are important but fail to highlight how this relationship was understood and used by contemporary actors, particularly those involved in generating medical knowledge. my analysis critically examines ways in which the epidemic-and-war couplet was constructed through laboratory practices and their institutional organisation during and after the pandemic. in britain the mobilisation of the military medical machine against the pandemic bound the disease to the war. especially important were military pathology investigations, supported by the war office and mrc, into the bacteriology of the pandemic and, in due course, into the role of a 'filterable virus'. these investigations not only connected the pandemic to a filterable virus; they also connected virus research to the wartime organisation of pathology. both were used by the mrc as a rationale for developing virus research in the s. by tracing this process, my account counters the ahistorical tendency to retrospectively viralise the pandemic. ever since a virus was established as influenza's primary cause in , virologists and historians have used it to explain many aspects of the pandemic. such histories tend to gloss the important historiographical point that such knowledge was in-the-making between and , so that there has been a tendency to ignore the historicity of the relationship between influenza, the pandemic and virus research. to properly situate the making and uses of medical knowledge of the pandemic it is important recognize that this relationship has a history that goes right back to the pandemic itself. few histories of the pandemic explore its contemporary microbiology. historians now know that it struck britain in three distinctive waves. a relatively mild epidemic beginning in early spring was quickly followed by a lethal autumn epidemic after which developed a somewhat less virulent epidemic in spring . an estimated , died, with most succumbing in autumn . while each wave presented its own clinical and epidemiological characteristics, retrospective accounts have linked them together into a single cataclysmic pandemic. at the time, however, the identities and relationship between the waves baffled medical experts. the summer epidemic shared characteristics with previous visitations, particularly that of - , with doctors describing extreme body aches, prostration, fever, sore throat, dry cough, nausea and general lassitude in most patients. yet important aspects did not fit the established picture. much confusion stemmed from uncertainties about its aetiology. between early spring and late autumn , a loose bacteriological consensus built before the war fragmented, with competing pathologists and physicians backing different pathogens. resolving the aetiological problem was imperative to official pandemic strategies. the war office, army medical service, and mrc prioritised identifying the suspected influenza germ and developing preventive measures-particularly, vaccines-to control it. these strategies were initially based on the assumption that the germ was bacillus influenzae, an organism first identified in by the berlin bacteriologists, richard pfeiffer and bresalier . burnet and clark ; morens and taubenberger . tognotti . langford , johnson and mueller shibashuro kitasato. pfeiffer developed special methods for its cultivation, which, he found, required a substrate of blood-in particular, haemaglobulin. he promoted the bacillus as the primary cause of influenza and his blood-agar culture technique as necessary for establishing it. identification of the germ became widely accepted as the best way to distinguish 'true influenza' from other influenza-like conditions. in britain, pfeiffer's claims were first substantiated in by e. e. klein, a founder of british bacteriology, in investigations for the medical department of the local government board (lgb). pfeiffer's techniques were slowly incorporated into bacteriological practice and 'pfeiffer's bacillus' gained standing as the 'germ of influenza'. both became part of the general organization of pathology for war. british approaches to the pandemic were elaborated within the contexts of military medicine, and defined by military imperatives. like all facets of british society, medicine had been mobilised for total war. over half the medical profession was eventually enlisted, along with many hospitals. medical science was also mobilised and the mrc played a vital role in its coordination. most important for the subsequent fight against influenza, fletcher worked closely with william boog leishman, advisor on pathology to the war office, to link pathological laboratories to base and field hospitals in france and flanders. organised to collect, identify, and control pathogens, military pathology delivered therapeutic and preventive measures against a range of battlefield diseases, and its planners trusted that it could do the same with influenza. signs of the spring wave were first encountered in military garrisons in france and flanders in march . the epidemic perplexed medical authorities. it occurred in late spring instead of autumn. rather than the usual susceptible groups-the very young, aged, and infirm-it affected soldiers in the prime of life. few typical symptoms were evident. but most importantly, when pathologists ran bacteriological tests they rarely found b. influenzae. so seldom were pathologists' efforts successful that many concluded that b. influenzae was at best associated with, but not essential to, the epidemic. bacteriological evidence did not support classifying the epidemic as influenza. rather, it generated controversy. through the summer of , two camps of pathologists clashed over the causal agent: the 'pfeiffer school' argued that the epidemic was influenza, and attributed failures to find b. influenzae to technical failures; the 'anti-pfeiffer school' argued that its absence indicated either that the epidemic was not influenza or that influenza was caused by another organism. they proposed a range of known bacteria, including streptococci, staphylococci, and streptodipplococci. but no one could agree which played the primary role. coles , pp. - . prüll . bresalier . prüll . leishman . tanner abrahams et al. ; french . ludwik rajchman, editor of the mrc's medical supplement, used the term 'pfeiffer school' those who believed that 'true influenza epidemics' were caused only by b. influenzae, 'influenza', medical supplement, october , p. . fildes and , used the term 'anti-pfeiffer school' to describe its critics. this situation jeopardised official prevention strategies. without an agreed agent against which to develop a specific vaccine, army medical authorities decided to produce a 'mixed' vaccine, which incorporated bacteria associated with secondary complications, including b. influenzae. generally viewed as unsatisfactory, the decision added official backing to doubts about pfeiffer's bacillus. these doubts also prompted leishman and his colleagues to organise research into other agents. fletcher argued that, 'on the hypothesis that b. influenzae, no less than pneumococci and streptococci, are secondary [infections]', official strategy would be better served by exploring the possible role a so-called 'filter-passing virus.' in november , the mrc and the war office put in train a 'search for an unrecognized virus'. little was known about the basic nature of filter-passing viruses. the category only emerged at the turn of the century, when it became popular among some bacteriologists as a way to explain diseases for which causes could not be ascertained by standard bacteriological methods. filter-passing viruses were operationally defined by the fact that they were neither retained by standard bacteriological filters, nor susceptible to cultivation on artificial media, nor visible by available methods of light microscopy. a pathogen was characterised as filterable when clinical material passed through the smallest of available filters still induced disease in a host. the new category included a number of important human and animal diseases: foot-and-mouth, fowl pox, rabies, swine fever, measles and poliomyelitis. the first filterable virus theory of influenza was proposed in by the german bacteriologist, wilhelm kruse, in studies of the common cold. british interest grew in as a possible explanation for bacteriological failures. between october and december , the mrc helped s. l. cummins, advisor in pathology to the ams, organise filterpasser studies in abbeville, flanders and etaples, france. major howard graeme gibson led the abbeville team. with major f. b. bowman and captain j. i. connor, gibson's team followed the work of two french bacteriologists, charles nicolle and charles lebailly, who claimed in early that they had discovered a filter-passer. as proof, nicolle and lebailly reported that they had reproduced the disease in healthy monkeys and men by serial inoculation of filtered bronchial secretions from infected cases. seeking to repeat these experiments, gibson's team relied on the mrc for expertise and materials, including macaque monkeys and baboons shipped from london zoo. within weeks, gibson reported that his team had isolated a filterable agent and produced an experimental disease. in december , they described how they had reproduced a characteristic lung haemorrhage in monkeys, similar to that seen in clinical cases, 'the utilisation of vaccine for the prevention and treatment of influenza', lancet, october , p. . matthews horder . na, fd / fletcher to fildes and october ; na, fd / mrc, influenza general research, , november na, fd / fletcher to fildes, october . ibid. van helvoort . rivers , pp. - . rivers , p. . waterson and wilkinson . kruse . mrc gibson et al. . cummins a . thomson and thomson , p gibson et al. , pp. - . and isolated and cultured minute 'coccoid bodies' from the tissue. since the agent passed through filters and produced 'experimental influenzal' lesions, they reckoned that it was a 'filterable virus' and 'in all probability the cause of influenza' (figure ). tragically, gibson died from influenza, leaving the team's work unfinished, but not before pathologists at etaples, headed by major-general john rose bradford and captain james wilson, claimed to have isolated similar coccoid bodies (figures and ) . although preliminary, the studies won support from the lancet and the bmj. cummins argued that the 'two series of observations, carried out independently, should confirm each other, [and] greatly strengthen the case for the new organism.' f. w. andrewes, director of the department of pathology at st bart's, who had assisted on klein's investigations of pfeiffer's bacillus, pointed to gibson's experiments as providing the best evidence for the primary role of a filterable virus. these endorsements did not allay criticisms. in , j. a. arkwright, then working with the war office on trench fever, levelled a devastating critique of the etaples research. arkwright demonstrated that the coccoid bodies were identical to those found in uninoculated tubes and that the cultures were contaminated with ordinary bacteria. in effect, the bodies were not pathogens, but either benign globoids or bacteria. this analysis forced rose bradford and wilson to reconsider, and in a stunning move, they publicly retracted their claims. bradford et al. , pp. - . gibson et al. , p. . andrewes , pp. - . arkwright , pp. - . arkwright , pp. - . arkwright's criticisms coincided with renewed support for pfeiffer's bacillus. in , paul fildes and james mcintosh, two leading london pathologists, published new evidence for its primary role and launched their own attack on the virus theory. former colleagues at the london hospital's department of bacteriology, they shared the view that the bacillus was the agent of the pandemic, and that new techniques made it possible to demonstrate its role. mcintosh wrote: [t]he epidemic can be divided into two stages, a first in which b. influenzae was seldom demonstrated, and a second, in which this bacillus was demonstrated with great regularity. this fact is not attributable to any alteration in the epidemic itself, but to the application of new methods for the demonstration of the bacillus of influenza. most important among these techniques were 'selective media', which inhibited the overgrowth of cultures by other microorganisms. fildes and mcintosh argued that selective culture enabled them to establish a causal link between the bacillus and influenza. first, they could regularly identify the bacillus from large numbers of cases. second, they could isolate it from broncho-pneumonia lesions clinically associated with the disease. finally, they could use pure cultures to reproduce the disease in experimental animals. for fildes and mcintosh, the evidence generated with selective media was enough to counter the 'great revolt against the view … that b. influenzae … was the cause of the disease.' building on arkwright's criticisms, they argued that filter-passing work was 'unconvincing'. gibson's research was symptomatic of its shortcomings. neither the identity of the virus nor its relationship to influenza had been demonstrated. moreover, inoculation experiments on humans-of the kind used by nicolle and lebailly-were easily discredited fig. etaples filter-passer films. in films the organism had 'the appearance of a minute, rounded, or slightly oval, undifferentiated coccus-like body, arranged in colonies of twenty to sixty elements'. source: bradford, bashford and wilson b, p. . fildes and mcintosh , p. . fildes and mcintosh , p. . . fildes and mcintosh , p. . because, as other observers noted, researchers either failed to isolate subjects from previous infections or simply produced a different illness. the alleged filter-passer thus failed to meet any of koch's postulates. most damningly, they reckoned that researchers used the filter-passer as an alibi: 'the invisible virus concept absolves the discoverers from the necessity of producing evidence of a characteristic microbe.' these criticisms cast doubt on whether influenza's cause could ever be determined. in a review, the bacteriologist robert donaldson observed that there were no good empirical grounds to support either agent. while he agreed with criticisms of the filterable virus, he found little to support pfeiffer's bacillus. while frequently found in post-mortem studies, most inoculation experiments failed to produce a characteristic lesion in laboratory animals. this strongly suggested that it was a secondary infection. donaldson challenged the idea that a causal inference could be drawn from its apparently high incidence during the autumn epidemic. 'we are not at liberty to claim, that because an organism is always present, it is therefore necessarily the cause.' proponents of pfeiffer's bacillus had mistaken an association for a cause. some critics took the conflicting aetiological claims as indication of the failure of bacteriologically-based approaches. andrew mendelshon has shown that a contingent of british physicians and epidemiologists used these conflicts to attack reductive approaches to disease causation and to promote multifactorial models to examine the role of hereditary and environmental factors in variations in the severity, incidence and susceptibility to infectious diseases. but while such models found support in epidemiology, pubic health and clinical medicine, the mrc countered them. contrary to critics, official pandemic strategies were widely lauded. while war conditions may have contributed to the virulence of the epidemic, british mortality, in both the services and amongst civilians, was lower than in other countries. fletcher insisted that the rapid organisation of coordinated strategies was testimony to the merits of military pathology. translating military pathology's success into peace time became an mrc goal, and both the pandemic and virus research were enrolled in this mission. the pandemic highlighted the need for a new direction in laboratory pathology, towards filterable viruses. at the same time, virus research represented a way to preserve a specific aetiology as the guiding principle of pathological research and to counter challenges to the authority of laboratory medicine. mrc interest in virus research was intimately tied to its larger goal of modernising the organisation of basic sciences in medical training, practice and research. for fletcher, the pandemic demonstrated the particular need to modernise pathology. this was not the old pathology of the mortuary, rather the microbiology-inspired enterprise, previously fostered in germany and france, that linked aetiologies with patho-physiology and immunology, and focused preventive and therapeutic measures on them. while the maitland et al. . fildes and mcintosh , p. . donaldson , pp. - . maitland and cameron , p. ; maitland et al. . donaldson , p. . crookshank hamer . mendelsohn . johnson . austoker , pp. - . mrc war confirmed its importance, fletcher attributed the failure to control the pandemic to a general lack of institutional support for basic pathological research. he feared that peace time would mark a return to a pre-war pathology, defined by its subordination to clinical and public health interests. in his first annual report after the war, he characterised british pathology as an anathema to experimental science. unlike physiology, which had generated researchers, discoveries, and university departments of international importance, pathology lacked an experimental orientation and a place in universities. in hospitals, medical schools, and public health, its practitioners played a service role, generating income but little new research and few trained researchers. for fletcher, the pandemic underscored the need for an experimental approach to pathology located in dedicated laboratories, funded by the state, and coordinated by experts. this vision found support in the new british journal of experimental pathology. established in by a group of mrc researchers, led by fildes, the journal emphasized the multidisciplinary nature of experimental pathology and its role as a foundation for rational medical knowledge and practice. the bjep defined itself as providing an alternative to descriptive research in the morbid anatomy and epidemiology of infectious disease. it aimed to publish 'original communications describing the techniques and results of experimental research into the causation, diagnosis and cure of disease in man'. no one discipline defined this approach. for its founders, experimental pathology joined together 'bacteriological, biochemical, pharmacological, physiological, serological and other subjects' in the production of new pathological knowledge. multidisciplinary in character, it was bound together by the core principle of specific aetiology. fletcher built this broader vision into his campaign to create new pathology departments at oxford and cambridge. but it was at the nimr that it was most completely realised. the idea of the institute was born with the creation of the mrc in . only after the war was its position consolidated as the flagship for medical science. occupying mount vernon hospital in hampstead in north london, built between and and purchased by the mrc in , originally it was to be modelled on the rockefeller institute for medical research in new york (rimr) (figure ) . the mrc incorporated important aspects of the rockefeller approach-particularly the positioning of experimental sciences as foundational to medical education and to the production of medical knowledge and therapeutics-and depended on its patronage. yet the mrc research system was also adapted to the british context, where clinical medicine remained dominant. as in germany, the mrc made the state an active agent of change and used its own authority and institutions to pursue its agendas. the nimr was crucial to the mrc mission. an independent government institution, with no formal affiliation with hospitals, its primary function was to foster experimental medicine, with experimental pathology one of its cornerstones. experimental pathology lawrence , p. . mrc kohler , pp. - . alter . the first editorial board included fildes, mcintosh, j. a. murray and w. e. gye. fildes , p. i. ibid. weatherall , p. - . austoker , p. ; fisher a fisher , b wilkinson ; wilkinson and hardy . austoker and bryder , pp. -- ; thomson , pp. - . came under the remit of the department of bacteriology, headed by s. r. douglas. it shared the institute's broader organisational ideology of team work, which prioritised research that straddled disciplinary boundaries. douglas and henry hallet dale, the institute's acting director, decided that experimental pathology would be primarily constructed around filterable viruses. the need for a dedicated virus scheme became readily apparent in the first years after the pandemic, when fresh investigations into the identity of the filter-passer in influenza highlighted the underdeveloped state of the field. in , the mrc started funding new influenza 'virus' work by mervyn h. gordon at st barts department of pathology. an enthusiastic researcher, gordon had worked in the department since . early on, he was drawn to testing the idea that filterable agents might be causes of high-profile infectious diseases for which bacterial agents could not be found. in , he confirmed the claim made by the viennese pathologists karl landsteiner and erwin popper, that a filter-passer played a primary role in poliomyelitis. in , he announced that he had successfully isolated a filter-passer from cases of mumps. when he returned to filter-passer work after the war, he and andrewes decided to focus on influenza. a member of the mrc, andrewes became convinced during the pandemic that a filterable virus held the keys to its aetiology. an impetus for gordon's research was a widely reported discovery by two american pathologists, peter k. olitsky and frederick l. gates, of an alleged 'new' filter-passer from cases of influenza. working at the rimr in new york, olitsky and gates first identified the agent during the pandemic. in may they reported a new technique to produce and serially transmit a 'definite and characteristic' infection in rabbits. like human influenza, the experimental infection was localized in rabbits' lungs, from where they isolated an agent. to establish its filterability, they passed solutions of ground lung material through grades of berkefeld filters, and then used the filtrate to reproduce the infection in healthy rabbits. they also employed a special method for source: wellcome library. thomson , p. . gordon gordon - . gordon . andrewes , pp. - . olitsky and gates . cultivating the agent, developed in by their colleague, the japanese-born bacteriologist, hideyo noguchi. the so-called 'noguchi' medium was composed of fresh rabbit liver tissue, set in narrow glass tubes, sealed with wax and vaseline. rockefeller researchers had used it with the common cold virus, as had british researchers at etaples in their influenza work. according to olitsky and gates, the medium enabled them to make pure cultures of the agent, to photograph it, and to study its properties. in a further fourteen papers published between and , they detailed its morphological, pathogenic, and serological characteristics. they concluded that it was a minute organism, with particular affinity for the lungs, and accordingly named it, bacterium pneumosintes-'a bacterium that injures the lung' (figure ). the american researchers's animal model and culture system appeared to solve the two fundamental methodological problems that had hampered filter-passer studies in . yet, the reception of their research among supporters of the filter-passer theory was mixed. gordon, for one, believed they had identified an agent similar to that identified by british workers in , and had only 'added precision' to these original studies. he tested their claims during an epidemic in late december . using the noguchi method, he ran bacteriological tests on nasal and throat washings of staff at st barts hospital and the ministry of health. in early january he reported to landsborough thomson, assistant secretary to the mrc, that 'something very like [olitsky and gates'] filter passer is coming up in my cultures.' the agent also looked like the one identified by british workers. but gordon admitted that, 'rendering the filterable organism was difficult,' because its presence was only indicated by a rather vague 'cloudiness near the piece of kidney at the foot of the [noguchi] tube.' it became visible when gordon made films of material that had been fixed, stained, and chemically differentiated. these procedures yielded 'swarms of minute round bodies', but they were so small that they could be 'very easily missed unless especially looked for', and might be dismissed 'by an inexperienced fig. olitsky and gates' bacterium pneumosintes. two stained cultures from a rabbit's lung into which were infected nasal secretions from a case of 'epidemic influenza'. on the left, the culture was magnified × . on the right, it was magnified × . infectivity was tested by inoculation into the lungs of rabbit. source: olitsky and gates a, p. . noguchi . olitsky and gates b, p. . gordon a, p. . gordon a, pp. , . na, fd / gordon to thomson, january ; gordon to fletcher, february . observer'. unable to induce an experimental disease, gordon used ultraviolet photomicrographs of the culture and slide preparations to compare their morphology with those described by the british and americans ( figure ). it was unclear if the agent was a 'coccoid' or a 'baccilloid', and he was unable to induce disease in experimental animals. moreover, when he consulted existing literature on filter-passers, he was struck by their similarity with bodies found in other diseases. these problems raised rather than settled questions about the filter-passer's identity. the most vexing centred on its nature and classification. gordon and his american counterparts reckoned that it was a filterable organism. its 'prodigious multiplication' convinced them that it was living and not, as earlier critics had claimed, a protein. this aligned it with the dominant view of filterable viruses in medical and veterinary pathology: their ability to multiply, demonstrated by pathogenesis, was evidence of their biological nature. yet this was not the only theory available for explaining filterable viruses; an important alternative approached them as chemicals. this theory had various roots, but in british pathology it was most influenced by f. w. twort's characterization of the bacteriophage as a chemically-induced lytic phenomenon, a concept he introduced in . h. m. woodcock, head of the department of protozoology at the lister institute, enlisted twort's concept to challenge gordon. woodcock accepted the existence of a pathogenic 'virus', but not that it was living. he argued that those who viewed influenza virus as a microorganism had no direct method for distinguishing it from protein particles. gordon's microphotographs did not support this conclusion. quite the contrary, on their staining properties gordon a, p. . gordon b, p. . creager , pp. , - . twort . chick et al. and physical appearance in microphotographs, they looked more like typical 'protein enzymes', the result of the breakdown of cell material by a ferment. if these bodies were indeed the cause of influenza, woodcock argued, they were not exogenous organisms but by-products of an endogenous chemical process occurring within human cells. these debates spilled into a general debate about viruses and virus diseases, and the experimental methods used to elucidate them. through the early s, the mrc organised several discussions on the state of virus research. the many research challenges were brought to the fore in july at a special panel on the 'bacteriology of influenza' at the annual meeting of the british medical association. concentrating on gordon's work, discussants probed his filter-passer. james mcintosh recapitulated the view that all studies had failed to establish its pathogenic identity. sympathetic researchers observed that neither adequate methods nor criteria were available for determining its nature. charles ledingham, head of bacteriology at the lister, argued that the greatest difficulty remained 'the lack of any animal, other than man, [that] was readily susceptible to the causal agent'. poor filtration methods were identified as yet another obstacle. j. h. dible, junior lecturer in pathology at manchester university, noted that filtration was 'extremely crude'; bacterial filters were 'not reliable' and the criteria for deeming an agent 'filterable' depended on an inexact calculation of the relationship between the largest hole in the filter and the smallest bit of protoplasm being filtered; much of the process was left to chance. f. w. twort stressed the need for standard methods of filtration. w. b. leishman, who chaired the meeting, concluded that, the status of the filter-passer remained a 'big unsolved problem'. the bma meeting made one thing clear: too little was known about the general category of viruses to determine the identity of one. '[the] search for the primary infective agent in influenza,' agreed gordon, 'has led us into the realm of filter-passers', a realm rife with new problems. supporters of the filter-passer theory viewed these problems as features of an emerging field, the boundaries of which urgently needed to be defined. preferring the bench to the office desk, he embraced the opportunity. the nimr programme was moulded around two lines of work. the first employed physical and biochemical methods to create new instruments and techniques for exploring the creager , pp. - . 'bacteriology of influenza ', lancet, , pp. - . gordon a , p. . mrc , p. . na, fd / , june dale , p. . cameron fundamental nature of viruses. its main locus was the department's division of applied optics, created in for j. e. barnard, a west-end hatter and amateur microscopist, who built the first ultra-violet microscope in britain. bernard worked with william gye on the role of a filterable virus in cancer sarcomas, and explored physical problems associated with rendering viruses visible and with filtration methods for purifying and determining their size. the second line of work concentrated on creating pathological and 'immunological devices' for the investigation, identification and control of virus diseases. familiar to bacteriologists, these techniques included new media and experimental animals for growing viruses, and serological assays, therapeutic sera, and vaccines for typing and controlling them. both research lines were closely entwined. but it was the applied aspects of the programme that linked it to medical and public health concerns with influenza and other disease with unknown aetiologies. the public and medical profile of influenza remained high throughout the s. prior to the pandemic it had been viewed as an inescapable part of modern life; but after it was viewed as a major threat. no one knew if, or when, it would again become a deadly pandemic. influenza typically ranked highest amongst cases reported by general practitioners and amongst patients' complaints. there was greater awareness of the disease, with , , and designated epidemic years. among infectious diseases, only diphtheria and scarlet fever accounted for higher annual morbidity. although influenza rarely killed on its own, 'influenzal pneumonia' accounted for stunning levels of mortality killing on average nearly ten times more people than diphtheria or measles. resolving influenza's aetiology remained a paramount problem. the mrc considered including it on the nimr programme. but it was deemed unsuitable for basic work because of its aetiological complexity and the lack of a viable experimental animal. rather, it would be studied indirectly through a model disease. when plans were settled in june , three 'virus' diseases were chosen: measles, chicken sarcoma-a model for human cancer-and dog distemper, which served as the model for influenza. according to fletcher, distemper's apparent analogies made it 'peculiarly suitable for working out methods by which human diseases of this class might be subsequently investigated.' distemper addressed a broad range of mrc interests. as fletcher highlighted in his annual report for - , its relevance as a potential model for influenza was most important: there is good reason to think that [dog distemper] offers a close parallel to human influenza. it seems probable that the infective agent is a filterable virus, and that here also the severity of the resulting disease depends largely upon secondary infections, facilitated by the primary infection. there is ground for hope that the study of dog's distemper under strict experimental conditions may throw important light austoker and bryder ; thomson , pp. - . austoker , pp. - . mrc , p. . honigsbaum bresalier , ch. . digby , pp. , . deutschman , p. . ministry of health , p. . na, fd / , may upon analogous problems of human disease, and at least suggest new clues for investigation or new technical methods for the investigator. it is with the primary object of gaining knowledge of human disease that the council decided to support further study of distemper in dogs. on that ground alone they find complete justification of the expenditure of part of their funds in this direction. concerned that the focus on distemper might be seen as being at odds with the nimr's mandate to work on human diseases, fletcher framed it as good for exploring influenza and general problems associated with virus infection and immunity. important analogies also existed between the state of research on distemper and on influenza. veterinary pathologists were divided on whether a filterable virus or a bacillus caused distemper. its virus aetiology had been first proposed in , but then widely disputed by researchers who aligned themselves behind b. bronchisepticus, identified in . crucially, neither side had access to dogs bred under controlled conditions, so results could always be contested. without accurate means to isolate and test either agent, there was little hope for a vaccine. the prospect of resolving these problems drew together british virus researchers, veterinarians and dog owners. in late , as the nimr was assembling its programme, the country magazine, the field, and the veterinary journal launched a dog distemper fund, with the aim of raising £ , for new research. in early , sir theodore cook, the field's editor and honorary secretary of the fund approached fletcher about collaborating on 'research into the causation, prevention, and treatment of distemper'. while a government body, funded through the national health insurance system, the mrc actively sought out patrons for its schemes. its close collaborations with wealthy benefactors, the rockefeller foundation and dunn trust, are well known. but its collaboration with the distemper fund involved a different form of patronage, one that depended on a large and varied group rather than a mighty patron. the fund relied on voluntary contributions from hunt packs, kennels, breeders, associations and middle class dog owners from across britain and the empire, and from the united states. support went well beyond monetary contributions, as many made their own dogs available as research subjects. managing these different interests required a novel research organisation. a distemper research council was created to oversee fund-raising and publicity, and an expert distemper research committee was created to coordinate scientific work at the nimr. all participants shared the belief that experimental research was the best way to develop effective control technologies against distemper. the nimr's department of bacteriology and experimental pathology was made the fulcrum and laidlaw made dog distemper his main research object. mrc , pp. - . mrc na, fd / fletcher to c.j. martin, october laidlaw and dunkin , pp. - ; laidlaw and dunkin a, pp. - . laidlaw and dunkin a, pp. - . worth nearly £ , . in . the fund eventually raised over £ , (£ , , ). for details, see na, fd / , 'saving the living of our dogs ', the field, february , pp. i-xiii. mrc , p. . kohler lawrence . na, fd / , the cure and causes of distemper, november . between and laidlaw worked with the resident veterinary pathologist, g. w. dunkin, on all aspects of the disease. the single most important obstacle was access to purpose-bred dogs. the need for controlled supplies of experimental animals was a general problem for the institute. with the support of the fund, the mrc built a large-scale, animal-breeding and research facility at mill hill, an agricultural site north of hampstead. completed in , the 'farm laboratories' made provision for breeding and housing dogs and other large animals, a well-equipped laboratory, and an isolation compound for quarantining distempered dogs. the facility put laidlaw and dunkin in a unique position to carry out distemper studies under controlled conditions and to settle the dispute over the causative agent. by , they had ruled out b. bronchisepticus and had established a strong foundation for a filterable virus. the telling piece of evidence came when they used bacteria-free filtrates to reproduce an experimental infection in their dogs. this enabled them to determine the pathogenesis and role of the agent. its filterability partially confirmed its identity, but equally important was its resistance to cultivation. the result was in line with the emerging view that failure to grow filterable viruses in artificial culture was evidence of their unique dependence on living tissue. while the nature of this dependence was not well understood, it was increasingly taken as a defining property. laidlaw and dunkin could thus legitimately claim that, 'from these three [factors] it follows that the infecting agent of dog-distemper belongs to the class of filter-passing viruses.' once they settled the aetiology, they turned their attention to a vaccine. but dogs were not ideal for tackling the problem. dog experimentation was the target of vociferous antivivisection campaigns. moreover, the animals were ill-suited for the work. in particular, distemper varied considerably in dogs, making it difficult to diagnose. searching for a solution, laidlaw and dunkin found that the ferret was highly susceptible to distemper, and easily reproduced and identified in the animal. ferrets were also easier to manage. they bred readily and quickly. unlike the dog, they were known to thrive in small spaces, which made them well suited to confinement in laboratory cages. they were ideal research animals. by , laidlaw and dunkin developed and tested experimental vaccines for ferrets and dogs. their key innovation was a two-step immunisation process. it involved first administering a killed virus, waiting - days until the animal developed sufficient antibodies, and then injecting it with live virus. following successful trials, in the distemper council arranged for its commercial production by burroughs wellcome & co in britain and two american companies in the united states. the vaccine became widely available in . kirk . mrc dunkin a, p. . laidlaw and dunkin . dunkin and laidlaw . van helvoort, . dunkin , p. . tansey . dunkin and laidlaw , p. . na, fd / third report of the distemper research committee-'ferrets ', october , thomson , pp. - . laidlaw laidlaw and dunkin , pp. - . laidlaw and dunkin , pp. - ; b, p. ; a, pp. - ; b, pp. - . laidlaw and dunkin a, p. . the american houses were lederle laboratories, in new york, and murphy laboratories in philadelphia. the distemper campaign was hailed as an enormous success. dale described it as an exemplar of 'a complete and systematic investigation of a virus disease', and its culmination of a vaccine for the nation's dog owners made it symbolise the efficacy of the nimr's approach to virus research. a number of aspects ensured that it had wide-reaching significance. it established a style of virus research that linked together fundamental and applied research in ways that broke down that distinction. the innovation and commercial manufacture of a vaccine demonstrated the practical relevance of virus research. from a professional standpoint, the campaign was crucial to legitimising virus research. when laidlaw and his colleagues started their work in , the challenges of rendering viruses by established bacteriological techniques of filtration, in vitro cultivation and light microscopy were well known. not only did these challenges spur technical innovations, they also gave rise to new ways of thinking, the most important of which was the concept of viruses as obligate parasites-entities dependent on living tissue for their multiplication. while this concept had been proposed as early as , as researchers ran up against limitations of culture techniques in the s, it grew into a basic framework. investigating viruses in experimental animals became a necessary condition of medical and veterinary virus research. work at the nimr became organised around this biological concept of viruses and the experimental approach it demanded. the distemper campaign proved that the approach was immensely productive of both expert knowledge and practical tools for tackling virus diseases. when laidlaw and dunkin handed over responsibility for their distemper vaccine to burroughs wellcome in , virus research was becoming an established medical scientific field. through distemper, nimr workers fashioned their scientific identities as virus researchers and the authority of their institute. laidlaw was knighted for his distemper work in . virus research was funded in universities and hospitals, and the lister had created its own programme. in , the mrc devoted an entire volume of its system of bacteriology in relation to medicine to 'viruses and virus diseases', and dog distemper occupied a key place in the category. the mrc had always justified distemper research in terms of its potential applications to influenza. fletcher returned to the theme in , writing that, it is already clear that the usefulness of this work is not to be limited to the prevention and cure of canine distemper. in the field of medical research the work has at many points aided the development of technical methods for the study of viruses in general. by the end of the campaign, the mrc had rallied much support for its convinction that virus research could conquer influenza. through the s, medical and public concern about influenza's toll grew. a times editorial in observed that, at more or less regular intervals, influenza breaks out and marches across the world, claiming millions of victims and causing grievous dislocation of human enterprise. immense sums of money are spent on sickness benefits and on the care of the sick, and heavy losses are incurred by the majority of industrial undertakings; while numberless men and women lose their health permanently and become dependent on others. the following year, the same paper bemoaned '[t]he sad state of unpreparedness in which the world finds itself ought to awaken determination to discover, if possible, some means of prevention.' yet, hope was at hand. the editorial went on to note that an effective approach had been demonstrated with dog distemper: is it too much to ask that work on similar lines be undertaken on the cause of influenza? the work on distemper has opened a way; general studies organized by the medical research council on virus diseases have made parts, at any rate, of that way smooth. has not the time arrived to launch a campaign and to come to grips with the enemy? these comments suggest that the mrc's strategy of linking influenza to distemper research had been successful in redefining the disease as a problem for virus research. fletcher's rhetoric, now echoed in the times and other general press, raised expectations that tools created to control distemper could be applied to influenza. in early , sir halley stewart, an important mrc patron, offered fletcher £ , to launch an 'influenza campaign'. linkage to the distemper campaign was both symbolic and pragmatic. the campaign's success legitimised the nimr approach to virus diseases. its primary goal had become virus identification and control through the production of serological assays, therapeutic sera and, ultimately, vaccines. new interest in applying this approach to influenza was ignited in , when the american veterinary pathologist, richard e. shope announced that a combination of haemophilus bacillus (suis) and a filterable virus produced a disease in pigs-'hog flu'-that was analogous to human influenza. shope's work prompted speculation that a similar type of infection might be the cause of human influenza and re-opened the possibility of finding an animal model for the disease. in late , the mrc decided to concentrate its influenza efforts at the nimr, under the control of laidlaw. within months, the decision paid off. in early , laidlaw and two young researchers, c. h. andrewes and wilson smith, succeeded in using the ferret to isolate a filter-passer from patients in london, including smith himself, and they rapidly identified the agent they called 'w.s.' virus as the primary cause. while shope's work provided an incentive for the research, its organisation, material foundations, personnel and general reception were directly shaped by the distemper campaign. by end of the s, influenza's virus identity had become part of medical and public health knowledge, and the nimr collaborated with researchers around world in developing diagnostic techniques and vaccines. the nimr's success came so quickly only because so much had been put in place in the previous decade. influenza's virus identity is now taken for granted. i have shown that the link between the virus and the disease has many histories. its genesis needs to be traced not to its 'discovery' in but to the battlefields of the first world war, to military pathology, and to debates over influenza's aetiology during and after the - pandemic. most crucial to this process was the strategic use of the pandemic by the mrc to justify construction of virus research, which created the material conditions for laidlaw and his team to establish influenza as a virus disease. historians of virology conventionally suggest that medical and veterinary virus work of the kind pursued at the nimr was bound to a 'bacteriological paradigm', which acted as an obstacle to the development of a modern biochemical virus concept. my account challenges this view. contrary to the notion that nimr virus research was defined by bacteriology, i have shown that viruses and virus diseases were construed as complex research problems requiring a multidisciplinary and collaborative approach. virus workers were committed to the principle of specific aetiology and bacteriology provided an ontological understanding of viruses and methods for tackling them. but the nimr framed virus work as a form of 'experimental pathology', and nimr reseachers, and their colleagues, identified themselves as 'experimental pathologists' rather than as bacteriologists. this distinction took on important meaning as it became evident that viruses resisted bacteriological methods. work done at the nimr, and elsewhere through the s, slowly yielded a concept of viruses as obligate parasites, which set viruses apart from bacteria and forced workers to develop specific skills and knowledge, and new scientific identities. my account of the nimr programme supports angela creager's observation that, rather than being bound by one disciplinary framework, interwar virus work is better understood in terms of its dependence on the interchange of bacteriological, pathological, physical and biological practices. as the distemper case shows, this collaborative enterprise extended beyond the laboratory walls, involving relations with other professionals and lay constituencies. the mrc's ability to rally different groups to the cause of virus research was tied to how it mobilised the experience of the - pandemic. my analysis challenges the standard historiographical view that the pandemic had little lasting impact on medical or social institutions. the mrc's virus scheme shows that the pandemic played key roles in the creation of a research system that became an emblem of scientific modernity. rather than leading to the abandonment of laboratory-based pathology, failure to master the pandemic spurred the mrc to improve and expand it. the mrc's ability to mobilize the pandemic points to the existence of a broad consensus about the threat of influenza. van helvoort . laidlaw rivers . creager . johnson the story told here suggests we rethink how we write the history of the pandemic. rather than use virology as an explanatory resource, we need to examine its construction as a multidisciplinary field and how influenza-and other diseases-figured into this process. my analysis of the mrc's virus programme underscores the need for accounts of how different actors and institutions used the pandemic to pursue different agendas. historians have acknowledged government support for the mrc during the pandemic but not how the mrc subsequently mobilised the experience in its post-war plans. in this context, virus research emerged as a new domain for tackling infectious diseases and a vehicle for constructing a new experimental pathology. ignoring this dimension of virus research means we ignore its crucial role in shaping the history and 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global public health: the rockefeller foundation, the london school of hygiene and tropical medicine, and the "hookworm connection prevention and cure: the london school of hygiene & tropical medicine: a th century quest for global public health many thanks to michael worboys, carsten timmerman, john pickstone and colleagues at chstm for valuable comments and suggestions. national archives, kew, surrey. key: cord- - hbqgdrb authors: Øvretveit, john title: implementation researchers can improve the responses of services to the covid- pandemic date: - - journal: nan doi: . / sha: doc_id: cord_uid: hbqgdrb this article describes a rapid implementation research project with the stockholm health care system to assist the system to respond to the covid- pandemic. it uses this example to illustrate some ways in which implementation research and knowledge can contribute to improving service responses to the pandemic and its consequences as these evolve over the coming months. a sub-specialty of rapid implementation science is proposed to provide practical assistance and as one way to develop implementation research. plain language abstract: this article describes a rapid implementation research project with the stockholm health care system to assist the system to respond to the covid- pandemic. it uses this example to illustrate some ways in which implementation research and knowledge can contribute to improving service responses to the pandemic and its consequences as these evolve over the coming months. a sub-specialty of rapid implementation science is proposed to provide practical assistance and as one way to develop implementation research. when a pandemic arrives, service delivery systems make many changes. some are evidence based, many are not. managers and front-line practitioners implement and modify the changes. can implementation scientists help them make the changes more quickly and effectively? what can researchers offer in the next months? for many services, changes in service delivery are directed from above and the details of implementation are left to the service. their aim is rapid implementation using fast and effective ways to make the changes. research into implementation is low priority for managers and front-line practitioners. they do not usually seek help from researchers, for various reasons. in the case presented here, we, the researchers, had a long running partnership with the service delivery system. we were assertive in "selling" implementation science to management. our experience was that we could use implementation methods and research to help our health system's response to the pandemic. but also, that we found limitations to the science that we and others can now address. the purpose of this article is to share our experience and encourage colleagues to contribute to improving the responses of our services as the pandemic and its consequences evolve over the coming months. a sub-specialty of rapid implementation science is proposed for providing practical assistance and developing implementation research. the article describes the emergency response implementation project that we undertook. we concentrate on the challenges we faced as implementation researchers and our resolutions. findings from the first months of the project are summarized, and the interim report will be available in september (Øvretveit et al., ) . stockholm healthcare about . million people live in stockholm. most hospital and many community health services are publicly provided and owned by the stockholm regional government. about % of primary care services are private and there are a few private hospitals. many health-related services are provided or purchased by the municipalities in the region, including many services for older people, such as private home care visiting services and residential care homes. stockholm is unique in that all the public non-acute health care services are owned by the region and managed within the "stockholm healthcare" organization (stockholms läns sjukvårdsområde [slso], ). this primary and community health care organization includes a diverse range of service delivery units and medical home care services, including primary care centers, services for psychiatry, rehabilitation, addiction, and specialist "hospital at home" and palliative care. the public acute care hospitals are managed by the regional government, separately to the slso. region stockholm government activated a full emergency management system for the first time on february , , in response to the rising number of our residents infected with sars-cov- . the first covid- death was reported on march , the same day that the world health organization (who) declared a global pandemic. although we had little information about the best prevention and treatment of the disease, our contacts with italian colleagues showed that we could be overwhelmed, not least because the swedish government did not institute a "lock-down" as did other european countries (our world in data, b ). yet covid- mortality per capita each day between march and july was almost the same as the united kingdom, which did have a strict "lock-down." what explains this? how did our health services respond? what did we learn about implementation? the author works % time as a research and development officer for the slso primary and community health care organization, and % time as a professor of implementation, improvement, and evaluation at the nearby karolinska institutet, a medical university; there are m between the slso and the medical university offices. with a background in these fields, i saw a role for applied research and to help implement the changes being made. although retrospective implementation studies are needed, i was not willing to stand on the side-lines as my clinical colleagues prepared for the expected increase in infections and illness. my plan for rapid implementation research was welcomed by the managing director of the slso primary and community service delivery organization, and he and i submitted an ethics application that was fast tracked and approved in days. the research started in the last weeks of march and will continue until next year. this partnership and action research approach gave us access to information and interviews that would not have otherwise been available as this historic series of events unfolded. challenge : defining which research is useful, but feasible, given the resources the first step was to define more precisely the scope and objectives following the "start with the end in mind" principle. the karolinska medical management center approach to applied research views practical and scientific contribution as mutually reinforcing and of equal importance. our methods are designed to deliver both, although there are sometimes challenges in balancing the different time perspectives. one objective of the covid- rapid implementation response project is to contribute to implementation science. the other is to provide timely researchinformed assistance and reports to the emergency and operational management teams of slso. our most valuable resources were our health system digital data bases and the national data bases, as well as our established -year working relationships with health system staff. our other resources for the period described here from march to july were seven "volunteer" researchers. all had other full-time work and added this work and paused other tasks where possible. this team was a joint academic/health system team, so several of us, including myself, were employees of the health system. as principal investigator, one of my leadership challenges was to maintain the enthusiasm for the project, honor the high ambitions of the team, and recognize all the different possibilities for the study, but limit the data and analysis to what was feasible and useful. i did this by summarizing the options raised in the team discussions and then itemizing the hours and data we had available and the target dates for reports to management. how did we focus the research and decide which data to collect, given these resources, time frames, and assets? given the aims of providing implementation researchinformed assistance to the management team and contributing to the science, we found two sets of ideas useful for focusing the research. the first was a version of the learning or improvement science testing iteration cycle. rapid learning experimental cycle. we found a generic rapid learning experimental cycle (rlec) useful for our study, and our service providers found it useful to track and improve their changes. this generic method has five steps: ( ) define problem; ( ) decide data indicating problem solved; ( ) design and implement solution; ( ) review data and revise solution; and ( ) repeat as situation changes. we used this " d- r" model to guide our documentation and assessment of the adaptations made to the intervention and to the implementation actions of the different changes that staff made to their practices, service organization and facilities, and to support systems. the difference between this model and the more well-known plan-do-study-act (pdsa; see leis & shojania, , for an updated narrative review) quality improvement cycle is the emphasis on the first two steps of defining the problem and deciding the data that would indicate it as solved. implementation science concepts. we found the simple distinction between intervention and implementation useful both to define the project and to continually remind ourselves where we could most add value with our knowledge about implementation. our practitioner colleagues assumed "intervention" meant only a new treatment or diagnostic method. we found the term, "the new better way (perhaps)," useful for identifying and communicating the many types of changes that were being made, and quickly. these changes included new ways to practice telemedicine, infection control practices, physical changes to buildings and people flows, as well as changes to the content of it systems. the word "perhaps" provided a reminder that some changes were not supported by strong evidence or were adapted to the service and population from proven versions tested elsewhere. the terminology made it easier to separate actions or methods used to establish the "new better way" into everyday operations-the implementation actions. methods used to help people take up the new better way in everyday work typically included training, practicing the training (e.g., for the many steps to don and doff protective personal equipment [ppe]), feedback on performance, and highlighting incentives to perform the new better way. these concepts also highlighted that many different changes were being made at the same time. should we choose a sample of primary care centers and document and evaluate how they implemented changes? or should we document and assess how all the service within the slso organization implemented different changes? which of these or other changes should we concentrate on, given our limited resources and the objectives of the research? we chose to focus on the implementation of the emergency management system (the intervention): what was done to implement the new emergency management groups and establish their working with the routine operations management system (implementation actions). the idea was that, if emergency management needed to be re-established later, we would have documented how it was implemented for the first time, and what worked well and less well. this could help in august to prepare for the winter. in addition, there is limited research into implementing new management structures and systems, and even less knowledge about implementing emergency management systems. implementation science has found that contextual influences help and hinder implementation of the intervention and its performance (mccormack et al., ; Øvretveit et al., ) . also, research suggests that some context influences are specific to certain interventions: for example, for a computer application intervention, the type of hardware and computer system and privacy rules are important contexts helping or hindering the implementation and operation of the computer application intervention. other contextual influences are important for implementing practices to reduce the number of falls experienced by older people in a care home. the focus on the emergency management system as the intervention helped us to theorize which contextual influences would help and hinder implementing and operating the system. the emergency response highlighted that the context mostly influences the implementation actions. then, over the longer term, context has a greater effect on sustainment or continual operation of the intervention. the consolidated framework for implementation research website (https://cfirguide.org/) provided ideas and guidance that help to specify the inner and outer contextual influences that we needed to consider. what then would be the outcomes of a successful or unsuccessful implementation of an emergency management system? we found an implementation outcomes model useful to differentiate different outcomes of the emergency management system for the primary and community health services (proctor et al., ) . the immediate outcomes of implementation are whether and how quickly changes to work practices, organization, and support systems are accomplished and sustained. we found useful reminders of the many different implementation actions that could be used from the compilation of implementation strategies (powell et al., ) and the list of behavior changes (michie et al., ) . these implementation science concepts helped us to outline a logic model of context, implementation, and outcomes that helped us to decide which data to collect about the emergency management system for the slso primary and community health services. we then developed a data collection strategy that started on march , , with our first weekly survey of all our service delivery managers ( units). illustrations of how the implementation science concepts helped us structure questions are as follows: • • context, which includes the regional government requirements of the slso organization to establish emergency management and to coordinate all other community services, and the hindering context of inadequate and timely data about service utilization and prognosis of likely demand on services, as well as changes to patient portals to enable easier use of smart phones for telemedicine; • • implementation actions to establish emergency management, which included formal instructions to service delivery unit managers to make certain changes, formulating guidance to service delivery unit managers and clinical staff (e.g., when to refer patients to acute hospital), establishing a special purchasing and supply center with volunteers to obtain and distribute ppe; • • implementation outcomes of the implementation of emergency management included whether managers reported receiving clear instructions and guidance as well as sufficient ppe, as evidenced in the weekly surveys to the managers. which data to collect. our resources, the project focus, and the concepts described above helped us to concentrate on using already collected data and to ensure low burden data collection from the weekly survey and interviews by avoiding collecting data not essential for achieving the project objectives. one set of data was the weekly survey reports from heads of service delivery units. this included what helped and hindered implementing the changes they needed to make, as well as questions about implementing actions to maintain staff health. we used the internal secure health system intranet to ask questions about context and outcomes of the changes, using nine multiplechoice questions with free text at the end of each and taking min to complete. response rates varied from % to % for weeks analyzed so far. interviews were carried out with all members of the emergency management team and included questions about preparations for the winter and how to establish emergency management and systems more quickly and effectively in the future. other data sources were documents recording the twice daily emergency management team meetings and the one daily operations management team meeting, as well as emergency plans and other documents. many implementation science projects collect data to quantify implementation outcomes. in this rapid implementation study, we needed to collect data for this purpose, but also to provide reports to the management team to enable them to track the care provided and predict demand on the units. we found the national data about these subjects was not sufficiently up to date and was inconsistent with our local data. we established a system for combining various information sources to provide frequent reports. these data included hospital admissions sourced from hospital data systems, intensive care unit occupancy, and infection rates in older peoples' homes. to estimate predicted demand, we worked with one local hospital center that was using the penn university open-source modeling system and were able to compare the projections from this model over time with actual data from all the hospitals that we worked with and adjust the model (weissman et al., ) . we found limitations to implementation science in methods available researchers to use in partnership with health care services for rapid implementation research of the type we sought to carry out. drawing on quality improvement project work, we found our version of iterative testing using the rlec was useful. we also discovered the value of a project team that combined university researchers and health care system staff with a principal investigator employed % time with both organizations. one area to develop implementation science is through reporting and assessing different rapid implementation research methods and structures: others may have responded like we did and developed other methods and structures and we would hope this and other implementation science journals could report these. implementation outcomes: infection and mortality?. the focus of this article was on implementation of emergency management and our research continues. one question we are investigating is the possible impact of effective emergency management on covid- mortality rates. as with most implementation research, there are attribution challenges in estimating how much implementation actions influenced implementation outcomes and then later health outcomes. the article noted earlier that sweden was unusual in not instituting a "lock down," as shown in the covid- government response stringency index (our world in data, b). did sweden's "life as normal, with precautions" result in high infection and excess mortality compared to other european countries? evidence shows covid- deaths per million as significantly higher than other nordic countries, but consistently equal to the united kingdom from march to july (our world in data, a) . at present, the explanations of why covid- deaths were not considerably higher in sweden are speculative. it is possible infection rates were comparable, but an effective primary and community health care response with e-health and other support for self-care at home with monitoring and fast transfer to hospital when necessary may have reduced further infection and mortality. one hypothesis we are investigating is whether what appears to be an effective implementation of emergency management in primary and community care may have prevented higher mortality. there is evidence that it did prevent our hospitals from being overwhelmed. one finding from our study was to highlight again the importance of a systems understanding: what happens in hospitals depends on what happens in primary and community health services. this was starkly demonstrated by the high infection and death rates in long-term care homes. there are simple evidence-based methods to prevent this (cdc, ). the failure is in implementation. the ethical and economic imperatives are for a "zero infection in longterm care homes" program. implementation research traditionally studied how empirically tested changes to practice, organization, and policy have been taken up in different health and welfare services. more recently, this research field has studied how changes proven elsewhere have been adapted for different situations. the covid- pandemic raised the question of whether knowledge within implementation science could help with implementing, planning, or evaluating the many changes made during in response to the pandemic. our rapid implementation research with region stockholm health care system revealed useful concepts and methods from implementation science for designing a study and assisting the response. it also showed limitations in the science for understanding and assisting an emergency response and in the methods within implementation science for rapid research into proven and unproven changes in a rapidly changing situation. one of the aims of this article is to invite discussion in different forums about how implementation science can contribute new and useful knowledge and practice for emergency responses, and especially for the evolving crises that our services are experiencing in the next months. one view is that we are best equipped to carry out systematic research, using existing methods and concepts which may mean retrospective research. another view is that rapid implementation research is needed with an implement-ology of emergency response and a sub-specialty of rapid implementation research. both may be required. the author, john Øvretveit, is the named principal investigator in the ethics application and acknowledges the contributions of the following colleagues to the project: mats brommels, associate karolinska institute, mmc, vibeke sparring, senior administrator, research and development, slso, clara hellner, professor karolinska institute and director of research and development, slso, mikael ohrling, director slso, karin solberg carlsson, karolinska institute, mmc, hakan uvhagen, development officer slso, and editors cara lewis and sonja schoenwald. the author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: the author and some research team members were employed by the stockholm region government and by the karolinska institutet medical university and conducted the research in addition to their other funded projects and work assignments. one research member was retired and not employed by either organization. because the author and some of the researchers were dually employed by the university and the stockholm region government, there is a potential conflict of interest and potential for bias in their investigation of how their employers implemented emergency management. this commentary offers the author's perspectives on the project and process but does not present findings, and procedures to manage potential bias will be described in a forthcoming research report. the author(s) received no financial support for the research, authorship, and/or publication of this article. nursing homes and assisted living, long-term care facilities: infection prevention tools a primer on pdsa: executing plan-do-study-act cycles in practice, not just in name getting evidence into practice: the meaning of "context the behavior change technique taxonomy (v ) of hierarchically clustered techniques: building an international consensus for the reporting of behavior change interventions confirmed covid- deaths per million covid- government response stringency index implementation of emergency management in stockholm healthcare: report to management team how does context affect interventions to improve patient safety? an assessment of evidence from studies of five patient safety practices and proposals for research a refined compilation of implementation strategies: results from the expert recommendations for implementing change (eric) project outcomes for implementation research: conceptual distinctions, measurement challenges, and research agenda. administration in policy and mental health stockholms läns sjukvårdsområde (slso) health care services stockholm county locally informed simulation to predict hospital capacity needs during the covid- pandemic key: cord- - k kf authors: al-janabi, hareth; coles, jenny; copping, john; dhanji, nishit; mcloughlin, carol; murphy, jacky; nicholls, jean title: patient and public involvement (ppi) in health economics methodology research: reflections and recommendations date: - - journal: patient doi: . /s - - - sha: doc_id: cord_uid: k kf patient and public involvement (ppi) can be used in methods research, as well as applied research, in health economics. however, methods research goals may seem quite abstract when compared to the lived experiences of lay participants. this article draws on years of ppi in a research project to develop methods for including family carer outcomes in economic evaluation. key challenges in using ppi for health economics methods research relate to ( ) training and preparation, ( ) maintaining involvement, and ( ) selecting suitable tasks. we suggest three criteria for selecting a research task for ppi input based on task importance, professional researcher skills gap, and potential ppi contribution. health economics research is beginning to benefit from patient and public involvement (ppi) [ ] [ ] [ ] . this research can be 'methodological' or 'applied' in nature, and both forms of research raise important challenges in using ppi. in this article, we reflect on ppi input alongside a health economics project to develop methods to include informal (family) carer outcomes in economic evaluation. the article starts with a brief overview of ppi and methods research in health economics. we then describe our research project and experience of using ppi. drawing on our experience, the remainder of the article discusses issues in ppi in health economics methods research. ppi has grown substantially over the last decades and is increasingly required for health research in the uk [ ] and elsewhere [ , ] . it can be used in various stages of the life cycle of a research project from prioritising research questions through to impact evaluation [ ] . ppi has been used in various ways in health economics, including in support of priority setting [ ] , setting the scope of costs and outcomes [ ] , selecting health states for valuation [ ] , and identifying approaches to collecting cost and outcome data [ ] . it is important to distinguish ppi from qualitative research (an important parallel development in health economics [ ] ) in the sense that ppi is research done in partnership with the public, rather than using the public as participants. ppi covers 'the experience of patients, service users and carers [as] a fundamental and valued source of knowledge' [ ] . various benefits of using ppi in health economics have been noted, including covering gaps in knowledge, embedding the voice of the public in research, ensuring tasks are presented in an appropriate format, and providing an economic explanatory framework [ ] [ ] [ ] . methods research in health economics potentially covers a wide terrain, but a key focus of health economics methods research is improving the techniques of economic evaluation. this includes, for example, the development and testing of new outcome measures [ , ] , the development of costing tools [ ] , devising theoretical or practical frameworks for economic evaluation [ ] , and methods advances, such as in decision modelling [ ] , that are 'nested' in applied studies. good methods research is crucial to ensure costs and outcomes are accurately measured, synthesised, and modelled; economic analyses are comprehensive and reflect society's values; and innovation and scientific rigour are promoted in economic evaluation research. two important features differentiate methods research from applied research in health economics. first, the intended impact is different. methods research seeks to develop techniques for application across the discipline, and therefore the intended impact is improved science conducted by the research community. conversely, applied health economics research focuses on directly informing how health and social care services can be efficiently and equitably delivered. thus the intended impacts are on the provision of services by care professionals for members of the public. while both methods and applied research can ultimately impact on the delivery of care, the impact of methods research is indirect, and therefore the research goals may be more abstract from the public perspective. second, methods research is often undertaken as a standalone project, and therefore there may be a need to establish a bespoke ppi group. conversely, applied health economics research is often nested within a clinical study. as such, it can benefit from ppi which is undertaken as part of the wider clinical study. in the next section we discuss the input of a bespoke ppi group (the lived experience advisory panel [leap]) as part of a standalone health economics methods project. the aim of the research project itself was to generate (and test) research methods to measure and value the quality of life of family carers. a leap that consisted of five people with current or recent experience of family care was recruited. the group included individuals who had fed into the design of the project, particularly in terms of how ppi would be used and how the project was communicated. the leap members were recruited through charity organisations in mental health and dementia and, in one case, via a colleague. as far as possible we sought diversity in caring experiences (e.g. care for spouses, adult children, and parents, and in the home and outside). the group's size was based on a desire to reflect the diversity of caring experiences while ensuring participants felt involved and able to contribute throughout the process. ultimately, there were three work packages of the research project that the leap contributed to: -understanding the mechanisms behind carer spillovers this work package comprised interviews, focus groups [ ] , and a delphi survey to understand the mechanisms by which health and social care interventions may affect carer wellbeing. -measuring carer quality of life this work package comprised postal questionnaires [ ] and think-aloud interviews to study the validity of different tools to measure the quality of life for carers. -valuing carer quality of life this work package comprised a person trade-off (pto) study [ ] and econometric analysis [ ] to value carer quality-of-life improvements to use in economic evaluation. the work was funded through a national institute of health research career development fellowship. to support the work, the leap and academic research team met times with specific meeting dates timed to coincide with relevant work. twelve full-day meetings represented a significant commitment for all concerned, but it was felt this was needed, given the multiple work packages, different phases of work within each work package (recruitment, survey design), and need to maintain contact throughout a -year project. the leap meetings consisted of discussions and specific tasks related to the work packages of the project. table summarises the details of the leap involvement in the work, in the form of the guidance for reporting involvement of patients and the public-version (gripp ) short form, the recommended system for documenting ppi within health research [ ] . below, we discuss the range of tasks undertaken by the leap over the -year period in more detail. understanding the mechanisms behind carer spillovers involved qualitative research with family carers and care professionals to investigate how health and social care delivery may impact on family carers [ ] . in early meetings involving the leap, we discussed which individuals would have insight into these issues, how to ensure different perspectives could be understood, where potential participants could be located, and how individuals should be approached. we discussed the process of the interviews and focus groups, how participants could be made to feel comfortable, and how we could structure the session to best meet the research objectives. once interviews and focus groups started, the leap reviewed initial transcripts and identified issues that contributed to the process of open-coding the transcripts [ ] . once the work had been completed, it became apparent that the research findings on 'spillover mechanisms' would potentially have wider relevance (beyond health economics and the research community). the leap helped to design a summary of the findings and identify organisations to share this research with. another major area of leap involvement was in designing surveys across the work programme, where it was important to achieve satisfactory response rates and meaningful answers. the leap completed early versions of the surveys. terminology, in relation to mental health issues, was amended to improve clarity. the timescales and format the aim of the programme was to develop a framework for measuring and valuing carer quality of life for economic evaluation. this comprised investigations of the mechanisms behind carer quality-of-life spillovers (work package ) the validity of different quality-of-life measures with carers (work package ) the valuation of carer quality of life (work package ) inclusion of carer and patient quality of life in economic evaluation (work package ) the main clinical contexts for the work were mental health, dementia, and stroke, three areas associated with major, though differing, challenges for family carers methods a leap was established to engage individuals with recent experience of family care. leap members were recruited via charities in each of the three clinical areas. the leap included individuals with experience of caring for adult children, spouses, and parents, within the home and outside the home. four of the five individuals had been involved in research before as a lay participant and two were involved in conducting research themselves. twelve -day meetings ( - ) were held to coincide with relevant work and led by ha. panel members were reimbursed for their time and expenses using involve guidelines. the meetings focused on recruitment strategies (e.g. for the interviews and delphi), the language and content of research materials (including questionnaires and participant information sheets), the process of interviews and focus groups, the interpretation of study findings, and dissemination strategies. each meeting had a major focus (e.g. participant recruitment), with supplementary issues discussed. some preparation was needed for the meetings. detailed notes and actions were written up by the academic researchers. a member of the leap also sat on the scientific advisory panel for the project four core members attended all meetings, and one further member attended one meeting. the ppi work resulted in the following outcomes: . participant recruitment recruitment to focus groups and interviews was expanded and facilitated by contacts in the leap . interview process the process of the focus groups and interviews was influenced by the leap; this included the location, terminology, socio-demographic mix, prompts (to ensure focus), brevity of questions, use of flip charts, and 'magic bullet question' . coding the early coding of transcripts built on the perspectives of the leap . questionnaire design survey questions were amended in various ways, including to ensure appropriate terminology and clarity (especially around mental health). changes were made to response categories, question ordering, and question content (including removal of question on 'strain'). the timescales and format of reminders to complete the survey was also informed by the leap . delphi study design in the delphi survey, a number of elements were amended, including the instructions (e.g. in relation to how long the survey was likely to take), language, use of open responses, formatting of feedback information, and the 'prioritisation task' . think-aloud interview in the 'think-aloud' study, the number of instruments included (maximum of three), the handling of upsetting questions, and ways of handling the presence of the care recipient was informed by the leap . pto design the pto tasks were made more comprehensible. the leap encouraged us to use pto rather than dce, and estimate equivalence values through iterative rather than direct methods. instructions were amended to standardise information. graphical methods (scales and stick people), worked examples, and the scale and duration of benefit were all informed by the discussion with the leap . dissemination the work on 'impact' mechanisms was disseminated to various health and care organisations, nhs trusts, and charities on the recommendation of the leap. the format and language used in dissemination was influenced by the leap. further contact and face-to-face presentations have resulted from the dissemination discussion and conclusion the leap played a particularly important role in opening up new avenues for recruitment and dissemination and making research surveys more accessible for participants. the latter was challenging because the surveys required participants to make difficult trade-offs, between, for example, improving the lives of patients and carers. an important objective of the research was to ensure carers could answer questions about their own quality of life openly and honestly, and the input of the leap was important in achieving this reflection and critical perspective members of the leap commented on feeling useful in helping to shape surveys and understanding more about the research process (putting them in a good situation to contribute to other studies). the leap made numerous contributions to ensuring the research project was more effective (see study results). however, there were also important lessons in terms of how to best prepare for the work, maintain involvement over a period of time, and ensure tasks are appropriate for us to work on jointly. the leap involvement gave the participants the experience and motivation to become involved in future ppi and research activity. this is worth stressing because the expansion of ppi nationally will require a greater number of such participants of reminders to complete the survey were also informed by the leap. in later meetings, the leap piloted and helped to refine the delphi survey. this influenced the instructions (e.g. in relation to how long the survey was likely to take), language, use of open responses, formatting of feedback information, and the 'prioritisation task'. the leap also contributed in a similar way to designing the pto study (to value carer quality of life) [ ] . in this case, the leap completed a lengthy initial survey with both discrete choice experiment (dce) and pto tasks, formatted in various ways to try to identify the most feasible method. this work was followed by in-depth discussion between the leap and academic team, which helped us identify that an iterative pto task was the most viable option. graphical methods (scales and stick people), worked examples, and the scale and duration of benefit were all informed by the discussion with the leap. towards the end of the project, the leap completed an online version of the pto task, as part of the process of moving to the final experiment. our work over the years has (presented in table ) suggested three important lessons in making best use of ppi in a health economics methods project. first, lay participants and professional researchers need training and preparation. second, practical measures (such as reimbursement) and 'soft' skills (such as maintaining a welcoming atmosphere) are needed to ensure ongoing engagement in a long research project. third, care needs to be taken to select appropriate research tasks for ppi. we discuss these three lessons in more detail in the next two sections. a key issue raised in our experience of using ppi for methods research is the need for prior training and clear objectives. in our study, the team (i.e. leap and academics) had initial discussions about the purpose of the project and how ppi could inform this. however, some lay participants reflected that more upfront training would have helped them feel more reassured about their input. discussions within our team also revealed differing expectations of timescales and understanding about how ppi would fit into a research project. training needs include research methods (and timescales), as well as health economics, with specific training (e.g. in outcome valuation) added on as needed. written materials that came to light during the project [ , ] could also be a useful supplement to more interactive training. to avoid duplication, it might be helpful to develop online training modules that all ppi participants could have access to. making ppi work also requires efforts from academic researchers. two of the academic team trained in qualitative research methods. however, in common with most health economists, the academic team had not been exposed to ppi through their training and career development. as with lay participants, a combination of written materials and interactive training would undoubtedly help. specific areas of training could include the following: the practicalities of recruiting and communicating with lay participants, running tasks, facilitating group discussion, safeguarding welfare, handling different perspectives, and ending a research project. as with other academic projects, methodology research projects can often last several years and the project's impact on the field may be gradual. as such, there is a need for practical measures to ensure lay participants feel engaged throughout. in our project, we had a range of activities that the leap supported (table ) which helped to vary the meetings, we met every - months, and we distributed agendas and actions from the meetings, to ensure a clear trail of what we were doing and how the leap was contributing. leap members were reimbursed for full day meetings in accordance with involve guidelines [ ] . diaries (i.e. keeping a record of tasks and comments), remote participation and individualised feedback on impact [ ] might be additional ways to maintain involvement through a long project. thought also needs to be given to how to end the project. we used the final few meetings to discuss follow-on projects and dissemination. it will make sense to disband the group in some cases and to maintain it in other cases, to support dissemination or follow-on work. using 'soft skills' to maintain a positive group dynamic was also important in maintaining involvement throughout a long project. ppi meetings can be intimidating, even if they feel fairly informal from the perspective of the academic team. we were fortunate in that four of the group stayed throughout the -year project. this created a sense of cohesion and familiarity that encouraged involvement. however, when people do drop out of ppi groups, this needs to be carefully handled, recognising there may be a trade-off between replacing a lost perspective in the group and maintaining the group's identity. we found it particularly useful that several of the panel had experience of research before either as a lay member or through other means. this enabled them to bridge the lay and academic worlds to some extent, helping others in the group to feel at ease. of course, having lay participants with research experience may not always be helpful, particularly if some members feel alienated through a lack of research experience. this reinforces the need for training prior to the start of the research. as detailed in table , we undertook a wide range of tasks with the ppi group. in general, these tasks worked well. but not all aspects of a research project are equally amenable to ppi. reflecting on the process of selecting tasks, we suggest three elements that are needed for ppi to add real value to a research task. first, the task must be necessary in meeting the research goals of the project. this may sound obvious, but it means that tasks should not be invented for the purposes of ppi. this creates unnecessary work for everyone and undermines the legitimacy of ppi. second, the task should be one where the academic research team alone have a knowledge or skills gap. it would not really make sense, for example, for ppi participants to execute analysis that the researchers are trained and experienced in, simply for the sake of it. third, the lay participants should have some additional knowledge or skills, in relation to the task, above that which the professional researchers possess. this means a task will not necessarily be suitable for ppi just because the academic team are struggling with it-there should be a reasonable expectation that ppi participants can bring additional insights to the task. we would argue all three criteria need to be fulfilled for ppi to be valuable in relation to a research task. qualitative coding of transcripts is one task where careful consideration of the relevance of ppi may be needed. coding is a vital stage of the qualitative research process. in our experience, involvement of the leap enriched initial discussions about the issues that were raised in interviews. however, once the formal (or axial) coding was underway, academic researchers were best placed to do this, given their professional training and arguably greater objectivity. of course, lay participants bring many skills, which may include relevant research skills, but in general, data preparation and analysis is an area where lay participants ought not to be replacing professional researchers. conversely, identifying organisations for sampling is an activity that can really benefit from ppi. for the research to happen, participants need to be identified, approached, and recruited. while professional researchers will be expected to know the sampling criteria, they will not always be aware of the voluntary and professional groups that exist in each disease context and how open such groups are to participating in research. as documented in sect. , the leap members were helpful in identifying carers and care professionals we could include, where to access them, and how to approach them. ppi participants will often have first-hand experience of these organisations, personal networks, and an awareness of how open such organisations might be to research involvement. relating this to our framework in fig. , this is certainly an area where a professional researcher may have a knowledge gap, and lay participants can make an important contribution. a second research activity that is likely to benefit from ppi is what we might term 'deep piloting' of surveys and other research materials. over the course of the work, the leap assisted in developing a postal survey to assess the validity of quality-of-life measures, a delphi study of carer/care professional attitudes, and a person-trade-off experiment. good practice in outcome measurement and valuation of course involves piloting measures and experiments before use [ ] [ ] [ ] . however, ppi is capable of going further than conventional piloting. in our experience, this process encompasses several elements. in box , we have focused on what we did in the pto task, but we also used a relatively similar process to develop our delphi survey and quality-of-life survey. the real benefit of deep piloting with a ppi group is the ability to work with a group of people who have a 'foot in both camps' (as researchers and participants) in identifying problems and generating solutions through several iterations. step : initial pilot. lived experience advisory panel (leap) members completed paper-based pto and discrete choice experiment (dce) scenarios as if they were a participant, to generate information on response patterns, time taken, and completion rate. step : reflective survey questions. at the end of the exercise, leap members completed survey questions about feasibility, ease, attractiveness, confusing questions, and so forth. step : group discussion on initial pilot. following steps and , we had a group discussion about the feasibility and problems with the task; we then co-developed solutions to these problems. step : redesign task. redesign task based on feedback from steps - . repeat steps - with version of pilot. the academic team redesigned the preference elicitation study (and it was put into an online format) for a second similar phase piloting a few months later. the final pto survey built on these two phases of piloting and redesign. ppi contributed in many important ways to the quality, timeliness, and relevance of our methodology research project. we found that effective ppi required careful preparation, steps to maintain involvement in the project, and the careful selection of tasks. research tasks will only add value if they are central to the research objectives and in areas where academic researchers have a knowledge or skills gap that the ppi can fill. this is particularly important in health economics methods research where the steps and tasks might be quite abstract to the lay public. a significant feature of our lay panel was that it was composed of family carers. this is a group whose voice is often under-represented, and their role in this research study was especially relevant, given its focus on the quality of life of family carers. the leap provided a valuable opportunity for members to discuss the impact on their own well-being, rather than their role as a provider of care to a patient. these discussions were facilitated by a smaller ppi group, which we felt led to carers feeling more comfortable sharing their own concerns. the group size may need to be larger for projects where a high level of diversity is needed or for certain research tasks, for example, if extensive cointerviewing is needed. global and national pressures on family carers, including coronavirus disease (covid- ), inadequate social care provision, and ageing populations, mean the importance of involving family carers in ppi will remain and is likely to grow. because health economics methods research may not occur alongside a clinical project, it is less likely there will be an available ppi group to access. this means specific efforts will be needed, on behalf of the health economists, to engage and maintain members (as discussed in sect. ). the point is well made in other studies that researchers need to work sensitively [ ] , see ppi as a two-way education process [ ] , and avoid exploitation [ ] . our work adds to a small but growing amount of literature on ppi in health economics. many of the principles for best practice will be relevant regardless of the focus of the research project. others have highlighted the need for clear aims and objectives and to ensure appropriate tasks are selected [ , ] . the fact that ppi is most successful when members can contribute actively is also underscored by gibson et al. [ ] , who highlight ppi as a dynamic interaction between lay and professional knowledge. however, health economics methods research involves perhaps paying even more careful attention to preparation and task selection, given the more abstract nature of the work. ppi requires significant resources, both from lay participants and academics, and ultimately the funder and society. it is therefore legitimate to ask how value-for-money can be maximised. where ppi is needed for a methodology project, we suggest devoting resource to upfront training and careful selection of tasks. fewer meetings, with well-briefed participants, focused on key tasks will contribute more to the research than more frequent, tokenistic discussions. in conclusion, ppi in health economics methods research has much potential, but making the best use of ppi requires careful consideration. ppi contributed in many important ways improving the quality, timeliness, and relevance of our work. we hope the tools developed through the work, in addition to the ideas in this article about how to make best use of ppi will be helpful to the broader research community. we look forward to further discussion and debate about this important topic. involving members of the public in health economics research: insights from selecting health states for valuation to estimate quality-adjusted life-year (qaly) weights. appl health econ health policy working with patients and members of the public: informing health economics in child health research. phar-macoeconomics open knowledge of public patient involvement among health economists in ireland: a baseline audit ppi resources for applicants to nihr research programmes gripp reporting checklists: tools to improve reporting of patient and public involvement in research guidance for researchers on ppi qualitative methods for health economics. london: rowman & littlefield going the extra mile: improving the nation's health and wellbeing through public involvement in research development of a self-report measure of capability wellbeing for adults: the icecap-a developing a descriptive system for a new preferencebased measure of health-related quality of life for children core items for a standardized resource use measure (isrum): expert delphi consensus survey a framework to include family health spillovers in economic evaluation alternative decision modelling techniques for the evaluation of health care technologies: markov processes versus discrete event simulation six mechanisms behind carer wellbeing effects: a qualitative study of healthcare delivery validity and responsiveness of preference-based quality of life measures in informal carers: a comparison of measures across conditions valuing carer quality of life for economic evaluation: a person trade-off (pto) study. discussion paper at health economist's study group meeting newcastle valuing care-related outcomes for economic evaluation: an application of the wellbeing valuation method. discussion paper at health economist's study group meeting a research handbook for patient and public involvement researchers a researcher's guide to patient and public involvement is it worth it? patient and public views on the impact of their involvement in health research and its assessment: a uk-based qualitative interview study qualitative research and content validity: developing best practices based on science and experience reporting formative qualitative research to support the development of quantitative preference study protocols and corresponding survey instruments: guidelines for authors and reviewers using qualitative methods for attribute development for discrete choice experiments: issues and recommendations a framework for public involvement at the design stage of nhs health and social care research: time to develop ethically conscious standards patient and public involvement: how much do we spend and what are the benefits? evaluating patient and public involvement in health research: from theoretical model to practical workshop the authors thank eve wittenberg (harvard university) and james hall (university of birmingham) for comments on an earlier draft of the manuscript. funding this research was funded by a national institute for health research (nihr) career development fellowship awarded to ha (award number: cdf- - - ). this paper presents independent research funded by the national institute for health research (nihr). the views expressed are those of the authors and not necessarily those of the nhs, the nihr or the department of health and social care. the first draft of the manuscript was written by ha, and all authors reviewed previous versions of the manuscript. all authors read and approved the final manuscript. key: cord- -aeyf yu authors: joshi, bhrugesh; bakarola, vishvajit; shah, parth; krishnamurthy, ramar title: deepmine - natural language processing based automatic literature mining and research summarization for early-stage comprehension in pandemic situations specifically for covid- date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: aeyf yu the recent pandemic created due to novel coronavirus (ncov- ) from wuhan, china demanding a large scale of a general health emergency. this demands novel research on the vaccine to fight against this pandemic situation, re-purposing of the existing drugs, phylogenetic analysis to identify the origin and determine the similarity with other known viruses, etc. the very preliminary task from the research community is to analyze the wide verities of existing related research articles, which is very much time-consuming in such situations where each minute counts for saving hundreds of human lives. the entire manual processing is even lower down the efficiency in mining the information. we have developed a complete automatic literature mining system that delivers efficient and fast mining from existing biomedical literature databases. with the help of modern-day deep learning algorithms, our system also delivers a summarization of important research articles that provides ease and fast comprehension of critical research articles. the system is currently scanning nearly , , , english words from , research articles in not greater than . seconds with multiple search keywords. our research article presents the criticality of literature mining, especially in pandemic situations with the implementation and online deployment of the system. the recent pandemic of coronavirus disease [ ] is caused by novel human coronavirus, initially referred to as the wuhan coronavirus (cov), which is currently designated as a severe acute respiratory syndrome (sars)-cov- as per the latest international committee on taxonomy of viruses (ictv) classification [ ] and was suggested to have a possible zoonotic origin [ ] . as an outbreak of pneumonia started in wuhan, china and the first case was found on december . by the end of march , it has infected more than , individuals in nearly countries and causes more than , deaths worldwide [ ] . sars-cov- belongs to the family of coronaviridae. it has an envelope and maintains a single-strand, positive-sense rna genome ranging from to kb in length [ ] . these viruses can be classified into four genera: alpha, beta, delta, and gamma. from which alpha and beta coronavirus (covs) are known to infect humans [ ] . they are circulated among humans, other mammals, and birds. they can cause respiratory, hepatic, enteric, and neurologic diseases [ ] as well. despite the fact majority of human coronavirus infections found to have a mild effect, the epidemics of two beta coronaviruses (betacov) -namely severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov) have caused more than ten thousand cumulative cases in the past years with mortality rates about percent for sars-cov and percent in case of mers-cov [ ] [ ] . as the situation is getting worst turning from epidemic to pandemic with an increasing number of confirmed cases and its related deaths with each passing day. science community around the world has joined their hand to fight against this deadly disease in all the possible ways including, making the vaccines, repurposing the existing drugs and designing diagnostic kits for the detection of the presence of virus or disease. varieties of works also includes sequencing the virus genome to identify the origin of the virus and the possible mode of transmission, to allocate the resource for the patients and applying the statistical models to predict how fast the disease can be spread. to begin any new research it is very essential to know what information is already available. as we know the fact that the similar virus has already been known, it becomes crucial to collect and organize this information from the ocean of research articles. the average reading speed of a human is roughly wpm (words per minute) [ ] , that means that it will take a substantial amount of time to comprehend and find useful information from thousands of research articles published. to help the research community for screening the thousands of published research and making the comprehension in a short amount of time we have designed the ai-nlp based system which can mine the relevant articles and give a short article summary, that can make ease in fast and efficient comprehension for researchers. as various experts are working in a different domain they search for the relevant information which lies in a varied domain. data mining is becoming a fundamental component of the global world with verities of applications. data mining assists a quick and efficient decision-making process that enhancing the accuracy of an outcome. we already have a huge amount of data or information in forms of research articles published in the last decades or more. with the application of the literature mining system, meaningful research articles can be extracted from this huge set and brief technical summary can be produced for research articles user interested with. in computer science, text summarization is a process of shortening the large text document(s) in order to generate short and meaningful piece of text. the objective is to create fluent natural language text keeping major insights or technicality of the source data. the automatic text summarization is an ordinary problem in the field of natural language processing and machine learning. the task was first carried out in form of generating automatic literature abstracts in [ ] . over the past half a century, the problem of text summarization has been addressed with verities of perspectives. primarily, the task of summarization is divided into two major categories as extractive summarization and abstractive summarization [ ] . as the name itself suggests, the extractive method involves pulling of key phrases from the source document in order to generate the targeted summary. the abstractive summarization works similar as we human do [ ] . it involves the end-to-end deep learning technique called sequence-to-sequence learning to derive the understanding about the association between words. the primary objective of the our system is to deliver quick and efficient search from a huge amount of available literature. by entering a keyword coron-avirus we are getting number of research articles that contain the keyword in the title. this is even time consuming to go through the abstract of each literature, here we are interested in. hence we found that searching for an article is not serves the ultimate purpose of serving important research articles to a user so that the researcher can speed up the research in epidemic situations. to overcome this limitation we developed a research text summarizer that can generate a technical summary by scanning all the research articles derived from user-entered keyword(s). in the demanding situation of covid- , we applied the literature mining with user entered keyword(s) and automatic generation of brief summary of research articles, that user searches for. the ultimate objective of our system deepmine, is to provide quick and efficient access of the openly available research articles. for the initial starting purpose, we have used the covid- open research dataset (cord- ) [ ] . the dataset has been published by allen institute for ai on th march . the detailed category wise description of the cord- dataset is given in table- . the deepmine is primarily performing two major functions namely mining of articles from available open data sources using user-entered keywords and generate brief technical summary in natural language for a quick review of articles that user interested with. the entire system has been developed using python programming language with the support of various scientific and natural language processing libraries available. as represented in figure- , a researcher can submit the keyword(s) he or she is interested with and the mine article process of the system returns article titles and available links by scanning more than , , , words from , research articles openly provided by cord- . a user of the system can separately make requests for generating a summary of the research articles with article summarization process. the system has used the deep natural language processing-based text summarization for generating detailed technical summary given the research article as an input. the deepmine system uses the natural language processing based text mining from available data sources. one user enters keyword(s) related to articles he or she is interested with, system returns all the articles having the entered keyword(s) in the article title. for returning more precise articles, we have currently focused on the title of the available research articles. the system returns found research articles having the keyword(s) in the title with total count and the user can explicitly visit to abstract and summary to the research article. the deepmine service is openly available on url https://deepmine.in/ as discussed under section - , cord- dataset contains nearly , , , words in title, abstract and article body. the below table- represents the dataset category wise title word count. while mining through the deepmine given a keyword "coronavirus" we found number of research articles as shown in figure- it is utmost significance for a researcher to take a quick idea of research with the abstract of the research article. deepmine system derived abstract for all mined articles from the dataset for a quick review. the below figure- shows the abstarct of one of the research articles mined suing keyword "coronavirus". from the homepage of the system as shown in figure- , we mined research articles using multiple keywords i.e. "coronavirus" and "rna". this mining returns us total results. statistics of country wise research articles as shown in figure- and abstract of one of the research articles is shown in figure- . our primary contribution majorly focuses on the design and development of the system that can able to automatically mine the literature, provided the keyword(s) as an input. the system is able to generate the brief summary of the research article, that user interested with. our ultimate objective is to provide a quick and easy literature mining service so that the researchers working for fighting current pandemic situation of covid- can get early-stage comprehension. currently we have used the covid- open research dataset (cord- ) , publicly made available by allen institute of ai on th march . our system deepmine is providing mining from , research articles with keywords by scanning nearly , , , english words available in literature dataset in not greater than . seconds. from experiments performed on the live deepmine system we can observer that the system is helpful for the primary mining of the huge amount literature available in various openly available datasets. however, in many of the research articles of the dataset the affiliation and/or location has not been provided. we are in the process of cleaning and improving the dataset with automatic and manual processes. currently, we are working on the collection of various research articles openly available from reputed publishers. afterward, the system will be upgraded with a wide range of research literature especially of the bio-medical field. an equal amount of work is going on to improve the process of text summarization with the latest deep learning techniques to provide more accurate and human like text summarization on research articles. the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- a novel coronavirus from patients with pneumonia in china prevention: novel coronavirus ( -ncov), wuhan, china epidemiology, genetic recombination, and pathogenesis of coronaviruses host factors in coronavirus replication, in roles of host gene and non-coding rna expression in virus infection coronavirus pathogenesis, in advances in virus research summary of probable sars cases with onset of illness from middle east respiratory syndrome coronavirus (mers-cov standardized assessment of reading performance: the new international reading speed texts irest the automatic creation of literature abstracts a review on neural network based abstractive text summarization models text summarization with tensorflow covid- open research dataset key: cord- -nmmz tke authors: verma, surabhi; gustafsson, anders title: investigating the emerging covid- research trends in the field of business and management: a bibliometric analysis approach date: - - journal: j bus res doi: . /j.jbusres. . . sha: doc_id: cord_uid: nmmz tke the covid- pandemic has been labeled as a black swan event that caused a ripple effect on every aspect of human life. despite the short time span of the pandemic—only four and half months so far—a rather large volume of research pertaining to covid- has been published ( articles indexed in scopus and the web of science). this article presents the findings of a bibliometric study of covid- literature in the business and management domain to identify current areas of research and propose a way forward. the analysis of the published literature identified four main research themes and sub-themes. the findings and propositions of this study suggest that covid- will be the catalyst of several long- and short-term policy changes and requires the theoretical and empirical attention of researchers. the offered propositions will act as a roadmap to potential research opportunities. the coronavirus disease (covid- ) is a human social and economic crisis that has attacked the core of human existence. it continues to spread uncontrollably around the world; as of may , , , , people had been infected globally (who, ) . the outbreak is predicted to reach its peak in june , declining only from july onwards (who, ) . the covid- pandemic has affected all segments of the population, especially vulnerable groups such as the old, the infirm, the disabled, the marginalized and the poverty-stricken (donthu & gustafsson, ) . in response to the pandemic outbreak, the leaders of many countries decided to save lives before saving the economy, declaring sudden or phased lockdowns in their countries. policies like "social distancing" and "stay-at-home" were implemented overnight, which severely damaged several businesses across industries (donthu & gustafsson, ; leite, hodgkinson, & gruber, ) . according to the world trade organization (wto, ), world trade was already experienced a slump in , and then the covid- pandemic precipitated a global financial crisis. early estimates have predicted that major economies will lose around . to . percent of their gross domestic product (gdp) during due to the covid- pandemic (wto, ) . it is becoming challenging for most businesses across the world to keep their financial wheels rolling, given reduced revenues and the high level of uncertainty. it is thus of the utmost importance for businesses to conduct proper assessment and feasibility analyses of their business models (donthu & gustafsson, ) . covid- has also pushed businesses across the world to rapidly operate in newer and more resilient ways. as firms change their priorities in response to old challenges like real-time decision-making, workforce productivity, business continuity, and security risks, newer challenges introduced by the pandemic are testing businesses' resilience as they attempt to lay a foundation for the future (ivanov, ) . to help business practitioners and researchers understand the impact of the pandemic on future economic growth, this study analyzed a corpus of covid- and business and management articles to address questions like the following: what are the growth trajectories and trends of publications in the early phase of the covid- pandemic outbreak? what are the topical foci for research regarding covid- and businesses? this review captures the current state of research about covid- through a systematic literature review and develops propositions to guide future research. it can be described as a prescriptive study that reviews articles pertaining to covid- and aims to provide a reference point for practitioners and researchers alike. the paper includes sections delineating the methodology, results, discussion, conclusion, and future research avenues. bibliometric analysis is an expedient approach to examine the evolution of research domains, including topics and authors, based on the disciplines' social, intellectual, and conceptual structures (donthu, kumar, & pattnaik, ) . researchers have used this technique in a range of disciplines, like strategic management (ferreira, fernandes, & ratten, ) , corporate social responsibility (bhattacharyya & verma, ) , medicine (liao et al., ) , and corporate universities (singh, verma, & chaurasia, ) . although a few recent bibliometric studies have addressed the impact of covid- (chahrour et al., ; hossain, ; park, cook, lim, sun, & dickens, ) , each study has its own set of limitations. chahrour et al. ( ) only examined the most influential observational studies and therapeutic trials described in articles published in the pubmed and world health organization (who) databases. also, they did not study the intellectual structure of the research area. the bibliometric analysis performed by park et al. ( ) included only the pubmed database and the therapeutic aspects of the pandemic. hossain ( ) collated articles published only in the web of science (wos) database and reported that researchers largely targeted biological topics, such as those related to genetics, epidemiology, and zoonosis. importantly, hossain ( ) pointed out the necessity of extending the covid- pandemicrelated research to the field of socioeconomics. to overcome the limitations of these earlier works, this study utilized the science mapping approach to understand the topical foci of the field of covid- and business research ( figure ). the science mapping approach helps researchers to understand the extent of a topic, its emergent trends, and its evolution over time (singh et al., ) . it is a holistic approach that provides better insights than a traditional literature review because it minimizes the potential subjectivity of the researcher (bhattacharyya & verma, ) . this bibliometric study is novel in several respects. first, it applied the science mapping approach (consisting of bibliometric literature and scientometric analyses) in the emerging research domain of covid- and business to minimize subjectivity and bias. second, the research extended the science mapping approach with an in-depth analysis of the identified research themes. third, it analyzed the research topics, identified significant research gaps, and provided future avenues in the research domain of covid- and business. to address the research questions of this study, we identified several research articles published within the business and management field. figure summarizes the research design. the publications was obtained using the scopus and wos databases. data acquisition from existing literature is crucial in the science mapping approach since it determines the dataset of articles from which pertinent conclusions will be drawn (singh et al., ) . we selected both the scopus and wos databases to ensure a wider range of high-quality peer-reviewed journals in the domain of business and management compared to ebsco, google scholar, or others (ferreira et al., ) . we retrieved existing literature related to covid- in the business management domain from the scopus and wos databases using keywords related to covid- (hossain, ) : " -ncov" or "covid- " or "coronavirus disease " or "novel coronavirus pneumonia" or "ncp" or " novel coronavirus" or "sars-cov- " or " novel coronavirus diseases" or "novel coronavirus" or "pneumonia." the keyword search in scopus and wos was set to include titles, abstracts, and keywords in order to retrieve all relevant publications. the search period was set to include articles published between january , , and may , . only english-language publications were considered for the review process. [insert figure here] the initial search yielded over , papers, but narrowing the results to only the business research area yielded papers published in the scopus and wos databases. of these, articles were duplicates (i.e., indexed in both databases) and thus were excluded from further analysis (homrich, galvao, abadia, indicates the adaptability and openness of researchers across the world to understand contemporary issues like the impact of covid- on business. further, the results indicate that this field is still evolving and is in its initial stage, as there are no dominant authors yet. also, researchers with different backgrounds have contributed to the field. in order to identify emerging themes related to covid- in the articles identified in the business research area, we performed a co-word analysis of keywords using vosviewer. co-word analysis applies text-mining techniques to the titles, abstracts, and keywords of articles (van eck & waltman, ). coword links identify multiple keywords that occur together in the same articles. the relationship between keywords is determined based on the number of articles in which the keywords occur together (van eck & waltman, ) . to perform a co-word analysis, we combined the datasets retrieved from the scopus and wos databases and converted them into a microsoft word file for data cleaning and preprocessing. we manually eliminated the duplicate articles from the file and removed coding errors in the sources, affiliations, and cited references for further analysis. for example, we corrected duplication errors such as two different forms of the same author's name (e.g., "lee s." and "lee s.j.") through the data cleaning process. further, we standardized keywords to ensure unification and consistency (i.e., singular/plural). after data cleaning, we conducted data analysis to understand the evolution of the corpus of covid- literature in the business and management domain during using bibexcel and vosviewer. bibexcel is a data analysis tool used for descriptive analysis of data (ferreira et al., ) . we used it in this study because of its high flexibility for data modification in databases like wos and scopus. further, we employed the visualization tool vosviewer, which collected all the keywords from the dataset and created a co-word network. this network helped us to understand the research interests and relationships among keywords. it was from this analysis that the prominent research themes emerged. the study identified themes and sub-themes ( figure and table ). each theme and sub-theme is discussed in detail in the findings and discussion section. [insert figure here] in comparison with the global financial crisis, the covid- pandemic is associated with several new challenges, given that major economic activities have been stifled. the extent of covid- spread across the world has heightened uncertainty regarding consumption and investment among different stakeholders, like consumers, trade partners, suppliers, and investors (donthu & gustafsson, ) . increasing the length of the lockdown and travel restrictions (national and international) are severely affecting the general economy. the covid- crisis is generating spillover effects throughout global and regional supply chains, disrupting demand and supply (pantano, pizzi, scarpi, & dennis, ) . additionally, social distancing policies have almost destroyed service industries, like travel and tourism and hospitality, which could trigger a recession (donthu & gustafsson, ) . the severe global challenges posed by covid- can be tackled using a range of digital technologies, like the internet of things, artificial intelligence, big data analytics, and drones (donthu & gustafsson, ) . this study tried to examine the impact of covid- on different facets of businesses through bibliometric analysis and identified four major discourses ( table ) including impact of covid- on overall business, technologies, supply chain management, and the service industry. [insert table here] the covid- pandemic has already had dramatic, rippling effects across global economic activities in every region of the world (bofinger et al., ) . in order to flatten the curve of infection rates, several countries across the world have imposed widespread restrictions (e.g., lockdowns, quarantines, and closure of physical shops and businesses) to protect the functioning of healthcare systems (michie, ) . understandably, these closures have had an immense, immediate impact on the economic activity in almost every sector. for example, activities involving direct contact between consumers and service providers have been adversely affected by restrictions on movement and social distancing (giritli & olofsson, ) . the closure of the economy has also increased the risks associated with investments by households and businesses. many companies are either facing bankruptcy or reducing their production capacity, which has led to higher unemployment and underemployment (bofinger et al., ) . a prolonged lockdown period also increases the risk of a massive increase in corporate and governmental debts, leading to fundamental financial imbalances that could prolong the recovery period from the covid- crisis (donthu & gustafsson, ) . during the covid- crisis, businesses are working faster to ensure a competitive advantage (lee, venkataraman, heim, roth, & chilingerian, ). in order to solve emerging problems, firms are embracing methods and processes that are responsive rather than reactive to the crisis (chesbrough, right governance (graves & karabayeva, ; lee et al., ) . they are quicker to decide where to invest and reallocate their resources. in addition, firms are pushed to create new product/services and radically adapt in order to remain visible, agile, and productive (chesbrough, ; kim, ) . the covid- crisis has seriously threatened the potential for innovation and discouraged start-ups that could have been viable under normal circumstances (kuckertz et al., ) . a high percentage of startups are poised to go out of business in a few months as the capital and revenue required to sustain them are quickly drying up (bofinger et al., ) . therefore, until the covid- pandemic is over, survival must be the primary focus of start-ups and regulators. further, the crisis has brought about a change in the investment patterns of venture capital firms, which are shifting their focus to start-ups operating in sectors like online grocery delivery, healthcare, fast-moving consumer goods, and home entertainment. (kuckertz et al., ). the creativity and experimentation of start-ups is highly dependent on intense personal and social exchanges between stakeholders. the pandemic-induced lockdown has reduced the opportunities for direct communication and spontaneous encounters, resulting in deleterious impacts on start-ups' growth curves (bofinger et al., ) . according to the wto ( ), trade in will plunge by - % or - % according to the optimistic and pessimistic scenarios, respectively. further, the recovery in is uncertain and will depend on the duration of the covid- pandemic and the effectiveness of policy responses (evenett, ) . despite the closure of international borders, maintaining trade flow is crucial to ensure access to essential goods like medicine and food in addition to supporting jobs and economic activities (ozili & arun, ) . covid- is severely affecting service trades, threatening permanent closure (garvey & carnovale, ) . however, some service trades, like information technology services, are booming because employees are able to work from home (evenett, ) . the covid- crisis has affected around . billion employees (monitor, ) . people across the world are unable to work because they are being asked to isolate or quarantine themselves. due to covid- lockdowns, businesses have increased layoffs and working hours and reduced wages (dey & loewenstein, ) . many employees have been affected by low wages, or loss of income. sectors like travel and tourism, food services, retail, manufacturing, and business and administrative activities are at the highest risk of unemployment and under-employment (bell & blanchflower, ) . covid- is disrupting the economies of many countries by reducing economic activities across multiple sectors, leading to decreases in employment, and reducing revenue streams for several businesses (dey & loewenstein, ; woodside, ) . during this crisis, understanding people's risk perceptions is critical for risk communication (aven & bouder, ) . the risk perception of covid- is influenced by several societal, cultural, and psychological factors and affect one's preparedness and planning (giritli & olofsson, ) . therefore, risk communication should be based on factors like risk attitude, risk perception, and trust in communicating authorities. further, risk perceptions can affect how individuals evaluate threats and information communicated by authorities (aven & bouder, ) . during the covid- pandemic, big data and advanced analytics are helping to detect surface indicators related to the pandemic (guo et al., ) . specifically, real-time big-data-driven insights have helped policymakers and researchers to comprehend and forecast the reach and impact of the covid- outbreak. real-time covid- trackers are helping epidemiologists, scientists, health workers, and policy-makers make more informed decisions to fight the pandemic by aggregating and synthesizing incident big data (hancox, ). further, real-time analysis of gps data indicating people's movement within a certain locality is helping the government understand the population's compliance with social distancing mandates (chen, ) . big data analytics are also helping many leaders to make difficult decisions that affect staff, customers, and operational capacity. for example, firms are leveraging their internal and external data on, for example, customers' contact history, employees' information, business operations monitoring, and social media to understand different scenarios for sustainable development during and after the covid- pandemic (donthu & gustafsson, ) . during the covid- pandemic, digital innovations quickly became the buttresses of personal and professional life (panigutti, perotti, & pedreschi, ) . connected digital devices enable both remote work and education. additionally, chatbots are providing instant life-saving information, partially relieving overwhelmed health systems (leite et al., ) . further, geolocation information systems are helping health workers and researchers to track and map the spread of the virus. firms and governments are designing and piloting fast, responsive frameworks to implement emerging technologies, like data policy, digital trade, iot, ai, drones, autonomous vehicles, blockchain telemedicine, and environmental innovations (panigutti et al., ) . the public health emergency of covid- confronting global healthcare systems has prompted the development of digital health solutions to mitigate the impact of the pandemic (panigutti et al., ) . these digital healthcare systems include telehealth; robust surveillance systems; technology-driven diagnostic and clinical decision-making tools; wearable tracking devices to measure physiological parameters like temperature, heart rate, and sleep-duration; and interactive chat services providing information about covid- (leite et al., ) . telemedicine administered through virtual chatbots and webbots is emerging as a viable option for communication and safe medical care. digital healthcare solutions are helping to detect, report, and provide surveillance and rapid response for covid- cases (panigutti et al., ) . further, they are helping with the creation of geospatial dashboards that display pertinent information at the national and international scales to track covid- statistics in real time (panigutti et al., ) . according to the who director general, the world is fighting not only an epidemic but also an "infodemic" (who, ). this infodemic is causing an over-abundance of information related to the covid- situation, not all of which is accurate (yu, li, yu, he, & zhou, ) . such uncertainty can create confusion and distrust among people and, ultimately, hamper an effective public health response. the covid- infodemic has largely dealt with the cause and origin of the novel coronavirus, its symptoms and transmission patterns, available treatments and cures, and the effectiveness of interventions by health authorities (krause, freiling, beets, & brossard, ) . in response to this, social media and search engine giants, like facebook, google, pinterest, twitter, and youtube, are filtering out unfounded medical advice, hoaxes, and other false information that could risk public health (krause et al., ) . along with several challenges, the infodemic is also creating opportunities to identify and adopt new preparedness and response tools to fight covid- . according to wells et al. ( ) , the covid- pandemic has led to a global socio-technical crisis and several alternative futures. it is globally, rapidly, and pervasively disrupting existing practices (huynh, ) and is quickly emerging as a catalytic and meta-transition event that challenges and reshapes the legitimacy and efficacy of existing political and economic structures (sendak et al., ; woodside, ) . these socio-technical changes will redefine future business activities and ecological burdens (huynh, ) . the covid- crisis continues to globally disrupt manufacturing and supply chains, with severe consequences for consumers, businesses, societies, and the global economy (ivanov, ) . the global production system has been badly affected due to surges in demand for essential goods, factory shutdowns, panic-buying, and shifts in consumer preference (e.g., online over physical shopping ; ivanov, ) . companies are modifying the supply chain by carefully managing interdependent factors, like localization, complexity reduction, dual-sourcing, and investing in advanced manufacturing technologies (garvey & carnovale, ) . apart from these initiatives, manufacturing firms are taking measures to ensure business continuity. these measures include cross-functional controls and coordination on a global and regional level, increase in safety stocks and shift inventories, pivot freight models, improved customer support programs, rebound measures for demand returns, and quick reactions to changing sources of demand in order to secure revenue streams (ivanov & dolgui, ) . the viral outbreak has paralyzed the tourism industry, leading to job losses and devastating economies that are highly dependent on tourism (boulos & geraghty, ) . tourism and cities have strongly intertwined economic, social, and environmental relationships, which shape localities, landscapes, and regions. according to the united nations world tourism organization (untwo), this pandemic has led to a potential loss of usd - billion for the travel and tourism industry (unwto, ), as several countries and regions have imposed entry and exit bans and other restrictions, which have decreased willingness to travel (higgins-desbiolles, ). closure of educational institutes due to the pandemic has not only affected students and teachers but also created economic and social consequences related to digital learning, internet facilities, childcare, food insecurity, healthcare, and housing (arora & srinivasan, ) . as of may , , . billion (approximately . percent) students across countries have been affected by the closure of educational institutes (zhang, wang, yang, & wang, ). educational institutes are opting to conduct classes through internet, digital devices and online platforms (like group video programs) (arora & srinivasan, ) . the covid- crisis is affecting all service sectors due to either spikes or surges in demand. for example, while the retail, tourism, and aviation sectors have been badly affected due to the closure of services to mitigate the risk, food retailers and grocery stores are struggling with rising demand as consumers are trying to stock up for long periods of isolation (addo, jiaming, kulbo, & liangqiang, ) . essential service workers are working in grim and testing working environments, continuously exposed to the virus with no choice but to work (frazer, merrilees, nathan, & thaichon, ) . therefore, essential services need to consider the direct and indirect impacts on employees' occupational safety and health. as the covid- crisis continues to impact businesses in the next few years, economic, societal, and technological changes will become unavoidable for survival. given this backdrop, the current study presents a bibliometric review of papers on the ramifications of covid- in the domain of business and management in order to delineate this emerging research field and summarize the available knowledge. this sub-section provides a synopsis of the four main clusters discussed in section . the synthesis of four distinct research streams in the covid- domain reveals several new opportunities for future practice and research work (table ). due to fears of a financial collapse and new recession, resilient businesses and sturdy government leadership are urgent and critical (giritli & olofsson, ). short-, medium-, and long-term plans are required to rebalance and re-energize the economy (michie, ) . in addition, socioeconomic risk assessments and strategies for robust and sustainable business models are required across every sector. the covid- pandemic is radically changing the demand pattern for products/services, which has in turn increased the risk of fragility in global and regional supply chains and networks (lee et al., ) . to sustain and position themselves for the "new normal," firms should improve their operational resilience, accelerate end-to-end value chain digitization, rapidly increase the transparency of capital and operating expenses, embrace remote work, reimagine sustainable operations, and ensure competitive advantage. in addition, start-ups need to be more flexible and adapt their business models to dynamic markets. policy measures will only be successful if they are complemented by an entrepreneurial ecosystem. further, policy-makers need to implement measures to protect start-ups and adopt or discard policies in the future based on the knowledge derived from crisis situations (kuckertz et al., ) . based on this, we propose the following: proposition a: the covid- crisis not only requires short-, medium-, and long-term plans to rebalance the economy but also raises a clarion call for robust and sustainable business strategies across every sector. proposition b: the covid- crisis demands new operating models to meet changing demand patterns and remain agile and productive. [insert table reforms in trade policy during the covid- pandemic will help to reduce the negative economic and social impact of the virus, which will eventually help to build resilience and ensure economic recovery. for instance, reforms in trade policy can reduce the need for close contact between transporters, traders, and officials, helping to maintain social distancing and limiting the spread of the virus (carnevale & hatak, ) . further, to mitigate disruptions in the regional and global value chain, interventions in logistics operations are required. the impact of this pandemic on employment is unprecedented and will continue to increase; therefore, economies urgently need to implement policy measures to boost the demand for labor. the new labor policies should effectively consider health protection measures and economic support on both the demand and supply sides (bell & blanchflower, ) . also, businesses should pay special attention to building capacity and updating employees' technological skills (carnevale & hatak, ) . based on this, we propose the following: proposition c: reforms in trade policies are required to reduce the negative impact of the covid- crisis. proposition d: changes in existing labor policies are needed to boost the demand for labor. in the covid- crisis, digital technologies, like the internet of things, ai, and blockchain are becoming essential for economic and social functioning (huynh, ) . drones can be used for surveillance to ensure safety guidelines are being followed and to spray disinfectants on affected areas (leite et al., ) . ai-powered tools could be used to obviate the need for manual temperature checks and distance analysis panigutti et al., ) . data-driven insights have the potential to facilitate accurate prediction of impacts and can make the difference between a misstep and a strong continuity response in these uncertain times (hancox, ) . although these emerging technologies have immense potential as equalizers, without the right governance, they could intensify the digital divide in society. further, these technologies are associated with critical privacy and security issues and urgently require a global baseline consensus on security (panigutti et al., ) . the right information related to the pandemic is key for the success of mitigation measures, but the infodemic has exacerbated the crisis, propagating misinformation through social media platforms and other channels (huynh, ) . it should not be doubted that the covid- pandemic has affected relationships within socio-technical systems at the landscape and regime levels. global stabilization agents, such as the wto, world bank, and united nations, can help to create checks and balances and attempt to ensure a return to business as usual (sendak et al., ) . based on this, we propose the following: proposition b: resisting the social-technical meta-transition is essential in the covid- crisis. this sudden shift in the global production system has raised questions about the resilience of global and local value chains and has caused firms to rethink and transform their overall approach to manufacturing and the supply chain model. companies should launch strategic initiatives to create more resilient supply chains. the operation model should be adjusted to enable more flexible and decentralized manufacturing with a consistent risk management system. also, cross-industry collaboration models need to be redefined, and product portfolios require thorough reviews to reduce complexity. based on this, we propose the following: proposition a: the covid- crisis demands resilient strategies to reduce manufacturing complexities. proposition b: modification of existing supply chain measures is required to ensure business continuity while dealing with the covid- crisis. the covid- pandemic will have a long-lasting effect on international tourism due to travel restrictions and changes in people's risk perception of overseas travel. this global epidemic has impacted tourist destinations, leading hotels, restaurants and bars, theme parks, museums, trade fairs, and cultural and sports events to be deserted (boulos & geraghty, ) . the authorities responsible for providing different services should provide clear and consistent guidance to workers to ensure compliance (frazer et al., ) . in the education sector, sudden switching of teaching styles is creating several challenges, like changes to learning plans, acclimatization issues with new online platforms, and conversion of lessons and hands-on learning materials to remote learning and communication (zhang et al., ) . along with these technical challenges, many students and parents do not have access to the proper technologies for distance learning, like the internet and digital devices, or the necessary skills to handle them. based on this, we propose the following: proposition a: the covid- crisis requires robust strategies in different service industries to ensure compliance. proposition b: switching from existing teaching styles is required to reduce the challenges of learning while dealing with the covid- crisis. the objective of this paper was to explore emerging research trends regarding the impacts of covid- on business and management using bibliometric and science mapping approaches. this article contributes to research on covid- by elucidating the theoretical evolution of covid- research and its linkages with multiple economic, social, and technological factors. broadly, the findings and propositions of this article contribute to the epistemological discourse on collective knowledge about the impact of covid- on business and management by examining the most productive authors. in addition, the results show how knowledge is evolving over time based on the use of keywords. importantly, an understanding of the contributions of the most productive scholars and their research helps other researchers build on their work by choosing and following a line of inquiry. ivanov, the most productive researcher in this area, has mainly focused on the short-and long-term impacts of epidemic outbreaks on global supply chains and intertwined supply networks' resilience. ivanov's publications help identify different elements of risk preparedness, mitigation, and recovery policies. this study outlines several research propositions that can serve as a foundation for future research in the area of covid- . there are several theoretical, conceptual, and empirical research opportunities to understand the development of a new paradigm and advancement of existing theories within the business domain due to the disruption caused by the covid- pandemic. additional empirical research opportunities have been created by the emergence of a body of knowledge regarding covid- and subsequent modifications to this knowledge. this is the first academic work to recommend a set of propositions for future research work intended to advance this body of knowledge. this study could benefit managers interested in adopting a proactive approach to understand which changes in strategies, services, and products are required to meet unprecedented demands and develop sustainable business practices. a major practical lesson is that the covid- crisis is quite complex and has caused not only changes in existing business models but also a need to understand and observe transitions in the economy, business, and society. the propositions discussed above suggest that, to mitigate the covid- crisis, managers require forward thinking, new strategies, and re-planning on several fronts. the propositions give managers and decision-makers a variety of practical insights into the challenges posed by covid- and actions and reforms that must be carried out at the economic, social, and technological levels. the propositions also help managers to predict the need for advanced technologies, supply chain resilience, and organizational agility to achieve the right growth trajectory for growth. the economic consequences of the covid- crisis require urgent policy responses from the government to support individuals and businesses alike. properly designed policy reforms are critical for reducing global market distortions. governmental and industrial policy-makers play a paramount role in formulating short-, medium-, and long-term plans. governments must be adaptable as the circumstances of the covid- crisis evolve. the findings and propositions of this study demonstrate that governments should reform existing economic policies to meet individuals' immediate health, food, and other basic needs; maintain political and economic stability; and protect social cohesion. the propositions could help governments and policy-makers to respond effectively to future crises by reevaluating rules and regulations, increasing their digital footprint, and revisiting supply chains. the propositions could also help government agencies identify potential changes to policy tools to resolve medical and economic issues and restore shuttered services, like education, trade, hospitality, and travel and tourism. tables and provide synopses of the new research sub-areas related to covid- and business management. these synopses should provide a solid basis for advancing research in these domains. the evolution of the covid- literature in the business domain exhibits a distinct pattern. first, the diversity of topics and sub-topics addressed by scholars related to the covid- crisis is exponentially increasing, indicating that the virus has impacted our present and future way of life on various fronts. the impact of covid- on business and management is continuously attracting researchers, who are bringing new perspectives on research. second, while the diversity of research areas is increasing, a few broad research sub-topics are emerging more significantly. these core topics include the impacts of covid- on the economy, value chain, supply chain management, innovation, service industry, and employment. thus, the bibliometric results of this study provide evidence that covid- is gradually emerging as a discourse in the business area. the identified core topics serve as pathways for practitioners and academicians aiming to conduct future research. this bibliometric study revealed that, in a short time span (four and a half months), unique documents were published in scopus and wos from different journals, different institutes, and different countries. the findings identify the most active authors and sub-areas of research on business aspects related to covid- . thus, this study has helped identify potential covid- knowledge domains in the field of business and management. lastly, co-word analysis based on keywords provided insights into the main research themes/sub-areas related to the impact of covid- . the covid- crisis will have short-, medium-, and long-term effects on various aspects of society and businesses. this study is an early attempt to gain perceptive insights into the intellectual structure of covid- research in the arena of business using bibliometric analysis. the findings of this study complement existing subjective and evaluative literature reviews on covid- research. journals covered in the scopus and wos databases are published and reviewed each year to ensure their high quality. this bibliometric study utilized only these databases to collect data, but the absence of other databases, like google scholar, ebsco, and pubmed, may have influenced the data representation. therefore, future studies need to amply cover these databases in order to collect more comprehensive data and avoid bias. covid- : fear appeal favoring purchase behavior towards personal protective equipment impact of pandemic covid- on the teaching-learning process: a study of higher education teachers the covid- pandemic: how can risk science help us and uk labour markets before and during the covid- crash the intellectual contours of corporate social responsibility literature economic implications of the corona crisis and economic policy measures geographical tracking and mapping of coronavirus disease covid- /severe acute respiratory syndrome coronavirus (sars-cov- ) epidemic and associated events around the world: how st century gis technologies are supporting the global fight against outbreaks and epidemics employee adjustment and well-being in the era of covid- : implications for human resource management a bibliometric analysis of covid- research activity: a call for increased output covid- : a revelation-a reply to ian mitroff to recover faster from covid- , open up: managerial implications from an open innovation perspective how many workers are employed in sectors directly affected by covid- shutdowns, where do they work, and how much do they earn? effects of covid- on business and research forty-five years of journal of business research: a bibliometric analysis sicken thy neighbour: the initial trade policy response to covid- a co-citation bibliometric analysis of strategic management research creating effective franchising relationships: challenges of managing mature franchisees the rippled newsvendor: a new inventory framework for modelling supply chain risk severity in the presence of risk propagation managing the covid- pandemic through individual responsibility: the consequences of a world risk society and enhanced ethopolitics managing virtual workers-strategies for success a hybrid machine learning framework for analyzing human decision-making through learning preferences robustness in machine learning explanations: does it matter? socialising tourism for social and ecological justice after covid- the circular economy umbrella: trends and gaps on integrating pathways current status of global research on novel coronavirus disease (covid- ): a bibliometric analysis and knowledge mapping the covid- risk perception: a survey on socioeconomics and media attention predicting the impacts of epidemic outbreaks on global supply chains: a simulationbased analysis on the coronavirus outbreak (covid- /sars-cov- ) case viability of intertwined supply networks: extending the supply chain resilience angles towards survivability. a position paper motivated by covid- outbreak the impact of covid- on consumers: preparing for digital sales i'll trade you diamonds for toilet paper: consumer reacting, coping and adapting behaviors in the covid- pandemic fact-checking as risk communication: the multi-layered risk of misinformation in times of covid- new development: 'healing at a distance'-telemedicine and covid- a bibliometric analysis and visualization of medical big data research the covid- crisis-and the future of the economy and economics covid- and the world of work spillover of covid- : impact on the global economy doctor xai: an ontology-based approach to blackbox sequential data classification explanations competing during a pandemic? retailers' ups and downs during the covid- outbreak a systematic review of covid- epidemiology based on current evidence the human body is a black box" supporting clinical decision-making with deep learning mapping the themes and intellectual structure of corporate university: co-citation and cluster analyses international tourism and covid- software survey: vosviewer, a computer program for bibliometric mapping impact of international travel and border control measures on the global spread of the novel coronavirus outbreak interventions as experiments: connecting the dots in forecasting and overcoming pandemics, global warming, corruption, civil rights violations, misogyny, income inequality, and guns who announces covid- outbreak a pandemic trade set to plunge as covid- pandemic upends global economy communication related health crisis on social media key: cord- -yjn sja authors: o'connor, daryl b.; aggleton, john p.; chakrabarti, bhismadev; cooper, cary l.; creswell, cathy; dunsmuir, sandra; fiske, susan t.; gathercole, susan; gough, brendan; ireland, jane l.; jones, marc v.; jowett, adam; kagan, carolyn; karanika‐murray, maria; kaye, linda k.; kumari, veena; lewandowsky, stephan; lightman, stafford; malpass, debra; meins, elizabeth; morgan, b. paul; morrison coulthard, lisa j.; reicher, stephen d.; schacter, daniel l.; sherman, susan m.; simms, victoria; williams, antony; wykes, til; armitage, christopher j. title: research priorities for the covid‐ pandemic and beyond: a call to action for psychological science date: - - journal: br j psychol doi: . /bjop. sha: doc_id: cord_uid: yjn sja the severe acute respiratory syndrome coronavirus‐ (sars‐cov‐ ) that has caused the coronavirus disease (covid‐ ) pandemic represents the greatest international biopsychosocial emergency the world has faced for a century, and psychological science has an integral role to offer in helping societies recover. the aim of this paper is to set out the shorter‐ and longer‐term priorities for research in psychological science that will (a) frame the breadth and scope of potential contributions from across the discipline; (b) enable researchers to focus their resources on gaps in knowledge; and (c) help funders and policymakers make informed decisions about future research priorities in order to best meet the needs of societies as they emerge from the acute phase of the pandemic. the research priorities were informed by an expert panel convened by the british psychological society that reflects the breadth of the discipline; a wider advisory panel with international input; and a survey of psychological scientists conducted early in may . the most pressing need is to research the negative biopsychosocial impacts of the covid‐ pandemic to facilitate immediate and longer‐term recovery, not only in relation to mental health, but also in relation to behaviour change and adherence, work, education, children and families, physical health and the brain, and social cohesion and connectedness. we call on psychological scientists to work collaboratively with other scientists and stakeholders, establish consortia, and develop innovative research methods while maintaining high‐quality, open, and rigorous research standards. the global impact of the coronavirus disease (covid- ) is unprecedented. by the june , in excess of million cases of covid- worldwide had been confirmed and covid- -related deaths were close to half a million. however, its impact should not only be measured in terms of biological outcomes, but also in terms of its economic, health, psychological, and social consequences. the covid- pandemic is unique with respect to the ongoing risks associated with the large numbers of infected people who remain asymptomatic, the impacts of the countermeasures on societies, the likelihood of second or third waves, and the attention it has received due to its global reach (particularly in high-income countries). the effects of the covid- pandemic will likely shape human behaviour in perpetuity. psychological science is uniquely placed to help mitigate the many shorter-and longer-term consequences of the pandemic and to help with recovery and adjustment to daily life. the immediate research response to covid- was rightly to focus resources on the transmission of covid- , identify biologics with which to treat those infected with the virus, and develop vaccines to protect populations. however, biomedical science can only go so far in mitigating the severe negative health, economic, psychological, and social impacts of covid- . the future availability of a vaccine currently remains uncertain; therefore, the primary weapons to mitigate the pandemic are behavioural, such as encouraging people to observe government instructions, self-isolation, quarantining, and physical distancing. even if a vaccine becomes available, we will still require changes in behaviour to ensure its effective delivery and universal uptake, so we need to prioritize research that will make the greatest contributions to our understanding of the effects of, and recovery from, the pandemic. the important contributions made by psychological scientists to understanding the impact of previous pandemics, including the ebola disease outbreak, severe acute respiratory syndrome (sars), and the middle east respiratory syndrome (mers), are welldocumented and mean we knew already a lot about public messaging and stress among frontline workers when the covid- outbreak began (e.g., brooks et al., , holmes et al., rubin, potts, & michie, ; tam, pang, lam, & chiu, ; thompson, garfin, holman, & silver, ; wu et al., ) . however, the unique features of covid- , including its virulence, the large proportions of people who remain asymptomatic but may still spread the virus (centre for evidence-based medicine, ), the stringent lockdown procedures imposed at pace on whole societies, and its global reach mean there is an urgent and ongoing need for social science research (world health organisation, ) . the collective and individual responses to severe acute respiratory syndrome coronavirus- (sars-cov- ) and to the introduction of measures to counter it have fundamentally changed how societies function, affecting how we work, educate, parent, socialize, shop, communicate, and travel. it has led to bereavements at scale, as well as frontline workers being exposed to alarming levels of stress (e.g., british medical association, ; greenberg, docherty, gnanapragasam, & wessely, ) . there have additionally been nationwide 'lockdowns' comprising physical distancing, quarantines, and isolation with the associated effects on loneliness, forced remote working, and homeschooling (e.g., hoffart, johnson, & ebrahimi, ; holmes et al., ; lee, ) . however, as well as having adverse psychological effects, the measures introduced to fight the pandemic may have led to positive social and behavioural changes. most obvious are the remarkable levels of compassion and support that have developed among neighbours and within communities as well as positive changes in behaviours such as hand hygiene, homeschooling, and physical activity. therefore, in addition to mitigating the negative effects of the pandemic, it is important to understand how any positive effects can be maintained as restrictions ease. there are, and will undoubtedly continue to be, inequalities in the effects of the pandemic and its aftermath; recognizing these vulnerability and resilience factors will be key to understanding how the current situation can inform and prepare us for dealing with future crises. of course, while we, as psychological scientists, are interested in the general effects of the pandemic, we are acutely aware of the fact that these effects disproportionately impact on different groups (box ). the issue of inequality is of central importance and runs through the research priorities that we describe below and it is a picture is emerging of covid- not as a single pandemic, but multiple parallel pandemics with some people facing numerous severe challenges and others experiencing few or none (williamson et al., ) . for those most vulnerable groups, the social, economic, and consequent psychological challenges of the pandemic are likely to be far-reaching and sustained. a clear priority for psychological scientists is to understand how best to help those in need and to consider the following factors in their research efforts. in western europe and the united states, the death rate among people with black, asian, and minority ethnic backgrounds is substantially higher than that of the general population. it is not known what is causing the disproportionate impact nor how it can be mitigated. psychological science is in a good position to explore the biopsychosocial antecedents and consequences of having a black, asian, or minority ethnic background in the context of covid- . individuals living in poverty face disproportionate challenges in relation to education, work, income, housing, and physical and mental health. for these most vulnerable groups, the social, economic, and consequent psychological challenges of the pandemic are likely to be far-reaching and sustained. moreover, an impending financial crisis means that people who have never before experienced hardships may suddenly find themselves in precarious circumstances. a quarter of people in the uk experience mental health problems every year, with particularly high levels in young people (mental health foundation, ) . the changed social conditions of the pandemic may increase the severity of mental health challenges, particularly when standard (face-toface) treatment and support are difficult to access. at the same time, pregnant women and those with existing long-term conditions such as transplant patients, cancer patients, and chronic obstructive pulmonary disease patients have been designated 'extremely vulnerable' and asked to self-isolate for long periods of time with uncertainties over access to support. those individuals who have recovered from covid- might also have new biological vulnerabilities, uncertainty over immunity post-covid- , and risk stigma arising from infection. individuals with disabilities, learning disabilities, special educational needs, and developmental disorders may also be more vulnerable due to the increased psychological challenges associated with shielding and self-isolation. the challenges generated by the pandemic vary markedly across the lifespan and will influence the nature of current and future psychological needs of different groups. many young people have struggled with reductions in direct social contact, decreased motivation, and uncertainty caused by disrupted training and education. adults have experienced multiple stresses as a consequence of intensified caring responsibilities, financial concerns, job uncertainty, and health conditions. for many older people, the greatest challenges have been social isolation, disruptions in access to health and social care, and coping with bereavement. in addition to the challenges surrounding age, there are emerging data to suggest that the effects of covid- may exacerbate existing inequalities for women. for example, women are more likely to be key workers and primary caregivers, thereby being exposed to higher levels of psychological and financial stress (fawcett society, ). the covid- pandemic is likely to have had a disproportionate impact on groups with low levels of social inclusion and/or those who traditionally have declined support services, such as people living in poverty, traveller communities, and people who are homeless. being separated from wider support networks may also be particularly difficult for those living in hostile households such as victims of domestic abuse and lgbt people living with family members who are unaccepting of their identity. many of those detained in secure settings have been exposed to marked changes in service delivery and reduced social contact, increasing their vulnerability to the psychological effects of the pandemic. surely not a coincidence that the murder of george floyd during a global pandemic prompted a global civil rights movement drawing attention to inequalities. in this position paper, informed by a group of experts and a survey (box ), we highlight the many ways in which psychological science, its methods, approaches, and interventions can be harnessed to help governments, policymakers, national health services, education sectors, and economies recover from covid- (box ) and other future pandemics (if they occur). specifically, we have identified the shorter-and longerterm priorities around mental health, behaviour change and adherence, work, education, children and families, physical health and the brain, and social cohesion and connectedness in order to ( ) frame the breadth and scope of potential contributions from across the discipline, ( ) assist psychological scientists in focusing their resources on gaps in the literature, and ( ) help funders and policymakers make informed decisions about the shorter-and longer-term covid- research priorities to meet the needs of societies as they emerge from the acute phase of the crisis. the methodology we employed to develop the main research priority domains is described in box , and the seven priority domains are outlined below and summarized in table . how does collective identification impact on social responsibility and adherence to anti-pandemic measures? one of the most striking aspects of the covid- pandemic has been the importance of social psychology to the outcomes. given the highly differentiated nature of susceptibility to the virus (box ), one might have expected many (especially the young and fit) to conclude that they have more to lose than gain by observing the rigours of lockdown and other preventative measures. if they had acted on such an individualistic calculus, then far more people would get infected and far more (especially the old and infirm) would die. however, on the whole, people did not act on the basis of such narrow self-interest, and the vast majority supported the lockdown (e.g., duffy & allington, ) . what is more, conversely, well-functioning social support is likely to confer resilience against the negative psychological impacts of the pandemic. finally, it is important that psychological scientists consider the interconnectedness of the above factors. for example, individuals who are young and from a bame background who are also from a less affluent socio-economic background may be disproportionately impacted by the educational, economic, and other consequences of the measures taken to contain and recover from the pandemic. similarly, many of the solutions to the problems posed by the pandemic involve the use of new technologies that assume the requisite skills, access to devices, and internet connectivity meaning that the 'digital divide' will likely have been exacerbated by the pandemic (ons, ). this paper outlines research priorities for psychological science for the covid- pandemic. in april , the british psychological society convened a core group of nine experts who met regularly for weeks in order to develop the research priorities. the nine experts represent broad areas of the discipline, namely biological, clinical, cognitive, developmental, educational, health, occupational, and social, and were assisted by a wider advisory group of psychological scientists (n = ) drawn from a range of uk higher education institutions and areas of research expertise. we also received input from two international experts. briefly, we used an iterative expert consensus procedure (e.g., merry, cooper, soyannwo, wilson, & eichhorn, ) to elicit and distil the judgments of experts on the research priorities for psychological science. unlike other consensus methods, which typically start with a list of priorities that are then ranked over the course of or meetings (e.g., fitch, bernstein, aguilar, burnand, & lacalle, ; mcmillan, king, & tully, ) , the present approach both generated and judged the priorities over hour long face-to-face meetings of the core group. consensus was achieved through discussion, and the experts were encouraged to discuss with the wider advisory group and their professional networks in between meetings. given the need to establish the priorities rapidly, a lengthy consultation process or an extensive review of all relevant scientific literatures was not possible. however, a brief online survey of psychological scientists was launched early in may with the aim of ensuring that the core and advisory groups had not missed any key research priorities, and to identify the highest ranked priorities in each of the broad areas of psychology to help inform the final wider-ranging research priority domains. the online survey had two components: first, participants were asked the open-ended question, 'please can you tell us what are your priorities for psychological science research in response to the covid- pandemic?' second, participants were asked to rank order the top five research priorities identified by the core group in each of the eight broad areas of the discipline (i.e., biological, clinical, cognitive, developmental, educational, health, occupational, social). the survey was distributed to psychologists via heads of uk psychology department email lists, the social media outlets of professional psychology networks (including the british psychological society), and snowball email methods by the expert and advisory group members. we received replies from psychological scientists representing all of the main areas of the discipline. respondents were . % female, . % were aged between and years, and . % self-identified as being from a minority group. the highest ranked research priorities in each of the broad areas are presented in table (see appendix for the full list of priorities). as a result of the time constraints, a detailed qualitative analysis was not possible for inclusion in this paper; nevertheless, the core group gave consideration to all of the free responses provided. overall, there were differing degrees of specificity, and respondents provided numerous, additional, and wellspecified research questions. however, at the broadest level, respondents' priorities coalesced around the question of how do we address the negative biopsychosocial effects of the covid- pandemic? the degrees of specificity related to population (e.g., people with black, asian, and minority ethnic backgrounds, children, people with low socio-economic status, people living with long-term conditions), type of intervention (e.g., service provision, environmental/social planning), methodology (e.g., qualitative, online, survey, laboratory-based), and setting (e.g., workplace, school, prison), but there was broad agreement. perceived personal risk bears no relation to whether people adhere to government instructions: whether or not one identifies with the broader community and hence acts on the basis of the risks to the community as a whole is the key driver (jackson et al., ) . so, getting people to think in collective rather than personal terms is critical to controlling the pandemic (reicher & drury, ) . or, in the rather more forceful terms of new york governor andrew cuomo: 'yeah it's your life do whatever you want, but you are now responsible for my life. you have a responsibility to me. it's not just about you . . . we started saying, "it's not about me it's about we." get your head around the we concept. it's not all about you. it's about me too. it's about we'. how can we nurture the development and persistence of mutual aid and pro-social behaviour? the significance of such 'we-thinking' is not limited to issues of adherence and social responsibility. the literature on behaviour in disasters and emergencies (drury, ) suggests that the experience of common fate in such events leads to a sense of shared social identity that in turn underpins solidarity and cohesiveness between peopleeven strangers. we have seen numerous examples of 'we-thinking' in the time of pandemic, which have played a key role in sustaining people through difficult circumstances. these range from neighbours knocking on doors to see whether people need help to over three million people contributing to more than four thousand mutual aid groups across the uk (butler, ) . so, how can we nurture such we-thinking in order to build mutual aid in communities and ensure it endures even after the acute phase of the covid- pandemic is over? what is the relationship between group membership, connectedness, and well-being? there is growing evidence of the role of group membership in sustaining both physical and mental health (haslam, jetten, cruwys, dingle, & haslam, ) . in addition to asking in general terms about how group identities are created, sustained, or else undermined in times of crises, we also need to investigate further the interface between group processes and health during and after periods of crisis. in other words: how can we keep people psychologically together even when they are physically apart and what is the relationship between face-to-face and virtual groups in terms of their health effects? more generally, that is not to say that all research priorities were covered in the original survey. two issues in particular stood out from the comments we received. the first was the importance of dealing with inequalities and differences between groups in the experience of the pandemic. the second was the need to address the positive as well as the negative developments coming out of the response to covid- . these were both incorporated into revisions of the paper and now occupy a much more central place than before. we are thankful to all those anonymous respondents whose comments helped improve our argument. a more rigorous, thematic analysis of these data is now available (see bps, c). the picture was very similar when respondents were asked to place research priorities identified by the expert group into rank orders. that is, broadly speaking, the priorities that received the highest rankings, irrespective of area of subdiscipline, were related to the need to address the negative biopsychosocial effects of the covid- pandemic. box : psychological science: methods, approaches, and interventions to help meet the immediate and longer-term covid- research priorities the future research landscape will be challenging due to the ongoing physical distancing requirements; however, psychological scientists are equipped with a broad range of methods, approaches, and interventions that will allow these research priorities to be met. some examples are as follows: internet-mediated research will be an important approach utilized by psychological scientists to collect data in the immediate post-pandemic phase and at longer-term follow-ups. internet-mediated research can be reactive (e.g., online surveys, online interviews) and non-reactive (e.g., data mining, observations from screen-time apps) and can be integrated with objective assessments of behaviour as well as with biological and social markers of physical and mental health. internet-mediated research can also be used to run experiments with online software available such as gorilla, psychopy, and e-prime. recent work has summarized the range of software for building behavioural tasks, and their efficacy in being used online (sauter, draschkow, & mack, ) . changes in the use of research methodologies may provide a catalyst for the formation of new collaborations and training to develop research skills in the psychological science community. at the same time, trust around data security and confidentiality will need to be built between researchers and the general public from whom we sample. however, in , more than an estimated million people aged - years in the european union reported they had not used the internet in the preceding months (eurostat, ) , and researchers will need to think creatively about conducting research projects remotely. for example, participants can have study materials delivered by post (e.g., salivettes for cortisol sampling or asking participants to self-sample), replacing face-to-face communication with telephone and/or video calls, and the use of personal protective equipment when collecting data. psychological therapies and behaviour change interventions can already be delivered remotely and evidence suggests that remote delivery does not necessarily mean inferior delivery (e.g., irvine et al., ) . urgent research is needed to translate interventions that are typically delivered in-person to telephone and online delivery modalities. psychologists are well-positioned to collect valuable qualitative data concerning people's relevant experiences, perspectives, and practices associated with covid- , which could inform psychologybased interventions to improve well-being and social cohesion. multiple participant-centred qualitative research methods can be rapidly deployed to elicit first-hand accounts from members of different communities, including (online) interviews, focus groups, and qualitative questionnaires, focusing on the psychological and social impact (jowett, ) . beyond the immediate term, qualitative data can be gathered longitudinally so that insights can be generated into the experiences of diverse groups over time, identifying salient crisis points and effective resolutions. implementation science is a branch of psychological science that is dedicated to the uptake and use of research into clinical, educational, health care, organizational, and policy settings. principles of implementation science can be used to help stakeholders navigate the extensive and unwieldy psychological science research literature. to inform policymakers and support professional decisionmaking about implementation, psychological research needs to be disseminated in an accessible format. one example of a well-regarded translational system is the us institute of education sciences what works clearinghouse (https://ies.ed.gov/ncee/wwc/), which provides reviews and recommendations about evidence-based practices for professionals working in educational settings. can we learn from this in order to improve the plight of socially isolated people as we emerge from the acute phase of the pandemic? under what conditions does unity and social solidarity give way to intergroup division and social conflict? finally, in addressing the positive potential of social psychological processes, we must not forget their darker side. 'we' thinking can all too easily slip into 'we and they' thinking, where particular groups are excluded from the community and then blamedeven an important feature of the covid- pandemic has been requested by government to provide psychological science expertise at pace. the inclination of many psychological scientists is to begin designing a new study or conducting a systematic review following preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines, but this does not meet the needs of policymakers. it would be valuable for psychological scientists providing expert advice to acquaint themselves with the terminology and procedures that are familiar to civil servants who are more likely to have use for a quick scoping review or rapid evidence assessment (collins, coughlin, miller, & kirk, ) rather than embarking on a time-consuming systematic review of systematic reviews (keyworth, epton, goldthorpe, calam, & armitage, ) . there are many challenges involved with conducting covid- -related research including dealing with vulnerable groups, giving due consideration to ethical concerns, as well as issues around running studies in the light of physical distancing requirements. therefore, having relevant patient and public involvement and including individuals with lived experience (as appropriate) in designing studies will be of paramount importance. psychological science has been leading the way in promoting and adopting open science principles and practices. nevertheless, psychological scientists need to ensure they balance the urgency of conducting covid-related research (during and in the recovery period) with ensuring research quality and open research practices. therefore, in order to help maintain quality, openness, and rigour, we urge researchers to endeavour to use registered reports, where possible (e.g., https://osf.io/rr/), or preregister their research hypotheses and analysis plans (e.g., https://aspredicted.org/) and make their data findable, accessible, interoperable, reusable (fair) recognizing the principle of 'as open as possible; as closed as necessary' (bps, a (bps, , b norris & o'connor, ) . moreover, we urge researchers to utilize pre-print servers, such as psyarxiv, in order to ensure their latest research findings are made publicly available rapidly and at no cost. we hope that openness will drive quality, but as yet there is no substitute for articles being peer-reviewed prior to wider acceptance by the scientific community. psychological science has responded swiftly to the covid- pandemic, but there is a danger of duplication of efforts and participant fatigue in the proliferation of online surveys, experiments, and focus groups that have arisen. we need to harness the ongoing efforts of psychological scientists worldwide in a coordinated effort on the scale of the large hadron collider (cern, ) to deliver truly evidence-based interventions to help societies emerge from the covid- pandemic. this will include cross-cultural research to understand why mortality rates, mitigation measures, and adherence to government instructions have differed so markedly between countries. finally, we urge researchers to register their research studies and findings on international repositories (https://osf. io/collections/coronavirus/discover). attackedfor the crisis. thus, un head antonio guterres has warned of a 'tsunami of hate' unleashed by the pandemic (davidson, ) . this hate and violence can take different forms: of anti-authority riots as in france (willsher & harrap, ) , or of racist violence against minorities as in india (mazumdaru, ) . in sum, insights from social psychology can be a valuable resource in a crisis; it can bring people together and generate constructive social power. but equally, it can set people apart and create problems that endure well beyond the crisis itself. it is evidently of the greatest importance to understand the processes that determine whether people unite or divide in hard timesand notably to understand the role of leadership, which has been so significant and so diverse in different countries during covid- . work environment and working arrangements consistent with previous pandemics (e.g., rubin et al., ) , the work-related challenges of the pandemic have been particularly high and widely recognized for health and social what is the impact of remote and flexible working arrangements on employee health, mental wellbeing, teamwork, performance, organizational productivity, and colleague/client relationships? what is the impact of social distancing in the workplace on employee health, mental well-being, teamwork, performance, organizational productivity, and colleague/client relationships? how can organizational resilience be developed to deal with the impact of covid- whilst supporting employees and protecting jobs? how will the covid- pandemic affect children's development? how will the covid- pandemic affect family functioning? how do school closures influence children's educational progress and well-being? what kinds of support improve long-term outcomes for children and young people? how can support services be effectively delivered to vulnerable children and young people, families, and schools? what are the immediate and longer-term consequences of covid- for mental health outcomes? what changes in approaches resulting from the pandemic need to be harnessed for the future? . physical health and the brain does covid- have neurological effects on the brain with consequences for mental health? what are the psychobiological impacts of the covid- pandemic on physical and mental health? how do we best apply existing theories and tools to promote sustained behaviour change among policymakers, key workers, and the public/patients? how do we develop new theories and tools to promote sustained behaviour change? care workers in direct contact with patients suffering the effects of covid- , leaving them vulnerable to trauma, fatigue, and other manifestations of chronic stress. what is unique about covid- is that changed working conditions and anxiety about infection have affected almost all employees, with particular challenges being faced by delivery workers, shop assistants, teachers, emergency services personnel, care home staff, transport staff, and social workers. the full economic severity of the covid-related restrictions is uncertain, although up to two million people could lose their employment in the uk alone (wilson, cockett, papoutsaki, & takala, ) . for those people still working, and those about to return to work, there are notable changes that will likely affect working practices in the foreseeable future. therefore, understanding the impact of the covid- pandemic on the work environment and new working arrangements is paramount to kick starting the economy and adjusting to daily life. what is the impact of remote and flexible working arrangements on employee health, mental wellbeing, teamwork, performance, organizational productivity, and colleague/client relationships? for many workers, particularly those in white-collar occupations, work took place entirely from home during the lockdown. it is possible that the lockdown will accelerate the general increase observed in home working practices (ons, ). a move to greater levels of remote working has clear economic benefits for employers (e.g., reduced estates costs). the flexibility to balance work and family life is also attractive to many employees (cf. strategic review of health inequalities in england, ). overall, the evidence points to positive benefits of remote working in terms of well-being (charalampous, grant, tramontano, & michailidis, ) , although these effects are not consistent. for example, it may lead to greater levels of professional isolation (golden, veiga, & dino, ). an increase in remote working will likely occur with a concomitant increase in the use of online technology to support communication and aspects of collaborative working. this has the potential to blur boundaries between work and home domains, resulting in negative impacts on well-being and productivity from work-home interference (van hoof, geurts, kompier, & taris, ) . greater use of technology may also be associated with different perceptual and cognitive demands that may affect productivity and wellbeing including social connections with work colleagues (e.g., mark et al., ) . what is the impact of physical distancing in the workplace on employee health, mental well-being, teamwork, performance, organizational productivity, and colleague/client relationships? until an effective vaccine is available, physical distancing rules will need to continue to be in place in work environments and we may experience multiple stay-at-home versus return-towork cycles. there is very little research exploring physical distancing and its effect on the general workplace, but returning to work will likely be both a welcome change and a potential stressor. while we have research from teams working in difficult and extreme environments (power, ; smith, kinnafick, & saunders, ) and research on professional isolation (golden et al., ) , this is an unprecedented opportunity to study adaptation across a breadth of individuals and organizational settings. how can organizational resilience be developed to deal with the impact of covid- whilst supporting employees and protecting jobs? the unprecedented demands that the pandemic has placed on organizations also offer a unique opportunity to understand how organizational resilience and preparedness for dealing with disruptions and emergencies can be developed. while a pandemic of this nature is rare, we can anticipate increasing periods of disruption due to covid- flareups and additionally, for example, in response to climate-induced events (e.g., recent australian fires, uk flooding), which are predicted to occur more frequently (banholzer, kossin, & donner, ) . although we know a lot about individual resilience, we know relatively little about organizational resilience, especially in the context of well-being and performance (taylor, dollard, clark, dormann, & bakker, ; fasey, sarkar, wagstaff & johnston, under review) and the ingredients such as the structures, processes, culture, and leadership that are essential for developing organizational resilience. parenting can be a challenging and anxiety-provoking experience at any time, but the covid- pandemic has brought these challenges and anxieties into sharp focus. for most families, the lockdown will represent the longest period of parenting they have experienced without ( ) the support of extended family members, friends, and childcare professionals; ( ) the routine of school and out-of-school activities; and ( ) any face-to-face social life outside the home. these changes in the social environment may have both negative and positive impacts on children and their families. at the most extreme end of the spectrum, the restrictions in place to combat the spread of the virus have been associated with worrying increases in domestic violence and child abuse. however, all families are likely to have experienced greater levels of stress (social care institute for excellence, ). the majority of carers with school-age children are dealing with homeschooling for the first time, and many carers are having to adapt to working from home while also looking after their children and older relatives. these pressures will be particularly acute for single-carer families. of course, such multi-tasking concerns apply only to carers fortunate enough to have maintained employment. it is important to support families during the current crisis, but also to understand the implications of these unprecedented changes in family life for family functioning and children's development as we emerge from the pandemic. how will the covid- pandemic affect family functioning? many effects of the pandemic on children's development are likely to be indirect, functioning through its impact on caregiving and family functioning. it is crucial for this research to include family members such as grandparents and non-resident parents and siblings. children in families who are already vulnerable due to domestic violence or abuse, social or economic disadvantage, or physical or mental ill health are likely to be most adversely affected. there is an urgent need for research to examine how these vulnerabilities moderate changes in family functioning post-pandemic and their impacts on the child. the ability to regulate behaviour and emotional responses is a key aspect of successful social interaction in individuals of all ages (e.g., baumeister & heatherton, ; kochanska, murray, & harlan, ) . family members may develop new self-regulation strategies as a result of having extended contact with the same restricted group of people. while such strategies may be adaptive, individuals facing extreme social or financial challenges may cope by psychologically distancing themselves from family members, ruminating on negative events, or engaging in behaviours that are harmful. understanding how adaptive and maladaptive self-regulation strategies change post-pandemic may prove useful in identifying individuals who need additional psychological support. school closures and social restrictions may provide a unique opportunity for family members to gain insight into each other's lives, potentially reducing disagreements and improving family functioning. research should investigate whether reporting such improvement during the crisis is associated with lower caregiving stress and better mental health. it is also important to study how families can maintain any positive aspects of functioning that have resulted from the pandemic as restrictions are eased. how will the covid- pandemic affect children's development? the effects of the pandemic will undoubtedly vary as a function of the child's age. while carers with young infants may have concerns about the negative impact of the lockdown on their babies' development, the infants themselves will be unaware of the abnormal nature of their social environment. optimal later development is predicted by caregivers' ability in the first year of life to see the world from the infant's point of view and respond appropriately to their cues (e.g., fraley, roisman, & haltigan, ; zeegers, colonnesi, stams, & meins, ) . the social restrictions do not obviously impede this type of infantcaregiver engagement, and young infants may therefore be least affected by the pandemic. older children who recognize the drastic changes in social contact may find transitioning back to pre-pandemic social behaviour difficult. it is therefore important to study how children and young people manage this transition and investigate whether the lockdown has raised the incidence of emotional and behavioural difficulties. studying the effects of the pandemic and its aftermath on particular groups that are known to be vulnerable to educational and health disadvantage (e.g., looked after children or children with developmental disorders) should be prioritized. positive effects of the pandemic on children's behaviour and social interaction are also anticipated. many children and young people will have found new ways to communicate with friends, entertain themselves, and keep themselves physically active. time away from school may have been spent learning new skills, developing new hobbies, or helping or supporting others. investigating changes in children and young people's empathy, altruism, theory of mind, creativity, innovation, problem-solving, and cognitive flexibility post-pandemic will help shed light on potential positive outcomes of the social restrictions associated with the pandemic. the challenges posed by the covid- pandemic have never been more evident than for the education and well-being of children and young people. in april , a third of the world's population were experiencing extended periods of lockdown with closure of schools and nurseries. parents, many of whom had work and other family responsibilities had to adopt the additional role of educator in home environments not set up for formalized learning. ad hoc arrangements were put in place at speed by schools with limited opportunities to develop clear definitions of learning activities, provide access to learning resources, and establish effective home-school communication. early surveys have shown wide variation in homeschooling arrangements, including stark differences between state and private schools in access to online learning and pupil-teacher communication (sutton trust, ) . there is a wealth of evidence about the factors that facilitate effective learning in schools, such as curricula and teaching strategies (hattie, ) . other studies have established that children's academic attainment and adjustment are predicted by higher caregiver education (erola, janolen, & lehti, ) and engagement in schooling (harris & goodall, ) . however, little is known on how to set up and deliver home education effectively under the unique conditions of the pandemic. while for some children the extended period at home is likely to have distinct positive benefits, research prior to covid- on substantial externally driven disruptions in schooling has shown adverse effects on child achievement and well-being (meyers & thomasson, ; sunderman & payne, ). the outcomes for the individual child are likely to depend on the capacity of families to step in and effectively support curriculum delivery at home. studies of other severe unplanned disruptions to schooling and family lives such as long-running strikes and natural disasters have shown greatest impacts on long-term educational and emotional outcomes for the most disadvantaged children (jaume & will en, ; masten & osofsky, ) . at particular risk of disproportionate adverse outcomes are children from families living in poverty, those receiving social care support, individuals with special educational needs and disabilities, and young people with mental health problems. there are high levels of concern that the recognized attainment gap for children from disadvantaged families (education in england: annual report . education policy institute) could be magnified by the pandemic conditions. there is an urgent need to identify and understand both the positive and negative factors that influence children's educational outcomes during and after the pandemic, and to use this knowledge to target support to those who need it most. the unanticipated consequences of the pandemic pose challenges for conventional designs depending on pre-intervention assessments. understanding its impacts on children's lives will require a robust body of research that draws on the diverse research methods of psychological science. this will require large-scale multidisciplinary data collection in addition to smaller-scale quantitative and qualitative approaches that will be vital for understanding the experiences of children, families, and professionals. some key questions to be addressed by this research are outlined below. in addition to collecting data on home-based support for learning, detailed contextual data are needed about social and environmental factors that are likely to interact in determining positive educational outcomes at particular educational phases (e.g., reading, writing, and maths in primary schools), as well as a range of mental health outcomes (e.g., anxiety, depression, self-harm, resilience). this will include research into the effect of social distancing on a range of social outcomes in children and young people (e.g., inclusion/ exclusion, friendships). what kinds of support improve long-term outcomes for children and young people? knowledge about the impacts of school disruptions on all children and young people will allow evidence-based interventions and resources to be targeted at those with greatest need. robust evaluations are required to scrutinize how interventions are accessed, by whom and with what degree of success. how can support services be effectively delivered to vulnerable children and young people, families, and schools? with reduced resources and restricted movement, professionals (such as practitioner psychologists) have had to adapt and develop new ways of delivering services. researchers in psychological science have a key role to play in working with practitioners and service providers to evaluate systems put in place for monitoring and delivering professional support during and in the aftermath of the pandemic. what are the immediate and longer-term consequences of covid- for mental health outcomes? there is expected to be an increase in mental health problems as a result of the covid- pandemic and the measures used to counter it. we already have evidence for the long-term mental health effects of previous pandemics and disasters (e.g., tam et al., ; thompson et al., ; wu et al., ) and an emerging literature on the near-term effects of covid- (e.g., ahmad & rathore, ; williamson et al., ) . but previous pandemics have been more localized and circumscribed making covid- different. social distancing, school closures, self-isolation, and quarantine have lasted longer than anything previously experienced. we know that these factors, together with financial uncertainty and concerns about health, are predictive of mental health difficulties, particularly anxiety. the current pandemic amplifies these factors and not only exacerbates problems in those with pre-existing mental health difficulties, but also increases the chance of new onset in those with no previous contact with mental health services. concerns about mental health effects may be particularly heightened for children, who have experienced high levels of disruption to normative developmental opportunities (including opportunities for social and outdoor play) and education, and potentially high levels of family stress (https://emergingminds.org.uk/cospace-study- ndupdate/). various poor mental health outcomes are also potentially associated with the disease itself. information about the long-term consequences comes from similar viruses such as sars and the mers. for example, many people who suffered from sars seemed to experience detrimental psychological effects even a year later (rogers et al., ; tam et al., ; thompson et al., ; wu et al., ) . therefore, we need to establish the immediate and long-term consequences of covid- on mental health outcomes in the population generally, but also in vulnerable, shielding, and self-isolating groups (box ). we urgently need to understand how all these factors interact and whether these consequences will require psychological interventions and supports not currently available. what changes in approaches resulting from the pandemic need to be harnessed for the future? even if the mental health consequences of this pandemic are not as predicted, we still expect increases in mental health problems. we know that mental health accounts for an increasing proportion of sick leave and that one in eight children and young people experience a diagnosable mental health problem (nhs digital, ) . childhood mental health problems often recur in adulthood (kessler et al., ) and are associated with physical health difficulties, poor academic, and occupational functioning, and are the primary predictor of low adult life satisfaction (layard, clark, cornaglia, powdthavee, & vernoit, ) . the increased prevalence will place a further burden on a mental health system that was already stretched and will increase waiting times and accentuate gaps in care. during the pandemic, mental health services rapidly changed. inpatients were discharged, even if they were detained in hospital because they were a risk to themselves or others. some people benefited, but we do not know how this reduction in bed use was managed. was it because the right supports and accommodation were provided? the move to remote contact in mental health services had been slow and of varied quality prior to covid- with challenges for both staff and service users. but the shift during the pandemic was swift, and although undoubtedly nhs staff felt pressure during the changeover, there now seems to be a steadier state. again, some service users may have benefited from this change with reductions in travel and, for some, better access to care and treatment. however, although the digital divide is reducing (robotham, satkunanathan, doughty, & wykes, ) , it remains highest in those who already have high unmet needs, including people in rural areas, those on lower incomes, people with lower levels of formal education, and older people. if remote working is tobe abeneficial part of an evolved mental health service, then we need to understand how to provide that 'webside' manner that will increase adherence and promote a therapeutic alliance. we also urgently need to evaluate the effectiveness of remotely delivered, digital interventions in the immediate and longer term. future interventions will need to be deliverable remotely, depending on local resources. for example, from an international perspective, many low-to-middle-income countries do not have high broadband penetration; hence, optimizing digital delivery that depends strongly on good internet connections will further widen the welfare gap. physical health and the brain the effects of covid- on health outcomes will be far-reaching and complex. for those falling ill, there are the direct consequences of the disease symptoms, such as respiratory failure in severe cases, alongside potentially direct viral effects on the brain. there are also more indirect population-wide effects of covid- pandemic-related stress and anxiety on physical and mental health, not only from the disease itself but also from changes in lifestyle including delayed treatment and screening for other known or suspected conditions. moreover, it is also likely that from an international perspective, in many lowto-middle-income countries, the pandemic will result in greater hunger/starvation, which will have severe impacts upon health. does covid- have neurological effects on the brain with consequences for mental health? at one level, covid- might alter mental health by the direct actions of the specific virus (severe acute respiratory syndrome coronavirus- ; sars-cov- ) on the brain. while neurological dysfunction is often described in covid- , including dizziness, and loss of taste and smell, these conditions are common to other respiratory tract infections and need not reflect a neurological disease per se (needham, chou, coles, & menon, ) . data from cerebrospinal fluid and post-mortem analyses will help resolve issues over the penetrance of sars-cov- . it is, however, known that the target receptor for sars-cov- is the angiotensin-converting enzyme- receptor (ace ). disruption of the blood-brain barrier during illness might enable entry of the virus, potentially aided by the presence of ace receptors in glial cells and brain endothelium. other potential routes of entry include the cribriform plate and olfactory epithelium, as well as via peripheral nerve terminals, permitting entry to the cns through synapse connected routes (ahmad & rathore, ) . at the same time, there is an array of immunological responses, including the cytokine 'storm' in severe cases, alongside non-immunological insults to the central nervous system provoked by covid- . the latter include hypoxia, hypotension, kidney failure, and thrombotic and homeostatic changes involving neuroendocrine function (needham et al., ) . together and separately, they may contribute to brain dysfunction in ways that vary with the severity of the infection, other underlying conditions (needham et al., ) , and the treatment for those other conditions (south, diz, & chappell, ) . largescale studies help confirm differential clinical risk factors for death following infection (williamson et al., ) , prompting genotype analyses, while noting that covid- might also induce epigenetic changes, including ace demethylation (sawalha, zhao, coit, & lu, ) . additional health concerns include post-viral fatigue and whether it might provoke a long-lasting syndrome. research consortia are initiating comparisons between populations that have or have not contracted covid- . challenges for psychological scientists include how to assess impacts on cognition and mental health, both in the short term and long term. a part of this challenge is how to deliver effective, online psychological testing (e.g., for 'shielded' populations), or to help follow-up large population cohorts, while not biasing the sample away from those least likely to use these platforms. an integral part of some investigations will be the inclusion of multiple neuroimaging methods, despite the era of distancing. just one of many questions would be the impact of covid- on mild cognitive impairment and its conversion to dementia. there is a premium on studying pre-existing cohorts (e.g., uk biobank, alspac), where retrospective, baseline data exist. such data are especially precious in the present landscape where everyone is, to some degree, affected by the pandemic. the power of these pre-existing cohorts will, however, be heavily influenced by the proportion of the population who contract covid- . what are the psychobiological impacts of the covid- pandemic on physical and mental health? despite the umbrella term 'stress' covering many different things, there is agreement that in its different forms, stress can lead to physiological changes (e.g., neuroendocrine, cardiovascular), with negative consequences for health (o'connor, thayer & vedhara, in press). three principal research questions can be identified: ( ) to what extent does pandemic-related stress, anxiety, and worry impact on biological mechanisms that influence health (i.e., hypothalamic-pituitary-adrenal axis regulation and cortisol dynamics, the autonomic nervous system, and gene expression) as well as on health behaviours (e.g., eating, sleep, alcohol consumption)? ( ) how best to counter their adverse effects? and ( ) how might such stress exacerbate existing medical and mental health conditions, and for how long? for all three questions, there will be considerable variations between groups and individuals (box ). one challenge will be to collate and verify relevant information, including that from 'smart' devices that can provide daily physiological data, activity information, and other measures of diurnal patterns, including sleep. one of the groups most likely to be negatively affected by stress is health care professionals. the pandemic may exacerbate the already high prevalence of secondary traumatic stress, burnout, and physical exhaustion among health care professionals, as well as impact on patient safety and medical error (e.g., dar & iqbal, ; figley, ; hall, johnson, watt, tsipa, & o'connor, ) , due to excessive workload and workplace trauma (e.g., itzhaki et al., ) . while resources such as support from managers and colleagues can help protect health care professionals against traumatic stress, the longerterm impact is likely to be substantial on individuals, their families, on the national health services and the wider care industry. amongst other groups of concern (box ) are those caring for a vulnerable relative or partner at home. one novel feature of daily life in the wake of the covid- pandemic in countries around the world are near-daily government briefings. one focus of these briefings is government instructions to the public as to how to behave. adherence to these and future instructions will be key to dealing with future crises. moreover, many sections above share in common the requirement that people adhere to instructions, whether it is practitioners delivering psychological therapies effectively over the telephone or employees continuing to maintain physical distancing at work. in the initial response to the pandemic, many governments instructed people to ( ) stay inside as much as possible; ( ) stay > m away from other people at all times; and ( ) maintain hand hygiene, among other measures such as wearing face coverings. the evidence suggests that public adherence to government covid- -related instructions worldwide has been high (ons, ), but it is not clear for how long people will continue to adhere to instructions that impinge on personal freedoms. what is clear is that there is a dearth of workers sufficiently trained to advise policymakers and to implement behaviour change interventions rapidly and at scale. the british psychological society's guidance on behaviour change is a good starting point for ensuring that instructions and messaging is clear (british psychological society, a). appointing chief behavioural science advisers to governments would ensure that cuttingedge psychological science advice is placed at the heart of policymaking. as people begin to emerge from the acute phase of the pandemic and the changes that were made to tackle it, it is important that psychological science is at the heart of ensuring that health-enhancing behaviours are sustained and that health-damaging behaviours are changed or prevented. there are numerous approaches to developing such interventions, including the behaviour change wheel (michie, atkins, & west, ) and intervention mapping (bartholomew eldrigde et al., ) , but they require the expertise of psychological scientists to deliver and to evaluate them (west, michie, rubin, & amlôt, ) . one of the main challenges now, and in the future, will be to ensure there is a workforce equipped with the competencies to develop behaviour change theory and tools that will bring about sustained changes in behaviour. taught post-graduate courses exist that could be scaled up and/or adapted to continuing professional development qualifications to meet this demand and help ensure that the changes in behaviour that will be required for the foreseeable future are sustained. how do we best apply existing theories and tools to promote sustained behaviour change among policymakers, key workers, and the public/patients? we sometimes forget that we have the theories and evidence for solutions that can be applied at pace to address novel problems. although we have never seen a lockdown before and so cannot predict what the outcomes will be directly, we do know what processes underpin adherence to instructions, and so can advise on the levers that can sustain adherence. in unprecedented and uncertain times now and in whatever the future might bring, the nature of psychological science allows us to make unique and invaluable contributions. if the covid- pandemic teaches us one thing, it is on the need to accelerate the translation of evidence from psychological science into practice. how do we develop new theories and tools to promote sustained behaviour change? at the same time, we should not forget the 'slow' approach to research (armitage, ) that involves addressing key research questions with multiple perspectives and methodologies, and accumulating such knowledge in prisma-guided systematic reviews. it is vital that continued investment is made into behaviour change research. only with this can we refine and develop the theories that best explain human behaviour (e.g., michie et al., ) . key research priorities include identifying which behaviour change techniques work best, for whom, in which contexts, and delivered by what means (e.g., epton, currie, & armitage, ) as well as how to counter the conspiracy theories and misinformation that arise during crises that seem to be aimed at derailing the very behaviours required to keep us safe and to reduce risk. in this position paper, we have set out seven research priority domains in which psychological science, its methods, approaches, and interventions can be harnessed in order to help governments, policymakers, national health services, education sectors, economies, individuals, and families recover from covid- . these are mental health, behaviour change and adherence, work, education, children and families, physical health and the brain, and social cohesion and connectedness. we have also highlighted that a clear overarching research priority relates to understanding the inequalities in the effects of the pandemic and recovery; recognizing the vulnerability and resilience factors that will be key to understanding how the current pandemic can inform and prepare us for dealing with future crises. we call on psychological scientists to work collaboratively with other scientists in order to address the research questions outlined, refine them and to adopt multidisciplinary working practices that combine different disciplinary approaches. an important next step will be to engage with wider stakeholders, potential users, individuals with lived experience, and beneficiaries of the research. addressing each of the research priority domains will benefit enormously from larger scale working and coordinated data collection techniques and the establishment of research consortia with their associated economies of scale. we also call on psychological scientists to further develop and adapt innovative research methodologies (e.g., remote testing and intervention delivery, online data collection techniques), while maintaining high-quality, open, and rigorous research and ethical standards in order to help with the recovery as we emerge from the acute phase of the crisis. how can we use biological markers to facilitate people's return to work? how do we link covid- -related biomarkers to existing population cohort databases? how do we address the negative biological impacts of the covid- virus on mental health? what are the impacts of covid- infection, treatment, and recovery on the brain? how do school closures influence educational progress, and physical and mental health outcomes for all children and young people? what 'homeschooling' practices are associated with positive educational and psychological outcomes? what is the effect of social distancing on a range of social outcomes in children and young people? what methods are used to track, monitor, and deliver local authority support services to vulnerable children and young people, families, and schools during lockdown, at transition back to school, and after return to school? how are educational and psychological interventions allocated, structured, delivered, and evaluated for children and young people in need, after schools have reopened? what is the impact of remote and flexible working arrangements on employee health, mental well-being, teamwork, performance, organizational productivity, and colleague/client relationships? what is the impact of social distancing in the workplace on employee health, mental well-being, teamwork, performance, organizational productivity, and colleague/client relationships? what managerial behaviours are most effective to manage remote working, possible mental health issues, job insecurity, and productivity? what is the risk of longer-term mental ill health among frontline staff after the immediate crisis? how can organizational resilience be developed to deal with the impact of covid- whilst supporting employees and protecting jobs? neurological manifestations and complications of covid- : a literature review changing behaviour, slow and fast: commentary on peters, de bruin and crutzen the impact of climate change on natural disasters planning health promotion programs: an intervention mapping approach self-regulation failure: an overview stress and burnout warning over covid- behavioural science and disease prevention taskforce. behavioural science and disease prevention: psychological guidance position statement on open data covid- research priorities for psychological science: a qualitative analysis the psychological impact of quarantine and how to reduce it: rapid review of the evidence covid- mutual aid: how to help vulnerable people near you. the guardian lhc the guide systematically reviewing remote e-workers' well-being at work: a multidimensional approach the production of quick scoping reviews and rapid evidence assessments: a how to guide beyond linear evidence: the curvilinear relationship between secondary traumatic stress and vicarious posttraumatic growth among healthcare professionals global report: virus has unleashed a 'tsunami of hate' across world, says un chief. the guardian the role of social identity processes in mass emergency behaviour: an integrative review the accepting, the suffering and the resisting: the different reactions to life under lockdown. the policy institute, king's college london unique effects of setting goals on behavior change: systematic review and meta-analysis parental education, class and income over early life course and children's achievement what do you use the internet for? defining and characterising organisational resilience in elite sport coronavirus: urgent call for uk government to support women and girls compassion fatigue: coping with secondary traumatic stress disorder in those who treat the traumatized the rand/ucla appropriateness method user's manual the legacy of early experiences in development: formalizing alternative models of how early experiences are carried forward over time the impact of professional isolation on teleworker job performance and turnover intentions: does time spent teleworking, interacting face-to-face, or having access to communication-enhancing technology matter managing mental health challenges faced by healthcare workers during covid- pandemic healthcare staff wellbeing, burnout, and patient safety: a systematic review do parents know they matter? engaging all parents in learning the new psychology of health: unlocking the social cure visible learning, a synthesis of over meta-analyses relating to achievement loneliness and social distancing during the covid- pandemic: risk factors and associations with psychopathology multidisciplinary research priorities for the covid- pandemic: a call for action for mental health science are there interactional differences between telephone and face-to-face psychological therapy? a systematic review of comparative studies mental health nurse's exposure to workplace violence leads to job stress, which leads to reduced professional quality of life the lockdown and social norms: why the uk is complying by consent rather than compulsion the long-run effects of teacher strikes: evidence from argentina carrying out qualitative research under lockdown -practical and ethical considerations. lse impact blog age of onset of mental disorders: a review of recent literature delivering opportunistic behavior change interventions: a systematic review of systematic reviews effortful control in early childhood: continuity and change, antecedents, and implications for social development what predicts a successful life? a life-course model of well-being mental health effects of school closures during covid- email duration, batching and self-interruption: patterns of email use on productivity and stress disasters and their impact on child development: introduction to the special section coronavirus: eu fears a rise in hostile takeovers how to use the nominal group and delphi techniques mental health statistics: children and young people an iterative process of global quality improvement: the international standards for a safe practice of anesthesia paralyzed by panic: measuring the effect of school closures during the polio pandemic on educational attainment (no. w ) the behaviour change wheel: a guide to designing interventions neurological implications of covid- infections mental health of children and young people in england - science as behaviour: using a behaviour change approach to increase uptake of open science stress and health: a review of psychobiological processes exploring the uk's digital divide coronavirus and the social impacts on great britain extreme teams: toward a greater understanding of multiagency teamwork during major emergencies and disasters the two psychologies and coronavirus. the psychologist do we still have a digital divide in mental health? a five-year survey follow-up psychiatric and neuropsychiatric presentations associated with severe coronavirus infections: a systematic review and meta-analysis with comparison to the covid- pandemic the impact of communications about swine flu (influenza a h n v) on public responses to the outbreak: results from national telephone surveys in the uk building, hosting and recruiting: a brief introduction to running behavioral experiments online epigenetic dysregulation of ace and interferonregulated genes might suggest increased covid- susceptibility and severity in lupus patients coping strategies used during an extreme antarctic expedition domestic violence and abuse: safeguarding during the covid- crisis covid- , ace , and the cardiovascular consequences strategic review of health inequalities in england post- does closing schools cause educational harm? a review of the research. information brief covid- impacts: school shutdown severe acute respiratory syndrome (sars) in hong kong in : stress and the psychological impact among frontline healthcare workers psychosocial safety climate as a factor in organisational resilience: implications for worker psychological health, resilience, and engagement distress, worry, and functioning following a global health crisis: a national study of americans' responses to ebola work-home interference: how does it manifest itself from day to day? applying principles of behaviour change to reduce sars-cov- transmission opensafely: factors associated with covid- death in million patients in a paris banlieue, coronavirus amplifies years of inequality. the guardian getting back to work: dealing with the labour market impacts of the covid- recession. institute for employment studies coronavirus disease (covid- ) the psychological impact of the sars epidemic on hospital employees in china: exposure, risk perfection, and altruistic acceptance of risk mind matters: a three-level meta-analysis on parental mentalization and sensitivity as predictors of infant-parent attachment christopher armitage's contribution is supported by the nihr manchester biomedical research centre and the nihr greater manchester patient safety translational research centre. the views expressed in this publication are those of the authors and not necessarily those of nihr. armitage would like to thank professors madelynne arden and alison wearden for their support in writing. til wykes would like to acknowledge the support of her nihr senior investigator award. the set of priorities utilized for the survey of the psychological community.how do we increase adherence (and ability to adhere) to uk government covid- related instructions? how do we promote maintenance of positive behaviour changes and reverse negative behaviour changes resulting from covid- -related lockdown? how do we address the negative psychological impacts of the covid- pandemic? how do we maximize recovery from covid- for those infected with the virus? what is the impact of covid- -related stress on biological processes and health outcomes? what makes people adhere to anti-covid measures? what are the bases of anti-social behaviours such as stockpiling? how do mutual aid groups form and what makes them endure? when does social cohesion give way to scapegoating, prejudice, and intergroup conflict? what creates (or prevents) the potential for protests and collective disorder in the crisis? what are the long-term mental health effects of covid- ? what coping mechanisms are useful in reducing mental health problems during a pandemic? how do we provide beneficial remote psychological therapy and maintain therapeutic alliance? has discussion of mental health during the pandemic reduced stigma and discrimination in the community? people detained in hospital under the mental health act were discharged to free up bedshow was this possible? what are the impacts of covid- infection, treatment, and recovery on cognition, behaviour, and the brain? what are the drivers of covid- -related stress and its cognitive, neural, and physiological mechanisms and consequences? what are the perceptual and cognitive demands of digital and other alternative forms of communication and how do they impact on work and social connectivity? what factors influence the effectiveness of communication of scientific evidence and national guidance, and how do they influence behaviour? how do restrictions of movement, communication, and social support influence the cognitive, physical, and mental health of older individuals, and what factors lead to improved outcomes? how has the covid- pandemic affected parenting? how has the covid- pandemic affected children's development? how has the covid- pandemic affected family functioning? which factors moderate family members' response to the covid- pandemic? what support is most effective for families during the covid- pandemic? how do we assess biological markers of health and well-being remotely?continued key: cord- - jzkdu authors: bickman, leonard title: improving mental health services: a -year journey from randomized experiments to artificial intelligence and precision mental health date: - - journal: adm policy ment health doi: . /s - - - sha: doc_id: cord_uid: jzkdu this conceptual paper describes the current state of mental health services, identifies critical problems, and suggests how to solve them. i focus on the potential contributions of artificial intelligence and precision mental health to improving mental health services. toward that end, i draw upon my own research, which has changed over the last half century, to highlight the need to transform the way we conduct mental health services research. i identify exemplars from the emerging literature on artificial intelligence and precision approaches to treatment in which there is an attempt to personalize or fit the treatment to the client in order to produce more effective interventions. in , i was writing my first graduate paper at columbia university on curing schizophrenia using sarnoff mednick's learning theory. i was not very modest even as a first-year graduate student! but i was puzzled as to how to develop and evaluate a cure. then, as now, the predominant research design was the randomized experiment or randomized clinical trial (rct). it was clear that simply describing, let alone manipulating, the relevant characteristics of this one disorder and promising treatments would require hundreds of variables. developing an effective treatment would take what seemed to me an incalculable number of randomized trials. how could we complete all the randomized experiments needed? how many different outcomes should we measure? how could we learn to improve treatment? how should we consider individual differences in these group comparisons? i am sure i was not insightful enough to think of all these questions back then, but i know i felt frustrated and stymied by our methodological approach to answering these questions. but i had to finish the paper, so i relegated these and similar questions to the list of universal imponderables such as why i exist. in fact, i became a committed experimentalist, and i dealt with the limitations of experiments by recognizing their restrictions and abiding by the principle "for determining causality, in many but not all circumstances, the randomized design is the worst form of design except all the others that have been tried " (bickman and reich , pp. - ) . for the much of my career, i was a committed proponent of the rct as the best approach to understanding causal relationships (bickman ) . however, as some of my writing indicates, it was a commitment with reservations. i did not see a plausible alternative or complement to rcts until recently, when i began to read about artificial intelligence (ai) and precision medicine in . the potential solution to my quandary did not crystallize until , when i collaborated with aaron lyons and miranda wolpert on a paper on what we called "precision mental health" (bickman et al. ) . with the development of ai and its application in precision medicine, i now believe that ai is another approach that we may be able to use to understand, predict, and influence human behavior. while not necessarily a substitute for rcts in efforts to improve mental health services, i believe that ai provides an exciting alternative to rcts or an adjunct to them. while i use precision medicine and precision mental health interchangeably, i will differentiate them later in this paper. toward that end, i focus much of this paper on the role of ai and precision medicine as a critical movement in the field with great potential to inform the next generation of research. before proposing such solutions, i first describe the challenges currently faced by mental health services, using examples drawn almost entirely from studies of children and youth, the area in which i have conducted most of my research. i describe five principal causes of this failure, which i attribute primarily, but not solely, to methodological limitations of rcts. lastly, i make the case for why i think ai and the parallel movement of precision medicine embody approaches that are needed to augment, but probably not replace, our current research and development efforts in the field of mental health services. i then discuss how ai and precision mental health can help inform the path forward, with a focus on similar problems manifested in mental health services for adults. these problems, i believe, make it clear that we need to consider alternatives to our predominant research approach to improving services. importantly, most of the research on ai and precision medicine i cite deals with adults, as there is little research in this area on children and youth. i am assuming that we can generalize from one literature to the other, but i anticipate that there many exceptions to this assumption. according to some estimates, more than half ( . %) of adults with a mental illness receive no treatment (mental health in america ) . less than half of adolescents with psychiatric disorders receive any kind of treatment (costello et al. ) . over % of youth with major depression do not receive any mental health treatment (mental health in america ). several other relevant facts when it comes to youth illustrate the problem of their access to services. hodgkinson et al. ( ) have documented that less than % of children in poverty receive needed services. these authors also showed that there is less access to services for minorities and rural families. when it comes to the educational system, mental health in america ( ) estimated that less than % of students have an individual education plan (iep), which students need to access school-supported services, even though studies have shown that a much larger percentage of students need those services. access is even more severely limited in in low-and middle-income countries (esponda et al. ). very few clients receive effective evidence-based quality mental health services that have been shown to be effective in laboratory-based research (garland et al. ; gyani et al. ). moreover, research shows that even when they do receive care that is labeled evidence-based, it is not implemented with sufficient fidelity to be considered evidence-based (park et al. ) . no matter how effective evidence-based treatments are in the laboratory, it is very clear that they lose much of their effectiveness when implemented in the real world (weisz et al. (weisz et al. , . research reviews demonstrate that services that are typically provided outside the laboratory lack substantial evidence of effectiveness. there are two factors that account for this lack of effectiveness. as noted above, evidencebased services are usually not implemented with sufficient fidelity to replicate the effectiveness found in the laboratory. more fundamentally, it is argued here that even evidencebased services may not be sufficiently effective as currently conceptualized. a review of published studies on school-based health centers found that while these services increased access, the review could not determine whether services were effective because the research was of such poor quality (bains and diallo ) . a meta-analysis of studies of mental health interventions implemented by school personnel found small to medium effect sizes, but only % of the services were provided by school counselors or mental health workers (sanchez et al. ) . a cochrane review concluded, "we do not know whether psychological therapy, antidepressant medication or a combination of the two is most effective to treat depressive disorders in children and adolescents" (cox et al. , p. ) . another meta-analysis of studies on school-based interventions delivered by teachers showed a small effect for internalizing behaviors but no effect on externalizing ones (franklin et al. a) . similarly, a meta-analysis of meta-analyses of universal prevention programs targeting school-age youth showed a great deal of variability with effect sizes from to . standard deviations depending on type of program and targeted outcome (tanner-smith et al. ) . a review of rcts found no compelling evidence to support any one psychosocial treatment over another for people with serious mental illnesses (hunt et al. ) . a systematic review and meta-analysis of conduct disorder interventions concluded that they have a small positive effect, but there was no evidence of any differential effectiveness by type of treatment (bakker et al. ) . fonagy and allison ( ) conclude, "the demand for a reboot of psychological therapies is unequivocal simply because of the disappointing lack of progress in the outcomes achieved by the best evidence-based interventions" (p. ). probably the most discouraging evidence was identified by weisz et al. ( ) on the basis of a review of rcts over a -year period. they found that the mean effect size for treatment did not improve significantly for anxiety and adhd and decreased significantly for depression and conduct problems. the authors conclude: in sum, there were strikingly few exceptions to the general pattern that treatment effects were either unchanged or declining across the decades for each of the target problems. one possible implication is that the research strategy used over the past decades, the treatment approaches investigated, or both, may not be ideal for generating incremental benefit over time. (p. ) there is a need-indeed, an urgent need-to change course, because our traditional approaches to services appear not to be working. however, we might be expecting too much from therapy. in an innovative approach to examining the effectiveness of psychotherapy for youth, jones et al. ( ) subjected rcts to a mathematical simulation model that estimated that even if therapy was perfectly implemented, the effect size would be a modest . . they concluded that improving the quality of existing psychotherapy will not result in much better outcomes. they also noted that ai may help us understand why some therapies are more effective than others. they suggested that the impact of therapy is limited because a plethora of other factors influence mental health, especially given that therapy typically lasts only one hour a week out of + waking hours. they also indicated that other factors that have not been included in typical therapies, such as individualizing or personalizing treatment, may increase the effectiveness of treatment. i am not alone in signaling concern about the state of mental health services. for example, other respected scholars in children's services research have also raised concerns about the quality and effectiveness of children's services. weisz and his colleagues (marchette and weisz ; ng and weisz ) described several factors that contribute to the problems identified above. these included a mismatch between empirically supported treatments and mental health care in the real world, the lack of personalized interventions, and the absence of transdiagnostic treatment approaches. it is important to acknowledge the pioneering work of sales and her colleagues, who identified the need and tested approaches to individualizing assessment and monitoring clients (alves et al. (alves et al. , elliott et al. ; alves , ; sales et al. sales et al. , . we need not only to appreciate the relevance of this work but also to integrate it with new artificial intelligence approaches described later in this paper. i am not concluding from such evidence that all mental health services are ineffective. this brief summary of the state of our services can be perceived in terms of a glass half full or half empty. in other words, there is good evidence that some services are effective under particular, but yet unspecified, conditions. however, i do not believe that the level of effectiveness is sufficient. moreover, we are not getting better at improving service effectiveness by following our traditional approach to program development, implementation, research, and program evaluation. while it is unlikely that the social and behavioral sciences will experience a major breakthrough in discovering how to "cure" mental illness, similar to those often found in the physical or biological sciences, i am arguing in this paper that we must increase our research efforts using alternative approaches to produce more effective services. a large part of this paper, therefore, is devoted to exploring what has been also called a precision approach to treatment in which there is an attempt to personalize treatment or fit treatment to the client in order to produce more effective interventions. in some of my earliest work in mental health, i identified the field's focus on system-level factors rather than on treatment effectiveness as one cause of the problems with mental health services. the most popular and well-funded approach to mental health services in the s and s, which continues even today, is called a system or continuum of care (bickman (bickman , bickman et al. b; bryant and bickman ) . this approach correctly recognized the problems with the practice of providing services that were limited to outpatient and hospitalization only, which was very common at that time. moreover, these traditional services did not recognize the importance of the role played by youth and families in the delivery of mental services. to remedy these important problems, advocates for children's mental health conceptualized that a system of care was needed, in which a key ingredient was a managed continuum of care with different levels or intensiveness of services to better meet the needs of children and youth (stroul and friedman ) . this continuum of care is a key component of a system of care. however, i believe that in actuality, these different levels of care simply represent different locations of treatment and restrictiveness (e.g., inpatient vs. outpatient care) and did not necessarily reflect a gradation of intensity of treatment. a system of care is not a specific type of program, but an approach or philosophy that combines a wide range of services and supports for these services with a set of guiding principles and core values. services and supports are supposed to be provided within these core values, which include the importance of services that are community-based, family-focused, youth-oriented, in the least restrictive environment, and culturally and linguistically proficient. system-level interventions focus on access and coordination of services and organizations and not on the effectiveness of the treatments that are provided. it appeared that the advocates of systems of care assumed that services were effective and that what was needed was to organize them better at the systems level. although proponents of systems of care indicated that they highly valued individualized treatment, especially in what were called wraparound services, there was no distinct and systematic way that individualization was operationalized or evaluated. moreover, there was not sufficient evidence that supported the assumption that wraparound services produced better clinical outcomes (bickman et al. ; stambaugh et al. ) . a key component of the system is providing different levels of care that include hospitalization, group homes, and outpatient services, but there is little evidence that clinicians can reliably assign children to what they consider the appropriate level of care (bickman et al. a ). my earliest effort in mental health services research was based on a chance encounter that led to the largest study ever conducted in the field of child and youth mental health services. i was asked by a friend to see if i could help a person whom i did not know to plan an evaluation of a new way to deliver services. this led to a project that cost about $ million to implement and evaluate. we evaluated a new system of care that was being implemented at fort bragg, a major u.s. army post in north carolina. we used a quasi-experimental design because the army would not allow us to conduct a rct; however, we were able to control for many variables by using two similar army posts as controls (bickman ; bickman et al. ) . the availability of sufficient resources allowed me to measure aspects of the program that were not commonly measured at that time, such as cost and family empowerment. with additional funding that i received from a competitive grant from the national institute of mental health (nimh) and additional follow-up funding from the army, we were able to do a cost-effectiveness analysis (foster and bickman ) , measure family outcomes (heflinger and bickman ) , and develop a new battery of mental health symptoms and functioning (bickman and athay ). in addition, we competed successfully for an additional nimh grant to evaluate another system of care in a civilian population using a rct (bickman et al. a, b) and a study of a wraparound services that was methodologically limited because of sponsor restrictions (bickman et al. ) . i concluded from this massive and concentrated effort that systems of care (including the continuum of care) were able to influence system-level variables, such as access, cost, and coordination, but that there was not sufficient evidence to support the conclusion that it produced better mental health outcomes for children or families or that it reduced costs per client . this conclusion was not accepted by the advocates for systems of care or the mental health provider community more generally. moreover, i became persona non grata among the proponents of systems of care. while the methodologists who were asked to critique on the fort bragg study saw it as an important but not flawless study (e.g., sechrest and walsh ; weisz et al. ) that should lead to new research (hoagwood ) , most advocates thought it to be a well-done evaluation but of very limited generalizability (behar ). it is important to note that the system of care approach, almost years later, remains the major child and youth program funded by the substance abuse and mental health services administration's (samhsa) center for mental health services (cmhs) to the tune of about a billion dollars in funding since the system of care program's inception in . there have been many evaluations funded as part of the samhsa program that show some positive results (e.g., holden et al. ), but, in my opinion, they are methodologically weak and, in some cases, not clearly independent. systems of care are still considered by samhsa's center for mental health services to be the premier child and adolescent program worthy of widespread diffusion and funding (substance abuse and mental health services administration ), regardless of what i believe is the weak scientific support. this large investment of capital should be considered a significant opportunity cost that has siphoned off funds and attention from more basic concerns such as effectiveness of services. sadly, based on my unsuccessful efforts to encourage change as a member of the cmhs national advisory council ( - ), i am not optimistic that there will be any modification of support for this program or shift of funding to more critical issues that are identified in this paper. in the following section, i consider some of the problems that have contributed to the current status of mental health services. my assessment of current services led me to categorize the previously described deficiencies into the five following related problem groups. the problems with the validity of diagnoses have existed for as long as we have had systems of diagnoses. while a diagnosis provides some basis for tying treatment to individual case characteristics, its major contribution is providing a payment system for reimbursement for services. research has shown that external factors such as insurance influence the diagnosis given, and the diagnosis located in electronic health records is influenced by commercial interests (perkins et al. ; taitsman et al. ) . other studies have demonstrated that the diagnosis of depression alone is not sufficient for treatment selection; additional information is required (iniesta et al. ). moreover, others have shown that diagnostic categories overlap and are not mutually exclusive (bickman et al. c) . in practice, medication is prescribed according to symptoms and not diagnosis (waszczuk et al. ) . in their thematic analysis of selected chapters of the diagnostic and statistical manual of mental disorders (dsm- ), allsopp et al. ( ) examined the heterogeneous nature of categories within the dsm- . they showed how this heterogeneity is expressed across diagnostic criteria, and explained its consequences for clinicians, clients, and the diagnostic model. the authors concluded that "a pragmatic approach to psychiatric assessment, allowing for recognition of individual experience, may therefore be a more effective way of understanding distress than maintaining commitment to a disingenuous categorical system" (p. ). moreover, in an interview, allsop stated: although diagnostic labels create the illusion of an explanation, they are scientifically meaningless and can create stigma and prejudice. i hope these findings will encourage mental health professionals to think beyond diagnoses and consider other explanations of mental distress, such as trauma and other adverse life experiences. (neuroscience news , para. ) finally, a putative solution to this muddle is nimh's research domain criteria initiative (rdoc) diagnostic guide. rdoc is not designed to be a replacement of current systems but serves as a research tool for guiding research on mental disorders systems. however, it has been criticized on several grounds. for example, heckers ( ) states, "it is not clear how the new domains of the rdoc matrix map on to the current dimensions of psychopathology" (p. ). moreover, there is limited evidence that rdoc has actually improved the development of treatments for children (e.g., clarkson et al. ) . as i will discuss later in the paper, rush and ibrahim ( ) , in their critical review of psychiatric diagnosis, predicted that ai, especially artificial neural networks, will change the nature of diagnosis to support precision medicine. if measures are going to be used in real world practice, then in addition to the classic and modern psychometric validity criteria, it must be possible to use measures sufficiently often to provide a fine-grained picture of change. if measures are used frequently, then they must be short so as not to take up clinical time (riemer et al. ) . moreover, since there is a low correlation among different respondents (de los reyes and ohannessian ), we need measures and data from different respondents including parents, clinicians, clients, and others (e.g., teachers). however, we are still lacking a systematic methodology for managing these different perspectives. since we are still unsure which constructs are important to measure, we need measures of several different constructs in order to pinpoint which ones we should administer on a regular basis. in addition to outcome measures, we need valid and reliable indicators of mediators and processes to test theories of treatment as well as to indicate short-term outcomes. we need measures that are sensitive to change to be valid measures of improvement. we need new types of measures that are more contextual, that occur outside of therapy sessions, and that are not just standardized questionnaires. we lack good measures of fidelity of implementation that capture in an efficient manner what clinicians actually do in therapy sessions. this information is required to provide critical feedback to clinicians. we also lack biomarkers of mental illness that can be used to develop and evaluate treatments that are often found in physical illnesses. this is a long and incomplete list of needs and meeting them will be difficult to accomplish without a concerted effort. there are some resources at the national institutes of health that are focused on measure development, such as patient-reported outcomes measurement system information (promis) (https ://www.healt hmeas ures.net/explo re-measu remen t-syste ms/promi s), but this program does not focus on mental health. thus, we depend upon the slow and uncoordinated piecemeal efforts of individual researchers to somehow fit measure development into their career paths. i know this intimately because when i started to be engaged with children's mental health services research, i found that the measures in use were too long, too expensive, and far from agile. this dissatisfaction led me down a long path to the development of a battery of measures called the peabody treatment progress battery riemer et al. ). this battery of brief measures was developed as part of ongoing research grants and not with any specific external support. for over a half century, i have been a committed experimentalist. i still am a big fan of experiments for some purposes (bickman ). the first independent study i conducted was my honors thesis at city college of new york in . my professor was a parapsychologist and personality psychologist, so the subject of my thesis was extrasensory perception (esp). my honors advisor had developed a theory of esp that predicted that those who were positive about esp, whom she called sheep, would be better at esp than the people who rejected esp, whom she called goats (schmeidler ) . although i did not realize it at the time, my experimentalist or action orientation was not satisfied with correlational findings that were the core of the personality approach. i designed an experiment in which i randomly assigned college students to hear a scripted talk from me supporting or debunking esp. i found very powerful results. the experimental manipulation changed people's perspective on the efficacy of esp, but i found no effect on actual esp scores. it was not until i finished my master's degree in experimental psychopathology at columbia university that i realized that i wanted to be an experimental social psychologist, and i became a graduate student at the city university of new york. however, i did not accept the predominant approach of social psychologists, which was laboratory experimentation. i was convinced that research needed to take place in the real world. although my dissertation was a laboratory study of helping behavior in an emergency , it was the last lab study i did that was not also paired with a field experiment (e.g. bickman and rosenbaum ) . one of my first published research studies as a graduate student was a widely cited field experiment (rct) that examined compliance to men in different uniforms in everyday settings (bickman a, b) . the first book i coedited, as a graduate student, was titled beyond the laboratory: field research in social psychology and was composed primarily of field experiments (bickman and henchy ) . almost all my early work as a social psychologist consisted of field experiments . i strongly supported the primacy of randomized designs in several textbooks i coauthored or coedited (alasuutari et al. ; bickman and rog ; bickman and rog ; hedrick et al. ) . while the fort bragg study i described above was a quasi-experiment (bickman ) , i was not happy that the funding agency, the u.s. army, did not permit me to use a rct for evaluating an important policy issue. as i was truly committed to using a rct to evaluate systems of care, i followed up this study with a conceptual replication in a civilian community using a rct (bickman et al. b ) that was funded by a nimh grant. while i have valued the rct and continue to do so, i have come to the conclusion that our experimental methods were developed for simpler problems. mental health research is more like weather forecasting with thousands of variables rather than like traditional experimentation, which is based on a century-old model for evaluating agricultural experiments with only a few variables (hall ) . we need alternatives to the traditional way of doing research, service development, and service delivery that recognize the complexity of disorders, heterogeneity of clients, and varied contexts of mental health services. the oversimplification of rcts has produced a blunt tool that has not served us well for swiftly improving our services. this is not to say that there has been no change in the last years. for example, the institute of education sciences, a more recent player the field of children's behavioral and mental health outcomes research, has released an informative monograph on the use of adaptive randomized trials that does demonstrate flexibility in describing how rcts can be implemented in innovative ways (nahum-shani and almirall ). the concerns about rcts are also apparent in other fields. for example, a special issue of social science and medicine focused on the limitations of rcts (deaton and cartwright ) . the contributors to this incisive issue indicated that a rct does not in practice equalize treatment and control groups. rcts do not deliver precise estimates of average treatment effects (ates) because a rct is typically just one trial, and precision depends on numerous trials. there is also an external validity problem; that is, it is difficult to generalize from rcts, especially those done in university laboratory settings. context is critical and theory confirmation/disconfirmation is important, for without generalizability, the findings are difficult to apply in the real world (bickman et al. ) . scaling up from a rigorous rct to a community-based treatment is now recognized as a significant problem in the relatively new fields of translational research and implementation sciences. in addition to scaling up, there is a major issue in scaling down to the individual client level. stratification and theory help, but they are still at the group level. the classic inferential approach also has problems with replication, clinical meaningfulness, accurate application to individuals, and p-value testing (dwyer et al. ) . the primary clinical problem with rcts is the emphasis on average treatment effects (ates) versus individual prediction. rcts emphasize postdiction, and ates lead to necessary oversimplification and a focus on group differences and not individuals. subramanian et al. ( ) gave two examples of the fallacy of averages: the first was a study to describe the "ideal woman," where they measured nine body dimensions and then averaged each one. a contest to identity the "average woman" got responses, but not a single woman matched the averages on all nine variables. in a second example, the u.s. air force in measured pilots on body dimensions to determine appropriate specifications for a cockpit. not a single pilot matched the averages on even as few as dimensions, even when their measurements fell within % of the mean value. as these examples show, the problem with using averages has been known for a long time, but we have tended to ignore this problem. we are disappointed when clinicians do not use our research findings when in fact our findings may not be very useful for clinicians because clinicians deal with individual clients and not some hypothetical average client. we can obtain significant differences in averages between groups, but the persons who actually benefit from therapy will vary widely to the extent to which they respond to the recommended treatments. thus, the usefulness of our results depends in part on the heterogeneity of the clients and the variability of the findings. the privileging of rcts also came with additional baggage. instead of trying to use generalizable samples of participants, the methodology favored the reduction of heterogeneity as a way to increase the probability of finding statistically significant results. this often resulted in the exclusion from studies of whole groups of people, such as women, children, people of color, and persons with more than one diagnosis. while discussions often included an acknowledgment of this limitation, little was done about these artificial limitations until inclusion of certain groups was required by federal funding agencies (national institutes of health, central resource for grants and funding information ) . the limitations of rcts are not a secret, but we tend to ignore these limitations (kent et al. ) . one attempt to solve the difficulty of translating average effect sizes by rcts to individualize predictions is called reference class forecasting. here, the investigator attempts to make predictions for individuals based on "similar" persons treated with alternative therapies. however, it is rarely the case that everyone in a clinical trial is influenced by the treatment in the same way. an attempt to reduce this heterogeneity of treatment effects (hte) by using conventional subgroup analysis with one variable at a time is rejected by kent et al. ( ) . they argue that this approach does not work. first, there are many variables on which participants can differ, and there is no way to produce the number of groups that represent these differences. for example, matching on just binary variables would produce over a million groups. moreover, one would have to start with an enormous sample to maintain adequate statistical power. the authors describe several technical reasons for not recommending this approach to dealing with the hte problem. they also suggested two other statistical approaches, risk modeling and treatment effect modeling, that may be useful, but more research on both is needed to support their use. kent et al. ( ) briefly discussed using observational or non-rct data, but they pointed out the typical problems of missing data and other data quality issues as well as the difficulty in making causal attributions. moreover, they reiterated their support for the rct as the "gold standard." although published in , their article mentioned machine learning only as a question for future research-a question that i address later in this paper. i will also present other statistical approaches to solving the limitations of rcts. there is another problem in depending upon rcts as the gold standard. nadin ( ) pointed out that failed reproducibility occurs almost exclusively in life sciences, in contrast to the physical sciences. i would add that the behavioral sciences have not been immune from criticisms about replicability. the open science collaboration ( ) systematically sampled results from three top-tier journals in psychology, and only % of the replication efforts yielded significant findings. this issue is far from resolved, and it is much more complex than simple replication (laraway et al. ) . nadin ( ) considered the issue of the replicability as evidence of an underlying false assumption about treating humans as if they were mechanistic physical objects and not reactive human beings. he noted that physics is nomothetic, while biology is idiographic, meaning that the former is the study of the formulation of universal laws and the latter deals with the study of individual cases or events. without accurate feedback, there is little learning (kluger and denisi ) . clinicians are in a low feedback occupation, and unlike carpenters or surgeons, they are unlikely to get direct accurate feedback on the effects of their activities. when carpenters cut something too short, they can quickly see that it no longer fits and have to start with a new piece, so they typically follow the maxim of measure twice, cut once. because clinicians in the real world of treatment do not get direct accurate feedback on client outcomes, especially after clients leave treatment, then they are unlikely to learn how to become more effective clinicians from practice alone. clinical practice is thus similar to an archer's trying to improve while practicing blindfolded (bickman ) . moreover, the services research field does not learn from treatment as usual in the real world, where most treatment occurs, because very few services collect outcome data, let alone try to tie these data to clinician actions (bickman b) . there are two critical requirements needed for learning. the first is the collection of fine-grained data that are contemporaneous with treatment. the second is the feedback of these data to the clinician or others so that they can learn from these data. learning can be accomplished with routine use of measures such as patient outcome measures (poms) and feedback through progress monitoring, measurementbased care (mbc), and measurement feedback systems (mfs). these measurement feedback concepts have repeatedly demonstrated their ability to improve outcomes in therapy across treatment type and patient populations (brattland et al. ; bickman et al. ; dyer et al. ; gibbons ; gondek et al. ; lambert et al. ) . despite this evidence base, most clinicians do not use these measurement feedback systems. for example, in one of the largest surveys of canadian psychologists, only % were using a progress monitoring measure (ionita et al. ) . a canadian psychological association task force (tasca et al. ) reinforced the need for psychologists to systematically monitor and evaluate their services using continuous monitoring and feedback. they stated that the association should encourage regulatory bodies to prioritize training in their continuing education and quality assurance requirements. moreover, lewis et al., in their review of measurement-based care ( ), presented a -point research agenda that captures much the ideas in the present paper: ( ) harmonize terminology and specify mbc's core components; ( ) develop criterion standard methods for monitoring fidelity and reporting quality of implementation; ( ) develop algorithms for mbc to guide psychotherapy; ( ) test putative mechanisms of change, particularly for psychotherapy; ( ) develop brief and psychometrically strong measures for use in combination; ( ) assess the critical timing of administration needed to optimize patient outcomes; ( ) streamline measurement feedback systems to include only key ingredients and enhance electronic health record interoperability; ( ) identify discrete strategies to support implementation; ( ) make evidence-based policy decisions; and ( ) align reimbursement structures. (p. ) it is not surprising that the measurement feedback approach has not yet produced dramatic effects, given how little we know about what data to collect, how often it should be collected, what feedback should be, and when and how it should be provided (bickman et al. ) . regardless, every time a client is treated, it is an opportunity to learn how to be more effective. by not collecting and analyzing information from usual care settings, we are missing a major opportunity to learn from ordinary services. the most successful model i know of using this real-world services approach is the treatment of childhood cancers in hospitals where most children enter a treatment rct (o'leary et al. ) . these authors note that in the past years, the survival rates for childhood cancer have climbed from % to almost %. they attribute this remarkable improvement to clinical research through pediatric cooperative groups. this level of cooperation is not easy to develop, and it is not frequently found in mental health services. most previous research shows differential outcomes among different types of therapies that are minor at most (wampold and imel ) . for example, weisz et al. ( ) report that in their meta-analysis, the effect of treatment type as a moderator was not statistically significant but there was a significant, but not clearly understood, treatment type by informant interaction effect. in addition, the evidence that therapists have a major influence on the outcomes of psychotherapy is still being hotly debated. the fact that the efficacy of therapists is far from a settled issue is troubling (anderson et al. ; goodyear et al. ; hill et al. ; king and bickman ) . also, current drug treatment choices in psychiatry are successful in only about % of the patients (bzdok and meyer-lindenberg ) and are as low as - % for antidepressants (dwyer et al. ) . while antidepressants are more effective than placebos, they have small effect sizes (perlis ) , and the choice of specific medicine is a matter of trial and error in many cases. it is relatively easy to distinguish one type of drug from another but not so for services, where even dosage in psychosocial treatments is hard to define. according to dwyer et al. ( ) , "currently, there are no objective, personalized methods to choose among multiple options when tailoring optimal psychotherapeutic and pharmacological treatment" (p. ). a recent summary concluded that after years and studies, it is unknown which patients benefit from interpersonal psychotherapy (ipt) versus another treatment (bernecker et al. ) . however, to provide a more definitive answer to the question about which treatments are more effective, we need head-to-head direct comparisons between different treatments and network meta-analytic approaches such as those used by dagnea et al. ( ) . the field of mental health is not alone in finding that many popular medications do not work with most of the people who take them. nexium, a common drug for treating heartburn, works with only person out of , while crestor, used to treat high cholesterol, works with only out of (schork ) . this poor alignment between what the patient needs, and the treatment provided is the primary basis for calling for a more precise medicine approach. this lack of precision leads to the application of treatments to people who cannot benefit from it, thus leading to overall poor effectiveness. in summary, a deep and growing body of work has led me to conclude that we need additional viable approaches to a rct when it comes to conducting services-related research. an absence of rigorous evaluation of treatments that are usually provided in the community contributes to a gap in our understanding why treatments are ineffective (bickman b) . poor use of measurement in routine care (bickman ) and the absence of measurement feedback systems and clinician training and supervision (garland et al. ) are rampant. there also a dire need for the application of more advanced analytics and data mining techniques in the mental health services area (bickman et al. ). these and other such challenges have in turn informed my current thinking about alternative or ancillary approaches for addressing the multitude of problems plaguing the field of mental health services. the five problems i have described above constitute significant obstacles to achieving accessibility, efficiency, and effectiveness in mental health services. nevertheless, there is a path forward that i believe can help us reach these goals. artificial intelligence promises to transform the way healthcare is delivered. the core of my recommendations in this paper rests on the revolutionary possibilities of artificial intelligence for improving mental healthcare through precision medicine that allows us to take into account the individual variability that exists with respect to genetic and other biological, environmental, and lifestyle characteristics. several others have similarly signaled a need for considering the use of personalized approaches to service delivery. for example, weisz and his colleagues (marchette and weisz ; ng and weisz ) called for more idiographic research and for studies tailoring strategies in usual care. kazdin ( ) focused on expanding mental health services through novel models of intervention delivery; called for task shifting among providers; advocated designing and implementing treatments that are more feasible, using disruptive technologies, for example, smartphones, social media such as twitter and facebook, and socially assistive robots; and emphasized social network interventions to connect with similar people. ai is currently used in areas ranging from prediction of weather patterns to manufacturing, logistic planning to determine efficient delivery routes, banking, and stock trading. ai is used in smartphones, cars, planes, and the digital assistants siri and alexa. in healthcare, decision support, testing and diagnosis, and self-care also use ai. ai can sort through large data sets and uncover relationships that humans cannot perceive. through learning that occurs with repeated, rapid use, ai surpasses the abilities of humans only in some areas. however, i would caution potential users that there are significant limitations associated with ai that are discussed later in this paper. rudin and carlson ( ) present a non-technical and well written review of how to utilize ai and of some of the problems that are typically encountered. ai is not one type of program or algorithm. machine learning (ml), a major type of ai, is the construction of algorithms that can learn from and make predictions based on data. it can be ( ) supervised, in which the outcome is known and labeled by humans and the algorithm learns to get that outcome; ( ) unsupervised, when the program learns from data to predict specific outcomes likely to come from the patterns identified; and ( ) reinforcement learning, in which ml is trial and error. in most cases, there is an extensive training data set that the algorithm "learns" from, followed by an independent validation sample that tests the validity of the algorithm. other variations of ai include random forest, decision trees, and the support vector machine (svm), a multivariate supervised learning technique that classifies individuals into groups (dwyer et al. ; shrivastava et al. ). the latter is most widely used in psychology and psychiatry. artificial neural networks (anns) or "neural networks" (nns) are learning algorithms that are conceptuality related to biological neural networks. this approach can have many hidden layers. deep learning is a special type of machine learning. it helps to build learning algorithms that can function conceptually in a way similar to the functioning of the human brain. large amounts of data are required to use deep learning. ibm's watson won jeopardy with deepqa algorithms designed for question answering. as exemplified by the term neural networks, algorithm developers appear to name their different approaches with reference to some biological process. genetic algorithms are based on the biological process of gene propagation and the methods of natural selection, and they try to mimic the process of natural evolution at the genotype level. it has been a widely used approach since the s. natural language processing (nlp) involves speech recognition, natural language understanding, and natural language generation. nlp may be especially useful in analyzing recordings of a therapy session or a therapist's notes. affective computing or sentiment analysis involves the emotion recognition, modeling, and expression of emotion by robots or chatbots. sentiment analysis can recognize and respond to human emotions. virtual reality and augmented reality are human-computer interfaces that allow a user to become immersed within and interact with computer-generated simulated environments. hinton ( ) , a major contributor to research on ai and health, described ai as the use of algorithms and software to approximate human cognition in the analysis of complex data without being explicitly programmed. the primary aim of health-related ai applications is to analyze relationships between prevention or treatment techniques and patient outcomes. ai programs have been developed and applied to practices such as diagnosis processes, treatment protocol development, drug development, personalized medicine, and patient monitoring and care. deep learning is best at modeling very complicated relationships between input and outputs and all their interactions, and it sometimes requires a very large number of cases-in the thousands or tens of thousands-to learn. however, there appears to be no consensus about how to determine, a priori, the number of cases needed, because the number is highly dependent on the nature of the problem and the characteristics of the data. ai is already widely used in medicine. for example, in ophthalmology, photos of the eyes of persons with diabetes were screened with % specificity and % sensitivity in detecting diabetes (gargeya and leng ) . one of the more prolific uses of ai is in the diagnosis of skin cancer. in a study that scanned , clinical images, the ai approach had accuracy similar to that of board-certified dermatologists (esteva et al. ) . cardiovascular risk prediction with ml is significantly improved over established methods of risk prediction (krittanawong et al. ; weng et al. ). however, a study by desai et al. ( ) found only limited improvements in predicting heart failure over traditional logistic regression. in cancer diagnostics, ai identified malignant tumors with % accuracy compared to % accuracy for human pathologists (liu et al. ). the ibm's watson ai platform took only min to analyze a genome of a patient with brain cancer and suggest a treatment plan, while human experts took h (wrzeszczynski et al. ) . ai has also been used to develop personalized immunotherapy for cancer treatment (kiyotani et al. ). rajpurkar et al. ( ) compared chest x-rays for signs of pneumonia using a state-of-the-art -layer convolutional neural network (cnn) program with a "swarm" of radiologists (groups connected by swarm algorithms) and found the latter to be significantly more accurate. in a direct comparison between radiologists on , interpretations and a stand-alone deep learning ai program designed to detect breast cancer in mammography, the ai program was as accurate as the radiologists (rodriguez-ruiz et al. ). as topol ( b) noted, ai is not always the winner in comparison with human experts. moreover, many of these applications have not been used in the real world, so we do not know how well ai will scale up in practice. topol describes other concerns with ai, many of which are discussed later in this paper. finally, many of the applications are visual, such as pictures of skin or scans, for which ai is particularly well suited. large banks of pictures often form the training and testing data for this approach. in mental health, visual data are not currently as relevant. however, there is starting to be some research on facial expressions in diagnosing mental illness. for example, abdullah and choudhury ( ) cite several studies that showed that patients with schizophrenia tend to show reduced facial expressivity or that facial features can be used to indicate mental health status. more generally, there is research showing how facial expressions can be used to indicate stress (mayo and heilig ) . visual data are ripe for exploration using ai. although an exhaustive review of the ai literature and its applications is well beyond the focus of this paper, rudin and carlson ( ) present a well-written, non-technical review of how to utilize ai and of some of the problems that are typically encountered. topol ( a) , in his book titled deep medicine: how artificial intelligence can make healthcare human again, includes a chapter on how to use of ai in mental health. topol ( b) also provides an excellent review of ai and its application to health and mental health in a briefer format. buskirk et al. ( ) and y. liu et al. ( ) provide well-written and relatively brief introductions to ml's basic concepts and methods and how they are evaluated. a more detailed introduction to deep learning and neural networks is provided by minar and naher ( ) . in most cases, i will use the generic term ai to refer to all types of ai unless the specific type of ai (e.g., ml for machine learning, dl for deep learning, and dnn for deep neural networks) is specified. precision medicine has been defined as the customization of healthcare, with medical decisions, treatments, practices, or products being tailored to the individual patient (love-koh et al. ) . typically, diagnostic testing is used for selecting the appropriate and best therapies based a person's genetic makeup or other analysis. in an idealized scenario, a person may be monitored with hundreds of inputs from various sources that use ai to make predictions. the hope is that precision medicine will replace annual doctor visits and their granular risk factors with individualized profiles and continuous longitudinal health monitoring (gambhir et al. ) . the aim of precision medicine, as stated by president barack obama when announcing his precision medicine initiative, is to find the long-sought goal of "delivering the right treatments, at the right time, every time to the right person" (kaiser ) . both ai and precision medicine can be considered revolutionary in the delivery of healthcare, since they enable us to move from one-size-fits-all diagnoses and treatment to individualized diagnoses and treatments that are based on vast amounts of data collected in healthcare settings. the use of ai and precision medicine to guide clinicians will change diagnoses and treatments in significant ways that will go beyond our dependence on the traditional rct. precision medicine should also be seen as evolutionary since even hippocrates advocated personalizing medicine (kohler ) . the importance of a precision medicine approach was recognized in the field of prevention science with a special issue of prevention science devoted to that topic (august and gewirtz ) . the articles in this special issue recognize the importance of identifying moderators of treatment that predict heterogeneous responses to treatment. describing moderators is a key feature of precision medicine. once these variables are discovered, it becomes possible to develop decision support systems that assist the provider (or even do the treatment assignment) in selecting the most appropriate treatment for each individual. this general approach has been tried using a sequential multiple assignment randomized trial (smart) in which participants are randomized two to three times at key decision points (august et al. ) . what i find notable about this special issue is the absence of any focus on ai. the articles were based on a conference in october , and apparently the relevance of ai had not yet influenced these very creative and thoughtful researchers at that point. precision medicine does not have an easy path to follow. x. liu et al. ( b) describe several challenges, including the following three. large parts of the human genome are not well enough known to support analyses; for example, almost % of our genetic code is unknown. it is also clear that a successful precision medicine approach depends on having access to large amounts of data at multiple levels, from the genetic to the behavioral. moreover, these data would have be placed into libraries that allow access for researchers. the u.s. federal government has a goal of establishing such a library with data on one million people through nih's all of us research program (https ://allof us.nih.gov/). recruitment of volunteers who would be willing to provide data and the "harmonization" of data from many different sources are major issues. x. liu et al. ( b) also point to ethical issues that confront precision medicine, such as informed consent, privacy, and predictions that someone may develop a disease. these issues are discussed later in this paper. chanfreau-coffinier et al. ( ) provided a helpful illustration of how precision medicine could be implemented. they convened a conference of veterans affairs stakeholders to develop a detailed logic model that can be used by an organization planning to introduce precision medicine. this model includes components typically found in logic models, such as inputs (clinical and information technology), big data (analytics, data sources), resources (workforce, funding) activities (research), outcomes (healthcare utilization), and impacts (access). the paper also includes challenges to implementing precision medicine (e.g., a poorly trained workforce) that apply to mental health. ai has the potential to unscramble traditional and new diagnostic categories based on analysis of biological/genetic and psychological data, and in addition, more data will likely be generated now that the potential for analysis has become so much greater. ai also has the potential to pinpoint those individuals who have the highest probability of benefiting from specific treatments and to provide early indicators of success or failure of treatment. research is currently being undertaken to provide feedback to clinicians at key decision points as an early warning of relapse. fernandes et al. ( ) describe what the authors call the domains related to precision psychiatry (see fig. ). these domains include many approaches and techniques, such as panomics, neuroimaging, cognition, and clinical characteristics, that form several domains including big data and molecular biosignature; the latter includes biomarkers. the authors include data from electronic health records, but i would also include data collected from treatment or therapy sessions as well as data collected outside of these sessions. these domains can be analyzed using biological and computational tools to produce a biosignature, a higher order domain that includes data from all the lower level techniques and approaches. this set of biomarkers in the biosignature should result in improved diagnosis, classification, and prognosis, as well as individualized interventions. the authors note that this bottom-up approach, from specific approaches to domains to the ultimate biosignature, can also be revised to a top-down approach, with the biosignature studied to better understand domains and its specific components. the bottom of the figure shows a paradigm shift where precision psychiatry contributes to different treatments being applied to persons with different diagnoses and endophenotypes, producing different prognoses. endophenotypes is a term used in genetic epidemiology to separate different behavioral another perspective on precision psychiatry is presented by bzdock and meyer-lindberg ( ). both models contain similar concepts. both start with a group of persons containing multiple traditional diagnoses. bzdock and meyer-lindberg recognize that these psychiatric diagnoses are often artificial dichotomies. machine learning is applied to diverse data from many sources and extracts hidden relationships. this produces different subgroups of endophenotypes. machine learning is also used to produce predictive models of the effects of different treatments instead of the more typical trial and error. further refinement of the predictive ml models results in better treatment selection and better prediction of the disease trajectory. an excellent overview of deep neural networks (dnns) in psychiatry and its applications is provided by durstewitz et al. ( ) . in addition to explaining how dnns work, they provide some suggestions on how dnns can be used in clinical practice with smartphones and large data sets. a major feature of deep neural networks is their ability to learn and adapt with experience. while dnns typically outperform ml, the authors state that they do not fully understand why this is the case. in mental health, dnns have been mostly used in diagnosis and predictions but not in designing personalized treatments. dnn's ability to integrate many different data sets (e.g., various neuroimaging data, movement patterns, social media, and genomics) should provide important insights on how to personalize treatments. regardless of the model used, eyre et al. ( ) remind us that consumers should not be left out of the development of precision psychiatry. in my conceptualization of precision medicine, precision mental health encompasses precision psychiatry and any other precision approach such as social work that focuses on mental health (bickman et al. ). there has not been much written about using a precision approach with psychosocial mental health services. possibly it is psychiatry's close relationship to general medicine and its roots in biology that make psychiatry more amenable to the precision science approach. in addition, the use of the precision construct is being applied in other fields, as exemplified by the special issue of the journal of school psychology devoted to precision education (cook et al. ) and precision public health (kee and taylor-robinson ) . however, in this paper i am primarily addressing the use of psychosocial treatment of mental health problems, which differs in important ways from psychiatric treatment. for example, precision psychosocial mental health treatment does not have a strong biological/medical perspective and does not focus almost exclusively on medication; instead, it emphasizes psychosocial interventions. psychosocial mental health services are also provided in hospital settings, but their primary use is in community-based services. these differences lead to different data sources for ai analyses. it is highly unlikely that electronic mental healthcare records found outside of hospital settings contain biological and genomic data (serretti ) . but hospital records are not likely to contain the detailed treatment process data that could possibly be found in community settings. the genomic and biological data offer new perspectives but may not be informative until we have a better understanding about the genomic basis of mental illness. in addition, the internet of things and smart healthcare connect wearable and home-based sensors that can be used to monitor movement, heart rate, ecg, emg, oxygen level, sleep, and blood glucose, through wi-fi, bluetooth, and related technologies. (sundaravadivel et al. ) . with wider use of very fast g internet service, there will be a major increase in the growth of the internet of things. i want to emphasize that applying precision medicine concepts to mental health services, especially psychotherapy, is a very difficult undertaking. the data requirements for psychosocial mental health treatment are more similar to meteorology or weather forecasting than to agriculture, which is considered the origin of the rct design. people's affect, cognition, and behavior are constantly changing just like the variables that affect weather. but unlike meteorology, which is mainly descriptive and not yet engaged in interventions, mental health services are interventions. thus, in addition to client data, we must identify the variables that are critical to the success of the intervention. we are beginning to grasp how difficult this task is as we develop greater understanding that the mere labeling of different forms of treatment by location (e.g., hospital or outpatient) or by generic type (e.g., cognitive behavior therapy) is not sufficiently informative. moreover, the emergence of implementation sciences has forced us to face the fact that a treatment manual describes only some aspects of the treatments as intended but does not describe the treatment that is actually delivered. nlp is a step in the right direction in trying to capture some aspects of treatment as actually delivered. data quality is the foundation upon which ai systems are built. while medical records are of higher technical quality than community-based data because they must adhere to national standards, i believe that the nascent interest in measurement-based care and measurement feedback systems in community settings bodes well for improved data systems in the future. moreover, although electronic hospitalbased data may be high quality from a technical viewpoint (validity, reliability) and be very large, they probably do not contain the data that are valuable for developing and evaluating mental health services. the development of electronic computer-based data collection and feedback systems will become more common as the growth in ai demands large amounts of good-quality treatment and finer grained longitudinal outcome data. there is a potential reciprocal relationship between the ai needs for large, high-quality data sets and the development of new measurement approaches and the electronic systems needed to collect such data (bickman a; bickman et al. a bickman et al. , . to accomplish this with sufficiently unbiased and valid data will be a challenge. ai can bypass many definitional problems by not using established diagnostic systems. ml can use a range of variables to describe the individual ml classifier systems (tandon and tandon ) . moreover, additional sources of data that help in classification are now feasible. for example, automated analysis of social media including tweets and facebook can detect depression, with accuracy measured by area under the curve (auc) ranging from . to . compared to clinical interviews with aucs of . (guntuku et al. ). as noted earlier, dnns have been shown to be superior to other machine learning approaches in general and specifically in identifying psychiatric stressors for suicide from social media (du et al. ). predictions of , adolescent suicides with ml showed high accuracy (auc > . ) and outperformed traditional logistic regression analyses ( . - . aucs) (tandon and tandon ) . saxe has published a pioneering proof of concept that has demonstrated that ml methods can be used to predict child posttraumatic stress (saxe et al. ) . ml was more accurate than humans in predicting social and occupational disability with persons in high-risk states of psychosis or with recent-onset depression (koutsouleris et al. a) . machine learning has also been used in predicting psychosis using everyday language (rezaii et al. ) . another application of ai to diagnosis is provided by kasthurirathne et al. ( ) . they demonstrated the ability to automate screening for , adult patients in need of advanced care for depression using structured and unstructured data sets covering acute and chronic conditions, patient demographics, behaviors, and past service use history. the use of many existing data elements is a key feature and thus does not depend on single screening instruments. the authors used this information to accurately predict the need for advanced care for depression using random forest classification ml. milne et al. ( ) recognized that in implementing online peer counseling, professionals need to participate and/or provide safety monitoring in using ai. however, cost and scalability issues appeared to be insurmountable barriers. what is needed is an automated triage system that would direct human moderators to cases that require the most urgent attention. the triage system milne et al. developed sent human moderators color-coded messages about their need to intervene. the algorithm supporting this triage system was based on supervised ml. the accuracy of the system was evaluated by comparing a test set of manually prioritized messages with the ones developed through the algorithm. they used several methods to judge accuracy, but their main one was an f-measure, or the harmonic mean of recall (i.e., sensitivity) and precision (i.e., positive predictive value). regression analysis indicated that the triage system made a significant and unique contribution to reducing the time taken to respond to some messages, after accounting for moderator and community activity. i can see the potential for this and similar ai approaches to deal with the typical service setting where some degree of supervision is required but even intermittent supervision is not feasible or possible. another use of ml as a classification tool is provided by pigoni et al. ( ) . in their review of treatment resistant depression, they found that ml could be used successfully to classify responders from non-responders. this suggested that stratification of patients might help in selecting the appropriate treatment, thus avoiding giving patients treatments that are unlikely to work with them. a more general systematic review and meta-analysis of the use of ml to predict depression are provided by lee et al. ( ) . the authors found qualitative and quantitative studies that qualified for inclusion in their review. while most of the studies were retrospective, they did find predictions with an average overall accuracy of . . kaur and sharma ( ) reviewed the literature on diagnosis of ten different psychological disorders and examined the different data mining and software approaches (ai) used in different publications. depending on the disorder and the software used, the accuracy ranged from to %. accuracy was defined differently depending on the study. only % of the articles exploring diagnosis of any health problem were found to be for psychological problems. this suggests that we need more studies on diagnosis and ai. a very informative synthesis and review are provided by low et al. ( ) . they screened studies and reviewed the that met the inclusion criterion: studies from the last years using speech to identify the presence or severity of disorders through ml methods. they concluded that ml could be predictive, but confidence in any conclusions was dampened by the general lack of cross-validation procedures. the article contains very useful information on how best to collect and analyze speech samples. another innovative approach using ml focused on wearable motion detector sensors, in which these devices were worn for s during a -s mood induction task (seeing a fake snake). these data were able to distinguish children with an internalizing disorder from controls with % accuracy (mcginnis et al. ) . this approach has potential for screening children for this disorder. a problem that seemingly has been ignored by most studies that deal with classification or diagnosis is the gold standard by which accuracy is judged. in most cases, the gold standard is human judgment, which is especially fallible when it comes to mental health diagnosis. we can clearly measure whether the ai approach is faster and less expensive than human judgment, but is the ultimate in ai accuracy matching human judgment with all its flaws? i believe that the endpoint that must also be measured is client clinical mental health improvement. a system that provides faster and less expensive diagnosis but does not lead to more precise treatment and better clinical outcomes will save us time and money, which are important, but they will not be the breakthrough for which we are looking. a solution to the problems described above will involve the integration of causal discovery methods with ai approaches. ai methods are capable of improving our capacity to predict outcomes. to enhance predictability, we will need to identify the factors in the predictive models that are causal. thus, there is the need to identify techniques that provide us with causal knowledge, which currently is based primarily on rcts. but, for real-world and ethical reasons, human etiological experiments can rarely be conducted. fortunately, there are newer ai methods that can be used to infer causes, which include well validated tests of conditional independencies based on the causal markov condition (pearl ; aliferis et al. ; saxe ) . these methods have been successfully used outside of psychiatry (sachs et al. ; ramsey et al. ; statnikov et al. ) and have, in the last five years, been applied in research on mental health, largely by the team of glenn saxe at new york university and constantin aliferis and sisi ma at university of minnesota. this group has reported causal models of ptsd in hospitalized injured children (saxe et al. (saxe et al. , , children seen in outpatient trauma centers (saxe et al. ) , maltreated children (morales et al. ) , adults seen in emergency rooms (galatzer-levy et al. ) , and police officers who were exposed to trauma (saxe et al. in press ). saxe ( ) recently described the promise of these methods for psychiatric diagnosis and personalized precision medicine. new measures need to be developed that cover multiple domains of mental health, are reported by different respondents (e.g., child, parent, clinician), and are very brief. cohen ( ) provides an excellent overview of what he calls ambulatory biobehavioral technologies in a special section of psychological assessment. he notes that the development of mobile devices can have a major impact on psychological assessment. he cautions, however, that while some of these approaches have been used for decades, they still have not progressed beyond the proof of concept phase for clinical and commercial applications. ecological momentary assessment (ema) is a relatively new approach to measurement development. ema is the collection of real-time data collected in naturalistic environments. this approach uses a wide range of smart watches, bands, garments, and patches with embedded sensors (gharani et al. ; pistorius ) . for example, using smartphones, researchers have identified gait features for estimating blood alcohol content level (gharani et al. ). other researchers have been able to map changes in emotional state ranging from sad to happy by using a movement sensor on smart watches (quiroz et al. ) . others have described real-time fluctuations in suicidal ideation and its risk factors, using an average of . assessments per day (kleiman et al. ) . social anxiety has been assessed from global positioning data obtained from smart watches by noting that socially anxious students were found to avoid public places and to spend more time at home than in leisure activities outside the home (boukhechba et al. ) . a review of studies using ema concluded that the compliance rate was moderate but not optimal and could be affected by study design (wen et al. ). this review is also a good source of descriptions of different approaches to using ema. another good summary that focused on ema in the treatment of psychotic disorders can be found in bell et al. ( ) . for ema use in depression and anxiety, schueller et al. ( ) is a good source. ema has been used to measure cardiorespiratory function, movement patterns, sweat analysis, tissue oxygenation, sleep, and emotional state (peake et al. ) . harari et al. ( ) present a catalog of behavior in more than aspects of daily living that can be used in studying physical movement, social interactions, and daily activities. these include walking, speaking, text messaging, and so on. these all can be collected from smartphones and serve as an alternative to traditional survey approaches. however, it is still not clear what higher-level constructs are measured using these approaches. a comprehensive and in-depth review of studies that have used speech to assess psychiatric disorders is provided by low et al. ( ) . they conclude that speech processing technology could assist in mental health assessments but believe that there are many obstacles to this use, including the need for longitudinal studies. another interesting application for children is the use of inexpensive screening for internalizing disorders. mcginnis et al. ( ) monitored the child's motion for s using a commercially available and inexpensive wearable sensor. using a supervised ml approach, they obtained an % accuracy ( % sensitivity, % specificity) compared to similar clinical threshold on parent-reported child symptoms that differentiate children with an internalizing diagnosis from controls without such a diagnosis. in a systematic review of ema use in major depression, colombo et al. ( ) evaluated studies that met their criteria for inclusion. these studies measured a wide variety of variables including self-reported symptoms, sleep patterns, social contacts, cortisol, heart rate, and affect. they point out many of the advantages of using emas such as realtime assessments, capturing the dynamic nature of change, improving generalizability, and providing information about context. they believe that the use of emas has resulted in novel insights about the nature of depression. they do note that there are few evaluations of these measures, and there is not much use in actual clinical practice. mohr et al. ( ) note that most of the research on ema has been carried out primarily by computer scientists and engineers using a very different research model than social and behavioral scientists. while computer scientists are mostly interested in exploratory proof of concepts approach (does it work at all?) using very small samples, social/behavioral scientists are more typically theory driven and investigate under what conditions the intervention will work. mental health care, apart from medication, is almost exclusively verbal. several approaches have been tried to capture the content of treatment sessions. my colleagues and i have tried by asking clinicians to use a brief checklist of topics discussed after each therapy session . although this technique produced some interesting findings such as the identification of topics that the clinician did not discuss but that were believed to be important by the youth or parent, it is clearly filtered by what the clinician recalls and is willing to check off as having been discussed. while recordings provide a richer source of information, coding recordings manually is too expensive and slow for the real world of service delivery. the content of therapy sessions, including notes kept by clinicians, is pretty much ignored by researchers because of the difficulty and cost of manually coding those sources. however, advances in natural language processing (nlp) are now being explored as a way of capturing aspects of the content of therapy sessions. for example, tanana et al. ( ) have shown how two types of nlp techniques can be used to study and code the use of motivational interviewing in taped sessions. carcone et al. ( ) also showed that they could accurately code motivational interviewing (mi) clinical encounter transcripts with sufficient accuracy. other researchers have used ai to analyze speech to distinguish between what they called high-and low-quality counselors (pérez-rosas et al. ). some colleagues and i have submitted a proposal to nimh to refine nlp tools that can be used to supervise clinicians implementing an evidence-based treatment using ai. as far as we know, using nlp to measure fidelity and provide feedback to clinicians has not been studied in a systematic way. while ai appears to be an attractive approach to new ways of analyzing data, it should be noted that, as always, the quality of the analysis is highly dependent on the quality of the data. jacobucci and grimm ( ) caution us that "in psychology specifically, the impact of machine learning has not been commensurate with what one would expect given the complexity of algorithms and their ability to capture nonlinear and interactive effects" (p. ). one observation made by these authors is that the apparent lack of progress in using ai may be caused by "throwing the same set of poorly measured variables that have been analyzed previously into machine learning algorithms" (p. ). they note that this is more than the generic garbage in, garbage out problem, but it is specifically related to measurement error, which can be measured relatively accurately. as described earlier, our privileging of rcts has contributed to a lack of focus on a precision approach to mental health services. this has resulted in the problem of ignoring the clinical need for predicting for an individual in contrast to establishing group difference, the approach favored by the experimentalist/ hypothesis testing tradition. ai offers an approach to the discovery of important relationships in mental health in addition to rcts that are based on singlesubject prediction accuracy and not null hypothesis testing (bzdok and karrer ) . saxe et al. ( ) have demonstrated the use of the complex-systems-causal network method to detect causal relationships among variables and bivariate relations in a psychiatric study using algorithms. a comprehensive review and meta-analysis of machine learning algorithms that predict outcomes of depression showed excellent accuracy ( . ) using multiple forms of data (lee et al. ) . it is interesting to note that none of the scholars commenting on the rct special issue in social science and medicine (deaton and cartwright ) specifically mentioned the use of ai as a potential solution to some of the problems of using average treatment effects (ates). kessler et al. ( a) noted that clinical trials do not tell us which treatments are more effective for which patients. they suggested that what they label as precision treatment rules (ptrs) be developed that are predictors of the relative treatment effectiveness of different treatments. the authors presented a comprehensive discussion on how to use ml to develop ptrs. they concluded that the sample sizes needed are much larger than usually those found in rcts; observational data, especially from electronic medical records (emrs) can be used to deal with the sample size issue; and statistical methods can be used to balance both observed and unobserved covariates using instrumental variables and discontinuity designs. they do note the difficulty in obtaining full baseline data from emrs and suggest several solutions for this problem, including supplemental data collection and links to other archival sources. they recommend the use of an ensemble ml approach that combines several algorithms. they are clear that their suggestions are exploratory and require verification, but they are more certain that if ml improves patient outcomes, it will be a substantial improvement. wu et al. ( ) collaborated with kessler on a proof of concept of a similar model called individualized treatment rules (itr). in a model simulation, they used a large sample (n = , ) with an ensemble ml method to identify the advantages of using ml algorithms to estimate the outcomes if a precision medicine approach was taken in prescribing medication for persons with first-onset schizophrenia. they found that the treatment success was estimated to be . % under itr compared to . % with the medication that was actually used. wu et al. see this as a first step that needs to be confirmed by pragmatic rcts. kessler et al. ( b) conducted a relatively small randomized study (n = ) in which soldiers seeking treatment were judged to be at risk for suicide. they were randomly assigned to two types of treatment but not on the basis of any a priori ptr. the data from that study were then analyzed using ml to produce ptrs. these data were then modeled in a simulation to see if the ptr would have produced better outcomes. the authors did find that the simulated ptr produced better effects. lenze et al. ( ) address the problems of rcts from a somewhat different perspective than i have presented here and suggest a potential solution that they call precision clinical trials (pcts). the authors propose that the problem with most existing rcts is that they measure only the fixed baseline characteristics that are not usually sensitive to detecting treatment responders. moreover, treatment is typically not dynamically adapting to the client during treatment, and measures are not administered with sufficient frequency. instead, the pcts would: ( ) first attempt to determine whether short-term responses to the intervention could determine who was a likely candidate for that specific treatment; ( ) initiate the treatment in an adaptive fashion that could vary over time, using stepped care or just-in-time adaptations that are responsive to the client's changing status, and frequently collect data possibly using multiple assignment randomized trial methods; and ( ) use frequent precision measurement, possibly using ecological momentary assessments described earlier. coincidently, they illustrate the application of pcts using repetitive transcranial magnetic stimulation (rtms), a form of brain stimulation therapy used to treat depression and anxiety that has been in use since . rtms will be described later in connection with what i call a third path for services and ai. it is disappointing that i could not find any examples of published research that used a rct to test whether an ai approach to an actual, not simulated, delivery of a mental health treatment produces better clinical outcomes than a competitive treatment or even treatment as usual. this is clearly an area requiring further rigorous empirical investigation. imel et al. ( ) provide an excellent overview on how ai and other technologies can be used for monitoring and feedback in psychotherapy in both training and supervision. imel et al. ( ) used ml to code and provide data to clinicians on metrics used to measure the quality of motivational interviewing (mi). a prior study (tanana et al. ) established that ml was able to code mi quality metrics with accuracy similar to human coders. they conducted a pilot study using standardized patients and -min speech segments that was designed to test the feasibility of providing feedback to clinicians on the quality of their mi intervention. the feedback was not in real-time but was provided after the session. they were able to establish that clinicians thought highly of the feedback they received. the authors anticipate that further developments in this technology will lead to its widespread use in supervision and in real-time feedback. it would seem that the next step is evaluating the enhanced ai feedback procedure in a real-world effectiveness study. another example of the use of nlp application is the use of a bot that was trained to assess and provide feedback on specific interviewing and counseling skills such as asking open-ended questions and providing feedback (tanana et al. ) . after training the bot on transcripts, non-therapists (using amazon mechanical turk recruits) were randomly assigned to either immediate feedback on a practice session with the bot or just encouragement on the use of those skills. the group provided the feedback were significantly more likely to use reflection even when feedback was removed. the authors consider this to be a proof of concept demonstration because of the many limitations (e.g., use of non-therapists). a plan for using nlp to monitor and provide feedback to clinicians on the implementation of an evidenced program is provided by berkel et al. ( ) . they provide excellent justification for using nlp to accomplish this goal, but unfortunately it is only a design at this point. rosenfeld et al. ( ) see ai making major contributions to improving the quality of treatment through efficient continuous monitoring of patients. until now, monitoring was limited to in-session contacts or manual contacts, an approach that is not practical or efficient. the almost universal availability of smartphones and other internet active devices (internet of things) makes collecting data from clients practical and efficient. these various data sources provide feedback to providers so that they can predict and prevent relapse and compliance with treatment, especially medication. the authors note that there is not a large body of research in this area, but early studies are positive. one concrete application of ai to providing feedback is described by ryan and his colleagues . their article only describes how such could be done; unfortunately, it is not an actual study but a suggestion on how to apply ai for feedback to physicians to improve their communications with patients. they note that routine assessment and feedback are not done manually because of the cost and time requirements. however, ai can automate these tasks by evaluating recordings. they suggest using already existing ai approaches that are in use by call centers to categorize and evaluate communication along the following dimensions: speaker ratio that indicates listening, overlapping talk that are interruptions, pauses longer than two seconds, speed, pitch, and tone. the content could also be evaluated along the dimensions of the use of plain language, clinical jargon, and shared decision making. ai could also explore other dimensions such as the meaning of words and phrases using nlp, turn taking, tone, and style. many technical difficulties would have to be overcome to assess many of these variables, but the field is making progress. an actual application of ml to feedback, but not in mental health, is provided by pardo et al. ( ) in a course for first-year engineering students. instructors developed in advance a set of feedback messages for levels of interaction with learning resources. for example, different feedback messages were provided depending on whether the student barely looked at video, watched a major portion, watched the whole video, or watched it several times. an ml algorithm selected the appropriate message to send the student through either email or the virtual learning environment. compared to earlier cohorts who did not receive the feedback, those who did were more satisfied with the course and had better performance on the midterm. i can see how such a protocol could be used in mental health services. an indication of the work that needs to be done in becoming more specific about feedback is a study conducted by hooke et al. ( ) . they provide feedback to patients with and without a trajectory showing expected progress and found that patients preferred the feedback with the expected change over time. they found that these patients preferred to have normative feedback with which they could compare their own ideographic progress. two systematic reviews that focused on implementing routine outcome measurement (rom) concluded that while rom has been shown to produce positive results, how to best implement rom remains to be determined by future research (gual-montolio et al. ; mackrill and sorensen ) . the authors of both reviews note several interesting points but focus on these two: how to integrate measurement into clinical practice and how organizations support staff in this effort. they highlight the importance of developing a culture of feedback in organizations. neither review includes any studies using ai. while they call for more research to move this field forward, i do not think there will be much change until either measurement feedback systems are required by funders or service delivery organizations are paid for providing such systems. probably the most advanced work in this area that includes ml is being done by lutz and his colleagues (lutz et al. ) . they have developed a measurement feedback system that includes the use of ml to make predictions and to provide clinicians with clinical decision support tools. they are able to predict dropouts and assign support tools to clinicians that are specific to the problems their clients are exhibiting, based on the data they have collected. lutz and his colleagues are currently evaluating the system to influence clinical outcomes in a prospective study. this comprehensive feedback system provided clinical support tools with recommendations based on identification of similar patients to the treatment group but not to the control group. they already have some very promising results using three different treatment strategies (w. lutz, personal communication, september , ) . almost all the research in this area has been on prediction and not in actually testing whether precision treatments are in fact better than standard treatments in improving mental health outcomes. even these predictive studies are on extant databases rather than data collected specially for use in ai algorithms. with a few exceptions to be discussed later, this is the state of the art. to establish the practical usefulness of ai, we need to move beyond prediction to show actual mental health improvements that have clinical and not just statistical significance. there are some scholars who are carefully considering how to improve methodology to achieve better predictions (e.g., garb and wood ) . in addition, zilcha-mano ( ) has a very thoughtful paper that describes traditional statistical and machine learning approaches to trying to answer the core question of what treatments work best for which patients, as well as the more general question about why psychotherapy works at all. nlp has been used to analyze unstructured or textual material for identifying suicidal ideation in a psychiatric research database. precision of % for identification of suicide ideation and % for suicide attempts has been found using nlp (fernandes et al. ) . a meta-analysis of studies of prediction of suicide using traditional methodologies found only slightly better than chance predictions and no improvement in accuracy in years (franklin et al. b ). recent ml decision support aids using large-scale biological and other data have been useful in predicting responses to different drugs for depression (dwyer et al. ). triantafyllidis and tsanas ( ) conducted a literature review of pragmatic evaluations of nonpharmacological applications of ml in real-life health interventions from january through november , following prisma guidelines. they found only eight articles that met their criteria from citations screened. three dealt with depression and the remainder with other health conditions. six of the eight produced significantly positive results, but only three were rcts. there has been little rigorous research to support ai in real-world contexts. accuracy of prediction is one of the putative advantages of ai. but the advantage of predicting outcomes is not as relevant if a client prematurely leaves treatment. thus, predicting premature termination is one of the key goals of an ai approach. in a pilot study to test whether ai could be beneficial in predicting premature termination, bohus et al. ( ) were not able to adequately predict dropouts using different ml approaches with responses to the borderline symptom list (bsl- ). however, they obtained some success when they combined the questionnaire data with personal diary questionnaires from patients, although they note that the sample is too small to draw any strong conclusions. this pilot study illustrates the importance of what data goes into the data set as well as our lack of knowledge of the data requirements we need to have confidence in as we select the appropriate data. duwe and kim ( ) compared statistical methods including ml approaches on their accuracy in predicting recidivism among , offenders. they found the newer ml algorithms generally performing modestly better. kessler et al. ( ) used data from u.s. army and department of defense administrative data systems to predict suicides of soldiers who were hospitalized for a psychiatric disorder (n = , ). within one year of hospitalization, ( . %) of the soldiers committed suicide. they used a statistical prediction rule based on ml that resulted in a high validity auc value of . . kessler and his colleagues have continued this important work, which was discussed earlier. another approach to prediction was taken by pearson et al. ( ) in predicting depression symptoms after an -week internet depression reduction program using participants. they used an elastic net and random forest ml ensemble (combination) and compared it to a simple linear autoregressive model. they found that the ensemble method predicted an additional % of the variance over the non-ml approach. the authors offer several good technical suggestions about how to avoid some common errors in using ml. moreover, the ml approach allowed them to identify specific module dosages that were related to outcomes that would be more difficult to determine using standard statistical approaches (e.g., detecting nonlinear relationships without having to specify them in advance). however, not all attempts to use ai are successful. pelham et al. ( ) compared logistic regression and five different ml approaches to typical sum-score approaches to identify boys in the fifth grade who would be repeatedly arrested. ml performed no better than simple logistic regression when appropriate cross-validation procedures were applied. the authors emphasize the importance of cross-validation in testing ml approaches. in contrast, a predictive study of people with first-episode psychosis used ai to successfully predict poor remission and recovery one year later based only on baseline data (leighton et al. ) . the model was cross validated on two independent samples. a comprehensive synthesis of the literature of studies that used ml or big data to address a mental health problem illustrated the wide variety of uses that currently exist; however, most dealt with detection and diagnosis (shatte et al. ) . a critical view of the way psychiatry is practiced for the treatment of depression and how ai can improve that practice is provided by tan et al. ( ) . they note that most depression is treated with an "educated-guess-and-check approach in which clinicians prescribe one of the numerous approved therapies for depression in a stepwise manner" (p. ). they posit that ai and especially deep learning have the ability to model the heterogeneity of outcomes and complexity of psychiatric disorders through the use large data sets. at this point, the authors have not provided any completed studies that have used ai, but two of the authors are shareholders in a medical technology company that is developing applications using deep learning in psychiatry. we are beginning to see commercial startups take an interest in mental health services even though the general health market is considerably bigger. entrepreneurially motivated research may be important for the future of ai growth in mental health services, with traditional federal research grants to support this important developmental work, including such mechanisms as the small business innovation research (sbir) program and the r and r nih funding mechanisms. one of the few studies that go beyond just prediction and actually attempt to develop a personalized treatment was conducted by fisher et al. ( ) . in a proof of concept study, the authors used fisher's modular model of cognitive-behavioral therapy (cbt) and algorithms to develop and implement person-by-person treatments for anxiety and mood disorders for adults. the participants were asked to complete surveys four times a day for about days. the average improvement was better than found in comparison benchmark studies. the authors state that this is the first study to use pre-therapy multivariate time series data to generate prospective treatment plans. rosenfeld et al. ( ) describe several treatment delivery approaches that utilize ai. woebot, for example, is a commercial product to provide cbt-based treatment using ai. the clients interact with woebot through instant messaging that is later reviewed by a psychologist. it has been shown to have short-term effectiveness in reducing phq- scores of college students who reported depression and anxiety symptoms. the authors are optimistic that approaches like the ones described will lead to more widely available and efficacious treatment modalities. applications of ml to addiction studies was the focus of a systematic review by mak et al. ( ) . they did an extensive search of the literature until december and could find only articles. none of the studies involved evaluating a treatment. i want to distinguish between the use of computer-assisted therapy, especially that provided through mobile apps, and the use of ai. in a review of these digital approaches to providing cbt for depression and anxiety, wright et al. ( ) point out while many of these apps have been shown to be better than no treatment, they usually do not use ai to personalize them. thus, they are less relevant to this paper and are not discussed in depth. ecological momentary interventions (emis) are treatments provided to patients between sessions during their everyday lives (i.e., in real time) and in natural settings ). these interventions extend some aspects of psychotherapy to patients' daily lives to encourage activities and skill building in diverse conditions. in the only systematic review available of emis, colombo et al. ( ) found only eight studies that used emis to treat major depression, with only four different interventions. the common factor of these four interventions is that they provide treatment in real-time and are not dependent on planned sessions with a clinician. the authors report that participants were generally satisfied with the interventions, but there was variability in compliance and dropout rates among the programs. with only two studies that tested for effectiveness with rcts, there is clearly a need for more rigorous evaluations. momentary reminders are typically used for behaviors such as medication adherence and management of symptoms. the more complex emis use algorithms to optimize and personalize systems. they also can use algorithms that changes the likelihood of the presentation of a particular intervention over time, based on past proximal outcomes. schueller et al. ( ) note that emis are becoming more popular as a result of technological advances. these authors suggest the use of micro-randomized trials (mrts) to evaluate them. an mrt uses a sequential factorial design that randomly assigns an intervention component to each person at multiple randomly chosen times. each person is thus randomized many times. this complex design represents the dynamic nature of these interventions and how their outcomes correspond to different contextual features. ai is often used to develop algorithms to optimize and personalize the mrt over time. one interesting algorithm, called a "bandit algorithm," changes the intervention presented based on a past proximal outcome. as an example, schueller et al. describe a hypothetical study to reduce anxiety through two different techniques-deep breathing and progressive muscle relaxation. the bandit algorithm may start the presentation of each technique with equal frequency but then shift more to the one that appears to be most successful for that individual. thus, each treatment (a combination of deep breathing and progressive muscle relaxation) would be different for each person. unlike rcts, this method does not use group-level outcomes of average effect sizes but uses individual-level data. in the future, we might have personal digital mental health "therapists" or assistants that can deliver individualized combinations of treatments based on algorithms developed with ai that are data driven. of course, this approach is best suited for these momentary interventions and would be difficult if not impossible to successfully apply to traditional treatment. i consider explicating the relationship between ai and causality to be a key factor in understanding whether ai is to be seen as replacing or as supplementing rcts. toward that end, i first consider whether observational data can replace rcts using ai. second, should a replacement not seem currently feasible, i explore ways to design studies that combine ai and rcts to evaluate whether the ai approach produces better outcomes than non-ai enhanced interventions. the journal prevention science devoted a special section of an issue to new approaches for making causal inferences from observational data (wiedermann et al. ). an example is the paper by shimizu ( ) that demonstrates the use of non-gaussian analysis tools to infer causation from observational data under certain assumptions. malinsky and danks ( ) provide an extended discussion of the use of causal discovery algorithms to learn causal structure from observational data. in a similar fashion, blöbaum et al. ( ) present a case for inferring causal direction between two variables by comparing the least-squares errors of prediction in both possible directions. using data that meet some assumptions, they provide an algorithm that requires only a regression in both causal directions and a comparison of the least-square errors. lechner's ( ) paper focuses on identifying the heterogeneity of treatment effects at the finest possible level or identifying what he calls groups of winners and losers who receive some treatment. hassani et al. ( ) hope to build a connection between researchers who use big data analysis and data mining techniques and those who are interested in causality analysis. they provide a guide that describes data mining applications in causality analysis. these include entity extractions, cluster analysis, association rule, and classification techniques. the authors also provide references to studies that use these techniques, key software, substantive areas in which they have been used, and the purpose of the applications. this is another bit of evidence that the issue of causality is being taken seriously and that some progress is being made. however, because of the newness of these publications, there is a lag in publications that are critical of these approaches; for example, d'amour ( ) provides a technical discussion about why some approaches will not work but also suggests that others may be potentially effective. clearly, caution is still warranted in drawing causal conclusion from observational data. chen ( ) provides a very interesting discussion of ai and causality but not from the perspective of the rct issue that i raise here but as a much broader but still relevant point of view. he advances the key question about whether ai technology should be adopted in the medical field. chen argues that there are two major deficits in ai, namely the causality deficit and the care deficit. the causality deficit refers to the inferior ability of ai to make accurate casual inferences, such as diagnosis, compared to humans. the care deficit is the comparative lack of ability of ai to care for a patient. both deficits are interesting, but the one most germane to this paper is the causality deficit. chen notes that ai represents statistical and not causal reasoning machines. he argues that ai is deficient compared to humans in causal reasoning, and, moreover, he doubts that there is a feasible way to deal with this lack of comparability in reasoning. he believes that ai is a model-blind approach in contrast to a human's more model-based approach to causal reasoning. thus, causation for chen is not an issue of experimental methodology (he never mentions rcts in his paper), but a characteristic associated with humans and not computers. chen does recognize that ai researchers are attempting to deal with the causality issue, for example, by briefly describing pearl's ( ) directed acyclic graphs and nonparametric structural equation models. but chen is skeptical that either the causality or care deficits will be overcome. he concludes that ai is best thought of as assisting humans in medical care and not replacing them. the relationship between ai and humans is a major concern of this paper. caliebe et al. ( ) see big data, and i would assume ai, as contributing to hypotheses generation that could then be tested in rcts. the critical issues they see are related to the quality and quantity of big data. they quote an institute of medicine (iom) report that refers to the use of big data and ai in medicine as "learning healthcare systems" and states that these systems will "transform the way evidence on clinical effectiveness is generated and used to improve health and health care" (institute of medicine , p. ). moreover, in , the iom suggested that alternative research methodologies will be needed. they do not acknowledge the conundrum that i have raised here; moreover, they do not see any need to consider changing any of our methodology or analyses. i have found many individual papers that describe how to solve the causality problem with ai (e.g., kuang et al. ; pearl ) . although these papers are complex, their mere existence gives me hope that this problem is being seriously considered. in addition to the statistical and validity issues in trying to replace rcts with observational data, there is the feasibility question. although the data studied in much of the research reported in this paper are in the medical domain and deal primarily with medications, the characteristics of these data have some important lessons for mental health services. bartlett et al. ( ) identified trials published in the top seven highest impact medical journals. they then determined whether the intervention, medical condition, inclusion and exclusion criteria, and primary end points could be routinely obtained from insurance claims and/or electronic health data (ehr) data. these data are recognized by the fda as what they term real-world evidence. they found that only % of the u.s.-based clinical trials published in highimpact journals in could be feasibly replicated through analysis of administrative claims or ehr data. the results suggest that potential for real-world evidence to replace clinical trials is very limited. at best, we can hope that they can complement trials. given the paucity of data collected in mental health settings, the odds are that such data are even less available. suggestions for improving the utility of real-world data for use in research are provided in an earlier article by some of these authors (dhruva et al. ). pearl ( ) posits causal information in terms of the types of questions that, in his three-level model, each level answers. his first level is association; the second, intervention; and the third, counterfactual. association is simply the statistical relationship or correlation. there is no causal information at this first level. the higher order levels can answer questions about the lower levels but not the other way around. counterfactuals are the control groups in rcts. they represent what would have happened if there had been no intervention. to pearl, this unidirectional hierarchy explains why ml, based on associations, cannot provide causal statements like rcts, which are based on counterfactuals. however, as noted earlier, pearl does present an approach using what he calls structural causal models to "extract" causal relationships from associations. pearl describes seven "talks" and accompanying tools that are accomplished in the framework provided by the structural causal models that are necessary to move from the lower levels to the counterfactual level to allow causal inferences. i would anticipate that there will be direct comparisons between this approach to causality and the randomized experiments like those done in program evaluation (bickman and reich ; boruch et al. ) . theory development or testing is usually not thought of as a strength of ai; instead, its lack of transparency, that is, the lack of explanatory power that would enable us to identify models/mechanisms that underlie outcomes, is seen as a major weakness. coutanche and hallion ( ) present a case for using feature ablation to test theories. this technique involves the removal or ablation of features from algorithms that have been thought to be theoretically meaningful and then seeing if there is a significant reduction in the predictive accuracy of the model. they have also studied whether the use of a different data set affects the predictive accuracy of a previously tested model in theoretically useful ways. they present a very useful hypothetical application of their approach to test theories using ai. it is clear that ai can be very useful in making predictions, but can it replace rcts? can ai perform the major function of rcts, that of determining causality? the dependence on rcts was one of the major limitations i saw as hindering the progress of mental health services research. while rcts have their flaws, they are still considered by most as the best method for determining causal relationships. is ai limited to being a precursor in identifying those variables that are good candidates for rcts because they have high predictive values? the core conceptual problem is that while it is possible to compare two different but theoretically equivalent groups, one receiving the experimental treatment and the other the control condition, it is not possible to compare the same individuals on both receiving and not receiving the experimental treatment. rcts produce average effect sizes, but the ultimate purpose of precision mental health is to predict individualized effects. how do we reconcile these two very different aims? one approach is to use ai to identify the most predictive variables and then test them in a randomized experiment. let us take a group of patients with the same disorder or problem. there may be several alternative treatments, but the most basic concept is to compare two conditions. in one condition, call it the traditional treatment condition in the rct, everyone in that condition gets the same treatment. it is not individualized. in the second condition, call it the ai condition, everyone gets a treatment that is based on prior ai research. the latter may differ among individuals in dosage, timing, type of treatment, and so on. the simplest is medication that differs in dosage. however, a more nuanced design is a yoked design used primarily in operant and classical conditioning research. there have been limitations associated with this design, but these problems apply to conditioning research and not the application considered here (church ) . to separate the effects of the individualization from the differences in treatment, i suggest using a yoked design. in this design, individuals who would be eligible to be treated with either the standard treatment or the ai-selected treatment would be yoked, that is, paired. which participant of the pair received which condition would be randomized. first, the eligible participants would be randomly divided into two groups. the individuals in the ai group would get a treatment that was precisely designed for each person in that group, while those in the yoked control group would not; instead, those in the control group would receive the treatment that had been designed for his or her partner in the ai group. in this way, each participant would receive the same treatment, but only the ai group participants would be receiving individualized treatment. if the ai approach is superior, we would expect those in the ai group to have a superior average treatment effect compared to the control group, who received a treatment matched not to their individual characteristics but to those in the ai group. we could also use an additional control group where the treatment is selected by a clinician. while this design would not easily identify which characteristics were responsible for its success, it would demonstrate whether individualized ai-based treatment was the causal factor. that is, we could learn that on the average, a precision approach is more effective than a traditional approach, but we would not be able to identify from this rct which particular combination of characteristics made it more effective. of note is that the statistical power of this design would depend on the differences among the participants at baseline. for example, if the individuals were identical on measured covariates, then they would get the same personalized treatment, which practically would produce no useful information. instead of yoking participants based on randomly assigning them as in the above example, we could yoke them on dissimilarity and then randomly assign each individual in the pair to ai-based treatment or a control condition that could be the same ai treatment or a clinician-assigned treatment. however, interesting this would be from a methodical point of view, i think this would also bring up ethical issues that are discussed next. of course, as with any rct, there are ethical issues to consider. in many rcts, the control group may receive standard treatment, which should not present any unusual ethical issues. however, in a yoked design, the control group participants will receive a treatment that was not selected for them on the basis of their characteristics. moreover, the yoked design would make the formulation of the informed consent document problematic because it would have to indicate that participants in the control group would receive a treatment designed for someone else. one principle that should be kept in mind is equipoise: there should be consensus among clinicians and researchers that the treatments, a priori, are equivalent. in a yoked design, we must be assured that none of individualized treatments would harm the yoked control group members, and moreover, that there is no uniform agreement that the individualized treatment would be better for the recipient. that is, the research is designed to answer a question about relative effectiveness for which we do not know the answer. almost all of the research previously cited in this paper has dealt with psychosocial interventions, along with some research on interventions with medications. clearly these are the two main approaches taken in providing services for mental health problems. however, in the last decade, a new approach to understanding mental illness has emerged from the field of psychoneuroimmunology. this relatively new field integrates research on psychology, neuroscience, and immunology to understand how these processes influence each other and, in turn, human health and behavior (slavich ). i want to explore this relatively new approach to understanding mental health because i believe that it is a potentially rich field in which to apply ai. slavich and irwin ( ) have combined diverse areas to show how stressors affect neural, physiologic, molecular, and genomic and epigenetic processes that mediate depression. they labeled this integrative theory the social signal transduction theory of depression. in a recent extension of this work, slavich ( ) proposed social safety theory, which describes how social-environmental stressors that degrade experiences of social safety-such as social isolation and rejection-affect neural, immunologic, and genomic processes that increase inflammation and damage health. a key aspect of this perspective is the role of inflammatory cytokines as key mediators of the inflammatory response (slavich ) . cytokines are the biological endpoint of immune system activity and are typically measured in biobehavioral studies of stress and health. cytokines promote the production of c-reactive protein, which is an inflammatory mediator like cytokines, but which also is a biomarker of inflammation that is assessed with a blood test. cytokines also interact with the central nervous system and produce what have been labeled "sickness behaviors," which include increased pain and threat sensitivity, anhedonia, fatigue, and social-behavioral withdrawal. while the relationship between inflammation and depression is well-established in adults, a systematic review and meta-analysis of studies with children and adolescents concluded that because of the small number of studies, more evidence was needed before drawing a similar conclusion for youth (d'acunto et al. ) . in contrast, a major longitudinal study of more than adults followed over years found that participants who had stable high c-reactive protein levels were more likely to report clinically significant late-life depression symptoms (sonsin-diaz et al. ) . chronic inflammation has been shown to be present in many psychiatric disorders including depression, schizophrenia, and ptsd, as well as in many other somatic and physical disease conditions (furman et al. ) . chronic inflammatory diseases have been shown to be a major cause of death. a typical inflammatory response occurs when a threat is present and then goes away when there is no longer a threat. however, when the threat is chronic and unresolved, systemic chronic inflammation can occur and is distinct from acute inflammation. chronic inflammation can cause significant damage to tissues and organs and break down the immune system tolerance. what is especially interesting from a behavioral health perspective is that inflammatory activity can apparently be initiated by any psychological stressor, real or imagined. thus, social and psychological stressors such as negative interpersonal relationships with friends and family, as well as physical stressors, can produce inflammation, which leads to increased risk of mental and physical health problems. this inflammatory response initially can have positive effects in that it can help increase survival in the short term, but it can also lead to a dysfunctional hypervigilance and anxiety that increases the risk of serious mental illness if chronic. the "cytokine storm" experienced by many covid- patients is an example of the damage an uncontrolled immune response can cause (konig et al. ). although we do not know a great deal about how this process operates, it is clear that there is a strong linkage between inflammatory responses and mental disorders such as depression. the role of the immune system in disease, especially brain inflammation related to brain microglial cells (i.e., neuroinflammation), is also receiving attention in the popular press (nakazawa ). psychoneuroimmunology research has explicated the linkage between the brain and the immune system, showing how stress affects the immune system, and how these interactions relate to mental illness. the relationships between these constructs suggest interventions that can be used to improve mental health. but much research remains to be done to identify specific processes and effective interventions. research will require multidisciplinary teams to produce personalized interventions guided by each patient's specific level of neuroinflammation and genetic profiles. this process will need to be monitored by continuous feedback that i believe will be made more feasible with the application of ai. at present, there are some existing interventions that appear to be aligned with this approach that are being explored. these include the following. three anti-inflammatory medications have been found to reduce depressive symptoms in well-designed rcts. these agents include celecoxib, usually used for treating excessive inflammation and pain, and etanercept and infliximab, which are used to treat rheumatoid arthritis, psoriasis, and other inflammatory conditions (slavich ) . however, there has not been a great deal of research in this area, so caution is warranted. a recent well-designed rct with depressed youth tested aspirin, rosuvastatin (a statin), and a placebo and found no significant differences in depression symptoms (berk et al. ). a meta-analysis explored the possible link between different types of psychosocial interventions, such as behavior therapy and cbt, and immune system function (shields et al. ) . the authors examined eight common psychosocial interventions, seven immune outcomes, and nine moderating factors in evaluating rcts. they found that psychosocial interventions were associated with a . % improvement in good immune system function and a . % decrease in detrimental immune function, on average. moreover, the effects lasted for at least months and were consistent across age, sex, and intervention duration. the authors concluded that psychosocial interventions are a feasible approach for influencing the immune system. repetitive transcranial magnetic stimulation (rtms) has been found to be an effective treatment for several mental illnesses, especially treatment-resistant depression (mutz et al. ; somani and kar ; voigt et al. ) . while the literature is not clear on how rtms produces its effect (noda et al. ; peng et al. ) , i was curious about its relationship to neuroinflammation. i could find little in the research literature that addressed the relationship between inflammation and rtms; therefore, i conducted an informal survey of rtms researchers who have published rtms research in peer-reviewed journals and asked them the following: i suspect that rtms is related to inflammation but the only published research that i could find on that relationship was two studies dealing with rats. are you aware of any other research on this relationship? in addition, do you know of anyone using ai to investigate rtms? i received replies from all but of the researchers. about half said they were aware of some research that linked rtms to inflammation and supplied citations. in contrast, only % were aware of any research on rtms and ai. the latter noted some research that used ai on eegs to predict rtms outcomes. a most informative response was from the author of a review article that dealt with several different nontraditional treatments including rtms on the hypothalamic-pituitary-adrenal (hpa) axis and immune function in the form of cytokine production in depression (perrin and parianti ) . the authors found relevant human studies ( studies using rtms) but were unable to conduct the metaanalysis because of significant methodological variability among studies. but they concluded that non-convulsive neurostimulation has the potential to impact abnormal endocrine and immune signaling in depression. moreover, given that there is more information available than on other neurostimulation techniques, the research suggests that rtms appears to reduce cytokines. finally, there is some support from animal models (rats) that rtms can have an anti-inflammatory effect on the brain and reduce depression and anxiety (tiana et al. ). moreover, four published studies showed that the efficacy of rtms for schizophrenics could be predicted koutsouleris et al. ( b) . three other studies were able to use ml and eeg to predict outcomes of rtms treatment for depression (bailey et al. ; hasanzadeh et al. ) . the existing literature indicates that metabolic activity and regional cerebral blood flow at the baseline can predict the response to rtms in depression (kar ) . as these baseline parameters are linked to inflammation, it is worth studying responses to rtms that predict inflammation. as noted by one of the respondents, "in summary, it is a relatively new field and there are no major multi-site machine learning studies in rtms response prediction" (n. koutsouleris, personal communication, march , ) . one of the significant limitations of measurement in mental health is the absence of robust biomarkers of inflammation. furman et al. ( ) caution us that "despite evidence linking sci [systemic chronic inflammation] with disease risk and mortality, there are presently no standard biomarkers for indicating the presence of health-damaging chronic inflammation" (p. ). however, some biomarkers that are currently being explored for inflammation may be of some help. for example, furman et al. ( ) are hopeful that a new approach using large numbers of inflammatory markers to identify predictors will produce useful information. a narrative review of inflammatory biomarkers for mood disorders was also cautious in drawing any conclusions from extant research because of "substantial complexities" (chang and chen ) . it is also worth noting the emerging area of research on gut-brain communication and the relationship between microbiome bacteria and quality of life and mental health (valles-colomer et al. ) . however, there is need for more research on the use of biomarkers. the area of inflammation and mental health offers an additional pathway to uncovering the causes of mental illness but also, most importantly for this paper, potential services interventions beyond traditional medications and psychosocial interventions. given the complexity, large number of variables from diverse data sets, and the emerging nature of this area, it appears that ai could be of great benefit in tying some potential biomarkers to effective interventions designed to produce better clinical outcomes. however, some caution is needed concerning the seemingly "hard data" provided by biomarkers. for example, elliot et al. ( ) found in a meta-analysis of experiments that one widely used biomarker, task-fmir, had poor overall reliability and poor test-retest reliability in two other large studies. they concluded that these measures were not suitable for brain biomarker research or research on individual differences. as noted in several places in this paper, ai is not without its problems and limitations. the next section of the paper discuses several of these problems. ai may force the treatment developer to make explicit choices that are ethically ambiguous. for example, automobile manufacturers designing fully autonomous driving capabilities now have to be explicit about whose lives to value more in avoiding a collision-the driver and his or her passengers or a pedestrian. should the car be programmed to avoid hitting a pedestrian, regardless of the circumstances, even if it results in the death of the driver? mental health services do not typically have such clear-cut conflicts, but the need to weigh the potential side effects of a drug against potential benefits suggests that ethical issues will confront uses of ai in mental health. some research has shown that inherent bias in original data sets has produced biased (racist) decisions (obermeyer et al. ; veale and binns ) . an unresolved question is who has the responsibility for determining the accuracy and quality of original data set (packin and lev-aretz ) . data scientists operating with data provided by others may not have sufficient understanding of the complexity of the data to be sensitive to its limitations. moreover, they may not consider it their responsibility to evaluate the accuracy of the data and attend to its limitations. librenza-garcia ( ) provides a comprehensive review of ethical issues in the use of large data sets with ai. the ethical issues in predicting major mental illness are discussed by lawrie et al. ( ) . they note that predictive algorithms are not sufficiently accurate at present, but they are progressing. the authors raise questions about whether people want to know their risk level for major psychiatric disorders, about individual and societal attitudes to such knowledge and the possible adverse effects of sharing such data, and about the possible impact of such information on early diagnosis and treatment. they urge conducting research in this area. related to the ethics issue but with more direct consequences to the health provider is the issue of legal responsibility in using an ai application. it is not clear what the legal liability is for interventions based on ai that go wrong. who is responsible for such outcomes-the person applying the ai, the developer of the algorithm, or both? price ( ) points out that providers typically do not have to be concerned about the legal liability of a negative outcome if they used standard care. thus, if there are negative outcomes of some treatment but that treatment was the standard of care, there is usually no legal liability. however, currently ai is probably not seen as the standard of care in most situations. while this will hopefully change as evidence of the effectiveness of ai applications develops, currently the healthcare provider is at greater risk of legal liability in using an ai application that is different from the standard of care. i have previously discussed the insufficient evidence for the effectiveness of many of the interventions used in mental health services. this lack of strong evidence has implications for the use of ai in mental health services. in an insightful article on using ai for individual-level treatment predictions, paulus and thompson ( ) make several key observations and suggestions that are very relevant to the current paper. the authors summarize several meta-analyses of the weak evidence of effectiveness of mental health interventions and come to conclusions similar to those i have already stated. they also identify similar factors i have focused on in accounting for the modest effect sizes found in mental health rcts. they point out that diagnostic categories are not useful if they are not aggregating homogenous populations. they suggest that what i call the diagnostic muddle may result from the nature of mental disorders themselves, for which there are many causes at many different levels, from the genetic to the environmental. thus, there is no simple explanatory model. paulus and thompson note that prediction studies rarely account for more than a very small percentage of the variance. they recommend conducting large, multisite pragmatic rcts that are clearly pre-defined with specific ml models and variables. predictive models generated by this research then need to be validated with independent samples. this is a demanding agenda, but i think it is necessary if we are going to advance mental health services with the help of ai. treatments are often considered black boxes that provide no understanding of how and why the treatment works (kelley et al. ; bickman b) . the problem of lack of transparency is compounded in the use of deep neural networks (samek et al. ) . at present we are not able to understand relationships between inputs and outcomes, because this ai technique does not adequately describe process. deep neural networks may contain many hidden layers and millions of parameters (de choudhury and kikkoman ). however, this problem is now being widely discussed, and new technologies are being developed to make ai more transparent (rauber et al. ; kuang et al. ). i do not believe it is possible to develop good theories of treatment effectiveness without this transparency. this is an important limitation of efforts to improve mental health services. but how important is this limitation? early in my program evaluation career, i wrote about the importance of program theory (bickman (bickman , . i argued that if individual studies were going to be conceptually useful, beyond local decisions such as program termination, then they must contribute to the broader goal of explaining why certain programs were effective and others not. this is in contrast to the worth and merit of a local program. a theory based evaluation of the program must add to our understanding of the theory underlying the program. while i still believe that generalizing to a broad theory of why certain interventions work is critical, at present it may be sufficient simply to increase the accuracy of our predictions, regardless of whether we understand why. as stephens-davidowitz ( ) argues, "in the prediction business, you just need to know that something works, not why" (p. ). however, turing award winner judea pearl argued in his paper theoretical impediments to machine learning with seven sparks from the causal revolution ( ) that human-level ai cannot emerge from model-blind learning machines that ignore causal relationships. one of the positive outcomes of the concern over transparency is the development of a subfield of ai that has been called explainable artificial intelligence (xai). adai and berrada ( ) present a very readable description of this movement and show that it has been a growing area since . they are optimistic that research in this area will go a long way toward solving the black box problem. large data sets are required for some ai techniques, especially deep neural networks. while such data sets may be common in consumer behavior, social media, and hospitalbased electronic health records, they are not common in community-based mental health services. the development and ownership of these data sets may be more important (and profitable) than ownership of specific ai applications. there is currently much turmoil over data ownership (mittelstadt ) . ownership issues are especially important in the mental health field given the sensitivity of the data. in addition to the size and quality of the data set, longitudinal data are necessary for prediction. collecting longitudinal data poses a particular problem for community-based services given the large treatment drop-out rate. in addition to the characteristics of the data, there is the need for competent data managers of large complex data sets. the data requirements for mental health applications are more demanding than those for health in general. first, mental health studies usually do not involve the large samples that are found in general health. for example, the wellknown physicians' health study of aspirin to prevent myocardial infarction (mi) utilized more than , doctors in a rct (steering committee of the physicians' health study research group ). they found a reduction in mi that was highly statistically significant: p < . . the trial was stopped because it was thought that this was conclusive evidence that aspirin should be adopted for general prevention. however, the effect size was extremely small: a risk difference of . % with r = . (sullivan and feinn ) . a study this size is not likely to occur in mental health. moreover, such small effects would not be considered important even if they could be detected. it is unlikely that very large clinical trials such as the aspirin study would ever be conducted in mental health. thus, it is probable that data will have to be obtained from service data. but mental health services usually do not collect sufficiently fine-grained data from clients. while i was an early and strong proponent of what i called a measurement feedback system for services (bickman a) , recent research shows that the collection of such data is rare in the real world. until services start collecting these data as part of their routine services, it is unlikely that ai will have much growth with the limited availability of relevant data. there is, of course, a chicken and egg problem. a major reason why services do not collect data is the limited usefulness of data in improving clinical care. while ai may offer the best possibility of increasing the usefulness of regularly collected data, such data will not be available until policy makers, funders, and providers deem it useful and are willing to devote financial resources to such data collection analysis. at present, there are no financial incentives for mental health providers to collect such data even if they improved services. moustafa et al. ( ) made the interesting observation that psychology is behind other fields in using big data. ai and big data are not considered core topics in psychology. the authors suggest several reasons for this, including that psychology is mostly theory-and hypothesis-driven rather than data-driven, and that studies use small sample sizes and a small number of variables that are typically categorical and thus are not as amenable to ai. moreover, most statistical packages used by psychologists are not well-equipped to analyze large data sets. however, the authors note that the method of clustering and thus differentiating among participants is used by psychologists and is in many ways similar to ai, especially deep neural networks, in trying to identify similar participants. using ml methods such as random forest algorithms, the investigator can identify variables that best explain differences among groups or clusters. instead of the typically few variables used by psychologists, ai can examine hundreds of variables. as a note of caution, rutledge et al. ( ) warn that "there is no silver bullet that can replace collecting enough data to generate stable and generalizable predictions" (p. ). while there are techniques that are often used in low sample size situations (e.g., the elastic net and tree-based ensembles), researchers need replications with independent samples if they are to have sufficient confidence in their findings. moreover, since big data are indeed big, they are easily misunderstood as automatically providing better results through smaller sampling errors. it is often not appreciated that the gain in precision drawn from larger samples may well be nullified by the introduction of additional population variance and biases. finding competent big data managers, data scientists, and programmers is a human resource problem. in my experience, ai scientists who are able and want to collaborate with mental health services researchers are rare. industry pays a lot more for these individuals than universities can afford. moreover, even within the health field, mental health is a very small component of the cost of services, so it is often ignored in this area. difficulty and resistance are encountered in the implementation of new technologies. clinicians are reluctant to adopt new approaches and to engage clients in new approaches and data collection procedures. community mental health services have been slow to successfully adopt new technologies (crutzen et al. ; lattie et al. ; yeager and benight ) . in their mixed methods study of community clinicians, crutzen et al. ( ) found there were concerns about privacy, the wide range of therapeutic techniques used, disruptions in trust and alliance, managing crises, and organizational issues such as billing and regulations contained in the privacy rule established by the health insurance portability and accountability act of (hipaa) that inhibited the use of new technologies. moreover, our current reimbursement policies do not support greater payment for better outcomes. thus, there is little or no financial incentive for hard-pressed community services to improve their services at their own expense. in fact, i would argue that there is a disincentive to improve outcomes since it results in increased costs (at least initially), organizational disruption and potentially a loss of clients if it takes less time and effort to successfully treat them. an interesting meta-issue has emerged from the widespread and ever-increasing investment in ai in healthcare. in a perceptive "viewpoint" published in jama, emanuel i would be happy to serve as a "matchmaker" for any ai programmers, data scientists (etc.), or behavioral scientists who are interested in collaborating on mental health projects. just contact me describing your background and interests and i will try to put together likeminded researchers. and wachter ( ), argue that the major challenge facing healthcare is not that of obtaining data and new analytics but the achievement of behavior change among both clinicians and patients. they point out the major failures of google and microsoft in not recognizing the problems in translating evidence into practice in connection with their large, web-based repositories for storage of health records, google health and microsoft healthvault, both of which have been discontinued. they indicate that the long delays in translation are due not primarily to data issues or lack of accurate predictions, but to the absence of behavioral changes needed for adoption of these practices. for example, the collection of longitudinal data has been problematic. another problem they note is that about half the people in the united states are nonadherent with medications. there is a huge gap between knowing what a problem is and actually solving it that "data gurus" seem to ignore. while this translation problem is evident in the sometimes narrow focus of ai promoters, it also represents an opportunity for the behavioral scientists engaged in ai research to marshal their skills and the knowledge gained from years of dealing with similar behavioral issues. the emergence of translational and implementation sciences, the latter more often led by behavioral scientists, can be of great service to the problems of applying ai to healthcare. the field of translational sciences has been developed and well-funded by the nih in recognition of the difficulty in using (i.e., translating) laboratory studies into practice. in , the budget for the clinical and translational science awards (ctsa) program was over a half billion dollars from to . however, as director of evaluation for vanderbilt's medical center's ctsa program for many years, i became very familiar with the difficulties in applying medical research in the real world. mental health is determined by multiple factors. it is unlikely that we will find a single vector such as a virus or a bacterium that causes mental illness. thus, data demands can include multiple systems with biological, psychological, sociological, economic, and environmental factors. within many of these domains, we do not have objective measures such as the lab tests found in medicine. subjective selfreports are prone to many biases, and many of the symptoms are not observable by observers. the lack of a strong theory of mental disorders also makes it difficult to intelligently focus on only a few variables. even with such apparently simple measures that include observations or recordings from multiple informants, we do not have a consensus on how to integrate them (bickman et al. a; martel et al. ). however, i would expect that research generated with ai will contribute not only to improved treatment but also to enhanced theories by including heterogeneous clients and many data sources. confidentiality and trust are key issues in mental health treatment. how will the introduction of ai affect the relationship between client and clinician? as noted earlier, there are problems, especially with deep learning, in interpreting the meaning of algorithmic solutions and predictions. our ability to explain the algorithms to clients is problematic. while many research projects outside of mental health show that combining ai with human judgment produces the best outcomes, this research is still in its infancy. a great deal has been written about ai in the context of medicine, but we need a reality check about the importance of ai in clinical practice. ben-israel et al. ( ) addressed the use of ai in a systematic review of the medical literature from to . the authors focused on human studies that addressed a problem in clinical medicine using one or more forms of ai. of the studies, only % were prospective. none of the studies included a power analysis, and half did not report attrition data. most were proof of concept studies. the authors concluded that their study showed that the use of ai in daily practice of clinical medicine is practically nonexistent. the authors acknowledge that use was defined by publication and that many applications of ai may be occurring without publication. regardless, this study suggests that there are many barriers that must be overcome before ai is more widely used. the self-help industry can provide perspective on digital apps, including some that use ai. it has been estimated that this sector was worth $ . billion in and is expected to be worth $ . billion in (la rosa ). part of that big dollar market is in digital mental health apps, although their precise monetary value is unknown. more to the point is that we know little about the effectiveness of digital apps in the marketplace (chandrashekar ) . moreover, many have warned that these unregulated and untested apps could be dangerous (wykes ) . in the united states, the publication of books is protected by the constitution, so there are no rules governing what can be published in the self-help sector. the market determines what gets accepted and used, regardless of effectiveness or negative side effects. but publication is limited by the cost of publishing and distribution. this is not the case for digital programs, where marginal costs of adding an additional user are negligible. unlike other mental health interventions, there are no licensing or ethical standards governing their use. there are no data being uniformly collected on their use and their effects. although there are u.s. government rules that can be applied to these apps (armontrout et al. ) , the law has many exceptions. the authors note that they could not find a single lawsuit related to software that diagnoses or treats a psychiatric condition. an interactive tool is provided by the federal trade commission to help judge which federal laws might apply in developing an app (https ://www.ftc.gov/tips-advic e/busin ess-cente r/guida nce/mobil e-healt h-apps-inter activ e-tool). it is clear that digital mental health apps will continue to grow. it is critical that services research and funding agencies do not overlook this development that might have potentially positive or negative effects. these are but a few of the many areas or ai needing additional research and potential limitations to be addressed. an excellent discussion of these and other relates issues regarding the potential hype common in the ai field is provided in the national academy of medicine's monograph on the use of ai in healthcare (matheny et al. ) . a thought-provoking paper by hagendorff and wezel ( ) classifies what ai can and cannot do. some of the authors' concerns, such as measurement, completeness and quality of the data, and problems with transparency of algorithms, have already been discussed, so i will describe those that i feel are most relevant to mental health services. the authors describe two methodological challenges, the first being that the data used in ai systems are not representative of reality because of the way they are collected and processed. this can lead to biases and problems with generalizability. second is the concern that supervised learning represents the past. thus, prediction can be based only on the past and not on expectations of change; thus, in some respects, change is inhibited. hagendorff and wezel ( ) also note several societal challenges. one such challenge they cite is that many software engineers who develop these algorithms do not have sufficient knowledge of the sociological, psychological, ethical, and political consequences of their software. they suggest this leads to misinterpretations and misunderstandings about how the software will operate in society. the authors also note the scarcity of competent programmers. i noted earlier that this is especially the case in academia and particularly in the behavioral sciences. the authors highlight that ai systems often produce hidden costs. this includes hardware to run the ai systems and, i would add, the disruptive nature of the intrusion of ai into a workflow. among the technological challenges discussed by hagendorff and wezel, i believe the authors' focus on the big differences between human thinking and intelligent machines is especially relevant to mental health. machines are in no way as complex as human brains; even ai's powerful neural networks, with more than a billion interconnections, represent only a tiny portion of the complexity of brain tissue. in order to obtain better convergence between machines and humans, hagendorff and wezel suggest that programmers follow the three suggestions made by lake et al. ( ) . first, programmers should move away from pattern recognition models, where most development started, to automated recognition of causal relationships. the second suggestion is to teach machines basic physical and psychological theories so that they have the appropriate background knowledge. the third suggestion is to teach machines to learn how to learn so that they can better deal with new situations. the comparison between ai and human thought is the only aspect of their paper where hagendorff and wezel mention causality issues. they note the challenge related to the inflexibility of many algorithms, especially the supervised ones, where simply changing one aspect would result in processing errors because that aspect was not in the training data. machines can be vastly superior to humans in some games where there are very specific inputs for achieving specific goals, but they cannot flexibly adapt to changes like humans. the authors suggest that promising technical solutions are being worked on to deal with this weakness in transferability. all these challenges will affect how well ai will work in mental health services. most problems will probably be solved, but the authors believe that some of these challenges will never be met, such as dealing with the differences between human and computer cognition, which means that ai will never fully grasp the context of mental health services. the machine's construction of a person may lead to a fragmented or distorted self-concept that conflicts with the person's own sense of identity, which seems critical to any analysis of the person's mental health or lack thereof. i do not have a sense of how serious this and the other challenges will be for us in the future, but it is clear that there is a lot more we need to learn. yet another set of concerns, specifically about the variation in ai called deep learning (dl), was enumerated by marcus ( ) , an expert in dl. in a controversial paper in which he identified limitations of dl, he noted that dl "may well be approaching a wall" (p. ) where progress will slow or cease. for example, he noted that dl is primarily a statistical approach for classifying data, using neural networks with multiple layers. dl "maps" the relationships between inputs and outputs. while children may need only a few trials to correctly identify a picture of a dog, dl may need thousands or even millions of labeled examples before making correct identifications without the labels. very large data sets are needed for dl. this is not the case for all ml techniques. i will not attempt to summarize the nine other limitations he sees with dl since many of them are noted elsewhere in this paper. he concludes that dl itself is not the problem; rather, the problem is that we do not fully understand the limitations of dl and what it does well. marcus warns against excessive hype and unrealistic expectations. i am taking this advice personally, and i am not expecting my tesla to be fully autonomous in as predicted by elon musk (woodyard ) . wolff ( ) provided an overview of how some of the problems of deep learning can be ameliorated. he responds to marcus using many of the subheadings in marcus's paper. he calls his framework the sp theory of intelligence, and its application is called the sp computer model (sp stands for simplicity and power). the theory was developed by wolff to integrate observations and concepts across several fields including ai, computing, mathematics, and human perception and cognition, using information compression to unify them. despite these and other concerns previously described, i do think that the advantages of ai for moving mental health services forward outweigh its disadvantages. however, this summary of advantages does not attempt to balance in length or number the disadvantages described above. i do not think it is necessary to repeat the already described numerous applications and potential applications of ai that can be used to improve health services. rather than repeating the numerous applications and potential applications of ai that can be used to improve health services, i highlight only a few key advantages. one of the main advantages is the way ai deals with data. it can handle large amounts of data from diverse sources. this includes structured (quantitative) and unstructured (text, pictures, sound) data in the same analyses. thus, it can integrate heterogeneous data from dissimilar sources. as noted earlier, the inclusion of non-traditional data such as those obtained from remote sensing (e.g., movement, facial expression, body temperature) will be responsible for a paradigm shift in what we consider relevant data. ai, if widely adopted, has the potential to have a major impact on employment. while most of the popular press coverage has been on the potential negative effects of eliminating many jobs, there also are potential positive effects. ai can reduce the costs of many tasks, thus increasing productivity. on the human side, it can streamline routine work and eliminate many boring aspects of work. it thus can free up workers to engage in the more complex and interesting aspects of many jobs. previous innovations have caused job dislocations. the classic loss of jobs in making buggy whips after the advent of automobiles is just one example. the inventions of the industrial age, such as steam engines, displaced many workers but also created many more new jobs. we know that many unskilled or semi-skilled jobs will be affected by ai in a major way. the elimination of cashiers with automated checkouts is now being implemented by amazon. in these stores, you scan your phone, and then ai and cameras take over. you just put products in your bag or cart and leave when you are finished. self-driving cars and trucks will greatly disrupt the transportation industry. we have weathered these disruptions in the past, but even the experts are unsure about how ai will influence jobs. probably the area in which there is the most positive potential in healthcare is when humans and machines collaborate in partnership. here, ai augments human tasks but keeps humans in the center. thus, physicians will no longer be separated by a laptop when speaking to a patient because ai will be able to record, take notes, and interpret the medical visit. we have documented the shortage of mental health workers and the immense gap between mental health needs and our ability to fill them. yes, we can train more clinicians, but our society seems unwilling to offer sufficient salaries to attract and keep such individuals. we have been experimenting with computers as therapists for more than years, but now we finally have the technological resources to develop and implement such approaches. we have started to use chatbots to extend services, but in the near future, ai may allow us to replace the human therapist under some conditions (hopp et al. ). in , the computer scientist and science fiction author vernor vinge developed the concept of a singularity in which artificial intelligence would lead to a world in which robots attain self-consciousness and are capable of what are now human cognitive activities (vinge ) . advocates and critics disagree on whether a singularity will be achieved and whether it would be a desirable development (braga and logan ) . braga and logan, editors of a special issue of information on the singularity and ai, conclude that although ai research is still in the early stage, the combination of human intelligence and ai will produce the best outcomes, but ai will never replace humans and we cannot fully depend on ai for the right answers. while these authors are well-informed, their crystal ball may not be clearer than anyone else's. the relevance of the singularity for healthcare lies in asking whether there will there be a time when ai-based computers are more effective and efficient than clinicians and will replace them. it is a question worth considering. i have presented a comprehensive, wide-ranging paper dealing with ai and mental health services. i have described major deficiencies of our current services, namely the lack of sufficient access, inadequate implementation, and low efficiency/effectiveness. i summarized how precision medicine and ai have contributed to improving healthcare in general and how these approaches are being applied in precision psychiatry and mental health. the paper then describes research that shows how ai has been or can be used to help solve the five problems i noted earlier. i then described the disadvantages and advantages of ai. in reviewing all this information, i believe there is one factor that i have not discussed sufficiently that clearly differentiates the way mental health services have been delivered and the way i expect they will be delivered in the future. i want to focus this last section of the paper on what i believe is the most important and significant change that can occur. this change is reflected in a simple question: is a human clinician necessary to deliver effective and efficient mental health services? i believe the answer to this question does not depend on the occurrence of the singularity but lies in the growth of ai research and its application to mental health services. i think there is widespread agreement that there are significant problems with diagnoses and the quality of our measures. moreover, most will probably agree that if ai can improve diagnoses and measures, then we should use utilize ai and let the results speak for themselves. the dependence on rcts will probably not be resolved by ai research, but ai can clearly help inform what should be tested in rcts. however, our current services overwhelmingly depend on human clinicians to deliver treatment. the problem with learning and feedback is that it requires clinicians to learn how to improve treatment over time with feedback. we are still uncertain about how well clinicians can learn from experience, training, and education (bacon ) . we also lack evidence of the best way to provide feedback to enhance that learning (bickman a; dyason et al. ). the problem of treatment precision is also currently tied to having the clinician deliver the treatment. while we can expect ai to deliver more precise information about treatment planning, we still depend on the clinician to interpret and deliver it with fidelity with some evidence-based model. a precision approach requires the clinician to systematically deliver treatment that is most appropriate to a specific client. we do not have good evidence that most clinicians can do that. i believe no other issue generates a bigger emotional response than the idea of the changing the role of the clinician. no other issue has the economic impact on services as the position of the clinician. i believe this issue is the most critical to the future of mental health services and will be most affected by ai. i note that in in writing an introduction to an extensive special issue of this journal called "therapist effects in mental health service outcome" (king ) , the authors of the introduction to that issue not did not note the potential role of ai in affecting clinicians (king and bickman ) . change is happening rapidly. mental health services are not alone in facing the issue of the role of humans, although human clinicians are probably more central to the provision of mental health services than other health services. a similar issue of the role of humans in the provision of services is being played out in surgery. surgery has been using robots for over years (bhandari et al. ), but the uptake has been slow for a variety of reasons. the next iteration of robot use is a move from using robots guided by surgeons to using robots assisted by ai and guided by surgeons. the use of ai may be seen as an intermediate step to fully autonomous ai-based robots not guided by surgeons. however, it is very clear that this progression is speculative and will take a long time to happen, if ever, given the consequences of errors. closer to our everyday experience is the similar path that the development of autonomous driving involves as we move toward the point at which a human driver is no longer needed. will mental health services follow a similar path? since we do not currently have a sufficient amount of research on using ai in treatment alone to inform us, we must look elsewhere for guidance. two bodies of literature are relevant. one deals with the use of computers and other technologies that do not include the use of ai at present, the second with self-help in which the participation of the clinician is minimal or totally absent. first, let us consider the existing literature that contrasts technology-based treatments with traditional face-to-face psychotherapy. then i will present some reviews of self-help research, followed by a description of the small amount of research using ai in treatment. a review of studies of internet-delivered cbt (icbt) to youth, using waitlist controls, supports the conclusion that cbt could be successfully adapted for internet-based treatment (vigerlan et al. ) . in a meta-analytic review of meta-analyses, containing studies of adult use of internet delivered via icbt, the authors concluded that icbt is as effective as face-to-face therapy (andersson et al. ) . hermes et al. ( ) include websites, software, mobile aps, and sensors as instances of what they call behavioral intervention technologies (bit). in their informative article, dealing primarily with implementation, they note that these technologies (they do not mention ai) can relate to a clinician in three ways: ( ) when intervention is delivered by the clinician and supported by bit, ( ) when bit provides the intervention with support from the clinician, or ( ) when intervention is fully automated with no role for the clinician. this schema clearly applies to the ai interventions and the role of clinicians as well. their conceptual model is helpful in understanding the parameters of implementation. they present a comprehensive plan for research to fill in the major gaps in the literature that addresses the question of comparative effectiveness of bit and traditional treatment. carlbring et al. ( ) conducted a systematic review and meta-analysis of eligible studies of ibct versus face-to-face cbt and reported that they produced equivalent outcomes, supporting the conclusions drawn by previous studies. it is also important to consider the issue of therapeutic alliance (ta) and its relationship to internet-based treatment. ta, to a large extent, is designed to capture the human aspect of the relationship between the clinician and the client. there are thousands of correlational studies that have established that ta is a predictor of treatment outcomes (flückiger et al. ) ; however, there are few studies of interventions that show a causal connection between ta and outcomes (e.g., hartley et al. ) . moreover, the very nature of ta as trait-like or state-like, which is central to causal assumptions, is being questioned and is subject to new research approaches (zilcha-mano ) as well as to questions about how it should be measured regardless of my doubts about the importance of ta, the fluckiger et al. ( ) meta-analysis found similar effect sizes (r = . ) for the alliance-outcome relationship in online interventions and in traditional face-to-face therapies. however, most of these studies were guided by a therapist, so the human factor was not totally absent. penedo et al. ( ) , in their study of a guided internet-based treatment, showed that it was important to align with the client's expectations and goals because these were related to outcomes, but no such relationship existed with the traditional third component of ta, bond with the supporting therapist, implying that ta might play a different role in internetbased treatments. i was trained as a social psychologist and was a graduate student of stanley milgram (of the famous obedience experiments), so i was curious about the research on the relationship between technological virtual agents and humans beyond the context of mental health treatment. several studies cited by schneeberger et al. ( ) showed that robots could get people to do tiring, shameful, or deviant tasks. the authors found that participants obeyed these virtual agents similarly to the way they responded to humans in a video-chat format. the participants did the same number of shameful tasks regardless of who or what was ordering them. moreover, doing the tasks produced the same level of shame and stress in the participant. they concluded that virtual agents and humans appear to have the same influence as human experimenters on participants. of course, there are many limitations associated with generalizing from this laboratory study, which was conducted with female college students in germany, but it does suggest that a great deal of research needs to be done on how humans relate to robots and virtual agents. miner et al. ( ) suggest that use of conversational ai in psychotherapy can be an asset for improving access to care, but there is limited research on efficacy and safety. can we learn about the role of the therapist from therapies that do not involve any therapist or technology? there is substantial research on self-help approaches from written material or what some call bibliotherapy. in general, research has supported the effectiveness of bibliotherapy before the advent of digital approaches. in , cuijpers et al. published a review of the literature that compared face-to face psychotherapy for depression and anxiety with guided selfhelp (i.e., with some therapist involvement) and concluded that they appeared comparable, but because there were so few studies in this comparison, this conclusion should be interpreted with caution. has the situation changed in the last decade? in a comprehensive review and meta-analysis almost years later, bennett et al. ( ) conducted a review and meta-analysis of studies. they concluded that self-help (both guided and unguided) had significant moderate to large effects on reducing symptoms of anxiety, depression, and disruptive behavior. however, there was also very high heterogeneity among the outcomes of these studies. compared to face-to-face therapy, self-help was better than no treatment but slightly worse than face-to-face treatments, guided therapy was better than unguided, and computerized treatment was better than bibliographic treatment. it is important to note that none of the studies were fully powered noninferiority trials, which would be a superior design. the authors concluded that their study showed potential near equivalence for self-help compared to faceto-face interventions, and their conclusions were consistent with several other reviews of self-help for mental health disorders in adults. the paper makes no mention of ai. cuijpers et al. ( ) conducted a network meta-analysis of trials of cbt addressing the question of whether format of delivery (individual, group, telephone-administered, guided self-help, or unguided self-help) influenced acceptability and effectiveness for these adult patients with acute depression. no statistically significant differences in effectiveness were found among these formats except that unguided self-help therapy was not more effective than care as usual but was more effective than a waitlist control group. the authors concluded that treatments using these different formats should be considered alternatives to therapist-delivered individual cbt. as in the previous publication, there was no mention of the use of ai, but cuijpers believes that few if any of the studies reviewed in his publication used ai (p. cuijpers, personal communication, march , ) . there is an emerging area of the use of ai in treatment that is informative. tuerk et al. ( ) , in a special section of current psychiatry reports focusing on psychiatry in a digital age, describe several approaches to using technology in evidence-based treatments. most relevant is their discussion of the use of ai in what has been called "conversational artificial intelligence" where there is a real-time interchange between a computer and a person. they note research that shows that this approach is low risk, high in consumer satisfaction, and high in self-disclosure. they suggest that there is a great deal of clinical potential in using ai in this manner. in a review of the literature from to on conversational agents used in the treatment of mental health problems, gaffney et al. ( ) found only qualifying studies out of an initial , with four being what they called full-scale rcts. they concluded that the use of conversational agents was limited but growing. all studies showed reduced psychological distress, with the five controlled studies showing a significant reduction compared to control groups. however, the three studies that used active controls did not show significant differences between the waitlist controls and use of a conversational agent, although all showed improvement. the authors concluded that the use of conversational agents in therapy looks promising, but not surprisingly, more research is needed. a similar conclusion on conversational agents was reached in another independent review (vaidyam et al. ) . i have little doubt that more research will be forthcoming in this emerging area. in summary, previous research using digital but not aipowered icbt, self-help (bibliotherapy), and ai-powered conversational agents suggests that effective treatment can be delivered without a human clinician under certain circumstances. i want to emphasize that these studies are suggestive but far from definitive. rather, they suggest that the role of the clinician is worth more exploration, but they do not establish the conclusion that we do not need clinicians to deliver services. we need to know a great deal more about how ai-supported therapy operates in different contexts. a survey of psychiatrists from countries asked about how technology will affect their future practice (doraiswamy et al. ) . only . % felt their jobs would become obsolete, and only a small minority ( %) felt that ai was likely to replace a human clinician in providing care. as much of the literature on the effects of ai on jobs suggests, those surveyed believed that ai would help in more routine tasks such as record keeping ( %) and synthesizing information, with about % believing their practices would be substantially changed. about % thought ai would have no influence or only minimal effect on their future work over the next years. another % thought their practices would be moderately changed by ai over the next years. more than three quarters ( %) thought it unlikely that technology would ever be able to provide care as well as or better than the average psychiatrist. only % of u.s.-based psychiatrists predicted that the potential benefits of future technologies or ai would outweigh the possible risks. some of the specific tasks that psychiatrists typically perform, including mental status examination, evaluation of dangerous behavior, and the development of a personalized treatment plan, were also felt to be tasks that a future technology would be unlikely to perform as well. i do not think many psychiatrists in this study are prepared for the major changes in their practices that are highly likely to occur in the next quarter century. in a thoughtful essay on the future of digital psychiatry, hariman et al. ( ) draw a number of conclusions. they predict major changes in practice, with treatment by an individual psychiatrist alone becoming rare. patients will receive treatment through their phones, participate in videoconferencing, and converse with chatbots. clinicians will receive daily updates on the patients through remote sensing devices and self-report. ai will be involved in both diagnosis and treatment and will integrate diverse sources of information. concerns over privacy and data security will increase. this is not the picture that the previously described survey of psychiatrists anticipated. brown et al. ( ) present the pros and cons of ai in an interesting debate format. on the pro side, the authors argue that while there are current limitations, the improvements in natural language processing (nlp) will lead to better clinical interviews. they point to research that shows people are more likely be honest with computers as a plus in obtaining more valid information from clients. they expect the ai "clinician" will be seen as competent and caring. they do note the danger that non-transparent ai will produce unintended negative side effects. those arguing against the use of ai clinicians acknowledge the technical superiority of ai to accomplish more routine tasks such as information gathering and tracking, but they point out the limitations even in the development of ai therapists. the lack of data needed to develop and test algorithms is critical. i have noted this in the discussion of the diagnostic muddle as a problem that ai can help solve, but these anti-ai authors argue that because psychiatrists disagree on diagnoses, there is no gold standard against which to measure the validity of ai models. this seems to be a rather unusual perspective from which to challenge change. they insightfully note that ai is different from human intelligence and does not perform well when presented with data that are different from training data. but the anti-ai authors acknowledge that more and better data may lead to improvement. brown et al. ( ) argue that common sense is something that ai cannot draw on; however, this seems to be a weak argument when common sense has been demonstrated to be inaccurate under many situations. they conclude with the statement that psychiatry "will always be about connecting with another human to help that individual" (p. ). this may be more wishful thinking than an accurate prediction about the future. those arguing the pro position state that the "the advance of ai psychiatry is inexorable" (p. ). on the other hand, the opponents of ai correctly point out that there is not yet sufficient evidence to draw a conclusion about the effectiveness of ai versus human clinicians. while there is disagreement about the potential advantages and disadvantages of ai, both sides agree that we need more and better research in this area. simon and yarborough ( ) present the case that ai should not be a major concern for mental health. they argue that ideally, our field would abandon the term artificial intelligence in regard to actual diagnosis and treatment of mental health conditions. using that term raises false hopes that machines will explain the mysteries of mental health and mental illness. it also raises false fears that all-knowing machines will displace human-centered mental health care. big data and advanced statistical methods have and will continue to yield useful tools for mental health care. but calling those tools artificially intelligent is neither necessary nor helpful. (p. ) the authors further take the position that despite the buildup around artificial intelligence, we need not fear the imminent arrival of "the singularity," that science fiction scenario of artificially intelligent computers linking together and ruling over all humanity. . . a scenario of autonomous machines selecting and delivering mental health treatments without human supervision or intervention remains in the realm of science fiction. (p. ) a more balanced approach to the role to the issue of replacement of clinicians by ai is presented by ahuja ( ) . after his review of the literature on medical specialists who may be replaced or more likely augmented by ai, his pithy take on this question is "or, it might come to pass that physicians who use ai might replace physicians who are unable to do so" (ahuja , p. ) . clearly, ai research will have to provide strong evidence of its effectiveness before ai will be accepted by some in the psychiatric community. there are several pressing questions about how mental health services should be delivered and about the future of mental health services. doubts about how much clinicians contribute to outcomes, our seeming inability to differentiate the effectiveness among clinicians except at the extremes, the lack of stability of employment of most community based clinicians, the poor track record on implementation of evidence-based programs, the cost of human services, the very limited availability of services especially where resources are inadequate-all lead to strong doubts about continuing the status quo of using clinicians as the primary way in which mental health services are delivered. in contrast, alternative approaches have many advantages. if scaled, ai therapists could be available to patients / and would not be bound to office hours. these ai therapists could represent any demographic or therapy style (e.g., directive) that the client preferred or that had been found to be more effective with a particular client. they can be specialists in any area for which there is sufficient research. in other words, not only can a personalized treatment plan be developed, but a personalized clinician (avatar) can be constructed for the best match with the client. of course, all these are putative advantages. as noted earlier, the application of ai is not without its risks and challenges, especially in putting together the interdisciplinary teams needed to accomplish this research. while i am optimistic about the potential contribution of ai to mental health services, it is just that-a potential. extensive research will be needed to learn whether these approaches produce positive outcomes when compared to traditional face-to face treatment, while also dealing with the ethical issues raised by ai applications. moreover, the quality of research needs significant improvement if we are going to have confidence in the findings. however, as exemplified by the rapid and uncontrolled growth of therapy apps, the world may not wait for rigorous supporting research before adopting a larger role for ai in mental health services. while my brief summaries of findings of ai in the medical literature are supportive of the application of ai, i do not want to give the impression that these positive findings are accepted uncritically. a deeper reading of many of these studies exposes methodological flaws that temper enthusiasm. for example, in reviewing comparisons between healthcare professionals and deep learning algorithms in classifying diseases of all types using medical imaging, x. liu et al. ( a) conclude that the ai models are equivalent to the accuracy of healthcare professionals. this review is the first to compare the diagnostic accuracy of deep learning models to health-care professionals; however, only a small number of the studies were direct comparisons. the authors also caution us by indicating what they labeled as the poor quality of many of the studies. the problems included low external validity (not done in a clinical practice setting), insufficient clarity in the reporting of results, lack of external validation, and lack of uniformity of metrics of diagnostic performance and deep learning terminology. however, the authors were encouraged by improvement in quality in the most recent studies analyzed. in commenting on the study, cook ( ) noted other limitations and concluded that it is premature to draw conclusions about the comparative accuracy of ai versus human physicians. if we are not more cautious, she warns that we will experience "inflated expectations on the gartner hype cycle" (p. e ). the latter refers to the examination of innovations and trends in ai. she cautions us to "stick to the facts, rather than risking a drop into the trough of disillusionment and a third major ai winter" (p. e ). many issues are raised in cook's paper, and the need to avoid the hype often found in the ai field is reiterated in the national academy of medicine's monograph on the use of ai in healthcare (matheny et al. ) . mental health services are changing. there are more than , mental health apps on the internet that are being used without much evidence of their effectiveness (marshall et al. ; bergin and davis ; gould et al. ) . the explosion of mental health apps is the leading edge of future autonomous interventions. however, there is pressure to bring some order to this chaos. probably the next innovation that will involve ai is its use in stepped therapy in which clients are typically triaged to low-intensity, low-cost care, monitored systematically, and stepped up to more intensive care if progress is not satisfactory . in this schema, the low-cost care could be ai-based apps with little risk to the client. if more confidence is gained in the safety and effectiveness of this type of protocol, the use of ai-based treatment would be expected to increase. the covid- pandemic will produce a major impact on mental health services. first, it is expected that the stresses caused by the pandemic will increase the demand for services (qiu et al. ; rajkumar ) . already poorly resourced mental health systems will not be able to meet this demand (Ćosić et al. ; ho et al. ; holmes et al. ) , especially in low resourced countries. however, the biggest change will be in the service delivery infrastructure. because of social distancing requirements, in-person delivery of therapy is being severely curtailed. while the major change at this time appears to be a shift to telemedicine (shore et al. ; van daele et al. ) , which is being adopted across almost all healthcare, there will need to be changes instituted in how clinicians are trained and supervised (zhou et al. ) . i have little doubt that ai will be adopted in order to increase efficiency and address the change in the service environment caused by the pandemic. in addition to changes initiated by the pandemic, there appear to be some changes in funding as a result of the protests concerning george floyd's killing. there is reconsideration of shifting some funding from police services to mental health and conflict reduction services to be delivered by personnel outside law enforcement (stockman and eligon ) . it will be difficult to meet this potential demand using the current infrastructure. the literature on ai and medicine is replete with warnings about the difficulties we face in integrating ai into our healthcare system. as a program evaluator, i appreciate the position paper describing the urgent need for well-designed and competently conducted evaluations of ai interventions as well as the guidelines provided by magrabi et al. ( ) . more suggestions for improving the quality of research on supervised machine learning can be found in the paper by cearns et al. ( ) . celi et al. ( ) describe the future in a very brief essay that is worth quoting: clinical practice should evolve as a hybrid enterprise with clinicians who know what to expect from, and how to work with, what is fundamentally a very sophisticated clinical support tool. working together, humans and machines can address many of the decisional fragilities intrinsic to current practice. the human-driven scientific method can be powerfully augmented by computational methods sifting through the necessarily large amounts of longitudinal patientand provider-generated data. (p. e ) however, research on ai, data science, and other technologies is in its infancy if not the embryonic stage of development. i am fully immersed in the struggle to implement several types of technologies in practice. changing the routine behavior of clinicians and clients is a major barrier to using new technologies, regardless of the effectiveness of these approaches. emanuel and wachter ( ) argue that the most important problem facing healthcare is not the absence of data or analytic approaches but turning predictions and findings into successful accomplishments through behavior change. alongside the investment in technology and analytics, we need to support the research and applications of psychologists, behavioral economists, and those working in the relatively new field of translational and implementation research. the emphasis on practical and implementable digital approaches requires a methodology that departs from the traditional efficacy approach, which does not focus on context and thus is difficult to translate to the real world. mohr et al. ( ) suggest a solution-based approach that focuses on three stages that they label create, trial and sustain. creation focuses on the initial stages of development, although not exclusively, and takes advantage of the unique characteristics of digital approaches that focus on engagement rather than trying to mimic traditional psychotherapy. trial must be dynamic because digital technologies rapidly change; rapid evaluations are required, such as continuous quality improvement strategies (bickman and noser ) . sustainability requires more from investigators and evaluators than publication of results; they must also produce sustainable implementation that no longer depends on a research project for support. we are currently in an ai summer in which there are important scientific breakthroughs and large investments in the application of ai (hagendorff and wezel ) . but ai has had several winters when enthusiasm for ai has waned and unreasonable expectations have cooled. we were confronted with the reality that ai could not accomplish everything that people thought it could and that investors and journalists had hyped. ai, at least in the near term, will not be the superintelligence that will destroy humanity or the ultimate solution that will solve all problems. enthusiasm for ai seems to run in cycles like the seasons. ai summers suffer from unrealistic expectations, but the winters bring an experience of disproportionate backlash and exaggerated disappointment. there was a severe winter in the late s, and another in the s and s (floridi ) . today, some are talking about another predictable winter (nield ; walch ; schuchmann ). floridi ( ) suggests that we can learn important principles from these cycles. first is whether ai is going to replace previous activities as the car did with the buggy, diversify activities as the car did with the bicycle, or complement and expand them as the plane did with the car. floridi asks how acceptable an ai that survives another winter will be. he suggests that we need to avoid oversimplification and think deeply about with we are doing with ai. in the june issue of the technology quarterly of the economist ( ), it is suggested that because ai's current summer is "warmer and brighter" than past ones because of widespread deployment of ai, "another fullblown winter is unlikely. but an autumnal breeze is picking up" (p. ). i have traced a path my career has taken from an almost exclusive focus on randomized experiments to consideration of the applications of ai. i have identified the main problems related to mental health services research's almost sole dependence on rct methodology. i have linked the problems with this methodology with the lack of satisfactory progress in developing sufficiently effective mental health services. the recent availability of ai and the value now being placed on precision medicine have produced the early stages of a revolution in healthcare that will determine how treatment will be developed and delivered. i anticipate that in the very near future, a first-year graduate student will be contemplating the same questions that i raised years ago, because they are still relevant, but this time he or she will realize that there are answers that were not available to me. acknowledgements this paper is part of a special issue of this journal titled "festschrift for leonard bickman: the future of children's mental health services." the issue includes a collection of original children's mental health services research articles, this article, three invited commentaries on this article, and a compilation of letters in which colleagues reflect on my career and on their experiences with me. the word festschrift is german and means a festival or celebration of the work of an author. there are many people to thank for their assistance in both the festschrift and this paper. first, i want to acknowledge my two colleagues and friends, nick ialongo and michael lindsey, who spontaneously originated the idea of a festschrift during a phone conversation with them. the folks at the johns hopkins bloomberg school of public health were great in supporting the daylong event held on may , . the many friends, family, former students and colleagues who traveled from around the country to attend and present made the event memorable. i am grateful to the committee that helped put this special issue together, which included marc atkins, catherine bradshaw, susan douglas, nick ialongo, kim hoagwood, and sonja schoenwald. this paper represents more than a yearlong effort for which many contributed including the scholars who provided email exchanges and ideas throughout the conceptualization and writing process. i thank the two editors of this special issue, sonja and catherine, who spent much of their valuable time on this project during a very difficult period. the manuscript was greatly improved through the efforts of my copy editor, diana axelsen. most of all i thank corinne bickman, who has been my partner in life for almost years and has managed this journal since its inception. without her support and love none of this would have been possible. funding no external funding was used in the preparation or writing of this article. conflict of interest from the editors: leonard bickman is editorin-chief of this journal and thus could have a conflict of interest in how this manuscript was managed. however, the guest editors of this special issue, entitled "festschrift for leonard bickman: the future of children's mental health services," managed the review process. three independent reviews of the manuscript were obtained and all recommended publication with some minor revisions, with which the editors concurred. while the reviewers were masked to the author, because of the nature of the manuscript is was not possible to mask the author for the reviewers. from the author: the author reported receipt of compensation related to the peabody 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alliance really therapeutic? revisiting this question in light of recent methodological advances major developments in methods addressing for whom psychotherapy may work and why key: cord- -cdpsy f authors: dobler, claudia c. title: poor quality research and clinical practice during covid- date: - - journal: breathe (sheff) doi: . / . - sha: doc_id: cord_uid: cdpsy f breathe chief editor @claudiacdobler on how #covid amplifies flaws in clinical research and practice https://bit.ly/ cx jpo the covid- pandemic has turned our lives upside down. health services have adapted to the challenges posed by the pandemic at eyewatering speed. telemedicine has seen a rapid uptake in order for patient-physician encounters to comply with social distancing regulations. elective surgeries have been put on hold to make room in hospitals for patients with covid- and save valuable personal protective equipment. many pre-pandemic research projects have been put on hold, and legions of medical researchers are now dedicated to researching covid- . the number of publications on covid- , many published on pre-print servers that allow sharing of a research publicly before it has been peer reviewed, is skyrocketing. many journals have seen a drastic increase in manuscript submissions. the demand for quick distribution of information on a disease that we still know very little about is understandable and justified, and rapid dissemination of knowledge that can be accessed freely without a paywall is desirable. covid- , however, also highlights and amplifies some of the challenges clinical research and practice are facing in general. studies published on pre-print servers before peer review allow unrestricted and quick access to research results. there is, however, a significant danger that flawed results are picked up and disseminated by the media [ ] . studies with positive results, indicating effectiveness of an intervention, are likely to receive more attention than studies with negative results, even if the latter are of higher quality. there are numerous trials on different drug interventions competing for the same pool of covid- patients, many with poor study design (e.g. small sample size, no comparator group, not randomised, single centre and no study protocol publicly available) [ ] . trying to draw conclusions about the effectiveness of interventions based on (biased) nonrandomised studies can be grossly misleading. in the rush to make the latest science available, editorial scrutiny may fall short of usual benchmarks, as evidenced by the publication of large numbers of poorly designed studies, but also poor editing including increases in clunky, awkward or otherwise poor phrasing, grammatical errors, and mislabelling of tables and figures. some covid- investigators have never conducted research in patients with respiratory infections before and are not embedded in research networks with the required expertise to deliver high-quality trials. this will likely lead to wasteful, poor-quality research. national research funding bodies in most countries have missed the opportunity to streamline research efforts and focus on a small number of large, high-quality trials supported by extensive clinical research networks rather than a large number of small (and likely underpowered) studies. during any pandemic, evidence is rapidly developing and treatments might no longer be of interest by the time a trial starts recruiting patients. bayesian adaptive trial designs are therefore particularly suited to these circumstances. they will allow us to assess the most promising treatments based on the state of knowledge at the time and will increase the probability of patients being randomised to the best-performing treatments [ ] . there is a substantial imbalance in trial topics with only a small proportion of covid- trials registered on clinicaltrials.gov focusing on nondrug interventions [ ] . this pertains to important preventive non-drug interventions, such as hand hygiene and wearing of masks, but also non-drug interventions to treat patients with covid- , such as noninvasive positive pressure ventilation (nippv). the use of nippv is discouraged in some settings in favour of early intubation to minimise viral aerosolisation (and therefore the risk of viral transmission to healthcare workers). it is, however, unknown whether the use of nippv is associated with increased viral aerosolisation and transmission compared with oxygen therapy delivered via nasal prongs, hudson mask or high-flow nasal cannula. patients who overcome covid- with nippv as opposed to intubation will likely make a quicker recovery, but it is unknown whether delayed intubation in patients with covid- in whom nippv fails is associated with a higher risk of complications. on may , studies were registered on clinicaltrials.gov using the term "covid", of which only three were randomised trials to assess nippv, despite the urgent need for evidence to inform clinical practice in this area. physicians are frequently using experimental therapies in critically ill patients with covid- in the absence of proven effective treatments. they may, however, significantly underestimate potential harms of these drugs, for example, the potential cardiotoxicity of hydroxychloroquine, ritonavir, lopinavir, interferon-α β, azithromycin and methylprednisolone [ ] . clinicians must resist the impulse "to do something" and only use unproven treatments within clinical (ideally adaptive) trials [ ] . the tendency to prefer action over inaction is also known as commission bias and is motivated by avoiding regret about a missed opportunity when a treatment is not given, even if its efficacy is unclear [ ] . commission bias is a potential barrier to the implementation of evidence-based practice, and drives overtreatment and "low-value care" (care that provides little or no benefit and may cause harm to patients) [ ] . as clinicians, we must reflect on the biases that may influence our clinical decisionmaking, critically appraise the evidence of covid- treatments, and act with caution and reason [ ] . there might be exceptions to the rule, especially for non-drug interventions, where the use of the "precautionary principle" is sensible. this principle is a strategy to "adopt precautionary measures when scientific evidence about an environmental or human health hazard is uncertain and the stakes are high" [ ] . it has been proposed that this principle should be applied to the wearing of face masks in public, because covid- is a serious threat and the potential benefits of wearing a mask likely outweigh potential downsides, including a false sense of security and reduced compliance with other infection control measures [ ] . in a similar way, the precautionary principle should be applied to hand hygiene to reduce the spread of coronavirus. it is likely that covid- will pose a challenge to our healthcare systems for the foreseeable future, and we will have to find ways to treat patients with covid- while not neglecting patients with other diseases. with this in mind, i hope that the june issue of breathe on the topic of "rare and orphan lung diseases" will be informative and inspirational. claudia c. dobler , waste in covid- research adaptive clinical trials: a partial remedy for the therapeutic misconception sars-cov- : first do no harm clinicians' cognitive biases: a potential barrier to implementation of evidence-based clinical practice covid- amplifies flaws in clinical research and practice covid- -a reminder to reason the precautionary principle: definitions, applications and governance face masks for the public during the covid- crisis the author would like to thank prof. christine jenkins (the george institute for global health, sydney, australia), prof. guy marks (university of new south wales, sydney, australia), dr zinta harrington (liverpool hospital, sydney, australia) and dr hima vedam (liverpool hospital, sydney, australia) for many lively discussions on the topic of poor quality research and clinical practice during covid- . c.c. dobler has nothing to disclose. key: cord- -qt pu wp authors: varadarajan, rajan title: relevance, rigor and impact of scholarly research in marketing, state of the discipline and outlook date: - - journal: ams rev doi: . /s - - -x sha: doc_id: cord_uid: qt pu wp this commentary presents a dissenting point of view on some of key et al.’s (ams review, ) observations concerning the relevance and rigor of scholarly research in marketing, impact of scholarly research in marketing on research in other disciplines, and influence of the marketing function in firms at the top management level. the value proposition and values proposition of the marketing academy science "an objective, logical, and systematic method of analysis of phenomena, devised to permit the accumulation of reliable knowledge." (lastrucci , p. ) . marketing science objective, logical and systematic methods of analysis of marketing phenomena devised and used for the creation and accumulation of reliable knowledge. value proposition of the marketing academy members of the marketing academy create reliable knowledge on marketing phenomena and disseminate the knowledge by posting it in the public domain for use by individuals and organizations, worldwide. constituencies in a society who may find reliable knowledge on marketing phenomena to be of value include decision-makers in for-profit, not-for profit and governmental organizations, researchers and educators in marketing and allied disciplines, students of marketing and allied fields of study, journalists, and consumers. values proposition of the marketing academy members of the marketing academy are committed to respecting, upholding and abiding by the university's core mission, namely, the creation and dissemination of objective knowledge, and their grand compact with society. they consider society as the ultimate client for the knowledge they create, and specific constituencies such as marketing educators, practitioners and students as intermediate clients. [adapted from hunt on the responsibilities of the marketing discipline]. the grand compact of the academia with society: the state grants academic freedom to faculty-the privilege of conducting research free from the dictates of the church and state. in exchange, the state demands from faculty a commitment to the ideal of objective knowledge (see: hunt ) . in the opening paragraph of their article, key et al. ( ) highlight the importance of the academic value proposition of the marketing discipline founded on theoretical and conceptual research. two other value propositions that also merit our collective thought are the value proposition and the values proposition of the marketing academy to society. in that spirit, the above position statements on the value proposition and the values proposition of the marketing academy to society are proposed for consideration and deliberation by the marketing academy. as highlighted in the proposed definition of marketing science, its principal focus as a scientific endeavor is the creation and accumulation of reliable knowledge on marketing phenomena (i.e. knowledge based on which marketing decisions can be made and actions taken with confidence). also, as highlighted, a commitment by the marketing academy to society that is enshrined in the grand compact between academia and society is the ideal of objective knowledge. issues relating to the relevance and rigor of scholarly research in marketing and allied business disciplines are sometimes framed and debated as relevance versus rigor or balance between relevance and rigor. that is, the pros and cons of a tradeoff of some level of rigor for greater relevance or vice-versa. in this regard, a caveat that should be borne in mind is that marketing knowledge generated by research studies that tradeoff some level of rigor for greater relevance would be less reliable compared to studies that employ the current state-of-the-art of conceptual and empirical rigor. the theoretical and conceptual value proposition of the marketing discipline key et al. ( ) describe their article on the theoretical and conceptual value proposition of the marketing discipline as a dissenting perspective on the current course of scholarship in marketing. they note that a discipline-wide drift and myopic approach to rigor and relevance is a central premise that emerges from a critical selfexamination of the field of marketing. they enumerate a number of concerns regarding the current state of the field and outlook, and the need for theoretical and conceptual advances to move the discipline forward, increase its impact, create value, and preserve its overall relevance. further, they note that the purpose of their dissenting opinions is not to cast a disparaging shadow on the contributions by countless scholars over several decades to advancing marketing knowledge, but to stimulate critical self-reflection at the discipline level in order to combat an uninformed march into an unintentional future that could result in lack of relevance. they note that questioning the current state and trajectory of the discipline are crucial for creating potentially impactful marketing knowledge and disseminating them through scholarly journals, in classrooms and to organizations. this commentary strives to contribute to the conversation initiated by key et al. ( ) on the current state of the marketing discipline and its outlook. in light of space considerations, the scope of this commentary is limited to only some of the issues that are the focus of key et al.'s article. specifically, some of their observations concerning the relevance and rigor of scholarly research in marketing, reliance by researchers in the marketing discipline on theories developed in other disciplines to research marketing phenomena, influence of the discipline on scholarly research in allied disciplines, and influence of the marketing function in firms at the top management level. conjectures on the current state of the discipline and outlook: a cautionary note key et al. ( ) describe the current state and outlook of the marketing discipline as a discipline that is ( ) adrift and myopic in its approach to rigor and relevance, ( ) on an uninformed march into an unintentional future that could result in lack of relevancy, and ( ) finessing its way to irrelevance. two caveats should be borne in mind regarding the above and other discipline level generalizations about marketing's shortcomings by key et al. first , the marketing discipline, over the course of several decades, has evolved into a number of specialized fields. most marketing academics, in an attempt to stay current with the state-of-the-art in their chosen field of specialization, are likely to read only articles published in scholarly journals that are aligned with their research and teaching interests. given our limited knowledge or lack of knowledge of research and scholarship in other specialized fields, there is a need for caution when making generalizations about marketing's shortcomings at the discipline level. the second caveat is the number of observations that are the basis for key et al.'s ( ) discipline level generalizations about marketing's shortcomings. a very conservative estimate (guestimate) of the scholarly research output of the marketing discipline published in a select list of journals and business magazines listed in footnote # would be well over articles each year, , articles during the decade of to , and , articles during four decades from to . the above estimates are indicative of the vastness of the cumulative body of knowledge of the marketing discipline. the titles of the journals that the estimates are based on are indicative of the breadth of the discipline (i.e. specialized streams of research such as advertising, consumer behavior, innovation, interactive marketing, marketing and public policy, and strategic marketing). issues concerning the relevance of scholarly research in marketing, or lack thereof, are the focus of the essays by clark and pitt in key et al. ( ) . clark's essay is titled, "finessing our way to irrelevance." pitt notes that most scholarly research in marketing is ignored, because researchers in marketing ignore ( ) artifacts and technologies as shapers, ( ) biology, ( ) information technology, until it is too late, ( ) the shadow of the field, ( ) new ideas, and ( ) good writing. it should be noted that the nature and scope of relevance of marketing relevance is multifaceted, and managerial relevance is only one of its many dimensions. broadly construed, marketing relevance encompasses (but is not limited to) the following. & managerial relevance: research of relevance to marketing practitioners-research that contributes to (has the potential to contribute to) better marketing decision-making in for-profit and not-for-profit organizations (i.e. enable decision-makers to make more informed marketing decisions). & research relevance: research of relevance to researchers in marketing-research that contributes to improving the quality of scholarly and applied research in marketing. & public policy relevance: research of relevance to decision-makers in gover nment al and quasigovernmental agencies (e.g. department of justice, federal trade commission and securities and exchange commission in the us, and similar institutions in other countries)-research that contributes to improving laws and regulations governing the practice of marketing and welfare of the public as customers and consumers. & marketing education relevance: research of relevance to marketing educators-research that has an impact on curriculum content, pedagogical material, manner of delivery of marketing knowledge in the classroom, etc. & consumer welfare relevance: research of relevance to the public as customers and consumers-research that enables the public to make informed decisions on matters relating to the acquisition, possession, consumption and disposal of products. in addition to the above constituencies based perspective of the scope of marketing relevance, certain other facets of relevance should also be borne in mind. they include ( ) direct versus indirect relevance, ( ) articulated relevance by the researcher versus discovered relevance by readers, ( ) firstorder versus second-order relevance, ( ) conceptual versus instrumental relevance, ( ) enduring relevance versus relevance during a specific time frame, ( ) latent relevance, ( ) serendipitous relevance, ( ) immediacy of relevance, and ( ) breadth of relevance (varadarajan ) . a brief discussion on some of these facets of relevance follows. alleviation of poverty and hunger afflicting mankind were the principal relevance considerations behind the pioneering research by the late dr. norman borlaug (recipient of the nobel peace prize) to develop genetically improved highyield disease-resistant varieties of seeds for growing rice, wheat, corn and other food grains. on a scale of research relevance for the wellbeing of humanity, only a few other research endeavors in the annals of human civilization rise to the level of borlaug's research. a study lists the green revolution as one of human civilization-transforming innovations since the wheel (fallows ) . by increasing the a conservative estimate of the total number of articles focusing on marketing related topics published in the above marketing journals, three broad based business journals and four business magazines would be over articles each year, articles during the decade from to , and , articles during the four decades from to . collectively, in all of the journals and business magazines listed above, a very conservative estimate of the annual output of scholarly marketing knowledge would be well over articles. the cumulative body of knowledge output would be well over , articles during the decade of to , and well over , articles during the four decades from to . the year was the th year of publication of marketing science and the th year of publication of the international journal of research in marketing. the other five journals have a publication history longer than years. similarly, some of the journals in the list of marketing journals have a publication history that is less than years. some of the text presented in this section draws on varadarajan ( ) . yields of various food grains more than four-fold, borlaug's research was instrumental in alleviating hunger and poverty that afflicted hundreds of millions of people in developing and less developed countries during the last century. in addition to the intended relevance (alleviation of poverty and hunger), borlaug's research also resulted in a number of other unintended (un-envisioned/serendipitous) socially beneficial outcomes. for instance, the green revolution has been instrumental in preventing the destruction of hundreds of thousands of acres of forestland and their conversion into agricultural land in order to increase food output to feed a growing world population. the effect of the green revolution in preventing the destruction of vast stretches of forest green cover precedes our recent and growing awareness of the adverse environmental impact of deforestation. universities are home to both the passionates and the agnostics about relevance and research impact universities are (should be and always will be) home to both researchers passionate about the relevance and potential impact of their research as well as those who are agnostic. in his book, "the mathematician's apology," hardy ( , p. ) described number theory, his area in mathematics, as useless. he noted: "if useful knowledge is, as we agreed provisionally to say, knowledge which is likely, now or in the comparatively near future, to contribute to the material comfort of mankind, so that mere intellectual satisfaction is irrelevant, then the great bulk of higher mathematics is useless." he also noted (p. - ): "no discovery of mine has made, or is likely to make, directly or indirectly, for good or ill, the least difference to the amenity of the world." almost six decades later, in a review of a book on cryptography by singh ( ) , osserman ( ) noted that although number theory did initially seem useless, in , three mathematicians came up with an ingenious method to put it to practical use. according to osserman, their simple but clever use of a littleknown elementary result from number theory evolved into a multimillion dollar software business and the installation of a software program being on millions of computers worldwide. rigor: the virtue of prizing rigor and truth above all else key et al. ( , section "leyland pitt", paragraph ) note, "at the same time however, much of academic marketing research seems to prize rigor and truth above all else; whether it is actually interesting or relevant seems to matter less and less." in a later section, they allude to the management discipline as having achieved a remarkable balance between rigor and relevance. it is debatable as to whether researchers in the management discipline strive for a balance between rigor and relevance. however, should this indeed be the case, a shift to both rigor and relevance may be in the best long-term interests of the discipline. a number of considerations justify prizing rigor and truth above all else, regardless of whether the questions that are the focus of a research study are interesting and/or relevant. first, as highlighted in the introductory section, pursuit of truth through rigor in research is the hallmark of science (lastrucci ) , and integral to the grand compact between the academia and society (hunt ) . second, stewart, in his essay in key et al., draws attention to the credibility crisis currently faced by social sciences in the aftermath of the findings of the reproducibility project (i.e. differences between the findings of the replication research studies and the findings reported in previously published research). any dilution of rigor in scholarly research (i.e. the current state-of-the-art of conceptual and empirical rigor) will only further erode the credibility of academic social sciences. third, while there is always a possibility of a research endeavor being serendipitously relevant, it is inconceivable that a research endeavor would be serendipitously rigorous. cole's ( , p. ) characterization of a research university as an institution with tolerance for unsettling ideas and insistence on rigorous skepticism is instructive in this regard. he notes: "the university ought to be viewed in terms of a fundamental interdependence between the liberality of its intellectual life and the conservatism of its methodological demands. …we permit almost any idea to be put forward-but only because we demand arguments and evidence to back up the ideas we debate and because we set the bar of proof at such a high level. these two components, tolerance for unsettling ideas and insistence on rigorous skepticism about all ideas-create an essential tension at the heart of the american research university. it will not thrive without both components operating effectively and simultaneously." bernard ( , p. ) points out the knowledge will be closer to the truth in certain disciplines than in other disciplines. he notes: "in his an essay concerning human understanding ( [ ] ), locke reasoned that since we cannot see everything, and since we cannot even record perfectly what we do see, some knowledge will be closer to the truth than other knowledge. prediction of the behavior of planets might be more accurate than the prediction of human behavior, but both predictions should be based on better and better observation, measurement, and reason…" all else being equal, marketing knowledge created by employing scientific research methods will be farther from truth compared to knowledge created in the physical sciences and natural sciences by employing scientific research methods. however, as bernard ( ) alludes to, greater conceptual rigor (better and better reasoning through development of new theories and refining existing theories) and empirical rigor (better and better observation, measurement, modeling, analysis, etc.) is the proven pathway for creating more reliable marketing knowledge (i.e. knowledge that is closer to truth). overbye's ( ) observations concerning the values that science imparts in scientists also is also insightful concerning the virtue of prizing rigor and truth above all else. he notes: "science is not a monument of received truth but something that people do to look for truth… that endeavor, which has transformed the world in the last few centuries, does indeed teach values. those values, among others, are honesty, doubt, respect for evidence, openness, accountability and tolerance and indeed hunger for opposing points of view. these are the unabashedly pragmatic working principles that guide the buzzing, testing, poking, probing, argumentative, gossiping, gadgety, joking, dreaming and tendentious cloud of activity …nobody appeared in a cloud of smoke and taught scientists these virtues. this behavior simply evolved because it worked... and indeed there is no leader, no grand plan, for this hive. it is in many ways utopian anarchy." advances in research rigor, both conceptual and empirical rigor, are hallmarks of progress in scientific method, and indispensable from the standpoint of creating reliable knowledge. as researchers in a discipline become aware of weaknesses in their current methods of inquiry, refinements in them and new research methods take root. consequently, the current research rigor thresholds (conceptual and empirical rigor threshold), for doctoral dissertations and journal articles in the marketing discipline are considerably higher than they were a decade ago, and will be substantially higher a decade from now than they are currently. the following illustrations from other disciplines provide additional insights into the virtue of prizing rigor and truth in research, above all else. at a number of laboratories worldwide, several thousands of researchers are currently working on developing a vaccine for the coronavirus. in the absence of rigor during every step in the development and testing of the vaccine, it would be an exercise in futility, and a waste of precious time and resources. it would not result in a vaccine that can be safely administered to humans and bring to an end the pandemic. the concerns voiced by a number of leading scientists in the us about the specter of compromises in research rigor in the development of a safe and effective vaccine for the coronavirus in the face of political pressure to speed development of the vaccine is instructive in this regard (lafraniere et al. ) . as noted in an earlier section, alleviation of poverty and hunger were the principal relevance considerations behind borlaug's pioneering research to develop genetically improved high-yield disease-resistant varieties of seeds for growing rice, wheat, corn and other food grains. however, in the absence of rigor during every stage of research that spanned well over a decade, the development of genetically improved high-yield disease-resistant varieties of seeds to alleviate hunger and poverty would not have materialized. impact of scholarly research in marketing on scholarly research in other business disciplines key et al. ( ) draw attention to a study based on a bibliometric analysis of leading journals in accounting, finance, management, and marketing that found marketing scholarship to be the least influential of the four (clark et al. ) . during the period of their study ( to ), they found finance to have had the greatest volume of total citation exports ( , ), followed by management ( ), accounting ( ), and marketing ( ). clark et al. further note that marketing's largest volume of citation exports ( ) and imports ( ) are with management, and it had virtually no citation exports to either finance or accounting. regarding the use of the term, "export" in the context of patterns of knowledge flow across disciplines, the following caveat should be borne in mind. while researchers routinely draw insights from other disciplines (i.e. they import), they do not proactively engage in exporting knowledge. export of knowledge from a discipline is merely an artifact of researchers in other disciplines importing the knowledge. a plausible explanation for the differences in the citation patterns across business disciplines reported by clark et al. ( ) is differences in the propensity of scholars in different disciplines to explore other disciplines for knowledge insights. beginning with the doctoral program, in the marketing discipline, there is considerable emphasis on looking outward into allied disciplines for conceptual, theoretical and methodological insights. a cursory review of articles published in marketing journals would show references to books and journal articles in other fields such as accounting, anthropology, biology, computer science, economics, finance, law, management, management information systems, political science, psychology, sociology, statistics, etc. in fact, in marketing, a doctoral dissertation proposal (manuscript) submitted for review to the dissertation committee (journal) that is void or weak in respect of building on scholarly insights from other disciplines is likely to be viewed as shallow. marketing scholarship benefits from the receptivity of its research community to build on insights from other disciplines, and relatedly, the research community being not afflicted by the "not invented here" syndrome. influence of the marketing function in organizations at the top management level key et al. ( ) highlight certain trends as cause for concern. they include the ( ) decline in the influence of the marketing function in organizations, ( ) elimination of the position of the chief marketing officer (cmo) in a number of organizations, ( ) acquisition of advertising agencies by management consulting firms and information technology firms, and ( ) decline in the influence of the academic discipline of marketing in universities. the first three pertain to developments in the world of business. even if the empirical evidence in support of the above were to be overwhelming, marketing academics should view them only as interesting research questions worthy of scholarly research, and not as developments to be alarmed about. our focus as marketing academics should be disinterested pursuit of truthdescribing, understanding, explaining and predicting marketing phenomena of interest. the following are some research questions that may be worthy of investigation in our quest for disinterested pursuit of truth: ( ) what explains the relative influence of different organizational functions in firms? ( ) what explains changes in the relative influence of organizational functions in firms? ( ) what explains the emergence of dominant coalitions in firms (certain organizational functions emerging as more dominant and wielding greater power than other functions)? ( ) what explains the marketing function wielding more versus less influence in certain types of firms, industries, and country markets, than in others? ( ) what do various macro environmental trends portend for the relative influence of the marketing function in organizations in the future? (varadarajan ) . the findings of the following studies serve to illustrate the essence of disinterested pursuit of truth in scholarly research. in their study, homburg et al. ( ) report a decline in the influence of marketing department in organizations. they further caution that this trend could have an adverse effect on firm performance, in light of their finding that an influential marketing department makes the greatest contribution to firm performance. based on analysis of data from up to publicly traded firms over a -year period ( - ) , germann et al. ( ) report that firms benefit financially from the chief marketing officer (cmo) being a member of the top management team [the chief executive officer (ceo), and executives directly reporting to the ceo]. specifically, they report that the performance of firms with a cmo was about % higher than that of firms without one. instead, let us suppose that the findings of the above research studies were as follows: ( ) homburg et al. finding the degree of influence wielded by the marketing department to have a negative effect or no significant effect on firm performance, ( ) germann et al. finding no significant difference between the performance of firms with a cmo versus without a cmo, or firms in which the cmo is a member versus not a member of the top management team. in the spirit of disinterested pursuit of truth, they would still constitute important research findings worthy of dissemination through scholarly journals. in closing key et al. ( , conclusion section) conclude their article by noting: "all disciplines need periodic reflection and self-assessment. the ultimate goal is to have other scholars' critique, challenge, or add more support for our positions. in this way we can advance and have a positive impact on the field of marketing." this commentary presents a perspective on the relevance, rigor and impact of scholarly research in marketing, the current state of the field and outlook. it also complements key et al.'s article by drawing attention to certain other considerations that are pertinent in the context of some of the issues they focus on. at the same time, the commentary voices disagreement with their positions on some of the issues. as key et al. note, engaging in constructive debate and dialog on important issues is crucial to the advancement of the discipline. understandably, when challenging a point of view of a fellow scholar or voicing disagreements, it is important that we adhere to the norms of civility in scholarly discourse. research methods in anthropology: qualitative and quantitative approaches the intellectual ecology of mainstream marketing research: an inquiry into the place of marketing in the family of business disciplines academic freedom under fire the greatest breakthroughs since the wheel. the atlantic monthly the chief marketing officer matters a mathematician's apology the loss of the marketing department's influence: is it really happening? and why worry marketing is … marketing's theoretical and conceptual value proposition: opportunities to address marketing's influence scientists worry about political influence over coronavirus vaccine project. the new york times the scientific approach: basic principles of the scientific method an essay concerning human understanding cryptanalyze this. the new york times elevating science, elevating democracy. the new york times the code book: the evolution of secrecy from mary queen of scots to quantum cryptography musings on relevance and rigor of scholarly research in marketing musings on the need for reform in marketing musings on interesting and impactful theory and research publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -w cg b authors: nan title: spotlight on osaka date: - - journal: nature doi: . /nj sha: doc_id: cord_uid: w cg b nan spotlight on osaka n orthern osaka is rapidly becoming one of the world's most successful biomedical clusters through active collaboration and the pursuit of research that is attractive to young scientists, says renowned immunologist tadamitsu kishimoto. following approvals in japan last year, japan's first antibody drug, tocilizumab (sold as actemra in japan), this year also received government approval in europe for its application to be extended as an immunosuppressive agent. it can now be used to treat rheumatoid arthritis and articular inflammation in addition to the rare castleman's disease. the drug is a humanized monoclonal antibody that inhibits the receptor of interleukin (il)- , a protein known for its involvement in immune and inflammatory responses. tocilizumab is now expected to become a highly popular treatment for rheumatoid arthritis. the development of tocilizumab is perhaps the best example reflecting the underlying strengths of medicinal and biological research and the businessoriented mindset in northern osaka, the site of one of asia's largest biomedical clusters. soon after il- was discovered by immunologist tadamitsu kishimoto at osaka university in the mid- s, it was swiftly combined with the technological prowess of a pharmaceutical company. their persistent work led to the successful market introduction of the drug two decades later. "osaka embraces a culture that allows unconventional thinking, which has led to a bloom in numerous achievements of translational research. and it still does, " says kishimoto, a professor of the graduate school of frontier biosciences at osaka university. "in the early s, for example, many people thought small molecules would be the mainstream of drug development, but we looked to large molecules like antibodies, " says the native of osaka who is also one of the university's former presidents. northern osaka has been a centre of life sciences research dating back to the early nineteenth century, when the predecessor of osaka university's faculty of medicine -the school known as tekijuku -was first established. the university has since become a global frontrunner in research on proteins, microbiology and immunology. the region's pharmaceutical tradition in fact started much earlier: as early as the seventeenth century, a district known as doshomachi began to flourish with the arrival of hundreds of merchandisers from around japan to participate in the burgeoning pharmaceutical trade. the merchants included the predecessors of takeda pharmaceutical and other major modern companies. despite the recent challenging conditions of the global economy, the biomedical cluster in northern osaka has progressed largely unimpeded thanks to government-sponsored economic stimulus packages, which have greatly increased financial support for promoting those research fields in which japan has been particularly competitive. the northern osaka cluster is also accelerating its unified efforts towards translating research achievements into practical uses, kishimoto says. the need for a practical focus was recognized as far back as , when the senri life science foundation was first established. the foundation provides research grants, organizes seminars and supported the creation of the biomedical cluster. the saito life science park was opened in as the core of the research cluster. the osaka prefectural government also established the osaka bio headquarters in the cluster in to coordinate various activities and facilitate progress on life sciences strategies. kishimoto now heads the foundation and the bio headquarters, among his other public roles. many of the significant advances at the biomedical cluster are the products of impressive work by young researchers, kishimoto says. as an example, naoki hosen at the graduate school of medicine at osaka university, supported by the foundation through the japanese government's knowledge cluster initiative ( nd stage), is developing therapeutic antibodies targeting antigens specific to cancer stem cells. in another example, ken ishii at the university's research institute for microbial diseases is working on an influenza vaccine adjuvant that is activated by innate immune signalling pathways. "the most important thing is not money, but the fostering of the next generation of researchers, " kishimoto says. "our biomedical cluster can provide young researchers with a promising career pathway if they come and work here for a few years. " just kilometres from downtown osaka, the northern osaka biomedical cluster is a concentration of the world's foremost academics and japan's leading pharmaceutical companies. the well-balanced combination of academics and industry is key to driving product innovation and research success through active collaborations. the cluster centres around the saito life science park. several of japan's top biomedical research institutes are located within five kilometres of the park, including the suita campus of osaka university, the osaka university hospital, the national institute of biomedical innovation (nibio), the national cardiovascular center (ncvc), the osaka bioscience institute and the senri life sciences center. the tradition of modern academic research in osaka is derived from tekijuku, a school that taught western studies and medicine in the nineteenth century and which became the predecessor of osaka university's faculty of medicine. many of the scientific activities undertaken in the biomedical cluster are highly evaluated internationally. the institutes in northern osaka play a critical role in a number of large-scale national projects. for example, last year the japanese government included four projects from osaka university, nibio and the ncvc in its five-year super-tokku initiative. the program provides a platform for the swift evaluation of pharmaceuticals and medical equipment through the flexible use of public funding in order to accelerate the research and development of advanced medicine and medical equipment in japan. the cluster lies south of saito and encompasses the district of dosho-machi, where more than pharmaceutical companies have been established. five of japan's top ten pharmaceutical companies are headquartered in this district and neighbouring areas. osaka is also known as a home for electronics companies such as panasonic and sharp, as well as a number of manufacturing companies involved in the development of advanced medical equipment. these companies receive dedicated support from the osaka pharmaceutical manufacturers association and the osaka medical instruments association. in the biomedical cluster, industry, academics and policymakers coordinate effectively to promote biomedical activities. the senri life science foundation plays a key role in these activities, and also takes charge of the regional administration of the japanese government's knowledge cluster initiative ( nd stage). in , the osaka prefectural government commissioned the establishment of the osaka bio headquarters within the cluster to coordinate industry-academic collaborations more effectively. in its most recent move, the bio headquarters in collaboration with some of the top brains in osaka developed the biomedical strategy , which set the goal of making osaka the world's fifth most influential biomedical city over the next decade. to this end, osaka is ready to increase support for venture companies, accelerate deregulation and speed up clinical trials. saito life science park -leaping ahead how to better join forces s et among the green hills of northern ibaraki city and southern minoh city in osaka, the saito life science park has established itself as a leading hub of life sciences research in japan. the -hectare site currently hosts six major research institutes, including nibio, as well as three commercialization incubator facilities with capacity to support start-ups and laboratories. occupants are eligible for a range of publically funded subsidies and tax incentives. more than researchers and staff working in the park benefit from its close location to osaka university and the ncvc, providing a basis for close research collaboration. anges mg is one of the innovative biopharmaceutical companies headquartered at the saito bio-incubator, and is currently developing two gene therapy medicines as mainstay projects. another company, soiken, is developing various biomarkers and assay systems as well as implementing clinical trials for the development of drugs and functional food. both companies are using research outcomes from osaka university and are recognized as frontrunners in japan's biotech industry. the bio-incubator is not restricted solely to japanese companies: it also houses a japanese subsidiary of san diego-based anticancer. the life science park opened in as the first part of osaka's large-scale urban redevelopment plan to create the international culture park, an area that has been nicknamed saito. next year, development will commence in the central area, which will also focus on life sciences research and the innovation industry, with completion scheduled for fiscal . the life science park offers not only a superb research environment, but also convenience -it is located about minutes by train from downtown osaka. the grand opening of the park in was marked by the inauguration of an extension of osaka's monorail service to the area. m atchmaking is a common practice undertaken at biotech events worldwide to introduce laboratory seeds to companies -but the effectiveness of such introductions for creating real outcomes is often questioned. the osaka chamber of commerce and industry (occi) has proved to be one of the few successful coordinators of such introductions. since , the occi has organized the forum for the industrialization of next-generation medical systems, an open platform for the joint development of medical instruments for clinical applications. every month, doctors and scientists make proposals for collaborations based on their ongoing research. if companies participating in the forum become interested in a proposal, the occi sets up working groups or individual meetings to promote collaboration. so far, working groups have been established, covering % of presenters, and a quarter of the presenters have subsequently gone on to launch a joint development project in collaboration with a commercialization company. in one example, a laboratory of fumio miyazaki at osaka university and the company daiken medical are currently working to develop a robot capable of holding and manipulating an endoscope during surgery, which could one day replace a human assistant. the forum is the most successful activity in the industry in japan and has become so popular that there is now a waiting list for making presentations. behind the high success rate of the forum is the comprehensive hands-on support of the occi's coordinators and administrative staff, who assist in many ways, from advising presentation topics, to offering follow-up support before and after the forum. participation in the forum is not limited to osaka or the greater kansai area, and the number of participants has been on the rise, now totalling research organizations and companies. the occi is looking to partner with overseas clusters and biomedical associations, and the first such move was made recently with the formation of the biobusiness alliance of minnesota. in osaka, globally recognized institutes like the ncvc are situated beside the accumulation of medicalrelated companies. by setting research institutes and medical companies as centripetal forces, the occi continues to contribute matchmaking capabilities to create a globally competitive industrial cluster in osaka. osaka's collaboration focus has led to the creation of one of the more ambitious projects to come out of the biomedical cluster -the pharmaceutical innovation value chain, which is run by the non-profit organization biogrid center kansai in osaka. the value chain allows stakeholders in any stage of drug development to participate in the creation of innovative drugs. the latest innovation to emerge from the biomedical cluster is the protein mall kansai, which was inaugurated in may this year. the protein mall acts like a catalyst: it searches for member and non-member companies interested in collaboration, and shares information among them to spur interaction. it also provides expert advice on protein-related business and potential collaboration. all the services supporting participation are provided free of charge. the protein mall is at present considering supporting technology for a protein synthesis developed by the institute for protein research at osaka university, and for a process of human protein production using vegetable chloroplasts developed by a collaboration between the nara institute of science and technology and the institute for virus research, kyoto university. "a unique protein production method and a unique protein itself would also be interesting to members, " says katsube. whereas some institutions are preoccupied with history and traditions, osaka university, or handai as it is colloquially known, prefers to talk about the future and seems to take delight in a tradition of being ahead of the times. 'live locally, grow globally' is the kind of motto you'd expect from osaka university. tracing its roots back to the tekijuku school established by the legendary pioneer of western medicine koan ogata in , osaka imperial university was formally established in as the sixth of japan's seven imperial universities and the only one to be built largely with private funds. from this start, the university has grown rapidly into a world-class educational institution of over , students and staff with formal links to universities in countries around the world. these unique beginnings -a combination of longstanding commercial roots and an early pedigree in medicine -have produced a seat of learning that thrives on continuous development, expansion and amalgamation with other local institutions, most recently with the osaka university of foreign studies in . the university was an early adopter of an interdisciplinary approach to learning when it created the school of human sciences in . they also recognized before most the importance of academic and industry partnerships and established the institute of scientific and industrial research way back in at a time when conventional wisdom viewed such liaisons with suspicion. now in the sixth year of its latest mediumterm plan, the university is looking to the next phase of development in its research capabilities under three themes stressing 'fundamentals' , 'curiosity' and 'responsibility' . and as one might expect, cooperative research with industrial partners features heavily. this translates into the concept of 'industry on campus' , which will see parts of the largest of the three sites of osaka university redeveloped as a cluster of high-tech facilities including the nanotech incubator, the technological alliance institute and a centre devoted to imaging, photonics and photovoltaics research, all in time for the university's th anniversary in . these facilities will provide an environment where academia and industry can work together on basic research as well as projects of commercial interest. whilst key initiatives are being implemented in high-tech science and technology, it is probably in the field of medicine more than any other that the strength of osaka university is most keenly felt. from its inception, the school of medicine has been a key driver in the growth of the university as a whole, as well as being at the forefront of many important developments in japanese clinical medicine and basic medical research. early on it established a reputation as a centre of excellence in the treatment of infectious diseases, notably for smallpox and tuberculosis, in the process becoming the site for the first popularization of smallpox vaccinations and bcg inoculations in japan. the osaka university school of medicine subsequently became known as the leading institute for transplant surgery in japan, beginning with the first kidney live locally, grow globally whilst noting strength in organ transplantation, hirano is quick to point to other areas at which the university excels -immunotherapy and translational research aimed at bringing the potential benefits of treatments such as cancer vaccines and stem cell therapies out of the lab and into the clinic. the university also boasts state-of-the-art medical imaging resources including a magnetic resonance imaging facility powered by an . tesla magnet -currently the world's largest -as well as a number of advanced positron emission tomography sources allowing lab animal experiments to be performed in real time. hirano is convinced that the university's achievements spring from a solid base of all-round excellence in general medicine and basic research. however, maintaining this strong foundation is not without its problems. "these days fewer and fewer medical students are going into basic medical research after graduation, " observes hirano, himself an osaka university phd, with regret, "and the result is a shortage of talent in those areas. " to tackle this problem, the school implemented a number of changes to the undergraduate curriculum to make it easier for students to gain experience in areas of medical-related research outside the school, and also created an elective course allowing students to carry out medical research for a few hours each week outside of normal class times. introduced in april of this year, the 'after five' option already has a healthy take-up rate, and enquiries from prospective future students for the course in future years are growing steadily. it is a typically forward-looking, flexible approach to a challenging problem. but that's the way they like to do things at handai. in , the japanese government announced the recipients of its world premier international (wpi) research center initiative program. the wpi program focuses government financial support on five priority projects to create elite research institutes that attract the world's top scientists. the ifrec at osaka university is one such institute, selected for its mission to "unveil the whole picture of the dynamic immune system" using a systems biology approach. immunology has long been one of osaka university's greatest scientific strengths, beginning with former presidents yuichi yamamura and tadamitsu kishimoto and now exemplified by the director of the ifrec, shizuo akira, who is a pioneer of innate immunity and currently the world's most cited scientist in the field. to take immunology to its next level, the ifrec seeks to move beyond the test tube where immune cells work in isolation and observe these cells live in the body, leading to a better understanding of the dynamics driving immune cell communication and cooperation. akira explains, "in vitro experiments only allow snapshots of immune cells. we want to capture the movement of cells dynamically. therefore, we need to develop new research techniques to understand immune responses in the body. " this means the ifrec will be the leader of a new systems immunology field that combines immunology, imaging and informatics. in addition to gathering several of the world's top immunologists, the ifrec has assembled an extraordinary imaging team led by one of the world's leading researchers in bio-imaging, toshio yanagida. the large number of high-impact publications produced by akira and yanagida and their impressive list of accomplishments and awards made the ifrec an obvious choice for the japanese government when it considered where to commit at least million yen annually for ten years or more. "our selection for one of these highly competitive programs was recognition of our research and the potential to achieve the goals of this challenging project, " says akira. just assembling a multidisciplinary team with international standing, however, is not enough. in order to accomplish its stated goals, the ifrec is also changing the way scientific research is undertaken in japan. this means more independence for young principal investigators, and emphasis on collaborative efforts -an absolute necessity for bringing together disparate fields like immunology and imaging, and researchers with different educational and cultural backgrounds. as akira notes, "we have around people involved in this program, % of whom are from overseas, including % of the principal investigators, with backgrounds ranging from engineering and physics to medicine. " one of the more recent principal investigators to join the ifrec is diego miranda-saavedra, who previously worked on developing bioinformatics techniques to study transcriptional networks in blood cell development. "i felt like i could accomplish more in ten years here than i could anywhere else. i saw that the ifrec is made up of a very enthusiastic and collaborative community of outstanding scientists, and that the science done here is not only world-class, but also worldleading. i quickly understood that the ifrec's goal is to answer very important and difficult questions in immunity and that this is achievable with the amazing setup here. my lab at the ifrec consists of a bioinformatics core with access to supercomputing facilities and wet bench space to validate our in silico predictions. " atsushi kumanogoh was attracted to the centre by its strong emphasis on imaging, which he believes will significantly advance his team's work on semaphorins and their role in the cross-talk between the nervous system and the immune system. having already isolated the first semaphorin (sema d) by subtractive cdna cloning in , kumanogoh has seen his research benefit from the imaging facilities at the ifrec. "our images offer a deeper insight into how semaphorins regulate cellular migration in the nervous and immune systems, " kumanogoh says. it is this unique environment that allows the ifrec to pursue its ambitious projects. "at present, we are not able to predict the outcome when a certain pathogen invades the body, " explains akira. ultimately, the ifrec seeks to do just that by visualizing how the body reacts when a pathogen invades. "by integrating immunology and imaging, we seek to understand the dynamic interactions of immune cells and their activation. this will allow us to manipulate the immune system, leading to the development of vaccines. " whole body imaging of the immune system a chimeric mouse generated from embryonic mouse cells and genetically modified embryonic stem cells. such mice are essential for generating knockout mice -important tools for immunology studies at the ifrec. advertiser retains sole responsibility for content |naturejobs| september such advances rely on allowing researchers to focus on their science. "a genuine international environment is critical for this world-class centre, " says takao kodama, administrative director of the ifrec. this means not only aiding researchers in their grant applications and other matters that relate to their science, but also facilitating a smooth transition for them and their families when they arrive to live in japan. this is one reason why cevayir coban brought her family to osaka to work at the ifrec, where she now leads a project investigating innate immune responses to malaria. "malaria affects millions of people each year and kills mostly children and pregnant women, yet no vaccine is available to prevent it. " almost immediately, she was able to form collaborative projects with imaging and informatics groups to create a new generation of drugs and vaccines to combat malaria. while immunology is at the forefront of this project, yanagida sees other significant benefits. "i am interested in the dynamics of living cells, " says yanagida, who as an engineer takes a strong interest in the design of the immune system. "an immune cell is an amazingly efficient machine superior to anything we can engineer. unravelling how living cells handle large amounts of information and function in tandem has the potential to allow us to design machines that use far less energy. " it is this component of the ifrec that should appeal to researchers without a direct background in immunology. "for me, the immune system is a model to better understand how cells function and how to build better machines, " adds yanagida. "the ifrec may be an immunology centre, but that most certainly does not mean it should only consist of immunologists. in fact, if we are to achieve our stated goals, then it can't. " the imaging team is aggressively applying new combinations of various imaging and labelling techniques, like multiphoton-excited fluorescent microscopy and nano-environmentsensitive probes, to conduct single-molecule imaging, nanometry tracking and magnetic resonance imaging of immunological systems. this gives physicists and engineers an unusually direct role in systems-based immunology research. this also applies to bioinformatics. "it doesn't matter if you have been working in another computational field. if you're at the top at what you do, then you can make a positive contribution here, " says daron standley, leader of the structural bioinformatics lab. "my team consists of people from backgrounds in bio-engineering, computer science and even financial modelling. when recruiting someone, we're not concerned with what you did at your last job. we are more concerned with finding smart people who have a strong desire to work with a variety of scientists in other fields in order to develop a more complete view of immune function. " it is this notion of building a collaborative immunology centre, made up of top scientists with diverse backgrounds, that will help the ifrec to attain its ambitious goals. through state-of-the-art facilities, guaranteed investment and an exceptional group of scientists, the ifrec is creating the necessary environment to take immunology to a new level. in vivo two-photon microscopy of bone tissue. microscopic blood vessels in the bone marrow of a transgenic mouse visualized by masaru ishii's group at the ifrec. - yamadaoka, suita, osaka - , japan the rimd was set up in after a donation from gendo yamaguchi, an osaka merchant. "we conduct research on infectious diseases, immunology and cell biology, " says hitoshi kikutani, director of the institute. "we also have a brother institute at osaka university, the research foundation for microbial diseases, known as biken. it is japan's largest manufacturer of vaccines and a major exporter as well. biken is currently developing a vaccine to fight the h n pandemic flu virus. " profits from the sales of vaccines are used to fund research at the rimd. "we accept graduate students for multidisciplinary research on microbiology, oncology and molecular biology, " says kikutani. "we have more than research staff here. " rimd researchers have made major breakthroughs, such as the discovery of cell fusion, which led to the development of monoclonal anti-bodies -important tools in biochemistry, molecular biology and medicine. the rimd's most notable research achievements include the discovery of vibrio parahaemolyticus, a bacterium that causes food poisoning, and the development of a vaccine for varicella (chickenpox), which is now manufactured by biken and licensed worldwide. "these discoveries were possible because this institute has been a core facility within osaka university from the outset, " says kikutani. "we have many young researchers from all over the world, and from backgrounds ranging from medicine to chemistry and other physical sciences. " with funding from the japanese government, several national universities including the rimd are maintaining research centres overseas to investigate emerging and re-emerging infectious diseases in asia and africa. an example is the thailand-japan research collaboration center on emerging and re-emerging infections (rcc-eri). "we established the rcc-eri in bangkok in , " says kikutani. "the rimd is focused on disseminating information to prevent epidemics, developing medicines and vaccines, and extending our network with other asian countries. " the rcc-eri, set up in partnership with thailand's national institute of health, is equipped with state-of-the-art facilities including 'bio-safety level ' laboratories for conducting research on hiv/aids, avian influenza and other zoonotic infections, and intestinal infections. "the international network of overseas research centres supported by the japanese government is similar to the network maintained by the centers for disease control and prevention in the us or the pasteur institute in france, " says kikutani. the rcc-eri supports ten japanese researchers in bangkok, who conduct research as well as train local staff. toshihiro horii at the rimd is developing a pioneering vaccine for malaria -known as the 'neglected disease' . "malaria is a huge burden for the human race, " says horii. "about % of the world's population lives in malaria-endemic areas. there is increasing urgency for a malaria vaccine and anti-malarial drugs. " horii has developed a vaccine based on serine repeat antigen (sera), a protein produced by the plasmodium falciparum parasite responsible for malaria. "an epidemiology survey conducted by us in uganda showed that people with high concentrations of antibodies against sera did not show symptoms of malaria, " says horii. "i have developed artificial sera in the form of the se vaccine, which when injected into people will produce antibodies against malaria. " transforming this discovery into a product requires adherence to strict international guidelines for good manufacturing practice (gmp). "the gmp rules must be obeyed for the manufacture of vaccines, " says horii. "the rules even include requirements for the number of dust particles allowed per cubic metre in the manufacturing facilities. the executives at the kanonji institute of biken are interested in manufacturing the se malaria vaccine, and have provided their gmp-compatible facilities for producing test material for the initial trials. this is our contribution to society. " the se malaria vaccine has passed safety trial tests in japan, and clinical trials in uganda are scheduled for this year. the vaccine is expected to be available for general use within five years. like adjacent strands of a β-sheet, the study of proteins and the development of osaka university are intimately linked. the relationship dates right back to the earliest days of the university as a modern institution in , although it was not until that a laboratory dedicated to the investigation of the organic chemistry of proteins and amino acids was set up in the faculty of science. the current institute was opened two years later and has since grown into one of the premium centres for protein research in the world. although the ipr is active in all areas of protein science, it plays a particularly prominent role in the development and management of the worldwide protein database (wwpdb), of which it was a founding member in . currently the protein data bank japan (pdbj: www.pdbj.org) located at the ipr and led by haruki nakamura acts as the first point of reference for scientists from the asia-pacific. in this role, the pdbj processes - % of the total world depositions to the wwpdb, amounting to around , structures each year. as well as curation and registration of data, the pdbj develops tools that allow users to access the data in a range of different formats and to search the protein structures in the database for points of structural similarity, such as functional sites, backbone shape and molecular surface. the ipr also carries out ground-breaking research in ab initio computational chemistry relating electronic states to the three-dimensional structure of proteins and related systems. this has provided important information about questions of fundamental importance in biosystems, such as asymmetric electron transfer in the photosystem ii reaction centre -one of the key mechanisms in photosynthesis. whilst the majority of the data in the wwpdb is generated by x-ray diffraction analysis -a technique that allows the structure of a molecule to be obtained directly by analysing x-rays that have passed through a crystal of the test substance -not all proteins can be examined using this method. "some proteins just don't crystallize nicely enough for x-ray studies, " explains toshimichi fujiwara, head of the laboratory for molecular biophysics at the ipr, "but fortunately in many cases they are suitable for analysis by nmr. " nuclear magnetic resonance (nmr) is a technique that uses differences in the way radiation is absorbed by atoms within a molecule when placed in a strong magnetic field to gain information about the molecule's structure. fujiwara's group has already deployed solid-phase nmr techniques extensively in the study of membrane proteins that are either not sufficiently soluble for solution-based nmr or that undergo drastic structural change during dissolution, which renders the structural data obtained of little use. "it's all about sensitivity, " explains fujiwara. "the more sensitive the atoms in the sample are to radiation, the better the data we can obtain. " however, in many cases the sample is not sufficiently sensitive to the low-energy radio waves of conventional nmr to allow the collection of detailed structural information. to get round this problem, fujiwara's group is developing new 'terahertz' technology using high-field dynamic nuclear polarization. in this technique, electron spins, which are very much more sensitive to irradiation, are energized by terahertz-frequency radiation and used indirectly to excite nuclear spins in the sample at very low temperature. this has allowed accurate measurements of microgram quantities of protein, rather than the tens of milligrams normally needed, at sensitivities up to - times that previously possible. the terahertz project is carried out in collaboration with academic and industrial partners including jeol, at which fujiwara worked for seven years before moving into academic research. currently at the experimental stage, the plan is to place the technology at the heart of an international nmr resource centre along with existing high-powered conventional nmr instruments such as the mhz machine scheduled for delivery in early . "we want to see what is really possible with nmr, " says fujiwara. the electrostatic molecular surface of the rat liver vault. an example of the complex computational chemistry performed at the ipr by haruki nakamura's group. - yamadaoka, suita, osaka - , japan the oral cavity is responsible for many crucial activities that most of us take for granted: breathing, eating, defence against pathogens, language and facial expressions. however for many people -those suffering severe periodontal disease -these basic functions become a challenge that dramatically affects their quality of life. the ability to apply the most effective preventive and therapeutic approaches to periodontal care should be a top priority, but far too many dentists are satisfied with conventional techniques. not enough energy goes into introducing new biomedical methods based on the latest molecular and cellular biology research. the osaka university graduate school of dentistry aims to correct this situation. "many people still see dentists as practicing the art of pulling teeth, filling cavities or putting in dentures, but that image is completely outdated, " says toshiyuki yoneda, dean of the graduate school of dentistry. yoneda argues that discoveries concerning the function of genes, the immunological system and the nervous systems have opened the door to an era of new treatments. "molecular biology has revolutionized dentistry, " he says. one obstacle to this advance had been the divide between the clinic and the scientists' workbench. "dentists don't really understand research and researchers don't really understand dentistry, " says yoneda. "many dentists are clinically oriented and they don't care about science. " the graduate school of dentistry is an attempt to resolve this problem. of the school's staff, are researcher/practitioners who hold both doctor of dental surgery (dds) and doctor of philosophy (phd) degrees, showing their ability to bridge the two cultures. "there is no doubt that this is important for the development of high-level healthcare, " says yoneda. the school was given a vote of confidence for its approach in from the japanese government, which bestowed on the school a center of excellence grant worth up to million yen annually. the grant allowed yoneda to establish the frontier dentistry/oral biology (fdob) program. for the past seven years, the program has been at the forefront of integrating molecular and cellular biology into dentistry practice. the program has focused on three topics: management of oral infection, support of oral development and biological stimulation of the oral cavity. the results have been impressive. publications originating from the school have had a scientific impact comparable with the world's strongest programs, including harvard. an important outcome of this research from the patients' perspective is progress on a treatment for regenerating bone based on fibroblast growth factor (fgf- ). this treatment, pioneered at the osaka university school of dentistry hospital and now in phase-three clinical trials, has already helped many patients in the late stages of periodontal decay to regrow the bone that holds their teeth in place. "some of these people can't eat, they can't bite and the gums get infected with bacteria, " says yoneda. he hopes such examples will help convince the general public that 'advanced dentistry' can play a vital role in improving their quality of life. seminars and open forums held by the fdob program show people what the latest research can do. "what we' d like them to see is that our basic science supports the clinic. people are very interested and come with a lot of questions, " he says. another key role of the fdob program and the graduate school of dentistry is education, with a mission to train dentists capable of deepening our understanding of the biological phenomena of the mouth at the molecular level. the educational scope is widened through the exchange of graduate students with foreign countries, and by hosting international symposia and establishing joint research programs with foreign institutions. these international activities, along with english educational programs at the school, will enable japanese dental researchers to build names for themselves. efforts by japanese dental researchers to interact with their peers from other countries, present scientific findings at international conferences and publish their findings are often hampered by inadequate english language ability. yoneda's program aims to train the next generation to undertake research, be active in the clinic and be involved in the international research community. by doing so, the school will help to give japanese dentists the respect they deserve and their patients the high-quality treatments they need. "people underestimate japanese dentistry at the moment, but we are world leaders in the field, " he says. regeneration of tooth-supporting bone weeks after topical application of fgf- . this type of pioneering research, directed by shinya murakami, has direct applications in dental care. akemichi baba has his own perspective on finding treatments for psychiatric disorders. such disorders are complex, and those researching treatments have a hard time knowing where to start. baba has provided a solid foundation. he has spent nearly two decades characterizing pituitary adenylate cyclase activating peptide (pacap), a polypeptide first isolated in and known to play an important role in the camp pathway. baba and his team of researchers soon took the lead in examining pacap by cloning it, carrying out genomic analyses, identifying its receptors, tracing its involvement in molecular pathways and localizing its distribution. it turns out that the peptide is mainly expressed in the brain -an exciting finding that suggested a potentially important role in brain function. that interpretation turned out to be correct. using modern gene-targeting methods, baba's team found that when the pacap gene was knocked out, mice suffered the tell-tale signs of schizophrenia: hyperactivity, reduced ability to adjust to disturbances (lower pre-pulse inhibition), a tendency to jump around explosively, cognitive dysfunction and depression-like behaviour. it was a triumph of 'reverse pharmacology' , an approach in which scientists study phenotypic effects of a disturbance to a complex system. baba knows that the phenotypic expression of genes also depends on certain environments, so he has also been studying relevant environmental factors. baba's pacap knockout mouse is on its way to becoming a new model for human schizophrenia. the mice show improvement when given anti-psychotics. and analogies with humans are supported by the fact that a certain mutation in the pacap gene, found to be significant in schizophrenic patients, is associated with smaller hippocampal volume and poor memory. what causes schizophrenia and how should it best be treated? with his mice, baba is on his way to finding out. hiroyuki mizuguchi is perfecting methods for introducing genes into cells at will -a technology that offers the unprecedented ability to manipulate cells for biomedical research -to produce vectors for gene therapy and potentially to carry out life-saving clinical therapies. adenovirus vectors accomplish this feat, with the added advantage that they express the gene of choice without disturbing the host's dna. but conventional adenovirus vectors also have problems, such as being unable to penetrate cells that lack a certain type of receptor. mizuguchi has experience in solving such tough problems. "we want to find a way to take advantage of the merits, limit the problems and find a new generation of adenovirus vectors that can serve even more functions, " says mizuguchi. mizuguchi pioneered an efficient way to make adenovirus vectors through a patented approach that is now commonly used worldwide. and with several publications and patents on a new kind of adenovirus vector, he is well positioned to make even more breakthroughs. satoshi obika found a recipe that works. twelve years ago, he pioneered a technology that stabilizes nucleic acids, whether dna or rna, by adding an extra bridge in the molecule's sugar. the 'bridged' nucleic acid (bna) outdid his expectations, binding to complementary rna with an affinity more than , times that of normal dna. "i was very surprised, " says obika. bna can be used to increase the specificity of detection in common dna microarrays or polymerase chain reactions. the specificity will also allow 'antisense' blocking of dna transcription or rna translation, which could make dna-based clinical therapies possible. obika designs a successful bna roughly once per year, each with its own rna or dna targets, paving the way to a future full of discoveries. yasuo tsutsumi, a toxicologist, thinks people should be worrying more about the smaller things, and his animal studies back up that position. he studies silica materials composed of molecules smaller than nanometres in diameter, which are increasingly being used in cosmetics, food and drugs. his first question: do they get into the body? the answer he found in his mouse and pig studies is 'yes' -even when applied to the skin, which is supposed to be the toughest barrier to pass. the second: where do these nanoparticles go in the body? the answer: into many vital organs, including the brain. the last question: are they safe? "we don't know yet, " says tsutsumi, "which is exactly why we have to extend our studies on nanotoxicology as quickly as possible. " tsutsumi wants to bring these facts to our attention. "we believe these products are safe, without any evidence, " he says. "i want to grasp the risk involved. " "basic science is essential to advances in human understanding, " emphasizes kiyoshi higashijima, dean of the graduate school of science. "and it is our chief focus in research and in education. " higashijima is quick to note that the emphasis on basic science is just as productive in spawning real-world applications as in illuminating scientific fundamentals. "our faculty and students tackle issues in bioscience from diverse perspectives, including macromolecular science, physics and even mathematics, as well as biology and chemistry. we expect our people to do world-class research, and they have responded impressively. multidisciplinary initiatives leverage our strengths in individual disciplines, and multilateral international exchange broadens our horizons further still. " epitomizing the world-class work in bioscience at osaka university is innovative research by three of the graduate school's professors of biology: hiroki nishida, tatsuo kakimoto and hisao masukata. all three are leaders in their fields, and each has captured worldwide attention with their discoveries. nishida's research on ascidians (sea squirts) has yielded insights into embryonic development in chordates. "ascidians are among the simplest of chordates, " notes nishida, "and scientists have elucidated the fates of nearly all of their embryonic cells. so those humble marine creatures are a superb laboratory for studying embryonic development, including patterns of gene expression, determinants of cell fate and cellular interaction. we have learned how to manipulate the fates of embryonic cells in ascidians artificially. our findings fortify the foundation for work in developmental medicine, including stem cell therapies. " kakimoto, meanwhile, has contributed greatly to parsing the mechanism of intercellular communication. he discovered a biosynthesizing enzyme and receptor for cytokinin, which figures prominently in intercellular communication and in regulating cellular differentiation in plants. and he has gone on to identify regulators of cell multiplication and cellular positioning. "i'm interested, " kakimoto explains, "in the basic principles that determine form in plants. so i am studying the communication and the signalling networks that fulfil those principles. " masukata describes his pioneering work on chromosomal replication as the result of a long-standing fascination. "the replication, " he observes, "is subject to different structural control mechanisms, depending on the stage of the cell cycle and even on the position of the chromosomes. that mystified me early on, and i was determined to solve the mystery. " masukata's quest has illuminated important aspects of chromosomal replication. for example, he has helped identify the action of heterochromatin protein , the chief determinant of chromosomal structure in fission yeast. and masukata is broadening his research with an eye to achieving a more comprehensive grasp of chromosomal replication. active in cutting-edge chemistry at osaka university is akira harada, a professor of macromolecular science. he has received plaudits for his work on artificial molecular machines. "organisms, " harada comments, "exhibit different kinds of motor movement, including the linear motion of muscles and the rotary motion of atp synthesis and of flagella. scientists have devoted immense time and effort to analysing biological motor functions with an eye to harnessing them for various applications. i have built self-assembling structures by combining the ring-shaped molecule cyclodextrin with linear macromolecules, and i have succeeded in controlling the movement of the molecules relative to each other. the next step in my research is to develop applications for my molecular machines in sensors, catalysts and other applications by combining them with bioactive molecules, such as antibodies. " koichi fukase, a professor of chemistry at osaka university, regards organisms as communities of molecules. "my work unfolds, " he quips, "in the realm of biomolecular society. " fukase works to verify the action of biologically active molecules in observable functions. his approach has led him into a diversity of projects, including research on the synthesis and functioning of molecules that regulate innate immunity; analysis of the synthesis and functioning of sugar chains on cell surfaces and in glycoproteins; and the imaging of sugar chains, macrobiomolecules and cells. he pioneered the application of positron emission tomography (pet) to analysing glycoproteins, and his 'glycopet' imaging technique has provided visual verification of the decisive effect of sugar chains on protein dynamics. an ascidian tadpole larva, just before hatching. hiroki nishida's group uses ascidians as a laboratory for embryonic research. advertiser retains sole responsibility for content |naturejobs| september "sugar chains and proteins are just the beginning, " declares fukase. "we could assemble those and other biomolecules into structures for performing useful functions, such as recognizing patterns in the communities on cell surfaces and regulating the molecular communities inside cells. that approach could even provide an effective means of correcting autoimmune disorders and targeting anticancer drugs at tumours. " michio murata, a colleague of fukase in osaka university's department of chemistry, is also doing important work in the realm of biomolecular society. murata works to elucidate biological phenomena by reconstituting the biomolecular communities required for various functions. for example, he analyses the interaction of biomolecules and pharmaceutical compounds inside a liposome membrane as a model for the cell membrane. using this approach, murata has demonstrated that membrane lipids figure prominently in the pharmaceutical action of antibiotics. "the cell membrane is a classic biomolecular society, " remarks murata. "i look forward to developing methodologies for grasping the exact roles that lipids, proteins and other biomolecules play in that society. " these achievements are but a small sample of the cutting-edge work under way in bioscience at osaka university. some of the other bioscience researchers at the university who have earned international acclaim include hitoshi watarai in analytical chemistry, yasuhiro kajihara in organic biochemistry and yasuhisa mizutani in biophysical chemistry. the mei centre is a major osaka university initiative that brings together researchers from various fields to collaborate on projects in medical sciences, bioengineering and bioinformatics. the centre is funded, mostly externally, to the tune of around one billion yen per year and houses principal investigators who also maintain primary affiliations with other divisions of the university. "in one sense it's a 'virtual' department, " says yoshihisa kurachi, director of the mei centre. at the heart of this virtual department is the global center of excellence for an in silico medicine-oriented platform that forms the basis for collaboration with leading institutions from new zealand, the us and the eu. the aim of the platform is to compile physiome databases of dynamic mathematical models describing biological systems -from the molecular and cellular level right through to organs and individuals -based on experimental data on the fundamental chemical and physical properties of physiological entities. these models, whether a liver, a heart or even an entire human skeleton, are made available for download from a morphology database (www.physiome.jp) for integration into biological modelling software, allowing the effects of given stimuli on the structure-based physiological function to be modelled and investigated. current applications include a study on the risk of drug-induced cardiac arrhythmia for pharmaceutical drug candidates, and the use of morphology data to predict optimal parameters for joint replacement surgery and rehabilitation aftercare packages. the mei centre offers tuition in medical engineering and informatics to postgraduate students from osaka university and other institutes in the region. although the classesunique in japan -are currently only an elective course module, there are plans to develop a fully accredited postgraduate degree program in the near future. and what of the future? the dream is to construct a complete in silico human from mathematical data, but it's a dream -admits taishin nomura, leader of the in silico medicine project -that may never be realized. "building a model of a human being is probably impossible, " he says candidly, "but at least we can continue to improve human understanding. " an in silico human. the mei centre's in silico medicine project aims to build an open platform for multi-scale mathematical models of the human body and its functions. in , fbs established an integrated five-year graduate school course to nurture students with multifaceted skills in the biosciences. "this course is extremely popular, " says fujio murakami, dean of the fbs school. "we usually receive around applicants for only places. " the success of research and education at fbs has been underscored by its selection for one of the japanese government's global center of excellence (g-coe) programs. the program supports doctoral students by arranging summer schools, international exchanges and courses on improving their english language skills. in addition, the g-coe invites leading researchers from overseas to give lectures and seminars to doctoral students affiliated with the program. fbs is also collaborating with the national institute of information and communications technology (nict). "in , we will launch a project with the nict on imaging the flow of information in the brain, " says murakami. the new , m interdisciplinary research center will house members of this project working on such areas as bio-communications and dynamics, imaging technology and computation. key tools for this project include magnetic resonance imaging, magnetoencephalography and two-photon microscope imaging. "fbs is responsible for cell imaging at the immunology frontier research center, a world premier international research center, " says murakami. "we have a lot of expertise to offer the biosciences. " murakami is using neural imaging to study the movement of cells in the brain. "cell movement in the brain is extremely important, " says murakami. "lack of cell movement can lead to schizophrenia, for example. we use a combination of in vitro electroporation, green fluorescent proteins and confocal fluorescence microscopy to image labelled cells. " murakami encourages students to "enjoy the discovery" of science. "we recently started in vivo experiments for a firsthand look at cell movement and brain functions, " he says. yasushi hiraoka pioneered fluorescence microscopy for imaging the structure of living cells. he holds concurrent posts at fbs and the kobe advanced ict research center of the nict. "i succeeded in the fluorescence imaging of living cellular structures because of the strong involvement of nict researchers. in particular, i have worked with tokuko haraguchi in kobe for over two decades, where i covered engineering and she, the biological aspects of this research. " hiraoka's microscopy allows information to be procured from individual living cells, a major advance compared with conventional methods that yield only 'averaged' information. in , hiraoka published a high-profile article on high-resolution, stereoscopic, three-dimensional time-lapse fluorescence microscopy to monitor chromosomes. his work established fluorescence microscopy as a tool for the observation of single living cells. hiraoka emphasizes that the research environment and human resources were critical for success. his group of researchers at the nict consisted of % male and % female members. over two decades, the female staff continued their research after maternity leave. "this is an exceptional research environment in this country, " says hiraoka. "our success was based on the accomplishments of a minority group. " "tokuko haraguchi and i have organized courses on live cell imaging biannually for the last seven years, " says hiraoka. "we usually have two to three times more students applying than we can handle. " "we had many challenges over the last years, " says hiraoka. "irradiating cells with strong light killed them. we resolved this by developing a high-sensitivity automated imaging system. synthesis of low-toxicity dyes was another milestone, with one immediate application of monitoring the effects of anti-cancer drugs on individual cells and related drug screening. " "i want to exploit the wealth of specialties at fbs for in vivo, functional imaging of cell movement in the whole body, " says hiraoka. live images of dividing human cells. triple staining shows chromosomes (blue), centromeres (magenta) and microtubules (green). drug discovery is getting costly and time consuming: it can now take a decade or so from the discovery of candidate compounds to the marketing of a new drug, with total investments of us$ million to us$ billion. the introduction of highperformance computers, however, can drastically shorten the time for research and development in experimental biology, which remains one of the most important fields for drug development. from to , the japanese government sponsored the biogrid project with the objective of making it possible for users anywhere to access cutting-edge data processing power by networking and coordinating the vast databases, software and other information technology resources hosted on supercomputers at geographically distributed research institutions. the main purpose of the initiative was to speed up the research and development of drug design by introducing high-performance computing into the steps of cell modelling, drug screening and investigations of drug efficacy. to take full advantage of the project, biological and computational researchers established a non-profit organization called the biogrid center kansai in osaka in . the centre is presided over by shinji shimojo, an executive researcher of the national institute of information and communications technology (nict). members of the biogrid center -biotechnology venture firms, osaka university, research institutes and pharmaceutical companies -have access to powerful computing tools such as 'mypresto' for modelling of proteins and drug docking, and ' amoss' for quantum mechanics calculations. members also have access to spring- in kobethe world's most powerful synchrotron radiation facility -and supercomputers at osaka university. the centre aims to accelerate technology transfer, strengthen collaborations between academia and industry, and ultimately enhance the opportunities for drug innovation and the creation of start-ups. the picture looks bright, but collaborations rarely work well without strong commitments from each of the participants. "that's why we decided to design attractive projects on our own, " says tsuneaki sakata, a director of the biogrid center and visiting professor at the cybermedia center of osaka university. the flagship scheme is called the pharmaceutical innovation value chain, an open platform that allows members and non-members around the world to contribute their expertise in any stage of drug development towards the common goal of generating innovative drugs. currently, a venture company called kringle pharma is taking advantage of technologies provided by participants in the value chain for the development of cancer treatments. "it is important to prompt researchers -particularly younger ones -to support the value chain and develop their skills without expecting immediate returns. " says sakata. last year, the value chain produced kyoto constella technologies, a spin-off from kyoto university. initiated in , the value chain is funded by the knowledge cluster initiative ( nd stage) of the japanese ministry of education, culture, sports, science and technology, and the 'consortium r&d projects for regional revitalization' program of the ministry of economy, trade and industry. as part of the biogrid center's expansion of the value chain beyond national borders, the center has created an opportunity to form a memorandum of understanding between the biomedical cluster kansai, which includes contributors in kyoto and kobe, and a biotech cluster in alsace, france. in three years, the biogrid center is expected to benefit from the next-generation supercomputer being built near kobe with funding from the japanese government. the new supercomputer will be able to perform at a rate of petaflops, or operations per second. the government has identified simulation of the human body as one of the 'pillar' projects for this new facility. in august, the biogrid center held the first workshop discussing suitable applications for the advanced supercomputer, such as predictions of drug toxicity. yet there still remain many gaps between computational scientists and biologists, and also between industry and academics, concerning intellectual property and the best approach to research. "we must increase the opportunities and incentives for interactions for everyone, " says sakata. the pharmaceutical innovation value chain. venture companies and research institutes interested in commercializing drugs can seek collaboration partners from the biogrid center kansai's value chain project. the ncvc has pioneered medical care and research in areas including revascularization of the heart and brain during acute and superacute phases, less-invasive heart surgery, artificial heart and lung technology, and genetic diagnosis and therapy. "our research is based on close collaboration among industry, academia and government, " says hashimoto. "this translational approach to research is important for conducting clinical trials, for drug development, and for a myriad of medical instruments and devices. " highly acclaimed breakthroughs by the ncvc researchers include the discovery of the novel hormone 'ghrelin' , the rapid diagnosis of thrombosis, peptidomics/proteomics and the development of artificial hearts and lungs. new buildings and initiatives are in the pipeline for . "next year sees some major administrative changes at the ncvc, " says hashimoto. "we will reorganize into an 'independent administrative institution' from april . this will give us more autonomy from the japanese government to hire specialists -including highly motivated scientists from overseas -and to define new areas of research. " kenji kangawa is an internationally acclaimed biochemist, director general of the ncvc and a researcher who was pivotal in the discovery of the hormone ghrelin, publishing a series of high-profile papers on the topic in - . "most researchers were looking for this hormone in brain extracts, " says kangawa. "the assumption was that this hormone was produced in the hypothalamus, and hence related to the brain. but quests to find this hormone in the brain were not fruitful. " kangawa and his group decided to look in the stomachs of rats and humans. "if our competitors, especially at large industrial labs, had looked in the stomach, then they would have found it before us, " says kangawa. kangawa was the first in the world to isolate other important bioactive peptides, including brain natriuretic peptide (bnp), c-type natriuretic peptide (cnp), and atrial natriuretic peptide (anp). the discovery of anp, which is isolated from the heart, showed that the heart acts as both a pump and an endocrine organ that secretes hormones. kangawa stresses that the unique research environment at the ncvc -combining basic research within a busy hospital -has played a major role in his search for peptides. "new peptides lead to new areas of research. " translational research on hormones has advanced rapidly, with anp and bnp already being used for treatment and diagnosis by physicians. ghrelin is also undergoing clinical trials for the treatment of anorexia nervosa and related illnesses. the director of the department of pharmacology, naoto minamino, is searching for new bioactive peptides by a strategy based on peptidome analysis. "we use mass spectrometers to identify peptides in tissue extracts, " says minamino. "but there are still many issues to resolve because the peptides are easily degraded. " two approaches, 'activity-first' and 'peptide-first' strategies, are being investigated. "the key to indentifying peptides and proteins accurately is to keep cells and tissues intact. " minamino and his colleague kazuki sasaki applied peptidome analysis to the conditioned media of cultured cells -a procedure known as 'secretopeptidome analysis' . using this approach, they successfully acquired high-quality secretory peptide data by reducing the degradation fragments of cellular proteins. "it is possible to propose processing pathways for precursor proteins, and to predict the peptides generated from precursor proteins, " says minamino. these studies based on the peptide-first strategy will allow the identification of new bioactive peptides and biomarkers, and will increase the probability of computer-based prediction of bioactive peptides and biomarkers. the results of research on the functions of peptide hormones are critical for the development of new drugs for the treatment of heart failure, hypertension and metabolic syndrome. another example of the basic-to-translational research being conducted at the ncvc is toshiyuki miyata's work on thrombosis -the clogging of blood vessels. miyata is director of the department of etiology and pathogenesis, and has been investigating the diagnosis and treatment of the life-threatening systemic disease known as thrombotic thrombocytopenic purpura (ttp). effective diagnosis of ttp requires detection of adamts -an enzyme that cleaves the protein responsible for blood clotting (von willebrand factor; vwf). adamsts regulates diagnosing ttp using frets-vwf . cleavage of frets-vwf by adamts results in fluorescence of the intact peptide. normal blood will thus fluoresce increasingly over time, while adamts -deficient plasma will not produce fluorescence. advertiser retains sole responsibility for content |naturejobs| september spotlight on osaka the molecular size of vwf in the blood, and an absence of adamsts leads to the build-up of vwf, with subsequent dangers of ttp. "monitoring the activity of adamsts is critical for early diagnosis of ttp, " says miyata. "with my colleague koichi kokame, we determined that the minimum region recognized as a specific substrate by the adamts enzyme was only amino acids, from d to r , of vwf. " this discovery led to the development of the fluorescence resonance energy transfer (fret) assay, which allows the activity of adamts to be checked within one hour, instead of the - days required using conventional methods. "we worked with industrial partners to chemically synthesize a fluorogenic peptide, frets-vwf , containing the amino acids, " says miyata. "the fret design is a fluorescent molecule attached to a quenching group. if the molecule is cleaved by adamts , then fluorescence is observed, but in the absence of adamts , cleavage does not occur and fluorescence is quenched. " frets-vwf and related fluorogenic substrates are now sold commercially by vendors including osaka-based peptide institute inc. taking discoveries from the lab to the bedside is critical in translating scientific discoveries into real-world medical applications. "the ncvc was selected as a super-tokku region by the japanese government, " says yoshiyuki taenaka, director of the advanced medical engineering center. "super-tokku refers to a region designated to pursue high-level research and development based on close industrial, academic and government collaboration. " taenaka's speciality is mechanical circulatory support systems, such as artificial hearts and lungs. "our success in translational research at the ncvc paved the way for our selection by the government for the super-tokku project, " says taenaka. "the project is for five years, with people each being funded with about two million dollars per year. " industrial collaboration is imperative for developing implantable artificial organs. "our interaction with mitsubishi heavy industries, bridgestone, dic corporation, toyobo corporation and nipro corporation is important to get through the ' critical path' , " says taenaka. the director of the department of artificial organs, eisuku tatsumi, in collaboration with taenaka, is a pioneer of artificial hearts and lungs. "worldwide, there is a move towards the longterm use of artificial hearts, 'destination therapy' as it is known, as opposed to temporary support during recovery from heart disease, " says tatsumi. ventricular assist devices (vads) are examples of temporary support devices that are commonly used for short periods after heart attacks. "in japan, the toyobo vad is widely used, " says tatsumi. the ncvc is focused on developing a portable pneumatic vad system and an implantable axial flow pump vad system. "the size and weight of vad systems are important factors for improving quality of life, " says tatsumi. "our portable mobart-ncvc pneumatic vad driver, which has been widely used in clinical cases, is compact and weighs only . kg. " an even more compact vad driver that weighs less than kg and fits in a wearable shoulder bag is expected to be commercially available in two years. the implantable axial flow pump vad is being developed by the ncvc in collaboration with mitsubishi heavy industry and aist. this vad is less than the size of a d-type battery and weighs only g. "our device is durable because of its contact-free hydrodynamic bearings. importantly, the small size enables it to be used by children as well as adults, " says tatsumi. commercial products are expected within three years. there is also demand for artificial lung systems (pcps/ecmo systems) for use in emergency life support and over longer periods of time for individual patients. however, current systems are complex to set up and operate, are bulky, and require the use of anticoagulants. the biocube ncvc series of ecmo systems developed by the ncvc in collaboration with dic, toyobo and nipro solves these problems. "the use of anticoagulants can be minimized, or even made unnecessary, with the biocube ncvc series. they are compact, mobile platforms that will find applications in the out-of-hospital arena, such as at road accidents resulting in lifethreatening lung damage, " says tatsumi. the results of translational research at the ncvc will enhance the quality of life for patients, and act as seeds for new areas of medical research. "when i started at nibio, few people were interested in vaccine development. immunology progressed, but manufacturers are too small to do much r&d, " says yamanishi, a virologist with decades of experience studying herpes and other viruses. following panics caused by the sars coronavirus, the h n avian flu and most recently the h n pandemic flu, governments and industry have started paying attention. yamanishi leads a super special consortium in which universities, national research centres, pharmaceutical companies and hospitals aim to create next-generation vaccines for hiv, malaria, influenza and other infectious diseases. the consortium's goal is to improve vaccines by studying new vectors that will enable single vaccines to convey immunity to multiple infectious diseases. nibio has significant capabilities in the genetic recombination technology needed to create these vectors, which will make vaccination programs easier and cheaper. the consortium is also developing nasally, dermally and orally administrated vaccines as safer and more convenient alternatives to injections. "this type of immunization is closer to our natural immunity, " says yamanishi. the consortium will develop new adjuvants to enhance the effect of vaccines and allow the use of smaller doses. uncertainty about how adjuvants work has been an obstacle to their implementation, but japanese scientists have led the analysis of toll-like receptors and other related components of the immune system. "a new generation of effective adjuvants will come from japan, " says yamanishi with confidence. pharmaceutical companies are increasingly using embryonic stem (es) cell-derived cell lineages for in vitro testing of drugs. but es cells are in short supply. induced pluripotent stem (ips) cells, first created in japan in , offer an alternative. ips cells are made by reprogramming somatic cells to an embryo-like state. scientists can do this with anyone's cells, offering an infinite source of fresh cells with defined genetic backgrounds. research groups by the dozens have embraced ips cell technology. before they can be used in drug development, however, they need to be standardized and quality controlled in a manner that facilitates clinical studies by pharmaceutical companies. "japan is leading the examination of ips cells, " says nibio's hiroyuki mizuguchi, "but most university groups stop at the research level. " mizuguchi leads a super special consortium to establish a drug toxicity testing system based on ips cells. such a system will limit controversial a drug revolution in the making the unique role of nibio in japan. nibio combines extensive collaboration with basic research, valuable biological resources and project funding to support the pharmaceutical industry. advertiser retains sole responsibility for content |naturejobs| september spotlight on osaka animal testing, rein in ballooning drug development costs and put japan at the forefront of highly competitive global efforts to understand the genetic background of adverse drug reactions. nibio is creating a cell bank that will eventually hold ips cell lines. standardizing the protocol for banking ips cell lines is a labour-intensive process requiring analysis of cell morphology, cell surface antigens and gene expression profiles, and monitoring for unexpected changes. "we need to be able to grow the cells in a strictly defined medium so that we can differentiate them stably and with a high level of reproducibility, " says miho furue-kusuda, who is in charge of the bank. analyses using the cells will benefit from nibio's toxicogenomics database, the largest in the world, which collects data on the effects of more than drugs in rats. standardized protocols for producing ipsderived cells will be the foundation for new guidelines on their use in pharmacological testing. mizuguchi hopes to have the protocols in place within two years. "japan is the only country moving forward so quickly towards the use of ips cells to validate new drugs, " says mizuguchi. nibio is developing other resources that will give a crucial boost to pharmaceutical studies. for example, nibio' s dna bank has started to make available quality-controlled resources derived from patients suffering from rare diseases. the bank distributes cdna clones from chimpanzee and crab-eating macaques along with related genetic sequences. a unique experimental mouse has also been established at nibio. whereas conventional severe combined immunodeficiency (scid) mice can only hold transplanted human tissue for a few weeks, nibio's 'super-scid' mice maintain human tissue transplants, such as lung, thyroid and skin, for a year or more, a boon for drug development and the study of environmental hazards. in addition, nibio holds mice that develop various diseases spontaneously. "we can watch the natural development of chronic diseases rather than making artificial disturbances with mutagenesis or transgenes, " says nibio's tohru masui. the resource bank also includes a cell bank of cultures of human brain, lung, liver, kidney, gut and blood cells, as well as many samples of mesenchymal stem cells and cells derived from patients, which are important for drug development and regenerative medicine. through extensive quality control and monitoring, nibio ensures that there is no contamination by microorganisms or other cell types. this is not easy considering a recent study by nibio's arihiro kohara showing that % of cell cultures in japan contain mycoplasma. "these figures are similar to those seen all over the world, " says masui. "it shows the importance of quality control. contamination has a huge impact on the reliability and repeatability of experiments. " the vital experimental resources available through nibio also include primate and medicinal plant collections. the research center for medicinal plant resources has four bases across the japanese archipelago, representing different climatic areas. new restrictions on crude drug exports in china as well as quality-control problems prompted the cultivation of several important medicinal plants locally. the centre cultivates and preserves over , plant species to supply seeds and seedlings, and offers guidance on cultivation technology to research institutes. "it's the most comprehensive collection in japan, " says yamanishi. the tsukuba primate research center, with , cynomolgous macaques, also offers a unique resource. the monkeys are mated in specifically selected pairs so that hereditary factors can be traced. the centre carries data on the monkeys' age, laboratory test results and family histories stretching back years, enabling researchers to predict, for example, which monkeys might experience high blood pressure. "we know the entire history of these monkeys, " yamanishi says. the monkeys, used at nibio for vaccine and other research, are also available to scientists and pharmaceutical companies throughout japan for research on disorders such as hereditary diseases, diabetes and cardiovascular disease. yamanishi hopes that these resources, maintained at high levels of quality, and the research that nibio supports will give the pharmaceutical industry a push. "these kinds of research and resources are essential for drug development, but neither the universities nor the drug companies are doing it. we've only been around for four years, but we're already starting to see results. " a n international group of young, talented and highly motivated scientists at the osaka bioscience institute (obi) is at the forefront of research on systems neuroscience, olfactory mechanisms, retinal systems and sleep mechanisms. the obi is a non-profit organization conducting research in the biological and medical sciences. with around researchers in five departments, the obi has been at the forefront of research in bioscience, with breakthroughs that include elucidating the mechanisms governing cell death, sleep and consciousness. the obi has a unique history, being established in following an initiative by former osaka mayor yasushi ohshima as part of the th anniversary celebrations of the osaka municipal government. "oshima wanted to create something for the future of osaka, " says osamu hayaishi, former director of the obi and present chairman of the obi's board of trustees. "osaka is the commercial heart of japan. oshima asked a panel of experts from academia, industry and government to recommend a way of creating something for the people of osaka. bioscience was considered important because many local companies made products such as pharmaceuticals and alcoholic beverages based on bioscience and medical science. this proposal led to the birth of the obi. " research at the obi is funded by osaka city, the japanese government and private foundations. this scheme for funding research is unique. "we could say this system is 'too unique' , " says hayaishi. "almost all research in japan is funded by the national government. even in the us, the national institutes of health are supported solely by the federal government. our approach is different. many companies have donated funds to build and run this institute. our research is based on a solid industrygovernment-academia relationship. " for an international perspective, the obi advisory board asked arthur kornberg -the noble laureate in physiology or medicine who clarified mechanisms in the biological synthesis of deoxyribonucleic acid -to be a consultant to the obi. the first chairman of the board was kenzo saji, former president of suntory, who had a doctorate in chemistry. "i was appointed the first director of the obi, " says hayaishi. "i think i was chosen because of my extensive experience in international research. i had spent ten years in the us as section chief at the national institutes of health and teaching at universities. after returning to japan, i held faculty positions at the universities of kyoto, tokyo and osaka -my alma mater. i also had visiting positions at harvard, vanderbilt and the karolinska institute, amongst others. so i knew many young and active researchers all over the world. " in contrast to other research institutes in japan, which offer permanent tenured positions, the obi employs young scientists on fixed-term contracts ranging from three to ten years. "this was my idea, " says hayaishi. "it was and still is an unorthodox approach to research in japan. we offer well-paid positions, with an excellent research environment hosting state-of-the-art facilities. a turnover of scientists is important for innovative research. during my directorship i allowed our researchers to choose their own areas of research. " at the time, hayaishi's approach was thought to be too idealistic by some administrators at monbusho -the former japanese ministry of education -and some doubted whether this approach would succeed. as if to disprove their critics, obi scientists have made many major breakthroughs over the last years. examples include identification of the basic mechanisms of apoptosis by shigekazu nagata and clarification of the control mechanism of sleep by osamu hayaishi. the quality of obi research was highlighted in when the obi was ranked at the top in scientfic impact factors for publications on molecular biology and genetics from to . internationally recognized as an institute at the leading edge of research " we provide an excellent research environment for ambitious scientists to spend % of their time doing research, " says hayaishi. "i personally interviewed all applicants. i believe that people wishing to conduct research at the obi have great courage and confidence for considering working in this small, private institute. " shigetada nakanishi is the present director of the obi. himself an accomplished scientist, he is renowned for elucidating the molecular nature of the glutamate receptor combining xenopus oocyte expression systems and electrophysiology. "i studied under dr hayaishi at kyoto university, and years ago i was a member of the obi advisory committee, meeting annually with the directors of the obi, " says nakanishi. "when i was an advisor, i was impressed by the fact that the researchers at the obi were tackling problems in completely unknown fields -sleep and cell death were new fields back then. the discovery of the mechanism and inhibition of cell death by obi scientists is acknowledged as being a major advance in understanding cell cancer. " hidesaburo hanafusa, who worked at rockefeller university, was the second director of the obi between and . "unfortunately, dr hanafusa became ill, and i was appointed director four years ago, " says nakanishi. "our situation has changed recently because we now have to compete with large universities who are using the same approach towards research as us. my main mission now is to devise ways of competing with major universities such kyoto, tokyo and osaka. " given the changing research landscape, nakanishi is making changes to compete with new approaches to research in bioscience. "i have decided to define certain areas of research and find talented principal investigators for these areas, " says nakanishi. "one of the new areas of research i want to expand is brain science, or more precisely, systems neuroscience. we know that many molecules are involved in brain functions, but we still do not know how the brain actually functions. we need to clarify the dynamic mechanism governing information processing and integration in the brain. this is systems neuroscience. " nakanishi has recently appointed a young scientist who is now working with her husband at the obi on olfactory systems -the sense of smell. "these scientists found that olfactory memory is divided into areas, " says nakanishi. "one area is the genetically controlled olfactory mechanism. for example, certain animals show a fear response towards the smell of a fox; this is a hereditary response. but animals also learn smells by training. our scientists have found that these two fear responses are divided, and they have elucidated the neural information paths responsible for each individual type of memory recognition. " "i studied molecular biology before coming to the obi, " says nakanishi. "now i have shifted towards systems biology, and in particular, systems neuroscience. " research in the future will focus on mechanisms of memory learning in the brain using systems biology, as well as the olfactory mechanism, visual retinal systems and information processing. sleep and consciousness are also major topics, such as how caffeine acts on the brain. the obi's unique approach to basic research is reflected in the construction of its spacious and eye-catching main building, designed by the famous architect, kenzo tange. the institute is equipped with state-of-the-art facilities including recombinant dna laboratories, genetic analysers and laser confocal microscopes. the well-stocked library and comfortable meeting spaces provide an ideal research environment. a mouse lacking dorsal olfactory sensory neurons. by targeted expression of the diphtheria toxin gene, researchers at the obi successfully bred a mouse without odour-evoked innate fear responses. - - furuedai, suita, osaka - , japan tel: + - - - web: www.obi.or.jp/index .html advertiser retains sole responsibility for content |naturejobs| despite pioneering research by many japanese biologists, the application of this expertise has proceeded slowly. but change is coming quickly.nibio was established in april by the japanese government to fill crucial gaps in the path from discovery to drug development. a unique institute, nibio carries out basic science aimed at developing shared technologies at its laboratories in the saito life science park, standardizes and shares biological resources throughout japan, and funds external research in a similar fashion to the national institutes of health in the us. "it's the only place like it in japan, " says koichi yamanishi, director general of nibio.the japanese government has recognized nibio's special mission in several ways. in , nibio was awarded two coveted 'super special consortium' designations for the development of cutting-edge medical care -two of only that were approved nationwide. nibio has also taken a leadership position in the japanese government's knowledge cluster initiative -a five-year project to stimulate cooperation between research institutes, universities and industry in biomedical sciences. nibio maintains a host of joint research projects with pharmaceutical manufacturers in fields ranging from toxicogenomics and biomarker exploration to animal models and vaccine development. "we meet regularly with companies to ask what they need from us, " says yamanishi. t he national institute of advanced industrial science and technology (aist), with a network of research centres across japan, promotes the creation of innovative ideas for society through 'full research' -a multidisciplinary approach to research from basic studies through to product realization. aist kansai is no exception.the aist institute is japan's largest public research organization, employing about , researchers at nine locations throughout the country and hosting almost , visiting scientists from universities and industry in japan and overseas."i was appointed president of aist in april after serving as chairman of the board of mitsubishi electric corporation, " says tamotsu nomakuchi. "needless to say, sales and profits are important in industry. but aist's role is to define and solve problems, anticipate future industrial needs and develop innovative technology for a sustainable society. " 'medicine' and 'engineering' are keywords that define research conducted at aist kansai on maintaining and promoting good health in an ageing society. "life sciences, and in particular, medical engineering, form the basis of multidisciplinary research at aist kansai, " says nomakuchi. this research is carried out at the research institute for cell engineering (rice), the health technology center and the institute for human science and biomedical engineering (hsbe)."at aist, i want to use my experience of working in industry, and i hope to act as a bridge connecting industry and academia in osaka as well as other aist centres in the rest of japan, " says nomakuchi.masayuki kamimoto, director of aist kansai, is proud of his centre's multidisciplinary capacity. "in addition to life sciences, we also have research projects in areas including the environment, energy, information technology and materials science, " he says. recent internationally recognized breakthroughs by researchers at aist kansai include cell cultivation protocols, carbonnanotube molecular actuators and biomarkers for parkinson's disease. self-repair of damaged bone and cartilage is extremely difficult in the elderly. hajime ohgushi, director of the research institute for cell engineering, is addressing this problem using stem cells for 'regenerative medicine' ."i use mesenchymal stem cells, which exist in bone marrow, to repair damaged bone tissue, " says ohgushi. "this is my contribution to regenerative medicine. "in , ohgushi and his group reported pioneering work on the regeneration of bone tissue using tissue-engineered bone derived from the patient's own mesenchymal stem cells (mscs).however, in spite of the tremendous advances as a result of this work, clinical applications of this approach were limited because the ability of the cells to proliferate and differentiate declined dramatically over time."we succeeded in activating the mscs by introducing nanog or sox -genes expressed by embryonic stem cells -into the mscs, " says ohgushi. introduction of only the sox gene was ineffective, and a combination of sox and a protein called basic fibroblast growth factor (b-fgf) was required."in , shinya yamanaka reported that introducing - transcription factors into the skin cells of an adult led to these skin cells gaining pluripotency like embryonic stem cells, " says ohgushi. "this report was the birth of induced pluripotent stems cells, or ips cells. we also succeeded in generating ips cells from mscs, therefore the technology may be available for regenerative medicine based on mscs. " kinji asaka and his group at the research institute for cell engineering is developing low-voltage electroactive polymer (eap) actuators to produce light-weight, directly fitting artificial muscles for rehabilitation. eap actuators are operated by applying a voltage to move and bend sheets of polymer actuators. "conventional polymer actuators rely on chemical reactions within liquid electrolytes to convert the applied voltage into mechanical motion, " says asaka. "they have short lifetimes and slow response times. "in asaka's approach, movement is produced by changing the internal volume of a polymer composite. "we mix millimetre-sized, single-walled carbon nanotubes with an ionic liquid to produce a gel that is dried to form highly conductive, free-standing sheets, " says asaka.the actuators were produced by cutting fifteen mm strips from the carbon nanotubes, sandwiching a soft polymer film between the strips, and finally applying an alternating voltage across the carbon-nanotube electrodes. low-voltage electroactive polymer (eap) actuators. these devices form the basis for light-weight, directly fitting artificial muscles. the actuator works at frequencies of - hz without notable deterioration of the actuator structure or range of motion over more than , continuous operations.these ' dry' actuators are extremely robust and flexible, and show rapid response to alternating voltages of as low as . v. "we could use roll-to-roll manufacturing for mass production of arbitrarily shaped actuators, " says asaka. the development of diagnostics technology for chronic diseases is a priority at aist kansai. parkinson's disease is a neurodegenerative disorder in which dopamine-producing brain cells are lost. the disease affects millions of people worldwide, and comes with symptoms that include trembling hands, stiff limbs and inability to balance."we are developing protocols for early diagnosis and treatment of parkinson's disease based on the detection of biomarkers, " says yasukazu yoshida at the health technology research center.yoshida's team found the gene park (dj- ) to be critical for early diagnosis of parkinson's disease. the next step for the researchers was to develop a means of detecting this biomarker in blood samples. "we successfully prepared a monoclonal antibody and developed a competitive enzyme-linked immunosorbent assay system for an oxidized form of dj- , known as cys- -oxidized dj- , " says yoshida.the concentration of oxidized dj- in blood samples of unmedicated parkinson's disease patients was about ten times greater than that detected in control and medicated patients. "future plans include improving the performance of the measurement system and introducing this technology into hospitals for actual clinical applications, " says yoshida. "we also want to establish our approach as an international standard for diagnosing parkinson's disease. " sunao iwaki is a group leader at the institute for human science and biomedical engineering. his group is at the forefront of developing technology for diagnosis and sensory prosthesis support for people with higher brain dysfunctions, such as dementia and language difficulties. "we are trying to unlock the mysteries of the brain using our integrated non-invasive brain imaging technique, " says iwaki. "it is extremely important to elucidate not only 'where and when' but also 'how' various parts of the brain interact. "iwaki's group has combined magnetoencephalography (meg), electroencephalography (eeg) and functional magnetic resonance imaging (fmri) with innovative information algorithms to allow non-invasive imaging of the brain with high spatiotemporal resolution. meg detects magnetic signals from neural networks in the brain, eeg maps changes in electric potential over the scalp due to neuron activity, and fmri detects brain activity by the degree of oxygenation of the blood. a system integrating these three analyses has been used to identify the neurophysiological mechanism of bone-conducted ultrasound (bcu) -the detection of sounds above khz not directly discernable by humans. "the bcu experiments led to the development of a bcu hearing aid, " says seiji nakagawa, senior research scientist at the hsbe institute. "our trials showed that % of profoundly deaf people could hear simple words, and more than half perceived some form of sound. in the future, we hope to commercialize this technology. "the bcu hearing aid could replace cochlear implants, which require time-consuming and physiologically taxing surgical procedures.non-invasive brain imaging system. a combination of meg, eeg and fmri analyses allows visualization of spatiotemporal neural dynamics (top) and neural interactions (bottom). key: cord- -vfl i p authors: largent, emily a; lynch, holly fernandez title: paying participants in covid- trials date: - - journal: j infect dis doi: . /infdis/jiaa sha: doc_id: cord_uid: vfl i p trials are in development and underway to examine potential interventions for treatment and prophylaxis of coronavirus disease (covid- ). how should we think about offering payment to participants in these trials? payment for research participation is ethically contentious even under ideal circumstances. here, we review functions of research payment—reimbursement, compensation, and incentive—and identify heightened and novel ethical concerns in the context of a global pandemic. we argue that covid- trial participants should usually be offered reimbursement for research-related expenses, and compensation for their time and effort, as for other types of research under usual circumstances. given increased risk of undue influence against pandemic background conditions, incentive payment should be avoided unless essential to recruitment and retention in important trials whose social value outweighs this risk. where essential, however, incentives can be ethically permissible, so long as reasonable efforts are made to minimize the possibility of undue influence. with much of the globe rushing to respond to coronavirus disease (covid- ) , clinical trials to evaluate safe and effective options for treatment and prevention are critical. the trials are diverse, examining new and repurposed drugs and vaccines at various stages of development, and involving a variety of designs with a range of opportunity for direct benefit. a wide variety and number of research participants are needed to enroll in these trials, reflecting a spectrum of experience with covid- -including healthy individuals, individuals exposed to the virus, individuals experiencing different levels of disease severity, and recovered individuals-as well a diversity of age, sex, race and ethnicity, socioeconomic status, medical comorbidities, and more. we have already seen that many individuals are eager to try nearly anything that has exhibited some promise against this disease, which may facilitate trial recruitment [ ] . but what role will payment play in encouraging trial participation-and what role should it play? paying research participants is ethically contentious under ideal circumstances and pandemic circumstances are far from ideal. concerns about offering payment are therefore likely to be heightened in this context [ ] . existing frameworks for evaluating the ethics of paying research participants offer a strong foundation for assessing the acceptability of payment in covid- trials. nonetheless, consideration must be given to the unique medical, financial, and institutional circumstances wrought by the pandemic. we argue that participants in covid- trials should be offered reimbursement for research-related expenses and compensated for their time and effort, just as they should be for any other type of trial. given the pandemic's devastating economic effects, as well as the fact that risks may be higher or more uncertain in covid- trials than in nonpandemic research, there is an increased likelihood of undue influence stemming from incentive payments. still, it may be permissible to offer incentives when necessary to recruit and retain participants for important trials offering the possibility of social value sufficient to outweigh these concerns, so long as steps are taken to minimize the risk that participants will be unduly influenced. paying research participants serves several discrete functions [ ] . first, participants may be reimbursed for out-of-pocket expenses incurred as a result of research participation, such as copayments or travel costs. second, payment can compensate participants for the fair value of their time and effort expended in research participation. third, incentive payments go beyond what is necessary to either reimburse or compensate, with the intention of improving recruitment or retention. offers of payment that fall into any of these categories can be ethically acceptable; however, each function raises distinct ethical considerations influenced by the pandemic. offering reimbursement for reasonable, research-related expenses to restore participants to their financial baseline should be viewed as the ethical default in covid- trials, as in research more broadly. this treats participants fairly by preventing them from having to pay to contribute to socially valuable research that may not, or is not expected to, benefit them directly. where direct benefit is possible, covering expenses can also promote distributive justice by making those potential benefits more widely accessible without regard to participants' financial need or wealth [ ] . considering that a high percentage of the population is likely to be infected with severe acute respiratory syndrome coronavirus (sars-cov- ), the current absence of curative therapies or a vaccine, and the widespread financial hardship and exacerbation of economic disparities caused by the pandemic, covid- trial participation must not be limited only to those who can afford it. making participation widely accessible is especially important in light of the disproportionate impact this virus is having on minority communities [ , ] . while there are of course many barriers to trial participation, financial barriers are among the most easily modified [ ] . although reimbursement should be the rule, there will be exceptional circumstances when it can be ethically acceptable to proceed without it. in the context of covid- , for example, investigators and institutions may be initiating trials without traditional sources of public research funding or with only limited support, such as the provision of product, by commercial companies. in these instances, there may not be adequate funding to reimburse participants. lack of funding should not preclude important trials from proceeding, so long as participants are made aware of the financial implications of enrollment and reasonable efforts are made to minimize financial burden. if a limited budget is available for reimbursement, it may be acceptable to reimburse only select participants-such as those with the greatest financial need-or to cover expenses up to a prespecified limit without necessarily reimbursing them all [ ] . compensation also should be viewed as the ethical default because it helps to minimize the chance participants will be exploited by receiving benefits that are disproportionately low compared to the burdens they undertake and the value they contribute to research. while the prospect of direct benefit is relevant to avoiding exploitation, research benefits are not always present and are never certain. thus, it often makes sense to compensate participants for their work via a wage-payment model, using a fair local wage for similarly burdensome nonresearch endeavors as a benchmark [ , ] . this is not intended to make participants better off as compared to their financial baseline or even to fully compensate for participants' opportunity costs but rather to acknowledge the value of their time and effort. compensation can also help distinguish research activities, with their distinct goals and risks, from clinical care, signaling that participants are contributing to science and that individual benefit may not result from their research participation [ ] . this is likely to be particularly important for covid- patients, because the dearth of compelling treatment options means that clinical care will often incorporate experimental methods and research. nevertheless, it may be ethically acceptable to proceed without compensation when reasonable budget constraints preclude it, as above. when compensation is offered, ethical concerns can arise, which sometimes point in opposite directions. first, if the amount is unfairly low, it will not adequately address the possibility of exploitation; this concern can be addressed by offering more compensation. yet, relatively higher compensation may be linked to a second concern, which arises when circumstances extrinsic to a trial transform payments intended as compensation into de facto incentives. this is especially likely in the context of covid- . compensation is intended to render research participation as financially attractive as other, nonresearch opportunities that demand a similar amount of time, burden, and skill. however, the availability and type of those nonresearch opportunities can be impacted by a variety of factors. even for those who have remained healthy, the covid- pandemic is causing a human tragedy with significant economic impact, including widespread layoffs, small business closures, hiring freezes, and dramatic market fluctuations [ ] . these challenges have exacerbated longstanding economic inequalities and laid bare the fragility and inadequacy of the social safety net in many countries, including the united states. these economic and social challenges are only likely to increase as the pandemic continues. against this backdrop, many prospective covid- research participants will no longer have meaningful alternatives for paid work, which may contribute to a perception-and perhaps reality-that paid research participation is their best opportunity to make money. this perception may be heightened if compensation is increased to the extent necessary to avoid exploitation, even if the total amount of money remains relatively modest. these difficult background circumstances do not make offers of payment to covid- research participants impermissible. it would be perverse to withhold fair compensation simply because participants are facing economic hardship [ ] . rather, in light of pandemic circumstances-similar features of which may be replicated in other contexts, including research conducted in low-and middle-income countries or with participants whose nonresearch options are limited even in the absence of a pandemic-offers of compensation may raise ethical concerns akin to incentives [ ] . thus, institutional review boards (irbs) should consider whether it is sometimes appropriate to treat them as such. rather than aiming to satisfy obligations of fairness to participants or to set research on a par with other payment-generating activities, incentives aim to push trial enrollment higher on an individual's choice set-or incidentally have that effect based on available alternatives. as such, incentives are not ethically obligatory. however, to the extent incentives can contribute to recruitment and retention, and thus to efficient trial completion with adequate statistical power, they can be ethically important [ ] . incentives may also advance distributive justice by increasing willingness to enroll, thereby spreading the burdens of research participation over a larger swath of the population and avoiding concentrating those burdens exclusively on individuals who are the least well off financially by making research more broadly attractive. yet, incentives can be ethically fraught. some people worry that incentives may coerce research participants. the prevailing view is that coercion entails a threat to violate an individual's rights or not fulfill an obligation to her unless she complies with some request, in a circumstance in which she has no reasonable alternative but to comply [ ] . by this definition, genuine offers of payment are not coercive because they are not threats [ ] . importantly, feeling that there is no reasonable alternative to make a similar amount of money-a feeling likely to arise for at least some prospective covid- trial participants-is not the same as being coerced. a more salient ethical concern is that incentives may unduly influence participants, although this is more complex than just making a decision motivated by a desire for financial benefit. undue influence occurs when an excessive reward leads the recipient to make a choice that is unreasonably against her selfdefined values, interests, or responsibilities [ , ] . a decision would be objectively unreasonable if it reflected a level of risk an irb would not or should not approve for participants in the target population. for example, it would be objectively unreasonable for persons with a known hypersensitivity to hydroxychloroquine to participate in a trial assessing that drug's effect on the progression of covid- [ ] . approval of a trial protocol by a well-functioning irb should generally eliminate concerns that incentives will lead to objectively unreasonable decisions. in contrast, research participation may be subjectively unreasonable if it is discordant with an individual's particular values and interests or if the risks (should they materialize) would be particularly burdensome for the individual to bear [ ] . because irbs are tasked with making population-level judgments, they cannot be expected to assess the subjective reasonableness of trial participation for every individual. thus, irb review cannot eliminate the possibility that that incentives will lead to subjectively unreasonable decisions-and, therefore, to undue influence. for instance, a jehovah's witness considering participation in a trial of intravenous immunoglobulin-a blood plasma product-for covid- might have consciencebased concerns. similarly, someone who is highly risk-averse may prefer to avoid the uncertainties associated with research participation. either of these individuals could be encouraged by offers of incentive payment to set aside those concerns. while this may seem worrisome, in practice, it is quite hard to distinguish between cases in which an offer of payment has unproblematically tipped the balance in favor of an otherwise undesirable but not unreasonable choice and those in which it has problematically tipped the balance in favor of an unreasonable or irrational choice. in many cases, a decision to enroll in research will reflect a participant's reasoned judgment that, in these circumstances, participation is aligned with her overall interests, even if certain considerations might have weighed in the other direction. decisions are often multifactorial, with various pros and cons; the fact that all cons have not been resolved does not necessarily render a decision subjectively unreasonable. acknowledging this challenge, the best irbs can do is to minimize the possibility of undue influence for trial participants on the whole by making it unlikely for research participation to constitute an objectively unreasonable choice for members of the target study population. they should also make sure the consent process alerts participants to factors that might make participation subjectively unreasonable [ ] . although careful irb review generally constitutes a critical bulwark against undue influence, there are additional considerations when evaluating the potential for undue influence in covid- trials. first, given that there is still much to be learned about sars-cov- and so little is known about the various interventions under study in this context, we may justifiably be concerned that it will be difficult for irbs to make risk-benefit determinations confidently. therefore, participation even in irb-approved studies may not always be truly objectively reasonable for the target study population, or there may be disagreement about what is objectively reasonable. moreover, the challenge of evaluating this research may be exacerbated by the heightened burdens currently facing irbs. the sheer volume of covid- research proposals being put forward [ ] and the dire need for clinical advancement mean that members are being asked to review a tremendous number of protocols as quickly as possible, often while meeting remotely to promote social distancing, all of which may influence the quality and nature of review. second, the global scale of the pandemic and associated morbidity and mortality may render even quite risky research objectively reasonable on the basis of high social value. for example, we have already seen vaccine trials proceed without the usual animal trials, and there is increasing discussion of using challenge trial design to speed vaccine development, a design far riskier for sars-cov- than for viruses used in other recent challenge trials given the lack of a proven cure [ ] [ ] [ ] [ ] . it is widely accepted that trials can be justified on the basis of benefits to society; in the absence of direct benefits, it may be desirable to offer larger incentive payments similar to hazard pay offered to emergency workers or others performing dangerous work-as noted above, this can help make participation attractive across a wider socioeconomic swath [ ] . yet, larger incentives might also increase the likelihood of undue influence by making it more likely that people will make subjectively unreasonable decisions [ ] . a third concern is that incentives, because they go beyond amounts available through participating in other activities, may cause individuals "to lie, deceive, or conceal information that, if known, would disqualify them as participants in a research project" [ ] . such obfuscation can have effects [ , ] . first, it may expose individuals to research-related risks that exclusion criteria were designed to shield them from. second, it may jeopardize the scientific integrity of the research. this can be of particular concern when it is not possible to rely on objective, independently verifiable measures to confirm trial eligibility [ ] . in response to these challenges, one might consider the most conservative course of blocking incentives for covid- trials. however, this approach could inhibit recruitment, retention, or both, impeding the conduct of critically important research, in turn creating a greater possibility that participants will be exposed to research risks without realizing the social benefits that initially justified them. there are potential errors here: ( ) inhibiting ethically acceptable trials out of an abundance of caution, or ( ) risking undue influence and deception by incentivizing participation in covid- trials [ ] . given the importance of research in response to the pandemic, as well as limited, albeit encouraging, empirical data suggesting that higher offers of payment increase participants' perceived risk as well as time spent reviewing research-related risks [ , [ ] [ ] [ ] , our view is that incentive payments can be ethically permissible, despite the residual risk of undue influence and deception. rather than blocking incentives for covid- trials due to the concerns raised above, the better approach is to differentiate those circumstances in which incentive payments are truly essential to boost recruitment and retention for important research. if they are not, as may be the case for covid- research that offers participants other benefits or that can rely on altruism born of social solidarity, it is best to avoid incentive payments. this is also the more efficient approach; why spend resources on incentives that are unneeded? however, incentives sometimes will be critical. for example, covid- trials that offer no or low potential for direct benefit and impose substantial burdens and risks, such as early phase trials focused on dosing and safety, may otherwise fail to adequately enroll participants. of course, the fact that participants may have qualms about participating that need to be overcome by offering incentives might suggest that concerns about undue influence are highest in these circumstances. yet these are also the covid- trials for which irb attention is likely to be most intense and focused, potentially reducing concerns about objectively unreasonable decisions to enroll. risks of subjectively unreasonable decisions remain, but should be viewed in a similar context to other research risks, meaning that they can be justified when both minimized and reasonable in relation a study's potential for benefit. thus, incentives should be limited to covid- trials with adequate time and research personnel to facilitate robust informed consent processes that can help prospective participants carefully consider the risks, burdens, and discomforts of participation, as well as those that can adopt objective measures of eligibility and adherence. they should also be limited to those trials with sufficient importance to the battle against covid- that their potential benefits can overcome residual concerns about undue influence. relatedly, care should be taken to avoid incentive payments being used to draw participants into lower priority trials [ ] . irbs are tasked with minimizing-not eliminating-the possibility of undue influence; we should accept that minimization may look different for covid- trials compared to other research. offering payment for trial participation intended to combat a pandemic that is coupled with economic distress raises unique considerations. we argue that reimbursement and compensation should be offered in covid- trials as a matter of fairness, as is true for other types of clinical research. yet the economic stressors of the pandemic may cause compensation to be experienced as an incentive, raising concerns about undue influence, while the usual protections against undue influence may also be weakened by pandemic circumstances. rather than abandoning the utility of incentives, we recommend that they be limited to those covid- trials that truly need them, that will permit undue influence to be minimized, and whose social value and importance can outweigh residual risks of undue influence. this suggests that financial incentives will be most appropriate for covid- trials without the prospect of direct benefit, although in the face of such a massive global threat, altruism and a call to duty may render incentives even less critical. hospital orders for old malaria drugs have spiked amid coronavirus pandemic conventional wisdom versus actual outcomes: challenges in the conduct of an ebola vaccine trial in liberia during the international public health emergency fernandez lynch h. a framework for ethical payment to research participants what makes clinical research ethical covid- and african americans evidence mounts on the disproportionate effect of covid- on ethnic minorities addressing financial barriers to enrollment in clinical trials differential payment to research participants in the same study: an ethical analysis what's the price of a research subject? approaches to payment for research participation human research subjects as human research workers informative inducement: study payment as a signal of risk kff health tracking poll -early economic vulnerability and payment for research participation precarity, clinical labour and graduation from ebola clinical research in west africa the continuing unethical conduct of underpowered clinical trials paying research participants: regulatory uncertainty, conceptual confusion, and a path forward payment for research participation: a coercive offer undue inducement: nonsense on stilts? hydroxychloroquine in outpatient adults with covid- paying research participants: the outsized influence of "undue influence a real-time dashboard of clinical trials for covid- [published online ahead of print researchers rush to test coronavirus vaccine in people without knowing how well it works in animals human challenge studies to accelerate coronavirus vaccine licensure ethical considerations for zika virus human challenge trials ethics of controlled human infection to address covid- compensation for cures: why we should pay a premium for participation in 'challenge studies on paying money to research subjects: 'due' and 'undue' inducements association between financial incentives and participant deception about study eligibility exploring ethical concerns about human challenge studies: a qualitative study of controlled human malaria infection study participants' motivations and attitudes making the case for completion bonuses in clinical trials empirical assessment of whether moderate payments are undue or unjust inducements for participation in clinical trials perceptions of financial payment for research participation among african-american drug users in hiv studies the influence of risk and monetary payment on the research participation decision making process when clinical trials compete: prioritising study recruitment key: cord- - ablrwuo authors: guintivano, jerry; dick, danielle; bulik, cynthia m title: psychiatric genomics research during the covid- pandemic: a survey of psychiatric genomics consortium researchers date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: ablrwuo between april , and june , we conducted a survey of the membership of the psychiatric genomics consortium (pgc) to explore the impact of covid- on their research and academic careers. a total of individuals responded representing academic ranks from trainee to full professor, tenured and fixed-term appointments, and all genders. the survey included both quantitative and free text responses. results revealed considerable concern about the impact of covid- on research with the greatest concern reported by individuals in non-permanent positions and female researchers. concerns about the availability of funding and the impact of the pandemic on career progression were commonly reported by early career researchers. we provide recommendations for institutions, organizations such as the pgc, as well as individual senior investigators to ensure that the futures of early career investigators, especially those underrepresented in academic medicine such as women and underrepresented minorities, are not disproportionately disadvantaged by the covid- pandemic. since the initial outbreak in december , the novel coronavirus sars-cov- (covid- ) has mushroomed into a global pandemic affecting every aspect of life. in an effort to reduce transmission, many governments, universities, and other research institutions issued work from home orders for non-essential workers. interruptions were widespread on both work and home fronts. researchers or their family members were infected by covid- , schools were closed leaving little time or space for work, and the unpredictability of the course of the pandemic led to persistent anxiety and distress for most people in the world. specifically, many clinician-researchers were seconded to covid-related clinical duties; patient facing research was halted or postponed; many basic science researchers were mandated to halt all laboratory-based activities; and academic medical centers faced enormous financial consequences (colenda, applegate, reifler, & blazer, ; kim et al., ; weissman, klump, & rose, ) . in person teaching was suspended requiring rapid adaptation to remote teaching platforms. in addition, other academic activities, such as conferences and face-to-face meetings, were cancelled or transitioned to virtual formats, interrupting training, networking, and other means of scientific information exchange. several studies have documented challenges that have been faced by academics at different career levels, different genders, and different family structures, and concerns have been raised, especially for early-career researchers and women regarding the long-term impact of the pandemic on their career progression (denfeld et al., ) . as a service especially to our early career researchers, the psychiatric genomics consortium (pgc) wanted to better understand the effects of covid- on its members and their research. we modeled our questionnaire after weissman et al (weissman et al., ) and assessed: (a) the impact of covid- on pgc research now and in the foreseeable future; (b) the level of concern about covid- -related disruptions of research and the potential impact of these disruptions on individuals' careers; (c) strategies that respondents thought to be effective in coping with covid- -related research disruptions; and (d) respondents' suggestions for how the pgc or the field should respond to help researchers move through and beyond the current crisis. to address these aims, we administered an anonymous survey containing both quantitative and free text responses to pgc researchers. quantitative questions focused on the perceived impact of covid- -related research disruptions. we hypothesized that respondents in secure employment positions (e.g., with tenure or permanent contracts) would report less stress and less concern about potential adverse impact of covid- on their research and career, than respondents at earlier stages in their career (e.g., tenure-track or fixed-term contracts, postdoctoral fellows, graduate students). the primary goal of the qualitative, free text questions was to describe participants' strategies or suggestions, and highlight issues that they felt were important that we had not addressed. we had no hypotheses regarding the free text responses. all members of the psychiatric genomics consortium (pgc) identified by the consortium listserv were invited to participate in the survey. invitations were sent through the main pgc listserv, through individual workgroup listservs, and were promoted during regular pgc videoconferences. the survey was launched on april , and closed on june , . qualtrics was used to administer and store the survey. this study was approved by the university of north carolina institutional review board committee for the protection of human subjects. this mixed-methods survey was composed of likert-scale items measuring concern about covid- disruptions (rated from = no concern to = extreme concern) and highest level of stress experienced since the outbreak of the pandemic ( = no stress to =highest level of stress imaginable). one item asked respondents to characterize the proportion of their pgcrelated research that had to be completely shut down due to covid- , using six options (from to up to % in % increments). three categorical items (yes, no, do not know/does not apply) addressed whether studies had been transferred to online; whether researchers were anticipating making changes to their research practices; and whether their institution had made policy changes in response to demographic data were collected in a final set of questions. participants were asked to report their gender (female, male, gender variant/nonconforming, choose not to answer), current position ("faculty appointment (> years post training)", "faculty appointment (up to years post training) (early career researcher)", "graduate student", "post-doctoral fellow", "resident", "other"), type of position ("a position that could lead to tenure or a permanent contract, but i have not yet reached this status", "a position that is not in the tenure track or permanent contract system", "a tenured or permanent contract position", "other"), department/institution/organization of their primary appointment (genetics, government organization, medicine [other than psychiatry], non-academic hospital or clinic, other, psychiatry, psychology, public health, research institute), and the country where they hold their primary research appointment (a drop-down menu of countries in the world). descriptive statistics were calculated for quantitative items using r (r core team, ). chisquare tests and analyses of variance (anovas) were used to test hypotheses that individuals would differ on the basis of type of academic position and gender. for analyses on gender, only male and female respondents were used due to small sample sizes of other respondents. a stringent significance threshold of p < . was used to account for multiple testing throughout the study. in addition, cohen's d were used to estimate effect sizes. responses to open-ended questions were grouped into thematic categories, as follows. for each open-ended question, the first author independently developed a set of themes to capture the responses. each theme was required to capture at least three responses to the open-ended question. because the goal of reporting the open-ended responses was strictly descriptive, we made no attempt at establishing reliability of the coding of major themes. a total of individuals completed the survey, with a majority of respondents being female (n = , . %) compared to male (n = , . %) and gender variant/nonconforming or unreported (n = , . %) ( table ). the majority of respondents more held permanent/tenured positions (n = , . %) compared to non-permanent faculty (n = , . %), trainees (n = , . %) or "other" positions (n = , . %). six respondents ( . %) did not specify their academic position ( table ) . of those who did report their position, a majority held primary research appointments in psychiatry (n = , . %) or genetics (n = , . %). the remaining respondents indicated various other departments (medicine, psychology, public health), research institutes, or nonacademic hospitals. academic appointments were most commonly in the united states (n = , . %), followed by united kingdom, n = ; germany, n = ; sweden, n = ; australia, n = ; denmark, n = ; canada, n = ; brazil, n < ; greece, n < ; italy, n < ; mexico, n < ; netherlands, n < ; spain, n < ; switzerland, n < ; afghanistan, n < ; austria, n < ; estonia, n < ; japan, n < ; new zealand, n < ; norway, n < ; romania, n < ; south africa, n < . ten individuals ( . %) not report the country of their appointment. a significantly greater proportion of men (n = , . % of male sample) reported holding a permanent appointment compared to women (n = , . % of female sample) (c ! ( , ) = . , p = . ). as shown in table , the largest proportion of participants ( . %) reported that % of their research was shut down due to covid- . a total of . % of respondents reported no shutdown of their research, many respondents in this category emphasized that their work was mostly on archived data. a total of . % of respondents reported their research to be totally (up to %) shut down. among those who reported any research disruptions (n = , . % of the total sample), some reported being able to transition their studies to online settings (n = , . %), whereas others (n = , . %) reported not having the need to move their studies online due to the nature of their work being theoretical or secondary data analyses. further, the majority of respondents who indicated "up to %" or "up to %" of their research was shut down due to covid- restriction (n = ) were more likely to also report they were unable to move their research work to an online setting (n = ). tables and present research related concerns stratified by academic position ( table ) and gender ( table ) . on average, the highest levels (mean ³ . ) of research-related concern were related to cancellation of career opportunities, securing future funding, recruitment, and data collection. intermediate levels of concern ( . > mean ³ . ) were disruptions from having to work from home (both technological and domestic concerns), staffing, transferring teaching/supervision to remote/online, budget, and obtaining institutional approvals. specific problems related to supply procurement (mean = . ) and animal research (mean = . ) ranked lowest among respondents. there were no significant differences in research-related concerns between appointment groups. however, when stratified by gender, women report significantly greater concerns regarding domestic issues around disruptions from having to work from home (p = . ). overall, the impact of covid- on career was an intermediate concern (mean = . ). however, very large and highly significant differences emerged in career concerns, with trainees and nontenured faculty reporting higher levels of concern compared to those with permanent positions (p = . e- , d = . ). moreover, women reported greater levels of career concerns, of medium effect size, compared to men (p = . e- , d = . ). stress levels displayed similar patterns as career impact. stress levels were high among all respondents (mean = . ), with higher levels reported among those with non-tenured positions (p = . , d = . ) and by females (p = . e- , d = . ). a minority (n = , . %) of respondents expected no changes would be made to their research moving forward as a result of covid- , while the majority (n = , . %) thought it was "too soon to tell" if any changes need to be made ( table ) . there was no significant difference across positions regarding the future practices of pgc research (p = . ). a full . % of respondents reported that there would be no changes in performance evaluations at their institutions related to covid- , with approximately one-third of respondents ( . %) indicated changes, and . % reporting that it was too soon to tell ( table ). respondents were asked to provide " - effective strategies for dealing with covid- in terms of your pgc research," which yielded responses. four main themes characterized the comments: maintain team dynamics (e.g., utilizing videoconferencing for regular team meetings, being flexible with deadlines, use clear communication) ( . % of responses); maintain good personal habits (e.g., keeping in mind productivity may be reduced, practicing self-care, keeping work and personal areas separate) ( . %); reprioritize research goals (e.g., spending more effort on dry-lab projects rather than wet-lab, using available time to complete analyses or manuscripts, utilizing existing data for new projects) ( . %); and shift recruitment to online approaches (e.g., phone interviews rather than face-to-face, development of online recruitment and consent protocols) ( . %). two themes emerged from the responses to the prompt, "effective strategies for transitioning your pgc research to online settings." both themes employ technological approaches to: maintain contact with research participants (e.g., using online questionnaires and other remote means for recruitment and consent) ( . %) and support activities of the research team (e.g., establishing connections to remote databases, regular teleconferencing with colleagues) ( . %). the responses to the prompt "describe - changes you expect to make in your pgc research practices" could be grouped into five themes: move to remote recruitment (e.g., phone/online recruitment, saliva rather than blood sample collection) ( . %); increased use of virtual meetings (e.g., video conferencing for in lieu of lab or scientific meetings) ( . %); organizational changes (e.g., expectations for decreased budget, reduced personnel) ( . %); increased protective measures when collecting samples in-person (e.g., increased ppe for blood draws) ( . %); and shifting research priorities (e.g. add covid- as a research focus) ( . %). when asked to "describe - changes the pgc should make to support pgc researchers during and after covid- " responses were provided. these comments fell into six common themes: continue to support remote meetings (e.g. virtual world congress of psychiatric genetics annual meeting, worldwide lab meetings) ( . %), facilitate more secondary analyses (e.g. making data access easier, increasing time frame on existing proposals, prioritize data access for junior researchers) ( . %), offer greater support to early career researchers (e.g. advocate for junior researchers, create support groups for early career researchers) ( . %), provide support for researchers directly impacted by covid- (e.g. leniency for those who see patients and other caregivers, support career advancement for those directly affected by covid- ) ( . %) , provide funding to support researchers (e.g. bridge funding to sustain pgc research especially among junior researchers and those not part of core pgc funding) ( . %), and provide online training (e.g. statistical genetics and other key topic courses) ( . %). of the answers to the prompt, "briefly describe your institution's policy changes about performance evaluations of researchers," by far, the most reported change was an extension of performance evaluation periods ( . % of responses), specifically extending the tenure clock by one year. seventeen comments were made in response to the question "is there a question about covid- 's impact on your pgc research that we should have asked but didn't?" there were two themes that met a minimum of three comments per theme. first, respondents indicated that we should have asked questions regarding clinical researchers and how their practice has shifted to support covid- patients ( . % of responses). second, respondents wanted more questions into how broader shutdowns (e.g., school closings, unemployment, work from home) have affected productivity, specifically childcare ( . % of responses). overall, our survey revealed high stress and concern about the impact of covid- on their careers especially in individuals with non-permanent positions and in women. our results are consistent with those reported across various academic fields (andersen, nielsen, simone, lewiss, & jagsi ; brubaker, ; denfeld et al., ; kibbe, ; weissman et al., ) , and highlight steps that can and should be taken to ensure the ability of early career researchers and female academics not only to survive but to thrive post-pandemic. results of the pgc survey align with other surveys reported in the literature that reveal the disproportionate impact that covid- -related interruptions have had on female researchers. from having primary responsibility for childcare at home while trying to work from home to concerns about an advancing tenure clock when their productivity is hampered by pandemicrelated disruptions, women do appear to be more stressed and more directly impacted than men. this augments the already disproportionate burden of domestic and emotional labor shouldered by female academics (brubaker, ; jolly et al., ; rao, ) . other studies confirm this observation including a disproportionate number of male first authors in papers submitted to journals on covid- (andersen et al., ) , journal submissions and productivity in general (viglione, ) , and projections of serious interruptions of career progress for women that could adversely affect progress toward gender equity in academe (sheikh et al., ) . a surprising number of institutions were not intending to make allowances on performance evaluations or tenure clocks. this is of significant concern, especially given the documented differential burden placed on junior female faculty in their childbearing years who are entrusted with the majority of childcare duties (jolly et al., ) . senior mentors, institutions, and scientific organizations like the pgc should actively develop and deploy measures to support the careers of junior researchers and those of all genders who have had to take on additional child-or elder-care burdens during this time. likewise, although we did not assess ancestry, it has been widely documented in the united states that individuals from underrepresented minority groups have been disproportionately affected by covid- (moore et al., ) , which means that not only might more minority researchers be directly affected by covid- , but they are also more likely to have connections in socially vulnerable communities and have family members and members of their communities impacted (nayak et al., ) . this can divert both time and emotional energy away from career progress and further perpetuate existing systemic inequities in academe (davis & fry, ) . in many years to come, evaluations of productivity and hiring decisions should explicitly address and account for disruptions encountered during this time. although many institutions have implemented a single-year extension of tenure clocks, that may not suffice given the prolonged nature of the pandemic. applications should include the opportunity to describe the impact of the pandemic on an applicant's life such that it can be factored into the evaluation of the candidate. it is critically important that covid- not set back progress toward equity in science and academe in general, but definitive action must be taken in order to ensure that outcome. some caveats and limitations should be considered when interpreting the results. first, the extent to which our sample represents the larger pgc is unknown as we are unable to calculate response rate or representativeness as the survey link was shared widely across pgc groups and subgroups. the composition of the sample, namely primarily female ( . % of total sample) and in a permanent/tenured position ( . %), does not necessarily reflect the overall composition of the pgc and may reflect selective participation. second, given our goal of providing strictly descriptive results, we did not undertake formal efforts at establishing a coding scheme for the free text responses. third, the survey was deployed relatively early in the pandemic when it was not yet clear how long the disruption to research would go on. responses could change as the duration of the home-and work-related disruption continues and researchers become increasingly fatigued by the pervasive and persistent disruption. finally, our failure to assess race and ethnicity was a missed opportunity to capture specific concerns faced by researchers from underrepresented minority groups. as a field, genetics already has considerable underrepresentation of researchers from diverse ancestral backgrounds; our findings for other historically disadvantaged groups (e.g., women, early career investigators) suggest that the pandemic may further exacerbate this underrepresentation. recurring themes that emerged focused on the cancellation of career opportunities in terms of networking, but also the financial impact of covid- on job availability as many institutions have implemented hiring freezes. this along with personal economic instability, and concerns about the availability of sources of future research funding lead many researchers to question their future job prospects and the viability of remaining in academe. many respondents expressed desire for the pgc and senior investigations to devise ways to help boost productivity and success in publications and grant applications-basically devoting greater energy to ensuring the success of early career researchers during this time. the disproportionate underrepresentation of women at higher academic ranks (carr et al., ) is a known phenomenon in many fields of academic medicine, especially for women. it is a critical juncture to ensure that we can shore up promising young investigators such that we can retain them in science and not erase the albeit slow and incremental advances that we have seen in striving for equity in academe (wingard, trejo, gudea, goodman, & reznik, ) . bulik is supported by r mh , r mh , r mh , r mh , r mh , r mh , u mh , and h sm . she also acknowledges funding from the swedish research council (vetenskapsrådet conflict of interest: j guintivano -none. cm bulik reports: shire (grant recipient pearson (author, royalty recipient). dm dick -none covid- medical papers have fewer women first authors than expected women physicians and the covid- pandemic gender differences in academic medicine: retention, rank, and leadership comparisons from the national faculty survey covid- : financial stress test for academic medical centers college faculty have become more racially and ethnically diverse, but remain far less so than students covid- : challenges and lessons learned from early career investigators gender differences in time spent on parenting and domestic responsibilities by high-achieving young physician-researchers consequences of the covid- pandemic on manuscript submissions by women one academic health system's early (and ongoing) experience responding to covid- : recommendations from the initial epicenter of the pandemic in the united states disparities in incidence of covid- among underrepresented racial/ethnic groups in counties identified as hotspots during impact of social vulnerability on covid- incidence and outcomes in the united states. medrxiv influences for gender disparity in academic psychiatry in the united states are women publishing less during the pandemic? here's what the data say conducting eating disorders research in the time of covid- : a survey of researchers in the field faculty equity, diversity, culture and climate change in academic medicine: a longitudinal study responses to free text questions number of comments recorded and themes and illustrative (shortened, paraphrased) statements identified by open-ended questions %) utilizing videoconferencing for regular team meetings, being flexible with deadlines, use clear communication . maintain good personal habits ( / , . %) keeping in mind productivity may be reduced, practicing self-care, keeping work and personal areas separate %) spend more effort on dry-lab projects rather than wet-lab, use available time to complete analyses or manuscripts, utilizing existing data for new projects other caregivers, support career advancement for those directly affected by covid- we should have asked questions regarding clinical researchers and how their practice has shifted to support covid- patients ( / school closings, unemployment, work from home) have affected productivity, specifically childcare ( / , . %) please share your - most effective strategies for dealing with covid- in terms of your pgc research. ( comments) please share your - most effective strategies for transitioning your pgc research to online settings please describe - changes you expect to make in your pgc research practices as a result of covid- . (respondents were instructed to skip the question if they did not anticipate making changes or had checked "it's too soon to tell") ( comments) is there a question about covid- 's impact on your pgc research that we should have asked but didn't? ( comments) please describe - changes the pgc should make to support pgc researchers during and after covid- dr. jerry guintivano is supported by k mh dr. danielle dick is supported by nih r aa (finnish twin study), p aa (alcohol research center), r aa (vcu great), r aa (personalized risk assessment), and u aa (coga) from the national institute on alcohol abuse and alcoholism (niaaa), and by r da (externalizing consortium) from the national institute on drug abuse (nida). key: cord- -s jrtbb authors: head, michael g; brown, rebecca j; newell, marie-louise; scott, j anthony g; batchelor, james; atun, rifat title: the allocation of us$ billion in global funding from g countries for infectious disease research between and : a content analysis of investments date: - - journal: lancet glob health doi: . /s - x( ) - sha: doc_id: cord_uid: s jrtbb background: each year, billions of us$ are spent globally on infectious disease research and development. however, there is little systematic tracking of global research and development. we present research on investments into infectious diseases research from funders in the g countries across an -year time period spanning – , comparing amounts invested for different conditions and considering the global burden of disease to identify potential areas of relative underfunding. methods: the study examined research awards made between and for infectious disease research from g -based public and philanthropic funders. we searched research databases using a range of keywords, and open access data were extracted from funder websites. awards were categorised by type of science, specialty, and disease or pathogen. data collected included study title, abstract, award amount, funder, and year. we used descriptive statistics and spearman's correlation coefficient to investigate the association between research investment and disease burden, using global burden of disease study data. findings: the final – dataset included awards for infectious disease research, with a sum investment of $ · billion (annual range · billion to · billion) and a median award size of $ (iqr – ). pre-clinical research received $ · billion ( · %) across ( · %) awards and public health research received $ · billion ( · %) from ( · %) awards. hiv/aids received $ · billion ( · %), tuberculosis received $ · billion ( · %), malaria received $ · billion ( · %), and pneumonia received $ · billion ( · %). funding for ebola virus ($ · billion), zika virus ($ · billion), influenza ($ · billion), and coronavirus ($ · billion) was typically highest soon after a high-profile outbreak. there was a general increase in year-on-year investment in infectious disease research between and , with a decline between and . funders based in the usa provided $ · billion ( · %). based on funding per disability-adjusted life years (dalys), hiv/aids received the greatest relative investment ($ per daly), compared with tuberculosis ($ per daly), malaria ($ per daly), and pneumonia ($ per daly). syphilis and scabies received the least relative investment (both $ per daly). we observed weak positive correlation (r= · ) between investment and disease burden. interpretation: hiv research received the highest amount of investment relative to daly burden. scabies and syphilis received the lowest relative funding. investments for high-threat pathogens (eg, ebola virus and coronavirus) were often reactive and followed outbreaks. we found little evidence that funding is proactively guided by global burden or pandemic risk. our findings show how research investments are allocated and how this relates to disease burden and diseases with pandemic potential. funding: bill & melinda gates foundation. large amounts of funding are allocated to research in infectious diseases each year, spanning pre-clinical science, clinical trials, product development, and public health, including implementation research. these allocations involve numerous stakeholders across the global health community, including funders, researchers, policy makers, and clinicians. however, there is little systematic tracking or detailed analysis of investments into research and development for infectious diseases to support how to make the best funding decisions. nor is there systematic coordination between stakeholders involved in funding research and development, despite efforts such as the who global observatory on health r&d to achieve better coordination. funders differ in their approaches to commissioning research, from the curiosity-driven approaches of the wellcome trust, to the focused data-driven strategies of the bill & melinda gates foundation, which creates a heterogeneous landscape of research priorities. thus, there is a need for an in-depth and comprehensive review of the global research and development landscape to identify what research has taken place, where the research was done, and which institutions were involved in the research. such research on research is crucial for priority setting, informing funding decisions, and improving efficiency in allocating funds. we present research done by the research investments in global health (resin) study group on research investments into infectious diseases from funders in the g countries across an -year time period spanning - , comparing amounts invested for different conditions and considering the global burden of disease to identify potential areas of relative underfunding. this study considered research awards related to infectious disease research from public and philanthropic funders in the g countries (appendix p ), made between jan , , and dec , . the methods used were similar to those described in detail elsewhere. [ ] [ ] [ ] [ ] data collected included study title, abstract, award amount, funder, and year. data were manually collated from multiple sources. awards to institutions in the uk between jan , , and dec , , have been previously analysed. , most data (> %) from and were sourced from the uberresearch dimensions database, which includes · million financial awards across health and nonhealth research and development sectors from global funders. us national institutes of health (nih) data from between jan , and dec , , was sourced directly from the project reporter database. other data were sourced from the websites of individual funders, funder databases such as the world report, the uk national research register (a now-archived website owned by the uk department of health), or by contacting the funder directly and requesting data. we applied keyword searches and filters (appendix p ) to identify studies of human-related infectious disease. awards purely focused on plant pathology or veterinary science were excluded, unless there was a clear zoonotic component. excluded studies were manually reviewed to identify any false negatives. the included financial awards were individually scrutinised to assess their relevance to infection. mgh assessed all financial awards for inclusion and categorised infection-related awards, applying keyword labels as appropriate (appendix pp - ). secondary checks on included and excluded awards were made (by rjb and other), as per the study protocol. , ( · %) of awards were double checked, with a review of the award inclusion in the dataset and the applied labels indicating the disease areas of the research. where there was disagreement, study information was provided to a third co-author for consensus. research award amounts were adjusted for inflation in original currency and converted to us$ using the mean exchange rate in the award year. award amounts were missing for ( · %) of awards, from funders (appendix p ). in these cases, estimates to our knowledge, this is the first study to describe in depth the global landscape for all infectious disease research from public and philanthropic funders. our study covers years of funding data, so captures long-term time trends and fluctuations. we combined and categorised awards using the classification system developed by the resin study. this strategy allowed us to provide a comprehensive overview of how infectious disease funding has been allocated, and compare findings with the global burden of disease, an important variable to consider when setting research priorities. this information can be used by global health research funders in decision-making processes. our findings show that between and , hiv received significantly more research funding than similar high-burden diseases such as tuberculosis, malaria, and pneumonia. the usa provides much of the global funding, particularly the us national institute for health. there are several infections that appear neglected compared with their burden of disease, such as syphilis and scabies. thus, the global health community could use our findings to inform discussions, alongside other drivers for research prioritisation. were made using maximum award amounts for that funding stream as per the funder's website, by asking principal investigators for an approximate or exact award amount provided, or by asking in-country researchers who had knowledge of the research and development landscape for typical award amounts. datasets and analyses were circulated to all authors for review and comment. included award types comprised project and programme grants, fellowships, and pump-priming or pilot projects. excluded award types were conference and infrastructure grants and funding focused on operational activities rather than research. labels applied to each award included pathogen, disease areas and specialty (eg, antimicrobial resistance, respiratory, oncology, and paediatrics), and type of science along the research continuum (pre-clinical, phase - clinical trials, phase and product development research, public health [ focusing on populations], and cross-disciplinary studies across multiple stages of the research continuum). we defined cross-disciplinary as a study that covered more than one stage of the research continuum (for example pre-clinical research that progressed to a phase study). antimicrobial resistance included antibacterial, antiviral, antiparasitic, and antifungal resistance. the diagnostics category included research into screening. sexually-transmitted infections excluded hiv, which had its own category. neglected tropical diseases were based on the who definition (as of oct , ). burden of disease data were sourced from the global burden of disease study online tool. disease burden data are reported from for all infectious diseases, and additional examples are presented using hiv/ aids, malaria, tuberculosis, and pneumonia from years and (six-year time intervals during the period covered by the investments dataset). measures of disease burden analysed were mortality, disabilityadjusted life years (dalys), and years lived with disability (yld). comparison between awards and their associated observed disease burden were made by calculating investment per mortality, daly, or yld observed. we computed the investment relative to the burden of infection using the following equation: cumulative research investment up to the year of burden measure or number of deaths, dalys, or yld at that timepoint. for example, for assessment of hiv dalys in , the sum of hiv research investment from jan , , to dec , ($ · billion), was divided by dalys in ( ), to get an investment per daly metric of $ . descriptive statistics and spearman's correlation coefficient were used to investigate the relationship between research investment and disease burden, using global burden of disease study data. we used microsoft excel for data preparation stata se version for data analysis. the funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. the hiv/aids was the pathogen or disease with the greatest amount of funding ($ · billion [ · %]) across ( · %) awards (tables , ). funding for tuberculosis totalled $ · billion ( · %) from ( · %) awards, funding for malaria was $ · billion ( · %) from ( · %) awards, and funding for pneumonia was $ · billion ( · %) from ( · %) awards. funding for ebola virus-related research was $ · billion ( · %); $ · billion ( · %) of all ebola virus-related research investment was awarded between and , following the high-profile outbreak of ebola virus disease in west africa in . similarly, $ · billion of funding was allocated to research on zika virus, of which · % was awarded in or after the zika virus epidemic. of the $ · billion ( · %) of funding for influenza, $ · billion ( · %) was awarded in - , with the highest annual funding amount awarded in ($ · billion [ · %]). there were global outbreaks of h n influenza in (so-called bird flu) and (so-called swine flu). funding for coronavirus-related research was $ · billion ( · %) from ( · %) grants, with a median award size of $ · million (iqr · million to · million; , the year after the international sars outbreak, by disease area, $ · billion ( · %) was awarded for antimicrobial resistance from ( · %) awards, $ · billion ( · %) was awarded for neglected tropical diseases, $ · billion ( · %) for sepsis, and $ · billion ( · %) for health-care-associated infections. in areas relating to hard-to-reach groups, $ · billion ( · %) was awarded for infections related to drug use and addiction and $ · billion ( · %) for infectious diseases in prison (table ) . awards for comorbidities and non-communicable diseases included $ · billion ( · %) for mental health and $ · billion ( · %) for cardiovascular disease. funders from the usa provided $ · billion ( · %) of the investment, which covered ( · %) of the awards. within this, the us nih was the largest funder, providing $ · billion ( · % of total us funding) and the greatest number of individual awards ( [ · %]). the bill & melinda gates foundation provided $ · billion ( · %) in ( · %) awards. uk funders provided $ · billion ( · %) in ( · %) awards. when the awards had a clear geographical focus, $ · billion ( · %) of the funding was focused on africa and $ · billion ( · %) on asia (appendix p ). when ranking investment levels on the basis of burden of disease by dalys across infectious diseases (appendix p ), african trypanosomiasis ($ per daly) and genital herpes ($ per daly) were ranked first and second, respectively (table ). hiv/aids ($ per daly) was ranked eighth, tuberculosis ($ per daly) was ranked seventeenth, malaria ($ per daly) was ranked twenty first, enteric infections ($ per daly) were ranked twenty fourth, and pneumonia ($ per daly) was ranked twenty eighth. scabies and syphilis were ranked joint last with $ per daly. when comparing investment for individual infections alongside dalys, spearman's correlation coefficient was · (p= · ), suggesting weak positive correlation between research investment and global burden of disease ( figure ) . infections within the shaded area of the graph showed a stronger correlation between investment and burden of disease. infections below the shaded area were relatively underinvested, and infections above the shaded area were relatively well-invested, compared with their dalys burden. when comparing investment by mortality, syphilis ($ per death) and tetanus ($ per death) were ranked the lowest of the infections for which mortality data were available (appendix p ). the highest-ranked infections by investment per death were those for which associated mortality is typically low, specifically chlamydia ($ per death) and african trypanosomiasis ($ per death). hiv was ranked seventh ($ per death), malaria was ranked thirteenth ($ per death), tuberculosis was ranked fifteenth ($ per death), and pneumonia was ranked twenty fourth ($ per death). across different timepoints of the study, hiv-related research consistently received greater investment than did malaria, tuberculosis, or pneumonia (figure ). pneumonia-related research consistently received far less funding during the study period compared with hiv, tuberculosis, or malaria. by type of science, · % of research funding for hiv was for pre-clinical research, · % for phase - trials, and · % for public health research (appendix p ). pneumonia had the greatest proportion of funding allocated to pre-clinical science ( · %) and the lowest amount for public health research ( · %) compared with hiv, tuberculosis, and malaria (appendix p ). in this study, we provide an analysis of $ billion of research investment as public and philanthropic awards for infectious disease research covering the years - . over half of this investment was for pre-clinical science and over a quarter for public health research. by type of infection, hiv-related research received more than double the investment for tuberculosis, malaria, and pneumonia combined. infections that are relatively less well-funded include some sexually-transmitted infections (syphilis and gonorrhoea) and neglected skin infections, such as scabies. funding for coronavirus-related research was $ · billion from grants, of which · % was for preclinical research. however, in there has been a huge reactive effort to support the response to the covid- pandemic, which includes substantial financing for research. as of aug , , the resin study has tracked $ · billion of global public and philanthropic research funding, which already exceeds the total investment in hiv research ($ · billion; appendix page ). viral respiratory infections are known to be one of the most likely causes of a pandemic, but despite this and the existing high levels of mortality in young children and older people due to such infections, systems for pneumonia research and advocacy are not well established. confusion over the definition of pneumonia, few experienced researchers to make a strong case to funders, and few high-profile public figures championing the cause have led to pitifully low levels of funding compared with the disease burden. the bill & melinda gates foundation, which is guided by childhood deaths, is the main funder of pneumonia-related research. the metrics used to compare investment by burden of disease are misleading for pathogens such as ebola virus and zika virus, which at first appear to be relatively wellfunded compared with their burden of disease (appendix p ). however, for these conditions, which are public health emergencies, dalys are not a fair metric to use. outbreaks of this nature are not necessarily high-burden in terms of numbers of cases but are high-risk given the potential for rapid spread to cause widespread outbreaks, an important factor that influences research investment decisions. as illustrated by the evolving covid- pandemic, there is a public health need to support outbreak responses and research should very much be part of such a response. such outbreaks create uncertainty and fear, with media promoting a need to do something and urging political circles to respond rapidly. , historical funding for coronavirus research was very low, even after the high-profile outbreaks of severe acute respiratory syndrome (sars), due to sars coronavirus, and middle east respiratory syndrome and the potential for the rapid spread of such infections. other analyses highlight how funding for neglected infectious disease research (distinct from neglected tropical diseases) is increasing. our analysis supports this conclusion, for example, showing that research on neglected tropical diseases or with a focus on africa is increasing (appendix p ). there have been substantial declines in hiv funding, primarily in higher-income settings (and thus not captured under neglected disease definitions). the coalition for epidemic preparedness innovations, founded in , has received substantial research investment from multiple funders to research selected high-threat pathogens. for example, there are several ongoing studies to develop a universal influenza vaccine to reduce pandemic risk, as well as vaccine in development against coronaviruses. antimicrobial resistance, which continues to be a serious worldwide threat, has led to the introduction of the global amr r&d hub with a remit to address challenges and improve coordination and collaboration in global antimicrobial resistance research and development using a one health approach. antimicrobial resistance is also an important contributor to sepsis mortality, which is responsible for million deaths annually with most of the burden in sub-saharan africa, but receives just · % of the funding. research investment analyses can be a valuable audit of a system that has perhaps maximised scientific efficiency through peer review of curiosity driven research and provide a direction for revision of research on under-investigated diseases and subject-based opportunities. the covid- pandemic has shown the fragility of national and global infrastructures, and pandemic preparedness will surely be a focus for highprofile global health research stakeholders in years to come. sustainable tracking of how research funding is spent is vital to ensure that all priority areas and knowledge gaps are addressed, and there must be adequate translation of that new knowledge into policy and practice, with findings that can be feasibly adopted in resource-poor settings. multiple factors other than the current and projected burden of disease influence research funding decisions, such as political drivers of decision making (notable in our study given the major funder was the us government), advocacy and lobbying, emergency preparedness for emerging infectious diseases with pandemic potential, and public health research for conflicts and other humanitarian responses. applying a globally recognised label to a disease can be important. who oversees the designated list of neglected tropical diseases, which helps to raise the profile of these conditions and support arguments for research funding. as an example, african trypano somiasis, which has been at the forefront of efforts to tackle neglected tropical diseases, has been described as an extraordinary success story, with a decline in the daly burden by % between and . african trypanosomiasis has received twice the amount of research funding compared with lymphatic filariasis or schistosomiasis, and more than non-neglected tropical diseases, such as meningitis or the respiratory syncytial virus. researchers who study african trypanosomiasis have elimination and eradication in sight, although this will probably require further substantial investments. investment in other neglected areas might help produce similar effective responses, although the type of research investment must be appropriate. for example, our analysis highlighted scabies and syphilis as particularly underfunded. effective treatments are available for scabies, so the most useful research might be around an effective drug supply chain or addressing stigma. other factors beyond the burden of disease also influence the direction and amount of investment. the geographic focus of research investments affects the likelihood of knowledge being translated into policy and practice, particularly in the country or sector where the research was undertaken. it is important to consider where research capacity should be created or enhanced, rather than simply which research areas to prioritise and fund. the uk invests greater resources in former colonies, influenced by historical ties and a shared language. investments in different sectors will also be affected by diplomatic considerations, for example, funding countries seeking cooperation from recipient countries or regions in response to security threats and terrorism. this study has several limitations. there will be missing data, particularly when data could not be accessed from public and philanthropic funders. we propose that the effect of this should not be substantial and should not greatly influence our findings, as the included data relate to of the top leading investors in research. , the focus on g countries means that funders from countries who are not in this group but are proactive in global health research, such as switzerland and norway, were not included. a key challenge was integrating data that were presented in numerous different formats. future analyses would be simplified considerably if funders could adopt a minimum dataset of required information, perhaps recommended by the who global observatory on health r&d, which would require that applicants add standard labels (eg, the type of science along the research pipeline) to their project at time of submission. applying categories to an award retrospectively is time-consuming and subjective, although errors were reduced by observations from a second author and consensus. automated categorisation based on keyword searches is problematic, since the title and abstract of many awards contain references to diseases that are not the study areas of focus. furthermore, separating out awards for operational or implementation research and activities that are nonresearch based implementation (ie, not generating new knowledge) is a subjective process. our study lacks data from the private sector, particularly concerning tools and products such as vaccines, diagnostics, and therapeutics. the analyses use global burden of disease study data, which are themselves modelled estimates and will on occasions be based on imputations due to missing data, and have been subject to criticism. selection of timepoints for research investments and disease burden will affect our findings as both of these change over time. more than $ billion has been spent globally on infectious disease research between and , but this funding does not correlate with current levels of burden or the level of risk posed by infections with pandemic potential. since priority setting for research must consider many different factors, our analysis should be used to support decision making rather than providing clear-cut answers. it is worrying that the funding allocated to infectious disease research is declining during a period in which there are concerns surrounding antimicrobial resistance and the pandemic potential of many pathogens. in conclusion, our findings show where research funding resources are allocated and how this relates to disease burden and diseases with pandemic potential. we anticipate that our results could be an invaluable resource to global health stakeholders (eg, who, research funders, or national governments) who define research strategy and make decisions about the allocation of restricted research and development resources. mgh created the study and led data collection, analysis, and reporting. rjb supported data collection, analysis, and reporting. jb, ra, jags, and m-ln provided contributions to methodology and expert comment and overviews on global health, research investment, and health financing. mgh wrote the first draft. all authors read subsequent drafts and contributed revisions. all authors approved the final manuscript and the and decision to submit for publication. biomedical research; what gets funded where? money and microbes: strengthening research capacity to prevent epidemics how we've defined what success looks like for wellcome's work bill & melinda gates foundation. global health strategy overview sizing up pneumonia investment global funding trends for malaria research in sub-saharan africa: a systematic analysis 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resistance-a manual for developing national action plans. version global, regional, and national sepsis incidence and mortality, - : analysis for the global burden of disease study london declaration on neglected tropical diseases the elimination of human african trypanosomiasis is in sight: report from the third who stakeholders meeting on elimination of gambiense human african trypanosomiasis research, evidence and policymaking: the perspectives of policy actors on improving uptake of evidence in health policy development and implementation in uganda infectious disease research investments follow colonial ties: questionable ethics the largest public and philanthropic funders of health research in the world: what they fund and how they distribute their funds health research funding organizations the evolution of the disability-adjusted life year (daly) we thank all funders who directly or indirectly provided data and those who provided information about and links to the funders. much of the data awarded from onwards was sourced by accessing the dimensions database, owned by uberresearch (london, uk). we also thank joseph fitchett, for his previous help and support in data collection and analysis and for assisting with secondary checks on included and excluded awards, and pat oxford, who provided support for developing the figures and images. jags is funded by a fellowship from the wellcome trust ( ). the study received funding from the bill & melinda gates foundation (opp ). key: cord- - opgzgcj authors: jia, hongpeng; wu, min title: sustained research fund and dedicated research center for preparing next pandemic date: - - journal: precis clin med doi: . /pcmedi/pbaa sha: doc_id: cord_uid: opgzgcj the current covid- pandemic caused by the sars-cov- virus is imposing a great threat to human lives and international panic that is not seen since wwii, resulting in financial crisis, daily life disturbance, transportation shutdown, industry disruption, and countries/cities lockdown in every corner of the globe. the inability to effectively contain the virus indicates that our investment and attention in research, prevention, and treatment development for this type of deadly viruses is insufficient, considering it has been years since the brother coronavirus, sars-cov outbreak. the biggest lesson learned from the acrimonious past experiences is that humans quickly lose memory and do not continue to support related research when a pandemic is gone. it is the very time for the government, industry, and private foundations to work together to respond to this wake-up call and to take extraordinary measures to sustain the research support and establish comprehensive research centers. only this level response may give us a hope to prepare the future and adequately deal with the next potential pandemic caused by emerging devastating viral infections. the new coronavirus (sars-cov- ) leads to severe lung infection and unique pneumonia disease, namely the covid- that was found first in december and has quickly spread in the world, which has caused international concern for healthcare and interfered with the normal lives of a large portion of the human population due to extensive quarantine and separation. this catastrophic disturbance may serve as a wake-up call for governments, policymakers, philanthropic organizations, healthcare systems, and industries to the reality of extraordinarily dangerous infections caused by emerging pathogens, especially viruses. admittedly, the government, healthcare institutes, and hospitals as well as medical professionals in china have swiftly taken extraordinary steps and implemented well-coordinated measures to contain the disease by locking down wuhan city, sharing research results, supplying all emergent materials and sending medical aids to the hospitals at ground zero in hubei province, particularly wuhan city. similar approaches have been applied in italy, usa, spain, uk, india, etc. however, the measures taken to control this rapidly spreading disease were insufficient in most of these countries, even with intense efforts, resulting in quick dissemination throughout the world. this is a testament that our awareness and preparation of these types of diseases, even in relatively well-prepared countries such as the usa, italy, the u.k., spain, germany and china, are extremely insufficient. over the past few decades, humans have experienced continued attacks by various deadly infectious diseases caused by newly found or evolved viruses, including sars, mers, zika, ebola, influenza illness, etc. due to a lack of continued funding support and other intervention measures, human societies are entering into a miserable cycle. when one emergency occurs, we have a knee-jerk response and quickly allocate emerging funding to reduce the potential spread, alleviate the disease suffering, support research on the pathogenesis and develop vaccines and drugs for the new pathogen. however, the dramatic urgency to respond to the pandemic each time has imposed humongous costs, damaged our economy, and disrupted routine life, including study, work, business, sports, and music events. however, in each instance, we tend to rather quickly forget the pain it caused once the disease is relieved. there is no sustained funding available for continued research into the disease pathogenesis and development of tools for diagnosis, prevention, and treatment. as a result, the number of researchers in the field quickly dwindles after the initial enthusiasm at the disease outburst due to funding shortages, and industries are reluctant to invest in the development of effective vaccines and drugs. as there is no way to predict when the next pandemic will occur accurately, hence financially, a weak business sector cannot help in keeping the profit-driven industries continuing the development of drugs and vaccines. therefore, when a new infectious pathogen emerges, or an old pathogen evolves, such as the current sars-cov- , we are not well prepared regarding the vaccines and drugs. the lesson learned from the bitter past experiences is that as humans, we just drop the ball in the related research after each of the many recent pandemics, such as sars, ebola, mers, and zika, the last two still biting us. around - , a series of clinical trials of vaccines against sars showed somewhat efficacy, not convincing results but had to be abandoned because of a lack of continued funding. to avoid the sudden death of promising research towards diagnostics, vaccines, and treatments for pandemic causing bugs, we have to do a lot more with an extraordinary, unconventional response from all levels to sustain interest and funding from all sectors, such as government, industry, and philanthropy. we hope we have now learned enough. people did not realize that the real costs to us for missing this much research are humongous and unmeasurable, just thinking about the rescue $ trillion bills for this disaster approved by the u.s. congress in march . if we have only had a tiny fraction of this funding but sustainable, humans will have more weapons to combat this type of emerging diseases. sars-cov virus is a sister of the current sars-cov- , meaning some drugs for sars-cov would be potentially effective for the sars-cov- and have been quickly used as compassionate treatments ( , ). due to the meager funding of research investment, many of the previous vaccines have been abandoned ( , ) , and no effective vaccines for mass protection are available. hence, after years of the outbreak ( ), we are still crying for no countermeasures to this very horrible virus. furthermore, the current and future economic damage and human life disruption are enormous, and we all cannot travel and work. companies are sacking employees. even our staff who need to work on the research to combat this virus are not allowed to access to the laboratories due to some viral phobias/fear or restriction/shutdown/lockdown. alarmingly, the trend of viral evolution, antibiotic resistance growing, social-political development, economic pattern transition, and human behavior change (much more frequent global travels, human interactions and intercontinental economy dependence than years ago), this type of pandemic is highly likely to threaten and slaughter us sooner and/or more often. to solve this problem, we recommend two major reformations: ) invest heavily and continuously into the development of countermeasures to control emerging pathogens that cause massive population infections, and ) build multiple international and state-level research centers to deal with next pandemics. the international communities thoroughly rethink their policies and allocate sufficient funds even after the initial outbreak to continue at least for years (preferably non-stopping) and sustain the research on these emerging pathogens and their diagnosis, prevention and treatment to better control future infections and avoid pandemics. a large scale of infrastructure, resources, and the task force is desperately needed at state, national. even international levels build many-dedicated research centers to develop diagnostic, vaccines, and drugs as well as basic research to understand the molecular and cellular pathogenesis and host-pathogen interactions. the national institutes of health (nih) set up the office of emergency care research (oecr) in to recognize the importance of emergency care. however, this office has no funding to support scientific research. it is not explicitly designed to control rapid and widely spreading infectious diseases. through the biodefense & emerging infectious diseases program, the national institute of allergy and infectious diseases (niaid) has played a vital role in supporting research involving emerging infectious diseases/pathogens in recent years; however, the level of funding and extent of intention is far less than needed to combat these new pandemic diseases effectively. for example, the annual support for coronavirus researchthe brother of the current outbreak pathogen, in the u.s. is a meager $ million last year, which is rather small compared to many other diseases such as cancer, heart diseases, diabetes, and even different types of infectious diseases, to name a few. we suggest that the niaid or oecr of the us nih, the natural science foundation (nsf), the nsf of china, european molecular biology organization (embo), european union (eu), etc. establish a funding branch termed "sustained support for emerging infections" with multiple billion-dollar budgets yearly to continuously fund research into the emerging infections caused by viral diseases that have recently emerged to anticipate new outbursts. we need to vigorously encourage young and established scientists to join these research frames to strengthen the efforts to combat these horrifying diseases. in addition, the government should allocate some funds to set aside for industries that work on prevention and therapy. these approaches are advised to all governments worldwide. we call for the unification of funding sources from governments, industries, private foundations and philanthropic sectors such as the bill melinda foundation, chan zuckerberg initiative, etc., to invest heavily in this emerging viral infection. this has to be a serious creative input and significant investment and has a specific office to devote their efforts to quickly develop tools and novel strategies to invent vaccines, drugs, and diagnostic devices and kits by nurturing creative ideas and innovative technologies. we call for governments, the world health organization (who), and the united nations to rethink this need as a concerted effort to maximally prevent and curb the future infectious pandemic. we need not only research funding but also infrastructure, hospitals, containments, and medical disposables. all should be prepared and manufactured rapidly to contain the colossal pandemic, including continued stocking in case an unexpected outbreak such as this one. the preparation also needs all governments to establish universal and standard regulations to reinforce our awareness and swift response mechanisms by the leaders at every government level, not just the countries leaders to any potential emerging epidemic or pandemic. when an emerging epidemic or pandemic occurs, the international communities need to communicate timely and often, interact and take a consortium approach to tackle the shortage of prevention and treatment including beds and medical professionals to unify to combat the disease, which will be much more useful than each country acts alone without others' support and cooperation. simple banning airlines, distance separation, social distancing, or geographic locking down are helpful and sometimes may be necessary but not the right approach and causing a substantial cost. in summary, the critical lesson learned from past experiences and the new pandemic infection is that we cannot continue to do the same as we have done before. instead, we genuinely need an urgent, radical, and fundamental change. immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates first case of novel coronavirus in the united states safety and immunogenicity from a phase i trial of inactivated severe acute respiratory syndrome coronavirus vaccine a sars dna vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial key: cord- -knlc bxh authors: holmes, emily a; o'connor, rory c; perry, v hugh; tracey, irene; wessely, simon; arseneault, louise; ballard, clive; christensen, helen; cohen silver, roxane; everall, ian; ford, tamsin; john, ann; kabir, thomas; king, kate; madan, ira; michie, susan; przybylski, andrew k; shafran, roz; sweeney, angela; worthman, carol m; yardley, lucy; cowan, katherine; cope, claire; hotopf, matthew; bullmore, ed title: multidisciplinary research priorities for the covid- pandemic: a call for action for mental health science date: - - journal: lancet psychiatry doi: . /s - ( ) - sha: doc_id: cord_uid: knlc bxh the coronavirus disease (covid- ) pandemic is having a profound effect on all aspects of society, including mental health and physical health. we explore the psychological, social, and neuroscientific effects of covid- and set out the immediate priorities and longer-term strategies for mental health science research. these priorities were informed by surveys of the public and an expert panel convened by the uk academy of medical sciences and the mental health research charity, mq: transforming mental health, in the first weeks of the pandemic in the uk in march, . we urge uk research funding agencies to work with researchers, people with lived experience, and others to establish a high level coordination group to ensure that these research priorities are addressed, and to allow new ones to be identified over time. the need to maintain high-quality research standards is imperative. international collaboration and a global perspective will be beneficial. an immediate priority is collecting high-quality data on the mental health effects of the covid- pandemic across the whole population and vulnerable groups, and on brain function, cognition, and mental health of patients with covid- . there is an urgent need for research to address how mental health consequences for vulnerable groups can be mitigated under pandemic conditions, and on the impact of repeated media consumption and health messaging around covid- . discovery, evaluation, and refinement of mechanistically driven interventions to address the psychological, social, and neuroscientific aspects of the pandemic are required. rising to this challenge will require integration across disciplines and sectors, and should be done together with people with lived experience. new funding will be required to meet these priorities, and it can be efficiently leveraged by the uk's world-leading infrastructure. this position paper provides a strategy that may be both adapted for, and integrated with, research efforts in other countries. it is already evident that the direct and indirect psychological and social effects of the coronavirus disease (covid- ) pandemic are pervasive and could affect mental health now and in the future. the pandemic is occurring against the backdrop of increased prevalence of mental health issues in the uk in recent years in some groups. , furthermore, severe acute respiratory syndrome coronavirus (sars-cov- ), the virus that causes covid- , might infect the brain or trigger immune responses that have additional adverse effects on brain function and mental health in patients with research funders and researchers must deploy resources to understand the psychological, social, and neuroscientific effects of the covid- pandemic. mob ilisation now will allow us to apply the learnings gained to any future periods of increased infection and lockdown, which will be particularly important for front-line workers and for vulnerable groups, and to future pandemics. we propose a framework for the prioritisation and coordination of essential, policy-relevant psychological, social, and neuroscientific research, to ensure that any investment is efficiently targeted to the crucial mental health science questions as the pandemic unfolds. we use the term mental health sciences to reflect the many different disciplines, including, but not limited to, psychology, psychiatry, clinical medicine, behavioural and social sciences, and neuroscience, that will need to work together in a multidisciplinary fashion together with people with lived experience of mental health issues or covid- to address these research priorities. the uk has powerful advantages in mounting a successful response to the pandemic, including strong existing research infrastructure and expertise, but the research community must act rapidly and collaboratively if it is to deal with the growing threats to mental health. a fragmented research response, characterised by smallscale and localised initiatives, will not yield the clear insights necessary to guide policy makers or the public. rigorous scientific and ethical review of protocols and results remains the cornerstone of safeguarding patients and upholding research standards. deploying a mental health science perspective to the pandemic will also inform population-level behaviour change initiatives aimed at reducing the spread of the virus. international comparisons will be especially helpful in this regard. in this position paper, we explore the psychological, social, and neuroscientific effects of covid- and set out clear immediate priorities and longer-term strategies for each of these aspects. we also surveyed the public and people with lived experience of mental ill-health (panel ). the general population survey, done by ipsos mori, revealed widespread concerns about the effect of social isolation or social distancing on wellbeing; increased anxiety, depression, stress, and other negative feelings; and concern about the practical implications of the pandemic response, including financial difficulties. the prospect of becoming physically unwell with covid- ranked lower than these issues related to the social and psychological response to the pandemic. the mq: transforming mental health stakeholder survey of people with lived experience of a mental health issue likewise highlighted general concerns about social isolation and increased feelings of anxiety and depression. more specifically, stakeholders frequently expressed concerns about exacerbation of pre-existing mental health issues, greater difficulty in accessing mental health support and services under pandemic conditions, and the effect of covid- on the mental health of family members, especially children and older people. both surveys are reported online. these findings, combined with the published scientific literature, informed the development of our research priorities. the surveys represent a snapshot of the current situation, but they will need to be repeated more rigorously over the course of the pandemic, and the research priorities reviewed. in this section, we focus on the psychological processes and effects in individual people related to covid- , such as cognition, emotion, and behaviour, that affect mental health (table ) . although a rise in symptoms of anxiety and coping responses to stress are expected during these extraordinary circumstances, there is a risk that prevalence of clinically relevant numbers of people with anxiety, depression, and engaging in harmful behaviours (such as suicide and selfharm) will increase. of note, however, is that a rise in suicide is not inevitable, especially with national mitigation efforts. the potential fallout of an economic downturn on mental health is likely to be profound on those directly affected and their caregivers. the severe acute respiratory syndrome epidemic in was associated with a % increase in suicide in those aged years and older; around % of recovered patients remained anxious; and % of health-care workers experienced probable emotional distress. [ ] [ ] [ ] patients who survived severe and life-threatening illness were at risk of post-traumatic stress disorder and depression. , many of the anticipated consequences of quarantine and associated social and physical distancing measures are themselves key risk factors for mental health issues. these include suicide and self-harm, alcohol and substance misuse, gambling, domestic and child abuse, and psychosocial risks (such as social disconnection, lack of meaning or anomie, entrapment, cyberbullying, feeling a burden, financial stress, bereavement, loss, unemployment, homelessness, and relationship breakdown). [ ] [ ] [ ] a major adverse consequence of the covid- pandemic is likely to be increased social isolation and loneliness (as reflected in our surveys), which are strongly associated with anxiety, depression, self-harm, and suicide attempts across the lifespan. , tracking loneliness and intervening early are important priorities. crucially, reducing sustained feelings of loneliness and promoting belongingness are candidate mechanisms to protect against suicide, self-harm, and emotional this position paper summarises the priorities put forward by an interdisciplinary group of world-leading experts, including people with lived experience of a mental health issue, from across the bio-psycho-social spectrum of expertise in mental health science in march and april, . the experts were convened by the uk academy of medical sciences and the mental health research charity, mq: transforming mental health. members participated in an individual capacity, not as representatives of their organisations. a coordinating group of seven experts met daily over a period of two weeks to develop the research priorities, informed by input from the expert advisory group. given the need to develop the research priorities rapidly to inform immediate funding priorities, extended evidence gathering and consultation was not possible. however, we are confident that the wide breadth of expertise on the expert group and their leading roles in their respective fields provide a wide-ranging and comprehensive view of the mental health and neuroscience research priorities now; priorities which should be reviewed and should evolve with the pandemic. lived experience of a mental health issue was incorporated by four mechanisms. first, three representatives with lived experience provided input as part of the expert advisory group. second, an online survey collected data on people's two biggest concerns about the mental health and wellbeing implications of the coronavirus disease (covid- ) pandemic and the coping strategies used by patients. the survey was promoted via email to mq's supporter network and via social media. in total, people completed the survey, submitting concerns about the mental health effects of the covid- pandemic and responses about what has helped to maintain mental health and wellbeing during the pandemic. a thematic analysis of the full dataset was done. third, two questions were asked on ipsos mori's online omnibus survey to collect data on people's concerns about the effect of covid- on mental wellbeing and what is helping people's mental wellbeing at this time. in total, interviews were completed with adults aged between and years from across england, wales, and scotland. quotas were set and data were weighted to the offline population to ensure a nationally representative sample by gender, age, and region. statistical analysis was done and any subgroup differences included are statistically significant at a % confidence interval unless stated otherwise. a summary report of the findings of both surveys and further methodological details can be found online. the ipsos mori tabular data can be found on its website. finally, the manuscript was peer-reviewed by a reviewer with lived experience of a mental health issue. we acknowledge the limitations of our surveys, including the representativeness of the mq sample, the short timescale for input, and the representativeness of online populations. we also acknowledge the restricted evidence gathering and opportunity for wider consultation of people with lived experience. however, combined, these four mechanisms of collecting input from people with lived experience provide important insight into people's concerns about the effect of covid- on mental health and coping strategies within the very short timeframe. problems. , social isolation and loneliness are distinct and might represent different risk pathways. to inform management of covid- , it is vital to understand the socioeconomic effect of the policies used to manage the pandemic, which will inevitably have serious effects on mental health by increasing unemployment, financial insecurity, and poverty. , involvement of people with lived experience and rapid qualitative research with diverse people and communities will help to identify ways in which this negative effect might be alleviated. achieving the right balance between infection control and mitigation of these negative socioeconomic effects must be considered. the immediate research priorities are to monitor and report rates of anxiety, depression, self-harm, suicide, and other mental health issues both to understand mechanisms and crucially to inform interventions. this should be adopted across the general population and vulnerable groups, including front-line workers. monitoring must go beyond nhs record linkage to capture the real incidence in the community, because self-harm might become more hidden. we must harness existing datasets and ongoing longitudinal studies, and establish new cohorts with new ways of recording including detailed psychological factors. , techniques assessing moment to moment changes in psychological risk factors should be embraced. given the unique circumstances of covid- , data will be vital to determine causal mechanisms associated with poor mental health, , including loneliness and entrapment. to optimise effectiveness of psychological treatments, they need to be mechanistically informedthat is, targeting factors which are both causally associated with poor mental health and modifiable by an intervention. a one-size-fits-all response will not suffice because the effectiveness of interventions can vary across groups. , [ ] [ ] [ ] [ ] digital psychological interventions that are mechanistically informed, alongside better understanding of the buffering effects of social relationships during stressful events, are required in the long term. the digital response is crucial, [ ] [ ] [ ] not only because of social isolation measures but also because less than a third of people who die by suicide have been in contact with mental health services in the months before death. digital interventions for anxiety, depression, self-harm, and suicide include information provision, connectivity and triage, automated and blended therapeutic interventions (such as apps and online programmes), telephone calls and messages to reach those with poorer digital resources (digital poverty), suicide risk assessments, chatlines and forums, and technologies that can be used to monitor risk either passively or actively. the digital landscape extends beyond apps and requires an evidence base. artificial intelligence-driven adaptive trials could help to evaluate effectiveness, while digital phenotyping could be helpful to ascertain early warning signs for mental ill-health. looking beyond digital interventions (as not everyone has access to them), and ascertaining what other mechanistically based psychological interventions are effective and for whom is important. , risks and buffers for loneliness should be a focal target in interventions to protect wellbeing. the longer-term consequences of covid- for the younger and older generations (and other groups at high risk, including workers, those with existing mental health conditions, and caregivers) are also unknown and must be a priority. how do individuals build optimal structures for a mentally healthy life that works for them in the wake of covid- and social and physical distancing? the optimal structure of a mentally healthy life for individuals in the wake of covid- needs to be mapped out. structure will vary as a function of background and individual circumstances. changes in sleep and lifestyle behaviours influence our mental health and stress response. understanding the effective, individualised ways of coping in such a situation is of paramount importance. [ ] [ ] [ ] the social and personal resources (eg, seeing family and getting sufficient sleep) available to individuals can be important resiliencerelated factors for mitigating mental health difficulties under particularly stressful circumstances. we need what is the effect of covid- on risk of anxiety, depression, and other outcomes, such as self-harm and suicide? improve monitoring and reporting of the rates of anxiety, depression, self-harm, suicide, and other mental health issues; determine the efficacy of mechanistically based digital and non-digital interventions and evaluate optimal model(s) of implementation determine the mechanisms (eg, entrapment and loneliness) that explain the rates of anxiety, depression, self-harm, and suicide; understand the role of psychological factors in buffering the effect of social context on mental health issues; ascertain the longer-term consequences on wellbeing of covid- for the young and older generations (and vulnerable groups) what is the optimal structure for a mentally healthy life in the wake of covid- and social or physical distancing? determine what psychological support is available to help front-line medical and health-care staff and their families; understand the psychological (eg, coping), physiological (eg, sleep and nutrition), and structural (eg, work rotas and daily routines) factors that protect or adversely affect mental health the immediate research priorities are to understand how front-line health and social care staff and their families can be supported to optimise coping strategies to mitigate symptoms of stress, and facilitate the imple mentation of preventive interventions in the future. , during the covid- pandemic, it is important that health and social care workers are supported to stay in work, the health, personal, social, and economic benefits of which are vast. personalised psychological approaches are likely to be a key component to address complex mental health conditions, coping mechanisms, and prevention. given the association between sleep disturbance and mental health, and the effect of sleep disturbance on the risk of suicide, research on mitigating the effect of such changes on mental health and stress response is required. the longer-term strategic research programmes are to develop novel interventions to protect mental wellbeing, including those based on positive mechanistically based components (ie, causal, modifiable factors), such as altruism and prosocial behaviour. this could include increased opportunities to elicit community support, , exercise, social activities, training in assertiveness and conflict resolution, and group interventions that provide support through peers. the inclusion of altruism in uk government health messages has likely had a positive effect on wellbeing compared with compulsory orders to stay at home. key research questions include "what positive mechanistically based psychological interventions can be developed for mental wellbeing derived from theories of altruism and prosocial behaviour?" and "what can be learned from the large-scale roll-out of volunteer-based psychological interventions that will optimise the benefits to individuals and society?" working from home, loss of employment, and social and physical distancing have abruptly interrupted many social opportunities important to physical and psychological health. it is important to research the mental health dimension of online life and investigate how changes in engagement with gaming and online platforms might inform interventions aimed at improving mental health. we must rapidly learn from successful existing strategies to maintain and build social resources and resilience and promote good mental health in specific populations moving forward. population-level factors, such as the effect of social distancing measures (more recently being redescribed as physical distancing) and other necessary public health measures, affect mental health within a syndemics approach (table ) . by syndemics we mean intersecting global trends among demographics (eg, ageing, rising inequality) and health conditions (eg, chronic diseases and obesity) that yield resultant comorbidities. these longer-term strategic programmes what are the mental health consequences of the covid- lockdown and social isolation for vulnerable groups, and how can these be mitigated under pandemic conditions? determine the best ways of signposting and delivering mental health services for vulnerable groups, including online clinics and community support; identify and evaluate outreach methods to support those at risk of abuse within the home; ascertain which evidence-based interventions can be rapidly repurposed at scale for the covid- pandemic, and identify intervention gaps requiring bespoke remotely delivered interventions to boost wellbeing and reduce mental health issues; swiftly provide interventions to promote mental wellbeing in front-line health-care workers exposed to stress and trauma that can be delivered now and at scale on the basis of the intervention gaps identified, design bespoke approaches for population-level interventions targeted at the prevention and treatment of mental health symptoms (eg, anxiety) and at boosting coping and resilience (eg, exercise); develop innovative novel universal interventions on new mechanistically based targets from experimental and social sciences (eg, for loneliness consider befriending) that can help mental health; assess the effectiveness of arts-based and life-skills based interventions and other generative activities including exercise outdoors what is the effect of repeated media consumption about covid- in traditional and social media on mental health, and how can wellbeing be promoted? understand the role of repeated media consumption in amplifying distress and anxiety, and optimal patterns of consumption for wellbeing; develop strategies to prevent over-exposure to anxiety-provoking media, including how to encourage diverse populations to stay informed by authoritative sources they trust; mitigate and manage the effect of viewing distressing footage inform evidence-based media policy around pandemic reporting (eg, clearly identify authoritative sources, encourage companies to correct disinformation, and policies on traumatic footage); mitigate individuals' risk of misinformation (eg, improve health literacy and critical thinking skills and minimise sharing of misinformation); understand and harness positive uses of traditional media, online gaming, and social media platforms what are the best methods for promoting successful adherence to behavioural advice about covid- while enabling mental wellbeing and minimising distress? understand how health messaging can optimise behaviour change, and reduce unintended mental health issues; track perceptions of and responses to public health messages to allow iterative improvements, informed by mental health science synthesise evidence base of lessons learned for future pandemics, tailored to specific groups as required; motivate and enable people to prepare psychologically and plan practically for possible future scenarios; understand the facilitators and barriers for activities that promote good mental health, such as exercise; promote people's care and concern for others, fostering collective solidarity and altruism covid- =coronavirus disease . interacting health effects and societal forces that fuel them combine to form syndemics, or complex knots of health determinants. research priorities around covid- require us to embrace complexity by deploying multidimensional perspectives. what are the mental health consequences of the covid- lockdown and social isolation for vulnerable groups, and how can these be mitigated under pandemic conditions? we do not yet know the acute or long-term con sequences of the covid- lockdown and social isolation on mental health. although worries and uncertainties about a pandemic are common, for some they can cause undue distress and impairment to social and occupational functioning. , , across society, a sense of loss can stem from losing direct social contacts, and also range from loss of loved ones, to loss of employment, educational opportunities, recreation, freedoms, and supports. existing evidence suggests some measures taken to control the pandemic might have a disproportionate effect on those most vulnerable (panel ). vulnerable groups include those with pre-existing mental or physical health issues (including those with severe mental illnesses), recovered individuals, and those who become mentally unwell (eg, in response to anxiety and loneliness surrounding the pandemic; panel ). , , therefore, loss of access to mental health support, alongside loss of positive activities, might increase vulnerability during covid- lockdown. increased feelings of anxiety and depression in response to the outbreak have been highlighted already. health workers who come in close contact with the virus and are exposed to traumatic events, such as death and dying, while making highly challenging decisions, are particularly at risk of stress responses. the pandemic intersects with rising mental health issues in childhood and adolescence. , , ascertaining and mitigating the effects of school closures for youth seeking care is urgent and essential, given that school is often the first place children and adolescents seek help, , as is considering vulnerabilities, such as special educational needs and developmental disorders, and finding therapeutic levers. for the older population, promoting good mental health is important during self-isolation, which can be compounded by lifestyle restrictions, exacerbated loneliness, comorbidities (such as dementia), and feelings of worry and guilt for using resources. there is an acute need to identify, in consultation with people with lived experience, remotely delivered interventions that support those at risk of abuse. , the immediate research priorities are to reduce mental health issues and support wellbeing in vulnerable groups in particular. a coordinating mechanism for pandemic mental health interventions is required for the agile identification of interventions that can be repurposed, alongside the identification of intervention gaps that will require bespoke de novo design, and the evaluation and roll-out of remotely delivered interventions. by the term intervention, we mean interventions of all sorts that make a difference to mental health, including populationlevel policy, occupational guidelines, and psychological interventions. we need to gather high-quality data rapidly to ascertain the effects of lockdown and social isolation over time. innovative research is needed to establish ways to mitigate and manage mental health risks and inform interventions under pandemic conditions. research to support vulnerable groups needs to consider cross-cutting themes (such as the physical absence of schools and clinics) to create methods to provide connectivity and support; promote rapid innovation in mental although the whole population is affected by the coronavirus disease pandemic, specific sections of the population will experience it differently. children, young people, and families will be affected by school closures. they might also be affected by exposure to substance misuse, gambling, domestic violence and child maltreatment, absence of free school meals, accommodation issues and overcrowding, parental employment, and change and disruption of social networks. older adults and those with multimorbidities might be particularly affected by issues including isolation, loneliness, end of life care, and bereavement, which may be exacerbated by the so-called digital divide. people with existing mental health issues, including those with severe mental illnesses, might be particularly affected by relapse, disruptions to services, isolation, the possible exacerbation of symptoms in response to pandemic-related information and behaviours, and changes in mental health law. front-line health-care workers might be affected by fears of contamination, moral injury, disruption of normal supportive structures, work stress, and retention issues. people with learning difficulties and neurodevelopmental disorders might be affected by changes and disruption to support and routines, isolation, and loneliness. society might experience increased social cohesion and communitarianism, but also be negatively affected by increased health inequalities, increased food bank use, increased race-based attacks, and other trauma. rural communities might also be affected differently to urban communities. socially excluded groups, including prisoners, the homeless, and refugees, might require a tailored response. people on low incomes face job and financial insecurity, cramped housing, and poor access to the internet and technology. health services that can be remotely signposted and delivered (including online clinics and community support); identify and evaluate means to support those at risk of abuse within the home (eg, online outreach); and swiftly provide interventions to promote mental wellbeing in front-line health workers. by identifying cross-cutting research themes, interventions to help specific vulnerable populations should be leveraged to help other vulnerable groups. with regard to the longer-term priorities, health services research must reliably and iteratively inform remotely delivered mental health resources, such as digital clinics, to efficiently manage mental health issues in an adaptive and flexible manner. this requires a coordinating mechanism to prioritise and streamline efforts, working with service users to optimise signposting and delivery and define therapeutic targets that matter from a user perspective (eg, loss, loneliness). such a mechanism requires a range of disciplines, including psychology, digital science, and social sciences. , international collaboration will ensure the necessary research skills and expertise. research should harness internet-based social media and gaming using existing platforms and be cognisant of the so-called digital divide, which leaves % of britons without internet access. research for population-level interventions will require rapid evolution of approaches, starting with testing whether existing digital interventions can be repurposed, such as physical activity, sleep, and stress management programmes, as well as targeted approaches for the prevention and treatment of established mental health symptoms (eg, anxiety and worry). , tailoring of such universal interventions will need to be informed by exper imental and social science (eg, for loneliness, befriending, and physical activity). , the effectiveness of arts-based interventions also needs to be assessed as do other generative activities that boost positive coping and resilience throughout society, from community-based activities, to life-skills classes, to exercising outdoors. the effectiveness of all interventions requires rigorous evaluation and implementation to avoid recommending a plethora of apps with no evidence base. interventions at the population level should be repurposed, developed, and tested in a virtuous loop to create the necessary evidence base. what is the effect of repeated media consumption about covid- through traditional media and social media on mental health, and how can wellbeing be promoted? people seek trusted information via the media, which can provide swift, critical guidance regarding the pandemic. media consumption can be adaptive and positive for mental health. however, reports of infectious diseases often use risk-elevating messages, which can amplify public anxiety. social media can be a source of rapidly disseminated misinformation, amplifying perceptions of risk. repeated media exposure to information about an infectious disease particularly can exacerbate stress responses, amplify worry, and impair functioning. anxiety and uncertainty can drive additional media consumption and further distress, creating a cycle that can be difficult to break. media-fuelled distress can promote behaviours that negatively affect the health-care system (eg, visits to emergency departments and hoarding of face masks), with downstream mental and physical health consequences. the immediate research priority is to better understand the role of repeated media consumption around covid- in amplifying distress and mental ill-health in various groups, and the optimal patterns of consumption to promote wellbeing. research is needed to inform future approaches, including strategies to help individuals to stay informed by authoritative sources, prevent overexposure to media, and mitigate and help manage the effect of viewing images with traumatic content. longer-term research priorities should inform evidencebased guidelines for media around pandemic reporting (eg, clearly identifying authoritative sources, limiting graphic footage, and encouraging social media companies to flag or correct disinformation and rumours). research should also help to develop strategies to mitigate an individual's risk of exposure to misinformation and amplification of anxiety by minimising sharing of misinformation, and promoting strategies for managing the emotional consequences. adaptive and positive uses of traditional media and social media, such as influencers, should be understood and harnessed. understanding the effect of pandemic media on various vulnerable groups is essential. behavioural change-such as the three personal protective behaviours of handwashing, not touching the t-zone of the face, and tissue use, and social or physical distancing required to control the pandemicnecessitates ensuring people know what to do, are motivated to do it, and have the skills and opportunity to enact the changed behaviours. , messaging is key for good knowledge, but public health messaging needs to draw on behavioural science if it is to be effective and avoid unintended consequences. we know that the more concerned people are in pandemics, the more likely they are to adhere to advice. however, increasing concern experienced by the public might heighten distress, which could undermine adherence or exacerbate existing mental health issues. anxiety can be fuelled by uncertainty and by fears of risk of harm to self or others. for example, feelings of paranoia can be heavily influenced by anxiety, and symptoms of obsessive compulsive disorder can be associated with fear of contagion and rigid handwashing. increasing people's confidence and clarity in what they need to do fosters position paper adherence to health behaviours, and can help people to manage psychological distress. immediate research on covid- health messaging is urgently required to both optimise health behaviour change and to reduce unintended mental health issues, which will be required in the event of a second wave of infection. research should prioritise message content, format, and delivery modes and behavioural change alongside risk communication, and consider how this might need to vary for diverse groups. a virtuous cycle that tracks perceptions of and responses to public health messages during this pandemic will enable iterative improvements. it must be informed by mental health science to close the knowledge-to-implementation gap (eg, between effective behaviour messages and maladaptive consequences). longer-term research priorities are to create an evidence base of lessons learned to plan for future pandemics-that is, detailing how to foster a rapid and coordinated response regarding health messaging from governments and simultaneously to develop effective systems embedded in communities to reach out and access the most vulnerable groups in our society, including how to motivate and enable people to prepare psychologically and plan practically for possible future scenarios, and how to promote people's care and concern for others, fostering a sense of collective solidarity and altruism. the optimal messaging should be tailored (including digitally) to different social groups to connect diverse segments of the population to appropriate mental health information resources. almost nothing is known with certainty about the effect of sars-cov- infection on the human nervous system. sars-cov- is a zoonotic virus and a review from suggested that about half of zoonotic virus epidemics have been caused by neurotropic viruses that invade the cns. the closely related coronaviruses responsible for the severe acute respiratory syndrome epidemic in and the so-called middle east respiratory syndrome in are biologically neurotropic and clinically neurotoxic, causing mental health and neurological disorders. [ ] [ ] [ ] sars-cov- has a similar receptor-binding domain structure to sars-cov and probably shares its neurotropism and neurotoxicity (panel ). neurological symptoms of covid- infection are common, diverse, and often severe. in a retrospective study of patients in wuhan, china % had cns symptoms or disorders and the subgroup of patients with severe respiratory disease had significantly increased frequency of cns problems ( %). the problems reported include dizziness, head ache, loss of smell (anosmia), loss of taste (ageusia), muscle pain and weakness, impaired consciousness, and cerebrovascular complications. similar reports have begun to emerge from italy. some of these acute neurological presentations could reflect systemic aspects of infection, such as disseminated intravascular coagulation causing strokes or intense inflammation and hypoxia causing delirium. sars-cov- infection of the brain could be a contributor to the core medical syndrome of respiratory distress and failure in patients with covid- . viral infection of the lung alveoli is the immediate cause of severe acute respiratory syndrome; but viral infection of key brainstem nuclei could disrupt the normal rhythms and homoeostatic control of respiration. this idea needs to be tested rapidly because if brainstem infection does contribute to the severity of sars and the need for treatment in an intensive care unit, it could be directly relevant to the immediate covid- crisis in the nhs and other health-care systems. in the longer term, it is possible that sars-cov- will have persistent direct neurotoxic effects and immunemediated neurotoxic effects on the brain. the spanish flu epidemic of - was linked to a spike in incidence of post-encephalitic parkinsonism. currently, it is not known if sars-cov- infection could cause mental health or neurodegenerative disorders immediately or years after the acute respiratory phase of covid- has passed, but action is needed now to build the research capacity to test these potentially important biological causes of covid- -related mental illness. immediate actions include the development of a neuropsychological database of covid- cases to bring together standardised, longitudinally repeated data at scale both from the clinic for those needing hospital facilities for sars-cov- -infected tissue handling need to be expanded to examine human brain tissue post mortem, which is crucial to understanding the neurotropic and neurotoxic properties of the virus. facilities equipped to safely handle human (or animal) brain tissue infected with sars-cov- are currently very few in number. we recommend building pathology and molecular neuroscience networks to enable brain and other tissue to be collected at autopsy and examined for viral infection and damage. this will require protocols for tissue collection and examination in appropriate laboratory facilities to protect researchers and other staff at all times. the longer-term research priorities are to understand the mechanisms by which sars-cov- might enter the brain. there are two conceivable pathways: neuronal or vascular. the neuronal pathway, used by other coronaviruses, , is to invade a specialist sensory receptor in peripheral tissue, travel by the axonal transport systems to the brainstem, and propagate between neurons by transsynaptic mechanisms. it is not known whether sars-cov- can follow the same path to infect the human brain or whether it invades nerve cells by hijacking angiotensin converting enzyme (ace ), - despite neurons expressing low amounts of the protein, as described in a preprint and two other published studies. , alternatively, sars-cov- might invade the brain from the blood, if circulating particles of the virus were transported across the blood-brain barrier by binding to ace receptors expressed by endothelial cells, or if infected leucocytes could carry the virus with them as they migrate into the tissues as part of the immune response to infection. better understanding of how the intense systemic immune response to sars-cov- infection affects mental health and neurological symptoms, , , and of the mechanisms of immune clearance of sars-cov- , is also needed. , post-infectious fatigue and depressive syndromes have been associated with other epidemics, and it seems possible that the same will be true of the covid- pandemic. longitudinal studies, especially if commenced before or soon after the start of the current pandemic, will be crucial in establishing the often complex biological pathways between infection and mental health outcomes. [ ] [ ] [ ] candidate biomarkers need to be evaluated to measure the effects of sars-cov- infection on the human brain and brainstem in living patients, including structural and functional mri, diffusion-weighted mri, quantitative cerebral blood flow imaging, and magnetic resonance spectroscopy. the tesla mri technique has sufficient spatial resolution to measure functional connectivity between subcortical structures that constitute networks for respiratory control and distress. other methods could include sampling cerebrospinal fluid or use of pet to measure brain inflammation; patient self-reporting or behavioural testing of smell, taste, and other cranial or vagal sensory functions; electrophysiological methods to measure brainstem function; and computerised tests of cognitive and emotional processing. informed by greater understanding of the effects of viral infection on the nervous system and by more accurate biomarkers of brain function in patients with covid- , interventions need to be developed to interrupt or prevent the adverse biological effects of sars-cov- on brain function and mental health. potential drug targets include putative mechanisms for neuronal invasion, interneuronal propagation, and immune clearance of sars-cov- . biological and clinical validation of these or other targets would enable experimental medicine studies or early clinical trials of repurposed drugs. for example, the ace inhibitors already licensed for treatment of hypertension, and a licensed drug for reflux oesophagitis, camostat mesylate, that blocks the serine protease tmprss (which operates with ace to facilitate viral entry into cells) have already been advocated as repurposable drugs. there are many other potential candidates for drug repurposing described in a preprint, which could be a faster route to effective treatment for cns infection than development of entirely new drugs or vaccines. partnerships between researchers in academia and industry will be vital. many of the immediate priorities are for surveillance of general and specific populations for effects of sars-cov- infection on health, ranging from health behaviours, psychological symptoms, neuropsychiatric disorders, and mortality, including, but not limited to, suicide. the other immediate priority is to assemble cohorts to determine longer-term outcomes and provide a resource for nesting intervention studies, and a resource of interventions to monitor their effectiveness. we recommend three main routes. for each of these routes, there is a need to coordinate existing research infrastructure through shared protocols, research measures, and data assets, and to uphold the highest standards of scientific and ethical review. we urge the mental health science community to combine agility in initiating new or adapting existing research with collective scrutiny and collaboration. first, administrative data assets principally derived from existing electronic health records, with systems in place to interrogate these for research purposes, provide a means of identifying health effects at scale. for general hospital settings, which provides near realtime information from health records (eg, to provide feedback on neurological consequences of severe covid- ). these systems should be linked between mental health services, acute medical services, and community health services to identify patterns and trends both in clinical populations and in individuals with confirmed or suspected covid- . second, surveillance through recruitment platforms and existing cohorts has the benefit of embedding research on covid- into studies where participants' mental or cognitive health has previously been ascertained. existing cohorts or data platforms that can be rapidly deployed for covid- research are likely to be particularly valuable. examples include the national institute for health research national bioresource, a platform that already includes clinical and genetic data on participants, and could be deployed for rapid characterisation of mental health and neurological symptoms. uk biobank has successfully done a webbased mental health survey of individuals, and the ongoing neuroimaging studies of individuals with some repeat imaging, provide an ideal opportunity to image the effect of sars-cov- infection on the brain and the brainstem via a before-and-after imaging comparison. third, novel population-based studies on mental health and covid- should be established, using appropriate epidemiologically robust survey methodology for both the whole population and specific groups of particular interest (eg, children and young people, front-line staff in health and social care, and people who have survived severe . priority should be given to assembling representive populations using explicit sampling frames. finally, many other disciplines will be establishing similar studies and it is vital that the ascertainment of mental health should be embedded wherever possible. whether using established or new cohorts, priority should be given to methods that can ascertain covid- status, symptoms, and behaviours in as close to real-time as possible, providing a dynamic picture of change in illness status, social circumstances, and behaviours. questions regarding covid- and mental health symptoms and social stressors can readily be disseminated through smartphones. passive data from smartphones can also give high temporal resolution to behaviours related to the pandemic. cohorts should gain permissions for the linkage of records, including serological status, when mass testing becomes available, and consent for recruitment into nested substudies, including randomised trials of interventions. patient and public involvement in research is a critical underpinning component to research. given that the entire population has lived experience of the covid- pandemic, researchers will need to be particularly mindful of consulting and collaborating with patient and public groups that reflect the diverse groups being studied when developing protocols, conducting research, and interpreting results (panel ). multidisciplinary mental health science research must be central to the international response to the covid- researchers must continue to describe the patient group or population and the research question under study. a priori research questions are crucial. sample size, sources of bias, participant characteristics (including sex, age, and ethnicity), and study design need to be carefully considered and must be appropriate to the research questions. research on human participants should maintain high standards of ethical practice, including seeking research ethics committee approval. committees now have fast-track procedures to expedite study start up. ethical considerations for doing coronavirus disease (covid- )-related research have been published. , vulnerable groups researchers should recognise the capacity of the pandemic to exacerbate health inequalities within populations, particularly affecting people with established mental health issues (including severe mental illnesses) and physical disability. those with precarious or no employment or housing, or other forms of social inequality, such as digital poverty, should also be considered. researchers should continue to engage and involve patients, people with lived experience, the public, and service providers in their work by mutually setting research questions, testing the acceptability of protocols and questionnaires, and interpreting results. researchers should ensure that they discuss their research findings with participants. there is an obvious need for researchers to use and share full study protocols and measures, where possible. this will facilitate comparisons between data and projects. the urgency of the research effort should be a strong driver for the principles of open science, reproducibility, and data sharing. the ready availability of analysis code and data is essential to verifying findings. broad adoption of the registered reports publication model, including rapid peer review of study protocols before data collection, will help to minimise waste and ensure conclusions are empirically sound. the challenge of the covid- pandemic requires imaginative collaborations between disciplines, including, but not limited to, psychology, psychiatry, neuroscience, virology, intensive care medicine, and respiratory medicine. previous experience with epidemics has shown the "essential role that the humanities and social sciences play in information, reduction of fear and stigma, prevention, screening, treatment adherence, and control policies". where possible, research protocols should be deployed at scale harnessing existing research infrastructures, including the clinical research networks, biomedical research centres, mental health translational research collaboration, mq data science group, charities, service user groups, and professional bodies. to avoid waste and protect against participant fatigue, it is essential that there is national coordination across research groups. international collaboration and a global perspective would also be beneficial. pandemic, given the potential effects on individual and population mental health, and its potential effect on the brain function of some of those affected by the disease. there are important immediate insights to be gained, which could provide evidence-based guidance on responding to this pandemic and on how to promote mental health and wellbeing, and safeguard the brain, should future waves of infection emerge (panel ). the research priorities across the social, psychological, and neuroscientific aspects of this pandemic should be coordinated at a national and international level. we urge uk research funding agencies to work with researchers, people with lived experience, and others to establish a high-level coordination group to ensure that the mental health science research priorities are addressed swiftly, and that a firm evidence base is established for long-term studies. we need rigorous, peer-reviewed, ethically approved research codeveloped with people with lived experience that can be translated into effective interventions, rather than the current uncoordinated approach with a plethora of underpowered studies and surveys. the immediate priority is the collection of high-quality data on the mental health and psychological effects of the covid- pandemic across the whole population and in specific vulnerable groups, and on brain function, cognition, and mental health for patients with covid- at all clinical stages of infection and illness. these datasets must be brought together under a national data portal for rapid access and use. there is an urgent need for the discovery, evaluation, and refinement of mechanistically driven interventions to address the psychological, social, and neuroscientific aspects of this pandemic. this includes bespoke psychological interventions to boost wellbeing and minimise mental health risks across society, including in vulnerable groups, and experimental medicine studies to validate clinical biomarkers and repurpose new treatments for the potentially neurotoxic effects of the virus. there is an urgent need for research to address the effect of repeated pandemic-related media consumption and to optimise health messaging around covid- . rising to this challenge will require integration across disciplines and sectors, including industry and health and social care. new funding will be required to meet these priorities, and it can be efficiently leveraged by the uk's worldleading neuroscience and mental health research infrastructure. the uk must connect with international funders and researchers to support a global response to the mental health and neurological challenges of this pandemic. in these challenging times, mental health science should be harnessed to serve society and benefit both mental and physical health in the long term. eb, eah, mh, rco'c, vhp, it, and sw contributed to the literature review, conceptualisation, design and interpretation of surveys, and writing and editing of the manuscript as part of the core advisory group. cc contributed to and coordinated the writing and editing of the manuscript. kc analysed the qualitative data gathered via the stakeholder survey. la, cb, hc, rcs, ie, tf, aj, im, sm, akp, rs, cmw, and ly contributed to the drafting and formulation of the manuscript as part of the expert advisory group. tk, kk, and as contributed to the drafting and formulation of the manuscript as part of the expert advisory group and by including lived-experience expertise. all authors approved the final version for submission. cb reports grants and personal fees from acadia and lundbeck; personal fees from roche, otsuka, biogen, eli lilly, novo nordisk, aarp, and exciva; and grants from synexus, outside the submitted work. eah reports serving on the board of trustees of the charity mq: transforming mental health and as chair of the research committee, but receives no remuneration for these roles. eah receives royalties from books and occasional fees for workshops and invited addresses; receives occasional consultancy fees from the swedish agency for health technology assessment and assessment of social services; and reports grants from the oak foundation, the lupina foundation, and the swedish research council. rco'c is a member of the national institute of health and care excellence's guideline development group for the management of selfharm; is co-chair of the academic advisory group to the scottish government's national suicide prevention leadership group; receives royalties from books, and occasional fees for workshops and invited addresses; and reports grants from medical research foundation, the mindstep foundation, chief scientist office, medical research council, nhs health scotland, scottish government, and national institute for health research (nihr). kk has received meeting attendance payments from the department of health and social care, nhs england and nhs improvement, and the royal college of psychiatry (rcpsych) over the last year for service user representative work, and payment for a training session she facilitated for rcpsych; and received a pass and accommodation for the rcpsych annual conference in . akp reports financial support from uk taxpayers, the uk's economic and social research council, the british academy, the diana award, the john fell fund, the leverhulme trust, barnardo's uk, and the huo family foundation in the past five years. as part of science communication and policy outreach activities; and served in an unpaid advisory capacity to the organization for economic co-operation and development, facebook, google, and the parentzone. it is a trustee of mq: transforming mental the stakeholder survey was funded by mq: transforming mental health. activity costs for this work, 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ethical considerations in responding to the covid- pandemic what's next for registered reports? toward a global health approach: lessons from the hiv and ebola epidemics we are grateful to all staff at the academy of medical sciences and mq: transforming mental health for their work in coordinating and supporting this project's secretariat and communications. special thanks to rachel quinn, nick hillier, helen munn, neil balmer, angeliki yiangou, fern brookes, holly rogers, claire bithell, naomi clarke, melanie etherton, tom livermore, dylan williams, and daisy armitage. we also extend our sincere thanks to katie white, carolin oetzmann, valeria de angel, and sumithra velupillai at king's college london, and norman freshney from norman freshney consulting, for their tremendous efforts in data analysis, and beau gamble at uppsala university and seonaid cleare at university of glasgow for their support with referencing. we are also grateful to the team at ipsos mori for their work on the online omnibus. special thanks also to everyone who participated in the mq: transforming mental health and ipsos mori surveys for sharing their views and personal experiences during challenging times-we are hugely grateful to them for their openness and honesty about mental health and wellbeing. we are grateful to peter jones from the university of cambridge and a member of the mq: transforming mental health board of trustees for comments on an earlier draft. key: cord- -mehijkzo authors: guo, shuaijun; yu, xiaoming; okan, orkan title: moving health literacy research and practice towards a vision of equity, precision and transparency date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: mehijkzo over the past two decades, health literacy research has gained increasing attention in global health initiatives to reduce health disparities. while it is well-documented that health literacy is associated with health outcomes, most findings are generated from cross-sectional data. along with the increasing importance of health literacy in policy, there is a lack of specificity and transparency about how to improve health literacy in practice. in this study, we are calling for a shift of current research paradigms from judging health literacy levels towards observing how health literacy skills are developed over the life course and practised in the real world. this includes using a life-course approach, integrating the rationale of precision public health, applying open science practice, and promoting actionable knowledge translation strategies. we show how a greater appreciation for these paradigms promises to advance health literacy research and practice towards an equitable, precise, transparent, and actionable vision. health literacy underpins everyday health behaviours and health-related decisions. defined as an individual's ability to find, understand, and use health information to promote and maintain good health [ , ] , the term "health literacy" has been widely used in healthcare, disease prevention, and health promotion since the s [ ] . health literacy is a context-and content-specific concept; this means that researchers need to define and measure it within a specific context for a particular purpose [ ] . from a public health perspective, health literacy is regarded as a personal asset that evolves over the life course and promotes empowerment in health decision-making [ ] [ ] [ ] . in the context of the coronavirus disease of (covid- ) , an individual's health literacy supports his/her decisions on washing hands, maintaining physical distance, adopting protective behaviours, seeing a doctor, and complying with quarantine policies, thus contributing to a more likely successful public health response strategy [ ] [ ] [ ] . health literacy also helps to navigate the infodemic-the overabundance of valid and invalid information that is circulating on the internet-that is attached to the covid- pandemic [ , ] . low health literacy is a global public health concern. internationally, it is estimated that . % to . % of adults have low health literacy [ , ] , costing national governments at least $ billion annually [ ] . mounting evidence suggests that low health literacy is associated with adverse health outcomes [ ] [ ] [ ] , including frequent use of emergency care, prolonged hospital stays, and high mortality rates, which in turn lead to health disparities [ ] . national and international health programs have shown promising outcomes (e.g., improved health knowledge, healthier behaviours, self-management of chronic illness, access to healthcare) when intervening to improve health literacy [ ] [ ] [ ] . most recently, the world health organization's shanghai declaration on promoting health in the agenda for sustainable development highlighted health literacy as an integral part of the skills developed over a lifetime and recognized it as a critical driver of achieving an equitable world [ , ] . enhancing health literacy requires a systems approach to understanding its risk factors and its impact on health outcomes [ , , ] . the social determinants framework suggests that health literacy is an interactive product of an individual's health literacy skills and the broad environment and culture [ , ] . empirical studies show that health literacy levels differ substantially across age groups and countries. based on the demographic and health surveys, mcclintock et al. [ ] found that the prevalence of poor health literacy among respondents aged - years ranged from . % in namibia to . % in niger across sub-saharan countries. as for children and adolescents, the health behaviour in school-aged children study shows that, in countries (e.g., austria, england, finland), a total of . % of participants have low levels of health literacy, ranging from . % to . % across countries [ ] . there is a social gradient in health literacy for children [ ] , adolescents [ ] , and adults [ ] . the lower the socioeconomic status an individual has, the lower the health literacy level is likely to be. health literacy can affect health outcomes at each life stage. prior to childbirth, low health literacy in pregnant mothers has a significant impact on the health and development of their offspring, including prematurity, infancy death, and child vaccination participation [ ] . low health literacy in children and adolescents is associated with poor health behaviours, such as smoking, alcohol use, and obesity [ ] [ ] [ ] [ ] . when children and adolescents transit into adulthood and older age, health literacy is closely linked with healthcare outcomes, such as prolonged hospitalization and poor medication adherence [ , ] . while health literacy research has gained momentum in the global context [ , ] , it is predominated by cross-sectional studies, with less than % of all published papers focusing on health literacy interventions, including randomized controlled trials [ , ] . unlike time-series data, cross-sectional data make it impossible to make a valid statement regarding the change. health literacy is a life-course personal asset [ ] , which progresses as a child grows up with different characteristics and health needs at each life stage [ , ] . for instance, children's and adolescents' health literacy rely heavily on their developmental ability and their parents and peers [ ] . when they transition into adulthood, they become more independent in making their own decisions in healthcare, disease prevention, and health promotion [ ] . as cognitive function declines with age, older adults are an especially vulnerable group, with low self-management ability for everyday health-related decisions [ , , ] . currently, there is a lack of holistic ways to look at the impact of health literacy over the life-course due to a lack of longitudinal studies. there is promising evidence showing the effectiveness of health literacy interventions on health outcomes at the individual and community level [ , [ ] [ ] [ ] . however, there remain substantial gaps. in practice, health literacy interventions vary in terms of their study designs, measurement tools, and types of outcome measures [ , ] . besides, there is a lack of specificity in the intervention targets (e.g., individual level, organizational level, community level), content (e.g., functional health literacy, interactive health literacy, critical health literacy), timing (e.g., antenatal, preschool, adolescence), and formats (e.g., universal, intensive, low-threshold). it remains unclear about which interventions are the most effective in improving health literacy, related health behaviours, and associated health outcomes. when translating health literacy evidence into practice, researchers should design interventions that are specifically tailored to people with different health literacy levels and needs [ , ] . there is a need to use precise and transparent approaches to improving health literacy and reducing health inequities in the end. in response to low health literacy levels in the population, many countries have developed national action plans to strengthen health literacy for achieving sustainable development and health equity (e.g., the national actional plan to improve health literacy in the usa [ ] , the national statement on health literacy in australia [ ] , the national action plan health literacy in germany [ ] ). common features in these policy documents include a response to perceived deficiencies in health literacy, the importance of professional education in improving the quality of communication, and a need for responsive healthcare systems [ , ] . policy responses to health literacy are important public statements of priorities by governments and provide a mechanism for public accountability [ , ] . however, in contrast to the increasing number of evidence generated from empirical studies, discussions on the knowledge translation and implementation process are scarce. there remains a lack of specificity in the implementation process and monitoring systems for progress. this perspective is a proposition for four new research paradigms to address the aforementioned knowledge gaps, expecting to move health literacy research and practice towards an equitable, precise, transparent, and actionable vision. this includes using a life-course approach to health literacy [ ] , integrating the rationale of precision public health [ ] , applying open science practice [ ] , and promoting actionable knowledge translation strategies [ ] . in what follows, we will discuss the life-course approach to health literacy as a starting point, and then the necessity of integrating the rationale of precision public health. we are calling for a shift of current research paradigms from judging health literacy levels (low versus high) towards observing how health literacy skills are practised and developed over the life-course. based on these new paradigms, we expect a nuanced understanding of how health literacy develops over the life-course, how it influences health behaviour and decision-making, and thus how it informs specific interventional opportunities-especially in the early life stages across educational and healthcare settings-for a precise policy recommendation. we also highlight the importance of applying open science and considering knowledge translation strategies from the beginning of research planning to generate or replicate policy-relevant findings rapidly and cost-effectively across different cultural contexts, and thus facilitate the process of knowledge dissemination. we need to extend the current concept of "health literacy" from cross-sectional to longitudinal studies. health literacy is a personal asset that develops dynamically over time [ ] . a life-course approach to health literacy will assist researchers in discovering opportunities for optimizing health development and reducing health inequities, and explaining how health practices and policies can go beyond the avoidance of disease to the promotion of health at the early life stages [ , , ] . as shown in figure , we recognize potential intervention levers (both upstream and downstream) for giving all children the best start to life. a life-course approach to health literacy aligns with national and international health initiatives that aim to reduce inequities (e.g., the national action plan for children and young people in australia [ ] ). a life-course approach is well-recognized in public health research and practice to close the gap in health inequities [ , ] . using life-course data from the wisconsin longitudinal study - , clouston et al. [ ] found that life-course predictors of health literacy among older adults included parental educational attainment, and adolescent cognitive and non-cognitive skills. findings from this life-course analysis add to our understanding of how health literacy might change over time through adolescent cognitive and non-cognitive skills. depending on the research purpose and available data sources, researchers could also propose other specific research questions using one of the life-course models exemplified in table . for example, early life represents a sensitive period of health and development. exposure to stressors associated with disadvantages during this time can exert adverse effects on health over the life course [ ] . using the sensitive period model, researchers can examine and compare the effect of parental health literacy on children's health behaviours and health outcomes at different ages of children (e.g., pregnancy, infancy, toddler age). a life-course approach to health literacy (hl) and its impact on health and social outcomes. table . applying life-course models to health literacy research. the sensitive period model to examine timing effects in which exposures during sensitive periods of development have stronger effects on health, social, emotional, and cognitive development outcomes than they would have at other life stages [ ] . • to examine and compare the effect of parental health literacy during pregnancy and infancy on infant and child health outcomes. • to examine and compare the different timing effects of risk or protective factors (e.g., socioeconomic status) in early years on health literacy in later years. the accumulation model to examine the role of persistent advantage or disadvantage over time-in both specific life stages and over life stages-on health and development [ ] . • to examine the role of persistent advantage or disadvantage (e.g., socioeconomic status, ethnic minorities) on health literacy in a specific life stage and over the life course. to examine the effect of persistent high or low health literacy (e.g., using the growth-based trajectory modelling method) on health outcomes over the life course. the pathway model to examine the pathway effects whereby early experiences set in motion a chain of events that put individuals on paths differentiated by types and levels of exposures to social and biological factors [ ] . • to examine the mediating role of health literacy (e.g., adolescent health literacy) in the relationship between socioeconomic disadvantage and health outcomes. the social mobility model to examine the unique importance of social mobility in explaining the early-life and later-life socioeconomic status and health link [ ] . • to examine whether the effect of later-life exposure (e.g., socioeconomic status, immigration status) on health literacy is stronger than the effect of early-life exposure. we are entering an era of "big data" and "precision". big data has enabled extensive and specific research and trials of stratifying and segmenting populations at risk for a variety of health problems, including poor health literacy [ ] . in the field of big data and public health, machine learning is a fundamental component of data analytics that provides data-driven insights, decisions, and predictions [ , ] . machine learning techniques have been broadly adopted for researchers to answer a series of public health research questions (e.g., identifying leading dietary determinants in children [ ] , predicting the development of type diabetes [ ] ). using different machine learning approaches, researchers can also address health literacy research questions, such as identifying elderly people at high risk of low health literacy. particularly, the breadth of longitudinal data available in existing cohorts enables researchers to generate policy-relevant findings quickly [ ] . similar to the precision medicine initiative of providing the right treatment to the right patient at the right time [ ] , a precision public health approach to health literacy calls for harnessing the power of resourceful life-course data to inform the right intervention to the right population at the right time [ , ] . in the context of covid- [ ] , precision public health is particularly useful to design targeted interventions for populations by person, place, and time to promote better navigation of health care and disease prevention [ ] . if a population has a higher proportion of persons with low health literacy, public messages could be provided to educate persons on where to obtain trustworthy information and when to seek health professionals [ ] . integrating the rationale of precision public health aligns with the relation-and context-specific nature of health literacy [ , ] . currently, there is a lack of specificity to inform clear health literacy policy decisions [ ] . figure shows that there are substantial opportunities for researchers to generate specific recommendations between personal and social determinants and health literacy (i.e., upstream intervention levers), and between health literacy and health and social outcomes (i.e., downstream intervention levers). for example, the education sector is a critical platform for health literacy interventions, and education for health literacy is a fundamental process and outcome across the life course [ ] . precision evidence is needed, such as at which time point, at what dosage, and which delivery approach is likely to have the most significant impact on improving population health literacy and reducing health inequities. we need to identify precise policy levers (either upstream or downstream) and build an evidence base with sufficient specificity to generate actionable policy implications. open science refers to a range of practices that promote transparency, openness, and reproducibility in research [ ] . efforts to reproduce published findings have yielded a concerningly high failure rate (e.g., only % replicated in nature and science [ ] ) [ , ] . in response to concerns about this "reproducibility crisis", the open science practice has been increasingly recognized across disciplines [ ] . [ ] . however, in practice, null results are less frequently published than statistically significant results and are more likely to be inaccessible and lost in the "file drawer" [ ] . to reduce publication bias, we need to move the current evidence of health literacy from an era of "publish or perish" to "visible or vanish" [ ] . transparency, openness, and reproducibility are central principles of open science practice [ ] . examples of open science practice include a preregistered report, detailed analytic plan, and publicly shared coding via the open science framework (table ) [ , ] . a future vision for health literacy research is to increase its clarity, credibility, and transparency, which can help to provide reliable evidence that can serve as a basis for making decisions about clinical or population-health interventions [ ] . for example, the health literacy tool shed is an online, publicly accessible database of health literacy measures [ ] . currently, more than measurement tools are available. healthcare providers and researchers can search and select the most appropriate instrument according to a specific research purpose [ ] . adoption of open science practice in health literacy research is effective to replicate studies across different cultural contexts. it also provides researchers with a system structure in documenting their work and improving workflows, and offers a path to publication irrespective of the null conclusions [ , ] . knowledge translation is the exchange, synthesis, and ethically sound application of research findings within a complex set of interactions among researchers and knowledge users [ ] . while a number of knowledge translation frameworks has been developed for researchers [ ] , there is a well-known gap between research and practice [ , ] . it is estimated that it takes years for just % of medical research to be implemented [ , ] . this is the same case in the field of health literacy [ , ] . while the importance of health literacy is increasingly recognized in national and global health initiatives [ , , ] , there is still a long way to go when applying health literacy into current practice [ , ] . the evidence synthesis shows that, of the existing and developing health literacy policies in european regions, the main barriers influencing the successful implementation of health literacy policy include cultural barriers, budget restrictions, and the difficulty obtaining high-quality evidence. besides, there is also a lack of engagement in policy evaluation by the academic community [ ] . translating the best available research evidence into evidence-based practice and policy is a complex process which confronts multiple barriers at the individual, organizational, and political level [ ] . there has been a range of efforts to reduce these barriers. for example, the optimizing health literacy and access (ophelia) is a whole-of-system approach to developing and implementing health literacy research [ ] . this approach is widely accepted in high-income and low-and middle-income countries, and uses health literacy profiling and community engagement to create and implement health reforms, thus improving health and equity [ ] . the ophelia approach has also been adapted for different populations and contexts, such as the healthlit kids [ ] . another well-established whole-of-system approach is organizational health literacy, which is widespread in north america and europe [ ] . organizational health literacy is based on the assumption that health literacy is a relational concept in which not only individual skills must be addressed, as well as system-level complexities. this concept has also been used in the helit-schools project, interlinking the organizational health literacy as applied to the school setting with the who health-promoting school framework [ , ] . there are four main benefits if the above research paradigms are applied in current health literacy research and practice. first, we can monitor and evaluate population health literacy levels over time by implementing routine data collection. this allows us to look at health literacy levels among different age groups as well as vulnerable groups, such as those from different ethnic minorities, backgrounds, and migrants, children and young people, chronically ill, and older people. we can also examine the protective and risk factors of health literacy and its impact on health outcomes from a longer-term perspective, thus informing policy opportunities at the best time. second, we can investigate a specific research question about health literacy from a precision public health perspective. we can use modern epidemiological methods, such as causal inference to explore the ideal time point to intervene in low health literacy of a specific population [ , ] . when a randomized trial is not available, we can use the emulated target trial to investigate the causal effect of improving health literacy on a specific health outcome [ ] . a most valuable approach to better understanding real-life health literacy is to focus on ethnographic research exploring the social practices when health information and knowledge are the action focus [ ] [ ] [ ] . third, through the open science movement initiative, it is cost-effective and time-efficient to measure, collect, and analyze health literacy data via existing or linked datasets. for example, the covid health literacy consortium (covid-hl) is a timely project in the context of covid- [ , ] . covid-hl aims to establish a global network on digital health literacy and increase global awareness on health literacy as a critical tool to help protect from communicable diseases. this international platform makes it possible for a health literacy comparison across countries and enables collaborations and data access for researchers. further examples include the who action network on measuring population and organizational health literacy (m-pohl), which aims at the routine measurement of different types of health literacy in the european adult population [ ] . fourth, the knowledge translation and engagement process moves the generated health literacy evidence towards the real world. knowledge end-users, such as policymakers or parents of young children, can benefit from interaction with researchers through reflections on their own daily activities, enhanced health knowledge, and skills to protect health. researchers can also benefit as they gain a nuanced understanding of the practice and policy environment, and develop health literacy research questions that have real-world applicability and benefits [ ] . there are also several challenges. first, health literacy measurement is a complex phenomenon across the life stage, even at a particular time point. the assessment of health literacy varies depending on the setting, research purpose, and the scope of health literacy definitions [ , ] . given that different age groups have different characteristics and health needs, researchers may consider using a core measurement tool plus a variety of add-on modules that target varying age groups [ ] . eventually, this will also make the measurement much more complex and time-consuming. second, it is complicated for life-course data planning and analysis using modern epidemiological methods. dropouts, missing data, and other study deviations (e.g., low response rates) are a common occurrence in both population research and clinical studies [ ] . it is important to consider the power analysis strategies to estimate the sample size, thus enabling researchers to detect a significant effect of health literacy on the outcome of interest. researchers also need to consider critical questions commonly encountered in longitudinal data analysis, such as confounding bias, selection bias, measurement bias, and whether to include an interaction term in a parametric model [ ] . in this case, informed by expert knowledge, researchers can use the directed acyclic graph [ ] to visually represent the hypothesized causal pathway from health literacy to a specific health outcome. the lifecourse analysis plan template is another useful toolkit that can strengthen the quality of observational epidemiological studies [ ] . as for big data in public health, while it provides opportunities to make causality inferences based on chains of sequence, it also introduces challenges to machine learning, such as high data dimensionality, model scalability, and distributed computing [ ] . third, using open science practice in health literacy often requires more time and effort for archiving, documenting, quality controlling of codes, and data security [ ] . open science is changing how research and practice are conducted, and it takes time to consolidate in the mainstream [ ] . currently, the majority of open-source datasets do not adhere to data principles, such as being findable and accessible [ ] . besides, data access and sharing are recurring challenges attributed not only to privacy concerns, but also ambiguous data ownership and unaligned incentives [ ] . there is a need for researchers to adhere to principles of research partnership and data governance models to prevent the breaches of privacy that obstruct ethically justified data access. fourth, knowledge translation barriers are common in practice, especially in the context of covid- [ ] . for example, when disseminating health literacy information in a multi-cultural setting, how could we engage with culturally and linguistically diverse families for the first time and get them to understand the right information? along with challenges related to information overload and an ongoing infodemic [ , ] , researchers and policymakers should be aware of the main facilitators that drive successful health literacy policy implementation, such as intersectoral working, political leadership, and overcoming cultural barriers [ ] . in addition, specific knowledge translation plans are needed in advance when implementing relevant strategies in the real world [ , , ] . there remains much work to be conducted to understand how to implement health literacy evidence into practice. when applying the above paradigms into practice, researchers need to be aware that we are not calling for a "one size fits all" solution to fill the gaps in current research. instead, we are calling for a more equitable, precise, transparent, and actionable way to advance health literacy in research and practice. the four paradigms mentioned above cover a broad range of considerations, ranging from a theoretical approach for individual research to empirical studies generating information using big data for policymaking. researchers can integrate one or two into their research planning and implementation. for example, when a researcher is exploring personal experiences of health literacy at the micro levels, it is more suitable to consider using open science practice and knowledge translation strategies to enhance the rigour of reporting studies and disseminate research findings to a range of stakeholders. health literacy is a crucial driver to health equity. while the evidence base shows a significant impact of health literacy on health outcomes, we need to move this field towards an equitable, precise, transparent, and actionable vision. a life-course approach to health literacy will allow for a better understanding of the mechanisms linking health literacy to health outcomes. a precision public health rationale corresponds with the specific nature of health literacy, and will enable us to generate specific policy recommendations to improve population health. open science practice will assist with minimizing publication bias and motivating researchers to share resources to produce more reliable and cost-effective evidence. finally, actionable knowledge translation strategies will bridge the gap between the academic world and the real world, leading 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hickman, d.l.; johnson, j.; vemulapalli, t.h.; crisler, j.r.; shepherd, r. title: commonly used animal models date: - - journal: principles of animal research for graduate and undergraduate students doi: . /b - - - - . - sha: doc_id: cord_uid: oovkoiyj this chapter provides an introduction to animals that are commonly used for research. it presents information on basic care topics such as biology, behavior, housing, feeding, sexing, and breeding of these animals. the chapter provides some insight into the reasons why these animals are used in research. it also gives an overview of techniques that can be utilized to collect blood or to administer drugs or medicine. each section concludes with a brief description of how to recognize abnormal signs, in addition to lists of various diseases. the mouse is a small mammal that belongs to the order rodentia ( fig. . ). the house mouse of north america and europe, mus musculus, is the species most commonly used for biomedical research. it is likely that the mouse originated in eurasia and utilized its commensal relationship with humans to spread through to other continents as humans explored and colonized. mouse fanciers around the turn of the th century are the source of the majority of the laboratory mice that are in use today. a summary of the overarching categories of mouse models that are available is presented in table . . physiology; and the possibility for breeding genetically manipulated mice and mice that have spontaneous mutations. mice have been used as research subjects for studies ranging from biology to psychology to engineering. they are used to model human diseases for the purpose of finding treatments or cures. some of the diseases they model include: hypertension, diabetes, cataracts, obesity, seizures, respiratory problems, deafness, parkinson's disease, alzheimer's disease, various cancers, cystic fibrosis, and acquired immunodeficiency syndrome (aids), heart disease, muscular dystrophy, and spinal cord injuries. mice are also used in behavioral, sensory, aging, nutrition, and genetic studies. this list is in no way complete as geneticists, biologists, and other scientists are rapidly finding new uses for the domestic mouse in research. mice are mammals and their organ systems are very similar to organ systems in humans in terms of shape, structure, and physiology. basic physiologic data are presented in table . . mice have very long loops of henle in the kidneys, thus allowing for maximal concentration of their urine. as a result, urine output in mice usually consists of only a drop or two of highly concentrated urine at a time. they also excrete large amounts of protein in their urine with sexually mature male mice excreting the largest levels of protein possibly as pheromones. mice have only two types of teeth, incisors and molars. the incisors are openrooted and erupt (i.e., grow) continuously throughout their lives. this predisposes mice to malocclusion if not given feeds or objects such as nylon bones to help wear down the teeth during mastication. the molars are rooted and, thus, do not continuously erupt. the stomach has two compartments with the proximal portion completely keratinized and the distal portion entirely glandular. their intestines are simple, but the rectum is very short ( e mm) and hence is prone to prolapse, especially if the animal has colitis. the gastrointestinal flora consists of more than species of bacteria that form a complex ecosystem that aids digestion and health of the mouse. mice have no sweat glands but have a relatively large surface area per gram of body weight. this results in dramatic changes in physiology and behavior in response to fluctuations in ambient temperature. when too cold, mice will respond by nonshivering thermogenesis (i.e., metabolism of brown adipose tissue). in addition to the lack of sweat glands, they cannot pant or produce large amounts of saliva to aid in cooling their body temperature. therefore, when exposed to very hot situations, mice increase the blood flow to their ears to maximize heat loss; and in the wild, they move into their burrows which are at cooler temperatures. the thermoneutral zone, the range of ambient temperatures at which the mouse does not have to perform regulatory changes in metabolic heat production or evaporative heat loss to maintain its core temperature, is about . fe . f ( . ce . c). the female reproductive system is comprised of paired ovaries and oviducts, uterus, cervix, vagina, clitoris, and paired clitoral glands. pregnant female mice have hemochorial placentation, similar to humans (i.e., maternal blood is in direct contact with the chorion, the outermost layer of the fetal placental membranes). the female mouse also has five pairs of mammary glands. the male reproductive system consists of paired testes, penis, and associated sexual ducts and glands. the inguinal canals are open in the male mouse, and the testes can retract easily into the abdominal cavity. both sexes have well-developed preputial glands, which can become infected. males have a number of accessory sex glands, including large seminal vesicles, coagulating glands, and a prostate. secretions from these glands make up a large part of the mouse's ejaculate. when mice ejaculate, the semen forms a coagulum or copulatory plug. mice breed continuously throughout the year unless conditions are very unfavorable to them (e.g., lack of food). their reproductive potential can be affected by a number of external influences such as noise, diet, light cycles, population density, or cage environment. genotype also can affect reproductive performance as it is common knowledge that some inbred strains of mice are poor breeders, and if pups are born they may receive poor maternal care. additional reproductive physiologic data are presented in table . . mice can be bred using a one-on-one system (one male to one female; monogamous) or in a harem mating system (polygamous mating). in a monogamous system, the male and female are always left together, but at weaning the pups are removed from the cage. this system allows for maximal use of the postpartum estrus and the maximum number of litters for the females involved, and facilitates recordkeeping and monitoring of specific breeders in the colony. in a harem mating system, multiple males are placed with multiple females, usually at a ratio of one male to two to six females. usually, females are removed to separate cages just before parturition and the postpartum estrus is underutilized. mouse pups are born hairless, blind, and deaf and require extensive parental care that is provided mainly by the mother. due to the ruddy coloration of the skin of the hairless pups, they are also known as "pinkies." while mouse pups can increase their body temperature through the metabolism of brown fat stores, they are unable to adequately conserve body heat until they develop an adequate fur coat. thus, inclusion of nesting materials in the cage is highly recommended as huddling inside the nest can provide much needed warmth and safeguard against temperature-associated neonatal losses. the most reliable method for determining the sex of a mouse is by measuring the length of the anogenital distance, i.e., the distance from the anus to the genitalia. this distance can be measured with a ruler or animals assessed side by side with the rear ends held up by their tails. the anogenital distance is longer in males than females. in sexually mature animals one can also determine the sex of mice by the presence or absence of testicles in a testicular sac. mice are social creatures and can be group housed easily. their main method of communication is via pheromones. they use these olfactory cues to establish a pecking order (i.e., a hierarchical system of social organization). these chemicals are so important that when cage environments are changed, such as by simple cleaning or with bedding changes, a bout of fighting may occur until scent marking of the cage is completed as a way to reestablish the pecking order and social organization in that cage. pheromones also play a vital role in reproduction of these animals. this is demonstrated by the whitten effect and the bruce effect. the whitten effect occurs when a group of female mice that are not cycling are exposed to male urine, which contains a large quantity of pheromones. the females will all resume cycling as a group soon after the introduction of the male. in contrast, the bruce effect is characterized by abortion of litters when pregnant females are exposed to the urine of a strange male. as with most rodents, mice are nocturnal animals exhibiting peak levels of activity at night. because mice are a prey species, they display thigmotactic behavior or wall hugging. they avoid open spaces where they might be easily caught by predators. despite this, mice are very curious about any new objects in their territory and will often examine them at length. mice not only have poorly developed eyesight but they are also color blind. a number of inbred strains (e.g., fvb/n and c h/he) are functionally blind by weaning. they rely on their very sensitive hearing to escape detection and on their sense of smell and taste to detect food (and possibly avoid poisons). mice can hear over a range of frequencies between . and khz; however, normal mice are most sensitive to frequencies of e khz. it is important to note here that some inbred strains of mice, e.g., c bl/ , suffer significant hearing loss before year of age. mice can climb, swim, and jump (up to a foot), though they normally prefer to avoid swimming, if possible. under certain conditions they display stereotypies, which are obsessiveecompulsive behaviors. the behaviors may be strain-related, environment-related, or study-related and include wire gnawing, circling, jumping, and aggression. the use of environmental enrichment items such as cardboard tubes or other structures offer the animal an area for retreat from cage mates and add complexity to the environment. aggression is another important behavior that commonly occurs in group-housed male mice. it can also occur in group-housed females and mixed-sex cages. indications that there is an aggressive animal in the cage include bite wounds on the tail, rump, ears, and shoulders of mice ( fig. . ). the wounds can be so severe as to cause significant blood loss and abscess formation at bite sites. aggression has been shown to be influenced by strain, age, and prior encounters. in terms of strains, the more aggressive strains are balb/c, c bl/ , c bl/ , dba/ , and outbred swiss. methods to prevent or reduce aggression include use of properly designed enrichment devices; provision of adequate space and shelter for each animal; grouping of mice before they reach puberty; use of docile strains; and removal of dominant animals as soon as possible. a common manifestation of social organization in group-housed mice is barbering, a behavior in which a dominant mouse will trim, by chewing, the hair or whiskers of other mice in the cage. barbering is also sometimes referred to as the dalila effect. it is usually instigated by a dominant male or female mouse. the dominant animals retain their whiskers and full hair coats, while their cage mates have "shaved faces and bodies" (fig. . ) . although barbering does not generally result in any physical harm to the animal, removing the dominant mouse (the nonbarbered one) from the cage is a good approach to control. general types of housing mice in a laboratory setting include: conventional, specific pathogen free (spf), and germ free. in conventional housing, no attempt is made to keep out adventitious microbial and parasitic organisms. mice housed in this manner can be found in open-topped cages. room air, along with any airborne contaminants, is allowed to freely circulate into the mouse's cage. in addition, the food and water are not sterilized, though it should be noted that microbial contaminants may enter into the mouse population in this way. spf mice are raised in barrier conditions to ensure that they remain free of a specific list of pathogens. care is taken to ensure that adventitious microbes and parasites are excluded from the animals. spf mice are typically raised in specialized caging such as microisolator cages. these cages contain a . mm filter top that aids in the exclusion of microbes and parasites. individually ventilated caging systems include a rack of microisolator cages, each of which receives a filtered air supply ( fig. . ) . under spf conditions, everything that comes into contact with the animal should be sterilized or disinfected. this includes, but is not limited to, the water, food, bedding, and caging. special care must be taken by anyone handling the mice, including researchers, to ensure that handling and experimental procedures do not introduce potential pathogens into the colony. thus, all handling and procedures done on spf mice are often performed under hepa-filtered air conditions, such as within a biosafety cabinet. placing the mouse in an unfiltered environment ("room" air), even for a moment, is enough to potentially colonize the mouse with a whole host of adventitious microorganisms, thus destroying its spf status. once a contaminated mouse is placed back into the colony, the entire colony is at risk for infection. germ-free, or axenic, mice are raised to contain no microbes or parasites whatsoever. raising germ-free mice requires strict barrier maintenance. usually, this requires the use of flexible film isolators which provide hepa-filtered air to the mice within the isolator. additionally, any materials (e.g., food, water, and bedding) must be sterilized or thoroughly disinfected prior to being moved into the isolator unit so as not to contaminate the living space of the germ-free rodents with adventitious microorganisms. all procedures performed within the flexible film isolator must utilize strict aseptic technique for the same reason. in addition to the microbiological environment of the animal's housing systems, mice need to be housed at specific environmental parameters otherwise they may experience stress. the guide for the care and use of laboratory animals, th edition (national research council, ) is an internationally accepted document that outlines and discusses globally accepted environmental parameters for housing different species of animals including the mouse. table . outlines the specific environmental requirements listed in this document for housing mice. mice are omnivorous and coprophagic with at least one-third of their diet being the consumption of their feces. in the laboratory setting mice are fed a clean, wholesome, and nutritious pelleted rodent diet ad lib. there are many commercially formulated diets for the various stages of life and for animals with specific induced diseases such as diabetes mellitus or hypertension. these diets may be available as autoclavable or irradiated forms to prevent transmission of disease via contaminated feed. there are also a variety of "pet" treats available for mice. however, the treats should not make up more than e % of the daily diet. mice should be provided with a continuous supply of water daily. if the animals do not get enough water daily, their food consumption will decrease. the animals will also look scruffy and unhealthy. mice can be provided with water from water bottles or pouches, automatic watering systems with nipples, or water-based gel packs. some general signs of ill health include: weight loss, depression or lethargy, anorexia, obesity, diarrhea, scruffiness or ruffled coats, abnormal breathing, sneezing, weakness, dehydration, enlarged abdomen, discolorations (e.g., yellow for jaundiced animals or very pale for anemic animals), masses or swellings, and abnormal posture or gait. body condition scoring is an objective measure to truly assess how fat or thin the animal is and can be used for accurate determination of endpoints in studies where animals are expected to lose or gain weight ( fig. . ) (ullman-cullere and foltz, ) . some of the more commonly found diseases of mice are presented in table . . noninfectious disorders are presented in table . . based on their genetic and physiologic makeup, mice can be either immunocompetent or immunodeficient. immunocompetent means that the mouse has a normal functioning immune system and can stage an immune response to any insult or injury. in contrast, immunodeficient means that some component or components of the mouse's immune system is not working or functioning normally, and so they cannot stage an adequate immune response and are more susceptible to infectious disease. immunosuppressed mice are mice that have a complete immune system but because of a drug or chemical or disease state, the immunological response is attenuated. rats and humans have a long history of coexistence. the origins of the laboratory rat, also known as the norway rat, stretch back centuries to the areas of modern day china and mongolia (burt, ; song et al., ) . the dispersal of the norway rat has occurred across the centuries and its natural habitat stretches from the mediterranean across southeast asia and down into australia and new guinea . unfortunately, most people associate rats with disease and destruction. throughout history, outbreaks of bubonic plague, typhus, and hantaviruses have had an unwitting accomplice in the rat (zinsser, ; benedictow, ; firth et al., ) . over the centuries, rats have also been used for food (e.g., in imperial china), companionship, and sport (gorn and goldstein, ; hopkins et al., ; burt, ) . ratting, a vicious blood sport where people laid bets on the dog that could kill the most rats in a given period of time was especially popular in both the victorian england and american underworld (thomas and mayhew, ; gorn and goldstein, ) . at the turn of the th century, breeding rats as a hobby or for companionship (i.e., "fancy rats") was recognized by the addition of "rat" to both the name and mission of national mouse club in the united kingdom (american fancy rat and mouse association, ). however, as interest in pet rats waned over the following years, the club reorganized and dropped "rat" from its name. a similar club, the american fancy rat and mouse association, was founded in the united states in (american fancy rat and mouse association, ). the first recorded use of rats as research subjects occurred in (hedrich, ) , and the first known rat breeding experiments occurred in the late s (lindsey and baker, ) . the first major effort to perform research in the united states using laboratory rats occurred at the wistar institute of philadelphia, the oldest independent research institute in the united states, in (lindsey and baker, ) . rattus norvegicus constitutes one of the most commonly used laboratory species ( fig. . ), second only to the laboratory mouse. because rats and mice are not included under the animal welfare act regulations, the precise number of these species used per year within the united states is unavailable. however, examining the data collected within the european union can give some indication of their use relative to other common laboratory animal species. in , rats accounted for just fewer than % ( . million) of the total animals ( . million) used in research within the european union (european commission, ) . this contrasts to mice, which constituted % ( . million) of the total animals used within the european union (european commission, ). rats possess a number of qualities which make them a highly suitable and much preferred animal model. like mice, these traits include relatively small size; known genetic background; short generation time; similarities to human disease conditions; and known microbial status. their tractable nature makes them easier to handle in a laboratory setting than many other rodents. rats rarely bite their handlers unless extremely stressed or in pain. rats have been used as animal models in numerous areas of research from space exploration to answering more basic scientific questions regarding nutrition, genetics, immunology, neurology, infectious disease, metabolic disease, and behavior. perhaps their largest use is in drug discovery, efficacy, and toxicity studies. in the united states, the approval of any new drug for use in humans or animals usually necessitates that toxicity testing be done in at least one small animal species (e.g., rodents) and one large animal or target species (e.g., dog, nonhuman primate). there are known physiologic differences between the numerous outbred stocks and inbred strains of rats. the rat genome database (rgd) is an extensive, free resource filled with information regarding the different phenotypes, models, and genomic tools used in rat research (laulederkind et al., ) . vendors of commercially available rat stocks and strains are often good resources for normal physiologic data of these strains. many provide stock-and strain-specific data directly on their websites such as growth curves, complete blood count and serum biochemistry panels, and spontaneous lesions seen on histopathology. a summary table of normal physiologic references is in table . . sexual dimorphism exists between male and female rats. sexing of adult rats is most easily done by examining the perineal area of the rat and identifying the external reproductive structures such as the penis, testes, or vagina. in addition, male rats are typically larger and weigh significantly more than their age-and strain- matched female counterparts. sexing of rat pups is most easily performed by examining the distance between the anus and genital opening in the pup. males have a greater anogenital distance than females. male rats possess paired testicles that descend from the abdomen into the scrotal sac at approximately days of age (russell, ) . due to the lack of closure of the inguinal rings, the testes may be retracted into the abdominal cavity even as an adult. the male rat also possesses a number of accessory sex glands. a four-lobed prostate is present along with four other paired glands, including: the seminal vesicles, coagulating glands, ampullary glands, and bulbourethral glands (noted in some texts by the older name, cowper's gland) (popesko et al., ) . due to the unusual bihorned shape of the closely associated coagulating gland and seminal vesicles, individuals unfamiliar with rodent male anatomy may initially mistake these structures for the female uterus. however, the apices of these glands are freely mobile and easily exteriorized from the abdominal cavity unlike the uterus, which is attached to the dorsal body wall bilaterally via paired ovaries and their respective ovarian ligaments. the reproductive anatomy of the female rat contains some distinct features. the uterus of the female rat is classified as a duplex uterus, because the vagina is separated from the uterus by two individual cervices with each cervix leading to a separate uterine horn (popesko et al., ) . the placentation of the pregnant rat is hemotrichorial (three layers) rather than the hemomonochorial (single layer) placentation present in humans (wooding and burton, ) . the rest of the reproductive anatomy (e.g., ovary, oviduct) is structurally and functionally similar to other mammals. a summary of basic reproductive physiology is presented in table . . rat pups are born hairless, blind, and deaf and require extensive parental care that is provided mainly by the mother. as with mice, the skin of the hairless rat pups has a pink coloration, thus they are also referred to as "pinkies." the inclusion of nesting materials in the cage is recommended to assist the rat pups with thermal regulation until they have a full hair coat (whishaw and kolb, ). like other rodents, rattus norvegicus is a nocturnal species with the highest level of activity occurring during the dark phase. behaviors exhibited by rats include grooming, nesting, eating, and other social behaviors. nesting behavior serves several purposes among rats and mice (gaskill et al., (gaskill et al., , a . nests allow for better thermoregulatory control within a given environment as well as protection against predation (gaskill et al., c) . recent work in mice suggests that energy not diverted to thermoregulation can be shunted to other activities as seen via improved feed conversion and breeding performance (gaskill et al., c) . however, anecdotal evidence suggests that nest building in rats is largely a learned behavior, and it appears that there is a developmental period in young rats whereupon if exposed to nesting materials during this time they will begin using the materials to build at least rudimentary nests (gaskill, ) . at a minimum, rats benefit from having a structural shelter or nest box into which they may rest away from prying eyes ( fig. . ). rats emit alarm vocalizations during times of distress. however, these negatively associated vocalizations typically register in the ultrasonic wavelengths (approximately khz), well outside of the human hearing range (burman et al., ; parsana et al., ) . rodents may also emit high-pitched audible vocalizations when extremely alarmed, distressed, or in pain (jourdan et al., ; han et al., ) . as discussed in chapter , rats exhibiting abnormal behaviors and stereotypies can create variables in the research findings and should not be used in a study unless abnormal behavior is the object of the study subjects (baenninger, ; callard et al., ; garner and mason, ; cabib, ; ibi et al., ) . examples of stereotypies seen in rats include: bar-gnawing, pawing behavior, repetitive circling, and backflipping. it is critical that rats should be provided some form of environmental enrichment to stimulate positive species-typical behaviors. the housing of rats in a laboratory setting is similar to that described previously for mice: conventional, spf, and germ free. as rats are social animals, at minimum, they should be housed in pairs whenever possible. there is a preponderance of evidence that shows the differences in affiliative versus aggressive behavior, biochemical changes, and changes in learning between rats raised and housed in social isolation versus those housed in social groups (baenninger, ; einon and morgan, ; robbins et al., ) . enrichment items, such as a hut, nesting box, or similar type of shelter may be included in the cage to provide a visual barrier between the rat and the rest of the animal room. evidence suggests that rats prefer shelters made from opaque plastic (patterson-kane, ) . rats also spend a lot of time in the wild chewing either for eating or for manipulating objects for nest building. providing objects made of safe materials in the cage allows the rats to exhibit this natural behavior and encourage the normal wear of the rat's incisors, minimizing the incidence of malocclusion of the teeth. rodents can benefit from frequent gentle handling by the researcher and animal care staff. this concept is also known as "gentling" and has been demonstrated to reduce the stress experienced by rats during experimental handling and procedures (hirsjarvi et al., ; van bergeijk et al., ) . another positive interaction between humans and rats is found in the "tickling" of rodents. based on ultrasonic vocalization data, rodents find tickling a pleasurable experience (burgdorf and panksepp, ; panksepp, ; hori et al., ) . tickling may also decrease the stress response seen in rodents after experimental manipulations like intraperitoneal injections (cloutier et al., ). a multilevel cage with an intracage shelter. this style of caging provides opportunities for exercise for the rats. photo provided by melissa swan. as for mice, some general signs of ill health would include: weight loss, depression or lethargy, anorexia, obesity, diarrhea, scruffiness or ruffled coats, abnormal breathing, sneezing, weakness, dehydration, enlarged abdomen, discolorations (e.g., yellow for jaundiced animals or very pale for anemic animals), masses or swellings, and abnormal posture or gait. when assessing animals, a body condition score can be used as an objective measure or scale to truly assess how fat or thin the animal is; and it allows for the accurate determination of endpoints in studies where animals are expected to lose or gain weight (hickman and swan, ) (fig. . ) . some of the more commonly found diseases of rats are presented in table . . the ancestral home of the european rabbit (oryctolagus cuniculus) is the iberian peninsula (hardy et al., ) . the earliest archeological evidence of the coexistence of humans and rabbits can be found in excavation sites dated at approximately , years bce in nice, france (dickenson, ) . in antiquity, romans used rabbits as a food source and are thought to be responsible for their dispersal throughout europe, although there is no evidence that they attempted to actually domesticate them (dickenson, ) . domestication and selective breeding is thought to have begun in france in the middle ages where monks began to breed rabbits in their monasteries (dickenson, ) . the rabbits were kept confined in enclosures called "clapiers"(dickenson, ). they were kept largely as a source of food for the monks especially since ce when pope gregory i officially classified them as "fish" and thus eligible to being eaten during lent. however, rabbit wool production soon became a welcome by-product of these domestication efforts. european rabbits have been used in research since the middle of the th century. early work with the species was concentrated on the comparative anatomy of the rabbit with other species, such as the frog, and the unique features of the rabbit's heart and circulatory system (champneys, ; roy, ; smith, ) . louis pasteur used rabbits in a series of experiments that led to the development of the world's first rabies vaccine (rappuoli, ) . while there are numerous so-called "fancy" breeds of rabbits available in the pet trade, the list of breeds routinely used in research is much shorter. the new zealand white (nzw) rabbit is the most frequent breed of used in research (fig. . ). the california and dutch-belted rabbit breeds are also occasionally used. researchers have developed genetically inbred rabbit strains for particular research applications. for example, the watanabe heritable hyperlipidemic (whhl) and the myocardial infarction-prone whhl rabbit (whhlmi), both developed by researchers in japan, are used to explore diseases associated with dyslipidemia such as atherosclerosis (shiomi et al., ; shiomi and ito, ) . rabbits have been used as a model of human pregnancy and for the production of polyclonal antibodies for use in immunology research (hanly et al., ; ema et al., ; ito et al., ; fischer et al., ) . rabbits are routinely used in (southard et al., ; arslan et al., ; mcmahon et al., ; castaneda et al., ; habjanec et al., ; manabe et al., ; xiangdong et al., ; panda et al., ; sriram et al., ; wei et al., ; zhou et al., ) . the production of polyclonal antibodies is preferentially performed in the rabbit due to its relatively large blood volume compared to rodents (hanly et al., ) . their tractable nature and larger body size also make them suitable for surgical implantation of biomedical devices (gotfredsen et al., ; swindle et al., ; ronisz et al., ) . additionally, rabbits are a favored model in pharmacologic studies for teratogenicity testing of novel pharmaceutic compounds (gibson et al., ; lloyd et al., ; foote and carney, ; jiangbo et al., ; oi et al., ) . while much of the anatomy of the rabbit is similar to other mammalian species, it should be noted that there are a number of key differences. for example, the skin of the rabbit is quite thin and fragile. care should be taken when restraining a rabbit or shaving a rabbit's fur (e.g., in preparation for surgery) to avoid tearing the skin. unlike rodents and other laboratory animals, rabbits do not have pads on their feet; rather, the plantar surface is covered with fur (quesenberry and carpenter, ). new zealand white rabbits are commonly used in research. photo provided by kay stewart. the long ears of the rabbit serve several purposes. the most obvious is for hearing. in addition, the central ear artery and marginal ear veins are easily accessible for both intravenous administration and blood sampling (diehl et al., ; parasuraman et al., ) (fig. . ) . the ears also serve as a means of thermoregulation, as excess heat may be exchanged across the large surface area of the ears (sohn and couto, ) . the skin of rabbits lacks sweat glands and is therefore unable to sweat; panting is insufficient to dissipate the excess heat (sohn and couto, ) . thus, the ears play a vital role in maintaining proper body temperature. other unique features of the skin and adnexa are the presence of chin and inguinal glands used in scent marking. additionally, the female rabbit (doe) is noted by the presence of a large skin fold filled with subcutaneous fat just under the chin (the dewlap) (sohn and couto, ) . the skeleton of rabbits makes up only % of the body weight by mass (brewer, ) . this is in contrast to other similarly sized mammals. for example, the cat skeleton makes up e % of body weight (brewer, ) . the small skeletal mass of the rabbit coupled with strong back muscles mean that the back is prone to traumatic fracture (meredith and richardson, ) . proper holding and restraint techniques are necessary to avoid this undesirable outcome. there are several unique features of both the respiratory and cardiovascular systems of rabbits. for example, rabbits are obligate nose breathers (varga, ) . this is especially important during procedures involving anesthesia and placement of an endotracheal tube. with respect to the cardiovascular system, the rabbit heart is unique in that the right atrioventricular (av) valve has only two leaflets instead of three (brewer, ) . additionally, due to the similarity to humans with respect to the neural anatomy of the ventricles, the rabbit is the species of choice for purkinje fiber research (brewer, ) . rabbit teeth are "open-rooted" meaning that they continue to erupt and grow throughout life. this applies to all of the teeth in the rabbit dental arcade (i.e., incisors, premolars, and molars; rabbits do not have canines). this contrasts with rodents, where the incisors are the only open-rooted (or hypsodontic) teeth (sohn and couto, ) . thus, rabbit teeth are subject to overgrowth. another unique feature of rabbit dentition that sets them apart from rodents is the presence of a second set of incisors just behind the first set of upper incisors known as "peg" teeth (sohn and couto, ) . they are thought to aid in tearing off the succulent leaves of plants while grazing. as an obligate herbivore, the gastrointestinal tract of rabbits differs greatly from that of carnivores and omnivores. rabbits require a high fiber diet of between and % (sohn and couto, ) . the small intestine is divided up into three main sections: the duodenum, jejunum, and ileum. the ileum connects to the cecum via a structure called the sacculus rotundus. the presence of lymph follicles suggests that the sacculus rotundus has immunological functions. it is sometimes referred to as the ileocecal "tonsil" (jenkins, ) . in the rabbit, gastric associated lymphoid tissue is also present in the small intestine and the vermiform appendix (lanning et al., ) . the cecum, a large distensible outpouching of the large intestine, holds up to an estimated % of the total ingesta (sohn and couto, ) . rabbits are considered to be "hindgut fermenters." bacteria present in the cecum ferment the digestible fiber found within the diet. the product of this fermentative process becomes cecotrophs (also known as "night feces"). cecotrophs are excreted roughly h after the initial foodstuffs are ingested (sohn and couto, ) . they are softer and more mucoid in appearance than the hard, dry "day feces" produced just h after consuming food (sohn and couto, ) . the bulk of day feces consists of the indigestible fiber found in the diet. the sorting of foodstuffs destined to become either day feces or cecotrophs and the timing of their relative excretion is largely dependent upon the neural input of the fusus coli, also termed the "pacemaker" of the colon (sohn and couto, ) . the fusus coli is anatomically located between the ascending and transverse colons of the rabbit (popesko, ) . consumption of cecotrophs by rabbits is an important part of the digestive process in rabbits as they are rich in b vitamins, such as niacin and b , and vitamin k (hörnicke, ) . while cecotrophs are known colloquially as "night feces," rabbits produce and eat them at all hours of the day (sohn and couto, ) . rabbits are agile enough to eat these night feces directly from their anus (sohn and couto, ) . those researchers performing digestive research (e.g., fecal collection via metabolism cages) should keep this in mind. sexing of adult rabbits is aided by the sexual dimorphism present in the species. mature females are readily identified by the presence of the dewlap (sohn and couto, ) . females have e nipples, while in males these nipples are present, but rudimentary (sohn and couto, ) . nzw rabbits reach sexual maturity between and months (suckow et al., ) . reproductive data are summarized in table . . mature bucks have paired testicles enclosed in paired hairless scrotal sacs (sohn and couto, ) . like rodents, the inguinal rings do not close. accessory sex glands include several bilobed organs: the seminal vesicle, vesicular gland, prostate, and paraprostatic gland. the bulbourethral glands of bucks are paired (sohn and couto, ) . female rabbits are induced ovulators (dal bosco et al., ; sohn and couto, ) . that is, the egg does not ovulate spontaneously from the ovary, rather manual stimulation via copulation is required. ovulation occurs approximately h postcopulation (sohn and couto, ) . because they are induced ovulators, does do not have a defined estrous cycle. rather, they have periods of sexual receptivity lasting approximately e days followed by e days of nonreceptivity. nonfertile matings may result in a period of pseudopregnancy of up to e days (sohn and couto, ) . fertile matings result in pregnancy lasting e days (sohn and couto, ) . the placenta of rabbits is classified as hemodichorial; this is in contrast to humans which have a hemomonochorial placenta (furukawa et al., ) . birthing (i.e., parturition; also known as "kindling") occurs most often during the early morning hours (sohn and couto, ) . kits are born deaf and blind. by and days of age they can hear and see, respectively (quesenberry and carpenter, ) . amazingly, does suckle their young ones daily, usually during the dark phase, and for approximately only e min (sohn and couto, ) . the kits are able to drink about % of their entire body weight in that time. wild and domesticated does both follow this nursing behavior. rabbit kits may be weaned between and weeks of age (suckow et al., ) . earlier weaning should not be attempted as there may be profound detrimental effects on the functioning of the gastrointestinal system (bivolarski and vachkova, ). rabbits are very social, nocturnal creatures. scent marking is a normal and important part of their behavior repertoire. rabbits will rub the secretions from their chin scent glands against inanimate objects, other rabbits, and human handlers in a process called "chinning" (sohn and couto, ) . dominance hierarchies are established behaviorally. dominant animals may mount, "barber," or scent-mark subordinates (sohn and couto, ) . barbering is the act of chewing the hair of a subordinate animal, usually on the neck and back in the case of rabbits, very close to the skin giving the appearance of having been cut or "barbered" (bays, ). rabbits will "thump" one or both back feet on the ground when frightened or as an alarm call to other rabbits (bays, ) . highly stressed rabbits may actually emit a loud, piercing scream, especially when roughly caught by an untrained individual (bays, ) . it is important to approach rabbits calmly and quietly. relaxed, content rabbits may be heard making a purring sound (bays, ) . rabbits benefit by repeated, positive interactions with people similar to the concept of "gentling" in rats (see section . ). changes in behavior are often first indication that an animal is in pain. given that rabbits are a prey species, it is evolutionarily speaking not in a rabbit's best interest to display signs of pain. thus, these behaviors are most often quite subtle in nature and may be easily missed if particular attention is not paid. the first sign often seen in a rabbit experiencing pain is a decreased appetite resulting in little to no food intake (sohn and couto, ) . rabbits will often grind their teeth (i.e., bruxism) when experiencing pain (sohn and couto, ) . other rabbits may simply appear very dull and inactive. as with rodents, rabbits can develop stereotypies. due to the sensitivity of the rabbit's nose and lips many stereotypies involve chewing behaviors. bar chewing, chewing on the water bottle, and self-barbering are all stereotypical behaviors seen in rabbits (gunn and morton, ; chu et al., ) . in addition, rabbits may engage in "nose sliding" against solid surfaces like the cage walls and head swaying (sohn and couto, ) . animals that exhibit stereotypies do not make good research animals. efforts should be made, where possible, to prevent these behaviors through the use of environmental enrichment. enrichment may be in the form of chew-resistant objects (such as plastic dumbbells and stainless steel rattles) and food treats (poggiagliolmi et al., ). as a prey species, rabbits benefit from the inclusion of a hut in the cage or at least a visual barrier into which they may retreat when psychologically stressed (baumans, ) . breeding females should always have access to a nest box to allow for the necessary expression of normal nesting behavior (baumans, ) . being social creatures, ideally rabbits should be housed in compatible pairs or trios unless contraindicated by the research objectives or by incompatibility of the animals (sohn and couto, ) . stable social groups formed shortly after weaning, where animals are not added or removed, is most beneficial (boers et al., ) . structurally, rabbits benefit from housing that has both adequate vertical and horizontal space (boers et al., ) . one recommendation on the space requirements of laboratory rabbits stipulates in. as the minimum vertical cage height (national research council, ) . at a minimum, rabbits must be able to comfortably sit upright in the cage without their ears bending over (national research council, ) . laboratory rabbits are typically housed in easily sanitized stainless steel cage racks ( fig. . ). slatted flooring allows for urine and feces to fall through the slats onto special pans fixed below the cage, thus providing for easier sanitation of the cages. however, care must be taken that the slats are of sufficient width so as to prevent a condition known as bumblefoot (see section . . ). dog runs with elevated, slatted flooring or a solid floor with bedding have also been used by some investigators in group-housed rabbits with success (personal observations). again, attention should be paid to the flooring and its effect on foot health. commercially available research diets specifically formulated for rabbits are available. these diets are preferred to so-called "natural diets" and feeding individual vegetables. this is because rabbits tend to be very selective eaters which can lead to nutritional imbalances (fraser and girling, ). additionally, the use of fresh vegetables may lead to the introduction of unwanted pathogens like salmonella (varga, ) . commercial diets are available in maintenance and reproductiveperformance dietary formulations as well as presterilized diets for rabbits housed under spf conditions. rabbits are very easily heat stressed and thus must be kept at significantly lower temperatures than other laboratory animals like rats and mice. noise is another significant stressor to rabbits (verga et al., ) . sudden, high-pitched, sharp noises are most disruptive. however, in general, noise within the animal rooms should be avoided as much as possible. for this reason, rabbits should not be housed, even temporarily for short procedures, near areas of high noise. problems in rabbits related to the gastrointestinal system are relatively common. these problems can become serious very quickly. therefore, it is critical that abnormalities seen (e.g., rabbit not eating or abnormal feces) be reported to the veterinary care staff immediately. even if a researcher is unsure if there is a problem, it is best to report suspicions because without prompt intervention, seemingly minor problems can escalate to potentially life-threatening conditions. some of the most commonly seen clinical conditions in rabbits are summarized in table . . the zebrafish, danio rerio of the cyprinidae family, is a small, dark blue and yellow striped, shoaling, teleost fish, popular among aquarium enthusiasts, and increasingly among the research community (fig. . ) . the adult fish are e cm in length, with an incomplete lateral line and two pairs of barbels (laale, ) . males have larger anal fins and more yellow coloration; females have a small genital papilla just rostral to the anal fin (laale, ; creaser, ) . zebrafish are hardy, fresh water fish originating from a tropical region with an annual monsoon season. the fish are generally found among slow moving waters of rivers, streams, and wetlands, across the south asia region of india, bangladesh, example of rabbit caging for a laboratory setting. photo provided by deb hickman. and nepal (engeszer et al., ; spence et al., ) . the waters tend to be shallow, relatively clear with substrates of clay, silt, or stone of varying size (mcclure et al., ; engeszer et al., ) . the fish feed mostly on insects and plankton, with evidence of feeding along the water column as well as water surface (mcclure et al., ; engeszer et al., ; spence et al., ) . gastrointestinal upset generally secondary to the use of broad spectrum antibiotics, such as penicillin. misalignment and subsequent overgrowth of the continuously growing teeth pododermatitis ("bumblefoot") infection of the underside of the feet pasteurella multocida common cause of respiratory infections and abscesses male zebrafish have a more stream-lined body with darker blue strips while the females have a white protruding belly. photo provided by kay stewart. the small size of zebrafish, the ease of keeping large numbers, frequent spawning, large egg clutches, translucent nonadherent eggs, rapid development and complex sequencing of the zebrafish genome are all key components that make the zebrafish an attractive research model. interestingly, approximately % of zebrafish genes have at least one orthologous human gene (howe et al., ) . publications on the use of zebrafish in research are cited as early as the s (creaser, ) . until the early s, the use of zebrafish stayed fairly low, with the number of articles published staying below per year. in the mid- s, publications increased to about per year, doubling again in the s, increasing to almost articles per year in the early s, and rapidly expanding to publications by . developmental biology was the initial focus of zebrafish research use. however, in recent years, use of the zebrafish in research related to biochemistry and molecular biology, cell biology, neurological sciences, and genetics has been rapidly increasing. zebrafish are known to live for only a year in the wild (spence et al., ) . for most of the year, the fish reside in shallow streams. with the onset of monsoon rains, they move to flooded, highly vegetated shallow wetlands and floodplains, including rice paddies, with little to no current and often silt bottoms for spawning (engeszer et al., ) . the offspring then develop in these waters until the seasonal waters diminish (engeszer et al., ) . zebrafish rapidly mature, reaching sexual maturity as early as months postfertilization . the zebrafish continues to grow throughout life, which is much longer in captivity, with a mean lifespan of . years in captivity (gerhard et al., ) . in nature, spawning behavior occurs within small groups of three to seven fish. males within the group pursue females, with spawning occurring along the substrate (spence et al., ) . similar behaviors are noted in laboratory zebrafish, with spawning often occurring with the first light of day. courtship behavior involves a rapid chase of the female, the male swimming around the female, nudging her, or swimming back and forth working the female to the spawning site. interestingly, zebrafish prefer spawning near artificial plants. once there, the male remains close to the female, extending his fins to bring his genital pore in line with the female. the male may also rapidly undulate his tail against the side of the female to initiate egg release by the female, coinciding with sperm discharge by the male. the female produces eggs in batches of e over several encounters with the male for up to an hour. most eggs are released within the first min, with a peak in production during the first min (darrow and harris, ; spence et al., ) . zebrafish produce large clutches of eggs, from to eggs per clutch (laale, ) . the eggs, approximately . mn in diameter, are transparent and protected within a chorionic membrane (kimmel et al., ) . first body movements and beginning stages of organ development occur e h postfertilization (kimmel et al., ) . as development continues, the larva hatches from the egg two to three days postfertilization (kimmel et al., ) . the early larva has special secretory cells within multicellular regions of the head epidermis that allow the larvae to attach to various hard surfaces and plants until the swim bladder inflates or days postfertilization (laale, ; kimmel et al., ) . once the air bladder inflates, the fish can maneuver through the water column. in captivity, zebrafish can breed year round. the presence of males, or even just the male pheromones, is needed to induce ovulation (gerlach, ) . if females are housed away from males for an extended period, they can retain the eggs resulting in egg-associated inflammation, which can be lethal (kent et al., a) . to accommodate the fish life cycle, zebrafish are typically housed in static spawning cages to allow for fertilized egg production. spawning cages include a housing tank containing a clear slotted bottom insert, and a plastic plant. the insert is often placed in the holding tank at an angle to create a shallow region for spawning and the slotted bottom of the insert allows for ease of egg collection (lawrence and mason, ; nasiadka and clark, ) (fig. . ). the embryos are then incubated at around . c in a petri dish for at least e days postfertilization (wilson, ) . the fish are then kept in static or slow water flow containment and can be fed paramecium, rotifer, a powdered food, or a combination of these feed types. unfortunately, other than the need for essential fatty acids in their diet, little is yet known on the nutritional requirements of zebrafish. zebrafish in research settings are typically fed live feed like artemia (brine shrimp), rotifers, bloodworms example of a zebrafish spawning system. the system is designed to allow eggs to fall beneath a slotted insert to the bottom of the tank as a way to prevent the adult fish from consuming the eggs. photo provided by robin crisler. (chironomid larvae), commercial feed, or a combination of all (lawrence, ) . the size of the feed is necessary to suit the gape size of the larvae, approximately mm (lawrence, ; wilson, ) . water flow and feed size increase with development, with transition of the feed to artemia (brine shrimp), and/or use of a larger particle commercial feed during the e days postfertilization (wilson, ) . once the juvenile stage is reached, around days postfertilization, the fish are housed more like adult fish, with more frequent feeding and slower water flow to accommodate their remaining development and smaller size, respectively (wilson, ). as early as months of age the fish are sexually mature . adult zebrafish can be housed in traditional glass aquaria or elaborate computerized and automated systems that monitor and control water quality parameters such as temperature (typically . c), ph, water hardness, salinity, dissolved oxygen, and nitrogenous wastes (lawrence, ; lawrence and mason, ) . whether maintained manually or computerized, these parameters are important to monitor and maintain at appropriate levels to maximize the health of the fish. poor water quality can lead to disease in the fish (kent et al., b) . many of the organisms that cause disease in zebrafish are opportunists in the environment and remain subclinical until the fish is stressed, often due to problems with husbandry. appropriately maintained housing, combined with a healthy water quality, avoidance of overcrowding, and a functional quarantine and health surveillance program are key components to avoiding stress and disease. to date, there are currently no known viruses documented in zebrafish as naturally occurring disease concerns (kent et al., b) . mycobacterium infections are the most frequently documented bacterial infections (kent et al., a,b) . class reptilia is made up of four orders classified as chelonia, rhynchocephalia, squamata, and crocodilia (frye, ) . in class amphibia, animals more commonly encountered in research setting are in the order anura, containing frogs and toads (such as xenopus, bufo, rana, hyla, and dendrobates spp.); and in the order caudata, containing salamanders such as the tiger salamander, ambystoma tigrinum and the axolotl, ambystoma mexicanum (national research council, ) (figs. . and . ). snakes and lizards are in class reptilia, order squamata; chelonians (turtles, tortoises, terrapins) are in order chelonia; and alligators, caimans, and crocodiles are in order crocodilia. in contrast to research in mammals, there is a tendency for reptile and amphibian research to be more oriented to studying evolution and ecology as opposed to basic science evaluating models of human disease (pough, ) . salamanders and frogs are important for studying embryonic development, metamorphosis, regeneration, figure . a commonly used amphibians in research is the axolotl, ambystoma mexicanum. provided by chris konz. african clawed frogs, xenopus laevis, a commonly used amphibian. provided by randalyn shepherd. physiology, and climate change (burggren and warburton, ; hopkins, ; pough, ) . reptiles are often studied because of their more simple cardiovascular systems as well as for evaluating mechanisms of immune responses, hormonal controls, and unique reproduction methods such as parthenogenesis (frye, ) . of the amphibians, xenopus laevis (south african clawed frog) and xenopus tropicalis (western clawed frog) are commonly studied in the research setting. x. laevis is a prominent research model in comparative medicine and developmental studies, and is the most commonly studied species in the genus xenopus (denardo, ; schultz and dawson, ; o'rourke, ) . advantages include large-sized eggs for ease of observing embryo development, as well as the wealth of published literature in areas of research such as evolution, neurobiology, regeneration, endocrinology, and toxicology (koustubhan et al., ; gibbs et al., ) . rana catesbeiana (bullfrogs) have been used for developmental and toxicological studies, and for infectious disease study of the chytrid fungus batrachochytrium dendrobatidis (alworth and vazquez, ) . a. mexicanum, in particular, is studied to understand the regenerative ability of the blastema of amputated limbs at the molecular level (gresens, ; rao et al., ) . ambystoma tigrarium has been studied in regard to general amphibian decline in north america, environmental contaminants such as pesticides and effects of infection with a. tigrarium virus (sheafor et al., ; kerby and storfer, ; chen and robert, ; kerby et al., ) . a variety of snakes, crocodiles, lizards, and turtles have been studied in research. for example, anolis carolinensis (the green anole) has been used for the study of reproduction biology (lovern et al., ) . caiman crocodilus and alligator mississippiensis (crocodiles), trachemys scripta elegans (red-eared sliders) represent a few other examples of reptiles used in research (o'rourke and schumacher, ). amphibians and reptiles are considered to be ectotherms (greene, ) . unlike mammals and birds, ectotherms are unable to internally regulate body temperatures above that of the ambient environment through metabolism and require complex behavioral and thermoregulatory adaptations to regulate temperature (pough, ; seebacher and franklin, ) . in captivity, ectotherms typically require supplemental sources of heat to mimic the thermoregulatory effects of basking in the sun. some amphibians and reptiles are aquatic (xenopus frog spp.) whereas others are semiaquatic or terrestrial. x. laevis and x. tropicalis are from geographically distinct areas and have different temperature requirement depending on life stage, with x. tropicalis adults generally around c in their natural habitat versus about c for x. laevis (tinsley et al., ) . the skin of amphibians is permeable to water and some adults (semiterrestrial tree frogs in family hylidae, arboreal and terrestrial toads in bufonidae) may receive a significant portion of their daily water requirement via absorption through a vascular-rich area on the pelvic area termed the pelvic patch (pough, ; ogushi et al., ) . the skin of some amphibians contains toxins which can cause arrhythmias in human handlers, for example, alkaloids from dendrobatid frogs, and bufotoxins from toads of the genus bufo (denardo, ) . the toxins serve to keep predators away but, as with xenopus, may harm the animals themselves by continued direct contact or diffusion through the water (tinsley et al., ; chum et al., ) . the skin of amphibians is easily damaged thus to protect the animal during handling powder-free gloves should be worn (gentz, ) . researchers and animal care providers should investigate the natural environment of each species within their care and critically evaluate what features are required for normal behavior and physiology to provide the essential elements in the research setting (pough, ) . in the wild, amphibians and reptiles live in ecological environments that span a range of diversity from topical forest areas to dry desert. they may be arboreal, aquatic, or terrestrial. they are often secretive and hide when in natural habitats, preferring to hide under vegetation or in crevices. parameters from the natural habitat to evaluate include temperature, humidity, nutritional requirements, natural diet, nocturnal versus diurnal behavior, and housing density. temperature and lighting gradients should be established so animals can choose to move toward or away from the heat source as a way to avoid overheating. most amphibian species in the wild are nocturnal (pough, ; tinsley et al., ) . amphibians and reptiles are sensitive to chemicals in the environment. water quality parameters (such as ph, hardness, ammonia, nitrate/nitrite, salinity, conductivity) should be regularly monitored. chloramine and chloramines are often present in municipal water supplies and are toxic to aquatic species. water should be treated prior to use for aquatic species with an agent like sodium thiosulfate, since chloramine does not readily dissipate (browne et al., ) . ammonia is a breakdown product between the chloramine and sodium thiosfulfate reaction and is a concern for aquatic animals (browne et al., ; koustubhan et al., ; o'rourke and schultz, ) . a wide variety of caging materials may be used for housing such as glass, plastic, stainless steel, or fiberglass but should be free of contaminants or harmful chemicals like bisphenol a that could leach from the caging into the water (levy et al., ; browne et al., ; bhandari et al., ) . agents used to sanitize caging should be chosen to minimize likelihood of harmful residues. environmental enrichment should be provided to encourage natural behaviors and can include providing cage mates for social interaction, cage accessories that serve as hiding spots or shelters (fig. . ) as well as providing a variety of food treats in changing locations for foraging opportunities (hurme et al., ) . scents, sounds, and color choices may also be incorporated into enrichment strategies provided that they are carefully evaluated to ensure that they are beneficial and do not cause stress. for example, the tortoise, chelonoidis denticulata, may show a color preference for red-colored enrichment items (passos et al., ) . pvc tubes are another example of enrichment that has been provided to x. laevis for use as hiding cover (koustubhan et al., ) . some species may require haul-out ramps, areas for sun basking, floating rest areas, or enrichment devices along the water's surface to help prevent drowning. one should consider the possibility of ingestion, as reptiles and amphibians may attempt to consume the substrates provided to them. the degree to which amphibians are social varies significantly depending on the species and is not always well understood. they use visual and olfactory discrimination to help them find food, forage, and avoid predators (vitt and caldwell, ) . both in the wild and in captivity, reptiles and amphibians may exhibit excitatory behavior when fed (sometimes described as a "feeding frenzy") which may result in animal injury where animals are in close proximity (divers and mader, ; tinsley et al., ) . overcrowded tanks can result in competition for food and subsequent trauma. thus, when placed together for the first time, animals should always be observed for compatibility; and only members of the same species should be housed together. many reptiles and amphibians are escape artists and prevention of escape and injury is a critical factor when considering housing design. species that are prone to jumping must have secured lids on their enclosures. the diets of amphibians and reptiles are highly variable in the wild and are species dependent. commercially prepared pelleted diets may be available and accepted by reptiles and aquatic amphibians, however, terrestrial amphibians and many use of a rabbit feeder for xenopus enrichment. photo by randalyn shepherd. reptiles may prefer live diets (pough, ) . it is not unusual for some species to go several days of fasting between meals in nature (pough, ) . consultation with those experienced at successful housing and feeding the species in question (zoos, nutritionists, herpetologists) is recommended. there are many different types of infectious agents such as bacteria, viruses, fungi, and parasites that can cause health problems in amphibians and reptiles in addition to noninfectious conditions such as those resulting from nutritional imbalances, metabolic disease, neoplasia, trauma, and other spontaneous maladies. although significant advances in knowledge have been made over the past years regarding disease in these species, much still remains unknown. it is not possible to go into detail here, but there are excellent reference texts for diseases in amphibians and reptiles that can be consulted (jacobson, ; frye, ; wright and whitaker, ) . from a taxonomic standpoint, birds are placed into class aves which includes multiple orders based on anatomical, physiological, and genetic characteristics. passeriformes is the largest order and contains songbirds and perching birds such as the finch, canary, and cardinal ( fig. . ). order columbiformes contains pigeons and doves; order psittaciformes contains budgies and parrots such as the african gray; and order galliformes contains domestic fowl such as the chicken and quail (proctor and lynch, ; ritchie et al., ) (fig. . ). birds have been used as research models of human disease and are important in evaluation of aging, memory, parasitology, atherosclerosis, reproduction, and infectious disease among other topics (austad, (austad, , maekawa et al., ) . the genomes of several avian species have now been sequenced (jarvis et al., ) . historically, chickens (gallus domesticus) are the most common bird species studied in biomedical and agricultural research and are a classic model in areas such as immunology, virology, infectious disease, embryology, and toxicology (scanes and mcnabb, ; kaiser, ) . chickens are also studied to evaluate reproductive development and retinal disease. embryonated chicken eggs have been used to commercially produce vaccines (such as for human influenza), studied for developmental analysis, and are now being treated with viral vectors like lentivirus to produce transgenic embryos. inbred lines with improved disease resistance are being developed and transgenic technology in the future may allow embryos to be used as bioreactors to produce therapeutic proteins of interest and potentially to generate transgenic chickens which have improved resistance to pathogens (bacon et al., ; scott et al., ) . because chickens develop spontaneous ovarian cancers at an incidence of up to %, they are also a prominent model of ovarian cancer in humans (bahr and wolf, ; hawkridge, ) . quail the zebra finch is a common avian species used in research. from http://www.redorbit.com/news/science/ /male-zebra-finches-fake-song- /. the domesticated chicken commonly used in research. provided by kay stewart. (coturnix coturnix and coturnix japonica) have been studied in many of the same research disciplines as chickens, but offer advantages because of their smaller size and because they are among the shortest-lived bird species (austad, ) . japanese quail (c. japonica) have been selected as a model to evaluate reproductive biology and social behaviors such as mate selection because they readily show sexual behavior in captivity (ball and balthazart, ) . as with the chicken, methods to study transgenic quail are now becoming available and offer a useful tool to study gene function (seidl et al., ) . of the psittaciformes, amazon parrots and budgies (melopsittacus undulates) are among the most commonly studied, with research topics including veterinary medicine, diagnostics, behavioral, cognition, aging, and sensory studies (austad, ; kalmar et al., ) . the african gray parrot has been studied for its cognition and communication abilities (hesse and potter, ; harrington, ) . of the passerines studied in laboratory research, the most commonly evaluated include the zebra finch (taeniopygia guttata), european starling (sturnus vulgaris and sturnus roseus), and house sparrow (passer domesticus) (bateson and feenders, ). zebra finches and other songbirds are commonly studied in regard to aging and neurogenesis in addition to speech, learning, and memory because of their ability to learn and communicate intricate bird songs (harding, ; scott et al., ; austad, ; mello, ) . the most popular songbird species for neurobiological research include the zebra finch, canary, and other types of small finches such as lonchura striata domestica (schmidt, ) . zebra finches are favored in research settings since they are easy to house due to their small size, for their compatibility in groups, and proclivity for breeding. they are also studied for their biologic features such as sexual dimorphism, year-round singing in captivity, age-dependent period of song-learning propensity, and for ease of measurement with respect to their bird song (fee and scharff, ; mello, ) . pigeons (columba livia) have been evaluated in areas such as comparative psychology, neuroanatomy, neuroendocrinology, and atherosclerosis (santerre et al., ; austad, ; shanahan et al., ) . they are studied to understand their navigational skills and memory which allow homing, vision and discrimination ability. barn owls (tyto alba) are an example of a nocturnal avian species and are studied for neuroanatomy, vision, hearing, and for understanding learning mechanisms during auditory space mapping (pena and debello, ; rosania, ) . birds are warm-blooded vertebrates that have feathers for the purpose of flight and plumage. their respiratory system includes avascular air sacs, some of which attach to the lung and bronchi, but do not serve as sites for gas exchange as does the lung (maina, ; ritchie et al., ) . air sacs serve as internal compartments which hold air and facilitate internal air passage to allow birds to have a continuous flow of large volumes of air through the lungs as a way to increase oxygen exchange capacity and efficiency. birds lack a functional diaphragm and use muscles of the thorax to assist with respiration (ritchie et al., ) . care must be taken to ensure that use of physical restraint does not interfere with respiratory movement, cause the bird to struggle, or become stressed. the skeletal system includes pneumatic bones which are lined with air sac epithelium and are considered pneumatized by connection to the respiratory system (frandson et al., ). the specific bones which are pneumatized depend on the species but typically include the humerus, cervical vertebrae, sternum, sternal ribs, and sometimes the femur (ritchie et al., ) . the esophagus in birds leads to the crop, which is an outpocketing where food is held temporarily, and then continues to the proventriculus (also called the true stomach) which produces enzymes to break down food. food travels from the proventriculus to the ventriculus (gizzard) and then on into the small and large intestines. the presence or absence of a gallbladder is species dependent (tully et al., ; kalmar et al., ) . the rectum and urinary tract terminate in the cloaca, resulting in excreta where the fecal portion of waste is mixed with urate (white and/or creamy component). there are many additional unique and complex anatomic and physiologic adaptations of birds. other excellent references are available in the literature (scanes, ). housing requirements of birds held in captivity vary significantly depending on the particular species. basic parameters that apply to all birds include the necessity to provide an enclosure which is safe and permits species-specific behaviors to the greatest extent possible. consideration should be given to ensure that the type of structure is nontoxic, as some birds such as parrots have a powerful beak with the ability to chew through substrates. enclosures may be made of metals or durable plastic, but it is important to note that zinc wire, as well as leaded paint, can be toxic to birds and is best avoided. bar spacing on caging should be appropriate to prevent escape and injury based on the size of the bird. caging size varies and can include large aviaries where full short-distance flight is possible, to individual housing in smaller sized cages where flight may not be feasible. use of environmental enrichment and provision of opportunity for interaction is important to include as part of the cage structure, complexity, and social dynamic. some types of birds are considered social, polygamous, and benefit from group housing, whereas others such as those that pair-bond (such as new world quail) may prefer housing with a single mate (ritchie et al., ) . some species, genders, or individuals show aggression and may not be compatible. for example, sexually active male quail may injure each other and are generally considered incompatible (huss et al., ) . to help reduce aggression, housing densities should be kept low and multiple points of access to resources, such as feed and perches, should be provided. enrichment in the form of manipulanda can take the form of toys and food items. some types of birds may demonstrate foraging behavior in nature and may like to manipulate their feed. parrots, for example, typically grasp their food with their feet and may peel or strip the outer portion of the foodstuff prior to ingesting. toys should be size appropriate for the species, easily sanitized, free from sharp edges, and replaced once wear shows. birds can become easily caught in items that hang from the cage and as toys deteriorate they can become a hazard. for example, rope toys may begin to fray and become a hazard, causing entrapment; and some types of toys contain weights which pose a choking hazard or may be made of toxic materials such as lead. some types of birds spend considerable time perching and require perches, which vary in diameter, for comfort and to prevent pressure sores from developing on their feet. the respiratory system of the bird is very sensitive and caution must be taken by animal care staff to avoid exposure of birds to aerosols from chemicals that may arise from disinfectants used in the laboratory animal facility. scented cleaners, perfumes, hairspray, and emissions from teflon-coated materials are all examples of products which can be especially harmful to birds and may cause death. feeding requirements vary by species and life stage, but commercial pelleted diets designed to meet the nutritional needs can generally be provided. although many birds are seed eaters, a diet of seeds alone is unlikely to provide adequate or balanced nutrition. many birds have a requirement for dietary calcium, especially those that are reproductively active, and should be provided with calcium supplementation in the form of soluble grit such as cuttlebone or crushed oyster shells (sandmeier and coutteel, ; tully et al., ). birds often display neophobic behavior and may require long acclimation periods before fully accepting novel foodstuffs. for this reason, dietary changes should not be made abruptly and daily intake should be closely monitored. for birds in the laboratory setting, clean, fresh water should be provided daily either by use of nonbreakable bowls or sipper tubes. water intake will vary by species and environmental housing conditions. birds can mask disease and are easily stressed. it is best to first observe the bird in its normal home environment whenever possible and only perform restraint for physical exam or collection procedures when indicated. general indications of sickness may include decreased appetite, depressed behavior, loose stools, distended abdomen, ruffled feathers or unkempt appearance, skin lesions, openmouth breathing, abnormal respiratory sounds such as wheezing or sneezing, or signs of dehydration such as reduced skin turgor and sunken eyes. a healthy bird should have well-groomed feathers, appear alert, active and inquisitive, and should show species-typical behaviors. its eyes should be clear and bright. no evidence of discharge should be present from the eyes, nares, mouth, or urogenital area. numerous types of infectious (example, fig. . ) and noninfectious disease presentations are described in birds. additional reference resources should be consulted for in-depth information (ritchie et al., ; tully et al., ; doneley, ) . to provide the reader a broader view of animal use in research, descriptions of some less commonly used small mammal models follow. guinea pigs (cavia porcellus) are rodents, related to porcupines and chinchillas in the suborder hystricomorpha (fig. . ) . they originate from the mountain and grassland regions along the mid-range of the andes mountains in south america. they are small, stocky, nonburrowing, crepuscular herbivores with short legs and little to no tail, ranging from to g, females being smaller than males (harkness et al., ) . guinea pigs have a long-standing historical role in research stretching as far back as the s, when they were first used in anatomical studies (pritt, ) . further, they were used by louis pasteur and robert koch in their example of skin pox on the feet of a dark-eyed junco (junco hyemalis). photo from randalyn shepherd. investigations of infectious disease, and have contributed to the work of several nobel prize worthy studies (pritt, ) . specifically, the guinea pig has been used as a model for infectious diseases such as tuberculosis, legionnaires disease, sexually transmitted diseases such as chlamydia and syphilis, and one of the more common causes of nosocomial infections in people, staphylococcus aureus (padilla-carlin et al., ) . guinea pigs have also been useful tools in researching cholesterol metabolism, asthma, fetus and placental development and aspects of childbirth, as well as alzheimer's disease (bahr and wolf, ) . guinea pigs have many similarities to humans hormonally, immunologically, and physiologically. unlike other rodents, and more like primates (including people), guinea pigs are prone to scurvy if they do not receive adequate vitamin c, typically in their diet (gresham et al., ) . guinea pigs are housed similarly to other rodents, although they require more room than the smaller rodents. hamsters are of the rodentia order, suborder myomorpha along with the mouse and the rat. there are over species of hamsters described in the literature, with the most common hamster used in research being the golden or syrian hamster, mesocricetus auratus (harkness et al., ) (fig. . ) . originating from the northwest region of syria, golden hamsters are thought to be descendants of only three or four littermates collected from syria in (adler, ; smith, ) . as their name implies, the typical wild-type coat is reddish gold along their dorsum, with a gray underside. they are granivores and insectivores, weighing e g, females weighing more than males, with short legs and short tail, and large cheek pouches (harkness et al., ) . specific anatomical and physiological features including their susceptibility to disease and infection make them a useful model for study. initially hamsters were utilized in studies of infectious disease, parasitology and dental disease, transitioning into cancer research in the s (smith, ) . hamsters are still used in many areas of research, including investigations into metabolic diseases like diabetes mellitus (hein et al., ) , cardiovascular disease (russell and proctor, ) , reproductive endocrinology (ancel et al., ) , and oncology (tysome et al., ) . guinea pigs have also been used as models for infectious disease associated with bacteria, parasites, and viruses, such as leptospirosis (harris et al., ), leishmaniasis (gomes-silva et al., , and severe acute respiratory syndrome (sars) and ebola viruses (roberts et al., ; wahl-jensen et al., ) . other species of hamsters used have been used in research. for example, chinese and african hamsters have been used for investigations into diabetes mellitus (kumar et al., ) ; european and turkish hamsters have been useful to evaluate aspects of hibernation (batavia et al., ) ; and siberian and turkish hamsters have been used to study circadian rhythm and pineal gland activity (butler et al., ) (fig. . ) . chinchillas (fig. . ) are in the order rodentia, suborder hystricomorpha, as are the guinea pig and the degu. there are the long-tailed chinchilla, chinchilla lanigera, and the short-tailed chinchilla, chinchilla chinchilla. chinchillas originate from the andes mountains of south america (martin et al., ) . they are e g in size, females weighing more than males, with compact bodies and long, strong hind limbs and dense fur coats (alworth et al., ) . the lushness of the coat is what led them close to extinction in the wild due to excessive hunting siberian hamsters. photo from greg demas. chinchilla. photo from bill shofner jr. in the early to mid- s (jimenez, ) . the chinchilla has a large head, large eyes and ears. the large inner ear anatomy is of specific note as chinchillas are the traditional model for auditory studies (shofner and chaney, ) and otitis media (morton et al., ) . the gerbil is a rodent, suborder myomorpha, used in research. there are over species of gerbil-like rodents documented, but the mongolian gerbil (meriones unguiculatus) is the species most commonly used in the united states (fig. . ). mongolian gerbils originate from a desert terrain in mongolia and northeast china. they are long-tailed, burrowing, herbivorous rodents, e g in size, males being larger than females (harkness et al., ) . due to anatomical variations in the blood supply to the brain in an anatomical region known as the "circle of willis," gerbils have been used most notably as a model for cerebral ischemia or stroke (small and buchan, ) . an interesting animal model to note among the small mammals is the nine-banded armadillo (dasypus novemcinctus), a new world mammal ranging from the southeastern half of north america, extending south through the americas to the northern region of argentina (balamayooran et al., ) . armadillos have a banded carapace, and, importantly, a low core body temperature of e c. the breeding season is in the summer, but embryo implantation is delayed until late fall, at which gerbil. photo used with permission of american association for laboratory animal science. point identical quadruplicates are always formed (balamayooran et al., ) . the armadillo's low body temperature, and susceptibility and physiologic response to the infectious organism, mycobacterium leprae, have made it an ideal model for studying leprosy (balamayooran et al., ) . the consistent polyembryony of the species has also made the animal a model of interest in understanding various aspects of twinning (blickstein and keith, ) . choosing the correct animal model is an essential component 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amniotic epithelial cells and repair of limbal deficiency in rabbit models rats, lice, and history key: cord- -safr z authors: alexander, paul elias; debono, victoria borg; mammen, manoj j.; iorio, alfonso; aryal, komal; deng, dianna; brocard, eva; alhazzani, waleed title: covid- research has overall low methodological quality thus far: case in point for chloroquine/hydroxychloroquine date: - - journal: j clin epidemiol doi: . /j.jclinepi. . . sha: doc_id: cord_uid: safr z what is new? key findings: clinical decision-makers must be informed by the best, most trustworthy, highest-quality, robust evidence. this translates into how much confidence we can have in the research findings and thus be optimally informed for decision-making. the estimates of effect in clinical research depends on the underlying research methodology. covid- disease is presenting global health systems, clinicians, and patients grave challenges. no treatment or prophylaxis currently exists for covid- . the overall body of covid- research is very flawed methodologically. an examination of hydroxychloroquine-azithromycin research findings due to the recent media focus revealed very low-quality methodology underpins the research. vast amounts of time and resources are being allocated to covid- research, and being potentially squandered. what this adds to what was known: flawed methodology and sub-optimal reporting of research findings could lead to biased estimates of effect. this could lead to treatment decisions that are not optimal based on biased estimates which could harm the patient. this article provides specific suggestions for improving on the covid- methods and reporting with a focus on the issues that researchers must consider in their methodology and reporting if we are to have confidence in the estimates of effect. failure to consider harms in research could be detrimental to the patient. this article focuses on the potential harms when therapeutic agents such as hydroxychloroquine, are being considered. what is the implication and what should change now: research thus far on finding an optimal therapeutic agent (s) for covid- could be hampered by methodologically flawed research. covid- researchers must immediately and acutely focus on improving their methodology and reporting. no current treatment or prophylaxis has proven to be effective in coronavirus disease , and patients receive either symptomatic treatment for milder presentations or more advanced life-support strategies in moderate to severe cases. as the global community eagerly awaits credible scientific solutions for this pandemic, researchers and scientists are under much pressure to identify effective therapeutic and preventive strategies for covid - . this commentary focuses on the quality of current covid- research. we used published clinical studies on chloroquine/hydroxychloroquine as an example to demonstrate some of the methodological concerns around research currently conducted in the field. in recent weeks, academic journals and public media published and disseminated information on the use of quinine derivatives (i.e., chloroquine and hydroxychloroquine) ( , ) for the treatment of covid- . chloroquine has long been and still is used to prevent and treat malaria. whereas, hydroxychloroquine was first used to prevent and treat malaria and is currently used to treat rheumatoid arthritis, some symptoms of systemic lupus erythematosus, juvenile idiopathic arthritis, and other autoimmune diseases. although ongoing clinical trials are testing the efficacy and safety of several treatments for covid- , including chloroquine and hydroxychloroquine, there is not yet credible evidence from clinical trials on the efficacy and safety of those agents in covid- . most of the data released or published thus far on chloroquine/hydroxychloroquine, and covid- research in general, are imprecise and at high risk of biased estimates of effect. here lies our concern. this pandemic emergency is fraught with tremendous uncertainty about the evidence on treatment or prophylaxis. there are many unknowns, and the massive demand for evidence on the treatment of a novel disease like covid- may be unintentionally affecting studies' design and conduct. furthermore, it may inadvertently affect the peer-review and publication process, leading to significant methodology gaps and overall lower quality evidence on covid- . these gaps lead to less informative studies, loss of precious time, and valuable resources. therefore, current research should balance feasibility and efficiency against methodological rigor and carefully address methodology gaps as much as possible. in order to support universal clinical decision-making and minimize harm, the research community should focus on conducting and publishing trust-worthy evidence. hypothesis generating studies are welcome and essential but should be explicitly framed as such, acknowledging their limitation, and not be used for critical decisions, at the national or global level. furthermore, experimental studies aiming to confirm or refute these observations should be supported. until there is a certainty, patients deserve, for themselves and for the sake of future patients, to be randomized in ethical and well-designed clinical trials. our concerns on the state of the chloroquine and hydroxychloroquine research thus far expand to the quality of research methodology we see spanning across covid- research. we sought to draw attention to this. to optimally inform our appraisal of the quality of covid- research, we searched the medline and embase electronic databases to identify clinical studies on the use of these agents in covid- ( to april rd , ), finding initial citations that were screened for eligibility, yielding six clinical studies judged eligible ( ) ( ) ( ) ( ) ( ) ( ) . based on the quality of the evidence to date, can a clinical practice guideline issue any recommendations on the use of chloroquine or hydroxychloroquine alone or combination with other treatments? the case has not been made either way for these quinine derivatives. the body of evidence thus far has been from both in vitro studies ( ) ( ) ( ) and clinical studies ( ) ( ) ( ) ( ) ( ) ( ) of sub-optimal methodological quality. the emerging clinical studies such as case-series, cohort studies, and randomized control trials (rcts), are all inconclusive; while some may suggest some benefit, the entire body of evidence does not reach the level of certainty and confidence that is required to justify the use of quinine derivatives as a treatment for covid- ( ) ( ) ( ) ( ) ( ) ( ) . as exciting as some of these results may appear, the underlying research methodologies are often flawed, and as such, the reported results cannot be trusted. a most recent french prospective study ( ) on the use of chloroquine in combination with azithromycin completely contradicts a prior french study asserting the benefit of the combination ( , ) . when critically appraised using the appropriate risk of bias tools, all studies ( ) ( ) ( ) ( ) ( ) ( ) have been classified as high risk of biased estimates of effect ( , ) (table ) . overall, the methodologies of the published studies are not robust, and the results are tempered by selection bias and residual confounding bias. at the design level, most studies lack the randomization and blinding/masking needed to generate sound evidence, when they are rcts and not observational uncontrolled single arm case-series. at the analysis level, they lack the standard steps taken to minimize confounding such as prospective design, statistical adjustment for prognostic factors, (propensity) matching, or stratification ( ) . the reporting is very sparse, and patient-important outcomes needed for decision-making are often not studied or not reported. we do understand the urgency to identify effective treatments, as well as the barriers to perform rigorous research in health care settings overwhelmed by an unprecedented workload and a novel deadly disease. however, these unprecedented and unfortunate circumstances do not transform flawed data into sound results. the adequately powered, comparative, rigorous effectiveness research that is needed for optimal evidence-informed decision-making remains absent. researchers need to prioritize minimizing bias by randomizing a large enough number of patients and masking the treatment allocation as much as it is feasible, and fully accounting for all the patients enrolled in the study following the intention-to-treat analysis principle ( table ). these critical components of high-quality, trustworthy research are required to generate high confidence that the estimates of effect reflect the true effects. at this time, more than ever, the high-quality, robust, comparative evidence from ethical randomized controlled trials (rcts) is urgently needed to assess patient-important outcomes, including mortality, morbidity, need for life support, safety, and toxicity, informing on the safe use of chloroquine or hydroxychloroquine (with and without azithromycin) in people with covid- . again, we make this clarion call across the breadth of covid- research, and across all research in general. of course, these studies will need to be fast, and even better, conducted in a flexible framework (such as adaptive trials) able to accommodate the adding of and switching to different treatments as soon the ones under study are proven ineffective or more promising alternatives are suggested. consideration of master protocols (harmonization of efforts) and adaptive trial designs become very important ( ) . the world health organization (who) has advocated for randomized multi-center adaptive clinical trials to evaluate the efficacy and safety of investigational therapeutic agents in combination with standard-of-care for the treatment of hospitalized patients with novel coronavirus disease (covid- ) ( ) . researchers aligning their methodology to who's master protocol will surely improve quality of the covid research ( , ) . additionally, global research groups conducting clinical trials would help in disseminating their results by adhering to the consort (consolidated standards of reporting trials) checklist for optimal clinical trial reporting ( ) and the strobe (strengthening the reporting of observational studies in epidemiology) statement that outlines the guidance for optimal reporting of observational studies ( ) . this is critically important and especially in this covid- urgency when very serious national and global public health decisions are being made based on what information is shared (or not shared). evidence exists to show that clinical trial results are biased when the trials utilize inferior methodology or report findings without satisfactory description of the methods used. failing to conceal allocation of the generated sequence has been linked to an exaggeration of the effectiveness of % or greater ( ) . the urgency of the covid- situation would also make it appropriate to be creative and move beyond the classical modalities and boundaries of academic research: what if a mobile app was made available by a respectable institution to allow randomizing any small number of consenting patients, collecting a small set of relevant covariates (age, sex, days since diagnosis, relevant comorbidities), by any doctor willing to participate in a chloroquine or hydroxychloroquine trial wherever the treatment is available for compassionate prescription (i.e. most of the world)? what if mortality in the two groups, masked as being treatment and control, was posted on a website every patients reaching the outcome (recovered or dead) to transparently show if equipoise persists? what if the dataset at patients, or every patients if needed, was made publicly available for highly skilled statisticians to propose their interpretation? we would get patients every few days, and we would be receiving clinically sound results faster than any traditional study framework. of course, this flexibility may not warrant publication in a top tier journal but could save thousands of lives. we need to use the most optimal methodology and not compromise on rigor but be willing to think outside of the box. the outcomes being reported in the covid- research thus far are informative but are not ideally patient-important that could help in patient and clinician decision-making. in addition, research question gaps are glaring and future methodologically strong comparative research of quinines for covid- (as well as other drug treatments) should assess the following: ) the net benefit ratio of chloroquine or hydroxychloroquine alone or in combination with other interventions; ) specific subgroups classified by age, stage and severity of illness, and other potential effect modifiers; and ) optimal dosing and timing of dosing. we do believe there is still full equipoise justifying the continued investigation of the role of chloroquine/hydroxychloroquine in studies in patients hospitalized with covid- . caution is urged regarding large scale uptake of the treatment as open-label use of the drug. why extreme caution is urged in using chloroquine, hydroxychloroquine, or any drug? we could be doing more harm than good. primum non nocere -first, do no harm. potential adverse effects, toxicities, and medication interactions must remain key considerations when using any drug, and chloroquine or hydroxychloroquine, in combination with azithromycin, are no exception. indeed, the evidence suggests that both drugs prolong the qt interval leading predisposing patients to serious arrhythmias ( ) ( ) ( ) ( ) . this is of particular concern when co-administering macrolides (azithromycin) ( ) , also known to affect cardiac electrophysiology. indeed, the use of doxycycline ( ) has been proposed in azithromycin's place. the u.s. food and drug administration (fda) has even warned the public that azithromycin (zithromax or zmax) can potentially cause irregular changes in the electrical activity of the heart and could lead to a potentially fatal irregular heart rhythm ( ) . in this regard, researchers recently looked at covid- infected patients who were administered a hydroxychloroquine/azithromycin combination ( ) . they found that the qtc was prolonged maximally after - days from the beginning of treatment, and in patients the qtc increased more than ms. they also found that in patients ( %), the qtc increased to > ms, indicative of a high-risk group for arrhythmia. this issue of potential harmful effects and the urgent need for high-quality, methodologically robust studies also comes from a recent pre-publication (not yet peer-reviewed) of in vitro activity of hydroxychloroquine or chloroquine in combination with metformin (used in treatment of type diabetes to lower blood sugar in humans) in mice ( ) . researches reported that when hydroxychloroquine or chloroquine was combined with metformin as a possible anti-cancer drug, - % of all mice died. until more high-quality evidence is available and particularly that excludes harm, caution is urged especially with combination therapy outside rcts. there must also be acute pharmacovigilance and monitoring of adverse drug reactions with regards to these and other drugs in covid- patients given so much in unknown. some countries have chloroquine or hydroxychloroquine readily available, in some cases as an overthe-counter medicine as they are required to treat common conditions. national authorities should be mindful of these situations and take measures to govern the use of these medicines and prevent dangerous self-medication. on the equity side, there is a concern of massive purchasing, including for both personal use and research, leading to possible shortage of supply. this can lead to treatment shortages for malaria and autoimmune diseases where there is confirmed benefit. the use of existing drug treatments such as chloroquine and hydroxychloroquine outside of current guidelines and recommendations may result in adverse effects, including serious illness and death, affect patients with other diseases who may benefit from its use, and hinder the ability to conduct clinical trials if there are high demands to sue these agents by clinicians and patients. toxic results from in vitro studies may not translate to toxicity in humans, but we caution that care must be exercised in extrapolating in vitro results before establishing clinical efficacy and safety ( ) . in summary, while the massive effort in generating and disseminating evidence globally in response to covid- has to be applauded, the accumulated body of evidence thus far can at best be considered hypothesis generating due to the methodological flaws across covid clinical research. the methodological quality of ongoing and planned clinical research has to be urgently upgraded if the vast amount of time and resources are not to be squandered in this emergency. until availability of sound results from one or more covid- clinical trial (s) showing a favorable risk-benefit, caution is urged in considering indiscriminate use of these two drugs, alone or in association. public health authorities are urged to prioritize resources on those interventions that are currently recommended as standard of care and ensure that empirical clinical use or research on these medication does not endanger adequate supply for the patients that need it for conditions in which the efficacy is already known. declaration of interest: none to declare but note, most of the co-authors are heavily involved in guideline development as well as the grade working group, including the guide methods group out of mcmaster university, hamilton, ontario. roles in the study and manuscript: none for the project what is new? clinical decision-makers must be informed by the best, most trustworthy, highest-quality, robust evidence. this translates into how much confidence we can have in the research findings and thus be optimally informed for decision-making. the estimates of effect in clinical research depends on the underlying research methodology. covid- disease is presenting global health systems, clinicians, and patients grave challenges. no treatment or prophylaxis currently exists for covid- . the overall body of covid- research is very flawed methodologically. an examination of hydroxychloroquine-azithromycin research findings due to the recent media focus revealed very low-quality methodology underpins the research. vast amounts of time and resources are being allocated to covid- research, and being potentially squandered. flawed methodology and sub-optimal reporting of research findings could lead to biased estimates of effect. this could lead to treatment decisions that are not optimal based on biased estimates which could harm the patient. this article provides specific suggestions for improving on the covid- methods and reporting with a focus on the issues that researchers must consider in their methodology and reporting if we are to have confidence in the estimates of effect. failure to consider harms in research could be detrimental to the patient. this article focuses on the potential harms when therapeutic agents such as hydroxychloroquine, are being considered. research thus far on finding an optimal therapeutic agent (s) for covid- could be hampered by methodologically flawed research. covid- researchers must immediately and acutely focus on improving their methodology and reporting. none to declare but note, most of the co-authors are heavily involved in guideline development as well as the grade working group, including the guide methods group out of mcmaster university, hamilton, ontario. roles in the study and manuscript: none for the project chloroquine and hydroxychloroquine as available weapons to fight covid- breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease- (covid- ) efficacy of hydroxychloroquine in patients with covid- : results of a randomized clinical trial url: med rxiv preprint doi pre-publication. not peer-reviewed treating covid- with chloroquine hydroxychloroquine and azithromycin as a treatment of covid : results of an open label non randomized clinical trial clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in covid- patients with at least a six-day follow up: an observational study; prepublication no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid- infection risk of bias in rcts risk of bias in non-randomized studies confounding and adjustment /chapter_ / _ _ _ _confounding_and_adjustment.htm (accessed on the strengthening the reporting of observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies empirical evidence of bias. dimensions of methodological quality associated with estimates of treatment effects in controlled trials the need for novel trial designs, master protocols, and research consortia in transplantation who r&d blueprint novel coronavirus covid- therapeutic trial synopsis suspected hydroxychloroquine-associated qt-interval prolongation in a patient with systemic lupus erythematosus torsade de pointes and cardiorespiratory arrest induced by azithromycin in a patient with congenital long qt syndrome azithromycin and the risk of cardiovascular death azithromycin and risk of sudden cardiac death: guilty as charged or falsely accused? long island doctor tries new twist on hydroxychloroquine for elderly covid- patients drug safety communication: azithromycin (zithromax or zmax) and the risk of potentially fatal heart rhythms the qt interval in patients with sars-cov- infection treated with hydroxychloroquine/azithromycin. pre-publication (non-peer reviewed) fatal toxicity of chloroquine or hydroxychloroquine with metformin in mice chloroquine is effective against influenza a virus in vitro but not in vivo. influenza other respir viruses key: cord- - g jebq authors: nan title: in the realm of opportunity: the kaiser wilhelm institute for anthropology, human heredity and eugenics during world war ii, / – date: journal: the kaiser wilhelm institute for anthropology, human heredity, and eugenics, - doi: . / - - - - _ sha: doc_id: cord_uid: g jebq on march , , eugen fischer wrote a long, confidential letter to otmar von verschuer, director of the institute for genetic biology and race hygiene at the university of frankfurt at that time. in this letter fischer expressed critique — and certainly also self-critique — about the scientific development of his institute since the mid- s. speakers for public lectures, as representatives of the new germany at international congresses, all of which were performed at the cost of the scientific work. more serious was that the emphases of research had shifted as a consequence of the interconnections with politics -and not necessarily in the direction fischer would have wished. in , under pressure from arthur gütt and with a view to the genetic health policy of the new rulers, fischer had placed the stress on genetic pathology. with verschuer's departure in , fischer had ceased forcing genetic pathology research, although the projects in progress were continued, and the emphasis was shifted to strengthening genetic psychology instead. the dual course shift had the result that the research program was visibly fragmenting into unrelated, individual projects. fischer recognized that the institute was in danger of losing its scientific focus. as he wrote in his letter to verschuer of march : aside from the problem of demarcation from the university clinics and institutes working in the area of genetic pathology, fischer probably foresaw that a clinical orientation of his institute would cause conflicts of competence with two other kaiser wilhelm institutes: the kaiser wilhelm institute for brain research, which, under the direction of hugo spatz since , had turned increasingly to questions of genetic pathology in neurological disorders, mental illnesses and mental disability, and above all with the german research institute for psychiatry in munich under rüdin. in munich the very founding of the department for genetic psychology had been taken as an affront, and the vehement exchange of blows concerning diabetes research in had shown that the two institutes were bound to get in each other's way in the field of genetic pathology. against this background it seemed to fischer, who had never worked purely clinically anyway, that a onesided orientation of research at the kwi-a toward genetic pathology -while neglecting fischer's own original research field, the heredity of "normal" (not pathological) attributes -to be a strategic mistake, although he certainly held genetic pathology to be a constitutive element of his further research strategy. but as fischer established self-critically, his own research in the field of physical anthropology did not have the potential to constitute a new paradigm either: for my own (personal) work, of course, i have plans that include my doctoral students. the one project is, as for many years, the research of the conditions for the shape of the skull; the other the bastardization problem. but these two topics cannot fill an entire institute, and assistants from the field of medicine, who want to pursue practical activity later, cannot, or at least not exclusively, be set to work on such subjects. and since you have been gone i feel both a great void and a sense of being orphaned. the department for human genetics had been dissolved in , as mentioned above. in addition to the department for anthropology led by fischer himself, the only pillar of the institute that remained was the department for race hygiene headed by fritz lenz. however, fischer lamented, it could hardly be expected that new impulses would come from there. for lenz does not take care of any of this. he works without planning, assigns themes to pupils without a plan. these themes are individual questions of a genetic pathology or race hygiene biological nature, which occur to him while editing baur-fischer-lenz or during his critical perusal of the literature. such occasionally arising, isolated themes were to be pursued in the future as well, and the work of the department for genetic psychology under kurt gottschaldt was to be continued according to fischer's wishes. but as to the main point, such an institute needs an ambitious plan. and because i can not receive it from lenz's sphere of interest, let alone through his initiative, i do it alone. but such a plan is conceived for a number of years, and certainly -not only presumablylonger than i will be in office here. because i do not doubt in the least that you will be my successor someday, in truth i would like to begin with a long-term plan only if i have the hope that you like it enough to pursue it further. term for all factors affecting phenogenesis that did not lie directly in the genes. fischer emphasized that the "series of forces" triggered by genome and peristasis do not simply complement each other cumulatively, but are related in a very complex system of interdependence and synergy that is subject to constant change. the task of phenogenetics was thus to disentangle the networks of effects exerted by genes and peristatis for analytical purposes and to pursue their effects and interactions all the way up to the complete phenome. methodologically this task was to be approached through a combination of classical genetics, embryology and developmental mechanics, anthropometry and clinical diagnostics, whereby fischer repeatedly emphasized the utility of combining the animal model with observation of humans. at this juncture it is worth taking another look across the atlantic. since the early s the interest of geneticists there, too, encouraged in part by the rockefeller foundation, shifted increasingly to physiological genetics. in sharp competition with the research group around kühn, the geneticist george beadle and the embryologist boris ephrussi worked in pasadena to bridge the gap between classical genetics and developmental physiology. they may have lost the race to identify kynurenin, but in the early s beadle and edward lawrie tatum succeeded in finding evidence for the "one-gene-one-enzyme" hypothesis they had advanced during experiments on the neurospora mold -a pioneering success, for which they were awarded the nobel prize in . while this had directed attention to the effects of genes, the embryologist, geneticist, and evolutionary biologist conrad h. waddington broadened the horizon in with his concept of "epigenetics," in which he -in nearly the same words as eugen fischer years previously, and also with reference to valentin haecker's phenogenetics -shifted the complex developmental processes from the genotype to the phenotype into the focus of interest: for the purposes of a study of inheritance, the relation between phenotypes and genotypes can be left comparatively uninvestigated; we need merely to assume that changes in the genotype produce correlated changes in the adult phenotype, but the mechanism of this correlation need not concern us. yet this question is, from a wider biological point of view, of crucial importance, since it is the kernel of the whole problem of development. many geneticists have recognized this and attempted to discover the processes involved in the mechanism by which the genes of the genotype bring about phenotypic effects. the first step in such an enterprise is -or rather should be, since it is often omitted by those with an undue respect for the powers of reason -to describe what can be seen of the developmental processes. for inquiries of this kind, the word "phenogenetics" was coined by haecker. the second and more important part of the task is to discover the causal mechanisms at work, and to relate them as far as possible to what experimental embryology has already revealed of the mechanisms of development. we might use the name "epigenetics" for such studies […] . the concept of epigenetics has since undergone a series of transformations, but has managed to persist -off the track of mainstream genetics. indeed, in the most recent bioethical debates many hopes are pinned on the concepts of epigenesis and epigenetics, as they promise to break open the "reductionist approach" of classical genetics. from fischer's perspective, the new paradigm of phenogenetics offered several advantages. first, it permitted a whole series of projects that had been performed at the kwi-a and its periphery in the s, which were rather loosely connected, to be related to each other under a complex of issues that applied to all of them. this was true first of all for a great number of works by wolfgang abel, georg geipel, bernhard duis, and others on the methodology of dermatoglyphics, that is, the genetics of epidermal patterns, on the increased frequency of characteristic epidermal ridge patterns for certain human races, and on the connections between defective epidermal ridge patterns and physical disability or mental diseases. fischer attributed a central position in the field of phenogenetics to the works of his earlier pupil konrad kühne on the genetics of the variations of the spinal column, which had been continued at the kwi-a in maria frede's work on rats. great value was also ascribed by fischer to the embryological studies by rita hauschild on the skulls of negroid and caucasion fetuses and by baeckyang kim about race differences in embryonic pig skulls -even at the time, both studies were understood explicitly as contributions to phenogenetics. according to fischer, various other works originating from the kwi-a on morphology and the genetics of human hair growth, the auricle, on asymmetries in body structure, on the heritability of stature, on miscegenation and on genetic pathology could also be classified under the umbrella of phenogenetics. on the other hand, second, the paradigm of phenogenetics demarcated a broad research area that the existing works had barely begun to cover, and which was underdeveloped in terms of both breadth and depth. at the congress in würzburg, fischer established retrospectively in , it became apparent that the "true course of development" was only really known for coloboma, the congenital gap in the eye area due to the insufficient closure of the fetal eye cleft, on the iris, choroid, lens, or lid. just as we have a history of development of every normal organ, we should have an exact history of development of every hereditary disease. everything has yet to be done here. much is also missing on the heredity of normal things. the paradigm of phenogenetics was thus open enough to provide the foundation for a comprehensive research program with questions covering all of its areas. a further advantage of turning toward phenogenetics was that, third, the research focused on a form of human genetics that was compatible with the orientation of developmental genetics predominant in german animal and plant genetics at the time. in his lecture in würzburg, fischer referred to the work of the developmental physiologist hans spemann , who had performed experiments on amphibian embryos in the s, which proved that some parts of the embryo, such as the primitive roof of the mouth and the eye socket, act as "organizers" to "induce" the formation of other structures in the embryo. spemann had not attempted to explain the inductive effect of the organizers by investigating the genes. alfred kühn made more progress on this with his experiments on the flour moth ephestia kühniella, to which fischer referred several times in his comments. kühn had found a mutant ephestia with red eyes rather than the usual black ones, and his doctoral student ernst wolfgang caspari ( * ) was able to prove that, by injecting tissue from wild moths into the larvae of the red-eyed mutants, the eye color of the mutant could be adapted to that of the wild type. a substance missing in the mutants was apparently added through the injection. genes, it was concluded, obviously work through enzymes. if an enzyme is lost through mutation, this can block the transformation of a certain substance into another. through artificial implementation of the missing enzyme -in the case of the light-eyed ephestia this was the tryptophan derivate kynurenin, as two assistants to adolf butenandt , erich becker and wolfhard weidel, were able to prove -it was possible to generate a "phenocopy" of the wild type, an idea that apparently fascinated eugen fischer. yet, even more often than he referred to kühn, fischer brought up hans nachtsheim's work on the genetic pathology of rabbits. consequently, in march fischer presented to verschuer the idea of bringing nachtsheim to the kwi-a as director of a new department for experimental genetic pathology, in order to supplement his studies on the phenogenesis of genetic illnesses of the rabbit with "parallel studies of a clinical nature on humans" as a way of connecting animal and human genetics. fischer's remark, "i have no idea whether he [nachtsheim] would want to," indicates that fischer had not yet negotiated with nachtsheim at this time. he first wanted to await his designated successor's opinion of this plan, and further of the plan associated with it -quite explosive in terms of institute politics -to "completely dismantle" the department for race hygiene headed by fritz lenz, as "race hygiene could then be taken care of in the university institute." in other words, the kwi-a was supposed to give up race hygiene as a field of research, and fritz lenz gradually be forced to the margins. fischer closed his letter to verschuer with the request that he not answer in writing, for he hoped that there would be opportunity at easter to discuss the complex of topics in person. hence we know nothing about verschuer's immediate reaction. yet the further course of events suggests that verschuer fully agreed with "drosophila genetics has been our pacemaker until now. this appears to be nearly over; it no longer teaches us anything new." (ibid.). this can be interpreted as a renunciation of the drosophila genetics at the genetic department of the kwi for brain research under nikolaj v. and elena a. timoféeff-ressovsky. of course, it must be remembered that the timoféeff-ressovskys, before turning to the genetics of mutations and populations, made significant contributions to developmental genetics. cf. harwood, styles, pp. f. the research plan developed by fischer. the two most important conceptual works by verschuer from the year -his lecture about "the genotype of humans" to the main assembly of the kaiser wilhelm society in breslau on may , and his presentation "on the genetic analysis of humans" for the th international congress for genetics in edinburgh (which he was not able to give himself due to the early departure of the german delegation, but was read to the audience on august , ) -first of all show that verschuer picked up on fischer's impulses immediately, and also illuminate the background against which fischer's and verschuer's new conception must be viewed: over the course of the s, classical mendelian genetics was undergoing a dramatic and extensive process of transformation. the idea generally accepted up to that time, that every attribute was simply transmitted as dominant or recessive, monofactorial genetic information, did not hold up to the results of mutation research, population genetics and developmental physiology. thus mendelian genetics was giving way, in the words of the day, to "higher mendelism, which presumed much more complicated mechanisms of heredity. it became generally accepted that genes could not be observed in isolation from each other, but only in the context of the genotypical setting -the effect of one gene was always influenced by other genes, and even by the genome as a whole. it was acknowledged that the genes on the chromosomes are not just pearls strung on a string in any order, but that the effect of each gene depends on its position in the genome. with increasing clarity it became apparent that these mutual effects within the genome, but also prenatal influences on the intra-uterine environment during maturation of the embryo, and even influences from the external environment, had modifying effects on the way genes were manifested in the process of phenogenesis. the phenomenon of "weak genes" made its first appearance. the team of the kwi for brain research around nikolaj vladimirovich timoféeff-ressovsky and elena aleksandrovna timoféeff-ressovsky had attempted to grasp the phenomenon of the variations in how such genes were manifested in terms of the three concepts "penetrance" (the frequency with which a genetically conditioned attribute develops in the phenotype), "expressivity" (the degree to which it develops) and "specificity" (the nature of its development depending on the part of the body the gene must affect for this development) -a terminology that was picked up everywhere, including by fischer and verschuer. it was acknowledged that in many cases a single gene is involved in the development of several attributes (pleiotropy) and, inversely that the development of a single attribute can be influenced by multiple genes (polygeny). furthermore, the advances in differential diagnostics showed that one and the same clinical picture can be caused by both genetics and environment (heterogeny). finally, the results from radiation and verschuer, erbbild vom menschen; idem., bemerkungen zur genanalyse. cf. roth, schöner neuer mensch, pp. - . the term was coined in , probably by günther just, and picked up on immediately by verschuer. cf. just, probleme des höheren mendelismus; verschuer, genetic pathology, nd edn., p. . cf. weß, humangenetik, pp. - . population genetics suggested that the rates of mutation were higher than originally presumed, but that the heterozygotic mutants did not become visible because the gene did not necessarily develop in their phenotype. verschuer, as his lecture texts indicate, was completely up to date in the contemporary specialized discourse; his institute in frankfurt had even made a significant contribution to theoretical research in human genetics in , when one of verschuer's staff members, bruno rath, on the basis of a family study of a "bleeder clan," succeeded in finding the first proof of a crossing-over (exchange of genes or gene sections through the recombination of chromosome fragments) in humans. against the background of higher mendelism, verschuer was fully aware that the previous conception of human genetics required greater differentiation, an expanded catalog of questions, and a larger arsenal of methods. fischer's suggestions could hardly have come at a better time. in his breslau lecture verschuer first related the success story of human genetics. by that time around , of the estimated , - , genes in humans were known, along with several hundred hereditary diseases, for most of which the heredity had been illuminated. "the human being is an object of the human sciences that has been examined in manifold ways […] ." yet there remained much to be done. genetic analysis could no longer be content with using the methods of family and twin research to reveal the dominant or recessive mode of inheritance of a gene. on the contrary, a whole bundle of new questions had to be posed: in summary it can be stated that the cognitive advances in the field of human genetics in the s practically forced an expansion of classical mendelian genetics. theoretically, expansion was conceivable in various directions -from mutation research, to the "synthetic theory of evolution," all the way to molecular genetics. however, considering its own resources, and also with a view to the orientations of competing research institutions, the paradigm of phenogenetics seemed most promising for the kwi-a. moreover, it was quite advantageous for verschuer, because his personal research interest in clinical genetic pathology dovetailed perfectly with the new paradigm. as such -another point that must not be overlooked -it was practically tailored to verschuer and provided him a weighty advantage over lenz as a potential rival for fischer's succession. at this juncture our discussion turns to the thesis by niels c. lösch, who goes into great detail about the changes that took place under the banner of phenogenetics at the kwi-a in the years - . however, because he is fixed too one-sidedly on personnel-policy strategies, he interprets the development as a kind of "false label" designed to "prepare the ground for verschuer." there is no question that fischer, who celebrated his th birthday on june , , had been building up his pupil and friend verschuer as his successor for a long time already, and that the establishment of the new paradigm of phenogenetics was linked intimately with verschuer's person. it is also indisputable that fischer had long since begun taking precautions to nip in the bud any aspirations fritz lenz might have for the post of the institute director -although, it must be added, there are no indications that lenz pursued ambitions in this direction. in september , when fischer, much to his dismay, learned of the plans to call verschuer to frankfurt, he immediately began considering whether the chair in frankfurt could serve verschuer as a stepping stone on the path to succeeding fischer. in november -that is, while the würzburg lecture was being printed -fischer then set the course for the future development of the institute in a talk with ernst telschow, general secretary of the kaiser wilhelm society: "for the case of his departure upon reaching the age limit in - years, professor fischer nominated professor verschuer of frankfurt as his successor." in march fischer informed verschuer of this conversation, after congratulating him for the most recent evidence of the kaiser wilhelm society's favor -"exchange professor in london! speaker at the general meeting […] some time ago mr. telschow and i had a long talk, during which we also discussed you in great detail. he is informed for now and for the future and was entirely of my opinion." in his exchange of opinions with telschow, fischer had indicated that he rejected the plan to set up a major institute for anthropology at the friedrich wilhelm university in berlin, advising telschow "to pare this institute down considerably and plan it […] merely as an institute for race hygiene. as director prof. lenz would then be suitable on a full-time basis." this is yet another indication of fischer's strategy of strengthening lenz's role at the university and pushing him to the margins of the kwi-a, even though at this point in time he was still advocating lenz's appointment as deputy director of the institute. in july -by this time fischer had coordinated his plans for reorganizing the institute with verschuer -the departing director expressed himself more clearly to telschow: in repetition of earlier conversations, prof. eugen fischer designated prof. von verschuer in frankfurt as a suitable successor. it would then be appropriate to grant prof. lenz the title of "director" because of his age, without entrusting him with the direction of the institute. prof. fischer held it even more appropriate to transfer prof. lenz to the institute for race hygiene at the university of berlin, which -at present consisting of two rooms in the hygiene institute -would have to be expanded. then, in october fischer got down to brass tacks: within his institute prof. fischer wants to have a special institute for race hygiene under prof. lenz, who thus would receive the title of "director," as it were, but without becoming prof. fischer's deputy. on the contrary, prof. fischer wants to prevent this, in consideration of the proposed succession to his position by prof. von verschuer. at the same time fischer conveyed his intention to rename his institute. in the future it was to be called the "kaiser wilhelm institute for genetic and race science" (kaiser-wilhelm-institut für erb-und rassenkunde) and thus in its very name express the demarcation from race hygiene. fischer's attitude toward race hygiene was expressed quite clearly year later, in october , when he argued -much more aggressively than lenz, who lacked the requisite tact -for the expansion of the university's race hygiene institute: […] today race hygiene [has] become a state policy, it no longer requires propaganda. race hygiene is a required lecture and examination subject for medical students. i can no longer recognize race hygiene as such as a research subject; rather, the research subject is its substrata, first of all human genetics and then demographics. […] for these reasons i hold the expansion of a university institute for race hygiene at the greatest german university to be a quite self-evident necessity. […] because of the auspicious historical development in the third reich, at the kaiser wilhelm institute this race hygiene department must be dismantled rather than expanded. race hygiene, as the theory of the practical implementation of the knowledge in human genetics and demography, one could summarize fischer's argument, had a right to exist at the kwi-a during the weimar republic, but in the third reich race hygiene seemed to him unnecessary baggage that distracted the institute from its theoretical research. by no means did fischer want to force lenz out of the institute entirely -as an astute critic he made a major contribution to the conceptual foundations of the research at the kwi-a. the fact that he did not want to see lenz in the director's post concerned not only his lack of qualities as a science manager, and was not founded only in personal animosities between the two scientists -that, too -but above all lenz was far too much a proponent of classical race hygiene, which fischer did not believe had much potential for innovation. the result is indisputable: fischer machinated behind the wings in order to guarantee that verschuer would be appointed director of the kwi-a and to prevent lenz from offering himself as an alternative candidate. in contrast to niels lösch, however, this author advocates the thesis that the paradigm of phenogenetics was also, but by no means only a means to an end in order to prejudice an impending personal-policy decision. the realization of the newly developed research conception presupposed a kind of package solution: the new orientation of research in progress; opening up new areas of work, but also relinquishing areas that could not be fit into the new paradigm in a meaningful way; integrating scientists who fit into the new research profile; changes to the internal structure of the institute; the creation of an infrastructure to implement new methods in practice, and finally the solicitation of the additional financing these tasks would require. in the years - fischer resolutely pushed ahead in all of these directions, but initially he met with considerable resistance. this setback opened up a precarious phase in the reorganization of the institute, for the new department for experimental genetic pathology was to become one of the pillars supporting phenogenetic research, if not the supporting pillar. fischer needed political protection to succeed in implementing his plan by circumventing the general administration -but this strategy was also shaky, as hans nachtsheim was not exactly considered a convinced national socialist. nevertheless: fischer sought and found the necessary patronage of a functionary located high in the machinery of the national socialist regime: leonardo conti. it was convenient that a connection to conti already had been established, as mentioned above, albeit a loose one. in december conti had taken over the duties of the medical councilor of berlin. in this capacity he was entitled to a seat and vote on the boards of the kwi for brain research and the kwi-a. the kwi for brain research must have interested him less -in any case in november he appointed one of his closest staff members, director of the department for the care of genes and race in the main health office of berlin, dr. theodor paulstich ( * ), as his permanent representative on the board of this institute, although he reserved the right to participate in future board meetings himself. at first conti did not have anything to do with the kwi-a, either -in the years from to no board meetings took place. conti had since moved up to the pinnacle of civilian health care: in april hitler had appointed him as director of the main office for national health (hauptamt für volksgesundheit) and "führer of physicians of the reich (reichsärzteführer) and awarded him the title of reichsgesundheitsführer ("reich health leader"). in august conti was also appointed state secretary for health care in the reich ministry of the interior and thus held all of the reins to steer the health matters of the state and the party. when fischer endeavored to call a board meeting in january , he discovered that the previous chairman of the board, the premier of saxony, landeshauptmann richard otto, who had resigned his office as senator of the kaiser wilhelm society in "in quite an abrupt manner," no longer considered himself to be in office. because the general administration -after consulting with ministry director mentzel -did not regard the option of convincing otto to remain on the planck to conti, / / , mpg archive, dept. i, rep. a, no. , p. . max planck solicited conti's interest insistently: "because the next sessions of the two boards will probably not be held until the coming spring, perhaps you might first find an opportunity to tour the two institutes." for a biography: klee, personenlexikon, p. . cf. schmuhl, hirnforschung, p. . kater, conti; labisch/tennstedt, weg, vol. , pp. - . fischer to otto, / / , mpg archive, dept. i, rep. a, no. , pp. - . justifying the long interruption in board meetings, fischer stated that there had "never occurred anything in particular and on the other hand the years were so eventful politically that one wanted to dispose of the time of such very busy men as sparingly as possible." note by telschow institute's board to be opportune, the question of a successor was raised, an issue fischer and telschow discussed in a meeting on march , . apparently telschow's first suggestion here was leonardo conti, followed by walter groß. on the following day fischer expressed his opinion on these suggestions in writing, declaring himself completely agreeable to state secretary dr. conti. i find this proposal of yours especially good. of course, i would have nothing against dr. groß either; on the contrary, i would be pleased. but here my good personal relationship with groß should not be the crucial factor. as a responsible representative of race policy, groß is not as professionally close to the objectives of my institute as conti, the director of the medical and race hygiene department of the reich ministry of the interior. the connection to him would presumably be more important to the institute; in any case, i already have a connection with mr. groß. it would soon become apparent that this was quite a clever move, especially since fischer's strongest ally in the nazi health leadership up to that point, arthur gütt, had been ousted by an intrigue in , clearing the way for conti. but fischer needed strong political protection to realize his ambitious -and exceedingly costly -plans for the reorganization of the institute under the banner of phenogenetics. on october , the general administration of the kaiser wilhelm society officially filed fischer's proposal to offer conti the chairmanship of the board of trustees of the kwi-a. fischer, it was recorded there, wanted "first to personally approach [conti] on this matter." this personal meeting between fischer and conti took place on november , . it can be presumed that fischer took this opportunity to relate his plans for reorganizing the institute to the new strong man in the health policy leadership and acquire his support. in any case conti declared himself willing to accept the chairmanship of the board of trustees and call a board meeting immediately, which was initially scheduled for december , , but then postponed to january , due to conflicts with conti's schedule. telschow to fischer, / / , ibid., p. . fischer to telschow, / / , ibid., p. . the situation was all the more piquant because gütt remained a member of the board. conti proved magnanimous. he informed fischer that he woud find it "especially nice if gütt were retained on the board without further ado." fischer, as he let telschow know, had "the sense that he, too, wanted to avoid the appearance of having forced him [gütt] out." fischer himself spoke for gütt's remaining on the board: "since mr. gütt always had especially friendly interest in the institute and did much for it, i, too, would be very pleased if he remained on the board. of course, this is only possible if, first, the number of members would not be raised to beyond that allowed by any existing regulation, and second, if mr. gütt is not expressly nominated as a representative of his ministry." (fischer to telschow, / / , ibid ., pp. - v.) both were not the case, and consequently gütt remained a member of the board. telschow attending this decisive meeting were -besides fischer and conti -from the side of state and party, walter groß and hans reiter, further the medical councilor of berlin, theobald sütterlin ( * ); and -as representative of the german council of municipalities -from kiel, dr. klose; then the inspector of the army medical corps, general and chief staff physician siegfried handloser; from the side of the kaiser wilhelm society, general secretary ernst telschow and friedrich schmitt-ott; as representatives of science, finally, fritz von wettstein, director of the kwi for biology, otmar von verschuer, and -as a guest -fritz lenz. the new chairman of the board set a political signal right in his welcome message, by pointing out that activity and research of the kaiser wilhelm institute for anthropology, human heredity and eugenics is of particularly great importance for the state and that it would be wrong if -as it sometimes seems -interest in the meaning of issues of the heredity and race of our nation were to decline. the new greater germany needs such knowledge urgently, the next generation of scholars in this area must be provided for. the kwi-a as the "first and most outstanding" in this area must "serve and influence other institutes as a model." for this reason he, conti, had accepted the chairmanship of the board. telschow's comments about the institute's budget plans from to turned out to be considerably more sober. cuts of , rm from the regular budget had been necessary. the reich education ministry was not able to refrain from this cut, "although the other ministries relevant for the kaiser wilhelm institutes had not made such cuts in consideration of the institute's acknowledged status as essential for the war." on this subject, fischer elaborated that the institute had been able to "get over" the reduction due to the decline in the personnel budget, which had been eased as staff members were called up for military service, and thanks to savings in the material budget achieved by the "restriction of experiments" -although, fischer emphasized, at "the detriment to scientific achivement." however, a glance at the revenue and expenditure accounts of the institute and the auditing reports of the kwg for the fiscal years and indicate that telschow and fischer painted an exaggeratedly gloomy picture of the institute's financial situation, which did not correspond to reality -more on this later. the fifth agenda item, the "director's report about the erection of a new department for experimental genetic pathology" was the sensation. in a speech explaining the entire framework of his proposal, fischer submitted to the board his plans to reorganize the institute under the paradigm of phenogenetics. he started by for a biography: klee, personenlexikon, p. . the invitation was declined by arthur gütt providing detailed reasons why, in the middle of the war, he was submitting a research plan that pointed so far into the future. "the impending victorious conclusion of the war and the vast expansion of the greater german reich," fischer claimed, would also pose "great and new challenges" to the research institutes. while until now the institutes directly important for the war had stood at the foreground, like those in the areas of physics, chemistry, and technology, in the "near future" all institutes that dealt with "questions of genetic health, race, human selection, environmental influences" would become more important, as these were "of consequence for leadership." one could never know "how pure scientific research, often of a seemingly completely theoretical nature, will work out in practice in the future." and thus, fischer added somewhat less than humbly, there had been no way of knowing that his bastard studies of "one day could lay a foundation for race legislation." until his institute had transformed "the young field of human genetics into a securely founded, widely developed theory, […] which measured up to all demands of practical application in genetic consulting, genetic legislation, and as a basis of race theory and race legislation." by this time human genetic research was so far, fischer proclaimed boastfully, that the genes for all essential normal and pathological attributes were known "in principle," the external phenotype could be related to these genes and "to some extent […] the approximate scope of the environmental effects" was known. then came the transition to fischer's project of phenogenetics: but one large area here is still quite dark. this is the question of how a given genetic disposition actually develops, how it works, how the gene "does its thing" (metaphorically speaking) to obtain the external appearance it is due. the path from the finished genetic disposition to the completely developed genetic attribute is still unknown. to legitimate the new research program, fischer's argumentation stressed applicability. phenogenetics was not only of "greatest scientific interest;" beyond this it promised "practical medical utility, the direction of which i can only hint at: differentiability of genetic conditions, prophylaxis for the genetically encumbered and corresponding marriage consulting, treatment of symptoms." embedded in this context of justifications, fischer concretized his ideas for reorganizing the institute: first he emphatically championed the hiring of hans nachtsheim. for because "human embryonic material with certain pathologically determined genetic dispositions [could be] received only in very restricted amounts," one had to rely on "model experiments" on animals -and nachtsheim's rabbit breeding was the most suitable model by far. the study of genetic conditions of the rabbit must be linked closely with clinical research. iv. genetic illnesses: fetuses, newborns, and organs from families with certain genetic pathological dispositions (later sorting by illnesses) v. domestic animal races: fetuses and organs vi. animal genetic illnesses: fetuses, newborns, and organs from breeds with certain genetic pathological dispositions lösch advances the thesis that this central collection was "a new label for the institute's already existing, extensive collection of specimens." this is a misinterpretation, however -there had not been an embryologically oriented collection of fetuses, premature births and stillborn children at the institute before this time. back in fischer had placed appeals in the deutsche medizinische wochenschrift and the wiener klinische wochenschrift ("vienna clinical weekly"), asking practical physicians to supply the institute with such material. moreover, verschuer the only problem left was what should become of fritz lenz and his department for race hygiene. this was to "remain linked securely to the whole," but granted autonomy as an "institute for race hygiene" under "director" fritz lenz. to the board of trustees, fischer sang lenz's praises as a race hygiene pioneer. his strength lay in "positive suggestions, consulting with the responsible offices and oral and written instruction for students, physicians and the general public." due to his "unique character," however, he could "not be considered […] for the organization of the institute -a fritz lenz needs and has received unreserved relief and liberation from simple administrative and other institute activities in the interest of his theoretical work." this was an extremely elegant formulation to express that fischer held his department head to be unsuited for the post of director. the fact that lenz attended the meeting and did not contribute to the discussion again confirms the impression that lenz was altogether satisfied with the solution of an "institute in the institute" to which fischer aspired. as the negotiations concerning the extension of the race hygiene institute commencing later that year showed, lenz pinned his hopes on his institute in the institute receiving its own budget and the right to hire its own staff, and, if this would be guaranteed, was even willing to relinquish any claim to a larger institute for race hygiene at the planned university clinic. in closing fischer addressed the delicate issue of financing. he offered to finance the equipment and furnishings of the new department for experimental genetic pathology from institute funds, since the budget offered some latitude as a consequence of the restrictions to its work necessitated by the war. but additional finances were required for future operating costs, of which the personnel costs of , rm comprised the lion's share, as fischer intended to hire not only the department head, but also an assistant with experience in anatomy, pathology, and histology, a technical assistant, and an animal keeper. fischer estimated the additional material costs incurred by keeping animals at , rm, so that the future additional requirements amounted to , rm annually, and fischer wanted this sum in the form of a regular budget increase rather than as a special allocation. finally, new land was also required for the construction of stalls for the rabbits, as no more room was available on the grounds of the institute. lösch presumes that such a comprehensive concept for the reorganization of the institute "was expected by hardly any of those attending," and that it was "new in this dimension" even for telschow. however, this is not the case. in fact, fischer had sent a written draft of his talk to both telschow and conti back on december , . telschow thus would not have felt "affronted," as lösch presumes; rather, his comments about necessary budget cuts simply made clear once and for all whence the funds for the institute's modernization would not come. in so doing he had hit the ball back into the politician's court, where it was readily received. the game was rigged. for back on october , , in the very same conversation between fischer and telschow in which fischer officially made the proposal to give conti the chairmanship of the board of trustees, and offered "to personally approach [the reichgesundheitsführer] on this matter," fischer had laid out to the general secretary of the kwg his plan to equip the department for experimental genetic pathology, applied for a , rm increase in the personnel budget and , rm in the material budget, and for an investment of , rm for the rabbit hutches. fischer even brought with him to this meeting hans nachtsheim, who used the opportunity to negotiate with telschow about his future salary. his appointment was slated for january , . at the same time, according to a file note by telschow, agreement was reached that the additional funds would "of course not be demanded in professor fischer's budget request until after conclusion of the war." since, as we showed above, telschow did not believe that the war would be over within the year , only one conclusion is possible: fischer and telschow had agreed to ask conti for help in procuring the missing money from other sources for the time being. the course of the board meeting makes unmistakably clear that fischer had done precisely this in his meeting with conti on november , , and that conti had pledged his support. under conti's direction, the board thus recorded in the protocol that the discussion had reiterated for the record "the special importance of the new department"; the provision of , rm was also "designated as urgently necessary." as regards the purchase or leasing of property for the rabbit hutches, the reichgesundheitsführer pledged his "active support." conti's confidant sütterlin seconded the motion, signalizing the interest of berlin's city medical administration and promising its support as well. at the same time, sütterlin stated for the record "his satisfaction with the cooperation with the institute achieved in the working group with dr. diehl in sommerfeld." even should the hospital change leadership, sütterlin ensured, diehl's research could be continued without restriction. finally, army medical inspector handloser also wished to have "his special interest in the work on rabbit tuberculosis" written in the protocol. yet again it must be emphasized that fischer's push surprised neither conti and his right hand sütterlin nor telschow, nor verschuer and lenz -in a sense, the roles had already been distributed in the preliminary talks, and the course and result of the consultations set beforehand. the entire meeting was completed in just min, and the society retired to harnack house for a snack. on the very day after the board meeting, conti turned to telschow to coordinate the next steps. the general secretary of the kwg drew up two letters in the name of the reichgesundheitsführer, which he forwarded for conti's signature. one was directed to rudolf mentzel, the president of the german research association. it contained a request to the dfg to approve fischer's application for a research grant of , rm for the / fiscal year to finance the department for experimental genetic pathology -the sum had increased over fischer's original estimate, as fischer now wanted to hire a clinical physician for the department as well. to the kwg fischer justified this decision with the board of trustee's express wish "to bring the experimental […] results in as rapid and lively connection with the humanclinical questions as possible." "from the close cooperation between the zoologist and theoretical genetic researcher nachtsheim and a clinical physician [he hoped for] an acceleration of the results and and adaptation of the formulated questions to the burning questions of medicine." fischer submitted the application heralded by conti to the german research association on march , , and the grant was issued by the reich research council on march without further ado. "the influence of conti," lösch established correctly, "was worth its weight in gold […] ." but conti's patronage did more than make the money sources gush forth: the second letter telschow prepared for conti in january was directed to the responsible district economic office (bezirkswirtschaftsamt) and applied that the kwi-a be recognized as strategically important for the war because of the research to be performed at the new department for experimental genetic pathology in the course of formation. since the beginning of the war it had been a formidable obstacle to the work of the institute that it was the only one of the total of kaiser wilhelm institutes (including the general administration of the kwg) in the region of the mark brandenburg province not to be classified as a "w" concern (for wehrwirtschaft, army economy). here, too, conti sought to remedy the problem, just as he also supported telschow's request for the classifying the pathologist otto baader as "indispensable" with the responsible military district command (wehrkreiskommando) after such an application by fischer had been rejected. conti supported the undertaking as best he could, even after fischer had finally secured the appointment of abel as his successor to the professorship for anthropology, and verschuer as his successor for the directorship of the institute in winter / . verschuer came to berlin on may , and put his ideas and demands on record. in the case of his appointment, he guaranteed, he would continue the research under the banner of phenogenetics according to the wishes of eugen fischer, but in doing so would stick to his own research profile, shifting the emphasis to genetic pathology. further, he and his pupils would continue the twin and family studies already begun. accordingly, a small polyclinical and a small clinical department were to be created at the institute, which was to employ two national social workers (volkspflegerinnen) and two nurses. his own department for human genetics, for which verschuer requested two further assistant positions, would continue to work closely with the department for experimental genetic pathology. "as a central, connecting node between these, a new department for embryology should be set up." the planned changes, so verschuer calculated, necessitated an increase of , rm in the personnel budget and , in the material budget. besides this, verschuer's plan earmarked non-recurring expenditures -for a new stall building for nachtsheim's rabbit breeding, a laboratory for the animal breeding, equipment of the clinical and polyclinical departments, etc.totaling , rm. verschuer also requested, if possible, a full professorship at the medical faculty of the university of berlin. despite the strained financial situation, the kwg accepted verschuer's ambitious plans surprisingly readily -after verschuer had conducted a conversation with the president of the kwg on may , , the fulfillment of his demands was approved, initially orally; and this approval -upon his express wish -was confirmed in writing shortly thereafter. there is no indication that the kwg, and be it "even only pro forma," had been on the lookout for another candidate for the post of director. correctly, lösch assesses: "fischer had been successful with his tactics of making verschuer out to be the only sufficiently qualified candidate." in july fischer received the message that his son hermann had been killed in action on the eastern front -he lost any interest in the work of the institute and moved to freiburg in august. due to an illness he was not able to resume the business of the institute. since verschuer was not able to take over direction of the institute until october , , in september -irony of fate -fritz lenz was appointed interim director. on october , verschuer continued his negotiations with the kwg. during these negotiations he appeared full of self-confidence and demanded that the grants from the reich and the prussian state be increased considerably in the next fiscal year. yet finances were not the decisive problem -although the increase in public grants demanded by verschuer was rejected, he ultimately received the money from the dfg and from the "sponsorship association of german industry" (förderergemeinschaft der deutschen industrie). more difficult to master, as the negotiations on october , evince, were the restrictions on facilities and personnel due to the war. here, too, verschuer pinned his hopes on conti. with the assistance of the reichgesundheitsführer, the new director hoped to win back the lower rooms of the institute, which had been used as sanitary facilities up to that time. further, conti was to procure the construction permit for extending the attic -until then the skull collection had been kept there, which now was to be transferred to the university -into a sickroom and rooms for the nurses. further, verschuer hoped to achieve with conti's help that gottschaldt and his colleague from frankfurt hans grebe ( - ) be classified as "indispensable." originally conti was to be addressed in a board meeting, but since this never took place, walter forstmann ( - ) from the general administration visited conti at his office on november , . the reichgesundheitsführer willingly pledged his support on all points, inquired as to the works in progress and promised to tour the institute over the course of the next months. under these circumstances the genetic research of tuberculosis had to be of immense interest for the reichgesundheitsführer, especially as it can certainly be presumed that fischer played this card in the decisive conversation with conti. back at the beginning of world war ii he had justified his application to the general administration for feed for the rabbits in beetz by claiming that there was "no doubt that these studies from the area of one of the worst national epidemics promise to be of great importance for fighting human tuberculosis" -a justification that was forwarded by the general administration to the responsible food office (ernährungsamt) almost word for word. that conti was familiar with diehl's and verschuer's tuberculosis research can be proved on the basis of an (undated) typed lecture manuscript on the subject of "genes and performance" (erbgut und leistungsfähigkeit): the genetic disposition also plays a role in infectious diseases. the views about heritability have oscillated extremely. first it was observed that tuberculosis occurred in certain families, then the pathogen was discovered and the way it befalls the diseased, namely in earliest childhood; the disease is then carried forth and does not break out until puberty and professional life and even later: at that point no one considered that the disease in question might have been acquired in childhood. it was twin research that illuminated us to the fact that this congenital inferiority is important in tuberculosis. it was possible to establish that identical twins who grew up separately nevertheless got tuberculosis. if a person is resistant he will not become ill if he is only susceptible. it is clear that someone who may be absolutely resistant, but becomes a tuberculosis doctor or nurse, ultimately does take in the bacillum, which then spreads in the body. a doctor who may come from a tuberculous family, but so far has remained entirely healthy, may not become a tuberculosis doctor, for the risk of infection is too great. in other respects the environmental influences are important in fighting tuberculosis; reasonable living conditions must be created. if any further proof is needed that tuberculosis research was irresistible bait for the reichgesundheitsführer, it is provided by a letter which verschuer wrote on january , -that is, just weeks after the decisive board meeting -to his friend karl diehl. the latter had asked for advice about whether he should set about expanding the rabbit hutches in sommerfeld. verschuer's advice ran as follows: i would undertake absolutely everything that is at all possible. so build with all of the money and material you get! since your research activity was acknowledged at the board meeting of the institute in dahlem by the relevant people, above all by the city medical councilor and reichgesundheitsführer, and its continuation declared to be urgently necessary, you need not have any concern about your future. your tendency toward moving forward is thus altogether correct. in the end, verschuer's assessment turned out to be right. diehl's project enjoyed high priority up to the end of the third reich. upon verschuer's application, the reich research council classified diehl's research on tuberculosis as "important for war and state" and issued a corresponding research contract on august , . achim trunk is correct to emphasize that diehl's project was the only one of all of the research projects being conducted at the kwi-a in to be granted the higher priority of "ss." since the costs of the project ultimately consumed a large portion of the institute budget, in february verschuer submitted an application for funding of , rm to the reich research council, which was also approved without a hitch. "it is truly unpleasant for me to be the greatest consumer of the institute's funds," diehl commented about the application. "couldn't money be saved? but where? everything i have is still so meager and yet so much money. it embarrasses me. and if anything is to come of it, this is only the beginning." conti's interest in tuberculosis research is easy to understand. but here the thesis will be advanced that conti was interested by no means only in genetic pathology research, but also in research under the banner of phenogenetics. this thesis is supported by a source from the estate of leonardo conti, which indicates that, at the time when the negotiations about the reorganization of the kwi-a were under way, the reichgesundheitsführer was fervently interested in issues of "ethnic cleansing" in occupied poland, the "germanization" of poland and the resettlement of german nationals. conti was concerned with this complex of subjects because of the danger of epidemics associated with the resettlement of german nationals. at himmler's request, in december the rusha had presented the draft of a "selection system for the settlement of the new reich districts" (ausleseordnung für die besiedlung der neuen reichsgaue), which also entailed the participation of the reichsgesundheitsführer. the reorganization under the banner of phenogenetics had significant consequences for the institute budget. the size of the budget grew continuously in the war years. fiscal year on, the institute lived beyond its means. in order to be able to continue working in the same order of magnitude as it had until then, it was dependent on the constant flow of third-party funds of considerable scope. this, in turn, had effects on the research program and practice. the initial financial situation when fischer set about to reorganize his institute was not as bad as he had portrayed it to the board of trustees. it had been possible to stop the gap left by the cuts at the start of world war ii through the significant surpluses amassed in the years from to . in september the subsidies from the reich and prussia planned for / were cut to , rm, despite fischer's vehement protest -the shortfall of nearly , rm resulting from this cut ate up around half of the credit balance from the previous years. in the / fiscal year the subsidies from the reich and from prussia were cut by another , rm to , rm, but the expenditures dropped so sharply due to the drafting of nearly all scientific staff and "the cessation of research works associated therewith" that a surplus of nearly , rm remained at the close of the fiscal year. this, together with the remaining surpluses from the previous years, yielded a credit balance of almost , rm, which was transferred to the new budget in view of the research projects the institute had been forced to defer because of the war. added to this was a travel fund of , rm, leaving the cf. fischer to generalverwaltung, / / , mpg archive, dept. i, rep. a, no. , pp. - v: "a certain reduction as a result of the war situation is certainly understandable. but the overall situation is not such that the institute will be closed. […] all of our operations, which, of course, were greatly restricted in the past, first weeks of the war, are coming back into gear. it would be entirely wrong to perform exclusively chemical and physical science because these can be put directly in the service of military economy. our studies about hereditary diseases are at least as important for the volk." thus fischer rejected -successfullyhuge cuts to his budget. kwi-a with "secret reserves" of around , rm in april . in other words: the balances could barely conceal that fischer was again hoarding money in his institute for future research projects. in comparison to the balances for the / and / fiscal years, however, it becomes apparent that funds flowed even faster now. the subsidies from the reich and prussia increased by about , rm. they reached the level of , rm and thus more or less that of the late s. despite the dramatically increased personnel costs -a consequence of founding the department for experimental genetic pathology -at the end of the fiscal year a new surplus of over , rm remained, so that the surpluses, including the full-to-bursting travel fund, totaled over , rm -and this although of the , rm earmarked for nachtsheim's department from the german research association, only , rm were called in right away. and because this subsidy could not be spent in any reasonable way, with the consent of the general administration it was used for the purchase of the library and collection of specimens from the private property of the departing director -the money thus flowed into fischer's pockets. the rest could be transferred to the next accounting year. in other words: at the start of the budget year the institute had "silent reserves" of , rm at its disposal, more than a third of the entire kwi-a budget. not until the / fiscal year did the unchecked expansion thrust result in a hefty deficit. the subsidies from the reich and prussia diminished to , rm, and although the oustanding payment of , rm from the dfg balanced this out, the revenues were not sufficient to cover the dramatically increased expenditures. the personnel costs were the largest post -in this fiscal year alone, three new scientific assistants and eight technical and administrative employees were hired. the deficit ultimately amounted to , rm, through which the accumulated reserves dwindled to just under , rm; however, this did not include the travel fund of over , rm, which remained untouched. the new director otmar von verschuer was faced with a weighty problem. the personnel costs had exploded so greatly as a consequence of creating the departments for human genetics and experimental genetic pathology that they far surpassed the level of the usual grants. in the negotiations with the general administration about his appointment on october , verschuer thus submitted a cost estimate of , rm for the / fiscal year. in oral negotiations telschow made clear that, while an increase had been requested from the reich education ministry, it could not be expected in such an order of magnitude. this assessment was to prove correct: the allocations from the reich and the prussian state did increase back to , rm in the / fiscal year, however, under the condition that the remaining funding gap of , rm be covered by another source. in this situation, alfred kühn, deputy director of the kwi for biology, who had followed the reorganization of the kwi-a around phenogenetics since with interest, leapt into the breach. in his capacity as chairman of the biology and medicine section of the academic council of the kwg, he took part in the meeting of the kwi-a board of trustees on may , , which was dominated by the financial crisis. kühn suggested turning to the association of sponsors of german science (stifterverband der deutschen wissenschaft), which had free funds at its disposal at the time. to this effect friedrich schmidt-ott, the chairman of the association of sponsors, was to be addressed, who also belonged to the board of trustees, but had not attended the meeting on may . also missing was reichsgesundheitsführer leonardo conti, who had supported all of the institute's financial requests so effectively before. conti's star was waning by this time, and it is striking that fischer, verschuer, telschow, and kühn, who were alone at the meeting on may , , no longer included conti in their calculations, but rather decided to arouse the interest of conti's former rival, karl brandt , who had since overtaken conti in importance as the "accompanying physician" of the führer, one of the two figures responsible for the "euthanasia" program, and since july also hitler's authorized representative for the medical and health service, in the institute's work. telschow took immediate action. just day after the board meeting, on may , , he addressed schmidt-ott -with express reference to kühn. since the association of sponsors no longer had such a high sum at its disposal, schmidt-ott forwarded the letter from telschow to albert vögler, president of the kwg since , who suggested directing a petition to the "sponsorship association of german industry" (förder[er]gemeinschaft der deutschen industrie), to request a nonrecurring grant of , rm. verschuer kept this possibility under his hat for the moment. for in the meantime, on may , , the german research association -in response to an application by verschuer on march , -had approved , rm for the institute in dahlem, for "studies in the area of comparative genetic pathology." in june verschuer reported that kühn was negotiating with schmidt-ott about the remaining deficit of , rm. these negotiations ultimately resulted in resorting to vögler's offer: on september , the association of sponsors of german industry approved a -year research grant of , rm annually for the kwi-a. the association of sponsors of german industry had been founded officially in november . the motives that led to the founding of this organization were located on two levels: for one, in view of the profit restrictions imposed upon business by the national socialist regime, sponsoring research was simply a possibility for "investing the considerable war profits, when the traditional possibilities for reducing profits, that is, increasing share capital and increasing capacity, no longer appeared interesting." second, leaders in industrial circles were concerned about theoretical research and the sponsorship of young scientists -at a point in time that coincided with the "first disillusionment about germany's chances of military success" and in view of "the future existence of business and research in a postwar period." extremely interesting -and until now disregarded -is that the economic leaders assembled in the association of sponsors accorded such great importance to phenogenetic research in dahlem in this context that they approved quite a considerable amount for the kwi-a. by way of comparison: in spring the association of sponsors had an endowment of million rm, of which a total of around , rm in interest yields were available for distribution. in the / fiscal year, besides the subsidies from the reich and prussia, the kwi-a received third-party funds from the association of sponsors and the dfg amounting to , rm. in the / fiscal year this total even increased, to , rm, as the dfg not only renewed its grant of , rm, but also, as mentioned above, responded to verschuer's petition by providing an additional , rm for diehl's tuberculosis research. in august verschuer was able to state with satisfaction, in a letter to his friend bernhard de rudder: surprisingly, the cutbacks i expected in my institute have not come to pass; on the contrary, great value is placed on continuing the research important to the war. and so the cogs remaining in my institute machine are turning at full speed, as if the entire machine were still running. but i am glad that so much remains in operation, and that thus still quite a bit of productive work can be performed. since the subsidies from the reich and prussia had been fixed at , rm, the ratio of public subsidies to third-party funds was : in the final budget year. in other words: the subsidies from the german research association and the association of sponsors of german industry were of vital importance for the institute from on. the at the congress of the german society for genetics in , which took place at verschuer's institute for genetic biology and race hygiene in frankfurt, nachtsheim introduced his breeding experiments to the genetic community, and also aroused the attention of those geneticists working on the genetic pathology of humans. after the war, fischer admitted that the idea of winning nachtsheim for his institute occurred to him at this congress. when fischer approached nachtsheim in september , the latter did not hesitate. in the very next month he gave notice that he would be leaving his senior assistantship. his professional status was precarious. since , when the institute for genetic research, along with the agricultural academy, had been integrated into the agricultural faculty of the friedrich wilhelm university, his room for maneuver had been severely restricted. although nachtsheim had been appointed associate professor of the university in , there was no prospect of a regular professorship, especially since he was considered suspect in party circles. he never joined the nsdap. in he had been dismissed as chairman of the reich league of german rabbit breeders. for nachtsheim entirely new possibilities for continuing to advance his research on comparative genetic pathology opened up with the switch to the kwi-a, on a secure material foundation, shielded by the kaiser wilhelm society. the new orientation presented no difficulty for him, since his research on the genetic pathology of mammals had been conceived from the outset as a model for human genetics. in nachtsheim then also switched from the agriculture to the mathematical-natural sciences faculty of the university of berlin -he could not bring himself to decide to switch to the medical faculty. as in the cases of kurt gottschaldt and karl diehl, in hans nachtsheim, too, fischer opted for a scientist whose career seemed to have hit a dead end in the third reich -and who was rather distanced from national socialism. this proved to be a skillful move, for nachtsheim, too, justified the trust placed in him and built the new department for experimental genetic pathology into a supporting pillar of the kwi-a in a very short time. in fischer's and verschuer's plans for reorganizing the institute in dahlem, the triangle of clinical genetic pathology of humans, the animal model and embryology was assigned decisive importance in terms of research strategy. the high value placed on embryology in this concept is often overlooked, because the planned department for embryology was never founded due to the war. this was not for verschuer's lack of trying. in june -that is, a full three months before he took over as director of the institute in dahlem -he began asking around in his circle of colleagues in order to find candidates for the position of director of the new "department for embryology or genetic developmental physiology" in planning. since the new department was not only to study animal embryos from nachtsheim's department for experimental genetic pathology, but also "to build the bridge […] to humans" and to work "on human material" as well, verschuer elaborated to inquiries that no zoologist, but only an "embryologist coming from the field of anatomy" came in question. preferably, verschuer was searching for a young scientist who was nevertheless well-versed in embryological methods -postdoctoral qualifications were not required. the survey produced very few indications of any utility. an acute lack of young anatomists was a problem at the time, and most of the few younger scientists in this area had been drafted, so that ultimately only one of the candidate's names seemed at all suitable to verschuer: the university lecturer dietrich starck ( * ), prosector and senior assistant at the anatomical institute of the university of cologne. verschuer entered into detailed negotiations with starck and his superior franz stadtmüller, the director of the anatomical institute. starck indicated that he was interested, but expressed from the very beginning reservations because he was "an anatomist, body and soul," and could find that a move to dahlem could " 'sideline' [his chances as] an anatomist" and end up doing himself out of a chair in anatomy. in early verschuer and starck agreed to put the negotiations about the appointment on ice for the time being. in may , verschuer invited the still hesitant starck to hold a lecture at one of the upcoming "biological evenings" in harnack house. these evenings were presided over by alfred kühn -the invitation to starck underlines an earlier indication by verschuer that "through the close cooperation with the neighboring kaiser wilhelm institutes, above all with von wettstein, kühn and butenandt, quite special working possibilities are presented" for the new department for embryology, especially "in joint colloquia and team projects." as becomes apparent in the correspondence between verschuer and his friend de rudder, in summer semester starck actually did appear "at a dahlem biological evening" and held a talk about "the importance of developmental physiology for comparative anatomy, explained on the example of the head of vertebrates," which, according to verschuer, was "outstanding in form and content." personally, too, starck had "made the best impression." ultimately he rejected the appointment to dahlem, however, because he did not wish to "endanger his anatomical career." "despite the high qualification of mr. starck," verschuer continued, he was "not unhappy about the rejection," as he had since believed to have found "another and […] apparently more suitable candidate for the position of department director." the person in question here was the university lecturer wouter frans hendrik ströer, prosector at the anatomical-embryological institute of the university of groningen, who had worked as a guest scholar at the kwi-a for several months in . "ströer is a dutchman, but entirely on our side." he was "an outstanding researcher personality" and "decidedly the best man i could think of for my institute." nevertheless it was open to question whether his move to dahlem would take place. ströer himself had "doubtlessly the greatest inclination." however, "by order of the reich commissioner for the netherlands," he was supposed "to take over a professorship at the new reich university in groningen." the decision was still open. verschuer had informed the reich education ministry of his intention to appoint ströer to departmental director on july , -mediated by the general administration of the kwg: dr. ströer is a scientist known for his superior research work in the field of developmental history and genetic pathology […] . he has been occupied with phenogenetic studies as a guest assistant at my institute for some time […] . politically i hold him to be altogether reliable and pro-german -he is a storm-trooper of the germanic ss in the netherlands. in the end verschuer was not able to get his way. as late as september he reported to de rudder that ströer was "still being held back by the reich university of groningen (by now one must write 'former'!) for the time being," but his wife and three small children had been "sent here into my protection, as their lives were threatened directly by their fellow countrymen." "emergency quarters" had been set up for them at the institute. in a further letter by verschuer written a short time later, this time to fischer, he stated that ströer was "stationed with the ss in arnheim" and "certainly took part in the heavy fighting there." the total number of "working scholars" remained -on paper -nearly unchanged during world war ii: from ( / ) it fell slightly to ( / ) and finally to ( / ), and then rose again over the course of verschuer's takeover, to ( / ), then ( / ). yet this impression of relative stability is misleading, for the numerous drafts into the wehrmacht thinned out the scientific personnel extremely. as fischer's activity report for the / fiscal year shows, this began as early as spring : the activity of the institute was restricted, for even in the first five months of the year covered by this report, which were before the outbreak of the war, all assistants but one had been drafted for military drills, sometimes alternately, sometimes simultaneously. at the beginning of world war ii, in addition to the department director kurt gottschaldt, all assistants of the kwi-a and the majority of the male doctoral students were drafted. until late three assistant positions remained unfilled, and the remaining assistants -wolfgang abel, otto baader, heinz lemser, and siegfried tschamler -were in the wehrmacht and held contact with the institute only sporadically. thus, the "central block" was lost, so to speak, which not only had the consequence that all of the assistants' research projects lay idle. the supervision of the foreign guest scholars and the remaining doctoral students suffered as well. the fact that the assistants were drafted also meant that eugen fischer and fritz lenz had to take on more duties in academic instruction -in winter semester / , fischer himself had to hold the practical course in anthropology at his university chair, which he had been able to load off to his assistant abel until that time. demands on lenz's time were made by academic instruction duties, but primarily through the supervision of a great number of dissertations. further, due to the loss of their assistants, fischer and lenz had to take on an even higher degree of activities in producing expert opinions and evaluations. the situation remained unchanged in the / fiscal year -despite leonardo conti's intervention there was no success "in freeing up even a single assistant from military service, through which the scientific activity of the institute is greatly limited," fischer lamented. in the first draft of his activity report the final clause read as follows: "[…] since even among the doctoral students only one foreigner and two ladies remained, the scientific activity of the institute, aside from professor lenz, professor gottschaldt and the institute director, was completely extinguished." fischer's final activity report as director of the kwi-a, which referred to the / fiscal year, began with the resigned observation: through the further duration of army service of all assistants, one departmental director [abel] and at times a second [gottschaldt] , through the lack of nearly all male doctoral students, the institute has not been able to carry out scientific activities on a larger scale. not until the change in institute leadership did the personnel situation improve. wolfgang abel, by now director of the department for race science, was finally classified as "indispensable" in october , as was kurt gottschaldt, so that all departmental director positions were filled. in the first round of negotiations with the kwg in may , verschuer had also, as mentioned above, managed to acquire two further assistantships for his own department of human genetics to be reestablished, which he wanted to occupy with his closest colleagues from frankfurt. in july verschuer reported to the race biologist wolfgang lehmann of strasbourg, a member of the "dahlem circle": "i will take almost all of my newly founded institute for genetic biology and race hygiene at the university of rostock. siegfried liebau ( * ) had worked at the rusha and as an adjutant of the ss medical academy of berlin since , from may to september he was a personal consultant in the ss paramedical office in berlin. from december to october he was detached to the kwi "for professional training in the areas of anthropology, human genetics and race hygiene." the posting of liebau, whose wife ingeborg, née von ekesparre, was a close friend of the verschuer family, apparently can be traced back to a request by verschuer on november , . as will be shown below, liebau carried out twin studies in auschwitz during his time at the institute. in the further course of the war he became chief physician for the higher ss and police leadership of the adriatic coastal region and italy. the two assistants grebe and liebau were joined in the / fiscal year by two auxiliary assistants: karl joachim hene, who had entered gottschaldt's department for genetic psychology as an auxiliary assistant in and taken his ph.d. in with a genetic psychology dissertation about twins in early childhood, returned from military service. further, the teacher hans ritter ( * ), who had begun a second university degree in zoology, anthropology and psychology in , but then had been drafted into military service, started work as an auxiliary assistant in abel's department for anthropology, where he dedicated himself to "gypsy twin research." additional reinforcements came in the course of : karin magnussen ( magnussen ( - , working at the kwi-a on a scholarship since , was promoted to an assistantship -during the war period she was the only woman to hold this status. finally, heinrich schade was also hired. schade, member of the nsdap and sa since , had taken part, as already mentioned elsewhere, in the first yearly course held at the kwi-a for physicians from the ss in / . in he collaborated in the sterilization of the "rhineland bastards." in the same year he started at the frankfurt institute as verschuer's assistant and senior physician. in he submitted his postdoctoral dissertation about the genetic biological inventory of the population of the schwalm region, located between treysa and alsfeld in hesse. in december he moved -on paper -to the kwi-a as verschuer's senior physician. however, because he had been drafted into the wehrmacht, he was not able to start his new position right away. not until the turn of the year / did schade come to berlin, in the course of a military command, where at times he was able to continue his work of evaluating the genetic biological inventory of the schwalm region. he must have been detached to the front again later, for he experienced the end of the war as a yugoslavian prisoner of war. of the veteran scientific staff, only georg geipel remained at the institute over the entire period of the war. otherwise german scientists could only be recruited sporadically, like the "lateral hire" karin magnussen, and -as a convalescent -the physician gerhard koch ( koch ( - . the ranks were filled instead with foreign guest scholars from neutral or allied states. piebenga was supposed to become director of an institute "for the execution of certificates of ancestry and race" in . to make inquiries about him, l. ten cate, a consultant for questions (erik hug), spain (jésus cabeza), turkey (senhia tunakan) and hungary (mihali malán, lajos csik, anton steif, ladislaus apor). the number of doctoral students at the kwi-a oscillated between ( / ) and eleven ( / ), whereby the number of those who worked under fritz lenz - in / -is not included. the comparatively high numbers are deceiving in this case, too, however. numerous doctoral students had been called up to the wehrmacht -those who were able to work at the institute with any continuity were generally only the foreign doctoral students and the female doctoral students, whose number oscillated between two and four. finally, a glance at the nonscientific personnel, which also grew considerably in the course of reorganizing and expanding the institute. at the beginning of world war ii four technical assistants, eight secretaries, one nurse, and five "wage earners" (gardener, driver, keeper, cleaning ladies) had worked at the kwi-a; in / there were five technical assistants, secretaries, one photographer, one laboratory technician, one nurse, one auxiliary technical assistant, one caretaker, plus the married couple who worked as caretakers in the external department for tuberculosis research in sommerfeld, as well as four "wage earners" (keepers, cleaning ladies) and several "temps." despite verschuer's fears to the contrary, the institute was able to maintain all of its personnel in the second half of as well, as impending drafts were postponed for the time being: apparently it is primarily thanks to the vigorous action of prof. osenberg [werner osenberg ( - ] of the reich research council that research is so protected at the moment and should be continued to its full extent. thus verschuer still expected that abel and gottschaldt would be called up again, and perhaps nachtsheim as well, who had been ordered for a physical examination. hours of the institute -in keeping with the times -but only to such a degree that overstraining is avoided […] . verschuer's private household had initially employed foreign civilian workers. in the move from frankfurt to berlin in november , the verschuer family had a croatian maid. at the beginning of the family appears to have employed an additional female "eastern worker." in late february, verschuer reported in a letter to bernhard de rudder, that there had been "all kinds of sagas with our russian east worker." "it turns out there had been minor thievery, with which she provided provisions for all kinds of male compatriots […] ." yet she had been "kept again on probation." shortly thereafter verschuer again complained of "troubles at home with our russian (bolshevik!)." besides this, since at the latest, an additional female "eastern worker" was working at the neighboring institute. in august , after the renewed proclamation of "total war," verschuer feared that he would probably "have to give up the two eastern workers from the house and the institute." yet it never came to this. in late september verschuer wrote to fischer in freiburg that the "russian woman" in service in his household had "run away" -as once before in , and this time "the russian woman from the institute […] ran away with her." between september and november most of the scientific work at the kwi-a -aside from the external department for tuberculosis research in sommerfeld and (from ) the department for experimental genetic pathology -came to a standstill due to the fact that so many departmental directors and assistants had been drafted. this was not immediately apparent to the outside world, however. between and the institute still published scientific papers; however, this was the result of a "publication backlog." most of the publications from this period -to the extent that they were not simply overview papers, conceptual or methodological discussion papers -resulted from research projects that had been concluded before the outbreak of the war. only very few papers, such as a paper by karl diehl and eugen fischer about the tuberculosis experiments on rabbits in sommerfeld and the papers by hans nachtsheim about "the state of convulsion readiness and genotype," referred to current projects. the change came with the new director. verschuer was successful in getting the departmental director kurt gottschaldt and wolfgang abel classified as "indispensable," filled the ranks of the assistants with hans grebe, siegfried liebau, hans ritter, and karin magnussen and obtained a larger number of foreign guest scholars. and -not to be forgotten -verschuer achieved a budget hike and solicited considerable third-party funds. thus research resumed on a large scale from december on. of course, this was not immediately reflected in the publication lists. even so, between and the institute in dahlem produced another publications, whereby -in addition to the general intensification of the war situation -it must be taken into consideration that nearly all of the publications that had accumulated in the prewar period were published at this time. the papers published in the last two years of the war were almost without exception minor works presenting intermediate findings from projects in progress, and some of them were based on material that had been collected previously. a number of publications that had been available in manuscript form or were even in print were lost in the chaos of the war's end; others were not completed before the collapse of the third reich. some of these papers were still published after world war ii, for others this did not seem opportune because they were all too closely associated with the state crimes of national socialist germany. in the final years of the war, the shift in emphases between the fields of research of the institute in dahlem, observable since , continued at a faster pace. genetic pathology moved to center stage with publications -and this was, so to speak, only the tip of iceberg, as several large-scale projects in the field of genetic pathology never found their way to publication. this dominance of genetic pathology had various reasons: first, the two departments that had been able to keep up their operations in the first war years, that is, the external department for tuberculosis frankfurt to dahlem, verschuer had an assistant who, because of a project for the collection of stillborn fetuses in progress since the prewar period, and because of his postdoctoral dissertation about chondrodysplasia, had a rich fund of pathological material at his disposal, which could be evaluated without any great cost. fourth, through the activities of preparing assessments and evaluations, individual cases of genetic pathological interest (including all of the important genealogical information required for their genetic pathological evaluation) came to the attention of the researchers in dahlem. fifth and finally, the findings of genetic pathology promised a direct practical utility with regard to the measures of both genetic health policy, as well as eugenic sterilization, marriage bans in accordance with the "marriage health law," the allocation of "matrimony loans" and so on. the kwi-a extolled this practical aspect of genetic pathology research quite audibly, which was evident in the mere fact that the research application which covered the major portion of the work in this area bore the keyword "race hygiene." in total it can be asserted that the research field of genetic pathology increasingly pushed its way into race hygiene over the course of world war ii: there was hardly a genetic pathology study that was not oriented to genetic health policy, and hardly a race hygiene paper without clear references to genetic pathology. the trend toward specialization observed in the final years before world war ii, which inclined to lead race hygiene and genetic pathology (and race biology) away from each other, was reversed at the kwi-a from on. for a concrete example, in the years - there were nine research contracts with the keywords: wehrmacht contract numbers: pp. - ( / ) -iii/ ("genetic pathology research"), ss - ( / ) -iii/ ("tuberculosis"), s - ( / ) -iii/ ("specific proteins"), k ro/rfr- / / -iii/ ("eye color"), k ro/rfr- / / -iii/ ("twin camps"), k ro/rfr- / / -iii/ ("race hygiene"), k ro/rfr- / / -iii/ ("genetic biological inventory"), k ro/rfr- / / -iii/ ("stillborn fetuses") and k ro/rfr- / / -iii/ ("pneumoconiosis"). five of these nine research contracts -"genetic pathology research," "tuberculosis," "race hygiene," "stillborn fetuses" and "pneumoconiosis" -were located directly in the field of genetic pathology, one further -"genetic biological inventory" -had strong bearings on genetic pathology. this illustrates the dominance of genetic pathology even more strongly than the analysis of the publication list. the genetics of normal attributes, even and especially under the aspect of race was relegated down to second place, with two research contracts -"specific proteins" and "eye color" -and a total of publications. weighting the individual research projects according to their financial, political, and research-strategic value, it becomes apparent that from on, four areas were of fundamental importance for the future of the institute: comparative genetic pathology (nachtsheim), research on the heredity of tuberculosis (diehl), the project on the phenogenetics of eye color (magnussen) , and the project to develop a serological race test (verschuer) . their progress determined whether the budget could be fixed at a high level, whether sources of financing outside of the regular budget kept flowing, and whether research operations could be maintained in their entirety. they decided whether the project of phenogenetics, above all its integration into general genetics and biology, would succeed. and they were eminently important for genetic health and race policy. verschuer energetically pushed ahead with the concept of phenogenetics developed by fischer, but placed the emphasis on genetic pathology research, whereby, of course, he consequently conceived of genetic pathology as "medical genetics," thus embedding it in general human genetics. besides, verschuer understood genetic pathology as a principle encompassing and penetrating all subdisciplines of medicine and urged -in keeping with his concept of the "genetic doctor" -that it be indulged generously in both specialized and general medical practice. with his attempts to influence the medical students' conditions of study and examinations, his house journal der erbarzt, and -since -fortschritte auf dem gebiet der erbpathologie, rassenhygiene und ihrer grenzgebiete ("advances in the field of genetic pathology, race hygiene and their boundary areas"), and finally with the remaining areas had nearly no importance at all: in the area of geographic and paleoanthropology six papers appeared (mainly connected with colonial science research on "white africa"); four papers dealt with subjects that were decidedly race hygiene; four works were dedicated to conceptual and methodological issues. no papers appeared on genetic psychology. his leitfaden der rassenhygiene ("manual of race hygiene"), verschuer contributed to the process of making the findings of genetic pathology research flow into practice. the orientation on genetic pathology was also expressed in the erection of a genetic biological examination office in the attic of the kwi-a, which was to be expanded to a "clinical and polyclinical station […] in order to be able to continue the activities of consulting and producing expert opinions and also genetic clinical and genetic pathology research." verschuer had already operated such an office in frankfurt -the model for it had been the "polyclinic for the care of genes and race" in berlin-charlottenburg, which verschuer had run in / . from frankfurt he brought nurse emmi nierhaus ( * ) from the protestant social services association (evangelischer diakonieverein), who not only took over the administration of the institute as his "right hand," but also provided nursing care for the examination office. "for as intensive specialized study of the research material as possible" verschuer further founded a "genetic pathology working group," to which he invited -besides the staff of the institute -prominent representatives of "pathological anatomy, radiology and all clinical specialities." this working group, which convened for the first time in march , was also supposed to "discuss difficult questions in the practical care of genes and race and prepare the basic decisions for the state offices." in his journal erbarzt, too, verschuer emphasized the genetic pathology working group's orientation to practice: over and again he was "enlisted for genetic medical consultations and evaluations, by the health offices as a genetic biology consultant, and by the hereditary health courts and appellate the nd edn. of leitfaden der rassenhygiene appeared in . in verschuer reported that a french edition was in printing, and a portuguese one in preparation. cf emmi nierhaus started at verschuer's institute in frankfurt in september , followed him to dahlem in december and from there to the lay-by in solm. after the war nierhaus continued to work as verschuer's assistant, from july on, officially in the service of protestant social services (evangelisches hilfswerk). after a short interruption in / she joined verschuer at the university of münster. her responsibilities proceed from a letter by verschuer from the year : "i would like to assign nurse emmi the same group of duties she used to perform for me in frankfurt and then in berlin in such an excellent manner: it means a great deal to me that those people who come to use for scientific examination (e.g. twins), for evaluation (e.g. paternity certificates) or for their own consulting and examinations (e.g. marriage counseling), enjoy nursing care. the help of a nurse during the examinations currently in progress at the institute would thus have the highest priority. added to this would be the economic direction and administration of the institute, along with the many individual tasks associated with these duties, in which nurse emmi has proved particularly invaluable in the past." verschuer to oberin sprenger, evangelischer diakonieverein, / / , archiv des evangelischen diakonievereins zehlendorf, w (pre-archive). courts and other offices contracted to carry out race hygiene measures, as a chief evaluator." in frankfurt, whenever a specialized medical examination became necessary, he had turned to his specialist friends and their clinics -the working group in dahlem was supposed to serve an equivalent function. in his report about the / fiscal year verschuer reported that the genetic pathology working group had held "several sessions […] at which not only scientific cases from the field of genetic pathology were presented and discussed, but also practical issues of the care of genes and race debated, in order to provide to the reich ministry of the interior and the hereditary health appellate courts a position on evaluations." the erbarzt published the protocols of two meetings of the working group, those held on march and may . in his memoirs, published in , gerhard koch stated that he attended a further meeting of the genetic pathology working group in july or august , in which the subjects were hip luxation and club foot and whose participants included the internist friedrich wilhelm bremer, the orthopedic surgeon lothar kreuz ( - ), director of oskar helene heim and the orthopedic clinic of the university of berlin, and the pathologist robert roessle ( - ). on this occasion kreuz claimed he advocated the elimination of these two congenital disabilities from the catalog of indications in the gzven; his proposal had been agreed to, even "by the high-ranking medical officers of the army and waffen-ss attending this session, whose names were not known to me." koch presumes that the publication of the protocols of the meeting was "suppressed by the censors." this could in fact be the reason why -in contrast to the original proclamation -after the first two, no further protocols of meetings by the genetic pathology working group were published. however, it must be taken into consideration that koch's account is not confirmed by any other source and that koch has a tendency to exaggerate the frictions between genetic pathology research at the institute in dahlem and ns genetic health policy, not to mention the importance of censorship. a key role in the area of genetic pathology was played by hans grebe, who came to berlin from frankfurt with verschuer. it is essentially due to his influence that a new emphasis on the field of the differential diagnosis of congenital defects developed at the kwi-a from on. in grebe had begun with comprehensive studies on chondrodysplasia (hereditary disproportional dwarfism). he wrote a circular to health offices in southern, western, and northwestern germany, with which the institute in frankfurt had already been in contact regarding further professional training for medical officers. eighty-five health offices responded to this survey and reported a total of people with "dwarfism," nearly all of whom grebe visited personally and subjected to a thorough clinical and radiological examination, together with the members of their families (parents, siblings, children, uncles, aunts, nieces, nephews, and cousins). family tables were produced on the basis of registry office and church records, whereby particular attention was paid to the question of whether the parents were related by blood. for the purpose of comparison, grebe consulted the specimens of miscarried and stillborn chrondodysplastic fetuses that had been dissected at the pathological institute of the university of frankfurt in the years from to . grebe had to discontinue his work after the beginning of the war because he was called up for military service. in summer -as previously mentioned, grebe had been discharged from the wehrmacht because he was seriously wounded -the "main part" of the work performed at the university of frankfurt was submitted as his postdoctoral thesis. the manuscript was sent to thieme-verlag in leipzig for publication, but the proofs were destroyed there by a bombing -not until was the work published in analecta genetica, largely unchanged, by luigi gedda , the founder and director of the mendel institute in rome. grebe had examined a total of families with around , persons, frequently against the bitter opposition of the subjects. one of the probands, who had been sterilized at the age of in , as grebe reports casually in , had reacted to "a clinical and radiological examination and especially the production of photographs […] with the greatest resistance." among these probands, he continued, there was the "highest degree of mental sensitivity, which was also shared by most of the members of the family." of one -year-old subject he writes that she was "very sensitive mentally" and seemed "decidedly depressive. for instance, during the examination, against which she put up vehement resistance, she began to cry. during a later visit, too, her mental behavior seemed melancholy." only in the case of a -year-old girl, who had been sterilized in spite of an appeal to the hereditary health court, did grebe express a degree of sympathy: "the resistance brought against our examination was particularly great under these circumstances." the boundaries between voluntariness and compulsion were blurred to the extent that some health offices used grebe's survey to request an opinion as to whether a marriageability certificate could be issued for certain probands. occasionally a hereditary health grebe, chondrodysplasie, pp. - . ibid., pp. vii-viii. ibid., pp. - . ibid., p. . grebe did not even hesitate to secretly take a picture of a female subject whose behavior was guarded and suspicious. ibid., p. . ibid., p. . ibid., p. . further indications of resistance on, e.g., pp. , , p. , , . cf. e.g. ibid., p. : "with the negative family finding and the particular professional prowess of the proband, who also successfully graduated from a rural vocational school," in this case court requested that the frankfurt institute for genetic biology and race hygiene provide an evaluation in accordance with the gzven -in these cases it was not possible for the subjects to refuse an examination. as grebe adhered strictly to his analysis of the conditions of heredity, he sometimes took a position against sterilization, yet this did not change the fact that in this situation he confronted his subjects in compulsory proceedings as an officially empowered evaluator with comprehensive powers of attorney. this constituted a transgression of the boundaries of scientific ethics of major importance, both potential and in principle. the material grebe had collected in the course of his study of chondrodysplasia constituted the basis for a series of publications in the years - , as he had run across an abundance of additional physical defects, mental disabilities, and mental disorders in his comprehensive genealogical studies. this material increased when, after completing his postdoctoral dissertation, grebe set about recording miscarried and stillborn fetuses on a large scale. by march he had examined nearly families who had produced a stillborn child with a serious defect. verschuer's activity report for the / fiscal year relates: grebe concluded a major family research project using a non-selected series of deformed stillborn fetuses. generally speaking he was able to prove that heritability plays a much greater role in the problem of stillbirths than was previously presumed. for the first time he was able to prove that certain forms of congenital defects are hereditary. thus grebe, proceeding from his collection of stillbirths, described three families in which multiple intestinal deformities had occurred (stenoses, atresias, ventricles, cysts). in he published a paper about the problem of a genetic disposition for hernias (inguinal and umbilical), based on observations of twins and families. grebe attempted to prove that a hereditary factor was involved in the etiology of both cases. in other cases he endeavored to illuminate the hereditary precisely. in , for instance, he published an essay that proceeded from the "stillbirths" project, on the emergence of arhinencephaly (absence of olfactory tract, olfactory bulbs, and frequently the frontal lobe of the brain), whereby he presumed an grebe took a position for issuing a marriageability certificate. a case with similar circumstances is depicted on p. . cf., for instance, ibid., pp. , . perusing the book, indications of more than twenty sterilization trials are found, whereby in one case (p. ) the application was supposed to have been submitted by the subject herself. cf. grebe, fistula; idem., struma; idem., erblichkeit; idem., akrocephalosyndaktylie; idem., lipomatosis. "irregularly dominant genetic disposition." in the same year he published a major paper on acrocephalosyndactylia (a syndrome characterized by skull deformation and webbing of the fingers). with reference to the etiology of this syndrome he rejected all "exogenous attempts at explanation (above all deficient amnion, lues and hypophysis damage)." grebe assumed a specific mutation and concluded that "no race hygiene measures proceed from acrocephalosyndactylia at this time." in all of his research grebe endeavored to make as precise a differential diagnosis as possible. he assumed that one congenital defect could have very different genetic or even exogenous causes (heterogeny). verschuer emphasized the importance of grebe's research in this direction in his activity report for the / fiscal year: in the area of the typical clinical picture [of chondrodysplasia] it was possible to establish several gene types that could be differentiated clinically and genetically. on the margins of the typical complex of symptoms there are many other genetic conditions of the cartilageskeletal system, some of which could be observed and described for the first time. the project thus yielded a very far-reaching heterogeny, which is of fundamental importance. this finding made it seem very important to demarcate the different clinical occurrences as precisely as possible, to explain the genes responsible in each of the hereditary forms, and to reveal genes that were manifested to a hardly perceptible degree or not at all. in the context of his study about chondrodysplasia grebe published a family study in which he pursued the question as to whether the heterozygotic carriers of the recessive gene for chondrodysplasia could be recognized on the basis of minor, nonpathological varieties. in x-rays he established that the heterozygotic family members showed minimal changes in the bone structure of the hands and feet. "but should it not be possible," grebe asked at the close of his article, "to find a way to recognize the heterozygotes in the future, for other recessive genetic conditions as well?" for the "practical care of genes and race" the importance of this question "could not be underestimated." yet another study by grebe of the year must be viewed against the background of his search for stigmata. this particular work dealt with a family with an increased frequency of lipomatosis (painless symmetrical diffuse deposits of fat), but also "mental anomalies (schizophrenia, schizoid psychopathy, feeble-mindedness to greater or lesser degree, suicide, epileptic-type fits, melancholy)" as well as physical deformities (chondrohypoplasy, microcephaly, wryneck, hernias). in this case, however grebe discarded the hypothesis of a genetic connection. rather, he traced the coincidence of the various anomalies back to "sifting by mating." moreover, it was possible "that the effect of one or more pathological genes on the manifestation of other genes resulted from the particular frequency of anomalies in the family described." in a further essay entirely tailored to practical race hygiene, in grebe discussed the question of how high the risk should be estimated that a mother who already had experienced a miscarriage or stillbirth would give birth to yet another child with defects. in the case of very serious defects that made survival impossible, grebe summarized his considerations, "more or less complete destruction" resulted on its own. however, a "complete elimination" of all genes responsible for defects was not possible, "first of all because only some of the carriers of very frequent, irregularly dominant and recessive genes become phenically ill, and further, because constant new generation through mutation is possible." after all in many cases the probability that a further deformed child would be born after the birth of a non-viable, seriously deformed child was so low that there was no need to advise against a new pregnancy. and even for minor changes "that can hardly be addressed as defects" there was no need to take action. "on the other hand, great misgivings about the conception of additional children must be expressed in cases of defects which allow the affected child to survive and reproduce, but reduce to some degree the capability of the adult to work or perform military service." in every consultation, however, "the total value of the given family [must be] considered." methodologically speaking, genetic pathology research at the kwi-a was committed to higher mendelism at the time of the world war ii, and its objectives thus differed from those of practical race hygiene. in terms of contents a clear emphasis on the area of physical defects emerged, due above all to grebe's research interests. in the department for race hygiene the research "about the heritability of deaf-muteness and the race hygiene prospects of its prevention" begun in the prewar period was continued. hereditary blindness, too, remained an object of interest. finally, these were joined by the research on epilepsy in the department for experimental genetic pathology. this constitution of emphases entailed a clear division of labor with the german research institute for psychiatry and the kaiser wilhelm institute for brain research in berlin-buch, both of which, closely connected with the nazi "euthanasia," were concerned with the differential diagnostics of the various forms of mental disability, schizophrenia, and neurodegenerative diseases at this time. this area played no further role at the kwi-a during world war ii. grebe, mißbildung, pp. f. yet the withdrawal from the areas of psychiatry and neurology was only in part the result of a conscious demarcation of the fields of work. it was much more a result of the fact that the large-scale project on the genetic biological inventory of peasant villages in the schwalm region of hesse, which was begun in frankfurt and was supposed to be continued in dahlem, was not making any headway. originally, the project was one of those taken on by walter scheidt as part of the "german race science" campaign. at scheidt's request church records had been catalogued, scholars had begun to compile family tables from the around , excerpts from church records -the declared objective was to establish the genealogy of the peasant population of schwalm from to the present. in scheidt had turned the project over to the frankfurt institute for genetic biology and race hygiene, which used funds from the reich committee for national health service to hire an assistant to complete the family tables. further, verschuer's institute set about recording the living population in these villages, whereby not only the usual anthropometric examinations took place, "but rather beyond these also comprehensive clinical-physiological and pathological findings [were to be] recorded" -this was the reason for handing the project's direction over to the physician heinrich schade. as the counterpart of the long-time resident population of the schwalm region, a parallel genetic biological inventory of the city of frankfurt south of the main was to be carried out using the same methodology. the objective of the genetic biological inventories was to link together the fields of race anthropology, genetic pathology, and race hygiene: schade and his staff had been working in the schwalm region since winter / -with the active support of the district administration, the mayors, the health offices, the schools, and the party offices. in february they had concluded their studies in two villages. in addition they evaluated the patient files of the relevant institutions of the treatment and care, hospitals, welfare offices, and practical physicians. all findings were recorded in the family tables and files, which were made accessible by a personal card index. in late schade -with financial support from the dfg -had over , excerpts from the case histories of the university clinics in marburg and the files of the ziegenhain health office, the state insurance institute in kassel and from army physicals. schade and his colleague günter burkert had personally examined , patients. by march over , excerpts from patient files and health office certificates had been produced. in the framework of the project, schade's interest was directed primarily to the distribution of "feeble-mindedness" in the "inbreeding area" of the schwalm region, with a strong practical orientation to race hygiene. burkert dealt with "acts of selection" through immigration to and emigration from the schwalm region. a study about the "character and aptitude of the schwalm population" apparently was never concluded. the project staff member heinz koslowski performed anthropological studies in one of the region's communities, which had been founded as a hugenot settlement, establishing there "demonstrable differences with regard to the population of purely german descent." schade submitted his postdoctoral thesis about the genetic biological inventory of the schwalm region in -it appeared in print in . at the beginning of the war the evaluation of the daunting mountain of material was far from concluded, however. schade was drafted into military service. in december -on paper -he followed his mentor verschuer to dahlem, but continued to serve as a surgeon major on the front and was not able to work at the institute himself. a new "auxiliary statistical assistant" continued to evaluate the genealogical, anthropological and medical data. not until the turn of the year / could schade, as mentioned above, come to berlin in the course of a military command and resume his work at the institute. in his activity report for the fiscal year / verschuer reported that schade had [t]he essential task of processing the great amount of material on the genetic biological inventory for an old-established peasant population (from the pre-war period) continues to be sponsored. the population movements over years have been established, the average burden with numerous illnesses determined and the question as to the importance of heredity for early invalidity investigated. two publications that had been announced never came to be, however. consequently there was a great deal of material available at the kwi-a that could have been evaluated with regard to aspects of genetic pathology, in particular with regard to mental disabilities, had there not been a dearth of personnel. in other areas, too, such as internal medicine, for practical reasons it was hardly possible to perform genetic pathology research during the war. back in grebe had begun a large-scale study in frankfurt on the question of a "constitutional conditionality" of pneumoconiosis ("black lung" disease) on behalf of the reich labor ministry. through his service at the front this study was interrupted for years and was supposed to be brought to its conclusion in dahlem in . but the air war made it impossible "to perform systematic examinations in the ruhr area at this time, whence the majority of the cases originated," such that completion of the study was delayed even further. by then grebe had recorded over , cases of black lung, which had been treated in social miner's hospitals or discovered during the series of x-ray examinations performed by the ss. the twins had been determined by means of inquiries at the offices of vital statistics. grebe had contacted over twins and requested file data and photographs. the clinical examination of the twins was interrupted by the start of the war, however, and the study had run aground in the second half of the war. in the area of infectious diseases, genetic pathological research in the last years of the war concentrated exclusively on tuberculosis research, after another longterm project had remained without any concrete results. around verschuer, together with the biologist richard prigge ( - ) of the state institute for experimental therapy in frankfurt, had begun a "heredity experiment" on the "natural resistance of the guinea pig to diphteria toxin." in their final report published in , prigge and verschuer reached the conclusion that "the question of hereditary differences in resistance to diptheria toxin in the guinea pig clearly must be answered in the negative." of the guinea pigs tested, only two survived, which had been "taken into breeding." the production of a diptheria-resistant erkrankungsstatistik einer wohnbevölkerung ("illness statistics of a residential population") and bevölkerungsbewegung in drei jahrhunderten in acht dörfern ("population movement over three centuries in eight villages"). cf guinea pig through pure breeding was not successful, however. instead, it seemed that the breeding of a tuberculosis-resistant rabbit was within reach. since tuberculosis research using the twin method had hit a dead end in the s, karl and anne diehl -in close collaboration with verschuer and fischer -had been experimenting with rabbits in the "waldhaus charlottenburg" since . diehl infected his experimental animals with a constant strain of the bovine tuberculosis bacillum by means of intravenous injection, but not until sufficient progeny were available for further breeding. the infected animals were held in a secluded stall and dissected after their death. clear differences, interpreted as conditioned by heredity, became apparent as regarded the time of survival after injection and the infestation of the individual organs. interest was directed primarily to two breeding lines -the one, "central" type developed a serious tuberculosis of the lung, while the other organs were hardly affected at all; in the other, "peripheral" type, by contrast, the sources of infection emerged primarily in the peripheral tissues, like in the kidneys or the nerve tissue. "a heritability of this organ specificity in the reaction to tuberculous infection," fischer announced in his / annual report, was "thus proven experimentally for the first time." in the next annual report fischer added that the results of the rabbit experiments could be "conferred without further ado […] to humans. this also yields important prospects for combatting tuberculosis in humans." according to fischer, at the tuberculosis congress in baden-baden diehl "held a lecture that aroused great attention and was received with much applause." from on fischer and diehl presented the results of the rabbit experiments in sommerfeld to the experts. the two different manifestations of tuberculosis could be bred constantly and in a pure form through eight generations of rabbits. the hereditary character of the clinical picture remained completely preserved, "even when animals were pre-treated with human tuberculosis bacilla, which are avirulent for rabbits." now diehl attempted to get to the bottom of the riddle of organ resistance in animal experiments: it was attempted to modify the type of tuberculosis manifestation by inbreeding specimens. for this ink blocking, re-infections and organ transplants were performed. the persistence of the way in which tuberculosis is manifested appears to be very great in the bred specimens. these experiments will be expanded further, since it is possible that their result can be of fundamental importance for the medical therapy of humans. the endeavors to obtain clarity about the status of individual organs in the process of infection in the bred specimens aimed in the same direction. what was particularly interesting here was the status of the liver. in addition to these experiments, diehl began crossing the two pure breeds with each other. when in the period from april to july he set about infecting the animals proceeding from the crossbreeding experiments with tuberculosis, he believed that his research was entering a decisive phase: crossing the two pure breeds has now yielded a large f . seventeen animals from the pure breeding experiments were taken as the point of departure. the f amounts to around animals. from the f , which we generated from animals born the previous year, we unfortunately lost quite a few because of the wet weather and the consequently wet feed. now we have only about animals. in the coming year the f will then appear in full force. i am glad that these animals were "vaccinated away"without having been able to reproduce. only the desired "immune" animals will reproduce. i believe that if i aim for an f of about animals that should be sufficient. the approach was clear: through crossing the two pure breeds diehl hoped to be able to cultivate "tuberculosis-resistant" rabbits. thus, he continued working as if obsessed, although he felt miserable and exhausted at the time, since the late consequences of a lung tuberculosis contracted in his youth became noticeable. diehl and verschuer were feeling time pressure, too, not least because they had heard about rabbit experiments by the american tuberculosis researcher max bernhard lurie ( * ). in january -the catastrophe of stalingrad was imminent -diehl was still filled with hope by the sight of the dying rabbits: i go into the stalls in sonnenberg often. biological events are taking place there with a cruel consequence. it seems obvious that i hold the key in my hand. the decision will be made this summer! the hope for tuberculosis-resistant rabbits was not fulfilled, however. nevertheless, diehl continued working doggedly on crossing the two pure breeds until the end of the third reich. in october he had dissected nearly rabbits originating from verschuer, tätigkeitsbericht / , mpg archive, dept. i, rep. , no. . in diehl's draft the final point was explained in more detail: "in order to obtain clear results, pieces of the liver were surgically removed from members of the last two inbred generations, these pieces tested in terms of their antibacterial power and the animals, after recovering from surgery, tested as usual. the antibacterial power of the liver proved to be very different among the animals. the lung is currently being examined in the same direction." diehl's draft for the these crosses and performed statistical analyses of the results -but without attaining any final certainty. nonetheless, diehl -and verschuer as well -still believed that this was the way to achieving a breakthrough in tuberculosis research. biochemists were also interested in diehl's experiments. the specifically genetic resistance of the organs had to be effected through the production of a substance in the organism, which prevented or hindered the tuberculosis bacillus from settling in certain organs. thus the search was on for a biochemical compound. if they succeeded in isolating and identifying it, this would yield far-reaching consequences for tuberculosis therapy as well. this is the background of the lateral contacts between the external office of the kwi-a in beetz and the kwi for biochemistry under adolf butenandt. the connections extended all the way back to . to keep from distorting the results of his study, diehl required for his injections an emulsion that was completely dispersed, i.e. the tuberculosis bacilla had to be distributed as regularly as possible without any clumping. to solve this problem, diehl had arranged with gerhard schramm ( * ) of the kwi for biochemistry to use the colloid mill located there, which could liquidify the tissue by rotating it at high speeds, in late august or early september . verschuer made adolf butenandt himself aware of diehl's experiments in july , who proved to be "extraordinarily" interested. "unfortunately," verschuer reported to his friend diehl, "we were interrupted, so that our conversation did not come to a conclusion. thus i cannot give you any result yet today. yet i will come back to the matter upon the next opportunity." in a telephone conversation just a few days later, butenandt expressed his wish to meet diehl personally as soon as possible and learn about his experiments. verschuer asked diehl to bring "some of his family tables, tables, pictures or specimens." the meeting was supposed to take place in july or august, but apparently was delayed until october. this is in keeping with the comment in a report by verschuer to the dfg of september , that contact had been established with butenandt in connection with the tuberculosis project in order to accomplish the biochemical analysis. the collaboration between diehl and butenandt survived the third reich, but diehl's research ultimately fizzled out. through diehl's rabbit experiments, butenandt became aware of the entire range of genetic pathological research performed under the banner of phenogenetics at the kwi-a. on november , verschuer held a lecture to the prussian academy of sciences about "heredity in infectious diseases," in which he attempted to link the results of the phenogenetically oriented research -especially diehl's rabbit experiments -with the work by alfred kühn and adolf butenandt on gene action chains. it was no longer possible to publish the text of the lecture before the imminent collapse of the third reich, but it appeared -unaltered, as far as we can judge -in under the title die wirkung von genen und parasiten im körper des menschen ("the effect of genes and parasites in the human body"). verschuer's argumentation is still entirely fixed upon the problem of the interaction between infection and hereditary disposition, but also picked up on some thoughts of butenandt's to touch on issues that are highly relevant today. for instance, verschuer emphasized the importance of infectious diseases that jump from animals to humans, which is nothing short of prophetic in the age of bse and sars. he further indicated the similarity between viruses and genes -today we know that a significant part of the human genome consists of incorporated viral material. in butenandt verschuer found an attentive listener; the two even had "an especially pleasant (post-)meeting over a cup of tea at home." on january , the department for experimental genetic pathology under the direction of hans nachtsheim began its work. in the meeting of the board of trustees on january , fischer explained -presumably above all for leonardo conti -the key role of the new department. in the science of genetics, fischer claimed, "the animal experiment [had] always been in the lead"; "human genetic research" had "always [received] directions and stimulation from the former." nachtsheim's great service had been "to have recognized the fundamental importance of this wonderful research material and to have set about its evaluation […]. this new ground he has broken must become ours." this ground was not entirely new, as since the institute's founding in fischer had occasionally provided for experiments to be performed on rats, rabbits or guinea pigs in order to get to the bottom of the genetics of normal attributes, especially race attributes, through the study of embryos. but now nachtsheim's preliminary works opened up the possibility of continuing this research on a grand scale and, what was even more important, expanding it to the area of genetic pathology. although nachtsheim's papers since fit into the leading research paradigm of phenogenetics and were not designed for rapid and direct application to race hygiene, fischer never tired of emphasizing the practical importance of experimental genetic pathology before the board and also to the dfg, to whom he applied for , rm in research funds for nachtsheim's department on march , : what is most important is the corresponding examination of those genetic diseases that are important for humans. these are then model experiments for human genetic pathology. they will teach us why the same genetic condition often occurs in such different intensities; it will give us tips as to whether the development can be steered by external influences. at the same time, fischer stressed again that there was no alternative to the animal model: such studies are practically impossible on humans, because it is never possible to know with any certainty what would have become of a dead embryo. as fischer elaborated to the german research association, nachtsheim and his staff were to perform three parallel series of experiments on the diseases and anomalies to be studied: the first were breeding experiments. nachtsheim's group of scientists was to detect rabbits with pathological genes, to propagate these "in pathologically pure culture" to the extent that pregnant females could be killed at all stages of embryonic development, and finally the heredity of the pathological genes be elucidated in crossbreeding experiments. second, the dead embryos -in close collaboration with the department for embryology, still to be founded -were to be examined pathologically and histologically, in order to be able to study the inception of pathological processes during ontogenesis by comparing findings from various embryonic stages. third and finally, it was to be attempted to influence the outbreak of disease by environmental stimuli (poisons, chemicals, feeding), not least in order to be able to differentiate between a "general" and a "genetically increased" susceptibility, which, according to fischer, was "of particular importance in view of the most modern methods of treating diseased humans." at the close of his application fischer stated his conviction "that these theoretical and experimental studies will be of benefit to suffering humanity and serve the preservation of the genetic health of our volk." mentioning discreetly that the reichsgesundheitsführer shared his views, fischer guaranteed that he could "carry the full responsibility" for the importance of nachtsheim's research "even now at a time of war." when nachtsheim started in dahlem, he had at his disposition, as fischer informed the dfg, a series of rabbit strains that exhibited genetic diseases or disabilities: these were "genetic epilepsy […], shaking palsies and other nervous diseases; glaucoma and other eye diseases; deformation of the limbs, the external sex organs (similar to those of humans), harelip and cleft palate and many others." from nachtsheim's report to the german research association of january , -the first he submitted from his new position in dahlem -proceed the work emphases of the group of scientists around nachtsheim in , that is, still at the institute for genetics and breeding research. at the very foreground was epilepsy research. nachtsheim had bred from vienna white rabbits a pure "strain of epileptics" and shown "through crossing with strains free of epilepsy […] that one recessive gene [was] responsible for the increased convulsion-readiness." however, this gene was subject to certain fluctuations in manifestation: "in the pure-bred epileptic strain the condition becomes manifest in about % of individuals." nachtsheim had also made some progress in the search for a genetic marker, although no real breakthrough had been achieved. according to nachtsheim's observations, the gene responsible for the increased convulsion-readiness must also grant leucism (the white color of the coat), although nachtsheim was forced to admit that not every form of leucism could be traced back to this gene. moreover, the influence of other genes, such as those for albinoism or "sooty coloring," could suppress the occurrence of leucism. the breeding experiments were complemented by a largescale series of experiments on nearly rabbits of different races, both from the "epileptic" and the "non-epilectic" lines, in which an injection of cardiazol induced convulsions to test their convulsion readiness. in addition to epilepsy research, pathogenetic research on eye diseases, especially on the progressive heredity of certain forms of cataracts, constituted a second working emphasis, in collaboration with hellmuth gürich of the charité ophthamalogical clinic. a third and final emphasis emerged from the work of the two doctoral students christian schnecke ( * ) and harry suchalla , who concerned themselves with growth anomalies. schnecke's studies on the "lethal dwarfism in rabbits led to the result, also important for the assessment of corresponding conditions in humans, that while the recessive dwarf factor in general may lead to a pathological form only in the homozygotes, but that there are genes that are harmless in and of themselves, which, when linked with the dwarf in his report to the board (mpg archive, dept. i, rep. a, nr. , p. ) fischer further mentioned rabbits "with a kind of st. vitus' dance." "genetic st. vitus' dance," one of the indications in the gzven, was the contemporary term for huntington chorea. it is unclear whether this might have indicated the rabbits with "shaking palsy," which is a general lay term for morbus parkinson. in his activity report for the / fiscal year fischer also mentioned rabbits with "skin diseases" (fischer, tätigkeitsbericht factor in the homozygous form, yield a combination with lethal effects, even if the dwarf factor is only present in a single dose." suchalla crossed giant and dwarf varieties of rabbits. this paper, nachtsheim emphasized, represented "the first attempt to achieve an analysis of skull genetics by performing experiments with modern methods on large amounts of material." here the research projects of the department for experimental genetic pathology overlapped with hans grebe's studies on chondrodysplasia -nachtsheim and grebe did, in fact, work together closely, for instance, on a genetic biology dictionary. in , the first year for the department for experimental genetic pathology, the group around nachtsheim was able to continue its research only on a very restricted scale. because it was increasingly difficult to obtain feed for the experimental animals, it was necessary to reduce their number and restrict the "consumptive research." the apparatus applied for arrived only after major delays -as, for instance, the "convulsator" for the generation of electric spasms -or were not delivered at all, as was the case for a slit lamp, a zeiss microscope and a binocular eyepiece. the greatest problem was that all of the staff was called up for military service, such that the experiments could only be continued by nachtsheim on his own. the fact that epilepsy research remained at the focus, although it had come to a preliminary conclusion in , was grounded first of all in pragmatic reasons: the research on eye diseases could not be continued because hellmuth gürich, the partner in this collaboration at the ophthamalogical clinic, was drafted to the wehrmacht. the same was true for research projects on growth anomalies. by this time christian schnecke and harry suchalla had also been drafted. the planned genetic pathological studies on a syndrome observed in dachhunds (characterized by hypodactyly or hyperdactyly, respectively, and hereditary blindness) never really got in gear. the resumption of research on the "pelger anomaly" (today: pelger-huët nuclear anomaly), an autosomal-dominant hereditary anomaly of the leucocytes that occurs in both humans and rabbits, was just getting started -the first task that kept nachtsheim busy was breeding a "pure pelger-huët strain," on the basis of which the characteristics of the gene it was based on could be studied. when an epidemic broke out among the laboratory rabbits in summer of , which necessitated halting the epilepsy experiments temporarily, the research on the pelger anomaly shifted far into the foreground. moreover, in this area nachtsheim was able to present a sensational finding. while up until that time it had been assumed that "this deviation of the blood count from the normal [was] to be observed in both humans and animals as a harmless variety of blood without any further clinical manifestations," nachtsheim produced evidence that in a rabbit which inherited the pelger gene from the maternal and paternal side, and was thus homozygous with reference to the pelger gene, "most serious impairments" were to be expected. according to nachtsheim's observations, most of the homozygous pelger rabbits died in the womb. the few survivors -the "über-pelger," as nachtsheim called them -showed not only a changed blood count, but also a whole bundle of other clinical symptoms: "meager growth, serious deformation of the limbs, especially the forelegs, with shortening and twisting of the long, hollow bones and synostoses [fusion of bones], rashes of scurf around the muzzle and nose, salivation, anorexia." these findings, nachtsheim explained, were of extraordinary importance for humans. true, no human "homozygous pelgers" had been encountered as yet. yet, trusting in the soundness of the animal model, nachtsheim predicted that in humans, too, the homozygous carriers of the pelger gene, if they were able to survive at all, would "thus certainly be greatly weakened in their vitality and deformed." in any case it is clear that the pelger anomaly did not constitute a "harmless 'play of nature'" in humans either, but was an "erroneous mutation […], whose propagation, in terms of race hygiene, [was] altogether undesirable." here nachtsheim opened up a new race hygiene perspective. the only way to be able to follow this perspective was to link the research on animal models with the genetic pathology of humans. the mission of the research would be, in nachtsheim's words, "to carry out exhaustive surveys about the propagation of the pelger gene in human populations." at the same time it would have to be investigated "whether among the stillbirths or behind an already familiar clinical picture, especially among cases with certain deformations of the limbs, homozygous pelgers are to be found." this suggested building a bridge from experimental genetic pathology to the genetic pathological research by heinrich schade and hans grebe, namely to grebe's series of studies on stillbirths. in his report about the fiscal year nachtsheim remarked that work in this direction had been "initiated," but had "not yet led to positive results." he further announced embryological studies in order to clarify in "which embryonic stage the homozygous pelgers die and what the cause of this death" and "what, on the other hand, [is] the cause of the survival of individual homozygous pelgers of certain parents." the clinical and histopathological diagnostic picture of the homozygous pelgers also demanded closer study. since these formulations were repeated word for word in nachtsheim's final report, which was dated march , , it must be assumed that the studies never picked up speed. in nachtsheim turned his attention to another hereditary blood anomaly of the rabbit, which had its parallel in humans: erythroblastosis, which occurs in rabbits as hereditary, general dropsy (hydrops universalis congenitus). today we know that this form of newborn jaundice in cases of incompatible rhesus factors in the blood of mother and child is caused by the formation of antibodies in the mother and their transition into the circulatory system of the fetus, where they destroy red blood cells. in , however, nachtsheim traced erythroblastosis in rabbits back to a gene that was "inherited recessively." but, nachtsheim continued, this was "not a case of simple heredity" -indeed, it appears "that a wide variety of factorsbesides the remaining genotype, also those of a peristatic nature -had an influence on the manifestation of the condition." in nachtsheim presumed that "several genes" were involved. perhaps there must also be "a certain conditional factor present […] so that the actual dropsy gene [could] become effective." possibly, however -and nachtsheim was on the right track here -"in addition still other factors located in the mother but outside the embryo [played] a role." as far as erythroblastosis in humans was concerned, nachtsheim's judgement in was more cautious, stating that it was "still quite contested," whether a hereditary condition was involved or not -much spoke against, some for heredity. in his penultimate report of march , nachtsheim suggested that "certain observations on humans [made] probable a connection between the fetal blood diseases and certain serum characteristics of the blood." thus it appeared desirable "to test experimentally for existing connections of this kind in animals, too." again, he states that experiments in this direction had been "initiated." by the way, the serological studies on hydrops universalis congenitus in rabbits were conducted in collaboration with the serological department of the reich health office, and the histopathological studies by hans klein while the research on fetal blood diseases of the rabbit was an emphasis of the work in the department for experimental genetic pathology in , in the research on the growth anomalies of the rabbit swung into full gear, when wouter ströer, the designated director of the planned department for embryology, took on the histological study of the rabbits with "lethal dwarfism" during his residency in dahlem. in addition to these working areas, in the final years of the war, epilepsy research moved back up to the top of the agenda of the department for experimental genetic pathology. in further breeding experiments nachtsheim investigated the heredity of genuine epilepsy. here it had become apparent, he reported in , that the "epilepsy gene," although its behavior was "generally recessive," and thus had to be inherited homozygously in order to take effect, was also able "to let the diagnostic picture of epilepsy develop" even in cases of heterozygous heredity, "in combination with certain genes." as such, "the carrier of two albino genes and one epilepsy gene can become an epileptic." the situation was similar for the allele closest to the albino gene, the "black factor." in addition to his breeding experiments, from nachtsheim performed a great number of experiments on producing spasms through oxygen deprivation. since these experiments led him directly into the research accompanying the nazi "euthanasia" program, they are described in detail in another section. this field of research now lagged behind, also and primarily because of problems acquiring material. much of genetic pathology research was based on clinical material, which verschuer and his staff had brought with them from frankfurt, and which could be supplemented continuously through individual cases brought to the institute for evaluation. the genetic pathology research by diehl and nachtsheim also used the animal model -and the rabbit stocks were safeguarded by the elevation of the kwi-a's status into that of a military economic enterprise. general human genetic research, in contrast, was based essentially on the combination of twin and family research. yet these methodological approaches were nearly completely obstructed in the second phase of the war. as fritz lenz lamented in his annual report for the "institute for race hygiene" in / : the work of research has been quite impeded by the circumstances of the war, especially since summer . it is very difficult and frequently impossible to acquire sufficient observation material for certain essential scientific and practical problems. as a consequence of the evacuation of women and children, family research and twin studies are practically impossible. not even surveys can be conducted any more. conventional twin research in this area apparently did not come to complete standstill, but the difficulty in acquiring subjects for both twin research and family research soon became an obstacle that could hardly be surmounted. thus it is no coincidence that the research in the area of the genetics of normal attributes in the year was restricted to two projects -"specific proteins" and "eye color"which made use of the unfettered access to subjects in the auschwitz concentration and extermination camp -more on this later. in the department for genetic psychology, gottschaldt, who was called up to the wehrmacht for a time, and his staff continued even after the start of world war ii with the evaluation of the enormous amount of material they had compiled in the twin camps in / , and working through it "in a new methdological way. removed to stavenhagen castle in mecklenburg in september . these tasks of evaluation were extremely elaborate: in the months from october to march , gottschaldt reported, "around psychological analyses [were dictated], each of which was pages long." by october gottschaldt and his staff had put to paper around psychological analyses, "which cover extraordinarily comprehensive material, prepared for statistical evaluation, of more than , individual findings." and for the coming months gottschaldt requested another , sheets of writing paper. during the war period, gottschaldt could not simply retire to his ivory tower. more and more he worked together with state and party offices, and endeavored to make the methods of genetic psychology useful for genetic health, race and colonial policy -be it voluntarily or under the pressure of the conditions must remain an open question. even today, almost nothing is known about most of these projects. in the / fiscal year, gottschaldt's department, in collaboration with the department for the protection of children and youth (kinderund jugendschutz) of the nsv, began with "catamnestic surveys of children formerly under the care of state welfare." the / business report also stated that the "polyclinic for nervous and difficult children," whose resources "increasingly [were] claimed for the scientific evaluation of the very extensive material on families that accumulates there," and that this would continue. from / gottschaldt held lectures and training courses, connected with the german labor front (deutsche arbeitsfront), the department for professional training and the improvement of efficiency (abteilung für berufsausbildung und leistungsertüchtigung) in the reich chamber of commerce and the colonial policy office of the nsdap. a deeper collaboration arose from the contact with the colonial policy office -more on this later. with the excursion into colonial science, gottschaldt set out on the field of race psychology, which he had only skirted before world war ii. thus it was fitting that he prepared an article about "race psychology" for the fifth edition of "baur-fischer-lenz." also to be viewed in this context are the untersuchungen über den rassenruf mongolider völker im rassenbewußtsein von japanern ("studies about the race reputation of mongoloid nations in the race consciousness of japanese"), which were carried out in the department for genetic psychology in collaboration with the cultural department of the japanese embassy -presumably by the two japanese guest scholars, the doctoral student masataka takagi and professor masaji kamitake. the fact that gottschaldt participated in a "german-japanese science camp" together with the two guest scholars in summer suggests that he, too, was actively involved in these obscure studies. finally, it must be added that a doctoral student of gottschaldt's, inez the parallel progression of political and scientific thought is no coincidence, but an internal necessity. […] we genetic biologists and race hygienists […] remain in the peace of our scientific research activity from the interior conviction that on this field, too, battles of major importance are being fought for the continuity of our volk. however, even as director of the kwi-a he was sometimes subjected to political pressures. with his very first lecture as a newly appointed member of the prussian academy of sciences on november , he offended party circles. under the title erbanlage als schicksal und aufgabe ("genetic disposition as fate and function") verschuer took his audience on a tour d'horizon through the regions of higher mendelism and phenogenetics. certainly: verschuer criticized the naive dogma of heredity predominant in higher mendelism. "for in many cases genotype and race were regarded far too simplistically in terms of materialistic determinism -as the sole source of all life performance, even of intellectual power, especially for culture and history." but at the same time verschuer made it perfectly clear that it was hardly his intention to explode the structure of genetic determinism: after these results of genetic and race research, is it justified to assert that genetic disposition is fate? yes and no! through genetic disposition, certain fateful limits are determined for the development of each individual. a negro cannot produce any white children, the genetically feeble-minded have predominantly feeble-minded children, certain defects are passed down according to familiar rules, etc. these are limits that are becoming ever more clearly and definitely demarcated through our research. they cannot be transcended. what could appear offensive, however, were the social and moral conclusions verschuer drew from the insights of higher mendelism and phenogenetics: in terms of their genetic dispositions, verschuer grouped people into a three-level model, arrayed between the two poles of "fate" and "function." verschuer located the majority of people on the third and highest level: in their genetic dispositions lay "a fateful predetermination only very weakly […] concealed," they had a "great breadth of possibilities for development." the shaping of the phenotype on this third level was the task of the individual and of society. the people on the second, intermediate level may carry the disposition for serious diseases and disabilities with them, but these appear either not at all or only weakly in the phenotype due to the oscillation of manifestation, and in any case can be compensated for by measures of prevention or rehabilitation. on this second level the molding of the phenotype lay between fate and function. as examples verschuer named club foot and congential hip luxation. from his comments clearly proceeded that he believed that all possibilities for orthopedic rehabilitation must be exhausted -he was well advised to factor out the question of race hygiene sterilization at this juncture, since both conditions were officially considered to be indications for sterilization, a position that verschuer and his colleagues, as we will show later, did not share. on the first and lowest level, finally, verschuer placed people with serious genetic defects, whose manifestation was not mediated by other factors -"association with other genes," "course of development," "external influences." on this level the phenotype was "to be accepted as determined by fate." nevertheless, with a view to these humans as well, verschuer argued in terms of the dualism of fate and function. although their genetic dispositions had to be "accepted as given by fate," the affected confronted a double function: first, even with a serious genetic defect, it is possible to give one's own life higher value and deeper meaning. just think of the extraordinary achievements of the blind and deafmute. yes, even a mentally retarded person can still carry out useful work and distinguish himself through loyalty, love and the spirit of sacrifice. second -and this demands a selfless readiness to make sacrifices -for the welfare of the volk, the serious genetic defect must be eliminated by forgoing propogation. this passage could be understood as a criticism of the "euthanasia" under way since , which had already claimed the lives of over , mentally ill and mentally disabled by this time -and it appears that party circles understood it as such. even more important: it was probably so intended. at this juncture verschuer made clear that he would continue to actively support eugenic sterilization that could be legitimated with the moral philosophy and theology of the idea of sacrifice -a position which verschuer had advocated since the final years of the weimar republic -, but rejected for ethical reasons the murder of the mentally ill and mentally disabled. what's more, he openly repudiated the "breeding of the Übermensch" in friedrich nietzsche's terms as the basic motif for race hygiene -he wanted to restrict race hygiene to its function as "custodian of the genotype of the race." as demonstrated, verschuer's lecture included some critical tones that could not have pleased the makers of national socialist genetic health policy. but an entry in the diary of ulrich von hassell ( - ) shows that verschuer's lecture could be interpreted differently as well: for me, a lecture on race policy for the berlin circle of the german academy was indicative of the level of some sectors of german science. the speaker was prof. von verschuer, the man whom e. fischer dared to propose as his successor in the mittwochs-gesellschaft. superficial prattle tailored to the purposes of party politics, truly a disgrace. nevertheless: what appeared as pseudoscientific party propaganda to a member of the resistance provided for unrest in sectors of the party. on april , , ibid., pp. f. ibid., p. (original emphases). ibid., p. . diary entry of / / , hassell-tagebücher, p. . that is, a considerable time after the lecture, verschuer related in a letter to fischer: yesterday afternoon i visited [walter] groß in his new office in babelsberg, a country house in a beautiful setting. we conversed for ½ hours in a very friendly and mutually obliging tone. he confirmed that he found nothing objectionable in the content of my lecture to the academy, and that my depiction and my standpoint were irreproachable. incorrect reporting and the misleading interpretation of individual passages have caused political turbulence. however, i got the impression that he will put an end to the matter. i made an agreement with him to submit to him any publications that encroach upon the area of race policy for fine tuning. so i hope that our friendly terms are restored, and that in future he will not be so easily disquieted by such yapping and put the over-zealous curs back on their chains. the incident is further exemplary evidence of the fact that, within the alliance between science and politics, it was ultimately the political decision makers who made the rules. hans-peter kröner's interpretation must be endorsed, that verschuer's account of his meeting with groß promoted his own self-deception: with the arrogant gesture of the academic, he required every effort to conceal from himself and his mentor that he -the director of a kaiser wilhelm institute -had been "muzzled" by one of the national socialist satraps. after the war, verschuer, together with his "whitewashers," greatly exaggerated the danger that threatened him from this direction. politically, he deviated from the line of state and party only in part. aside from the issue of "euthanasia," broad consensus predominated in genetic health and race policy. as will be shown in the next section, verschuer and his staff legitimated and propagated this policy, tended to the scientific substrate, provided practical support and did not hesitate to use the national socialist politics of genocide in order to acquire scientific "material." until well into the year , the staff of the kwi-a, hardly hampered by the circumstances of the war, undertook lecture trips all over germany and europe. the lectures at universities and to scientific societies were attended by "science camps" thus it is hardly possible to say that verschuer was "invited to report" to groß. according to kröner, von der rassenhygiene zur humangenetik, p. . in addition to their foreign travels, in february fischer and verschuer held lectures at the führerschule der deutschen Ärzteschaft ("leadership school of the german medical fraternity") on the occasion of a "joint camp" for physicians from alsace, luxembourg and the netherlands in alt-rehse. beyond their general foreign policy function, many of the lectures abroad apparently had the additional task of bringing functional elites from the field of medicine in the occupied and allied states "on course" with national socialist genetic health and race policy. thus in a dual sense they were a "service" the institute performed for the political rulers. however, they were also in the institute's own interest, as they can be regarded as part of a strategy to shape a continental european research alliance under german leadership after the collapse of the international scientific community. in this view, utilizing a large number of foreign guest scholars at the kwi-a, too, made a virtue of necessity. beyond this fischer and verschuer endea- contacts to scientists from the "antagonistic foreign countries" were disrupted as a natural course of the war. however, otmar von verschuer regarded science, too, as part of the military campaign. while he was at pains to keep himself up-todate on the scientific production of the "enemy states," he did his best to conceal this from foreigners. this became particularly clear in march , when he rejected fischer's proposal to publish the swiss guest scholar erik hug's summary of the last volumes of the most important anthropological and eugenic journals from the great britain and the usa in one of the journals he edited: , which were loaned out to me for days. the time was just sufficient for me to have the works most important for us photocopied." after all, he was a subscriber to the journal fortschritte der medizin ("progresses in medicine"), which had developed into a "reference work of the foreign medical press." "however, this journal is only dispensed for on may , , fritz lenz addressed an extensive letter to the editorship of das schwarze korps. he took reference to an article of april , entitled eine frau hat das wort ("a woman has her say"). the anonymous author had demanded, in view of the surplus of women after world war i, that the state should create incentives for women of who were still single to become unmarried mothers: such single mothers should receive a higher income than childless married women of the same age; their dual role as mother and career woman should be accommodated by flexible working hours; they should receive a one-off benefit similar to the marriage loan; "for the less well off," further, "current state supports [were] to be guaranteed." prerequisite for this benefit was the "genetic health" of the mother and the father -sperm donations were to come from single young men who had not yet started their own families. finally, the anonymous author, "for the protection of the honor" of the single mothers, had demanded that "anyone who reproached the morals of a single mother" be sentenced to prison on principle. lenz responded to these proposals with sharp critique. higher income for single mothers which, as lenz emphasized, would have to be financed by state subsidies, was not only economically intolerable, especially since, for reasons of equal treatment, the demand would "as a consequence […] would have to amount to ongoing support for all mothers." under aspects of race hygiene, too, it would always make more sense to support married mothers. the particular displeasure of the nestor of the race hygiene movement was evoked by the proposal to guarantee ongoing support for single mothers who were less well off: "a similar demand was raised in the reichstag of the weimar system by the communists." lenz warned that measures of this kind would "encourage the propagation of elements that [could not] demonstrate any sufficient performance as a result of mental or physical weakness and thus also [could not] exhibit any sufficient income." there was the danger of "adverse selection," which, as lenz argued with reference to possible concealed genetic dispositions, could not be avoided by making the "genetic health" of the men and women involved a prerequisite, either: "by no means can the danger of a preferential propagation of inferior race elements be averted in this manner." under quasi educational aspects lenz pointed out that the proposals would make necessary "special legislation" to expressly exempt the fathers of the children from support payments, "while in recent years the tendency has been to increase the responsibility of the father, in economic terms as well." the whole scheme boiled down to "state-approved temporary marriages of uncertain duration, which moreover would even be privileged by the fact that the state would take over the costs of official use and under the obligation that it be kept under lock and key, to certain subscribers who must pledge their confidentiality with a signature on the back cover." at another juncture verschuer reported that he received, "from the exchange service," the eugenics review, of which he had photocopies made. verschuer bringing up the children." this would have to weaken marriage as such. here lenz argumented quite conservatively and fundamentally, but not consistently in terms of race hygiene. he did see the childlessness of many women from the generation of the world war i as a race hygiene problem, but to him illegitimate motherhood did not seem a suitable solution under moral aspects -when, then polygamy instead: "purely objectively" he would hold the "permission of a […] limited number of second wives to be the relatively best solution; but the moral tradition of the german occident appears to virtually exclude such a solution." "breaking the moral tradition of a nation" was, however, "always perilous." lenz did not insist that his reply be printed in das schwarze korps. but he did call upon both the editorship and the author to enter into a critical dialog -to no avail, as the editorial board of das schwarze korps did not react at all. lenz addressed copies of his letter to the rusha, the race policy office and the german family league of the reich (reichsbund deutsche familie). at the close of his letter lenz illustrated his conception of state measures to increase the birthrate among married couples. a "state obligation to bring up children" should be introduced, in keeping with the principle "every member of the nation capable of living has the duty to bring up at least four children." anyone who did not fulfill this obligation should ante up "substitute payments in percentages of his income," which corresponded to "approximately the cost of bringing up children." lenz elaborated on this basic idea in an exposé about "ways to further advance in population policy," which he wrote at the same time as the protest letter to das schwarze korps. the french campaign had not yet entered its decisive phase, but lenz appeared optimistic that the end of the war was immediately imminent and would offer "a unique opportunity for generous population policy." because the birth cohorts since , which were already not terribly strong as a consequence of world war i, had been weakened further by the losses in the war from to , the idea was to induce the birth cohorts before to bring up as many children as possible. "against birth premiums and child subsidies," lenz announced yet again apodictically, there were "serious race hygiene objections." the experiences with marriage loans were, under quantitative aspects alone, "by no means encouraging." each marriage supported by a marriage loan accounted for "slightly less than one child." what is more: "from the perspective of race hygiene there is hardly a reason to regret that only meager funds are available for such benefits at this time." in contrast, lenz expressly advocated burden sharing for families through higher taxes for families with no or only few children. the tax increases dictated by the war seemed to offer a convenient opportunity to engineer such a burden-sharing scheme; the idea was "to make a demographic policy virtue out of the financial necessity of the war." the core of the tax policy concept worked out to the last detail by lenz was the proposal that the war surtax on income be eliminated after the end of the war only for families with four or more children. tax advantages for childless married couples were to be omitted, just as the temporary tax relief for young couples and the enduring tax break for couples with grown children. both parents of illegitimate children were to be allowed to deduct a child from their taxes, but only by half. only families with many children were to enjoy full deductions from property and inheritance taxes. finally, lenz developed a mandatory savings system for peasant families, to finance the compensation paid out to the daughters and sons who did not inherit property -if a peasant family had fewer than four children, part of the money saved would fall to the state. in fact, lenz's proposals -measured against the tax level before the beginning of world war ii -amounted to a constant tax burden for families with many children and enormous tax increases for everyone else. what is interesting is that verschuer, to whom lenz sent both of his documents for his perusal, responded immediately and declared himself in complete agreement with the contents. he expressly subscribed to lenz's thesis that the propaganda for illegitimate children evoked the "race hygiene danger" of "adverse selection." he proposed publishing lenz's tax policy exposé in the erbarzt. yet lenz had misgivings, since his proposals collided with the tax policy of the relevant state secretary in the reich finance ministry, fritz reinhardt ( - ), whom he did not want to provoke by publishing the exposé. however, lenz reported, it had been forwarded to reinhard via the race policy office. in lenz decided to publish his ideas after all, in the archiv für rassen-und gesellschaftsbiologie under the title gedanken zur rassenhygiene (eugenik) ("thoughts on race hygiene (eugenics"). with this he spurred into action the press department of the reich government in the reich ministry for propaganda and enlightenment of the nation, which ordered that the passages about relieving families of the tax burden be struck. new in this version of was that lenz demanded compulsory employment for childless and "child-poor" women. while lenz continued his efforts on the path of scientific policy consulting to secure recognition in population policy for a program of positive eugenics, verschuer and his staff continued to be in demand as experts and evaluators whenever questionable cases arose in the application of the gzven. as has been shown above, after the changing of the guard at the head of the institute, genetic pathology research in dahlem was consistently oriented toward this practical application, in order to "clear the complex jungle of the activity of producing expert opinions." due to the incomplete knowledge about the heredity of the diseases and disabilities listed in the catalog of indications of the gzven, since a jumble of contradictory decisions had resulted, and the initial enthusiasm of the race hygienists had given way to a kind of "hangover." in accordance with the insights of higher mendelism, since verschuer had urged that the hereditary health court proceedings be based not on the clinical diagnosis, but solely on the genetic diagnosis, to be reached through intensive genealogical studies. this had the consequence that in some cases which had fallen under the gzven as a matter of course up to that point, verschuer and his staff advised against sterilization. in one case this consistently pursued line went too far for even verschuer's friend and teacher fischer. when in verschuer's employee heinrich schade advocated the interpretation that certain defects of the limbs were not genetic and thus, must be excepted from sterilization, fischer lodged vehement protest with verschuer: with a degree of shock i read the paper by schade about the defects of the limbs. i do not hold to be correct the conclusions that heredity was not present in general, presented here in apodictic form. here goethe is wrong!! all fun aside. schade is, of course, right, that heredity is not proved in these cases. but for schizophrenia we also do not know what kind of and how many genetic factors are the basis. for the limbs there could hardly be any single, separate gene for each form and each location of defects, but rather different kinds, such as those which govern the development processes chemically. […] there is not only this theoretical side to the matter, however. in terms of praxis, schade arrives at the conclusion that one may not sterilize these cases unless another identical case was found to have occurred in the family. and this is extremely rare for today's small families. then we would have the situation, which seems intolerable to me, that conditions like cleft palate, club foot and hip luxation are sterilized as serious physical defects, but when an entire extremity is missing, or when both hands are completely crippled, sterilization does not take place. the public knows both groups as congenital. now the one defect, in fact the lesser, is regarded as congenitally inherited and thus to be sterilized; the other, more serious one, is regarded as congenital but not inherited, and thus not to be sterilized. the volk does not understand this. and for us, too, it goes against every feeling of justice. perhaps we were really somewhat hasty with the presumption of heredity in the case of defects. if one believes that, then the consequences must also be drawn for luxation, harelip, etc. i do not believe it personally. i am of the opinion that schade went too far and allow only that while we do not know the individual genetic process, genes are the cause. here fischer pursued the dual logic of an applied science, which must always attempt to combine the logic of science with the logic of politics. its recommendations to politics are always the product of several factors: scientific knowledge, the consideration of the practical utility of a measure, the expectation of its political feasability and its cultural acceptance, and finally its ethical admissibility. thus, weß, humangenetik, p. . ibid. cf. e.g. verschuer, unfruchtbarmachung. many scholars of the human sciences in the third reich advocated large-scale eugenic sterilizations, although it was quite clear to them that they were on shaky ground scientifically. the heritability of one or the other clinical picture constituted a plausible assumption, but one that in many cases required further empirical confirmation. that this assumption was sometimes presented to the outside world as a fact -to the public, but also to the state -is by no means unusual, but rather is part of everyday science even today. they believed that the empirical proof for the seemingly evident facts of the case could be presented afterward, sooner, or later. fischer's stated opinion is practically paradigmatic for this position. yet verschuer did not follow his mentor on this path. on the contrary: he countered fischer's argument that if one did not want to sterilize people with deformed limbs, then "cleft palate, club foot and hip luxation" would also have to be deleted from the gzven's catalog of indications, by aspiring to that very end. as regarded cleft lip, cleft jaw, and cleft palate, since the late s verschuer had proceeded from the assumption of a high degree of heterogeny -based on josef mengele's dissertation, by the way. but the consequence of this was that heredity had to be checked for in each individual case. and according to the testimony of gerhard koch mentioned above, in july or august the genetic pathology working group under verschuer's direction argued that club foot and congenital hip luxation should no longer be recognized as "genetic conditions" in the sense of the gzven. in the first, still-documented sessions of the genetic pathology working group, the participating scientists had also been extraordinarily reluctant to recommend sterilization in individual cases -in principle they were in agreement that the heritability of a condition had to be proved with certainty on the basis of family anamnesis in the individual case, whereas the logic of the gzven had saddled the subject to be sterilized with the burden of proof: he or she had to prove that in his or her case the general assumption did not hold that the condition was hereditary. in the judgement of concrete cases, verschuer consistently held fast to the genetic diagnosis. as a rule, he was extremely reluctant to acknowledge nonscientific considerations, even when they spoke for the subject. this became apparent, for instance, in the different judgements of the very first case dealt with in the genetic pathology working group, by verschuer and lenz. it involved a -yearold man, who had gone completely blind at the age of due to hydrophthalmus congenitus (congenital glaucoma), but had nevertheless graduated from secondary school, worked as a music teacher and piano tuner and led the association of the blind of his district, and finally studied law. in he wanted to marry a healthy teacher, but he was denied the marriageability certificate and an exemption from the regulations of the marriage health law. an application for sterilization in accordance with the gzven was rejected, however. the case had been submitted to verschuer for his expert opinion. in the ensuing discussion about the race hygiene consequences, lenz argued that the risk of rare, recessive genetic conditions occuring in the couple's progeny was so low that the couple, which consisted of "personalities of above-average talent and prowess," should not be deprived of the possibility of having children. verschuer argued against sterilization as well, but believed that according to the gzven the man "would have to be sterilized because of genetic blindness, if the hydrophthalmia was definitely genetic in his case, which he [verschuer] personally did not hold to be proved." by contrast, lenz was steadfast in his judgement "that even if the heritability of the condition was presumed, the man was not be sterilized, because there was no high probability that genetically ill individuals were to be expected among the progeny. the probability of this was less than %, and one may not do without healthy children because of one that might be genetically ill." in the end lenz and verschuer came to an agreement, "albeit for slightly different reasons," that in the given case neither sterilization nor a marriage ban was justified. the salient point is that in his argumentation lenz adhered closely to the letter of the law, to his official interpretation, and above all to the jurisdiction of the hereditary health courts, which had long since begun to grant broader latitude to the criterion of "preservation of life" in their judgements. verschuer's standpoint that upon proof of genetic blindness the subject was to be sterilized in any case, regardless of all other aspects, amounted in fact to an intensification of the existing legislation and administration of justice. that he decided against sterilization in this concrete case was due solely to the fact that he did not consider the proof of heritability to have been adduced. here it becomes apparent how misleading it is to use the individual cases in which verschuer and his staff advised against sterilization as an indication for the fact that the scientists of the kwi-a attempted, as a rule, to exert a moderating influence on the praxis of sterilization. they wanted to place the sterilization program on a new scientific basis that did justice to the insights of higher mendelism: some of the subjects to be sterilized, who up until that time had been sterilized without any hesitation, were thus spared from sterilization. in other cases verschuer and his staff judged even more harshly than the hereditary health courts. what is more: as a consequence of verschuer's position, sterilization legislation had to be extended to the heterozygotic bearers of recessive genes, who had no grebe, hydrophthalmus, p. . so koch, humangenetik, p. . in a case of paramyotonia congenita (eulenburg syndrome), a dominant autosomal genetic disorder with occasional muscular rigidity, primarily induced by physical excercise and cold, gerhard koch indicated that sterilization was not appropriate. in his memoirs koch emphasizes that this work "despite my critical opinions on the 'gesetz zur verhütung erbkranken nachwuchses,' [was] released for publication in the erbarzt by the military censors." (ibid., p. ). of course, from the perspective of nazi genetic health policy, there could hardly have been anything objectionable about this article. the passage in question reads: "[heinz] boeters holds an application of the g.z.v.e.n. to be unnecessary due to the rarity of myotonical clinical pictures ( of every , - , affected). in the case before us now, too, sterilization does not appear suitable because of the intellectual abilities of the subject. but since it is not to be expected that the disorder will become extinct through self-selection, as a rule marriage and having children should be urgently advised against. […] patients with myotonic apraxia are, of course, of no use for military service." koch, paramyotonia, p. . clinical symptoms. verschuer -in contrast to lenz -was too cautious to make such a demand publicly, for after all -to the disappointment of many a eugenicist and race hygienist -hitler and the national socialists had excepted the healthy bearers of genetic dispositions in their formulation of the gzven (which the prussian draft of had not, by the way). under conditions of war it was not to be expected that they would consent to such an explosive expansion of the sterilization program, which had been geared down in anyway. nonetheless, verschuer and his staff attempted to lay the scientific foundations for such an expansion, as shown by research like grebe's search for stigmata that would allow healthy bearers of the genes for chondrodysplasia to be identified. in principle and potentially, verschuer's position amounted to a further intensification of the sterilization legislation. while the kwi-a continued to fulfill an important consulting function as regarded the sterilization program, as mentioned above, verschuer distanced himself publicly from the nazi "euthanasia." in contrast, fritz lenz cooperated with the "euthanasia" planning staff to create a legal foundation for the mass murder of the mentally ill and mentally disabled. it has been asserted on several occasions that lenz had changed his opinion on the question of the "annihilation of life unworthy of life" at the beginning of world war ii -but this is only partially true. in the third edition of his work menschliche auslese und rassenhygiene (eugenik) -that is, the second volume of "baur-fischer-lenz," which appeared in -lenz had expressed his views on the issue of "euthanasia" at great length, after having made the impression on the public that he unreservedly advocated "euthanasia." on the contrary, lenz emphasized "that so-called euthanasia [is out of the question] as an essential means of race hygiene." taking reference to intensified postwar discussion about medically assisted suicide, killing on request and the "annihilation of life unworthy of life," lenz pled for the view that "euthanasia" was "preeminently a question of humanity. even the ancient spartan abandonment of deformed children is still incomparably more humane than today's practice of rearing even the most unfortunate creatures in the name of 'compassion' […] ." with reference to race hygiene, however, "euthanasia" had no great importance to the extent that the circle of those affected would hardly have the opportunity to propagate -if this danger existed, then it could be prevented by sterilization. what did speak for the painless killing of disabled children from the standpoint of race hygiene is that it would enable the parents to bring another, healthy child into the world. this would also mean that "the question of the marriageability of encumbered persons [could be] judged much more generously than it is today." for instance, there would be lenz, gedanken zur rassenhygiene, p. . at this juncture lenz also openly demands the inclusion of "asocial" subjects in the sterilization legislation. ibid fewer misgivings about permitting a marriage between partners who were healthy themselves but known to be bearers of a recessive gene for deaf-muteness, because the deaf-mute children from such a marriage could be killed and thus the parents given the opportunity to have as many healthy children as their economic situation allowed. although lenz thus indirectly attributed a eugenic function to "early euthanasia," he persisted in his opinion that "euthanasia" was "hardly […] so effective a means" under race hygiene aspects "that race hygiene must advocate it." decisive for lenz was that through "euthansia" the "respect for individual life, which is an essential foundation of our social order, would experience a critical loss." although more than a few infanticides occurred for the purpose of family planning even in the western cultures, "the moral consciousness in the occident [excluded] a legal license for infanticide." in other words: lenz did hold the killing of disabled newborns to be justifiable in principle as an act of "humanity," but in the early s he still believed that deregulating "early euthanasia" would shock the "moral consciousness" and the "social order." it is presumably not incorrect to presume that lenz feared "early euthanasia" could become the gateway to the deregulation of abortion for social indications. in principle lenz adhered to his position. after including the passage about "euthanasia" unchanged in the fourth edition of his work in , in the preface to the publication by wolfgang stroothenke about erbpflege und christentum ("care of genes and christianity") published in , in which "early euthanasia" was demanded on as a measure of caring for the genetic pool, lenz reaffirmed his standpoint that "euthanasia" was primarily a question of "humanity." what lenz did not write openly, however: apparently he believed the time had come to set about resolving this question. in any case lenz -presumably in his capacity as a member of the expert council for population and race policy -proved ready to participate in a commission, which probably convened in october , to debate and finalize a draft law to legalize the "euthanasia" program under way since the change of years / . a number of the physicians represented in this commission belonged to the medical staff of the "euthanasia" program -among them georg renno, who had passed through the kwi-a's first annual course for ss trainees -as well as several representatives of the medical administrations of the states, and, finally, the chief of the security police and the sd, reinhard heydrich , who was interested in the procedure because at this time he was concerned with the planning for a "community alien law" (gemeinschaftsfremdengesetz). the draft law lenz, auslese, rd edn, p. . cf. the preface in stroothenke, erbpflege. on the previous history: roth/aly, "gesetz über die sterbehilfe"; gruchmann, euthanasie; klee, "euthanasie," pp. f.; schmuhl, rassenhygiene, nationalsozialismus, euthanasie, pp. - . cf. on this: ayaß, "asoziale," pp. - . otmar von verschuer also got involved in this discussion. he approved the establishment of a register of "community aliens" in order to attain a "differentiation between those to be eliminated and those to be supported" (erbarzt , , pp. f.). ultimately worked out by this body then presumably bore the title proposed by fritz lenz, "law about euthanasia for the incurably ill." the final version of the draft was not preserved, but the contents of its six articles can be reconstructed on the basis of some of the surviving commission protocols. apparently the preamble states that people "who because of an incurable disease long for [an end to their suffering]" or "as a consequence of an incurable chronic condition are incapable of productive life," were to be afforded assisted suicide. the first two articles represented slightly modified versions of a draft law debated by the official criminal law commission on august , : § : anyone who suffers from an incurable disease [that presents a great burden to himself or others or is certain to lead to death] can receive euthanasia upon his express request with the approval of a specially authorized physician. § : the life of a patient who otherwise would require lifelong custody as the consequence of incurable mental disease can be ended through medical measures unnoticeable for the patient. the following four articles regulated the process. the patients were to be registered -unless the patient himself submitted the petition -by the public health officers and institutional physicians. thereupon an evaluation was to take place by "expert committees," each of which was to include a "specially authorized" public health officer and two medical assessors (psychiatrists). the committees were to be assembled by a "reich deputy" to be appointed for the execution of the law. this special agent, vested with far-reaching powers, was also to make the final decision about the petitions for "medical assistance" on the basis of the expert opinions submitted by the expert committees, and to appoint the physicians to perform the procedure. if an executing physician stated that he did not agree with the vote of an expert committee, he could submit a detailed written explanation of his reasons and apply for a new expert opinion by another expert committee. what role did lenz play on this commission? according to the protocol he was one of the most eager discussants, and many of the essential formulations of its content were based on his proposals. this began with the title of the draft law. upon lenz's suggestion, the word "deliverance" was struck, "which, originating from the world of christian ideas, would evoke negative feelings against the law." the wording of article also came from lenz. he had rejected the original term of "abnormal disposition" as "too indefinite and vague." in some cases it was "not at all clear whether abnormal genes or external damage was the basis"; "idiotic or seriously deformed children" would thus not be included by the concept of abnormal disposition -for these, moreover, "a special legal determination was required." therefore lenz pled for restricting the law only to the mentally ill for the time being. presumably with a view to heydrich and his interests, lenz added that one would also have to define expressly "that criminal psychopathy is a mental illness in the sense of the law." lenz did not want to make exceptions, but he held the inclusion of "senility of the mind" to be unsuitable. all in all the impression arises that lenz was one of the driving forces in the discussion and that he left his mark on the draft law. repeatedly he pushed for precise specifications -in the interest of legal certainty. it was also due to this interest that lenz wanted to make sure that the law would be applied initially only to cases of serious mental disease, whereby he urged a regulation of "early euthanasia" at a later point in time. that it was by no means his concern to check the "euthanasia" program in progress is apparent in the fact that he was ready to include the "criminal psychopaths" in the sense of the "community alien law" planned by heydrich. in the end the draft law discussed remained nothing more than paper. hitler rejected a legal enclosure for the "annihilation of life unworthy of life." "euthanasia" continued to proceed in the unlawful cavity of the national socialist "prerogative state," flanked by an ambitious program of genetic psychiatry and genetic pathology research. in one case the kwi-a, too, profited from the unfettered access to human subjects in sanatoriums and hospitals. back in hans nachtsheim, as mentioned above, had performed a large-scale series of experiments on almost rabbits of different races, both from the "epilectic" and the "non-epileptic" breeds, in which convulsions were induced through cardiazol injection to check their compulsion-readiness. as explained, the point was first to theoretically illuminate "the connections between convulsion-readiness and genotype." the series of experiments also pursued a second, entirely practical purpose, however: they were supposed "to provide a contribution to the question so debated in psychiatry, as to whether a genuine epileptic responds to a lower dose of cardiazol with convulsions than does a symptomatic epileptic or a non-epileptic, and thus whether inducing convulsions by cardiazol is of value for differential diagnostics." in this respect the experiments ended in failure. it became apparent that the convulsion-readiness of the rabbits was dependent on their age: young rabbits convulsed at a lower dose of cardiazol than older animals; convulsion-readiness in response to cardiazol thus, appeared to diminish with increasing age. this alone would not have debased cardiazol convulsions as a differential diagnostic instrument, as the rabbits of the purely bred "epilectic strain" reacted more sensitively to cardiozol at all ages than did other rabbits. however, at the same time it turned out that the convulsion-readiness of the "epileptic" rabbits was subject to frequently occurring, strong individual oscillations, so that the animals sometimes did not respond to a high dosage -at which even a high percentage of "non-epileptic" animals convulsed, and at other times convulsed even at low doses that would never have triggered convulsions in "non-epileptic animals." in view of these findings nachtsheim had to admit that cardiazol convulsions possessed "only limited differential diagnostic value." although the hope for a direct practical use of the convulsion experiments had not been fulfilled, nachtsheim continued to grant high priority to his research on the "epileptic" vienna whites even after starting at the kwi-a on january , . from this point on the research projects on epilepsy pursued modified research questions: the direct perspective on differential diagnostics was abandoned, and epilepsy research oriented instead entirely toward the paradigm of phenogenetics. what appeared as a mere disruptive factor in the experimental arrangement under the aspect of differential diagnostics -the modifying influence of age, time of year and season on the convulsion-readiness of the rabbits-, became the actual object of research when embedded in the paradigm of phenogenetics, for apparently the convulsion-readiness of the experimental animals was the result of an interplay among genetic and peristatic factors, which intertwined to cause, enable and induce. if research succeeded in exposing the complex reciprocal actions of genetic disposition, maturation, and environment, science would be much closer to illuminating the process of the pathogenesis of epilepsy. in his activity report about the / fiscal year, eugen fischer quite skillfully referred to nachtsheim's "investigations about the epilepsy of rabbits, which corresponds completely to that of humans." the experiments "to use cardiazol to induce epileptic convulsions like those in humans" were "in full swing," and promised "a more precise analysis of the genetic and non-genetic conditions of the convulsion-readiness of vessels in the brain." here fischer did imply that the studies in progress could contribute to the demarcation between genuine and symptomatic epilepsy, yet he painstakingly avoided the term "differential diagnostics," selecting a more open formulation. on the other hand, he left no doubt as to the applicability of the animal model. however, nachtsheim had to struggle with some exceptions voiced from the ranks of his critics on precisely this point. concerns, such as the fact that the structures of the human and rabbit nervous systems were too different for the findings obtained with rabbits to be applied to humans without further ado, were forestalled by nachtsheim himself, who raised the conjecture that the pathological processes which also occurred in humans might possibly be better studied on rabbits, because in the rabbit the convulsion takes place in a much more primitive form, "without all of the accessory parts that have accrued in humans," -an argument based on an abridged mechanistic concept of the organism, which was not terribly solid. after brashly asserting the applicability of the animal model at the outset, over the course of time he sought refuge in more careful formulations: in this we are certainly aware that a result in an animal experiment can be translated to humans only with caution, especially when the substrates in question are as different as the rabbit brain and the human brain. however, a result for rabbits can be regarded as at least pointing the way to the conditions in humans. nachtsheim also had to struggle with the clinicians' critical pointer to the polymorphy of the various clinical pictures subsumed under the concept of epilepsy. he thus toned down his pretense of using the animal model to explain the epilepsy of humans and hence aspired only to relate "rabbit epilepsy to a certain 'variety' of human epilepsy." the most serious was the objection of leading psychiatrists that cardiazol convulsions in rabbits were not comparable to spontaneous convulsions in humansthis critique was aimed straight at the experimental arrangement, which made the animal model organism available for human genetic research. nachtsheim and his staff therefore, also tested other possibilities such as insulin and acetylcholine shock as well as electric spasms. in the course of these tests they concluded that the convulsions in vienna white rabbits artificially triggered by cardiazol most closely approximated the spontaneous convulsions of the human epilepsy victim. according to the concept of convulsion-readiness, every human could suffer convulsions -in the case of epilepsy nachtsheim imagined the boundaries between health and illness to be fluid. epileptics were different from other humans, he presumed, in that their convulsion threshold was significantly lower. now, under the banner of higher mendelism, this was no longer simply regarded as genetic, but rather -in terms of phenogenetics -as the result of a causal chain, the first cause of which is to be sought in the genotype, but which was also influenced by factors in the internal and external milieu. when an organism in a condition of heightened convulsion-readiness was subjected to an adequate environmental stimulus, this would trigger a convulsion -the epilepsy became manifested clinically. his rabbits by changing the intervening peristatic variables that modified the effect of the gene. the comparison of young and mature animals was an obvious choice for the first series of tests, as nachtsheim believed he had determined in his cardiazol experiments, performed to resolve the differential diagnostic problem, that the convulsion-readiness was dependent on age. therefore, in a paper for the zeitschrift für altersforschung in nachtsheim reevaluated his previous experimental results under this aspect, and in so doing also introduced them into the still young discipline of geriatrics. in the field of psychiatric research it was controversial at what point in time and in what form the various types of epilepsies became manifest, whether the clinical picture changed over the course of life, and whether such age differences occurred only in symptomatic epilepsy or in genuine epilepsy as well. nachtsheim wanted to attempt to pursue these questions using a comparative experimental system on the animal model. in summer an epidemic raged among nachtsheim's experimental animals and forced him to temporarily suspend the experiments on epilepsy, because the stocks had to recover before this form of "consumptive research" could be continued -in the artificially provoked convulsions, especially when cardiazol was used, it was not seldom for the experimental animals to suffer broken bones or collapse. when nachtsheim resumed the experiments in early , he changed the method. he no longer resorted to cardiazol, which had the disadvantage in nachtsheim's view that its toxic effect was superposed upon the convulsion events and made their observation more difficult. therefore, it must have been easy for him to give up this method, especially since cardiazol was difficult to obtain during the war as it was urgently needed for therapeutic purposes. the electric spasm attempts conceived as an alternative to the cardiazol experiments in had "not proceeded beyond certain preliminary tests," not least because the "convulsator" by the siemens-reiniger plant procured in proved unsuitable for animal testing. a change in the experimental arrangement was thus essential, and this led nachtsheim to high altitude medicine. crucial for the further development was the incipient collaboration between hans nachtsheim and gerhard ruhenstroth-bauer . after completing his studies of physics, in september ruhenstroth-bauer had come to adolf butenandt at the kwi for biochemistry to write a dissertation in the area of hormone chemistry. when this dissertation project hit a snag due to the war, ruhenstroth-bauer turned to research on the regeneration of red blood cells (hemopoiesis). he had been forced to interrupt this research when he was drafted into the luftwaffe as a military physician and sent to the eastern front. butenandt lobbied erich hippke ( - ), head of the luftwaffe medical corps, to have ruhenstroth-bauer reassigned to berlin, and hippke -it is not clear whether upon butenandt's urging or on his own initiative -ordered the young military physician to berlin in june , in order to perform special research on hemopoiesis. ruhenstroth-bauer was searching for a substance that was capable of effecting a prolonged propagation of the red blood cells (erythrocytes) -he assumed that it would be a hormone, which he intended to name hemopoietin. the potential military importance of the project for air warfare was obvious: pilots who were injected with the blood-enriching substance before takeoff would be able to fly at higher altitudes in air with less oxygen, without any decrease in performance. in a series of preliminary tests, ruhenstroth-bauer endeavored to research the process of generating blood cells in various experimental animals in different test arrangements, and in so doing also experimented with oxygen deficiencies and low air pressure. at this point the research interests of ruhenstroth-bauer and nachtsheim overlapped. convulsions are a characteristic symptom of altitude sickness -ruhenstroth-bauer's research practice was oriented around raising the threshold for altitude convulsions by increasing the number of red blood cells. for its part, epilepsy research was close to altitude research because oxygen deprivation had long been discussed as a possible trigger for epileptic convulsions. the possibilities of high altitude medicine to generate oxygen deprivation experimentally in vacuum chambers thus also opened up new ways for nachtsheim to move his experiments with the "epilectic" vienna whites forward. nachtsheim was interested in collaborating with ruhenstroth-bauer because the latter experimented with rabbits deprived of oxygen, was well familiar with the physiology of blood and respiration and brought along biochemical expertise. for ruhenstroth-bauer's part, nachtsheim's research on the phenogenetics of convulsion-readiness must have been of fundamental importance -and added to this was the fact that nachtsheim developed an interest in blood and hemopoiesis starting around mid- . the initiative for collaboration probably came from hans nachtsheim, although it can be presumed that the two men were already acquainted due to the tight net of reichsluftfahrtministeriums) , for assistance in conducting the low-pressure experiments involved in his hemopoietis project. nachtsheim, too, through his former doctoral student harry suchalla, who had found a position on the "top floor" of the institute, had contacts to strughold, whose institute, which was housed in the military physicians' academy (militärärztliche akademie) along the bank of the spandauer schiffahrtskanal on scharnhorststrasse in berlin, had several vacuum chambers at its disposal. around june nachtsheim and ruhenstroth-bauer began with their rabbit experiments in the vaccum chamber of the research institute for aeronautical medicine. yet these low-pressure experiments constituted only a small portion of the around experiments that nachtsheim and ruhenstroth-bauer performed in , with support from the reich research council and the third-highest priority rating of "s," for the purpose of depriving their test subjects of oxygen in various ways. the two scientists advanced a concise justification for their experimental program: the results of the cardiazol experiments on epileptic and non-epileptic rabbits in previous years made it seem desirable to investigate the importance of oxygen deprivation for the inducement of the epileptic attack in special experiments on young and mature animals. through the experiments in the vacuum chamber nachtsheim saw his view confirmed that convulsion-readiness depended on age. "normal mature animals" subjected to oxygen deprivation in the vacuum chamber, which corresponded to a height of , - , m, showed no reaction at all, and this was also the case for "normal young animals" and "mature epileptic animals." in contrast, "young epileptic animals aged - months" nearly always suffered at least a rudimentary epileptic attack under these conditions, and in cases of a "generalized attack with all phases of spontaneous convulsions" the frequent result was "the sudden death of the animals." further series of tests "proved" to nachtsheim that it was the oxygen deprivation ensuing from the low pressure that induced the convulsions: for one, the same result could also be attained when the test subjects were subjected not to low pressure, but to a mixed nitrogen-oxygen atmosphere that corresponded to a height of around , m. second, it turned out that an epileptic attack could also be induced in the rabbits by interrupting the flow of blood to the brain, again, particularly "promptly and impressively" in the "young epileptic animals." for a more precise analysis of the effect of oxygen deprivation, in further tests the rabbits were "set in part into a proconvulsant, in part into an anticonvulsant condition." so some animals were tested in the condition of alkalosis or acidosis (shift in the acid-base balance in the blood toward the alkaloid or acidic side, respectively). others were placed in a mixture of air and carbonic acid, or treated with bromural, luminal, or caffeine before the oxygen deprivation test. it proceeded from all tests, nachtsheim proclaimed, "that in the epileptic rabbit oxygen deprivation is the root cause for the inducement of the epileptic attack." the term "myoclonic threshold" is largely identical to the term "sensitivity to oxygen deprivation of the brain cells inducing the attack." this very assertion was disputed from an influential quarter. a group of scientists around alois kornmüller , director of the department for the experimental physiology of the brain at the kwi for brain research, had been studying epilepsy for a long time -also in collaboration with strughold -and was already looking into the connections between epilepsy and altitude sickness. the junior physician j. gremmler, who belonged to the "brain research office of the air force" (gehirnforschungsstelle der luftwaffe) under hugo spatz, performed a series of experiments in which (adult) epileptic patients from sanatoriums and hospitals were experimentally put into a condition of hypoxemia and then their brain waves measured. this experiment brought gremmler to the conclusion that oxygen deprivation must be excluded as the trigger for epileptiform convulsion fits. this result constituted a double challenge to nachtsheim: not only were gremmler's findings on the importance of oxygen deprivation diametrically opposed to his own, but gremmler also cast doubt as to whether the convulsions in altitude sickness could be equated with the epileptic attack at all. in so doing, he also questioned the very foundations of the animal model developed by nachtsheim and ruhenstroth-bauer, for if the convulsions generated in rabbits by low pressure were not epileptiform, then the results on varying convulsion thresholds in young and mature animals could not be translated to human epileptics. unless nachtsheim and ruhenstroth-bauer wanted to call gremmler's findings into question in principleand they did not, because they saw nothing objectionable in the experiments -there was only one way for them "to salvage" their own research findings: elsewhere it has been proved for humans that adult epileptics do not respond to oxygen deprivation with an attack. since a significant difference in the behavior of mature and young epileptics was yielded in our animal experiments, we tested epileptic children at low pressure in a similar manner. if they were successful in inducing epileptic attacks in epileptic children through low pressure, gremmler's negative findings would be relativized -in gremmler's experimental arrangement, it could be argued, the oxygen deprivation was simply not great enough to induce a convulsive attack in the adult test subjects -and the hypothesis of oxygen deprivation as the trigger of the epileptic attack would be saved. beyond this, if the epileptic children reacted to low pressure in the same way as the young epileptic rabbits, this would furnish impressive evidence of the animal model's applicability. paradoxically, in this case the human experiment was to function as the confirmation for the animal experiment, which was originally conceived of as a substitute for human experiments. there are only two written sources on the further course of events, both of them quite meager -a report by nachtsheim to the reich research council of september , or march , , respectively, and a short letter from nachtsheim to gerhard koch of september , -as well as several testimonials put down in writing by gerhard ruhenstroth-bauer at great intervals of time. these sources document without a doubt that at least one such human experiment took place. however, we know hardly anything about how this experiment came about and how it proceeded in detail. for the present we also remain in the dark about what happened to the human "guinea pigs" later and whether further experiments of this kind followed. apparently nachtsheim, in his search for epileptic children for the planned tests, turned to gerhard koch, who was convalescing in berlin from june to august and worked as a guest scholar at the kwi-a during this period. at the time koch's research included work on "residual epilepsy." as he wrote in his memoirs, he and nachtsheim "repeatedly [conducted] instructive and useful conversations about the etiology and heritability of the various epileptic convulsive conditions in humans and animals and about the convulsion-readiness behind these conditions which is so different for each individual." it was presumably koch who drew nachtsheim's attention to the berlin-wuhlgarten sanatorium and hospital (heilund pflegeanstalt berlin-wuhlgarten), in which a large number of epileptics were housed. koch had worked there from to on "family studies" in the context of his dissertation about sturge-weber disease (today: sturge-weber-krabbe syndrome). he had maintained contact afterward -as late as julius hallervorden sent koch the pathological report of a test subject who died after the family study was concluded. while still working in dahlem in summer , koch, assisted by hans grebe and and mature epileptic animals in response to oxygen deprivation made it appear desirable to investigate on humans a comparison of young and adult epileptics. gremmler investigated only adults, and was not successful in inducing an epileptic attack in them through hypoxemia. after conclusion of our own studies of young epileptics, which are also interesting to the clinic, we intend to report about the detailed results." -on the human experiment described in the followiing, cf. chamber. yet the tests came out just as negative as those gremmler performed on adult epileptics. but at the moment it is not possible to say that rabbits and humans respond differently to low pressure, for the children we tested were aged - , which corresponds to a rabbit aged - months. however, epileptic rabbits of - months do not show the reaction-readiness of - -month-old animals, which nearly always had attacks. we would have to be able to test epileptic children of - years of age, but this is not possible at the moment because this age group is not present at görden. so from wuhlgarten, ruhenstroth-bauer and nachtsheim had been referred to the state institute in brandenburg-görden directed by hans heinze, which played an important role in the nazi "euthanasia" program. it remains unclear who ultimately established contact with görden. in the s ruhenstroth-bauer claimed that nachtsheim had enjoyed good contacts to görden and was involved in the treatment of epileptic children there, so that he addressed the children by their first names and elucidated their anamneses, while he -ruhenstroth-bauer -met the children for the first time in the vacuum chamber on september , , had never seen them before and did not even know where they came from. considering the letter from nachtsheim to koch, there is certainly reason to regard this testimony as an attempt at self-justification, but it is indeed conceivable that nachtsheim had been in contact with görden for some time previously. this could have come about via the kwi for brain research, which was, for its part, linked closely with görden through julius hallervorden, who was both director of the department for histopathology at the kwi for brain research and prosector of the brandenburg state psychiatric institutes from onward -in fact the department of pathology located in görden from had been officially transferred to the kwi for brain research in berlin-buch, and the laboratory in görden was run as an outpost of the kwi. through the department of pathology in görden and other channels, over brains of "euthanasia" victims made their way to the kwi for brain research, where they were subjected to pathological examination by julius hallervorden and hugo spatz. nachtsheim had good contacts to the kwi for brain research -for years he had sent his rabbits from the epilepsy experiments to gerd peters ( - ) for postmortem examination. as mentioned above, nachtsheim also had close contact with the pathologist hans klein, who performed postmortem examinations on rabbits with dropsy for him starting in , but also participated in the autopsies of the victims of the "special children's department" at wiesengrund. what should not be forgotten is that fritz lenz was familiar with a number of physicians from the staff of the "euthanasia" program from his consulting activities on the draft law for euthanasia, including hans heinze. pointing out these entanglements is important to the extent that it can be presumed with a high degree of security that nachtsheim was aware of the "euthanasia" program still in progress. as the available sources testify unanimously, the experiment did not produce any tangible result -it did not succeed in inducing an epileptic fit in the children through low pressure. consequently it did not cause them any suffering -but ruhenstroth-bauer and nachtsheim could not have foreseen this. according to nachtsheim's account, the children were subjected to a low-pressure situation that corresponded to an altitude of , m (not to mention the mental strain of being locked into the vacuum chamber). according to the knowledge available to altitude medicine at the time, at this altitude the onset of threatening conditions had to be expected even for adults -all the more so for children. moreover, there was no possibility of resorting to any previous experience with epileptic humans in lowpressure situations. furthermore, ruhenstroth-bauer and nachtsheim knew from the animal experiments that young epileptic rabbits reacted to low pressure with violent, often fatal convulsions -and they expected (and hoped!) that the children would react like the rabbits. in other words: the scientists knowingly accepted the risk that the children could be placed in fatal danger. ruhenstroth-bauer's reassuring statement that he himself, nachtsheim and an additional physician of the luftwaffe had been in the vacuum chamber with the children and had been able to abort the experiment at any time -as could the children themselves -thus fails to get at the root of the matter. there is no doubt that ruhenstroth-bauer and nachtsheim planned further tests with younger children after the failed first experiment. whether these came about cannot be determined with any certainty. however, it is probable that it was no longer possible to realize these tests. of the six children in the first experiment, there is proof for only one having survived the third reich; the fate of the other children must remain an open question. perhaps they fell into the gears of the "euthanasia" program -in contrast to the clinical examinations and tests in the two "research departments" of the "euthanasia" apparatus in brandenburg-görden and wiesloch/heidelberg, however, it went against the logic of the experiment to kill the children and examine them pathologically as long as they had not suffered an epileptic attack. nonetheless: the low-pressure experiments by nachtsheim and ruhenstroth-bauer ignored the reich health council's regulations on human experiments from the year as a matter of course. for the most part, these regulations, as adduced elsewhere, had already been ignored by research back in the s. yet this experiment marked a further boundary crossing, as the experimenters unscrupulously subjected the children to an incalculable health risk, even accepting a potentially fatal outcome of the test -and all of this needlessly, for the utilization of the vacuum chamber was by no means imperative. oxygen deprivation could have been effected in other ways, especially as gremmler, upon whose work the experiment was based, had not worked with low pressure. apparently the standards of scientific ethics had shifted further. a comment with which nachtsheim and ruhenstroth-bauer preceded their short report about the low-pressure experiments on rabbits implied as much: for the clinician working on patients experimentally, the possibilities are always restricted, for he has to take the welfare of his patients into consideration. only in exceptional cases will a researcher dare to perform an experiment on a patient in the interest of future patients, the outcome of which cannot be predicted with any certainty. here a method assists the field of medicine, which allows these difficulties to be circumvented at least for a few genetic illnesses, the model experiment on animals. alexander von schwerin is correct to emphasize that this opens up a new moral dimension. while up to this point nachtsheim had designated the human experiment as morally inadmissable without restriction, and recommended the animal experiment as a morally unobjectionable alternative, he now no longer categorically excluded the possibility of research on humans for the benefit of others, even if the outcome was uncertain. in this case human and animal experiments no longer appear as mutually exclusive alternatives; on the contrary, it suggests a complementary relationship. schwerin lists a number of factors that contributed to the erosion of the ethical standards of science: the objectifying linguistic usage, which not only blurred the boundaries between humans and animals (nachtsheim, for instance, referred to both as simply "epileptics") and transformed both into "material," but also elevated the "genotype epilepsy," detached from the human patient, to the actual scientific object; and also the "militarization" of altitude research. two other aspects deserve special emphasis: first it must be kept in mind that the newly developed coma and shock therapies (insulin coma treatment, cardiazol convulsion treatment, and electric shock therapy in the first years of world war ii) had been widely adopted in german institutional psychiatry since the mid- s, although these "heroic therapies" put the patients in horrible states of anxiety, often inflicted serious injury to their health, and in some cases even resulted in their deaths. therapeutic ambition was willing to accept high risks -thus, it is no wonder that artificially inducing convulsive fits in epilepsy research was not questioned. second it must be considered that by , somewhere around , mentally ill, epileptic, or mentally disabled patients from the sanatoriums and hospitals of the german reich already had been murdered in the course of "euthanasia" -and thousands of infants, children, and teenagers had also been killed in the course of the children's "euthanasia," the "aktion t " and "decentral euthanasia" ruhenstroth-bauer/nachtsheim, bedeutung des sauerstoffmangels, p. (original emphasis). schwerin, experimentalisierung, pp. f., f. cf. kersting/schmuhl, einleitung, pp. f. since august . this undermined the moral status of the children from görden. now they were little more than readily available, not terribly valuable "material" for "consumptive research." with the entry of the german reich into the circle of colonial powers, german anthropology and ethnology -like the other sciences -felt challenged to make their knowledge useful for the justification and legitimation, execution, and consolidation of colonial rule. a relationship of mutual engagement emerged: the sciences aligned themelves with colonial interests in their selection of subjects and objects, their theoretical and methodological approaches, and made the knowledge thus obtained available to the colonial administration. in return, the colonial state furnished the colonially oriented scientific disciplines and subdisciplines with financial resources, granted them privileged status in the institutional structures and raised their value in the public. colonial interest groups mediated between state and science. "in this system of mutual obligations between state, political parties, interest associations, and sciences after , a spectrum of new areas of knowledge developed in the german science landscape, which was known as the 'colonial sciences' in the contemporary diction […] ." fischer's study on the "bastards of rehoboth" of the year was conceived and intended as a contribution to colonial science, apparent in the fact that the author drew practical consequences for colonial policy from his research findings in its concluding chapter, die politische bedeutung der bastards ("the political importance of the bastards"). despite his heterosis thesis, according to which a "population of bastards" is located between the "source races" as regarded their physical, mental, and intellectual characteristics, he took a clear position on the ban on mixed marriages in the colonies so hotly debated at the time: every european nation without exception […] that has assimilated the blood of inferior races -and that negroes, hottentots and many others are inferior can be denied only by dreamers -has paid for this assimilation of inferior elements with intellectual, cultural decline. at the end of his colonial policy conclusions, fischer designed a system of apartheid for german southwest africa, long before such a system was introduced in south africa: the ovambo and herero were to be deployed as agricultural laborers, the hottentots as herders. the "bastards of rehoboth," in contrast, were assigned an important function as a privileged intermediate class, "as native craftsmen and manual laborers […], as policemen, i.e. minor officers, foremen, and leaders of the entire supply lines and vehicle pool of the government, troops and private persons, in part as small farmers in their bastard country, to which everyone returns after serving their time." despite his paternalistic attitude toward the "little nation of bastards," fischer regarded the rehoboths from the perspective of the colonial masters: so they will be granted just that degree of protection which they need as a race inferior to us, in order to endure, no more and only as long as they are useful to us -otherwise free competition, i.e. in my opinion, here downfall! this last comment by fischer reads like a retrospective justification of the war of extermination the german colonial troops had led against the rebellious herero and nama from to . fischer had profited from this genocide directly, for he apparently brought skulls and skeletons of "hottentots" with him from southwest africa, which may have come from the internment camps on shark island, where people died like flies. the skeleton of the nama leader cornelius frederiks ( † ) also supposedly came into fischer's collection in this way. as mentioned above, fischer continued his studies of the "bastards of rehoboth" until . yet the "bastard studies" by fischer and his pupils were no longer embedded in a colonial science and colonial policy context, but rather in the concept of anthropobiology: with its particular methodology, which combined anthropometry, genealogy, genetics, and ethnology, they were supposed to bring together anthropology and human genetics. however, "bastard research" had not lost its practical application, as the role of the institute in dahlem in the sterilization of the "rhineland bastards" quite impressively evinced. after world war i the colonial sciences became part of the "colonialism without colonies," which blossomed so lushly in germany between and . colonial research did not simply cease after the loss of the colonies. on the contrary, in view of a future german colonial empire, it was even intensified. until the german defeat in stalingrad, when colonial planning was officially discontinued, a perfect colonial empire had been designed on the drawing board. "one can only ask with astonishment," in the words of wolfe w. schmokel, "whether at any point in history a non-existent empire had ever been so well administered […] ." increasingly, colonial planning was based on a scientific foundation. tropical medicine, tropical technology, geography, regional development, demography, anthropology, social hygiene, and eugenics dealt intensively with colonial policy issues. at this time there is no indication that the kwi-a was included in colonial policy planning in the late s -be it by the race policy office of the nsdap, which was closely linked with the institute in dahlem through its director walter groß, and which had presented the main features of a future national socialist race policy in the colonies in with a tract entitled kolonialfrage und rassegedanke ("the colonial question and race theory"): the plan was for strict race segregation, a ban on mixed marriages, the restriction of contact between blacks and whites to a minimum, and so on. in an article for the periodical rassenpolitische auslandskorrespondenz (race policy foreign correspondence), eugen fischer legitimated such forms of apartheid with reference to "indisputable and provable facts, to the fact that mental attributes are based on genetic dispositions, that race differences are genetic differences, that mental attributes are different for each races, and that there are thus mental differences between races." fischer's admonition to investigate such "mental race differences" scientifically and to lay a scientific foundation for race policy fell on deaf ears, however. that the "law for the protection of colonial blood" drafted by the colonial policy office in equated the "half-breeds with an admixture of native blood" with the population of color as regarded the ban on mixed marriages was certainly in accordance with fischer's wishes, but the notion upon which this passage was based, that the "halfbreed" was under both "source races" in terms of his or her mental and psychic attributes, stood in blatant opposition to fischer's theory of heterosis. in the course of world war ii, however, as mentioned above, collaboration developed between the colonial policy office and gottschaldt's department for genetic psychology. in september gottschaldt took on the article about "psychological problems and methods in colonial science" for the afrika handbuch der angewandten (kolonialen) völkerkunde ("africa manual of applied (colonial) ethnology") contracted by the colonial policy office of hugo adolf bernatzik ( - ). the manuscripts were ready for printing in fall , but were destroyed as a result of a bombing, so that the handbuch der angewandten völkerkunde could not be published until -including the article by gottschaldt along with a "questionnaire for the psychological evaluation of native workers" he had developed. hecht, kolonialfrage. fischer, geistige rassenunterschiede, p. . in fischer felt compelled to protest vehemently in volk und rasse, the organ of the national committee for the national health service, against an article by the catholic theologian theodor gentrup (berlin), who had advocated "racially mixed marriage" in the colonies on the authority of fischer's work on the "bastards of rehoboth." fischer, frage "rassenmischehe." another, entirely unexpected possibility to reestablish himself in the field of colonial science emerged from one of fischer's other research interests: his search for the "cro-magnon race," whose traces he believed to have discovered back in upon his return journey from southwest africa, and then on a further research trip in in the population of the canary islands, and finally also in the contemporary european "phalian type." on a research journey to spanish morocco, planned for the / fiscal year, fischer apparently had intended to track down the cro-magnon type in northern africa as well, yet this research plan was delayed indefinitely because of fischer's heavy workload at the rectorate. with the formation of the german africa corps in january and the conquest of cyrenaica in march/april , when the plans for founding a german colonial empire in northern africa took on more concrete shape, cro-magnon research, little more than a hobbyhorse of fischer's for so many years, quite surprisingly took on political importance. the virtuosic research strategist eugen fischer immediately recognized the emerging possibilities. on may , he lectured to the prussian academy of sciences about "the problems of white africa." proceeding from the term "white africa," coined by dominik josef wölfel ( - ), fischer claimed that the part of africa located north of the sahara, in terms of climate, geology, zoology, and botany, but above all "according to human races and cultures, clearly and fundamentally departs and stands out from the remainder of africa, from the africa of the negroes, from black africa." fischer presumed that the entire mediterranean region was settled by a "mediterranean race," in which shares of other races had been incorporated in the historical era -arab, nordic, alpine, negroid. fischer saw one of the roots of the "mediterranean race" in the prehistoric cro-magnon race, which was characterized by "blondness and blue eyes." the line of attack is clear: through the anthropological-ethnological differentiation between black and white africa, fischer supplied the scientific basis to legitimate pushing forward the borders of the emerging greater european empire under the hegemony of national socialist germany to the northern edge of the sahara, without any race policy scruples. it can come as no surprise that fischer, in return, demanded funds to accelerate the advancement of the scientific exploration of northern africa. following his lecture, fischer -along with the africanist dietrich westermann ( - ) and the egyptologist hermann grapow ( - ) -thus proposed to the prussian academy of sciences the establishment of an interdisciplinary research commission on white africa. in their proposal the three scholars urged for haste, for "after the war the development of the sahara areas with automotive and aeronautic routes, and through the construction that has just commenced of a […] trans-saharan railway, will certainly restart in full strength, and thus an increasing cf. also ritter, cro-magnon-merkmale. quoted in lösch, rasse, p. . reworked version of the lecture: fischer, weißafrika. ibid., p. . destruction of the remaining witnesses of the white african past set in." the academy approved the proposal immediately. in / fischer held "soliciting lectures" on the topics surrounding white africa. the commission instigated by fischer had no opportunity to develop any activities of note -germany's colonial dreams were over too soon. when the commission's three subject groups convened for the first time at the invitation of the colonial science department of the reich research council and the german research association on january , -a few days before the defeat in stalingrad -for a -day conference about "colonial ethnology, colonial linguistic research and colonial race research" in leipzig, all of the colonial science plans were already scrap. the speakers in the "colonial race research" section -besides otto reche, director of the institute for race science and ethnology at the university of leipzig, and egon von eickstedt ( - ), director of the institute for anthropology at the university of breslau -were eugen fischer and wolfgang abel. based on a reworked version of his lecture for the academy, fischer outlined the anthropological concept of white africa once again. abel dealt with "race problems in sudan and its borderlands." this harmless title concealed extremely explosive subject matter. abel presented numerous photographs of anthropological types from the sahel zone, most of which depicted french prisoners of war. as abel mentioned in passing, he had been detailed to the "inspection of the personnel controlling of the army (army psychology)" (inspektion des personalprüfwesens des heeres (heerespsychologie), to perform series of anthropological examinations of french colonial soldiers in a number of war prison camps. "hereby the residents of different areas or different tribes of sudan were put together in large groups and the number of the persons best rendering the type were always photographed." thus, "good illustrative material" was created, comprising the photographs of around persons. according to statements made in the s, in the context of this activity abel was also at a "leper station in bordeaux" -what was probably meant was the special military hospital for colonial medicine in st. médard near bordeaux -in order to examine the changes in the pattern of fingerprints caused by the disease. the footprints of "guinea negroes" from the special military hospital for colonial medicine in georg geipel's estate were quite probably taken by abel. abel was not alone: otto baader, too, combed through the war prison camps in france in his search for cro-magnon types. both scientists presented their findings to the berlin anthropological society. those involved apparently had no grasp of the fact that such examinations in prison war camps signified a subtle, but nevertheless fundamental boundary crossing -for the first time, scientists of the kwi-a researched on people who were capable of giving consent, but whose possibilities for refusing the examination were at least restricted because they were imprisoned. even though the examinations as such were harmless, and the probands had to suffer neither pain and fear nor abasement and were not subjected to any health risks, abandoning the principle of informed consent signified a deep rupture. the war prison camps of the french campaign amounted to a sort of laboratory for race anthropology research. the special conditions of such research resulted in a process of radicalization, which is to be illustrated with a further example: robert stigler ( - ), director of the institute for the anatomy and physiology of domestic mammals at the university of vienna, and his five assistants, performed a series of race anatomy and race physiology tests in a war prison camp near vienna in july . in the camp, besides around , white french, alsatians, flemish and walloons there were also around , moroccans, tunesians and algerians, tonkinese, annamese, negroes, among them from west africa, from tropical america, and european jews, among them several diamond merchants from antwerp. as in the examinations by abel and baader, here to the question as to the consent of the probands was not posed at all. even so: our examinations met with no resistance at all from the prisoners. the colored were intially very shy, but soon began to trust us and many cheerful scenes ensued. i had the negroes perform their dances and sing their songs for us. the moroccans, tunisians and algerians were much more negative, the little yellow tonkinese were the shyest of all […] . recorded in the examinations was the clotting time of the blood, the sinking speed of the blood, the viscosity of the blood, blood pressure, pulse rate, respiration, the upper hearing limit, the threshold of the sense of touch, reaction speed, right-handedness and left-handedness, hair growth on the genitals, and sexual characteristics -the last of these substantiated by numerous photographs. in comparison to the examinations by abel and baader, further boundary trangressions can be determined: not only would the measurements of the naked body and the photographing of the genitals have been perceived by the probands as humiliating and a violation of modesty. in taking blood samples the scientists had gone a step further -this was a first, albeit minimal, invasive approach. the examinations of the group of researchers around stigler were thus positioned between those of abel and baader and the examinations and experiments of karl horneck, which will be depicted at a later juncture. even after the start of world war ii, eugen fischer and otmar von verschuer brought their national and international reputations to bear in order to provide a scientific foundation to legitimate the "total solution to the jewish question" tackled by the national socialists, which by late early had taken on the character of the "final solution" once and for all. for fischer and verschuer there could hardly have been a doubt as to what the measures aimed to achieve. they were guests of honor to a working congress at the inauguration of the "frankfurt institute for the investigation of the jewish question" (frankfurter institut zur erforschung der judenfrage) on march / , . the aspired goal of the "total solution" to the "jewish question," as was bluntly stated here, was the volkstod ("death of the nation"). the economist peter-heinz seraphim pointed out for consideration that the deportation for forced labor in camps in poland or an overseas colony could also have the consequence of "social pauperization and upheaval," but "by no means the physical self-disintegration of jewry, for the death of a nation is never a fast death." the logical conclusion from these comments was, as benno müller-hill emphasizes correctly, that the "physical self-disintegration" would require some assistance. when the deportation of the german jews began in october , nobody who had participated in the congress in march could have been in doubt as to what was in store for the jews deported to the east. this did not prevent fischer from making an appearance in late /early as part of a lecture series organized by the german institute in paris. in his lecture about "race and german legislation," fischer certified that the "bolshevist jews" were of "monstrous mentality" and assigned them to a "different species." fischer himself emphasized in a report about his trip to paris that he had found much acknowledgement among the attendant french scientists for his discussion held of the "negro problem" and the "jewish problem" in a "very candid, but in purely scientific form" -and this right before the deportation of , jews from france was discussed at the wannsee conference. as mentioned previously, in fischer and the theologian gerhard kittel published a book about the "world jewry of antiquity," essentially a selection compiled by kittel of ancient sources with a decidedly anti-semitic perspective. kittel supplemented the written sources with illustrations of egyptian mummy tablets, which supposedly constituted further evidence for the worldwide propagation of jewry. at kittel's request, fischer undertook to determine the "race type" of the persons illustrated. this was not the first time fischer had done something like this (for instance, he had studied the illustrations on etruscan tombs and the masks found during excavations in mycenae), yet in this publication it was practically tangible that fischer's interpretations of the pictures completely abandoned the basis of precise anthropometry and relied only on intuition -and that his intuition was distorted by anti-semitism: granted, the expert sees for all races, and also for the basic races of the jews, a number of physiognomic details which we cannot name and fit into the usual model: shape of nose, shape of face, shape of skull, etc. often a jew is recognized as a jew with complete certainty even though he does not have […] a so-called "jewish nose." there is something […] in the jewish physiognomy that cannot be measured, and can hardly be described in detail such that the reader or listener can visualize it clearly. but no one will doubt that very many jews can be picked out from groups of non-jews with complete certainty. […] it is not permissible to disqualify as unscientific the statement of a general "impression" of "jewish" in the evaluation of the pictures. the attempt by niels c. lösch to play down fischer's participation in this anti-semitic pamphlet as an expression of senility deserves vehement contradiction -fischer, years old at the time, was of remarkably fresh intellect, and his scientific publishing activities extended well into the s. it must also be kept in mind that fischer's studies on "jewish physiognomy" were by no means the concern of an individual scholar in retirement, but rather were based on preliminary work performed at the kwi-a at the beginning of world war ii. in late /early -probably in the first months of -one assistant and three students made several trips to Łódž ("litzmannstadt") on fischer's behalf, where the group -in a cauldron of executions, pogroms, and synagogue cf. also heiber, walter frank, p. . upon fischer's request, verschuer had copies of the "jew pictures of egypt" slides made for the collection of the kwi-a. cf fischer explained the costs, stating that "all four gentlemen occasionally traveled and worked independently, so that i was not able to give the entire sum of the expenses to the assistant, so that he could pay out the individual amounts, but occasionally had to pay all gentlemen individually. […] and in so doing the account between their own funds and those of the institute occasionally were somewhat mixed up." fischer to generalverwaltung, / / , desecrations, while at the same time ten thousands of jews were deported from the city and carried off to concentration camps -performed series of anthropological examinations on more than jews. among the students were also harry suchalla and christian schnecke, who were still working as doctoral students of nachtsheim's at the institute for genetic and breeding research and were "loaned out" by fischer, presumably for want of manpower of his own. this circumstance indicates that the opportunity was favorable and time was pressing. it can be presumed that herbert grohmann, a graduate of the first annual ss course at the kwi-a and an assistant to fischer in the years / , made the "field research" in Łódž possible, having held the position of senior medical councilor at the newly founded health office of "litzmannstadt" since september . in not only photographs taken by suchalla and his comrades on their trips to Łódž were included in fischer's and kittel's book about "ancient world jewry." after the war the fingerprints and handprints from the Łódž ghetto were recovered in hans nachtsheim's institute for comparative genetic biology and genetic pathology of the german research academy in dahlem, where they were discovered by georg geipel, who, as elaborated elsewhere, had worked at the kwi-a as an expert for dermatoglyphics (and who had introduced suchalla to the technique of dactyloscopy in ). as late as the end of , the publication of this material was discussed in all earnestness in the context of dermatoglyphic race research, but was stopped, presumably upon fischer's advice. the fingerprints and handprints themselves are untraceable today. however, the anonymized fingerprint formulas of "litzmannstadt jews" are included in geipel's scientific estate. the incident shows that fischer was willing to use the "total solution of the jewish question" at short notice in order to obtain research material, and in return ibid., pp. a- a v, quote: p. a. -the trips must have taken place between september , , the day on which Łódž was occupied by the wehrmacht, and the close of accounts on march , . in retrospect, harry suchalla dated his stay in Łódž to the year . the incident shows that a close connection between fischer and nachtsheim must have existed before october . to place the results of his research, as dubious as they might have been, unquestioningly at the service of "jewish policy." that fischer regarded his anthropological studies as a contribution to the "total solution of the jewish question" was demonstrated quite clearly in june , when alfred rosenberg ( - ), minister for the occupied eastern territories, invited fischer to act as one of the presidents of an international "anti-jewish congress" to be convened in kraków. fischer accepted the invitation, explaining: i hold […] your intention to found a scientific front to defend against the influence of jewry on european culture and to call together the scientists of all of the nations in conflict with jewry to be very good and altogether necessary. yes, it is high time for such an action, for jewry has been battling us for decades not only politically, but quite certainly in terms of pure intellectural history as well. the congress never took place. nevertheless the incident shows how loyally fischer supported the "final solution" even at a point in time when the collapse of the national socialist state was already clearly imminent. this was also true for his successor, friend and pupil otmar von verschuer. in late /early -the deportation of german jews had begun a few months previously -he wrote in the erbarzt: never before in history has the political importance of the jewish question emerged so clearly as today: the whole of europe in alliance with japan-led east asia is battling against the english-american-russian world power jointly led by jewry. the nations unified with us recognize more and more that the jewish question is a question of race, and that they therefore must find a solution like the one we initially introduced for germany. this was open approval for the deportation of jews from the third reich, and pled for its expansion to german-dominated europe. in evaluating this statement it must be kept in mind that the mass murder of mentally ill and mentally disabled people in the gas chambers of the "aktion t " in / was known to large sectors of the german population, and that information had leaked quickly about the massacres committed by the task groups of the security police and the sd in the occupied territories of poland and the soviet union. when the systematic deportation of german jews began in october , a significant degree of self-deception was needed to accept the official version of "resettlement" and "work assignment in the east." as late as verschuer, as mentioned above, demanded a "new total solution of the jewish problem," now that the "historical attempts at solution" -"absorption of the jews," "seclusion of the jews through the ghetto" and "the emancipation of jewry" -had failed. as to what the "total solution" looked like in the ghettos and extermination camps, there was hardly a scientist in the german reich who had such profound information as otmar von verschuer. yet, with his research on the development of a serological race test all the way into the final months of the war he made his contribution to this "total solution." fischer to rosenberg, / / , quoted in müller-hill, tödliche wissenschaft, p. . at fischer's suggestion lothar loeffler also took part in the organization of the congress. verschuer, erbarzt an der jahreswende, p. . verschuer, leitfaden, nd edn., pp. f., . in addition, under verschuer's directorship the kwi-a continued, albeit to a diminished degree, to contribute practical legwork on "jewish policy" in the form of certificates of race and descent. in / , for instance, verschuer and his staff members schade, grebe, mengele, fromme, baader, and liebau produced evaluations bringing in a total of , rm. "as a special war service" the institute also provided "certificates of wehrmacht members (racial descent, marriage permits)." that verschuer used his activities as an evaluator to help those suffering racial persecution methodically and systematically, as a war legend claimed, must be challenged on the basis of today's state of knowledge. it is indisputable that he delivered a judgement advantageous for the individual involved in individual cases. it is also obvious that the test subjects enjoyed his sympathy in these cases. yet for the assertion that one of his closest friends, the frankfurt pastor otto fricke, made in his denazification testimony of october , that verschuer had gone "to the limit of scientific credibility […] in order to prevent people from fall victim to the methods of the national socialist state," there is no believable source evidence. important in this context is a letter by verschuer to karl diehl of february , . the subject was the case of the "half-jewish" physician werner wund , to whom approbation was denied in national socialist germany and who had found employment in may as an intern in the remote eckardtsheim branch institute, one of the von bodelschwingh bethel institutes. his situation had become precarious in september , when the reich ministry of the interior had rescinded the employment permit it had initially granted for wund. thereupon bethel endeavored to procure a certificate of exemption from the reich chamber of physicians. in this context a file on the "wund case" must have made its way via the channels of the deaconry to the practicing catholic karl diehl. diehl had submitted the case to his friend verschuer, requesting his assistance. however, in a letter of february , verschuer expressed his regret that he could not undertake anything, "as the question of the racial descent is undisputed." "for such applications a race biology certification plays no role. for it is of no consequence whether or not the individual involved looks jewish." verschuer recommended a "clemency plea to the reich chancellory," whereby he was skeptical about the success of such a petition from the outset. interesting in this context is a passage of his letter in which verschuer went into his role as an assessor: only in those cases in which doubts exist as to the correctness of the blood descent am i consulted as an expert, and in many such cases i have been able to help the people involved decisively. just recently, for instance, a physician from stuttgart came to me, whose wife was hitherto supposed to be a full-blooded jew. from her appearance alone doubts as to this descent were justified. the couple also had four children, who now were supposed to be taken out of school and who would be banned from all higher professions as " st degree mixed-race." in this case i could supply evidence that the woman was not descended from her jewish father, but had a german physician, since deceased, as her biological father. this just as an example of the cases in which my involvement can be successful. here verschuer was probably alluding to the case of the professor's wife luise s., in which he had been consulted as an assessor. verschuer's expert opinion had in fact been successful in declaring mrs. s., who had been considered a "full jew" until that time, to be a "half-jew," by abnegating the biological paternity of her legal father. by no means did this close the case, however, for the husband of mrs. s. fought for the recognition of his wife as "german-blooded" by questioning the biological maternity of her legal mother as well. the race policy office, to which he addressed his petition, called in the race biologist wolfgang lehman from strasbourg, who, as already mentioned, was a member of the "dahlem circle." lehman was to examine photographs to ascertain whether they yielded "indications for a jewish descent" of mrs. s. since he gathered from the files that verschuer had already submitted an expert opinion, lehmann turned to verschuer first before delivering an opinion himself. the characteristic style of lehmann's letter to his former colleague makes apparent that he was disposed to agree with the standpoint of professor s., and that he proceeded from the assumption that verschuer would agree as well. the response was different than expected, however. while verschuer allowed that mrs. s. belonged to the cases "in which nobody would suspect a jewish influence. as such one can concede to her husband that she appears to be a 'pure german woman' […] ." but in his expert opinion at the time he had not been able to "lend support for the assumption that she was not the child of her mother. she received notification from the reich heritage office that she was […] degree mixed race, and i believe," verschuer added with slight irritation, "professor s. professor for race biology and the race policy office of the nsdap -to relieve mrs. s. of the stigma of being a "half-jew," and her children "quarter-jews" -had he only been willing, despite his scientific conviction, to depart from the result of his first expert opinion in the interest of this human being. this case confirms the judgement hans-peter kröner made about verschuer as an evaluator on the basis of a case of "race treason" from : verschuer was neither one of those scientists who provided incorrect opinions knowingly and deliberately in order to save people, nor one of those who interpreted the race laws extensively to the disadvantage of their subjects. verschuer was the type of the "correct, law-abiding but merciless evaluators." of course, verschuer abetted the emergence of his legend by portraying to his friend diehl his first expert opinion as emergency assistance for a subject suffering racial persecution, although he did not claim at this juncture to have falsified the findings of the paternity examination. through this it was possible for the impression to emerge in verschuer's circle of friends -and also among the affected -that he used his position as an evaluator to help the persecuted. however, all cases documented by sources prove that verschuer followed the exact letter of the law and that "scientificity" was the only criterion for his expert opinions. with his appointment to "reich commissioner for the fortification of german volkstum" himmler was entrusted with the "ethnic cleansing" of the occupied eastern territories. consequently he set a mighty population transfer in motion, whereby the settlement of german nationals in the conquered areas and the deportation of poles and jews from these spaces drove each other like cogs in a powerful machine. yet the forced migration, which was effected starting in winter / , constituted only a fraction of the planned resettlement program, which was worked out by himmler's accomplices between and and entitled the generalplan ost ("general plan for the east"). the original generalplan ost, which was reworked several times, has not survived. but through two written position papers from the pen of the head of the race division in alfred rosenberg's reich ministry for the occupied eastern territories, erhard wetzel , we know its contents down to the details. within years at least million germans were to be resettled in the east. the territories slated for settlement were the occupied areas of poland, the baltic countries, belarus, parts of russia, ukraine and crimea. the population in these areas was estimated at about million, including - million jews, whose extermination wetzel presupposed as a matter of course in his position paper of april , . in total, of the million people who lived in the territory destined for german settlement were categorized as "racially undesirable." they were to starve or be expelled to siberia. according to generalplan ost, - % of the population of poland, % of the population of western ukraine and % of the population of belarus was to disappear. the remainder was to be "germanized" or to serve the german "master race" as "helot folk." as such, generalplan ost was the blueprint of a gigantic program of extermination, expulsion and enslavement. three scientists from the kwi-a were involved directly or indirectly in elaborating the plan. eugen fischer took part in a meeting in the ministry of the east protocolled by wetzel on february , "about the questions of germanization, especially in the baltic countries," in which a draft of generalplan ost, presumably worked out by group iii b of the rusha in late was discussed -by the way, side by side with his old nemesis bruno k. schultz, by now head of the race office in the rusha. according to the protocol, in this meeting fischer gave one of the introductory position papers and spoke once during the discussion, when wetzel asked the group to consider "whether through the industrialization of the baltic region it might not be possible to scrap the racially undesirable sectors of the population," rather than forcibly deporting them to siberia. with the formulation "scrapping through industrialization" wetzel did not mean "extermination through labor." by way of explanation he added, namely: "if they [the 'racially undesirable' sectors of the population] were given suitable pay, in particular, if their cultural condition were to be raised, a drop in the birthrate would be expected." wetzel thus set his hopes in the regularly observed drop in birthrates in the industrialized states as a means of making the sectors of the baltic population that were not to be "germanized" vanish in subsequent generations. in opposition fischer expressed misgivings: the "better standard of living" could, contrary to wetzel's expectation, "easily lead to a rise in birth rates." wetzel admitted that fischer's view was "correct to the extent that […] those concerned are unmistakably anti-social." in short the brief exchange between fischer and wetzel amounted to the scholar coming out against the representatives of the ministry of the east and with the representatives of the ss for large-scale deportations from the baltics to siberia, and thus giving preference to a more radical variant of "ethnic cleansing." in spite of this, fischer's consulting activity continued to enjoy high estimation in the ministry for the east. he played a key role in rosenberg's plans for the founding of a "reich headquarters for research on the east." in a file note for hitler of march , rosenberg informed the führer that he had "thought of" fischer to fill the position at the reich headquarters, "as a representative personality for biological research and a leading member of the kaiser wilhelm society." quoted in heiber, generalplan ost, p. . quoted in müller-hill, tödliche wissenschaft, p. . in a detailed position paper on generalplan ost of april , , wetzel took reference to fritz lenz and eugen fischer in the section about "german settlement issues." this section concerned the question as to whether southern ukraine and crimea would come into question for german settlement because of the climate there. lenz, as wetzel reported, had "taken the standpoint that the climatic conditions in these regions were detrimental for the settlement of the nordic-phalian appointed race." in this wetzel must have referred to lenz's exposé submitted to the rusha in january , bemerkungen zur umsiedlung unter dem gesichtspunkt der rassenpflege ("remarks on resettlement under the aspect of the care for the race"). lenz continued to concern himself intensively with the issues involved in "east settlement," and advised the ss physician hellmuth thieme ( * ), who had been involved with the processing of marriage applications at the rusha since , on his dissertation on the topic of "the selection of new peasants and their importance for a race hygenic population policy." in december , eugen fischer, too, expressed his opinion on the question of german settlements in southern ukraine and on the crimean peninsula. wetzel cited him with the words "that a settlement of german people in these regions could only be considered if there was a conscious effort to create rich wooded regions all over and thus bring about a change in climate." in his exposé of april , wetzel finally cited a third scholar from the institute in dahlem: wolfgang abel. after being called up for military service, abel had first seen action in the luftwaffe, but after he was wounded he was transferred to the department for army personnel controlling as a consulting anthropologist. as mentioned above, in this capacity he had examined colonial soldiers held in war prison camps in occupied france in . in winter / , accompanied by two army psychologists, he then visited various war prison camps in which soldiers of the red army were crowded together in close quarters. on behalf of the superior command of the wehrmacht, he subjected the russian prisoners of war to crude anthropological evaluation. his findings, which he presented to a larger public in a lecture at the "east conference of german science" (osttagung der deutschen wissenschaft) flowed directly into wetzel's exposé. according to wetzel, abel had reached the conclusion: [t]hat in the russians much stronger nordic race elements are present than had been presumed up to this time. in addition to these truly nordic race elements, the great mass of which probably have been located in this region for some time now, especially in the northwestern areas of russia, and which cannot be traced back solely to germanic, especially varangian immigrants, there are a predominant number of light-skinned, primitive caucasian, more or less long-headed race types, who by no means fall under the races of günther's system, and cannot be explained as caucasian-mongoloid hybrid forms either, but rather constitute the undoubtedly ancient caucasian race forms that have yet to be described in detail. also present among the russians, primarily in the western regions, are eastern baltic influences. however, these eastern baltic race characteristics are by no means as strong as was previously presumed. at the congress, wetzel continued to relate, abel had proposed two different "solutions" to this delicate race question, which showed how abel's advantageous judgement about the racial composition of the russian nation ultimately cut both ways: the "very serious comments by abel," according to wetzel, deserve "the very greatest attention." the "path of liquidating russian volkstum" suggested by abel, however, aside from the fact that its "execution [was] hardly possible," was "out of the question for political and economic reasons." however, the strategy that wetzel himself developed in the following -fragmentation of the russian population, "racial lixiviation of russian volkstum," the "singling out the nordic clans present in the russian nation and gradual germanization," sinking of the russian birthrates -was largely oriented to abel's biologistic perspective. what is more: because abel in all seriousness posited the idea of physically exterminating many millions of people, he set a negative precedent against which all other proposed solutions, no matter how radical they were, seemed moderate. abel continued his anthropological examinations of russian prisoners of war, intensifying his connection to the ss ahnenerbe ("ancestral heritage society") at the beginning of -presumably not least with a view to his own uncertain future prospects, as the call to fischer's vacated professorial chair met with hans f. k. günther differentiated between the nordic, phalian, mediterranean, dinaric, alpine and eastern baltic races. wetzel, stellungnahme und gedanken zum generalplan ost des reichsführers ss, / / , reprinted in: heiber, generalplan ost, pp. - , quotes: p. . ibid., p. . unexpected resistance. abel sought cover with the ornithologist and ss sturmbahnführer (major) ernst schäfer . after three expeditions to tibet -he had just returned from the last in august , schäfer had taken over the "department for the central asian research and expeditions" of the ahnenerbe society in , which developed to become a "reich institute" of its own under his direction. the "sven hedin institute for central asia and expeditions," soon the largest department of the ahnenerbe, with its own domicile in the medieval castle of mittersill in pinzgau, had been opened on january , on the occasion of the th anniversary of the university of munich and the awarding of the honorary doctorate to sven hedin ( - ) . one week later, on february , , abel, who had performed anthropological examinations of around , soviet prisoners of war by this time, turned to schäfer with a request for support -a clever move, as schäfer had since encroached on the entire area of the natural sciences within the ahnenerbe organization. abel's concrete request was that the anthropologist and ss hauptsturmführer (captain) bruno beger ( * ) be assigned to him. beger had originally belonged to the rusha, then was transferred to himmler's personal staff, took part in schäfer's expedition to tibet in / as an anthropology student, entered schäfer's department for central asian research and expeditions in the ahnenerbe in , and took his doctorate in anthropology with ludwig ferdinand clauss. on march , schäfer forwarded abel's remarks to himmler's personal assistant rudolf brandt , with the request that he report them to the reichsführer ss. after intial skepticism, himmler's staff received abel's research plans quite positively. not wanting to make a decision without consulting the directors of ahnenerbe, however, the managing director of this organization, wolfram sievers ( sievers ( - was called in. sievers first consulted with a number of staff members at the "institute for military science application research" (institut für wehrwissenschaftliche zweckforschung) under his direction, which, founded in , functioned like a "state within the state of the 'ahnenerbe'," which also approved abel's research plans. on may , he wrote to brandt that he held lösch, rasse, pp. f. even though abel had been drafted into military service, he was still a departmental director at the kwi-a and maintained constant contact with the institute. as such, his examinations of soviet prisoners of war and the demands and recommendations derived from these studies cannot be factored out of the kwi-a's responsibility, especially since abel's anthropological studies in war prison camps were a direct continuation of his prewar work in the context of the institute. for a biography: kater, "ahnenerbe," pp. f., - available until the analysis has been completed. if the reichsführer-ss approves the application, however, then we must come to a precise agreement with prof. abel as to how long the designated anthropologists will have to be available for the evaluation. what was the "caucaus project" mentioned here? on august , , days after the wehrmacht had captured the oil fields of the caucasus, heinrich himmler ordered the "ahnenerbe" to prepare a scientific expedition led by schäfer in order to explore the caucasus under the aspects of botany, zoology, entomology, geophysics, and also anthropology. the planning for this unternehmen k as michael h. kater establishes, "exceeded in scale everything that came before it." with the defeat of stalingrad the plan for an ss expedition to the caucasus may have lost any basis in reality, but unternehmen k was not abandoned for good until january . thus it was from the pool of scientists involved in this planned caucasus expedition that three anthropologists were detached temporarily for abel's project of an anthropological study of russian prisoners of war. in a further letter to brandt of may , sievers stated more precisely that the evaluation of the material from the study was "extraordinarily important, because labor is to be assigned, and also for demographic, economic and cultural reasons. […] however, prof. dr. abel should be disposed to concentrate his work above all on the question of the individual groups' treatment and utilizability for labor in the war and to orient his work toward the solution of these questions." as such, abel's examinations were embedded in a new context. after the defeat at stalingrad, the labor administration under the direction of the "general deputy for the employment of labor," fritz sauckel ( - ), made every effort to effect the deployment of foreign forced laborers under the banner of "european workers against bolshevism." anthropological expertise was welcome in the attempt to differentiate from the giant army of "eastern workers" individual "racially more valuable" groups, who were supposed to be spurred on to higher performance by offering them better living and working conditions, or so the apparent calculation of the ahnenerbe. by way of precaution, sievers had rübel, endres, and fleischhacker exempted from the staff of unternehmen k on the very same day. sievers further proposed in his letter to brandt of may , that the three anthropologists to be detached to abel could take care of an additional mission on this opportunity: once access to the auschwitz camp is possible again, these anthropologists could also perform the examination there for that collection of persons of which you are familiar. since at this time, as ss obersturmbannführer ( st lieutenant) [adolf] eichmann informed me, there is especially suitable material available, the time would be particularly opportune for this examination. in this abel's project was linked with another one that had been pursued for quite some time by the ahnenerbe: the erection of a "jewish skeleton collection." the first impetus for this project proceeded from bruno beger in december . on the search for a scientist who was to take control over the setting up of the collection, the organization quickly hit on august hirt ( - ), who held the chair for anatomy at the newly founded "reich university" in strasbourg. from late /early he was courted by sievers, brandt, and himmler, so that he took over a primary role in the framework of the natural science research empire that was to emerge under the protectorate of the ss. on december , brandt passed on to sievers a generally formulated directive of himmler's that hirt should be "given the possibility to engage in experiments of every kind that could support his research work, using prisoners, professional criminals who will never be released anyway, and persons awaiting execution." initially the jewish skeleton collection was an issue. rather, the hope was to win over hirt for the directorship of a planned institute for entomology. the anatomist had made a name for himself with his work in the fields of the sympathetic nervous system and intravital microscopy, and especially this latter area was to be used in the research of new possibilities for pest control. in the end, hirt's criminal experiments with poison gas (mustard gas) on prisoners at the natzweiler concentration camp emerged from these plans. yet back in january there was also talk of "anthropological studies" hirt was supposed to perform. probably the jewish skull collection was meant with this. in any case, a report by hirt about his research fields, which sievers forwarded to the reichsführer ss on february , , was appended by an exposé in which the plan for establishing the jewish skull collection was explained in greater detail: comprehensive skull collections exist for nearly all races and nations. only of the jews are there so few skulls available to science that their processing does not permit any certain results. the war in the east now offers us the opportunity to remedy this lack. in the jewish-bolshevist commissars, who embody a disgusting, but characteristic class of sub-humans, we have the possibility to acquire a concrete scientific document by securing their skulls. the plan was frustrated by the reality of the war. hirt, sievers, and beger thus agreed to procure the material not from the front, but from a concentration campand then not just skulls, but entire skeletons. on november , , in a secret letter to brandt, sievers wrote, "for certain anthropological examinations […] skeletons of prisoners (jews) [were] required, which are supposed to be provided from the auschwitz concentration camp." the head office for reich security was instructed to issue a corresponding directive. brandt forwarded this request to the ss obersturmbannführer adolf eichmann , head of the department for jews (judenreferat) iv b in the head office for reich security. as the letter from sievers to brandt of may , indicates, eichmann had just sent word that "at present especially suitable material" was available in auschwitz for the jewish skeleton collection. on june , bruno beger arrived in auschwitz, surveying technician willi gabel having been sent ahead. on june fleischhacker followed, temporarily detached from the rusha to the ahnenerbe. by june beger had selected and, assisted by fleischhacker, gabel, and several prisoners, measured the victims. in total beger had selected, as sievers wrote eichmann on june , " persons, of which were jewish men, polish men, central asian men, and jewish women." the unfortunate were deported to the natzweiler concentration camp in august and murdered there in a specially furnished gas chamber under hirt's direction, and some parts of their bodies conserved, others preserved. so how was abel's project of anthropological examinations of soviet prisoners interlocked with this complex of crimes? and how did it continue? at present these questions can be answered only in part due to the fragmentary sources available. what is clear is that abel, armed with a research contract from the reich research council, continued working on his "race biological studies of eastern nations." in september , with sievers' help, he managed to extend his "indispensable" quoted in mitscherlich/mielke (eds.), medizin, p. . quoted in kater, "ahnenerbe," p. . quoted in mitscherlich/mielke (eds.), medizin, p. . beger's own interest focused on the four "central asian" prisoners. "two usbeks, usbekian-tadjik mixed-race and chuvash from the kasan region [were] measured and cast," beger reported to his superior schäfer on june , . "in addition, just for our institute" (quoted in kater, "ahnenerbe," p. ). status. this was justified with the fact that it was absolutely necessary that the examinations of war prisoners be concluded, "since it is imperative that the racebiological selection and evaluation of the great russians be clarified for later deployment, for up to this point we knew almost nothing about them and were misguided by incorrect conceptions." sievers' intercession can be interpreted as an indication that abel's research on russian prisoners of war continued to be performed in cooperation with the ahnenerbe. this fits in with the fact that in october , beger suggested continuing the anthropological studies begun in auschwitz on the "mongoloid" types among the soviet prisoners of war "by taking advantage of the material handed to us by this war in the form of prisoners." with the help of schäfer and sievers, in spring beger succeeded in deploying the wounded anthropologist rudolf trojan ( * ) to various camps in order to measure "central asian" prisoners of war. another question is whether and to what extent the ss was involved directly in abel's anthropological examinations. in response to his letter of may , , in which he suggested providing abel with three assistants, the anthropologists rübel, endres, and fleischhacker, sievers received an answer on june , . brandt had presented the plan to himmler and now imparted the decision of the reichführer ss: one of the anthropologists can be detached for the short term, for , or weeks, while instead of the other two suitable inmates of the sachsenhausen concentration camp should assist. for this it would be necessary that prof. dr. abel and one of the three ss führer move out to sachsenhausen for this period to take care of their work there […]. whether fleischhacker -for only he came in question under the circumstances, as endres and rübel were no longer available -was actually dispatched to abel's project after his assignment in auschwitz, and whether abel actually set up a base in the sachsenhausen concentration camp, must remain an open question at the current state of knowledge. the assumption that abel's examinations of soviet prisoners of war took place in the sachsenhausen camp is highly plausible. for one, sachsenhausen was very conveniently located, not only near berlin, but more importantly, not far from the kwi-a's external department for tuberculosis research in sommerfeld/beetz. secondly, a large number of soviet prisoners of war were held in sachsenhausen, such that this would open up a further field of activity for abel's ambitions. since abel had pursued the idea "of an instructive collection for the race history of europe and the world, the development of race, population movements domestic and international, etc." should this idea have taken on shape over the course of the war, a portion of the material could have come from sachsenhausen. from the testimony of witnesses we know that skulls, skeletons, and other body parts were sent from the sachsenhausen concentration camp to universities and other anatomical institutes. but specimens could also have come from auschwitz -at least the former prison physician miklos nyiszli ( nyiszli ( - ) mentions in his memoirs that jewish skeletons were sent from auschwitz to berlin for a "race exhibition." in the first years of the third reich, "gypsy policy" for the most part remained in the trails blazed for it in the weimar republic. the outlines of a new "gypsy policy" began to emerge as individual sinti and roma were subjected to compulsory sterilization in accordance with the gzven. from fall on they also fell under the "blood protection" law, which enacted bans on marriage between "germans" and "members of alien races" -besides jews, as the commentators of the nuremberg race laws emphasized expressly, this meant above all the sinti and roma -and also under the "marriage health law," which prohibited marriage for the "inferior," regardless of their ethnic heritage. this complex of laws signaled a shift in "gypsy policy." had the "gypsy question" been conceived as a problem of regulatory policy up to that time, now it was reinterpreted, like the "jewish question," as a "race problem." as such the sinti and roma found themselves doubly suppressed: like the jews they were stigmatized as an "alien race" in terms of race anthropology; as mentally ill and mentally disabled they were also considered to be "genetically inferior aliens to the community" in terms of race hygiene. the supposed "anti-sociality" of the sinti and roma was interpreted to be the consequence of a genetic defect, which, in term, was traced back to the interbreeding of the "genuine gypsy lineage" with the "german-blooded" lower classes. the racist conception of national socialist "gypsy policy" necessitated the collaboration of scientific functionary elites. the scientific center to "combat the gypsy nuisance" was the "race hygiene and population biology research office," which was founded in spring at the reich health office in berlin-dahlem. it was headed by robert ritter ( - ) , who was chief physician in the youth department of the tübingen university psychiatric clinic before turning to the research of "vagabond stock" and "gypsy half-breeds" full time in / . from spring on, the research office dispatched "mobile working groups," which sought out sinti and roma at gathering places, in camps, prisons, and institutions, subjected them to anthropometric examination and interrogated them -even under the application of threats and violence -to ascertain their family backgrounds. this information was supplemented by genealogical material from church and civic registries, private and state archives, as well as communications from the police, the courts, community authorities, welfare institutions, prisons, and penitentiaries. the information was compiled into family tables at the "gypsy clan archive" of the research office, which, in turn, served as the data source for the expert opinions produced by the research office. by march the research office produced almost , such expert opinions, in which the subjects were classified according to a sophisticated system as "gypsies" or "gypsy half-breeds" of various degrees. the staff of the research office was aware of the deportation of the german sinti and roma to the auschwitz concentration and extermination camp in march . in spite of this they continued to write their certified expert opinions, which constituted a decisive foundation for internment in auschwitz. the research office also took on consulting duties. it advised the offices of the criminal police on the application of the "gypsy legislation," the wehrmacht and the reich labor service on physical inspections, the groupings of the nsdap on the admission and expulsion of members, the school boards on school admissions, factory managers on hiring and labor offices on the provision of labor, rural authorities on the issuing of peddling licenses, mayors, national socialist welfare offices in welfare questions, and tax offices regarding the granting of child subsidies. above all, however, ritter and his staff instructed medical officers and registry officials about how to behave when sinti and roma applied for banns and marriage loans. by the second half of the war, the research office also delivered recommendations for sterilizations and abortions among sinti and roma. finally, by at the latest, ritter intervened in the discussion about a "reich gypsy law," but this legislation was never introduced. shortly after the founding of the race hygiene and population biology research office, close connections developed with the nearby kwi-a. wolfgang abel, who, as mentioned above, had undertaken a "study trip" to romania in / in order to examine the roma living there with regard to "the question crossbreeding," established contact with ritter in march in order to draw his attention to the supposed importance of fingerprints in differentiating between "purebred gypsies" and "gypsy mixed-breeds". at abel's instigation, from this time forth the anthropological files of the sinti and roma collected by the "mobile working groups" of the research office also included fingerprints, which were registered by the police as a matter of routine. at the beginning of world war ii, two doctoral students and scientific staff members of the kwi-a moved to ritter's research office: adolf würth, as mentioned above, had earned his doctorate under eugen fischer in with a dissertation on the emergence of flexion creases on the human palm. immediately thereafter, würth, who had also been interested in the "gypsy ques , ritter's "right hand," obtain her ph.d. in . after training as a nurse, justin had begun work as an intern in ritter's genetic biology laboratory at the university of tübingen clinic in . in the race hygiene and population biology research office she effectively acted as ritter's deputy. in she registered as a student at the university of berlin, where she supposedly studied anthropology, genetic psychology, race hygiene, criminal biology, and ethnology -although she could not provide evidence of a methodical program of study when she registered for her doctorate in . she had accepted a dissertation topic proposed by kurt gottschaldt, but then changed it without consulting gottschaldt, and then on her own, so to speak, written her dissertation about "the fates of gypsy children raised as aliens and their progeny." upon fischer's recommendation, on the basis of this dissertation she was permitted to register for the doctorate with a major in anthropology and minors in ethnology and criminal biology. fischer, ritter, and the ethnologist richard thurnwald ( - ) passed the dissertation, which quite obviously did not meet basic scientific standards. the oral examination by fischer, abel, thurnwald and ritter took place on march , in ritter's private residence. why fischer, abel, and thurnwald were willing to issue positive evaluations as an obvious favor to ritter becomes clearer upon perusal of justin's references: the documents include letters of recommendation from hans reiter, president of the reich health office; herbert linden, hitler's reich deputy for sanatoriums and hospitals, one of the key figures in the ns "euthanasia" program; as well as the ministry official paul werner , deputy director of the reich criminal police department, responsible for "preventative crime-fighting," "gypsies," "juvenile delinquency" as well as concentration camps for juveniles, and who had assisted the "euthanasia" planning staff by procuring medications for the murder of patients. justin's dissertation picked up directly on the debates about the "limits of educability" underway since the s in the area of corrective training. she subjected sinti children, who were accommodated in the catholic st. josefspflege home in mulfingen/württemberg, because their parents were interned (most of them in the concentration camps at buchenwald and ravensbrück), to "psychological" tests. on may , , months after justin's dissertation appeard in print, the children were deported to auschwitz-birkenau -only four survived. also in , georg wagner ( * ) submitted his dissertation about "race biology observations on gypsies and gypsy twins." the trained farmer had spent the years from to abroad, where he apparently worked as a correspondent for german newspapers and as a "nationalist political writer." in he began studying natural sciences at the university of berlin. he must have joined up with ritter's research office shortly thereafter, for the material upon which wagner's dissertation was based had been collected in the framework of the total inventory of the sinti and roma in germany and the occupied territories initiated by ritter. as such, wagner -like eva justin -was at the same time an employee of ritter's research office and a doctoral student at the kwi-a. his doctoral research was advised by verschuer, although verschuer requested that fischer step in as the official doctoral advisor for wagner, whom he described as "a somewhat peculiar fellow." in the introduction to his dissertation wagner proudly remarked "that for the examinations of the probands around , km had to be traveled, and over locations of the old reich and the protectorate had to be visited." he had examined persons and categorized them according to ritter's classification system. he characterized the "pure gypsies" as the descendants of the aryans. thus, wagner was the right man for the ss ahnenerbe, which was searching for a scientist to "research the gypsy attributes derived from aryans" in november . on behalf of the ahnenerbe and with the consent of arthur nebe ( - ), head of the reich criminal police department until , wagner settled in königsberg, intending to survey the "gypsies" in latvia, estonia, lithuania, and finnland first, and to visit the "gypsy settlements" in the białystock district. hence, wagner, despite an unmistakable fondness for the "pure gypsies," was party to creating the scientific foundations for the extermination of the sinti and roma. wagner was still working on his research project in march/april . on this joachim s. hohmann fittingly remarks, "apparently wagner was to merely record the evidence of life of an ethnic minority sentenced to extinction, before its genocide was completed. that he obstinately continued working on this just days before the end of the war is presumably one of the many paradoxes of the racist 'third reich'." wagner drew the attention of twin researchers to the sinti and roma, as he had examined "gypsy twins" himself as part of his dissertation, and had reached the conclusion that twin births occur nearly twice as often among sinti and roma than in the remaining population. wagner reported to his colleague karin magnussen about "gypsy twins" among whom he had noticed certain eye anomalies -wagner's scientific curiosity brought these people directly to their death. dahlem and auschwitz josef mengele, with a dissertation about "family examinations in cases of cleft lip, cleft jaw, and cleft palate." mengele proceeded from a group of children with cleft lip, cleft jaw and cleft palate, who had undergone surgery at the surgical clinic of the university of frankfurt/main between and . for these children mengele produced "family tables" covering a total of , "clan members," of whom mengele visited personally. he had the remainder examined by their local health offices. the genetic evaluation of the genealogical material, as mengele summarized his results, made it possible "to recognize an irregular, singly dominant heredity of the disposition, whereby the manifestation depends on other developmental disorders" -among others, mengele mentioned serious defects of the limbs, the lack of a closed spine and closed cranial bones, "as well as feeble-mindedness and mental disorders." in addition, mengele established the frequent occurrence in the families he examined of rudimentary forms of clefts in the area of the lips, the jaw and the palate, which suggested strong variations in the manifestation of the gene. mengele's work made an important contribution to the elucidation of the disputed question as to the heredity of cleft lip, cleft jaw, and cleft palate, whereby the evidence of variations in manifestation fit in well with the recent findings of higher mendelism. moreover, the work was located in the area of arrested development malformations, in which a certain embryonic state of development remains intact, even when development ceases prematurely. such arrested development malformations were of central interest under the aspect of phenogenetics, however -mengele's later attachment to the kwi-a was due not only to his personal relationship with verschuer, but also predisposed by his research emphasis. the "cum laude" dissertation met the scientific standards of the time and was published in in the renowned zeitschrift für menschliche vererbungs-und konstitutionslehre. it immediately attracted considerable attention, not only on the national, but also on the international level, after verschuer referred to mengele's findings in his paper at the international congress for genetic science in edinburgh. well into the s mengele's dissertation was well-received internationally and considered to be the standard work on its topic. until mengele published several short articles and reviews in verschuer's journal der erbarzt. interesting to note is that he worked not only in the field of hereditary defects, but also undertook an excursion into neurology: after the death of the assistant ottwil reichert in , mengele completed reichert's genealogical study "on the heritability of thrombangitis obliterans"(today: thrombangiitis obliterans, winiwarter-buerger disease), which was oriented toward the question of the heritability of rheumatism. mengele also produced expert opinions in frankfurt. verschuer even entrusted him with the scientific evaluation of the comprehensive material that accrued in the "certificates of race and descent." in one case this yielded a short genealogical study on the heredity of fistula auris congenita (branchiogenic syrinx, a special form of the cervical syrinx). whether mengele was actually verschuer's "pet pupil," as hans grebe asserted in the s, remains to be seen. certainly verschuer saw in mengele great promise for the future. it was verschuer's suggestion that mengele attend the international congress for anthropology and ethnology in copenhagen in , and the international congress for genetics in edinburgh in -and that in both cases mengele was not able to participate was due to foreign exchange difficulties, but changed nothing about verschuer's special esteem, which left no doubt that the young scientists included in his list of proposals were the only ones who came into question for him as future university instructors. the judgement of benoît massin, that mengele, had there been no war or had germany not lost the war, in all probability would have made the leap to a professorial chair -like his associates in frankfurt, ferdinand claußen, heinrich schade, and hans grebe -must be confirmed wholeheartedly. it was probably mengele's tremendous ambition that led him into the temptation to take a shortcut against the background of world war ii to drive his career forward more quickly and further than his associates, by unscrupulously taking advantage of the unfettered access opened up to him by the world of the national socialist camps. his close connections to the ss constituted free admission to this world. from to mengele was a member of the stahlhelm; after this organization was subsumed by the sa he remained a member until october . in he joined the nsdap, in , the ss. called up to the wehrmacht in , mengele volunteered for the waffen-ss, where he was assigned to the medical corps inspection office. in november he was transferred to the rusha, where he worked in department ii of the family office, responsible for "care of genetic health" and "genetic health tests." what his job was and where he was deployed has yet to be clarified conclusively. presumably, for a time at least, he wrote expert opinions about the "germanizationability" of "german national" resettlers at an office of the reich commissioner for the fortification of german volkstum in posen. in late /early mengele was sent to the eastern front with the ss division "viking," after he had been promoted from ss-untersturmführer (lieutenant) to obersturmführer ( st lieutenant). during his military deployment he received the iron cross, nd and st class, the eastern campaign / medal and the kriegsverwundetenkreuz (germany's purple heart), nd class with swords. in july -probably due to a wound -mengele was transferred to the office of the "reich physician ss and police" ernst grawitz in berlin, i.e. to the office responsible for oversight of the concentration camps and the human experiments performed there. it is questionable whether he actually reported for service there, however, for he apparently remained with the "viking" division -perhaps he was posted to the "viking" division by the reich physician ss and police. in any case he was still on the rolls of this unit as physician for the troops in october and recommended for a further promotion. he also participated in the battle of stalingrad. as proceeds from a letter by verschuer to fischer of january , , mengele did not return to berlin until early : a few days ago my assistant mengele flew days long from salsk [a city east of rostow on the don] to germany. he took part in all of the battles with the ss division viking, was decorated with the iron cross and has been transferred to an office here in berlin for the time being, so that he can also be active at the institute in addition to his duties there. in february mengele was assigned to the ss infantry substitute batallion "east," which was stationed in berlin. he used his time in berlin -from late january to late may -to consolidate his relationship with his doctoral advisor verschuer. as mentioned above, verschuer already had the intention of bringing mengele to dahlem once he had established himself there. at the institute mengele was apparently regarded as a guest scholar, although he did not sign a regular employment contract with the kwg. his name appeared on an internal list of birthdays, apparently as a matter of course. as also mentioned above, verschuer again entrusted him with expert opinions. however, the official version was that mengele was on combat leave from the university of frankfurt/main until the end of the war -the position as a regular assistant under verschuer's successor heinrich personnel command of / / (signed by siegfried liebau), ibid., p. . recommendation for promotion of / / by battallion commander schäfer of ss pioneer dept. , a sub-division of the ss division "viking," ibid., p. . mengele was not deployed in stalingrad (as stated in kröner, von der rassenhygiene zur humangenetik, p. ), but took part in the battles around stalingrad. wilhelm kranz was reserved for him. nevertheless one may presume that mengele saw his future in berlin. verschuer certainly regarded him as a candidate for professorship, and it probably can be assumed that the two discussed possible topics for his postdoctoral dissertation during mengele's stay in berlin. on may , mengele, who had been promoted to captain of the reserves of the waffen-ss shortly before, was transferred effective may , to the ss main economic and administration office, group d iii (medical care and camp hygiene for concentration camps) and sent to the auschwitz concentration and extermination camp, where he worked as executive camp physician in the "gypsy camp" (section b ii e auschwitz-birkenau). whether mengele was assigned to auschwitz through no fault of his own, or, as verschuer claimed after the war, against his will, or whether, on the contrary, he took steps himself to effect a transfer to auschwitz, and whether verschuer pulled some strings -these questions cannot be answered conclusively based on today's state of knowledge. ulrich völklein argues that mengele ended up in auschwitz more or less by coincidence: the ss physician initially assigned to the "gypsy camp" at auschwitz-birkenau, benno adolph , had fallen ill with scarlet fever in april and was unable to work until november -thus a short-term replacement was sought, and mengele was available at the time. völklein can support his argumentation with the fact that mengele's transfer orders expressly noted "reference: none." this can be assessed as a certain indication that no written transactions existed. in other words: a voluntary enlistment by mengele in written form was in all probability not submitted. but this was not absolutely necessary. it cannot be excluded that mengele -possibly with verschuer's support -contrived behind the scenes for a transfer to auschwitz. this is the gist of benoît massin's argument, whereby he assigns a key role to siegfried liebau and even alleges that there was an "alliance between verschuer and the ss" arranged by liebau. it is documented that liebau, in his capacity as head of the personnel division in the office of the waffen-ss medical corps, signed the order of july , which provided for mengele's transfer from the "viking" division to the "reich physician ss and police." also documented is the fact that liebau, at verschuer's request, was detached to the kwi-a for specialized training from december to october and thus present there in the period when mengele was a regular guest at the institute. finally, it is also documented that liebau spent the first half of there before mengele's transfer to auschwitz -and brought with him photographs of a "gypsy clan" with heterochromous eyes for karin magnussen. massin finds support for his theory in a statement by hans münch ( - ) , who was the director of the ss hygiene institute in auschwitz from to and worked closely with mengele in this capacity. münch, as he stressed later in an interview with robert jay lifton, had the impression that mengele had "requested his transfer to auschwitz, apparently because of the great research possibilities." münch further testified that mengele had worked on a postdoctoral project in auschwitz -and this claim, regardless of how mengele ended up at auschwitz, can arrogate a high degree of probability. in retrospect münch described mengele's mentality with the words, "it would be a sin, it would be crime … that it was irresponsible not to take advantage of the opportunity presented by twin research in auschwitz. if they were going to be gased anyway … this comes around only once, this chance." regardless of whether mengele caught wind of this chance on the basis of information from the office of the "reich physician ss and police" and thus actively instigated his transfer to auschwitz, or whether he did not recognize this chance until he reported for duty -it is clear that mengele unscrupulously exploited the opportunities presented to him there. before long he built up his own research empire. from among the prisoners, he recruited a group of medical specialists for pathology, pediatrics, gynecology, ophthamology, ear, nose and throat medicine, and dentistry, along with technical assistants, nurses, kindergarden and nursery-school teachers, and secretaries. mengele's laboratory barracks in the "gypsy camp" -after its liquidation the laboratory was moved to block in section b ii f of the camp -was directed by the internationally respected pediatrician berthold epstein ( - ) from the university of prague, supported by charles sigismund bendel from the university of paris. for the analysis of blood, urine, feces, saliva and tissue, mengele had the ss hygiene institute in rajsko at his disposal. but above all, the selection of new arrivals on the platform gave him unlimited possibilities to access humans completely devoid of rights and protection. from the endless stream of deportation trains he could single out any human "guinea pigs" he pleased -jews, "gypsies" and other "alien nationals," people with so lifton paraphrased münch's testimony. lifton, Ärzte, p. . quoted in ibid., p. (original omissions). this also explains mengele's obsession, who -in contrast to his colleagues -often came to the platform even when he was not on duty. ibid., pp. - . cf. kieta, hygiene-institut. physical anomalies, entire families and, best of all, twins. mengele created a "twin camp" in auschwitz, the sheer population of which exploded all dimensions previously known. the exact number of twin pairs that fell into his hands in auschwitz is unknown -massin estimates that at least children went through mengele's "twin camp." moreover, twin research under the conditions of the auschwitz concentration and extermination camp presented the unique opportunity to supplement the clinical and anthropological examination of twins with the pathological examination of their corpses, as mengele could murder, or have murdered, both twins at any time. miklós nyiszli already pointed out this circumstance: an event never before experienced in the history of medicine worldwide is realized here: twins die at the same time, and there is the possibility of subjecting their corpses to an autopsy. where in normal life is there the case, bordering on a miracle, that twins die at the same place at the same time? […] a comparative autopsy is thus absolutely impossible under normal conditions. but in the auschwitz camp there are several hundred pairs of twins, and their deaths, in turn, present several hundred opportunities! massin characterizes mengele's research empire at auschwitz as the "auschwitz branch office" of the kwi-a. i find this analysis problematic for two reasons. first, it suggests a formal institutional connection, which certainly did not exist in this form -mengele's position in auschwitz did not at all correspond to diehl's position in sommerfeld. secondly, massin's interpretation constructs all too great a dependence of mengele's on verschuer. certainly: mengele's interests in twin research, in chondrodysplasia, in physical defects and in eye anomalies were oriented toward the model of the frankfurt institute for genetic biology and race hygiene, according to which the institute in dahlem was also reorganized from on. even his interest in noma facies (gangrenous stomatitis, water cancer), a rare deficiency disease caused by extreme hunger, which raged among the children in the "gypsy camp," presumably had a genetic pathology background. in this case mengele probably continued with his mentor verschuer's research strategy of scrutinizing all kinds of forms of disorders -from cancer to tuberculosis, to diabetes, to diptheria, and pneumoconiosis -to see if they were hereditary. this orientation is ultimately not surprising. mengele shaped his own research empire in accordance with the institutes at which he had worked before, but in auschwitz he was his own master. on the other hand it is indisputable that mengele, at his outpost in the no-man's-land of the world of national socialist camps, was interested in being integrated into the scientific community and sought contact and collaboration with other scientists and research institutions -consider, for instance, his pharmacological investigations for massin, mengele, p. , points out that auschwitz, in contrast to all other concentration camps, had sections of the camp in which entire families were imprisoned together: the "gypsy camp" (from february until late july ) with around , inmates and the "family camp" for the jews from the theresienstadt ghetto (from september to july ) with more than , inmates. this was an essential aspect for a scientist interested in "family research." ibid., pp. f. i.g. farben. his most important cooperation partners by far, however, were and remained verschuer and his group of researchers in dahlem (all the more so if the assumption is correct that mengele intended to write his postdoctorate dissertation under verschuer). in any case mengele upheld contact with verschuer from auschwitz, and paid at least one visit to the institute in dahlem during this time -in his memoirs based on his diary of the time, gerhard koch reports meeting mengele sometime around july in the kwi-a library. in at least two cases this contact resulted in concrete collaboration: in the first case, in / , mengele delivered the heterochromous pairs of eyes belonging to several members of a sinti family to karin magnussen, on the other, between october and märz , he joined in verschuer's "specific proteins" project, providing his mentor with around blood samples from persons of various races. as the prisoners' physician miklós nyiszli reported, mengele was also interested in inmates with growth anomalies ("dwarfism" or "gigantism") or physical defects. according to nyiszli, mengele picked out such persons during the selections on the platform, and then had his assistants examine, kill and dissect them. mengele ordered that some of the specimens obtained from these autopsies be sent to dahlem: the scientifically interesting parts of the corpse are preserved, so that dr. mengele can take a look at them. i have to keep anything that could be of interest to the institute in dahlem. these specimens then come into a package for the journey, and a special stamp sees to it that it is dispatched more quickly: 'urgent, contents of strategic importance for the war.' during my stay at the crematorium i expedited countless packets of this kind to berlin-dahlem, in response to which extensive replies with scientific opinions or instructions came in. i put together a special dossier for the purpose of storing this correspondence. for the rare materials we sent, the institute almost always expressed its deepest thanks to dr. mengele. elsewhere nyiszli depicts the case of two jews, father and son, who were deported to auschwitz on a train from the Łódž ghetto and had piqued mengele's scientific interest. the father suffered from scoliosis as a long-term consequence of rachitis, the son from hypomelia (a disorder that affects the development of the limbs). after a clinical examination, mengele had them murdered and commanded that "the skeletons [must be] dissected and sent to the anthropological museum in berlin'." on the basis of this testimony, the authenticity of which is estimated to be very high, there is the suspicion that mengele's deliveries to dahlem took place on a large scale, and that not only eyes and blood, but also internal organs and skeletons found their way to the kwi-a. the most likely recipients are hans grebe and wolfgang abel: grebe as a specialist for chondrodysplasia and physical defects, abel with his plan for an "instructive collection for the race history of europe." in addition to mengele, two other scientists connected with verschuer's institute worked at auschwitz. one of them was siegfried liebau, who performed research on "gypsy" twins in auschwitz during the period when he was detached to dahlem for further training -as mentioned above, he may have been the one who initiated the contact to auschwitz and arranged for mengele's transfer there. the other was the ss-obersturmführer and physician at the ss military hospital in berlin-lichterfelde, erwin von helmersen , who took his doctorate with fritz lenz in august , with a dissertation on "the descendants of an armenian family in a village of the bukovina settled by germans." helmersen had been listed as a doctoral student since , and thus was connected with the kwi-a at the same time mengele was spending time as guest scholar there. after receiving his ph.d., followed by a short assignment in the oranienburg concentration camp, helmersen went to auschwitz, where he worked as camp physician in the "gypsy camp" in section b ii e and in the prisoners' hospital b ii f. helmersen, who was also involved in medical experiments at auschwitz, was thus one of mengele's subordinates for a time. consequently, a network of lines emerge connecting dahlem and auschwitz, which cannot yet be tracked down into its finest nooks and crannies on the basis of the contemporary state of research. clear is that magnussen received from mengele a series of pairs of eyes for her "eye color" project, and verschuer around blood samples for his "specific proteins" project. it is also clear that the two "not only knew of the provenance of these specimens, but that, in this knowledge, they used their contact to mengele in order to obtain these specimens." what is not clear, however, is the question as to whether they knew under what circumstances and in what manner the specimens were extracted in auschwitz. during interrogation by the american military authorities on may , , verschuer denied that he had known about the events in auschwitz, but did admit to having heard the rumors abounding at the time. during a visit by mengele, verschuer testified further, he had asked mengele "just to explain if there was actually anything true about these rumors." in response mengele had reported "about factories located there, his camp hospital, the excellent harmony that existed between him and his patients." "he knew absolutely nothing about inhuman treatment or any other kinds of atrocities." whether mengele completely denied the industrially mass murder perpetrated in auschwitz, or simply let the matter rest with these sinister intimidations, it is highly improbable that he confronted his collaborative partners at the kwi-a with the whole truth. however, it can also be assumed that they did not harry him to do so. they certainly knew enough to not want to know more. in general it can be said -with all due reservations -that only a few germans knew everything about the "final solution," but likewise, only very few knew nothing at all. those working at the kwi-a certainly did not know everything, but quite a bit indeed: "in hardly any other scientific institution in germany," in the judgement of carola sachse and benoît massin fittingly, "could access to knowledge about the crimes in auschwitz have been so easy […] ." as discussed extensively elsewhere, the genocidal character of the "total solution of the jewish question" must have been altogether clear to fischer and verschuer. further, fischer was familiar with the details of the generalplan ost, for which the extermination of the eastern european jews was a prerequisite. beyond this, the kwi-a enjoyed excellent connections to the rusha through herbert grohmann, günther brandt and helmut thieme. at least two scientists who worked at the kwi-a, harry suchalla and christian schnecke, knew about the crimes against jews in occupied Łódz´ . siegfried liebau, josef mengele, and erich von helmersen amounted to three scientists with contact to the kwi-a who actually worked at auschwitz. wolfgang abel, too, had indirect contacts at auschwitz, and perhaps at the sachsenhausen concentration camp as well, but in any case he had insight into the stalags for soviet prisoners of war. karin magnussen was born in bremen in . her mother was a sculptor, her father a teacher at the bremen school for applied arts. after graduating from secondary school, in she began studying biology, chemistry, geology, and physics -still quite unusual for a woman at the time -at the university of göttingen. in she spent two semesters at the university of freiburg, where she was influenced above all by the lectures of the developmental physiologist hans spemann. in , back in göttingen, she took her doctoral examinations in the subjects zoology, botany, and geology. her dissertation, entitled untersuchungen zur entwicklungsphysiologie des schmetterlingsflügels ("studies on the developmental physiology of the butterfly wing") was advised by alfred kühn and published in . this work concerned artificially induced defects in the rudimentary origins of the organs in caterpillars and their effects on the patterns and pigmentation of the fully developed butterfly wing -the influence of spemann and kühn is obvious. göttingen had been a stronghold of national socialism of the first hour, and the student body in göttingen was particularly involved in the earliest successes of the nsdap in the university town. walter groß, who had been involved in the very sachse/massin, forschung, p. . for the basics on the following: hesse, augen, pp. - ; klee, medizin, pp. - . magnussen, untersuchungen zur entwicklungsphysiologie. tollmien, nationalsozialismus in göttingen; dahms, universität göttingen, p. . founding of the göttingen branch of the nsdap back in , one of the first in northern germany, established a university group of the national socialist league of german students (nationalsozialistischer deutscher studentenbund) in göttingen in / . the students in göttingen who were active in the party included rudolf mentzel -as district leader of the nsdap -, who became president of the german research association in ; and achim gercke, the organizer of the "archive for race science statistics on professions" (archiv für rassenkundliche berufsstatistik) who later became "race science expert" (sachverständiger für rasseforschung) to the reich minister of the interior before advancing to the top of the reich genealogical office. magnussen found admittance to this network during her days as a student in göttingen. in she became a member of the nsdap -this, too, highly unusal for a woman, as only very few women joined the party at this time -and was active in the national socialist league of german students. her associates later remember that magnussen had attracted notice in göttingen as a "fanatic national socialist." after the nazis took power she resolutely pursued her party career. in she became a member of the bdm, for which she held training lectures about race and demographic issues in the district of bremen. from on she was also an employee of the race policy office of the nsdap in the district of hanover. magnussen apparently also had contact with the bremen branch of the german society for race hygiene under its chairman, the lecturer hans duncker ( - ) since the weimar republic. in this framework, magnussen may even have experienced the greats of weimar eugenics first hand -fritz lenz, hermann muckermann, eugen fischer, and otmar von verschuer -all of whom held lectures to the bremen branch. there is no doubt that karin magnussen was a fervent national socialist, race hygienist and anti-semite. in -barely years of age -she published her -page book rassen-und bevölkerungspolitisches rüstzeug ("the munition of race policy and population policy"). the third edition appeared in , now swollen to pages. the tract, which propagated "population statistical and race statistical material" and discussed relevant "legislative measures," was conceived -as stated in the preface to the first edition -as an overview for "biology teachers and trainers for instruction in the graduating classes and for race biology training." in the author designated the solution of the "jewish question" as the "core racial problem in europe": emigrants merely breed unrest and incite the völker ("nations") against each other. […] the race policy goal of this struggle of nations thus must be: the spatial separation of the european races and nations from all aliens (jews, gypsies, negroes) […] . with her book magnussen supplied an apparent basis of scientific legitimation to the national socialists' gigantic deportation program, which reckoned with the decimation of the deported from the very outset. in december magnussen passed the state examinations for secondaryschool education in the subjects biology, chemistry, and geology and began teaching. her last position -from to -was as a secondary school teacher in hanover, in line for a civil service post. on september , she began her work at the department for experimental genetic pathology at the kwi-a -initially as a scholarship student. none of the details of the circumstances of her hiring are known. hans hesse conjectures that she was hired "because of her old connections and early party membership." walter groß, whom she knew well from her days in göttingen, had played an important role on the board of trustees of the kwi-a since , as discussed previously. hesse further assumes that the drafting of male employees to the wehrmacht had created gaps in the scientific staff that were difficult to close, so that eugen fischer had been forced to appoint a "woman and not particularly established scientist." this thesis can be concurred with only in part. that political protection played a role in magnussen's appointment may well be true. that a woman was hired and entrusted with a research project central to the conception of the institute, however, was not as rare an exception as hesse apparently assumes, even before -remember rita hauschild. what is least true of all is the assumption that karin magnussen was not sufficiently qualified for her post at the kwi-a. true: she had worked years as a teacher, far removed from research. but for eugen fischer she was nevertheless a very interesting candidateespecially against the background of the paradigm shift to phenogenetics: the complex interplay of genes and environmental factors in the pigmentation of the iris constituted one of the central research fields in fischer's conception of phenogenetics, and he oriented his focus above all on alfred kühn's and adolf butenandt's research on the flour moth ephestia. a pupil of kühn, magnussen had worked on the influence of genes and pharmacologically effective agents on eye color, and after earning her ph.d. in july with butenandt as her advisor, worked on pituitary hormones. according to later testimony by magnussen, back in she was already researching the pigmentation of the eyes, and the phenomenon of heterochromia (the occurrence of two irises with different colors) in particular. in other words: magnussen was well-versed in a research field that was of the greatest interest to fischer in the course of restructuring his institute, and she had collaborated with the researchers to whom he had sought closer contact since . magnussen, rüstzeug, rd edn., pp. f. hesse, augen, p. . moreover, as a student in freiburg she had also learned from hans spemann the techniques of producing, dyeing and embedding microtome cuts. the new institute director otmar von verschuer also thought the world of magnussen, and gave her an assistantship in november , when she was unable to extend her leave from the school system. from that point on, magnussen was listed in nachtsheim's official reports as a staff member of his department for experimental genetic pathology, but nachtsheim neither went into her research in his annual report of / , nor did he include her works in the list of publications from his department -verschuer did this instead in his own report. it seems that nachtsheim and magnussen followed different paths in their research, although magnussen took up work in one of nachtsheim's main areas of research -in fact, she started at the kwi-a shortly after nachtsheim's eye research had begun stagnating as a consequence of his collaboration partner hellmuth gürich's being drafted. there are also numerous potential points of contact: both used rabbits as their animal model, both were interested in the pigmentation of the rabbit eye (nachtsheim's "epileptic" vienna whites had blue eyes as a consequence of a pigment deficiency), both directed their attention to the effects of the aging process on genetic attributes. yet their research projects, as far as we can tell, proceeded in parallel and without any connection: nachtsheim worked on genetic pathology in the strictest sense, while magnussen understood her work as a contribution to the phenogenetics of normal attributes. nachtsheim stated after the war that he had not wanted to work with magnussen because of her political views. he also claimed to know about her connections to auschwitz, which was the "greatest shock" he experienced during the third reich. in three of magnussen's progress reports of september , march and october , the contours of the research project "on the investigation of the heritability of the development of eye color as the basis for examinations of race and descent" became clear, which covered six different, clearly demarcated subareas: first, in preparation for all other subareas, as it were, magnussen dealt with methodological questions on the determination of the structure, color, and pigment distribution of the human iris. she published the results of this work in in der erbarzt. this clarification of preliminary methodological questions pertained directly to application: "in the paternity expert opinions, new experiences are being collected constantly and already obtained experience evaluated." in the very heading of her first research report magnussen had emphasized that her research, as applied genetics, was of importance for the praxis of national socialist race policy. it can be presumed that in magnussen's case this was more than the rhetoric necessary to get a research application approved, and that she placed her research at the service of national socialist race policy out of true conviction. but this was only one side of the coin: her research on eye pigmentation was also, and above all, conceived of as theoretical research, as an important building block of phenogenetics. second, magnussen bred strains of rabbits with certain eye colors "for the purpose of determining the influence of certain hereditary dispositions on eye pigmentation." the animals were under constant observation so that the development of pigmentation could be followed over time. from the report of october it is clear that magnussen was also busy with "breeding for the investigation of heterochromia." third, magnussen performed experiments on rabbits "to physiologically influence pigment development." this project was initially delayed by the war. "the series of experiments planned," magnussen reported in september , "could not be performed, since the compound required, which is manufactured in hamburg, was lost in the terrible attack […] ." in october magnussen was able to report on the first results of these experiments: during the main breeding period in the summer months, several series of examinations on the physiology of pigment development were performed, in which the action of several hormones and pharmacologically effective substances on the development of pigment in the eyes of different races was studied. here certain races whose pigment development showed certain similarities with that of humans, the influence of such substances was determined and thus the foundation laid for larger series of experiments in the coming year. it is no longer possible to reconstruct which substances were used in these series of experiments -possibly a conversation which magnussen conducted with adolf butenandt on december , concerned the question as to which hormones should be applied in the animal experiments. it proceeds from an essay fragment -which presumably originated in -that magnussen dropped adrenaline into the eyes of several chinchilla rabbits in experiments performed privately from to , as well as the extraneous substances physostigmine, atropine, and doryl. magnussen, bericht über die durchführung der arbeiten zur erforschung der erbbedingtheit der entwicklung der augenfarbe als grundlage für rassen-und abstammungsgutachten, / / , ibid., p. . magnussen, bericht über die durchführung der arbeiten zur erforschung der erbbedingtheit der entwicklung der augenfarbe als grundlage für rassen-und abstammungsgutachten, / / , ibid., p. . magnussen, bericht über die durchführung der arbeiten zur erforschung der erbbedingtheit der entwicklung der augenfarbe als grundlage für rassen-und abstammungsgutachten, / / , ibid., p. . in summer the studies about the pigment development in childhood and the required genetic inquiry among the families could be performed only on a smaller scale than previously, due to the drafting of fathers for military service and the evacuation of children. yet the observations are still in progress and, as soon as the situation in the air permits, will be continued on a larger scale. […] as the start of a larger series of observation series, serial examinations about iris structure and eye pigmentation were performed on over , children in holstein. half a year later she sounded less optimistic: "the remaining observations of humans had to be discontinued for a time for reasons concerned with the war, but are to be resumed in summer, to the extent possible." in addition to the serial examinations in holstein (eutin), two further were performed in baden (wolfach) and upper bavaria (mittenwald), and moreover "in eutin and mittenwald family studies to investigate the heredity of certain structural attributes […] (especially important for opinions on descent)." further studies of schoolchildren planned "in several cities of the reich" had to be discontinued in september , since they were not categorized as of strategic importance, and because "by no means [were] they to interfere with lessons." magnussen's research report of october shows that, once the serial examinations of the german population had been disrupted, magnussen intended to start a parallel study in the world of the national socialist camps: "the first series of observations of alien nations in a prisoner of war camp, planned as a comparison with the german population, was prevented by enemy operations. further series of magnussen, bericht über die durchführung der arbeiten zur erforschung der erbbedingtheit der entwicklung der augenfarbe als grundlage für rassen-und abstammungsgutachten, / / , barch. koblenz, r / . , p. . magnussen, bericht über die durchführung der arbeiten zur erforschung der erbbedingtheit der entwicklung der augenfarbe als grundlage für rassen-und abstammungsgutachten, / / , ibid., p. . magnussen, bericht über die durchführung der arbeiten zur erforschung der erbbedingtheit der entwicklung der augenfarbe als grundlage für rassen-und abstammungsgutachten, / / , ibid., p. . on this also, verschuer to fischer, / / , mpg archive, dept. iii, rep. a (münster), no. : "miss magnussen was just here. she performed iris examinations of schoolchildren in eutin (schleswig-holstein) and collected very interesting findings. it is necessary for her to examine populations in germany of different racial composition in the same manner. presumably she will best find the alpine groups in the black forest. i am writing to my sister in wolfach, which seems to me a suitable location. studies of this kind are planned for the coming months." at the current state of knowledge we have no more details about this first serial study in a war prison camp, for which there were already concrete plans, but which never took placewhich camp was involved, who the prisoners were whose eyes were to be measured, in what manner and with whose help magnussen intended to gain access to the camp. neither do we know whether such studies ultimately did take place in other camps and, if so, what happened. the fifth subarea of magnussen's project was histological, in which she dissected the eyes of rabbits, and of humans as well, in series of microtome cuts. as proceeded from the progress report of september , this area had high priority: at the moment, primarily the histological works are being performed, so that the irreplaceable material is processed and thus not subjected to the risk of loss due to long storage. from this emerged a paper "about the relationships between iris color, histological pigment distribution and the pigmentation of the bulbus in the human eye," which was completed in , but not published until . for this study magnussen used the eyes of " subjects from central europe" and compared them with "specimens from the institute's collection, of the dissected eyes of colored races and of a papuan eye." magnussen failed to make any mention of who those people from central europe were. hans hesse suspects that they could also have been concentration camp inmates. in procuring her material, magnussen had few scruples: according to benoît massin's account, magnussen also worked on the eyes of people who were murdered by the gestapo and made available to the anatomist hermann stieve ( - ) in berlin. by the way, it is equally unclear where the dissected specimens of "races of color" contained in the institute's collections came from, which magnussen studied comparatively. sixth and finally, magnussen was interested in anomalies of the eye, such as corneal conjunctivalization, but above all heterochromia. in october she magnussen, bericht über die durchführung der arbeiten zur erforschung der erbbedingtheit der entwicklung der augenfarbe als grundlage für rassen-und abstammungsgutachten, / / , ibid., p. . magnussen, bericht über die durchführung der arbeiten zur erforschung der erbbedingtheit der entwicklung der augenfarbe als grundlage für rassen-und abstammungsgutachten, ibid., p. . magnussen, beziehungen. cf. also idem., beitrag. magnussen, beziehung, p. . massin, mengele, p. . the "papua eye" had been provided to magnussen by eugen fischer. magnussen had established corneal conjunctivization in several animals of one strain of rabbits from nachtsheim's breeding experiments. at the same time, georg wagner, in his examinations of "gypsy twins" apparently discovered in east prussia two "clans" of "gypsy half-breeds of predominantly gypsy descent" in which this anomaly occurred with unusual frequency (wagner, partielle irisfärbung, pp. , ). thereupon magnussen systematically investigated a series of human eyes provided by hermann stieve for the occurrence of this anomaly and found several cases. she also found "during the systematic investigation of the eye specimens of colored races at the institute […] a corresponding tissue fold in a negro." she concluded that the conjunctivization "is propagated more widely announced: "a paper about the histology of total heterochromia in humans is about to be concluded." this work had become possible through one of the most monstrous medical crimes by josef mengele in the auschwitz concentration and extermination camp. in "a member of the institute's staff who worked on criminal biology issues" -from another source it is clear that this meant georg wagner -, in one of the "mixed-breed gypsy clans" he had examined, which also included several pairs of twins, established an increased frequency of heterochromia, "in addition to other supplementary findings interesting in terms of genetic biology." it is highly probable, as hans hesse convincingly portrayed, that the family in question was that of the sinto otto mechau of oldenburg. verschuer commissioned magnussen with the task of taking on the "genetic biological analysis" of this case of heterochromia. to the institute director, this sinti clan must have seemed a rare godsend in several respects at once: first, as mentioned above, heterochromia represented an extremely interesting anomaly within the phenogenetic concept, which promised information about the way gene action chains worked in phenogenesis. second, in this case -as a consequence of the complete inventory of sinti and roma aspired to by the "reich central office of gypsies" and the "race hygiene research office" -it was possible to create a complete family table, clarify the genealogical relationships of the "clan" and thus and occurs in various races" (magnussen, hornhautüberwachsung, p. possibly obtain conclusions about the heredity of heterochromia. third, because the sinti and roma were "locked up" in "gypsy collection points," the test subjects were together at close quarters and -even more important -in the completely lawless area in which sinti and roma were now compelled to live, there were practically unlimited possibilities for access. the scientists could thus perform all examinations and collect all data they liked -even against the will of the affected. fourth, the circumstance that this family included several pairs of twins with heterochromous eyes presented a truly unique opportunity to combine family and twin research in order to investigate the developmental physiological events in the emergence of heterochromia. for this, however, an imperative prerequisite was the histological examination of the heterochromous eyes of the twins -and that meant: the death of these children. fifth, it must have seemed an advantage that the affected family was also the object of criminal biology research -as such, supposedly genetic physical stigmata like heterochromia or deaf-muteness potentially could be linked with supposedly genetic social deviance. sixth, the circumstance that the family was classified in the group of "gypsy half-breeds" built a bridge between phenogenetics and race research, and race diagnostics in particular. thus it is no surprise that verschuer strived to obtain additional financing from the german research association for this central research project. clear is that magnussen performed eye examinations of members of the mechau family before their deportation to auschwitz in march . after the war she gave contradictory accounts about the exact course of events. in her interrogation by the bremen denazification commission on may , she testified: "in spring i took my own photographs of the eyes of such twins at the institute in dahlem, before the twins ended up at auschwitz." a short time before, on march , , in contrast, magnussen had written in a letter to viktor schwartz, an associate of alfred kühn's: the only thing i got to see of the entire clan was two young boys without an eye anomaly, for all of the clans were interned around that time, namely in auschwitz. since civilians were not admitted, any inspection of the people was made impossible during the period of their prophylactical internment. i had a "criminal" twin researcher, who was allowed to visit the camp in his capacity as a officer of medical corps, bring me back color photographs of a number of the people at the time, so that i had a small foundation, but it was very imprecise." the officer of the medical corps described here ironically as a " 'criminal' twin researcher" was, as mentioned above, siegfried liebau. at the time i had seen only one pair of twins from this clan, which had come to the institute in dahlem for the doctoral student [wagner] . i was able to photograph the eyes of both children on this occasion. at the time it was said that these twins (like a major portion of this clan already) were supposed to be sent to an internment camp. at the time i was told that antisocial clans were to be interned in this camp for the duration of the war as a preventative measure. thus, it is probable that magnussen herself photographed the (homochromous) eyes of one pair of twins from the mechau family, which georg wagner had brought to the institute in dahlem for examination before their deportation to auschwitz in march , and that she received additional photographs of the (heterochromous) eyes of members of the family in auschwitz from liebau. through had i not heard from prof. von verschuer that a previous associate of his [mengele] had been ordered to the camp as a physician, i would have been able to base the further genetic biology of the attribute only upon police files. through this colleague, who recorded the attributes precisely and compiled the family tables, i learned that a part of the clan was contaminated with pulmonary tuberculosis, above all a family with children. in her written testimony from magnussen went into this point in greater detail: my demand to be able to visit the remaining members of the clan in the camp was rejected as impossible. at that time i was forced to conclude that women were strictly forbidden mengele , who had been his assistant in frankfurt, and who had been ordered to the camp as a physician (officer of the medical corps). i did not know dr. m. at the time; i only knew from the literature that he worked in the field of genetics. during a visit to the institute in dahlem (summer ?) i made his acquaintance and discussed with him the possibility of performing the research task. i asked him first of all to make sure that this clan not be lost track of after the war, so that the research on this very rare mutation could then be continued intensively. the work of the geneticist is entirely dependent on the life of the carriers of the gene, who hand their genes down to the next generation. at the time dr. m. told me that a particularly important family was subject to tuberculous contamination (with children). everyone knew what that meant in those days, when there was still no remedy for pulmonary tuberculosis, especially for young people under years of age. thus i asked dr. m., whenever any of the carriers of heterochromia should die, to send me protocols of the autopsy and the pathological eye material for examination if possible, -just as i would have in any other case. magnussen's account cannot be confirmed, supplemented, or contradicted by other sources. as far as the framework of facts is concerned, it appears to be coherent and fits in logically with the reconstruction of the project on heterochromia: by this account magnussen, when the project slowed down as a consequence of the deportation of the mechau family, was alerted to mengele by verschuer. had mengele made efforts of his own to be ordered to auschwitz, and had verschuer known of these or even actively encouraged mengele's assignment to auschwitz, the tip to magnussen might have been issued before the posting was ordered on may , ; on the other hand, had verschuer been surprised by mengele's transfer he might have informed magnussen immediately after the command was issued. in either case it is conceivable that magnussen, even before menegele's departure from berlin to auschwitz, reached an agreement with him that he would compile the "family table" of the mechau family on location in the "gypsy camp" andpresumably using the eye-color table developed by magnussen -determine the eye color of the family members. otherwise such a deal must have been made during one of mengele's visits to dahlem on leave. at this or a further meeting then, the arrangement must have been made concerning the family members supposedly suffering from pulmonary tuberculosis. with some degree of certainty it can be presumed that magnussen rendered the events by and large correctly in her postwar testimony. her interpretation of what happened, however, can be scrutinized with a critical view to the sources. in her defense after world war ii she made every effort to portray her arrangement with mengele as entirely normal cooperation among colleagues. in , she stated on record to the bremen denazification commission: in her letter to schwartz, magnussen vested this argument in the form of a rhetorical question: in cases of death should i thus dispense with the histological analysis of the unique, abnormal material just because the people happened to die in the camp? otherwise i could have arranged to get the material from the responsible pathological institute. in her statement from she added that in she had also received from the charité hospital the heterochromous eyes of a deceased patient. the linchpin of this argumentation is magnussen's assertion that she did not know that auschwitz was an extermination camp. in her testimony to the denazification commission she claimed to have been unsuspecting: according to the impression i had of the case histories and of the extremely responsible and humanly decent attitude of dr. mengele toward his interned patients and staff (after the war, he hoped to win over for the institute a certain female polish physician interned in auschwitz, as he told me), the idea would never have entered my mind that anything could occur at the auschwitz camp that might violate state, medical or human laws. to schwartz, too, magnussen asserted that "nothing awful [was] known" to her; "on the contrary -from the case histories, the colleague's stories and from his human attitude to the inmates i could only have the impression of proper and decent treatment." nevertheless, it seems more than improbable that karin magnussen, a scientist at an institute whose leading members had been involved in the discussions about the generalplan ost and who made no secret of their attitude toward the "final solution" of the "jewish question" and the "gypsy question," and, moreover, an active national socialist with close contacts to the race policy office of the nsdap, had no knowledge at all of the genocide of jews, sinti and roma and no conception at all of the conditions in a concentration camp; accordingly, this must be dismissed as nothing more than an attempt to rationalize her behavior. rather, it can be assumed that it was altogether clear to magnussen that the sinti and roma had hardly a chance of survival in birkenau, and that this came in handy due to her interest in the eye specimens. mengele's indication that a family was "contaminated" by tuberculosis could even be interpreted as a discreet offer to assist in their demise, and magnussen's request to send her the specimens as consent. at this juncture magnussen also claimed that she did not know that other categories of prisoners besides "gypsies" and "mixed-race gypsies" were held at auschwitz. quoted in klee, medizin, p. . magnussen went so far as to assert that even former prisoners from auschwitz had "nothing awful" to report. the following testimony in her defense from must be relativized in this respect: "my work about the genetic biology of heterochromia was not performed on this clan because it was in a concentration camp, but rather even though it was interned in a camp, which made my work extraordinarily difficult, and almost impossible, had not a scientist coincidentally been assigned as a physican there. of the twelve children from the one family, initially two died in the years and (one of erysipelas and one of a pulmonary tb on both sides). a child from a related family died of pulmonary tb as well. the case histories and autopsy reports i received were painstakingly recorded. at this juncture the testimony of two surviving prison physicians can be drawn upon for further information. in his memoirs, miklós nyiszli reports that on several occasions he was required to dissect the corpses of "gypsy twins." once the corpses were of four pairs of twins, that is, eight children who were under years of age. nyiszli established the cause of death to be injection of chloroform to the heart -therefore the children were murdered systematically. nyizli related the further events as follows: of the four pairs of twins, three have eyes of different colors. one is blue, the other brown […] . i dissect the eyes out and lay them, each separately, in formalin, whereby i note precisely all information about them, so that they cannot be mixed up. […] in two pairs i also find an active pulmonary tuberculosis. […] in the afternoon dr. mengele makes his rounds of the ward. […] he is very interested in the heterochromia of the eyes […]. right away he instructs me to prepare the entire material for dispatch, along with the protocols, but i should also list the causes of death. he leaves it up to me what i write, but the causes of death must be different. almost by way of apology he says that these children, as i could see myself, suffered from syphilis or tuberculosis and would not have been able to survive anyway, so … he does not say anything more. this account is confirmed in its entirety by testimony from the ss commander erich mussfeld. whether the children's corpses autopsied by nyizli were members of the mechau family must remain an open question. the children of the mechau family may also have been autopsied by the jewish romanian prison physician iancu vexler, who worked in the "gypsy family camp" from june , . at least, this is what hermann langbein, himself a prisoner at auschwitz reports: vexler also reports about mengele's laboratory in the sauna block of b ii e, in which anthropological examinations took place, especially on hair and eye color. […] once a gypsy family by the name of mechau with striking heterochromia of the eyes was brought in. mengele drew vexler's attention to the family and ordered him: 'well, good, when it's time -when that happens, understand? -you will carefully take out the eyes and put them in bottles prepared for you. they will go to berlin for the investigation of the iris pigment. you understand, genetic issue, heredity dominant, recessive, etc. highly interesting. the parallel account suggests that several prison physicians were occupied with the autopsies of (twin) children with heterochromous eyes from the "gypsy family camp" at auschwitz-birkenau -and there are additional indications that point in this direction. in any case the reports from the prison physicians confirm the suspicion that mengele killed many more twin children from the "gypsy family camp" because of their eyes, and delivered many more eyes to the kwi-a than magnussen testified in her defense after world war ii -in she spoke of three, in of five pairs of eyes that she received from mengele. nyiszli's report further suggests that several heterochromous pairs of eyes from twins ended up in dahlem simultaneously. by this time at the latest it must have been clear to magnussen that the children had to have died a violent death. benoît massin is justified to establish: morphologie und anthropology, which was then edited by hans weinert -failed due to misgivings about the origin of the material investigated. in auschwitz, mengele also performed experiments "about the possibility of a change in iris color," by dropping substances into the eyes of numerous children. the consequences of these experiments ranged from slight itchiness to swelling, inflammations, and suppurations of the eyes, in some cases the subjects lost their eyesight. mengele even performed such experiments on newborn infants -with fatal results. according to the prison physician ella lingens , in a newborn died after mengele injected a substance into its eye "because he was attempting to induce a change in eye color. little dagmar was to get blue eyes." in irmgard ludwig had her newborn child taken away from her. when she saw it again, the eyes looked "like a crude clump." it is not known what substance mengele dropped into the children's eyes. according to a report by the polish prison physician rudolf diem, mengele claimed that the drops he had administered to persons with heterochromous eyes contained adrenaline: "he believed that the application of these drops would cause the iris color to change." what at first glance seems to be a mad, pseudoscientific experiment to instantly "aryanize" brown-eyed "gypsy children" by injecting substances like methylene blue takes on a new meaning against the background of magnussen's project on heterochromia. these experiments investigated the pigmentation of the human eye under phenogenetic aspects. mengele did not perform these experiments "singlehandedly […], but rather in 'teamwork' with magnussen. mengele was thus more than a passive supplier of dead 'human material,' and by no means did magnussen research on dead objects only; she was actively involved in mengele's human experiments." after the war magnussen confirmed that she had taken part in mengele's eye experiments, whereby she boldly redefined heterochromia as a disease and passed off the human experiments performed by mengele in coordination with her project as an attempt to cure the subjects: the histological investigation succeeded in obtaining an indication of the presumptive cause of the disturbing anomaly. -we decided to apply the results immediately in the interest of the same family as an attempt to cure the anomaly in one of the children. -since animal experiments of this kind had already been performed with success by other scientists, and since we had received previous assurance from the university ophthamological clinic in berlin that no unpleasant side effects of any kind were to be expected from the treatment planned (adding drops of a bodily substance for the purpose of restoring a disturbed function), the treatment was started. -since the child, unfortunately, died after a few months, it was not possible to perform the treatment for a sufficient length of time to yield an externally visible success. -shortly thereafter i received the eyes of this child for histological analysis, along with the eyes of another deceased child from this family contaminated with tb, -(i.e., of children) and performed the histological analysis of these eyes as well, although the histological work on the first three pairs of eyes had been langbein, menschen in auschwitz, p. . quoted in hesse, augen, p. . for instance, lifton, Ärzte, p. . in opposition: massin, mengele, p. . ibid. concluded long ago. -the possibility that an advantageous effect of the treatment is present after two months is given in the histological examination, so that i would advise this treatment even today in a case with similar conditions. also closely linked with the auschwitz concentration and extermination camp was that secretive research project of verschuer's, funded by the dfg and listed in the files of the reich research council as "experimental research on the determination of the heredity of specific proteins as the foundation of genetic and race research." this project was long regarded as a contribution to genetic pathology research under the aspects of race and implicated with diehl's tuberculosis research in the external office of the kwi-a in beetz. one of the connections existed on the organizational level, for verschuer's project used rabbits from diehl's breeding; their sensitivization with human blood sera took place in the kwi-a "reception center" in haus am see in beetz, that is, in the direct vicinity of the external department for tuberculosis research, and technical problems were resolved in part thanks to the know-how of the kwi for biochemistry, which also provided consulting for genetic tuberculosis research. yet there was also a connection on the conceptual level, to the extent that both projects fit in to the paradigm of phenogenetics. there may have been a practical connection beyond this, as it cannot be excluded that blood samples of sick patients were also targeted for use in the "specific proteins" project -more on this later. in this case there would have been the prospect of genetic pathology findings becoming available as a kind of "byproduct" of the "specific proteins" project. however, it must be emphasized that the two projects did not constitute a single unit and that it is by no means correct to conceive of the tuberculosis project as a preliminary phase of the "specific proteins" project. benno müller-hill and many others after him advanced the theory that the "specific proteins" project concerned the investigation of race-specific susceptibility or resistance to tuberculosis, and this, in turn, was connected with the theory that josef mengele had purposely infected inmates of the auschwitz camp with tuberculosis on behalf of the kwi-a. in contrast to this, bernd gausemeier formulated the theory that the project pursued the goal of developing a serological race test. achim trunk recently subjected both positions to meticulous critique, reaching the conclusion that gausemeier's theory can be reconciled with the scant source material available much better than that of müller-hill. i concur with gausemeier's position, whereby i can submit a document not yet taken into consideration that provides magnificent confirmation for trunk's analysis. the "specific proteins" project was presumably launched in spring . it was acknowledged as strategically important and thus received special funding as regarded material procurement, but yet -in contrast to the tuberculosis project -it was rated as the lowest priority s. the first indications as to what the project was about appeared in verschuer's interim report to the dfg of september/october : once all materials for the performance of this research had finally been delivered, the first preliminary examinations were begun and the methods tried out in consultation with privy councilor abderhalden, halle. an interruption in the work occurred when this branch of research was moved to the reception center of the institute in beetz, but by now the laboratory there is completely equipped. work can be continued. two things can be taken from this report: the project had been temporarily moved to haus am see in beetz, and it used a method that was widespread in germany at the time, although controversial, "aberhalden's reaction." this procedure, developed by the swiss biochemist emil abderhalden starting in , proceeded from the basic assumption that an animal organism can recognize and destroy a foreign protein that penetrates it -such as those of bacteria in the case of an infection -by manufacturing enzymes (at that time they were known as "ferments") that catalyze a defensive reaction against the foreign protein. the "protective ferments," the presence of which abderhalden and his pupils believed they could demonstrate in blood, and since in urine, ultimately turned out to be chimerical. the entire edifice of teachings constructed by abderhalden was founded on either fraud and/ or -scholars are still arguing about this -on error and self-deception. in the early s abderhalden had faced increasing criticism, but his method had not yet been clearly disproved. broad hopes were still linked with the method; it was believed that it might open up new possibilities for the diagnosis of infectious diseases, cancer, and even psychoses. what is more: in the second half of the s, abderhalden and his pupils attempted to use protective ferments for the determination of race in sheep and pigs. "this important research," verschuer commented in his review, "finds the greatest interest of the genetic biologist […] ." in abderhalden and verschuer exchanged several letters, in which it was abderhalden's idea to investigate the reactions of protective ferments in twins. verschuer rejected this for the time being, referring to the difficulties of obtaining blood samples. the correspondence between verschuer and abderhalden shows that in the latter was training one of verschuer's medical-technical assistants to use his methods in halle -müller-hill's investigations produced evidence that the assistant in question was irmgard eisenlohr (from : married haase). the second interim report by verschuer to the dfg of march confirms clearly that abderhalden's method was applied in the "specific proteins" project: in the trials of the methods new difficulties have come to light, which were resolved in consultation with privy councilor abderhalden, halle. series of rabbits are being subjected to thorough testing in order to find animals free of spontaneous ferments and thus suitable for the experiments. my assistant dr. med. et dr. phil. mengele has come in as an associate in this branch of research. he is stationed in the auschwitz concentration camp as hauptsturmführer and camp physician. with permission of the reichsführer ss, anthropological examinations are being performed on this camp's many different racial groups and the blood samples sent to my laboratory for analysis. this is the first evidence that the "specific protein" project used blood samples from people of different races, which came from josef mengele. in two letters by verschuer to the pediatrician bernhard de rudder, a close friend of his and diehl's, he goes into greater detail about the blood samples supplied by mengele. in october verschuer wrote: plasma substrates were produced from over people of various races, pairs of twins and a number of families. and a letter of january reads: what these "actual comparative studies" involved and what purpose they pursued is a matter of great controversy in historical research. achim trunk reconstructed the course of events as follows: from the blood samples serum was extracted and sent to berlin. from this, dry preparations were produced in the laboratory, which were then supposed to serve as the "substrate" converted by the protective ferment in the defense reaction. meanwhile, the search was on for suitable test rabbits, i.e. ones that did not already have the capability to decompose the foreign protein before they were inoculated with this protein. this is what verschuer was referring to with his comment that "series of rabbits [had been] subjected to thorough testing in order to find animals free of spontaneous ferments." when suitable rabbits were found, researchers injected them subcutaneously with a portion of the dried substrate and waited until they developed protective ferments against the race-specific human proteins. in the next step the protective ferments supposedly created had to be isolated. to do this, the urine of the rabbits was collected in special apparatus, as it was believed that the protective ferments were excreted with the urine. all substrates were then subjected to cross-reactions by adding the ferment solutions extracted from the rabbit urine in order to determine similarities and differences between the substrates. the final objective was to identify a panoply of protective ferments, each of which reacted with the proteins from the blood of a very specific human race. this would indeed have opened the way for a biochemical race test that would have eclipsed all anthropometric methods of race diagnostics attempted up to that time. a letter from verschuer to karl diehl of july , presents impressive confirmation for trunk's version of events: the experiment about the serum proteins with abderhalden's reaction has proceeded to the point where i have copious material at hand in the form of substrates. a conversation i conducted a few days ago with one of butenandt's assistants gives me occasion to begin now with the actual experiment, i.e. the reaction with the protective ferments generated in the rabbit. to do this, first of all, as last fall, the rabbits must be subjected again to thorough tests for the presence of spontaneous ferments, so that we can finally arrive at an animal that tests negative for ferment. therefore i request of you, just as you did last fall, to allow that a few animals from each of your normal breeds be placed into the special cages so that urine can be collected for the examination. this clearly proves that which trunk held to be very probable: the "specific proteins" project quite clearly did not concern protective ferments against tuberculosis or any other infectious disease in the blood samples taken by mengele in auschwitz. rather, these served without a doubt as substrates, which were to be converted by protective ferments extracted from rabbits. also worthy of emphasis in this letter by verschuer is the term normal breeds. what must be kept in mind here is that diehl held not only rabbits from the two pure breeds in his stalls -the ones he called "lung and belly rabbits" -and from the cross of these two breeding lines, but beyond these also a great number of other rabbits, of which many gave their lives for "preliminary experiments," for instance, by using glycerine to extract from their lungs a culture medium for tuberculosis bacilla. the normal breeds were very valuable for diehl -in december he answered the verschuer family's inquiry as to whether he could spare a rabbit or two for the christmas feast in the negative. presumably with a heavy heart, he declared himself willing to make rabbits from the "normal breeds" available for the "specific proteins" project, in return for verschuer's submitting a dfg application to obtain funding for his "tuberculosis" project. the point here is that the "specific proteins" project had nothing to do with diehl's pure breeds and the crosses between these pure breeds. thus, we can exclude with a high degree of probability that the human tuberculosis bacilla, with which a portion of the pure breeds were pretreated according to verschuer's statement, came from auschwitz. in verschuer's letter to diehl of july , the "specific proteins" project's connection with the kwi for biochemistry becomes clear for the first time. the report by verschuer to the dfg of october provides further explanation: "it is too bad that our shared work plans cannot be continued at the time being, but i hope it will be possible later." from what was said it should have become obvious that while the "tuberculosis" and "specific proteins" projects were closely linked together on the practical level, they pursued different objectives and were located in different fields of research: diehl's tuberculosis research fit into the long series of genetic pathology projects at the kwi-a, while the "specific proteins" project concerned human races. nevertheless verschuer, as proceeds clearly from his letters to de rudder, saw a close connection between the two projects. this is evinced particularly by a letter of october , , in which verschuer named the two projects in one breath with regard to his impending lecture to the academy: diehl obtained new, and as i believe, fundamentally very important research in his tuberculosis research. […] i believe that my research about the question as to the heritability of specific proteins is also connected to the problem as a whole. […] the goal of my different endeavors is now not only to establish that the influence of heredity is important in some infectious diseases, but in what manner it takes action and what kind of processes occur in these cases. at first glance this passage appears to speak for müller-hill's reconstruction of events, according to which the "specific proteins" project did have the object of race-specific dispositions or resistances to tuberculosis. it is clear that verschuer was interested in such issues. in his lecture to the academy on november , he hit on the gradual differences in the susceptibility and frailty of various human races with regard to infectious diseases -here he also mentioned the supposedly greater resistance of jews to tuberculosis -but he added, with reference to a publication by de rudder, that it had yet to be elucidated "whether these differences are truly conditioned by the different genetic disposition of the races and not by other influences." the "specific proteins" project actually promised to contribute to the clarification of this question indirectly, and thus there was an internal connection to tuberculosis research. the conceptual brackets around the two projects were constituted by the paradigm of phenogenetics. while each of the projects had a practical application in mind: diehl's attempt to breed rabbits resistant to tuberculosis was borne by the hope of discovering a biochemical substance that could also give humans protection from tuberculosis -and this without inoculation. at the forefront of verschuer's protein project was the development of a biochemical race test. however, it must not be overlooked that both projects were apparently understood as complementary contributions to theoretical research in the area of phenogenetics, as they both aimed at the level of the proteome, where the gene action chains proceeding from the genome are set into action and shape the phenome, where race attributes are developed and many of the dispositions for diseases were also established. the "specific proteins" project shows -as did karin magnussen's project on heterochromia, by the way -that research guided by the paradigm of phenogenetics, although it left behind the genetic determinism of the old race hygiene and race anthropology, was by no means invulnerable to drifting into the zone of crime. my theory is that one can conclude from this that in the area of the science of humans there can be no security against crossing scientific boundaries inherent in the paradigm guiding research -whatever shape it may take -. every form of research on humans takes place in the tug of war between the researcher's interest in scientific knowledge and the human and civil rights of the person researched, regardless of their idea of man, that is, no matter whether they regard the individual as a pure product of his or her genetic information, as result of the interplay between heredity and environment, or as a tabula rasa that is marked by the environment. drawing science-ethics boundaries takes its basis of legitimation from values held beyond the sphere of science. otmar von verschuer was neither the only one nor the first to work on a serological race test. the anthropologist theodor james mollison had long been concerned with the question as to whether serological race diagnostics was possible. mollison attempted to reach his goal using the "precipitine reaction." this reaction involved the formation of precipitation that occurred when blood serum from another animal, for example, from a chimpanzee, was injected into a rabbit, and the antiserum, which was produced from the blood of a rabbit immunized in this fashion, was mixed with the original serum of the chimpanzee. if the same antiserum was allowed to react with sera of related species -like macaques, gibbons, orangutans, or humans -the precipitin reaction varied in strength. mollison traced this back to proteins in the blood serum specific to each species. what was true for animal species, mollison deduced, must also be applicable to human races. therefore, since the s he had been attempting to develop serological race diagnostics on the basis of the precipitin reaction. other scientists in the third reich picked up on this approach. werner fischer ( - ) from the scientific department of the institute for experimental cancer research in heidelberg under ernst rodenwaldt, who collaborated with benno raquet in to submit a paper "on the question of the proof of a perform studies on the "question of generating mutations in drosophila through x-rays." "in these studies [work was performed] in very close coordination with timoféeff-ressovsky, in order to achieve as great a consistency as possible in work methods and work orientation." in june loeffler was able to report that the studies, which were part of the joint project sponsored by the german research association and the reich health office, already covered , cultures with over , animals. yet loeffler had strong competition in this field he had originated, for instance, from the genetic biology department of the reich health office. thus, it was altogether logical that he assigned his right hand horneck to the explosive problems surrounding a serological race test, which -as loeffler had recognized clearly -touched on not only "important fundamental issues of our science," but also was aimed "at purely practical matters." thus, loeffler could open up a new field of research, which was not only of scientific interest, butmore importantly -also of immense importance for national socialist race policy. in this he attached himself to werner fischer, who had been working on the complex of themes for some time and had both the required know-how and the necessary infrastructure at his disposal. for his part, fischer was happy to accept loeffler's advances, and bound horneck's project to his institute, as in this manner he could take advantage of loeffler's far-reaching political connections in the national socialist state. this was of particular interest to fischer because, at around the time horneck published his first results, he had begun with serological examinations of concentration camp inmates. on this, a report by the reich physician ss ernst grawitz to reichsführer ss heinrich himmler of july , states: wittenau sanatoriums, but perhaps through wolfgang abel, who, we recall, also had spent time at the special colonial medicine military hospital in st. médard, possibly worked in the sachsenhausen concentration camp as well, and could have stumbled over horneck's and fischer's tracks. whatever the sources from which verschuer obtained his knowledge about the competition's project: with the "specific proteins" project he attempted to regain the upper hand in this field of research. second, the experimental design of the "specific proteins" project, as reconstructed by achim trunk, corresponded to fischer's and horneck's approach down to the last detail -only that verschuer pursued his goal using abderhalden's protective ferments rather than the precipitin reaction. verschuer was familiar with mollison's attempts to make the precipitin reaction useful for race diagnostics, just as he was with abderhalden's works, but, as his reviews show, since the late s he granted the better chances to abderhalden's method. since he enjoyed a direct connection to abderhalden, he believed his hand held a decisive trump over fischer and horneck. third it becomes apparent that the competition between the scientists corresponded to the rivalry between the politicians involved -conti and blome. it was all the easier for verschuer to win over conti for his plans because the competing undertaking was located in blome's sphere of influence. fourth and finally, against the background of horneck's project it cannot be excluded that the "specific proteins" project utilized the blood of subjects who were ill, perhaps even that of humans who were made ill. since horneck had researched on blood samples of diseased subjects of various races to investigate whether the serum of sick members of a race behaved differently in the precipitin test than did that of healthy members of the same race. this question was also posed in principle by verschuer in his application of abderhalden's reaction -in competition with horneck and fischer he could not really afford to leave this question unanswered. thus, it is possible that the blood samples mengele sent to dahlem include some originating from diseased inmates. similarly, against the background of horneck's research, the suspicion that mengele purposely could have infected humans with infectious diseases, such as typhus, cannot be dismissed completely in the context of the "specific proteins" project. moreover, verschuer was interested in questions of "race pathology," as his paper from already demonstrated. in this paper he presented "as certain pathological facts" that "numerous genetic disorders like diabetes, deaf-muteness, endogenous psychoses [occurred] in germany more frequently among jews than non-jews and "amaurotic idiocy [had] been observed only in polish jews. as we have seen elsewhere, he was still concerned with "race pathology" in and -to that extent it is quite conceivable that verschuer followed horneck's experimental design and opened up the "specific proteins" project toward a genetic pathology orientation. as heavy air attacks on berlin increased in mid- , verschuer began to look around for possibilities to relocate part of the institute. he managed to rent a house in sommerfeld, on lake beetz, in the direct vicinity of the external office for tuberculosis research in waldhaus charlottenburg, which had been expanded and equipped as an auxiliary hospital, but had never been moved into. from july this haus am see had been used as a "receiving office for the institute" -it was staffed by hans grebe with his secretary, nurse emmi nierhaus, karl diehl's technical assistant charlotte gruetz and the technical assistant irmgard eisenlohr, who was involved with the "specific proteins" project. verschuer had thought about relocating the entire institute to beetz, but for tactical considerations he dispensed with this idea. if the institute were vacated voluntarily, verschuer wrote in a letter to fischer, it might be lost, even if it were not destroyed by air raids. "i could not take responsibility for being at fault myself." in order "to prevent the destruction of our scientific body of thought" he had a wagon load brought to beetz and sommerfeld, and in beetz a hutch for nachtsheim's rabbits was to be built as well. in the dahlem institute, which officially bore the name "eugen fischer institute" since its founding director's th birthday in june , of the valuable goods only the "photograph collection" remained, which was stowed in the air raid shelter. the only staff that continued working in dahlem were the department heads, karin magnussen and a few auxiliary assistants. "this dispersion of items is not ideal," verschuer wrote, "but provides great reassurance." in order to preserve coherence, he set up a standing "courier service" between dahlem and beetz. in february the institute was damaged in a heavy bombing attack. yet verschuer still regarded the situation to be "by no means so grave that a relocation of the complete institute would come into question." in the provisionally repaired building he kept operations afloat for the time being. in september , however, first signs of deterioration became apparent. lenz, as verschuer reported, after having brought his wife and children to relatives in obernfelde near lübbecke in westphalia, fell deeper and deeper into depression and could hardly work any longer -shortly thereafter he took leave for reasons of poor health and followed his family to the west, such that the institute for race hygiene ceased to exist in fact. abel had "left his people more or less to themselves and consumes aerated baths in bad ischl." according to verschuer, abel managed "excellently to put his personal affairs in order as advantageously as possible. now he is shifting ever further away from the institute and has become a rare guest." karl and anne diehl, despite health problems, continued their rabbit research, "albeit often by summoning their last strength;" the same was true of hans nachtsheim, who had been declared fit for combat in his army physical, so that it was only a matter of time before he was called up. gottschaldt was the only one who exhibited "an active demeanor loaded with energy," and pushed ahead "the evaluation of his twin findings with extremely hard work and great energy." however, he was often in rottmannshagen, where he had lodged his wife and children. the situation there, verschuer warned, was "by no means harmless, for in the sparsely settled land the foreign workers constitute a majority, which could easily seize power for themselves if enemy pilots were to furnish them with ringleaders." on february , a directive was issued by the reich minister for armaments and war production, albert speer , to the operations staff of the kwg, instructing that the institutes under its control be relocated from endangered areas. ernst telschow forwarded this directive to the kwi-a, where it arrived on february , delivered by a courier. had it been, up to new year's , verschuer's express goal to hold out in dahlem as long as possible and await the further course of events in order to defend the institute building against competing claims, by february it must have been clear to him that the fall of berlin was merely a matter of time. relocating the institute appeared imperative, and in secret verschuer already had begun the preparations for a move. so speer's directive came at just the right time, although initially appearances suggested that it was already too late, for an execution of the directive appeared "impossible." in addition, between february and , telschow, as he claimed angrily after the fact, informed verschuer orally that speer "in retrospect [had] not desired" the "application of the relocation directive" to the kwi-a. although verschuer later vehemently denied ever having received such a communication, telschow's account is confirmed by other sources. thus, it can be presumed that verschuer was quite aware that he had received a green light to relocate his institute neither from the general administration nor from the armaments ministry. however, when engelhardt bühler, who had been assigned to the institute a short time before, managed to organize a trailer truck around february , -to everyone's surprise, verschuer acted without delay, supported by speer's written command to relocate, abruptly overrode the oral counter-command communicated by telschow and set the relocation in motion. on february , , when part of the material sent to beetz had already been loaded on the truck, he sent a circular to the department heads abel, diehl, gottschaldt, lenz, and nachtsheim, officially informing them that the majority of the institute's inventory was to be relocated to his family estate in solz near bebra. the department for experimental genetic pathology remained in dahlem, since the extensive animal breeds could not be taken with the institute. nachtsheim was appointed verschuer's deputy and entrusted with the oversight of the institute building and the inventory remaining there. the external department for tuberculosis research stayed in sommerfeld, as diehl was indispensable as the senior physician of the waldhaus charlottenburg hospital, and he, too, did not want to leave his animal breeds behind. by contrast, the alternative location rottmannshagen, under gottschaldt's direction, was to be dissolved and also relocated to solz as soon as possible. some of the "followers" were supposed to remain in dahlem, some were to move to solz, and some sent home on leave. on february , the inventory of the dahlem institute was loaded on the trailer truck provided. in a letter of february , verschuer informed the general administration in writing of the relocation already in progress. immediately before his departure, on the afternoon of february , , verschuer must have had another meeting with telschow, in which the general secretary vented his anger, but he was not able to stop the operation in progress. quite obviously, verschuer used the chaos predominant in the final phase of the war, above all "the nearly complete collapse of the standard paths of communication," to remove himself from berlin and in this manner present the general administration of the kwg with a "fait accompli." how hasty verschuer's departure was is apparent in the fact that he did not even find the time to contact günther hillmann to discuss the continuation of the "specific proteins" project. he left the inventory of the laboratory with the "special rabbit cages for the collection of urine" in butenandt's institute in dahlem. "i brought with me only the especially valuable and irreplaceable protein substrates," verschuer reported from solz -thus it is possible that some of the sera that ended up in solz came from the blood samples taken by mengele in auschwitz. on the other hand, the written documentation on the "specific proteins" project may have been left in dahlem. on march , nachtsheim wrote to verschuer: verschuer confirmed that some of the material involved was "secret files, which by no means may fall into enemy hands," asked nachtsheim to attend to the matter and to give the caretaker the order to burn the material "in good time." on february , verschuer laconically informed the general administration that the relocation of the institute to solz had been completed "without significant inconvenience." on march , gottschaldt arrived in solz as well, with the material that had been stored in rottmannshagen. in a letter to his friend karl diehl of march , verschuer appeared optimistic that the institute would be able to continue its scientific work in solz: from here i have good news as far as it goes. it is certainly an enormous luxury not to have any sirens in the village and not to feel like a direct target of enemy pilots. as such i manage more positive work than was possible during the last phase in berlin. the establishment of my small institute here is making progress, although all sorts of difficulties must be overcome. someday i would like to give you a tour of my facilities here, my director's study (also living room and bedroom for erika and me); the library, in which all of the books brought from beetz have been arranged, which is also the study for miss sesselberg (not to mention the group dining room); to the church hall in the manse, which i have furnished as a study for miss lüdicke and nurse emmi, in which thus the twin files are being analyzed and the institute's administration and treasury are located; and, finally, in a restaurant hall where the institute property is stacked (including that which gottschaldt has since brought here from rottmannshagen). shortly before christmas eugen fischer and his wife had fled from freiburg before the approaching allied troops to their daughter gertrud in sontra, near bebra, so that fischer and verschuer found themselves just a few kilometers distance from each other at the end of the war. in berlin nachtsheim had to struggle with increasing signs of dissolution. in fear of the approaching red army, many staff members refused to work. most of nachtsheim's rabbits had to be slaughtered once the plan to bring the animals to switzerland had been discarded. some of the institute's rooms had to be yielded to the reich office for land use planning, the reich ministry for church matters and to a department of the university of posen. finally, on march , the institute building was requisitioned as a reserve military hospital. the general administration of the kwg, angered by verschuer's going it alone, undertook nothing to prevent the requisitioning. the general administration also took a passive stance in the conflict about the haus am see that broke out in march , when the responsible local group leader requisitioned the building to accommodate refugees. in the end, the kwi-a was left with two rooms of the haus am see, in which institute bericht über die im jahre durchgeführten und für das jahr geplanten forschungen cf. koslowski, einfügung. the genetic psychology part of the project was to be carried out by a "miss dorer erbbiologische bestandsaufnahme rep. a (münster), nr. for the continuation of these studies using biochemical methods contact with professor butenandt has been established tuberculosis (bericht für den zeitraum vom . april bis zum r i/ formulation proposal by ernst wentzler, in square brackets: formulation proposal by lenz. wentzler's version read: "who long for deliverance because of an incurable disease formulation proposal by lenz, in square brackets: formulation proposal by kurt pohlisch . formulation proposal by lenz tätigkeitsbericht / , mpg archive eickstedt himself was not present in leipzig, his lecture must have been read by a deputy. cf. verschuer to fischer, / / , mpg archive on hitler's orders, a large portion of the african prisoners of war -around , men -had been deported to southern france (bordeaux) in an interview with benno müller-hill, abel stated that he was "in a leper station in bordeaux visiting dr. weddingen in the tropical medicine military hospital pictures for the work on pygmy soles of the foot the same formulation is included in verschuer's tätigkeitsbericht for / (mpg archive here: p. ; dekan der medizinischen fakultät frankfurt to hessisches staatsministerium für kultus und unterricht kirchliches urteil über die persönlichkeit und die wissenschaftliche arbeit von herrn professor dr on this in detail: schmuhl, Ärzte in der anstalt bethel jüdische mischlinge von der rassenhygiene zur humangenetik denazification certificate general heiber generalplan ost * ) worked at the rusha in and served as an aptitude tester for the germanization of poles, especially in Łódž sievers to brandt, / / , quoted in lösch, rasse falkenburg am krössinsee" addressed the kwi-a in june , requesting that it make available "for preparatory works for deployment in the future reich commissariat in the caucausus […] data and material about population density, races, nations and religions in the caucasian and central russian areas, respectively sievers had expressed himself quite similarly in a letter to richard korherr (* ), the inspector for statistics at the reichsführer-ss office / / , quoted in lösch, rasse on the following ahnenerbe bdc, wi a- . cf. sievers to hirt, / / , barch on the "auschwitz-dahlem connection untersuchungen des vorderen unterkieferabschnittes review: lothar stengel von rutkowski, grundzüge der erbkunde und rassenpflege; idem., review: gerhard venzmer, erbmasse und krankheit; idem., review: gottfried pressler, untersuchungen über den einfluß der großstadt; idem., review: georg von knorre on the discussions about the naming of this disease in the third reich, cf. schmuhl, Ärzte in der westfälischen diakonissenanstalt sarepta verschuer and his staff had produced paternity opinions. verschuer, vaterschaftsgutachten, pp. f. cf. also idem there is no evidence of collaboration between mengele and kranz, however after the re-integration of the auschwitz ii camp into the main camp in november he became executive camp physician in the men's hospital area b ii f. kubica stellungnahme zu den angaben, die sich auf meine person beziehen und in der "neuen zeitung" nr. vom . . unter der rubrik "kunst und kultur in kürze" in der notiz "vertriebene wissenschaft" erschienen sind on this, the letter by wilhelm r. mann, the director of i.g. farben, to verschuer from the european standpoint the jewish question is not solved by the circumstance that jews emigrate from the racially thinking states to the other states only - % of the new members of the nsdap before were women. cf. falter, hitlers wähler so georg melchers ( - ) in an interview with müller-hill, tödliche wissenschaft esp. pp. ff also walter groß and the later "gypsy researcher extensive case histories and dissection protocols were sent to dahlem with the eye specimens for inspection. after processing they had to be returned, as case histories are the property of the hospital or the treating physician as a matter of principle ) gave the "race biology and anthropological institute in berlin-dahlem" as the address for dispatch, this could -if it is not simply a mistaken memory -have been because one of the possible addressees, wolfgang abel, was both director of the department for race science at the kwi-a and, since , director of the institute for race biology at the university of berlin. abel used letterhead with the address kumpania according to ernst klee, the mother of the hungarian composer györgy ligeti also performed such autopsies in that magnussen, in view of possible fluctuations in manifestation verschuer reports to fischer that he now had the technical assistent irmgard eisenlohr and that "with her the research with abderhalden's reaction, now finally picking up steam i have the substrates from the blood sera of over persons of various racial descent and also of pairs of twins and a few families ready, so that it is now possible to start the actual comparative studies müller-hill, tödliche wissenschaft spezifische eiweißkörper to perform abderhalden's protective ferment reaction in order to study the individual specificity of the serum proteins, i received blood samples sent from several hospitals, like those taken for most clinical investigations (wassermann's reaction, the erythrocyte sedimentation rate [esr]), around - ccm, without harming the health of the patient in any way. among these were also blood samples from the sick bay where that assistant from frankfurt worked in auschwitz / / , ibid. at this point in time diehl had around live rabbits at his disposal, although around young animals had died in the previous weeks spezifische eiweißkörper adolf butenandt. on the friendship between verschuer and butenandt cf. also sachse, adolf butenandt from mollison's work: mollison, serodiagnostik; idem., verwandtschaftsforschung; idem he believed that he had established quantitative differences in the abilities of "white serum" and "negro serum" to react with a certain "white serum-antiserum," which could be demonstrated using precipitation. fischer qualified his conclusion, however, adding that "before the potential perspective of a serological race diagnosis using such antisera can be considered at the same time he was also active in the "protection of the steyr homeland" (steirischer heimatschutz) and joined the austrian national socialists. in he found a position at the medical clinic of the university of graz, from early , however, only as an unpaid assistant. in he applied for a position as railway physician, but his application was denied due to his membership in the nsdap. his application as a panel doctor was not processed for the same reason personnel questionnaire on the request for a research stipend microscopic studies about the structure of the capillaries using infrared photography bericht über die von mir im januar begonnenen untersuchungen über die serologische verschiedenheit der menschlichen rassen loeffler to dfg, / / / , ibid. with von wettstein, butenandt, heisenberg silicate research] (i name only those with whom i actually spoke) we are in agreement that we must defend our institutes here gottschaldt, as mentioned above, sent the materials from the "twin camps" from dahlem to rottmannshagen castle near stavenhagen in mecklenburg, at the same time further research materials were sent to haus am see in beetz verschuer to fischer, / / , mpg archive on this celebration in detail: lösch, rasse rep. a (münster) the broken window panes could not be replaced by september ; the empty window frames were sealed with cardboard. verschuer to fischer, / / , ibid von der rassenhygiene zur humangenetik, p. , is presumably correct in viewing lenz's " 'vacation' " as a "move to withdraw from berlin rep. b rep. a (münster / / , mpg archive, dept. i, rep. a, ni. von der rassenhygiene zur humangenetik in bühler had submitted a postdoctoral thesis, but this had been rejected by the anatomist hermann stieve after fritz lenz had refused to head the examination committee. verschuer endeavored in vain to obtain a professorial qualification for bühler on the basis of the works he had published so far rep. b one of the institute's secretaries] i learned that many files remained here, which should, or must, be destroyed before falling into enemy hands. while i have not yet taken a look to see what and how much is concerned, i presume that miss jarofki knows this exactly. you did not speak about this with me, otherwise i would have advised that the things be taken to solz. in any case we may not choose too late a point in time for their destruction von der rassenhygiene zur humangenetik v. on verschuer's news that a trailer truck had been "made available" to the institute, a marginal comment reads v. here verschuer also claimed that he had been in contact von der rassenhygiene zur humangenetik no. . nachtsheim to verschuer, / / , quoted in kröner, von der rassenhygiene zur humangenetik / / , mpg archive, dept. i, rep. a, no. von der rassenhygiene zur humangenetik rep. a (münster von der rassenhygiene zur humangenetik at the end of the war abel withdrew to his estate at mondsee and dropped out of sight. heinrich schade was still a prisoner of war in yugoslavia. lenz initially remained in eastern westphalia, and -as the first of the "dahlem circle" -was appointed associate professor for human genetic theory at the university of göttingen in october . with this it appeared that the institute's "political baggage" had been swept under the carpet. verschuer indulged himself in the hope that he would be able to reestablish the kaiser wilhelm institute for anthropology, human heredity and eugenics, whereby of the former department chiefs he wanted to reappoint only his friend verschuer to an bürgermeister der gemeinde beetz, / / , mpg archive, dept. i, rep v. cf. also verschuer to generalverwaltung in the course of the change in leadership at the institute, michaelsen had been displaced from her position as executive secretary by nurse emmi nierhaus, had taken a long leave of absence for health reasons, and had found herself in fierce conflict with verschuer in december , who charged her publicly with kleptomania and forced her to resign from the institute (cf. michaelsen to generalverwaltung, / / , mpg archive, dept. i, rep. a, ). magnussen had sided with her friend (verschuer to fischer, / / ; fischer to verschuer that verschuer, and later, fischer as well, broke off contact with magnussen was clearly because of this "micha case" (cf., for instance, an undated postcard by fischer to verschuer until late magnussen was officially listed as an assistant at the institute (cf. notetat für das kwi-a für das rechnungsjahr / , mpg archive returned from captivity," was supposed to take magnussen's place from rep. a (münster), no. . extensively on this: kröner, von der rassenhygiene zur humangenetik his past caught up with him when the physicist robert havemann ( - ), who had spent the final years of the third reich as a political prisoner in the brandenburg penitentiary and been appointed by the city council of east berlin (magistrat) as the provisional director of the kaiser wilhem institutes remaining in berlin in , exposed verschuer's connections to national socialism and his state crimes in . although at times he was in danger of criminal prosecution and temporarily banned from professional activity kaiser wilhelm institute for applied anthropology" in dahlem in / . muckermann's application for admission to the max planck society was dragged out by the general administration. while the "research office for applied anthropoligy" received financial support from the max planck society, it did not receive the title of a max planck institute. the "institute for natural science and humanities anthropology" (institut für natur-und geisteswissenschaftliche anthropologie), as it was known from on, never developed noteworthy activities; it was dissolved without further ado in . in the end verschuer was appointed to the newly created chair for human genetics at the university of münster in , which long remained the only one of its kind. even though the dahlem institute fell apart after the end of the war: the "dahlem circle" of verschuer the case of the three heterochromous pairs of eyes from twins, which died more or less on the same day, must have been conspicuous and surprising. the very case that two twin siblings die "a natural death" on the same day and in the same place is a statistical rarity. moreover, twins with heterochromous eyes are extremely seldom. but the death of six twin children with heterochromous eyes on the very same day or in the very same week is well outside the bounds of statistical probability and clearly points to a crime. the above-mentioned publication of her research results about the "heredity and histology of a total heterochromia of the iris in humans" failed in late /early , because from the article it was clear -at least, according to testimony by georg melcher ( - ) of the kwi for biology and coeditor of the zeitschrift für induktive abstammungs-und vererbungslehre at the time, in an interview with benno müller-hill in the early s -that all subjects died at the same time and thus it stood to reason to suspect that they had fallen victim to a crime. in karin magnussen herself claimed that her essay did not appear because the printing plates, ready to go to press at the time, were destroyed in an air raid -this was probably much closer to the truth. then, after world war ii, magnussen's persistent attempts to place the manuscript after all -perhaps in the zeitschrift für the research has continued to enjoy intensive support. blood samples of over persons of various racial descent were processed and substrates of the blood plasma produced. the further research will be continued in collaboration with dr. hillmann [günther hillmann ( - ], a staff member of the kaiser wilhelm institute for biochemistry. dr. hillmann is a biochemical specialist for protein research. with his help abderhalden's original method has been perfected, so that now the actual experiments on the rabbits finally can be started. official visits" with werner fischer, by this time director of the serological department of the robert koch institute for infectious diseases in berlin, he had arranged to participate, under fischer's "guidance," in the control experiments fischer had declared necessary in his essay of . possibilities for this of which he never could have dreamed presented themselves in occupied france. horneck took blood samples from two "moroccans," one "annamese" and one "senegalese negro" from the ranks of the colonial troops held in war prison camps, and conducted serological investigations on these and other blood samples while on leave, assisted by a french laboratory technician in the serological-bacteriological laboratory of the hospice générale du havre. from the blood samples taken in the war prison camp, horneck produced "moroccan, annamese and negro sera." these he compared to various "european sera." over a period of two months, horneck injected five to seven intravenous injections of these sera in five rabbits, in order to immunize each of them against a specific serum. then he killed the animals, let them bleed to death and in this manner obtained a "precipitating antiserum" for each serum injected. a precipitin reaction was induced for each antiserum by combining them with all sera -i.e. with "white, annamese, senegalese negro, and moroccan serum." horneck arrived at the conclusion that the "white serum" reacted more weakly in the two cases portrayed in detail -both in the precipitation with "moroccan serum-antiserum" and with "white serum-antiserum" -and thus possessed less "precipitating antigens" than the other sera. this, horneck stated, could mean "that the differences present were not actually of race, i.e. based on the circumstance that whites, besides the antigen for the human species, also possess a white antigen, while the moroccan, negro, annamese also possess a moroccan, negro, or annamese antigen in addition to the antigen for the human species; rather, there may merely exist certain differences between whites and the other races in the amount of precipitatable antigens." the "determination of the protein content" and the determination of the composition of the protein were thus an imperative prerequisite "for a serological race diagnosis." at werner fischer's urging, for this research in horneck also began "to attempt the immunization of human to human in different races." in a later research report horneck mentions incidentally that these first immunization horneck, nachweis, p. . ibid., p. f. two rabbits who were treated with "senegalese negro serum" perished of peritoneal tuberculoses during this procedure. since there was not sufficent "senegal negro serum" available, horneck dispensed with immunizing the third animal, so that no antiserum was available for this serum. ibid., p. . ibid., pp. f. (original emphasis). it could be that other races possessed more easily precipitatable (lyophobic) serum protein (euglobulins), while europeans had more strongly lyophilic serum protein (pseudoglobulins, albumins). ibid., p. . ibid., p. . attempts, which had not produced any "usable results," were performed on himself. however, this account must be cast in doubt -horneck, who had worked as a general physician for years, after all, must have been aware of the great risks involved with such immunization experiments. it is highly improbable that he bore this risk himself."as a consequence of his […] deployment on the eastern front," in horneck was forced to discontinue his experiments for the time being. he published his results in a paper, which he submitted to the editorial board of the zeitschrift für menschliche vererbungs-und konstitutionslehre on april , , and was published in october of that year.even before this paper appeared in print, lothar loeffler submitted an application for research funding to the german research association, in order to allow horneck to continue his project. the objectives of future investigations were, according to loeffler, "absolute exclusion of individual differences, especially diseases, within one and the same race," as well as "determination of the protein factions of the antigens." independent of this, the "experiments about immunization from human to human" were to be continued. research on "negroes" was to be continued, "as initially only significantly different races come into question." the medical faculty of the university of königsberg, he stated further, soon will apply for a military exemption or "working leave" for horneck, which had good prospects for success. since the institute in königsberg was not equipped for such extensive examinations, and the required apparatus could not be procured during the war either, and because the race biology institute being set up in vienna (loeffler was just about to move from königsberg to vienna at the time) did not yet have a serological workplace, werner fischer expressed himself willing to grant horneck a temporary workplace at the robert koch institute.since , loeffler had first propelled ahead with his research in the field of radiation genetics. called upon by alfred kühn to take part in a joint project for the investigation of genetic damage through x-rays, in december he had requested a considerable sum from the emergency committee of german science for radiation genetics experiments on mice, which loeffler wanted to perform in collaboration with paula hertwig ( - ) of the kwi for biology and nikolaj timofféeff-ressovsky of the kwi for brain research. the emergency committee actually approved a credit of up to , rm for this project. de crinis ( - ) , a national socialist emigrated from austria whose curriculum vitae exhibited many a parallel to horneck's, and who had succeeded karl bonhoeffer as full professor for psychiatry and neurology at the charité hospital in berlin. loeffler bestowed particular urgency upon his application by following it with a letter to kurt blome ( - ), deputy director of the main office for national health at the nsdap, a liaison of the german research association for the subject area "population policy, care of genes and race," who was certain to be one of the people evaluating horneck's research plan. loeffler supplied a short synopsis of horneck's biography, summarizing that he had "proved his worth both politically and in the war." according to report, loeffler continued, horneck had been listed in third place for two pending appointments, and it was to be expected that he would move up to positions with more prospects in later appointments, so that, also in view of the "lack of truly good new blood," it was important to give horneck the opportunity to perform scientific work in the future. moreover, horneck was "almost the only race biologist performing serological work at this time." blome actually did send loeffler's letter immediately to the reich research council with a request for review. one month later -in november -the reich research council approved the application for a grant of , rm. in january horneck, who was working in the special colonial medicine military hospital in st. médard near bordeaux at this time, resumed his research. in his first preliminary report, horneck once again described the point of departure of his study: the purpose was to establish whether the varying intensity of precipitin reactions to human sera was influenced by individual factors, especially by diseases, in such a way that the race differences were blurred. therefore sera of both healthy and sick "negroes" as well as of whites -for the purpose of comparisonwere tested using the precipitin method (optimal precipitation), whereby the same blood groups were used in each test. "with the enormous material" horneck had "at his disposal an abundance of the most varied diseases, some of which hardly occur at all in our country (like leprosy the blood group to which each of the rabbits belonged was also taken into consideration. there were "as we know, rabbits -known as 'a rabbits,' who possess an anti-a factor. upon pretreatment with a serum, these rabbits give a much more strongly precipitating antiserum, and that is why this fact must be taken into consideration." karl horneck, bericht über die von mir im januar begonnenen untersuchungen über die serologische verschiedenheit der menschlichen rassen, n.d. (april ) , ibid. tuberculosis, typhus abdominalis and the worm infection filaria bancrofti. horneck summarized his preliminary results as follows:to the extent that anything at all can be said about them, these studies showed that individual differences do exist, but that they are expressed only in the time and intensity of flocculation. thus in such a manner that the serum of a certain subject, e.g., a typhus patient, flocculates earlier and more intensively than the serum of a healthy subject of the same race. however, in all experiments it could be confirmed that with regard to the concentration at which the best (optimal) flocculation occurs, fundamental differences exist between white serum and negro serum. in a later, brief interim report horneck portrayed this preliminary result as already proven and declared categorically: "differences in the optimal stage of flocculation may thus be based only on race differences." the studies to "determine the protein factions" had not been tackled yet in the first quarter of . on the other hand, horneck had resumed his experiments on immunization from human to human:this time i began the immunization experiments on several negroes with various blood groups. before the first injection, about - ccm blood was taken from each of the negroes, in order to obtain a serum of the species before treatment. then the negroes received a total of - cmm white serum in four intravenous injections. twenty-four hours after the final injection and one week after the last [sic, presumably must mean: first] injection, another - ccm blood was taken from the negroes. the sera of the pre-treated and those of the non-pre-treated were evaluated for their optimal precipitation and interesting differences were established in this anaylsis. a portion of these sera was filled into sterile test tubes and sent by courier to fischer in berlin, where control tests were to be undertaken. ernst rodenwaldt showed animated interest in the immunization experiments in particular. he visited horneck on location in the special colonial medicine military hospital -as mentioned, werner fischer had worked as rodenwaldt's assistant at the institute for experimental cancer research in heidelberg from to . in his later report horneck noted with disappointment that "extensive attempts at a direct immunization from human to human [proceeded] completely in vain." "the proof of an immunization can only be furnished indirectly via rabbits […] ." with the immunization experiments on war prisoners of color, horneck clearly transgressed the boundary to criminal human experiments, for hereby he not only disregarded his proband's right to self-determination -as in taking blood samples for the precipitin reaction, but he also subjected them to serious health risks. for with the injection of the foreign serum horneck assented to hazard the potential occurrence of allergic ibid. karl horneck, bericht über die arbeit "serologische differenzierung der menschlichen rassen," n.d., ibid. ibid. karl horneck, bericht über die arbeit "serologische differenzierung der menschlichen rassen," n.d., ibid.shock, of hemolysis (dissolution of the red blood cells), of intravascular clotting events and thromboembolism with consequent circulatory failure and death.apparently horneck's research came to a standstill when he was transferred from france to italy in . in november loeffler reported to the reich research council that horneck "has received a command from the wehrmacht, which now puts him in the position to continue his scientific work despite his continued military service status." horneck intended to travel to france in the near future to resume the interrupted research, loeffler continued. in february horneck applied for a further grant of , rm, which was approved in march. in october he informed the reich research council that he had completed a paper "about the possibility of a serological race differentiation" and sent it to fischer for appraisal -"with consideration of the fact that this paper contains many new aspects," fischer expressed the wish to talk through it with horneck personally before it went to print, a plan that was frustrated for the time being by the fact that horneck was denied special leave. the account ends abruptly at this point; the project must have run aground.horneck's and fischer's project is of fundamental importance with respect to verschuer's project for several reasons:first, it temporally preceded the "specific proteins" project. verschuer had dealt with the proteins of human serum back in his dissertation in and showed his lively interest in the possibilities of serological race diagnostics in the late weimar republic. in a short paper about "physiology and pathology in anthropology" of , verschuer had regretted that there was still no success in using the precipitin reaction to "establish with certainty protein differences between the human races." one must presume that he observed the developments in this field of research attentively. when he succeeded eugen fischer in , the race to develop a race test on a serological basis was in full swing -and the institute in dahlem had not left the starting blocks. engelhardt bühler's project begun in , on the heritability of the isoagglutinin content of human blood serum, which -as eugen fischer had implied to the german research association -also was to open up possibilities for a serological race test, had come to a complete standstill when bühler was called up to the wehrmacht at the beginning of world war ii. certainly it can be assumed that verschuer had taken notice of fischer's and horneck's work, and it can also be assumed that he knew about the series of experiments in progress at the sachsenhausen camp and in the special colonial medicine military hospital, perhaps from lothar loeffler, who was, after all, a member of the "dahlem circle," and -as portrayed elsewhere -probably remained in constant contact with the kwi-a because of the fingerprints and handprints from the key: cord- - n wroj authors: devinney, timothy; dowling, grahame title: what are the strategies of australia’s universities? arenas, vehicles, differentiators, staging and economic logic date: - - journal: the strategies of australia&#x ;s universities doi: . / - - - - _ sha: doc_id: cord_uid: n wroj because australia’s universities have multiple missions, they also have a broad scope of operations. notwithstanding this, there are a set of institutional mechanisms that ensure a large degree of sameness across the academic footprint of the universities. they lack fundamental differentiation. also, the cost structure of the universities requires some faculties (cash cows) to generate free cash flow to fund the research operations of other faculties. because university research is expensive, universities are trying to convince industry to fund more of this activity. however, universities have many obstacles to overcome in this endeavour. what are the strategies of australia's universities? arenas, vehicles, differentiators, staging and economic logic . arenas figure . earlier suggests that each of australia's universities is probably two enterprises-one for education and the other for research. each university differs somewhat according to the range of arenas (faculties) over which it practises these activities. for most universities their mix of faculties seems to be the result of circumstance as much as a grand design. in contrast, in the post-war years when stanford university started its rise to stardom, the decision was made to concentrate on postgraduate programmes (where national reputations were forged), and a relatively few areas where government funding signalled national importance-one of which was electronics research. the aim was to do a few things with distinction rather than 'creating excellent programs in fields no one cares about'. a strategic decision that is very costly for a university is its choice of arenas. as just noted, stanford chose some of these because the government of the day was prepared to underwrite much of the cost. if a university chooses to do world-class teaching and research in the natural and physical sciences, it can involve significant investments in infrastructure. in contrast, research in many fields of the humanities is far less costly. the number, variety and size of arenas define the scope and academic footprint of a university. from a strategic perspective, when the logic guiding the choice of arenas is not clear it • makes positioning the institution more complicated, • increases the costs of administration (due to complexity) and • complicates decisions about which areas to prioritise for funding. a criticism of australia's public universities is that their scope is too broad. to illustrate this point, consider the profiles in fig. . . each circle represents an academic faculty scaled to size. their relationship is depicted by their distance from each other. an issue we will raise in chap. is that each of australia's universities looks more like comprehensive university, one with a scattered academic footprint, than focused university where the logic of its collection of faculties is more apparent. and if the numerous research centres and institutes that a comprehensive university has were added to fig. . , the picture would look much more complex. the major vehicles used to codify education, such as the common range of degrees; deliver education, such as teaching philosophy, curriculum design, lectures, tutorials and web-based materials; assess learning, such as assignments and examinations; and do research, such as academic time and research equipment, are similar across all of australia's universities. thus from both a student and an academic perspective, 'going to university' means that they are joining a fairly well-defined type of institution and committing to a broadly similar experience. yes, each university has some unique aspects, but universities have a high degree of 'sameness'. the vietnamese have a saying that captures this situation: history and what is called isomorphism (imitation) create this sameness. this is the way that universities were organised in the past (history), and this is the way that a university's peers are currently organised, and so each tends to conform (isomorphism). also, the rankings are designed to reflect this structure. so to look like a university, this is the dominant design. the value of this structuring of operations is that it clearly positions all universities as 'a university'. stakeholders, both internal and external, are comfortable with this model. consultants as we noted earlier suggest that it should be shattered. it is interesting to delve deeper into the issue of sameness for two reasons. one is to expose the weakness of some consulting suggestions for change. at the present time many are simply infeasible. the other is to uncover the structural mechanisms that will impede any significant attempts to change. four institutional mechanisms cement the essential sameness of australia's universities. the first is the overwhelming reliance on government funding. it is anchored to the grand bargain mentioned earlier, which comes with strings attached, namely, to be public institutions committed to the three traditional missions of teaching, research and community engagement. no university has the courage to publicly jettison any of these missions. the second factor creating sameness is three powerful agencies. one is the tertiary education quality and standards agency (teqsa). stemming from the bradley review of australian higher education, this government regulator now imposes quality assurance mechanisms to set and review sector standards in the areas of teaching, learning, scholarship, research and research training. the second agency is the australian research council's excellence in research australia (era) programme. as noted earlier, this initiative produces an australian university league table that encourages a degree of sameness of research across the universities by recognising discipline-based journal ranking lists which identify the set of de facto target journals for much university research. these are then used in the individual performance appraisal of staff. thus, the requirements of these journals create the parameters for what is judged to be the type of research one should do and what quality means. the third agency is the national tertiary education union. this union negotiates the basic workplace agreements employed throughout the university system. while each university negotiates a different agreement with this powerful workforce union, they are very similar in structure and content. we would argue that these union agreements are designed more to protect the 'average' employee than to foster excellence across the system. one of their key conditions illustrates this opinion, namely, the duration of the paid working week. currently this is typically . hours per week. for an academic . hours is far too short! it is impossible to be an excellent teacher and world-class researcher and then do some university service, and then some industry and community engagement by working . hours per day. so here is the problem: • work . hours per week and do part of the job, or • work . hours per week and do all the job at average quality or • the university is deliberately underpaying its academic staff (and it should be taken to the fair work commission). when one of us first joined the australian university system, our terms and conditions of employment would easily fit a . hours per week. the employment contract was one page in length. it had three main paragraphs: . welcome to the university. . your level is xyz and your pay is $pqr pa. . if you have any questions please talk to your head of school. in essence, the deal was 'you pretend to work and we will pretend to pay you'. there were no formal university expectations (regular performance appraisals), and some academic staff treated their employment as a form of paid social welfare. and yes, mediocrity was prevalent. in contrast, the last union-negotiated employment contract for one of us was pages long. it was dense with terms and conditions of employment. the third factor cementing sameness is the rise of npm / professional management in universities. much of this is directed at responding to the requirements of the teqsa, the era and the legislation under which each university receives its charter. also, there now seems to be a need to manage an ever-growing number of risks within the university environment, such as oh&s, staff welfare, student welfare, student malpractice, research malpractice, cyber risk, data risk, ethical risk, operational risk, financial risk, regulatory risk, crisis risk, climate risk, reputation risk. professional risk consultants have targeted universities to help them assess their vulnerability, design programmes to manage risk and recover from risk failures. a rich and successful history plus these institutional mechanisms have combined to produce a higher-education sector with few meaningful differences. in commercial situations where there is an inherent sameness across members of a strategic group, such as the big australian banks, one often observes artificial attempts to differentiate one institution from another. corporate identity, branding and communication are often used for this purpose. the websites of the universities are revealing here. they are marketing devices. the first screen looks more like the package of a supermarket product than the front door to an organisation with a deep purpose. and from here they explode to reveal all the complexity of the institution's operations. at our last university there was a group of administrators tasked with managing the institution's marketing and communications, and a further responsible for brand strategy and engagement. as old marketers, we think that people are not necessary to tell students about the university, especially when research suggests that many students get most of their information via wom-word-ofmouth and word-of-mouse. the strategic narratives of most universities suggest that they are constantly making changes to their operations to exploit opportunities and overcome obvious problems. explore the website of any university and it is easy to find examples of new programmes being launched and new people being appointed to run such initiatives. there is no shortage of activity. for example, in the area of education each university seems to have adopted a different approach to learning. their websites reveal learning philosophies like blended learning, block learning, flipped learning, group-based learning, case-study learning, student-led learning, workbased learning, problem-based learning, active learning, active and inquiry-based learning, flipped classrooms, design thinking, moocs and so forth. long gone it seems is the era of 'chalk and talk'. in the area of research while most activity still occurs within faculties, these endeavours have been supplemented with cooperative research centres, university and industry partnerships, and cross or multi-disciplinary research centres. the easiest way to explore new areas of interest seems to be to bolt-on a new research centre. the university of western australia's annual report notes it has of these. from an npm perspective these are an interesting idea because they allow the university's administrators to measure efficiency and performance. it is clear what they cost to run, what independent money they attract and what they produce. and because of their insular nature, they can be closed down if performance does not meet expectations. another staging mechanism is the branch office. some are local, such as a regional university with an office in the near-by capital city, and some are located in a foreign country, such as dubai, hong kong, singapore, thailand, vietnam. many overseas universities have also followed this approach. all sorts of reasons are provided for why such endeavours are a good idea. however, many are not strategically sound. for example, some years ago both of us attended a meeting where the senior administration of the university suggested that a branch office of our school be opened in hong kong. the idea originated with an industry advisory group-'asia is booming, there are lots of students, why aren't you up there!' at the end of the meeting one old strategy professor (who was an exmanagement consultant) summed up the idea as follows: as a small institution with limited financial resources let's be a late entrant into a highly competitive market where it is not clear about the regulatory requirements for accreditation; where some local universities are very good, and where recent overseas entrants like us are not making any money. and, to succeed we will have to redeploy from our current programs some of our best resources in order to establish our quality. this comment was all it took to bring sanity back to the discussion. it was also a good example of the value of harsh academic scrutiny of the 'free advice' from people without deep knowledge of higher education and no skin in the game-the industry advisors were not willing to bankroll the venture. on a number of occasions we have been critical of the lack of a clear line-of-sight between key aspects of strategy and the financial management of the university. while we were at the university of new south wales, one of the vice chancellors (fred hilmer) made the effort to inform the senior academics about the revenues and costs of the university. at our last university this information appears in the annual business plan. also, each university publishes a set of accounts in its annual report. given that the annual report is a public document, we will use it to raise some important strategic issues in the next section. chapter noted that australia's universities are happy to argue that they are under-funded. so a key question is whether this is true? a second question is whether our universities waste some of the funds they receive? answers to both questions help to expose the business model and the profit and loss statement of a university. the university of south australia is one of the few universities to discuss some of these factors. for example, by their plan is to: • grow the student body to , , with % of these learning online. unisa online is the core asset that will support this (ad)venture. • generate % of their revenue from research activities, % of which will come from industry partnerships and % from research grants. now how will they grow student numbers and change the mix of research funding? presumably, the student body will grow via the introduction of online courses. hopefully it will not grow by lowering entry standards. so the first challenge is to make sure that these online courses do not cannibalise the revenue from their current courses. the second challenge is to do this without reducing quality. the third challenge is that they will have to compete against the online programmes of other universities in australia and overseas. on the research front the idea is-'by growing the scale and focus of research by building capacity in areas that have demonstrated excellence and shown potential for growth'. with a large budget this might be feasible, but do they have these funds? they don't say. on the teaching front what puts grand plans like this at risk is price discounting. for example, there is an emerging trend for universities to offer substantial tuition fee discounts, especially to attract international students. phil honeywood, president of the international education association of australia, said that many of these discounts were dressed up as scholarships. so, there is a downward pressure on fees-revenue. competitors are just as desperate as unisa to grow their numbers. on the research front unisa is exposed by its lack of reputation. a quick look at the era rankings doesn't put the university in the top echelon, and as noted elsewhere, many australian academics and their universities do not have a very good reputation for their commercial acumen. so while universities would like to generate more money from industry, many research academics simply don't care what industry wants. getting favourable recognition from peers is far more important than getting research money from industrialists that is targeted to specific projects, and some of which the university may tax. because the economic logic of a university can be partly deciphered from its annual accounts, this is where we turn to next. at the australian financial review higher education summit, anthony walker, a director of sovereign and international finance ratings at s&p global, said that there is a game being played by the government and the universities. because of budget pressures, the government wants to reduce its funding of universities while they want more funding, so the truth about what is an appropriate level of funding is likely to be somewhere in between. his analysis suggests that many universities have operating margins of between and % and quite healthy cash flows. yes, some universities might be in less than good financial health, but there is a moral hazard here-the government is highly unlikely to let one of them become insolvent. so, maybe the overall financial health of the universities is not as woeful as often claimed. at this summit philip clark, a former federal government advisor on education, makes another point about the funding of the universities, namely that relying on the high numbers of overseas students, many of whom come from china, is a risky strategy. there are two financial risks here. one is that if overseas student revenue is used to 'balance the books', a downturn in demand puts the university's short-term revenue at risk. this was dramatically illustrated during the covid- pandemic. the other is that if this revenue source is used to fund long-term investments in infrastructure or research, a downturn here puts these at risk. so, even though it is a luxury good in china to have an education from a groupof-eight university, if the chinese government or other circumstances mean that these students stop coming to australia, this revenue steam will stop very quickly. also, an adverse change in the exchange rate or more restrictive student visa requirements quickly reduces the flow of international students. strategists like to look at what they call the revenue model of a business enterprise. in the university context, the focus is on the principal sources of revenue. to illustrate this, we will look at the annual report of the university of western australia (uwa). here we see that uwa had $ million income for an underlying result (sustainable surplus) of $ million. approximately % of this revenue was sourced from the federal and western australian state governments. fees and charges were $ million ( %), other income was $ million ( %), research income was approximately $ million ( %) and interest was $ million ( %). in summary, uwa is a big enterprise that relies for about half of its income from government funding, and it generates about one-third of its income from student fees. hence, government relations, cost control and financial management are key skills. there is nothing surprising here. however, think about research. the reputation of the university is only strong enough to generate % of income from this source. this is the same amount generated by the investment committee of council. what these figures suggest is that the external demand for uwa research seems to be very small. a little further digging through the annual report reveals another interesting observation. the university had , students of whom , were fee-paying undergraduates, and , were fee-paying coursework postgraduates. these students are serviced by five faculties (one of which is very small). one of the super-faculties comprised the cheap-to-run faculties, namely, arts, business, law and education. this group educates % of the university's undergraduates, % of coursework postgraduates and % of research postgraduates. this pattern suggests that this faculty is the cash cow for the rest of the university. it is like a giant atm from which other parts of the university withdraw their weekly allowance. as a member of this super-faculty, you could feel proud-your efforts fund much of the rest of the university, or unappreciated-your efforts are unrecognised by others. getting the economic mix of this core funding cross-subsidy game correct can make or break the finances of a university. it can also damage the reputation of the university if in a faculty like education, student entry standards are lowered to fill up the numbers. at the time of writing, this was a topic of conversation in the media. when compared to the great universities in the usa, the piece of the revenue model that is largely missing from australia's universities is its endowment. while there are many reasons that might account for the paucity of australian university endowments, the stark fact is that these institutions do not have access to a significant amount of this revenue. much of the money given to universities comes with constraints, such as to be used for a specific piece of infrastructure or type of research. and even sponsored academic positions, such as named chairs, are rare. as government funding becomes more constrained, the need for this type of income becomes greater. imagine the opportunities that could be pursued by an australian university with a couple of billion dollars in its war chest! the cross-subsidy game all australian universities have a group of low-cost faculties and degreesthe cash cows that are used to fund the high-cost degrees-the stars. the star disciplines are generally the ones that underpin the major university reputation ranking systems. they are also the disciplines that tend to make the major scientific breakthroughs that get publicity on the nightly news and in television natural science documentaries. the deans of these disciplines are generally the most powerful academic voices inside the university. at uwa these are all sciences-science, health and medical sciences, engineering and mathematical sciences. notwithstanding the economic significance of the cash cow faculty to uwa finances, in the annual report, the exploits of the sciences are given much more prominence than law, business and education. cross-subsidies lie at the heart of university growth. if an institution decides to grow by increasing the size of its high-cost faculties, it will generally have to generate more revenue from its less costly faculties. this can be done by enrolling more students, raising tuition fees, reducing the delivery costs of teaching and research or clawing back more of the current revenue from these faculties. however, if the institution grows only the less costly faculties, it has free cash flow to spend as it pleases. thus, the economic modelling of these institutions is a critical component of strategy. this is seldom mentioned outside the executive suite. cross-subsidies, however, come at a price to reputation and social equity. consider the following paradox-or as ross gittins, economics editor of the sydney morning herald, calls it 'short-changing their students'. step is to attract students to cheap-to-provide occupational degrees-that have good job prospects like commerce, or a good image but fewer job prospects like law. step is to teach these undergraduates in large classes or substitute technology for face-to-face teaching. step is to agree to charge them a premium price. step is to use the free cash flow to pay the university administrators and cross-subsidise research and teaching in other areas. now the paradox here is that even though our universities wish to project themselves as model social citizens, an unemployed or underemployed student from a cash cow faculty soon discovers that they have been sold a degree that is not worth as much as they thought. just like most of their business counterparts, transparency is not generally one of a university's core values. in big business, and this is what our universities are, you keep your business model out of public view. the alternative to cross-subsidy is internal privatisation. for example, some us and european universities have allowed their business schools to retain their revenue and pay only a minimal contribution to the rest of the university. as david kirp describes for one us school, this allowed it to raise salaries and improve facilities while salaries were frozen and faculty needs were not met in other parts of the university. any faculty with strong student demand and/or demand for its research could advocate for such a strategy. so from a strategic perspective, should a university allow any or some or all its faculties to 'privatise'? you will not find this strategic question discussed in any public statement. however, the pressure to (partially) privatise can be seen in the internal budgeting practices of the university. when universities adopt performance as opposed to activity budgets, it encourages their units to become (more) self-supporting. chief financial officers argue that it brings more discipline to the budget process and greater internal transparency. academics in smaller faculties might argue that it diminishes the overall contribution of the university. vice chancellors need to make some tough strategic choices here. public universities by their nature have adopted an open, distributed model of research transfer through publishing research and educating people who then enter the workplace. while education provides revenue, publishing gives away much of the university's new knowledge to the publishing houses, which then sell it back to them (through subscriptions to their journals). to try to get some return from their research, some universities have entered the publishing game via their university press or by publishing a journal or two or three. if successful, both revenue and reputation flow back to the university. however, in australia the more likely outcome is a university press that publishes short production runs and seeks not to put too much drain on the parent institution's finances. the open model of research transfer puts a considerable strain on university finances. to help counter this, the universities now ask their academics to raise more of their own research funding. securing australian research council competitive research grants is highly prized, both for revenue and for reputation. however, this and other forms of funding come at a cost. the grant writing and peer-review process are very timeconsuming and highly problematic. they generate considerable amounts of stress for the people forced to seek these funds. and then when funds are secured, most academics need help navigating the university accounting, reporting and intellectual property policies. at a national press club address professor ian jacobs lamented about the underperforming state of business-university collaboration in australia. his model is that the entire research pipeline moves from discovery, to translation, to application, to commercialisation. our universities tend to focus on discovery, and industry on commercialisation. his strategy to help fill the gap was to ask government for more help in the form of things like better tax incentives for university-business projects, government co-investment in research projects and specific funding for translational research. the strategy here is to move down the value chain. along these lines, in the british commercialisation company ip group committed $ million to help some the group-of-eight universities commercialise their research. because this is such an important issue, it is worthwhile thinking about alternative models of research transfer and the roadblocks to such initiatives. the different initiatives respond to the different needs of organisations. here are some further ideas. one was noted earlier in our description of working in the australian graduate school of management. business (and engineering) schools are closer to industry than many other faculties on a university campus. in the business school where industry collaboration was easiest to achieve, and where it seemed to have most impact, was when we were invited into a company to educate its managers. this generally involved customising our mba course material to address a specific internal need. one crucial design element of many of these in-house courses was that the presenter would emphasise the role of evidence and research as inputs to management decisions. this was a good way to get research inside a company and to get decision-makers comfortable with using research in management practice. this is a good example of industry collaboration based on the perceived strength of the academic community (teaching). however, unlike the harvard business school we did not leverage this activity into writing case studies and selling them to other business schools. our performance appraisal kpis did not reward such 'clinical' activity. they do at harvard. a second approach is for the university to encourage and help its students to start their own enterprises, using what they have learned and discovered at university. this goes beyond offering courses in entrepreneurship. it requires a venture-capital approach to funding and mentoring new enterprise development. the idea here is for universities to formally invest in the new enterprises created by their students. the ip group has also put $ . million into australian university research start-ups. a third approach is for universities to promote to their researchers the benefits of working more closely with national laboratories (like the csiro), and government departments and agencies (like the australian institute of sport). there are hundreds of these that span many areas of interest to the academic community. they also have their own funding that will support various types of research projects. these alliances are generally perceived as 'safer' than the next option. a fourth approach is for the universities to develop strategic partnerships with key innovative companies. old companies like cisco, exxon, general electric, hoechst a.g., ibm, monsanto, rolls-royce and siemens have collaborated with universities for years, and new companies like amazon, facebook and google also work with universities. because many companies adopt an open innovation approach to new product development, the basic research done in universities extends the workbench of both parties. the challenge is to get both parties to work together. what will often impede the development of a close working relationship is different goals, expectations and rewards of the parties and, more recently, squabbles over intellectual property rights. a fifth approach is to start a new academic hub. for example, the australian national university has created its a innovation institute (autonomy, agency and assurance) to create new applied science focused on managing the artificial intelligence and big data revolution. many universities are 'playing' in this space with similar academic-industry collaborations. many are also trying to entice students to join these initiatives by offering specialist (master's) degrees. a sixth approach is to start or expand university consulting units. this type of work tends to focus more on problem solving than innovation. for example, the university of new south wales has a professional enterprise called unisearch that provides expert witness services. the university of south australia has the ehrenberg-bass institute for marketing science that collects money from companies to gather data on patterns of consumer behaviour, which are analysed to provide insights back to these companies. other universities do sponsored research for specific industrial companies. while this is not without controversy, especially in some fields like pharmacy, in other fields like market research, there are fewer pitfalls. where difficulties arise is when working with industry becomes transformative and some vocal staff claim that the institution's academic values may be compromised. then contracts are required to provide confidence that conflicts can be managed, and leadership is required to endorse the principle that externally funded research is permissible and desirable. a strategic advantage of building up industry engagement is that it often provides insight into future research opportunities. thus, engagement becomes a dynamic capability focused on sensing research opportunities and seizing other-peoples' money to explore these issues. in the usa one of the biggest sources of funding for such future-oriented research is the military. from a goal-setting and incentive point of view, industry engagement activities need to align with university and individual key performance indicators. three current metrics are funding, impact and engagement. so research that is of interest to industry can secure new funding. then to create impact, the university and its research teams need to add an advocacy element to their research. this can be done by a public relations effort to explain who needs to know about the research and what policy is necessary to implement the findings. finally, engagement can be facilitated by the research team 'consulting' with the parties who will use the research findings. they help organisations use the findings to solve their problems. research funding without specific advocacy and engagement often falls short of its promise. while all these initiatives could be helpful, they don't address some of the key reasons why too few academics work directly with industry and why many business enterprises are somewhat reluctant to work with academics and their universities. here are some roadblocks that need to be addressed before a university's goal of significantly greater industryuniversity collaboration is likely to be met: • we don't understand each other: business often find it difficult to identify if a university has any knowledge of interest to them. research academics have their own interests that may or may not have any practical implications. so the base condition for both parties is to politely ignore each other. • training and focus: doing a phd can be a brutal process that imprints a certain way and timescale for doing research. also, the motivation for doing this piece of research is often grounded in the existing scholarly literature rather than in the field of practice. so in many disciplines phd students follow the university mission outlined earlier by brian schmidt, namely, they want to add to the stock of knowledge rather than solve a problem that is troubling industry. • because approximately % of new phd graduates now enter industry, various programmes have been designed to help these graduates migrate to a non-university workforce. for example, the apr.intern programme focuses on getting students internships across all sectors of industry. what both employers and graduates are finding is that doctoral training provides many transferable skills that enhance workplace performance. however, many of these skills often need some reshaping before they 'work' in the workplace. • in academic performance appraisals, working with industry tends to rate behind research publications in the top-rated journals. also, few of these journals focus on industry problems. and excellent teaching evaluations are often not related to industry engagement. so if universities really want their academics to work more with industry partners, they might consider rewriting the contracts of some chosen academics. the harvard business school case noted earlier has solved this problem. • taxing industry funding: in order to pay for overheads, there are a number of ways in which universities can 'tax' research money from outside sources. hence, from the point of view of the patron, not all their money goes to the nominated research topic. this can detract from the perceived value of their contribution. • academic reputations: in australia many academics have a reputation for being not very commercially astute. they live and work 'in an ivory tower'. their research is 'just academic'. they are too slow. doing linkage research with an industry partner is too bureaucratic. and so the list goes on. so many australian academics are handicapped by their reputation and that of their parent university. many organisations would rather seek help from dedicated research institutions like the csiro or hire a freshly minted phd who has not been too 'corrupted' by the academic environment. in many cases what makes academic research so powerful is also what makes it less than timely in a commercial setting. good research especially big-bang breakthroughs, takes time and is plagued by missteps, things that industry can't easily accommodate. • track record: over many years we have seen repeated intellectual failures across many sciences. so if open research transfer is laudable but expensive, and industry collaboration is somewhat problematic, there is a lot of work to be done before industry will come rushing to seek help from australia's universities. yes, there will be ad hoc pockets of success, but universities need to take a realistic look at their strategic and financial goals for this activity. like every large organisation universities have a diverse cost base. however, a quick look at the university of western australia's (uwa) annual financial statements reveals that the biggest cost is people. about % of total expenditure is classified as 'employee-related expenses'. so the people in administrative roles outnumber the front-line academics ( . - . %). this situation is slightly more in favour of the administrators if we add some of the . % of professional staff in the academic units to the administration count. these percentages reveal direct administrative costs. but it gets worse. because nearly every academic will be involved in some form of faculty administration, then part of their salary should also be allocated to the total university administration cost. for example, in in the university of sydney there were more than faculty committees involved in administration. another interesting picture emerges if we look at non-salary costs. for example, in the non-salary budget was just under $ million. then add to this-student expenses of $ million, materials and supplies of $ million, repairs and maintenance of $ million, finance costs of $ million and other expenses of $ million and we get a total of $ million before a single class is taught or a single research paper published. this is a lot of money to run a medium-size university. most other australian universities look similar. multinational corporations and other large organisations spend millions of dollars and millions of hours of employee time each year on compliance. the belief is that rigorous compliance programmes reduce the motivation for employee wrongdoing. also, if an employee does something wrong, the regulator and the institution's insurance provider will note that at least the organisation actively tried to avoid such a problem. the aims of these programmes are (a) to get employees to behave ethically and adhere to best practice, and (b) to eliminate the time-consuming and distracting regulatory and legal processes that accompany regulatory failure. but as is all too evident, these programmes don't always stop employee wrongdoing. an aggressive organisational culture and financial incentives will often drown out compliance training, especially when meeting stretch goals determine salary and promotion. for example, there are still periodic cases of administration (admissions scandals), academic (falsifying research) and student (cheating) misconduct. all universities require that their staff do compliance training. for an administrator this is part of what they are paid to do. however, for a student or an academic it is not clear what compliance training crowds out of the daily agenda. for example, as most academics will acknowledge, teaching crowds out research and service. anything to do with delivering or administering a course will take priority over other activities, even sometimes one's family. however, what comes next in the priority list depends on who asks for help. in the latter part of our academic careers, we have seen the people in central administration jump up the priority list, not by asking for help, but by demanding compliance. for example, this occurs when say a new teaching or reporting protocol is launched and everybody has to make changes to what they were doing previously. another example of a compliance cost was noted earlier. recall that the university of technology sydney requires training for all staff on issues such as equal opportunity, mental health awareness, privacy essentials, communication and collaboration, consent (unwanted sexual behaviour), health and safety, legal affairs, preventing bullying and safety and well-being. at the time of writing, the consent training was being pushed throughout the university-for all staff and all students. it was mandatory for everybody. the online course took approximately one hour to complete and required a % pass on the quiz that followed the training module. students were threatened with the withholding of their grades if they did not successfully complete the course; casual academics were offered one hours' worth of salary for their participation, and old professors were sent escalating emails (first by the hr system, then the head of school, then the dean of the business school, then the vice chancellor) when the system reported that they had not completed the course. leaving aside the legal and moral issues of such a request, the costs of this programme can be estimated as follows. from the university website we see that there were now if we take an hourly rate of $ , we see that this exercise has cost the university more than $ , for staff. and at, say, a notional $ per hour for the students, it also has a cost of $ , for the students (minus one hour of their study time). so here is an administrative initiative with about a $ million price tag. all this to address a social issue that the senior administrators in the university thought would signal the institution's commitment to social justice. to cover themselves, they sought legal advice before embarking on this course of action. there is an important ethical issue here. imagine the cost to a finalyear student who can't get a job because there is an embargo on their grades because they did not complete and pass a non-curriculum course on a social issue for an organisation that they are about to leave. second, there is no research that supports the claim that sexual harassment training actually works. so here we see the university imposing a dead-weight economic cost on the institution and a dead-weight social cost on some of its members. another largely hidden cost in our universities is due to quality assurance programmes. they are used to signal institutional quality. for example, there are a number of organisations that have developed best practice protocols for education. these organisations promote their programmes, and once a couple of institutions 'sign on', they chase other similar universities to 'catch up'. many university faculties sign onto such programmes with little evidence that they work and without due consideration for the total compliance costs. for example, the business schools of many of australia's universities have signed up to various accreditation programmes. the university of new south wales business school advertises its membership of two such accrediting bodies, namely, the aacsb (association to advance collegiate schools of business) and equis (european foundation for management development). the school pays to join, pays to be certified and pays to be recertified every few years. inside the business school, degree programmes need to be (re)shaped to fulfil the accrediting scheme's standards and someone is tasked with checking compliance. then information has to be collected to report compliance each period. the costs easily accumulate into the hundreds of thousands of dollars for each such certification. however, there is little empirical evidence to suggest that these sorts of accreditation lead to more effective or efficient education, that they attract more students or that they allow the institution to charge higher course fees-especially when all competitors have signed onto the same programmes. however, what these programmes do is to lead to a lack of differentiation across the schools who sign on. all of australia's universities are plagued with hidden compliance costs. the key question is whether they deliver noticeably better outcomes. one of the great paradoxes of modern times is that as information technology has advanced in leaps and bounds, it is much harder to find if these advances have had a general effect on increasing productivity. within the university context, the productivity gains from new technology have been patchy. many gains are the result of faster communication between people rather than better communication. but as the overuse of social media has demonstrated, new or upgraded information technology doesn't necessarily lead to better outcomes. on a university-wide basis, many of the changes in technology are introduced because the suppliers of a platform have upgraded their offer. so the university 'keeps up to date' by adopting the latest version. however, the benefits of these upgrades are seldom realistically estimated relative to the compliance costs of requiring thousands of staff, and students make changes to accommodate the new system. hence, an upgrade usually causes some pockets of frustration and costly time in compliance, especially for the people who are not as it proficient as their colleagues. universities could save hundreds of thousands of dollars in cost if they had more staff and student input into what really needs to be upgraded as opposed to being 'led' by their suppliers. is the australian taxpayer getting good value for money from its public universities? if a typical university like the university of western australia costs hundreds of millions of dollars annually before it produces a single key output, is this money well spent? and if not, where might cost savings be found? these are difficult questions. and because universities know more about universities than governments or the media, the universities are best placed to provide the answers. our analysis suggests that the problem is of two parts, namely, that the strategies of the institutions are underdeveloped and thus complexity and coordination costs accumulate. the other related cost is that the institutions become overstaffed and too bureaucratic. so one strategy for the government is to tighten up its funding and make each university find its own solution. universities would do well to avoid this brute-force solution by seeking greater efficiency and effectiveness in their operations. . towards the end of a report to the council revealed that many students found that the course was inappropriate to their concerns and that many had yet to complete it. so teaching staff were asked to remind the students that their grades would be withheld! . for example, on the unsw website you can't find what the letters aacsb actually stand for. you have to go to the association website and then search beyond the first page to find what they stand for. so how can these five letters really affect the decision-making of a new student if they are essentially meaningless? campus leadership and the entrepreneurial university: a dynamic capabilities perspective discounts catch on as international lure unis banking upside to defy funding cuts and lift margins', australian financial review we've turned our unis into aimless, money-grubbing exploiters of students for example, while a degree in medicine costs much more than a degree in commerce (at the time of writing down payment made on $ m start-up bet how to create productive partnerships with universities developing successful strategic partnerships with universities advancing australia's knowledge economy: who are the top phd employers? a note to the annual financial statements indicates that this group's salary is slightly less than that of the academics improving our organizational design some people might want to add $ million of asset depreciation, amortisation and impairment to this figure at this time, the pay rate for the casual staff was either $ . or $ . per hour (depending on their qualification) while sexual harassment training is the most traditional approach to preventing sexual harassment, it has not been shown to do so key: cord- -opuwyaiv authors: amram, denise title: building up the “accountable ulysses” model. the impact of gdpr and national implementations, ethics, and health-data research: comparative remarks date: - - journal: computer law & security review doi: . /j.clsr. . sha: doc_id: cord_uid: opuwyaiv abstract the paper illustrates obligations emerging under articles and of the eu reg. / (general data protection regulation, hereinafter “gdpr”) within the health-related data processing for research purposes. furthermore, through a comparative analysis of the national implementations of the gdpr on the topic, the paper highlights few practical issues that the researcher might deal with while accomplishing the gdpr obligations and the other ethical requirements. the result of the analyses allows to build up a model to achieve an acceptable standard of accountability in health-related data research. the legal remarks are framed within the myth of ulysses. ulysses, according to the homer's epic poem the odyssey, was king of ithaca, son of laertes and anticleia, and father of telemachus. ulysses was described as a man of outstanding intelligence, wisdom, and endurance. ulysses is cunning as he is able to overcome insurmountable obstacles to shape reality. dante, the latter admired his skills and competence, courage and smartness. in the nineteenth century, ulysses became the intellectual hero, who is far away from the current society, looking for a safe harbor but always in trouble. in twentieth century, ulysses is the modern hero bringing the anxieties and sufferings to find the true sense of things. nowadays, the ulysses . has been interpreted as a human person in his complexity of skills and feelings. the fils rouge in the several interpretations of the myth is how ulysses faces new challenges during his journey, combining his technical and organizational skills in order to deal with (and sometimes overcome) the vulnerabilities and limits of human beings. this model is particularly relevant today that scientific efforts are addressed to facing covid- pandemia through new technological solutions aimed at both supporting the early-detection and treatment of the disease as well as at managing its social and economics consequences in light of a needed balance between fundalmental rights. without pretending to contributing to literature, the idea of ulysses as a researcher, who develops technical skills and ethical competence to properly achieve his scientific goals, appears suitable in order to build up a model of ethical-legal compliance within research and development activities in light of the gdpr principle of accountability. in the following paragraphs, we will discuss the ethicallegal obligations that the researchers have to deal with while processing personal data, and in particular, health data, during their journey (i.e. the life cycle of the research), highlighting through a comparative analysis the critical profiles emerging from the current legal framework. after the gdpr entered into application, a strong debate arose in light of the impact that new regulation had on research. the balance between the protection of fundamental rights and the free circulation of data makes the researchers responsible of a series of obligations for the whole duration of the research lifecycle. this could be seen as an obstacle, at least in terms of time and resource allocation. the need to adapt current practices to the new paradigm of privacy by design and privacy by default approach with respect to the whole research architecture includes the necessity to deal with the adoption of proper technical and organizational measures. however, they cannot be standardized for every project, as they should be appropriate to the specific activity and they can be replaced during the developing of the research, considering the possible introduction of new risks or new technologies to mitigate it. according to the principle of accountability, in fact, the researcher has the burden to prove the implementation of the mentioned measures not only because the project proposal has to satisfy a given check list of conditions, but as the research methodology itself has to be ethical-legal compliant by design. therefore, the first skill of our ulysses . . is the openmindness to consider the ethical-legal compliance as a necessary step despite of the given area of research and regardless (as well as beyond) the existence of a given check-list section in the project proposal template. in the daily routine, these profiles might constitute a new combination of procedures, contacts, administrative activities which take time and should be supported by the research institutions. the great challenge that the gdpr launched is to create the opportunity of sharing ideas, models, and options in a continuous interdisciplinary dialogue aimed at addressing research and innovation towards the eu values and fundamental rights. to protect dignity and fundamental rights is the compass to improve society and enhance its values: science serves human beings, not viceversa , despite the technological progress goes faster than legal positivism and it has already put (personal as well as non-personal) data analysis as the first step of the research. this is true in the case of research dealing with health data, since the information connected to such a processing make the data subjects particularly vulnerable. the gdpr has standardized the legislation at the european level, but at the same time it allowed member states to nationally specify conditions and requirement in the context of data processing for scientific and statistical research purposes. this might create an obstacle while the activities (and therefore the processing of personal data related to them) are frequently conducted by partnerships belonging to several nationalities. from this viewpoint, it is certainly useful to compare different national interpretations in order to compare the implemented/proposed national models to find out possible best practices which may address the discussion towards a specific code of conduct. for this reason, considering the new ethical-legal issues emerging from the scientific-technological progress that involves a daily use of health-related data, our comparative analysis will firstly discuss the legal bases for health data processing for research purposes in order to identify the critical profiles as well possible practical solutions that might help ulysses . . to develop the "accountability" virtue. health-related data are those information which are relevant for health conditions, i.e. reproductive outcomes, causes of death, and also the quality of life. for this reason, they are included in the list of personal data considered as sensitive under article gdpr. this article regulates the legal bases to process data belonging to the so-called "particular categories". first of all, to allocate the data subject "responsibility" of the whole data processing, national implementations prefer to asking for the consent to data processing, even if other legal bases might be applied. this tension reflects a sort of confusion between the informed consent for volunteers who participate to a research experiment and the information about personal data processing as well. the involvement of human being in a research, both for clinical and not clinical trials, in fact, requires to ask for their consent, therefore the overlap of the procedure is quite common. however, instead of asking for consent for data processing under article sub a) or , para , sub a) gdpr, data might be processed in the public interest of the controller under article sub e) and art. , para , sub i) or j) gdpr) or pursuing a legitimate interest (article sub f) and art. , para , sub j) gdpr), in light of article gdpr, which regulates the data processing for archiving purposes in the public interest, scientific or historical research purposes or statistical purposes. according to the gdpr system, in fact, the data subject's consent could be seen as a residual legal basis, considering that transparency and awareness are in any case achieved by the information under article ff. gdpr, which are due regardless the legal basis of the data processing. moreover, to collect consent does not mean only to sign a form, but it means to be responsible that the obtained consent is informed, relating to a specific purpose, unambiguous, freely given, as stated by article gdpr and explained in the recitals ff. the right to revoke it should be always guaranteed. this might constitute an issue, if we consider that the favor for data processing for research purposes, and the presumption of conformity of re-using for research purposes under article sub b) and e) gdpr are strictly connected to the article gdpr paradigm, which recalls the necessity to implement appropriate safeguards that the researcher has to adopt to minimize any risks. some critical profiles emerge from article , para , gdpr which allows member states to decide whether or not maintaining the legal bases provided by the eu regulation or introducing further conditions, including limitations, with regard to the processing of particularly sensitive data, like the genetic data, the biometric ones, or those concerning health. this reduces the room of harmonization of the legal basis to process health-related data, also for research purposes. in fact, article gdpr offers the opportunity to member states to provide derogation to data subjects' rights while processing personal data under the proper safeguards (such as the pseudonymization and anonymization). at this regard, the spanish ley orgánica de protección de datos personales states that limits to the exercise of data subjects' rights are valid only if addressed to the researchers that process anonymized or pseudonymized data. in light of this provision, some systems have introduced new rules or updated the previous legal framework, identifying specific conditions and requirements applicable also in case of health-related data processing for research purposes. the topic has been highlighted in the last weeks while na- tional legislators have been introducted specific provisions to deal with the covid- emergency. for example, the irish data protection act , which replaced the previous data protection acts in light of the gdpr, states under the article that the suitable and specific measures for processing sensitive data also for research purposes should be regulated in a further specific act: the health research regulation (hereinafter "hrr"). according to the hrr, the data controller who is processing or further processing personal data for the purposes of health research shall ensure that the collection of an explicit consent from the data subject, before starting the health research. the consent could be obtained "either in relation to a particular area or more generally in that area or a related area of health research, or part thereof ". the favor for the application of article , para , sub j) is anyway recovered when there is a public interest in the research as declared by a specific committee appointed by the health ministry. in that case, the data protection impact assessment under article gdpr should be performed together with the positive approval from the ethics committee. likewise, the italian ethics rules adopted by the italian data protection authority to process personal data for scientific research and statistical purposes, namely regole deontologiche per trattamenti a fini statistici o di ricerca scientifica pubblicate ai sensi dell'art. , comma , del d.lgs. agosto , n. , refer to the consent as a legal basis to process sensitive data under article gdpr. for medical, biomedical, epidemiological research, article of the above-mentioned ethics rules states that data subjects/patients should be able to distinguish through proper information data flows for healthcare purposes and data flows for research purposes, but consent seems to be maintained as the principal legal basis to process data. this might be misled if it is not compared with article of the privacy code legislative decree n. / , as amended by the legislative decree / , a higher ranking rule. it states, in fact, that consent it is not necessary if the legal basis is article , para , sub j) and a data protection impact assessment has been performed and published. the provision is quite cryptic as it is not evident which are during the publication of this paper, the scientific community focused on the ethical and legal issues emerging from the covid- emergency management. in particular, the necessity to balance individual and collective health protection and personal data protection stimulated an interesting and inditersciplinary debate, that we cannot avoid. the cases where article , para , sub j) is not applicable and it does not explain how the data protection impact assessment should be published to fulfill the requirements. furthermore, according to the mentioned article, the consent is not required whether there is a positive approval from an ethical committee and a prior consultation before the data protection authority has been performed. also this exception may create some practical issues if we consider that data protection is an ethical profile that an ethical committee should face in its opinion. the relationship between data protection compliance and ethical compliance is, again, recalled within the article of the ethics rules. its para , indeed, states that the informed consent under the oviedo convention and helsinki declaration shall include information about incidental findings, while in the previous paragraphs the topic was the legal basis for data processing. this combination of provisions on privacy information and informed consent misleads the gdpr paradigm which promotes data circulation, under the principles of data protection by design and by default, instead of requiring the data subject's consent. another group of legal systems, instead, did not introduce the consent as a further condition under article , para , gdpr, but established additional measures to allow health data (as well as genetic or biometric ones) processing. this is the case of the belgian loi relative à la protection des personnes physiques à l'égard des traitements de données à caractère personnel , which for example provides specific confidential obligations for those who processes, at any title, sensitive data. research purposes conditions are included in article ff. of the mentioned act. further organizational measures, like the necessity to appoint a data protection officer (dpo) even if not required for the data controller according to the article gdpr, are stated in case the data protection impact assessment considers a high risk. the data processing record under article gdpr must include (i) the reasons that justifies the public interest in pursuing the research or in further processing data and how the possible lack of information might be justified by the anonymization (the pseudonymization or the reasons to avoid it) or the risk to compromise the research, and (ii) the agreement between who firstly collected data and the further processing actors. the belgian approach addresses the debate on the role of the legal basis compared to a series of other ethical-legal requirements while processing sensitive data for research purposes. indeed, pursuing the data protection by design and by default in a project means to build up a complex system of checks and balance, through organizational and technical measures discussed between the several expertise involved in the research. their implementation ensures that the research output will be aligned with the eu values and fundamental rights. loi relative à la protection des personnes physiques à l'égard des traitements de données à caractère personnel , . . , https://www.ipnews.be/wp-content/uploads/ / / -loi-belge-adaptant-réglementation-belge-au-rgpd. pdf. according to the above-discussed system, the data controller (i.e. the university/research institute in person of the legal representative) shall involve the principal investigator in the data management activities, authorizing to data processing under article gdpr, in order to proactively guarantee the adoption of those technical and organizational measures aimed at safeguarding the rights and freedoms of data subjects in her project. this first organizational measure that a research institute has to apply is to appoint a role in the privacy orgchart to the principal investigator of each research. at the same time, this helps to sensitize, trains, makes each ulysses . responsible of a data protection by-design research, and let the data controller achieve the compliance with the principles of correctness, transparency, and minimization under article gdpr. however, this might not be sufficient since the principal investigator might not have an ethical-legal background able to identify those proper safeguards that would make her research compliant with the current legal framework. at this regard, a principal investigator non-gdpr expert may consider a double level of ethical-legal experts' involvement. the first level concerns the data protection officer appointed by the data controller under articles ff., who -in light of the principle of proximity -shall be able to deal with research issues and be a key-person between the principal investigator, the data controller, the ethics committee, the it services, and the data protection authority. this supposes a strong collaboration with the administrative staff that supports the research. the second level refers to the increasing role played by an ethical-legal unit as a partner of the developed project. its task is to help the principal investigator to design and implement the research in compliance with the whole ethical-legal framework during the entire duration of the project. considering the ethical-legal challenges emerging from the current legal-ethical framework, to address an impact assessment on the basis of the risk for the shared values and fundamental rights could enhance the given research not only in terms of innovation, but also for the consequences on the society, economy etc. the involvement both of a dpo and an ethical-legal unit could make the difference in terms of achieving the goal of an ethical-legal compliant research by design and by default in a given system. in fact, it strengthens the interdisciplinary dialogue and helps the cross-fertilization between different domains. according to the current national implementations of the gdpr, in fact, many systems, like the above-illustrated belgian one have introduced check-lists and protocols to properly address the data protection compliance activities. for example, in the spanish system, firstly, an impact assessment must be carried out; secondly, the research must be subjected to quality standards according to the international directives and clinical best practices; thirdly, it is necessary to implement tools aimed at avoiding the re-identification of the data subjects; finally, the spanish law requires the ap-pointment of a legal representative in the european union under the article eu reg. / and gdpr in case of extra-eu partnerships. the provision sub g), indeed, states that in case the ethical committee cannot express an approval, the principal investigator may ask the data protection officer's one. however, it specifies that ethical committees should add specific competence on data protection by one year. from this perspective, as far as health data are concerned, data protection compliance walks together with the ethical one. therefore, the ethical committees should empower their competence on data protection and the data protection officer shall be able to address the researcher to an ethical-legal unit or legal-ethical advisor. the proactive risk-based approach which has been implemented by the gdpr for data processing could be potentially applicable to all the ethical issues emerging in the research. to this end, the already mentioned irish hrr takes the opportunity to deal with the ethical profiles related to research (e.g. informed consent, voluntary research, cost-benefits assessment with respect to the clinical trial, conflict of interests etc.) answering the need to develop a coherent paradigm to properly process health data for research purposes. the hrr firstly provides a definition for "health research", listing scientific areas for the purpose of human health, including: "(i) research with the goal of understanding normal and abnormal functioning, at molecular, cellular, organ system and whole body levels; (ii) research that is specifically concerned with innovative strategies, devices, products or services for the diagnosis, treatment or prevention of human disease or injury; (iii) research with the goal of improving the diagnosis and treatment (including the rehabilitation and palliation) of human disease and injury and of improving the health and quality of life of individuals; (iv) research with the goal of improving the efficiency and effectiveness of health professionals and the health care system; (v) research with the goal of improving the health of the population as a whole or any part of the population through a better under-standing of the ways in which social, cultural, environmental, occupational and economic factors determine health status ". then the hrr recalls the ethical issues to be assessed by the ethical committees for approval: data protection is one of them, but it finds a specific development in article and ff. article states that personal data can be processed for research purposes as long as it is necessary to achieve the research purposes and it does not cause any damage or distress to the data subject and if the organizational measures stated sub b) are in place. the check-list that follows is a sort of data protection plan, which includes the ethical approval by an ethical committee, the identification of the privacy governance structure (including joint controllers, data processors, and recipients), the training programme for those who are involved in the research, the data protection impact assessment for higher processing risks. the italian ethics rules issued by the data protection authority, instead, identify the field of application under a subjective requirement: "to all the data processing carried out for statistical and scientific purposes -in accordance with the methodological standards of the relevant disciplinary sector -which are held by universities, other research institutions and research institutes and scientific societies, as well as researchers operating within the scope of said universities, institutions, research institutes and members of these scientific societies ". to this end, marketing purposes hidden under "research or statistical" purposes are excluded since private companies are included only if in their company bylaws research activities are mentioned. pseudonymization, anonymization, and data re-using for research purposes according to the article gdpr data processing for archiving purposes in the public interest, scientific or historical research purposes or statistical purposes, shall be subject to appropriate safeguards. the same article refers to pseudonymization as the first measure aimed at achieving the minimization purposes. for further processing, it states that a further level "which does not permit or no longer permits the identification of data subjects" should be gained. technically, it does not exist a unique criterion of anonymization. data can be considered anonymized having regard to any methods reasonably likely to be used by the data controller (or any third party) to reverse the process and allow the re-linking of the data subject. the assessment is based on the risk of re-identification through a rational effort. therefore, the pseudonymization standard could be always obtained through technical separation of information, considering several levels (e.g. scrambling, encryption, masking, tokenization, data blurring, etc.) while the anonymization could be achieved by the combination of technical and organizational measures as well, considering the features of the data controller. some national implementations of the article gdpr focused on this profile. the belgian law establishes, in case of health data processed for research purposes, that pseudonymization could not be performed by the data controller, but by an independent body, who is subject to specific confidentiality obligations (i.e. professional secrecy). at this regard, the spanish law requires a "technical separation" between who performs the two activities and an explicit obligation for the ones who pseudonymized to avoid re-identification. these provisions arise two issues. firstly, when the data controller is a research hospital, healthcare purposes and scientific ones might be performed at the same time by the same team. in this context, despite of the application of pseudonymization techniques, clinicians might be always able to recognize and refer to their patients, even if committed to professional secrecy. secondly, to always identify a partner or team for pseudonymization could constitute an expense for the research: perhaps it could be sufficient to appoint such a task to it services of the data controller and establishing granular accessibility to the token (e.g. only the principal investigator, but not the research team). the article bis of the italian privacy code refers to the reusing of data by third parties. first condition is that data subjects must be informed. otherwise a prior authorization from the data protection authority is needed. this approach is not applicable whereas personal data are collected for healthcare purposes and used for research ones by the same research clinics, considering the functional link between the two purposes. the provision seems to refer to patients' personal data before being pseudonymized or anonymized for research purposes, as stated under article gdpr. another profile of the gdpr compliance consists of the system security: data flows are usually in a digital format, therefore proper measures shall be implemented to guarantee the availability, integrity and confidentiality of data. as far as the security of data is concerned, the irish act, for example, refers to the criterion of the "effectiveness" of the adopted measures. so far, the interdisciplinary dialogue touches the fields of the data management, including ip rights. if we consider that open access is becoming the new standard to manage research data, the role of the data protection officer/expert/advisor becomes essential to establish which data can be shared or not. therefore, skills required to ulysses . and his crew become everyday more specialized ones. in the context of the covid- pandemia, for example, during the so called lockdown, governments opted for establishing interdisciplinary task forces aimed at identifying effective, ethical-legal, suistanable solutions to plan a safe re-starting of the activities. this strategy appears in line with the ulysses . model. as shown in the previous paragraphs, the "accountable ulysses" is a standard which might be achieved only estab-lishing an interdisciplinary dialogue between the researcher in different fields. starting from this principle, some common features can be identified to reach an acceptable level of compliance. i) whereas health data are processed, to involve ethicallegal experts, who could play either an institutional role (as the data protection officer) or being a partner of the research, since the beginning of the project proposal is an added value to design an ethical-legal compliant ecosystem. ulysses cannot avoid from including an interdisciplinary support in her crew. ii) research purposes are functional to empower human dignity and values. considering the strong impact on individuals as well as on groups, as the research could identify new vulnerabilities, or classify individuals (as more exposed to a given risk), ulysses processing health data shall adopt suitable and effective technical and organizational measures to ensuring the ethical-legal compliance, in order to avoid possible misuse or dual use. iii) the it infrastructure and data management strategy should be designed in order to guarantee the availability, confidentiality, integrity of data. iv) if ulysses is also a physician, the combination of data processed for healthcare and scientific research should be clearly distinguished and therefore following possible different data protection plans: risks, technical protocols, access, time retention, level of pseudonymization might be different. v) data flows should be regulated between partners as well as within the given research teams. vi) data flows should be recorded and described in the information given to data subjects. vii) data protection is one of the several ethical issues that might arise from a research. the coordination between different legal constrains, protocols, and requirements should be analyzed in terms of risk assessment and monitored during the whole life-cycle of the research. the gdpr introduced a new proactive approach to dataintensive research. its handling supposes the cross fertilization between different domains, where the legal one plays the role to establish boundaries between lawful and unlawful, contributing to identifying possible tensions under the ethical framework. in order to sensitize ulysses to this new approach, which necessarily includes the allocation of time and resources, a coherent ethical-legal support to the core-research should be promoted by the research institutes. in this perspective, ulysses does not represent only the principal investigator of a given research, but the university/research institute per se , which as the data controller, should firstly train the research staff and the administrative staff to ethical-legal compliance, inform on duties and responsibilities, organizing and introducing specific support. in other terms, be accountable both within the technical and the organizational activities. the anonymisation of research data-a pyric victory for privacy that should not be pushed too hard by the eu data protection framework? passenger name records, data mining & data protection: the need for strong safeguards, report for the council of europe consultative committee on data protection therefore, ulysses . is the one who embraces a new way of working, as it has been stressed during these last weeks within the analysis of the solution to combat open to assess together with the technical specifications the ethical-legal consequences not only in order to mitigate the eu commission, joint european roadmap towards lifting covid- containment measures, https://ec.europa.eu/info/sites/ info/files/communication _ -_ a _ european _ roadmap _ to _ lifting _ coronavirus _ containment _ measures _ .pdf. risk to compromise fundamental rights, but to empower human dignity as main core of her research. i hereby declare that there is no conflict of interests in publishing this paper. key: cord- -raznn k authors: nelson, joni d.; marshall, julie; kelly, abigail; vuthiganon, jompobe title: dental student research mentorship in the era of covid‐ date: - - journal: j dent educ doi: . /jdd. sha: doc_id: cord_uid: raznn k nan in response to coronavirus disease (covid- ), the medical university of south carolina (musc) college of dental medicine has modified clinical, research, and educational operations. this included adapting the predoctoral dental research course that consists of in-person class sessions and -on- mentoring meetings. during this course, third-year dental students are required to complete an independent research project supervised by a faculty mentor; these projects are presented as part of a studentfocused research symposium. - beginning march , , evolving safety precautions, such as social distancing and reduced access to research facilities, have presented challenges. human and animal subjects research has drastically declined, resulting in projects that are now retrospective, literature reviews, or survey-based. current examples of topics include benefits of botulinum toxin a in dentistry: a systematic review and assessing access to care for special needs patients amid covid- through survey analysis. the pandemic and its associated long-term uncertainty have changed the types of projects that are feasible and how we provide student research mentorship. we addressed common barriers students might encounter in receiving quality research mentorship. [ ] [ ] [ ] the key barriers prioritized were transitioning the research experience to a virtual environment, adapting our mentorship approach, and creating virtual research opportunities. metrics of success include students matching with mentors and research completion. we used the full extent of musc's learning management system (lms), harbor, a product of blackboard open lms, which is accessible through a university account. we held virtual office hours and shared a repository of research resources via harbor. through harbor, blackboard collaborate, and microsoft teams were used to create group discussions for student engagement and videoconferencing features provided a synchronous format for mentoring. we recognized the need for adaptability due to working remotely. our cohort of mentors spend - hours weekly working with - students (n = ) per project starting june and concluding in the spring semester. we do not expect changes in research time commitment but recognize varying levels of experience in working remotely by both mentee and mentors. therefore, we modified research timelines to accommodate students transitioning to remote interaction. we adjusted our availability to accommodate students' revised schedules, including communication outside typical work hours and concurrently meeting with multiple student groups with similar project topics. we created opportunities for students to retrieve publicuse data for secondary data analyses and codified a list of evidence-based topics that dental students can research virtually, this information is accessible through harbor. we are also using skilled experts from multiple campus units to virtually present research-relevant material, via harbor, including: library resources and literature search tools, statistical methods, and submitting to musc's institutional review board. between march and june , all students (n = ) selected mentors, students and mentors have adapted to virtually engaging, and research activity has commenced. although covid- imposes "socially-distant mentorships," we expect all students will complete and present their projects at the spring dental student research symposium and anticipate continuing components of virtual research mentorship following the pandemic. o r c i d joni d. nelson phd, ms https://orcid.org/ - - - x musc dental research scholar's day. available at: https: //dentistry.musc.edu/departments/oral-health/scholars-day effects of a research requirement for dental students: a retrospective analysis of students' perspectives across ten years instant mentoring: sharing wisdom and getting advice online with e-mentoring medical student education in the time of covid- key: cord- - l c my authors: rochwerg, bram; parke, rachael; murthy, srinivas; fernando, shannon m.; leigh, jeanna parsons; marshall, john; adhikari, neill k. j.; fiest, kirsten; fowler, rob; lamontagne, françois; sevransky, jonathan e. title: misinformation during the coronavirus disease outbreak: how knowledge emerges from noise date: - - journal: crit care explor doi: . /cce. sha: doc_id: cord_uid: l c my although the amount of information generated during this most recent coronavirus disease pandemic is enormous, much is of uncertain trustworthiness. this review summaries the many potential sources of information that clinicians turn to during pandemic illness, the challenges associated with performing methodologically sound research in this setting and potential approaching to conducting well done research during a health crisis. data sources: not applicable. study selection: not applicable. data extraction: not applicable. data synthesis: not applicable. conclusions: pandemics and healthcare crises provide extraordinary opportunities for the rapid generation of reliable scientific information but also for misinformation, especially in the early phases, which may contribute to public hysteria. the best way to combat misinformation is with trustworthy data produced by healthcare researchers. although challenging, research can occur during pandemics and crises and is facilitated by advance planning, governmental support, targeted funding opportunities, and collaboration with industry partners. the coronavirus disease research response has highlighted both the dangers of misinformation as well as the benefits and possibilities of performing rigorous research during challenging times. d uring times of uncertainty, it can be challenging to decipher which information is credible. watching major news channels early during the course of an evolving and breaking story, it is usually clear that the newscasters do not have complete information; however, this does not stop the constant flow of discourse to viewers. to fill this void, it is common for broadcasters to rely upon information that is not fully vetted, much of which ends up being incorrect once the entire story becomes clear. these same themes may occur during medical crises, most clearly demonstrated during infectious pandemics that elicit a primal fear in people, bringing forth images of blockbuster films in which novel viruses wipe out large swaths of the global population. a combination of fear and a lack of credible information in the early phase of an outbreak are the largest contributors www.ccejournal.org • volume • e to public hysteria. information is the best tool to combat hysteria, and as illustrated in the current infectious outbreak of severe acute respiratory syndrome coronavirus , in our digital media era, information is everywhere. the more important concern for clinicians and patients, similar to watching a breaking news story, in which information to believe and which to ignore. the ongoing coronavirus disease pandemic has demonstrated the volume of information that can be produced in a short period of time; this has been associated with both benefits (easier access for clinicians) and risks (misinformation). media sources including newspapers, magazines, and news shows have been covering this story with fervor. although the objectives of corporate news media include informing the public of the latest medical updates, they have an obligation to shareholders or private owners of selling more newspapers, magazines, or advertisements and the natural inclination to therefore stoke the fires of hysteria. certainly, some sources are worse offenders when it comes to this than others who take the time to more carefully vet sources. the amount of print and news media dedicated to covid- in the last few months is huge. tangible risks of misinformation should not be ignored, as they may lead to ill-informed health decisions ( ) including isolation orders, travel bans, population quarantines and even discrimination against travelers from certain countries or persons of certain ethnic origins. the use of unproven therapeutic or prophylactic interventions also introduces unnecessary risks and, unless they are used carefully in the context of an approved clinical study, increase the amount of noise thereby limiting our collective ability to discover new ways to treat patients. there are however benefits to digitalization of health media. based on experiences with previous outbreaks, for example, influenza a(h n ) pdm pandemic in , the world health organization (who) and other governmental organizations are better prepared. the who maintains a live and up-to-date covid- website which contains credible information on the outbreak (www.who.int/ health-topics/coronavirus). the u.s. centers for disease control and prevention website includes updates on virus status in the united states, travel restrictions, and a world map highlighting areas with covid- cases (www.cdc.gov/coronavirus). johns hopkins runs a website (www.gisanddata.maps.arcigis.com) that provides up-to-date and credible data describing the number of those infected broken down by severity and separated by country, as well as the number of deaths. these governmental and public health organizations sources of information should be considered most trustworthy, as they can be relied upon to avoid misinformation, and as such the public should be going here as their main source of information during the health crisis. perhaps unique to this pandemic, compared with others, has been the response from the medical community. although bedside practitioners are in need of data that will help them to better identify, risk-stratify, and treat affected patients, medical research often takes time. traditionally, research is deliberate, and producing trustworthy and methodologically sound results may not be as rapid as what is required. for example, according to pubmed, although over , citations related to h n influenza have been published since , the large majority ( , of these) were published after , over years following the major phase of the pandemic. major contributors to research delays include competing interests of investigators, regulatory barriers, time taken for protocol development, ethics approval, peer review and delays related to the publication process. this classic research model does not fit well with pandemic research, where there is a need for rapid information to fill gaps and address public concern. for covid- , some of these traditional delays have been circumvented (we will discuss how shortly), and as such, many of the major general medicine journals, including journal of american medical association (jama), new england journal of medicine (nejm), and the lancet have prioritized publications related to covid- . this has been facilitated at medical journals through invited content and expedited peer review processes. jama, the lancet, and nejm, for example, maintain a coronavirus resource center including research and multimedia content (www.jamanetwork.com/journals/jama/pages/coronavirus-alert), most as free online content. providing peer-reviewed and easily accessible content has helped to overcome some of the misinformation rampant in lay media. as of march , , unique citations related to covid- have been indexed in pubmed, in , and , in . this represents an enormous amount of scientific content for a disease that was first discovered in wuhan, china in mid-december. it remains to be seen, how valid and trustworthy the data from these publications will turn out to be, given the rapidity in which they were produced and the expedited peer review and editorial decision-making required to publish so quickly. there have already been some highly visible examples of dubious and scientifically questionable reports, even some that have been published and now corrected in highly reputable journals ( ) . the lesson is that not everything posing as trustworthy research truly is, and it is important to both support high-quality work, but also discourage and prevent work that is not trustworthy. also unique to this outbreak is the role social media has played in information dissemination and at times, propagation of misinformation ( ). twitter has become entrenched as an information source for both patients and clinicians ( , ) . although the platform is unique in allowing for engagement with experts and rapid discourse, the lack of scientific vetting and peer review ( ) can contribute to hysteria, rather than alleviating it. each day over , tweets are sent using the #covid hashtag (www. symplur.com) and this is increasing exponentially. filtering the knowledge from the misinformation in social media is extremely challenging, and probably a strategy to be avoided in times of pandemic. at the very least, if using social media, the focus must be on reliable sources presenting vetted information and avoiding conjecture and opinion. even this rule is not absolute, as we have seen dramatic cases of prominent individuals advocating for specific unproven therapies (e.g., hydroxychloroquine and azithromycin) leading to drug shortages and increased rates of toxicity. examples such as this reinforce the necessity of consistent and well-informed communication strategies in times when the risks of misinformation are significant. research during a pandemic or health crisis presents challenges beyond the usual difficulties surrounding research in the critically ill ( ) . most obviously, pandemic preparedness, planning and management requires time, resources and personnel. clinical researchers may be diverted to the bedside caring for affected patients or working with government and public health organizations to contain the outbreak. preliminary data from china suggests that mortality in wuhan (the center of the covid- outbreak with the largest number of cases) has been higher (> %) compared with other regions in china (around . %) and this has been hypothesized to be at least partly due to a shortage in healthcare providers ( ) . not only is researchers time diverted to clinical care but so are other resources including funding. governments are usually the largest research funder, especially in developed nations; however, these funds may be required during an outbreak to augment capacity through infrastructure or human resources investment. the most dramatic example of this was the government of china's investment in building a new , square foot hospital with , beds and icu beds, built in only days to care strictly for covid- patients. organizational stress has other collateral impacts on research capacity. research involving humans often requires regulatory or governmental support, especially if there are significant ethical, public health or safety concerns ( ) . more than likely, during times of institutional pressure, these regulatory pathways will be delayed, limiting the ability to get the approvals necessary to proceed. research ethics boards may mistakenly consider the emotional pressure on patients and caregivers during a health crisis as an unsuitable environment to conduct research, thereby enacting further delays and barriers to timely investigation ( , ) . safety concerns for research staff may keep them out of hospitals or limit their ability to enroll patients and capture study-related information. unfortunately, the greatest impact of health crises and pandemics and the most significant challenges with outbreak tracking occur in low-or middle-income countries (lmics), regions that are already well below capacity in terms of health and research infrastructure ( ) (fig. ) . lack of local or regional expertise in conducting methodologically rigorous research may require external collaboration, which is challenging in the setting of travel restrictions and which runs the risk of ignoring scientific input from lmic investigators and clinicians. this is all further complicated by a rapidly evolving landscape. within pandemics, the clinical situation often evolves day-to-day or week-to-week, a pace uncommon in the setting of traditional epidemiologic and clinical research. a research question or medical intervention that was relevant weeks ago may no longer be relevant by the time approval and funding are secured. as such, research priorities and approaches must be capable of responding nimbly and rapidly. this need for rapid information and rapid dissemination of trustworthy results is daunting and uncomfortable for most clinical researchers who are used to operating within extended timelines. the exponential increase in pubmed citations related to covid- over the last months is a testament to this rapid evolution in information. a we've learned time and time again, true salvation figure . countries judged to be most at risk for originating pandemic illness (red = high risk, orange = moderate risk, yellow = low risk). reference: global health security index (www.ghsindex.org). from pandemic times (e.g., ebola) will come only from well-conducted research informing prevention of disease with vaccines, use of prophylaxis, improving treatments, and mitigating diseaserelated consequences. although these barriers are significant, and clinical research during the health crisis is enormously complicated, this is the first outbreak in which rapid, potentially clinically useful research is being conducted alongside the pandemic response. at this time, approximately randomized controlled trials have been registered in clinicaltrials.gov, and more than in the chinese trials registry, investigating interventions such as antivirals (multiple), iv immunoglobulin (nct ), corticosteroids (nct ), antibiotics (e.g., azithromycin) (multiple), tocilizumab (nct ), sildenafil (nct ), thalidomide (nct ), immunotherapy (nct ), chloroquine (nct ), recombinant angiotensin-converting enzyme (nct ), thalidomide (nct ), biologic agents (nct ), mesenchymal stem cells (multiple), convalescent plasma (nct ), nitric oxide (nct ), vitamin c (nct ), traditional chinese medicine (multiple), and vaccines (nct ) in the treatment of covid- related illness. research funding bodies can help by prolonging funding periods, augmenting funding envelops to help overcome the barriers mentioned above, and considering funding pandemic research even outside pandemic times. over the coming months, the most significant issue facing clinicians caring for covid- patients will be to critically appraise the multiple research outputs and decide which to apply in clinical practice. for researchers conducting these trials, it is important to balance rapidity along with sound methodologic principles. this can be facilitated in a number of ways, which will be discussed next, including some direction on how best to incorporate new data into pandemic-based patient management. how might health researchers go about pursuing this in a timely manner? there are a number of strategies that have been employed to overcome some of the challenges associated with conducting research in this setting ( ) . pandemics related to respiratory viruses have occurred at regular intervals throughout history ( ) (fig. ) ; it is not a matter of if they will recur, but rather when. as such, rather than waiting until a pandemic occurs to build infrastructure, researchers may develop collaborative networks, initiate study protocols, and begin regulatory and ethical approval processes in anticipation of the next outbreak. then, when the inevitable pandemic occurs, research capacity will already be in place allowing for a facilitated response. the international severe acute respiratory and emerging infections consortium (isaric), a group which was formed in collaboration with international forum for acute care trialists (infact) umbrella, has followed this model ( ) . the group, which includes clinical research networks worldwide, was launched in following the h n pandemic with the plan to be ready for the next viral pandemic and with the goal of ensuring timely and efficient research in the setting of health crises related to emerging infection. as introductory work has been ongoing over the last few years, with the emergence of covid- , isaric is already prepared with whoendorsed case report forms, clinical characterization protocols to enable harmonious clinical and biological sample data collection, and clinical trial protocols that have been collated and endorsed by the entire research network (www.isaric.tghn.org). infact is also guiding the who on supportive and adjuvant care in severe viral disease through leadership within who committees, a great example of intensive care physicians leading the global response and research initiatives related to this pandemic. randomized, embedded, multi-factorial, adaptive platform trial for community-acquired pneumonia (remap-cap) is another example of an infact-led initiative that has positioned itself well to answer timely research questions during pandemic illness such as covid- (nct , www.remapcap.org). the unique study design allows for sequential investigation of a number of different interventions targeting pneumonia including specific antibiotics, antivirals, or corticosteroids, for example ( ) . in the setting of a pandemic, the adaptive design allows for evaluation of new interventions and multiple treatment options, even those specifically targeted to new or emerging viruses. this adaptive feature allows for trial infrastructure to be established and to even begin enrolling patients examining traditional interventions for pneumonia, while providing opportunity to change intervention mid-trial to more specific or relevant agents, targeted to specific emerging pathogens. for these reasons, the adaptive trial design is likely the optimal methodology for investigating different anti-covid interventions within the same design. in fact, the remap-cap team has already evolved their protocol to address covid- and will focus on treatment domains in study centers affected by the virus, including the evaluation of prolonged macrolide therapy, corticosteroid administration strategies, antiviral use, and interferon-beta. through central administration and wide-scale international recruitment, remap-cap is well-positioned to enroll a large number of geographically diverse patients; both crucial components to study a global pandemic. through the adaptive randomization, treatment arms that show the most promise or benefit along with the least amount of toxicity will see increased allocation of trial participants, while those with less efficacy or more toxicity will see decreased allocation ( fig. ) . similarly, the who has an adaptive trial planned assessing multiple interventions which may be efficacious in the setting of covid- and has developed a core outcome set to be used during pandemic research (www.who.int). given the rapidity of new research data associated with the covid- pandemic, the next question for bedside practitioners becomes which data are of sufficient quality and trustworthiness that it should inform clinical practice ( table ) . might we accept a lower threshold in the setting of health crises, as opposed to other settings ( ) ? clinical practice guidelines (cpgs) are often considered the gold standard for informing healthcare decisionmaking; however, traditionally, cpgs take years to produce, limiting their ability to impact knowledge translation during pandemic illnesses. to address this, guideline developers have attempted to provide rapid guidance documents, still produced using rigorous methodology, but often addressing questions of smaller scope, using larger teams to facilitate expedited recommendations, and frequently updated ( ) ( ) ( ) . there are a number of these rapid guideline efforts, some done using grading of recommendations assessment, development and evaluation methodology, currently underway addressing covid- with a couple having just recently been published such as the australia and new zealand intensive - ) . one of the risks associated with this constantly evolving research landscape, is that evidence, and subsequently best practice, is constantly changing. as such, it is not a surprise that these guidelines are not entirely consistent with one another, and risk quickly becoming out of date. this can be overcome through frequent reassessment of recommendations based on emerging evidence (living guidelines), which although crucial in this setting, represents an added challenge to be addressed. pandemics and healthcare crises provide extraordinary opportunities for the rapid generation of reliable scientific information but also for misinformation, especially in the early phases, which may contribute to public hysteria. the best way to combat misinformation is with trustworthy data produced by healthcare researchers. although challenging, research can occur during pandemics and crises and is facilitated by advance planning, governmental support, targeted funding opportunities, and collaboration with industry partners. the covid- research response has highlighted both the dangers of misinformation as well as the benefits and possibilities of performing rigorous research during challenging times. dr. rochwerg is supported by the hamilton health sciences early career research award. dr. lamontagne is supported by a fonds de recherche du québec -santé award. dr. sevransky's institution has received funding from the marcus foundation for a sepsis clinical trial. the remaining authors have disclosed that they do not have any potential conflicts of interest. for information regarding this article, e-mail: rochwerg@mcmaster.ca clinical decision making during public health emergencies: ethical considerations transmission of -ncov infection from an asymptomatic contact in germany social media and emergency preparedness in response to novel coronavirus tracking the flu pandemic by monitoring the social web twitter hashtags for health: applying network and content analyses to understand the health knowledge sharing in a twitter-based community of practice twitter as a tool for communication and knowledge exchange in academic medicine: a guide for skeptics and novices clinical research during a public health emergency: a systematic review of severe pandemic influenza management potential association between covid- mortality and health-care resource availability clinical research ethics for critically ill patients: a pandemic proposal social value, clinical equipoise, and research in a public health emergency ethics of clinical science in a public health emergency: reflections on the role of research ethics boards major issues and challenges of influenza pandemic preparedness in developing countries influenza pandemics: a historical retrospect isaric council: open source clinical science for emerging infections adaptive designs for clinical trials national ebola training and education center's special pathogens research network (sprn)'s medical countermeasures working group: evaluating promising investigational medical countermeasures: recommendations in the absence of guidelines developing who rapid advice guidelines in the setting of a public health emergency development of rapid guidelines: . gin-mcmaster guideline development checklist extension for rapid recommendations introduction to bmj rapid recommendations key: cord- -ho m nqn authors: nguyen, van thu; rivière, philippe; ripoll, pierre; barnier, julien; vuillemot, romain; ferrand, gabriel; cohen-boulkia, sarah; ravaud, philippe; boutron, isabelle; alawadhi, solaf; amer-yahia, sihem; Ávila, camila; bafeta, aïda; baudry, julia; bollig, claudia; bonnet, hillary; bouet, marinette; cabanac, guillaume; chaimani, anna; chavalarias, david; chen, yaolong; chevance, astrid; cohen-boulakia, sarah; coquery, emmanuel; conil, francoise; davidson, mauricia; de nale, laura; devane, declan; diard, elise; doreau, bastien; evrenoglou, theodoros; fabri, alice; feron, gilles; fezeu, leopold; fouet, mathilde; el chall, lina ghosn; graña, carolina; grasselli, giacomo; grolleau, françois; hacid, mohand-said; haddy, loubna; hansen, camilla; hohlfeld, ameer; hróbjartsson, asbjørn; julia, chantal; mavridis, dimitris; meerpohl, joerg j.; meyer, brice; naidoo, nivantha; thu, van nguyen; oikonomidi, theodora; pienaar, elizabeth; quirke, fiona; rada, gabriel; riveros, carolina; sauvant, marie; schmucker, christine; toumani, farouk; tovey, david; xia, jun; yu, xuan; zoletic, emina; zweigenbaum, pierre title: research response to covid- needed better coordination and collaboration: a living mapping of registered trials date: - - journal: j clin epidemiol doi: . /j.jclinepi. . . sha: doc_id: cord_uid: ho m nqn background researchers worldwide are actively engaging in research activities to search for preventive and therapeutic interventions against covid- . our aim was to describe the planning of randomized controlled trials (rcts) in terms of timing related to the course of the covid- epidemic and research question evaluated. method we performed a living mapping of rcts registered in the who international clinical trials registry platform. we systematically search the platform every week for all rcts evaluating preventive interventions and treatments for covid- and created a publicly available interactive mapping tool at https://covid-nma.com to visualize all trials registered. results by august , , , trials for covid- were registered worldwide. overall, the median ([q -q ]; range) delay between the first case recorded in each country and the first rct registered was days ([ - ]; - ). for the countries with the highest number of trials registered, most trials were registered after the peak of the epidemic (from % trials in italy to % in the united states). most trials evaluated treatments ( , trials; %); only ( %) evaluated preventive strategies and post-acute period intervention. a total of trials were planned to assess different regimens of hydroxychloroquine with an expected sample size of , patients. conclusion this living mapping analysis showed that covid- trials have relatively small sample size with certain redundancy in research questions. most trials were registered when the first peak of the pandemic have passed. researchers worldwide are actively engaging in research activities to search for preventive and therapeutic interventions against covid- . our aim was to describe the planning of randomized controlled trials (rcts) in terms of timing related to the course of the covid- epidemic and research question evaluated. we performed a living mapping of rcts registered in the who international clinical trials registry platform. we systematically search the platform every week for all rcts evaluating preventive interventions and treatments for covid- and created a publicly available interactive mapping tool at https://covid-nma.com to visualize all trials registered. by august , , , trials for covid- were registered worldwide. overall, the median ([q -q ]; range) delay between the first case recorded in each country and the first rct -there is a notable redundancy in research questions. -we have created a living mapping that visualizes all clinical trials of covid- . -the living mapping supports researchers and decision makers in identifying research gaps, thus planning research of high priority. -research community needs a better coordination in research planning to ensure that all potential treatments for covid- are evaluated with robust methodology. -the living mapping provides a tool to monitor status of research, enhance research collaboration and interaction in medical and scientific community to avoid research waste. j o u r n a l p r e -p r o o f in december , an outbreak of pneumonia caused by a novel coronavirus started in wuhan, hubei province in china. the disease was later determined to be sars-cov- infection, or covid- ( ). in early march , the disease had spread to more than countries and territories ( ). on march , , the world health organization (who) declared the outbreak a pandemic ( ) . to respond to this emergency, researchers all over the world began to actively engage in research activities to develop and evaluate preventive and therapeutic agents for covid- . given this unprecedented context, we aimed to inform decision makers and researchers in near real-time about current research efforts, research gaps and overlap. a mapping of all research efforts is imperative to support researchers and decision makers to monitor status of research response to the epidemic and integrate emerging evidence in research planning timely to ensure that all potential treatments are evaluated, whilst avoiding waste in resources invested. for this purpose, we performed a living mapping of all registered randomized controlled trials (rcts) investigating interventions to prevent and treat covid- . this living mapping is updated every week and results are publicly available at https://covid-nma.com/. this paper describes the planning of rcts in terms of timing related to the course of the pandemic and research questions. this mapping is part of the covid-nma project, which also includes living systematic reviews and living network meta-analyses of studies of covid- . the protocol of this project is available at https://zenodo.org/record/ #.xwlasubui x. our data are obtained from the who international clinical trials registry platform (ictrp) (https://www.who.int/ictrp/en/), an international registry that assembles information on clinical trials registered in primary registries ( ) . who ictrp has created a database dedicated to all clinical trials evaluating interventions to prevent and treat covid- . the database is updated weekly and is publicly available. whenever the database is updated, we use php programming language to identify studies that are newly registered in the database. two researchers (vn and gf) systematically search the platform every week to identify new eligible rcts for data extraction. all rcts assessing the efficacy and safety of interventions for preventing or treating covid- and patients in the postacute period are included. we exclude observational studies, case series, non-randomized or single arm studies (i.e., diagnostic tests studies). we also exclude studies ) evaluating interventions to reduce psychological distress caused by the covid- outbreak or ) assessing herbs, homeopathy therapy, traditional chinese medicine (tcm) (with only tcm in two groups, or tcm plus standard of care). a standardized data collection form is used to collect data describing the rcts. several data items are available from the who ictrp database, such as registration number, countries where trials are conducted, recruitment status, inclusion and exclusion criteria, primary outcomes, and sample size. a team of trained data collectors independently retrieve other information from j o u r n a l p r e -p r o o f the trial registration such as study aim, number of arms, type of participants, and information related to experimental treatments and comparators (i.e., treatment name, treatment type). two researchers (vn and gf) verify the quality of the data and ensure the consistency of data entered in the database. we classify study aims as evaluation of prevention interventions, covid- treatments and post-acute period interventions. in rcts evaluating preventive interventions, participants are classified as healthy volunteers, health workers, and high-risk patients. patients in rcts assessing covid- treatments are classified by disease severity (i.e., mild, moderate, severe and critical). clinical criteria for classifying disease severity are provided in appendix . the full list of treatment types is provided in appendix . when the database of who ictrp is updated every week, we use php programming language to identify rcts with changes in recruitment status (e.g., from not recruiting to recruiting) and update our database accordingly. the covid- database maintained by our world in data (https://ourworldindata.org/coronavirus-source-data) was used to visualize the evolution of the pandemic over time. the database is updated daily and includes the number of confirmed cases, deaths, and testing data. we considered only data related to the number of confirmed cases and deaths. we created an online interactive mapping tool to visualize the data of trials registered. the interactive mapping was developed with d .js ( ) as an observable notebook ( ) . the projection for the map used was implemented in javascript ( ). we also used time series plotting to visualize the evolution of covid- research over time. this visualization was performed in r v . . (the r foundation statistical computing, vienna, austria). up to august j o u r n a l p r e -p r o o f in certain countries, the sample size is relatively small for trials evaluating covid- treatments ( timing of research response to the evolution of the pandemic in europe, spain registered only / trials ( %) before the peak (i.e., march , . in france, the first trial was registered only days before the peak (i.e., april , ). eight trials ( %) registered before the peak in france accounted for % of the total number of patients to be recruited in all trials. in the united kingdom, / ( %) trials were registered before the peak (i.e., april , ), representing % of the total number of patients to be recruited in all trials. in italy, no trial was registered before the peak on march , . furthermore, trial results will probably be published after the epidemic has passed, and countries where the trials were conducted might not have the direct benefits to improve clinical practice at the time of the epidemic ( - ). the planning of trials in response to covid- has a notable redundancy in research questions. in march , hydroxychloroquine received tremendous attention after the results of an observational study in france were published that generated a huge debate ( ) . following this publication, the us president highlighted this treatment as being a "game changer", despite the regulatory and health authority should provide timely guidance to clinicians to avoid off-label drug uses based on anecdotal evidence which might cause difficulties to trial planning and recruitment ( , ) . this study highlights the importance of clinical trial registries, an underused resource, to monitor the state of research for improving the organization of research efforts ( ) ( ) ( ) . our interactive living mapping of covid- research was designed to help decision makers use data from clinical registries for an up-to-date picture of all research questions being investigated so as to prioritize research and avoid waste in research ( ) . furthermore, this interactive mapping tool might also enhance collaboration in research to reduce redundancy and competition in trial organization ( , ) . in this analysis, we visualized trial registration over time by using the registration date rather than the actual starting date of recruitment because these data were not available on the ictrp database. additionally, investigators might not regularly update the status of recruitment on trial registries. for example, the trial chictr was reported as "not recruiting" on the registry, but the results of the trial were published ( ) . furthermore, the structure of reporting is heterogeneous across the primary registries, which affects the quality of reporting ( ) . investigators might register one trial in more than one registry under different titles or investigator names, such duplicates are almost systematically detected by the ictrp while a very few may remain undetected. lastly, we did not assess the risk of bias for each trial registered as information in trial registration is inadequate to enable a comprehensive assessment. we have created a living mapping tool to keep track of the evolution of research on covid- for supporting decision makers in prioritizing and planning research. this mapping analysis showed that many covid- trials were registered after the first peak had passed and a need to j o u r n a l p r e -p r o o f improve the organization of research efforts to avoid research redundancy. visualizing ongoing research can enhance the collaboration and interaction between research communities that can go beyond the covid- crisis. funding: this study received funding from the agence nationale de la recherche (anr). the funder had no role in the design, analysis and reporting of this study. statement regarding cluster of pneumonia cases in wuhan director-general's opening remarks at the media briefing on covid- - who. who announces covid- outbreak a pandemic 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prioritising study recruitment covid- : the inside story of the recovery trial covid- : recovery trial will evaluate "antiviral antibody cocktail advancing preparedness for clinical research during infectious disease epidemics covid- clinical trials: learning from exceptions in the research chaos future of evidence ecosystem series: . introduction evidence synthesis ecosystem needs dramatic change impact of searching clinical trial registries in systematic reviews of pharmaceutical treatments: methodological systematic review and reanalysis of meta-analyses clinical trials registries are underused in the pregnancy and childbirth literature: a systematic review of the top journals how to increase value and reduce waste when research priorities are set against pandemic research exceptionalism clinical outcomes and plasma concentrations of baloxavir marboxil and favipiravir in covid- patients: an exploratory randomized the quality of registration of clinical trials: still a problem data sharing: the dataset will be publicly available after the paper is published. key: cord- -v icun z authors: kozlowski, hannah n.; farkouh, michael e.; irwin, meredith s.; radvanyi, laszlo g.; schimmer, aaron d.; tabori, uri; rosenblum, norman d. title: covid‐ : a pandemic experience that illuminates potential reforms to health research date: - - journal: embo mol med doi: . /emmm. sha: doc_id: cord_uid: v icun z covid‐ has halted research around the globe and forced researchers out of their labs. non‐emergency medical appointments were cancelled. ongoing clinical trials were challenged to create new modes of operation while public pressure mounted to find therapeutic options against covid‐ . yet, the inability to conduct research during covid‐ was overcome with cooperation, resource sharing, and compassion, which provides important lessons on how to improve health related research as we enter a new normal. this article is protected by copyright. all rights reserved to meet the covid- challenge, the global scientific community focused human and material resources to develop new drugs, therapies, vaccines, diagnostics and so on. however, it takes to years to turn a basic discovery into a new drug via the typical translational path (mohs & greig, ; paul et al, ) , which would not be compatible with the goal of rapidly developing diagnostics and therapeutics for covid- . this situation created a need to overcome the many hurdles along the translational research pathway -financing, business development, regulation, product development, commercialization and different cultures-in order to satisfy an urgent societal need. here, we discuss the lessons learned from the toronto academic health sciences network's (tahsn) response to covid- . we highlight the critical importance of cooperation and compassion in maintaining productivity during the pandemic and identify opportunities for shaping a new era of research with increased patient engagement, fewer silos and a shared goal of improving health and decreasing disease burden. the development of ready-for-use clinical products during the covid- pandemic has engendered cooperativity, engagement and investment in a common goal (figure ), enabled by a shared view of the problem and a genuine desire to contribute to its solution. the fruits of these efforts can be seen around the world as pharmaceutical and biotech companies and research groups team up to develop, test and manufacture vaccines (liu, ) . this created a real 'open science' environment, in which equipment, manufacturing capabilities, patient cohorts and results were shared in real time (rouleau, ) . prior barriers that obstructed cooperation and sharing were dismantled, facilitating the development of novel ideas into solutions that benefit patients. to expand covid- testing capacity and reduce redundancy, four medical research institutions in toronto agreed to a resource-sharing approach. this is not necessarily new: many philanthropic organizations including the gates foundation and the wellcome trust have already made open this article is protected by copyright. all rights reserved data-sharing mandatory for their grants (kiley et al, ) . in toronto, this new sharing agreement went further to include samples and reagents to allow free movement between institutes without any attendant intellectual property (ip) agreements. two major lessons emerged. first, focusing on the shared goal, rather than first negotiating ip rights, increased productivity. early conversations around ip are expensive, slow negotiations between institutes and have stymied innovation. second, sharing resources such as reagents, equipment, staff and expertise increased the number of problems that could be solved. without such agreements, hospitals would only test their own patients and the majority of these basic tests indicated only viral load. instead, cooperation leveraged equipment, protocols and expertise external to hospitals. the ontario institute of cancer research, normally a cancer-focused genomics institute, sequenced indeterminate and positive covid- samples. they shared the sequences in real time to further the scientific community's understanding of covid- , which exemplifies how resource-sharing between institutions can increase testing capacity and support scientific discovery. new partnerships were built on a foundation of resource-sharing initiatives and the motivation to contribute during the crisis. in toronto, new collaborations between academia and industry resulted in multi-center clinical trials that transitioned from idea to approval in as few as days. the scale and speed of these agreements was only possible because academic researchers and private industry worked together with a shared understanding of the problem and jointly identified their collective goals, expertise, available resources and clinical networks. in one instance, a conversation between two colleagues on the use of immunomodulators to protect cancer patients from the severe side effects of covid- infection became a phase three, multicentre clinical trial. the total time from initial conception to protocol drafting to approval by health canada was only six weeks. we have also seen international pharmaceutical companies share their manufacturing facilities, supply chains and expertise in scaling (liu, ; the association of the british pharmaceutical industry, ). these joint commitments led to further cooperation between researchers, private industry, research ethics boards and national regulators. this article is protected by copyright. all rights reserved during covid- , regulators worked closely with scientists to quickly translate scientific discoveries into medical interventions. health canada released guidance documents for approval of covid- medical devices, including diagnostic tests, to simplify and streamline the application process, without impacting post-market safety standards (reid, ) . similarly, the us fda issued an emergency use authorization that expedited the review of covid- diagnostics (reid, ) . this sort of response by regulators around the world led to increased cooperation with researchers, which boosted productivity. compassion and support for the people behind the research fostered sustained productivity. the research community also realised that supporting research meant supporting each other. behind each study are researchers with unique values, expectations and experience. attention to individuals and their circumstancesdelays in student graduation, personal comfort in the workplace, safe commuting, family responsibilities, challenges associated to working from home and uncertainty due to job securitybecame central to discussions on restarting research after covid- restrictions are loosened. for example, flexible working times and shift work allowed for physical distancing, avoiding rush hours and benefited families with working parents. beyond any particular research program and institution, tahsn provided virtual resources, seminars, social interactive space and training to promote support research staff and foster a positive working environment, all of which strengthened the research community and sustained productivity. during the pandemic, several non-covid- trials were also forced to adapt. running these trials without compromising rigor or the safety of patient safety required cooperation between researchers, local research ethics boards (rebs), patients and patient advocates to address patient anxieties and clinical priorities by developing new protocols. in toronto and medical clinics worldwide, clinicians adapted by moving medical visits and clinical trial appointments online. using video calling, physicians saw their patients in their home environment as well as other this article is protected by copyright. all rights reserved family members who joined the call. although these interactions provided a broader view of the patient, their health and their environment, careful retrospective analyses of the safety, reliability and effectiveness of virtual patient assessments are still needed to determine if this approach is sustainable. harnessing lessons learned during covid- to improve health research requires joint action to further improve research infrastructure, integrate parts of the translational pathway and support the people who do the work. here, we build upon those lessons to propose a framework for the "new normal" after the first phase of covid- (table ) . this includes creating infrastructures that promote cooperation, support team science and prioritize the wellness of people. the proposed framework can help to break down research silos and efficiently move scientific discoveries from the lab to the clinic. to foster cooperation we need to assemble team members of the broader research and translation community around a shared goal. during covid- , researchers, industry professionals and regulators were each determined to find solutions that benefit patients right away. maintaining this shared focus going forward requires that we rebuild activities to support cooperation. in toronto, we started by restructuring hospital grand rounds, the institute-wide clinical seminars to educate the community on a relevant medical topic. clinicians and researchers from different fields now give joint talks to highlight the larger problem and possible solutions from their perspective. this was particularly helpful during covid- as infectious-disease clinicians discussed the newfound pediatric presentation of covid- and immunologists explained the immune response and how that informs vaccine development. moving forward, collaborative research should become a model for trainees and other researchers to strive for as they see the fruitful outputs of teamwork. this article is protected by copyright. all rights reserved to further support cooperation and build partnerships, we need to invest in research clusters that are grouped by disease or clinical question instead of methods. each cluster would include basic science, health services, implementation, engineering and clinical expertise. seminars within each cluster would describe different facets of the problem, as per the expertise of the speaker. this provides researchers and trainees a shared view of the big picture so that they can readily come together to solve the problem. these collaborative clusters should also include patient advocates and industry to open communication and help different groups to understand each other and facilitate a shared culture. in such a disease-focused research cluster, the role of each member will be clearly understood, reducing the number of barriers to producing research that benefits patients. to encourage resource sharing and to move research discoveries closer to patients, research institutions need to value and reward teamwork. yet, the current academic structure does not always reward team science or the building of collaborative networks. in the medium-term, we should change how we evaluate scientific contributions and create promotional metrics that recognize team science. in toronto, we acknowledge that success may not always result in a publication but in the adoption of a new policy, better clinical guidelines, a new partnership with industry or the clinical validation and implementation of a diagnostic test. in the long-term, we should provide more grants that promote collaboration akin to the european union's research programmes that provide long-term funding for collaborations between multinational research groups. supporting long-term development relieves the pressure to publish quickly and often and encourages teams to tackle big problems. given the time and resource needed to bring new interventions to the clinic, the funds for these grants should come from government-industry partnerships, and there should be an option of applying for additional funding if pre-specified milestones are met (van dijk et al, ) . this article is protected by copyright. all rights reserved producing research that is meaningful to patients also requires that we humanize research. during covid- there was a shift from thinking about research as something that eventually trickles down to a "patient", to understanding that we all may benefit. this makes it more natural to think of patients as the reason and focus of our scientific inquiries. in toronto we previously started shifting towards patient-centred research by involving patients and families in advisory committees, ethics boards and research teams as charitable foundations have been doing for years (stevens, ) . we can further strengthen this partnership by creating pathways for students, the researchers-oftomorrow, to learn from patients directly. in these pathways, students will spend more time in the clinic, meet patients and caregivers, write reb proposals, analyze the flow of information between health professionals and observe medical procedures (delnero & mcgregor, ) . for example, asking patients why they continue/ discontinue their treatments and what quality of life indicators/outcomes matter to them can inform study design and give students a sense that they are contributing to improve patient health. this experience also breaks down barriers between research and clinic. for researchers it provides a broader understanding of a disease and the challenges of implementation. for patients and caregivers it is an opportunity to share their wisdom and learn about the research process. now is the time to harness the lessons learned from covid- and shape a new era of innovation that more effectively brings the benefits of research to patients. we cannot let the positive changes be lost. first, we need to continue to create joint goals between all members of the translational path. sharing must continue to extend beyond reagents and funding. these actions can be strengthened by restructuring our institutions to reinforce 'open science', developing broad datasharing agreements and creating collaborative work spaces. second, we need to value and reward teamwork. this involves creating new measures of academic success and collaborative grants. the this article is protected by copyright. all rights reserved validation of new diagnostic tests, the implementation of a new workflow or pharmacokinetic studies for a new treatment is not considered high-impact science, but it is needed to improve quality of life and clinical practice. third, funding must be shared between immediate clinical need and continuous support for basic science to enable the biological understanding of disease. the rapid development of diagnostics and vaccines for covid- were based on years of basic research in virology, therapeutics and engineering. lastly, we must remember that people are at the heart of research. we must work to make sure all team members are supported and that patients are engaged in research. together we can create a new era of medical research, where knowledge moves fluently between patients, researchers, industry, regulators and policy makers in order to generate solutions that are used in the clinic. h.n.k. and n.d.r. conceived the idea, wrote and edited the manuscript; all authors contributed content and reviewed the final manuscript. the experiences and framework discussed in this commentary were based on a town hall given by medical innovations toronto (mito), a translational research hub within the tahsn. in this discussion tahsn leaders discussed how the pandemic changed the research landscape in ontario, the lessons they learned from the response to covid- and the implications their this article is protected by copyright. all rights reserved from patients to partners. science ( -. ) building a professional identity and an academic career track in translational medicine data sharing from clinical trials -a research funder's perspective pfizer offers up talents, tools and manufacturing capabilities in call for wide covid- collabs drug discovery and development: role of basic biological research how to improve r&d productivity: the pharmaceutical industry's grand challenge how regulators are addressing covid- related medical device approvals the key to finding a cure for covid- ? open science medical philanthropy pays dividends the association of the british pharmaceutical industry ( ) what are pharmaceutical companies doing to tackle covid- ? available at this article is protected by copyright. all rights reserved accepted article key: cord- - uo hsst authors: sepúlveda-vildósola, ana carolina; mejía-aranguré, juan manuel; barrera-cruz, carolina; fuentes-morales, natxieli alexandra; rodriguez-zeron, carlos title: scientific publications during the covid- pandemic date: - - journal: arch med res doi: . /j.arcmed. . . sha: doc_id: cord_uid: uo hsst • covid- pandemics has shocked our world in a few months, not only by attacking individual health, but also public health and economic systems, the way people relate to each other, but has also changed scientific and editorial practices. méxico, méxico; e-mail: ana.sepulvedav@imss.gob.mx covid- pandemics has shocked our world in a few months, not only by attacking individual health, but also public health and economic systems, the way people relate to each other, but has also changed scientific and editorial practices. by now, more than . million persons have been infected by sars cov virus, and more than , have died worldwide. as there is no vaccine to prevent the disease, or a specific therapeutic drug to treat patients, health care systems treat the sick with supportive measures, hoping that each persons' immunologic system can confront the disease. but the rush of scientists to quickly understand the virus and its behavior, and to design proper prevention and therapeutical interventions must not sacrifice rigorous science, as vital decisions must be taken daily not only by health care workers but also by national policymakers. clinical research and evidence based medicine have been the tools by which physicians and public health policymakers take informed decisions. both strategies follow strict rules in order to make strong scientific observations and recommendations. retrospective analysis of uncontrolled clinical experience often leads to erroneous conclusions about the efficacy of a treatment. thus, solid scientific conclusions must be derived from randomized controlled studies. furthermore, systematic reviews and meta-analysis confirm valuable findings. validity then, in therapeutic trials, depends on the power of the methods and the degree in which they can be generalized in clinical settings ( figure ). evidence based medicine refers to the process of systematically reviewing, appraising and using clinical research findings to deliver optimal clinical care to patients. the combination of principles and methods ensure that medical decisions, guidelines and policies are based on the current best evidence. covid- has quickly spread globally, causing countries health systems to collapse due to the great number of simultaneous patients with moderate and severe disease. daily, doctors and administrators must urgently decide on the best treatment or recommendation in the field of public health, with very scarce information, as it is a new disease ( ). the possibility of making mistakesincreases. for example, treatments based on what is known about the pathogenesis of the disease led very early to point out that the use of steroids should not be recommended due to the possibility of disease spreading. however, the role of cytokine storm as a complication was later identified and the use of steroids is now known to improve patients' conditions and prevent the use of ventilators ( ) . from the ethical point of view, it is considered that, given the imminent possibility of a patient's death and the lack of proof that a treatment is useful, but having the possibility that it will produce some benefit, treatment should be offered. the problem is that it may become routine to treat in that way without having clear evidence of its benefit or even exposing patients to unnecessary risk. furthermore, many of these studies are either not properly reviewed in a research ethics committee or are poorly designed ( ). scientists have relentlessly rushed to analyze information, but the strongest evidence flows very slowly. in the past few weeks, we have seen an exponential growth of publications related to covid- . torres-salinas d, ( ) recently reported , documents retrieved by dimensions by april th , , with an exponential growth (r ) of . , with more than new documents published daily. an analysis by our team revealed , documents in scopus and , by web of science by april th , (table ). more than % of the published information is in english language, followed by co-occurrence of keywords for published articles shows that "covid- ", "coronavirus", "pandemic", "outbreak", "wuhan", "coronavirus disease ", "viruses", "china" are the most frequent. co-occurrence is an indicator of semantic proximity in which it is observed that the keywords have coincidences between the analyzed documents (larger clusters). (figure ). ( ), their concern about the quality of the research that is being done and published, principally related to low quality of trials (low sample size, non-randomization or patients, poor outcome measures, etc.), repeated trials and poor reporting. measurement errors are increasingly evident due to the lack of sensitivity and specificity of tests to diagnose sars-cov- , either by molecular biology or by antibody measurements, and possible confusion biases generated by a lack of control of all the potential variables that can influence the results in most studies. a big problem with what is being published is the lack of original findings, as almost half correspond to editorials, opinions, letter to the editor, commentaries, news, proceedings/conference or data paper, book chapter, short survey or reprint ( figure ). unfortunately, another problem that cannot be neglected is that a lot of information is supported by the pharmaceutical industry. there is a clear intention to help, but it is known that it is an unfair race, where some powerful companies that have a greater potential to disseminate the results of a study, which favor their products, and on the other hand, smaller companies find it more difficult to get their information properly and quickly to users ( ) . publication during the pandemic has also become complicated as scientific journals have had to adapt to manage regular submissions along with an increasing amount of manuscripts related to covid- , in many cases with a shortage in personnel and a shortage of experts available for peer review, as many of them are attending doctors in covid hospitals. the flaws of peer review, slow traditional publication times, and the urgent need to share information have led to the rise of pre prints, (manuscripts submitted to publicly accessible repositories, which may or may not be later submitted to a formal scientific journal). covid- has promoted the use of repositories such as biorxiv and medrxiv to make communication more agile, open and accessible. outbreak science rapid prereview, an open-source platform for rapid review of preprints related to covid- ( ) has been recently created. but the quality and scientific robustness of some of these articles has led to further retraction of papers, (like the one posted on biorxiv in late january claiming that the similarities between sars cov coronavirus and hiv- were "unlikely to be fortuitous in nature", which led to a conspiracy theory that the virus was a man-made bioweapon, and was later retracted after thousands of scientists cleared out that "although there were some genetic similarities between the two viruses, these similarities are shared by many other viruses as well"), creating confusion among information consumers. also, a problem is that dissemination of the information occurs as if they were final results accepted by the scientific community. editorial processes have been modified in response to covid- pandemic, for sarscov papers and for regular submissions. the journal of clinical investigation ( ), cell systems ( ) and elife ( ) , for example, relaxed some of their policies on regular submissions, as many laboratories have closed or established social distancing policies during the pandemic, and will allow authors more flexible times to respond to reviewers, flexible times for reviewers, or curtail requests for additional experiments. for covid- papers, many journals offer expedite peer review. research has adapted to this crisis by speeding up editorial processes for covid- manuscripts. editors daily analyze newly arrived documents and make a first editorial decision. daily follow up of "in process" manuscripts are done and peer reviewers are urged to speed their analysis. "accepted for publication" reviews and original research (biomedical, clinical or epidemiological) are all peer-reviewed, as we are engaged with maintaining our editorial quality. we know peer review is not perfect, but is still better than the alternatives. we want to thank the scientific community as an increasing amount of manuscripts have arrived to our journal in the last two months, from countries worldwide (figure ), and we countersign our commitment to fulfill our authors and our readers expectations. as to april th , , one third of the incoming manuscripts were accepted and sent to on-line publication (average of . d), and % are still under peer review. figure shows the type of articles that have been received. in a scenario like the one that we are experiencing nowadays, the responsibility of all actors to ensure that the published information is useful is very important ( ) . researchers must reflect on their responsibility and remember that, although we are experiencing an emergency, there must be robust scientific results. this is a good time to search for the interaction between the need to do (treat patients) and the need to learn (try treatments) ( ). universities, institutions, hospital centers where the research studies are being carried out must supervise that the projects are being properly evaluated by the research and ethics committees and not arc med res e _ be carried away by the pressure of who should publish first, but who is doing better research, and whose evidence will be more useful to patients. financial institutions that support research, in addition to guaranteeing that the research carried out complies with all the appropriate ethical and methodological requirements, must avoid duplication of information and over investing. open and accessible databases must be generated in different languages for researchers´ consultation. priority should be given to research that has the greatest application in the shortest term. journals must continue to ensure that published articles comply to methodological and ethical quality standards, and have no conflict of interest. impartiality, transparency, objectivity and confidentiality must always be observed. the "urge to publish" must never prevail over good editorial practices. . subcategories "psychology", "agricultural and biological science", "computer science and decision sciences" and "economics", econometrics and finance" were excluded. figures legend figure . type of studies and force of evidence. need for transparency and reliable evidence in emergency use authorizations for coronavirus disease (covid- ) therapies pharmacologic treatments for coronavirus disease (covid- ): a review finding effective treatments for covid- : scientific integrity and public confidence in a time of crisis ritmo de crecimiento diario de la producción científica sobre covid- análisis en bases de datos y repositorios en acceso abierto. el profesional de la información waste in covid- research systematic review evidence: cochrane's response to covid- fast peer review for covid preprints changing the editorial process at jci and jci insight in response to covid- pandemic keeping the wheels of the scientific endeavor turning during the covid- publishing in the time of covid increasing value and reducing waste in research design, conduct, and analysis covid- pandemics has shocked our world in a few months, not only by attacking individual health, but also public health and economic systems, the way people relate to each other, but has also changed scientific and editorial practices. key: cord- -upv o f authors: prior, sarah jane; mather, carey; ford, karen; bywaters, danielle; campbell, steven title: person-centred data collection methods to embed the authentic voice of people who experience health challenges date: - - journal: bmj open qual doi: . /bmjoq- - sha: doc_id: cord_uid: upv o f nan the patient or consumer voice in healthcare has evolved from an aspiration to becoming an expectation, which in some developed countries is respected by inclusion in nationally auditable standards (australian commission on safety and quality in health-care. inclusive and appropriate research about patient perspectives requires skills and resources to ensure that sound quality assurance processes are designed, delivered and evaluated. understanding the characteristics of the population is key to ensuring appropriate representation of consumers in person-centred or patient involvement research methods. vulnerable patients, those with ongoing health and literacy challenges, can benefit from advocacy, and often are not used to being valued in terms of their own views, lacking empowerment to present their own opinions. additionally, traditional methods need to be used more sensitively to ensure that these consumers can be included and participate equitably in the quality assurance process. meaningful person-centred engagement is occurring more often in healthcare settings through the process of codesign. originating from design science, codesign is defined as the engagement of patients and other consumers, to capture their experiences in the design or redesign, of healthcare services and is a central concept of health improvement initiatives. codesign includes core principles of equity, understanding of experience and service improvement and provides an avenue for person-centred participation, recognising that consumer experience and knowledge is increasingly being considered important to complement professional knowledge. direct engagement of patients and other consumers in research and health service improvement activities requires careful methodological planning around desirability and feasibility and the practical implications for the involvement of patients and consumer as participants. the following examples of person-centred research methods enable the authentic voice of individuals who experience health challenges to be collected. the five approaches provide opportunities for the voice of consumers to be heard. each of the methods can be used to emphasise active participation by consumers in the design, delivery and evaluation of health services safety and quality systems. a review of person-centred research methods in the literature was undertaken to scope for current techniques to ensure currency of the information. patients or consumers were not involved in this review of methods as the focus was on different strategies, rather than recruitment of participants in any study. the search strategy employed a pragmatic approach as it allowed the research methods to be the focus. the authors initially searched individually for articles using multiple databases including cinahl, medline and scopus. articles that described personcentred strategies for the methods within their area of expertise were sought. a further search using references of articles and updates over the time period of development of the manuscript was also undertaken. the research group met monthly over a -month period to discuss the research methods, literature and develop the article. the authors included research that specifically related to methods deployed for researching personcentred issues, rather than reports of findings from consumer involvement in research studies. interviews can be used to gather information as part of qualitative and quantitative data collection for research, education or quality assurance purposes. understanding the characteristics of the group of interest, identifying potential issues with recruitment, ethical concerns such as consent, privacy and confidentiality need to be addressed. the development of the interview schedule usually depends on the methodology. for example, semistructured questions with prompts may be more valuable for encouraging narratives, whereas a more structured schedule could promote eliciting specific information from participants. a previous study used face-to-face interviews to engage with immigrant patients and ascertain factors influencing their ability to trust healthcare providers. the findings outlined the complexity in developing rapport and creating trust in intercultural healthcare, a valuable outcome for improving person-centred care and overall quality in healthcare. additionally, sandvik and mccormack highlighted the importance of deploying a person-centred framework that promoted mutuality of understanding between the researcher and interviewee. skill in interviewing relies on the interviewer being able to prompt and elicit required information in partnership with participants without creating bias or leading the voice of the participant's opinions, experiences or descriptive accounts. initially, it is important to develop rapport and ensure the participant understands the purpose of the data collection and willingly consents. the setting of this type of interview may also alter the patient-researcher relationship. conducting an interview in the patient's home may differ from being carried out in healthcare environments and should be considered when planning. it is also important to ensure that the participant knows there are no right or wrong answers. studies have shown telephone or digital technology can be as effective as face-to-face interviews. [ ] [ ] [ ] however, other considerations are needed when telephone interviews are selected as a cost-effective method of data collection. as visual cues are lacking, it is important that patients can hear adequately, that there are no distractions and that there is an understanding of what the interviewer is asking. the participant may divulge information such as using a hearing aid or hands-free telephone that could be useful for ensuring a successful interview. preparation is important for both parties including confidence in using the technology for interviewing or providing assistance to the participant. a previous study comparing the use of telephone and face-to-face interviews found that there were some advantages to the telephone method. the authors suggest that participants who agree to be interviewed about sensitive topics may prefer the anonymity; it is easier to reach a wider participant group and that interviewer and participant safety is more easily controlled. following completion of an interview, it is important to ask the participant whether they would like to add any other information. this opportunity may be where participants provide vital information in their own words without the constraints of scheduled questions. similarly, asking participants whether they would like to be sent a copy of the transcript, so they can verify the discussion during the interview is important if the participant has difficulty with expression or other comorbidities, such as deafness or poor digital voice connection that may have hindered understanding during the interview. focus groups use group discussions, facilitated in a specific manner to gather informal information on a selected topic. in healthcare, focus groups can assist in generating a rich understanding of consumer experiences, beliefs and values, and developing strategic goals aligned with these outcomes. while one-on-one interviews can be suitable to obtain a measure of personal feelings and opinions, which can be beneficial for eliciting the opinions of minority groups, focus groups may be useful for obtaining opinions that are likely to reflect the majority. codesign using focus groups is an opportunity for patients and other consumers to consider and discuss their experiences within healthcare systems and services in a collaborative, participatory setting. theis et al held focus groups to determine what factors were important to patients during their healthcare journey. this information was then utilised to develop 'report cards' as a strategy for consumer choice in healthcare, commonly used in the usa to compare providers and health plans as well as incentivise quality improvement. the size of a focus group can determine the progression of discussions and, although sometimes difficult and unpredictable, ensuring optimum number of participants can assist with achieving the desired outcomes. six to eight people per group are generally recommended, however, focus groups can work with as little as or up to participants. to encourage respectful and active conversations among all participants, the moderation of focus groups requires a complex set of skills. the moderator needs to be able to guide the discussions without introducing bias such as leading the group or unconsciously providing positive or negative cues. they need to be able to encourage a relaxed and comfortable space for participants to engage. the composition of focus groups plays an important role in determining the data that will be gained from the discussions. for example, purposive sampling, considering the socioeconomic status of potential participants, has been shown to be beneficial in focus groups with chronically ill participants where it was demonstrated that societal norms are flexible and can be challenged and reformulated during focus group discussions. however, forming a representative focus group does not ensure participants will be equally represented in discussion. participants with higher education, health literacy and better knowledge of health systems have the potential to dominate conversations because they are better equipped to do so. individuals participating in focus groups of this nature often construct their 'patient view' by establishing themselves as knowledgeable, or by validating or challenging another's claims, depending on the dynamics of the group. a further limitation that can hinder focus group participation is poor moderation. tausch and menold describe inadequate moderation as a major barrier to successful focus group discussion for a variety of reasons including poor group dynamic, inability to use various communication methods, concentrated attention and poor preparation. there has been recent interest in the use of online focus groups which due to the covid- and social distancing measures may become more accepted by groups who could be impacted by potential exposure to pathogens by leaving home. additionally, consumers who are less mobile, lack transport or have difficulty attending a face to face focus group could join remotely to participate in data collection processes. conversely, the use of online focus groups may also preclude citizens who do not have access, or know how to use digital technology. additionally, some people with specific health challenges such as hearing loss may not be able to fully participate or prefer to use a traditional face to face approach where visual cues are more easily interpreted. a citizen jury is a participatory action research method that draws on the symbolism of a jury trial. it is a deliberative and inclusive approach for community engagement that is increasingly being used to gain understanding about issues of health concern and policy. key tenets of citizen juries are inclusivity, deliberation and active citizenship. it is these features that indicate citizen juries can allow representation and give a voice to those who might normally be less visible. in a citizen jury, evidence and opinions of experts are presented to a representative group (jury) who then deliberate to reach an outcome-for example, a consensus or priority list. citizen juries are based on the notion that any person, given the opportunity, time, support and resources is capable of decision making about complex technical, health, scientific and ethical issues, including diverse subjects such as population ethical issues; resource allocation; health policy; environmental health and community well-being. citizens' juries acknowledge representation is not just for the very literate and advantaged, but is, by intent, inclusive of people who experience wide-ranging disadvantage and inequity. the approach provides a presence or 'voice' of marginalised or minority groups so that their interests and perspectives are included. the process is overseen by a steering committee to guide question development, evidence presentation, oversight, stakeholder engagement and dissemination or implementation. recruitment occurs by word of mouth; via community or government organisations; electoral role or random digit dialling or more deliberative. jury size may be - citizens who meet for a period of time, usually between and days to 'hear, question, challenge and clarify expert witness testimony from a range of perspectives'. in healthcare, expert witnesses may include clinicians, policy-makers and health consumers who provide testimony about their personal experience of the aspect of healthcare under deliberation. a study involved a citizen jury to assess health needs in an area with high levels of social deprivation and social exclusion, including poverty; poor housing; high levels of death, illness and disability; alcohol and drug abuse; poor access to health services and low literacy. rejecting traditional methods of jury selection, recruitment occurred by way of talking to people at post offices, supermarkets, outside schools and at bus stops to ensure a diverse group reflecting the needs and interests of the community. this example of citizen juries shows how, with careful planning regarding recruitment and support during the period of participation, the method can be inclusive of some of the most marginalised in communities. photo elicitation interview (pei) refers to the use of photographs to trigger dialogue in the context of research interview. in pei, interview is stimulated and guided by images. these images can be chosen from archives or magazines, or created by the researcher or the participant. when photographs are taken by the participant, the method is referred to as native, reflexive or autodriven pei. autodriven pei shifts the balance of control over generation of data from the researcher to the participant. the increased control autodriven pei gives participants makes it a person-centred data collection method. images change the focus and energy of conversation and can lead to information and understandings that traditional oral interviews may not. language processing uses different areas of the brain compared with processing visual information, so the use of image-based research methods enables ways of creatively expressing thoughts, concepts and experiences. in doing so the methods can present a different way of telling as well as a different way of knowing. participatory interview activities can be more engaging and can facilitate a more relaxed atmosphere, reduces the power imbalance that can exist between participant and researcher. this participatory method is an effective way of achieving partnerships with patients and consumers within health services and provides a way for inclusion of those less visible and lacking representation. this was demonstrated in a study where children took photos to explain things that were important to them, thereby facilitating children's control over the data. when photos taken by patients form part of the data there are specific ethical considerations as participants are more visible, including, but not limited to, their physical open access appearance and representation. details of their lives are revealed making them much more identifiable than they might be in traditional research. robust approaches and clear guidance are required about the taking of and use of photographs and specific consent obtained around the use of images in papers, conferences and other forms of dissemination. video-reflexive ethnography (vre) is a participatory research method that regards consumer and clinician participants as experts and encourages their involvement in the research process. vre studies tend to start with participant observation and interviews to build relationships with participants. the method involves video recording episodes of clinical care, conducted in healthcare settings in real time, known as video ethnography and showing these video recordings back to participants, known as video-reflexivity to elicit their responses and collectively identify practice change. vre is a strength-based method and is about opening up or exnovating healthcare and recognising what works well in practice. exnovation is more than visualising a clinical episode of care, as its impact is evident in the videoreflexive sessions where clinical care is made explicit. wyer et al used vre to explore how patients nursed in isolation identify infection risks. wyer et al showed the patient a video recording of an episode of their care, and the video recording produced of the video-reflexive session was then shown to clinicians. video-reflexivity can act as a catalyst as clinicians become privy to how the patient experienced their care, triggering clinicians to identify strategies to change their practice to improve health service provision. this type of active patient involvement is vital for ensuring quality, person-centred healthcare initiatives. australian standards suggest that health service organisations must develop, implement and maintain systems to partner with consumers. capturing the consumer voice and experience is an important part of quality assurance across many facets of healthcare. the five methods described highlight a variety of useful tools for engagement with individuals who experience health challenges. the research method chosen to involve and represent consumers depends on the type of data that is necessary to guide maintenance of safety and quality assurance processes. collaborative management, including patient involvement in design, delivery and evaluation of health services has been shown to drive quality improvement as well as improve health economics and satisfy the legal and moral rights to person-centred care and autonomy in health. patients need to know they are a valuable part of the healthcare team and that their experiences matter. person-centred research methodologies capture the patient. interview methods such as semistructured interviews or the use of interview charts are supported for participants with health challenges as they can be a flexible and personal way of eliciting information for validation or as an experiential account. similarly, focus groups can provide a level of social cohesiveness and support while building relationships with participants to allow them to share their experiences. the use of citizen juries has capacity to involve the wider community in decisionmaking, regardless of their status, while pei and vre methods provide alternative participatory approaches enabling improved communication in healthcare. patient or consumer involvement can take multiple forms in healthcare and there is no single strategy that can be considered to reflect best practice. improving the health status and promoting the quality of life for individuals with ongoing health challenges necessitates cultural change at an individual, organisation and systems level. incorporating a variety of person-centred approaches in routine quality assurance activities provides a means of capturing the experiences of representative consumer groups for the benefit of all stakeholders to ensure high safety and quality in healthcare is maintained. twitter sarah jane prior @snezzajane contributors all authors were involved in the conception of this paper. each author led one section. a final draft of the paper was prepared by sjp and cm. all authors provided feedback for revisions of the paper and a final edited copy was prepared by sjp and cm. funding the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. competing interests none declared. patient and public involvement patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research. patient consent for publication not required. provenance and peer review not commissioned; externally peer reviewed. data availability statement all data relevant to the study are included in the article. open access this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by-nc . ) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by-nc/ . /. sarah jane prior http:// orcid. org/ - - - australian commission on safety and quality in health care. patient-centred care: improving quality and safety through partnerships with patients and consumers australian commission on safety and quality in health care. national safety and quality health service standards co-design of a patient experience survey for arthritis central intake: an example of meaningful patient engagement in healthcare design co-design for implementing patient participation in hospital services: a 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nan title: ecb : th european congess on biotechnology date: - - journal: j biotechnol doi: . /j.jbiotec. . . sha: doc_id: cord_uid: i reanan nan in the last years biotechnology has made tremendous progress in its different application fields: red biotechnology, the use of biological methods for medical purposes, is firmly established in the development of new drugs. the use of plant or green biotechnology is under controversial discussion in politics and public. nevertheless, genetically modified herbicide and insect resistant crops are cultivated to a large extent. industrial biotechnology, now often named white biotechnology, seems widely underestimated in the public perception. it includes all industrial processes for the production of chemical products and enzymes, which fully or partly rely on the biological toolbox of nature. white biotechnology processes are carried out in a contained environment, typically in a bioreactor in a dedicated industrial plant. well-known examples are the fermentative productions of antibiotics, amino acids, vitamins and enzymes, products related to medical, food and feed applications. many products like the amino acids glutamic acid, lysine, threonine and tryptophane are exclusively produced using microbes in large scale industrial processes. in other cases, like the water soluble vitamin b , biotechnological processes successfully replaced chemical productions, due to lower costs and improved ecoefficiency. in contrast to this, most industrial chemicals and polymers are produced by chemical synthesis based on oil and gas. however, there are some examples for bioproducts among industrial chemicals. the solvents acetone and butanol, for instance, were manufactured by fermentation for several decades in the last century. since the s these fermentations have been replaced by more efficient and cheaper chemical synthesis. recently, new pilot and production processes for biopolymers like pha or biomonomers like , -propanediol or lactic acid were announced by different companies. currently, ethanol is by far the largest white biotech product by volume. in brazil, where bioethanol is used as liquid transportation fuel, the annual production is in the range of mio m . bioethanol is of growing importance also in the united states. business consultants predict a tremendous growth of biotechnological products within the chemical industry. high prices for crude oil, dropping prices for renewable resources, and scientific progresses nourish the expectation that industrial biotechnology will replace many bulk chemicals. is this realistic? will we switch from a petrochemical industry to a biobased chemistry within the next years? based on economic considerations it can be stated that this is a long term goal. to achieve this it remains a scientific challenge to make renewable raw materials available for competitive bioproduction of bulk chemicals at low costs. conversion of lignocellulosic material to fermentation sugar may be a solution. also green biotechnology can contribute to the supply of cheap fermentation raw materials. innovative ideas for downstream processing or further chemical conversion of fermentation products are required to enter the chemical value chains. furthermore, the identification of new higher value bioproducts is a chance for short term successes in white biotechnology. enzyme and protein engineering has the potential to create new biomolecules, metabolic engineering can contribute to develop new metabolic pathways, may be even for unnatural compounds. by continuously increasing the efficiency and throughput of dna sequencing we, together with colleagues, have sequenced the human genome and the genomes of all the major model organisms. the challenge now centers on understanding these vast instruction sets. our ability to read these instructions must be enhanced through collection of key additional data sets. one productive path for delineating the functional sequences and inferring their function is comparative sequence. the mouse genome sequence, for example, led to estimates that only % of the human genome is functional. sequencing of an extensive set of additional mammalian genomes promises to define these functional sequences with a resolution of less than base pairs. on a different course, we have sequenced the chimpanzee genome to learn what has changed in the evolution of humans. beyond providing for the first time a catalog of the differences between the two genomes, the comparison of the chimpanzee and human genomes reveals the patterns of neutral mutation and regions that deviate from that. the talk will summarize these and related findings. the sequence of additional primate genomes will help delineate what has changed specifically in humans and add power to the analysis. ultimately, capturing human sequence variation and correlating with phenotypic variation will be required to understand function. but learning what these functional elements do requires new sets of experimental data. for this, we have turned to the nematode c. elegans. in this simple system most of the ∼ k genes have been defined and experimentally confirmed. beyond the hundreds of genes with already known mutants, two centers are systematically producing gene knockouts or rnai can be used to inhibit any gene temporarily. sequences of three caenorhabditis species are already available, and two more are underway. expression data has been collected for all the genes under many conditions and time points through development. to enhance the resolution of expression data and to simplify phenotypic analysis of embryonic mutants, we are developing a system that will automatically trace the cell lineage and assign gene expression to precise cells with high temporal resolution. the latest results with the system will be described. in the longer term, this and similar datasets should provide an understanding of how the genome specifies the form and behavior of the worm. uhlen department of biotechnlogy, albanova university center, royal institute of technology (kth), stockholm, sweden here, we present a new protein atlas database (www.proteinatlas.org) showing the expression and localization of human protein in normal and cancer tissues. the atlas is based on the use of antibodies (agaton et al., ) to generate high-resolution immunohistochemistry images representing normal tissues and different cancer types (uhlen and ponten, ) . each antibody is used to generate more than individual images and each image has been annotated by a pathologist (kampf et al., in press) . the database has been created by the swedish human proteome resource (hpr) and the program has been set-up to allow the exploration of the human proteome with antibody-based proteomics (nilsson et al., in press) . the basic concept is to generate, in a systematic and high-throughput manner (uhlen and ponten, ) , specific antibodies to all human proteins, and subsequently used these for functional analysis of the corresponding proteins in a wide range of assay platforms, including (i) a protein atlas for tissue profiles (kampf et al., in press) , (ii) specific probes to evaluate the functional role of individual proteins, and (iii) affinity reagents for purification of the specific proteins and their associated complexes for structural and biochemical analyses. ments, most effective source of variation was perturbation in growth medium, followed by perturbation in growth rate. effect of gene deletion on data variation was found to be less apparent when compared to other perturbations. a significant similarity in variation of metabolome and mrna data was observed, which may be used as the key point for integration of these two sets of data in functional analysis of genes. projection to latent structures (partial least squares, pls) is used for integration of transcriptome and metabolome data. comparison of pca and pls shows that linear model constructed via pls to predict the metabolome data does not make use of all the variation in transcriptome data. thus, pls allows the discrimination between the portion of gene expression change that affects the metabolome profile and the portion that is not directly effective on metabolome. both pca and pls can be used to detect the open reading frames (orfs) which are the main sources of variation in transcriptome data and/or effective on metabolome profile. extracellular metabolomics to accelerate the discovery of key genes involved in fibre degradation silas g. villas-bôas, geoffrey lane, graeme attwood, adrian cookson agresearch limited, grasslands research centre, tennent drive, private bag , palmerston north, new zealand. e-mail: silas.villas-boas@agresearch.co.nz (s.g. villas-bôas) the genome of the hemicellulose-degrading microbe clostridium proteoclasticum is been sequenced and an array of candidate genes with diverse activity relevant to fibre degradation have been identified by automated gene annotation methods. c. proteoclasticum falls within the butyrivibrio-pseudobutyrivibrio assemblage of rumen bacteria which are though to play an important role in the degradation of plant hemicellulose-lignin complexes which limit fibre degradation in the rumen. for new zealand it makes strategic sense to invest in microbial genomics efforts applied to agriculture where the country holds a strong competitive advantage and where ruminants constitute the vast majority of farmed animals. in conjunction with dna sequencing, proteomics and transcriptomics (micro-array analysis) we are using metabolomics as an additional functional genomics tool for gene discovery. we have established a footprinting approach for microbial metabolome analysis focused mainly on metabolic intermediates of polysaccharide degradation to provide quantitative information on end products of fibre-degrading enzymes. a gc-ms method has been developed that is able to resolve complex biological mixtures containing mono-, di, and oligosaccharides, in addition to a series of organic acids. we are currently phenotyping a series of c. proteoclasticum mutants to validate our analytical methodology and we are going to fully characterize the fibrolytic ability of c. proteoclasticum to be compared with other fibre-degrading microbes. we believe that our metabolomics data will complement current proteomic analysis of fibre-degrading enzymes and micro-array analysis of gene expression from a series of mutants by providing direct evidence of the metabolic function of key genes involved in fibre-degradation processes. many gram-negative bacteria utilize cell-to-cell communication systems that rely on diffusible n-acyl homoserine lactone (ahl) signal molecules to monitor the size of the population in a process known as quorum sensing (qs). in human pathogens this form of gene regulation ensures that the cells remain invisible to the immune system of the host until the pathogen has reached a critical population density sufficient to overwhelm host defenses and to establish the infection. the qs regulon of pseudomonas aeruginosa and burkholderia cepacia, two important pathogens for patients suffering from cystic fibrosis, has been studied by proteome analyses. comparative twodimensional gelelectrophoresis of pre-fractionated protein mixtures (extra-, surface-, and intracellular proteins) coupled to mass spectrometry analysis or n-terminal sequencing has been employed to recognize and identify qs-controlled proteins. our findings strongly support the importance of ahl-mediated cell-cell-communication as a global regulatory system and suggest that qs control also operates via post-translational mechanisms. as qs has been proven to be a central regulator for the expression of pathogenic traits and biofilm formation in opportunistic human pathogens it represents a highly attractive target for the development of novel anti-infective compounds. functional genomics technologies (transcriptomics and proteomics) have been exploited to validate the target specificity of natural and synthetic qs inhibitors, thus having a great potential as alternative therapeutics for the treatment of bacterial infections. modeling cell cycle complex formation from high-throughput data sets lars juhl jensen european molecular biology laboratory, meyerhofstrasse , heidelberg, germany. e-mail: jensen@embl. de to analyze the dynamics of protein complexes during the mitotic cell cycle, we integrated data on protein interactions and gene expression. the resulting time-dependent interaction network for the first time places both periodically and constitutively expressed proteins in a temporal cell cycle context, thereby revealing novel components and modules. we discover that most complexes consist of both periodically and constitutively expressed subunits, suggesting that the former control complex activity by a mechanism of just-in-time assembly. consistent with this, we show that additional regulation through targeted degradation and phosphorylation by cdk (cdc p) specifically affects the periodically expressed proteins. alessandra luchini, andrea callegaro, silvio bicciato department of chemical engineering processes, university of padova, padova, italy. e-mail: alessandra.luchini@unipd.it (a. luchini) since transcriptional control is the result of complex networks, analyzing dynamical states of gene expression is of paramount importance to detect the multivariate nature of biological mechanisms. although hundreds of studies fully demonstrated the relevancy of microarrays in describing different physiological conditions, to reconstruct complex interaction pathways it is necessary to analyze the temporal evolution of transcriptional states. however, a robust experimental design for identifying differentially expressed genes over a temporal window would require large amounts of microarrays. unfortunately, replicates for each time point and experimental condition are not always available, because of cost limitations and/or biological samples scarcity. in addition, common data analysis tools, like anova, require replicates and disregard correlation structure among times. we present a method for the identification of differentially expressed genes in un-replicated time-course experiments. the procedure does not assume any model or distribution function, takes into account the correlation of data, and does not require sample replicates at the various time points, other than the presence of an initial time point for all analyzed conditions. the identification of differentially expressed genes as the result of a system perturbation is formally stated as a hypothesis testing problem in which a defined statistic is used to rank transcripts in order of evidence against the null hypothesis. specifically, (i) data are structured so that measurements are correlated in time, within the same biological condition; (ii) the null hypothesis is formulated so that changes in expression levels at different time points are equivalent; (iii) time point t represents the system before the perturbation. therefore, modulated genes are detected testing the statistical significance of expression differences between physiological states at each time point, once corrected by the variability at t , and given an empirical null distribution constructed using permutations. statistical significance is assessed by the q-value. the method has been tested on time-course microarray experiments aimed at studying the temporal changes of gene expression in: (i) skeletal muscle cells treated with a histone deacetylase inhibitor (iezzi et al., ) and (ii) immature mouse dendritic cells (dc) exposed to larval and egg stages of s. mansoni (trottein et al., ) . differentially expressed genes, identified using the proposed algorithm, have been compared with results obtained from anova model and sam paired test. the biological significance and soundness of selected transcripts was also verified using global functional profiling by means of ontotools. results demonstrate that this novel procedure allows the identification of biologically relevant genes using half of the replicates required by standard model-based approaches. carbon sources. interesting data on the expression profile of the sty and paa genes in pseudomonas sp. y have been obtained, and have raised new questions on styrene and paa degradation by this bacterium. the calcium-dependent antibiotic (cda) is a lipopeptide synthesised non-ribosomally and produced by streptomyces coelicolor a ( ). cda contains several non-proteinogenic amino acid residues. hydroxyphenylglycine ( -hpg) is one of the unusual amino acids in the structure of the cda and vancomycin groups of antibiotics. for the members of the vancomycin group of antibiotics, the -hpg residue plays crucial roles in the structure and function of the final glycopeptide antibiotic. to reveal the putative biosynthetic pathway of this amino acid in cda, a standard "double crossover replacement strategy" was used to delete -hydroxymandelic acid synthase ( -hmas, encoded by hpd) from s. coelicolor mt and , using the delivery plasmid pzmh . there was no cda production in the disrupted strains. plates containing a gradient of hydroxymandelic acid were used to restore cda production in both s. coelicolor mt hpd and hpd. exogenous supply of -hydroxyl phenylglyoxylate and -hydroxyphenylglycine reestablished cda production by the hpd mutant. feeding analogs of these precursors to the mutant resulted in the directed biosynthesis of novel lipopeptides with modified arylglycine residues (mutasynthesis). a cxcl tandem repeat promoter polymorphism is associated with susceptibility to severe sepsis in the spanish population n. maca-meyer , c. flores , l. pérez-méndez , r. sangüesa , e. espinosa , j. villar : research institute, hospital universitario n.s. de candelaria, s/c tenerife , spain; department of anesthesiology, hospital universitario n. s. de candelaria, s/c tenerife , spain. e-mail: nmacame@ull.es (n. maca-meyer) sepsis describes a complex clinical syndrome resulting from a systemic inflammatory response to bacteria, and remains an important cause of mortality in the intensive care unit. cxcl chemokine (or mip- ) exhibits a pivotal role in the immune response, and several functional studies in animal models of sepsis have catalogued cxcl as a candidate gene for the development of sepsis. we have performed a case-control association study of cxcl gene variants and susceptibility to severe sepsis in hospitalised patients and healthy individuals. after the examination of linkage disequilibrium in the region, we analysed whether two promoter polymorphisms (snp rs and a newly described polymorphic short tandem repeat d s ) were associated with the syndrome. we found a significant association of common variants at d s with the development of severe sepsis (heterozygote carriers or . ; % ci . - . , and homozygote carriers or . ; % ci . - . ; mantel-haenszel χ test for linear trend p = . ). the risks remained significant even after a genomic control adjustment, based on additional genotyped polymorphisms not linked to the candidate gene. these preliminary results suggest that cxcl gene variants may contribute to the development of severe sepsis. kasper møller , ana paula oliveira , jens nielsen , mark johnston : center for microbial biotechnology, biocentrum, technical university of denmark, denmark; department of genetics, school of medicine, washington university, st. louis, usa glucose is the preferred carbon and energy source for most cells. in saccharomyces cerevisiae, a complex regulatory network ensures that s. cerevisiae ferments glucose to ethanol even in the presence of oxygen. to obtain a better understanding of this crabtree effect and the logic of the glucose signalling network in s. cerevisiae, we are analyzing glucose sensing and signalling in the related species saccharomyces kluyveri, which exhibits much less of a crabtree effect (it prefers not to ferment glucose when oxygen is available). we show that there are only two major glucose transporters in s. kluyveri, and that these are regulated in response to the availability of glucose via a glucose sensor and a signalling pathway similar to the glucose induction (rgt /snf -rgt ) pathway in s. cerevisiae. we have used dna-microarrays for s. kluyveri to find targets of the s. kluyveri glucose induction pathway, as well as to evaluate the global response to a change in environment from growth on ethanol to growth on glucose. this study identifies a number of differences in the regulation of glucose uptake and global responses to glucose between s. kluyveri and s. cerevisiae, which may contribute to their different glucose metabolism. detection and analysis of microrna using lna probes nana jacobsen, christian lomholt, peter mouritzen, peter stein nielsen, mikkel noerholm exiqon a/s, bygstubben , dk- vedbaek, denmark. e-mail: mouritzen@exiqon.com (p. mouritzen) micrornas are a class of short endogenous rnas that act as post-transcriptional modulators of gene expression. growing evidence suggest that micrornas exhibit a wide variety of regulatory functions and exert significant effects on cell growth, development, and differentiation. recent studies have shown that human microrna genes are frequently located in cancer associated genomic regions and perturbed microrna expression patterns have been observed in many malignant tumors. we have exploited the significantly improved hybridization properties of lna oligonucleotides against rna targets to design lna-modified dna probes for detection of different micrornas in animal and plants by northern blot analysis, microarray hybridization and in situ hybridization. we will describe the results obtained from detection and analysis of different micrornas in c. elegans, zebrafish, mouse, and plants. in addition, we will describe a novel lna-based method for expression profiling of mature micrornas by quantitative rt-pcr. expression profile of the sty and paa genes in pseudomonas sp. y by means of dna microarrays david bartolomé-martín , david juck , m a teresa del peso-santos , charles w. greer , julián perera : departamento de bioquímica y biología molecular i, facultad de ciencias biológicas, universidad complutense de madrid, madrid, spain; environmental microbiology group, biotechnology research institute, national research council canada, montréal, que., canada h p r . e-mail: perera@bio.ucm.es (j. perera) dna microarrays are a new and powerful tool to study gene expression in very diverse systems. environmental biotechnology and biodegradation are some of the fields of research where this technology may be very promising. pseudomonas sp. y is a bacterium able to grow in minimal medium plus either styrene (sty) or phenylacetic acid (paa) as the sole carbon and energy sources. this bacterium is the only organism where the genes that code for both the upper (sty genes) and the lower (paa genes) catabolic pathways for the styrene degradation have been described till now. it is unique in having two active copies of the genes encoding the lower pathway (paa and paa gene clusters). we have designed a dna microarray with the sty and paa genes in order to analyse their expression in the wild type pseudomonas sp. y , in p. sp. y t (a paa deletion mutant) and in p. sp. y c (a crc gene mutant). this analysis has been performed on bacterial cultures grown in media with different carbon sources. interesting data on the expression profile of the sty and paa genes in pseudomonas sp. y have been obtained, and have raised new questions on styrene and paa degradation by this bacterium. dynamics in induced repression of phosphomannose isomerase pmi gene of saccharomyces cerevisiae anssi törmä , , juha-pekka pitkänen , , laura huopaniemi , risto renkonen : medicel ltd., haartmaninkatu , helsinki, finland; rational drug design program, department of bacteriology and immunology, haartman institute and biomedicum, university of helsinki, p.o. box , helsinki, finland. e-mail: juhapekka.pitkanen@medicel.com (j.-p. pitkänen) gdp-mannose is the precursor of cell wall biosynthesis in s. cerevisiae. to understand the system level role of gdp-mannose, we studied a conditional knock-out strain of the key enzyme in its synthesis; pmi . the experimental procedure allowed us to study the order of mechanisms the cells launch in order to adjust to a sudden malfunction in the metabolic machinery. we collected samples from continuous cultivations over h and measured genome-wide gene expression levels, enzyme activities, and concentrations of intracellular metabolites. for sampling we have built a sample robot, which automatically takes and preserves the samples. in order to carry out this magnitude of experimentations and generated data, we have constructed a proprietary software platform to handle all the phases from project management in wet-lab to workflow and pathway management in in silico. after normalization and clustering, significantly changed genes and metabolites were searched for enrichment in biological processes and molecular complexes. further, gene expression levels, metabolite concentrations, and enzyme activities were searched against each other for causality over time. overall, we focused on thorough analysis of our own data and known database data in order to reward our efforts with knowledge. at the transcriptome level, repression of pmi led to two major types of activation profiles, one peaking at the time when pmi p activity and gdp-mannose were depleted and the other later during recovery from the perturbation. the primary response was most enriched with genes known to play roles in mating and filamentous growth and associated with the transcription factors ste p, tec p, dig p, and mcm p, whereas the secondary response consisted of genes involved in carbon metabolism and associated with the general stress response regulators msn p and msn p. skn p, a high-level transcription factor was associated with both the primary and the secondary response, consistent with its suggested role of coordinating environmental responses and developmental processes. transcriptome of pig ovarian cells: discriminant genes involved in follicular development bonnet a., le cao k.a., low-so g., san cristobal m., tosser-klopp g., hatey f. laboratoire de génétique cellulaire, centre inra de toulouse, castanet-tolosan , france in order to identify genes and gene networks involved in pig ovarian follicular development, we built subtractive suppressive hybridization libraries (ssh) from granulosa cells of healthy follicles (small, medium or large). the rna isolated from these cells was used to hybridize cdna nylon micro-arrays. data analysis using a gaussian linear mixed model showed that % of the variability is due to the genes. two hundred fifty one regulated genes (from the expressed) were identified and clustered into three groups according to the follicle size. moreover, we found previously identified genes such as aromatase, igfbp which supported the validity of our experimental model. ramdom forest analysis put forward the most discriminant genes between the three follicle classes. this study put forward gene sets such as those involved in cell modeling, regulation of transcription, apoptosis during follicle growth. the next step will be to describe more precisely the spatio-temporal expression patterns at the mrna levels of the genes identified by these experiments. microalgae constitute a significant source of valuable natural products, e.g. sulfated polysaccharides, polyunsaturated fatty acids, and phycobiliproteins that find applications in wide range of industries, including food, pharmaceutical, agricultural and cosmetics. however, genomic and molecular genetic studies of microalgae lag far behind those of higher plants. in order to accelerate red microalgal genomic studies by taking advantage of current genomics and post-genomic technologies, we have generated expressed sequence tag (est) databases of two red microalgae porphyridium sp. and dixoniella grisea grown under various physiological conditions. to date we have sequenced and ests of porphyridium sp. and d. grisea, respectively. the sequence assembly resulted into, ca. non-redundant unigenes for each microalga, only % of which were identified by similarity to sequences in the public databases. porphyridium sp. and d. grisea unigenes were compared with the whole-genome predicted proteomes of three microalgae and those of representative eukaryotic and prokaryotic organisms. both microalgae have highest similarity to the red microalga cyanidioschyzon merolae. the order of sequence similarity to other organisms examined was arabidopsis thaliana, oryza sativa, chlamydomonas reinhardtii, thalassiosira pseudonana (diatom), saccharomyces cerevisiae, caenorhabditis elegans, archaea and cyanobacteria. although red microalgae are considered as phylogenetic bridge between prokaryotes and eukaryotes, our data show that the red microalgae have strong similarity to eukaryotes and only distant similarity to prokaryotes. gene expression profiles were studied by analyzing cdna and subtraction libraries constructed from algae grown under various physiological conditions. we observed that top three most abundant ests in the stationary phase of porphyridium sp. were adp ribosylation factor like- , flavohemoglobin and adp ribosylation factor- . in addition, we have identified several genes which were specific to nitrate-and sulfate starvation. the sarco(endo)plasmic reticulum ca + -atpase (serca, "the calcium pump"), is responsible for pumping the ca + released into the cytoplasm during muscle contraction back into the sarcoplasmic reticulum store while proton are pumped the opposite way as counter-cations. these transport processes go against the concentration gradients and are therefore energy consuming. the energy is derived from atp hydrolysis via formation and break-down of a phospho-enzyme intermediate. over the last year a number of new crystal structures have been published which have added to our understanding of how this task is accomplished, which provides an impressive insight to the mechanism of a molecular pump. rhomboids are a family of intramembrane serine proteases that are widely conserved throughout evolution. among diverse functions discovered so far, rhomboids participate in intercellular signalling, parasite invasion, membrane dynamics and bacterial quorum sensing, making them potentially valuable therapeutic targets. the identification of physiological substrates and of selective inhibitors will be key towards their evaluation as drug targets. we have developed an in vitro cleavage assay to monitor rhomboid activity in the detergent solubilised state, enabling the first isolation of a highly pure rhomboid with catalytic activity. analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad, and gives insights into subsidiary functions like ligand binding and water supply. this work was supported principally by embo and the mrc. structure and target-specificity of thioredoxin h kenji maeda , anette henriksen , per hägglund , christine finnie , birte svensson : biochemistry and nutrition group, biocentrum-dtu, technical university of denmark, dk- kgs. lyngby, denmark; biostructure group, carlsberg laboratory, dk- valby, denmark. e-mail: kenji@biocentrum.dtu.dk (k. maeda) thioredoxins are ubiquitous small proteins with protein disulphide reductase activity. thioredoxins can alter the structures and the activities of various target proteins by reducing their disulphide bonds. seeds of several plants are abundant in cytosolic thioredoxins referred as h-type. in barley, two thioredoxin h isoforms, hvtrxh and hvtrxh that share % sequence identity but differ in temporal and spatial distributions were previously identified and characterised. in the present study, the relationship between structures and targetspecificities of h-type thioredoxins are analysed. the d-structures of hvtrxh and hvtrxh are determined by x-ray crystallography as the first crystal structures of thioredoxin h. comparison of the structures shows that the majority of solvent exposed residues near the active sites are conserved between the two isoforms. this is in agreement with previously observed similarity in target-specificity of the two isoforms. thioredoxins from organisms distantly related to barley, such as e. coli, have highly similar folds but different surface charge distributions compared to barley thioredoxins. a comparison of the target-specificities of hvtrxh , hvtrxh , e. coli thioredoxin and several thioredoxin mutants will be attempted to reveal the structural features that influence specificity of barley thioredoxin h isoforms. enbrel is a dimeric fusion protein consisting of the extracellular ligand binding portion of the human kda (p ) tumor necrosis factor receptor (tnfr) linked to the fc portion of human igg . the cho-expressed molecule contains both n-and o-linked oligosaccharides with a total carbohydrate content of % by mass. the o-linked oligosaccharides were released by hydrazinolysis and their structure determined by exoglycosidase sequencing and maldi-tof mass spectrometry. to locate precisely the o-linked sites, the glycosylation heterogeneity of tnfr:fc was simplified by treatment with n-acetyl neuraminidase and n-glycanase. the remaining molecule, which only carries core o-linked glycan structures, was cleaved by trypsin and analyzed by lc-ms. precise localization of o-glycosylation sites was determined based on the concept of a specific modification of the o-glycosylated serine into aminopropenoic acid and o-glycosylated threonine into -amino- butenoic acid. the deficit in mass resulting from this transformation was the marker used to localize the modified residues on the peptides by tandem mass spectrometry sequencing (ms-ms). ms-ms spectrum of enbrel glycopeptides were interpreted based on the presence of -aminopropenoic acid and -amino- -butenoic acid, resulting in a complete map of o-linked glycans precisely located at different sites. anu mursula, beatrix fahnert, sari krapu, eija-riitta hämäläinen, ritva isomäki, peter neubauer bioprocess engineering laboratory and biocenter oulu, university of oulu, oulu, finland. e-mail: anu.mursula@oulu.fi (a. mursula) wnt proteins form a highly conserved family of secreted glycoproteins important in cell-cell signaling events during embryogenesis and adult tissue maintenance. impairments within this complex signaling pathway can lead for example to developmental defects in embryos, degenerative diseases and cancer. respectively, wnt proteins can be used as tools in basic research concerning wnt function, developmental biology, screening for interacting compounds, and for medical applications (e.g. therapeutics, stem cells). hence, recombinant wnts provide a valuable basis for these purposes. however, production of recombinant wnt proteins is challenging, because they contain multiple disulfide bonds making the folding very difficult. a process for production of murine wnt- in e. coli has been developed in our laboratory. the knowledge obtained from this research has also been applied to the expression of other wnts, namely wnt- and wnt- , and can be used to approach other cysteine-rich proteins as well. since the expression level of wnt proteins is rather low so far, tools for monitoring and optimizing the production process have been established. by means of this sandwich hybridization method the level of target (wnt) mrna can be measured. the technique has already been applied to analyzing wnt- mrna levels. probes also for wnt- and wnt- have been generated. thus, transcription of wnt genes in all kind of cells (e.g. tissue, recombinant hosts) in general as well as kinetics of transcription can be studied using these tools. in growth factor signaling, stimulation of cell-surface receptors first triggers activation of the receptor itself and then of a large number of intracellular effector molecules. the stimulus is integrated with a host of other cellular processes, leading to cytoskeletal changes, activating transcriptional programs in the nucleus and ultimately resulting in cell proliferation, differentiation or motility. classical signaling pathways and networks depict potential protein-protein interactions only in a static form. in the cell, these interactions are dynamic and occur in an ordered fashion. here, we apply a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance in order to study the global dynamics of signaling events. briefly, three cell populations are metabolically labeled with either normal arginine or a c substituted form, or a c n variant (stable isotope labeling by amino acids in cell culture, silac). each population was then stimulated with egf for a different time period and tyrosine phosphorylated proteins were affinity purified with anti-phosphotyrosine antibodies. the proteins from the precipitated complexes were quantitatively analyzed and identified using lc-ms/ms. arginine containing peptides occurred in three forms, directly indicating protein activation at the corre-sponding time point. combination of two experiments sharing one common time point of activation then generated five-point dynamic profiles. from the proteins quantified, we identified signaling proteins, including virtually all known egfr substrates and novel effectors, and the time course of their activation upon egf stimulation. discriminating proteins involved in the signaling network from unspecific binders was straightforward as these presented an activation profile. we have now further extended this study by directly measuring in vivo phosphorylation sites in response to growth factor stimulation and monitoring the time evolution of the phosphorylation events. finally, we determined and quantitatively compared the global egf and pdgf tyrosine phosphoproteomes in human mesenchymal stem cells and revealed a control point in their differentiation into bone-forming cells. such global activation profiles provide a novel perspective in cell signaling and will be crucial to model the highly dynamic signaling networks in a systems biology approach. klaus schneider , dave g smith , steven skaper , alastair d. reith : discovery research, glaxosmithkline, coldharbour road, harlow, essex, uk; neurology & gastrointestinal cedd, glaxo-smithkline, coldharbour road, harlow, essex, uk over the last years, progress in signal transduction research has revealed an astonishing degree of complexity in cell signalling which is manifested in positive and negative regulations and feedback loops within signalling pathways and by cross-talks between pathways, all of which are highly cell-type dependent. it has become evident that protein phosphorylation by protein kinases plays a major role in this complex regulation of cell signalling (hunter, ) . due to the importance of signal transduction in disease processes, many protein kinases may constitute key targets for disease intervention. yet, the lack of a full understanding of the regulation, the activation and, importantly, of downstream substrates of particular protein kinases requires often more detailed studies before initiation of resource-intensive efforts to find disease-modifying molecules. technologies for the study of protein kinase signalling include p labelling, mutational and knock-out studies and more recently rna interference. these tools are complemented by approaches that are based on proteomic technologies developed over the course of the last years. in this presentation, the scope of proteomics technologies to contribute to an understanding of kinase signalling will be discussed. an overview of available technologies will be given and results will be presented from a proteomic study of glycogen-synthase kinase (gsk ) signalling (coghlan et al., ) . novel findings will be presented on a study of gsk inhibition in a primary neuronal cell line by differential d gel electrophoresis, which resulted in the identification of more than proteins that were significantly regulated. proteome analysis is typically done by nanospray lc/ms in order to achieve higher sensitivity and thus a greater number of protein identifications. however, nano-scale lc systems can be more challenging to use and maintain. to obtain the best chromatographic performance, connections must be made carefully to minimize band broadening. improved chromatographic performance can enhance the mass spectrometric results by tandem ms as a greater number of peptides can be detected. a microfluidic chip-based system has been developed (yin et al., ) that minimizes the number of connections and the delay volumes. this work evaluates the performance of this device against the traditional nanospray approach. a yeast extract sample was separated by sds-page and bands were excised from the gel for further analysis. after in-gel digestion, the sample was analyzed by both traditional nanospray and the microfluidicbased chip device. after protein database searching, the identified proteins and the protein sequence coverage's were compared for the two approaches. the microfluidic device was demonstrated to be equivalent or better compared to the traditional approach. yin, h., killeen, k., brennen, r., et al., . anal. chem. , - . plant cytochromes p (p s) play key roles in the biosynthesis of most bioactive compounds with agronomic and therapeutic applications. a collection of about plant p s was expressed in yeast. the cdnas were isolated from the model plant with a sequenced genome arabidopsis thaliana, some others from wheat, helianthus tuberosus or vicia sativa. they were expressed under the control of a galactose-inducible promoter in an engineered strain of saccharomyces cerevisiae in which the gene of the native p reductase was replaced with the gene of a p reductase from a. thaliana under the control of the same galactose-inducible promoter, in order to provide an optimal context for plant p expression and activity (pompon et al., ) . an original procedure was designed for the high-throughput functional screening of this enzyme collection. it is based on the detection of oxygen consumed during the catalytic reaction by a fluorochrome embedded in the bottom of the microwell plates. this method was validated using several recombinant p s of known activity. it also allows for a very efficient screening for enzyme inhibitors. the advantages and limits of the method will be discussed. this work was carried out with the support of génoplante programme (no ) . reference pompon, d., louerat, b., bronine, a., urban, p., . methods enzymol , - . folding of a bacterial membrane protein studied by protein engineering daniel e.otzen, pankaj sehgal, peter a. christensen department of life sciences, aalborg university, sohngaardsholmsvej , dk - aalborg. e-mail: dao@bio.aau.dk (d.e. otzen) we have carried out a kinetic analysis of the folding of the -helix transmembrane protein dsbb in a mixed micelle system consisting of varying molar ratios of sodium dodecyl sulfate and dodecyl maltoside. this analysis incorporates both folding and unfolding rates, making it possible to determine both the stability of the native state and the process by which the protein folds. the analysis also takes into account the composition of the mixed micelles, which is different from the bulk detergent composition. refolding and unfolding are consistent with a three-state folding scheme involving the sdsdenatured state, the native state and an unfolding intermediate that accumulates only under unfolding conditions at high mole fractions of sds. the temperature-dependence of the folding reaction displays an unusual decrease in heat capacity accompanying unfolding, which probably reflects the amphiphilic environment of the membrane protein. destabilization of dsbb by different short-chain alcohols correlates very well with the alcohols' respective hydrophobicities. data from a series of ala-scanning mutants tentatively identify a nucleus for folding, which is relatively diffuse and involves all four helices. we are currently complementing this work with studies of the association of peptides corresponding to individual transmembrane segments of dsbb. the reca protein of e. coli plays a crucial role in homologous recombination and dna repair. the recombination process takes place in a filamentous complex, in which the protein monomers are arranged in a helical manner around a single-stranded dna (ss-dna). in the presence of atp the filament can accommodate a second, double-stranded dna (ds-dna) and the strand exchange reaction can occur. the three-dimensional structure of reca itself and its complex with adp have been determined by x-ray crystallography. the active nucleoprotein filament, however, has only been studied at low resolution. both electron microscopy (em) and small-angle neutron scattering (sans) indicate significant differences between the structures of the active nucleoprotein filament and the compressed, inactive filament of only reca. we have presented a structural model of the reca protein in its active filament with ss-dna, using data obtained by linear dichroism (ld) polarized-light spectroscopy, based on a technique, we call "site-specific linear dichroism", which allows the orientation of a set of amino acids to be determined from ld data by systematic modification of the protein. here, we show that ld data of the nucleoprotein filament with ds-dna is over all similar to the data of the complex with ss-dna, indicating that the orientation as well as internal structure of reca in the active filament is not significantly altered when the bound dna is changed from single-stranded to double-stranded. this result supports the idea that the strand exchange reaction occurs without large conformational change of the reca protein. the choline-binding modules: a powerful biotechnological tool jesús m. sanz instituto de biología molecular y celular, universidad miguel hernández, elche, spain choline-binding modules (chbms) are present in some virulence factors of streptococcus pneumoniae (pneumococcus). the most extensively studied chbm is c-lyta, the carboxy-terminal domain of the pneumococcal cell-wall amidase lyta. the three-dimensional structure of choline-ligated c-lyta is built up from six loop-hairpin structures ("choline binding repeats", chbrs) forming a left-handed ␤-solenoid with four choline binding sites. although the structure of the ligand-free form is not yet known, our folding studies suggest that it is more loosely packed, with a partially unfolded amino-terminal region and a stable carboxy-terminal moiety that is extremely resistant to chemical denaturation (maestro and sanz, ) . the affinity of c-lyta for choline and other structural analogues allows its use as an efficient affinity tag for overexpression, immobilization and single-step purification of proteins of biomedical interest (c-lytag fusion protein purification system). this system presents many advantages when compared to current commercial methods, namely simplicity, compatibility with buffers and robustness. the availability of multiple supports that specifically bind chbms (such as multiwell plates) has recently allowed the development of a new procedure for the immobilization of c-lyta-containing hybrid proteins that may be used in proteomics, diagnostics and peptide display. in this communication, we present our last results about the stability, folding and engineering of c-lyta, together with a compendium of the current biotechnological potential of this protein, and highlight the productive link between basic molecular studies and their application. many of the modern approaches for studying disease compare steady state functions, such as repair, growth, and regulated gene expression within the various biological compartments organised by specialized function, be it mitochondria or blood vessels. the assignment of protein identities, which are linked to key biological mechanisms, which are associated with disease processes and disease progressions are an important area of this work (marko-varga and fehniger, ) . today, the technology available for studying proteome expression and resolving exact protein and peptide identities in complex mixtures of biological samples allows global protein expression within cells, fluids, and tissue to be approached with confidence. this confidence is due in part to reproducible repetitive sampling and analysis technologies including robotics data acquisition and high level mass spectrometry including both laser-desorbtion and electro spray ionisation. the precision in defining differences between normal and diseased steady states is aided by the creation of compiled reference and master data sets and by new methods for multiplexing the analysis of samples in groups. the establishment of key representative reference proteome systems representing the dynamic changes in protein expression during disease will be vital to the interpretation of changes observed in specific samplings of disease states and specific cells obtained from these samples. the creation of reference databases of proteins linked to disease pathways will play an important role in furthering our understanding of the "proteome of disease". examples will be given where protein expression patterns have been generated from compartments within tissue sections. marko-varga, g., fehniger, t.e., . j. proteome res. , - . adaptation of the saccharomyces cerevisiae proteome to nutrient limitations studied by metabolic stable isotope labeling and mass spectrometry albert j.r. heck netherlands proteomics centre and utrecht university, the netherlands. e-mail: heck@npc.genomics.nl. url: www.netherlandsproteomicscentre.nl one of the major aims of proteomics is to provide quantitative data on differential protein expression levels. recently, mass spectrometry-based methods have been introduced that can provide quantitative data on differential protein expression, mostly using stable isotope labeling (goshe and smith, ) . we opted for metabolic labeling as this provides efficient means to quantify differential protein expression, and has the advantage that all proteins are labeled universally (romijn et al., ) . in their natural habitat microorganisms encounter non-optimal growth conditions and often growth is limited by one nutrient. microorganisms need to respond rapidly to changes in the environment in order to survive. in the present study, we investigate the proteome response of chemostat cultivated wildtype saccharomyces cerevisiae to two different nutrient limitations, namely carbon and nitrogen limitation. yeast was metabolically labeled in well-controlled chemostat cultures. n and n labeled proteins were separated using d gel electrophoresis followed by rp-lc-esi-ms on a lc-q. relative quantification was performed by using relex software (maccoss et al., ) . we quantified proteins, using on average peptide peak pairs per protein. this analysis revealed that proteins showed a significant increase/decrease in expression level. the functional annotation of these proteins revealed that the yeast cells change expression levels of enzymes involved in metabolism of the growth-limiting compound. the protein expression ratios were compared with corresponding transcript levels. moreover, we compared the accuracy of quantifica-profiles mainly reflected differences in cellular origins in addition to different functional roles. mass spectrometric analysis identified proteins pertaining to several functional classes, i.e. acute phase proteins, antioxidant proteins and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization and in lipid metabolism. several proteins not previously implicated in nerve regeneration were identified, e.g. translationally-controlled tumor protein, annexin a / , vitamin d-binding protein, ␣-crystallin b, ␣-synuclein, dimethylargininases and reticulocalbin. real-time pcr analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. in conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants. yeasts plasma membrane macromolecular components involved in stress resistance paola branduardi, paola paganoni, danilo porro dipartimento di biotecnologie e bioscienze, università degli studi di milano-bicocca, piazza della scienza, - milano, italy. e-mail: paola.branduardi@unimib.it (p. branduardi) the plasma membrane is a universal structure of living cells constituting an essential barrier dividing and defining the intracellular from the extracellular environment. it is consequently easy to deduce the crucial role played by said structure for any cell of any living organism, and especially for unicellular organisms, since all the information deriving from the external environment as well as many of the consequent cellular responses have to pass through this barrier. unicellular organisms, thanks to easy manipulation and cultivation techniques, can represent a very useful model for studying the plasma membrane function and response. in addition microorganisms, and among them yeasts, can be considered advantageous cell factories for recombinant productions. in this contest, the implementation of any process of production has to take into account, among others, the response and the tolerance of the host to the external environment. from these considerations derives the interest of our group to analyse the main macromolecular components of yeasts plasma membranes (proteins, lipoproteins and lipids), isolated from cells grown under different stress conditions, with particular attention to acidic environments. here, we present our recent data about separation and identification (by sequencing analyses) of lipoproteins isolated from the conventional yeast saccharomyces cerevisiae as well as from the non-conventional and acid tolerant yeast zygosaccharomyces bailii cell cultures grown in different conditions. in parallel, the protein fraction is under evaluation through a differential d proteomic approach and consequent analyses. effect of fungal polysaccharides on the expression of pancreatic proteins in streptozotocin-induced diabetic rats sang woo kim , hye jin hwang , kwang bon koo , jang won choi , jong won yun * : department of biotechnology; department of bioindustry, daegu university, kyungsan, in an attempt to search novel biomarkers for monitoring diabetes prognosis, we examined the influence of the hypoglycemic fungal extracellular polysaccharides (eps) on the differential expression of pancreatic proteins in streptozotocin-induced diabetic rats. the results of diabetic study revealed that orally administrated eps exhibited excellent hypoglycemic effect, lowering the average plasma glucose level of the diabetic rats to . %. pancreatic proteome were analyzed by -de system, which separated more than individual spots. the -de analysis demonstrated that thirty-four proteins from a total of about matched spots were differentially expressed, of which spots were identified as the proteins whose expression has previously been associated with diabetes. twenty-two overexpressed and twelve underexpressed proteins were significant (p < . ) between the healthy and diabetic rats, and the altered proteins were restored (p < . ) upon eps treatment. it was first found that carbonyl reductase ( . -fold, p < . ) and mawdbp ( . -fold, p < . ) were surprisingly upregulated upon diabetes induction, and then those two protein concentrations were completely restored by eps treatment. moreover, we obtained eight unidentified proteins that have not been reported to be related with diabetes mellitus. these results evidenced the effect of fungal eps on searching potential markers for diagnosis and therapeutic manipulation of diabetes mellitus. although molecular basis of protein modulation after eps administration in diabetic rats was not verified in this study, the results of the proteomic analysis provide impetus for further studies of mining the biomarkers for diabetic therapy. the model established in our experiment is expected to mimic human diabetic status, which will help us to interpret the roles of biomarkers in diabetic state. the use of polyol-responsive monoclonal antibodies in immunoaffinity chromatography and as a probe for unfolding of wild-type and altered (t i) amidase from pseudomonas aeruginosa s. martins , j. andrade , a. karmali , a.i. custódio , m.l. since immunoaffinity chromatography is a powerful protein purification technique of interest in proteomics, monoclonal antibodies (mabs) against mutant (t i) amidase from p. aeruginosa were raised by hybridoma technology. in order to identify mabs that bind t i amidase tightly but release under gentle conditions, hybridoma clones secreting polyol-responsive mabs (pr-mabs) were previously screened. nearly % of elisa assay-positive hybridoma produced clones secreting pr-mabs with potential application as ligands for immunoaffinity chromatography. to select the optimal conditions for amidase elution, an elisa-elution assay was carried out, with two of these clones (f g ; e a ). the dissociation of ag-ab complex required % of propylene glycol and either . m (nh ) so or . m nacl. the binding of purified mab of igm class (e a ) to wild-type and mutant amidases was investigate by direct elisa, which revealed that it recognised specifically a common epitope on both amidases. conformational changes on antigen molecule were studied. mab e a showed a higher affinity for heat denatured forms than for native forms as revealed by affinity constants suggesting that the mab recognizes a cryptic epitope. the effect of mab e a on amidase activity was also investigated. the binding of mab to wild-type and mutant amidases exhibited an inhibition and activation of % as a function of time, respectively. this pr-mab is useful as a probe to detect conformational changes in native and denatured amidases as well as a ligand in immunoaffinity chromatography, which is of great interest in protein purification and proteomics. fragility and solubility of non-classical inclusion bodieš s. peternel , a. ristič , , v. gaberc-porekar , v. menart , : national institute of chemistry, ljubljana, si- ; lek pharmaceuticals d.d., ljubljana, si- . e-mail: spela.peternel@ki.si (Š. peternel) human granulocyte colony stimulating factor (g-csf) is a pharmaceutically important cytokine. when overexpressed in escherichia coli, it is usually accumulated in the form of inclusion bodies (ibs) . when produced at • c classical insoluble ibs are formed while at • c non-classical ibs containing a high amount of correctly folded g-csf are formed. as higher fragility and solubility of non-classical ibs were noticed, we decided to check whether bacterial cell disruption method has any influence on their mechanical stability and solubility. enzymatic lysis, sonication and homogenization, methods often used for disruption of bacterial cells during the isolation of ibs were compared. lysozyme treatment of bacterial cells appears to be mild enough disruption method not influencing the integrity of ibs. homogenization of bacterial cells at high pressure ( . - . kpa) shows no impact on classical ibs while some loss of target protein from the non-classical ibs is observed. sonication seems to be most harmful as even at rather low sonication altitudes, noticeable disassembling and solubilization of non-classical ibs occurs while no effect on classical ibs is perceived. our studies show that non-classical ibs are much more fragile and soluble than classical ones. therefore, one should extremely carefully choose the method for cell disruption to avoid undesirable loss of the target protein. the danish tick ixodes ricinus parasitize three different hosts both mammals and birds during the -year life cycle. the aim of this study was to identify the last blood host being the host, which the nymph had parasitized before molting to the adult instar. the reason for the study was to reveal the origin of the host contributing the most to the life cycle of the tick and thereby the maintenance of tick-borne diseases in denmark. the most common tick-borne diseases are lyme borreliosis and tick-borne encephalitis (tbe) causing illness in both animals and humans. we analyzed adult ticks, which were collected from known hosts. the analysis was performed at different heat stable proteins, which could be detected during the off host period by elisa. we found that heat stable proteins could be used as identification markers for host recognition. mushroom polysaccharides alter the expression of diabetesassociated proteins in the liver of streptozotocin-induced diabetic rats hye-jin hwang , sang-woo kim , kwang-bon koo , jang-won choi , jong-won yun * : department of biotechnology, daegu university, kyungsan, kyungbuk - , korea; department of bioindustry, daegu university, kyungsan, kyungbuk - , korea. e-mail: jwyun@daegu.ac.kr (j.-w. yun) in the present study, we investigated the influence of the hypoglycemic fungal extracellular polysaccharides (eps) on the differential expression of liver proteins in streptozotocin (stz)-induced diabetic rats. the results of diabetic study revealed that orally administrated eps exhibited an excellent hypoglycemic effect, lowering the average plasma glucose level in eps-fed rats to . %. in the next step, we analyzed the differential expression patterns of rat liver proteins from each group, to discover potent candidates for diabetesassociated proteins. a total of proteins of the -de gel were expressed differentially between diabetic and healthy rats. among them, proteins were upregulated and proteins were downregulated upon diabetes induction. many of these changes were in accordance with observations in previously published studies. surprisingly, the altered levels of most proteins in diabetic group were fully or partially restored to those of non-diabetic control group by eps treatment. moreover, we obtained unidentified proteins that have not been reported to be related with diabetes mellitus. although molecular basis of protein modulation after eps administration in diabetic rats was not verified in this study, the results of the proteomic analysis provide impetus for further studies of mining the biomarkers for diabetic therapy. potential is still limited by the differences observed in the structure of plant and mammalian n-glycans. indeed, theses differences and particularly the presence of ␤ , -xylose and ␣ , -fucose glycoepitopes are responsible for the immunogenicity of plant n-glycans. in order to reduce the structural differences between plant and mammalian n-glycans, current strategies are to knock out plant-specific glycosyltransferases or to humanize plant n-glycans by expression of mammalian glycosyltransferases in plants. in the present study, we have expressed a human ␤ , -galactosyltransferase in alfalfa. in order to further increase the efficiency of the human ␤ , -galactosyltransferase in the plant golgi apparatus, we have exchanged the endogenous targeting signal of this human glycosyltransferase for the ones from plant glycosyltransferases recently characterized in our laboratory. we will illustrate this approach of targeted expression with the results obtained by fusion of the catalytic domain of human ␤ , -galactosyltransferase with the n-terminal sequence of a plant glycosyltransferase that targets the fusion to the very early compartments of the golgi apparatus. the efficiency of natural versus targeted expression of human ␤ , galactosyltransferase in alfalfa will be compared in term of n-glycan humanization. altogether, our results clearly illustrate that we are now on the way to get perfect copy of mammalian glycoproteins in alfalfa plants. construction of recb-recd gene fusion and analysis of fusion enzyme activities oytun portakal , gerald r. smith , pakize dogan : department of biochemistry, hacettepe university medical school, , ankara, turkey; divisions of basic sciences, fred hutchinson cancer research center, seattle, wa - , usa. e-mail: oytun@hacettepe.edu.tr (o. portakal) protein folding is a fundamental process to gain protein function. in an oligomeric protein, the interaction between polypeptides affects the folding process and assembly to the holoenzyme. recbcd is a heterotrimeric and multifunctional enzyme that plays an essential role for major pathway of homologous recombination in e. coli. it is composed of recb, recc and recd gene products. recd is the fast motor unit of the recbcd enzyme. recd also plays a role for high affinity dsdna binding, nuclease activity and chidependent regulation. this study was designed to test the hypothesis that recd polypeptide regulates the essential reca loading activity. the approaching of the study was to fuse recd gene to subsequent recb gene and to observe the changes in enzyme activity and structure. for these purpose two genetic fusion mutations, two-nucleotide deletion and three-codon substitution were created at the overlap sites (ta) of recb and recd genes. fusion mutations were constructed by phage-mediated recombination system, which is called recombineering. this technology requires red function but not host reca protein function. here, we showed the recbd fusion polypeptides in crude extracts. genetic characterization tests were revealed that both fusion enzymes are recombination proficient and have wild-type phenotype. biochemical assays demonstrated that recbdc fusion heterotrimers have dsdna exonuclease, unwinding and chi cutting activities. they were also resistant to dna damaging agents. western blot analysis also detected a wild type length recd polypeptide together with recbd fusion polypeptides and a decreased heterotrimer compared to wild type. our findings suggest that recb-recd genetic fusions may affect recd assembling to the heterotrimer, but not affect it's native folding. sandwich immunoassay-a simple strategy for enhancement of the sensitivity and the specificity in prostate specific antigen detection based on surface plasmon resonance cuong cao, sang jun sim department of chemical engineering sungkyunkwan university, chunchun-dong, jangan-gu suwon, prostate cancer is a deadly disease in men. prostate specific antigen (psa) has been proved to be the most reliable and specific biomarker in preoperative diagnosis, monitoring and followup of patients with prostate cancer. in this study, a biochip based on surface plasmon resonance was fabricated to detect psa at concentrations ranging from to ng/ml. to reduce nonspecific binding, the chemical surface of sensor was constructed by using various ethyleneglycol mixtures of different molar ratios of hs(ch ) (och ch ) cooh and hs(ch ) (och ch ) oh. we also biotinylated the sams surface to enhance the orientation of protein immobilization. by using this surface, spr-based psa detection gave a positive ru value at the fist response in the whole range of psa concentrations. however, this ru value could get better and more reliable by simply applying a secondary interactant, the psa polyclonal antibody, in sandwich immunoassay. the results shown this approach could satisfy our purpose without modify the secondary interactant, which has usually been done by the other report. expression of epitopic domains of human coagulation factor viii in escherichia coli amir amiri yekta , , naser amirizadeh , alireza zomorodipour * , fariba ataei : department of mol genet. national institute for genet eng & biotechnol tehran-iran p.o. box: - , tehran, iran; islamic azad university of jahrom, jahrom, iran; department of hematol, faculty of med, tarbiat modarres university, tehran, iran. e-mails: amir amiriyekta@yahoo.com (a.a. yekta), * zomorodi@nrcgeb.ac.ir (a. zomorodipour) human factor viii (hfviii) plays major role in the intrinsic pathway of blood coagulation and is used to treat individuals with hemophilia a for bleeding episodes. many researches have been focused on the molecular aspects of this protein. in this regard, epitopes of hfviii as well as their corresponding antibodies have many important applications. bacterially produced fviii-epitopes are capable to neutralize the alloantibodies that inhibit hfviii activity. the purpose of present study was to over-express two epitope-containing fragments of fviii in e. coli under t promoter (novagen). two dna fragments from light-and heavy-chains of hfviii ( bp-c c and bp-a a , respectively) were subcloned in the expression vector. the use of his -tagged tail was also considered for detection and purification purposes. in each of the examined clones, a protein of expected size was detectable. in the c c -expressing clone the specificity of the over-expressed protein was confirmed by its reaction with the rabbit serum directed against native hfviii as well as anti-his-tag antibody. in the heavy chain-related-expressing clone, the expression level was low, but it was detectable by immunoblotting experiments. manipulations of the growth as well as induction may be required. the over-expression of the other epitopes reported in the heavy chain may be achievable by the expression of (a) sub-fragment(s) of this region. the over-expressed his-tagged c c -related protein was appeared to be trapped in the cell as non-soluble inclusion bodies. therefore, after homogenizing of the induced recombinant cells, the nonsoluble fraction was dissolved in a solution of denaturant (guanidine hydrochloride) and subjected for the purification, using a ni-nta resin (qiagen) followed by protein measurement. accordingly, an expression level of mg/l (of culture) of the purified c c -related peptide was obtained. the recombinant hfviii c c -derived peptide has provided useful mean for further experimental and medical applications. isotachophoresis has almost exclusively been applied for contracting and stacking samples ions before zone electrophoretic separation of proteins. this study attempts to apply microfluidic isotachophoresis (itp) as a high resolution analytical method for proteins. beta-lactoglobulin and other milk proteins with slightly different pi were labelled with fluorescent red and analysed by the micralyne tk system using microfluidic glass chips, either with simple cross (sc) or double cross (tt) injection or designed d-itp-cze chips with double tt injection and sc for transfer to the second dimension cze channel, efficiently non-covalently coated with . % w/v epoxy-polydimethylacrylamide to lower electroendoosmosis. capillary zone electrophoresis (cze) in borate or phosphate buffer was reproducibly perform for more than consecutive runs using upchurch tm reservoirs glued to the wells to enable larger buffer volumes and greater run-to-run stability. finally, isotachophoretic anionic separation of the proteins were done using phosphate (ph . ) or chloride (ph . ) as leading ion and -amino-caproic acid (ph . ) as terminating ion. the effect of narrow cut ampholytes as spacers needs further investigations. the perspective aim is to combine the migrating itp separated zones with second dimension capillary zone electrophoresis as a new microfluidic proteomic danalysis. development of strategies for heterologous expression of glucose dehydrogenase from the halophilic archaeon halobacterium sp. nrc- juan carlos cruz-jiménez , lorenzo saliceti-piazza , rafael montalvo : chemical engineering, university of puerto rico-mayagüez campus, mayagüez , puerto rico; biology, university of puerto rico-mayagüez campus, mayagüez , puerto rico. e-mail: juancruzj@hotmail.com (j.c. cruz-jiménez) halophilic archaea are excellent model organisms and valuable for biotechnology applications; they are easy to culture in the lab, genetically tractable, and exhibit a variety of interesting and useful characteristics. most halophilic archaea require . m nacl to sustain growth and structural integrity. among the . genes in halobacterium, we are studying the gene encoding a glucose dehydrogenase, gene id is , located between the and bases (halobacterium sp. nrc- genome project). this extremozyme is bioengineerable, and its use as a model for studying biocatalysis in aqueous/organic and nonaqueous media has not been explored to date. the utilization of enzymes in organic solvents has several potential advantages over aqueous systems. a major benefit is the increased solubility of many substrates, resulting in higher concentrations of reactant and products, hence reducing and purification costs and simplifying recovery protocols. cells were grown aerobically during seven days at • c in a complex medium, harvested by centrifugation and their genomic dna extracted. for cloning of the gene, primers were designed based on the sequence recently published by the halobacterium sp. nrc- genome project. the forward ( -ccgcatgcgcc cacagtccc- ) and reverse ( -ccggcctctagaacggcctgg- ) primers were designed to incorporate restriction sites for sph i and xba i, respectively (in bold). we are pcr amplifying the genomic dna and developing methods for the heterologous expression using the mesophilic escherichia coli, as well as purifying the enzyme. the purification procedure will be carried out using high resolution methods based on the protein's halophilicity. bioinformatics methods will be used to facilitate conforming of protein function and for comparison with a native enzyme. quantitative measurements by mass spectrometry of hundreds of proteins simultaneously using the new proteinchip systemseries p. iversen, e. fernvik ciphergen biosystems inc., symbion research park, fruebjergvej , dk- copenhagen, denmark. e-mail: piversen@ciphergen.com (p. iversen) most mass spectrometry methods used in proteomics allow for the identification of multiple proteins in a limited number of complex samples, but lack the ability to assess the quantity of the proteins and their modifications. however, mounting evidence shows specific cleavage of well-known proteins as being strong candidates for specific biomarkers, and in order to discover these biomarkers one has to be able to monitor the quantity and mass of hundreds of proteins from hundreds of complex samples reproducibly. the new series instrument in connection with proteinchip arrays ® from ciphergen biosystems enables this. the new series instrument is optimized for sensitivity, reproducibility and quantitation. new ion optics allows the use of higher acceleration voltages thus increasing the sensitivity, but without lowering the resolution. a new method of blanking the detector in connection with a non-linear gain of the detector also increases the sensitivity to the effect that igg can be detected down to . fmol. furthermore, the unique design of the instrument permits the detection of proteins with great variation in both mass and concentration and thus making it ideal for proteomics studies. a unique feature of the series instrument is the possibility to normalize the output by controlling the laser and detector so that results can be read with equal precision on different instruments, which is not often possible in mass spectrometry where individual instruments yield different results. this feature is vital in the validation of research results beyond individual laboratories. the coupling of liquid chromatography with mass spectrometry is now firmly established as a routine method for the identification of proteins that have been subjected to enzymatic digestion. in an on-line lc-ms experiment, the column eluent is coupled to the electrospray source via an emitter and any tryptic peptides present in the mixture are mass analyses as they elute from the hplc column. should there be any co-eluting species in the eluent, these will be separated in the mass analyser by their mass-to-charge ratio. it has become increasingly clear that relative quantification of protein expression changes is important in modern biology and medicine. several current approaches have been developed that utilise stable isotope labelling of samples in combination with separation and subsequent analysis by mass spectrometry. however, we have recently described an lc-ms strategy where quantification is achieved via normalisation of the ms datasets and comparison of the peptide intensities (of the observed tryptic peptides) across samples is performed. in this case, it is desirable to perform replicate injections and hence reduce statistical errors. this approach places a requirement upon good chromatography, especially in terms of retention time reproducibility. in addition exact mass measurement of the eluting ions is required as well as the ability to generate reproducible and reliable peak intensity, or area, calculations for the eluting tryptic peptides. the ability to measure the mass to charge ratios of ions accurately, across injections and across samples, increases confidence that the same ions have been matched from each sample injection. in this presentation our current strategy for the relative quantification of proteins will be discussed using, as examples, complex protein mixtures from salmonella enterica, eschericia coli and human serum. proteomic analysis for the production of rhctla ig in transgenic rice cell cultures using dige ji-suk cho, song-jae lee, inha university, difference in gel electrophoresis (dige) technology using fluorescent dyes is a novel method, which simplifies the process of detecting and matching proteins between multiple gels by allowing the separation of up to three separate protein samples in the same gel. it provides accurate quantitative and reproducible differential expression values for proteins in several samples. recombinant human cytotoxic t lymphocyte-associated antigen -immunoglobulin (rhctla ig) was produced in the transgenic rice suspension cell cultures using ␣-amylase promoter system. this system is efficient for the production of recombinant proteins, as it secretes target proteins into culture medium under sugar-depleting condition. in this study, the intracellular proteins expressed at both growth and induction stages of culture were separated and analyzed using -d dige. each sample from different conditions and internal standard were labeled with n-hydroxy succinimidyl ester-derivatives of cy , cy and cy dyes and run within a single dige gel. using decyder tm software, spots were detected with two-fold thresholds with % confidence and it was found that proteins underwent significant change during the production of rhctla ig with normalization method improving data distribution. a pooled sample mixture for the picking gel was prepared with deep purple staining and analyzed with mass spectrometry. in addition, the intracellular rhctla ig spots were identified with western blot analysis using goat anti-human igg (fc) antibody after dige gel was transferred to pvdf membrane. study of substrate specificity of rnr-exoribonucelases using hybrid proteins ana barbas, mónica amblar, cecília m. arraiano instituto de tecnologia química e biológica, ean, - oeiras, portugal. e-mail: ab@itqb.unl.pt (a. barbas) the ribonucleases are essential enzymes responsible for the regulation of gene expression and have shown to be important for biotechnology purposes. for instance, commercial mutants deficient in ribonucleases have been quite relevant for the over-production of recombinant proteins. escherichia coli rnase ii is a processive - exoribonuclease prototype of the rnr family that has homologues widespread in the majority of the sequenced genomes. by sequence alignment it has been proposed for the rnr type proteins the existence of three different domains: an n-terminal cold shock nucleotide binding domain (csd), a rnb catalytic domain, and a c-terminal s nucleotide binding domain. we have constructed several rnase ii deletion mutants to enable the characterization of each domain. these studies have allowed us to determine that both csd and s are involved in the binding of the enzyme to the rna substrate, being the s domain the most important. in rna-binding proteins it has been shown that the s domain's conformation is highly conserved. however, it is not known whether the substrate specificity is s -dependent. in order to characterize the s domain and verify if it is directly related to substrate specificity, we have constructed rnase ii hybrid proteins in which the s domain was substituted by the s of two other exoribonucleases, rnase r (rnii-rnr) and pnpase (rnii-pnp). preliminary results have demonstrated that both quimeric proteins are capable of binding and degrading various rna substrates. in addition, studies are currently being carried out to verify the possibility that s domain of pnp in the hybrid protein might be involved in multimerization and/or interaction with other proteins. the murine monoclonal antibody igg , anti-digoxin was produced in a rolling bottle fermentor. purification was performed on a protein g column. cd spectra were recorded on a jasco- spectropolarimeter. protein concentrations of - g/ml and path length of cm were used for measurements in a far uv region. all measurements were performed in a cell holder thermostand with an accuracy of ± . at • c. at this temperature the predominance of ␤-strands is indicated. large conformational changes occur at • c. at this temperature the spectra tense to irregular ␤-strands and unordered structures. these evidences confirming temperaturedependent conformational changes of protein and also high thermal stability of mentioned monoclonal antibody. generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling janni kristensen, kim kusk mortensen, hans peter sørensen laboratory of biodesign, department of molecular biology, aarhus university, gustav wieds vej c, dk- aarhus c, denmark. e-mail: hans.peter.sorensen@teknologisk.dk (h.p. sørensen) we recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of escherichia coli. recombinant proteins were covalently coupled to the e. coli ribosome by fusing them to ribosomal protein (rpl ) followed by expression in an rpl deficient strain of e. coli. this allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpl and the target protein. to assess the efficiency of separation of target protein from ribosomes, by site specific proteolysis, we required monoclonal antibodies directed against rpl and gfp. we therefore purified rpl -gfp-his, rpl -his and gfp from e. coli recombinants using affinity, ion-exchange and hydrophobic interaction chromatography. these proteins could be purified with yields of , and g per gram cellular wet weight, respectively. however, rpl -gfp-his could only be expressed in a soluble form and subsequently purified, when cells were cultivated at reduced temperatures. the purified rpl -gfp-his fusion protein was used to immunize balb/c mice and the hybridoma cell lines resulting from in vitro cell fusion were screened by elisa using rpl -his and gfp to select for monoclonal antibodies specific for each protein. this resulted in antibodies directed against rpl and antibodies directed against gfp. antibodies were screened for isotypes and their efficiency in western immunoblots. the most efficient antibody against rpl and gfp were purified by protein g sepharose affinity chromatography. the purified antibodies were used to evaluate the separation of ribosomes from gfp, streptavidin, murine interleukin- , a phagedisplay antibody and yeast elongation factor a by centrifugation, when ribosomes with covalently coupled target protein were cleaved at specific proteolytic cleavage sites. proteomic analysis for the production of rhctla ig in transgenic rice cell cultures using dige ji-suk cho, song-jae lee, inha university, difference in gel electrophoresis (dige) technology using fluorescent dyes is a novel method, which simplifies the process of detecting and matching proteins between multiple gels by allowing the separation of up to three separate protein samples in the same gel. it provides accurate quantitative and reproducible differential expression values for proteins in several samples. recombinant human cytotoxic t lymphocyte-associated antigen -immunoglobulin (rhctla ig) was produced in the transgenic rice suspension cell cultures using ␣-amylase promoter system. this system is efficient for the production of recombinant proteins, as it secretes target proteins into culture medium under sugar-depleting condition. in this study, the intracellular proteins expressed at both growth and induction stages of culture were separated and analyzed using -d dige. each sample from different conditions and internal standard were labeled with n-hydroxy succinimidyl ester-derivatives of cy , cy and cy dyes and run within a single dige gel. using decydertm software, spots were detected with two-fold thresholds with % confidence and it was found that proteins underwent significant change during the production of rhctla ig with normalization method improving data distribution. a pooled sample mixture for the picking gel was prepared with deep purple staining and analyzed with mass spectrometry. in addition, the intracellular rhctla ig spots were identified with western blot analysis using goat anti-human igg (fc) antibody after dige gel was transferred to pvdf membrane. similarity searches and multiple alignment of s and s protein of sars-cov for modeling d structure and its evolution (origin) mohammad soltany rezaee rad, iman tavassoly, negar mottaghi, banafsheh rezaee. e-mail: mohammad.soltany@gmail.com (m.s.r. rad) aims: the exact origin of the cause of severe acute syndrome (sars) is still an open question. nowadays recombinant origins for this virus have been found. s and s subunit of spike protein of this virus are the most important proteins responsible for severe acute respiratory syndrome. in fact they are glycoproteins of this virus exist on its surface. they are responsible for mediating fusion of viral and cellular membrane. the classification and modeling d structure of this virus can help us to suggest new ideas about its charististics and function, which may lead to new therapeutic and preventing modalities. methods: we used nucleotide sequence of s and s subunit of s (spike) protein for multiple alignments. we have done multiple alignments with different bioinformatics software (clusterx, entrez) for comparing the sequence with the other viruses and, we used weblab view software for modeling and identifying d structure of these proteins. findings: the similarity searches on nucleotide sequence of this protein with the single strands rna (ssrna) shows the virus belong to a known classification named coronaviridae. these d structures show the responsibility of s protein in this syndrome. another findings based on these alignments is an important similarity between these subunits and genome of hiv- showing they have familiar mechanism in pathogenesis. discussion: multiple alignments are powerful tool in classification of new recombinational virus and emerging infection. d structure model of this virus is an important guide to understand the mechanism of this virus. the shape of glycoprotein that modeled with bioinformatics software can help us in understanding mechanism of binding this virus to human cells. this fact can be used in designing drug and vaccine to cure and prevent the sars. blocking these origins and sites leads to inhibiting the virus attachment. also the similarity between this virus and hiv- shows us that both of them have similar proteins that cause pathogenesis of these viruses. the simulated moving bed (smb) technology is a continuous countercurrent chromatographic separation technique that has been applied successfully in the last years to a number of significant problems. an smb consists of a series of fixed bed chromatographic columns connected in a loop, and outperforms column chromatography in terms of productivity and solvent consumption. the use of smb instead of batch processes for bioseparations, i.e. separations involving large and rather complex molecules with multiple d configurations depending on parameters such as ph, temperature, etc., is becoming of greater and greater interest. examples of these are therapeutic proteins, antibodies and plasmid dna among others. for all chromatographic processes in this field, one of the most crucial issues is the cleaning of the chromatographic media with a special solvent system, an operation usually referred to as cleaning in place (cip). in single column chromatography this is easily done off-line, but this is not compatible with standard smb operation. in order to overcome this limitation, the standard smb configuration has been modified by adding a dedicated section plus an additional section for the re-equilibration of the freshly cleaned column with the working solvent before it is re-inserted into the smb loop. in such a way, cip according to gmp criteria can be incorporated into the smb unit and operation, which is then called cip-smb. this new smb configuration is also related to the three fraction separation unit called f-smb that has been recently introduced and applied to the separation of nucleosides. in this work we apply cip-smb using a size exclusion stationary phase to the separation of plasmid dna from the filtered cell lysate solution. plasmid purification has become a key issue in the last years as a result of the advances in gene therapy, whereas traditional laboratory methods are not always suitable for therapeutic purposes. we report about separation performances, which are then discussed in the light of smb design criteria and compared to column chromatography performance. computer guided optimization of adsorptive bioseparation processes bernt nilsson department of chemical engineering, lund university, lund, sweden. e-mail: bernt.nilsson@chemeng.lth.se separation processes like chromatography can be highly nonlinear and the behavior can sometimes be hard to predict. optimization of preparative chromatography is often done experimentally, which is both time consuming and expensive. a model-based approach to optimization is therefore an attractive and challenging way to overcome some drawbacks in the traditional working procedure in biotechnical industry. efficient model-based optimization for industrial needs requires three parts; models, methods and tools. model-based methodology requires a set of chromatography column model structures, which can capture the phenomenon of interest. for instance they have to capture column load variations, elution profile changes, operation condition disturbances, column configurations and stationary phase properties. to derive a reliable model for optimization it has to be calibrated and validated to experimental data, which requires an efficient calibration procedure. different calibration procedures are discussed and compared. after validation the model can be used in the design of a separation step. to do a robust design a set of requirements have to be fulfilled. the column size and operation conditions are used to optimize the performance of the step, which requires a constraint nonlinear optimization method. the choice of objective function for optimization and corresponding constraints are not obvious and the resulting operation conditions are often not robust. therefore there have to be additional methods available for analysis of the performance, like sensitivity and robustness analysis. optimization of the purification of antibodies is discussed and exemplified. the work with mathematical models and numerical methods has to be supported by a set of computer tools of different kinds in order to solve industrial problems effective. there is a need for different kinds of tools; customized tool to solve specific problems by a non skilled user and general toolbox for the expert. an example of a toolbox is presented. tina tarmann, alois jungbauer department of biotechnology, university of natural resources and applied life sciences, vienna, austria plasmids and viruses are the contemporary vehicles for genetherapy and genetic vaccination. extremely promising results have been reported from in-vitro, in-vivo and clinical studies. currently a lot of these compounds are manufactured with a technology which has been directly transferred from laboratory to pilot scale without further engineering. membrane based separations, chromatographic separations and precipitation have been employed for separation of plasmids and viruses. chromatographic separation have been designed with aim of protein separation. thus such processes suffer from either mass transfer limitations or low capacity. monoliths without intraskeleton mesopores and chromatography particles with giga pores are excellently suited for adsorption and separation of plasmids and viruses. low mass transfer resistance and high capacity compared to conventional beaded materials can be observed. adsorption kinetics were derived from infinite and finite bath methods and isotherms were constructed. these data also suggest that a conformational change of the plasmids takes place upon adsorption. discussion of the mass transfer properties and an example of scale up of a chromatographic separation process using these novel materials will be shown and discussed in respect to already existing processes. the recent developments in molecular therapies such as non-viral gene therapy and dna vaccination have fostered the development of efficient plasmid dna (pdna) purification processes. the separation of supercoiled (sc) and open circular (oc) isoforms is one of the key steps in the large scale purification of pdna vectors intended for a therapeutic use. although escherichia coli produces mainly the more compact sc pdna isoform, oc, linear and denatured pdna isoforms are usually present and are likely to be less efficient in transferring gene expression. for this reason, regulatory agencies specify that more than % of pdna in a therapeutic product is in the sc isoform. in this work histidine-base recognition is explored as a mean to separate pdna isoforms. the agarose gel used here combines the mild hydrophobic characteristics of an epoxy spacer arm with a pseudo-affinity histidine ligand. chromatographic profiles were obtained by injection of native plasmid (sc + oc) samples in the histidine-agarose support showing an efficient and baseline separation of both isoforms. the high resolution obtained with this support indicates that the method is potentially applicable to the separation of pdna at preparative and analytical scale. affinity ligand development with a novel encoded bead screening technology ib johannsen, versamatrix a/s, gamle carlsberg vej , dk- valby, denmark. e-mail: www.versamatrix.com the presentation describes a new invention for fast development of affinity ligands, where up to , ligands can be screened onbead and identified in a few hours. combinatorial synthesis by the split and mix procedure is a powerful technique for generating vast numbers of diverse chemical compounds on polymer beads with relatively little effort. traditionally, the technique is hampered by the laborious spectroscopic and chemical analysis, needed to determine the exact structures of the ligand on selected beads. in this way, - month analysis time could easily be spent just to analyze a tiny fraction of the library. in the versaffin tm technology each bead is encoded, individually tracked, and identified during the synthesis and screening. this decreases the whole ligand development time from months to weeks and increases the amount of information significantly. the bead code further enables evaluation of the ligandprotein binding under varying binding and elution conditions. the instrument for reading the encoded beads and for quantifying the amount of bound protein is presented. the encoded beads we use are based on functional cross-linked polyethylenglycol (peg), which is compatible with water as well as most organic solvents. thus, the combinatorial synthesis can be carried out in organic solvents and the resulting compounds can be evaluated, still bound to the parent beads, under aqueous conditions. a further advantage of using peg based beads for on-bead screening is the fact that peg is biologically inert and therefore does not interfere in a bioassay. in the biopharmaceutical industry, pressure is mounting to shorten development times and thereby time to market, e.g. in the field of monoclonal antibodies, generic processes have been established which allow for more rapid development from gene to production of pre-clinical and proof of concept/phase i material. for non-mab products from various expression systems, productspecific approaches still prevail. however, for most product types similar issues like clearance of process-and product-related impurities, overall purity and yield, or manufacturing issues have to be dealt with in downstream process development. integrated and timely approaches based on process science and developed orthogonal analytical tools are often hampered by tight time frames and limited resources available. on the other hand, thorough understanding and analytical characterization of product characteristics but also of (process-related) impurities are pre-requisites for fully exploiting separation power and for achieving final purities way above % in a robust and cost-effective manner. from primary separation to polishing steps, we have made several attempts to improve the efficiencies of process steps themselves but also of ways to develop them. the strategies applied comprise implementation of innovative processing tools, rational streamlining and optimization of a sequence of unit operations, tech transfer and scale up considerations. also in this context, the applicability of scale down and ultra scale down models for process development and optimization purposes, their potential for speeding up and their limitations will be discussed. chromatographic monoliths are rather new chromatographic stationary phases. they consist of a single piece of a highly porous material. the pores are interconnected forming a network of channels. since the transport mechanism is predominantly based on convection, mass transfer between mobile and stationary phase is significantly enhanced resulting in short separation times. because of that they seem to be an ideal support for separation and purification of extremely large molecules like proteins, dna or even viruses. in this talk, various features of the monoliths like high porosity, fast mass transfer, surface accessibility and dynamic binding capacity will be described. effect of each feature on the separation and purification efficiency will be discussed in terms of molecular size and properties. while chromatographic monoliths are already widely accepted in microchip fluid devices, capillary columns as well as analytical columns, very few reports about preparative monolithic columns can be found. reasons for lack of preparative chromatographic columns will be elucidated and preparation strategy for construction of several liter volume methacrylate based monoliths will be presented. finally, several examples of biomolecule purification like large proteins, plasmid and genomic dna and viruses on cim convective interaction media ® monolithic columns will be given. further, their application as bioreactors and supports for solid state synthesis will be demonstrated. hubbuch institute of biotechnology, forschungszentrum juelich, juelich, germany. e-mail: m.schroeder@fz-juelich. de (m. schroeder) the intraparticle diffusion coefficient is an important parameter for modeling of chromatographic separation processes. a new method based on dynamic measurements of intraparticle concentration profiles of proteins in process chromatographic media with a confocal laser scanning microscope is presented. the diffusion coefficient is determined by fitting experimental data to a spherical diffusion model. excellent agreement of experimental data with simulation results is obtained. the diffusion coefficient is measured for seven proteins in sepharose ff, spanning molecular weights from . to kda. in addition, multicomponent diffusion processes for combination of differently sized proteins are analyzed and the influence of adsorbed proteins on the diffusion coefficient is measured in sp or q sepharose ff. taken together the presented method allows measuring the diffusion coefficient of proteins in process chromatographic media in a packed column. use of automated docking for predicting chromatographic behavior of proteins in hydrophobic interaction chromatography andrea mahn, m. elena. lienqueo contreras university of chile, santiago, chile in the present work, we have extended and automated the methodology proposed by mahn et al., for predicting protein behavior in hydrophobic interaction chromatography (hic). this methodology is based on the good correlation level between the average surface hydrophobicity of the interfacial zone (local hydrophobicity lh) and protein retention time in hic, for only three different ribonucleases. for determining the lh it is necessary to select the most probable protein-ligand conformation. in this work, we have determined the most probable conformation, of more than proteins, using (i) first, the module insight ii affinity by accelrys for providing automated docking (grid method) of ligands (phenyl) to the proteins ( conformations); (ii) then, the different probable docked protein-ligand conformations were automatically scored using the module insight ii ludi by accelrys; (iii) after that, each conformation was clustered and each cluster was scored by using the average score of each cluster (iv) finally, the most probable conformation was selected using a function based on the number of cluster components, and the average score value. then, when the most probable conformation was selected, the local hydrophobicity (lh) was calculated using the graphical representation and analysis of structural properties (grasp) program. the results have shown an acceptable correlation level (r > . ) between lh and the experimental dimensionless retention time (drt). in view of these results, we consider that this methodology could be used to adequately represent the chromatographic behavior in hic for all kinds of proteins (with a heterogeneous and homogeneous surface hydrophobicity distribution) and without a large number of tedious experiments, but only using computational simulation and adequate score criterion. potato tuber proteins are nutritious and show potential as functional ingredient in food systems. however, the present bulk processing technology can only recover byproduct protein for animal feed use. an expanded bed adsorption (eba) process for isolating functional food-grade protein from crude potato starch effluent was previously developed. moderate capture efficiency ( - %) of the total crude protein was most likely caused by diffusion limitations and aggregated protein, inaccessible for adsorption. we employed the same adsorption ligand attached to agarose-tungsten carbide beads to create stable beds of . - . × expansion using flow rates at - cm h − . a pilot scale eba process was run in a commercial processing plant over a three month campaign of starch production from potatoes of mixed variety. fresh crude effluent ( - l/cycle) was applied to a column ( cm × m) containing l of resin. protein capture by eba was reliable in operation, producing a refined protein material, which after dewatering and gentle drying, showed improved functionality over heat-coagulated protein produced at the same plant. overall productivity increased. however, finding a robust operating window of predictable productivity is challenging since the potato fruit water is complex and deteriorates easily. from breakthrough curves, it is observed that the major bulk protein, patatin, displays non-langmuir adsorption behavior. this may indicate a range of interactions for different species of the same protein. chlorogenic acid (ca), the main polyphenolic substance in potato tuber, causes enzymatic browning and undesirable flavor changes, but polyphenols can also react with protein. assessing the effects of interacting cell components therefore applies to the bioprocessing of plant material. at present the acceptance of biochip technology for on site use, e.g. diagnosis or environmental control is hindered by rather expensive and complex instrumental systems. there is a need to provide reliable and cost-effective systems that can be operated with minimal training. the construction of electronic biochip microarrays using semiconductor technology enables the construction of compact systems with high integration at acceptable production costs. the key feature of the fully electrical biochip technology are micro arrays made in advanced si-technology and carrying several array positions with interdigitated nanometer gold electrodes on its surface. the chips are fabricated by standard silicon fabrication methods allow-ing high volume production and to minimise the cost per chip. the advantage of fully electronic microarrays is the intrinsic high spatial resolution and direct signal coupling of the biosensing element and the transducer. the function of fully electronic biochips is also based on the electrochemical transduction and quantification of the formation of affinity complexes on the chip surface. a portable device for field applications and point of care diagnosis have been designed and manufactured. the amperometric device enables the recognition of biomolecular interactions by measuring the redox recycling products of elisa enzyme labels. the highly sensitive signal transduction is achieved with a -channel interdigitated ultramicroelectrode array. one major advantage of fully electronic microarrays is the direct signal coupling of the biosensing element and the resulting robustness and opportunity for miniaturisation. those electrical biochip arrays have been adapted for the detection of all types of affinity complexes, such as for dna, rna, proteins and haptens. self assembling of capture oligonucleotides via thiol-gold coupling have been used to construct the array chip nucleic acid interface. thus e.g. pathogenic micro organisms have been identified and quantified via their genomic dna or ribosomal rna respectively. another application based on immobilized antibodies is shown to sense extreme low concentration of bioagent toxins. for processing the assay formats and the electrical read out of the detection of affinity complexes a modular fully automated measurement system has been developed. it is manufactured in industrial lines and available at market. dynamics and self-assembly of organic molecules on surfaces revealed by high-resolution, fast-scanning stm flemming besenbacher interdisciplinary nanoscience center (inano), university of aarhus, dk- aarhus c, denmark. e-mail: fbe@inano.dk in the interdisciplinary area of nanoscience and nanotechnology, the adsorption and self-assembly of organic molecules on singlecrystal surfaces have attracted much attention lately due to the potential applications in fields ranging from molecular electronics to biocompatible interfaces. the supramolecular structures formed upon deposition of molecular species on solid surfaces depend on the molecular architecture and the distribution of functional groups on one hand, which determines the thermodynamically stable molecular arrangement, and on the other hand, on kinetic factors like thermal diffusion, spontaneous rotations and conformational dynamics. i will show how the unique aspect of our aarhus stm and the time-resolved, high-resolution stm imaging can be used to obtain important new insight into the dynamics, and can provide very important new information on the atomic-scale realm and on the dynamics of molecular nanostructures. the time-resolved stm data are visualized in the form of stm movies (see www.inano.dk/spm) which can subsequently be analyzed in order to extract quantitative information on the activation energy, the prefactors and the adsorbate-promoted diffusion. i will specifically discuss: (i) the self-assembly of guanine quartets on au( ) and the influence of cooperative hydrogen bonds, and (ii) the molecular recognition in binary mixtures of dna bases. g molecules are found to self-assemble into a hydrogen-bonded network of g-quartets, whose structure corresponds perfectly with the quartet structure of telomeric dna determined by x-ray crystallography. the strong preference of g molecules to form quartets can be explained by a cooperative effect that strengthens the hydrogen bonds within the g-quartet network over the hydrogen bonds in isolated dimers. by means of a combination of stm experiments and dft calculations we compare the d molecular networks formed on deposition of the binary mixtures g-c (purine-pyrimidine pair of complementary bases) and a-c (purine-pyrimidine pair of non-complementary bases). we find that the non-complementary bases segregate into islands of pure a and a network of pure c, whereas the complementary bases g and c form a network that cannot be separated by annealing up to the desorption temperature for c. high-resolution stm images allow us to identify the structures for the enhanced thermal stability as structures that contain g-c bonds, possibly with the same structure as the watson-crick pairs in dna molecules. kühnle, r., et al., . nature , . otero, r., et al., . angewandte chemie int. ed. , . otero, r., et al., . nat. mater. , . otero, r., et al., . angewandte chemie int. ed. , . rosei, f., et al., . science , . schunack, m., et al., . biomedical and pharmaceutical companies are using an increasing number of carbohydrate polymers in the formulation of drugs. one such polymer with highly attractive features is chitosan that can be produced from crustacean shells. chitosan is non-toxic, biocompatible and biodegradable. chitosan can be formulated as nanoparticles or membranes and have enhanced several bioprocesses. among the well-documented features are enhanced drug uptake by tight junction relaxation and enhanced in vivo uptake and protection of nucleic acid formulations. chitosan research has increased throughout this decade. research programs are addressing the potential of chitosan applications but preparations with variable molecular size and charge are not easily available. specifically companies working with the development of new drugs and enhancement of drug functionality are in need of formulation technology that provides well characterized biocompatible material. in the chitosan innovation consortium the danish companies, coloplast, novozymes biopolymers, pipeline biotech, and zgene, together with the research center inano (aarhus university) and bioneer a/s have developed a series of chitosan preparations suitable for research of biopharmaceutical applications (www.chitosan.dk). the ability to obtain functional formulations is currently being tested in both in vitro and in vivo experiments. the consortium participants have established a platform from which chitosan processing, characterization and formulation technology can be extracted. by providing specified chitosan preparations the polymer feature can be adjusted to fit specialized biopharmaceutical applications. high throughput bioprocessing govind rao center for advanced sensor technology, umbc, baltimore, md , usa the post genome era holds a great deal of promise. an enormous number of new proteins await study. these will require sophisticated culture techniques, as cells will have to be grown under large numbers of environmental conditions to elucidate expression triggers. unfortunately, unlike molecular biology, bioreactor technology is little changed since its inception. the primary reason has been a lack of sensor technology that can be readily employed to monitor the cellular environment. we will take a look at the current status of the technology and report on promising optical sensor technology that permits low-cost high throughput cell culture and fermentation. noninvasive sensors that monitor ph, po and pco and high sensitivity solutions for glucose and glutamine measurements will be presented. in addition, the mixing characteristics that determine bioreactor performance will be examined at the small scale and their relevance to the large scale will be demonstrated. on-line monitoring and fed-batch operation in shake flask and micro titre plate cultures jochen büchs , frank kensy , markus jeude , tibor anderlei , barbara dittrich , doris klee : biochemical engineering, rwth aachen university, aachen, germany; ac biotec, jülich, germany; textile chemistry and macromolecular chemistry, rwth aachen university, aachen, germany although methods of molecular biology has led to rational design of micro-organisms to suit our requirements, screening of large numbers of strains and media is still one of the most important tasks in biotechnology. batch operation of shaken bioreactors and absence of on-line monitoring is still the general state of the art for that purpose. it is also a very common practice in screening projects that only the final product titre is measured at the end of the culture for the evaluation of the "best performers". in the recent years several approaches were introduced to follow microbial cultures also in shaken bioreactors like shake flasks or micro titre plates (mtp's). it became obvious that the cultures can behave quite unexpected and most relevant and essential information is lost, if only the final product titre at the end of the cultures is utilised for evaluation. as a result, the screening may be directed to an unknown and non-intended direction or may even fail. this is demonstrated with some examples in this contribution. new methods and techniques are introduced to measure the oxygen and carbon dioxide transfer rate and the respiratory quotient in shake flasks and the optical density, nadh fluorescence, ph and do in mtp's. if the desired product can be fused to a fluorescence protein, like gfp or yfp, also the product formation can be monitored on-line in mtp's. it is of utmost importance that the operating conditions of the applied shaking bioreactors are suitable and shaking motion is not stopped during the measurement. otherwise, e.g. power input, mixing and oxygen supply is interrupted and the micro-organisms will ongoingly rearrange their metabolism to cope with these disturbances of their environmental conditions. another problem of screening is the commonly applied batch operation mode. a lot of microbial systems display an overflow metabolism, substrate or osmotic inhibition or are characterised by a catabolite repressed product formation. in all these cases, batch operation is not the preferred operation mode and, therefore, these cultures are run in fed-batch in larger scales. in particular, it is nearly impossible to screen systems, which are catabolite repressed by the carbon source, in batch mode in defined mineral media. after initial growth has led to a nearly complete consumption of the carbon source, the product formation is derepressed. but then no more carbon source is available to continue with production. it is quite questionable whether in batch operation mode suitable strains can be selected for later fed-batch operation or not. this consideration has resulted in the development of a new technique which allows to run the screening in fed-batch operation mode. this technique is applicable in shake flasks as well as in mtp's. dramatic increases in product titre between -and -folds were observed under these conditions compared to conventional batch screenings. mikkel nordkvist, john villadsen center for microbial biotechnology, technical university of denmark, dk- lyngby. e-mail: mnq@biocentrum.dtu.dk (m. nordkvist) efficient mixing and mass transfer are highly important in the chemical industry and in the fermentation industry. poor mixing can result in low yield and variable product quality in a number of cultivation processes, and mass transfer can easily become the limiting step in aerobic cultivations, especially at high cell density. we have tested a new tank reactor system, where liquid is withdrawn from the bottom of a tank, rapidly circulated, and injected back into the bulk liquid through the nozzles of rotary jet heads. liquid feed as well as gas is added in the recirculation loop and thereby distributed via the rotary jet heads. solid feed in powder form can also be added in the loop with advantage, and heat is efficiently removed in a plate-type heat exchanger, which is part of the loop. the system has a very simple design with no internal baffles or heat exchange area, and between batches the rotary jet heads are used for cleaning in place. a number of applications ranging from dispersion of liquid and powder to mass transfer will be presented. mass transfer applications include baker's yeast cultivation and oxidation of lactose to lactobionic acid by a carbohydrate oxidase. fast and accurate analytical information that can be used to rapidly evaluate the interactions between biological systems and bioprocess operations is essential for optimization of biological production processes. we have researched and developed a multiplexed microbioreactor system for the parallel operation of multiple microbial fermentations. the microbioreactors have working volumes from to l, and are instrumented for real-time monitoring of dissolved oxygen, ph and optical density. the growth profiles obtained with escherichia coli compare favorably to results obtained from conventional ml batch bioreactors. we also demonstrate the use of our microbioreactors coupled to dna microarray analy-sis, as a tool for accelerated discovery and elucidation of metabolic pathways and gene expression profiles. the multiplexed system represents a significant step towards high-throughput data acquisition and has the potential to replace current instrumented bioreactors, which are bulky, expensive to run, and require many mechanical manipulations. design of a laboratory scale bioreactor to study solid-state tobacco fermentation m. di giacomo, l. nappi, d. silvestro, m. paolino, d. parente r&d biology department, british american tobacco italia, naples , italy italian toscano cigar production is based on the fermentation of dark fire-cured tobacco. the process starts with the rise of leaf moisture to levels of water activities assuring development of the wild phylloplane microflora in the absence of free water. the intense growth of microorganisms modifies leaf characteristics (ph rise from acidic to alkaline condition) contributing to define the toscano typical smoke profile. tobacco fermentation takes place in great bulks of kg which cause considerable amount of heat evolution as a function of the metabolic activities of the microorganisms. this heat accumulates and temperature can rise to as high as • c. a laboratory cylindrical packed-bed bioreactor was designed to work under isothermal conditions. the reactor was ideal to ferment small quantities of tobacco ( g) and was made up of a column aerated from the bottom with humidified air and placed in a thermoregulated room. experiments were conducted with constant temperature and air flow. moreover, bioenrichment experiments were conducted in the presence of different microbial starter cultures. fermentation courses were monitored by measuring microbial counts and chemical/physical modification of the substrate. with this laboratory scale system we obtained different kinds of information on the role and dynamics of the microorganisms involved in the fermentation process and on the influence of different environmental conditions. for the future, the design of an adiabatic device for tobacco fermentation is planned. on-line liquid chromatography as a process analytical technology for monitoring and control of biotech processes rick e. cooley , : process analytics center of excellence, dionex corporation, sunnyvale, ca, usa; eli lilly and company, indianapolis, in, usa (retired) biotech processes, used to produce an active pharmaceutical ingredient (api), generally differ from small molecule api manufacturing processes in that the starting materials tend to be more variable, more complex, and the product of interest in lower concentration due to the fact that they originate from a biological rather than a chemical process. this complexity and low starting concentration has generated a high level of interest in developing technologies that can be utilized to increase yield and reduce variability in the initial bioreactor phase, as well as, downstream isolation and purification operations. the use of process analytics, or on-line analytical measurements, is a technology approach that can contribute to increased process understanding and control. this presentation will provide examples of how various analytical measurement technologies have been utilized to monitor typical bioprocess unit operations leading to increased automation and control. examples of the use on-line liquid chromatography to monitor bioreactors, process scale chromatography columns, and enzymatic reactions will be presented in more detail. application of advanced monitoring strategies for recombinant protein production karl bayer department of biotechnology, university of natural resources and applied life sciences, vienna a- , austria high yield in combination with the required quality are the key objectives of large-scale production of recombinant proteins using different host/vector systems. however, in the past the efficient production of recombinant proteins was frequently limited due to inadequate exploitation of the cell factory and deficiencies in process design. to achieve optimal exploitation of microbial cell factories the key requirement is to enhance the monitoring capabilities to improve the insight into the host metabolism dynamics and to cope with limited understanding of the interaction of recombinant protein synthesis with host cell metabolism. since each protein exerts an individual influence on the host/vector system, the selection of appropriate analytical methods is even more important. in order to overcome these problems high throughput technology platforms, such as dna microarrays for transcriptome and differential -d electrophoresis (dige) for proteome analysis provide extensive data to screen for significant analytes and provide an appropriate basis for in silico modelling and reverse engineering of regulatory networks. taking advantage from such an iterative process experimental design will be improved and further aid to increase the performance of modelling. in addition, transcriptome data are used to screen fast stress responsive promoters to set up gfp based reporter gene fusions for in-situ monitoring of the metabolic load due to recombinant gene expression. moreover chemometric methods are frequently applied to model complex data from easily obtainable on-line data sets to overcome the limited monitoring capabilities due to the high complexity and nonlinearity of biological reactions and reaction networks. this strategy has been successfully applied to model bdm (bacterial dry matter), pcn (plasmid copy number) and the amount of recombinant protein using data sets acquired from off-gas analysis (o consumption, co evolution), alkaline consumption rate, in-situ capacitance and multi-wavelength fluorescence measurements. at-line monitoring of bioprocess-relevant marker genes using an electric dna-chip britta jürgen , daniel pioch , le thi hoi , jörg albers , rainer hintsche , stefan evers , karl-heinz-maurer , michael hecker , thomas schweder : institut für pharmazie, ernst-moritz-arndt-universität, greifswald, germany; ebiochipsystems, itzehoe, germany; vtb-enzymtechnologie, henkel kgaa, düsseldorf, germany; institut für mikrobiologie, ernst-moritz-arndt-universität, greifswald, germany. e-mail: britta.juergen@uni-greifswald.de (b. jürgen) the gram-positive bacteria bacillus licheniformis and bacillus subtilis represent important industrial hosts for the production of enzymes (e.g., proteases and amylases) or antibiotics (e.g., bacitracin). both organisms are attractive for this purpose because of their apathogenity and their classification as gras organisms (generally regarded as save). moreover, their easy cultivation and their high natural capacity to secrete proteins into the growth medium qualify them for the industrial overproduction of homologous or heterologous proteins (simonen and palva, ) . for the control of the physiological state and the productivity of the production cells efficient analysis tools are of great interest. a prerequisite for the evaluation of the physiological state of cells during industrial fermentation processes is the analysis of so-called process-relevant marker genes, the expression of which indicates unfavorable growth and production conditions (schweder and hecker, ) . by means of proteome and transcriptome analyses we have identified critical process-relevant genes of b. licheniformis and b. subtilis cells under different nutrient limitation conditions and during industrialclose bioprocesses. dna-chips with probes for such process-relevant marker genes could be valuable diagnostic tools for the monitoring of the cellular physiology during microbial bioprocesses. in order to provide reliable tools for the monitoring of the cell physiology during microbial bioprocesses, we have developed a fast mrna analytical approach, which allows an at-line monitoring of the transcriptional activity of selected marker genes during bioprocesses. this approach is based on an easy, fast and reliable rna isolation procedure and the measurement of specific mrnas by means of an electric dna-chip barken et al., a robust bioprocess is crucial to ensure the consistent process performance and provide the high quality of product for drug manufacturing in biopharmaceutical industries. existing methodologies for bioprocess design do not involve establishing mechanisms to achieve the desirable robust bioprocesses and have low capacity in handling uncertainty in the product manufacturing. also, the solutions are often obtained step wise and do not account for interactions between the steps. despite its importance, the robustness of a bioprocess has not been properly defined and studies carried out in statistic sense are often retrospective. in addition, the computational cost is expensive due to using a line search algorithm for finding an optimal operating solution. finally, the existing methodologies are difficult to apply to the whole bioprocess in biopharmaceutical industries. this paper attempts to define rigorously a measure for process robustness and presents a new methodology for evaluating the robustness of bioprocess operations and their performance. the methodology is based on the concept of 'windows of operation' which shows the whole range of possible operating regions. the methodology also establishes a lower bound for the largest variation of a design variable to ensure the performance. these bounds are achieved by min-max optimization techniques. a direct search algorithm has been developed and its computational cost is much lower than the line search algorithm. results include visualization of robust operating regions and a set of indices which compare the performance of different operating strategies. the capabilities and efficiency of this methodology are illustrated by applying it to the centrifuge selection for the clarification of high solids density cell broths. the research work will impact considerably upon robust bioprocess operation. when grown on methanol, pichia pastoris is able to synthesize proteins to high titres as well as secreting and glycosylation, thereby making this organism a very interesting host for the production of recombinant drugs at large scale. the methanol residual concentration has been reported to strongly influence the specific productivity, the optimum concentration being around g/l. a suitable monitoring and control technique is therefore necessary to study and improve the productivity of p. pastoris fermentations. the current research aims at showing how a mid-infrared spectrometer (atr-ftir) can be calibrated in-situ in order to monitor and control p. pastoris fermentations. this method is simple and fast, and eliminates the need of both standards preparation and off-line calibration. it is based on the observation that during fed-batch processes, only substrate and biomass concentrations vary significantly. the method therefore consists in adding a known amount of methanol at the beginning of the process, just after inoculation, and subsequently calibrating the instrument. financial support for the swiss national science foundation is gratefully acknowledged. implantable biomaterials are subjected to inflammatory responses mediated by adherent phagocytes such as monocytes and macrophages. these cellular responses and behavior have been shown to be dependent on the type of protein that adsorbs to the surface. surface modification is necessary to control and prevent protein adsorption, and thus modulate the inflammatory responses. hydrophilic surfaces that adsorb minimal amounts of protein are considered useful for minimizing the inflammatory reactions to biomaterials. in this study we have used two routes to modify polyethylene terephthalate (pet) films: ( ) a wet-chemical method for attachment of linear polyethylene glycol chains (mpeg); and ( ) gas-phase plasma polymerisation of diethylene glycol vinyl ether (degve) to generate peg-like surfaces. the surface chemistry was assessed by x-ray photoelctron spectroscopy (xps), fourier transform infrared spectroscopy (ftir) and time-flight secondary ion mass spectrometry (tof-sims). the two pegylated surfaces were compared for their ability to minimise both fibrinogen adsorption and the adhesion and activation of macrophage-like human leukocytes. adsorbed fibrinogen has been shown to be one of the key proteins in stimulating inflammatory responses to biomaterials. adsorption was investigated quantitatively using i-radiolabeled human fibrinogen. in addition, the conformation of the adsorbed protein was tested using an antifibrinogen monoclonal antibody in an enzyme-linked immunosorbent assay. the results showed that pegylated surfaces adsorbed up to % less fibrinogen, and that unfolding of adsorbed fibrinogen was more pronounced on the linear mpeg layers than on the peg-like plasma polymer surfaces. adhesion of in-vitro differentiated macrophage-like u cells was reduced on both the peg-like plasma polymer surfaces and the linear mpeg layers compared to the unmodified pet surface, but cells adhering to the peg-like plasma polymer surfaces secreted less tumor necrosis factor-␣ (tnf-␣) than cells adhering to the linear mpeg layers. thus, the linear mpeg surface is relatively efficient at reducing adhesion of macrophage-like cells, but those cells that do attach are in a more activated and proinflammatory state. analysis of ceramides from biological samples m. budvytiene , j. liesiene , b. niemeyer , n. ceramides in human skin play an important role in the regulation of cell growth, differentiation and apoptosis. moreover, they are involved in the numerous signaling pathways. the growing interest in the investigations of ceramides physiological functions requires efficient separation methods of ceramides from biological resources and sensitive analytical methods. in this work some sensitive and selective methods, involving thin layer chromatography (tlc), high performance liquid chromatography (hplc), mass spectrometry (ms) and nuclear magnetic resonance spectroscopy (nmr) were employed for the separation and characterization of ceramides from human foreskin. epidermal lipids were extracted from human foreskin for h at room temperature using three solvent mixtures (chloroform/ethanol/water : : . , v/v/v); (chloroform/ethanol : , v/v), and (chloroform/ethanol : , v/v). ceramides retention char-acteristics in tlc and hplc were compared with the retention of commercial standards. the best separation was obtained using normal phase column packed with hilic silica m. the elution was performed using mixture of chloroform and ethanol / (v/v) as an eluent. two commercial standards n-stearoyl-sphingosine cer(ns), r f = . , and n-palmitoyl-sphingosine cer(np), r f = . , were selectively separated with hplc system in above mentioned conditions. the retention time of cer(np) and cer(ns) was . and . min, respectively. the same lipids were detected by hplc in the human foreskin extracts. the structure of the lipids from collected fractions was confirmed by means mass spectrometry and nmr. the physiological functions of the separated ceramides are investigated. production recombinant human thymosin-␣ overexpressed as intein fusion protein in e. coli roman s. esipov, vasily n. stepanenko, anatoly i. miroshnikov, shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, ul. miklukho-maklaya / , moscow, russia. e-mail: esipov@ibch.ru (roman s. esipov) medicines based on polypeptides consisting of and more amino acid residues are widely spread in pharmaceutical market at the present time. practically all polypeptide medicines known are prepared by general chemical synthesis that caused high cost of their production. that's why biotechnological way of polypeptide medicines preparation using recombinant gene expression in bacteria seems to be promising. during our work we design hybrid construction allowing solving a problem of specific cleavage of target polypeptide from the hybrid protein using system of protein splicing from new england biolabs. in case of thymosin-␣ production system intein mediated purification with affinity chitin (impac system)-binding tag has been used, where the modified sce vma from s. cerevisiae has been applied as intein. in contrast to other systems, impact allows the preparation of target protein without using of serine protease and other factors that may cleave the hybrid protein. in the presence of thiol reagents, such as dithiothreitol, mercaptoethanol, or cystein the hybrid protein can be site-specifically cleaved to give intein, the target protein and small fragment of nextein. using of this system does not allow to obtain the target protein with ser, cys or thr on n-terminal of protein, because in those cases target protein and n-extein ligation product will be formed. ser is n-terminal amino-acid in thymosin-␣ . it was recently found that some metal ions essentially affect the splicing. we tried to use zncl and we have found that, in case of intein-thymosin-␣ , the maximal yield of target polypeptide and the minimal yield of splicing products are observed in the absence of dithithreitol and in the presence of . - mm zinc chloride in buffers on all stages of thymosin ␣ isolation. the structure of recombinant thymosin-␣ of human was confirmed by the determination of n-and c-terminal amino-acid sequences and by maldi tof mass-spectrometry. we present a microbioreactor with thermoelectric cooling to inactivate cellular metabolism by cell culture freezing. small-scale cultivation methods have gained increased importance and their development has been supported by advances in bioprocess monitoring methods. yet efficient sampling methods for off-line analysis remain important where in-situ real-time measurements are difficult, for example for intracellular metabolite concentration or enzyme activity measurements, and for all methods which are invasive by nature, such as protein purification. freeze-stop measurements of metabolite levels are ideal, because they are inert, i.e. do not require the addition of a chemical. in large systems, the chilling time is often the limiting factor, and alternative methods for cell metabolism inactivation are required, such as the spraying of the cell suspension in % methanol at a temperature of − • c, or the use of boiling buffered ethanol. due to the small thermal mass of microsystems, shorter chilling times can be expected. sample cooling to • c in a microbioreactor has been presented previously. in this contribution, we investigate sample freezing to completely inactivate the cell metabolism from microbioreactor working volumes of approximately l. the use of multi-parameter flow cytometry for characterisation and monitoring of insect cell-baculovirus fermentations in a mechanically-agitated bioreactor bojan isailovic , alvin w. nienow , ian w. taylor , ryan hicks , christopher j. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf- cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp coding for am-cyan coral protein, which emits natural green fluorescence. the optimization of a fermentation process requires the organism to be cultivated under desirable conditions, which depends on how well the fermentation process is controlled. inadequate mixing and mass transfer are responsible for the heterogeneous environment at large scale in terms of nutrient concentration and ph profile, resulting in lower product yields. these have been associated with, inadequate control of ph and the production of acetate or formate in response to over-feeding of glucose and oxygen deficiency. rapid analyses of substrates and indicator metabolites in a fermentation process is critical for optimal control. this can be achieved in real time with nir spectroscopy. in this study, nir-spectroscopy has been applied to monitor the concentrations of glucose, acetate, formate, ammonium hydroxide and biomass in the cultivation of e. coli (w ). a comparison of partial least square models built using water standards, synthetic medium standards and fermentation samples has been made. the control of the brewing process is important for improving product quality, and lowering costs. infrared spectroscopy is a technique that can be used at-line and on-line to rapidly measure component concentrations of unprocessed whole broth samples in real time. in this study, both mid -infrared (mir) and near-infrared (nir) spectroscopy have been used and compared for the monitoring of ethanol, flavour components, wort sugars, biomass and specific gravity during the brewing. partial least-squares regression (pls) was used to model relationships between component concentrations and spectra. the performance of these models was evaluated in terms of the standard error of prediction (sep), number of pls factors and the correlation coefficient (r). calibration models were constructed using spectra acquired for multi-component mixtures, intended to simulate brewing fermentation conditions, and actual brewing fermentation samples. chemometric results indicated that nir is a powerful tool for accurately measuring sugar, ethanol and biomass concentrations. when choosing an expression host for production of a specific recombinant protein, one can essentially select from a multiplicity of different systems. while e. coli bacterium is usually the starting point for any cloning and expression effort, there is no universal expression host that would work optimally for all proteins. practical issues to consider include e.g. need for post-translational modifications and protease activity of the host. we have produced recombinant hiv- nef in different host cell systems: e. coli, p. pastoris yeast and stable drosophila s insect cells. using strain/cell line development, production and purification data from practical experiments, we were able to conduct a techno-economical comparison of the different host cell systems. the annual production goal was set at mg of high-purity nef. this was supposed to be produced campaign-wise in - batches using laboratory-scale bioreactors and other equipment. in this study, it was shown that although the production costs of the different systems were in the same range, the production in e. coli was most inexpensive, and the s cell system was the most expensive. regardless of the selected host system, the labour costs incurred the most expenses. when comparing different stages of the work (strain/cell line development, bioreactor production and down-stream processing), the strain development, the most man-hours demanding stage, involved approximately half of the costs of every production system. although e. coli was the most inexpensive host system for producing nef, it has some definite disadvantages: e.g. the production of endotoxins and the disability to perform post-translational modifications. if these disadvantages are of importance, the production must be done using more expensive system. modelling and control of industrial fermentation j.k. rasmussen, s.b. jørgensen capec, department of chemical engineering, technical university of denmark, dk- lyngby, denmark. e-mail: jkr@kt.dtu.dk (j.k. rasmussen) fed-batch processes play a very important role in chemical and biochemical industry. fermentations in biochemical industry are most often carried out as fed-batch processes. present control schemes do not utilize the full potential of the production facilities and may fail to achieve uniform product quality and optimal productivity. the introduction of model based control strategies is considered difficult because suitable models are not readily available. first principle engineering models can be used but the usually limited knowledge of the regulatory network in the micro-organism makes model development very time consuming. another strategy is to use a purely data-driven approach where only limited prior knowledge of the process is required. a framework for generation of such blackbox models is used in this project. this framework is called "grid of linear models" (golm), it uses a large number of linear models which each describes the behaviour of the process within a certain time interval. the combination of these models results in a model which covers the entire time span of the fermentation and approximates the nonlinear time varying behaviour. a procedure for deriving golm models from operational data has been developed and because they consist of a large set of linear models it makes them suitable for model predictive control implementation with iterative learning capabilities from batch to batch. iterative learning model predictive control (ilmpc) based on a golm model is being implemented on a fermentor at novozymes a/s. the results will be evaluated in terms of the controller's capability to ensure uniform product quality and reject both intra and inter batch process disturbances. the model based approach renders optimization of the process recipe possible by using the ilmpc capability. this opportunity provides a great potential for increase of productivity and reduction of cost. j.m. viader-salvadó, j.a. fuentes-garibay, l.j. galán-wong, m. guerrero-olazarán institute of biotechnology, biological science school, autonomous university of nuevo león, san nicolás de los garza, n.l., méxico, the production of recombinant trypsin and trypsinogen has been reported as difficult due to a probably toxicity on host or its instability. in an effort to attain high-level production of shrimp trypsin for aquaculture applications, we have evaluated shrimp trypsinogen production by recombinant pichia pastoris strains in -l bioreactors. a p. pastoris gs mut+ containing in its genome the litopenaeus vannamei trypsinogen cdna fused in frame to saccharomyces cerevisiae ␣-factor secretion signal, previously constructed in our laboratory, was used. four three-step fermentations (glycerol batch, glycerol and methanol fed-batch) were carried out. the glycerol batch step ( l of basal salts medium, g/l glycerol, . ml biotin . %, . ml ptm , l antifoam, ph adjusted to with % nh oh) was carried out until glycerol was completely exhausted ( h). the glycerol fed-batch step was carried out feeding with % glycerol ( ml/l biotin . % and ptm ) at . ml/min by h and min of posterior starvation. the methanol fed-batch step was carried out feeding with % methanol ( ml/l biotin . % and ptm ) by h using a methanol concentration on/off feedback control to maintain constant the methanol concentration in the culture medium to g/l. in all the fermentations the air flow rate and the agitation were set at l/min and - rpm, respectively. with the four fermentation assays, the influence of the ph and temperature in the production phase to the recombinant shrimp trypsinogen production were evaluated. in the four fermentations, at the end of the second step a biomass of g/l wet weight were obtained (od ). the methanol demand in the four fermentations surprisingly was not increasing rather initially it was . ml/min, after h decreased . times for h, increased to . ml/min for h and afterwards it was decreased manually to a constant value of . ml/min for that the dissolved oxygen will not decrease to values less than % (last h). the total protein amount in the culture medium supernatant increased during the production step until values of . g/l (assay at ph ), . times more than the worst assay (ph ) recombinant shrimp trypsinogen production was confirmed by sds-page (about mg/l) and trypsin enzymatic activity was detected using bapna as substrate after trypsinogen activation with shrimp hepatopancreas extract. large conformational change on giant dna induced by ascorbic acid: a nobel scheme on its antioxidative activity yuko yoshikawa, emi sakai, yoshiko oda department of food and nutrition, nagoya bunri college, nagoya - , japan ascorbic acid is often regarded as an antioxidant in vivo, where it protects against dna damage by scavenging reactive oxygen species. in the present, we will show another potent scenario on the protective effect of ascorbic acid through a significant structural change of giant dna. recently, we examined the effect of ascorbic acid on the higher order structure of dna through single molecular observation with fluorescence microscopy, and found that ascorbic acid generates a pearling structure in giant dna molecules, where elongated and compact parts coexist along a molecular chain. the results of observations with atomic force microscopy indicate that the compact parts assume a loosely packed conformation. as the extension, here we study the protective effect against double-strand breaks by reactive oxygen at different concentrations of ascorbic acid, in relation to the change of the higher order structure of giant dna. we have performed a real time observation on the doublestrand breaks on individual dna molecules by use of fluorescence microscopy. we have found that the double-strand break is markedly protected when ascorbic acid exists over millimolar concentrations. it is found that such a protective effect of ascorbic acid corresponds well to the above mentioned change on the higher order structure of dna. it has been reported that human circulating immune cells, such as neutrophils, monocytes and lymphocytes, accumulate ascorbic acid in millimolar concentrations. therefore, it is expected that the ascorbic acid concentration that induces the large conformational change on dna may be of physiological significance. , y., et al., . febs lett. , - . yoshikawa, y., et al., . plant proteins are increasingly being used as an alternative to proteins from animal sources and substantially contribute to the human diet in several developing countries. there are many process both industrial and food based which employ protein hydrolysis and hydrolytic products have a wide variety of applications from industrial fermentation media to food additives. traditionally, proteins are hydrolysed by chemical means. acid hydrolysates of protein are used to produce food ingredients and flavour compounds. however, hydrolysis by chemical reagents produce potentially hazardous byproducts and these non-selective hydrolysis products cannot easily be defined. the use of enzymes allows for selective hydrolysis of protein and thus produces a potentially safer and more defined material. the present investigation describes the effects of substrate, enzyme and hydrolysate concentration on the hydrolysis of corn gluten. the corn gluten was hydrolysed by a commercial protease preparation neutrase. the protein hydrolysis reactions were carried out in . l of aqueous solutions at the temperature of • c and ph and were monitored by using ph-stat method. the degree of hydrolysis (%) and soluble protein concentration depending on time were investigated by using , , , , and % (w/v) substrate concentrations; and . , . , . , . and . , % (v/v) enzyme concentrations; and , , and % (v/v) this paper describes medium optimization for urease production by aspergillus niger ptcc by one-factor-at-a-time and orthogonal array design methods. the one-factor-at-a-time method was used to study the effects of carbon and nitrogen sources on urease production. among various carbon and nitrogen sources used, sucrose and yeast extract were the most suitable for urease production, respectively. subsequently, the concentration of sucrose, yeast extract and mineral sources were optimized using the orthogonal array method in two stages. the effects of nutritional components for urease production by a. niger ptcc in the first and second stages were in order of urea > niso > sucrose >kh po > k hpo > cacl > yeast extract > mgso and yeast extract > sucrose > k hpo > kh po > urea > cacl > niso , respectively. the optimal concentrations of nutritional components for improved urease production were determined as g/l sucrose, . g/l urea, . g/l yeast extract, . g/l niso · h o, . g/l mgso · h o, . g/l cacl , . g/l kh po , and . g/l k hpo . these results showed that urea, niso , yeast extract and sucrose had significant effect on urease production by a. niger ptcc . tween and mgso had negligible effect on urease production by this strain. the subsequent confirmation experiments determined the validity of the models. maximum urease activity in optimized media by onefactor-at-a-time and orthogonal array methods were about . and . times greater than with the basal medium, respectively. carbon sources create fingerprint fermentation characteristics pınar Ç alık , güzide Ç alık , tunçer h.Özdamar : bre lab, department of chemical engineering, ankara university, ankara, turkey; ib lab, department of chemical engineering, metu, ankara, turkey. e-mail: calik@eng.ankara.edu.tr (g. Ç alık) this work reports on a systematic investigation of the interactions between the single-carbon sources, i.e. glucose and citric acid, and complex-medium components, i.e., carbon and nitrogen sources, in enzyme fermentation processes, i.e. serine alkaline protease (sap; ec . . . ),) and ␤-lactamase (ec . . . ), with the oxygentransfer and ph conditions to demonstrate the influences of carbon sources that create the fingerprint fermentation characteristics, moreover, their influences on the product and by-product formations and the intracellular reaction rates. the influence of the medium composition i.e. citric acid-, glucose-, molasses-and soybean-based media together with the oxygen transfer (ot)-and ph-conditions applied, on the product and by-product distributions and ot characteristics were investigated in batch bioreactors. for sap, in general, under uncontrolled-ph operations the variation of the medium ph in sap fermentation process has a tendency to increase in the sap production phase; however, depending on the carbon source used, its behaviour changes in the early stages of the fermentation as the consequences of the directed functioning of the intracellular bioreaction network. the loci of the dissolved oxygen (do) curves also strongly depend on the carbon source(s) utilised, in addition to the applied ot conditions. the complex media profiles are significantly different compared to the defined media as the ph and do profiles are interrelated owing to the bioreactor operation conditions affecting the metabolic reaction network. the highest volumetric oxygen uptake rates were obtained with soybean-based medium that was ca. three-fold higher than the values reported in citrate-based and glucose based media, and ca. . - -fold higher than the values reported in molasses-based medium. the significant changes, moreover, the drastic change observed with the use of soybean-based complex medium are due to the compositions of the fermentation media used, and its influence on the intracellular bioreaction network. thus, we conclude that the change in medium composition based on the carbon source changes the fermentation characteristics under the designed bioreactor operation conditions that appear as the fingerprints of the bioprocess. effects of oxygen transfer on ␣-amylase production by b. amyloliquefaciens nurhan güngör, güzide Ç alık bre lab, department of chemical engineering, ankara university, ankara, turkey ␣-amylase (e.c. . . . ) a commercially important enzyme used in food, textile, detergent, brewing, paper, and animal feed industries; hydrolyses ␣- , glucosydic bonds in amylose, amylopectin, and related polysaccharides. optimum medium composition and influence of bioreactor operation parameters on ␣-amylase production with high yield and selectivity were determined together with the metabolic flux distributions using b. amyloliquefaciens (nrrl b- ) which is found to be a good producer of the enzyme. systematic investigation of oxygen transfer in relation with the metabolic fluxes for ␣-amylase is not available in the literature. shake-flask experiments were conducted in . dm airfiltered bioreactors in orbital shakers with agitation and heating controls (b. braun, certomat-bs ). laboratory-scale bioreactors were composed of agitation, heating, foam, dissolved-oxygen and ph-controlled . and . dm systems (b. braun, biostat q; and chemap). after separation of the cells with a sorval rc s ultracentrifuge, ␣-amylase activity was measured by the dns method (bernfeld, ) . amino acid concentrations were determined with a hplc (waters), protein and organic acid concentrations were measured with a hpce (waters, quanta e) (Ç alık et al., ) . oxygen transfer characteristics in the bioreactor systems were calculated using the dynamic method (rainer, ) . in the mass flux balance-based analyses, a pseudo-steady state approximation for the intracellular metabolites and the accumulation rates of the extracellular metabolites measured throughout the fermentations in considerations of the biochemical feature of the system were used to acquire the flux distributions. in laboratory scale, the effects of different c sources i.e., glucose, fructose, maltose, lactose and soluble starch; n sources i.e., (nh ) hpo , (nh ) so , and nh cl and/or their concentrations; and the operation parameters, ph and temperature on cell growth, substrate utilization, ␣-amylase and byproduct concentrations, and ␣-amylase activity were investigated. in pilot scale, the fermentation and oxygen transfer characteristics of the bioreaction system together with the metabolic fluxes were determined. oxygen transfer regulates benzaldehyde lyase production in e. coli pınar Ç alık iblab, department of chemical engineering, metu, ankara, turkey. e-mail: pcalik@metu.edu.tr the effects of oxygen transfer rate on benzaldehyde lyase (bal) production by puc ::bal carrying recombinant e. coli on a defined medium with . kg/m glucose were investigated in order to finetune the bioreactor performance, in v = dm batch bioreactors at five different conditions with the parameters at, i.e. q /v r = . vvm and n = , , and min − and; q /v r = . vvm and n = min − . the concentrations of the product and by-products amino acids and organic acids were determined in addition to bal activities. medium oxygen transfer rate conditions and uncontrolled ph operation at ph . are optimum for maximum bal activity, i.e. u/cm at h, and productivity and selectivity. on the bases of the data, response of the intracellular bioreaction network of r-e. coli to oxygen transfer conditions were analysed using a mass-flux balance based stoichiometric model that contains metabolites and reaction fluxes. the results reveal that metabolic reactions are intimately coupled with the oxygen transfer conditions. oxygen transfer rate showed diverse effects on the product formation by influencing metabolic pathways and changing metabolic fluxes. metabolic flux analysis was helpful to describe the interactions between the cell and the bioreactor by predicting the changes in the fluxes and the rate controlling step(s) in the metabolic pathways. therefore, knowing the distribution of the metabolic fluxes during the growth, and bal and by-product formations provide new information for understanding physiological characteristics of the r-e.coli, and reveals important features of the regulation of the bioprocess and opens new avenues to successful application of metabolic engineering. the detection and diagnosis of pathogenic bacteria causing many diseases to the human body is an area of important research to public welfare. food is the most important energy source to humans, but it can give rise to disease caused by pathogenic bacteria not performing adequate detection tests. oligonucleotide-based microarrays are becoming increasingly useful for the analysis of expression profiles and polymorphisms among interested genes. here, we checked the possibility of development of oligonucleotide-based microarrays for detection and diagnosis of foodborne pathogenic bacteria. the oligonucleotide chip technology was applied to one control strain and seven foodborne pathogenic bacteria strains. it was designed repeated spots of eight hyperspecific and two highly conserved (control) capture probes from s rdna sequences. in order to validate experimental quality and to certificate specificities among specific spots at a glance by d and d views, quantitative visualization tool was developed. using the proposed oligonucleotide chip, we could classify and diagnose species and even subtypes of some pathogens. induction of in vitro neuro-muscular junctions using neuroblastoma and fibroblast cell lines for facilitating receptor-binding studies with botulinum toxin arindam chaudhury, bal ram singh department of chemistry and biochemistry, university of massachusetts dartmouth, old westport road, north dartmouth, ma - , usa. e-mail: g achaudhury@umassd.edu (a. chaudhury) botulinum toxin (bont), the most potent biological toxin known, is responsible for botulism, a fatal paralytic disease of the neuromuscular transmission. it blocks the release of acetylcholine at the neurotransmitter junction of the synapse. the objective of the current study was to induce in vitro neuro-muscular junctions through co-culturing of nerve and precursor-muscle cell lines. presently no known primary cultures or cell lines are available for nerve-muscle co-culture, thus validating the current work. j - t fibroblast cell line was first adapted to grow in media conducive for growth of sh-sy y neuroblastoma cell line. the two cell lines were then splitted and co-cultured and observed for junction formations. light and fluorescent microscopic studies revealed en plaque (twitch-type) and en grappe (bulbous nerve endings) nerve-muscle junctions. growth rate of j - t cells decreased substantially when the media was initially changed. structurally they were more spindle-shaped than the normal reticular shapes of j - t cells, when grown in a media tailor-made for them. the formation of nerve-muscle junctions were confirmed using markers specific for each cell type. future work is focusing on receptor identification for the botulinum toxin in the established in vitro neuro-muscular junctions and also the transcellular translocation of the toxin. fourier-transform infrared (ftir) spectrometers have recently enjoyed widespread popularity in bioprocess monitoring applications due to their non-invasiveness and in-situ sterilizability. their online applicability creates an interesting opportunity for process control and optimization. however, the precision and accuracy of the predicted analyte concentration values directly depend on the quality of the measured signal and the robustness of the calibration model. instability and time drift in the measured spectra are currently one of the main obstacles in ftir monitoring. the intensity of the detected signal is influenced both by random noise and structural drifts and offsets. as a result, it is often necessary to scale the measured spectrum with respect to a constant reference spectrum, a technique similar to the internal standard approach used in analytical assays, such as hplc. applying this technique has lead to a noticeable decrease in the standard error of prediction in the monitoring of an anaerobic s fermentation of the saccharomyces cerevisiae yeast. in order to test the robustness of the calibration model and to increase its resistance to signal instability, random spikes of known amounts of analytes were introduced into the measured medium. this approach can be used to fine-tune the calibration model on-line and is currently one of the aspects investigated in this laboratory. the effect of the stringent response induction on l-valine biosynthesis by corynebacterium glutamicum ilze denina , , longina paegle , liga zala , , maija ruklisha : faculty of biology, university of latvia, kronvalda blvd. , riga lv- , latvia; institute of microbiology and biotechnology, university of latvia, kronvalda blvd. , riga lv- , latvia. e-mail: ilzede@hotmail.com (i. denina) the present study was focused on methods of the stringent response induction and on investigation of its effect on valine overproduction by isoleucine auxotrophs of corynebacterium glutamicum. the intracellular level of guanosine -diphosphate diphosphate (ppgpp) increased and bacterial growth rate (µ) decreased during the short-term experiments when the exponentially growing cells were exposed to isoleucine limited conditions. the induction of the cellular stringent response resulted in a drastic increase in the activity of acetoxydroxy acid synthase (ahas), also by a significant increase in valine production. in contrast, an increase in ahas activity and valine synthesis by c. glutamicum was not achieved when bacterial growth was down-regulated in a ppgpp-independent manner. these results demonstrated that induction of the ppgpp-mediated stringent response might be significant in order to increase valine overproduction by c. glutamicum. infections with human cytomegalovirus (hcmv) continue to be an important health problem in certain patient populations, such as newborns, graft recipients of solid organs, or bone marrow and aids patients. in these groups, hcmv is a major cause of morbidity and mortality. the complex biology of hcmv necessarily begins with an initial interaction between the envelope of the infectious virus and the host cell. glycoprotein b (gb) is the major antigen, on the envelope of hcmv, for the induction of neutralizing antibodies. the region between aa and of hcmv gb (termed antigenic domain , ad- ) has been identified as the immunodominant target for the humoral immune response following natural infection. screening methods for detection of neutralizing antibodies have not been used because they are costly and labor intensive and thus far are not feasible for use on a large scale. for the development of reliable and inexpensive serodiagnostic tests the ad- of hcmv glycoprotein gp , which are known to bind neutralizing antibodies, was expressed in prokaryotic systems. in this work, one prokaryotically expressed fusion protein which codifies ad- with ␤-galactosidase was used. the influence of different process conditions, on the production of the fusion protein containing the ad- as well as sugars addition to the fermentation medium was investigated. in order to analyze the expression of fusion protein, the ␤-galactosidase activity was followed throughout the fermentation. lysis process was also optimized and some final confirmation tests about protein antigenicity were performed. polyenzymic systems for the preparation of drugs based on modified nucleosides d. chuvikovsky, r. esipov, t. muravyova, a. miroshnikov shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, ul. miklukho-maklaya / , moscow , russia. e-mail: esipov@ibch.ru (r. esipov) considerable progress in the preparation of nucleoside analogues was achieved by combination of chemical and biochemical transformations. enzyme-catalyzed chemical transformation is now widely recognized as practical alternative to traditional organic synthesis in pharmaceutical and chemical industries. pentofuranosyltransfer reaction catalyzed by nucleoside phosphorylases was successfully employed for the synthesis of a variety of base-and sugar-modified nucleosides. enzymes involved in the metabolism of ribose phosphate and deoxyribose phosphate, such as ribokinase and phosphopentomutase, were used for the preparation of sugar-modified nucleosides. nucleosides phosphorylases (thymidine phosphorylase (tp), uridine phosphorylase (up) and purine nucleoside phosphorylase (pnp)), ribokinase and phosphopentomutase from e. coli have been cloned and overexpressed in e. coli. fast and efficient methods for the purification of nucleosides phosphorylases have been developed. the amount of purified protein was about mg/l of cell culture, corresponding to , , and units, respectively, of the up, tp and pnp. synthesis of medicinal drugs ribavirin ( -(␤-d-ribofuranosyl)- , , -triazole- -carboxamide), cladribine ( -chloroadenine- -␤-d- -deoxyribofuranoside) and fludarabine ( -fluoroadenine- -␤-darabinofuranoside) with the use of recombinant enzymes were studied. several important factors affecting the modified nucleosides production (ph, temperature, enzyme concentration, donor/acceptor ratio) were investigated and optimized. under optimum conditions ribavirin, cladribine and fludarabine produced in the reaction mixture in yields of , and %, referred to , , -triazole- -carboxamide, -chloroadenosine and -fluoroadenosine, respectively. aggregation and adsorption of fibroin molecules in aqueous solution won hur school of biotechnology and bioengineering, kangwon national university, chunchon - , korea. e-mail: wonhur@kangwon.ac.kr fibroin, the structural protein from bombyx mori, is composed of heavy chain (generally called 'fibroin') and light chain polypeptides of about and kda, respectively. this study investigated the aggregation of fibroin and the adsorption between fibroin and surfaces. the variations of particle size and zeta potential were investigated by electrophoretic light scattering spectrophotometer (els). the adsorption of fibroin on surface was investigated in a continuous flow system by biacore applied surface plasmon resonance(spr) technique. the particle size and zeta potential range of aqueous fibroin were nm, ± mv, respectively. iso-electric point(ph iep )of fibroin was ph . . the amount of fibroin adsorbed on a gold surface was less than . g/ml even in the presence of high concentration of fibroin. the modification of gold surface was accomplished by applying chemicals known to form self-assembled monolayer, those are carrying nh + , coo − , benzene ring and peptide that similar structure with fibroin. the adsorbed amount of fibroin on the self-assembly monolayers (sams) increased in the following order: nh + > benzene ring > coo − > peptide surface. the deposition of fibroin in aqueous solution on non-waven fabric was affected by nacl and high temperature. ph influences metabolite profiling of ␤-lactamse producing b. licheniformis nazarİleri , pınar Ç alık , ali Şengül : ib lab, department of chemical engineering, metu, ankara, turkey; gülhane sch med, dept immunol, ankara, turkey. e-mail: e @metu.edu.tr (n.İleri) ph conditions in the bioreactor affect product and by-product formations by influencing metabolic pathways and changing metabolic fluxes, based on its influence on, i.e. dna transcription, protein synthesis, transport mechanism, atp generation and cellular energetics. whereupon, some fermentation processes favours uncontrolled-ph conditions while others favours controlled-ph conditions. on the bases of the interactions between the cell and the bioreactor through a process, carried out at either uncontrolled-or controlled-ph conditions, intracellular ph can be widely different and variable during the fermentation process. consequently, if one aims towards a quantitative understanding of the cell metabolism, one has to take into account the time variations of the intracellular ph and its effects on the in-vivo kinetics of the metabolic steps involved. moreover, since the presence of dormant or dead cells in the cultivation medium have negative effect on the synthesis of the production of desired product; the physiological state of the culture has great importance. in this context, the effects of ph on the regulation of intracellular ph, transport mechanism, and metabolic activity of b. licheniformis during production of ␤-lactamase (ec . . . ), an industrial enzyme catalyzing the hydrolysis of beta-lactam ring in beta-lactam antibiotics, was investigated. in addition, the physiological state of the organism and its effect on the production were observed. the optimal controlled-ph operation was found to be ph . with u/cm enzyme activity. the intracellular and extracellular na + , k + ion concentrations increased significantly throughout the process with increasing ph. on the other hand, the intracellular nh + ion concentration was relatively constant. isolation and characterization of angiotensin-i converting enzyme inhibitory peptides by use of anti-peptide antibody fida hasan , megumi kitagawa , yoichi kumada , naoya hashimoto , masami shiiba , shigeo katoh , masaaki terashima : graduate school of science and technology, kobe university, nada, kobe - , japan; department of human science, kobe college, okadayama, nishinomiya - , japan inhibitory peptides against angiotensin-i converting enzyme can be promising bio-functional peptides as natural alternatives for the non-peptide ace inhibitory drugs. these peptides are inactive within sequences of parent proteins and can be released during enzymatic digestion or food processing. immunointeraction is very effective for the purification of proteins and peptides with high purity. in this study, ace inhibitory peptides from hydrolysate of bonito meat were isolated by an anti-peptide antibody column and hplc, and kinetics of production of these ace inhibitory peptides was studies. an anti-peptide antibody against an ace inhibitory peptide, which was found by kohama et al. from tuna was obtained by immunization of the antigen peptide pc-iace (kkpthikwgd). water extract of bonito meat was digested at • c in a modified gastric juice, . mg/ml nacl containing g/ml pepsin (ph ). peptides in hydrolysates were purified by use of an affinity column coupled with the antipeptide antibody and separated by hplc equipped with a reverse phase column (cosmosil c -ms-ii, . cm × cm). amino acid sequences and ic values of the potent ace inhibitory peptides were determined. sds-page and western blotting experiments clarified that bonito protein contained peptides having similar sequence to the antigen peptide. a fraction of retention time - min in hplc purification samples showed high inhibitory activity, and several peptides in this fraction were separated. after h digestion, two major inhibitory peptides, herdpthikwgd and pthikwgd, were found to be relatively stable in the gastric juice. kluyveromyces marxianus physiology on several levels of carbon, nitrogen sources and oxygenation during inulinase production silva-santisteban yépez, o. bernardo, francisco maugeri department of food eng./unicamp, - , campinas, sp-brazil. email: maugeri@fea.unicamp.br (f. maugeri) inulinase produced by yeasts is an interesting alternative compared with the one produced by filamentous molds, as culture conditions can be better controlled. during the assays, it was observed that inulinase production levels varied with nutritional conditions in batch culture. kluyveromyces marxianus atcc culture is described by two main phases, the first one being the growth phase, where substrate consumption and basal inulinase production were performed, and the second one being the phase where some metabolites are uptaken and high inulinase production is observed. the metabolic fluxes analyses were used to describe the cell physiology in the first phase, in a variety of conditions of sucrose and ammonium sulfate concentration and aeration condition. the metabolic network included the main metabolic pathways such as glycolisis, pentose phosphate pathway, krebs cycle, oxidative phosphorylation and biomass biosynthesis. the physiology in this phase was correlated with high inulinase production in the second phase. it was also noticed that inulinase production diminished when sucrose was in high concentration, leading, additionally, to ethanol production. in these terms, it was unveiled a kind of crabtree effect performed by this strain. forward extraction of l-aspartic acid from fermentation broths by reverse micelles and backward extraction by gas hydrate methodÖ. aydogan , e. bayraktar ,Ü. mehmetoglu , m. parlaktuna , t. mehmetoglu : department of chemical engineering, faculty of engineering, ankara university, tandogan, ankara , turkey; department of petroleum and natural gas engineering, middle east technical university, ankara , turkey. e-mail: mehmet@eng.ankara.edu.tr (Ü. mehmetoglu) recently gas hydrate method has been applied as a technique for backward extraction of amino acids from reverse micelle systems. in this study, backward extraction of l-aspartic acid was investigated by gas hydrate method. at the first stage, production of l-aspartic acid was carried out using ml of . m ammonium fumarate (ph . ) as substrate at circc in an orbital shaker at rpm for h. e. coli (atcc ) was used as biocatalyst. at the end of reaction excess fumaric acid was extracted in reverse micelle phase. then forward extraction of l-aspartic acid was carried out with injection method in reverse micelle phase. for back extraction, co is used to form gas hydrates crystalline structure. back extraction experiments were carried out between - bar g pressure and at circc. at the end of the back extraction l-aspartic acid was obtained in crystalline form. the results indicate that recovery of l-aspartic acid from reverse micelles by forming gas hydrate can be achieved with a yield of . %. consequently, gas hydrate method can be used as a new technique for backward extraction of amino acids from reverse micelles. aerobic and anaerobic cultivations of aspergillus niger on different nitrogen sources susan meijer, gianni panagiotou, lisbeth olsson and jens nielsen center of microbial biotechnology, biocentrum-dtu, technical university of denmark, dk- lyngby, denmark in this study, we aim at creating a succinic acid producing strain of a. niger. a. niger is known to be a strictly aerobic organism, meaning it is not able to use the reductive part of the tca cycle to produce succinate. during aerobic growth a. niger uses oxygen as electron acceptor in the respiratory chain, thereby reoxidizing the produced nadh to nad + . however, under anaerobic conditions other compounds than oxygen, such as no − are required as electron acceptor (denitrification). this process consists of no − reduction to nh + coupled to substrate-level phosphorylation that supports growth under anaerobic conditions. in the present study, our aim was to investigate the effect of different nitrogen sources on the physiology of a. niger during growth under aerobic and anaerobic conditions. aerobic growth experiments on three different nitrogen sources; ammonium, nitrate and nitrite, showed that ammonium and nitrate could be consumed by the filamentous fungus. nitrite on the other hand could not facilitate growth, indicating the absence of a nitrite uptake system. however, under anaerobic conditions notable growth was only observed on nitrate. these data support the hypothesis of the existence of an alternative electron acceptor that might facilitate anaerobic growth of a. niger. among the therapeutic proteins derived from mammalian cells, recombinant antibodies received a great deal of attention as a prominent product through biotech pipelines toward the marketplace. they now occupy about % of the estimated medicines in clinical development and many more antibodies which lead the value of the market going forward are reported. there are various environmental factors affecting rcho cell cultures such as medium components, temperature, ph, and byproducts (ammonia, lactate, and, etc.) . because most of mammalian cells are very sensitive to their environmental change, appropriate control of environmental parameters is a very important consideration to enhance cell growth and production of target proteins. balanced addition of limiting medium components plays an essential role on improvement of cell density and product concentration. temperature and ph are easily adjustable process parameters, being reported to influence cell growth and recombinant protein production. ammonia and lactate are well-known byproducts which have an inhibitory effect on cell growth when their concentrations exceed a specific level. in this work, effects of various environmental factors including temperature, ph, amino acids, vitamins, hormones, and metabolic byproducts on cell growth and recombinant antibody production were investigated in the cultivation of recombinant chinese hamster ovary cells. the most suitable condition of each environmental condition was proposed for enhancement of the cell growth and the productivity of recombinant antibody. the present study was carried out in order to assess the protective effects of calycosin- -o-␤-d-glucopyranoside isolated from astragali radix (ar) on hyaluronidase (haase) and the recombinant human interleukin- ␤ (il- ␤) induced matrix degradation in human articular cartilage and chondrocytes. we isolated the active component from the n-butanol soluble fraction of ar as haase inhibitor and structurally identified as calycosin- -o-␤-d-glucopyranoside by lc-ms, ir, h nmr, and c nmr analyses. the protective effect of arbu on the matrix gene expression of immortalized chondrocyte cell line c- /i treated with haase was investigated using a reverse transcription polymerase chain reaction (rt-pcr). its effect on haase and il- ␤-induced matrix degradation in human articular cartilage was determined by using a staining method and calculating the amount of degraded glycosaminoglycan (gag) from the cultured media. pretreatment with calycosin- -o-␤-d-glucopyranoside effectively protected against matrix degradation of the human chondrocytes and articular cartilage. therefore, it would appear that calycosin- -o-␤-d-glucopyranoside from ar is a potential natural ant-inflammatory or anti-osteoarthritis agent and can be effectively used to protect against proteoglycan (pg) degradation. catechol-o-methyltransferase (comt) is an enzyme that catalyses a variety of endogenous and exogenous catechol substrates by transferring a methyl group from s-adenosylmethionine (sam) to either the meta-or the para-hydroxyl group of the catechol ring. the enzyme has a physiological role in the metabolism of the catechol estrogens, inactivation of the catecholamine neurotransmitters such as dopamine and epinephrine and detoxification of a variety of xenobiotic catechols. comt activity has been identified in various tissues; however with the developments in molecular biology and gene technology, the production and purification of large amounts of recombinant comt is a good option for biochemical, pharmacological and structural studies. in this work, cultures of recombinant e. coli harbouring a model plasmid were grown in a ml shake-flask containing ml of complex medium. the influence of medium composition and induction time on comt production, recovery and clarification by sonication, ammonium sulphate precipitation and purification by hydrophobic interaction chromatography onto a butyl-sepharose column will be presented and discussed. bioactive bacterial exopolysaccharides corinne sinquin , karim senni , jacqueline ratiskol , farida guéniche , jean guézennec , gaston godeau , sylvia colliec-jouault : ifremer, nantes cedex , france; ea université rené descartes, montrouge, france. e-mail: corinne.sinquin@ifremer.fr (c. sinquin) interest in mass culture of microorganisms from the marine environment has increased considerably, representing an innovative approach to the biotechnological use of under-exploited resources. marine bacteria associated with deep-sea hydrothermal conditions have demonstrated their ability to produce in an aerobic carbohydrate-based medium, unusual extracellular polymers (guezennec, ; colliec-jouault et al., ) . these exopolysaccharides (eps) present original structural features that can be modified to design innovative bioactive compounds and improve their specificity. with the aim of promoting biological activities, chemical modifications (depolymerization and substitution reactions) of one eps produced by vibrio diabolicus have been undertaken (raguenes et al., ) . the structure of the native eps has been described (rougeaux et al., ) the potential of the eps derivatives as therapeutical agents will be presented. physiological responses of e. coli to glucose and oxygen shifts in fed-batch fermentations jaakko soini , christina saarimaa , arne matzen , peter neubauer : bioprocess engineering laboratory, department of process and environmental engineering, university of oulu, oulu fi- , finland; sanofi-aventis, germany. e-mail: jaakko.soini@oulu.fi (j. soini) in high-cell density fermentations e. coli cells are often subjects of transient changes in microenvironment around them. these changes can be, for example, medium component gradients or differences in oxygen availability. we have studied the physiological response of e. coli w cells to simultaneous oxygen limitation and overfeeding of glucose. the aim is to obtain more information of physiological changes for better understanding of the bottlenecks in such processes. the response of the cells for glucose and oxygen shifts was studied by analyzing key metabolites and proteins and mrna transcript levels. the transcript levels were measured using a sandwich hybridization technique (rautio et al., ) proteomic analysis was carried out by d-electrophoresis and the metabolite analysis by hplc. the main focus of this study is to monitor the expression patterns of marker genes involved in mixed acid fermentation, glycolytic pathway and tricarbonic acid cycle. rautio et al., . sandwich hybridisation assay for quantitative detection of yeast rnas in crude cell lysates. microb. cell fact. influence of ner on genetic instability of the (ctg/cag) tracts in bacterial chromosome sylwia szwarocka , paweł parniewski : department of microbiology and immunology, university of Łódź, - Łódź, banacha / , poland; centre for medical biology, polish academy of sciences, - Łódź, lodowa , poland many human hereditary neurological diseases, including fragile x syndrome, myotonic dystrophy and friedreich's ataxia, are associated with expansions of triplet repeat sequences (trs) (cgg/ccg, ctg/cag and gaa/ttc) in or near specific genes. mechanisms that mediate the expansions and deletions of trs include dna replication, repair and recombination. many investigations suggest that the structural properties of the trs play a consequential role in their genetic instabilities. nucleotide excision repair (ner) is the major cellular system in both prokaryotes and eukaryotes and recognises damages due to distortion of the dna helix. involvement of ner in the hairpin loop repair that can form within ctg tracts has been reported. the participation of this repair systems in the trs instability was investigated in e. coli only on multicopy plasmids. the results showed that deficiency of some ner functions dramatically affects the stability of long (ctg/cag) inserts. in this work we present a chromosomal model to study the instability of the trs in e. coli. we introduced the (ctg/cag)n tracts into the chromosome of e. coli and used strains with some deficiency of the ner and investigated genetic stability of these tracts after multiple recultivations. in general, our results show that the (ctg/cag)n repeats are much more stable in the chromosome than in plasmids. these data may suggest that instability of trs in plasmids is associated with interaction between repetitive tracts on different plasmid molecules inside the cell. however, mutations of ner genes may increase (uvra and uvrb mutants) or decrease (uvrc and uvrd mutants) stability of the trs in the e. coli chromosome. this study was partially funded by the kbn grant p a . performance analyses of a multi-stage integrated fermentation process for lactic acid production hsun-tung lin, feng-sheng wang department of chemical engineering, national chung cheng university, chia-yi - , taiwan. e-mail: chmfsw@ccu.edu.tw (f.s. wang) in this work, we considered a multi-stage integrated continuous fermentation process for producing lactic acid. each stage consists of a mixing tank, a fermenter, a cell recycle unit and an extractor. the generalized kinetic model is first applied to formulate the integrated process. we have compared the overall productivity and conversion of the integrated process with those of two simplified processes. from the design equations, we obtain that three processes have the identical overall conversion. however, the proposed process has the greatest overall productivity. the specific kinetic model for lactic production (youssef et al., ) was applied to the integrated process in order to find the maximum overall productivity. two optimization problems are respectively considered to determine the optimal stages, operating conditions and design variables. the first problem supposes that the integrated process has the equal working volume ratio for each fermenter. such a process requires four stages to yield the maximum overall productivity and the nearly complete overall conversion. however, if the working volume ratio for each stage is considered as the decision variables in the second optimization problem, three stages is enough to achieve the identical overall productivity. youssef, c.b., guillou, v., olmos-dichara, a., . contr. eng. pract. , - . modelling of the binding of ligands to macromolecules jørgen m. mollerup department of chemical engineering, building , dtu, lyngby, denmark a variety of factors that govern the properties of proteins are utilized in the development of chromatographic processes for the recovery of biological products including the binding and release of protons, the non-covalent association with non-polar groups (often hydrophobic interactions), the association of small ions (ion exchange) and the highly specific antigen-antibody interaction (affinity interactions). the fulcrum point in the understanding and modelling a chromatographic separation is the adsorption isotherm that determines the peak shape at preparative load. to enable an efficient chromatographic process development strategy it is necessary to conduct theoretical and experimental investigations of the adsorptive behaviour of proteins. thermodynamically consistent models for ion exchange chromatography and hydrophobic interaction chromatography have been developed and can be utilised in the simulation of a chromatographic separation. besides, measurements on hic media can be utilised to determine the cohn salting-out coefficient. the lig-and binding process can frequently be coupled to associated structure changes in the protein, the ligand or both. this gives rise to nonlinear adsorptive behaviour known as cooperativity which cannot be modelled using conventional models which displays convex behaviour. examples of cooperative behaviour are the reversible binding of oxygen and carbon monoxide to haemoglobins and the binding of nad + to yeast glyceraldehydes -phosphate dehydrogenase. in the paper we discuss the modelling of reversible binding of mobile as well as immobilised ligands to macromolecules and compare modelling to experiment. comparative analysis of the temperature policy for processes with a deactivating native enzyme i. grubecki, m. wojcik department of chemical and biochemical engineering, university of technology and agriculture, - bydgoszcz, ul. seminaryjna , poland a comparative analysis of the temperature policy for an enzymatic reaction with michaelis-menten kinetics in a batch reactor has been carried out. both isothermal and optimal temperature policies for processes with deactivating native enzyme have been considered. in the model, the thermal deactivation was described by a first-order reaction, and the arrhenius-type dependence between rate parameters and temperature was assumed. as an indicator for a direct comparison between the isothermal and optimal temperature policies the quotient of conversions under identical initial and final condition was used. a method was presented to calculate this indicator, which is based on the analytical and numerical solutions. this method can be of great importance for the industrial practice. application of changeable temperature policy could result in significant increase in conversion when ratio of activation energy for deactivation and activation energy for reaction is high. studies on the impact of mixing during brewing using near and mid-infrared spectroscopy georgina mcleod , alvin w. nienow , graham poulter , reg wilson , henri tapp , christopher j. the control of the brewing process is important for improving product quality, and lowering costs. infrared spectroscopy is a technique that can be used at-line and on-line to rapidly measure component concentrations of unprocessed whole broth samples in real time. in this study, both mid -infrared (mir) and near-infrared (nir) spectroscopy have been used and compared for the monitoring of ethanol, flavour components, wort sugars, biomass and specific gravity during the brewing. partial least-squares regression (pls) was used to model relationships between component concentrations and spectra. the performance of these models was evaluated in terms of the standard error of prediction (sep), number of pls factors and the correlation coefficient (r). calibration models were constructed using spectra acquired for multi-component mixtures, intended to simulate brewing fermentation conditions, and actual brewing fermentation samples. chemometric results indicated that nir is a powerful tool for accurately measuring sugar, ethanol and biomass concentrations. the optimization of a fermentation process requires the organism to be cultivated under desirable conditions, which depends on how well the fermentation process is controlled. inadequate mixing and mass transfer are responsible for the heterogeneous environment at large scale in terms of nutrient concentration and ph profile, resulting in lower product yields. these have been associated with, inadequate control of ph and the production of acetate or formate in response to over-feeding of glucose and oxygen deficiency. rapid analyses of substrates and indicator metabolites in a fermentation process is critical for optimal control. this can be achieved in real time with nir spectroscopy. in this study, nir-spectroscopy has been applied to monitor the concentrations of glucose, acetate, formate, ammonium hydroxide and biomass in the cultivation of e. coli (w ). a comparison of partial least square models built using water standards, synthetic medium standards and fermentation samples has been made. template refolding utilizing biospecific interactions shigeo katoh , yoichi kumada , nanae maeshima , daisuke nohara : graduate school of science and technologykobe university, kobe - , japan; department of biomolecular sciencegifu university, gifu - , japan. e-mail: katoh@kobe-u.ac.jp (s. katoh) recombinant proteins over-expressed in e. coli are often accumulated as insoluble particles called inclusion bodies. proteins in inclusion bodies must be solubilized by a denaturing agent, such as urea and guanidine hydrochloride, and refolded to recover their native structures having biological activities. in bioprocesses it is important to obtain high refolding efficiencies and high throughputs at high protein concentrations. in refolding operation, a denatured protein solution is usually added batch-wise into a large volume of a refolding buffer in order to start refolding by reducing the concentration of a denaturant and to prevent aggregate formation of renaturing molecules. thus, a large volume of a stirred tank is required, and the concentration of protein after renaturation becomes low. biointeractions between a pair of biomolecules, such as enzyme-inhibitor, antigen-antibody and hormone-receptor, are highly specific and have been used for detection and separation of biomolecules. these interactions may be used as templates for refolding of target molecules, which can be captured with the templates and are prevented from aggregate formation and, in the case of proteases, from autoproteolysis. the specific interaction might promote refolding of the target molecules. these might improve the refolding efficiency. the biointeractions between antigen-antibody and enzyme-inhibitor were used for efficient refolding in packed columns, in which template ligands (antibody, inhibitor) were coupled on gel support. denatured solutions of target molecules (carbonic anhydrase and s. griseus trypsin) were mixed with refolding buffer and supplied to the affinity column coupled with the template ligands for refolding. with refolding in the column, higher refolding efficiencies were obtained than those by the batch dilution method with relatively low concentrations of denaturants. by increasing the adsorption capacity of the column, throughput of refolding can be increased without decrease in the refolding efficiency. the use of multi-parameter flow cytometry for characterisation and monitoring of insect cell-baculovirus fermentations in a mechanically-agitated bioreactor bojan isailovic , alvin w. nienow , ian w. taylor , ryan hicks , christopher j. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf- cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp coding for am-cyan coral protein, which emits natural green fluorescence. carbon sources create fingerprint fermentation characteristics pınar Ç alık , güzide Ç alık , tunçer h.Özdamar bre lab, department of chemical engineering, ankara university, ankara, turkey; ib lab, department of chemical engineering, metu, ankara, turkey. e-mail: calik@eng.ankara.edu.tr (g. Ç alık) this work reports on a systematic investigation of the interactions between the single-carbon sources, i.e., glucose and citric acid, and complex-medium components, i.e., carbon and nitrogen sources, in enzyme fermentation processes, i.e., serine alkaline protease (sap; ec . . . ),) and ␤-lactamase (ec . . . ), with the oxygentransfer and ph conditions to demonstrate the influences of carbon sources that create the fingerprint fermentation characteristics, moreover, their influences on the product and by-product formations and the intracellular reaction rates. the influence of the medium composition i.e. citric acid-, glucose-, molasses-and soybean-based media together with the oxygen transfer (ot)-and ph-conditions applied, on the product and by-product distributions and ot characteristics were investigated in batch bioreactors. for sap, in general, under uncontrolled-ph operations the variation of the medium ph in sap fermentation process has a tendency to increase in the sap production phase; however, depending on the carbon source used, its behaviour changes in the early stages of the fermentation as the consequences of the directed functioning of the intracellular bioreaction network. the loci of the dissolved oxygen (do) curves also strongly depend on the carbon source(s) utilised, in addition to the applied ot conditions. the complex media profiles are significantly different compared to the defined media as the ph and do profiles are interrelated owing to the bioreactor operation conditions affecting the metabolic reaction network. the highest volumetric oxygen uptake rates were obtained with soybean-based medium that was ca. three-fold higher than the values reported in citrate-based and glucose based media, and ca. . - -fold higher than the values reported in molasses-based medium. the significant changes, moreover, the drastic change observed with the use of soybean-based complex medium are due to the compositions of the fermentation media used, and its influence on the intracellular bioreaction network. thus, we conclude that the change in medium composition based on the carbon source changes the fermentation characteristics under the designed bioreactor operation conditions that appear as the fingerprints of the bioprocess. kinetic resolution of racemic benzoin with different lyophilized microorganisms Ç . babaarslan ,Ü. mehmetoglu , a.s. demir : ankara university, faculty of engineering, department of chemical engineering, , ankara, turkey; middle east technical university, department of chemistry, ankara, turkey. e-mail: barslan@eng.ankara.edu.tr (Ç . babaarslan) the biocatalytic resolution of racemates is valuable tool for enantioselective synthesis and proved to be a convenient method for obtaining optically enriched compounds from their racemic form. in this work, enantiomerically pure benzoin which is one of the -hydroxy ketones was synthesized by kinetic resolution of racemic benzoin using different lyophilized microorganisms as lipase sources. the effect of lyophilized microorganism type, solvent type and acyl donor type on enantioselectivity were studied. in kinetic resolution experiments, lyophilized microorganism was resuspended in the media containing solvent, racemic benzoin and acyl donor at • c and rpm on orbital shaker. the reaction was followed by tlc during the experiment and the enantiomeric ratio of benzoin was determined by hplc analysis using chiral cell ob column. the best enantioselectivity value was obtained with lyophilized rhizopus orayzae cbs - as ee s = % and ee p = % (conversion = %) using thf as solvent and vinyl acetate as acyl donor. otimizing the fermentation broth for tanase production by a new isolated strain paecilomyces variotii vania battestin, gláucia pastore, gabriela macedo department of food science, unicamp, p.o. box , campinas, cep - , são paulo, brazil tannase is an inducible enzyme that catalyses the breakdown of ester linkages in hydrolysable tannins, resulting in gallic acid and glucose. the fermentation broth can use by-products as wheat bran, rice or oats, adding tannic acid. the use of by products or residues rich in carbon source for fermentation purposes an alternative to solve pollution problems that can be caused by an incorrect environmental disposal. in the present study we have optimized the production of an extracellular tannase by a new isolated paecilomyces variotii using response surface methodology. the first step was identify the variables having a significant effect on enzyme production. the variables evaluated were temperature, residues ratio (coffe: wheat bran), concentration of tannic acid, salt solution during , and days of fermentation time. results showed that temperature, residues ratio (coffe: wheat bran) and tannic acid had significant effects on tannase production. commercial wheat bran (cwb) and coffe rusk residues (cr) were used as solid substrate. for fermentation the mediun was composed by, cwb:cr were mixed with distilled water and transferred into ml capacity erlenmeyers flasks and autoclaved at • c for min. the medium was then inoculated with spores ( . × ) and the flaks were incubated at • c. tannase was assayed according to the methodology of mondal et al. ( ) . acording to the statist analyses, the optimum conditions to produce tannase was the range of temperature ( - • c); tannic acid ( . - %); residues % (coffe: wheat bran) ( : ) and days fermentation time. the enzyme production increased . times more enzyme production than that was obtained before this optimization. how to cope with fda's pat-initiative with respect to fermentation process monitoring and control marco jenzsch , andreas luebbert , rimvydas simutis : institute of bioengineering, martin-luther-university halle-wittenberg, halle (saale), germany; process control department, kaunas university of technology, kaunas, lithuania with its pat initiative, fda forces drug manufacturers to increase their activities in innovative manufacturing techniques, and, more than previously, to focus on quality assurance. the agency particularly places emphasis on making use of modern process supervision and control techniques such as up-to-date process analytics, multivariate data acquisition and analysis tools in order to improve process monitoring and control. in this contribution we show by means of practical examples how this guidance can be applied to cultivations of genetically modified microorganisms. a comparison of different multivariate state estimation techniques will be presented and compared with more knowledge-based techniques such as the extended kalman filter. the comparison was made for the model system gfp expressed from e. coli bacteria (bl /de /gfp) for which more than full data sets are available. all these techniques have already been used during real protein formation at productionscale fermenters, with the same success. hence, the requirements expressed in the pat initiative can immediately be put into practice. feedback control of the recombinant protein production processes based on such estimations is show for several cultivation systems. simple parameter adaptive controllers are compared with model supported controllers, for instance, generic model controllers and model predictive controllers. the results clearly show that we have at hand a rather extended arsenal of feedback control procedures that can be used successfully to tightly control the processes even along set-point profiles of physiological variables such as the specific growth rate (µ). again, fda's suggestion with respect to "control in the engineering sense" can be applied immediately to reduce batch-to-batch variances and thus to increase process quality. extending life by alternative respiration? alexander kern, franz hartner, anton glieder institute of biotechnology, graz university of technology, a- graz, austria. e-mail: a.kern@tugraz.at (a. kern) alternative oxidase transfers electrons directly from the ubiquinol pool in mitochondria to oxygen, allowing cell respiration in presence of complexs iii and iv inhibitors like antimycin a or cyanide. electron transfer by alternative oxidase is not coupled with proton transfer across the mitochondrial membrane, thereby uncoupling the supply of small metabolic intermediates by the central metabolic pathway from energy production in the cell. alternative oxidase is present in mitochondria of plants, many fungi and a few, mostly crabtree-negative yeasts, but not in p. angusta (hansenula polymorpha) and s. cerevisiae. alternative oxidase has multiple functions in different organisms. it is involved in stress answers, in programmed cell death, maintenance of the cellular redox balance, and also citric acid accumulation in a. niger. we isolated the alternative oxidase gene from the methylotrophic yeast p. pastoris in order to study its effects on the cellular energy content, respiratory activity, its protective role against oxidative stress. our results indicate the importance of an exact regulation of the alternative oxidase due to its impact on many cellular functions. new types of energy efficient fermenters with better mass transport, mixing and cooling properties than the current crop of rushton turbine derived tank bioreactors are likely to be required in the future. such fermenters will be needed in order to meet the increasing pressure on costs for low price commodity type products such as single cell protein or food and technical grade enzymes, and to meet the demands of the new wave of white biotech, in which bio-produced chemicals must be made at prices competitive with those of the traditional chemical industry. with this in mind, a prototype pilot scale ( l) u-loop fermenter has recently been commissioned at biocentrum-dtu. in this fermenter, liquid circulation is driven by a propeller pump through a vertical u-shaped pipe, which is connected at the top with a de-gassing tank. we present here a study of liquid mixing and dispersion in the prototype u-loop fermenter. sub-sequently we show that the results can be described with the tanks in series model. mixing was characterised using pulses of nacl tracer, which were detected with a conductivity probe in various parts of the fermenter. bodenstein numbers (bo) were determined for flow rates corresponding to a linear fluid velocity of . m/s in the 'legs' of the reactor and showed that the majority of the mixing occurred in the top degassing part (bo = ) rather than in the u-loop section (bo = ). it was also observed that the time for mixing to % homogeneity after tracer pulse addition was a function of the number of cycles through the reactor ( - . ) within the range of flow velocities (u) studied (u = . m/s to u = . m/s). the mixing time to % homogeneity was between . s (at u = . m/s) and s (at u = . m/s). today many biotechnological processes are operated at suboptimal conditions and according to best practice. however, the current industrial development is towards analyzing more parameters and in particular there is a large interest in analysis of biological/biochemical variables. the quality of products and also the possibility to optimize production in submerged cultivations would be greatly enhanced if more on-line/real-time information were at hand. the present investigation was undertaken with the aim of evaluating the potential in using multi-wavelength fluorescence for monitoring and control of filamentous fungi fed-batch cultivations. a recombinant a. oryzae expressing a heterologous lipase was applied as model system. spectra of multi-wavelength fluorescence were collected every five minutes with the bioview ® system (delta, denmark) and both explorative and predictive models, correlating the fluorescence data with the important biological parameters cell mass and lipase activity, were built. the models will be presented, furthermore, advantages and disadvantages of multiwavelength fluorescence for monitoring of cultivation processes will be discussed. moving from r&d to pharmaceutical development is a costly process. it is therefore of paramount importance to design a manufacturing process that combines robust and well-documented technological platforms. therapeutic recombinant proteins designed for human administration should be as close to the authentic product as possible. here, the use of a scalable process and an economically sound affinity tag can be a relevant choice. the tagzyme tm system has been designed to allow for the precise removal of amino terminal affinity tags. the system is based on the use of recombinant aminopeptidases including dipeptidyl peptidase i (dapase tm ). dapase tm is currently produced under cgmp providing a suitable strategy for its use in pharmaceutical production. the tagzyme tm system is superior to other methods since: ( ) it is based on exopeptidases, precluding, e.g., unspecific protein cleavage reported when using so-called site-specific endoproteases. ( ) it has been tested for production of more than recombinant proteins. ( ) it is easily scalable from lab scale to kg of processed protein. ( ) it allows the use of his-tags for commercial production without patent infringment, due to our ipr position. ( ) the commercial use of tagzyme tm does not require any licensing, only purchase of the enzyme(s). ( ) the use of aminopeptidases for pharmaceutical production has been extensively documented for approved drugs. ( ) a number of therapeutics is currently being developed using tagzyme tm . ( ) unizyme can assist in the optimization of the dsp to enable further cost reduction in the process. these aspects will be discussed and illustrated in the presented poster. website sphingolipids are biologically active molecules involved in the regulation of a large quantity of biological responses. they function in cell proliferation, survival and death (apoptosis) as messengers. dysregulation of apoptosis has significance in numerous pathological conditions including cancer. several anticancer agents act by increasing tumor cell ceramide (a kind of sphingolipid) content. so, a novel approach to cancer therapy would be the pharmacological manipulation of sphingolipid metabolism. in this study, sphingolipid metabolism in baker's yeast s. cerevisiae is used as a model system as many of its sphingolipid related genes and proteins have been characterized. gepasi-biochemical kinetics simulator was used for metabolic control analysis (mca) of the above-specified system. the concentration control coefficients (ccc), flux control coefficients (fcc) and elasticity coefficients were calculated, and their significance in identification of anticancer drug targets is determined. elementary flux modes were also identified and metabolic pathway analysis (mpa) was performed. quantitatively, control effective flux (cef) values were used for potential drug target identification. the results from mca and mpa indicate almost the same potential drug targets: serine palmitoyl transferase, ceramide synthase and ceramidase. drugs against these targets are in preclinical and clinical development. for the identification of new potential drug targets, the cccs, fccs, cefs and elasticity coefficients were examined with an objective function of maximizing the cell ceramide concentrations. it was found that manipulation of inositol- -phosphate synthase and phosphoinositide kinase activities have considerable effects on ceramide concentrations. if a drug targeting the two enzymes at the same time is designed, it might give a better outcome in terms of cancer therapy. in recent years, there is a growing interest in utilization of airlift reactors (alrs) to biotechnological processes. nevertheless, their industrial application still remains limited because of a lack of reliable studies on transfer phenomena and mixing enabling a suggestion of suitable scale-up procedure. the way to more widely utilization of alrs to biological processes lies in experimental research (on a model medium as well as on a real fermentation medium) followed by mathematical modelling and scaling-up of the processes. this paper deals with a modelling of a glucose-gluconic acid fermentation by a. niger in an internal loop airlift reactor. knowledge of the stoichiometric relationship in the key reaction provides a good opportunity for estimation of substrate and product concentration. the model is based on material balance equations and has been adjusted to experimental data obtained from three internal loop airlift reactors ( . , and l) . in the model, the alr is divided into ideal stirred tanks in series. in each zone (tank) of the alr the material balance is calculated in two phases (the gas and the liquid phase). this work was supported by the slovak scientific grand agency, grant number vega / / alkaline phosphatase (ap, e.c. . . . ) is a thermolabile enzyme which is indigenous to all dairy products. it has an inactivation temperature slightly above the value that is required to destroy the most resistant pathogenic microorganism likely to be found in milk. due to that feature, this enzyme is used as an indicator of proper pasteurization. the effect of temperature treatment on the activity of ap was investigated in raw cow's and goat's milk. the stability of alkaline phosphatase in raw milk was compared with the stability of this enzyme in a . m potassium phosphate buffer with ph . . the ph value of the buffer was approximately the same as that of raw milk. the inactivation curves were measured in the temperature range from to • c. ap in cow's milk was completely inactivated at • c during s but approximately % of activity remained at • c after min of treatment. the time required for a complete inactivation of the enzyme in the raw cow's milk was reduced from to min as the temperature increased by • c. heat treatment of goat's milk caused the decrease of activity of the enzyme in the same temperature range as in the case of cow's milk. the increase of temperature from to • c reduced the inactivation time from min to s. the study of thermal stability of the alkaline phosphatase in the buffer solution showed that the time required for inactivation of enzyme was significantly shorter than in milk. milk thus had a protective effect on the activity of alkaline phosphatase. the experi-mental curves were fitted simultaneously using kinetic models where the initial heating period was considered. this work was supported by a grant of th framework program of eu, project foodpro, no. sme- - - . during the process of separation, purification and concentration of monoclonal antibodies (mabs) at industrial scale, the chromatographic unit operations have an important role. three different protein-binding modes are employed: ion-exchange, hydrophobic and affinity binding. two adsorbent properties are of uppermost importance: a high selectivity and adsorption capacity. in the case of ion-exchange/hydrophobic chromatography, the binding of charged proteins can be affected by ph and ionic strength. in this work, the adsorption capacity of eight commercially available adsorbents designed for separation of mabs (mabselect, rprotein a sepharose ff, poros a, prosep-va, fractogel emd se hicap (m), sp sepharose ff, mep hypercel, s ceramic hyperd f) was measured as a function of ph. as a model mab and contaminant proteins, human immunoglobulin (igg), human serum albumin (hsa) and horse skeletal muscle myoglobin (myo) were used. the resin properties were investigated within the range of ph - . the experiments were conducted in a batch-mode, individually for each model protein. the results showed that ion-exchange and hydrophobic resins provided the best selectivity for igg at ph . the selectivity of affinity adsorbents was essentially unaffected by ph, however, the highest capacity for igg was at ph . another investigated aspect was the dynamics of protein binding. the solution of individual protein in contact with tested adsorbent was circulated through an uv spectrophotometer, what enabled the measurement of time-dependent decrease of protein concentration. the results indicated that affinity adsorbents with a rigid matrix needed approximately four times shorter time to reach the adsorption equilibrium with igg in comparison with a gel. the gels, however, provided higher adsorption capacity. at ion-exchange resins, the time necessary to adsorb % of total amount of igg was about . - h. the affinity adsorbents were highly selective and therefore they adsorb very small amount of tested contaminant proteins (hsa, myo). the adsorption capacity was saturated by % in less than min in all cases of dynamic adsorption measurements. this work was supported by a grant of th framework program of eu, project aims, no. nmp -ct- - . microtechnology has for several years been applied within chemical reaction engineering. the advantages of microtechnology are that it makes it possible to develop light weight and compact systems, and the systems enable large surface-to-volume ratio, which results in low mass-transfer distances. in addition, parameters like pressure, temperature, residence time, and flow rate are more easily controlled. the use of microtechnology is also beginning to find its ways into the field of biotechnology. what we are aiming at is the development of a microreactor that can be applied as a production tool in industry as an alternative to conventional enzymatic reactors. our strategy is to use a small plate of a suitable material with microchannels fabricated into its surface, and the approach is to covalently couple enzymes into the microchannels. substrate can then be pumped through the channels and the enzymatic conversion will take place within the channels. as model enzyme in the development of the microreactor we are applying celb, a thermostable ␤-glycosidase from pyrococcus furiosus. kinetic resolution of racemic benzoin with different lyophilized microorganisms Ç . babaarslan ,Ü. mehmetoglu , a.s. demir : ankara university, faculty of engineering, department of chemical engineering, , ankara, turkey; middle east technical university, department of chemistry, , ankara, turkey. email: barslan@eng.ankara.edu.tr (Ç . babaarslan) the biocatalytic resolution of racemates is valuable tool for enantioselective synthesis and proved to be a convenient method for obtaining optically enriched compounds from their racemic form. in this work, enantiomerically pure benzoin which is one of the -hydroxy ketones was synthesized by kinetic resolution of racemic benzoin using different lyophilized microorganisms as lipase sources. the effect of lyophilized microorganism type, solvent type and acyl donor type on enantioselectivity were studied. in kinetic resolution experiments, lyophilized microorganism was resuspended in the media containing solvent, racemic benzoin and acyl donor at • c and rpm on orbital shaker. the reaction was followed by tlc during the experiment and the enantiomeric ratio of benzoin was determined by hplc analysis using chiral cell ob column. the best enantioselectivity value was obtained with lyophilized rhizopus orayzae cbs - as ee s = % and ee p = % (conversion = %) using thf as solvent and vinyl acetate as acyl donor. chromatography is one of the most important unit operations at separation, purification and concentration of monoclonal antibodies (mabs) at industrial scale. since these proteins belong to the group of immunoglobulins, their molecular weight (about , g/mol) or hydrodynamic radius ( . nm), respectively, is relatively large. the adsorbents used in ion-exchange/affinity chromatography of these biomolecules should thus provide a high pore accessibility coupled with a high value of specific surface area in order to ensure a sufficient ligand density and a high binding capacity. in this study, the pore accessibility of eight commercially available adsorbents designed for separation of mabs (mabselect, rprotein a sepharose ff, poros a, prosep -va, fractogel emd se hicap (m), sp sepharose ff, mep hypercel, s ceramic hyperd f) was investigated via size exclusion of standard-sized molecules (glucose, sucrose and dextrans with molar weight range - × g/mol) at nonbinding conditions. the experiments were conducted in a batch and column-mode. the batch experiments provided absolute partition coefficients, which were calculated from a mass balance and represent the fraction of pore water accessible to a solute. it was found that several adsorbents contained a small fraction of very small pores (less than nm), whereas some adsorbents contained a significant fraction of pores larger than nm. the column measurements provided relative partition coefficients, which were calculated from the retention volumes of solutes and represented the relative accessibility of pores, scaled between the accessibility of the smallest and largest solute used. when the absolute partition coefficients were recalculated into the relative form, it was found that the coefficients obtained by both methods correlated very well. the relative partition coefficients of solutes with the hydrodynamic radius of nm (corresponding to mabs) was about . - . at ion-exchange and hydrophobic adsorbents and . - . at affinity adsorbents. the relation between hydrodynamic radius of the solutes and their partition coefficients was successfully described with the giddings random plane model. this work was supported by a grant of th framework program of eu, project aims, no. nmp -ct- - . the transfer of laboratory results to a larger scale is often a critical step in process development to industrial application. the objective of this study was to scale-up the bioreactor for the fructosyltransferase production based on the results obtained in a dm stirred bioreactor. the investigations made in this bioreactor provided a clear picture of the effect of medium composition on the obtained fructosyltransferase (ftase) activity but the influence of mixing intensity was unequivocal. the increase of agitation rate had a positive effect up to a certain level where both fructosyltransferase and biomass production increased. the final optimal yield factor of ftase per dry cell mass obtained in the laboratory bioreactor was , u g − . we studied the effect of oxygen transfer on the process of ftase production at a larger scale, in and dm mechanically stirred bioreactors and in air-lift bioreactors, and dm whilst the medium composition was kept constant. the yield factors of ftase were comparable in both mechanically stirred bioreactors and they were about u g − . this decrease compared to that in the laboratory bioreactor could be explained by a slower cell growth. this fact was also confirmed by that glucose was not depleted till the end of fermentation and free fructose concentration was also lower. the yield factor of ftase was u g − in the dm air-lift reactor and u g − in the dm air-lift bioreactor. the lower yield of ftase in dm bioreactor was caused by a better biomass growth. this work was supported by slovak scientific grand agency, grant numbers vega / / and / / and by science and technology assistance agency, grant number apvt- - . the poster gives an overview of the objectives and achieved results of an interdisciplinary project on direct product isolation from crude feedstocks using magnetic micro adsorbents in combination with suitable magnet technology. the project was funded by the deutsche bundesstiftung umwelt and was running between august and november . in the course of the project several milestones could be met, which can be looked at as critical key points on a route towards an industrial realization of the process. among these milestones are: (i) the production of magnetic micro adsorbents with high capacity and selectivity in batches up to - g; (ii) the proof that the micro adsorbents can be reused many times; (iii) generation of a variety of recombinant tagged, active enzymes; and (iv) the design, assembly and operation of a fully automated pilot plant capable of generating approx. g/h (≈ % purity) protein. the process was also simulated by help of the software tool superpro designer and simple mass balance and sorption equilibrium approaches were used to derive rules for estimating optimum process parameters and productivities. finally an environmental performance evaluation was conducted externally by the german dechema. in this study the effect of fed-batch cysteine addition to a culture of a high-gsh-accumulating yeast strain on the metabolism of glutathione was investigated. it is known that cysteine is the rate limiting amino acid in the biosynthesis of gsh. the influence of the consumption rate of cysteine on glutathione metabolism and growth of s. cerevisiae mt- was determined. the results show that for rates of consumption below a critical value the microorganism growth is similar to a culture without feed of cysteine, but glutathione production is increased two-fold. on the other hand, if cysteine consumption rate is above the critical value the changes of cell metabolism implies ethanol accumulation in the extracellular media which diminishes biomass synthesis. the maximum specific glutathione production in this case is maintained at two-fold; however, gamma-glutamylcysteine accumulation is increased. cysteine present in culture media directs cell metabolism to a greater synthesis of ammonia and amino acids. hydrophobic interaction chromatography (hic) exploits the hydrophobic properties of protein surfaces for separation and purification by performing interactions with chromatographic sorbents of hydrophobic nature. in contrast to reversed phase chromatography this methodology is less detrimental to the protein and is therefore more commonly used in industrial scale as well as in bench scale when the conformational integrity of the protein is important. hydrophobic interactions are promoted by salt and thus proteins are retained in presence of a cosmotropic salt. when proteins are injected on hic columns with increasing salt concentrations under isocratic conditions only, a fraction of the applied amount is eluted. the higher the salt concentration the lower is the amount eluted protein. the rest can be desorbed with a buffer of low salt concentration or water. it has been proposed that the stronger retained protein fraction has partially changed the conformation upon adsorption. this has been also corroborated by physicochemical measurements. the retention data of five different model proteins and different stationary phases were evaluated. partial unfolding of proteins upon adsorption on surfaces of hic-media were assumed and a model describing the adsorption of native and partial unfolded fraction was developed. furthermore we hypothesize that the surface acts as catalyst for partial unfolding, since the fraction of partial unfolded protein is increasing with length of the alkyl chain. stationary phases for bioseparation of glycoproteins j. aniulyte , j. liesiene , b. niemeyer : department of chemical technology, kaunas university of technology (ktu), radvilenu pl. , kaunas, lithuania; institute of thermodynamic, helmut-schmidt-university/university of the federal armed forces hamburg, holstenhofweg , hamburg, germany d- nowadays glycomics raise new challenges for affinity chromatography related with an abundance of glycoconjugates in living organisms and with scaling-up of the preparative processes. economics, efficiency and practicality dictate the search of novel chromatographic biospecific adsorbents that could contribute to enhancing the productivity of the affinity separation process. the purpose of the work was to prepare cellulose-and silica-based biospecific adsorbents with immobilized lectins and to evaluate them for the affinity chromatography of glycoproteins. cellulose-based matrix granocel and silica coated with hydrophilic polymers were used as a support. the effect of support characteristics, such as pore size, chemistry of active groups and their density on the support' surface on lectin immobilization and on the efficiency of adsorbents obtained were evaluated.three different methods were used for the activation of the support: oxidation with sodium periodate, modification with pentaethylenhexamine (spacer arm of atoms) and carbonyldiimidazole activation.cona and wga two lectins of different molecular weight and shape were selected to notice differences resulting from the size and diffusion behaviour. chromatographic performances of the adsorbents were studied applying two different glycoproteins (god and fetuin) carrying specific terminal glycomoieties of mannose (god), and n-acetylglucosamine (fetuin) for specific interaction with cona and wga, respectively. the adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to . mg per ml support and a high recovery (up to %). it was shown that spacer arm affected ligand coupling kinetic as well as the chromatographic behavior of the adsorbents obtained. the adsorption isotherms of god onto cona adsorbents reveal an adsorption behavior with high and low affinity binding sites. the dissociation constant k d of the ligand-sorbate complex is approximately × − m, and . × − m, respectively. it was supposed that the second step is related to the sorption of solvated god onto already adsorbed god forming sorbate dimers. cell disruption and chromatography are key unit operations in the downstream processing of an intracellular product. the cost involved in the extraction and purification of intracellular products can be reduced by selective release of proteins and reduction in the number of steps involved in the purification. the extent of disruption can be varied to provide a selective release, limiting the release of the contaminant proteins. the particle size distribution of the cell debris in the resulting suspension depends on the extent of disruption. expanded bed adsorption chromatography allows for the direct capture of the proteins from an unclarified suspension. this technique allows for the integration of solid-liquid separation, concentration and preliminary purification in one unit operation. a perfectly stable expanded bed can be obtained by choosing the appropriate flow conditions and a suitable adsorbent. the difference in the density between the adsorbent and the cell debris in the suspension, permits the cell debris to flow through the column without blocking, whilst the protein molecules in the suspension are adsorbed onto the adsorbent. after sample application, the bed is washed with buffer and the proteins eluted from the column in the packed bed mode. the presence of the cell debris in the feedstock influences the expansion of the bed and the adsorption of protein molecules. the physical properties of the suspension obtained after cell disruption depends on the extent of disruption. the particle size distribution of the cell debris, the viscosity and the release of soluble proteins and other intracellular components are influenced by the extent of disruption. the influence of the extent of disruption of e. coli on the expansion of the bed and the adsorption of ß-galactosidase is presented in the current study. e.coli cells were disrupted at different operating pressure using a high pressure homogenizer. the resulting crude homogenate is subjected to expanded bed adsorption chromatography using streamline deae as adsorbent. the disrupted suspension was characterised in terms of viscosity, density, particle size distribution of the cell debris and the extent of protein and ß-galactosidase released. the interaction between cell debris and adsorbent was quantified as the cell transmission index (ratio of the amount of cells present in the sample before and after passing through the bed). the expansion of the bed at a constant settled bed height and flow rate was measured. the influence of the cell debris on the extent of adsorption of ß-galactosidase has been quantified in terms of dynamic binding capacity (dbc) at % of the inlet concentration. the dbc of ß-galactosidase that was released by disruption at psi ( %, w/v, w/w, pass) was found to be u/ml of adsorbent while dbc of samples disrupted at psi ( % w/v, w/w, pass) was u/ml of adsorbent. the extent of disruption of e. coli over a wide range and its effect on the expansion and adsorption will be presented. study of dna binding during expanded bed adsorption and factors affecting adsorbent aggregation ayyoob arpanaei , niels mathiasen , timothy hobley , owen rt thomas , : center for microbial biotechnology, building , biocentrum-dtu, technical university of denmark, , lyngby, denmark; department of chemical engineering, university of birmingham, edgbaston, b tt, uk. e-mail: aa@biocentrum.dtu.dk (a. arpanaei) the adsorption of sonicated calf thymus dna (as a model dna molecule) to biosepra q hyper z adsorbents was evaluated in batch and expanded bed modes. stability of the expanded bed during feedstock loading was also studied. two batches of prototype q hyper z (batch and ) were examined, which had ionic capacities measured to be and mmol cl − /ml support respectively. in all adsorption experiments a mm tris-hcl ph buffer was used. maximum static binding capacities of adsorbent batches and were determined to be . and . mg dna/ml particle, respectively. dynamic binding capacity at % breakthrough (dbc % ) was measured in a -cm diameter eba column containing . ± . cm settled bed with a feed of g/ml dna. dbc % of the adsorbents were . and . mg dna/ml support for batches and , respectively in buffer containing no salt. however, the maximum dbc % for batch ( . mg dna/ml support) and ( . mg dna/ml support) were obtained in buffers containing . and . m nacl, respectively. further increases in salt concentration led to a decrease in dbc % for both adsorbent batches. the bed compression during loading that was observed in experiments at high conductivities (achieved by adding salt) was less than that seen with low conductivity ( ms/cm) solutions. aggregation of adsorbent particles and channeling of flow were not observed in the presence of salt concentrations more than . m. the effect of different concentrations of dna during loading in the presence of . m nacl was studied. it was found that increasing dna concentrations in the feed from to g/ml, to g/ml resulted in a decrease of dbc % by , and %, respectively. the bed compressed slower during loading of feedstock with low dna concentrations compared to that for higher concentrations. the expanded bed showed a partly reversible compression behavior during feedstock loading. this is attributed to the electrostatic interaction between dna adsorbed on the particles surface and rearrangements of dna strands as the number of free ligands on the adsorbent surfaces decrease during loading. large-scale production of plasmid dna for gene therapy and dna vaccination applications has become necessary as a result of the increasing number of approved protocols using non-viral vectors for gene delivery. a major challenge of large-scale plasmid production is to establish a robust cgmp manufacturing capable of producing hundreds of milligrams or grams of a pharmaceutical grade product. alcohol and salt precipitation are operations largely used in the early steps of plasmid downstream processes. however, there are few systematic studies on the influence of these precipitation agents in the final plasmid recovery and purity. in this work, alcohol and salt precipitation steps used in a plasmid purification process developed by our group have been optimized aiming at large-scale production. the optimization of alcohol precipitation indicated that almost % of the pdna precipitated when . vol. of isopropanol were used. the studies also indicated that the precipitation profile was strongly influenced by pdna initial concentration. finally, the final plasmid recovery and purity after a sequential alcohol and salt precipitation were strongly dependent on the concentrations of these precipitation agents. thus, a commitment between high recovery and purity level should be made during the development of the downstream processes. comparison of novel and conventional processes for protein refolding and initial purification h. ferré , , u. jørgensen , l. scale down of downstream processing unit operations is convenient for assessing process alternatives, particularly if feedstock is scarce. in this study it was imperative to use the smallest possible scale for comparison of a new system for continuous protein refolding and direct expanded bed adsorption (eba) capture with a traditional process composed of discrete operations of batch renaturation, centrifugation, microfiltration and packed bed chromatography (pbc). minimisation of the scale was restricted by the eba step: the smallest practical scale being a cm diameter column with - cm of settled bed, expanded two fold. in order to permit a fair comparison a similar column diameter and adsorbent volume was used in the packed bed process. in both alternatives, chelating media charged with cu + was used and a feedstock of denatured hat-tagged human beta- microglobulin (hat-h␤ m). following batch refolding and clarification, the performance of the packed column was severely hampered due to fouling of the top adapter. reducing the protein loaded to the packed bed to % of dbc working lead to a recovery of . % at a purity of % and . -fold concentration. the eba-based process performed unimpeded and productivity was calculated to be % higher than for that employing a packed bed. however, due to the severe scale restrictions placed on the eba process, which limited optimisation, significant productivity improvements of eba over packed bed are expected at larger scale. high gradient magnetic filtration has the potential for rapid processing of large volumes of crude bioprocess liquors when magnetic adsorbents are employed. the binding of a protein to a superparamagnetic solid support provides a unique selective 'handle'. typically the focus is placed on using the magnetic handle for direct capture of a protein from a fermentation broth. however, magnetic adsorbents may provide solutions to a range of downstream processing problems and in this presentation we illustrate this with a number of case studies. using whey as a model system, we show that the extent of the tryptic hydrolysis (ca. . mg/ml added enzyme) of proteins could be controlled by adding benzamidine-linked magnetic adsorbents after a given period ( - min), followed by removal of the loaded adsorbents using a magnetic filter. hydrolysis was stopped effectively and approx. % of the added trypsin could be recovered. a coupled process was devised for the refolding and purification of inclusion body proteins. solubilised (in m urea) inclusion bodies of recombinant histidine affinity tagged human beta microglobulin (hat-h-beta m), were refolded by dilution in a pipe reactor ( s), then captured directly on cu(ii) charged magnetic immobilised metal affinity adsorbents in a second pipe reactor ( s residence time). loaded adsorbents were retained in a magnetic filter, then washed and the protein eluted. a generic framework for the prediction of scale-up when using compressible chromatographic packings r. tran , j. joseph , a. sinclair , y. zhou , n. packed bed chromatography is the pre-eminent technique in the downstream purification of many biological products. the aspect ratio of a packed bed has a significant effect on the column pressure drop by virtue of wall support which is reduced at low aspect ratios. this can result in unexpectedly high pressures during manufacturing caused by the compression of the matrix via drag forces due to fluid flow through the bed. the need for an accurate model to predict flow conditions at increasing scale is essential for the scaling-up of chromatographic processes and for avoiding bed compression during operation so that maximum throughput can be achieved. several studies have generated correlations which allow for the prediction of column pressure drops but have either been mathematically complex, which makes their practical use unfeasible, or they have used highly specific empirical constants and hence require a large amount of experiments to be performed before they can be used. in this study, we have established relationships to link the critical velocity of operation, to bed height (l), column diameter (d c ), feed viscosity (µ) and also to the matrix rigidity through the level of agarose cross-linking (a%). the correlation is straight forward to use and involves very few system-specific constants thus significantly reducing the need for any preceding laboratory-scale experimentation. this paper describes the series of experiments that were performed to establish the correlation, using a range of cross-linked agarose matrices ( - %), at various aspect ratios, fluid flow rates and varying viscosities ( . - . mpas). a mathematical model was developed where parameter estimation for multi-variables was achieved by least squares optimisation. the model can be used to predict the extent of compression in industrial chromatography applications and will be useful in the development of chromatographic operations and for column sizing. institute of process engineering, swiss federal institute of technology, zurich. e-mail: makart@ipe.mavt.ethz.ch (s. makart) simulated moving bed (smb) technology receives increasing attention in biotechnology and in the biopharmaceutical industry as it enables an increase in productivity per unit mass of stationary phase, reduced solvent consumption, and fast and reliable scale-up. combining continuous chromatographic separation unit and reactor should enable the production of biopharmaceuticals and fine chemicals with high purity and yield at the same time. due to the increasing demand of enantiopure intermediates in the pharmaceutical industry, biocatalytic processes gain more and more importance because of their excellent enantioselectivity. yet the application of biocatalytic carbon-carbon bond formation on process scale is often hampered by an unfavourable equilibrium position and difficult downstream processing due to substrate/product mixtures. coupling a continuous separation unit to such a process would improve the feasibility by driving the reaction to completion and thus increasing the overall yield. we will discuss the design of such an integrated biocatalytic/smb process, taking the formation of l-allo-threonine from glycine and acetaldehyde, catalysed by the glycine-dependent aldolase glya from e. coli, as a model reaction. the enzyme exhibits absolute stereoselctivity at the c-alpha atom, whereas selectivity is less strict at c-beta. in situ product removal, by the integration of an smb unit, would aid to maintain a high diastereomeric excess as it shortens the residence time of the products in the reactor, in addition to shifting the reaction to the product side. the in-line coupling of the chromatographic unit to the enzyme reactor requires the use of the same solvents for reaction and separation, so the choice is limited to aqueous solutions close to physiological ph, limiting in turn the possible stationary phase materials. in a screening of different cation exchangers, amberlite cg- ii gave promising results: threonine is more retained than glycine, acetaldehyde is poorly and the cofactor plp is not retained. adsorption isotherms were determined by the retention time method and a smb under process conditions was simulated. by improving the packing of the column, i.e. achieving a more even particle size distribution, we tried to further increase the efficiency of the separation step. the application of enzymes for the synthesis of optically active substances is nowadays of growing importance in the pharmaceutical industry. this requires a proper cultivation of the microorganism as well as a posterior isolation process yielding a constant catalyst quality at high purity. goal of this project is the development of an integrated process for the production and isolation of a lipase from trichosporon beigilie and its posterior application for the enantioselective synthesis of pharmaceutical products. the cultivation of the microorganism is optimised in a laboratory and pilot-scale fermenter in a fed-batch mode. parameters like media composition, temperature, ph and aeration rate are set up. taking advantage of the localisation of the enzyme (covalently attached to the cell membrane) the first step of product isolation consists of a continuous cell disruption. optimal results are achieved with the continuous bead mill disruption process ( % enzyme release with a specific activity of . u/mg of protein). the non disrupted cells are recycled as inoculums for a new cultivation, increasing the yield of the overall process (by -times in the pilot-scale fermenter). in order to isolate the product two different process sequences are considered. the first one consists of an extraction (peg and phosphate buffer) coupled to an ion-exchange chromatography (q-sepharose ff). the second one applies a precipitation step with ammonia sulphate followed by a hydrophobic interaction chromatography (sepharose-hic) provid-ing a lipase yield of % ( -times higher than the one provided by combining extraction-chromatography). an ultrafiltration process is used in order to concentrate the lipase and its final properties (molecular weight, isoelectric point, activity, stability and kinetic data) are studied using p-nitrophenylacetate as model substrate. the relevance of the obtained product for its application in the pharmaceutical industry is proven by transforming (r,s)-naproxen-methylester into (s)-naproxen acid with an enantiomeric excess of > % (after h). biotensides (sugar fatty acid esters, sfaes) find nowadays a wide range of applications in pharmaceutical, personal care and food industry because of their biocompatibility, biodegradability and special surfactant properties. goal of this project is the development and optimisation of an integrated process for the enzymatic synthesis of sfaes from renewable sources to be used in cosmetic formulations. the following figure shows the scheme of the overall process. commercial and also new screened lipases are applied in the reaction between sugar and fatty acid. the mixture grade of the initial reaction system is increased by ultrasounds taking into account the influence on the catalyst characteristics and also the necessity of an organic solvent as adjuvant. the reaction takes place in an enzymatic membrane reactor (emr) equipped with an ultrafiltration membrane which retains the catalyst. the separation of the by-product (water) from the rest of the components can be achieved by means of a pervaporation unit which coupling to the emr allows the semi-batch process. in order to separate the esters from the fatty acid a stepwise elution chromatography method is developed using silica as adsorbent and ethyl acetate and methanol as eluents. with this system % of the dimer is isolated with purity (hplc) of %. the application of a dialysis membrane technique allows the separation of % of the fatty acid by building ester micelles changing the polarity of the organic solvent used as eluent. solubility and crystallisation properties of recombinant bacillus halmapalus ␣-amylase cornelius faber centre for microbial biotechnology, biocentrum dtu, building , lyngby, denmark a comprehensive knowledge of solubility properties is a prerequisite for the efficient design and operation of bulk enzyme recovery processes, however, complete phase diagrams are only available for very few proteins, in particular lysozyme of high purity. here, we present the results of detailed solubility studies in aqueous solutions of an industrially relevant ␣-amylase of technical grade. experiments were conducted in small scale batch mode (working volume of ml). the influence of selected cations and anions from the hofmeister series on the stability of the ␣-amylase was examined. the hofmeister series for anions was followed in the correct order at all salt concentrations studied, i.e. from to m, whereas the series was reversed for monovalent cations at concentrations up to . m, with the exception of lithium. to further investigate why the position for lithium was different to the hofmeister series established for lysozyme, the zeta potential of protein solutions at low concentrations of selected salts was determined. the results of these measurements indicate a pronounced effect of lithium on the zeta potential, as compared to other salts. in particular, the ph of zero zeta potential (i.e. the pi) was shifted approximately . ph units towards alkaline conditions in the presence of lithium, whereas the pi stayed almost constant for sodium and potassium. since the solubility exhibits a minimum at ph-values at or near the protein's pi, shifts in ph caused by salt addition are important to identify and quantify to avoid uncontrolled phase separation. the measurement of the zeta potential of proteins in solution holds significant promise as an attractive tool for understanding and controlling processes that are operated close to the solubility limit and which are often plagued by uncontrolled precipitation or crystallisation and thus rely on carefully chosen operating conditions. polyphenolic interactions with potato proteins during industrial expanded bed adsorption processing sissel løkra , knut olav straetkvern , bjørg egelandsdal , gerd vegarud : department of natural science & technology, hedmark university college, n- hamar, norway; norwegian university of life sciences, as, norway. e-mail: sissel.lokra@lnb.hihm.no (s. løkra) in plant extracts it has been shown that polyphenols have a tendency to react with proteins, either by covalent or non-covalent interactions. these reactions can induce changes in the surface properties of the proteins, and, e.g. cause proteins to be insoluble and precipitate at ph-values below their isoelectric point. potato proteins have a high nutritional quality and show interesting functional properties in food systems. moreover, chlorogenic acid (ca) and caffeic acid constitute about % of the total polyphenol content of potato tuber. we have experienced expanded bed adsorption (eba) chromatography to be a method well suited for recovering industrial proteins from potato starch effluent. the process separates proteins from polyphenolic pigments, fiber and minerals. during the adsorption step, patatin, the major potato tuber protein shows complex binding kinetics demonstrated by breakthrough curves. in addition to diffusion limitations in the eba resin, changes in protein structure and surface properties probably are likely to affect this adsorption behavior. reactions between ca and patatin might result in a range of interactions for different species of the same protein. this project therefore aims to assess the interactions between ca, patatin and other major tuber protein fractions and how these changes affect the protein capture in eba. changes in size and charge are screened in -d electrophoresis and analyzed further. samples of different protein fractions are taken from breakthrough curves and dynamic binding capacity experiments in model systems with real feedstock. sandwich hybridisation assay for analysis of brewery contaminants s. huhtamella , m. leinonen , t. nieminen , a. breitenstein , p. neubauer : bioprocess engineering laboratory, university of oulu, finland; scanbec gmbh, halle, germany. email: peter.neubauer@oulu.fi (p. neubauer) here we describe the development of a sensitive, cultivationindependent analytical method for the analysis of brewery contaminants which can be performed within three hours in crude sample extracts. the method is based on s rrna detection by a paramagnetic bead based sandwich hybridization assay (sha) with two oligonucleotide probes designed to either detect the species or a group of contaminants. the signals were read out either by a fluorimeter (rautio et al., ; leskelä et al., ) or potentiometrically with an electric biochip instrument (ebiochip systems) . this assay is advantageous over rt-pcr becasue it only detects viable cells and the method can be directly applied to crude cell extracts without prior purification. we describe the principle of designing and evaluating a series of groupspecific lactobacillus probes and the optimisation towards effective cell lysis and high assay sensitivity. the applicability of the sha was evaluated with real brewery samples and the results were compared to routine tests. in all steps of the evaluation the reliability and usability of the method was prioritised. the optimised method combined with a h pre-enrichment period gave reliable results, had a detection limits of about - cells per assay and was easily applicable in a brewery environment. biodesulfurization is one of the possibilities studied by the researchers to attain the maximum sulfur levels imposed for a near future by governments (european directive, ) . rhodococcus erythropolis igts is a natural and strictly aerobic microorganism able to remove the sulfur atom from dibenzothiophene (dbt) in a selective way ( s route (oldfield et al., )), obtaining hydroxibifenyl (hbp) and sulfate. growth is carried out using the experimental procedure performed in previous works dealing with the inoculum built up, media composition and operational conditions (del olmo et al., a (del olmo et al., , b . this work is focused to determine the oxygen uptake rate during the production of the biocatalyst. four experiments were carried out at a biostat b fermentor (braun biotech.) using as only variable the constant stirrer speed used: , , and rpm. oxygen uptake rate have been determined by means of two methods: dynamic technique at different times during growth for few seconds to ovoid influences and from oxygen profile when the term dealing with oxygen transfer rate is known (predicted by the model proposed in a previous work (garcía-ochoa and gómez, )). our values obtained from the techniques used present the same tendency in all the runs carried out: our values from dynamic technique is always lower than our values obtained from the oxygen profile. these values are modeled and the difference observed is explained due to the cellular economy principle: during the seconds employed in the dynamic technique determinations microorganism do not produce s route enzymes. it was studied different methods to recover a p. salmonis antigenic protein from recombinant e. coli cells. this protein has shown be highly effective in vivo vaccine. it has the ability to stimulate salmon immune system protecting them against of aggressive disease salmonid rickettsial syndrome resolving by this way a great problem of salmonid aquaculture. biomass obtained from iptg induced e. coli bl (de ) codon plus culture was used for soluble and insoluble antigenic protein recovery. it was evaluated recuperation by glass bead mill, freezing and thawing, osmotic shock and lisozyme/edta treatments, all of them applied in single or combined way. biomass was measured by dry weight of cells, soluble protein concentration was quantified according to bradford method, and antigenic protein was identified by sds-page and western-blot analysis. cells treated with lisozyme/osmotic shock and then glass bead mill the soluble protein was a . % of the dry weight cell mass whereas using lisozyme/edta and glass bead mill as a single treatment only a . and . % were obtained respectively. the freezing and thawing disruption treatment released less than % of soluble protein, as well as the osmotic shock procedure too. the sds-page and west-ernblot analysis revealed that the antigenic protein must be purified from the insoluble cell fraction when physical or mechanical disruption methods were employed and from the soluble cell fraction when chemical or enzymatic treatments were used. we propose investigate in further studies the inclusion bodies formation to design an efficient purification procedure for the target protein. the iso-peroxidase pox from garlic bulb allium sativum that represented the major peroxidase activity was purified to homogeneity. the enzyme is monomeric and has a molecular mass of kda, and a pi around . the optimum temperature ranged between and • c, while optimum ph was around . pox appeared remarkably thermostable since it retained % of its activity at • c for at least h. in addition, the enzyme was stable at a ph range from , to . kinetic constants were calculated, the apparent k m values were and m for gaïacol and h o , respectively. the high thermostability of pox may represent clear advantages in a number of processes including immobilizing peroxidase and use it as a biosensor to detect oxidant component as h o and other peroxides. immobilization of pox was achieved by binding covalently the enzyme to a sepharose matrix (bead and membrane va epoxy). the immobilized peroxidase showed great stability at heat and storage than the soluble enzyme. the native enzyme retained % of its activity at • c for mn while the immobilized pox retained full activity for mn at the same temperature. in other side, the free enzyme retained full activity for at least one month and a half during storage at • c, and lost m of its activity after months. the immobilized form of pox retained complete activity for months at the same temperature. the immobilized enzyme was used to detect h o in some food components such as milk and fruit juices. in a second study, same experiments were performed in order to detect the smaller quantity of added h o to the farming milk. purpose: a new research field has been created to begin to address protein function at level of regulation of enzyme activity. this new area has been given the name chemical proteomics, or activity-based proteomics (abps), and makes use of small molecules that can covalently attach to catalytic residues in an enzyme active site. the selectivity of the chemically reactive group allows specific proteins or protein subset to be tagged, purified and analyzed. methods: this molecule (abps) has three subsets: tag, linker, and warhead. warhead is a nucleophile and attach to active site. linker is a polypeptide that makes a simple connection between warhead and tag. tag is fluorcent or radioactive material that facilitates the detection of drugs. findings: several diseases such as cancer, rheumatoid arthritis and osteoporosis are associated with elevated levels of protease activity. serine hydrolyses abps have been used to profile enzyme activity in a diverse range of cancer cell lines. in studies comparing metastatic and non-metastatic human breast cancer models, it was shown that the former exhibited a higher activity of a ␥-glutathione-s-transferase, an enzyme that has not previously been associated with breast cancer. discussion: additionally, abps can be used to develop robust screens for small molecule inhibitors of a specific enzyme target within a large family of related enzymes. this method of inhibitor screening allows compounds to be assayed for both potency and selectivity against a set of related in complex biological samples. this technique is able to identify novel enzymatic proteins and drugs and has the potential to accelerate the discovery of new drug target. a cyclodextrin glycosyltransferase (cgtase) from a new isolated strain from bacillus clausii e , was purified through q-sepharose, gel filtration chromatography and deae-sephadex a- . the mw of the pure enzyme was kda with sds-page. the enzyme displayed optimum ph value and ph stability at ph . and in range of . - . , respectively. the optimum temperature and thermostability were at • c and up to • c by h, respectively. the k m and v max were . mg/ml and . mol/min mg and . mg/ml . mol/min mg using maltodextrin and soluble starch, respectively. the isoeletric point was . and the n-terminal region of the pure enzyme was sequencing by maldi-tof-ms. the ratio of ␣-, ␤and ␥-cd was . : . : . and : : with maltodextrin and soluble starch at . %. application of magnetic separation technology for recovery of immobilised lipases nadja schultz , anke neumann , george metreveli , matthias franzreb , christoph syldatk chair of technical biology, university of karlsruhe, engler bunte ring , d- karlsruhe; forschungszentrum karlsruhe, institute of technical chemistry, water-and geotechnology; inst. für wasserchemie, engler bunte ring , ka. e-mail: nadja.schultz@ciw.uni-karlsruhe.de (n. schultz). url: www.fzk.de/itc-wgt (m. franzreb) first results on the development of the magnetic separation technology for the recovery of immobilised lipase from a -phase-system, which should be suitable for a large scale use in future, known as high gradient magnetic separation (hgms) are presented. the application of immobilised lipases makes the reuse of the enzyme in a process possible and is therefore interesting for industrial applications. in this study immobilised lipase is used in a -phase-system. here the new approach to recycle and reuse the lipase, immobilised on magnetic particles, from a -phase-system with the help of the new high gradient magnetic separator (hgms) is examined in ml scale. as model enzyme for the immobilisation on magnetic microparticles (polyvinyl alcohol (pva), - m) the commercially available (novonordisc) lipase a (cala) from candida antarctica was used. necessary for screening of immobilisation methods and characterisation of the immobilised lipase (candida antarctica) was the development of a robust, simple and rapid chromophoric activity assay. therefore the pnpp-lipase assay was optimised for direct application on immobilised lipases (in preparation schultz et al., ) . further more a ph-stat-assay for measuring the activity of free and immobilised cala in a -phase-system of tributyrate and buffer was optimized for this system. another important basis for the realisation of the recovery of immobilised lipases was to optimise the immobilization technique of the lipase. furthermore approaches for the explication of generally empirical based immobilization techniques on insoluble support were made. hereby we successfully applied the zeta potential measurement on the immobilization behaviour of the lipase cala. for to determine the operating temperature for the biomagnetic separation procedure we studied stability analysis of free and immobilised lipase cala at different temperatures ( , , , − • c) and at different ph values (ph , and ). the optimal temperature and ph value for the free and immobilised lipase was determined. presently and constructively on the so far developed methods and techniques we intensively work on the demonstration and feasibility of the recovery of immobilized lipase from a -phase-system. challenging approaches and first results on the recovery of immobilized lipase from a -phase-system in ml scale were shown already. nowadays glycomics raise new challenges for affinity chromatography related with an abundance of glycoconjugates in living organisms and with scaling-up of the preparative processes. economics, efficiency and practicality dictate the search of novel chromatographic biospecific adsorbents that could contribute to enhancing the productivity of the affinity separation process. the purpose of the work was to prepare cellulose-and silica-based biospecific adsorbents with immobilized lectins and to evaluate them for the affinity chromatography of glycoproteins. cellulose-based matrix granocel and silica coated with hydrophilic polymers were used as a support. the effect of support characteristics, such as pore size, chemistry of active groups and their density on the support' surface on lectin immobilization and on the efficiency of adsorbents obtained were evaluated. three different methods were used for the activation of the support: oxidation with sodium periodate, modification with pentaethylenhexamine (spacer arm of atoms) and carbonyldiimidazole activation. cona and wga two lectins of different molecular weight and shape were selected to notice differences resulting from the size and diffusion behaviour. chromatographic performances of the adsorbents were studied applying two different glycoproteins (god and fetuin) carrying specific terminal glycomoieties of mannose (god), and n-acetylglucosamine (fetuin) for specific interaction with cona and wga, respectively. the adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to . mg per ml support and a high recovery (up to %). it was shown that spacer arm affected ligand coupling kinetic as well as the chromatographic behavior of the adsorbents obtained. the adsorption isotherms of god onto cona adsorbents reveal an adsorption behavior with high and low affin-ity binding sites. the dissociation constant k d of the ligand-sorbate complex is approximately × − , and . × − m, respectively. it was supposed that the second step is related to the sorption of solvated god onto already adsorbed god forming sorbate dimers. quantitative methods in high throughput screening of aqueous two phase systems matthias bensch, björn selbach, jürgen hubbuch institute of biotechnologie , forschungszentrum jülich, germany purification of biopharmaceuticals is one of the most expensive and at the same time least understood steps in bioprocesses. during the process development for protein production, short time to market and the demand for cheap processes dominate today's process development. one way of reducing process costs is to implement integrative processes. aqueous two phase systems (atps) combine the advantages of removing cell debris and simultaneously purifying and concentrating the target protein, however, to the cost of highly complex systems which are difficult to predict and optimize. using high throughput screening techniques in the development of atps processes thus seems to be an ideal candidate for achieving both a reduced development time and an economical process without the need for preliminarily well characterized systems. in this study, we use the robotic system tecan freedom evo tm as an automation platform for the evaluation of aqueous two phase systems. central to this workstation are the integrated hardware as liquid handler, gripper, reader and centrifuge. we have created high throughput methods for rapid parameter estimations. as a first step, pipetting and mixing had to be calibrated for the use of highly viscous polymer solutions which are common in atps. the focus of the current work lies on the integration of the automatic preparation and analysis of two phase systems in microtiter plate scale. the robotic platform can now automatically create aqueous two phase systems and measure characteristic values such as binodal curves, protein concentrations and protein distributions between the two phases. the major bottleneck of hts processes, namely the rapid analysis of impure systems, is tackled by using automated elisa tools. depending on the intended use, the high number of measured partition coefficients and yields can be used for modelling or rapid process design. today, most optimisations of chromatography separations are based on experimental work and rule of thumb. the pat initiative has opened up for a model-based approach for downstream processing of pharmaceutical substances. this work uses a nonlinear chromatography model to optimize an ion exchange separation step. the general rate model with langmuir mpm kinetics described the behaviour of the components in the column. the optimal operating points using both productivity and yield as objective functions were found. the optimizations were run with both igg and bsa as target proteins respectively to compare their optimal operating points. the requirement on the optimal operating point was a purity of at least %. this requirement was added to the optimization problem as a nonlinear inequality constraint. flow rate, loading volume, start salt concentration in elution, elution gradient and cut points were used as decision variables in the optimization. the more retained component, bsa, was much easier to separate from igg with a gradient elution than igg from bsa while still retaining a high productivity and yield. the higher load volume at the optimal operating point, with bsa as target protein, causes a displacement of igg and thereby improving the separation. a high productivity at the yield optimum was still possible with bsa as target protein. both a lower productivity and yield was obtained with igg as target protein. optimisation and robustness analysis of a hydrophobic interaction chromatography step niklas jakobsson, marcus degerman, bernt nilsson department of chemical engineering, lund university, p.o. box , se- lund, sweden process development, optimisation and robustness analysis for chromatography separations are often entirely based on experimental work and generic knowledge. the present study proposes a method of gaining process knowledge and assisting in the robustness analysis and optimisation of a hydrophobic interaction chromatography step using a model-based approach. factorial experimental design is common practice in industry today for robustness analysis. the method presented in this study can be used to find the critical parameter variations and serve as a basis for reducing the experimental work. in addition, the calibrated model obtained with this approach is used to find the optimal operating conditions for the chromatography column. the methodology consists of threes consecutive steps. firstly, screening experiments are performed using a factorial design. secondly a kinetic-dispersive model is calibrated using gradient elution and column load experiments. finally the model is used to find optimal operating conditions and a robustness analysis is conducted at the optimal point. the process studied in this work is the separation of polyclonal igg from bsa using hydrophobic interaction chromatography. department of biochemistry and microbiology, ict prague, technicka , prague cz- , czech republic the display of novel metal binding sites on the surface of the biosorbent represents potent tool to increase its binding capacity and improve selectivity. in this study, the . kda transcriptional regulator merr of mercury-inducible mer operon of tn exhibiting high affinity and selectivity towards hg + , was displayed on the surface of s. cerevisiae. to achieve this, merr was genetically fused with gene encoding c-terminal domain of ␣-agglutinin which resulted in covalent attachment of the of the fusion protein on the cell wall glucan via glycosylphosphatidylinositol anchor. to evaluate the performance of such modified whole-cell biosorbent with specific regard to hg + , we constructed a new biosensor e. coli strain, which utilizes kanamycine resistance gene as a reporter under the control of mer promoter. it allowed determination of hg + in a range of - nm by simply monitoring the growth in the hg + /kanamycine-containing media. the effect of genetic engineering of s. cerevisiae surface by merr became significantly pronounced in biosorption experiments with solutions containing m hg + when modified cells accumulated . -fold more hg + than the control strain expressing mere anchoring domain. sensitivity analysis of amino acids in simulated moving bed chromatography ju weon lee, chong ho lee, yoon mo koo center for advanced bioseparation technology, inha university, inchon - , korea. e-mail: ymkoo@inha.ac.kr (y.m. koo) the difficulty of simulated moving bed (smb) design is that the optimization of the operation conditions relies on the determination of accurate adsorption isotherms. most smb chromatograph is carried out under nonlinear conditions, and the nonlinear behavior should be considered properly in the equilibrium isotherms. the other difficulty is the smb operation which has the characteristics of continuous process, all flow rates and switching time of valves should be maintained during the operation of smb. if the disturbances of operating conditions and isotherm parameters are occurred, it affects the zone flow rates and the migration velocity of the solutes, and these effects change the internal profiles of the solutes. therefore, it is the reason of decreasing the purity and the yield of products the objective of this work is to consider the sensitivity of isotherm parameters and operating parameters in smb chromatography process. two amino acids, phenylalanine and tryptophan, separation by smb process is selected as control system. application of ph and po probes during bacillus caldolyticus fermentation: an additional approach in improving a feeding strategy johannes bader , boris neumann , karima schwab , milan popovic , rakesh bajpai : studiengang biotechnologie, fachbereich v, tfh-berlin, seestr., berlin, germany proteome factory ag, dorotheenstr. , berlin, germany; department of chemical engineering, university of missouri-columbia, w ebe, columbia, mo, usa. e-mail: popovic@tfh-berlin.de (m. popovic) bacillus caldolyticus,a thermophilic microorganism, is a good producer of thermostable liquefying ␣-amylase. during optimisation of initial and feeding media for fed-batch fermentation a two component feeding containing starch and casitone was found advantageous. to approach the optimal feeding rate the method published by akesson et al., was extended to two component feeding. the key idea, discussed in this presentation, was using the po and ph probing signals to determine if the feeding of one or the other component should be increased or decreased. each of the probes offers information of different areas of feeding condition. to prevent excessive feeding of starch the ph probe is preferable. in case of excessive casitone feeding the po probe responds in very authentic way enabling together with the ph signal reliable and reproducible evaluation of feeding strategy. however a congruent response of po and ph probes means the approaching of the optimum feeding rate for both components. akesson, m., hagander, p., axelsson, j.p., . probing control of fed-batch cultivations: analysis and tuning. contr. eng. pract. , - . antibody immobilization by using the plasma polymerized acrylic acid r. jafari, m.tatoulian, f. arefi-khonsari laboratoire de génie des procédés plasmas et traitement de surface, enscp, upmc, rue pierre et marie curie, paris, france the objective of this work is therefore to produce a surface containing a high density of cooh functions on the polymer beads (ps) for the covalent immobilization of antibodies. we have investigated the plasma polymerization of acrylic acid in a fluidized bed reactor the polystyrene (ps) beads. for such application, there is a strong need to obtain stable plasma polymerized acrylic acid (ppaa) coating, resistant to washing with water. different physico-chemical analyses have been used (water contact angle measurements (wca), xps and sem analysis) to characterize the ppaa coating deposited on ps beads under different experimental conditions. the xps results showed that the pretreatment of surface of the beads before deposition of acrylic acid plays an important role on the stability of ppaa layer. the instability of the coating is partially due the fact that under certain conditions the coatings are soluble in water and secondly due to the bad adhesion of the polymer beads which are hydrophobic to the growing ppaa coatings. xps as well as tof-sims gives evidence of the immobilization of the antibody. xps results as well as static sims allows to detect nitrogen on the surface of the treated beads which proves the presence of the immobilized antibodies. under optimum condition the ppaa coatings provides the possibility to show a nitrogen uptake which varies between . and % of the apparent stoicheiometry of the surface. holst department of medical physiology, the panum institute, university of copenhagen, dk- copenhagen, denmark glp- (glucagon-like peptide- ), a peptide of amino acids secreted by endocrine cells in the gut in response to meal ingestion, was discovered during a systematic search for gut factors capable of enhancing insulin secretion. it turned out to be the most efficacious insulin releaser known, and unlike other factors, was shown to retain its insulinotropic activity also in patients with type diabetes. subsequent research has documented that the peptide not only releases insulin from the beta cells, but also enhance all steps of insulin biosynthesis, up-regulates beta cell gene transcription, and has trophic effects on the beta cells. the latter includes both proliferation of existing cells, neogenesis from ductal precursor cells, and inhibition of apoptosis. the peptide also inhibits glucagon secretion, reduces gastric emptying and reduces appetite and food intake. because of these actions, glp- administered to patients with type diabetes dramatically lowers blood glucose as well as glycated hemoglobin levels, and reduces body weight. however, natural glp- is extremely rapidly metabolized in the body, and the problem has been how to convert the unstable peptide into a clinically useful agent. the two main problems are its susceptibility to enzymatic degradation by ubiquitous dipeptidylpeptide peptidase iv (dpp-iv) and its rapid renal elimination. a related peptide, isolated from the saliva of a lizard, exendin- , was found to be a full agonist of the glp- receptor, to be resistant to dpp-iv and to be cleared more slowly by the kidneys. this peptide was highly effective in clinical studies and has now ( / ) been approved for diabetes treatment by the fda. other approaches include acylation of glp- whereby it attaches to albumin in the body and acquires resistance to dpp-iv as well as a slow renal elimination. also this analogue (liraglutide) has favourable clinical effects. fusion proteins of glp- and larger, slowly eliminated proteins in the body are currently being evaluated. small molecule, orally available inhibitors of dpp-iv have been demonstrated to protect endogenous glp- from degradation and to be efficacious in both experimental and clinical diabetes, and numerous inhibitors are currently in clinical development. the incretin hormones are released from gut endocrine cells upon meal ingestion. they enhance glucose-induced insulin secretion and nay be responsible for up to % of postprandial insulin secretion. the incretin hormones are glucagon-like peptide- (glp- ) and glucosedependent insulinotropic polypeptide (gip). in patients with type diabetes ( dm) the incretin effect is severely reduced or absent. in dm patients the secretion of gip is normal, but its effect on insulin secretion is almost completely lost. glp- secretion, on the other hand, may be impaired, but its insulinotropic actions are preserved and it may restore insulin secretion to near normal levels. substitution therapy with glp- might therefore be possible. glp- is a product of the glucagon gene and its actions include: ( ) potentiation of glucose-induced insulin secretion; ( ) stimulation of the expression of ␤-cell genes essential for insulin secretion, including the insulin gene; ( ) stimulation of ␤-cell proliferation and neogenesis (by enhancing endocrine differentiation of duct cells) and inhibition of ␤-cell apoptosis; ( ) inhibition of glucagon secretion; ( ) inhibition of gastrointestinal secretion and motility, notably gastric emptying; and ( ) inhibition of appetite and food intake. these actions make glp- particularly attractive as a therapeutic agent for dm. thus, continuous subcutaneous administration of glp- for weeks resulted in a mmol/l reduction in mean plasma glucose and a reduction in hgba c of . %; a weight loss of kg; improved insulin sensitivity; improved ␤-cell function; and the treatment was associated with no significant side effects. unfortunately, glp- is rapidly destroyed in the body by the ubiquitous enzyme, dipeptidylpeptidase iv (dpp-iv). clinical strategies therefore include: ( ) the development of metabolically stable analogues of glp- viz. activators of the glp- receptor; and ( ) inhibition of dpp-iv. orally active dpp-iv inhibitors have proven successful in experimental diabetes and several companies are now trying to develop clinically suitable inhibitors. so far the clinical experience is limited, but recent clinical studies have provided proof of concept. metabolically stable analogues/activators include the structurally related lizard peptide, exendin- or analogues thereof, as well as glp- derived molecules that bind to albumin and thereby assume the pharmacokinetics of albumin. these molecules are effective in animal experimental models of type diabetes, and have been employed in clinical studies of up to weeks' duration. on the basis of these studies it can be concluded that a therapy of type diabetes mellitus based on stimulation of glp- receptors is likely to be effective and to become a clinical reality within the not too distant future( - ). recombinant activated coagulation factor vii (rfviia) was developed to treat bleedings in hemophilia patients, who have developed inhibitors against fviii or fix, and has been demonstrated to have an efficacy rate of - % in major surgery as well as in serious bleedings in such patients. to use rfviia as a hemostatic agent in severe hemophilia is a new concept of treatment, not being a substitution therapy, but using a pharmacological dose of exogeneous rfviia to compensate for the lack of fviii or fix. the administration of extra rfviia has been found not only to bind to tissue factor (tf), but also to the negatively charged phospholipids surface of thrombin activated platelets. hemostasis occurs on surfaces being initiated on the tf-expressing cells as a result of exposure of tf, not normally exposed to the circulating blood, following an injury to the vessel wall. tf is a true receptor protein with an intramembraneous part and an intracellular tail. its ligand is fvii/fviia. as soon as tf is being exposed to the blood, it forms complexes with fviia already present in the circulation. these complexes activates fx and provide the initial limited amount of thrombin molecules activating the co-factors, fviii and fv, as well as fxi and platelets. following the thrombin activation of platelets, negatively charged phospholipids are being exposed on the outer surface of the platelets. on this surface most coagulation proteins bind tightly, facilitating the conversion of fx into fxa and the full thrombin burst, necessary for the formation of a tight fibrin hemostatic plug resistant against premature lysis. in hemophilia patients the initiation of hemostasis is essentially normal, but, since they lack fviii or fix, they do not form the fviiia-fixa complex necessary for full thrombin generation on the activated platelet surface. as a consequence the fibrin plug formed in hemophilia is loose, fagile and easily dissolved resulting in continuous bleeding. pharmacological doses of rfviia have been demonstrated to mediate direct binding of rfviia to the negatively charged thrombin activated platelet surface, thereby generating thrombin formation in the absence of fviii/fix. through this mechanism hemostasis is generated in hemophilia patients independent of fviii/fix. furthermore, by generating more thrombin at an increased rate the formation of stable, tight fibrin hemostatic plugs are facilitated. such fibrin plugs are more resistant against premature lysis and help not only to initiate but also to maintain hemostasis. based on its capacity of enhancing thrombin generation locally on the activated platelet surface, rfviia has been used to ensure hemostasis also in other situations than hemophilia, such as platelet defects including thrombocytopenia. recently, rfviia was shown to be hemostatically effective in patients with profuse bleedings as a result of vast trauma and tissue damage. in these patients with a complex hemostasis pattern including a host of changes leading to an impaired hemostatic function, extra rfviia seems to help generate a burst of thrombin resulting in the formation of a stable hemostatic plug more resistant against the ongoing lysis. in patients with intracerebral haemorrhage, one single dose of rfviia recently was found to limit the expansion of the haemorrhage and thereby leading to improved functional outcome. institute for medical microbiology and immunology, panum . . , blegdamsvej , dk- copenhagen n, denmark. email: s.buus@immi.ku.dk complete genomes from several species including many pathogenic microorganisms are rapidly becoming available along with the corresponding "proteomes". even at the peptide level, the diversity of proteome is enormous and easily represents a unique imprint of the originating organism. it is perhaps not surprising that the immune system considers peptides as key targets. recent immunological advances have shown that mhc molecules act as peptide selectors for immune recognition. we have proposed to generate accurate predictions of peptide binding to mhc and used these to identify immunogenic epitopes directly from genomic data. we have developed an iterative data-driven immunobioinformatics approach where data is used to generate predictors, and predictors are used to select new and complementary data for the next iteration. we have demonstrated the superior performance of this approach compared to a random data selection approach. we have also developed an efficient approach to select the most informative mhc molecules to investigate. the resulting, immunobioinformatics resource represents an immediate and powerful application and interpretation of genomic data, and will enable a rational approach to immunotherapy in the future. allergen specific immunotherapy is a causal treatment for igemediated allergic diseases such as hay-fever, and it has relied traditionally on preparations derived from aqueous extracts of various natural allergenic source materials. the cloning and production of an increasing number of allergens through the use of dna technology has not only facilitated the characterisation and analysis of the allergenic proteins, but also provided the opportunity to use these recombinant proteins instead of natural allergen extracts for the diagnosis and therapy of allergic disease. detailed physicochemical, biochemical and immunological characterisation are essential for the comparison of natural and recombinant proteins, and also provide a basis for developing derivatives. chemically modified allergens with attenuated ige-reactivity are currently used for immunotherapy in order to enable high doses to be achieved with a minimized risk of inducing allergic side reactions. dna technology provides the opportunity to develop and produce hypoallergenic allergen variants using strategies including gene mutation. the design of such variants must ensure that t cell reactivity and immunogenic activity are retained in order to preserve therapeutic potential. the recombinant allergens and their derivatives have several advantages over natural allergen extracts. they are relatively easy to produce in consistent pharmaceutical quality; the problems of natural extract standardisation can be avoided completely; the relative concentrations of the individual allergens can be controlled to obtain optimal dosages; nonallergenic proteins are excluded; the possible risks of contamination are avoided. the first clinical trials with grass pollen allergens and birch pollen hypoallergenic variants have yielded very encouraging results. the use of recombinant polyclonal antibodies (pabs) may improve the treatment of disease caused by complex targets such as infectious agents, when compared to monoclonal antibody therapy. symphogen has developed a method for reproducible production of target-specific fully human pab compositions, so-called symphobodies. the antibody genes are first isolated from donors with an immune response against the target and antibodies are screened for specificity. subsequently, the pabs are expressed in mammalian cells using the sympress technology, which is based on site-specific integration. this procedure ensures that each of the expression constructs encoding the antibody genes are stably integrated at the same site in each of the host cells, thereby eliminating genomic position effects and differential growth and production rates. further, the sympress technology comprises the generation of a polyclonal working cell bank (pwcb) which is used as inoculation material for the manufacturing. these cells display sufficient genetic stability to enable a controlled gmp production of recombinant polyclonal antibodies. symphogen's first product, sym , is a recombinant human polyclonal rhesus d-specific symphobody preparation consisting of different anti-rhesus d antibodies. this product is intended to be used for treatment of idiopathic thrombocytopenia purpura and prevention of hemolytic disease in newborns. recombinant anti-rhesus d symphobodies were produced and shown to be biologically active against rhesus d. the expression technology provided a compositional reproducibility between batches which is sufficient for manufacturing of such a polyclonal product for clinical use. scaledup production for clinical trials is currently ongoing. stem cells play an important role in renewing tissues such as skin and cornea. they are responsible for the continuous generation of the differentiated epithelium. we have characterized stem cells of the skin and cornea in situ and their fate in vitro in human skin reconstructed by tissue engineering using keratin (k) . in the outer root sheath of the hair follicle, stem cells (label-retaining cells) present in the basal layer of the bulge area express k and present a loosely arranged keratin filament network and low levels of k protein in their cytoplasm. in addition, another stem cell population (also labelretaining) is present in the first suprabasal layers. these cells exhibit a very dense keratin network and express k . three-dimensional tissue constructs (dermis and epidermis) obtained by the self-assembly approach of tissue engineering allow the preservation of k positive stem cells in the basal layer of the epithelium. in the eye, the stem cells are located in the limbal part but not in central cornea and they express k . the epithelium of reconstructed cornea is thinner compared to reconstructed skin and more transparent. the characterization of stem cells in reconstructed tissue is essential to evaluate the long-term survival of these tissues in vitro but also after grafting. these human reconstructed tissues are developed for fundamental (physiological, toxicological studies) and clinical applications such as transplantation for the permanent replacement of damaged organs. lg is holder of the canadian research chair (cihr) on stem cells and tissue engineering. alessandra gliozzi physical department, university of genoa, genoa, italy hollow nanometer-sized capsules can be prepared by means of different techniques. first "nanocapsules" were liposomes, however they are too unstable for many medical or pharmaceutical applications. in contrast, recently developed polyelectrolyte capsules prepared by means of the layer-by-layer technique are much more stable and seem to be a very promising way for coating living cells or tissues in order to prevent or reduce their immune rejection after implantation. several observations on single living cells encapsulated by the alternative adsorption of oppositely charged polyelectrolytes will be presented. the most relevant result is that cell preserve their metabolic activity, are still capable of dividing and performing specific functions. moreover, a technique to immobilize in defined arrays coated cells expressing green fluorescent protein by using a microcontact printing of polyelectrolytes will be presented. finally, tests performed to study the induction of fibrosis and vascularization by nanocapsules implanted in rat kidney and liver will be presented. over the past decade we have developed methods to generate spontaneously and synchronously beating tissue equivalents from neonatal rat heart cells in the culture dish. these tissue equivalents display the key morphological and functional features of intact myocardium and have been termed engineered heart tissue (eht). to generate ehts, heart cells are mixed with freshly neutralized, liquid collagen i, matrigel and growth supplements and grown in a circular casting mold around a central cylinder, which subjects the cells to a continuous mechanical load. this process is enforced by cyclic mechanical stretch. we use eht mainly for two purposes, as a test bed for the effects of pharmacological or genetic manipulations and for cardiac repair. as a cell culture model, ehts compare with standard d monolayer cultures of neonatal rat cardiac myocytes and freshly isolated adult cardiac myocytes. advantages of ehts are their functional similarities with intact heart muscles, the ability to easily measure force of contraction under mechanical load, the pos-sibility to transfect cardiac myocytes inside ehts with adenovirus at high efficiency and the reproducibility in large series. a disadvantage is that contractile function as measured at the end of the culture period also integrates influences on tissue development, cell-cellconnections, extracellular matrix production and on non-myocytes. at present we are working on downscaling the eht method to a well format for screening purposes. to use ehts for cardiac repair we created multi-looped ehts from five circular ehts large enough to cover the infarct scar days after coronary artery ligation in rats. ehts survived and formed a layer of muscle tissue on top of the infarct scar. ehts restored undelayed anterograde impulse propagation over the scar, prevented further ventricular dilatation, normalized enddiastolic pressure and relaxation, and partly restored contraction of the scar. thus, the study provides evidence that implanting ehts onto infarcted hearts can improve cardiac contractile function after myocardial infarction. the goal of tissue engineering is the development of skin, bones and even organs to restore, maintain and improve tissue function within the body. the current paper focuses on the investigation of invitro growth of osteoblast cells in different types of scaffolds. three of the scaffolds were made of pcl (polycaprolactone) % glycerol and % hca(hydroxylapetite). two of the scaffolds were made by compression molding, and one was made by fused deposition modeling utilizing the stratasys. the fourth cerabio was a commercially available product totally ceramic. the pores in compression molding were obtained by putting in % volume of sugar either and m which was later removed by leaching. the fused deposition scaffold was made by placing the filaments in a predetermined arrangement. the stratasys system was a computer designed model. the scaffolds were seeded with hfob . human fetal osteoblast cell line with vigorous shaking overnight and incubating at • c and % co . observation of the seeded scaffolds was made after days and days of incubation. the seeded cells were stained with bcip/nbp at • c overnight. the cell proliferation in the and m scaffolds appeared approximately the same with a possible advantage of m. the cerabio sample demonstrated the greatest proliferation among the four scaffolds studied and the stratasys sample exhibited a different type of cell adhesion with the cells were clustered in the interstices of the structure. denise freimark, ruth freitag, valérie jérôme chair of process biotechnology, university of bayreuth, d- bayreuth, germany. e-mail: denise.freimark@uni-bayreuth. de (d. freimark) tissue engineering is emerging as an alternative to bone grafts for the regeneration of defects that do not heal spontaneously. ultimately, the development of an optimum carrier and the identification of ideal inductive factors and cells may enable tissue engineering to provide an improvement over bone grafts in the future. bone formation and repair require a complex cascade involving growth factors, cytokines and angiogenesis. at present the complexity of the molecular mechanisms that control gene expression in bone forming cells in embryo as well as in adult is not fully understood. several factors like bone morphogenetic proteins (bmps), transforming growth factor beta (tgf-␤), vascular endothelial growth factor (vegf) and insuline-like growth factor (igf) have been identified and their ability to stimulate bone formation in vitro and in vivo has been investigated. while much is known about these factors per se, less is known about genetic regulation of artificially stimulated osteogenesis. interestingly, some in vitro investigations showed that only optimal growth factors concentrations lead to effective bone formation whereas higher concentrations had deleterious effects which suggest some variation in the activated regulation pathways. therefore, one of our goals is to analyze kinetics, dose-dependence and synergistic effects of growth factors and cytokines on regulation pathways of bone formation. moreover, the optimal vascularization of the scaffold is a major hurdle in the development of engineered bone. it is well known that: (i) vascular invasion precedes bone growth and (ii) osteogenesis takes place in the vicinity of newly formed vessels. thus, inadequate bone vascularization is associated with decreased bone formation. further analysis of the intercommunication between endothelial cells and osteoprogenitors in co-culture systems could provide key information that could be thereafter used to solve this problem. we propose to add some new knowledge to this complicated puzzle. a first step in our investigation is the production of some of the growth factors mentioned above in recombinant form. these factors are expressed in a novel vector (ptriex tm ; novagen) which allows recombinant protein production in prokaryotic or in eukaryotic systems with a single plasmid. afterwards, we analyze potential synergistic effects of these factors as well as kinetic and dose-depend parameters on the genetic regulation of downstream pathways in osteoblasts culture. in parallel, we develop an in vitro system allowing us to investigate the intercommunication between endothelial cells and osteoprogenitors. there has been significant interest in the therapeutic and scientific potential of human embryonic stem (es) cells since they were first isolated in . if human es cells could be differentiated into suitable cell types, stem cells might be used in cell replacement therapies for degenerative diseases such as type i diabetes and parkinson's disease, or to repopulate the heart following myocardial damage. however, there is a significant shortage of high quality human es cell lines and few research groups have experience in the propagation and manipulation of these cells. we are addressing this important issue using the combined expertise of the stem cell biology laboratory and the assisted conception unit at king's college, london. with local ethical approval and under licence from the uk human fertilisation and embryology authority, we have been establishing high quality human es cell lines from a novel source of human embryos. to date, we have derived three human es cell lines and are now focused on the generation of therapeutically important cell populations, including cells that may have clinical application in degenerative and traumatic injury to the brain and spinal cord, heart, retina and other target organs. wallenberg neuroscience center, department of physiological sciences, lund university, bmc a , s- lund, sweden cell replacement therapy for parkinson's disease is based on the idea that implanted dopamine neurons may be able to substitute for the lost nigrostriatal neurons. in rodent and primate models of parkinson's disease it has been shown that transplanted dopamine neuroblasts can re-establish a functional innervation and restore dopaminergic neurotransmission in the area of the striatum reached by the outgrowing axons; that the grafted neurons are spontaneously active and release dopamine in an impulse-dependent manner, at both synaptic and non-synaptic sites; and that they can reverse or ameliorate some of the parkinson-like motor impairments induced by damage to the nigrostriatal system. clinical trials in patients with advanced parkinson's disease have shown that dopamine neuroblasts obtained from fetal human mesencephalic tissue can survive and function also in the brains of pd patients, restore striatal dopamine release, and ameliorate impairments in motor behavior. the principal limitation of this approach is the problems associated with the use of tissue derived from aborted human fetuses, and the large numbers of donors needed to obtain good therapeutic effects. until now, transplantation of dopamine neurons has focused primarily on differentiated neuroblasts and young postmitotic neurons, at the stage of neuronal development that is optimal for survival and growth of the grafted cells. however, progenitors taken at earlier stages of development might prove more effective. efforts are now made to expand multipotent neural stem-or progenitor cells in vitro, and control their phenotypic differentiation into a dopaminergic neuronal fate. initial results suggest that in vitro expanded cells can survive and function after transplantation to the striatum in the rat pd model, but the overall yield of surviving dopamine neurons has been very low. with further development, expanded progenitors or dopamine neuron precursors, possibly in combination with cell engineering techniques, may offer new sources of cells for replacement therapy in pd. stem cell therapy has been very much in vogue for several years now. like gene therapy before it, it has raised unrealistic hopes of cures being available imminently. unlike gene therapy, it can cite proof of principle in the well established practice of bone marrow transplantation which is actually a good example of stem cell therapy. however, most of the uses that are now touted as targets for stem cell therapy, envisage the conversion of the stem cells into lineage restricted progenitor cells or more commonly, the final differentiated cell type. such conversions are extremely difficult to initiate and control. the procedures involve the manipulation, differentiation, and expansion of cell cultures in the laboratory, with unknown long term effects on the genetics and physiology of the cells. these issues are compounded when one considers as source material, human embryonic stem cells, where not only the final cell product requires significant scrutiny, but also there are safety issues surrounding the persistence of undifferentiated cells. on top of all these challenges are commercial (for stem cell companies), clinical, and regulatory pressures which will impact heavily on the pace of progress. nonetheless, despite all these hurdles, various academic groups and companies are making significant progress and examples of such developments in diabetes and cardiovascular repair will be given stem cells have the unique ability to perpetuate themselves while continually replenishing tissues throughout the life of an organism. the era of cellular and tissue regeneration for the treatment of disease and the effects of aging has indeed begun. it has been known that mechanical factors play an important role in the regulation of cell physiology. it is therefore reasonable to believe that mechanical factors also play a significant role in the metabolic activity and differentiation of mscs. in this study, we investigated the viscoelasticity of individual bone marrow-derived adult human mesenchymal stem cells (hmscs), and the role of specific cytoskeletal component -f-actin microfilaments on the mechanical properties of individual hmscs. the mechanical properties of hmscs were determined using the micropipette aspiration technique coupled with a viscoelastic solid model of the cell. for the hmscs under control conditions the instantaneous young"s modulus e was found to be ± (pa), the equilibrium young"s modulus e ∞ ± (pa), and the apparent viscosity ± (pas). after exposed to m of chemical agent-cytochalasin d that disrupt the f-actin microfilaments, the young's moduli of hmscs decreased by up to % and the apparent viscosity increased by %. these findings suggest that microfilaments are crucial in providing the viscoelastic properties of the hmscs, and changes in the structure and properties of them may influence the mechanical properties of hmscs significantly. pharmacologic transcription control of desired transgenes is essential for gene-function analysis, drug discovery, biopharmaceutical manufacturing, design of complex artificial regulatory networks, precise and timely reprogramming of key cell characteristics for gene therapy and engineering of preferred cell phenotypes for tissue engineering. capitalizing on our recent advances in the design of small molecule-responsive transcription control modalities we have used conditional molecular interventions for (i) improvement of specific productivity in biopharmaceutical manufacturing, (ii) transdifferentiation of therapeutically relevant cell phenotypes and (iii) design of artificial microtissues. we will also report on a completely new dimension of transgene control as well as engineering of hysteretic and epigenetic gene networks in mammalian cells. chronic diseases are a growing burden for the individual and society alike. one key factor driving this increase is an ageing population. currently, there are million persons aged years or over and this number is predicted to triple by the middle of the st century. effective prevention of irreversible damage to major organ systems requires early diagnosis and treatment yielding significant quality of life to the individual and sparing valuable health care resources. even when safe and effective medicines are available, a remaining problem to successful therapy are issues of patient compliance. historically, vaccines have been one of the major advances towards the longevity we enjoy today, with compliance rates close to %. hence, vaccines for early treatment of chronic diseases are ideally positioned for long-term therapy, and will take away the burden of self-medication associated with orally active drugs. here we will discuss a new generation of therapeutic vaccines based on virus-like particles (vlps). by displaying target molecules in a highly repetitive manner on vlps, it is possible to break b cell unresponsiveness in experimental animals as well as in humans. using such vaccines in animals, chronic diseases such as hypertension, alzheimer's disease, obesity and rheumatoid arthritis could be treated. furthermore, vaccination against nicotine resulted in high nictotine-specific antibody titers in humans, greatly facilitating smoking cessation in immunized individuals. interdependence of the impact of methanol and oxygen supply on protein production with recombinant pichia pastoris n.k. khatri, f. hoffmann martin-luther-university halle-wittenberg, institute for biotechnology, halle d- , germany. e-mail: f.hoffmann@biochemtech.uni-halle.de (f. hoofmann) the methylotrophic yeast pichia pastoris is a potent expression system for secretion of recombinant proteins. methanol as inductor of the foreign gene expression is also a substrate with high oxygen demand, which can lead to sudden oxygen depletion upon induction. thus, supply rates of methanol and oxygen are major process parameter during protein production with recombinant pichia pastoris. limiting dosage of methanol allowed maintenance of oxygen sufficient conditions during production of a single chain antibody fragment, but the product was degraded from the c-terminal end. in contrast, full-length product accumulated with controlled methanol concentrations despite oxygen limitation. the volumetric methanol uptake rate are limited by the oxygen transfer capacity of the reactor. higher methanol concentrations decreased the biomass yield and thereby increased the specific methanol uptake rate. this enabled prolonged production and yielded fivefold higher product concentrations. at the same time, the accumulation of small molecular weight contaminants was reduced. dosage of pure oxygen accelerated methanol uptake, grow and production. switching to dostat mode upon oxygen depletion led to an arrest of product accumulation, in contrast to persistent methanol feeding. the productivity was tenfold higher than without oxygen. combined with high methanol concentrations, however, fast methanol uptake led to toxication of the cells and early stop of production. flow cytometry revealed that perturbation of oxygen metabolism was followed by partial lysis of the culture. recombinant production of therapeutic proteins poses severe challenges due to their complexity (cystines, subunits, size). formation of the correct disulphide bridges is a prerequisite for activity but difficult to achieve in a prokaryotic host. there proteins mainly fold post-translationally as opposed to eukaryotic co-translational folding. thus the recombinant products are often not soluble and/or active. that is why different production parameters (e.g. host, induction conditions, temperature, compartment, proteinaceous fusion partners, co-expression of chaperones, foldases) are applied in order to gain functional recombinant proteins. the impact of all these strategies cannot be predicted and every problem of the production (expression, solubility, activity) might need to be solved for every target protein separately. nevertheless, much effort has been put into this for almost decades, because they are important targets for the pharmaceutical industry. human growth factors influencing cellular proliferation and/or differentiation are one example. this case study gives an overview of strategies tested within the development of two processes leading to an optimised yield of active protein. murine wnt- (wnt family) possesses conserved cysteines (most likely all involved in disulphide bridge formation and one in posttranslational modification) and could only be successfully produced in e. coli (fahnert, ) recently despite various attempts for many years. the other target protein is human collagen prolyl- -hydroxylase being a heterotetramer consisting of two ␣-subunits and ␤-subunits each. the ␣-subunit strongly aggregates if produced separately whereas the ␤-subunit is pdi and is suggested to have a chaperone function. therefore a sequential induction strategy was proposed for this protein . the moss physcomitrella patens has been recently recognized as an ideal producer of recombinant proteins with respect to glycosylation. due to the elaborated post-translational capabilities of moss cells, the glycosylation patterns have been manipulated to obtain similar proteins to those found in animal cells. the protein expression using moss in suspension offers important advantages in comparison to other systems e.g. cho cells. the recombinant proteins can be targeted into the mineral medium, simplifying the down stream processing. moreover, there are neither known moss viruses nor plant viruses that are pathogenic for humans. the moss are cultivated axenically in a filamentous stage, the so called protonema. a pilot l tubular photoreactor is used to characterize the response of p. patens to variations on the culture conditions. the phototrophic culture in bioreactors is systematically investigated, where light quantity and quality, stress, concentration of phytohormones, and moss morphology influence the differentiation, growth, and protein expression. a tight control of the moss morphology in suspension, quantified by image analysis, has shown to be advantageous in order to delay the cell differentiation and maintain the carbon dioxide uptake in long bioreactor runs. the introduced perfused culture system by means of cross flow filtration allowed for a continuous product separation and concentration, and feed back of the productive cells. the characterization of this highly controlled culture system is presented and the potential of p. patens as an alternative tool for molecular farming is discussed. (rnai) is an evolutionarily conserved, endogenous mechanism for sequence-specific gene silencing that uses small double-stranded rnas (called short interfering rnas or sirnas) to direct cleavage or prevent translation of homologous mrnas. harnessing rnai for therapy presents an opportunity for potentially treating a wide variety of diseases. the main obstacle is delivering sirnas into the cytosol of target cells in vivo. although we were able to protect mice from autoimmune hepatitis by hydrodynamic tail vein injection of sirnas targeting fas, this delivery method is unlikely to be adaptable for human use. alternate strategies to deliver sirnas in vivo as small molecule drugs using currently available, clinically acceptable injection methods that have shown promise in mouse models will be discussed. these include local delivery to mucosal surfaces and delivery into specific cells via cell surface receptors using an antibody fragment fused to protamine. these sirna complexes silence gene expression in vivo only in cells bearing the targeted receptor. experiments showing efficient, effective and cell-specific delivery in a mouse tumor model will be discussed. in addition, encouraging preliminary data using rnai for a microbicide to prevent sexually transmitted infection will be presented. rna interference (rnai) holds significant progress as a therapeutic approach to siolence disease-causing genes, particularly those that encode "non-druggable" targets. the key hurdle for rnai therapeutics is in vivo delivery. a critical requirement for achieving systemic rnai in vivo is the introduction of "drug-like" properties, such as stability, cellular delivery and tissue biodistribution, into synthetic sirnas. our progress in achieving in vivo silencing of endogenous genes with chemically modified sirnas will be discussed. hiv- replication in human t cells can be inhibited by stable expression of a short hairpin rna targeting the viral nef gene (shrna-nef). however, hiv- escape variants emerge after prolonged culturing, and all but one escape mutant acquire a mutation in the shrna-nef target sequence. we observed single and multiple nucleotide substitutions, but also partial or complete deletion of the target sequence. these results demonstrate the sequencespecificity of this antiviral approach. we observed an inverse correlation between the level of resistance and the stability of the shrna/target-rna duplex for most of the escape mutants. however, two escape variants did not follow this pattern, including an escape mutant with a single point mutation at position − upstream of the target sequence. these mutants provide a much higher level of resistance than expected based on duplex stability, which is obviously not affected in the − mutant. we demonstrate that these mutants adopt an alternative rna secondary structure that occludes the target sequence. this results in reduced shrna-nef binding and provides a novel mechanism for rnai-resistance. to avoid viral escape, one should ideally target hiv- with multiple effective shrnas against conserved genome sequences. we performed a large scale screening to identify such targets, and we have identified at least nine genome segments that can be targeted effectively with shrnas. these potent antivirals are currently being assembled in a lentiviral vector for gene therapy applications in hiv-infected individuals. furthermore, we will describe approaches to forecast viral escape routes and to effectively block such evolutionary paths with additional rnai measures. in this study we analyzed the effect of antibodies against electronegative ldl on the development of atherosclerotic lesions in low-density receptor-deficient (ldlr −/− ) mice. two groups of (ldlr −/− ) mice (eight females) fed . % cholesterol-enriched chow were used. the first group received a monoclonal antibody against electronegative ldl ( g) and the second one received pbs (controls). additionally, other two groups (eight males) of (ldlr −/− ) mice were treated with a polyclonal antibody against electronegative ldl ( g) or pbs (controls). antibodies were administered by intravenous route one week before starting the hypercholesterolemic diet and then every week over an experimental time of days. afterwards, quantification of atherosclerotic plaque area of heart and aortic arch was done by analysis of the slices stained with oil red/hematoxolin/light green with the image propus software. the passive immunization with either monoclonal or polyclonal antibodies against electronegative ldl significantly reduced the atherosclerotic plaque areas in atherosclerosis-prone ldlr −/− mice. in conclusion, antibodies against electronegative ldl administered by intravenous route may play a protective role in atherosclerosis. supported by fundação de amparoà pesquisa do estado de são paulo (fapesp, scholarships to d.m.g., l.s. and a.b. and grants to m.h.k. and d.s.p.a.). the enzyme asparaginase from the procaryote escherichia coli or erwinia crysanthemi is used for the treatment of lymphoblastic leukaemia. the drug causes immunological reactions in despite of the treatment efficiency. asparaginase may also be obtained from saccharomyces cerevisiae and this enzyme could provide an alternative to its bacterial counterparts. in this study, the periplasmic nitrogen regulated asparaginase ii from s. cerevisiae, that is coded by the asp gene, was cloned and expressed in the methylotrophic yeast pichia pastoris under the control of the aox gene promoter. the recombinant p. pastoris strain was cultured in shake flasks and in a l instrumented bioreactor. in both cases it was observed specific enzyme yields seven fold higher in comparison to that using a nitrogen derepressed strain of s. cerevisiae, reaching u/g dry cell mass. high cell density cultures carried out in the l bioreactor, in which it was attained g dry cell mass/l, resulted in a dramatic improvement in asparaginase fermentation. as such, it was measured enzyme yields of , u/l and productivities of u/l h. department of biochemistry and food chemistry, biotechnology, university of turku, tykistokatu , biocity th floor, turku, finland. e-mails: lorenzo.galluzzi@utu.fi; deadoc@libero.it; deadoc@aliceposta.it (l. galluzzi) the bacterial luciferase operon from the bacterium photorhabdus luminescens has been used, since its first description, for exceptionally different applications. these ranged from the environmental monitoring to the cell tagging, from the analysis of cellular metabolism to the high-throughput screening of novel compounds. the wild-type luxcdabe operon has been engineered in countless ways (for instance by changing the order of the constituent genes, by optimizing the codon usage and by coupling it to many promoters) and has been expressed in prokaryotic and eukaryotic organisms in order to meet precise research and commercial needs. upon the operon expression light is emitted as the side product of a chemical reaction catalyzed by the luciferase enzyme, an ␣␤ heterodimer encoded in luxa and luxb genes. the reaction involves the oxidation of a long-chain aliphatic aldehyde and reduced flavin mononucleotide (fmnh ) with the liberation of excess free energy in the form of a blue-green light at nm. the luxcde genes code for the polypeptides (transferase, synthetase, and reductase) forming the fatty acid reductase complex that catalyzes the conversion of fatty acids into the long-chain aldehyde required for the luminescent reaction. noteworthy is that for the production of the substrates for the luciferase both atp and nadph are required, while neither is involved in the actual light emitting reaction (wilson and hastings, ) . recently, the coupling of the luciferase operon to regulated promoters lead to the construction of genetically modified bacteria able to sense the presence of chemicals and to respond, in a dosespecific manner, with light emission. this approach has been applied to the detection of antibiotics in samples from the food industry as well as to the detection of heavy metal ions in environmental samples (kurittu et al., ; bechor et al., ) . in addition, it opened the possibility of screening wide libraries of new compounds looking for molecules with pre-determined features, able to induce the bioluminescent response by de-repressing the lux operon transcription when incubated with the appropriate bacterial sensor. the high throughput and low costs are the main advantages of this system, which shows as well a certain degree of specificity (galluzzi and karp, ; galluzzi et al., ) . nevertheless, since the in vivo bioluminescence relies upon a complex network of biochemical reactions, under certain circumstances the light emission is not a direct consequence of the lux operon transcriptional induction but it more likely originates at a posttranslational stage. as a matter of fact, one can suppose that a change in the light emission will be observed whenever the concentration of one or more substrates for the lux␣␤ reaction occurs. consequently, all the molecules sharing the ability to impair the delicate chemical equilibrium regarding the compounds involved in bioluminescence will be sensed by the bacteria as inducing compounds. this, in turn, will result in a loss of specificity of the assay. we investigated this aspect of the whole-cell assays based upon the bacterial luciferase operon for drug discovery by means of a reporter plasmid in which the luxabcde genes, rearranged and optimized for the after min (black downward arrow) of incubation at • c under vigorous shaking the following concentrations of trimethoprim were added to the growing cells: g/ml (triangles), g/ml (circles) and g/ml (rumbles). water was administered to control cultures (squares). light emission was quantified every min by means of the wallac victor multilabel counter (perkin-elmer, turku, finland) and normalized to the absorbance, measured at nm with the same device. multi- white-walled transparent-bottomed plates were used for the assays (nalge nunc, usa). between the measurements the plates were kept at + • c under vigorous shaking. filled symbols refers to the absorbance values (left side y-axis); open symbols to the normalized count per seconds (right side y-axis). expression in gram + organisms, are under the control of the qacr regulatory region from staphylococcus aureus . non-pathogenic s. aureus rn cells bearing the pqaclux plasmid (cultivated in lb broth supplemented with , % d-glucose and g/ml chloramphenicol) emit light upon the specific transcriptional induction with quaternary ammonium compounds, widely used as surface disinfectants and in many over-the-counter drugs . however, the incubation of the same cells with an inhibitor of the dihydrofolate reductase enzyme, trimethoprim (sigma-aldrich chemie, steinheim, germany), enhanced in a dosedependant manner the light emission observed upon the induction with the optimal concentration ( g/ml) of benzalkonium chloride (bc). the extent of this increase in luminescence varied from - % to more than %, according to the trimethoprim concentration and to the measurement time. interestingly, when the same plasmid is carried by escherichia coli xl cells, the lux operon is expressed constitutively (since the qacr regulatory region is not functional in xl cells) and at much higher levels than in induced rn cells. also in this experimental system the incubation with trimethoprim results in a dramatic increase of the luminescent signal from the cultures. the explanation for these observations can be found in the molecular mode of action of trimethoprim. the inhibition of dihydrofolate reductase, indeed, directly leads to the accumulation of its substrates, among which is nadph, deeply entangled in the biochemical network of reactions centred on the light emission from the lux operon. nadph provides the reducing power to restore the reduced flavin mononucleotide pool and it is as well involved in fig. . xl /pqaclux growing cells were incubated with the following concentration of trimethoprim: g/ml (triangles), g/ml (circles) and g/ml (rumbles). water was administered to control cultures (squares). light emission and absorbance measurements were performed as previously described for rn cells. filled symbols refers to the absorbance values (left side y-axis); open symbols to the normalized count per seconds (right side y-axis). the diverse level of growth observed for rn and xl cells can be accounted by the different antimicrobial activity exerted by trimethoprim towards gram− and gram+ cells and by the lower activity of the promoter which in pqaclux plasmid drives the transcription of the selection marker (chloramphenicol acetyl transferase). the production of the long-chain aldehyde, both substrates of the lux␣␤ heterodimer (wilson and hastings, ) . in conclusion, here we demonstrate that the use of light emission from the bacterial luciferase as a transcriptional reporter has to be very carefully controlled, since some molecules (here trimethoprim) could mimic to some extent a specific promoter activation by impairing the delicate intracellular biochemical equilibrium. on the reverse side of the coin, fig. . simplified scheme depicting the bacterial folate metabolic pathway. only part of the reactions and compounds are reported. trimethoprim inhibits the nadph-dependant reduction of dihydrofolic acid into tetrahydrofolic acid catalyzed by dihydrofolate reductase. this results in the accumulation of both substrates, which become available for other reactions, and in the depletion of tetrahydrofolate, the major c carrier in the synthesis of purines, thymidine, glycine, methionine, and pantothenate in bacteria. for antimicrobial purposes trimethoprim is often associated with sulfonamides, with which it displays a synergistic activity (since they act on the same pathway but at an earlier reaction) (scholar and pratt, ) . however, it has to be noted that the lux reporter system could be used in many different in vivo experimental setups, where the change in the intracellular concentration of nadph, atp or other metabolites would be sensibly detected. we have carried out the construction of five pichia pastoris strains harboring in their respective genome three different growth hormones cdna's ( and kda human growth hormones and bovine growth hormone), a shrimp (litopenaeus vannamei) trypsinogen cdna and a bacterial (bacillus subtilis) phytase gene. in all the cases, the same kind of expression vector and the same transformation technique were used. each dna was fused in frame to saccharomyces cerevisiae alpha-factor secretion signal to lead the secretion of the foreign protein into the culture medium. the induction of each recombinant strain, all with his + and mut + phenotype, was carried out in shake flaks using methanol buffered minimal medium (bmm) and growth rates on methanol determined. production level of secreted proteins was evaluated by protein analysis by sds-page. furthermore, the expression with three of the constructed strains was performed in a -l bioreactor and the level of secreted protein determined in each case. both human growth hormones (hghs) were secreted into the culture medium with a high degree of purity obtaining up to % of hghs of total proteins in crude fermentation medium and - mg/l of hghs. neither bovine growth hormone, shrimp trypsinogen or bacterial phytase were detected in methanol induced cultures of the respective strains, in spite of the same culture conditions were used for all p. pastoris strains. the growth rates on methanol were different between some strains. in fermentor cultures the hghs production were increased and shrimp trypsinogen was produced and secreted into the culture medium after improving the culture conditions. bovine growth hormone was detected only when the culture conditions were modified. the protein production level for each strain was affected by the proprieties of each recombinant protein produced. competitive advantages of the diagnostic method for invasive amoebiasis using preserved antigenic extracts without using enzymatic inhibitors m.s. flores , * , e. tamez we have patented a method to diagnose invasive amoebiasis using a novel assay that preserves antigenicity of extracts with high protease content without using enzymatic inhibitors (ic:mc). the available tests for serologic diagnosis of invasive amoebiasis like elisa and indirect haemaglutination (iha) do not have consistent results in endemic zones. here we show the advantages of the assay and the validation of this diagnostic test for invasive amoebiasis. we demonstrated the reduction of proteolytic activity of ic:mc compared with the proteolytic activity of crude extract and crude extract with enzymatic inhibitors using assays. we displayed the ic:mc sds-page pattern and the western blot (wb) pattern useful for diagnosis. to search the clinical utility of this test we examined the wb obtained with sera from patients with different liver diseases; patients had invasive amoebiasis and patients had other liver diseases. the results were compared with those of iha test. also we have tested the accuracy of wb using sera from people with multiple intestinal parasites, like giardia lamblia, hymenolepis nana, blastocystis hominis, entamoeba coli, etc. the sensibility of the wb using the preserved amoebic antigens was %, specificity was %, positive predictive value was %, negative predictive value was % and accuracy was %. the wb did not exhibit cross reactions with sera from persons with intestinal parasites. our test was better than the (iha) test commonly used in endemic zones. these results show the improvement of using the preserved amoebic antigens in diagnostic tests. also they prove the diagnostic accuracy of our new wb test. antibacterial and antifungal activity of heracleum sphondylium subsp. artvinense yasemin kaçar , sema tan , aysun ergene , perihan güler , semra mirici , ergin hamzaoglu , ahmet duran , sinem yildirim : mersin university, faculty of science and literature, department of biology, mersin, turkey; kırıkkale university, faculty of science and literature, department of biology, yahsihan-kırıkkale, turkey; akdeniz university, faculty of education, department of biology education, antalya, turkey; selcuk university, faculty of education, department of biology education, konya, turkey turkey is covered yearly with a huge number of plant species. about species are condenced on the region that between asia and europe. many plant species have been used in folkloric medicine to treat various ailments. heracleum l (apiaceae) is include over than species on the world. this variety is represent with species in turkey that seven species are endemic. heracleum sphondylium subsp. artvinense is endemic species for turkey. ethanolic and aquous extract of heracleum sphondylium subsp. artvinense were investigated for their antimicrobial activities against six bacterial species (e. feacalis, e. coli, s. aureus, p. aeruginosa, l. monocytogenes, shigella) and two yeast (c. albicans, c. krusei). both ethanolic and aqueous extract of heracleum sphondylium subsp. artvinense showed antimicrobial activity against the gram negative bacterium (shigella) and gave the best activity against c.albicans. to develop artificial vectors allowing nucleic acid to transfect into mammalian cells are crucial for extending gene therapy. synthetic vectors based on lipid molecules are particularly attractive because of their potential safety. however, the low encapsulation efficiency of nucleic acid is one of the problem to be solved. recent advances in dna-lipid complex have improved this drawback, and now some lipid molecules are used as transfection agents. in particular, cationic lipids interact with negatively-charged cell surfaces and nucleic acids. the former interaction results in delivery of the nucleic acids directly across the cell membrane. on the other hand, the latter interaction improves the efficiency of nucleic acids entrapment. in this study, we developed a preparation method of "nanovesicle" containing nucleic acids by using reverse micellar solubilization. since dna interacts spontaneously with cationic amphiphiles, complete extraction of the dna molecules into an organic phase using dimethyl distearyl ammonium bromide ( c ab), an oil-soluble cationic surfactant, is achieved. the re-encapsulation of the reverse micellar droplets solubilizing dna by water-soluble surfactants facilitates the formation of nanovesicles. a high salt concentration at the re-encapsulation step promotes the production of nanovesicles containing dna molecules. eventually, more than % of dna was encapsulated in this nanovesicles under the condition of m nacl and % (w/v) cetyltridecyl ammonium bromide (ctab). in addition, the nanovesicles prepared at • c was smaller than that prepared at room temperature. the resultant c ab/ctab nanovesicle was ca. nm. asymmetric and chemically-modifiable vesicle surface are effective to design gene delivery system. moreover, such a small dna (or rna) carrier has a potential for novel gene therapy application from skin. the key players in clinically important inflammatory diseases, endothelial cells and leukocytes, communicate through membranebound cell adhesion molecules (cam's). obvious strategies for therapeutic intervention include specific means to affect the expression of cam's on the cells involved. rna-interference (rnai) is a well known means to achieve specific gene-inhibition. for this study, two cam's were chosen, namely vascular cell adhesion molecule- (vcam- ) as a target for inhibition, and intercellular adhesion molecule- (icam- ) as a non-target reference. we designed short interfering rna (sirna) oligos for vcam- in order to selectively inhibit the expression of this cam in cultured human vascular endothelial cells (huvec). real-time rt-pcr showed an % down-regulation of vcam- mrna while the expression of icam- remained unaffected. neither cam was affected by non-specific sirna. in order to further substantiate the potential use of sirna for therapeutic purposes, we have set out to investigate two things: ( ) does vcam- -specific sirna affect the amount of vcam- on the surface of huvec? ( ) is adhesion between leukocytes and endothelial cells affected by vcam- -specific sirna? the manufacturing of plasmid dna (pdna) is crucial to obtain a consistent product for gene therapy applications. although flowsheets for pdna production are established on the basis of experience, simulation tools provide a valuable help for evaluating alternatives. a process designed to produce kg pdna/year is analysed with the superpro designer tool. the target pdna is amplified in escherichia coli. after harvest, alkaline lysis is used to disrupt cells and release pdna and impurities. precipitations with isopropanol and ammonium sulphate are performed to concentrate/pre-purify pdna prior to hydrophobic interaction chromatography. experimental data is used as input for simulation. inventory analysis identified water, yeast extract, tryptone, isopropanol and ammonium sulphate as the major raw materials. major raw material costs ( %) are related to fermentation components. economic analysis indicates a unit production cost of $ /g pdna. for a selling price of $ /g, the payback time was . years and the roi was . %. an environmental analysis highlighted the replacement of the isopropanol precipitation for a microfiltration step as a benefit which would: (i) reduce the cost of raw materials ( . %), (ii) reduce the environmental impact associated with isopropanol ( %) and (iii) reduce costs associated with the treatment/disposal of liquid waste ( . %). preparation of chitosan microspheres for controlled release of somatotropin s. simsek , j. introduction: proteins and peptides have received extensive interest for their therapeutic applications in clinical applications. in order to achieve high administration efficacy of proteins, polymeric particulate carriers have been developed as an effective way to control the drug release profile and to protect the protein molecules from degradation. somatotropin also known growth hormone is a protein hormone of about amino acids. growth hormone is of considerable interest as a drug used in both humans and animals. chitosan a natural linear biopolyaminosaccharide is obtained by alkaline deacetylation of chitin. properties such as biodegradability, low toxicity and good biocompatibility make it suitable for use in biomedical and pharmaceutical formulations. the aim of this study was to prepare chitosan microspheres containing somatotropin and to investigate these microsphere formulations in-vitro release properties. methods: somatotropin-chitosan microspheres were prepared as follows: chitosan was dissolved in acidic solution ( %) containing polysorbate . sodium sulphate solution ( % w/v) containing somatotropin was added into the chitosan solution and mixed rpm for a hour. resulting suspension was centrifuged , rpm min at • c. the formed microspheres were freeze-dried and sieved. in-vitro release studies were performed in ph . phosphate buffer and time interval samples were removed and analysed by bradford protein assay method. results: microspheres were obtained by using chitosan. protein encapsulation efficiency was between and %. average particle size of microspheres was between and m. during to in-vitro release studies burst effect was observed with chitosan microspheres. for decreasing the burst effect gluteraldehit, betacyclodextrin and poly ethylene oxide were added to formulations. conclusion: according to our results modified chitosan microspheres are promising vehicles for controlled release somatotropine delivery. cancer immunotherapy using hyperthermia with magnetic nanoparticles and dendritic cells k. tanaka, a. ito, t. kobayashi, t. kawamura, s. shimada, k. matsumoto, t. saida, h. honda department of biotechnology, school of engineering, nagoya university, nagoya, aichi - , japan. e-mail: h d@mbox.nagoyau.ac.jp (k. tanaka) our hyperthermia system utilizes magnetic nanoparticle covered with lipid layer including cationic lipid (magnetite cationic liposomes, mcls) as a heating mediator and necrotic cell death is induced by means of locally generating heat. in this process, heat shock proteins (hsps) are strongly induced and released. dendritic cells (dcs) are potent antigen-presenting cells (apcs) that play a pivotal role in regulating immune responses in cancer, which is in carrying antigens to apcs and in the maturation of dcs by acting as a danger signal. in the present study, we investigated the therapeutic effects of dc therapy combined with mcl-induced hyperthermia on b melanoma. in an in vitro study, when immature dcs were pulsed with b cells heated at • c for min, mhc class i/ii, costimulatory molecules cd /cd , and chemokine receptor ccr in the dcs were up-regulated, thus resulting in dc maturation. c bl/ mice bearing a b melanoma nodule were subjected to combination therapy using hyperthermia and dc immunotherapy. mice were divided into four groups: group i (control), group ii (hyperthermia), group iii (dc therapy), group iv (hyperthermia + dc therapy). complete regression of tumors was observed in % of mice in group iv, while no tumor regression was seen among mice in the other groups. increased ctl and nk cell activity was observed on in vitro cytotoxicity assay using splenocytes in the cured mice treated with combination therapy, and the cured mice rejected a second challenge of b melanoma cells. this study has important implications for the application of mcl-induced hyperthermia plus dc therapy in patients with advanced malignancies as a novel cancer therapy. fermentation of a marine bacterium for the production of cytotoxic compounds vicky webb , els maas , eiichi akaho , hiroto kambara , debbie hulston , anna kilimnik : marine biotechnology, national institute for water and atmospheric research ltd, kilbirnie, wellington new zealand; faculty of pharmaceutical sciences, kobe gakuin university, kobe - , japan marine bacteria are a potential source of novel compounds for the pharmaceutical industry. new zealand marine bacteria were isolated from a variety of sources and initially bacterial supernatants were screened using a cell based mtt cytotoxic assay. two bacteria were chosen for further fermentations in different media to optimise cytotoxic compound production. the media used were selected for their diverse ingredients. they were either carbon rich, nitrogen rich, starch rich or a basic seawater medium. the fermentations were extracted using ethyl acetate and methanol prior to assaying. the results showed that cytotoxic compound production was enhanced ten fold in the starch rich medium compared to the other media. the levels of cytoxicity appeared to be depended on the cell line used, with the epithelial lung carcinoma cell line a being more sensitive to the cytotoxic compounds than the human fibroblast cell line mrc- . isolation and identification of marine bacteria from deep-sea sediments els maas , cara brosnahan , vicky webb , helen neil , phil sutton : marine biotechnology, national institute for water and atmospheric research ltd., kilbirnie, wellington new zealand; oceanography, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand polystyrene micro plate activated by utilizing a common co- gamma source with a crotonic acid. the well surface have been studied in term of optical quality, protein (h-igg) binding capacity and stability. a significantly enhanced total capacity and strength of binding to grafted surfaces was demonstrated as compared to passive adsorption of the proteins to untreated surfaces.the majority routine laboratory tests for the measurement of rheumatoid factor (rf) are semi quantitative and in order to achieve accurate, sensitive and specific rf assay, we are developed enzyme linked immuno sorbent assay (elisa) for human igm-rf kit. stable and reproducible binding of antigen (human-igg) to well of micro titer plate is a prerequisite for rf-elisa kit. the principle of the assay was that the antibodies in serum of patients with rheumatoid arthritis (ra), commonly rheumatoid factor (rf) are directed to the fc part of human igg. in most cases rf belong to the igm class, the well of micro titer plate are coated with antigen (h-igm), and antibodies (h-igg) binding to immobilized antigen is detected by adding enzyme conjugate (anti human-igm-hrp-enzyme) to the wells, substrate was used for color reaction.the performance characteristics of the assay was, the coefficient of variation (c.v) of intra and inter assay was . % and . % respectively, the linearity is ranging from to %, and the recovery for three different sera is ranging from to %. the data presented in this paper indicated that the activation of polystyrene micro titer plate by the gamma rays could be use for preparation of igm-rf kit and may be others immunoassay techniques. overcoming the nuclease barrier to gene expression during trafficking of plasmid dna vectors a.r. azzoni , a. tavares , g.a. monteiro , d.m.f. prazeres : centro de engenharia biológica e química (cebq), instituto superior técnico, - lisboa, portugal; instituto gulbenkian de ciência, rua da quinta grande , - oeiras, portugal. e-mail: azzoni@ist.utl.pt (a.r. azzoni) inefficient nuclear delivery of plasmid dna (pdna) vectors is thought to be a bottleneck to gene transfer in gene therapy and dna vaccination utilizing non-viral delivery systems. one of the main barriers found by pdna vectors during trafficking to the nucleus is degradation by a population of endo/exo-nucleases. this barrier may be partially circumvented by shielding the pdna from the nucleaserich cell environment with adjuvants or by using nuclease inhibitors. a different approach that has been studied at the cebq is the generation of pdna vectors that are more resistant to nuclease action a priori. in this work, the nuclease barriers to gene expression are being studied aiming at the generation of pdna with improved resistance to nucleases and thus higher transfection efficiency. by engineering the plasmid labile sequences, new plasmid vectors with an improved resistance to physical-chemical and biological degradation are being generated. this was indicated by an extended half-life of the supercoiled isoforms during storage and when the plasmids were exposed to nucleases present at eukaryotic cell lysates and mice plasma. the intracellular trafficking of the new plasmid vectors through the cytosol of mammalian cells was then assessed by fluorescence in situ hybridisation (fish) and the expression of the reporter protein (egfp) was detected by fluorescence microscopy. identification and evaluation of antibacterial phytochemicals of fishbone fern (nephrolepis cordifolia) rikhia chakraborty , , promod kumar verma : department of cancer biology, lerner research institute, euclid avenue cleveland, oh , usa, department of biotechnology, guru nanak dev university, amritsar, punjab , india. e-mail: riar @rediffmail.com (r. chakraborty) in this study, different aqueous and organic extracts from the fern, nephrolepis cordifolia, were used for screening tests for antibacterial effects. protein and lipid extracts were first tested for antibacterial activity. subsequently, crude extracts of leaves, roots, and stems were prepared in methanol, ethanol, chloroform, hexane, petroleum ether, diethyl ether, and water using optimized standard protocols. each fraction was tested for anti-microbial effect through agar well-diffusion assay, and paper disc method. the antibacterial spectrum against which the fern is active was thus determined. dosagedetermination for optimum activity was also determined for each of the extracts. bacillus and staphylococcus were used as the indicator test-organisms. the results were very encouraging; being effective even at the th day, thus showing that the antibacterial properties were not due to any changes in external factors and physiological effects. ethanol, methanol and chloroform extracts from the subaerial portions had strong anti-microbial properties. agar-well diffusion assay and paper-disc diffusion assay done for different solvent fractions were giving comparable results. . mg was adequate for maximum effect against b. circulans, s. aureus, s. epididermis, and streptococcus sp. . mg was adequate as effective dosage for kleb-siella pneumoniae. mg was required for mycobacterium bovis, e. coli. pseudomonas was not showing any susceptibility. given the broad spectrum of activity, especially towards the gram-negative bacteria, nephrolepis cordifolia is definitely a promising plant having pharmacological importance. for a full interpretation of the present results further investigations are necessary to elucidate the different physical and chemical parameters of the active principles and also to determine the mechanism of action. the present work highlights n. cordifolia as a plant having a broad spectrum of antimicrobial activity, a phenomenon very rarely observed in the plant kingdom. bacteria (escherichia, salmonella, proteus, staphylococci and bacillus) were isolated from hospital soil using selective enrichment and growth on selective and differential medium, viz. macconkey's agar, clyed medium and baird parkers medium. confirmation and species identification was carried out by biochemical and serological tests. from these isolates, three different pathogens were used to study multiple antibiotic resistances. e. coli bj showed resistance to ampicillin, streptomycin and cefurixime. s. typhi and s. aureus showed resistance to ampicillin and cefuroxime. assay using octadisc using e. coli and s. typhi showed a broader resistance pattern to antibiotics including amoxicillin, clavulanic acid, cephalexin, chloramphenicol, ciprofloxacin, and cotrimoxazole. the r plasmid profile was studied to understand the mechanism of drug resistance. to combat the problem of drug resistance, a strategy of combined antibiotic response of cultures were studied. such a synergistic combination would possibly have the effect of overcoming multiple antibiotic resistances. for e.g. kanamycin resistance strains were inhibited in presence of kanamycin and cefotaxime. further, the bactericidal activities of antimicrobials in honey and garlic were also tested. the mic of honey was observed to be %, while that of garlic was between . and %. honey and garlic were also found to inhibit the growth of organisms in the presence of antibiotics. kanamycinresistant e. coli was unable to grow in presence of kanamycin and . % garlic. these traditional agents, long used in ayurvedic system of medicine in india, could be further explored for potent antimicrobial properties. hepatitis b virus (hbv) infection is s global health problem. assays for hbv antigens and antibodies are widely available and standardized. extremely sensitive qualitative pcr kits are also available for detection of hbv in serum. hbv pcr kit may be useful in assessment of occult hepatitis b in hbcab positive alone subjects and carriers. a positive pcr results show presence of virus articles in serum without considering serologic results. but there are differences in efficiency of hbv dna amplification kits. in this study we compare two commercially available hbv pcr kits for evaluation viremia of hbsag positive carriers and hbcab alone positive subjects. material and methods: of the randomly selected subjects serologically examined for hbv, and were positive for hbsag and hbcab alone respectively. both later groups were tested for hbv dna by two commercial kits, hbv pcr detection kit (cinnagen, iran) and hbv pcr test (pazhohesh azma, iran). dna extraction kits recommended by each manufacturer were used for hbv dna extraction. amplicons in both kits were a highly overlapped fragment in conserved sequence of viral genome. results: of the hbsag positive carriers, hbv dna was detected in . and . % using kit and kit respectively. only . % were positive in both kits. in hbcab positive subjects (n = ), . % were positive by kit and all samples were negative when tested by kit . there was a significant difference between two kits. sensitivity of kit and kit were . and . %, respectively. overall, kit increased the detection rate of hbv dna by . %. discussion: our study show there is a significant variation between these two commercial kits especially in hbcab positive subjects that the copy of virus is very low. from these results, it can be concluded that the unstandardized kits have not compatible results, and pcr test interpretation should be done with great care. . the antibacterial activity of the extracts was evaluated based on the inhibition zone using plate diffusion method. most of the extracts were active against both gram positive and gram-negative bacteria, but pseudomonas aerugenosa, bacillus subtilis and sarcina marcescens, were more susceptible to almost all the extracts. finally, further research will be done to elucidate the nature of the active compound and investigate for peptides by using protein gel immobilization bioassay. short interfering rna delivery and gene silencing using polymeric nanocarrier systems k.a. howard , x. liu , d. oupicky , f. besenbacher , j. kjems : inano, university of aarhus, denmark, department of pharmaceutical sciences, wayne state university, detroit, usa the effectiveness of a drug is determined by the ability to migrate through the body and reach target sites in therapeutically relevant levels. nanocarriers for delivery of bioactive agents are being developed at inano to maximise drug payload at target sites. the inclusion of "biological triggers" into the nanocarrier design is used for modulation of cellular nucleic acid trafficking and increased target interaction. chitosan and peptide-based polymers were used to formulate nanocarriers in the size range - nm containing small interfering rnas (sirnas) for gene silencing applications. page analysis showed the structural integrity of the sirna was maintained during particle formation. in systems composed of bioresponsive polymers, nanocarrier disassembly and sirna release under cellular conditions were shown, using atomic force microscopy. the time course for sirna uptake into nih cells was visualised using confocal microscopy. in addition, sirna localisation within cells could be modulated by the composition of the polymer used. the ability of the nanocarrier system to mediate gene expression was investigated in a cell line stably expressing enhanced green fluorescent protein (egfp). furthermore, the various delivery systems were tested in a mouse model stably expressing the egfp protein using both nasal and intravenous delivery routes. the use of animals as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human organ donors. however, the presence of xenoreactive antibodies in humans directed against swine gal antigen present on the surface of xenograft donor cells leads to the complement activation and immediate xenograft rejection as a consequence of hyperacute immunological reaction. the graft of genetically modified organ of a swine depleted of enzyme ␣ , -galactosyltransferase that is responsible for gal antigen origin, would be tolerated with simultaneous administration of medicines decreasing other less severe immunological reactions. to prevent hyperacute rejection it is also possible to modify swine genome by human genes controlling enzymatic cascade of complement or modifying the set of donor's cell surface proteins. for this purpose genetic constructs containing inactivated ␣ , -galactosyltransferase gene, human cd , cd and cd genes controlling complement activation and human genes encoding ␣ , -fucosyltransferase and ␣-galactosidase enzymes modifying cell surface proteins were prepared. these genetic constructs were transfected into the pig foetal fibroblast using lipofection method. after selection, molecular and cytogenetic characteristic of cells with transgene integrated into the host genome were performed. introduction: protease a( a-pro) of coxsackievirus b (cvb ) plays major role in viral replication. in case of infection, viral proteins are being synthesized from viral mrna using host biosynthesis machinery. a-pro of virus, after being synthesized, exhibit two critical functions, cleavage of viral proteins and breaking eif g (eukaryotic initiation factor g-formerly called p ) which leads to host cell translational system shot-off. the enzyme plays essential role in viral replication and cellular damage. to understand pathogenicity of infection and also developing potent and selective inhibitors against picornavirus infection, it is necessary to prepare pure apro enzyme. in this study an expression system with efficient and high yields was obtained. methods: cdna of apro was synthesized using in vitro infection of permissive host through reverse transcription process and was cloned in pet b(+) and since a-pro is a toxic product, naturally before induction its expression will act on the host and damage the cells. for this different hosts were checked and finally, blr(de ) plyss which carries an extra-plasmid for lysozyme expression that minimizes unwanted target protein production (leakage) was selected. on the other hand for biological activity assay, polyclonal antibodies against antigenic sites of p was prepared by synthesizing small peptides, corresponding to antigenic site of p coupling to klh and injecting subcutanously to rabbit. then, the enzyme and its substrate (hela cells lysate that contain p ) were incubated together for different times intervals. results: the recombinant product was confirmed by sds polyacrylamide gel electrophoresis and immunoblot analysis. also p cleavage by apro was assessed by sds-page and western blot analysis. cleavage of p by r a-pro was prominent after h. so recombinat a-pro with good activity was prepared. application of bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system on production of glycoprotein in larvae of silkworm ayano kageshima, tatsuya kato, misun kwon, enoch y. park department of applied biological chemistry, shizuoka university, ohya , suruga-ku, shizuoka - , japan bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system was applied on production of glycoprotein in larvae of silkworm. the bacmid system of autographa californica nuclear polyhedrosis virus (acnpv) has already been established and widely used. since the acnpv does not have a potential to infect silkworm we developed the first practical bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system directly applicable for the protein expression of silkworm. by using this system, the green fluorescence protein and glycoprotein, human ␤ , -n-acetylglucosaminyltransferase were successfully expressed in silkworm larvae not only by infection of its recombinant virus but also by direct injection of its bacmid dna. three different kinds of signal sequences were tested for the secretion of glycoprotein into hemolymph of silkworm. signal peptides of prophenoloxidase-activating enzyme and bombyxin: insulin-like brain secretory peptide showed the highest secretion ratio, % of total expressed ␤ , -n-acetylglucosaminyltransferase was secreted into hemolymph of silkworm. using bacmid system mu/ml of ␤ , -n-acetylglucosaminyltransferase was expressed in hemolymph of silkworm, which was -three times higher than that of insect cell. silkworm is one of the most attractive hosts for large-scale productions of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. this method provides the rapid protein production in silkworm, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses. we have established an efficient system for foreign gene expression in lily (lilium longiflorum) pollen in a transient mode. pollen was transformed using agrobacterium via vacuum filtration for min. the pollen germinated for h was analyzed to confirm its foreign gene expression in molecular analysis. mouse fed the transgenic pollen culture for weeks showed immune reaction specific for pollen-derived recombinant protein. and the igg level was highly elevated by one time boosting injection afterwards. the lily pollen system may be suggested as a novel type of biofactory for producing edible vaccine with rapidity. anti-apoptosis engineering with the kc gene obtained from silkworm shin sik choi, won jong rhee, tai hyun park school of chemical and biological eng., seoul national university, seoul - , korea the chinese hamster ovary (cho) cell line producing recombinant human erythropoietin (epo) was manipulated to express the kc gene, which was originally obtained from a silkworm. the expression of kc inhibited serum deprivation-induced apoptosis and increased the cell density and epo expression level per unit cell by five-and two-folds, respectively. an increase in these two factors resulted in a -fold increase in the volumetric productivity of epo. compared with the controls, the oligosaccharide structures of the epo synthesized by the cells expressing kc showed greater homogeneity. the terminal sialylation of the glycans of epo were promoted by the expression of kc . the positive effects of kc expression on the cell viability and productivity were attributable to the stable maintenance of the mitochondrial activity. these results demonstrate that the cho cell line genetically engineered with the kc gene has a great potential for use in the production of therapeutic proteins. evaluation of fucoidan-chitosan hydrogels on superficial dermal burn healing in rabbit: an in vivo study a.d. sezer , f. hatipoglu , z. ogurtan , a.l. baş , j. introduction: healing of dermal wounds with macromolecular agents such as natural polymers is one of the research areas of the pharmaceutical biotechnology. fucoidan is a sulphated polysaccharide which is commonly obtained from seaweeds. although a great number of studies on the different pharmacological properties of fucoidan are present, there is very limited information on the fucoidan-based system used in dermal burns. the aim of this study was to prepare chitosan hydrogel containing fucoidan and to investigate this hydrogel formulation for treatment of dermal burns on rabbits. methods: fucoidan-chitosan hydrogels were prepared as follows: the polymers were dissolved in acidic solution and sonicated for removing the air-bubbles then the gels were stored at + • c for in vivo studies. seven adult male new zealand white rabbits (mean weight, . ± . kg) were used for the evaluation of the gels on superficial dermal burns. the back of the rabbits were depilated and sedated. the wounds were made by circular stamp aluminium caps ( . cm ) at • c. four wounds were formed for each rabbit; (a) was treated with fucoidan-chitosan gel, (b) was treated with fucoidan solution, (c) was treated with chitosan gel (without fucoidan) and (d) as a negative control group. biopsy samples were taken at , and st days at the beginning of the study and each wound site was macroscopically observed and evaluated histopathologically. results: oedema was not observed in all groups after days treatment except controls. after days treatment, fibroplasia and scar were observed on wounds treated with fucoidan-chitosan gel and fucoidan solution. the best regenerate dermal papillary formation and the fastest closure of wounds were observed in group a after days treatment. the wound epithel elongation and thickness values were measured; a ( and m), b ( and, m), c ( and, m), d ( and, m) at the end of the study. conclusion: re-epithelization and contraction of the wound area which was treated with fucoidan-chitosan hydrogel were faster than the other groups. the fucoidan-chitosan hydrogel formulations can be suitable for the treatment of dermal burns. aim: to analyze the neutralizing -related activity of antibodies against e region of hcv, specific polyclonal antibody was raised by immunized rabbits with synthetic peptide that had been derived from e region of hcv with the amino acid sequence e antibody [ghrmawdmm). materials and methods: hyper-immune hcv e antibodies were incubated over night at • c with serum samples from patients positive for hcv rna, with different viral load, ranged - million copes/ml, then incubated min to hepg cells. rt-pcr and flow cytometry were used to study the inhibition binding and entry effect of e antibody. direct immunostaining of e antibody conjugated with fitic and flow cytometry analysis showed reduced the mean fluorescence intensity in the samples pre-incubated with e antibody compared with samples without e . of positive serum samples, ( %) samples showed completely inhibition of infectivity as detected by rt-pcr. conclusion: in house produced e antibody, blocks binding and entry of hcv virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. isolation of these antibodies that block virus binding and entry will be useful in providing potential therapeutic reagents and for vaccine development. tumor necrosis factor beta (tnf␤) is a pleiotropic cytokine mediating its activity through the same receptors as the structurally related tnf␣. shortening of the n-terminal part has been reported to enhance its cytotoxic activity, with the explanation that removal of the flexible n-terminus reduces steric interferences in the receptor binding process. for studies of relationship between the n-terminal protein structure and physicochemical properties, three different forms mettnf␤, his tnf␤ and n tnf␤ were expressed in e. coli. high solubility of n tnf␤ was expected, however, both analogs his tnf␤ and n tnf␤ were predominantly obtained in the form of inclusion bodies (ibs). on the other hand, mettnf␤ was equally distributed between the soluble and insoluble fraction. most probably, the composition of the n-terminal part, such as the exposure of hydrophobic residues in the case of n tnf␤, or a mildly hydrophobic stretch of seven histidines appended to the natural hydrophobic n-terminus in the case of his tnf␤, lead to incorrect folding and force aggregate formation. solubilization of ibs was performed under native and denaturing conditions using various concentrations of nls, urea and gndhcl. mettnf␤ ibs were easily dissolved with . % nls, while his tnf␤ and n tnf␤ demanded denaturing conditions. structural differences in the nterminal part of various tnf␤ proteins were also reflected in protein refolding characteristics and chromatographic behavior. dilution, ultrafiltration and dialysis were used for refolding, and imac was chosen as the main chromatographic step. our results suggest that not only the length of the n-terminal part of tnf␤ but also the composition and exposure of certain amino acid residues affect its physicochemical properties. enzymatic modification of sphingomyelin long zhang, lars hellgren, xuebing xu biocentrum-dtu. e-mail: lz@biocentrum.dtu.dk (l. zhang) due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potential in cosmetic and pharmaceuticals such as hair and skin care products. currently, chemical synthesis of ceramide is a costly process, and developments of alternative cost-efficient, high yield production methods are of great interest. in the present study, the potential of producing ceramide through enzymatic hydrolysis of sphingomyelin (sm) have been studied. sm is a ubiquitous membrane-lipid and rich in dairy products or by-products. in present study, we have optimized the production of ceramide from sm using phospholipase c from clostridium perfringens. water and enzyme amount had the biggest influence on sm hydrolysis in the system. botulinum neurotoxins constitute a family of bacterial toxins for botulism syndrome in human. the toxins bind with high affinity to nerve cells where they cause a complete inhibition and release of neurotransmitters and thereby produce flaccid paralysis. in this work we have reported isolation of the binding domain of type e neurotoxin by pcr and expressed in a proper expression vector. the output of this investigation can be used as a tool to study the mechanism of binding of holotoxins and also can be useful to study the antibody production against botulism syndrome. the synaptobrevin (vamp ) a protein which play a key role in the fusion and exocytosis of the vesicle of mammalian nerves terminals this protein is substrate different serotypes of botulinum neurotoxins. light chain of clostridium botulinum type b, d, f and g cleave end of neurons. due to intraction of clostridium botulinum neurotoxin with vamp , this protein can be used as one of the toolsin the detection of poisings cause by this bacteria in the clinical laboratory using the enzyme with proof reading activity the above gene amplified by pcr technique for the production of the recombinant protein, the prokaryotic expression vectors (pet system) was used. a recombinant protein developed on poly acrylamide gel analyzed by western blotting and eliza. most children with down syndrome (ds) are born to younger mothers (< years). recent reports linking down syndrome (ds) to maternal polymorphisms at the methylenetetrahydrofolate reductase (mthfr) gene locus have generated great interest among investigators in the field. in this study, forty mothers with their affected outcomes and control mothers were included. all mothers were subjected to complete medical and nutritional history with special emphasis on folate intake through food or oral supplementation. estimation of blood homocysteine level was done. also we examined the two polymorphisms in genes encoding the folate metabolizing enzyme methylenetetrahydrofolate reductase (mthfr), namely, c > t and a > c. folic acid intake from food and from vitamin supplements was significantly low (below the recommended daily allowance) in the group of mothers with ds children compared to control mothers (p < . ) using student t-test. blood homocysteine was normal in both control and ds mothers. the prevalence of the two polymorphisms, namely, c > t and a > c in mothers of ds children (case mothers) (n = ) was compared with controls (n = ). frequencies of mthfr genotypes (cc, ct, and tt) at position demonstrated no difference between the case and control groups. genotype frequencies of mthfr at position a (aa, ac, and cc) were different among the case and control mothers. we here report the first study on a possible relation between ds with mthfr a > c genotypes in egypt. our results showed that mthfr a > c polymorphism is a remarkable genetic entity among egyptian females with d.s. children. sufficient folate intake and supplementation is an important preventive strategy in overcoming the risk of nondisjunction. homologous recombination (hr) is the mechanism that permits the creation of genetically engineered strains through gene targeting. in order to further develop gene targeting techniques, notably for higher eukaryotes such as filamentous fungi, it is of crucial importance to fully understand the molecular mechanisms behind mitotic hr. in saccharomyces cerevisiae, a dna double strand break (dsb) is an essential intermediate in meiotic hr. however, as hr occurs at a low rate in mitotic cells, it has been difficult to determine the nature of the event(s) that triggers it. rad is a key protein involved in hr and is evolutionarily conserved from yeast to human. to shed light on the molecular events in hr, we have generated a large collection of defined rad mutant strains in the yeast s.cerevisiae. a screen of these mutants led to the identification of strains that fail to repair dna dsbs, yet are proficient for homologous recombination. this result strongly suggests that dsbs may not be the major cause of spontaneous mitotic hr and gives new perspectives in respect to novel potential gene targeting substrates. we have analyzed these separation of function mutants in a variety of new assays to obtain a more detailed understanding of their controversial phenotype. our latest results will be presented. the outcome of interferone plus ribavirine treatment of hepatitis c virus (hcv) genotype is unfortunately poor. development of alternative therapy for this genotype is of a paramount importance. inhibition of hcv gene expression in vitro by the use of antisense phosphorothioate oligodeoxynucleotides (s-odn) against internal ribosomal entry site (ires) elements were associated with favorable results. to assess s-odn activity, previous studies utilized viral subgenomic or full cdna fragments linked to reporter genes and transfected into adhered cells or in a cell free system. in the present study we utilized hepg cells infected with native hcv rna of genotype . the culture system presented herein was shown to support hcv replication on the following bases ( ) consistent detection of both plus and minus rna strands for weeks in cells and in fresh culture supernatent, ( ) ability of supernatent to infect naive hepg cells ( ) consistent expression of core and e envelope proteins in infected cells throughout the week culture. s-odns against aug translation start site (s-odn- , nt - ) of the viral polyprotein precursor and stem loop iiid within the ires region (s-odn , nt - ) were added to infected cells. intracellular viral replication was monitored by nested rt-pcr of plus and minus strands. the results of these experiments demonstrated that intracellular replication of hcv genotype was completely arrested after h in culture using either s-odn molecule (with more efficacy of s-odn than s-odn ) at concentrations as low as m. the inhibitory effect of s-odn appeared to be specific to hcv replication since equal levels of human glyceralehyde -phosphate dehydrogenase (gapdh) gene expression were noted pre and post supplementation of s-odns. in conclusion, the present study provides evidence antisense phosphorothioate oligonucleotides have potent inhibitory effect on genomic replication of hcv genotype , the most common type in egypt. a sensitive and specific pcr-elisa was developed to detect shigella dysentery in food. the assay was based on the incorporation of degoxigenin-labeled dutp and a biotin-labeled primer specific for shiga toxin genes during pcr amplification. the labeled pcr product were bound to streptoavidin-coated wells of a microtiter plat and detected by an elisa. the elisa detecting system was able to increase the sensitivity of the pcr assay by up to -fold, compared with a conventional gel electrophoresis. the detection limit of the pcr-elisa was . - cfu dependent upon shigella dysentery serotypes and genotypes of shigatoxin. the entire procedure took about h. isolation, cloning, expression and purification of snap- as a substrate of botulinum neurotoxin type a and e m.l. mossavi , f. ebrahimi , j. amani * , h. basiry , z. ahmadi : department of biology, faculty of science, imam hussein university, tehran, iran; department of biology, faculty of medicine, bageatallah university, tehran, iran. e-mail: kpjamani@ihu.ac.ir (j. amani) clostridial neurotoxin inhibit neurotransmitter relase by selective and specific intracellular proteolysis of synaptosomal associated protein of kda (snap- ); synaptobrevin/vamp- and syntaxin. snap- is one of the components that form docking complex in synaptic ends. this protein is substrate for botulinum neurotoxins type a and e. each of these toxin serotypes specifically cleave snap- in particular position and there by block docking and synaptic vesicle membrane fusion and finally prevent neurotransmitter exocytosis and transition of neurotic signals. in order to use the protein as a substrate for detection of different type of clostridium neurotoxin in vitro test, the protein was produced by recombinant technique. the dna encoding snap- was isolated from rat brain by pcr using the two primer the amplified fragment colonel into expression vector pet a.the expression protein was purified by affinity chromatography. confirm by different method the his tag fusion protein was digested with entrokinase. the present work deals with the preparation of pure alpha- antitrypsin (aat) protein from healthy subjects, which can be used in preparing its corresponding monospecific antibody in albino rabbits. this antibody was found very useful in the immuno-diagnosis of pulmonary emphysema. this study has been also concerned with the biochemical changes associated with aat deficiency in pulmonary diseases. to fulfill this work, a group of healthy blood donors was selected for separation of pure aat antigen from blood. the pure aat was used for the preparation of anti-aat, the purity and potency of antibody was checked by titration methods. the biochemical changes were studied in thirty subjects clinically divided into three groups including, control, heavy cigarette smokers with pulmonary emphysema, and non-smoking subjects with pulmonary emphysema. the activity of elastase and hydroxyproline (hp) level as a marker of elastin and collagen breakdown were assayed in bronchoalveolar lavage (bal) fluid. the activity of aat and to inhibit proteases as represented by tryptic inhibitory capacity (tic) was evaluated. serum ceruloplasmin, transferrin and iga level as well as thiobarbituric acid reactive substances (tbars) were estimated in all groups. our data revealed that aat and its tic showed very highly significant decreased levels in all patients with emphysema as compared to control, while the elastase activity and hp level in bal fluid were significantly increased in these patients. serum tbars was significantly increased in such patients associated with increasing level of both ceruloplasmin and transferrin. while, serum iga was significantly increased. furthermore, the biochemical changes were markedly changed in smokers with emphysema than non-smoking subjects. in conclusion, the preparation of anti-aat on the local level is very important where less expensive and less time consuming and can be useful in immuno-diagnosis and prognosis of pulmonary diseases. a new method combined with boosting and projective adaptive resonance theory for analysis of gene expression data from cancer patients hiro takahashi, yasuyuki murase, hiroyuki honda department of biotechnology, school of engineering, nagoya university, nagoya - , japan. e-mail: h d@mbox.nagoyau.ac.jp (h. takahashi) an optimal and individualized treatment protocol based on accurate diagnosis is urgently required for the adequate treatment of patients. for this purpose, it is important to develop that a sophisticated algorithm that can manage large amount of data, such as gene expression data from dna microarray, for optimal and individualized diagnosis. in our previous study, we developed the projective adaptive resonance theory (part) as a gene filtering method and boosted fuzzy classifier with sweep operator (bfcs) as a modeling method. in the present study, we applied the combination of part and bfcs (part-bfcs method) to microarray data of brain tumor (central nervous system tumor) obtained from the website. the method enabled the selection of important genes related to the prognosis of the tumor, i.e., sensitivity for combined therapy with surgery, radiotherapy, and chemotherapy mainly with vincristine, cisplatin, and cytoxan. the constructed model showed about % higher accuracy than that of the conventional method. genomic signal analysis of hiv variability based on rt gene p.d. cristea, rodica tuduce d. otelea biomedical engineering center, university "politehnica" of bucharest, romania. e-mail: pcristea@dsp.pub.ro (p.d. cristea) dna sequences genotyped from clade f hiv- isolates from romanian patients in the laboratory of the national institute of infectious diseases "matei bals", bucharest, romania, have been studied. the symbolic sequences have been converted into digital genomic signals by using a complex quadrantal representation of the nucleotides described earlier. the cumulated phase and unwrapped phase of a complex genomic signal reflect the statistical distribution of bases and base-pairs, respectively. independent component analysis of the genomic signals has been used to characterize the variability of the f subtype hiv strains isolated in romania. the sequenced segment is of (about) base pairs, approximately aligning with the standard sequence of hiv- (accession nc in genbank) over the interval - bp. this segment, which is currently used for the standard identification and assessment of hiv- strains, comprises the protease (pr) gene and two thirds of the reverse transcriptase (rt) gene. only results referring to the analysis of the rt gene region are presented here and used for extracting features of virion isolates and for establishing phylogenetic trees of the studied strains. the analyzed rt encoding segment has the length bp and is located in the second interval ( - bp) of the analyzed dna segment, respectively along the - bp region of nc . taking into account the mutations identified in these sequences, the samples were classified in three groups from the point of view of their resistance to current antiretroviral compounds: sensitive, resistant and multiresistant. the paper presents results for the isolates in which mutations leading to multiple drug resistance have been identified. over expression of secb protein in e. coli enhances the periplasmic expression of human growth hormone m. ghafari , , a. zomorodipour : islamic azad university of jahrom, tehran, iran; national institute for genet eng and biotechnol., tehran, iran. email: maryamghafari @yahoo.com (m. ghafari) among several proteins involved in the secretion pathway of proteins in e. coli, secb plays a key-important role in solubilization of preproteins before processing. in order to increase processing of a human growth hormone precursor (pelb::hgh) which appeared to have problem in processing efficiency, as a possible solution a regulated co-expression of a secb gene was considered. in this regard, we designed an arabinose-regulated secb expressing plasmid compatible with an iptg/lactose-regulated pelb::hgh expressing plasmid. for the construction of the secb expressing plasmid the origin of replication and antibiotic resistant gene (amp) of a pbad vector was replaced by a p a-ori and a kanamycin resistant gene, respectively. the expression and processing of pelb::hgh preprotein in the two-plasmid containing bacteria in a secb over-expression state was compared to that of the pelb::hgh expression in normal bacteria. although a decline in total expression level of hgh during the overexpression of secb was observable, probably due to presence of two different expressing plasmids, but both the processing efficiency of pelb::hgh and the transport of mature protein into the periplasmic space was enhanced during prolonged arabinose induction. current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts. natural allergen extracts contain a mixture of allergenic and non-allergenic components that are difficult to standardize. recombinant allergens can improve diagnosis and de-sensitization against single component. major cat allergen is a heterodimer composed of disulfide linked . and . kda polypeptide chains. both chains of feldi protein were obtained in e.coli system. purification of recombinant proteins was performed in denaturing conditions using immobilized metal affinity chromatography specific for proteins with histidine tag. from ml culture approximately mg protein for feldi chain and mg of feldi chain were obtained. after purification histidine tag was removed by hydrolysis with thrombin. immunological activity of feldi against serum of patients allergic to cat was narrowed to subgroup of patients allergic to feldi protein by surface plasmon resonance. immunological activity of each chain and renatured heterodimer was also tested using immunoprecipitation techniques against serum of population group. subdoligranulum variabile -a novel member of the human gut micro flora with a high prevalence kim holmstrøm, trine møller bioneer a/s, hørsholm, dk- , denmark in we isolated and cultured for the first time a bacterium from a human fecal sample representing a hitherto unknown member of the clostridium leptum rrna supra generic cluster. the c. leptum rrna supra generic cluster represents one of the major phylogenetic lineages within the human gut microbiota, and is characterized by having only a small proportion of its members actually identified by cultivation compared to the estimated numbers of bacteria contained in this group from culture-independent gut flora analyses. s. variabile is an obligate anaerobe gram negative bacterium with a characteristic pleiomorphic coccoid-droplet-like cellular morphology. its closest previously cultivated relative based on a s rdna phylogenetic analysis is faecalibacterium prausnitzii, a rod-shaped and therefore easily distinguishable gram negative bacterium present in high numbers in the human fecal micro flora. based on s rdna sequence we designed a s. variabile specific oligonucleotide probe for use in fish analysis to estimate the prevalence of this "new" bacterium in fecal samples collected from healthy human beings. interestingly, we observed a high proportion of s. variabile present in all tested samples, and in some instances we observed a higher prevalence than the more well-known group of bifidobacteria equally estimated by fish analysis. documentation of these results will be presented. several species of sea cucumbers, long an incumbent of traditional medicines were selected as the source of animal based antibiotic compounds. swabs of the inner surface and coelomic fluid (inner fluid) samples from sea cucumber (holothuria atra jaeger) were taken. thirty strains of bacteria were isolated. these strains were grown in different antibiotic production media. nine human pathogenic bacterial species were used as test agents and they are, k. pneumoniae, mrsa, m. luteus, s. thyphimurium, s. epidermitis, s. saprophyticus, b. subtillis, p. aeruginosa and s. pyogenes; only four bacterial strains showed mild antibiotic activity against s. pyogenes and s. thyphimurium. similar testing on two other species, h. scabra and s. variegatus will be carried out. different media, especially antibiotic production enrichment media will also be used. characterization will be done upon obtaining an antibiotic compound, which shows moderate to high activity against at least one of the nine human pathogens used. for plant based medicines, three rhizomes, "cekor," "jerangau" and "bonglai" were analyzed. solvent extraction using ethanol, methanol and acetone was carried out, at a concentration of - mg per ml solvent. the filtrates were used for the antibiotic testing stage. all the three plant species showed moderate antibiotic activity against m. luteus, s. epidermitis, s. pyogenes and s. saprophyticus. interestingly, the antibiotic activity increased when combinations of the herbal extracts were used. cell-based therapy continues to be a promising avenue for the treatment of duchenne muscular dystrophy, an x-linked skeletal muscle-wasting disease. recently, we have demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (sp) can engraft into dystrophic fibers of non-irradiated mdx cv mice after intravenous transplantation. engraftment rates, however, have not been therapeutically significant, achieving at most % of skeletal muscle myofibers expressing protein from donor-derived nuclei. to improve the engraftment of transplanted myogenic progenitors, an intra-arterial delivery method was adapted from a previous procedure. cultured, lentivirus transduced skeletal muscle sp cells were transplanted into the femoral artery of non-injured mdx cv mice. based on the expression of microdystrophin and gfp transgenes in host muscle, sections of the recipient muscles exhibited % to % of skeletal muscle fibers expressing donor-derived transgenes. further, donor muscle sp cells, which did not express any myogenic markers prior to transplant, express the satellite cell transcription factor pax and the musclespecific intermediate filament desmin after extravasation into host muscle. the expression of these muscle-specific markers indicates that progenitors within the side population can differentiate along a myogenic lineage after intra-arterial transplantation and extravasation into host muscle. given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step towards the improvement of cell-based therapies for dmd and other myogenic disorders. metabolic engineering design of an extracellular hgh synthesis system birgül Şentürk , pınar Ç alık , güzide Ç alık , tunçer h.Özdamar : bre lab, department of chemical engng, ankara university, ankara, turkey; iblab, department of chemical engineering, metu, ankara, turkey. e-mail: ozdamar@eng.ankara.edu.tr (t.h.Özdamar) metabolic engineering design of an extracellular human growth hormone (hgh) synthesis system is based on cloning the dna encoding human therapeutic protein together with the signal dna sequence of an extracellular enzyme gene into a host-vector system. in this context, extracellular protease (subc) signal dna sequence, i.e. pre-signal dna sequence, was fused into the frame in front of hgh mature dna sequence, by the use of four primers designed using pcr-based gene splicing by overlap extension method. b. licheniformis chromosomal dna and plasmid carrying hgh cdna were used as templates in pcrs, respectively, for the amplification of the subc signal dna sequence and hgh mature peptide sequence. for the fusion of two target genes, i.e. mature peptide sequence of hgh and, signal dna sequences were amplified separately by pcrs. the primers used at the ends to be joined were designed as complementary to one another by including nucleotides at their ends that are complementary to portion of the other primer. these products were mixed in the next pcr reaction, where one strand from each fragment contains the overlap sequence at end. extension of this overlap by dna polymerase has yielded the recombinant hybrid-gene; and hybrid-gene serve as template for the continuation of reactions for the increase of the concentration in the microreactors. the hybrid gene fragment was first cloned into puc , and then sub-cloned to pmk e.coli-bacillus shuttle plasmid. thus, a new expression vector with high stability and high copy-number was obtained and transferred into host b. subtilis (spo − ). the metabolic flux distributions calculated by the mass balance based stoichiometric model based on the proposed metabolic reaction network for r-b.subtilis were determined by using time profiles of the substrate, dry-cell, hgh, amino acids and organic acids concentrations as the constraints. on the basis of the intracellular bioreaction rates and the interactions with the bioreactor operation parameters, an in-depth insight will be provided for further metabolic engineering design for the extracellular hgh production in r-b.subtilis. medicines based on polypeptides consisting of and more amino acid residues are widely spread in pharmaceutical market at the present time. practically all polypeptide medicines known are prepared by general chemical synthesis that caused high cost of their production. that's why biotechnological way of polypeptide medicines preparation using recombinant gene expression in bacteria seems to be promising. during our work we design hybrid construction allowing solving a problem of specific cleavage of target polypeptide from the hybrid protein using system of protein splicing from new england biolabs. in case of thymosin ␣ production impac system (intein mediated purification with affinity chitin-binding tag) has been used, where the modified sce vma from s. cerevisiae has been applied as intein. in contrast to other systems, impact allows the preparation of target protein without using of serine protease and other factors that may cleave the hybrid protein. in the presence of thiol reagents, such as dithiothreitol, mercaptoethanol, or cystein the hybrid protein can be site-specifically cleaved to give intein, the target protein and small fragment of nextein. using of this system does not allow to obtain the target protein with ser, cys or thr on n-terminal of protein, because in those cases target protein and n-extein ligation product will be formed. ser is n-terminal amino-acid in thymosin ␣ . it was recently found that some metal ions essentially affect the splicing. we tried to use zncl and we have found that, in case of intein-thymosin ␣ , the maximal yield of target polypeptide and the minimal yield of splicing products are observed in the absence of dithithreitol and in the presence of . - mm zinc chloride in buffers on all stages of thymosin ␣ isolation. the structure of recombinant thymosin ␣ of human was confirmed by the determination of n-and c-terminal amino-acid sequences and by maldi tof mass-spectrometry. mature embryos of five t. aestivum and five t. durum cultivars formed embryogenic callus on two different media. embryos were removed from surface sterilised seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of murashige skoog and mg/l , -dichlorophenoxyacetic acid ( , -d) or mg/l naphtalenacetic acid (naa). the developed calli and regenerated plants were maintained on , -d or naa free ms medium. wheat plants can be regenerated via two different systems. there were significant differences in percentage of callus induction and regeneration capacity on the different initiation medium. among the t. aestivum cultivars, yakar had the highest regeneration capacity in both induction medium. in t. durum cultivars, kiziltan gave the highest regeneration capacity in ms + , d medium and yilmaz gave the highest regeneration capacity in ms + naa medium. a strong genotypic effect on the culture responses was found for both induction medium. tumor necrosis factor beta (tnf␤) is a pleiotropic cytokine mediating its activity through the same receptors as the structurally related tnf␣. shortening of the n-terminal part has been reported to enhance its cytotoxic activity, with the explanation that removal of the flexible n-terminus reduces steric interferences in the receptor binding process. for studies of relationship between the n-terminal protein structure and physicochemical properties, three different forms mettnf␤, his tnf␤ and n tnf␤ were expressed in e. coli. high solubility of n tnf␤ was expected, however, both analogs his tnf␤ and n tnf␤ were predominantly obtained in the form of inclusion bodies (ibs). on the other hand, mettnf␤ was equally distributed between the soluble and insoluble fraction. most probably, the composition of the n-terminal part, such as the exposure of hydrophobic residues in the case of n tnf␤, or a mildly hydrophobic stretch of seven histidines appended to the natural hydrophobic n-terminus in the case of his tnf␤, lead to incorrect folding and force aggregate formation. solubilization of ibs was performed under native and denaturing conditions using various concentrations of nls, urea and gndhcl. mettnf␤ ibs were easily dissolved with . % nls, while his tnf␤ and n tnf␤ demanded denaturing conditions. structural differences in the nterminal part of various tnf␤ proteins were also reflected in protein refolding characteristics and chromatographic behavior. dilution, ultrafiltration and dialysis were used for refolding, and imac was chosen as the main chromatographic step. our results suggest that not only the length of the n-terminal part of tnf␤ but also the composition and exposure of certain amino acid residues affect its physicochemical properties. high level expression of recombinant growth hormones in e.coli faces common problems such as protein aggregation and inclusion body formation. discussions are raised whether it is more beneficial to obtain soluble protein but to loose expression rates. here we describe an experiment based on the hypothesis that slower expression should result in at least partially soluble recombinant protein. experiments were performed on bovine, chicken and mink growth hormones. expression rate was controlled externally by adjusting cultivation temperature, media, inducer amount, and both induction and cultivation times. another approach to the problem was performed through genetic manipulation. we changed strong t promoter to e. coli promoter consensus sequence thus reducing expression rate. recombinant growth hormone was still found to form aggregates, even when expressed at extremely low levelsseveral ( - ) percent of total intracellular protein. we developed optimization scheme of insoluble protein production and showed that expression rate minimization is not influencing recombinant growth hormone solubility in vivo thus suggesting an idea of sequence specific aggregation. to optimize the recombinant protein production in small-scale shake-flask system and high cell density fermentation, new tools have been developed at our laboratory which helps to get knowledge about the physiological state of the cell culture. these tools include (i) a quantitative monitoring system for cellular mrnas based on a sandwich hybridization technique, and (ii) a wireless online monitoring tool (senbit), applicable for standard sensors such as ph, po and temperature for the continuous data collection from shake flasks. the senbit system is a new tool supplying valuable information for the optimisation of the expression of recombinant genes in shake flasks and allowing conclusions towards the reproducibility of shake flask cultures. the presentation will focus on use of the sensitive sandwich hybridization technology for the quantitative analysis of process relevant marker genes in different kind of microbial cell cultures with a focus on the production of recombinant proteins. samples from shake flask cultures and high cell density fed-batch fermentations of the yeast pichia patoris, have been analysed. additionally the mrna analysis was combined with the application of the senbit wireless system to study the production of a recombinant protein in shake-flask cultures of p. pastoris. aside from p. pastoris, mrna sandwich hybridization also was used to monitor product expression in fed-batch fermentation of e. coli for the production of a protein with two subunits by sequential induction. quantitative rna analysis as a tool for optimization of tetrameric collagen prolyl -hydroxylase production in e. coli a. neubauer , m. bollok , j. myllyharju , p. collagen prolyl -hydroxylase (p h) involved in the biosynthesis of collagens is an ␣ ␤ tetramer. recombinant expression of p h in e. coli was described recently . the construct for cytoplasmic expression contains the genes of both subunits in one plasmid under control of different promoters. the ␣ subunit forms inactive aggregates, when expressed separately. in mammalian cells the ␤ subunit is available in large excess and keeps the ␣ subunit in a soluble active form. to mimic this in the bacterial system, we induced both genes sequentially. after induction of the ␤ subunit with iptg, expression of the ␣ subunit was initiated with anhydrotetracycline. here we use the analysis of the product mrnas with a bead based sandwich hybridisation assay (sha) (rautio et al., ) for optimization of the fermentation procedure. a high p h activity was obtained if a high mrna level of the ␣ subunit could be maintained over a longer time. the obtained results illustrate the importance of the second induction for a high level expression of the p h tetramer. the cells need to be in a "healthy state" with low metabolic load to react efficiently to the second induction. the data illustrate the optimization of a fermentation process by monitoring mrna levels which is of general interest for optimization of products which are difficult to detect. cell-based therapy continues to be a promising avenue for the treatment of duchenne muscular dystrophy, an x-linked skeletal muscle-wasting disease. recently, we have demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (sp) can engraft into dystrophic fibers of non-irradiated mdx cv mice after intravenous transplantation. engraftment rates, however, have not been therapeutically significant, achieving at most % of skeletal muscle myofibers expressing protein from donor-derived nuclei. to improve the engraftment of transplanted myogenic progenitors, an intra-arterial delivery method was adapted from a previous procedure. cultured, lentivirus transduced skeletal muscle sp cells were transplanted into the femoral artery of non-injured mdx cv mice. based on the expression of microdystrophin and gfp transgenes in host muscle, sections of the recipient muscles exhibited - % of skeletal muscle fibers expressing donor-derived transgenes. further, donor muscle sp cells, which did not express any myogenic markers prior to transplant, express the satellite cell transcription factor pax and the muscle-specific intermediate filament desmin after extravasation into host muscle. the expression of these muscle-specific markers indicates that progenitors within the side population can differentiate along a myogenic lineage after intra-arterial transplantation and extravasation into host muscle. given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step towards the improvement of cell-based therapies for dmd and other myogenic disorders. collagen and its derived product gelatin are attractive mammalian proteins to be used as model for the production of complex heterologous proteins in plants. the availability of a recombinant product will provide a safer, more homogeneous product than the current animal-derived material. the aim of the project is to investigate the feasibility of a production system for the accumulation of recombinant collagen for conversion to gelatin using barley. the -end of the cocksfoot mottle virus (cfmv; genus sobemovirus) genomic rna sequence, called cfmv -element, has been shown to enhance recombinant protein synthesis in barley (wo / , mäkinen et al., ) . the -element will be used to study whether accumulation levels of complex mammalian proteins can be further increased, using collagen as a model that will serve as basis for exploring the expression of other complex proteins. this system can be study the production of barley-derived recombinant collagen for conversion to gelatin. classical swine fever virus (csfv) is an animal pestivirus which can be used as a surrogate model to elucidate the role of envelope glycoproteins of closely related human hepatitis c virus (hcv). the necessity to use the surrogate models for hcv is due to the fact that this virus cannot be grown in vitro cultures. csfv genome codes for three major antigenic glycoproteins which are located in the same cluster of genes; they are designated as e and e (e rns ) and e . glycoproteins form heterodimeric and homodimeric complexes on the external part of viral particles. it is generally accepted that envelope glycoproteins play a major role in the initial stages of viral infection both for csfv and hcv. formation of complexes is needed to effectively infect host cells. we have investigated the formation of glycoprotein dimers by immunoperoxidase monolayer assay and by immunoblotting (western blotting). immunoblotting is a very useful technique in these studies because the complexes are formed via cysteine-cysteine disulphide bonds and they are retained during sds-page under non-reducing conditions. by modifying the glycoprotein genes and by arresting n-glycosylation of e and e we have investigated which factors influence the formation of complexes. it has been found that some glycosylation inhibitors which act at the early stages of glycan chain processing influence, not only glycosylation, but also the stability of e protein, effectively inhibiting the formation of glycoprotein complexes and the yield of the virus. these inhibitors are potential agents for arresting the multiplication and spread of csfv, and its relative -human hcv. recombinant proteins have been produced in a variety of heterologous protein expression systems. eukaryotic unicellular algae have distinct advantages, e.g. it can synthesize complex protein that requires post-translational modification. furthermore, microalgae can be grown in confined environment and thus prevents leakage of genes to the environment. our group has developed a platform technology for the production of recombinant proteins in red microalga porphyridium sp. we have constructed algal transformation plasmid vectors containing a camv s promoter and polya signal site. a streptoalloteichus hindustanus bleomycin-resistant gene was used as the selective marker. we have expressed ovalbumin and hepatitis-b surface antigen (hbsag) as model proteins. transformation was carried out by agitating algal cells and vector dna with glass bead. transgenic lines were selected by growing algal cells on agar plate containing g/ml zeocin. positive transgenic lines were selected by screening the colonies by pcr and confirmed by dna sequencing. expression of ovalbumin and hbsag protein was examined by western blot analysis. ovalbumin was found to be expressed inside the algal cells while small hbsag was secreted into the medium due to presence of signal peptide. these findings indicate that red microalgae are capable of producing heterologous proteins. life-material exhibition as ethical interpretation chang shih-lung biotechnology industry study center, taiwan institute of economic research, taipei , taiwan. email: schang@tier.org.tw in this thesis we aim to explore the medical-related life-material exhibitions within ntu hospital-the humanity building, and taipei mackay hospital-the historical showroom as abecedarian clues to understand the burgeoning phenomenon-medical museums in taiwan. following these clues, we treat the whole context of medical museums, which transform values through situational construction, as the background to interpret the ethical implications of group val-ues that are transformed in the medical profession. in this thesis, we see medical museums, as the social prescription transforming medical profession, format the ritual context of situation ethics with the cultural construction of life-material. multi-disciplinary interactions and visiting itinerary can be transferred to the exploring horizon of research approach through description and interpretation. among them, we observe that humanistic elements have become essential equipment (or mat'eriel) of medical profession. though humanistic equipment (or mat'eriel) has its bottleneck in the museum situation, it can also unblock new possibilities for museum exhibitions, ethical practice or life ethics. as part of a wide research program aimed at developing new antitumoral agents, we present herein a series of stereoisomeric derivatives of fused tetrahydrofuranes (fthf) substituted with diverse protecting groups either at the primary or secondary hydroxyl groups. unprotected derivatives were also synthesised to investigate the influence of substituents on the in vitro activity of fthf. data on the synthesis, chemosensitivity and apoptosis induction by this series of fthf will be correlated to substitution pattern, stereochemistry and protecting groups, as an aid to the rational design of novel antitumour drugs. establishment of in vitro test systems for pulmonary edema resorption by peptide drug candidates dominik geiger, aswin mangerich, rudolf lucas, klaus p. schäfer, inge mühldorfer department of biotechnology, altana pharma ag, konstanz, germany; imc university of applied sciences, krems, austria tnf-␣ was found to up-regulate the rate of lung liquid clearance (llc) in several animal models by the activity of its lectin-like tip domain. this activity can be mimicked by a circular peptide designated tip. tip was shown to induce llc and consequently pulmonary edema resorption in different animal models. in order to study the mechanism of action of tip, we established two in vitro test systems for edema resorption with the human lung epithelial cell line calu- : ( ) the ability of calu- cells for spontaneous dome formation within confluent monolayers was utilized for quantitative examination of tip's activity on active transepithelial fluid transport ("dome assay"). ( ) the effects of tip on bioelectrical properties of polarized cell monolayers were studied by using the transepithelial electrical resistance (teer) technology ("teer assay"). dome assay experiments confirmed that dome formation is a sodium dependent process and that tip is able to increase this process. teer assay experiments proofed that tip acts in a polarized and dose dependent manner. in conclusion, there is strong evidence that the dome and the teer assays are suitable systems for in vitro activity testing of tip and other anti-edema peptide drug candidates and are useful for studies on their mechanism of action. increasing safety concerns in gene therapy result in more stringent regulatory requirements. those cover the complete process chain of cell banking, fermentation, and purification. a wide range of applications for pdna requires gram to kilogram amounts for clinical trials and market supply. economic, productive and robust processes are a prerequisite for low cost of goods (cogs). therefore manufacturer of biopharmaceuticals need to address these considerations by developing new production processes meeting the new standards. boehringer ingelheim austria developed a novel pdna production process suitable for large-scale cgmp production. the process is based on e. coli fermentation. the process contains no components from animal origin. the optimized fermentation process yields up to g pdna/l fermentation volume. for isolation of pdna from e. coli alkaline lysis in glass bottles or stirred tanks is commonly used. cell wall structure is destroyed by a combination of alkaline ph and detergents. in our process alkaline lysis is operated in a closed, continuous system directly connected to clarification without using enzymes. we developed a scalable process for pdna purification based on different chromatographic principles. the capture step is carried out by hydrophobic interaction chromatography followed by anion exchange chromatography as intermediate step. final polishing is carried out by conventional size exclusion chromatography in a group separation mode providing also buffer exchange and desalting for the final formulation. the process results in a pdna drug substance of highest quality containing a low level of impurities (genomic dna, rna, proteins endotoxines) suitable for therapeutic applications. depending on the conditions during fermentation and the used host homogeneities of greater than % or even % are possible, while a high over all yield can be achieved (∼ %). during the development monolithic chromatography supports (cim ® ) were compared with conventional resins and evaluated as potential alternatives. the complete process is monitored by a set of analysis covering cell banking to final purification. new sensitive methods based on hpce, hpiex and fluorometric measurement were developed. whereas many natural amino acids are currently produced by very cost efficient biological processes, the manufacture of methionine is still performed by traditional chemical synthesis. this was mainly due to the poor performances of the currently available producing strains that inhibited the development and commercialization of a biological process to l-methionine. metabolic explorer has recently reinvestigated and developed an efficient biological process that employs an engineered microorganism and utilizes a renewable starting material (corn sugar) as its feedstock and converts glucose into l-methionine. we will describe (a) the general scheme for the engineering of the host organisms and (b) the general approach to maximize carbon and reducing equivalent throughput to l-methionine. to highlight this effort, we will present the global approach developed to improve the process combining metabolic flux analysis, traditional protein biochemistry, molecular biology and fermentation optimisation. saccharomyces cerevisiae is an established 'work horse' of the fermentation industry and modern biotechnology has led to a spectacular expansion of the range of products that can be produced by this yeast. however, for the large-scale sustainable production of chemicals, it is equally important that the range of carbohydrate feedstocks be expanded. especially relevant in this respect is the ability to consume the pentose sugars d-xylose and l-arabinose, which make up a substantial part of plant carbohydrates. wild-type s. cerevisiae strains cannot metabolise d-xylose, but are capable of slowly metabolising its keto-isomer, d-xylulose. therefore, efficient conversion of d-xylose into d-xylulose has long been a key issue in yeast metabolic engineering. non-saccharomyces yeasts that is capable of growing on d-xylose use two enzymes, xylose reductase and xylitol dehydrogenase for this purpose. while both enzymes have been successfully expressed in s. cerevisiae, this is not always compatible with efficient product formation. for example, in the case of ethanol production, the different cofactor specificities of these two oxidoreductases cause massive byproduct formation. theoretically, introduction of a xylose isomerase, which catalyses the interconversion of xylose and xylulose, might circumvent these problems. however, it is notoriously difficult to express bacterial and archaeal xylose isomerases in s. cerevisiae and, until recently, activities of heterologous xylose isomerases expressed in s. cerevisiae were vanishingly low, at least under physiological conditions. a breakthrough was reached when, in , a xylose isomerase gene from the anaerobic fungus piromyces was expressed in s. cerevisiae. while this led to high activities of xylose isomerase, these were not enough to enable fast growth or product (ethanol) formation. in this presentation, we will discuss how a combination of metabolic and evolutionary engineering led to fast and efficient xylose utilization by engineered saccharomyces cerevisiae strains under aerobic as well as anaerobic conditions. furthermore, we will illustrate how evolutionary approaches can be applied to facilitate the utilization of mixed substrates. hyaluronic acid (ha) is a natural and linear polymer composed of ␤- , -n-acetyl glucosamine and ␤- , -glucuronic acid repeating disaccharide units with a molecular weight (mw) up to mda. it is a major constituent of the extracellular matrices and the synovial fluid. in the last decades, various fields of application including cosmetics, ophthalmology, rheumatology, tissue engineering and drug delivery have been explored, owing to the many important biological functions of ha and its unique physico-chemical properties. however, for some specific applications, the relatively high mw of ha is a limiting factor and the availability of low mw fractions would be highly desired. for food applications, low mw ha has been shown to penetrate the gastrointestinal barrier, thereby increasing the ha bioavailability. moreover, low mw ha fractions are able to re-establish the ha content in the skin and can thus be used in cosmetics as anti-aging and anti-wrinkle agents. finally, low mw ha has shown to prevent oxygen free radical damage in granu-lation tissue during wound healing. a range of methods has been described for the depolymerization of ha to low mw fractions. these techniques involve heat treatment, ultrasonication, uv/gamma irradiation, chemical and enzymatic degradation. we present results on a degradation process of ha originating from bacillus subtilis fermentation into well-defined low mw fractions. the process developed in lab scale is safe, well-controlled and produce low mw ha fractions with narrow polydispersity. moreover, the process is readily up-scalable. the low mw ha fractions are being evaluated in various cosmetic applications. metabolic pathway manipulation for improving the properties and productivity of microorganisms is becoming an established concept. metabolic engineering can be defined as the directed improvement of product formation or cellular properties through the modification of specific biochemical reactions or introduction of new ones with the use of recombinant dna technology. a detailed analysis of the physiological means of the different pathways is needed to be able to introduce modifications aimed to the production of not only important metabolites, but also to understand the fundamentals of cell biology. aimed to produce single compounds, metabolic engineering necessarily includes the modification of the cellular pathway(s) as well as the redirection of the energy toward the production itself. the existing metabolic engineering applications are the culmination of more than two decades of global experience developing processes for the production of fine chemicals, vitamins, nutraceuticals and animal nutritional aids such as amino acids. based on the relative low complexity, the first biotechnological applications have been developed from microorganisms. our laboratory has been engaged in this field since different years. yeasts like saccharomyces cerevisiae, kluyveromyces lactis and zygosaccharomyces bailii have been developed for the production of fine chemicals like lactic and ascorbic acids from d-glucose. in this contribution, we will present the last data obtained. since years amino acid production is in the focus of industrial microbiology. l-glutamate and l-lysine are produced with corynebacterium glutamicum, while escherichia coli is used for l-threonine production. the worldwide market of threonine drastically increases: in the amount of threonine produced worldwide was , t and raised to , t in and , t in . concerning predictions of experts the demand of threonine will rise with a two-digit rate of economic growth within the next few years. meanwhile the prices declined. these conditions enforce very efficient production processes. beside the strain development and an optimised downstream procedure the fermentation process is an important target for productivity improvement. strain development is dependent on detailed knowledge of the production strains. with innovative methods we are able to get a close look inside the cells under different culture conditions. these methods have been called 'omics' in recent literature. knowledge about genome, transcriptome, proteome, phosphoproteome, metabolome and fluxome leads to new ideas for strain improvements. data generated by these methods must be based on clearly defined culture conditions. therefore, highly parallel fermentations have to be performed to generate biological parallel samples. cutting-edge technical equipment is the basic requirement for experiments like this. other requirements are optimised sampling for different analysis, technical parallels of analytical steps and a detailed statistical analysis of data. these procedures guarantee distinction between real data and data noise. integration of all these data to a holistic model of the cell is the challenge for the future. by combination of a new strain, process and downstream improvements the plant productivity was increased drastically. we ended up in an optimized, fast and high yield process to scope challenges worldwide market comes up with. rieping, m., hermann, t. ( ); fermentation process for the preparation of l-threonine; wo/ . ) are potentially quite useful biocatalysts, as they allow for the regioselective and stereoselective hydroxylation of activated as well as non-activated carbon atoms. in addition, the large number of members of the p superfamily exhibits a wide diversity of specificities from which a useful biocatalyst may be selected. from a technical point of view, however, they have significant drawbacks. thus, they usually cannot be produced in large quantities nor recovered or stored without a severe loss in activity. their catalytic activity is mostly quite low, and their operational stability leaves much to be desired. most p enzymes require a complex protein/phospholipid machinery for activity, and the final electron donor in the reaction cascade, usually nadph, does require extensive recycling to arrive at a commercially satisfactory process. recently, the use of bacterial cytochromes as hydroxylation biocatalysts has received considerable attention. some of them are natural fusion proteins, which contain the heme and the reductase domains on a single polypeptide chain. they are catalytically much more active compared to, e.g. cytochromes occurring in human tissue. we thus have set out to further improve the technical applicability of these enzymes, and have centered our activities around several bacterial cytochromes. it proved very useful to apply rational mutagenesis and directed evolution to these enzymes, leading to a surprising compatibility of mutant enzymes with a wide variety of substrates. mechanisms for the limited stability of the enzymes were explored, leading to hybrid enzymes with enhanced stability, and the cofactor problem was alleviated using auxiliary enzymes or mediator-based technologies. as a result, a bioreactor based on microbial cytochromes was built and operated for several days. baeyer-villiger monooxygenases represent useful biocatalytic tools as they can catalyze reactions, which are difficult to achieve using chemical means. however, so far only a limited number of these monooxygenases were available in recombinant form kamerbeek et al. ( ) . using a recently described protein sequence motif fraaije et al. ( ) and the available genome sequence information, we were able to identify and overexpress a number of novel bacterial bvmos. one of the overexpressed bvmos was found to be relatively stable as it originates from thermobifida fusca, which grows at ∼ • c. the enzyme was shown to be active on a broad range of substrates, preferring aromatic ketones fraaije et al. ( ) . the best substrate discovered so far is phenylacetone, hence its name: phenylacetone monooxygenase. we have solved the crystal structure of phenylacetone monooxygenase, which represents the first structure of a bvmo malito et al. ( ) . the crystal structure provides insight into the complex mechanism of catalysis mediated by fadcontaining bvmos. by site-directed mutagenesis we have probed the role of several active-site residues. a crucial role is played by an arginine residue. as phenylacetone monooxygenase shares significant sequence identity (> %) with all known nadph-dependent bvmos, many of the observed structural features seem to be conserved within this class of atypical monooxygenases. by homology modeling using the phenylacetone monooxygenase structure, catalytic properties of other baeyer-villiger monooxygenases can be explained or predicted. screening for fungal baeyer-villiger monooxygenases l. butinar , j. friedrich , v. alphand : laboratory of biotechnology, national institute of chemistry, ljubljana si- , slovenia; groupe biocatalyse et chimie fine cnrs fre , université de la méditerranée, marseille, france the asymmetric form of the baeyer-villiger (bv) oxidation (transformation of ketones into lactones) is an important challenge for organic chemistry since the obtained lactones are valuable building blocks for synthesis of countless biologically active products. to date, enzymatic or microbial bv oxidations appears as more successful than their chemical counter-parts. (ten brink et al.) whereas most active bv monooxygenases are produced by bacteria (among which the well-studied enzyme of acinetobacter calcoaceticus), only a few fungal strains expressing bvmo were described (alphand et al., carnell and willetts) . in order to increase the number of available biocatalysts which perform such an asymmetric biotransformations, a screening of fungi belonging to major groups of zygo-, ascoand basidiomycetes was conducted using bicycloheptenone as testsubstrate. surprisingly, a large number of the tested fungi were able to transform the substrate into one to four different lactone isomers. the yields, the enantio-and regio-selectivity of the reaction depended on the fungal strain. alphand, v., furstoss, r., . j. mol. catal. b , - . carnell, a., willetts, a., . biotechnol. lett. , - . ten brink, g.j., et al., . pyranose oxidase (p ox) is a periplasmic enzyme that widely occurs in basidiomycetes. it catalyses the c- oxidation of several aldopyranoses to the respective -keto derivatives, transferring electrons to molecular oxygen to yield h o . p ox is of interest for carbohydrate conversions, as its reaction products ( -keto sugars) can be attractive intermediates in the production of food ingredients. we cloned the gene encoding p ox, and subsequently amplified a cdna clone by rt-pcr. the cdna was inserted into a bacterial expression vector and successfully expressed in e. coli. properties of the heterologous protein were compared to those of the native enzyme showing that they are essentially identical. both the native as well as the recombinant enzyme were used in biotransformations of sugars. recently, the d-structure of this tetrameric enzyme was elucidated. based on structural information, several enzyme variants containing point mutations were constructed and further characterised. two of these variants (e k and e r) displayed improvements in stability and certain kinetic properties thus making them attractive for biocatalytic applications. lactones are important compounds for the fragrance and flavour industry. right now the production of lactones is dependant on the import of crude materials from tropical countries. in this project, we want to tackle the manufacture of lactones via a biocatalytic route using p monooxygenases. cytochrome p monooxygenases catalyse the oxyfunctionalization of non-activated c-atoms. cyp a from bacillus megaterium, cyp a and cyp a from bacillus subtilis are soluble fusion proteins comprising p monooxygenase and fad/fmn reductase domains in one polypeptide chain. all three enzymes are highly homologous fatty acid hydroxylases. especially, cyp a also known as p bm- is well characterized and shows high activity compared to other p monooxygenases. the aim of the work is to change selectivity but conserve the high activity that is typical for those enzymes. using methods of structure modelling, rational protein design and directed evolution new mutants of these enzymes with changed regioselectivity are obtained. products of conversion with monooxygenases are intermediates in the production of lactones. the interface of biology and materials science has led to new materials with unique structural and functional properties, and new process technologies with the ability to produce, from "bottoms up", a wide range of biomimetic structures. these materials and their designs have broad application as catalysts, sensors, and devices for use in synthesis, cell and tissue engineering, bioanalysis and screening, and nanoelectronics. we have focused on the generation of sugar-based nanostructures, complete with tailored selectivities and biocatalytic activities at the molecular and nanoscales. these include biocatalytically-generated carbohydrate derivatives that selfassemble with high precision to give novel architectures with functional and responsive properties. izumoring: a strategy for total production of rare sugars ken izumori rare sugar research center, kagawa university, kagawa - , japan. e-mail: izumori@ag.kagawa-u.ac.jp we found a new enzyme, d-tagatose -epimerase (dte), that epimerize all ketohexoses at c- position. this epimerase catalyze not only between d-tagatose and d-sorbose, but also d-fructose = dpsicose, l-sorbose = l-tagatose, and l-psicose = l-fructose. this new enzyme offered us a useful key tool to connect all ketohexoses using hexitols as intermediates. the figure shows that all eight ketohexoses can be connected with dte and polyol dehydrogenases (pdh) in a ring. using this ring, we can easily find the pathway to transfer d-fructose to d-tagatose via d-psicose using dte and pdh. various aldose isomerases transform ketohexoses to the corresponding aldohexoses. so, we can connect all aldohexoses with ketohexoses using the enzyme. finally, all hexoses, ketohexoses, hexitols and aldohexoses are connected using enzyme reactions in a ring structure (not shown). this kind of strategy is effective also on transformation of tetroses and pentoses. now, we can produce all monosaccharides; tetroses, pentoses and hexoses by enzyme reac-tions. the bioproduction strategy of all rare sugars (monosaccharides that are rare in nature) is illustrated using ring form structures named as izumoring. we have already succeeded to produce d-psicose in large scale and are now in the progress of mass production of various rare sugars from natural and cheap sugars using izumoring. bioprocess development for chiral intermediates christian wandrey institute of biotechnology, forschungszentrum jülich gmbh, jülich d- , germany chiral alcohols, diols, amino alcohols and chiral acids (e.g. hydroxy acids and amino acids) play an important role in pharma and agro synthesis. in the past such chiral intermediates were obtained by racemic resolution via chiral reductions using prochiral precursors. here the problem of cofactor regeneration arises. this problem could be solved by enzyme-coupled or substrate-coupled cofactor regeneration using formate or isopropanol as reducing agent. alternatively, whole cell bioreductions were developed where glucose is used as the reducing agent. in recent years "designer microorganisms" were developed in which oxidoreductases (e.g. alcohol dehydrogenases) were over-expressed in escherichia coli. in such cases, cofactor regeneration was achieved intracellularly with isopropanol as the reducing agent or by coexpression of a formate dehydrogenase, so that once again format could be used for reduction. another route to obtain chiral intermediates is a fermentative approach using classical pathways (like the aromatic amino acid pathway). here, the pathway is interrupted after the intracellular production of chorismate. new chiral intermediates can be obtained by over expression of additional genes, which catalyzed the production of chorismate derivatives leading to cyclohexadiene-transdiols and the corresponding amino cyclitols. the last example can be regarded as an example of industrial biotechnology where glucose is used as starting material (white biotechnology). here bioprocess development is carried out in an integrated approach, in which molecular biochemical engi-neering cares for the optimal intracellular metabolic network and the classical biochemical engineering cares for the optimal environment of the cell in a fermenter. examples will be given which reach (in cooperation with industrial partners) up to kilogram scale. biotransformations are usually involved in just one or very few separate reactions in organic syntheses. the development of a cell-free "system of biotransformations" (sbt), in which a set of enzymes acts in a coordinated fashion in a one-pot synthesis, lead to increased catalytic complexity, selectivity and yield, as well as facilitated operation at reduced costs. the example chosen to prove the usefulness of the sbt-approach is the production of dihydroxyacetone phosphate (dhap). dhap, a c -compound from glycolysis, is an important precursor for asymmetric c-c-bond formation. so far, the production of dhap is difficult and expensive. for the construction of the dhap-producing sbt, e. coli's glycolysis is isolated from the metabolism to an as large as possible extent by the construction of a multi-ko-mutant. a culture is grown in an appropriate medium, homogenized in the production buffer, and used as the catalytic system. high production yields can be achieved since the production pathway is almost completely isolated from the metabolic network. the employed dhap-producing sbt provides not only a path from glucose to the product, but also an integrated atp-regeneration and nad-recycling system. in first experiments with a tpi-ko-mutant, a dhap-production yield on glucose of % could be achieved, without optimizing the system. the system remained active for more than h. up to now, atp cannot be applied in catalytic concentrations, but has to be present in equimolar amounts to glucose. the production yield could be increased by % through the addition of phosphate ions as substrate to the reaction, enabling the system to utilize atp more efficiently. these experiments indicate that the sbt-approach is viable and a large potential remains to improve the dhap-producing sbt. for some years novozymes have manufactured a pectate lyase for scouring of textile as an ecological alternative to the traditional harsh chemical treatment, and recently, we at novozymes discovered additional applications for pectate lyases. however, to be commercially attractive more robust pectate lyses had to be developed. in this paper, we will demonstrate how we for two different pectate lyases have improved their stability significantly. as the two enzymes are quite similar in sequence and structure, it was a new discovery for us to find that different concepts of protein engineering had to be used in our attempt to stabilize each individual pectate lyase. the stability of one enzyme was improved by substitutions in the internal of the structure whereas the stability of the other pectate lyase was primarily improved by changing surface residues. starting with knowledge from structural analysis, we have applied rational based protein engineering resulting in few selected variants. also random based protein engineering combined with screening of hundreds of thousands variants was used. in conclusion: the project team showed that by synergistic use of the two approaches, we were able to move faster towards a solution and eventually we succeeded finding new stabilized pectate lyase variants, applicable for new business areas. the importance energy independence as a national goal equals or exceeds that of the moon landing in . the development of a new industry to produce fuel ethanol from woody biomass would increase national security, improve employment and the environment, and provide substantial relief from the debt of imported petroleum. costs associated with the rapid development of this new industry (∼$ . billion per year) could be paid by re-assigning cent per gallon from existing federal gasoline taxes, a small price to pay for future energy independence. the corn-to-ethanol industry continues to make a remarkable contribution to our liquid fuel needs through expansion. today, one row of every six rows of corn is converted into fuel ethanol in the u.s. however, this expansion will be limited to - % of total automotive fuel by the economics of corn costs and production. corn can do more! corn stover is the single most abundant agricultural residue in the us and can be used as a feedstock to produce - gallons of ethanol per dry ton. further expansion with other biomass feedstocks such as agricultural and municipal residues (lignocellulose, woody biomass) could produce over billion gallons of fuel ethanol annually according at a recent joint report by the usda and doe (april, ) . current technology has been demonstrated at pilot scale for the production of fermentable sugars from hemicellulose by dilute acid hydrolysis and for the hydrolysis cellulose using fungal cellulose enzymes. biocatalysts such as recombinant escherichia coli have been developed and demonstrated for the efficient conversion of all sugar constituents of biomass to ethanol. a national goal for the full-scale deployment of current technology to produce biomass-based fuel ethanol will allow the us to reduce imported petroleum by %. together with increased efficiencies of hybrid vehicles, energy independence could be achieved within - years. similar gains could be realized by many nations around the world to provide new manufacturing and employment, redistributing wealth and ensuring a cleaner, healthier environment. bioethanol production using thermophilic bacteria marie just mikkelsen, birgitte k. ahring emab, biocentrum-dtu, lyngby, denmark the industry of bioethanol production is facing the challenge of redirecting the process from fermentation of relatively easily convertible but expensive starchy materials, to complex but inexpensive lignocellulosic biomass. on lignocellulosic hydrolysates, gram-positive thermophilic bacteria have unique advantages over the conventional ethanol production strains. the primary advantages are their natural broad substrate specificities, and in some strains, a high tolerance to lignocellulosic hydrolysates. moreover, ethanol fermentation at high temperatures also has the advantages of high productivities and substrate conversions, low risk of contamination and facilitated product recovery. some thermophilic bacteria naturally produce primarily ethanol from most sugar monomers present in lignocellulosics, but modifications are still necessary to increase ethanol yields. the release of useable sugars from lignocellulose biomass for industrial fuel-ethanol fermentation is often facilitated by a weak acid hydrolysis step. as a consequence, inhibitors such as furfural and -hydroxymethylfurfural (hmf) are formed as degradation products of xylose and glucose, respectively. moreover, the fermentative end-product of ethanol is also inhibitory. these, and other inhibitors present an environment, which elicits the expression of stress-related genes in saccharomyces cerevisiae. recently, s. cerevisiae genes have been identified as important in furfural stress tolerance. when furfural is present, yeast with these genes disrupted grows poorly compared to wild-type yeast. a sub-class of these genes suggests that yeast grown under furfural-induced stress may rely upon similar pathways as cells grown under various other stresses, including oxidative, heat, and sorbate. to investigate this link further, we analyzed stress-induced phenotypes such as ros activity, dna damage, and membrane damage in wild-type and mutant yeast exposed to furfural or hmf stress. moreover, we investigated whether overexpression of this sub-class of genes would provide protection from furfural-induced stress and oxidative damage. micro-organism to be used in fermentation of lignocellulose hydrolyzates should preferably have three characters: (a) high ethanol tolerance, (b) resistance to inhibitors found in the hydrolyzate, and (c) a broad substrate utilization range, since the hydrolyzate contains several sugars. in addition to the possibility of controlling the level of potential inhibitors, fed-batch fermentations also permit the parallel uptake of several different monomeric sugars. two strains of saccharomyces cerevisiae, cbs (a commonly used laboratory strain) and tmb (a strain isolated from a spent sulfite liquor fermentation plant), were characterized in batch and fed-batch fermentation of a dilute-acid hydrolyzate from spruce. the strains had different abilities to ferment spruce hydrolyzate. the study suggests that the furan reduction capacity of a yeast strain is a key factor for its performance in fermentation of lignocellulosic hydrolyzate. polyketides constitute a structurally highly diverse group of natural products that possess broad ranges of pharmacological properties and represent a major source for novel cancer therapeutics. however, these compounds may be sub-optimal in regard of activity, selectivity, availability and unwanted side effects. in addition, the sustainable production of these valuable metabolites can be a challenge. studying the molecular basis of the biosynthetic pathways may set the basis for improving the production and for rationally engineering derivatives with altered bioactivity profiles, e.g. through targeted knockouts, mutasynthesis ziehl et al. ( ) , and swapping of pathway genes. our results in elucidating and manipulating the biosynthesis of selected antitumoral polyketide metabolites from bacteria (aureothin, chartreusin) and fungi (cytochalasines, rhizoxin) are presented. analyses at the genetic and biochemical levels provided new insights into several unusual biosynthetic features, e.g. non-linear polyketide assembly for the nitroaryl-substituted polyketide aureothin he and hertweck ( , ) , an oxidative rearrangement cascade in the chartreusin pathway xu et al. ( ) , and a fungal iterative pks-nrps hybrid synthase schuemann and hertweck ( ) involved in cytochalasin biosynthesis. the most surprising result was obtained from elaborating the biogenesis of the antimitotic agent rhizoxin from rhizopus sp., which allowed for a significant improvement in large-scale production partida-martinez and hertweck ( ). he, j., hertweck, c., chem. biol. , , - chem. bio. chem. , , glycopeptides such as vancomycin and teicoplanin are the drugs of last resort for the treatment of severe infections caused by antibiotic resistant gram-positive bacteria. glycopeptides inhibit the peptidoglycan biosynthesis by binding as dimers to the d-ala-d-ala termini of the cell wall precursors. amycolatopsis balhimycina synthesizes the vancomycin-type glycopeptide balhimycin, whose structure and biological properties greatly resemble vancomycin and which only differs by its glycosylation pattern. using a "reverse genetics" approach we have identified the -kb gene cluster encoding the biosynthesis of balhimycin. by a combination of genetics, biochemistry and analytical organic chemistry, we were able to elucidate the biosynthetic pathway and to assign functions to almost all genes of the cluster. the biosynthesis starts with the pathway-specific provision of the non-proteinogenic amino acids ␤-hydroxytyrosine (␤-ht), hydroxyphenylglycine (hpg) and dihydroxyphenylglycine (dpg) which form together with (n-methyl)-leucine and asparagine the heptapeptide backbone of balhimycin. dpg is synthesized via a polyketide synthase mechanism (pksiii) similar to that known from plant chalcon/stilben synthases (pfeifer et al., ) . for the ␤-ht synthesis three genes are essential which form an operon (puk et al., ) : bpsd, an nrps binds a tyrosine molecule, which is then hydroxylated by the p monooxygenase oxyd. the perhydrolase bhp is required for the release of ␤-ht. subsequently bhaa, a nadh/fad-dependent halogenase catalyzes the chlorination of ␤-ht to form chloro-␤-hydroxytyrosine (puk et al., ) , which is needed to stabilize the dimerization. the amino acids are linked by non-ribosomal peptide synthetases (recktenwald et al., ) , and the aromatic side chains are interconnected by p monooxygenases; a series of reactions which lead to the first antibiotically active intermediate. inactivation of the oxygenase genes revealed the order of the cyclization steps (bischoff et al., ) : the oxygenases act in a stepwise fashion in the sequence oxyb, oxya and oxyc. the resulting cross-linked heptapeptide is then finally modified by methylation and glycosylation. the biosynthesis is regulated by the strr-type regulator bbr, which was shown to bind in front of different operons of the balhimycin gene cluster. this ensures the coordinated expression of the biosynthetic genes. the non-producing mutants, defective in the supply of the non-proteinogenic amino acids, were used as recipients in cloning experiments as well as in approaches of precursor-directed biosynthesis by feeding chemically synthesized alternative precursors. thus, novel balhimycin derivatives were generated (weist et al., (weist et al., , . bischoff et al., . angew. chem. int. ed. , - . pfeifer et al., . j. biol. chem. , - . puk et al., . j. bacteriol. , - . puk et al., . chem. biol. , - . recktenwald et al., . microbiology , - . weist et al., . angew. chem. int. ed. , - . weist et al., spectroscopy guided discovery of novel bioactive microbial natural products thomas ostenfeld larsen, michael edberg hansen cmb biocentrum-dtu, technical university of denmark, lyngby, denmark the task of finding novel bioactive natural products is usually bioassay driven. often a certain type of compound (e.g. polyketide, alkaloid) turns out to be active in an assay. when having generated a promising hit in a bioassay the normal procedure in the drug discovery process usually is to produce a large number of structurally analogous compounds either by traditional chemical synthesis or by combinatorial chemistry in order to study structure activity relation-ships and to find even more active lead compounds. alternatively to chemical synthesis of analogues nature can be explored for structurally similar compounds by uv-spectroscopy guided screening. this work will present a new method for the systematic and automated computer assisted search of full uv spectra in large number of datafiles for both dereplication of known and discovery of new natural products based on the use of the new mathematical algorithm x-hitting. exploring the substrate spectrum of the antibiotic producing bacteria saccharopolyspora erythraea p. krabben, p. oliveira, f. baganz, j. ward department of biochemical engineering, university college london, london wc e je, uk knowledge of substrate utilisation capabilities play an important role in the development of genome scale metabolic models (borodina et al., ) and refinement of first generation annotations. furthermore, knowledge of the product formation during catabolism of different substrates provides valuable information about the distribution of metabolic fluxes and thereby forms a basis for rational strain improvement. we present here, data on the substrate utilisation capabilities and the corresponding product formation of s. erythraea. this analysis will help in improving the production of erythromycin and provide clues to the activation of the cryptic secondary metabolic pathways present in the s. erythraea genome. reference borodina, i., et al., ( ) . genome-scale analysis of streptomyces coelicolor a ( ) modeling of growth and product formation on complex media containing multiple substitutable substrates is a challenge. complex media offers the organism multiple choices of carbon and nitrogen substrates including free amino acids, peptides, soluble and insoluble proteins in addition to the defined sources such as glucose and ammonium sulfate. we present a structured model that accounts for growth and product formation kinetics of rifamycin b fermentation in a multi-substrate complex medium. the model considers the organism to be an optimal strategist with a mechanism to regulate the uptake of the substrate combinations. further, we assume that the uptake of a substrate depends on the level of a key enzyme, which may be inducible. the model also considers control parameters as fraction of flux through a given metabolic branch. the control parameters are obtained using a simple multi-variable constrained optimization. the model parameters were rigorously estimated via a specifically designed experimental plan. the model correctly predicts the experimentally observed growth and product formation kinetics and the regulated simultaneous uptake of the substitutable substrates under different fermentation conditions. the model and the model parameters provide useful insights into the growth and product formation strategy of this industrially important process. this presentation will describe the experimental results, the model development and the relevant model parameters for a. mediterranei s . the recent surge in oil price and the increasing concern on our environment have generated much interest in the production of chemicals from renewable resources. succinic acid, also called as amber acid, is a four-carbon dicarboxylic acid, which can be used as a precursor of numerous products including biodegradable polymers, green solvents, pharmaceuticals, and bulk and fine chemicals. a new capnophilic bacterium named mannheimia succiniciproducens mbel e was isolated from the rumen of korean cow. this bacterium can produce large amounts of succinic acid along with some other metabolites such as lactic, formic and acetic acids. we have completely sequenced the genome of m. succiniciproducens and charaterized its genome content in the context of metabolic pathways. we then constructed the genome scale in silico metabolic network for metabolic flux analyses, and carried out metabolic flux analysis under varying environmental conditions. based on the in silico analyses results, we selected several target genes to be manipulated for enhanced succinic acid production. detailed results of metabolic engineering based on genome-scale information will be reported. we have been developing tools for inverse metabolic engineering in order to identify gene targets that improve the phenotype of industrial strains and cells for medical applications. to this end, we create genomic fragment libraries from a source organism and use it to transform the host organism. cells are properly selected in environments that favor the phenotype of interest and genes enriched in these cells are sequenced and used in follow up transformations of cells with specific genetic backgrounds. this overall strategy is complemented with additional tools for modulating gene over-expression, gene deletion, and high throughput clone isolation. we will demonstrate applications of this strategy to the identification of gene targets for improved xylose assimilation in recombinant saccharomyces cerevisiae and improved lycopene production in escherichia coli. based on assumed reaction network structures, nadph availability has been proposed to be a key constraint in ␤-lactam production by penicillium chrysogenum. in this study, nadph metabolism was investigated in glucose-limited chemostat cultures of an industrial p. chrysogenum strain. enzyme assays confirmed the nadpspecificity of the dehydrogenases of the pentose-phosphate pathway and the presence of nadp-dependent isocitrate dehydrogenase. pyruvate decarboxylase/ nadp-linked acetaldehyde dehydrogenase and nadp-linked glyceraldehyde- -phosphate dehydrogenase were not detected. although the nadph requirement of penicillin-gproducing chemostat cultures was calculated to be . - . -fold higher than that of non-producing cultures, activities of the major nadph-providing enzymes were the same. isolated mitochondria showed high rates of antimycin a-sensitive respiration of nadph, thus indicating the presence of a mitochondrial nadph dehydrogenase that oxidizes cytosolic nadph. the presence of this enzyme in p. chrysogenum has important implications for stoichiometric modelling of central carbon metabolism and ␤-lactam production and may provide an interesting target for metabolic engineering. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf- cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp coding for am-cyan coral protein, which emits natural green fluorescence. complete elucidation of the genetic control of a metabolic flux requires the availability of fine-grained expression levels of the gene(s) of interest. we developed a collection of promoters of varying strength for tuning gene expression in the yeast s. cerevisiae. engineered promoters were obtained through random mutagenesis of the constitutive tef promoter. eleven mutated promoters were selected by fluorescence-activated cell sorting (facs) spanning gradually increasing activities between about and % compared to the native tef promoter. data were also confirmed at the level of mrna via rt-pcr. by introducing selectable markers in front of the different tef promoter mutations, we provided plasmid collections, which can be directly used to amplify promoter replacement cassettes for genomic integration of the fine-grained promoter collection in front of any yeast gene. l-arabinose, widely distributed in plant kingdom, is a component of plant cell wall. l-arabinose does not abundantly exist in free state in plants, but usually in corn hull, sugar beet pulp, gum arabic, mesquite gum, as the polysaccharide such as arabinoxylan and arabinogalactan. to produce arabinose from agricultural wastes, we screened arabinogalactan degradable strain from compost. thereafter, putative arabinase gene from this strain was cloned (b ts - ). as a result of spectrometric assay using -nitrophenyl ␣l arabinofuranoside, recombinant showed - -fold higher activity than wild type e. coli strain. after enzymatic reaction with corn fiber, b ts - produced . g/l of l-arabinose, which was detected on hplc. however, the enzyme activity was very low. so, we are transferring the gene into expression vector system. further characterization study and enzyme engineering to enhance the activity toward corn fiber will be presented in poster. there are only a limited number of hypersaline areas all over the world, which include several locations in turkey such as van lake located in eastern region of turkey. isolation and identification of halophilic and hyperhalophilic microorganisms from such locations is essential for the determination of biodiversity in turkey. high-level production of extremozymes from these microorganisms has also many economical advantages due to their stability at extreme reaction conditions. proteolytic enzymes are the most important group of enzymes produced commercially. of these, proteases produced by alkalophilic microorganisms are investigated not only in scientific areas such as protein chemistry and protein engineering but also find wide application in food, pharmaceutical, leather and detergent industries. in this study, microorganisms isolated from van lake were screened for the presence of extracellular alkaline protease activity. the optimum screening temperature and ph were determined as • c and ph . , respectively. one of the isolates that could grow at - % salinity reached highest levels of extracellular alkaline protease activity. this best producer, which was identified as the halotolerant bacillus pumilus, was found to produce alkaline protease both in the presence and absence of nacl. to improve enzyme production yields, culture conditions such as medium composition, growth ph and temperature were optimized. the effect of different carbon sources, organic and inorganic nitrogen sources on the production of alkaline protease was studied. whereas a mixture of inorganic and organic nitrogen sources induced high protease production, use of only an organic nitrogen source supported poor enzyme production. halotolerant bacillus pumilus produced maximum alkaline protease activity when maltose, yeast extract and sodium nitrate were used as carbon source, organic and inorganic nitrogen sources, respectively. this project was supported by tubitak through project tbag - t . in the market of biochemical products a very important role is played by heterologous proteins production, and despite recent advances in mammalian cells exploitation, yeasts can still present advantages as host systems. among them, the spoilage yeasts belonging to the zygosaccharomyces genus have become, due to some peculiar properties, significantly attractive. in particular, z. bailii is characterized by acid resistance, osmotolerance to high sugar and ethanol concentration combined with high biomass yield. despite still little is known about its genetics and cellular biology, our group is working on its development and exploitation for recombinant productions with an integrated approach coupling physiological study with the creation of molecular tools for heterologous proteins production. we previously described and did a patent application regarding the first techniques necessary to transform this yeast and to express and secrete different proteins derived from different sources. here we present and discuss the last advances in optimization of heterol-ogous protein expression in particular, on one side we present a reproducible strategy for target gene deletion, leading to the first z. bailii auxotrophic mutant, and on the other we show the improvement of gene dosage and plasmid stability by building a set of multicopy expression vectors based on the sequences of the z. bailii -like endogenous plasmid psb and an integrative plasmid. all the known ␥-butyrolactone autoregulator receptors are highly conserved in the dna binding motif present in their n-terminal portions and have been proposed to play roles as transcriptional regulators in antibiotic production and/or morphological differentiation. previously, kim et al. reported that the cloned scar in streptomyces clavuligerus has several characteristics of the autoregulator receptors in the genus streptomyces. in this study, to clarify the in vivo function of scar, a scar-disrupted strain was constructed by means of homologous recombination after introducing a scar-disruption construct via transconjugation from e. coli. no difference in morphology was found between the wild-type strain and the scar disruptant. however, the scar disruptant showed a . -fold higher production of clavulanic acid than the wild-type strain. the phenotype was restored to the original wild-type phenotype by complementation with intact scar. therefore, the autoregulator receptor, scar, acts as a negative regulator of biosynthesis of clavulanic acid but plays no role in cytodifferentiation of s. clavuligerus. lactate dehydrogenase catalyses the production of lactate from pyruvate. it is the first target for many researches on lactic acid producer microorganisms like rhizopus oryzae. in the present study based on the known sequences of r. oryzae ldha and ldhb genes skory ( ), they were cloned and expressed in a citric acid producer fungus aspergillus niger. the aspergillus niger strains expressing ldha or ldhb gene resulted in increased production of lactate in aspergillus niger. among transformants tested ldha and ldhb expressing strains were found to have higher lactate dehydrogenase activity compared to wild type in the conditions tested. the highest specific activity obtained with ldha transformants was only . times of the wild type while this was times for one of the transformants expressing ldhb. in addition to increased lactate production citric acid production was also increased. however, gluconic acid production ceased in the ldha or ldhb expressing a. niger strains. the production of lactic acid in a. niger transformants and lactate dehydrogenase a and lactate dehydrogenase b enzymes are being investigated in the chosen strains. selection of n source suitable for production of rhodococcus sp. biomass for the purposes of microbial transformation of ␣h-epoxypregnanolone ( ␣h) and -epoxy-pregnenolone ( ) into their ␣-hydroxy-derivatives was carried out. three dehydrated and three non-dehydrated n sources were tested. the transformation reaction was carried out in phosphate buffered medium containing g l − of the steroid substrate and . g l − cells. the steroids were determined by hplc. the transformation resulted in formation of three derivatives appearing in the reaction medium in the sequence: - -keto-; ␣-hydroxy-and ␣, ␤-hydroxy-epoxy-pregnenolone. a strong influence of the n source on the hydroxylating activity of the biomass was observed. triptose (difco) gave a cell depot actively hydroxylating ␣h without any significant accumulation of the - -keto-derivative. the most effective accumulation of hydroxylated derivatives of was observed with biomass grown on freshly prepared meat extract while the commercial products triptose (difco), meat extract (difco) and lactalbumin (flika) gave valuable information about the dynamics of the transformation process. biobleaching of kraft cellulose pulp by poliporus versicolor aysun ergene , nazif kolankaya : kırıkkale university, faculty of science and literature, department of biology, yahsihan, kırıkkale, turkey; hacettepe university, faculty of science, department of biology, beytepe, ankara, turkey the suitability of culture supernatant from poliporus versicolor for use in the biobleaching of kraft cellulose pulp was investigated. p. versicolor was found to grow on mycological broth ( % soytone, % d-glucose and . % cellulose pulp). maximal extracellular ligninase production was detected after day ( nkat). the optimum biobleaching conditions are • c and ph: . , with days. in this condition p. versicolor decreased the kapa number from . to . and increased brigthness from to . in day treatment. xylanase production, purification and characterization from a soil isolate, bacillus m- ayşegül ersayin , aytaç kocabaş , b. zümrüt ogel , ufuk bakir : biotechnology department, middle east technical university, ankara, turkey; food engineering department, middle east technical university, ankara, turkey; chemical engineering department, middle east technical university, ankara, turkey, . e-mail: ubakir@metu.edu.tr (u. bakir) xylan is a major component of the plant cell wall, representing up to % of the dry weight. xylan molecule is a complex polymer consisting of a ␤-d- , -linked xylanopyranoside backbone substituted with acetyl, arabinosyl and glucuronosyl side chains. hydrolysis of the xylan backbone is mainly catalysed by endo-␤- , -xylanases (ec . . . ). many bacterial and fungal species are able to utilize xylan as a carbon source. interest in the enzymology of xylan hyrdolysis has increased because use of xylanases in bioconversion of agricultural wastes to valuable products like single cell protein, xylo-oligosaccharides and fuel, in bio-bleaching processes, food and animal feed industries. in this study, xylanolytic nature of a soil isolate bacillus spp., bacillus m- , has been shown. bacillus m- produced multiple xylanases when grown on a liquid medium containing agricultural wastes as the sole carbon source. various agricultural wastes including corn-cobs and cotton waste, with and without pretreatments were used to maximize enzyme production. the major xylanase having molecular weight of kda upon sds-page and a pi of . upon ief was partially purified by liquid chromatographic techniques -fold with % recovery, including gel filtration, ion exchange and hydrophobic interaction chromatography. enzymes are important constituents in the laundry detergents due to their contribution to shortening washing times, reduction of energy and water consumption by lowering washing temperatures, provision of environmentally friendlier wash-water effluents and fabric care. however, they can loose a significant part of their activity in the chemically-hostile detergent matrix over a time period of several weeks. therefore, improving the storage stability of enzyme granulates is the main challenge in the development of a new product. the complexity of the detergent matrix implies the presence of a complicated mechanism involved in the inactivation of the enzymes. a combination of factors, such as oxidation by h o released by the bleaching agents, humidity, high temperature, autolysis of enzymes, high local ph in a granule, oxygen, and other detergent components, plays a role in the activity loss. an experimental investigation on the inactivation of the solid-state enzyme during storage has been initiated. the release rate of h o from the bleaching agent, sodium percarbonate, was determined using a simple and accurate method for measuring the gas phase h o concentration. the deactivation kinetics of pure enzyme was determined as a function of gas phase h o concentration and humidity. the preliminary results indicate that humidity plays a significant role in the inactivation mechanism of the detergent enzyme due to a possible increase in the mobility of the enzyme molecule and the surface area exposed to destructive agents. the effect of main granulate ingredients on the stability of the enzyme was investigated and the extent of the protection of each component was estimated. the study is important for the revealing of the phenomena occurring in the detergent matrix during storage. understanding the inactivation mechanism provides a valuable tool for the development of more effective protective coatings and stabilizers. the use of biosurfactants in cosmetic industry has attracted great attention of biotechnological researchers because they consist of two inexpensive, renewable and easily accessible starting agricultural materials: sugar and oil/fat. carbohydrate based products are non-toxic, biocompatible and biodegradable. in addition, the enzymatic processes present many advantages in comparison with the chemical methods, which employ high temperatures in the presense of alkalin catalysts, high-energy consuption and low selectivity of products. sugar esters present vast application, such as for antibiotics, biomaterials, surfactants, cosmetics and so on. we investigated the synthesis of sugar vinyl esters, using protease-catalysed transesterefication method applying protease from bacillus subtilis. sucrose . m and vinyl ester . m has been mixed in dimethylformamide at rpm of agitation. at first, we have studied the effects of protease from bacillus subtilis concentrations ( , , and mg/ml) as catalyst. afterwards, the influence of the temperature ( and • c). after that, the influence of the molar ratios ( : ; : ; : m/m) between vinyl laurate (ch (ch ) cooch ch ) and sucrose. subsequently, we investigated the effects of water amount, using , , and % of water in dmf. the conversion ratios of sucroseto-sucrose esters were determined decreasing sucrose measurement with hplc. the results showed that the best conditions to produce high activity on the enzymatic reaction was by using mg/ml of protease from bacillus subtilis at • c, molar ratio of : (vinyl laurate:sucrose) and adding % of water in dmf. finally, we succeeded in the characterization of vinyl sugar ester, which was produced after hours of reaction by c nmr. the results confirmed the c substituted sugar mono-ester ( -o-vinyl lauroyl sucrose). effects of oxygen transfer on ␣-amylase production by b. amyloliquefaciens nurhan güngör, güzide Ç alık bre lab, department of chemical engng, ankara university, ankara, turkey ␣-amylase (e.c. . . . ) a commercially important enzyme used in food, textile, detergent, brewing, paper, and animal feed industries; hydrolyses ␣- , glucosydic bonds in amylose, amylopectin, and related polysaccharides. optimum medium composition and influence of bioreactor operation parameters on ␣-amylase production with high yield and selectivity were determined together with the metabolic flux distributions using b. amyloliquefaciens (nrrl b- ), which is found to be a good producer of the enzyme. systematic investigation of oxygen transfer in relation with the metabolic fluxes for ␣-amylase is not available in the literature. shake-flask experiments were conducted in . dm air-filtered bioreactors in orbital shakers with agitation and heating controls (b. braun, certomat-bs ). laboratory-scale bioreactors were composed of agitation, heating, foam, dissolved-oxygen and ph-controlled . and . dm systems (b. braun, biostat q; and chemap). after separation of the cells with a sorval rc s ultracentrifuge, ␣-amylase activity was measured by the dns method bernfeld ( ) . amino acid concentrations were determined with a hplc (waters), pro-tein and organic acid concentrations were measured with a hpce (waters, quanta e) Ç alik et al. ( ) . oxygen transfer characteristics in the bioreactor systems were calculated using the dynamic method rainer ( ) . in the mass flux balance-based analyses, a pseudo-steady state approximation for the intracellular metabolites and the accumulation rates of the extracellular metabolites measured throughout the fermentations in considerations of the biochemical feature of the system were used to acquire the flux distributions. in laboratory scale, the effects of different c sources, i.e. glucose, fructose, maltose, lactose and soluble starch; n sources, i.e. (nh ) hpo , (nh ) so , and nh cl and/or their concentrations; and the operation parameters, ph and temperature on cell growth, substrate utilization, ␣-amylase and by-product concentrations, and ␣-amylase activity were investigated. in pilot scale, the fermentation and oxygen transfer characteristics of the bioreaction system together with the metabolic fluxes were determined. bernfeld, p., . methods in enzymol. : - . Ç alık, p., Ç alık, g.,Özdamar, t.h., . enzyme microb. technol. , - . rainer, b.w., . geldanamycin is a benzoquinone ansamycin produced as a secondary metabolite by the actinomycete, streptomyces hygroscopicus var. geldanus, in submerged culture. it is a broad-spectrum antibiotic and exhibits an anti-tumour activity through its interaction with the heat shock protein family of chaperone proteins. the optimal recovery of geldanamycin from fermentation broths is the focus of the presented work. the application of adsorbent resins was assessed and the viability of developing a solid phase extraction process for geldanamycin was determined. it was found that recovery of geldanamycin from fermentation broth was possible using adsorbent resins and the use of resins facilitated the recovery of a product stream of high purity. the composition of the fermentation broth had an impact on the performance of the resins and it was found that assessing performance on the basis of experimentally derived data was more apt than studying the kinetics of adsorption alone. adsorptive processes are, by their nature, difficult to optimise and this was found to be the case when optimising the recovery of geldanamycin from partially clarified fermentation broth. considerable effort was required to optimise geldanamycin adsorption, via examining the effect of environmental conditions and process system configuration, and geldanamycin desorption, via examining the effect of environmental conditions and investigating selective elution patterns. it is well known that halophilic eubacteria synthesize compatible solutes in order to face the high ionic strength environment in which they proliferate. these biomolecules are gaining more and more importance as biotechnological tools in a wide array of applications, and the recently developed novel bioprocesses enabled large-scale production of these compounds and therefore commercial distribution. however, there is still interest in the optimization of the production process of sole hydroxyectoine that was demonstrated to have a superior stabilization capacity. in this research project, we optimized growth conditions of marinococcus m to obtain high yield of hydroxyectoine. their production proved faster in the batch experiments at a higher oxygen supply, even if the stationary phase was comparable in all cases. the mf experiments showed a final biomass which was -fold that obtained in the corresponding batch process. in addition the monitoring of compatible solutes production showed that in the last h experiment hydroxyectoine accounted for - % of the total content, accumulating up to - % of the cell dry weight. studies for improving downstream process for ectoine and hydroxyectoine recovery showed that short permeabilization cycles in water are effective in a temperature range between • c and • c using a ratio : /biomass:water. moreover, we evaluated the ability of ectoine to stabilize lactic acid bacteria during freeze-drying and to protect human cells from heat stress. in particular, the compatible solutes were added to the medium of confluent keratinocytes before subjecting the cells to heat stress, or lps insult. rt-pcr and western blot analysis demonstrated the hsp b' gene over-expression in heat stressed human keratinocytes treated with ectoine. finally, we demonstrated that even at low concentration ( mm) these compatible solutes are able to diminish cell death in lactic acid bacteria due to lyophilization procedure. among all existing alternative energy sources, biomass-derived bioethanol is especially advantageous since it is clean, sustainable and potentially inexpensive. the actual production of bioethanol is divided into a pre-treatment step, an enzymatic hydrolysis step and a fermentation step. while fermentation has been practiced by humans for centuries, our knowledge of enzymatic hydrolysis is still limited. nevertheless, it is well accepted that hydrolysis is a synergism among three classes of enzymes, ␤-glucosidase, endoglucanase and cellobiohydrolase. furthermore, a complete and efficient hydrolysis is only achieved when the enzymes are in the correct proportion. the common enzyme proportions have so far been based on the natural enzyme abundance as produced by the microorganism or mainly been determined by a trial-and-error approach. in this study, however, we used metabolic control analysis (mca) as a modelling tool to gain fundamental knowledge about enzymatic hydrolysis and to design an optimal enzyme mixture. using gepasi, a free software, the degree of control of each reaction step or each enzyme towards the overall hydrolysis can be calculated. our hypothesis is that the amount of each enzyme used for hydrolysis should be proportional to the degree of control of the enzyme. with mca, a significant amount of time, labour and reagents can be saved on developing hydrolysis enzyme mixture. furthermore, this study should demonstrate the usefulness of mca on understanding enzyme-catalyzed reactions outside the cell. process optimization for fed-batch fermentation of bacillus thuringiensis subsp. israelensis arindam chaudhury, gopinathan c department of biotechnology, university of calicut, calicut, kerala india. e-mail: g achaudhury@umassd.edu (a. chaudhury) bacillus thuringiensis (bt) is a desirable biopesticide because of its low cost and lack of toxicity. the use of bt in developing countries is limited due to process complications and economic non-feasibility of the fermentation process. in the present study, we have shown how regional production, using inexpensive alternatives for carbon and protein sources, can effectively reduce the cost of mass production of bt. while using alternative media supplements, the biomass production, nor the larvicidal activity was hampered. in addition, the positive effects of sparged aeration and the indispensable role of yeast extract were also proved. this work provides the first experimental proof of delineating the sporulation process and delta-endotoxin production. the role of various buffering agents and additives in increasing biomass production and early sporulation were also investigated. for the production of coenzyme q (coq ), an electron carrier in the respiration chain with antioxidant activity. with decrease of dissolved oxygen level from to %, the intracellular coq content increased about -fold, yielding mg per g-dry cell weight at % dissolved oxygen level. azide significantly increased the intracellular coq content, with the highest value of . mg per g dry cell weight in the presence of . mm of sodium azide. however, dnp (up to m) and h o (up to m) did not affect the intracellular coq content, indicating proton gradient release and oxidative stress do not affect the synthesis of coq . these results show that restricted electron flux by limited oxygen supply and the addition of azide increases the intracellular coq content. fourier transform infrared spectroscopy (ft-ir), combined with in situ heat sterilizable attenuated total reflection (atr) probes, constitutes a promising and versatile technique for on-line monitoring of bioprocesses. the ft-ir enables rapid determinations of the medium composition without the requirement of sample withdrawal and preparation. in this work the concentration levels of the substrates glycerol and methanol were monitored on-line in pichia pastoris cultivation. partial least squares (pls) models were used for obtaining the concentration readings. the glycerol concentration measurement proved to be very reliable and reproducible in the glycerol batch phase. however, the on-line information regarding the glycerol concentration was not utilized for any process control purposes. on the other hand, the availability of on-line information about the methanol concentration proved to be crucial for the successful implementation of the cultivations. the temperature strategy in the methanol fed-batch phase utilized temperatures as low as • c. in order to keep the metabolic activity at a reasonable level the culture was therefore pushed towards the maximal substrate consumption rate, rather than being a conventional substrate limited fed-batch. as a consequence methanol accumulation occurred on occasions. without on-line information about the concentration this accumulation, if sustained, would have resulted in a poisoning of the culture, either from methanol itself, or perhaps more importantly from formaldehyde. therefore, it can be concluded that the ft-ir/atr instrument was very useful in this application. jørgensen department of chemical engineering, denmark technical university, building , dk- lyngby, denamrk. e-mail: fpd@kt.dtu.dk (f.p. davidescu) modeling biochemical reaction network in microorganisms still represents a challenge due to the very large number of enzyme catalyzed biochemical reactions, to the very complex system and to the many feed-forward and feedback regulation mechanisms. the presented approach to model such a system is based on the stochastic grey-box modeling framework proposed by kristensen et al. ( ) . this methodology consists of parameters estimation based on a prediction error method followed by different statistical tests for parameter significance and for model (in-) validity. the methodology furthermore allows estimation of unknown functional relations, e.g. kinetic rates. a set of experimental data zangirolami ( ) obtained during continuous cultures of a high enzyme producing aspergillus oryzae strain. the oxygen concentration was decreased stepwise and the substrate concentration was modified from one experiment to other. a model proposed by agger et al. ( ) is investigated on these data. the primary interest is to develop a physiologically feasible model, also at the low oxygen concentrations often found in industrial practice. microbially produced secondary metabolites such as antibiotics have tremendous economic importance. streptomyces spp. have long been identified as sources of antibiotics and chemotherapeutic compounds, synthesising over bioactive compounds. geldanamycin is a novel chemotherapeutic agent produced by streptomyces hygroscopicus var. geldanus in submerged fermentation. initial studies have focused on optimisation of media design through understanding and controlling metabolic routes of biosynthesis within the cell. geldanamycin is a by-product of the shikimate or aromatic amino acid biosynthesis pathway. stimulation of this pathway and concomitant production of geldanamycin is achievable through amino acid control. increasing concentrations of primary carbon source greatly influence biomass generation and product formation, as does the inclusion of cations such as magnesium and calcium to the fermentation media. optimisation of production media through balancing minerals, nitrogen, and carbon sources has significantly improved antibiotic yields in shake flask cultures and the development process will be extended into pilot scale through the use of bioreactors. microbiology and biotechnology research group, school of life sciences, napier university, edinburgh, eh dt, scotland. email: m.el-mansi@napier.ac.uk (m. el-mansi) synopsis: during growth of corynebacterium glutamicum on glucose or other glycolytic intermediates, pep carboxylase fulfils an anaplerotic function as it replenishes intermediary metabolism with biosynthetic precursors that are essential for growth and glutamate production. under these conditions, pep carboxylase plays a central role and this in turn is characterised by a high flux control coefficient thus rendering this enzyme an ideal target for metabolic interventions. further analysis in silico revealed that any increases in the concentration of the enzyme was accompanied by increases in flux through the enzyme itself as well as glutamate formation, presumably as a consequence of sustaining a high intracellular level of ␣-ketoglutarate; the immediate precursor for glutamate biosynthesis. a combined approach to enhance periplasmic expression of human growth hormone in escherichia coli, using a modified signal peptide from alpha amylase gene of bacillus licheniformis s.k. falsafi , , a. zomorodipour : islamic azad university of jahrom, iran; department of mol genet. national inst for genet eng & biotechnol., tehran, iran. e-mail: soheil falsafi@yahoo.com (s.k. falsafi) the alpha amylase gene signal peptide, originated from a strain of bacillus licheniformis, was shown to be able to transport its native protein, when expressed in e. coli the competence of the fusion protein being processed and translocated through the inner membrane is highly dependent on the amino acid sequences in the signal peptide. therefore, in order to increase the expression efficiency of bla signal peptide, we reconstructed the bla signal peptide coding fragment with the following modifications. two rare codons of arg (cgg) and arg (cga) and codons for leu (tta) and pro (cct), in the signal peptide were substituted with their corresponding e. coli major codons. two other changes, including phe (ttc) → leu (ctg) and ala (gcg) → met (atg), were also introduced to increase the processing efficiency. the hgh-expressing plasmid equipped with the modified bla (blaf ) was subjected for further expression analysis in a t -based expression system. the results obtained from the protein patterns of the induced bacteria indicates in high expression level of hgh preprotein (hgh::blaf ) followed by efficient transfer of the mature hgh to e. coli periplasm. (ip ) has been demonstrated to have a wide range of health benefits such as prevention and therapy of various cancers, amelioration of heart disease, and prevention of renal stone formation as well as complications from diabetes. on the hand, lower phosphorylated forms of inositol, especially inositol trisphosphate (ip ) and inositol tetrakisphosphate (ip ) are important signal transduction molecules within the cells both in plants and the animal kingdom. it has been hypothesized that at least the anticancer function of ip is mediated via these lower inositol phosphates. the diversity and practical unavailability of the individual myo-inositol phosphates preclude their investigation. phytases, which catalyze the sequential hydrolysis of phytate, render production of defined myoinositol phosphates in pure form and sufficient quantities. different phytases may result in different positional isomers of myo-inositol phosphates and therefore different biochemical properties. phytases differing in ph optima, substrate specificity, and specificity of hydrolysis have been identified in plants and microorganisms. in this paper the dephosphorylation pathway of the novel phyfauia was compared to other bacterial phytate degrading enzymes. preliminary results have shown that phyfauia converted ip into ip (myoinositol , , , , pentakisphosphates) and another isomer, which is yet to be elucidated. characterization of the novel ␤-peptidyl aminopeptidase (bapa) from sphingomonas sp. - w that cleaves synthetic ␤peptides birgit geueke, hans-peter e. kohler environmental microbiology, eawag, duebendorf, switzerland. e-mail: birgit.geueke@eawag.ch (b. geueke) non-natural peptides, which are capable of evoking a specific biological response, are currently receiving much attention. oligomers of ␤-amino acids (␤-peptides) are representing a group of pharmaceutically interesting peptides because of their very high stability towards enzymatic degradation and their ability to mimic the structure of naturally occurring biologically active peptides. the pharmaceutical potential on the one hand and the high stability on the other hand aroused interest for studies on the environmental fate and the degradation behaviour of this class of compounds. a novel bacterial strain (sphingomonas sp. - w ) that was capable of degrading short ␤-peptides was isolated from an enrichment culture. the ␤peptide degrading enzyme was purified and its gene sequence was determined (bapa). the gene encodes a ␤-peptidyl aminopeptidase (bapa) of amino acids that is synthesized as preprotein with a signal sequence of amino acids. it belongs to the n-terminal nucleophile (ntn) hydrolase superfamily and is the first peptidase that is capable of cleaving amide bonds in ␤-peptides composed of synthetic ␤-amino acids. the biochemical properties of recombinant bapa were investigated regarding its substrate specificity and possible application in the synthesis of ␤-peptides. to produce efficient strains of agaricus bitorquis (quel.) saccardo, which are resistant to high temperatures p. guler, a. ergene, s. tan kirikkale university, faculty of science and literature, department of biology, yahsihan-kirikkale in this study, the culture mushroom agaricus bitorquis (quel.) sacc. the growth of the mycelium and the fructifications under high temperature is examined. the spores taken from the mushrooms that were collected from nature were grouped as a, b, c, d, e. the spores were inoculated into malt extract agar and incubated at • c and primer mycelium was produced. the mycelium discus taken from primer mycelium in mm diameter were inoculated into the center of malt extract agar and incubated at , , , and • c separately. during the incubation period the growth of the mycelium were measured. during the growing period the radial growth speed of the mycelium were taken as criteria. the best mycelium growth for all groups was seen at • c. at • c the e group mycelium and at • c other group's mycelium did not grow. these temperatures were determined as thermal lethal point for the groups. from all the mycelium produced from all temperatures spawn was prepared and with the results taken from these, spawn calendar is prepared. in this research, the spawn was inoculated to compost with mixing system and separately put in culture rooms, temperatures as and • c. at this level the culture mushroom production techniques were used. the harvested mushrooms were inspected morphologically. at this morphological inspections the cap width, cap tissue thickness, stalk thickness and stalk long ness was taken as criteria. in the study the best growth was seen at d group mushrooms and this group mushrooms tyrosinase's activities were measured and graphics were made. introduction: viral contamination of biological products; cause many problems in viral diagnostic laboratories, blood transfusion organizations, and biological producers. bovine viral diarrhea virus (bvdv), from the pestivirus genus, is the most common viral contamination in (fetal) bovine serums (fbs). also, bvdv used as a module, for study hepatitis c virus inactivation due to its similarity in structure and genome. pulsed uv lights (puvls) have this potential to inactivate known and unknown or reemerging viruses as well as prions. two puvl with the wavelengths of and nm, were produced by q-switched nd + :yag laser in its third and forth harmonic, respectively. the energy of each pulse for nm was . mj/cm and for nm was . mj/cm . bvdv were produced and titrated in mdbk cell line. mdbk and fbs were already checked for non-cytopathic or cytopathic pestiviruses, using related ag-elisa kit. bvdv suspended in solution with the dilution of : before exposure. the quartz tube with the minimum uv-absorption in compare with air, used as a container for exposed solutions. calculation of the virus titer, . tcid /ml, was done based on the reed and muench method. bvdv suspended in pbs was exposed into the . - j/cm of puvls with the wavelength of nm and also, was exposed into the . - . j/cm of puvls with the wavelength of nm. furthermore, bvdv suspended in fbs was exposed into the , , and j/cm of puvls with the wavelength of nm and also, was exposed into the . , . , . , and . j/cm of puvls with the wavelength of nm. results: the minimum dose for inactivation of bvdv suspended in pbs with the and nm wavelengths of puvls, were and . j/cm , respectively. also, the minimum dose for inactivation of bvdv suspended in fbs with the and nm wavelengths of puvls, were and j/cm , respectively. to evaluate the fbs quality to support cell culture, treated fbs with the dose of . j/cm of nm puvls was used to grow vero cell line in successive passages. the viability of cells in two study groups was identical. the statistical evaluation of two treated groups showed no significant difference, in passages. conclusion: because inexpensive equipment can be used to produce puvls capable of handling different volumes of biologics with operational ease, this viral inactivation technique is cost effective for relevant industries. the procedure has the potential to be combined synergically with other inactivation method. puvls offer a new, nonadditive and chemically safe alternative for the treatment of fbs to inactivate adventitious viruses and to preserve the biological activity necessary for the propagation of cell culture. characterization and gene cloning of the g-resorcylic acid decarboxylase for application to selective production of g-resorcylic acid y. iwasaki , y. ishii , k. kino , k. kirimura : dept. appl. chem., sch. sci. eng., waseda univ., tokyo, japan; bme, asmew, waseda univ., tokyo, japan. e-mail: iwasaki@moegi.waseda.jp (y. iwasaki) for selective production of ␥-resorcylic acid (␥-ra, , -dihydroxy-benzoic acid) from resorcinol (re, , dihydroxybenzene) under mild conditions, we screened various microorganisms and found the reversible ␥-ra decarboxylase (rdc) as a novel enzyme applicable to carboxylation of re to form ␥-ra, in a bacterial strain rhizobium radiobacter wu- ) . rdc catalyzed the decarboxylation of ␥-ra, and regio-selective carboxylation of re to form ␥-ra, without formation of ␣-ra and ␤-ra. the molecular weight of rdc was estimated to be kda by gel-filtration, and that of the subunit was determined to be kda by sds-page, suggesting that rdc is a homotetrameric structure. the gene encoding rdc was sequenced, and a site-directed mutagenesis study revealed that the two histidine residues at positions of and in rdc are essential for the catalytic activity of rdc. through the reactions using e. coli cells highly expressing rdc, . mm ␥-ra was produced from mm re at • c for h, with a yield of %. ishii, y. et al., . biochem. biophys. res. commun., , - . laccase biosynthesis in stirred fermenters teresa jamroz, stanislaw ledakowicz, barbara sencio department of bioprocess engineering, technical university, lodz pl - , poland industrial applicability of enzymes is closely related to development of efficient methods of their production. currently, significant interest in lignolytic enzymes, including laccase, has been observed. laccase is an enzyme applied in various industrial branches and environmental processes. broad laccase applicability induces researchers to develop urgently efficient methods for its commercial production. laccase (ec. . . . . p-diphenol oxidase) is produced by cap mushrooms from the class basidomycetes. this is the so-called white rot fungi which in natural conditions appears on both living and dead wood. as shown in the practice of biotechnological processes, high-efficient strains have low resistance to destructive factors in bioreactors. hence, to preserve a proper morphology and physiological state of an organism, strictly determined culture conditions must be obeyed. this is very important in the case of basidomycetes for which submerged culture in the liquid phase is not a natural habitat. results of studies on laccase production from cerrena unicolor family are discussed. cultivation of active biomass was carried out in stirred tank and rotating disc bioreactors of different volume (b. braun of working volume dm ; fas- of working volume . dm ). experiments in both fermenters were made at impeller revolutions and min − , on a modified lindberg substrate. significant differences in the rate and yield of laccase production were reported. an almost three times higher values of laccase activity were obtained in the b. braun fermenter, at rotational speed min − . to retain suspended cells in bioreactor a filtration process can be used. the biomass is concentrated by withdrawing cell-free culture broth. if the desired product is dissolved in the broth (extracellular production), the procedure enables the continuous harvest in the cellfree permeate. an application test of filtration system for suspended biomass of aspergillus niger in submerged single stage continuous culture was presented in this report. the system is easy to construct and there is a possibility of its sterile exchange during cultivation. the culture medium contained the following substances (g/dm ): white sugar, . ; nh no , . ; mgso · h o, . ; kh po , . ; feso · h o, . . fermentations were carried out in the lab bioreactor biomer . the bioreactor was a standard cstr (continuous stirred tank bioreactor) with working volume of dm . high citric acid concentration in culture medium (p = . g/dm ), high yield of citric acid (y p/s = . %) and high efficiency coefficient (k ef = . ) were observed in single stage continuous culture with biomass retention. oxygen transfer regulates benzaldehyde lyase production in e. coli pınar Ç alık, iblab, department of chemical engineering, metu, ankara, turkey. e-mail: pcalik@metu.edu.tr the effects of oxygen transfer rate on benzaldehyde lyase (bal) production by puc ::bal carrying recombinant escherichia coli on a defined medium with . kg/m glucose were investigated in order to fine-tune the bioreactor performance, in v = dm batch bioreactors at five different conditions with the parameters at, i.e. q o /v r = . vvm and n = , , , min − and; q o /v r = . vvm and n = min − . the concentrations of the product and by-products amino acids and organic acids were determined in addition to bal activities. medium oxygen transfer rate conditions and uncontrolled ph operation at ph o = . are optimum for maximum bal activity, i.e. u/cm at h, and productivity and selectivity. on the bases of the data, response of the intracellular bioreaction network of r-e. coli to oxygen transfer conditions were analysed using a mass-flux balance based stoichiometric model that contains metabolites and reaction fluxes. the results reveal that metabolic reactions are intimately coupled with the oxygen transfer conditions. oxygen transfer rate showed diverse effects on the product formation by influencing metabolic pathways and changing metabolic fluxes. metabolic flux analysis was helpful to describe the interactions between the cell and the bioreactor by predicting the changes in the fluxes and the rate controlling step(s) in the metabolic pathways. therefore, knowing the distribution of the metabolic fluxes during the growth, and bal and by-product formations provide new information for understanding physiological characteristics of the r-e. coli, and reveals important features of the regulation of the bioprocess and opens new avenues to successful application of metabolic engineering. saprophytic mycobacterium strains belong to the best known microorganisms which have been applied to the pharmaceutical industry for the production of steroid drugs. the mycobacterial cell wall is the permeation barrier to chemical compounds, including lipophiles. using isoniazid (inh), the inhibitor of the mycolic acids biosynthesis, we were able to demonstrate increased ad production and susceptibility to antimicrobial agents. the process of sterol transformation and products accumulation was monitored using gas chromatography. isoniazid was shown to intensify ␤-sitosterol side-chain degradation by mycobacterium sp., and accumulation of -androstene- , -dione (ad) and , -androstadien- , -dione (add), which are the starting materials in the biotechnology of medically important steroids. to confirm these results, the sensitivity of the bacteria to antimycobacterial drugs was performed. the minimum inhibitory concentration mic of rifampicin and erythromycin decreased markedly in the presence of inh. this work was supported by grant nr p c of the committee for scientific research. for the purpose of high-throughput screening and to reduce experiments with animals in pharma biotechnology biosensor systems gain importance. the principle of a biosensor is the combination of cultured cells and a sensorchip device, which allows the monitoring of cellular activity. in contrast to traditional analytics with a biosensor you can measure on-line cellular activity change caused by an effector as well as the restored activity after privation of the effector (re-native activity). cmos technology can be used for the realisation of various biological sensorchips such as adhesion sensorchips, metabolical sensorchips and electrophysiological sensorchips. the standard cmos technology allows a high reproducibility of the chips, the integration of electronic components on the chip, which reduces the amount of external devices and the combination of different sensors on one chip. in cooperation with the semiconductor company micronas and the biotech company bionas we have realised different types of cmos sensorchips to measure adhesion of a cellular monolayer with interdigitated electrodes (ides), metabolical activity via acidification with ion-sensitive field effect transistors (isfet) and sponteneous neuronal network activity with passive palladium electrodes. microbial agents have been applied to the different stages of pulp and paper processing. the work presented describes a study on the effect of applying ligninolytic enzymes, such as a laccase plus mediator system, on a variety of different types of pine and eucalyptus pulps and subsequently subjecting these to different ageing processes. industrial pulps were obtained from different portuguese pulp and paper companies. the pulps used were ( ) unbleached pine pulp from portucel tejo; ( ) unbleached eucalyptus pulp from portucel setúbal; ( ) bleached eucalyptus pulp from portucel setúbal; and ( ) pulp made from recycled paper from renova s.a. several types of handsheets were produced with different grammage namely, and g/m . the prepared handsheets were subject to an aging sequence in three different chambers: ultraviolet radiation (wavelength of nm), temperature ( • c) and moisture ( %); and thick saline fog at a concentration of % and temperature of • c. in order to evaluate the effect of moisture cycles and temperature, two aging sequences were used for each type of handsheet. in the first, the moisture varied ( , and %), while the temperature was held constant ( • c); in the second the temperature varied ( , and • c) and the moisture was held constant ( %). following the aging phase, the handsheets were subject to several chemical (viscosity and index kappa) and physico-mechanical (colour, tensile breaking strength, stretch and the bursting strength) tests in order to characterize the effect of the aging conditions. results will be presented describing the effect of application of the laccase-mediator system on the optical and mechanical properties of the prepared handsheets. fundação para a ciência e a tecnologia, project pocti/ agr/ / . aspergillus niger is a filamentous fungi widely used in industry. its growth as freely dispersed hyphae leads to an increase in the medium viscosity and to problem of mass transfer, especially oxygen transfer. oxygen acts both as final electron acceptor in the mitochondrial chain and as nutrient for the biosynthesis of unsaturated fatty acids and sterols. thereby, a lack of oxygen affects the nadh/nad ratio, the atp production, the growth and has a strong influence on the physiology of the microrganism. in the present study, the metabolic changes of a. niger in response to a lack of oxygen was investigated using oxygen limited chemostats combined with nitrogen pulse. under these conditions, the main consequence of a sudden decrease of oxygen availability is an increase in the mannitol production. this work showed that the mannitol biosynthesis, involving the enzyme mannitol- -p dehydrogenase, helps the reoxidation of nadh when the final electron transport acceptor, oxygen, is limiting. investigation of the lipase activity of the bacteria isolated from olive mill wastewater sevgi ertugrul , nur koçberber , gönül dönmez , serpil takaç department of biology faculty of science ankara university beşevler ankara turkey; department of chemical engineering faculty of engineering ankara university tandogan, ankara, turkey the bacteria that could grow on media containing olive mill wastewater (omw) were isolated and their lipase production capacities were investigated. the strain possesing the highest lipase activity among strains grown on tributryin agar medium was identified as bacillus sp. the effect of ph on the lipase activity of the strain was investigated in tributryin medium and ph was found to be the optimal. the liquid medium composition was improved by adding different carbon sources and fatty acids into tributryin medium -omitted tributryin -to increase the enzyme activity. the cultivations were performed at • c and ph . lipase activity of the bacillus sp. was measured spectrophotometrically through the hydrolysis of p-nitrophenol palmitate. among the media containing different compositions of tricapryn, trimyristin, tributyrin, triacetin, tween , glycerol-trioleate, glycerol-trioctanoate, glycerol-tridodecanoate, omw, glucose, and whey; the medium consisted of % whey + % glycerol-trioleate was found to give the highest lipase activity. cultivation of bacillus sp. in the optimum medium at ph = and • c for h was resulted in the extracellular and intracelluar lipase activities of and u/ml, respectively. this study was supported by ankara university biotechnology institute (project no: - and - ) . under different abiotic stresses, cell growth and metabolic activity are highly influenced in all types of living organisms medium osmolality is usually one of those factors affecting different types of biological systems in different ways. however, even in the same organ of higher eukaryotes the degree of osmoregulation mechanism is highly variable in different types of cells. therefore, studying the effect of osmotic stress on mammalian cell is very important subject for particular cell line. the effect of hyperosmotic pressure on the kinetics of cell growth of and metabolic activity of mesenchymal stem cells (mscs) and two industrially important cell lines, hybridoma cells and human embryonic kidney cell (hek) were investigated in batch cultures at different osmotic pressures in the range from to mosm kg − . in case of mscs cells, the maximal specific growth rate [] of . [h − ] associated with the highest specific glucose consumption rate [-q gluc ] of . × − [mol cells − h − ] was obtained in medium of mosm kg − . in case of hybridoma cells, osmotic pressure showed not only influence on the kinetics of cell growth and metabolism but also on the monoclonal antibody production. the maximal mab production was obtained in case of cells cultivated under osmotic pressure of mosm kg − . further increase in osmotic pressure resulted in significant reduction in growth rate as well as mab production. on the other hand, hek cells were more sensitive to osmotic pressure in industrially used serum free medium and the addition of serum decreased the inhibitory effect of high osmotic pressure on the cells. gustavo g. fonseca , , andreas k. gombert , elmar heinzle , christoph wittmann : biochemical engineering, saarland university, saarbrücken, germany; chemical engineering, são paulo university, brazil kluyveromyces marxianus cbs is a potentially interesting yeast strain characterized by a high capacity of conversion of substrate into biomass. however, this yeast has been only marginally studied so far. therefore, we performed a metabolic characterization in batch and chemostat cultures at dilution rates of . , . and . h − . the specific rate of o consumption (qo ) increased with dilution rate from . to . mmol (g dw) − h − . the respiratory coefficient remained almost stable around . for all metabolic states investigated. even at the dilution rate of . h − , which is close to the maximum growth rate of the strain of . h − , no significant overflow metabolism was observed. the concentration of extracellular metabolites increased with the dilution rate, but remained below % of the carbon consumed as glucose. all carbon balances closed near % underlining the consistency of the data. in contrast to s. cerevisiae the respiratory capacity of k. marxianus cbs is not strongly influenced by the dilution rate in aerobic chemostat or batch cultures, indicating its high potential for biomass-directed applications. a thermostable l-arabinose isomerase for enzymatic production of d-tagatose o. hansen, f. jørgensen, p. stougaard department of enzyme technology, bioneer a/s, kogle allé , dk- hørsholm, denmark. e-mail: och@bioneer.dk (o. hansen) d-tagatose, an isomer of d-fructose, is a low-calorie bulk sweetener with a sweetness equivalent to sucrose. d-tagatose has obtained gras approval for use as a food ingredient, and is currently produced by chemical isomerization of d-galactose, which may readily be obtained by hydrolysis of lactose. structurally, d-galactose is closely related to l-arabinose, and it has previously been shown that some variants of l-arabinose isomerase (araa) may catalyze the conversion of d-galactose to d-tagatose, in addition to the metabolic conversion of l-arabinose to l-ribulose. we have screened a number of bacterial araa enzymes for their ability to catalyze the isomerization of d-galactose to d-tagatose. the best enzyme was found in the thermophilic bacterium thermoanaerobacter mathranii (dsm ). the araa gene of t. mathranii was cloned, sequenced and expressed in e. coli. amino acid sequence comparisons of the t. mathranii sequence and other known araa sequences showed a relatively low sequence identity of about %, indicating a distant phylogenetic relationship to the other members of the l-arabinose isomerase group. the t. mathranii enzyme was thermostable with optimal activity at • c and it required manganese ions. unlike other araa variants, the t. mathranii enzyme showed k m values in the same order of magnitude for l-arabinose and d-galactose, suggesting that this enzyme is a versatile isomerase capable of isomerizing structurally related aldoses. the enzyme was immobilized by chemical cross-linking of a crude e. coli cell homogenate, and the immobilized enzyme efficiently converted d-galactose into d-tagatose. currently, we are developing an enzymatic method for industrial production of d-tagatose using the immobilized enzyme. the agricultural production can be negatively affected by different pest insects (pi) and the use of chemical insecticides (chi) has been the traditional method for controlling pi during decades. nevertheless, there are various ecological implications due to the extensive application of chi. a viable alternative for the use of chi in certain agro-systems, is the use of entomopathogenic nematodes (epn) of the genera steinernema and heterorhabditis that are natural pathogens for different pi; besides, the presence of a symbiotic bacterium is necessary for an effective entomopathogenic activity can take place. the nematode/bacterium complex does not represent a risk for the environment. different authors propose that the best alternative for the massive production of epn is the submerged culture within bioreactors; nevertheless, more research is required to have really robust processes. particularly, information regarding the actual hydrodynamics during epn production and its relation with the epn productivities are scarce, among other aspects. the present study deals with the hydrodynamic characterisation during the production of the epn steinernema carpocapsae and its symbiont bacterium xenorhabdus nematophilus, in submerged monoxenic culture in an internal-loop air-lift bioreactor (v l = . l) using two culture media: one of them containing whey and the other one, agave juice, aguamiel, (agave spp.). process viscosity of the culture broth was determined along the time, exhibiting a maximum value of mpa s. moreover, it was determined that the hydrodynamic conditions were always located within the laminar region (re < ). at the experimental conditions tested, it can be inferred that the epn productivity are more sensitive to changes in the culture medium composition than on the prevailing hydrodynamic conditions during the fermentations. bacillus thuringiensis is a gram-positive bacterium used as a biological pest control agent. moreover, it is able to produce, several biologically active molecules such as bacteriocins and hydrolytic enzymes among which chitinases that play double roles, fungicide and improving the insecticidal effect of b. thuringiensis deltaendotoxins. a newly isolated b. thuringiensis subsp. kurstaki strain bupm , was shown to produce a novel bacteriocin named bacthuricin f . the highest bacteriocin activity was found in the growth medium and evidenced in the late exponential growth phase. upon purification of bacthuricin f , the specific activity was increased -fold. this bacteriocin was heat-stable up to • c and resisted up to ph . . its molecular mass, determined by mass spectrometry was . da. direct n-terminal sequencing of bacthuricin f revealed the following sequence: dwtxwsxl. the latter was unique in the databases. bacthuricin f was active against bacillus species while it had little or no effect on gram-negative bacteria. the bacteriocin produced by the b. thuringiensis strain bupm respond to both criteria of thermostability and stability to low phs. thus, it could be used as a source of bacteriocin active against related species of bacillus harmful for agricultural products and as food preservative. the other example of antimicrobial compound produced by b. thuringiensis is a chitinase. we describe the selection of b. thuringiensis high chitinase-producing strain bupm , and the characterization and the heterologous expression of a novel chitinase encoding gene. the cloning and sequencing of the corresponding gene named chi showed an open reading frame of bp, encoding a amino acid residue protein. both nucleotide and amino acid sequences similarity analyses revealed that the chi is a new chitinase gene, presenting several differences from the published chi genes of b. thuringiensis. the identification of chitin hydrolysis products resulting from the activity, exhibited by chi through heterologous expression in e. coli revealed that this enzyme is a chitobiosidase. the addition of the sequence of chi to the few sequenced b. thuringiensis chi genes might contribute to a better investigation of the chitinase "structure-function" relation. cloning and characterization of s-adenosyl-l-methionine synthetase from pichia ciferrii dscc - kwon-hye ko , gee-sun yoon , gi-sub choi , joo-won suh , yeon-woo ryu s-adenosyl-l-methionine(sam) has an important role for dna methylation and cell signaling. sam was synthesized from methionine and atp by sam synthetase and play an pivotal function in the primary and secondary metabolism of cells. recent studies have revealed in the effect of sam in case of morphological differentiation in both eukaryotes and prokaryotes. the p. ciferrii produces large quantities of sphingoid base. tetraacetylphytosphingosine(taps), which is a precursor of sphingolipid, could be used for the production of pharmaceuticals and cosmetics. we isolated sam gene from p. ciferrii and cloned it into expression vector for e. coli and p. pastoris, respectively. an . kb sam-s gene fragment was isolated by low-strigency pcr using degenerated primer. by the analysed primary sequence deduced from dna sequence, this gene included conserved domains similar with other well-known sam synthetase. first of all, sam synthetase gene cloned pgem-t vector and subcloned into histidine tagging system to purify the expressed protein using metal chelating resin. typical characteristic analysis of this enzyme is underway. metabolic networks offer a large variety of different synthesis pathways starting from cheap substrates and leading to interesting high-value compounds, i.e. metabolites. in case an interesting pathway can be disconnected from the remaining metabolic network, the perforated cell or the crude extract could be used for a one-pot multi-step synthesis of the desired compound. pathway isolation, achieved by deletion of genes encoding gene products enabling side reactions, interferes with the viability of the organism, which is a requisite for the production of the system of biotransformation (sbt). in this work, a rational systems biology-derived approach is presented for the design of a sbt. it is illustrated for a sbt allowing for production of dihydroxyacetone phosphate. the design procedure comprises three steps: (i) a production pathway is identified in the metabolic network of e. coli. the e. coli pathway is complemented by two additional enzymes in order to obtain a fully energy and redox balanced production pathway. (ii) an optimal combination of gene knockouts is designed and a suitable growth medium composition is identified, both by a model-based approach: flux balance analysis of a genome-scale metabolic network is used to predict enzyme expression within the wild-type organism on different media, while a mixed-integer optimisation is employed to identify viable mutants as this approach strongly depends on the quality of the fba prediction, available regulatory information on the usage of metabolic pathways and thermodynamic constraints were taken into account. (iii) thermodynamic analysis of the obtained, partially branched sbt reaction cascade revealed the extent of loss in yield by the remaining side reactions. in summary, this systems biology-driven approach potentially enables the substitution of a elaborative multi-step synthesis process by a one-pot enzyme reaction cascade. institute of industrial biotechnology, inha university, incheon - , korea. e-mail: leecg@inha.ac.kr (c.-g. lee) algal biotechnology is drawing increasing interest due to its potential as a source of valuable pharmaceuticals, pigments, carbohydrates, and other fine chemicals. currently, its application is being extended to the areas of wastewater treatment and agriculture. however, lack of suitable photobioreactors (pbrs) makes the cost of algally-derived compounds higher than those derived by chemical synthesis and thus has prevented widespread use of algal cultures. the culture of algae prior to the late s was apparently restricted to laboratory scale operations. experiments on outdoor algal mass production began in the late s with nearly concurrent development of experimental culture facilities in germany and the united states. for the next two decades, outdoor mass culture of algae was undoubtedly the hottest topic in the algal biotechnology area. recent developments of high-density pbrs enable the production of valuable biologically active compounds by algal mass cultures. however, light is almost always the limiting factor in high-density photobioreactors. key factors for successful photobioreactors will be discussed and various photobioreactors will be analyzed and compared for their advantages and disadvantages. the new techniques, such as pigment redcution and application of flashing light and lumostatic operation, will be discussed for possible solutions to overcome the light limitation in high-density microalgal cultures. a quick and reliable method for screening fungal transformants for specific genetic modifications is essential for many molecular applications, for example when one tries to develop a transformation system for a new fungal host. southern analysis is laborious and time consuming. several colony hybridisation methods have been developed for the analysis of a large number of transformants. unfortunately these methods suffer from different disadvantages such as non-specific binding, a limited usability to screen for specific integration and the fact that these procedures always take a few days (van zeijl et al., ) . recently, methods for pcr-based analysis of fungal transformants have been described. most of these methods require high quality dna. many methods for dna extraction from fungi have been described in the past few years. these methods often are tedious, time consuming, costly or limited to a small number of samples each time. most of the available protocols include the growth of mycelium in a liquid culture, followed by freeze-drying or maceration in liquid nitrogen and grinding of the frozen material to break the cell walls (cassago et al., ) . lately a few methods have been described to isolate dna from fungi suitable exclusively for pcr and appropriate for the simultaneous treatment of a large number of samples. some methods also describe the direct use of mycelium (cooke et al., ) in the pcr-reaction mixture. we compared the applicability of a few rapid dna extraction methods for myrothecium gramineum and tested the resulting dna samples on there suitability for pcr-applications. myrothecium gramineum is a filamentous ascomycete used in ongoing research as a new cloning and expression host. five methods were tested. in four of these methods dna was extracted from mycelium (goodwin et al., and aljanabi et al., ) or spores (ferreira et al., and xu et al., ) prior to pcr. a fifth assay used mycelium straight in the pcr-reaction mixture. only this last method seemed useful for myrothecium to isolate dna suitable for pcr. fragments up to bp were amplified. cheese whey is a liquid effluent from cheese-making processes. there is an increased interest in the economic utilization of whey produced by the dairy industries, because the whey is a pollutant, due mainly to its lactose content. the goal of this work was to find the most suitable values of some fermentation parameters for lactic acid production from whey by a lactic acid bacterium, lactobacillus helveticus (atcc ). the effects of lactose content, temperature, ph and the supplementation with yeast extract were investigated using surface response methodology. a composite central design was used with three center points, making a total of operational conditions. the region of maximum production is outlined by the following intervals: temperature around • c; lactose concentration between and g/l; concentration of yeast extract between and g/l; ph between and . . fermentation studies on a continuous fermentative process coupled to a vacuum flash evaporator were carried out in lab scale equipment. the phases of this work consisted in an assembly and instrumentation of the prototype and elaboration of a supervisory system coded in labview . , which allows the data acquisition and control through personal computers. the experiments in continuous fermentation used saccharomyces cerevisiae and sugar cane molasses as substrate. the analytical follow up was done through analysis of total reducing sugars, ethanol, glycerol, dry mass and viable cells. the system worked for months uninterruptedly, producing an alcoholic solution at the condenser with • gl. the fermentation operated with concentrations of ethanol at • gl, which is a weakly inhibitory value for the yeast of the process, even when fed with concentrated cane molasses, containing up to g/l of sugar. the result meets the initial goal, which was to operate the system with low level of ethanol and to guarantee high productivity, even in high concentrations of sugar in the feeding. the results showed that system productivity was superior to that of the conventional continuous process. lactic acid (la) is a versatile chemical, used as an acidulant, flavor and preservative in the food, pharmaceutical, leather and textile industries, and for production of biodegradable poly lactic acid (pla). l(+)lactic acid is the only optical isomer for use in pharmaceutical and food industries because human body is only adapted to assimilate this form. in this research, lactic acid production was improved on l fermentor. in our experience, among six strains of lactobacillus were examined for the production of l(+) lactic acid, lactobacillus casei ssp. casei atcc was selected as a highest l(+) lactic acid producer. optimized medium used for lactic acid production contained (per l) g glucose, g whey powder and g corn steep powder. for a homofermentative process, ph . was found to be optimal. in order to avoid product inhibition, the produced lactic acid was neutralized using calcium hydroxide. maximum production and productivity of lactic acid in batch system, were g and . g/lh, but in fed batch system, after feeds of glucose, production and productivity increased up to g and g/lh. saleh a. mohamed molecular biology dept., national research centre, cairo, egypt an extracellular polygalacturonase (pgii) from trichoderma harzianum was purified to homogeneity by two chromatography steps using deae-sepharose and sephacryl s- . the molecular weight of t. harzianum pgii was , da by gel filtration and sds-page. pgii had isoelectric point of . and optimum ph of . . pgii was very stable at the ph . . the extent of hydrolysis of different pectins by enzyme was decreased with increasing of degree of esterification (de). pgii had very low activity toward nonpectic polysaccharides. the apparent km value and kcat value for hydrolyzing polygalacturonic acid (pga) were . mg/ml and s − , respectively. pgii was found to have temperature optimum at • c and was approximately stable up to • c for min of incubation. all the examined metal cations showed inhibitory effects on the enzyme activity. , phenanthroline, tween , tween , triton x- and sds had no effect on the enzyme activity. the rate of enzyme catalyzed reduction of viscosity of solutions of pga or pectin was higher three times than the rate of release of reducing sugars indicating that the enzyme had an endo-action. the storage stability of the enzyme in liquid and powder forms was studied, where the activity of the powder form was stable up to one year. these properties of t. harzianum pgii with appreciable activity would be potentially novel source of enzyme for food processing. tarek m. mohamed, biochemistry division, chemistry department, tanta university, tanta, egypt preparation of peroxidase from horseradish, which could be used for commercial applications such as diagnostic kits, was occurred through a simple reproducible method consisting of extraction, ammonium sulphate precipitation, filtration through non-binding protein filter and lyophilization. the purification method was developed allow the preparation of mg of enzyme from kg of horseradish roots. the one mg of enzyme contains units of peroxidase. this value is similar to that produced by sigma ( - unit mg − powder). the final preparation is salt free reddish brown powder with free ammonia content less than . g − units. the rz value (a /a ) of the enzyme, which is a good criterion of purity and heme content, was . . the lyophilized enzyme was stable at − • c for at least one year. the liquid form of the enzyme in presence of . % sodium azide was stable up to days at • c, while it lost most of activity at room temperature in the same period. the properties of horseradish peroxidase including km, optimal ph and temperature, activation energy, thermal stability and effect of different compounds were studied. the applicability of this enzyme in determination of serum glucose was performed. the analysis of glucose in human sera gave results using the kit containing the prepared peroxidase similar to those obtained with a commercial glucose kit. lactobionic acid production using lactose oxidase: from laboratory to l scale mikkel nordkvist , per munk nielsen , peter budtz , john villadsen : center for microbial biotechnology, technical university of denmark, dk- lyngby, denmark; novozymes a/s, dk- bagsvaerd, denmark; chr. hansen a/s, dk- hørsholm, denmark. e-mail: mnq@biocentrum.dtu.dk (m. nordkvist) currently, lactobionic acid is mainly a high-price specialty product used, e.g. in solutions for organ stabilization. however, lactobionic acid can also be used as a biodegradable cobuilder in detergents, and it has several applications in food technology. with lower production costs it has the potential to become a bulk chemical. the kinetics for the oxidation of lactose to lactobionic acid by a new carbohydrate oxidase was studied in a l bio-reactor with control of ph, temperature, and dissolved oxygen. the byproduct hydrogen peroxide has a negative influence on the lactose oxidase enzyme, and hence additional experiments were made with addition of catalase to remove hydrogen peroxide, thereby also providing extra oxygen. on the basis of the experiments in l scale, experiments were performed in a l reactor equipped with a new system for dispersion of air to supply the necessary oxygen for the oxidation. the aeration system in the large scale reactor was able to supply oxygen sufficiently fast to give the same production rate, at low values of the air flow rate and the energy input, as was obtained in the high-performance laboratory reactor. the non-characterized gene previously proposed as d-tagatose -epimerase from agrobacterium tumefaciens was cloned and expressed in escherichia coli. the expressed enzyme was purified by affinity chromatography on histrap hp, desalting chromatography on hiprep / , and gel filtration chromatography on sephacryl s- hr with a final specific activity of . u/mg. using maldi-tof-ms, the native protein was estimated to have a molecular mass of , da and a monomeric structure. the purified enzyme exhibited maximal activity at • c and ph . without the addition of metal ions and at • c and ph . with . mm mn + . among various metal ions, mn + was the most effective divalent cation for d-fructose epimerization activity. the addition of mn + significantly increased the thermal stability and the epimerization activity with other ketoses such as d-psicose, d-tagatose, d-ribulose, d-sorbose, and d-xylulose. the activity, substrate affinity, maximum velocity, and catalytic efficiency (k cat /k m ) of the enzyme for d-psicose were higher than those for d-tagatose, which suggests that the enzyme is not d-tagatose -epimerase but d-psicose -epimerase. the equilibrium ratio between d-psicose and d-fructose was : at • c with . mm mn + . when the enzyme was used at u/ml, dpsicose was produced at g/l from g/l d-fructose containing mm mn + after min, corresponding to a conversion yield of . %. the role of ammonium ions in glucosamine formation during the citric acid fermentation process by aspergillus niger m. papagianni , f. wayman , m. mattey : department of hygiene and technology of food of animal origin, school of veterinary medicine, university of thessaloniki, thessaloniki , greece; department of bioscience, university of strathclyde, glasgow, g xw, uk. e-mail: mp @vet.auth.gr (m. papagianni) stoichiometric modeling of the citric acid fermentation process by aspergillus niger, in -l stirred tank reactor, indicates that nh + ions combine with a c-containing metabolite inside the cell to form a nitrogen compound which is then excreted by the mycelium. the close correlation between calculated and experimental profiles indicates that this metabolic process is rapid and takes place before the c-structure of the glucose has been greatly altered by glycolysis or the pentose phosphate pathway. hplc analysis identified glucosamine as the product of this relationship. a clear effect of medium concentration of nh + on glucosamine formation was observed when fermentations carried out with optimal and sub-optimal ammonium concentrations. (nh ) so addition when medium nitrogen was depleted, enhanced the formation of new cells from the tips of fragmented hyphae and led to glucosamine accumulation in amounts depending to pulse concentration. the fungus reacts in excess ammonium by converting it to glucosamine, to be utilized later when a regeneration process takes place with fragmentation of vacuolated hyphae and subsequent regrowth, depending always on the culturing conditions. however, depending on carbon and ammonium concentration in medium, glucosamine can be secreted in concentrations as high as g/l. about microorganisms originated from traditional korean food origin were screened for efficient palatinose production. an isolate designated fmb was exceptionally efficient in sucrose-palatinose conversion activity. conversion of sucrose into palatinose by fmb was much faster than a reference strain of erwinia rhapontici. fmb is a gram negative, facultatively anaerobic, motile, noncapsulate, and straight rod-shaped bacterium producing acid from glucose. based on api and s rdna analyses, fmb was determined to be enterobacter sp. the maximum conversion of % sucrose to palatinose and trehalulose by enterobacter sp. fmb was achieved within h. the preliminary dna sequencing result of the gene corresponding to sucrose isomerase of enterobacter sp. fmb revealed that it showed % similarity to that of klebsiella sp. (??). within the scope of an r&d project developed in collaboration with leather tanning portuguese industrial partners a screening of new proteases to be used in the industrial process was performed. a bacillus subtilis strain isolated from alkaline spent purge liquor was shown to be a promising protease producer. microorganism growth was studied for optimisation of temperature, agitation, ph and medium composition either for biomass or proteases production. optimal growth temperature is different for maximum biomass growth ( • c) and optimal proteolytic activity ( • c) yielding biomass specific growth rate of . and . h − , respectively. the achieved proteolytic activities were . and . u/ml of protease, respectively. optimised medium composition ( g/l beef extract, g/l yeast extract, g/l peptone and . g/l cacl ) yielded a specific growth rate of . h − and . ku/l of protease, in shake flask experiments. bioreactor experiments (from to l) with the selected medium were performed at • c in order to test aeration rate ( and vvm), stirring ( - rpm) and ph (uncontrolled, controlled at and ). best protease activity was u/ml in l bioreactor without ph control at rpm and vvm. the proteolytic extract was characterized and compared to commercial bates. results indicate that these proteases can be employed in the purge phase of the leather tanning process in industry. the gram positive bacterium bacillus megaterium is known for its capacity to produce exoenzymes including amylases, proteinases, and penicillin amidase at industrial scale. here, we describe the development of various vectors for the production and export of recombinant heterologous proteins employing b. megaterium signal peptides. the target gene can be cloned directly adjacent to the signal peptide coding sequence (bart et al., ) . this arrangement allows for a correct n-terminal sequence of the mature protein after processing by the signal peptidase sipm. using this newly developed protein production and export system lactobacillus reuteri levansucrase (van hijum et al., ) was secreted in significant amounts (∼ mg/l) into the growth medium. fusion of the recombinant levansucrase to affinity tags allowed one-step purification of the recombinant protein from the growth medium. however, fused peptide tags resulted in a decreased secretion of the fusion protein. . mg his -tagged levan-sucrase were purified per litre of culture. the system was further enhanced via coexpression of a gene for the signal peptidase sipm (malten et al., a) and deletion of the gene for the extracellular protease nprm. developed new tools allow for various strategies of integrated high level production, export and purification of heterologous proteins in b. megaterium. methods for high-throughput screening of secreted enzymes are under development. the determined sequence of the b. megaterium genome, studies using high-cell density cultivations (malten et al., b) and proteome data from batch fermentations implicate new targets for directed genetic optimization of b. megaterium production and secretion strains. novel strong and inducible promoters are currently under investigation. toru matsui , naoya shinzato , hisashi saeki , hitoshi matsuda : center of molecular biosciences, university of the ryukyus, okinawa - , japan; japan energy co., japan. e-mail: tmatsui@comb.u-ryukyu.ac.jp (t. matsui) optically active epoxides are considered as the potential intermediate for chiral drugs synthesis. although s-styrene oxide (so) have been extensively investigated using styrene monooxygenase from pseudomonas sp., microbial production of r-so with high enantiomeric excess was hardly examined. in this study, r-so producing bacteria from styrene was screened using various alkene assimilating bacteria. r-so with the highest ee (ca. %ee) was obtained using ethene utilizing bacteria, identified as mycobacteirum sp., while produced relatively lower at around %ee when using propene utilizing bacteria. the alkene monooxygenase gene homologue sequence amplified from the genomic dna revealed a significant similarity to that of etnabc. these bacteria also showed stereoselective degradation of racemic so, suggesting that the produced so might be further stereoselectively degraded to increase the ee. the ethene utilizing bacteira produced not only r-so but also s-epichrolhydrin at high ee.when using arylchloride as the substrate. this research was supported by nagase science and technology foundation. the secretion efficiency of the escherichia coli sec pathway is dependent on the growth phase but not on protein size f.j.m. mergulhão, g.a. monteiro centro de engenharia biológica e química, instituto superior técnico, av. rovisco pais, - lisbon, portugal. e-mail: filipem@alfa.ist.utl.pt (f. mergulhão) the secretion efficiency of the escherichia coli sec pathway was evaluated through the expression of green fluorescent protein and human proinsulin fusion proteins. translocation to the periplasm is dependent on the growth phase of the bacterial culture and the highest secretion efficiency is attained in mid-exponential phase. secretion performance is independent of protein size ( - kda) and even when the amino acid composition of the secreted proteins is very similar, the amino acid distribution within the protein can affect translocation. in silico prediction analysis suggests that proteins that are prone to form ␣-helix structures are more efficiently translocated. culture medium composition plays an important role on secretion performance with the highest secretion results being obtained in minimal medium. streptokinase is a common fibrinolytic drug. that is used in thrombolytic therapy for long time. to compare with another thermbolytic drugs like tpa, it has lot of advantages. in this present research dna was extracted from s. equisimilis h a for the first time in iran. streptokinase gene was amplified by using two forward primers and one reverse primer. a common restriction enzyme, bamh-i, was used for cloning. both ends of the pcr products (full length: bp and mature section: bp) and the restriction site on mcs of pqe- vector were digested. in this study, pqe- expression vector was used with high level expression ability for production of recombinant fusion streptokinase with simplifying the purification by employing affinity-metal chromatography method. in addition, the cloning results were controlled by double digestion and sequencing. takesono oxidation of short-chain iso-alkanes was studied with propanegrown resting mycelia of scedsporium sp. a- . isobutane was oxidized to tert-butanol, but not to isobutanol. isobutanol was used for growth, but both isobutene and tert-butanol were not used for growth. isopentane was oxidized to -methyl- -butanol, -methyl- -butanol, and -methyl- -butanol but not to -methyl- -butanol. -methylpentane was oxidized to -methyl- -pentanol, -methyl- pentanol, and -methyl- -pentanol but not to -methyl- -pentanol or -methyl- -pentanol. -methylpentane was not oxidized. oxidation of branched alcohols was also studied. application of nadph-dependent . -diketo-gluconic acid reductase for production of l-ascorbic acid claudia pacher , : division of food biotechnology, department of food sciences and technology, boku, university of natural resources and applied life sciences, muthgasse , a- vienna, austria; research centre applied biocatalysis, petersgasse , a- graz, austria. e-mail: claudia.pacher@boku.ac.at ascorbic acid is an organic acid with various applications in the food and pharmaceutical industries. at present, the majority of commercially manufactured vitamin c is synthesized via the reichstein process, which is highly energy-consuming, involves considerable quantities of organic solvents and gives an overall yield of about %. therefore, during the past decades a lot of research was done to develop biotechnological alternatives for the synthesis of reichstein intermediates by enzymatic or fermentative means, which show some advantages regarding costs and environmental friendliness. one of the fermentation routes runs via . -diketo-d-gluconic acid ( . -kdg), produced by pectobacter (erwinia) cypripedii. this compound has been reduced by a nadph-dependent . -diketogluconic acid reductase (dkr) from corynebacterium glutamicum to the key intermediate -keto-l-gulonic acid ( -klg) before chemical rearrangement leads to the final product. for economical reasons we wanted to express dkr heterologously. based on our long term experience with coenzyme regeneration we wanted also to perform the reaction in homogeneous solution. the spent coenzyme of nadph-dependent dkr has been regenerated by a second isolated nadp-dependent enzyme like glucose dehydrogenase. we describe here the recombinant production, purification and characterization of dkr and the results of enzymatic -klg formation by using the recombinant enzyme in a homogeneous system with conjugated coenzyme regeneration. grp residing in endoplasmic reticulum (er) functions as a molecular chaperon by associating transiently with incipient proteins as they traverse the er and aiding in their folding and transport. furthermore, the protein can also be induced under various stress condition such as glucose starvation, inhibition of protein glycosylation by tunicamycin, blockage of vesicular trafficking by brefeldin a and er-calcium-atpase pump inhibition by thapsigargin. thus, substances that directly down and up-regulate grp transcription are expected to be useful for treatment of cancer and alzheimer's disease, respectively. in the course of our screening program to obtain substances, which regulate grp expression, we first constructed an assay system monitored by the expression of a reporter gene. hela cells, which are transformed with luciferase gene under the control of grp promoter designated as hela c cells, respond sensitively to luciferase grp induction by er stress such as treatment of tunicamycin. by using this screening system, we isolated pyrisulfoxin as an up-regulator of grp , and valinomycin, citreoviridin and alternariol as down-regulators. detailed studies on other biological activities were now under way. pdh is an enzyme that was described only several years ago in a number of ecologically related litter-decomposing fungi (agaricales, gasteromycetales). it catalyzes the c- and/or c- oxidation of several aldopyranoses to the respective keto sugar derivates. pdh shows a very broad substrate range, oxidizing almost all major sugar components of wood polysaccharides, and is implicated to play a role in lignocellulose degradation. agaricus bisporus, the white button mushroom, is an economically significant agricultural crop. the cultivation, which is done by solid-substrate fermentation on straw-and hay-based composted substrate, is sometimes seen as one of only few economically feasible methods for bioconversion of lignocellulosic agricultural waste material. deeper insight in the physiological role of pdh may provide help for mushroom growers to increase yield, improve quality or make new sources of raw materials utilizable. we amplified a fragment of the pdh gene with degenerated primers derived from internal peptide sequences. the screening of a genomic library led to the isolation of the pdh gene. subsequently we amplified a cdna clone by rt-pcr and investigated the transcriptional regulation by different carbon sources on a defined minimal medium. optimization of monoclonal antibody production processes with simulation and scheduling tools demetri petrides, charles siletti intelligen inc., scotch plains, nj , usa. e-mail: dpetrides@intelligen.com (d. petrides) this presentation will review the state of the art in batch process simulation and scheduling tools and their applications in the design and debottlenecking of integrated biopharmaceutical processes. a systematic methodology will be presented for identifying and eliminating size, time, and throughput bottlenecks that limit production in single and multi-product facilities. the methodology will be illustrated with an industrial case study dealing with the optimization of a multi-product facility that produces therapeutic monoclonal antibodies (mabs). mab processes are characterized by a long bio-reaction time (e.g., . - weeks for fed-batch operation and - months for perfusion operation). the cycle time for processing a lot in the recovery and purification train typically takes - days. consequently, one way of increasing throughput is by installing extra bioreactors that operate in staggered mode and utilize the same recovery train. the result is that multiple batches may be at different stages of completion at any given time. since cleaning equipment (e.g., cip skids) and buffer preparation and holding tanks are shared by multiple steps across many batches, this type of operation leads to time/scheduling bottlenecks that constrain the cycle time and the throughput of a process. the problem becomes more challenging in the context of multi-product facilities and when constraints imposed by the limited availability of resources are considered. our methodology and its computer implementation will illustrate how to systematically identify and eliminate such bottlenecks. the industrial case study will provide a real world example of the methodology. application of two stage continuous cultures of aspergillus niger for citric acid biosynthesis jerzy j. pietkiewicz, malgorzata janczar, wladyslaw lesniak food biotechnology department, university of economics, wroclaw - , poland. e-mail: jerzy.pietkiewicz@ae.wroc.pl (j.j. pietkiewicz) the aim of the work was application test of submerged two stage single stream continuous cultures of aspergillus niger for citric acid production from sucrose. studies were carried out in lab fermenters with working volume of dm . the bioreactors were standard cstrs. in two stage continuous cultures (tscc) high mycelium growth and high citric acid production were observed in the first bioreactor. there was almost four times lower growth of biomass rate and about three times lower citric acid production rate in the second bioreactor. studies on influence of dilution rate in race from . to . dm /(dm h) on course and efficiency of tscc showed, that the highest citric acid yield (y p/s = . %), high volumetric rate of its production (r pc = . g/(dm h)) and the highest biosynthesis efficiency coefficient (k ef = . ) were obtained with dilution rate d = . dm /(dm h). there was also high citric acid concentration (p = . g/dm ) and low residual sugar concentration (s k = . g/dm ) in the medium flowing out the second bioreactor in those cultures. the beta-galactosidases in commercial use are of different origins and yeast and fungal lactases present the greatest interest. the yeast lactases present neutral optima ph and are suitable for the hydrolysis of lactose in milk. in this work, the aim was to study the influence of aeration in the production of beta-galactosidase in batch fermentations with kluyveromyces marxianus atcc . the medium composition for culture was as follows (in g/l): lactose pa , yeast extract , (nh ) so and kh po . the fermentations was carried out at • c, ph . , at rpm starting with an initial cellular concentration of × cels/ml, with different aeration rates. the cells were disruped with chloroform % (v/v) as solvent. the enzymatic activity was determined as initial rate of lactose hydrolysis at defined conditions. the studies have revealed the importance of aeration on kluyveromyces marxianus in the growth and beta-galactosidase synthesis. the enzymatic activity of fermented medium with . vvm was % higher than one without aeration. furthermore, the cellular growth was faster in the aerobic fermentation than in the anaerobic one. the aeration has taken an important place in the enzymatic synthesis and in the cellular growth, however the results have shown that the aeration rate increase of . - . vvm has not implied a increase in the cellular growth neither in the enzymatic reached activity. the lactose presents in the milk has a solubility of only % at • c, and a high percentage of the world population presents intolerance to this sugar, due to the low or absence of the activity of the lactase enzyme in the organism. to minimize such problems, the most viable alternative for nourishing dairy products is the enzymatic hydrolysis of milk, although it is an expensive process due to the high cost of the beta-galactosidase enzyme. an alternative that has been greatly studied is the immobilization of this enzyme, originated from many different sources. there are several immobilization procedures for this enzyme, however, a procedure considered ideal was not obtained yet. the objective of this work was to study the immobilization process of beta-galactosidase from aspegillus oryzae in sodium alginate with commercial gelatin. in the immobilization process was studied the glutaraldehyde influence for , and %, in the presence of commercial gelatin at the concentration of % at the immobilization medium. the activities of the immobilized enzymes were obtained in a stirred micro-reactor, at the temperature • c, ph . with a gl − lactose solution in acetate buffer. the experimental results showed that the immobilized biocatalyst that presented the larger initial activity was the one obtained at the immobiliza-tion medium that contained % of glutaraldehyde. after daily determinations of enzymatic activities, a fall of , and % was verified in the enzymatic activities for the immobilized biocatalysts using glutaraldehyde at , and %, respectively, however, in all cases, the enzymatic activity reached the half of their initial activity after determinations. hydrolysis of sucrose by immobilized beta-fructofuranosidase in silica eloízio júlio ribeiro, ubirajara coutinho filho faculdade de engenharia química, universidade federal de uberlândia, uberlândia - , brazil. e-mail: ejribeiro@ufu.br (e.j. invertase, known as beta-fructofuranosidase (ec . . . ), plays a catalytic role in the conversion of sucrose into glucose and fructose. it is largely used in the food industry to prevent the crystallization of sucrose in sugar mixtures and can be used in enzyme reactors for hydrolysis of sucrose. the objective of this work was to study the kinetic of sucrose hydrolysis by immobilized betafructofuranosidase in a continuous recirculation reactor, evaluated the enzyme stability and determine the effective half-life of immobilized enzyme. invertase was covalently immobilized on sillanized controlled pore silica. nonlinear fitting were used to determine the kinetic parameters for substrate and product inhibition observed in the enzymatic hydrolysis of sucrose. the kinetics studies of immobilized invertase were carried out in a continuous recirculating reactor. the half-time of enzyme inactivation (t / ) was calculated from the initial rates of the remaining enzyme activity. the model of inhibition by substrate and product adequately represented the enzymatic hydrolysis. the fructose effect was competitive inhibition (k f = . . − mol/ml) and the glucose effect was noncompetitive inhibition (k g = . . − mol/ml). the effective diffusivity of sucrose into the support was shown to be the same as for sucrose in dilute solution ( . × − cm /s at • c). the half-time of enzyme inactivation (t / ) was h. controlled pore silica showed to be an excellent immobilizing support. the immobilized invertase was very stable at temperatures lower than • c. the intrinsic parameters (k i , k f , k g and v m ) were shown to be similar to the apparent values. the low permeability of mycobacterial cell wall envelopes is a result of the unique composition and organization of the cell wall lipids. the permeability of mycobacterial cell wall can be changed by means of partial disintegration of its compounds. the aim of present work was to characterize the changes in the cell wall skeleton (cws) and non covalently bound free lipids under the influence of isoniazid, the inhibitor of mycolic acids biosynthesis. fatty acid (fames) and mycolic acid methyl esters (mames) obtained from all tested preparations were analyzed by gc/ms analysis. the analysis of free lipids and cws revealed distinct changes in the composition of the frac-tions obtained from the cells exposed to action of the isoniazid. the changes in the quantity of fatty acids in the inh-treated cells indicates that inh interferes with the synthesis of lipidic compounds of the mycobacterial cell wall also. the decreased amount of covalently bound mycolic acids in the cws is responsible for the enhanced penetration of hydrophobic compounds through the cell wall. this work was supported by grant nr p c of the committee for scientific research. barbara sencio, teresa jamroz, stanislaw ledakowicz department of bioprocess engineering, technical university, lodz, poland the enzyme laccase (ec. . . . . p-diphenol oxidase) is a subject of research in many centres dealing with improvement of bioprocesses with the use of different white rot fungi species. most strains that produce this enzyme in vitro require inductors initiating its biosynthesis. when cerrena unicolor was applied, it was found that the strain produced laccase very efficiently without additional toxic compounds. to specify optimum conditions for laccase production in a submerged culture, research was undertaken to obtain the most efficient inoculum c. unicolor. the goal of this research was to determine the effect of form and incubation time of inoculum on enzymatic activity of the laccase producing strain. the experimental inoculum was the mycelium prepared on a solid and liquid substrate. basing on results obtained, it was found that the laccase yield was the highest in the cultures where the mycelium was grown on a solid substrate. maximum activity of the c. unicolor strain was achieved on the th day of culture, and the amount of laccase produced was higher by, ca. % as compared to the mycelium obtained from the liquid substrate. results of these experiments were used to continue studies on the impact of inoculum age. experiments were carried out using an inoculum incubated for - weeks at the temperature • c in a certomat bs shaker at rpm. the best results in the c. unicolor strain culture were achieved using a -day-old inoculum. effect of alcohol treatment on hydrolytic activity of candida rugosa lipase serpil takaç, a. ezgiÜnlü department of chemical engineering, institute of biotechnology, ankara university, tandogan, ankara, turkey candida rugosa lipase (crl) was treated with , , and % concentrations of methanol (m), ethanol (e), -propanol ( p) and -butanol ( b) to investigate the changes in its hydrolytic activity toward p-nitrophenylacetate. the treatment included the following steps at + • c: (i) stirring crl with phosphate buffer for h; (ii) treating the solutions with alcohols; (iii) stirring treated-crl for h; (iv) centrifugation at , rpm for min; (iv) dialysis the supernatant against bidistilled water for h. the activity of crls was followed for h at • c in the presence and absence of isooctane. the enzyme activity was measured spectrophotometrically and the protein concentration was measured by lowry's method. it was found that the recovered protein did not change considerably with the type of alcohol; however, decreased with alcohol concentration. in the presence of isooctane, specific activities of the untreated and treated-crls increased compared with those obtained in the absence of isooctane. b-crls and e-crls showed higher activities than m-crls and p-crls whereas untreated-crl exhibited higher activity than m-crls, e-crls and p-crls. the highest and the lowest activities were obtained with % b-crl and % p-crl, respectively. the changes occur in the structure of crl after treatments were investigated by electrophoretic analysis. this study was supported by ankara university biotechnology institute (project no: ) . different genera, species and strains of microorganisms were found to posses different cryoresistance. optimal ways for cryopreservation of microorganisms-producers of antibiotics, microorganisms, used in food industry, agriculture and veterinary have been developed. it was demonstrated, that non-lethal damages could occur in cryopreserved microorganisms after their returning to physiological culture conditions, which were manifested in streptomyces' hypha fragmentation, that of cyanobacteria's, streptococci's chains as a result there was an increase in a number of colony-forming units, a reversible inhibition of microorganisms' proliferative activity in bacillus thuringiensis and lactic streptococci, stimulation of the enzyme processes and antibiotic production. non-lethal damages are repaired during microorganism culturing in the first passage. the cause of non-lethal damages is a reversible inhibition of biosyntheses of protein and nucleic acids respiratory activity. the repairing of non-lethal damages is accompanied by the production of stressproteins, different from heat shock proteins. effect of ph in the -propanol treatment of candida rugosa lipase on its enantioselectivity in the hydrolysis of racemic naproxen methyl ester serpil takaç, a. ezgiÜnlü department of chemical engineering, ankara university, tandogan, ankara, turkey candida rugosa lipase (crl) was treated with -propanol ( p) at the ph values of . , , , . , and to investigate the changes in its enantioselectivity in the hydrolysis of racemic naproxen methyl ester. the treatment included the following steps at + • c: (i) stirring crl with different buffer solutions to maintain the desired ph values for h; (ii) treating the solutions with % p; (iii) stirring treated-crls for h; (iv) centrifugation at , rpm for min; (iv) dialysis the supernatant against bidistilled water for h. hydrolyses of racemic naproxen methyl ester to form s-naproxen were performed in shaking flasks at rpm and • c for h in isooctane-phosphate buffer solution biphasic system using treated-crls with the activity of . u. the concentrations of the enantiomers of naproxen methyl ester and naproxen were determined with hplc. it was found that the treatment ph has an important role on the enantioselectivity and conversion. the highest enantiomeric excess for the substrate, for the product, enantiomeric ratio, and con-version were obtained with crl treated at ph . as , , and %, respectively. these values were followed with p treated crl at ph as , , and %. however, lower enantiomeric excesses, conversions and enantiomeric ratios were obtained at the treatment ph values between . and . the effects of fatty acids, nitrogen (as nh no ), phosphorus (as kh po ), ph value, manganese (mn + ), iron (fe + ) and methanol concentration on growth and production of oxalic acid from post refining fatty acids by a mutant of aspergillus niger xp in submerged fermentation experiments was studied. of the a. niger strains screened, a. niger xp was identified as the best oxalate producer on lipids. the influence of the ph on oxalic acid formation shows that the maximum production rate and higher concentration of product are observed at the ph ranging from to . with a medium containing g fatty acids/l, the production reached a maximum of g oxalic acid/l after days. the addition of . % (w/v) methanol to seed culture increased the product yield and concentration of oxalic acid but decreased the amount of an undesired by-product (citric acid). under this condition, the maximum oxalate productivity ( - g/l days) was maintained for - days of fermentation. other results of the experiments show that supplementation of the production medium with manganese and iron enhances oxalate production. fatty acids proved to be a very good substrate for oxalic acid production by a. niger xp giving excellent yields and productivity at low ph. the results are very promising as they may lead to cheap alternative processes for oxalic acid production from renewable lipid resources. department of food engineering, middle east technical university, ankara , turkey. e-mail: banuy@metu.edu.tr (b. yalcindag) laccase (e.c. . . . , p-benzenediol:oxygen oxidoreductase), which is an enzyme belonging to the multi-copper oxidase family, catalyzes the oxidation of a broad variety of polyphenols with a preference for p-isomers, which are converted to p-quinones. fungi generally contain several laccases which have been found to be involved in delignification, melanin synthesis and pathogenesis. laccase has also important potential application areas especially in food and chemical industries. after aspergillus fumigatus genome data were released, research on functional analysis of laccases has been initiated in our laboratory. laccase genes of aspergillus nidulans, ya and tila, and laccase and multi-copper oxidase genes of aspergillus fumigatus, abr and abr , were used to analyze a. fumigatus genome for laccases. this sequence analysis resulted in probable laccase genes, one of which was the previously cloned abr gene. in this study, one of these genes (aflac ) was further characterized. after sequence alignment and characterization studies, aflac was predicted to have bp having six introns, which makes the protein amino acid long, and the predicted protein sequence showed % homology with the dihydrogeodin oxidase of aspergillus terreus and % homology to the laccase of botryotinia fuckeliana. aflac gene is found within an uncharacterized gene cluster containing genes with homology to glutathione-s-transferase, polyketide synthase, o-methyl transferase, and others. the information obtained from sequence analysis was employed in designing pcr-primers to amplify the aflac gene, followed by cloning onto pan - and pan - vectors for heterelogous expression in aspergillus sojae. in addition, by the use of rt-pcr, aflac cdna will be cloned and expressed in escherichia coli. furthermore, gene silencing studies will be performed to enlighten the function of aflac and associated gene cluster. stability of growth rate of photosynthetic cells is an important factor in designing of effective photobioreactors especially in long term operations. in our experiments, in order to keep operational parameters almost constant, a semi-continuous culture method was developed. in this method, a part of culture broth containing grown cells was repeatedly replaced by fresh medium at a predetermined time interval. the replacement of broth with fresh media could keep the cell concentration, volume of broth and distribution of light intensity constant at initial values throughout the cultivation. it was shown that in one side illumination with a halogen lamp, if the ratio of the light intensity at the front side of a flat plate photobioreactor to that at the rear side was kept lower than , the growth rates was sustained in constant levels. however, at higher ratios the growth was followed by rapid decrease after - h. supplemental illumination with a fluorescent lamp from the rear side of the flat plate photobioreactor could sustain almost stable growth rate. beside of the illumination conditions, increased ferrous ion concentrations in medium could keep the stability of growth rate even in unstable illumination conditions, while consumed ferrous ion was slight. glutathione (gsh) plays a pivotal role in protecting cells from by-products generated by oxidative metabolism. these characteristics make this active tripeptide an important drug for the treatment of liver diseases and is of interest in the food additive industry, therapeutics and sport nutrition. in the first part of the research, a screening was carried out among yeast strains, to find out those able to accumulate higher gsh intracellular levels. two saccharomyces cerevisiae strains proved to be the best gsh producers ( . %dw), in every samples the presence of s-adenosyl-methionine in traces ( . % dw) was also evidenced. s-adenosyl-methionine (sam) plays a role in the immune system, maintains cell membranes, participates in detoxification reactions and in the manufacture of brain chemicals and cartilage. the second part of the research was aimed at increasing, in a post fermentative procedure, gsh levels present inside the cells at the end of the growth phase. moreover, time course of sam intracellular levels, to be related with accumulated gsh, was also monitored. cells were then suspended in an appropriate solution containing mineral salts, glucose and the aminoacids, precursors of the two studied molecules. according to this procedure, gsh intracellular levels reached . % dw after h incubation. moreover, gsh levels can be related to sam production (up to . % dw). the presence, in several samples, of intermediate metabolites, such as cystathionine and omocysteine, proved the establishement of an intracellular equilibrium between gsh and sam; this behaviour represents a promising starting point for the set-up of a microbial process for the simultaneous production of the two studied molecules. three acetate mutants of y. lipolytica yeasts, which varied in colony morphology (rough and smooth), were employed for continuous citric acid production from glucose and fructose syrup in a membrane reactor with cell recycle. the strains were compared for their product yields, specific acid production rates and ratios of citric acid to isocitric acid. experiments shoved that glucose syrup was a better substrate for citric acid production by y. lipolytica. citric acid concentration in the effluent ranged from to g/l, depending on the yeast strain used. all y. lipolytica strains produced very low amounts of isocitric acid. its concentration did not exceed g/l. based on the results of these experiments, smooth strain awg- was found to be the most suitable for citrate production both from glucose and fructose syrup during long time continuous processes ( h). in the steady state, the highest citrate productivity ( . g/lh) was obtained with this strain, when the feed medium contained g/l of glucose and dilution rate (d) was d = . /h. supplementation of the feed medium with bacto-pepton improved the productivity, citric acid yield and stability of the continuous process in the cell recycle fermentation system. for textile dyeing with natural dyes, indigo has an almost unique position as the most blue natural dye. due to the importance of indigo, considerable research has been conducted to replace the chemical synthesis of the dye by an application of biotechnological methods. therefore, we investigated several characteristics of natural indigo derived from polygonum tinctorium and its dyeing properties using silk fabrics, such as washing, perspiration, and light fastnesses. this work was financially supported by program for cultivating graduate students in regional strategic industry from korea industrial technology foundation. glycine oxidase is the product of the yjbr gene of bacillus subtilis that was predicted by sequence homology to be a flavoprotein similar to sarcosine oxidase. glycine oxidase catalyzes the oxidative deamination of various primary and secondary amino acids (e.g. sarcosine, n-ethylglycine, and glycine) and d-amino acids to form the corresponding ␣-keto acids and hydrogen peroxide. previous investigations reported on the cloning and production of the glycine oxidase gene in escherichia coli was up to u/g cell. the present works has improved the expression of the recombinant his-tagged glycine oxidase by -fold by using pet a and rosetta cells under the optimal iptg, temperature and time of induction. the protein obtained represented % of total soluble proteins in crude extract. the enzyme was purified to near homogeneity using imac with a % recovery and with and specific activity of . u/mg protein. the enzyme was active towards glycine, sarcosine and different d-amino acids, having in general, a basic ph optimum. the kinetic parameters were also studied, showing a km range from . to mm. the enzyme was immobilized, and used to obtain pyruvic acid (␣-keto acid) from d-alanine with a good yield. the enzymatic synthesis of lipophilic derivatives of various natural antioxidants including flavonoid glycosides, as well as derivatives of cinnamic acid, was performed using various immobilized lipases in ionic liquids such as -butyl- -methylimidazolium tetrafluoroborate (bmim-bf ) and -butyl- -methylimidazolium hexafluorophosphate (bmim-pf ). the influence of various reaction parameters on the catalytic behavior and the selectivity of lipases was pointed out. a response surface methodology was applied for the optimization of the yield and the productivity of the biocatalytic process. the antioxidant activity of the biocatalytically prepared lipophilic derivatives of natural antioxidants, as expressed on cu + -induced oxidation of low-density lipoprotein (ldl) and total serum, was investigated. process strategy for reduction of proteolysis in pichia pastoris fermentations jan weegar , john dahlbacka , noora sirén , niklas von weymarn , kaj fagervik : faculty of chemical engineering,Åbo akademi university, finland; laboratory of bioprocess engineering, helsinki university of technology, finland. e-mail: jan.weegar@abo.fi (j. weegar) the yeast pichia pastoris is a popular host organism for production of recombinant proteins. it is, however, common that the products are degraded by proteases towards the end of the fermentation, resulting in productivity and purity decreases. proteolysis of recombinant proteins in p. pastoris fermentations is affected by the temperature and ph of the growth medium. in this study, it was shown that decreasing the temperature from to • c during the induction phase effectively prohibited proteolysis. on the other hand, the temperature decrease resulted in a reduced maximal methanol consumption rate, which subsequently resulted in a culture highly sensitive to residual methanol. the temperature was slowly decreased according to a predetermined trajectory. as the temperature reached values below • c, the methanol concentration had to be closely monitored and the substrate feed rate adjusted in order to prohibit methanol poisoning as well as to maintain the culture as a substrate limited fed-batch. measurement of protease concentrations revealed that proteases were present at • c, but at this temperature the proteolysis rate was evidently effectively reduced. the recombinant protein produced could almost totally be recovered (i.e. high purity) with this process strategy compared to a constant high temperature culture ( • c) where severe breakdown of the product was observed. with the growing of an ecological conscience in the public opinion, more and more industrial processes are analyzed for a possible ecologically beneficial alternative. for the production of ascorbic acid, which is nowadays done by the reichstein process, a lot of research was done to develop biotechnological alternatives for the synthesis of reichstein intermediates by enzymatic means, which show some advantages regarding costs and environmentalfriendliness. besides of two-stage fermentation, our approach is to design a tailor-made organism that produces -keto-l-gulonic acid, which is the direct precursor of ascorbic acid from glucose or gluconic acid and which can easily be converted to the final product by conventional methods. erwinia (pectobacter) cypripedii, which is a natural producer of , -diketo-d-gluconic acid, was selected as a suitable host for a , -diketo-d-gluconate reductase from corynebacterium glutamicum. to increase the yield of the desired compound we investigate two -keto-reductases in the host organism that diminish the yield of -keto-l-gulonate by reducing the compound to l-idonic acid, or by metabolisation of intermediates. these two enzymes were investigated with vari-ous biochemical and molecular biological methods, which will be presented. overproduction of bioinsecticides by heat and salt stress and control of dissolved oxygen in cheap media of bacillus thuringiensis nabil zouari, dhouha ghribi, samir jaoua laboratoire des biopesticides, centre of biotechnology of sfax, tunisia, bp:k, sfax, tunisia bioinsecticides based on preparations of spores and insecticidal crystal-proteins (icps) produced by the bacterium bacillus thuringiensis (bt) proved to be a high tool for fighting some agricultural pests and vectors of diseases. however, the use of bt preparations as commercial insecticides would be prohibitively expensive because it is not easy to reach cheap overproduction of icps during large-scale fermentation. here, we report possibilities to improve delta-endotoxins production as a consequence of responses of bt strains to low levels of heat and salt stress. each stressor results differently in the improvement of delta-endotoxins production, but both were shown to be most efficient at the beginning or the midexponential phase of the cultures which become resistant at the stationary or the sporulation steps. heat stress caused increase of % of synthesis yields of the sporulating cells, in contrast, salt caused increase of % of spores counts, corresponding to % toxins production improvement. combined effects of both stressors lead to toxins production improvement of %, yield improvement of %. we focused on the overcome of carbon repression catabolite, closely related to oxidative metabolism, by an adequate control of dissolved oxygen in the cheap media we formulated for bt insecticides production. we showed that an equilibrium between the high density of vegetative cells and their ability to synthesize toxins during their sporulation was necessary to take into account. % increase of icps production was reached into l fermenter combination of mutagenesis, heat and salt stress and oxidative metabolism control allowed more than % improvement of delta-endotoxins. these results are of great importance in practical point of view, since high bioinsecticides concentrations could be produced without decrease of the yields of their production. mechanism and function of the intramembrane-cleaving protease rhomboid marius lemberg , javier menendez , christopher koth , matthew freeman : mrc laboratory of molecular biology, cambridge, uk; ontario center for structural proteomics, toronto, canada rhomboids are a family of intramembrane serine proteases that are widely conserved throughout evolution. among diverse functions discovered so far, rhomboids participate in intercellular signalling, parasite invasion, membrane dynamics and bacterial quorum sensing, making them potentially valuable therapeutic targets. the identification of physiological substrates and of selective inhibitors will be key towards their evaluation as drug targets. we have developed an in vitro cleavage assay to monitor rhomboid activity in the detergent solubilised state, enabling the first isolation of a highly pure rhomboid with catalytic activity. analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad, and gives insights into subsidiary functions like ligand binding and water supply. oligosaccharides and -keto-glycosides. availability of the enzyme is, however, hampered by the very slow growth and low production rates of the fungus. cloning of the encoding gene and production of the protein by heterologous expression are therefore a prerequisite not only for any biotechnological application, but further scientific investigations as well. on the basis of peptide sequences degenerated primers were designed, and the resulting pcr fragment was used as a probe to isolate the gene from a genomic library. two very similar genes encoding previously uncharacterized proteins were also found, and flanking regions were amplified using rage-pcr. furthermore cdna clones of all genes were isolated by rt-pcr. for textile dyeing with natural dyes, indigo has an almost unique position as the most blue natural dye. due to the importance of indigo, considerable research has been conducted to replace the chemical synthesis of the dye by an application of biotechnological methods. therefore, we investigated several characteristics of natural indigo derived from polygonum tinctorium and its dyeing properties using silk fabrics, such as washing, perspiration, and light fastnesses. this work was financially supported by program for cultivating graduate students in regional strategic industry from korea industrial technology foundation. the behavior of phytate degrading enzymes isolated from malaysian zea mays root in rice bran media anis shobirin meor hussin , abd-elaziem farouk , hamzah mohd salleh , ralf greiner : biomolecular engineering research group, department of biotechnology engineering, kulliyyah of engineering, international islamic university malaysia, jalan gombak, kuala lumpur, malaysia; centre for molecular biology federal research centre for nutrition and foods, haid-und-neu-straße , d- karlsruhe, germany phytate degrading enzymes catalyze the step-wise release of phosphate from phytate, the principle storage form of phosphorus in plant seeds and pollen. they are widespread in nature, occurring in plants and microorganisms, as well as in some animal tissues. phytate-degrading enzymes have been studied intensively in recent years because of the great interest in such enzymes for phytate degradation and their application for animal feed and human health. from over isolate screened isolates of phytate degrading enzymes, three isolates from the root malaysian maize plantation have shown phytate degrading activity. the production of phytate degrading enzyme was studied using different concentrations [%, w/v] of rice bran media during different stages of cultivation. the dephosphorylation of phosphate from rice bran phytate has shown regulatory effect on the secretion of bacterial phytases. in this conference, we will present data for the characterization of the enzymes. in this paper we present the properties of a phytase purified from a bacterium isolated from malaysian wastewater, which might find application as an animal feed supplement. the phytase described herein is a periplasmic enzyme. the phytase was purified about fold to apparent homogeneity using ion-exchange chromatography and gel-filtration with a recovery of % referred to the phytatedegrading activity in the crude extract. the enzyme exhibits an activity of about u mg − . gel filtration of the native enzyme on a calibrated sephacryl s- column gave a molecular mass of the phytase of , ± da with elution position being measured by determination of enzyme activity. lower molecular mass species or higher molecular mass aggregates were not observed. the phytase appeared homogeneous by polyacrylamide gel electrophoresis under non-denaturing conditions at ph . and . and gave a single protein band upon sds gel electrophoresis after coomassie staining of the gels. these results indicate that the phytase could be regarded as homogeneous. the estimated molecular mass after sds-page indicated that the phytase having a molecular mass of , ± da. consequently, this enzyme is a monomeric protein. the purified enzyme had a single ph optimum at ph . and was virtually inactive above ph . . at ph . , % and at ph . , % of the activity at optimal ph was observed. the effect on enzyme stability was studied in the ph range . - . at • c. within days the phytase did not lose any activity in the ph range from . to . , but at ph values below . a rapid decline in activity was observed. at ph . , % and at ph . , % of the initial activity was lost during h. in the range of temperatures studied, - • c, the optimum temperature for the enzyme was found to be • c. the apparent activation energy was estimated at ph . from the slope of log v max versus /t. the data showed excellent linearity from to • c. the arrhenius activation energy for the hydrolysis of phytate was calculated to be . kj/mol. in order to check thermal stability, the purified enzyme was incubated at different temperatures, cooled to • c and assayed using the standard phytase assay. the enzyme lost no activity in min at temperatures up to • c. when exposed for min at • c, it retained % and at • c % of the initial activity. in order to determine the substrate selectivity of the purified phytase, several phosphorylated compounds in addition to phytate, were used for k m and v max estimation by detecting the release of the phosphate ion during hydrolysis using formation of a soluble phospho-molybdate complex in an acidic water-acetone mixture. only phytate was identified as a substrate. the kinetic parameters for the hydrolysis of phytate were determined to be k m = . mmol l − and k cat = s − at ph . and • c. like other bacterial phytatedegrading enzymes, the purified enzyme showed substrate inhibition. the activity of the purified enzyme was inhibited at substrate concentrations > mm. the study of the effect of metal ions on enzyme activity showed that none of them had an activating effect when used at a concentration between − and − m. mg + , ca + , mn + , co + , ag + , hg + , and cu + had little or no effect on enzyme activity, while zn + , fe + , and fe + showed strong inhibitory effects. the reduced phytate-degrading activity in the presence of fe + and fe + is attributed to a lower phytate concentration in the enzyme assay because of the appearance of a fe-phytate precipitate. when compounds which tend to chelate metal ions, such as o-phenanthroline, edta, oxalate, citrate or tartrate, were tested for their effect on enzyme activity, it was noted that none of them was inhibitory at a concentration from − to − m. fluoride, a known inhibitor of different phytate-degrading enzymes from bacteria and the hydrolysis product phosphate as well as its structural analogs molybdate, wolframate and vanadate were found to be strong inhibitors of the purified enzyme. flouride inhibited the hydrolysis of phytate with a k i value of mol l − . several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (amps) in recombinant bacterial expression systems. in the present work, we investigated the use of the baculoviral polyhedrin (polh) protein as a novel fusion partner for production of a model amp (halocidin subunit; hal ) in escherichia coli. the recombinant hal amp could then be hydroxylamine cleaved from the fusion protein and easily recovered by simple dialysis and centrifugation. this was facilitated by the fact that polh was soluble in the alkaline cleavage reaction but became insoluble during dialysis at a neutral ph. importantly, recombinant and synthetic hal peptides showed nearly identical antimicrobial activities against e. coli and staphylococcus aureus, which were used as representative gram-negative and -positive bacteria, respectively. these results demonstrated that baculoviral polh can provide an efficient and facile platform for production or functional study of target amps. extensive industrial and food additive applications of succinate have attracted much effort towards finding an environment-friendly alternative to the petrochemical production processes. it is very attractive to engineer s. cerevisiae for succinate production because of its generally regarded as safe (gras) status, ease of genetic manipulation and fermentation. we approached this metabolic engineering problem with a two-step methodology combining modern computational as well as molecular biology tools. in the first step we identified potential metabolic engineering targets leading to high succinate yield and productivity, with the aid of genome scale metabolic model and a bi-level optimization framework using flux balance analysis and quadratic programming. in the next step, various deletion mutants are being constructed and characterized for physiology and succinate production. results from these experiments then will be used to improve the predictions in computational models. so far, we have constructed a saccharomyces cerevisiae mutant deleted in sdh , which encodes a major subunit of sdh-complex converting succinate to fumarate in mitochondria. the physiology of sdh mutant has been characterized in aerobic and anaerobic batch cultivations and in glucose limited chemostat at dilution rate as low as . h − . in aerobic batch fermentations, the mutant showed reduced maximum specific growth rate as compared to the wild-type, and it was incapable of growing on ethanol as sole carbon source, as predicted from the model. interestingly, the mutant showed much higher specific growth rate in anaerobic conditions, close to the wildtype strain. moreover, the chemostat cultivations indicate that the critical dilution rate of the mutant is below . h − . this opens further opportunities to investigate interesting behavior of the mutant and the underlying regulatory processes to improve our understanding of yeast mitochondrial metabolism. department of chemical engineering, yıldız technical university, davutpaşa campus, esenler/istanbul, turkey. e-mail: dkilic@yildiz.edu.tr (d.k. apar) lactose is the dominant carbohydrate in milks which are, in turn, the only significant natural sources of lactose. a large number of people do not digest lactose properly due to a lack or inactivity of the intestinal beta-galactosidase and they suffer from intestinal dysfunctions -gas, abdominal pain and diarrhea -if their diet contains lactose. moreover, lactose is a sugar with a high bod, low sweetness, low solubility, when compared to the products of its hydrolysis (glucose and galactose) and being a hygroscopic sugar has a strong tendency to adsorb flavours and odours. the hydrolysis of this sugar is very attractive towards the improvement of processes for the production of ice cream and other refrigerated dairy products and it could be very interesting for the development of additives for animal and human alimentation. the enzymatic hydrolysis of lactose is carried out by beta-galactosidases, enzymes that are widely distributed in nature, appearing in micro-organisms, plants and animal tissues. the present investigation describes the effects of the sonication process parameters on enzymatic hydrolysis of milk lactose and enzyme stability. bandelin sonopuls sonicator was used for the lactose hydrolysis experiments. ␤-galactosidase enzyme used is produced from kluyveromyces marxianus. the reactions were carried out in ml of milk. the process variables for the sonicator are duty cycle, acoustic power and enzyme concentration. the amount of residual lactose concentration (g/l) and residual enzyme activity (%) against time were investigated versus process variables. beside of this; the mathematical models depending on the operating conditions were also derived by using the experimental data of lactose concentration and enzyme activity. kinÖzbek department of chemical engineering, yıldız technical university, davutpaşa campus, esenler/istanbul, turkey. e-mail: dkilic@yildiz.edu.tr (d.k. apar) over the last decade, the use of plant protein hydrolysates alternative to animal protein hydrolysates in human nutrition has broadly expanded. protein hydrolysates are often used in different nutritional formulations, such as supplementation of drinks to enhance their nutritional and functional properties, or special medical diets. there are many processes which employ protein hydrolysis and hydrolytic products. among these processes, the use of enzymes allows selective hydrolysis of protein and produces a potentially safer and more defined material. in the present study, the effect of the temperature, ph and viscosity on the hydrolysis of corn gluten was investigated using a stirred batch reactor system. the corn gluten was hydrolysed by using neutrase enzyme, a bacterial protease produced by a selected strain of bacillus amyloliquefacien. the reactions were carried out in . l of aqueous solutions containing % (w/v) corn gluten and . % (v/v) enzyme. the degree of hydrolysis (%) and soluble protein concentration depending on the time were investigated at the temperatures , , , , and • c; and at the ph values . , , . , and . to investigate the effect of viscosity, the various amounts of glycerol was added to the reaction solutions to produce viscosities in the range of . - . cp. the degree of hydrolysis (dh) was computed by using ph-stat method. for the soluble protein determination in the hydrolysates samples, the folin-lowry method ( ) was used. polyhydroxyalkanoates (pha), one of the most promising bioplastics for the partial replacement of synthetic polymers like polypropylene, are polyesters produced by bacteria as intracellular storage reserves of carbon and energy. the industrial production of pha is achieved by pure cultures in its natural state or using genetically engineered organisms. the main obstacle to the replacement of synthetic plastics by biopolymers is their great cost difference. research on the field of biopolymers synthesis using mixed cultures and waste organic carbon sources as substrates prove to decrease substantially the production costs of pha. the optimization of pha production under aerobic feeding conditions (adf) was achieved recently in our group, obtaining the highest value of pha content stored by mixed cultures ( . % of cell dry weight). in this work only a homopolymer of polyhydroxybutyrate (phb) was obtained and since it is a highly crystalline and brittle material its application field is limited. the mechanical and thermal properties of pha can be varied to a great extend by adjusting the monomer composition. the incorporation of different monomeric units, other than hb, in the polymer chain, originates copolymers with improved mechanical properties. optimization of pha production from propionate by a mixed culture was studied varying the carbon and ammonia concentrations. propionate only, acetate alone or a mixture of acetate and propionate were tested. copolymers of hydroxybutyrate and hydroxyvalerate, p(hb/hv), with different compositions were obtained. consequently polymer properties could be manipulated by feeding the selected volatile fatty acid composition. the pharmaceutically important plant species of glycyrrhiza sp. (called licorice) is an important commercial product used as a natural sweetener, anti-inflammatory, anti-cancer and anti-diabetic agents. agrobacterium rhizogenes transformation system was used for the hairy root cultures of licorice g. uralensis. after inoculation of aseptic stem segments the ability of hairy root formation was scored for a period of weeks. mean transformation frequency ranged from % (for up to % (for ). some transformed genotypes showed significant differences in roots weight, flavonoids and glycyrrhizin (gl) production. the cotransformation rate of licorice intact explants cultivars with lba tl-dna and the s gus gene showed an average of more than %. these obtained root cultures were additionly elicited with extracts of biotic elicitor acremonium sp. (endomycorhizal fungus), and were used as an in vitro system to metabolites production. the transformed and elicited hairy roots of g.uralensis were obtained by infection of a. rhizogenes have produced gl at an yield of . % dry weight on the period of culture as a days. according to tentative analyses the hairy roots cultures of glycyrrhiza species produced flavonoids (liquiritigenin and liquiritigen). more high levels ( . g/l) of the total flavonoids production have been identificated on the strains which transformed by lba . this study involved any difference among elicitor treatments and incubation periods for the optimal meabolites production. clearly, the selection of an effective agrobacterium strain for the production of transformed root cultures is highly dependent on the plant species, and must be determined empirically. production, purification and characterization of scytalidium thermophilum phenol oxidases didem sutay , ufuk bakir , zumrut b. ogel : chemical engineering department, middle east technical university, inonu bulvari, ankara, turkey; food engineering department, middle east technical university, inonu bulvari, ankara, turkey. e-mail: ubakir@metu.edu.tr (u. bakir) phenol oxidases (pos) are a group of enzymes which are responsible for oxidation of various phenolic compounds in the presence of molecular oxygen. there are different types of pos present in nature and three major groups of these enzymes are laccases (e.c. . . . , p-benzenediol: oxygen oxidoreductase), catechol oxidases (e.c. . . . , o-diphenol oxidoreductase) and tyrosinases (e.c. . . . , monophenol monooxygenase). another group of enzymes, peroxidases (e.c. . . . ), can also be considered as a member of po family. pos have very wide substrate range and final oxidation products of these substrates are quinones, which are highly reactive molecules and polymerize into brown, red or black waterinsoluble compounds. pos are very common in nature, they can be found in almost all plants, animals and microorganisms. pigmentation and protection from the environment are main functions of these enzymes. pos have different applications in food, pharmaceutical, textile industries and waste-water treatment systems. the objective of this study was po production by the thermophilic fungus, scytalidium thermophilum, purification and characterization of the enzyme. for this purpose, enzyme production was performed either in a shaker-incubator or a temperature, ph and dissolved oxygen controlled l bioreactor (probiotem) to optimize enzyme production medium composition and bioreactor parameters. as the carbon and nitrogen sources, % glucose and . % yeast extract were determined as the optimal concentrations, respectively. copper, gallic acid and tannic acid were determined to increase enzyme production. purification was performed by using membrane and chromatographic techniques. hydrophobic, ion exchange and gel filtration columns were used by using a fplc system;Äkta prime (amersham biosciences). especially the phenyl sepharose tm high performance column appeared to be very efficient for po purification from scytalidium thermophilum. purified po have been characterized by electrophoretic techniques and kinetic studies. isolation of lipolytic microorganisms from subtropical soils. cloning, purification and characterization of a novel esterase from strain pseudomonas sp. cr- núria prim, cristian ruiz, cristina bofill, f.i. javier pastor, pilar diaz department microbiology, university of barcelona. av. diagonal , microorganisms or their enzymes are used in a wide range of biotechnological activities such as polymer hydrolysis, synthesis of added-value compounds, sample decontamination, etc. thus, there is an increasing interest for isolating new enzymes and new enzymeproducing organisms for their use in industrial conversions (cherry and fidantsef, ) . among these enzymes, lipases, esterases, cellulases, xylanases and pectinases play an important role in many biotechnological processes like those related to pulp and paper processing. three samples of subtropical forest soil from puerto iguazú (argentina) were used for the isolation of autoctonous microorganisms growing in an organic matter-rich environment. a total of pure cultures of bacteria and fungi were obtained and their hydrolytic activities on polysaccharide and lipidic substrates were assayed using olive oil, tributyrin, cholesterol esters, xylan, cellulose and pectin as substrates. among the isolates analysed, were active on one or more of the substrates evaluated, and of them degraded all substrates. nearly half of the strains displayed lipolytic activity, whereas the number of strains active on xylan, cellulose, pectin and cholesterol esters, was much lower. the strains bearing the highest hydrolytic activities were selected and stored for further characterization (ruiz et al., ) . among them, strain cr- , one of the most active isolates on tributyrin, was selected for identification and characterization of its lipolytic system. lipolytic strain cr- was identified by morphological, physiological and phylogenetic tests, as a pseudomonas sp., closely related to p. fluorescens. sds-page and zymogram analysis (diaz et al., ) of cell extracts and supernatants from the strain revealed a complex lipolytic system consisting of at least two lipolytic enzymes. sequence alignment and clustering of previously described pseudomonas lipases and esterases allowed the design of different sets of primers for the isolation of the lipase/esterase coding genes. a gene coding for an esterase with homology to family vi bacterial lipases (arpigny and jaeger, ) was isolated and cloned in escherichia coli. the cloned enzyme was further purified and characterized, showing preference for short fatty acid esters and displaying a typical michaelis-menten kinetics, with no interfacial activation. the substrate profile, together with the kinetic behaviour and sequence similarity of the cloned enzyme to family vi bacterial esterases, allowed to identify this enzyme as an esterase and was named esta . maximum activity was achieved on muf-butyrate at • c and ph . , suggesting that it could be of interest for biotechnological purposes. microbial xylitol production from agricultural wastes has recently attracted much attention from industries because it has potentials to realize the cheaper production of xylitol with low environmental impact (tada et al., ) . in order to realize the effective xylitol production by a xylose utilizing yeast, the oxygen supply is a key for maximizing xylitol yield over consumed xylose (y xl ) because the intracellular xylitol metabolism is strongly influenced by the amount of available oxygen. in the present work, we tried to apply a metabolic reaction model in order to determine the optimal oxygen transfer rate (otr) in a fermentor for maximizing xylitol yield. corn cob hydrolysates containing g-xylose/l was employed as medium for xylitol production by computer-controlled batch cultures using candida magnoliae (ferm p- , aist). a metabolic reaction model considering main xylitol metabolisms including glycolysis, pentose-phosphate pathway, tca cycle and cell synthesis was developed. the model allows to estimate various intracellular metabolic flux distributions including a xylitol production rate. the oxygen uptake rate to maximize the ratio of xylitol production rate over xylose consumption rate corresponds to the otr condition to maximize a xylitol yield over xylose consumed. based on the metabolic reaction model, the otr was optimized by a linear programming, the optimal otr and the maximum xylitol yield were estimated as . mmol o /l h and . g-xylitol/g-xylose, respectively. the experimental verification using the optimal otr demonstrated that the xylitol yield was greatly improved to . g-xylitol/g-xylose from . g-xylitol/g-xylose in our previous study. expression of a bacterial sugar phosphate transporter in s. cerevisiae to release l-glycerol -phosphate accumulated by metabolic engineering almut popp, huyen thi thanh nguyen, ulf stahl, elke nevoigt department of microbiology and genetics, university of technology, berlin, germany. e-mail: a.popp@lb.tu-berlin. de (a. popp) in contrast to glycerol, its phosphorylated precursor l-glycerol- phosphate (l-g p) is retained by the plasma membrane. therefore, engineered yeast strains accumulate l-g p in the cytosol resulting in low overall yield of the desired product and laborious downstream processing. a suitable sugar phosphate transporter in the yeast plasma membrane would overcome these limitations. the glycerol- -phosphate transporter (glpt) of e.coli is an antiporter and naturally mediates the uptake of l-g p in exchange with inorganic phosphate. we assume that this transporter would also mediate the excretion of accumulated l-g p into a phosphate-rich medium if it was present in the plasma membrane of engineered yeast. despite many inconsistencies in codon usage, we were able to express the bacterial glpt gene in yeast. expression was monitored by a c-myc tag added to the c-or n-terminal hydrophilic tail, respectively. the quantity of the construct with n-terminal tag clearly exceeds the quantity of the construct with c-terminal tag. both gene products are located in the endoplasmic reticulum, as shown by immunofluorescence microscopy. obviously, yeast's transmembrane protein sorting machinery does not recognise it as a substrate for the secretory pathway. metabolic flux analysis of c-and p-limited shikimic acid producing e. coli gaspard lequeux , louise johansson , jo maertens , peter vanrolleghem , gunnar lidén : biomath, ghent university, coupure links , gent, belgium; department of chemical engineering, lund university, p.o. box , lund, sweden. e-mail: gaspard.lequeux@biomath.ugent.be (g. lequeux) metabolic flux analysis (mfa) was applied to decipher why plimited e. coli fermentations are more optimal for shikimic acid production in comparison with glucose-limited fermentations. as mfa allows obtaining insight in the intracellular flux distribution over different pathways by only measuring net production and consumption rates of metabolites, under the condition that parallell pathways are removed. to this end a detailed metabolic network model was created and checked for consistency, dead-ends, and parallell pathways. several fermentations were performed at different dilution rates (ranging from . to . h − ) and different limitations (phosphate and glucose). the e. coli strain used was w with genetic modifications in the aromatic amino acid pathway to enhance shikimic acid production. for each dilution rate, a metabolic model was solved. this way, the evolution of each flux can be followed with respect to the dilution rate. the p-limited cultures showed a better yield which can be explained by the diminished excretion of dehydro-shikimic acid (as is known from literature) and a reduction of the hydrolysis of atp. takasumi hattori, kuniki kino, kohtaro kirimura department of applied chemistry, school of science and engineering, waseda university, tokyo, japan. e-mail: takasumi@suou.waseda.jp (t. hattori) alternative oxidase is a terminal oxidase in respiration chain, which is a branched chain of cytochrome pathway, and inhibited by salicylhydroxamic acid (sham), but not by cyanide. the citric acid-producing fungus aspergillus niger wu- l has a cyanide-insensitive and sham-sensitive respiration catalyzed by the alternative oxidase (kirimure et al., ) and did not produce citric acid when cultivated with sham (kirimure et al., ) . in this study, the transcript levels of alternative oxidase gene (aox ) (kirimure et al., ) and activities of alternative oxidase under the conditions of citric acid production were examined during days-cultivation. the amount of aox mrna was determined by northern blot analysis, and the specific activity of alternative oxidase as that of duroquinol oxidase. the transcript level and the activity were highest at days at log-phase, decreased during and days, and thereafter maintained at low levels. however, the transcript and alternative oxidase activity was constitutively detected during whole the cultivation periods under the conditions of citric acid production. the sequence analysis of aox chromosomal dna revealed the presence of potential binding site of cyclic amp responsive element (cre), stress responsive element (stre) and heat shock factor (hsf) in its upstream region. these results indicated that the expression of alternative oxidase was regulated in the transcription level and alternative oxidase contributes as the main respiration chain during citric acid production. kirimura, k., et al., . curr. genet. , - . kirimura, k., et al., . biosci. biotechnol. biochem. , - . trichoderma strains are considered to be among the most useful fungi in industrial enzyme production, agriculture and bioremediation. metabolic versatility displayed by these fungi makes it a very amenable source of new gene products. functional analysis of candidate genes goes by two complementary ways: gene overexpression and loss-of-fuction mutants generation. a few expression systems are available mainly to direct constitutive gene expression in catabolite repression conditions. following a genomic approach, we have recently cloned some gene promoters with high expression in glucose in trichoderma harzianum cect . a main goal in a gene functional analysis is the generation of knock-out mutants. up to date, there is no reference about successful gene disruptions in t. harzianum mainly due to a very low homolog recombination frequency. rna-mediated gene silencing has been shown as an efficient tool to diminish or totally abolish specific gene expression. especially those strategies based on the use of hairpin constructs allow rapid and easy generation of strains with reduced levels of mrna from genes of interest. we have used the t. harzianum cect tss promoter to direct the expression of a hairpin dna construct that induced the appearance of small-interfering rnas (sirnas) and the silencing of the uida reporter gene in a previous uida overexpressing strain. reduced levels of mrna and gus activity correlated with the presence of sirnas. this is the first report on rna-mediated gene silencing in trichoderma and constitutes a useful and promising tool for functional genomic studies in fungal systems. analysis of the metabolic response of escherichia coli to quantitative modulations of the glucose- -phosphate dehydrogenase based on c-labelling experiments cécile nicolas, fabien létisse, stéphane massou, philippe soucaille, jean-charles portais. e-mail: fabien.letisse@insa-toulouse.fr (f. létisse) microorganisms have an efficient capacity for adapting their metabolism in response to genetic or environmental changes, and understanding metabolic robustness has become an emergent issue. part of the robustness originates from the network organization of metabolic systems, where the interplay between all available biochemical reactions provides alternative mechanisms for compensating the perturbations. recently, c-metabolic flux analysis ( c-mfa) has been applied to escherichia coli knock-out mutants lacking key enzymes to determine the phenotypic effects of structural changes in the metabolic network, providing further evidences for compensatory phenomena. the aim of our on-going work is to understand how the central metabolism in e. coli responds to quantitative alterations at a specific key-point of the metabolic network. the glucose- -phosphate dehydrogenase (g pdh), a key enzyme in the central metabolism for which the effects of deleting the gene (zwf) has been already described (zhao et al., ) , was chosen as the target. to this aim we have generated a set of expression mutants, i.e. mutants having each a fixed level of expression of the zwf gene. four different levels of expression, leading respectively to g pdh activity ; . ; . and times higher than in the wt strain, have been obtained. for each mutant, transcriptomics analysis will be carried out and compared to both the zwf-and wt strains to detect changes in the network structure, and the distribution of fluxes will be measured using c-mfa. the flux maps obtained for the various strains will be compared to evaluate the quantitative response of the central metabolic network to imposed and increased g pdh activity. metabolic control analysis will be applied to provide insights onto the sensitivity of the measurable metabolic fluxes to g pdh activity. combination of transcriptomics and fluxomics approaches will provide information on the nature and extent of the compensatory mechanisms. because the activity of a single enzyme is tuned at different levels in knock-out and expression mutants, this investigation provides a situation that mimics gene-level regulation of metabolism. , j., et al., , metab. eng., , . with the depletion of the world's petroleum supply, there has been an increasing worldwide interest in ethanol as an alternative, nonpetroleum source of energy. this fact caused increased interest in the new ethanol technology fermentation process research. as reported before, bacteria zymomonas mobilis possesses more advantages than saccharomyces cerevisiae, microorganism used for ethanol production in industrial scale. for that reason, we have focused on the fermentation studies of this facultative bacterium in free and immobilized form. the immobilization of the cells into polyvinylalcohol (pva) hydrogel lens-shaped capsules lentikats, improved the batch fermentation process efficiency nine times. starch, the substrate considered as one of the best of renewable energy source is considered as fuel ethanol feedstock. due to z. mobilis disability of maltose, maltotriose and dextrin utilization, the starch has to be converted into glucose monomers. this pre-fermentation step can overcharged whole ethanol production process. this ineffective part of the process was resolved with immobilization of glucoamylase into lentikats. the system with immobilized enzyme and cell was stabile in continuous mode for days without any significant change in the system efficiency. the combination of cell and enzyme immobilization can significantly improve the efficiency and the cost of ethanol production in industrial scale. fermentation of an inhibitory dilute acid-hydrolysate from spruce using a fed-batch procedure combined with cell-reuse andreas rudolf, gunnar lidén department of chemical engineering, box , lund university, se- lund, sweden. e-mail: andreas.rudolf@chemeng.lth.se (a. rudolf) a well-controlled addition of hydrolysate to the fermentation has proved very efficient in reducing yeast inhibition due to compounds formed during lignocellulose hydrolysis. furthermore, using high cell mass concentrations has been another way of avoiding the negative impact of the inhibitory compounds. if possible, the yeast should therefore be re-used in the process. in the present work a dilute-acid hydrolysate from spruce was fermented using a fed-batch procedure with reutilization of yeast. the fermentation procedure worked satisfactorily, with more than % of fermentable sugars consumed in each of the four consecutive fed-batch fermentations performed. the ethanol yields on fermentable sugars reached . g/g. there was continued cell growth in the repeated fed-batch experiments, with an average cell yield on fermentable sugars of . g/g. in contrast, only about % of the fermentable sugars were consumed within h, when the fermentation of the hydrolysate was run in a batch process. the work shows the potential to re-use the yeast in a suitably designed process. metabolic engineering is defined by bailey in his seminal paper as "the improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant dna technology". the manipulation of these functions ultimately results in the manipulation of metabolism, which is the purpose of many biotechnological processes. metabolic engineering has sought its methods and tools in mathematics and the physical sciences, and later in information technology (it) leading to the proliferation of bioinformatics. in this work we propose a novel approach to metabolic engineering that regards it as a business process reengineering (bpr) endeavour. hammer and champy in their celebrated book define bpr as "the fundamental rethinking and radical redesign of business processes to achieve dramatic improvements in critical contemporary measures of performance, such as cost, quality, service and speed". our thesis is that metabolic engineering with its goal of reengineering the metabolism of the microorganism, is equivalent to business process reengineering (bpr) in business and management. indeed this is essentially what metabolic engineering does to the cell through the use of recombinant dna technology, which can be viewed as a radical redesign of the metabolic process. after all, it causes changes that cannot be attained otherwise, and whose purpose is to achieve dramatic improvements in cellular activities such as the increase in production of some metabolites by orders of magnitude. the cost incurred by the process, which is metabolism in this case, can be, for example, energy requirements or change to a cheaper substrate. in this study we elucidate this parallelism between the two with emphasis on modelling of metabolism and how the concepts of business process modelling can be applied to it. the purpose is not to produce a model, but rather to introduce the modelling methodology and demonstrate its utilisation and benefits and outline its limitations and challenges. we believe that the novelty of our work lies in applying a new paradigm in approaching metabolic engineering that has not been considered previously. thermophilic ethanol production from wheat straw hydrolysate in continuous culture tania i. georgieva, birgitte k. ahring biocentrum-dtu, technical university of denmark, building , dk- lyngby, denmark. e-mail: tig@biocentrum.dtu.dk (t. georgieva) ethanol production from lignocellulosic biomass has attracted widespread attention as an unlimited low cost renewable source of energy to transportation fuels due to increasing petroleum use and air pollution towards greenhouse gases. wheat straw available as agricultural residue has been considered as a potential lignocellulosic substrate for industrial bioethanol production. a major technical obstacle to commercialize bioethanol production form lignocellulose (such as wheat straw) is associated with a lack of microorganism able to rapidly and efficiently ferment both hexose and pentose sugars into ethanol and to tolerate the inhibitors present in undetoxified hydrolysates. currently used industrial mesophilic microorganisms (saccharomyces cerevisiae and zymomonas mobilis) are excellent ethanol producer from glucose, however, they are not able to ferment other sugars such as xylose, which is the second most abundant sugar in lignocellulose. thermophilic anaerobic bacteria have been considered for ethanol production from lignocellulosic biomass as an alternative to mesophilic ethanol producing strains, predominantly because of their abilities naturally to ferment the whole diversity of sugars found in lignocellulosic biomass. an increase attention to thermophilies for ethanol production have also arise from broad spectrum of advantages regarding industrial scale ethanol fermentation such as high growth and metabolic rates, low oxygen solubility, reduced risk of reactor contamination, and cost savings via mixing, cooling and facilitated product recovery. in addition, simultaneous co-fermentation of glucose and xylose in a single operation unit could substantially reduced the ethanol production cost. research has been attempted to study the potential of using a thermophilic anaerobic bacterium for continuous ethanol fermentation of lignocellulosic biomass, with particular emphasis on effectiveness of our strain to ferment undetoxified wet oxidized wheat straw hydrolysate with respect to sugar (glucose and xylose) conversion and ethanol yield. the experiment was carried out in a lab-scale reactor operated at • c with wheat straw hydrolysate as a substrate in concentrations from to wt.% equivalent to total sugar mixture of - g/l. wheat straw hydrolysate (woh) [ g/l wheat straw, . % dry matter (dm)] was prepared using wet oxidation pretreatment process followed by enzymatic saccharification with commercial enzymes mixture of celluclast . , and novozym (novozymes, denmark). both xylose and glucose sugars were simultaneously converted to ethanol. the sugar utilization was higher than %, and high ethanol yields were achieved. reactor shows good long-term performance ( days) in terms of operation stability and reactor contamination. maltotriose is the second most abundant fermentable sugar in wort and due to incomplete fermentation, residual maltotriose in beer may cause problems in the brewing industry. to study genes that might improve utilization of maltotriose we used a library with dna from brewer's strains and a laboratory strain and identified a new transporter encoded by mtt . mtt gave lager strain a the ability to grow on yp/ % maltotriose in the presence of mg/l of the respiratory inhibitor antimycin a. this transporter gene shares % similarity with mph and mph , % similarity with agt and % similarity with mal and mal . moreover, mtt shares even higher similarity ( %) with the s. pastorianus mty gene (m. salema-oom, unpublished, ncbi accession number aj ). purified radiolabeled maltotriose and radiolabeled maltose were used to study sugar uptake of lager strains a and ws / , and of a containing mtt or mtt alt, a more efficient, altered version of this gene lacking the basepairs from the end and containing base-pairs of vector sequences. these transport studies show that mtt and, especially, mtt alt encode maltose transporters with relatively high activity towards maltotriose compared to maltose. this study is part of a multi-disciplinary project, funded by the european union (contract no. qlk -ct- - ) focusing on the development of high-gravity resistant brewer's yeast strains. metabolic engineering of l-phenylalanine pathway in bacillus subtilis yasemin demirci , pınar Ç alık , güzide Ç alık , tunçer h.Özdamar : bre laboratory, department of chemical engineering, ankara university, ankara, turkey; iblab, department of chemical engineering, metu, ankara, turkey. e-mail: ozdamar@eng.ankara.edu.tr (t.h.Özdamar) metabolic engineering design of a recombinant l-phenylalanine (phe) production system is based on coordinated overexpression of the flux-controlling genes in the aromatic-amino acid pathway. based on the insights gained by the work carried out in our laboratories (Özçelik et al., ) , aroh for the reaction r at the branch-point chorismate that connects the preceding reactions of the aromatic group amino acid pathway to the proceeding reactions towards phe, was predicted as the first-, and aroa for dahp synthase (r ) predicted as second-metabolic engineering sites. aroa gene was cloned next to aroh, by the use of four primers designed using pcr-based gene splicing by overlap extension method. the genes were amplified separately by pcrs. the primers used at the ends to be joined were designed as complementary to one another by including nucleotides at their ends that are complementary to the portion of the other primer. these products were mixed in the next pcr reaction, where one strand from each fragment contains the overlap sequence at end. extension of this overlap by dna polymerase has yielded the recombinant two-gene product; and the two-gene product serve as template for the continuation of reactions for the increase of the concentration in the micro-reactors. the two-gene fragment was first cloned into puc , and then sub-cloned to pmk e. coli -bacillus shuttle plasmid. the new expression vector with high stability and high copy-number was obtained and transferred into host b. subtilis. the metabolic flux distributions calculated by the mass balance based stoichiometric model based on the metabolic reaction network for r-b.subtilis were determined by using time profiles of the substrate, dry-cell, phe and other amino acids, and organic acids concentrations as the constraints. on the bases of calculated intracellular fluxes of recombinant b. subtilis carrying pmk ::aroa::aroh, an in-depth analyses of the metabolic engineering design will be presented. ozçelik,İ., Ç alık, p., Ç alık, g.,Özdamar,t.h., . metabolic engineering of aromatic group amino acid pathway in bacillus subtilis for l-phenylalanine production. chem. eng. sci. ( - ), - . the % respiratory-deficient nuclear petite amylolytic saccharomyces cerevisiae npb-g strain capable of excreting a hybrid protein possessing both ␣-amylase and glucoamylase enzyme activities was generated and its employment for direct fermentation of starch into ethanol was investigated under both shake flask and controlled bioreactor cultivation conditions. when compared with a standard host strain, higher ethanol concentrations and yields were achieved with the nuclear petite strain under both cultivation conditions. further improvement in ethanol production was achieved by the use of an initial glucose supplement. comparison of the ethanol fermentation performances of the respiratory-deficient npb-g and the parental respiratory-sufficient wtpb-g strain showed an increase of, ca. % in both ethanol production yields and ethanol productivities with the respiratory-deficient strain. response surface methodology (rsm) was used as a statistical tool to optimize the initial yeast extract and starch contents of the medium, which resulted in a substantial increase in the stability of the expression plasmid in both strains with concomitant improvement in their amylolytic potentials. high ethanol yields on substrate values of the bioreactor cultures, that were very close to the theoretical yield, indicated that the amylolytic respiratory-deficient strain developed in this study was very effective in the direct fermentation of starch into ethanol. establishing a biotechnology educational framework to support a knowledge-based economy in puerto rico rosa buxeda, lorenzo saliceti-piazza industrial biotechnology program, university of puerto rico, mayagüez campus, mayaguez , puerto rico. e-mail: rbuxeda@uprm.edu (r. buxeda) industrial biotechnology has been identified as a major thrust area of economic development within the past five years for the island of puerto rico. the portfolio of biotechnology manufacturing investments in the island has passed the two billion dollar mark. world known companies like amgen, abbott and eli-lilly lead these investments, which have catalyzed a strong technology transfer to the island. a strong collaboration between industry and academia was needed to provide a well-trained workforce for these company startups. as a result, the university of puerto rico, mayagüez campus (upr-m) developed four initiatives which are part of the educational pipeline in biotechnology. these are: (i) an industrial biotechnology program, a -year bs degree which contains a novel curriculum with courses from science and engineering with undergraduate research and industrial internships as part of the degree requirements; (ii) an industrial biotechnology learning center, which provides customized biotechnology and bioprocessing training modules, including lectures and hands-on experiences to train and develop the workforce needed in the biotechnology manufacturing plants; (iii) a biotechnology summer camp, which addresses the high school student population and its main purpose is to educate and advise on the different career paths that can be followed in the field of biotechnology; and (iv) a biotechnology center for research and training in bioprocessing, that will address the development of corporatesponsored research projects to strengthen links between industry and academia in order to build up a knowledge-based economy. our paper will describe each initiative in detail as well as its outcomes and impact on puerto rico's knowledge-based economy goals. isolation of acid phosphatase from sweet potato and immobilization using different adsorbent d. omay, y. güvenilir, n. deveci istanbul technical university, department of chemical engineering, maslak , istanbul, phosphatase enzymes occur in a wide range of plant and animal tissues. they catalyze the hydrolysis of phosphate bonds in organic phosphates, between the phosphate group and rest of the molecule. immobilization of enzymes and biological compounds is currently gaining importance due to its wide variety of applications in the food and pharmaceutical industries and also its biomedical applications. it was reported that enzymes can be activated by complexation with polysaccharides such as chitin or chitosan. the aim of this experimental study was determined as partial purification and isolation of acid phosphatase enzyme and its immobilization. the purification was realized by applying centrifugation, ammonium sulfate precipitation and dialysis respectively. the specific activity of the supernatant was . u/mg and after % saturation this value increased . u/mg. furthermore, acid phosphatase was investigated using different adsorbent (chitin, chitosan, synthetic zeolite and raw zeolite) and evaluated the storage stability and re-usability of the immobilized acid phosphatase. it was estimated that, acid phosphatase activity was shielded the ratio of , , , and % by using raw zeolite, synthetic zeolite, chitin and chitosan respectively under h operation condition. Øyvind m. jakobsen , , michael c. flickinger , svein valla , trond e. ellingsen , trygve brautaset : department of biotechnology, norwegian university of science and technology, norway; sintef applied chemistry, sintef, norway; biotechnol. institute, department of biochemistry, molecular biology and biophysics, university of minnesota, usa aerobic methylotrophs can utilize one-carbon (c ) compounds such as methane and methanol as a sole c-source for growth and energy. the majority of research on these organisms has focused on their biochemical novelity and commercial viability. for the industrial production of bulk products such as the amino acids lysine and glutamate raw material costs and abundance are important, and c sources are thus attractive compared to sugars. bacillus methanolicus can secrete up to g/l of glutamate upon methanol growth at • c (thermotolerant and methylotroph) and mutants producing - g/l of l-lysine have been selected. we study the genetics for conversion of methanol into biosynthesis of glutamate and lysine, and in the present report we unravel the regulation of genes impor-tant for the consumption and tolerance level for c compounds by b. methanolicus. b. methanolicus has a methanol dehydrogenase gene (mdh) for oxidation of methanol into formaldehyde and a ribulose monophosphate (rump) pathway for assimilation of formaldehyde. we recently discovered that mdh and five rump genes are carried by natural plasmid pbm in this bacterium and this represented the first documentation of plasmid-dependent methylotrophy in any microorganism. we here use real-time pcr to analyse the regulation of plasmid-and chromosomally located rump genes, in cells upon methylotrophic and non-methylotrophic growth. high methanol concentrations in the growth medium is cell toxic and the mechanisms for this sensitivity of b. methanolicus is poorly understood. our results indicate that plasmid pbm plays a fundamental role for this trait as well and the impact of our results on the biotechnological applications of this bacterium is discusssed. department, national research centre, dokki, cairo, egypt adding a proteolytic enzyme extraction from jack fruit (artocarpus integrifolis) in combination of fermentation process in low fat yogurts manufacture was tried to improve yogurt flavour and rheological properties. experimental yogurts milk contained control, . (t ), . (t ) and . (t ) units/ml milk from crude extracts of plant proteinase. the ph of the product treated with crude proteinase was lower than the control. however, the rate of acidity development during storage slightly increased with increasing the addition of crude proteinase level and progress of storage period of yogurt. the proteolytic activity of all yogurts gradually increased until the end of storage period ( days). yogurts made from milk treated with crude proteinase preparations were less firm compared with control at all storage periods, where t showed more less firm after days of storage being . g/ g. generally, increasing units of plant proteinase preparations decreased the firmness. on the other hand, yogurt made from milk pretreated with plant proteinase had higher syneresis, and apparent viscosity than the untreated product. the greatest viscosity was found in t and t of and mpa s respectively, compared with control of mpa s at days storage. the results indicated that there is an inverse relationship between the amount of units of crude proteinase preparations and susceptibility of yogurt to syneresis. the t gained the highest scores ( points) followed by the control ( . points) after days of storage, while yogurt of t showed a low scoring being . from the foregoing results, it is recommend to use jack fruit (artocarpus integrifolis) as a source of plant proteinases and utilize it to develop a high quality yogurt at a level of . units of plant proteinases/ml milk. the penicillium chrysogenum oat gene encoding a class iii omega-aminotransferase has been cloned and characterized. this enzyme that converts lysine into -aminoadipic semialdehyde is important in providing -aminoadipic acid, a precursor of penicillin and other ␤-lactam antibiotics. the enzyme is related to ornithine- -aminotransferases and to lysine- -aminotransferases encoded by the lat gene located in the bacterial cephamycin gene clusters. expression of oat is induced by lysine, ornithine and arginine and repressed by ammonium ions. area-binding consensus sequences and an -bp direct repeat associated with arginine induction in emericella (aspergillus nidulans) have been found in the oat promoter region. deletion of the oat gene resulted in the loss of omegaaminotransferase activity. the deletion mutants were unable to grow on ornithine or arginine as sole nitrogen sources and showed a reduced growth on lysine. complementation of the deleted mutant with the oat gene restored growth on ornithine, arginine and lysine to the levels of the parental strain and omega-aminotransferase activity. the strong expression of oat gene after induction by the basic amino acids may provide additional -aminoadipic acid for the formation of the -aminoadipyl-cysteinyl-valine tripeptide for ␤-lactam biosynthesis. morphological characterisation of two high producing strains of penicillium chrysogenum carrying a disruption in the nadph-dependent glutamate dehydrogenase k. rueksomtawin, j. thykaer, h. noorman, j. nielsen center for microbial biotechnology, biocentrum-dtu, technical university of denmark, dk- lyngby, denmark. e-mail: kr@biocentrum.dtu.dk (k. rueksomtawin) metabolic engineering has proven to be useful in optimisation of ␤-lactam production, e.g. constructing superior strains with multiple copies of the ␤-lactam gene cluster. it is however, of equal importance to gain insight into other aspects of the metabolism to establish a general overview in order to apply metabolic engineering for further improvement of the production strains. in that context, the redox metabolism is essential, as it functions as a tightly controlled connection between the different parts of the metabolism. in order to investigate this role of the redox metabolism in more detail, the gdha-gene, encoding the nadph-dependent glutamate dehydrogenase, was disrupted in two industrial strains of penicillium chrysogenum. during physiological characterisation of the two strains it became apparent that considerable changes in the morphology had occurred due to the genetic alteration. since the morphology is an important parameter in process optimisation, an examination of the morphology of the two strains was undertaken. in this work, the morphological differences between the gdha-disrupted strains and the reference strains were comprehensively investigated both during growth on solid media and submerged growth in a flow-through growth chamber. with the advance development of computerized image analysis techniques, the key morphological properties of the individual hyphal elements were quantified. in comparison to the reference strains, the disruption of the gdha gene resulted in a morphological change from short hyperbranched hyphal elements to long elongated hyphal elements with less branches. in the parallel studies with aspergillus nidulans and its corresponding gdha-deleted strain, no difference in the morphology was observed. polyketides (pk) represent one of the largest groups of natural products and are found in fungi, bacteria and plants. since many useful polyketides either originate from sources that are difficult or even impossible to cultivate or are produced in inadequate amounts, we are interested in expressing polyketide synthases (pkss) in heterologous hosts. saccharomyces cerevisiae, aspergillus niger and aspergillus nidulans were chosen as initial hosts, because the techniques necessary to cultivate and manipulate these strains genetically are well established. -methylsalicylic acid synthase ( -msas) was chosen as a model pks. replicative plasmids carrying the genes encoding -msas from penicillium patulum and phosphopantetheinyl transferase (pptase), respectively, were transformed into s. cerevisiae. in addition, an integrative vector was designed and the gene encoding -msas was integrated in the yeast chromosome. batch cultivations on galactose minimal media were performed. the results are presented and in particular the effect of expression mode and type of pptase (bacterial versus fungal) is discussed. furthermore, the progress of the work on expressing -msas in a. niger and a. nidulans using an integrative vector system is presented and discussed. the valorisation of functionalized chemicals from biomass resources compared to the conventional fossil production route ben brehmer, wageningen ur agrotechnology & food sciences, workgroup: valorisation of plant production chains, p.o. box , aa wageningen, the netherlands. e-mail: ben.brehmer@wur.nl at some undisclosed point in the foreseeable future, cheap fossil fuels resources will become depleted and the industry will be forced to pursue more difficult reserves with increasingly high extraction costs. most alternatives available and under research do not consider price as the main motivation for replacement, but focus solely on sustainability and environmental benefits. sustainability is an interesting word as there are enough fossil resources scattered around the world to be sustainable in quantity but not sustainable in price. seeing that fossil fuels are derived from prehistoric biomass it is not at all presumptuous to assume that every application and product can be replaced by the biomass of today. in fact, many highly specialised pharmaceutical chemicals already have a biomass origin. yet, not all of the uses of fossil fuels need to be replaced by a comparable carbon based source, such as biomass. energy and transportation in particular do not necessary need to rely on carbon cleavage, whereas practically all of the petrochemicals contain a carbon backbone. the main stipulation in substituting fossil-based chemicals with bio-based chemicals is availability and cost. it is proposed that already today, by utilising existing, recently developed and developing technology, it is economically advantageous for many chemicals to derive from biomass, in particular the functionalized chemicals. the only way to validate this conjecture is to develop a complete comparative life cycle analysis. as opposed to a traditional lca, the "multicriterion" developed here will revolve around energy flows and process efficiency in terms of exergy. the aim is to assess the optimum route with the best production options along the whole production chain while determining any possible limiting factors. using this tool, a systematic production matrix relating several logical source crops and a few key chemicals of varying derivative levels can be created and compared to the conventional fossil routes. combined with economic considerations and some unambiguous environmental factors, the investigation will provide all the information relevant to the industry. the goal is to create an objective and reliable simulation system ratifying the economic and environmental feasibility of exploiting biobased chemicals today and indicate the steps necessary for further improvement. biosynthesis of multi-enzymatic preparation from aspergillus niger ibt- useful in textile fabric treatment rita pyc , jadwiga sojka-ledakowicz , tadeusz antczak , joanna lichawska : institute of technical biochemistry, the technical university of lodz, lodz - , poland; textile research institute, lodz - , poland among many methods of producing enzymatic preparations, i.e. by liquid surface fermentation -lsf, submerged fermentation -smf or solid state fermentation -ssf, this last is most advantageous. cultivation in solid state means fermentation on a matrix formed by industrial and agricultural wastes. most often filamentous fungi -due to the lack of available water in the foundation -are the efficient, competitive microorganisms applied in solid state bio-conversion. the aim of research works carried out at the institute of technical biochemistry of the technical university of lodz and at textile research institute, lodz was defining optimal conditions for biosynthesis and testing the possibility of applying multi-enzymatic preparation from aspergillus niger ibt- in the treatment of woven fabrics made of natural cellulose fibres. as the result of biosynthesis optimization process, malt sprouts, wheat barn and beet pulp were selected as the best media to obtain enzymes of high pectinolytic activity maintaining at the same time high activity of cellulolytic enzymes and xylanase. the highest obtained activities reached: pm for total pectinolytic activity, j/ml for endoglucanase and j/ml for endoxylanase and they were . - . times higher than those achieved before optimizing process. performed research works demonstrated that the optimum activity of applied enzymatic system is obtained in the range of ph . - . . woven fabrics made of flax and cotton fibre blends, subjected to bio-pre-treatment, were evaluated with reference to their sorption properties. comparative evaluation of liquid sorption by woven fabrics allowed to notice efficient enhancement of fibres' sorption capabilities after pre-treatment using enzymes system from aspergillus niger ibt- . this offers the possibility of substitution of alkali scouring of linen-cotton woven fabrics before their bleaching by bio-treatment. the phylum actinobacter includes many bacteria of industrial importance both for accumulation of primary and secondary metabolites. both primary and secondary metabolites are dependent on precursors and cofactors that are provided by the central carbon metabolism of microorganisms. there are alternative pathways for catabolism of carbon either via the embden meyerhof parnas (emp) and pentose phosphate (pp) pathway or through the entner doudoroff (ed) pathway. the emp pathway is energetically more favorable and has therefore been presumed to be the dominating route for carbon metabolism in bacteria producing secondary metabolites. however, primary metabolism is poorly studied for most actinobacter species as focus traditionally has been on secondary metabolism. with the aim to gain more knowledge about the diversity of central carbon metabolism within the phylum actinobacter, different strains were collected from various sources and strain collections. the strains were grown in minimal medium with supplement of standard vitamin solution and [ - c] glucose as carbon source. the c-labeling patterns of proteinogenic amino acids were determined by gc-ms analysis. through this method, the fluxes in the central carbon metabolism during balanced growth were estimated and pathways for carbon metabolism were determined. in particular the labeling patterns of alanine and valine were of interest as they are derived from pyruvate and therefore can be used to distinguish between whether glucose is metabolized through the ed pathway or the emp pathway. nobacter, amycolatopsis balhimycina produces the glycopeptide balhimycin. the balhimycin aglycone is identical to the aglycon of vancomycin, which is a commercial glycopeptide. as a. balhimycina appears to be accessible to genetic modifications and the biosynthetic cluster responsible for balhimycin production is published, this genetically well-characterised academic strain can serve as a platform strain for production of vancomycin analogues derived through combinatorial biosynthesis. the understanding of the physiology of this microorganism is essential for the efficient accumulation of potentially commercial secondary metabolites. the bacterium is capable of growth in a fully defined minimal medium, with the production of balhimycin. the strain was grown at either nitrogen or phosphate limitation and balhimycin accumulation was followed at these conditions. flux analysis of balhimycin production by amycolatopsis balhimycina based on c labelling experiments was performed. the zygomycetes blakeslea trispora and phycomyces blakesleeanus accumulate beta-carotene, ubiquinone (coenzyme q), various different sterols, and other terpenoids, all of them produced via the mevalonate pathway. these fungi are used or could be used for the industrial production of these terpenoids, edible oil, chitosan, and various organic acids. by measuring the specific radioactivity of terpenoids made from radioactive mevalonate, leucine or acetate in the presence of excess glucose in wild types and mutant strains we have concluded that these fungi have separate subcellular compartments for the production of carotene, sterols and triacylglycerols. the terpenoid moiety of ubiquinone is synthesized in the same compartment as ergosterol. these compartments contain separate pools of all their common metabolites, beginning from acetyl-coa. mevalonate carbon atoms do not find their way back to general metabolism, i.e., these fungi lack the "shunt" pathway. the compartments are regulated independently. the very large variations in carotene content caused by many environmental and genetic changes are not accompanied by variations in the ubiquinone content. the ubiquinone content increases when the cultures grow on leucine or acetate as carbon sources and is not affected by illumination. phycomyces, but not blakeslea, increases the production of ubiquinone in presence of oligomycin. lincomycin, produced by streptomyces lincolnensis, is important, clinically used antibiotic. its gene cluster consists of putative open reading frames with biosynthetic or regulatory functions (lmb genes) and three resistance genes (lmra, lmrb, lmrc). the organization of transcription units was determined. the analysis of the lincomycin biosynthetic gene transcripts in various cultivation stages revealed the genes with putative regulatory functions which are transcribed in early stages of cultivation. previous analysis of biosynthetic pathways of lincomycin and functionally different anthramycin antibiotics (anthramycin, sibiromycin, tomaymycin, mazetharmycin and porothramycin) indicates that the genetic information on the lincomycin and anthramycin biosynthesis should share common elements (genes), both biosynthetic and regulatory. hybridization experiments demonstrated presence of several analogues of lmb genes involved in the biosynthesis of anthramycin produced by streptomyces refuineus and porothramycin produced by streptomyces albus. effect of various calcium salts on the erythromycin production by saccharopolyspora erythraea m. rostamza , a. noohi , j. hamedi * : department of biology, faculty of science, science and research branch, islamic azad university, tehran, iran; microbial biotechnology lab., department of biology, faculty of science, university of tehran, tehran, iran. e-mail: jhamedi@khayam.ut.ac.ir (j. hamedi) calcium carbonate has the positive effect on the erythromycin, however, because of its low water solubility, caused to clogging the spargers of fermenters and fouling the microfilters. in this research, various soluble calcium salts was added to the fermentation medium and their effect the growth of saccharopolyspora erythraea and erythromycin production was studied in the complex medium consisted soy bean meal, dextrin and starch as major ingredients. the fermentation conditions were rpm, days at • c. the results obtained showed that there is no significant difference between erythromycin concentrations in the medium containing calcium lactate and calcium carbonate. however, erythromycin concentrations in the other calcium salts containing media were less than to calcium carbonate containing medium. optimum concentration of calcium lactate for erythromycin production was g/l. lincosamides and its derivatives are clinically important antibiotics. comparison of gene cluster coding for lincomycin biosynthesis and newly identified cluster of genes for celesticetin biosynthesis revealed new information on functions of several genes common for both biosynthetic pathways. the celesticetin gene cluster was identified by screening the cosmid library of streptomyces caelestis with heterologous probes based on lincomycin biosynthetic genes involved in a part of biosynthesis shared by both antibiotics. sequence analysis of the cluster revealed putative orfs, out of which are lincomycin biosynthetic genes analogues, four are specific for celesticetin biosynthesis and one codes for resistance. the gene cluster is bounded with transposase genes on both sides. in order to clarify function of three putative regulatory genes of lincomycin biosynthesis, the insertional inactivation with the pcr targeting system in streptomyces lincolnensis was done and resulted in differently reduced production of lincomycin. larsen cmb biocentrum-dtu, technical university of denmark, kgs. lyngby, denmark we present a new algorithm called "x-hitting" for automatic identification of novel bioactive compounds based on full spectroscopic characters of highly complex mixtures of natural product. one of the most dramatic advances in recent drug discovery has been the increase in screening capacity throughput and data handling. therefore, analysis has become the bottleneck in the drug discovery process. the algorithm presented here is investigated and demonstrated on identifying potentially new bioactive compounds. in addition method is shown to have a high performance for automatic identification of known structures. these tasks are referred to as new-hitting and cross-hitting, respectively. finally, the receiver operating characteristics (roc) is introduced to the research field as an important tool for evaluating the performance of the "compound predictor". through examples it is shown, that known cross-hits are identified with high proficiency, and that the new-hitting works on finding new targets represented by analogues and structurally new compounds. a gene encoding a ␥-butyrolactone autoregulator receptor that have a common activity as dna-binding transcriptional repressors, controlling secondary metabolism and/or morphological differentiation in streptomyces was cloned from a natamycin producer, streptomyces natalensis, and its function was evaluated by in vivo. pcr using the primers designed from two highly conserved regions of streptomyces autoregulator receptors gave a -bp band, the sequence of which revealed high similarity to the expected region of a receptor gene. by genomic southern hybridization with -bp insert as a probe, a -bp intact receptor gene (sngr) was obtained from s. natalensis. in vivo to clarify the function of sngr, a sngrdisrupted strain was constructed, and a phenotype was compared with the wild-type strain. the sngr disruptant started natamycin production h earlier and showed a . -fold-higher production of natamycin than the wild-type strain. in addition, sporulation was earlier and -fold abundance. the phenotype indicates that the autoregulator receptor protein of s. natalensis acts as a primary negative regulator of the biosynthesis of natamycin and is related to regulation of sporulation. exploring the biocatalytic potential of the novel thermostable baeyer-villiger monooxygenase: phenylacetone monooxygenase daniel e. torres pazmiño , gonzalo de gonzalo , gianluca ottolina , giacomo carrea , dick b. janssen , marco w. fraaije , biochemical laboratory, groningen biomolecular sciences and biotechnology institute, university of groningen, nijen- baeyer-villiger monooxygenases (bvmos) represent useful biocatalytic tools as they can catalyze reactions which are difficult to achieve using chemical means. however, only a limited number of these atypical monooxygenases are available in recombinant form. using a recently described protein sequence motif, a putative bvmo was identified in the genome of the thermophilic actinomycete thermobifida fusca. the nadph-dependent and fad-containing monooxygenase is active with a wide range of aromatic and aliphatic ketones and sulfides. genetic and kinetic data suggest that phenylacetone is the physiological substrate of the enzyme. previously, it was reported that this bvmo exhibits only a moderate enantioselectivity with (r,s)-␣-methylphenylacetone. this poster we will show an overview of the biocatalytic potential of the enzyme as explored so far. interestingly the enzyme has been found to perform highly enantioselective oxidations with a range of ketones and sulfides. this again indicates that this novel thermostable oxidative biocatalyst can be a useful tool for the synthesis of chiral building blocks. the enzyme s-adenosylhomocysteine hydrolase (adohcyase) catalyzes hydrolysis of s-adenosylhomocysteine (adohcy), an inhibitor of transmethylation reactions, into adenosine (ado) and homocysteine (hcy). the catalysed reaction is reversible, and the equilibrium is strongly displaced in direction of the synthesis of adohcy when reaction occurs in vitro. nevertheless, its biotechnological application resides in the synthesis of antivirals. for it, we selected between different producing microorganisms of the enzyme, the gram positive bacterium corynebacterium glutamicum. after designing the specific oligonucleotides, the gene was expressed in escherichia coli using the expression system pet a+ with iptg. the electrophoretic analysis under denaturing conditions, shows a clear induction and over-expression of a protein with a mw of kda. on the other hand, the immobilization of this recombinant enzyme in a solid support allows to use it as a catalyst for the synthesis of adohcy. the enzyme was purified by imac thanks to the presence of n-terminal × his tag end, and immobilized in eupergit c for the optimization of the production of adohcy, a product of high value. sequencing, cloning, expression, purification and characterization of a novel cytochrome p monooxygenase from rhodococcus rubber luo liu, rolf d. schmid, vlada b. urlacher institute of technical biochemistry, stuttgart university, allmandring , stuttgart, germany. e-mail: itbvur@itb.unistuttgart.de (l. liu) the cytochrome p monooxygenases are heme-containing proteins, which catalyze a wide range of oxidative reactions (werck-reichhart and feyereisen, ) . a monooxygenation activity was observed for the strain rhodococcus ruber dsm . a p -like gene fragment was amplified by pcr using degenerated primers. for identification of regions that flank this p -like dna fragment, the method "directional genome walking using pcr" was applied (mishra et al., ) . the full size gene encoding a cytochrome p enzyme was amplified by pcr from genomic dna and cloned into the vector pet a(+) for heterologous expression in escherichia coli bl (de ) cells. the enzyme was purified using metal affinity chromatography. the primary protein structure suggests, that this enzyme is a natural self-sufficient fusion protein consisting of a ferredoxin, a reductase and a p monooxygenase. the reductase activity was determined using an exogenous electron acceptor cytochrome c. the reductase domain of this p monooxygenase showed a strong preference for nadph over nadh. the substrate spetrum was investigated. in the presence of nadph the p enzyme shows hydroxylation activity towards -ethoxycoumarin, naphthalene, indene, acenaphthene, toluene and fluorene. mishra, r.n., singla-pareek, s.l., nair, s., sopory, s.k., reddy, m.k., . directional genome walking using pcr. biotechniques , - . werck-reichhart, d., feyereisen, r., . cytochromes p : a success story. genome biol. ( ), . - . . selective production of monoglyceride consisted of conjugated linoleic acid by penicillium lipase yomi watanabe , , yoshie yamauchi-sato , toshihiro nagao , satoshi negishi , tadamasa terai , takashi kobayashi , rolf d. schmid , yuji shimada : osaka municipal technical research institute, osaka, japan; stuttgart university, stuttgart, germany; the nisshin oillio group, ltd, yokosuka, japan; osaka institute of technology, osaka, japan conjugated linoleic acid (cla) is a group of c fatty acid (fa) containing two conjugated double bonds. it is expected to prevent cancer, adipositas, atherosclerosis etc, and is commertially available in the primary form of free fa, containing almost equal amounts of cis, trans-and trans, cis-cla. it is therefore desired to be converted to a palatable form. for this purpose, we have previously proposed two enzymatic ways to convert cla to monoglyceride, an emulsifier, with penicillium camembertii lipase; ( ) sequencial esterification-glycerolysis, ( ) esterification at low tem-perature. these methods, however, are time-and energy-consuming. esterification of cla with glycerol under ambient pressure by the lipase produces equal amounts of mono-and diglycerides. in contrast, it was newly found that the reaction under reduced pressure supressed the formation of diglycerides and achieved to produce % monoglyceride at % esterification. improving the thermal stability of cellobiohydrolases i (cel a) from t. reesei by site directed evolution frits goedegebuur , lydia dankmeyer , peter gualfetti , brad kelemen , edmundo larenas , paulien neefe , pauline teunissen , colin mitchinson : genencor international bv, archimedesweg , cn leiden, the netherlands; genencor international inc., page mill road, palo alto, ca , usa genencor international has been working to produce improved enzyme products for economic conversion of ligno-cellulosic biomass to fermentable sugars. most of this work was performed under a subcontract with the u.s. department of energy for cellulose cost reduction for biomass conversion. cellulolytic biomass conversion is performed in nature by a complex mixture of enzymes. cellobiohydrolases play a key role and all effective cellulase mixtures contain a large excess of cellobiohydrolases over endoglucanases, suggesting that it is the exoglucanase activity that is limiting. the fundamental dependence of reaction rate on temperature predicts that large increases in performance, and decreased enzyme cost, would be achieved if the enzymatic conversion could be operated at elevated temperatures. industrial strains of trichoderma reesei produce cellulases at very high levels and low cost. however, t. reesei cbh (hypocrea jecorina cel a) does not have sufficient stability to survive and perform at high temperatures. this poster shows the thermal stability improvement in t. reesei cbh by site directed evolution. sites with increased thermal stability properties were combined and evolved in high temperature stable cbhi variants. the evaluation of lipases as biocatalysts for organic chemistry can be carried out, at laboratory scale, by using soluble enzymes for biotransformations in aqueous media. however, the industrial exploitation of such an enormous potential should require a suitable protocol for immobilization of lipases. the binding of lipases on suitable pre-existing supports should greatly improve the performance of industrial reactors allowing us a continuous use or re-use of such interesting biocatalysts. in addition, lipases, like most enzymes, are not perfect chemical catalysts. lipases may be unstable and they may not have the optimal activity nor the optimal enantio or regioselectivities. in this way, immobilization of lipases, together with its relevance for the performance of each different industrial reactor, could be also used as a tool to improve and optimize some of these parameters. that is, immobilization of lipases, far from an already solved problem, constitutes an exciting field of research in the promising area of industrial bio-organic chemistry. in this work we would like to present useful immobilization methods of several lipases. the lipase immobilised were used for enzymatic hydrolysis of peptidomimetics of structure a. type of immobilzation used can changed the enantioselectivity of biocatalyst prepared. the absolute configuration of products b and c obtained in enzymatic reactions were assigned by chemical correlation. two-component flavin-dependent monooxygenases form an interesting class of flavoenzymes. they consist of two separate proteins; a monooxygenase component, which catalyses an oxygenation reaction in the presence of reduced flavin, and a flavin reducing component, which reduces flavin (fad or fmn) using nad(p)h as an electron donor. a well-known example of this class of monooxygenases is styrene monooxygenase (otto et al., ) . due to the ability to form enantiopure epoxides, which are relevant building blocks for the pharmaceutical industry, styrene monooxygenases form a valuable class of enzymes for biocatalysis. while screening a metagenomic library for oxidative enzymes, an indigo-producing clone was found. sequencing the particular clone revealed an inserted fragment of environmental dna encoding a two-component monooxygenase ( many investigations over the recent years have been directed to the production of natural aroma compounds. through biotransformation and bioconversion, aroma compounds considered as "natural" can be produced starting from monoterpenes, generating high value products as rose oxide. rose oxide is found in small amounts in some essential oils such as bulgarian rose oil and geranium oil. (−)-rose oxide is an impacting flavor compound and has a small threshold: . ppb. application of agro-industrial residues in bioprocess on one hand provides alternative substrates, and on the other hand helps solving pollution problems, that might be caused by the disposal of this residue in nature. the liquid cassava waste, is originated by the pressing of cassava roots. it is considered as a "harmful" pollutant waste due to its high organic charge and presence of cyanide. on the other hand, can be considered rich in nutrients that can be used in other applications. it was found that sporulated surface cultures of penicillium sp. were able to convert citronellol into cis-and trans-rose oxides. other bioproducts were , -dimethyl- -octen- , diol and , -dimethyl- , -epoxy- -octanol. no chemical oxidation or auto-oxidation products were detected in liquid control broths. the experiments were conducted at • c and rpm. when the medium was cassava, the production of rose oxide, , -dimethyl- octen- , -diol and , -dimethyl- , -epoxy- -octanol were insignificant reaching trace amounts. but when the mycelium developed in cassava medium and than transferred to mineral medium (citronellol as c-source) the concentrations of rose oxide increased dramatically, reaching mg/l for the cis-isomer and mg/l for trans-isomer. a mechanistic mathematical model of enzymatic degradation of avicel and phosphoric acid swollen cellulose (pasc) has been proposed. the model is based on the degree of polymerization (dp) of starting substrate, and follows its decline with time, to the final end product -glucose. three enzyme classes, namely, endoglucanase (eg), cellobiohydrolase (cbh) and ␤-glucosidase (bg) are all individually incorporated in the model. the model is, additionally, taking into account cooperative action of the involved enzymes, as well as effects of enzyme inhibition by end-products, cellobiose and glucose. to be able to describe the complex process of enzymatic hydrolysis with a set of differential equations certain assumptions needed to be introduced. those assumptions represent the simplification of an up-to-date knowledge of both substrate and enzyme structure, but also enzyme mode of action. for example, one of the often asked questions is: "what is happening with shorter cellooligosacharides (dp and up) laying on the surface of cellulose chain? are they being adsorbed to the core cellulose chain or partly solubilized to a hydrolysis broth?" to give answers to these questions and confirm mathematical modeling real enzyme hydrolysis data are needed. in this work, four well characterized, highly purified mono-component enzymes from humicola insolens (two eg and two cbh) and one bg from aspergillus niger were used to hydrolyze avicel and pasc. by careful choice of catalyst, some enzyme specific characteristics like presence or absence of cellulose binding domain will also be incorporated into the model. hydrolysis experiments were initially performed by distinct mono-component enzymes, to confirm the basic characteristics of each of the enzyme classes. soluble hydrolysis products (dp - ) were analyzed by hplc and detection of non-soluble, higher-dp polysaccharides was performed by technique of polysaccharide analysis using carbohydrate gel electrophoresis. optimisation of halogenase enzyme activity k. muffler , m. retzlaff , k.-h. van pée , r. ulber : technische universität kaiserslautern, germany; technische universität münchen, germany; technische universität dresden, germany. e-mail: muffler@rhrk.uni-kl.de (k. muffler) halogenases provide the opportunity of a regioselective and stereospecific halogenation of organic subtrates in contrast to the class of haloperoxidases. these enzymes allow a gentle synthesis of halogenated organic molecules and are capable to halogenate in specific positions (hammer et al., ; keller et al., ) , whereas traditional organic synthesis often failures or mainly leads to byproducts (hasegawa et al., ) , e.g. halogenation of tryptophan in other positions than position . our current research is focussed on tryptophan- -halogenases, because the -br/cl-tryptophan could be applied as a pharmacologically attractive precursor of serotonin. in our work we describe the optimization of an enzyme assay respectively the enzyme activity. for this purpose we use a genetic algorithm. the responsible gene of the fadh dependent enzyme was cloned from streptomyces sp. origin into a pseudomonas fluorescens strain. however, the optimization procedure was done with the purified his-tagged tryptophan- -halogenase, which was easily obtained from the crude extract of the lysed cells by application of immobilized metal affinity chromatography. the application of algorithms allows an optimization of the multidimensional search problem leading to a global optimum in the search space in contrast to the traditional used one-factor-at-a-time method. latter often failures, because this method does not reflect possible influences the parameters can have on each other. effect of organic solvent type on the enantioselectivity of candida rugosa lipase in the hydrolysis of racemic naproxen methyl ester in biphasic reaction system serpil takaç, deniz mutlu department of chemical engineering, institute of biotechnology, ankara university, tandogan, ankara, turkey hydrolysis of racemic naproxen methyl ester to produce s-naproxen was carried out in the biphasic system using isooctane, cyclohexane, hexane and toluene with candida rugosa lipase after stirring in the phosphate buffer (ph . , . m) for h at + • c and centrifuging. the hydrolyses were carried out in shaking flasks for h at rpm and • c with the initial substrate concentration of . m. the concentrations of the enantiomers of racemic naproxen methyl ester in organic solvents and those of naproxen in buffer solution were determined with hplc. it was found that the enantiomeric excess for substrate (ee s ), enantiomeric ratio (e) and conversion (x) decreased in the following order: isooctane > cyclohexane > hexane > toluene. the enantiomeric excess for product (ee p ) was found to be the same for isooctane, cyclohexane and hexane where the lowest ee p was obtained in toluene. the highest ee s , ee p , e and x values achieved in isooctane at the residence time of h were , , , and %, respectively. this study was supported by ankara university biotechnology institute (project no: ). fusarium fujikuroi (gibberella fujikuroi mating group c) produces multiple secondary metabolites such as gibberellins and bikaverin. gibberellins are terpenoid hormones that induce growth and regulate various stages of development in plants. they have numerous applications in agriculture industry. bikaverins are polyketides that have toxicity against different organisms because they inhibit respiration. regulation of polyketide and gibberellin synthesis by nitrogen has been intensively studied in fusarium but little is known about their regulation by carbon source. our main interest is to understand the regulation of biosynthesis of these compounds in f. fujikuroi imi . to investigate this regulation the organism was grown in high nitrogen medium under submerged conditions and then transferred to nitrogen-free media having various concentrations of different carbon sources. the gibberellin production was not affected significantly. on the other hand bikaverin was synthesized enormously when sucrose was used as the only carbon source. high production in sucrose required a minimal amount of the sugar, but did not change appreciably above this threshold along a wide range of concentration. the bikaverin synthesis was repressed when glucose coexisted with sucrose in the medium. the effect of the c source on the expression of key genes, cps/ks (copalyl diphosphate synthase/kaurene synthase) for gibberellin and pks (polyketide synthase) for bikaverin biosynthesis is currently under investigation. ment of dormancy and loss of viability in seeds with the passage of time, it lacks any systematic propagation from seeds and is typically propagated through rhizomes. this restricts large scale cultivation of this plant. in vitro propagation of plants is an effective means for rapid multiplication of species, in which conventional methods have limitations. in the pesent study we have analysed the role of various growth promoters and the effects of dark and light incubation on germination of n. alba seeds. the results indicate that in vitro propagation of n. alba from seeds can be applied as an efficient method of multiplication. the study was funded by the state planning commisson (dpt) turkey and the university of ankara vide projects no. . this research was performed for developing of biological treatment process of odor gas such as mek, h s, and toluene, which is generated from the food waste recycling process. to establish the operational conditions of odor gas removal by small-scale biofiltration equipment, it was continuously operated by using toluene as a treating odor object. when the odor treating microorganisms were adhered to fibril form biofilter, high removal efficiency over % was obtained by biofilm formation. at ppm of inlet odor gas concentration and s of retention time, the removal efficiency was and % in first stage reactor and second stage reactor, respectively. however, the removal efficiency remained over % at the operational conditions above s of retention time. post soviet countries are going through the transition stage and are extremely sensitive to new technology, economics or social changes and globalization processes. that is why decision-making and system of regulation of the use of gmo's are very sensitive to number of factors. three levels of factors, the most influential to decision-making: global, regional and local; are identified at the research paper. global level depends on external policy of leaders of gmo regulations. usa and eu have the biggest influence on transition countries, though their positions completely differ. as usa was leader in inventing gmos, it is lobbing newly created biotechnological industry. in eu and other european countries lobbing of biotechnological industry was not as strong as in usa. thus, their national law is stricter. world trade organization and international agreements are also part of global level. regional level. geographical position of country is also very important because every regulation system depends a lot on regulation that is implemented in neighboring countries. countries do not exist in vacuum; they are linked territorially, politically, economically and socially with neighboring states. regulation systems of the transition countries in the eastern europe can be divided into three types: those who have no system of regulation of gmo (belarus, romania, hungary and ukraine); who approved some variety that are treated as safe to the market (poland, moldavia and georgia) and who approved all of the gmos (bulgaria, croatia and russia [before ]). but even if a country declares not to use gmo it is rather difficult to control import of such products, because of the lack of the testing laboratories, corruption of the state employees, agreements on intellectual property, and institutional country problems. in spite of global and regional tendency of gmos related regulation the most important part is the local level, namely the national regulation system. depending on national level we are choosing priorities at higher levels. as an example of post soviet countries ukraine is taken, as ukraine is one of the largest countries and one of the biggest exporters of agricultural products in europe. research includes the analysis of attitude to gm product, their potential risks and benefits of three categories that influence decision-making the most: farmers, gm experts and non-governmental organizations. recombinant microorganism development for extracellular benzaldehyde lyase production hande kaya , pınar Ç alık , tunçer h. ozdamar : iblab, department of chemical engineering, metu, ankara, turkey; bre laboratory, department of chemical engng, ankara university, ankara, turkey. e-mail: e @metu.edu.tr (h. kaya) in this study, the extracellular production of the benzaldehyde lyase (bal, ec . . . ) that catalyses the synthesis the enantiopure -hydroxy ketones for drug syntheses, by bacillus sp., was aimed. for this purpose, the signal dna sequence of an extracellular bacillus enzyme, i.e., serine alkaline protease, was fused in front of the bal gene (accession number ax ) from pseudomonas fluorescens biovar i, using pcr-based gene splicing by overlap extension (soe) method. b. licheniformis (dsm ) chromosomal dna was used as sap gene (accession number x ) template for the synthesis of sap signal sequence. bal gene was amplified by using the plasmid carrying bal gene, puc ::bal. thereafter, the signal peptide of sap with its own promoter was fused in front of the bal gene by soe method. the hybrid gene first cloned into puc plasmid, thereafter sub-cloned into pbr , pmk and phv shuttle vectors. the escherichia coli-bacillus plasmids carrying the hybrid gene pre(subc)-bal was transferred into bacillus subtilis npr− apr−, and bacillus licheniformis. the influence of the host bacillus species on bal production on a defined medium with glucose was investigated in bioreactor systems. for each of the recombinant (r-) bacillus species, effects of initial glucose concentration on cell growth and bal production were investigated; and, physiological differences and similarities between the wild-type and r-bacillus species are discussed. thereafter, the benzaldehyde lyase production capacities of recombinant e. coli and b. subtilis are compared in terms of cell concentration and bal volumetric and specific activities. for the comparison bal gene was cloned into prseta vector which is under the control of strong t promoter and expressed in e. coli bl (de ) plyss strain. the variations in by-product distributions with each recombinant organism and yields are also discussed. phosphoketolases (ec . . . ) are thiamine diphosphate (thdp)-dependent enzymes, that play a crucial role in the pentose phosphate pathway (ppp) of heterofermentative and facultative homofermentative lactic acid bacteria, and of the d-fructose phosphate shunt of bifidobacteria. reports affirm that cellulomonas flavigena can use the ppp when is cultured under anaerobic conditions. a genomic library of c. flavigena constructed in -zap express vector was screened. four positive clones were isolated, in vivo excised and the resulting pbk-cmv phagemids, each containing a . kb insert, were characterized by restriction analysis and dna sequencing. the open reading frame (orf) of the dxylulose -phosphate/d-fructose -phosphate phosphoketolase gene, xpkl, was located from nucleotide to . the xpkl orf encoded a amino acids-residue polypeptide (xpkl) with a calculated molecular mass of , da, a value coincident with that estimated by comparative sds-page (about , da). a putative ribosome binding site (gggagc) is present - nucleotide upstream of the translational start of the xpkl polypeptide. the c. flavigena xpkl polypeptide sequence was % identical to dxylulose -phosphate/d-fructose -phosphate phosphoketolase from bifidobacterium sp., bifidobacterium gallinarum, gloeobacter violaceus and bifidobacterium adolescentis. this analysis also revealed highly conserved regions. lactococcus lactis is a main diacetyl-producing bacteria by citrate metabolism in dairy products. the transport of citrate in these bacteria is dependent on citrate permease that is encoded by citp gene. previous studies of the citqrp operon in escherichia coli mutants showed that citp message is considerably stabilized in rnase iii mutant. so, in the context of the citrate metabolism research, the characterization of the lactococcal rnase iii enzyme is very important for the dairy industry. rnase iii is an endoribonuclease which has an important role in rrna processing and control of gene expression. with the aim of studying lactococcal rnase iii we have cloned the rnc gene from l. lactis ssp lactis il in the broad host range pls rgfp vector. this plasmid includes the gfp gene, encoding the green fluorescent protein (gfp), cloned under the control of the pm promoter, that is inducible by maltose. maltose induction of the lactococcal rnc expression showed a -fold increase of rnc transcription from this plasmid. activity assays for lactococcal rnase iii were standardized using crude extracts and a substrate specific for b. subtillis rnase iii. the results showed that this substrate was specifically cleaved by lactococcal rnase iii and its activity induced by maltose. lac-rnc was cloned in pet vector and the corresponding six-histidine-tagged rnase iii protein was overproduced in e. coli bl (de ) strain by iptg induction. the protein was purified by affinity chromatography using hplc system and was shown to be active by in vitro activity assays using the lac-rnase iii specific substrate mentioned above. we have also cloned lactococcal rnc gene and studied its expression in an e. coli rnc deletion mutant ( rnc). complementation assays performed in e. coli demonstrate that the lactococcal rnase iii (lac-rnase iii) is able to process rrnas and to regulate the levels of polynucleotide phosphorylase (pnpase). these results demonstrate that the lactococcal enzyme is able to substitute the ec-rnase iii not only in the rrna processing, but also in the processing of mrnas. the amount of lactococcal rnc transcript in an e. coli rnc strain was . -fold higher than in the wild type strain, suggesting that the e. coli rnase iii triggers the degradation of the heterologous rnc mrnas. the results obtained have shown that lac-rnase iii is an interesting enzyme for biotechnological purposes. objectives: the pharmaceutical and food industry has an increasing demand for selectively glycolized active agents. in our application isomaltose can be synthesized by immobilized dextransucrase, which transfers a glycosyl residue from sucrose (substrate) to glucose (acceptor). as the reaction proceeds, isomaltose can act as an acceptor and is converted into undesired follow-up products called isomalto-oligosaccharides, imos. we investigate on two approaches to avoid imo formation, selective adsorption of isomaltose (ergezinger et al., ) and the co-entrapment of dextranase adsorbate, which breaks imos down to isomaltose. results and conclusions: the first part of our research concerns the adsorption of dextranase on bentonite, which complies with langmuir model. at complete saturation ( . g g − ) our immobilisate exhibits an activity of , u g − . a kinetic analysis does not reveal significant differences between the adsorbed and free form of enzyme (k m,bentonit : . ± . m versus k m,free : . ± . m). thus, bentonite displays a high binding capacity paired with favorable kinetic properties. beyond that we investigate the activity of dextransucrase in co-immobilisates, which is reduced during coimmobilization due to interactions with the adsorbate. among various co-immobilisates, the one containing dextranase bound to preblotted bentonite imparts the highest activity ( % as compared to control: immob. dextransucrase). the molar yield coefficient of coimmobilisates y isomaltose/sucrose surpasses coefficient of control by %. further on we will characterize mass transfer of dextranase substrate into alginate matrix as well as bentonite-dextransucrase interactions. and kefir. a second approach is to use yeast as a production organism to produce natural folates for fortification. here we investigate and discuss the folate content in skq n, a diploid strain of saccharomyces cerevisiae, when cultured in different media and at different stages of growth. the aim is to gain a basal knowledge of the folate production profile, forms of folate produced and degree of leakage to the surrounding medium, in relation to the culturing medium and physiological state of the cells. danisco innovation, danisco a/s, langebrogade , po box , dk copenhagen k, denmark we at danisco a/s (copenhagen, denmark) have revealed a new starch degrading pathway by the discovering several new enzymes and metabolites in fungi and algae. these new enzymes include glucan lyases, dehydratases and tautomerases, which proved to be useful in biocatalysis. these new metabolites proved to be useful as both antioxidants and antimicrobials for food and non-food applications. this pathway is named as anhydrofructose pathway of starch and glycogen degradation. this technology is referred to as the anhydrofructose technology. diet is evolving from nourishing populations via providing essential nutrients to improving health of individuals through nutrition. modern nutritional research focuses on health promotion and disease prevention, on protection against toxicity and stress, and on performance improvement. as a consequence of these ambitious objectives, the disciplines "nutrigenetics" and "nutrigenomics" have evolved. nutrigenetics asks the question how individual genetic disposition, manifesting as single-nucleotide polymorphisms (snps), copy-number polymorphisms (cnps) and epigenetic phenomena, affects susceptibility to diet. nutrigenomics addresses the inverse relationship, i.e. how diet influences gene transcription, protein expression and metabolism. the mid-term objective of nutrigenomics is integrating genomics (gene analysis), transcriptomics (gene expression analysis), proteomics (global protein analysis) and metabolomics (metabolite profiling) to define a "healthy" phenotype. the long-term deliverable of nutrigenomics is personalised nutrition for maintenance of individual health and prevention of disease. the major challenges for -omics in nutrition and health still lie ahead of us, some of which apply to -omic disciplines in general while others are specific for -omic discovery in the food context: (i) the integration of gene-and protein expression profiles with metabolic fingerprints is still in its infancy as we need to understand how to (a) select relevant sub-sets of information to be merged, and (b) resolve the issue of the different time-scales, at which transcripts, proteins and metabolites appear and act; (ii) the definition of health and comfort is less of a clear-cut case than the one of disease; (iii) -omics in nutrition must be particularly sensitive: it has to reveal rather many subtle than a few abundant signals to detect early deviations from normality; (iv) in the food context, health cannot be uncoupled from pleasure, that is, food preference and nutritional status are interconnected. transcriptomics serves to put proteomic and metabolomic markers into a larger biological perspective and is suitable for a first "round of discovery" in regulatory networks. metabolomics, the comprehensive analysis of metabolites, is an excellent diagnostic tool for consumer classification. the great asset of this platform is the quantitative, non-invasive analysis of easily accessible human body fluids like urine, blood and saliva. this feature also holds true to some extent for proteomics, with the constraint that proteomics is more complex in terms of absolute number, chemical properties and dynamic range of compounds present. proteomics in the context of nutrition and health has the potential to (a) deliver biomarkers for health and comfort, (b) reveal early indicators for disease disposition, (c) assist in differentiating dietary responders from non-responders, and, last but not least, (d) discover bioactive, beneficial food components. independent of the context of application, proteomics represents the only platform that delivers not only markers for disposition or condition but also targets of intervention: the only way to intervene in a biological condition and to modulate its outcome is interfering with the proteins involved. it is evident that not only comprehensive analyses with one discovery platform (lateral integration of information) are required but also vertical integration between different -omic levels are indispensable for a deeper understanding of disposition, health, environment and diet (desiere, ) . a major "vertical integration issue", to date unresolved, is given by different timescales of transcript production, protein expression and metabolite generation (nicholson et al., ) . the transcript machinery usually responds fast to an external stimulus (seconds to minutes), the proteins may be expressed within minutes to hours (and have a halflife from minutes to even months) and metabolites vary significantly during the day and depend on latest dietary input. this means that data, which seem to correlate qualitatively (e.g. reflecting the same pathway), may not necessarily be related time-wise. rather, they may represent different responses at different time points and, possibly, to different stimuli. comprehensive -omic analyses is an essential building block of "systems biology", which can be defined as follows (clish et al., ) : systems biology is the comprehensive analysis of the dynamic functioning of a biological system (cell, organ, organism or even ecosystem) at gene, protein and metabolite (or higher organizational) level, achieved by comparison of two defined biological states of this system, typically before and after perturbation. while a comprehensive list of components (genes, proteins, metabolites) of a given biological system is a pre-requisite for this kind of research, the main reasoning for the "system view" is that only information on the interactions between the components gives clues to function of the entire network. a systems biology approach has recently demonstrated the power of proteomics to dissect immunity and inflammation. toll-like receptor recognition and signalling was elucidated and showed, how bacterial "barcodes" are read and interpreted in order to trigger an adapted immune response (aderem and smith, ) . in order to address some of the challenging objectives of -omics-driven nutritional research, we have addressed (a) the effect of early antibiotic administration on the maturation of intestinal tissues, (b) protein discovery in human milk, (c) the effects of polyunsaturated fatty acids on gene expression and lipid profile in the liver, and (d) biomarkers for intestinal stress. (a) antibiotics and gut maturation: the effects of early administration of antibiotics on intestinal maturation were assessed at the gene expression level in a rat model. (b) human milk: rapid enrichment and iterative, consolidated identification of immunologically relevant milk proteins was achieved through the employment of restricted-access media and a tailored proteomic strategy (labéta et al., ; lebouder et al., ; panchaud et al., ) . (c) fatty acids and liver transcriptome/lipidome: epidemiological studies have correlated higher intakes of poly-unsaturated fatty acids (pufas) with lower incidence of chronic metabolic disease. the molecular mechanisms regulated by pufa consumption were examined assaying the liver transcriptome and lipid metabolome of mice fed a control and a pufa-enriched diet (mutch et al., ) . (d) gut stress markers: as a first step, we catalogued protein expression along the jejunum, ileum and colon of the rat intestine and found gut segment-specific proteins (marvin-guy et al., ) . the innovative combination of a neonatal separation model with proteomic analysis allowed us to study, whether early life psychological stress may impact the adult gut neuromuscular protein expression and the approach revealed specific protein biomarkers. omics for engineering lactic acid bacteria willem m. de vos wageningen center for food sciences, wageningen university, the netherlands. e-mail: willem.devos@wur.nl. url: http://www.wcfs.nl/ lactic acid bacteria (lab) are high at-rich gram-positive bacteria that have a well-established record in industrial food fermentations where they contribute to conservation, flavour and texture. in addition, several lab are used as food-grade hosts for the production of enzymes, peptides or metabolites. finally, lab are exploited in functional foods that contribute to the health and well-being of the consumer. a variety of metabolic engineering approaches have allowed for the improvement of many attributes of lab. these approaches have been facilitated by the possibility of uncoupling of growth and metabolite production in lab, the wealth of genetic tools that allow modulation of gene expression in a dynamic range, and the determination of several complete lab genomes (de vos et al., ) . we have developed lactobacillus plantarum as a paradigm for lab engineering by experimental and modelling approaches, the application of functional and comparative genomics, and the implementation of other post-genomics avenues (kleerebezem et al., ; smid et al., ; de vos et al., ) . examples of optimizing the production of vitamins and other cofactors, the impact of these engineering approaches on the global transcription and metabolite profiles, and determining the l. plantarum activity in the human host will be discussed. solanum tuberosum (potato) is the fourth major crop worldwide and used for food, feed and biotechnological applications. to fully realize the biosynthetic potential for production of starch, protein and metabolites, we conducted an extensive quantitative profiling of the expressed genes of mature potato tuber. a total of , sage (serial analysis of gene expression) tags of nt representing , different tags were analyzed. the tags seen or more times were assigned a tentative function by comparison to homologous genes. contrary to the transcript profile of rice seedlings (gibbings et al., ) the storage organ of potato is not dominated by transcripts encoding storage proteins. transcripts for four types of protease inhibitors, a metallothionein and a lipoxygenase were more prominent than patatin isoforms. the lactic acid bacterium lactococcus lactis is used extensively in the production of fermented milk products. during cheese production the bacterium experiences many changes in its immediate environment, as a result of its own reactions. the most severe change is the accumulation of lactic acid, which changes the ph of the medium until growth is totally inhibited. we have focused upon a survey of these dairy related stress responses, as a means of constructing more robust strains. when l. lactis starter cultures are produced in rich media, they will experience an initial period with purine limitation after being added to the milk substrate, a stress condition that in several studies have been found to induce cross resistance towards a number of other stresses. we have analyzed both general purine nucleotide (atp and gtp) and specific gtp limitation in chemi-cally defined medium, using both proteomics and transcriptomics. the differential expression analyses were performed with a custom designed dna microarray of pcr amplified probes. the two stress conditions resulted in very different stress responses, both at the transcriptomic and proteomics level. from a new study on the temporal expression pattern of l. lactis during growth in milk, we present preliminary data showing differential expression of genes and proteins of the purine stress stimulon as well as other stress stimulons. cell physiology of the yeast saccharomyces cerevisiae glucose repression mutants ∆snf , ∆snf and ∆snf ∆snf was studied in batch and glucose limited chemostat cultivations. detailed physiological studies were performed on cells grown in batch using glucose, galactose, or glucose-galactose mixture as a carbon source. during growth on glucose-galactose mixtures it was shown that after glucose was consumed, galactose consumption remained repressed for about h in ∆snf or ∆snf mutants, and for more then h in ∆snf ∆snf mutant, whereas it only lasted h in wild-type cells. the global transcriptional response in the glucose repression mutants was studied using chemostat cultures. s. cerevisiae wild type and the mutants were grown in glucose limited aerobic chemostats at a dilution rate of . h − . biological triplicates were performed for each strain. to identify transcriptional responses of the glucose repression mutants, statistical tests, clustering method and a model-driven analysis method were used. the global transcription data analysis experiments showed that genes involved in hexose transport, carbohydrates metabolism, respiration, and signal transduction were differently expressed in ∆snf and ∆snf mutants comparing to wild type cells. combination of gene expression data and gene-scale metabolic model indicated changes in the metabolic sub-networks among studied glucose repression mutants. genomics technologies have recently been introduced into food and nutrition science for identifying targets of molecular actions of nutrients as well as non-nutrient components of foods. changes in the transcriptome, proteome and metabolome have been determined for assessing the molecular actions of zinc as an essential micronutrient and of flavonoids in processes such as colon carcinogenesis and atherosclerosis. zinc is essential for the structural and functional integrity of cells and plays a pivotal role in the control of gene expression. zinc deficiency effects in human cells and an animal model (rats) were analyzed by microarrays and showed that a low intracellular zinc concentration caused major alterations in the steady-state mrna levels of several hundred target genes-dependent on the tissues studied including liver, brain, muscle, intestine and kidney. proteome analysis from the same samples by d-page followed by peptide mass fingerprinting via maldi-tof-ms identified similarly a large set of proteins with altered expression level but allowed a common theme of action to be identified. although pleiotropic in first view, the obtained pattern of zinc-affected genes/proteins may represent a reference for defining the zinc regulon in mammalian cells. flavonoids occurring in large number in plant species are considered protective agents in a variety of processes including inflammation and cancer development. we have studied the effects of around selected flavonoids in a screening program and identified for compounds such as flavon, genistein or quercetin by genomics technologies their putative mode of action in colon cancer models and endothelial cells. as part of a collaborative effort employing human endothelial cells and blood mononuclear cells from a human intervention trial with soy isoflavones (genistein/daidzein) the effects of the flavonoids on the stress-response to oxidized ldl and homocystein was analyzed. a set of markers of anti-inflammatory and anti-apoptotic activity was identified for genistein and daidzein and cell biological studies confirmed that both compounds prevented programmed cells death in stressed endothelial cells. in the food processing industry, unwanted presence of extremely heat-resistant bacterial endospores creates major problems due to their capability to survive classical thermal treatments and their ability to subsequently germinate and form actively growing vegetative cells. screening of spoilage isolates using genomic typing techniques to visualise putative genome-based biomarkers allowed us to classify strains according to the degree of thermal resistance of their spores. in addition, we showed that sporulation in the presence of ingredients rich in calcium ions promotes thermal resistance of developing spores and correlates with the expression of specific (marker) genes (see oomes and brul, ) . finally, the molecular program that forms the basis of spore germination has been assessed using genome-wide expression analysis. noticeably genes involved in dna-repair were transiently expressed in germinating wild-type spores. also, surprisingly, it was found that spores contain significant levels of ribosomal and messenger rnas. degradation of these rna molecules upon spore thermal injury was found to be characteristic for their thermal resistance and predictive for their subsequent outgrowth behaviour. this finding is currently being patented. the information on spore presence, predictions of their thermal resistance and process survival chances, is used to structure a process management system to facilitate optimal food quality assurance and allow for real time analysis in case of the need for quality control. the information on spore presence, predictions of their thermal resistance and process survival chances is now being integrated. this is used to formulate the conditions for a process management system with state of the art food production quality assurance, which allows for real time analysis in case of the need for quality control. the interplay of the pectinase spectrum of aspergillus niger as revealed by dna microarray studies elena martens, jac benen, johan van den berg, peter schaap fungal genomics group, laboratory of microbiology, wageningen university, dreijenlaan , ha wageningen, the netherlands. e-mail: elena.martens@wur.nl (e. martens) the saprobic fungus aspergillus niger is an efficient producer of extracellular enzymes many of which show carbohydrate modifying activities. these enzymes have gras status and therefore are widely used in the food and feed industry. after determination of the genomic sequence of a. niger by dsm, it was estimated that only a fraction of the potential of secreted enzymes is currently characterised. database mining using the proprietary genome sequence has resulted in the identification of more then genes encoding enzymes involved in the depolymerisation of the back bone and the site chains of the complex polysaccharide pectin. additional enzymatic activities required to remove methyl and acetyl esters, present in pectin were also observed. by using dna microarrays we have sought to gain insight into the complex regulation of all the genes involved in pectin degradation. a. niger was cultivated on sugar beet pectin and on the monomeric constituents of pectin, viz. galacturonic acid, rhamnose and xylose. subsequently the corresponding transcriptomes were analysed. we will report on our findings concerning the regulation of the expression of the genes involved in the degradation of pectin and its main constituent-galacturonic acid and the consequences for the interplay of the encoded (novel) enzymes. since reactive oxygen species (ros) are formed in all living organisms a wide range of antioxidative enzyme systems are present to keep the system in balance. when an animal is slaughtered the cellular anti-oxidative capability is reduced, resulting in an accumulation of ros followed by an increased oxidation of dna, lipids and proteins. generally lipid oxidation is a well-known problem, causing increased rancidity during prolonged storage, of especially fatty fish. the implications of protein oxidation are, however, more unclear also in respect to quality decay of fish. protein oxidation causes a wide variety of amino acids modifications, where of the most studied is carbonylation of proline, argenine, lysine or threonine. these carbonyl groups can be labelled with , -dinitro-phenylhydrazine. combining two-dimensional gel electrophoresis with immunoblotting enables the detection of carbonyl groups for each single protein. the results presented here, reveal that both during frozen storage and tainting of rainbow trout protein oxidation/carbonylation increases, furthermore there is an increase in oxidation/carbonylation for distinct proteins. anne-marie neeteson european forum of farm animal breeders, benedendorpsweg , wl oosterbeek, the netherlands society is concerned about food, animal welfare, food safety, new technologies, scientists and industry. these elements are all present in genomics for farm animals. therefore, it is important to build awareness in scientists and industry, start a dialogue with stakeholders and society, and to be transparent in a pro-active way. this paper will address the issues at stake for scientists and industry, when it comes to genomics and animal health. it will combine the results of imperical, ethical and sociological efforts in three eu funded projects. (c) the proper use of genomics in relation to infectious diseases in production animals, and the role of the scientist in the development in new technologies in this field, are being addressed in european animal disease genomics network of excellence (ead-gene, http://www.eadgene.org/). some observations are that: ( ) genomics does not concern changing the gene. however, acceptability of any discovery dealing with living beings and edible products, is not obvious just like that! animals have a symbolic and emotional load. ( ) genes are still related to eugenics, in the mind of people. genes as such cause reluctance, but it is seen as positive if the use of medication can be reduced, and if animals will be better resistant to disease. ( ) consumers are in favour of consumer education, compulsory labelling and the imposition of minimum standards. the inclination to pay more for foods produced according to desired standards relates closely to income level. ( ) animal welfare is the major issue citizens mention as a concern. the focus of breeding organisations on productivity should be counterbalanced by serious attention to the animal's needs in order to avoid unnecessary negative impact on the welfare of the animals. ( ) when technical specialists and lay people communicate, they tend to use different languages: they use the same words, but with rather different interpretations. so transparency of breeding practices and clear definitions of terminology will be essential for effective communication among all stakeholders. ( ) food safety and human health are the major concern for most people, when it comes to making a choice. during the latest decades research within the field of animal genomics has in general been following the same strategies as those used within the field of human genomics, although with much less resources. the porcine genome has been characterized intensively through the development of linkage maps, comparative maps and physical maps. until a few years ago it had not been anticipated that it would be possible to embark on whole genome sequencing of animals genomes. however, because of technological developments and much lower costs for sequencing, several animal genomes have now been assembled/are on the way to being assembled. the initial step towards sequencing the porcine genome was taken by the sino-danish pig genome project. the efforts within this project have now generated approximately . million genomic shotgun sequences and . expressed sequence tags (ests). the shotgun sequences have been included in a three-species alignment to make an initial evolutionary analysis. the results show that pig is much closer to human than mouse is. the ests represent -end sequences from a total of non-normalized cdna libraries. based on assembly and annotation of the ests the structure of the porcine transcriptome has been analysed. the relevance of assembling the porcine genomic sequence is justified both from the perspective of sustainable animal breeding and from the fact that the porcine model is an important research platform because of the anatomical, physiological, biochemical and metabolically similarities with man. examples of functional genomic studies both aimed at sustainable animal breeding and aimed at exploiting the pig as a model for medical studies will be discussed. genomics refers to global, systematic and high throughput approaches that allow collecting large amount of data and thus offer new possibilities for analysis and understanding biological processes. we will present some new knowledge related to reproduction in farm animals resulting from three different strategies. ( ) functional analysis of gene and protein expression: the transcriptome and the proteome analysis allowed to identify new genes and proteins whose expression is associated with processes of ovarian follicular growth and atresia as well as oocyte maturation in bovine and porcine species, maturation of spermatozoa in the different compartments of epididymis. farm animals produce food as cost effectively as possible, however this may have negative side effects for their health and welfare. trade off processes between production on one hand and reproduction and health on the other hand play a crucial role. the principles of selective breeding for the best of naturally occurring variation has proven to be able to balance an increased level of production and quality of life for the animal. every year, the economic value of the genetic gain achieved by the breeders and carried over to the producers is . % of the economic value of eu farm animal production. consequently, a conservative estimate of the gain from animal breeding is, every year d . billion in europe. recent developments, such as the sequencing of the genomes of the human, chicken and cow, together with high throughput laboratory techniques, means that there are new opportunities to enhance quality of life. the goal of this paper is to give an overview of the options offered by genomics for enhanced quality of life with focus on identifying relevant gene variants and technologies for large scale tracking and tracing. selective breeding for the best of naturally occurring variation remains the same as in traditional systems, but by pinpointing the relevant gene variants along genomics it is possible to identify directly the animals best selected for high production without comprising health and welfare. the combination of full genome sequences, software tools, study of functional physiological processes cost-effective high-throughput snp genotyping and comparative mapping have the (proven) potential to identify relevant gene variants, e.g. pork color, boar taint, general disease resistance. functional mutations have direct option of application in breeding programs. unfortunately this is not the case for genetic markers due to cost of genotyping and inconsistent phenotypic effects. new technologies for snp genotyping are cost effective and enable large scale genotyping ( . of animals/day). a selection of the best technology and strategic use of these opportunities enable tracking and tracing. the application of this technology offers new opportunities for quality of life, both for animal and humans. background: studies have shown that prebiotic and probiotic consumption alters the gastrointestinal flora, modulates the immune system, inhibits genotoxicity and has a protective effect on colon carcinogenesis. however, the effect of synbiotic consumption on these parameters in subjects at risk of colon cancer has not until now been investigated. aim: to determine if a synbiotic (prebiotic and probiotics together) modulates cancer risk biomarkers in human subjects at risk of colon cancer. methods: a -week randomised, double blind, placebo controlled, ethically approved trial of a food supplement containing lactobacillus gg, bifidobacterium bb- and raftilose synergy (prebiotic) was performed in colon cancer subjects who had undergone 'curative resection'. faecal and blood samples were obtained before (t , week ) midway through (t , weeks) and following intervention (t , weeks). rectal biopsies were obtained at t and t . the effect of synbiotic consumption on the faecal flora was assessed using standard plate count techniques. genotoxic damage was measured in single cells derived from biopsies using the comet assay. fw was prepared by diluting faeces : in dmem, ultracentrifugation and sterile filtration. the genotoxic (comet assay) and cytotoxic potential (almar blue assay) of fw was determined. peripheral blood mononuclear cells were isolated from blood and cytokine production in vitro assayed by elisa. natural killer cell cytotoxic activity and the phagocytic and respiratory burst activity of monocytes and granulocytes in whole blood were determined by flow cytometry. results: in the synbiotic group faecal numbers of bifidobacteria significantly increased (p < . ) and lactobacilli increased although not significantly (p = . ) while coliforms decreased (p < . ). enterococci, clostridium perfringens and bacteroides were unaffected. in the placebo group bifidobacteria decreased (p < . ), the other bacterial groups were unaffected. in biopsies genotoxic damage was increased in the placebo group at t versus t (p = . ) but was unchanged in the synbiotic group. the genotoxic and cytotoxic potential of fw was unaltered. synbiotic consumption significantly increased (p < . ), ifn-␥ production by pbmcs but il- , il- , il- , and tnf-␣ production was unaffected. natural killer cell, phagocytic and respiratory burst activities were unaltered. conclusion: synbiotic consumption did not have a strong immunomodulatory effect on the systemic immune system in this study, nor did it influence the genotoxic and cytotoxic potential of fw. however, synbiotic consumption altered the composition of the gut flora to a more beneficial composition as well as protecting against genotoxic damage in vivo, suggesting a protective effect of synbiotics against colon carcinogenesis. emmaÅrsköld, malin svensson, halfdan grage, peter rådström, ed w.j. van niel applied microbiology, lund institute of technology, lund university, p.o. box , sweden lactobacillus reuteri is used today in a variety of dairy products as a probiotic bacterium. several lactic acid bacteria have the ability to produce different kinds of exopolysaccharides (eps), which have the potential to be used as an alternative biothickener. however, the yield of eps is too low to be profitable in the food industry. to optimise the environmental conditions for eps formation a l. reuteri strain was chosen for a factorial design study. the factors used in this experiment were temperature ( - • c), ph ( . - . ) and sucrose concentration ( - g/l); for each factor three different values were chosen. the strain was grown in batch mode using a semi-defined medium at constant ph and sucrose as the carbon source. the results obtained with fermentations revealed that the highest eps formation was found at • c, ph . and g/l of sucrose. sucrose did not further affect the eps formation above a concentration of g/l. temperature and ph were significant for the eps formation, but only temperature was significant for growth. a central composite design study was chosen for further optimization of the ph and sucrose concentration for maximum eps formation. also the gene expression of the sucrase enzyme responsible for eps formation was investigated using qrt-pcr. the data were used to develop a model for growth and eps formation. dendritic cells (dc) play a pivotal immune regulatory role in the th , th and treg cell balance. dc are present in the gut mucosa and may thus be target for modulation by gut microbes. here, we screened a large panel of human gut-derived lactobacillus and bifidobacterium spp. for dc polarizing capacity: bone marrow-derived murine dc were exposed to lethally irradiated bacteria and cytokine and dc surface markers were analyzed. substantial differences were found among strains in their capacity to induce proinflammatory cytokines, while the differences for anti-inflammatory cytokines were less pronounced. bifidobacteria were weak il- , il- and tnf-␣inducers, while both strong and weak cytokine-inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- , il- and tnf-␣ production induced by otherwise strong cytokine-inducing strains, while il- production remained unaffected. those lactobacilli with greatest capacity to induce il- were also most effective in up-regulating surface markers. surface marker up-regulation was however reduced in the presence of weak il- -inducing strains. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. cell surface-associated glycolytic enzymes from lactobacillus plantarum v mediate adhesion to human epithelial cells and extracellular matrix proteins s.m. madsen, j. glenting, a. vrang, p. ravn, h.k. riemann, h. israelsen, m.r. nørrelykke, a.m. hansen, m. antonsson, s. ahrné, h.c. beck bioneer a/s, hørsholm dk- , denmark among the main selection criteria of lactic acid bacteria for probiotic use, the ability to adhere to intestinal epithelial cells, mucus, or extracellular matrix proteins is considered important. using a proteom based approach we identified a group of novel surface proteins that are non-covalently bound to the cell wall of the probi-otic bacterium lactobacillus plantarum v. surface proteins were extracted and analysed by gel electrophoreses followed by mass spectrometry analysis. the surface proteins included glycolytic enzymes like, e.g glyceraldehyde -phosphate dehydrogenase, which usually is a typical intracellular enzyme. a collection of lactobacillus species was screened and the phenomenon of surface-associated glycolytic enzymes was found in many of the analyzed species. this is to our knowledge the first example of surface-associated glycolytic enzymes in probiotic bacteria. however, in pathogenic bacteria these enzymes are well known and their surface localization is involved in adhesion to human epithelial cells and invasion. we suspect that the presence of these enzymes on the surface of probiotic bacteria could prevent adhesion of pathogenic bacteria and possibly also involve other probiotic activities such as immune modulation. binding studies showed that the surface-associated glycolytic enzymes of lb. plantarum v were able to bind to caco- intestinal cells and extracellular matrix proteins like fibronectin. scientific and industrial interest on exopolysaccharides (eps) synthesized by microorganisms and on their chemico-physical properties has been quickly growing in the last years. many strains of lactobacilli produce eps allowing them to adhere to human mucosae and therefore to have probiotic effects such as the stimulation of immune response and even antitumoral activity of these molecules has been claimed. futhermore prebiotic actions of eps beneficially affect the human host health improving the properties of indigenous microflora. in this research we have studied two novel interesting lactobacilli strains: lactobacillus plantarum dsmz and a particular human isolated strain of lactobacillus crispatus that have probiotic potentialities and are good eps and l(+)-lactic acid producers. the aim of this work has been to characterize these strains and their metabolites (eps, organic acid and bacteriocins), to state them as probiotics and to study their adhering ability on human cells. we have studied the physiology of l. plantarum and l. crispatus in shaking flasks as well as their optimal fermentation conditions to obtain high cell density cultures suitable for use as starters in the food industry and eventually for probiotic preparations. fermentation experiments have been performed in a bioreactor equipped with microfiltration (mf) modules using a semidefined medium and various carbohydrates in different culture conditions (aeration, temperature). the kinetic of eps production has been followed and according to results both strains have shown a growth-related production ranging from to mg/l. in vitro studies concerning the ability of these strains to adhere to human mucosae are in progress as well as the structural characterization of the exopolisaccharides. previous studies have shown that compression coating improves the storage stability of freeze-dried lactobacillus acidophilus, although this stability is related to the degree of cell injury, which in turn is related to the compression pressure used. compression coating has also been found to improve the survival of freeze dried l. acidophilus during exposure to simulated gastric fluid (sgf). the aim of the present work is to create a compression coated l. acidophilus formulation, with targeted release at the terminal ileum and beginning of the colon in the human gastrointestinal tract. dissolution studies were performed using a phosphate buffer with a ph of and . , to simulate gastric fluid and intestinal fluid (sif), respectively. cell viability was monitored using multi-parameter flow cytometry (mpfc), together with traditional cfu/ml counts. mpfc was used to identify live, dead and stressed cell populations, using the fluorescent stains propidium iodide (pi), , -dihexylocarbocyanine iodide (dioc ( )) and to-pro- . results show that an enteric coating material, eudragit l - , is both suitable for compression coating, and enhancing the survival of cells when exposed to sif. pectin usp has also been shown to promote targeted release of the cells. the opium poppy papaver somniferum contains more than tetrahydrobenzylisoquinoline-derived alkaloids. it is the source of the narcotic analgesics codeine and morphine, which accumulate in specialized internal secretory cells called laticifers. in the aerial parts of the plant, the laticifer cells are anastomosed, forming an articulated network. laticifers are found associated with the vascular bundle in all plant parts. the morphinan alkaloids morphine, codeine and thebaine are found both in roots and in aerial plant parts and specifically accumulate in vesicles within laticifers. the benzo[c]phenanthridine alkaloids sanguinarine and -hydroxysanguinarine are found in root tissue. the syntheses of sanguinarine and of the tetrahydrobenzylisoquinoline latex alkaloid laudanine are completely understood at the enzyme level. nearly all enzymes of morphine biosynthesis have also been described. in more recent years, cdnas encoding enzymes of alkaloid biosynthesis in p. somniferum have been isolated and characterized. the cell-specific localization of several of the enzymes of morphine, sanguinarine and laudanine biosynthesis has also been described. with knowledge of many of the gene of alkaloid formation and their sites of expression, the metabolic engineering of p. somniferum for tailored alkaloid profiles is now being undertaken. an agrobacterium tumefaciens-based transformation and a regeneration protocol have recently been developed specifically for narcotic tasmanian cultivars. the various cdnas encoding genes of alkaloid biosynthesis in p. somniferum are being systematically reintroduced into the plant to achieve engineered plants with altered alkaloid profiles. the first results have now been obtained with sense and antisense genes stably expressed in a regenerated tasmanian cultivar. the ultimate goal of exploiting the genes of alkaloid biosynthesis is to produce transgenic medicinal plants of specific alkaloid content that would facilitate commercial production and improve our understanding of the factors that regulate biosynthesis as well as provide experimental systems with which to investigate the ecological role of alkaloids in planta. integrated transcript and metabolite profiling for gene discovery in plant natural product pathways richard a. dixon, lahoucine achnine, bettina deavours, mohammed farag, marina naoumkina, lloyd w. sumner plant biology division, samuel roberts noble foundation, ardmore, ok , usa the rich diversity of chemical structures found in the plant kingdom arises in large part from a limited number of basic chemical scaffolds (e.g. terpene, polyketide) that are modified by a limited number of chemical substitution types (hydroxylation, glycosylation, acylation, prenylation, o-methylation, etc.) . much of the diversity is brought about by the substrate-and/or regio-specificities of the substitution enzymes. in contrast to the large collections of gene sequence and transcript level data available on-line, little detailed information exists on the plant (secondary) metabolome. promiscuity of substrate specificity in vitro may complicate attempts to assign functions to genes of secondary metabolism accessible to researchers through various cdna library collections. using the isoflavonoid and triterpene pathways in medicago species as examples, we describe how integrated metabolite and transcript profiling approaches can aid functional genomics, help explain metabolic regulation, and provide tools for assessing the impacts of genetic modifications in plant secondary metabolism. focused and non-targeted approaches were used to assess the impact associated with introduction of new high flux pathways in arabidopsis thaliana by genetic engineering. transgenic a. thaliana plants expressing the entire biosynthetic pathway for the tyrosine derived cyanogenic glucoside dhurrin as accomplished by insertion of cyp a , cyp e , and ugt b from sorghum bicolor accumulated % dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome and metabolome. in a similar manner, plants expressing only cyp a accumulated % dry-weight of the novel tyrosine derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleitropic effects. in contrast, insertion of cyp a plus cyp e resulted in stunted plants, transcriptome alterations, accumulation of numerous new glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae specific uv protec-tants sinapoyl glucose and sinapoyl malate as well as kaempferol glucosides. the accumulation of new glucosides in the plants expressing cyp a and cyp e , was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify novel xenobiotics. the pleiotrophic effects observed in plants expressing sorghum cyp a and cyp e were complemented by retransformation with s. bicolor ugt b . accordingly, insertion of high flux pathways directing synthesis and intracellular storage of high amounts of natural products is achievable in transgenic plants with marginal inadvertent effects. arabidopsis thaliana-distinct function in gene transcription dynamics? jeppe madura larsen, brian stougaard vad, søren mølgaard, kell andersen, mads n. davidsen, klaus d. grasser department of life sciences, aalborg university, aalborg, denmark in recent years it has been shown that introns to some extent can regulate expression in the eukaryotic cell. insertion of introns in the utr in arabidopsis genes has shown to increase gene expression at both rna and protein level. in order to investigate if utr introns have distinct characteristics, we analyse these in the well annotated arabidopsis thaliana genome published by the arabidopsis genome initiative. , loci annotated with full-length cdnas were analysed and loci ( . %) containing utr introns were isolated. we studied if the genes containing these introns showed different patterns in alternative splicing and gene function (gene ontology classification) compared to the remaining genes not containing this intron type. of the isolated loci ( . %) contained only one utr intron and these were used for further analysis, where it was investigated if the utr introns had a characteristic size distribution. genes containing transcripts with utr introns were more subjected to alternative splicing ( . % versus . %) and had a tendency to be more involved in cell regulatory functions compared to genes without this intron type. it was also found, that utr introns was characteristically size-distributed. we identified thee predominant sizes of approximate , and bp compared to only one for orf introns. this suggests widespread multiple splicing events in utr introns. the results presented here suggest that utr introns have distinct characteristics and function in gene transcription dynamics. cytochrome p monooxygenases appear to be involved in the biosynthetic pathways of a large variety of primary and secondary metabolites in microbial, animal and plant cells. in particular, cytochrome p scc catalyzes the conversion of cholesterol into pregnenolone-the precursor of all steroid hormones in mammalian steroidogenic tissues. cytochromes p are also involved in the biosynthesis of different plant steroid derivates that play important role in regulation of plant growth and development. therein, investigation of possible influence of cytochrome p scc expression on plant regulatory system is of a great interest. this report devoted to the investigation of transgenic tobacco plants, which have been generated by the transformation with recombinant plasmid pgbp f constitutively expressing cyp a cdna of the bovine cytochrome p scc . the transgenic state of the plants was confirmed by southern blot analysis. transgenic plants are phenotypically different from the control ones. in particular, they obtained a substantially higher growth rate and are larger than wild type plants. we have demonstrated that incubation of fragments of the transgenic plants leaves in [ c]-labeled cholesterol containing medium results in formation of the radioactively labeled product with chromatographic mobility corresponding to pregnenolone. the presence of this metabolite in the steroid fraction of lipid extracts obtained from the transgenic plants leaves was confirmed by gas chromatography mass spectrometry (gc-ms) method. the data obtained indicate that cytochrome p scc synthesized in transgenic plants displays its specific catalytic activity. biotechnological production of glucose isomerase enzyme with streptomyces olivochromogenes for production of fructose syrup from hydrol m. hashemiravan, a. sadat barikani department of food science and technology, azad university (pishva, varamin unit), institute of food science and agriculture, tehran, iran the use of glucose isomerase for isomerization and production of fructose syrup was performed by selected industrial strain of streptomyces olivochromogenes ptcc . growth of microorganism and production of enzyme in different culture media was studied, and effects of different parameters such as phosphate and aeration was evaluated. the growth of microorganism at • c, caused a production of high amount of enzyme. the production of enzyme was considered in two culture media (a and b). medium (a) was selected for the higher production amount of enzyme. the highest amount of enzyme production was seen in medium a, which was . giu/ml, after h. the use of baffles in culture flasks, increased the amount of enzyme production, four times more. the production of enzyme was increased, . times more, in phosphate deficient medium. the cells containing enzyme (intra cellular glucose isomerase) was separated by centrifuge, and extraction and release of enzyme was performed by ultra sonication, that is a physical-mechanical method. this method released about . % of total intracellular enzyme. the best length of time for sonication was found to be min. experiments showed that optimum ph and temperature of the enzyme were . - and • c, respectively. the highest activity of the enzyme was observed at ph . for up min. at the time of isomerization reaction the existence of magnesium ions showed to be necessary and omission of this ion cause a decrease of enzyme activity in isomerization process, but this effect was not necessary for the enzyme activity results showed that treatment of glucose syrup at temperatures of , and • c, by the enzyme, caused %, % and % of isomerization, respectively. efficiency increase of high acetic acid production with the use of acetobactereace iranian native strains mutation m. hashemiravan , a. alirezasadat barikani : department of food science and technology, azad university (pishva, varamin unit) institute of food science and agriculture, iran; young iranian researchrer's club, tehran, iran the first step in the present research is the isolation of acetobactereace native strains from fruits (such as grape or apple) and fresh vinegar. this separation has been done with the use of effective isolation methods and optimized mediums. ten strains were isolated effectively, the bio chemicals test were performed, each of them were detected, classified and nominated with a special code. two methods have been used for mutation: (a) mutation with the ultraviolet radiation; (b) mutation with nitrous acid. in the first method the microbial cells were treated with us radiation for different periods of , , and s, and the effect of the uv mutation was assessed. as a result, the period of s was determined as the optimum mutation periods. in the second method, the microbial cells were first washed with the acetate buffer . m with the ph of . , then ml nitrous acid . m was added and was mixed for - min. finally the sampling was done in the periods of , , , and min and was transferred to a plate containing the medium of ethanol-phenol red-agar. the two methods have been compared with each other. each method has its own advantages and disadvantages. the mutation pathway in method (b) is more stable and conducted, while mutation with the uv radiation method, change the position of thiamine-cytosine bases absolutely randomly. finally, the best mutant site was inoculumed with the medium containing alcohol %, acetozyme gz . g/l, acetozyme d g/l, acetic acid . % and l double distilled water. the acetic acid was produced in the rate of g/ ml. also the mutant strains were detected with scanning electron microscope (sem) and interesting photographs were taken from mutant cell. the performed experiments were planned with the use of taguchi statistical method (qualitek ). this research has been performed in the irost as the thesis of ph.d. in food biotechnology industry. isotachophoresis has almost exclusively been applied for contracting and stacking samples ions before zone electrophoretic separation of proteins. this study attempts to apply microfluidic isotachophoresis (itp) as a high resolution analytical method for proteins. beta-lactoglobulin and other milk proteins with slightly different pi were labelled with fluorescent red and analysed by the micralyne tk system using microfluidic glass chips, either with simple cross (sc) or double cross (tt) injection or designed d-itp-cze chips with double tt injection and sc for transfer to the second dimension cze channel, efficiently non-covalently coated with . % (w/v) epoxy-polydimethylacrylamide to lower electroendoosmosis. capillary zone electrophoresis (cze) in borate or phosphate buffer was reproducibly perform for more than consecutive runs using upchurch tm reservoirs glued to the wells to enable larger buffer volumes and greater run-to-run stability. finally, isotachophoretic anionic separation of the proteins were done using phosphate (ph . ) or chloride (ph . ) as leading ion and -amino-caproic acid (ph . ) as terminating ion. the effect of narrow cut ampholytes as spacers needs further investigations. the perspective aim is to combine the migrating itp separated zones with second dimension capillary zone electrophoresis as a new microfluidic proteomic danalysis. genotypical differences affecting the response of pisum sativum to differing boron/iron applications e.e. hakki, u. zeynep, m. hamurcu, a. tamkoc, m.b. babaoglu, s. gezgin department of field crops, faculty of agriculture, selcuk university, kampus, konya , turkey. e-mail: eehakki@selcuk.edu.tr (e.e. hakki) boron and iron are among the microelements required for the proper development of the vegetative and generative tissues of plants. though iron is present in high amounts in almost all soil types, its bioavailability to crops is extremely reduced, hence most of the plants face an iron defficiency problem and while on one side crop productions effected, on the other hand the nutrition problems come up to human through contagious nutritional chains. boron is also among the most problematic micronutrients of the major crop plantation areas of turkey. both defficiency and toxicity problems exist in a total of about % of the central anatolian soil where pea is among the legumes cultivated. application of varying levels of boron and iron combinations in greenhouse and the analysis of plant aquisition via icp-aes as well as determination of the effects of the element combinations both in morphological and molecular levels are the aim of our studies. the genetic bases of the response differences of plant genotypes to b and/or fe, were investigated through the applications of molecular marker techniques. considerable growth rate and stem size differences were detected within the parents (wild-type versus cultivar) and the f plants. the presence of efficient genotypes to high micronutrient levels are expected to help us increase the cultivation of the crop in problematic areas as well as in exploring the molecular bases of the microelement uptake mechanisms. increase in sulfite production by accelerating sulfate uptake in brewing yeast t. fujimura, y. kodama, y. nakao, n. nakamura, w. miki institute for advanced technology, suntory ltd., mishima-gun, osaka - , japan sulfite plays a role as an antioxidant, which stabilizes beer flavor. therefore, it is important to control the sulfite concentration during fermentation. sulfite is produced as an intermediate in the sulfate assimilation pathway in yeast. we have already reported that over-expression of a lager yeast-specific ssu gene, encoding a sulfite efflux pump, leads to increase of sulfite production (fujimura et al., ) . in the present work, we have clarified that there are two types of sul genes (scsul and non-scsul ) each encoding a high affinity sulfate permease in lager brewing yeast. eighty percent and % identity are found by comparing the dna sequences and the deduced amino acid sequences, respectively. a comparative functional analysis of the two genes has been performed aimed at achieving further increases in sulfite production by accelerating sulfate uptake. over-expression of scsul and non-scsul has been achieved by transformation of lager brewing yeast, saccharomyces pastorianus. experiments have been done with and without expression of non-scssu . the resultant transformants have been evaluated by fermentation tests in wort. over-expression of either scsul or non-scsul failed to show significant effect on sulfite formation. a combination of over-expression of non-scsul and non-scssu resulted in two-fold higher sulfite production compared with overexpression of only non-scssu and four-fold higher compared with the parental strain. these results suggest that the non-sc-gene types significantly contribute to sulfite production in lager brewing yeast. , t., et al., . functional phytases (myo-inositol hexakisphosphate phosphohydrolase) catalyse the release of phosphate from phytate (myo-inositol hexakisphosphate), the predominant form of phosphorus in cereal grains, oilseeds and legumes. possible applications of phytases have been suggested in animal nutrition to increase mineral bioavaliability and to decrease phosphate pollution in area of intensive life stock management and in human health. zea mays is one of the cereals that contain high amount of phytate as the major phosphate storage compound. over bacteria were isolated and screened for phytases from the halosphere, rhizosphere and endophyte of malaysian maize plantation. the phytase activity of the isolates was screened by a modification of the ammonium molybdate method. the highest extracellular phytase activity was detected from bacteria that isolated from the endophyte of the maize root. in this paper, results for isolates chosen for media, temperature and ph optimization will be presented. production of plant proteinase from jack fruit seeds (artocarpus integrifolis) and its influence on rheological and sensory characteristics of low fat yogurt el-sayed el-tanboly dairy sciences department, national research centre, dokki, cairo, egypt adding a proteolytic enzyme extraction from jack fruit (artocarpus integrifolis) in combination of fermentation process in low fat yogurts manufacture was tried to improve yogurt flavour and rheological properties. experimental yogurts milk contained control, . (t ), . (t ) and . (t ) units/ml milk from crude extracts of plant proteinase. the ph of the product treated with crude proteinase was lower than the control. however, the rate of acidity development during storage slightly increased with increasing the addition of crude proteinase level and progress of storage period of yogurt. the proteolytic activity of all yogurts gradually increased until the end of storage period ( days). yogurts made from milk treated with crude proteinase preparations were less firm compared with control at all storage periods, where t showed more less firm after days of storage being . g/ g. generally, increasing units of plant proteinase preparations decreased the firmness. on the other hand, yogurt made from milk pretreated with plant proteinase had higher syneresis, and apparent viscosity than the untreated product. the greatest viscosity was found in t and t of and mpa s, respectively, compared with control of mpa s at days storage. the results indicated that there is an inverse relationship between the amount of units of crude proteinase preparations and susceptibility of yogurt to syneresis. the t gained the highest scores ( points) followed by the control ( . points) after days of storage, while yogurt of t showed a low scoring being . from the foregoing results, it is recommend to use jack fruit (artocarpus integrifolis) as a source of plant proteinases and utilize it to develop a high quality yogurt at a level of . units of plant proteinases/ml milk. an effective process for the chemical-biotechnological utilization of distilled white lees was studied. a first treatment with hydrochloric acid allowed the solubilisation of tartaric acid. the influence of temperature, amount of hcl and reaction time were considered through an experimental design. under the optima conditions g/l from white distilled lees and . g/l from red distilled lees were recovered. the tartaric acid was precipitated as calcium tartrate so that it can be isolated from the rest of the raw material compounds. the solid residue was used as an economic nutrient for lactic acid production by lactobacillus pentosus using trimming wastes as substrate. the lactic acid concentrations and volumetric productivities achieved were similar to those obtained using distilled lees without tartaric acid recovery as nutrient. toasted wine was traditionally produced in galicia, northwest of spain. nowadays this technique is being recovered. grapes after harvesting are air dried in order to concentrate sugars, acids and flavor compounds. raisings are pressed to obtain a must with high sugars concentrations. two different grape wines were prepared concentrating the sugars up to and brix, respectively. in order to get a better knowledge of the problems involved, synthetic media simulating the grape musts were prepared. theses musts were used to optimize the initial sugar concentration, the amount of nutrients required, the optimum temperature to carry out the fermentation and the influence of the type and amount of yeast. under the best conditions some fermentations with grape must were carried out to produce wines with intense aroma and flavor notes and high residual sugar concentrations. in this studies, bacillus sp. e strain was isolated from koreanstyle fermented soybean paste and it was producing the biological response modifier (brm). the brm activated the b cell selectively. it was identified the bacillus licheniformis e . the brm was purified by ion-exchange chromatography and gel filtration. chemical properties of brm: molecular weight of brm was estimated to be about , , da. sugar content of brm was . % (w/w) and glucosamine ( . mol%) was the high level. protein content of brm was . % (w/w) and serine ( . mol%) was the high level. infra-red absorption spectrum was showed the characterization of glycoprotein. biological properties of brm: the brm which isolated from fermented soybean paste was similar to that of bacillus licheniformis e by immuno-fluorescence assay. we confirmed that the brm was capsular substance of b. licheniformis e . potato nitrogen concentrate (pnc) is a highly viscous liquid with high complex nitrogen content produced from the protein-fraction in potato starch extraction. the concentrated extract is rich in minerals and ␣-amino nitrogen. although ␣-amylase nowadays is mainly produced exploiting bacillus production systems there is still considerable demand for fungal ␣-amylase from aspergillus oryzae origin. the aim of the experiments to be reported here was to investigate, if pnc can replace commonly used complex nitrogen sources in the production of fungal ␣-amylase. the following data have been measured in pnc pretreated by diluting to / and clarifying by centrifugation. total-n: . g n/l; ␣-amino-n: . % (w/v) (as glycine); soluble protein (bradford): . mg/l (as bsa); total carbohydrates: . g/l; reducing sugars: . g/l; dry weight: . % (w/w). in the following experiments nitrogen sources were replaced on the basis of their ␣-amino nitrogen content. the carbon source for all experiments was maize starch. the formation of ␣-amylase by a. oryzae atcc in shake flasks -using pnc (centrifuged or not), yeast extract, malt extract, casein hydrolysate or meat extract -was compared to "standard" cultivation with corn steep liquor. the experiments showed only small differences in ␣-amylase titers using complex nitrogen sources. no remarkable differences were observed in the resulting biomass. in general no differences in enzyme productivity and biomass formation could be seen after h of incubation. especially the bench top bioreactor experiments indicated an optimal fermentation time of about h. cultivations of a. oryzae atcc were carried out in bench top bioreactors. comparing cultivations in a medium with pnc as the sole complex nitrogen source to one containing csl as such no significant differences both in the formation and amount of ␣-amylase and the fungal growth were observed. thus pnc might be able to replace complex nitrogen sources such as csl or even the more expensive yeast extract and casein hydrolysate in fungal amylase production systems. . ) is involved in the metabolism of inositol and catalyzes the conversion of d-glucuronic acid to l-gulonic acid with nadph as a cosubstrate posterior to the oxidation of inositol to glucuronic acid by the enzyme inositol oxygenase. although the yeast sporobolomyces oryzicola (nakase and suzuki, ) is not able to grow on inositol as the sole carbon source, intracellular glucuronate reductase can be found in cells grown in a medium containing d-glucuronic acid. the enzyme could be a useful tool in the design of a specific quantitative assay for glucuronic acid, e.g. in so called energy drinks. the organism was grown in media containing either glucose and glucuronic acid or only glucuronic acid and difco yeast nitrogen base. whereas growth on both media was similar in shake flask culture, hardly any growth in either medium was observed in bench top bioreactors. the influence of dissolved oxygen tension was investigated and the relevant data will be shown. the formation of intracellular glucuronate reductase activity by sp. oryzicola is inducible by media containing glucuronic acid. no activity is found in cells grown in a medium containing only glucose as the carbon source. besides the activity against d-glucuronic acid, activities against -ketogluconate and -at very low levelsagainst galacturonic acid and the lactone of glucuronic acid were detected. the enzyme activity is stable up to • c. the ph has relatively low influence on the activity against glucuronate, whereas the reduction rate of -ketogluconic acid is optimal at ph . - . with significantly lower values at ph . and . , respectively. data on the kinetics of the conversion of both glucuronate and -ketogluconate will be shown. nakase, t., suzuki, m., . j. gen. appl. microbiol. , - . the multiple nutritional and functional impacts of food fermentation on human health have been widely accepted (reddy and pierson, ; hugenholtz et al., ) . however, the related role of the involved microorganisms to the nutritional effect from the fermented food is still not well defined and the mechanisms involved are still largely unknown. the present study was to investigate iron bioavailability in carrot juice fermented by two selected lab strains, l. pentosus fsc and ln. mesenteroides fsc . after digestion by gi enzymes, the juice was supplied to fully differentiated caco- cells to study iron uptake and transepithelial transport by caco- cells from the digested juice. our data revealed strain specified changes in iron bioavailability in carrot juice fermented by these two strains. after in vitro digestion with pepsin and pancreatic-bile enzymes, the best yield of soluble iron was from ln. mesenteroides fsc fermented juice. surprisingly, the l. pentosus fsc fermented juice yielded about five times higher uptake iron as compared to fresh juice, while ln. mesenteroides fsc fermented juice was not significantly different from the fresh juice. interestingly, the transepithelial transferred iron across the cell line was however better from ln. mesenteroides fsc fermented juice than from l. pentosus fsc fermented juice. to summarise, our study showed that level of soluble iron after in vitro digestion does not necessary indicate iron absorption, especially in the case of lab fermented food. data on improved iron uptake from l. pentosus fsc fermented juice indicated exiting of promoter(s) for iron absorption in such juice that is not related to the production of organic acids and lowering ph effect. peng zhang, herve vanderschuren, martin stupak, wilhelm gruissem institute of plant sciences, zurich, the tropical root crop cassava (manihot esculenta crantz) is a major source of food for approximately billion people worldwide. in sub-saharan africa, more than million people rely on cassava as their major source of dietary energy. in many parts of africa and latin america, cassava leaves are a vegetable source for daily uptake. cassava is grown mostly by poor farmers under marginal environmental conditions and in areas where few other crops can sustain competitive yields. the crop is therefore fundamental for subsistence farming and food security, but it is also very susceptible to stresses common in the areas and conditions where it grows. in many parts of africa, reliable cassava production is strongly impacted by infections with the african cassava mosaic geminiviruses (cmgs), a rapidly spreading disease that causes large yield losses. in the coastal areas of east africa, cassava production now is threatened by another devastating disease, cassava brown streak disease (cbsd). cassava plants are also frequently attacked by many pests, such as cassava hornworm and stemborers. several reports also indicate that greater leaf longevity, especially under drought conditions, could be important for increasing yields and/or the stability of production in cassava, as well as improve the access to an important nutrient source. conventional breeding efforts have attempted to address the constraint to cassava production, but with limited success. the new tools of biotechnology can change this situation by offering new approaches to the challenges of cassava. these new technologies have the potential to make cassava much more productive, a better source of nutrients, and profitable to grow, hence, greatly contributing on the sustainable development of tropical agriculture. recently we have developed biotech cassava with value-add traits, including resistance to cassava mosaic virus, prolonged leaf life and insect resistance. new strategies are also explored to increase protein content of cassava storage roots. we are currently undertaking pilot studies with two teams of leading scientists and experts for projects to test acmv-resistant transgenic cassava lines in africa and lines with extended leaf retention at ciat, colombia under field conditions. this development of substantially equivalent improved transgenic cassava lines is part of a larger study to analyze the need, effectiveness and biosafety of biotech cassava for agricultural production. the goal of the pilot studies will be the development and coordination of a broader project that produces important and novel scientific results, valuable information on the need and impact of biotechnology at the subsistence farming level, and a sound scientific basis for the development of guidelines for biosafety assessments and release of transgenic organisms into the environment and agricultural production in africa and latin american countries. this study was conducted to reveal the effects of different pretreatments on obtaining haploid plants by using the anther culture in pepper capsicum annum l. cultivars demre sivrisi and sirena. buds were collected at uninucleate microspore stage. anthers collected from buds were cultured in ms medium containing different hormones and hormone concentrations. experiment results revealed that when sirena anthers were pre-treated cold at + • c for h and kept in darkness at • c for a period of week gave good results. in the case of demre sivrisi anthers were pre-treated cold at + • c for h and kept in darkness at • c for a period of week gave good results. on the other hand no cold pretreatment to anthers resulted with low embryo formation. similar results were also observed on the anthers kept at • c for week as callus was produced in some petri dishes but no regeneration was observed. as a conclusion, since no cold pretreatment to anthers resulted with low embryo formation it is possible to say that cold pretreatment should be applied to anthers in pepper another culture studies. the yeast d. hansenii ufv- was tested in this work in batch experiments in synthetic media at constant initial substrate concentration ( g l − ) under variable oxygenation conditions. to get additional information on its fermentative metabolism, a stoichiometric network was proposed on the basis of the general knowledge available in the literature on xylose metabolism in pentose-fermenting yeasts and the specificities of xr and xdh activities in d. hansenii and checked through a bioenergetic study performed using the experimental data of product and substrate concentrations. it can be stressed that under strongly oxygen-limited conditions xylitol production was negligible, whereas under semi-aerobic conditions maximum xylitol production (p max = . g l − ) and yield (y p/s = . g g − ) were obtained. a progressive decrease in these parameters was observed under fully aerobic conditions, suggesting that xylitol-producing yeasts require limited oxygen conditions, which is species-dependent. the proposed model, which utilizes the experimental specific rates of substrate consumption and product formations, allows estimating the main bioenergetic parameters. besides, it proved to be an effective tool to investigate different metabolic situations and showed how they can influence the flux distribution of the carbon source and the bioenergetics of this biosystem. the effect of disinfectants on fungi anne svendsen, pernille skouboe bioneer a/s, hørsholm dk- , denmark prevention of mould spoilage of foods can only be carried out successfully, if the species, which are actually spoiling the food product, are known. a very limited number of fungal species has been associated with the spoilage of each food category. proper disinfection of production facilities is very important to avoid mould spoilage. resistance of moulds to disinfectant treatments are known and different species have shown different response to the same disinfectant. to obtain proper disinfection it is important to know the resistance of the spoilage fungi against different disinfectants. in this study the effect of disinfectants on the spoilage fungi of cheese, rye bread, liver paté and fruit juice was investigated. in collaboration with five food companies the dominating species responsible for spoilage of each food product were isolated and used for testing. commercial disinfectants and disinfectants "under development" were tested. tests were performed in suspension and on surfaces, the methods used were modified after en and en . considerable variability in fungicidal effect among the species was observed. some disinfectants were ineffective at low temperature. some disinfectants showed different effect in suspension and on surfaces, resulting in an effective kill in suspension and almost no effect on surface. the identification of effective disinfectants in the food industry includes: ( ) testing against the specific spoiling species of the food product, ( ) testing on surface, not only in suspension, ( ) test parameters adapted to the food manufacturing plant. intensified research efforts in recent years confirm the major importance of the microbial flora in the gastro-intestinal tract for human health. ingestion of prebiotic oligosaccharides increases the number of the desirable bacteria like bifidobacteria and lactobacilli in the colon. we are looking at beta-galactosidases from lacto-bacillus spp. for the production of galacto-oligosaccharides (gos) because we speculate that the enzymes of probiotics will form gos with high prebiotic potential. in this present study, purified betagalactosidases of selected lactobacillus strains were used for the production of gos from lactose. different enzyme reactor set-ups, both discontinuous and continuous, were tested and compared. temperatures up to • c and ph values between and . were required for satisfactory enzyme stability during the process. enzyme source, substrate concentration and the level of substrate conversion were found to be critical process parameters for gos yields and composition. yields of up to % (w/w) of total sugars were achieved when the initial lactose concentration was g/l. capillary electrophoresis (ce) and hplc with pulsed amperometric detection were the analytical tools for investigating the influence of reactor type, enzyme source and conversion level on gos composition. the prebiotics market is increasing rapidly and is expected to more than double until to about million d world-wide. therefore, the development of enzymatic processes on an industrial scale is a high priority goal of our research. starter addition does not always succeed in improving standardisation and quality of the complex sensory properties of traditional fermented foods. in many cases the added strains do not grow as well as the environmental strains present in the production plant. here, a method of geometric simplification (by dichotomy) of a complex ecosystem found on a raw milk livarot ( strains) was tested on cheese curd. by a limited number of cultures, successively, out of , out of and out of strains were selected on the basis of two criteria (i) respect of the taxonomic proportion, (ii) generation by the daughter ecosystems of an odour close to the one of the mother ecosystem. finally a sub-ecosystem of strains gave an odour similar to the one of the more complex mixture. the use of molecular methods (pcr-sscp) permitted to follow the main species growing. mother and daughter ecosystems were characterized by sensory analysis and gc-ms. probably because of an important redundancy of the strain functions, the method was very efficient. this method may permit to improve a lot the set up of mixture of strains and species used in fermented food industry. effect of the dilution rate on the exopolysaccharide production by bifidobacterium longum atcc c. shene, m. rubilar, s. bravo universidad de la frontera, chemical engineering, av. francisco salazar , casilla -d temuco, chile exopolysaccharides (eps) producing lactic acid bacteria are used in dairy industry (cheese and yogurt) due to the rheological properties that these compounds confer to the products. preliminary results also suggest the use of eps as health-promoting (anti-tumor and immunostimulatory actions) ingredients. bifidobacteria are grampositive bacteria natural inhabitants of the gut of warm-blooded animals and man. a number of investigations have shown that bifidobacteria promote host health mainly because of the reduction proliferation of some pathogenic bacteria through acid synthesis. in this work results obtained in the experiments carried out to test the capability of b. longum atcc to synthesize eps are presented. continuous culture fermentations were carried out at dilution rates between . and . h − . composition of the culture media was that of the mrs broth. biomass concentration presents higher values ( . - . g l − ) at dilution rates between . and . h − . biomass growing at these rates is difficult to pellet and adheres to the fermentor walls behavior that was not observed at other growth conditions. eps from cultures grown at these rates were preparated and fractionated. authors wish to thank the chilean conicyt for the economical assistance given through the project fondecyt . high pressure-low temperature (hplt) inactivation processes were performed on bacillus subtilis vegetative cells at various conditions. at atmospheric pressure, lowering the temperature to as low as − • c was found to have minor anti-microbial effects. upon application of high pressure various phase transitions occurred in the microbial suspensions under study. after pressure treatment at - mpa, cells were plated under optimal conditions to assess cell viability. treatments at - mpa and − • c were the most effective in inactivation. in these cases, ice i-iii solid-solid phase transition was observed. in addition, we hypothesised that intracellular thawing (solid-liquid phase transition) had already occurred while the extracellular surrounding was undergoing solid-solid phase transition. this double effect is suggested to be key in mediating the observed large drop in viability. we speculate that more cells survived after treatment at − • c compared to the same treatment at − • c because both the extra-and intracellular surrounding remained fully frozen. at − • c a solid-solid phase transition was observed when pressure was higher than mpa. a metastable state of ice i was observed at mpa treatment. results from the current study will be presented (see also shen et al., ifset in press) . the data call for a mechanistic evaluation of the effects of hplt as an anti-microbial treatment. such data are currently being gathered and will be used in defining optimal hplt process conditions for the food industry. the influence of saccharomyces cerevisiae, kloeckera apiculata and candida pulcherrima mixed cultures on the selected alcohols formation during model fermentation pawel satora, tadeusz tuszynski department of fermentation technology and technical microbiology, food technology faculty, agricultural university, cracow, poland. e-mail: psatora@ar.krakow.pl (p. satora) for the study five yeast species were chosen, isolated from successive stages of plum fruits spontaneous fermentation: from the beginning (candida pulcherrima, kloeckera apiculata, saccharomyces cerevisiae w ), middle (s. cerevisiae w ) and final fermentation (s. cerevisiae k ). to characterize the potential influence of yeast mixed cultures on the selected alcohols formation, wick-erham synthetic medium ( % glucose) was fermented by mixed cultures of two and three yeast species. after distillation, ethanol, propanol, isobutanol, isoamyl alcohols, hexanol and -phenylethanol were determined using gas chromatography. findings were compared with the results obtained after monoculture fermentations. the use of mixed cultures resulted in increasing of glucose utilization rate, ethanol and fusel alcohols formation (except propanol) and decreasing of methanol synthesis. the samples fermented using two yeast species characterized higher (about %) amount of volatile compounds in relation to monocultures. it takes note of especially high level of ethanol (av. . g/dm ), methanol ( . mg/dm ) and isoamyl alcohols ( . mg/dm ). the positive feature of triple cultures using was limitation of methanol and fusel alcohols synthesis that was accompanied by relatively high ethyl alcohol production (av. . g/dm ). the consumption of sugar syrup becomes increasingly significant in industrial processes due to economic advantages and the easy of use. the production of sucrose syrup using enzymatic hydrolysis represents the safest alternative, once the reaction does not produce any toxic or undesirable substance. this work consists on the production of sugar syrup by immobilized inulinase from kluyveromyces marxianus, with two alternatives process: (a) syrup enriched with fructooligosaccharides or solely with glucose and fructose. the process is comprised by the following stages: production and purification of the enzyme in optimized conditions, immobilization of the enzyme in solid support and the conversion of sucrose in a fixed bed bioreactor with the immobilized enzyme. the final composition of the product can be a mixture of glucose, fructose, sucrose and fructooligosaccharides or a mixture of fructose and glucose, according to the operational conditions. the bioreactor can be operated continually for approximately months with the same biocatalyst. the product from this process is ideal for applications in the food products such as sweet, candies, chocolates, yogurts, etc. besides, the prebiotics properties of the fructooligosaccharides, is a beneficial stimulant of the intestinal flora, which gives to the product a functional property. studies on plant microbial interactions using azotobacter sp. as bio-inoculants towards soil fertility baljeet singh saharan faculty of biotechnology, jcdm college of engineering, sirsa , india. e-mail: baljeet.saharan@gmx.de, baljeet br@yahoo.co.uk (b.s. saharan) high nitrogen fixing, phytohormone producing isolates of azotobacter, azospirillum, acetobacter and pseudomonas were used as inoculants on wheat and cotton with varying doses of nitrogen under field conditions. bio-inoculants were selected on the basis of yield, dry weight and survival rate of bacteria under field conditions. seeds of wheat variety wh were treated with different biofertilizers using nitrogen level of , and kg ha − and one level of p, i.e. kg ha − in field along with control. under field conditions, maximum yield was obtained with azotobacter chroococcum e at ( ± . kg ha − ) as well as kg ha − ( ± . kg ha − ) followed by a. chroococcum ht ( ± . kg ha − ) and avk ( ± . kg ha − ). whereas, with kg ha − highest yield was observed with mac ( ± . kg ha − ) followed by e ( ± . kg ha − ) and avk ( ± . kg ha − ). maximum height at kg ha − was observed with mac inoculation ( . ± . cm) followed by avk ( . ± . cm) and ht ( . ± . cm). various chosen strains were tested with desi (hd ) and american cotton (h ) under similar pot and field conditions as for wheat in the following season. plant height and yield were determined at the time of harvesting whereas survival rate was monitored at various intervals of time. survival rate of inoculated bacteria was determined after , , and days. highest survival rate was observed in mac (( . ± . ) × ), which decreased after and days, respectively. ( . ± . ) × , ( . ± . ) × with mac and ( . ± . ) × and ( . ± . ) × with ht , respectively. maximum boll weight was with avk ( . ± . g boll wt. plant − ) followed by pseudomonas ( . ± . g), ac ( . ± . g) and ala ( . ± . g) boll no. plant − was maximum with ala and avk ( ± . plant − ) followed by pseudomonas ( ± . plant − ). maximum height and dry matter was obtained with pseudomonas ( . ± . cm) and avk ( . ± . cm) with variety hd under field conditions. net saving of % nitrogen was observed using a. chroococcum (e and avk ) bioinoculants for wheat and cotton, respectively. to characterize the antioxidative properties of tempeh-fermented food prepared from vicia faba (l. kontu) with the use of rhizopus oligosporus, the sulfhydryl groups content and surface aromatic hydrophobicity of albumins were investigated. the results obtained for tempeh albumins were compared with raw vicia faba and bovine serum albumin (bsa). these results indicate that tempeh fermentation increased antioxidative activity of albumins. the measurements of antioxidative activity were carried out with the use of , -diphenyl- -picrylhydrazyl (dpph) and , -azinobis-( ethylbenzothiazoline- -sulfonic acid (abts). the albumins of faba bean-tempeh have possessed much higher activity for scavenging free radicals as measured with the dpph and abts ( . % and . %) than raw seeds ( . % and . %) and bsa ( . % and . %), respectively. it has been also found that tempeh fermentation process increased . times sulfhydryl groups content ( . m/mg of albumins) as compared to raw seeds ( . m/mg of albumins). the tempeh albumins have possessed lower surface aromatic hydrophobicity than raw seeds ( . fi and . fi, respectively). orange peel characterization and generation of fermentable sugars solutions for the biotechnological production of food additives b. rivas , j.m. domínguez , p. torre , j.c. parajó : department of chemical engineering, vigo university (campus of ourense), polytechnic building, as lagoas, ourense, spain; department of chemical and process engineering, genoa university, via opera pia , genoa, italy. e-mail: brivas@uvigo.es (b. rivas) the citrus processing industry generates in mediterranean area around millions tonnes of orange peel as byproduct from the extraction of citrus juices in industrial plants. in order to avoid ecological problems and provide an extra profit, this residue was studied in order to generate a suitable substrate for the fermentation process oriented to the production of food additives. orange peels were characterized and the data collected allowed quantifying a % of this waste. soluble sugars ( . %), cellulose ( . %) and pectin ( . %) were identified as more important fractions. this material was submitted to two hydrolysis techniques, prehydrolysis (with diluted sulfuric acid) and autohydrolysis (with water) under different experimental conditions. autohydrolysis was selected as the most appropriate technique for the production of suitable fermentation media. finally, the liquors obtained at • c and liquid:solid ratio of g/g, containing . g/l of sugars, without additional nutrients, were employed to citric acid production by aspergilus niger cect (atcc , nrrl ) . the influence of the addition of calcium carbonate and methanol were studied. under the best conditions an effective conversion of sugars into citric acid was attained, showing the viability of the production of fermentable solutions from this industrial waste. today there is an increasing interest in using high gravity fermentation in brewing. high-gravity fermentation involves production of beer wort of up to • p or even higher and results in beer that has more consistent product quality. the main aim of this study is an increased understanding of how brewer's yeast respond to the various stress factors imposed during high gravity beer fermentation and the consequences these stress factors have on the gene regulation and its consequences on the metabolite levels (both intra-and extracellular). higher attenuation of the wort will be achieved by two different techniques: by the addition of highly fermentable adjuncts such as sucrose or glucose syrups and by mashing with addition of microbial enzymes such as pullulanases and glucoamylases. in the first part of the study model fermentation conditions are established, where the sugar uptake and product formation can be studied in details. characterization of the carbohydrate profile is analyzed by hplc. as flavour changes may occur at higher gravities, it is important to study changes in formation of secondary metabolites, especially esters. transcriptome and metabolome analysis will be used to establish how the stressful conditions prevailing under high gravity fermentations may influence the secondary metabolism in saccharomyces cerevisiae. furthermore, analytical aroma characterization of final beer will be studied by spme and gc-ms. detailed analysis of the effect of different stress factors on the cellular response using dna arrays and metabolite profiling will be carried out. dna arrays will be employed to evaluate if specific metabolic pathways are up-regulated or down-regulated as a consequences of the stress factors. naringin, a bitter compound that occurs in citrus fruit juices, may be converted to a nonbitter form by enzyme hydrolysis. the enzymatic complex naringinase was produced in aspergillus niger cect cultures with naringin as inducer (pérez-mateos et al., ) . crude extracts from a. niger and purified naringinase from penicillium decumbens were immobilized into a polymeric matrix of polyvinyl alcohol (pva) hydrogel cryostructured in liquid nitrogen. the operating stability of the pva-naringinase beads was tested using synthetic citric juice (gray and olson, ) . immobilized enzymes reduced % the naringin content at • c and ph . . furthermore, immobilized preparations from aspergillus and penicillium could be re-used through six cycles ( h) remaining % and % catalytic efficiency, respectively. financial support from "ministerio de ciencia y tecnología" and feder (no. agl - /ali). gray and olson, . j agric. food chem. , - . pérez-mateos, et al., ( ), . otimizing the fermentation broth for tanase production by a new isolated strain paecilomyces variotii vania battestin, gláucia pastore, gabriela macedo department of food science, unicamp, p.o. box , campinas, cep - são paulo, brazil tannase is an inducible enzyme that catalyses the breakdown of ester linkages in hydrolysable tannins, resulting in gallic acid and glucose. the fermentation broth can use by-products as wheat bran, rice or oats, adding tannic acid. the use of by products or residues rich in carbon source for fermentation purposes an alternative to solve pollution problems that can be caused by an incorrect environmental disposal. in the present study we have optimized the production of an extracellular tannase by a new isolated paecilomyces variotii using response surface methodology. the first step was to identify the variables having a significant effect on enzyme production. the variables evaluated were temperature, residues ratio (coffe: wheat bran), concentration of tannic acid, salt solution during , and days of fermentation time. results showed that temperature, residues ratio (coffe: wheat bran) and tannic acid had significant effects on tannase production. commercial wheat bran (cwb) and coffe rusk residues (cr) were used as solid substrate. for fermentation the medium was composed by, cwb:cr were mixed with distilled water and transferred into ml capacity erlenmeyers flasks and auto-claved at • c for min. the medium was then inoculated with spores ( . × ) and the flaks were incubated at • c. tannase was assayed according to the methodology of mondal et al. ( ) . according to the statist analyses, the optimum conditions to produce tannase was the range of temperature ( - • c); tannic acid ( . - %); residues percent (coffe: wheat bran) ( : ) and days fermentation time. the enzyme production increased . times more enzyme production than that was obtained before this optimization. yeast and lactic acid bacteria are two major microbial groups of the most fermented products. a large variety of fermented foods and beverage are made by the activities of both yeast and lactic acid bacteria, simultaneously or successively. during the spontaneous mixed fermentation of lactic acid bacteria and yeast population, it is extremely difficult to control microbial species due to the complexity of the microorganism involved. therefore, we have compared the antimicrobial activity of chitosan against two lactic strains, lactobacillus plantarum and lb. brevis, and yeast strains, saccharomyces cerevisiae to investigate the possible use of non toxic biopolymer chitosan for selective control in mixed culture. the lactobacilli were more sensitive to the inhibitory activity of chitosan than s. cerevisiae. the results suggest the possible use of low molecularweight-chitosan for the control of food fermentation in which both groups of organisms frequently occur together. the effect of vegetable oils on astaxanthin production of phaffia rhodozyma and xanthophyllomyces dendrorhous csaba vágvölgyi, gyöngyi lukács, miklós takó, Árpád csernetics, tamás papp department of microbiology, faculty of sciences, university of szeged, p.o. box , astaxanthin ( , -dihydroxy-␤,␤-carotene- , -dione) is one of the most important carotenoid product. it is used primarily as food colorant and animal feed additive. their effective antioxidant properties linked to a preventive action on various types of cancer and an enhancement of the immune response could lead to expanded commercial applications. among the natural microbial source available, the closely related red pigmented yeasts phaffia rhodozyma and xanthophyllomyces dendrorhous are of great biotechnological interest. these yeasts have desirable properties as biological sources of pigment, including rapid metabolism and producing high cell densities in fermentor, but the commercial production of astaxanthin is limited by the relatively low content in wild-type strains. the purpose of this study was to determine whether the different vegetable oils had an effect on the carotenoid production in p. rhodozyma. effects of media supplemented with corn germ oil, wheat germ oil, sesame-seed oil, palm oil, pumpkin-seed oil, coconut grease, olive oil (extra virgin), olive oil (sanza), sunflower-seed oil and cottonseed oil were tested. studies were performed on both a phaffia and a xanthophyllomyces strain. yeast was grown in yeast-pepton-glucose liquid medium complemented with the appropriate vegetable oil in different concentrations ( . - , v/v, %) . after four days the total carotenoid production was determined spectrophotometrically, and it was referred to dry cell mass. palm oil increased significantly the carotenoid production of the phaffia strain, while a similar effect on the xanthophyllomyces strain could be observed with coconut grease. composition of carotenoid compounds in the strains was determined by thin layer chromatography. lutein is considered a nutraceutic compound that has developed an increasing interest since it is one of the two carotenoids that are located in the macula of the human eye. its consumption is associated with the prevention of age related macular disease (amd). industrially, lutein may be produced using a saponification step of a mixture of lutein diesters that are previously extracted with hexane from natural sources. our proposal is to improve the process by catalyzing the same reaction using microbial lipases during the extraction step with hexane. additionally, the use of supercritical fluids represents an extension of enzymology in non-conventional media with process and environmental advantages. this work was developed using extracts from marigold flower (tagetes erecta) in hexane and supercritical carbon dioxide (sc-co ) where the lutein esters were hydrolyzed by two commercial lipases: lipase b from candida antarctica (novozym ) and lipase from mucor miehei (lipozyme rm m). in particular, we focused our interest in the role of water in the system. interestingly our results show an inverse dependence of the initial reaction rate with respect to the initial water activity (awi) for both lipases, a phenomena that seems to be related to the partition of substrates and products in the solid support)and the hexane phase as a function of water. when sc-co was used as solvent an increase in the consumption rate of lutein diesters occurred, reaching conversions of % in h. for hexane, the same conversion was reached after h. this result suggests a significant effect of the media on the reaction that can be related to shifts in the partition of compounds that bring the substrates in closer contact with the enzyme. this work also demonstrates that lutein hydrolysis seems to be another potential application of commercial immobilized lipases in the food/nutraceutical market. the enzyme of interest in this work is ␤-galactosidase from lactobacillus sp. (ec . . . ). ␤-galactosidases catalyze the hydrolysis and transgalactosylation of ␤-d-galactopyranosides (such as lactose). an attractive biocatalytic application is found in the transgalactosylaction potential of these enzymes which is based on the catalytic mechanism of ␤-galactosidases. the products of transgalactosylation, galacto-oligosaccharides, are non-digestible carbohydrates which meet the criteria of 'prebiotics' and therefore have attracted increasing attention. to produce these 'prebiotic' galactooligosaccharides, an inexpensive and efficient process is desired. immobilization of the enzyme ␤-galactosidase on an insoluble support is an attractive tool to make the process of lactose conversion more economical because the enzyme can be recovered and reused during continuous operation. in this present study, we aimed at immobilizing ␤-galactosidase from lactobacillus sp. by covalent linkages on two solid supports which are commonly used for protein immobilization: chitosan and eupergit c. the protein-binding capacity, the immobilization yield, ph and temperature dependency of activity and stability, and the kinetic parameters of immobilized enzymes were studied. higher activity retention of the immobilized enzymes over a broader ph range and at higher temperatures compared to those of the free enzyme was observed. the immobilized enzymes were evaluated in terms of transgalactosylation activity and stability for a to introduce of foreign genes for the important crop plants such as rice, we need a reproducible efficient procedure for regeneration of the calli through somatic embryogenesis. for this intention, we established the best callus induction medium for tarom mahalli and deilamani cultivars and created the method that the regeneration frequency was reached to %. calli were induced from scutellar tissues of mature seeds on ms medium supplemented with three level of , -d ( , . and mg l − ) and n medium supplemented with five level of , -d ( . , , . , and . mg l − ). for deilamani cultivar the best medium was n with . mg l − , -d and for tarom mahalli the same medium with mg l − were the best. in subculture media, sucrose was used instead of maltose. for regeneration analysis of plantlets, we used two-factorial experiment in base of crd; one factor was regeneration media with six levels (ms medium supplemented with five amount of kinetin and: naa (mg l − ) [( : ), ( : ), ( : . ), ( : ), ( : ), respectively] and . mg l − , -d and mg l − bap). other factor was dehydration process with three levels (without dehydration, dehydration with two layers of filter paper for min [prior to transfer to the regeneration medium], and third factor was factor of with substitution of sucrose with maltose [after weeks; sucrose: maltose]). we conclude that maltose due to changing in osmolarity proceeding can elevate the regeneration frequency to %. therefore, type of carbon source is critical in callus induction and regeneration. márová ivana, hrdličková jana, kubešová jitka, kočí radka, vidláková tereza faculty of chemistry, brno university of technology, purkyňova , brno, carotenoids are the most widespread natural pigments with important biological activities and applications mainly in food and feed industry. at present many ways including genetic engineering are developed to reach higher production of naturally formed carotenoids using microbial producers. in this work cloning and expression of crt gene cluster from pectobacterium carotovorum in recipient bacterial strain e. coli dh ␣ as well as in yeast strain s. cerevisiae was tested. plasmid vector phsg with inserted crt genes was used for transformation of chemically competent e. coli dh ␣ cells, while in s. cerevisiae shuttle vector paur was used. transformants were selected based on resistance to antibiotics, formation of orange-coloured transformant colonies, analysis of recombinant plasmid size and lc/ms analysis of carotenoids produced by recombinant cells. the yield of individual carotenoids (lutein, beta-carotene, lycopene) obtained from various bacterial transformants was several fold higher than in natural producer (lutein: . - . g/g of d.w., beta-carotene: . - . mg/g of d.w.). the highest yield obtained in transformed strain was . g/g of lutein and . g/g of beta-carotene. the yield of biomass and carotenoids in. transgenic s. cerevisiae was comparable to some industrial red yeast strains ( . mg of total carotenoids + mg ergosterol/l; g/l of biomass). so, transgenic yeasts could be suitable for large scale production of carotenoids and/or enriched biomass, while transgenic bacterial producers are perspective above al for high production of rare carotenoids as lutein or lycopene using transformation by specific genes of crt gene cluster. this work was supported by the project msm of the czech ministry of education, youth and sports. two forms of grape seeds, whole and powdered forms, were heated at four different temperatures- , , and • c. after heating, grape seeds were extracted with % ethanol ( . g grape seed/ ml of % ethanol), and total phenol contents (tpc), radical scavenging activity (rsa) and reducing power of the extracts were determined. thermal treatment of grape seed increased the antioxidant activity of extracts. the maximum tpc and rsa of whole grape seed extract (wgse) were achieved when the seeds were heat-treated at • c for min, while that of powdered grape seed extract (pgse) were at • c for min, and were greater than that of the non-treated control. according to the gc-ms analysis, several low-molecular-weight phenolic compounds were newly formed in the wgse heated at • c for min. these results indicated that antioxidant activity of gse was affected by heating conditions (temperature and time) and physical conditions of grape seeds at the time of heat treatments. analysis of the unexpected phenotypic consequences associated with plant transformation jonathan latham, allison wilson, ricarda steinbrecher econexus, , canon frome court, ledbury hr td, uk. e-mail: jrlatham@gn.apc.org (j. latham) transgenic plants often exhibit unexpected phenotypes. such phenotypes could arise from pleiotropic effects associated with the transgene, or they could arise from other sources. a recent econexus report underlined the potential for the process of plant transformation to result in genetic damage to the transformed plant (genome scrambling-myth or reality? transformation-induced mutations in transgenic crop plants: http://www.econexus.info/). the report showed that mutations arising at the site of transgene insertion are often substantial, frequently resulting in loss or rearrangement of chromosomal dna and insertion of multiple superfluous dna fragments. unintended mutations were also documented at other locations in the plant genome. such transformation-induced mutations could provide an explanation for unexpected phenotypes in transgenic plants. we decided to survey regulatory documents and the scientific literature for instances of unexpected phenotypic consequences arising in transgenic plants. this poster documents the preliminary results of our survey. it is intended to assess the range and frequency of unexpected consequences and to examine whether there is sufficient data available to determine their origin. it is our belief that investigating the origin of these unexpected phenotypes should be a principal aim of biosafety research. biotechnological production of metabolites such as carotenoids could be of high interest because of their antioxidative and antimutagenic activities in human body. production of these metabolites by microbial cells is dependent on cultivation conditions. so, presence of exogenous stress in cultivation environment could stimulate biosynthetic pathways of desired metabolites. two non-conventional yeast strains, rhodotorula glutinis and sporidiobolus salmonicolor, were chosen for study of carotenoid production useful in feed industry. hydrogen peroxide, sodium chloride and/or their combinations were used as exogenous stimulators of carotenoid pathway. presence of exogenous stress led to important overproduction of pigments as well as of supplementary studied substances (ergosterol, glycerol). higher adaptability of yeast cells was observed not only in cultivations with one type of stress. combination of stress factors in cultivation media induced significant increase of pigment formation. moreover, under controlled conditions in laboratory fermentor s. salmonicolor produced about eight-fold amount of ␤-carotene ( g/g) in medium with % nacl and mm h o than in control sample. similar result was observed in r. glutinis cultivated in presence of % nacl in inoculation medium only ( g/g of ␤-carotene). the use of stressed biomass of red yeasts in feed industry could have positive effect not only in animal and fish feeds because of high content of physiologically active substances, but it could influence nutritional value and organoleptic properties of final products for human nutrition. this work was supported by the project msm of czech ministry of education. the culture ph significantly affects mycelial growth and morphology, exopolysaccharide (eps) formation, and their molecular properties during submerged cultures of a medicinal mushroom ganoderma lucidum. when the culture ph shifts from to , mycelial growth ( . g/l) and eps production ( . g/l) were favorable compared with other ph-control strategies. the mycelial morphology was also significantly varied upon culture ph: a feather-like pellets were found when the ph was controlled shifting from to at day , which was regarded as undesirable morphological form for eps production. compositional analyses revealed that the ratios and chemical compositions of the eps formed in bottom or top fractions of ethanolic precipitates were significantly different upon culture ph. the molecular characteristics of the eps were further investigated using a size exclusion chromatography/multi-angle laser light scattering (sec/malls) system. plant ␤-n-acetyl-hexosaminidase (hex) (ec . . . ) is reported to have diverse physiological roles like fruit ripening, degradation of reserved glycoproteins in germinating seed and chitin-elicited lignification. in this paper we report the purification and characterization of hex from korean ginseng roots. after extraction with citrate-phosphate buffer, hex was purified to homogeneity using ion exchanger chromatography, hydrophobic interaction chromatography and gel filtration. its molecular weight was determined using gel filtration and mass spectrometer. enzymatic parameters were studied with -methyl-umbelliferyl-n-acetylglucosaminide as substrate. the effect of heat stress and weak organic acids on escherichia coli and a comparison of its recovery by the plate count method and flow cytometry monica s. talsania due to the importance of microbiology for human health, methods have been developed to enumerate viable bacteria. dilution plating is seen to be the 'gold standard' for proof of a cells viability. however, the success of this method relies on post sampling growth, which is limited by our ability to grow cells in the laboratory. additionally, stressed or sub-lethally damaged cells, remain undetected. single cell measurements can provide rapid detailed physiological information, and the assessment of population heterogeneity. this work compared the recovery of stressed e. coli as measured by the number of cfu/ml and by multi-parameter flow cytometric analysis. weak organic acids and high temperature-short time processing (htst) were used to stress the cells both methods commonly used during food preservation. it was shown here that flow cytometry is a powerful tool for the enumeration and detailed analysis of any non-culturable microbial population, which is important because cytotoxic compounds and heat stresses used in food preservation often have a growth inhibiting effect but not necessarily a lethal one. paul g. kovalenko molecular biology & genetics nasu, zabolotnogo str. , kyiv, ukraine the pharmaceutically important plant species of glycyrrhiza sp. (called licorice) is an important commercial product used as a natural sweetener, anti-inflammatory, anti-cancer and anti-diabetic agents. agrobacterium rhizogenes transformation system was used for the hairy root cultures of licorice g. uralensis. after inoculation of aseptic stem segments the ability of hairy root formation was scored for a period of weeks. mean transformation frequency ranged from % (for up to % (for , ). some transformed genotypes showed significant differences in roots weight, flavonoids and glycyrrhizin (gl) production. the cotransformation rate of licorice intact explants cultivars with lba tl-dna and the s gus gene showed an average of more than %. these obtained root cultures were additionally elicited with extracts of biotic elicitor acremonium sp. (endomycorhizal fungus), and were used as an in vitro system to metabolites production. the transformed and elicited hairy roots of g. uralensis were obtained by infection of a. rhizogenes have produced gl at an yield of . % dry weight on the period of culture as a days. according to tentative analyses the hairy roots cultures of glycyrrhiza species produced flavonoids (liquiritigenin and liquiritigen). more high levels ( . g/l) of the total flavonoids production have been identificated on the strains which transformed by lba . this study involved any difference among elicitor treatments and incubation periods for the optimal meabolites production. clearly, the selection of an effective agrobacterium strain for the production of transformed root cultures is highly dependent on the plant species, and must be determined empirically. ayse gul nasircillar akdeniz university, biology, akdeniz univ. faculty of art-science, biological department, antalya, turkey mature embryos of five t. aestivum and five t. durum cultivars formed embryogenic callus on two different media. embryos were removed from surface sterilised seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of murashige skoog and mg/l , -dichlorophenoxyacetic acid ( , -d) or mg/l naphthalenacetic acid (naa). the developed calli and regenerated plants were maintained on , -d or naa free ms medium. wheat plants can be regenerated via two different systems. there were significant differences in percentage of callus induction and regeneration capacity on the different initiation medium. among the t. aestivum cultivars, yakar had the highest regeneration capacity in both induction medium. in t. durum cultivars, kiziltan gave the highest regeneration capacity in ms + , -d medium and yilmaz gave the highest regeneration capacity in ms + naa medium. a strong genotypic effect on the culture responses was found for both induction medium. the glycolytic enzyme triosephosphate isomerase (tpi), which catalyses the interconversion of the triosephosphates dihydroxyacetone phosphate (dhap) and glyceraldehyde- -phosphate (gap), was studied for its control on glycolysis and mixed acid production in lactococcus lactis il . we constructed a number of l. lactis strains in which the tpi activity was modulated from % to % of the wild-type level. the enzyme was found to be present in high excess with % tpi activity supporting % of the wildtype glycolytic flux, and with % of the wildtype tpi activity the glycolytic flux was essentially unchanged. measurements of the upstream metabolites glucose- -phosphate (g p), fructose- , bisphosphate (fbp) and dhap were essentially unchanged for tpi activities from % to %, and only in the strain with % tpi activity we observed a significant increase in the intracellular dhap concentration. homolactic product formation was preserved throughout the interval of tpi activity studied, though a small increase in the amount of acetate and formate production was observed in the strain expressing tpi at the lowest level ( % tpi activity). the finding of an increased mixed acid pattern under intracellular conditions with a high dhap concentration is in contrast to earlier data from literature, which indicated that the triosephosphates play an important role in regulation of pyruvate metabolism in l. lactis with a negative effect on the mixed acid flux. we have recently shown that alcohols induce the adhesion of l. monocytogenes at low temperatures, presumably accompanied by enhanced exopolysaccharide (eps) production. however, little is known about the mechanisms involved in the formation of biofilm and eps by l. monocytogenes. in the present project, we show that deletion of selected regulatory and up-regulated genes did not abolish attachment, though the degree of alcohol-induction in some cases was affected. we are applying bioinformatics to search for homologues in l. monocytogenes of known eps genes from various gram positive bacteria. this has revealed candidate genes involved in the synthesis of eps, such as genes encoding glycosyltransferases. moreover, we are at present performing dna microarray analysis for the egde strain grown at • c in the presence of . % isopropanol. this data should, combined with the bioinformatic results, give us a good indication of the genes involved in alcohol-induced surface attachment. repetitive-pcr (rep-pcr) was applied in research on non-starter lactic acid bacteria (nslab) in cheese. we first showed that strains previously differentiated by pulsed field gel electrophoresis (pfge) also could be differentiated by rep-pcr. this was partially due to slight changes in the pcr conditions that allowed reproducible amplification of - kb bands. more than bands were obtained for most strains. a clear differentiation was also obtained between lactobacillus paracasei, lactobacillus plantarum, lactobacillus curvatus and lactobacillus danicus (a new species found in danish and estonian cheeses and traditional starter cultures). we found that this technique is highly reproducible, e.g. identical profiles in three different pcr-machines, two different dna isolation procedures, and different trained personnel. we applied the developed rep-pcr technique to confirm that survivors after heat treatment, were the actual strains introduced and not due to post-pasteurization contamination. we also showed that when we added a cocktail combination of five strains as protecting cultures to cheese, two to three members of this cocktail was dominating the cheese nslab microflora. in control cheeses without the cocktail in most cases other strains dominated, but in a few cases we were able to show cross-contamination between cheese vats. these data indicate that the rep-pcr will be useful to follow development of adjunct cultures as well as provide a reproducible subspecies (e.g. strain) differentiation. rep-pcr is a much quicker and less labour requiring procedure than pfge, and is apparently a much more reproducible technique than what has been seen for rapd. dynamic modeling of lactococcus lactis metabolism and its dynamic behavior for lactate secretion and regulatory characteristics jinwon lee, ui sub jung, hye won lee department of chemical and biomolecular engineering, sogang university, seoul, south korea, - . e-mail: jinwonlee@sogang.ac.kr (j. lee) dynamic metabolic model for lactococcus lactis has been developed in order to analyze a time-dependent behavior of lactate secretion mechanism and probe its regulatory roles. the model was used to compare and analyze the lactate metabolism through in silico simulation and in vitro experimental measurements most of all pyruvate branch point seems to play a major role in producing lactate, and the results of metabolic control coefficient analysis recommend to increase lactate dehydrogenase activity and to decrease nadh oxidase activity. for obtaining more realistic data, we have added some measured flux data including some intermediate metabolites. by combining the simulation results and experimental measurements, we could establish more reliable and robust systematic lactate secretion model. in addition, an efficient parameter estimation method was used to test the exactness of the reported kinetic parameters. what to choose -the fast or the detailed -strategy to get informative profiles of secondary metabolite produced by fungi in culture. chemo-diversity and lead discovery calls for high throughput techniques, but do we need columns will direct infusion esi-ms (dims) do the job. the latter may give matrix effects and lacks resolution resulting in loss of information, while lc-ms analysis takes time and challenge the data processing. results from nano-esi dims and lc-ms analyses of the same extracts important penicillium species are compared. these results illustrate advantages and problems using these techniques for rapid profiling of fungal secondary metabolites, reviling that matrix effects in dims do not seriously hampers detection of important metabolites while the specificity and certainty, for e.g. de-replication is much higher in lc-ms. phenotypic classification of fungi is essential in food biotechnology ulf thrane center for microbial biotechnology, biocentrum-dtu, søltofts plads , technical university of denmark, dk- kgs. lyngby, denmark. e-mail: ut@biocentrum.dtu.dk fungi are of great importance in food and food production. the intended use of fungi as cell factories for production of food ingredients is an upcoming issue in food biotechnology; however, this brings up a possible contamination with mycotoxins as a major issue. a reliable identification of the producer strains is crucial as a correct identification at species level following an updated taxonomy is the key to information on functional characters, e.g. useful metabo-lites and potential mycotoxins, growth conditions, resistance, etc. unfortunately, many mycological reports do not specify the taxonomy used or do not pay sufficient attention to taxonomical systems based on classification by functional characters-in contrast they are using a nucleotide sequence based phylogeny, which conveys little -if anything -about function of the organism. this situation is a major challenge for biotechnologists and mycologists in the years to come and will be highlighted by illustrative examples. the commercial interest in functional foods containing sufficient amounts of living probiotics is paralleled by the increasing scientific attention to the beneficial effect in the digestive tract. a daily intake of viable cells is proposed to ensure probiotic effect on consumer's health. one of the approaches which seems to be feasible to enhance probiotic viability and stability is to improve the fermentation conditions. during batch fermentation the viability of lactobacillus gasseri decreases after reaching a maximal value apparently indicating cell death. in this work, the apparent loss of viability can be avoided during fed-batch fermentation. a three-fold increase in viability is obtained when nutrient concentration was controlled compared with the viability reached in batch cultures. as a consequence, higher biomass concentration and lower specific lactic acid production were obtained. a mathematical model was developed to simulate and describe the effect of nutrient limitation on growth, viability, glucose consumption and lactic acid production. contribution to the metabolic adaptation to food restriction in rabbits (preliminary results) s. van harten , s. borges , p. cravo , l.a. cardoso : instituto de investigação científica tropical, cvz, lisboa, portugal; instituto de higiene e medicina tropical, lisboa, portugal. e-mail: svharten@gmail.com (s. van harten) in order to understand metabolic differences between two breeds of rabbits (halop ab and oryctolagus cuniculus algirus) during food restriction, the activities and expression of key enzymes and hormones of the rabbit were studied. animals from each breed were divided in two groups (ad libitum and restricted), revealing the results a similar difference in glycemic levels between fed and underfed rabbits, with a restriction of % of ad libitum feeding in the wild animals (decrease of % lw) and % of that ingestion in the halop breed (decrease of % lw). the activities of glutamine synthetase and glutaminase show a higher reduction of these enzymes in the wild animals superior to that of the halop breed, compromising, in this way, the ammonium detoxification and the entry of residual carbonated groups of the protein catabolism into the krebs cycle. in the latter animals, a rapid mobilization capacity of triacylglycerols (tga) appears to exist, with a rapid catabolism of fatty acids leading to their oxidation. the wild breeds' results reveal a rise of circulating tga, reflecting difficulties in the lipolysis and mobilization of nefa for oxidation. in these underfed animals, phosphoenolpyruvate and pyruvate suffered a large increase and oxaloacetate a decrease. the halop breed revealed results that indicate a diminution of glycolisis, being glucoses' energy substituted by carbonated chains of lipolysis and protein catabolism. hormone results showed a higher decrease in insulin, t and igf- in the underfed halop animals. in order to confirm the biochemical results, relative quantification of enzyme expression was studied by real time-pcr. since the introduction of genetically modified (gm) crops in , the area under their cultivation has globally increased from . million hectares in to . in . the number of countries adopting gm crops also rose from one country, the usa, in to in . despite numerous successes public opinion still questions the ecological, moral, ethical considerations and issues concerning altering the natural state of the organisms. in this study, a survey of food shoppers' knowledge, attitudes and perceptions of gm foods was carried out in food outlets in nairobi. the food outlets were determined by simple random sampling. using systematic sampling, shoppers were interviewed at targeted imported food products. focus group discussions were also conducted with farmers at city markets. the survey reflected views of a systematic sample of shoppers in seven food outlets between november and december of . it revealed knowledge at %, with positive attitudes and good perceptions towards gm foods (χ = . , d.f. , p < . ). seventy nine percent of shoppers were willing to buy and consume gm foods (χ = . , d.f. , p < . ). cross-tabulation of shopper's position on various issues raised in the survey showed a strong correlation between the respondents' respective knowledge, attitudes, and perceptions (r = . ). nineteen percent of food sampled tested positive for gms. poisson statistics were used to calculate the number of sample sequences. the statistical tools were obtained from spss version . . the results of this study will be of great interest in determining the use and adoption of gm crops in kenya. it will also guide the development of national foreign food policy on gm foods. the technology should be embraced as soon as it is acceptable to alleviate, drought, famine and hunger estimated to be affecting . million kenyans today, mostly children. consumers and gm foods: the case of turkeyÖzlenÖzgen , mustafa yildiz : department of family and consumer sciences, school of home economics, university of ankara, ankara , turkey; department of field crops, faculty of agriculture, university of ankara, ankara, turkey the future development of food biotechnology depends on consumer acceptance. scientists are aware that consumer attitudes will have a crucial impact on the process of the food biotechnology. because, food is one of the central features in human life. consumers' attitudes and trusts in the institutions will determine how gene technology will be used in food sector, in the future. recently, research concerned with consumer aspects of gm foods accelerated. but in turkey, the literature that deals with this subject is very limited and sparse. therefore, this research was carried out on the turkish consumers with the purpose of analyzing the consumers' awareness, assessments about benefits-risks, market place and labelling, and trusts in institutions, towards gm foods. this study was based on interviews with consumers who have recently purchased from major malls, during shopping hours. of the four major malls, voluntary male and female consumers were included in the research if they had main or secondary responsibility for household shopping. the questionnaire form was applied to subjects through face-to-face individual interview. the data were analyzed by using statistical methods according to explanatory variables, including age, gender and educational level. findings indicated that consumers' awareness and views about gm foods were connected to selected demographic characteristics. the results of this study can be important for consumer educators, marketing managers and policy makers. benefit-risk perceptions and moral beliefs of turkish consumers towards transgenic productsÖzlenÖzgen , haluk emiroglu , mustafa yildiz , ayşe sezen taş : department of family and consumer sciences, school of home economics, university of ankara, ankara, turkey; faculty of law, university of bilkent, ankara, turkey; department of field crops, faculty of agriculture, university of ankara, ankara, turkey the use of biotechnology in production has generated considerable debate involving the benefits-risks and moral beliefs associated with its use. consumer acceptance of genetically modified product is a critical factor will affect the future of this technology. this study was planned and conducted to determine the relationship between product/process related benefit perceptions, product/process related risk percentions and moral beliefs of consumers towards transgenic products. a total of university educated consumers, consisting of males and females, employed at the ministries selected by random sampling method in ankara, were included into study. interview techniques were used in the gathering the research materials. the interview instrument had been prepared considering previous research and literature. answers given to sentences typed likert were scored, used "varimax analysis technique" for validity. in order to test the reliability of questionnaire were calculated "cronbach alpha" as inner consistency coefficient. the t-test were performed for determining the differences dependent on gender and age variables between product related benefit perceptions, process related benefit perceptions, product related risk perceptions, process related risk perceptions and moral beliefs of consumers. the examination of relationships between product/process related benefit perceptions, product/process related risk perceptions and moral beliefs of consumers was made by correlation analysis technique. it is thought that the results of this study are important both for scientists and social scientists. the application of the dna recombinant technology for food production is generating a great debate in our society with the participation of scientists trying to explain the way of obtaining these new foods and which are their implications; environmentalist groups and anti-biotechnology associations that systematically are against to the application of this technology; legislatives bodies and the public in general, represented by consumers' organizations that expresses their right to be informed. considering that university students will be the future professionals and consumers their opinion on this topic will be decisive in its success or failure, this research is aimed to performed a global and comparative study of the agrobiotechnology perception by students from different areas of knowledge and studies. this study was carried out during the academic years - , being analyzed a total of valid surveys. the designed questionnaire included questions relatives to: evaluation of the own knowledge and interest on the topic; evaluation of the information sources mainly used by university students to obtain nutritional information; the opinion about gm food labelling; risks/benefits perception; purchasing intention and support of biotechnology. results obtained showed that % of the university students interviewed have a clear positive perception of biotechnology, mainly the students of the health sciences area. these students understand the scientific terminology and they use the university as the main source of information. they support the development of the biotechnology and they consider that in a future it will report them benefits. other group ( %) has a clear negative perception, they are mainly students from law and art history. they do not understand the scientific terminology, they consider that biotechnology will cause them risks, and as a consequence they don't have intention of buying these foods. the technology of the dna recombinant can be also used to introduce in the plants genome the gene that codes a protein of interest for their use as antigen. the application of agrobiotechnology has allowed the development of a new generation of vaccines that try to reduce or to eliminate the inconveniences of the classic ones. for the new vaccines design, the detailed knowledge of the biology of the pathogen is considered. with this knowledge the genes implied in virulence can be inactivated or modified selectively. the term "edible vaccines" it is usually applies to the use of edible parts of the plants (tubers, fruits, leaves, etc.) genetically modified with the purpose to produce specific components (antigens) of a pathogen (virus, bacteria, etc.) against which is wanted to protect a person or animal. however, oral is not the best vaccination route since the quantity of antigen for an efficient immunization it is usually high, being also needed, the co administration of an adjuvant that stimulates the immune answer. on the other hand, it is also important to highlight that the levels of antigen accumulation in transgenic plants is usually lower than the necessary ones. another problem is the irregular accumulation of the antigen in the different parts of the plants, thus difficult the appropriate control of the doses. tannin is polyphenolic component having some antioxidant properties and exists in many plants and fruits. in pomegranate juice this component causes turbidity and haze. during fruit juice clarification by conventional gelatin method, all poly phenolic substances which are responsible for antioxidant activity are removed and as a result the quality of the product is reduced. in the present study tannase enzyme (tannin acyl hydrolase; ec . . . ) was used to decompose tannin to gallic acid and glucose and as the result the amount of turbidity of the juice is decreased, however, the antioxidant properties remain unchanged since tannin is not decomposed and not separated in the juice as it occurs in the gelatin method. the amount of gallic acid in pomegranate juice samples before and after addition of tannase was measured using hplc tests and the optimum temperature, the enzyme and juice contact time, ph, and solvent concentration for clarification of pomegranate juice were obtained as • c, ph = . , h and mm citrate buffer, respectively. the potential benefits of enzymatic clarification of pomegranate juice, that is preservation of antioxidant activity and hence increasing the quality of the fruit product, in comparison to that of conventional clarification method by gelatin introduce a new technique in turbidity and haze removed in tannin containing fruit juices. the objectives of this study are to investigate the inhibitory effect of low molecular weight chitosan for its use as biopreservatives of foods. the inhibitory activity of chitosan against escherichia coli and staphylococcus aureus was investigated by determining the effect of chitosan treatment on bacterial growth during culture and its effect on the viability of non-growing cells. the activity of chitosan was decreased considerably for both strains when sucrose was added to ts broth containing chitosan. the addition of ethanol affected little on the inhibition of chitosan against s. aureus. the influence of nacl on the activity of chitosan was similar to the influence of sucrose and ethanol. however, the experiments with non-growing cells showed that the ethanol enhance drastically the antibacterial activity of chitosan on s. aureus. the results presented in this paper demonstrate that its antibacterial activity may be affected considerably by common food additives such as sucrose, ethanol and nacl. in lactococcus lactis the enzymes phosphofructokinase (pfk), pyruvate kinase (pk) and lactate dehydrogenase (ldh) are uniquely encoded in the las operon. we have applied metabolic control analysis to study the role of this organisation. earlier work showed that ldh at wildtype level has zero control on glycolysis and growth rate but high negative control on formate production (c j formate ldh = − . ). we find that pfk and pk have zero control on glycolysis and growth rate at the wildtype enzyme level but both enzymes exert strong positive control on the glycolytic flux at reduced activities. pk has high positive control on formate (c j formate pk = . − . ) and acetate production (c jacetate pk = . − . ), whereas pfk has no control on these fluxes. decreased expression of the entire las operon resulted in a strong decrease in growth rate and the glycolytic flux. increased las expression resulted in a slight decrease in the glycolytic flux. at the wildtype level the control was close to zero on both glycolysis and the pyruvate branches. the sum of control coefficients for the three enzymes individually was comparable to the control coefficient found for the entire operon at the wildtype level; the strong positive control by pk almost cancels out the negative control by ldh on formate production. the analysis suggests that co-regulation of pfk and pk provides a very efficient way to regulate glycolysis, and co-regulating pk and ldh allows the cells to maintain homolactic fermentation during regulation of glycolysis around wildtype level. bovine chymosin is used extensively in cheese production because of its specificity and low proteolytic activity. we are interested in the caprine chymosin as an alternative because in the canary islands cheese has traditionally been made using goats milk with extract from the abomasum of newborn goats as coagulating factor. we isolated and characterized the prochymosin cdna from the abomasum of milk-fed kid goats. this cdna predicts a polypeptide of amino acid residues, with a signal peptide and a proenzyme region of and amino acids, respectively. the caprine preprochymosin has % and % identity with the corresponding lamb and calf sequences. the cdna fragment encoding prochymosin was fused in frame to the killer toxin signal sequence in a constitutive vector, and to the ␣-factor signal sequence-flag in an inducible expression vector. kluyveromyces lactis pm - c, k. lactis sel , characterized by a "supersecreting" phenotype, and saccharomyces cerevisiae bj were transformed with the recombinant plasmids. activated culture supernatants of yeast transformants showed milk-clotting activity. the flag-prochymosin fusion was purified from bj culture supernatants by affinity chromatography. after activation at acid ph, proteolytic activity assayed toward casein fractions showed that the recombinant caprine chymosin specifically hydrolyzed -casein. the recombinant caprine enzyme could be an alternative milk coagulant in cheese making. lipid accumulation in schizochytrium g / s was studied under batch and continuous culture. different glucose and glutamate source concentrations were supplemented in a defined medium. during batch cultivation, lipid accumulation occurred towards the end of the growth phase but ceased when cell proliferation stopped. under continuous culture, as dilution rate decreased from . to . h − , both cell dry weight and total fatty acid content (tfa) of the cell increased. with a constant dilution rate of . h − , nitrogen limitation induced lipid synthesis ( % tfa) as described for other lipid-accumulating organisms. however, with carbon-limited conditions, some lipid accumulation was still possible, the tfa being %. finally, the batch and continuous culture methods are compared for docosahexaenoic acid ( : , n − ) production. the objectives of this study is to investigate the inhibitory effect of low molecular weight chitosan for its use as biopreservatives of foods. the inhibitory activity of chitosan against e. coli and staphylococcus aureus was investigated by determining the effect of chitosan treatment on bacterial growth during culture and its effect on the viability of non-growing cells. the activity of chitosan was decreased considerably for both strains when sucrose was added to ts broth containing chitosan. the addition of ethanol affected little on the inhibition of chitosan against s. aureus. the influence of nacl on the activity of chitosan was similar to the influence of sucrose and ethanol. however, the experiments with non-growing cells showed that the ethanol enhance drastically the antibacterial activity of chitosan on s. aureus. the results presented in this paper demonstrate that its antibacterial activity may be affected considerably by common food additives such as sucrose, ethanol and nacl. nitrite-oxidizing bacteria catalyze an essential step of nitrogen elimination in biological wastewater treatment. recently, novel and yet uncultured nitrite-oxidizing nitrospira-like bacteria were found to be abundant in municipal and industrial wastewater treatment systems where they outcompete nitrobacter, which has long been considered as the organism responsible for nitrite oxidation in bioreactors. despite the importance of nitrospira-like bacteria for wastewater treatment and for nitrogen fluxes in natural ecosystems, little is known about their ecophysiology and interactions with other organisms. cultivation-independent molecular techniques were applied to investigate the diversity, distribution, and physiological and genetic features of nitrospira-like bacteria in nitrifying activated sludge and biofilm. a surprisingly high diversity of these organisms was found to exist in these engineered and in natural habitats. moreover, significant physiological differences could be identified among various phylogenetic sublineages in the genus nitrospira. quantitative co-localization analyses performed by novel image analysis software revealed that these metabolic features are reflected by the spatial organization of nitrifiers living in biofilm and activated sludge flocs. based on an environmental genomics approach the genome of a nitrospira-like bacterium found in activated sludge is being analyzed. results obtained so far point at unexpected physiological capabilities of this organism, and allow us to propose that nitrospira-like bacteria may also play roles in the bioremediation of (per)chlorate and chlorite. the activated sludge process is the most common way to remove organic matter, nitrogen and phosphorus from wastewater by microbiologically means. knowledge about the microorganisms involved is fundamental for optimisation of existing plants and development of new plants and process designs. many of the bacteria believed to be involved in nitrification, denitrification, biological phosphorusremoval, and removal of organic matter in full scale plants are now identified by use of molecular methods. recent developments in experimental approaches have allowed the study of the ecophysiology of these uncultured and potentially important bacteria, thus providing a better understanding of their function in full-scale activated sludge ecosystems. relatively few dominant species in each functional group (e.g. denitrifiers and polyphosphate accumulating organisms) seems to be present. some species appear to be very specialized regarding nutrient requirements while others are more versatile. a new method for mercury remediation from industrial wastewater based on the enzymatic reduction of mercury by live mercury resistant bacteria immobilized on the pumice particles has been developed in gbf, germany, and implemented in the industrial scale (unknown). the experience gained during operation of this instalation led to the idea, that the process of bioremediation may be integrated in one bioreactor with the sorption of mercury from wastewater, by immobilization of the bacteria directly on the activated carbon. for this it was necessary to define several significant parameters of the activated carbon used and the sorption process itself. the paper presents results of the equilibrium and kinetics investigations of the process of mercury sorption from aqueous solutions onto seven different types of activated carbon. the effective diffusion coefficients in the particles were obtained from the transient-state experiments and the sorption isotherms, saturation capacity of the sorbents and its dependence on the temperature and ph were identified. then the hydrodynamic and sorption characteristics of the activated carbon bed in a laboratory-scale fixed-bed bioreactor were investigated in different process conditions (mercury concentration, volumetric flow rate, temperature, ph). the results (effective capacity of the bed, dispersion and diffusion coefficients, mass transfer coefficient) enable implementation of this bioreactor for modified, integrated process of mercury bioremediation from industrial wastewaters. research supported by the grant kbn t c . bacterial cr(vi) reductases convert the very mobile toxic cr(vi) to the less toxic and less mobile cr(iii). the ability to reduce cr(vi) was studied on cell extracts of ochrobactrum tritici strain bvl and microbacterium sp. strain a. both microorganisms were isolated from the same sample of chromium-contaminated sludge, taken from a wastewater treatment plant. while in the first case activity was found to be associated with the intracellular soluble extract, in the second case it was a process occurring extracellularly. cr(vi) reduction by the intracellular soluble extracts of strain bvl required the presence of nadh or nadph as electron-donor, while the extracellular fraction of strain a only used nadph. several studies were made on strain bvl intracellular soluble extracts. a k m of . m cr(vi) and a v max of . ± . nmol cr(vi) min − mg − protein were estimated from the lineaweaver-burk plot and michaelis-menten non-linear regression. the temperature and the ph optima for cr(vi) reduction were . • c and . , respectively. hyperthermus butylicus is an anaerobic hyperthermophilic crenarchaeon, isolated from the solfataric sea floor off sáo migel island, azores (zillig et al., ) . h. butylicus grows at up to • c (optimally between and • c) at ph . it can utilize peptides, polysaccharides, and other substrates, as carbon sources to produce acetate, butyrate, and n-butanol. the capability to produce enzymes (e.g. hydrolases, dna and rna polymerases, etc.) that can tolerate and function at temperatures • c higher than most other thermophilic archaea, renders h. butylicus of particular interest to the biotechnology industry. the complete genome sequence of h. butylicus was determined and it contains , , bp on a single circular chromosome. protein encoding genes were identified which use a high level of uug and gug start codons. many of these were assigned functions on the basis of sequence comparisons. our analyses revealed some unusual metabolic properties in h. butylicus. several sugar transporters were identified, although the set of genes required for glycolysis is incomplete. moreover, genes encoding enzymes converting glucose to trioses are absent and no genes encoding enzymes of the pentose phosphate cycle or the kdpg pathway were detected. the h. butylicus genome encodes many proteases and peptidases although the lon proteases, encoded in all other archaeal genomes, are absent. although it was reported that h. butylicus does not utilize free amino acids in the media, genes for amino acid transporters were identified, and several proteins involved in di-or oligo-peptide transport are encoded. genes encoding signal peptidases are absent. we will summarize gene products of special biotechnological interest. reference zillig et al., . j. bact. , - . hot genomics: insights in the thermophilic lifestyle of thermus thermophilus from its complete genome holger brüggemann , , anke henne , gerhard gottschalk : göttingen genomics laboratory, institute of microbiology and genetics, university of göttingen, germany; institut pasteur, unité de génomique des microorganismes pathogènes, paris, france thermus thermophilus is an extremely thermophilic, halotolerant bacterium, which was originally isolated from a natural thermal environment. recently completed genome sequences of two strains, hb and hb , provide a solid foundation for investigating many aspects of thermophilic lifestyle; these range from molecular stability determinants to key elements of organismic physiology. in addition, the species has considerable biotechnological potential; many thermostable proteins isolated from members of the genus thermus are indispensable in research and in industrial applications. the closely related genera thermus and deinoccoocus belong to a distinct branch of bacteria called the deinococcus-thermus group. genome comparison of t. thermophilus and d. radiodurans, a mesophilic organism, which exhibits high resistant to radiation, oxidative stress, and desiccation, is of particular interest for the identification and exploration of thermophilic determinants. a large number of orthologs with a high degree of sequence identity are shared between the two species. this opens the opportunity for comparative studies of conformational and chemical thermostability of proteins, as well as for the identification of specific traits for each organism, explaining their unique physiological properties and their intriguing differences in stress tolerance. although strains hb and hb share a highly conserved chromosome, striking differences can be found between their megaplasmids, which encode a huge proportion of genes not found in the genome of d. radiodurans. possible contributions made by the megaplasmids to a thermophilic lifestyle will be discussed. microorganisms that can live in high temperatures, extreme ph and high salt concentration are called extromophiles. extromophilic microorganisms have extended our knowledge and understanding of fundamental questions such as the origin of life. the ability to grow in extreme conditions and to produce stable proteins makes extremopliles very attractive for the researchers and also for the industry. extremozymes from extremophiles have a great economic potential in many industrial processes, including agricultural, chemical and pharmaceutical applications. concurrent development of protein engineering will increase the application of enzymes from extremophiles in industry. turkey has vast and various ecologi-cal areas, and so it has a broad microbial diversity. based on the extremophilles which defined in the scope of this project, halophilic microorganisms produced industrially important proteins were isolated from Ç amaltı saltern area in izmir, turkey. in this work, growth of isolates at different temperature, salinity and ph values were investigated to determine the effects of various growth conditions. eight isolates grow at ph between . and . and two isolates at . - . . they grow at temperature between and • c and salt concentration between % and %. the results of some phenotypic characters showed that they are gram (−) and oxidase,ürease, dnase and nitrate reduction are (−), and catalase (+). they used d(+) glucose, maltose, lactose, sucrose, l(+) arabinose, d(+) mannose, glycerol and four of isolates used d(+) xylose as a carbon source. the isolates resistant to erythromycine, ampicilin sulbactam, cefoxitin, penicillin, bacitracin, novabiocin, amikacin and sensitive to ceftazidine, ciprofloxacin, amoxycillin/clavulanic acid, imipenem, chloromphenicol, ceftazidime/clavulanic acid, aztreonam, cefepime, cefotaxime, cefoperazone amoxicillin. this project was supported by tubitak through project tbag - t . the technology of producing renewable energy sources such as ethanol, methane and hydrogen from biomass holds the potential of creating in-house energy resources while lowering the emission of greenhouse gasses as demanded by the kyoto protocol. recently, goals were defined for the european union determining that . % of the transportation fuel has to come from biofuels in year . a large-scale implementation of biofuels into the transportation sector will demand that lignocellulosic biomass, which is found in a surplus throughout the world is used as the raw material for the production process. the presentation will include a comprehensive description of the special bio-refinery concept developed in denmark for production of biofuels and other valuable products from straw. the concept includes several innovative steps such as a pre-treatment method using wet oxidation, on-site production of enzymes and a continuous fermentation process using a genetic modified thermophilic bacterium. by co-producing several biofuels in the plant optimal use of the biomass has been assured and the price of for instance of bioethanol is getting close to conventional oil-based fuels. optimizing each step in the bio-refinery, while having the full integration in mind, will be the way to make an economical viable biofuel production. in the presentation we will present our road map for achieving this goal in the nearest future. replacement of gasoline by liquid fuels produced from renewable sources is a high-priority goal in many countries worldwide. one such fuel, which has been found well suited, is ethanol. it may be produced from various lignocellulosic materials, such as forest and agricultural residues, which are fairly inexpensive. to compete with gasoline the production cost must be substantially lowered. ethanol production from lignocellulose comprises the following main steps: hydrolysis of hemicellulose, hydrolysis of cellulose, fermentation, separation of lignin, recovery and concentration of ethanol and wastewater handling. the enzymatic hydrolysis and fermentation can either be run separately (shf) or combined into a simultaneous saccharification and fermentation (ssf). the latter has been shown to result in higher ethanol yields than shf. some of the most important factors to reduce the cost are: efficient utilisation of the raw material by high ethanol yields, high productivity, high ethanol concentration in the feed to distillation and process integration in order to reduce capital cost and energy demand. in the last years we have performed several studies on the hydrolysis and fermentation of various forest and agricultural residues in a mini-pilot to improve the overall yield of ethanol and to reduce the energy demand and production cost. steam pretreatment, with small addition of acid catalyst, has resulted in sugar yields close to % of the theoretical for various types of raw materials, e.g. spruce, salix and corn stover. the ssf has been developed and optimized to give high yield of ethanol. for spruce an ethanol yield of about % of theoretical based on the composition of the raw material has so far been obtained using a two-stage steam-pretreatment of so impregnated raw material followed by ssf. improvements of the ssf step, in the form of high dry matter content, recirculation of process streams and adapted yeast have resulted in ethanol concentrations around g/l leading to substantial reduction in energy demand and production cost. these improvements have been assessed by techno-economic evaluation to determine the effect on the ethanol production cost. the process has been further optimised by process integration to further reduce the energy demand. the ethanol production cost was estimated to be around . - . euro/l ethanol assuming a yearly capacity of tonnes raw material (dry matter). production of bioethanol from spent grain, a by-product of beer production sho shindo, tadanori tachibana, akita research institute of food and brewing, akita-city, akita - , japan. e-mail: shindo@arif.pref.akita.jp (s. shindo) the breweries generate one million tons of spent grain every year, and about % of the spent grain is recycled in japan. therefore, it is environmentally and economically significant to consider the production of ethyl alcohol as biomass energy using the spent grain from the breweries industry. ethyl alcohol production from spent grain with immobilized yeast cells was investigated. spent grains were liquefied by a steam explosion treatment to obtain liquefied sugar. when kg of wet spent grain was treated under the kg/cm pressure for min using a l steam explosion reactor, g of total sugar was obtained from the liquefied spent grain. furthermore, . % (w/v) of glucose, . % (w/v) of xylose, and . % (w/v) of arabinose were produced when the liquefied spent grain was treated with glucoamylase, cellulase, and hemicellulase enzymes. ethyl alcohol production was carried out by immobilized sacchromyces cereviseae and immobilized yamadazyma stipitis simultaneously from liquefied spent grain. both yeast cells were immobilized on the glass beads carrier. xylose and arabinose were consumed after glucose was consumed completely during ethyl alcohol production. . % (v/v) ethyl alcohol was produced from liquefied spent grain that was adjusted % of initial sugar concentration after days. the vegetable oils constitute a resource of renewable potential for the production of fuels, becoming a viable alternative when compared to the diesel from petroleum. among the vegetable oils, the extracted oil of the castor plant seeds is a promising alternative source because it is constituted mainly of the ricinoleic acid ( -hydroxy- octadecenoic) that represents % of the total constitution of the oil approximately. the biodiesel obtained from castor oil can be defined chemically as being a mixture of methyl esters or ethyl esters of carboxylic acids synthesized by transesterification reaction of the existent triglyceride and an alcohol of little chain through the use of alkaline or enzymatic catalysts. in this work, we described the results found in castor oil with different degrees of purity. initially, it was made a rheological characterization followed by structural characterization (rmn c, rmn h and infrared) and thermal characterization (dtg, dta and dsc) of the crude and refined castor oil. it has been also measured the hydroxyl tenor, acidity index, saponification index and iodine index in different oils. later, these results were used to evaluate possible differences in the quality of the biodiesel (ethyl esters) produced in the enzymatic alcoholysis of the castor oil catalyzed by lipases (novozym , liposyme rm im and lipozyme tl im). the degree of substitution of castor oil derivative was performed by titration with . n hcl and confirmed by tlc analysis and the results showed conversion rates about %. has been demonstrated to have a wide range of health benefits such as prevention and therapy of various cancers, amelioration of heart disease, and prevention of renal stone formation as well as complications from diabetes. on the hand, lower phosphorylated forms of inositol, especially inositol trisphosphate (ip ) and inositol tetrakisphosphate (ip ) are important signal transduction molecules within the cells both in plants and the animal kingdom. it has been hypothesized that at least the anticancer function of ip is mediated via these lower inositol phosphates. the diversity and practical unavailability of the individual myo-inositol phosphates preclude their investigation. phytases, which catalyze the sequential hydrolysis of phytate, render production of defined myoinositol phosphates in pure form and sufficient quantities. different phytases may result in different positional isomers of myo-inositol phosphates and therefore different biochemical properties. phytases differing in ph optima, substrate specificity, and specificity of hydrolysis have been identified in plants and microorganisms. in this paper the dephosphorylation pathway of the novel phyfauia was compared to other bacterial phytate degrading enzymes. preliminary results have shown that phyfauia converted ip into ip (myoinositol , , , , -pentakisphosphates) and another isomer, which is yet to be elucidated. in a denitrifying pilot plant reactor, a new obligately anaerobic ammonium oxidation (anammox) process with great potential for nitrogen removal for high strength wastewater was discovered. after transfer of the complex microbial community to a laboratory sbr system, a highly enriched population, dominated by a single anaerobic chemolithoautotrophic bacterium related to the planctomycetes was obtained. the bacterium was purified via percoll centrifugation and characterized as 'candidatus brocadia anammoxidans'. survey of different wastewater treatment plants using anammox specific s rrna gene primers and anammox specific oligonucleotide probes revealed the presence of at least four other anammox bacteria, tentatively named 'candidatus kuenenia stuttgartiensis', 'candidatus brocadia fulgida', 'candidatus scalindua wagneri' and 'candidatus scalindua brodae'. a close relative of the two scalindua species, 'candidatus scalindua sorokinii' was found to be responsible for about % of the nitrogen conversion in the anoxic zone of the black sea and in the benguela upwelling system along the namibian coast, making anammox an important player in the global nitrogen cycle. electron microscopic studies of all five anammox bacteria showed that several prokaryotic membrane-bounded compartments are present inside the cytoplasm, which are surrounded by unique ladderane lipids. hydroxylamine oxidoreductase, a key anammox enzyme, was present exclusively inside one of these compartments, named the 'anammoxosome'. unique peptides fragments of the purified hao were used to locate the hao gene in genome assembly of 'candidatus kuenenia stuttgartiensis'. the implementation of the anammox process in the treatment of wastewater with high ammonium concentrations was started at the treatment plant in rotterdam, the netherlands, where it is combined with the partial nitrification process sharon. the estimated price for nitrogen removal with partial nitrification and anammox is about . euro/kg n. gas lift reactors could sustain the highest anammox capacity at . kg n removed/m reactor per day. an alternative configuration of anammox is the oxygen-limited canon process in which aerobic ammonium-oxidizing bacteria protect anammox bacteria from oxygen and produce the necessary nitrite. maximum nitrogen removal with canon in gas lift reactors was . kg n/m reactor per day. using several different conditions and parameters, the competition and co-existence of aerobic and anaerobic ammonium-oxidizing bacteria were modeled. in addition to ammonia, urea was also converted after a -week adaptation in the canon system. recently it was shown that anammox bacteria can use organic acids as additional energy source. murray moo-young, wa anderson department of chemical engineering, university of waterloo, waterloo, ont., canada n l g bioreactors are central to the bioremediation of contaminated environments of water, air or soil. in all three areas of application, bioreactor design is critical to the development of new or improved processes. this overview focuses on the physical limitations of bioreactors caused by biological requirements. the information is based on our own research findings. the need for more applicationsoriented bioremediation research becomes apparent. for technoeconomic reasons, the airlift type has often been the bioreactor of choice for most bioremediations. however, lack of adequate understanding of the quantitative effects of operating conditions on its performance has been an ongoing concern. these effects have been characterized for engineering implementation. to enhance productivity, innovative pretreatment techniques of the polluted sources have also been developed using photocatalytic and chemical oxidation methods. case studies on petrochemical-contaminated water and soil reveal significant enhancement potentials. other studies on microbial biofilters for air bioremediation indicate that the active mass of the biological consortia is not sufficiently understood for rational design. analysis and retrofit design of wastewater treatment facilities using process simulation tools demetri petrides, alexandros koulouris, intelligen, inc., scotch plains, nj , usa. email: dpetrides@intelligen.com (d. petrides) process simulators have been used in the petroleum and chemical industries for over four decades to facilitate the design of new processes and optimize the performance of existing ones. similar benefits can be derived from the use of such tools in the environmental arena, particularly in the field of physical and biological treatment of municipal and industrial wastewater. specifically, process simulators can be used to evaluate and improve options for: ( ) more efficient removal of nutrients (e.g., organic nitrogen and phosphorous) that cause eutrofication, ( ) estimation and control of volatile organic compound (voc) emissions from open tanks, and ( ) more efficient removal and control of hazardous compounds. the potential benefits will be illustrated with cases studies involving both municipal and industrial wastewater facilities. the microbial reduction of metals has showed recent interest as these transformations can play crucial roles in the cycling of both inorganic and organic species in a range of environments and, if harnessed, may offer the basis for a wide range of innovative biotechnological processes. under certain conditions, however, microbial metal reduction can also mobilise toxic metals with potentially calamitous effects on human health. some effluents present heavy metals as soluble compounds, several microorganisms have the capacity to precipitate these metals as insoluble compounds, and this fact allows the collection and separation of these metallic precipitates from contaminated medium. sulfate-reducing bacteria (srb), under anaerobic conditions, oxidize simple organic compounds (such as acetic acid and lactic acid) by utilizing sulfate as an electron acceptor and generate hydrogen sulfide. hydrogen sulfide reacts with heavy metal ions to form insoluble metal sulfides that can be easily separated from a solution. the purpose of this work was study the capacity of desulfovibrio sp. cultures to reduce mixtures of the heavy metals in presence or not of petroleum. for it the experimental design k (k = ) was carried out. the five studied factors were cr, cu, mn, zn and petroleum. the study was carry out with desulfovibrio sp. batch studies were performed in ml sealed bottles with different concentrations (cr(iii)- ppm, cu(ii)- ppm, mn(ii)- ppm, zn(ii)- ppm) of metal sulfate and g l − of petroleum. during batch incubation the dissolved concentration of metal studied in supernatant were decreased to undetectable levels for zn ( - %), however with cu ( - %), mn ( - %) and cr ( - %). the development of continuous process with sulfatereducing bacteria seems to be a suitable alternative to reduce metals in solution from contaminated media such as industrial or mine effluents. after these preliminary results, some experiments in course are focused to study that purpose. reduction of odour emissions from livestock buildings using a bioscrubbing system morten Øgendahl, nawaf abu-khalaf, jens jørgen lønsmann iversen department of biochemistry and molecular biology, university of southern denmark, dk- odense m, denmark. e-mail: tvede@bmb.sdu.dk (m. Øgendahl) a bioscrubbing system for reducing odour emissions from livestock buildings is presented. the bioscrubbing system consists of two separate units; an absorption column and a water purification module. the absorption column is mounted in the ventilations stacks in the livestock buildings absorbing odorants in the effluent air flow. the odorants are absorbed in a spray of droplets formed by a grid of high pressure nozzles in the inlet of the absorption column. the spray of droplets is extracted from the air flow and pumped to a centrally located water purification module, an inverse three phase fluidised bed bioreactor, where the bio-degradation of the absorbed odorants occurs. the bioreactor features a split sparging system for maximum mixing and aeration. the cleaned water is recirculated to the absorption column. an electronic tongue will quantify key odorants in the bioreactor. the absorption column is designed to be retrofitted into existing livestock building ventilation systems. the water purification module is constructed in standard size units simplifying scaling to match the requirements of individual applications. the total bioreactor volume is increased by increasing the number of standard bioreactors. this work describes a "light off" toxicity bioassay sensor based on whole cell genetically modified bioluminescent bacteria. the biosensor was constructed by mating between the environmentally isolated phenol-degrading acinetobacter sp. strain df and the plasmid putk that is an inc p␤ plasmid with the bioluminescence genes luxcdabe inserted into a genetic region involved in plasmid replication and transfer. subsequently, the bioreporter designated df /putk and used to investigate phenolics toxicity. among examined phenolics, pentachlorophenol, catechol and nitrophenol recorded the fastest effect on the bioluminescence of bioreporter df /putk over incubation period of min. the effect of various concentrations of phenol and its derivatives either in an individual, duple or triple mixture forms on the bioluminescence response of the constructed bioreporter df /putk were also examined. significant reduction of the bioluminescence was observed whenever a mixture contained pentachlorophenol, catechol and nitrophenol, respectively. to develop a system appropriate to commercialize, the constructed bioreporter df /putk was subjected for immobilization in microtiter plates using several entrapment gels. after a selection of materials was tried, lb/agar was chosen as the most suitable candidate material. characterization of key odour compounds in an air wet scrubber is presented. the key odour compounds represent five chemical groups, i.e. sulphide, alcohol, volatile fatty acids (vfas), phenol and indole. direct aqueous injection (dai) and solid phase extraction (spe) methods were used before injection of key odorants into the gas chromatography-flame ionisation detection (gc-fid). the dai and spe methods were efficient in the identification of odour compounds in the wet scrubber. the spe method had a high recovery and can be more effective in the identification of compounds at low initial concentration. however, dai showed a better linearity and a lower limit of detection (lod) than the spe method. the dai method was the method of choice for characterization, as it is cheaper, easier to handle and highly applicable. at least two odorants, phenol and -butanol, were quantified successfully using the dai method. their lod was less than their odour detection limit in the wet scrubber. dai method can be used as a reference measurement method for any further analytical application, e.g. electronic tongue. recent developments in biotechnology enabled the widespread use of microbial enzymes in textile, detergent, food and dairy industries and also in various environmental applications. microorganisms which live at extremes of temperature, ph and salinity, produce extremozymes that offer many exciting opportunities for their use in clean production. in this study, microorganisms were isolated from camaltı saltern area inİzmir, turkey. effect of medium salinity on the growth of these microorganisms was determined. seven out of isolates required salt for growth. the salinity ranges at which growth was detected were: - % for two isolates, - % for one isolate, - % for two isolates and - % for one isolate. the isolates were also screened for their capability of producing industrially important enzymes such as amylase, protease, lipase, xylanase and cellulose which are widely used not only in textile, detergent, food and dairy industries but also in various environmental applications. all of the isolates were found to be producers of both amylase and xylanase enzymes at varying salinity array within - % salt concentration range at ph . . extracellular protease activity was detected in the medium of all isolates grown at , , , and % salinity at both ph . (optimum growth ph) and ph . . out of isolates, , and were found to produce cellulase enzyme when the salt concentrations were , and %, respectively. at % salt concentration, only one isolate was found to be cellulase enzyme producer. none of the isolates were found to produce lipase enzyme at - % salt concentration range. this project was supported by tubitak through project tbag - t . chemical engineering department, middle east technical university, ankara , turkey. e-mail: ubakir@metu.edu.tr (u. bakir) glass and ceramic tiles are very widely used industrial materials. in most cases, periodical cleaning is required to maintain their optical properties such as transparency and visual aspects. because of the ever-growing demand for healthy living, there is a keen interest in materials capable of killing harmful microorganisms. the application of these tiles in care facilities to reduce the spread of infections, in public and residental places to improve hygienic conditions are of general interest. in this study the aim is developing methods to apply thin film coatings on glass tiles to make them anti-bacterial by utilizing photocatalysis and investigating their anti-bacterial properties. semiconductors because of their reasonable band gap energies find great attraction through this purpose. the photocatalytic property of semiconductors are used in this process. oxidising radicals are formed on the coated surfaces and these radicals attacks the organic pollutants and bacteria on contact with the surface. titanium dioxide (tio ) coated surfaces are considered to be very effective against organic and inorganic materials, as well as against bacteria. in the experimental procedure coating solution is prepared by sol-gel technique. after pretreatment of surfaces, the coating solution is applied on the surfaces by dip-coating method. after appropriate thermal treatments, to achieve thin, dense and strong coatings, indicator microorganism is directly applied on the coated surfaces and illuminated under solar simulater light source. finally, the number of surviving microorganisms are determined. in this study, the effects of titanium dioxide (tio ), tin oxide (sno ) solutions and metal doping to these coating solutions on anti-bacterial function were investigated. as a result of this study, the number of escherichia coli that is used as indicator microorganism, on tio and sno coated glasses with respect to the control glass reduced by - % and - %, respectively. doping with metals increased the activity of the coatings, hence the number of surviving microorganisms decreased. activity of a methanogenic ecosystem during the primary contact with a solid support s. michaud, n. bernet, p. buffière, j.p. delgenès inra-lbe, avenue des etangs, f- narbonne, france in this paper, the biological activity during the first initial contact between a methanogenic sludge and a solid support was investigated in batch experiments, at different solid concentrations, using two different granular solid materials and with glucose as the main organic substrate. in all cases, the introduction of a solid material in a methanogenic suspended biomass induced a response of the anaerobic microorganisms, after a lag phase during which biological activity was not detected. this lag phase could be the consequence of a physical stress induced by the first contact between microbial cells and the solid surface. this lag phase was not observed when the biomass used originated from a biofilm reactor, i.e. using a biomass previously exposed to a solid material. a change in the metabolism of organic matter from catabolism and methane production toward production of other compounds could be observed, characterised by a sharp decrease of the methane yield in the anaerobic system. analyses of the gas and liquid phases did not show the production of any new gaseous or soluble compound as the biological end product of this activity. this suggests the production of non-soluble compounds by an anabolic pathway, which could indicate the initiation of biofilm formation. this metabolic activity was shown to be directly correlated to the ratio between the solid surface introduced and the microorganism concentration in the anaerobic culture (m g vs − ). from kinetic observations, it could be observed that acetogenic methanogenesis recovered more rapidly than syntrophic propionate and butyrate degradation. evaluating microbial diversity of hydrocarbon degrading bacteria cleantis braithwaite, howard rosser, tawfiq al-ibrahim, hussain, al-bandi research and development center, saudi aramco, dhahran, saudi arabia the analysis of microbial diversity with molecular methods is central to isolating and identifying new and potential biocatalysts resources for research and industry. the ability to degrade hydrocarbon components of petroleum is widespread among bacteria, and is an effective method for remediation of a variety of ecosystems. due to the high carbon content of oil and the low levels of other nutrients essential for microbial growth, treatment of oil with phosphorus and nitrogen is generally required to enhance the growth of hydrocarbon-degrading bacteria and to stimulate oil sludge degradation. in this research study, three types of oily sludges from a gas plant, refinery, and terminal facilities were treated with nutrients. to assess the microbial diversity, both biolog culture method and culture independent polymerase chain reaction (pcr,) denaturing gradient gel electrophoresis (dgge) methods were used. nutrient addition significantly improved oil sludge degradation. we identified and characterized several hydrocarbon degrading bacterial strains that have the ability to convert petroleum. these bacteria included representatives both gram positive and gram-negative genera. there were slight difference in the quantity and type of hydrocarbon degrading bacteria found in the three sites. this is the first molecular analysis of hydrocarbon degrading microbial population in saudi arabian operations. mussel adhesive proteins, including the -plus variants of foot protein type (fp- ), have been suggested as potential environmentally friendly adhesives for use in aqueous conditions and in medicine. here we report the novel production of a recombinant mytilus galloprovincialis foot protein type variant a (mgfp- a) fused with a hexahistidine affinity ligand in escherichia coli, and its ∼ % purification with affinity chromatography. recombinant mgfp- a showed a superior purification yield and better apparent solubility compared to those of the previously reported recombinant m. galloprovincialis foot protein type (mgfp- ). the adsorption abilities and adhesion forces of purified recombinant mgfp- a were compared with those of cell-tak (a commercial mussel extract adhesive) and mgfp- using qcm analysis and modified afm, respectively. these assays showed that the adhesive ability of recombinant mgfp- a was comparable to that of cell-tak but lower than that of recombinant mgfp- . collectively, these results indicate that recombinant mgfp- a may be useful as a commercial bioadhesive or an adhesive ingredient in medical or underwater environments. cresol, a monomethylated phenol, is an aromatic compound. the environmental protection agency (epa) has determined the carcinogenic potential of cresol. various options are being examined for the degradation of cresol because of their unavoidable large scale production and toxicity. many aromatic hydrocarbons can be used as electron donors aerobically by species of pseudomonas, thus leading to the ring cleavage of these compounds. in the present study, pseudomonas strains were isolated from activated sludge collected from sewage treatment plant. it was repeatedly transferred onto nutrient agar plate to check the purity of the culture. the organism was grown aerobically in an inorganic medium with p-cresol as the solitary carbon source. pseudomonas was confirmed by the expression of green pigment, gram staining and biochemical tests including koh, catalase, nitrate reduction and carbohydrate fermentation reaction. inoculation status was used to determine the rate of degradation of p-cresol. the effect of temperature on p-cresol degradation was studied. moreover, the effect of different concentrations of the aromatic compounds on pseudomonas as well as substrate variability was also documented. phenolic intermediates were estimated colorimetrically using -aminoantipyrene, folin-lowry method, uv spectrophotometry and hplc. the results indicated that pseudomonas could degrade up to mg/l of p-cresol within h. pseudomonas sp. exhibited good metabolic versatility and degraded other aromatic compounds including m-cresol and p-hydroxybenzoic acid. we conclude that this strain of pseudomonas has excellent potential for bioaugmenting the degradation of p-cresol-containing waste water treatment units. a considerable amount of waste cooking oil is produced by the restaurant industry worldwide. this poses a significant environmental and economic problem, since high oil and grease concentrations in the sewage system could lead to pipes occlusion and decreased efficiency in water treatment operation plants. therefore, sending these wastes to recycling companies or hazardous waste processors is usually required. yarrowia lipolytica, a well-known lipase producer, requires the presence of lipidic compounds (i.e. vegetable oils) to boost enzyme biosynthesis. in this work, the suitability of waste cooking oil as lipase inducer in submerged cultures of this yeast has been assessed. if successful, this procedure could allow both the degradation of an abundant waste and its valorisation as a raw material for the production of a high added value product. the microorganism was grown in a liquid medium to which various amounts of waste cooking oil were added. biodegradation degrees up to % (measured as decrease in cod) were obtained after days of treatment. also, initial glucose concentration in the basal medium seemed to influence the efficiency of the process. on the other hand, addition of waste oil led to a significant increase in lipase production (more than two-fold), compared to oil-free cultures. moreover, chain-length specificity of the produced enzymes was significantly different: high activity towards medium chain length esters was found, which hinted to the occurrence of both lipases and esterases. biodesulfurization: a documental review j. ferrer, simon bolivar university, environmental engineering lab. caracas, venezuela a documental review about larger interest aspects in biodesulfurization technique is showed. especifically, the investigation is related to general framework and the justification of this technique, degradatives pathways elucidated up to now, involved microorganism, important elements in development of bacterial desulfurization and progress areas, and future tendency. in situ bioremediation of a p-nitrophenol contaminated site and assessment of its community structure debarati paul, gunjan pandey, sumeet labana, rakesh k. jain institute of microbial technology, sector a, chandigarh , india. e-mail: rkj@imtech.res.in (r.k. jain) biodegradation of p-nitrophenol (pnp), a priority pollutant, was studied as a model system for bioremediation of sites contaminated with nitroaromatic/organic compounds. bioremediation studies were carried out in pnp-spiked soil in small plots under natural field conditions using arthrobacter protophormiae rkj . role of carrier material was examined by immobilizing the bacteria on corncob powder prior to adding them to soil. these studies demonstrated successful removal of pnp by immobilized cells that were able to deplete pnp completely in days, whereas free cells were able to deplete % pnp in the same time period. monitoring the fate of released bacteria revealed fairly stable population of the cells when they were immobilized on corncob powder throughout the period of study. on the other hand, there was a decrease of . log units in colony forming units of free cells at the end of the study ( days). bacterial community structure and diversity was also studied for the pesticidecontaminated site wherein the effect of addition of an exogenous strain on the existing soil community structure and on soil functionality was determined using molecular techniques. as revealed by restriction fragment length polymorphism (rflp) studies different phylotypes could be identified on the basis of similar banding patterns. sequencing of representative clones of each phylopyte showed that the community structure of the pesticide-contaminated soil mainly constituted of proteobacteria and actinomycetes. terminal fragment length polymorphism (t-rflp) analysis showed only subtle changes in community structure during the process of bioremediation. bacteriocins encompass an array of structurally different molecules produced by a number of phylogenetically distinct bacterial groups and trigger the killing of the same or closely related species. the recombined escherichia coli strain harboring a bacterocin coding region of xanthomonas campestris pv glycines ra was disrupted to obtain cell homogenate. peptidic xanthomonas bacteriocins (pxb) were separated by lowering ph and adding salt. the resulting pxb's were partially purified using ion exchangers, gel filtration. two final active fractions, a and b, were obtained with a yield of . % and - -fold purification. the activity of pxb was stable at the ph ranging from . to . . andreja kresal, vanja kokol, vera golob textile department, university of maribor, , slovenia wastewater from textile dyeing industries is characterized by high chemical and biological oxygen demands (cod and bod) and intense color due to the extensive use of synthetic dyes. as dyes of complex aromatic structures are resistant to removal by the typical microbial population and may be toxic to the microorganisms present in the treatment plants, discharge of the wastewater to the treatment plants may lead to its failure. beside, direct discharge of these effluents into municipal wastewater plants and/or environment may cause the formation of toxic carcinogenic and/or unhealthy breakdown products. different chemical and physical methods (adsorption, coagulation-flocculation, oxidation, filtration and electrochemical treatments) for color removal have been proposed, but due capital costs and slow operating speed as well as huge amounts of sludge creation there is still a great need to develop an economic and effective method. the use of lignin degrading white-rot fungi and their enzymes (laccase, lignin peroxidase, manganese peroxidase) has attracted increasing scientific attention due their ability to oxidative degrade a wide range of recalcitrant organic compounds. in the contribution, the decolorization efficacy of different commercial textile reactive dyes (anthraquinone, azo, triphenylmethane) will be investigated after the treatment by laccase from trametes versicolor. in order to examine the reuse of enzymatically decolorized liquors, the ecological suitability and the toxicity of the degradation products after different time of enzyme exposure will be studied. this work was carried out within the scope of research project e! cawab. influence of heavy metals on growth and extracellular enzyme production of a trichoderma harzianum strain with biocontrol potential l. hatvani , l. kredics , a. szekeres , z. antal , l. manczinger , a. nagy , c. vágvölgyi : department of microbiology, university of szeged, p.o. box , h- szeged, hungary; hungarian academy of sciences, university of szeged, microbiological research group, hungary; pilze-nagy ltd. kecskemét, p.o. box , hungary. e-mail: kredics@bio.u-szeged.hu (l. kredics) trichoderma species are common soil inhabiting asexual filamentous fungi with teleomorphs belonging to the hypocreales order of the ascomycota division. besides the industrial and clinical importance of the genus, certain strains have been found to cause great losses in mushroom cultivation while other strains are well known to possess high antagonistic activity against several plant pathogenic fungi and therefore used as biocontrol agents. important mechanisms of antagonism include competition and mycoparasitism, which -among others -can be related to the fast growth of trichoderma strains and the production of several extracellular enzymes. the influence of certain, soil-occurring heavy metals on mycelial growth and the secretion of extracellular enzymes involved in competition and mycoparasitism was examined in this study regarding an effective, potential biocontrol isolate of trichoderma harzianum. the metal ions zinc, manganese, copper, iron, lead and mercury were applied at the concentrations of , , , , , and m, and dry mycelial weight as well as the activities of extracellular ß-glycosidase, cellobiohydrolase, trypsinand chymotrypsin-like protease and n-acetyl-glucosaminidase enzymes were determined. it was found that mercury totally blocked mycelial growth, while other metal ions exerted a much lower influence on growth. the presence of heavy metals did not have a significant effect on the activity of the examined extracellular enzymes with the exception of trypsin-like protease, which showed a four-to six-fold rise in activity in the presence of certain sublethal concentrations of copper. based on these results, our further aim is to develop copper-resistant derivatives by mutagenesis from trichoderma strains with biocontrol potential. since proteases play an important role in mycoparasitism, these strains could be applied within the frames of integrated pest management in combination with copper-containing fungicides, resulting in an enhanced level of crop protection even with reduced amounts of fungicides. this work was supported by grants f of the hungarian scientific research fund and grant omfb- / of the hungarian ministry of education. the significance of biocontrol agents (bcas) is that some of them possess good antagonistic abilities against plant pathogenic fungi. a significant number of the most prominent fungi for the purposes of agricultural application belong to the genus trichoderma. in previous studies, in vitro assays on agar plates were reported as the generally used method for the evaluation of antagonistic abilities, as the results of these assays are well transferable to the practical application. the aim of the present study was to develop an accurate, image analysis-based method for the evaluation of the biocontrol characters of bcas. randomly selected trichoderma isolates were tested against fusarium culmorum. in the currently developed method, the areas of the fungal colonies were calculated on petri dishes by measuring the occupied surface of the medium on digital images. the inhibition effect was recorded as the value of biocontrol index (bci), which was calculated from the ratio of the area of the trichoderma colony and the total area occupied by the colonies of trichoderma and the plant pathogen. the proposed method was tested for numerous parameters, and the results revealed that bci proves to be capable for the accurate measurement and scale of the biocontrol abilities of fungal isolates. this work was supported by grants f of the hungarian scientific research fund and grant omfb- / of the hungarian ministry of education. the effect of advanced oxidation processes and recirculation on biodegradation of leachates from aerobic landfills liliana krzystek, anna zieleniewska-jastrzębska, stanisław ledakowicz department of bioprocess engineering, technical university of lodz, - lodz, poland modern landfills are built and operated in a way which allows us to treat them as a special type of bioreactor. simulation of municipal waste biodegradation in lysimeters provides knowledge on basic processes that take place in an aerated landfill. the aim of aeration is to stabilise mainly biodegradable and nitrogen containing components and to reduce methanogenic potential. stabilised leachates from old landfills contain big quantities of refractive carbon compounds that cannot be removed by biological methods. in such case most advantageous is to apply advanced oxidation processes (aops). the objective of this study is an experimental simulation of a landfill aerobic stabilisation and the impact of aops and recirculation of leachate on the reduction of organic load. the performance of the processes was monitored by the reduction in time of basic indices of organic load (bod , cod, toc, vfa, tkn, n-nh + ) and changes in biogas composition. the simulation of aerobic landfill processes was carried out in lysimeters with a fixed bed of household solid waste stabilised during months in anaerobic conditions. leachates taken from the lysimeters were recirculated and subjected to advanced oxidation processes, i.e. ozonation and uv radiation with the addition of h o . experimental studies showed that the aerobic waste stabilisation was a very quick process. during a month the bed was stabilised, reaching a significant reduction of organic load indices. aeration of the lysimeters caused a quick reduction of mainly degradable organic substance (in terms of bod ) and n-nh + and vfa. the reduction of methanogenic potential of the landfill was even faster. the composition of gas at the outlet from the lysimeter changed and after one day already its content was similar to atmospheric air. a more frequent recirculation of leachates enhanced greatly the aerobic biodegradation. it was found that application of advanced oxidation processes (especially ozonation) contributed to a growing reduction of the organic load in the leachates from aerated lysimeters. the application of leachate ozonation resulted in a very high degree of reduction of organic compounds (up to %). the objective of the experimental study was to assess the effect of temperature on the extent of aerobic batch biodegradation of potato stillage with a mixed culture of bacteria of the genus bacillus. the experiments were performed at , , , , , , , , and • c, at ph , in five l l working volume stirred tank reactor (str) (biostat ® b, b. braun biotech international). the duration of the process was h. initial cod of the stillage amounted to . g o /l, the main carbon sources being reducing substances ( . g/l), organic acids (determined as their sum) ( . g/l) and glycerol ( g/l). at • c, no cod reduction or biomass increment was found to occur. at the other investigated temperatures, the reduction in cod measured after suspended solids (ss) separation varied from . % ( • c) to . % ( • c). without ss separation, cod reduction ranged between . % ( • c) and . % ( • c). this indicates that, in terms of the extent of cod reduction, the optimal process temperature was • c and that there was a local optimum at about • c. according to the temperature applied, the content of reducing substances decreased by . - %, that the organic acids by . - . %, and that of glycerol by . - %. the experiments also produced the following two findings: ( ) the rise in temperature brought about a decrease of biomass concentration in the str (measured as ss and bacterial number), and ( ) temperature was a factor affecting the demand for ammonia nitrogen (n-nh ), which was the highest at and • c. the high n-nh demand observed both over the higher and lover ranges of the investigated temperature should be attributed to the release of n-nh and to the large amounts of the biomass produced, respectively. the results obtained imply that the extent of potato stillage biodegradation with a mixed bacterial culture was high over a wide range of the investigated temperature. polychlorinated compounds such as tetrachloroethylene (pce) have become serious environmental pollutants. considerable attention has been paid to these organochlorine compounds. this paper describes the molecular analysis of dechlorinating gene in halorespirating bacterium and efficient bioremediation process. an anaerobic bacterium, that dechlorinates pce to tce, was isolated and identified as a species of the genus desulfitobacterium. a novel pce reductive dehalogenase (prda) gene from the desulfitobacterium sp. strain kbc- was identified. these prd genes, including membrane anchor protein, were classified as a novel type of pce reductive dehalogenase (approximately % homology with the general pce dehalogenase). according to the substrate utility of this strain kbc- and phylogenetic analysis of prda, the type of this microorganism may be expected to play the role of a primary degrader of pce in the environment. high efficient bioremediation process so called the restricted aeration system which means microaerobic/aerobic reciprocal bioremediation process was developed. strong modifications take place, as ammonia production with a subsequent rise of the ph value and a rapid heat evolution leading to temperatures of up to • c. little is known about the microbial community in the toscano cigar fermentation and its development as fermentation proceeds. the aim of this study is to investigate the microbial community composition, its dynamic and its influence on the toscano cigar production process. our results show that the fermentation could be divided into three different phases: initially yeasts are the predominant microorganisms while bacterial growth is partially inhibited; the middle phase is characterized by exponential growth of bacteria while yeasts disappear. in the final phase the microorganism population is mostly represented by sporigen microbial species. the occurrence of yeasts in the first phase could be attributed to their ability to grow at low temperature and low ph levels. the bacterial population flourishes after the yeast cells have reached a stationary phase and probably grows on residual nutrients and autolysing yeast cells. yeasts and bacteria involved in the fermentation process were isolated and characterized. the microbial community was investigated by a combination of phenotypic and molecular approaches. the phenotypic characterization was based on both colony and cell morphology. the isolates were then identified by rrna genes sequence analysis. finally, in order to clarify the role of the identified microorganisms in the production process, a preliminary biochemical characterization was carried out. biosensors have undergone rapid development over the last few years; in particular, in environmental field many biosensors using microorganisms and purified enzymes as biological component, were recently studied. benzene is present everywhere with high levels in the cities and sometimes, in petroleum processing plants. it is classified as carcinogenic compound of first class able to cause leukaemia. because the evaluation of benzene requires complex instruments and quite long analysis times, it is required to study alternative systems for benzene detection simple, fast and highly sensitive, such as biosensors. from pseudomonas putida mst, strain previously isolated in our laboratory and able to degrade benzene, we isolated genes encoding for benzene , -dioxygenase and cis- , -dihydrodiolbenzene dehydrogenase to use in the development of two different hydrocarbon biosensors based on microorganisms and on purified enzymes. the genes isolated were cloned in pvlt and we developed three microbial systems carrying: ( ) benzene dioxygenase, ( ) dihydrodiol dehydrogenase and ( ) benzene dioxygenase-dehydrogenase modified by pcr to obtain enzymes with histidine tag. the cloning was planned to construct recombinant strains able to overproduce the enzymes; the enzymatic activities will be evaluated both using whole cells and purified enzymes. study of operation condition of biofilter using fibril-form matrix for odor gas removal don-hee park, chonnam national university, this research was performed for developing of biological treatment process of odor gas such as mek, h s, and toluene, which is generated from the food waste recycling process. to establish the operational conditions of odor gas removal by small-scale biofiltration equipment, it was continuously operated by using toluene as a treating odor object. when the odor treating microorganisms were adhered to fibril form biofilter, high removal efficiency over % was obtained by biofilm formation. at ppm of inlet odor gas concentration and s of retention time, the removal efficiency was % and % in first stage reactor and second stage reactor, respectively. however, the removal efficiency remained over % at the operational conditions above s of retention time. ozonated water is produced using an ozone generator in a container filled with cold water. it is useful for sanitizing the surfaces of various products for which heat or chemical treatment is inappropriate, such as fresh food products. in this study, we investigated the antimicrobial effects of ozonated water and electrolyzed ozonated water against escherichia coli, s. aureus, bacillus subtilis and yeast, saccharomyces cerevisiae for practical use in sanitizing various products. the results demonstrated that the electrolyzed ozone water was effective for the reduction of microbial population at relatively low concentration of ozone. also, the electrolyzed and the ozonated water showed synergistic antimicrobial effects. many xenobiotics can react spontaneously with thiol moieties of glutathione (gsh), forming gsh-conjugates, or via glutathione s-transferases (gst). these enzymes participate in detoxification of potentially harmful compounds from endo or xenobiotic origin. using saccharomyces cerevisiae as experimental model, we observed that cells mutated in the gtt or gtt genes showed twice as much cadmium absorption than the control strain. we proposed that the formation of the cadmium-glutathione complex is dependent on those transferases, since it was previously demonstrated that the cytoplasmic levels of this complex affect cadmium uptake. the addition of glutathione monoethyl ester (gme), a drug that mimics glutathione (gsh), to gtt ∆ cells restored the levels of metal absorption to those of the control strain. however, with respect to gtt ∆ cells, addition of gme did not alter the capacity of removing cadmium from the medium. taken together, these results suggest that gtt p and gtt p play different roles in the mechanism of cadmium detoxification. by analyzing the toxic effects of this metal, we verified that gtt ∆ and gsh ∆ cells showed, respectively higher and lower tolerance to cadmium stress than control cells, suggesting that although gsh plays a relevant role in cell protection, formation of the gsh-cd + conjugate is deleterious to the mechanism of defense. furthermore, analyzing the harmful effects of other xenobiotic, menadione ( -methyl- , -napthoquinone), we have also observed that gtt p and gtt p isoforms play distinct functions in the process of cell protection as well as in drug remove, since both strains showed lethal phenotypes after direct exposure to mm menadione. however, after adaptive treatments (mild-heat or exposure to a lower menadione concentration), cells acquired tolerance to menadione stress, although the gtt ∆ mutant had still shown a higher sensitivity against drug toxicity. by analyzing the malondialdehyde (mda) produced in response to menadione, we observed that gtt ∆ cells exhibited increased levels of lipid peroxidation, indicating that, during menadione exposure, gsh-conjugates are formed by the same transferase isoform, gtt p, involved in cadmium stress. financial support: stint (sweden), cnpq and faperj (brazil). polycyclic aromatic hydrocarbons (pahs) are ubiquitous and persistent throughout the environment. they are generally distributed from both natural and industrial sources. many pahs can have a detrimental effect on the flora and fauna of affected habitats through uptake and accumulation in food chains, and in some instances, they induce serious health problems and/or genetic defects in humans. many research efforts have been expended to find a suitable method for remediation of soil and water environments contaminated with pahs. amongst them, the use of ligninolytic fungi is particularly suitable for the development of such processes, since they produce extracellular lignin-degrading enzymes (mnp, lip, laccase, . . .) which degrade a wide range of organic pollutants. coriolopsis rigida has been reported to produce extracellular laccase as the sole ligninolytic enzyme. this makes this fungus particularly suitable for the study of xenobiotics degradation by laccase. the purpose of this research was to obtain high laccase activities by c. rigida in solid state cultures and to determine their ability to degrade anthracene (typical pah). both in vivo and in vitro assays were performed. the former led to - % degradation in days depending on the culture conditions, whereas the latter showed a degradation percentage above % in days when low mediator concentration (hbt) was added to the reaction mixture. focus will be given on pressure-driven membrane bioreactors, gastransfer membrane bioreactors and the novel ion exchange membrane bioreactor (iemb). the latter concept has been developed and currently studied by our group. this process, based on integration of donnan dialysis with bioconversion of one or more target pollutants to harmless products, has been modeled and experimentally verified for the removal of various charged inorganic pollutants such as nitrate, perchlorate and bromate by mixed microbial cultures under anoxic conditions. tests of up to months showed a very good operational stability. the essential role of the microbial membraneattached biofilm, which develops naturally in this type of systems, will be also demonstrated and discussed. poly(lactic acid) (pla), which is one of biodegradable plastics, is depolymerized by hydrolysis and releases soluble monomer or oligomer of lactic acid. many bacteria can use the monomer and oligomer as an energy source or a carbon source. in this study, we applied pla to an electron donor for denitrification process of the previously developed bioreactor, which could remove ammonia from wastewater by simultaneously carrying out two biological processes, aerobic nitrification and anaerobic denitrification. a bench-scale bioreactor was constructed with a gel-plate containing pure-cultured cells of nitrosomonas europaea and paracoccus denitrificans and a pla-plate. the pla-plate was prepared by mixing three kinds of plas with different molecular weight and tricalcium phosphate to keep the constant release of the electron donor for a long term. batch treatment experiment with the bioreactor was repeated with an artificial wastewater containing ammonia for days. the bioreactor could remove nitrogen from the artificial wastewater at nitrogenremoval rate of approximately g n/day per square meter of gel-plate surface during the experiment period without an additional electron donor. the performance was equivalent to that obtained with our bioreactor using ethanol as electron donor for denitrification. the bioreactor using pla dose not need an additional pump for serving an electron donor (e.g., ethanol) and a hollow space for serving. therefore, the concept using solid electron donors like a pla would be effective our bioreactor to compact and simplify, and would be possible to develop a portable or disposable bioreactor. leucosporidium antarcticum as a source of enzymes for biotechnology arkadiusz wojtasik , , marianna turkiewicz , jaroslaw dziadek , pawel parniewski : centre for medical biology pas, lodowa street, - lodz, poland; faculty of biotechnology and food sciences technical university of lodz, stefanowskiego / street, - lodz, poland. e-mail: awojtasik@cbm.pan.pl (a. wojtasik) leucosporidium antarcticum is a psychrophilic yeast able to growth at low temperature. these microorganisms live in antarctic marine waters and are endemic to that cold environment. furthermore, l. antarcticum is also isolated from the digestive tract of antarctic krill euphausia superba. enzymes isolated from coldadapted microorganisms such as l. antarcticum having a specific activity at low temperatures ranging from to • c are considered for utilization at biotechnological applications such as bioremediation, production of polyunsaturated fatty acids of dietary significance and might be a source of industrially useful enzymatic proteins. the main goal of this study was to construct a cdna library of l. antarcticum. the partial cdna library was obtained and some of the clones were analysed. the sequencing analyses allowed us to find an approximately base pair nucleotide sequence which displayed a very high homology to disulfide bond chaperone belonging to the hsp family from psychrobacter sp. high similarity of that heat shock protein was found on an amino acid sequence level and was reaching nearly %. the main object of our further research is to clone hsp family protein gene and to obtain its expression in a mezophilic host strain. also, further clones will be analysed to find other interesting genes encoding the psychrophilic proteins. this work was partially funded by the kbn grant i / / . out of plant species found in the flora of turkey, about are endemic. beautiful flowering (geophytes) bulbous plants form an important part of this rich biodiversity. besides use as ornamental plants, these have great potential in perfume and pharmaceutical industry. genera of fritillaria, ornithogalum, muscari, bellevalia, tulipa, galanthus, sternbergia, crocus, arum and biarum have important and critically endangered species with high export potential that enters into this group. most of these are endangered and their collection from wild and export has been banned to conserve them. large scale production and conservation of these species could also be achieved by in vitro techniques. therefore bulb scale and immature embryo explants of sternbergia candida, s. fischeriana, muscari muscarimi, fritillaria imperialis and f. persica were cultured on different nutrient media supplemented with various concentrations of plant growth regulators using different culture applications. large numbers of bulblets were produced (over bulblets/explants) from single immature embryos on nutrient media in most species tested after months of culture initiation. regenerated bulblets were kept at • c for weeks and then transplanted to soil successfully. to our knowledge the present study is the first report for in vitro bulblet production from immature embryos of geophytes. the procedure described here provides a prolific bulblet production system that may form the basis of bioreactor culture and conservation of endemic and endangered geophytes. the commercial use of organofluorine compounds in industrial, pharmaceutical and pest-control applications has dramatically increased over the past few years, resulting in the introduction of numerous new organic compounds into the environment. organofluorine compounds are chemically very stable and are assumed to be resistant to biological degradation. given the chemical inertness of fluorinated organics, their bioactivity, and their potential for accumulation in the environment, it is important to understand their environmental fate and the mechanisms by which they might be degraded. examples of the biodegradation of fluorinated compounds in literature are scarce, being fluorobenzoic acids the most commonly reported. information on the cleavage of carbon-fluoride bonds in synthetic compounds is limited to fluoroacetate dehydrogenase. in this project we try to obtain more insight in the defluorination mechanisms by investigating the diversity of degradation routes for these compounds in several soil bacteria by making use of modern genetic tools. a gram-positive strain capable of aerobic biodegradation of -fluorophenol ( -fp) as the sole source of carbon and energy was isolated by selective enrichment from soil samples collected near an industrial site. batch cultures were set up and substrate consumption, accumulation of intermediates and product formation were monitored. the consortium was able to use -fp up to concentrations of . mg l − and was able to utilize a range of other organic compounds. stoichiometric release of fluoride ions was measured in batch cultures suggesting that there is no formation of dead-end products during -fp metabolism. biobleaching of kraft cellulose pulp by poliporus versicolor aysun ergene , nazif kolankaya : kırıkkale university, faculty of science and literature, department of biology, yahsihan, kırıkkale, turkey; hacettepe university, faculty of science, department of biology, beytepe, ankara, turkey the suitability of culture supernatant from poliporus versicolor for use in the biobleaching of kraft cellulose pulp was investigated. p. versicolor was found to grow on mycological broth ( % soytone, % d-glucose and . % cellulose pulp). maximal extracellular ligninase production was detected after days ( nkat). the optimum biobleaching conditions are • c and ph . , with days. in this condition p. versicolor decreased the kapa number from . to . and increased brightness from to . in -day treatment. boron and iron are among the microelements required for the proper development of the vegetative and generative tissues of plants. though iron is present in high amounts in almost all soil types, its bioavailability to crops is extremely reduced, hence most of the plants face an iron defficiency problem and while on one side crop productions effected, on the other hand the nutrition problems come up to human through contagious nutritional chains. boron is also among the most problematic micronutrients of the major crop plan-tation areas of turkey. both defficiency and toxicity problems exist in a total of about % of the central anatolian soil where pea is among the legumes cultivated. application of varying levels of boron and iron combinations in greenhouse and the analysis of plant acquisition via icp-aes as well as determination of the effects of the element combinations both in morphological and molecular levels are the aim of our studies. the genetic bases of the response differences of plant genotypes to b and/or fe, were investigated through the applications of molecular marker techniques. considerable growth rate and stem size differences were detected within the parents (wild-type versus cultivar) and the f plants. the presence of efficient genotypes to high micronutrient levels are expected to help us increase the cultivation of the crop in problematic areas as well as in exploring the molecular bases of the microelement uptake mechanisms. butachlor is one of the selective systemic herbicide toxins that are act by inhibition of protein synthesis. this toxin is used exclusively in the rice, barely, cotton and wheat farmlands. butachlor is belonging to chloroacetanilide herbicide group, which are consisting of butachlor, alachlor, acetochlor, metolachlor and poropachlor. in the view of bioenvironmental, butachlor is degraded in the soil by microbial activity. its stability is about - weeks. it is converted to the water-soluble derivatives in soil or water, with a slow evolution of carbon dioxide. because of butachlor is one of the herbicide toxin, it is inhibitor factor against growth of bacteria and microorganisms. microorganisms can be continuing their activities in the limited concentration of butachlor. therefore treatment of industrial wastewater consist of concentrated butachlor by the biological treatment is impossible and it is necessary chemical or physico-chemical treatment are used. in this research, biological treatment methods are used. in the biological treatment, an activated sludge system with volume of . l is used. in this method, butachlor with concentration of mg/l are treated. removal percent of butachlor for concentration of . and mg/l are calculated to . % and . %, respectively. removal percent of cod is also calculated to %. olive oil mill wastewater (omw) as the effluent of the concern of olive industry has high organic load. the conventional biological treatments despite of their simplicity and rather suitable performance are ineffective for the omw treatment since phenolics possess antimicrobial activity. in order to carry out a proper treatment on omw, use of microorganism able to degrade the phenolics thus, is necessary. the ability of phanerochaete chrysosporium immobilized purification and downstream process of xylitol obtained biotechnologically from hemicellulosic hydrolyzate of corncobs b. rivas , p. torre , j.m. domínguez , j.c. parajó , a. converti : department of chemical engineering, vigo university (campus of ourense), polytechnic building, as lagoas, ourense, spain; department of chemical and process engineering, genoa university, via opera pia , genoa, italy. e-mail: brivas@uvigo.es (b. rivas) biotechnological production of xylitol from lignocellulosic materials has been widely studied in the recent years with promising results that confirming the possible industrial application of this technology. xylitol purification from fermented broth is the limiting stage of this process. previous works suggest crystallization procedures in order to recovery xylitol from fermented synthetic solutions. the complexity of fermented hydrolyzate not allows direct crystallization. in this work, corncobs hydrolyzate obtained with autohydrolysis-posthydrolysis techniques, detoxificated with activated charcoal and concentrated was fermented to xylitol by d. hansenii. the fermented broth composition was . % of xylitol ( g/l), . % of other sugars and . % of other compounds that interferes in the crystallization process (as dry matter of the liquor). the fermented media was submitted to an absorption process with activated charcoal and concentrated until a xylitol concentration of g/l. the liquor was then submitted to a second step of precipitation with ethanol, the best results achieved in this study were obtained with an ethanol/liquor ratio of . in these conditions this treatment allows to remove a . % of the impurities. the resulting solution was evaporated and crystallized containing % of ethanol and a xylitol concentration of g/l. crystallization was performed at t = • c with slightly agitation. after h were separated xylitol crystals with a recovery yield of % and a purity degree of %. numerous publications have documented that only a minor number of the indigenous prokaryotic organisms found in complex environments such as the human intestine, biogas reactors, and soil are known, and probably only a fraction of this diversity can be accessed using traditional culturing techniques. some of the reasons for this are the lack of knowledge of specific growth conditions, specific nutrients, and obligate coculture requirements. also growth on a solid surface directly exposed to the atmosphere puts a very strong selective pressure on single cells supposed to develop into visible colonies. therefore, the knowledge of these microorganisms is scarce and generally limited to the s rrna genes that have been extracted from different environments and cloned for phylogenetic analyses. an obvious approach to circumvent these problems was the development of techniques based upon micromanipulation for isolation of single cells from complex mixtures. continuous development of modern microscopes in combination with the precision of a servo-powered micromanipulator and the development of the modern microscopic micro injectors used in ivf techniques has further aided the manipulation of single cells. this technique, however, does not solve the problems of the non-culturable cells, and other approaches are needed to gain more information about these organisms. an approach to the non-culturable cells could be genomic analysis of isolated single cells without preceding cultivation. this pcr-based technique is widely used for genetic analysis of human cells, but due to the small amounts of dna present in prokaryotic cells it has so far not been possible to produce identifiable amounts of dna from single cell amplification using conventional polymerases. a promising alternative used for amplification of small amounts of dna is the f dna polymerase operating under isothermic conditions. applying random hexamer primers, this polymerase carries out a multiple displacement amplification (mda) of high molecular weight dna template. in this study we demonstrate the successful application of mda for selective amplification of genomic dna from a single prokaryotic cell. the yield was > mg of amplified genomic dna corresponding to about a billion-fold amplification from a single cell. the technique was used to approach a large group of non-thermophilic archaea found in agricultural soil. our results show that combining mda with fluorescent in situ hybridization and cell isolation by capillary micromanipulation enables an unprecedented ability to investigate new species without cultivation. also this combination of techniques opens for studies of genetic heterogeneity within populations and processes such as horizontal gene transfer. precipitation of zn + , cu + and pb + at bench scale using biogenic hydrogen sulphide produced from the utilization of volatile fatty acids by sulphate reducing bacteria maria teresa alvarez , , carla crespo , bo mattiasson : department of biotechnology, center for chemistry and chemical engineering, lund university, p.o. box , s- lund, sweden; instituto de investigaciones fármaco bioquímicas, universidad mayor de san andrés, la paz, bolivia biological production of hydrogen sulphide (h s) from sulphate using sulphate reducing bacteria (srb) is popular within environmental biotechnology. srb require absence of oxygen, presence of nutrients required for growth and oxidizable organic substrates (to supply hydrogen atoms for reduction of sulphate). many organic wastes have been used as electron donors for the sulphate-reducers in the treatment of acid mine drainage (amd) including straw, hay, sawdust, peat, spent mushroom compost and whey, however, other wastes such as municipal organic waste can be used. the aim of this work was to study the possibility of using srb for the treatment of amd at bench-scale. this process involved three stages: the volatile fatty acid (vfa) production by hydrolytic bacteria from the degradation of vegetables and fruits, the production of h s through the utilization of the produced vfas by sulphate reducing bacteria and the precipitation of metals by using the biologically produced h s. the substrates used for vfa production consisted of tomato, papaya, apple and banana. the h s produced from the degradation of vfas was utilised for the precipitation of an artificial effluent simulating the heavy metal concentrations of a mine located at bolivian andean region, containing approximately mg/l of zn + , mg/l of cu + and mg/l of pb + . the maximum concentration of hydrogen sulphide obtained was approximately mm. removal efficiencies of %, % and % for zinc, cooper, and lead, respectively, were achieved in the present work. at • c during days. bacterial population was determined by counting in a neubauer chamber with optical microscope. sulfate concentration was measured by turbidity method and metal concentrations in the filtered supernatant were measured by icp-aes. the first part of study consists of determine the maximum concentration of each metal at which d. vulgaris and desulfovibrio sp. grow in similar way than control culture (without metal). both cultures tolerate: cr(iii) ppm, ni(ii) . ppm, zn(ii) ppm. the maximum precipitation percentages were approximately: % ( ppm cr(iii)), % ( . ppm ni(ii)) and % (for d. vulgaris- ppm zn(ii) and desulfovibrio sp.- ppm de zn(ii)). time to reach the highest precipitation was minor for mixed culture (desulfovibrio sp.) y all the cases. the next part was focused to study the precipitation percentage when metals are present in combination in the same metal levels (cr(iii)-ni(ii), cr(iii)-zn(ii), ni(ii)-zn(ii) and cr(iii)-ni(ii)-zn(ii)). the combination of metals does not affect significantly the bacterial growth and precipitation percentage of metals. this fact supposes an importance advantage so metals are commonly found together in the environment. future experiments are focused in development of this process in continuous operation mode. biosolubilisation and depolymerisation of coal has potential to produce a clean energy source or high value organic products from low rank coals such as lignite or sub-bituminous coal. these complex soluble phenolic compounds are of value as starting materials for biotransformation to value-added compounds such as antioxidants and flavourants. the bioprocess is carried out at ambient temperature and pressure and is perceived to be environmental benign. in the evaluation of coal solubilisation an important quantity for the assessment of process feasibility is the yield, i.e. the determination of the mass of product obtained per unit mass of coal solubilised. to date, results for coal biosolubilisation reported in the literature are qualitative or at best semi-quantitative, indicating trends with operating variables. the process kinetics has not been determined rigorously because measurement of fungal growth during coal solubilisation is hindered by the presence of the solid coal substrate. knowledge of the profile of biomass growth is required for the rigorous determination of the kinetic parameters necessary for process design and optimisation. in this paper, the use of an indirect method for the estimation of the growth and metabolism of fungal biomass by measuring co evolution and o consumption using an off-gas analyser is reported in the study of fungal coal solubilisation. coal determined rigorously because measurement of fungal growth during coal solubilisation is hindered by the presence of the solid coal substrate. knowledge of the profile of biomass growth is required for the rigorous determination of the kinetic parameters necessary for process design and optimisation. biosolubilisation was carried out in a stirred tank slurry bioreactor with working volume of . l. complete suspension of the coal particles of - m mean diameter was achieved at an agitation rate of rpm. growth yield coefficients based on coal and oxygen as well as maintenance coefficients were calculated from growth of the fungus under the same conditions using a non-coal carbon source such as glucose. these data were used to determine the stoichiometric coefficients for biomass growth, enabling the biomass production rate to be quantified in terms of co production rate and o consumption rate. a dna-chip platform for parallel detection of microorganisms related to biofilm in industrial systems and drinking water systems pernille skouboe, dorte lauritsen, kim holmstrøm bioneer a/s, kogle allé , dk- hørsholm, denmark. e-mail: psk@bioneer.dk (p. skouboe) an oligonucleotide microarray for simultaneous detection and identification of pathogenic bacteria related to technical water systems as well as drinking water has been developed. the approach is based on the use of a tandem hybridization technique with two ribosomal s rdna-pcr products, bp and bp long, generated from two consensus pcr reactions using conserved ribosomal primers end-labeled with cy and cy , respectively. the tandem hybridization technique implies an internally quality control for discrimination between target and non-target signals. the current prototype of the dna-chip platform includes oligonucleotide probes representing different genera (and subgroups of species), e.g. legionella, mycobacterium, aeromonas, campylobacter, vibrio and enterococcus. the platform has been used for detection and identification of species from pure cultures, and initial experiments with water samples from industrial systems have been performed. the potential as well as the limitations of using a dna-chip based detection format in its present form will be documented. particularly, its potential application as a rapid method for initial screening of environmental or food samples will be addresses. the aim is to reduce and optimize the number of samples required for traditional microbiological identification tests. in the course of a project for the development of a novel kind of a mycotoxin inactivating feed additive, the aim of this study was to isolate and characterize microorganisms with the specific ability to enzymatically break down and detoxify fumonisins, a group of structurally related fungal toxins, with fumonisin b (fb ) being the most abundant and -with respect to toxicology -also the most important representative of this group. these toxins are produced as secondary metabolites by some fusarium species such as fusarium verticillioides and f. proliferatum and are naturally occurring contaminants of cereal grains worldwide. they are found especially in maize and maize based products, and are known to be hazardous to human as well as to animal health. a natural feed additive, based on microorganisms and/or enzymes, should ensure the detoxification of fumonisins during feed uptake and digestion via microbial or enzymatic break down of these compounds, by that protecting the animal from the harmful effects of these mycotoxins. besides an extensive screening of microbial strains derived from strain collections, various different natural habitats were investigated for the presence of fb degrading microbial activity, such as intestinal contents of pigs, soil samples, and naturally fumonisin contaminated maize. while testing of nearly organisms from strain collections did not show positive results, fumonisin transforming activity could be detected in one soil sample and a number of maize samples. trials in order to isolate the respective fumonisin degrading microorganisms resulted in a number of strains, whose fb degrading activity could be proven. the most promising bacterial and yeast strains were further characterized with regard to a general taxonomic description, and to different aspects of their toxin degradation behaviour. approaching a more relevant in vivo situation, fb degradation trials in food-and feed-stuffs were conducted. further on, the applicability of the respective organisms as stabilized lyophilisates was investigated. arsenic is one of the most important global environmental pollutants and the toxicological effects are related to its chemical form and oxidation state. arsenite [as(iii)] is reported to be on average times more toxic than arsenate[as(iv)]. this work shows the ability of one strain of the species ochrobactrum tritici to grow in presence of several metals including arsenite, arsenate, selenite, selenate, tellurite and antimonite. its arsenite mic was determined as mm, whereas for arsenate, this bacterium could resist to concentrations upper than mm. we report the identification of two loci involved in high-level arsenic resistance. sequencing of the first locus identified four complete genes in the following order: arsr, arsd, arsa, arsb. the second locus containing genes for arsenic resistance was also characterized. each sequence has been compared with nucleotide and protein databank (blast programs) and significant homology with known orfs coding for arsenic resistance has been found. it is also possible that the phenomenon of high-level arsenic resistance in o. tritici could evolve other genes or loci. the ability of the ␣-proteobacterium o. tritici to tolerate high levels of arsenic in addition to other oxyanions has considerable potential for detoxification and bioremediation of contaminated environments. this work is based on the mathematical modeling of kinetics of a thermophilic bacteria cultivation system. the cultivation proceeded by way of batch and continuous on the synthetic medium with the main carbon source-lactose. this medium simulated an industrial waste approximately. mixed thermophilic aerobic bacteria popula-tion, applied to the wastewater treatment (sludge v&k bystrice pod hostynem), was used to the inoculation. the cultivation system consisted of the laboratory fermentor biostat b (b. braun biotech) with working volume l and the control unit connected with a computer. it is possible the temperature, ph, aeration, stirring and foaming regulation. physical and chemical cultivation conditions were optimized. the chemical oxygen demand (cod), generally expressive the impurity level, was selected as the main parameter for the cultivations run classification. but into the mathematical model also the kinetics of biomass growth, lactose consumption, production of choice metabolites (acetate, lactate, succinate) and dissolved oxygen concentration was included. the modeling was located to two head distinguishable growth phases of the microorganisms. an optimization and identification of mathematical model parameters was practised in the software language psi/c. the difference between simulated curves and experimental data is not statistically significant on the relevancy level . (f-test). it took place the cod degradation at . % with the average yield coefficient y chsk/x . g cod/g biomass in the batch process with the air aeration only. there is a better way to the cod elimination (> . %)-the aeration of air enriched by the pure oxygen. in experiments with the continuous system was obtained the . % cod decrease after the steady state stabilization. this work was supported by project msm . fluorescence in situ hybridization (fish) of whole cells using oligonucleotide probes was applied to study the influence of low temperature and temperature reduction on the bacterial community of biofilm reactors for the removal of chlorophenols (cps). two packed bed reactors were set up for degradation of a mixture of -cp, -cp, , -dicp, and , , -tricp as sole source of carbon and energy at • c (ra) and • c (rb) and were inoculated with bacterial consortia adapted to these respective initial temperatures. the performance of the reactors was studied under different conditions of pollutant loading, aeration rate, and hydraulic retention times over months. total chlorophenol removal capacities of and mg l − day − were achieved in the bioreactors ra and rb, respectively, under a total pollutant load of mg l − day − . the population of ␤-proteobacteria was the major bacterial community of the biofilm ( - %) followed by the ␥-proteobacteria ( - . %). two bacteria with the ability to mineralize mg chlorophenols l − were isolated from the bioreactors and characterized as ralstonia basilensis and alcaligenes sp., both belonging to ␤-proteobacteria. decreasing the temperature by • c (in two steps of • c each) resulted in an increase in the population of ␥-proteobacteria and a decrease in the population of ␤-proteobacteria in both reactors. application of genus specific probes showed an increase in the pseudomonas population from % of the ␥-proteobacteria at • c to % at • c. the pollutant removal capacity decreased to and mg l − day − in ra ( • c) and rb ( • c), respectively. the ␣and ␦-proteobacteria, cytophaga-flavobacteria and actinobacteria the survey was carried out in urban and rural areas of two cities (ankara and isparta). this paper is only analysing urban people by excluding villagers. urban sample is consisted of urban consumers and professionals. professionals were selected amongst pharmacists, doctors, agricultural engineering's and industrialists that is thought are affective in the process of developing new technologies and also the development of biotechnology in the society. basic data was gathered by a questionnaire including both structured and open-ended questions besides deep interviewed. workforce development for life sciences-the scottish experience carol booth scottish entreprise, uk the presentation will look at, the background and definition of workforce development for scottish enterprise, examine information available to support scottish enterprises economic intervention and where and when to intervene. conclusions emerging from the evidence base will be used to outline scottish enterprises approach to workforce development and look at which actions might be required to address identified issues. moving on to reasons for integrating workforce development into business development and how the life sciences cluster team at scottish enterprise, stakeholders and partners in scotland have approached their current and future contributions to workforce development for life sciences using a variety of projects. the national institute for bioprocessing research and training (nibrt) in ireland is a proposal that will be a state-of-the-art training, research and pilot plant service facility that brings together institutions with complementary expertise and state-of-the-art research technology, and industry partners. these include university college dublin, trinity college dublin, institute of technology, sligo and dublin city university. nibrt is an innovative collaboration between academic institutions at the forefront of biotechnology, cell biology, engineering and pharmacy and industry. for training, two separate training labs, for upstream and downstream training, in addition to research labs are planted. it will also include a state-of-the-art pilot plant fermentation facility for fermentation optimisation, fermentation scale-up, product separation and purification, regulatory aspects and automation. by aligning with industrial demands, the new institute will tailor its training programmes while remaining on the cutting edge of biotechnology research and technologies. the fermentation facility will offer hands-on training workshops and educational modules for outside researchers and companies. these workshops cover the fundamentals of small-scale fermenta-tion, scale-up considerations, and fermentor design and set-up. the training and educational philosophy underpinning the nibrt will focus on the needs of industry with an emphasis on providing training for accreditation of existing industry staff and prepare technicians and graduates for the technical, business, regulatory and professional aspects of the industry. the strategy is to provide specialised modules in nibrt in support of courses established in the higher education institutions, which will provide the certificates, diplomas and degrees. modules will be offered for all categories of students and will be given credits respected by other third level institutions in ireland. the role of professional graduate degrees in meeting current and future biotechnology industry workforce needs a. stephen dahms san diego state university, usa the presentation will review the status of new graduate training models designed to meet the unique needs of the biotechnology industry as it transitions to commercialization. emphasis will be on professional master's degree programs in biotechnology and their various versions, with a focus on operational and funding strategies and industry acceptance. discussion will also centre upon the creation and operation of industry-validated, specialized and highly targeted professional masters degrees in various refined aspects of the drug development process, including regulatory affairs, biomedical quality systems, clinical affairs, management of drug development, management of reimbursement affairs, bioinformatics, etc. the eurodoctorate in biotechnology, new combined mba/phd combined degrees in the molecular life sciences, the u.s. professional doctorate in chemistry and the proposed u.s. professional doctorate in biotechnology will be also discussed. data will also be presented on the current workforce and the industry's projected needs. genetic studies show that mankind is a rapidly expanded population of closely related individuals with very similar disease sensitivity. bad nutrition and infections dominate among the main health problems in the world. apart from malnutrition, the overeating habits of the developed world are now creating problems in the developing world as well. infectious diseases are also a global problem since new contagious agents like hiv, sars and avian flew do not recognize borders. thus, the global responsibilities of modern health care are obvious. many research scientists from and in developing countries find it nearly impossible to use their talents for the benefit of their own countries. some struggle to develop research and education programmes with poor facilities, some leave science completely, and others migrate to more developed countries. the talents of such people are either being wasted or lost completely to their home countries just at the time they are most needed to combat the great humanitarian challenges of hunger, illness and lack of knowledge. europe must strengthen programmes which allow third world scientists to work to their full potential in their home countries or regions. yang beijing genomics institute, chine academy of sciences, beijing, china europe has all the reasons to be proud of being the cradle of modern science and of its achievements and resources in life sciences. as a model of having solved many of the problems that many other counties are now facing, europe is expected by the whole world to make its further contribution to a better future of mankind and to play a more important role in the international community of life sciences. tropical diseases and public health basilio valladares director of the university institute of tropical diseases and, public health, university of la laguna, la laguna, tenerife, spain. e-mail: bvallada@ull.es the presentation will look at the main research interests of the university institute of tropical diseases. these are the following: . immunology and molecular biology of parasites. we express and purify recombinant proteins from leishmania sp. which have been shown to act as immunomodulators and protect against disease such as l , hsp , hsp . the study of acanthamoeba pathogenic factors has also resulted in the isolation and silencing of extracellular proteins related to their pathogenecity, which has a great potential in the development of novel chemotherapeutics. . diagnosis of parasites. the immunological diagnosis of leishmaniasis has been one of the main research interests in our laboratory for several years. as a result, we have identified peptides which could be used to develop kits for the immunological diagnosis of leishmaniasis such as hsp c-end, l n-end, etc. we have also developed some dna based methods for the identification of acanthamoeba species from biological and environmental sources. . water quality. biological parameters. our water research group has the expertise to identify and characterise bacterial, viral and parasitic indicators of faecal contamination in diverse water sources including tap water, rivers, reservoirs, sea, etc. this research area has been developed in collaboration with the local sewage treatment plant and reservoir managing authorities. currently, we are establishing a conjoined project with the center for disease control and prevention (cdc) in atlanta, usa for the identification of water-borne emerging pathogens. . development and formulation of chemotherapeutic antiparasitic agents. in this field, we evaluate the leishmanicidal activity both in vivo and in vitro of natural and synthetic drugs and synthetic peptides. in a later stage, the drugs which have shown the highest antiparasitic activity have been subjected to cytotoxicity assays and their molecular targets dissected. some of the drugs tested in the last few years have been submitted to patent due to their outstanding activity. finally, in order to allow the commercialization of these drugs, both in vivo and in vitro assays are being carried out to predict their chemical stability and degradation pathways. this will be followed by the use of liofilization and controlled crystallisation strategies for the development of efficient and safe treatments. . human and population genetics. tachykinins and their receptors in different tissues and groups of patients and their association with molecular polymorphisms is another one of our research interests. the knowledge of ligand and receptor sequences and their similarities will allow the rational development of drugs with specific activity against these receptors. furthermore, we are also interested in the interspecific variation along the evolutionary scale of these markers. . nitrate assimilation group. research in our group is focused on understanding nitrate assimilation in the yeast hansenula polymorpha. several biotechnological companies use this yeast to produce heterologous proteins (hepatitis b vaccine). genetic manipulation techniques for h. polymorpha are available in our laboratory. head of the department of biotechnology, technological institute of canary islands, pozo izquierdo santa lucía, las palmas, spain single cell analysis by flow cytometry has proved to be a tool to perform simultaneous and rapid measurements related to cell morphology and physiological state. previous studies showed the possibility of quantifying neutral and polar lipids spectrofluorometrically using a lipid specific fluorescent dye, nile red (nr), however the existence of inter and intraspecific variations in the fluorescent response had not been clearly established. in this work, two strains of marine microalgae: crypthecodinium cohnii and tetraselmis suecica, characterized both by high contents of polyunsaturated long chain fatty acids (dha and epa, respectively) and an hypersaline microalgae: dunaliella salina, characterized by a high ␤-carotene production, were grown under different conditions and collected at different growth phases to be used for in vivo lipid quantification with nr by flow cytometry. our results showed a high correlation between the mean fluorescence signal of nr stained cells and the neutral and polar lipid content measured by gravimetry for each strain. in this respect, these data make feasible the development of a rapid method for lipid quantification in monoalgal cultures. however, differences in the dye uptake related to specificity were detected. in this communication we also assess the possibility of use this cytometric technique to select microalgal strains with high lipid and polyunsaturated long chain fatty acids content from mixed samples. performance of such technique would be a good alternative to the time-consuming traditional screening protocols based on gravimetry and gas chromatography and would optimise the search of new commercial strains of microalgae. claverie-martín head of research unit, biomedical research institute, hospital universitario, de n.s. de candelaria, santa cruz de tenerife, spain our group is involved in the cloning and production of proteins of interest to the food and pharmaceutical industry. we have recently expressed in yeast the cdna that encodes the precursor of caprine chymosin. chymosin is the enzyme responsible for the coagulation of milk in the abomasum of unweaned calves. this enzyme is secreted by gastric mucosa cells as an inactive precursor, known as prochymosin. in the acidic conditions of the lumen, prochymosin is converted into the active form by autocatalytic cleavage of the n-terminal prosequence. chymosin is used extensively in cheese production because it cleaves -casein in a specific manner with low proteolytic activity. several biotechnology companies are producing the bovine recombinant enzyme for commercial use in the process of cheese making. we are interested in the caprine chymosin as an alternative because in the canary islands cheese has traditionally been made using goats milk with extract from the abomasum of newborn goats as coagulating factor. it is well known that the activity of these types of extracts varies depending on the age of the animal and the type of food ingested. these difficulties should be overcome using a recombinant caprine chymosin. the caprine mrna used for the synthesis of the cdna was obtained from the abomasum of milkfed kid goats. the cdna fragment encoding the mature portion of caprine prochymosin was fused in frame to a signal sequence in yeast expression vectors. culture supernatants of yeast cells transformed with the recombinant plasmids showed milk-clotting activity after activation at acid ph. proteolytic activity assayed toward casein fractions indicated that the recombinant caprine chymosin specifically hydrolysed -casein (patent ). the recombinant caprine chymosin could be an alternative milk coagulant in cheese making. work is underway to optimise the expression of the new recombinant prochymosin for further purification and characterization. smart molecules for health victor martín head of research, university institute of bio-organics "antonio gonzález", avda. astrofísico francisco sánchez , la laguna, tenerife, spain the instituto universitario de bio-orgánica "antonio gonzález" (iubo) is a multidisciplinary research centre that belongs to the university of la laguna. the iubo is located at the town of la laguna, inscribed on unesco's world heritage list in , and former capital of the canary island of tenerife. the geographical location in addition to its mild climate has made the canary islands to posses a variety of ecosystems with unique plants and animals. the institute was started up in the s with the need to study the natural products and secondary metabolites produced by those marine and terrestrial organisms, thus providing a new source of bioactive products. the main research lines that are being developed at iubo are summarised in the following paragraphs: anticancer agents from natural sources: several natural products and their semisynthetic derivatives are produced at the iubo in diverse joint projects for the development of new antitumour drugs with novel mechanisms of action. as an example we can mention natural products from the mevalonic, shikimic or polyketide pathways. some products have recently shown in vitro reversion of the resistance in multidrug resistant (mdr) tumour cell lines. genetic engineering: in vitro cultures of the plants atropa baetica, maytenus amazonica and m. macrocarpa are developed in order to manipulate their biosynthetic pathways and induce the production in large scale of the secondary metabolites for diverse applications, including arthritis, rheumatism, and back pain. marine organisms and toxins: dinoflagellates are marine organisms responsible for the red tides and food poisoning episodes. among others, okadaic acid and yessotoxin are the most common toxins present in european shellfish. the isolation of these products is best done from the microorganism cultures, since they are present in very low amounts in the natural sources. at the iubo we develop culture systems to provide us with amounts of toxins large enough to perform biological, metabolic and bioactive studies. insecticide and repellents: natural products are being isolated for their use against plagues, specially those affecting agriculture. these projects are run in collaboration with a number of public institutions and agrochemical companies throughout europe and latin america. fine chemicals and pharmachemicals: our institute possesses large expertise in the field of organic synthesis devoted to the synthesis of medicinal substances, with special focus on asymmetric processes. of particular interest is the development of new methodologies for the total synthesis of biologically active substances like polyether toxins, (un)natural amino acids, sphingosine analogs, alkaloids, etc. with an annual source of s of new compounds, the fine chemicals and medicinal chemistry branch at iubo have recently started and anticancer screening program in collaboration with the biomedical research unit at the hospital universitario n.s. de la candelaria. the program is committed to the discovery of novel drugs for application in cancer treatment. the outcome of this project in its first year has been outstanding, leading to the finding of several leads that form the basis for current and future projects. institute of canary islands, biotechnological department, playa de pozo izquierdo s/n, santa lucía, gran canaria, spain the presentation will look at the main research interests of the biotechnological department of the technological institute of canary islands. these are the following: nutrition and feeding in aquaculture: we conduct studies on digestion, absorption, transport, and utilization of the different nutrients applying also different techniques such as histology, enzymology, genetic or immunology among others. aquaculture feeding is the main important cost in fish farms, being higher that the prize of fries, personnel cost or energetic costs. thus, studies conducted on the improvement of diet formulation and the use of different dietary ingredients is one of our main research lines (such as vegetable oils and meals to be used as alternative to fish meal and oil, or carotenoid sources to improve fish colour). not only the use of the different ingredient is studied, but also the effect of these ingredients on fish health, flesh quality, flesh healthy aspects related with human consumption, are being studied. different formulae are being developed and patents of different diets are being obtained. studies on nutritional requirements are also conducted allowing us to patent different formulae in larvae studies, developing microdiets to substitute the high-cost processes associated with live prey in larval nutrition. development of immunostimulants, anti-stress diets, immune techniques to be applied as bio-indicators of fish health and welfare, as well as use of dietary ingredients derived from bio-reactors are other research lines in our group. genetics: genetic techniques applied to aquaculture are being an important tool. microsatellites are being used to determine genealogy of the fish, allowing to decreases important problems in aquaculture such as fish deformities. genetic techniques such as micro-arrays and gene expression are being applied to obtain indicators of stress and health in fish. these technologies also permit to make different selective breeding programs, increasing the accuracy to estimate genetic parameters and evaluating brood-stock. furthermore, this technology allows to obtain a procedure to increase the productivity and quality of fish hatcheries. new species for aquaculture. development of new culture techniques: the diversification of species cultured is one of the main objectives of european aquaculture, since nowadays only four marine species are commercially produced: gilthead sea bream. european sea bass, turbot and salmon. we have been developed rearing techniques for new species such as red porgy or canarian abalone and also we are conducting studies on different new species, such as different sparids species, octopus or yellowtail. new rearing technology: the election of adequate systems for fish growth for each species and site of production and the localization of more appropriate sites for farms, using gis technology are also of special importance in our research team. besides, new larval rearing techniques such as semi-extensive hatchery (mesocoms) were development and nowadays is being used to increase fry quality (survival, no-deformities, better growth). this technology is being exported to other countries in order to offer new technology easy to manage to be implanted in developing countries. alejandro cañeque project officer, canary islands special zone, ministry of finance, c/leon y castillo - a planta, edificio urbis, las palmas, spain. e-mail: acaneque@zec.org the canary islands special zone is the newest tax instrument within the canary islands economic and fiscal regime (ref). it offers a reduced tax rate of between and % of corporate income tax for companies setting up a business. the companies must cover the following minimum requirements: create employment and make a minimum investment. the zec offers other tax advantages such as the exemption from paying transfer tax and stamp duty and the canary islands general indirect tax (igic). this tax scheme particularly fosters the biotechnology and pharmaceutical sectors. references fahnert references bechor the antimicrobial drugs high-level production of human collagen prolyl -hydroxylase in sandwich hybridisation assay for quantitative detection of yeast rnas in crude cell lysates microbial processes and products press. biotech. bioeng. . van hijum microbial xylitol production from corn cobs using candida magnoliae the role of bacillus methanolicus citrate synthase gene, city, in regulatiing the secretion of glutamate in lysine-secreting mutants plasmid-dependent methylotrophy in thermotolerant bacillus methanolicus , -anhydrod-fructose; a versatile chiral building block: biochemistry and chemistry detailed dissection of a new mechanism for glycoside cleavage: the ␣- , -glucan lyase ␣- , -glucan lyase, a new class of starch and glycogen degrading enzyme ␣- , -glucan lyase, a new starch processing enzyme for production of , -anhydro-d-fructose efficient purification, characterization and partial amino acid sequencing of two ␣- , -glucan lyases from fungi ␣- , -glucan lyases producing , -anhydro-d-fructose from starch and glycogen have sequence similarity to alpha-glucosidases enzymatic description of the anhydrofructose pathway of glycogen degradation i. identification and purification of anhydrofructose dehydratase, ascopyrone tautomerase and ␣- , -glucan lyase in the fungus anthracobia melaloma a systems approach to dissecting immunity and inflammation integrative biological analysis of the apoe* -leiden transgenic mouse innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble cd soluble forms of toll-like receptor (tlr) capable of modulating tlr signaling are present in human plasma and breast milk proteomics of the rat gut: analysis of the myenteric plexuslongitudinal muscle preparation an integrative metabolism approach identified stearoyl-coa desaturase as a target for an arachidonate-enriched diet the challenges of modeling mammalian biocomplexity rapid enrichment of bioactive milk proteins and iterative, consolidated protein identification by mudpit technology post-genomics of lactic acid and other food-grade bacteria to discover gut functionality genetics, metabolism and application of lactic acid bacteria. fems microbiol complete genome sequence of lactobacillus plantarum wcfs functional ingredient production: application of global and metabolic models metabolic engineering of lactic acid bacteria for the production of nutraceuticals reduction in antinutritional and toxic components in plant foods by fermentation the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no k - ). the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no. k - ) and the microorganisms given to this work by prof. dr. francesco molinari at university of milano, and prof. dr. leyla aÇ ikel at gazi university. the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no k - ) and the microorganisms given to this work by prof. dr. francesco molinari at university of milano, and prof. dr. leyla aÇ ikel at gazi university. mrs. lynnette fernandez, johanna mäkeläinen, m.sc. and olli rämö, m.sc. are gratefully acknowledged for their help. this work was supported by the health science council, the academy of finland and the tekes-neobio program. supported by the mec of spain (ppq - -c - ) and the canary islands government. jmp thanks icic for a postdoctoral fellowship. frpc thanks cajacanarias for a fpi fellowship. tm thanks the spanish mcyt-fse for a ramón y cajal contract. this work was supported by the korean systems biology research grant from the ministry of science and technology, lg chem chair professorship, ibm sur program and bk program. this study was supported by ankara university biotechnology institute (project no: ). this work was partially supported by mec and feder (bio - ), and fundación séneca carm ( /pi/ ). this work was supported by grants f of the hungarian scientific research fund and grant omfb- / of the hungarian ministry of education. keto sugars have long been implicated as attractive intermediates or substrates for further chemical or enzymatic reactions, to generate a number of synthetic sugar derivatives and fine chemicals. the quinone-dependent pyranose dehydrogenase (pdh) purified of the basidiomycete fungus agaricus meleagris catalyzes with high specificity the oxidation of the c- of glycosidically bound d-glucose, whereas in contrast it oxidizes simultaneously the c- and c- of free d-glucose. considering the broad substrate tolerance, pdh provides a new convenient tool for high yield production of -keto- this work was partially financed by the scientific and technological research council (cicyt, spain), grant ren - , by the iii pla de recerca de catalunya (generalitat de catalunya), grant sgr- , and by the generalitat de catalunya to the "centre de referència en biotecnologia" (cerba). this work was supported by mega a.s. (czech republic) (www.mega.cz) and following vega grants: / / and / / . this work was partially supported by mec and feder (bio - ), and fundación séneca carm ( /pi/ ). financed by a marie curie re-integration grant merg-ct- - and consejería de educación y ciencia de la junta de andalucía. this project is part of the collaborative research centre sfb "development of biotechnological processes by integrating genetic and engineering methods", which is supported by the german research foundation dfg. this research was supported in part by grants from the hungarian scientific research fund (otka t , f and d ) and the hungarian-spanish intergovernmental s & t cooperation programme (omfb / ). this research was supported in part by grants from the hungarian scientific research fund (otka t , f and d ) and the hungarian-spanish intergovernmental s & t cooperation programme (omfb / ). this research was supported in part by grants from the hungarian scientific research fund (otka t , f , d ) and gvop- . . .- - - . the authors thank dr. hamid narjiss (director of morocco inra) for the instruction to identify nadorcott mandarin by molecular markers and helpful discussions regarding this paper. this work was partially funded by the program for scientific cooperation cnrst (morocco) and iccti (portugal), the international foundation for science (ifs) support in stockholm (sweden). this work has been financed by xunta de galicia (pgidt pxib pr). sevgil sadettin, gönül dönmez department of biology, faculty of science, ankara university beşevler, ankara, turkey this study was supported by ankara university biotechnology institute. demet Ç etin , sedat dönmez , gönül dönmez : department of biology, faculty of science, ankara university, beşevler, ankara, turkey; department of food engineering, faculty of engineering, ankara university, dışkapı, ankara, turkey sulfate-reducing bacteria (srb) that could grow on modified postgate c medium (pc) containing chromium(vi) were isolated from industrial wastewater and their chromium(vi) reduction capacities were investigated as a function of changes in the initial ph values, chromium, sulfate, nacl concentrations and carbon source. the optimum ph value at mg l − initial chromium(vi) concentrations was determined as . chromium(vi) reduction by srb was investigated at . - . mg l − initial chromium(vi) concentrations. at the end of the experiments, the mixed cultures of srb were found to reduce more than % of the initial chromium(vi) levels which ranged from . to . mg l − within - days period. the effects of initial - . g l − concentrations of sulfate and - % (w/v) concentrations of nacl to chromium reduction were showed that, the lowest concentrations of sulfate and nacl, were the best for chromium reduction in the pc media including mg l − chromium(vi). when the % whey was used as carbon source in the pc medium, . % of the . mg l − initial chromium(vi) concentration was reduced within this study was supported by ankara university biotechnology institute. this study was carried out as a part of the project for analyzing and controlling the mechanism of biodegrading and processing entrusted by the new energy and industrial technology development organization (nedo). this work has been financed by xunta de galicia (pgidt pxib pr). lead ions are considered a high pollutant of different waters. in our previous work were selected seven potential sorbent-strains rhodotorula mucilaginosa , rh. aurantiaca , rhodotorula sp. , williopsis californica , candida krusei t, cryptococcus sp. wt of lead ions. it was determined their stability to high concentration (up to mg/l) of lead ions in medium. the ph changes and yeast physiologies of growth were studied in medium with these heavy metal ions. the influences of environmental factors such as ph of solution, age of microbial culture, biosorbent concentration in suspension, alive or living state of biosorbent, time of contact on sorption were investigated. the levels of maximal sorption ability and biomass affinity to heavy metal ions were established by experimentally received sorption isotherms with mathematical modeling of biosorption process separately for everyone researched yeast strain. the sorption isotherms obtained in these experiments for non-living yeast biomass showed that the maximal sorption capacity was and × − mol (g sorbent) − for rh. aurantiaca and s. cerevisiae , respectively. in the case of living biomass, the high- this work has been financed by the spanish ministry of science and technology and european feder (project ctm - ). the authors wish to thank dra. m.j. martínez (cib, csic, madrid, spain) for providing coriolopsis rigida. this work was supported principally by embo and the mrc. fed-batch cultivation of haematococcus pluvialis under illumination with leds for production of astaxanthin abdolmajid lababpour, tomohisa katsuda, shigeo katoh department of molecular science and material engineering, kobe university, kobe, hyogo - , japan photosynthetic microalga haematococcus pluvialis is a most promising microorganism for production of astaxanthin, which has powerful antioxidant activity and is used both for human being as a food supplement and cosmetic; as well as animal farming such as salmon and poultry. the deficiency of nutrients in batch culture of h. pluvialis decreases the growth of cells and increases the induction of astaxanthin as an induction factor. therefore, it is impossible to reach to high cell concentrations in batch cultures. in previous experiments, the medium replacement increased the cell concentration, while accumulation of astaxanthin was not induced without other factors. in this work, the effects of fed-batch addition of culture medium on the cell growth and astaxanthin production in h. pluvialis cultures were studied. h. pluvialis was cultivated in cm culture medium containing sodium acetate, yeast extract, l-asparagines, mgcl · h o, feso · h o and cacl · h o (ph . ). light was supplied by panels of blue or red led lamps. the temperature was kept at • c and the culture was mixed with a magnetic stirrer. in fed-batch cultures of h. pluvialis, the cell concentration and production of astaxanthin increased in comparison with those in batch culture. in addition, the operation in feb-batch manner is easier than medium replacement from industrial viewpoints for production of astaxanthin. simvastatin and similar compounds are wide used as antihypercholesterolemic agents. simvastatin is obtained by c-methylation of side chain of lovastatin. this process is not perfect and some unreacted lovastatin is present in the reaction mixture. simvastatin is separated from lovastatin using fungi clonostachys compactiuscula. using of living microbials has some disadvantages such as high cost, difficult product separation from the reaction mixture. we work on overcome these difficulties by separation and immobilization of enzyme that hydrolyses lovastatin ammonium salt in presence of simvastatin ammonium salt. our results of purification and immobilization lovastatin hydrolase will be presented. investigation of peptide antibiotics produced by trichoderma strains isolated from winter wheat rhizosphere a. szekeres , l. kredics , l. hatvani , z. antal , l. manczinger , a. nagy , c. vágvölgyi : department of microbiology, university of szeged, p.o. box , h- szeged, hungry; hungarian academy of sciences, university of szeged, microbiological research group, hungry; pilze-nagy ltd. , kecskemét, p.o. box , species of the imperfect filamentous fungal genus trichoderma with teleomorphs belonging to the hypocreales order of the ascomycota division are of great economic importance as sources of enzymes, antibiotics, as plant growth promoters, decomposers of xenobiotics, and as commercial biofungicides. peptaibols and related peptaibiotics (prps) are secondary metabolites constituting a family of fungal peptide antibiotics which is constantly growing since alamethicin was isolated from cultures of trichoderma viride. these compounds are linear, amphipathic polypeptides composed of - amino acids and usually containing several non-proteinogenic amino acid residues, which are representing characteristic building blocks of the structure. one hundred and twenty trichoderma strains were isolated from roots of winter wheat grown in agricultural fields of southern hungary. the identity of species was examined based on morphological and molecular characters. the presence of prps-producing strains among the isolated trichoderma strains was detected by biological tests and the antibiotics were partially purified using a multistep chromatography procedure involving exclusion chromatography, adsorption chromatography and thin-layer chromatography. about % of the isolates proved to be able to produce prps. the antibacterial activity of the compounds was tested against staphylococcus aureus, bacillus subtilis, micrococcus luteus and escherichia coli, while the antifungal effect was recorded against fusarium oxysporum, f. culmorum, rhizoctonia solani and pythium debaryanum. ergezinger, m., bohnet, m., berensmeier, s., buchholz, k., . integrierte enzymatische synthese und adsorption von isomaltose in einem mehrphasenbioreaktionsreaktor. cit , - . glucansucrases from family of glycoside-hydrolases are transglucosidases that produce ␣-glucans from sucrose, a very cheap substrate, without any use of nucleotide activated sugars. based on sequence analyses, these enzymes have been classified in two families, the family and the family of glycoside hydrolases. among the natural diversity existing in family in which are found the glucansucrases produced by lactic acid bacteria, three enzymes have been selected for their distinctive specificities: dextransucrase from l. mesenteroides nrrl b- f (dsr-s), which catalyses almost essentially the synthesis of ␣- , -linkages, alternansucrase from l. mesenteroides nrrl b- (asr), which produces alternan polymer formed of ␣- , and ␣- , -alternated linkages and finally dextransucrase from l. mesenteroides nrrl b- (dsr-e), which is responsible for the synthesis of a branched dextran composed of about % of ␣- , -linkages in the main chain and % of ␣- , -branched linkages. for all these enzymes, the natural polymerase activity can be shifted towards oligosaccharide production or gluco-conjugate syntheses by introducing acceptors in the reaction medium. a number of sugar acceptors have been successfully glucosylated with the view of developing new functional food products. acceptor glucosylation yield as well as acceptor reaction product structures were shown to be highly dependant on the enzyme specificity. consequently, using glucansucrases of distinctive specificities and varying the acceptors give access to a large variety of applications. amylosucrase, the sole glucansucrase found in family of glycoside-hydrolases is also of great interest for functional food applications. this enzyme is able to synthesize highly resistant amylose from sucrose. again the reaction conditions can be used to modulate the yield and the size of amylose. the aim of our work is to further develop the applications of these enzymes via rational and combinatorial engineering. the most recent results obtained in this field will be discussed. novel food structure engineering concepts with enzymes johanna buchert vtt biotechnology, espoo, finland food structure is a very important quality attribute in food choice, since it affects not only the sensory perception of texture, but also release of flavour. enzymes offer specific means to engineer food structure by creating cross-links to food biopolymers, i.e. to proteins and/or carbohydrates. enzymatic cross-linking of food biopolymers can be exploited to create novel types of structures to foods without any need of added food ingredients. laccases and peroxidases can be used to crosslink ferulic acid containing carbohydrates, such as sugar beet pectin or arabinoxylan. proteins can be crosslinked by different oxidative or transferase type of enzymes. transglutaminases can crosslink protein via formation of isopeptide bond between glutamine and lysine residues. laccase and peroxidase can oxidize tyrosine residues to corresponding radicals, which in turn can further react with different groups in proteins. tyrosinases, on the other hand, oxidize tyrosine to a quinone, which can further react with aromatic ring, amine and thiol groups present in proteins. the biopolymer networks formed can be further engineered by combining adequate processing to the enzyme treatment. in this work the potential of enzymatic food structure engineering is reviewed. asparaginase-mediated reduction of acrylamide formation in baked, fried, and roasted products hanne vang hendriksen, beate kornbrust, steffen ernst, mary stringer, hans peter heldt-hansen, peter Østergaard novozymes a/s, dk- bagsvaerd, denmark. e-mail: hvhe@novozymes.com (h.v. hendriksen) in , it was discovered that acrylamide is formed in several potato and grain-based foods (e.g. chips, french fries, toasted bread, biscuits, cereals) and in coffee, all of which have been prepared at high temperatures. the level of this potential carcinogen in the final food appears to range from to ppb. later that year, the mechanism of acrylamide formation was unraveled, demonstrating that asparagine and reducing sugars are the precursors for acrylamide. this pointed to several potential enzymatic approaches to remove the root cause of the problem by degrading the precursors in situ. here, we demonstrate that asparaginase treatment leads to a more efficient reduction in acrylamide than alternative enzymatic treatments. asparaginase from aspergillus oryzae is used to reduce acrylamide formation significantly in laboratory models of a range of common food products. examples are french fries, biscuits, crisp bread, and fabricated chips. the sensory qualities appear to be constant. the implications for scaling up the processes for industrial food production are discussed. effect of cultivation conditions on folate content in yeast: exploring the potential of yeast as a bio-enrichment vehicle for folate in foods sofia hjortmo , johan patring , jelena jastrebova , thomas andlid : department of chemical and biological engineering, chalmers university of technology, po box , gothenburg, sweden; department of food science, swedish university of agricultural sciences, po box , uppsala, sweden. e-mail: sh@fsc.chalmers.se (s. hjortmo) over the past years, the interest in health benefits of the b vitamin folate has increased considerably. a good folate status may hinder progression of several diseases such as neural tube defects and downs syndrome in foetus, as well as cancer, dementia, alzheimer's disease and cardiovascular disease in adults. it is however not easy to reach the recommended intake and new strategies have to be developed to increase the folate status. in this project we explore the use folate producing microorganisms for this purpose. many yeasts have the ability to synthesise folate de novo and can thus serve as a source for humans. folate enrichment in fermented foods could be much improved by using starter cultures better at producing folate compared to traditional strains. this is, e.g. applicable to bread making fb over-expression of isoprene biosynthetic enzymes in the ␤-carotene producer zygomycete mucor circinelloides tamás mucor circinelloides has been involved to study the carotene biosynthesis genesis of fungi. this fungus is more amenable to molecular techniques than the others traditionally used in carotenogenic studies (e.g. blakeslea trispora and phycomyces blakesleeanus). moreover, mucor has a great advantage: it is a dimorphic organism. this type of morphology is preferred by the fermentation industry because yeast-like growth allows the submerged culture, when usually higher biomass production can be achieved and cells can be more easily separated from the media. β-carotene is a terpenoid-type chemical compound likewise to sterols, quinones or chlorophylls. the production can be increased by improving the non-carotene specific terpenoid biosynthesis. this can be carried out by the overexpression of the genes responsible for the ratelimiting steps of these pathways. in this study, polyethylene glycol mediated transformations of m. circinelloides protoplasts were performed with autoreplicative expression vectors containing the known terpenoid genes of m. circinelloides (e.g. isoa encoding farnesyl pyrophosphate synthase and carg encoding geranylgeranyl pyrophosphate synthase). carotene production of the transformants and the wild-type strains were analysed by high-performance liquid chromatography (hplc). transformants harbouring plasmids with isoa or carg produce about . times more carotene than the recipient strain, while carotene production increased about two times in the co-transformants containing both type of plasmids. members of the genus rhizopus are important from biotechnological aspects in consequence of their effective extracellular enzyme, alcohol and organic acid production. moreover, rhizopus strains are used for fermentation of various foods, because they are capable of transforming soybeans into edible products. the high affinity iron permease (ftr ) contains both highly conservative and variable regions applicable for phylogenetic comparisons. the aim of this study was the comparative analysis of this gene of different rhizopus species in order to elaborate a simple and fast method to identify these fungi at a species and subspecies level. conserved regions of candida albicans and rhizopus oryzae ftr genes (fu et al., ) have been analysed to design degenerate primers for polymerase chain reaction. they were used to amplify the homologous regions from different strains of r. oryzae, r. microsporus, r. stolonifer and r. niveus. isolates of the similarly thermophilic rhizomucor miehei and r. pusillus, as well as a strain of m. rouxii were involved in the study as outgroups. deduced protein sequences were aligned and phylogenetic analysis was performed. surprisingly the r. oryzae isolates formed a group completely different with a significant distance from the r. microsporus isolate. r. niveus is currently not distinguished from r. stolonifer var. stolonifer because of morphological considerations. however, phylogeny of ftr gene sequences, in agreement with earlier results based on rapd data (vágvölgyi et al., ) , raise the need to handle r. niveus as a separate species. sequences and pcr primers useful for identification of all tested rhizopus strains were elaborated. potential application in a lactose conversion process. the prebiotics market is at high demand therefore the development of the process to produce 'prebiotic' galacto-oligosacharides efficiently and inexpensively is our particular interest. citrus, particularly mandarins and clementines, are among the most economically important fruit crops in morocco. besides morphological traits multiple molecular markers have been used for the caracterisation of citrus germplasm. the main aim of this study was to evaluate the moroccan mandarin germplasm and to identify specific polymorphisms among accessions sharing identical name. eighty mandarin and two sweet orange varieties were analyzed by dna markers. issr markers were amplified using anchored primers and analysed by agarose gel electrophoresis. aflp markers analyses were performed using three primer combinations. the dice coefficient was used to estimate genetic similarities and the upgma algorithm was utilised to generate a phenogram depicting the genetic relationships among the acessions. the selection of primers out of the issr primers primarily assayed, allowed us to maximize the average number of amplified fragments analyzed per reaction ( . ), and the percentage of informative polymorphisms ( %). the three combinations of ecori/msei primers revealed reliable aflp markers, ( %) of witch were polymorphic. the range of fragment sizes varied from to bp. contrasting with the phenotypic diversity for agronomic and fruit quality traits, very low variability at the dna level has found among mandarins, which always showed a high (s > . ) coefficient of genetic similarity. the molecular marker analyses allowed the clarification of ambiguous denominations and the establishment of phenological relationships. the mandarin cultivars have been clustered into several different sub groups. this study allowed the identification of one issr marker, distinct and specific for the clementine sidi aissa and some aflp markers specific to maroc late and w. navel. many hybrids used in this study presented high coefficient of the similarity with one of their parents, such as siamelo and king of siam (s = . ), fortuna and clementine (s = . ), kara and king of siam (s = . ). this work was partially funded by the program for scientific cooperation cnrst (morocco) and iccti (portugal), the international foundation for science (ifs) support in stockholm (sweden). the new morocco mandarin variety nadorcott became very important in the international market because of high quality, good size, easy peeling and absence of seeds. also known as afourer or w. murcott, this variety was selected in - at the afourer experimental station, inra, located near to beni mellal city, as an original tree among several -year-old murcott honney (c. reticulata × c. sinensis) seedling trees. in order to shed additional light on the genetic origin of this variety, we have carried out isozyme and dna fingerprinting analyses. for better interpretation of the nadorcott molecular profiles, others mandarin cultivars, among which murcott honney, were also analyzed by molecular markers. three enzymatic systems (idh, pgm and pgi) permitted the discrimination between nadorcott and his female parent murcott honney. the molecular patterns displayed by these cultivars point out the sexual origin of nadorcott and discard the previously assumed hypothesis for its origin as a mutation of a nucellar zygote. the issr and rapd markers analyses allowed the identification of kinnow; du japon, vietnam; and swett lime as the genetically most closely related mandarins (s ∼ . ) to nadorcott. strong genetic similarity was also found with the clementine group, a possible male parent of nadorcott. the analysis by aflp markers confirmed the hybrid origin of nadorcott and the high genetic relatedness (s = . ) of this mandarin to its putative female parent murcott honey, and other cultivars as kinnow (s = . ) and clementine (s = . ). the possibility of an accurate molecular identification of nadorcott by specific molecular markers is of paramount importance for the protection and management of this original moroccan citrus variety. an effective process for the chemical-biotechnological utilization of distilled white lees was studied. a first treatment with hydrochloric acid allowed the solubilisation of tartaric acid. the influence of temperature, amount of hcl and reaction time were considered through an experimental design. under the optima conditions g/l from white distilled lees and . g/l from red distilled lees were recovered. the tartaric acid was precipitated as calcium tartrate so that it can be isolated from the rest of the raw material compounds. the solid residue was used as an economic nutrient for lactic acid production by lactobacillus pentosus using trimming wastes as substrate. the lactic acid concentrations and volumetric productivities achieved were similar to those obtained using distilled lees without tartaric acid recovery as nutrient. thermophilic cyanobacterial strains that could grow in the bg media was isolated from hot springs and their reactive dye bioaccumulation was studied under thermophilic conditions in a batch system, in order to determine the optimal conditions required for the highest dye accumulation. in the experiments performed with newly isolated synechocystis sp. and phormidium sp., the optimum ph values at about mg l − initial reactive dye concentrations was determined as . lipases are extremely versatile enzymes, that catalyze both hydrolysis and synthesis reactions. they have a wide range of industrial applications, among which the manufacture of detergents, pharmaceuticals and fine chemicals are outstanding. the fungus rhizopus oryzae has been reported to synthesize a number of commercially interesting enzymes. in this work, its ability to produce extracellular lipases when grown in solid state culture has been assessed. cultures were carried out in erlenmeyer flasks, using a complex medium and several supports, both synthetic (nylon sponge) and natural (barley bran, ground walnut or peanut). the latter appeared to be more suitable for lipase production. since the best results were initially obtained with lipid-containing supports, barley bran cultures were supplemented with a vegetable oil, in an attempt to optimise lipase production and design an efficient procedure for reusing this agroindustrial waste. surprisingly, this strategy did not improve enzyme synthesis. however, when a surfactant (triton x- ) was added to the basal medium, a dramatic increase in extracellular activity was detected (up to -fold). the results agreed with those previously obtained in submerged cultures of r. oryzae, in which addition of olive oil did not increase lipase production, while the presence of triton x- had a remarkably beneficial effect. also, enzyme concentration in solid state cultures was up to two-fold that of the submerged ones. est maximal sorption capacity was found for yeasts cryptococcus sp. wt and rh. aurantiaca . these cultures also demonstrated the high sorption affinity, which makes them especially efficient biosorbents at low concentrations of lead ions. the high efficiency of lead elution was shown with . n edta. isolation and identification of marine bacteria from deep-sea sediments els maas , cara brosnahan , vicky webb , helen neil , phil sutton : marine biotechnology, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand; oceanography, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand marine sediments were obtained using a piston corer with associated trigger core ( . m, . m diameter). cores were collected from depths of to m, along norfolk ridge and across challenger plateau. sediments ranged from coarse carbonate sands in the north to sandy and silty hemipelagic mud with increasing depth and latitude. all samples sites underlie subtropical surface water masses associated with, and south of, the tasman front. sediment samples were aseptically taken from the triggers cores upon recovery. samples were stored in sterile tubes at • c on board the vessel for - days. the core samples were plated on several different agar types and incubated aerobically for weeks at • c. individual colonies were sub-cultured and purified using standard microbiological techniques. morphological and molecular taxonomy revealed that the bacteria isolated from the sediments were closely related to novosphingomonas, halomonas, stappia, glaciecola, pseudoalteromonas and leeuwenhoekiella. phylogenetic trees constructed using s rrna gene sequence data showed that two other isolates were unrelated to known genera. the bacterial isolates are currently being investigated for their biotechnological potential. olive oil mill wastewater (omw) as the effluent of the concern of olive industry has high organic load. the conventional biological treatments despite of their simplicity and rather suitable performance are ineffective for the omw treatment since phenolics possess antimicrobial activity. in order to carry out a proper treatment on omw, use of microorganism able to degrade the phenolics thus, is necessary. the ability of phanerochaete chrysosporium immobilized on loofa was studied. the basal mineral salt solution along with glucose, ammonium sulfate and yeast extract were used to dilute omw properly. the fungus did not grow on the concentrated omw. therefore, omw diluted by % was used thought this study. the extent of removal in this biotreatment, of total phenolics (tp) and cod were and %, respectively. while the color and aromatocity decreased by and %, respectively. the kinetic behavior of the loofa immobilized fungus was found to follow monod equation. the maximum growth rate was . h − while the monod constant based on the consumed tp and cod (mg/l) were and , respectively. the control of water pollution has become of increasing importance in recent years. the release of dyes into the environment constitutes only a small proportion of water pollution, but dyes are visible in small quantities due to their brilliance. many dyes are difficult to decolourise due to their complex structure and synthetic origin. the adsorption of reactive dye remazol brilliant blue r (rbbr) on polyelectrolyte complex (pec) was studied in a batch systems. the adsorption parameters determined were: effect of the different values of ph on the adsorption of dye by pec, and the effect of contact time on the amount of rbbr adsorbed (in mg g − ). the data indicates that the adsorption capacity of rbbr by pec is dependent on ph. the maximum adsorption at ppm was . % equal to . mg of dye/g of polymer. the results show a tendency towards greater adsorption for reactive dyes (ph range of - ). the effect of contact time was studied at initial concentration ( ppm) of dye, the amount of rbbr adsorbed for these pec increased and reached a constant value with the increase in contact time. the increase in the extent of removal of dye after min of contact time is less and hence it is fixed as the optimum contact time. the pec show their capacity to remove rbbr to aqueous solutions by adsorption. flocculation of saccharomyces cerevisiae (diastaticus) ifo was studied. cells of ifo did not flocculate even in the stationary phase without mg + ("mg + -deficient cells") although they began to flocculate strongly h after inoculation in the presence of mg + ("complete cells"). cycloheximide completely inhibited induction of floc-forming ability of "mg + -deficient cells". co-flocculation between "complete cells" and "mg + -deficient cells" was investigated by chemical modification. treatment of "mg + -deficient cells" by proteolytic enzymes did not affect the co-flocculation with "complete cells". photo-oxidation or mercaptoethanol-reduction of "mg + -deficient cells" failed to weaken the co-flocculation with "complete cells" while treatment of "mg + -deficient cells" by periodate brought about a significant loss of the co-flocculation. on the contrary, "complete cells" deflocculated by proteolysis or chemical modification of proteinaceous component failed to co-flocculate with "mg + -deficient cells". these findings suggest that "mg + -deficient cells" are non-flocculent because of lack of proteinaceous component essential for flocculation of cells of ifo . the industrial toscano cigar production starts with the dark firecured kentucky tobacco fermentation process. during this phase the present work is a trial to study the portal serum factors which stimulate the cell proliferation of the schistosomules, aiming to find ways to block or inhibit their effects. our previous studies showed that portal serum of human and hamster (highly susceptible hosts) and a - kd fraction separated from human portal sera by ultrafiltration stimulate cell proliferation in immature schistosomules ( days old) in vitro. for further identification of the portal serum factors in the range of - kd that stimulate cell proliferation, schistosomules were incubated in vitro in medium containing % fetal calf serum, % portal human serum or % peripheral human serum or their fractions separated by native electrophoresis followed by electroelution, incubations were performed in presence of bromodeoxyuridine (brdu) in order to measure differences in cell proliferation. the results showed that human portal sera enhanced cell proliferation of schistosomules compared to the peripheral serum. this stimulatory effect was substantially reproduced by fraction separated from human portal serum with molecular weight . kd. these results may help in designing a drug or antibody therapy to block the stimulating effect of the portal serum fraction and subsequently to disturb the life cycle of the parasite at early stage of development. center of molecular biosciences, university of the ryukyus, okinawa - , japan. e-mail: naoya-s@comb.u-ryukyu.ac.jp (n. shinzato)oil strage tank sludge, mainly composed of water and solid hydrocarbons (waxes) needs to be treated when harvesting the stored oil. although the sludge treatment by microbial surfactant or microbial cracking are considered as the feasible method, microbial degradation of the waxes (ca. solid n-alkanes) have been reported in a very limited species such as acinetobacter. in addition, long-chain n-alkanes (so called paraffin waxes) are one of the major components of oil, and their resistant properties to biological attack hold up the recovery of oil-polluted environments. in this report, we have screened n-tetracosane (c ) degrading bacteria from soils in okinawan island, an unique sub-tropical area in japan, to know the bacterial diversity and their degrading mechanism. srdna phylogenetic analysis of the isolates, totally ca , showed they were not only acinetobacter and pseudomonas, but also other proteobacteria (alcaligenes), actinomycetes (gordonia, nocardia, and leifsonia), bacillus, staphylococcus, and unidentified ones. they also grew not only the solid n-alkanes but also iso-alkanes, mid-chain n-alkanes as the sole carbon source. results for the biosurfactant production will also be shown. the properties of environmental enterococci were studied. the strains were isolated mainly from surface and waste waters and several strains from sheep manure were also included. species identification was provided by combination of phenotypic (micronaut system, merlin) and molecular detection methods (automated its-pcr, ddl-pcr). several discrepancies were observed when comparing molecular and biochemical identification. six enterococcal species were overall identified; e. faecium and e. hirae were the most abundant ones, almost % of isolates belonged to these two species. the distribution of selected genes conferring virulence to enterococci (cyla, gele and esp) was investigated, the positive signal was obtained mainly for e. faecalis strains. the strains were also characterized for the possession of enterocin genes (enta, entb, entp, ent , entl ab) and high frequency of enterocins was observed. biosorption of three different dyes (reactive black , cibacron brilliant yellow, cibacron brilliant red) onto immobilized scenedesmus obliquus a microalga was investigated in a batch sys-tem. the immobilized alga exihibited the highest dye uptake capacity at the initial ph value of . for all dyes. the effect of temperature on equilibrium sorption capacity indicated that maximum was obtained at • c for rb , cby and cbr biosorption. the freundlich, and langmuir adsorption models were used for the mathematical description of the biosorption equilibrium and isotherm constants were evaluated. biocontrol properties of microbially-treated sugar beet wastes in presence of rock phosphate n. vassilev, i. nikolaeva, m. vassileva department of chemical engineering, faculty of sciences, university of granada, c/fuentenueva s/n, granada- , spain. e-mail: nbvass@yahoo.com (n. vassilev) the effect of soil application of sugar beet wastes (sb) treated with aspergillus niger in the presence of rock phosphate (rp) on the control of fusarium wilt of tomato were studied. two treatments and a control were used: inoculation with glomus intraradices (am), further inoculation with a. niger grown on sb + rp medium, and the control (c). application of the am fungus increased plant growth, p and n uptake and reduced disease caused by fusarium oxusporum f. sp. licopersici (fol) as compared to non-mycorrhizal control plants. soil amendment with sb + rp + a. niger resulted in % and % (versus c) higher plant shoot biomass in plant-soil experiments contaminated or not with fol, respectively. in this case, disease severity and number of fol cfu reached the lowest levels while soil phosphatase and beta-glucosidase activities increased compared to all other treatments. fol negatively affected plant root mycorrhization determined in the am treatment while the difference between the mycorrhization of plants grown in the presence and absence of f. oxysporum in sb + rp + a. niger-amended soil was insignificant ( % versus %, respectively). in conclusion, the fermentation mixture containing mineralized organic matter, partially solubilized rp, and a. niger biomass could be efficiently used not only in improving plant growth, nutrient uptake and properties of degraded and polluted soils, as previously reported (vassilev and vassileva, ) , but also in environmentally-mild management of fusarium wilt. vassilev, n., vassileva, m., . appl. microbiol. biotechnol. , - . biological treatment processes allow for the effective elimination of charged inorganic micropollutants, e.g. a number of oxyanions, heavy metals, etc. from contaminated drinking water supplies. however, dedicated technologies have to be implemented in order to eliminate the target pollutants without changing the quality of treated water, avoiding its secondary pollution by cells, nutrients and metabolic by-products. some innovative technologies, which combine the use of membranes with the bioconversion of charged micropollutants in order to deal with the secondary water contamination problem, will be presented and critically compared. a special on loofa was studied. the basal mineral salt solution along with glucose, ammonium sulfate and yeast extract were used to dilute omw properly. the fungus did not grow on the concentrated omw. therefore, omw diluted by % was used thought this study. the extent of removal in this biotreatment, of total phenolics (tp) and cod were and %, respectively. while the color and aromaticity decreased by and %, respectively. the kinetic behavior of the loofa immobilized fungus was found to follow monod equation. the maximum growth rate was . h − while the monod constant based on the consumed tp and cod (mg/l) were and , respectively. advanced start-up strategy of an anaerobic three-phase turbulent bed reactor treating winery wastewaters r. cresson, h. carrère, n. bernet, j.p. delgenès laboratoire de biotechnologie de l'environnement, institut national de la recherche agronomique (inra), avenue des etangs, narbonne, france. e-mail: cresson@ensam.inra.fr (r. cresson)the objective of our study was to compare two start-up strategies for an anaerobic biofilm process, to create an effective biofilm and increase the organic loading rate (olr) as quickly as possible. two methanogenic three-phase biofilm reactors have been started, using the same operational parameters (solid hold-up ratio, gas velocity of mm s − ), in order to test two different strategies:• maximal load strategy (reactor a): the olr is increased as long as the global amount of removed cod (biogas production) increased. • maximal removal strategy (reactor b): the olr is increased stepwise as soon as the cod removal rate reaches %.both reactors have been operated for days, until a volumetric olr of g cod l − j − , with more than % of carbon removal. the total amount of cod removed and methane produced were higher in reactor b ( . and . %, respectively). in both reactors, the short hydraulic retention time (hrt) applied during all the experiment caused a rapid wash-out of planktonic bacteria and an exclusive use of the substrate by the attached micro-organisms, which accelerates the biofilm growth. the lag-phase was reduced to approximately days. the reactor submitted to repetitive disturbance by the maximal removal strategy appeared to be more robust when confronted to perturbation like organic overload or nutritional deficiency. experiments have demonstrated capability and the efficiency of the aggressive strategy for controlling anaerobic bioreactor start-up. sustainability is the generally accepted paradigm for future industrial development. the re-integration of waste products into production processes is a major aspect of environmental sustainability. in this study the use of sugar cane molasses is being investigated for the production of bioplastics by mixed microbial cultures, with the added possibility of parallel biohydrogen production. polyhydroxyalkanoates (phas) are polyesters synthesized by bacteria and accumulated as granules in the cytoplasm. studies conducted by this group have shown that mixed microbial cultures subjected to dynamic feeding conditions may accumulate phas up to % cell dry weight, a value close to that obtained for pure cultures. volatile fatty acids are good substrates for the production of phas by mixed cultures. on the other hand, sugar molasses, with a very high sugar content (about % dry weight), can produce organic acids by fermentation. the two-stage process being implemented in this study includes a molasses fermentation step, in which the high sugar content of the molasses is converted into volatile fatty acids (vfas), and a pha production step, in which the vfas serve as the precursors to the formation of phas under dynamic feeding conditions. moreover, hydrogen can be produced by anaerobic bacteria from carbohydrate-rich substrates giving organic fermentation end products, h and co . to optimize the production of both high-value products, design of experiments (doe) is being used to elaborate a set of experiments to study the effect of ph, hydraulic retention time and organic loading on both the organic acids distribution (which will serve as precursors for pha production in the second step) and h production in the acidogenic fermentation reactor (a l cstr). preliminary results show that the effluent of the acidogenic reactor fed with g/l total sugars and operated at ph and d = . h − (composed mainly of acetate and propionate) can be successfully fed to a polymer-accumulating mixed culture. under these conditions, the h production yield has been estimated at . mol h /mol sucrose. vanillin is a flavour compound used in food industry, fragrances and pharmaceutical preparations, which is nowadays mainly produced by chemical synthesis. the increased demand of natural products for the food industry as well as the high cost of natural vanillin extracted from vanilla pods has recently stimulated the research for alternatives to produce this compound by a natural way. the microbial transformation of ferulic acid, a phenolic compounds from lignin degradation, is recognized as being the most interesting alternative to produce natural vanillin. the combined effects of initial ferulic acid concentration (s ) and biomass concentration (x ) on vanillin production by resting cells of escherichia coli strain were investigated using response surface methodology. e. coli jm /pbb a recombinant strain producing key enzymes of ferulate catabolic pathway from p. fluorescens bf (feruloyl-coa synthetase and feruloyl-coa hydratase/aldolase) was utilized in this work. a full-factorial design was employed for experimental design. the results shown a possible inhibition phenomena at a vanillin concentration of about . g l − leading to the accumulation in the fermentation media of secondary compounds like vanillic acid and vanillin alcohol. removal of dissolved nutrients from wastewater using a microalgae biofilter line christensen, suvina sooknandan, jens jørgen lønsmann iversen department of biochemistry and molecular biology, university of southern denmark, odense, a microalgae biofilter can be used for treatment of wastewater from landbased fish farms in order to remove excess amounts of dissolved nutrients such as nitrate, ammonium and phosphate. a bubble column bioreactor has been developed for cultivation and characterization of microalgae. this type of bioreactor is equipped with a control system that enables online determination of the photosynthetic quotient and optimization of light intensity. furthermore the bioreactor has a dualsparging system simultaneously allowing adequate mixing and high gas-liquid mass transfer coefficients. different species of microalgae have been cultivated in batch and fed batch cultures to characterize growth and ability to take up the different dissolved nutrients. the specific growth rate and substrate uptake rate have been determined to compare and select the algal species most suited for use in a biofilter. additionally the composition of lipid, protein and carbohydrates has been measured to determine the nutritional quality of the algae when used as animal feed. at present, biological nitrogen-removal is mostly carried out through several complicated steps. to simplify the present systems for nitrogen-removal, we have investigated a new nitrogenremoval bioreactor using packed gel envelopes capable of simultaneous nitrification and denitrification. the envelope consists of two plate polymeric gels with a spacer in between. ammonia oxidizer, nitrosomonas europaea and denitrifier, paracoccus denitrificans are co-immobilized in the plate gels. when the envelopes are exposed to wastewater containing ammonia, the immobilized n. europaea oxidizes ammonia to nitrite in the outer aerobic surfaces of envelopes. at the same time, as ethanol solution is injected into the internal anaerobic spaces of envelopes, the immobilized p. denitrificans reduces the nitrite to nitrogen gas using the ethanol solution as an electron donor for denitrification. in this way, the envelopes can remove ammonia from wastewater in a single step. we have already reported advantages of our bioreactor in laboratory-scale experiments. in this study, we show our large-scale bioreactor (water volume . m ) could treat three kinds of wastewater derived from coal power plants. ammoniacontaining wastewater that occurred regularly in a coal power plant was continuously treated with the bioreactor using thirty envelopes for over a year. the bioreactor could remove more than % of total nitrogen at hydraulic retention time (hrt) of h. at hrt of h, the bioreactor accomplished a maximum rate (the transformation of nh + to n ) of . g n/day m of the envelopes' surface. the performance was equivalent to that obtained in the laboratory-scale experiments. furthermore, our bioreactor showed similar nitrogen-removal performances when it treated nitrate-containing wastewater occurring regularly and condensed ammonia-containing wastewater occurring at irregular intervals in coal power plants. these results show that our bioreactor can treat various wastewater containing nitrogen in coal power plants. thus, our concept is effective to simplify the large-scale systems in coal power plants and the other plants. in order to establish an environmentally friendly process for the treatment of metal containing waste, in a portuguese refinery a process involving sulphur oxidizing acidophilic microbes is being considered. bioleaching of metal containing bottom ash, from fluidised bed incineration of sludge resulting from the refinery water treatment station, was performed using a sulphur oxidising acidophilic culture isolated from an acid pool resulting from the weathering of sulphur piles from the claus plant. this sample served as inoculum for liquid medium cultures with % sterile sulphur flowers as source of energy. application of monod kinetics to adapted culture growth of free cells presented a value of µ = . day − . yield of sulphur conversion to sulfate after days was η = %. in the presence of bottom ash from the incineration of refinery sludges µ = . day − and the yield of sulphur conversion was η = . %. a η fe = % removal of iron is obtained from the treated ash. x-ray fluorescence spectroscopy of the solid residue revealed a total removal of metals namely, v, cu, ni, zn and most of the fe after days of bioleaching. the present of heavy metals in the environment is a serious problem. they are commonly present in effluents from mining and industrial activities. usually, chemical conventional methods are very expensive and they have limitations when heavy metals are in low concentrations. at the moment the interest increases for processes that involve microorganisms as alternative method. some effluents present heavy metal sulphates which are soluble compounds. sulphate-reducing bacteria (srb), under anaerobic conditions, oxidize simple organic compounds (such as acetic acid and lactic acid) by utilizing sulphate as an electron acceptor and generate hydrogen sulphide. hydrogen sulphide reacts with heavy metal ions to form insoluble metal sulphides that can be easily separated from a solution. the purpose of this work was to evaluate the ability of srb to reduce cr(iii), ni(ii) and zn(ii) in artificial contaminated solution. desulfovibrio vulgaris and desulfovibrio sp. strains has been tested in this study. batch cultures was carried out in ml sealed bottles with different concentrations of studied metals ( - mg/l), with % of inoculum bacterial and adapted to postgate's medium c. a gaseous nitrogen current was employed to purge oxygen and obtain anaerobic conditions. the assays were incubated statically represented very low portion (less than - %) of the total bacterial community at all temperatures tested. schistosomules of schistosoma mansoni ( days old) were incubated in rpmi medium containing % fetal calf serum, % hamster portal venous or % hamster peripheral venous serum (highly susceptible host) or % rat portal venous or % rat peripheral venous serum (poorly susceptible host) in presence of bromodeoxyuridine (brdu) in order to measure differences in cell proliferation. also the rate of cell proliferation of s. mansoni were assessed in vivo in hamster to study the cell proliferation in the natural ontogeny of the organism. the rates of cell proliferation as expressed by brdu labeling indices (blis) were determined as a function of time of incubation by immunohistochemistry using monoclonal antibody to brdu. compared to schistosomules cultured in presence of rpmi plus % fetal calf serum, blis were increased by % in the presence of hamster portal, but not in peripheral serum. while in case of rat, no significant changes were observed in the blis in both portal and peripheral sera. the experiment was repeated using hamster portal and peripheral sera containing different schistosomal igg antibody titres. the results showed decreased values of blis compared to sera which did not contain the schistosomal antibody(ies). the in vivo results revealed that there was no cell proliferation of s. mansoni schistosomules ( days old) in the lungs. cell proliferation was detected in schistosomules of days old and the results revealed a significant decrement in the brdu labeling indices (blis) with the increase of the age of schistosomules in vivo. the results indicated that hamster portal venous serum (highly susceptible host) could have stimulating factor(s) for schistosomule cell proliferation which is not found in rat (poorly susceptible host) and the presence of antibody(ies) greatly inhibit the cell proliferation. this could be due to the blocking of some portal serum factors, which stimulate the cell proliferation by the antibody(ies). the release of dyes into the environment constitutes only a small proportion of water pollution, but dyes are visible in small quantities due to their brilliance. many dyes are difficult to decolourise due to their complex structure and synthetic origin. the adsorption of reactive dye remazol brilliant blue r (rbbr) on polyelectrolyte complex (pec) was studied in a batch systems. the adsorption parameters determined were: effect of the different values of ph on the adsorption of dye by pec, and the effect of contact time on the amount of rbbr adsorbed (in mg g − ). the data indicates that the adsorption capacity of rbbr by pec is dependent on ph, the maximum adsorption at ppm was . % equal to . mg dye/g of polymer. the results show a tendency towards greater adsorption for reactive dyes (ph range of - ). the effect of contact time was studied at initial concentration ( ppm) of dye, the amount of rbbr adsorbed for these pec increased and reached a constant value with the increase in contact time. the increase in the extent of removal of dye after min of contact time is less and hence it is fixed as the optimum contact time. the pec show their capacity to remove rbbr to aqueous solutions by adsorption. compared to other european nations, the austrian population shows a low level of knowledge in biosciences and a strong denial of gene technology ( ). the austrian non-profit organisation dialog<>gentechnik, a scientific society, organizes various activities to raise awareness for the "hot topics" in the life sciences. according to its principle of independence, all activities are funded publicly. projects on behalf of the austrian authorities and international cooperations demonstrate credibility and trust in dialog<>gentechnik ( ). a few examples will be presented. dialogue with the public: on the occasion of the first anniversary that the gmo labelling rules became effective, the action "gene technology on my plate" is performed austrian wide in shopping centres. here, consumers are informed about health and labelling aspects of gm food. two days of open discussions were organized in the context of the austrian genome research program gen-au ( ): topics were "gene diagnosis" ( ) and "genome research-what is in it for me?" ( ) . an open lab is currently set up in vienna to offer "hands on" experience in life sciences for everybody. motivating students: in the very successful gen-au summer school ( ), high school students spend - weeks in the lab and work with scientists. the best documentations are awarded. in an innovative project, student groups (age - ) work on the topics stem cells and cloning and develop units of an elearning course which will be accessible for all austrian schools in the near future. engaging stakeholders: dialog<>gentechnik manages interdisciplinary wor king groups that develop leaflets, brochures and questionnaires on various aspects of gene diagnosis. four products are currently distributed to the public and to health services. ( ) european commission. eurobarometer . , december ; ( ) www.dialog-gentechnik.at; ( ) www.genau.at. the aim of this paper is to compare the attitudes of the urban consumers with professionals towards to new technologies, especially to biotechnology. it was tried to find out in which area, medical, agriculture or industry, people can accept biotechnological developments and in which area not accept. key: cord- - rfb b authors: fell, michael j.; pagel, laura; chen, chien-fei; goldberg, matthew h.; herberz, mario; huebner, gesche m.; sareen, siddharth; hahnel, ulf j.j. title: validity of energy social research during and after covid- : challenges, considerations, and responses date: - - journal: energy res soc sci doi: . /j.erss. . sha: doc_id: cord_uid: rfb b measures to control the spread of coronavirus disease (covid- ) are having unprecedented impacts on people’s lives around the world. in this paper, we argue that those conducting social research in the energy domain should give special consideration to the internal and external validity of their work conducted during this pandemic period. we set out a number of principles that researchers can consider to give themselves and research users greater confidence that findings and recommendations will still be applicable in years to come. largely grounded in existing good practice guidance, our recommendations include collecting and reporting additional supporting contextual data, reviewing aspects of research design for vulnerability to validity challenges, and building in longitudinal elements where feasible. we suggest that these approaches also bring a number of opportunities to generate new insights. however, we caution that a more systemic challenge to validity of knowledge produced during this period may result from changes in the kinds of social research that it is practicable to pursue. the coronavirus disease (covid- ) pandemic is having tragic health consequences around the world, and measures to combat it are impacting people's lives in unprecedented ways. there is, as yet, no clarity on when and how measures such as suspension of certain businesses and physical distancing might end completely, or need to be reintroduced. the timescales required to develop a vaccine and deploy it globally suggest this could be well into and possibly later. during this time, the validity of energy social science research faces additional threats. validity generally refers to the truth of a knowledge claim or inference [ ] . national and global events continuously shape social worlds. but the magnitude, speed, and reach of the changes to our lives are of a different order to anything that most people alive today have experienced. given the scale and rapidity of change, how can we ensure that conclusions drawn from data collected during the pandemic are valid, representative, generalisable to a post-pandemic world, and comparable to the pre-pandemic one? while the answer is inherently unknowable [ ] , our aim in writing this paper is to highlight principles that we believe energy social science researchers can take to help mitigate this uncertainty, and ease future interpretation of research findings in the context of the progressing pandemic. broadly speaking, these principles involve giving consideration to possible impacts of the pandemic and associated response measures on findings; adjusting research design and data collection to reflect this; and reporting extra contextual detail. we argue that researchers who take reasonable steps in these areas will be able to ensure greater confidence in the validity of the work they conduct during this period. through consciously enhanced transparency for the 'extended peer community' that post-normal science scholars have long espoused [ , ] , their contributions will be better positioned to help address future challenges on the validity of findings by reviewers and users. we co-produced these principles as energy researchers who represent a variety of relevant disciplinary perspectives and subject interests, and are based in a range of institutions and countries. this allowed us to balance the will to draw on a breadth of input across the field, with the need to share these principles in a timely fashion. we set them out in the hope that researchers will find them helpful, but recognise that applicability will vary across energy social science research. our recommendations are likely to be most applicable to researchers employing quantitative research methods that are often restricted in the amount of contextual data they are able to collect [ ] . however, we hope that as a set of considerations they will be helpful to a broad range of energy social science scholars to employ as they see fit. the next section of this paper sets out the key challenges we identify for validity during the covid- pandemic, and justifies our focus on social science research in energy. we then expand on steps that researchers can take to address these challenges, and provide a simple checklist that can be applied by scholars in order to address the impacts of the pandemic on their research. we finally highlight additional opportunities these steps can yield, but also point out important potential implications for the nature of knowledge generated by contemporary research. decisions about validity inherently concern tradeoffs and priorities of a given research study [ ] . for instance, a researcher might prioritize internal validity (or "the degree to which a study establishes the cause-and-effect relationship" [ ] ) by conducting a randomized controlled laboratory experiment. artificial laboratory conditions enable strong experimental control, but limit generalizability across diverse, complex real-world situations. a field study, in contrast, might prioritize external validity (or "the generalization of research findings [...] to settings and populations other than those studied" [ ] ), but surrenders some ability to control and measure variables. both of these forms of validity are important. if we cannot trust the findings of a study because of methodological problems or unaccounted-for variables, generalisability is irrelevant. and findings that only apply in exceptionally narrow circumstances offer very limited value in applied research settings. external validity tends to be given special weight in applied research, including most energy research, where the generalisability of findings, and therefore any resulting conclusions and recommendations for action, often has primacy. our point of departure here is the impact of the covid- pandemic and associated response measures on internal, external, and subsidiary forms of validity. we argue that the pandemic merits explicit consideration for validity for a number of reasons. first, the response to covid- represents a departure from ordinary circumstances that is unprecedented in terms of its global nature, rapidity, diversity, and severity of impacts. at the time of writing in may , over countries and several billion people were under some form of lockdown, with restricted rights to movement and public assembly. in many cases, schools, non-essential businesses and hospitality venues were closed. evidence of prevalent psychological distress and anxiety had begun to emerge [ , ] . such a situation is far removed from the conditions under which knowledge is ordinarily produced and applied, and questions around the validity of findings generated during this circumstance are inevitable. second, an important consideration for external validity is how stable findings are over time. while there is always uncertainty about how closely the future will resemble the present, we argue that this uncertainty is now especially high. movement restrictions have already left millions of people unemployed, with millions more at risk of losing their jobs as businesses contract or close [ ] . governmental support packages are building up unprecedented levels of national debt that will have to be paid for, with little clarity around the effect this will have for public services and taxes. while some effects such as quarantine measures will be shorter-term, it is unknown whether the pandemic itself and associated consequences will result in long-term effects on the individual and societal level. realistic and symbolic threats induced by the pandemic are likely to affect individuals' values, identity, and worldviews and thus could exert long-term effects on various dimensions [ ] . moreover, research on past societal crises has shown that pandemic-related effects such as large-scale unemployment can lead to long-term effects on mental health [ ] ). taken together with the scale of current impacts, we believe this increased uncertainty in the short, medium, and long term justifies special consideration of validity of social research and, furthermore, a higher burden of proof on claims to such validity. why is a particular focus on energy studies important? energy use plays a prominent role in many aspects of human life. any changes on the scale being experienced during the pandemic have significant impacts not only on patterns of interaction with energy systems, but also on how people relate to and prioritise those systems. much energy research conducted today aims to inform transitions to clean, low-carbon energy systems that work for people and society. although research conducted now can shed light on how the extraordinary measures in place might impact energy use (such as evidence of reduced weekday electricity use [ ] and changing usage patterns [ ] ), it is challenging to disentangle these impacts from those that result from measures deployable absent a pandemic. moreover, the impact of such a drastic, globally shared experience impacts discursive and normative registers, with undetermined implications for public commitment to low-carbon energy transitions that become interwoven with other drivers of change pathways. although many of the principles we set out next could simply be viewed as good research practice, we think that they merit explicit attention during this pandemic and its aftermath. we argue that they are especially important for those domains of energy social research that claim broad generalisability to their findings and insights, with limited focus on context. for example, we think the points raised here are generally more applicable to survey-based than ethnographic research. by bringing these recommendations together here, we hope to stimulate a more consistent response by social researchers, allowing greater commensurability and comparability across studies in the future. furthermore, we recognise that scholars using social research approaches in energy have a wide variety of backgrounds and levels of experience. what we suggest may be self-evident to some, although for these we hope it will be helpful to have a checklist to compare their own responses against. to others, we hope it will provide both a prompt to consider challenges to validity, and a handy set of responses to consider. we have argued that challenges to the validity of social science energy research presented by the covid- pandemic warrant special recognition. we now lay out a set of principles for researchers to consider bringing to their practice for the duration of the pandemic period and its aftermath to help bolster the validity of their work, and to ensure that future use of their findings and recommendations is facilitated by requisite information to aid correct interpretation. our recommendations address data collection and the reporting of study conditions and context, as well as considerations for study designs in order to ensure high validity of energy social science research conducted during and after the pandemic. given the large number of possible new factors to be taken into account, we propose a 'core and consider' approach, allowing researchers in the field to prioritise and justify the measures they want to take to account for potential pandemic-related influences. where possible, we have drawn on existing good practice guidance, which itself has developed through conventional processes of cross-field engagement [ ] . while we think the validity challenges we have raised here are important, we also recognise that any responses to them must fit within existing research plans, budgets, timelines, labour constraints, and the heightened need for affective care, including researchers' own wellbeing under personal stress-inducing conditions. any response must be both proportionate to the anticipated vulnerability to validity challenges of the kind set out in the previous section. ethical and data protection concerns, while not directly related to validity, must be borne prominently in mind. any changes to planned research should not, unless it is explicitly justified, introduce collection of categories of data that are more sensitive than those that were originally (or would ordinarily be) planned and/or approved. this means, for example, that researchers should not (without careful thought and justification) begin to collect data on physical or mental health unless this was intended anyway. researchers should be mindful of the extra burden to participants that introducing additional data collection could bring. extra sensitivity is called for on the part of researchers to the potential impacts of collecting data on topics which may be more upsetting now than would ordinarily be the case. we suggest that additional and/or modified variables may need to be collected and reported for studies carried out during or after the covid- pandemic in order to account for the impact of the pandemic on research validity. already, researchers shouldand many doreport contextual factors of any study, and consider how these might impact the study findings [ ] [ ] [ ] . given the large number of possible new factors to be taken into account, we suggest researchers take a 'core and consider' approach. government restrictions and relevant demographic variables at the level of the unit of analysis (e.g., individuals or households) are core additional variables that should be reported and discussed. other factors should be considered for additional reporting depending on the precise topic of research. as in all studies, reporting of contextual factors should encompass date(s), place(s), and duration of data collection. as a core concern, we suggest that this should now be supplemented with information on pandemic-related national and local policies that were in force at the time and place of data collection. this could include factors such as levels of restriction of people's freedom to move around outside the home, including self-imposed precautionary behaviour, and the open/closed status of specific relevant services such as schools and certain businesses. significant changes in any of these measures during data collection should also be reported. researchers may consider it to be important for context to give a sense of the severity of the pandemic (including health, social and economic impacts, as relevant). we suggest using official government references for a description of such policies and impacts where possible, in ways that are cognizant of their rapid temporal evolution. a further core consideration is that local and national pandemic response measures affect individuals and households in diverse ways; specifying the national policies during data collection alone does not explain effects at the individual (or other analytical) level. more specific effects can be captured by measuring application of and compliance with response measures on the respective analytical level, and/or through collection of additional demographic variables from which application could be inferred. the nature and detail of measures will differ by locality, but could include whether someone is considered a 'key worker' (and hence still regularly leaves the home during lockdown) or comes under a highrisk category and has to observe stricter measures. other standard demographic variables may need amendment depending on the study aims. for instance, employment status can include categories such as being placed on government-subsidised furlough, working reduced hours, or working fully from home. other variables that might ordinarily have been judged as having limited importance, might gain relevance. impacts of the covid- pandemic are thought to be exacerbating existing inequalities in many societies, such as energy poverty issues [ ] . a key variable in many studies will likely be the financial situation of the individual, household, or other unit of interest. capturing information on recent (and risk of future) changes in factors such as income (including transfer payments), changes in employment status, increased receipt of benefits, or self-reported financial satisfaction may take on greater importance. unexpected deprivation from work income may have differential effects on energy-related measures, relative to foreseeable prolonged unemployment periods; while this is a consideration in samples at any time, it is likely to be especially common now. differentiated impacts on variables such as health, income or employment situation are already evident across individuals, notably across ethnicities, gender and income groups [ , ] . disaggregating on the basis of such variables, while always beneficial, may now be of more acute importance given heightened inequalities. at the individual level, we anticipate that covid- response measures will be associated with important changes in behaviour, as well as cognitive, affective, and other social and material dimensions [ ] . changes in energy-related behaviours and decision-making due to changes in daily routines, work and mobility might be more apparent and measurable, but changes in decisions and actions triggered by pandemic-related shifts in energy-related beliefs, attitudes, emotions, and judgments may be just as important to apprehend. for energy social science research focusing on the aforementioned dimensions it is important to assess to what extent these variables are different from a "normal" scenario and whether potential changes are durable or ephemeral. epidemiological research demonstrates the effect that pandemic response measures and consequences such as unemployment exert over time on personal well-being [ ] ). while empirical research on specific covid- measures is emergent, existing theoretical research on the psychological consequences of the crisis indicates that the fallout on current generations will linger in complex ways over time [ ] .the inability to accurately predict how such changes might be associated with energy-relevant outcomes, or which changes might be more or less enduring [ ] , makes it all the more important to capture and consider them in the long run. where additional measures are included, we suggest the use of standardized approaches to the extent possible, such as widely used and validated scales employed in regular national surveys. this will allow commensurability with pre-covid- levels, while minimising construct and instrument validity challenges and the resource-intensive efforts associated with developing new measures (which require substantial testing to ensure scale reliability and validity). we show an initial mapping of variables as 'core' and 'consider' , in table . we also provide a checklist (see appendix) suggesting where and how to report those additional variables (and other considerations) in studies. when thinking about the potential effects of the covid- pandemic on the validity of research findings, it is also important to consider how it might affect research design. in this section, we briefly introduce issues relating to study design, sample selection and recruitment, and data collection methodology, as well as implications for interpretation of findings. suggestions on ways to report such considerations are also provided in the checklist (see appendix). it is likely that the pandemic will affect non-experimental research in different ways than it will affect experimental research. research focused on identifying associations might be especially vulnerable to threats to internal and external validity. more specifically, if the pandemic affects both the independent and dependent variables of interest, it can induce a spurious correlation (confounding; [ ] ). for instance, the pandemic might harm mental health and increase energy usage, making it appear as though the variables are related when they might not be, absent the pandemic. researchers can address this concern in the way that is typically recommended for addressing confounding: anticipate how the pandemic might affect your variables of interest, measure this set of variables, and test whether they affect the study's primary results [ , ] . the idea that 'correlation is not causation' is wellknown --but worth keeping salient especially at times when non-experimental research is being planned or altered at short notice. experimental designs are still potentially vulnerable to other pandemic-induced issues. experiments, by design, manipulate a specific variable of interest. for example, an experiment aiming to improve people's motivation to purchase or support renewable energy by means of messaging strategies might focus on the harm caused by fossil fuels to increase people's fossil fuel risk perceptions. however, the salience of such risks, and therefore their malleability, may be substantially decreased if people are preoccupied with other worries related to covid- . thus, researchers should consider such influences and, if possible, take measures to ensure that they can indeed manipulate the causal variable of interest in an effective and meaningful way. this is an empirical question for each manipulated variable, but we advise that researchers attempt to anticipate such issues and design their research accordingly. a clear consequence of the pandemic is that it will make it more difficult to conduct betweenand within-country comparisons where covid- impacts and restrictions are different. for example, home energy usage will be higher in places where people are required to stay at home. a useful rule of thumb is, wherever reasonably possible, researchers should contextualize their research by considering how political and cultural circumstances might affect their results (see section "capture and report on extra relevant data"; [ ] ). it would be even better to anticipate how such factors might affect results and design the study to mitigate them, such as collecting a sample that is relatively homogeneous in orders to stay at home, limit travel, or any variable that might substantively affect the results. if substantial heterogeneity of restrictions is anticipated within a sample, increasing sample size to maintain statistical power should be considered. independent of study type, a powerful way to get a measure of stability and validity of findings over time is to build longitudinal elements into the research design. first, researchers could consider building replications into their research plan. this can be done by intentionally splitting data collection over waves separated by a period of time. this allows for comparison of the variables of interest over the two waves. variables that remain constant over this period are likely to be relatively less affected by pandemic response measures than those which show variation. this approach lends itself particularly well to collaborations between research groups, which could consider teaming up to add variables of interest reciprocally onto the end of each other's studies, saving on budget and potentially introducing opportunities for new analyses. please refer to the section "capture and report extra relevant data" for more information on variable dimensions to consider. another possible approach to demonstrate the robustness of research findings over time could be through attempting to reproduce previous research findings --either of related research by the researcher themselves, or of previously well-reproduced effects. the extent to which previous findings are reproduced, or change, could help 'calibrate' the more recent research and give some insight into whether or not the domain of interest is more or less impacted by the pandemic and the corresponding response (also accepting that failure to reproduce findings is not an unusual occurrence even under normal circumstances [ ] ). data collection with a given research method could produce different findings now compared to before pandemic-related restriction measures were put into place. for example, research conducted online could be more heavily influenced by distracting factors of the participant's environment. where people are confined to their homes, completing a survey or conducting an interview in a standardized way might be more difficult than before. this consideration is especially important, since persisting restrictions of contact might result in a shift towards more research being conducted online versus in person. it is thus recommended to explore the possibility of using more than one method to investigate the same research question, and to record potential limitations specific to a data collection method to account for their influence on the validity of the findings. this is another area where collaboration between research groups with complementary interests could bring significant additional value by allowing testing of the same research question through different approaches and in different settings [ ] [ ] [ ] . while elaborating on additional or alternative methods, it is again important to consider ethical aspects. as mentioned earlier, the extra sensitivity of collected data has to be thought through, and in terms of data collection methods, researchers and analysts should make sure that data privacy and confidentiality is not undermined by new approaches [ ] . in the previous subsection we already highlighted the importance of giving due consideration to contextual factors. in respect of covid- , this means paying particular attention to the extent to which pandemic response measures (and changes in them across time and the sample) might have contributed to the observation of particular results. if possible, researchers should attempt to communicate and justify their best estimate as to the impact such factors could have had on findings. for example, if little systematic difference is observed in an outcome variable across groups who were substantially differently impacted by pandemic response measures, this could be offered in support of a case that the impact of covid- of that particular variable could be small. as in many areas of research, transparency is likely to be key in allowing users to make informed judgements of their own. any recommendations for policy, practice, or further research should be similarly transparent and include appropriate caveats on the context of the findings to which they relate. employing the principles set out above presents a number of opportunities that go beyond simply mitigating threats to validity, and could help generate new insights or improve research practice in general. the introduction of longitudinal elements can provide important insights on stable and dynamic determinants of energy-relevant outcomes, especially if combined with new contextual, behavioural, and other data that may not previously have been collected. such longitudinal studies could moreover contribute to the research question whether observed changes on the individual and societal level are caused by the pandemic itself (e.g., due to perceived threats and vulnerability) or by associated measures and consequences (e.g., due to lockdown and job loss) and thus provide insights into short-term and long-term effects of the pandemic. moreover, where collected data suggests that different groups of people have been (or will be) systematically exposed to different conditions as a result of the pandemic, natural experiments could be possible. natural experiments provide a powerful opportunity to investigate causal associations which may otherwise be difficult or impossible to control for (for an example see: [ ] ). these fleeting windows of opportunity can provide novel research opportunities and should be considered by energy researchers. the same window of opportunity will likely extend to policy interventions introduced in the wake of the pandemic to aid economic recovery. we already highlighted the possible benefits that could accrue from collaboration with other groups to facilitate replication and support validity, but there is also a wider convergence research opportunity in energy social studies during and after covid- . convergence research is a way of addressing complex problems through highly integrated interdisciplinary approaches [ ] . given the range and scale of current and anticipated impacts of the pandemic, such an approach is likely to be especially valuable, and opportunities to build inter-and transdisciplinary collaborations should be proactively sought. such collaboration may also provide a route to adding in important contextual data, for example through matching datasets. finally, we suggest that responding to validity challenges presented by the covid- crisis is an opportunity for the energy research field to step up and embrace practices around transparency and reproducibility that are now seen as standard practice in other areas of research. for reasons likely connected with the multidisciplinary and applied nature of most energy research, tools such as reporting guidelines and pre-registration of analysis plans are still rarely employed [ ] . it is possible that the particularly pressing need to demonstrate validity at present will result in familiarity with, and adoption of, tools that subsequently become standard practice for an increasing number of energy researchers, potentially enhancing the overall validity of research in the field. in much of the social sciences, knowledge on the most severe and pressing problems is often difficult to create and therefore constitutes a smaller proportion of thematic scholarship than its implications merit. the flip side of this is that 'low-hanging fruits' can suffer from excessive coverage. this impacts the 'body of knowledge' validity, which we define as the representativeness of research in a field relative to the real-world problems the field is concerned with [ ] . energy social science research, with its diverse methodologies, spatial and scalar foci, and associated differences of requisite time and effort, is no stranger to these tendencies. consider, for instance, the wealth of scholarship on local and urban energy initiatives in the uk, home to many energy research scholars, versus the relatively thin body of work on energy practices in rural sub-saharan africa. both issues merit attention and are generative for conceptual insight, but the latter affects over a billion people, many of whom experience relatively severe degrees of energy poverty, and yet hardly registers in terms of volume in relevant energy social science research. we detect a risk that curtailment of field-based empirical research, especially in regions that face severe energy challenges and may be heavily impacted by the epidemic, will exacerbate existing biases in representation in terms of volume (more desk study over ethnographic research than usual), methodology (potentially more conceptual work over evidence-based research) and regional coverage (less pandemic-impacted areas over more pandemic-impacted areas). to some extent, this is a perennial problem in any interdisciplinary or transdisciplinary field of study: ethnographic work in challenging regions with marginalised populations takes time and the classics on such topics that have accumulated over the years (in quite large numbers) consequently receive considerable attention. it is similarly evident in other fields of energy research, such as modelling, and outside of the energy domain. yet research today is heavily metricised, and most scholars with access to most global peer-reviewed scholarship are based in global north institutions and typically urban contexts, often with pressure to publish frequently. this leads to the double jeopardy of being pressed for time to focus on short-term impact, and of being far more likely to access highly-cited and highvolume segments of the scholarship one engages with. since the pace of research outputs has escalated, few scholars are positioned to navigate a body of knowledge with adequate care to balance its in-built biases of representation. already, we see moves to run online surveys and study social perceptions; even with all the appropriate caveats and the best of informed intentions, these contribute to a likely disbalance by volume of the sort of concerns that will get platformed in energy research journals in the short-to medium-term. how much coverage of marginalised, hard-to-access concerns -such as migrants cast adrift with little energy access, subsistence farmers with crop loss and inability to pay for fuel costs -will be lost and substituted by low-hanging fruit? such exacerbation of an existing bias can cloud future accounts and understandings of the true effects of a pandemic on the subject of energy research, i.e., on the global lived experience of energy. but it is not inevitable -it is an artefact of choices we make as an epistemic community. informed by recognition of likely biases, our choices (and those of funders, who can prioritise research on marginalised research areas) can embody normative commitment to proportionally match research coverage to real-world problems. we can productively draw on approaches such as convergence research highlighted above. this drive captures the essence of our contribution, which is to work toward a reflexive understanding of our role as a scholarly community at this time of crisis and opportunity. in this paper we have set out what we see as important challenges to the validity --internal, external, and of other forms --of social research in energy associated with the covid- pandemic and measures put in place to control it. we have suggested a number of principles we think researchers should consider applying to give themselves and the users of their work confidence that the findings and recommendations they present will still be valid in the years to come. these focus mainly on the collecting and reporting of additional contextual data, and the review of research design elements to ensure they are as robust as possible to pandemic-related impacts. we think that these principles can be employed with relatively minimal impact on resources and timescales required for research. they even present some opportunities both to enrich insight into social aspects of energy, and draw attention to measures to improve research transparency that are still as-yet under-used in the energy field. however, we also need to be mindful that due to limits on the kind of research approaches that can be employed during the pandemic, there are likely to be important gaps in the knowledge generated during this period. we all hope that the period of direct applicability of this paper will be as short as possible, and that measures to control the spread of covid- will soon no longer be needed. nonetheless, we also think that the considerations we raise here have enduring relevance for energy social science in general, and the potential to contribute to more widespread use of transparent, contextually aware and valid research practices in the longterm. the authors declare no conflicts of interest. table : checklist of items to report or consider reporting in relation to covid- pandemic validity challenges. report: main details of covid- response measures in action at the time/place of data collection, at least including: level of freedom to move around in public; degree to which schools and businesses are open. methods "at the time of data collection, public movement in the uk was severely restricted by government measures to combat the covid- pandemic. people were instructed to stay at home at all times, except for doing essential shopping, one period of daily exercise, working outside the home if work at home was impossible, and providing support to vulnerable people. all schools, hospitality venues and nonessential shops were closed." consider reporting: how covid- restrictions are applying to individual participants. results "in our sample, % of participants reported staying at home at all times except for when conducting essential shopping and exercise. a further % also reported leaving home to undertake work or volunteering. % of the sample reported staying at home at all times." consider tailoring of the following aspects of the research: research design methods "in response to the rapidly changing circumstances connected with the response to the covid- pandemic, we introduced a longitudinal element to our data collection. the survey was administered over two waves separated by two months, allowing us to check whether any of the key independent variables changed over this time, and whether this was associated with any change in the outcome." data collected (see table for suggested dimensions) methods "in addition to employment status, we also collected data on the extent to which those in employment were working from home." consider possible implications for: findings discussion "we found a strong association between altruism and stated willingness to participate over both waves of the study. however, the association was weaker in the second wave, which, combined with the change in reported application of covid- response measures (while other variables remained stable), suggests that conditions surrounding the covid- pandemic could have affected this finding." conclusion "our findings suggest that policymakers should prioritise energy saving messaging framed in terms of benefits to the local environment. however, our participants reported spending more time in their local area as a result of covid- control measures which could have influenced our result. we therefore recommend that the effectiveness of such messaging be carefully monitored." experimental and quasi-experimental designs for generalized causal inference uncertainty, complexity and post-normal science extended peer communities and the ascendance of post-normal politics european energy poverty metrics: scales, prospects and limits establishing the internal and external validity of experimental studies theory-testing, generalization, and the problem of external validity a nationwide survey of psychological distress among chinese people in the covid- epidemic: implications and policy recommendations americans' risk perceptions and emotional responses to covid- covid- ) from pandemic to recovery: local employment and economic development conducting social psychological research in the wake of covid- long-term effects of youth unemployment on mental health: does an economic crisis make a difference? new data suggest covid- is shifting the burden of energy costs to households promoting novelty, rigor, and style in energy social science: towards codes of practice for appropriate methods and research design the importance of heterogeneity in large-scale replications the importance of social relations in shaping energy demand using in-home displays to provide smart meter feedback about household electricity consumption: a randomized control trial comparing kilowatts, cost, and social norms measures to tackle the covid- outbreak impact on energy poverty: preliminary analysis based on the italian and spanish experiences ethnicity and covid- : an urgent public health research priority how are americans coping with the covid- crisis? key findings from household survey survey tool and guidance: behavioural insights on covid- , who what sticks? ephemerality, permanence and local transition pathways, environmental innovation and societal transitions three conditions under which experiments and observational studies produce comparable causal estimates: new findings from withinstudy comparisons can nonrandomized experiments yield accurate answers? a randomized experiment comparing random and nonrandom assignments estimating the reproducibility of psychological science many labs : investigating variation in replicability across samples and settings crowdsourcing hypothesis tests: making transparent how design choices shape research results scientific utopia iii: crowdsourcing science on the responsible use of digital data to tackle the covid- pandemic mask-wearing increases after a government recommendation: a natural experiment in the us during the covid- pandemic convergence research in the age of big data: team science, institutional strategies, and beyond how to improve transparency when theories become tools: toward a framework for pragmatic validity while this work did not receive any dedicated funding in its own right, the individual authors gratefully acknowledge funding as follows. key: cord- -tkp tifx authors: saberi, parya title: research in the time of coronavirus: continuing ongoing studies in the midst of the covid- pandemic date: - - journal: aids behav doi: . /s - - - sha: doc_id: cord_uid: tkp tifx nan a novel human coronavirus called severe acute respiratory syndrome coronavirus (sars-cov- ) was identified in late in wuhan, china, resulting in a pandemic [ ] . due to the need for physical distancing and isolation, universities closed and most medical clinics were ordered to continue with "essential" in-person visits only. in addition to a wave of medical appointment cancellations, many medical research activities, such as participant recruitment, study visits, and outcomes assessments were halted. in light of rapid societal change in response to the pandemic, this paper summarizes our considerations for conducting remote research. the discussion provided here is not exhaustive, but rather a starting place for a fundamental shift in how research is conducted. even without a pandemic, over % of trials close without meeting target accrual rates [ ] , underscoring the need to investigate new methods for research recruitment. reports from the pew research center show %, %, and % of individuals ages - , - years, and - , respectively, use some form of social media [ , ] . this indicates a wide reach for online advertisement and recruitment for research, with the added potential to recruit populations typically considered hard-to-reach [ ] . compared to traditional recruitment methods (e.g. print and television), studies using advertisements via social media have been shown to be financially feasible, attract large numbers of individuals, and have condensed recruitment periods. they have also been shown to provide opportunities for connecting with individuals with specific health conditions, living in remote geographic locations, or who may have been difficult to recruit due to stigma or medical mistrust [ , ] . some examples of social media to advertise and recruit participants include: social networks (such as facebook, instagram, and twitter), dating apps (grindr, scruff, and jack'd), and online listservs for various medical societies or health conditions. the main disadvantage of using social media for study recruitment is the lack of reach to those with limited or no internet access. this can result in reduced generalizability of research findings [ , ] . researchers concerned about generalizability, however, can supplement online advertisement with traditional methods. another remote recruitment method involves networkbased online referral strategies. one example is respondentdriven sampling for online research which involves recruiting initial participants (i.e. "seeds") who are then asked to recruit friends to enroll in the study, and so on [ ] . finally, a host of specialist medical research recruiting agencies now exist that tout increased recruitment efficiency. screening (or pre-screening) can be conducted via simple web forms on a study website. this can increase web traffic to the study website, decrease the workload for research staff, and increase a potential participant's trust in the legitimacy of the study (e.g. if the webpage is part of a reputable university with an "edu" domain). the main disadvantage of online screening is that it may either deter some to participate (e.g. if the form is too complicated or lengthy) or increase inappropriate participation (e.g. an individual or bot responding repeatedly to be eventually deemed eligible). one option to minimize these barriers is to include a simple pre-screening form on the study website and to contact the interested individual by phone call or text message to complete the screening process. to ensure certain key study inclusion criteria, participants can be asked to text message photographs of documents. for example, an identification card bearing their name and date of birth to verify age or a photograph of a letter of hiv diagnosis or antiretroviral medication vial bearing their name to verify hiv serostatus [ , ] . signing consent and medical release forms can also be conducted online using various tools such as docusign or a qualtrics survey [ , ] . detailed review of the consent process is critical to ensure an individual's understanding of the study. there are numerous ways of providing remote interventions. text messaging has been used frequently for motivational messages, reminders, and ecological momentary assessments [ ] [ ] [ ] . many text messaging platforms have been developed and evaluated for hipaa-compliant communication with participants [ ] . telehealth via videochat platforms have also been used for the provision of study interventions [ ] . this modality lends itself well to interventions that do not require physical contact but are enhanced by face-to-face communication, such as interventions for mental health, smoking cessation, and medication adherence counseling. participants in prior studies noted high acceptability levels in using videochat for research intervention delivery [ ] . benefits included being able to speak more candidly and being less intimidated, and experiencing reduced barriers for research participation (e.g. financial barriers related to travel expenses or time off from work, stigma associated with research participation, and physical disabilities precluding mobility) [ , ] . in addition to providing interventions, these platforms can be used for one-on-one qualitative interviews and focus groups. they allow for video-or audio-recording of the session (with consent from the participant) which can be used later for interview transcription and analysis. other remote methods of delivering an intervention include mobile health applications [ ] , computers-based programs [ ] , and the internet [ ] . in addition to self-reported outcomes, which may be subject to recall and social desirability biases [ , ] , objective monitoring of study outcomes can be conducted remotely in numerous ways. for example medication adherence can be evaluated by drug hair concentrations using mail-in hair samples [ ] [ ] [ ] and text messaged photographs of pill counts or refill dates [ ] [ ] [ ] . adherence has been monitored using medication event monitoring system (mems) caps, ingestion sensors, and "wise"-products [ ] [ ] [ ] [ ] [ ] . some labs provide infectious disease testing services which can examine th generation hiv antibody testing using dried blood spot and chlamydia and gonorrhea testing using mail-in swabs, as well as mail-in samples for hepatitis b and c testing [ ] [ ] [ ] . alcohol use can be remotely monitored using bluetooth-enabled breathalyzers that estimate breath alcohol concentration or wrist-worn alcohol biosensors that continuously measure transdermal alcohol content [ ] [ ] [ ] . study assessments can be conducted via online surveys which can be emailed or text messaged to participants. when surveys are conducted online, research has shown less social desirability bias, especially related to sensitive health information [ ] . in cases of limited literacy, research staff can read questions to study participants or use survey platforms that include the ability to audio-record questions. qualitative research can also be conducted using telephone, videochat, or remote focus group platforms currently typically used for marketing. study incentives can be provided remotely in numerous ways. for cross-sectional surveys or one-time assessments, provision of e-gift cards is the most convenient remote incentive. for longitudinal research, reloadable debit cards are a convenient method of payment and have been shown to be highly feasible and acceptable by research participants [ ] . these debit cards can be mailed to participants without any funds loaded until confirmation of receipt and funds can be transferred from one card to another in case of lost or stolen cards. these are important benefits of reloadable debit cards over mailed gift cards. in the era of pandemics, such sars-cov- , there is a need to continue research activities, while keeping research participants and staff safe. aligning research activities with remotely-conducted research methodology has the potential benefits of reducing time and cost for conducting the study, improving ease of participation for many individuals, enhancing the generalizability of findings, and increasing the speed of publication of study findings, all while preventing potential viral 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protocol wyz: a pilot study protocol for designing and developing a mobile health application for engagement in hiv care and medication adherence in youth and young adults living with hiv meta-analysis on the effect of text message reminders for hiv-related compliance social inequity and structural barriers to completion of ecological momentary assessments for young men who have sex with men and trans women living with hiv in san francisco mobile phone text messaging for promoting adherence to antiretroviral therapy in patients with hiv infection scoping review and evaluation of sms/text messaging platforms for mhealth projects or clinical interventions overcoming technological challenges: lessons learned from a telehealth counseling study. telemed e-health a pilot study to engage and counsel hiv-positive african american youth via telehealth technology use of technology for delivery of mental health and substance use services to youth living with hiv: a mixed-methods perspective computerized counseling reduces hiv- viral load and sexual transmission risk: findings from a randomized controlled trial test of a web-based program to improve adherence to hiv medications practical and conceptual challenges in measuring antiretroviral adherence assessing the association between self-report items for hiv pill adherence and biological measures moving antiretroviral adherence assessments to the modern era: correlations among three novel measures of adherence feasibility and acceptability of novel methods to estimate antiretroviral adherence: a longitudinal study novel methods to estimate antiretroviral adherence: protocol for a longitudinal study real-time and wireless assessment of adherence to antiretroviral therapy with co-encapsulated ingestion sensor in hiv-infected patients: a pilot study cognitive behavioural therapy for adherence and depression in patients with hiv: a three-arm randomised controlled trial consortium for ring a biomarkers and biometric measures of adherence to use of arv-based vaginal rings feasibility, performance, and acceptability of the wisebag (tm) for potential monitoring of daily gel applicator use in durban real-time adherence monitoring for hiv antiretroviral therapy innovative approach for enhancing testing of hiv, hepatitis b, and hepatitis c in the general population: protocol for an acceptability and feasibility study evaluation of the 'colli-pee', a first-void urine collection device for self-sampling at home for the detection of sexually transmitted infections, versus a routine clinic-based urine collection in a one-to-one comparison study design: efficacy and acceptability among msm in belgium acceptability and feasibility of self-collecting biological specimens for hiv, sexually transmitted infection, and adherence testing among high-risk populations (project caboodle!): protocol for an exploratory mixed-methods study temporal dynamics of transdermal alcohol concentration measured via new-generation wrist-worn biosensor the feasibility of using smartphones and mobile breathalyzers to monitor alcohol consumpti on among people living with hiv/aids. addiction science & clinical practice wristworn alcohol biosensors: strengths, limitations, and future directions a comparison of web and telephone responses from a national hiv and aids survey acknowledgements special thanks mr. nikolai caswell and ms. albaloo saberi for their help on this paper. research reported in this publication was supported by the california hiv/aids research program (chrp) award number hd -sf- (saberi) and the national institutes of health award numbers r mh (saberi) and r mh (saberi). key: cord- -r y hz q authors: lavretsky, helen title: scientific autobiography of a spiritual seeker in the year of hindsight's / .: “was i deceived, or did a sable cloud turn forth her silver lining on the night?” john milton “comus” ( ) date: - - journal: am j geriatr psychiatry doi: . /j.jagp. . . sha: doc_id: cord_uid: r y hz q nan activities are suspended indefinitely, and i have plenty of time to reflect upon the meaning of life. we are living during unprecedented and difficult times when the entire world is asked to find its spiritual center and resilience in order to find collective solutions to its many problems. the surreal nature of the covid- pandemic and the global lockdown makes each of us look back and ask "how did i get here?" followed by "and how do i move forward?" reflections upon our life choices that resulted in the situation at hand can be very revealing and can help define our next chapter. perhaps, we have been caught in the pre-programmed game of rules and expectation, and we can use this imposed pause to create a new game with the new rules. for the first time we are asked to consciously consider our choices that define our daily lives and determine our survival that is a truly global existential crisis. the covid- pandemic pushed us to "go within" and to take personal responsibility for our existence. however, unfolding events have made us also more hopeful in our ability to create the new order that will support our lives in a new way. we are already witnessing high-paced scientific and technological innovation ( ) that will require united trans-disciplinary efforts to meet the demands of the world"s mental health post-covid- that are described in our upcoming book co-edited with harris eyre, michael berk and charles reynolds "convergence entrepreneurship" that will be published later this year ( ) . my "scientific activities" have been a big part of my spiritual journey focused on seeking to understand the true nature of human mental and emotional suffering and resilience, where all life events are assumed to provide valuable lessons and "silver linings" that ensure individual and collective evolution of consciousness. i look forward to this unprecedented opportunity for reinvention of ourselves, our world, our science, and the global evolution of consciousness as a result of our collective search for peace and alleviation of suffering. i grew up in moscow, russia. i often say that i was born to become a psychiatrist, in part, because, my mother was (and still is) a psychiatrist and my father was a neurologist and i had an easy access to the large library that introduced me to the workings of the mind and brain. before i turned , i read books about cerebral palsy and down syndrome and was strangely attracted to the pictures of young children who were clearly suffering. a natural empath, i wanted to help the suffering. my first "patient" was my little friend, a three year old boy who was completely mute. i was able to understand his wishes intuitively and translated them to the adults. although later, i went through decades of medical and psychiatric training, the essence of what i do for patients is not different: empathically understanding their suffering, translating it to the world, and helping in alleviating suffering by empowering them to change their lives. during high school, i developed an interest in psychiatric research, and my first summer job was in a psychiatric hospital" pharmacy. i watched patients who wandered in the beautiful gardens of the psychiatric hospital where they worked in the green houses as a part of their vocational rehabilitation program (the main staple of psychiatry in russia) and tried to imagine what was going on in their minds. for high school science projects, i chose to perform hypnosis on my classmates, as was described in the book on hypnosis, using a pendulum, and described and classified their responses. the following year, after reading an old french phrenology book, i examined their skulls and made phrenological descriptions of their personality based on the skull topography, which gained me some popularity among my schoolmates and was a precursor of my current interest in mindbody interventions and brain biomarkers. psyciatry. at that time, he suggested that i write a paper on the russian concept of schizophrenia because nothing was known about it in the us ( ). this paper was published in the schizophrenia bulletin, and also served as a cathartic nine-month-long journey that helped release all of the "soviet" experiences. in the process of working on it, i discovered my passion for academic writing. this was the only paper of to-date that received a single line review "a magnificent contribution". this was more of a "passion project" that one of my supervisors called "avocational," but i highly recommend this type of experience to learn about moving beyond your comfort zone and pushing your own limits. later, i also learned that merging personal interests (e.g., in yoga) with professional activities can eradicated burnout and lead to the ultimate job satisfaction. all you always wanted to know about "academic ladder," but were afraid to ask despite a relatively "smooth sailing" through the academic system, the intricate details and secrets of academic success were still elusive, and the road was rather "bumpy". i attended workshops put together by the american psychiatric association (apa) appropriately entitled "swimming with sharks" explaining unwritten and untold "rules of engagement" and the relational hierarchy that seemed intentionally complex and biased. in the s, there were very few women mentors and role models to share their wisdom and the "operating manual." i used to say that i was well prepared to be "a woman in academia" because i had been "a jew in russia," and after i had read that women in academia had . children, i felt that i was a close match with my . son. many academic institutions and scientific societies have recently recognized that the implicit biases that we all harbor can be barriers to fairness and progress and have since provided greater resources and training. also, the landscape of academic medicine and psychiatry has been rapidly changing and becoming more diverse, with over % of the psychiatry residents being women and minorities and % foreign medical graduates. these difficult experiences at the early stages of my professional journey have made me more sensitive to the implicit and explicit biases within academic medicine, and i now use the knowledge and skills i gained in mentoring others. the main source of professional support, collaborators, mentors and mentees, and simply, in the single year of . i called that the trifecta of , the year of cancelled meetings, which took place in the virtual sur-reality of the covid- pandemic. i am certainly hoping that many more will be able to achieve these honors in years to come. i am forever indebted to the numerous mentors i met through these programs who became my colleagues and friends, and later, close collaborators. many former scholars like myself went on to become the sri/cima/ari mentors that also cemented crossgenerational lines of the organizational wisdom transmission. this was also a good way to learn the "do"s and don"ts" of mentoring that helped in shaping up my own mentoring identity. today, i learn as much from my mentees as they learn from me as a result of this training. the big discovery early in my career was the need to participate in the competitive peer- modern research is highly complex and requires collaborative work to develop novel ideas and utilize individual talents and the cutting edge technologies in order to advance the field. research community forms a "group-consciousness" that defines the direction of the field"s development and benefits from the advanced "laboratory" that is a research depression that was recently challenged by the covid- pandemic. we have proceeded with collaborative problem-solving, learned from each other, and supported each other, making the challenges much easier to cope with. "a greater truth" about that nature of late life mood and cognitive disorders will emerge from this collaboration and will be the source for new research ideas for years to come. another bit of advice regarding developing research ideas and securing funding that i received was to have an idea that is novel "enough" but not too far "ahead of the curve" in order to be accepted as a "fundable idea." the original impetus of studying brain- in the later years, new research questions originated from my clinical experience. i wanted to answer clinically relevant questions on the behalf of the entire field. for example, the decades-old question of whether depression and cognition improve with the addition of methylphenidate has been of interest to many psychiatrists and primary care physicians. with the help of the r- grant funding, i conducted a study on methylphenidate augmentation of citalopram and the findings of this study put "a nail in the coffin of the decades-old question" proving that the addition of methylphenidate could accelerate and improve treatment response in older adults. because of its high clinical relevance, this paper was named among the top ten articles in psychiatry by the new england journal of medicine in ( ) . i also learned to use the intervention studies to understand brain mechanisms of treatment response, while developing novel pharmacological and behavioral interventions and mastering advanced research tools like neuroimaging, genetics, inflammatory markers ( ) ( ) ( ) ( ) . in the early stages of careerdevelopment, my traditional and "expected" neurobiological direction in academic psychiatry was easy to adopt and keep "ahead of the curve" to identify the next important question to answer. at the stage of mid-to-senior career development, one is encouraged to take risks in order to move forward and retain a sense of purpose. it is important to re-invent oneself periodically to avoid repetition. my "dizzying" turn-around shift occurred when i encountered a kundalini yoga practice that captured my imagination by its observed health benefits. it occurred during a very stressful time in my life, and i was looking for tools for stress reduction. everything about this yoga practice seemed amazing: learning about mind-body connections via yogic body postures, breathing, chanting, community, gathering, and vegetarian food. i pursued yoga teacher training and certification followed by years of rigorous practice resulting in the complete makeover of my body, mind, immune system, and discovery of my own spirituality. my colleagues were amused by the transformation of my reductionistic "neurobiological self" into my newly discovered "spiritual self" that emerged, along with my desire to study the brain, health, and consciousness effects of mind-body practices. i turned my attention to developing studies of yoga, tai chi and meditation in older adults and stressed dementia caregivers. the initial studies preceded "the curve" by - years and were some of the earliest studies of mood and brain effects accompanied by profound epigenetic changes, anti-inflammatory effects, and most importantly, direct neuroplastic effects and cognitive improvement ( ) ( ) ( ) ( ) ( ) ( ) . the findings were novel and well-received by the medical and research communities, and especially, by the yoga communities around the world. this was also an opportunity to turn a passion project into a professional, evidence-based one. a number of new collaborations outside of the comfort zone and the field now include mouse biologists, stress biologists, respiratory physiologists, anthropologsits, and neuroscientists. our goal is to develop a translational center dedicated to the study of mechanisms of breathing control of emotion regulation in the mouse and human models of anxiety and panic. our hope is that this work will take the field of mind-body medicine even further in promoting understanding of how our breath can help regulate our emotions during stress or panic. this has become particularly relevant given the recent global distress emphasizing the importance of promoting personal wellbeing using ancient breath practices as the simplest and all-encompassing solution. in addition, after thirteen years of conducting mind-body research, we have started an integrative psychiatry clinic that uses mind-body, lifestyle, and spirituality-based approaches to help patients with neuropsychiatric symptoms. we hope to empower our patients to take control of their own health and learn resilience-building tools to allow for self-regulation during these difficult times and for stress-related psychiatric disorders. now, more than ever before, the healthcare system is ready to endorse integrative medicine that has accumulated evidence of its low-cost effective therapies for stress-related disorders. experimenting with mind-body research and with my own yoga immersion led me to shift from the medical disease models to the health-promotion and wellbeing models, with resilience being a mechanism of maintaining wellbeing ( ) . antidepressants and psychotherapy can improve resilience and prevent depression recurrence. however, providing patients with the ability to learn about their own strengths and utilize their lifestyle choices and spirituality to improve treatment outcomes is another powerful therapeutic and preventive approach to neuropsychiatric diseases of late life, such as depression, caregiver stress and dementia. during this pandemic, the entire world population can benefit from this knowledge and skills of stress reduction that may continue to be beneficial post-pandemic as well. as a clinician and a researcher, it is much more gratifying to understand patients from the point of their own strengths and to empower them to take charge of their health by learning and using the tools of selfregulation of their choice. these resilience-boosting techniques can be powerful additions to the traditional psychiatric practices. the best way to master the subject is to write a book. most of these ideas are expressed in laird, adrienne grzenda, and beatrix krause-sorio, who shared my scientific passions and co-authored many published papers that made writing them so much more fun. all successes and challenges proved to be useful lessons, silver linings and blessings indisguise that led to a greater wisdom and growth. i am certainly looking forward to the new adventures and progress in next years. author contribution: helen lavretsky was the sole contributor to this manuscript. there are no conflicts. rebooting geriatric mental health innovation and entrepreneurship with convergence science convergence mental health: a roadmap towards transdisciplinary innovation and entrepreneurship the russian concept of schizophrenia: a review of the literature programs for developing the pipeline of early-career geriatric mental health researchers: outcomes and implications for other fields optimizing 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cord- - zq tln authors: vaz, manjulika; timms, olinda; johnson, avita rose; s., rathna kumari; ramanathan, mala; vaz, mario title: public perceptions on controlled human infection model (chim) studies—a qualitative pilot study from south india date: - - journal: monash bioeth rev doi: . /s - - - sha: doc_id: cord_uid: zq tln research using controlled human infection models is yet to be attempted in india. this study was conducted to understand the perceptions of the lay public and key opinion makers prior to the possible introduction of such studies in the country. respondents from urban and rural bangalore district were interviewed using qualitative research methods of focus group discussions and in-depth interviews. the data was analyzed using grounded theory. safety was a key concern of the lay public, expressed in terms of fear of death. the notion of infecting a healthy volunteer, the possibility of continued effects beyond the study duration and the likelihood of vulnerable populations volunteering solely for monetary benefit, were ethical concerns. public good outcomes such as effective treatments, targeted vaccines and prevention of diseases was necessary justification for such studies. however, the comprehension of this benefit was not clear among non-medical, non-technical respondents and suggestions to seek alternatives to chims repeatedly arose. there was a great deal of deflection—with each constituency feeling that people other than themselves may be ideally suited as participants. risk takers, those without dependents, the more health and research literate, financially sound and those with an altruistic bent of mind emerged as possible chim volunteers. while widespread awareness and advocacy about chim is essential, listening to plural voices is the first step in public engagement in ethically contentious areas. continued engagement and inclusive deliberative processes are required to redeem the mistrust of the public in research and rebuild faith in regulatory systems. a chim is a 'controlled human infection model' study in which a well-characterized strain of an infectious disease agent is administered at a controlled dose by a specified route to healthy adult volunteers to evaluate the disease itself, its treatment, or the efficacy of candidate vaccines, among others. chim studies can lead to better understanding of the mechanism of infection, pathogenesis of disease, contributing host factors, microbial virulence, treatment protocols and vaccine induced responses; and can evaluate potential vaccines. (gordon et al. ; gopichandran ; who ) . chim studies have a varied lexicon and are also known as 'challenge studies' as the body is challenged with a microbial pathogen as part of the research model, and more recently as 'experimental infection' (langenberg et al. ; todnem et al. ) . animal studies may be inadequate in predicting human responses to interventions. aided by advances in immunology, functional genomics, microbiomics, pharmacogenetics, pharmacokinetics and pharmacodynamics, a chim study has evolved as a new methodology in modern infectious disease research (balasingham et al. ; porter et al. ) . however, only % of chim studies are carried out in low, middle income countries (lmic), where the need for research on infectious diseases and vaccines is higher (gordon et al. ; elliott et al. ; selgelid and jamrozik ) . further, cross immunity and biological variability in different populations can alter host-pathogen dynamics and response to vaccines, making extrapolations of study results from other populations difficult. since % of the global infectious disease burden is contributed by india, there is clearly a need for infectious disease research in india and the possible introduction of chim, in particular. chim studies are currently not done in india. chim studies are believed to accelerate vaccine and drug development, making the process efficient and less costly (balasingam and wilder-smith ) , a benefit that could be important for india (gordon et al. ; gopichandran and kang ; dholakia ) . inadequate infrastructure, training and resources, and the absence of a clear regulatory framework are some of the reasons why chim studies are not done in india (gopichandran and kang , timms ). the history of atrocities associated with experimentation in humans could be a cause for apprehension with human testing in chim. (pollard et al. ) as chim studies intentionally infect healthy human volunteers and cause disease, it challenges the bioethical principle of 'primum non nocere' or 'non-maleficence'. thus, these studies would be viewed differently from human studies that typically evaluate therapeutic interventions (balasingham et al. ; rose ; johari ; vaz ) . at present, chim studies are typically conducted in non-endemic, high-income countries where volunteers are usually college students. apart from difficulties with extrapolations due to the genetic profile and environmental factors, it is ethically questionable for a population to be exposed to risk when there is little personal or community benefit. in india, where the understanding of biomedical research itself is low (vaz ) , acceptance of a study design that includes purposeful infection and quarantine, could be a challenge. in addition, research participants here are often less educated or socially disadvantaged with poor 'health literacy' (vaz ) . the validity of chim studies can be questioned in light of the more pressing need to address the social determinants of health. from a social justice viewpoint, chim studies may appear to promote immediate medical/technological solutions over mandates for long-term, sustainable provision of basic human living and environmental conditions that can prevent disease. the scholarly article by bambery et al ( ) , suggests four requirements for human challenge studies to be ethical '(i) conduct independent expert reviews, including systematic reviews; (ii) ensure a publicly available rationale for the research; (iii) implement measures to protect the public from the spread of infection beyond the research setting; and (iv) develop a new system for compensation for harm' (bambery et al. ). there is scarce empirical bioethical literature currently available in india to guide researchers and research ethics committees in navigating the complex ethical issues of purposefully infecting healthy volunteers. it appears that the central ethical dilemma is of individual risk vs public (public health) benefit. deliberative public engagement is recommended for policy development in contested ethical areas (molster et al. ) . interactions with the public on biomedical research, benefits of participation, and protective bioethical guidelines and laws is likely to promote social acceptance and empowerment within the community. it is also important that ethics regulations evolve 'bottom up' and are not exclusively 'expert-based' as the latter risks being "one-sided, biased or ideological-thus illegitimate" (schicktanz et al. ) . listening to the voices of the people also ensures that the notion of 'public good' encompasses multiple perspectives and standpoints (london ) . key outcomes of public engagement are greater transparency of purpose and procedures, the ability to understand and anticipate problems at individual and societal levels, issues of vulnerability and methods to address them; fears and concerns around risks of participation and trust building between the scientific community and the public. public engagement can improve people's participation, trust and confidence in the researcher, and understanding of the safeguards in research. public participation in the development of bioethical guidelines that protect their interests, establishes the public as a key stakeholder in research (blom and de vries ) . since chim research studies have yet to begin in india, it presents an opportunity to do things differently this time around, creating guidelines, processes and infrastructure in a transparent and inclusive manner, to dispel apprehensions born of past clinical trials, and advance vaccine science in a sustainable, safe and cost-effective manner. this paper is the outcome of a study aimed to engage with the public and local stakeholders on their perceptions of acceptability, concerns and participation in chim studies, in the indian context, and thereby influence guidelines to be more people-centered. qualitative research methods and the grounded theory approach (corbin and strauss ) were used in this exploratory study of public perceptions, to generate insights into this new area of medical research. focus group discussions (fgds) and in-depth interviews (idis) were the methods of data collection. the details are described below. the study was conducted in the urban metropolis of bengaluru and in the surrounding villages of bengaluru district. bengaluru is the fastest-growing indian metropolis after new delhi, with an estimated population of . million in its urban area (as of ). about % of residents in bangalore live in slums. much of the population growth in bengaluru is due to migration from other states in india (bangalore population ). bengaluru was selected as the study setting as it is the location of the primary study team and is also a hub of biotechnology and clinical research (pulakkat ) . . % of bangalore district is urban (census - data), with a small proportion remaining rural. mugalur village in the sarjapur hobli (cluster), of anekal taluk (administrative block), bangalore urban district, kms from bangalore city was chosen as the area for collection of rural data. the medical college and the department of community health (arj, rk) conduct an outreach/ community health program at that location. participants were of two types-the general public and key informants. they were selected from bangalore city and mugalur village (anekal block, bangalore district). participation was completely voluntary. participant selection was purposeful and aimed at accessing a cross section of society. the key strata of general public covered were males and females, youth and middle aged, members of the public belonging to specific work categories-college students, information technology professionals (bangalore is the it hub of the country), un-organized sector workers, and mid-income school teachers in the urban areas and members of women's self-help groups, farmers collectives, community health workers and youth groups in the rural areas. local contacts were used to identify potential participants. table provides the number of participants in each category, their age groups and work profiles. fgds were the method of data collection from the general public as it was more practical to meet them as a group. a focussed, guided discussion also makes lay people more comfortable with sharing, as they are not experts in this area. in addition, decision making among these communities in real life is usually through consultation and discussion. the fgd participants were between and years. of the participants, there was nearly equal representation of younger (< years) and older participants (> years). about two thirds were married. majority of the urban respondents were graduates and post graduates while the rural respondents had completed secondary or higher secondary education. people belonged to different religions and caste groups, though many urban respondents were unwilling to disclose religion and caste. they came from various occupational backgrounds: students, salaried employees or daily wage labourers in urban areas. most of the rural women were homemakers, while the rural men were farmers or weavers. many of the urban fgd participants refused to disclose their monthly income. where reported, income levels between the rural and urban participants were extremely disparate. the socio-economic profile of the rural participants was closer to the urban poor. key informants (kis) were in all. in the rural area they were people who understood the community, were influencers in decision making and were engaged with community health outreach work. in the urban area, the kis were those knowledgeable about communicable diseases, infectious disease researchers, ethics committee members and those likely to influence public opinion. they included microbiologists, clinicians, public health workers, social workers, research ethics committee members, media representatives, lawyers, and human rights activists. kis were between and years of age. the in-depth interview method was used for this category, as each was an independent professional with recognised expertise. table has a list of the kis. the research team included the principal investigator (mv), a social scientist with many years of experience working with communities and qualitative research, an ethicist (ot), two public health researchers (arj, rk) and a medical researcher (last author) with many years of experience in epidemiology and social science research. mr is an experienced qualitative researcher and a public health scientist who joined the team at the stage of data analysis and interpretation of data. a focus group discussion guide was developed, pilot tested and fine-tuned. prior to the fgd, participants were reassured that the discussion was not aimed at recruiting subjects for chim research, but intended to understand their views and opinions, there being no right and wrong answers. the discussions flowed naturally and followed the narratives of the respondents and were not limited by the topic guide. a warm up section of the fgd covered a discussion on medical research and ethics. to trigger the discussion on a chim study, a generic case scenario was presented as follows: "a chim study involves giving an infectious agent (a carefully identified low infective form of a germ) at a controlled dose and controlled conditions to carefully selected healthy adult volunteers. the main reasons to conduct a chim study are to understand the exact way that the infection affects humans, human responses and the length for which the germ remains in blood and body fluids…". the three areas of focussed probe included ( ) perceptions of benefit and concern, ( ) understanding who a potential volunteer could be and the nature of consent needed, ( ) issues around compensation and its influence on participation. the last section of the guide focussed on the subject of public good and individual altruism. the topic guide for the idis was similar to that of the fgds but did not include basic awareness of research and ethics. (copies of the topic guides are available with the first author). data collection was done by rk in the rural areas with mv and arj as note takers and was done by mv and arj in the urban areas. an audio recorder was used after the respondents' consent. while english was the language of most fgds and idis in urban areas, kannada, the local language of the region, was used for the rural fgds and idis and the lower socio-economic groups in the urban areas. between june and september , eleven focussed group discussions ( rural, urban) and in-depth interviews ( rural, urban) were completed covering a total of respondents. data collection continued till data saturation was reached. audio recorded data was translated and transcribed into textual documents by an external agency. the transcripts were read by data collectors arj and mv for errors in content or meaning. all the data collected were analysed using the qualitative method of grounded theory and constant comparison. data analysis was an iterative process and began when the first transcript was received. open inductive coding was done by mv and arj, a coding framework was developed consisting of a priori codes (those from the topic guides) and de novo codes (those emerging new from the data) using nvivo software, version . . and was an ongoing process. even when mv and arj were coding independently, both researchers would recheck the codes, and modify or add if necessary, to the coding framework. the coding framework is provided in table . themes have been delineated and presented in the results section. all the study team members were involved in mapping the emerging themes into a conceptual model and this followed a reflexive process. the study was designed following the ethical guidelines of the indian council of medical research. approval of the study protocol, the subject information sheets and consent forms in english and kannada was received from the institutional ethics committee of the primary research team's institution. (iec study ref no / dated th may . after reading and/or discussing the study information sheet, all respondents provided written consent for participation, audio recording and for de-identified quotes to be included in publications. participants were assured of confidentiality, freedom from coercion to speak or participate, and sharing of findings if the participant wished. the perceptions of the general public towards chims have been presented under six major themes (see fig. ). views of the key informants-the health and research experts and the opinion influencers-the media, activists and legal experts have been used to explain the public perceptions or to provide contrasting perspectives. illustrative quotes (with affiliation codes) have been provided to support the themes and sub themes. as a backdrop to the perceptions of the public to controlled human infection trials, we have two subsidiary themes-the basic perception of 'research' among the lay public and their perceptions of ethics and its place in research. there was a clear socio-economic and geographical divide in the understanding of the meaning of research in general. there was less understanding of research among the rural poor. urban groups and individuals had a clearer understanding-some of the notions they expressed were related to the idea of clinical trials, a focus on 'medicines' and the long timeframe for the outcomes of research to reach the public. the ideas about who did research included 'pharma' companies; i know that people go through clinical trials …there are pharmaceutical com-panies… (urban fgd ;); …there are clinical trials [and] all these various aspects, to ensure it [a drug] is completely safe for the public… …you realise it is just a preliminary indication and that whatever the benefit to the public, is around - years later. (urban-idi- ). the idea of ethics went beyond medicine and emphasized desired behavior in everyday life. words that were used to indicate the idea of ethics in the local language included: sabyatha (decency, civilized), sampradaya (traditions, spirituality), maryada (respect, uprightness), naithika (ethical), sambanda (relationship with others), gaurava (honor, prestige), maanavyamoulya (human values), ganathe ( dignity),, vyathiyanadathe (behavior), naithika moulya (following ethics), and siddhantha (principle, tenet) (ventakatasubbaiah ; asian educational services ) . in addition to words, understanding of ethics was also expressed in phrases that appeared to be linked to some of the principles of ethics-for instance, • 'it is about considering others before self' (urban-fgd )-beneficence, • 'no intentional harm to anyone' (urban fgd )-non-maleficence, • 'i think ethics is more like moral rights among the people' (urban fgd )autonomy of choice, • 'we should not underestimate any person or degrade' (urban fgd )-respect and dignity, among others. ethics was seen as important in research to reduce harm-'ethical guidelines should try to avoid collateral damage' (urban fgd- ); 'medical research team should be confident that there will not be harm to that person' (rural idi- ); and, to regulate the processes and purpose of research-'all the procedures and norms in a proper way'(urban fgd ). they did, however, also identify the gap between intent and practice-they [researchers] will concentrate only on research they don't consider anything about ethics (rural fgd ); pharmaceutical companies recruit people from poor backgrounds for human trials (urban fgd ); commercialization has outgrown the benefit of medical research. (urban fgd ). a chim was seen as ethical if there was a clear benefit. this was echoed across both rural and urban settings. ..i feel it is good if it helps in the prevention of diseases (rural fgd ); we can find the treatment for disease… (urban fgd ). however, many felt that they could not understand the benefit of chim clearly. respondents felt that if chim studies had their knowledge base in data from animals it would be more acceptable-this concern seemed to be overwhelmingly about safety. there was also the strong perception that infecting a healthy person was wrong and that studying the natural course of the disease of an already infected person would be the more ethical, alternative option. an exception was during an epidemic when 'to save those many lives this action should be taken… until then i think this is not required.' (urban fgd ). it was also considered important that participation in such studies should be with voluntary consent and with a complete understanding of the study and what it involved. as school teachers in a group discussion said, it should be [done] voluntarily and it should not be the people who don't understand consequences and just do it for the money…they should have the knowledge and decide on their own. so, it depends on the explanation, … time and effort taken to explain how it can be done. (fgd-u- ). the legal expert also cautioned against the enrollment of economically and psychologically vulnerable persons into such studies as it raised ethical and legal suspicions of exploitation. similarly, the legal position of an individual to voluntarily accept self-harm was perceived as being contentious under the law. if economically vulnerable strata are subjected to this … it raises all the wrong alarms… if someone wants to look at a public interest litigation or let us say criminal prosecution… the first question would be to look at psychologically have you misled somebody to self-harm … (u-idi- ). participants in general had no prior knowledge about chim, except for a few key informants who were microbiologists and immunologists. the lay public were able to echo the idea of an infection with a germ, but the extent to which they were able to explain what was communicated to them depended on their education, and prior understanding of medical research, among others. one of the questions they raised, important in the context of advocacy for chim, was why naturally infected persons were not studied? another was, while chim studies were said to be 'controlled', individual response was likely to be unpredictable, so was it really 'controlled'? among non-english speaking communities, the word 'controlled' was misunderstood, while health workers associated the word 'control' with 'prevention'. comparisons with clinical trials were made by key informants with research expertise, who likened the issues of a chim study to a phase i clinical trial. in some ways it is analogous to a phase one clinical trial where you have normal healthy people and give increasing doses of new drugs and study the pharmacokinetics, dynamics, toxicity etc., basically we are doing the same thing except using micro-organisms (urban idi- ). another similarly trained informant felt that chim studies posed less of a risk than drug trials because the infecting agents that were being used were weakened and it was within a controlled (safer) environment. i think that with a chim, germs are being weakened. i think subjects hold a better chance [than a clinical trial]. so, i think the risk factor is slightly less [for chim], because it is controlled, the germs are weakened, and it is in a controlled atmosphere. (urban idi- ). there were concerns and fears across all age groups and in both the rural and urban areas. fears appeared to be greater among those least knowledgeable about the idea of chims and less among the more health literate and technical experts. these included: • fear of the potential consequences to self and those near and dear it is highly dangerous, and it involves something … there are unknown side effects on your body. (urban fgd- ). … we are the earning persons for the family, i may be confident enough that nothing will happen but still i have to take care of my family and i should be saved to take care [of them]. confidence has to be given that nothing will happen to them… (rural fgd ); …but what if something happens to my next generation (urban-idi - ). • fear of something hazardous entering the body where the risk of death was imminent, or the nature of the risk was unknown, …but when it comes to being infected everyone has second thoughts… (rural idi ). what if something happens and if i die… (urban-idi- ); …it may give negative effects to the brain or it may damage organs… (rural fgd - ); we also don't know the long-term repercussions, we actually don't know if the small virus is going to mutate, we want to know what the outcome is (urban fgd- ); but what if that remains in our body till, we die (urban fgd- ); …we are scared that something might happen, so if it is done on the animals initially it would be good. (urban idi- ). • fear of exploitation of the socio economically less endowed groups, … those who are economically less privileged are more likely to take part in a study whether it is risky or not because they really require the benefit that you are providing, that is monetary……not ethical because you are taking advantage of (their) current situation (urban idi- ). • fear and mistrust of a non-responsive health system in case on long term side effects, it might be possible that for months nothing will happen and later on it might start reacting in their life, so that has to be taken care of." (rural fgd- ); after completion of the research will it create any problems in the future and who do we go to then? (rural fgd- ); …there is no guaranteed healthcare, and healthcare not being a standardized thing can be a problem for things that were not diagnosed in that period but later on… (urban idi ); …the frequency with which some of these things go wrong in the indian context is much higher… (urban idi- ); what has happened with vaccine research in india in the past has been so murky and it has been unethical. how does one create an ethical study in such an unethical space…? (urban idi- ). when probed if the risk perception would change if the pathogen was known or unknown, health workers in the rural areas gave examples from their own interactions with local communities, where they implied that the facts didn't matter, but perceptions were what influenced responses. one parallel was drawn to methods of family planning that were advocated and another to any health intervention being prescribed, where negative associations were made with what was actually safe. among the urban respondents, the iec lay member felt that it was not about the germ being known but about the perception of danger: one thing is about a known and unknown pathogen and the other is which is known and known to be …dangerous… (urban-idi ). a health activist suggested that people indulge in riskier behaviours in their daily lives -and seemed to suggest that individuals may be overestimating the risk of chim-…there are so many things that healthy individuals do, there are so many risky behaviours that that healthy individuals indulge in. one cannot say that this is riskier than anything else that you are already doing. (urban idi ). while participants expressed doubts and fears regarding chim, they also perceived some benefits of chim, primarily in relation to the prevention and treatment of diseases. some drew parallels with vaccinations 'i feel it is good, it helps in the prevention of diseases… so we can say it is helpful…like we give vaccine to children' (rural fgd- ), 'the benefits would be in one of two ways, to improve the immune system or to treat similar pathogens' (urban idi- ). perceived benefits were personalised based on how it could help prevent or cure a disease in their own family. at a broader level, the value of the benefit was towards public good, where 'lives could be saved'. there was a clearer articulation of the benefits of conducting chim studies in india among the technically educated, which focused on the opportunity that the method provided to advance science and medical research, relevant to local populations. what would be the objective of the research, broadly one is to test vaccines because you can expose the person to the infective agent with or without achieving illness, and the second is that it could be used for medical treatment, thirdly it could be used to study the natural course of the infection to know how people will behave with the infections (urban idi- ). …it is not like a conventional vaccine trial where you have to take large number and wait for years for natural incidence in the community, here if you have to do to the challenge [chim] method, i can take [volunteers] and i challenge them and i need to follow all hundred and not depend on the natural incidents happening in the community. another advantage is that we have access to multiple geographies and a very large ethnic diversity in india (urban idi- ). …we almost never know what the premorbid state of the individual is, which is something that in the absence of the controlled infection model we have not been able to assess, by the time we get into the picture and start collecting data, especially in the indian context it is fairly late into the infection. (urban idi- ). participants identified people who would likely participate in a chim study and included those in need of money, 'risk takers', those who were impulsive, those without dependents or responsibilities, and those with a heightened sense of altruism and social concern. table provides the details. in identifying potential participants for a chim study, there was a great deal of deflecting-where each group felt that people other than themselves were ideally suited to be participants. it is unclear whether this reflected an underlying fear of a novel method which participants perceived as potentially dangerous. participation was determined by the existing knowledge of the infective agent i.e. a priori evidence and treatment options. another aspect that determined participation was trust; both in individual researchers or medical professionals doing the study, and the overall regulatory environment for support in case of harm. depends on the history of the organization … how they treat patients, how they look at money (rural idi ); if doctor is giving guarantee that nothing will happen then people will agree. (urban fgd- ). research has to be done, if it is done under the protection of government then it will be good… (urban fgd- ); the deflection of the possibility of participation could be indicative of the low levels of trust in individuals and systems for protection of those who are socially and economically vulnerable. therefore, potential participants identified included those in positions of power and authority; such as gram panchayat (village table who will volunteer for a chim study? perceptions of rural participants • those who need money 'people who are desperate for money would come for this, but still we should not take that as an advantage and start doing things like this' (urban fgd- ) there was also a sense that enrolment of the poor should be avoided for this very reason • those who need money 'if they are struggling…if they are paid some money …then they will agree for that' (rural fgd- ) and the contrary position, 'rich people will not come… because they don't accept anything' (rural fgd- ) • those who are 'impulsive' 'more impulsive people will be more willing to take this risk, they may be seeking fame…' (urban idi ) • those who were 'risk takers' • • those who are altruistic and with heightened social concern 'whoever has kind heart…' (rural idi ); 'people who think that when we do something it will be benefited to other people in society' (rural fgd- ); 'if social motive is there then people may come forward…. if someone thinks that it will be helpful for others council) members and politicians, or those with the capacity to deal with adverse consequences; such as medical researchers, wealthy or educated persons. financial incentives for participation had contrary responses across the spectrum of participants-rural women felt that altruism should be encouraged. in their words, 'money should never be there in such kind of a trial…', 'financial incentives would be like a bribe… monetary payments should be avoided, i mean that would then make it into money making thing.' at the other end of the spectrum, urban male youth took a pragmatic view that participants should be paid-but such payments should not be so large as to induce risk taking. college students were seen as particularly prone to such risk taking-all for a bit of 'pocket money'. on the other hand, compensation to participants for time and other costs, was acceptable. in order to ensure fairness in compensation, it was suggested to be commensurate with the engagement-in terms of age of the participant, time or wages lost, social status and number of dependents. even the urban youth were conscious that such compensation should not become undue inducement; they suggested it should not be mentioned at the recruitment stage to pre-empt this possibility. …we should not be giving them anything [in the beginning] so that. will make them real volunteers and in the end, when it is done you can pay them … that would become a reward (urban, fgd ). alternatively, insurance mechanisms that provide not only for persons harmed but for all participants, for a sufficiently long period of time, was also seen as a way of compensation. although the idea of chim was novel to the general public in this study, they identified many ethical issues and specific responsibilities that they believed researchers needed to bear; which could build confidence in people. as specific regulatory guidelines were not yet in place when this study was being conducted, participants' wish list for desired regulations have provided a framework for possible regulations [refer fig. ]. the first issue was the validity of the chim method as opposed to alternate research methods, and the choice and safety of the germ, including the availability of suitable treatment-…let the virus be checked properly before injecting to human being … check if it is not harming and there is treatment…,' (rural fgd ); participants were clear that the research team was ultimately responsible for issues related to the participant, in case there is a sudden side reaction or side effects, providing the person with the medical care required. (urban fgd- ) . …who is doing research must take the responsibility. they should see that nothing will happen to the person who is participating. (rural fgd- ) . to ensure complete voluntariness of participation and protection of every individual's right to life and free choice, the participant in a chim study must do so with a free will, with thorough comprehension of purpose and risks, and without the enticement of financial gain. in the words of a college student, it is the fundamental right of every healthy human being to reject it if they are scared. (urban fgd- ). while consent was unanimously seen as integral to participation in chim, the responses obtained indicated that the public see the current system of consent as inadequate-'…it doesn't mean saying yes or putting a thumbprint there… it means they understand what their body will undergo' (urban fgd- ). the idea of relaying back an understanding of the information received, was a way of testing comprehension. assent from a family member was considered an added protection to the participant's informed consent, as there were implications for the family; not only risk and uncertainty but also confined stay in the research facility away from family and work. any dependents of the participant, may be a wife or children or parents should give their consent… they should be aware of what the individual is going through (urban idi ). this was not meant to take away from the autonomy of the individual, as it was emphasized that the ultimate decision lay with the research participant. the idea of self-harm, even if consented to, appears to be a legal and ethical issue according to the legal expert. there is an underlying perception of having to protect the researcher from legal liability and moral impropriety-we still need to show objectively from our research that what we're doing is not something shocking to the conscience of public morality. contracts are void if the intention is self-harm. sooner or later this can't be left to contract as it must be regulated through legislation. (urban-idi- ). complete information was a repeated request. widespread public interaction and awareness through mass media-television, newspapers and websites were emphasized as a prerequisite, with convincing information about 'the injection', 'why it is given' and 'the benefit that is expected'. public good and the benefit of disease prevention emerge as a required justification from the public's point of view. participants believed that widespread media coverage and public discussion of chim would ensure greater transparency and more ethical conduct of chim. the role of the government in adding credibility to the messaging was also implied. this did not refer to advertisements for recruitment of participants, but an engaged information sharing with the wider public. awareness has to be given to people through media like radio and television by government," (rural fgd- ). of importance to the public was the quality of the research, researcher and institution-and these are judgements that people made, at least partly, based on prior knowledge and interactions with people and institutions-…who is doing the study should take responsibility and should give me confi-dence… (rural idi- ). thus, the responsibility of researchers was beyond what routine clinical trials regulations prescribe and should empower volunteers of chims. the importance of ensuring that the individual was disease and germ free at the end of the study was emphasized as an important regulatory issue, to avoid spread of infections on the one hand, and social ostracization on the other. this was brought up by key respondents through their insights of social and community dynamics. … they are scared if the subject comes back into the community and infects all of them. so, they might not allow the subject to come back or the second thing is that subject might get ostracized in that area and the future of the children would be stake. (urban idi- ). several issues emerged in relation to financial transactions during research protocols. compensation for participation in chim protocols was expected to be formulated and mandated by the government. the focus of compensation for injury and inconvenience needed to include the health of the individual not just during the time of the study, but even afterwards. not only were life insurance and care of family members in case of extreme adverse events required, but health insurance was also advocated. calculation of compensation was not to be based on who the participant was, but on the risk of the chim and the related insurance cover, insurance should be more because of the risk. insurance is compulsory, i think it depends on the risk of the study not on the person who joins the study (urban fgd- ). this was however a highly debated issue among community participants. on the other hand, participants generally agreed that financial incentives to participate needed to be discouraged. 'who should regulate these studies?' evoked much interest among the general public, perhaps because such a question was not generally asked of them and it was left to 'people in authority'. there were varied and interesting responses that emerged, ranging from 'the medical team taking full control and being responsible' (rural fgd- ) to 'a neutral committee with scientists and experts and people from various strata of society… and definitely a few lay people from the public to be involved' (urban it youth-idi- ). rural respondents suggested the presence of government representatives on these committees and mentioned 'gram panchayat' (village self-government body) members. there was however some cynicism of having the government as sole regulator of such research. not many participants in group discussions were aware of ethics committees and their role in overseeing research in the country. there was some skepticism on the motives of such committees-we don't know whether ethics committees are there or not in india. we are not aware whether they are doing it for profit, or they are genuinely concerned. they say watch dog but finally it becomes the dog itself. we have various associations, but we don't know how far all this works. (urban fgd- ). experts from the research fields expressed their doubts on whether the present ethics committees in the country would be equipped to handle these additional areas of oversight and monitoring. an ethics committee member as a key respondent in this study had a contrary view, 'ethics committee do the ground work and icmr (indian council of medical research) is the second layer. i think these two regulators are perfectly fine'. a key regulatory requirement would be to ensure that ethics committees are appropriately trained and that an infectious diseases specialist is brought into the review process as an expert. for controlled human infection studies with its contentious ethical positions, the value of public perceptions in identifying regulatory requirements and researcher responsibilities emerges as critical in the process of ensuring transparency and building trust. studies in africa have shown that understanding perceptions of communities on research activities helps in addressing misconceptions, rumours and concerns which can undermine the ethical underpinnings of a well conceptualized study done by credible medical research institutions gikonyo et al. , ogobara-doumbo . for the lay-person in this study, ethics and being 'ethical' went beyond the word and its usage in purely research contexts. ethics was expected to embody higher virtues of integrity, decency, uprightness, good behavior and relationships with others. this, with the backdrop of people's limited understanding of research, implies that ethical research is not just the way research is done but how it is perceived to be done. the main messages emerging from this exploration of public perceptions of controlled human infection model studies in india reflect that, safety is a key concern of the general public with regard to 'being infected' as has already been established that the protection of participants is an important concern for chims (timms ). a healthy person 'being infected' purposefully appears unethical. the various fears expressed by the public could be a reaction to the novelty of chim and the absence of chim studies in india so far. there was no outright rejection of chims. however, there was an expectation that public good should emerge from such research. indeed, a clear justification of public good would make such research ethically acceptable. the disease chosen was expected to have public health relevance to india and it would be desirable if earlier research had been done on animals or humans in other countries. there was also considerable 'othering' that took place when readiness to participate was explored, with each constituency feeling that people other than themselves may be ideally suited as participants. they created an alternative response to 'the other' than what would be suitable for themselves, thus following a process of 'distantiation' (brons ) . by positioning persons unlike oneself as 'others' and giving them characteristics, seen as unacceptable for oneself demonstrates this 'othering' which also reflects existing or perceived unequal relationships (brons ) , common in indian society. the rural folk identified others whom they perceived to be better off than them as potential participants. urban respondents considered it appropriate for those 'without responsibilities' to volunteer, even the elderly and prisoners. this reflects a utilitarian mindset where the outcome of good for the greatest number makes a certain action acceptable even if it is at the expense of some people i.e. the end justifies the means (khan ) . those who would willingly volunteer were seen to be the economically poor, who would do so to gain monetary benefit. but this motivation was seen as interfering with the person's ability to discern risk and hence it was recommended that such persons be avoided as participants. it was also the legal opinion that such participation would indicate undue exploitation of the vulnerable. comprehension of the procedures and risks emerge as essential pre-requisites for participation. relaying back the explanation of a chim by a potential participant could be a useful means of assessing comprehension. other methods such as q&as and opportunities to discuss concerns and doubts over multiple sittings as suggested in the kenyan malaria studies (njue et al ) could be considered. in addition, psychological wellness was an important criterion for a person to assess risks and provide consent. a gradation of opinion was apparent between the lay public and rural communities on the one side, and health professionals on the other, with the former responding to information provided, while the health professionals and health workers based their responses on their experiences in the field or with similar research. this hierarchy of understanding is dependent not just on education, but technical expertise in research and science. those with health or medical expertise appear more discerning of the risks, benefits and safeguards in research. monetary compensation (seen as a key motivator in the kenyan malarial studies- njue et al. ) emerged as a contested area for chim studies across all groups and strata of respondents. there was a moral rationale presented, especially in the key informant interviews, for compensation not as cash but as 'care', moving the risk-benefit analysis from the immediate to the long term, and from the individual to the family and/or community (third party). it can be argued that in a country like india where comprehensive universal health coverage is absent, a commitment to care can be considered an inducement as well. this, therefore, appears to be a conflicting imperative. however, the duty to care, for the treating physician or the study investigator is an ethical obligation, especially in the context of a chim where the intention to harm is a strong perception, with legal implications in india. these views were also reinforced in recent deliberations on the feasibility and ethics of chims in india, held among wider stakeholders , and also from deliberations in other countries such as vietnam (kestelyn et al. ) . it emerges that in the present context of indian society (of which this study was a small subset), if such research was to begin, the following would be suitable criteria for selection of participants to such studies: • educated persons with reasonable health literacy -for example, science graduates or students, health professionals. • persons not in an economically vulnerable situation for whom financial compensation would not be the sole motivation for participation. • persons who can not only give free, informed, understood consent, but can also get informed assent from a next of kin or close family member. it could be argued that these selection criteria perpetuate a paternalistic approach and that all decision making in a liberal democratic society should be autonomous and free. however, indian culture like that of many other asian countries, tends to be more communitarian. this is reflected in how respondents described ethical research-"a long term relationship with others", the stated need to obtain assent from family, and the concern of social ostracism following participation. if the protection of potentially vulnerable persons requires paternalistic rules, so as to ensure ethical conduct in the early days of chim trials in india, it would be reasonable for regulations to be paternalistic in this regard (timms ) . scholars such as resnik ( ) , support this contention with the reasoning that healthy volunteer participants are not always in a position to understand and assess risk completely, hence need some mechanism, such as a guideline or a committee that can restrict the risk to which healthy volunteers may inadvertently subject themselves to (resnik ) . public engagement begins at the stage of conceptualizing such a study, by listening to diverse voices across a range of people and proactively understanding the public's fears, concerns and expectations (vaz et al. ; vaz ) . the findings of this study highlight areas where public awareness and advocacy are required. the systemic failures of health care delivery, conduct of clinical trials and overall public utilities in india seem to have made the urban, educated sections cynical and wary of government regulation (bhan ) . if chims and perhaps any medical research needs to be successful, it needs to begin with an engaged and empowered public (schicktanz et al. ; cioms ) to develop trust at multiple levels (gopichandran )-at an individual level of the treating doctor and researcher, since a significant amount of clinical research is done in hospitals and not stand-alone research institutions/contract research organisations; at the level of the institution; and at a higher level involving systems related to health delivery and the research regulatory process. the data from the fgds and urban interviews reveal a great distrust-the practice of medicine itself is seen as a commercial enterprise and medical research as problematic, given the understandings of people about 'unethical research'. perceptions of ethics emerge based on 'people's lived experiences and the contexts of their lives and not on a preconceived set of ethical principles (mcintosh ). public engagement for chim studies is therefore required, not to convince the public to participate in chims or facilitate recruitment but to develop public trust in the system and inform regulatory authorities about perceived areas of contention. trustworthiness of the system emerges as an overriding issue. it has three key components in the public engagement paradigm, -consultation where listening to diverse opinions is central (hodgson et al. ) -collectivism where altruism of the participant is reciprocated by altruism of the researcher in terms of benefit sharing and achieving public good (molyneux et al. , lairumbi et al. ) -stake holding, where different players-the lay public, the technical experts, diverse professionals and regulators come together to review regulations and develop contextually appropriate guidelines (vaz ; regulations that are in the best interest of the public and address areas of mistrust and skepticism, are needed for chims in india (srinivasan and johari ; vaz ; ). this will help to redeem the poor public image of medical research, clinical trials and ethics committees in india. regulation that is responsive to public perceptions needs to be widely disseminated (blom et al. ; vaz ) . it would be of value to have the public participate in a watchdog neutral body that can question, review, audit and advise the regulator and researcher on the conduct of the study, and also be the eyes and ears for any community repercussions. this could be on the lines of a community advisory board (cab) that augments the functioning of an institutional ethics committee . the cab's structure and functioning would be mainly to liaise between the community and the researchers, offering a mechanism of contact and mediation. it does not take on the responsibility of recruitment for the study nor is it a gate keeper offering such access. at best, it can be an enabler of responsible decision making. the institutional ethics committee on the other hand has the responsibility of determining risk vs benefit of the study, assessing participant safety and ensuring free and fair participant recruitment. at the end of this study, no simple answer emerged to the question of whether india is ready for a chim or not; that was not the focus of our enquiry. what it does confirm is that the general public has strong views of safety, expectation of safeguards and ideas of how such research can be regulated. these perceptions not only indicate who are likely to participate in chim studies but provide underlying motivations and concerns about these studies being ethical or exploitative. many of the findings that have emerged from this study support the findings of research elsewhere , molyneux et al. , gordon et al. , bambery et al. , njue et al , kestelyn et al. . as this study was qualitative-we cannot conclude that these represent the views of the public everywhere in india-and there may well be a place for an expanded study across geographies, using a quantitative tool. a more nuanced understanding may also be obtained by presenting specific chim scenarios to people and seeking their responses, as was done in a multi-stakeholder workshop in march where a malaria, typhoid and chikangunya chim were discussed . in the light of the covid pandemic and the race to arrive at a safe and efficacious vaccine, additional research in public response to chims in a pandemic and readiness to participate to save lives needs to be explored. what is additionally clear, is that there is a felt need for sustained public engagement, and building awareness and advocacy regarding chims. kittle kannada-english 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cgmrc-wellcome trust research programme maori and cross-cultural research: criticality, ethicality and generosity an australian approach to the policy translation of deliberated citizen perspectives on biobanking benefits and payments for research participants: experiences and views from a research centre on the kenyan coast ethical considerations in controlled human malaria infection studies in low resource settings: experiences and perceptions of study participants in a malaria challenge study in kenya human microbial challenge: the ultimate animal model bangalore population developing and utilizing controlled human models of infection how bengaluru became the biotech capital of india. the economic times limits on risks for healthy volunteers in biomedical research paternalism and utilitarianism in research with human participants the-ethic s-of-volun teer-selec tion-and-compe nsati on-in-contr olled -human -infec tion-model s-in-india the ethics of 'public understanding of ethics'-why and how bioethics expertise should include public and patients' voices ethical challenges posed by human infection challenge studies in endemic settings consultations on human infection studies in india: do people's voices really count? protecting challenge study participants in low-and middle-income settings experimental infection of human volunteers with the heat-stable enterotoxin-producing enterotoxigenic escherichia coli strain tw public engagement in the context of a chim study listening to the voices of the general public in india on biomedical research-an exploratory study the views of ethics committee members and medical researchers on the return of individual research results and incidental findings, ownership issues and benefit sharing in biobanking research in a south indian city consultation on the feasibility and ethics of specific, probable controlled human infection model study scenarios in india: a report kannada-english dictionary acknowledgements this publication was supported by a grant from the translational health science and technology institute (grant no. ). its contents are, however, solely the responsibility of the authors and do not necessarily represent the official views of the translational health science and technology institute. we acknowledge the time and views shared by the participants of this study and the attendees of the chim pre congress workshop of the th world congress of bioethics , where the findings of this study were shared. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -asg qtr authors: beasley, lana o.; ciciolla, lucia; jespersen, jens e.; chiaf, ashleigh l.; schmidt, mallory; shreffler, karina m.; breslin, florence j.; bakhireva, ludmila n.; sanjuan, pilar m.; stephen, julia m.; coles, claire d.; chambers, christina d.; kable, julie a.; leeman, lawrence; singer, lynn t.; zellner, jennifer; morris, amanda s.; croff, julie m. title: best practices for engaging pregnant and postpartum women at risk of substance use in longitudinal research studies: a qualitative examination of participant preferences date: - - journal: advers resil sci doi: . /s - - - sha: doc_id: cord_uid: asg qtr there are significant barriers in engaging pregnant and postpartum women that are considered high-risk (e.g., those experiencing substance use and/or substance use disorders (sud)) into longitudinal research studies. to improve recruitment and retention of this population in studies spanning from the prenatal period to middle childhood, it is imperative to determine ways to improve key research engagement factors. the current manuscript uses a qualitative approach to determine important factors related to recruiting, enrolling, and retaining high-risk pregnant and postpartum women. the current sample included high-risk women who participated in focus groups or individual interviews. all interviews were analyzed to identify broad themes related to engaging high-risk pregnant and parenting women in a -year longitudinal research project. themes were organized into key engagement factors related to the following: ( ) recruitment strategies, ( ) enrollment, and ( ) retention of high-risk pregnant and parenting women in longitudinal research studies. results indicated recruitment strategies related to ideal recruitment locations, material, and who should share research study information with high-risk participants. related to enrollment, key areas disclosed focused on enrollment decision-making, factors that create interest in joining a research project, and barriers to joining a longitudinal research study. with regard to retention, themes focused on supports needed to stay in research, barriers to staying in research, and best ways to stay in contact with high-risk participants. overall, the current qualitative data provide preliminary data that enhance the understanding of a continuum of factors that impact engagement of high-risk pregnant and postpartum women in longitudinal research with current results indicating the need to prioritize recruitment, enrollment, and retention strategies in order to effectively engage vulnerable populations in research. there are significant barriers to recruiting and retaining individuals with overlapping vulnerabilities (i.e., substance use disorders (sud)) in the pregnancy or postpartum period (davis, yao, & bierer, ; mchugh votaw, sugarman, & greenfield ( ) wetherington & roman, ) . this may result in challenges for generalizability and therein create a relatively sparse knowledge base about the long-term outcomes for these women and their children including the environmental, mental health, physiological, and neurological factors. filling these knowledge deficits and gaps requires ongoing assessment because research tools including those for recruitment and retention change; in addition, substance exposures in pregnancy change (e.g., prescription opioid exposure, cannabis use), thereby shifting methods to reach target populations of interest and methods to measure outcomes of interest. it is imperative for the field to identify and address engagement in research, to ensure representation of pregnant and postpartum women that use substances. engagement in longitudinal studies will allow a more complete understanding of maternal and child health outcomes as a result of new and emerging trends in prenatal substance exposure. enhanced understanding of participants' perspectives on engagement and study participation will allow researchers to more fully address this pressing research and public health need. prenatal exposure studies began in earnest in the s, after the identification and diagnosis of fetal alcohol syndrome (jones & smith, ) . careful participant selection and comparison selection were and are necessary to classify effects of prenatal exposures. protectionist and paternalistic regulations (e.g., the national research act of , and federal regulations designating pregnant women as a vulnerable population) excluded women from health research and limited the field's understanding about how sex and gender shape substance use and sud (davis, et al. ; wetherington & roman, ; institute of medicine, ; institute of medicine, a) . research studies on substance exposures during pregnancy expanded rapidly in the past years, in recognition of the cocaine epidemics of the s, and the current increases in prenatal opioid and methamphetamine exposures (gabrhelík, et al. ) . indeed, research that focused specifically on prenatal exposures and other women's health issues has been encouraged by journal editors, policymakers, and funding agencies including the nih helping end addiction longterm (heal) initiative. despite bioethical, legal, and social concerns regarding the risks and benefits of research participation for pregnant and postpartum women who use alcohol and drugs (davis, et al. ) , the inclusion of vulnerable populations who are marginalized or stigmatized in research on sensitive topics has not demonstrated undue harm or exposure to unacceptable risk, and in fact, has been associated with potential benefits, such as altruism, catharsis, and gained knowledge (alexander, pillay, & smith, ) . of course, it is important for researchers to adopt careful experimental design and safeguards that will uphold the principal of non-maleficence and protect vulnerable participants from harm (alexander et al., ; sikeweyiya & jewkes, ) . exclusion of substance using populations may violate important bioethical principles of human subjects research, particularly the principles of autonomy, beneficence, and justice (alexander et al., ) . exclusion from research not only strips individuals from making decisions about their own autonomy and denies them potential benefits of participating, but also exposes them to greater societal marginalization and may ultimately place them at increased risk of harm due to deficits in critical health knowledge and exposure to inappropriate or ineffective treatments (alexander et al., ; johnson et al., ; institute of medicine, b) . unfortunately, prenatal exposures to alcohol, tobacco, and other drugs are rising (substance abuse and mental health services administration, ), with in pregnancies exposed to tobacco ( . %), alcohol consumption ( . %), or illicit drug use ( . %) (ebrahim & gfroerer, ; havens, simmons, shannon, & hansen, ). specifically, opioid exposed pregnancies have increased from . to . per pregnancies (haight, ko, tong, bohm, & callaghan, ). yet, cannabis exposures are the most prevalent drug exposure, with nearly - % reported exposure in the first trimester (alshaarawy & anthony, ) . rising rates of substance exposure correspond to increasing health risks and adverse outcomes at great societal cost and burden to systems of health care and social services, as well as criminal justice. notably, researchers involved in the nida-funded perinatal- treatment research demonstration program that focused on sud treatment for pregnant and postpartum women identified seven clinical factors that contributed to significant difficulty and complexity in the recruitment and retention of women in substance use treatment research, including as follows: ( ) severity of sud, ( ) legal system involvement, ( ) housing instability, ( ) interpersonal relationship challenges, ( ) parenting responsibilities, ( ) employment challenges, and ( ) need for more intensive services. these difficulties with recruitment and retention contribute to additional complications for research, including biased samples of convenience recruited through referrals from social and health agencies, limited sample diversity, deviations from the research design, and ethical issues associated with risk and benefits of participation and involvement with the criminal justice or child welfare system. in particular, when research designs do not involve the possibility of direct benefit due to participation (i.e., observational versus intervention studies), it is important to understand the unique reasons and motivations that drive decision-making about research participation (hallowell et al., ) . due to all of the aforementioned factors that potentially inhibit the inclusion and engagement of high-risk participants (i.e., participants previously or currently using substances), it is imperative to understand the motivations for engagement in research among high-risk participants, focusing specifically on understanding motivation for research participation, factors that influence decision-making about participation, and barriers to participation. the current study reports results from a qualitative research study conducted as part of an -month, multi-site pilot study aimed to develop and demonstrate feasibility of an experimental design for a -year, prospective, longitudinal investigation of normative childhood brain development, beginning in pregnancy. a major aim of the -year study will be to determine factors that alter brain development including prenatal exposure to opioids and other psychoactive substances, as well as other prenatal and childhood environmental exposures. this goal necessitates recruiting pregnant women previously or currently using substances, as well as a large group of pregnant women who are at low risk of prenatal substance use. two of the primary aims of the pilot are ( ) developing and testing recruitment and retention strategies and ( ) addressing ethical and legal challenges of conducting research with a stigmatized and vulnerable population. the current qualitative study is one arm of the -site consortium to improve understanding, from a qualitative perspective, the continuum of engagement of low-and highrisk participants in research. this manuscript focuses on the results of the distinct needs and responses of high-risk participants. specifically, the objectives in this analysis were to address important factors that impact best practices in ( ) promoting longitudinal research to high-risk participants, ( ) enrolling high-risk participants in research, and ( ) retaining high-risk participants in research studies. individual interviews and one focus group were conducted with a total of women (five participated in a focus group). women were at high-risk of prenatal or postnatal substance use and were identified through medical clinics, other research study involvement, or sud treatment programs. recruitment took place across five sites in the usa located in california, georgia, new mexico, ohio, and oklahoma (see table for demographic information). high-risk pregnant and postpartum women were defined in the current study as a parenting or pregnant woman who had used alcohol and tobacco and/or had a current or past history of sud. some participants were currently receiving sud treatment. contact was made through trained research personnel located at each specific site with total participants taking part in the current study (california, n = ; georgia, n = ; new mexico, n = , ohio, n = ; oklahoma, n = ). only one focus group that included five women was combined with the individual interviews. the one focus group was conducted in new mexico prior to group restrictions imposed due to covid- . qualitative methods for the research team, study design, and analysis followed the guidelines recommended by tong, sainsbury, and craig ( ) . qualitative study recruitment began with sites contacting participants in person or by phone and describing the current study and qualitative interview process. all women who expressed interest in participating were scheduled for either a focus group or individual interview depending on whether the interview took place prior to or following covid- restrictions regarding in-person gatherings. interviews conducted during the covid- restrictions were conducted individually by phone. all participants gave oral informed consent. during the consent process, a brief overview of the qualitative study and all safety measures taken to ensure confidentiality were discussed. trained qualitative research assistants collected all qualitative data from march through june . before engaging in focus groups/individual phone interviews, all participants completed an in-person or online survey that included a demographic questionnaire and watched a short video describing the protocols planned for the larger, longitudinal study including neuroimaging (e.g., mri), neurodevelopmental, and biospecimen collection. for the focus group, snacks were provided. participants received a $ - incentive for their participation, and this varied based on site. all focus groups and individual interviews were audio-recorded and lasted approximately - min. transcription work was conducted by qualitative team members or a transcription company, with team members crosschecking all transcripts to verify accuracy. during the transcription process, all identifying information was removed to ensure privacy. all procedures were approved by the sirb for the -site consortium. focus group and individual interview guides for the current project were developed by the first author, in conjunction with the evaluation team and other sites within the research consortium reviewing and revising the guide as needed. focus group and individual interviews were coded individually and combined for data analysis. all coding and data analysis was conducted at one site. recordings were transferred securely according to irb-approved methods. it is important to note that focus group and individual data themes were examined a priori and themes were congruent and therefore data were merged. qualitative data was analyzed using the nvivo © software. five qualitative researchers worked together to develop a codebook focused on broad themes influenced by the semistructured interview guide. thematic analysis was used to define specific themes within the broader categories (braun & clarke, ) . the codebook was developed using an agreed upon coding scheme with themes not being predetermined but rather emerging from the data. upon completion of the codebook, two teams consisting of two qualitative researchers coded all transcriptions using developed coding templates. cleaning of data took place as needed (broader codes enveloping smaller codes). once coded, inter-coder reliability was established using simple percent agreement, which is a commonly used method for assessing reliability in qualitative studies (lombard, snyder-duch, and bracken, ; stemler, ) . average inter-coder reliability was over %. in the "results" section, themes are described in more detail. the validity of the current research findings are enhanced by several design factors such as the calculation of salient factors using percentage of comments and the team-based approach used for coding. specifically, calculating the percentage of comments from participants related to specific themes allowed the research team to ensure that themes discussed in the current paper were saturated or were discussed frequently in focus group/individual interviews. therefore, relying on percent of comments strengthens demonstration of saturation in the current study. further, the majority of qualitative data were collected from individual interviews (n = ) rather than a focus group (n = ), which allows for a more in-depth conversation. specifically, during individual interviews, comments were able to be probed deeply with rich content emerging throughout the qualitative data, rather than simple agreement or disagreement that often emerges from focus group data collection. additionally, the fact that both primary researchers as well as consortium partners were involved in developing the focus group/individual interview guide is a strength, increasing the likelihood that the items on the interview guide validly and comprehensively captured the intent of the research aims. qualitative results for the current study focused on a continuum of engagement of high-risk participants in a multi-year prospective longitudinal, cohort design research study. specifically, themes were organized into key engagement factors related to the following: ( ) promoting research to high-risk participants, ( ) enrollment of high-risk participants in research, and ( ) retention of high-risk participants in a multi-year prospective longitudinal, cohort design research study. key socioeconomic and demographic variables for the study participants are presented in table . the current sample was more racially/ethnically diverse than the general us population, with most participants identifying as white ( %), followed by hispanic/latino ( %), black/african american ( %), multi-racial ( %), american indian ( %), and asian ( %). many participants reported they had never married ( %) with other participants reporting they were currently married ( %), living with a significant other ( %), or divorced ( %). many participants reported completing high school ( %) with an additional % reporting obtaining a ged. the average monthly income was $ . per month (sd = $ . ) and most participants reported receiving some form of government assistance ( %). best ways to promote research studies to high-risk participants participants were asked about the best ways to promote a longitudinal research study to recruit participants and their children into studies with participants explaining the best recruitment locations, ideal individuals to share research study information, and the best type of recruitment material. participants reported that social media is an ideal platform to reach participants ( % of comments). specific social media locations include facebook, instagram, and online mother groups. one participant explained, "a lot of people use social media. so, if it got posted to somebody's account, then you could share it with all of your friends. so, i think that would be great." participants also reported that medical offices ( % of comments) and community/state agencies ( % of comments) were good locations to share information about research studies. an example quote includes: and maybe … when i go to (local agency) for counseling and you know, my mental health needs for me and my kids, therapy anything like that. they always have all kinds of [information] … are you struggling with this, are you interested in this, here's a study to help you earn extra money, are you a single mom, do you have this or that, well then you may qualify for this study that we're doing. other suggested locations included bus stops ( % of comments), billboards ( % of comments), and grocery/ convenience stores ( % of comments). participants also reported that child-friendly locations (i.e., library, parks; % of comments) and educational settings ( % of comments) were good locations to share study information. one participant explained, "maybe flyers at childcare centers and stuff like that, where they have the younger school-aged kids from infant to whatever. i know a lot of moms frequent those places." when participants were asked about locations they would trust the most to receive information, it was disclosed that medical offices such as doctor offices/clinics, state agencies (e.g., women and children food programs, other human services), and educational settings were considered most trustworthy. participants also discussed the type of material they would recommend using to promote research studies with participants explaining they would use commercials ( % of comments), brochures ( % of comments), and radio ( % of comments) to share information about research studies. it was also reported that using news/newspapers ( % of comments), online marketing strategies ( % of comments), and sharing information through word of mouth ( % of comments) would be most effective. in terms of the individuals that would be best to share research study information, participants stated that study information would best come from medical personnel ( % of comments), friends ( % of comments), family members ( % of comments), other participants ( % of comments), and professionals ( % of comments). professionals included counselors/therapists, daycare personnel, clergy, and staff at resources centers. participants were also asked who they would find the most trustworthy in sharing information with them and disclosed doctors ( % of comments), family/other mothers ( % of comments), and friends ( % of comments) would be most trusted. an example quote regarding who participants would trust the most was: …when you think of doctors you think you can trust them more, because they're there to help you take care of your kids or take care of yourself … the doctor isn't going to try to scam you out of something so when you get information from them about a study … you tend to want to read about what they are giving you. research enrollment decision-making participants were asked if any information could be provided during recruitment to help them make the decision of whether or not to enroll in a -year research project that includes data collection from them and their children. the most frequent request for more information involved study procedures ( % of comments). specifically, participants wanted to know more information about data collection, including procedures related to neuroimaging such as magnetic resonance imaging (mris), biosampling, and frequency of procedures. an example quote of wanting more information on study procedures was: …looking at everything that might be required of participants, it seems kind of … a bit invasive and like kind of a large commitment in regards to [sic] coming for mris and sending blood work, the diapers, giving hair, things like that. participants also explained that they wanted to know additional information about the purpose of the study ( % of comments) with participants explaining that they wanted an explanation of the research study in its entirety and how it related to child development. another area that participants explained they needed more information about was study logistics ( % of comments) with participants explaining they would like to know the location of the study and commitment involved with being in the study. in terms of research commitment, one participant shared needing to know "… times and dates to make sure everything is scheduled right … trying to get a couple of days in at the job and … just timing stuff." participants were also asked if there was anyone they would need to speak with to make a decision about enrolling in research involving themselves and their child(ren). participants indicated they would speak to a variety of key individuals, including their significant other or the biological father of the child ( % of comments), family members ( % of comments), and medical personnel ( % of comments). participants reported that several things would make them interested in joining a research study with the most frequent being understanding the research benefits ( % of comments) to others ( % of comments) and themselves ( % of comments). an example quote regarding research benefits to others was: i think if participating contributes to information that can help other moms in the future, if there's more information that can be gained or developed out of this study that can be provided to other moms, before they get pregnant, as they're pregnant, to help in their baby's development. i feel like that's, in some part a small contribution i can make. participants also reported that for research studies that involved their children, there was a strong interest in being provided study results about their children ( % of comments). this included information on brain development and overall child development. one participant explained, "it seemed like something that would be really interesting is to find out more information about your own child …not? just brain development. that's really something that interests me." participants also reported that compensation would impact their interest in joining a research study ( % of comments) with several participants indicating that a potential increase in compensation over time would be attractive. specifically, one participant explained, "i would say maybe increase the compensation as the years go on because … it's hard to stick with a program." barriers to joining a research study participants also discussed barriers to joining a research study. participants reported that a busy schedule ( % of comments) could make it difficult to join a research study. this includes challenges regarding the time commitment for the study and also balancing their work schedule around research study demands. other barriers included transportation difficulties ( % of comments), travel time and location of the study ( % of comments), and having childcare while participating in research activities ( % of comments). many comments from participants regarding barriers were concerns about taking part in a research study. primary concerns included potential risks to the fetus/child ( % of comments) both during pregnancy and after the child is born. one participant explained, "just making sure no harm to the baby … i mean i understand the blood samples but just making sure it's % safe." other concerns included ensuring that participant personal and research information was kept confidential ( % of comments) as well as understanding the invasiveness of biospecimen collection ( % of comments). in terms of confidentiality, an example quote from a participant was: …a reassurance that the information would stay private and … the only thing that made me a little uncomfortable was when you were like "we will have to take your identifying information but then it will be destroyed." i feel like there might be something to substantiate that it's going to be destroyed, and not just a word of mouth thing. supports needed to stay in a research study additional advice was collected from participants regarding what would help them stay in a research study after enrollment. participants reported a variety of research supports that would be helpful to stay in a research project ( % of comments). specific research supports included providing childcare during data collection, compensation for time spent in research, and provision of transportation as needed for families. related to this theme, one participant explained that "… childcare on site is probably a huge one" and another participant reported that "transportation would be very helpful." it was also explained that specific research study logistics are important to consider in supporting participants to stay in a research study ( % of comments). these included receiving regular research updates including the benefits of the study, having well-trained research staff, research staff engaging in regular contact with participants, families being close to the research/data collection site, and families being provided advanced notice of data collection. with regard to receiving research updates, a participant explained: …getting kind of feedback about … here's what you contributed and here's what … we're gonna use … information on what we found, about your baby's development ... for me that would be important. flexibility was another key area of importance for participants ( % of comments). specifically, it was reported that flexibility of the participant schedule, when research appointments are scheduled, and who brings the child to research appointments are important to support ongoing research engagement. an example quote regarding flexibility was: so, i don't know how long an appointment would take. but if appointments could be, quick, able to work around my schedule, flexible, and maybe offer something like what we're doing now (phone interview). if we're not able to meet, like some kind of tele-health option. that would make it so much easier. participants also reported that home visits could potentially increase their ability to stay in a research study ( % of comments) as it would make visits more convenient. it is important to note that some families reported concerns with home visits indicating that not all families feel comfortable with research personnel coming into their home. another theme that emerged included the importance of becoming familiar with research staff ( % of comments) as it creates comfort in staff working with children involved in the research study. participants explained barriers to staying in a research study after enrollment with the most frequent barrier being family-related barriers ( % of comments) including work schedule, family moves, generally busy schedule, lack of childcare, and child(ren) not being interested in the study (when older). with regard to family moves, a participant shared "… it would depend on where i moved and if you guys had the same research study where i moved to." specific research study logistics ( % of comments) were also reported as a potential barrier to staying in a research project with participants explaining barriers related to transportation, time commitment, and frequency of data collection. specific to time commitment/frequency of data collection, one participant explained wanting more information on "how often do you want me to come in? how long are we gonna be there?" best ways to stay in contact with high-risk participants participants were also asked about the best way to stay in contact with participants during a longitudinal research study with participants reporting that collecting contact information from family members is recommended ( % of comments). specific to information important to collect from participants, personal information such as phone number ( % of comments), email ( % of comments), social media information ( % of comments), and home address ( % of comments) were reported as ideal. the current qualitative data describes a continuum of factors that impact engagement of high-risk participants (i.e., those currently or in the past using substances) in a -year complex longitudinal research study. this continuum included factors that impact research promotion strategies, enrollment, and retention of high-risk participants in research studies (see fig. ). recruiting a representative sample of pregnant high-risk participants for a longitudinal study is challenging. however, considering promotion strategies, it is evident that there are a number of avenues whereby researchers may have success both finding and creating contact points with high-risk participants. our findings illustrate how the construct of research promotion is multifaceted. responses from interviews included three major factors: ( ) the location where research material is shared, ( ) the type of material used, and ( ) the person sharing the research material all potentially impact marketing success. considering recruitment locations, participants frequently recommended social media, followed by medical offices, and community/state agencies. in regard to social media, studies have increasingly indicated social media as a key location to gain access to individuals who may be harder to reach, including high-risk individuals (betsch, ; frandsen, walters, & ferguson, ) . additionally, marketing through social media can contribute to reduced recruiting costs, shorter recruiting periods, and better population representation (maloni, przeworski, & damato, ; whitaker, stevelink, & fear, ) . social media has become increasingly common as a major component of research recruitment strategies in recent years (fusar-poli et al., ; whitaker, et al. ) . television commercials, brochures, and radio ads were the three most often recommended mediums. these more traditional marketing methods have been used in research for decades; however, barriers in the use of television and radio advertisements have become more relevant in recent years. for example, many of those who watch television have begun resorting to streaming services. these streaming services often allow users to skip ads, or even remove ads all together. additionally, online radio services with ad-free options have become increasingly common (wlömert & papies, ) . however, although these trends are occurring in the general population, less is known regarding how changes in television and music consumption have changed specifically for pregnant and parenting women with sud. medical personnel, family members, and friends comprised the limited sources for trusted information on research studies for participants. this suggests close interpersonal relationships are important to consider during recruitment. moreover, doctors and nurses may be successfully engaged in the recruitment process where feasible. this is consistent with research newington and metcalfe ( ) showing that forming collaborations with trusted medical professionals aided in both identifying and gaining access to eligible, hard-to-reach participants. adding to support this, when asked who they would trust the most to receive research recruitment information materials from, the most common answer among participants was doctors. the decision to enroll in a long-term research study with few or no direct benefits for participants is complex and multifaceted (hallowell et al., ) . for pregnant and parenting women who use drugs and/or alcohol, the decision can be further complicated by concerns about privacy and safety, logistics related to participation, and details about study procedures and how they relate to child development. the interview and focus group responses from our participants indicated that comprehensive information about specific study procedures and the purpose of the study was most important for informing research participation, with particular interest in understanding the commitment and burden (i.e., time, effort) associated with participation. additionally, almost a third of participants reported concerns about safety and invasiveness of study procedures, particularly the risks to their child, as well as concerns about confidentiality and maintaining the privacy of their personal information. in addition to the concerns about privacy, participants expressed a desire for reassurance that they could trust the research team, and to that end, it was fig. recruitment, enrollment and retention strategies of high-risk participants in research important that study procedures were clearly explained so that they could weigh the risks of participating with potential benefits. this speaks to the importance of autonomy in decisionmaking and informed consent practices, whereby participants' ability to weigh the costs and (potential) benefits of participation and to make the decision for themselves should be valued and respected (alexander et al., ) . interestingly, when participants were asked about factors that would influence motivation to participate in nontherapeutic research, a number of participants spoke about understanding the perceived benefits for others as well as for themselves. consistent with previous research showing that altruism was a key motivator for research participation (alexander et al., ; hallowell et al., ) , current participants indicated that "helping other moms in the future" was an important factor in driving motivation. specifically, participants explained that related to others, they wanted to know more about how research knowledge could support other participants and children as well as how physicians could support young children and families. although altruism has been associated with positive health benefits (post, ) , the indirect benefits of charitable helping, such as positive mood and enhanced meaning, can be difficult to articulate and capture, especially when the help provided does not have a clearly defined beneficiary or observable impact (i.e., helping an unknown other, sikeweyiya & jewkes, ) . notably, altruistic motivation was more likely to occur among participants who were better informed about research generally and what might be gained through research participation at the level of the community or society more broadly (sikeweyiya & jewkes, ) . these findings suggest that altruism is a motivating factor that could be more directly addressed in marketing and informed consent processes, with additional information provided to potential participants about the value of research participation more generally, as well as how participants' specific data will help others, as much as it can be known or anticipated. direct compensation for participating was also identified as an important factor in deciding whether to participate in research. the decision to participate in research is shaped by personal situations and life factors, in particular, money and time. even among participants who reported altruism as a motivating factor, many expected a mutually beneficial interaction that both contributed to the betterment of society and compensated them directly (owen-smith et al., ; sikeweyiya & jewkes, ) . previous research has shown that participants who are older or have financial hardship were more likely to expect a direct material compensation for participation (sikeweyiya & jewkes, ) . if the real costs of study participation are not covered, then study participation can add to an already financially burdened household. in attempting to recruit high-risk or vulnerable populations into research, it is important to consider material compensation as both a motivating factor and a factor that reduces barriers to participation in the context of socioeconomic hardship without introducing undue coercion. a number of participants spoke of concerns about balancing research participation around their work schedules, as well as the costs associated with participation in terms of time, transportation, and childcare. thus, consistent with previous research with vulnerable populations (owen-smith et al., ) , compensation that adequately compensates time (especially if any work needs to be missed or childcare must be obtained), effort, and inconvenience is an important factor for motivating enrollment, with special attention given to unique participant needs and preferences regarding type of incentive (george, duran, & norris, ; owen-smith et al., ) . transportation has also been identified as a barrier in multiple studies involving high-risk families (e.g., mendez, ) , and providing transportation is necessary to ensure a diverse sample. one of the most common threats to internal validity to any longitudinal research is attrition and loss to follow-up bias. therefore, the thoughtful implementation of retention strategies can prove critical for conducting research among highrisk populations (dumka, garza, roosa, & stoerzinger, ) . in regard to such strategies, three major themes emerged when considering how to enhance study retention: ( ) specific supports that can help participants remain in the research study, ( ) barriers to be aware of that can potentially make it difficult to continue study participation, and ( ) the best channels for staying in contact with participants over the course of the study. participants most often reported needs of support in areas of childcare, transportation, and being compensated for their time. these findings align well with previous research, as childcare and transportation needs tend to be more common among vulnerable populations (dilworth-anderson, ; haley et al., a, b) . as such, offering support in terms of transportation assistance and compensation could prove beneficial in terms of retention. additionally, many participants recommended home visits by research staff as a potential solution to transportation and childcare barriers. however, it should be noted that while home visits may aid in reducing potential barriers, a number of participants in the current study voiced that they would not feel comfortable with individuals coming in their home. therefore, prior to the use of home visits, researchers must ensure that families feel comfortable with visits taking place within the home or consider giving participants an option of laboratory-only visits. these options support participant decision-making, a recurring theme among participants. logistical factors mentioned to increase retention included providing participants with regular research updates, advanced notice for study appointments, and a sense of familiarity with research staff. in line with these recommendations, studies that consistently engage with participants via appointment reminders and research updates can foster a sense of anticipation and progress in participants (kim, hickman, gali, orozco, & prochaska, ) . moreover, a number of researcher characteristics can contribute to participant engagement and retention including being well experienced with the services provided/research protocols, flexibility, being nonjudgmental, and being culturally competent (beasley et al., ; o'brien et al., ) . high-risk participants often face a disproportionate number of barriers to remaining engaged in research studies (kim et al., ) . indeed, it has been documented that retention rates for vulnerable families are often mitigated by higher instances of unpredictable negative life events (e.g., car problems, unreliable phone access, eviction) while possessing fewer resources to compensate for them (heinrichs, bertram, kuschel, & hahlweg, ; nicholson et al., ) . additionally, it has been found that low-income, high-risk families tend to move more often than those in elevated ses categories, while also being at a higher risk for experiencing evictions and homelessness (phinney, ) . participants in the current study mentioned barriers related to work schedules, inconsistent daily routines, a lack of childcare, and the potential lack of child interest when children are older. despite these barriers, a number of studies have made attempts to work around some of these issues. for example, studies have been successful in addressing schedule barriers by allowing participants to designate appointment times that would be most convenient for their family (dumka et al., ) , by meeting participants in-person at a hospital or clinic appointment (kim et al., ) or by offering services within the home to reduce transportation and child care needs (fifolt, lanzi, johns, strichik, & preskitt, ) . moreover, it has been found in vulnerable populations that providing childcare and an environment that is child-friendly can ease parents' burden as well and boost young children's motivation and interest in participating (chaffin et al., ; dumka et al., ) . the most common methods for maintaining contact with participants throughout the -year study period that were recommended by participants were personal phone number, email, social media, and home address, in that order. while personal phone numbers can provide the most immediate access to an individual, mobile phone numbers have been found to change more often than other modes of contact, such as email or even social media accounts (haley et al., a, b) . therefore, gathering as many contact modalities as possible, as well as contact information from two close friends or family members, can contribute to better participant tracing and retention rates (haley et al., a, b; nicholson et al., ) . this study may be limited in generalizability by sample demographics. caution should be used in applying the findings of these high-risk participants to all women at-risk of substance use in pregnancy. this study was also limited because the research method changed during the course of the study due to covid restrictions, resulting in the combination of a focus group with individual interviews. in addition, almost half of the women interviewed were from the oklahoma site. findings should be interpreted in light of these limitations. overall, the current study adds vital information to understanding the complexities of marketing, enrollment, and retention strategies when conducting research with participants that are at high risk for substance exposed pregnancies. several key factors proved to be important across a variety of areas related to enrollment and retention. specifically, transportation was found to influence enrollment decision-making, as a barrier to joining a research project and as a support that was needed to stay in a research project. childcare is another area that was reported to impact enrollment and retention. these results indicate the importance of understanding transportation and childcare availability during data collection and to consider ways to support participants in accessing the study location with child supports in place. other key areas that were discussed within enrollment and retention were benefits of the study and compensation. specifically, participants reported that understanding the benefits to self and others was important. these findings indicate the importance of reporting potential benefits and compensation not only when recruiting participants to enroll in a research project, but also to continue this conversation to retain research participants. lastly, across enrollment and retention, busy schedule of participants is an important consideration. leaning on another theme within the retention strategies, it is important to remain flexible with scheduling data collections so that participants are able to work appointments around a potentially chaotic schedule. a potential research barrier in the current study is the possibility that participants might have difficulties in conceptualizing the continuum of engagement in a longitudinal research study without actually being enrolled and experiencing the study. to mediate this barrier, many of the participants in the current study had been involved in other research in an effort to include participants that had some research experience. additionally, answers to qualitative questions were varied and robust which indicates that participants had a wealth of ideas regarding engagement in research. another potential limitation of the current study was that all participants were considered "high-risk." to remediate this limitation, researchers from the current study are currently analyzing qualitative data to determine potential key differences in engaging low-risk versus high-risk participants in longitudinal research. it is important to note that although it was not specifically examined in this study, it is crucial for researchers and staff to be trained in, and understand, culturally competent methods for recruitment, data collection, and retention. this is particularly the case for building trust among researchers and participants from different cultural and high-risk backgrounds. for example, bicultural research staff and bilingual team members are needed, as well as specific training regarding cultural norms and sensitivity (see mendez, ; beasley et al., ) . overall, researchers need to be aware of barriers to enrollment and study engagement strategies for recruiting and retaining high-risk participants in research. future research should focus on understanding further behaviors and techniques in supporting high-risk participants, as engaging this population is essential for understanding developmental trajectories of risk and resilience among children starting already at risk for mental and physical health difficulties. funding this work was supported by the national institute on drug abuse (nida) research grant numbers r da (lob, jmc, and asm) and r da (lnb, js, and ll). conflict of interest the authors declare that they have no conflict of interest. disclaimer the views and opinions expressed in this manuscript are those of the authors only and 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cord- -u l jv authors: bao, yinyin; bossion, amaury; brambilla, davide; buriak, jillian m.; cai, kang; chen, long; cooley, joya a.; correa-baena, juan-pablo; dagdelen, john m.; fenniri, miriam z.; horton, matthew k.; joshi, hrishikesh; khau, brian v.; kupgan, grit; la pierre, henry s.; rao, chengcheng; rosales, adrianne m.; wang, dong; yan, qifan title: snapshots of life—early career materials scientists managing in the midst of a pandemic date: - - journal: chem mater doi: . /acs.chemmater. c sha: doc_id: cord_uid: u l jv nan ■ yinyin bao, group leader, institute of pharmaceutical sciences, eth zürich i never expected that a simple assembly of certain macromolecules could have such a huge worldwide impact on human life, which is what we as polymer scientists endeavor to achieve but cannot. ironically, the coronavirus succeeded in making it happen. working in the second most densely infected country, as a father of two little kids and a junior group leader, i would not say that lockdown life is easy. with our lab closed, i can only focus on the papers, reports and other writing work, and communications with students can only be done online. what is more complicated is that i have to shift my working time mainly to the night, so that i can have "bulk time" to concentrate on one thing. although with less productivity, fortunately i have more time to teach my daughter mathematics, read story books for my son, make handicrafts with them, and have other fun. this greatly eases my anxiety due to the suspended research, which makes my lockdown life better than expected. during the spread of coronavirus, another thing i did not expect was the huge difference between east and west regarding people's reactions. a typical example is the big debate on whether uninfected people should wear masks. since the outbreak of sars in , chinese and other asians have become very sensitive to unknown viruses, and wearing masks has been considered as an effective method to prevent the spread. however, this is treated as a sign of sickness and overreaction for most europeans, and is thus not socially acceptable. three weeks ago, when i wore my first mask just before the lockdown of eth, i only saw asian people wearing masks in switzerland. surprisingly, along with the situation of covid getting worse, i start to see europeans wearing masks in the stores and on the street. i even read the news that austrians are required to wear masks in supermarkets. it is interesting to see this transformation due to the reconciliation between eastern and western culture, and i will continuously follow this trend during this special period. ■ amaury bossion, post-doctoral while the new coronavirus pandemic is dominating headlines worldwide and the french president, mr. emmanuel macron, just extended the stay-at-home order, here are my thoughts on the surreal atmosphere. as a young postdoctoral scientist, i must admit it is quite harsh not only sacrificing laboratory time but also socializing only remotely. it is even more frustrating when this crisis put an instantaneous halt to promising ongoing experiments. this bizarre and heavy atmosphere is even more present in that i became, partly against my will, a troglodyte in my small parisian apartment. while lots of my friends and colleagues returned home to spend more time with their families at the beginning of the crisis, a mixture of logic and reflection advised me to stay home for the greater good, despite my family's call to come back. although, being organized and rigorous, covid- is putting a strain on me mentally as remaining focused upon writing ongoing articles and reviews all day is really demanding with all the other distractions surrounding me. physically, it has considerably reduced my daily physical activity to the point where the most exercise i get is the few walking steps needed to move around my flat. although, i sincerely hope this horrific time will end soon, and we will learn lessons for future preparedness, i definitely believe that we can take advantage of it to grow mentally and learn new things. ■ davide brambilla, assistant professor, facultéde pharmacie, universitéde montreÁl as a son of a health professional in the red zone in italy, i was rapidly aware of the seriousness of the infection. nonetheless, the pandemic materialized as a storm when, from one day to the other, we had to shut down the laboratory and stop all nonessential experiments. after the initial phase of disorientation, the first work-related thoughts went to the teaching, the graduate students and their projects. while for undergraduate classes the universitéde montreál rapidly reacted and provided support to generate online classes, for the research, the initial stress slowly converted in the recognition that this could be a great opportunity to review and better plan our projects. now, after a month of the home-office, i feel that this forced shutdown brought me out of a working routine, and made me appreciate even more the importance of our profession. scientific research is the only actual weapon we have to fight this infection and to prevent or give a rapid response to future ones. deeply, i hope this pandemic will teach us all something, and that the opinions of scientists will receive higher consideration by the society and the decisionmaking institutions. ■ kang cai, post-doctoral fellow, department of chemistry, northwestern university i am experiencing my second "stay-at-home" period in the us now. i am a postdoctoral researcher at northwestern university in the us. three months ago, i went back to china to attend an academic forum, and then stayed at my hometown in hunan province to spend chinese spring festival with my family. on jan. , the covid- outbreak happened, and i experienced my first "stay-at-home" period. my return flight to the us was on january st, which turned out to be united airline's last flight from china to the us. after a two-week self-quarantine, i worked hard in the lab and tried to get as many results as possible, since i realized that universities in the us could also be shut down in the near future, which happened one month later. now, i have been staying in my apartment for three weeks i work on manuscripts, read papers, think about proposals, do "reactions" in the kitchen, and watch tv. the group meeting is held every week via zoom video, which is good, because it becomes the only "social activity" every week. currently i have plenty of work to do, which makes the "stay-at-home" days not that boring. but if the days continue for another one-to-two months, or even longer, which seems very likely to happen, i am not sure whether i will become anxious or not. it is a tough year for all of us. i am supposed to be on the job market this year, but now the situation has changed a lot. the future is full of uncertainty. ■ long chen, professor, department of chemistry, tianjin university during the locked-down period in our city since february, although the laboratories are still closed and all the students keep staying in their hometown, we all have great confidence that our country, and the entire world, can win this covid- pandemic crisis. we keep in touch with our group members via wechat, and also continue to hold the group meetings online, with a focus on literature reports. although from the early times of this pandemic we were not allowed to enter the office in the campus, all the online resources of the university can be conveniently accessed via a vpn connection. it is also an opportunity for the principal investigator and graduate students to analyze and summarize their research work. we recently managed to finish an invited review contribution and several manuscripts. with the situation improving and becoming better and better in china, some universities have already announced their gradual reopening time schedule. it is my hope that humanity pulls together and builds a future in which we are united to fight together against this virus and bring about the fastest possible victory. ■ joya cooley, post-doctoral fellow, materials research laboratory, university of california santa barbara i've read plenty that says that a routine is the most helpful for maintaining overall sanity. i've been keeping a routine, but have found that keeping a flexible routine with some goals works better for me. some of those goals include: sit down at my desk and get some writing done, read some literature, do yoga, make meals, check on my parents. i find that variation in my routine helps with maintaining sanityif i stray from a rigid routine, i start to create more inner turmoil when i cannot keep up. however, if i just try to set some daily/weekly goals, i can tackle them based on how i'm feeling that particular day. try to practice yoga at least times a week; some days it happens in the morning, some days in the evening, some days not at all. write for at least one hour a day; some days that happens all at once, some days it is broken up into shorter sessions. i feel i'm in a precarious position as i'm wrapping up my postdoctoral work and gearing up to begin my independent career, but all i can do for now is take it one day at a time. our laboratories closed unofficially on march th, , as i worried about the health of my students and postdocs. it coincided with spring break and some students were traveling abroad to see their families. i was hesitant to advise them on travel and the institute had not officially closed, making it difficult to issue strong recommendations. georgia tech officially closed all nonessential activities one week later. the weeks after were tough, as students got stranded abroad and experiments were not finished to the extent we wanted. our laboratories only opened in october , and we spent the past months ramping up. it took one day to ramp everything down. nonetheless, this has presented itself as an opportunity to come up with strong experimental plans, revisit the literature, and compile and analyze data that will be going into future manuscripts. the week of april , , finally started feeling normal and we are trying to make the most of it. i have a plan for each of my students and postdocs for the next two months, after that, i will have to reevaluate! will we turn into a computational group? we will see! as for teaching, transitioning to online format for this millennial (me) was easy and my students adapted quite wellthey ask more questions than ever. we are a team at lawrence berkeley national laboratory working with colleagues from the biomedical research community to make text mining and search tools specifically tailored to covid- research with the goal of helping to accelerate our colleagues' research. our team is made up of a number of graduate student researchers and postdocs from lbnl and uc berkeley who specialize in natural language processing methods for analyzing materials science literature, but we were approached about a month ago by colleagues from the innovative genomics institute about applying some of our techniques to the covid- literature. since then, we have been working around the clock to build covidscholar.org, a knowledge portal designed to help researchers stay on top of the covid- literature. to our knowledge, our database is the most comprehensive and current source for covid- papers available today with more than , papers and we are expanding to include patents and clinical trials in the near future. our site also includes features that leverage machine learning models that extract knowledge from the literature and help researchers make new connections that they might have missed due to the sheer volume of research coming out every day (we are seeing more than papers published on covid- daily.) this project has been extremely motivating for everyone on the team and we have been able to make rapid progress as a result. ■ miriam fenniri, undergraduate student, university of british columbia i am a soon-to-be fourth (senior) year undergraduate student at the university of british columbia (ubc). i was fortunate enough to spend last summer in an organic chemistry lab at universitélaval, where i was working on the synthesis of low band gap conducting polymers in the laboratory of prof. mario leclerc. this summer, i was planning on staying on ubc campus doing research and continuing my work as a teaching assistant until covid- got in the way. not only is the course that i was going to assist with canceled, but the research center where i was going to spend these next and a half months is closed until further notice. i am still living on-campus. many of my colleagues and classmates have returned homesome are still here either by choice or because of travel restrictions. classes were moved online on march th, and shortly thereafter, laboratories closed and research was halted. the transition to online learning has been smooth, however i never expected to have to write a midterm, much less three midterms, in my living room. at least i am now prepared to do the same regarding my final exams. to combat loneliness (and boredom), my family has been hosting weekly video-chats and strangely enough, i look forward to them! ■ matthew horton, materials project staff, lawrence berkeley national lab i work in computational materials science, a field for which i'm enormously grateful that myself and my colleagues can continue to make contributions to remotely. however, this can only happen because of people out there who are still performing the necessary maintenance and support for the high-performance computers and servers that we use. for myself, these are the people who are still keeping the facilities at the national energy research scientific computing center (nersc) running smoothly here in berkeley, but i know that my colleagues greatly appreciate similar efforts all across the world. it is important we recognize all of these people, and the personal risk they're taking on, as well as everyone working to keep laboratories in a safe and stable condition. beyond computation, much of my job is working to share the data we generate online at the materials project so that it is accessible to as broad a group of people as possible, and part of that is working to try to build a community. i have many open questions for how best to do this, but it is feeling necessary now more than ever that we better understand what steps we can take to make our community stronger and more inclusive. for my part, i'm enjoying talking to scientists on an online materials science discussion forum we recently launched, as well as help welcome in new developers to make contributions to the open-source codes we work on. as this situation evolves we can challenge ourselves to become more inventive in finding ways to connect and collaborate together, and carry these lessons forward. ■ hrishikesh joshi, graduate student, i have been living in germany for . yrs, pursuing a doctorate in chemistry. presently, i am preparing for a big day in my career, my ph.d. defense, "remotely." if i had to describe my current state in one word, it would be "uncertain" on all accounts. will the internet hold up during the defense? will my online presentation be good enough? will i find a job after my defense? the global economy is headed toward a recession, and most companies are downsizing. being an international candidate, it will be more challenging to find a job now than before. on the one hand, i am thrilled as i get to at least defend my ph.d. on the other hand, i am disappointed as i miss out on the opportunity to share this day with people. working remotely has made me appreciate personal interactions even more. every thursday, i am very excited as i get to go to the lab (reduced-workforce regulations) and feel a bit normal again. nevertheless, these times have also been productive as i am working on overdue programming projects and experimenting a lot with cooking. i feel these times are uncertain and disappointing but opportunistic in some ways. the enactment of unfamiliar public health measures and the rapid breakdown of our status quo are two major emotional stressors associated with the ongoing covid- pandemic. as an early career scientist, it is easy to fall into the rut of futility that comes with leaving experiments half-finished with looming deadlines. instead of focusing on events outside of my control, i found it more productive to reframe the current situation as a unique opportunity to work on myself, whether it be through reading up on current literature or investing more time into hobbies. in the past month, i've invested more time to properly care for my existing houseplants, repurposing a garment rack to create a diy grow light setup. if you are in need of a hobby, i recommend cultivating common, inexpensive vining plants such as pothos or philodendron! both plants display rapid growth and thrive even when you occasionally forget to water them, and with enough care and time they will grow into respectable, climbing foliage. houseplants are also inspiring metaphors for how we should live our lives; by constantly reaching for the sky while taking care of our essential needs, we can succeed and flourish in the face of unexpected change. ■ grit kupgan, postdoctoral researcher, as a member of a theoretical/computational group, i feel grateful that our research does not have to come to a halt. i cannot imagine the frustrations of researchers who have to delay their work as laboratories become inaccessible. for me, the transition from working in the lab to working from home has been a smooth one. at the beginning of march, our advisor requested a meeting to develop a contingency plan. as expected, the plan was invoked within a week due to the worsening pandemic. because of this preparation, everyone in the group was ready. our data were backed up, and some of us even took our workstations home. indeed, working from home has several advantages. i get to spend more time with my family who lives in a different state, and eliminate daily commutes. to keep the research momentum going, our group holds the research meetings twice a week online. additionally, i try to maintain my usual routines, such as work hours and breaks, and attend online seminars whenever possible. unfortunately, in my opinion, working from home reduces the sense of camaraderie slightly. in the lab, we can have research discussions, ask questions, get suggestions, and share personal stories and thoughts about politics (domestic and international) with our colleagues daily, which is always a pleasure. ■ henry s. la pierre, assistant professor, my professional and personal life move on different frequencies. my research group is in a rush to publish and to meet grant deadlines. even with the closure of laboratories and the cancellation of a busy travel schedule, leaving synchrotron and magnet lab experiments indefinitely suspended, our scientific progress is still planned on the order of days. while i am building plans for my students to safely return to the lab, these may very well not be implemented: there is no justification to rush back without effective and organized testing. as my wife, daughter, and i prepare for the arrival of her baby brother in july, i am acutely aware that the changes and dangers of this new world will not abate soon. it is exhausting simultaneously meeting the demands of the moment and mitigating the risks of a nebulous "return." these risks are particularly worrisome in the vacuum of federal and state leadership. as we rebuild our institutions, scientists, engineers, and academics must demand the integration of our technical and organizational expertise to the structure and function of our governments. one bright spot in this debacle has been the competent and measured response of scientific leadership across disciplines and institutions. ■ chengcheng rao, graduate student, with respect to my own research, my ongoing experiments needed to be postponed, accompanied by a shift of focus to literature, writing, and paperwork. i had been wondering if it would become possible to execute my experiments automatically and/or remotely? currently, the answer is no, but i cannot help but observe that with the growth of ai and breakthroughs underpinned by intelligent robots, some experiments will be doable by machines with fewer hands-inthe-lab. it is an eye-opening moment to think about how to bridge and transfer this advanced technology to my research as well. for interpersonal communication, our group meetings have moved online to keep social distancing and self-isolation, which is a new format for me. hence, we need some time to get familiar with this new communication method/software as face-to-face communication is more productive. all graduate courses are all online as well, for graduate students who need to take courses, and this is a challenge. meanwhile, so much information is shared by email, and it sure feels like we are receiving literally tons of emails every dayit is very hard to follow every email as some have too much/little information, and some are duplicated or even conflicting. it is another challenge to obtain useful information effectively through the information explosion. for my graduation, as i approach the end of my ph.d., my defense was expected at the end of the summer of . will i have an online defense, and a virtual graduation convocation? i hope not. hence, i am always thinking about when the coronavirus outbreak will come to an end. wuhan's shutdown was lifted on april , and it shows that the epidemic will be brought under control if effective controls are taken. but will it manifest itself as a second wave? this is the part i am most worried about. after thinking about all aspects, it is necessary to create and maintain a routine during this ever-changing time. do some work on paper or computer, avoid going out unnecessarily, and be sure to get some exercise to strengthen immunitythese are the things that i am doing. although the temperature is still below zero celsius in edmonton, i do believe spring is on its way. ■ adrianne rosales, assistant professor, almost exactly one year ago, i was working from home for a different reason: the birth of my daughter. while i was over the moon to be a new mother, there was a part of me that struggled with the anxiety of staying productive. my research group was only two years old, and i had watched others continue to submit grants, write papers, and advise students soon after the births of their children. whether that productivity was real or not, i held myself to an impossibly high standard on very little sleep. when covid- began to spread, i was acutely aware of the implications of shutting down my lab space and working from home again, not even one year later. nap time only goes so far, and my group was d still mostly firstand second-year graduate students. things were finally starting to work! ultimately, it was clear that much larger consequences were at hand and that many would suffer. while it was still voluntary, i decided to pause our lab activity. our university mandate came the following week, but in the meantime, it felt like a decision i made every day. and although i still hold high expectations for my group, we have worked together to make sure those expectations are also realistic. this will not be the last challenge my lab faces, and i hope that in addition to research productivity, i am training my lab on leadership and resiliency. the covid- pandemic is now well under control in china as of recent. most of our regular daily life has recovered, although the institute is only open to permanent staff. the "good" thing is i have much more time to concentrate on researchi reviewed several papers during this time, probably more than i usually do, and i feel that i have read each manuscript more carefully than before. senior graduate students have not yet come back from home, which will certainly have a big impact on the progress of their thesis and the production of the group as a whole. as a group of experimental chemistry, we urgently await, with great anticipation, the start of normal experiments and research. social media on the web have played an important role during this special period. i taught a course to first-year graduate students online, in spite of having no experience with respect to online teaching, and with relief i can say finally that it went well. many conferences have been canceled or postponed to next year, but on the other hand, online conferences became increasingly popular. i have already received several invitations for online ph.d. defenses, which is critical for our young people to progress. web-based conferences can significantly save time, and may become more and more popular even after the pandemic. i am confident that the pandemic will be under control worldwide, probably starting around the summer. however, the impact of the pandemic on the economy already shows, and i hope that it will not affect the funding situation in the future. during this winter vacation, i went to wuhan to visit my family on th of january, one week before the lockdown of the city caused by the covid- outbreak. i stayed at my parents' home with my family for more than two months, wondering about the fates of my entire family. the virus infected several of my relatives, four of whom were hospitalized. luckily, they all recovered, except my -year-old grandfather. during the most dire of days, like the people of wuhan, i could not sleep in the night and was looking for the slightest hope in the news, while planning to limit exposure of uninfected family members. with strong backup from the chinese people, especially medical personnel, wuhan survived and reopened on th of april. i returned to shanghai by flight on the first day wuhan reopened, and was greeted by media instead of medical teams upon arrival. during the city lockdown, a journal article authored by a student of mine and me was submitted in spite of difficulties caused by a lagging internet connection. after peer-review, a reviewer raised stability issues, which required further experimental data. luckily, my collaborators were able to provide relevant results. i sincerely cannot anticipate the outcome, had i wrote to the editor explaining that we could not provide any further experimental data in the near future due to the coronavirus outbreak, or if i were to ask for an unlimited extension of time to revise. as of now, we still do not have a schedule to reopen the laboratories. i am still waiting for another days upon arrival in shanghai before the university can clear me to return to campus. almost all my group members were in their respective hometowns, longing for a notice to return to shanghai, especially the ones who are graduating this year. online meetings were possible but were difficult due to slow internet connections. i have individual discussions regarding literature with each one of them, hoping they are coping well with the current situation. chemistry of materials we wish the best to all of our authors, readers, and reviewers. we are in this together, and we look forward to another set of snapshots in a month amaury bossion orcid grit kupgan orcid key: cord- -j m authors: imperiale, michael j.; howard, don; casadevall, arturo title: the silver lining in gain-of-function experiments with pathogens of pandemic potential date: - - journal: influenza virus doi: . / - - - - _ sha: doc_id: cord_uid: j m half a decade after the contentious “gain-of-function” (gof) debate of that followed experimentation showing that highly pathogenic avian influenza virus could become mammalian transmissible, it is possible to reflect on the arguments for and against this type of research. in this essay we argue that gof-type experiments have already produced important information not available from any other source while also providing information on pathogenesis and the requirements for optimizing strains for vaccine production. we analyze the moral arguments against gof and find them less compelling for a variety of reasons ranging from the uncertainty of risk-benefit analysis to the reduced likelihood of accidents given the enhanced biosafety and biosecurity protocols currently in place. in our view the most important consequence of the gof debate is that it brought renewed attention to biosafety protocols and ushered innovation in answering the relevant biological questions with greater safety. we conclude that gof experiments should go forward provided that necessary biosafety and biosecurity conditions are in place. at the turn of the twenty-first century, public health officials began to notice an increase in the number of human infections with highly pathogenic avian h n influenza virus originating from birds in close proximity to people. the possibility of influenza pandemics is always a concern, but this zoonotic jump seemed particularly worrisome because the case fatality rate (cfr) was approximately %. fortunately, the virus did not acquire mammalian transmissibility, and there were no confirmed examples of human-to-human spread. nevertheless, there was great concern that if the virus did acquire human transmissibility and maintained such a high cfr, the world could face a public health emergency of unprecedented danger. for comparison, the cfr during the influenza epidemic, which many believe to be the worst influenza pandemic in history, was approximately . %. another development in the mid- s was the creation in the usa of the national science advisory board for biosecurity (nsabb), whose charge was to advise the us government on so-called "dual-use" problem in biomedical research: research being performed with beneficial goals in mind, but the results of which could be misused for nefarious purposes. the nsabb delved into the dual-use controversy early in its tenure when the us government asked it to review the paper describing the reconstruction of the influenza virus strain responsible for the epidemic [ ] . although the nsabb voted to recommend publication, the editor of science made it clear that the journal would have published the article irrespective of the nsabb vote unless the paper was classified [ ] . for the next half decade, the nsabb struggled with the problem of how to deal with dual-use research in the biological sciences and proposed identifying a small subset of science known as "dual-use research of concern," or durc, as that domain on which to focus efforts. while the nsabb was formulating durc definitions and devising recommendations, two laboratories set out to experimentally test whether h n virus could become transmissible in what is thought to be the best animal model for such studies, the ferret. research groups led by yoshi kawaoka in the usa and ron fouchier in the netherlands took similar approaches albeit with different starting strains, first engineering the ability of the virus to bind to human receptors and then serially passaging it through ferrets. both obtained the same answer: the h n virus could attain the ability to be transmitted via respiratory droplets. they wrote manuscripts and submitted them for publication in nature and science, respectively [ , ] . in late the nsabb had the opportunity to evaluate its policies and recommendations when the us government learned of the two submitted manuscripts describing these high pathogenicity avian influenza viruses (hpaiv) that had been made mammalian transmissible: it asked the nsabb to advise it whether publication was wise given potential biosecurity concerns. the details of the deliberations of the nsabb and its ultimate decision to recommend publication have been described in detail elsewhere, but briefly, the nsabb determined that the benefits of the research outweighed the biosecurity risks [ ] . hence, the first round of the controversy was focused primarily on biosecurity issues. after the decision on the two influenza papers, the situation quieted for a couple of years until a series of biosafety lapses at us government laboratories at the cdc and the nih received rekindled interest on the problem. these laboratory incidents received a high degree of public attention, spearheaded by the reporting of alison young at usa today. this conjunction of laboratory accidents together with additional follow-up publications [e.g., ] puts the h n story in a new light, namely, whether similar experiments in which new phenotypes are added to pathogens, gain-of-function (gof) studies, could be conducted safely and whether they should be pursued at all. confronted with a public outcry combined with a serious scientific debate on the benefits and risks of gof-type experiments, in the nih, which has administrative responsibility for the nsabb, imposed a moratorium on us-funded gof experiments with "pathogens of pandemic potential (ppp)," those being influenza virus, severe acute respiratory syndrome (sars) coronavirus (sars-cov), and middle east respiratory syndrome (mers) coronavirus (mers-cov). while sars-cov had disappeared from the human population due to a highly successful public health containment endeavor, the mers-cov outbreak had emerged in the middle east and was (and still is) ongoing at the time. the us government also charged the nsabb with making recommendations about the future of gof research (https://www.phe.gov/s /dualuse/documents/ gain-of-function.pdf). we have summarized the events of in a series of papers, and those details will not be recounted here [ , ] . instead, our focus will be to make the case for the scientific and moral value of gof-type research provided that it can be conducted safely. hence, the second round of the controversy was focused less on biosecurity and intentional release and almost entirely on biosafety. we have previously argued that the h n experiments, and similar gof studies, provide benefits at many levels, from the practical to the epistemological [ , ] . some tangible benefits of gof studies involving influenza virus include: [ ] , extending the threat horizon to h viruses. . the results of gof experiments can inform on important biological questions. for example, gof experiments yielded mutants that when analyzed showed that higher and lower ph optima for the hemagglutinin were associated with enhanced virulence in birds and mammals, respectively [ ] . this information is important for knowing how influenza viruses jump from birds to humans, which is a critical step in the emergence of new pandemic strains. . gof experiments can be used to produce new viral strains to improve vaccine production. one of the hurdles in vaccine preparation is the adaptation and growth of vaccination strains in eggs for efficient production. gof-type of experiments can be used to identify mutations that facilitate replication of influenza strains in eggs, and this information could facilitate vaccine production [ ] . are there other claimed benefits that have been derived from the h n studies? at the time of publication, the authors and others argued that the results would inform surveillance efforts and vaccine development. it is unclear to us how much surveillance has benefitted although the mutation information has been used in monitoring [ , ] . as we and others have pointed out, there is a danger of focusing on the exact mutations discovered in the ferret experiments because there may be other genetic routes to human transmissibility [ ] . some have noted that by the time such mutations have been detected in avian populations, it might be too late to stop the spread due to the relative rates with which we can identify those mutations using current technology and the rate of spread of influenza virus [ , ] . increased awareness of the threat of h n , however, must certainly have led to better isolation of patients who have been exposed to the virus. in this regard, it seems reasonable to assume that any onward transmission of an avian strain would occur largely in the healthcare setting, as has been the case with sars-cov and mers-cov, and rapid intervention could prevent a widespread outbreak. while monitoring the mutations discovered in the laboratory from gof experiments is not sufficient to predict dangers from environmental isolates, it is important to note that while both groups obtained viruses with different mutations, they both achieved similar phenotypes of enhanced stability of the ha protein. as our ability to predict phenotype from genotype improves, this becomes increasingly important. a big caveat to overstating the importance of this, however, is the contribution of epistasis to any given phenotype. for example, the same mutations identified by the fouchier group, when introduced into a different h n background, yielded a different ha phenotype [ ] . this implies that insights gained from one set of mutations in one strain are unlikely to be generalizable to other strains given sequence differences. hence, with the hindsight of half a decade of work since the original controversy in , it appears that gof-type experiments are very informative with regard to big questions such as whether mammalian virulence and transmissibility potential exists in hpaiv but may be less useful in making fine-scale molecular predictions. as mentioned above, in the us government mandated a moratorium on gof-type experiments involving ppp such as influenza, mers-cov, and sars-cov. to the best of our knowledge, us-supported experiments to examine changes in transmission of avian influenza viruses have largely stopped. the fouchier lab did publish a follow-up report to their original science manuscript in which they narrowed down the exact mutations that enabled transmission in the ferret model, but based on the publication date, it appears that this work was completed prior to the moratorium [ ] . in the mers-cov field, the development of a small animal model that faithfully reproduces human transmission and disease has been slowed significantly by the moratorium. mers-cov uses the dipeptidyl peptidase (dpp ) protein as a receptor, and the human and murine proteins differ enough such that the virus cannot use the mouse molecule. transgenic mice that express human dpp ubiquitously experience a broader set of symptoms than do humans when infected with wild-type mers-cov [ ] [ ] [ ] [ ] . this problem was recently overcome by developing a transgenic mouse expressing a mutant dpp in which two key amino acids were mutated from the mouse allele to the human allele. however, the authors still needed to passage the virus through these transgenic mice to derive a gof variant that recapitulated human disease [ ] . this gof virus was subsequently used to show the efficacy of a promising nucleotide prodrug, originally initially developed for ebola virus, for treating mers [ ] . continuing efforts to produce a mouse-adapted mers-cov in wild-type mice have been prohibited [ ; r. baric, personal communication]. the question remains, how does society at large, and the scientific community and regulatory agencies in particular, weigh the risks and benefits? the difficulty lies largely in trying to apply quantitative risk assessment measures to the problem. the benefits of all biological research, not just gof research, often do not make themselves apparent until years or even decades after the experiments are performed. on the other hand, the risks, even when theoretical, manifest themselves in the present when the experimentation is done. fortunately, we have not had real examples of laboratory accidents leading to significant morbidity or mortality. the reintroduction of h n into human circulation has been attributed to a laboratory mishap, although not all agree since other explanations are possible [ ] [ ] [ ] . as discussed below, some authorities have presented worst-case scenarios based on hypothetical calculations, whereas others have used similar types of data to come up with numbers at the extreme opposite end of the spectrum, arguing that the likelihood of such events is extremely low. perhaps the greatest benefit of this controversy has been an increased attention to biosafety when working with dangerous pathogens. in , the nih proposed a biosafety stewardship month as a means to promote increased attention to biosafety, and many institutions took advantage of this opportunity. indeed, while it can be misleading to draw strong conclusions from the absence of events, the types of events that found their way into the news media earlier in this decade seem to have decreased significantly. another benefit of the controversy was that it has stimulated efforts to gain comparable information in systems that do pose the same biosafety or biosecurity risks as working on the wild-type virus. for example, a recent study reporting that only three mutations were needed to switch h n tropism to human cells was performed using an attenuated virus [ ] . gof research, especially that involving a ppp, has become the focus of intense ethical debate in the wake of the moratorium. the ethical concerns have been formulated along three main axes of criticism. the first concern is the potential for misuse of any information or product generated by gof research by bad actors. the worry here is that, once the results of gof studies are published, bad actors could replicate the work for nefarious purposes, such as a terrorist attack. such concerns were expressed, for example, in connection with the aforementioned publication of studies of engineered avian flu virus transmissibility in ferret models [ , , ] . as noted above, the question whether to permit publication was considered by the nsabb, which eventually allowed publication, judging that the risk of misuse was outweighed by the potential benefits. this is neither the first nor the last occasion on which questions have been raised about the risks of dual-use research, with examples ranging far beyond biomedical research to include such technologies as unmanned aerial vehicles being used as weapons and hacking being used for the stuxnet cyberattack. cyberwarfare tools can be used to enhance national security by disarming opponents while at the same time finding employment in crime such as occurred recently when the leaked national security agency code was used in spring for global ransomware attacks. such misuse of gof research is certainly possible. but one serious problem in an ethical evaluation of possible misuse is the difficulty of estimating the likelihood and potential impact of misuse. one can imagine numerous different scenarios involving everyone from major state actors and non-state terrorist organizations to freelance mischief-makers working in a home basement lab. there is no way to quantify over so many possibilities, let alone to assay systematically the effects that such a diverse array of actors might achieve. the misuse concern is further complicated by the fact that the cost and complexity of using relevant tools, such as crispr/cas , continues to decline, making ever easier the replication of even unpublished research. indeed, the risks of the democratization of crispr/cas technology go well beyond gof research, and there has been a call for a much more concerted, international, public debate about monitoring and possibly regulating access to some of those tools [see ]. given these difficulties, what is the responsible way for gof researchers to proceed with respect to potential misuse? this is a place where prudence might be a better guide than cost-benefit analysis. thus, one notes that, for the researcher's own safety, gof research with especially dangerous pathogens must be done in labs with very high biosafety standards, at least biosafety level (bsl)- . that means that, except for the rare suicidal individual, such research will only be feasible in countries with the necessary biosafety facilities and protocols, and the list of such facilities today includes mostly nations with proven records of responsible research conduct, nations which also have the technical expertise to conduct such research entirely on their own. of course one could imagine a nation like north korea aspiring to a bioweapon capability, and as they have demonstrated with their successes in offensive cyber operations, ballistic missiles, and nuclear weapons, there is no lack of technical talent in north korea. for all of these reasons, the risk of publication of gof research related to misuse of biotechnology seems to have receded from the forefront of concern. in addition, the scientific publishing community is much more aware of these issues, and many journals have instituted internal reviews for papers that include durc [e.g., , ] . the more prominent worry today is about accidently unleashing the very kind of global pandemic that one was seeking to prevent. a number of critics have argued that the risk of inadvertently creating a global pandemic through accidental release of an engineered, human-transmissible pathogen with high virulence and case fatality rate vastly outweighs any benefits that might be obtained from such research [e.g., ]. thus, one source claims a . % to . % probability per year of research in a bsl- lab of an accidental release of highly transmissible influenza virus that would kill between , and million people [ , ] . if this is a reliable estimate, that's a scary prospect. on the other hand, one of the authors of the original h n studies has calculated the risks to be much, much lower [ ] . given a claimed risk on that high a scale, how shall we think about the balance between benefit and risk? first, we must ask some tough questions about the risk analysis itself. many factors go into such an analysis, including historical data on accidental exposure in bsl- and bsl- labs and simulation studies of disease spread after accidental exposure. we consider each of these factors separately. to date, only one reasonably sophisticated simulation study for the spread of a potentially pandemic influenza virus has been reported [ ] . this is the study cited by lipsitch and inglesbsy [ , ] . the researchers who conducted the simulation study had to make many assumptions about such variables as virulence, case fatality rate, latency time, demographics, geography, response capabilities of public health authorities, and monitoring of lab personnel for symptoms of exposure. the simulation model was robust against variations in many of these parameters, but there were considerable differences in outcome with some variations, such as early detection of initial exposure. still, on the whole, the numbers are grim. with the right combination of factors, it is theoretically possible for an accidental release to wreak havoc. that brings us to the question of how probable such a catastrophic event is and the science of risk-benefit analysis. the other major component of the risk calculation-the chance of accidental exposure or release-is generally estimated on the basis of historical data, which, with its foibles, is far less robust. the spate of accidents in us bsl- labs a few years ago rightly aroused concern among experts but also generated in the public mind a perception of risk that might not accurately represent the current state of affairs after more stringent monitoring and review procedures were implemented. the annual rate of accidental exposure today could easily be ten to one hundred times smaller than it appeared to be in . of course, , to million deaths per lab year is still reason for worry. but then there are other factors to consider. the reported accidental exposures aggregate data on all organisms that require high levels of biosafety containment. none of those cases involved gof research, and, given the attention being paid to gof by comparison with research on, say, natural pathogens, it is not at all clear that we can reasonably extrapolate from the historical data on accidental exposure to the risk of accidental exposure or release in gof research. there are still deeper problems with a cost-benefit approach to assess the ethics of gof research. first, assessing the risk of accidental release and an attendant global pandemic is only one small part of a more comprehensive cost-benefit analysis, which must always compare the costs and benefits of alternative courses of action. proponents of gof research argue that it can play a crucial role in preventing or lessening the effects of a global pandemic by enabling mitigation factors such as early detection and the rapid development of vaccines. one obvious additional risk, then, is the risk of a global pandemic that might have been prevented or mitigated by continuing gof research. that risk is even more difficult to quantify than the risk of pandemic through accidental release. still, it must be part of a comprehensive analysis, and since its major consequence, e.g., a global pandemic, is just as severe as a pandemic caused by accidental release, such a scenario would loom just as large as the accidental release scenario in a thorough cost-benefit analysis. another deep problem with conventional cost-benefit analyses is that they usually do not include the intrinsic benefit of new knowledge [ ] . one might think that "knowledge" is something too ephemeral to be quantified, or one might argue that, if knowledge counts as a "benefit," it does so only through the consequences of such knowledge for human flourishing, which means that it does enter our calculations indirectly though measures of the costs and benefits of human health and human suffering. one does not want to indulge uncritically in the mantra of knowledge for knowledge's own sake, a trope that has too often been used to justify research, but the fact remains that, on the whole, increased knowledge brings increased ability to promote the good and to mitigate suffering. new knowledge can always be used for evil ends, but how we use that knowledge is a moral choice, and if we don't have that knowledge in the first place, then we cannot use it for the good. exactly how new knowledge will contribute to future human flourishing in a complex and rapidly changing technical and social world is usually unforeseeable at the time when the new knowledge is first generated. technology ethicist shannon vallor dubs this the problem of "technosocial opacity" [ ] . thus, it would seem impossible to include the long-term effects of new knowledge production in a cost-benefit analysis of the research generating that knowledge. the only way to fairly assess the benefits of such research is to put a premium on knowledge production per se. how to do that is the hard problem, and that difficulty must be added to the list of reasons suggesting that cost-benefit analysis is a seriously limited tool for policymaking about scientific research. perhaps the deepest problem with the cost-benefit approach to policymaking is what we have termed elsewhere the "apocalyptic fallacy" [ ] . imagining an unintentional, global pandemic as one possible outcome of gof research is tantamount to assigning infinite negative utility to such research in a cost-benefit analysis. thus, it makes no difference how low the probability of such an outcome might be. infinite negative utility, multiplied by any finite probability, totally swamps every other term in a cost-benefit analysis, meaning that no imagined benefits of alternative courses of action, however great (excepting eternal salvation for all of humankind), can make a difference in the calculation. what this means is that cost-benefit analysis is, effectively, useless in such settings, at least with regard to the moral assessment of something like gof research. if cost-benefit analysis is the epitome of reason and rationality in policymaking, then reason fails us in cases where the risks include the extermination of a significant fraction of all human life. nevertheless, there is still value in risk analysis. as the abovecited simulation study of disease spread after accidental exposure and release amply demonstrates, a careful risk analysis can point us toward those factors that are critical for minimizing risk, such as enhanced biosafety protocols and rapid public health responses. but when promoted to the public and policymakers as forecasting a doomsday scenario, such analyses risk inducing panic that overwhelms practical reason. similarly, comparisons of the risks of gof research to the events leading to the development of the nuremberg code do not serve a useful purpose in the discussion [ ] . the use of the word "nuremberg" connotes an association with war crimes that is simply inappropriate for use in a rational discussion about experiments that are ostensibly being done by well-meaning scientists trying to prepare humanity for confronting a potential pandemic. although we are well aware that the nuremberg code and the war crimes of the high echelons of the nazi hierarchy are very different things, the problem lies in the symbolism of the phrase and how it may be perceived by the public. we urge that it not be used and in fact, it is possible to discuss these important principles without invoking the name of the city with its historical baggage. if cost-benefit analysis is not the optimal tool for policymaking with respect to gof research, what is? earlier, we mentioned the virtue of simple prudence in thinking about dual-use research and technology. perhaps that is the answer here as well. technology ethicists have given the name "precautionary principle"-policymakers prefer sophisticated names like this-to the homespun idea of prudence. but prudence is really a simple idea, familiar to us all. it means, "think before you act" and "do not act if you are not sufficiently secure in thinking that you will do more good than harm." thinking before acting should include risk analysis in settings such as gof research. but a risk analysis is not the end of clear thinking; it is only the beginning. where does this leave us? one major conclusion is this: for almost every form of human activity, there is a nonvanishing probability of catastrophic outcomes. any casual individual act could set in motion a chain of events that would lead to the rise of the next world war. this is highly improbable but possible, and the consequences would be cataclysmic, outweighing any imaginable good that might derive from that action. therefore, the precautionary principle would suggest that we do nothing. but doing nothing can equally well engender a sequence of events leading to human extinction. we conclude, therefore, one should act. this dilemma highlights a fatal contradiction lurking within any attempt to rationally assess actions that might entrain consequences with infinite negative utility via cost-benefit analysis. how otherwise should we proceed? this is not a hard question. think about both risks and benefits, take obvious precautions, and then make the prudent choice. with enhanced biosafety protocols and improvements in the public health response, we should not ban gof research but monitor it. the relevant research communities should insist upon stringent norms for the conduct of the research and in safety protocols. provided that these conditions are met, there is no obvious reason why gof type of experimentation should not go forward. reconstruction of the influenza virus: unexpected rewards from the past better never than late airborne transmission of influenza a/h n virus between ferrets experimental adaptation of an influenza h ha confers respiratory droplet transmission to a reassortant h ha/h n virus in ferrets public health and 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two dozen risky virus studies the reemergent h n strain and the gain-of-function debate reply to "the h n influenza virus reemergence demonstrated gain-of-function hazards the h n influenza virus reemergence demonstrated gain-offunction hazards three mutations switch h n influenza to human-type receptor specificity ethical issues in genome editing using crispr/cas system dual-use research of concern (durc) review at american society for microbiology journals biosecurity and the review and publication of dual-use research of concern the ethics of biosafety considerations in gainof-function research resulting in the creation of potential pandemic pathogens moratorium on research intended to create novel potential pandemic pathogens erratum for lipsitch and inglesby, moratorium on research intended to create novel potential pandemic pathogens studies on influenza virus transmission between ferrets: the public health risks revisited ) pandemic influenza a/h n pdm in italy: age, risk and population susceptibility technology and the virtues: a philosophical guide to a future worth wanting the apocalypse as a rhetorical device in the influenza virus gain-of-function debate key: cord- -zzkqb u authors: moore, jason h.; barnett, ian; boland, mary regina; chen, yong; demiris, george; gonzalez-hernandez, graciela; herman, daniel s.; himes, blanca e.; hubbard, rebecca a.; kim, dokyoon; morris, jeffrey s.; mowery, danielle l.; ritchie, marylyn d.; shen, li; urbanowicz, ryan; holmes, john h. title: ideas for how informaticians can get involved with covid- research date: - - journal: biodata min doi: . /s - - -y sha: doc_id: cord_uid: zzkqb u the coronavirus disease (covid- ) pandemic has had a significant impact on population health and wellbeing. biomedical informatics is central to covid- research efforts and for the delivery of healthcare for covid- patients. critical to this effort is the participation of informaticians who typically work on other basic science or clinical problems. the goal of this editorial is to highlight some examples of covid- research areas that could benefit from informatics expertise. each research idea summarizes the covid- application area, followed by an informatics methodology, approach, or technology that could make a contribution. it is our hope that this piece will motivate and make it easy for some informaticians to adopt covid- research projects. the coronavirus disease (covid- ) pandemic has had a significant impact on population health and wellbeing. research efforts are underway to identify vaccines [ ] , improve testing [ , ] , understand transmission [ ] , develop serologic tests [ ] , develop therapies [ ] , predict risk [ ] , and develop mitigation and prevention strategies [ , ] . biomedical informatics is central to each of these research efforts and for the delivery of healthcare for covid- patients. critical to this effort is the participation of informaticians who typically work on other basic science or clinical problems. the goal of this editorial is to highlight some examples of covid- research areas that could benefit from informatics expertise. each research idea summarizes the covid- application area followed by an informatics methodology, approach, or technology that could make a contribution. this is followed by some practical suggestions for getting started. these are organized under sub-disciplines for biomedical informatics including bioinformatics that focuses on basic science questions, clinical informatics that focuses on the delivery of healthcare, clinical research informatics that focuses on research using clinical data, consumer health informatics that focuses on the use of mobile devices and telemedicine, and public health informatics that focuses on research questions at the population or community level. it is our hope that this piece will provide motivation and make it easy for some informaticians to adopt covid- research projects. we present here two applications of bioinformatics approaches to the basic science aspects of severe acute respiratory syndrome coronavirus (sars-cov- ) and covid- . these focus on sequencing the virus, in order to understand the genomics of sars-cov- with the goal of informing treatment regimens and vaccine development. the genome sequences of sars-cov- are essential to design and evaluate diagnostic tests, to track the spread of disease outbreak, and to ultimately discover potential intervention strategies. phylogenetics is the study of the evolutionary connections and relationships among individuals or groups of species. these relationships can be identified through phylogenetic inference methods that evaluate the evolutionary origins of traits of interest, such as dna sequences. similar to tracing your ancestry through a dna test, a phylogenetic analysis approach can be used to help map some of the original spread of the new coronavirus and trace a sars-cov- family tree based on its rapid mutations, which creates different viral lineages. note that many countries have shared an increasing number of sars-cov- genome sequences and related clinical and epidemiological data via the global initiative on sharing all influenza data or gisaid (https://www.gisaid. org). gisaid has generated a phylogenetic tree of sars-cov- genome samples between december and april . in particular, nextstrain, an open-source software package (https://nextstrain.org), uses sars-cov- genome data to help track the spread of disease outbreaks. for example, it could be applied to tell researchers where new cases of the coronavirus are coming from. this can be crucial information for investigating whether new cases arrived in given countries through international travel or local infection. one caveat is that the number of genetic differences among the sars-cov- genomes is close to the error rate of the sequencing process. thus, there is a possibility that some of the observed genetic differences may be artifacts of this process. however, rapid data sharing for sars-cov- is the key to public health action and has led to faster-thanever outbreak research. with more data sharing of the sars-cov- genomes, more genetic diversity will become apparent making it possible to better understand how the coronavirus is being transmitted. while exploring the genome sequence of the sars-cov- virus is anticipated to provide scientists a better understanding of viral evolution and aid in the development of vaccines and treatments, evaluation of host genetics in response to covid- is of similar importance. for other viruses, we know that some individuals have a natural immunity whereby even when exposed to the virus, they do not develop infection. for example, the well-known ccr -delta allele has a variation that protects individuals who have been exposed to the human immunodeficiency virus (hiv); they are protected from developing aids (acquired immunodeficiency syndrome) [ ] . because of this, researchers are gearing up to study the genomes of covid- positive patients in comparison to controls (covid- -negative patients). for example, stawiski et al. investigated coding variation in the gene, ace . ace , the human angiotensinconverting enzyme , is a cell surface protein that the viral spike coat protein sars-cov- engages to invade the host cell [ ] . what would be optimal for these and other genome-wide analyses, to identify potential risk and protective variants, are individuals who test positive for the virus but remain asymptomatic. these individuals will be more difficult to identify because of the lack of widespread testing (most individuals without symptoms are not being tested). however, the research community is building large, international, collaborative consortia to address this challenge, such as the covid- host genetics initiative (https://www.covid hg.org/). much like understanding the viral genome will be useful for drug development, identifying the genetic variation in the host dna that is either increasing the risk of, or protection from, sars-cov- infection will enable us to identify putative targets for therapeutics and vaccines. we present here three topics relevant to the diagnosis and management of covid- patients. these include imaging, suggestions for the roles that informaticians can assume in the pandemic, and the need for novel approaches to delivering patient care and learning from in-practice data. imaging provides a powerful tool for covid- diagnosis and patient monitoring given the impact on lung physiology and anatomy. for example, chest computed tomography (ct) has been shown to have promising sensitivity and early detection power compared with the standard reverse transcriptase polymerase chain reaction (rt-pcr) test [ ] . in addition, imaging plays an important role in assessing patients with worsening respiratory status [ ] , which is crucial for monitoring and treatment planning. given the fast-growing volume of covid- cases, to help alleviate the huge manual evaluation burden on clinicians, there is an urgent call for researchers in imaging informatics (or radiomics) to work on developing automated image analysis and artificial intelligence (ai) methods and tools. to achieve these goals, major efforts have been initiated to address two critical research foci. the first is to create large-scale high-quality imaging data repositories (e.g., radiological society of north america (rsna) covid- imaging data repository, https://www.rsna.org/covid- ) to accelerate collaborative research on image-based covid- diagnosis and treatment. the second is to develop innovative ai methods for automatic image analysis for covid- diagnosis and severity assessment. to get started on supporting these efforts, below we suggest a few relevant resources for interested imaging informaticians. several covid- resource and initiative web portals have been created by major organizations such as american college of radiology (acr), radiological society of north america (rsna), and european society of medical imaging informatics (eusomii). these portals offer important information on policies, guidelines, discoveries, initiatives, data sets, and/or other relevant resources. given the rapidly growing ai-based imaging literature on covid- , it is worth noting a recent review article [ ] , which provides comprehensive coverage on a variety of interesting topics, including ai-empowered contactless imaging workflow, ai in lung image segmentation, ai-assisted diagnosis and severity assessment, ai in follow-up studies, public imaging datasets for covid- , and future directions. to effectively address the ever-growing surge of covid- patient cases, informatics solutions are being developed to help care providers and healthcare institutions manage patients from symptoms to recovery. symptom screening tools have been developed to aid patients in distinguishing covid- symptoms from common colds and flu. telemedicine is helping keep patients at home by deploying chatbots to answer patient covid- questions and providing virtual visits and consultations to limit the number of individuals exposed to covid- and to manage patients with mild covid- symptoms. this reduces resource utilization and overburden on the care delivery system. capacity and resource management tools can generate projects based on regional infection counts and current patient admissions to estimate the number of patients that will require hospitalization, intensive care unit beds, medications, and mechanical ventilation. these projections can improve clinical response times and inform triage care strategies. donation and resource inventory tools can be helpful for identifying, cataloging, and distributing personal protective equipment (ppe), homemade masks, and other critical medical supplies to those fighting on the front lines. informaticians can support these efforts by ) educating patients and care providers about data science resources and electronic health record (ehr) platforms for building point-of-care solutions, ) joining the open-source community efforts to develop these technologies, and ) volunteering with the information services divisions within their healthcare organizations to deploy telehealth tools and engage in patient management projects. the covid- pandemic has been an unprecedented stress test for clinical information systems. the scramble to develop and implement new clinical practices has in many cases outpaced our ability to effectively use standard tools for building, testing, and monitoring these practices. for instance, clinical laboratories have rapidly implemented several different methods for sars-cov- diagnostic testing and have also needed to send out testing to multiple reference laboratories. these complex practices have made it non-trivial to collect even the most basic information, including who is being tested and who is positive. these data are essential both to the care of individual patients and to health providers who need to design these care systems and plan for what is coming next. these data are also being reported to government agencies in multiple new manual processes. the work that has been done to build these data collection systems is extraordinary and commendable. but, for our clinical informatics and public health communities, these challenges highlight the need for developing modern, flexible clinical information systems and robust infrastructure for inter-institution data sharing. the implementation of novel clinical practices has also been notable for how much we still do not know about their clinical utility. as a consequence, there is a great need to learn about clinical utility from in-practice data. for example, the precise clinical sensitivity and clinical specificity of the sars-cov- diagnostic testing being used are currently unclear [ ] . this is critically important because false-negative results could lead to the inappropriate non-use of ppe or insufficient clinical and epidemiological monitoring. the rate of such false-negatives is also highly variable across time, as the disease prevalence changes, and across multiple patient, provider, and geographic factors. to fill in these knowledge gaps, there is a big need for the design and application of methods for estimating such parameters from in-practice data. these approaches must be robust to the many sources of bias in these kinds of retrospective data and must be applied to datasets of large enough sample sizes, to generate meaningfully precise estimates. we present here four clinical research informatics domains related to the generation, integration, and use of clinical and other data that could be leveraged in addressing the pandemic in various settings. the domains include a well-developed informatics infrastructure that encompasses a large healthcare landscape, the potential for systematically and cautiously repurposing drug treatments, the leveraging of existing clinical and biospecimen data, and the role of advanced statistical, integrative, and machine learning (ml) tools for diagnosis and treatment. one critical need to support covid- -related clinical and translational research studies is the development of informatics infrastructure that contains accurate and timely clinical data from the electronic health records of the covid- population. as a first step, healthcare institutions can create patient registries to maintain reliable lists of covid- patients and cases (e.g., confirmed, ruled out, uncertain). these data must be updated regularly (daily or several times each week) and contain a broad set of data elements representing demographics, prior medical histories, current medications, comorbidities, diagnoses, procedures, outcomes, etc. to serve a broad base of clinical investigators and scientific inquiries. to adequately code all patient data, image processing will be needed to encode salient radiological findings, and natural language processing will be needed to extract symptom onset, severity, and duration among other variables. secure informatics platforms such as integrating bench to bedside (i b ) and the shared health research information network (shrine), trinetix, and atlas play an important role in standardizing and harmonizing clinical data to common data models (cdms) including i b , patient-centered outcomes research network (pcornet), fast healthcare interoperability resource (fhir), and observational medical outcomes partnership (omop). once covid- patients are indexed within the patient registry and their clinical data has been extracted, transformed, and loaded into these frameworks, clinical researchers can execute secure, privacy-preserving, and federated queries across all participating sites using any framework to identify patients for clinical trials, generate scientific hypotheses, and conduct observational studies. both aggregate and individual-level information can be made available with appropriate data governance, ethical review, and institutional agreements. informaticians can support these efforts by ) developing technologies and algorithms for extracting, encoding, and mapping raw ehr data to emerging covid- -specific cdms, ) engaging in existing and emerging consortiums, both grass roots and nationally-sponsored efforts, across clinical and translational science awards (ctsas) and informatics networks, and ) connecting with clinicians to develop and share informatics tools and predictive models that identify clinically-formative, actionable insights from heterogeneous, temporal data. one of the major challenges with emerging diseases, such as covid- , is that evidence for effective drugs and treatments is sparse. while vaccine development is important, vaccines are only helpful to prevent individuals from becoming infected in the first place. for those that have covid- , the main strategy for treatment with drugs (while the disease is still emerging) is to reuse those that have been approved for other purposes. there are several drugs that may therapeutic use in covid- , namely: hydroxychloroquine sulfate, chloroquine phosphate, remdesivir, carfilzomib, eravacycline, valrubicin, lopinavir and elbasvir. these medications were designed for treatment of various diseases, including lupus, malaria, cancer and hiv. therefore, the use of these medications to treat covid- is termed 'drug repurposing' and one avenue for studying the potential for a drug to be repurposed is through informatics. informatics methods have been developed for both drug repurposing and pharmacovigilance (studying the adverse effects of a drug). the advantage of using existing ehrs for studying drugs as candidates for drug repurposing is that it enables risk assessment profiles to be generated for each candidate drug. since the drugs have been prescribed previously during routine clinical care, it is possible to study their effects on human health in a variety of situations that may not have been included in the original clinical trials. for example, the birth and pregnancy outcomes following drug exposure can be assessed using ehrs for drugs potentially useful in treating covid- , such as hydroxychloroquine. this is important as the hydroxychloroquine clinical trials for covid- specifically exclude pregnant women from enrolling in their studies. informatics methods can also be designed, which use more sophisticated machine learning and artificial intelligence methods to study the effects of medication exposure during pregnancy on fetal and maternal outcomes [ ] . with the aggregation of clinical and medication data from ehrs, along with the recruitment of covid- positives and negatives for genetic studies (as described above), there is an opportunity to explore genetic data in combination with this ehr data to improve our understanding of the covid- disease's severity and outcomes. early research has suggested that individuals, who have medical conditions such as heart disease, diabetes, obesity, or asthma, may be at higher risk for severe disease and/or worse outcomes from covid- . additionally, early data suggests that some medications such as ace-inhibitors, angiotensin release blockers (arbs), or non-aspirin nonsteroidal anti-inflammatory drugs (nsaids) may be linked to worse health outcomes due to covid- . however, these reports are primarily based on small, observational datasets without rigorous, epidemiological study designs. as such, these associations are met with much controversy in the literature. with the accumulation of covid- positives and negatives, along with access to ehr data, including comorbid conditions and medications, researchers will be able to develop more thorough studies of which medical conditions are associated with poorer covid- outcomes and/or which medical conditions place individuals at higher risk for hospitalization due to covid- . additionally, if these data are paired with genetic data from ehr-linked biobanks, we may be able to determine if some of these differences in covid- severity and/or outcomes related to comorbidities and medications are also related to host genetics. fortunately, there are several efforts to establish data-sharing consortia that provide an opportunity for informaticians to assist with analyses. for example, the consortium for clinical characterization of covid- by ehr, or ce (https://covidclinical.net/), has released summary-level covid- data from several countries including france, germany, italy, singapore, and the united states along with a preprint of the initial analyses [ ] . presently, there is much to be learned regarding how best to treat covid- patients when sufficient resources are available, as well as how to optimize operational decisions such as the triage of patient testing and care when they are not. as accessible, cleaned, and structured ehr data become available for covid- patients at both the institutional and multi-site consortium levels, there will be increased opportunity to apply machine learning to better understand and make risk predictions on a variety of clinically and operationally relevant outcomes. the accessibility of data science and ml packages (e.g. pandas, scikit-learn, and tensorflow python libraries), paired with widely available high-powered computational hardware offers significant opportunity for researchers to get involved in data analysis and modeling. however, many caveats need to be taken into consideration in order to develop and apply effective, rigorous ml analysis pipelines for replicable covid- investigation. some key considerations and targets of research include: ( ) feature engineering, transforming raw data into features (i.e. variables) that ml can better utilize to represent the problem/target outcome, ( ) feature selection, applying expert domain knowledge, statistical methods, and/or ml methods to remove 'irrelevant' features from consideration and improve downstream modeling, ( ) data harmonization, allowing for the integration of data collected at different sites/institutions, ( ) handling different outcomes and related challenges, e.g. binary classification, multi-class, quantitative phenotypes, class imbalance, temporal data, multi-labeled data, censored data, and the use of appropriate evaluation metrics, ( ) ml algorithm selection for a given problem can be a challenge in itself, thus strategies to integrate the predictions of multiple machine learners as an ensemble are likely to be important, ( ) ml modeling pipeline assembly, including critical considerations such as hyper-parameter optimization, accounting for overfitting, and clinical interpretability of trained models, and ( ) considering and accounting for covariates as well as sources of bias in data collection, study design, and application of ml tools in order to avoid drawing conclusions based on spurious correlations. advanced tools may be necessary to deal with data analytic challenges, properly analyze these data, and accurately extract the knowledge embedded in them. some key challenges include: ( ) accounting for correlation structure induced by multi-level, spatial, and longitudinal designs, ( ) adjusting for biases emanating from the observational data using causal approaches, ( ) accounting for privacy-induced limitations on the resolution of data that can be shared, and ( ) discovering and characterizing interpatient heterogeneities in incidence, progression, or response through stratified or latent class models. some of these challenges can be handled by aptly chosen existing methods, while others require new methodological development. the covid- crisis and the extensive data resources that it will produce will provide an excellent opportunity to develop such methods, including privacy-preserving integrative analytical tools as well as advanced causal inference tools that also account for these other data complexities. we present here two related approaches to using informatics solutions, which directly involve the public who are not physically situated within a healthcare setting. the first focuses on using smartphones and other technology for educating the public about the pandemic and ways to avoid infection as well as monitoring, and the second explores the use of sensors in this domain. consumer health informatics, focusing broadly on tools and systems that engage and empower patients and more general health consumers in health delivery and decision making processes, has a substantial role to play in the context of a pandemic. specific areas that consumer informatics researchers and system designers can target include consumer education, self-triage, monitoring, and social engagement. in a time when behavioral guidelines are continuously adjusted based on new data, consumer education is essential to conveying and disseminating actionable and timely information. patient portals and other web sites can provide educational content that can be tailored to individual information needs as well as literacy and health literacy levels. furthermore, systems can include an interactive component that can facilitate decision support and selftriage. one such example is a patient portal for self-triage and scheduling that was created at the university of california san francisco to enable asymptomatic patients to report exposure history and for symptomatic patients to be triaged and paired with appropriate levels of care [ ] . the system is already being used extensively and performs with high sensitivity in recommending emergency-level care for symptomatic patients. it also prevents unnecessary visits. tools that have been traditionally used for patient monitoring at home and the community can also be useful in generating data that provide insight into disease spread and health needs. an example is that of a smart thermometer vendor that has created an app which allows users to record their temperature and other symptoms with a health insurance portability and accountability act (hipaa) compliant platform; data are aggregated and demonstrate how the virus moves from one county to another, providing a detailed visualization map that highlights areas with an unusually high number of recorded prevalence of fever (https:// healthweather.us/). other mobile health tools that track aspects of daily living including activity levels, sleep quality, or symptom self-management can facilitate better monitoring of health and wellness and potentially lead to effective symptom management at an individual level, and contribute to disease surveillance at a population level. examples include activity tracker data that can inform surveillance of social distancing patterns, and home spirometer and pulse oximetry data that can generate a trajectory of symptom progression in various communities. finally, in times of "social distancing", vulnerable populations such as older adults living alone are at greater risk of increased social isolation, which is often referred to as a silent epidemic and great health risk [ ] . digital tools have the potential to connect individuals for the delivery of social services, and creation of virtual peer support groups and connected communities including friends and family members. this current pandemic has highlighted the need for accessible and secure tools that may include video-conferencing, synchronous and asynchronous communication, and even more sophisticated features such as virtual reality and augmented reality, designed for audiences with diverse abilities with the goal to promote social connectedness in times of physical distancing. smartphones and other wearable smart devices contain research-grade sensors that are capable of shedding light on at least a subset of covid- symptoms which include fever, fatigue, dry-cough, and shortness of breath. for example, the temperature recorded by fingerprint sensors, which are now standard on most modern smartphones, has previously been used to successfully predict fever [ ] . in addition, activity sensors such as the accelerometer have been used to detect fatigue. while high resolution computed tomography (ct) images of a patient's lung may provide a more reliable indicator of infection, the high cost and low scalability make this approach infeasible to apply widely at the general population level. on the other hand, smartphones are currently pervasive with high penetrance even in low and middle-income countries and their high-quality sensor data can be used at next to no cost to measure a subset of important covid- symptoms as a screening tool to identify individuals that may require more extensive evaluation or testing. we present here six considerations of the role of public health informatics in the covid- pandemic. these represent a broad range of topics, from information systems for the monitoring and dissemination of accurate information to the public, to leveraging existing evidence currently available in a huge corpus of virus infection-and pandemic-related research, to building more realistic models of disease risk, spread, and effect of societal interventions, to as-yet poorly understood post-pandemic effects on public health. a critical need for any strategy that addresses covid- is adequate disease monitoring. at the level of cases and deaths, several efforts around the world have arisen to maintain and display official counts, including by researchers at johns hopkins university (https:// coronavirus.jhu.edu/map.html) and reporters at the new york times (https://www. nytimes.com/interactive/ /world/coronavirus-maps.html). these and other efforts rely on reports obtained from heterogeneous sources, many of which capture and store data differently, requiring that informaticians process and display data effectively. case and death counts are helpful and widely used by healthcare systems, policy makers, governmental institutions, and the general public. however, they are notoriously biased given the differing availability and use of lab-based tests to determine covid- case status at various locations. more comprehensive efforts to track the true impact of covid- necessitate appropriate wide-scale testing of sars-cov- . knowledge of who carries the virus regardless of symptom or disease status enables efficient prevention of further transmission, the proper identification of risk factors that lead to divergent symptoms, and adequate preparation of healthcare systems to treat patients who are carriers while minimizing risk to providers and patients who have not been infected. design and deployment of population-level testing should be a primary goal for the effective containment of covid- . in conjunction with apps developed by informaticists, contact tracing along with case isolation can proceed effectively to control outbreaks [ ] . such efforts are thought to have curtailed the spread of covid- in singapore and south korea. because it is unlikely in countries like the u.s., that the federal or local governments, or many citizens would use contact tracing without ensuring individual-level data is safeguarded, various informaticists are engaged in efforts to create privacy-preserving contact tracing apps. sars-cov- containment was not successful in most countries, due in part to lack of appropriate wide-scale testing which contributed to its undetected transmission. ultimately, nothing can replace appropriate lab-based viral testing to understand disease transmission, but informatics solutions are helpful to partly overcome testing inadequacies. in the u.s., canada, and mexico, covid near you (https://covidnearyou.org/) is a citizen participation platform via which any person can contribute their current health status as it relates to covid- symptoms and test results. aggregation of this individual-level data is being used to track population-level health in real-time. other data that can be used to fill monitoring gaps includes search engine data (e.g., google queries for covid- -related terms), and to a lesser extent, social media data (e.g., twitter posts related to . informaticists are leading and contributing to such efforts around the world. as results of sars-cov- tests, along with serological assays to detect its seroconversion, become more widely available, retrospective studies can proceed to more accurately determine how covid- spreads and how many true cases existed prior to widespread testing. informaticians can participate in these efforts that require accounting for test characteristics (sensitivity/specificity) and comparing the characteristics of patients who were actually tested versus those of the underlying population. ongoing retrospective analyses such as these are critical to gain knowledge necessary to avoid future resurgences of covid- . systems for disseminating accurate information related to covid- to the public an emerging issue that concerns the prevention of covid- is the widespread dissemination of speculation, rumors, half-truths, disinformation, and conspiracy theories by means of popular social media platforms. in order for policies, guidelines, and mandates, that may be updated on a weekly or even daily basis, to reach and be adopted by the general public it is important for relevant, vetted information sources to be clearly identified and potentially pointed to in response to misleading posts. in recent years there have been many exciting efforts to combine natural language processing (nlp), machine learning, and social media scraping to monitor clinical outcomes of interest such as foodborne illnesses [ ] . there may be an opportunity to work towards adapting such informatics approaches to monitor and perhaps even combat the dissemination of 'bad' information through automated responses that redirect individuals to sources identified as reliable within the scientific community. rule-based systems such as 'expert systems' could be combined with nlp technologies to construct such monitoring and response frameworks. equally important is the consumer health informatics task of developing clear, concise, and easily navigable informational resources for covid- , that summarize up-to-date information and guidelines but also link summary information back to relevant primary sources, attempt to quantify the certainty/ reliability of available information, and offer explanations of reasoning whenever such information or guidelines need to be updated. the spread of infectious diseases such as covid- provides a unique opportunity to assess the regional spread and progression of disease at a population level. differences in pathogenic mechanisms of different diseases responsible for past pandemics imply that the spread of covid- may not be completely predictable based on the observing historical rates of disease transmission. data on the cumulative number of covid- cases is available at country/regional/city levels and by studying the progression and spread of disease in regions affected close to the time of the initial outbreak, meaningful projections of infection rates can be made for areas which will be affected later. for example, by modeling daily regional cumulative covid- cases, regional differences in the trends can illuminate the comparative effectiveness of different policy decisions and can identify countries and policies that have succeeded in slowing the rate of covid- spread, providing evidence for the adoption of effective public health policies by areas still in the early phases of the pandemic. presenting this information to the public using data visualization methods in an important informatics activity. synthesizing evidence to understand covid- origins, spread, and prevention as of april , , there are more than manuscripts published or posted at pubmed, biorxiv, and medrxiv on covid- from researchers all over the world (https://www.ncbi.nlm.nih.gov/research/coronavirus/). these manuscripts cover a wide spectrum of important topics that can help us to understand the critical aspects of clinical and public health impacts of covid- , including the disease mechanism, diagnosis, treatment, prevention, viral infection, replication, pathogenesis, transmission, viral host-range, and virulence. on the other hand, the amount of information is increasingly overwhelming for stakeholders, policymakers, researchers and interested parties to comprehend. a systematic review, which is a type of literature review that uses systematic methods to collect secondary data and critically appraise research studies, can be useful in synthesizing the existing evidence of covid- related research findings. in particular, meta-analysis plays a central role in the systematic review in quantitatively synthesizing evidence from multiple scientific studies which address related questions. manual literature review is time consuming and, more importantly, it is challenging to keep up-to-date with the rapidly increasing volume of literature. medical informatics tools can improve the efficiency and scalability of up-to-date evidence synthesis for covid- related research. for example, clinical natural language processing (nlp) tools can be used for literature screening and information retrieval. software such as abstractr [ ] [ ] [ ] and distillersr (https://www.evidencepartners.com/) has been used to reduce manual effort in literature screening. beyond literature screening, distillersr is also a useful tool for the management of the multi-step workflow of systematic review process. recently, distillersr made its tool freely available for systematic reviewers and researchers to conduct systematic reviews related to covid- . for meta-analysis, tools such as comprehensive meta-analysis (cma) (https://www.meta-analysis.com/), revman (https://training.cochrane.org/online-learning/core-software-cochrane-reviews/ revman), and macros in stata (https://www.stata.com/), are available for standard metaanalyses. however, for covid- related research, more sophisticated methods are needed in order to address unique features related to this topic. for example, the quality of the reported findings in the above-mentioned manuscripts is expected to be highly heterogeneous, especially for those manuscripts that have not been peerreviewed. it is critically important to properly account for such heterogeneity across studies. furthermore, the reported findings may be subject to more severe publication bias and outcome reporting bias [ ] , as the analysis of the data and reporting of the analysis results are likely to be based on different protocols. visualization tools, sensitivity analyses, and inference based on bias correction models can be useful in evaluating the quality of the evidence [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition, novel visualization tools, such as the tornado plot in a cumulative meta-analysis [ ] , will be valuable for presenting how the cumulative evidence on answering a covid- related question evolves over time. r packages including 'meta', 'metafor', 'metasens', 'netmeta', 'mvmeta', mada' and 'xmeta' are useful for advanced meta-analyses with these needs. finally, online platforms for meta-analysis, such as programs with shiny interfaces, are in great need for offering convenience to covid- researchers in summarizing and synthesizing results. advanced, more realistic models of disease spread to guide policymaking differential equation-based epidemiological models such as the susceptible-infected-recovered (sir) or susceptible-exposed-infected-recovered (seir) models and their variants are key workhorses for studying infectious disease dynamics. these models have been widely used in making projections and informing policy-makers in constructing mitigation strategies for the disease. one weakness of these models is that they treat individuals in a given population as homogeneous, with constant risk rates, exposure rates, infection rates, and recovery/death rates throughout the larger group. this is a gross oversimplification which is a primary factor of the models' limited predictive accuracy. statisticians have been engaging in covid- efforts with statistical models using functional data or time series modeling techniques. these models often use covariates or latent factors to account for population heterogeneity and provide uncertainty quantification, thus improving on a weakness of the seir models. however, these models do not present the dynamic infectious disease process which may limit their interpretability and accuracy in forecasting. one key area of quantitative research that can emerge from this covid- crisis is hybrid epidemiology-statistical models. that is, models based on sir or seir frameworks that stochastically show the transition probabilities as differing according to person or environmental covariates, accounting for clustering effects, and effectively propagating uncertainty in the forecasting. these can combine the strengths of each type of model, and given the broad availability of large scale data on mobility, density, demographics, etc. that vary in different communities, they can produce much more realistic models and more accurate projections to guide policymaking. the covid- pandemic has resulted in unprecedented disruption to the healthcare system. in addition to understanding the direct health impacts of the disease, there is a public health need to understand the secondary effects of covid- -related healthcare disruption on access to and timeliness of care for other urgent conditions, and resultant effects on health outcomes. prioritizing healthcare resources for covid- patients and efforts to depopulate healthcare settings in order to reduce healthcare-related disease transmission has resulted in reduced access to care for patients across the spectrum of clinical need and severity including delayed access to surgery for cancer patients, organ transplant recipients, and others with time-sensitive conditions. public health informatics can play an important role in informing our understanding of how the effects of healthcare disruption propagate across a community, affecting access to care, and population health. answering questions about the effect of healthcare disruption on population health requires three components: ( ) access to data on healthcare utilization and outcomes, ( ) data on timing and types of public health and hospitallevel interventions, and ( ) causal inference methodologies that support our ability to draw conclusions about the causal effects of these interventions. data on health care utilization and outcomes can be obtained from a variety of sources including individual and multi-institutional ehr data and claims databases. data on public health interventions are already being compiled by researchers, including national and international databases of policy changes (https://is.gd/cqs th, https://is.gd/lvvuiz, https://is.gd/ mlcu i). finally, disentangling the causal impacts of covid- itself; interventions at the local, state, and federal level; and interventions and innovation at the individual health system level requires the rigorous implementation of study designs and analytic methods for causal inference. a number of techniques in common use in health services and econometrics research can be harnessed for this purpose including interrupted time series and difference-in-difference designs [ ] . the total number of users of social media continues to grow worldwide, resulting in the generation of vast amounts of data. popular social networking sites such as facebook, twitter, and instagram dominate this sphere. about million tweets and . billion facebook messages are posted every day (https://www.gwava.com/blog/internet-data-created-daily). a pew research report (http://www.pewinternet.org/fact-sheet/social-media/) states that nearly half of adults worldwide and two-thirds of all american adults ( %) use social networking. the report states that of the total users, % have discussed health information, and, of those, % changed behavior based on this information and % discussed current medical conditions. advances in automated data processing, machine learning, and nlp present the possibility of utilizing this massive data source for biomedical and public health applications, if researchers address the methodological challenges unique to this media. when events such as the covid- pandemic sweep the world, the public turns to social media. while there is a general belief that most of the content is not useful, adequate collection, filtering, and analysis could reveal potentially useful information for assessing public sentiment. furthermore, given the delay and shortage of available testing in the united states, social media could provide a near real-time monitoring capability (e.g. the penn covid- u.s. twitter map, https://is.gd/l gga), giving insights into the true burden of disease. preliminary work in this direction is under review. the archived version of the paper, with a training dataset and annotation guidelines as supplementary material, is available [ ] . although social media text mining research for health applications is still incipient, the domain has seen a surge in interest in recent years. numerous studies have been published of late in this realm, including studies on pharmacovigilance [ ] , identifying user behavioral patterns [ ] , identifying user social circles with common experiences (like drug abuse) [ ] , monitoring malpractice [ ] , and tracking infectious/viral disease spread [ , ] . population and public health topics are most addressed, although different social networks may be suitable for specific targeted tasks. for example, while twitter data has been utilized for surveillance and content analysis, a significant portion of research using facebook has focused on communication rather than lexical content processing [ , ] . for health monitoring and surveillance research from social media, the most common topic has been influenza surveillance [ , ] . from the perspective of informatics and nlp, proposed techniques have typically been in the areas of data collection (e.g., keywords and queries) [ , ] , text classification [ , ] , and information extraction [ ] . while innovative approaches have been proposed, there is still a lot of progress to be made in this domain. effective utilization of the health-related knowledge contained in social media will require a joint effort by the research community, and bringing together researchers from distinct fields including nlp, machine learning, data science, biomedical informatics, medicine, pharmacology, and public health. the knowledge gaps among researchers in these communities need to be reduced by community sharing of data and the development of novel applied systems. the covid- pandemic presents a myriad of challenges and opportunities for research across virtually every scientific discipline, and biomedical informatics is no exception. from the molecular and genetic sciences to population health, researchers in the five domains of biomedical informatics stand to make substantial contributions to addressing these challenges. we hope, through the numerous examples of research we have considered in this editorial, informatics researchers and practitioners can see possible avenues for their work. there is no dearth of opportunities related to covid- for those working in informatics, and it is our hope that informaticians will vigorously explore these as they arise. furthermore, we hope that those who are not informaticians will appreciate the contributions that informatics researchers can bring to their respective fields as we all seek to address the covid- pandemic and its effects around the world. the covid- vaccine development landscape diagnostic testing for severe acute 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imaging data acquisition detection of sars-cov- in different types of clinical specimens enabling pregnant women and their physicians to make informed medication decisions using artificial intelligence international electronic health record-derived covid- clinical course profile: the ce consortium. medrxiv rapid design and implementation of an integrated patient self-triage and self-scheduling tool for covid- national academies of sciences e. social isolation and loneliness in older adults: opportunities for the health care system transparent and flexible fingerprint sensor array with multiplexed detection of tactile pressure and skin temperature machine-learned epidemiology: real-time detection of foodborne illness at scale. npj digital med toward systematic review automation: a practical guide to using machine learning tools in research synthesis semi-automated screening of biomedical citations for systematic reviews deploying an interactive machine learning system in an evidencebased practice center: abstrackr publication bias in meta-analysis: prevention, assessment and adjustments meta-analysis, funnel plots and sensitivity analysis a sensitivity analysis for publication bias in systematic reviews: statistical methods in medical research bias in meta-analysis detected by a simple, graphical test the case of the misleading funnel plot maximum likelihood estimation and em algorithm of copas-like selection model for publication bias correction funnel plots for detecting bias in meta-analysis: guidelines on choice of axis misleading funnel plot for detection of bias in meta-analysis cumulative meta-analysis of therapeutic trials for myocardial infarction designing difference in difference studies: best practices for public health policy research a chronological and geographical analysis of personal reports of covid- on twitter towards internet-age pharmacovigilance: extracting adverse drug reactions from user posts to health-related social networks the role of facebook in crush the crave, a mobile-and social media-based smoking cessation intervention: qualitative framework analysis of posts an exploration of social circles and prescription drug abuse through twitter malpractice and malcontent: analyzing medical complaints in twitter national and local influenza surveillance through twitter: an analysis of the - influenza epidemic you are what your tweet: analyzing twitter for public health please like me: facebook and public health communication facebook advertising across an engagement spectrum: a case example for public health communication using social media to perform local influenza surveillance in an inner-city hospital: a retrospective observational study evaluating google, twitter, and wikipedia as tools for influenza surveillance using bayesian change point analysis: a comparative analysis phonetic spelling filter for keyword selection in drug mention mining from social media scoping review on search queries and social media for disease surveillance: a chronology of innovation text classification for automatic detection of e-cigarette use and use for smoking cessation from twitter twitter catches the flu: detecting influenza epidemics using twitter pharmacovigilance from social media: mining adverse drug reaction mentions using sequence labeling with word embedding cluster features publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. all authors contributed equally to the writing and editing of the editorial. all authors read and approved the final manuscript. not applicable.availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. key: cord- -jgae r q authors: armstrong, melissa j.; gamez, noheli; alliance, slande; majid, tabassum; taylor, angela; kurasz, andrea m.; patel, bhavana; smith, glenn title: research priorities of caregivers and individuals with dementia with lewy bodies: an interview study date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: jgae r q background: funding bodies are placing increased emphasis on patient and public involvement in research, but the research priorities of individuals and caregivers living with dementia with lewy bodies (dlb) are unknown. method: investigators conducted telephone interviews with individuals living with dlb and caregivers. participants were recruited from a lewy body dementia association research center of excellence. interviews employed a semi-structured questionnaire querying research needs in different categories and then asking participants to select their top priorities. investigators used a qualitative descriptive approach to analyze transcripts and identify themes. results: twenty individuals with dlb and caregivers participated. seventeen from each group participated as part of a patient-caregiver dyad. twenty-three of the caregivers were spouses, two were daughters. individuals with dlb and caregivers identified research needs relating to focusing on awareness, determining the cause of dlb, improving diagnosis, and investigating what to expect/disease stages. participants also highlighted dlb symptoms needing additional research, therapies to prevent, cure, or slow the progression of dlb, and research targeting daily function and quality of life, caregiving, and improving education. conclusions: these findings support the research priorities defined in the national institutes of health dementia care summits in addition to adrd priority-setting summits. research is needed across all domains of dlb. funding should be informed by the priorities of all relevant stakeholders and support research investigating causes, natural history, biomarkers, and treatment in addition to research targeting themes regarding living with disease (e.g. independence, quality of life, caregiving, and education). funding bodies including the national institutes of health (nih) are placing increased emphasis on patient and public involvement in research. patients can have a role throughout the research process, from study design to dissemination [ , ] . before studies are even initiated, patients, caregivers, and the public have a role in identifying research priorities [ ] . this is critical given a mismatch between the research priorities of patients, caregivers, clinicians, and researchers [ ] . the advisory council on alzheimer's research, care and services recommends that all funders "should establish the engagement of the ad/adrd [alzheimer disease/ alzheimerdisease related dementia] community as a standard practice in. . . participating in setting national research priorities for ad/adrd. . ." [ ] . the nih has adopted multiple strategies to increase the role of patient and public involvement in dementia priority setting. adrd national research priority summits include sessions led by nongovernmental organizations to increase non-expert stakeholder input and public-private partnerships [ ] . the national research summit on care, services, and supports for persons with dementia and their caregivers included "persons living with dementia" and family caregiver stakeholder groups as two of the six stakeholder groups informing summit planning and recommendations [ ] . one aim of the planned national research summit on care, services, and supports for persons with dementia and their caregivers was to develop recommendations for research priorities to inform federal agencies and other research funders, but the in-person meeting was cancelled due to the global covid- pandemic. the summit will now occur via a series of virtual meetings in summer . involvement of patient representatives alongside experts in priority setting reflects a "participation" engagement strategy. through participation, representatives have an active and equal voice in the process. participation strategies usually rely on small numbers of representatives, potentially missing other perspectives. consultation strategies (e.g. surveys, interviews, focus groups) collect a variety of views from larger groups of people, but fail to give patient representatives an active voice in the process. employing participation and consultation approaches as complementary may be the optimal approach to incorporate the views of a diverse group while also giving patient representatives an equal and active voice in decision making [ ] . stakeholders representing individuals with lewy body dementia participated in nih ad/ adrd priority setting [ ] and the care summit [ ] . however, no prior research investigates the research priorities of individuals and caregivers living with dementia with lewy bodies (dlb). dlb is a subset of lewy body dementia, the nd most common neurodegenerative dementia in the united states [ ] . dlb is a dementia with clinical and pathological overlap with parkinson disease (pd) and both fall in the pathological category of lewy body diseases. however, dlb has important differences from both pd and ad/other dementias which could meaningfully impact research priorities. for example, symptoms such as cognitive fluctuations, hallucinations, and dream enactment behavior distinguish individuals with dlb from ad [ ] . individuals with clinically-diagnosed dlb survive a median of - years after presentation [ ] [ ] [ ] , shorter than individuals with pd [ , ] or ad dementia [ , ] . caregiver burden in dlb may be similar to pd dementia [ ] , but it is higher in dlb compared to ad dementia [ ] [ ] [ ] . quality of life is also worse in dlb compared to ad dementia [ , ] . given these and other important differences between dlb and other dementias and parkinsonisms, prior publications reporting on research priorities in pd [ , ] and dementia in general [ ] [ ] [ ] [ ] may not capture the priorities of individuals with dlb. we thus aimed to identify the research priorities of individuals with dlb and caregivers, particularly to guide research at our center. the study used telephone interviews to investigate the research priorities of individuals living with dlb and caregivers of individuals living with dlb. investigators used a qualitative descriptive approach [ ] to analyze interview transcripts. a qualitative descriptive approach involves reporting and summarizing straightforward accounts of participants' views without an intent to generate or test theory. a qualitative descriptive approach was employed given the aim of identifying research priorities, rather than investigating theories underpinning those priorities. consolidated criteria for reporting qualitative research guided study reporting (s checklist) [ ] . individuals with dlb and caregivers of individuals with dlb were recruited from the uf health norman fixel institute for neurological diseases, a lewy body dementia association research center of excellence. inclusion criteria were: ( ) patient or caregiver of a patient followed at the fixel institute, ( ) personal diagnosis of dlb [ ] or caregiver for an individual diagnosed with dlb, ( ) if a person with dlb, clinician-judged mild-moderate severity such that the individual could understand the study and provide personal opinions, and ( ) willingness to participate in a telephone interview. individuals and caregivers were not required to participate as a dyad. participants were recruited when presenting for routine clinical visits or through a consent-to-contact research database at the institute. the university of florida institutional review board provided approval (irb ). the study used a waiver of documentation of informed consent. the pi (mja), a dlb specialist, drafted the semi-structured interview guide (s file) and revised it based on suggestions from a neuropsychologist (gs), a dementia specialist (not further involved in the project), phd specializing in qualitative research (tm), and former caregiver of an individual with dlb (at). the interview included questions regarding clinical care priorities, which will be analyzed separately. research questions asked about participant priorities for dlb research in general and then specifically queried participant priorities regarding research on dlb symptoms, daily challenges, caregiving/family life, and diagnosis. finally, the interview guide asked the participant to identify how they would divide $ to spend on dlb research and to prioritize what research was most important to them. a research assistant with qualitative research experience conducted the telephone interviews (sa). the research assistant had no relationship with participants prior to the study. interviews occurred via telephone to allow individuals to participate from home. participants selected the interview times. the preferred approach was to interview individuals with dlb and their caregivers separately (if both participated), but the interviewer accommodated requests for the caregiver to be present during the patient interview. any caregiver opinions offered during a patient interview were coded as belonging to the caregiver, not the individual with dlb. audio recording started after allowing for participant questions and verbal consent at the beginning of the call. a professional service transcribed the interviews verbatim, so member checking was not performed. investigators used tables in microsoft word and excel to organize data and a qualitative descriptive approach to identify and organize themes [ ] . broad topics/categories were defined by interview questions, but themes were identified from interview transcripts. the pi and two research assistants independently analyzed interview transcripts to create a codebook and then reached consensus regarding emerging themes (open coding). the research assistants analyzed remaining transcripts using a constant comparative technique, revising themes and subthemes with the pi if needed (axial coding) (s file). coders assessed saturation during analysis. co-investigators gave feedback after the initial coding. participants were numbered in the analysis such that participants who enrolled in the study as a dyad shared participant numbers (with "p" indicating patient participants and "cg" indicating caregiver participants). interviews occurred between / / and / / . twenty individuals with dlb and caregivers participated (table ) . seventeen from each group participated as part of a patient- well, maybe the breaker tripped. maybe the bulb burned out. maybe the wire broke." you know, there's a lotta maybes. well, you've got to find the root cause. . . you know, you find the cause or the problems, and you can treat it or correct it. (cg , daughter) in addition to desiring to know causes in general, multiple participants wanted further research regarding whether other health problems, prior injuries (e.g. head trauma), exposures (e.g. from mining), a history of shift work, or a traumatic event could contribute to developing dlb. multiple individuals with dlb and almost half of caregivers discussed the importance of research to improve early diagnosis ( table ). while these respondents discussed the need for earlier diagnosis, particularly in circumstances where a diagnosis was delayed, one individual (cg , husband) multiple individuals with dlb and caregivers described needing more research to help individuals with dlb, caregivers, and healthcare professionals recognize the signs of dlb (table ). caregivers wanted research to focus on improving the accuracy of diagnosis, including distinguishing dlb from pd and dementia generally, particularly given widespread confusion regarding lewy body terminology. several individuals with dlb and caregivers desired more research into tests and biomarkers to assist with dlb diagnosis ( participants highlighted the need for more research for many of the core and supportive features of dlb (table ) [ ] . many discussed that research should investigate the symptoms and also medications to help: medicine-trying to find the right combination. anything you can give him to tamp down his symptoms-anything you can give to make it so it's easier for him to exercise, it's easier for him to live his daily life, is very important. participants also desired research on non-pharmacologic strategies to address dlb symptoms, including therapy, exercise, meaningful activities, "natural" therapies, changes in diet and nutrition, and unconventional approaches such as hyperbaric chambers. multiple individuals with dlb and caregivers discussed the need for pharmacologic or nonpharmacologic disease-modifying therapies that will prevent, cure, or slow dlb: how it could possibly be prevented or mitigated or slowed down in its decline. (p ) individuals with dlb and caregivers offered many topics pertaining to daily life that they felt were deserving of more research, including how to maintain socialization, maintain the ability to drive, identify emergencies, keep individuals with dlb safe, communicate well between couples (patient-caregiver), find support (e.g. good healthcare providers, external programs), handle non-dlb healthcare issues, make medical decisions, and strategize longterm planning. research aiming to improve quality of life was another common theme, particularly from caregivers: several individuals with dlb and most caregivers identified the need for additional research addressing different realms of caregiving, including caregiver burden and support, depression and how to handle impatience and frustration, balance external work and caregiving, identify alternative care options and ways to keep the individual with dlb at home, and identify resources to pay for caregiving needs. almost all caregivers and half of participants with dlb described a need for improved education surrounding dlb and how research could improve educational efforts. while these opinions were provided in response to questions about research, it was sometimes unclear whether participants were expressing a need for more education clinically or a specific desire for research on this topic. participants described a need for education in general and also specifically for physicians, medical trainees, therapists, patients, and caregivers/families. the most commonly described needs were education for non-specialist healthcare professionals and for caregivers. i think there should be probably some more, uh, i guess they have to have yearly training or whatever, you know. i think that should be something that's sort of at the forefront, because so many doctors don't even know what you're talkin' about when you tell 'em your disease. and, you know, all of them, a lot of 'em. education for regular doctors would be really good. (cg , wife) [it] would be helpful for everybody to have a caregiver course, to help take care of the person, but also to learn how to take care of themselves. most of the research topics queried in the interviews or mentioned by interviewees were identified by at least some participants as their top priorities for dlb research (table ). symptoms described as priorities for research included cognition, hallucinations, movement, mobility, sleep, and mood. a few participants mentioned that obtaining the patient and caregiver voices is the most important thing to them. (cg , wife). the study guide queried a variety of research categories and individuals with dlb and caregivers identified topics important for research in all of them-focusing on awareness, determining the cause of dlb, improving diagnosis, investigating what to expect and disease stages, dlb symptoms needing additional research, therapies to prevent, cure, or slow the progression of dlb, targeting daily function and quality of life, caregiving, and improving education. furthermore, when participants were prompted to assign virtual money to research topics and identify their highest research priorities, at least one participant endorsed each of these areas. this highlights that research is needed across the dlb spectrum-basic science, translational, biomarker, natural history, drug development/therapeutic, quality improvement, and outcomes. published lewy body dementia research priorities were developed primarily by experts and focus on disease mechanisms and processes (including animal model development), diagnostic criteria, terminology, risk factors/prodromal stages, longitudinal cohorts, pathological staging, imaging, biomarkers, genetics, new treatment targets, and preventative/disease-modifying and symptomatic treatments [ , ] . the individuals with dlb and caregivers in this study supported and prioritized research on many of these topics. however, many topics proposed in this study were outside the national research priorities, possibly because the adrd summit aimed to inform prevention and effective treatment of ad/adrds [ ] . individuals with dlb and caregivers described needing symptomatic treatments but also more research on topics pertaining to how families should handle dlb symptoms in daily life, improving daily life in general, targeting quality of life, caregiving research, understanding disease progression, and improving education regarding dlb across populations (public, patients, caregivers, healthcare professionals). recommendations form the national research summit on care, services, and supports for persons with dementia and their caregivers address some of the themes from current study participants-the need for research to understand different dementia trajectories, determinants of behavioral and psychological symptoms, caregiving, decision-making, and support and financial considerations, as well as research investigating pharmacologic and nonpharmacologic treatment strategies and studying complex, multicomponent programs accommodating care needs [ ] . the care summit also identified the need to understand what outcomes are important to persons living with dementia and to engage persons living with dementia and caregivers as part of research teams [ ] . nearly half of the final research recommendations expressed ideas contributed by the persons living with dementia summit working group [ ] . this emphasizes the need for funders (federal and otherwise) to consider research priorities from both dementia summits (adrd and caring) when developing funding announcements and selecting projects to support. in this study, individuals with dlb and caregivers described several topics as needing research which professionals might put in the category of clinical care. for example, the need for more education across populations was a common theme. many participants mentioned needs relating to daily life (decision making, independence including driving, socialization), practical caregiving considerations, when and how to obtain and use extra support, and finances. these practical research priorities are consistent with previously published research priorities of individuals with dementia and their caregivers. the top six overall research priorities identified through modified delphi consensus involving participants from dementia advocacy organizations were ( ) cure and treatment, ( ) caregiving, ( ) education and training, ( ) quality of life, ( ) complementary therapies, and ( ) care settings [ ] . similarly, dementia priority setting through the james lind alliance identified research priorities, including maintaining independence, optimal care, pharmacologic and non-pharmacologic care, and caregiver support [ ] . a more recent dementia priority setting process in canada identified research priorities including investigating stigma, supporting emotional well-being, early treatment, health system changes, caregiver support, connection to education and support, necessary dementia-related skills and knowledge for healthcare professionals, dementiafriendly communities, best dementia practices, and non-pharmacologic treatments [ ] . research priority setting for people with pd identified needed research on physical functioning, symptoms, coping, stress, socialization, relationships, support, autonomy, and good care and communication alongside prioritizing research addressing pd causes, diagnosis, subtypes and medication for both motor and non-motor symptoms [ , ] . differences in research priority-setting studies likely reflect the populations participating (e.g. country of origin; combination of patients, caregivers, advocates, healthcare professionals), the types of dementia represented, and the focus of the organizers or project leadership. the current study is also distinct from prior formal priority-setting work, as the goal of the current project was to investigate the views of individuals with dlb and caregivers at a single center, and not to use survey or formal consensus-building processes to build a top-ten list. the current results could serve as background for such a project specific to dlb. regardless of methodology, it is clear from the current study and prior work that individuals with dementia and caregivers of individuals with dementia, including dlb, prioritize research targeting issues relating to living with disease alongside efforts to better understand the cause of dlb and identify a cure. indeed, research into the topics discussed by participants is sorely needed. it is estimated that that in cases of dlb may be missed [ ] and initial misdiagnosis is common [ , ] . it takes over a year for half of individuals with dlb to receive a diagnosis [ ] . family members of individuals who died with dlb described both lack of knowledge of what to expect and negative experiences relating to lacking healthcare professional education and knowledge [ , , ] . while α-synuclein deposition has been the presumed pathogenic cause of dlb for years, recent re-analysis questions this assumption [ ] . there are no biomarkers for dlb and no treatments for dlb approved by the u.s. food and drug administration. caregiver burden in dlb is high [ ] [ ] [ ] and quality of life is negatively affected by dlb [ , ] . this study is the first to evaluate the research priorities of individuals living with dlb and caregivers of individuals with dlb. this study can inform survey development to identify research priorities of a larger group of individuals living with dlb, serve as the background for formal research priority-setting in dlb, and help guide research planning. the study recruited individuals with dlb and caregivers from a single united states-based center of excellence, affects generalizability, though the identified topics were consistent with publications enrolling stakeholders with other dementias and pd. it is plausible that a different cohort could have identified different or additional themes. for example, because enrolled individuals with dlb had to have mild-moderate dementia (such that they could participate), they and the caregivers who enrolled with them as part of dyads may not have considered end of life research questions, which could potentially be a priority for families dealing with advanced dlb. furthermore, interview questions were open-ended within categories and additional prompting regarding research topics could have resulted in different responses. the study specifically sought the opinions of individuals with dlb to give them an active voice and role, but several of the participants struggled to understand and answer the questions or perseverated on certain answers, limiting the information that could be gained from those interviews. most participants were of white non-hispanic backgrounds and had high educational attainment, potentially affecting generalizability. similarly, most participating caregivers were wives and it is plausible that other caregiving roles (e.g. husbands, children caring for parents) would have different priorities. individuals with dlb and caregivers of individuals with dlb identified areas needing more research related to dlb awareness, causation, diagnosis, prognosis and dlb stages, core and supportive symptoms, disease-modifying and symptomatic treatments, issues relating to daily function and quality of life, caregiving, and education across populations. these findings support the research priorities defined in the nih care summits in addition to the nih adrd summits. further research is needed across all domains of dlb. research funding should be informed by the priorities of all relevant stakeholders and support research investigating causes, natural history, biomarkers, and treatment in addition to research targeting themes regarding living with this disease (e.g. independence, quality of life, caregiving, and education). continuous patient engagement in comparative effectiveness research patient involvement in clinical research: why, when, and how patients', clinicians' and the research communities' priorities for treatment research: there is an important mismatch. research involvement and engagement department of health & human services alzheimer's disease-related dementias summit : national research priorities contributions of persons living with dementia to scientific research meetings. results from the national research summit on care, services, and supports for persons with dementia and their caregivers participation and consultation engagement strategies have complementary roles: a case study of patient and public involvement in clinical practice guideline development relative frequencies of alzheimer disease, lewy body, vascular and frontotemporal dementia, and hippocampal sclerosis in the state of florida brain bank diagnosis and management of dementia with lewy bodies: fourth consensus report of the dlb consortium three years survival in patients with a clinical diagnosis of dementia with lewy bodies mortality in dementia with lewy bodies compared with alzheimer's dementia: a retrospective naturalistic cohort study cause of death and end-of-life experiences in dementia with lewy bodies prognosis of parkinson disease -risk of dementia and mortality: the rotterdam study the sydney multicenter study of parkinson's disease: the inevitability of dementia at years survival and mortality differences between dementia with lewy bodies vs alzheimer disease care burden and mental ill health in spouses of people with parkinson disease dementia and lewy body dementia caregiver burden in family carers of people with dementia with lewy bodies and alzheimer's disease clinical findings, functional abilities and caregiver distress in the early stage of dementia with lewy bodies (dlb) and alzheimer's disease (ad) examining carer stress in dementia: the role of subtype diagnosis and neuropsychiatric symptoms patients with dementia with lewy bodies have more impaired quality of life than patients with alzheimer disease trajectories and determinants of quality of life in dementia with lewy bodies and alzheimer's disease priority setting partnership to identify the top research priorities for the management of parkinson's disease living with parkinson's disease: priorities for research suggested by patients dementia priority setting partnership with the james lind alliance: using patient and public involvement to inform the research agenda national priorities for dementia care: perspectives of individuals living with dementia and their care partners priorities for people living with dementia: education, counseling, research patient and public involvement in identifying dementia research priorities qualitative descriptive methods in health science research consolidated criteria for reporting qualitative research (coreq): a -item checklist for interviews and focus groups lewy body dementias national research summit on care, services, and supports for persons with dementia and their caregivers: final summit report development of assessment toolkits for improving the diagnosis of the lewy body dementias: feasibility study within the diamond lewy study lewy body dementia: the caregiver experience of clinical care caregiver-reported barriers to quality end-of-life care in dementia with lewy bodies: a qualitative analysis end-of-life experiences in dementia with lewy bodies: qualitative interviews with former caregivers revisiting protein aggregation as pathogenic in sporadic parkinson and alzheimer diseases key: cord- -jop rx authors: vignais, pierre v.; vignais, paulette m. title: challenges for experimentation on living beings at the dawn of the (st) century date: - - journal: discovering life, manufacturing life doi: . / - - - - _ sha: doc_id: cord_uid: jop rx “we can talk endlessly about moral progress, about social progress, about poetic progress, about progress made in happiness; nevertheless, there is a type of progress that defies any discussion, and that is scientific progress, as soon as we judge it within the hierarchy of knowledge, from a specifically intellectual point of view.” introduction of new exploratory methods such as biocomputing or bioinformatics and high-throughput screening, which involves the simultaneous processing of hundreds or even thousands of samples. this approach is in contrast with traditional biology, in which the research strategy is based upon the observation of effects obtained as a function of experimental parameters that are modified one by one. another aspect of modern times is that, with the irresistible trend in genetic manipulation towards a focus on human beings, certain areas of fundamental research are finding themselves locked into philosophical dilemmas that are matter for ethical and sociocultural consideration, and the subjects of fierce debate. instead of setting out to discover unknown mechanisms by analyzing effects that are dependent on specific causes, with some uncertainty as to the possible success of the enterprise being undertaken, which is the foundation stone of the bernardian paradigm of the experimental method, many current research projects give themselves achievable and programmable objectives that depend upon the means available to them: sequencing of genomes with a view to comparing them, recognition of sequence similarities in proteins coded for by genes belonging to different species, with the aim of putting together phylogenetic trees, synthesis of interesting proteins in transgenic animals and plants, analysis of the three-dimensional structure of proteins, in order to find sites that are likely to fix medicinal substances, and synthesis of molecular species able to recognize pathogenic targets. the facilities that are called into play include instruments that are often sophisticated, the performance of which, in terms of miniaturization, computerization and robotization, is far beyond that of apparatus that was in use a few decades ago. these facilities, applied to research into living beings, have entered the framework of a methodology that has been given the label biotechnology. proceeding handin-hand with applications that have become more and more meaningful in the domains of medicine, pharmacology, agronomy and animal husbandry, the biotechnological process has come to the fore as a new paradigm for the experimental method as applied to living beings. in addition to new discoveries, the driving forces behind biotechnologies are related to economic imperatives as well as the interest and support they receive from the political powers-that-be. the academic spirit that presides over fundamental science gives way to the entrepreneurial spirit that implements a rational programming of facilities and an efficient organization of scientific collaborations. as an example, the sequencing of the human genome, which includes three billion nucleotide base pairs, required the coordination of several dozen scientific teams around the world and the matching of several tens of thousands of results. research on dna provides a typical illustration of the way in which research has become divided, over the last few decades, between an approach and an interest that had previously been purely academic, and the increasing role of technology, which can be justified by the results that arise in the life of society at large, but which, because of these results, also gives rise to questions concerning how wellfounded some of these results are, particularly in the health domain. the experimental method, which had been confined to the laboratory, is now a matter for public debate. before it won acclaim, dna, which was isolated under the name of nuclein by johann friedrich miescher ( - ), at the end of the th century, had to undergo a series of structural evaluation tests that were spread out over the first five decades of the th century. an overall conclusion then came to the fore. dna is a polydeoxyribonucleotide that carries four cyclic bases, adenine, thymine, cytosine and guanine. each base is involved in the structure of a mononucleotide where it is itself associated with a sugar, deoxyribose, which is associated with a phosphate residue. dna was compared to a ladder, the rungs of which (mononucleotides) were linked by ester bonds between an acid group of a phosphate residue of a nucleotide and the free hydroxyl group of the deoxyribose of the following nucleotide. research committed them to this path. it was the curiosity of each, a new way of considering old problems, that led a few men and women to solve the problems of heredity." the middle of the th century saw an accumulation of experimental evidence showing that dna carries genetic information, and because of this, that it controls the transmission of hereditary characteristics: the proof provided in by oswald avery ( - ) , colin macleod ( - and maclyn mccarthy ( - of the transforming power of dna in pneumococcus, the highlighting by alfred hershey ( - ) and martha chase ( - , in , of the role played by bacteriophage dna as an infectious agent for bacteria, the revelation by erwin chargaff at the beginning of the s of the equivalence of molar concentrations of adenine (a) and thymine (t), on the one hand, and of cytosine (c) and guanine (g), on the other hand, in dnas arising from a multitude of sources, animal, plant and microbial, thus suggesting a complementary pairing of adenine and thymine, and cytosine and guanine. based on the pairing of a/t and c/g bases, the model of the double helix structure of dna, formulated in by james watson and francis crick, made it possible to understand the identical synthesis of double strands of dna by replication during cell division (figure iv. ) and, as a consequence, the conservation of hereditary characteristics in descendants. afterwards, it was found that the information contained in the dna base sequence determines the amino acid sequence in proteins. then the roles played by messenger rna and transfer rnas were elucidated, the former acting as a carrier of information between dna and the proteins being synthesized and the latter acting as double-headed adaptors, able to recognize nucleotide triplets (codons) in messenger rna and to specifically fix amino acids in order to position them on the ribosomes, the final result being the synthesis of a protein chain. in , the genetic code was deciphered. the veil of mystery that had covered the mechanism of the synthesis of proteins was lifted, and the decisive role played by nucleic acids in this synthesis was shown. later on, there were a few adjustments. although, in bacteria, proteins are coded for by a continuous sequence of nucleotide triplets in dna, in the s the surprising discovery was made that in eukaryotic organisms, genes are discontinuous and made up of coding dna sequences (exons) interrupted by non-coding sequences (introns). from the end of the s, françois jacob and jacques monod had postulated the existence of a dual determinism for protein synthesis and shown that, next to structural genes expressed as proteins, there are regulatory genes able to control the expression of the structural genes. the importance of the differential regulation of gene expression in cell differentiation in higher organisms was quickly recognized. from this point on it was possible to explain why a particular species of protein is more specifically expressed in a given tissue and another species of protein is more particularly expressed in another tissue, each type of tissue finding its specificity in its molecular components. this fantastic framework of knowledge, which was built up over a couple of decades, has been used as the foundation stone for the so-called central dogma of molecular biology, which explains the transcription of dna sequences into messenger rnas and the translation of messenger rnas into proteins and which, with only a few variants, is the same throughout the living world (figure iv. ) . the fascinating history of molecular biology was well described by james d. watson in molecular biology of the gene ( rd edition ). there are now many works concerning this subject and how it has progressed, including two well-documented books in french: the secrets of the gene by françois gros ( ) and histoire de la biologie moléculaire by michel morange ( ) . new strand a -double helix structure of dna and simplified representation of its self-replication. each strand of the parent molecule of dna acts as a matrix for the synthesis of a daughter molecule of complementary dna, in conformity with the rules of pairing: adenine (a) with thymine (t) and guanine (g) with cytosine (c). the double strands that appear are identical to each other and identical to the parent dna molecule. b -transcription of dna into messenger rna and its translation into an amino acid chain. diagram of gene expression in a eukaryotic cell. one of the strands of dna (the coding strand) has coding sequences (exons) and non-coding sequences (introns). it is said that the gene is split. the transcription of the exons, accompanied by their splicing leads to the formation of a messenger rna, the codons (nucleotide triplets) of which are translated into amino acids that are linked to each other by covalent bonds in order to form a protein chain. in prokaryotic cells (bacteria), the genes do not contain introns and are not split. a: adenine; c: cytosine; g: guanine; t: thymine; u: uracil. met: methionine; his: histidine; tyr: tyrosine; gly: glycine; phe: phenylalanine. interlinked with the epic rise of molecular biology, there was a succession of technical innovations that led to the synthesis of dna by chemical or enzymatic means, and to its being cleaved at specific locations, with the pieces that were obtained being joined together again . in , in geneva, werner arber (b. ) and daisy dussoix highlighted the restriction phenomenon, which involves the degradation of bacteriophage dna by a recipient bacterium. they discovered that an extract of e. coli has a restriction activity, and that this activity is of an enzymatic nature, caused by a nuclease that breaks the phosphodiester bonds in dna. in , the americans hamilton smith (b. ) and kent wilcox purified the first restriction enzyme from a strain of haemophilus influenzae. in , daniel nathans ( - and kathleen danna (b. ) at johns hopkins university in baltimore (usa) drew up the first restriction map based on the circular dna of the monkey sv virus, using a restriction enzyme that was named hindiii and a follow-up of the sequential appearance of shorter and shorter fragments resulting from the partial digestion of dna. in the following years, dozens of restriction enzymes were isolated, all of them endowed with a surprising specificity with respect to specific base sequences in dna ( figure iv . ). these enzymes were to be indispensable tools in genetic recombination experiments. the transformation of rna back into dna was observed by howard temin ( - ) and s. mizutani , in experiments on the rous sarcoma virus, a virus with rna that, when it proliferates in host cells, is able to synthesize a dna that is complementary to its rna. the enzyme responsible, reverse transcriptase, was purified by both h. temin and david baltimore (b. ) . starting with a determined messenger rna, it then became possible to work back to the dna, i.e., to the gene, by a simple enzymatic reverse transcription operation. dna that has been synthesized in this way is called complementary dna (cdna). in eukaryotic organisms, reverse transcription has proved to be all the more useful as a technique in that all cdna is coding, unlike the situation in vivo in which the genes are divided up into portions that are coding (exons) and portions that are non-coding (introns). the ability to cleave dna and to join together the fragments obtained in a deliberately chosen order, or, in other words, to manufacture previously unseen dna sequences by making new combinations, led to the dawning of recombinant dna technology and caused scientists to come to the sudden realization that the pandora's box that contains the secrets of life had been opened, that uncontrollable catastrophes might arise from this, and that there was a potential danger of causing tumorigenic viruses to reproduce in commensal bacteria such as the enterobacterium escherichia coli. in , around a hundred molecular biologists gathered together at the asilomar conference center near monterey in california, in order to discuss the dangers of the new dna technology. they proposed strict regulation to govern genetic manipulation. time and experience have shown that the risks being run were very low. in , dna sequencing methods were published. one of them made use of chemical techniques , the other made use of enzymatic techniques . applications were not slow in appearing. from , the team led by frederick sanger (b. ) in cambridge (uk) determined the first sequence of a genome, that of bacteriophage phix , which is nucleotides long. this was the beginning of an audacious adventure, the apparently senseless challenge being met with unbelievable rapidity thanks to the innovative methods of bioengineering, resulting, during the first years of the st century, in the sequencing of the human genome. analysis of the human dna sequence involved the participation of two rival groups, one of them being academic, coordinated by francis collins (b. ) , and bringing together dozens of laboratories around the world, and the other being a private californian company directed by craig venter (b. ) . at the beginning of the s, when everyone was persuaded that the rnas could be placed into three well defined categories, messenger rnas, transfer rnas and ribosomal rnas, it was with great surprise that it was learned that there were rnas that have catalytic properties (see thomas cech [b. ] ). these rnas, called ribozymes, have, in keeping with enzymatic proteins, structured catalytic sites that are able to catalyze rna or dna cleavage or ligation reactions. recently, engineering techniques have been used to obtain artificial ribozymes that have been found to be able to catalyze reactions as varied as oxidations or the synthesis of peptides and nucleotides, thus opening up wide-ranging possibilities of applications in molecular therapeutics, and, in addition, reinforcing the famous theory of the "world of rna" at the beginning of the appearance of life on earth . another discovery of the s was the role of methylation of dna bases, cytosine and adenine, and its deregulation in a certain number of pathologies: fragile x syndrome, scapulohumeral dystrophy, certain forms of cancer … in the past decade or so, basic proteins known as histones that are associated with the nuclear dna of eukaryotes in the form of a complex called chromatin and which had previously been assigned a structural role, have now acquired the status of functional partners. thanks to specific modifications of certain amino acids (acetylation, methylation, phosphorylation), histones control the state of condensation of the chromatin and the efficacy of transcription of dna contained in the chromatin, to such an extent that we now speak of the "histone code". the development of our understanding of histones is a good illustration of the complexification of a concept, the dna code, into an entity that comes closer to living reality, the dna code in partnership with the histone code. there has also been the discovery of interfering micrornas, small polymers made up of around twenty nucleotide units, the role of which is to control protein synthesis (chapter iv- . . ). methylation of dna, structural modifications of the chromatin histones, and blocking of transcriptional activity by interfering micrornas are a few of the major areas of research in a scientific domain that is in full expansion, epigenetics, which could be said to have "pipped the science of genetics at the post," and which explains the plasticity of the functions of living beings. with the arrival of restriction enzymes and reverse transcription, the foundation stones of genetic engineering have now been laid, and are ready to be used, this being all the easier in that synthetic chemistry is now able to manufacture dna chains that are several hundreds of nucleotides long, and progress in robotics and computing techniques made it possible for chemists to avoid carrying out tedious routine tasks by using completely-programmable machines. the hope that it would be possible to experiment on living beings by means of the manipulation of dna became a reality when the american researchers paul in the first work carried out on the expression of foreign genes, the use of plasmids as vectors was preferred, particularly that of plasmid pbr , because of its considerable replication capacity. in , a first success was obtained by herbert boyer and his co-workers, with the expression of the gene for somatostatin, a peptide hormone comprising twelve amino acids that negatively regulates the secretion of growth hormone, in the bacterium e. coli. because of its small size, the somatostatin gene was synthesized by chemical means. the expression of somatostatin in e. coli was verified using immunological and physiological criteria, thus demonstrating the validity of the procedure that was used. the following year, human insulin was produced in e. coli. fairly soon, yeast was substituted for this bacterium because, as a eukaryotic organism, it has enzyme systems that are able to carry out chemical finishing operations on neosynthesized proteins that bacteria are unable to do, for example the formation of disulfide bridges in insulin. genetic recombination is used in order to cause bacteria to manufacture a foreign protein of animal or plant origin. this involves the insertion of the fragment of animal or plant dna that codes for this foreign protein into a plasmid. the plasmid, a small ring of bacterial dna, acts as a vector for the foreign dna. in order to carry out insertion, plasmid dna is cleaved by an appropriate restriction enzyme. the foreign dna is obtained by reverse transcription from a useful messenger rna. its duplication is catalyzed by a dna polymerase. the s nuclease makes it possible to break the covalent bond between two strands of dna. in the following step, a terminal transferase is used to add four nucleotides for which the base is a cytosine (c) to each of the two dna strands. the same lengthening operation is carried out on the bacterial plasmid, but, in this case, the addition involves a sequence of four nucleotides for which the base is a guanine (g) (complementary to the cytosine c). the bacterial plasmid is hybridized in vitro with foreign animal or plant dna and then introduced into the bacterium which, using its own machinery, perfects the junction between the integrated dna and the plasmid dna. a very small volume of dna ( . - ml) is injected under the microscope into eukaryotic cells (hela cells in inset) using a micropipette with a very fine end that pierces the cell membrane. the swelling of the cells at the moment of injection can be seen (inset). supported by these successes, genetic engineering started to come to the fore as an application-oriented discipline. levels of performance that would never have been imagined half a century before were achieved, such as the production of growth hormone, interferons, blood coagulation factors and vaccines. in the final decades of the th century, phenotype transformations using genetic modifications that had previously been carried out in bacteria and yeasts were successfully attempted in animals and plants. it was observed that a mutated dna integrated into a plasmid and introduced into a fertilized mouse egg (by micromanipulation) modifies the mouse's genetic inheritance, which affects first the embryo and then the adult mouse with phenotype modifications. such mice, which are said to be transgenic because of the stable integration of a foreign dna into their genome, are now widely used as animal models in studies that aim to understand the mechanisms involved in high-incidence human pathologies such as cancer, diabetes, and rheumatoid conditions. in , two american researchers , ralph brinster (b. ) and richard palmiter (b. ) carried out a spectacular transgenesis experiment in mice. using microinjection, they introduced the growth hormone gene (obtained from the rat) into oocytes of mice from a "little" germ line. once the transgeneic mice had reached adulthood, they were giants. at present, the transgenesis technique is being applied both to the animal kingdom and to the plant kingdom. in the s, kary mullis (b. ) perfected an ingenious technique, the polymerase chain reaction (pcr), which makes it possible to produce several tens of thousands of copies of a fragment of dna. using this technique, it is possible to detect traces of a fragment of dna of a given sequence down to an attomolar concentration, i.e., one billion billion ( - ) times smaller than molar concentration. by the end of the th century, genetic engineering had become well-established and wide-spread, thanks to a mastery of techniques involving the manipulation of dna such as the accurate cleavage of a gene into fragments using commercially available restriction enzymes, the covalent assembly of two fragments of dna by ligases, the automated chemical synthesis of fragments of dna of more than one hundred nucleotides and the possibility of manufacturing a complementary dna (cdna) from a messenger rna by using a reverse transcriptase and automated dna sequencing. given this particularly well-equipped toolbox, the molecular biologist is now able to manipulate dna, that is to say, the chemical material that contains the information that is central to the functioning of living structures (microorganisms, plant and animal organisms), and thus to modify, at will, the genotype of these structures that the selective pressure of evolution has previously favored. genomics has produced enormous quantities of data that are stored in databanks. automated procedures have been invented to make the information contained in these data intelligible, these procedures forming the basis for a new discipline, biocomputing, or bioinformatics, which develops programs, or algorithm-based strategies, that are able to solve specific problems, of which the annotation of genomes, i.e., the identification of coding and non-coding sequences. while the annotation of the prokaryotic genomes is relatively easy, because of the absence of introns, that of the eukaryotic genomes is considerably more difficult because of the alternating exons and introns and the small proportion of coding exons (fewer than % in the case of the human genome). this explains why, at the time of writing, several hundred genomes of prokaryotes (around at the beginning of ) have been sequenced, as opposed to only a few dozen genomes of eukaryotes. annotation was carried out manually at first, but it has become automated and it is now possible to analyze thousands of items of genomic data. the comparison of nucleotide base sequences in dnas and of amino acids in proteins of different origins involves biocomputing. the identification of similar or identical regions that provide information about functional similarities and phylogenetic proximity involves the use of alignment methods. one of these methods, which is in current use, is called blast (base local alignment search tool). comparison of protein sequences has been particularly instructive in the science of evolution. it has highlighted evolutive processes in the phylogenesis of proteins and linked these processes to precise functions. it has been possible to deduce that, over time, different families of proteins with similar functions appeared independently and evolved along different routes. this is the case for membrane proteins whose polypeptide chain crosses the thickness of the membrane six or twelve times; thus, the mitochondrial membrane proteins that transport metabolites are formed by triplication of an element with two transmembrane segments, while proteins located in other membranes of the cell are derived from duplication of an element with three transmembrane segments. from this academic context arose the study of paleogenetics, a new discipline that compares dna sequences extracted from fossils and amplified by pcr with dna sequences of current species. in addition to being of immense interest to fundamental biology, genetic bioengineering has led to innumerable industrial applications making use of genetically modified microorganisms that are able to synthesize molecules with a high added value that can also be used in xenobiotic depollution operations. so much data has already been deposited, equivalent to the sequencing of more than one hundred billion nucleotides, that it is inevitable that there have been errors, some of which might prove prejudicial for future use (comparison of sequences, screening of drugs…). nevertheless, the ever-increasing numbers of genome sequencing projects for animal, plant and microbe species show the interest that is shown in understanding the genetic information present in different types of cells, in order to be able to exploit their potential. dna chips appeared in the last decade of the th century, and came to the fore as part of a new technical revolution, the "high throughput" revolution ( figure iv. ). an article written by the group headed by ronald davis and patrick brown (b. ) at the university of stanford gives a precise description of the hybridization technique used in dna chips. thus, around a hundred short dna strands, corresponding to portions of genes of the plant arabidopsis thaliana, commonly known as mouse-ear cress, a small plant in the brassicaceae (formerly cruciferae) family, are synthesized. a robot is used to deposit microquantities of these dnas in solution in a dot pattern on a small glass slide coated with poly-l-lysine, thus comprising a "chip" on which the covalently fixed dnas act as probes for specific molecules. a later step involves both the use of reverse transcription to produce complementary dnas (cdnas) from messenger rnas arising from the expression of genes in the same plant and the labeling of these cdnas with fluorescent ligands for use in screening. once these fluorescent cdnas have been denatured, i.e., after separation into single strands, they are brought into contact with the dna chip. the unhybridized molecules are removed by washing. a -the term dna chip corresponds to a small, chemically-treated glass (or sometimes silicon) plate on which a robot has deposited dna strands of known sequence in a pre-determined order. b -the dna chip may be used for different types of diagnostic procedures. in the differential diagnosis experiment represented here, messenger rnas are prepared from two cell samples that have been treated in parallel, the control sample (normal cell) and the experimental sample (pathological cell). these messenger rnas are reverse transcribed into complementary dnas (cdnas) by means of a reverse transcriptase. each of these two types of cdna, corresponding to the two types of messenger rna, is labeled by a chemical reaction with a specific fluorescent ligand (cy , which emits at nm and cy , which emits at nm). they are then hybridized with chip dna strands. after hybridization and then washing, the fluorescence emitted at nm and nm under laser irradiation are analyzed using an appropriate detection system. the differential expression of the genes in the control cell sample and the experimental sample (shown by a color difference) can thus be analyzed. hybridization between the fluorescent dnas called targets and the complementary nucleotide probes fixed to the dna chip is detected by means of an automated fluorescence detection system. at the beginning of the s, stephen fodor and his group, who were working at the affymax research institute (palo alto, california), developed an ingenious procedure for microphotolithography that led to the synthesis of a network of a thousand peptides on chemically pre-treated glass microscope slides. the resolution of the network was shown by epifluorescence microscopy after fixation of specific antibodies labeled with fluorescent probes. soon after this, microphotolithography was used for the manufacture of dna molecule networks on solid supports. from then on, two competing techniques for the preparation of dna chips became well-established, either the depositing on a solid support of cdna obtained by gene amplification (technique used by davis and brown), or the synthesis in situ of oligonucleotides carried out directly on a solid support (technique used by the american affymetrix company, arising from affymax). one considerable advantage of dna chip technology is that it provides information on the level of transcription of thousands of genes into messenger rnas (mrnas), in a simultaneous manner and in a relatively short lapse of time. experiments that previously required weeks, months or even years to be completed can now be carried out in a matter of hours. we therefore have a sort of instantaneous, precise, freeze-frame picture of the state of a cell at a given moment, with a great number of parameters explored in a semi-quantitative manner. dna chips are a typical example of the application of high throughput technology to the study of living beings. the panoply of mrnas produced by the transcription of dna is called the transcriptome. the method by which transcriptomes are obtained is called transcriptomics. it should be added here that there is not necessarily a correlation between the abundance of a mrna, evaluated on a dna chip, and the functionality of the corresponding protein, which depends on multiple factors that particularly involve post-translational modifications: phosphorylation, glycosylation, hydroxylation, and so on. at the end of the s, dna chips were being used extensively in the research programs of many biology laboratories. they are used in a variety of domains: human pathology, to differentiate between forms of cancer linked to multiform mutations; microbiology, to identify pathogenic germs; comparative genomics, to look at model eukaryotic organisms that have in common a certain number of genes; or even in populations genetics, to detect polymorphisms linked to a change in a single base in a dna sequence. as a complement to the dna chip technique, the fish (fluorescent in situ hybridization) method holds a key position in the study of cytogenetics. this method is based on hybridization between fluorescent nuclear probes of known nuclear sequence and of complementary motifs located in the dna of the chromosomes. it allows the detection of chromosomal modifications with a gain or loss of genetic material, such as those that are found in certain tumors. it is used in prenatal diagnostics to diagnose such modifications. protein chips, which are used to characterize the reactivity of proteins with respect to specific ligands, are another example of the application of high throughput technology. dozens of proteins of different types (antibodies, for example), as well as derivatives of nucleic acids or even molecules capable of being ligands of proteins that might arise from combinatorial chemistry (chapter iv- . ), are arranged in a network on small glass plates that are chemically treated to act as hooks to entrap specific proteins present in a tissue extract or serum ( figure iv . a). this procedure, which is essentially analytical in nature, is complemented by a functional study in which proteins that have been isolated in their native form, i.e., those that are capable of expressing the same functions that they posses in vivo, are deposited on a glass microplate. this type of biochip makes it possible to analyze the reactivity of the proteins that are fixed to it with respect to a multitude of targets (proteins, nucleic acids or pharmaceutical substances) (figure iv. b). antigens peptides a -biochip for the identification of proteins. different types of ligands, antibodies, antigens, dna or rna, small molecules with a high affinity and specificity, are deposited on a reactive surface. these biochips can be used to determine the level of expression of proteins and the type of proteins expressed in cell extracts. they can be used for clinical diagnostics. b -biochip for the functional study of proteins. native proteins or peptides are arranged in micronetworks on a reactive medium. biochips produced in this way are used to analyze the activities of proteins and their affinities as a function of posttranslational modifications. they are useful for identifying drug targets. analytical proteomics, which was still in its infancy in the last decades of the th century (chapter iii- . . ) has become a vigorous discipline. the association of liquid nanochromatography and of mass spectrometry allows the identification of peptides obtained by the trypsin hydrolysis of samples of proteins of around one picomole in size. applied to fundamental and pathological cell biology, the aim of analytical proteomics is not only to decipher the list of proteins on the scale of the cell, but also to highlight variations in the abundance of synthesized proteins as a function of environmental conditions. it also aims to determine the posttranslational modifications that are undergone by the proteins inside the cells, which, for a large part, control the specificity of their operation. in parallel with the study of proteomics, the study of peptidomics, or the study of all of the peptides (peptidome) present in animal and plant cells and in the fluids that bathe these cells, has developed. for example, several hundred different peptides have been found in the cerebrospinal fluid. structural proteomics, which deals with the three-dimensional structure of proteins, has also become a domain in which the activity has been increasingly dominated by high-throughput techniques. this is partly due to the fact that the pharmaceutical industry has given considerable, sustained attention to the understanding of the structure of proteins that could play the role of therapeutic targets. this is the case for protein kinases that catalyze, via atp, the phosphorylation of endocellular proteins, of proteases involved in hydrolysis reactions, or even of cell surface receptors that are able to combine with ligands such as hormones. the use of automated crystallization systems has become common in structural biology. from the classical technique of the hanging drop, in plates comprising wells, we have moved on to plates with , and, recently, wells. obviously, this increase in dimensions requires the use of an automated system that includes a robot in charge of transferring microaliquots of the protein solution into the wells and adding media that differ according to their ph, ionic force and molecular composition to these wells. the crystallization process is followed by an automated microscopic examination coupled with video photography. making use of the recent development of genomics and of proteomics, and a detailed inventory of the structures and functions of the different protein species of living beings, contemporary biology is now able to sketch out a scheme of molecular systematics, including a classification into phylla, families, and classes that echoes those of the zoological and botanical systematics of the th and th centuries. however, modern systematics does not tell us how protein macromolecules interact within dynamic networks. there is still an enormous amount of work to be done in order to achieve an understanding of the meaning of the dialogue between macromolecules in a normal or pathological cell context. this work will require a detailed analysis of metabolic pathways and of how they are controlled, and their evaluation in kinetic and thermodynamic terms. it will be accompanied by modeling (chapter iv- ). there is no doubt that it will be successful. making use of subtle differences in the qualitative and quantitative expression of genes, it will become possible to understand the molecular principles that modulate differences in morphology and in function between neighboring animal, plant or microbial species. the science of evolution should benefit from this. in medicine, the forecasting of predispositions for certain diseases should be made easier (chapter iv- . ), opening up the perspective of prevention strategies. using recombinant dna technology, metabolic engineering applied to microorganisms and plants should make it possible to improve the production of molecules that are of economic interest or can be used for drugs. the diversity of bacteria is amazing, much greater than might be supposed by looking at the number of bacterial species identified by culturing on appropriate media. in fact, the number of bacterial species that can be cultivated only represents % of the total number of existing bacterial species on the surface of the earth. there are two major reasons for this: we do not know the appropriate conditions for culturing these bacteria; a certain number of environmental bacteria live in symbiosis, acting as commensal organisms that benefit from the products secreted by other organisms. nevertheless, the study of the bacterial genome, without any clonal culture, has been carried out, and comprises a branch of genomics known as metagenomics. instead of looking at an isolated, well-identified bacterial species, in order to analyze the sequence of its dna, as has been done traditionally, researchers look at a heterogeneous bacterial sample from which the dna is extracted, amplified, and then sequenced by high throughput methods. computer processing of the data provides information about individual germs. craig venter, who had already gained notoriety with the sequencing of the human genome, recently applied "metagenomic" procedures to the study of the sequence of the "metagenome" of the bacterial species of the sargasso sea . he came up with nucleotide sequences corresponding to approximately million kilobases of non-redundant nucleotides, attributable to more than two thousand different genomes. the challenge to be met by metagenomics is to connect a function to its phylogenetic source and to extend this information to specific species within a bacterial community. group . in human biology, a metagenomics approach has been applied to the study of the population of bacteriophages present in the intestinal flora. approximately genotypes have been identified, a number that greatly exceeds the bacterial species of this flora . this result leads us to think that the luxuriant community of bacteriophages which cohabits with that of the intestinal bacteria may influence the diversity of the latter by selective bacterial lysis and also by promoting the exchange of genes between bacteria. a rapid overview of the history of the exploration of genomic dna over the last fifty years shows the rapidity with which a traditional experimental paradigm can move thanks to modern computing and robotics procedures. in less than twenty years, we have moved from the manual sequencing of dna that was developed at the end of the s to automated high throughput sequencing. at the turn of the st century, the sequencing of communities of genomes (metagenomics) has been substituted for the sequencing of individual genomes. dna and protein chips have become objects of everyday use in fundamental and applied biology. transgenesis is widely practiced. dna, a molecule that remained mysterious for a long time after it was discovered, delivered some of its secrets during the second half of the th century. the purpose of the first experiments on dna was to understand how dna, detector of the genetic code, transmitted its message. after having questioned dna, researchers moved on to manipulating it. the current aim is to use oligonucleotides to build nanoscale constructions with original and, if possible, useful, properties. in addition, the possibility that has recently become available of being able to interfere with the expression of the genome in living cells, with the intervention of small rna molecules, allows the programmed manipulation of the genome. another challenge, the extending of the coding power of the genetic code, now appears to be achievable. from the fact that each of the strands of the double helix overhangs in one direction, and in the other the strand with which it is paired, thus leaving a few bases free ( figure iv. ) . if two strands of dna with sticky ends are brought into contact, when the bases of these ends are complementary, a branched structure will appear spontaneously. using this principle as a basis, cube-shaped nanometric constructions that make it possible to encage molecules of interest have been built. the opening of the cage by appropriate devices liberates the encaged molecules, which can act as substrates in specific reactions. the cutting of a double strand of dna using restriction enzymes able to create fragments with cohesive ends (a) has been used to "build" an artificial construction (b), which, in this case, is a cube (c), but which could be an object of a different geometrical type. a dna nanomachine that is capable of movement is becoming a reality. one dna nanomachine, which is admittedly still rudimentary, has been put together based on the structural difference that exists between b-dna, the classical double helix that twists to the right, and z-dna, a double helix that twists to the left. a propensity to adopt the z-form is triggered when there is an alternating sequence of cytosine (c) and guanine (g) (cg sequence) in the dna. the experiment illustrated in figure iv. makes use of a duplex formed of b-double helices. the dna nanomachine constructed by n.c. seeman comprised a duplex of double strands of dna. one of the double strands, of the classical b-form of dna (right-hand twist), has been cleaved in such a way as to fix fluorescent molecular probes onto the cleavage zones. facing this cleavage zone, a short nucleotide sequence, in which the cytosine (c) guanine (g) motif is repeated, can be found in the other dna double strand, which is also of b-form. the addition of cobaltihexammine induces the transition of the cg segment from a right-hand twist (b-dna) to a left-hand twist (z-dna), which leads to a rotation of this segment and to a rotation of the assembly, which can be detected using fret (fluorescence resonance energy transfer) spectroscopy. one of the double helices has a short cg segment. facing the cg segment, the other double helix is interrupted, and its ends, where the interruption is, carry fluorescent molecular probes. the simple fact of adding a cationic substance such as cobaltihexammine, which neutralizes the negative charges of the phosphate groups, triggers a conformational transition, with the cg segment taking the z-form, causing a rotational movement of the assembly that is detected by the movement of the probes. there is no doubt that the use of dna strands in order to build nanomolecular constructions that are capable of programmed movement marks the beginning of an adventure that we may imagine will be rich in outlets for domains such as computer technology, nanomechanics and even the life sciences. in addition, the discovery that dna conducts electrical current gives rise to dreams of a revolutionary technology in which dna may be used in the design of electrical circuits, in competition with classical electronics . interfering rnas are non-coding rnas of around twenty nucleotides that control gene expression at post-transcriptional level. as with many discoveries, that of rna interference was the result of serendipity. it began during the s with observations made by two american research groups, that of victor ambros now at darmouth medical school, hanover, and that of gary ruvkun (b. ) at boston's massachusetts general hospital, that a gene named lin- , which is involved in the post-embryonic development of the nematode c. elegans, did not code for a protein, but for a small size rna that played an antisense role. this odd discovery was supported and made more explicit a few years later by the research groups of andrew fire (b. ) at baltimore's carnegie institute and craig c. mello (b. ) at the university of massachusetts in worcester . in order to block the production of certain proteins in the nematode c. elegans, the researchers used synthetic antisense rnas. the control involved the use of sense rnas according to a classical protocol. unexpectedly, protein synthesis was blocked in both cases, suggesting that a contaminant was present in the sense and antisense rna preparations. this contaminant was identified as a double strand rna (dsrna -double strand) that is, an rna that is folded back on itself in a "hairpin" loop because of the pairing of complementary bases (adenine vs uracil and guanine vs cytosine). in order to verify the mechanism by which the translation of messenger rnas into proteins is silenced, the nematode c. elegans was injected with a synthetic dsrna, part of the sequence of which was complementary to that of the gene unc- , known to code for a protein involved in muscular contraction. within a few hours, the worm was making disordered movements, suggesting that the dsrna interferes with the production of proteins in the process of muscular contraction. the mechanism of action of dsrna was quickly unraveled: dsrna gives rise to two single strand rnas after cleavage by a specific enzymatic mechanism. one of the single strand rnas (sirna -small interfering rna) is paired thanks to a complementarity of bases with a short sequence of messenger rna transcribed from the gene unc- . the result is a blockage of the translation of messenger rna into a protein, followed by the destruction of messenger rna. this phenomenon was named rna interference ( figure iv the dicer cleavage enzyme, which has a ribonuclease activity, cuts the double strand rna into two strands. in the presence of the risc (rna-induced silencing complex) protein complex, one of the rna strands finds a complementary nucleotide sequence in a messenger rna (mrna) and associates itself with this rna, making it unable to be translated into protein. we now know that eukaryotic cells from animals and plants produce and host interfering rnas that are said to be "natural" . natural interfering rnas, of around twenty nucleotides, are called micrornas (mirnas). although a few details differ between the modes of formation and action of natural interfering rnas and those of synthetic interfering rnas, in particular the fact that messenger rnas are not destroyed by mirnas but blocked in their translation, the effect of negative regulation on the production of specific proteins comes to the same thing ( figure iv. ). there is a far from negligible number of genes that code for mirnas. already, several hundred mirnas have been identified in the genomes of plants and animals. the amount of interest that they arouse, and the feverishness of the research being carried out on them, are in keeping with the major mechanisms that they control: embryogenesis, hematopoiesis, neuronal differentiation, etc. given an understanding of the genome sequence in man, the rat and the mouse, trials have begun that aim to achieve an understanding of how the expression of mammal genes of known sequence might be manipulated by the interplay of interfering rnas (chapter iv- . ). the treatment of viral infections such as aids or hepatitis b, which are worrying public health problems, could benefit from this new technology. it appears that interfering rnas have much more to give to us in the near future than they have taught us up until now. the deciphering of the genetic code in the middle of the s was the end of a first step in elucidating the mechanism by which a sequence of nucleotides in dna is translated into a sequence of amino acids in a protein (chapter iv- . ). during the years that followed, the subtleties of the transcription of dna into messenger rna and of the translation of messenger rna into protein via transfer rnas were explored in hundreds of laboratories around the world. particular attention was paid to the understanding of how a given amino acid is activated and bound to a transfer rna (trna) after being picked up by an aminoacyl-trna synthetase. nevertheless, the idea remained of a code in which triplets of purine and pyrimidine bases of messenger rnas are translated into natural amino acids. recently, methods have been developed that give more flexibility to the action of the aminoacyl-trna synthetases, or, in other terms, relax their specificity . synthetases that have been manipulated in this way are able to recognize non-natural amino acids and to incorporate them into proteins by working together with the ribosomal machinery. it is in this way that, at the time of writing, around thirty non-natural amino acids, obtained by insertion of different types of chemical residue (photoactivable, fluorescent or radioactive residues capable of acting as probes for structural and functional analyses) (figure iv. ) have been incorporated into protein structures. with such an innovation, an unexpected field of exploration has opened up to research in domains as far apart as pharmacology and the science of evolution, giving rise to burning questions: could such non-natural proteins have therapeutic properties? could they give a selective advantage to the organisms that host them? with the addition of non-natural amino acids to the genetic code and the demonstration that proteins containing such amino acids can function in living cells, in sum, with the transgression of the potentialities of the natural genetic code, the experimental method appears to challenge the order of living beings. the triumph of genetic engineering via the study of dna is not unique to biology. many other sectors are undergoing changes in their type of experimental approach, dictated by the technosciences and making use of computer sciences, robotics and high-throughput screening. however, given the many questions that its operation continues to raise, and its central position at the heart of scientific ethics, the study of dna remains a typical example of the way in which the experimental life sciences and the techniques that underlie them are evolving nowadays. "i perfectly agree that when physiology is sufficiently advanced, the physiologist will be able to make new animals or plants, as the chemists produces substances that have potential, but do not exist in the natural order of things." more than a century after claude bernard predicted a genetically manipulated world, it has come to pass. the molecular biologist, having original, highperformance methods for "tinkering" with dna, has moved on to the application and use of his technical expertise for utilitarian ends. during the s, with transgenesis, research on bacteria (chapter iv- . . ) opened up a new biological domain, that of genetically modified organisms (gmos). results led to predictions that it would be possible to transfer a fragment of dna corresponding to a gene of a certain species into the genome of another species and have this foreign gene express itself as a protein in the host cell. in , the successful trans-genesis of a gene for resistance to an antibiotic, kanamycin, in tobacco plants, signaled the beginning of the technology of the first plant-type genetically modified organisms, still called gmps (genetically modified plants). in , the birth of dolly the ewe unveiled the era of reproductive cloning in mammals, i.e., the identical reproduction of an already-existing organism. an additional step was taken with the first tests on the differentiation of embryonic stem cells towards different types of lines that are characteristic of well-defined tissues, such as nerve tissue, cardiac tissue or the hepatic parenchyma, thus opening up promising perspectives in regenerative medicine. the frontiers of the experimental method continue to be pushed back to the limit of what is feasible and sometimes into the realm of fiction, as in immunology, for example, with the idea of xenotransplantation, using "humanized" animal organs. given the universality of the genetic code, any gene that is introduced into the genome of a plant, whether that gene is of animal, plant or microbial origin, is able to replicate itself and be expressed as a specific protein. thus plant gmos or genetically modified plants (gmps) are able to express specific foreign proteins from another plant, a bacterial microorganism or an animal organism. in the s, a short time after fundamental research had revealed the feasibility of plant transgenesis, the first transgenic plant, the flavr savr tomato, was marketed in the usa. since this time, numerous other plant gmos have been cultivated on a large scale and become available on the world market, including corn, soya, rice, cotton and the poplar. one of the desired aims is to produce modified plants that are able to resist destruction by the herbicides that are commonly used to eliminate weeds, while another is to prevent predation by harmful insects. in the first case, transgenesis involves the insertion of a herbicide-resistant gene, and in the second case, the inserted gene codes for an insecticidal toxin. recently, plant gmos that produce proteins with a therapeutic effect have appeared, ranging from antibiotic peptides to antibodies or proteins as unexpected as human hemoglobin. current projects aim to create plants that are resistant to adverse conditions such as the dryness of arid climate zones. the preferred procedure for producing a plant gmo is to use a bacterium, agrobacterium tumefaciens, a microorganism that is able to insert fragments of its own dna into plant cells ( figure iv . ). the useful gene that we wish to transfer into the plant may be a gene for resistance to a pesticide such as glyphosate, marketed under the name of roundup, phosphinothricin (basta) or glufosinate (liberty). the plasmid is reintegrated into a. tumefaciens. during infection of a plant cell by a. tumefaciens the t-dna carrying the gene of interest is inserted into one of the chromosomes of this cell. the ti plasmid is isolated and cut using a restriction enzyme. a foreign gene, known as a gene of interest, is inserted into the t-dna of the plasmid. a plant is generated from a modified clone. all of its cells carry the foreign gene. transgene of interest in the case of the fight against insect predators, the useful gene is carried by a fragment of dna contained in the genome of the bacterium bacillus thuringiensis. this gene, called bt, expresses a toxin responsible for the insecticidal capability of b. thuringiensis. a current application involves the protection of bt corn with respect to the corn-borer, a devastating insect whose caterpillars are particularly destructive. another, more direct, gene transfer method, known as biolistics, involves bombarding plant cells with tungsten microbeads covered with modified dna. with the implementation of large-surface-area experimental fields and the first marketing of gm soya, in , the question of whether or not the advantages achieved with respect to crop yields are counter-balanced by risks for the environment and for consumers came to the fore. food risks could arise from the toxicity or allergenic power of artificially synthesized proteins. at the time of writing, this question remains unanswered, due to the lack of epidemiological studies carried out rationally over several years. when the first creations of gmos took place, the transfer of the gene of interest was carried out by means of the co-transfer of an antibiotic resistance gene. the transformed cells were selected according to the criterion of their resistance to this antibiotic, which involved a risk of dissemination of the resistance gene. this selection technique has been abandoned. in practice, it is difficult to evaluate the theoretical ecological risk of wild plants being invaded by genes that have been inserted artificially into gmos. as a precaution, zones used for experimentation of plant gmos are now surrounded by refuge zones, i.e., fields in which the same species of plants, in non-gmo form, are cultivated. there has been a much fiercer and completely legitimate debate concerning the presence of the terminator gene in seed from the first gmos marketed by the monsanto company in the usa. the terminator gene blocked germination of the seed from the cultivated plant, so it was necessary for the farmer to buy more seed from the company each season, thus creating a state of dependency. this technique is no longer in use, but the fact remains that most transgenic seed is patented, and therefore farmers who use such seed are dependent on the companies that posses this genetic know-how. the culture of plant gmos has spread around the world, covering more than a billion hectares of our planet, more than half of which are in the united states of america. this type of culture is used on a large scale for soya and in a less extensive way for corn, rape seed and cotton, but there are many other applications of plant transgenesis. among the countries that are actively involved we may mention argentina, brazil, canada and china, and more recently india, paraguay and south africa. while the policies of these countries are based on the fact that gmo products do not differ fundamentally from non-gmo products with respect to checks carried out a posteriori, and that there is thus no reason to prohibit them, european policy has taken refuge behind a principle of precaution, and it remains basically restrictive. although the moratorium on the culture and marketing of plant gmos that was put in place in was lifted in , mandatory labeling for any consumable product containing more than . % gmo remains dissuasive. the united states of america has refused to use such labeling. the worries that are aroused by plant transgenesis, which are often exacerbated by the diktats of ecology groups, must be analyzed in a reasoned manner. common sense and lucid thought dictate that the debate should be situated within a scientific perspective in which the main role is played by the experimental method in long-term applications. simple reflection leads us to think that with time parasites and self-propagating plants will develop a resistance to the most drastic treatments, as was the case for bacteria confronted with antibiotics. the perspective of an acquisition of uncontrolled resistances, which gives rise to so much passionate debate, is, in fact, only the first stage of a technology with promising applications. the mastery of plant transgenesis that was acquired through the first experimentations should, in fact, allow the emergence of plant gmos that are assigned to the production of molecules with therapeutic effects (drugs, vaccines, human proteins, vitamins…). in this domain, there have already been creations that include golden rice, which carries β-carotene, the precursor of vitamin a, banana plants that express a vaccine against hepatitis b and tobacco that produces human lactotransferrin and hemoglobin. if we just look at the production of golden rice as a palliative for vitamin a deficiency, it should be remembered that, in certain countries of our planet, this deficiency affects people's sight and is a frequent cause of blindness, that it generates problems with development and the immune response to infections, that it affects more than a hundred million children around the world and that it is responsible for the death of three million of them each year. if these plants are considered to be a material of choice for the production of proteins with a therapeutic effect, this is partly due to the yield of such crops over large surface areas, and also partly due to the low risk of transmission of viral pathogens to man, because of the species barrier, a risk that is less negligible when animal productions are involved. genetically modified plants are also potential factories for the manufacture of chemical products with an industrial impact, for example lubricants, perfumes and aromas. given the unpredictable outlets that plant gmos may have in human medicine and the different domains of the economy, plant gmo technology should be considered in a manner that is free of any pressure or passion, and, as far as the political authorities are concerned, it should be subject to appropriate measures to surround and protect certain strategic experiments. when looking at the worries being expressed by the european society, it should be remembered that the genetic inheritance of plants has never ceased changing, not only in the most of natural of manners, over millions of years, particularly with the mobility of transposable elements located in the genome, but also artificially, at the hands of farmers from ancient times onwards, with their methods of hybridization and selection. the nervousness of european authorities, showing an ignorance of basic scientific ideas, with the pretext of a principle of precaution, and sometimes political compromises that are exemplified by fluctuating and contradictory positions, runs the risk, in the short term, of causing their countries to lag disadvantageously behind the united states of america, which holds the majority of plant biotechnology patents. the principle of gene therapy is simple: the introduction of an appropriate gene into the cells of a patient who carries a mutation can correct the phenotypical consequences of this mutation, or, in other terms, cure the disease affecting the patient, or at least slow down its evolution. the technical difficulty involved in gene therapy is that of finding an appropriate vehicle or vector for the transfer of the gene and addressing it to an appropriate location in the genome of the host cell. the most commonly used vectors in human gene therapy are viral. a certain number of criteria are necessary for a transfer to be efficacious, including a high concentration of viral particles carrying the gene to be transferred (more than a billion viral particles per milliliter) and a good capability on the part of the foreign gene to be integrated into the host's genome. the patient's immune response remains a major worry in the use of viral vectors: at cell level it often leads to a proliferation of cytotoxic lymphocytes and, especially at humoral level, to the synthesis of antibodies directed against the viral proteins. in order to minimize its immune response, the genetic material of the viral vectors is modified. for ethical reasons, gene therapy is currently only applied to somatic cells, germinal gene therapy being rejected. somatic gene therapy has been experimented in the treatment of hereditary illnesses linked to hematopoiesis. one of the technical reasons for this choice is easy access to the progenitor cells of the bone marrow, with the aim of transfection. it was with this in mind that mouse gene therapy models were developed a few years ago. the sickle cell mouse is one of these models. human drepanocytosis (sickle cell anemia) is a serious disease that is caused by a mutation in the β protein chain of normal human hemoglobin a. the molecules of sickle cell hemoglobin s tend to aggregate and form fibers that obstruct the blood capillaries of the microcirculation. somatic gene therapy has been applied to these sickle cell mice. this involves an autograft of bone marrow hematopoietic cells transfected with a retrovirus hosting the gene coding for the β subunit of normal hemoglobin. encouraging results have shown the validity of this approach. in , a gene therapy protocol that had been applied with success to man was described by the group of alain fischer (b. ) and marina cavazzana-calvo at the necker hospital in paris (science, vol. , pp. - ). the purpose of this therapy was to bring about a long term remission in the case of an immune disease known as scid-x (severe combined immunodeficiency linked to a mutation on the x chromosome). because of their susceptibility to microbial and viral infections, babies who are affected can only survive in sterile rooms. they are known as bubble babies. in this illness, the hematopoietic progenitor cells of the bone marrow are unable to differentiate into t and nk (natural killer) lymphocytes because of a mutation that affects a cytokine receptor. previous experiments carried out on model mice show that scid can be corrected by in vivo transfer of the cytokine receptor gene into hematopoietic progenitors. the transfer of the gene of interest paired with a retroviral vector was carried out first in march , in two babies, one of them eleven months and the other two months old. progenitor cells from their own bone marrow, cultured and modified genetically, were injected into them. these were therefore autografts, without any risk of immune rejection. a remission of symptoms over a period of nearly a year, shown by the almost normal behavior of the babies' immune cells, encouraged the application of the same therapy to other babies. in total, ten babies were given this therapy. the enthusiasm that greeted the successes that were recorded was nevertheless tempered by fact that in the spring of , and again in the following year, a child who had undergone the gene therapy developed a leukemia characterized by an anarchical proliferation of lymphocytes, necessitating chemotherapy. these two occurrences were explained by the random character of the insertion of the gene of interest into the patients' genomes: insertion into a site close to a proto-oncogene had led to activation of this proto-oncogene and the proliferation of the lymphocytes. while the trial carried out at the necker hospital gave rise to great hopes, it nevertheless showed that there is still a long way to go before we achieve a targeted transfection of genes so that no undesirable consequences follow. here we have a typical example of the limits of an experimental method that is based on an in-depth technological know-how, but also on a still imperfect understanding of the complex arcana of the mechanisms that regulate the positioning and interaction of genes in the chromosomes of eukaryotic cells. this example highlights a harrowing ethical dilemma: should we not treat a patient whose illness is likely to be fatal, or attempt a therapy that may save the patient, without having any formal assurance of its success? an experimental medicine that has the power to modify the human organism via its genetic material is now able to take over from the experimental method that up until now operated on animals and plants. we can easily understand, given the progress that has already been accomplished and that which is to come in the domain of gene therapy, that the temptation will be great, in the future, to consider manipulations of the human germ cell genome as being licit, insofar as such manipulations make it possible to eradicate a handicapping defect in our descendents. at present, the idea of any attack on the germinal genetic inheritance has been rejected unconditionally on the basis of ethical considerations. nevertheless, the history of science shows that prohibitions that were once considered to be untouchable finish by being contravened. this was the case for abortion. in a text entitled why genetic engineering should continue its battle , james watson writes of his confusion when faced with a choice that is likely to become more and more insistent over the years: "dare we be entrusted with improving on the results of several million years of darwinian natural selection? or do the human germ cells represent on the contrary rubicons that geneticists will never dare to cross?" a mastery of the differentiation of stem cells and of cloning are two essential weapons in the biotechnological arsenal, the use of which for utilitarian ends, particularly in human medicine, gives rise to hope and disquiet, agreement and disapproval. at the beginning of the s, experiments carried out by the canadian biologists ernest mcculloch (b. ) and james till (b. ) attracted attention to the particular properties of cells in the bone marrow, the stem cells, which would subsequently be found in other tissues . the experimental protocol is simple. bone marrow cells from a mouse are injected into another mouse that has previously been irradiated in order to destroy its stem cells. the injected cells go to the spleen where they divide and form colonies that take the form of nodules of different sizes. the researchers realized that the cells of these nodules present differences in their potential for renewal, which is more or less rapid. they reinjected the nodule cells into mice from a second batch. the reinjected cells showed themselves capable of multiplying and generating several types of blood line. these observations suggest the presence in the nodules of progenitor cells that have a strong potential for self-renewal and self-differentiation. in the following years, these observations were confirmed and explained by two characteristic criteria of stem cells; self-renewal and differentiation into multiples cell lines with specific characteristics. from this point on, it was possible to understand the enigma of the amputated hydra in the experiments carried out by trembley, two centuries beforehand (chapter ii- . . ). we now understand why, like the hydra, organisms like the flatworm, the salamander, the starfish and the zebrafish are able to recreate an amputated or damaged part of their bodies. the hydra mobilizes stem cells that it has preserved since its birth. in the case of the salamander, regeneration involves the reprogramming of cells that have already been differentiated. like all stem cells, embryonic stem cells (or es cells) are able to self-renew and differentiate into the different types of known adult cell line, giving rise to different types of cell such as neurons, cardiac cells that are able to contract, or hepatocytes ( figure iv . ). this potential has led to the hope that es cells could be used in regenerative medicine. in a fertilized egg that has developed to the blastocyst stage, it is possible to distinguish a cell mass (inner cell mass, icm) which protrudes inside the blastocyst. the icm cells are removed and placed on a mat of irradiated (and thus unable to divide) fibroblasts that provide them with a support and nutrients (steps and ) so that they can proliferate. the stem cells arising from the icm cells, placed in a medium that has been specifically conditioned to provide cytokines and other biomolecules, are able to differentiate into various cell types (step ). at what stage of embryo development is it possible to remove es cells for experimental purposes? after fertilization by a sperm cell, the ovum undergoes a series of divisions that give rise to a microstructure, the blastocyst, the cells of which are called blastomeres. each isolated blastomere remains capable of producing an entire organism of fetus and placenta, by division and differentiation. at this stage, blastomeres are totipotent. five days after fertilization, the embryo has the form of a hollow sphere. an external layer of cells, the trophoectoderm, surrounds a cavity, the blastocele, inside which a small mass of cells, the inner cell mass, protrudes. from the beginning of the implantation of the blastocyt in the uterus, the trophoectoderm evolves to form the placenta. the cells of the inner cell mass take part in the process of differentiation that generates all of the tissues of the future adult organism. these are called embryonic stem cells (es cells). es cells are said to be pluripotent. isolated, they have lost their ability to give rise to a complete individual, but they have maintained the possibility of differentiating, according to their environment, into any of the two hundred cell types that make up animal tissues. during their division, es cells evolve from a stage of being pluripotent to a stage of being unipotent, passing through a stage of multipotency beyond a hundred cells. a state of multipotency characterizes cells that give rise to a restricted number of cell lines in the tissues in which they nest. this is the case for of the hematopoietic stem cells of the bone marrow that form the red blood cells and the white blood cells. the term unipotent refers to the progenitors, which give rise to a single type of cell, for example the hepatocyte of the liver or the cardiomyocyte of the heart. when es cells are cultivated for to days in a conventional nutritive medium, they multiply and aggregate. if the culture medium is supplemented with certain biomolecules such as insulin, retinoic acid, transferrin or fibronectin, the differentiation of the es cells is oriented towards cells of different types, such as neuron cells, glial cells or muscle cells. there are many publications about experiments concerning the grafting of differentiated stem cells in the mouse or the rat. for example, neuron precursors derived from the spinal cord or the brain are grafted into rats whose spinal chords have been injured. five weeks after the grafts are carried out, the transplanted cells have filled the area of the injury and differentiated into oligodendrocytes, astrocytes and neurons. what is more, after about twelve weeks, locomotive function has been partially restoredirradiated . other experiments involving the grafting of differentiated stem cells have been carried out on rats in which the dopaminergic neurons of the "substantia nigra" of the brain that secrete the neurotransmitter dopamine have been selectively destroyed by injection of -hydroxydopamine. the problems found in the rat as a result of this neuronal degenerescence mimic those found in man in patients suffering from parkinson's disease. dopaminergic neurons obtained by the differentiation of mouse es cells are grafted into the striatum of each of these rats, a region of the brain whose neurons communicate with those of the substantia nigra and play a fundamental role in the control of movement. this results in a significant improvement in the motor deficit, coupled with the establishment of functional synapses between the injected neurons and those of the host , . a recent publication bringing together the results of two french research teams, that of michel pucÉat (b. ) in montpellier and that of philippe menaschÉ (b. ) in paris, provides interesting information about how mouse embryonic stem cells, grafted into sheep cardiac tissue where an infarctus has been artificially induced, are able to colonize the infarct zone and regenerate cardiac contraction in a functional manner. moving from the mouse to the sheep constitutes a considerable species leap, and the absence of any immune rejection leads us to say that embryonic stem cells have an "immune privilege" . the use of es cells in regenerative medicine necessarily requires that their differentiation be regulated in an exhaustive manner into well-defined pathways, in order to produce homogeneous cell lines with a view to implanting them in damaged tissues. in fact, contamination with non-differentiated es cells is likely to cause tumors (teratomas) over the long term. the mastering of the use of es cell culture and differentiation, as well as of cloning, in such a way as to overcome problems of histocompatibility, is still in its infancy. for a long time, the mouse was the preferred animal model for experimental studies on the differentiation of es cells. in , the first es cells from mouse blastocysts were isolated and successfully cultured by two groups of researchers in great britain and the usa. it was only in that human embryonic stem cells (hes) were isolated for the first time and held in culture, on a nutritive layer of fibroblasts from irradiated mice. this delay with respect to the ability to culture animal es cells can be explained by the fact that the molecular machinery that activates replication and cell differentiation programs is not completely identical in man and the mouse . for example, a cytokine called lif (leukemia inhibitory factor), which is indispensable for the renewal of es cells in an undifferentiated state in the mouse, has no effect on human es cells. there are several other differences concerning the control of proliferation and differentiation in human and murine es cells by growth factors. briefly, the conclusions obtained from experiments carried while there is a highly promising future for the use of es cells, this future is littered with obstacles, and rigorous checks and balances need to be put in place. nevertheless, research on such cells is mandatory if we wish to move on to a regenerative medicine that aims to be a new frontier in the art of healing. after specific differentiation, hes cells could provide unlimited quantities of the tissues needed to replace damaged tissues responsible for handicapping illnesses (dopaminergic neurons in parkinson's disease, cardiomyocytes in myocardial infarction, pancreatic islets of langerhans cells in diabetes, fibroblasts in skin grafts, chondrocytes in rhumatoid arthritis). in addition, metabolic analysis of hes cells carrying defective genes whose phenotypical expression is known in human pathology should improve our understanding of the perturbed mechanisms, and could lead to pharmacological advances. as well as the technical difficulties involved, which have not yet been adequately overcome, the handling of hes cells is subject to much ethical debate in many countries, with those who object to it holding to their prejudices, which are linked to religious or cultural traditions. this is the case in france, where, nevertheless, a few timid dispensations had begun to appear at the time of writing. in contrast, in great britain, the law authorizes the isolation of hes cells for therapeutic purposes, using embryos of less than one hundred cells, produced by in vitro fertilization, and surplus to requirements. the british response to the burning question of whether an isolated hes cell may be considered as a potential human embryo is clearly "no", for, in order to be able to develop in utero, such hes cells would need to have the placental progenitor cells. an alternative to the use of es cells is to make use of adult stem cells. however, the proliferation capacity of adult stem cells is considerably lower than that of their embryonic homologues. the hematopoietic stem cell is the paradigm of the adult stem cell. it can differentiate into all known types of cells. in the last decade of the th century, several publications concerning the plasticity of the adult stem cell awakened a hope that these cells could transform the treatment of degenerative illnesses. certain of these publications stated that adult bone marrow stem cells, implanted into different types of tissues, differentiate into hepatocytes, cardiomyocytes or neurons, depending on the specific environment. careful re-examination of the techniques used revealed that, in certain cases, interpretation of the results as showing cell transdifferentiation was an erroneous one, and that the fusion of the bone marrow stem cells with cells from other tissues was a more plausible explanation. in any case, while not ignoring the use of adult stem cells, experimentation on hes cells remains a judicious choice, given our current state of understanding. in france, the law application decree that was issued on the th of february , revising the restrictive bioethical standards of , opens up the possibility of using human embryonic stem cells for scientific purposes, with certain ethical reserves being maintained. one of the obstacles to the stabilization over time of a stem cell graft in a receiver involves the phenomenon of rejection for reasons of histocompatibility. considered to be foreign by the receiver (host), grafted stem cells coming from a donor are rejected. this obstacle could be overcome by using the technique of cloning. based on experiments on several animal species, it is now accepted that the transfer of the nucleus of an adult somatic cell from a host into an enucleated oocyte makes it possible to obtain from this oocyte, which is once again nucleated, and which is the equivalent of a zygote and able to divide, es cells whose genome is identical to that of the host. because of this, the es cells are immunologically compatible with the tissues of the host. in man, such cells could be directed by differentiation towards stable cell lines creating well-defined tissues and organs (liver, muscle…) that could be used in regenerative medicine. this is the principle of therapeutic cloning. in march , korean veterinary researcher woo suk hwang (b. ) and his co-workers, who were recognized experts in animal cloning, announced in the american review science that they had succeeded for the first time in obtaining around thirty human blastocysts by cloning, i.e., by the transfer of nuclei of somatic cells into enucleated ova. this first experiment involved autologous cloning (ovum nuclei and enucleated somatic cells taken from the same woman). hwang and his team used ova, and the yield from the experiment was close to that obtained at that time for the cloning of mammals. using the inner cell mass of one of the blastocysts, they isolated a line of embryonic stem cells able to maintain a normal karyotype after several dozen divisions. this publication, which appeared in a highly prestigious scientific review, triggered an enthusiasm in the media that was in keeping with the spectacular nature of the team's exploit, tempered here and there by a few comments that were mainly linked to questions of medical ethics. in , there were numerous other articles by the same team on the same subject, reinforcing the first results with a heterologous cloning technique (ovum nuclei and enucleated somatic cells taken from different people), thus giving rise to great hopes that the era of regenerative medicine was near. at the beginning of , professor hwang's retractation of all his work, and a public confession of a spectacular fraud, were even more dramatic, offering certain media an occasion for a disproportionate level of fury against therapeutic cloning. however, despite such rear-guard combats, it is obvious that one day these technical difficulties will be overcome. human cloning, in order to obtain stem cells for therapeutic purposes, cannot escape the future. once this aim has been achieved, it will be spoken of as the outcome of a long story. the adventure of animal reproductive cloning began in . in developmental biology , two american researchers, robert briggs ( - ) and thomas king ( - described experiments involving the transfer of cell nuclei of embryos from a frog (rana pipiens), at the blastula and gastrula stages, into enucleated eggs of the same species. a high percentage of the clones obtained in this way were able to reach the tadpole stage when the transferred nuclei came from the early blastula stage, but only mediocre success was achieved when the nuclei came from the later gastrula stage. these experiments emphasized both the totipotency of the embryo somatic cells and the equivalency of the somatic cell nucleus and the nucleus of the fertilized egg in cell division and differentiation. briggs and king's publication did not arouse any particular interest. it is true that the s were dominated by the saga of molecular biology, which would reach its culmination in the deciphering of the genetic code. from the s onward, the first attempts to clone mammals (rat, mouse, pig) began. moving from the amphibian egg, which was a millimeter wide, to a mammal egg that was one hundred times smaller, presented a technical difficulty that would be overcome by a technique of cell-to-cell electrofusion. cloned embryos were thus obtained by nuclear transfer and then implanted into the uterus of a surrogate female. however, in all cases, the nucleus came from embryo cells. in february , the announcement made by ian wilmut (b. ), keith h. campbell (b. ) and their collaborators at edinburgh's roslin institute of the birth of the cloned lamb dolly had an immediate effect in the media. in fact, this was not only the cloning of a higher mammal, but, above all, the cloning by insertion of an adult somatic cell, in this case a mammary tissue cell, into an enucleated oocyte. this went far further than the experiment carried out by briggs and king, which essentially involved the transfer of embryo cell nuclei into enucleated frog eggs. the trick that gave wilmut and campbell their success was to bring the cells providing the nuclei to a quiescent state corresponding to the interphase stage of the cell cycle, by impoverishing their culture medium, before electrofusion with enucleated oocytes. although we should be aware that attempts were made before a positive result was achieved, this does not make it any less astonishing that the nucleus of a cell in its adult state, i.e., completely differentiated, was able to behave as if it were totipotent. despite being committed to a program of differentiation that is considered to be more-or-less irreversible, and which will give it a specific identity, the nucleus of an adult cell can be reprogrammed and become totipotent. since dolly, many other mammals have been cloned from nuclei of adult cells; mice, cows, goats, pigs, rabbits, cats, dogs, rats and horses. as far as ethical discussion about cloning is concerned (chapter iv. ), it is essential to note that the demarcation line between reproductive cloning and therapeutic cloning is situated where decisions are made concerning the destiny of the cloned blastocyst ( figure iv . ). the transfer of the nucleus of a somatic cell (liver, epidermis, muscle) containing n chromosomes into an enucleated oocyte gives rise to an egg ( n chromosomes) that is able to divide and to produce a blastocyst. the cells of the blastocyst inner cell mass (icm) can be used as stem cells that can differentiate into different types of cell line (therapeutic cloning). on the other hand, if the whole blastocyst is implanted into a uterus, it will produce an embryo which, after birth, will grow into an adult animal (reproductive cloning). reproductive cloning and therapeutic cloning therefore differ because of the fact that in reproductive cloning, the whole blastocyst is used, while in therapeutic cloning, only certain cells, corresponding to the inner cell mass (icm) of the blastocyst, are used. the structural and functional identity of the cells of a given tissue in an adult organism involves a basic mechanism: while each cell has the same set of genes, only some of the genes are expressed as proteins and the genes that are expressed differ according to the tissue involved. the key to the mechanism responsible is in the epigenetic type chemical modifications of cell dna, for example methylations, which repress the expression of certain genes without altering the expression of others. these modifications of the dna, which control cellular specificity (muscle, liver, brain…) are not very reversible, but, in certain circumstances, they can become so. this is what happens from time to time when the nucleus of an adult cell is inserted into an enucleated oocyte. we are thus able to assume that in the molecular arsenal of the oocyte cytoplasm there are substances that can cancel the epigenetic modifications of the dna present in the nucleus of an adult somatic cell and recreate a state of pluripotency in this nucleus, or, in other words, provoke the reprogramming of the somatic cell nucleus. in the long term, it is to be hoped that biochemical technology will be able to find and purify the molecules responsible for the nuclear reprogramming of somatic cells. the use of human oocytes for the purpose of therapeutic cloning is still subject to severe criticism. certain groups wish it to be prohibited, because of a fear of a drift towards reproductive cloning. to obviate this risk, the idea has been to make use not of human oocytes but of those of animals, transferring the nuclei of human somatic cells into them. even supposing that the technical difficulties involved could be overcome, the cells that would result, a sort of man-animal chimera, would also be the subject of an ethical debate, even if the purpose of this type of cloning were to be solely therapeutic. some japanese researchers have succeeded in creating mice according to a parthenogenetic process that involves adding the nucleus of an oocyte that is haploid ( n chromosomes) to another haploid oocyte, the result being the equivalent of a fertilized egg ( n chromosomes). this exploit is achieved by the invalidation of one of the genes (h ) involved in the control of the parental imprint. it is known that sexual reproduction in mammals involves a phenomenon called the parental imprint, which, by means of the methylation of dna and perhaps also of histones, allows the expressing or silencing of certain genes in male and female gametes. a single copy of a given gene, originating either from the oocyte or from the sperm cell, is therefore expressed, while the other is inactive. in the japanese experiment, if the mouse h gene had not been invalidated, the result would have been an anarchical development of the responsible genes involved in the parental imprint with overexpression in the case of some and an absence of expression in the case of others. these disturbances would have been incompatible with the viability of the embryo. however limited its application might be, the manipulation of the germinal genome poses the problem of the mechanism by which the parental imprint intervenes in the viability of the egg, a parameter that at the time of writing is still not completely understood, but is being actively explored. in boston, massachusetts, in , a kidney was transplanted from a healthy boy into his twin brother, who was suffering from a fatal renal anomaly. the success of this graft ushered in the era of transplantations of such organs as the heart, liver and kidney in man. in order to try to prevent the rejection of grafts, caused by an immune incompatibility between the receiver and the graft from the donor, different immunosuppressing treatments were tried, one by one, involving corticosteroids or cytostatic agents such as -mercaptopurine. in the s, a decisive step forward was made with the fortuitous discovery of the powerful immunosuppressive effect of the cyclosporin a, a cyclic polypeptide isolated from the mold tolypocladium inflatum. each year, human organ transplants into patients make it possible to save many lives. however, for some time now, organ transplantation has been suffering from penury of donors. one alternative to the homograft is the grafting of animal organs, or the xenograft, and, at the dawn of the st century, this type of graft has entered an active, promising phase, with the creation of pigs that have been partially "humanized" and are thus, as a consequence, immunocompatible. for reasons of genetic and physiological similarity, the first choice for such grafts was to use apes or monkeys. however, this idea was quickly abandoned, for several reasons; a non-negligible risk of viral infection due to the phylogenetic kinship of human and simian species; a slow growth rate; a low reproduction rate and, finally, laws that protect primates. these disadvantages are not found, or are at least minimized, in the pig: the risk of a viral infection passing from the pig to man should be low because of the species barrier (but nevertheless, it ought to be evaluated), the pig growth rate is relatively rapid, pig litters are large and pig organs are of a size close to those of man. hyperacute rejection of grafts is the critical obstacle that must be overcome before it is possible even to envisage the feasibility of xenotransplantation. hyperacute rejection is caused by the presence in man of natural antibodies (xenoantibodies) that accumulate throughout a lifetime and are directed against antigenic motifs carried by the products of the digestion of food or dust that is breathed in. xenoantibodies are mobilized when a xenograft occurs, and when they combine with xenoantigens brought by the graft this activates immune proteins such as the complement proteins. the catastrophic effect of this xenoantibody/xenoantigen combination is a vascular thrombosis followed by necrosis and rejection of the graft. the pig xenoantigen that is considered to be the one mainly responsible for the phenomenon of rejection in man is a sugar molecule, galactose α- , -galactose, located on the plasma membrane of endothelial cells. synthesis of this molecule requires the enzyme α- , -galactosyltransferase, which is present in most mammals, but absent in man and the primates. this enzyme disappeared in man around twenty million years ago, following a double mutation of the gene. in , cloning by nuclear transfer, associated with the invalidation of the gene coding for galactosyltransferase, made it possible to create pigs without galactose α- , -galactose . this performance shows that the xenotransplantation objective, although it can only be envisaged over the long term, is not based on false hopes. plant gmos, gene therapy, embryonic stem cells, therapeutic cloning, and xenotransplantation are a few of the many examples that show how far experimentation on living beings has progressed in just a few decades, from inquiries into the operating mechanism of an organ or a cell, in the interests of pure understanding, to a programmed process, planned with an objective in mind, the chances of success of this objective being analyzed and counted in terms of impact and cost-effectiveness. during the renaissance, ecclesiastical authorities, worried by the libertarian forces that were assailing them, applied the brakes to audacious questioning of dogma such as the circumterrestrial revolution of the sun that had, since ancient times, placed man at the center of the universe. nowadays, civil authorities, conscious of the potential but also of the possible misuses of genetic manipulation, insist on having the right to oversee such procedures. in truth, since the th century, governments have been interesting themselves in research on living beings and encouraging it, as long as its applications have allowed improvements in human health. this has been the case for vaccinations against infectious diseases or for prevention of microbial infections by means of aseptic or antiseptic methods. with the breakthroughs made in genetic manipulation at the end of the th century, it was more than just the results of experiments on living beings that attracted the attention of the political authorities, it was, above all, the manner in which the experimental method, with all its hazards, made use of living material, sometimes of human origin, in order to unlock mysteries. conscious of the social impact of emerging discoveries that are subject to considerable media coverage and are sensationalized in both the written and audiovisual media, the state, with the help of researchers and philosophers, has laid down a code of bioethics, applied through strict or even restrictive legislation. it remains to be seen whether the rules of this code will continue to be an inviolable absolute or will be modified according to the evolution of the moral codes and the cultures of nations. university teaching and the education of a society must now take into account not only the content of successive discoveries, but also the fallout of these discoveries, insofar as they concern man, and even the ethical justification of the methods that have allowed these discoveries. in "remaking" living beings according to imposed norms, and in scheduling, in a certain fashion, the manufacture of life according to new codes, certain questions move from the "how" to the "why", i.e., from the scientific domain that is accessible to human thought to the metaphysical sphere, with its problems of the limit of what is surmountable and tolerable in terms of ethics. in his birth of predictive medicine, jacques ruffiÉ ( - ) reminds us that medicine has evolved through three stages over the course of time: curative medicine, which has been practiced since ancient times and is still being practiced; preventive medicine, which is more recent, and is designed to prevent people from falling ill, either by vaccinating them, in the case of infectious diseases, or by recommending an appropriate diet and medication in the case of metabolic disorders such as diabetes or arterial hypertension that have been detected by means of systematic examination; and, finally, predictive medicine, a branch of medicine that is still in its early phases, and which is based on modern technology and is able to predict situations of risk because of anomalies detected in the genetic inheritance or because of exposure to environments that are reputed to be dangerous (for example, carcinogenic smoke, asbestos). about one and a half centuries ago, the publication of the introduction to the study of experimental medicine ( ) provided proof, based on scientific arguments, that the time had come to transfer the experience that had been acquired through the experimental method practiced on animal models to the ill person. after claude bernard, attentive to the progress made by ideas and techniques in the physical and chemical sciences, and making use of its own advances in the understanding of the living cell, both normal and pathological, experimental medicine was to live through a development that was without precedent in the history of humanity. to understand the causes of epidemics, nutritional deficiencies, metabolic deviations of hereditary origin and degenerative illnesses, and then to translate these causes in cellular and molecular terms, this was the process undertaken by medicine once it began to use the experimental method. in fact, for several decades, from the beginning of the th century, medicine had already undergone some major revisions of outdated practices and had inaugurated a new era in diagnosis. for example, the differential diagnosis of pulmonary ailments became possible because of the invention of the stethoscope by rené laennec ( - ) and the practice of auscultation and percussion by joseph skoda ( - ), the uncontested master of the vienna school. in france, pierre louis ( - ) used statistical methods to evaluate the efficacy of different treatments. armand trousseau ( - ), a pupil of pierre bretonneau ( - ) wrote a famous treatise on the hôtel-dieu medical clinic in paris. in great britain, chronic nephritis, with its identifying symptoms, was described by richard bright ( - ), paralysis agitans by james parkinson ( - ) and addison's disease, which affects the adrenal glands, by thomas addison ( - ). during the th century, many other famous names signaled the arrival of a medicine that was resolutely anatomoclinical in nature, in line with bernardian doctrine. "experimental medicine is thus a medicine that claims to understand the laws of the organism in sickness and in health, in such a way that it not only predicts phenomena, but also in such a way that it can regulate and modify them, within certain limits." in the introduction to the study of experimental medicine, claude bernard stigmatizes the relics of an empirical medicine that was still being practiced in his day and was forgetful of rationalism. the terms that he uses are without leniency: "i have often heard doctors who, when asked the reason for a diagnosis, reply that they don't know how they recognize such a case, but it is obvious, or who, when asked why they administer certain remedies, reply that they don't really know how to put it exactly, and that anyway they are not required to give a reason, because it is their medical tact and their intuition that guides them. it is easy to understand that doctors who reason in this way are denying science. what is more, it is impossible to be too forceful in rising up against such ideas, which are bad not only because they stifle any scientific seed in the young, but also, above all, because they favor laziness, ignorance and charlatanism." in order to evaluate the meaning of these words, it should be remembered that in claude bernard's time, the medical profession was far from considering the microscope as a useful instrument for the study of cell structures and that the cause and effect relationship between bacterial germs and infections was still to be shown. with the development of increasingly effective instruments for exploration, and of methods for microanalyses concerning a wide range of blood and humoral constants, throughout the th century, medicine, which was once empirical, has now become scientific. claude bernard's dream, experimental medicine, is now operative. this medicine is no longer content simply to determine the cause of an illness and to locate the affected organ, which was the major objective of clinical medicine, but it seeks to detect the mechanisms of pathological processes by means of histological and physicochemical explorations. this medicine is no longer willing to passively monitor the evolution of an infectious disease. after having identified the responsible germ, it tries to target this germ with the chemical weapon that is able to selectively destroy it. this medicine is no longer content simply to find remedies, it aims to understand the mode of action. it sets itself the goal of meeting challenges such as finding the genetic cause of degenerative illnesses or of cancers and developing appropriate therapies. it is supported by statistical data. when a new drug is implemented, the results are now evaluated by the double blind method: neither any of the patients (treated and non-treated) nor any of the investigators are aware of who has been administered with the drug and who has been administered with a placebo. in the surgical domain, audacious techniques have also led to considerable progress, particularly in neurosurgery and in cardiovascular surgery. thanks to robotics and to computer technology, remote surgery or telesurgery has become practicable, although up until not that long ago, it was only to be found in fiction. faced with emerging problems in public health, the task undertaken by experimental medicine is immense. in the middle of the th century, the spectacular recovery from high-incidence infectious diseases such as pneumococcal pneumonia, meningococcal meningitis or acute forms of tuberculosis, which that was brought about by antibiotics, gave rise to the idea that medicine had won a battle against the microbial world and that, from then on, it would be able to control the evolution of infectious diseases and to offer rational treatments. the gradual appearance of a microbial resistance to antibiotics has brought an end to this euphoric era. penicillin, for example, which was put on the market at the end of the s, was active on practically all strains of staphylococcus aureus. sixty years later, more than % of the strains of this same microbe are resistant to penicillin. the incidence of nosocomial infections, which are contracted in health care facilities, never ceases to rise. at present, around % of the hospitalizations that take place are complicated by the patient developing a nosocomial infection. equally worrying are the re-emergence of diseases that were once considered to be under control, such as tuberculosis or poliomyelitis in africa, and the emergence of new diseases such as aids, whose hiv virus (human immunodeficiency virus), which was identified at the beginning of the s, has generated a pandemic that has spread throughout the planet. infectious diseases are currently responsible for more than a quarter of human deaths. the koch bacillus responsible for tuberculosis and the pneumococcus kill three to four million people a year, around the world. in , hiv killed more than three million people, and more than forty million people are infected. one person is infected every seconds. in viral diseases, the role of vectors (insects, various animals) as well as the notions of contagiousness and aggressivity have been emphasized. we have only to remember the dreadful contagiousness and aggressivity of the spanish flu virus (influenzavirus ah n ) which, in - , killed more human beings around the world than the first world war that preceded it. in contrast, the sars (severe acute respiratory syndrome) epidemic of , the vector of which was doubtless the civet, a small carnivore raised in china and desired for its meat, was rapidly contained because of its low contagiousness and also because of the isolation measures that were taken. human behavior is not without its effect on the emergence of viral diseases. the growth in intercontinental travel and human migration, as well as intensive deforestation in africa and south america, which bring virus vectors into contact with man, are factors concerned in the emergence of viral diseases that risk being explosive and devastating. in this context, the history of the ebola virus and of the marburg virus, which cause violent hemorrhaging, is edifying. in , in the german village of marburg, an epidemic of unknown origin broke out, the illness manifesting itself with brutal suddenness by vomiting, diarrhea, a high fever and an increased tendency to bleed. this pathology, which was contained rapidly by means of drastic isolation measures, was found to be of viral origin. the pathogen concerned was a filovirus (filiform virus). a brief enquiry showed that the origin of the epidemic was contact between technicians of a pharmaceutical company and monkeys that had been imported from uganda and that were carrying the virus. in , two other epidemics, characterized by severe and often fatal hemorrhagic fevers, were reported in the sudan and the republic of the congo. here again, the illness was caused by a filovirus, the ebola virus. at the time of writing, only public health organizations, including the nih (national institutes of health) in the usa, have attempted to set up vaccination and therapeutic strategies. research on these dangerous viruses requires high security installations that are particularly costly, so that private companies are reticent about investing in work that is only targeted on poor regions and which concerns epidemics that have so far been contained successfully, although one day the ebola and the marburg virus could quite well escape their african niches. experimental medicine must also understand the colossal challenge of the five thousand hereditary diseases that are currently listed, the most handicapping of which are myopathies and neuropathies. given the means that are available to the contemporary clinician in order to assign each of these diseases to a genetic defect, one can only be amazed by the mass of information about them that has accumulated over a century, since the first diagnosis of a hereditary disease, alcaptonuria, which was made in by archibald garrod ( - ), a doctor at london's st bartholomew's hospital. alcaptonuria is a non-serious genetic flaw that can be detected easily by a blackening of the urine. it is the result of a blockage caused by the mutation of an enzyme involved in the catabolism of an amino acid, tyrosine, this blockage leading to the accumulation of homogentisic acid, the polymerization of which gives rise to a brownish color. the patient examined by garrod was a young boy. investigation of the family history revealed that transmission of the flaw was correlated to cross-cousin marriages and followed mendel's laws for recessive traits. garrod demonstrated other hereditary-type anomalies, cystinuria, porphyria and pentosuria. in , these observations were published in a work that became a classic: inborn errors of metabolism. in , the specific molecular defect of a metabolic anomaly linked to a mutation was identified for the first time by the german-born british biochemist vernon ingram. this was the hemoglobin defect responsible for drepanocytosis or sickle cell anemia: a glutamic acid in the β chain is replaced by a valine. the consequence of this simple change is a modification of the structure of the hemoglobin, leading to a sickle-shaped deformation of the red blood cells, the increased fragility of these cells and also a tendency towards cell lysis. this discovery made use of the electrophoresis and chromatography techniques that had just been introduced in biochemistry (chapter iii- . . ): such a discovery would not have been possible without these techniques. because of the progress made in molecular biology, the nosological framework of hereditary diseases has been greatly enriched over the last twenty years. for example, at present, more than one hundred hereditary-type myopathies have been identified by accurately locating molecular lesions in the genomic dna and characterizing the structural and functional modifications of the mutated proteins. certain health problems present real challenges for experimental research. this is the case for the spongiform encephalopathy caused by a prion (proteinaceous infectious particle) , which has all the more impact on the imagination because its etiology remains a mystery. it is also the case for degenerescence of the central nervous system correlated with aging, alzheimer's disease being a striking example, although, as far as familial forms of this illness are concerned, i.e., those of the hereditary type, it has been possible to link the invasion of the brain by a so-called amyloid peptide, which accumulates in plaques, on the one hand, and, on the other hand, the absence, due to a mutation, of an enzyme, a peptidase, which normally degrades the amyloid peptide. contemporary scientific medicine sometimes acquires a revolutionary aspect. here again, as with other disciplines involved in the study of living beings, it arises from discoveries resulting from the principle of serendipity (chapter iii- . . ). this was the case when, in january , a team in grenoble, france , led by the neurosurgeon alim-louis benabid (b. ) and the neurologist pierre pollack (b. ) discovered by accident that in patients affected by parkinson's disease a beneficial effect was achieved by deep, high-frequency electrical stimulation of the brain. the three major symptoms of parkinson's disease are muscular rigidity, a tremor when at rest and a slowing down of the execution of movements. in the s, the swedish team of arvid carlsson (b. ) , who won the nobel prize for physiology and medicine, demonstrated a relationship between the parkinson syndrome and a deficit in the secretion of a neurotransmitter, dopamine. a group of neurons that is limited to half a million (of the billion contained in the brain) produces this neurotransmitter in a small structure located in the midbrain, called the substantia nigra. the neurons of the substantia nigra have elongations that interact with different nerve formations (called nuclei) including the subthalamic nucleus. in , bergman et al. published an article that describes a curious relationship between a provoked lesion of the subthalamic nucleus and the disappearance of the signs of parkinson's disease in a monkey which had been made parkinsonian by chemical treatment. this publication led the team in grenoble to target their electrical stimulation on the subthalamic nucleus. this was completely successful. this electrical stimulation procedure, which is now well-codified, involves using stereotactic neurosurgical techniques, controlled by magnetic resonance imaging, to implant an electrode into the subthalamic nucleus. the electrode is connected to a generator that is implanted under the patient's clavicle. the generator sends brief electrical impulses of frequencies from to hz. under the effect of this stimulation, the characteristic symptoms of the illness, particularly the static tremor and bradykinesis, regress in a spectacular manner. the mechanism by which this stimulation acts is not yet understood. no doubt this has to do with complex phenomena involving the inhibition of certain neuron relays near to the substantia nigra, which remain to be deciphered. here we have a typical case of a progression from an experimental fact, discovered by accident, towards the analysis of its cause. from the point of view of the experimental method, it is interesting to make parallels between this discovery by serendipitous means of the beneficial role of electrical stimulation of the midbrain in parkinson's disease and cartesian style programmed research that aims to graft into the brain of parkinson's disease sufferers embryonic stem cells differentiated into dopaminergic neurons . civilian society and its armed force, the political authorities, have understood that experimental science has the tools, the method and the thought processes necessary to develop strategies for prevention and healing. immune cells of this person and induces the synthesis of immune proteins. finally, it seems relatively certain that hopes concerning gene therapy for hereditary diseases will be fulfilled within before too long (chapter iv- . . ). one of the traits that is characteristic of the period we live in, and which arises partly from the economic stakes involved, is the shortening of the time that elapses between a discovery being made and the application of that discovery. for example, interfering rnas, which were discovered in the s (chapter iv- . . ) are already the subject of therapeutic investigation. more than a hundred biopharmaceutical companies around the world are using them with a view to producing drugs from them . in mice, a certain number of synthetic interfering rnas have proved their efficacy in silencing genes which, following mutation, have acquired carcinogenic potential. however, the use of interfering rnas as therapeutic agents requires them to be stabilized, because they are fragile molecules. the group headed by achim aigner (b. ), at the school of medicine in marburg, germany, managed to stabilize a synthetic interfering rna by complexing it with polyethyleneimine, and this interfering rna is able to block the expression of a receptor involved in cancerization (c-erbb /neu(her- ) receptor). used in mice, such a drug appears promising. despite the undeniable progress that has been made, experimental medicine is still some way from finding solutions to some of the enigmas that it meets along the way, and which underline the complexity of living beings. some time ago, it was thought that after having invalidated a gene coding for a protein that is indispensable to a function, we would discover the secret of a cause-and-effect relationship. experimental practice has shown that, generally, this is far from being the case. another example of the complex relationships that exist in living beings is the interference of the mental and the organic. one experiment that suggests this interference was carried out on mice who had acquired a form of pathology similar to huntington's chorea, by transgenesis. mice from the same line were separated into two batches, one acting as a control, and the other being subjected to daily mental stimulation, including memorization tests. unexpectedly, the appearance of symptoms was noticeably slowed down in mice who had been subjected to mental gymnastics , as if the brain, by intentionally mobilizing its neuron activity, was able to secrete substances able to alleviate its own defects. in short, by means of possible retroactive mechanisms that are called upon by the mind, the brain appears to act as actor and spectator. at the turn of the st century, experimental medicine was being nourished by techniques inherited from experimental physics, chemistry, and even mathematics and computer technology, in the same way as the other sciences of living beings. the progress made in medical imaging techniques has been particularly impressive since the time, at the end of the th century, when the x-rays discovered by wilhelm rÖntgen made it possible to view the structure of the human skeleton. the saga of x-radiation continued through the th century (chapter iii- . . ). for the last few decades, new imaging techniques have come to the fore. they have spread rapidly, and been refined. ultrasound imaging, which is based on the principle of the reflection of ultrasound waves off of different kinds of surfaces, has become an everyday technique for viewing blood flow in blood vessels and the heart. however, it is mainly in the study of the brain that medical imaging has benefited from technical advances in the domains of physics and computer technology, and it has been innovative in assigning cognitive activities to well-identified anatomical structures. this functional neuroanatomy makes it possible, in a non-trauma-inducing manner, to monitor and locate the operation of neuron networks with great temporal and spatial precision, during various cognitive tasks such as reading and the written or oral expression of thought. the middle of the th century saw the gradual development of two methods for exploring zones of cerebral activity, electroencephalography and magnetoencephalography. at present, these techniques are being taken over by mri (magnetic resonance imaging) ( figure iv. ) . the principle of mri is based on the detection of hydrogen nuclei and their differentiation according to their environment. functional mri leads to the location of the areas of the brain that are active during calculation exercises, the perception of sounds, language and objects, and memorization, with a resolution of just a few millimeters. its power of exploration is such that it has been possible to analyze the brain response, in sleeping or awake babies who are only three months old, to auditory stimuli from language that either makes sense or does not make sense . the response, located in the left hemisphere and the prefrontal cortex, leads to the conclusion that, from the first months of life, there are zones of the brain that are potentially active before the first attempts at language appear. both in france (cea-saclay and the frederic joliot hospital at orsay) and abroad, recent mri performance has encouraged projects concerning the manufacture of instruments able to produce magnetic fields of around ten teslas, which allows an unequaled definition in the identification of areas of the brain assigned to specific cognitive functions and in the highly accurate determination of the location of pathological lesions. a technique that is complementary to mri is positron emission tomography (pet). this generally uses water labeled with oxygen ( o), a radioactive isotope of natural oxygen that has a very short lifetime ( s), produced extemporaneously in a cyclotron by bombardment of an n target with protons. the radiolabeled water is injected into the blood flow of the patient. it is found in greater concentration in the zones that are the most irrigated by blood capillaries. the positrons that it emits collide with the surrounding electrons and give rise to photons that can be detected by the appropriate apparatus. affected by a stimulus (whether this stimulus results from talking, writing or listening), the blood irrigation of the zones of the brain that have been specifically excited increases noticeably. the location of the positron emission provides information about the location of these zones. within a few dozen minutes, it is possible to locate a highly vascularized cerebral tumor. pet can use molecules other than water, such as organic molecules labeled with positron-emitting atoms, ( f) fluorine (half life min) and ( c) carbon (half life min). around twenty years ago, in canada, an analogue of l-dopa, the precursor of dopamine in the brain, f- -l-fluorodopa, was synthesized, and was found to be an excellent probe for determining the capture capability of the endings of the dopaminergic neurons in the striatum. in patients suffering from parkinson's disease, this capture capability is noticeably reduced. at present, pet involving f- -l-fluorodopa is being used to evaluate the survival of dopaminergic cells grafted into the striata of parkinson's disease sufferers , . nowadays, brain imaging techniques can be used to explore the electromagnetic anomalies of neurological or neuropsychiatric illnesses such as huntington's chorea, the different forms of alzheimer's disease or even autism, the genetic origin of which is in the process of being deciphered. a bridge has now been built between the molecular defects identified by genetics and the electromagnetic anomalies that result, analyzed by functional cerebral imaging. it was not so long ago that descartes considered that human thought was unconnected to a material support (chapter ii- . . ). we are not far from the era when broca located the language area in a specific zone of the brain after the autopsy of an aphasic patient (chapter iii- . ), thus opening the door to another scientific domain, neuropsychology, which had previously only been the subject of speculation. the consequences, from the societal point of view, were far from being insignificant. thus autism, which was once suspected of being caused by errors in the mother's behavior with respect to her child, has been shown to be a disturbance in the development of the fetal nervous system, in the temporooccipital region. while the neurosciences occupy a preponderant position in the medicine of the beginning of the st century, because of the development of techniques that aim to analyze even the functions of thought, emerging methodologies of another order, such as gene therapy (chapter iv- . ), are in the process of completely modifying our ways of treating and curing a range of previously incurable human diseases, from incapacitating immune disorders to cardiovascular diseases and cancer. "it is in the domain of thought about the future that man is singled out. we are beings who have an imagination. not content to live in the present, to profit from past experience, we remain haunted by a future that we are conscious of constantly entering. this obsession with the future has been a powerful driving force in cultural evolution. we seek to predict in order to avoid the worst and to better prepare for our tomorrows." by predicting potential dangers in subjects who are in good health, predictive medicine aims to provide the means of avoiding these dangers. these dangers can be intrinsic in nature, being written, for example, into a certain genome dna sequence, or they can be extrinsic in nature, linked to an unsuspectedly deleterious environment. in each generation, mutations occur, certain of which can lead to so-called genetic diseases; between and % of newborns are affected. besides these spontaneous mutations, there are also mutations arising from the genetic inheritance of the parents. the purpose of genetic counseling is to warn parents when the existence of a potentially serious genetic flaw is suspected. the highlighting of genes that give a predisposition for cancer (proto-oncogenes) is a convincing illustration of the power of predictive medicine. this involves genes that control the synthesis of growth factors, the activity of which is essential to embryogenesis and to the repair of damaged tissue. while they are normally subject to strict control by anti-oncogenes, proto-oncogenes are able to become active in an anarchical manner, under different influences, and to transform themselves into cancer-generating oncogenes. recently, mutations have been found in two genes, brca and brca , these mutations giving a predisposition for cancers of the breast and of the ovary. thanks to genetic exploration, it will soon be possible to predict whether a cancer of the breast will have a rapid progression leading to uncontrollable metastases or a slow progression. depending on the case patients will be subject to heavy chemotherapy or to a less aggressive treatment. in this context, targeted therapy with monoclonal antibodies is a source of great hope. while genetic inheritance has a role in cancer, the environment plays a notinsignificant role as well. this is the case, for example, in lung cancer sufferers who smoke tobacco, cancer of oesophagus in those who drink alcohol and job-related cancers in those working in factories producing colorants or materials derived from asbestos or tars. cardiovascular diseases are the primary cause of death in the more developed countries, involving either an infarctus, or a stroke. many risk factors for these diseases are known, i.e., metabolic deviations affecting cholesterol or the blood serum proteins involved in the transport of lipids. these metabolic anomalies result in a syndrome known as atherosclerosis, which is characterized anatomically by the deposit of fats in the form plaques in the arteries. while genetic factors are at the origin of these metabolic problems, the latter are clearly amplified by an inappropriate diet. the role of predictive medicine is to recognize the genes that are responsible, warn individuals of the risks they are running and to advise them about the types of lifestyle and diet that do not increase these risks. being able to predict, predictive medicine should be able to prevent by means of targeted drugs. within this context, it gives rise to reflection upon polymorphism linked to variation in a single nucleotide in the dna of the genome of an individual. known as snp (single nucleotide polymorphism), this polymorphism has proved to be a very useful auxiliary in molecular medicine. hundreds of thousands of snps are present in the human genome and several tens of thousands in genes coding for the proteins. where they are located differs according to ethnic backgrounds. among these snps, some appear to be linked to certain pathologies, such as certain forms of cancer or degenerative illnesses such as alzheimer's disease. in addition, in a small number of patients, the location of certain snps has been connected with previously-inexplicable drug incompatibilities. in line with these observations, pharmacogenomics, a branch of pharmacology that deals directly with genome sequence data, is trying to evaluate the impact of "snp variants" on the efficacy and toxicity of drugs and to understand the genetic bases that explain the differences that are observed in the responses of different individuals to the same medication . rather than using a standard drug that is not very efficacious or causes adverse side effects, it might be possible, depending on the genetic profile of the patient, to use a drug that is more appropriate to his or her genetic map. it is doubtless not just a fantasy to imagine that, in or years' time, a patient visiting the doctor will be offered a genetic map thanks to cells taken from the buccal mucosa. finding snp variants that are known to be responsible for drug incompatibilities in such a map will make a targeted prescription possible. it will allow the detection of genes for susceptibility to an illness, at the same time uncovering targets for new drugs. pharmacogenomics, which is still called new pharmacogenetics, contrasts with old pharmacogenetics in which, having found an adverse clinical response to a certain therapy, an attempt was made to identify the protein target of the incriminated drug, and then to go back to the gene coding for this protein, and to look for the mutation responsible for the aberrant response to the drug. the existence of customized predictive medicine, which would read the destinies of individuals in their genes, would not be without its consequences in the life of a citizen. by registering each citizen with a genetic map, matched with a named identity card, predictive medicine might begin to take on the aspect of a janus, with his beneficent face warning subjects of potential risks of metabolic problems, and guiding them towards the actions to be taken to lower the risks, but also with his evil face delivering each individual's intimate details to the indiscrete inquisitiveness of investigators who are operating towards their own ends (insurance companies, employers…). no less worrying would be the sly but predictable transformation of the individuality of the repaired or even doped human being within a system of imposed, docilely-accepted assistance. in the th and th centuries, the methodology for biological experimentation underwent a revolution caused by the progress made in the domain of chemistry, both analytical chemistry, with the deciphering of increasingly complex molecular structures, and also in synthetic chemistry, with the large-scale production of tens of thousands of new molecules. the effects of these molecules, which might eventually be used as drugs, were tested directly on animals. it was thus that in the german chemist paul ehrlich discovered salvarsan, a derivative of arsenic, which was active against a type of treponeme, the agent of syphilis. this was the result of a systematic analysis of the effect of synthetic products, aromatic derivatives of arsenic acid, on syphilis in rabbits. salvarsan was the th derivative that was tested, and this is why it was called for a long time before it was given the name salvarsan. sometimes, lucky chance shows surprising and unexpected properties in synthetic molecules. this was the case for chlorpromazine, which was initially used as an antihistamine. it was luck that led to its antipsychotic activity being discovered in . a new era opened up in psychiatry with the arrival of synthetic narcoleptics like chlorpromazine. a new chemical science known as combinatory chemistry, which dates from the s, has aroused an increasing amount of interest in pharmacology. this involves making two or more species of organic molecules that carry reactive functional residues react in solution or in the solid phase in such a way as to synthesize, by means of all possible combinations, a number of final and intermediary products that is situated in the hundreds or even the thousands, and which makes up chemical library or drug library. we can directly test all of the products formed on a sample of eukaryotic cells, in order to verify their effects (for example the inhibition of an anarchical proliferation of cancerous cells), or on microorganisms in order to evaluate an antibiotic capability. we can also proceed straight away with the fractioning of the reaction products and the testing of each of the fractions. if the response is positive, fractioning is continued until the molecular species responsible for the desired effect is obtained. other evaluation parameters for this molecule, such as its absorption, its toxicity and its metabolic future (distribution in the organs, chemical modifications and excretion) are then explored, first in cells, and then in animals (rats, mice), thus comprising pre-clinical tests. these screening operations, which are said to be high-throughput, require automation and robotization aided by powerful computer technology. each year, pharmaceutical companies screen tens of thousands of different molecules on hundreds of targets. complementary to combinatory chemistry, in silico chemistry works by molecular modeling and uses computer programs for the rational design of new drugs that are able to fix onto specific protein targets. the purpose of this is to provide a virtual follow up to modifications in the reactivity of a given drug molecule as a function of the modifications imposed on its structure, for example, the addition of residues that differ according to their electrophilic or hydrophilic properties, or according to the length of their side-chain. provided there is a chemical library and we know the three-dimensional structure of a macromolecule, for example an enzyme, as well as the nature of the residues that define its active site, we can hope to select and chemically modify a substance that is able recognize the active site of this enzyme and to make an almost perfect ligand out of it which is able to efficiently block the operation of the target enzyme. this method, which is based on computer-aided chemistry, is called "structure-based drug design", and has had some notable successes. it has made it possible to develop an inhibitor capable of blocking a protease involved in the replication of the aids virus. however, both in combinatory chemistry and in molecular modeling, the many successes that have been achieved remain modest in number compared to the means that have been deployed to achieve them. in terms of statistics, out of ten thousand molecules that are recognized as being efficacious for a given target in vitro, around one hundred are chosen for preclinical trials on animals, around ten are chosen for preclinical trials in man and only one will come out as a drug. the financial and economic effect is far from being negligible. it has even become a preoccupation in a system where merciless competition is the rule. in addition to synthetic chemistry, preparative chemistry, which is based on the isolation of natural molecules, is now the subject of renewed interest, due to the introduction of high-throughput techniques. high-throughput screening, which is an essential tool in combinatory chemistry, is also carried out to ensure the systematic detection and isolation of natural substances having interesting pharmacological activities such as antibiotic activities or anti-cancer activities, based on marine animals, microscopic fungi, prokaryotic organisms and various plants. for example, among the substances that have been isolated recently are cibrostatin, a specific cytostatic of melanoma cells, from a marine sponge, mannopeptimycin, a bacterial antibiotic from an actinobacterium streptomyces hydroscopicus and a whole set of alkaloids with a cytostatic activity with respect to human tumor cells from an exotic plant of the genus daphniphyllum. the molecular diversity of the living world is such that the reserves of natural products having pharmacological activities are far from being exhausted. so far, only a small percentage of the microbial species populating the earth have been listed. the depths of the oceans harbor many unknown species. thousands of insect species remain to be discovered in the canopies of tropical rainforests. exploration of the plant kingdom is far from being complete. the listing of natural molecules having a therapeutic activity has only just begun. the hunt promises to be a fruitful one, all the more so because the highthroughput screening methods that can now be used greatly increase the efficiency of the search. high-throughput screening, applied to natural molecules, has overturned the methodological procedures that were in use until recently, which progress through logical steps, using relatively simple artisanal analytical methods, from observation, often resulting from serendipity, to the isolation of the active substance. thus, in the th century, using inherited traditional knowledge that a decoction of cinchona officinalis bark calms malaria crises, pierre joseph pelletier ( - ) and joseph bienaimé caventou ( - ) decided to isolate the active substance of this bark. from the raw extract, they purified an alkaloid, quinine, which proved to be the anti-malarial substance they were looking for. more recently, the starting point of florey and chain's isolation of penicillin from the microscopic fungus penicillium notatum was the fortuitous observation made by fleming that this penicillium secretes an antibiotic factor (chapter iii- . . ). there are many examples in which serendipity has been the principle factor involved in the discovery of a drug, and this will no doubt continue to be the case. the appearance of a lucky chance, after all, is not incompatible with highthroughput practices. also, it is not impossible that in the future there will be a conjugation of the discovery of new natural substances and the use of combinatory chemistry, with the aim of manufacturing derivatives having a much greater power of action and quality of specificity from these substances . to sum up, the experimental method has caused contemporary medicine to take a giant leap forward, with the discovery of increasingly high-performance functional exploration techniques, the development of therapies using molecules that are already present in nature or are manufactured by synthesis and the more and more advanced understanding of molecular mechanisms that takes into account the basic idea of the pioneers of molecular biology was that the function of a macromolecule depended on its structure. thus, perutz's elucidation of the tetrameric three-dimensional structure of hemoglobin, and of its modifications depending on the degree of oxygenation, shed a considerable amount of light on the cooperative mechanism of the transition from the hemoglobin state to the oxyhemoglobin state (chapter iii- . . ). in the same way, an understanding of the structure of many enzymes, receptors and transporters of metabolites has shed light on their mechanisms. in a parallel manner to the exploration of the structures and functions of proteins, that of genomes has made remarkable progress. the subtle entanglements of genomics and proteomics that have become accessible to the experimental method are the order of the day. one major challenge for post-genomics is to understand how proteins, expressed by genes, interact with one another to generate functions that characterize cellular specificity. even more ambitious are attempts to understand the operation of organs or even of living organisms, based on mechanisms that are implemented at molecular level. these attempts lead straight to an integrated biology, that is, a biology that aims to understand the overall functioning of living beings. taking as its purpose the access to emerging functions, resulting from interactions between macromolecules, integrated biology first tries to invent methods that make it possible to detect these interactions. strengthened by the information obtained, it tries to integrate this information with a mathematized language into modules that attempt to simulate living beings. from the simplistic procedures of the middle of the th century, which were justifiable within the reductionist context of this period, and which involved considering each species of proteins as an autonomous functional entity, we have moved on to the idea that the different species of protein that inhabit a cell have a dialogue with one other, and that they may move from one endocellular organelle to another, depending on post-translational modifications (for example, phosphorylations) that change their conformation and, at the same time, their reactivity and their behavior. thus, an enzyme protein is not only defined by its catalytic performance with respect to a given substrate, but also by its place in a metabolic network where it interacts in a dynamic and transitory manner with a multitude of other protein species (figure iv. a) . the concept of cell signaling has also evolved. instead of considering that a cell membrane receptor, activated by fixation of an extracellular ligand (a hormone, for example), addresses the information received to an endocellular effector protein via a linear cascade of individual proteins, it has come to be postulated that communication between an activated receptor and its effector is mediated by proteins organized into interactive networks ( figure iv. b) . this machinery provides more flexibility in the addressing of messages to effectors. the diagram on the right shows that besides its catalytic function, protein a interacts with other proteins in the cell. b -case of the transduction of a signal that is external to the cell (a hormone, for example). the diagram on the left refers to the classical idea of signaling from a receptor r according to a linear cascade of protein-protein interactions inside the cell, leading to an effector z. the diagram on the right shows that the signal is spread through proteins organized into interactive networks. another subject to be considered is the density of macromolecules of all types, such as proteins, nucleic acids, lipids and polysaccharides, contained by a microorganism or a eukaryotic cell, which reaches values of to g/liter, denoting a semi-solid state or a considerable degree of compacting. however, for technical reasons, kinetic studies carried out in vitro on isolated enzymes have been carried out with solutions that are or times more dilute. conscious of this difference in scale between information obtained from in vitro studies and the in vivo reality, the biology of today is trying to re-evaluate molecular dynamics within the context of a cell. this is why we are seeing the birth of an integrated (or integrative) biology of functions, which, using modeling procedures, aims to achieve an understanding of the temperospatial dynamics of the interactive components inside cells. this holistic conception of biological systems ("systems biology ") has been made possible by progress in technological expertise in domains as varied as biochemistry, molecular biology, physical optics, electronics, nanomechanics, physical and mathematical modeling and computer technology. it is a necessary complement to the classical experimental method based on bernardian determinism which, in order to connect an effect with a cause, explores living beings in a manner that is often monoparametric and is inevitably reductionist. this signals a change in paradigm in the experimental approach to living beings. a particularly effective investigative method used to explore the dialogue between proteins is the double-hybrid by genetic construction, two proteins, p and p , whose interaction is to be tested, are expressed in the form of fusion proteins in yeast. protein p is fused with the binding domain (gal -bd) to the dna of gal , a protein that regulates the transcription of the β-galactosidase gene. protein p is fused with the activation domain of gal (gal -ad). insofar as p interacts with p (b), the gal transcription regulation activity is re-established, which is verified by the transcription of the reporter gene. if the opposite occurs, i.e., in the absence of any interaction between the two domains of gal (a), the reporter gene is not transcribed. the principle of this method is based on the modular nature of numerous transcription factors in eukaryotes. these factors contain both a dna-binding domain that includes a specific dna-binding site and a transcription activation domain that starts up the machinery for transcribing dna into messenger rna. these two domains can be dissociated and then re-associated in a functional manner, by forming hybrids with interacting proteins. a first protein, p , is fused with the dna-binding domain of a transcription factor by genetic manipulation, and a second protein, p , is fused with the activation domain of the same transcription factor. if p is able to interact with p , the transcription factor is reconstituted and the reporter gene upon which it depends can be expressed. the trapping technique, which is complementary to the double-hybrid system, was developed to make it possible to identify a set of interactive proteins within a cell. a protein that is included in this set (protein of interest) is fused by genetic engineering techniques to a short polyhistidine chain (called a tag). using this tag, the protein of interest is fixed to a solid medium containing nickel ions, a material that is reactive with respect to the polyhistidine chain. in the presence of a soluble cell extract, the protein of interest binds the cognate proteins of this extract, making it possible to retrieve a complex whose components, corresponding to interactive proteins, can be resolved after denaturing gel electrophoresis and then characterized ( figure iv . ). the techniques described above are backed up by cell imaging techniques that make use of confocal microscopy, which is more directly in keeping with living reality. the optical performance level of confocal microscopes has improved lately, with the arrival of biphotonic and multiphotonic lasers that illuminate precise points of the cell. as we have seen previously (chapter iii- . . ), it is possible to create a protein chimera made up of a protein of interest fused with a fluorescent protein, in this case gfp (green fluorescent protein), inside a cell. there are currently several variants of gfp that are able to emit fluorescent light at different wavelengths. this has allowed the development of a technique known as fret (fluorescence resonance energy transfer ) which explores the interaction between two fluorescent proteins. in practice, two gfp variants that have neighboring emission spectra are fused, by genetic engineering inside the cell, to two proteins of interest, p and p , that are suspected of being interactive. if this is the case, the fluorochromes that they carry are sufficiently close that the result is a modification in the intensity of the emission fluorescence of the donor fluorochrome (decrease) and of the receiver fluorochrome (increase), which is readily detectable. all of these studies, taken together, have given rise to the idea that endocellular proteins are organized into networks, that these networks are interactive and that their location in defined compartments of the cell is dependent on epigenetic events such as phosphorylations. two attributes can be found in integrated systems: firstly, the presence of modules, i.e., interactive motifs, which, like the pieces of a jigsaw puzzle, fit together to produce a complex, coherent structure, and, secondly, the emergence of functional properties due to the newly created interactions. the protein of interest p is expressed in the form of a protein fused to a protein "tag" t that is able to bind to a solid support with a specific affinity. the assembly is brought into contact with a cell extract. certain proteins of this extract, a, b and c, which are able to interact with the protein p, become fixed to the latter. in a second step, the tag t is freed from its attachment to protein p by means of a specific cleavage enzyme. the pabc complex that is recovered from the solid medium in soluble form is subjected to polyacrylamide gel electrophoresis, in order to separate and identify its components. given an analytical description of the basic building blocks that are used to construct living systems, and an understanding of their modes of association in defined circumstances, it is normal to try to reconstruct, in their entirety, mechanisms that show the functioning of these systems. this idea was first applied to the yeast saccharomyces cerevisiae for different reasons, such as cell homogeneity (in principle, and, in any case, statistically speaking, all yeast cells have the same genome and the same proteome), an in-depth understanding of the genome and the proteome and the presence of a vast directory of well-characterized mutants. the use of techniques for the detection of interactions between proteins has revealed the existence of a potential dialogue of unexpected richness between a multitude of proteins ( figure iv. ) , in the yeast cell. it is necessary to reflect upon this evidence, which leads to the postulate that, for a given protein, there are mechanisms that restrict and select the many partners able to react with it at a precise moment.faced with a situation in which chance has the upper hand, leading to uncontrollable anarchy, it is necessary to have regulation, which is underpinned by darwinian logic. example of an interaction network involving the yeast sup prion protein. the line of dashes refers to experimental data concerning the interaction of sup with another protein, sup . the lines in bold refer to interactions taken from experimental data; while the fine lines refer to predictions, particularly phylogenetic ones. this logic arises from a choice of the most efficient reaction path, which is first of all dictated by the speed constants involved in the association and dissociation of molecular partners, without, however, neglecting any stochastic events that may arise. chemical modifications of proteins participate in this regulation, such as phosphorylation, glycosylation and acylation. the result at cell level is a coherent channeling of the information that is carried from a molecular signal. thus, fixation of a hormone onto a receptor induces a series of modifications to the intracellular proteins that channel information towards an effector terminal, for example an enzyme responsible for the production of a metabolite with a strategic function. how can the sum of the scattered experimental data that we have concerning the catalytic capabilities of a multitude of enzymes of cellular origin be integrated into the operation of a cell? how can we envisage the gene-enzyme relationship according to current evidence concerning the complexity of the genetic message? biocomputing, or bioinformatics, a science that emerged towards the end of the th century, proposes to try to answer these questions. at the turn of the th century, with the development of increasingly powerful computer microprocessors that are able to carry out complex operations with amazing rapidity, the hope arose that it would be possible to simulate processes as varied as the regulation of the cell cycle, molecular flow in metabolic pathways and the reception of molecular signals, for example from hormones by living cells, as well as the transmission of the messages that result. the dream of an in silico virtual biology has become achievable. the first mathematical theory of simple enzyme reaction kinetics was put forward approximately a century ago, by victor henri ( - ). born in marseilles to russian parents, victor henri studied in saint petersburg and then spent time at the universities of göttingen and leipzig before becoming established in paris. having an eclectic mind, studying both psychology and physicochemistry, he had the wonderful intuition that enzyme catalysis arises from a specific mechanism, different from that implemented in a chemical reaction. the study carried out by henri concerned the cleavage of sucrose (table sugar) into fructose and glucose by the action of an enzyme called invertase (sucrase). the term invertase was used because during reaction there was a change in the rotatory power of the sucrose solution, shown by a polarimeter. analysis of the reaction suggested that an enzyme-substrate complex is formed, which breaks down to regenerate the enzyme and liberate the product of the reaction. this analysis gave rise to an equation for the speed of the enzyme reaction as a function of substrate concentration. henri published the results of his experiments both in his thesis, which he presented to the paris faculty of sciences in , and in two articles that appeared in the reports of the academy of sciences , . in , in biochemische zeitschrift (vol. , pp. - ), leonor michaËlis and maud menten ( - ) confirmed the results of victor henri and formulated an equation that became a classic, describing the speed of formation of a product from a substrate in enzyme catalysis. since the period of these first studies, the concepts involved in enzyme kinetics have evolved considerably. the first metabolic pathway be deciphered was that of the degradation of glucose (glycolysis) , either into ethanol in yeast, or into lactic acid in muscle tissue. after this, researchers became aware that the activity of enzymes could be modulated as a function of covalent modifications of amino acid residues of their protein chain (phosphorylation, dephosphorylation, acylation…). metabolic flow analysis led to the idea of the limiting reaction. in the s, the idea that there is a single limiting reaction in a chain or a cycle of reactions gave way to the idea that metabolic control is distributed over several reactions, and that each reaction has its own, more-or-less intense control force. another complexity factor came to light with the discovery of allostery . allosteric enzymes have the particularity that they can fix reversibly onto a site that is different from the active site (allosteric site) molecules that are often the terminal products of a chain of reactions: the consequence of this is a conformational modification of the structure of the enzyme that has repercussions on the geometry of the active site and modifies its reactivity with respect to the substrate. in the s, faced with the complexity of the tangle of listed metabolic and signaling networks, attempts were made to use mathematical modeling to show the progress of the traffic of molecules inside a cell in relatively simple metabolic pathways such as glycolysis. in the modeling procedure, the concentrations of the different molecular species are considered to be variables whose variations over time depend on their speed of production and their speed of disappearance, which leads to a set of paired differential equations. with this procedure, we entered the domain of virtual biology. thanks to the creation of increasingly powerful software, the aim of virtual biology is to simulate signaling and metabolic pathways. in the longer term, the aim is to understand the molecular and cellular processes that direct embryo development, or to test the effects of drugs of known target on the metabolic behavior of the cell. metabolic engineering (which is already well developed) comprises two types of models, stoichiometric models and kinetic models. stoichiometric models describe metabolic networks in the stationary state, based on analytical data. kinetic models combine stoichiometric information and that concerning the catalytic capabilities of the enzymes in a metabolic network. in canada, the cyber-cell project plans to model the overall functioning of the machinery which, in the bacterium, includes its metabolism and its proliferation. the aim of the afcs (alliance for cellular signaling), which was launched in the usa, is to understand how signaling occurs in cells such as the b lymphocyte, the macrophage or the cardiac cell in response to different types of stress. the techniques that are used range from identification of all signaling network proteins to the evaluation of the flow of circulating information and to the integration of the data acquired into theoretical models. the european nerve synapse project makes use of similar procedures, with its long-term hope of linking the functioning of nerve cells with the cognitive and behavioral functions of living beings. this is a sizable challenge. in fact, there is far from being a real consensus concerning the principle of a demarcation between, on the one hand, cognitive functions such as language or memory, which are located in precise zones of the brain, and which could be reduced to physicochemical processes, and, on the other hand, forms reflective thought that are expressed through the creative imagination or by judgements concerning ethics or esthetics, the notion of personal responsibility, or even pictorial, architectural or musical beauty. should we see the human soul as the programmer of a superb computer that never ceases to develop from the embryonic state onwards, like john eccles ( - ) and others, or should we admit, like jean-pierre changeux (b. ), stanislas dehaene (b. ), daniel dennett (b. ) and others, that thought is not transcendent, and that it is intrinsically dependent on the brain, which is considered to be a neurochemical system, and thus look for the secret of the individuation of the human being in brain information storage mechanisms with retrocontrol loops associated with subtle neuron architectures, or, in short, refer to a sort of turing machine? whatever the case, in this domain, as in others, simple animal models are used in order to identify elementary processes that are able to explain easily-tested functions such as the memory in anatomical and physiological terms. this is the case for the sea slug or sea hare (chapter iii- . ) which, despite its rudimentary cognitive capabilities, provides information that can be used to reconstruct higher cognitive functions, present in the brains of mammals. it is clear that the cognitive sciences have reached a stage in which they are emerging from their infancy (chapter iv- . ). now, ingenious computing methods and a basis for reflection that has spread beyond the confines of psychology and philosophy, are available to them. they have set themselves the goal of producing an artificial intelligence, using ultrarapid computers as well as software that is able to model the operation of neural networks and to come close to the performance of human intelligence in terms of the power of their reactivity and their memorization. at present, many other biological systems are being subjected to multiparametric exploration, with the aim of producing models. this is the case, for example, with the program of the differentiation of certain white blood cells, the neutrophils (chapter iii- . . ), from precursors located in the bone marrow, a differentiation that leads to the emergence of functions such as phagocytosis that are implemented in the fight against microbial infections . in a domain that is closer to mechanical science, hydrodynamics, the digital simulation of the cardiovascular system has already made it possible to represent the physical phenomena associated with the propagation of a wave in deformable arteries during a cardiac contraction, in the form of equations, with a good approximation . in short, from a monoparametric approach that often began as being essentially and necessarily reductionist, the experimental method, applied to living beings, has become a "globalized", or synthetic, multiparametric approach, the aim of which is to understand the dynamics of molecular interactions in defined biological systems. making use of data obtained, the hope is to use mathematical processing to simulate the overall functioning of a cell, organ or organism. this new paradigm of the experimental method ("systems biology" ) is not limited to a simple accumulation of observations concerning a given biological system and their abstraction in mathematical form. the originality of this approach is that it formulates predictions of changes in the behavior of a system as a function of the manipulation of parameters such as substrate concentration, the presence of inhibitors, and so on. the mathematical processing of experimental data, with a view to learning about the functioning of complex systems by modeling, is supported by the technosciences, particularly biocomputing. it is linked not only to the enormous sum of accumulated knowledge concerning the structures and functions of living beings in the post-genomic era, and to the notion that the life of a cell depends on multiple networks of molecular interactions and thousands of enzyme reactions located in its different organelles, but also to the information that comes to it from its environment. a first type of modeling is based on observations made or experiments carried out on an easy-to-study model system. laws are drawn up from this. this so-called "bottom-up" (or synthetic) procedure, which proceeds from the simple to the complex, makes it necessary to have a set of very precise biochemical data. this great precision is all the more imperative in that any deviation, even a minimal one, in the integration of an experimental result can generate a mathematical model that is apparently plausible but which is unconnected to the living reality. the reverse, "top-down" (analytic) procedure proceeds from the overall operation of an organ and its theoretical analysis towards the specific mechanisms of its components. it takes into account the functioning of complex integrated systems such as the nerve and endocrine systems, immune and reproduction systems and the system controlling homeostasis, descending in stages towards the cellular, molecular and genetic levels. in the end, an understanding of living beings involves the management of an amazing capital of experimental data. this makes it necessary to consider all of the genes (genome), all of the transcripts coding for the proteins (transcriptome), all non-coding rnas (rnaome), all proteins expressed in a particular cell type (proteome) and all metabolites (metabolome) as a function of the enzyme catalyzers expressed and the energetics that underlie the catalyzed reactions, and, finally, to connect upstream events (mutations of genes and interference of messenger rnas, chemical modifications to amino acid residues in the proteins) to phenotypical modifications on the scale of the whole organism (phenome) (figure iv. ) . the goal of integrated or integrative biology ("systems biology") is therefore to put living beings into equations, that is, to represent them in virtual systems for which the behavior, accessible by means of calculation, can be predicted as a function of modifying parameters. in addition to the possibilities that are opened up in terms of a deeper understanding of physiological mechanisms, such virtual systems could be used for the design of new drugs or for the manufacture of economically valuable biomolecules. the diagram illustrates the different levels of complexity in the pathway that goes from all the genes together (genome) to all of the expressed characteristics (phenome) in the living being, passing via coding rnas (transcriptome) and non-coding rnas (non-coding rnaome), all the proteins (proteome), all addressing systems in the cell compartments (localisome) and all of the metabolic pathways (metabolome). at a scientific meeting held in sheffield, england, in january of , with the theme systems biology: will it work?, an argumentative discussion of the advantages as well as the disadvantages of an integrated, mathematized biology was useful in that it included a reminder that most of the parameters used in "systems biology" come from studies that are carried out in vitro on purified enzymes, and that it is not sufficient to know the value of the michaelian parameters (v max and k m ) in order to reach biological reality. in fact, in vivo, many enzymes record variations in activity that are hard to control due to allosteric type regulation or interenzyme contact; several enzymes of a metabolic pathway being able to interact to form a metabolon. however, by compacting several enzymes that catalyze contiguous reactions in a metabolic pathway, a metabolon considerably increases the catalytic efficiency of this pathway. another element of uncertainty arises from the protein density of the cell medium, and also from the fact that covalent modifications of enzymes can introduce a change in endocellular location (nucleus, organelles of the cytoplasm…). nevertheless, an approximative approach could limit itself to dealing with biological systems in modular terms, i.e., to considering them as being made up of a number of black boxes, each black box containing a series of reactions being processed mathematically together, with an input and an output. there is still a long way to go if we place ourselves on the cellular scale, but the end of the pathway seems even further away if we envisage the organism as a whole, taking into account the remote interactions between organs involving the interplay of chemical mediators. the brain plays a critical role in the dialogue between different organs, and in the regulation of the energy equilibrium in higher animals. this equilibrium can be disturbed by fasting or intense, prolonged muscular activity, or by an overabundant diet. the corrective response comes from a deep region of the brain, the hypothalamus, via the secretion of different types of peptides, some of which stimulate the appetite and others of which suppress it . while taking into account the multitude of parameters that affect the complexity of living beings on an individual level, the theoretical approach to the study of cell function by modeling has the advantage that it produces predictions and provides information about the validity of conclusions and of theories based on experiments that are old and accepted in the absence of contradictory elements. this was the case for the theory that stated that the state of activation of a gene is determined only by the presence in its environment of transcription factors. recent studies concerning the level of gene transcription in isolated cells have shown that there are probabilistic-type factors which mean that a given gene in a given cell can be activated at any moment. a review which came out in sums up this subject. in this review, the authors use modeling to analyze the behavior of cells in the process of differentiation during embryogenesis. their darwinian model, which associates contingency and selectivity, competes advantageously with the determinist (or instructive) model, based on an all-or-nothing logic, that has been implicitly accepted up until now. the darwinian model takes into account the occurrence of stochastic events at gene expression level, events that are partially linked to the structural modifications to the chromatin that depend on covalent modifications of an epigenetic nature (phosphorylation, methylation…). by basing itself on the existence of mutational fluctuations that arise by chance, associated with a selfregulation of gene expression, the model that is obtained shows that during embryogenesis a cell has a choice either to differentiate into another cell type or to remain in its initial state. differentiated cells stabilize their own phenotype and, in their surroundings, stimulate the proliferation of foreign cell phenotypes. a harmonious equilibrium between these two processes is the necessary condition for the setting up of the steps that lead to the arrangement of different cell types during organogenesis, which take place in an apparently inescapable order, in the absence of disturbances. a break in this equilibrium leads to an anarchical cell proliferation. generally speaking, from the point of view of experimental science, the lesson that can be drawn from current modeling experiments is that the bernardian determinism that has prevailed as the essential foundation stone of the methodology applied to the study of living beings may find itself being requalified by the taking into account of stochastic phenomena. this is the case when the number of reacting molecules is low and the probability of stochastic events is non negligible. the modeling of such systems necessitates having recourse to a complex mathematical formalism. it remains true that determinist models for simulation of the dynamics of living beings, represented by classical differential equations, are more-or-less valid when the number of reacting molecules involved is high and the reactions supposedly take place in a homogeneous medium. should "systems biology" be regarded as a resurgence of a physiology that has been somewhat neglected over the last few decades, but has been reinvigorated by a salutary hybridization of biologists and model-makers? in any case, this is the intention of the "physiome" project which has recently been launched on an international scale. it is also doubtless due to this state of mind that a trend which had gone out of fashion, involving the simulation of the performance of living beings by very elaborate concrete models, robots, is being reborn. an immense distance has been covered in just over two centuries, since the time when vaucanson presented automata in the forms of human figures, moved by ingenious springs and cogs, and giving the illusion that their movements were controlled by an intelligence, to a marveling public (chapter ii- . ). in the last decades of the th century, considerable progress was made in the understanding of the operation of the nervous system and in the development of technologies in which miniaturized electronics have come to the aid of already high-performance micromechanics. the brain being considered as an information processing machine, the aim is to understand the logic of this information machine by means of simulations on computers and, based on the results obtained, to construct robots whose electrical circuits take their inspiration from the operation of animal neurons. these robots are called biorobots or animats. insects have been chosen as a reference for the construction of such creatures because of the relative simplicity of their nervous systems: several hundred thousand neurons, in comparison with the billions of neurons present in mammals ( billion in man). the fly's system of vision has been favored as a subject of study because of the possibility it offers of being able to record the electrical response of neurons that can be identified one by one. in the middle of the s, in france, this inspired the pioneering work in biorobotics carried out by nicolas franceschini (b. ) and his team , (figure iv. ) . their objective was to study how an animal can avoid obstacles by means of its ocular perception and its movement-detecting neurons, the operation of which the team just analyzed using microelectrodes and a microscope-telescope specially built for the purpose. the fly's composite eye has elementary units or ommatidia, each carrying eight light receptor neurons. the electrical signals emitted by these neurons in response to captured light (at most a few dozen millivolts) are sent to subjacent neurons that are organized into three levels that correspond to the optical ganglions called the "lamina", "medulla" and "lobula". the lobula is a strategic decoding center which, because of the small number of neurons contained in it (sixty), has been the subject of in-depth electrophysiological investigation. each of the sixty neurons of the lobula operates as a signal integrator. the neurons of the lobula send their messages to motor neurons involved in the contraction of small muscles that control the guidance and stabilization of the fly's flight. based on an exhaustive study of the neuron wiring of the fly's eye, franceschini and his colleagues were able to reconstruct a facetted artificial eye that can retranscribe the light signals received optoelectronically. this artificial eye, the electronic components of which correspond to around one hundred movement detectors in the fly, was incorporated into the head of a robot. the recorded light signals were transmitted to the moving components of the robot. a -head of the blowfly, calliphora, seen from the front, showing the two compound eyes with their multifacetted array. each eye hides , photoreceptors that drive various image processors based on a few hundred thousand neurons. b -"elementary motion detector" (emd) neuron and its evolution over fifteen years: on the left, first generation ( ), using surface mounted device (smd) technology, compared to a one franc coin from that period; on the right, the version of the highly-miniaturized hybrid (analog + digital) emd circuit (mass . grams), compared with a one euro coin. c -autonomous vehicle ( kg) able to move around in a field of obstacles that it does not know about in advance. its vision is based on a genuine compound eye, whose circuits are inspired by those of the fly. it includes a network of "motion detecting neurons", transcribed electronically according to the principle analyzed in the fly's eye by means of microelectrodes and a specially-constructed microscope-telescope. this network is arranged around a ring that is about thirty centimeters in diameter. the recently-constructed roboflies, oscar and octave, only weigh around one hundred grams. d -routing of the electronic components (resistances, condensers, diodes and amplifiers that operate in their thousands) soldered onto the six-layer printed circuit-board that provides the connection between the sensors and the steering motor on board the autonomous mobile robot shown in (c). figure iv . illustrates the neuromimetic biorobofly constructed according to this principle. completely autonomous because of its on-board power supply, this robot was able to move around at high speed ( cm/s) in a cluttered area, avoiding the obstacles. this first "terrestrial" robofly, which was completed in , was followed by several much lighter brothers and sisters: fania, oscar and octave are aerial roboflys . constructed in , oscar is a captive robot that weighs around one hundred grams. it is equipped with an eye that reproduces the retinal microscanning of the fly's eye discovered by franceschini, oscar is able to rotate around a vertical axis because of its two diametrically opposed helices and can thus orient its view towards an object. if this object moves, oscar follows it with its eye, up to an angular speed comparable to the tracking speed of the human eye. produced in , octave is another aerial robofly that is able not only to take off and distinguish a relief, but also to land automatically and to react sensibly to a contrary wind in a turbulent atmosphere. on board, it has an electronic visuomotive self-regulation system, the operation of which is based on the signal processing operations that, in the insect, carry out the automatic pilot functions . the age of biorobotics, in which robots take their inspiration from animals, has only just begun . if specimens are still so rare, this is because behaviors for which we have a good understanding of the underlying neuronal bases are also rare. at the time of writing, a robot rat named psikharpax, with artificial muscles and a vision system that enables it to perceive objects in three-dimensional space, is being developed at the university of paris vi. almost in the realm of science fiction, we find hybrid robots obtained by hybridization of the living and the non-living. this is the case for the hybrid robot produced by japanese researchers, based on the silkworm moth. control of the nervous system of this insect is spread throughout its body. if its head is cut off, it continues to fly, which gave rise to the idea of replacing the head with an electronic transistor system . using a remote measurement device, it was possible to explore certain behavioral aspects of the insect. although the construction of hybrid robots may raise ethical objections, such technology is capable of giving rise to spectacular applications in the domain of prostheses. the neurological prostheses of the future will nevertheless require that a contact be made between living neurons and the electronic chips that are able to improve the inadequate processing of the physio- logical signal. such a contact was produced recently in a german laboratory directed by peter fromherz (b. ) . a small network of snail neurons, chosen because of their large size, was cultured on the surface of a silicon chip. a signal emitted at one location of the chip was able to be transmitted to another location via the synapse connection between two neurons ( figure iv. ) . on the molecular scale, mitochondrial atpase or atp synthase, with a size of around ten nanometers (chapter iii- . . ) was used recently for the manufacture of a biorobot that made its mark in the media as the smallest known rotating molecular motor. the membrane-type enzyme catalyzes the reversible reaction atp + h o adp + pi. this enzyme therefore has a double function; hydrolysis and synthesis. for this reason it is called atpase or atp synthase depending on the physiological context in which it is involved. in the mitochondria that oxidize metabolites, the enzyme operates like atp synthase. it catalyzes the synthesis of atp coupled to oxidation reactions. in the absence of respiration or of oxidizable substrates, the enzyme operates like atpase; it catalyzes the hydrolysis of atp. for ease of language, the enzyme will be designated here by the term atpase. it should be remembered that mitochondrial atpase includes two sectors, a hydrophobic sector, fo, characterized as a proton channel located inside the mitochondrial membrane, and a hydrophilic sector, f , carrying catalytic subunits that are arranged as if they were on a turret (see figure iii . c). fo contains two master parts of the atpase motor, i.e., a rotor comprising an assembly of around ten so-called "c" subunits and a stator that corresponds to the "a" subunit. the "c" subunit assembly is attached to the "γ" subunit of the catalytic sector f , which thus functions as a rotor. in , the british biochemist peter mitchell ( - ) showed that phosphorylative oxidation in the mitochondria is associated with a transmembrane transfer of protons. the mechanism involved is said to be chemiosmotic. the most important experiment involved an almost serendipitous observation, carried out with a simple ph meter. when a current of oxygen was passed through a suspension of mitochondria in an unbuffered saline medium, in the absence of adp and phosphate, an instantaneous acidification of the extramitochondrial medium occurred, shown by means of the ph meter electrode immersed in this medium. it was concluded that the sudden switch from anaerobiosis to aerobiosis, i.e., the start up of respiration, is correlated with an ejection of protons from the mitochondrial matrix to the extramitochondrial medium. afterwards, this fact was linked with several others, the whole leading to the formulation of the chemiosmotic theory. briefly, mitochondrial respiration generates a vectorial movement of protons from the interior to the exterior of the mitochondrion. because of this, a proton concentration difference is established on either side of the mitochondrial membrane. the electrical potential that is created in this way is used by the mitochondrial atpase in order to synthesize atp from adp and mineral phosphate. this process involves two correlated events: return movement of protons towards the inside of the mitochondrion across the fo sector of the atpase; rotation of the assembly of c subunits and the γ subunit that is interdependent with it. we have therefore moved from electrical to mechanical energy. during its rotational movement, the γ subunit establishes contacts with the three catalytic subunits of the f sector, in succession. one after the other, each of the three catalytic subunits in contact with the γ subunit undergoes a change in the conformation of its active site, which is at the origin of the synthesis of atp. in the absence of mitochondrial respiration, the reverse process occurs. the atp is hydrolyzed into adp and mineral phosphate, and the energy released at each of the three catalytic subunits is used to rotate the γ subunit in the reverse direction to that which accompanies the synthesis of atp. the existence of a rotational movement of mitochondrial atpase, which had been suggested on the basis of biochemical arguments and of structural data was authenticated by masasuke yoshida and his co-workers in japan in , thanks to an imaging technique . in a first step, the molecular system was simplified by being limited to the catalytic f sector of the enzyme. a methodological trick was employed: genetic engineering was used to modify the α and β subunits of this sector by fixing polyhistidine chains to them. because of the strong affinity between polyhistidine and nickel ions, the f sector α and β subunits were immobilized on a medium covered with nickel ions (carried by an organic molecule). an actin filament labeled with a fluorescent ligand was attached to the end of the f sector γ subunit. this assembly made it possible, under a fluorescence microscope, to display a rotational movement of the actin arm carried by the g subunit affected by the addition of atp and by its hydrolysis into adp and mineral phosphate. a similar rotational movement of the γ subunit carrying a metal microbar was observed by an american research group . remarkably, in , after having fixed a metal microbead onto the γ subunit, the japanese researchers demonstrated synthesis of atp from adp and mineral phosphate by rotating the γ subunit by means of rotation of the magnetic bead, induced by magnets. thus, the experimental coupling of a mechanical force and a chemical synthesis was demonstrated. in , the japanese research team succeeded in photographing the rotational movement of the enzyme powered by atp under the microscope, this time looking at the entire atpase complex, f fo. after having attached a gold microbead onto the "c" subunits of the fo sector, to act as a probe, the researchers were able to confirm that the rotational movement of these subunits depended on the hydrolysis of atp into adp and phosphate ( figure iv. ) . the whole of the mitochondrial atpase (atp synthase) does, in fact, function as a molecular rotational motor powered by a proton flow, rather like an industrial rotational motor powered by a fossil fuel or electricity. the analogy is a striking one; the γ subunit of the enzyme corresponds to the motor driveshaft and the "c" subunits correspond to the motor itself. because of its association with non-living structures, for example metal bars or gold beads, which are carried along in the rotational movement of the enzyme, it is possible to speak of molecular biorobots. this domain, in which nanomachines use macromolecules from the living world, has only just opened up, but its future is full of promise. the story of scientific progress made with respect to the mechanisms of phosphorylative oxidation via the functioning of mitochondrial atpase, from the time of mitchell's experiment with the ph meter until the time of the manufacture of yoshida's biorobots, is an exemplary one. it is typical of the way in which a mode of thought evolves over time, from a primary discovery resulting from serendipity or an experiment "to see what happens", leading to the proposal of the existence of a mechanism, to a carefully programmed project which, because of its inventive technicity, shows the validity of the proposed mechanism, and, in addition, demonstrates its future utilitarian value. nowadays, certain biotechnologists dream of being able to "synthesize life" in terms of cells that are able to imitate the performance of living cells. the concept of the "lab-in-a-cell " is coming to the fore , . nevertheless, it would be necessary to design an artificial cell that is an authentic replica of a living cell, and which benefits from all the attributes of a living cell. this is not achievable at the moment. thus, the current aim of nanobiotechnology is limited to scheduling the construction of artificial cells that are relatively simple both in composition and in function, for example, a microvesicle edged with a lipid membrane, containing a system of protein synthesis expressed from a short sequence of dna, as well as a system of atp synthesis able to supply the energy necessary for this protein synthesis. demonstration of a rotational movement of f fo mitochondrial atpase (atp synthase) , induced by atp. atpase or atp synthase (reversible catalysis enzyme that hydrolyzes or synthesizes atp) has two sectors (see figure iii . ). the membrane sector, fo, comprises an assembly of a dozen so-called c (rotor) subunits and an a (stator) subunit. the other, extra-membrane sector, f , is catalytic. it comprises three β catalytic subunits and three α non-catalytic subunits arranged in a ring, in alternating order. at the center of the ring is the γ subunit which is attached to the c subunits of the fo sector. subunits δ, ε and b stabilize the whole of the molecular complex. in the experiment illustrated in this figure, subunits α and β of the f sector of the enzyme have been genetically modified to include polyhistidine chains (his-tag, artificial ligand). due to the interaction of these chains with nickel ions (linked to an organic molecule) covering a solid support medium, the α and β subunits are immobilized. in addition, a gold microbead is fixed onto the ring of the fo sector c subunits by means of a chemical device (streptavidin molecule, artificial ligand). following the addition of atp, rotation of the microbead attached to the ring of fo sector c subunits is observed by microscopy on a black background. this rotation is dependent on (and at the same time an indicator of) the rotation of the c subunits, itself led by the rotation of the γ subunit in contact with the catalytic β subunits. note the ejection of protons. when the enzyme functions as atp synthase, the proton movement takes place in the opposite direction. "progress in biology is possibly mainly tributary to the drawing up of concepts or principles […] . in the process of elaborating concepts, which marks scientific progress in biology, there is sometimes a crucial step, when we realise that a more-or-less technical term that we had previously considered to cover a given concept, in fact covers a mixture of two (or more) concepts." ernst mayr translated from a french translation entitled history of biology. diversity, evolution and heredity - on the margins of the modeling and the difference in mathematized systems that comprise theoretical biology, particularly in silico biology, concepts are mental representations, often image-filled and idealized ones, of fundamental mechanisms that are deduced on the basis of experimental results. from the imaginary domain of the probable, they extrapolate constructions of the mind that are in phase with the facts and experimental data, within a reflective projection that gives them their meaning and makes it possible to make certain predictions. there are premonitory concepts. this was the case for the concept of the reflex arc that associates movement with sensation. this concept was already present in the ideas of descartes (chapter ii- . ), but it took more than a century before the theory of the existence of a reflex arc was supported by bell and magendie's demonstration of the existence of relay centers for sensory and motor nerves in the spinal chord (chapter iii- ). there have been premonitory concepts that, while they were demolished at the time they were first proposed, were shown to be completely accurate a few decades later. in the middle of the th century, the german pathologist jacob henle ( - ) needed a healthy dose of imagination and audacity in order to oppose the theory of the "miasma", a theory that was taught as a dogma, with a new theory that not only explained the spread of contagious diseases by microscopic beings, but also formulated the criteria for validating this theory, i.e., isolation of the pathogenic agent and its development in culture away from the diseased organism, then reproduction of the original pathology after injection of the pathogenic agent, which has been isolated, characterized and multiplied in a culture, into a model animal. thirty years would go by before the formulation of koch's postulates, based on experimental evidence (chapter iii- ). we may ask ourselves whether or not history is currently repeating itself in the case of spongiform encephalopathies that affect humans and animals, for which, according to the thesis of stanley prusiner (b. ) , the prion, as an infectious protein, is responsible. other evocative concepts hold the keys that open doors to domains that are unknown, but are potentially rich in information. it is thus that the double helix dna structure proposed by crick and watson, based on the complementarity of adenine-thymine and cytosine-guanine bases (chapter iv- . . ), led to the concept of dna replication with reconstruction of a double strand that is identical to the original double strand. the concept of dna replication spurred on matthew meselson (b. ) and franklin stahl (b. ) to develop an experimental protocol based on the labeling of the dna nucleotide bases of the enterobacterium e. coli with a heavy isotope of nitrogen, n, and on the differentiation of monocatenary dna strands in the process of synthesis by measurement of their density, as analyzed by centrifugation in cesium chloride gradients. in the same vein, jacob and monod's discovery of regulatory genes (chapter iv- . . ) gave rise to the concept of the operon which, in the bacterium, defines a genetic unit comprising structural genes and regulatory genes. the concept of the regulation of gene expression, extended to higher eukaryotes, makes it possible to explain the phenomenon of differentiation in cells with specific activities (muscle cells, nerve cells, epithelial cells…) by the silencing of certain genes and the activation of others. within the framework of bioenergetics, the chemiosmotic theory put forward by mitchell, in order to explain the coupling of mitochondrial respiration with atp synthesis (chapter iv- . ), gave rise to consideration of the concepts of transmembrane transport of metabolites and of vectorial metabolism. some generalizing concepts that carry a unifying virtue within them are known. one such is the concept of compartmentation. the cell is no longer considered to be a bag of enzymes, as used to be the case. it is now considered to be a compartmented structure in which each type of compartment corresponds to a type of organelle delimited by a membrane and characterized by specific functions. thus, because of the genetic material that is present in it, the nucleus of the cell holds the information necessary for the manufacture of proteins. the mitochondria, which are called cell power plants, are in charge of oxidizing the products of cell catabolism and using the resulting energy for the synthesis of atp from adp and mineral phosphate. the lysosomes are the garbage collectors of the cell. among the functions carried out by peroxisomes is the partial breakdown of very long chain fatty acids. the endoplasmic reticulum and the golgi apparatus are involved in the maturation and the secretion of proteins. the ribosomes represent the machinery upon which messenger rnas are displayed in order to be decoded into proteins. a sign of the extreme sophistication of this setup is that the membranes of the endocellular compartments are not sealed common walls. they contain proteins that act as selective transporters of metabolites or highly specific ion channels, allowing the exchange of messages throughout the cell. thus, each organelle, informed of the condition of the others, is able to adjust its own activity to ensure the greatest harmony of the whole. this conditioned compartmentation at cell level may be compared to the socialization of human communities. while endocellular organelles are compartments delimited by membranes, there are non-membrane-bound compartments in the cell, such as protein complexes in which two, three or even more proteins are closely linked. often, these are enzymes that catalyze reactions that are contiguous in a metabolic pathway. being compacted into a complex known as a metabolon results in an increased efficiency of the flow of metabolites by facilitating the channeling of this flow. concepts evolve, often adjusting their representations according to accumulated knowledge. a good example of this is the evolution of the concept of the gene since its formulation at the beginning of the th century. the term "genetics" was created in by the english naturalist william bateson ( - ) . the term "gene" was introduced three years later by the dane wilhelm johannsen. this term designated a principle which, in the chromosomes of fertilized egg, and in an intentionally vague manner, was supposed to have an influence on the phenotype of the progeniture. during the same period, the term "locus" appeared out of the experiments carried out by the american thomas hunt morgan on the drosophila, a locus being defined as a region of a chromosome which, when altered by a mutation, leads to a modification of the phenotype of the living organism. based on cross-breeding experiments carried out on hundreds of drosophila mutants, morgan and his co-workers drew up the first genetic maps. by chance, the salivary glands of the drosophila have a particular characteristic; the nuclei of their cells contain giant chromosomes called polytenes, which result from the association of a hundred replicate copies of chromosomes that, after staining, are visible under the optical microscope. on these chromosomes, it is possible to distinguish colored bands separated by clear bands. it was observed that specific mutations had specific effects on the arrangement and number of these bands. the material contained in the bands was therefore the site of mutations. in the middle of the s, the listing of more than bands made it possible to construct a cytological map that was already highly detailed. the concept of the gene, the material basis of inheritance, took root. the sporadic mutagenic effect of x-radiation in the drosophila, which was shown by the geneticist and biophysicist hermann mÜller ( - ), led the austrian physicist erwin schrÖdinger to question the sporadic event which, at the level of a target of a few dozen atoms, determines a mutation. he postulated that the target is located in the chromatin of the chromosomes, organized as an aperiodic crystal. the chemical nature of this target was identified with dna, following bacterial transformation experiments (avery, macleod and mccarthy, ) and experiments concerning bacterial infection by the bacteriophage (hershey and chase, ) (chapter iv- . . ). this is how the idea that gene = dna was born. the simple and reassuring idea that one gene → one enzyme, which was deduced from mutation experiments carried out by beadle and tatum on the mold neurospora crassa (chapter iii- . ), had only a limited lifetime. a first stumbling block appeared when it was shown that the activity of a gene, and in consequence its contribution to the phenotype, depends on nucleic elements outside the gene. the definition of the term "gene" was then extended to include promoting and regulatory sequences. in the case of the lac operon of escherichia coli, these sequences are located just upstream of the site where transcription begins. however, in eukaryotes, a regulatory sequence may be distant from the gene that must be transcribed and sometimes it may be involved in the regulation of several genes (chapter iv- . . ). in the s, the idea of the existence of the mosaic gene in eukaryotes appeared. a gene was now thought of as an assembly of several exons that originally in the chromosome are separated by introns. the alternative splicing of these pieces of genes gives rise to numerous possibilities for reconstitution, i.e., many messages coding for many different proteins. thus, however useful the concept of the gene has been with respect to its ability to generate discussion and to provoke experimentation concerning the molecular machinery responsible for the transmission of the hereditary characteristics, we can see that the term itself has not ceased to be the subject of readjustments, since the time it was first formulated. certain concepts are matched with metaphors. while some metaphorical concepts, particularly those that make use of images designed to grab the imagination, and to be easy to understand, tend to take liberties with the realities of living beings, they can also shed light on unsuspected mechanisms in sectors that have been neglected. metaphorical concepts are not a current fashion. it should be remembered that in his passions of the soul ( ), descartes, when asked "how limbs can be moved by objects of the senses and by the mind without the help of the soul," responds that this takes place "in the same way as the movement of a watch is produced only by the force of its spring and the arrangement of its cogs." later on, with lavoisier's clear vision of the vital role of oxygen, and his comparison of respiration with combustion, the concept of the chemistry of life, combined with that of bioenergetics, came to the fore, and was at the heart of studies on the metabolism. chemical reactions that liberate and absorb heat were substituted for the cogs of cartesian mechanics. the second half of the th century saw the birth and development of the concept of the program, a concept with computer technology connotations, which was destined to explain the phenomena of inheritance. this concept began to fill out from the moment when it became certain that, in its nucleotide sequence, dna contains the necessary information for the construction of the protein material of cells. for a certain period of time, the passion for molecular genetics eclipsed the interest that had previously been given to metabolic chemistry. the powerfulness of the metaphorical concept may be measured according to the effect it has in pushing scientific research in particular directions, with the results this has on society. thus, during the th and th centuries, the study of human pathology was impregnated with a strong iatromechanical current. physiological chemistry and its corollary, pathological chemistry, which emerged as disciplines in their own right in the th century and achieved full expansion in the th century, are our inheritance from lavoisier and the concept of discussion about concepts necessarily leads to a brief discussion of scientific semantics, as shown by the few examples given in the previous pages. as we have just seen, the word gene that was put forward by johannsen around one century ago did not have the same meaning at that time as it has now, a meaning that still remains fluid. the gmo, an acronym meaning genetically modified organism, which has been the subject of vehement diatribes over the last few years, becomes much less of an object of passion if it is considered within the context of evolution. after all, for the last two to three billion years, living organisms have been genetically modified constantly by spontaneous mutations, which is why the human beings that we are today are able to discuss them! the term cloning is another example of a semantic misunderstanding that leads to inaccurate interpretation and arouses the passions. the primary meaning of the term cloning is the multiplication and the identical reproduction of a living cell. the simplest and most unambiguous example is that of bacterial cloning, a bacterial cell producing millions of cells that are identical to the original cell by its multiplication in a nutritive medium. the term animal reproductive cloning does not carry exactly the same semantic weight. it should be remembered that, in eukaryotes, the preliminary act of the cloning procedure involves the injection of the nucleus (with n chromosomes) from a somatic cell into an enucleated oocyte (chapter iv- . . ; see also chapter iv- . ). the somatic cell nucleus, by providing its genetic equipment, gives the being that will develop in the uterus a phenotype that is practically identical to that of the somatic cell donor, but nevertheless not completely identical, as the cytoplasm of the enucleated ovum, with its mitochondria, provides a small but non-negligible fraction of genes, the mitochondrial genes. as for therapeutic cloning (in the absence of uterine implantation), this is used for the manufacture of differentiated cells that may be grafted into the individual who has donated the original somatic cell, with no immune-related rejection occurring. this is non-reproductive cloning. the passionate argument that has arisen because the term cloning is bandied about in an ill-considered fashion illustrates the confusion that can result from a lack of precision in the use of certain terms with a high level of media impact. "all the major problems of the relations between society and science lie in the same area. when the scientist is told that he must be more responsible for his effects on society, it is the applications of science that are referred to […] . no government has the right to decide on the truth of scientific principles, nor to prescribe in any way the character of the questions investigated." the progress of science is linked to that of civilization. it is in keeping with the state of mind, the beliefs, the lifestyle and the thought patterns of societies. in ancient greece, where manual work was considered to be servile, science remained essentially theoretical, confined to logic and dialectics, and strongly attached to questions of philosophy. the birth of experimental science in the th and th centuries went hand-in-hand with the rehabilitation of manual work. the technical side dominates in modern biology, which seeks to solve problems concerning the "how", rather than to address philosophical problems concerning the "why". as ian hacking (b. ) says in representing and intervening ( ), nowadays engineering, and not theorizing, is the greatest proof of scientific realism, which leads to the minimization of philosophical thought. in a skeptical biochemist ( ) , the polish-born american biochemist joseph fruton ( fruton ( - emphasizes the contrast between the th century and the first half of the th century, when eminent scientists were still interested in the ideas of the professional philosophers of the history of the sciences concerning the progress of experimental research and, in contrast, the end of the th century, when philosophy and the experimental sciences pretended to ignore one another. this is doubtless partly because the history of biology has become the history of biotechnologies to such an extent that, according to some, the objects being explored are so familiar that they are now part of the life of society. in pandora's hope ( ), bruno latour (b. ) considers that the current confrontation between subject and object, in which the researcher-subject explores the structure and function of the object, is being transformed into a human-nonhuman dialogue, in which the nonhuman-object becomes "socialized". taking yeast as an example, latour writes that it has been "working for millenia in the brewing industry, but now it works in a network of thirty laboratories where its genome is mapped, humanized and socialized like a code, a book, or a program of action that is compatible with our ways of coding, counting and reading […] . non-humans have become automatons, admittedly without rights, but much more complex than material entities." latour visualizes the human-nonhuman associations in the form of collectives that are organized into strata that implement the technical, the political, the social, the ethical, the ecological… the technosciences correspond to one of these strata, the sociotechnical stratum that is directly linked to the stratum of political ecology. in the same spirit, the belgian philosopher gilbert hottois (b. ), in his philosophies of the sciences, philosophies of techniques ( ) remarks that "laboratories produce things that go off to live their lives in society and in nature." thus, bacteria, yeasts or genetically modified plants are able to produce drugs such as insulin, growth hormone and vaccines for human medicine. these drugs become part of and indispensable to life in society. they are evaluated according to their market value by the companies that patent, manufacture and sell them, and according to the comfort they bring to the patients to whom they are administered. the financial management that results from their consumption becomes a worry for those responsible for public health, while their manufacture by specialized companies generates industrial activity and economic growth which may be measured according to how fashionable they are and how they sell. for a long time, society, while benefiting from scientific progress, remained indifferent to the experimental method, that is to say, the way in which knowledge progresses. in the last decades of the th century, society became aware, via information concerning the occasionally demonized exploits of genetic engineering, that science can "take liberties" with the human being. populations were well informed about the effects that genetic engineering could have on the mortality rates of pathologies such as cancer and diabetes or on degenerative illnesses of the nervous system, and about the closeness of possible solutions. however, they were also warned about the risks to which science was exposing humankind. remembering certain tragic episodes concerning hiv-contaminated blood transfusions, growth hormone and mad cow disease, and certain cassandra-like predictions, such as a catastrophic epidemic of spongiform encephalopathy that has happily yet to appear, society shows reservations when the media inform its members of new feats of modern technology. political authorities, for their part, afraid of potential problems, tend to follow the principle of precaution, which in fact hides a fear of risk. however, evaluating risk involves not being afraid of it but understanding it in a lucid and courageous fashion. informed by the media, which often use sensationalism, the citizen is increasingly calling into question whether certain practices involving the biosciences, such as cloning, or certain mercantile transactions such as the taking out of patents concerning gene sequences, or even experimentation on live animals, are well-founded. "the problem of experimentation on man is no longer a simple problem of technique. it is a problem of value. from the moment that biology concerns man no longer simply as a problem, but as instrumental to the search for solutions concerning him, the question arises of deciding whether the price of knowledge is such that the subject of the knowledge is able to consent to become the object of his or her own knowledge. we have no difficulty here in recognizing the still open debate concerning man as a means or an end; an object or a person. this is to say that human biology does not contain in and of itself the answer to questions concerning its nature and its significance." knowledge of life - written at a time when people were far from imagining how molecular biology was going to expand, the prophetic words of georges canguilhem ( - ) have maintained their philosophical validity. manipulation of the human embryo, whether this involves its creation by cloning or the modification of its genetic inheritance, obviously leads to the need to consider the societal, religious and political points that arise from the domain of bioethics and are a reflection of the period in which we are living. until recently, advances made in biology left moralists indifferent. this ceased to be the case when scientific experimentation began to look at the human embryo with a view to utilitarian ends in the health domain. the specter of cloning was brandished without any clear distinction being made between reproductive cloning and therapeutic cloning. biology became demonized. however, as biologist pierre chambon (b. ) said in an interview in the french journal biofutur: "in absolute terms, biology is unable to tell us whether the cloning of a human being is moral or immoral, it simply tells us whether it is biologically possible." the birth dolly the sheep in (chapter iv- . . ) triggered a virulent debate because now that the cloning of an animal had become possible, that of a human being became envisageable. the media sensationalized this debate all the more in that it was exacerbated by debate concerning gmos (chapter iv- . ). the dolly affair became a problem of society. up until then, the biosciences had been happy just to try and understand the mechanisms that explained the functions of living beings, but now, with the advent of gmos and cloning, it became obvious that a forbidden barrier had been crossed and that man had the power not only to transform but also invent himself. faced with this desacralisation of nature, the need arose for some philosophical reflection. this was given the name of bioethics, which is the title of the book, bioethics, a bridge to the future, which was written by the american biologist van rensselaer potter ( - ) in . the term bioethics covers philosophical considerations that range from the biosphere to the human person. bioethics tries to give a wider meaning to the moral codes which, in human societies, depend on ancestral traditions. it aims to prescribe that which is desirable according to the kantian maxim of the categorical imperative. in his what is bioethics? ( ), the belgian historian gilbert hottois reminds us that bioethics are above traditional morals, the latter being a set of norms that are most often spontaneously respected as good habits, without any critical reflection being involved, while bioethics, on the other hand, arises out of critical thought, analysis, discussion and the evaluation of established mores. over the last few years, the problems that are targeted by bioethics have moved towards today's burning issues. human cloning is an example. while allegations of the transcendence of man in nature may lead to human reproductive cloning being considered as a crime, strictly scientific considerations lead to an emphasis on the lack of responsibility shown by a few zealots, given the hazards involved in cloning in animals, such as the need to use a large number of oocytes in order to achieve success in cloning, the very low viability of the cloned embryos and the development of serious functional anomalies in the clones that survive. even supposing that scientific progress will one day overcome these difficulties, human reproductive cloning will come up against an insurmountable obstacle, the cloned subject's fear of finding that he or she is identical to the relative from whom his or her genetic inheritance comes. after all, the notion of manipulation of the human ovule with the aim of serial reproduction has often haunted science fiction stories. in brave new world ( ), aldous huxley ( - ) gives an apocalyptic vision of the budding of human eggs that produce hundreds of identical twins which are conditioned into classes and subclasses while being raised in jars, depending on the quality of the nutritive substances they are given. in the artificial uterus ( ), henri atlan (b. ) predicts that the raising of human fetuses in jars could well become an alternative to uterine gestation in a distant future. let it be understood that human reproductive cloning, which is no longer part of the domain of science fiction, as it has become feasible, must be considered as being reprehensible because it goes beyond the limits of reason, and is a denial of human transcendence. man as subject cannot be considered as an object. the problem of therapeutic cloning is quite different, although it leads to reticence and prohibition because the demarcation between therapeutic and reproductive cloning depends mainly on whether a cloned embryo is implanted in a uterus. while, at the time of writing, therapeutic cloning has been prohibited in france, germany and other countries, it is tolerated in great britain. in the usa, the prohibition only applies to publicly-financed researched, while each state has its own legislation, which is relatively flexible. the objective of therapeutic cloning is to provide patients with tissues that arise from their own selves, and are therefore immunocompatible and able to be grafted without there being any risk of rejection (chapter iv- . . ). it is based on the removal of somatic cells from the subject to receive the graft and the transfer of the nuclei of these cells into enucleated oocytes. the stem cells that are obtained after the first divisions are stimulated using appropriate growth factors. depending on the factor used, the stem cells differentiate to form a type of tissue (hepatic, muscular, nerve…) that can be used as a graft. such a procedure may be envisaged for patients who have suffered a serious, invalidating trauma, for example section of the spinal chord. a graft of immunocompatible nerve cells might make it possible to re-establish nerve continuity. a similar type of therapy has been considered for parkinson's disease, the cause of which is a degenerescence of certain cells of the encephalon (chapter iv- . . ). given the hopes that are raised by the possibility of such therapies, and the fact that, after all, such therapeutic cloning is the equivalent to an autograft, even if the ways in which the graft is obtained are slightly tortuous, the demonization and rejection of such practices should be reconsidered, calmly and coolly. another option for therapeutic cloning is the correction of mutations identified in the mitochondrial genome of a woman wishing to have children. it is, in fact, the mother's ovum that provides the fertilized egg with its complement of mitochondria that are indispensable for its viability. the manipulation involves inserting the nucleus of a fertilized ovum from the mother, obtained by artificial insemination, into an enucleated oocyte taken from a woman who is not suffering from the mitochondrial defect. the cytoplasm of the enucleated oocyte provides the stock of functional mitochondria that are indispensable to normal cell function in the future embryo. in a domain of the bioethics, in which rational objectivity comes up against deliberately technophobic religious and cultural considerations, it is useful to remember certain legal and legislative paradoxes. thus, in france, after having been considered to be a criminal act that was subjected to severe repression by the law up until , the right to have an abortion before the end of the third month of pregnancy became not only authorized but also protected by law. it is interesting to note that in the th century, thomas aquinas, the father of the church, had acknowledged that a fetus only becomes "animated" by the implantation of the soul by holy will in the third month after fertilization. another subject to be considered is pre-implantation genetic diagnosis (pgd), in which human embryos that have been fertilized in vitro are sorted in order to find those that are without defects, a practice which is on the verge of being a deviation in the direction of eugenics. nevertheless, pgd is the basis of a practice that is either already legalized or is in the process of being so in several european countries, the creation of so-called designer babies. a typical example is that of a designer baby arising from an embryo whose immune profile to that of an older sibling who is suffering from leukemia. in this case, there is good reason to hope that a graft of immunocompatible blood cells from the designer baby into the sibling who is suffering from leukemia will save the latter from death. out of the disharmony of opinions that arising from cultural tradition, religious conviction or simply scientific pragmatism, the american biologist and philosopher h. tristram engelhardt (b. ) , in the foundations of bioethics ( ) proposes a lay bioethics that is based upon the principle of permission. lay bioethics advocates tolerance while admitting that this tolerance in no way prevents anyone from taking up a personal position; it means that each human being has a moral sensitivity as well as the ability to reason and to choose within a defined limit of non-harmfulness and of justice. the individual is free to modify his or her destiny, or to manipulate his or her nature by genetic interventions because, adds engelhardt, "there is no lay moral foundation to prohibit such an intervention." when a researcher or the research organization that the researcher belongs to files for a patent for an invention with a patent office, it is necessary to demonstrate the novel and utilitarian nature of this invention. if a patent is accepted, this gives the person or body that filed it the exclusive right to make use of the invention over a pre-determined period of time, generally years, which is a means of protection, or, if desired, to allow others to make use of the invention by issuing a license to do so. in the domain of living beings, there has sometimes been confusion between invention and discovery. in , craig venter, known for his participation in the sequencing of the human genome, filed a demand for a patent covering the sequences of fragments of recombinant dna (cdna) called est (expressed sequence tags) that are obtained by reverse transcription from human brain messenger rnas, in the name of the nih (national institutes of health) at bethesda (usa). the patent specified that ests could be used as probes to characterize genes that are potentially involved in neurological ailments. the resulting outcry led the nih to withdraw its patent demand. in fact, the patenting of living beings has a long history that goes back to the patent that was filed in in france by louis pasteur, and then in in the usa, for the use, in brewing, of a yeast culture that was free from pathogenic bacteria. from this historical perspective, the case of ananda chakrabarty (b. ) set a legal precedent. in , chakrabarty filed a demand with the us patent office for a patent relating to a pseudomonas type bacterium which, by genetic modification, had acquired the ability to digest crude oil. his demand was refused. after an appeal and many legal battles, the united states supreme court overturned the patent demand refusal, the basis of the judgement being that any modified microorganism is a product of human ingenuity and has a specific name, characteristics and use. thus, from onwards, the arrival of an era of patents derived from genetic engineering was indicative of how this discipline was growing. in december of that year, stanley cohen and herbert boyer, acting on behalf of the university of stanford, patented a nucleic chimera comprising a recombinant dna carried by a vector. in , a patent concerning the growth hormone gene was awarded to the university of san francisco. in , the university of california at berkeley obtained a patent for the human insulin gene. in , the american company pioneer hi-bred succeeded in patenting a variety of corn in which genetic modification has led to an increased synthesis of tryptophan, an amino acid that is indispensable for animal feed. in , the genentech company acquired a patent for the gene coding for human gamma interferon. this was followed in japan by a patent for the gene coding for beta interferon. in the same year harvard university patented the oncomouse, a transgenic mouse whose susceptibility to cancer is greatly increased. after this, several species of transgenic animals were patented for utilitarian purposes, such as the production of human alpha- -antitrypsin taken from the milk of transgenic goats and used for the treatment of cystic fibrosis. the frenetic patenting of living beings has reached the domain of natural products arising from the plant world in tropical regions, the immensely varied essences arising from these plants being full of pharmacological potential. the potential for producing drugs of a considerable commercial value from such plants is very high. here we return to the problem of the patenting of genetically modified, cultivatable plants (gmps) (chapter iv- . ). thus, the experimental method, the principle of which is to acquire pure knowledge, finds itself led astray in its applications. whatever the motives that are given, particularly for manipulations that give rise to the manufacture of marketable products, the patenting of genomes for mercantile ends shows the regrettable, but unfortunately inevitable, direction in which the very spirit of a science, molecular biology, which half a century ago wished to be at the heart of an understanding of living beings, has drifted. the suffering of animals that are being experimented upon gives rise to a moral problem. the end of the th century saw large-scale demonstrations against vivisection and repeated demands for it to be abolished. today, there is renewed vigor in the call for the abolition of vivisection, without any real coherent basis. this desire to stop experimentation on animals ignores the imperatives of contemporary medicine, which must meet the challenge of pathologies whose increasing incidence is worrying, such as cardiovascular diseases, diabetes, cancer, and the degenerative illnesses that are linked with aging or are of genetic origin. it is true that animal experimentation inevitably leads to questions. are the stakes involved in a particular experiment, in terms of the acquisition of new knowledge, worth the suffering of an animal used in that experiment? is it not necessary to ensure that the experimental protocol is well-documented, that it is not redundant, or even that it has been the subject of previous studies carried out on cells in culture? it is easy to see the size of the methodological chasm that separates contemporary physiology from that of the time of claude bernard, when cell culture techniques were not yet being used, when the main instrument used was the scalpel and the researcher, using his or her imagination and creativity, had to develop specific protocols that were able to validate or refute a working hypothesis. each period in history operates in its own way according to its moral laws and its technical capabilities. the bloody operations carried out by magendie and by claude bernard in the th century, which were tolerated at this time despite criticisms from antivivisectionists, would not be permitted today. nevertheless, it is true that the physiologists of the th century, by means of the results of their experiments, wove a tapestry of new knowledge on which contemporary biologists were going to work and without which the level of understanding the modern science would be much lower than it is. animal experimentation remains indispensable in many areas of physiological investigation, in genomics, in toxicology and in pharmacology. it is a precondition for clinical trials of any new drug, being used to test for the drug's efficacy, its metabolism and any toxicity. however, not all data arising from animal experi-mentation can be extrapolated to man. the margin of uncertainty can be reduced by means of comparative trials on several animal species. because of their phylogenetic proximity to man, primates may seem to be the solution for experimentation prior to the application of a drug in man. this was the case for the development of a vaccine against hepatitis b. it has been proposed the grafting of stem cells in man should be preceded by experimentation in apes, in order to ensure the absence of tumorization over the long term. however, the researcher is confronted with a dilemma: should he or she ensure the safety of man with respect to possible deleterious effects or respond to ethical demands that recognize the very great genomic similarities between man and the chimpanzee. a consideration of cloning, patenting and animal experimentation practices illustrates the excesses of the experimental method in domains where political authorities consider themselves able to legislate. administrative decisions, often made in the absence of any dialogue with scientific authorities, can have serious consequences. thus, given the pretext of strict obedience to the principles of bioethics, which are a matter of tradition, and while certainly respectable, are nevertheless arguable, and also given the pretext of a sickly and unconsidered fear of the risk involved in certain experimental practices, and the absence of an intelligent evaluation of this risk, research, which until recently took place in a motivating atmosphere of liberty, may, over the long term, be weighed down with a highly prejudicial handicap and a limitless sense of discouragement. in the th and th centuries, experimental research, which was still in an emergent phase, was mainly artisanal, and in the hands of rare scholars. it took form during the th century in the west, particularly actively in germany, and became operational in the th century, under the aegis of governmental authorities, with the creation of institutes, the programmed recruitment of researchers and the allocation of renewable budgets. modern science, based on the principles of the experimental method, came to the fore much later in the east than in the west. the globalization of knowledge has meant that at present experimental science, in all domains, including that of the life sciences, has spread throughout the world, with even those countries that had become relatively backward in these domains because of their isolation catching up rapidly. nevertheless, it is true that the progress of the experimental sciences in the usa and in the united kingdom has been distinguished by pragmatic management of these countries' science policies, based on the excellence and the high degree of autonomy of their universities and research institutes with respect to recruitment and choice of subjects of study. the efficacy of this policy in the life sciences may be judged by the number of researchers who have won nobel prizes since the second world war (at the time of writing, more than in the usa and twenty or so in great britain as opposed to only in france). in france, research on living beings is carried out in the laboratories of universities, in institutes connected with higher education and in laboratories that are run by large organizations such as the national scientific research center (cnrs), the national institute of health and medical research (inserm), the national institute of agronomic research (inra), the atomic energy commission (cea), the national institute of research in computer processing and automation (inria), the national center for space studies (cnes) and the french institute of research on the seas and oceans (ifremer). equivalent bodies exist in countries other than france, some of them being institutes that are dedicated solely to research, and some being university laboratories that associate research and teaching. at the beginning of the th century, the function of researcher was most often associated with that of a professor occupying a chair at a university, surrounded by a few assistants, the professor directing the research work in his area of specialization. now, within a period of a few decades, the status of researcher has been modified greatly. today we talk of research careers classified according to level of expertise and technicality. management, or the supervision of career paths and the control of financing, is carried out by an administration that is itself highly hierarchical. the scientific process has undergone a metamorphosis, shown by changes in the behavior of researchers not only within the institutions in which they work but also in their relationships with the media, the political sphere and society. the teaching of the life sciences needs to take this into account. "long ago, there was a time when scientists recounted the exact circumstances of their discoveries, without shame, even when their recital showed up the fragility of their forecasts or an indecent collaboration on the part of every bit of luck. such times are past, and the researchers of today often like to make us believe that they only find what they are looking for. the thousands of pages pasteur's lab books provide an opportune reminder to us (and to program-makers or impatient users) that it is just as difficult to ask a question as to answer it, that a scientific discovery often occurs after a long, winding path, that rather than following the fashion, it is preferable to follow one's ideas, particularly if they are good ones, and are in advance of the fashion." jean jacques molecular dissymmetry, in "pasteur, workbooks of a scholar" - current technological progress, the accumulation of the scientific knowledge, the institutionalization of the public research and many other factors are disrupting a ritual of the experimental process that had survived until the middle of the th century, and even beyond. the experimental life sciences of the st century will necessarily see themselves remodeled with respect to their objectives and procedures. faced as it is by an increasingly tough international competition, the scientific community is also subject to restrictions in terms of operation and prospectives. an organization into small teams of a few researchers gathered around a boss, working in friendly interaction, is increasingly giving way to large groupings that sometimes seem like consortiums. focused on research subjects that are deemed to be "cost-effective", these superstructures are encouraged, or even imposed, in the sadly illusive hope that the will lead to greater efficacy. the person in charge of such large groups is taken up with everyday management tasks and by maintaining good relations with the administrative bodies on which his or her organization's survival depends. he or she may become distanced from the experimentation and forget the intellectual motivations that in the past caused his or her competence to be recognized. it should be emphasized that the secret of future successes lies in situations where young researchers are in direct contact with their bosses, and where friendly interaction with a known master teaches the apprentice researcher how to learn, how to think and how to experiment in a critical fashion. preoccupied by the rapid expansion of the scientific population, accompanied by the creation of laboratories whose operation necessarily requires financing, often on a large scale, political authorities, giving way to the requirements of media-fed public opinion, are interfering more and more, via administrative relays, in the control of the objectives of experimental research. short-term objectives, considered to be "visible", are favored. a priori, the viability of a project is judged according to the scientific context of the period and its impact on society, insofar as the project looks at health problems with a high degree of media coverage (cancer, degenerative illnesses, viral infections…) and often in agreement with a consensus that avoids going against the orthodoxy of the moment. this leads to a rigid management of projects that are financed and controlled according to objectives that have been fixed in advance, and that are all the more easily accepted by state authorities when they are somewhat fantastic in character. however, fundamental research proceeds from a playful activity, and for this reason, its efficacy is dependent on the passion of the researcher for the problem that he or she is studying. in contrast to what is believed by the narrow-minded, the effectiveness of a researcher in terms of discoveries depends upon the liberty that is given to this researcher, assuming, of course, that this liberty is underpinned by criteria of confidence such as the researcher's scientific past, his or her motivation, and judgements made concerning the researcher by impartial peers. it should not be forgotten that the determination of the three-dimensional structure of hemoglobin by max perutz (chapter iii- . . ) took around twenty years of solitary, uninterrupted and untiring labor. the theoretical and technical tricks that led to this success helped to open up the domain of the structures of giant macromolecules, several dozen kilodaltons in size, which no-one had dared study before. anyone who uses the experimental method realizes that while fundamental research must be organized, it cannot be scheduled. such a person knows that the pathways to discovery are convoluted, and that an inexplicable observation that appears unexpectedly during an experiment can sometimes, if the researcher is sufficiently perspicacious, be the beginning of an adventure that leads to a discovery. it was to just such a convoluted path that the belgian biologist christian de duve (b. ) alluded in his speech when he received the nobel prize for medicine and physiology in . after working at the university of saint louis in the usa, de duve, who had taken up a post at the university of louvain, belgium, decided to look at a research theme that had received a great deal of media coverage, diabetes and insulin. it was while operating on one of the subcellular fractions obtained from ground rat's liver, and analyzing certain of the enzyme activities of these fractions, that he was surprised to find, in one of them, enriched with mitochondria, a phosphatase activity that, paradoxically, increased with time, while the enzyme activities specific to the mitochondria declined. this was an activity belonging to organelles that were contaminating the mitochondria. dropping all research on diabetes, de duve set out to identify and characterize these unknown organelles. he discovered that they were involved in the breakdown (lysis) of molecules that are undesired by the cell and, for this reason, he called them lysosomes. the discovery of lysosomes helped to open a new chapter in cell biology and to attribute a molecular cause to diseases with serious prognoses whose etiology had remained a mystery up until then. these diseases were given the label lysosomal diseases. these diseases result from the absence of a lysosomal enzyme that is responsible for the breakdown of a given metabolite. the accumulation of this non-broken-down metabolite in the lysosomes leads to cell malfunction, which causes the lysosomal disease. as de duve said jokingly, if he had carefully followed the experimental process laid down in his diabetes research project, and if he had not given way to the temptation of "playing hooky" or "playing truant" he would never have mounted the podium in stockholm. in the same way, henri-gèry hers ( - ), a cell pathologist at the internationally renowned louvain school, remarked in an article published in the review médecine/sciences: "i believe we would obtain maximum value for the money devoted to research if we were willing to distribute it to those who have been shown to be productive, according to their needs, and without asking them for a program." hers concluded, in a tone that was deliberately playful, but thought-provoking, "such a simple system would lead to unemployment for a large number of administrators, which is why i suspect that it will never be adopted." research has its own set of ethics, driven by anticonformity and the creative imagination, capable of shaking up firmly-anchored ways of thinking and established hierarchies, and of leaving the researcher the freedom to express him or herself and to experiment off the beaten paths. as eccles says in evolution of the brain and creation of the conscience, it is important to distinguish between intelligence and imagination. intelligence is measured according to the rapidity and depth of understanding and clearness of expression. it may be measured and even given a numerical value. the same is not true for the imagination, a more subtle, unmeasurable phenomenon that cannot be learned. the imagination is one of the levers that is able to lift the boulder that hides scientific truth. the imagination is the ultimate weapon of research, which shakes up the knowledge acquired by the intelligence. nevertheless, the imagination must be tempered by a good critical sense that is able to perceive potential sources of artifacts, both in sophisticated instruments that act as so many black boxes from which already manufactured information emerges and in genetic or chemical cell exploration methods whose specificity must be carefully checked. the benefits that can sometimes be gained from prospective research that is far from dogma that is rooted in sterilizing tradition, the way in which knowledge progresses, most often by moving away from any orthodoxy, the way discoveries appear unexpectedly on the fringes of carefully put together projects, all of these points are matters for reflection for those in power in the worlds of politics, economics and industry. publication is an essential tool for communicating scientific knowledge, and is the judgement criterion for committees in charge of evaluating the creativity of a researcher. in order to have meaning, a publication must provide information that is sufficiently innovative with respect to parallel work carried out in other laboratories. here again, media coverage has quietly infiltrated the scene. its role is all the more perverse in that the rating of a publication is estimated according to its impact index, or, roughly speaking, the renown of the scientific journal in which it is published. curiously, it has happened that articles that would later be considered to be of primary importance have been rejected by highly prestigious journals, simply because the facts mentioned in the article and the conclusions made have not coincided with the orthodox opinions of the period and the traditionalist spirit of the journal's editorial committee. this was the case for an article which the biochemist hans krebs ( krebs ( - submitted to the british journal nature in . in this article krebs described a series of experiments showing that an endocellular metabolite, pyruvate, product of glycolysis, is completely degraded during a cycle of enzyme reactions. this degradation cycle would later be recognized as the central pivot of the intermediate metabolism. called upon to judge revolutionary scientific considerations, and unable to perceive their importance, nature's editorial committee rejected the article. krebs then sent his article to a journal with a relatively restricted audience, enzymologia. it was accepted and published in the two months that followed. the importance of the concept that was put forward in the article ensured that its author gained international recognition, leading to his winning the nobel prize for physiology and medicine in . for the researcher, publication is a way of making his or her work known. it is also the way in which the researcher learns about the work of others. while the rhythm at which publications in the life sciences appeared increased slightly in the first half of the th century, the second half of that century saw a great acceleration in this rhythm, leading to a difficult-to-manage proliferation of reviews and books. it has been estimated that in the last thirty years the volume of publications in the biological domain has increased five-fold; in the preceding twenty years it had already doubled. this accumulation of publications makes it harder for the researcher to judge the quality of the huge mass of published articles, even in the highly targeted domains that are within his or her area of expertise. the researcher, therefore, will deliberately choose a particular article according to the prestige of the journal in which it is published, which is not an inviolable criterion of quality. in addition, any judgement concerning the pertinence of a scientific article necessitates a dissection of the subtleties of the methodology, the well-groundedness of the experimental protocol and the validity of the results, by means of a careful examination of tables of results and graphs, and, finally, the logic of the discussion. this restrictive yet absolutely necessary requirement limits the number of articles that are likely to be screened. however, this is not the worse fault of publication today. there is another problem that is much more worrying. many documentation centers have reacted to this inflation in the scientific press by equipping themselves with computing facilities that are able to find, in data banks, articles that have been selected on the basis of a key word index, and to display them on screens. while acknowledging that this constitutes an inescapable change in the transmission of scientific know-how, it should be recognized that in browsing through the pages of a highquality scientific review, it is possible to come across an article containing an innovative idea or a useful technique, an advantage that is less available when using the on-line system of scientific publication that is most prevalent nowadays. mention should also be made of the requirement to publish frequently and within short time frames, for reasons of competitivity, when aspiring to obtain jobs or promotions, or even just to obtain recognition, this requirement being another factor that is prejudicial to fundamental research. it is the cause of worrying excesses, such as experiments that are hastily published and non-reproducible, or even the falsification of experimental results, occasionally within a context of considerable media coverage. although such practices, which are the exception rather than the rule, are rapidly detected and condemned in a scientific culture where information circulates freely, the publicity that they incite, which reaches society at large via the media, leads to an overall discrediting of experimental research. at present, one of the most noticeable trends in scientific publication is that of collectivism. while, in the th century, scientific articles were usually published in the name of a single author, occasionally two authors, and very rarely more than two, nowadays publications are often co-authored by several people, and when the work involves the analysis of structures, or the sequencing of genomes, several dozen researchers may be co-authors. from being the work of individuals, research has become collective. in domains whose complexity requires a wide selection of techniques that may range from physics to genetics, the hybridization of specific areas of expertise is certainly indispensable, and this requires the collaboration on a particular project of researchers who are sometimes physically remote from one another. the downside for the researcher, particularly one who is young, is that this requires him or her to abandon individuality and creativity. both collectivism and inflation in scientific publication are facts that are an integral part of contemporary science, facts which reflect an irreversible trend that it would be difficult to obviate. over the last few years, scientific publication has been subject to a type of restraint, in that certain "sensitive" data in the domain of molecular biology might be used for the manufacture of biological weapons in a form of terrorism known as bioterrorism. thus, the means of synthesizing de novo viruses (influenza virus, poliomyelitis) and the possibility of modifying their tropism by "directed molecular evolution" (change from a sexual tropism to a respiratory tropism for the aids virus) have been the subject of publications in prestigious journals. given sufficient means, terrorist pharmacists could well make use of such data in order to carry out malicious actions with catastrophic consequences . in order to please a public that is eager for progress and the sensational, politicians favor, by means of targeted financing, the types of organization that appeal to their sensibilities, such as the technological platforms. while recognizing that such platforms are now an integral part of the landscape of research on living beings, and that they must therefore be taken into account, and while acknowledging that projects which implement the latest technologies in different domains need to be federated, it is nonetheless vital not to underestimate the potential creativity of small groups of researchers, a point that was expressed by one of the greatest of contemporary biologists, arthur kornberg ( kornberg ( - , winner of the nobel prize for physiology and of medicine, in a speech given in : "as i view the steady growth of collective science and big science, the greatest danger i see is a dampen-ing of individual creativity and reversion to the old politics -the inevitable local politics that infects every group and institution." however, conscious of the metamorphosis that is occurring in the experimental method, and faced with a particularly inventive and all-conquering technology, fundamental research in the life sciences must come to terms. a century ago, fundamental research and technological research interacted all the more directly because they were both in their infancy. this is no longer the case. management of the ever-increasing amount of knowledge in the life sciences, and the degree of sophistication achieved by bioengineering techniques and instruments, is widening a gap that makes dialogue increasingly laborious. however, dialogue appears to be a guarantee of future progress. the solution can only come from an increase in cross-disciplinarity, which should begin with university teaching and the establishment of a recruitment policy that advocates the cohabitation of talents from different educational backgrounds in the same laboratory. fortified by such hybrid expertise, while maintaining its share of originality and liberty in the choice of problems to be studied, fundamental research on living beings can only be enriched by a marriage of reason with biotechnology. convinced of the necessity for such a marriage, stanley fields, the inventor of the double hybrid method (chapter iv- . ), in an article entitled "the interplay of biology and technology" (proceedings of the national academy of sciences, usa, , vol. , pp. - ), concludes,: "it is at the interfaces of biology and other sciences that many of the future discoveries will be made, at the interfaces of biology and engineering that these discoveries will come to be exploited, and at the interfaces of biology and ethics and law that their consequences for society will be decided." the desired dialogue between biology and technology also implies the breaking down of barriers that too often isolate fundamental research and so-called applied research, and the facilitating of consistent interaction between the discoveries made in the academic institutions and their application for utilitarian ends in private companies. this is where the twin demons of money and power raise their heads. already, at the turn of the s, a. bartlett giamatti ( - ) , who was then president of yale university in the usa, commenting on american university policies, spoke of a "ballet of antagonisms" between, on the one hand, commercial companies that are interested in the rapid cost-effectiveness of any new therapeutic advance and, on the other hand, non-profit university laboratories. recently, james j. duderstadt (b. ), emeritus president of the university of the michigan, argued that the university is a "counter-hierarchical" organism. in fact, its members are free to carry out the research that pleases them and to think in the ways that they wish to think, in any case within an academic norm that considers itself as being free from the constraints dictated by private interest groups. until recently, such behavior was considered as a sort of ethic which arose out of the university conscience and dignity. the crumbling away of this ethic in the final decades of the th century coincided with the rise of biotechnologies and the large-scale filing of patents relating to molecular genetics techniques that could be applied to the manipulation of living beings, by researchers in the public sector. the intrusion of the american private sector into public research laboratories, in the form of collaborations with transfer of "sensitive" information from the public to the private, has become such a worrying problem that drastic control measures have had to be taken. within this context, the american federal government, in february , issued a certain number of prohibitions targeting the national institutes of health (nih) of bethesda, particularly with respect to the retribution of researchers for services rendered to industry . these stands call for thought concerning the place that is currently held in universities with respect to fundamental research. without arguing against the efficacy of major research institutes, it is nevertheless necessary to remember the part played by the university in this domain. the university is not only the place where knowledge, both as it is now, in its current state of advancement, and as it has been, it is also the place where knowledge must be created by fundamental research. for the last few decades, under pressure from state policies, and also as a function of an improvement in social status, the world of the university has opened up to a wider public, leading to an influx of students that is sometimes so enormous that the task of teaching them has become overwhelming. because of this, the share of their time that university researchers can, in practice, devote to their research tasks has shrunk. this situation is highly prejudicial to the mission to innovate, which should be a priority. it is, in fact, during their university studies that the thought patterns of young students are forged by contact with teachers who not only instruct them, but also educate them by inspiring in them a motivation and an enthusiasm that gives rise to hope. how could this be true if the teaching faculty did not itself participate in scientific creation? "what can teaching, ex cathedra, do to guide the researcher? nothing, obviously. the researcher is trained in the laboratory. and the first stroke of genius on the part of a future researcher is to find a good boss. such a find will open up the royal road to success. the road will be opened -but the researcher must travel along it. a researcher may be taught many things. he or she can become familiar with techniques and with equipment. she or he can be assigned a problem to resolve. however, what is essential for the researcher is to know how to understand relationships between phenomena that seem unrelated, and to be able to progress from the particular to the general. a boss may develop such qualities in a gifted young researcher, but intuition is a gift; it cannot be taught." while the bernardian style experimental method, based on a working hypothesis aroused by an observation, followed by implementation of an experimental protocol, is still extant in the life sciences, and while "serendipity" is still the origin of great discoveries, "big science" , underpinned by sophisticated biocomputing or bioinformatics procedures, is intruding more and more, while genomics and proteomics are not far behind. the methods and instruments developed by the biotechnosciences have led to profound modifications in the ways that the structures and functions of living beings are investigated. for example, by varying multiple parameters in dna chips or protein chips, at the same time, the experimenter is able to ask questions that lead to grouped all-or-nothing answers (chapter iv- . . ). in combinatory chemistry, screening makes it possible to detect a molecule that is active for a given pathology from among a multitude of molecules (chapter iv- . ). the mathematical simulation of metabolic networks or of signaling chains is already well under way (chapter iv- . ). given this new technological outlook and the hope that it can provide rapid solutions to health problems subject to considerable media coverage, the teaching of biology in universities must not be limited to a description of current advances, no matter how brilliant and promising they may be. this teaching should return to its origins, be a reminder of history, and should not hesitate to use examples to illustrate how a major discovery can arise from a long period of wandering in the wilderness. in practical terms, while being conscious of the extraordinary complexity of living nature, and carefully avoiding the dangers of simplification, it is important to remember that the reductionist method was a necessary path to an understanding of the integrated, modelized biology that is emerging nowadays. at present, certain people call reductionism naive, but this is only the case insofar as we have faith in recent advances in integrated biology . with this in mind, it should be noted that the deciphering of the protein synthesis mechanism in prokaryotic microorganisms (chapter iv- . . ) was, along with the discovery of the genetic code, a jumping-off point for an inventory of similar, but noticeably more sophisticated, mechanisms in eukaryotic organisms. the reductionist "one gene, one enzyme" dogma, formulated on the basis of beadle and tatum's experiments on the mold neurospora crassa (chapter iii- . ) was a necessary prerequisite to a considerably more elaborate understanding of the relationship between the genotype and the phenotype. the way in which the nucleic acid and protein units in the tobacco mosaic virus spontaneously organize themselves (chapter iii- . ) acted as a basis for thought concerning the self-organization of macromolecular complexes in the cell. these few examples underline the fact that it is difficult to comprehend the scientific research process if we only refer to experiments carried out in the present, and if we do not have a clear idea not only of the way in which hypotheses, even false ones, were once formulated, but also of the way in which experimental work, which may have led to failures, was once carried out, or, in brief, if we do not look back at the past. let us add that it is occasionally good for us to show some humility when we take the trouble to examine the past. thus, the processes involved in the phagocytosis of bacteria by innate immune cells (neutrophils, macrophages), which are today studied in the greatest detail with particularly refined technical facilities, had already been perceived more than a century ago by metchnikoff, and even analyzed, admittedly with the clumsy means at his disposal, but with such accuracy that none of the conclusions formulated at that time have yet been disproved (chapters iii- . . and iii- . . ). the experimental method applied to the life sciences, the history of its birth and of its development, the way in which it is regarded by political and societal authorities, and, finally, the dependencies that are developing at present between the technosciences, human medicine and the different branches of the economic sector, all of these aspects should be covered by university teaching that includes not only the pure sciences, but also the human, political and economic sciences, as well as philosophy. the student should not be saturated with book-learning, but he or she should be taught to reason, to imagine and to criticize, not to accumulate knowledge in an indigestible catalogue, but to ask questions about the way in which certain, carefully chosen, items of knowledge have been acquired, and not to deliberately accept science in its current state without knowing what it was like in the past. he or she should understand what pathways of thought led to dogmas that were established and taught as truths being refuted, and favor experimentation, with its risks and questions, rather than well-smoothed, abstract theoretical presentations without rough edges. these should be the principles of teaching that is designed to open up young minds to creativity. in anglo-saxon countries, the worlds of industry and research that welcome the graduate manage to communicate with one another, but these worlds ignore one another in france, or at least remain reserved, a situation which is prejudicial from the economic point of view. if we look at the pharmaceutical industry in particular, we see that only half a century ago the pharmacopeia was limited to plant extracts or active agents isolated from these plants, with antibiotics quietly beginning to make their appearance. in the last decades of the th century, a great technological leap forward was made, with completely new methods in bioengineering, combinatory chemistry, and the finding of therapeutic targets in macromolecules, and this created a hiatus that severely handicapped countries that were unprepared for it. france, with its biological fundamental research training that is out of phase with that of the anglo-saxon countries, fell behind, and continues to be behind, a situation that is prejudicial for its economy. the remedy for this does not lie in incantatory speeches. it requires a volontarist policy for the management of experimental research. generally speaking, the fact that the major engineering schools in france, which recruit the scientific intellectual elite, students being chosen by competitive exams that select for intelligence rather than imagination, are unable to impose upon their students an end-of-course thesis that would authenticate their engineering degree, should not be tolerated. in contrast to other countries, in france only a small percentage of engineers have received doctoral training or had to present a thesis before entering their careers. the french dual system of major engineering schools and universities, which, a century ago, made sense for the economy of that period, has become completely obsolete, and deserves a courageous revision. "there is a question, much older than modern science, which has never ceased haunting certain men of science: that of the conclusions that the existence of science and the contents of scientific theories can lead to concerning the relationships that humankind has with the natural world. such conclusions cannot be imposed by science as is, but they are an integral part of the metamorphosis of this science." the new alliance. metamorphose of science - ( nd edition) in the s - s, the hybridization of the techniques of genetics, biochemistry and biophysics gave birth to molecular biology. with the resolution of the double helix structure of dna, the demonstration of its replication, the elucidation of the mode of expression of its nucleotide sequence as a sequence of amino acids in proteins and finally the deciphering of the genetic code, biology underwent a revolution of an amplitude similar to that which, at the end of the th century, saw a blossoming of the seeds of cell biology. the last decades of the th century represented the utilitarian era of molecular biology. the introduction of genetic engineering into biological experimentation dates to the beginning of the s. it was at this time that techniques were developed that made it possible to transfer a fragment of genomic dna from one species into the genome of another species. genetic engineering now fills a predominant position in the life sciences, supported by increasingly effective biocomputing or bioinformatics techniques. it is easy to understand that expertise and a high degree of knowledge about fundamental research is necessary in order to be able to master or even invent the genetic engineering techniques that are indispensable if we are going to produce biomolecules with a therapeutic impact, such as those that are currently being used in the pharmaceutical domain: insulin, growth hormone, blood coagulation factors, vaccines, etc. the engineering sciences that make up the greater part of contemporary biotechnology have now come to the fore in many domains of the life sciences. it is thus that a modernistic and original way of investigating nature has come into being. a multiparametric model, in which biocomputing or bioinformatics and high-throughput screening reign, is added to, or even substituted for, the bernardian model for the experimental method, based on observation, an a priori hypothesis, and experimentation to verify this hypothesis by varying a single parameter at a time. the aim of this globalized approach is to integrate the multiple reactions that take place almost simultaneously in different locations of a cell into a coherent whole, to rationalize the interpretation of the dialogue that operates between the different endocellular organelles, and finally to discover how the exchanges of information between cells in an organ and between organs in multicellular organisms are set up. we are therefore witness to the emergence of an integrated biology that has been labeled "systems biology". its long-term objective is to model the functioning of living beings and to theorize them. its development is encouraged by the perspective of consequences that could revolutionize certain sectors of the human economy and of public health. today, concrete, mechanical models, in the form of biorobots and hybrid robots, and, very recently, molecular motors are added to abstract models that are based on the logic of mathematics and algorithms, ushering in the era of nanobiomachines. becoming more utilitarian, the life sciences are imperceptibly detaching themselves from traditional philosophical concepts that try to explain the modes of reasoning of the researcher, or even to impose a framework for thought that is likely to orient his or her way of doing research. looking at genetic inheritance, contemporary experimentation has shown that at all levels of the tree of nature, including man, this inheritance can be modified. aware of his or her ability to influence the functioning and the destiny of living beings, the researcher is confronted with the dilemma of a desire for knowledge versus a questioning of the use to which discoveries may be put. there has never been such a real divorce between the world of phenomena that are understood by the experimenter and the world of noumena whose intelligibility is foreign to our senses. there has never been such a wide gap between the biotechnosciences, whose possibilities are coming to be seen as limitless, and a reflective analysis of thought, which wanders between freedom of action and prohibition. as society becomes aware of the potential applications of discoveries made concerning living beings, problems of bioethics, particularly those involving reproduction, have become problems of public interest. cloning and the production of stem cells are subjects that give rise to diatribes and passions. in the near future, genotyping, which is the result of progress in pharmacogenetics, could usher in a new form of customized medicine. elsewhere, the cognitive sciences that are bringing together philosophy and psychology in the domains of computer technology and artificial intelligence, and which are tackling the processes of thought, the creative imagination and memory, will no doubt be the subject of the considerable questioning concerning research on living beings with which the experimental method will be confronted in the st century. when faced with the way in which biotechnologies have erupted into the life of society, the mind travels back to the allegorical illustration that embellishes francis bacon's novum organum (see figure ii. ) , showing vessels returning from unknown lands, loaded with precious cargoes and returning to port having sailed past the pillars of hercules. at present, the challenge has been partially met, but a great deal remains to be done. innumerable cargoes have already reached port, but what will be the destiny of this precious merchandise? after all, the seeds of the idea of technoscience were already in place in the th century, in the philosophy of francis bacon and robert boyle (chapter ii- ). bacon recommended that the governments of the time promote experimental science by the creation of laboratories equipped with high-performance instruments and libraries, by the organization of researchers into teams and by appropriate financing. the utilitarian ends of scientific research were underlined. boyle imagined a situation in which laboratories were open to society and researchers were able to accept criticism. given innovations that upset tradition, protestations arose. the pneumatic machine or vacuum pump was the subject of the fameuse diatribe between boyle and the philosopher hobbes (chapter ii- . ). hobbes criticized the validity of boyle's conclusions, drawn from experiments that he qualified as doubtful. following his words, he came to see in the discoveries of experimental science a possible threat to the power of governments and the hierarchical layout of society. such overcautious opposition to the pursuit of knowledge is in no way anecdotal, it is still a reality, with the uprooting of genetically modified plants and the veto that has been placed in certain areas on stem cell research. this type of opposition is also shown when pressures or even vetoes are in operation that take into account more the opportunism of the moment than an in-depth understanding of science and of its history and that forget that freedom of the mind is a guarantee of its creativity, because, just as in the world of arts and letters, the world of scientific research is situated outside those norms that can be modulated by state decrees. the creativity of the researcher cannot be manufactured on demand. where it exists, it still needs to be detected and encouraged. the atp-synthase -a splendid molecular machine structure at . Å of f -atpase from beef heart mitochondria direct observation of the rotation of f -atpase powering an inorganic nanodevice with a biomolecular motor mechanically driven atp synthesis by f -atpase atp-driven stepwise rotation of fo-f atp synthase key: cord- -dojdlfrv authors: doerr, megan; wagner, jennifer k title: research ethics in a pandemic: considerations for the use of research infrastructure and resources for public health activities date: - - journal: j law biosci doi: . /jlb/lsaa sha: doc_id: cord_uid: dojdlfrv the number and size of existing research studies with massive databases and biosample repositories that could be leveraged for public health response against sars-cov- (or other infectious disease pathogens) are unparalleled in history. what risks are posed by coopting research infrastructure—not just data and samples but also participant recruitment and contact networks, communications, and coordination functions—for public health activities? the case of the seattle flu study highlights the general challenges associated with utilizing research infrastructure for public health response, including the legal and ethical considerations for research data use, the return of the results of public health activities relying upon research resources to unwitting research participants, and the possible impacts of public health reporting mandates on future research participation. while research, including public health research, is essential during a pandemic, careful consideration should be given to distinguishing and balancing the ethical mandates of public health activities against the existing ethical responsibilities of biomedical researchers. although public health research is undoubtedly essential during a pandemic, the line between research and public health activities is tricky in the best of times and can blur quickly in a public health emergency. elements common to both endeavors range from study design, to the collection and use of personally identifiable and protected health information, and to analysis techniques. many point to the a priori purpose of a given initiative as a way to distinguish between research and public health activities. yet, even while public health practice focuses on assurance, assessment, and policy development, these activities might contribute to generalizable knowledge-the hallmark of research. for example, in following the deepwater horizon oil spill in the gulf of mexico, the u.s. centers for disease control and prevention (cdc) tapped into the national poison data system (npds) for the purpose of monitoring health impacts of people in the region (ie, surveillance as a public health activity). nevertheless, the cdc's utilization of the npds post-environmental disaster also demonstrated the database's utility for advancing scientific understanding of how oil spill exposures affect human health (ie a resources for potential public health research with the primary purpose of contributing generalizable knowledge). additionally complicating the divide between research and public health activities is the now widespread practice of banking of data and samples for secondary research use. during a public health emergency, research repositories are attractive, ready-made data resources and communication channels with large, and, ideally, diverse cohorts through which public health activities could be pursued expeditiously. given that emergency responses 'tend to be nonresearch,' what risks are posed by repurposing research infrastructure for public health activities? the covid- pandemic has already provided case examples highlighting key questions about the public health activities that seek to leverage existing research infrastructure. for research participants and collected nasal swabs with the goal of improving detection, monitoring, and control of influenza outbreaks in greater seattle, washington. on march , , the new york times reported on sfs's ongoing efforts to assess retrospectively the prevalence of the novel coronavirus, sars-cov- , using nasal swab samples collected for research purposes during the - influenza season. in early february , sfs began petitioning the state, cdc, and u.s. food and drug administration (fda) officials for permission to use the sfs's existing sample bank to track covid- spread. sfs participants had consented to the testing of their swabs for influenza and 'other respiratory pathogens (germs)' and to receiving these research results back from the study team, as well as for the secondary use of their data for research purposes. through the consent process, sfs had alerted participants that washington state law requires reporting of infectious diseases, including influenza, but did not discuss the use of sfs's research infrastructure, including data or samples, for other public health activities. after about weeks of rebuff, and within the context of undeniable national spread of the virus and inadequate testing for it, the sfs team decided to test the samples without the explicit approval of public health authorities or regulators. the sfs team promptly identified a sars-cov- positive result and alerted local public health officials. the sample was rerun in the washington state laboratory, where the positive result was confirmed, and the research participant was subsequently notified by public health officials. despite this apparent successful use of existing research infrastructure for public health activities, cdc and fda regulators ordered sfs to stop retrospective testing of their existing samples immediately but indicated that, with additional consent language clarifying the use of research study materials for public health activities, sfs could prospectively test participants for sars-cov- . in the first few days of march, the university of washington's ethical review board determined that, given the public health emergency, sfs had an ethical obligation to test all samples for sars-cov- , citing that sfs already had consent from participants to test for another communicable diseases and return those results and, therefore, was already engaged in both research and public health activities. on march , , state regulators again shut down retrospective testing by sfs. sfs eventually completed its retrospective testing of samples, identifying positive results across participants, including the first documented case of community transmission of sars-cov- in the usa. the back and forth between federal and state authorities, the research team, and the overseeing ethics board, which eventually culminated in the seattle flu study turning its resources toward a joint public health initiative announced march , , illustrates the complexity of the boundary between federally regulated research and public health activities and highlights key concerns about the repurposing of research infrastructure and its use for public health activities. firstly, what are the points researchers must consider as they contemplate either mining already collected research data during a public health emergency, or, as in the case of the sfs, undertaking new analyses on already collected samples in the name of public health response? secondly, what are the considerations for reporting back to research participants types of information derived from public health activities not explicitly disclosed in the informed consent process? thirdly, given the uncertainty of risks and benefits posed by public health activities, are there any additional concerns raised by legal mandates to disclose information derived from research sources to public health authorities at different governmental levels? these questions are particularly worthy of contemplation given the number of large research initiatives' data and sample banks that could potentially be called upon by public health authorities during this pandemic-including, notably, the national institutes of health's all of us sm research program. most federally sponsored human subject research activities are governed by a set of regulations known as the common rule. however, while public health research is governed by the common rule, public health activities are among those deemed 'not to be research' and therefore entirely outside of common rule's reach. this regulatory exception specifically acknowledges that public health activities may 'use information and biospecimens from a variety of sources,' including, presumably, from existing research studies or data repositories. section . (d)( )(iii) further clarifies consent is not required for secondary use of research data or biospecimens for public health activities. so regardless of whether the data used for public health activities are data that have been previously generated for research or novel data generated from research samples, public health activities are legally considered 'not research.' following from this exemption, the use of research data/specimens for public health activities does not require consent from the individuals to whom those data and samples originated. from this perspective, the sfs would not have needed additional consent of participants for sars-cov- testing had the sfs's sars-cov- testing been designated a public health activity. arguments in favor of research data use for public health activities highlight the difference between the profound physical and emotional harms wrought by historical antecedents, such as the notorious u.s. public health service syphilis study at tuskegee, and the potential dignitary harms caused by data or samples previously derived from consented research participants being used for public health activities. and if the primary risk posed to research participants by public health activity use of their data is dignitary harm, researchers should correspondingly consider the privacy rights of participants (outside of those mutually agreed upon in informed consent) before proceeding with these activities. the most influential health data privacy protections in the u.s. are codified by the health insurance portability and accountability act (hipaa) privacy rule. all covered entities and their 'business associates' must follow the hipaa privacy requirements, which generally covers people/entities providing healthcare, health insurance, or related services. under hipaa, outside of their use for care delivery, anyone wanting access to a person's records must obtain their explicit consent with a few very specific exceptions. one of the exceptions that allows for no-signature release of protected personal health information is the request of a 'public health authority.' within the regulations 'public health authority' is broadly conceptualized as a federal, state, or other territorial division's agency or authority (or their designee), whose mandate includes public health matters. notably, the u.s. national institutes of health (nih), the largest funder of biomedical research in the world, is authorized by law to assist as a 'public health authority' based on u.s. department of health and human services (hhs) interpretation dating back to at least december . as a public health authority, one might argue that the entirety of the nih's research resources-whether nih-funded researchers or participants are aware or not-might be accessible for use in public health emergencies unless other restrictions would preclude such use. notably, the sfs, funded through the private brotman baty institute for precision medicine, under nih's public health authority designation and was not designated as a public health activity by state authorities as it initially pursued sars-cov- testing. of further interest with regard to privacy protections, during a pandemic, certificates of confidentiality-which are shields protecting identifiable sensitive research information from disclosure-are potentially penetrable, as disclosures are permitted if required by laws regarding the reporting of communicable diseases, necessary for the individuals' medical care, or done with the individuals' consent. although the human subjects research regulations are relatively clear-cut with respect to public health activities, the ethical considerations for the use of existing research infrastructure for public health activities might not be. past examples of unethical practice of public health research drove the development of the current regulatory structures intended to protect human research subjects. almost two decades ago, dr nancy kass set forth an ethical framework for public health practitioners to assess the implications of public health activities, distinguishing biomedical ethics (which relies heavily upon individual autonomy) and public health ethics (which emphasizes justice, among other principles). later lee, heilig, and white ( ) provided a compelling justification for the conduct of public health surveillance in the absence of explicit consent from individual patient-participants, recognizing an ethical obligation to put any public health data collected to use and, similarly, the need to justify nonuse of data that has been collected ['to use the data we collect for public health benefit; not using the data for improving health must be justified' (at )]. as felice batlan highlights in her analysis of national security claims from the lens of public health emergency, the power to define a 'public health emergency' and the ethical concerns raised by these powers are far from straightforward. these complexities are only compounded if individual researchers themselves-rather than designated public health authorities (such as the nih as a whole) who/which are, at least, politically accountable-take it upon themselves to engage in public health activities, as did the researchers of the sfs who quietly defied state and federal guidance to continue their testing program. when research resources have been funded by public tax dollars (such as nih grants), even decisions regarding the well-intentioned donation of supplies and equipment (redirecting such items from research labs that were wound down as nonessential during the pandemic to support emergency medical compromising individual rights and interests for public benefit has a fraught and contentious history. yet even the constitutionally protected right to privacy has long been recognized as not absolute but one that is (i) conditioned upon exercise of that individual right to privacy not interfering with another's enjoyment of the same right and (ii) subject to reasonable, proportional, and necessary constraints imposed by state and local authorities fulfilling their roles to ensure public health and safety and by federal authorities supplementing such public health responses when they are inadequate. moreover, there is a compelling argument that, although not yet widely recognized, there exists a constitutional right to public health. this argument builds upon an acknowledgment that health has individual and collective aspects, as individuals alone 'cannot achieve environmental protection, hygiene and sanitation, clean air and surface water, uncontaminated food and drinking water, safe roads and products, or control infectious disease.' in any case, considering vertical conflicts between local, state, and federal authorities and issues regarding preemption is essential to reconciling researcher obligations that seem to be inconsistent or in conflict within public health law and ethics: a reader ( nd ed. )) (emphasis added). the interaction of and relationship between the right of privacy and right of public health are both interesting and important considerations; however, given such a discussion requires advanced legal analysis and involves complex legal theory, it has been left for discussion elsewhere. the specific context of a public health emergency. research repositories that cross jurisdictional boundaries could be particularly complicated in this regard when trying to ensure a uniform research experience as well as equitable distribution of risks and benefits. another dilemma highlighted by the sfs case is the considerations of reporting back to research participants' information that is not explicitly described in the informed consent process. further complicating matters in the sfs's case was the fact that their sars-cov- test had not, at the time of their original proposal, undergone traditional regulatory review and approval. the majority of the sfs's laboratories, like many research laboratories, are exempt from the clinical laboratory improvement amendments of (clia) and therefore generally are not authorized to return individual research results by the fda. the fda is the oldest consumer protection arm of the federal government and works to ensure that food, drugs, devices, biologics, and others are trustworthy. nevertheless, since its inception, the fda has been criticized for 'slowing the progress' of medical innovation and for its perceived political bent. an emergency use authorization (eua) under section of the federal food, drug, and cosmetic act (fd&c act) allows for the special use of unapproved medical products during some types of emergencies. these are sometimes called 'medical countermeasures' (and include, for example, in vitro diagnostic tests, personal protective equipment, antivirals, vaccines, and biological therapeutics) that can be used 'to diagnose, treat, or prevent serious or life-threatening diseases or conditions' when there are 'no adequate, approved, and available alternatives.' for example, in the case of sars-cov- , hhs secretary alex azar issued a determination on february , , that covid- 'is a public health emergency and that circumstances exist justifying the authorization of emergency use of in vitro diagnostics for detection and/or diagnosis of the novel coronavirus.' on february , , the fda issued guidance to 'accelerate the availability novel coronavirus (covid- ) diagnostic tests developed by laboratories and commercial manufacturers during the public health emergency.' this guidance stressed the importance of test validation, limits of detection, accuracy, and inclusivity; recommended the inclusion of a transparency statement that the test has been validated but fda's independent review of this validation is pending on all results; and required laboratories to report positive results immediately to federal, state, and local public health authorities. the return of research results has been a catch- for this reason. if researchers share information that turns out to be inaccurate or misleading, they might be held liable for the erroneous disclosure. alternatively, if researchers withhold information that could be considered clinically relevant, they might be liable for failing to disclose this information. expert panels have recommended that research results be returned with clear disclaimers regarding their potential limited reliability and validity, but participants might not fully appreciate these limitations. liability concerns (at least those related to disclosing the information), however, seem reduced in the context of actions taken in immediate response to covid- , given the liability immunity declaration issued by the hhs. while this immunity declaration unequivocally includes testing for sars-cov- within its scope of covered countermeasures, researchers do not categorically fall within the scope of covered persons. for immunity protection to be applicable, researchers would need to be recognized as 'qualified persons.' it is possible, but not a given, that nih-funded researchers could be within this category. additionally, when considering the return of unexpected research results derived from public health activities, what, if any, considerations should be given to participants right not to know, for example, in the case of sars-cov- antibody testing? while 'right not to know' considerations within the specific context of an oft-fatal infectious disease might seem a stretch, reporting such results might seem contrary to the 'no surprises' principle in biomedical research, (which essentially means that researchers should avoid data practices that fail to align with participants' understanding and expectations). when asked, the many of participants from a variety of different types of research want and expect to receive results back from their research participation. given these expectations, is it necessary to obtain consent to return research results? in the past decade, 'right not to know' has been supported primarily in terms of incidental findings on genetic assay. for many genetic conditions, there are no treatments. however, in the case of results generated as the result of a public health emergency, an individual's right not to know might be supplanted by the public good of informing them. if research resources are later used for public health activities, a question not definitively answered and likely requiring a case-by-case determination is whether reporting of those results should be treated pursuant to research norms (which historically have required consent) or public health norms (which prioritize information access to control the spread of disease over individual preferences). although the return of results might seem like a minor consideration, as 'back to work' certificates are being contemplated by many governments, the implications of whether and which sars-cov- results are to be returned should not be summarily dismissed by researchers or policy makers. such concerns underscore the need for a system of ethical board oversight or other structured consultation, to aid researchers in assessing the risks and benefits of using research resources for public health activities. finally, are there any additional reporting concerns raised by legal mandates to disclose to public health authorities at different governmental levels if consent has not been obtained specifically? public health reporting varies from aggregate, potentially anonymous data (eg, disease prevalence) to fully identifiable data (eg, contact tracing). because public health response toolkits include police powers and the ability to infringe upon individual civil liberties, there are understandable concerns regarding the numerous potential uses for research data that might be generated or seized during a public health emergency. for example, because of the immigration law implications (such as the inadmissibility on public charge grounds final rule), undocumented immigrants might be unwilling to risk seeking health care during the covid- pandemic regardless of public statements from u.s. citizenship and immigration services (within the department of homeland security) that seeking services to test, treat, or prevent covid- would 'not negatively affect' any individual in the public charge analysis. the inclusivity of a research data set being contemplated for use as part of a response during a public health emergency might require careful consideration regarding whether doing so advances or impedes an equitable distribution of the benefits and risks not only of the public health surveillance itself but also (i) the actions taken and policies developed and implemented based on those results made possible with that research resource and (ii) the subsequent willingness to participate in research. one example to highlight this dilemma is contact tracing. public health authorities in other nations have adopted contact tracing to identify networks of exposed people. given that human subject research studies now commonly include connected devices which collect data that could be valuable in contact tracing, this is of particular concern. researchers themselves struggle with appreciating the scope and implications of privacy concerns raised by the scope of big data research, leaving ethics review boards and the participants they serve at a loss. in the public health emergency context, these powerful data might only further obscure variables in the delicate calculus of individual risk and public benefit, underscoring the benefit of establishing formal consultation and review processes for public health activities that would use research data. both the volume and granularity of data collected in research repositories are orders of magnitude greater than it has ever been. however, utilizing these data-as well as the research infrastructure that supports them-in the name of public health response is not without risk. the differing legal and ethical obligations for research and public health activities are worthy of researchers' careful consideration even in the face of a public health emergency imposing powerful urgency constraints on decision-making. to be clear, these tensions should not inhibit research from proceeding during a pandemic nor the transfer of research resources to public health activities per se. rather, it is incumbent upon the research community, including biomedical legal and ethical scholars and practitioners, to reflect upon the many tensions experienced during the covid- pandemic between public health initiatives (the infrastructure and support for which has been proven woefully inadequate in the u.s.) and biomedical research (the leveraging of which might be particularly useful in times of public health emergencies, regardless of the state of public health infrastructure) and consider the creation of a formal consultative process, so that, in the future, research infrastructure might be called upon both responsibly and swiftly to augment public health initiatives. further, as ever larger and more diverse datasets are amassed, the lines between research and public health activities-not to mention clinical care-will continue to blur. the current pandemic highlights the need for each of these communities-researchers, public health authorities, and clinicians-to reconsider the legal and ethical bounds of their mandates and critically examine areas of overlap. active engagement with policy makers is needed. finally, it would be particularly prudent for the research community, equipped with its robust resources and good intentions, to think critically about how to avoid the research enterprise being simply an enabler for the continued neglect of public health in the u.s. a lab pushed for early tests christening of new coronavirus and its disease name create confusion new coronavirus cases confirmed in snohomish, king counties scan: greater seattle coronavirus assessment network public health practice is not research, participatory disease surveillance systems: ethical framework, final rule: federal policy for the protection of human subjects. fed. reg. office for human research protections, activities deemed not to be research implementing a universal informed consent process for the all of us research program how to demand a medical breakthrough: lessons from the aids fight, npr fda moves to rein in drugmakers' abuse of orphan drug law, npr § - , as amended to add section by the project bioshield act of , pub. l. - , and as amended by st century cures act, pub. l. - , u.s.c. bbb- , bbb- a, and bbb- b. for example, during the h n pandemic, the fda issued an eua so an unapproved antiviral drug could be used as a treatment, but the drug was not ultimately approved until . see authorization of emergency use of the antiviral product peramivir accompanied by emergency use information lexi white & sarah wetter, from (a)nthrax to (z)ika: key lessons in public health legal preparedness emergency use authorization of medical products and related authorities: guidance for industry and other stakeholders determination of a public health emergency and declaration that circumstances exist justifying authorizations pursuant to section (b) of the federal food, drug, and cosmetic act, u.s. c. § bbb- policy for diagnostic tests for coronavirus disease- during the public health emergency: immediately in effect guidance for clinical laboratories, commercial manufacturers, and food and drug administration staff returning individual research results to participants: guidance for a new research paradigm declaration under the public readiness and emergency preparedness act for medical countermeasures against covid- , fed at - , as '(a) any person authorized in accordance with the public health and medical emergency response of the authority having jurisdiction...; (b) any person authorized...to perform an activity under an emergency use authorization...; and (c) any person authorized to prescribe, administer, or dispense covered countermeasures in accordance with section a of the fd&c act explaining the 'no surprises principle' as 'assert[ing] that an individual's personal information should never be collected, used, transmitted, or disclosed in a way that would surprise the individual were she to learn about it health research participants are not receiving research results: a collaborative solution is needed health research participants' preferences for receiving research results the right to know and the right not to know revisited: part one, covid- 'immunity certificates': practical and ethical conundrums privileges and immunity certification during the covid- pandemic immunity passports' could speed up return to work after covid- , the gaurdian coronavirus: scientists publish advice to government undocumented u.s. immigrants and covid- , new eng ethical and legal considerations for the inclusion of underserved and underrepresented immigrant populations in precision health and genomic research in the us, ethnic israel unveils open source app to warn users of coronavirus cases, harretz travel log' of the times in south korea: mapping the movements of coronavirus carriers, the washington post singapore says it will make its contact tracing tech freely available to developers, cnbc privacy and security in the era of digital health: what should translational researchers know and do about it? detecting the impact of subject characteristics on machine learning-based diagnostic applications. npj digit med reimagining human research protections for st century science the authors would like to express appreciation to kayte spector-bagdady for formative discussions that helped prompt the development of this manuscript and for constructive feedback on an earlier draft. the content of this article is the authors' responsibility and might not represent the official views of the authors' institutions, funding sources, or any other person or entity. the authors have no conflicts of interest to disclose. key: cord- - w x tq authors: kriz, anton; nailer, christopher; jansen, karen; potocnjak-oxman, camilo title: teaching-practice as a critical bridge for narrowing the research-practice gap date: - - journal: nan doi: . /j.indmarman. . . sha: doc_id: cord_uid: w x tq abstract management researchers and management practitioners increasingly appear to be talking past each other. a solution lies in understanding that interactive management education has an important role to play in bridging this divide, but for some reason this mode of academic exchange is often forgotten. our paper broadens the stakeholder value perspective to explore how and why the interests of researchers and practitioners have diverged, before going on to present illustrative cases of programs attempting to bridge such differences. current conditions suggest that the dissonance between different cycle-times of research and practice is not sustainable with the inevitable outcome of a shrinking commons. generating new knowledge and propagating it rapidly through education and teaching-practice is an important way of disseminating higher-order research and findings. in a world where academic relevance is under threat, enabling academics to better cross such a divide is critical. marketing-management researchers and teachers ironically have their own challenge of taking what can be a complex theory (the marketing academic equivalent of a “sausage”) and making it “sizzle”. marketing and management research and marketing and management practice have been acknowledged as losing touch with each other storbacka, ) . universities reward academics for publications while the broader industry domain questions the benefits of such narrow pursuits. such is the frustration of research outcomes coming at the expense of impact and relevance (storbacka, ) that many may be satisfied if academics did publish and perish. gimmicky marketing campaigns like being in "the top youngest universities" potentially alienate more discerning industry stakeholders. while challenging the value of universities internationally is not a new concern, few could deny a growing intensity around business schools having 'lost their way' (bennis & o'toole, ) , with ongoing tensions around academic rigor versus professional relevance (möller, ) . management academics generating superfluous theories missing societal and professional challenges adds frustratingly to a global phenomenon of constant questioning of the value of universities. this overall relevance (or lack thereof) of universities and their business and management schools satisfies stark's ( , p. ) definition of a 'perplexing situation' -a 'principled disagreement about what counts.' there are several alternative conceptions of what management research versus management educators and society see as valuable (boltanski & thévenot, ) . adding to this value perplexity are anecdotes of prominent businesspeople dropping out of university. jobs, gates and zuckerberg are common examples. overlooked is that the latter two made it to harvard, and all three were extraordinarily capable. for all the anecdotes, there are multiple counterpoints and interesting stories about the value derived from education. steve jobs made a conscious decision to save his adoptee parents the educational costs of completing his degree because he could not see any real benefit. however, he still attended courses like calligraphy which he noted later as serendipitously fundamental in apple's success. ray dalio of bridgewater fame in principles (dalio, , p. ix) discusses his own academic trials and tribulations: "i'm a 'dumb shit'" is how he puts it. his transformation came via a growing interest at college in stocks. eventually he made it to harvard and now advocates for case studies, the benefits of a prestigious alumni, and continuous learning. jack ma of alibaba fame never deviated from his early passion for education. it may come as a surprise to some that ma sat the chinese university entrance exam multiple times before acceptance into teacher training. this revelation is matched only by the shock announcement that he intended to step down at alibaba to return to teaching. warren buffet's harvard rejection led fortuitously to an op-school. graham's book the intelligent investor was crucial in buffet's development with graham going on to employ his prodigy. academics like graham are often under-rated, but he provides just one example of the value of effective teaching and diffusion of research. with total student debt now estimated in the us to be beyond us$ . trillion it is understandable why stories of dropping out resonate, with the opposite getting little attention. bok's law (former president of harvard) is worth remembering: "if you think education is expensive -try ignorance." the oecd education at a glance report highlights that technological change will exacerbate the difference between higher-educated "haves and have-nots" (oecd, , p. ) , 'those who have attained only upper secondary education will earn % as much as a tertiary graduate, on average.' the broader societal value of education turns the attention to the real focus of this paper: addressing a shrinking social-research-practice commons. we accordingly believe it's time the impact of teaching and learning (tushman, o'reilly, fenollosa, kleinbaum, & mcgrath, ) was even more vigorously added to the debate about research versus practice (storbacka, ) . in doing so we challenge disciplines like marketing and management to increasingly "practise what they preach". public commentary on the marketing academy now challenges not only the research relevance but the value of the discipline more generally (jaworski, ) . too often we preach accelerated change and disruption but are conservative in responding to such trends. in an age of twitter, blogs, podcasts, sound bites and ted talks, a "stand and deliver" andragogy no longer suffices. in continually discussing whether research conforms to science i.e. 'pure basic', 'userinspired', 'tinkering' or 'pure applied' (storbacka, , p. ) , we are often falling into our own potential production-oriented myopia. timely, better, and more contemporary results are what our consumers want. many urge early career academics to publish or perish and have done so since the first use of this term in the academic man: a study in the sociology of a profession in (moosa, ) . however, the education market demands much more. teaching, executive training, book and blog publishing may be low priorities in academia compared to peer-reviewed papers, but are effective ways for researchers to gain practitioner and societal relevance. our contemporary knowledge consumers see rigor as an order-qualifier while relevance is their orderwinner. deeper understanding of rigor and relevance is key here varadarajan, ) , as is research-related academic-practitioner engaged scholarship ( van de ven, ) . a key proposition of this paper is that research alone overlooks an array of interactive educational opportunities in our armoury, and that innovative teaching practice, processes and programs can be used to help bridge the research-practice gap. this paper outlines the causes of the shrinking commons and likely consequences of leaving this unchecked, before discussing potential solutions with reference to contemporary australian cases. these cases outline interesting variants that tap into an educational sweet spot: teaching-practice, highlighting that teaching-leading to the ultimate goal of learning-is an important way of realigning stakeholder interests and strengthening the commons (boltanski & thévenot, ) . extending tushman et al.'s ( ) intervention in executive education, we conclude that teaching-practice (whether undergraduate, post-graduate, mba or executive level) has important intrinsic and extrinsic value for improving research-practice. we suggest a-priori that our professionally related disciplines of marketing and management need to acknowledge that teaching is far from an add-on to a researcher's cause. identifying that we are all part of life's descent with modification is something for which we can thank charles darwin. existentially many of us are hoping to leave a greater legacy than descending simply into this adaptation pathway. fortunately, academics share a privileged position where passing on knowledge and extending a legacy is possible. potentially academics, as researchers and teachers, are therefore offered a different type of advantage: ascent with modification. in the increasing hurly-burly of publishing, teaching and service, it is easy to forget the academic vocation offers an important chance to add to the stock of ideas. there have been few better times for sharing knowledge than the current era of servitisation, digitisation, computerisation, virtual worlds and ai. inevitably the role of universities and academics is changing and for traditionalists, not all will be to their liking. concerns about what ought to be largely go beyond the scope here. however, in fields like management and marketing a truism remains: we must be relevant to our respective professions (bennis & o'toole, ) . avoiding such responsibilities (jaworski, ) is not a solution as reibstein, day and wind ( , p. ) identified in the journal of marketing, '…it is our responsibility to work on relevant problems, make a difference, and push for institutional changes.' the aphorism often misattributed to lewin, "there is nothing more practical than good theory" (bedeian, ) resonates here. irrespective of the misattribution, lewin was a great action researcher who truly understood the value offered by combining practice and theory. for management and marketing theory to ascend-to improve, advance and remain useful-we need to have authentic and embedded dialogue and more effective communication between stakeholders. the last few decades have seen the opposite with management education and research caught up in a rigor v. relevance debate and false premise that to 'gain more of one, we must lose some of the other, in an ongoing zero-sum game' (gulati, , p. ). arguments about whether business schools should focus on pure research as opposed to writing texts and teaching are not new. germany debated such elements in the th and th centuries, with textbooks often regarded as inferior by-products aimed at codifying and simplifying theoretical concepts (watson, ) . research rigor versus practice became hotly contested in the us around the mid twentieth century (gulati, ) with 'physics envy' (tapp, ) finding its way into western business schools. if business schools wanted to be taken seriously academically, it was thought that they needed to reduce practicality for more purist pursuits. the challenge of satisfying a "commons" between the need for (a) developing rigorous research, while (b) helping business and practice, but also (c) fulfilling societal needs, was emerging. fig. illustrates the ideal where social, researcher and practice stakeholders collaborate and balance interests to advance the commons. it provides an adaptation of the 'triple helix' parameters of university-industry-government put forward by etzkowitz ( ) . social stakeholders include the work of governments that fund universities (main providers of research and tertiary education in australia), regional communities with a stake in general economic prosperity, taxpayers and employees. research stakeholders include all activities conducted by tertiary institutions including research and higher education. practice stakeholders include all activities undertaken by management practitioners, namely deploying and managing assets and resources on behalf of corporate entities both large and small. each domain represents a complex network of actors joined by national interest: social stakeholders have a stake in how both research and practice advance social good; research has a stake in social stakeholders as subjects of inquiry and as funders of their efforts; practitioners have a stake in social stakeholders as markets (consumers), as resources (employees), as regulators (government), and as arbiters of corporate conduct (public interest). although the commons is broader than an industrial marketing setting, the intersection aligns with the marketing configuration of co-creation of value (vargo, ) . we support the grönroos ( ) view of value in the commons (improvement i.e. closer circles mean users are better off). the commons is a. kriz, et al. industrial marketing management xxx (xxxx) xxx-xxx different from firm-customer relationships and we also agree (grönroos, , p. ) 'from a value creation point of view, the fact that interactions do not include two parallel processes but one merged coordinated interactive process is key.' the researcher circle or sphere, like a firm, is responsible for sharing in the creation. a fully functioning triple or quadruple helix (with the added sphere of community) sees complementary interactions between stakeholders leading to a temporal expansion and strengthening of shared components (kriz, bankins, & molloy, ) . in management education at present, the reverse seems to be happening. parties pursue divergent goals and the common ground is shrinking (bartunek & rynes, ; gosling & mintzberg, ) . the co-creation of value between research and practice is ironically often neglected by marketing and management academics. fig. implies that research incorporates teaching, yet unfortunately instruction is often treated as a chore, whereby academics are trading-off between research and the business schools' lower priority of teaching (allan, ; bennis & o'toole, ) . none of this is helping the management research and education commons. what has largely been lost in this often us dominated debate is that stakeholders are not uniform. each country has its own unique characteristics, impacting how each country's commons operates. the embeddedness between university and industry in germany, for example, with institutes such as fraunhofer (audretsch & lehmann, ) has placed a greater emphasis on engagement over international university rankings. cambridge in the uk, and mit in the us have vibrant research-teaching-practice cultures with embedded commercial institutions built into their ecosystems. china has weathered a cultural revolution and reveres further education. their research-education commons is currently more open with businesses often spinning out of university (kriz, molloy, & denness, ) , an interesting by-product of more relaxed ip laws and fewer university-commercialisation caveats. australia regularly ranks highly among university systems for publications (ranking th in the - global competitiveness index) but has a woeful record for translating research into practice. in terms of university-industry collaboration in r&d, it ranks st, placed below indonesia, tajikistan and kenya. in this commons discussion it therefore is important not to see social-research-practice as an international "one-size-fits-all". denigration of researchers and teachers in western cultures has become a common pastime, irrespective of global rankings. the teaching-only path has been tried with moderate success but is tantamount to oblivion for aspiring long-term academics (storbacka, ) . nevertheless, like gulati ( ) we are hoping for a middle ground, avoiding trade-offs between relevance versus rigor and critically teacher versus researcher. gulati discusses the academic 'boundary spanner' in helping bridge such 'tribalism'. supporting we see interactive education as an underestimated, but fundamental process for diffusing and disseminating more of our most relevant research outcomes. improving incentives to help with such causes have been spasmodic. while the research-education-practice gap remains and indeed grows, the problem for a management research and education commons exacerbates. stakeholders in the commons may be surprised by the size of investment in academics-as-researchers over academics-as-teachers. funding for many universities emanates from student numbers but rewarding academics for publications dominates, dependent on each university's and country's policies. popular writer malcolm gladwell has been scathing of wealthy universities in the us seeking philanthropic endowments while struggling universities rely solely on public funding. finding common ground and shared value, linking the three stakeholders (social-research-practice) in an ideal system, seems increasingly difficult. no-one appears to be winning. signalling to job markets that you have a degree has been described as a lottery (spence, ) with graduand employment failing to align with skills sought by employers. in many countries, students pay high fees for the privilege of tertiary education, so little wonder there is dissatisfaction with outcomes. this more compromised commons process, with divergent pressures drawing away from shared value (worse off and declining cocreation), is illustrated in fig. . social stakeholders in this shrinking commons complain, like others, that research is not relevant and also that practitioners and researchers need to place more emphasis on community and social goals beyond economic ones. social stakeholders are cognisant of increased complexity and are looking to corporate practitioners to share the burden through taxation and pro-social actions, and to researchers for better solutions to such wicked issues. the perplexing situation and commons dilemma does align well to the criteria of wicked (alford & head, ) . researchers complain that social stakeholders are not investing sufficiently in research and development; at the same time, they complain that practitioners are satisfied with quick-fix fads and ignore fundamentally sound theories. institutions compete for global prestige and resources using their prominence in elite journals as their main quantum, with a limited and closed community as principal audience and gatekeepers. as a result, what is produced offers diminishing relevance to social stakeholders and practitioners. for their part, practitioners complain that social stakeholders over-regulate the domain with researchers providing theoretical answers, out of touch with contemporary exigencies. clearly few parties are winning in what seems a a. kriz, et al. industrial marketing management xxx (xxxx) xxx-xxx race to the bottom. all parties are justified in their concerns that a shock to the existing system (loss of international student funds, reductions in government support, further decline in confidence in the sector) is likely to create a tipping point with all commons stakeholders losing out. the coronavirus covid- offers one such example with australia particularly adversely impacted due to disproportionately high chinese student numbers. open innovation (huizingh, ) is an important concept relating to a shared commons. supplementing organisational ideas through research and development exchanges whether outside-in (oi), inside-out (io) or coupled (oi and io) makes increasing sense. however, again failing to practise what they preach, universities and researchers have been laggards themselves in harnessing the benefits of more open innovation. the level of siloing within and across universities is embarrassing and not new. redressing the institutional and cultural logics that have developed over decades is complex, requiring a trans-disciplinary approach. ultimately for a management research and education commons to thrive, we need to find and nurture more common ground. apart from tushman et al.'s ( ) exposé of executive education, the role of teaching seems too-often missing from such discussions. we suggest it is time to rethink the role of teaching in order to redress the shrinking commons. the research versus practice debate barely mentions education and teaching. in brennan, tzempelikos, and wilson ( ) and storbacka ( ) , the word 'education' appears only in the bibliography, in journal titles. bartunek and rynes ( ) only mention 'education' five times. it is interesting that researchers and educators appear to abdicate responsibility for knowledge propagation. the role of educators to profess has meant different things in different settings. however, when faculty members think that research and ensuing publications are the only route to passing on ideas and knowledge, then a new dark age may have dawned. little wonder practitioners have largely turned their backs on higher-order, more abstract research and are only interested in skills. in a report on the future of education by australia's peak business lobby group, the business council of australia (future proof: protecting australians through education and skills, ), the word 'skills' appears times (omitting page headers and the proper names of specific skill development programs) while the word 'research' appears only three times. practitioners no longer believe in it. as an example, one of the authors of this study was instructed in a government project not to mention the word 'research'; it was taboo. practice and social stakeholders increasingly juxtapose problems in the research domain with poor teaching quality and outcomes. indeed teaching has its own perceived failings, exacerbated by growing expectations of universities as vocational training centres. the authors of this paper importantly have no bias against deriving knowledge-throughtheory. we believe basic theory does have value and some concepts, even in business, can be derived through more abstract pursuits. we also concur with increasing calls for utilising the abductive paradigm, and acknowledge that it offers an important form of middle ground dubois & gadde, ) . proponents of more participatory academic intervention are not new and include kurt lewin, reg revans and more recently evert gummesson. gummesson's standing in marketing theory and advocacy of techniques such as action research has been fundamental in shining a light on the critical nexus between practice, research and teaching (gummesson, ) . he understands that methodologies bringing researchers closer to marketing experience are likely also to better reflect the real "truth". falsifying social or business-related phenomena is at best transitory with the likes of popper acknowledging that social sciences are human-centred with people-related systems dynamic and constantly changing (teece, ). flyvbjerg ( ) explains the difference between the theoretical researcher and expert practitioner, articulating the role of technical skills. flyvbjerg draws on the dreyfus model (dreyfus, ) of skills development incorporating: ( ) novices (programmed in basic tasks and operational rules); ( ) advance beginners (understand context specific operations needed to function adequately); ( ) competent performers (programmed in varying situational aspects but simplify complexity to function most effectively); ( ) proficient performers (tackle more advanced positive and negative situational challenges while still growing in competency); and ( ) experts (advanced understanding and can sense, seize and adjust at a skill level). one could argue that master be added as a sixth level, where experts take on a sage-like capacity to train others, such as master black belts in six sigma. flyvbjerg ( ) , drawing on aristotle, identifies that this mastery of knowledge in academic and professional pursuits requires phronesis or practical wisdom. the late polymath peter drucker is an example of more practical wisdom, rarely relying on logical empiricism. as a more rare counterpoint, joseph schumpeter created theoretical accuracy by embedding himself in extant and historical literature. however, equally it could be argued that schumpeter's underperformance as austrian finance minister and in banking was in part due to his lack of previous practical exposure. on that level he was at best a competent performer. the late clayton christensen made the combination of good theory and good practice part of his forte, and is one of the exemplars for others to follow. reclaiming the research centre in fig. requires researchers and practitioners to agree on a 'composite object' or shared goal that unites a. kriz, et al. industrial marketing management xxx (xxxx) xxx-xxx interests and acts as a bridge (boltanski & thévenot, ) . more interactive education and teaching provides an important andragogicallogic in the context of management research and practice that could help find such common ground. bennis and o'toole assert, as described in fig. , that academics have given up important elements: 'businesspeople are starting to sense that individuals in the academy are not engaged in the same profession they practice' ( , p. ). management education and its knowledge-creating interactions, such as van de ven's ( ) engaged research scholar, can enable innovative business strategies and sometimes even generate breakthrough theories. for the sake of the commons, and our social, practice and research stakeholders, let's hope that there is an opportunity to rekindle such value (a win-winwin). however, getting institutions to change takes effort and time and requires a major rethinking of policies and incentives. there is no doubt that we will need more than chance to secure such positive outcomes. academics, whether researchers and/or teachers, have always had an important knowledge diffusion and exchange role. defining what makes a great teacher versus researcher is beyond the scope of the current article, but it is recognised that some individuals are quality teachers while others have strength in research and publications. some institutions like london business school and harvard business school value both. ask a past or present marketing student to identify a prominent academic and philip kotler is commonly nominated. kotler influenced marketing theory and practice through teaching and effective text-book communication. communicating complex research into easily understood interactive prose and visuals is probably easier when your teacher (as in kotler's case) is paul samuelson. undervalued by many academic peers is kotler's impact on transferring complex marketing concepts to the masses. the best texts and academic manuscripts are often derived from a synthesis of higher-order research. nobel winner samuelson in economics was a standout. kotler will no doubt be remembered long after most marketing theorists have been forgotten and is an example of an educator capable of extracting theory that he made useful and more practical. wilson ( ) in consilience suggested the st century would be focused on those who can synthesise. kotler managed this incredibly well (over , citations on google scholar). drucker, as the father of management, is similarly recognised. his mckinsey award at the age of for the best article published in harvard business review in , was exceptional. explaining complex theory effectively and simply to students and managers is too often underrated. it's why journals like hbr and california management review focus increasingly on communication of ideas over complex explanations of detailed empiricism. teaching is currently experiencing similar change with the advent of moocs, block teaching and more online and blended learning. vyakarnam & hartman ( , p. ) identified: 'we also ring an alarm bell for educators. media…and private sector web-based organisations are gaining rapid influence by creating programmes and content that inform and inspire. it will not take long before these forms of media are able to replace what is currently offered by educators.' some of our more applied universities are now taking up this challenge with executive training direct to the workplace. singleloop learning (understanding more of the what and how), double-loop learning (understanding more of the how and why) and importantly sharing and transferring triple-loop learning (going more to a metatheoretical why) (tosey, visser, & saunders, ) is aided in such contexts. "ah-ha" theoretical insights often derive from these latter teaching-practice why moments yet this contribution often goes unrecognised. the four co-authors of this paper are researchers and educators. like their colleagues in management and business, they are mindful of kpis that are prevalent in the university domain and within business and management. all four have taught at undergraduate and post-graduate levels at the australian national university (anu), have been practitioners, consult to industry, are keen to see students succeed and are academically and vocationally driven. their specialty areas include marketing, entrepreneurship, innovation, leadership and change management. located in the australian capital canberra, anu is part of a group of eight (go ) leading australian universities. the four cases illustrated below (cases a to d) are built around the combined authors' educational teaching and training praxis. praxis is an apt description as it emanates from the greek for a theory or lesson being enacted, and reflects that these four cases are direct lessons from academics engaging and interacting in their field. most of the cases remain operational with continuous adjustments, refinements and recalibrations. student evaluation, peer reviews and feedback from practice and social domains inform improvements. cases a to d represent multiple offerings from undergraduate through to executive and industry educational programs and training. one of the most important attributes of the anu management school teaching approach is the introduction of evidence-based management (ebm) which puts emphasis on problem/solutions from multiple perspectives including scientific literature, organisations, stakeholders and practitioners. ebm (dietz et al., ) acknowledges that better business decisions require accurate data, facts and triangulation. such material can be derived from secondary sources, surveys, qualitative material, cases and/or business operations. ultimately ebm relies on a combination of theory and practice-with kahneman's ( ) systems or more reflective thinking (as opposed to systems , more automatic thinking)-illustrative of this approach (thaler & sunstein, ) . the four cases described are customised to the anu management school philosophy but are examples of initiatives increasingly offered by a range of australian universities in programs encouraging more interactive theory-practice work integrated learning (wil). the cases are supported by peer reviews and student feedback but for the purposes of this article the material is built on the diaries, memos, observations and reflective discussions of the academics involved (kriz et al., ) . two of the authors have developed programs applying action learning and action research for more participatory approaches with cases b, c and d offering examples closer to tushman et al.'s ( ) executive and industry training workshops. this latter case (d) has been subject to participant entry and exit surveys with some of the findings published in r&d management. since , anu post-graduate management programs have required final-year students to complete, as a capstone project, a live consultancy brief for a local company, focused on an innovative growth initiative and development. by , one author had mentored of these projects for local client organisations. the strategy challenges are posed as unstructured, complex problems requiring participants to deploy and adapt multiple frameworks learned across their entire management program. the projects have yielded rich research insights into the growth strategies of local companies across multiple industries as well as accelerating their development in practice by embedding novel strategic frameworks into the local business ecosystem. another problem-based interactive example is anu's global marketing course which incorporates an international business plan competition (ibpc). this brings together business, industry and government partners to create export-ready outcomes for local companies (approximately to partnerships per annum). student teams a. kriz, et al. industrial marketing management xxx (xxxx) xxx-xxx develop global marketing plans for the respective companies with each finalist pitching their outcomes to an expert panel of industry practitioners. companies benefit from well-developed internationalisation plans, students gain real-world insights and faculty members gain access to in-situ business activity and research data. the program has two offerings: one at undergraduate and one at post graduate level. the recruitment of companies is undertaken in close collaboration with industry and government stakeholders and the program has been responsible for multiple international export successes. an induction of companies into international marketing theory and process kick-starts the ibpc, conveying expectations and providing recruited smes with a snapshot of relevant theory. tools and modules have been developed to assist students in quickly understanding and applying theory to the live businesses, with standout students selected by the smes to enter internships. anu's college of business and economics (cbe) operates a growing and extensive internship program with approximately undergraduate and postgraduate students working in businesses each semester. two of the authors of this paper provide academic supervision, ensuring students apply suitable theoretical elements to their experience. projects are research related and monitored by academic supervisors for adequate rigor. successful interns are consistently hired by participating companies. this internship program now supports international students in acquiring positions in their home market. internships are an important way of assimilating and disseminating research concepts into the field, and are increasingly common in university settings. in , the anu launched innovationact, a network-based program encouraging students, staff, academics and industry mentors to collaborate in local venture initiatives. the program has gradually expanded to include other educational institutions in canberra. over the past four years, the program has awarded over aud$ , in seed funding and other resources. in , innovationact's online platform was viewed over , times and a record thirty teams completed the program which includes a series of practice-oriented workshops based on the anu's research into new venture processes (potocnjak-oxman, ) . in , the act government launched the canberra innovation network (cbrin) to further consolidate collaborations between local government, innovators and education and research institutions. innovationact has built strong ties to cbrin, the griffin accelerator which supports local high-growth firms, and the federal intellectual property agency-ip australia-linking social, research and practice stakeholders (potocnjak-oxman, ). anu's college of business and economics has now also piloted a venture lab for further integration of entrepreneurial ventures created by students, including the promotion of joint innovation efforts. student-centred activities linked to design thinking are central to this initiative. the start-up entrepreneurial portfolio is managed and developed by one of the co-authors who is currently undertaking research into entrepreneurial opportunities and design. two authors have delivered a pilot executive education offering at the anu in partnership with a professional association of transformational change practitioners. this program has combined academic theory, ebm, and practical expertise in transformational change. the project-based course has built on action-learning approaches to accelerated executive learning (tichy, brimm, charan, & takeuchi, ) . the intention from the outset was to use the educational context to go beyond current theory and practice to co-create new knowledge applicable to both domains. the course had initially been organized around three face-to-face modules of four days each, with a final module of two days, delivered over a nine-month period, with time between modules for application and practice. the first module entitled 'understanding' introduced participants to the current state of research in transformational change. two external program sponsors then posed wicked transformational challenges, on which participants worked as members of a team, for the entire program. the second module, 'activation', presented a theoretical lens for interpreting the wicked challenges and focused on scoping the project work. module , 'implementing and adjusting', deepened participants' understanding of transformational change by drawing upon previous insights of transformational leader-practitioners. dialectic debate through modules and surfaced conflicting cognitive frames between research and practice. in module , intensifying deadline pressure exposed participants to their assumptions and blind spots, which brought frustrations into the open as they grappled with tensions between theory and practice. as the date for delivery loomed, pressure to articulate a strategy resolved the tension into synthesis. module concluded the program with presentations of findings to sponsors and a broader audience of practitioners, and sharing of team and individual reflections. along the way, the anu and executive-based teams experienced their own version of a "pressure-cooker" challenge. what seems clear and straightforward at the outset creates unexpected tensions, taking learning in new directions. a shared goal is to collaborate on a common educational platform to co-create knowledge and tools to benefit both research and practice through transformational change. although there is general agreement on overall goals, strategies for achieving these vary significantly between teachers (who prioritise theory and learning assuming that practitioners find theory valuable) and practitioner students (who emphasise tools and solutions, believing academics are unaware of current practice and unable to respond quickly enough to emerging opportunities). much time in this program has therefore been spent on building a common language. the most useful tools are theoretically based but practically useful. for example, causal loop diagrams based upon systems theory are an abstract, yet applicable lens for understanding complex dynamics associated with transformational change; an ebm framework provides a practical context for methodological rigor in data gathering, critical appraisal and reflection. participants learn transformational skills while academics find new ways to explain value propositions. a new mba program has also been launched at anu with one of the authors responsible for developing an entrepreneurship and innovation course for this program. an important component of the program is the inclusion of weekly readings of key journal articles on boundary spanning, innovation champions, ambidexterity, opportunity recognition and bricolage. the major assignment is the development of an innovation plan and strategy for implementation in each mba student's business or government department. many of the executive participants acknowledge that they are implementing the innovation plans in their workplace. for example, a state innovation award and funding was recently received by one of the participating mba students based on their respective innovation project. course objectives are adjusted in early weeks, depending on the skill, knowledge and learning needs of the cohort to ensure students gain significant practical and theoretical benefits. two of the authors have been key contributors to a master of management (mom), a jointly accredited program taught in mandarin at tsinghua university in beijing, currently in its th year. the authors have taught approximately students per cohort in the final course, new venture creation. participants are senior executives and leaders from various provinces across china. the course focuses on business model and business plan development for new ventures, including potential spinouts and spinoffs. a recent addition to the program has been the incorporation of tools for applying innovation championing to a. kriz, et al. industrial marketing management xxx (xxxx) xxx-xxx enterprises. the mom has witnessed many changes in china with enrolling participants now often already successful independent entrepreneurs. in earlier cohorts, most participants came from government or state owned enterprises (soes). increasingly, the chinese students/executives in the program are now often creating ventures pitched at service opportunities and social innovations. the new venture creation course has provided important innovation skills and encouraged student teams to create and kick-start real ventures built around team capabilities, analysis of combined resources and clarification of a feasible opportunity. a number of these new ventures have successfully transitioned from the classroom to commercial reality. a key aim is to equip all students with a theoretical and practical capacity to kick-start a venture but there are important additional objectives. advancing understanding of teamwork, applying knowledge gained from the suite of earlier management courses, and sharing vignettes of existing chinese entrepreneurs (including those in the class) are other key elements of the course. the opportunity for australian academics to immerse themselves in beijing with executives from an array of china's leading businesses, including consulting, finance and government enterprises, has obvious two-way benefits. beijing's zhongguancun district, where tsinghua is located, is renowned as china's silicon valley and is a key centre for enterprise growth and innovation. tsinghua was recently ranked th on the qs rankings ( ) which places it close to the top in asia. tsinghua cohorts now visit australia both prior to commencing the mom and at its completion, and academics have utilised these opportunities to introduce students to canberra businesses to advance firsthand understanding of australian businesses and policy environments. one of the authors is now teaching a new course on innovation to the mom cohorts and already the results are highlighting interesting additional theory-practice anomolies and insights for those taught (chinese executives) as well as those teaching (australian instructors). one of the authors has had the opportunity to incorporate theory in educational training in regional research and practitioner interventions. these stemmed from a range of quadruple helix research undertakings mostly supported by grants from industry and policy stakeholders. the author has been responsible for several place-based interventions at enterprise, cluster and regional levels and has trained and supported two significant clusters in new south wales (nsw): hunternet ( businesses as cooperative members) and central coast industry connect (a plus database). activities with these member bodies have led to training and leadership initiatives, for example the development and implementation of training programs for china, japan and korea as part of the development of australia's recent free trade agreements. additional work by this author with place-based initiatives has extended to federal government grants and initiatives. these opportunities have led to benefits like the development of an innovation champions program (with over participants, approximately per session) and two follow-up regional innovation management (rim) training programs (north east and north west tasmania). the latter were supported by skills tasmania with several findings showcased at peak academic/industry international conferences. the author supports one of these international conferences (international society for professional innovation management -ispim) with wicked problem training programs (specific for each conference destination) and action research/learning skills. the wicked problem sessions attract over participants (policy, practitioners, academics), with action research/ learning sessions limited to approximately participants (practitioners and researchers). the success of the abovementioned quadruple-helix inspired endeavours has led to closer university-industry-government-community linkages. these interventions also have international implications, extending to markets like germany. the australian-german initiatives are aimed at advancing regional development and business growth in australia. a potential programs has been underway in bendigo, victoria, with training likely modelled on a combination of successful domestic rim approaches and regional transition lessons from fraunhofer institute in the kaiserslautern district of germany. the bendigo-fraunhofer program aims to link business, academics, students and communities in what could be described as a potential international cluster of innovation (engel, ) . workshops incorporating industry, policy and regional stakeholders were conducted in as part of this initiative. another project that emulates this much deeper exchange between educators and the business environment is an action learning intervention on the central coast, nsw. nine companies were involved in an endeavour to improve industry outcomes by upskilling participants, building family business leadership skills and helping small businesses unlock growth opportunities. reg revan's action learning process of combining the right mix of formal programmed knowledge (p) with questioning and actions (q) has been inspirational in these developments. the author's role has been to develop and facilitate the initial program (now in its rd year) which is an example of van de ven's ( ) engaged scholarship. bringing out the thoughts and reflections of industry peers is paramount here. indeed, these programs go beyond simply researchers crossing the rigor and relevance divide and acknowledge that engaged scholarship is increasingly possible through targeted educative initiatives. the latter cases suggest that academic boundary spanners are becoming harder to distinguish from practitioners. nevertheless, it is important to highlight that all four authors of this study have always been keen to keep their theoretical toolkits close-at-hand. these illustrative interactive educational cases provide narratives of academics crossing the threshold between research and practice: success relies on academics' business, research and practitioner strengths, combined with advanced educational and facilitation capacity. common to the first three cases (a, b and c) are the following four characteristics that appear necessary to increase the common ground. . each case uses overarching educational goals and processes to integrate mindsets, interests and approaches of social, research and practice stakeholders, bridging and advancing understanding across the three domains of research, practice and social stakeholder while delivering valuable outcomes to each. in so doing, a common basis for value is established (stark, ) . . relevant processes are multidirectional. they do more than apply established theoretical frameworks derived from management research into the social and practice domains; they integrate the value of practitioners' and social stakeholders' experiences as contributors to new knowledge. in doing so, they stimulate researchers and students to generate unique adaptations of theory that stretch beyond established foundations, reframing them into unique clientspecific outcomes. in the process, they also challenge social and practice stakeholders to engage with theory in hands-on interactions that deepen their portfolio of mental models. . they require a core team of research-practitioners willing to experiment across the three domains (posner, ). this is often at personal cost to individual careers because efforts devoted to the goals of 'other' domains, or leading initiatives that bridge domains, attracts little career recognition. but as the value of shared outcomes becomes more visible, bridging these domains is recognised as a solution to the individual pressures each domain faces, rather than as a competing resource demand. engagement is increasingly regarded as a key process for reigniting relevance, for generating research funds, and for enhancing graduate employability. a. kriz, et al. industrial marketing management xxx (xxxx) xxx-xxx generate knowledge that propagates and extends new insights beyond direct participants to other domain members through network interactions during and after programs. the experience of business innovation (von hippel, ) suggests that researchers who expose their emerging theories to early testing in an educational context will generate more novel and robust theories faster than those who try perfecting their work in research silos. over time, these illustrative experiments help catalyse the development of additional change agents across domains in a minor social movement that gradually influences others to shape common ground. case d is a good example of more extreme testing of theory within the regional field. this has considerable benefits for both educator(s) and beneficiaries and is important in countering the divide between university, business and society. case d has also initiated multiple research spinoffs and has significantly influenced regional outcomes. practitioners have participated in such programs as have government field workers, helping diffuse benefits of these interventions more broadly. regions targeted through the interventions have often underperformed in educational achievement, with many participants actually gaining exposure to higher levels of education for the first time. cases b and c have similarly high impacts albeit all lessons are conducted within a more contained educational environment. case a targets undergraduate students transitioning into industry. theory passed on to them has immediate impacts and longer-term diffusion advantages particularly when lessons around such theory are also incorporated by employing businesses. graduands who have learned to adapt theories and create unique client-specific frameworks rapidly create networks of advocates across surrounding business ecosystems. research-practice experiments in management education are thus a highly effective means of disseminating new knowledge more rapidly than via the conventional means of academic publication. table provides a synthesis and cross-case analysis and lists benefits of these educational interactions. the table reconfigures cases to align educational gain with the level of offering: undergraduates ( nd and rd year), postgraduate (minimal work experience), mba (prior extensive work experience), executive leadership (ceos), chinese mom (executive level), and regional industry training workshops (extensive regional business and industry stakeholder expertise). the table outlines social, practice and research gains from educational interactions, with the final column focused on the shared commons and benefits of academics engaging deeply with external stakeholders. the illustrative cases and educational praxis demonstrate that cocreating new knowledge with practitioners can result in significant achievements. the contribution that each program type can make to the domains of the commons varies, consequently instruction and teaching processes need to reflect these variations. the needs of participants in an mba class, and the commensurate value for the commons, will be different to that of an undergraduate program. when training industry partners or international postgraduate students, the context is different again. thaler and sunstein's ( ) notion of the role of a 'choice architect' is particularly vital here. educators accordingly need to be cognisant of the choices they are making with what they include/exclude in relation to guiding a cohort. the amount of programmed instruction with higher-order research is dependent on the levels and context, as illustrated in the range of illustrative cases. alternatively, choices may be made to embed more action-based inquiry to ensure more practical translation is occuring. the benefit for the commons is the architectural "osmosis" which encourages co-creation and a constant "drip-feed" of new knowledge through learning by doing, and an appropriate "dose" of programmed instruction and research. this is not limited to executive training but can be facilitated across all domains as the examples within case a highlight. this paper asserts that teaching is an important bridge between research and practice, with benefits across all domains within the commons. as outlined in the introduction-the more we isolate management researchers toward publishing, and either-or pursuits around rigor and away from social phenomena-the more we threaten the relevance and quality of what management and marketing schools produce. knowledge is most effectively created through the continuous interaction of ideas, theories, schemas and empirical data in a continual dialogue, not only among researchers but also through engagement with others in the social, communal and corporate domains where human capital interacts. exclusivity of the peer-review audience toward higher-ranked research publications makes research increasingly inward-looking and inaccessible. for those in business practice, prior experience, rules-of-thumb and intuition provide poor guidance for addressing uncertainty and wicked-style problems. linking theories to practice through educational exchanges, as detailed in the cases and summarised in table , is a mutually beneficial pathway for management and marketing. one characteristic common to all the cases is a willingness on the part of each of the authors to experiment, applying a spirit of inquiry, in the educational setting. fig. identifies the gains attributed to education and teaching becoming more aligned to others. the education sweet-spot derived from the discussion is where we expect research and practice to increasingly intersect. the calls by storbacka for more abductive studies and van de ven for engaged scholarship are platforms for increased researcherderived relevance. however, similar to , we believe that the potential and scope for educators to make a difference is understudied. fig. highlights an important implication for research arms of business schools. to remain relevant to practice and social stakeholders it will be beneficial to offer significantly increased value through effective teaching programs. table highlights benefits of more applied teaching to both practice and social stakeholders. direct benefits ensue through theory being disseminated in ways respective users can and want to learn. educators and their institutions should not be limited by student evaluations but be bolder in encouraging teaching that incorporates more interesting theory-practice pursuits; they have an important role in leading the commons as theoretical plus teaching experts. applied, relevant, purposeful, action-oriented learning, undertaken in a variety of ways (formal learning for executives and mbas, internships, masters level studies, problem-based learning, industrybased competitions) is a clear way to achieve this. increasing the understanding of social stakeholders will mean better outcomes and less resistance as depicted in fig. . one caveat in the process is the quality of the teacher. while beyond the scope of this paper, it must be recognised that not all researchers are capable of this type of instruction. drucker highlighted the importance of playing to strengths. inappropriately pushing researchers beyond their specialisation into training and facilitation roles may not result in ideal outcomes. however, there are ample educators and practitioners with skills to fill the sweet-spot void. professors of practice are one avenue, but universities and faculties should also look for additional boundary spanners and to team academics with practitioners to further bridge domains and diffuse the research benefits of the commons. as tushman et al. indicated and we have further developed, the domains of the commons can only come closer if genuine efforts to change behaviours are taken beyond rhetoric to real incentives and actions. rewarding academics for successful strategies around wil needs to be matched in workload adjustments and additional rewards. practise, publish and prosper is more representative of what we academics should be aspiring to achieve rather than the publish or perish mantra. a. kriz, et al. industrial marketing management xxx (xxxx) xxx-xxx table gains from educational interaction for various stakeholders. -advances leadership quality -advocacy for lifelong learning -positive influences on key decision makers -improved financial returns and leadership skills -increased ability to manage rapid change -immersion in ebm -complex problem solving through "wicked" methods -access to research in top companies -alumni networks -on-going research grant opportunities -increases currency in research and contributions -immediate feedback mechanism for educators to industry leaders -keeps educators savvy with higher-order needs of top management teams -adds executive insights to management and marketing schools regional industry training -broaden community exposure to education -adds value to regional systems and clusters -lifts quality of regional thinking -increased enterprise and regional value -growth for individual enterprises -experience of action learning to improve business outcomes -familiarisation with new frameworks -opportunities for action research -improved likelihood of successful linkage and arc partnership grants -opportunity to test current theory in practice -ensures needs of regions inform educators, management and marketing schools and universities -builds two-way links and bonds across respective domains a. kriz, et al. industrial marketing management xxx (xxxx) xxx-xxx all three domains (research, practice and social) need to reinvigorate their commitment to the pursuit of understanding, recognising that the complexity of the commons has taken us well beyond the capacity of any one domain to achieve its goals without interaction with others. fig. identifies that education provides one bridge to effective interaction. however, multiple perspectives have moved the commons beyond taming such dilemmas with simplistic linear solutions. incentives and rewards in all three domains are complicated and there will be many trial-and-error interactions without necessarily guaranteeing immediate and measurable benefits. young career academics are under increasing scrutiny to perform well on student evaluations even though teaching is often treated as a given, and not celebrated like high level publication. these younger academics increasingly game the teaching system and avoid risk in pursuit of a simple "tick" on their education-related tenure evaluations. this only exacerbates distrust of students and stakeholders. we cannot predict which experiments will lead to which outcomes (rice, o'connor, & pierantozzi, ) but our more interactive educational cases show that there are plenty of opportunities and plenty do work. the message of this paper is clear: we cannot continue to overlook the role of teaching in strengthening relevance and building connections between research and practice. the authors are cautious in using terms like "pracademic". if this implies a boundary spanner gifted in both practice and academia then we are supportive, knowing that improved translation across our stakeholder groups is critical (kuhn, ) . we have identified that one-size-does-not-fit-all and have offered a range of examples where early and mid-career researchers can fine-tune their educative abilities. like researchers in search of better methods to bring them closer to practice, teaching and learning need similar strategies. however, instruction through practice alone is insufficient, and evidence and rigor must be maintained. we suggest that well-designed action research and action learning are effective but currently underutilised ways of co-creating new knowledge within practical environments. embedded field-work teaching and instruction definitely has merit in leading to win-win-win outcomes. cases b and d are representative of typologies where academics not only fine-tune their craft but gain immediate and critical feedback from the market. sometimes the response can be blunt but that is what reflective inquiry is all about. anne cunliffe, andrew van de ven and the late donald schon, chris argyris and reg revans should all be on the reading lists of aspiring theory-practice reflexive academics. fortunately, as the authors of this paper and their anu colleagues in design science research (dsr) are showing, such action-based reflexive studies can be published. the commons could draw insights from the industrial marketing and purchasing (imp) group's actors-activities-resources model (aar) in terms of delving more deeply into how bonds, ties and links (håkansson & snehota, ) are stimulated across our three domains. the approach outlined in this paper correlates with a more applied philosophy of improving education/research processes in the broader social sciences (flyvbjerg, ) . the five stages in dreyfus' model of skills development of novice, advanced beginner, competent performer, proficient performer, and expert is important. how many proficient performers or true experts have we got in the disciplines of business? senior academics never practising their trade in pursuit of purist academic pathways, are not likely to pass the test. our purists are needed but they have a limited market with often long term horizons. turning to our graduands, the best skill level we can probably aspire to regarding teaching and learning is a novice with some reaching advanced beginners. this is reliant on practice-based exposure, and if we deprive our students of such opportunities, we have forsaken their chance of attaining applied skills readiness. this is an important gap that practice and social stakeholders are asking us to fill (jaworski, ) . mbas and executives are calling for a different type of training. they increasingly want more advanced capabilities that add to their unique value and individual brand. this means complementing industry experience and helping them advance to proficient performers or experts. cases b and c illustrate these aspirations. even a competent performing management academic would struggle at this level given they are often lacking exposure to practice. it is something that medical professions and universities like stanford and harvard do well. microcredentials and advances that support a growth mindset (dweck, ) are increasingly fundamental at such levels, as are the benefits of sharing experiences with peers and expert lecturers. only those with equivalent understanding of "real world" practices are likely to be able to teach in such environments. university settings not willing to invest in such innovations are likely to become increasingly redundant. the benefits of getting closer to practice and social partners cannot be underestimated but whatever tools we use as academics, it is important that we draw from quality evidence to make decisions. if we stand still it will only be a matter of time before the market and burgeoning industry competitors leave us behind. susskind ( ) in his confessions of a pracademic investigates in conceptual detail the theory of engagement, starting with a practical or perceived problem, followed by analysis then conceptualisation. such spirals of inquiry focus on a deeper understanding, new knowledge and ultimately improved outcomes. much of the criticism of theory a. kriz, et al. industrial marketing management xxx (xxxx) xxx-xxx development in marketing and management is about providing post priori results and in essence informing practitioners of what they already know. as our praxis cases identify, this can be overcome when stakeholders are more intertwined and co-creating together. susskind is proud of his engagement process that he suggests outsmarts the current academic system. this starts with documenting issues before theory building, which is then followed like many of our examples, with exchange through teaching, training and active educational partnerships. our regional industry training approach (case d) is closest to gaining direct societal and industry-wide impacts. while currently an exception for universities, such approaches are slowly becoming more popular. swinburne university of technology in australia with their approach to industry . and partners like siemens follow this style of engaged wil interactivity. firenze smart (first) working lab is a european attempt trying similarly to bridge the teaching-practice gap. incentivising professors to champion industry projects for students sounds easy, but is difficult when schools do not seriously acknowledge the individual academic's publishing trade-off and resource outlay. unlike business innovation, where there is a 'fuzzy front-end' (koen et al., ) , experiments in management education seem to have a 'fuzzy middle'. the 'fuzzy middle' makes direct measures of cause-effect relations between programs and outcome inconclusive. for example, entrepreneurship education programs often measure the number of new ventures launched by participants yet in our experience few of the ventures designed in undergraduate entrepreneurship programs move beyond prototyping. nevertheless, anecdotally, the same individuals who met, interacted, and learned together, are occasionally discovered two or three years later still connected and launching completely different ventures. skills development, as in the dreyfus model, do take time to perfect with good doses of experiential learning critical for graduand development. we agree with revans ( ) that a blend of 'programmed learning' and dose of real 'action' is important for growing into a profession. novice students are not totally dissimilar to entrepreneurial start-ups with wide and varied skills that effectuate before they narrow their capability and competences. this is different to the modern more causal mba student (more competent and proficient), who is more like the incumbent firm trying to reconfigure resources, perfect their craft, and exploit new opportunities. the experienced executives in cases b and c were increasingly wanting more than simply business smarts as they pursue high-order expertise and a rare combination of practical and theoretical wisdom. our executives are increasingly striving for currency and are conscious of the up-and-comers with their newly acquired mbas. success in our digital ai world is likely to be built around continuous learning and constant program improvement. we need better measures of the longerterm impacts of educational experiments when outcomes are mediated by indeterminate 'fuzzy middle' processes. a key aspect of this study is promoting education as a fillip for reversing what storbacka ( ) and others see as a continual research relevance decline in the commons. narrowing the gap between those that teach and/or those that research should be high on the agenda of university administrators. the sweet spot for a win-win-win scenario has potential to extrapolate the cocreated value harvestable from engaged scholarship that combines not only van de ven's better research-practice but emphasises teachingpractice as well. several practical implications stem from this research. firstly, a person with a phd with extensive real-world business expertise is a rare find in australia yet this research identifies that transdisciplinary and boundary-spanning academics are critically important in helping overcome the current divide between practice and research and a shrinking commons. we should start with phds as they enter the field, and train them in the science of teaching and communicating and not simply the science of research. pracademics crossing these boundaries are ideal but we need to be careful that we don't lose research credibility in such a process. practise, publish and prosper has merit and for marketing and management academics this means communicating theory more effectively while simultaneously investigating practical and management benefits. marketing and management are not alone here but it is ironic that we don't practise our communication skills as well as we preach them in theory. osterwalder and his co-designers (business model generation and value proposition design) are typical of a new breed of consultants turning academic tools upside-down. like eric ries, this popularist new breed is simplifying complex theoretical phenomena and making it more user-friendly (frederiksen & brem, ) . their refinements are now commonly seen in the curricula. adam grant from organisational psychology is particularly astute at using different mediums to communicate difficult concepts simply and humorously. it may be time to reconsider reward structures for academics and, as indicated by etzkowitz ( ) , appoint more industrycapable academics committed to mentoring researchers and teachers. an academic balanced scorecard and workload model that properly rewards research, teaching and practice would be a great start. this stream should not simply support professors of practice but also extend more commonly to lecturers of practice and associate professors of practice. a second practical implication is to carefully design programs around a strong theoretical base, cognisant of industry and practical elements. the programs discussed in our cases were offered at later undergraduate years or pitched at postgraduates, executives and external regional stakeholders. the practice elements of these courses are built around students having multidisciplinary and comprehensive theoretical foundations. the transfer of higher-order theory occurred as a result of these foundations not despite them, with ebm and rigor still central. as david epstein in range points out, we need to be careful to promote critical and diverse abstract thinking and not limit our students to simplistic over-structured thinking. this study has only touched upon opportunities for universities and a limited array of options. there is a suite of possibilities where educators can work with practitioners for similar benefit without falling into the potential trap of becoming vocational providers. we should not lose sight of the importance of being deep and unique in our research skills and offering that as a key point of difference to external practitioners and consultants. anu is a university particularly clear in its support of humboldtian research-teaching traditions. once again, one-size does not-fit-all, and there are many universities that should also pursue their own visions and traditions. the key is to ensure the commons is growing and strengthening. more can and needs to be done to reduce the dissonance and to build a shared overarching logic for valuing management research and practice that delivers benefits to all stakeholders. globally we must recognise the value of tertiary institutions. some countries are better than others with china respecting with reverence the role of professor or teacher. it is important that the current dialogue changes and policy supports the virtues of on-going learning. industry shares in this responsibility. where wins occur, these should be celebrated and publicly acknowledged to build positive momentum. australia is poorly performing on translating an excellent per capita patent and publication performance into commercial outcomes. australia has therefore had to recently introduce a national policy to lift engagement and impact. europe and the uk are designing similar policies. significant funds are now being allocated at the national top-down level to incentivise universities to pull change up through the system. this is an interesting nudge to a national system. internationally this could be supported by global and business school rankings that additionally reward universities with phds possessing and nurturing strong practice capability. we mentioned earlier that the commons aligned well to a wicked problem or challenge. these multiple stakeholder societal problems are messy, intractable, confusing and incorporate countervailing opinions (alford & head, ) . taming such challenges requires a. kriz, et al. industrial marketing management xxx (xxxx) xxx-xxx transformational insight and ultimately more wicked innovation. changing top-down national and international systems is complicated. the ams review has added a more robust theory and practice section which is one example of changing international efforts. many of the more effective instruments for positive change are likely to be tactical and bottom-up; i.e. at your own business school, faculty and university level. a simple start for each business school, at the regional commons and quadruple helix level, is more effective engagement with industry, government and community on a place-based level. some do this well, but most don't. the imp aar model combines well with regional innovation systems models (tödtling & trippl, ) as a way of mapping and understanding each local helix and commons stakeholders. key stakeholders can then be selected as part of an advisory board to help set kpis and monitor a new academic balanced scorecard. the advisory board should measure aspects like the number of interns and the number of courses where wil is operational and effective. rewards for practice around educational programs and impact need to at least match workload rewards for publishing. advisory boards should incorporate alumni and executives that have been involved in educational programs. monitoring skill performance of graduands for key capabilities around immediate employability could also be introduced as a broader regional commons initiative. instead of simply surveying students on their satisfaction, an omnibus of the region should measure effectiveness. this should incorporate international partners to assess global impact. rewards for educational excellence, including high quality theory, could be applied and then monitored by the advisory board. reviewing research quantum and other measures to ensure the commons remains connected should also form part of the brief. as suggested, this process is not new to some business schools, but its broader diffusion would add some competitive tension to the sector. signalling is indeed a two-way activity. knowing that a university ranks highly in skills development and employability signals to the best management and marketing students where to enrol. variations across cultures and across universities are noted in the literature review; such variations are likely to have strong implications. the commons portrayed through our cases in the australian context may be different to that of other countries. this requires further exploration and offers a strong opportunity for future research. additionally, the discussion in this study is limited to one university. it would be valuable to audit programs in other institutions both in and beyond australia to gather a fuller picture of the status quo. this links closely to our identification of the need for more research to measure impacts of effective teaching-practices and interventions. shining a light on teaching-practice as a way of bridging the growing gap in research-practice is a key aim of the paper. unfortunately, this coincides with what some are suggesting is a sector on the edge of disruption. improving teaching and education in business schools, as a science and as a practice, has therefore never been more pressing. fortunately, there are many ways forward. gamification, virtual reality, artificial intelligence and social media are all potentially ways to improve the effectiveness of teaching-practice outcomes. these instruments are already in use in many of our institutions and adapted appropriately, can help turn teaching into learning with andragogical rigor. a detailed study of these tools and their role within the commons is beyond this article's scope but such investigations will be fascinating going forward. what our senior executive praxis cases highlight is that wisdom seldom emanates solely from technology enablers. case d was all about tactile and sensory peer interactions and in these settings, like in cases b and c, even the educator becomes an "actor" in a virtuous knowledge "theatre". learning how to facilitate such theatres is an important horizon for academics traditionally used to standing and delivering. the evidence highlights that marketing and management is not lacking in theory-driven research and researchers. ironically a major research challenge going forward is to find ways of attracting and incentivising better communicators and more interactive educators to research ways of turning our current researchers into better teachers. we also need to realise that there is extraordinary scope for us as educators to diffuse our research into the broader community via a range of innovative teaching practices. ultimately, in the traditions of wilhelm von humboldt, we should not lose sight of the value of research and education combining to build the moral character of our students in what should be a better commons. maybe after all our perplexing situation is that we have forgotten the roots of why universities are actually here. wicked and less wicked problems: a typology and a contingency framework the seven secrets of germany: economic resilience in an era of global turbulence academics and practitioners are alike and unlike: the paradoxes of academic-practitioner relationships a note on the aphorism "there is nothing as practical as a good theory how business schools lost their way on justification: economies of worth improving relevance in b b research: analysis and recommendations theorizing with managers to bridge the theory-praxis gap principles: life and work teaching evidence-based management with a focus on producing local evidence the five-stage model of adult skill acquisition systematic combining" -a decade later global clusters of innovation: lessons from silicon valley the triple helix: university-industry-government innovation in action making social science matter how do entrepreneurs think they create value? a scientific reflection of eric ries' lean startup approach future proof: protecting australians through education and skills the 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how to achieve both academic rigor and practical relevance? education at a glance : oecd indicators the pracademic: an agenda for re-engaging practitioners and academics support role of information systems in action-based entrepreneurship education guest editorial: is marketing academia losing its way implementing a learning plan to counter project uncertainty signaling in retrospect and the informational structure of markets the sense of dissonance: accounts of worth in economic life does publish or perish lead to stylish rubbish? jbm confessions of a pracademic: searching for a virtuous cycle of theory building, teaching, and action research: confessions of a pracademic dynamic capabilities and strategic management: organizing for innovation and growth nudge: improving decisions about health, wealth, and happiness globalizing management: creating and leading the global organisation one size fits all?: towards a differentiated regional innovation policy approach the origins and conceptualizations of tripleloop learning: a critical review research and relevance: implications of pasteur's quadrant for doctoral programs and faculty development relevanceandrigor: executive education as a lever in shaping practice and research academic-practitioner engaged scholarship. information and organization musings on relevance and rigor of scholarly research in marketing customer integration and value creation: paradigmatic traps and perspectives democratizing innovation unlocking the enterpriser inside the german genius: europe's third renaissance, the second scientific revolution and the twentieth century consilience: the unity of knowledge key: cord- -qfjet sa authors: paparini, sara; green, judith; papoutsi, chrysanthi; murdoch, jamie; petticrew, mark; greenhalgh, trish; hanckel, benjamin; shaw, sara title: case study research for better evaluations of complex interventions: rationale and challenges date: - - journal: bmc med doi: . /s - - - sha: doc_id: cord_uid: qfjet sa background: the need for better methods for evaluation in health research has been widely recognised. the ‘complexity turn’ has drawn attention to the limitations of relying on causal inference from randomised controlled trials alone for understanding whether, and under which conditions, interventions in complex systems improve health services or the public health, and what mechanisms might link interventions and outcomes. we argue that case study research—currently denigrated as poor evidence—is an under-utilised resource for not only providing evidence about context and transferability, but also for helping strengthen causal inferences when pathways between intervention and effects are likely to be non-linear. main body: case study research, as an overall approach, is based on in-depth explorations of complex phenomena in their natural, or real-life, settings. empirical case studies typically enable dynamic understanding of complex challenges and provide evidence about causal mechanisms and the necessary and sufficient conditions (contexts) for intervention implementation and effects. this is essential evidence not just for researchers concerned about internal and external validity, but also research users in policy and practice who need to know what the likely effects of complex programmes or interventions will be in their settings. the health sciences have much to learn from scholarship on case study methodology in the social sciences. however, there are multiple challenges in fully exploiting the potential learning from case study research. first are misconceptions that case study research can only provide exploratory or descriptive evidence. second, there is little consensus about what a case study is, and considerable diversity in how empirical case studies are conducted and reported. finally, as case study researchers typically (and appropriately) focus on thick description (that captures contextual detail), it can be challenging to identify the key messages related to intervention evaluation from case study reports. conclusion: whilst the diversity of published case studies in health services and public health research is rich and productive, we recommend further clarity and specific methodological guidance for those reporting case study research for evaluation audiences. the need for methodological development to address the most urgent challenges in health research has been welldocumented. many of the most pressing questions for public health research, where the focus is on system-level determinants [ , ] , and for health services research, where provisions typically vary across sites and are provided through interlocking networks of services [ ] , require methodological approaches that can attend to complexity. the need for methodological advance has arisen, in part, as a result of the diminishing returns from randomised controlled trials (rcts) where they have been used to answer questions about the effects of interventions in complex systems [ ] [ ] [ ] . in conditions of complexity, there is limited value in maintaining the current orientation to experimental trial designs in the health sciences as providing 'gold standard' evidence of effect. there are increasing calls for methodological pluralism [ , ] , with the recognition that complex intervention and context are not easily or usefully separated (as is often the situation when using trial design), and that system interruptions may have effects that are not reducible to linear causal pathways between intervention and outcome. these calls are reflected in a shifting and contested discourse of trial design, seen with the emergence of realist [ ] , adaptive and hybrid (types , and ) [ , ] trials that blend studies of effectiveness with a close consideration of the contexts of implementation. similarly, process evaluation has now become a core component of complex healthcare intervention trials, reflected in mrc guidance on how to explore implementation, causal mechanisms and context [ ] . evidence about the context of an intervention is crucial for questions of external validity. as woolcock [ ] notes, even if rct designs are accepted as robust for maximising internal validity, questions of transferability (how well the intervention works in different contexts) and generalisability (how well the intervention can be scaled up) remain unanswered [ , ] . for research evidence to have impact on policy and systems organisation, and thus to improve population and patient health, there is an urgent need for better methods for strengthening external validity, including a better understanding of the relationship between intervention and context [ ] . policymakers, healthcare commissioners and other research users require credible evidence of relevance to their settings and populations [ ] , to perform what rosengarten and savransky [ ] call 'careful abstraction' to the locales that matter for them. they also require robust evidence for understanding complex causal pathways. case study research, currently under-utilised in public health and health services evaluation, can offer considerable potential for strengthening faith in both external and internal validity. for example, in an empirical case study of how the policy of free bus travel had specific health effects in london, uk, a quasi-experimental evaluation (led by jg) identified how important aspects of context (a good public transport system) and intervention (that it was universal) were necessary conditions for the observed effects, thus providing useful, actionable evidence for decisionmakers in other contexts [ ] . the overall approach of case study research is based on the in-depth exploration of complex phenomena in their natural, or 'real-life', settings. empirical case studies typically enable dynamic understanding of complex challenges rather than restricting the focus on narrow problem delineations and simple fixes. case study research is a diverse and somewhat contested field, with multiple definitions and perspectives grounded in different ways of viewing the world, and involving different combinations of methods. in this paper, we raise awareness of such plurality and highlight the contribution that case study research can make to the evaluation of complex system-level interventions. we review some of the challenges in exploiting the current evidence base from empirical case studies and conclude by recommending that further guidance and minimum reporting criteria for evaluation using case studies, appropriate for audiences in the health sciences, can enhance the take-up of evidence from case study research. case study research offers evidence about context, causal inference in complex systems and implementation well-conducted and described empirical case studies provide evidence on context, complexity and mechanisms for understanding how, where and why interventions have their observed effects. recognition of the importance of context for understanding the relationships between interventions and outcomes is hardly new. in , canguilhem berated an over-reliance on experimental designs for determining universal physiological laws: 'as if one could determine a phenomenon's essence apart from its conditions! as if conditions were a mask or frame which changed neither the face nor the picture!' ( [ ] p ). more recently, a concern with context has been expressed in health systems and public health research as part of what has been called the 'complexity turn' [ ] : a recognition that many of the most enduring challenges for developing an evidence base require a consideration of system-level effects [ ] and the conceptualisation of interventions as interruptions in systems [ ] . the case study approach is widely recognised as offering an invaluable resource for understanding the dynamic and evolving influence of context on complex, system-level interventions [ ] [ ] [ ] [ ] . empirically, case studies can directly inform assessments of where, when, how and for whom interventions might be successfully implemented, by helping to specify the necessary and sufficient conditions under which interventions might have effects and to consolidate learning on how interdependencies, emergence and unpredictability can be managed to achieve and sustain desired effects. case study research has the potential to address four objectives for improving research and reporting of context recently set out by guidance on taking account of context in population health research [ ] , that is to ( ) improve the appropriateness of intervention development for specific contexts, ( ) improve understanding of 'how' interventions work, ( ) better understand how and why impacts vary across contexts and ( ) ensure reports of intervention studies are most useful for decision-makers and researchers. however, evaluations of complex healthcare interventions have arguably not exploited the full potential of case study research and can learn much from other disciplines. for evaluative research, exploratory case studies have had a traditional role of providing data on 'process', or initial 'hypothesis-generating' scoping, but might also have an increasing salience for explanatory aims. across the social and political sciences, different kinds of case studies are undertaken to meet diverse aims (description, exploration or explanation) and across different scales (from small n qualitative studies that aim to elucidate processes, or provide thick description, to more systematic techniques designed for medium-to-large n cases). case studies with explanatory aims vary in terms of their positioning within mixed-methods projects, with designs including (but not restricted to) ( ) single n of studies of interventions in specific contexts, where the overall design is a case study that may incorporate one or more (randomised or not) comparisons over time and between variables within the case; ( ) a series of cases conducted or synthesised to provide explanation from variations between cases; and ( ) case studies of particular settings within rct or quasi-experimental designs to explore variation in effects or implementation. detailed qualitative research (typically done as 'case studies' within process evaluations) provides evidence for the plausibility of mechanisms [ ] , offering theoretical generalisations for how interventions may function under different conditions. although rct designs reduce many threats to internal validity, the mechanisms of effect remain opaque, particularly when the causal pathways between 'intervention' and 'effect' are long and potentially non-linear: case study research has a more fundamental role here, in providing detailed observational evidence for causal claims [ ] as well as producing a rich, nuanced picture of tensions and multiple perspectives [ ] . longitudinal or cross-case analysis may be best suited for evidence generation in system-level evaluative research. turner [ ] , for instance, reflecting on the complex processes in major system change, has argued for the need for methods that integrate learning across cases, to develop theoretical knowledge that would enable inferences beyond the single case, and to develop generalisable theory about organisational and structural change in health systems. qualitative comparative analysis (qca) [ ] is one such formal method for deriving causal claims, using set theory mathematics to integrate data from empirical case studies to answer questions about the configurations of causal pathways linking conditions to outcomes [ , ] . nonetheless, the single n case study, too, provides opportunities for theoretical development [ ] , and theoretical generalisation or analytical refinement [ ] . how 'the case' and 'context' are conceptualised is crucial here. findings from the single case may seem to be confined to its intrinsic particularities in a specific and distinct context [ ] . however, if such context is viewed as exemplifying wider social and political forces, the single case can be 'telling', rather than 'typical', and offer insight into a wider issue [ ] . internal comparisons within the case can offer rich possibilities for logical inferences about causation [ ] . further, case studies of any size can be used for theory testing through refutation [ ] . the potential lies, then, in utilising the strengths and plurality of case study to support theorydriven research within different methodological paradigms. evaluation research in health has much to learn from a range of social sciences where case study methodology has been used to develop various kinds of causal inference. for instance, gerring [ ] expands on the within-case variations utilised to make causal claims. for gerring [ ] , case studies come into their own with regard to invariant or strong causal claims (such as x is a necessary and/or sufficient condition for y) rather than for probabilistic causal claims. for the latter (where experimental methods might have an advantage in estimating effect sizes), case studies offer evidence on mechanisms: from observations of x affecting y, from process tracing or from pattern matching. case studies also support the study of emergent causation, that is, the multiple interacting properties that account for particular and unexpected outcomes in complex systems, such as in healthcare [ ] . finally, efficacy (or beliefs about efficacy) is not the only contributor to intervention uptake, with a range of organisational and policy contingencies affecting whether an intervention is likely to be rolled out in practice. case study research is, therefore, invaluable for learning about contextual contingencies and identifying the conditions necessary for interventions to become normalised (i.e. implemented routinely) in practice [ ] . at present, there are significant challenges in exploiting the benefits of case study research in evaluative health research, which relate to status, definition and reporting. case study research has been marginalised at the bottom of an evidence hierarchy, seen to offer little by way of explanatory power, if nonetheless useful for adding descriptive data on process or providing useful illustrations for policymakers [ ] . this is an opportune moment to revisit this low status. as health researchers are increasingly charged with evaluating 'natural experiments'-the use of face masks in the response to the covid- pandemic being a recent example [ ] -rather than interventions that take place in settings that can be controlled, research approaches using methods to strengthen causal inference that does not require randomisation become more relevant. a second challenge for improving the use of case study evidence in evaluative health research is that, as we have seen, what is meant by 'case study' varies widely, not only across but also within disciplines. there is indeed little consensus amongst methodologists as to how to define 'a case study'. definitions focus, variously, on small sample size or lack of control over the intervention (e.g. [ ] p ), on in-depth study and context [ , ] , on the logic of inference used [ ] or on distinct research strategies which incorporate a number of methods to address questions of 'how' and 'why' [ ] . moreover, definitions developed for specific disciplines do not capture the range of ways in which case study research is carried out across disciplines. multiple definitions of case study reflect the richness and diversity of the approach. however, evidence suggests that a lack of consensus across methodologists results in some of the limitations of published reports of empirical case studies [ , ] . hyett and colleagues [ ] , for instance, reviewing reports in qualitative journals, found little match between methodological definitions of case study research and how authors used the term. this raises the third challenge we identify that case study reports are typically not written in ways that are accessible or useful for the evaluation research community and policymakers. case studies may not appear in journals widely read by those in the health sciences, either because space constraints preclude the reporting of rich, thick descriptions, or because of the reported lack of willingness of some biomedical journals to publish research that uses qualitative methods [ ] , signalling the persistence of the aforementioned evidence hierarchy. where they do, however, the term 'case study' is used to indicate, interchangeably, a qualitative study, an n of sample, or a multi-method, in-depth analysis of one example from a population of phenomena. definitions of what constitutes the 'case' are frequently lacking and appear to be used as a synonym for the settings in which the research is conducted. despite offering insights for evaluation, the primary aims may not have been evaluative, so the implications may not be explicitly drawn out. indeed, some case study reports might properly be aiming for thick description without necessarily seeking to inform about context or causality. we recognise that definitional and methodological plurality is not only inevitable, but also a necessary and creative reflection of the very different epistemological and disciplinary origins of health researchers, and the aims they have in doing and reporting case study research. indeed, to provide some clarity, thomas [ ] has suggested a typology of subject/purpose/approach/process for classifying aims (e.g. evaluative or exploratory), sample rationale and selection and methods for data generation of case studies. we also recognise that the diversity of methods used in case study research, and the necessary focus on narrative reporting, does not lend itself to straightforward development of formal quality or reporting criteria. existing checklists for reporting case study research from the social sciences-for example lincoln and guba's [ ] and stake's [ ] -are primarily orientated to the quality of narrative produced, and the extent to which they encapsulate thick description, rather than the more pragmatic issues of implications for intervention effects. those designed for clinical settings, such as the care (case reports) guidelines, provide specific reporting guidelines for medical case reports about single, or small groups of patients [ ] , not for case study research. the design of case study research in health care (descarte) model [ ] suggests a series of questions to be asked of a case study researcher (including clarity about the philosophy underpinning their research), study design (with a focus on case definition) and analysis (to improve process). the model resembles toolkits for enhancing the quality and robustness of qualitative and mixed-methods research reporting, and it is usefully open-ended and non-prescriptive. however, even if it does include some reflections on context, the model does not fully address aspects of context, logic and causal inference that are perhaps most relevant for evaluative research in health. hence, for evaluative research where the aim is to report empirical findings in ways that are intended to be pragmatically useful for health policy and practice, this may be an opportune time to consider how to best navigate plurality around what is (minimally) important to report when publishing empirical case studies, especially with regards to the complex relationships between context and interventions, information that case study research is well placed to provide. the conventional scientific quest for certainty, predictability and linear causality (maximised in rct designs) has to be augmented by the study of uncertainty, unpredictability and emergent causality [ ] in complex systems. this will require methodological pluralism, and openness to broadening the evidence base to better understand both causality in and the transferability of system change intervention [ , , , ] . case study research evidence is essential, yet is currently under exploited in the health sciences. if evaluative health research is to move beyond the current impasse on methods for understanding interventions as interruptions in complex systems, we need to consider in more detail how researchers can conduct and report empirical case studies which do aim to elucidate the contextual factors which interact with interventions to produce particular effects. to this end, supported by the uk's medical research council, we are embracing the challenge to develop guidance for case study researchers studying complex interventions. following a meta-narrative review of the literature, we are planning a delphi study to inform guidance that will, at minimum, cover the value of case study research for evaluating the interrelationship between context and complex system-level interventions; for situating and defining 'the case', and generalising from case studies; as well as provide specific guidance on conducting, analysing and reporting case study research. our hope is that such guidance can support researchers evaluating interventions in complex systems to better exploit the diversity and richness of case study research. abbreviations qca: qualitative comparative analysis; qed: quasi-experimental design; rct: randomised controlled trial complex systems 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technology assessment in its local contexts: studies of telehealthcare trading quality for relevance: non-health decision-makers' use of evidence on the social determinants of health we can't be % sure face masks work -but that shouldn't stop us wearing them | trish greenhalgh. the guardian what are case studies? in: what's wrong with ethnography the case study approach case study: a bridge across the paradigms case study research and applications: design and methods: sage methodology or method? a critical review of qualitative case study reports developing the descarte model: the design of case study research in health care an open letter to the bmj editors on qualitative research a typology for the case study in social science following a review of definition, discourse, and structure judging the quality of case study reports care guidelines for case reports: explanation and elaboration document publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable authors' contributions jg, mp, sp, jm, tg, cp and ss drafted the initial paper; all authors contributed to the drafting of the final version, and read and approved the final manuscript. this work was funded by the medical research council -mrc award mr/ s / hcs: case study, context and complex interventions (triple c). sp was additionally funded by the university of oxford's higher education innovation fund (heif). not applicable (article based on existing available academic publications)ethics approval and consent to participate not applicable the authors declare that they have no competing interests. key: cord- - rcptor authors: perez-gracia, jose luis; awada, ahmad; calvo, emiliano; amaral, teresa; arkenau, hendrik-tobias; gruenwald, viktor; bodoky, gyorgy; lolkema, martijn p; di nicola, massimo; penel, nicolas; vera, ruth; sanmamed, miguel f; douillard, jean-yves title: esmo clinical research observatory (ecro): improving the efficiency of clinical research through rationalisation of bureaucracy date: - - journal: esmo open doi: . /esmoopen- - sha: doc_id: cord_uid: rcptor during the last years, there has been a dramatic increase in the administrative and bureaucratic burden associated with clinical research, which has clearly had an impact on its overall efficiency and on the activity of clinical investigators and research teams. indeed, the supervision of the adherence of clinical research to good clinical practice (gcp) guidelines and legal regulations is of the utmost importance. yet, while such regulations have remained largely unchanged during recent years, the number of administrative tasks and their complexity have grown markedly, as supported by the results of a survey performed among clinical investigators that we report in this manuscript. therefore, many investigators believe that it has become necessary to undertake a rigorous analysis of the causes and consequences of this issue, and to create a conduit to channel the advice from experienced investigators regarding clinical research procedures, in order to improve them. based on these premises, esmo has launched the esmo clinical research observatory (ecro), a task force that will analyse different aspects of clinical research. ecro will aim to provide the views of esmo on clinical research procedures based on the feedback from clinical investigators, under complete adherence to the declaration of helsinki, the gcp guidelines and any other applicable legal regulations, while at the same time showing profound respect for all the stakeholders involved in clinical research. this manuscript provides the background and rationale for the creation of ecro, its planned activity and an analysis of the current administrative burden in clinical research with recommendations to rationalise it. indeed, we expect that this effort shall lead to a relevant improvement in the care of patients and in the development of clinical research. during the last years, there has been a dramatic increase in the administrative and bureaucratic burden associated with clinical research, which has clearly had an impact on its overall efficiency and on the activity of clinical investigators and research teams. indeed, the supervision of the adherence of clinical research to good clinical practice (gcp) guidelines and legal regulations is of the utmost importance. yet, while such regulations have remained largely unchanged during recent years, the number of administrative tasks and their complexity have grown markedly, as supported by the results of a survey performed among clinical investigators that we report in this manuscript. therefore, many investigators believe that it has become necessary to undertake a rigorous analysis of the causes and consequences of this issue, and to create a conduit to channel the advice from experienced investigators regarding clinical research procedures, in order to improve them. based on these premises, esmo has launched the esmo clinical research observatory (ecro), a task force that will analyse different aspects of clinical research. ecro will aim to provide the views of esmo on clinical research procedures based on the feedback from clinical investigators, under complete adherence to the declaration of helsinki, the gcp guidelines and any other applicable legal regulations, while at the same time showing profound respect for all the stakeholders involved in clinical research. this manuscript provides the background and rationale for the creation of ecro, its planned activity and an analysis of the current administrative burden in clinical research with recommendations to rationalise it. indeed, we expect that this effort shall lead to a relevant improvement in the care of patients and in the development of clinical research. the only thing that saves us from bureaucracy is its inefficiency. eugene mccarthy ( mccarthy ( - growth of the burden of bureaucracy in clinical research in recent years, the administrative and bureaucratic burden associated with clinical research in oncology has grown along with its clinical success and technical complexity, generating a profound impact on the activity of investigators and clinical research teams. indeed, regulation and monitoring are fundamental to guarantee the safety and the rights of patients and the quality of the data, according to the high standards that characterise clinical research, as defined by the declaration of helsinki, the guidelines for good clinical practice (gcp) and the applicable regional and local legal regulations. nevertheless, in the current scenario, physicians dedicated to clinical research have begun to feel overwhelmed by such administrative tasks, and it has become difficult for them to understand the appropriateness of certain procedures, to set a limit to the amount of time dedicated to administrative tasks, or even to perform their clinical role with an adequate level of autonomy. while adherence to the declaration of helsinki, gcp guidelines and local regulations remains unquestionable, many experienced investigators believe that their overinterpretation and misinterpretation by clinical research organisations (cros), and their substitution by their own internal standard operating procedures have significantly increased the administrative burden. [ ] [ ] [ ] the number of processes that need to be documented and the complexity of the documenting procedures and templates have increased dramatically, creating an unsustainable pressure on the investigational site staff. another layer of complexity has been added by the incorporation of cumbersome online platforms which require intricate procedures just to access them, and which generate myriads of emails that overwhelm the capacity of investigators and research teams. exhaustive training courses for administrative processes, which frequently involve examinations, are imposed on clinical research teams to qualify as trial site staff. frequently, such trainings are requested even from individuals that are not related to those specific administrative tasks. the administrative overload even affects the medical records of the patients, which are frequentlyand wrongly-considered as the most suitable place to document administrative procedures, thus distorting their true function. the number of meetings required during the development of trials has also increased relevantly, and once again, the focus of such meetings is often administrative. regrettably, investigators are unable to overcome, or even to discuss these situations. the appropriateness of such procedures is justified by the 'necessity to comply with gcp and legislation', even though such regulations do not usually require such high levels of detail and complexity. yet, the decisions are non-negotiable because the flow of communication is unidirectional and does not consider other opinions, including those coming from experienced investigators. negative impact of the increased administrative burden on investigators, research teams and patients this increased burden of bureaucracy makes poor use of the limited time physicians have available, generating frustration, loss of motivation and complaints from experienced investigators as well as decreasing the interest of young physicians towards developing a clinical research career. in addition, non-essential administrative procedures significantly increase the economic costs of clinical research and contribute to delays in trial implementation, thus negatively impacting the flow of drug development and hampering patient access to new drugs. this is particularly relevant in the setting of independent academic clinical research, which is critical for patient-focused drug development. more importantly, there is no evidence that this increased complexity leads to greater patient safety. a relevant example are pharmacovigilance procedures, which commonly consist of submitting all the available individual serious adverse events to investigators and documenting their reception, without any intent to summarise, prioritise or classify them. sometimes, the events even include those observed during screening periods, when the patient has not yet received the investigational drug. this leads to an excess of information that becomes unmanageable and prevents investigators from being effectively updated on the safety of investigational drugs. another example is the high number of informed consent versions generated in some studies and their complexity, which are difficult to understand by patients, and may even generate distrust of the study and the research team. clinical interference of protocols with best medical practice finally, on limited occasions, discrepancies arise between investigators and sponsors or cros regarding the most appropriate clinical management for some patients participating in clinical trials. for example, some study protocols mandate discontinuation of the study treatments based on the strict application of response criteria, disregarding the medical judgement of the investigators in clinically complex situations (eg, the possibility of maintaining treatment in the case of persistent clinical benefit; or to radically treat oligometastatic progressive disease while maintaining systemic therapy). frequently, in these situations, the strict interpretation of the protocol prevails, generating clinical interference with the best clinical judgement of the physician, who is responsible for the care of the patient; and potentially compromising the rights, safety and well-being of trial participants, in clear contrast with the main goals of the declaration of helsinki and the gcp guidelines. conversely, many protocols allow physicians to take the final decisions about these complex cases, either directly or after discussion with the medical coordinator of the study, thus confirming the validity of this approach. in order to evaluate the opinions of clinical investigators on these issues, we developed an online survey that was distributed among esmo members, esmo faculty, oncologists selected for their wide experience in clinical research and investigators attending the esmo meeting in barcelona. the characteristics of the responders are presented on table and the responses are displayed on table . the results clearly support that there is high agreement among investigators about the excessiveness of administrative procedures on clinical research (mean score: . over a - scale), which they consider an obstacle for the development of clinical research (mean score: . ). the survey also shows wide consensus about the feasibility to limit such procedures without compromising the safety and the rights of the patients and the quality of the data (mean score: . ); and about the necessity to incorporate the feedback from physicians about the procedures related to clinical research (mean score: . ). interestingly, scores were higher among oncologists with more than years of experience in clinical research (table ) . while we acknowledge the limitations of surveys, we believe that these results accurately reflect the opinions expressed by the vast majority of oncologists dedicated to clinical research. consequently, they should lead stakeholders to perform a profound analysis of the current situation and to implement the appropriate changes. ecro will pursue the following areas of development, which are summarised in box : . rationalisation of the bureaucratic burden associated with clinical research, based on strict adherence to current legal regulations and to any future amendments, and on showing respect to the time and expertise of clinical investigators and research teams, who should be mainly focused on clinical and research issues. importantly, the ecro will not enter into a debate on which administrative procedures are necessary and which are ancillary, but will rather support the following recommendations: a. limiting the administrative documents required for trials to those required by gcp and legal regulations: we recommend that section of the gcp guidelines ('essential documents for the conduct of a clinical trial') is strictly followed, and that documents not included in that section are therefore considered non-essential. of note, gcp guidelines only request that a small number of the essential documents be signed by the investigator; and they consider it "acceptable to combine some of the documents, provided the individual elements are readily identifiable" (gcp . ). b. using simplified document templates: for example, while gcp guidelines merely-and reasonably-request that "the investigator should maintain a list of appropriately qualified persons to whom the investigator has delegated significant trial-related duties" (gcp . . ), most study delegation logs have become extremely complex and labour-intensive to complete, and box initial areas of development and methods pursued by ecro. initial areas of development usually the investigator must assume this increased complexity. instead, we recommend the use of simplified templates. c. avoid redundant documentation: a relevant example is the documentation process of the informed consent process. while gcp clearly state that the "informed consent is documented by means of a written, signed and dated informed consent form" (gcp . ), it has become routine practice to request that investigators duplicate this documentation in the medical records, frequently including administrative details, such as the version code of the document and so on. such a process is redundant and lacks any value, since it is not signed by the patient, as required by gcp. d. avoid complex electronic resources: cumbersome applications with intricate access procedures should be replaced by user-friendly and intuitive systems, which should not require unnecessary trainings (ie, those dedicated to performing simple procedures, such as signing a document; or those dedicated to a resource that a particular member of the staff will not use). in addition, such resources should rely on single access platforms, rather than on multiple ones. e. avoid repetitive and unnecessary meetings, and control their duration: start-up and monitoring meetings should focus on the core aspects of protocols and on clinical issues, and should avoid reviewing general aspects, ancillary administrative procedures and so on. f. optimise the delegation of trial procedures from investigators to other members of the research team: indeed, the training and preparation of all the members of the research teams (ie, data managers, research nurses and so on) allows them to assume complex responsibilities. therefore, optimising the delegation procedures will facilitate the development and monitoring of trials. . avoid the clinical interference of protocols with best medical practice: protocols describe the methodology that should be followed to conduct the trial, but they cannot foresee the whole variety of individual clinical situations that patients undergo and therefore they cannot substitute best medical practice. consequently, protocols should avoid dictating strict and non-personalised instructions to manage clinically complex situations, such as discontinuation of an anticancer treatment whenever clinical benefit may persist, or when oligometastatic progressive disease may be controlled with radical treatment; or deny delivery of determined supportive therapy, such as palliative radiation. protocols may consider such events as progression with regard to the interpretation of the study data, but treatment discontinuation should generally be decided by the investigators, after thorough discussion with their patients. indeed, sponsors' recommendations regarding management of relevant toxicities, based on the centralised collection of information, should not be considered as clinical interference, particularly in the setting of early drug development. neither should the decision to discontinue a study or a specific cohort of a study be considered as clinical interference. nevertheless, the sponsor must inform investigators timely to avoid miscommunication with patients. institutional review boards (irbs) should specifically examine the existence of clinical interference in new protocols and should carefully review any potential situation of clinical interference in ongoing protocols, requesting appropriate amendments, as required by gcp guidelines (gcp . . ). . pharmacovigilance administrative procedures: reporting the safety of medicines in the routine and clinical research settings is essential to maintain physicians updated about adverse events, with the objective of improving patient safety. in order to increase the efficiency and the effectiveness of the process, ecro believes that current logistical and administrative pharmacovigilance procedures for reporting events from the sponsor to the investigators should be thoroughly revised and simplified. while the development of the optimal process is beyond the scope of this manuscript, and should be defined through the collaboration and consensus of all stakeholders, we believe that the current practice of unstructured submission of all reports on individual serious adverse events to investigators is difficult to manage and may even dilute the desired effect of proper safety information. adequate procedures, such as interpreting and periodically summarising the available information, should be evaluated. ecro will employ the following methods to attain its objectives (box ; the occurrence and causes of excessive delays in starting treatment in cancer patients due to centralised molecular testing [ ] [ ] [ ] ; the broadening of eligibility inclusion criteria in clinical trials, to avoid disparities in care [ ] [ ] [ ] ; the consequences of outsourcing the monitoring of trials ; the need to simplify informed consents and so on. . issue recommendations regarding relevant topics related to clinical research. importantly, ecro will not evaluate specific cases, given the regional differences in legislations and patient management; and since decisions must depend on the structures that are legally in place in each country (eg, irbs and other ethics committees). ecro may contact local scientific societies to seek their advice about specific regional issues. . foster the involvement of experienced clinical investigators in the training of study monitors, to provide them with the clinical perspective of clinical research. . collaborate with other national and international associations related to clinical research, especially those in the area of oncology, in order to endorse, revise and improve this initiative. the ecro will analyse different aspects of clinical research and will provide the views of esmo on clinical research procedures based on the feedback from clinical investigators, under complete adherence to the applicable legal regulations, and showing profound respect for all the stakeholders involved in clinical research. we expect that this effort shall lead to a relevant improvement in the care of patients and in the efficiency of clinical research. indeed, we expect that all the stakeholders involved in clinical research will recognise the need to critically analyse these relevant problems and to assume their responsibility in implementing the necessary changes to overcome them. consequently, rather than attempting to audit and to control clinical trial procedures, ecro will publicly acknowledge the merits of those entities (ie, cros, sponsors, irbs, clinical research teams, regulatory authorities and so on) who succeed in reviewing and simplifying procedures without compromising-or even improving-the quality of clinical research, and consequently, the well-being of patients. we also expect that our fellow physicians will support this initiative and that, while fully endorsing the need to stringently monitor clinical trials, they will demand respect of their time and their expertise and leadership in the organisation and completion of research projects and clinical care of patients wma declaration of helsinki -ethical principles for medical research involving human subjects guideline for good clinical practice e (r ) advocating 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criteria and requirements continue to increase in number and complexity patients with advanced nonsmall cell lung cancer: are research biopsies a barrier to participation in clinical trials? lung cancer at the intensive care unit: the era of targeted therapy the challenge of molecular testing for clinical trials in advanced non-small cell lung cancer patients: analysis of a prospective database re-evaluating eligibility criteria for oncology clinical trials: analysis of investigational new drug applications in broadening eligibility criteria to make clinical trials more representative: american society of clinical oncology and friends of cancer research joint research statement modernizing clinical trial eligibility: recommendations of the american society of clinical oncologyfriends of cancer research minimum age working group open access this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by-nc . ) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, any changes made are indicated, and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by-nc/ . /. orcid ids jose luis perez-gracia http:// orcid. org/ - - - teresa amaral http:// orcid. org/ - - - key: cord- -z k cujs authors: liu, yali; avello, maria title: status of the research in fitness apps: a bibliometric analysis date: - - journal: nan doi: . /j.tele. . sha: doc_id: cord_uid: z k cujs fitness applications have undergone considerable development in the last few years and becoming popular and significant in both academic and practical areas. however, contributions to the systematic mapping of this field continue to be lacking. this paper constitutes the first bibliometric study in this field to better understand the current state of research. we examined records from databases scopus and web of science (core collection) using several bibliometric analysis methods. all the records on this emerging topic were published between and . we processed these records using statistical analysis and science mapping. the bibliometric analysis included the year of publication, journal name, citation, author, country, and particularly, research methodology. additionally, we used the vosviewer software to perform bibliometric mapping of co-authorship, co-citation of authors, and co-occurrence of keywords. this field of study, it was found, is currently in its precursor stage, contributing primarily to the fields of medicine, computer science, and health sciences. the united states appeared to have made the largest contribution to this field. however, author productivity, number of citations, and number of core journals all indicated a high degree of fragmentation of research in this filed. remarkably, scientific research in this area is expected to progress tremendously over time. overall, this study provides basic data and research classifications for the initial phase of research and research direction for future research in this area. with the global outbreak of the covid- pandemic in , almost every country is facing problems concerning the shortage of medical and healthcare resources, and people have become more aware of the importance of following a healthy lifestyle and incorporating physical exercise into their daily lives. as the most downloaded type of mobile health applications (mhealth apps), fitness apps can help people manage their nutritional intake, assist their participation in fitness and physical activities, and promote a healthy lifestyle. therefore, these apps are gradually occupying the commercial mobile app market (beldad & hegner, ) . nowadays, fitness apps are rapidly developing in the commercial application market and are attracting the attention of academia (beldad & hegner, ) . numerous studies have implemented empirical protocols to verify the results of using fitness apps for improving the level of physical activity and/or diet in users (schoeppe et al., ) . however, from the academic side, it is still a novel and young area of research. as a diverse field of research that is related to an emerging phenomenon, and with the integration of new technologies, the research available on fitness apps is still scarce. both empirical research and theoretical orientation reviews, mostly focus on summarizing the functions and features of fitness apps and user perspectives. as a result, there appears to be a lack of more macro and objective quantitative research in this field. and the various types of literature are not as substantial or abundant compared to other mature areas of research. it is necessary to carry out a bibliometric study to know the main empirical and theoretical orientations in this case. the data obtained from the bibliometric analysis will be essential to assess the intensity and orientation of new lines of research (bartoli & medvet, ) . moreover, it is essential to classify the existing research in the research field to track the research progress and research trends in the field (gaviria-marin et al., ) . bibliometrics study can achieve this objective. it helps display past academic research activities and achievements visually. to our knowledge, there is no bibliometric study in the field of fitness app research, even though this type of literature has been used widely in other fields in recent years (zanjirchi et al., ) . bibliometrics can supplement existing experiments and review studies, help researchers identify hidden research lines, hot issues, and research methods in the field, and reduce the problems of neglecting certain excellent articles due to the deviation of researchers' subjective judgments (zanjirchi et al., ; veloutsou & mafe, ) . therefore, this study offers a bibliometric study of the advancements in research on the mobile-fitness app. it is based on data from a bibliometric analysis. it seeks to assess the intensity and research topics dominant in the scientific community when it comes to this emerging phenomenon, focusing explicitly on the fitness segment of mhealth. this study also aims to provide relevant data and bibliometric indicators for the initial stage of fitness application research and provide primary data for advancing future research in this field. the data used in this study is obtained from two leading databases for scientific research: scopus and web of science. the research is organized as follows. first, a research background is provided. second, the research methods and the sources of research data are outlined. third, the results are presented and discussed. finally, the main conclusions, limitations, and further opportunities for research are stated. nowadays, mobile apps pertain to a wide range of topics and areas of users' personal and social lives and fulfill various purposes. the use of advanced medical information systems and telematics applications is one of them, which has resulted in the increased availability of medical services at lower overall costs (kao et al., ) . medical and sanitary institutions have begun to appreciate the potential of mhealth apps for communication with patients as well as for the utilization of mobile devices that are specifically designed to monitor specific biomedical data. mhealth is defined as the provision of medical care and health-related services through mobile communication devices that enable user-interaction capability (cummiskey, ; lupton, ) . "mobile health (mhealth) has become an essential field for disease management, assessment of healthy behaviors, and for interventions on healthy behaviors" (mas et al., , p. ). there are two main areas of implementation of mhealth apps: in professional medical practices (both on the side of doctors and patients; e.g., skyscape, mysugr), and selfmonitoring of healthy habits (e.g., myfitnesspal). the first area has a field of an app exclusively in the healthcare field, involving the relationships between doctors and their patients. the second area represents fitness apps, which is the subject of this study, is concerned with the personal monitoring of the activities of individuals within the framework of adopting healthy lifestyles or disease prevention habits, and this category is often implemented through commercial apps that are developed without the supervision of medical administrations. the term "fitness" has a wide semantic field: on the one hand, it refers to the practice of physical exercise to obtain or maintain good body shape and composition; on the other hand, more generally, it refers to a good state of vitality and physical well-being (corbin et al., ) . since the s, academic as well as medical attention to health-related physical fitness (hrpf) has increased considerably. fitness is understood within the hrpf framework, which is defined as a set of people's abilities to perform certain physical activities, their energy level to perform daily tasks, and their capacity to reduce the risk of diseases related to sedentarism (cheng & chen, ). the who warns of the development of non-communicable diseases, the pathologies of which are associated with unhealthy lifestyles and diets, as these diseases currently constitute a serious cause of death worldwide (who, ) . in particular, the who has established a set of minimum criteria for physical activity for different age groups as well as balanced dietary patterns to maintain optimal health conditions such that people can achieve a reduction in risk factors for non-communicable diseases, including cancer, cardiovascular ailments, and diabetes. the high rate of obesity is one of the most worrying factors for health globally, particularly in developed countries, but also in emerging countries, with a drastic growth among children (anderson et al., ) . for this reason, the who recommends avoiding a sedentary lifestyle and following balanced diets for all age groups. interventions for population self-management, based on changes in lifestyle, are effective in reducing risk factors and the incidence of non-communicable diseases (burke et al., ) . the use of applications on mobile devices has become a key factor in helping and advising people on the adoption of healthy lifestyles in the st century. although some clinicians lack confidence in the protocols and recommendations of fitness apps, these fitness apps have a great potential to be effective due to their ability to educate a large portion of the population on healthy habits at a low operating cost (blackman et al., ) . the methodology used in this research work is depicted in figure . it consists of four steps. we identified "fitness app" as the field for this study with the aim of finding as many articles as possible on fitness-related apps closer to health behaviors than to a professional medical approach. however, in the compilation of the final set of articles, we also included those that, without being strictly articles on fitness apps, contained relevant keywords linked to the subject of study, even though they were papers dealing with other types of mhealth apps. the data was obtained from two databases: scopus and web of science core collection (wos). these two databases are currently the leading sources for indexing scientific articles and allow for the collection of data from a large number of journals (adriaanse & rensleigh, ) . scopus owns high-quality and reliable coverage and complete data for each reference. it is the largest abstract and citation database for peer-review literature (zanjirchi et al., ) . the wos is also recognized by the scientific community as a digital bibliometric platform with high-quality literature, which can also provide metadata for bibliometric analysis and covers a wide range of disciplines (gaviria-marin et al., ; hew, ) . the combination of more than one database for mining scientific data can provide more robust results for the bibliometric analysis (de oliveira et al., ) even though it makes it necessary to integrate the information from both databases with different structures and review the articles one by one. mining the data is the most basic and crucial step to obtain valuable and credible research results. the search for this study was conducted in april and included all relevant publications until the end of december . the study focused on scientific research related to personal care applications of fitness, using the keywords "fitness app" and its plural form in english for searching through titles, abstracts, keywords, or topics. our search criteria are detailed in table . these two keywords represent the technological concept (app) associated with the lifestyle (fitness), whose specific relationship makes the object of the present investigation. no more keywords related to the fitness industry were used (e.g., weight loss/running, dieting) since we wanted to examine which other specific categories were reviewed under the category of fitness apps in general. our search does not have a low-time frame limit, and the aim is to learn about the starting time of research in this field. title-abs-key ("fitness" and "app" or "apps") publication period* until article, review, and conference paper article, review, proceedings paper, and meeting abstract language english english *no low time frame limit was set, but articles published before , while containing relevant keywords, were seen not to be relevant to the field. after searching in the two databases separately, we performed a manual review of the titles and abstracts (also full text if necessary), excluding articles whose topics did not meet the criteria of the study, and subsequently removing duplicate literature. when the same article appeared in both databases, we opted to keep the references in scopus because scopus provides broader bibliographic information than wos. the search returned records. we decided to keep the conference papers and meeting abstracts due to the youth and relative novelty of the field of study. after filtering out the irrelevant and incomplete records, we ended up with a total sample of records. the records were then analyzed using bibliometric analysis. bibliometrics is "the quantitative study of physical published units, or bibliographic units, or of the surrogates for either" (broadus, , p. ) . the bibliometric analysis allows us to understand the intensity of the research available on a topic as well as the different research fields explored by the academic community. the variables analyzed for the bibliometric study were the year of publication, author, country of institutional origin, language of publication, type of document, journal, number of citations, area of research, topics analyzed, and the research method used. additionally, bibliometric mapping was also conducted. the construction of bibliometric maps has always received attention in bibliometric studies (van eck & waltman, ) . we used vosviewer software to present the relation of co-citation, co-occurrence of keywords, etc. finally, we summarized the current research hotspots and trends in this field, based on the content of these articles and the information presented by the keywords of their authors, to inform and inspire further studies. the first article on fitness apps was published in , and until , the intensity of research was very low. . % of the articles are published from onwards. in , there was a significant increase in the number of publications, doubling the number of . these results represent a price's index of . % until the end of . price's index (price, ) refers to the percentage of references less than five-year-old. as the price index's value is relatively high, this area is considered to be novel and dynamic. price 's law ( ) proposes that the development of the scientific field follows an exponential growth, which doubles in size every - years. the development of the scientific field goes through four stages: the precursor stage, the exponential growth stage, the consolidation of the body knowledge stage, and the decrease in the production stage. as shown in figure , publications in related fields underwent a growth process from to . a linear mathematical adjustment of the measured values provided us with a correlation coefficient r = . , which implies that . % of variance failed to explain this fitting. in contrast, a mathematical adjustment to the exponential curve provides a coefficient r = . , indicating an unexplained variance of . %. this reveals that the data analyzed is more consistent with a linear fitting rather than an exponential one. while the third stage of growth also showed a linear trend, the first contribution in this field was produced in , and the exponential growth trend stage was not detected. so, research in this field is still in its precursor stage. additionally, the number of publications in - was close to % of the total, exhibiting rapid growth. although there was a small decline in compared to , we expect the scientific production in this field to enter the exponential growth stage in the coming years. articles on fitness apps are published in a wide range of journals, from medical and health-related ones to computer science-related ones. out of the records, were published in academic journals, and were published as conference proceedings. the publication source also indicates a great dispersion: there were journals and different conference proceedings in total. among all the relevant journals, eight journals have published three or more articles. however, only nine conference proceedings had more than one article. compared to other fields of study, this number seems very small and indicates a low level of source concentration. table presents the field's most productive and influential journals, and table outlines the nine most productive conference proceedings. besides, % of the publications were from conference proceedings. the first and second positions by the number of publications came from the field of computer science. the high proportion may be explained by the fact that, although the importance of conference proceedings in areas such as the natural sciences is decreasing, they still play an important role in computer science, with nearly % of citations also distributed in the proceedings (michels & fu, ; lisée et al., ) . it also shows the importance of the development of fitness apps in the domain of computer applications. braford's law ( ) is a tool used in bibliometric studies to evaluate the concentration/dispersion factor of a set of publications. in essence, it allows the determination of the most productive nucleus in a particular subject. it postulates the existence of a small nucleus of journals that address the topic more broadly as well as a vast peripheral region that is divided into several zones with journals that have a decreasing representation in the subject studied (alvarado, ) . the number of journals in the core and the number in the successive zones are in a ratio of : n: n . therefore, journals included in the core have a comparatively high concentration of publication, while those involved in the surrounding areas are increasingly dispersed. thus, we can see that there is an unequal distribution of articles in the journals. a large number of articles are found in a small number of journals. as shown in figure and table , within the core of the ring, only journals contained one-third of all published articles ( records). zone comprises journals, and zone comprises journals. zona contains a much smaller number of journals than the theoretical value ( ). this result suggests the innovative and youthful nature of the field under study, which has not been considered in depth by many journals. the number of citations is an important indicator of the influence and the attention presented by the scientific community. according to the results shown in table , a total of articles received more than citations-all from academic journals. this number is relatively low compared to other more mature fields of research. the most cited article ( citations a total of , authors have contributed to this field. the average number of authors per article was . , which indicates the trend towards multi-author contributions in the field and a wide dispersion of research. table summarizes the first authors in the list, with more than two contributions. in those cases where the information was not available at scopus, we used the information provided by wos. the most productive authors in terms of the number of articles published are oyibo k. and vassileva j., both from the university of saskatchewan (canada), with contributions. third and fourth-ranked gay v. and leijdekkers p. are co-authors. in the scope of the subject of our study, they co-authored a total of six articles. the work of the most productive authors does not attract the highest number of citations. the author, with the highest number of citations in the fitness apps field, is west j.h. his six articles have garnered a total of citations. three of them are ranked in the top ten most influential papers in table . they were all published in the journal with the most contributions in the field, journal of medical internet research. the author with the highest h-index ( ) is salmon j., from deakin university, whose research pertains to the fields of medicine, health professions, and nursing. however, the total number of citations for his three articles was only . no other author had an h-index above . the high inconsistency in the number of citations, the number of author contributions, and the h-index show that no scholar or team of scholars has yet had a decisive influence on the field, which is also related to the fact that the field is still in the precursor stage of research. additionally, the authors in table are not widely dispersed in terms of institutional affiliation, with several authors (and close rankings) being from the same institution. this suggests that a high proportion of the top productive authors are co-authors, as evidenced in figure . it highlights that only four authors did not co-author papers with others. the remaining authors make up the remaining nine clusters. moreover, members in each group usually come from the same institutions or countries, with less cross-national/interregional cooperation. the authors' productivity data are much lower than the values suggested by lotka's law ( ) . this law states that the number of authors making n contributions in a given period is approximately equal to the number of authors who make /n contributions. generally, the application of lotka's law gives the theoretical result that about % of authors make only one contribution in their field of study. in the field of research on fitness applications, the value of lotka's law is . %. this confirms the huge dispersion of the field, which can be explained either by the novelty of the phenomenon or by a multidisciplinary approach. additionally, the analysis of co-citation of authors shows the structure and connections of the co-cited authors, i.e., "which authors are cited together more frequently" (gaviria-marin et al., , p. ) . figure shows the results of the analysis conducted using vosviewer, and the number of citations for each author is indicated by the size of the colored dot. authors with more than citations were clustered in five groups. some of these authors did not contribute directly to our field. however, their articles are frequently cited by other authors in the fitness app research field. authors in cluster mainly tend to focus on research in the areas of social sciences, business, management and accounting, and mathematics. sub-topics of interest to them include behavior change, physical activities, etc. authors in cluster primarily devote their research to the field of biochemistry, genetics and molecular biology, and health professions. physical and health education is also one of the sub-topics they are interested in. in cluster , the main research interests include psychology, and besides, the authors have contributed to the areas of computer science, nursing, and decision making. the main research interests of the authors of cluster lie in the arts and humanities, social sciences, computer science, and psychology. they have also undertaken certain interpretative explorations of technological acceptance. cluster consisted of only two authors, richard m ryan and edward l. deci. they are also co-authors of articles with fairly high citations, and both of them have an h-index of no less than . their main areas of research are psychology, in which self-determination theory and motivation are also a point of interest. out of the records did not specify the country/region of origin. of the remaining records, the countries that contributed the most were the united states ( . %), the united kingdom ( . %), and australia ( %). it should be noted that almost half of the studies were carried out in english-speaking countries. among the asian countries, china, india, and south korea stood out. national/regional contributions are double counted when authors of the same article are affiliated with institutions from different countries. our results show that the main research areas of study are medicine ( . %), computer sciences ( . %), behavioral sciences ( . %), computer medicine ( . %), and psychology ( . %). most articles contribute to more than one field. it seems that research in fitness apps has flourished through its study in the medical area, followed by its computational features. however, the study from the point of view of consumer behavior, integrated into the field of social sciences, seems not to have taken off yet. we predict significant growth in this domain as fitness apps become more popular, and communication through social networking sites goes viral, particularly among young people. the applied research methods allow the collection of empirical data to contribute to scientific knowledge. it is an important variable to understand the empirical orientations of research in this field of knowledge. total* . % *out of the total articles, articles ( . %) used multiple methods. of these, articles used two methods and one article used three methods. the most frequently used research method was the experiment. the experimental design was used in . % of all research. most of them were "in the wild" experiments, implemented on a small group of participants (n < ) who were asked to use a fitness app, developed expressly for the research, for a short period. the second most used research method was the survey ( . % of the articles), which allowed the evaluation of the user perspective and behavior with self-reported data. the third-ranked research method was content analysis. the articles that used this method analyzed and evaluated the total or partial functionality of a range of fitness-related apps, their technical characteristics and the attributes that make them more valued by users, more effective in changing consumer behavior, etc. for example, cowan et al. ( ) calculated a theoretical score for each of the health and fitness applications to determine whether the applications included relevant aspects of the behavioral change theory. the content analysis articles allow us to understand how fitness-related apps have evolved over the years and how researchers' focus has changed over that same period. by reviewing relevant articles, we found that behavior change techniques, gamification features, and consumer engagement strategies have been attracting attention, as shown in figure . the analysis of the frequency of appearance of the keywords allows the reader to approach the main topics analyzed in the articles in this field. the analysis of the keywords selected by the authors allows the determination of which relationships are established between a field of research and others close to it (durán-sánchez et al., ) . as shown in table , the terms "physical activity" and "mhealth" appear in . % of all the contributions. both keywords are the conceptual core of fitness app research. physical activity is also related to the terms "exercise" ( . %), "obesity" ( . %), and "weight loss" ( . %). portability is a concept associated with new devices for self-monitoring of activity: the terms "wearables" and "fitness tracker(s)" appeared in . % and . % of articles, respectively. the principle of playful functions is reflected in the term "gamification," with . % of the articles, which is a factor that can increase user adherence to the programs. figure maps the correlation between the keywords. to make the map clearer, with more focus on the core of the field of study, we removed the keyword "app" and its various related forms from the mapping analysis. the most frequent keywords were located in five differentiated clusters. cluster , which we named "digital mhealth" is mainly related to mhealth and ehealth (electronic health). they are platforms for fitness apps. also included in this group are keywords such as privacy and security, which are all related to the technology and device issues of fitness applications. cluster , which we named "mhealth and fitness trackers," is pretty similar to cluster , with only an emphasis on fitness trackers and persuasive technology as well as health apps and wearable electronic devices. cluster , which we named "physical activity, motivation, and social support," comprises keywords such as physical activity, exercise, physical fitness, etc. social support and motivation are also included in this group, which may be since these two are also important factors that support people to stick to physical activity (tang et al., ) . cluster , which we named "generalistic keywords," is more macro in nature and contains a wide range of topics such as fitness, mobile, and public health. cluster , which we named "behavior change and gamification," includes keywords such as behavior change, gamification, wearables, and self-determination theory. finally, based on all the information obtained as well as our thorough review of the contributions that are part of this bibliometric study, we now describe the main topics of research on the subject of fitness apps: ) analysis of the quality and performance of the use of the apps concerning the objectives of the users. the performance is measured through an evaluation of different indicators, such as the level of physical activity or weight loss. in this criterion of research, the use of innovative features is particularly important. for example, mata et al. ( ) tested the performance of the training planning function of the relevant apps and confirmed the high performance of these app-generated training and nutrition plans through expert validation. ) analysis of the benefit of the use of fitness apps for the chronically ill. patients affected by severe chronic diseases can undergo improvement in their general condition through lifestyle improvements. for example, bonato et al. ( ) analyzed the possibility of using an app for monitoring physical exercise routines for people affected by hiv. the apps are used to encourage patients to exercise to improve their general condition. examination of the use of fitness applications to encourage people with a specific need due to their socio-demographic profile to follow the minimum physical activity requirements established by the who. this includes the specific physical exercise needs that can be implemented through apps for the elderly (mas, palou & conti, ) , children (tripicchio et al. ) , or people with disabilities (pérez-cruzado & cuesta-vargas, ). increasing user motivation is an integral part of a significant number of articles. very high abandonment rates are observed in the use of these applications, and there is a lack of user engagement (bardus et al., ) . among the factors that may influence the use of the apps, some researchers are interested in the aesthetics of the user interface (bardus et al., ) , social relations (lewis et al., ) and the personalization (zhou et al., ) . some articles focus on the problems related to fitness apps and the adherence to hegemonic beauty canons. in this line of research, honary et al. ( ) concluded that the use of these apps might increase social pressure to achieve unrealistic beauty ideals and could thus increase the incidence of eating problems, such as anorexia or excessive physical exercise. another issue of concern relates to the privacy of and the large amount of personal data collected by these apps (adhikari et al., ) . wearable devices provide more accurate and convenient data for measuring people's daily activity levels. however, they are usually associated with relevant mobile apps for health data visualization and analysis. for example, lee et al. ( ) this study aimed to present in detail the current state of research on fitness applications through an exhaustive bibliometric analysis and bibliometric mapping. the social function and health potential of fitness apps represent a recent and growing phenomenon, which justifies an increase in the intensity of scientific research in recent years. . % of the contributions were published onwards when the usage of these apps had already been an important trend in the commercial market for several years. several bibliometric indicators (e.g., distribution of years of publication, price's index, author productivity, bradford's law, h-index, number of citations, source of publication, research areas, research methods, etc.) were analyzed to understand the main features and patterns of research on fitness apps. moreover, the scientific mapping analysis of the co-occurring keywords, co-authors, and co-citing authors provided an additional analysis from a timedepth perspective. in general, it is important to note the great dispersion of research, with a very high number of authors who have only made one contribution being a characteristic of a field of research that has not yet reached maturity. research in this field is still in its precursor stage. moreover, many of the studies have a relatively high number of co-authors. this situation is reflected in the indicator of author productivity, which is relatively low (oyibo, k. and vassileva, j. being the most active author with eight published articles). however, the most productive authors are not the most influential authors. west. j.h. has gained citations for his four articles, ranking first for this field of study. this dispersion of research is also reflected in the source of the publications. although there is a specialized journal in mhealth (jmir mhealth and uhealth), it can be found that submissions on fitness apps are distributed across a large number of academic journals and conference proceedings. with this data and support from the analysis of scientific mapping, it can be concluded that authors or prestigious journals have not been integrated and the research references in this field are relatively fragmented, partly due to their novelty and multidisciplinary requirements but also due to the technical orientation of the developers to circumvent the basic health, social, and behavioral aspects of health, society, and behavior. as in many other areas, the united states remains a prominent contributor in this area. china and india are the most productive in developing countries. these two countries are increasing their productivity and expanding their influence in various fields of scientific research at present. the most common research method used in this field is the experimental procedure that measures behavioral changes or changes in health indicators after a period of use. the second most used method is the survey, followed by the analysis of content. a considerable amount of literature is related to medicine, computer science, and healthcare. many authors have also focused on this main area of research. additionally, physical activity was the most frequently occurring keyword. "behavior change" linked to "physical activity" is also an important keyword. specifically, it refers to concepts such as behavior change theory, behavior change techniques (e.g., goal setting, self-regulation), etc. however, relatively few studies on consumer behavior from a social science perspective have been found. it seems that consumer-related research has mainly focused on analyzing the optimization of the functionalities of mobile applications from a medical or computer science point of view and neglected the aspects intrinsic to consumer behavior such as the motivations for using fitness apps, the attitude towards them, or how social networks influence the choice of the app to be used. the fact that the keyword "motivation" appears only times and all after is a clear indication of this finding. based on the generalization of all the information obtained and the review of the abstract and some of the full text, we found that the performance and function of fitness apps, the benefits for chronic disease treatment, the influence of using fitness app for public health, and factors of motivations of using fitness apps are currently popular research topics in this field. future research could build on these directions and incorporate relevant issues from a social science perspective (e.g., consumer motivations, consumer engagement, consumer behavior, etc) to further investigate on fitness applications. this article is useful in understanding the early state of research in the fitness app field. however, it is necessary to consider several limitations. one of the limitations of this study is the delimitation of the sample search criteria. in essence, the concept of fitness serves as a central reference for the applications that users utilize to perform selfmonitoring of health-related factors, particularly the level of physical activity. the control of "diet" is another health factor that overshadows and is superimposed on the concept of fitness, but one that 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fitness apps non-communicable diseases four decades of fuzzy sets theory in operations management: application of life-cycle, bibliometrics and content analysis evaluating machine learning-based automated personalized daily step goals delivered through a mobile phone app: randomized controlled trial key: cord- -tvy uo u authors: brock, rebecca l.; laifer, lauren m. title: family science in the context of the covid‐ pandemic: solutions and new directions date: - - journal: fam process doi: . /famp. sha: doc_id: cord_uid: tvy uo u the coronavirus disease (covid‐ ) pandemic has precipitated substantial global disruption and will continue to pose major challenges. in recognition of the challenges currently faced by family scientists, we share our perspectives about conducting family research in the context of the covid‐ pandemic. there are two primary issues we address in this article. first, we present a range of potential solutions to challenges in research, resulting from the pandemic, and discuss strategies for preserving ongoing research efforts. we discuss approaches to scaling back existing protocols, share ideas for adapting lab‐based measures for online administration (e.g., using video chat platforms), and suggest strategies for addressing missing data and reduced sample size due to lower participation rates and funding restrictions. we also discuss the importance of measuring covid‐ relevant factors to use as controls or explore as moderators of primary hypotheses. second, we discuss how the covid‐ pandemic represents a scientifically important context for understanding how families adjust and adapt to change and adversity. increased stress precipitated by the pandemic, varying from acute stress associated with job loss to more chronic and enduring stress, will undoubtedly take a toll. we discuss ways that family scientists can contribute to pandemic‐related research to promote optimal family functioning and protect the health of family members. the novel coronavirus disease has profoundly impacted society at large. in addition to economic disruptions and significant burden placed on healthcare systems, the covid- pandemic has undermined -and will continue to undermine -the physical and mental health of individuals across the globe. as family scientists, we must navigate unforeseen challenges as we abruptly pivot to salvage ongoing research and make plans for the future of our research programs. moreover, we are uniquely positioned to investigate how to best build resiliency in families amidst large-scale systemic changes that can undermine family functioning. the primary aims of this article were to (a) present a range of potential solutions to problems threatening the rigor of ongoing research and (b) propose new directions in family science aimed at understanding how families adapt to change and adversity arising from the pandemic. one of the most pressing questions facing many family scientists is how to preserve the rigor of ongoing research that has been derailed by the pandemic (e.g., longitudinal studies put on hold, resulting in missed assessments during critical periods of development). there are numerous challenges to address that could require sweeping changes to research designs. in-person data collection might not be advisable when considering participant safety-or could be expressly prohibited at times-which limits the feasibility of certain methods commonly employed in family research (e.g., behavioral observations in controlled laboratory settings, neuropsychological testing, biological measures, in-person interventions or experimental manipulations). additionally, both researchers and participants might be coping with elevated stress and adversity while adapting to altered roles and routines. consequently, research participants might be less engaged in research or unwilling to participate altogether, thereby threatening the reliability and validity of scores and contributing to elevated rates of missing data. researchers may not have as much time or energy to devote to data collection efforts as in the past and may feel overburdened in anticipation of potential budget cuts or limits on extensions to grant funding in the upcoming months and years. another challenge faced by investigators is restricted access to offices, laboratory spaces, and on-site resources. as such, difficult decisions must be made about which aspects of carefully constructed research designs, if any, can be changed to accommodate new constraints and loss of resources. we this article is protected by copyright. all rights reserved now turn to a discussion of possible adaptations to consider for ongoing research, drawing largely from existing tools and approaches that are established in the literature. online data collection has become relatively mainstream, particularly for administering questionnaires. as such, a relatively straightforward adaptation to lab-based research involves asking participants to complete surveys from home rather than in the laboratory. of course, even this is not a simple transition, as it requires institutional review board approval, an appropriate device (e.g., smartphone) and reliable access to internet connection in participants' homes, and clear instructions to minimize confounds (e.g., asking participants to complete the survey in a private location with minimal distractions). however, research has demonstrated psychometric equivalence of paper-and-pencil and internet formats of questionnaires often used in family research (brock et al., (brock et al., , . as such, validity and reliability concerns associated with this shift in approach are minimal. for investigators who routinely use lab-based methods such as behavioral observation paradigms in carefully controlled environments, experimental manipulation, in-person interventions, or methods that require equipment that is not easily transported, new ways of measuring constructs of interest should be considered. of course, these changes will not be feasible in all circumstances. for example, neuroimaging in the home is not an option. but, by stepping back and thinking creatively, there is the potential for considerable innovation in family science. fortunately, many lab-based measures and protocols can be adapted for online administration. telehealth and video conferencing are increasingly used for clinical interventions and are viable options for investigators conducting treatment outcome research (e.g., arnberg et al., ; perle & nierenberg, ) . further, prior to the pandemic, researchers were already effectively transitioning to video chat platforms for administering assessment tools that require interactions with investigators (e.g., bridgers et al., ; sheskin & keil, ) . there are numerous benefits inherent to home-based assessments, including increased ecological validity, more diversity in samples, reduced barriers such as travel and parking costs, and enhanced participation rates. further, in the context of safety concerns related to covid- , participants might experience elevated stress and anxiety when attending in-person appointments, which could introduce significant confounds. further, it is unlikely that participants who are at greater risk of complications from contracting covid- (e.g., those who are immunocompromised or this article is protected by copyright. all rights reserved pregnant) will be receptive to attending lab appointments which could decrease generalizability of findings. as such, the benefits of remote assessments that allow for research participation from the comfort and safety of one's home are not trivial. of course, there are also drawbacks to consider when collecting data remotely and adapting lab-based protocols for use on video platforms. most notably, participants must have reliable internet access and a device that can be used with the required software. there are also extra steps that need to be taken in service of standardization and preserving the internal validity of scores derived from these approaches. for example, observational paradigms of family interactions can be implemented by stipulating where and for how long the interaction will take place, asking if the parent and child can be alone in a room together so that other family members do not interrupt or influence relationship dynamics, and standardizing the props used during interactions as much as possible (e.g., perhaps you mail or deliver a toy in advance of the interaction that the child can then keep as a gift). as is customary with lab-based paradigms, we carefully track and record any potential confounds (e.g., the presence of significant distractors, technology failures, etc.) that emerge and account for these in the data analysis stage; however, this becomes absolutely essential when altering study protocols midstudy. when analyzing data, it will also be important to control for where and when participants complete the study procedures (e.g., in the home post-covid versus in the lab pre-covid) if these changes are made in the context of an ongoing project. plan for higher rates of missing data and smaller sample sizes. careful planning goes into research to ensure (a) adequate power to test hypotheses and (b) maximum retention and participation rates. these considerations are particularly important to family scientists who often employ advanced quantitative techniques that require larger sample sizes. yet, in the context of the covid- pandemic, the reality is that we might not reach our original goals for recruitment and participation. fortunately, numerous scholars have laid valuable groundwork for navigating these challenges. planned missing data designs can help to guide this process (little & rhemtulla, ). an example with particular relevance to family science is the two-method missing design, an approach used when there is a gold standard measure of a construct (e.g., behavioral observations of family interactions) that cannot be administered to all participants due to time, money, resources, or, in the case of covid- , social distancing guidelines. consider whether there is a more feasible or this article is protected by copyright. all rights reserved inexpensive measure to administer (e.g., an online questionnaire) that is intended to assess the same construct. if so, a random subgroup of the sample will complete the more intensive, gold standard measure whereas the entire sample will complete the "inexpensive" measure. the gold standard measure completed by the subset of the sample enhances the validity of scores, and the inclusion of the inexpensive measure allows for a larger sample size and the corresponding benefits (e.g., power, generalizability). this design could be particularly useful if in-person data has already been collected from a subsample of a cohort using an intensive protocol (e.g., behavioral observations of parental responsiveness). if there is an inexpensive measure that was used with that subsample (e.g., a parenting questionnaire), and it is correlated with scores from the more intensive measure, the inexpensive measure can be administered remotely (e.g., online survey) with subsequent participants. in the context of longitudinal research, a wave missing design (little & rhemtulla, ) could help reduce participant burden and provide a solution for salvaging ongoing data collection that has been put on hold or now has insufficient funding. in these designs, some participants are intentionally omitted from certain waves; as such, not all participants are required to complete every time point of data collection. finally, a multiform design (little & rhemtulla, ) involves randomly assigning participants to complete different versions of a survey that contain different combinations of items rather than administering a full battery of questionnaires to everyone. this approach can minimize participant burden while still providing rigorous assessments of your study constructs by using otherwise lengthy questionnaires with strong psychometric properties. finally, in anticipation of reduced sample sizes, we must carefully consider which of our hypotheses will still be sufficiently powered (for a brief guide to power and a list of resources, see murray et al., ) . are there empirical questions we can adequately address with a smaller n? if not, are there analytic approaches that well-suited for smaller samples to which we can turn? for example, although bayesian methods are not currently mainstream in family science, they are effective in addressing small sample problems. specifically, bayesian methods may be better suited to producing accurate parameter estimates in smaller samples relative to more traditional frequentist methods. for a detailed discussion of considerations when implementing a bayesian analytic framework with small samples, please refer to mcneish ( ). this article is protected by copyright. all rights reserved recommendation for ongoing research is to routinely assess the numerous ways that families are impacted by the pandemic. these measures will provide important contextual information for including as controls in hypothesis testing and for determining whether study effects are altered (i.e., moderated) by features of the pandemic. for example, our lab has been conducting a large-scale longitudinal study of families who completed numerous waves of data collection prior to a national emergency being declared in the united states (where the research is being conducted) as a result of covid- . one of the primary goals of the study is to examine trajectories of change in family processes and the mental health of family members over several years, and identify factors predicting adaptive versus maladaptive trajectories. yet, the global nature of the pandemic-and corresponding stress and expected changes to family functioning-could greatly alter the nature of those trajectories if we include new data collected in the context of covid- . as such, we are asking parents to complete home surveys to assess the various ways they have been impacted as a family to measure potential confounds. we acknowledge that, in some ways, this recommendation contradicts our earlier suggestion to scale back protocols; however, accounting for potential confounds that could alter your results is so essential that making concessions in other elements of your study to accommodate these measures might be advisable. further, these covid- specific assessments do not necessarily need to be time-consuming or expensive. for example, the epidemic-pandemic impacts inventory (epii; grasso et al., ) is a relatively comprehensive measure of pandemic impacts that only takes - minutes to complete and assesses multiple domains of functioning (e.g., work and employment, education and training, home life, social activities, economic, emotional health and well-being, physical health problems, physical distancing and quarantine, infection history, positive change). if administering a questionnaire is not feasible, even a brief phone conversation with research participants about how they have been impacted by the pandemic could subsequently be used for a thematic analysis (braun & clarke, ) , and quantitative codes could be assigned to reflect degree of impact and adversity. we have presented several potential solutions for adapting to the current circumstances to preserve the quality of ongoing research. however, there might ultimately be certain research questions that are not well-suited to the current circumstances and, as such, it might be better this article is protected by copyright. all rights reserved to postpone data collection until life returns to a state of relative normalcy (e.g., routine access to facilities and resources is restored; social distancing guidelines are relaxed and research personnel and participants feel safe attending in-person appointments). if it is not feasible to collect data remotely, and you are navigating pauses in ongoing data collection, consider staying in touch with participants while things are on hold to keep them invested. something we have implemented in our research is maintaining a study website to keep participants in our longitudinal projects engaged, which has helped to minimize attrition. we share results as they are published, provide resources, and post answers to common questions. in the wake of the covid- pandemic, we adapted our website to include status updates on the study and a list of resources for helping families talk to their children about covid- and manage stress. finally, there might be ways to adapt and evolve a research program in new directions, pursuing empirical questions that had not been previously considered. indeed, we now turn to a discussion of how the covid- pandemic represents a scientifically meaningful context for studying families. covid- represents not only a global public health emergency, but also the beginning of a major mental health crisis (united nations, ). exposure to adversity and stress is a robust predictor of mental health difficulties across the lifespan (e.g., benjet et al., ; juster et al., ; mclaughlin, ) , and research examining causal pathways of risk and modifiable factors that interrupt those pathways (e.g., high quality support, access to resources) is vital to inform prevention and early intervention efforts. experimental manipulation is touted as the gold standard approach for examining causality; however, as family scientists, we abide by ethical standards that prohibit us from subjecting individuals to extreme levels of stress and adversity. in the absence of experimental manipulation, disaster-based research offers an alternate approach for understanding the impact of stress on the family system. because they emerge suddenly and are outside of human control, natural disasters approximate the randomization of a true experiment, with stress quasi-randomly "assigned" to individuals (king et al., ) . further, norris ( ) states that "disasters generate an array of individually and collectively experienced stressors of varying degrees of intensity that interact with accepted article multiple characteristics of the person and environment to produce diverse outcomes that evolve over time" (p. ). thus, the covid- pandemic represents a scientifically important context for elucidating temporal relations between stress and family functioning. before we explore potential avenues for research, it is important to acknowledge the factors that make the pandemic a unique context for studying the effects of stress and adversity. what is unique and significant about the covid- pandemic? relative to more isolated natural disasters (e.g., floods, tornadoes), the covid- pandemic will affect individuals across the world. as of july , , covid- had impacted nearly every country or region ( of ), with over million confirmed cases and half a million deaths worldwide (johns hopkins university, ; united nations, ). further, the pandemic has triggered a collective experience of acute stress and psychological distress. social distancing measures, albeit varied in scope, have been implemented in countries and territories to help prevent the spread of covid- (international energy agency, ). though certainly effective in minimizing the spread of the virus, these measures have also resulted in significant occupational, educational, and personal disruptions that warrant further attention. in particular, the covid- pandemic has prompted acute, unprecedented job loss and disruption. as of april , the international labour organization, a specialized agency of the united nations, reported that full or partial lockdown measures impacted % of the global workforce (international labour organization, ) . unemployment rates in the united states rose sharply to nearly % in april, reflecting the economic impact of the pandemic and efforts to contain it (u.s. bureau of labor statistics, ). lower socioeconomic status (ses) is associated with an increased risk for mental health problems across the lifespan (reiss, ; santiago et al., ) . in particular, childhood ses predicts long-term physical and mental health outcomes (cohen et al., ; conroy et al., ) . ses is also a robust predictor of relationship quality and stability, parenting, and child development (see conger et al., for a review). thus, as more and more individuals worldwide experience job disruption and financial instability, we should expect the economic challenges spurred by the covid- pandemic to exert a toll on both individual and family functioning (e.g., gilman et al., ; kingston, ) . further, certain populations might be at particular risk for adversity stemming from covid- . for example, black americans not only suffer from higher rates of this article is protected by copyright. all rights reserved unemployment, but they are also more likely to work in front-line jobs deemed essential (gould & wilson, ) . covid- has also disproportionately impacted black americans, and race is associated with mortality rates (ferdinand & nasser, ) . as such, underrepresented and marginalized populations warrant particular attention amidst the pandemic. beyond the acute stress and uncertainty associated with job disruption, the covid- pandemic has the potential to contribute to chronic stress burden (e.g., baum et al., ; lantz et al., ) . there are enduring sources of stress related to covid- that will vary across individuals and families, including fear of becoming infected or infecting others; inadequate supplies to meet basic needs; insufficient information from health and government officials; and the potential for stigma (brooks et al., ) . a robust body of literature demonstrates that stress and adversity impact family functioning (e.g., masarik & conger, ; randall & bodenmann, ; story & bradbury, ) . indeed, stress resulting from external (e.g., occupational) demands can spill over into the family system and undermine individual mental health, relationship satisfaction, and parenting behaviors (e.g., bakker & demerouti, ; bass et al., ; bolger et al., ; brock & lawrence, ) . for couples, stress experienced by one person may cross over to impact their partner. increased stress might also result in one person demonstrating increased family involvement to compensate for their partner's decreased involvement (nelson et al., ) . amidst the covid- pandemic, parents may be at increased risk for role strain as they adapt to changing -and potentially conflictingoccupational and family demands. for instance, some families may be navigating job loss and economic adversity, while others might have job security but are adjusting to new roles and expectations, such as working from home while providing childcare or homeschooling. for some families in dual parenting households, one parent may be leaving the home to work, potentially in a high-risk environment (e.g., hospitals, pharmacies), while the other parent navigates responsibilities at home. thus, the pandemic will generate high levels of between-subject variability in objective and subjective stress, as all families will experience adversity but to different degrees and in diverse ways. further, social distancing measures enacted to minimize the spread of the virus may interfere with important social connections that help us to regulate and cope with our emotions (van bavel et al., ; williams et al., ) . social distancing can also contribute to feelings of isolation and frustration (brooks et al., ) . in addition to the stress associated with major transitions and role this article is protected by copyright. all rights reserved changes, many families have been forced into proximity with one another for an extended period of time (van bavel et al., ) . in combination with elevated levels of stress, forced proximity may be detrimental to family functioning. indeed, research demonstrates that stress experienced by one partner often places a heavy burden on caregiving partners, who report poor marital adjustment and increased subjective distress (dekel et al., ) . the covid- pandemic may prevent family members from accessing external sources of social support that help mitigate distress (e.g., ergh et al., ; rodakowski et al., ) . in addition, partner support, which buffers the association between stress and mental health trajectories (brock et al., ) , may be compromised if both partners are experiencing high levels of subjective stress. conversely, research demonstrates that partners who boast strong support skills may experience greater relationship satisfaction, particularly during times of increased stress (brock & lawrence, ) . further, forced proximity may be beneficial for some families by increasing time spent together to establish family rituals that were not previously feasible due to competing demands. as such, it is important to identify the factors that contribute to adaptive versus maladaptive outcomes in the context of pandemic stress (e.g., communication, support processes, intrapersonal coping resources, division of labor, conflict management skills). further, the consequences of forced proximity of family members -and reduced connection with important social contacts outside of the household (e.g., grandparents, extended family members, friends, coworkers)represents a novel area of research worth closer attention. this might be of particular relevance among families with adolescent children given that adolescence is characterized by an increased need for autonomy and independence and, in turn, the potential for increased parent-child conflict (mcelhaney et al., ; steinberg, ) . finally, with no clear end in sight, the chronic nature of the covid- pandemic is particularly notable. indeed, longer durations of confinement are associated with poorer mental health, particularly trauma-related symptoms (brooks et al., ) . currently, there is a tremendous amount of ambiguity about the future, including which businesses will survive, future job prospects (e.g., for recent graduates), and how much the way we work and live will be altered for the long-term. this chronic uncertainty poses a significant risk to mental health and the family system more broadly (afifi et al., ) . finally, it has become apparent that there are diverse perspectives about the pandemic, the threat associated with the covid- disease, and what constitutes appropriate and this article is protected by copyright. all rights reserved reasonable safety measures. disagreements about the best way to proceed as a family could increase conflict, put strain on collective decision-making efforts, and undermine vital coping resources originating within the family (e.g., high quality support, a sense of belonging and shared values). though covid- is a biological disease by nature, mental health research has emerged as an immediate priority (holmes et al., ) , as evidenced by numerous calls for research by various funding agencies. in fact, the national institutes of health has devoted existing resources and emergency funding to support administrative supplements and competitive revisions that would allow active grants to investigate urgent research questions related to covid- , such as how remotely delivered stress management strategies impact mental and physical health outcomes, resilience to covid- infection, or symptom severity (not-at- - ). as family scientists, we are uniquely poised to contribute to pandemic-related research. drawing on past research and theory, we can investigate the specific impact of the stress and adversity arising from covid- on individual mental health and family functioning and identify modifiable risk and resiliency factors to target in interventions. leveraging ongoing longitudinal data collection will be vital to determine causal mechanisms associated with poor mental health outcomes (e.g., depression, anxiety) and family dysfunction (e.g., couple conflict, child neglect, decreased responsiveness during parenting). indeed, research utilizing existing cohorts, among whom mental health and family functioning have been previously assessed, offers a unique opportunity to examine the longitudinal impact of covid- and to identify a range of risk and protective factors that can be harnessed to promote family functioning. further, integrating quantitative and qualitative data, often staples in family science, allows researchers to explore the specific family processes and relationships that contribute to adaptive and maladaptive outcomes amidst the pandemic. in the absence of ongoing longitudinal studies, the covid- pandemic presents another avenue for family scientists to pursue new research on the impact of stress and adversity on family functioning. for example, prospective studies can be designed to study the immediate and long-term effects of exposure to varying levels of stress from the pandemic on family functioning, especially for high-risk populations. in sum, we have identified several key themes at the intersection of family science and the covid- pandemic. building off of these themes, there are a range of unique questions to consider this article is protected by copyright. all rights reserved in pursuit of a more nuanced understanding of family processes. some of these questions include, but are not limited to: how do stress and adversity impact couples in different relationship stages? are dating couples impacted in unique ways relative to more established, long-term couples? how has forced proximity and lack of access to social contacts outside of the family impacted sibling relationships? how are divorced and remarried family systems navigating custody agreements and shared responsibilities in the context of social distancing and travel restrictions? how are families impacted when a family member tests positive for covid- ? what if certain family members are at higher risk for complications resulting from covid- ? how are individuals working in essential jobs maintaining family relationships? what impact has this had on parenting? how has covid- uniquely affected pregnant women and their partners? how might this contribute to the intergenerational transmission of stress and psychopathology? in this article, we have presented (a) a series of potential solutions to problems arising when conducting ongoing research during the covid- pandemic and (b) ideas for new directions in research that explicitly address issues related to the experience of the pandemic for families. it is important to recognize that the time, energy, and resources available to devote to research will vary across investigators, and perhaps by region, as some areas of the world have been impacted by the pandemic more than others. thus, our intention was to present a series of potential solutions and ideas ranging from more intensive, time-consuming efforts to relatively small, but meaningful, steps that can be taken in family science in response to the pandemic. we also acknowledge that this is by no accepted article means an exhaustive list of strategies or approaches. we have shared some of the key considerations made in our own research, along with ideas shared by colleagues who are navigating similar challenges. we look forward to learning more about the new and innovative ways that family scientists respond to this crisis and move the field forward. the impact of uncertainty and communal coping on mental health following natural disasters internet-delivered psychological treatments for mood and anxiety disorders: a systematic review of their efficacy, safety, and cost-effectiveness the spillover-crossover model new frontiers in work and family research do job demands undermine parenting? a daily analysis of spillover and crossover effects socioeconomic status and chronic stress: does stress account for ses 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how psychological telehealth can alleviate society's mental health burden: a literature review accepted article this article is protected by copyright the role of stress on close relationships and marital satisfaction socioeconomic inequalities and mental health problems in children and adolescents: a systematic review role of social support in predicting caregiver burden socioeconomic status, neighborhood disadvantage, and poverty-related stress: prospective effects on psychological syndromes among diverse low-income families thechildlab.com a video chat platform for developmental research autonomy, conflict, and harmony in the family relationship understanding marriage and stress: essential questions and challenges frequently asked questions: the impact of the coronavirus (covid- ) pandemic on the employment situation for policy brief: covid- and the need for action on mental health using social and behavioural science to support covid- pandemic response key: cord- -lxhq ga authors: sharma, manoj kumar; anand, nitin; singh, priya; vishwakarma, akash; mondal, ishita; thakur, pranjali chakraborty; kohli, tavleen title: researcher burnout: an overlooked aspect in mental health research in times of covid- date: - - journal: asian j psychiatr doi: . /j.ajp. . sha: doc_id: cord_uid: lxhq ga nan the sars-cov- related lockdown has not only adversely affected the mental health but also behavioral and mental health research (torales, o'higgins, castaldelli-maia & ventriglio, ) . however, continued lockdown and physical distancing measures pose a challenge to conduct behavioral research as most of it requires in-person, detailed assessment, and clinical interviews. the challenges have been observed in the form of delay in getting expedited ethical clearance for conducting research (ma, wang, gao, he, he, yue, et al., ) . the choice of an appropriate sampling strategy either online or offline or a combination is getting affected due to difficulty in the collection of data because of the implementation of social distancing measures. as a result, behavioral science researchers find it very difficult to convince people to participate in their research studies. the covid- related lockdown has also decreased the accessibility to research participants, in addition to existing challenges such as funding not coming through in time, the pressure to achieve results, to write grants, revise grant applications, and face non-acceptance of research grants proposals. the studies which involve qualitative research methods are impacted j o u r n a l p r e -p r o o f even more significantly in comparison to quantitative research. the existing guidelines on social distancing is making it difficult for researchers to conduct interviews with the participants and even if the lockdown is being relaxed with certain conditions, people are continuing to wear masks that is interfering in collecting the nonverbal information as the face is understood to be the index of mind (mehta, venkatasubramanian, chandra, ) . to overcome, these existing challenges in research, there is a spurt in data collection via digital platforms. however, it has been criticized for the applicability of obtained results, low response rates, and privacy and confidentiality issues existing with open source software programs (ameen & praharaj, ) . it also contributed towards the excessive use of digital devices and digital platforms to manage research work in times of covid- . also the boundaries for work and leisure activities have also become blurred as for both activities the modality requires digital presence which can lead to the maintenance of stress and burnout. the on-going uncertain situations concerning covid- and uncertainties related to research progress appear to be a major contributor towards stress which is likely manifesting itself in researchers and their teams in terms of sleep disturbance, poor appetite, increased interpersonal problems, decreased motivation to continue with research demands, procrastination, absence of other offline leisure activities and experiencing guilt feelings for not completing work. the authors have presented here an experiential account of the challenges experienced by around researchers working in the field of behavioral research in the times of covid- . the likely ongoing stress among researchers of under accomplishment in the times of covid- can cause them to get trapped in the burnout cycle which starts with feelings of heightened and prolonged stress with decreased interest. it is followed by short-term enhancement of attention, energy levels, and experience of anxiety and exhaustion. if the stressful situation does not improve, the final stage usually starts with the experience of despair, bleak and pessimistic views which eventually contribute to a feeling of burn out (burisch, ) . it is understood that poor levels of workplace well-being can lead to reduced productivity among researchers via both absenteeism and more crucially through presenteeism, where despite present at the workplace the productivity levels are lower. when such concerns remain unresolved it can impact the quality of research and the levels of commitment towards their research and to their institutions (susan, lichten, belle, ball, knack, & hofman, ) . it is true that the covid- is presenting itself as an extraordinary threat to the psychological wellbeing of clinical and community population and its impact on mental health is just beginning to be understood. covid- pandemic has caused a transitory pause in a number of on-going or planned researches. the researchers and research funding agencies can consider taking a number of steps j o u r n a l p r e -p r o o f which can help the researchers avoid experience of researcher burnout and can help them to continue being efficient in conducting research. a few of these perspectives can include researchers reprioritizing their research and also view it as an opportunity to explore the interface between covid- and mental health. the researchers need to consider making pragmatic decisions to reprioritize the on-going clinical research. these decisions can be made by evaluating situations in terms of risk benefit ratios of continuing with research study visits, interviews, assessing risk of pandemic at their study site, optimizing the staff functioning and seeking local and national guidance from health departments to minimize risk to both participants and study teams. besides this research funding agencies and ethics committees to be approached for taking permission for revision of study protocols to allow for data collection using online applications such as skype, microsoft teams and other digital applications. however, if a participant visit is required then it should be conducted by implementing all protective measures as per the advisories of international and national health agencies (keshavan, ). the available evidence indicates that many participants appear to be supportive towards continuation of medical research during the epidemics (padala et al., ) . illness are coping during covid- ? whether social distancing is likely to increase the experience of loneliness and precipitate mental health symptoms (narita et al., ) . in addition, neuropsychiatric and neuropsychological research is also needed to understand how covid- affects the neurological and neuropsychological functions of the brain. at present, there is emerging j o u r n a l p r e -p r o o f evidence which indicates that sars-cov- impacts the brain . the research also suggests that neuropsychiatric symptoms were observed in % of hospitalized patients (mao et al., ) . besides, a mental health pandemic may emerge which results from the experience of trauma, grief, social isolation, loneliness and as well from secondary impact of covid- related quarantine and its associations with abrupt changes in lifestyle. emergence of this research data driven insights will help health professionals become prepared for an effective response to the ongoing as well as future pandemics. in addition to repositioning research by researchers, the journals need to encourage the researchers for submitting research articles reporting the mental health impact of covid- and interventions implemented to address its impact. besides this there can be special focus given to research emerging out of asia as the covid- pandemic started from asia and different asian countries have taken different approaches to manage this challenge. the outcomes across asian countries and from rest of the world are likely to vary and there is much to learn from each other. this will help the relevant information to be made available to researchers and the health professionals in a timely manner (tandon, ) . researcher's burnout and mental health needs unlike burnout among members of the health care and teaching professions, have likely not been studied in other epidemics as well like mers and sars and the evidence is limited (susan, lichten, belle, ball, knack, & hofman, ) . the authors in their search could not locate much research on researcher burnout. however, there is emerging evidence that covid- pandemic has an impact on the mental health of healthcare professionals responding to the covid- pandemic . this indicates that covid- is relevant to the field of mental health and mental health aspects of covid- need to be investigated among all kinds of health professionals including researchers working in the field of mental health research (tandon, ) . thus, there appears to be a need for novel and innovative research approaches to explore the burnout and mental health needs among mental health researchers and to actively create mechanisms for its management and especially during the challenging times of a pandemic like covid- . research focus in this aspect of mental health will be beneficial for enhancing researcher's well-being and productivity and will help in being better prepared during similar other unpredictable situations that may arise in the future. j o u r n a l p r e -p r o o f problems in using whatsapp groups for survey research das burnout-syndrom : theorie der inneren erschöpfung -zahlreiche fallbeispiele -hilfen zur selbsthilfe mental health care for medical staff in china during the covid- outbreak the brain, another potential target organ, needs early protection from sars-cov- neuroinvasion neurologic manifestations of hospitalized patients with coronavirus disease challenges and strategies to research ethics in conducting covid- research the "mind" behind the "mask": assessing mental states and creating therapeutic alliance amidst covid- loneliness and psychotic experiences in a general population sample conducting clinical research during the covid- pandemic: investigator and participant perspectives understanding mental health in the research environment: a rapid evidence assessment. rand corporation the covid- pandemic, personal reflections on editorial responsibility the outbreak of covid- coronavirus and its impact on global mental health satyam dst,india awarded the grant to dr manoj kumar sharma compliance with ethical standard:there was no conflict of interest in relation to present work as well as informed consent of the human subjects had been taken prior to inclusion in the study. disclosure of potential conflict of interest:not applicable conflict of interest: authors of the paper did not have any conflict of interest. statement of human right: the studies have been approved by the institutional and/or national research ethics committee . research involving human participants and/or animals:.all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the helsinki declaration and its later amendments or comparable ethical standards. key: cord- -i mndt c authors: ball, roberta scipioni title: issues to consider for preparing ferrets as research subjects in the laboratory date: - - journal: ilar j doi: . /ilar. . . sha: doc_id: cord_uid: i mndt c the domestic or european ferret (mustela putorius furo) has been domesticated for thousands of years. ferrets have been used for hunting and fur production, as pets, and as models in biomedical research. despite the relatively small numbers used in the laboratory, ferrets have some unique applications including study of human influenza and severe acute respiratory syndrome (sars)-associated corona virus. they have served as models for peptic ulcer disease, carotenoid metabolism, cystic fibrosis, and drug emesis screening, among others. most research ferrets are males, due to estrus-related health problems in females. they may be housed conventionally and are easy to care for when their biology and behavior are understood. due to the small number of ferret suppliers, animals are often shipped long distances, requiring air transport and intermediate handlers. it is important to minimize shipment stress, especially with weanling and pregnant animals. additional expertise is required for success with pregnant and whelping ferrets and for rearing of neonates. the animals have specific dietary requirements, and proper nutrition is key. successful housing requires knowledge of ferret behaviors including social behavior, eating habits, a general inquisitive nature, and a species-typical need to burrow and hide. regular handling is necessary to maintain well-being. a ferret health care program consists of physical examination, immunization, clinical pathology, and a working knowledge of common ferret diseases. various research methodologies have been described, from basic procedures such as blood collection to major invasive survival surgery. ferrets have a distinct niche in biomedical research and are hardy animals that thrive well in the laboratory. history, taxonomy, and uses of the domestic ferret t he domestic or european ferret (mustela putorius furo) has been domesticated for thousands of years (boyce et al. ) . thought to be a descendant of either the european or steppe polecat, the domestic ferret should not be confused with the indigenous black-footed ferret, mustela nigripes, which is an endangered species (cohn ) . the ferret belongs to the order carnivora, family mustelidae, and is related to mink, otters, and weasels. uses of the ferret have varied. for hundreds of years the animals were used to hunt rabbits and other small mammals. "fitch" ferrets, which are polecat-ferret crosses, were used for fur production (bell ) . the use of ferrets in biomedical research dates back to the early s. more recently, beginning in the late s, ferrets became popular as household pets, and it is estimated that there are currently several million pet ferrets in the united states (boyce et al. ). it is difficult to know exactly how many ferrets are used in biomedical research. according to the us department of agriculture (usda ) animal care report, a total of . million animals were used in biomedical research (aphis ) . of these, , were reported in the "all other covered species" category. it is likely that < % of this number represents ferrets. despite these relatively small numbers, ferrets have a distinct niche and some unique applications in research. one of the earlier ferret models involved the study of human influenza, a disease to which ferrets are highly susceptible. to date, this model continues to be an important use of the ferret and includes work on pathogenesis, treatment, vaccine development, and investigation of reye's syndrome (maher and destefano ) . the ferret is also used to study infection of the highly pathogenic avian h n viruses (zitzow et al. ) . it has been determined that ferrets are susceptible to another human respiratory pathogen, the severe acute respiratory syndrome (sars )-associated corona virus (martina et al. ). the first eruption of this disease affected thousands of people in asia, causing sars to be recognized as a serious new pathogen (stadler et al. ) . ferrets have been utilized to study this disease and to investigate development of a vaccine (weingartl et al. ) . investigation of airway physiology and pathophysiology is another application of the ferret in respiratory disease research. there is remarkable similarity in lung physiology and morphology between ferrets and humans. as a result, ferrets are used as a model for the study of cystic fibrosis, including a ferret airway xenograft model to study gene therapy (li et al. ) . in addition to the study of respiratory diseases, ferrets have been used as a model for human gastrointestinal disease. most notable is the ferret model for peptic ulcer disease, which has resulted from the anatomical and physiological similarity of the ferret stomach to the human stomach, and the fact that both species possess a helicobacter gastritis-causing organism (fox et al. ; whary and fox ) . ferrets have been used to study the epidemiology of this disease as well as its pathogenesis and treatment (patterson et al. ) . ferrets have also been useful as a model for nutrition research due to their similarity to humans in carotenoid metabolism (wang et al. ) . furthermore, ferrets have been used to study the influence of carotenoids on the development of certain cancers and cardiovascular disease (lee et al. ; raila et al. ) . in addition to many basic research applications, ferrets have also been investigated as a nonrodent test system in pharmaceutical drug development, especially as a surrogate for dogs when test material is in limited supply (gad ) . a ferret model for evaluating the emetic potential of drugs is another use of the ferret in the field of drug development (crawford et al. ) . finally, ferrets are used in cardiovascular research, including myocardial infarct models, in neural development and visual system studies, in skeletal research, for pediatric endotracheal intubation training, and for the investigation of renal disease secondary to toxin producing intestinal infection with escherichia coli (crawford et al. ) . these uses of the ferret comprise a variety of applications that dictate necessary knowledge about selection, husbandry, health care, research methodologies, and possibly reproduction. ferret selection criteria may vary depending on the model system or research application (e.g., the purchase of timed pregnant females for visual system studies in neonates). often, however, the only requirement is a healthy ferret within a particular weight range. it is advantageous to be familiar with ferret biology to facilitate decision making. sexually intact female ferrets are not often used in research. they breed seasonally, ovulation is induced, and females experience persistent estrus if induction of ovulation does not occur (quesenberry and carpenter , ch. ) . after several weeks of estrus, clinical signs of estrogeninduced bone marrow suppression may result. ovariohysterectomy before sexual maturity is preventive; however, we at marshall bioresources find that it is both common and expedient to simply choose males. whether or not age is a study-specific requirement, it is important for the researcher to be familiar with normal ferret growth in order to plan properly for housing needs. as depicted in figure , growth is rapid, with sexually intact males weighing twice that of females by wk of age. investigators sometimes request preshipment castration and/or "descenting" of male ferrets. surgical removal of the scent glands, or anal gland sacculectomy, is invariably performed for the purpose of odor reduction (mullen and beeber ) . researchers should be aware that neither procedure, alone or in combination, will entirely eliminate the mustelid scent. even neutered, descented ferrets produce a slight musky odor due to normal sebaceous secretions that can be attenuated through bathing. the natural or "wild-type" color of the ferret is sable, sometimes also called fitch. it is the color pattern requested by most researchers, in contrast with the many coat color variations that abound in the pet industry. one research study, however, reported the use of albino ferrets as pseudopregnant recipients of embryos from cinnamon-colored parents (li et al. ) . individual identification of ferrets is not mandated by the usda animal welfare act (awa ) regulations, but may be required by other oversight bodies or by the scientific protocol. as a small fur-bearing mammal, ferrets have a thick hair coat and small head and ears. these factors complicate not only identification but also procedures such as blood collection, as further described below in methodology. tattoos and neck collars are largely unsuitable compared with larger laboratory species such as the dog, cat, and rabbit. however, ear tags and microchips are reliable methods of identification, which can be applied or implanted before shipment. unless restricted by the research, it is recommended that ferrets be fully immunized against rabies and canine distemper virus. breeding facilities routinely vaccinate, but if ferrets are purchased before the animals have reached the designated vaccination age, immunizations become the responsibility of the research institution. it is appropriate to expect the breeding facility to provide a health history for each ferret. because the vast majority of ferrets used in research are reared conventionally, histories should include birth dates, sex identification, immunization dates, surgery dates if applicable, and any medications administered before shipment. once selection criteria have been established, arrangements must be made for transportation of the ferrets to the laboratory. the delivery of laboratory animals in a safe nonstressful manner is extremely important for their health and well-being and for the generation of scientifically valid research. virtually all laboratory animals are subject to some degree of transportation stress, and studies have documented the resultant impact (fox et al. , ch. ) . for this reason, optimal transportation conditions are extremely important and warrant careful consideration. animal transportation is regulated domestically by the awa and by the states of origination and destination (awa/awr, usda-aphis ). european transport is regulated by ets , the european convention on the protection of animals during international transport, and by the countries of origin, transit, and destination (swallow et al. ) . globally, the live animal regulations of the international air transport association (iata ) have been formally adopted by many countries including the united states. these regulations have also been accepted by the convention on international trade in endangered species of wild fauna and flora, the office international des epizooties, and the european union (iata-lar ) . in addition, the laboratory animal science association recently formed a transport working group with the goal of defining standards that ensure good practice and full compliance with national and international legislation governing laboratory animal transport (swallow et al. ) . this working group addressed all aspects of transport that may affect welfare including route plans, vehicle and container design, driver training, travel duration, and food and water supplies. it is often necessary to transport ferrets long distances, including internationally, because the number of research ferret breeding operations is limited. in light of this situation, it becomes especially important to optimize transportation arrangements and ensure adequate quantities of feed, water, and bedding. although the vendor is responsible for making appropriate arrangements, it is advisable for the researcher to be aware of the details of the transportation process. ferrets are delivered by ground transportation, air, or a combination of the two. ideally, ground transportation includes direct door-to-door delivery from the vendor to the research institution. this situation, however, is often confined to transport distances of several hundred miles or less. crosscountry or international shipments invariably include air transport carriers and intermediate handlers. delivery from the vendor to a local airport typically takes place in a vendor-owned dedicated vehicle, but an intermediate handler may provide ground transport from the airport to the final destination. in this circumstance, it is especially beneficial for the receiving institution to be aware of the specifics of airport pick-up and delivery. occasional unanticipated delays are inevitable, but these instances can be minimized through effective communications among the vendor, the carriers and intermediate handlers, and the research facility. timely awareness by the research facility of delays in the process will minimize any negative impact. the functional design elements of transport containers are regulated by the organizations described above. these design elements include the following: viewing ports, ventilation areas, feed and watering devices, structural components that promote hygiene, and proper labeling. stocking densities are not specifically regulated by usda and iata for ferrets, but the animals must be able to turn around, lie down, and make normal postural adjustments. because young ferrets are social animals, it is preferable to ship them in groups under most circumstances. exceptions are sexually intact adult or breeder male ferrets (staton and crowell-davis ) . in my experience, to wk of age should be considered the minimum age for shipment of young ferrets without the mother. twenty-eight days of gestation, which is the end of the second trimester, should be considered the latest time-point for shipment of pregnant jills. shipment containers for conventionally reared ferrets should be lined with contact bedding and equipped with containers for feed and water. wire mesh or vent openings must allow for sufficient exchange of air but not compromise safety or hygiene from spilled bedding and excrement. animals must be readily visible, and containers must be injury and escape proof. regardless of container design, additional safeguards must be applied during very warm weather (e.g., > °f) due to the heat sensitivity of ferrets (fox et al. , ch. ) . precautions include reduction of stocking densities, provision of additional water sources, inclusion of gel packs, and possibly even delay of the shipment. filtered containers designed for specific pathogen-free rodents are sometimes used for ferrets to prevent exposure to influenza virus while in transit. in warm weather, particular attention must be paid to ambient temperatures and stocking densities because of the reduced airflow within these containers. regardless of the duration of transportation, it is imperative to assess the health and general well-being of the ferrets as soon as they arrive. this assessment is especially important for young or pregnant animals, during times of climatic extremes, or in the event of shipment delays. early intervention is key to addressing potential complications such as dehydration and stress-related diarrhea or anorexia. an appropriate acclimation interval should reflect the particular research protocol as well as the age and status of the ferrets. young adults are resilient, but weanling and pregnant ferrets require close attention during the first to wk after arrival. for ferrets, a lethargic or dehydrated condition is a medical emergency. in such a condition, a balanced electrolyte solution may be administered subcutaneously. fluid consumption may be encouraged by offering highly palatable oral electrolyte products such as pedialyte®, or by using multiple methods of water supply such as a water bowl in addition to automatic watering. most ferrets are raised on automatic watering systems, so specific acclimation should not be necessary except in the case of complications or undue stress in transit. use of a familiar diet will ease the transition, especially with young or pregnant ferrets. it is beneficial to offer a moist supplement in addition to a dry diet during the transition (bell ) . a thin gruel is made by adding warm water or milk replacer (e.g., kitten milk replacer) to the pelleted diet and offering it to times daily to encourage both food and fluid consumption. questions regarding optimal caging systems for ferrets are common. in many circumstances, facilities desire to retrofit or adapt caging from other species for ferrets. rabbit cages are most commonly adapted because the height and square footage are generally suitable. rabbit and ferret cages may be different with respect to the grid size of enclosure surfaces, including walls and floors. to ensure the safety of weaned and older ferrets, grid openings for all surfaces should not exceed inch square (fox et al. , ch. ) . preweanling ferrets require a grid that does not exceed ⁄ × inch. this safeguard has prompted the retrofitting of some styles of rabbit caging. both suspended and solid flooring are suitable for ferrets. because ferrets have a strong natural tendency to defecate in the back corners of the cage, suspended floors with inch grids will facilitate passage of stools into the drop pan below. cages with solid walls, especially those that are designed with a bevel along the bottom, will promote wastebuild up and require frequent spot cleaning. some solid bottom floors may be used if slippage can be avoided or minimized (e.g., using cage floor liners). in this circumstance, it is necessary to provide a litter box, which ferrets can be trained to use relatively easily (quesenberry and carpenter , ch. ) . ferrets are curious and playful to the point of mischievousness, so it is important to ensure that the cage is escape proof. regardless of cage design, it is important to provide an area that accommodates a ferret's instinct for burrowing and hiding, which are examples of species-typical behavior, and its need for resting (quesenberry and carpenter , ch. ) . to accommodate these needs, it is necessary to provide an enclosure with a solid bottom and sides, with or without an overhead cover. many options exist, including large dog bowls, -to -inch diameter polyvinyl chloride pipes, fabric ferret hammocks or sleeping bags, and ferret tunneling toys. play toys may be provided, but due to the ferret's inquisitive behavior, it is imperative to scrutinize toys for safety and from potential ingestion. as discussed above (see arrival and acclimation), automatic watering is optimal because most ferrets are already acclimated to this system, and mess and wastage are reduced. if bowls are used, they should be heavy crocks to prevent spillage. feed may be offered via a j-feeder design. it is possible to minimize digging by providing a restricted access opening. if the feeder opens to the outside of the cage, the waist should be sufficiently narrow to preclude escape if the feeder is emptied. open bowls may be used; however, their use inevitably results in digging and wastage. in addition, the use of a suspended floor design could result in inadvertent food restriction. although aggressive behaviors may sometimes be observed in group housing situations (staton and crowell-davis ) , ferrets in general are social animals and benefit from being in a group (boyce et al. ) . ferrets should be housed in groups or in pairs, and solitary housing should be avoided. as mentioned above (see transport containers), exceptions to group housing are adult or breeder hobs (males) and whelping jills (females). even nonbreeding sexually intact males may be housed together with some supervision. although males have been observed grasping or biting each other by the nape of the neck, this is actually normal mating behavior and it should be tolerated if it is not excessive. if the animals develop lesions due to excessive biting, it may be necessary to separate them. social hierarchies exist, but usually not to the detriment of an individual animal. ferrets sleep together in groups, so the rest area that is provided should be sufficient to accommodate all of the inhabitants of a given enclosure. properly trained care staff should handle ferrets regularly. this human contact will increase the ability of the animals to remain relaxed and comfortable during subsequent research procedures. ferrets that are fed and watered daily but not handled regularly tend to become rambunctious and more easily frightened. such fearful behavior may be evidenced by a vocalization best characterized as a screech. chuckling or chortling sounds are more indicative of stimulation from play (boyce et al. ). as with housing, knowledge of dietary requirements is an extremely important element of ferret husbandry. ferrets have been described as obligate carnivores, and although their complete nutritional requirements are unknown, it is accepted that a high-protein, high-fat, low-fiber, meat-based diet is necessary to support good health (bell ) . dog food is not suitable for ferrets due to the amount of cerealbased protein in most commercial diets. high-quality kitten food is acceptable, but there are also numerous commercially available ferret diets, including laboratory chows (scipioni ball ) . it is important to note that the ferret intestinal tract is short, with a rapid transit time and a relatively simple gut flora (quesenberry and carpenter , ch. ) . in addition, the energy requirements of ferrets are high and have been documented to range from to kcal per kg body weight daily (fox , ch. ) . these factors underscore the need to select a palatable, high-quality diet. in addition, because ferrets may also resist dietary change, it is important to select an optimum diet at the outset, taking into account age, reproductive status, and experimental stresses. immunization an immunization program is an important aspect of preventive health care. ferrets should be protected against canine distemper and rabies. a typical primary immunization protocol for weanling ferrets consists of canine distemper virus vaccination at approximately , , and wk of age followed by annual booster immunization. rabies vaccination takes place after wk of age and is also followed by annual booster immunization (quesenberry and carpenter , ch. ) . the safety and efficacy of commercial modified live canine distemper vaccines have been studied (wimsatt et al. ) . although only a few vaccines are available, it is advisable to use only those that are licensed and approved for use in ferrets (greenacre ) . presently, a modified live chick embryo vaccine (fervac d, united vaccines, madison, wi) and a recombinant canary pox vectored vaccine (purevax ferret, merial, duluth, ga) are available. the first rabies vaccine approved for use in ferrets became available in (niezgoda et al. ) . current recommendations regarding rabies prophylaxis in ferrets are described (compendium ) . presently, there are two approved rabies vaccines available for use in ferrets (imrab and imrab tf, merial, duluth, ga). one safety concern relative to immunization is the occurrence of anaphylactic reactions to one or both of the vaccines described above (greenacre ; moore et al. ) . potential sequelae include vomiting, diarrhea, hypersalivation, and collapse. for this reason, it is important to consider exposure potential and to maintain an available supply of emergency medicines including antihistamines, steroids, and epinephrine. pretreatment with diphenhydramine has been used for ferrets that have demonstrated allergic signs to previous immunizations (moore et al. ). in addition to immunization, the performance of routine physical examinations should be part of the health care program of any facility. the procedure with ferrets requires training in proper restraint methods and knowledge of baseline physiological parameters. the vital signs of these animals reflect their high metabolic rate. normal body temperature ranges from . to ºc, normal heart rate is to bpm, and normal respiratory rate is to /min (fox et al. ch , ) . published data are available regarding normal values for hematology, serum chemistry, and urinalysis (fox , ch. ; morrisey and ramer ) . during the physical examination, it is useful to be aware of several ferret behaviors that may confuse the assessment of an animal's state of health. for example, if ferrets are approached while sleeping, it often takes approximately to sec for them to become fully roused. to an inexperienced observer, it may initially appear that the ferret is severely depressed or even comatose. however, this behavior should be considered normal unless it persists. in addition, ferrets often express a shivering behavior that does not necessarily indicate ill health. although the reason for this behavior is unknown, it may be observed when the ferret awakens from sleep or becomes excited. finally, if a ferret is restrained by the scruff of the neck, it will often yawn as its body relaxes. again, the significance of this particular behavioral response is unknown, but handlers should be aware that it is normal (boyce et al. ) . posterior paresis is a sign that is often interpreted as evidence of neurological disease. in ferrets, it is a presenting sign that may be observed in a variety of states of ill health. ferrets normally maintain an arched position with their backs, typical of other mustelids such as weasels. when weak or lethargic, regardless of cause, ferrets will lose this arch causing the back to become parallel with the horizontal plane. this posture results in some degree of ataxia of the rear quarters and may be a nonspecific sign. however, it also may be due to central nervous system involvement from systemic illness such as aleutian parvoviral disease (quesenberry and carpenter , ch. ). another integral part of the health care program is awareness of common ferret diseases. various infectious and inflammatory diseases may be observed in ferrets in the laboratory. most neoplastic diseases occur in older animals, but some are observed in young adults. ferrets are highly susceptible to canine distemper virus. this multisystem disease is virtually % fatal and necessitates immunoprophylaxis (wimsatt et al. ) . ferrets are also highly susceptible to human influenza viruses, as described above (see ferrets as animal models in research). signs may be similar to distemper but are less severe and with reduced mortality (fox , ch. ). because of the zoonotic potential, care should be taken to protect workers and animals from influenza through use of appropriate protective clothing and practices such as vaccination (maher and destefano ) . enteric infectious disease in ferrets is commonly observed and may be either viral or bacterial. in neonates, rotavirus infection may cause diarrhea with significant morbidity and mortality if left untreated (fox , ch. ). these infections typically occur within the first month of life. dehydration or secondary bacterial infections may complicate recovery. coronavirus infection, also termed epizootic catarrhal enteritis or ece, causes mucoid enteritis with a high level of morbidity and variable mortality. the disease tends to be mild or even subclinical in young kits, with older ferrets showing more severe clinical signs. supportive care is the only treatment (williams et al. ) . aleutian parvoviral disease has been described as a wasting syndrome in ferrets. this disease should be included in the differential diagnosis of neurological disease with signs of ataxia, paresis, or paralysis (palley et al. ; welchman et al. ) . helicobacter mustelae infection causes gastrointestinal disease of variable severity including subclinical, chronic, or acute gastritis, gastroduodenal ulceration, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma in ferrets (erdman et al. ; fox et al. fox et al. , patterson et al. ) . clinical signs include inappetance, weight loss, tarry stools, severe deterioration, and acute death. conventionally reared ferrets are presumptively h. mustelae positive, with disease becoming manifest as the result of stresses such as weaning, other illness, or experimental manipulations. treatment includes use of amoxicillin, metronidazole, and bismuth subsalicylate, often referred to as "triple therapy" (hillyer and quesenberry , ch. ) . lawsonia intracellularis, previously described as desulfovibrio spp., is the causative agent for a proliferative colitis syndrome in ferrets that is similar to syndromes observed in swine and hamsters (mcorist et al. ) . it presents as a wasting disease with progressive weight loss and deterioration and has been noted to respond to chloramphenicol (fox et al. ) . otodectes cynotis, a common ectoparasite in ferrets, causes ear mite infestation. treatments that have been described and compared include topical use of ivermectin in propylene glycol (patterson and kirchain ) . although infestations are usually subclinical, consequences such as otitis may occur. ferrets appear relatively resistant to helminth infestation; however, routine fecal examinations are useful to verify endoparasite status. protozoa including coccidia and giardia have been described but are usually present subclinically (fox , ch. ) . ferrets are susceptible to heartworm infestation; however, preventive treatment with ivermectin is highly effective (quesenberry and carpenter , ch. ) . various neoplastic diseases may be observed in older ferrets and include adrenal cortical hyperplasia/neoplasia and insulinoma (wagner et al. ; weiss et al. ). thymic lymphosarcoma occurs in young (usually < yr of age) adult ferrets and has been proposed to have a viral etiology (batchelder et al. ) . other neoplastic diseases have been described (li et al. ) . urinary tract diseases have also been observed and include cystitis and obstructive uropathy (coleman et al. ; li et al. ; orcutt ) . splenomegaly is a relatively common finding in ferrets and may reflect extramedullary hematopoiesis, anesthetic administration, lymphoid hyperplasia, or lymphoma (morrisey and ramer ) . gastrointestinal obstruction from foreign bodies, including hairballs, has been described. conservative treatment with laxatives may be curative; otherwise, surgery is indicated (quesenberry and carpenter , ch. ) . a polymyositis syndrome has recently been noted in ferrets. although there are no published studies in the scientific literature to date, various reports have characterized this disease by fever, leukocytosis, hind limb weakness, and death in ferrets less than yr of age (personal communication, b.h. williams, department of telemedicine, us armed forces institute of pathology, washington, dc, ) . as a proposed immune-mediated event, several potential triggers have been suggested including vaccination, viral disease, and genetic predisposition. to reiterate, maintenance of an immunization protocol along with regular physical examinations that reflect knowledge of ferret biology, behavior, and disease are integral to optimal health care. for some institutions, it is also necessary to develop a breeding program in support of their research. as described above (see research-related ferret selection criteria and biology), some investigators may require ferret neonates for studies such as visual system development. in this circumstance, they may purchase timed pregnant jills, although another alternative is to maintain in-house breeding. if the latter option is chosen, personnel must be knowledgeable and trained in all aspects of ferret breeding and neonatal care. breeding several aspects of ferret reproduction are unique. for example, it is well documented in the literature that a long day photoperiod is necessary for successful breeding, gestation, and lactation (fox et al. ) . a short day photoperiod suppresses onset of estrus in females and prevents development of breeding condition in males. when the light cycle is reversed to produce a long day photoperiod, breeding activities are initiated. estrus is determined by marked vulvar swelling in females; in males, breeding condition is determined by enlargement and full descent of the testes into the scrotum. estrus jills may be induced to ovulate through breeding or by chemical methods using gonadotropin-releasing hormone or human chorionic gonadotropin. ovulation is not reliably induced unless the jill has been in estrus for approximately to days, when the follicle responds (fox , ch. ) . successful ovulation is evidenced by a gradual decrease in vulvar swelling over a period of several days. as mentioned above (see research-related ferret selection criteria and biology), jills that remain in heat for longer than to wk develop risk for estrogen-induced bone marrow suppression. clinically, loss of body condition and pale mucous membranes are noted. clinicopathologically, leukopenia, anemia, and thrombocytopenia may be observed. the eventual mortality rate is significant and necessitates early intervention. whether ferrets are bred in-house or purchased as timed pregnant jills, researchers should be aware that pregnant ferrets are subject to pregnancy toxemia. the disease is characterized by anorexia, depression, ketosis, and fatty liver (batchelder et al. ; dalrymple ) . this metabolic syndrome is a medical emergency, and mortality of both the jill and kits can occur. anything that results in anorexia of the jill can precipitate this syndrome through development of a negative energy state, especially in late gestation or with a high fetal burden. in my experience when neonatal ferrets are required for research, pregnant jills are typically requested in late gestation. it is strongly recommended that these animals be received before day of gestation to avoid undue stress. it is also very important to ensure that the transition to a new environment is nonstressful. a change of diet is contraindicated during this period. the diet should be made available ad libitum; offering a wet mash supplement of the diet mixed with warm water to a soft consistency will encourage food and water consumption. this supplement may be offered twice daily and should be continued throughout lactation because it will also serve to initiate the weaning process for the kits. if pregnant jills are received at the end of the second trimester, they should be housed in a cage that is suitable for parturition and equipped with a bedded nest. the gestation period for ferrets is to days, with little variation. if parturition begins earlier than or later than days, the prognosis for a successful outcome is guarded. little can be done to prevent early delivery, other than avoiding stresses and subsequent complications such as pregnancy toxemia. surgical intervention should be considered if gestation reaches days. it is critical to have quiet surroundings during delivery, and the whelp nest should provide privacy for the jill. it is important to monitor parturition to prevent dystocia or entanglement of umbilical cords (quesenberry and carpenter , ch. ) . at the same time, monitoring should involve minimal disturbance to avoid causing the jill to leave the nest and, in extreme cases, to cannibalize her young. even once parturition is complete, it is best to continue to minimize disturbances for the next several days. the progress of the litter may be sufficiently ascertained without removing the jill from the nest or handling the kits. after several days, it is safe to perform a closer inspection that includes weight and sex determination of the young. cage changeover and other noisy or disruptive activities should be planned accordingly. ferret jills have strong maternal instincts, which make cross-fostering a simple procedure even when delivery dates are not coincident. cross-fostering is especially useful given the difficulty of hand rearing neonates. lactating jills tend to be very protective of the kits, so care should be exercised in their handling. lactation failure may occur but is usually secondary to problems such as inadequate food or water intake, stress, or illness. mastitis may also occur and is usually caused by ␤-hemolytic e. coli. systemic illness in both the jill and kits may occur. antibiotics and supportive care are often required, and cross-fostering may be necessary (marini et al. ) . vendors should provide only pregnant jills that have a history of successful delivery and lactation to minimize complications. ferret kits are altricial at birth. they weigh only to g, and their eyes do not open until approximately days of age (quesenberry and carpenter , ch. ). nevertheless, their growth is rapid, and ferrets weigh approximately g by wk of age. at this time, kits should be offered a moist supplement that is sufficiently diluted with water to achieve a soup-like consistency. this supplement will allow the young kits to begin eating solid food, which initiates weaning. after to wk of age, the supplement may be made with less moisture but should continue to be offered along with the dry diet for several weeks. weaning is complete by wk of age, when body weights are approximately g. as stated above (see ferret-related research selection criteria and biology), a significant divergence in body weight gain between sexually intact hobs and jills is observed later (figure ). proper handling and restraint techniques are crucial not only for husbandry (see social and behavioral needs) but also for the conduct of experimental procedures (moody et al. ) . ferrets are used in a wide variety of research applications, so handling requirements may vary. for example, when ferrets are used in an acute surgical procedure, it may be necessary to handle them only for a preanesthetic physi-cal examination and for injection of a sedative or anesthetic induction agent. for survival surgery, especially for major invasive procedures, extensive handling is required, which includes preoperative blood work and daily postoperative observations and treatments. regardless, the key to successful nonstressful handling is acclimation of the ferret to whatever method of restraint will be required. this goal should be accomplished well in advance of initiation of the experimental procedure. otherwise, especially with young ferrets, their rambunctious behavior may make handling difficult. ferrets are easily removed from their cage or other enclosure by being grasped by one hand around the thorax, although large males may require the use of both hands. for more complete restraint, the ferret may be grasped by the skin at the base of the neck, a procedure called scruffing. ferrets may be retrieved from their cage in this manner or carried to a treatment table and then scruffed because the flat solid surface of the examination table facilitates grasping the ferret. as stated above (see physical examination), this method of restraint often produces a relaxation response that is accompanied by a yawn. it is useful for examination, oral dosing, or administration of injections. another effective and nonstressful method of restraint involves tucking the ferret "head first" against one's waist using the crook of one's arm. hugging the ferret in this manner appears to offer them security, which minimizes resistance. this method of restraint is particularly useful for taking a rectal temperature or giving an intramuscular injection (scipioni ball ) . even with optimal handling methods, peripheral vascular access is more difficult in ferrets compared with other large laboratory animals due to the short appendages, small ears, and thick hair coat of ferrets. accessible blood vessels include cephalic, jugular, cranial vena cava, and lateral saphenous veins, as well as the ventral tail artery (morrisey and ramer ) . when larger quantity sampling is required (greater than approximately ml of whole blood), jugular or cranial vena cava sampling is preferred (fox , ch. ; quesenberry and carpenter , ch. ). for small sample quantities or injections, including catheter placement, cephalic or lateral saphenous venipuncture is indicated (hem et al. ; morrisey and ramer ) . access to these vessels may be achieved when the animal is either sedated or conscious. surgical placement of vascular access ports in the jugular vein and femoral artery has also been described (etheridge et al. ) . as outlined above (see ferrets as animal models in biomedical research), ferrets are used in a variety of research applications that require anesthesia for surgery or other experimental procedures. research includes cardiovascular, respiratory, and skeletal studies, among others. ferrets have been described as hardy animals that make excellent surgical candidates (mullen and beeber ) . isoflurane anesthesia is often preferred, but numerous sedative and anesthetic protocols, both injectable and inhalation, have been reported (fox , ch. ; mullen and beeber ; quesenberry and carpenter , ch. ) . perioperative considerations for ferrets are similar to those of other laboratory species, albeit with some exceptions. because of ferret gastrointestinal physiology (see dietary requirements) , the preoperative fast interval should be short (∼ hr), especially for compromised subjects. due to their small body size, pediatric endotracheal tubes are needed as well as a nonrebreathing system for inhalational anesthesia. intraoperative monitoring (e.g., pulse oximetry and capnography) has been described (olin et al. ) . postperative analgesics should be used preemptively and routinely, and a description of analgesic options is available (fox , ch. ). techniques such as epidural narcotic administration have been assessed (sladky et al. ) . ferrets are prone to hypothermia, especially during prolonged procedures. use of warmed fluids and circulating warm water heating pads help prevent loss of body temperature (fox , ch. ) . humane euthanasia may be achieved by using injectable sedation or anesthesia followed by barbiturate euthanasia. the report of the american veterinary medical association panel on euthanasia (avma ) should be utilized for guidance on appropriate and humane methods of euthanasia. the domestic ferret has been used in biomedical research since the early s. although the number of animals used may be small compared with other laboratory animal species, they occupy a distinct niche in research. ferrets are used to study the pathogenesis and treatment of a variety of important human diseases, including influenza, sars, peptic ulcer disease, and cystic fibrosis to name only a few. despite the possibility that researchers may be less familiar with ferrets than with other laboratory species, it is important for investigators to realize that ferrets may be selected, purchased, and transported in a manner similar to other animals. caging systems are readily accessible either commercially or through modification of pre-existing equipment. knowledge of ferret behavior will guide the use of social housing and environmental enrichment devices that encourage species-typical behaviors. high-quality ferret diets are commercially available and satisfy the unique nutritional needs of ferrets, including young or pregnant animals. as with other conventionally reared species, a good health care program is composed of an immunization protocol, routine health assessments, and knowledge of common ferret diseases. if breeding the animals is required for the research, all aspects of ferret breeding may be successfully accomplished in the laboratory with sufficient training in the particulars of ferret reproductive biology. successful use of the ferret in the laboratory also requires familiarity with their unique behaviors, training in proper methods of restraint, and a regular routine of handling. acclimation to research procedures is crucial to minimize stress and promote well-being. ferrets are friendly, curious, and playful. they have distinct behaviors and a unique biology. efforts to prepare ferrets as research subjects in the laboratory are successfully achieved only through in-depth study. such study ensures a thorough understanding of the needs of these interesting animals. animal and plant health inspection service report of the avma panel on euthanasia 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helicobacter mustelae-associated gastric adenocarcinoma in ferrets (mustela putorius furo) intracellular campylobacter-like organism from ferrets and hamsters with proliferative bowel disease is a desulfovibrio sp pigs and ferrets as models in toxicology and biological safety assessment incidence of adverse events in ferrets vaccinated with distemper or rabies vaccine: cases saphenous vein puncture for blood sampling of the mouse, rat, hamster, gerbil, guinea pig, ferret and mink ferrets, rabbits and rodents: clinical medicine and surgery manual of international air transport association-live animal regulations review of animal models in carotenoid research cystic urogenital anomalies in ferrets (mustela putorius furo) neoplastic diseases in ferrets: cases ( - ) conditions for in vitro maturation and artificial activation of ferret oocytes developmental capacity of ferret embryos by nuclear transfer using g /g -phase fetal fibroblasts the ferret: an animal model to study influenza virus characterization of hemolytic escherichia coli strains in ferrets: recognition of candidate virulence factor cnf virology: sars virus infection of cats and ferrets characterization of lawsonia intracellularis gen. nov., sp. nov., the obligately intracellular bacterium of porcine proliferative enteropathy laboratory management of the ferret for biomedical research incidence and risk factors for adverse events associated with distemper and rabies vaccine administration in ferrets ferrets-clinical pathology and sample collection miscellaneous surgeries in ferrets pathogenesis of experimentally induced rabies in domestic ferrets evaluation of noninvasive monitoring techniques in domestic ferrets (mustela putorius furo) ferret urogenital diseases parvovirusassociated syndrome (aleutian disease) in two ferrets comparison of three treatments for control of ear mites in ferrets failure of surface ring mutant strains of helicobacter mustelae to persistently infect the ferret stomach ferrets, rabbits and rodents: clinical medicine and surgery the ferret as a model for vitamin a metabolism in carnivores husbandry and management of the domestic ferret evaluation of epidural morphine for post-operative analgesia in ferrets (mustela putorius furo) sars-beginning to understand a new virus factors associated with aggression between pairs of domestic ferrets working group report: guidance on the transport of laboratory animals leuprolide acetate treatment of adrenocortical disease in ferrets intestinal uptake and lymphatic absorption of betacarotene in ferrets: a model for human beta-carotene metabolism immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets insulinoma in the ferret: clinical findings and treatment: comparison of cases aleutian disease in domestic ferrets: diagnostic findings and survey results natural and experimental helicobacter infections coronavirus-associated epizootic catarrhal enteritis in ferrets serologic evaluation, efficacy, and safety of a commercial modified-live canine distemper vaccine in domestic ferrets pathogenesis of avian influenza a (h n ) viruses in ferrets the author wishes to thank the management and staff at marshall bioresources for their assistance in the preparation of this manuscript. key: cord- - ecm ive authors: sharma, arun title: sustainability research in business-to-business markets: an agenda for inquiry date: - - journal: industrial marketing management doi: . /j.indmarman. . . sha: doc_id: cord_uid: ecm ive abstract corporate interest in the topic of sustainability has increased in prominence. most of the research on sustainability has a business-to-consumer focus, with limited research emphasizing sustainability in the context of business-to-business marketing. the paper examines research on sustainability, published in the journal industrial marketing management, where thirty-eight articles have appeared in the past twenty-seven years, the majority on supply-chain research. research on sustainability in business-to-business markets is sparse, and some of the fundamental research questions in this area have gone unaddressed. this paper addresses three interrelated research issues associated with sustainability in business-to-business markets. first, what research is available to researchers and practitioners? second, what framework can be used to examine sustainability research in business-to-business markets? third, what subareas of sustainability research need focus and deeper examination, and what are the associated research questions? overall, the findings from this paper suggest the need for additional research in the area because of its substantial implications for theory, research, and practice. in the past two decades, sustainability has become an essential aspect of a firm's strategy. most large global firms have a statement of social purpose, and many firms have signed the un global compact and support its sustainable development goals (young & reeves, ) . davos has added the pressure to disclose progress on environmental, social, and governance metrics, as well as climate response (young & reeves, ) . research and practice in this area largely focus on business-to-consumer markets, with limited attention to business-to-business markets. for example, extensive research exists on consumer demand for sustainable products, but research on such demand among business customers is very limited. in the growing discussion of climate change and resource depletion, research on sustainability in business-to-business markets is critical. in practice, extensive discussion of this area already occurs. consultants have argued that firms must focus on both social and business value, using their core businesses to deliver financial returns and help society meet its challenges (young & reeves, ) . they suggest that current approaches to sustainability have two major issues-first, an inordinate focus on compliance and reporting and, second, keeping strategy and sustainability separate (young & reeves, ) . they describe firm evolution from recognizing corporate social responsibility to focusing on compliance (i.e., process changes to meet demands of the regulatory environment), to reactive changes for sustainability (e.g., responses to market and investor pressure), and finally, to sustainable business-model innovation that combines strategy and sustainability (young & reeves, ) . due to the limited research in the area, an essential question pertains to the research focus on sustainability in business-to-business markets. this paper highlights three interrelated research areas associated with the topic. the first entails the basic question of what research is available to researchers and practitioners. therefore, this study examines prior research in the area, and the review confirms the sparseness of the research. second is the framework for examining research on sustainability in business-to-business markets. with no existing frameworks, this paper presents one and suggests using it to classify extant and future research. a third research question addresses the subareas that need greater focus and deeper examination, using a survey of academics who work in the business-to-business area to identify directions for future research. accordingly, the next section addresses the first research question, namely, that of the existing literature on sustainability in business-tobusiness markets. then, the discussion proceeds to the development of a framework for research and examining the research in the topic's subareas. the next section provides the details of an academic survey on areas for future research, before the conclusion describes the implications of this research. the present without compromising the ability of future generations to meet their own needs" (world commission on environment and development, ) . there is extensive literature on sustainability and marketing; chabowski, mena, and gonzalez-padron ( ) found that articles were published in this area between and . however, the majority of that research is on consumers and business-toconsumer strategies. as the current paper focuses on business-to-business markets, the literature review focuses on this area within the topic of sustainability. the literature search examined major journals (e.g., journal of marketing, journal of marketing research, management science) and business-to-business marketing-focused journals (e.g., industrial marketing management, journal of business and industrial marketing). the keywords for the search were "sustainability," "sustainable," "environment," "environmental," "green marketing," "industrial," "business-to-business," and "corporate social responsibility." we conducted the search in november and found more than five hundred articles in our search, but when we focused on business-to-business articles, we found forty-six articles, three on corporate social responsibility (csr), and forty-three on sustainability. the first area the literature review examined was corporate social responsibility (csr), which focuses on the responsibilities to society that a business should assume (bowen, ) . carroll ( ) developed a "csr pyramid" that included economic, legal, ethical, and philanthropic responsibilities. intuitively, sustainability should be part of a firm's csr. however, minimal research exists on the relationship between corporate-responsibility initiatives and sustainability in businessto-business marketing. for example, three articles address csr activities, from the perspectives of sales (vlachos, theotokis, & panagopoulos, ) , certification by nongovernmental organizations (stekelorum, laguir, & elbaz, ) , and the role of power in supporting csr activities (harness, ranaweera, karjaluoto, & jayawardhena, ) . however, research addressing the relationship between csr and sustainability was not found. the review next examined research on sustainability, identifying forty-three articles that focus on sustainability in business-to-business markets, of which thirty-eight were in the journal industrial marketing management. surprisingly, the majority of those articles (twenty-three) focus on supply-chain strategy (e.g., inward and outward logistics). the outcome of the search suggests extremely limited research in the area. the results are contrasted with other aspects of the business-to-business literature. paesbrugghe, sharma, rangarajan, and syam ( ) found articles discussing sales and the purchasing function; kumar, sharma, and salo ( ) found articles on key-account management ( to ); and kumar, sharma, vatavwala, and kumar ( ) found articles on digital mediation in business-to-business markets ( to ). the research on sustainability addresses a number of subareas, calling for the development of a basic framework for examining the research on sustainability in business-to-business markets. typically, the paper would have attempted to map the extant literature or match papers to extant schools of thought, but the limited number of articles made that very difficult. also, very limited proposals exist for categorizing sustainability research in business-to-business markets. the traditional input-process-output model (e.g., forbes & milliken, ) , similar to the s-o-r model (stimulus, organism, and response) has been used in business-to-business marketing (bonoma & johnston, ) , sustainability (kudla & klaas-wissing, ) and to understand firm strategies (ginter & white, ) . based on existing research on sustainability, the research is divided into three areas: antecedents (inputs or stimulus), firm strategy (process or organism and behavior), and output or outcome, which leads to the feedback and learning loop. the next section discusses research in these areas. in the examination of antecedents to sustainability strategy, no research focuses on all antecedents, but some focuses on a limited number of antecedents. peattie and ratnayaka ( ) suggest that drivers of green marketing are disasters, public opinion, legislation, and scientific evidence. similarly, after an extensive literature review, chabowski et al. ( ) suggest that the antecedents of sustainability strategies are the environment, social networks, and economics. in summary, the extremely limited research on the antecedents of sustainability strategy in business-to-business markets indicates that most researchers start by assuming a demand for sustainable products and services. few articles examining overall strategy were found. peattie and ratnayaka ( ) developed a model for examining the drivers (e.g., legislation, public opinion), barriers (e.g., costs, complexity, lack of expertise, time needed), and strategies (limited and short-term to extensive and long-term) associated with green marketing. sharma ( ) suggest enhancing sustainability by reducing surplus supply and reverse logistics through build-to-order manufacturing. mariadoss, tansuhaj, and mouri ( ) examines innovation-based strategies for sustainability. patala et al. ( ) develop a value-creation model for sustainable offerings and highlight the impact of such offerings, key customer-value creation mechanisms, key success indicators, life-cycle modeling, and life-cycle value. finally, lacoste ( ) develops a model that focuses on a sustainable supply chain, and his model integrates supplier and customer networks, products, raw-materials production, business-to-business, end consumer, and return of product. in the context of an overall strategy, the area of research on a sustainable supply chain is extensive, with articles that focus on supply chains and business networks. very limited research exists on elements of overall marketing strategy for sustainable products and services. peattie and ratnayaka ( ) provide the first discussion on overall marketing-strategy elements for green products. the area of customer behavior has received limited research attention. ramirez, gonzalez, and moreira ( ) identify costs, ease of use, credibility, creativity, and execution as supply-related barriers, and organizational structure and culture as intraorganizational barriers to the adoption of sustainable products. in the area of product development, brand, and positioning, sharma and iyer ( ) suggest resource-constrained innovation as a path to sustainable-product development. kumar and christodoulopoulou ( ) and sheth and sinha ( ) suggest strategies for integrating branding and sustainability. the limited research on communication strategies for sustainable products focuses on identifying sustainability claims in advertising (leonidou, leonidou, hadjimarcou, & lytovchenko, ) . finally, no research was identified in the areas of pricing and distribution. very limited research on outcomes emerged. ortiz-de-mandojana and bansal ( ) suggest that firms that follow sustainability strategies have less financial volatility, higher sales growth, and higher chances of survival over a -year period. jr, glenn, musgrove, gillison, and gabler ( ) found that the correlations of environmental strategic focus with market performance and financial performance were . and . , respectively. the research does not address other outcomes. in summary, minimal research exists on sustainable strategy in a. sharma industrial marketing management ( ) - business-to-business marketing. due to the importance of the topic, the need for additional research motivates this paper's attempts to derive a framework for future research. this section reports the creation of a classification framework for highlighting the topics in sustainability research on business-to-business marketing. more broadly, the current study examines the areas of future research that will have the greatest impact on the understanding of sustainability in business-to-business markets. it seeks to identify the research focus on sustainability, using three classification steps: . classify sustainability research on business-to-business markets into manageable areas and subareas; . examine the research in the areas and subareas; . identify focus areas of research that predict maximal impact, in terms of theory and practice; present sample research questions in the subareas to use in future research. because of the limited research in the area, this paper classifies possible areas of future research. for each area discussed earlier (i.e., antecedents, strategy, and outcomes), subareas are identified to aid in understanding the area. based on the work of such researchers as chabowski et al. ( ) , the antecedents include science and the environment; the policy, regulatory, and legal framework; social aspects; economic aspects; and customer demand. logically, the strategy area focuses on the overall strategy of the firm, marketing strategy, and such associated elements as customer behavior; product/service development, positioning, and branding; pricing; communications; and distribution. finally, the outcomes include the effect on the environment; revenue and profitability; customer perception, preference, and satisfaction; and shift in customer demand. fig. shows the framework, and each area is briefly discussed. this section examines the research in areas and subareas of sustainability in business-to-business markets and the framework in fig. . the focus of this area is on the drivers of sustainable strategy, and the subareas of this area include science and the environment; the policy, regulatory, and legal framework; social aspects; economic aspects; and customer demand. the subarea of science and the environment is well established as sustainability science (kates et al., ) and offers enough evidence to suggest that businesses must practice sustainability. the subarea of a regulatory and legal framework for sustainability offers industry research (e.g., payne, , on realestate development), but no overarching understanding of the area. regarding social aspects, the research examines social programs to enhance sustainability (ostrom, ) and apply social pressure to firms (de lange, busch, & delgado-ceballos, ). in the first three subareas, no research examines the context of the business-to-business marketplace. the subarea of economic aspects offers limited examination of the economic benefits of sustainability (e.g., sharma & iyer, ) and calls for deeper examination. finally, in the subarea of customer demand, no research accurately assesses the demand for sustainable products and services; rather, it simply assumes robust demand. the subareas include the overall market strategy of the firm, supply chain, and marketing-strategy elements, including the " ps." the subarea of sustainability strategy offers extensive research (see bansal, ) , but none on business-to-business firms. however, as discussed earlier, substantial research exists in the supply-chain area. the subarea of marketing strategy elements includes customer behavior; product/ service development, positioning, and branding; pricing; communications, distribution, and supply chain. as mentioned earlier, no are except the supply chain offers more than limited research. the subareas include the effect of sustainability strategy on the environment; revenue and profitability; customer perception, preference, and satisfaction; and shift in customer demand. an examination of the literature found minimal research in this area. specifically, in the business-to-business context, no single study comprehensively examining any of the subareas was found. one of the key objectives of this study was to develop directions for future research. the key informants surveyed were academic researchers in business-to-business marketing because they actively follow the literature. the research design of paesbrugghe et al. ( ) and kumar, sharma, and salo ( ) was initially used, particularly to identify the categories for providing directions for future research. the same question was used for all sixteen research topics: science and environment; policy, regulatory, and legal frameworks; social networks; economic returns; customer demand; overall market strategy; customer behaviors; products/service development, positioning and branding; pricing; communications and sales strategy; distribution; supply chain; the effect of sustainable strategy on the environment; the effect of sustainable strategy on revenue and profitability; the effect of sustainable strategy on customer perceptions, preferences, and satisfaction; and the effect of sustainable strategy on a shift in customer demand. a self-administered online questionnaire, as paesbrugghe et al. ( ) and kumar, sharma, and salo ( ) had used, was designed using the same sentence for all research categories. for each research topic, the importance of the topic and sample research questions was asked. for example, regarding customer demand, the survey stated: "we would like your opinion on the importance of studying 'customer demand' when examining sustainable business-to-business market strategy. please rate from to ( = not important and = very important)"; and "can you please share possible research questions in the area of 'customer demand' in the context of sustainable business-tobusiness market strategy?" the editorial board members list of industrial marketing management was used as the sample and the internet was used to determine the email addresses. the editorial-board members were invited to sharma industrial marketing management ( ) - participate in a survey on the importance of the subareas of sustainability in business-to-business markets. two reminders were sent, a week apart. the mailing encountered three issues. first, the email addresses could not be confirmed and a number of emails were inaccurate. second, since the email had a survey link, a number of emails went into spam folders. finally, the survey was conducted during the covid- pandemic, which reduced response rates. invitations were sent out, of which could not be delivered (incorrect emails and bouncebacks). responses were received and responses were dropped because of incomplete data, for a final total of respondents. the response rate is comparable to that of paesbrugghe et al. ( ) , who received responses, and of kumar, sharma, and salo ( ) , who received responses. the importance of each topic and the provided research questions was calculated (see table ). one of the major findings was that the range of scores was small, from . to . . this contrasts with paesbrugghe et al. ( ) , whose range was . to . , and kumar, sharma, and salo ( ) , with a range of . to . . the results suggest that researchers regard as important all research areas in sustainability in business-to-business markets. the six most important topics were the effect of sustainable strategy on revenue and profitability (mean = . ); policy, regulatory, and legal framework (mean = . ); economic returns (mean = . ); overall market strategy (mean = . ), and customer demand (mean = . ). academic researchers also provided possible research questions for each category. to reduce the number of questions, two assistant professors and two phd students with an interest in sustainability and business-to-business marketing were asked to reduce the number of research questions to five relevant research questions for each subarea. the researchers received descriptions of the areas and subareas and were asked to reduce the number of research questions to five, representing the subareas. the initial agreement on the top five sample questions was % percent, and the researchers discussed the results and identified the top five research questions by consensus. the top five research questions appear in table for antecedents, table shows sustainability strategy, and table shows outcomes. the top six areas are discussed next. limited research addresses the impact of sustainability strategy on revenue and profits in the business-to-business domain. sharma and iyer ( ) suggest that resource-constrained sustainable-product development reduces costs to the customer. jr et al. ( ) found that environmental strategic focus relates to financial performance. some issues that cronin, smith, gleim, ramirez, and martinez ( ) raise are essential to future research endeavors. they suggest investigating the relationship between sustainability strategies and firm performance, the relative effectiveness of various sustainability strategies, and the relationship between developmental processes and performance of sustainable products. the outcomes focus on conservation or sustainable products and services. research has shown the positive impact of conservation (e.g., the effect of sustainability strategy on the environment . the effect of sustainability strategy on revenue and profitability . the effect of sustainability strategy on customer perceptions, preferences, and satisfaction the effect of sustainability strategy on shift in customer demand . table sample antecedent research questions. research area sample research questions reducing the use of energy) on profitability. academic researchers suggested the importance of the following aspects of sustainabilitystrategy effect on revenue and profitability: • mediators, moderators of the relationship between sustainable product strategies and performance in business-to-business contexts; • financial outcome indicators that can help identify long-term benefits of sustainability strategy; • the costs of sustainability strategy and determining recovery strategies; • firm's sustainability strategy and revenue and profitability; • tradeoffs among people, profits, and planet in sustainability business-to-business market strategy. academic research on the effect of regulatory and legal frameworks for sustainability on sustainable business strategy was not found. research on the real-estate industry (e.g., payne, ) includes no formal frameworks. academic researchers suggested the importance of the following aspects of policy, regulatory, and legal frameworks' effect on sustainability strategy: • the nature of business-to-business firms' influence on policy, regulatory, and legal frameworks; • the effect of public-policy differences in various nations on sustainability strategy of global firms; • the collaboration between business actors and policymakers and the achievement of sustainability goals; • business-to-business firms following the spirit of the policy and regulations or prescriptions; • the effect of policy, regulatory, and legal frameworks on businessto-business relationships. there is very limited research on economic returns from sustainable strategy (e.g., sharma & iyer, ) , and academic researchers suggested the importance of a deeper examination of the following aspects of economic returns' effect on sustainable strategy: • nature of economic returns in business-to-business sustainability • the economic impact of sustainable business models on business-tobusiness ecosystems; • metrics for assessing the performance of firms committed to sustainable business-to-business market strategy; • the incremental profit potential of the transition to a circular economy; • customers' willingness to pay more for sustainable products and services. few articles examining overall strategy were found. peattie and ratnayaka ( ) develop a comprehensive model of green marketing, patala et al. ( ) a value-creation model, and lacoste ( ) a model that focuses on a sustainable supply chain. academic researchers suggested that the following aspects of overall market strategy on sustainability are important: • the best practice in overall sustainable market strategy; • creating a balanced and sustainable business-to-business market strategy where different stakeholders have different expectations; • market-shaping strategies for creating sustainable product and service markets; • market strategy that allows business-to-business firms to be both sustainable and competitive; • the advantage and disadvantages of being an early or a late entrant to sustainable markets. very limited research exists on customer demand for sustainable products and services by business-to-business firms. academic researchers suggest a deeper examination of the following aspects of customer demand's effect on sustainability strategy: • managers' prediction of customer demand for a sustainable businessto-business market strategy; • level of business-to-business customers' knowledge about sustainability issues; • the degree to which demand drives innovative sustainable business models; • the factors that increase customer demand for sustainable businessto-business products and services. • the nature of the demand for sustainable business-to-business products and services. academic research on the effect of sustainability business strategy on the environment was not found. academic researchers suggested that the following aspects of the effect of sustainability strategy on the environment are important: • the effect of business-to-business sustainability strategies on the environment; • reducing carbon-dioxide emission by different sourcing over a shorter supply chain; • the collaboration of business-to-business firms and experts in environmental technologies to measure the impact of sustainable business-to-business market strategy; • the appropriate environmental indicators to measure the effects of sustainable market strategy; • business-to-business firms' introduction of sustainable products and services and the impact on the environment. table sample outcome research questions. sustainability offerings are becoming more important for businessto-business firms. this paper addresses three interrelated research issues. the first entails the basic question of what research is available to researchers and practitioners. therefore, this study examines prior research in the area and finds that the research is inadequate and not commensurate with the importance of the topic. the second research question considers the framework for examining sustainability research in business-to-business markets. as there are no existing frameworks, one is presented. the framework has three areas-antecedents, strategy, and outcomes. the subareas of antecedents are science and environment; policy, regulatory, and legal framework; social aspects; economic aspects; and customer demand. the subareas of strategy are overall strategy of the firm, supply chain, and marketing strategy and associated elements-customer behavior; product/service development, positioning, and branding; pricing; communications; and distribution. finally, the subareas of outcomes are environmental effects; revenue and profitability; customer perception, preference, and satisfaction; and shift in customer demand. the framework provides guideposts for future research. the third research question addresses the subareas that need greater focus and deeper examination. the paper provides details of survey responses by academic researchers and suggests that academics researchers regard sustainability as an important are of research. the six most important topics identified by academic researchers were the effect of sustainable strategy on revenue and profitability; policy, regulatory, and legal framework; economic returns; overall market strategy; and customer demand. this paper thus represents a strong call for further research into sustainable business-to-business marketing. three areas demand attention. first, previous research has assumed customer demand for sustainable products and services, but continued research must examine the nature of that demand. second, the majority of prior research focuses on specific and narrow models, indicating a clear need for broader perspectives. there are issues of corporate citizenship, business ethics, management state, and the level of analysis (firm or network/ecosystem level) that are important and need to be examined. third, the proposed framework for classifying sustainability research should be tested and expanded in future research, to achieve a deeper understanding of the key questions and research issues. for example, what alternate schools of thought can be used to develop frameworks? similarly, what are the boundaries and boundary conditions of the attributes of the framework? also, what are the drivers of the attributes in the model? how do firms support external research to advance sustainability throughout society? how can business-to-business firms combine expertise in science and environment and sustainable business-to-business strategy? . how does environmental crisis (e.g., covid- ) influence business-to-business relationships? how do business-to-business firms use scientific evidence to create sustainability strategies? policy, regulatory, and legal framework . how do business-to-business firms influence policy, regulatory, and legal frameworks? . how do public-policy differences in various nations' markets affect business-to-business sustainability strategies of global firms? . how does collaboration between business actors and policymakers affect achievement of sustainability goals? . which business-to-business firms follow the spirit of policy and regulations or prescriptions? . what is the effect of policy, regulatory, and legal frameworks on business-to-business relationships? social networks . how do social networks mediate unethical business-to-business practices? . how do social networks affect the identification and selection of partners with similar values and sustainability goals? how do customer social networks influence business-to-business firms' sustainability strategy? how do internal (to the firm) social networks influence business-to-business firms' sustainability strategy? economic returns . how do sustainable business strategies provide economic returns to business-to-business firms? . what are the economic impacts of sustainable business models in a business-to-business ecosystem? . what metrics should be used for assessing the performance of business-to-business companies which business-to-business customers are willing to pay more for sustainable products and services? customer demand . how can managers predict customer demand for a sustainable business-to-business market strategy? . how knowledgeable are business-to-business customers about sustainability issues? . to what degree are innovative sustainable business models demand-driven? . what factors increase customer demand for sustainable business-to-business products and services? . what is the nature of the demand for sustainable business-to-business products and services? a what are the best practices in overall sustainable market strategy? how can business-to-business managers create a balanced and sustainable business-to-business market strategy wherein different stakeholders have different expectations? . what market-shaping strategies can be used to create sustainable product and service markets? . what market strategy allows business-to-business firms to be both sustainable and competitive? what are the advantages and disadvantages of being an early entrant or a late entrant to sustainable markets? supply chain . how should supply-chain networks be restructured for sustainable business-to-business market strategy? . how can business-to-business firms incorporate reverse supply chains in distribution channels? . how can sales and operations planning underpin sustainable market strategy? . how should contracts and negotiations between supply-chain members change in sustainable markets? . what is the role of supply-chain in sustainable business-to-business market strategy? customer behavior . what are buyers' sustainable practices in business-to-business markets? what drives business-to-business customers' preference for sustainable organizations? what are the differences in business-to-business customer decision-making when buying sustainable products and services? how do business-to-business customers measure sustainability impact when buying products and services? product/service development, positioning, and branding . what types of sustainable new-product development/innovation affect business-to-business firms' performance? . how should a business-to-business firm position its products and services in the current and emergent sustainable markets? . what are the benefits of co-branding for creating value in business-to-business sustainable markets? how can brand managers manage the brand journey of consumers in the context of sustainable business-to-business strategy? pricing . how should companies account for customers' willingness to pay for sustainable products in their negotiations with suppliers? . what is the price sensitivity of business-to-business customers for sustainable products and services? . what are business-to-business customers' responses to price promotions for sustainable products? . should business-to-business firms focus on value instead of pricing for sustainable products and services? . which elements of sustainability should be considered in the price definition of business-to-business products and services? what types of sustainable product and service pricing in business-to-business markets different from traditional pricing? communications and sales strategy . how can a business-to-business sales organization positively impact the environment? . what message frames are more effective in changing business-to-business customers' preference for sustainable products? how can sustainability policies and strategies be successfully transferred to the sales organization? how can business-to-business firms develop transparent communications and sales strategy in the context of sustainable market strategy? what types of sustainable products and service communications in business-to-business markets different from traditional communications? distribution . how can distribution strategy become an integrated part of sustainable business-to-business market strategy? . how does the business-to-business distribution function evolve to address sustainable products and services? . what strategies shape direct and reverse logistics to minimize the impact of distribution on the environment? evolving sustainably: a longitudinal study of corporate sustainable development 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movement barriers and bridges to the adoption of environmentally-sustainable offerings sustainability and businessto-business marketing: a framework and implications resource-constrained product development: implications for green marketing and green supply chains b b branding in emerging markets: a sustainability perspective cooperation with international ngos and supplier assessment: investigating the multiple mediating role of csr activities in smes sales force reactions to corporate social responsibility: attributions, outcomes, and the mediating role of organizational trust our common future the quest for sustainable business model innovation overall market strategy the effect of sustainability strategy on the environment . how do business-to-business sustainability strategies affect the environment? . how does different sourcing over a shorter supply chain reduce carbon-dioxide emission? . how should business-to-business firms collaborate with experts in environmental technologies to measure impact of sustainable business-to-business market strategy? . what are the appropriate environmental indicators to measure the effects of sustainable market strategy? . how does the introduction of sustainable products and services by business-to-business firms impact the environment? the effect of sustainability strategy on revenue and profitability . what are the mechanisms (i.e., mediators, moderators) that connect sustainable product strategies to performance in business-to-business contexts? . what are the financial outcome indicators that can help identify long-term benefits of sustainable strategy? . what are the costs of sustainable strategy and what are the recovery strategies? . how does a firm's sustainability strategy affect the business-to-business firm's revenue and profitability? . what is the tradeoff between "people, profits, and planet" in sustainable business-to-business market strategy? the effect of sustainability strategy on customer perception, preference, and satisfaction . what sustainable business-to-business market strategies are driven by competitive parity? . do business-to-business customers believe that sustainable options are better? . what types of sustainability strategy lead to long-term effects on business-to-business customer preferences? . how does a firm's sustainability strategy affect business-to-business customers' perception, preference, and satisfaction? . how is customer satisfaction with sustainable business-to-business products and services different than with traditional products and services? the effect of sustainability strategy on shift in customer demand . how does sustainable business-to-business market strategy provide protection from demand fluctuations? . how can an organization advance sustainable consumption through sustainable business-to-business market strategy? . what are the key components of sustainable business-to-business market strategy in shifting customer demands? . how does a firm's sustainability strategy affect business-to-business customers' demand? . how does increased availability of sustainable products and services affect the business-to-business customers' demand? key: cord- -osfpc px authors: mckinley, jim; mcintosh, shona; milligan, lizzi; mikołajewska, agata title: eyes on the enterprise: problematising the concept of a teaching-research nexus in uk higher education date: - - journal: high educ (dordr) doi: . /s - - - sha: doc_id: cord_uid: osfpc px existing research into the relationship between teaching and research in higher education is mainly normative and atheoretical, resulting in assumptions of a close and beneficial connection between them. we problematise the idea of a nexus by undertaking a critical examination of the concept through the lens of educational ideologies to theorise the changes over time that shape the ways teaching and research are practised. two hundred seven academic staff in the humanities and social sciences were surveyed in universities in england and wales; the universities were identified as having strength in teaching, research, or in both. along with analysis of interviews with senior managers at these universities, findings suggest that systemic forces which separate teaching and research are evident in institutional contexts with implications for the idea of a nexus. while the nexus may exist in theory, in practice, we argue that teaching and research can be pulled in different directions by institutional priorities. furthermore, in institutions which adopt an enterprise ideology, there are signs of a nascent nexus emerging between research and innovation. the concept of a 'teaching-research nexus' in higher education is one that is constantly in flux, yet persists as an academic ideal (fanghanel et al. ) . while argued that bringing cutting edge research into lectures and exposing students to the latest advances in the field in which they study are beneficial endeavours (smeby ), more recently, the strength of this connection has been questioned in light of its relevance or value (malcolm ) in the between knowledge production and teaching whereas 'nexus' (jauch and gentry ) is a term implying an immediacy between the teaching and research aspects of academic work. according to tight ( ) , the nexus emerged from a humboldtian ideal of higher education. dating from the nineteenth century, when scholars learned alongside masters that were experts in their field, this model of a symbiotic connection between teaching and research came to characterise the work of the european academic, reified in the standard academic contract in which teaching and research are equally apportioned. this gives rise to the view that a close coupling is fostered when there is a strong overlap between teaching and research in practice (clark ) . however, such symbiosis rests on evocations of teaching and research as ideal types (fanghanel et al. ) , overlooking inherent dilemmas (martin and berry ) and fostering normative views about the nature of the nexus. its existence was queried when no positive correlation was found between teaching and research (hattie and marsh ) leading to its being declared virtually dead soon after (newby ) . however, healey and jenkins ( ) note distinctions between research into student experiences of research and institutional practices to encourage a close connection between teaching and research. in doing so, they argue that the institution mediates the enactment of academic work in ways that can strengthen or weaken the relationship between teaching and research. the focus on student experiences of doing research, or learning from research-active faculty, described as 'research-led teaching' (e.g. zamorski ) or 'research-based teaching' (e.g. fuller et al. ) is distinct from concepts of a nexus in academic practice. such distinctions (teaching or research-led/research-based/research-informed) in discussions of the teaching-research nexus reflect scholars' efforts to categorise different 'dimensions' of relationships between teaching and research. krause ( ) identified seventeen such dimensions concerning a nexus on australian university websites, and trowler and wareham ( ) developed a seven-dimension model based on a review of the nexus literature that subsumed krause's seventeen. this work suggests the identification of nexus models can provide useful frameworks for research and curricular policy decisions. however, the enactment of teaching and research is subject to influence from various factors that change over time. following becher and trowler ( ) , trowler and wareham ( ) point out that the relationship between teaching and research is profoundly influenced by academic disciplines, a point recently underscored by findings that pedagogies and curricula are valued differently across disciplines (abbas et al. ) . disciplines are also differently valued under different ideological conditions in higher education. the field of the humanities and social sciences, for instance, is considered disadvantaged by commercially driven higher education priorities (benneworth and jongbloed ) . moreover, academic practice is influenced by global forces, and related trends such as the massification of higher education began in the usa after world war , which prompted a shift away from the humboldtian-style nexus (tight ) . additionally, teaching and research are practiced within a sector that places greater value on research than on teaching. although, occasionally, teaching is the lead activity (brew ; fanghanel and trowler ) , studies on the nexus most often lead with research, describing a 'researchteaching nexus' (e.g. farcas et al. ) . this implicit, sector-wide value hierarchy (bazeley ) may explain why the claims in many nexus studies rest on data collected in researchintensive universities (e.g. cadez et al. ). this limitation is joined by a perceived lack of empirical evidence grounded in academics' perceptions (turk and ledić ) that fail to recognise institutional variation (jenkins and healey ) . consequently, a recent review of existing research into the nexus concluded that, as a field, it is 'ambiguous and sometimes contradictory' (elken and wollscheid : ) . uncritical idealisation of the nexus, therefore, evokes a normative, value-devoid concept that overlooks situated and historical influences. it also fails to recognise that its enactment unfolds under disciplinary, institutional, and systemic conditions that are shaped by overarching political ideologies. when resources such as time and money are scarce, understanding how teaching and research play out under different circumstances is crucial to discern whether the nexus is a help or a hindrance in conceptualising academic practice. assumptions underpinning teaching and research in current academic practice one task, then, is to critically examine the assumptions underpinning the existing research and question the normative stance that presents the nexus as 'a good thing'. another is to ask how it plays out in the current, highly marketised and globalised higher education sector (marginson ) which is facing increasing financial uncertainty as a consequence of covid- . even before spring , the funding of uk higher education was under strain, with universities competing for limited monetary resources while simultaneously attempting to meet rising expectations about standards of teaching and research from students, managers, and funders (fanghanel et al. ) . indicators of success came to be understood in relation to performative measures of standards of excellence. some of these standards were enshrined in evaluative instruments which, notably, separate teaching and research. currently, the research excellence framework (ref), which is in its second iteration, has attracted criticism summarised by butler and spoelstra ( ) as contributing to a 'regime' of excellence that orientates researchers to work towards ref criteria. meanwhile, the more recent teaching excellence framework (tef) acts as a proxy assessment of undergraduate teaching quality. the tef has been criticised for being unlikely to achieve its stated aim of putting teaching at the heart of higher education (forstenzer ) and of introducing ofsted-style rankings likely to 'punish institutions outside london and threaten arts and humanities courses' (fazackerley : ) . separate evaluations of teaching and research at systemic level put pressure on the relationship between the two activities in daily practice (mcintosh et al. ) . academics, depending on the priorities of their institutions, may find the connection between teaching and research under strain (macfarlane ) and be faced with daily compromise (mcintosh et al. ) . the ideal of a symbiotic nexus is, therefore, undermined by structural separation of teaching and research. this separation raises the question about how and in what ways the nexus emerges in a marketised university sector, where teaching and research are evaluated, funded, and managed separately (locke ) . however, and in addition to the limitations noted above, the field also: … tends to be atheoretical. an empiricist ethic prevails, and underpinning this is a foundationalist ontological position, which assumes that a reality exists which can be apprehended by research which is sufficiently robust and extensive. an alternative position is a social constructionist one which stresses situational contingency. (trowler and wareham : ) we meet this critique of the existing shortcomings of research in the field by recognising that any nexus is sensitive to the forces that shape teaching and research practices at the institutional level, as well as the wider landscape in which the institution operates. for this reason, it is important to theorise higher educational institutions as shaped by dominant ideologies. the nexus: in theory? conceptualising the nexus as an ongoing process (mckinley ) helps identify it as emerging with different characteristics under different structural conditions. we build on jenkins and healey's ( ) argument that institutional-level strategies are one way in which the relationship between teaching and research is mediated, by contending that the perceptions academics have of these strategies must also be accounted for. this is because the organisational context and the culture within organisations cannot be assumed to support a close connection between teaching and research (trowler and wareham ) . organisations that emphasise research over teaching, for instance, bifurcate teaching and research at institutional level (burke-smalley et al. ), making it difficult for academics to meet the demands of an encroaching ideology of excellence (butler and spoelstra ) which manifest institutionally through managerialist adherence to criteria of systemic tools of evaluation, such as the tef and ref. additionally, it is important to acknowledge that teaching and research will be variably interpreted in institutions, according to their different remits, histories, priorities, and values. in the uk, the robbins report ( ), for instance, categorised the 'ancient' universities separately from london, with its federation of colleges, the older civic universities separated again from teaching colleges and so forth. differences in 'types' of higher education have also been categorised in accordance with their function (moodie ; tight ) ; and, under a 'binary' system of higher education (scott ) , teaching, rather than research, has been viewed as central to the post- universities (macfarlane and hughes ). where the institutional primary task is understood to be one or the other, the conditions for bringing both together will be more challenging than in institutions which value both equally and may jeopardise the existence of a nexus. however, publicly available information about university performance in the universities funding council and the times good university guide challenges ubiquitous university types based on, inter alia, age, location, research performance, and student intake (lysons et al. ) . although many university typologies are functional (moodie ) and rest on assumptions of cohesion around institutions' logics (shields and watermeyer ) , we see this as over-simplistic, and agree with trowler and wareham ( ) who argue that academics' practice in teaching and research are undertaken in relation to ideologies, that is, values, ideas, and beliefs that guide their working practices and shape the university as an institution. to discuss the nexus without recognising these shaping forces is to perpetuate the limitations in the field we noted above. trowler and wareham ( ) illustrate changing attitudes and values towards teaching and research through a typology with four ideological perspectives on education. the typologies are arranged in a broadly chronological order, from oldest to most recent in time, with our own definitions provided for each in relation to a nexus: we find that these are useful ways of conceptualising the systemic conditions under which teaching and research relate to each other and, thus, different potential enactments of a nexus. our adaptation of this typology evokes the nexus as a lens through which to analyse higher education practices of teaching and research in relation to a shift towards enterprise as the new dominant educational ideology. for example, in the traditional ideology, the humboldtian ideal suggests a symbiotic nexus flourishes with the research student studying from the master advancing his field of scholarship, whereas this closeness under a progressive ideology that emphasises the transformation of individuals and knowledge will depend on the extent of overlap between teaching and research activities. under social reconstructionism, the university is a place for developing human beings as critical, sceptical, and vigilant contributors to a more socially and politically just society (abbas et al. ) and, when aligned with research objectives, can result in a strong nexus. however, in the enterprise ideology of the marketised university sector where higher education is positioned as an economic commodity (naidoo ) , what happens when the pairing of research and teaching has to accommodate further activities (brennan et al. ) as evinced through the knowledge exchange framework? disciplinary differences further complicate the interplay of teaching and research under different ideologies. in the humanities and the social sciences, for instance, moving away from social reconstructionism towards an enterprise ideology may prompt new alignments with innovation and entrepreneurship in ways that could compromise historic agendas of criticality and social justice and, consequently, the relationship between teaching and research. and finally, different universities will interpret and prioritise all these demands differently. the logical conclusion is that under differing ideologies, the teaching and research nexus unfolds in complex and institutionally variable ways. in this paper, we contribute to debates about the teaching-research nexus in three ways. firstly, we theorise the practices of teaching and research as related to educational ideologies by analysing academics' perceptions of the relationship between teaching and research. secondly, we analyse the nexus in relation to differing institutional priorities through data collected from a range of universities: institutions with strengths in teaching, strengths in research, and those where strengths in teaching and research are balanced. we do this analysis not according to trowler and wareham's ( ) four ideologies, but instead use institutional differences (teaching-strong, research-strong, and balanced) to illustrate that there is evidence of a shift in these ideologies. thirdly, we suggest that the teaching-research relationship is affected through variations in responses to the growing demands of an enterprise ideology. the original contribution lies not only in the new and extensive data set but also in extending the contentious debate about the characteristics, value, or even existence, of a nexus (malcolm ; tight ) once it is considered in relation to these wider historical and systemic contexts. the study follows a concurrent, partially mixed design, with equal weight given to quantitative and qualitative data. data were collected by two means: a survey of practising academics in uk universities via an online questionnaire and qualitative, semi-structured interviews with senior managers at the same institutions. the sample was selected to focus on humanities and social sciences faculty in universities across a spread of geographic locations. through purposive sampling, we identified senior management in key positions such as pro-vice chancellors of teaching and/or research at universities in england and wales with a range of institutional priorities. the complete university guide that uses tef and ref scores served as an index to classify universities by their orientation: research-strong (n = ), teaching-strong (n = ), or balanced (n = ). we understand that tef and ref ratings are limited indicators (forstenzer ), but following moodie ( ) , the guide was considered useful in characterising universities in ways that extended dominant university typologies. universities' participation was further dependent on our ability to secure appropriate senior management participants within the project time frame. the questionnaire link was sent to academics working in the humanities and social sciences at each university, distribution assisted by the participating managers. questionnaire data were gathered from participants ( females, males, no specified gender). the majority of respondents were from balanced universities (n = ), followed by academics from research-strong (n = ) and teaching-strong (n = ) universities (table ) . the semi-structured interview participants were well-established academics who had worked in higher education for - years and were, therefore, well-positioned to provide insights into teaching and research at their universities. each participant was asked the same set of eight questions. interviews lasted between and min and were conducted largely by telephone, audio-recorded, and transcribed before analysis, with the exception of one interview in which field notes were used as the participant chose not to have the interview recorded. the survey was designed to investigate respondents' perceived connections between teaching and research and identify factors related to any differences. the content of the survey was brainstormed by three of the researchers. for instance, we listed activities that we most commonly do in our own work as academics. this led to developing answer options for question number . we also provided space for open-ended responses to collect options not krause ( ) or trowler and wareham ( ) , as this could have yielded insights to the situation at a granular level. however, we elected not to use such categorisations, opting for a less clear identification of a nexus. due to changes in such categorisations over time, we wanted to allow for the possibility that a nexus might not, in fact, exist for some respondents. while this may be a potential limitation to the study as a more comprehensive survey could have yielded more insights, the decision did allow us to draw our own conclusions based on university types. a draft survey was sent to an expert who suggested changes, which were applied to the final version of the survey. the questionnaire consisted of four parts: the quantifiable data from the questionnaire, such as 'contract type' or research activities a participant engages in, were analysed using descriptive statistics to gain insights into participants' perceptions of a teaching-research nexus as enacted in their institution, as well as how they understand the priorities towards teaching and research activities in their own work. the answers to open-ended questions (e.g. when participants are asked to explain how teaching and research overlap in their academic work) were analysed using thematic analysis to identify perspectives that aligned, or did not, with a nexus. the qualitative data gathered through the interviews were also analysed and coded using thematic analysis. because we recognise the institutionally specific context matters in the way the nexus is perceived, in this paper, we focus on analysis of data that both provides insights into institutional differences and illuminates different demands that are placed on the teachingresearch relationship. how do teaching and research overlap? responses to teaching and research activities showed fragmented perceptions of an overlap (fig. ) . where some see the kind of complementarity inherent in the ideal nexus, there was a minority who reported little or no overlap or support. further detail about this fragmentation can be seen when activities are broken down (table ) . respondents were asked 'which of these do you consider to be teaching activities ( … )', and which are '(...) research activities that you have undertaken in the / academic year?' as activities could be identified as both, answer options listed in table from to indicate overlapping classification. supervision and keeping up to date with current research are clear examples of activities understood to be closely connected and, therefore, may be where a nexus might flourish. this is in keeping with the humboldtian ideal under the traditional educational ideology. a nexus therefore exists as a close connection in a small number of specific activities. the activity with the highest number of responses, 'keeping up to date with research', which seems fundamental to an academic identity, is understood to be a holistic activity that is achieved via both teaching and research. this point is further supported by responses to open-ended questions: keeping up to date with current research -ensuring reading lists are updated so students are exposed to the latest debates/articles in the field. this also assists with research and engaging with recent debates. (balanced) another example of an activity that supports a nexus is the supervision of students. most often, participants commented that supervising students is a stimulating and thought-provoking process mostly when considering postgraduate students: supervision of doctoral students ( … ) can be quite stimulating for ideas ( … ) marking students' work at postgrad and higher level as you learn from them. (balanced) there was a strong emphasis on the exchange of knowledge between lecturers and students in the responses from all academics. however, only academics from teaching-strong universities expressed that they find support for their research from undergraduates: (table ) raises a question. is it the case that, in teaching-strong universities, there may be an emphasis on the first set of (teaching-focused) activities rather than the lower (research-focused) activities? the resulting institutional focus on teaching or research may serve to shape the activities undertaken by academics' work. however, interview data show that collaboration of established academics and academics-soon-to-be exist in pockets, or as 'examples': we've got applications for readerships where colleagues have published - papers in peer-reviewed journals, with their students, so there is, you know, really good examples of where the opportunities for students is fantastic. (teaching-strong) academics keeping up with current research and supervising research students, following a 'traditionalist' ideology and the humboldtian ideal, as shown in the overlapping activities (table ) , may very well perceive a nexus in their practice. however, academics whose workload tends towards teaching, or working on funded research, might struggle to identify a nexus. this supports our earlier point that the nexus must be understood within its contextual and historical context. it is also worth noting that public engagement, a relatively recent aspect of academic work, appears to be understood by many as connected to teaching and research, although elsewhere it is understood as closely connected with the innovation agenda (pinheiro et al. ) . meanwhile, the development of new ideas being associated only with research may assume importance under an enterprise ideology, something that some universities may be adopting more readily than others. institutional differences between perceptions of how teaching and research are supportive of each other, and how much they overlap, show broad differences in percentages when responses were collapsed into 'very much/supportive' or 'not at all/unsupportive' ( table ) . analysis of potentially significant differences using the kruskal-wallis test on each item for the three groups (teaching-strong, research-strong, balanced) found no significant difference (p = . ) regarding the extent to which teaching and research support each other. however, a kruskal-wallis test for the item concerning perceived overlap between teaching and research found significant difference according to university type (p < . ). the results for teaching-strong universities were p < . , se = . while at balanced and researchstrong universities, the result was p < . , se = . . pairwise comparisons of university type revealed that there was no significant difference between respondents at balanced and research-strong universities but participants in teaching-strong universities perceived significantly less overlap than those at balanced and research-strong universities. the conclusion is that, for academics in teaching-strong universities, teaching and research overlap much less. therefore, although there is an agreement that teaching and research are perceived to support each other, regardless of university type, differences emerge when respondents are specifically asked to consider how they overlap. in other words, an acceptance of a close connection may be strong in the abstract, but in practice, depends on the institutional type. we see further evidence to raise questions about the closeness of the connection in practice, and thus a possible drift toward an enterprise ideology, when examining different priorities at university, faculty, and departmental levels (fig. ) . figure shows that the further from practice, the more research is prioritised. the university is perceived to predominantly prioritise research, perhaps due to a stronger focus on enterprise, while at the department level, both teaching and research are perceived to be priorities. teaching is perceived to be the lowest priority at all university levels, potentially undermining the complementarity required for a strong connection between teaching and research in practice. in teaching-strong universities, we see evidence of pressure when demands of faculty and university differ: although research is increasingly prioritised at the faculty level ( … ), the university is still focused primarily on teaching. this means that i have to find time to conduct research outside of my teaching commitments. for example, it was impossible for me to pay the necessary attention to a research project i had funding for last term due to teaching commitments. i am now in a position where my teaching workload is lighter and i can try to catch up on the research i am conducting. (teaching-strong) similarly, in interviews with managers, teaching and research were separated structurally, reflecting their separation in the tef and ref; for instance, 'there's a director of teaching and a director of research' (balanced), or connection only very high up in the structures. however, one senior manager in a teaching-strong university disagreed with such structural separation: for the first time the management of research and teaching was put kind of in the same person (...) it should be that every single department is underpinned by excellent teaching and research so it's a very clear attempt to bring those two things together. (teaching-strong) this comment points to the university's drive to integrate quality research with teaching excellence. indeed, for some managers, the nexus was the subject of intentional institutional policy: there really is an expectation that all students have a really high-quality teaching experience and that they meet the professors as well as the junior lecturers; there is no kind of, you know, some people are too important to teach; all professors are expected to teach. (balanced) this manager clearly sees benefits in students being taught by researchers and suggests a close connection between the two in institutional policy. additionally, in sketching a pastiche of the distant professorial type, she recognises that some researchers may seek to escape teaching altogether; intentionally discouraging that expectation will shape resource-deployment decisions and priorities. for example, several teaching-focused initiatives were listed in this interview, including teaching pathway promotions, indicating an institutional commitment to balancing teaching and research. in such a context, it may be possible for a close connection between teaching and research to thrive. however, this may differ across institution types. this was also evident in the questionnaire data; for example, when a university has 'a lot of research-only people but it doesn't have many teaching-only people' (research-strong) whereas academics from balanced universities realise that they should be able to juggle the work associated with teaching and research activities and they are 'very sceptical of either research-only or teaching-only contracts' (balanced). the option for teaching and research balance in a work contract may be difficult in a teaching-focused university: teaching-only contracts makes it hard to catch up with the research demands and be able to move to a teaching and research contract (which has more prestige and security in my university). (teaching-strong) questionnaire respondents at research-strong universities also indicated awareness of this institutional difference: i am fortunate that this faculty values teaching as well as research. my answers would be different in different universities, where i suspect teaching is undervalued relative to research. (research-strong) thus, where teaching is undervalued, the teaching-research nexus may struggle to flourish. however, there was evidence that some hope the tef will enable a better institutional balance between teaching and research demands: academia is the only work where poor practice is rewarded. if you neglect teaching and students but write papers you will be rewarded. conversely, being committed to students simply leads to more unrewarded work. this university is attempting to mitigate this by developing a teaching-related pathway. until the political economy of the funding of he changes this will always be the case (tef may change this). (research-strong) one respondent from a teaching-strong university who has been working in academia for several decades suggests there has been an increasing interest in strengthening the research profile of universities, regardless of their type: having taught at both pre and post institutions i can say with some authority that the teaching load is on a completely different scale at post- institutions in terms of quantity. this has an impact on the time members of staff are able to devote to research and publications. while i have indicated above that teaching is prioritised and that is unlikely to change, there is no doubt that a research culture is developing at subject community, school and university levels. (teaching-strong) the separation of teaching and research through systemic evaluative tools is seen to be shaping perceptions of a nexus for those managing as well as those undertaking academic work. however, research remains more highly valued. thus far, analysis shows an institutional variation in the way the nexus is perceived. next, we examine the effects on the nexus under the introduction of innovation and its increasing intimacy with research. the idea of a teaching-research nexus came under scrutiny from six of the interviewed managers, who questioned whether the nexus involved a close connection between teaching and research. they came from all three of the research-strong and two of the four balanced universities; none of those in teaching-strong universities was openly questioning the concept. maybe we have gone beyond it? but i was thinking that means we need to redefine that word, we need to, well revitalise it … making it something we actually physically recognise. (balanced) here, the interviewee recognises a disconnect between the idea of a nexus and seeing it in practice. others were aware of the increasing incentives to partner research with industry: [the university] innovations unit which is all about commercial dissemination of ideas; you know they're number one with industry for dissemination of research." (researchstrong) other examples of hitching research to enterprise were evident in interviews with managers in all three types of universities. examples from research-strong institutions included universitybased incubation and entrepreneurial schemes with prize money given to research with marketable potential that were open to undergraduates as well as postgraduates and academics. these strategies were found woven into institutions' fabric, influencing teaching as well as research: one of the core values of the university is enterprise, and i think we're all aware that there's quite a lot of challenges for [humanities and social sciences] in terms of speaking to the industrial strategy etc etc so we are being asked to write programmes that respond to that but they aren't actually necessarily rooted in people's research interest. (teachingstrong) an explicit identification of enterprise as a core value is seen to drive teaching away from being research-driven and towards industrial goals, while the language of competition emerges as this manager describes the industrial strategy driving the direction of research: the whole idea of the industrial strategy is about finding the edge to everything and how do we you know how do we find the next grapheme and how do we in a sense capture the quantum market. (balanced) industry, enterprise, and the marketability of ideas are evident, although this is not yet the case everywhere: i feel lucky to have been working in an organisation which hasn't radically gone down the line of splitting teaching and research but i am aware that a lot of organisations have to now quite a striking degree. (balanced) a clear perception emerges that some 'radical' splitting of research from teaching is underway 'now' and the examples we share indicate that some universities are indeed drifting towards connecting research and innovation in pursuit of an enterprise agenda. this may be at the expense of the research teaching relationship, introducing further strain on the 'ideal' nexus. these insights into varying institutional and wider systemic demands show teaching and research being pulled in different directions. and, when institutions are understood in relation to different educational ideologies, they can differently emphasise teaching and research. when research is harnessed to innovation, there is a drift towards an enterprise ideology that may undermine the connection between teaching and research and consequently weaken the nexus in institutions that explicitly pursue an enterprise ideology. our analysis contributes to debates in the field by raising questions about the teaching-research nexus in uk higher education today. while we find the idea of a nexus is persistent, one major criticism raised by elken and wollscheid ( ) is the partial nature of existing nexus research. it is often focused within individual practice or parts of institutions, with the result that claims are necessarily limited. the national scope of this study sought to avoid this limitation but, due to the disciplinary nuances attributed to teaching and research (abbas et al. ; becher and trowler ) , maintained some consistency by focusing on the humanities and social sciences. furthermore, we extended existing conceptualisations of university types beyond function that recognise institutional responses to historical shifts in the nature and conditions of higher education. we found variations between teaching and research, or research and innovation, may foster a disconnect between teaching and research exacerbated by an ideological shift towards enterprise. the current study indicates that universities with strong research priorities can jeopardise a strong nexus. therefore, hattie and marsh's ( ) conclusion that teaching and research are, at best, loosely coupled may still hold where research is the institutional priority. universities that adopt an enterprise ideology and are beginning to connect research with innovation may fall into this category, while universities who do not may still be motivated to foster a close connection between the two. a limitation of the current study is the number of universities. while conducting the questionnaire in a larger number of universities could have potentially yielded more comprehensive insights, we kept the number to for two reasons. first, the most important balance to maintain in the study was between the three university 'types', which we also tried to maintain with geographic diversity in the uk (initially, we contacted universities in scotland and northern ireland as well, but none agreed to participate), and where there were willing senior managers to distribute the questionnaire and sit for the interview. second, this was a funded project with a timeframe of just a few months, so pursuing further university connections was not possible. furthermore, as the funder for the project specifically targets humanities and social sciences, we only included universities where senior managers in these fields agreed to participate. a valuable extension of this study would be to conduct a refined version of the questionnaire across the uk. we support elken and wollscheid ( ) : ) in asserting that the relationship between teaching and research is 'a highly complex and multidimensional picture'. furthermore, when they point out that existing research is inconclusive, they also identify an alignment with the traditional humboldtian view of higher education, and one which appears increasingly dated. mckenzie et al. ( ) : ) claim that in australian law schools, the nexus is a myth, driven from existence by cultures where research excellence frameworks create 'an individualistic, competitive, disunited workplace'. in the uk today, the research excellence framework has been joined by two others, one focused on teaching and one on building links with industry. the academic who is operating under the evaluation of each of these different criteria of excellence may experience the different demands as an erosion of the quality of academic work (butler and spoelstra ) . research by cadez et al. ( ) presents the results of a cross-discipline study of academic staff in one research-focused slovenian university. the university is characterised as 'modern' for its incentivisation of research output production, as opposed to the quality of the outputs or quality of teaching. under such conditions, pressure to prioritise some activities over others may tip the balance to the extent that practice changes. the adaptation of trowler and wareham's ( ) earlier higher education typology to include the teaching-research nexus serves to emphasise the effect of changing ideologies on academic practice. it further highlights implications for the humanities and social sciences, as well as providing yet more evidence that a shifting higher educational ideology towards marketisation and competition, and an ethic of neoliberal enterprise is changing the nature and purpose of the sector (naidoo ; marginson ; mcintosh et al. ) . one recent example is the proposal by the uk government to measure the success of university courses by graduate earnings, entirely driven by this ideological shift (fazackerley ). equating quality with monetary earnings favours the disciplines leading to well-paid careers, such as medicine and law, over those in the arts and humanities sectors (see benneworth and jongbloed for stakeholder perspectives). such an initiative has further implications for the priorities within an institution and the position of disciplines within it. under an enterprise ideology, the funding of disciplines may come to be increasingly closely linked to metrics promoting the characteristics of market enterprise over social reconstructionism in a way that will shape institutional priorities, destabilise the sector's existing value hierarchy (bazeley ) , and rewrite the rules. although it is not a new idea that students see their degree as a step towards employability (tomlinson ) , our analysis shows that these views emerge within institutions that adopt a particular ideology. the extent to which this will remain the case, however, is uncertain in the coming era of artificial intelligence, technological unemployment (brown et al. ) , and the covid- fuelled economic crisis. this raises pressing questions about the purpose, nature, and material worth of higher education. since the enterprise perspective signals transformation of the nexus to align with innovation, and since innovation initiatives are used to build relationships between research and business, promoting them as social justice initiatives (as part of a social reconstruction ideology) might ultimately be economy-driven. indeed, such a summation aligns with descriptions of a higher education sector in an increasingly fastchanging knowledge economy as 'crucial … for the production, dissemination, and transfer of economically productive knowledge, innovation and technology' (naidoo : ) . this is of particular relevance in research-strong and balanced universities that adopt enterprise values, where harnessing research to innovation serves to disconnect research from teaching. from this disconnect, we see some significant implications concerning a teaching-research nexus. as universities respond to the incentives of the enterprise era, innovation, and especially innovation that is uncritically linked with student employability, is refocusing priority. meanwhile, some institutional initiatives that intend to redress the disconnect, for example introducing teaching promotion pathways, may in fact be mirroring the systemic divide evident at national evaluation levels (forstenzer ) . similarly, organisational structures such as departments for international education and innovation are sowing the seeds for the demise of the teaching-research nexus in ways that may reflect the changing character of higher education. to close, we argue that while the teaching-research nexus may be thriving in the imagination of uk academics, in practice, it is fragile. further research 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case publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements we are grateful to heath rose and joy cranham who provided invaluable feedback at various stages in the development of this paper. we thank the british academy for initiating and supporting the project and for providing the opportunity to conduct the study. authors' contributions all authors have made substantial contributions to the conception or design of the work or the acquisition, analysis, or interpretation of data; drafted the work or revised it critically for important intellectual content; approved the version to be published; and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.funding information this research was financially supported by the british academy, reference: tr / . conflict of interest the authors declare that they have no conflict of interest. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/ . /. key: cord- - cuzmsll authors: duprex, w. paul; fouchier, ron a. m.; imperiale, michael j.; lipsitch, marc; relman, david a. title: gain-of-function experiments: time for a real debate date: - - journal: nat rev microbiol doi: . /nrmicro sha: doc_id: cord_uid: cuzmsll according to the who, dual use research of concern (durc) is “life sciences research that is intended for benefit, but which might easily be misapplied to do harm”. recent studies, particularly those on influenza viruses, have led to renewed attention on durc, as there is an ongoing debate over whether the benefits of gain-of-function (gof) experiments that result in an increase in the transmission and/or pathogenicity of potential pandemic pathogens (ppps) are outweighed by concerns over biosecurity and biosafety. in this viewpoint article, proponents and opponents of gof experiments discuss the benefits and risks associated with these studies, as well as the implications of the current debate for the scientific community and the general public, and suggest how the current discussion should move forward. w. paul duprex. first, we must recognize that the term 'gof experiment' is being used somewhat pejoratively to describe a small number of recent studies using avian influenza virus that led to increased viral transmission in mammals. rather like the viruses we work with, the term has taken on a 'life' of its own, 'infecting' debates and 'muddying the waters' for scientists, governments, policy makers, journalists and the public. although à la mode, it is totally imprecise to equate gof studies only with influenza transmission experiments. virology is founded on adaptation approaches, and these have broad utility because they provide phenotypic evidence of a genotypic change when combined with a discriminatory biological assay. phenotypes include resistance to a drug, alteration of host range, enhanced stability and replication, and not only transmission. dissecting the underlying genotype drives mechanistic studies, which in turn facilitate the study of host-pathogen interactions. virologists will be deprived of a powerful tool of human inquiry if they are unable to perform adaptation experiments. second, it is critical to realize that the benefits of basic research are often unanticipated and accrue over time. considering that these influenza transmission studies were performed relatively recently , , it is impressive that translatable benefits are already apparent, including the identification of mutations that increase virus replication (which is applicable to vaccine production) and changes that enhance stability of receptor-binding proteins (which is useful for surveillance). ron a. m. fouchier. we need gof experiments to demonstrate causal relationships between genes or mutations and particular biological traits of pathogens. in most cases, there are no alternative approaches that would provide similarly strong evidence as gof experiments. for example, loss-of-function approaches will show that modification or deletion of almost every gene of a pathogen can result in a reduction of replication, pathogenicity or transmission. bioinformatics and modelling approaches may be used to identify associations between genotypic and phenotypic traits, but will very rarely prove causality. in vitro experiments on genes in isolation and studies with attenuated strains may identify causal relationships between genes and some biological traits, but many phenotypes can only be investigated in the context of the wild-type pathogen. therefore, gof approaches are absolutely essential in infectious disease research; although alternative approaches can be very useful, these can never replace gof experiments. michael j. imperiale. gof experiments using these types of pathogens allow investigators to ascertain whether certain new phenotypes, such as the ability to transmit more efficiently, can be acquired by the pathogens. in my opinion, there are two main reasons for performing such experiments. first, it is possible that the information gained from these studies can be used to improve surveillance or to develop therapeutics. second, these studies often teach us interesting biology. there may be alternative approaches available for some studies, but that would need to be determined on a case-by-case basis. there are also ways to build in safety features, such as the incorporation of a microrna target sequence into the influenza virus genome that results in inhibition of replication outside the laboratory setting . all of us share the goal of preventing and mitigating pandemics. biologists and public health specialists have a portfolio of approaches to do that; these include working with viral subunits to understand molecular and biochemical interactions in detail, studying sequences of animal and human viral strains, developing therapies that improve the host response or kill the virus, developing universal vaccines, improving technology for faster vaccine production and many more . creating ppps -a subset of gof experiments involving creation of novel, virulent, transmissible viruses -is one of these approaches. unlike other gof experiments, the creation of ppps entails a unique risk that a laboratory accident could spark a pandemic killing millions. the question is not whether to carry out research on ppps or to do nothing; it is whether to have a portfolio of approaches to defeat viruses without creating a pandemic risk, or whether to include ppp experiments in that portfolio. for example, we should decide whether devoting our limited resources for flu research towards ppp creation experiments -which are expensive, often underpowered, low-throughput and often poorly generalizable , and which create pandemic risk -is better than using those resources to enhance the rest of the portfolio for flu preparedness. similarly, it has been suggested that we need to enhance pathogenicity of coronaviruses in order to develop a valid animal model for corona viruses. this might be true, and we need to examine that assumption, but perhaps we can modify the animal to reproduce the human disease (as has been done, for example, by developing an animal model of meningococcal disease ) rather than making a novel virus. amazingly, abstract | according to the who, dual use research of concern (durc) is "life sciences research that is intended for benefit, but which might easily be misapplied to do harm". recent studies, particularly those on influenza viruses, have led to renewed attention on durc, as there is an ongoing debate over whether the benefits of gain-of-function (gof) experiments that result in an increase in the transmission and/or pathogenicity of potential pandemic pathogens (ppps) are outweighed by concerns over biosecurity and biosafety. in this viewpoint article, proponents and opponents of gof experiments discuss the benefits and risks associated with these studies, as well as the implications of the current debate for the scientific community and the general public, and suggest how the current discussion should move forward. these types of experiments were commenced without asking that question in quantitative terms, and no quantitative case has been made for why the unique risks are justified by unique benefits. david a. relman. i view gof as a generic label for a broad class of experiments that lead to a genetically altered biological agent with new or enhanced functions. these experiments help to link genotype with phenotype and can therefore be valuable, although they can entail risk and are by no means the only approach for linking sequence with function. my early research career was dedicated to the study of pathogens. i continue to believe that naturally occurring pathogens, including those that have the potential for causing pandemics, deserve detailed investigation in order to understand their behaviour and interaction with hosts, as this can inform drug design, vaccine development, diagnostics and surveillance. however, gof experiments are just one of several approaches for studying pathogens. inactivating mutations and the manipulation of key functional domains in attenuated genetic backgrounds are alternative approaches that may be slightly less informative, but are much less risky. because there are alternatives, gof strategies should be used cautiously and only to achieve critical benefits when they clearly outweigh the risks and are realizable in the near-term. in general, it is unnecessary and inappropriate to create new infectious agents that are capable of causing widespread harm. genetic and biological contexts are important. as an example, genetic engineering that is intended and likely to endow a low-patho genicity, low-transmissibility agent with either enhanced pathogenicity or enhanced transmissibility may be appropriate if the benefits are substantial. conversely, creating a highly pathogenic, highly transmissible organism that does not already exist in nature is unnecessarily risky and potentially irresponsible. in the debate over whether or not to allow durc, the main concerns seem to be over biosecurity and biosafety. however, research on pathogenic organisms that are major health threats already happens worldwide and is deemed safe. are there reasons to believe that the current requirements for biocontainment are insufficient for gof experiments? and what is your reaction to the recent announcement of a "pause on funding" for gof studies by the us government? w.p.d. i am confident that biomedical research on potentially dangerous pathogens can be performed safely and is essential for a comprehensive understanding of microbial disease pathogenesis, prevention and treatment. from the moment scientists brought clinical samples into the laboratory and isolated wild-type viruses, they have developed biocontainment procedures to mitigate risk. in my opinion, self-interest spurs a keen interest in biosafety, and virologists have no wish to endanger their colleagues or themselves. pioneer virologists would probably be amazed to see the advances in biocontainment infrastructure, and how developments in engineering and technology have changed working practices. trust, good communication and transparency are vital between scientists, facilities staff and security personnel. every time i wear my training suit in the biosafety level (bsl- ) laboratory, i am secure knowing that there are multiple reasons why, if there is a malfunction in the air, if the electricity supply fails or if there is a fire, i will still be able to breathe, pipette or exit the building safely. i trust the highly professional team of security guards, electricians, research safety experts, occupational health professionals and external inspectors from the us centers for disease control and prevention (cdc) and the us department of agriculture (usda), as this underpins everything i do. likewise, i am confident that they trust me to perform experiments responsibly and, if an accident occurs, to adhere strictly to standard operating procedures that are in place to minimize risk. working with dangerous pathogens is already highly regulated, and i believe that the current requirements for biocontainment are fit for purpose. therefore, i am convinced that limiting virus phenotype adaptation experiments by means of an ambiguously worded 'pause' is not the answer. in laboratories (and elsewhere), people make errors and machines occasionally stop working properly. as a consequence, biocontainment measures are designed in multiple layers, such that if some layers fail, others exist to mitigate the risks. furthermore, the layers of biocontainment measures increase in number and stringency with the increasing risk of the experiments. for example, in the case of our h n virus transmission studies, if initial biocontainment measures fail, our personnel is vaccinated against h n , can be treated prophylactically with antivirals and can be quarantined in specialized wards to prevent potential onward transmission. there is no evidence that current biocontainment measures are insufficient; major laboratory-derived human outbreaks have not occurred during more than a century of scientific research on dangerous pathogens, even at times when biosafety measures were largely non-existent. recent inferences of the likelihood of pandemics occurring as a consequence of laboratory incidents are misleading; all laboratory incidents were interpreted as accidents with potential onward human transmission, which is incorrect. historical evidence has shown that even when there were human transmission events after laboratory accidents (such as the cases of severe acute respiratory syndrome (sars) in beijing, china), human cases were limited. some people have argued that the russian influenza epidemic was the result of a laboratory accident, but in influenza research was done on the bench (under conditions of limited biocontainment), and attenuated and wild-type strains were tested in humans; we do not know what happened in , but we cannot conclude that the virus escaped a laboratory that met biosafety standards. finally, the influenza field voluntarily paused h n gof transmission research in - to facilitate deliberation and checking of the facts on safety and security. in , the us government concluded that the work could continue in specialized laboratories, but with additional governmental oversight. as the facts have not changed since then, i am hopeful that the same conclusion will be reached in response to the current moratorium. m.j.i. laboratory accidents happen, even in high containment settings. the recent events at the cdc in the united states, in which a strain of highly pathogenic avian influenza was accidentally shipped to another laboratory and in which a pathogen was taken out of a laboratory without proper inactivation, are just two examples. theoretically, the cdc has some of the best biosafety protocols in the world. one can only imagine what might happen if gof experiments are performed in laboratories with lower biosafety standards. however, the possibility that additional rules and regulations might end up slowing down the exact research that we require to protect ourselves from these pathogens is a real concern. i think that the wording of the announcement by the us government of a "pause on funding" for gof studies serves as an example. the office for science and technology policy published the following statement. "specifically, the funding pause will apply to gain-of-function research projects that may be reasonably anticipated to confer attributes to influenza, mers [middle east respiratory syndrome], or sars viruses such that the virus would have enhanced pathogenicity and/or transmissibility in mammals via the respiratory route ... the funding pause will not apply to the characterization or testing of naturally occurring influenza, mers, and sars viruses unless there is a reasonable expectation that these tests would increase transmissibility or pathogenicity. " what do "reasonably" and "reasonable" mean in this context? this is too subjective, and the open-ended timeframe of the pause also troubles me. m.l. the pause on funding for gof experiments for quantitative deliberation on risks and benefits is long overdue. the pause covers not all research on pathogenic organisms but only research that enhances pathogenicity or transmission of flu, sars or mers. ultimately, after deliberation some of that research may resume, and that may be appropriate if the weighing of risks and benefits has been done well. what we consider as 'safe' must depend on what the impact of an accident would be. more than twice a week in us laboratories, there is a 'possible release event' or a 'possible loss event' , even if we look only at select agents -some of the most dangerous pathogens . for every , lab-years of work in bsl- laboratories in the united states with select agents, there are at least accidental infections . this level of safety may be acceptable if the risk is to the laboratory workers only, as it is with most pathogens that are not readily transmissible. however, the same probability of an accident that could spark a global pandemic cannot be called acceptably safe. although most advocates of ppp experiments have been responsible in their discussion of the issue, a few have inappropriately caricatured those of us who express concern. we do not advocate curtailing research on dangerous pathogens in general, but we support replacing a very specific category of work that is small in extent (the funding pause affects about projects ) but that is exceptional in its level of risk with safer approaches. the current debate is not about whether durc in general should be allowed; it is about whether there is a tiny subset of durc with unusual risks that ought not to be allowed. for this tiny subset, there are additional main concerns besides biosecurity and biosafety -namely, concerns about the moral and ethical responsibilities of scientists to the general public , and concerns about justice. furthermore, research on pathogens is not always safe; on the contrary, it always carries risks. in fact, recent accidental releases of dangerous pathogens by some of the most-respected laboratories in the world have demonstrated to the public that the risks may be greater than previously assumed and are due to the inherent failings of humans. nevertheless, this reality should not prevent research on pathogens from taking place. gof experiments that seek to create new, highly transmissible, highly pathogenic infectious agents pose special risks because of the greater likelihood that these agents will escape from the laboratory through either accidental or deliberate means and will lead to much greater harm than their naturally occurring counterparts. from a biosafety perspective, i believe that some of the work performed so far with highly pathogenic influenza viruses that have enhanced transmissibility in mammals has not been conducted at a sufficiently high enough biosafety level. from a biosecurity perspective, the unfettered dissemination of the complete genome sequence of a new, highly transmissible, highly pathogenic agent enables anyone skilled in the art to produce the agent de novo if a reverse genetics system for that class of agent is available, which could occur at locations that lack even basic biocontainment measures. i strongly support a funding pause for a narrow subset of experiments that entail unusual risks. the pause focuses attention on risk, benefit, governance and responsibility, and begs for seriousness and breadth of discussion that have been lacking so far. woefully insufficient input has been obtained from a wide variety of scientists and from many other stakeholders among the general public. it is unethical to place so many members of the public at risk and then consult only scientists -or, even worse, just a small subset of scientists -and exclude others from the decision-making and oversight process. a parallel debate has focused on how and when to report the results of studies involving durc. in your opinion, how should journals deal with these concerns? should sensitive information be redacted for publication, should publication be halted until safety concerns are addressed, or should all experimental details be made available at the time of publication? who do you think should be responsible for making these decisions? w.p.d. the scientific continuum can be divided into four steps: conceive an idea, conduct the experiments, present the data and publish a manuscript; the number of people involved in this pipeline increases with each step. nevertheless, even conception usually involves more than one person and, in an academic setting, strict confidentiality is difficult to achieve, as colleagues tend not to request that non-disclosure agreements are signed before going to lunch and discussing an idea. at the other end of this pipeline are editors and publishers and, in response to the recent influenza transmission studies, journals and professional societies have established internal review processes to evaluate papers containing potential durc. journals could be considered as the ultimate 'gatekeepers' , but redaction is a blunt instrument that rarely, if ever, should be used to limit access to publically funded, non-classified research; all details should be published. similarly, i find it impossible to imagine how laboratory meetings, seminars, poster sessions and conference presentations could be regulated. conducting the research, a step that requires institutional facilities and external funding, seems to offer the best opportunity for some oversight. universities have developed policies and established durc committees that can work with scientists who have conceived studies that are flagged during institutional review. funders could request that applications with a durc component be presented for review to a standing committee of scientific experts. in both cases, the responsibility would be on institutions and funders to ensure expeditious review, and the scientists who conceived the study should be intimately involved. with public funds, exchange (international) personnel extensively, are expected to act with maximum transparency and do not operate in highly secretive environments. as a consequence, unless research is done in a 'classified' environment from the beginning, academic research is considered to be already in the public domain by the us legal courts, and redaction of manuscripts would thus be ineffective. therefore, the default decision should be to make all experimental details available at the time of publication. scientists, along with their host institutions, have a huge responsibility in this decisionmaking process. when in doubt, scientists should seek advice from their peers. funding bodies can decide not to fund particular work, but this does not necessarily prevent scientists from doing and publishing the research, or from seeking alternative funding mechanisms. publishers have a moral obligation to publish responsibly, but because they are at the end of the chain and because scientists can disseminate results via alternative channels, this is not where the primary response to durc issues should be. in my view, the best option is to leave the primary responsibility with scientists and their institutions, with oversight and advisory roles for governments. m.j.i. i think that the life sciences research community has to accept the fact that we live in a very different world today than even - years ago, a world in which individuals and groups will engage in unethical behaviours that we would not have imagined in the past. taken together with the fact that the technologies required to produce dangerous pathogens are relatively easy to acquire, i believe that we are going to be faced with more and more examples of data that could enable those wishing to do harm to do so. i believe that we must develop a system that will allow selective sharing of information that has a high likelihood of being misused. i am not trying to be a 'fear-monger' by asking us to think about potential misuse. rather, i am stating that we cannot ignore the possibility. however, this is a complicated issue because ascertaining the likelihood of misuse is incredibly difficult. the question of who gets to make these decisions is also a tough one to answer because of the complexity. arguably, authors themselves are in the best position to realize the potential risks of publishing certain details; this was the opinion of the us national science advisory board for biosecurity (nsabb) before the submission of the manuscripts describing gof experiments that resulted in increased transmission of h n influenza virus in mammals. however, there is great pressure to publish, any given individual may not think that the risks are more than negligible, and the risks and benefits of scientific research are not always immediately evident. at the journal level, similar concerns apply. are reviewers and editors in a knowledgeable enough position to be aware of and to analyse the risks and benefits associated with such publications? given the international nature of research, do we get governments involved and, if so, which ones? i think one approach to consider is to have a committee, similar to the recombinant dna advisory committee in the united states, that acts nimbly; as it accumulates experience, it can delimit what needs review and what does not, similar to the way the governance of recombinant dna research has evolved. m.l. i think the concern about publication has two issues: do we make it easier for well-meaning scientists with poor biosafety standards to do unsafe experiments, and do we make it easier for potential bioterrorists to create novel bioweapons? both are important concerns but, in my mind, for experiments that create novel, virulent, transmissible ppps, the accident concern alone is enough to outweigh any of the purported benefits that have been mentioned to date, especially if we appropriately compare the resources allocated to ppp experiments with the alternatives we could fund with the same resources. a proper discussion about risky research is far more effective if it is held before the initiation of such research, rather than after the results have been obtained. however, unanticipated discoveries happen, and some will produce information that creates risks and vulnerabilities. meanwhile, research takes place in an increasingly interconnected global society, in which all deserve to share in the benefits and all rightfully expect to be protected from undue risk. the moral and social obligations of all scientists include the duty to first do no harm. when research findings directly pose potential risks of such magnitude that they greatly outweigh the associated benefits of these findings, scientists and those that oversee their research and its dissemination are obligated to minimize these risks, which might include temporarily limiting the dissemination of data. the important general principle that supports the free and open sharing of scientific knowledge does not trump our obligation to prevent undue harm in those unusual circumstances where direct misapplication of information can be reasonably anticipated to cause grave and widespread consequences. of note, restrictions on communication of research findings need only to be temporary; they should and can be lifted as soon as risks have been mitigated, for example, by creation of countermeasures against the newly identified threat. we need standardized and widely accepted mechanisms for identifying these rare circumstances where research findings ought not to be freely disseminated, as well as legally validated mechanisms for limiting information dissemination . such mechanisms, aside from national security classification, are not currently available. the role for limited dissemination was discussed in the corson report issued in by the us national academy of science . in fact, there are plenty of circumstances today in which experimental results or details are not fully disclosed, such as situations involving intellectual property, commercial secrets and privacy. concerns about security and safety should be at least as compelling as these other concerns. such decisions are most appropriately made jointly by the relevant investigator (or investigators) and local institutions, with guidance by national and international experts within and outside governments. the current debate has made headlines in the press. in your opinion, has the debate been beneficial, as it has raised public awareness and has the potential to make scientists work towards a consensus, or has it been harmful, owing to its potential to alarm the general public, which could result in additional regulatory guidelines that many microbiologists fear could hinder future research? w.p.d. what concerns me greatly is that owing to the use of imprecise definitions, rhetorical language and a paucity of personal engagement between individuals who disagree, no meaningful debate has occurred about the merits and risks of the adaptation of pathogens towards enhanced transmission or any other durc phenotype. opinions have been reactionary, and arguments have been played out in the media, on blogs and in podcasts, augmented by twitter discussions and op-ed pieces, in a process that seldom involves peer review. selfishly, i believe my discipline deserves better, and the efforts of arturo casadevall and michael j. imperiale to raise the level of debate are to be applauded [ ] [ ] [ ] . however, on the whole, communication has been poor. this is exacerbated by the fact that the groups with differing opinions (on microbiology, public health and bioethics) largely inhabit very different worlds, meaning that individuals with opposing opinions rarely meet. additionally, the media feels the need to frame the debate as a fight, which is also counterproductive and harmful, and is doing little to help the public to understand the key issues. the least beneficial outcome is the current moratorium which, from a virologist's perspective, also seems reactionary. invoking the apocalypse should not be used to drive debate, set agendas, decide policy or regulate experiments with dangerous pathogens out of existence. there has hardly been a real debate. i have participated in several public meetings, but opposition against gof research has been minimal in most of these cases. instead of a real debate, we have seen the sharing of tweets and one-liners that are copied by press outlets in search of sensation. the lay press and some scientific journals have blindly placed opinion pieces without checking the facts or seeking alternative opinions. the problem here is that much of the press and the public are interested in sensational news but are less interested in careful explanations of the (boring) facts related to the regulatory frameworks that are in place, the safety and security procedures that are in use, the purpose of particular research projects, the weighing of risks and benefits of research, and so on. while ringing the alarm bell is fast and easy, communicating the fact that the bell may have sounded a false alarm will take considerable efforts. i am worried that new regulatory guidelines may not contribute to what they were designed for, which is to make the world a safer place. extensively recently about the complicated nature of the debate and the dangers of the manner in which it is being conducted [ ] [ ] [ ] . i think the debate has been both harmful and beneficial. it has been harmful in that, as in any debate that becomes public, people will draw conclusions without necessarily learning all the facts or understanding all the nuances of the issue. it has been beneficial in getting the disparate views out, but not as beneficial as it could be because the discussion has largely been in print, in social media or on the internet without people actually sitting down in a room and discussing the issues. it is common sense that before embarking on a course of research that has even a low risk of sparking a global pandemic, there should be very careful consideration given to the risks and benefits. both risks and benefits of performing gof experiments on influenza viruses apply to human beings in general, as we are all susceptible to flu infection. therefore, this must be a discussion that moves beyond flu researchers, some of whom have personal interests at stake, and beyond microbiologists, to the whole scientific and medical community and others who would be directly affected -the general public. the natural order of events is deliberation, risk and benefit analysis, evaluation of the results of that analysis, and then a decision to go forward or not with each type of research. unfortunately, the initial discussions of this topic fizzled a decade ago . research on ppp creation went forward and was reported publicly in , then published in . the public debate is long overdue and necessary. an admirable example of how such work might have proceeded is given by the scientific leaders of work on gene drives, another area of biological research with good intentions but that also poses danger to human and animal populations. the leaders of that field have publicly announced what they are doing, how they are mitigating risks, and how the public can get involved in discussing risks and benefits . when scientific research potentially endangers large numbers of lives, the public (in this case, the global public) should know and have input. the debate has been largely beneficial for raising awareness of and clarifying the issues. if the discussion has been flawed, it is because the pros and cons of the work have both been slightly exaggerated, the tone of the discussion too personalized and emotional, and the diversity of participants too narrow. in many cases, conversations have only involved infectious-disease researchers, and conflicts of interests among participants have not been adequately acknowledged or addressed. as discussed above, it is unethical to put the general public at risk, as one does with the creation of new ppps, and then minimize inclusion of the public in discussions about the appropriateness and oversight of such research. it is our responsibility as scientists to explain the rationale behind our work, including its benefits and risks, to the general public in terms that are accessible to those with an average level of education, rather than to be dismissive. this is especially important when the work has important consequences for the whole of society. flexible, agile and adaptive oversight mechanisms are critical because of the rapidly evolving nature of this field of science and technology. several scientists have argued that one of the positive aspects of the controversy has been the initiation of a debate on the pros and cons of durc. however, a consensus on how, when and where to allow this type of research, and how to handle the release of sensitive data, has not been reached. how do you think the debate should move forward? in your opinion, can the two sides come to an agreement? w.p.d. scientists have the responsibility to engage and inform, not to entertain or scare. this led to the foundation of scientists for science, a group of international scientists who are convinced that only by engaging in open, constructive dialogue can we learn from one another's experience, understand genuine concerns and move from dogmatism to consensus. transparency and good communication are important in articulating why working with potentially dangerous pathogens is critical for society, and we will continue to argue for safe and sound science. this debate goes far beyond the single issue of influenza transmission studies and has implications for all of microbiology; therefore, it must be inclusive. policy makers, national academies, international organizations and governments should recognize that although influenza virologists have been at the forefront of this debate, as the recent studies involved altering the transmission and host range of influenza viruses, many other microbiologists perform comparable in vitro and in vivo evolution and adaptation studies. colleagues working on sars and mers viruses are acutely aware of this following the us government's call for a "pause in funding" and instructions to stop certain ongoing experiments. a wide net should be cast when meetings and symposia are arranged by scientific societies and national academies. furthermore, a substantial amount of resources have been invested globally to build and operate bsl- and bsl- facilities and to mitigate risk and share good practice, so that risk is minimized; this is our most important line of defence. i am convinced that limiting certain types of experiments is not the answer. however, neither is completely resisting the use of appropriate quantifiable risk-benefit analyses. i am optimistic that dialogue will help and that building a consensus is not impossible. supportive of open debates about durc, biosafety, biosecurity and policies on scientific publishing. i participated in many of these and will continue to do so. however, we should be realistic in that we may not reach consensus on some of these topics. for instance, in durc discussions there will be debates about weighing risks and benefits of research. since neither the risks nor the benefits are truly quantifiable, the weighing will remain a judgment call. for example, in the debate about release of sensitive data, bioterror risks as perceived by intelligence experts are weighed against the scientific benefits as perceived by scientists; this is like comparing apples to oranges. furthermore, trying to address the question of whether we should do a particular kind of gof research may be aiming a bit too high. perhaps we should address some more tractable questions initially, such as how has biocontainment improved from the asilomar conference to the present day, with the introduction of purpose-built biosafety laboratories? how should we interpret laboratory incident reports in light of public health risks? what is the relative likelihood of dangerous human pathogens emerging in nature versus in the laboratory? what is the value of basic scientific research on dangerous pathogens? what is the risk of abuse of scientific research by 'lone wolves' , terrorist organizations and rogue states? and how effective and feasible is redaction, classification and export control of manuscripts produced within academic institutions? answers to these simpler questions may help to move the debate forward. m.j.i. since the gof issue came to the forefront in , i have been of the belief that there has not been the type of discussion there ought to have been -one in which the scientific, biosafety, biosecurity and ethical issues are all on the table. i am therefore one of those who welcome the recent movement to have these discussions. i think that not only can the two sides come to an agreement, but they must do so. these are important issues with large implications for human, animal and plant health. i think that individuals who hold extreme viewpoints on either side may not be able to compromise, but that a vast majority of stakeholders will be able to agree on the best way forward. m.l. there may never be complete agreement. quantifying risks and benefits has the effect of taking the discussion away from personal comments -such as "this person's laboratory is safe" or "this person's science is important" -and looking at it objectively. i have argued that the risks for creating novel flu strains are so large (low probability but very high potential consequence) that we cannot justify such work when there are safer alternatives , . the answer might turn out to be different for other viruses. furthermore, scientists are ingenious and may be able to find ways to do the science in which they are interested with less or no risk. the previous regime of not properly accounting for risk is clearly not sustainable, and i expect that a lot of new approaches to risk mitigation and alternative methods will come to light during this period of deliberation. a.r. i agree that there have been positive aspects of the controversy, in so far as attention has been drawn to important issues that deserve careful and deliberate discussion, and i do believe that agreements can be achieved on important aspects of this issue. moving forward, leadership of the discussion process should be shared by governments and by key stakeholders, including domestic and international science organizations (for example, national academies of science); highly respected, dispassionate and trusted representatives of the general-science communities; key non-scientist thought leaders; and representatives of the security communities. principles of deliberative democracy should be incorporated. goals of this process should include establishment of a credible, objective and balanced governance scheme for the life sciences; establishment of norms and an understanding of the relationship of the scientific community with governments and with the general public; articulation and acceptance of responsibilities by the scientific community towards the general public and the ecosphere; and development of a consensus on whether and how risky work should proceed. we will also need ongoing review and oversight of risks in the life sciences and associated technologies, as well as ongoing engagement of life scientists across all sectors on these issues. these are challenging goals for which the life sciences research community has so far failed to demonstrate broad commitment, but goals that are more than deserving of serious effort. experimental adaptation of an influenza h ha confers respiratory droplet transmission to a reassortant h ha/h n virus in ferrets airborne transmission of influenza a/h n virus between ferrets microrna-based strategy to mitigate the risk of gain-of-function influenza studies ethical alternatives to experiments with novel potential pandemic pathogens can limited scientific value of potential pandemic pathogen experiments justify the risks? cd in meningococcal disease monitoring select agent theft, loss and release reports in the united states moratorium on risky virology studies leaves work at institutions in limbo the increasingly compelling moral responsibilities of life scientists inconvenient truths" in the pursuit of scientific knowledge and public health the apocalypse as a rhetorical device in the influenza virus gain-of-function debate an epistemological perspective on the value of gain-of-function experiments involving pathogens with pandemic potential risks and benefits of gain-of-function experiments with pathogens of pandemic potential, such as influenza virus: a call for a science-based discussion tiptoeing around pandora's box regulating gene drives scientists for science: www.scientistsforscience.org key: cord- - fghudac authors: qoronfleh, m. walid title: health is a political choice: why conduct healthcare research? value, importance and outcomes to policy makers date: - - journal: life sci soc policy doi: . /s - - - sha: doc_id: cord_uid: fghudac this paper offers the eastern mediterranean region (emr) viewpoint with qatar as a case for lasting transformation of health systems. the qatar case study illustrates the importance of research in the development of health policy. it provides description of a series of projects that have been undertaken in relevant national areas such as autism, dementia, genomics, palliative care and patient safety. the paper discourse draws attention to investment requirement in health research systems to respond to country national health priorities and to strengthen public health policies for improving health and social outcomes by narrowing the gap between research and politics. in short, the discussion highlights the following: i) health is a human right marching towards universal health care, with research underpinning every advance in health care and quality medical services; ii) evidence-based research is emerging as a critical tool to aid policy- and decision-makers; iii) investment necessity in healthcare research/systems to enable responding to a country’s national health priorities and to strengthen public health policies; and iv) need for multi-sectoral involvement of stakeholders to bridge the gap between research and politics. finally, atypical stakeholders’ engagement and bond to politics is a prerequisite to achieve healthcare objectives and policy success so as to reap the benefits of public health results. historically between the th - th century, the eastern mediterranean region (emr, as defined by the who) had a remarkable influence on health care research and systems for a string of many firsts in diagnosis, treatment and patient management including many scientific discoveries (majeed ; pallejà de bustinza ) . today, in the emr non-communicable diseases account for % of disease burden having the highest traits of risk factors as well such as obesity, smoking and sedentary lifestyle, thusly, increasing mortality amongst the population. indeed, the world health organization (who) statistics speaks vividly of this challenge (fouad et al. ) . definitely, there are major health and development challenges facing the emr. amongst these chief challenges are resource constraints, regional conflicts, and inadequate health systems research that compound and exacerbate many issues confronting emr, all of which make for uncertain future for the region. it is about time to re-envisage health systems. it is critical to breakdown silos and boundaries, re-orientate health systems to address public health and the broader ecosystem influencing health. this treatise presents an emr perspective with qatar as an example for long-term transformation of health systems. the discourse highlights investment in health research systems (hrs), how hrs respond to country healthcare priorities and discussing the broader influence on emr. equally important, how essential and imperative is research/gained knowledge in the development of health policy. health research is very important and has high value to both individuals as well as society. overall, healthcare research can contribute to information about disease trends and risk factors, outcomes of treatment or public health interventions, functional abilities, patterns of care, and health care costs and use. different healthcare research types provide complementary insights. for instance, clinical trials can provide critical evidence about the efficacy and adverse effects of therapeutic treatment; also, crucial post-market surveillance data for comparing and improving the use of drugs, vaccines, medical devices, and diagnostics. therefore, clinical experience at a population level is imperative for identifying relatively rare adverse effects and for determining the effectiveness in different subpopulation groups (stratification) leading to precision medicine and individualized therapy. ultimately, this allows the actual development of clinical guidelines for best practices and to ensure high-quality patient care. clinical healthcare research have led to significant discoveries, the development of novel therapies, and a remarkable improvement in health care and public health. economists have found that medical research can have an enormous impact on human health, wellbeing and longevity, and that the resulting increased productivity of the population contributes greatly to the national economy (hatfield et al. ; murphy and topel ) besides the obvious individual benefits of improved health. if the research enterprise is impeded, or if it is less robust, vital societal interests are affected. clinical research and clinical trials registry are yet to be fully established in most of the emr countries or have formulated laws governing the conduct of clinical trials. moreover, system maturity vary between emr countries. the middle east north africa (mena) region is predicted to be one of the leading regions for clinical research outsourcing due to the availability of essential infrastructure for the conduct of clinical trials, access to required patients and financial benefits. public healthcare research with a particular focus on health services research is another approach to provide information and practical knowledge to improve health outcomes and return on investment. for example, the development of herceptin, a breast cancer treatment, is a prime illustration of the benefits of healthcare research using biological specimens and patients' medical records (slamon et al. ) . other examples of findings from healthcare research have changed the practice of medicine as well. a case in point, research underlying estimated patient's death from medical errors in hospitals, which has provided valuable proof for reducing these medical errors by implementing health information technology like e-prescribing (bates et al. ) . furthermore, medical records research has demonstrated that preventive screening services (e.g., mammography) substantially reduce mortality and morbidity at very reasonable cost (mandelblatt et al. ) . similar kinds of investigation has also established a correlation between nursing shortage and patient health outcomes by documenting that patients in hospitals with fewer registered nurses stay in hospital longer, thus, are more likely to suffer complications the likes of urinary tract infections, upper gastrointestinal bleeding and nosocomial infections (needleman et al. ) resulting in increased hospitalization cost. these findings have all informed and influenced policy decisions at the national level with effective change to medical practice, cost containment and improved outcomes. as the use of electronic medical records increases, the pace of this form of research is accelerating, and the opportunities to generate new knowledge about what works in health care are expanding (coalition for health services research (chsr) ). poor health information system has been identified as a major challenge in the emr healthcare system. even though, emr countries have embraced healthcare technology advances in the last decade along with tremendous investment in healthcare it products and services in order to drive higher adoption of electronic and digital healthcare systems. however, computer and english literacy remain an issue. the available evidence indicate that the region still lags behind and that there were many factors that hindered the widespread adoption such as cost, procurement and maintenance. health is a human right, with research underpinning every advance in health care. the goal is to attain universal healthcare coverage. undeniably, better research, i.e., evidence-based yields better health. evidence-based research is emerging as a critical tool to policy makers. increasingly, quality research that is backed up by caliber data/information and superior analytics is being utilized by policy and decision makers to formulate, enact and improve government policies thus influencing a country's health status and population health. a case in point, europe leads in many areas of research and has developed powerful models of cross-border, cross-sectoral research cooperation. the scientific panel for health (sph) is a science-led stakeholder platform, which elaborates scientific input concerning this societal challenge. it assists the eu commission in the preparation of legislative proposals and policy initiatives. this expert led group has proposed the creation of european council for health research to provide a comprehensive policy for health research in europe and facilitate crossborder collaboration (the lancet ). it is anticipated that these actions would lead to improved overall health outcomes and health economics, in other words better outcomes related to efficiency, effectiveness, value and behavior in the production and consumption of health and healthcare. since the inception of the first global symposium on health systems research and the era of sustainable development goals (sdg) the goal of 'leaving no one behind' is falling behind. the intertwined complexities and issues facing emr require an evolution from primary focus on health to a broader focus on sdgs' and multi-sectoral involvement. therefore, strengthening science, research, policies and societal engagement for the emr countries is critical to ensure dialogue continuity and experience healthcare improvements at all levels. continued investment in hrs not only is essential but also is an enabler to equip countries to deliver on promises and resolve increasingly complex and interconnected health and development challenges of the twenty-first century. devoting time and resources to integrated knowledge translation and engaging atypical stakeholders is a fundamental paradigm shift in the way we think about health systems. this demands being self-critical of the systems, to engaging with the social determinants of health to appealing to health systems influencers such as political and financial intuitions. emr mandatory achievement pillars are strengthening public health policy and improving health outcomes by narrowing the gap between research and politics. finally, wish, its research team and the qatari health research community at large are working to communicate and educate the public about why healthcare research is important and how healthcare research is done. equally important, healthcare researchers must convey the value of health care improvements derived from public health research, quality of medical records, availability of biological samples and to stress the negative impact of incomplete datasets/analysis on research findings vis-à-vis public health. in this vein, the qatar ministry of public health (moph) organized its first biennial qatar public health conference (qphc) from to november at the ritz carlton hotel | doha, qatar. the moph public health department has organized qphc to convene every years to discuss distinct public health themes with insights from national, regional and international experts. in preparation for the fifa world cup , which will be held in qatar, qatar health a first of its kind healthcare conference aimed at improving healthcare professionals' understanding of mass gathering events and informing policies. the conference -a collaborative effort between hamad medical corporation (hmc) and moph -took place from to january at the sheraton grand doha resort and convention hotel | doha, qatar. the covid- pandemic incidence is a stark reminder of the risk threat posed by communicable disease outbreaks including spread at major sporting events. the world innovation summit for health (wish) and the research team are committed to positively influencing public health research and policies in qatar. these areas include autism, mental health, dementia, patient safety, non-communicable diseases (cancer, diabetes & obesity), precision medicine and bioethics. wish endeavors to address qatar's national healthcare priorities. the environmental context is described below. additionally, it elaborates on the importance of multi-sectoral engagement to galvanize the community to work around silos and boundaries demonstrating appreciation to the influence and proper understanding of the qatari culture and business norms. similar to other countries in the region, qatar has witnessed a rapid change in many aspects of life over the past four decades. primarily because of rapid urbanization and socioeconomic development following the region's "oil boom" that took place between and , qatar's population today enjoys a very different standard and way of life compared to earlier generations. qatar has recently emerged as one of the wealthiest countries in the world when measured in terms of gross domestic product (gdp) per capita (the heritage foundation ). among other indicators, great progress is being observed when it comes to the country's health data, which reveals an increasing life expectancy and a significant drop in infant mortality. qatar has achieved overall better health outcomes over the past several decades due to making significant investments in the healthcare infrastructure. the state boasts the highest life expectancy rate in the world health organization's eastern mediterranean region (emro), and globally ranks in the top th percentile for healthcare access and quality. qatar's healthcare expenditure and investment is also among the highest in the middle east, with qr . billion (usd $ . billion) invested in . these achievements have led some organizations to rank qatar fifth ( th) in the world for healthcare (legatum institute ). certainly, qatar has the financial prowess with a budding health care system. the healthcare system, healthcare infrastructure and public health policies continue to be on a developing trajectory towards an ambitious personalized healthcare goal. in , the moph-qatar launched its second national health strategy (nhs, - aka nhs . ), which signified an important step in the state's health system development. the second national health strategy was developed taking into account the united nations (un) agenda for development, the who's recommendations and objectives, and the regional context. the nhs . has established three main objectives: better health, better care, and better value. priority areas include the development of an integrated model of high-quality care and service delivery for the state of qatar; enhanced health promotion and disease prevention; enhanced health protection; health integrated across the country in all policies; and establishing effective systems of health governance and leadership. the legendary management consultant and writer peter drucker is famously quoted "culture eats strategy for breakfast". these developments and achievements would not have been possible without convening key opinion and thought leaders, developing a global health viewpoint, and committing to encourage on evidence-based research. a multisectoral approach that is culturally sensitive is powerful and empowering. it is a surer route to success. the qatar case study series illustrates the significance of research in the development of health policy. research is at the core of everything wish does. wish is continuing its contribution to improve qatar's healthcare system. research undertaken by wish has made a significant influence to national policies in qatar. few examples are highlighted below that address qatar's national priorities and are in alignment with moph national health strategies. the specific projects case studies show the importance of application of knowledge coming from research. further, they elucidate and emphasize the link between health, society and policy to improve both health and social outcomes. the diverse stakeholders' participation ensures alignment, endorsement and drives action. this is exemplified in the underneath narrative. the social and economic burden of dementia is clear and enormous even today. the staggering increase in the prevalence of autism spectrum disorder ( have provided the foundation of evidence based knowledge to frame the national dementia strategy and the national autism strategy, which were launched in and , respectively. in addition, the policy report on dementia has provided a vehicle for the country's involvement in the who global dementia observatory that continues to this day. wish has also implemented the report findings through pioneering community outreach and awareness programs such as providing more social and recreational services to qatar's autism community . estimates show that in high-income countries as many as in patients is harmed while receiving hospital care. patient death occurring due to a preventable medical accident, while receiving health care, is estimated to be in . approximately two-thirds of all adverse events occur in low and middle-income countries (sources who august ). one of the key policy recommendations from the wish report on patient safety [transforming patient safety: a sector-wide approach, ] was focused around the need and urgency of including patient safety in the curriculum of medical/clinical majors. following that, wish collaborated with medstar health, usa and moph to bring the academy for emerging leaders in patient safety to qatar on an annual basis and to conduct a capacity building program, an inter-professional intensive training for both faculty members and students on reducing medical errors in the hospital setting. since its inception in , over participants from all health science colleges around qatar have graduated from the program. wish patient safety framework and qatari experience were also on display at two international conferences. at the th international patient safety conference - september , novotel hicc, hyderabad, india and at the th international conference on patient safety november - december, organized by riphah institute of healthcare improvement & safety and hosted by rawalpindi medical university (rmu), rawalpindi, pakistan. hence, stressing the worth and benefit of this work beyond the local public to influencing the global healthcare community especially for recognized countries with resource constraints. health systems around the world are facing rising health expenditures due to aging populations with complex conditions, new medical technologies, and challenges in providing universal access to care. consequently, policymakers are developing more person-centered care models, and "value-based" payment reforms to support and sustain them. many health systems are implementing accountable caredefined as a group of providers who are held jointly accountable for achieving a set of outcomes for a defined population over a period of time and for an agreed costto adopt the necessary organizational competencies and health policies needed to implement innovations in care that support better population health while avoiding unnecessary costs. in , wish led the efforts in establishing a multidisciplinary task force to implement an integrated care model between primary health care corporation (phcc) and hmc, which was published in its forum report [implementing accountable care to achieve better health at a lower cost, ]. the pilot phase of the project resulted in the smart clinic project, a national diabetes screening and prevention program. the pilot demonstrated how a health system could begin implementing accountable care by reorganizing care delivery before addressing payment reform. this model has now been expanded and adapted by phcc and hmc to tackle screening and prevention of other chronic diseases evolving into the "the better together program" besides being incorporated into the nhs . strategy. qatar's national health strategy, - , highlights the need for creating quality, care delivery infrastructureso-called "high-value health systems"to enable such improvements in patient access, affordability, and outcomes. the latest policy and outcome developments facilitated by wish were discussed in a roundtable on the th of november at qncc in doha with duke university center for health policy and phcc leadership. given the foreseeable scientific leap in genomics in the gulf region, the need for a solid knowledge base pairing scientific research with cutting-edge research in islamic ethics is more urgent than ever. managing the promising, pioneering genomics outcome coupled with the potential ethical challenges is a conundrum, one that requires acknowledging and understanding the religio-cultural fabric of this region and the wider muslim world to which it belongs (al-dewik and qoronfleh ). religion and socio-cultural ethical conduct not only influence research but also shapes political/policy perspectives. in , the qatar national research ethics committee published the country's very first "genomic policy" that aims at assisting investigators in the design and conduct of genomic research, oversees genomic research activity and addresses the different associated risks. this was based on the policy recommendations that stemmed from the report that was published in by wish entitled genomics in the gulf region and islamic ethics, . palliative care (pc) is a relatively new medical specialization that embodies a number of universally shared values. its principal aims are to relieve pain and other distressing symptoms, improve quality of life for individuals living with serious illness, and provide patients with good end-of-life care. pc physicians face various ethical dilemmas. experts predict a rise in the demand for pc. there are various reasons for this, such as the increase of geriatric populations and prevalence of chronic and life-limiting diseases, which affect a population regardless of age. to provide culturally sensitive pc, patients' (religious) beliefs and moral worlds must be integral parts of the care package. pc is an integral part of healthcare, and many countries in the arab/islamic world are increasingly offering this care. this is an area of particular sensitivity and interest within the region including the state of qatar, and represents a growing need for both clinicians and family members to have access to culturally acceptable guidance, which would alleviate suffering and facilitate compassion. through the wish policy report [palliative care and islamic ethics: exploring key issues and best practice, ], the proposed guidelines are under consideration, and should stimulate and galvanize efforts at the national as well as the regional levels. wish has recently launched an awareness campaign around palliative care practice both regionally and nationally. wish and the pontifical academy for life (vatican city) jointly organized a two-day symposium on religion and medical ethics - december . this special symposium examined the role that religion plays in providing holistic care in the context of medical ethics. by focusing on the intersection of belief-based and evidence-based approaches to care, speakers and participants had the opportunity to highlight and explore the benefits of interdisciplinary and interfaith approaches to treating the body, mind and soul. the two main topic areas of healthcare were palliative care and the mental health of the elderly. the aim was to harness intellectual input, deliberations, and to produce a special report with a framework and/or action plan with policy recommendation to facilitate national policy composition or guidelines to healthcare facilities that are integral to holistic care including workshops development and training sessions for healthcare workers. a vivid example of robust cross-cultural interaction and interfaith dialogue. effective and high-quality health systems rely on multidisciplinary teams. nurses and midwives play a central role in all health systems. the who has declared that is the "year of the nurse". in , wish published a report on nursing and midwifery [nursing and midwifery the key to the rapid and cost-effective expansion of high quality universal health coverage, ]. this report is about the contribution of both nurses and midwives to universal health coverage (uhc). additionally, in partnership with the moph, wish has been instrumental in the establishment of nursing now qatar. indeed, nursing now qatar was one of the very first to be launched in the mena region and has firmly put qatar at the vanguard of this global campaign. while still in its relative infancy, this program will address significant workforce issues and capacity building including promoting the profession and encouraging retention, both now and in the future, around nursing and midwifery in qatar. wish also welcomed representatives from countries across asia, africa, and the middle east and conducted a two-day symposium for nursing leaders on health workforce data on april at qncc, doha, qatar. the workshop was in consultation with hmc and who. afterwards, wish has partnered with the global nursing now campaign and the international council of nurses (icn) offering a nursing symposium to train young nursing leaders from around the world to amplify their voices and positively influence healthcare policy, and underscore their role in uhc ahead of attending the annual world health assembly in geneva, switzerland. health is a human right, with research underpinning every advance in health care. undoubtedly, better research that is evidence-based yields better health. the emr is facing considerable challenges. a game changer approach is the broad, multi-sectoral involvement of stakeholders to bridge the gap between research and politics. this leads to strengthening public health policy and improving health/social outcomes. evidencebased research is emerging as a critical tool to policy makers. this type of research should be more accessible to policymakers. for instance, the sixth global symposium on health systems research (hsr ) served as a catalytic platform for sharing knowledge and experiences, raising awareness and advocating for change, building capacity, and developing strategic partnerships to address the challenges facing health and development today. wish, its research team, and the qatari health research community at large are working to educate the public about why healthcare research is important and how healthcare research is done. equally important, healthcare researchers must convey the value of health care improvements derived from public health research, ensuring a high quality of medical record keepings, and through the availability of biological samples, and to stress the negative impact of incomplete datasets/analysis on research findings vis-à-vis public health, and use of evidence-based research in policy formulation as well as intervention. wish is addressing qatar's national priorities that are in alignment with moph national health strategies. in sum, lessons learned to implement policy recommendations or enact successfully policies include: i) recognize that health is a human right to achieve universal health care, with research underpinning every advance in health care, ii) execute evidence-based research, it is a critical tool to policy-and decision makers, iii) invest in health research systems to respond to country national health priorities, strengthen public health policies including their outcomes and deliver quality medical services, and iv) involve multi-sectoral stakeholders to bridge the gap between research and politics. finally, it is our firm belief that atypical stakeholders' engagement and bond to politics is fundamental to achieve healthcare objectives and policy success so as to reap the benefits of public health results. genomics and precision medicine: molecular diagnostics innovations shaping the future of healthcare in qatar. advances in public health effect of computerized physician order entry and a team intervention on prevention of serious medication errors framework for health services research policy for scaling up prevention and control of noncommunicable diseases in the who eastern mediterranean region exceptional returns: the economic value of america's investment in medical research the legatum prosperity index: qatar how islam changed medicine: arab physicians and scholars laid the basis for medical practice in europe the cost-effectiveness of screening mammography beyond age : a systematic review for the u.s. preventive services task force the economic value of medical research nurse-staffing levels and the quality of care in hospitals how early islamic science advanced medicine. national geographic history magazine response to the burden and impact of dementia through policy and innovation autism in the gulf states: a regional overview human breast cancer: correlation of relapse and survival with amplification of the her- /neu oncogene index of economic freedom: qatar the lancet the author wants to thank his institution for their continued support.author's contributions mwq conceived, designed and implemented conceptual work, framework, writing and critical editing. author read and approved the final manuscript. availability of data and materials all data generated or analyzed during this study are included in this published article. none. the author declares that there are no conflicts of / or competing interests.ethics approval and consent to participate none applicable. received: may accepted: june publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -phi re g authors: weissman, ruth striegel; klump, kelly l.; rose, jennifer title: conducting eating disorders research in the time of covid‐ : a survey of researchers in the field date: - - journal: int j eat disord doi: . /eat. sha: doc_id: cord_uid: phi re g the covid‐ pandemic has impacted research around the globe and required shuttering of research programs and the implementation of procedural adjustments to ensure safety. this study sought to document covid‐ 's impact on eating disorders (ed) research, which may be particularly susceptible to such disruptions, given its focus on individuals who are physically and emotionally vulnerable. we invited ed researchers from editorial boards and scientific organizations to complete a quantitative/qualitative survey about: covid‐ 's current and future impact on ed research; areas of concern about research disruptions; and effective strategies for conducting and supporting research during and after covid‐ . among participants, many had moved studies online and/or shutdown part of their research. across position types (permanent, . %; temporary, . %), participants reported high concern about data collection, recruitment, and securing future funding. those holding temporary positions reported significantly greater concern about covid‐ 's impact on their career and greater stress than participants in permanent positions. strategies for dealing with research disruptions included: employing technology; reprioritizing goals/tasks; and encouraging collaboration. results underscore the high levels of stress and disruption caused by covid‐ . we echo calls by our respondents for support for early career scholars and advocacy for additional resources for research and scientists. its focus on individuals who are physically and emotionally vulnerable. we invited ed researchers from editorial boards and scientific organizations to complete a quantitative/ qualitative survey about: covid- 's current and future impact on ed research; areas of concern about research disruptions; and effective strategies for conducting and supporting research during and after covid- . among participants, many had moved studies online and/or shutdown part of their research. across position types (permanent, . %; temporary, . %), participants reported high concern about data collection, recruitment, and securing future funding. those holding temporary positions reported significantly greater concern about covid- 's impact on their career and greater stress than participants in permanent positions. strategies for dealing with research disruptions included: employing technology; reprioritizing goals/tasks; and encouraging collaboration. results underscore the high levels of stress and disruption caused by covid- . we echo calls by our respondents for support for early career scholars and advocacy for additional resources for research and scientists. coronavirus, covid- , eating disorders, methodology, online research, research methods, stress, telehealth the outbreak in december of the novel coronavirus sars-cov- (covid- ) and the subsequent pandemic has prompted numerous governments worldwide to temporarily stop or restrict "nonessential activities." governments have issued stay-at-home orders for nonessential workers, and "stay healthy" orders (e.g., wear protective clothing or gear and maintain safe distances) for essential workers, in efforts to reduce the risk of covid- infection. these orders have impacted research activities from the outright shuttering of many research facilities or programs to the implementing of numerous procedural adjustments (e.g., switching to online data collection or virtual intervention sessions) to ensure compliance with safety requirements. some scientists conducting animal studies have had to euthanize some or all of their animal colonies (parry, , april ) . academic conferences have been cancelled well into , reducing the opportunity for information sharing, networking, and technical training activities commonly offered at scientific conventions. finally, experts are warning about dire consequences of the pandemic for the academic job market, with outlooks expected to be especially grim for early career scholars such as graduate students or postdoctoral fellows (keslky, , april ; wood, , april ) . while there is a rapidly growing literature on the clinical presentation and correlates of covid- infection-indeed, a pubmed search on april , using the search term "covid- " identified over , articles-we were unable to find studies of how researchers (regardless of field of interest) are responding to and coping with the disruption of their research-related work. we recognize that many eating disorders colleagues have multiple responsibilities beyond their research role, such as teaching, clinical, or administrative duties. our interest in the present study, however, was on the research impacts of covid- . as editors of the international journal of eating disorders (ijed), we (rsw, klk) were interested in hearing from eating disorders researchers, and the individuals who generate (as authors) or evaluate (as reviewers) the research that gets published in the ijed, in regards to four overarching questions: (a) the impact of covid- on eating disorders research now and in the foreseeable future; (b) the level of concern about covid- -related disruptions of research and the potential impact of these disruptions on individuals' careers; (c) strategies respondents thought to be effective in coping with covid- -related research disruptions; and (d) respondents' suggestions for how the ijed or the field should respond to help researchers move through and beyond the current crisis. to address these aims, we conducted an anonymous survey of quantitative and qualitative items among eating disorders researchers. quantitative questions focused on the perceived impact of covid- -related research disruptions. we hypothesized that respondents in secure employment positions (e.g., tenure or permanent contracts) would report less stress and less concern about potential adverse research or career impacts, than would respondents in time-limited (e.g., tenure-track or non-permanent contracts) appointments. the primary goal of the qualitative, open-ended questions was to describe participants' strategies or suggestions, and we did not pose or test any hypotheses based on the qualitative data. | methods four groups of researchers were selected for inclusion in the survey. the first group included all editorial board members of ijed (n = ) and the european eating disorders review (eedr, n = ), as well as the "senior" board members of the journal of eating disorders (jed, n = ). the second group included all current members of the eating disorders research society (edrs, n = ), a global organization of eating disorder research experts. the third group included members of a special interest group (sig) (n = ) for early career scholars that is organized by the academy for eating disorders, the world's largest professional organization in the field. finally, the fourth group included ad-hoc reviewers (non-editorial board members) who had provided three or more reviews of an ijed manuscript in the past months and who (n = ). membership in these four groups was not mutually exclusive (e.g., edrs members serve on at least one of the three journal boards; and ad-hoc reviewers are also either a member of the edrs or of the sig, resulting in an estimated unique individuals who received at least one invitation. initial survey invitations to members of journal boards and to ad hoc reviewers were sent by the first author (rsw). sig members were invited by the co-leaders of the sig via email, followed by a reminder tweet with a link to the survey. edrs members received one survey invitation from the edrs office with no follow-up. ijed board members were reminded of the survey in the context of two follow-up messages containing informational material related to ijed board service. finally, all invitation emails encouraged recipients to forward the survey link to other eating disorders researchers. given the diverse recruitment methods and follow-ups, and the unknown number of individuals who received the survey link from colleagues, we did not attempt to calculate a response rate. of the individuals who answered "yes" to the consent question of the survey, did not provide any responses. an additional respondents did not complete the question about type of position (i.e., tenure/permanent contract position, nontenure/nonpermanent contract position), resulting in a final sample of participants (see sample demographics in results below). due to missing data on some questions, sample sizes vary for responses to specific questions. the invitation email asked for participation "…in a brief ijed survey about covid- related disruptions to eating disorders research. we hope you will share strategies you are employing to cope with these impacts; your concerns about the current pandemic's impact on your future research program or career; and your thoughts or suggestions on how ijed and the eating disorders research community at large could support research during and after covid- ." the invitation further specified that: the survey was anonymous; only one response was possible from an ip address (i.e., no multiple submissions permitted); and participation was voluntary, with all items being "skippable," yet completion of all items was encouraged. the survey opened with a consent statement asking respondents (no, yes) whether they agreed to complete the survey. the survey opened on april , and closed on april , . the survey was approved by wesleyan university's institutional review board. this mixed-method survey (a copy of the survey is available via an online supplement) included likert-scale items measuring concern about covid- disruptions (rated from = no concern to = extreme concern) or highest level of stress experienced since the outbreak of the pandemic ( = no stress to = highest level of stress imaginable). one item asked respondents to characterize the proportion of their eating disorder research that had to be completely shutdown due to covid- , using six options (from to up to % in % increments). the survey included three categorical items (yes, no, do not know/does not apply) that asked participants whether they had moved their studies online, whether they were anticipating making changes to their research practices, and whether their institution had made policy changes, in response to covid- . these responses to the question about changes ijed should implement are not reported here because they were the subject of a recently published ijed editorial . to describe the sample, in a final set of questions, participants were asked to report their gender (check one: male, female, transgender male, transgender female, gender variant/non-conforming, don't wish to answer), type of position (check one: position is not in tenuretrack or is non-permanent, position could lead to tenure or a permanent contract, but i have not yet reached this status, or position is tenured or permanent), institutional location of their research appointment (check one: examples such as psychology, nursing, etc., and "other"), and the country where they hold their primary research appointment (a drop-down menu of countries in the world). descriptive statistics were calculated for quantitative items. chi-square tests and analyses of variance (anovas) were used to test our hypothesis that individuals holding permanent positions would differ from those holding temporary positions. to adjust for multiple comparisons, bonferroni correction was applied to the anovas (p = . / = . ). in addition, number needed to take (nnt) (kraemer, neri, & spiegel, ) and cohen's d were used to estimate effect sizes for categorical and continuous variables, respectively. responses to open-ended questions were grouped into thematic cate- far more women (n = , . %) than men (n = , . %) participated in the survey (n = [ . %] respondents selected "don't wish to report"). more respondents held permanent positions (n = , . %) than held either a position that possibly could lead to a permanent appointment (n = , %), a non-permanent position (n = , . %), or an "other" position (n = , . %). the latter three position categories were combined for subsequent analyses ("non-permanent": n = , . %). of the respondents providing information about the type of institution where their research appointment was held, a majority reported positions in psychology (n = , . %) or psychiatry (n = , . %) departments; the next largest subgroup reported appointments in medicine (not psychiatry, n = , . %). the rest of the sample selected various other university departments (e.g., public health), research institutes, non-academic hospitals, or governmental appointments (a complete listing of departments is shown in table s ). about % ( / ) of individuals did not answer the question of where in the world they held their primary research appointment. of the respondents who provided information, participants ( . % of total sample, . % of sample responding to this item) selected "us" and participants ( . % of total sample, . % of those responding to this item) selected another country (australia, n = ; canada, n = ; germany, n = ; italy, n = ; the united kingdom, n = ; countries with < participants included argentina, austria, belgium, china, france, luxemburg, mexico, new zealand, norway, portugal, sweden, and the netherlands). as shown in table , men were more likely than women to report holding a permanent position (chi-square [df = ] = . , p < . , nnt = ). specifically, more men held a permanent appointment (n = , . % of male sample) rather than a temporary position (n = , . % of male sample); in contrast, among women, slightly fewer women (n = , . % of female sample) reported holding a permanent versus a temporary (n = , . % of female sample) position. because position type and gender were significantly correlated, we conducted unplanned post-hoc analyses comparing male and female respondents. due to small cell sizes, we did not test for an interaction term of sex by position type. about half of respondents ( . - . %; see table ) indicated that they had transitioned at least part of their research to an online setting. on average, respondents reported that about - % of projects (i.e., score = or ) had been shutdown due to covid- (see table ). interestingly, only % ( / ) of respondents reported that none of their current eating disorders research (i.e., score = ) needed to be shutdown. as shown in table , on average, the highest levels of researchrelated concern were related to data collection, recruitment, and securing future funding, intermediate levels of concern were found regarding staffing and budgets, and the lowest scores were found for concerns about procurement of research supplies or obtaining institutional approvals. of note, statistically significant differences (with a large effect size, d = . ) were found comparing respondents holding permanent positions versus those in time-limited positions on ratings of how concerned they were about the impact of covid- -related research challenges on their future career in terms of promotion and/or career advancement. those in temporary appointments reported far greater concern than those in permanent positions. unplanned post hoc analyses found statistically significant gender differences in two variables, each with medium effect sizes: concerns about future career impacts (p < . , d = . ) and concern about staffing (p < . , d = . ). specifically, women reported higher levels of concern about future career impacts, while men reported higher levels of concern about staffing than women. overall, respondents reported high levels of stress, and those holding non-tenured/temporary positions reported significantly higher stress levels than respondents with permanent positions (p < . , with a medium effect size of d = . ). only a few respondents ( %, / ) anticipated making no changes to their future research practices; more than half of the respondents ( %, / ) indicated that it was "too soon to tell," while about one-third ( %, / ; . % of those in permanent positions versus . % of those in temporary positions, n.s.) of respondents expected making changes to future research practices because of covid- . most respondents ( . %- . %; see table ) reported their institution had not yet made any changes to performance evaluations. chi-square test results by position type the n and percentages for gender do not total to or %, respectively, because three individuals ( . %) responded "don't wish to report". b respondents answering "no" or "too soon to tell" were combined into "no" for this item. t a b l e anova test results by position type . note: aside from the item asking "amount of research shutdown" (see below), all other items were rated on a -point scale from (no concern/no stress) to (extreme concern/extreme stress). abbreviations: m, mean; sd, standard deviation. . note: aside from the item asking about "amount of research shutdown" (see below), all other items were rated on a -point scale from (no concern/no stress) to (extreme concern/extreme stress). abbreviations: m, mean; sd, standard deviation. a the "amount of research shutdown" item was coded on a -point scale as follows: = % of research shutdown; = up to % of research shutdown; = up to % of research shutdown; = up to % of research shutdown; = up to % of research shut down; = up to % of research shutdown. number of comments recorded and themes and illustrative (shortened, paraphrased) statements identified by open-ended questions. please share your - most effective strategies for dealing with covid- in terms of your eating disorders research. ( comments) attending online conferences or webinar programs; creating a platform for virtual convening to share information or provide support; using crowd-sourcing for participant recruitment; switching to remote design implementation. . goal adjustments for one's work overall or for specific projects ( / , . %) prioritizing tasks that can be done remotely; adjusting study goals; writing more review papers; focusing on analysis of data already collected by the respondent or of data created by others; brainstorming ideas for future projects. distractions due to childcare responsibilities; potential differential impact of family responsibilities as a career detriment for women; missing out on learning opportunities via informal encounters that typically happen in the workplace; disruptions threatening students' opportunities for graduate school stipends; disruptions diminishing the educational opportunities for students currently on internship; worries about a bad job market for graduates. . | qualitative data of respondents' perspectives on covid- 's impact on their research, career and the field two themes were identified that captured most of the comments for "describe - changes you expect to make in your research practices." by far, the most common changes involved moving their research to (or maintaining it on) online platforms or preparing their work accordingly (e.g., exploring online interventions) ( % of responses). the second theme was modifying ongoing studies or changing future research directions (e.g., focusing on qualitative work) ( %). this survey sought to describe covid- -related impacts on eating disorders research as reported by researchers in the field, using quantitative and qualitative measures. respondents to our survey were predominantly female, likely reflecting the gender distribution among members of the groups targeted by our invitation. for example, anecdotally, we estimate that a large majority of sig members are women, about % of edrs members are female, and % of the members of the largest of the three journal editorial boards (ijed) are female. we found four major findings. one, for most respondents, the pandemic has disrupted at least part of their eating disorders research, yet approximately half of our respondents reported that they had been able to transition some or all of their studies to online settings. two, the research activities that were rated as most challenging or concerning because of covid- included recruitment, data collection, and securing future funding, and our sample reported high level of stress experienced since onset of the pandemic. three, with two notable exceptions, reported concerns did not vary significantly between participants in permanent (presumably secure) jobs versus those in non-permanent positions. however, higher stress and greater concerns about impacts of covid on respondents' future career were reported among people holding non-permanent positions as compared with researchers in permanent positions. moreover, women reported greater concerns about their future career than men; men reported greater concern about staffing than women. in addition, four, we received an impressively large number of comments in response to open-ended questions. across several questions, employing technology was named as an effective strategy. that covid- disrupted eating disorders research is, of course, not an unexpected finding; however, the extent of studies that needed to be halted is noteworthy. an estimated - % of respondents' eating disorders projects reportedly had to be stopped. our survey did not ask why projects had to be stopped. as a handful of respondents noted in open-ended responses, research methods requiring in-person assessments and/or highly specialized equipment or technical expertise (e.g., for neuroimaging) cannot be transitioned to remote implementation. in terms of other possible reasons, we speculate that it is also possible that some researches may have suspended studies out of concern that their results would be confounded to the point of being uninterpretable due to covid- -related impacts on research participants (e.g., increased levels of disordered eating, anxiety, or depression). another possibility is that some colleagues may not have had the staff resources to quickly convert their research to remote administration. although an impressive number of respondents ( . - . %-see table ) reported taskfocused strategies for dealing with covid- -related disruptions that focused on online settings, for certain projects, going online may have been difficult if they involved participants with inadequate online access or devices, such as people living in rural or disadvantaged areas, and/or families who must provide computer access for parents working online and their children completing their coursework online. because of these differences and disparities, the disruptive impacts of covid- likely will be felt unevenly across research areas in our field. about half of respondents in both tenured/permanent groups and non-tenure/temporary positions reported transitioning their lab-based studies to online settings. the longer-term impact on recruitment or on completeness and quality of data from studies that needed to be converted "mid-stream" is yet unknown. the high levels of concern reported about data collection and participant recruitment likely reflect both the realization of severe constraints for those engaged in methods that require in-person implementation, and the challenges in maintaining high rigor when research participants are left on their own for completing assessments. funding agencies have issued policies in support of design modifications (european commission, ) and scientific journals have begun to describe best practices for design modifications (mcdermott & newman, , march ) . scientific productivity may be adversely impacted by delays in recruitment and data collection and by diminution of methodological rigor (e.g., due to attrition or needing to replace objective measures with subjective reports). the high levels of concern expressed about securing future funding may reflect, in part, respondents' worries about how a decrease in scientific productivity or rigor may impact the merit scores of their grant applications. additionally, as reflected in some comments, concern was expressed that for a time, funders will prioritize covid- -related research over other topics, depleting already scarce funds and further increasing competition for grant funding. overall, our sample reported having experienced a high degree of stress since the outbreak of the coronavirus. as by fernández-aranda and colleagues (fernandez-aranda et al., ) and by touyz and colleagues (touyz, lacey, & hay, ) , pandemics give rise to high levels of stress both in the general population and in various high-risk groups (brooks, dunn, amlot, greenberg, & rubin, ; torales, o'higgins, castaldelli-maia, & ventriglio, ) . we did not measure at-risk criteria such as whether respondents themselves had become infected with or were directly exposed to individuals infected with coronavirus. anecdotally, we know that some researchers in the field oversee clinical service departments (e.g., inpatient units), work in hospital settings, or continue to treat patients. our survey's crosssectional design and lack of a comparison group comprised of nonresearchers preclude attributing the reported stress-levels to strains caused by research disruptions. still, considering the response range of our stress-item from to , with being "the highest level of stress imaginable," our mean of . and median of indicates that many respondents experienced a high degree of stress. as hypothesized, however, the burden of stress was not equal across all participants. participants holding temporary positions (including researchers on time-limited contracts with or without an opportunity to convert to lifetime contracts; and post-docs or graduate students) reported markedly higher levels of stress than respondents holding more permanent appointments (e.g., tenure). individuals with temporary appointments also reported disproportionately high levels of concern about their career future. women were more likely to hold temporary appointments than men; unfortunately, the sample size of men in temporary positions was very small (n = ), precluding detailed exploration of the intersection of gender and position type. we note that in our unplanned analyses, overall, women expressed significantly higher career concerns than men. our short survey did not collect data about factors that would help fully explain these findings. for example, we did not ask respondents with lifetime appointments whether they had already reached their highest rank and may therefore be less concerned about promotion. qualitative data about researchers facing disadvantages noted several challenges that would be expected to contribute to high levels of stress and career concerns, including limited financial resources, time limits for establishing scholarly productivity, and childcare responsibilities. resoundingly, harnessing technology was noted as key to continuing research during the pandemic and succeeding in research in a post-covid- world. many respondents indicated that they expected that online methods (e.g., telehealth implementation of interventions, online data collection) represent the future modus operandi for their research. as has been noted for other sectors (e.g., higher education), remote work, and working online likely will characterize a greater part of work because it can be a convenient and resource-saving means of performing one's duties. participants, by necessity, currently are getting used to participating in online settings and some may, in fact, prefer to continue this form of engagement to in-person activities in some areas of research (linardon, shatte, tepper, & fuller-tyszkiewicz, including in areas that do not readily lend themselves to online implementation (and may require some form of "blended" implementation). although the long-term impact of the expected massive shift of research from in-person to on-line implementation will remain unknown for months/years to come, being purposeful and empirical in our application and evaluation of on-line research is likely to yield the most benefit to our science. beyond reporting task-focused, on-line strategies for dealing with covid- -related disruptions, several other respondents suggested strategies that were other-focused, such as reassigning tasks (e.g., enlisting teachers for data collection), employing effective communication strategies (e.g., maintaining connection with research participants), or pursuing collaborations (e.g., jointly recruiting participants across research groups). for example, suggestions included that major professional organizations could be re-organized into "federations" to achieve greater synergies and maintain connections with others in their field. another suggestion was to co-convene the field's major conferences to maximize efficiency in regards to travel time and venue costs. although these strategies are likely to be most needed during crises like covid- , these are organizational shifts that will likely enhance research practices and strengthen research networks in our field beyond covid- . finally, a remarkable number of qualitative comments were selffocused, such as adjusting one's goals or reprioritizing tasks (e.g., focusing on analyzing data or grant writing), practicing acceptance (e.g., "this too shall pass", acknowledging the inevitability of setbacks but committing to moving forward with creative solutions), or pursuing opportunities for "brushing up on the literature" and skill building. some of this self-help career advice is echoed in a recent editorial on how to stay "productive while teleworking" (brown, montenegro, yeo, & brody, ; schiffman, , april ) . these coping efforts may mitigate some of the adverse impacts of current research disruptions (e.g., maintaining a positive outlook and carrying on with tasks that can still be done) and may increase the likelihood future success (e.g., learning new skills or grant writing). we echo a caution, however, expressed in some comments; while nothing is gained by dwelling on what cannot be done, there is an undeniable reality that for some colleagues, covid- has thwarted opportunities that cannot be easily replaced, or created hurdles that cannot be overcome, no matter the actors' good intentions or positive self-talk. both as individuals and as a community of scholars, we need to set realistic performance expectations for our colleagues and for ourselves. in this regard, we note that some institutions reportedly have already modified performance evaluation policies, most typically, by extending the time based upon which productivity is measured (e.g., extending the tenure clock by year for all faculty). nonetheless, it is striking that over two-thirds of respondents noted that their institution had not yet made any changes to institutional performance evaluations. this is worrisome, particularly given that the impacts of covid- may not be distributed evenly, and that those with fewer financial resources, with obligations such as caring for children, or with less established careers, may find it more difficult to implement their research projects as planned before the pandemic struck. before ending, we should note limitations of our informal study. our brief survey was limited by an uncertain response rate, inclusion of a sample of convenience, and a lack of detail regarding important demographic (e.g., race/ethnicity; family status; specific appointment rank) and contextual factors (e.g., whether participants' research required complex equipment). our sample included far fewer men than women, and men were especially underrepresented among those in temporary positions, limiting our ability to test for subgroup differences and generalize findings. as noted by the respondents (see table ), we also did not include questions that could increase understanding of the ways in which other responsibilities (e.g., childcare, home schooling, moving teaching on-line) impacted our respondents' ability to conduct their research during covid- , or the ways in which researcher disadvantage (e.g., lower socioeconomic status) may compound stress and resource inequalities. these omissions result in missing information about the experiences of individuals who may be most at-risk for experiencing covid- research disruptions. we are aware that others have adopted and revised our survey, with permission, for use with their own research groups (e.g., the psychiatric genomics consortium; bulik, personal communication, may , ) and have included additional questions tapping some of these important topics (e.g., difficulties working from home, including difficulties arising from child care responsibilities). we welcome inquiries from others to use/revise the survey and hope that our colleagues will conduct more rigorous and extensive surveys to document the impact of covid- on our fellow eating disorder researchers and the field at large. scientist mothers face extra challenges in the face of covid- training and post-disaster interventions for the psychological impacts on disaster-exposed employees: a systematic review rules for scientific progress while living with the covid- pandemic: from 'benchside' to 'fireside covid- : research executive agency business continuity measures covid- and implications for eating disorders what are men doing while women perform extra unpaid labor? leisure and specialization at the transition to parenthood the professor is in: stranded on the academic job market this year? wrangling with p-values versus effect sizes to improve medical decision-making: a tutorial a survey study of attitudes toward, and preferences for, e-therapy interventions for eating disorder psychopathology preserving clinical trial integrity during the corona virus pandemic in colleges euthanized lab animals to protect employees from covid- . now they face an onslaught of criticism: the chronicle of higher education digital technology can revolutionize mental health services delivery: the covid- crisis as a catalyst for change the outbreak of covid- coronavirus and its impact on global mental health eating disorders in the time of covid- cognitive-behavioral therapy in the time of coronavirus: clinician tips for working with eating disorders via telehealth when face-to-face meetings are not possible ijed support for eating disorders research in the time of covid- for would-be academics, now is the time to get serious about plan b conducting eating disorders research in the time of covid- : a survey of researchers in the field the authors gratefully acknowledge the assistance of annie haynos and kathryn coniglio in sending the survey to the special interest group for early career investigators of the academy for eating disorders. we thank all who completed the anonymous survey. this manuscript was not peer reviewed. data will be shared upon reasonable request. https://orcid.org/ - - - key: cord- -hnvf e authors: bandarian, fatemeh; namazi, nazli; amini, mohammad reza; pajouhi, mohammad; mehrdad, neda; larijani, bagher title: endocrinology and metabolism research institute from inception to maturity: an overview of -year activity date: - - journal: j diabetes metab disord doi: . /s - - - sha: doc_id: cord_uid: hnvf e endocrinology and metabolism research institute (emri) was founded in . emri progressed step by step from inception and reached to its maturation during the past years. emri has expanded and progressed in different aspects including human resources and infrastructures (laboratories and new technologies) and has obtained the first rank in the country in endocrinology research. it has also collaborated with regional and international organizations such as world health organization (who), international osteoporosis foundation (iof), and american association of clinical endocrinologists (aace). this article provides an overview of emri activities during a quarter of a century. endocrinology and metabolism research institute (emri) was founded for the first time years ago in in a small room in endocrine ward at third floor of dr. shariati hospital as one of the five research centers founded in the country at that time and entitled as "endocrinology and metabolism research center" (emrc). the research center was founded by four top endocrinologists university staffs in the country. eight to nine years earlier than establishment of emri ( ) ( ) , endocrine ward of dr. shariati hospital had been launched jointly with gastrointestinal ward with to beds and in the next years they increased to to beds (in ) . therefore, a primary background had been provided for research. following an increase in the numbers of bed for patients with diabetes, patients with thyroid and hypothalamic disorders were admitted in the ward as well. after a few years, thyroid cancer registry was launched in endocrine department and this department became a referral center for cushing disease. currently, endocrine ward w is working jointly with gastrointestinal ward and admits patients with different endocrine disorders. the purpose of this article is to review the activities of the endocrinology and metabolism research institute over a quarter of a century. after establishment, emrc started research with small epidemiological retrospective studies using hospital records with only one research fellow. most hospital records were incomplete at that time and there was no complete archive in the hospital. at the beginning of emri work, it faced with several limitations including space, budget and staff. when emri started, only five university staffs subspecialist in endocrinology were in emri and in the next years by graduation of more endocrinologists and joining to emri, the number of staffs increased. the first emri budget was allocated in by the university (governmental). the emri budget before that was provided by the institutes out of university. two years after establishment ( ), the center received official approval from the ministry of health and medical education. at the same year the center moved to a new place with - rooms out of endocrine ward at the th floor of dr. shariati hospital near the hormone and biochemical laboratory. in this new place, the center expanded for the first time and the number of research fellows and staffs increased to six to seven. seven years later (oct ), by providing a proper space at the same flat, a large new building was built and prepared and emri moved to the new building. this building contained a long corridor with multiple rooms that different research units were deployed there and required manpower was employed gradually. to expand research activities with limited budget and for more productivity, emri recruited young motivated newly graduated physicians covered by the human resources project (the ministry of health and medical education) with minimum payment that was supported by the ministry. by this policy, emri in order to advance the goals, used the potential of motivated young graduated human resources appropriately and expanded research activities. research products of emri in the first decade of its activity were limited [ ] [ ] [ ] [ ] [ ] [ ] [ ] and most of them published in local journals with persian language (fig. ) . in this decade, most activities were focused on building of infrastructures. activity of emrc in different subjects during those years was categorized in separate small research teams. the last two years of first decade ( and ) was associated with significant achievements that had decisive role in the future of emri. these important decisive achievements were as follows: evaluation of research centers launched by the ministry of health and medical education was one of the administrative events. at the first year of assessment, emri ranked th in razi festival. by further progress, emri obtained first rank in razi festival in the next year. afterward up to now (except one year), emri has obtained fist rank in the country consistently. allocation of an independent budget line to emri for the first time by the ministry was another important event in the same years that was the main achievement of emri. to facilitate multicenter studies and expand national research in the country, emri established "national diabetes research network" (ndrn) [ ] (http://emri.tums.ac.ir/ dmnet) and "iranian osteoporosis research network" [ ] (http://emri.tums.ac.ir/osteonet) that were endorsed by the ministry of health in the same years ( ). by cooperation fig. number of emri publications during the past -years of ndrn, local diabetes guideline was prepared and released for the country [ ] . currently, more than universities all over the country are member of these networks. at the same time, in the field of research expansion and internationalization, emri was approved as a collaborating center of world health organization (who) for diabetes and osteoporosis. collaboration with international osteoporosis foundation (iof) was also started. first emri journal entitled "diabetes and metabolic disorders" was published for the first time in in persian language in the first years and then changed to english in the next years and published by springer. notably, the journal has obtained many important achievements among local journals (fig. ). simultaneously ( - ), diabetes clinic affiliated to emri was launched in dr. shariati hospital to provide medical services for diabetic patients and to provide a research platform for emri as well [ ] [ ] [ ] [ ] . the second decade was the beginning of the emri jump and was associated with significant advances. at the beginning of second decade of activity, emri infrastructure, facilities, and human resources were acceptable and the related process was ongoing. by availability of such potentials in this space, research development was begun and accelerated. during the second decade of emri activity, improvement continued. following moving to new building and expansion of emri infrastructures and staffs, organizational chart of emri was developed and emri activities were categorized into education, research, administrative, and support sections. administrative and support section emri in promoted to research institute and research activities were organized in three main areas in three research centers including "diabetes research center", "osteoporosis research center" and "endocrinology and metabolism research center". other related research topics out of the aim and scope of the above mentioned research centers were considered as research groups and units within these research centers such as immunogenetic group [ ] , fasting group [ ] , nutrition unit [ , ] and so on. training ph.d by research students was started by emri for the first time in the country in and since then annually few talented interested eligible students are selected and admitted by emri for this program. these students have conducted practical research projects that lead to valuable outputs [ ] [ ] [ ] [ ] [ ] [ ] . in launching population sciences institute was a background for conducting population-based studies. by this background, emri collaborated in bushehr elderly health cohort for the first time in [ , ] and after that started and contributed in other population-based cohorts including imos [ ] , caspian study [ , ] , steps [ , ] and so on. during the last years of second decade diabetes clinic expanded to a sub-specialty diabetes clinic with multidisciplinary approach and moved to a new independent building. the main activity of emri was in research section as its intrinsic duty. during this period, an important policy in the research section was providing facility for theses of under graduate and post graduate students in the field of medicine that resulted in considerable improvement in research productions [ , [ ] [ ] [ ] [ ] [ ] . another policy of emri in the platform of diabetes clinic was collaboration with medicinal industries and taking research grants to support remarkable projects [ , , ] . at the beginning of second decade, publishing unit was launched in emri to provide professional counseling and facility, and to handle and support research product publication. this strategic policy caused a significant growth in research output of emri [ , , ] . during this period, staffs in basic sciences were employed by emri and following that policy, genetic, cellular, and molecular lab was setup and various molecular techniques including pcr, rt-pcr, epigenetic and methylation tests, etc., were launched. they provided a background for conducting invaluable research projects including genetics, molecular, and cellular studies [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . by these available facilities in emri, taking post doc students was started that helped to expand collaborations with other research institutes [ ] . after , using available facilities, emri moved to high tech research in the edge of the frontiers of knowledge. in , cell therapy project was conducted on patients with diabetes for the first time in iran [ ] . in this study, fetal liver-derived hematopoietic stem cell allotransplantation was performed in a small number of patients with type or diabetes. in the mentioned study, after one year of transplantation, none of the patients became insulin free, transiently, or permanently [ ] . safety of fetal cell transplantation was evaluated three years later that showed no significant complications and confirmed long term safety [ ] . pancreas transplant project was started for the first time in emri in and first pancreas transplantation was performed [ , ] . educational activities of emri in this decade were in the area of professional and public education. professional medical education was provided by holding local, national, and international workshops, courses, seminars, and congress in the field of endocrinology with special programs in anniversary dates such as world osteoporosis day (on october each year) [ ] and world diabetes day (on november each year) [ ] . moreover, diabetes guideline [ ] , diabetic foot care guideline (approved by international working group on the diabetic foot/iwgdf) [ ] for physicians, and other care providers were prepared and published. patient education was provided for patients with diabetes by local workshops and face to face education as well as general conferences, especially in world diabetes day [ ] . in addition, many different booklets and brochures as well as guidelines for diabetic patients were published that are updated every few years. in the third decade of emri activity, improvement of infrastructure continued in different areas. following expanding emri activities, six years later than second promotion ( ), three new research centers including personalized medicine research center, metabolomics and genomics research center, cell therapy and regenerative medicine research center, and one another research institute (translational endocrinology) were added to emri with the aim of moving toward high tech molecular and cellular advanced research in the field of endocrinology and multidisciplinary studies and conducting research on the edge of the frontiers of knowledge [ ] [ ] [ ] . these new research centers contributed in knowledge production in the field of metabolomics, personalized medicine, and cell therapy, significantly [ , , ] and these progresses still continue in various aspects. emri to extend metabolomics research in recent years has provided infra-structures such as ms/ms for metabolom analysis and has launched zebrafish lab as animal model for various diseases and for basic science studies as well. research output and the numbers of citation increased continuously during a quarter of a century of emri activity and reached maximum in (fig. ) . launching and establishing registry programs for diabetes, thyroid cancer, and pituitary adenoma are examples of emri outstanding achievements in the past years that provide a big data for the future analysis of disease progress status and outcome as well. in the third decade, the collaboration of emr with international organizations such as who, international osteoporosis foundation (iof), and american association of clinical endocrinologists (aace) continued and recently it was approved as who collaboration center for non-communicable diseases (ncds). collaboration with international working group on the diabetic foot (iwgdf) was also approved in this decade. in order to extend diabetes education in national and regional level, iranian diabetes academy was launched in emri in this decade. face to face and electronic diabetes education programs were provided for health care providers and patients [ ] . different educational modules for different care of diabetes and its complications as well as for diabetic foot care were prepared and released for professionals and patients that is advantageous in these days of covid- pandemics. in this decade, in line of who program to control ncds mortality in the world up to , iran committed to follow who program. in collaboration with the ministry of health, emri prepared "national document for non-communicable diseases (ncds)" [ ] and "iranian national service framework for diabetes" and clarified the national targets to reach who goals in . in conclusion, research and development is ongoing in emri from inception up to present. this path continues with the advancement of research, science, and knowledge and a bright horizon is ahead of emri. data availability not applicable. declarations not applicable. ethics approval not applicable. number of publications and citation during the past -years by emri j diabetes metab disord baradar-jalili r. treatment of hyperfunctioning thyroid nodules by percutaneous ethanol injection comparison of fine-needle-nonaspiration with fine-needleaspiration technique in the cytologic studies of thyroid nodules diagnostic value of frozen section examination in thyroid nodule-surgery at the shariati hospital clinicopathological features of thyroid cancer as observed in five referral hospitals in iran-a review of cases metabolic and endocrinologic complications in beta-thalassemia major: a multicenter study in tehran cost analysis of different screening strategies for gestational diabetes mellitus adjunctive estrogen treatment in women with chronic schizophrenia: a double-blind iranian national diabetes research network project: background, mission, and outcomes iranian osteoporosis research network: background, mission and its role in osteoporosis management developing a clinical diabetes guideline in diabetes research network in iran psyllium decreased serum glucose and glycosylated hemoglobin significantly in diabetic outpatients potential risk factors for diabetic neuropathy: a case control study effect of vitamin d on insulin resistance and anthropometric parameters in type diabetes; a randomized double-blind clinical trial serum uric acid levels and risk of metabolic syndrome in healthy adults vegf gene polymorphism association with diabetic neuropathy the effect of nutritional education program based on health belief model (hbm) on the knowledge of fasting type diabetic patients effects of black seed (nigella sativa) on metabolic parameters in diabetes mellitus: a systematic review the effects of supplementation with conjugated linoleic acid on anthropometric indices and body composition in overweight and obese subjects: a systematic review and meta-analysis global dna methylation as a possible biomarker for diabetic retinopathy epigenetic alterations and exposure to air pollutants: protocol for a birth cohort study to evaluate the association between adverse birth outcomes and global dna methylation aberrant dna methylation patterns in diabetic nephropathy gmp-grade human fetal liver-derived mesenchymal stem cells for clinical transplantation meta-analysis of promoter methylation in eight tumor-suppressor genes and its association with the risk of thyroid cancer liquid biopsy as a minimally invasive source of thyroid cancer genetic and epigenetic alterations bushehr elderly health (beh) programme, phase i (cardiovascular system) bushehr elderly health (beh) programme: study protocol and design of musculoskeletal system and cognitive function (stage ii) prevalence of hypertension in an iranian population association between body mass index and perceived weight status with self-rated health and life satisfaction in iranian children and adolescents: the caspian-iii study association of fruit and vegetable intake with meal skipping in children and adolescents: the caspian-v study insulin pen use and diabetes treatment goals: a study from iran steps survey is salt intake reduction a universal intervention for both normotensive and hypertensive people: a case from iran steps survey burden of diabetes and it's complications in iran in year coronary heart disease and associated risk factors in qazvin: a population-based study insulinoma in iran: a -year review primary thyroid malignancies in tehran comparison of different screening tests for detecting diabetic foot neuropathy the efficacy of silybum marianum (l.) gaertn. (silymarin) in the treatment of type ii diabetes: a randomized, double-blind, placebo-controlled, clinical trial the clinical investigation of citrullus colocynthis (l.) schrad fruit in treatment of type ii diabetic patients: a randomized, double blind, placebo-controlled clinical trial insulin production by human stem cells curcumin inhibits in vitro mcp- release from mouse pancreatic islets gender-specific differences in the association of adiponectin gene polymorphisms with body mass index adenosine deaminase gene polymorphism is associated with obesity in iranian population severe acanthosis nigricans in a year-old female with partial lipodystrophic syndrome associations between hla-c alleles and papillary thyroid carcinoma expression level of circulating cell free mir- gene in serum of patients with diabetic nephropathy expression level of circulating mir- in serum of patients with diabetic nephropathy clinical microbiology study of diabetic foot ulcer in iran; pathogens and antibacterial susceptibility the effect of fetal liver-derived cell suspension allotransplantation on patients with diabetes: first year of follow-up evaluation of fetal cell transplantation safety in treatment of diabetes: a three-year followup establishing a cgmp pancreatic islet processing facility: the first experience in iran in vitro modulation of tcf l gene expression in human pancreatic cells world osteoporosis day: celebrating two decades of progress in preventing osteoporotic fractures in iran world diabetes day: celebrating two decades of progress in combating diabetes and its complications in iran a multidisciplinary team approach in iranian diabetic foot research group personalized treatment options for thyroid cancer: current perspectives the pathway from gene therapy to genome editing: a nightmare or dream precision medicine in non communicable diseases co-transplantation of human fetal mesenchymal and hematopoietic stem cells in type diabetic mice model amino acid profiling in the gestational diabetes mellitus conceptual map of diabetes education: necessity of establishing iran diabetes academy national action plan for noncommunicable diseases prevention and control in iran; a response to emerging epidemic publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -ph vrba authors: de’, rahul; pandey, neena; pal, abhipsa title: impact of digital surge during covid- pandemic: a viewpoint on research and practice date: - - journal: int j inf manage doi: . /j.ijinfomgt. . sha: doc_id: cord_uid: ph vrba the covid- pandemic has led to an inevitable surge in the use of digital technologies due to the social distancing norms and nationwide lockdowns. people and organizations all over the world have had to adjust to new ways of work and life. we explore possible scenarios of the digital surge and the research issues that arise. an increase in digitalization is leading firms and educational institutions to shift to work-from-home (wfh). blockchain technology will become important and will entail research on design and regulations. gig workers and the gig economy is likely to increase in scale, raising questions of work allocation, collaboration, motivation, and aspects of work overload and presenteeism. workplace monitoring and technostress issues will become prominent with an increase in digital presence. online fraud is likely to grow, along with research on managing security. the regulation of the internet, a key resource, will be crucial post-pandemic. research may address the consequences and causes of the digital divide. further, the issues of net neutrality and zero-rating plans will merit scrutiny. a key research issue will also be the impact and consequences of internet shutdowns, frequently resorted to by countries. digital money, too, assumes importance in crisis situations and research will address their adoption, consequences, and mode. aspects of surveillance and privacy gain importance with increase digital usage. by late may , at the time of writing of this article, over countries and territories in the world were affected by the coronavirus pandemic. this included most urban clusters and even rural regions. with the spread of the pandemic, almost all regions have implemented lockdowns, shutting down activities that require human gathering and interactions -including colleges, schools, malls, temples, offices, airports, and railway stations. the lockdown has resulted in most people taking to the internet and internet-based services to communicate, interact, and continue with their job responsibilities from home. internet services have seen rises in usage from % to %, compared to pre-lockdown levels. video-conferencing services like zoom have seen a ten times increase in usage, and content delivery services like akamai have seen a % increase of content usage (branscombe, ) . cities like bangalore have seen a % increase in internet traffic. the lockdowns across countries have entailed a rise in the use of information systems and networks, with massive changes in usage patterns and usage behaviour. employees are adjusting to new "normals" -with meetings going completely online, office work shifting to the home, with new emerging patterns of work. these changes have come across all organizations, whether in business, society, or government. the changes have also come suddenly, with barely any time for organizations and people to plan for, prepare and implement new setups and arrangements; they have had to adjust, try, experiment, and find ways that did not exist before. though now, in late may , the pandemic is receding and stabilized in certain countries, it is still on the increase in many others, and with serious threats. experts in most countries are wary of the possibility of the disease spread re-emerging, and that lockdown norms may be relaxed carefully and slowly with social distancing at the core of the new normal. it is in this context that we see the use of information systems to continue in the same vein for some time in the foreseeable future as during the lockdown. we examine the possible scenarios in this surge in information technology usage during and post the pandemic. our estimation of these effects assumes that there was a digital transformation already underway, before the pandemic set in, and it will take certain forms owing to the impact of the lockdowns. in the next section, we examine the impact of the covid- pandemic on the use of digital technologies where we discuss some possible scenarios and research issues of the post-pandemic world. the next section summarizes the implications for research and practice, and in the last section, we present our conclusions. in this section, we discuss some of the most pressing issues regarding the post-pandemic digital surge. these themes reveal the multiple directions in which is research can focus in relation to impacts on technology. as the use of video-and audio-conferencing tools increases significantly, organizations will ramp up their technology infrastructure to account for the surge. this will lead to increased investment in bandwidth expansion, network equipment, and software that leverages cloud services. with employees becoming j o u r n a l p r e -p r o o f acclimatized with the idea of work-from-home (wfh), meeting and transacting online, firms will shift to wfh as a norm rather than as an exception. this is being adopted by many firms (akala, ; bbc news, ; khetarpal, ) , which have the digital infrastructure in place to handle the required load and bandwidth. education is another domain in which there a dramatic shift to the online mode of transacting. since the beginning of the lockdown, schools, colleges, and universities around the world have shifted their classes to video conferencing platforms like zoom and google meet. along with these synchronous modes of teaching, asynchronous platforms like edx and coursera have also seen an increase in enrolments (shah, ) . some institutions are now shifting entirely to the online mode for the forthcoming academic year, with the exception of sessions that require a physical presence, such as the university of cambridge in the uk and the california state system in the us (new york times, ). digital transformation technologies such as cloud, internet-of-things (iot), blockchain (bc), artificial intelligence (ai), and machine learning (ml), constitute a bulk of the of what is being adopted by organizations as part of their transformation effort. blockchain (bc) technology presents an opportunity to create secure and trusted information control mechanisms (upadhyay, ). as education and healthcare services witnesses a shift to the digital domain, bcs enable a way to secure and authenticate certificates, health records, medical records, and prescriptions. research on the design of such systems, along with maintaining their ease-of-use and usefulness will gain importance. another issue is that of designing systems that work with smart contractshow the contracts are authenticated, how these contracts will be designed in a complex chain of processes with many agents involved, and how arbitration related to contracts will be handled. further, is research may point to regulatory aspects of bcs with regard to what must be encrypted and shared (such as for authenticating news and information sources), and how security will be managed. for instance, governments demand access to private keys to view blocks for surveillance and monitoring, versus the requirements of privacy and protection from persecution. the gig economy is driven by online platforms that hire workers on an ad hoc, short-contract, and mostly informal basis. well-known examples of these include uber and airbnb globally and ola and swiggy in india. these platforms have grown immensely since the wide availability of smartphones from onwards. during the lockdown, workers employed by these platforms have suffered heavily, as the demand for their services, taxi rides, rentals, or skill work, has disappeared (bhattacharya, ) . further, since these workers had no guaranteed salaries, their incomes dropped dramatically. in the post-pandemic scenario, there is likely to be, in the short term, a slow return of gig economy workers, as manufacturing and service firms return to their old activities. however, we anticipate that in the longer term as the threat of infection and spread recedes, the gig economy will thrive. this will also be driven by the wfh culture. work-from-home and gig work has received attention in is research, through topics in telecommuting, digital nomads, and virtual teams. one key issue is that of work allocation and collaboration, across and inside teams, and across projects. this issue will face a rise in scale and importance in the post-pandemic world, as the numbers of wfh and gig workers increase. research may focus on aspects of the design of work norms, work contracts, trust-building, and team-building, amongst others. research on telecommuting and virtual teams (belanger, collins, & cheney, ; morrison-smith & ruiz, ) has a long history in is literature. issues include the nature of "distance" whether temporal, spatial, or cultural, and the psychological needs of workers, the technological support and design for this kind of work, and many others. this research is important for the post-pandemic period. we anticipate that the "dark side" of virtual teams and dispersed work also assumes importance in the postpandemic world. substantive issues related to technostress -particularly work overload and presenteeism arise in these situations. research will have to address issues of design of collaborative work, evaluation, team performance and motivation, stress, and the issue of continuous learning. another aspect of digital use by large sections of the working population is that of constant workplace monitoring and being on-the-job continuously. those working from home using video conferencing technology find themselves under intense scrutiny and all interactions are "hyper-focused" (kalia, ) . digital technology makes it easier for bosses and managers to call and locate subordinates at any time, knowing that they can be reached at all times. though there is anecdotal early evidence that this has led to an increase in productivity, it has also led to increased technostress (ayyagari, grover, & purvis, ; tarafdar, tu, ragu-nathan, & ragu-nathan, ) where employees must learn new technologies, be available for work at almost all times, stay with digital devices all the time, and cope with multi-tasking. post-pandemic, it is likely that workers' organizations will demand no-digital hours, where they will find refuge from the constant work pressure. research may address the concerns of work equity, balance, and managing stress. along with the surge in the use of digital technologies, we are now witnessing a rise in online fraud, scams, intrusions, and security breaches. the pandemic has created a scenario of insecurity that is inviting fraudsters to exploit the crisis situation by extracting money or information or by creating vulnerabilities (agarwal, sengupta, kulshrestha, anand, & guha, ) many users are beginning to rely on digital resources extensively, some for the first time, and are becoming targets for fraud and scams. organizations and governments are aware of this threat and are taking countermeasuresfor instance, some governments took a strong stand against zoom sessions for education, forcing the platform provider to upgrade security (yu, ) . it is likely that these scams and frauds will increase in intensity after the pandemic. organizations will implement massive security arrangements, along with extensive information campaigns by government departments. security innovations and firms that offer security services will rise. research will likely focus on managing security, assess the causes of breaches, and the economic and social loss from them. information technology, and particularly the internet, will remain central to the post-pandemic scenario, where innovations will drive the surge in use. a key aspect of this surge will be the management and regulation of the internet itself. though the internet is a global resource and no one country can control its protocols and features, its local access and availability remain an in-country issue. during the pandemic to some countries have restricted access to the internet (chhibber, ) , for certain reasons. the regulation of the internet will become crucial after the pandemic as it will remain a policy tool for governments. they can intercede on aspects of monitoring, bandwidth control, surveillance, intermediary liability, and e-commerce. the pandemic has brought the world to a situation where those not connected to the internet are facing total exclusion. with strict social and physical distancing measures in place, new routines require accessing the internet for most services. hence, those on the wrong side of the digital divide are completely left out. reasons for the divide are many: unaffordable device access, unaffordable internet access, content relevance, access skills or government ordered internet shutdowns (armbrecht, ; scheerder, van deursen, & van dijk, ) in developing countries, the condition is more serious. thus, it becomes extremely important to explore the possibilities of ensuring connectivity. although these issues have been researched and discussed earlier (warschauer, ) , covid- has brought about a situation where internet access seems to have become necessary for survival. as a few studies have suggested, access or no-access to icts may reinforce societal inequalities (ragnedda, ) , where the post-pandemic situation may enhance this further. with the substantial use of technology in accessing basic requirements like health and education, it is imperative to understand the impact of the digital divide on social equality. therefore, it calls for researchers to examine the impact of connectivity to draw policymakers' interest and, perhaps, offer ways to enhance it towards better inclusion. the heavy use of the internet during the pandemic, for various purposes, has raised people's data requirements. with a significant digital divide in societies, this surge in the internet data requirement has revived the discussion on zero-rating plans. zero-rating plans enable firms to let users access data from their sites and services, without having to bear data charges. usually, this is not strictly permitted as it violates the basic principles of net neutrality, where internet traffic has to have the same priority and cost. india, for instance, had an exemplary record of regulating zero-rating plans. although the government did not permit the implementation of such plans, in the aftermath of the pandemic, the telecom regulatory authority of india (trai) decided to allow waiving charges for data and voice for certain websites (coai, ) (coai, ). the list primarily consisted of the sites related to covid- -such as the world health organisation and india's ministry of health and family welfare. the list also included some private players. the principal aim was to allow people, across all socio-economic levels, access covid- related information. given that zero-rating plans can be useful in exceptional circumstances, as is evident from the example of india, research on the conditions on various parameters where allowing zr plans may increase social welfare has enormous practical implications, both for firms as well as regulators. the existing literature on net-neutrality regulations and zero-rating plans (belli, ; cho, qiu, & bandyopadhyay, ) forms the basis to enhance literature in this aspect. issues to be studied include: expanding telecom infrastructure, providing subsidized internet devices, free extra data, or waiving off users' subscription fees (shashidhar, ) in current times, when the productivity of people depends significantly on the internet, its shutdown can be extremely detrimental to societies(isoc, ) however, internet shutdowns are not uncommon even in times like these. the internet was shutdown in kashmir, a union territory in india, since august th, j o u r n a l p r e -p r o o f and continued till may , making it the longest ever imposed in a democracy (masih, irfan, & slater, ) basic internet services, such as filing for driving licenses, were accessed by locals using the internet express, which is a train that shuttles kashmiris to the nearest town where they can get online. the kashmir chamber of commerce estimates $ . billion in losses owing to the internet shutdown (masih et al., ) . similar events are regularly noted across various other countries, arab spring being the significant starting point. with the pandemic, when the internet has become the most important tool available to citizens the impact of internet shutdowns has become grimmer. shutdowns lead to severe implications for all aspects of life, and there are many issues that require research in this regard. the impacts resulting from a climate of uncertainty can potentially discourage foreign investors and spillover on a wide range of sectors, including education, healthcare, press & news media, and e-commerce (kathuria, kedia, verma, bagchi, & sekhani, ) . it is important to understand the far-reaching human rights impact of internet shutdowns, which are exacerbated in the current scenario. shutdowns have deep political reasons and in many cases the consequences are indeterminate. research can focus on aspects of domino-effect consequences leading to grave political crises. digital payments and digital currencies to have a key role in the post-pandemic situation. as digital payments are contact-less they will be encouraged by governments, will likely see a surge. this will also be boosted by the gig economy and wfh situations. there are two distinct phenomena related to digital money that has aided the fight during the pandemic. first, banknotes and coins were suspected to be carrying the virus and digital payment was preferred to the 'dirty money' (gardner, ; samantha, ) . online delivery services were encouraging customers to make payments through digital payment systems like a credit/debit card or mobile payments, with mandates by the government in several parts of india (bhandari, ) . this is likely to result in a surge in digital payment usage, which will lead to work on the diffusion of digital payment technology. second, during the lockdown, there was a loss of jobs, and governments provided aid through payment apps and digital payment modes. these are a convenient mode of fund transfer from donors to recipients, as seen in previous crisis relief cases as well (pollach, treiblmaier, & floh, ) . in various crisis and disaster events, where the mobility of civilians was restrained, many mobile payment service providers (e.g. vodafone in afghanistan, safaricom in kenya, and orange in africa) provided quick funds transfer of remittances from migrants to their homes, and relief aid from the government to victims (aker, boumnijel, mcclelland, & tierney, ; pega, liu, walter, & lhachimi, ; wachanga, ) . this is once again observed in the covid- crisis and needs further examination. issues of surveillance and privacy are gaining prominence with digital usage during lockdowns. commentators, such as yuval harari, have written about the potential for state surveillance "under the skin" (harari, ) as governments rely on digital means to monitor the spread of the pandemic. as many governments have started using apps on smartphones to monitor infected persons and trace their contacts, civil society organizations have raised privacy and state surveillance concerns (pant & lal, ) . postpandemic, these measures of monitoring populations for epidemiological reasons with digital means are likely to continue and become prevalent. though the concerns of privacy and surveillance are valid and have to be addressed, these digital platforms are the most reliable and efficient way of tracking disease spread. "surveillance is a distinctive product of the modern world" (misa, brey, & feenberg, , p. ) , and today we are living in a surveillance society where any internet-based activity using a mobile phone or other electronic gadgets can be monitored and accessed in unfathomable ways (gilliom & monahan, ; lyon, ) . this has resulted in a surge in is research on implications of such web or app-based surveillance in applications including mobile health apps (lupton, ) , environment monitoring and pollution control apps (castell et al., ) , self-tracking apps (barassi, ) , and parental surveillance (ghosh, badillo-urquiola, guha, laviola jr, & wisniewski, ) . covid- has introduced a new application of surveillance for tracking citizens with the symptoms of the virus. this includes the covid- tracker in china (davidson, ) , the aarogya setu app for tracking infectious citizens in india (shahane, ) , and contact tracking apps in the united states (guynn, ) . while these technologies are innovations for fighting the global pandemic today, the issue of government surveillance on citizens has evolved repeatedly. research can focus on the multiple benefits of these apps, but also should not ignore the potential social complications that are possible to arise, including the historic problem of bureaucratic control by the government, using it (gandy, ) . closely related to surveillance is the issue of privacy that mobile apps, including covid- trackers, often tend to threaten users' personal information (gu, xu, xu, zhang, & ling, ; joy, ) . for example, online classes during the pandemic lockdowns have suffered issues of 'intrusion of privacy' as students and teachers are on camera in the private spaces of their homes (garcia, ) . privacy in the digital age has remained a research topic of high priority for is researchers (belanger et al., ; smith, dinev, & xu, ) . privacy has also been considered by is adoption and usage researchers, with privacy risk as a dominantly recurring factor in studies on mobile payments (e.g., johnson, kiser, washington, & torres, ; luo, li, zhang, & shim, ) , location-based mobile services (zhou, ) , and social networking sites (aghasian, garg, gao, yu, & montgomery, ; youn & hall, ) . it would be interesting to examine the different privacy concerns of users while adapting both covid- tracking apps, and online classroom applications. the risks involved in the breach of privacy by these two technologies are unalike and must be investigated with adequate contextual references. in this section, we revisit some of the key issues that are important for research and practice. our discussion is based on the assumptions about the post-pandemic situation and the aspects of is research presented above. . while deploying security technologies like the blockchain, it will be important to understand the implications of smart contracts, their integration in workflows, and their effectiveness in complex resource-constrained settings, as in developing countries. further, understanding the implications of secure and non-erasable technologies like blockchains will become relevant for regulation. . many research issues arise with regard to work-from-home and gig work, which include aspects of trust, measurement of performance, communication effectiveness, and collaboration. j o u r n a l p r e -p r o o f . it can be expected that the dark side of virtual work and gig work, will raise questions of stress, presenteeism, work overload, surveillance, and monitoring. new and severe forms of digital surveillance will have to be understood and their implications gauged. . though much work has been done in understanding the parameters and impact of the digital divide, it will be important to understand how those without access suffer more from the consequences of the pandemic when the world survives on digital communications and operations. . management of the internet within countries is important, and aspects of enhancing networks include regulating zero-rating plans cautiously, seeing their implications for welfare, and how they can enhance access. . internet shutdowns during and after a pandemic lead to severe difficulties for citizens, who have come to depend on these services. research has to examine the direct, second-order, and thirdorder impacts of these shutdown measures. . research on digital payments and their impact in crisis situations, for providing aid and subsidies to affected populations, and for disaster management. . surveillance issues about the extent of data collection by contact tracing apps are important areas of research. issues of persistence and elimination of data, the expanse of data collection, sharing of data between apps, and the multiple trade-offs involved. . design of secure technologies, like blockchain-based applications, for the surge in online education and healthcare activities. . policy for regulating digital infrastructure needed for increased digital transformation. . design of technologies for managing secure online interactionsfor education, healthcare, payments. . design of apps for contract tracing and disease surveillance that balance privacy versus public health. . managers will have to understand resistance to technology and ways to manage change, both among employees as well as customers. . given the significant role which the internet is about to play in times to come, internet intermediaries will work with government and civil society to address privacy and surveillance issues for better adoption of technology. we understand that a pandemic can have severe consequences (keys, ) , including changing the political contour of the world, destroying empires, and creating nations. for the covid- pandemic, we envisage a dramatic shift in digital usage with impacts on all aspects of work and life. how this change plays out remains largely dependent on our responses to and shaping of the emerging trends. in this paper, we have outlined what we see as some key trends and research issues that need to be examined urgently. they will have substantial consequences in the future. internet users to touch million by scoring users' privacy disclosure across multiple online social networks more big employers are talking about permanent work-from-home positions payment mechanisms and antipoverty programs: evidence from a mobile money cash transfer experiment in niger. economic development and cultural change reasons billion people are still offline society for information management and the management information systems … babyveillance? expecting parents, online surveillance and the cultural specificity of pregnancy apps twitter allows staff to work from home "forever technology requirements and work group communication for telecommuters net neutrality, zero rating and the minitelisation of the internet ahmedabad says no to cash on delivery to stop spread of covid- coronavirus lockdown has exposed the serious flaws of 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and online privacy among teens: risk perception, privacy concerns, and protection behaviors singapore allows schools to resume zoom use for home-based learning examining location-based services usage from the perspectives of unified theory of acceptance and use of technology and privacy risk key: cord- -ghwhgkdm authors: ekundayo, temitope cyrus; okoh, anthony i. title: a global bibliometric analysis of plesiomonas-related research ( – ) date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: ghwhgkdm plesiomonas shigelloides is an emerging pathogen with damaging effects on human health such as gastroenteritis and extraintestinal infections. here, we carried out a bibliometric survey that aimed to examine publication trends in plesiomonas-related research by time and place, international collaborative works, identify gaps and suggest directions for future research. the search term “plesiomonas shigelloides” was used to retrieve articles published between and from the web of science database. only primary research articles were included in the analysis. a total of articles were published within the survey period, with an average of . ± . articles per year and an annual growth rate of − . %. research output peaked in and (each accounting for . % of the total). the united states ranked first in terms of numbers of articles (n = , . %) and total citations (n = ). cameroon, canada, cuba, switzerland and turkey co-shared the (th) position each with articles ( . %). research collaboration was low (collaboration index = . ). in addition to plesiomonas shigelloides (n = , . %), the top authors keywords and research focus included lipopolysaccharide and nuclear magnetic resonance (n = , . %). diarrhea (n = , . %), aeromonas species (n = , . %) and infections (n = , . %) were also highly represented in keywords-plus. authors’ collaborations and coupling networks formed two mega-clusters which nodes were shared solely by authors from high-income countries. the common conceptual framework in retrieved articles determined by k-means clustering revealed three clusters with sizes of , , and , representing research responses focused on extraintestinal and gastroenteritis, p. shigelloides lipopolysaccharide structure, and co-infections, respectively. our bibliometric analysis revealed a global diminishing research in plesiomonas; greater research outcomes from high-income countries compared to others and low collaboration with developing countries. plesiomonas shigelloides is a bacterium that has been labeled as an emerging pathogen for over three decades. there are many outstanding questions regarding its pathogenic potential, a a a a a despite evidence for its detrimental effects on human health such as gastroenteritis and extraintestinal diseases [ ] [ ] [ ] [ ] [ ] [ ] [ ] . also, some food and waterborne outbreaks have been traced to p. shigelloides [ ] [ ] [ ] [ ] [ ] [ ] , and the incidence of plesiomonas infections linked to immunocompromised health [ ] is increasing, especially in light of present-day lifestyles [ ] . climate change and global warming are also predicted to contribute to increased incidence of waterborne infectious diseases including plesiomonas infections [ ] [ ] [ ] [ ] . accurate estimates of the incidence of plesiomonas-related gastroenteritis and extraintestinal infections both globally and at the level of individual countries remain unknown [ , , [ ] [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . retrospective reviews of infections due to p. shigelloides in china and hong kong have been published [ ] , and cases of p. shigelloides co-infection with viral and bacterial diarrheal pathogens are common in the literature [ ] . prevalence of p. shigelloides gastroenteritis varies considerably across regions, with lower rates reported from north america and europe and higher estimates from southeast asia and africa [ ] . nonetheless, there is a general underestimation of p. shigelloides infection, in part because it shares some clinical manifestations with other pathogens [ ] . p. shigelloides is not routinely examined in clinical settings, and as such, awareness regarding this pathogen remains limited. bibliometric analysis is a statistical method for assessing both the quantitative and qualitative scope and adequacy of research efforts attained in an area of interest [ ] . it can be used to determine national and international research focus and evaluate research performance in order to identify future research priorities, funding sources, and interdisciplinary collaborations [ ] [ ] [ ] . it also provides a resource to policy-makers for implementing necessary prophylactic measures in case the analysis reveals a sharp increase in case reports or articles regarding a health issue in a particular geographic area [ , ] . bibliometric reviews can additionally help international health agencies to identify priorities (e.g. by nations) for disbursing aid [ ] and awarding research grants. there have been a few recent reviews on p. shigelloides [ , , ] but no comprehensive surveys of published studies on p. shigelloides have been yet conducted. on the other hand, bibliometric analyses have been applied to global disease research on viral agents such as dengue virus [ ] , ebola virus [ ] , john cunningham virus [ ] , mayaro virus [ ] , middle east respiratory syndrome coronavirus [ ] [ ] , yellow fever virus [ ] , west nile virus [ ] , and zika virus [ ] ; and bacterial agents such as campylobacter [ ] , leishmania species [ ] , and mycobacterium tuberculosis [ ] . other bibliometric analyses have addressed plasmodium species and resistant malaria vectors [ , ] , toxocara species [ ] , and antifungal triazole resistance (especially in candida and aspergillus species) [ ] . here we carried out a bibliometric analysis of studies on p. shigelloides published between to . the articles were evaluated in terms of annual and country-specific output, theme, domain clusters, international collaboration networks, citations, topical evolution related to keywords and co-occurrence networks, co-authorship, and funding. the aim of the survey was to evaluate international participation in p. shigelloides research-with a special interest in regions where plesiomonas infections have higher prevalence rates (i.e., africa and southeast asia)-in order to address knowledge gaps and provide a resource that can help identify present and future research priorities. publication dataset (e.g.; authors, words, countries, references, keywords) for coupling, cocitation, collaboration, conceptual framework and multiple correspondence analyses. bibliographic coupling occurs between two articles ⅈ and ⅉ when their reference lists cited at least one common source [ ] . but, in a collaboration network, the nodes comprise authors and the links co-authorships [ ] . the number of bibliographic coupling that occurs between articles ⅈ and ⅉ or co-authorship in scientific collaboration network denotes the strength of the network [ ] . a network depicts relationships in a system as a set of nodes (components) and links (relationships) [ ] . co-word or conceptual framework analysis explore k-means clustering and other dimensionality reduction techniques to identify clusters of common concepts known in a bibliographic collection. it relies on word co-occurrences in a publication dataset [ , ] . scientific productivity or an author's contributions in a field is evaluated in term of lotka's law [ ] . the lotka's law is an inverse square law that describes how often authors published in a field [ ] . published peer-reviewed articles on p. shigelloides were retrieved from the web of science (wos) database on august , . the wos is among the most reliable and comprehensive databases for bibliometric studies and hosts a wide range of quality and high-impact scientific studies ( million articles in over , journals) [ ] . we used the search term "plesiomonas shigelloides" to identify primary research articles published between and . all available information was retrieved. to obtain subject-specific results and for the sake of accuracy (in order to avoid false-positive results), only article titles were searched. a title-specific search has been reported to increase recovery and specificity with a minimal loss of sensitivity compared to a topic search [ , , ] . articles were downloaded in the bibtex file format. in order to account for variations in country population on rate of scientific production, the world population was retrieved from the world bank website (https://data.worldbank.org/indicator/sp.pop.totl) and the midperiod population corresponding to the top countries was extracted for calculation of article per million populations. england population data was retrieved from office for national statistics website (https://www.ons.gov.uk/peoplepopulationandcommunity/populationandmigration/ populationestimates/timeseries/enpop/pop). we analyzed the retrieved data for bibliometric indicators using rstudio v. . . software ( - - ) with bibliometrix r-package (http://www.bibliometrix.org) [ ] . data were imported into rstudio and converted to a bibliographic data frame and normalized for duplicate marching. the duplicated article was reduced to one record in the analysis. the data frame as typical columns named after the standard isi wos field tag codify. further, authors' names, authors' keywords (de), and keywords-plus (id) were extracted for standardization. authors' names were extracted twice as two different sets (a and b). each set was checked for variant names, spelling errors and matched with affiliations. we achieved normalized authors' names when |a \ b| � |a [ b|. for keywords (de) and keywords-plus (id), a primary term was assigned to words with similar meanings (e.g., "plesiomonas shigelloides", "plesiomonas", "shigelloides", "aeromonas-shigelloides", and "plesiomonas-shigelloides" were allotted to "plesiomonas shigelloides"). multiple occurrences of a keyword or a similar keyword in an article were regarded as one. co-occurrence of a term in authors' keywords (de set) and keywords-plus (id set) in the dataset was assessed as a set made of the intersect of the two sets (de \ id||). all set-based test was performed using a venn diagram software (http://bioinformatics.psb.ugent.be/webtools/venn/). an annual number of articles and total citations were also graphed. data were analysed for descriptive output, citation analysis, authors' h-index and scientific productivity using the relevant functions of the bibliometrix r-package. bibliometric networks (e.g., citation, author, country, author keyword, and keywords-plus networks) and bibliographic coupling (co-citation and keyword co-occurrences) were computed and visualized from bibliometric two-way (bipartite) network of rectangular matrices of articles × attributes. a typical bibliometric network is expressed as network(n) = x × n t where x is a bipartite network matrix composed of articles × attribute (e.g. authors, keywords, citations, and countries) and n is a symmetrical matrix n = n t . we created a graphic model of all networks using force-directed algorithms (fruchterman) implemented in the networkplot function of the bibliometrix r-package. all networks were standardized using the simpson's coefficient (inclusion index), proximity index (association strength), the jaccard's similarity index, and the salton's cosine coefficient among nodes of a network [ ] . in addition, k-means clustering were performed on keywords to evaluate concepts in plesiomonas field of research using the function conceptualstructure of the package. the function implements porter's stemming algorithm [ ] to modulate inflected words to their root form. for detailed search boolean for articles identification from wos, see supplementary file (s appendix). other bibliometric indicators such as language and affiliation were determined using the content search of bibtex file. a total of articles were published within the survey period; their attributes are presented in table . the studies involved authors, with . article/author ( . authors/article), . co-authors/article, and a collaboration index of . . with the exception of two authors publishing solo, all authors were involved in multi-author articles. an average of . citations/article was recorded during the study period. the scientific output related to p. shigelloides research by lotka's law showed a beta coefficient and constant of . and . , table shows the top most productive authors in the field. k. krovacek (sweden) ranked first, co-authoring ( . %) articles; and i. ciznar (slovak republic) was second with ( . %) articles. the h_index (total citations) was ( ) for k. krovacek, and ( ) for i. ciznar. it is worth noting that the topmost active authors were affiliated with institutions in developed nations, including sweden (n = ), usa (n = ), japan (n = ), spain (n = ), poland (n = ), czech republic (n = ), and slovak republic (n = ). the top cited articles on p. shigelloides are listed in s table. these studies spanned the fields of infection, immunity, clinical microbiology, and biochemistry. the total no. of citations of the top-cited articles ranged from to ; most of these were the result of funded research. research output related to p. shigelloides for the top most active countries is shown in table . united states ranked first in terms of total number of articles (n = , . %) and citations (n = ), followed by sweden (n = , . %) and germany (n = , . %). the frequency of publication varied among the top countries from . to . %. sweden had highest productivity ( . article/million population) when normalized for population size using mid-period population ( ) . the rank order of these countries changed when productivity was mea- the top journals with the most published articles on p. shigelloides are listed in s table. these journals cover a range of subjects including carbohydrates, microbiology, food science, infectious disease, immunology, and biochemistry, reflecting active areas in plesiomonas research. carbohydrate research ranked first (n = , . %), followed by journal of clinical microbiology, folia microbiologica and food biotechnology each with articles ( . %). table shows the most relevant keywords related to plesiomonas studies, including both author keywords (de) and keywords-plus (id). both author keywords (de) and keywords-plus (id) have keywords in common (lipopolysaccharide, aeromonas species, antigens, oligosaccharide, disease, children, diarrhea, shigella, virulence, and water). fourteen keywords were unique to author keywords (plesiomonas shigelloides, nuclear magnetic resonance ranking based on the number of articles; tc, total citations. (nmr), pcr, structure, maldi-tof, fish, pathogenicity, media, meningoencephalitis, resistance, enterotoxin, gastroenteritis, serotyping, and sepsis), and keywords were unique to keywords-plus (infections, escherichia, septicemia, environments, in-vitro, polysaccharide, vibrio species, bacteria, biological repeating unit, iron, meningitis, humans, and strains). the unique author keywords primarily described medium of transmission (fish) and methods involved in isolation and characterization of the microorganism (nmr, maldi-tof, pcr, enterotoxin, resistance, pathogenicity, serotyping, and structure) and specific infections (meningoencephalitis, gastroenteritis, and sepsis). author keyword terms associated with identification methods of p. shigelloides included polymerase chain reaction (pcr, n = , septicemia (n = , . % (id)), meningoencephalitis (n = , . (de)), sepsis (n = , . % (de)), and meningitis (n = , . % (id)), which ranked th, th, and th respectively. the common conceptual frames in retrieved articles determined by k-means clustering with three clusters of , , and elements showed research responses focused on neonates (children) extraintestinal infections and gastroenteritis, elucidation of cell wall structure (lipopolysaccharides, core oligosaccharide, o-specific polysaccharide etc.), and co-infections of p. shigelloides with other pathogens, respectively (fig ) . the -element cluster explained the co-occurrence and co-infection of p. shigelloides with other bacteria. other indicators of frequently represented concepts and frameworks related to p. shigelloides included co-occurrence of terms and keywords. s fig shows the co-occurrence network of the top terms associated with p. shigelloides studies, while s fig shows the co-occurrence networks of keywords. these concept-related frameworks or terms included virulence, meningoencephalitis, aeromonas, newborn, antigen, pathogenicity, sepsis, diarrhea, infections, escherichia coli, septicemia, biological repeating unit, diarrheal disease/gastroenteritis, lipopolysaccharide, water, iron, polysaccharide, bacteremia, meningitis, septicemia, lipid-a, strains, and aquatic environments. the top authors' collaboration and coupling networks on p. shigelloides studies were divided into two mega-clusters or spheres with nodes occupied solely by researchers from high-income countries (fig a and b ). the first sphere of the authors' network comprised nodes (authors) with no fewer than linkages, while the second sphere included nodes (authors) with the number of collaboration linkages ranging from nine to . similarly, the two separate authors' coupling network spheres included and authors' networks, respectively; collaborative conjugation ranged from eleven nodes (authors) in the former and in the latter. fig shows countries' collaboration networks on p. shigelloides studies. collaboration pathways ranged from to . the sweden had a high number of collaborations (n = ), followed by united states (n = ), slovakia (n = ), and italy (n = ). other countries had no collaboration networks. prominent network color code: green, usa network; purple, spain network; light green, sweden network; pink, cuba-brazil network; blue, japan-china network. the present bibliometric analysis of p. shigelloides examined global research trends between and based on data retrieved from wos. we found that the number of research articles on p. shigelloides increased non-linearly from to articles. however, a negative trend in rate of increase was noted (− . %) suggesting that research on p. shigelloides has not been of broad interest in the past years, likely due to discontinuation of plesiomonas-related research by certain authors and differences in regional distribution of the microorganism. furthermore, the function estimates and goodness-of-fit indicated that scientific output on p. shigelloides does not follow lotka's law, suggesting that the number of articles related to plesiomonas research will further decline in the future. in general, emerging and re-emerging bacterial pathogens are not accorded the same degree of attention as their viral counterparts. health emergencies (e.g., outbreaks of infection) relating to emerging viral pathogens including zika and chikungunya viruses have driven the generation of new scientific knowledge, resulting in a significant increase in the number of research articles on these subjects [ ] . for instance, in only eight articles were published on chikungunya virus, but by , the number had reached [ ] . similarly, only articles on ebola virus were published in prior to the ebola outbreaks in west africa, but this increased to more than articles in [ ] . as is the case with other research areas, most of the leading authors in the plesiomonas research field were from developed nations such as the united states, sweden, austria, japan, spain, poland, czech republic, and the slovak republic, with few from low-income countries, thus follow the similar trend of low productivity of the region in other research areas. it has been suggested that the economic strength (growth) of a nation influences the research output [ , [ ] [ ] [ ] . the higher prevalence of plesiomonas infections in developing countries [ ] should motivate researchers in countries most affected to carry out more studies on this pathogen. some of the most frequently cited studies pertained to health and identification of the pathogen-for example, efforts to produce a multivalent vaccine using o-specific polysaccharides from shigella spp. and p. shigelloides; iron uptake in plesiomonas shigelloides; and comparative analysis of p. shigelloides and e. coli (shigella) sonnei o-antigen gene clusters. others focused on p. shigelloides hemolytic expression; serological analysis of the plesiomonas core; and multilocus sequence typing of plesiomonas and its pathogenic potential. the united states and sweden dominated the list of top countries most actively researching plesiomonas in terms of numbers of articles and citations. in addition to economic strength and availability of research facilities and funding [ , [ ] [ ] [ ] , this productivity can be ascribed to a high level of intranational and possibly multinational collaboration with other institutions, which can impact research visibility and citation frequency [ , , ] . in particular, the dominance of the united states has been noted in other fields of research [ , , , ] . also, authors' multiple affiliations influence country collaboration network. conversely, the low contributions from developing countries including countries from africa characterized by a high frequency of selffunded or independent studies [ ] , mirror the situation of research in other fields. the shift in rank among the top nations most active in the plesiomonas research field when productivity was measured based on total citation per country ought not be regarded as a precise measure of productivity. citation rate does not reflect publication output of an author or country [ ] , since the smaller the number of articles used for estimation, the larger the impact of a few frequently cited articles [ ] . self-citations and inaccurate citations can also provide false quality metrics [ ] . the most frequently mentioned keywords and research areas (including publication outlets) associated with plesiomonas studies reflect the research hotspot during the survey period, which included cell wall (carbohydrate research), co-infection, and extraintestinal infections such as septicemia, bacteremia, and meningitis. these findings reveal the most pressing health issues related to plesiomonas-induced gastroenteritis and extraintestinal infections, and coinfections with other pathogens and effort to gain an understanding of the structural architect of the pathogen's cellwall; this was supported by other conceptual framework indicators such as co-words or keyword co-occurrence networks. however, important topics such as strainbased delineation and identification, including detection of pathogenic and non-pathogenic strains, that are necessary for infection management were lacking and were not evident from the bibliometric analyses. newer research themes such as molecular and genomic-level studies as an alternative or complementary to traditional experimentation (which has some limitations) necessary to clarify the pathogenesis of plesiomonas infection were not apparent throughout this study. a bibliometric survey complemented with a narrative review or metaanalysis may be beneficial in plesiomonas research. future research is needed to answer questions related to what particular strain of the microorganisms are pathogenic and how to differentiate pathogenic variants from non-pathogenic ones. the two mega-clusters or spheres in the top authors' collaboration and coupling networks on p. shigelloides studies showed collaboration pathways mainly among authors from highincome countries, which is similar to trends observed in collaboration network analyses of human immunodeficiency virus and human papilloma virus studies [ ] . alliances between developing and developed countries are rare in a number of scientific areas [ ] . among researchers in the united states, collaboration pathways were largely intra-national, as suggested by a large number of publications but multiple country publications. in contrast, collaborations by authors in cuba, finland, czech republic, italy, slovakia, and sweden tended to be multi-national, which is more valuable for the epidemiological control of pathogens. the absence of collaboration pathway in venezuela, india, nigeria, and other african nations is consistent with the low number of publications from these countries. intra-and international collaborations between developed and developing nations could provide opportunities for the division of labor and resources to address important scientific questions. there were some limitations related to the bibliometric survey adopted in this study, including the use of a single database (the wos), the low sensitivity and strictness of the search terms and search strategy used, and the exclusions of other document types (e.g., meeting abstract, note and proceeding papers etc.) and articles published in non-english language (e.g in chinese). also, the current analysis did not allow for a narrative review and judgment on contents and results of the articles [ ] . as noted earlier, emerging themes and recent research focus in plesiomonas are not easily recognized in bibliometric studies due to low frequency of appearance in keywords. notwithstanding these limitations, this is the first bibliometric study on plesiomonas-related research contributing to the evidence base and would help direct future research. also, the wos has a larger coverage compared to other database, reliable indexing technology that minimizes the ''indexer effect" and is well accepted among scientific communities [ ] . our bibliometric analysis revealed a global diminishing research in plesiomonas, greater research output from high-income countries compared to low-and middle-income countries and limited collaboration with developing countries. the low productivity in developing countries in plesiomonas research mirror the state of affairs in other research fields. a better understanding of the clinical features, epidemiology and plesiomonas-associated diseases is needed in countries with high infection rates. emerging themes and recent research focus in plesiomonas research are not easily recognized in bibliometric studies due to low frequency of appearance in keywords and, hence, the need for future studies guided by narrative reviews. supporting information s fig. co-occurrence network of top terms associated with p. shigelloides studies. each node in the network represents a different term. the node's diameter corresponds to the frequency of co-occurrence with other terms. lines depict co-occurrence pathways between terms. (tif) s fig. keyword co-occurrence networks related to p. shigelloides studies. each node in the network represents one of the top keywords. the node's diameter corresponds to the keyword's frequency of co-occurrence with other keywords. lines depict co-occurrence pathways between keywords. (tif) s treating spontaneous and induced septic abortions plesiomonas shigelloides periprosthetic knee infection after consumption of raw oysters isolation, genome sequencing and functional analysis of two t -like coliphages of avian pathogenic escherichia coli wound infection with plesiomonas shigelloides following a freshwater injury susceptibility of bacteria isolated from acute gastrointestinal infections to rifaximin and other antimicrobial agents in mexico plesiomonas shigelloides: an extremely rare cause of spontaneous bacterial peritonitis plesiomonas shigelloides meningitis in an adult in the ed diarrheal disease caused by plesiomonas shigelloides oyster-associated out-break of diarrhoeal disease possibly caused by plesiomonas shigelloides. the lancet occurrence of plesiomonas shigelloides in surface water: relationship with faecal pollution and trophic state an acute foodborne outbreak due to plesiomonas shigelloides in yaounde, cameroon plesiomonas shigelloides and salmonella serotype hartford infections associated with a contaminated water supply-livingston county plesiomonas shigelloides revisited listeriosis: a resurgent foodborne infection. clinical microbiology and infection impact of regional climate change on human health the ecology of climate change and infectious diseases climate change and infectious diseases: from evidence to a predictive framework non-cholera vibrios: the microbial barometer of climate change fatal plesiomonas shigelloides septicaemia in a splenectomised patient cellulitis and compartment syndrome due to plesiomonas shigelloides: a case report spontaneous bacterial peritonitis due to plesiomonas shigelloides a case of plesiomonas shigelloides cellulitis and bacteraemia from northern europe plesiomonas shigelloides infection in hong kong: retrospective study of laboratory-confirmed cases plesiomonas shigelloides sepsis and splenic abscess in an adolescent with sickle-cell disease not always a sexual transmitted disease? pyosalpinx cuased by plesiomonas shigelloides in an immunocompetent host plesiomonas shigelloides sepsis in a thalassemia intermedia patient plesiomonas shigelloides pneumonia plesiomonas shigelloides sepsis and meningoencephalitis in a surviving neonate infection after consumption of raw oysters septic shock caused by plesiomonas shigelloides in a patient with sickle beta-zero thalassemia the patient presenting with acute dysentery-a systematic review successful liver transplantation from donor with plesiomonas shigelloides sepsis after freshwater drowning: case report and review of literature on gram-negative bacterial aspiration during drowning and utilization of organs from bacteremic donors meningoencephalitis caused by plesiomonas shigelloides in a chinese neonate: case report and literature review mä tzsch tw. plesiomonas shigelloides in acute cholecystitis: a case report pseudoappendicitis caused by plesiomonas shigelloides serology, virulence, antimicrobial susceptibility and molecular characteristics of clinical vibrio parahaemolyticus strains circulating in southeastern china from diarrheal disease in northwestern ecuador: prevalence, pathogenicity, and transmission of enteric pathogens across the region plesiomonas shigelloides revisited the bibliometric analysis of scholarly production: how great is the impact? bibliometrics analysis and density-equalizing mapping global toxocariasis research trends from to : a bibliometric analysis global cocaine intoxication research trends during - : a bibliometric analysis of web of science publications bibliometric analysis of publications on campylobacter bibliometric analysis of literature on antifungal triazole resistance global research trends of world health organization's top eight emerging pathogens plesiomonas shigelloides seventy years of systematics and taxonomy in perspective of the present-day diagnostic demands dengue research: a bibliometric analysis of worldwide and arab publications during tracing the scientific outputs in the field of ebola research based on publications in the web of science mapping the history and current situation of research on john cunningham virus-a bibliometric analysis bibliometric assessment of the scientific production of literature regarding mayaro global research trends of middle east respiratory syndrome coronavirus: a bibliometric analysis yellow fever disease: density equalizing mapping and gender analysis of international research output. parasites and vectors global research trends in west nile virus from to : a bibliometric analysis a bibliometric analysis of global zika research. travel medicine and infectious disease bibliometric analysis of leishmaniasis research in medline ( - ). parasites and vectors density equalizing mapping of the global tuberculosis research architecture malaria research, - , and the burden of disease abu-taha as. a bibliometric analysis of literature on malaria vector resistance: ( - ) bibliometrix: an r-tool for comprehensive science mapping analysis bibliographic coupling between scientific papers national characteristics in international scientific co-authorship relations mapping the knowledge structure of research on patient adherence: knowledge domain visualization based co-word analysis and social network analysis the frequency distribution of scientific productivity mapping the scientific research in organic farming: a bibliometric review an algorithm for suffix stripping bibliometric survey of plesiomonas-related research bibliometric indicators of the zika outbreak bibliometric assessment of scientific production of literature on chikungunya a bibliometric analysis of global ebola research. travel medicine and infectious disease ecological engineering and ecosystem restoration global trends in dem-related research from to : a bibliometric analysis global biodiversity research during - : a bibliometric analysis. biodivers conserv plesiomonas shigelloides infection in southeast china bibliometric analysis on global parkinson's disease research trends during - influenza: a scientometric and density-equalizing analysis world-wide architecture of osteoporosis research: density-equalizing mapping studies and gender analysis international scientific collaboration in hiv and hpv: a network analysis policy and the mapping of scientific change: a co-word analysis of research into environmental acidification knowledge discovery through co-word analysis the authors thank the south africa medical research council (samrc) and the national research foundation, the world academy of science (nrf-twas) for financial support. conclusions arrived at and opinions expressed in this article are those of the authors and are not necessarily to be attributed to samrc or nrf-twas. key: cord- -g oes authors: nemzek, jean a.; lester, patrick a.; wolfe, a. marissa; dysko, robert c.; myers, daniel d. title: biology and diseases of dogs date: - - journal: laboratory animal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: g oes historically, the dog played an important role as a laboratory animal in biomedical research. although numbers are declining, the use of dogs continues to be common in pharmacokinetics and cardiovascular studies. the normal biology of the dog as both a laboratory and a companion animal has been well studied and reference values are presented here as a clinical and experimental resource. this provides the necessary background to discuss the spontaneous diseases, including infectious and neoplastic conditions, prevalent in purpose bred as well as random source dogs used in biomedical research. in addition, diseases and conditions that arise secondary to the housing and experimental manipulation of dogs is discussed with emphasis on treatment and prevention. laboratory animal medicine mellitus. a comprehensive but concise review of the use of the dog as a research subject is available in gay ( ) . the breed of dog most commonly bred for use in biomedical research is the beagle. some commercial facilities also breed foxhounds or other larger dog breeds for use in surgical research studies. some specific breeds with congenital or spontaneous disorders have also been maintained by research institutions (see examples below). random-source dogs used in research are most frequently mongrels or larger dog breeds (e.g., german shepherd, doberman pinscher, labrador and golden retrievers) that are used for surgical research and/or training. according to a computerized literature search for "beagle" for the years - , a significant portion of the biomedical scientific publications identified were in the fields of pharmacology or toxicology. especially common were studies focusing on pharmacokinetics, alternative drug delivery systems, and cardiovascular pharmacology. other common areas of research using beagles were dental and periodontal disease and surgery, orthopedic surgery, skeletal physiology, and imaging studies. other research areas that utilized beagles included canine infectious disease, prostatic urology, and ophthalmology. most large-sized dogs (either purpose-bred or randomsource) are used in biomedical research because of their suitability for surgical procedures. anesthetic protocols and systems for dogs are well established and the organs of larger dog breeds are often an appropriate size for trials of potential pediatric surgical procedures. surgical canine models have been used extensively in cardiovascular, orthopedic, and transplantation research. there are also some unique spontaneous conditions for which dogs have proven to be valuable animal models. a colony of gray collies had been maintained at the university of washington (seattle) for the study of cyclic hematopoiesis. this condition is manifested by periodic fluctuations of the cellular components of the blood, most notably the neutrophil population. these dogs can be used to study the basic regulatory mechanisms involved with hematopoiesis, as well as possible treatments for both the human and the canine conditions (brabb et al., ) . golden retrievers affected with muscular dystrophy have been used as models of duchenne muscular dystrophy in human children. duchenne muscular dystrophy is caused by an absence of the muscle protein dystrophin, inherited in an x-linked recessive manner. the dystrophy in golden retrievers is caused by the absence of the same protein and is inherited in the same way. the clinical signs (such as debilitating limb contracture) are also similar between the canine and human conditions (kornegay et al., ) . other genetic disorders studied in dog colonies include hereditary canine spinal muscle atrophy (cork, ) and narcoplepsy in doberman pinschers (ripley et al., ) . bedlington terriers have been used to study copper storage diseases (such as wilson's disease) and the development of spontaneous diabetes mellitus and hypothyroidism has been studied in several breeds of dogs for comparisons with the human conditions. although historically the dog has been a common laboratory animal, their use in research has waned over the past years. according to the u.s. department of agriculture (usda), animal and plant health inspection service ( , ) , the number of dogs used in research has declined from , in to , in (prior to the previous edition of this text) and , in . this decrease was caused by a variety of factors, including (but not limited to) decreased availability, local restrictive regulations, conversion to other animal models (such as livestock or rodents), increased cost, and shift in scientific interest from pathophysiology to molecular biology and genetics. dogs used for research are generally segregated into two classes: purpose-bred and random-source. purposebred dogs are those produced specifically for use in biomedical research; they are intended for use in long-term research projects and/or pharmacologic studies in which illness or medication would require removal from the study. usually these dogs are either beagles or mongrel foxhounds, although other breeds may be available. purpose-bred dogs typically receive veterinary care throughout their stay at the breeding facility. they are usually vaccinated against rabies virus, canine distemper virus, parvovirus, adenovirus type , parainfluenza virus, leptospira serovars canicola, icterohaemorrhagiae, grippotyphosa, and pomona, and bordetella bronchiseptica (jasmin, personal communication). purpose-bred dogs are also usually treated prophylactically for intestinal helminths and ectoparasites, and possibly given a heartworm preventative. random-source dogs are not bred specifically for use in research. they may be dogs bred for another purpose (e.g., hunting and racing) or stray dogs collected at pounds or shelters. the health status of these dogs can be the same quality as purpose-bred dogs, or it can be an unknown entity. random-source dogs that have been treated and vaccinated in preparation for use in research are termed conditioned dogs. these dogs are then suitable for long-term studies or terminal preparations that require unperturbed physiologic parameters. conditioned dogs are often tested for heartworm antigen because of the implications that infestations can have on cardiovascular status and surgical risk. nonconditioned random-source dogs are useful only in a limited number of research studies, such as nonsurvival surgical training preparations and tissue/organ harvest. options for procurement of dogs for biomedical research typically include purchase from a usdadesignated class a or class b licensed dealer or directly from a municipal pound. the requirements for usda licensure are detailed in code of federal regulations (cfr), title , chapter ( - - edition), subchapter a, animal welfare, . definitions, and . requirements and application (office of the federal register, ) . briefly, class a licensees are breeders who raise all animals on their premises from a closed colony. class b licensees purchase the dogs from other individuals (including unadopted animals from municipal pounds) and resell them to research facilities. there are additional regulations that apply to class b dealers (such as holding periods and record-keeping documentation) because of the public concern that stolen pets could enter biomedical research facilities in this manner. in december , the national institutes of health (nih) issued notice not-od- - entitled notice regarding nih plan to transition from use of usda class b dogs to other legal sources (national institutes of health, ) . this nih policy begins in the fiscal year and prohibits the procurement of dogs from class b dealers using nih grant funds. from that point forward, dogs on nihfunded studies will have to be obtained from class a vendors, privately owned colonies (such as institutional breeding colonies), or client-owned animals (e.g., animals participating in veterinary clinical trials). the best resource for identification of possible vendors are online 'buyer's guide' sites or 'buyer's guide' issues of trade periodicals. online sites include the buyer's guide of the american association of laboratory animal science (http://laboratoryanimalbuyersguide.com), and the trade journals lab animal (http://guide.labanimal. com) and animal lab news (http://www.alnmag.com/ content/buyers-guide). a 'buyer's guide' typically lists sources for both purpose-bred and random-source dogs, and denotes such features as pathogen-free status, health status, and availability of specific breeds and timed pregnant females. some suppliers also have separate advertisements within issues of the journals. federal regulations promulgated by the animal and plant health inspection service, usda, in response to the animal welfare act ( cfr . , . , and . [g] ) are described in cfr chapter ( - - edition), subchapter a, animal welfare (office of the federal register, ) . regulations pertaining specifically to the care of dogs used in research are found in subpart a, specifications for the humane handling, care, treatment, and transportation of dogs and cats of part (standards) of subchapter a. particular attention should be paid to section . c (primary enclosures-additional requirements for dogs) because the space required for housing dogs is calculated using body length rather than weight (a parameter used for other species and also for dogs in the national research council (nrc) guidelines). section . (exercise for dogs) describes the requirements that dealers, exhibitors, and facilities must follow in order to provide dogs with sufficient exercise. the institute for laboratory animal research (ilar) has written the guide for the care and use of laboratory animals (national research council, ) . the 'guide' is the primary document used by institutional animal research units to develop their programs and by animal care evaluation groups, such as the association for assessment and accreditation of laboratory animal care international (aaalac international), to facilitate site visits and inspections. the primary difference between the th and th editions of the 'guide' (national research council, regarding the care of dogs is the notation that "enclosures that allow greater freedom of movement and unrestricted height (i.e., pens, runs, or kennels) are preferable." the ilar committee on dogs authored dogs: laboratory animal management (national research council, ) . this publication describes "features of housing, management, and care that are related to the expanded use of dogs as models of human diseases" and includes "an interpretive summary of the animal welfare regulations and the requirements of the public health service policy on humane care and use of laboratory animals." the reader is encouraged to use these publications to obtain further information on care and husbandry of dogs in the biomedical research setting. the information presented in the tables represents a range of normal values that can vary depending on the analytical method, as well as the age, breed, and sex of the animal. cohen, covance laboratories, inc., cumberland, va ( ) . physiological data for a mixed population of dogs of both sexes. fig. . demonstrates the normal weights and corresponding ages for both male and female beagle and hound dogs. tables . and . feature hematology data from beagles of both sexes from two commercial facilities. tables . and . list serum chemical data for beagles of both sexes from two commercial facilities. representative blood gas, coagulation data, and normal urinalysis parameters can be found in tables . - . , respectively. finally, the reviews in arterial and venous blood gas anaylses (rieser, ) and the manual of canine and feline cardiology (tilley et al., ) are excellent resources. good nutrition and a balanced diet are essential to the health, performance, and well-being of the animal. the nrc of the united states national academy of sciences is the leading provider of nutrient recommendations for dogs and provides average requirements needed to maintain growth and prevent deficiencies (subcommittee on dog and cat nutrition, ) . the nrc publications form the basis for the association of american feed control officials (aafco) nutrient profiles, which are updated periodically (baldwin et al., ) . the aafco is an advisory body comprising state representatives from across the united states. it provides a mechanism for developing and implementing uniform and equitable laws, regulations, standards, and enforcement policies, and establishes nutrient profiles for cat and dog foods (dzanis, ; thatcher et al., ) . additional resources should be consulted for details on the nutritional requirements for dogs of all ages (dzanis, ; subcommittee on dog and cat nutrition, ; baldwin et al., ; thatcher et al., ; hand et al., ) . recommendations for feeding the appropriate amount of diet are determined by the dog's metabolic requirements. the maintenance energy requirement (mer) is the amount of energy used by a moderately active adult animal in a thermoneutral environment. the mer for most breeds may be calculated using the following equation: mer (metabolizable kcal/day) = bw × . × kj de, where bw = body weight (kg), kj = kilojoules, de = digestable energy (kienzle and rainbird, ) . in-depth overviews of diets used in biomedical research are available in diet-specific literature. open-formula diets have defined concentrations of all ingredients and the information is publicly available. this allows researchers to control for this important environmental variable and enables retrospective analysis of possible diet composition effects on research results (barnard et al., ) . open-formula diets occasionally may require changes in formulation to maintain nutrient composition or meet changing nutrient requirements. these changes in quantitative ingredient formulation are made public when open-formula diets are modified. in contrast, closed-formula diets are commercially available, balanced diets that meet and label the minimum requirements for protein and fat and the maximum values for ash and fiber; however, the exact composition of ingredients may vary from batch to batch. ingredient composition varies as the manufacturer applies a leastcost strategy, referring to formulating diets to maximize profit by using the least-expensive ingredients. although the ingredients are listed, the quantitative ingredient formulation is not publicly available and can vary without public disclosure, due to proprietary nature of commercial diets produced and marketed under vendor trade names. closed-formula diets have also been referred to as as 'fixed formula' or 'constant nutrition' (labdiets, pmi nutrition international, st. louis, missouri) by manufacturers (barnard et al., ) . in fixed-formula diets, the quantitative ingredient formulation does not change; however, this information is proprietary and therefore laboratory animal medicine not disclosed publically (barnard et al., ) . semipurified and purified diets provide the strictest control of ingredients and are formulated from purified components: amino acids, lipids, carbohydrates, vitamins, and minerals. although purified and semipurified diets do differ in the types of ingredients used, the terms are generally used to mean the same thing. purified-ingredient diets are generally 'open' formulas, meaning that they are published and available to the scientific community. the animal care provider should be aware of the manufacture date of the diet, which should be clearly visible on the bag. as a general rule, diets are safe for consumption up to months following the manufacture date when stored at room temperature. refrigeration may prolong the shelf-life, but the best strategy is to feed only fresh diets and use each lot based on the date of manufacture. specifications for feeding and watering of dogs are provided in the regulations of the animal welfare act. management of a breeding colony requires broad knowledge of the dog's anatomy, reproductive physiology, and behavioral needs during breeding, gestation, and parturition. although a comprehensive discussion of the biology of canine reproduction is beyond the scope of this chapter, essential features of the broad topics noted above are presented. from dr. asheley wathen, covance laboratories, inc., madison, wi, and dr. kimberley cohen, covance laboratories, inc., cumberland, va ( ) . overall health, body condition, nutrition, and age greatly influence reproductive efficiency (gavrilovic et al., ; johnson, ) . therefore, only normal, healthy animals in excellent body condition should be used in breeding programs. beagles between and . years of age have the best conception rates and litter size with the lowest neonatal mortality. after years of age, conception rates and litter size decline and neonatal mortality increases (johnson, ) . the vagina is a long, musculomembranous canal that extends from the uterus to the vulva. during physical examination, the gloved finger or examination instrument should be introduced through the dorsal commissure of the vulva, avoiding the deep ventral clitoral fossa. examination should proceed at an angle of approximately ° until the instrument or fingertip has passed over the ischial arch, after which it can be directed further craniad toward the cervix. the uterus consists of the cervix, uterine body, and uterine horns. the cervix is an abdominal organ, located approximately halfway between the ovaries and the vulva. when the bitch is in proestrus and estrus, the cervix can be distinguished during abdominal palpation as an enlarged, turgid, walnut-shaped structure. female dogs are monoestrous, typically nonseasonal, spontaneous ovulators that have a spontaneous luteal phase approximately days longer than the ± days of pregnancy followed by obligate anestrus. puberty (beginning of the first estrus) occurs between and months in most breeds. the time of onset positively correlates with the body size (concannon, ) . the canine cycle is divided into four phases: proestrus, estrus, diestrus, and anestrus. the duration of laboratory animal medicine proestrus is - days with an average of days and reflects the follicular phase rise in estrogen. during this stage, the vulva is enlarged and turgid, and a serosanguinous vaginal discharge is present (concannon, ) . estrus may be from to days in duration but generally lasts days. the endocrine feature of estrus is the first abrupt increase in progesterone (> ng/ml), which occurs concomitantly with the luteinizing hormone (lh) surge % of the time, followed by ovulation within - h. the vulva is softer and smaller than in proestrus. the vaginal discharge persists and may remain serosanquinous or become straw colored. diestrus begins approximately days after the onset of standing heat. the end of this stage is days later, which would be coincident with whelping if the bitch had become pregnant. defined behaviorally as starting when estrous behavior ceases (concannon, ) , diestrus represents the peak of serum progesterone. anestrous may last from to days and is the stage of reproductive quiescence. it is characterized by an absence of ovarian activity and serum progesterone levels of less than ng/ml. the onset of puberty in the male ranges from to months of age and is affected by breed, season, nutrition, and disease status. this process is initiated by the secretion of lh from the anterior pituitary, which stimulates the production of testosterone by the interstitial or leydig's cells. at this time, the testicular growth is rapid, the seminiferous tubules begin to differentiate, and sertoli cells form the blood-testis barrier. secretion of follicle-stimulating hormone (fsh) by the anterior pituitary stimulates the production of other key hormones by the sertoli cells, including, inhibin, androgen binding protein, and estrogen. fsh stimulates spermatogenesis in the presence of testosterone, whereas inhibin and estrogen provide negative feedback to the pituitary gland to decrease fsh production. spermatogenesis in the dog is completed in days, with subsequent maturation of sperm occurring in the epididymis for approximately days. thus, the entire process from the initiation of spermatogonial mitosis to the delivery of mature sperm to the ejaculate is days. a breeding soundness exam should be conducted to assess the probability of a male dog's successful production of offspring. factors affecting male fertility include libido, ability to copulate, testicular size, and quality semen production. supression of sexual behavior and problems with libido may occur in dogs due to early weaning, isolation, or inherited abnormalities. animals with poor hind limb conformation or trauma to the back may be unable to properly mount the female. there is a positive correlation between scrotal circumference and the number of sperm produced. finally, the quality of sperm is assessed by motility, morphology, volume, and concentration. an ejaculate ( ml) that contains approximately million progressively motile sperm without significant morphological abnormalities is a good indicator of normal male fertility. complete anatomy of the bitch and dog can be found in miller's anatomy of the dog (evans and de lahunta, ) . cells of the vaginal epithelium mature to keratinized squamous epithelium under the influence of estrogen. because of the rise in estrogen throughout proestrus, with peak levels occurring just prior to the onset of standing heat, the vaginal smear can be used as an indicator of the bitch's readiness for breeding. the smear will not confirm the presence of ovulation nor is it of prognostic value in normal bitches during anestrus. the percentage of vaginal epithelial cell cornification is an index of estrogen secretion by the ovarian follicles. cornification occurs approximately days prior to the estrogen peak and days prior to standing heat. as cornification of vaginal epithelial cells proceeds, the cells become larger, with more angular borders. the nuclear/ cytoplasmic ratio decreases until the nuclei reach a point where they no longer take up stain (coincident with the onset of estrus). the cells appear 'anuclear' and are classified as 'cornified' or 'anuclear squames.' the vaginal cytology smear of the bitch changes from predominantly cornified to noncornified days after ovulation. the day of this change is the first day of diestrus. other epithelial cell types noted on vaginal cytology include superficial cells (large, angular cells with small nuclei); intermediate cells (round or oval cells with abundant cytoplasm and large, vesicular nuclei); and parabasal cells (small round or elongated cells with large, well-stained nuclei, and a high nuclear/cytoplasmic ratio). based on vaginal cytology, the estrous cycle is classified as follows: although vaginal cytology is a useful tool, observation of behavioral estrus is the best criterion to use in breeding management. during proestrus, the male is attracted to the bitch and will investigate her hindquarters, but she laboratory animal medicine will not accept breeding. estrus is characterized by proactive receptivity to mounting by males and increased male-seeking behavior (concannon, ) . during this stage, the bitch will exhibit 'flagging,' or elevation of her tail with muscular elevation of the vulva to facilitate penetration by the male. in order to maximize the conception rate and litter size, it is recommended to breed the bitch on days , , and of the standing heat. due to the long life span of canine sperm, fertilization occurs in the oviduct up to days after coitus. the ovulated oocyte is a primary oocyte that must undergo two meiotic divisions before fertilization can occur. this overall maturation process takes approximately days. after maturation, the oocyte remains viable for - days. optimal conception rates tend to occur when the bitch is bred from days before to days after ovulation; best litter size is achieved when the bitch is bred days after ovulation. implantation is evident by areas of local endometrial edema - days after breeding. there is no correlation between the number of corpora lutea and the number of fetuses in the corresponding uterine horn, suggesting transuterine migration of embryos. the dog has endotheliochorial placentation. the endothelium of uterine vessels lies adjacent to the fetal chorion, mesenchymal, and endothelial tissues, so that maternal and fetal blood are separated by four layers. the canine placenta is also classified as zonary, indicating the placental villi are arranged in a belt, and deciduate, reflecting that maternal decidual cells are shed with fetal placentas at parturition. the length of gestation is - days. luteal progesterone is responsible for maintaining pregnancy and canine corpora lutea retain their structural development throughout gestation. serum progesterone rises from less than ng/ml in late proestrus to a peak of - ng/ml during gestation, and then declines to - ng/ml just prior to parturition. progesterone is essential for endometrial gland growth, secretion of uterine milk, attachment of the placentas, and inhibition of uterine motility (johnson, ; verstegen-onclin and verstegen, ) . pregnancy detection can be performed by several methods. abdominal palpation of the uterus may be most informative at approximately days after breeding. the embryos and chorioallantoic vesicles form a series of ovoid swellings and are approximately inches in length at - days. by day , the uterus begins to enlarge diffusely and the vesicles become difficult to identify by palpation. radiology can be used to confirm pregnancy and facilitate determination of gestational age, beginning days after the lh surge (lopate, ) . bitches in which a difficult whelping is anticipated should be radiographed in late pregnancy to determine the litter size and to evaluate the size of the fetal skulls in relation to the bony maternal birth canal. ultrasonography can be used to confirm pregnancy beginning on days - , at which point the gestational sacs will be approximately cm in diameter, and until parturition (shille and gontarek, ; lopate, ) . ultrasonography can assess fetal viability by visualizing fetal heartbeats and fetal movement beginning on gestational days - and , respectively (lopate, ) . it can also predict gestational age using the inner diameter of the chorionic cavity in early pregnancy and the biparietal diameter in late pregnancy luvoni and beccaglia, ) . however, ultrasonography for determination of gestational age is most accurate at day of pregnancy when using correction factors for small (< kg) and large (> kg) body weight dogs (kutzler et al., ) . thermal support should be provided prior to parturition. dogs housed on grated flooring should be provided with mats and those on solid floors would benefit from blankets placed in a corner of the primary enclosure. shavings are discouraged because they may adhere to the umbilical cord and predispose to ascending infections. heat lamps may be placed h prior to parturition and remain until all neonates demonstrate vigorous suckling behavior. however, the use of heat lamps necessitates strict supervision in order to prevent thermal burns. if possible, whelping bitches should be housed in a quiet corridor in order to decrease periparturient stress, especially in primiparous or young mothers. monitoring of parturition is important, but human intervention should be minimal in order to prevent stressinduced cannibalism. an abrupt drop in body temperature to less than °f indicates impending parturition within - h. the process of parturition has been divided into three stages. stage of labor lasts - h and is characterized by uterine contractions and cervical dilation. during this stage, the bitch may appear restless, nervous, and anorexic. other common clinical signs include panting and increased pulse rate (johnson, ) . fetal expulsion occurs during stage , which lasts approximately - h. as the fetus engages the cervix, there is release of oxytocin, referred to as the ferguson reflex, which strengthens the uterine contractions and may elicit abdominal contractions as well. the bitch is able to inhibit this stage of labor if disturbed. the chorioallantois ruptures either during passage of each neonate through the birth canal or by the bitch's teeth at birth. interestingly, posterior presentation is common in dogs but does not predispose to dystocia. the time interval between deliveries of each pup is irregular, but the average is less than h between pups. veterinary assistance laboratory animal medicine is necessary if the bitch remains in stage for more than h without delivering the first pup, or for more than h before delivering subsequent pups. during stage of labor, the placentas are expelled either immediately or within min of delivery of each pup. if two pups are delivered from alternate uterine horns, then the birth of both puppies may precede expulsion of the respective placentas. the bitch will lick the newborn vigorously to remove the membranes from its head and to promote respiration. she will also sever the umbilical cord. the bitch may ingest the placentas, although they confer no known nutritional benefit and may induce a transient diarrhea. the peripartum use of oxytocin is required only in the event of uterine inertia, stillbirths, or agalactia. oxytocin should not be used in the event of systemic illness or abnormalities precluding vaginal delivery. indications for its use include lack of delivery h after onset of stage labor, greater than h of unproductive stage labor, inadequate contractions, or abnormal vaginal discharge. in these cases, radiographs are recommended to assess fetal size in relation to the birth canal and any possible obstructions, followed by . - . iu of oxytocin intramuscularly or subcutaneously. the oxytocin can be repeated - min after the first dose for a total of two doses (plunkett, ) . in some cases, treatment with . - . ml/kg of % calcium gluconate, delivered slowly iv while monitoring closely for bradycardia, and % dextrose iv may be indicated. uterine involution occurs during anestrus within - weeks of parturition. during this time, a greenish to red-brown vaginal discharge, or lochia, is considered normal. the presence of an odiferous, purulent discharge, accompanied by systemic signs of illness, indicates metritis or pyometra. desquamation of the endometrium begins by the th postpartum week, with complete repair by months. newborn puppies are easily sexed by examination of the anogenital distance. in female puppies, the vulva is evident a short distance from the anus, whereas the prepuce of male puppies is nearly adjacent to the umbilicus. eyes are open at approximately days, and ears are patent at approximately - days. solid food can be introduced between . and weeks of age, and puppies can be weaned at - weeks. artificial insemination (ai) is indicated when the male is physically incapable of mounting or penetrating the bitch, when there are vaginal abnormalities such as a vaginal-vestibular stricture, narrow vagina, vaginal septum, and vaginal hyperplasia, or if there is a behavioral incompatibility between the male and female dogs (kutzler, ) . semen is collected using a plastic centrifuge tube and rubber latex artificial vagina. the male is introduced to the scent of an estrous bitch and manually stimulated. the first two fractions are collected followed by a sufficient amount of the third fraction (predominantly of prostatic fluid) to bring the total semen volume to - ml. the semen can be introduced into the cranial vagina or directly into the uterus either through trans-cervical catheterization with a norwegian ai catheter or utilizing fiberoptic endoscopy. use of the norwegian ai catheter for intrauterine insemination of frozen-thawed, fresh, and chilled-extended semen results in significantly higher whelping rates than intravaginal insemination (linde-forsberg et al., ; thomassen and farstad, ) . for trans-cervical insemination, the bitch is either standing on all four legs or standing with hindquarters raised. the ai catheter and guiding tube are inserted into the vestibulum as far as the pseudocervix. firm abdominal palpation is then used to locate and fix the cervix in the other hand, at which point the catheter is further inserted along the dorsal vaginal fold until the cervical opening is located and semen is deposited into the uterus lumen (thomassen and farstad, ) . surgical and laparoscopic ai has been used successfully for intrauterine and intratubal insemination; however, these techniques are invasive and require anesthesia. therefore, the nonsurgical techniques mentioned above are recommended, as these approaches are less invasive and can be completed without anesthesia in nonsedated or sedated dogs depending on the experience of the personnel and personality of the dogs. ai with freshly collected sperm can be done on days , , and of standing heat or on days of maximal vaginal cornification. the viability of frozen-thawed sperm is significantly reduced compared to fresh or chilled sperm that may live up to or days in the reproductive tract of the bitch; frozen-thawed sperm live only a few hours. therefore, the ova must be mature and insemination with frozen-thawed semen must be done - days after ovulation in the bitch as determined by serum progesterone concentrations (thomassen et al., ) . false pregnancy (pseudocyesis), a stage of mammary gland development and lactation associated with nesting or mothering behavior, is common in the bitch. the condition occurs after the decline in serum progesterone toward the end of diestrus. there is no age or breed predisposition. pseudopregnancy does not predispose the bitch to reproductive disease or infertility. a comprehensive review of canine pseudocyesis exploring its cause, clinical features, and treatments is covered by c. gobello (gobello et al., ) . reproductive performance in the bitch is optimal prior to years of age. cycling does not completely cease; however, after - years of age, bitches demonstrate significant decreases in conception rate and the number of live pups whelped. by - years of age, pathologic conditions of the uterus, such as cysts, hyperplasia, atrophy, and neoplasia, are extremely common. dogs prefer living in a social environment. dogs have well developed olfactory glands, vision, and auditory and tactile senses that allow them to gain environmental cues and information from other dogs and humans (field and jackson, ; joint working group on refinement, ) . much of their instinctive behavior is dependent on learning to interact with other members of their species. beagles have been a popular animal model because of their docile nature. they are easily handled and, for the most part, respond favorably to repetitive manipulations such as body weight measurements, physical examination, electrocardiograms (ecgs), oral gavage, and venipuncture. although sexually mature by - months of age, dogs are not socially mature until - months of age. the socialization process should begin early during development, when puppies are receptive to conspecific and human contact. for example, from to weeks of age, puppies are most capable of learning about how to interact with other dogs. between weeks and , puppies are most capable of learning how to interact with people. by - weeks of age, dogs voluntarily wander and explore new environments. thus, early handling and mild stress (such as vaccination) appear to be extremely beneficial components of a dog's social exposure. canid social systems use signals and displays that minimize the probability of outright aggression. these behavior patterns are most likely elicited during distressful situations, such as strange environments, being handled by strange people, or encountering new animals. an excellent, illustrated discussion of normal canine behavior patterns can be found in the canine behavior section of the manual of clinical behavioral medicine for dogs and cats (overall, ) . by virtue of the dog's status as a companion animal, there are many veterinary publications and reference texts on the diagnosis, medical management, pathology, and epidemiology of its disorders. the authors of this chapter have chosen to emphasize those diseases that are more frequently encountered in the research setting, especially infectious diseases associated with the use of random-source dogs and conditions seen frequently in the beagle. for more thorough and detailed discussion of these diseases, as well as those not discussed in this chapter, the reader should consult standard veterinary textbooks. etiology canine infectious respiratory disease (cird) is a highly contagious illness and several organisms have been incriminated including bordetella bronchiseptica; streptococcus equi subsp. zooepidemicus; canine parainfluenza virus (cpiv); canine influenza virus (civ); canine respiratory coronavirus; canine adenovirus type (cav- ); canine herpesvirus; canine reovirus types , , and ; and mycoplasma and ureaplasma. naturally occuring infection can result in coinfection by two or more organisms (garnett et al., ; ford, ) . clinical signs cird can be subdivided into mild or severe forms. the mild form is more common and is characterized by an acute onset of a loud, dry, hacking cough. increased formation of mucus sometimes results in a productive cough, followed by gagging or retching motions. cough may be elicited by tracheal palpation and may be more frequent with excitement or exercise. otherwise, dogs are typically asymptomatic. mild tracheobronchitis usually lasts - days, even when untreated. the severe form results from poor general health, immunosuppression, or lack of vaccination. secondary bronchopneumonia can occur and can be the determinant of severity (sherding, ) . animals are clinically ill and may be febrile, anorexic, and depressed. productive cough and mucopurulent naso-ocular discharge are more common than in the mild form. epizootiology and transmission the natural reservoir for b. bronchiseptica is the respiratory tract (bemis, ) , and it is very easily spread by aerosol and direct contact. transmission is heightened by confined housing of multiple animals. bordetella bronchiseptica is highly infectious with an incubation period of - days. pathogenesis the most common clinical isolates are cpiv and b. bronchiseptica (mochizuki et al., ) . however, b. bronchiseptica is often recovered from clinically healthy animals (chalker et al., ) . during clinical infection, b. bronchiseptica attaches to the cilia of the upper airway epithelium, causing suppurative tracheobronchitis and bronchiolitis. infections with cpiv laboratory animal medicine or cav- alone are usually subclinical but can cause necrotizing tracheobronchiolitis (dungworth, ) . diagnosis and differential diagnosis diagnosis is often based on clinical signs and known history; however, cough elicited by tracheal palpation may be inconsistent and should not be used for definitive diagnosis. presumptive diagnosis can be made by isolation of b. bronchiseptica or mycoplasma by nasal swabs. viral isolation or paired serology is often impractical and expensive. if cough persists for more than days, other disease conditions should be considered. differential diagnoses include civ, canine distemper virus, pneumonia, heartworm disease, tracheal collapse, mycotic infections, and diseases resulting in tracheal compression (johnson, ) . prevention prevention is best achieved by avoiding exposure to infected animals. dogs should be vaccinated prior to or upon admission to the animal facility. intranasal vaccines protect against infection and disease and can be given to dogs as young as weeks of age (greene and levy, ) . combination vaccines for b. bronchiseptica, cav- , and cpiv are preferred. vaccinations should be boostered every months when multiple animals are housed in a confined area. control staff must practice proper hygiene to prevent transmission by fomites. sanitation, proper ventilation, and proper humidity are critical for control. symptomatic animals should be isolated and kennels should be disinfected with agents such as bleach, chlorhexidine, or quaternary ammonium chloride. treatment bordetella bronchiseptica is sensitive to potentiated sulfas, chloramphenicol, quinolones, tetracyclines, gentamicin, and kanamycin. use of antibiotics is indicated when severe or persistent clinical signs occur and should be continued for days. for severe or unresponsive infection, treatment should be based on bacterial culture/sensitivity patterns. nebulized gentamicin or kanamycin may be helpful in severe cases. antitussives should be avoided if the cough is productive; however, their use is indicated if coughing is causing discomfort or interfering with sleep. bronchodilators such as aminophylline, theophylline, or terbutaline can be helpful in reducing reflex bronchoconstriction. research complications due to the altered respiratory tract physiology, infected animals should not be used for pulmonary studies. etiology β-hemolytic lancefield's group c streptococcus (s. equi ssp. zooepidemicus) is a gram-positive, non-spore-forming coccus that causes pneumonia and sepsis in dogs. clinical signs clinical signs vary based on the organ system affected. pneumonic disease is typically associated with sudden onset of clinical signs including coughing, weakness, fever, dyspnea, and hematemesis. the rapid progression of disease is similar to that seen in humans with toxic shock syndrome (tss) caused by streptococcus pyogenes. peracute death has been reported in research and shelter dogs (bergdall et al., ; pesavento et al., ) . epizootiology and transmission streptococcus equi ssp. zooepidemicus is not considered a commensal of healthy dogs as most of the β-hemolytic commensal organisms belong to group g, specifically streptococcus canis. asymptomatic carriers are suspected to be the route by which infection enters populations. streptococcus equi ssp. zooepidemicus is considered an opportunistic pathogen and stressful factors such as transport can predispose to disease (priestnall et al., ) . pathologic findings in peracute cases, hemorrhage from the mouth and nose and within the pleural cavity can be the most striking lesion. ecchymotic and petechial hemorrhages can be noted on several organs ( fig. . ). 'bull's-eye' lesions may be observed on the pleural surface of affected lung lobes. histologic lesions can include fibrino-suppurative, necrotizing, and hemorrhagic pneumonia. gram-positive cocci can be found in intracellular clusters throughout the lung ( fig. . ), tonsils, and spleen of affected animals (bergdall et al., ; priestnall and erles, ) . pathogenesis predisposing factors such as transport stress and viral coinfection have been shown to contribute to the virulence of s. zooepidemicus (priestnall and erles, ) . due to the similarities with the clinical signs seen in human cases of tss, superantigens are thought to contribute to the virulence of s. zooepidemicus in cases of acute hemorrhagic pneumonia. these superantigens work by bypassing the conventional mechanisms of antigen presentation and binding to major histocompatibilitity complex class ii receptors. as a result, there is a hyperactive proinflammatory response and an 'avalanche' of cytokines including interleukin β (il- β), interleukin (il- ), and tumor necrosis factor alpha (tnf-α). three novel superantigen-encoding genes have been identified from a case of acute fatal hemorrhagic pneumonia, szef, szen, and szep. however, it is currently unclear what effect these superantigens have in vivo priestnall et al., ) . while superantigens have been detected in some isolates, there is not enough data to determine if superantigens play a role in the pathogenesis (byun et al., ; kim et al., ) . diagnosis and differential diagnosis definitive diagnosis is based on bacterial culture of nasal swabs or transtracheal lavage. polymerase chain reaction (pcr) can be done on post-mortem lung tissue. bacterial pneumonias or bacteremias can be caused by other pathogenic streptococcus spp., staphylococcus spp., escherichia coli, pasteurella multocida, pseudomonas spp., klebsiella pneumoniae, and b. bronchiseptica. nonbacterial causes of respiratory disease include rodenticide intoxication, coagulopathies, heartworm disease, pulmonary thromboembolism, ruptured aneurysm, and left-sided congestive heart failure. prevention and control there is no vaccine for prevention of s. zooepidemicus. the organism has been isolated from the environment during active outbreaks (pesavento et al., ) , so dogs diagnosed with s. zooepidemicus should be quarantined and any potential fomites (e.g. food bowls, enrichment) should be properly disinfected. treatment antibiotic therapy should be based on culture and sensitivity. resistance to doxycycline and tetracycline has been demonstrated (garnett et al., ; pesavento et al., ) . research complications dogs with severe hemorrhagic pneumonia or systemic disease are not appropriate for research study. the association between epizootics of this disease and transportation supports operational policies that require adequate acclimation periods for animals upon arrival. etiology serovars canicola, bratislava, and grippotyphosa result in renal or hepatic disease, whereas serovars icterohaemorrhagiae and pomona predominantly result in hepatic disease . clinical signs canine leptospirosis can present as subclinical, acute, or chronic disease. clinical signs in acute infection can be nonspecific and include lethargy, depression, abdominal discomfort, stiffness, anorexia, vomiting, muscle tenderness, and pyrexia. clinical signs can be related to renal failure including polyuria and polydipsia, with or without azotemia, oliguria, or anuria. leptospirosis can also lead to hepatic failure with signs such as icterus or bleeding abnormalities. peracute leptospirosis is characterized by shock, vascular collapse, and rapid death. uveitis, abortions, stillbirths, and pulmonary hemorrhage have also been associated with leptospirosis (klopfleisch et al., ; van de maele et al., ) . bivalent vaccines against the most common canine serovars, icterohaemorrhagiae and canicola, have resulted in the increased prevalence of other serovars including grippotyphosa, pomona, bratislava, and autumnalis. increased movement of wild animal reservoirs (rats, raccoons, skunks, opossums) into urban/suburban areas have also contributed to the greater prevalence of previously uncommon serovars (sykes et al., ) . transmission occurs primarily through environmental contact, although direct transmisson between hosts may also occur. leptospires passing from urine into water is the most common route of contamination (goldstein, ) . leptospirosis is a zoonotic disease. pathologic findings the kidneys consistently have gross and microscopic lesions. in the acute phase, the kidneys are swollen with subcapsular and cortical ecchymotic hemorrhages. petechial or ecchymotic hemorrhages and swelling of the lungs may also be noted. hepatic lesions during the acute phase consist of diffuse hemorrhage and necrotic foci (searcy, ) . in chronic stages of leptospirosis, the kidneys become small and fibrotic. endothelial cell degeneration and focal to diffuse lymphocytic-plasmacytic interstitial nephritis are the characteristic histopathological findings. pathogenesis the severity and course of leptospirosis depend on the causative serovar as well as the age and immune status of the dog. infection occurs after the leptospires penetrate a mucous membrane or abraded skin. the organisms then invade the vascular space and multiply rapidly, reaching the renal tubular epithelium several days postinfection. acute or progressive renal failure leading to oliguria or anuria may occur. nephritis may or may not be accompanied by hepatitis, uveitis, pulmonary hemorrhage, and meningitis. disseminated intravascular coagulation is often a secondary complication. diagnosis and differential diagnosis paired serology for the microscopic agglutination test is the most reliable means of definitive diagnosis, and successive serum sampling should be done - days after the first sample. pcr can be used to identify active infection early in the disease when serologic testing is negative or in previously vaccinated animals (sykes et al., ) . differential diagnoses include other causes of acute renal failure and hepatitis. prevention and control according to the american animal hospital association's vaccination guidelines, vaccination for leptospirosis is recommended based on geographic location and exposure risk (welborn et al., ) . both quadrivalent and bivalent inactivated bacterins are available. quadrivalent bacterins protect against canicola, icterohaemorrhagiae, grippotyphosa, and pomona serovars, whereas bivalent bacterins cover only canicola and icterohaemorrhagiae. immunization does not prevent the development of the carrier state or protect against other serovars. control requires preventing contact with wildlife reservoirs as well as identification of carrier animals. treatment doxycycline is the drug of choice as it can eliminate renal colonization. if vomiting or allergic reactions prohibit treatment with doxycycline, ampicillin or other penicillins should be utilized. aggressive fluid therapy and supportive care may also be needed. research complications due to the zoonotic potential, dogs with clinical leptospirosis should not be used in research studies. etiology campylobacter spp. are thin, curved or spiral, microaerophilic, thermophilic motile gram-negative rods. many species of campylobacter have been isolated from normal and diarrheic animals; however, the most common pathogenic species include campylobacter jejuni ssp. jejuni and c. coli (marks et al., ) . clinical signs most adult animals infected with c. jejuni are asymptomatic carriers; clinical signs are most commonly noted in dogs that are less than months of age (greene, ; burnens et al., ) . in cases of clinical illness, mild and intermittent mucoid or watery diarrhea, with or without frank blood, is most commonly noted. signs typically last - days but can persist for several months. tenesmus, inappetance, vomiting, and a mild fever may accompany the diarrhea (marks et al., ) . bacteremia and cholecystitis secondary to c. jejuni have also been documented in dogs (fox, ) . epizootiology and transmission the role of campylobacter spp. as a primary pathogen has been questioned; it may require a coenteropathy to produce disease (sherding and johnson, ) . stress or immunosuppression may make animals more susceptible. transmission is via the fecal-oral route, mostly through contaminated food or water. campylobacter jejuni can be zoonotic with immunocompromised individuals at greatest risk. pathologic findings lesions depend on the mechanism of the enteropathy (van kruiningen, ) . enterotoxin production results in dilated, fluidfilled bowel loops, with little or no histopathologic alteration. cytotoxin-mediated disease results in a friable, hemorrhagic mucosal surface. histologically, the mucosa is ulcerated with lymphoplasmacytic infiltration. translocation can result in edema and congestion of the lamina propria with focal accumulation of granulocytes. epithelial hyperplasia and decreased goblet cell numbers are also noted. campylobacter jejuni may be visualized between enterocytes with warthin-starry silver-stained sections. pathogenesis clinical disease may be produced by several different mechanisms as campylobacter spp. have a variety of virulence factors including enterotoxins, cytotoxins, and adherence or invasion properties. campylobacter jejuni can cause an erosive enterocolitis by invasion of epithelium and production of the cytolethal distending toxin (cdt) (fox, ; van kruiningen, ) . in addition, c. jejuni can produce illness via translocation to regional lymph nodes causing a mesenteric lymphadenitis. diagnosis and differential diagnosis fresh feces (per rectum) can be used for presumptive diagnosis by demonstration of highly motile, curved or spiral organisms with dark-field or phase-contrast microscopy. gram-stained c. jejuni appear as gull-winged rods. definitive diagnosis requires isolation of the organism (sherding and johnson, ) . culture requires selective isolation media, and growth is favored by reduced oxygen tension and a temperature of °c. a pcr multiplex assay for differentiation of c. jejuni, c. coli, c. lari, c. upsaliensis, and c. fetus ssp. fetus has been developed (wang et al., ) . any disorder that can cause diarrhea in dogs should be considered as a differential diagnosis. prevention and control proper environmental sanitation, waste disposal, and food storage can prevent campylobacteriosis. in enzootic situations, group housing should be avoided. outbreaks are controlled by isolation and treatment of affected individuals. treatment antibacterial treatment should be considered in severely ill dogs. erythromycin, neomycin, enrofloxacin, clindamycin, and doxycycline are all effective. resistance to quinolones and ciprofloxacin has been documented (acke et al., ) . treatment should be a minimum of - days with bacterial cultures repeating and weeks after treatment. research complications dogs with clinical campylobacteriosis have temporary derangements to digestive and absorptive functions. etiology helicobacters are gram-negative, microaerophilic, spiral bacteria that infect the gastrointestinal tract. helicobacter spp. can be separated into gastric and enterohepatic groups. the gastric helicobacters commonly identified in dogs are referred to as non- (haesebrouck et al., ; joosten et al., ) . the most common enterohepatic species found in dogs include h. bilis, h. canis, and h. cinaedi (castiglioni et al., ; dewhirst et al., ; fox, (haesebrouck et al., ; fox, ) . clinical signs most infections are subclinical in the dog. gastric infections may present with vomiting, diarrhea, and fever, accompanied by anorexia, pica, or polyphagia. enterohepatic helicobacters have been linked with inflammatory bowel disease in experimental animal models. heavy infections in dogs have been associated with inflammatory lesions of the large intestine (castiglioni et al., ; nguyen et al., ) . epizootiology and transmission the epizootiology and transmission of helicobacter spp. in the dog remain unknown. both oral-oral and fecal-oral routes for transmission have been suggested in humans, but transmission via canine saliva is a less reliable source of infection (craven et al., ) . enterohepatic infections of pet dogs are as high as % (castiglioni et al., ) . prevalence of gastric helicobacter infections in colony or shelter dogs can be as high as - % (fox, ; hermanns et al., ) . pathologic findings gastritis is usually mild and characterized by reduced mucus content of the surface epithelium with vacuolation, swelling, karyolysis, and karyorrhexis of parietal cells. multifocal infiltrates of plasma cells and neutrophils occur around blood vessels and between gastric pits (hermanns et al., ) . intestinal lesions include mild to moderate lymphoplasmacytic infiltration as well as crypt dilation and crypt hyperplasia (castiglioni et al., ) . pathogenesis gastric helicobacters are urease positive, which assists with survival in the acidic environment of the stomach (kusters et al., ; uberti et al., ) . enterohepatic helicobacters are urease negative and typically reside in the lower intestine. the mechanism by which enterohepatic helicobacters colonize the liver is thought to be through portal circulation after uptake by enterocytes or through retrograde movement from the intestine into the bile duct (fox, ) . diagnosis and differential diagnosis organisms may be demonstrated with histopathology on endoscopic or surgical biopsy tissue samples. warthin-starry silver stain may increase the sensitivity for histopathologic diagnosis. culture may be difficult depending on the helicobacter spp. for species that produce urease, a positive urease test on a gastric biopsy specimen may give a presumptive diagnosis. the urea breath test has been successfully used to diagnose helicobacter spp. in laboratory beagles with a sensitivity and specificity of % (kubota et al., ) . western blot has been used to detect serum antibodies to enterohepatic species and pcr can be used to detect helicobacter spp. in fecal samples (oyama et al., ; wadström et al., ) . any causes of acute or chronic vomiting and diarrhea in the dog are differential diagnoses. prevention and control until more is known about the epizootiology and transmission of helicobacter spp., specific recommendations cannot be made for prevention and control. treatment for gastric species, combination therapy of amoxicillin ( mg/kg q h), metronidazole ( mg/kg q h), and sucralfate ( . - . mg/kg q h) has proven to be most effective (hall and simpson, ) . replacing sucralfate with famotidine, omeprazole, or bismuth subsalicylate may also be effective (marks, ; jenkins and bassett, ; denovo and magne, ) . recurrence rates within days of treatment can be as high as % (anacleto et al., ) . treatment of enterohepatic helicobacters may depend on species susceptibility. aminoglycosides have been successful in treating h. cinaedi, but resistance to fluoroquinolones has been documented (tomida et al., ) . combination therapy of amoxicillin, clarithromycin, metronidazole, and omeprazole in medicated chow has been successful in eliminating various enterohepatic helicobacters from mice (del carmen martino-cardona et al., ) . long-term antibiotic treatment at a minimum of days is suggested for enterohepatic and gastric helicobacters. research complication dogs used in gastrointestinal physiology or oral pharmacology studies should be free from helicobacteriosis. etiology parvoviral enteritis in dogs is caused by canine parvovirus strain (cpv- ) of the family parvoviridae, genus protoparvovirus, species carnivore protoparvovirus . currently, there are three antigenic variants, a, b, and c. parvoviruses are nonenveloped, single-stranded dna viruses. clinical signs while parvoviral infection can affect the gastrointestinal tract, bone marrow, myocardium, and nervous tissues, the most common manifestation of disease is acute enteritis. clinical signs usually appear days after fecal-oral inoculation and include anorexia, fever, depression, vomiting, and profuse intractable diarrhea which may become hemorrhagic. excessive fluid and protein losses through the gastrointestinal tract result in rapid and severe dehydration. dogs can develop severe leukopenia with a total leukocyte count of cells/μl or less. repeated hemograms may provide prognostic value, as rebounds in leukocyte counts are indicative of impending recovery. terminally ill dogs may develop hypothermia, icterus, or disseminated intravascular coagulation due to endotoxemia. epizootiology and transmission parvovirus can infect dogs of any age, but puppies between and weeks of age are particularly susceptible. puppies less than weeks of age are protected by passive maternal antibody. strain cpv- c has been associated with severe disease in adult vaccinated dogs (calderon et al., ) . pathogenesis canine parvovirus has an affinity for rapidly dividing cells of the intestine and causes acute enteritis with intestinal crypt necrosis and villus atrophy. the virus also has tropism for the bone marrow and lymphoid tissues; thus, leukopenia and lymphoid depletion accompany the intestinal destruction. diagnosis and differential diagnosis parvovirus can be detected with a commercially available fecal enzyme-linked immunosorbent assay (elisa). due to intermittent and brief shedding of the virus, fecal elisas can have false-negative results. pcr can be used to confirm an elisa result and to differentiate the viral strain. at necropsy, diagnosis is based on gross and histopathologic evidence of necrosis and dilatation of intestinal crypt cells with secondary villous collapse. prevention and control parvoviral-positive animals should be quarantined for at least days as the infectious virus is shed for several days after onset of clinical signs. although pcr has been used to detect viral dna in feces for up to weeks (decaro et al., ) , it is currently unknown if the material being shed at this time is still infectious. disinfection of exposed areas with dilute bleach ( : ) or a commercial disinfectant is essential for elimination of the virus. six-week-old puppies should be vaccinated every - weeks with a modified live vaccine until at least weeks of age. treatment treatment is largely supportive and aimed at restoring fluid and electrolyte balance. antimicrobial therapy is recommended due to intestinal compromise and risk of sepsis. early nutritional support continued throughout the disease has been shown to decrease recovery times (mohr et al., ) . research complications infection with parvovirus precludes the use of a particular dog in an experimental protocol. due to the significant discomfort of the animal, as well as the intensive therapy required, humane euthanasia is usually chosen in a research setting. etiology rabies virus is a lyssavirus belonging to the family rhabdoviridae. clinical signs clinical progression of neurologic disease occurs in three stages. the first, prodromal, stage is characterized by a change in species-typical behavior. the loss of the instinctive fear of humans by a wild animal is a classic sign of impending rabies. in the second, furious, stage, animals are easily excited or hyperreactive to external stimuli and will readily bite at inanimate objects. the third, paralytic, stage is characterized by incoordination and ascending ataxia of the hindlimbs due to viral-induced damage of motor neurons. death due to respiratory failure usually occurs after onset of the third stage. wild animals such as raccoons, skunks, and bats are common reservoirs of infection for domestic animals, which in turn are the principal source of infection for humans. transmission occurs primarily by contact with infected saliva, usually via bite wounds. pathogenesis the incubation period for rabies is - weeks to the onset of clinical signs but can range from week to year. bites to the head and neck result in shorter incubation periods due to the close proximity to the brain. following infection, the virus migrates centripetally via peripheral nerve fibers to neurons within the brain, resulting in neurologic dysfunction. on reaching the brain, the virus migrates centrifugally to the salivary glands, thus enabling shedding and subsequent transmission. diagnosis and differential diagnosis definitive diagnosis is based on immunofluorescence of the virus in negri bodies of hippocampal cells. submission of the whole, unfixed brain, including the cerebellum and proximal brain stem, should be done within h of collection. the tissue should be kept refrigerated as freezing can cause delays in testing. differential diagnoses laboratory animal medicine include pseudorabies, canine distemper, bacterial meningitis, and toxicants that affect neurologic function. prevention and treatment puppies should be vaccinated by weeks of age, again at year, and then annually or triennially, depending on state and local laws. research complications immuno-prophylaxis is recommended for animal care and research personnel who may have work-related risks of exposure. due to risk of human exposure, animals with suspected infection should be humanely euthanized and brain tissue should be submitted for confirmation. giardiasis giardia lamblia, also known as g. duodenalis and g. intestinalis, is a binucleate flagellate protozoan that usually causes subclinical infestation of the small intestine. clinical disease is usually seen in young dogs and the characteristic sign is voluminous, light-colored, foul-smelling, soft to watery diarrhea, which is the result of malabsorption and hypersecretion. giardia has a direct life cycle with infection resulting after consuming cyst-contaminated food or water. the change in ph between the stomach and duodenum activates excystation and trophozoites then attach to the enterocytes. for diagnosis, direct fecal smears are considered best for observing trophozoites and zinc sulfate centrifugation is preferred for detection of cysts. a commercial elisa kit is licensed for use in dogs, but the positive predictive value is poor and zinc sulfate centrifugation techniques should be used in conjunction with elisa (rishniw et al., ) . pcr assays are also available for diagnosing giardiasis. differential diagnoses for giardiasis include bacterial and protozoal enteritis, coccidiosis, and whipworm infestation. metronidazole at - mg/kg po q h for - days is effective at treating giardiasis as well as other enteric protozoans, which may be potential differential diagnoses or coinfections. albendazole, fenbendazole, pyrantel, and praziquantel are also effective. coccidiosis intestinal coccidia associated with enteropathy in dogs include isospora canis, i. ohioensis, i. neorivolta, i. burrowsi, and hammondia heydorni (dubey and greene, ) . coccidian oocysts can be found in feces of clinically healthy dogs, as well as animals with diarrhea. clinically affected animals are young or immunosuppressed and develop diarrhea, which can vary from soft to watery and may contain blood or mucus. vomiting, dehydration, lethargy, and weight loss can also be seen. coccidia oocysts are typically spread by fecal-oral transmission, but dogs can ingest monozoic cysts in intermediate host tissues. the coccidian life cycle is both sexual and asexual, and results in the release of unsporulated eggs, which sporulate under appropriate environmental conditions. other causes for diarrhea should be excluded before a coccidial etiology is implicated. treatment may not be necessary, as infections are typically self-limiting and clinically insignificant. treatment may help to limit the number of oocysts shed in a kennel-housing situation and may be necessary in cases of protracted clinical illness. possible choices for treatment include daily administration of sulfadimethoxine ( - mg/kg po q h for - days) or trimethoprim sulfa ( mg/kg po q h for days). ascarids roundworms of dogs are most often toxocara canis; however, toxascaris leonina can also affect dogs. clinical illness is usually only seen in young animals with large worm burdens. diarrhea, vomiting, dehydration, and abdominal discomfort with vocalization can be seen. puppies may have a classical 'potbellied' appearance. heavy infestations can cause intussusception and/or intestinal obstruction. puppies that experience lung migrations of larval worms can develop fatal pneumonia. toxascaris canis can infect dogs by transplacental migration, transmammary migration, or ingestion of infective eggs. the infective stage of t. canis is the third-stage larva (l ). in transplacental infections, puppies may be born with l larvae in their lungs (sherding, ) . for diagnosis, large ( - μm in diameter) and relatively round ascarid eggs can be seen by standard fecal flotation methods. monthly administration of milbemycin or ivermectin plus pyrantel pamoate is recommended for prevention (hall and simpson, ) . most anthelmintics are effective for treatment. puppies should be treated early and often (every other week until weeks of age) because of the possibility of prenatal or neonatal infection. pregnant bitches can be treated with extended fenbendazole therapy ( mg/kg po once a day from day of gestation through day of lactation). hookworms the most common and most pathogenic hookworm of dogs is ancylostoma caninum. ancylostoma braziliense can also be found in dogs, but only a. caninum infestation typically results in clinical illness. puppies with hookworm infections can present as anemic with bloody diarrhea or melena. other clinical signs include lethargy, anorexia, dehydration, vomiting, and poor weight gain. these signs are a direct result of the worms' consumption of blood and body fluids. infective larvae (l ) are ingested from the environment and develop directly in the intestinal tract. infestation can also be transmammary, from ingestion of a paratenic host, and, less often, by transplacental migration. on histological sections, embedded worms with mouthparts may be identified. diagnosis is made by identification of eggs or larvae by either fecal flotation or direct smear. a differential diagnosis of parvovirus should be considered for puppies with bloody diarrhea, and autoimmune hemolytic anemia should be considered in young dogs with anemia. pyrantel pamoate is the anthelmintic of choice because it is safest in young ill animals. monthly administration of milbemycin or ivermectin plus pyrantel pamoate is recommended for prevention and control (hall and simpson, ) . due to transplacental or milkborne infection, puppies should be treated q weeks from to weeks of age. whipworms trichuris vulpis, the canine whipworm, can cause acute or chronic large intestinal diarrhea. the adult worm resides in the cecum or ascending colon. most infections are subclinical, but in symptomatic cases, the typical clinical sign is diarrhea with blood and/or mucus. abdominal pain, anorexia, and weight loss may also be seen. dogs may have eosinophilia, anemia, and/or hypoproteinemia on clinical hematology. trichuris vulpis has a direct life cycle with eggs passed in the feces. the penetration of the adult worm into the enteric mucosa, and the associated inflammation, can lead to diarrhea. factors that influence development of clinical symptoms are the number and location of adult whipworms; the severity of inflammation, anemia, or hypoproteinemia in the host; and the overall condition of the host. whipworm infestation is diagnosed by the presence of barrel-shaped, thickwalled eggs with bipolar plugs on fecal flotation. adult worms intermittently release eggs; therefore, negative results do not exclude infection. differential diagnoses for whipworm infestation include giardiasis, coccidiosis, and bacterial enteritis. fenbendazole, oxibendazole, and milbemycin have all been recommended for treatment of whipworms. treatment for whipworm infestation should be at monthly intervals for months (jergens and willard, ) . several species of cestodes parasitize the small intestine of dogs. the most common is dipylidium caninum. other species include taenia pisiformis and, more rarely, echinococcus granulosus, multiceps spp., mesocestoides spp., and spirometra spp. most cestode infestations are subclinical, but severe infestations with dipylidium can cause diarrhea, weight loss, and poor growth. the cestode requires an intermediate host, which for d. caninum are fleas and lice. ingestion of these arthropods results in transmission of the tapeworm. definitive diagnosis is usually made by the identification of egg capsules or proglottids (tapeworm segments) on the surface of the feces or around the anus. the most significant means to limit cestode infestation is to control flea and/or louse exposure. praziquantel at - . mg/kg orally or subcutaneously is the standard treatment for cestodiasis, especially taenia or echinococcus species. fenbendazole, mebendazole, or oxfendazole may also be effective against d. caninum (hall and simpson, ) . demodicosis canine demodicosis is caused by demodex canis, a commensal mite that lives in the hair follicles and is passed from dams to nursing pups. localized demodicosis is typically asymptomatic, but disease can present with variable and nonspecific clinical signs, such as alopecia, erythema, pruritus, crusts, and hyperpigmentation. it can occur anywhere on the body but is often seen on the feet and face, and around the ears (demanuelle, a). generalized demodicosis can develop in juvenile or adult populations and is indicative of an underlying immunosuppressive disorder. demodex has a characteristic 'cigar shape' and can be identified from deep skin scrapings mounted on mineral oil (campbell, ; noli, ) . differential diagnoses include dermatophytosis, allergic contact dermatitis, and seborrheic dermatitis. the primary differential diagnosis for generalized demodicosis is primary bacterial pyoderma, which is also a common secondary complication of generalized demodicosis. ivermectin at - μg/kg and oral milbemycin at - mg/kg/day are effective treatments. treatment duration can be extensive and must be accompanied by repeated skin scrapings. sarcoptic mange canine sarcoptic mange is caused by sarcoptes scabiei var. canis, which is zoonotic. the most common clinical sign is an intense pruritus, usually beginning at sparsely furred areas of the ear pinnae, elbows, ventral thorax, and abdomen. lesions are characterized by alopecia and yellowish dry crusts with a macular papular eruption. these lesions may be exacerbated by excoriation due to the pruritic nature of the condition. adult mites, mite eggs, or mite feces may be observed on superficial skin scrapings, but diagnosis may be difficult because multiple skin scrapings may yield negative results. even if scrapings are negative, a therapeutic trial should be initiated if the clinical signs and history suggest a sarcoptes etiology. demonstration of anti-mite ige either in the serum or via an intradermal antigen test can be used as a diagnostic aid (campbell, ) . histologic examination is nondiagnostic; however, suggestive lesions include small foci of edema, exocytosis, degeneration, and necrosis . an important differential diagnosis is flea allergy dermatitis (fad). unless antiparasitic therapy would interfere with research objectives, all dogs with sarcoptic mange should be treated. in addition, their kennel mates should also be treated due to the contagious nature of the disease and its zoonotic potential. the usual means of treatment is either ivermectin at - μg/kg q days or milbemycin at mg/kg q days for three oral doses . ticks ticks are obligate arachnid parasites that require vertebrate blood as their sole food source. genera that more commonly infest dogs in the united states include species of rhipicephalus, dermacentor, amblyomma, and ixodes. the primary significance of tick infestation is vector-borne infectious diseases, including rocky mountain spotted fever (rickettsia rickettsii), lyme disease (borrelia burgdorferi sensu stricto), thrombocytic anaplasmosis (anaplasma platys), and canine monocytic ehrlichiosis (ehrlichia canis). ticks alone cause minimal signs unless the dog develops a hypersensitivity reaction leading to a more granulomatous response at the bite location (merchant and taboada, ) . some species (primarily dermacentor andersoni and d. variabilis) produce a salivary neurotoxin that causes an ascending flaccid paralysis (malik and farrow, ) . uncomplicated tick bites and tick-bite paralysis are diagnosed by identification of the tick and clinical signs of paralysis. dogs with tick-bite paralysis usually show improvement within h of tick removal, with complete recovery within h (malik and farrow, ) . formamidines (amitraz), pyrethroids, and phenylpyrazoles (fipronil) are available as spot-ons, collars, sprays, and foggers to treat tick infestations in both the animal and the environment (halos et al., ; beugnet and franc, ) . differential diagnoses for tick-bite paralysis include botulism, snakebite, polyradiculoneuritis, and idiopathic polyneuropathy (malik and farrow, ) . fleas the most common flea to infest dogs is ctenocephalides felis felis, the cat flea (sousa, ) . flea infestations usually cause foci of alopecia and pruritus. dogs that are hypersensitive to antigenic proteins in flea saliva develop severe fad, which features papules, crusting, and excoriations over the lumbosacral region, flanks, thighs and abdomen. these animals may require oral corticosteroids to relieve clinical signs (muller et al., ) . secondary bacterial and fungal infections can also develop. fleas can also transmit other parasitic diseases, such as dipylidium tapeworms. flea infestations and fad are definitively diagnosed by observing the fleas on the host's skin; however, the presence of flea excrement can support a presumptive diagnosis (demanuelle, b) . treatment of flea infestations should use an integrated pest management (ipm) approach that targets adult fleas, immature stages, and environmental contamination in order to limit the risk of chemoresistance. combining ovicidal treatments, such as lufenuron and selamectin, with adulticidal treatments, such as fipronil, spinosad, selamectin, and imidacloprid, is recommended (halos et al., ; beugnet and franc, ; dryden et al., ) . certain chemicals (i.e., imidacloprid and selamectin) have both adulticidal and larvacidal abilities, but the principles of ipm preclude the use of one product solely for both adulticidal and larvacidal properties (schwassman and logas, ). differential diagnoses include mite and louse infestations, bacterial folliculitis, and allergic or atopic conditions that present with skin lesions in dogs. canine dermatophytoses are commonly caused by microsporum spp., trichophyton spp., and epidermophyton spp. (moriello and deboer, ) . uncomplicated infections are characterized by circular areas of alopecia and crusting with or without follicular papules, usually around the face, neck, and forelimbs. dermatophytes infect the hair shaft and follicle, as well as the surrounding skin. infected hairs become brittle and broken shafts remain infective in the environment for months. dermatophytoses are zoonotic and easily transmitted to other animals through the environment or by direct contact. definitive diagnosis is made using dermatophyte test medium for culture. hair and crust material from infected sites can be plucked and placed on culture; however, the 'toothbrush' method is more effective for sampling multiple sites. the brush is used to comb hairs and scales from several infected sites and then pressed into the culture media. media plates should be visually inspected daily for days. positive cultures will become red at the same time as growth of a fluffy white colony. microscopic examination of hairs and scales to visualize fungal elements can be done using skin scrapings in % koh or mineral oil; however, this method is not very sensitive. topical and systemic therapy should be initiated together after all suspected areas are clipped to reduce spreading of contaminated fragile hairs. wholebody topical therapies with antifungal shampoos, rinses, and creams are recommended rather than spot treatment. systemic therapy can be achieved with griseofulvin, ketoconazole, itraconazole, or fluconazole. due to the highly infective nature of this disease, animals should be isolated and the environment thoroughly disinfected. chlorhexidine and virkon ® s are ineffective at clearing environmental spores, but lime sulfur ( : ), enilconazole ( . %), and bleach ( : ) are effective across many strains of microsporum canis (moriello and deboer, ) . although the incidence of hypothyroidism in the canine population is not high (kemppainen and clark, ) , deficiency in thyroid hormone can significantly laboratory animal medicine affect basal metabolism and immune function. because these factors are important in many biomedical research studies, it is imperative that laboratory animal veterinarians be able to recognize, diagnose, and treat this problem. etiology primary hypothyroidism affects the thyroid gland directly, whereas secondary hypothyroidism has indirect effects through dysfunction of the pituitary gland (seguin and brownlee ) . both of these causes result in a gradual loss of functional thyroid tissue (avgeris et al., ; kemppainen and clark, ) . the majority of cases of canine hypothyroidism are due to lymphocytic thyroiditis, an autoimmune disorder, or idiopathic atrophy of the thyroid gland. lymphocytic thyroiditis is the major cause of hypothyroidism in laboratory beagles and appears to be familial in that breed (tucker, ; beierwaltes and nishiyama, ; manning ) . rarely, congenital defects or nonfunctional tumors may cause hypothyroidism (peterson and ferguson, ; kemppainen and clark, ) . clinical signs because it affects metabolism in general, hypothyroidism can produce a large number of clinical signs referable to many organ systems. an individual dog with hypothyroidism may have one or any combination of clinical signs. hypothyroidism reduces the dog's metabolic rate, which then produces such signs as obesity, lethargy, cold intolerance, and constipation. additionally, hypothyroidism can produce several dermatologic abnormalities, including nonpruritic, bilaterally symmetrical alopecia, hyperpigmentation, seborrhea, and pyoderma (avgeris et al., ; peterson and ferguson, ; panciera, ) . several clinicopathologic abnormalities have also been reported in a large percentage of hypothyroid dogs. these aberrations include increased serum cholesterol and triglycerides due to a decrease in lipolysis and decreased numbers of low-density lipopolysaccharide receptors (peterson and ferguson, ; panciera, ) . normocytic, normochromic, nonregenerative anemia may be seen in approximately one-half of the cases (avgeris et al., ) . increased serum alkaline phosphatase and creatine kinase have also been reported in a significant number of hypothyroid dogs (peterson and ferguson, ; panciera ) . neurologic signs of hypothyroidism, which include lameness, foot dragging, and paresis, may be caused by several mechanisms such as segmental nerve demyelination or nerve entrapment secondary to myxedema (peterson and ferguson, ) . mental impairment and dullness have also been reported in hypothyroid dogs, secondary to atherosclerosis and cerebral myxedema (peterson and ferguson, ) . hypothyroidism has been implicated in other neurological abnormalities such as horner's syndrome, facial nerve paralysis, megaesophagus, and laryngeal paralysis; however, these conditions do not always resolve with treatment (bichsel et al., ; panciera, ) , and a true causal relationship with hypothyroidism has not been completely defined (panciera, ) . myopathies associated with hypothyroidism are caused by metabolic dysfunction and atrophy of type ii muscle fibers and can present with signs similar to neurological disease (peterson and ferguson, ) . hypothyroidism can also cause abnormalities of the cardiovascular system including bradycardia, hypocontractility, increased vascular volume, and atherosclerosis (seguin and brownlee ) . abnormalities that may be detected by ecg include a decrease in p-and r-wave amplitude (peterson and ferguson, ) and inverted t waves (panciera, ) . these ecg abnormalities are caused by lowered activity of atpases and calcium channel function. an association between hypothyroidism and von willebrand disease has been suggested. however, the relationship is probably one of shared breed predilection and not a true correlation. contradictory studies have shown either deficient (avgeris et al., ) or normal (panciera and johnson , ; avgeris et al., ) von willebrand factor antigen and bleeding times in hypothyroid dogs. most importantly, hypothyroidism does not appear to cause overt, clinical von willebrand disease. however, it may exacerbate existing subclinical von willebrand disease (seguin and brownlee, ) . epizootiology the prevalence of hypothyroidism in the general canine population is reportedly less than % (panciera, ) . the disorder occurs most often in middle-aged, larger breed dogs (avgeris et al., ) , and reports suggest a higher incidence of hypothyroidism in spayed, female dogs (panciera, ; peterson and ferguson, ) . doberman pinschers and golden retrievers appear to have a higher incidence of hypothyroidism compared with other breeds (panciera, ; peterson and ferguson, ; scarlett, ) . there have been several reports about hypothyroidism in laboratory colonies of beagles (manning, ; tucker, ; beierwaltes and nishiyama, ) . diagnosis and differential diagnosis because of the large number of clinical manifestations in dogs, the recognition of hypothyroidism is not always straightforward. likewise, the diagnosis of hypothyroidism can be difficult because of the lack of definitive diagnostic tests available for the dog. a complete understanding of the diagnosis of hypothyroidism requires a familiarity with thyroid hormone metabolism and function that is beyond the scope of this writing. for additional information, the reader is referred to one of several manuscripts available (peterson and ferguson, ; ferguson, ) . currently, the ability to diagnose hypothyroidism relies heavily on the measurement of serum total t (thyroxine) and free t (peterson and ferguson, ; ferguson, ) . t serves primarily as a precursor for t and is heavily protein bound. free t represents the laboratory animal medicine unbound fraction that is available to the tissues (peterson and ferguson, ) . the measurement of total t carries a sensitivity of around % and can be used as a good screening tool. with the measurement of both serum total t and free t , hypothyroidism can usually be ruled out if the values are within the normal range or higher. if both hormone concentrations are low, it is highly likely that the patient has hypothyroidism, and a therapeutic trial may be in order (peterson and ferguson, ) . however, nonthyroidal illnesses and some drugs (e.g., glucocorticoids, anticonvulsants, phenylbutazone, salicylates) can falsely lower these values (peterson and ferguson, ; ferguson, ) . therefore, low values do not always indicate that hypothyroidism is present and animals should not be treated solely on the basis of serum hormone levels if clinical signs are not present. if the clinical signs are equivocal or only total t or free t is decreased, further diagnostic testing is warranted (peterson and ferguson, ) . although t is the most biologically active form of thyroid hormone, the measurement of serum t levels is an unreliable indicator of hypothyroidism (peterson and ferguson, ; ferguson, ) . serum t can be falsely lowered by many nonthyroidal illnesses and many drugs (see above). in addition, t may be preferentially released and conversion of t to t may be enhanced by the failing thyroid (peterson and ferguson, ; ferguson, ) , particularly early in the disease. in one study, t was within normal limits in % of the hypothyroid dogs (panciera, ) . autoantibodies can be responsible for false elevations in the concentrations of t and t found in these respective assays. it has been recommended that free t , measured by equilibrium dialysis, be assayed in dogs that are suspected of hypothyroidism and have autoantibodies with normal or high t and t . autoantibodies have been found in less than % of the samples submitted to one laboratory (kemppainen and behrend, ) . other means of diagnosing hypothyroidism have been described. in humans, endogenous thyroid-stimulating hormone (tsh) levels provide reliable information on thyroid status, and an assay is available for dogs. however, endogenous tsh levels can be normal in some dogs with hypothyroidism and high tsh levels have been noted in normal dogs and sick animals that are actually euthyroid. it is therefore recommended that tsh levels be considered along with other information (clinical signs, t ) prior to diagnosis and treatment (kemppainen and behrend, ) . tsh stimulation testing using exogenous bovine tsh provides a good and reliable method for establishing a diagnosis. unfortunately, the availability and expense of tsh limit the use of this diagnostic tool (peterson and ferguson, ; ferguson, ) . another drawback of tsh testing is that the test must be postponed for weeks if thyroid supplementation has been given (peterson and ferguson, ) . when tsh is available for testing, there are several recommendations for dosage, routes of administration, and sampling times. one recommendation is . u of tsh per pound of body weight (up to a maximum of u) to be administered iv. for this protocol, blood samples are taken prior to administration of tsh and h after. a normal response to the administration of tsh should create an increase of t levels at least μg/dl above the baseline levels or an absolute level that exceeds μg/dl (peterson and ferguson, ; wheeler et al., ) . treatment the treatment of choice for hypothyroidism in the dog is l-thyroxine (sodium levothyroxine). a recommended dosing regimen is . - . mg/kg once a day (avgeris et al. ). if drugs that decrease thyroxine levels are being administered concurrently, it may be necessary to divide the thyroxine dose for twice daily administration. after the supplementation has begun, the thyroid hormone level should be rechecked in - weeks, and blood samples should be drawn - h after the morning pill. a clinical response is usually seen in - weeks and would include weight loss, hair regrowth, and resolution of other signs (panciera, ) . ecg abnormalities also return to normal (peterson and ferguson, ) . for dogs with neurologic signs, the prognosis is guarded, because the signs do not always resolve with supplementation (panciera, ) . weight gain and eventual obesity are frequent findings in dogs in the research environment. because obesity can adversely affect several body systems as well as general metabolism, the laboratory animal veterinarian must address obesity and its potential effects on animal welfare and research results. etiology obesity is defined as a body weight - % over the ideal. in general, obesity occurs when the intake of calories exceeds the expenditure of energy, the result of overeating or eating an unbalanced diet. overeating is a common cause of obesity in pet dogs and may be triggered by boredom, nervousness, or conditioning (macewen, ) . in addition, pet animals are often subjected to unbalanced diets supplemented with high-fat treats. in the laboratory animal setting, overeating is less likely than in a household because access to food is more restricted, and diets are usually a commercially prepared balanced ration. however, obesity can still be a problem if specific guidelines for energy requirements are not followed. in addition, the necessary caging of dogs in the research environment and limitation to exercise reduces energy expenditure. it is also important to realize that other factors may predispose dogs to obesity, even when guidelines for caloric intake and energy laboratory animal medicine expenditure are followed (butterwick and hawthorne, ) . as in humans, genetics plays an important role in the development of obesity in dogs, and certain breeds are more predisposed toward obesity. in a study of dogs visiting veterinary clinics in the united kingdom, labrador retrievers were most likely to be obese. other breeds affected included cairn terriers, dachshunds, basset hounds, golden retrievers, and cocker spaniels. the beagle was also listed as a breed predisposed to obesity in the household environment (edney and smith, ) . several metabolic or hormonal changes are also associated with obesity. it has been well established that neutering promotes weight gain. in one study, spayed female dogs were twice as likely to be obese compared with intact females (macewen, ) . the authors proposed that the absence of estrogen promotes an increase in food consumption. a similar trend toward obesity was found in castrated male dogs (edney and smith, ). in addition, hypothyroidism and hyperadrenocorticism may present with obesity as one of the clinical signs (macewen, ) . differential diagnosis the diagnosis of obesity is somewhat subjective and relies on an estimate of ideal body weight. the ideal body condition for dogs is considered to be achieved when the ribs are barely visible but easily palpated beneath the skin surface. when the ribs are not easily palpated and/or the dog's normal function is impaired by its weight, the animal is considered obese. there are few objective, quantifiable methods for establishing this diagnosis. ultrasound has been evaluated for measurement of subcutaneous fat in dogs, and measurements taken from the lumbar area can be used to reliably predict the total body fat (wilkinson and mcewan, ) . after a diagnosis of obesity has been made, additional diagnostic tests should be performed to determine if there is an underlying cause for the problem. a complete physical exam should be performed to look for signs of concurrent disease and to establish if obesity has adversely affected the individual. serum thyroid hormones should be evaluated (see above), and serum chemistry may reveal an increased alkaline phosphatase associated with hyperadrenocorticism. treatment restricting food intake readily treats obesity, and this is easily done in the research setting. it has been suggested that a good weight loss program involves restriction of intake to % of the calculated energy requirement to maintain ideal body weight. it has been shown that restriction of calories down to % produces no adverse health effects. however, t levels will decrease in direct proportion with caloric intake. ideally, weight loss will occur at a rate of - % of body weight per week (laflamme et al., ) . with more severe calorie restriction and more rapid weight loss, the individual is more likely to have rebound weight gain after restrictions are relaxed. there has been a great deal of attention in humans as to the correct diet to encourage weight loss. likewise, the type of diet fed to dogs has been examined. as mentioned above, the restriction of calories is most important, and feeding less of an existing diet can do this. alternatively, several diet dog foods are available, and there is some evidence that these diets are superior to simple volume restriction (macewen, ) . there has been much concern about the addition of fiber to the diet as a method for reducing caloric intake while maintaining the volume fed. studies in dogs have examined the addition of both soluble and insoluble fibers to calorie-restricted diets. these studies have shown that the addition of fiber does not have an effect on satiety in dogs and therefore does not have a beneficial effect in weight loss protocols (butterwick et al., ; butterwick and markwell, ) . research complications it is important to control weight gain in research animals because of the association of obesity with metabolism. although an association between obesity and reproductive, dermatologic, and neoplastic problems has been reported (macewen, ) , this relationship is not consistently apparent (edney and smith, ) . joint problems including osteoarthritis and hip dysplasia have also been related to obesity (macewen, ; kealy et al., ) . in addition, diabetes mellitus has been linked to obesity and obesity-induced hyperinsulinism in several experimental models (macewen, ) . a recent study demonstrated metabolic disease, typified by hyperinsulinemia and hypoadiponectinemia, in approximately % of obese dogs (tvarijonaviciute et al., ) . research that requires anesthesia may be complicated by a greater risk of cardiovascular diseases (edney and smith, ) including hypertension and compromise to the respiratory tract. etiology in the laboratory setting, the majority of traumatic wounds will be small in size and quickly observed. occasionally, dogs may sustain minor trauma during transport or have a small, previously undetected, chronic wound upon arrival at the facility. when dogs are group housed, they may sustain bite wounds during early socialization periods. under these conditions, proper initial treatment will lead to uncomplicated wound healing. complications such as infection and delayed healing arise when wounds are not noticed immediately or when the basic principles of wound management are not followed. clinical signs the signs and appearance of a traumatic wound will vary with the cause and the duration of time since wounding. abrasions, sustained by shear forces, are partial thickness skin wounds characterized laboratory animal medicine by minimal bleeding or tissue disruption. puncture wounds have a small surface opening but penetrate into deep tissues with the potential for contamination. lacerations are wounds caused by sharp separation of skin that may extend to deeper tissues. acute wounds are characterized by bleeding tissue, sharp edges and no obvious devitalization. they have variable degrees of contamination. chronic wounds generally do not exhibit active bleeding and will have curled or rounded edges. these wounds often have necrotic tissue and are considered contaminated. treatment to aid decision making about wound therapy, several classification systems have been developed for traumatic injuries. at one time, decisions about wound therapy were largely based upon the length of time since wounding, or the concept of a 'golden period.' it is now recognized that several factors must be considered prior to initiating wound care, including (but not limited to) the type and size of the wound, the degree of wound contamination, and the competence of the host's defense systems (swaim, ; waldron and trevor, ) . one of the most widely used classification systems is based upon wound contamination and categorizes wounds as clean, clean-contaminated, contaminated, or dirty (see table . ). the vast majority of the wounds seen in the laboratory setting will fall into the clean and clean-contaminated categories. these wounds may be treated with the basic wound care described below and primary closure of the wound. contaminated and dirty wounds require more aggressive therapy. postsurgical infections or complications of initial therapy would be considered dirty wounds. when in doubt as to the classification of a wound, the worst category should be presumed in order to provide optimal therapy and reduce the chance for complications. the initial treatment of a wound is the same regardless of its classification. when first recognized, the wound should be covered with a sterile dressing until definitive treatment can be rendered. bleeding should be controlled with direct pressure; tourniquets are discouraged because of the complications that may arise with inappropriate placement (swaim, ) . it is best to avoid using topical disinfectants in the wound until further wound treatment (culture, debridement, lavage) has been performed (swaim, ) . anesthesia or analgesia may be necessary and the choice of agent will depend on the size and location of the wound as well as the preference of the clinician. if the wound is contaminated or dirty, bacterial cultures, both aerobic and anaerobic, should be performed. then a water-soluble lubricant gel may be applied directly to the wound to prevent it from further contamination during the hair removal process. a wide margin of hair should be clipped and a surgical scrub performed around the edges of the wound. povidone-iodine alternating with alcohol or chlorhexidine gluconate scrub alternating with water is most often recommended for surgical preparation of the skin surface (osuna et al., a, b) . simple abrasions that involve only a partial thickness of the skin do not generally require further treatment. full-thickness wounds require further attention, including irrigation with large quantities of a solution delivered under pressure. several irrigation solutions have been recommended (lozier et al., ; waldron and trevor, ; sanchez et al., ) , but type may not be as important as the volume and pressure of delivery. it has been suggested that psi is required to obtain adequate tissue irrigation, and this may be achieved by using a -ml syringe with an -or -gauge needle (waldron and trevor, ) . for wounds that are contaminated or dirty, debridement is an important part of initial therapy. debridement usually proceeds from superficial to deeper layers. skin that is obviously necrotic should be removed. although it is often recommended to remove skin back to the point at which it bleeds, this may not be feasible with large wounds on the limbs. in addition, other factors such as edema or hypovolemia may reduce bleeding in otherwise viable skin (waldron and trevor, ) . if one is unsure about tissue viability in areas that are devoid of waldron and trevor ( ) . extra skin, the tissue may be left (swaim, ; waldron and trevor, ) , and nonviable areas will demarcate within - days (waldron and trevor, ) . necrotic fat should be resected liberally, because it does not have a large blood supply and will provide an environment for infection. often, resection of subcutaneous fat is necessary to remove debris and hair that could not be removed during wound irrigation. damaged muscle should also be liberally resected (swaim, ) . the wound should be irrigated several times during debridement and again after completion. after initial wound treatment, the options concerning wound closure must be weighed. the principles of basic surgery are discussed in several good texts, and readers are encouraged to pursue additional information. primary wound closure is defined as closure at the time of initial wound therapy and is the treatment of choice for clean and clean-contaminated wounds. closure is performed in two or more layers, carefully apposing tissues and obliterating dead space. if dead space will remain in the wound, a drain should be placed. subcutaneous closure should be performed with absorbable suture such as polydioxanone, polyglactin , or polyglycolic acid. it is best to use interrupted sutures and avoid leaving excess suture material in the wound. it may be necessary to choose tension-relieving suture patterns, such as horizontal mattress. skin closure is generally performed with nylon ( - or - ). in situations where gross contamination cannot be completely removed, closure of the wound should be delayed or avoided. after debridement and irrigation, the wound should be bandaged. the wound may be covered by a nonadherent dressing such as vaseline-impregnated gauze (swaim, ) . the contact layer is covered by cotton padding, and the entire bandage is covered by a supportive and protective layer. the bandages should be changed once or twice daily, depending upon the amount of discharge coming from the wound. wound closure within - days of wounding (prior to the formation of granulation tissue) is considered delayed primary closure. when the wound is closed after days, this is considered secondary closure (waldron and trevor, ) . second-intention healing involves allowing the wound to heal without surgical intervention. this type of healing is often used on limbs when there is an insufficient amount of skin to allow complete closure (swaim, ) . it is important to note that second-intention healing will take longer than with surgical repair, and, in the case of large wounds, it will be more expensive because of the cost of bandaging materials. several factors must be weighed when considering the use of antibiotics in traumatic wound care, including the classification and site of the wound, host defenses, and concurrent research use of the animal. when wounds are clean or clean-contaminated, antibiotics are seldom necessary unless the individual is at high risk for infection. when wounds have been severely contaminated or are dirty, antibiotics are indicated and the type of antibiotic will ultimately depend on culture and sensitivity results. until such results are available, the choice of antibiotic is based on the most likely organism to be encountered. topical application of bacitracin, neomycin sulfate, and polymixin b combinations may be used in wounds with minor contamination. in skin wounds with more extensive contamination, staphylococcus spp. are generally of concern, whereas pasteurella multocida should be considered in bite wounds. when systemic antibiotics are necessary, cephalosporins, amoxicillinclavulanate, and trimethoprim sulfas are often recommended for initial antibiotic therapy (waldron and trevor, ) . prevention in facilities with good husbandry practices and a diligent staff, potentially injurious equipment or surfaces are identified quickly. appropriate attention to surgical technique and to initial wound care will generally reduce the occurrence of postprocedure wound infection. etiology pressure sores (decubital ulcers) can be a problem in long-term studies and housing situations that require chronic skin contact with hard surfaces. decubital ulcers often develop over a bony prominence such as the elbow, tuber ischii, tarsus, or carpus. the compression of soft tissues between hard surfaces results in vascular occlusion, ischemia, and ultimately tissue death (swaim and angarano, ) . several factors that increase pressure at the site and/or affect the integrity of the skin will predispose an individual to develop pressure sores, including poor hygiene, self-trauma, low-protein diet, preexisting tissue damage, muscle wasting, inadequate bedding, and ill-fitting coaptation devices (swaim and angarano, ) . clinical signs initially, the skin will appear red and irritated. over time, constant trauma can result in full-thickness skin defects and can progress to necrosis of underlying tissues. the severity of the sores may be graded from i to iv according to the depth of the wound and the tissues involved, from superficial skin irritation to involvement of underlying bone (waldron and trevor, ) . epizootiology the problem usually occurs in large dog breeds, but any type of dog can be affected. prevention and control minimizing or eliminating predisposing factors is important to both the prevention and treatment of this condition. if a dog will experience long periods of recumbency, adequate bedding or padding must be provided. recumbent animals should be moved frequently, ideally every h, to prevent continuous compression on a specific laboratory animal medicine area (waldron and trevor, ) . skin hygiene is of the utmost importance when trying to prevent or treat pressure sores. the skin should be kept clean and dry at all times. if urine scalding is a problem, the affected area should be clipped, bathed, and dried thoroughly at least once or twice daily. finally, an appropriate diet to maintain body weight will minimize compressive forces experienced over areas susceptible to ulceration (swaim and angarano, ) . treatment the treatment of pressure sores must involve care of the wound and attention to the factors causing the wound. the extent of initial wound management will largely depend on the depth of the wound. for simple abrasions and small wounds involving the skin only, simple wound cleansing and openwound management provide adequate treatment. when wounds involve deeper tissues, including fat, fascia, or bone, more aggressive diagnostics and therapy must be performed. the affected area should be radiographed to assess bone involvement and the wound should be cultured. all of the damaged tissue should be debrided and basic wound management guidelines should be followed (see above). when a healthy granulation bed has formed over the entire wound, a delayed closure over a drain may be performed (swaim and angarano, ) . with extensive lesions, reconstruction with skin flaps may be necessary (waldron and trevor ) . bandaging should be performed on all full-thickness wounds; however, it is important to remember that illfitting or inadequately padded bandages or casts may worsen the problem. the area over the wound itself should not be heavily padded. the wounded area should be lightly covered and then a doughnut, created from rolled gauze or towel, should be fitted around the wound, in order to displace pressure over a larger area and onto healthier tissue. the doughnut is then incorporated into a padded bandage. if a cast has been applied to the area for treatment or research purposes, a hole can be cut over the wound to reduce pressure in that area and allow treatment of the wound (swaim and angarano, ) . bandages should be removed at least once or twice a day to allow wound care. etiology an acral lick granuloma is a skin lesion caused by self-trauma. in a few cases, the self-trauma is due to initial irritation caused by an identifiable neurologic or orthopedic condition (tarvin and prata, ) . allergy may also be a source of irritation that leads to self-trauma. however, the majority of cases begin because of repetitive licking by dogs that are confined and lack external stimuli (swaim and angarano, ) . it has been theorized that the self-trauma promotes the release of endogenous endorphins, which act as a reward for the abnormal behavior (dodman et al., ) . the laboratory environment could promote the abnormal behavior and lead to acral lick granuloma. epizootiology the lesions associated with acral lick granuloma are seen most often in large dog breeds, particularly dobermans. however, any type of dog can be affected (walton, ) . clinical signs early lesions appear as irritated, hairless areas usually found on the distal extremities (swaim and angarano, ) . the predilection for the limbs may be due to accessibility or possibly a lower threshold for pruritus in these areas. as the lesions progress, the skin becomes ulcerated and the wound develops a hyperpigmented edge. the wounds may partially heal and then be aggravated again when licking resumes. diagnosis and differential diagnosis acral lick granulomas must be differentiated from several other conditions, including bacterial or fungal infection, foreign bodies, and pressure sores. in addition, mast cell tumors and other forms of neoplasia can mimic the appearance of acral lick granuloma. many of the aforementioned problems can be ruled out by the history of the animal. however, a complete history may be unavailable in the laboratory setting. fungal cultures and allergy testing may aid in diagnosis. biopsy of the affected area would rule out neoplasia. an uncomplicated acral lick granuloma would feature hyperplasia, ulceration, and fibrosis without evidence of infection or neoplasia (walton, ) . prevention and control behavior modification and relief of boredom are important aspects of preventing (and treating) acral lick granuloma. environmental enrichment including exercise, co-housing and various toys is already a basic requirement and may be increased to combat self injurious behaviors. treatment several treatments have been reported for acral lick granuloma and the selection of a treatment should be based on the underlying cause. one of the most important aspects of treatment is to break the cycle of self-trauma. mechanical restraint with an elizabethan collar is one of the easiest methods to accomplish this goal. several direct treatments have been examined, including intralesional and topical steroids, perilesional cobra venom, acupuncture, radiation, and surgery (swaim and angarano, ; walton, ) . opioid antagonists have been applied as treatments for acral lick granulomas and self-injurious behaviors with the theory that this will block the effects of endogenous opioids. naltrexone and nalmefene have been used successfully to reduce excessive licking behaviors and resolve associated lesions. however, lesions did recur after the drugs were discontinued (dodman et al., ; white, ) . the topical administration of a mixture of flunixin meglumine, steroid, and dimethyl sulfoxide has also been shown to be effective (walton, ) . in addition, psychoactive drugs have been suggested to relief of laboratory animal medicine boredom or anxiety. these have included phenobarbital, megestrol acetate, and progestins. however, side effects have been reported (swaim and angarano, ) . other behavior-modifying medications such as clomipramine may be effective in the treatment of compulsive anxiety disorder. the potential for effects that could interfere with experimental results must be determined prior to initiation of treatment. it is important to note that none of the above-mentioned treatments have been successful in all cases. the overall prognosis for acral lick granuloma should be considered guarded since the lesions often recur when treatment is discontinued. etiology hygromas are fluid-filled sacs that develop as a result of repeated trauma or pressure over a bony prominence. the area over the olecranon is most frequently affected, but hygromas have been reported in association with the tuber calcis, greater trochanter, and stifle (newton et al., ) . epizootiology elbow hygromas are most frequently reported in large and giant breeds of dogs, less than years of age (johnston, ; white, ; cannap et al., ) . elbow hygromas are seen infrequently in the laboratory animal setting because the commonly affected breeds are seldom used in research. however, the housing environment of research dogs, especially cage bottoms and cement runs, may predispose them to hygromas. for this reason, laboratory animal veterinary and husbandry staff should be familiar with this condition. clinical signs the clinical presentation will depend upon the chronicity of the problem. a dog with an elbow hygroma usually presents with a painless, fluctuant swelling over the point of the elbow without signs of lameness. the condition may be unilateral or bilateral. over a long period of time, elbow hygromas may become inflamed and ulcerated. if the hygroma becomes secondarily infected, the animal may exhibit pain and fever (johnston, ; white, ) . pathology the fluid-filled cavity in the hygroma is lined by granulation and fibrous tissue. hygromas lack an epithelial lining and therefore are not true cysts. the fluid within the cavity is a yellow or red serous transudate. this fluid is less viscous than joint fluid and elbow hygromas do not communicate with the joint (johnston, ) . treatment the treatment of elbow hygromas should be conservative whenever possible. conservative management of the elbow hygroma is aimed at relieving the source of pressure at the point of the elbow. in early and mild cases, simply providing padding to cover hard surfaces will result in resolution of the hygroma. a soft padded bandage or doughnut bandage around the affected site may also be of benefit. neoprene/polyester sleeves that cover the elbows and fit over the shoulders are also available as an option for either prevention or treatment of hygromas (cannap et al., ) . more aggressive therapies, including needle drainage and injection of corticosteroids into the hygroma, have been described but are not recommended due to the risk of infection (johnston, ) . surgical options should be reserved for complicated or refractory cases. even simple excision can be associated with complications such as wound dehiscence and ulceration (johnston, ) due to the location of the bony prominence at the surgical site. this issue may be avoided by using a skin advancement flap (white, ) that allows intact, healthy skin to cover the boney prominence. a muscle advancement flap has also been described (green et al., ) . regardless of the method used to treat an elbow hygroma, recurrence of the problem is likely unless the predisposing factors are identified and relieved. etiology in the research environment, corneal ulcers are most often associated with direct trauma, contact with irritating chemicals, or exposure to the drying effects of air during long periods of anesthesia. chronic or recurrent corneal ulcers may also be associated with infection or hereditary causes in some breeds of dogs; however, these would be rare in the laboratory setting. clinical signs the signs of corneal ulceration are blepharospasm, epiphora, and photophobia. the eye may appear irritated and inflamed. in minor cases, the cornea may appear normal however, in cases of deeper ulceration, the cornea may appear roughened or have an obvious defect. in addition, the periocular tissues may be swollen and inflamed because of self-inflicted trauma from rubbing at the eye. diagnosis a tentative diagnosis of corneal ulcer or abrasion may be based on the clinical signs. a definitive diagnosis of corneal ulcers is made by the green appearance of the cornea when stained with fluorescein dye. when a corneal ulcer has been diagnosed, the eye should be inspected for underlying causes such as foreign bodies, abnormal eyelids, or abberant cilia. treatment the treatment of corneal ulcers will depend on the depth and size of the affected area, as well as the underlying cause. superficial abrasions are generally treated with topical application of antibiotics. a triple antibiotic ointment that does not contain steroids given three times a day for - days usually provides adequate treatment. simple corneal ulcers are restained with fluorescein after days and should show complete healing at that time. if the ulcer is not healed, this may indicate that the ulcer has an undermined edge impeding proper healing. topical anesthetic should be applied to the eye, and a cotton-tipped applicator can be rolled over the surface of the ulcer toward its edge. this will remove the unattached edge of the cornea and healing should progress normally after debridement. deep ulcers may require further debridement and primary repair. in such cases, a third eyelid or conjunctival flap may be applied to the eye until experienced help can be obtained. in all cases, an elizabethan collar or other restraint may be necessary to prevent additional trauma to the eye. ulcers caused by entropion, ectropion, or dystichiasis will not resolve until the condition is repaired, and descriptions for this can be found elsewhere. prevention the proper application of lubricant eye ointment at the time of anesthesia will prevent drying due to exposure and may also protect the eye from scrub solutions applied near the eye. early treatment of superficial ulcers should prevent self-trauma and progression of the wound. etiology research protocols often require the placement of chronic implants. implants such as cardiac or other biomedical devices may be the primary focus of the research study. implants may also be used as chronic monitoring devices, for delivery of compounds, or to collect serial samples. infection may occur at the time of implant. alternatively, the implant may serve as a nidus after hematogenous spread from other sources. one of the most common sources of infection is from colonization of the device from an external component, which is a frequent complication with indwelling catheters. the actual incidence of complications associated with indwelling vascular catheters in dogs is unknown. one study (hysell and abrams, ) examined the lesions found at necropsy in animals with chronic indwelling catheters, which included traumatic cardiac lesions, visceral infarcts, and fatal hemorrhages. these lesions were primarily associated with catheter-induced trauma or secondary to embolization of fibrin. in a veterinary clinical setting, infections in peripheral catheters were more likely when the catheters were used for blood collection immediately after placement and when a 't' connector rather than a 'y' connector was used. . intestinal access ports have been used to study the pharmacokinetics of drugs at various levels in the intestinal tract. these catheters are usually vascular access ports with several modifications to allow secure placement in bowel (meunier et al., ) . the most frequently reported complication associated with these catheters is infection around the port site (meunier et al., ; kwei et al., ) . clinical signs dogs with implant infections may not exhibit signs initially . localized swelling around the implant may occur. in the case of indwelling catheters, signs may include redness and swelling of the skin around the external port or discharge from the skin wound. vascular access ports may develop fluctuant subcutaneous abscesses. in more severe cases, systemic signs may be noted (bach et al., ; hysell and abrams, ) . the systemic signs of infection are covered elsewhere in this chapter. treatment the treatment of catheter infections almost invariably requires removal of the catheter, as demonstrated in both dogs and monkeys (ringler and peter, ; darif and rush, ). superficial wound irritation or infection may be treated locally with antibiotic ointment, sterile dressing changes, and efforts to minimize catheter movement; however, more extensive problems require aggressive therapy. localized abscesses or sinus tracts may be managed by establishing drainage and copious flushing. aerobic and anaerobic cultures of blood and locally infected sites should be performed prior to initial treatment (ringler and peter, ) . systemic antibiotic therapy should be initiated for a -day period. the choice of drug will ultimately be based on previous experience and culture results. if retention of a catheter is important, the catheter lumen may be safely disinfected with chlorine dioxide solution (dennis et al., ) . the solution is removed after min and replaced with heparinized saline. all of the extension lines and fluids used with an infected catheter should be discarded. the blood cultures should be repeated days after the antibiotic therapy has ceased. if bacteria are still cultured, the catheter must be removed. prevention it is highly desirable to prevent complications that may result in loss of an implanted device. catheters and other implants should be made of nonthrombogenic material and be as simple as possible. a catheter with extra ports or multiple lumens requires additional management and supplies more routes for infection. the initial placement of an indwelling catheter must be done under aseptic conditions by individuals who are familiar with the procedure. intravenous catheters that are used for delivery of drugs or blood sampling should be positioned in the vena cava and not in the right atrium, thereby minimizing trauma to the tricuspid valve. ideally, catheters are secured to reduce movement and irritation of the skin, which may predispose to infection around external ports. the use of vascular access ports that lie entirely under the skin eliminates many problems with infection. it has also been found that long extension tubing connected to the port may actually reduce the potential for infection of the catheter (ringler and peter, ) . for intestinal access ports, catheter security may be improved with a synthetic cuff added to the end of the catheter allowing better attachment to the intestine (meunier et al., ) . after any catheter placement, animals should be observed daily for signs of either local or systemic infection. the catheter entry site should be disinfected, coated with antibiotic ointment, and rebandaged every other day. once a month, the catheter line may be disinfected with chlorine dioxide. in addition, a solution of the antibiotic ceftazidime used on alternate days with the heparin locking solution has been shown to effectively reduce infections in indwelling vascular catheters (bach et al., ) . throughout the life of the catheter, injections into and withdrawals from the catheter should be done in a sterile manner, and the number of breaks in the line should be kept to a minimum. periodically, the placement of an indwelling catheter may be verified by radiography. when placed and managed correctly, catheters and ports of any kind may remain in place for months without complications. etiology sepsis is defined as the systemic response to infection caused by bacteria (gram negative and/or gram positive), fungi, or viruses. in laboratory animals, sepsis is most often seen as a complication of surgical procedures or associated with chronic implants. sepsis may also be seen as a complication of infectious diseases such as parvovirus. clinical signs the signs of sepsis can vary, depending on the source of the infection and the stage of the disease. early in the course, dogs may present with signs of a hyperdynamic sepsis, including increased heart rate, increased respiratory rate, red mucous membranes, and a normal-to-increased capillary refill time. systemic blood pressure and cardiac output will be increased or within the normal range. the animals will often be febrile. later in the course of the syndrome, the animals may show classic signs of septic shock including decreased temperature, pale mucous membranes, and a prolonged capillary refill time. cardiac output and blood pressure are decreased as shock progresses. peripheral edema and mental confusion have also been reported (hauptman and chaudry, ) . pathogenesis the pathophysiology of sepsis is complex and is mediated by immune responses involving mediators such as cytokines, eicosinoids, complement, superoxide radicals, and nitric oxide. the body responds to overwhelming infection with an attempt to optimize metabolic processes and maximize oxygen delivery to tissues. however, if inflammation is left unchecked, the system may be unable to compensate, and the result is cardiovascular collapse. diagnosis in general, a presumptive diagnosis of sepsis is made based on the occurrence of several in a group of signs, including altered body temperature, increased respiratory and/or heart rate, increased or decreased white blood cell (wbc) count, increased number of immature neutrophils, decreased platelet count, decreased blood pressure, hypoxemia, and altered cardiac output. however, extreme inflammation without infection (e.g., pancreatitis, trauma) may create similar signs. one study examined the diagnosis of sepsis in canine patients at a veterinary hospital based on easily obtainable physical and laboratory findings. that study found that septic individuals had higher temperatures, wbc counts, and percentage of band neutrophils than nonseptic individuals, whereas platelet counts were lower in the septic dogs. there were no differences in respiratory rate or glucose levels between the groups. using these criteria, the results had a high sensitivity and a tendency to overdiagnose sepsis (hauptman et al., ) . ultimately, the presence of a septic focus simplifies diagnosis greatly; however, the focus may not be obvious. if the signs of sepsis are evident but the focus is not, several areas should be evaluated for infection, including the urinary, reproductive, respiratory, alimentary, and cardiovascular systems, as well as the abdominal cavity (kirby, ) . treatment the treatment of sepsis has three aims. the first aim is to support the cardiovascular system. all septic animals should be treated with fluids to replace deficits and to maximize cardiac output. crystalloids are most frequently used to maintain vascular volume, primarily because of their low cost. colloids offer the advantage of maintaining volume without fluid overload and may have other positive effects on the cardiovascular system. acid-base and electrolyte imbalances should also be addressed. after the animal has stabilized, the treatment of sepsis should be aimed at removing the septic focus. obvious sources of infection should be drained or surgically removed. if an implant is infected, it should be removed. antibiotic therapy should also be instituted. the choice of antibiotic will ultimately depend upon the results of culture; however, the initial choice of antibiotics is based on previous experience, source of infection, and gram stains. the organisms associated with sepsis are often gram-negative bacteria of gastrointestinal origin or are previously encountered nosocomial infections. ideally, the antibiotic chosen for initial therapy should be a broad-spectrum, bactericidal drug that can be administered intravenously. second-or thirdgeneration cephalosporins provide good coverage, as does combination therapy with enrofloxacin plus metronidazole or penicillin. finally, the treatment of sepsis is aimed at blocking the mediators of the systemic response. this category of sepsis treatment is the focus of much research. several studies have examined the effects of steroids, nonsteroidal anti-inflammatory drugs, and antibodies directed against endotoxin, cytokines, or other mediators of the inflammatory response; however, none of these treatments have proven greatly effective in clinical trials. consequently, there is no 'magic bullet' for the treatment of sepsis at this time. successful therapy remains dependent on aggressive supportive care coupled with identification and elimination of the inciting infection. etiology in research animals, aspiration may occur accidentally during the oral administration of various substances or by the misplacement of gastric tubes. aspiration of gastric contents may also occur as a complication of anesthesia. in pet animals, aspiration is often seen as a result of metabolic and anatomical abnormalities; however, such occurrence would be rare in the research setting. pathogenesis aspirated compounds can produce direct injury to lung tissue, but more importantly, the aspiration provokes an inflammatory response, probably mediated by cytokines. the result is a rapid influx of neutrophils into the lung parenchyma and alveolar spaces. the inflammation leads to increased vascular permeability with leakage of fluid into the alveolar spaces and can eventually lead to alveolar collapse. if the condition is severe, it may result in adult respiratory distress syndrome and respiratory failure. it should be noted that infection is not present in the early stages of this condition but may complicate the problem after - h. clinical signs the severity and clinical manifestation of aspiration lung injury are dependent upon the ph, osmolality, and volume of the aspirate. the signs of aspiration lung injury may include cough, increased respiratory rate, pronounced respiratory effort, and fever. when respiration is severely affected, the oxygen saturation of blood will be decreased. the diagnosis of this problem is based on witness of aspiration, history consistent with aspiration, and/or the physical findings. classically, radiographs of the thorax demonstrate a bronchoalveolar pattern in the cranioventral lung fields. however, these lesions may not appear for several hours after the incident of aspiration. in addition, the location of the lesions may be variable, depending on the orientation of the animal at the time of aspiration. treatment the treatment of aspiration lung injury is largely supportive and depends upon the severity of the inflammation and the clinical signs. if the aspiration is witnessed, the mouth and, idealy, the upper airway should be cleared of residual material. when small amounts of a relatively innocuous substance (e.g., barium) have been aspirated, treatment may not be necessary. when severe inflammation is present, systemic as well as localized therapy may be necessary. oxygen therapy may be instituted; however, the concentration and time frame are controversial, because lung injury may be exacerbated by long-term administration of oxygen at high concentrations (nader-djahal et al., ) . fluid therapy may also be necessary in severe cases; however, cardiovascular support should be performed judiciously as fluid overload could lead to an increase in pulmonary edema. the use of colloids is also controversial because of the increase in vascular permeability that occurs in the lungs. several studies have addressed the use of anti-inflammatory agents to reduce lung injury associated with aspiration; however, none are used clinically in human or veterinary medicine at this time. corticosteroids are contraindicated (raghavendran et al., ) . in humans, antibiotics are reserved for cases with confirmed infection, in order to prevent the development of antibiotic-resistant pneumonia. it has been suggested that dogs should be immediately treated with antibiotics when the aspirated material is not acidic or has potentially been contaminated by oral bacteria associated with severe dental disease. amoxicillin-clavulanate has been recommended as a first line of defense, reserving enrofloxacin for resistant cases (hawkins, ) . the presence of pneumonia should be verified by tracheal wash and cultures. prevention aspiration of drugs and other compounds may be avoided through careful administration of oral medications by experienced individuals. likewise, gavage or orogastric administration of liquids should be performed by experienced individuals, and the procedure should be aborted if coughing or other respiratory signs occur. the aspiration of stomach contents can largely be avoided by appropriate fasting prior to anesthesia for at least h for food and h for water. if appropriate fasting times are not observed, anesthesia should be postponed whenever possible, particularly if intended procedures require manipulation of the viscera or head-down positioning of the dog. if anesthesia cannot be avoided, it should be rapidly induced and the dog should be intubated. during recovery from anesthesia, the endotracheal tube should be removed with the cuff partially inflated and with the dog in a head-up position (haskins, ) . based on the source of energy, burn wounds may be categorized into four groups: thermal, chemical, radiation, and electrical. in laboratory animals, accidental burns are usually the result of thermal injury (heating pads, water bottles), chemicals (strong alkalis, acids, disinfectants, drugs), or experimental irradiation protocols. etiology inappropriate use of external heating devices is the most common cause of burns in laboratory animal medicine. the insult to the skin results in desiccation of the tissue and coagulation of proteins. in addition, the severely injured area is surrounded by a zone of vascular stasis, which promotes additional tissue damage. even small burns can result in significant inflammation that could affect the outcome of some research investigations and cause considerable discomfort to the animal. the proper and immediate treatment of burn wounds can reduce the effects of the injury on both the individual and the research. clinical signs the clinical signs vary with the depth, location, and surface area of burn injury. classification systems for thermal burns are generally based on the depth of the injury, varying from superficial involvement of only epidermis to complete destruction of skin and subcutaneous tissues (bohling, ) . superficial burns appear erythematous and inflamed. in some cases, matting of the overlaying hair with exudate may be the first sign of a previously undetected skin lesion. progressive hair and skin loss may be evident over the first few days after injury (johnston, ) . although blistering is a characteristic of partial thickness burns in humans, this is rarely seen in dogs (bohling, ) . uncomplicated, superficial burn wounds heal by reepithelialization within - days. deeper burn wounds are characterized by a central area of nonviable tissue surrounded by edematous, inflamed tissues. a thick eschar, composed of the coagulated proteins and desiccated tissue fluid, develops over deep burn wounds. these wounds heal by granulation under the eschar, which will eventually slough. the amount of pain associated with burns depends upon several factors including the depth and area of the wound, procedural manipulations, and movement at the affected site (bohling, ) . pain associated with superficial burn wounds usually subsides in - days. theoretically, deep burns destroy nerve endings and result in less pain than superficial burns. however, inflammatory pain may still be present due to the tissue reaction around the necrotic site. in addition, sharp procedural pain and breakthrough pain have been described in humans during the healing phases of burn injuries and should be considered as potential complications in dogs as well (bohling, ) . severe and widespread accidental burn injury can result in clinical signs associated with multiple organs including the pulmonary, gastrointestinal, hematopoietic, and immune systems. in addition, extensive burn injury can predispose to infection and even sepsis. this type of injury with the associated complications would be extremely rare in the laboratory setting. treatment appropriate and timely treatment of a burn wound will reduce the extent of tissue damage and associated pain. thermal injuries should be immediately exposed to cool water ( °c) to reduce edema and pain. exposure to very cold water and ice does not improve outcomes (bohling, ) . topical wound dressings are recommended in the early stages of treatment for both partial-and full-thickness burns that are of small size. systemic antibiotics are unable to penetrate eschar and are not adequately distributed through the abnormal blood supply of burned tissues. therefore, a thin film of a water-soluble, broad-spectrum antibiotic ointment should be applied to the wound surface. silver sulfadiazine has a broad spectrum, penetrates eschar, and is often the preparation of choice for burn wound therapy. povidone-iodine ointment will also penetrate thin eschar and provides a broad spectrum. mafenide has a good spectrum that covers gram-negative organisms well and is often used to treat infected wounds, although it has been associated with pain upon application (demling and lalonde, ) . once a topical antibiotic has been applied, a nonadherent dressing should be placed on the wound. burn wounds covered in such a manner tend to epithelialize more rapidly and are less painful than uncovered wounds (demling and lalonde, ; bohling, ) . after the initial treatment, burn wounds should be gently cleansed two to three times a day, followed by reapplication of the topical antibiotic and rebandaging (demling and lalonde, ) . systemic antibiotics are indicated in cases where local or systemic infection is present and their ultimate selection should be based on culture results. burn wounds can be extremely painful, and analgesia should be instituted immediately and adjusted accordingly throughout the treatment period. surgical intervention may be necessary in some cases. with small or moderately sized wounds, the eschar over the burn wound may actually impede wound contraction and reepithelialization. in such cases, once the eschar has become fully defined, a complete resection may improve wound healing. with large and severe burn wounds, repeated debridement by surgery or other means might be necessary. in the laboratory setting, a decision to pursue extensive surgical intervention would be dependent upon full consideration of the effects on animal welfare and research results. prevention thermal burns can be prevented in the research setting. electric heating pads and heat lamps should be avoided if possible. only heated water blankets or circulating warm air devices should be used to provide warmth to the animals. in rare instances, heated water blankets have also caused burns; therefore, these devices should be carefully monitored. as a precaution, a thin towel may be placed between the animal and the water blanket. basic fire prevention precautions should be taken particularly around oxygen sources and flammable agents etiology chemical injury may be due to skin contact with concentrated solutions such as disinfectants or inadvertent exposure to laboratory chemicals. in addition, perivascular injection of certain drugs (pentobarbital, thiamylal, thiopental, thiacetarsemide, vincristine, vinblastine, and doxorubicin) have been associated with extensive tissue damage (swaim and angarano, ; waldron and trevor, ) . the mechanism of action will vary depending upon the ph, osmolality, and chemical composition of the agent and may include oxidation, reduction, disruption of lipid membranes, or other reactions (bohling, ; swaim, ; waldron and trevor, ) . clinical signs surface contact with chemicals may result in mild irritation and redness of superficial layers of the skin. however, many agents may cause progressive injury until the chemical reaction has been neutralized. this may result in tissue necrosis and secondary infection. the immediate signs of perivascular injection are withdrawal of the limb or other signs of discomfort and swelling at the injection site. the area may appear red, swollen, and painful as inflammation progresses. there may eventually be necrosis of the skin around the injection site. in cases of doxorubicin extravasation, signs may develop up to a week after the injection, and the affected area may progressively enlarge over a -to -month period. this is because the drug is released over time from the dying cells (swaim and angarano, ) . treatment in cases of skin contact with chemical agents, the affected area should be thoroughly and repeatedly lavaged with warm water to dilute or remove the substance. the material safety data sheet for the substance should be consulted for any possible neutralization protocol. additional treatment will depend upon the severity of the tissue damage and will follow the same guidelines as for the thermal injury described earlier. for the treatment of perivascular injections, dilution of the drug with subcutaneous injections of saline is recommended. in addition, steroids may be infiltrated locally to reduce inflammation. topical application of dimethyl sulfoxide (dmso) may also be helpful in reducing the immediate inflammation and avoiding the development of chronic lesions. the addition of lidocaine to subcutaneous injections of saline has been used in cases of thiacetarsemide injection (hoskins, ) . the local infiltration of hyaluronidase accompanied by warm compresses has been suggested for perivascular vinblastine (waldron and trevor, ) and for doxirubicin. the use of dmso or another free radical scavenger, dexrazoxane, infused at the site has also been suggested for doxorubicin toxicities. despite these treatments, necrosis of skin may be observed and would require serial debridement of tissues with secondary wound closure or skin grafting. in cases of doxorubicin extravasation, early excision of affected tissues is advocated to prevent the progressive sloughing caused by sustained release of the drug from dying tissues (swaim and angarano, ) . in all cases, the condition can be painful and analgesia should be addressed. prevention prior to the use of any substance, the investigator should be aware of its chemical composition and the potential for problems. the material safety data sheets should be available for all compounds and storage recommendations followed closely. for intravenous administration of toxic compounds, insertion of an indwelling catheter is extremely important. prior to the injection, the catheter should be checked repeatedly for patency by withdrawal of blood and injection of saline. any swelling at the catheter site or discomfort by the subject indicates that the catheter should not be used. access to a central vessel such as the cranial or caudal vena cava is preferred over the use of peripheral vessels. when peripheral catheters are used, the injection should be followed by a vigorous amount of flushing with saline or other physiological solution and removal of the catheter. additional injections are best given through newly placed catheters in previously unused vessels. the repeated use of an indwelling peripheral catheter should be approached cautiously and done only out of necessity. etiology radiation burns are generally a complication of therapeutic administration and are a result of free oxygen radical formation (waldron, ) . the severity of radiation burns and their treatment will depend upon the dose, frequency, total surface area, and location of the radiation. damage to epithelial layers of the skin can lead to desquamation. direct injury to fibroblasts results in decreased collagen production and poor wound healing. in addition, there may be fibrosis of blood vessels (pavletic, ) and subsequent hypoxia causing necrosis of deeper tissues. clinical signs the tissues most often affected are the skin and mucus membranes. with superficial injury, affected skin may exhibit hair loss and erythema, and produce a clear exudate. the intensity of the inflammation may increase for - weeks after the completion of radiation treatment. deeper and more serious injury manifests with subcutaneous fibrosis and can lead to disfigurement . the skin and underlying deep structures including the bone may become necrotic over several weeks (pavletic, ) . these deeper injuries are prone to infection due to their lack of blood supply. systemic signs such as vomiting are rare in dogs unless there has been direct radiation treatment to organs . treatment with superficial skin burns, the wound should be kept clean and should be covered if possible. in cases of oral mucous membrane damage, there may be special feeding requirements. when wounds are ulcerated, avascular tissues should be excised. treatments with silver sulfadiazine, mafenide acetate, or other topical agents are recommended to control infection. in addition, infection is avoided by closure of the wound as soon as possible. the goal of surgery is to cover the wound with healthy tissue to promote vascularization of the area. in some cases, this may require muscle and/ or skin grafts. prevention radiation burns can be limited by selection of appropriate, fractionated therapy and application of shielding to reduce exposure. prompt treatment of the injuries can reduce the occurrence of infection. since radiation is associated with poor wound healing, complications may arise when additional procedures are required. it is recommended to wait at least week (pavletic, ) or even longer (laing, ) prior to administering radiation to a surgical site. after radiation, routine surgeries should be avoided for - months (pavletic, ). the prevalence of cancer in the general canine population has increased over the years (dorn, ) . this can be attributed to the longer life spans resulting from improvements in nutrition, disease control, and therapeutic medicine. because of these changes, cancer has become a major cause of death in dogs (bronson, ) . in a lifetime cancer mortality study of intact beagles of both sexes, albert et al. ( ) found death rates similar to the death rate of the at-large dog population (bronson, ) . approximately % of the male beagles died of cancer. the majority of the tumors were lymphomas ( %) and sarcomas ( %), including hemangiosarcomas of the skin and fibrosarcomas. of the female beagles dying of cancer ( % of the population studied), three-quarters had mammary cancer ( %), lymphomas ( %), or sarcomas ( %). of the sarcomas in females, one-third were mast cell tumors. in addition to these tumors that cause mortality, the beagle is also at risk for thyroid neoplasia (hayes and fraumeni, ; benjamin et al., ) . because of the popularity of the beagle as a laboratory animal, discussion of specific neoplasms will focus on the tumors for which this breed is at risk, as well as tumors that are common in the general canine population. a complete review of clinical oncology in the dog is beyond the scope of this chapter but can be found elsewhere (withrow et al., ) . fine-needle aspirates are generally the first diagnostic option for palpable masses, because they can easily be performed in awake, cooperative patients. this technique allows for rapid differentiation of benign and neoplastic processes. in cases where cytologic results from fine-needle aspirates are not definitive, more invasive techniques must be used. needle-punch or core biopsies can also be performed in awake patients with local anesthesia. an instrument such as a tru-cut ® needle (travenol laboratories, inc., deerfield, illinois) is used to obtain a -mm × - . -cm biopsy of a solid mass. a definitive diagnosis may be limited by the size of the sample acquired using this technique. incisional and excisional biopsies are utilized when less invasive techniques fail to yield diagnostic results. excisional biopsies aid in histopathological examination and are the treatment of choice when surgery is necessary, because the entire mass is removed. surgical margins should extend at least cm around the tumor and cm if mast cell tumors are suspected (morrison et al., ) . incisional biopsies are performed when large softtissue tumors are encountered and/or when complete excision would be surgically difficult or life-threatening. when performing an incisional biopsy, always select tissue from the margin of the lesion and include normal tissue in the submission. etiology lymphomas are a diverse group of neoplasms that originate from lymphoreticular cells. canine lymphoma represents - % of canine tumors and a majority ( %) of canine hematopoetic disease (ettinger, ; vail and young, ) . whereas retroviral etiologies have been demonstrated in a number of species (e.g., cat, mouse, chicken), conclusive evidence of a viral etiology has not been established in the dog. in humans, data implicate the herbicide , -dichlorophenoxyacetic acid as a cause of non-hodgkin's lymphoma, but studies in dogs with similar conclusions have come under scrutiny (macewen and young, ) . in addition, tobacco smoke, environmental chemicals, and waste emissions are considered possible risk factors (marconato et al., ; gavazza et al., ) clinical signs multicentric high-grade lymphoma (mhgl) accounts for the majority of reported cases of canine lymphoma. depending upon grade, immunophenotype, and location involved, dogs with mhgl usually present with painless, enlarged lymph nodes and nonspecific signs such as anorexia, weight loss, polyuria, polydypsia, fever, and lethargy. when the liver and spleen are involved, generalized organomegaly may be felt on abdominal palpation. less commonly, dogs develop alimentary, mediastinal, cutaneous, and extranodal lymphomas. alimentary lymphoma is associated with vomiting and diarrhea, in addition to clinical signs associated with mhgl. dogs with mediastinal lymphoma often present with respiratory signs (dyspnea and exercise intolerance) secondary to pleural effusion or cranial vena caval syndrome. hypercalcemia is most frequently associated with this form of lymphoma and may result in polyuria, polydypsia, and weakness. cutaneous lymphoma is an uncommon epitheliotrophic form of lymphoma. it is often referred to as mycosis fungoides and is typically of a cd + t-cell immunophenotype. it varies in presentation from solitary to generalized and may mimic any of a number of other inflammatory skin disorders including oral mucosal lesions. the lesions may occur as erythema, plaques, erosions, scales, nodules, crusts, hypopigmentation, and alopecia (fontaine et al., ) . approximately half of the cases are pruritic. a number of extranodal forms of lymphoma have been reported, including tumors affecting the eyes, central nervous system, kidneys, or nasal cavity. clinical presentation varies, depending on the site of involvement (e.g., nervous system: seizures, paresis, paralysis). epizootiology the incidence of lymphoma is highest in dogs - years old, accounting for % of cases. although the neoplasm generally affects dogs older than year, cases in puppies as young as months have been reported (dorn et al., ) . no gender predilection has been reported. diagnosis and pathologic findings a fine-needle aspirate is initially performed on accessible lymph nodes. thoracic radiographs and abdominal ultrasound ± fine needle aspiration of the liver or spleen can be used if mediastinal or abdominal involvement is suspected. additional staging can be determined through complete blood counts, serum biochemistry, flow cytometry for immunotyping, bone marrow aspiration, or surgical lymphadenectomy and histology. enlarged neoplastic lymph nodes vary in diameter from to cm and are moderately firm. some may have areas of central necrosis and are soft to partially liquefied. the demarcation between cortex and medulla is generally lost, and on cut section, the surface is homogenous. the spleen may have multiple small nodular masses or diffuse involvement with generalized enlargement. the enlarged liver may have disseminated pale foci or multiple large, pale nodules. in the gastrointestinal tract, both nodular and diffuse growths are observed. these masses may invade through the stomach and intestinal walls. flow cytometry and lymphoblastic markers (cd ) can aid in diagnosis and subtyping of tumors. in addition, positron emission tomography is being explored for detection of extranodal and metastatic lymphoma (leblanc et al., ; marconato, ; elstrom et al., ) . classification of lymphoma types is based upon cytological, morphological, and immunological characteristics using the kiel classification criteria (vail and young, ) . histologically, the most common lymphomas are classified as intermediate to high grade and of large-cell (histiocytic) origin. the neoplastic lymphocytes typically obliterate the normal architecture of the lymph nodes and may involve the capsule and perinodal areas. lymphoma subtypes can be further characterized based upon genetic, molecular, and immunological criteria (ponce et al., ) . pathogenesis all lymphomas regardless of location should be considered malignant. a system for staging lymphoma has been established by the world health organization. the average survival time for dogs without treatment is - weeks. survival of animals undergoing chemotherapy is dependent on the treatment regimen as well as the form and stage of lymphoma (macewen and young, ) . median survival time with aggressive therapy is generally less than months. hypercalcemia is a paraneoplastic syndrome frequently associated with lymphoma. the pathogenesis of this phenomenon is not fully understood but may be a result of a parathormone-like substance produced by the neoplastic lymphocytes. differential diagnosis differential diagnoses for multicentric lymphoma include systemic mycosis; salmon-poisoning and other rickettsial infections; lymph node hyperplasia from viral, bacterial, and/or immunologic causes; and dermatopathic lymphadenopathy. alimentary lymphoma must be distinguished from other gastrointestinal tumors, foreign bodies, and lymphocytic-plasmacytic enteritis. in order to make a definitive diagnosis, whole lymph node biopsies and full-thickness intestinal sections for histopathologic examination may be needed. treatment therapy for lymphoma primarily consists of one or a combination of several chemotherapeutic agents. in addition, radiation therapy and bone marrow transplantation have been utilized. the treatment regimen is based on the staging of the disease, the presence of paraneoplastic syndromes, and the overall condition of the patient. although treatment may induce clinical remission and prolong short-term survival, most treatment is palliative and aimed at improving quality of life. a thorough discussion of therapeutic options for the treatment of lymphomas in the dog can be found elsewhere (chun, ; marconato, ) . future directions include development of molecular and cellular targeted therapies to enhance traditional chemotherapy treatment, prolong remission, and treat immunologic subtypes of lymphoma (e.g., t-cell lymphoma). research complications given the grave prognosis for lymphoma with or without treatment, euthanasia should be considered for research animals with significant clinical illness. etiology mast cells are derived from cd + bone marrow progenitor cells. neoplastic proliferations of mast cells are the most commonly observed skin tumor of the dog and may account for up to % of canine skin laboratory animal medicine tumors (bostock, ; welle et al., ) . mast cells are normally found in the connective tissue beneath serous surfaces and mucous membranes, and within the skin, lung, liver, and gastrointestinal tract. current research has linked mast cell tumor development to multifactorial causes including breed predisposition and a genetic component, chronic inflammation, and mutations in the surface growth factor, c-kit (ma et al., ; reguera et al., ; webster et al., ) . clinical signs well-differentiated mast cell tumors are typically solitary, well-circumscribed, slow-growing, -to -cm nodules in the dermis and subcutaneous tissue. alopecia may be observed, but ulceration is not usual. poorly differentiated tumors grow rapidly, may ulcerate, and may cause irritation, inflammation, and edema to surrounding tissues. mast cell tumors can be found on any portion of the dog's skin but frequently affect the trunk and hind limb extremeties along with perineal and preputial areas. the tumors usually appear to be discrete masses, but they frequently extend deep into surrounding tissues. abdominal organs are rarely involved but may be associated with anorexia, vomiting, melena, abdominal pain, and gastrointestinal ulceration. mast cell tumors have also been reported in extracutaneous areas such as the salivary glands, larynx, nasopharynx (london and thamm, ) and conjunctiva (fife et al., ) . mast cell tumors within the perineal, preputial, or inguinal areas are associated with a greater predilection for recurrence or metastasis (misdorp, ) . epizootiology these tumors tend to affect middleaged dogs but have been observed in dogs ranging from months to years (pulley and stannard, ) . pathologic findings because of the substantial variation in histologic appearance of mast cell tumors, a classification and grading system described by patnaik et al. ( ) has become widely accepted. in this system, grade i has the best prognosis and are well differentiated, with round to ovoid, uniform cells with distinct cell borders. the nuclei are round and regular, the cytoplasm is packed with large granules that stain deeply, and mitotic figures are rare to absent. grade ii (intermediately differentiated) mast cell tumors have indistinct cytoplasmic boundaries with higher nuclear-cytoplasmic ratios, fewer granules, and occasional mitotic figures. grade iii (anaplastic or undifferentiated) mast cell tumors have the worst prognosis. the cells contain large, irregular nuclei with multiple prominent nucleoli and few cytoplasmic granules. mitotic figures are much more frequent. cells are pleomorphic with indistinct borders. in addition to associated skin lesions (e.g., ulceration, collagenolysis, necrosis, and infection), mast cell tumors have been associated with gastric ulcers in the fundus, pylorus, and/or proximal duodenum, most likely secondary to tumor production of histamine. histamine stimulates the h receptors of the gastric parietal cells, causing increased acid secretion. gastric ulcers have been observed in large numbers (> %) of dogs with mast cell tumors (howard et al., ) . pathogenesis although all mast cell tumors should be considered potentially malignant, the outcome in individual cases can be correlated with the histologic grading of the tumor. grade iii tumors are most likely to disseminate internally. this spread is usually to regional lymph nodes, spleen, and liver, and less frequently to the kidneys, lungs, and heart. diagnosis and differential diagnosis using fineneedle aspiration, mast cell tumors can be distinguished cytologically from other round cell tumors (such as histiocytomas and cutaneous lymphomas) by using toluidine blue to metachromatically stain the cytoplasmic granules red or purple. mast cell granules can also be stained with wright's, giemsa, and romanowsky stains. in addition, mast cells may contain tryptase, chymase, or both (fernandez et al., ) . histological evaluation is generally required for grading. examination of regional lymph nodes may be warranted if metastatic or systemic disease is suspected. in addition, radiographs and ultrasound with guided aspirates of the liver, spleen, or sublumbar lymph nodes can be used to determine metastatic disease. treatment depending upon the grade, initial treatment for mast cell tumors is generally wide surgical excision ( -cm margins), which may be followed by radiation, chemotherapy, or glucocorticoid therapy. aspiration or surgical removal of regional lymph nodes is recommended if lymphatic tumor drainage is suspected. if the tumor is not completely resectable or is grade ii or iii (moderately to undifferentiated), then debulking surgery and adjunct therapy may be used. treatment algorithms are outlined elsewhere (withrow et al., ) research complications because of the potential for systemic release of substances such as histamine, vasoactive substances, heparin, eosiniphilic chemotactic factor, and proteolytic enzymes, along with the possibility of delayed wound healing and tumor recurrence, dogs with mast cell tumors are not good candidates for research studies. grade i mast cell tumors may be excised, allowing dogs to continue on study; however, monitoring for local recurrence should be performed monthly. grade ii tumors are variable; animals that undergo treatment should be monitored for recurrence monthly, and evaluation of the buffy coat should be performed every - months for detection of systemic mastocytosis. because of the poor prognosis for grade iii tumors, treatment is unwarranted in the research setting. etiology also known as infectious or venereal granuloma, sticker tumor, transmissible sarcoma, and contagious venereal tumor, the canine transmissible venereal tumor (ctvt) is transmitted horizontally to the genitals by coitus (nielsen and kennedy, ) . ctvt is a 'parasitic-like' tumor that appears to have originated from dogs or wolves thousands of years ago and despite immense mutation, ctvt adapted, survived, and spread across multiple continents making it the oldest known continuously passaged somatic cell line (rebbeck et al., ; murchison et al., ; murgia et al., ) . it has been described as a round cell tumor of histiocytic origin. although this tumor has been reported in most parts of the world, it is most prevalent in tropical or temperate climates (macewen, ) . clinical signs the tumors are usually cauliflowerlike masses on the external genitalia, but they can also be pedunculated, nodular, papillary, or multilobulated. these friable masses vary in size up to cm, and hemorrhage is frequently observed. in male dogs, the lesions are found on the caudal part of the penis from the crura to the bulbus glandis or on the glans penis. less frequently, the tumor is found on the prepuce. females typically have lesions in the posterior vagina at the junction of the vestibule and vagina. when located around the urethral orifice, the mass may protrude from the vulva. these tumors have also been reported in the oral cavity, skin, and eyes. epizootiology and transmission ctvts are most commonly observed in young, sexually active dogs. transmission takes place during coitus when injury to the genitalia allows for exfoliation and transplantation of the tumor. genital to oral to genital transmission has also been documented (nielsen and kennedy, ) . extragenital lesions may be the result of oral contact with previously traumatized areas. pathologic findings histologically, cells are arranged in compact masses or sheets. the cells are round, ovoid, or polyhedral, and have large, round nuclei with coarse chromatin. the cytoplasm is eosinophilic with small vacuoles arranged in a 'string of pearls' pattern. pathogenesis tumor growth occurs within - months after mating or implantation, and then growth generally slows. metastasis is rare (< - % of cases) but may involve the superficial inguinal and external iliac lymph nodes as well as distant sites. spontaneous regression may occur within - months of tumor development. diagnosis and differential diagnosis transmissible venereal tumors have been confused with lymphomas, histiocytomas, mast cell tumors, and amelanotic melanomas. however, cytological examination of impression smears, swabs, and fine-needle aspirates generally provide a definitive diagnosis. although not usually required, histopathology of a biopsy from the mass can aid in diagnosis. prevention thorough physical examinations prior to bringing new animals into a breeding program should prevent introduction of this tumor into a colony. control removing affected individuals from a breeding program should stop further spread through the colony. treatment surgery and radiation can be used for treatment, but chemotherapy is the most effective. vincristine ( . - . mg/m ) iv once weekly for four to six treatments will induce remission and cure in greater than % of the cases (macewen, ) . research complications experimental implantation of ctvts has been shown to elicit formation of tumor-specific igg (cohen, ) . this response may occur in natural infections and could possibly interfere with immunologic studies. etiology dogs are susceptible to a wide variety of mammary gland neoplasms, most of which are influenced by circulating reproductive steroidal hormones. clinical signs single nodules are found in approximately % of the cases of canine mammary tumors. the nodules can be found in the glandular tissue or associated with the nipple. masses in the two most caudal glands (fourth and fifth) account for a majority of the tumors. benign tumors tend to be small, well circumscribed, and firm, whereas malignant tumors are larger, invasive, and coalescent with adjacent tissues. inflammatory mammary carcinomas may mimic mastitis or severe dermatitis and must be ruled out to prevent misdiagnosis. epizootiology mammary tumors are uncommon in dogs under years of age with the incidence rising sharply after that. the median age at diagnosis is - years. a longitudinal study of a large beagle colony showed that significant risk for development of mammary tumors begins at approximately years of age (taylor et al., ) . mammary tumors occur almost exclusively in female dogs, with most reports in male dogs being associated with endocrine abnormalities, such as estrogen-secreting sertoli cell tumors. pathologic findings the t (tumor size), n (lymph node involvement), and m (metastasis) system is commonly used to stage mammary tumors. based on histologic classification of mammary gland tumors, approximately half of the reported tumors are benign (fibroadenomas, simple adenomas, and benign mesenchymal tumors), and half are malignant (solid carcinomas, tubular adenocarcinomas, papillary adenocarcinomas, anaplastic carcinomas, sarcomas, and carcinosarcomas) (bostock, ) . histopathologic grades are scored based upon tubule formation, nuclear pleomorphism, and mitosis (elston and ellis, ) . extensive discussions of classification, staging, and histopathologic correlations can be found elsewhere (moulton, ; sorenmo et al., ) . pathogenesis mammary tumors of the dog develop under the influence of hormones. receptors for both estrogen and progesterone can be found in - % of tumors. malignant mammary tumors typically spread through the lymphatic vessels. metastasis from the first, second, and third mammary glands is to the ipsilateral axillary or anterior sternal lymph nodes. the fourth and fifth mammary glands drain to the superficial inguinal lymph nodes where metastasis can be found. many mammary carcinomas will eventually metastasize to the lungs and extraskeleton. diagnosis and differential diagnosis both benign and malignant mammary tumors must be distinguished from mammary hyperplasia, mastitis, and severe dermatitis. cytological evaluation from fine-needle aspirates correlates well with histological examination of benign and malignant tumors (simon et al., ) . radiographs and possibly ultrasound should be performed to rule out metastatic disease prior to surgery. prevention the lifetime risk of developing mammary tumors can effectively be reduced to . % by spaying bitches prior to the first estrus (schneider et al., ) . this is commonly done in the general pet population at months of age. the protective effects of early spay rapidly decrease after several estrus cycles. dogs spayed prior to the first estrus had a risk of . %, whereas dogs spayed after the first and second estrus had risks of % and %, respectively. treatment surgery is the treatment of choice for mammary tumors, because chemotherapy and radiation therapy have not been reported to be effective. the extent of the surgery is dependent on the area involved. single mammary tumors should be surgically removed with -cm lateral margins or margins wide enough for complete resection. deep margins may include removing sections of abdominal fascia or musculature en bloc with mammary tumor. multiple mammary tumors should be removed via regional or unilateral chain mastectomies. bilateral, staged mastectomies are reserved for more aggressive tumors. there is insufficient evidence at this time to recommend routine complete unilateral or bilateral chain mastectomies. at the time of surgery, axillary lymph nodes are removed only if enlarged or positive on cytology for metastasis. sorenmo et al. ( ) provide a thorough review of canine mammary gland neoplasia. research complications treatment of early-stage or low-grade mammary tumors may be rewarding, allowing dogs to continue on study. if removed early enough, malignant masses could yield the same results. all dogs should be monitored regularly for recurrence and new mammary tumors. beagles are subject to many of the inherited and/ or congenital disorders that affect dogs in general. in a reference table on the congenital defects of dogs (hoskins, ) , disorders for which beagles are specifically mentioned include brachyury (short tail), spina bifida, pulmonic stenosis, cleft palate-cleft lip complex, deafness, cataracts, glaucoma, microphthalmos, optic nerve hypoplasia, retinal dysplasia, tapetal hypoplasia, factor vii deficiency, pyruvate kinase deficiency, pancreatic hypoplasia, epilepsy, gm gangliosidosis, globoid cell leukodystrophy, xx sex reversal, and cutaneous asthenia (ehlers-danlos syndrome). other defects observed include cryptorchidism, monorchidism, limb deformity, inguinal hernia, diaphragmatic hernia, hydrocephaly, and fetal anasarca. each of these other congenital defects occurred at less than . % incidence. etiology benign prostatic hyperplasia (bph) is an age-related condition in intact male dogs. the hyperplasia of prostatic glandular tissue is a response to the presence of both testosterone and estrogen. clinical signs bph is often subclinical. straining to defecate (tenesmus) may be seen because the enlarged gland impinges on the rectum. urethral discharge (yellow to red) and hematuria can also be presenting clinical signs. epizootiology and transmission bph typically affects older dogs (> years), although glandular hyperplasia begins as early as years of age. approximately % of inact male dogs will develop bph by years of age (smith, ) . pathologic findings in the early stages of bph, there is hyperplasia of the prostatic glandular tissue. this is in contrast to human bph, which is primarily stromal in origin. eventually, the hyperplasia tends to be cystic, with the cysts containing a clear to yellow fluid. the prostate becomes more vascular with a honeycomb appearance (resulting in hematuria or hemorrhagic urethral discharge), and bph may be accompanied by mild chronic inflammation. pathogenesis bph occurs in older intact male dogs because increased production of estrogens (estrone and estradiol), combined with decreased secretion of androgens, sensitizes prostatic androgen receptors to dihydrotestosterone. the presence of estrogens may also increase the number of androgen receptors, and hyperplastic prostate glands also have an increased ability to metabolize testosterone to α-dihydrotestosterone (kustritz and klausner, ) mediating bph. diagnosis and differential diagnosis bph is diagnosed in cases of nonpainful symmetrical swelling of the prostate gland in intact male dogs, with normal hematologic profiles and urinalysis that may be characterized by hemorrhage. a prostatic biopsy can be performed to confirm diagnosis. differential diagnoses include squamous metaplasia of the prostate, para-prostatic cysts, bacterial prostatitis, prostatic abscessation, and prostatic neoplasia (primarily adenocarcinoma). these differential diagnoses also increase in frequency with age and, except for squamous metaplasia, can also occur in castrated dogs. as such, these conditions do not necessarily abate or resolve with castration. prevention castration is the primary means for prevention of benign prostatic hyperplasia. treatment the first and foremost treatment for bph is castration. in pure cases of bph, castration results in involution of the prostate gland detectable by rectal palpation within - days. for most dogs in research studies, this is a viable option to rapidly improve the animal's condition. the alternative to castration is hormonal therapy, primarily with estrogens. this may be applicable in cases where semen collection is necessary from a valuable breeding male (e.g., genetic diseases). if the research study concerns steroidal hormone functions, then neither the condition nor the treatment is compatible. finasteride, a synthetic α-reductase inhibitor, has been used in dogs to limit the metabolism of testosterone to α-dihydrotestosterone. treatment at daily doses of . - . mg/kg orally for weeks was shown to reduce prostatic diameter and volume without affecting testicular spermatogenesis (sirinarumitr et al., ) . upon discontinuation of finasteride, the prostate generally returns to its pretreatment size within several months (smith, ) . gonadotropin-releasing hormone analogs such as desorelin inhibit production of testosterone and estrogen via negative feedback on the hypothalamus-pituitary axis. this is available in a sustained release subcutaneous implant, which has demonstrated efficacy in reducing prostatic size in dogs (junaidi et al., ) . however, medical therapy has not shown to be as advantageous as castration. research complications bph can cause complications to steroidal hormone studies, in that the condition may be indicative of abnormal steroidal hormone metabolism, and neither castration nor estrogen therapy is compatible with study continuation. the development of tenesmus as a clinical sign may also affect studies of colorectal or anal function. research model older dogs with benign protastic hypertrophy are used in research to evaluate the use of ultrasonic histotripsy as a precise nonsurgical urethralsparing alternative to prostate surgery (lake et al., ; hall et al., ; schade et al., ) . etiology juvenile polyarteritis syndrome (jps), also known as steroid-responsive meningitis-arteritis, is a painful disorder seen in young beagles (occasionally reported in other breeds) caused by a systemic necrotizing vasculitis. the cause of the vasculitis has not been established but appears to have an autoimmune-mediated component and may have a hereditary predisposition. clinical signs clinical signs of jps include fever, anorexia, lethargy, and reluctance to move the head and neck. the dogs tend to have a hunched posture and/ or an extended head and neck. most dogs seem to be in pain when touched, especially in the neck region. neurological examination may reveal proprioceptive deficits, paresis, or paralysis. the syndrome typically has a course of remissions and relapses characterized by - days of illness and - weeks of remission (scott-moncrieff et al., ) . there may be a component of this condition that is subclinical, given that vasculitis has been diagnosed postmortem in beagles that had no presenting signs. epizootiology and transmission jps typically affects young beagles ( - months), with no sex predilection. jps has been reported in other breeds including sibling welsh springer spaniels (caswell and nykamp, ) . pathologic findings on gross necropsy, foci of hemorrhage can be seen in the coronary grooves of the heart, cranial mediastinum, and cervical spinal cord meninges (snyder et al., ) . local lymph nodes may be enlarged and hemorrhagic. histologically, necrotizing vasculitis and perivasculitis of small to mediumsized arteries are seen. these lesions are most noticeable where gross lesions are observed, but they may be seen in other visceral locations. arterial fibrinoid necrosis leading to thyroid gland hemorrhage and inflammation was also reported (peace et al., ) . the perivasculitis often results in nodules of inflammatory cells that eccentrically surround the arteries. the cellular composition of these nodules is predominantly neutrophils, but it can also consist of lymphocytes, plasma cells, or macrophages (snyder et al., ) . fibrinous thrombosis of the affected arteries is also seen. a subclinical vasculitis has also been diagnosed in beagles post mortem; it is not known whether this subclinical condition is a different disorder or part of a jps continuum. this subclinical vasculitis often affects the coronary arteries (with or without other sites). pathogenesis the initiating factors for jps are unknown. it was once presumed to be a reaction to test compounds by laboratory beagles, but this may have been coincident to the fact that the beagle is the breed most often affected with jps. immune mediation of jps is strongly suspected, because the clinical signs have a cyclic nature and respond to treatment with corticosteroids, and the affected dogs have elevated α -globulin fractions and abnormal immunologic responses. there may be hereditary predisposition, given that pedigree analysis has indicated that the offspring of certain sires are more likely to be affected, and breeding of two affected dogs resulted in one in seven affected pups (scott-moncrieff et al., ) . diagnosis and differential diagnosis differential diagnoses include encephalitis, meningitis, injury or degeneration of the cervical vertebrae or disks, and arthritis. in the research facility, the disorder may be readily confused with complications secondary to the experimental procedure, or with postsurgical pain. beagles with jps that were in an orthopedic research study were evaluated for postsurgical complications and skeletal abnormalities prior to the postmortem diagnosis of systemic vasculitis (authors' personal experience). prevention and control no prevention and control measures are known at this time. treatment clinical signs can be abated by administration of corticosteroids. prednisone administered orally at . mg/kg, q h, was associated with rapid relief of clinical symptoms. maintenance of treatment at an alternate-day regimen of . - . mg/kg was shown to relieve symptoms for several months. however, withdrawal of corticosteroid therapy led to the return of clinical illness within weeks. research complications because of the potentially severe clinical signs and the need for immunosuppressive treatment, jps is often incompatible with use of the animal as a research subject. it is unknown whether subclinical necrotizing vasculitis causes sufficient aberrations to measurably alter immunologic responses. etiology interdigital cysts are chronic inflammatory lesions (not true cysts) that develop in the webbing between the toes. the cause for most interdigital cysts is usually not identified unless a foreign body is present. bacteria may be isolated from the site, but the lesions may also be sterile (hence the synonym 'sterile pyogranuloma complex'). clinical signs dogs with interdigital cysts are usually lame on the affected foot, with licking and chewing at the interdigital space. exudation may be noticed at the site of the lesion. the lesion appears as a cutaneous ulcer, usually beneath matted hair, with possible development of sinus tracts and purulent exudate. epizootiology and transmission interdigital cysts are common in a variety of canine breeds, including german shepherds. beagles have been affected in the research setting. interdigital cysts usually occur in the third and fourth interdigital spaces (bellah, ) . a retrospective study of beagles housed in a research industry setting, linked development of interdigital cysts to body codition score, age, location of cyst, and type of caging, and may result from chronic interdigital dermatitis (kovacs et al., ) . pathologic findings histopathologically, interdigital cysts are sites of chronic inflammation, typically described as pyogranulomatous. pathogenesis initial development of the cysts is unknown, except for those cases in which a foreign body can be identified. diagnosis and differential diagnosis bacterial culture swabs and radiographs should be taken of the cysts to rule out bacterial infection and radiopaque foreign bodies or bony lesions, respectively. a biopsy should be taken if neoplasia is suspected. treatment if a foreign body is associated with the lesion, then removal is the first order of treatment. if biopsy of the site provides a diagnosis of sterile pyogranuloma complex, then systemic corticosteroid therapy (e.g., prednisolone at mg/kg q h) can be initiated and then tapered once the lesion heals. interdigital cysts that are refractory to medical therapy require surgical removal. excision includes the removal of the lesion and the interdigital web, and a two-layer closure of the adjacent skin and soft tissues is recommended (bellah, ) . the foot should be put in a padded bandage and a tape hobble placed around the toes to reduce tension when the foot is weight-bearing. the prognosis for idiopathic interdigital cysts is guarded, because the cysts tend to recur (bellah, ) . research complications research complications from the cysts are minimal, unless the dogs need to be weight-bearing for biomechanic or orthopedic studies. treatment with systemic steroids could be contraindicated with some experimental designs. etiology 'cherry eye' is a commonly used slang term for hyperplasia and/or prolapse of the gland of the nictitating membrane (third eyelid). this is not considered a congenital anomaly, but there is breed disposition for this condition, including beagles. a specific etiology is not known. clinical signs the glandular tissue of the nictitating membrane protrudes beyond the membrane's edge and appears as a reddish mass in the ventromedial aspect of the orbit. excessive tearing to mucoid discharge can result, and severe cases can be associated with corneal erosion. pathologic findings typically, the glandular tissue is hyperplastic, possibly with inflammation. rarely is the tissue neoplastic. pathogenesis prolapse of the gland may be a result of a congenital weakness of the connective tissue band between the gland and the cartilage of the third eyelid (helper, ) . prevention hyperplasia of the third eyelid cannot be prevented, but dogs that develop this condition unilaterally should have the other eye evaluated for potential glandular prolapse. preventative surgical measures might be warranted. treatment corticosteroid treatment (topical or systemic) can be used to try to reduce the glandular swelling. however, surgical reduction or excision of the affected gland is typically required to resolve the condition. in the reduction procedure, the prolapsed gland is sutured to fibrous tissue deep to the fornix of the conjunctiva (helper, ). if reduction is not possible (as with deformed nictitating cartilage) or is unsuccessful, removal of the gland can be performed. such excision is fairly straightforward and can be done without removal of the nictitating membrane itself. the gland of the third eyelid is important in tear production; although the rest of the lacrimal glands should be sufficient for adequate tear production, keratoconjunctivitis sicca is a possible consequence after removal of the gland of the nictitating membrane. research complications in most cases, research complications would be minimal, especially if treated adequately. either the presence of the hyperplastic gland or its removal might compromise ophthalmologic studies. antimicrobial resistance profiles and mechanisms of resistance in campylobacter jejuni isolates from pets life span and cancer mortality in the beagle dog and human studies of distribution and recurrence of helicobacter spp. gastric mucosa of dogs after triple therapy plasma von willebrand factor concentration and thyroid function in dogs catheter-related infections in long-term catheterized dogs. observations on pathogenesis, diagnostic methods, and antibiotic lock technique aaha nutritional assessment guidelines for dogs and cats open-and closed-formula laboratory animal diets and their importance to research comparison of the accuracy of two ultrasonographic measurements in predicting the parturition date in the bitch dog thyroiditis: occurrence and similarity to hashimoto's struma surgical management of specific skin disorders bordetella and mycoplasma respiratory infections in dogs and cats associations between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in beagles diagnostic exercise: peracute death in a research dog insecticide and acaricide molecules and/ or combinations to prevent pet infestation by ectoparasites neurologic manifestations associated with hypothyroidism in four dogs veterinary surgery: small animal neoplasms of the skin and subcutaneous tissues in dogs and cats neoplasia of the skin and mammary glands of dogs and cats pancreatic adenocarcinoma in two grey collie dogs with cyclic hematopoiesis variation in age at death of dogs of different sexes and breeds comparison of campylobacter carriage rates in diarrheic and healthy pet animals advances in dietary management of obesity in dogs and cats effect of amount and type of dietary fiber on food intake in energy-restricted dogs effect of level and source of dietary fiber on food intake in the dog an outbreak of fatal hemorrhagic pneumonia caused by streptococcus equi subsp. zooepidemicus in shelter dogs molecular characterization of canine parvovirus strains in argentina: detection of the pathogenic variant cpv c in vaccinated dogs external parasites: identification and control orthopedic coaptation devices and small-animal prosthetics enterohepatic helicobacter spp. in colonic biopsies of dogs: molecular, histopathological and immunohistochemical investigations intradural vasculitis and hemorrhage in full sibling welsh springer spaniels respiratory disease in kennelled dogs: serological responses to bordetella bronchiseptica lipopolysaccharide laboratory animal medicine do not correlate with bacterial isolation or clinical respiratory symptoms lymphoma: which chemotherapy protocol and why? detection of humoral antibody to the transmissible venereal tumor of the dog laboratory animal management: dogs reproductive cycles of the domestic bitch hereditary canine spinal muscular atrophy: an animal model of motor neuron disease. can evaluation of the helicobacteraceae in the oral cavity of dogs management of septicemia in rhesus monkeys with chronic indwelling catheters clinical and virological findings in pups naturally infected by canine parvovirus type glu- mutant eradication of helicobacter spp. by using medicated diet in mice deficient in functional natural killer cells and complement factor d client information series: canine demodicosis client information series: fleas and flea allergy dermatitis management of the burn wound burn trauma chlorine dioxide sterilization of implanted right atrial catheters in rabbits current concepts in the management of helicobacter associated gastritis discordant s and s rrna gene phylogenies for the genus helicobacter: implications for phylogenetic inference and systematics use of narcotic antagonists to modify stereotypic selflicking, self-chewing, and scratching behavior in dogs epidemiology of canine and feline tumors epizootiologic characteristics of canine and feline leukemia and lymphoma evaluation of the ovicidal cctivity of lufenuron and spinosad on fleas' eggs from treated dogs enteric coccidiosis the respiratory system the association of american feed control officials dog and cat food nutrient profiles: substantiation of nutritional adequacy of complete and balanced pet foods in the united states study of obesity in dogs visiting veterinary practices in the united kingdom pathological prognostic factors in breast cancer. i. the value of histological grade in breast cancer: experience from a large study with long-term follow-up utility of fdg-pet scanning in lymphoma by who classification principles of treatment for canine lymphoma miller's anatomy of the dog update on diagnosis of canine hypothyroidism immunohistochemical and histochemical stains for differentiating canine cutaneous round cell tumors the laboratory canine canine conjunctival mast cell tumors: a retrospective study canine cutaneous epitheliotropic t-cell lymphoma: a review canine infectious respiratory disease helicobacter-associated gastric disease in ferrets, dogs, and cats enteric bacterial infections hemorrhagic streptococcal pneumonia in newly procured research dogs association between canine malignant lymphoma, living in industrial areas, and use of chemicals by dog owners reproductive patterns in the domestic dog-a retrospective study of the drever breed the dog as a research subject a review of canine pseudocyesis leptospirosis surgical treatment of an elbow hygroma utilizing microvascular free muscle transfer in a newfoundland bacterial diseases immunoprophylaxis leptospirosis gastric helicobacters in domestic animals and nonhuman primates and their significance for human health non-helicobacter pylori helicobacter species in the human gastric mucosa: a proposal to introduce the terms h. heilmannii sensu lato and sensu stricto diseases of the small intestine histotripsy of the prostate: dose effects in a chronic canine model flea control failure? myths and realities small animal clinical nutrition operating room emergencies shock evaluation of the sensitivity and specificity of diagnostic criteria for sepsis in dogs pulmonary parenchymal disease canine thyroid neoplasms: epidemiologic features magrane's canine ophthalmology helicobacter-like organisms: histopathological examination of gastric biopsies from dogs and cats current veterinary therapy x: small animal practice congenital defects of the dog gastric and duodenal ulcers in dogs with mastocytoma complications in the use of indwelling vascular catheters in laboratory animals helicobacter infection diseases of the large intestine pregnancy management in the bitch tracheal collapse. diagnosis and medical and surgical treatment hygroma of the elbow in dogs thermal injuries joint working group on refinement factors contributing to the contamination of peripheral intravenous catheters in dogs and cats diversity in bacterium-host interactions within the species helicobacter heilmannii sensu stricto morphological study of the effects of the gnrh superagonist deslorelin on the canine testis and prostate gland five-year longitudinal study on limited food consumption and development of osteoarthritis in coxofemoral joints of dogs cvt update: interpretation of endocrine diagnostic test results for adrenal and thyroid disease etiopathogenesis of canine hypothyroidism maintenance energy requirement of dogs: what is the correct value for the calculation of metabolic body weight in dogs? outbreak and control of haemorrhagic pneumonia due to streptococcus equi subspecies zooepidemicus in dogs kirk's current veterinary therapy xii: small animal practice an emerging pulmonary haemorrhagic syndrome in dogs: similar to the human leptospiral pulmonary haemorrhagic syndrome? tarsal joint contracture in dogs with golden retriever muscular dystrophy an epidemiological study of interdigital cysts in a research beagle colony value of the ( )c-urea breath test for detection of gastric helicobacter spp. infection in dogs undergoing endoscopic examination pathogenesis of helicobacter pylori infection prostatic diseases semen collection in the dog accuracy of canine parturition date prediction using fetal measurements obtained by ultrasonography chronic catheterization of the intestines and portal vein for absorption experimentation in beagle dogs evaluation of weight loss protocols for dogs problems in wound healing associated with chemotherapy and radiation therapy histotripsy: minimally invasive technology for prostatic tissue ablation in an in vivo canine model fdg-pet imaging in canine lymphoma and cutaneous mast cell tumor comparison of fertility data from vaginal vs intrauterine insemination of frozenthawed dog semen: a retrospective study withrow & macewen's small animal clinical oncology estimation of gestational age and assessment of canine fetal maturation using radiology and ultrasonography: a review effects of four preparations of . % chlorhexidine diacetate on wound healing in dogs the prediction of parturition date in canine pregnancy clustering of activating mutations in c-kit's juxtamembrane coding region in canine mast cell neoplasms transmissible venereal tumors kirk's current veterinary therapy : small animal practice canine lymphoma and lymphoid leukemias the staging and treatment of multicentric highgrade lymphoma in dogs: a review of recent developments and future prospects association between waste management and cancer in companion animals tick paralysis in north america and australia thyroid gland and arterial lesions of beagles with familial hypothyroidism and hyperlipoproteinemia bacterial gastroenteritis in dogs and cats: more common than you think enteropathogenic bacteria in dogs and cats: diagnosis, epidemiology, treatment, and control dermatologic aspects of tick bites and tick-transmitted diseases a chronic access port model for direct delivery of drugs into the intestine of conscious dogs mast cells and canine mast cell tumours: a review etiologic study of upper respiratory infections of household dogs effect of early enteral nutrition on intestinal permeability, intestinal protein loss, and outcome in dogs with severe parvoviral enteritis determination of strain variability of microsporum canis to disinfectants cutaneous fungal infections diagnosis of neoplasia tumors of the mammary gland transmissible dog cancer genome reveals the origin and history of an ancient cell lineage clonal origin and evolution of a transmissible cancer notice regarding nih plan to transition from use of usda class b dogs to other legal sources (not-od- - ) guide for the care and use of laboratory animals surgical closure of elbow hygroma in the dog colitis and colon cancer in waspdeficient mice require helicobacter species tumors of the genital system practical laboratory methods for the diagnosis of dermatologic diseases animals and animal products, subchapter a, parts , , and comparison of three skin preparation techniques in the dog comparison of three skin preparation techniques in the dog. part : clinical trial in dogs manual of clinical behavioral medicine for dogs and cats. mosby-year book identification of and screening for human helicobacter cinaedi infections and carriers via nested pcr identification of three novel superantigen-encoding genes in streptococcus equi subsp. zooepidemicus, szef, szen, and szep hypothyroidism in dogs: cases ( - ) plasma von willebrand factor antigen concentration in dogs with hypothyroidism plasma von willebrand factor antigen concentration and buccal bleeding time in dogs with experimental hypothyroidism canine cutaneous mast cell tumor: morphologic grading and survival time in dogs atlas of small animal wound management and reconstructive surgery what's your diagnosis? fever and leukocytosis in a young beagle. canine juvenile polyarteritis syndrome (beagle pain syndrome) a clonal outbreak of acute fatal hemorrhagic pneumonia in intensively housed (shelter) dogs caused by streptococcus equi subsp. zooepidemicus thyroid diseases 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differentiation markers of canine mammary gland neoplasms withrow & macewen's small animal clinical oncology nutrient requirements of dogs and cats (nutrient requirements of domestic animals) trauma to the skin and subcutaneous tissues of dogs and cats chronic problem wounds of dog limbs acvim small animal consensus statement on leptospirosis: diagnosis, epidemiology, treatment, and prevention lumbosacral stenosis in dogs mammary neoplasia in a closed beagle colony complete mitochondrial genomes of ancient canids suggest a european origin of deomestic dogs artificial insemination in canids: a useful tool in breeding and conservation artificial insemination with frozen semen in dogs: a retrospective study of years using a non-surgical approach manual of canine and feline cardiology comparative evaluation of agar dilution and broth microdilution methods for antibiotic susceptibility testing of helicobacter cinaedi thyroiditis in a group of laboratory dogs: a study of beagles obesity-related metabolic dysfunction in dogs: a comparison with human metabolic syndrome pro-inflammatory properties and neutrophil activation by helicobacter pylori urease of agriculture, animal and plant health inspection service annual report animal usage by fiscal year. available from: <www leptospirosis in dogs: a review with emphasis on clinical aspects thomson's special veterinary pathology, second ed. mosby-year book endocrinology of pregnancy in the dog: a review immunoblot analysis as an alternative method to diagnose enterohepatic helicobacter infections management of superficial skin wounds psychodermatosis colony multiplex pcr assay for identification and differentiation of campylobacter jejuni, c. coli, c. lari, c. upsaliensis, and c. fetus subsp. fetus the role of c-kit in tumorigenesis: evaluation in canine cutaneous mast cell tumors aaha canine vaccination guidelines canine mast cell tumours: a review of the pathogenesis, clinical features, pathology and treatment serum concentrations of thyroxine and , , ′-triiodothyronine before and after intravenous or intramuscular thyrotropin administration in dogs surgical treatment of specific skin disorders naltrexone for treatment of acral lick dermatitits in dogs use of ultrasound in the measurement of subcutaneous fat and prediction of total body fat in dogs mammal species of the world: a taxonomic and geographic reference withrow & macewen's small animal clinical oncology the authors thank dr. bambi jasmin of marshall farms for contributions on preventive health practices for class a dogs. key: cord- -v bqi c authors: turner, tari; el-jardali, fadi title: the crucible of covid- : what the pandemic is teaching us about health research systems date: - - journal: health res policy syst doi: . /s - - - sha: doc_id: cord_uid: v bqi c the global health crisis created by covid- is providing valuable insights into the strengths of our health research system and, perhaps even more clearly, displaying its weaknesses. much of what is being shown so plainly in the current context is not truly new. we are being reminded that health research systems are slow and noisy as well as that there is a desire for research to inform decision-making, that researchers are great collaborators, and that the walls we are so quick to erect between health research and health practice are unhelpful facades. it is our hope that the clarity with which these issues are being demonstrated by covid- might provide the impetus to address these challenges and seize these opportunities to improve our health research system, for the benefit for communities facing covid- now, and for the benefit of us all in facing the further health challenges that are sure to come. main text " we are only what we always were" -arthur miller, the crucible. if it is true that, under pressure, we all revert to type, then covid- is revealing quite a few flattering things about the true nature of the health research community. it turns out that, when the going gets tough, researchers get productive, collaborative and impact focused. it is as if the nature of this current and unprecedented crisis is reminding us of all the reasons for which we embarked on research careers; that we are now seeing afresh that research is vital, is valuable and can be truly lifesaving, and we are kicking research production up a gear. sadly, we are also seeing that the systems we have in place to translate the results of research into improvements in health practice and policy are not fit for purpose in a pandemic. if we are frank, there is little in our experience of knowledge translation with covid- that is truly new; mostly what we are seeing are the known strengths and weaknesses of our system held up to a very, very clear light. so, what is covid- revealing? research publication is slow the current system for publication of peer-reviewed research is slow. this is not news. however, in the light of covid- , it is very clear that the delays caused by standard workflows, which are both appropriately rigorous and inappropriately bureaucratic, are slowing the translation of new research evidence into practice, and that lives are being lost in those lost days, weeks and months. a now familiar example is a key paper on the effectiveness of face masks [ ] , for which nature medicine achieved what would have, in other times, been considered a sprightly turnaround, but which resulted in a -month delay between receipt of the paper and its publication. the window between acceptance and publication was a full weeks. realising the importance of publication speed, some journals and publishers are implementing rapid publication models and many research teams are implementing workarounds to make research available more quickly, making prepublication versions of reports available, creating online repositories of rapid reviews and sharing emerging data. these are useful and well-intentioned steps; it will be important that, in a post-pandemic world, we seize on these initiatives, refine them and, where possible, scale them up. however, we need to acknowledge that they are creating flow-on problems, research that has not been peer-reviewed is used as the basis of decisionmaking, finding and curating all of those duplicating and overlapping reviews is very challenging, and how data has been validated and analysed is often unclear. interestingly, research platforms that were designed to overcome some of these challenges in a pre-pandemic world, like f , have not been swamped by publications. if this was their moment, the research community does not seem to have realised it. on the other hand, the growing interest in 'living' continually updated evidence reviews does seem to have taken off in an environment where so little is certain and new evidence is available every day. over the first few months of , there has been an initially small, but dizzyingly rapid production of primary research and reviews of research related to covid- . these research studies have been broadly disseminated through the usual research publication systems as well as through social media, clinical networks and other channels. challengingly, this useful research has been accompanied by a blizzard of commentary, opinion and editorialising; often from the same publication platforms as the research, but also from everyone else, up to and including celebrity chefs. clinicians, managers and policy-makers (and journalists) who were valiantly trying to stay across all the research as it emerged were quickly overwhelmed by the confetti of competing, unsubstantiated, conflicting voices and views, all purporting to be providing 'evidence'. we need better strategies to remove the noise from our research publication and dissemination systems, and we need them now. simple flags for identifying research that has been peer reviewed or accessible, curated collections of rapid systematic reviews would be useful steps in the right direction. there is a desire for research having been increasingly disheartened by the cynical attitudes towards 'experts' and 'evidence' so sadly ubiquitous in the pre-covid- world, it has been hugely encouraging to see the widespread demand for research evidence to guide health decision-making at all levels, from individual patients to global multi-laterals. this presents a huge opportunity for the research community to demonstrate their ability and willingness to engage, and it has been encouraging to see our colleagues step up and into the conversation. there is no doubting the real-world impact of research on decisionmaking in the shadow of covid- , even if it will be months before we see the publication metrics. the real challenge will be converting these acute 'evidence hunger pangs' into an ongoing appetite in our governments, health systems and others to fund and use research evidence to guide decision-making beyond the pandemic. to achieve this, we need to ensure that we capture data and stories of the impact of research on decision-making during the current crisis, so we can build on this momentum following the pandemic. never before have so many researchers asked for access to pre-publication data from their colleagues on the other side of the globe and received a rapid and resounding 'yes'. covid- has provided an undeniable imperative to put aside concerns of being pipped to the publishing post or to the funder's wallet, to collaborate in the service of the global, common and immediate good. as elsewhere, our challenge as a research community is to translate this willingness to share beyond the bubble produced by this pandemic and beyond the espoused rhetoric; to build and implement models for research funding, conduct and publication that reward true collaboration and acknowledge the reality that the partnerships required for successful collaborative research are complex and time consuming as well as, ultimately, incredibly rewarding. the line between research and practice has always been marked in fuzzy pencil and covid has taken a big smudgy eraser to that already unclear demarcation. a novel virus, an unprecedented health emergency, a nonexistent evidence base. in this evidence-free zone, when does a clinical hunch, a series of cases, the experience of a hospital for a month, become the best available evidence? when do we wait for the results of the randomised trial possibly months away and when do we make recommendations based on early indications from an icu in china? covid- has already demonstrated that, sometimes, early indications are very misleading and at others they are crucial. hydroxychloroquine, which was touted early in the outbreak as a revolutionary treatment, is looking less and less promising as rigorous trials emerge [ ] and clinical experience has been key in identifying that the symptoms of covid- may deteriorate rapidly on days - of illness, life-saving information for ensuring patients are appropriately monitored even if apparently well [ ] . we need to stop pretending that there is clinical and public health expertise that is entirely distinct from research knowledge and we need to build our methods for integrating the two in pragmatic and transparent ways. we also need to accept that a range of contributors are all crucial in producing the knowledge we need to effectively deal with this most challenging foe. vital knowledge about covid- has been produced along the entire research spectrum from the very fundamental, 'basic' research to the most hands-on clinician researcher; the engine rooms of translation have been the multidisciplinary networks of clinical specialists, policymakers and researchers in formal discussion lists, on knowledge translation platforms and in professional societies. community organisations, citizens and the media have all played important roles in identifying issues and disseminating information in (socially distanced) corridor chats, tweets and whatsapp messages. as is always true, and is even more so now, there is no us and them, we are all us. it has already become a cliché that covid- presents us with a pivotal moment; there is no question that many decisions made now will have a huge impact on history, for better or for worse. for health research systems, this is also true. we are in a moment where, if we do not let it consume us, we can use the light cast by this inferno to see ourselves, our strengths and our weaknesses, clearly. this crucible of covid- highlights the fundamental need to institutionalize the use of research evidence in policy decisions at a time of crisis, and beyond. if we act now, we might even emerge stronger and with greater purpose than when we entered in this horrific conflagration. respiratory virus shedding in exhaled breath and efficacy of face masks outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid- clinical course of covid- : what gps need to know adherence to national and international regulations not applicable. both authors contributed to drafting the manuscript and approved the final manuscript. availability of data and materials not applicable.ethics approval and consent to participate not required. not required. the author's declare that they have no competing interests.author details monash university, melbourne, melbourne, australia. american university of beirut, beirut, lebanon.received: april accepted: may key: cord- -dg jg t authors: el achi, nassim; honein-abouhaidar, gladys; rizk, anthony; kobeissi, elsa; papamichail, andreas; meagher, kristen; ekzayez, abdulkarim; abu-sittah, ghassan s.; patel, preeti title: assessing the capacity for conflict and health research in lebanon: a qualitative study date: - - journal: confl health doi: . /s - - -x sha: doc_id: cord_uid: dg jg t background: conflicts pose new challenges for health systems, requiring rapid and practical approaches to meet emerging needs on the ground. lebanon has been highly influenced by surrounding conflicts in the middle east and north africa (mena) region, especially the syrian crisis. strengthening research capacity to collect evidence on conflict in the mena region and beyond is crucial to inform healthcare policy and practice. for targeted capacity strengthening interventions, the main objective of this paper is to present key findings of a needs assessment of conflict and health research in lebanon. this will support recent efforts to scale up context-specific policies, interventions to strengthen the country’s health system, and research capacity. methods: the study is based on semi-structured interviews with key informants such as specialist academics, humanitarian workers and public sector officials. results: despite being ranked third in the number of publications on biomedical and health research per capita in mena and in hosting reputable universities which are considered central academic hubs in the region, lack of nationwide research culture, insufficient funding and limited access to data were reported to be major challenges for health researchers in lebanon. even with the ongoing efforts, poor impact of research on policy continues to be a persistent gap. large disparities in research capacities and taught skills were reported between different universities in lebanon, with a disproportionate emphasis on quantitative over qualitative skills. most medical students are not trained to conduct research or to practice in conflict settings. concerns were also expressed regarding the ethics of research conducted, specifically by local non-governmental organizations. recommendations: to conduct contextualized trainings on research skills with a stronger focus on qualitative approaches, medical practice, and ethical research in conflict. to better involve policymakers in designing research agendas by organizing multiple stakeholder meetings. conclusion: the study indicates that health research in lebanon is characterized by considerable strengths in terms of human capital and research capacities of certain universities. however, the lebanese research infrastructure needs further development in terms of ensuring sustainable funding, providing access to data, teaching qualitative research skills, conducting ethical and multidisciplinary research, and promoting cross-sectoral knowledge transfer. research capacity strengthening has been a top priority for the world health organisation (who) since , paving the way for substantive investments from research funding agencies and international institutions [ ] . while evidence generated from health research is crucial for addressing health and development challenges in countries and regions affected by armed conflict [ ] , the capacity to conduct locally relevant health research faces specific challenges in contexts affected by acute and protracted conflict. in conflict settings, health research is often deprioritized as focus shifts to improving security, conflict resolution, short-term humanitarian responses and managing forced migration [ ] . however, the protracted nature of contemporary conflicts and their long-term impact on health provision has led to an increased demand and willingness to conduct and strengthen health research capacity in conflict-affected settings [ ] [ ] [ ] , including countries in the middle east and north africa (mena) region [ ] . the mena region is no stranger to conflicts, many of which are complex and protracted (see table for definitions). the first wave of the arab spring in included large-scale mobilizations and political upheavals across the region, with armed conflicts developing in libya, yemen, and syria and mass displacement leading to the worst humanitarian crisis since world war ii [ ] . in , % of . million people displaced worldwide originated from the mena region [ ] . this process continues as more recently in , the second wave of the arab spring emerged in algeria, sudan, iraq and lebanon, with anti-government protests table key definitions lmic (low and middle-income country): according to the world bank's definitions, drawing on figures, low-income economies have a gross national income (gni) per capita of $ or less; the gni per capita of lower middle-income is between $ and $ ; and upper middle-income economies like lebanon have a gni per capita of between $ and $ , [ ] . capacity strengthening: as a working definition, capacity strengthening can be understood as a process of developing, upgrading and/or expanding pre-existing capabilities at individual, organisational, and institutional levels to plan, conduct, and disseminate evidencebased knowledge [ ] . the individual level includes researchers and research teams; the organisational level is composed of university research departments, think tanks and similar organisations; and the institutional level encompasses the regulatory environment and includes governmental bodies and policymakers [ ] . installing transitional governments amid calls for major reforms [ ] . these protests have significantly changed in intensity, spread and frequency since onwards but got fully subsided due to government measures to control the spread of the covid- pandemic in february-may . although it is premature to predict the post lockdown trajectory across the mena region, it is speculated that protests will regain momentum, exacerbated by severe economic and fiscal crises compounded further by the viral outbreak, as it is the case in lebanon where violent protests erupted in late april in response to severe economic hardship [ , ] . such an endemic fragility results in a serious rise in health burdens and inequalities that place serious pressure on the region's health systems which are already coping with the protracted nature of conflicts [ ] . the mena region thus needs continuous strengthening of its health research, at the institutional, organizational and individual levels to address these highly volatile health and social needs (see table for definition) [ ] . in recent years, the mena region has been one of many sites of increased investment and interventions in health research capacity strengthening. programmes such as the eu-funded research capacity for public health in the mediterranean (rescap-med) [ ] , the uk-funded research capacity building and knowledge generation (recap) [ ] and research for health in conflict middle east and north africa (r hc-mena) [ ] projects are examples of recent and ongoing mena-focused health research capacity strengthening interventions. r hc-mena, focused entirely on strengthening research capacity in the conflict-affected mena region by implementing contextually relevant activities, is an interdisciplinary partnership led by king's college london with uk partners such as imperial college london and the university of cambridge, mena-based partners that include the american university of beirut (lebanon), birzeit university (palestine), hacettepe university (turkey) and king hussein cancer centre (jordan), and several humanitarian and policy organisations. such activities include developing and delivering accredited multi-disciplinary courses, mentoring senior leadership at national and global/multilateral institution levels, and developing innovative learning technologies and informatics platforms for distance learning [ ] . lebanon, which is the focus of this paper, is an upper middle income country in the middle east with the highest refugee density worldwide following the syrian crisis [ , ] . it has been politically and socioeconomically influenced by regional conflict and crisis, as well as its own civil war ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ), israel's second lebanon war ( ) and protracted internal strife (see table for definition). although there has been a long-standing engagement with conflict and health research in lebanon [ ] , research outputs in the country were negatively affected by instabilities in war times [ ] , but managed to quickly recover following the end of the civil war [ ] . however, recent regional mobility of conflict-injured patients and the high influx of refugees have stimulated a renewed focus on conflict-related health research [ , ] . note that lebanon has a long history of academic research compared to other mena countries and has one of the highest numbers of publications and researchers per capita in mena. it also has strong medical and health research provided mainly by university hospitals such as the american university of beirut medical center and hotel dieu de france, both respectively affiliated to the large private and prestigious universities-the american university of beirut (aub) and université st-joseph (usj). lately other universities, including the lebanese university, balamand university and beirut arab university, are also focusing more on enhancing research capacity and outputs. however, most of lebanon's research production comes from the aub which has made considerable efforts to promote research production and translation [ , ] . an analysis of the country's capacity for conflictrelated health research is currently lacking, even as evidence for designing contextualized capacity interventions becomes more urgent. building on a conceptual framework on capacity strengthening of health research in conflict-affected countries that focussed on the mena region [ ] (fig. ), we conducted a needs assessment in lebanon to answer three main interrelated questions: what is the current capacity for conflict and health research in lebanon? what are the challenges and gaps in conflict and health research in the country? and, lastly, what are the preferred tools and mechanisms of strengthening the country's conflict and health research capacity from the perspectives of academics, humanitarian actors and public sector officials? the main aim of this paper is thus to analyse the strengths, weaknesses, opportunities and threats to conflict and health research capacity strengthening in lebanon. we employed a qualitative approach using semistructured interviews with academics, humanitarian workers and public sector officials as the main method of data collection. due to scarcity of data sources on health research capacity in lebanon, we opted for a qualitative approach that would additionally contribute to developing and refining interventions [ , ] . in a previous related study, our research group designed a working conceptual framework on capacity strengthening of health research in conflict-affected settings, which was the basis for the topic guide for the semistructured interviews [ ] . we used purposive sampling followed by snowballing sampling to identify interviewees. to achieve maximum variation, we sought out research participants from diverse geographic areas, ages, genders, and specialties ( table ) . key informants consisted of three professional categories: ) academics from various universities across lebanon, ) humanitarian workers working with local or international non-governmental organizations (ngos) or united nations (un) agencies with operations in the health sector, and ) public sector officials working at ministry of public health (moph) and the ministry of social affairs (mosa). all research participants worked professionally in fields related to health, conflict and research in lebanon. we included academics working at universities that provide postgraduate degrees as these tend to be more inclined towards research than universities that only provide undergraduate programmes. out of the informants approached, refused to participate or did not reply to the invitation email and subsequent conceptual framework for health research capacity strengthening in conflict [ ] reminders, leaving a total sample size of research participants. we continued recruiting key informants until we reached thematic saturation. we conducted face-to-face interviews between february and may across lebanon. semi-structured interviews were conducted in locations specified by the interviewees and were recorded when permitted to do so by the participant (n = , %), otherwise taking detailed notes of the interview (n = , %). the interviews were conducted in either english (n = , %) or lebanese arabic (n = , %). most participants (n = , %) agreed to be quoted in anonymized form. using a topic guide (additional file ), questions addressed experts' perception of research in conflict settings, strengths and challenges in conducting research in lebanon, and recommendations to strengthen conflict and health research capacities in lebanon. to ensure clarity and relevance of questions, we consulted key experts from each professional category for the development of the topic guide. the interviews lasted approximately min. interviews were first transcribed into english and arabic and transcripts in arabic were then translated to english. all transcripts were anonymized using a unique identifier for each participant. the assigned code, used for citation purposes below, was composed of a letter that refers to the professional category of the participant [academic (a), humanitarian worker (ngo) and public sector official (m)] followed by a number that represents the chronological order of the interview. for example, a codes for the th academic and m for the second public sector official that we interviewed. we used nvivo ® for data management and thematic analysis. we adopted the phase process described by braun and clarke [ ] . first, two investigators (ne, ek) worked independently on coding two transcripts line by line. together, they discussed their coding approach to identify similarities and differences, thus avoiding interpretability bias, and created a thematic framework for data analysis. second, the same investigators completed the open coding and started identifying emerging categories. members of the research team (gha, ne and ek) met on several occasions to reflect on the findings and identify the candidate themes and sub-themes. the latter to further increase rigor, we focused on achieving both credibility and reflexivity. regarding credibility, all discussions, except , were audio recorded, transcribed verbatim, accurately translated into english as necessary, and utilized as the main data repository. once reaching data saturation, a decision was made by all team members to cease data collection. as for reflexivity, and to limit biases, all team members were involved in the analysis and interpretation of the results. the participants in this study were academics, humanitarian actors, and public sector officials ( table ) . they are based in four different regions of lebanon -beirut, mount lebanon, north lebanon and bekaaall of which are central areas where major ngos, education and research institutes with a focus on health research are located. the academics interviewed in this study collectively work at out of a total of accredited universities in lebanon that provide health research training at the postgraduate level [ ] . the main themes that emerged are in accordance with those specified by the conceptual framework previously designed by our research team: perception of research capacity strengthening, research culture, current capacities and strategies of universities, research skills, infrastructure (data availability & ethics), funding & sustainability, partnerships (local & international) and the role of women working in lebanon in health research [ ] . when asked about defining research capacity strengthening, the responses varied significantly ranging between considering it as a "training" mostly by local informants to a more detailed understanding targeted towards the individual, organizational and institutional levels. these variations were highly related to experience in previous capacity building interventions. nonetheless, the term "capacity building" was used regardless of the context. for instance, locals used this term more often than those working with international ngos who preferred to use the term capacity strengthening since "building means starting from scratch which is rarely the case" (ngo ). many informants pointed to a 'lack of research culture' as a major challenge for conducting health research in lebanon: "i don't think we are in lebanon so strong in terms of research, conducting research; we don't think about research as a major power" (a ) "we [lebanese] spend a lot of money on it [education] . but then when it comes to moving forward, making innovations, i don't think we are the culture that nurture this environment of research" (a ) few informants also described hierarchical and challenging working conditions that can be counter-productive. "i might be very negative but i see a lot of motivated young researchers whose work is repressed by people at powerful positions … this is a major barrier to the progress of research" (a ) however, at the individual level, all of the informants expressed willingness to strengthen their health research capacities by making the best of the available resources. career progression via promotion and improving job prospects were considered the main drivers for enrolling into trainings, courses, internships and volunteering opportunities; along with personal motivation. "i am not strong enough in qualitative research so when i knew about the course, i directly got involved despite that the university didn't cover the expenses. i like to improve my skills. it is something i have deep inside."(a ) it was clear from the interviews conducted that there were huge disparities between the universities in lebanon in terms of budget, qualifications, human resources and infrastructure. all of those interviewed agreed on the importance of research and considered it to be a major pillar of the universities' strategies. some universities, especially the highly ranked ones, allocate some of their budgets to fund research to complement external grant income. changes at the organizational level and pushing academics to conduct more research are favoured to obtain accreditation from the ministry of higher education, or to adhere with international standards of research and to gain a better reputation to attract students particularly for the private universities. "the university puts a lot of money in research, with a certain amount of the budget allocated specifically for this purpose" (a ) to incentivize academics to conduct research, most of the universities are considering publishing and research as major requirements for promotion and/or qualification for tenured positions. "so there's a promotion system. if you want to get promoted, you have to publish […] so there's a percentage. it's % research, % service and % teaching." (a ) one of the informants highlighted that one of the leading universities is also adopting a strategy of forming research institutes and centres within the university, which focus only on providing executive trainings, courses, and knowledge to policy schemes along with knowledge production and research that could enhance the learning experience at the university. "along with education, we have multiple interfaculty research centers and institutes which deal with emerging health issues at the national and global levels." (a ) however, there were complaints from academics in smaller universities about not having time to conduct research as they are overwhelmed with other duties so that they try to conduct research in their extra time. "if the department asks me to teach courses every semester, i don't think that i have much time left for research.." (a ) according to most of the informants, individual research capacity for postgraduates is relatively high in some areas (epidemiology, health interventions, biostatistics) focusing on quantitative skills, especially in the top five universities in lebanon. the research skills of undergraduates are considered by most of the respondents to be mostly introductory and insufficient for conducting rigorous research. nonetheless, all universities do provide courses on research and writing skills, but this tends to be at the postgraduate rather than the undergraduate level. altering the content of courses would be challenging, as it would require new accreditation from the ministry of higher education along with the internal institutional bureaucracy which is very time consuming. as a result, some informants admitted that they prefer to give extracurricular workshops to cover key gaps in research skills at the undergraduate level. "they don't have this course (research design), i think they take a statistics course and introductory research course but they don't know what it means on the undergrad level… you cannot rely on undergrads to collect data if data needs interaction with participants… you need to get someone at the graduate level" (a ) "so if we have a gap in our curriculum related to research or even to the theoretical courses we bring expert and we do workshops and conferences… we are trying to fill the gaps with some extracurricular activities" (a ) the gaps identified by most of the informants are in the social sciences, history, and political economy and mainly in qualitative research designs despite stating that mixed methods are important for a more holistic understanding of the health impacts of conflicts. "the other thing even on the postgraduate level, qualitative research is hardly taught. this is another main issue … we do quantitative blindly and the qualitative, which i think is much more significant for us … is not … i waited for my phd to take a qualitative course. and still, i took a summer qualitative course when i was already a faculty to go over the themes..." (a ) "…at times qualitative studies, which bring about a treasure of information that statistics do not often give …" (a ) when asking informants working in the humanitarian field, they emphasized that graduates from lebanese universities have excellent quantitative research skills (depending on the university attended). however, they mentioned that there is a high demand in qualitative methods and tools of analysis to understand impact evaluations which fresh graduates lack. "we do a lot of interventions in the field, but we never understood the impact of the intervention and we don't know if the impact achieved was the result of what we did or result of other factors… so i think in order for us to replicate some interventions and to understand the weaknesses or the failure of some interventions, it's very important to understand the impact. we need qualitative research" (ngo ) interviews also indicated that monitoring of research quality and ethical approval in lebanon are usually to meet the standards of international funders rather than local governments. thus, ethics was a major theme highlighted by all informants. most of the academics interviewed had institutional review boards (irb) at their institutions for granting approvals before conducting any research involving human subjects. the only downside of the irb was the delay and difficulty in obtaining approvals especially when working with vulnerable populations. however, an informant mentioned that having an irb is not enough to ensure ethical research. "all kinds of research with the proviso that the culture, context and participants full rights are respected… and that they 'benefit' from the research one way or the other" (a ) an informant also mentioned the wellbeing of researchers, which is mostly ignored, as a key ethical determinant that should also be considered along with that of participants. "at times qualitative studies can be a traumatizing experiences. thus, it is important to have good solid ethical review for the research and psychological support to both researchers and participants when needed." (a ) as for ngos, there is a huge difference between international and local humanitarian actors. for the former, there are strict rules when it comes to dealing with vulnerable populations, whereas for the latter, this concept might be lacking. due to the limited regulation of research in the field, there are serious concerns about researching vulnerable populations, with infringement of the international regulations of ethical research especially in conflict settings. "our main mandate is protection and protection of cases. confidentiality is for example one of the tolerance for [organization name]. we also have ethics guidelines where everyone, every student needs to read, sign and abide. it's very strict, it's tolerance." (ngo ) the absence of oversight on research ethics in lebanon was a recurrent concern. for some, governmental oversight on research ethics would be detrimental, for fear of it being restrictive, especially when 'sensitive' topics are researched. for others, the repetitiveness of the same research projects on the same populations (for example, syrian refugees) is detrimental, often being intrusive, dehumanizing, or inappropriate (an example given is asking people who are hungry to document their meals). as indicated by a public servant, "usually ngos working in the field are not affiliated to any irbs in hospitals or universities. […] . i'm sure there's a lot of research at the being conducting on syrian refugees. and unfortunately ngos working in the field are not aware of these requirements of having ethical approvals, of the informed consent process, and all of these. the research that i come countered with by accident because we have no mechanism to find out what's going on. the way we catch them is when they take biological samples and they want to export it outside the country and they need the approval of moph. there is a study that we ended up putting under control … it was done between lebanon and a university outside .. it was a dna testing. they considered it not a big deal since they collect saliva and cells with toothpicks. i forced them to get ethical approval and to understand what they're doing with the samples beside research." (m ) as indicated by m "we (local centres of mosa) don't conduct research but provide data for institutes following the permission from mosa", health research data if available can be provided by moph and mosa. private hospitals also have their own databases that can be used in research. however, as indicated by most academics, access to data is a huge challenge that limits the ability to conduct impactful research. "there is no databases so you know if you want to have information about the patient, about any health problem, it's really hard to get what you need to know about it." (a ) "so when you go to private hospitals, they have a different interests. if there is a conflict with business because they don't want to have their image tarnished because of certain quality indicators which are not optimal or because that will reduce the number of patients coming to their services, they're very resistant to conduct research. and if they do conduct research, they show only the positive side of it." (a ) interviews with humanitarian agencies indicated that data collected is for basic analyses to inform the programmatic direction of the agencies, and not necessarily for statistically advanced quantitative or in-depth qualitative studies and thus can be of minimal use to academic research. moreover, one informant highlighted the problems that arises from interpretation of available data especially in conflict-affected settings; "data though can be misleading depending on how it is interpreted and by whom." (a ) all academics interviewed thought that partnerships and collaborations at both the national and international levels were crucial and highly beneficial for the progress of work. they suggested that these partnerships could help to mitigate the gaps and barriers of health research in the country in terms of infrastructure such as expensive equipment. for instance, international partners provide an opportunity for the local academic institutes to get access to facilities and equipment which are unavailable in lebanon and/or are too expensive to purchase and maintain. similarly, several local universities have joint phd programs with international universities for local students to produce high quality research which is context-oriented while still getting access to facilities available in international academic institutions. informants also mentioned examples of exchange of faculty members between universities as a way of knowledge and strengthening experience for both faculty and students in both institutes. partnerships were also considered as an opportunity to bring grants to the local institutes to conduct health research as it is more appealing for international funders to fund consortia rather than a single university or institute in lebanon. informants mentioned projects funded by the european union, uk research councils, and national council for scientific research (cnrs) included partnering with other local and international universities. partnerships between academics and local ngos were considered to be important as they provide access to refugee camps and local communities and help with logistical arrangements and security of data collectors. "as an academic institution […] you need to collaborate with an ngo for them to provide access to camps […] it's also always better to have someone from the ngo to stay with you for security measures or if anything is to happen on the field as researcher" (a ) the moph and mosa informants also highlighted the importance of collaborating with un agencies such as who, united nations educational, scientific and cultural organization (unesco), united nations development programme (undp), united nations international children's emergency fund (unicef), united nations high commissioner for refugees (unhcr) … and other international ngos that sponsor and deliver capacity strengthening workshops, trainings and seminars for the public sector officials working in these ministries. "usually when there is a training that the ministry wants to conduct, we approach these organizations for two things: first for providing technical experts to conduct the trainings, because it provides a better credibility for the training, and second for logistic support… but these trainings are not for research skills" (m ) some of the topics that were mentioned by the informants include: "psychological support, service provision, finances, management, training of trainers (tot), communication for development (c d)… following the syrian crisis, more trainings were conducted on topics related to child protection and gender-based violence… the content of these trainings is regularly updated." (m ) despite highlighting the importance of partnerships with local and international actors, it was found that most of these partnerships were within the same category: academics-academics, or ministry with ngos. the lack of coordination between ngos for example, out of competition or lack of communication in a highly unregulated field was reported to be a major drawback. "i feel there is no universal coordination at least per country and the national level, to divide efforts in a way that's equitable on all levels so there could be too many interventions happening on one topic, let's say reproductive sexual health, just an example. and too little happening on mental health. so if efforts were combined and property divided, i feel it would be more beneficial." (a ) even within academia, an informant mentioned that the current 'academic silos' coupled with the "fragmentation of academic disciplines" and 'over-specialization' of research areas is a major limitation for interdisciplinary research in lebanon. on the other side, most of the informants considered that policymaking is not evidence-based, relying on what are called 'cold' experts (the opinions of ministers, public personalities, funders, etc.) rather than empirical research. however, an informant from moph blamed this lack of coordination on the academic institutes that "live in their own bubble" and disseminate their findings in publications that policy makers have no time to read. the informant highlighted the importance of involving policymakers in a research from the early stages to improve the collaboration. "for example will we (moph) know about the academic research when it is done? its recommendations? there is no link... they present their findings in conferences and then say policymakers are not responsive .. how would we know about the research, we don't have time… policymakers should feel that they are setting research agenda and are more involved in the research process" (m ) for many, the main challenge is not the lack of individual research capacities, especially when it comes to quantitative research skills, but more the infrastructural capacities (database) and the availability of research funding. funding and sustainability have been identified as two of the main interlinked challenges for capacity strengthening for conflict and health research. informants argued that sustainable capacity strengthening is largely determined by the sustainability of funding. for laboratory sciences specifically, building of individual capacity in microbiology, for example, is limited when basic research infrastructure and sustainable funding for technologically and labour-intensive laboratory-based research is lacking. at the core of this in lebanon is the near absence of local funding, and the high dependency on highly competitive international grants and schemes. researchers describe competing for rigid international grants and research funding, while working in highly unstable research settings, require a high level of flexibility and adaptability. most institutions struggle to pay competitive salaries for pharmacists or medical doctors to conduct and generate evidence-based research. most of the informants indicated that adapting research questions and methods to secure funding was fairly common. such a high level of instability is also a disincentive for long-term planning. "naturally that's what happen most of the time… we have to go work with whatever the market is." (a ) however, few informants mentioned that funding is not an issue if a well-designed project is prepared especially for internal funding provided by universities or by local industries. "if we prepare a decent research protocol. i think that we can get funds.. even the scientific associations, if there is a good protocol... money is not an issue" (a ) "my motto is if you have the will you have a way…i think the other factors become secondary…. so funding could be a factor, but again it's not the major barrier or challenge." (a ) as for the humanitarian sector, according to informants, funding for humanitarian relief and operational research has slowly decreased, potentially leading to increased competition among local ngos that would limit even more the coordination among these humanitarian actors. as for international ngos, fewer resources are allocated to them in lebanon and thus they are currently withdrawing slowly. when asked about women's involvement in health research in lebanon, most of the informants, regardless of their gender, indicated that discrimination is not a major issue as women are capable of reaching high positions in academia. "representation of women at our institute is fantastic, more than %; % of researchers and teachers are women, out of deans are women and out of are vice presidents" (a ) most of the respondents were reluctant to consider lebanon as a conflict setting as it is not struggling with ongoing armed clashes. but they considered lebanon as a conflict-affected setting due to the huge influx of syrian refugees. at the research and development level, most of the informants agreed that being a conflictaffected country has led to an increase in research funding and opportunities. however, most of the research funded was focusing primarily on refugees. "i guess what happened is that many people started doing research related to the crisis because there are opportunities or there is money with this coming in… it has brought some opportunities like the syria lancet commission, which has been great for aub…"(a ) host communities were also considered in both health research and humanitarian interventions which made them benefit from getting access to services that were not prioritized before the syrian crisis, especially services for child protection and those that address gender-based violence. despite providing research opportunities and employment for locals researchers who obtained field experience, an informant highlighted that locals "learned by doing, by being exposed, rather than learning principles of intervention in an academic normal pace setting" (ngo ), which due to the lack of local expertise led to "humanitarian efforts in all of the conflict affected settings in mena to be led by international non-arab experts who do not necessarily understand the context" (ngo ) which might lead to inconsistent interpretations. another factor that was raised by most informants but not considered in the topic guide, was that the basic medical training provided in lebanese universities, does not include treatment of war injuries, trauma, health provision in conflict settings, or health research in such settings. public health professionals are able to conduct research in conflict settings but most medical doctors have limited research skills needed for such a context. clear recommendations regarding more effective health research capacity strengthening interventions that target the individual, organizational and institutional levels were indicated by many informants. regarding better regulation of research, particularly research ethics; workshops on ethical research especially for local ngos were recommended. to address limited coordination between academic bodies and ministries to provide evidence-based policies, roundtables and working groups to discuss priorities of health research with the different stakeholders including policy makers, local ngos, international ngos and academics were thought to be a top priority going forward. "it is important to have a research group to work on setting priorities in order not to have irrelevant research. the results should be then summarized in a policy brief a maximum of pages with direct and simple recommendations to send to policy makers." (m ) in order to have more sustainable funding schemes, it was recommended by most of the informants for research groups to be involved in multidisciplinary and context-specific consortia or research networks like the reproductive health working group and the lancet palestinian health alliance [ ] . those networks would also provide the opportunity of capacity strengthening of researchers via training and mentorship as they go to other countries with advanced technologies to be trained with the latest research developments. capacity strengthening activities for the humanitarian actors and graduates with medical and health backgrounds need to focus on basic qualitative and advanced qualitative and quantitative methods. an informant also added that qualitative research especially going beyond focus groups and interviews and into 'cutting-edge' tools is needed to benefit both communities and researchers (the example given was story-telling as a research method that may also have benefits to research participants). when asked about the type of training to be provided, most informants suggested that accredited certificates in health research methods are highly desirable for all as an incentive for participation. similarly, introducing a module about medical practice in conflict that addresses blast injuries, trauma, and medical practice under severe conditions was also recommended and considered to be an urgent need given the geopolitics of the mena region. when asked about teaching modalities of the certificate, most of the informants highlighted the importance of e-learning which has less burden on both financial and human resources and would be highly appealing especially if it is accredited. however, most agreed on having a blended learning course, rather than purely elearning, which might not provide the most effective learning experience especially to future fieldworkers. an informant also mentioned the possibility of having training that is fully distant but with innovative tools that make the courses more interactive. "i think, it opens doors to expertise that maybe we don't have that you can get from other universities…. i think it is great when you're talking on the theoretical level… but to go from there to design a research study that is a big step …. it saves time, money and human powers and then we can focus the energy on things that need this human interaction." (a ) in this study, we have provided an overview of capacity needs for health research on conflict in lebanon based on strengths and challenges at individual, organizational and institutional levels which are summarized in table . however as these levels are strongly interconnected, any strength or weakness in one of these levels will directly impact the other two levels [ ] . most of the findings are consistent with the international literature on research capacity strengthening in low and middle income countries (lmics) including those affected by armed conflict and political unrest [ , ] . the key themes emerging from this work are categorized below as strengths and challenges of health research which would support the design of impactful research capacity strengthening interventions. one could argue that these strengths and challenges impact research in general and are not limited to health research, with few exceptions including qualitative research designs for health research topics, ethical conduct of health research, and preparedness to practice medicine in conflict settings. therefore, using the same approach, suggested in this study could also improve research in conflict-affected countries at the individual, organizational and institutional levels. however, further research is required to validate this assumption, especially as strengthening research requires more financial and human resources. this would result in additional challenges and constraints where the suggested approaches herein might prove invalid especially that the inclination to health research in low-resource humanitarian-oriented conflict settings might not be applicable to other types of research or for research as a whole. the needs assessment highlighted several enablers within the lebanese context that can contribute towards impactful capacity strengthening interventions. for instance, both researchers (especially early career researchers) and institutions are incentivized to improve their research capacities and outcomes for various reasons. the former is driven by the need to increase their job prospects in a highly competitive job market and the latter to improve their reputation as centers of research given that there are universities in lebanon, one public university hosting , ( %) students and private for-profit organizations hosting , ( %) students that mostly rely on student fees [ , , ] . therefore, despite being a relatively small country in terms of size and population, lebanon has recently been ranked as the third in the east mediterranean region (emr), and mena, after kuwait and tunisia in terms of the number of publications on biomedical and health research per capita [ ] . it is also important to note that there are huge disparities among the universities in lebanon with some being considered as centers of research excellence within the region by contributing most significantly to the country's research outputs and to setting considerable momentum to continuously improving their research capacities. for instance, most of the research outputs originate from the lebanese university, usj and aub with the latter contributing the most and is being ranked the th in biomedical and health research output in emr [ , , ] . lately, the lebanese highly privatized educational system has shown resilience to adversity, at least in the short term. following the country's lockdown to control the spread of covid- , academic institutes have shifted to online learning. vast efforts are underway to provide innovative distant learning approaches that could provide similar levels of academic experience as in face-to-face learning modalities [ , ] . the current attempts to create a reliable online learning platform is an opportunity that can be used for a capacity strengthening intervention to be more inclusive to include those residing in ongoing armed conflicts [ , ] . another opportunity which universities are taking advantage of is the increasing trend of partnerships between local and international academic institutions which increases bidirectional knowledge sharing. for instance the country, along with jordan, morocco and tunisia, has the highest levels of co-authorship in mena [ ] . partnership has already been considered a pillar in capacity strengthening of health research in other settings as it supports collaborative, multidisciplinary and multi-sectoral work which in theory is aimed at addressing power imbalances and inequities [ ] [ ] [ ] [ ] . although lebanon is ranked the th out of for the global gender gap index [ ] , it was interesting to learn that almost all informants, regardless of gender, indicate that the health sector, including research, provides a permissive environment for female researchers to work and advance in their careers (as several deans and vice chancellors of research are women). however, health and education are traditionally considered femalefriendly compared to other sciences technology engineering & medicine (stem) disciplines, so the results reflected in this study cannot be generalized or considered on a larger scale. indeed a recent report on women in academia in the arab world found that women lead fewer than % of higher-education institutions [ ] . thus more research needs to be done on this topic especially that another recent study highlighted a high level of exploitation and alienation of research assistants, who are mostly women, that are working on uk funded research projects focusing on syrian refugees in lebanon. the study emphasized on how the hierarchy in academia, which was also mentioned by few of our informants, could reinforce cultural and academic inequalities including gender [ ] . despite of the variation in the findings and building on the informants' responses, this factor can be considered as an opportunity for both the health research field and the cause of lebanese women in general as having more women in the field in leadership positions will put women's issues at the front of the health research agenda and advocate for more inclusive and diverse research [ ] . despite the opportunities and strengths in terms of human capital, research capacities of certain universities, and the perception of improvement within academia, there are several challenges, besides political and economic instability, that impact health research in lebanon. a factor which was highly expected was the lack of research culture at the country level which limits the impact of any health research despite its importance and relevance to local challenges. unfortunately, this is an issue which the whole region is struggling with as only . % of the global biomedical research output originate from the region [ ] . research culture could be improved and developed by joint efforts between policy makers and the institutes of higher education to provide opportunities for research grants, local and international collaborations, cross-sectoral knowledge transfer, and skill acquisition [ ] [ ] [ ] . it can also be promoted at the community level by publicizing research through the media including social media [ , ] . the issue of research culture leads to the other challenges of health research and its strengthening in lebanon. for instance, there is a problem in crosssectoral knowledge transfer in emr, including lebanon, where there is a lack of both demand for and supply of relevant research which could be attributed to political forces, political sensitivity of findings, limited funding and limited opportunities for interaction during the policy-making process [ ] [ ] [ ] . closing this gap, by engaging policy-makers in setting research agendas to meet their needs, is a key challenge for the health research system where even the best studies are rarely translated into action [ ] [ ] [ ] . in lebanon, there are a few centers that directly engage with policy makers to advocate for evidence-based policies like the lebanese center for policy studies, issam fares institute-aub, global health institute (ghi)-aub and knowledge to policy center (k p)-aub with the last two focusing primarily on health. these centers have been greatly involved lately in issuing policy briefs on political, economic and health system situation in lebanon following the october uprising and the covid- outbreak [ ] [ ] [ ] . the chronic care center (ccc)'s β -thalassemia prevention program that was launched in , in collaboration with mosa and moph, is one of the few examples highlighting the benefits of direct collaboration between policy makers and health and research centres [ ] . key recent evidence use for policy in the country include passing of tobacco control law and introducing a reporting mechanism for occupational violence as part of the accreditation criteria of private lebanese hospitals [ , ] . however, given that in crisis zones four systemspolitical, health, international humanitarian aid and health researchare involved in advocating for evidence use, there is a need for further national level action by engaging multiple stakeholders to set research priorities [ ] . achieving efficient multiple stakeholder coordination will also contribute positively to generating and sharing reliable data across the four systems and with providing additional funding opportunities which would mitigate both of these constraints to health research in lebanon. with regards to funding, according to the cnrs, the country allocated only . % of its gross domestic product (gdp) for research and development in [ ] . as a result, universities in the country rely primarily on international funding schemes to support their research activities along with allocating part of their budgets for this purpose. the amount of money allocated varies remarkably among the universities in lebanon with aub being the lead, followed by the lebanese university and usj [ ] . it is due to external funding that the universities in lebanon are focusing on conflict and health research as it is an agency research priority [ ] . given the current drastic economic situation of the country, it is expected that universities will depend even more on international funding resources as they might not be able to allocate the same percentages of their budgets to fund research given that the number of registered students in private universities will severely decline. thus, the issue of sustainable funding of health research remains unresolved until the lebanese government is capable of adopting a research strategy that supports health research at the national scale. another serious issue, ethics in research, which was highlighted in this study is also linked to cultural factors. in the mena region, and lebanon, there is a strong collective orientation where the community plays a significant role in an individual's life; resultantly achieving informational and decisional privacy of research participants could be challenging [ ] . obtaining consent, in its western notion, from populations with multiple vulnerabilities such as syrian refugees is also problematic given the coercive societal dynamics and context. so despite a thorough irb process, conducting refugee research in practice remains ethically challenging [ ] . nonetheless, lebanon lacks a unified system of research governance, and hence research regulation is greatly influenced by the policies of individual institutions and their irbs [ , ] . obtaining unified guidelines of ethical research in lebanon faces multifaceted complications so a direct and short-term mitigation plan would be to train fieldworkers working with local ngos to understand better the concepts of privacy, confidentiality and consent while still adhering to the lebanese context. interdisciplinary research is crucial in understanding and resolving complex public health problems especially in conflict [ , ] . qualitative and quantitative research methods are required to provide an enhanced understanding of the causes and consequences of conflicts while emphasizing personal experiences and narratives of those struggling from these conflicts is important [ , ] . however, interdisciplinarity is not a strength in health research in lebanon despite having few funding opportunities for such initiatives [ , ] . human and social science research is significantly ignored as a research study indicated that only % of cnrs and % of aub research support goes to this field [ ] . qualitative research skills, at both undergraduate and postgraduate levels, appears to be a central weakness in the lebanese educational system. in addition, students with social sciences background also struggle with quantitative research skills. such weaknesses vary significantly depending on the university's teaching and research capacity which, as highlighted earlier, vary tremendously. the concentration of research in few private universities, with relatively high tuition fees, limits the opportunity of graduates from other universities, and from more diverse socio-economic backgrounds, to be trained in conducting conflict and health research of high quality. this heterogeneity in health research training can be mitigated if graduates, regardless of their alma mater, are provided with extra-curricular/ stand-alone courses on interdisciplinary research methods in conflict that are suitable for a wide range of participants from different academic backgrounds. while the initial focus was mainly on health research in conflict, this study also exposed a new fragility within the medical curriculum in the country. medical graduates are not prepared to practice in conflict settings despite the fact that the region, including lebanon, is highly vulnerable to instability [ ] . taking into consideration that any changes in the medical curricula across lebanon would be challenging due to logistics and difficulties in reobtaining accreditation from the ministry of higher education, specific postgraduate training could fill this gap by introducing training that focuses on medical practice in conflict settings. this would be taken by individuals interested in practicing in conflict-affected humanitarian settings across the mena. the international committee of the red cross (icrc) has war surgery courses which could benefit local practitioners [ ] ; however, these courses are limited in scope as they focus exclusively on medical students at the lebanese university. despite this study's focus on lebanon, the challenges and strengths of the country's health research capacities presented herein are in accordance with similar studies that focus on health research in the mena/emr region especially in conflict-affected settings. the recent work of alkhaldi et al. that focuses on research capacity strengthening in palestine, also revealed that health research capacities in the country are relatively weak despite the recent increase in health research production. guidelines for health research quality and standardization are not adhered to due to a lack of policies and resources, and health research knowledge transfer is a major challenge that requires contextualization and adequate implementation [ ] . regarding research skills, a needs assessment focusing on the west bank, palestine, published by r hc-mena colleagues, found that most of the respondents considered that they have a significant gap in their abilities and understanding of coding and analysis of qualitative data, and planning and designing research [ ] . this is also supported by our study's findings in terms of the need to focus more on qualitative research designs in lebanon. moreover, a recent bibliometric study focusing on research activity on non-communicable diseases (ncds) in the mena found that there has been an increase in research activity in the region that is heterogenous with respect to wealth, population size and type of ncd but with no clear correlation to the corresponding disease burden. the study suggested that international collaborations can support overcoming problems such as a lack of suitably trained researchers, low political commitment, and poor financial support; all of which have been indicated in our study [ ] . similarly, the paucity in health research and training which are contextualized to conflict settings is also a wider mena region problem as "emergency preparedness" is one of the least studied fields in the emr/mena regions- . % journal papers published in the field of public health research between and [ ] . one of the main limitations is perhaps that the findings may not be generalizable outside the lebanese context, although a follow-up comparative study focusing on mena would enable broader interpretations of the key research objectives. however, the high level of consistency from the key informant interviews of the same participant type and the similarity between the findings of this work to those of quantitative studies focusing on health research in the mena provided meaningful context. another limitation is that most of the refusals or unanswered invitations were from the public sector informants. therefore, there might be other themes that would be more relevant to the public sector that this study did not reflect due to the low number of participants from the public sector. however, those interviewed provided similar feedback to that of the other categories. promoting inter-sectoral communication, which is one of the major recommendations of this study, would mitigate this problem in future research as more informants from the public sector will be more willing to participate in research activities. in summary, the study revealed that health research in lebanon is characterized by considerable strengths in terms of human capital and research capacities of certain universities. however, there was considerable concern regarding research ethics among most informants, and workshops on ethical research especially for local ngos were recommended. guaranteeing sustainable funding of conflict and health research which was highlighted as a major challenge is problematic given the current economic crisis of the country and its lack of research strategy at the national level. thus strengthening partnerships with international collaborators is the only way to secure funding at least in the short run. the impact of research on policymaking was also another recurring theme where organizing roundtables to discuss priorities of health research with the different stakeholders was suggested as a way forward. for strengthening research capacity at the individual level, a great amount of interest around research methodology, conceptualizing and design was expressed. moreover, providing knowledge about medical practice and dealing with war injuries was also considered as a need to fill the gap in the medical training provided at universities that do not currently focus on this aspect in medical training. accredited certificates were considered as an incentive for participation and a combination of elearning and mentorship was identified as having the most potential impact. these findings will be useful for guiding research capacity strengthening approaches within large international projects that will include developing academic courses, engaging with ngos and policymakers to improve capacities for conducting useful research, and delivering trainings on health research skills in conflict settings. the study herein, supported by qualitative research methods, provides comprehensive insight and complements the findings of several quantitative bibliometric analyses of health and biomedical research in lebanon and the wider mena region [ , , , ] . supplementary information accompanies this paper at https://doi.org/ . /s - - -x. world health organization, the who strategy on research for health an evidence review of research on health interventions in humanitarian crises. london: 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mixed methods research in the study of political and social violence and conflict daad. foreign + foreignness: interdisciplinary research in the bekaa. beirut: daad announcing the sheikh khaldoun bakri barakat-ibsar research fund for interdisciplinary research in the natural and social sciences medical schools in times of war: integrating conflict medicine in medical education training needs assessment for mental health research in war and conflict: the west bank, occupied palestinian territory. birzeit: r hc-mena the profile of non-communicable disease (ncd) research in the middle east and north africa (mena) region: analyzing the ncd burden, research outputs and international research collaboration we would like to thank ms. marilyne menassa for her technical assistance.authors' contributions ne has led the data collection, analysis, and interpretation and wrote the first draft of the manuscript. gha has contributed substantively to data analysis and interpretation. ar contributed to data collection and wrote sections in the manuscript. ek has contributed to data interpretation. ap designed the topic guide for the semi-structured interviews. ap, km and ae provided critical feedback and helped shape the manuscript. pp, gha and gas critically reviewed the manuscript. pp oversaw the project, contributed with literature, and provided analytical feedback for the discussion of the paper. all authors have read and approved the final manuscript. the datasets generated and analysed during the current study are not publicly available due to them containing information that could compromise research participant privacy/consent but are available from the corresponding author on reasonable request.ethics approval and consent to participate ethical approval to conduct key informant interviews for the study was granted by ethical review committee at the american university of beirut on january (reference number sbs- - ). we obtained consent from all participants either verbally or by email. we anonymized all transcribed interviews. the consent included the acceptance to participate in the study and for the data retrieved to be presented in publication in an anonymized format. not applicable. the authors declare that they have no competing interests. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - h pg b authors: klingelhöfer, doris; braun, markus; brüggmann, dörthe; groneberg, david a title: coronavirus: an insight into global research until outbreak of covid- and its implications for the future date: - - journal: journal of global health doi: . /jogh. . sha: doc_id: cord_uid: h pg b background: the currently prevailing global threat of covid- caused the publication numbers on coronaviruses to explode. the awareness of the scientific and public community is enormous. but what about the sense of all these undertakings and what can be learned about the future for a better understanding? these questions were answered with established bibliometric analyses of the time until the avalanche of publications unfolded. methods: chronological, geographical aspects of publication output on coronavirus were also evaluated under the influence of epidemiological and socio-economic parameters. results: the trend in publication and citation numbers shows the strong influence of the past pandemics sars and mers with an untypical decline afterward. research is becoming increasingly multidisciplinary over time. the usa and china, as the countries with the highest number of publications, are being displaced by other countries in the consideration of socio-economic and epidemiological aspects, which shows the effect of regional interest in corona research. a significant correlation was found between the number of sars cases per country and related publications, while no correlation was found for mers cases and articles. conclusions: the results underline the need for sustainable and forward-looking approaches that should not end with the containment of covid- . in line with the aphorism: "only those who know the past can understand the present and shape the future", the present study provides background information of research on coronaviruses (cov) as a basis for the scientific situation of the global pandemic covid- and a source for a better understanding of research patterns from the time before covid- . viewpoints research theme : were recognized as pathogens causing only mild infections such as common cold and their clinical significance was not recognized. by , sars had infected more than people in countries and killed people. the pandemic ended abruptly, and no cases occurred later [ ] . at an animal market in china, the sars-cov pandemic was found to be transmitted by palm civet cats [ ] . more than % of all cases worldwide occurred in china. the second cov pandemic mers (middle-east respiratory syndrome) occurred in , but unlike sars, mers did not end suddenly and cases continue to be recorded until now [ ] . mers affected more than people and caused deaths in countries by , according to heath authorities worldwide. more than % of the mers cases occurred in saudi arabia, transmitted by dromedary camels [ ] . later studies have shown that bats are reservoir hosts for both of these former cov diseases [ , ] . the current extremely rapid global spread of sars-cov- has led to the highly dangerous outbreak of the pandemic covid- with daily increasing numbers of new infections and deaths around the world. at this point in time, the peak of the infections has not yet been reached, so the final figures are not predictable -but it seems that they will reach devastating proportions and will certainly change world societies forever. there are no vaccines yet, and treatment options are still limited. only symptomatic treatment or support is possible [ ] . due to the enormous spread of infections and the severe course of many cases, the appearance of covid- is accelerating research, which is certainly unique in the history of science. almost real-time results provide new insights from all areas of science, from basic to applied research. the financing of cov research is also picking up speed. for the dissemination of this rapidly generated knowledge, international communication is obligatory, and the most common way of disseminating it is the publication of results [ ] . can we learn from previous research patterns regarding cov? what influence do they have on future research? how can we use past efforts, their intensification and the influences of research on cov positively to better understand the needs for sustainable and appropriate research? these are compelling questions in light of the currently exploding research output, which, in addition to interesting and meaningful approaches, appears in part excessive, arbitrary and not scientifically sound. although many scientists around the world are giving their best to solve and improve the pandemic, the current development also gives rise to the pressure to be the first to find the solution and not to be overtaken by colleagues. against this background, it is very important to know about previous research. therefore, we have analyzed the global research output on cov in the time before covid- . in the present study, established bibliometric parameters on chronological and geographical aspects were combined with state-of-the-art visualization techniques based on density equalization principles. additionally, socio-economic, scientific and epidemiological parameters were related to the publication numbers to obtain an even more meaningful picture of the global landscape of cov research. the results may help to find more adequate approaches for future research in the financing, planning, implementation and networking of research based on quality and sustainability. the present study belongs to the established bibliometric platform new quality and quantity indices in science (newqis) [ ] , which examines a broad range of biomedical questions with regard to their translational utility [ ] [ ] [ ] . the underlying methodology is constantly evolving and adapted to changing circumstances in terms of global scientific, political and socio-economic characteristics [ ] . standardized bibliometric parameters are combined with newly integrated indices and figures on the research topic in order to analyze and discuss the research landscape appropriately. in combination with state-of-the-art visualization, the results are provided convincingly. the default database for retrieving the needed metadata for all newqis studies is the core collection of web of science (wos). not only because of its status as one of the leading online databases for scientific literature, but also because of its listing requirements, which allow only quality work. moreover, the provision of the journal citation report (jcr) enables the analysis of citation-based parameters by specifying all citations received for each publication. viewpoints research theme : covid- pandemic the search was performed at / / . to record the respective articles dealing with cov, the elaboration of an adequate search term is mandatory. the aim is to include as many related entries as possible and to exclude the false entries from the analysis database. therefore, the term must be a combination of all variants or synonyms of the names of the virus or its transmitted diseases. in order to find as many entries as possible, the resulting term was: "*corona virus" or "*coronavirus" or "sars" or "mers" or "covid- " or "severe acute respiratory syndrome" or "middle east respiratory syndrome". wos offers different modes of search. we applied the title search mode here, because a lot of incorrect entries occur when searching with the topic search mode that also includes abstract and keywords. to further decimate unrelated entries, mostly due to different meanings of the abbreviations sars and mers, a specified topic search was added and combined with the boolean operator "and" searching for the terms: "virus" or "epidem*" or "cov" or "co-v" or "covid- " or "patient*" or "*coronavirus" or "severe acute respiratory syndrome" or "middle east respiratory syndrome". this strategy ensures content linkage by querying the occurrence of one of these terms either in the abstract or the keywords of the publication. the resulting entries were subsequently filtered by the document type "articles" to include only original papers in this study. the metadata of the articles found in the manner described above were downloaded and recorded in a database that provides a variety of parameters according to the key information encoded by the wos tags. their analyses refer to chronological and geographical parameters, using data on the number of articles in relation to, eg, publication year or country of origin. other advanced parameters include socio-economic [ ] [ ] [ ] and epidemiological data [ , ] . for the epidemiological evaluation, sub-analyses were performed using the same search term, with the only difference that it was reduced to either sars or mers. furthermore, institutions and research foci were analyzed and the international network was presented. it has to be noticed that the sum of all assigned subject areas of wos must be higher than the number of articles due to multidisciplinary journals assigned to more than one subject area. in this study an article with more than one subject area assignment to assigned to each area and therefore counts several times. citation-based analyses provide information on the recognition of the articles in the scientific community. a threshold was applied to all valuation ratios to reduce distortions of extremely low values, eg, citation rate, and socio-economic ratios. in this way, a concise picture of the global landscape of cov publications and their development could be created. the geographical results were partially visualized with density equalizing map projections (demp) that are distorted maps according to an algorithm developed by gastner & newman [ ] . depending on the value of the evaluation parameter, the countries were either enlarged or reduced in size according to the physical principle of density equalization. using the vosviewer application [ ] , the results of the keyword analysis to determine research priorities are visualized with a network diagram showing clustered nodes and connections for all terms that occurred at least times. every scientific methodology has some limitations that must be reported on. in this case, the first limitation to be mentioned is due to the characteristics of the data source, as wos does not list all publications in its core collection. wos requires special recognition for listed journals in order to ensure quality, but this led to the fact that some important articles on cov could not be included in the analysis as they are not provided by wos. the title search strategy applied in this study further reduces the database. the advantage, however, is that the included data represent a representative collection that can clearly be used for a valid evaluation. furthermore, the wos is said to give preference to english literature, so that the resulting dominance of english-speaking countries is supported by this fact. another point that should be mentioned is the manual correction method for metadata on institutions and authors. for this, a threshold had to be introduced to make the procedure feasible. as a result, the exact number of institutions and authors publishing on cov could not be given. however, the leading institutions and authors could be determined exactly in this way. viewpoints research theme : considering the limitations of citation analyses and their impact on meaningfulness in terms of the quality of publications, the use of several citation parameters is appropriate, which gives more weight to the importance of the analysis results for the resonance of the examined publications in the scientific community. in summary, the applied method has proven to be a valid strategy for the evaluation of bibliometric scientific questions. a total of articles (n) on cov could be added to the database and form the basis for all analyses except those valid for geographical analyses. analysis of the keywords used (threshold value: occurrences) revealed four thematic clusters of cov articles (figure , panel a) . first, the molecular and biological topics form a cluster (red). the second cluster (blue) outlines the articles dealing with the sars epidemic, and the third cluster (yellow) combines the articles dealing with the mers epidemic. the fourth cluster (green) forms an intermediate group that mainly focuses on the spike protein that is characteristic of cov, its pathogenesis, and its connection to the other clusters. in terms of subject areas, the most frequently assigned research fields are virology (n = ), infectious diseases (n = ), veterinary sciences (n = ), microbiology (n = ), and immunology (n = ). looking at the developments over time, it can be seen that the research has become increasingly multidisciplinary ( figure , panel b). especially since the beginning of the s, a change can be observed. veterinary sciences and virology were relatively declining. the relative frequency of articles assigned to infectious diseases increased from this time onwards. other areas of cov research also became more popular. the first article about cov found in this study was published in . from then on, articles were pub- it is obvious that the articles from deal with the new cov and the related disease called sars, which appeared year. working groups from the usa, china, southeast asian countries, germany, france and the netherlands identified and characterized the new virus and its association with sars [ ] [ ] [ ] [ ] . canadian scientists worked on the genomic sequence [ ] . viewpoints research theme : in , the mers coronavirus appeared, leading to this year' s high-ranking publication of dutch and saudi arabian scientists [ ] . the article, which ranks th , stated that bats are natural reservoirs for sars-covs [ ] . this was a collaboration between china, australia and the usa. all ten most frequently cited articles were published in renowned journals. the new england journal of medicine and science published four of them each. one each was published in the lancet and nature. the necessary information about the country of origin can be collected through affiliation data. before this information is not always available and the gaps are too large to be used. exactly n = articles could not be assigned to any country of origin, so that n = articles are included in the geographical evaluations. from onwards, the usa was the country with the highest number of publications for the entire evaluation period (n = ), followed by china (n = ). by a larger margin, germany ranked third (n = ), followed by canada (n = ) and the united kingdom (uk) (n = ) (figure , the leading countries were also represented by the leading publishing institutions ( table ). in terms of citation numbers (c), the landscape looks similar to that of article numbers (figure , when looking at the world map when socio-economic parameters are included in the analysis, the distortion of country sizes shifts again. regarding the ratio of the number of articles and population size in million inhabitants (rpop) of countries with at least articles on cov (threshold) the ranking another socio-economic parameter, the relation of the number of articles on cov to the gross domestic product (gdp) in billion us-dollars (rgdp) for countries with at least articles (threshold), shows a similar ranking. the rankings of both socio-economic analyses were compromised in table . the inclusion of parameters referring to the research infrastructure of the countries considers two values. first, the gross expenditure for research and development (gerd) in billion ppp$ (purchasing power parity in us-dollars) and the number of researchers in million fte (full time equivalents). again, the number of articles on cov for countries with at least articles (threshold) was set in relation to these parameter (rgerd, rres). table ). the socio-economic parameters gdp, gerd, and number of researchers were significantly correlated with the number of cov articles (p < . ), with correlation coefficients (spearman r) ranging from . to . . in order to show the publication performance of cov-affected countries in relation to the sars and mers epidemics, the relationship of articles on sars or mers to the respective cases per country was analyzed. according to who [ ] sars cases occurred until the epidemic came to an abrupt halt. the sub-analysis of the present study resulted in sars-related articles corresponding to the affected countries. table in total, the who reported cases of mers worldwide occurring with smaller boosts until today [ ] . articles could be clearly assigned to mers. table viewpoints research theme : of the total number of n = articles for the geographical analyses, n = articles were prepared in international cooperation. the maximum annual international partnerships for cov research took place in (n = , first publication peak) and in (n = , second publication peak) (figure , panel a). in principle, however, an upward trend can be observed. the usa was at the center of the international network and was involved in the five strongest international collaborations (figure ) . the most productive bilateral cooperation in cov research was between the usa and china with n = cooperation articles, followed by usa/canada (n = ), usa -uk (n = ) and usa -netherlands (n = ). the non-us partnership with the highest publication volume was between the netherlands and germany (n = ). especially since the beginning of the covid- pandemic, which is causing many serious cases and deaths in many countries, the importance of research on cov has become clear. basic research to date, the identification of novel viruses of the two pandemics sars and mers, has influenced global research efforts. as more is learned about the background, incentives and impact of research, the impact of more focused and improved focus, planning, implementation, collaboration and communication of scientific projects becomes clearer, for all parties involved. this is also shown by the identified research foci, which were analyzed by the keywords used in the articles on cov. however, the proportion of human-related research has increased significantly since the outbreak of sars, and multi-disciplinarity has also become increasingly widespread over time. the currently rampant covid- disease and its impact on economic, political and social spheres will provide new impetus for cov research in other scientific fields in the future. the development of publication output on the cov clearly followed a different trend than that of other biomedical topics in general, which usually increase exponentially over the evaluation period [ ] and could be shown in other studies on viral diseases [ , ] . instead, the increase in the number of articles and citations corresponds to the occurrence of the two pandemics sars and mers in the past with peaks at the beginning of each. in , the year in which sars emerged, publication numbers rose at an unprecedented rate in science, with an increase of about % within one year. chiu et al. also noted in a bibliometric study on sars publications that the high publication rate at the beginning of the pandemic resulted in high citation rates due to the immediate recognition in the scientific community [ ] . now, years later, the present study confirms this extremely high recognition by the scientific community at the time of the sars outbreak, which even shows a number of citations per publication year, even before the maximum peak in publication numbers is reached. in , the highest citation rate was achieved with cr = . , which means that each article on the cov was cited about times on average. this is certainly an extraordinary result. almost at the same pace, however, the number of publications and citations decreases thereafter, resulting in a value that is below the average biomedical topic. this shows the decline of the scientific interest with the abrupt end of sars. the awareness of the importance of cov research as a preventive measure for future virus infections does not seem to have been present at this time. the progression curves of both publication and citation numbers showed a minimum level at exactly the time when the cited half-life (chl) of publications in biomedical publications is generally reached. basically, the chl is given as - years that a publication needs to reach half of the expected citations, resulting in a maximum peak of citations up to that point [ ] . the outbreak of mers in caused a second increase, but this time it reached only about one third of the sars-related figures, due to the lower incidence rates compared to the sars pandemics. it also showed a declining trend, but only four years after the outbreak. the longer period during which mers cases occurred also led to a longer lasting interest, but at a significantly lower level. the timeframe of the evaluation was set until march , so that the first months of covid- were included in the analysis. the expected exponentially increasing numbers could even be shown in this small time-interval by a further significant increase in the number of publications. the short-term effect of research efforts of earlier cov research and the strengthening of international networking could also be shown with regard to another emerging virus epidemic that occurred in south america in : the zika virus infection. the publication patterns corresponded to those reported here and showed an unsustainable short-term effect of national and international efforts [ ] . this similar publication pattern was reinforced by the enormous research incentives of short-term funding and public recognition due to the acute threat. this is also evident in the case of cov research, and the same influences must be considered in future approaches. the most frequently cited publications highlight the outstanding impact of the two pandemics and the years of their outbreaks. most of the ten articles dealing with the identification and characterization of the novel sars virus were published in , which shows the importance of the first articles on sars in the scientific community. one article deals with the isolation of the mers virus, which was published in , and another with the function of bats as natural reservoirs. these articles were from the countries that are considered to be the most publishing countries throughout the assessment period. viewpoints research theme : what is also true for the publication output of most life science and biomedical topics is the leading position of the usa in terms of the absolute number of publications. the rapid catch-up trend and the now high numbers of chinese scientists is also not unique for cov research. other bibliometric studies confirmed the ranking of the leading countries in terms of absolute publication figures. bonilla-aldana et al. found the usa publishing . %, china . %, and germany . %. saudi arabia, as the country most affected by mers, contributed only . % to cov publications [ ] . in comparison, the percentages revealed in our study were similar: the usa ( . %), china ( . %), germany ( . %), and saudi arabia ( . %). in , kostoff et al. found in another bibliometric study on sars the declining share of chinese and the increasing trend of us-american studies. they stated a higher percentage of highly cited publication authored by china compared to other research fields [ ] . these findings have been confirmed by the present results showing also the percentage decrease of chinese articles. although, a relative downwards trend of us-american articles could also be shown. this is due not only to the decline in absolute figures but also to the efforts of an increasing number of countries worldwide. covid- will certainly continue to influence this trend enormously. although the study by chiu et al. showed a low level of international collaboration shortly after the outbreak of the sars pandemic in [ ] , the pace at which international collaboration on cov was established thereafter was remarkable [ ] and will certainly continue. as third most publishing country, germany has been involved in cov research from the very beginning in the s, when only the usa and canada was interested in this topic. although germany had its highest share at the beginning of the s, it has been able to maintain its position among the three leading countries to date. the most cited german article by drosten et al. from ranks second in the overall research on cov and identified a novel coronavirus in a patient with sars [ ] . this was a german-french-dutch partnership. the following ranking consists of canada, the uk, taiwan, and the netherlands. all are involved in prominent research on cov and have sufficient scientific resources and infrastructure not only for research on cov. the netherlands -ranked th in absolute numbers -took a leading position when it came to the results of citation-based, socio-economic and science-related parameters. it also participated in three of the most cited articles and thus also in the identification of sars-and mers-cov as a partner country of germany, the usa, and saudi arabia [ , , ] . with a relatively high proportion of articles in the field of molecular biology, the dutch scientists contributed the main research on the structure of the spike glycoprotein, eg, they uncovered the three-dimensional structure [ ] . nevertheless, other countries have led the way with respect to the inclusion of socio-economic and science-related. thailand received the highest citation rate, although with n = it was only slightly above the required threshold of articles. thailand' s participation in the most frequently quoted article is therefore responsible for this high value. the article by kziazek et al. [ ] , which demonstrates the etiological role of a novel cov in sars, is a joint effort of the sars working group: usa, vietnam, china, thailand, singapore and taiwan. scientists from the center for disease control and prevention (cdc) worked in addition to them from the international emerging infectious disease program in bangkok (thailand) as partners in this study. this program was founded by the ministry of public health (thailand) and the cdc (usa) to establish a prevention and detection system that responds to emerging public health threats. vietnam was also a partner country in this cooperation, but with n = it was just below the threshold and was therefore excluded from the analysis of citation rates. it should nevertheless be mentioned here. scientists from the vietnamese regional division of who were involved in the research. singapore, which was also involved in this successful cooperation via the singapore general hospital, could also be highlighted concerning the analysis of socio-economic and science-related parameters. it received the highest scores in terms of rpop, rgdp, rgerd, and rres. chahour et al. [ ] conducted a bibliometric analysis of the first covid- publications, which confirmed the leading position of singapore in terms of its demographic characteristics. without applying a threshold, this study ranked mauritius first in terms of economic strength. we could not find an article from mauritius until march , and the chahour study found one publication from mauritius. this shows how important threshold applied in our study is to avoid overestimating countries with such a low publication output. with sars cases, singapore was one of the most affected countries. the resulting scientific interest and the possible in-si-viewpoints research theme : covid- pandemic tu investigation of the cases caused the publication figures to rise at the beginning of the sars disease and to fall rapidly thereafter. at the time of the covid outbreak, however, the figures from singapore rose strongly again. tan tock seng hospital (ttsh) is at the heart of sars research in singapore, a place where secondary cases mainly affected health workers [ ] . the epidemiological impact and the incentive of in-situ research promoted research on both pandemics of the past and showed the clearly centered focus of the publishing countries. the exploding publication numbers in and the second peak starting after are clearly caused by the outbreaks of sars and mers. therefore, we defined both the number of sars-and mers-related articles and set them in relation to the number of disease cases of each country. in the case of sars, china had clearly the most cases worldwide and had also written the most articles about it. this was followed by the usa and taiwan. however, the ratio of sars articles from china and taiwan to cases was relatively low. in general, the countries with the most cases did not reach the top ranks, as case numbers varied widely among publishing countries, ranging from more than in china to only one case in eight countries. therefore, spain and switzerland reached the highest ratios. but with only one identified article, these high rates are not surprising, as both countries were among the most publishing countries. the contribution of literature from both countries to the cov literature was relatively stable and has fluctuated only slightly since the early s at an average level of about articles per year. therefore, the publication efforts of both countries could not be directly linked to the outbreak of sars or mers. with regard to mers, most of the contributions came from the usa, which was also found in a study [ ] . saudi arabia produced the second most articles on mers. as far as the relationship between mers publications and mers cases is concerned, the situation is similar to that of sars. saudi arabia also achieved a relatively low ratio with most mers cases. here, the usa and china are the highest-ranking countries, demonstrating their overall interest in cov research and also focusing on the mers pandemic, despite the relatively low case numbers. egypt also came into the focus of this analysis because of the occurrence of only one case and the associated high ratio. however, egypt' s interest is directly related to the outbreak of mers, as it has seen a significant annual increase in publication numbers from that point on. the occurrence of mers-cov in egyptian dromedary camels and its similarity to the human type influenced the regional research interest [ ] . another causal factor is the strong cooperation partnership with saudi arabia and usa in cov research. in addition, egypt ranked th in the inclusion of gerd as a marker of economic strength and was the only country that did not have a high-income status among the top countries. the analysis by chiu et al. from [ ] found no evidence of a correlation between the number of sars cases and the number of publications in the publishing countries [ ] , while our results showed a significant correlation. the reason for this discrepancy seems to be the timing of evaluation. in contrast to chiu et al. who consider the beginning of publication activity on sars, the present study shows the results of development years after the appearance of the pandemic. during this period, the affected nations seem to have created an awareness of the importance of sars research and the international interest in cov research that responds to it. however, with regard to mers, our study could not demonstrate a statistical correlation between the number of cases in countries and the number of publications. this could be due to the low number of mers-related cases and the resulting centered interest. taking into account the unusual patterns of previous cov research, the current situation of covid- -related publication output must be evaluated and discussed accordingly. a short note on the most recent publication figures, which include the output of , already shows publications on the cov that were found with the same search term (as of . . ). of these publications, only are articles ( . %), which means that almost more than two-thirds of the publication includes other types of documents, especially editorial materials and early access, which are almost invisible in the previous period. as a rule, articles make up the largest share of document types. the most frequently assigned subject area in was general and internal medicine, which pushed back the fields of virology and infectious diseases as the most frequently assigned areas of previous research, viewpoints research theme : thus providing an indication of publication in more interdisciplinary journals. the field of public, environmental, and occupational medicine moved on to third place. these figures imply the enormous interest of the scientific community and the enormous willingness to publish. but it leaves one questioning the priority given to quality and shows the attitude of publishers to value the rapid publication of articles related to the cov. china has the largest share of articles in , followed by the us and italy. this is due to the advance of chinese science and the high number of cases in both china and italy. the results of the present study show the need for sustainable, valid and high-quality research in global cooperation to address not only the current pandemic but also future threats. the increasing divergence of research areas allows for more interdisciplinary approaches, which should be completely free of any blinkered ambitions or dogmatic reservations about the big picture. what concrete effects the current covid- pandemic will have on the global landscape of cov research can currently only be estimated. funding is rising exorbitantly, and the first figures for show an equally exorbitant increase in publications. as a result, the development of vaccines and effective therapeutic methods can be expected in the near future. but the question arises whether this is a short-term effect which, once covid- is contained, will lead to an equally sharp decline in publication numbers as was observed with the earlier cov pandemics sars and mers? the need for continued interest and research efforts worldwide is characterized by the characteristics of the past, which lead to a lack of basic knowledge about cov, about advanced therapies and to difficulties in the search for vaccines. the present results show the development and incentives for research in the period before covid- and underpin the need for awareness and sustainable international relations for best possible strategies and the benefit of scientific projects. this is also important in view of the fact that the effects of the prevailing climate change will influence the incidence of zoonotic diseases caused by cov through human and animal accumulation. this will certainly lead to the need to combat new groups of viruses in the future. therefore, sustainable and climate-friendly approaches must also be pursued in science, especially if they focus on the newly emerging aspects of cov research in relation to political and economic issues. annual review of diseases prioritized under the research and develpment blueprint detail/ - - -statement-on-the-second-meeting-of-the-international-health-regulations-( )-emergencycommittee-regarding-the-outbreak world health organization. summary of prabable sars cases with onset of illness form isolation and characterization of viruses related to the sars coronavirus from animals in southern china middle east respiratory syndrome coronavirus (mers-cov) any researcher with access to the web of science database can obtain the data using the methods described in the paper. readers that do not have access to web of science should contact clarivate analytics to obtain a license draft: dk; writing -review & editing: dk, mb, db, dag; visualization: dk. conflict of interest: the authors completed the icmje unified competing interest form (available upon request from the corresponding author evidence for camel-to-human transmission of 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analysis of covid- research activity: a call for increased output the outbreak of sars at tan tock seng hospital -relating epidemiology to control global research trends of middle east respiratory syndrome coronavirus: a bibliometric analysis mers coronaviruses in dromedary camels key: cord- -onc uw authors: siddiqui, urooj; hawryluck, laura; muneeb ahmed, muhammad; brull, richard title: same-day consent for regional anesthesia clinical research trials: it’s about time date: - - journal: anesth analg doi: . /ane. sha: doc_id: cord_uid: onc uw nan c oronavirus disease (covid- ) has changed the way anesthesiologists engage and interact with patients. as we hopefully approach the backend of this crisis, plans for the resumption of "nonessential" anesthesia services, including providing anesthesia for elective surgeries, reopening of anesthesia preoperative assessment clinics (pac), and recruiting for regional anesthesia clinical research trials, will take shape. the covid- pandemic has, however, highlighted a significant challenge in the current approach to research and the advancement of scientific knowledge in the regional anesthesia field: the perceived need to obtain consent to participate in such research in advance of the actual day of surgery. notwithstanding the low-risk nature of participation in most regional anesthesia clinical trials, subject recruitment on the same day as surgery is often prohibited by local research ethics boards (reb) due to their concerns regarding patient autonomy and perceptions of patient vulnerability immediately before surgery that could impact the voluntary nature and the rigor of the informed consent process. in many centers, the anesthesia pac has long served as the sole permissible and fertile ground for subject recruitment to clinical research, presumably ensuring fully informed consent to participate in a clinical trial in the absence of any undue duress and facilitating the establishment of a mutually trustful relationship. with the covid- -related suspension of in-person assessments in anesthesia pacs across most academic centers, recruitment for ongoing regional anesthesia clinical research trials has come to an abrupt halt and brought the long-standing controversy of same-day informed consent for low-risk clinical trials squarely back to the fore. the widespread reb concerns regarding same-day informed consent for participation in regional anesthesia research trials have not been supported in the current literature. even though anxiety in the face of impending surgery is a normal human reaction, patients are still presumed to be capable to continue to consent or to revoke consent to surgery while they wait to enter the operating room. there is no evidence that carefully conducted assessments of capacity to understand information pertaining to a research study and to appreciate how a choice to participate or not would apply to them cannot be performed in this period. there is no existing literature to suggest that patients are so vulnerable during this period that they must be systematically protected by prohibiting any discussion of potential participation in research in the immediate preoperative period. arguably, such a systematic prohibition is ethically problematic in that, on its face, it appears paternalistic and can deny patients the benefits of research participation. the anticipation of a second wave of covid- and the high likelihood of future pandemics from other emerging pathogens requires a more rigorous examination of such reb assumptions as prohibiting same-day consent to participate in regional anesthesia research risks stymying research and growth of this important and innovative field and fail in its goal to benefit patients in the perioperative period. the declaration of helsinki outlined the tenets of informed consent: competency, disclosure, autonomy. to ensure autonomy, subjects must offer their participation voluntarily, specifically, without any element of force, fraud, deceit, duress, or coercion. these tenets remain the pillars on which clinical research involving humans is founded and inform the respective canadian and american anesthesiologists' society's guidelines for the ethical consult of clinical research. , despite adaptations and updates over several decades, a single truth has prevailed: consent is paramount and, as with treatment, no research can occur without consent. chief among the roles of rebs is to ensure the consent process is rigorous and the autonomy of clinical research participants is respected. while the meaning of consent is both uniform and clear, that is, research participants must be capable of decision-making, fully informed, with ample time for consideration of options without coercion, and their choice must be respected, what constitutes ample time is not as clearly defined. the world health organization states that "subjects must be given ample opportunity to enquire about the details of the trial… sufficient time, determined by the patient's health condition." the tri-council policy statement, representing canadian standards for ethical research involving humans, declares that "for consent to be informed, prospective participants shall be given adequate time and opportunity to assimilate the information provided." in the united states, the american medical association is even less explicit, stating only that a valid consent process includes, "reviewing the process and any materials to ensure that it is understandable to the study population." locally, the university of toronto's position on "ample time" is equally vague, only to consider "whether the contact person is known to the subject/ authorized third party, has access to the patient information as part of their normal professional duties, or is able to assess capacity to consent." while there is currently no uniform explicit recommendation or absolute quantification for what constitutes adequate time for patient reflection before consenting to participate in a clinical research trial, , , hospital-based rebs are also given some guidance regarding how much time is inadequate. specifically, the canadian national council on bioethics in human research likens the practice of same-day consent to intimidation, coercion, and breach of autonomy. ostensibly equating quantity with quality, some rebs have strongly discouraged same-day consent practices, instead opting for a minimum of - weeks for patient contemplation, irrespective of risk involved in study participation. outside of north america, guidelines and recommendations on consenting practices for clinical research trials are similarly varied. the table summarizes available recommendations from various international professional societies and government agencies regarding consent practices in the context of clinical research. recommendations range from the oft-repeated requirement for "adequate" or "sufficient" time , , to a more explicit demand for at least hours for patient consideration. a notable deviation is the proportionate approach to seeking consent for clinical trials advised by united kingdom's national health service (nhs). when seeking consent for patient participation in a clinical trial, the nhs recommends that "for research involving only minimal risks and/or little deviation from normal/standard clinical practice… it may be reasonable to accept a decision taken at the time of approach." additionally, the extent of information provided ought to be proportionate to the "nature and complexity of the research trial, risks, burdens, and potential benefits, the ethical issues at stake." most physicians and surgeons meet with their patients on multiple occasions, affording these investigators time to identify, recruit, and enroll suitable research participants and obtain informed consent. however, specialties, such as anesthesiology, critical care, interventional radiology, and emergency medicine, have a varied pattern of practice and patient acquaintance that does not typically afford the luxury of time or, in many cases, delayed consent to research. indeed, the initial encounter between anesthesiologists and patients undergoing elective procedures routinely occurs on the day of surgery. recognizing our specialty's unique practice patterns, the canadian anesthesiologists' society's guidelines on the ethics of clinical research state that "preoperative consent for clinical research in anesthesia may be obtained after admission to hospital, either before or on the day of the scheduled surgery." yet an impasse is occurring in regional anesthesia with clinical investigators working in a time-limited perioperative system yet prohibited by rebs, both locally and otherwise, from consenting patients for clinical trials on the same day as surgery. , the question of patient vulnerability and need for protection in practice concerns of inadequate patient comprehension, time for contemplation, and privacy, as well as undue duress, coercion, and anxiety, continue to undermine same-day consent for regional anesthesia clinical research trials. these concerns, however, have not been borne out in the literature. when consent is obtained on the same day as surgery, the vast majority of patients do understand the intent of the clinical anesthesia trials and recognize that participation is voluntary and that consent may be withdrawn at any time without consequence. , patients are capable of digesting consent form documents and making informed decisions about research participation in thirty minutes or less. , similarly, most patients feel that the perioperative setting offers adequate privacy for consent discussions. purported coercion of patients by their clinician investigators in the immediate preoperative setting has also been refuted , ; anesthesia study found that % of patients rated the preoperative setting as "ideal" for obtaining informed consent to participate in clinical anesthesia trials. "it is good practice where possible to seek the service user's consent to the proposed procedure well in advance, when there is time to respond to the service user's questions and provide adequate information." "where the research entails only minimal risk, it is sufficient if the research offers the prospect of benefits either to the participants directly or to the group which is the focus of the research and to which the participants belong." "asking a service user to provide consent just before the procedure is due to start, at a time when they may be feeling particularly vulnerable, or seeking consent from someone who is sedated, in pain or anxious, creates doubt as to the validity of the consent." "where the research poses more than minimal risk, it should … offer the prospect of direct benefits for the participants themselves and be commensurate with the level of foreseeable risk." new zealand auckland district health board ( ) "sufficient time should be allowed for the patient to read the written information, and discuss this and any verbal information with whomever they wish." "the higher the probability of risk or the greater the magnitude of harm, the more care and detail in giving information is required." "the patient must be informed of rare risks that are more likely because of their particular circumstances, or which would have greater significance for that particular patient, for example, the consequences of arm nerve damage for a carpenter." united kingdom nhs health research authority ( ) "there are no definitive guidelines or legislation regarding the appropriate amount of time (or minimum amount of time) that potential participants should be allowed to consider whether to take part in research or not. a proportionate approach (in a nonurgent scenario) means that for more complex or burdensome studies a longer time may need to be provided for potential participants to consider their decision than that provided for simpler studies involving lower risks…for research involving only minimal risks…it may be reasonable to accept a decision taken at the time of approach." "a proportionate approach to seeking consent, that is, adopting procedures commensurate with the balance of risk and benefits, should always be adopted so that potential participants are not overwhelmed by unnecessarily lengthy, complex, and inaccessible information sheets but instead are provided with succinct, relevant, truthful information in a user-friendly manner that better promotes their autonomy." "the methods and procedures used to seek informed consent and the level of information provided should be proportionate to: -nature and complexity of the research -risks, burdens, and potential benefits -ethical issues at stake" abbreviation: nhs, national health service. www.anesthesia-analgesia.org same-day consent for regional anesthesia research the latter is most likely explained by patient preference for physicians with whom they will and/or must establish a relationship; accordingly, same-day consent by the responsible physician is likely superior to that by any surrogate. moreover, concerns of patient anxiety have not been realized as anesthesia researchers found no incremental increase in patient anxiety with same-day versus day-before recruitment and consent. finally, increasing the quantity of time for patient contemplation as a means to increase the quality of the informed consent process for regional anesthesia research has not been substantiated. moreover, nowhere in medicine is the direct relationship between vulnerability, quantity of time for patient contemplation, and quality of consent more poignantly questionable than in the intensive care unit (icu). rarely are patients (and their substitute decision-makers) more vulnerable than when a person is admitted to an icu with life-threatening illnesses. nonetheless and until proven otherwise, icu patients (or their substitutes) are deemed capable of making life-altering, and sometimes life-ending, and participation in research decisions in one or more moments of time. furthermore, similar to academic regional anesthesiologists, emergency medicine and radiology clinician investigators have limited interaction with their potential study participants, often meeting on a single encounter with no opportunity to recruit and consent their patients in advance of that encounter. recognizing these limitations, rebs allow for deferred, targeted, or staged consent in order for patients to participate in emergency medicine clinical trials. while such urgent or emergent adaptations to the standard informed consent process are not justified for the elective perioperative setting wherein most regional anesthesia clinical trials occur, the same is not true for the radiology research experience. indeed, low-risk radiology studies are generally approved for enrollment, recruitment, and consent on the same day as the radiological investigation or intervention. the radiology ("x-ray") department may be unlike the operating room environment with respect to heightened patient anxiety; nonetheless, parallels are readily drawn between these settings, including limited time and privacy, the potential for coercion, as well as the low-risk nature of many radiology and regional anesthesia clinical trials. one workaround to ensure a robust consent process and patient protection, adopted by many anesthesia research programs, including those at the university of toronto, has been the anesthesia pac. principally purposed to mitigate or optimize patient-related factors that may increase risk of perioperative complications, anesthesia pacs also function as the sole permissible venue (by our local rebs) for subject recruitment by research staff to low-risk clinical anesthesia research trials wherein subjects can provide informed consent days to weeks ahead of surgery. unfortunately, however, this long-standing workaround is fraught with challenges in appropriate recruitment of participants in that patients attending pacs are likely to be sicker and thus ineligible for study inclusion than those fitter patients who do not attend pac and are more likely eligible for regional anesthesia clinical research. while the idea of coordinating with surgical colleagues to have healthy patients referred to pac for the secondary purpose of study recruitment may be convenient for investigators, when balanced against creating inconvenience and lost income for patients, the use of hospital resources, health care dollars, and pac time constraints, the idea quickly loses appeal. another makeshift solution is preadmission telephone calls, which have been used to introduce research protocols and initiate the informed consent process. however, many institutions consider these calls a violation of patient privacy as research personnel callers are not yet within the patient's circle of care. furthermore, scheduling of calls, anxiety provoked from unsolicited calls originating from the hospital, and constraints in time and manpower represent important ethical and logistical challenges. , conceivably, the covid- pandemic may alter patient and provider views on telephone or videoconference as means to identify, recruit, enroll, and consent for research protocols. though the pandemic has already rendered telemedicine more applicable and acceptable to patients and practitioners alike, whether or not it could or should penetrate clinical research programs to a similar degree, especially with respect to preserving the sanctity of privacy within the circle of care, will require ongoing consideration. , ongoing requirements for universal masking inside of hospitals may further complicate recruitment and consent for clinical trials as clinician investigators must first establish a trustful relationship with potential research participants. while it removes the physical face-to-face component of a patient-physician interaction, potential advantage of telemedicine is that it does allow unencumbered facial recognition and mutual awareness of affect. thus, the persisting effects of telemedicine on clinical research programs beyond this pandemic are yet to be seen and require further study, including the patients' understanding and appreciation of disclosed information, perceptions of the consent process, concepts of ample time for decision-making, patient perceptions of coercion, and ability to make decisionmaking voluntarily and research recruitment rates. yet, our current understanding of patients' ability to provide same-day consent, , the lack of evidence of perceived or actual coercion, the perceived value of the fiduciary relationship with the physician performing the procedure, [ ] [ ] [ ] and its low-risk nature would seem to mandate a reconsideration of the absolute prohibition on obtaining same-day consent for regional anesthesia clinical research instead of seeking to create more workaround solutions which may be more disruptive to patients and generate more patient anxiety. most regional anesthesia clinical research trials primarily strive to improve and prolong pain control in the acute and subacute postoperative settings. in comparing the risk-benefit ratio for typical regional anesthesia clinical research trials versus that of other anesthesia subspecialties, it is evident that a proportionate approach to consent protocols is warranted. prohibiting same-day consent practices threatens systematic exclusion of patients otherwise fit and competent who may benefit from participation in regional anesthesia clinical research trials. ostensibly, the issue of same-day consent and its implications for clinical research trials would apply to all fields of anesthesia, but this is not necessarily true. regional anesthesia is unique from other anesthesia subspecialties in its predilection for healthy and fit patients undergoing elective surgical procedures commonly in an ambulatory setting. in contrast, clinical trials in other anesthesia subspecialties (such as cardiac, thoracic, transplant, trauma, and obstetrical anesthesia) typically involve study of riskier interventions or care modifications with generally less resilient patients. we recognize that consenting practices for regional anesthesia research trials vary across north american institutions, and consent on the same day as surgery is permissible at some institutions that house leading regional anesthesia research programs. however, our governing institution-the university of torontopublishes the second most scholarly journal articles in our specialty, second only to harvard university, yet the esteemed research hospitals affiliated with both the university of toronto and harvard university do not allow same-day consent for recruitment of patients to clinical anesthesia research trials. such prohibitive regulations regarding same-day consent must not be the model for other institutions striving to develop their own regional anesthesia clinical research portfolios. it behooves all regional anesthesia investigators to learn from the covid- pandemic and identify opportunities for growth thereafter. the covid- pandemic has unceremoniously exposed the arranged and strained marriage between our heretofore proliferative clinical regional anesthesia research program and our anesthesia pacs. during an unprecedented time in which clinical research and knowledge are driving day-to-day political, economic and health care decisions with monumental impacts locally, nationally, and globally, regional anesthesia research has been brought to a halt. while the issue of consent is not one to be taken lightly, the validity of same-day consent for low-risk anesthesia research trials has been widely supported. , [ ] [ ] [ ] [ ] [ ] indeed, the nhs has responsibly acknowledged that the timing of consent can vary depending on the risk of study participation and that a universal "one-size-fits-all" approach to the timing of consent is not reasonable. it is the process, rather than the time, that is the central to the validity of informed consent and safeguarding subject autonomy. prohibiting same-day consent for low-risk regional anesthesia clinical trials is an overly burdensome exercise for both clinical investigators and research staff. and so, while we continue to practice physical distancing, it is, in our opinion, high time to distance ourselves from such a prohibitive practice. world medical association declaration of helsinki: ethical principles for medical research involving human subjects guidelines on the ethics of clinical research in anesthesia guidelines for the ethical practice of anesthesiology who guidelines for good clinical practice (gcp) for trials on pharmaceutical products: responsibilities of the investigator tri-council policy statement: ethical conduct for research involving humans www.anesthesia-analgesia.org anesthesia & analgesia same-day consent for regional anesthesia research . toronto academic health sciences network (tahsn). guidelines for research ethics review involving human subjects same day consent for anaesthesia research the national health and medical research council, the australian research council and universities australia clinical trials regulation: informed consent and information to patients. european patients forum guidelines about notification etc auckland district health board applying a proportionate approach to the process of seeking consent. nhs health research authority informed consent for clinical anaesthesia research consent for anesthesia clinical trials on the day of surgery: patient attitudes and perceptions are patients comfortable consenting to clinical anesthesia research trials on the day of surgery? surgical patients' attitudes regarding participation in clinical anesthesia research protocol understanding and anxiety in perioperative clinical trial patients approached for consent on the day of surgery say what? patients have poor immediate memory of major risks of interscalene block disclosed during the informed consent discussion issues of vulnerability and equality: the emerging need for court evaluations of physicians' fiduciary duties in high stakes end-of-life decisions targeted consent for research on standard of care interventions in the emergency setting an overview of informed consent for radiologists a preadmission telephone call to initiate the consent process for clinical anesthesia research perspective on covid- : finally, telemedicine at center stage trend of academic publication activity in anesthesiology: a -decade bibliographic perspective balancing the quality of consent key: cord- - um jmhk authors: imperiale, michael j.; casadevall, arturo title: a new approach to evaluating the risk–benefit equation for dual-use and gain-of-function research of concern date: - - journal: front bioeng biotechnol doi: . /fbioe. . sha: doc_id: cord_uid: um jmhk in the twenty-first century, biology faces a problem that has previously vexed other disciplines such as physics, namely the prospect that its knowledge domain could be used to generate biological agents with altered properties that enhanced their weapon potential. biological weapons bring the additional dimension that these could be self-replicating, easy to manufacture and synthesized with commonly available expertise. this resulted in increasing concern about the type of research done and communicated, despite the fact that such research often has direct societal benefits, bringing the dual-use dilemma to biology. the conundrum of dual use research of concern was crystallized by the so-called “gain-of-function” type of experiments in which avian influenza viruses were endowed with new properties in the laboratory such as increased virulence and the capacity for mammalian transmission. after more than a decade of intensive discussion and controversy involving biological experiments with dual-use potential, there is no consensus on the issue except for the need to carry out such experiments in the safest conditions possible. in this essay, we review the topic with the hindsight of several years and suggest that instead of prescribing prohibitions and experimental limitations the focus should be on the importance of scientific questions at hand. we posit that the importance of a scientific question for medical and scientific progress provides a benchmark to determine the acceptable level of risk in biological experimentation. revolution in the late twentieth century that the potential of biology, and in particular microbiology, in biological warfare and terrorism came into focus. these fears came true with a single act of terrorism involving the mailing of anthrax spores in the u.s. in , which caused several deaths and considerable disruption to government facilities such as congressional offices and the postal system (bush and perez, ) . this act of bioterrorism catalyzed a series of events that led to greater awareness of the potential of using biological knowledge in nefarious ways, new regulations in biological research in the u.s. such as the select agents and toxins list, and heighted concerns about some types of microbiology research. one of the immediate effects of the post- environment was increased scrutiny of published findings, and several papers were particularly noteworthy for eliciting discussion and concern. in , researchers in australia showed that insertion of the gene for il- into the ectromelia virus genome defeated vaccine immunity (jackson et al., ) , a report that drew immediate concerns because of its possible implication for similar effects with variola major virus. similarly, the finding that complement inhibition potentiated variola virus virulence suggested a mechanism for enhancing its pathogenicity (rosengard et al., ) . an analysis of the u.s. milk distribution system revealed vulnerable nodes where the introduction of botulinum toxin could lead to mass casualties (wein and liu, ) , leading to an outcry about journals publishing research that could be exploited by terrorists. advances in molecular biology led to the chemical synthesis of poliovirus (cello et al., ) and the reconstruction of the pandemic influenza virus (tumpey et al., ) , which greatly heightened concern about the re-introduction of extinct, controlled, and new viruses to naïve human populations. in this environment, the u.s. government created the national science advisory board for biosecurity (nsabb) in to provide guidance on issues relating to biological risk and security. both authors of this article were inaugural members of the nsabb and served on that board for years. when the nsabb first began their deliberations, one of the major early topics was that of dual-use research in the biological sciences. this issue had previously vexed the physics community in the twentieth century as advances in physics and associated technologies had led to radar, nuclear weapons, ballistic missiles, and other military hardware. however, there was little or no precedent for similar concerns in the biological research community, especially since biological weapons were outlawed by the biological weapons convention in . moreover, the term dual use, which had meant civilian and military use in the physics arena, was redefined in its application to biology by an nrc report in to mean technologies and information that had both beneficial and maleficent use (nr council, ) . nevertheless, among the early accomplishments of the nsabb was formulating a definition for dual use research of concern (durc), which limited the scope of worrisome work to a small subset of biological research. the nsabb also generated a set of recommendations for communicating durc in publications and other venues. the work of the nsabb proceeded quietly and in relative obscurity until when the u.s. government learned about two submitted manuscripts reporting that highly pathogenic avian influenza virus (hpaiv) could be made mammalian transmissible by laboratory passage in ferrets, and sought an opinion from the board as to whether publication should be permitted. before considering the gof controversy involving influenza virus, it is worthwhile reviewing what is meant by "gof" and the limitations of the lexicon in the controversy. at the most basic level, a gof experiment, as the name implies, is one that gives a biological entity any new property. gof experiments can be highly beneficial and can generate pest resistant crops, microbes expressing proteins for medical therapy such as recombinant human insulin, and new cancer therapies by enhancing lymphocyte function. the controversial aspects of gof experiments in microbiology arise when microbes are modified to have new properties that can enhance their virulence and transmissibility because such experiments raise worries about new diseases, outbreaks, pandemics, biosafety, and biosecurity. in the experience of the authors, the nsabb probably did not anticipate what a problematic dual-use paper would look like until they had the opportunity to consider the two papers that reported the gain of mammalian transmissibility for hpaiv (herfst et al., ; imai et al., ) . what made the two papers highly controversial was that they reported that only a few amino acid changes were sufficient to confer mammalian transmissibility to the h n hpaiv, and there was concern that this information could be used to generate a virus capable of pandemic potential with high mortality by anyone with basic molecular biology and virology skills. the nsabb initially recommended redacting the sequence information, but this was considered not feasible given the legal framework in the u.s., and the two papers (herfst et al., ; imai et al., ) were eventually published in after some modifications to the text. the arguments, counterarguments, and events surrounding that episode are described in various publications (berns et al., a,b; fouchier et al., a) , often by the participants themselves, and will not be repeated here. the major outcome of the great gof controversy of is that it defined and crystallized some of the issues of dual-use research in biology by providing clear examples of experiments that were of great scientific value while also raising biosecurity and biosafety concerns. although both papers were eventually published with full information, the debate and controversy did not solve the central questions of what work should be performed moving forward and how it should be reported. in fact, the controversy erupted again in the summer of following the publication of additional papers describing other gof experiments (e.g., watanabe et al., ) as well as a series of biosafety lapses at u.s. government laboratories, which led the government to impose a pause on these types of experiments with certain viruses until the nsabb could formulate new recommendations and guidelines. the pause was lifted in mid-december, , alongside policy guidance about how gof funding decisions should be made going forward. one of the most vexing questions of the problem of dual-use research is publication of scientific findings. in the u.s., the research community is guided by national security decision directive (nsdd) , formulated in by the reagan admini stration, which states that all fundamental research results could be published freely. in other words, there was either open publication or classification, and nothing in between. viewed from the context of nsdd- the papers involved in the gof controversy were either to be published in their entirety or classified, with the latter alternative being impractical and unfeasible since the work had been carried out in an unclassified environment. with this binary policy, redacting papers because they might contain information useful for nefarious purposes is not an option unless export control regulations are invoked [for a full discussion of the legalities involved, see nas em durc report (national academies of sciences engineering and medicine, )]. consequently, when faced with gof papers containing information that could conceivably be used to enhance the pathogenicity or transmissibility of a virus, editors and journals have almost always opted for full publication, usually requiring more details from the authors about biosafety and biosecurity methods, and often publishing an accompanying editorial emphasizing the scientifically useful aspects of the research [for examples, see dermody et al. ( dermody et al. ( , a ]. such decisions have sometimes elicited strong criticism from members of the virology community (dermody et al., b; wain-hobson, a) . a more recent situation illustrates the difficult issues faced by authors, editors, and journals when publishing papers that contain durc. in , the journal of infectious disease faced the quandary of receiving a paper reporting a new botulinum toxin that was resistant to neutralization by the then-available antisera. in response, the journal took the remarkable decision of publishing the paper without the toxin sequence and vowed to keep the data confidential until an antidote was available (casadevall et al., ; dover et al., ; relman, ; hooper and hirsch, ) . a major factor in withholding the sequence data was the realization that this toxin could be easily produced using standard techniques if the toxin sequence was available and that the availability of such a toxin might pose a major public health risk. this decision was highly controversial since without the sequence information other investigators could not verify the findings reported in the publication or begin to develop medical countermeasures. in fact, subsequent work revealed that the new toxin was indeed neutralized by available sera, thus diminishing the initial concerns (enserink, ) . on one hand, withholding the sequence information from public scrutiny was the responsible action given the potential threat, while on the other hand, such an action delayed verification of the findings and the generation of countermeasures had the threat been real. clearly, manuscripts containing durc pose vexing problems for journals that often must make such decisions alone. at american society for microbiology journals, there are protocols in place for reviewing such manuscripts and the journals have available many individuals with microbiological and safety expertise who can provide input (casadevall et al., ) . other situations are likely to pose different challenges, but it is clear that journals are often poorly equipped to handle the difficult problems involved in publishing durc content. in this regard, we have argued for the need of a national board that can help journals make publication decisions (casadevall et al., ) , although to date no such body exists. another major challenge to the control of durc information is the emergence of preprint servers in biology that allow the posting of research findings before peer review and the proliferation of predatory open access journals that will publish essentially any paper for a fee. consequently, there now exist alternative systems for publication even if standard journals decline to publish a particular article over durc concerns. bypassing traditional publication methods could also allow authors to avoid government scrutiny. it is not an exaggeration that the situation today regarding durc and gof experiments remains highly unsatisfactory. experimental work involving gof experiments on pathogens with pandemic potential is highly regulated with proponents of such work arguing that restrictions on experimental design are unwarranted (fouchier et al., b) while opponents of such experiments argue that such work cannot be justified (wain-hobson, a,b). proponents of gof experiments have noted that data generated in such experiments is useful in epidemiological surveillance (schultz-cherry et al., ), whereas opponents have argued that such work cannot be morally justified because of its inherent risk (lipsitch and galvani, ) . in the u.s., government-mandated pauses on certain types of experiments remain in effect for federally funded research but such prohibitions do not extend to other countries, or to work funded by other sources. although the nsabb continues to analyze and debate the issues involved with durc and gof experiments in the u.s., there is no comparable body on the international scene, where much of this type of research is done. there is no consensus in the scientific community on whether the value of some experiments justifies the risks involved. the central problem in finding a consensus is that the value of the research cannot be measured in real time, while assessments of risk involve assumptions that can lead to widely divergent estimates. for example, some risk-benefit calculations have suggested that a high consequence accident is likely to occur in the near future (lipsitch and bloom, ; lipsitch and inglesby, ) , while others have estimated such probabilities to be near zero (fouchier, ) . however, there is some evidence that the incessant debate, which shows no sign of resolution, is taking its toll on the virology community as shown by surveys suggesting that scientists in training may avoid these areas of research (pfeiffer, ) . this is of particular concern given that recent years have seen new viral threats in the forms of the west africa ebola outbreak, the emergence of zika virus in the americas, and middle east respiratory syndrome coronavirus in the arabian peninsula, highlighting the importance of virology in human preparedness against pandemic threats casadevall, a, ) . one of the most important developments in the past few decades has been a change in the zeitgeist of the field that concerns itself with durc research and gof experiments. in the early years of the twenty-first century, as the u.s. reeled from the / terrorist attacks, which were shortly followed by the anthrax spore attacks, the focus was primarily on biosecurity. at that time, the concern was that terrorists and state actors would use the tools of the molecular biology revolution to create new and more devastating biological weapons. however, as the years passed and no new attacks developed, combined with the attribution of the anthrax spores in mail attacks to a lone insider in a u.s. government laboratory (bhattacharjee and enserink, ) , this security threat appeared to recede. at the same time, a series of unfortunate biosafety lapses in government laboratories heightened concerns about biosafety. hence, today people (at least those outside the security community) appear to be more worried about an unintended release of a pathogen with pandemic potential than a deliberate biological attack. this change in emphasis from biosecurity to biosafety has developed slowly and could easily change if there is another deliberate biological attack or if becomes apparent that adversaries are developing biological weapons. a major problem contributing to the unsatisfactory nature of the current situation is that the nsabb definition of durc does not work well in day-to-day practice. the nsabb defined durc as "life sciences research that, based on current understanding, can be reasonably anticipated to provide knowledge, information, products, or technologies that could be directly misapplied to pose a significant threat with broad potential consequences to public health and safety, agricultural crops and other plants, animals, the environment, materiel, or national security. " although the crafting of this definition was a major achievement at the time in being able to limit the scope of durc to a relatively small portion of biomedical research, thus leaving most biological research to proceed unfettered, subsequent experience showed that the clause "reasonably anticipated" is so subjective in nature that it requires a risk assessment beyond the capabilities of most scientists, reviewers, and editors (casadevall et al., ) . in essence, the definition was helpful in keeping whole swaths of biological research outside the durc category, such as cancer and immunology research, but it difficult to apply in determining what is included in the durc definition. also contributing to an unsatisfactory situation are concerns whether such regulations as the select agents and toxins list help or hinder societal security. on one hand, placing great restrictions on the accessibility to a number of agents and toxins does increase security by making them more difficult to obtain, with the caveat that these are naturally occurring agents that could be obtained from nature by a determined actor. on the other hand, there is the concern that focusing on lists and a relatively short list of agents means that the overwhelming majority of microbial threats are not on the durc radar screen. for example, zika virus emerged as a pathogen of pandemic potential with little or no anticipation from experts. hence, making lists and focusing attention on those agents in the list has the paradoxical potential to reduce biosecurity since regulations are tightened for listed agents, possibly hindering research, while other dangerous agents are neglected (casadevall and relman, ) . a similar argument applies to the current durc oversight policy of the u.s. government, which is focused on a specific list of agents. this process, which is akin to searching under the lamppost, carries great danger because it ignores what could be significant threats. in this regard, it is noteworthy that fungal pathogens are seldom considered as biological threats despite the fact that members of this kingdom have significant weapon potential (casadevall and pirofski, ; casadevall, ) . against this backdrop of dissatisfaction is the fact that science continues to progress very rapidly, introducing new technologies such crispr/cas , gene drives, and more efficient synthetic biology, each of which brings with it new possibilities for research that improves the human condition as well as new tools for nefarious purposes. furthermore, as the technologies improve they are increasingly accessible to more individuals and countries for whom this type of research was previously beyond reach. hence, the problem of durc is likely to become significantly more urgent in the near future. in a world where new infectious agents that can rapidly spread among vulnerable populations are described on a regular basis, humanity needs a healthy research establishment focused on microbial threats. it is estimated that there are a minimum of , mammalian viruses (anthony et al., ) : some fraction of these are probably zoonotic events waiting to happen. as ian malcolm, the fictional character created by michael crichton in the novel jurassic park, stated, "life finds a way. " information and knowledge are the best defenses against these threats. in addition to terror from nature, a new crisis will almost certainly occur in the future arising from a deliberate attack, a new provocative paper, or another biosafety lapse. gof experiments and durc research are essential for preparedness and the question is not whether this research should be pursued but rather how to do it safely and mitigate risk. to date, each of the controversies has been reactive, with proponents and opponents of such experiments responding to a specific finding or study. after more than a decade of discussion on what constitutes durc, benefits and risks of gof experiments, regulations, pauses, and moratoriums, it is increasingly apparent that current approaches are inadequate for the challenges at hand. given these limitations, we have proposed a new framework for durc research that focuses on answering specific questions (imperiale and casadevall, b) . hence, instead of prohibiting certain types of experimentation, we suggest that the way forward is to focus on specific scientific questions and the problems that need answers. for example, if there is a need to determine whether a particular feral virus pathogen has the capacity for mammalian infection and transmission, then gof experiments performed in safe and controlled conditions can be justified. on the other hand, endowing hiv with new transmission properties is not a medically important gof experiment (national academies of sciences engineering and medicine, ). all human activities that involve probing the unknown, ranging from space exploration to tissue culture procedures, carry some degree of risk, and it is nature of humanity to accept risk to attain progress. opponents and proponents of this type of experimentation need to maintain open channels for continued discussion because the dialectic of ideas is likely to result in better decisions. institutional bodies such as the nsabb need to be supported 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influencing future plans of graduate students and postdoctoral fellows? inconvenient truths" in the pursuit of scientific knowledge and public health variola virus immune evasion design: expression of a highly efficient inhibitor of human complement influenza gain-of-function experiments: their role in vaccine virus recommendation and pandemic preparedness characterization of the reconstructed spanish influenza pandemic virus an avian h n gain-of-function experiment of great concern the irrationality of gof avian influenza virus research circulating avian influenza viruses closely related to the virus have pandemic potential analyzing a bioterror attack on the food supply: the case of botulinum toxin in milk biological warfare at the siege of caffa both authors contributed to the writing of this manuscript. references conflict of interest statement: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -qpgc qm authors: tiberius, victor; siglow, caroline; sendra-garcía, javier title: scenarios in business and management: the current stock and research opportunities date: - - journal: j bus res doi: . /j.jbusres. . . sha: doc_id: cord_uid: qpgc qm the scenario technique is widely used to cope with uncertainties plan for alternate future situations. the extensive research led to a scattered literature landscape. to organize the field quantitatively, we conduct bibliometric performance analyses and a bibliographic coupling analysis. results show an increased interest in scenario research since and clear distinctions between strategic and operational as well as methodological and applied research. future research can be expected to further enhance the method towards robust decision making and to combine it with methods searching for most likely scenarios, such as prediction markets, crowdsourcing, and superforecasting. additionally, cognitive and behavioral aspects of using the scenario technique might draw further attention. the scenario technique is expected to be applied across all industries and will probably play an increasing role in currently underrepresented business functions such as marketing and innovation. the scenario technique is an established method to cope with uncertainty by exploring multiple alternate future situations for strategic and operational planning purposes (godet, ; schoemaker, schoemaker, , wright & goodwin, ; tiberius, ) . the method allows firms to adapt to dynamic environments and to enhance innovativeness (rohrbeck & gemünden, ; worthington, collins, & hitt, ) and firm performance (phelps, chan, & kapsalis, ; rohrbeck & kum, ) . widely used in practice, it has been subject to intense scholarly discussion and application. as a consequence, the literature landscape related to scenarios can be described as extensive and fragmented and is therefore in need of systematic organization. our research goal is to systematize the research landscape on scenarios in business and management. to achieve this, we use bibliometric analyses, more specifically, performance analyses and a bibliographic coupling. focusing on the quantity of citations which are seen as the measure for scientific relevance and impact, bibliometrics objectively analyze publications in a field using statistical methods (zupic & Čater, ) . we explore the temporal distribution of scenario publications, paper types, productivity and impact of journals and authors, the most influential articles, and identify research themes to suggest future research opportunities. our research contributes to the literature on scenarios by providing a quantitative and objective perspective on published research and therefore mapping the research field. in the remainder of the paper, we explain which bibliometric analyses we conducted, present their results, highlight key interpretations, and provide future avenues for scenario research. we collected our dataset of scholarly publications on scenarios from the web of science (wos), which is considered as a comprehensive database of social science literature (norris & oppenheim, ) and has recently been used for bibliometric analyses (castillo-vergara, alvarez- noyons, moed, and luwel ( ) distinguish between performance analyses and science mappings as two distinct approaches in bibliometrics. performance analyses measure scholarly productivity by publication number and scientific impact by citation numbers. in particular, our performance analyses comprise a temporal analysis of scenario-related research and the paper types, the productivity and impact of journals and authors, and the impact of the most relevant articles. assuming a lifecycle of research, the temporal analysis allows for an assessment of the relevance of scenario research over time. for the analysis of the articles by type, we read all abstracts, and in case they were not informative, the articles, to identify whether a publication addresses methodological issues or develops specific scenarios in a practical field. the analysis of the productivity and impact of journals and authors and the impact of articles help to find the most relevant research. for the performance analyses, we narrowed the dataset using the following restrictions. first, we limited the dataset to publications which belong to the categories "business" and "management". second, we only included articles published in scholarly journals rather than books, proceedings, etc., because we wanted to include only highquality research, and only for journals clear criteria, such as journal rankings or impact factors, exist. third, we applied a quality threshold to the sample (kraus, breier, & dasí-rodríguez, ) such that articles published in journals assigned to the third or fourth quartile (q /q ), according to the scimago journal ranking (sjr), were excluded. we decided to use the sjr rather than the journal citation reports (jcr) because the former includes more journals and the evaluation period was three years rather than two. fourth, we only included research that was published before the end of because the temporal analysis would not have been representative for . fifth, we screened the abstracts of all remaining papers and excluded those which, despite the prior filtering, did not deal with scenario technique in business or management. as a result, the dataset was reduced to articles. science mapping techniques cluster a research field based on the idea that strong links between citing and cited articles suggest that they deal with similar topics (zupic & Čater, ) . more specifically, we conducted a bibliographic coupling analysis, for which we used vos-viewer version . . , to visually map bibliometric data (waltman, van eck, & noyons, ) . whereas co-citation analysis looks at an article which cites two older articles (small, ) , bibliometric coupling, reversely, looks at connections between two younger publications that jointly cite an older publication (kessler, ) . therefore, this method better suits our interest in the more recent rather than historical structure of the scenario research field. to focus on the most relevant research and to reach a manageable number of cited references, which allows clear conclusions, we set the citations threshold to a minimum of ten. as a consequence, the former list of documents was reduced to , of which were connected. since then the productivity dropped, but is still on a pre- level. more specifically, we find publications dedicated to methodological issues and publications applying the scenario technique. therefore, the research on scenarios is almost equally divided. a time analysis of the distribution between the two article types does not provide a clear picture (fig. ) . fig. lists the journals with the most published articles on scenarios in business and management, with at least three papers. it has to be noted again that we limited our performance analysis to the wos categories "business" and "management" and to the first and second quartile in the sjr. as a consequence, several journals, which are very productive in scenario research, were excluded due to its different categorization or multidisciplinary orientation. under this condition, the most productive journal in this field is technological forecasting & social change, which accounted for articles ( percent). it is followed by the european journal of operational research with ( percent) and long range planning with ( percent). these three journals together comprise % of all articles on scenarios. after the journal of the without the filtering of our dataset to journals that are assigned to the wos categories business and management and to sjr q /q , the list would show higher productivities and have a somewhat different order. according to this, george wright is the most productive author and has published articles thus far. his articles examine a broad range of aspects as they focus on the epistemological foundations (cairns, wright, & fairbrother, ; , the enhancement of the scenario methodology (cairns et al., ; derbyshire & wright, ; wright & goodwin, ; wright, bradfield, & cairns, ) , establishing a relationship of scenarios with other foresight techniques or the strategy process (goodwin & wright, ; rowe, wright, & derbyshire, ) , and practical context factors of using the scenario method (cairns, ahmed, mullett, & wright, ; cairns et al., ) . george cairns is the second most productive scenario scholar. all of his scenario-related articles in our dataset were co-authored with george wright. therefore, they have similar research profiles. thomas j. chermack, the third most productive author, also addresses the foundations of scenario planning (chermack, ) and enhancements of the scenario process by using scenarios (bodwell & chermack, ), but has a stronger focus on organizational and individual side-effects of the scenario technique (chermack & nimon, ; chermack, van der merwe, & lynham, ; chermack, coons, nimon, bradley, & glick, ; chermack, coons, o'barr, & khatami, ; haeffner, leone, coons, & chermack, ) . fig. shows the scholars with the highest citations. paul j. h. schoemaker is the scenario author with the highest citation number, , . he is both highly productive and impactful in decision sciences and strategic management. his two seminal articles on scenario planning (schoemaker, (schoemaker, , are the second and fourth most cited of his complete publication list. the energy and climate change scholar keywan riahi became second-most cited author with mainly one highly-cited article on greenhouse gas emission scenarios (riahi, grübler, & nakicenovic, ) , an applied paper. his two co-authors follow on the third and fourth place with that paper as their only scenario-relating article. george wright, the most productive scenario author, is the fifth most-cited author. table lists the articles with the highest average annual citations. the ranking by average citations ensures that older publications are not privileged over younger ones. several ranks are split by two articles because they have the same number of average citations. in the following, we briefly summarize the first ten articles which have more than citations per year on average. the already mentioned applied paper, by riahi et al. ( ) , ranks first place with . annual citations on average. in second place, lempert, groves, popper, and bankes ( ) paper follows, with . citations per year. the paper suggests a method for robust decision making. the method helps to identify possible robust strategies for sets of possible future states and assesses possible hedging against failures. schoemaker ( ) seminal paper on scenario planning reaches the third place with . citations per year. he addresses cognitive biases in decision making, such as overconfidence and tunnel vision, and provides scenario planning as a strategic tool to face these cognitive shortcomings. the author provides a step-by-step guideline on how to generate scenarios, and illustrates v. tiberius, et al. journal of business research ( ) - this with examples. in fourth place follows bryant and lempert ( ) , which was cited . times per year on average. the authors address the problem that the diversity of stakeholders' interests and values can complicate the scenario generation process. they introduce a scenario discovery method which is participatory but also computer-based, using statistical or data-mining algorithms. høyland and wallace ( ) , in their fifth placed paper with . citations on average per year, address the challenge of adequately representing uncertainties in a quantitative way. as a solution, they introduce a non-linear programming method, a scenario tree over several periods, which generates a manageable number of outcomes that can be used statistically. the sixth most-cited paper on an annual basis with . citations per year, is by kowalski, stagl, madlener, and omann ( ) . the authors argue for a combination of the scenario technique with participatory multi-criteria analysis (martinez, de castro-pardo, pérez-rodríguez, & martín, ) . the simultaneous application of both methodologies is considered to be resource intense but it allows for a participatory and robust decisionmaking process. kok, van vliet, barlund, dubel, and sendzimir ( ) account for the seventh place, with . citations on average. their paper applies the scenario technique to generate scenarios about europe's fresh water future. however, it can also be considered as a methodological paper as it combines the scenario development process with participatory backcasting in order to identify obstacles and opportunities regarding the previously formulated narrative scenarios. in eighth place follows schoemaker ( ) paper in which he describes the scenario technique as an innovative tool in strategic management, with its intellectual foundations. he discusses the benefits and obstacles including cognitive biases of using the scenario method in organizations. the author conducts experiments to test the effect of scenario planning on confidence and beliefs. postma and liebl ( ) who account for the ninth place with . citations per year on average, address methodological shortcomings of the scenario technique which v. tiberius, et al. journal of business research ( ) - stem from contradictory, paradoxical, and overlooked trends. they provide several recommendations to improve the scenario process. tenth place, with . annual citations, is taken by bent and van hentenryck ( ) . the paper applies a multiple scenario approach to solve the multiple vehicle problem with time windows. the bibliographic coupling analysis led to thirteen research clusters (fig. ) . as the clusters are based on the strength of the links between two younger publications that jointly cite an older publication, there are no precise categories that only contain articles of a clearly defined topic. for two clusters, we did not find a common thread. the other clusters can be described as follows. exploring the future: this cluster contains articles which mainly address the purpose of scenarios. compared to the other clusters, the articles are older and deal with the origin and evolution of the scenario technique (zentner, ) , its relationship to forecasting (van der heijden, ), how to develop multiple scenarios (schnaars, ) , and how to use them in strategic management (malaska, ) and turn them into opportunities (gausemeier et al., ) . (wright & goodwin, ) and uncertainty (kwakkel, auping, & pruyt, ) . the role of stakeholder interests in the scenario and strategy development process is also addressed (cairns et al., , trutnevyte, stauffacher, & scholz, ) . strategic radar for risk mitigation: this rather small cluster, more specifically than the prior one, covers the use of the scenario technique as an early warning system that increases managerial attention (ramirez, osterman, & gronquist, ) and strategic thinking and helps to identify weak signals (schoemaker, day, & snyder, ) . the general purpose is to mitigate strategic risks and to increase innovativeness (worthington et al., ) . scenario technique in practice: most of the articles in this cluster take a pragmatic look at scenarios by addressing ways to simplify the method (mercer, ) , heuristics (schoemaker, ) , tools and pitfalls (godet, ) , and uses and abuses (durance & godet, ) . also specific firms, such as shell (jefferson, ; mercer, ) and british airways (moyer, ) , are referred to. cognitive and behavioral aspects of the scenario technique: the largest cluster, with articles, addresses diverse aspects of scenarios. an identifiable common thread focuses on the human and social aspects of scenario processes. for example, articles deal with the impact of the scenario development on fostering strategic thinking (millett, ) , on cognitive biases and decision quality (meissner & wulf, ) , on decision-making style (chermack & nimon, ) , and on perceptions of organizational learning (haeffner et al., ) or of strategic conversation quality (chermack et al., ) . enhancing and combining the scenario technique with other methods: in this cluster, several of the articles discuss possibilities to refine the scenario technique, for example, by adding an evolutionary process (saritas & nugroho, ) , allowing participation, or the use of computers (bryant & lempert, ) . scenarios can also be combined with other methods such as bibliometrics (stelzer, meyer-broetz, schiebel, & brecht, ) , the delphi method (tseng, cheng, & peng, ), or road-mapping (amer, daim, & jetter, ) . models and simulations: this cluster consists of articles which are mainly published in operational research journals. their prevailing topics are models and simulations as ways to generate (operational) scenarios (e.g., islei, lockett, & naude, ) and to determine risk probabilities for decision-making (e.g., klibi & martel, ) . scenarios for stochastic portfolio optimization: this cluster containing articles also refers to operational research. scenario planning and stochastic programming models are used to optimize investment portfolios (e.g., hanafizadeh, kazazi, & bolhasani, ) ; kouwenberg, ; mulvey & ruszczynski, ) . scenarios for energy and sustainability: this cluster contains articles of which the majority deals with the application of the scenario technique in both interconnected fields of energy and sustainability (kowalski et al., ) , or one of them (silberglitt, hove, & shulman, ; turton, ) . scenarios for diverse industries: similarly, this cluster consisting of articles, mainly covers the application of the scenario technique in specific sectors. however, this cluster is broader and addresses sectors such as health insurance (gnatzy & moser, ) , logistics (hirschinger, spickermann, hartmann, von der gracht, & darkow, ) , or materials production (von der gracht & stillings, ) . scenarios for hr assessment and training: this very small cluster with two articles, which were both published in the same issue of the international journal of selection and assessment, addresses the use of (computer simulated) scenarios to assess and train human resources (funke, ; kleinmann & strauss, ) . the general long-term trend of annual publications shows an increased interest in scenarios in business and management research. interestingly, the detected increase of publications occurred shortly after the / financial crisis. even though not many articles specifically deal with this crisis, it can be assumed that the general perception of risks and uncertainty has risen. as a consequence, the scenario technique, designed for coping with uncertainty, might have drawn increased attention. it can be assumed that scenario-related research further gains momentum due to the covid- crisis . the fact that scenario research is almost equally distributed between methodological and applied papers is rather surprising. in comparison, just . percent of publications on the delphi technique is methodological (flostrand, pitt, & bridson, ) . however, it has to be noted that . percent of all publications belong to healthcare research (flostrand et al., ) where the method is mostly used as a consensus building tool without a forecasting orientation. business and management papers only account for . percent of all delphi publications. unfortunately, the authors did not specifically report on the distribution between methodological and applied papers for this discipline. a reason for the probably still comparatively low share of applied papers in scenario research could be that scenario planning is predominantly applied by organizations as an antecedent of their idiosyncratic strategy formulation process, which contains proprietary information for their internal use only. in contrast, delphi studies address rather publicly accessible topics on an industry or technology level. the list of the most productive journals already shows on its first places that scenarios are mainly discussed and applied in the field of strategic management and strategic/corporate foresight, as well as in the field of operational research. this suggests that scenarios have a strong relation to decision-making irrespective of whether it addresses strategic or operational issues. interestingly, the strategic management journal only published three articles on scenarios and other top-tier strategy, and general management journals hardly cover scenario research recently. these two categories clearly outpace forecasting-related journals, which usually focus on methodologies which try to predict the most probable future rather than explore multiple alternative futures (rohrbeck & schwarz, ) . the comparison of the productivity and impact of authors shows that both not necessarily go hand in hand. however, as riahi et al. ( ) article is probably highly cited not due to its application of the scenario technique, but rather because of its focus on greenhouse gas emissions, it can be considered as an outlier, practically making wright the second-most cited author. considering the average citations per year, he would replace schoemaker as first place because his publications are younger. george wright's frequent co-author george cairns and also thomas j. chemack, who are the second-and third most productive scenario authors, are, regarding citations, outpaced by other authors such as michel godet who wrote a highly-cited seminal paper (godet, ) . most of the most cited articles are methodological rather than applied articles, which suggest a strong interest in the scenario technique as a methodology. however, as the most cited article is an applied paper, it can be concluded that the scenario technique is also a recognized and generally accepted research method in which other scholars see a high value. additionally, a closer look at the content of the ten most cited articles reveal that two distinct conceptualizations of scenarios can be distinguished. generally speaking, strategic management and foresight scholars tend to work with narratives, thus leaning towards a qualitative formulation of scenarios. in contrast, researchers in operational research use a quantitative conceptualization. the bibliographic coupling analysis provides a differentiated picture of current scenario research, which also allows drawing conclusions for future research opportunities. the foundations and enhancement of the technique and its combination with other methods play an important role. however, a strong conceptual shift we expect to accelerate in research is from risk to uncertainty because of two reasons: first, the determination of the probabilities of individual scenarios might become more and more difficult. second, with today's computer power, the number of scenarios can potentially tend towards infinite, meaning that all scenarios are treated equally, without an assigned likelihood. as a consequence, a shift from searching for optimal to robust decisionmaking is appropriate, which does not aim to identify the best option, but the one suitable for many possible future situations (hall et al., ) . in contrast, we also expect a stronger focus on combining the scenario technique with methods which aim to identify the single most probable future. apart from the delphi method (höhne & tiberius, ; tseng et al., ; tiberius & hirth, ) , also prediction markets (arrow et al., ; tiberius & rasche, ) , crowdsourcing (flostrand, ) , and superforecasting (schoemaker & tetlock, ) could increase in relevance in this regard. as these techniques show high forecast accuracies, they can provide a focal scenario, which can be surrounded by alternate scenarios. another methodological extension would be that scenarios do not only depict what the future might look like, but also theorize about the path that leads to them (tiberius, ) . apart from mere theoretical considerations, scholars also specifically address the use of scenarios in practice, including its pitfalls and abuses. scholars also examine cognitive and behavioral aspects which can be observed when the scenario technique is used by (groups of) individuals. we see a potential to address further benefits and side-effects of scenarios in future research (tiberius, ) . especially, scenario-based foresight seems to enhance a firm's dynamic capabilities (haarhaus & liening, ; schwarz, rohrbeck, & wach, ; semke & tiberius, ) , which needs further exploration. a large section of research is dedicated to the application of the scenario technique to explore possible futures in several sectors. whereas the energy sector stands out and several other sectors are addressed by individual papers, it can be expected that the scenario technique will be applied in all sectors which are challenged by a high degree of risk or uncertainty. besides the sector-specific application of the scenario technique, scholars also have a functional focus. obviously, the relation between scenarios and strategy is paramount. operational research applies the scenario technique to optimize production processes. the bibliographic coupling also reveals clearer insights into additional uses in this field. especially, elaborations on mathematic modelling and simulations can be found, and stochastic, scenario-based portfolio optimization stands out as a specific research field. similarly, scenarios are used in finance to optimize portfolios. however, the application of scenarios in other business functions or departments seem to be given less attention. for example, the use of scenarios for hr assessment and training represents a research niche. as uncertainty depicts normality for almost all business functions and the thinking in alternatives proves to be beneficial, we see a potential to use scenarios across all functions and departments. especially in marketing and innovation, scenarios could play an increasing role. in marketing, scenario approaches can help explore new needs and wants of customers and thus new market opportunities (højland & rohrbeck, ; verdenhofa, afanas'jev, panchuk, kotelnykova, & chumak, ) . in innovation management, the range, number, and quality of ideas and concepts (lee & trimi, ; rohrbeck & gemünden, ; worthington et al., ) , and thus their agile implementation (brand, tiberius, bican, & brem, ) , can be enhanced. we structured the extensive and scattered research field of scenarios in business and management by using bibliometric performance analyses and science mappings. the scenario literature has been growing, especially since , possibly triggered by the preceding financial crisis. half of the publications are methodological, the other half applies the scenario technique to specific research questions. whereas the share of methodological papers might decrease over time, we expect an ongoing scholarly discussion. scholars publish in foresight and strategy or in operational management journals, splitting the research landscapes in two quite distinguishable sections. however, top-tier strategy and general management journals seem to be restrained regarding scenario papers. scenarios are also not a prevailing topic for forecasting journals. among the most cited journals, most are methodological, but applied papers with high citations show that the technique is highly accepted in other research fields. the bibliographic coupling shows what the various aspects scenario researchers have focused on recently. apart from basic methodological questions, the link to the strategy process is strong. the scenario technique is continuously enhanced and combined with other methods. we expect a stronger shift from risk to uncertainty and therefore from optimization to robust decision-making, but also an increasing focus on the combination with methods which search for the most probable scenario, such as prediction markets, crowdsourcing, and superforecasting. also cognitive and behavioral aspects of using the scenario technique will probably continue to draw attention. scenarios are used in many industries such as energy and sustainability. however, as most sectors increase in regards of dynamics and uncertainty, we expect scenarios to be applied almost everywhere. this is also expected regarding business functions. whereas the scenario technique is well-established in strategy, operations, and finance, other functions are still underrepresented, such as marketing, procurement, and others. as with all research, our study also comes with several (potential) limitations. first, the data was only collected from the web of science. while this database is considered to be comprehensive, it is probably not complete and might contain faulty entries and misspellings. therefore, future research might also include other databases such as scopus and/or google scholar. second, our selection criteria and the manual selection of publications could have led to biased results. future research might consider to be less selective. third, whereas the clusters derived from the bibliographic coupling are based on citations and therefore objective, their content is somewhat blurry and makes the v. tiberius, et al. journal of business research ( ) - identification of common research themes difficult. this process corresponds to a qualitative content analysis and is therefore partly subjective. as a consequence, other researchers might have labeled the identified clusters in a different way. future research should increase the efforts to employ both quantitative and qualitative methods to grasp the state of scenario research. as bibliometric analyses and literature reviews are only snapshots of previous research, they have to be repeated periodically. fourth, the matthew effect has to be kept in mind when interpreting bibliometric analyses (garcía-lillo, Úbeda-garcía, & marco-lajara, ; kruggel et al., ) , as highly cited articles could just be cited due to their high citations in the past or due to citation cartels. as a consequence, some publications would be considered as relevant or qualitative, from a bibliometric point of view, even if they contain little contributions. this also suggests the necessity of qualitative reviews. technology roadmap through fuzzy cognitive map-based scenarios: the case of wind energy sector of a developing country the promise of prediction markets scenario-based planning for partially dynamic vehicle routing with stochastic customers organizational ambidexterity: integrating deliberate and emergent strategy with scenario planning agility as an innovation driver: towards an agile front-end of innovation framework thinking inside the box: a participatory, computerassisted approach to scenario discovery scenario method and stakeholder engagement: critical reflections on a climate change 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past, present and future bibliometric methods in management and organization he holds an mba in innovation management and received his phd in economics from technische universität carolo wilhelmina zu braunschweig and a second phd in education sciences and futures studies from freie universität berlin she has a professional background in human resource management. she currently works in the human resource mobility team at capgemini. her research interests include corporate foresight including scenario planning mba at the instituto de empresa (madrid), and bachelor in business administration in the university of valencia. professor in sales and marketing in the mba of the universidad europea de madrid (uem) executive and member of the senior leadership committee of pepsico south west europe as franchise director in portugal and spain key: cord- -z okp authors: levine, ross l.; rathmell, w. kimryn title: covid- impact on early career investigators: a call for action date: - - journal: nat rev cancer doi: . /s - - - sha: doc_id: cord_uid: z okp the covid- pandemic has broadly impacted biomedical research and health care. here we discuss current challenges for the cancer research community as they apply to early career investigators (ecis). we propose a series of collaborative initiatives aimed to sustain ecis and preserve and accelerate the ability to innovate with long-lasting impact. the current covid- pandemic has had a pervasive effect, including an unprecedented toll on health, the economy and education worldwide. the impact of this crisis on medical care delivery, disrupting the health-care economy in unprecedented ways, and causing a near-complete halt to basic and translational biomedical research is substantive. while the damage is widespread, early career investigators (ecis) are at highest risk to experience a negative impact. however, this crisis also reveals opportunities to innovate in crea ting solutions that accelerate research programmes in the short and long term. from our perspective, the pause in laboratory and clinical research in the us owing to covid- has lasted more than months and is only resuming under careful, orchestrated guidelines with an uncertain future. this disruption to research presents itself in varied ways in regions across the world. across the cancer science space, this interruption has been creating uncertainty and suspended states of animation that are particularly detrimental to young investigators. ecis, including phd students, postdoctoral researchers, clinical fellows and junior group leaders, are in a time of steep acceleration with respect to training milestones, protected time, fellowship funding and promotion. reduced productivity during this time can produce an undesirable 'flattening of the curve' that is potentially unrecoverable. it is heartening to see ecis prioritizing data analyses, grant and/or manuscript preparation and reviewing journals during their time out of the lab as an effort to maintain momentum, as they balance their career responsibilities with a daunting set of demands related to increased personal responsibilities during the pandemic . family care and/or sustaining community ties may also be disproportionately carried by ecis, particularly impacting women, and the set of challenges faced by investigators who are from minority groups may be amplified. moreover, clinical trainees, with in-demand skillsets, are necessarily being pulled from protected research time to provide clinical care to patients with covid- . these challenges further threaten the careers and trajectories of the next generation of cancer investigators. it is also worrisome that job opportunities for ecis are rapidly diminishing during the pandemic. academic institutions are facing unprecedented financial challenges with reduction in clinical revenue and philanthropic support. efforts to counteract these challenges, for example hiring freezes, slowdowns in hiring and/or salary reductions may persist for years. moreover, the international nature of the covid- pandemic is adding barriers for those who require a visa and/or international travel to continue their careers. the resulting 'back-up' will cause ecis to stay longer in current positions than intended or end up in limbo between assignments. ecis may prematurely depart to pursue opportunities in industry or other arenas. the current landscape has the potential to result in irreparable harm to a generation of talented scientists and derail the impressive progress we have seen in cancer research in the past decade. the funding landscape for cancer research in the us context, which is likely applicable to other contexts, also presents daunting challenges. universities and academic research and/or clinical institutions will be operating with reduced resources to provide essential institutional support for hiring, internal grants and new programmatic initiatives in the context of reduced clinical and endowment revenues. moreover, non-profit organizations that provide foundation grants for research, which ecis often depend on, are seeing a precipitous reduction in donations. this has resulted in reductions in ongoing grant support and a decline in funding opportunities, which may continue for an extended duration. therefore, we foresee a major gap in support for salary, protected time and pivotal investigations needed for the next generation to develop robust and durable research programmes. lastly, we regret the unique impact of social distancing that this pandemic is having on mentoring and networking opportunities for ecis. scientific meetings are covid- impact on early career investigators: a call for action the covid- pandemic has broadly impacted biomedical research and health care. here we discuss current challenges for the cancer research community as they apply to early career investigators (ecis). we propose a series of collaborative initiatives aimed to sustain ecis and preserve and accelerate the ability to innovate with long-lasting impact. www.nature.com/nrc often the venue for ecis to present work, meet and network with current and future leaders in their field and learn about cutting edge science from across the world. this impact is particularly felt by ecis at institutions without robust scientific networks or career development programmes, such that it can disproportionately impact those with less institutional support and a less vibrant local research environment. what can we do in light of these headwinds? we see this as an existential moment to galvanize the international cancer research community to collective action. we believe every person can contribute and propose that collective efforts focus on these six core areas. there has been a heartening move to increased dissemination of scientific knowledge during the pandemic, including online lectures, increased use of preprints and online discussions of cancer science and career development issues. we need to see further extension of these opportunities to break down silos and ensure that young investigators have a chance to present their science and interact with international leaders in our field in silico. whether in person or virtual, scientific meetings have always been a tenuous balance between presentations by established leaders in the field and opportunities for ecis to make their mark. we need to acutely and permanently change this balance and increase opportunities for young investigators to give invited and selected talks at meetings with a commensurate reduction in presentations by senior researchers. this will result in increased diversity and exposure for investigators looking to secure their next training position, their first grant or promotion. local mentoring is critical and not easily replaced, and many ecis are suffering owing to reduced interaction with local mentors and no exposure to mentors at outside institutions. we need to develop innovative approaches to facilitate broader mentoring opportunities. for example, one of us (r. l. l.) has invited young investigators to join for virtual one-on-one career and/or science discussions or joint lab meetings. in addition, organizations such as the us department of defense kidney cancer research program have initiated national academy style mentorship programmes to support early career networks and distance mentoring . such programmes are nascent, and we must seek opportunities to extend mentoring and scientific networks, with feed-forward benefits to the entire community. increase support and flexibility thereof most foundations and government funders have shown remarkable flexibility given the pause in research, but we cannot forget that salary and core research expenses (for example, animal care) continue without commensurate productivity. as funding allows, we propose that career awards be extended in duration and in the period of support to allow investigators to recover scientific progress and achieve career development goals. transparency in the hiring process one of the hardest things about the academic hiring process is the relative opacity, with little information on the priorities, positions or resource availability for prospective applicants. we need 'radical transparency' in the hiring process, such that ecis can obtain a clearer sense of the number and type of opportunities available at each career stage with clear points of contact for open positions. moreover, institutions need to enable flexibility for ecis who cannot transition to their next position owing to hiring freezes or visa restrictions, or we will lose a generation of our best and brightest. on a broader scale, we are at a critical moment where the public has never been as cognizant of science and medicine as they are during the pandemic. we must seize this moment to engage lay people in our respective communities on the importance of science and medicine and the need for increased, longer-term investment into medical research to benefit all of society, to engage future generations to pursue careers in cancer research and to encourage active participation of people affected by cancer in research and clinical trials. we believe bold innovations are essential to not only preserve careers of the next generation of cancer innovators, but to have long-lasting impact that allows cancer science to emerge from this devastating crisis stronger, more inclusive and forward thinking to make even greater strides in cancer. the pandemic and the female academic summary from the first kidney cancer research summit we thank members of the levine lab (bobby bowman, aaron viny, linde miles, sheng cai and andrew dunbar) for their perspective as trainees/ colleagues and ushma neill for her comments and guidance. r.l.l. is supported in part by memorial sloan kettering cancer center support grant/ core grant p ca . w.k.r. is supported by nih grants k ca and t ca . r.l.l. is on the supervisory board of qiagen and is a scientific advisor to loxo/lilly, zentalis, imago, mana therapeutics, auron, ajax, syndax, c therapeutics and isoplexis. he has consulted for incyte, syndax, celgene and roche. he receives research support from prelude therapeutics and has reviewed grants for celgene and gilead. w.k.r. receives research support from incyte and has clinical trial support from merck, genentech, bms, pfizer, novartis, incyte and calithera. key: cord- -rjc b br authors: ritterson, ryan; casagrande, rocco title: basic scholarship in biosafety is critically needed to reduce risk of laboratory accidents date: - - journal: msphere doi: . /msphere. - sha: doc_id: cord_uid: rjc b br our firm conducted a risk/benefit assessment of “gain-of-function” research, as part of the deliberative process following a u.s. moratorium on the research (u.s. department of health and human services, u.s. government gain-of-function deliberative process and research funding pause on selected gain-of-function research involving influenza, mers, and sars viruses, ). due to significant missing but theoretically acquirable data, our biosafety assessment faced limitations, and we were forced to provide a relative, instead of absolute, measure of risk (gryphon scientific, llc, risk and benefit analysis of gain of function research, ). here, we argue that many of these types of missing data represent large and stunning gaps in our knowledge of biosafety and argue that these missing data, once acquired via primary research efforts, would improve biosafety risk assessments and could be incorporated into biosafety practices to reduce risk of accidents. governments invest billions in biological research; at least a small fraction of this support is warranted to prevent biological accidents. i n , the white house issued a moratorium on so-called gain-of-function research involving influenza virus and severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) coronaviruses and initiated a deliberative process to be managed by the national science advisory board for biosecurity (nsabb), a process which recently concluded with the release of the white house office of science and technology policy's (ostp) recommended policy guidance document ( , ) . in an earlier stage of the process, our firm was contracted by the national institutes of health to perform and deliver a risk/benefit assessment of this research to independently investigate its biosafety and biosecurity risks, as well as the scientific and public health benefits it may bring. as part of our biosafety risk assessment, we were asked to provide a measurement of the absolute risk of a biosafety incident. however, due to significant missing, but theoretically acquirable data, our work faced limitations, and we were forced to provide a relative, instead of absolute, measure of risk ( ) . many of these types of missing data represent large and stunning gaps in our knowledge of biosafety. these missing data, once acquired via relatively simple primary research efforts, would not only improve biosafety risk assessments but also could be immediately incorporated into biosafety practices to reduce the risk of accidents. governments invest billions of dollars to support biological research with the purpose of improving the human condition; clearly, at least a small fraction of this support should be used to prevent the biological accidents that imperil that basic goal. to the best of our knowledge, gryphon scientific's biosafety risk assessment was the first to comprehensively consider the probability and types of human error that play a role in laboratory accidents, and we identified it as the dominant component of laboratory biosafety risk. this dominance of human errors is based on multiple factors. first, most plausible pathways to laboratory-acquired infection or accidental release require a human error to precipitate. for example, spills and needlesticks begin with a person making a motor control mistake. second, humans fail more frequently than equipment. for example, in our aerosol release scenarios, the median chance of an exhaust fan failing was estimated to be . orders of magnitude less likely than the chance of someone improperly responding to an airflow alarm should the fan fail ( ). these relative probabilities align with our common sense-most laboratory scientists have likely experienced a simple human error, such as a dropped plate or tube, many times more often than we have experienced random mechanical failures. the last, but perhaps most important contribution of human error to laboratory incidents comes after an incident has already begun: human errors after an exposure, whether due to ignorance, panic, or expediency, can drastically exacerbate the consequences of that event. a laboratory worker who immediately notifies appropriate personnel and follows proper health surveillance and isolation protocols after an exposure is significantly less likely to cause secondary infections, limiting the consequences of the incident. despite the dominance of human error in determining risk, we could find limited data to support a quantitative estimate of human failure rates. publicly available quantitative biosafety risk assessments done by others focused primarily on detailed measurements of equipment and mechanical failure rates, and either omitted a quantitative treatment of human error entirely ( , ) or used a flat failure rate for all types of errors in all incidents ( ) . the available primary data were similarly limited, and we identified only a single source of human error measurements taken directly from biological laboratories ( ) . due to these data limitations, we instead analogized the rate of human failures in laboratories to that of similar errors in other industries, primarily aerospace and nuclear power ( ) . indeed, in contrast to the life sciences, these other industries invest heavily in the study of human error. for decades, many technical industries have turned to formalized assessments of human performance and mistake rates, termed human reliability assessments (hras) ( , ) . these hras provide analytical frameworks to rigorously identify the type and frequency of the mistakes humans make, as well as the circumstances in which they are most likely to make them. armed with the knowledge of how and when things may go wrong, risk and safety managers can work to redesign systems and update practices to best prevent common and/or serious mistakes before they occur. these investments are made even though the scale of potential consequences of an accident in the power and transportation industries is dwarfed by the consequences of a global infectious disease outbreak. not only is scholarship lacking on how mistakes may be made, data are not collected on how mistakes have been made either. despite a clear need for keeping these records, the united states has no standardized or comprehensive system for tracking laboratory incidents or near misses in high-containment laboratories. astoundingly, we appear to lack even the most basic knowledge of how many high-containment laboratories are currently in operation ( ) . although some partial reporting systems exist ( , ) , none of these systems are sufficiently standardized, complete, or of high-enough quality and detail to provide usable statistics about the type, magnitude, and kind of incidents that occur in biological laboratories. this lack of centralization and standardization hinders the spread of lessons learned and best practices between laboratories, which likely results in us suffering the same mistakes in multiple laboratories. other industries have discovered that centralized tracking of incidents can significantly reduce risk. in , twa flight crashed outside washington's dulles airport, killing people ( ) . during the investigation, it was discovered that a united airlines flight had a near miss due to the same cause just weeks earlier, and although united had alerted its pilots of the danger, the warning did not spread industry-wide. in the wake of the accident, the federal aviation administration (faa) created-and has maintained ever since-a no-fault system of reporting aviation incidents and mistakes, no matter how minor. this reporting correlates with a decrease in risk: airline industry sources show a substantial and continuing decrease in accident rates from to today (today's rate is about a third of the rate in the early s) ( , ) . similar measures have been undertaken in the nuclear power and chemical manufacturing sectors, which greatly improve the ability to predict potential accidents and prevent them ( , ) . although we note that these industries have had decades of additional time and experience beyond that of the relatively new life science industry to advance their state of risk assessment and prevention, we believe that the life sciences industry should strive, over time, to meet the same standards those industries have pioneered. also, although these industries are dominated by a few giant concerns, the fact that the government and a handful of companies are responsible for the large majority of research on dangerous pathogens enables a similarly few number of influential players to effect significant change. the combination of prospective human reliability primary data gathering to assess what might go wrong and historical incident record keeping to assess what did go wrong provides a powerful path to reducing biosafety risk. using these data, laboratory safety practices can be improved, lowering the risk to the researchers and to their surrounding communities. in addition, training, equipment, and safety systems can be redesigned to prevent common mistakes before they happen. beyond these benefits in risk reduction, gathering data on human reliability and biosafety incidents will also support the development of absolute biosafety risk assessments, which is desirable for several reasons. first, biosafety levels can begin to be defined by the maximum risk of an experiment allowable under that safety level, instead of being defined by organism phenotype, ensuring that the riskiest experiments remain tightly protected without unnecessarily burdening others. second, absolute numbers enable comparisons across industries and activities, which contextualizes the level of biosafety risk we are assuming. by making risk less subjective, policy debates can be more focused and targeted. in our experience with the gain-of-function debate, our inability to provide absolute numbers left unresolved questions about the baseline acceptable level of risk, distracting from the policy questions at hand. in sum, gathering these data is achievable and would provide immense benefits. as such, we believe significantly more funding is urgently and immediately needed to support three basic thrusts: (i) development of a national incident reporting system, (ii) primary research programs focused on hras, equipment failures, and decontamination efficiencies, and (iii) sharing of best practices. we believe the reporting system should be structured like the faa's database and should include not only consequential incidents but also reports of near misses, defined as situations that required a response but where no infections or consequences resulted. consequential incidents are often rare outcomes of a series of common mistakes or failures. by reporting near misses that may contain these same common mistakes, the mistakes can be identified and remedies can be put into place prior to the mistakes precipitating high-consequence events. the nsabb's recommendations for overseeing gain-of-function research includeddue to prompting by us and others-a recommendation to create such a database of incidents and near misses, and we strongly agree with their recommendation ( ) . ostp's guidance document in response to nsabb's recommendations ( ) does not address this data gap, and we fear momentum for the creation of the database is at risk of being lost. creation of such a database is not without challenges. currently, many institutions have expressed reluctance to share information about biosafety incidents, due to fears of how that information may be shared by organizations beyond their control, for example, in the series of articles usa today has published ( , ) . in addition, some institutions have hesitated to share data on incidents due to a lack of standard definitions of what qualifies as reportable, and no institution wants to be penalized as unsafe for the good deed of overreporting. although daunting, these challenges can-and should-be overcome. one first step would be to identify the largest current disincentives to reporting and potential approaches to removing them. a second step would be building consensus for a complete definition of what should be reportable. we believe efforts to accomplish these steps should be led by the biosafety and research communities, similar to how these same communities led the efforts at asilomar in to establish biosafety guidelines for work with recombinant dna ( ) . to address biosafety knowledge gaps, federal grant funds should be made available for primary research studies. although the federal government does fund biosafety education and training programs, there appears to be little spending on quantitative studies of laboratory biosafety practices and accidents, despite a national institute of allergy and infectious disease (niaid) operating budget of $ . billion in fiscal year ( ) . we note that a new annual allocation of a mere / th of % of that number-$ million per year-set aside for biosafety research and tracking could make significant strides to cover these gaps and greatly increase our ability to predict and prevent accidents. the funding for investigations of accidents and research into their causes by agencies whose primary mission is safety in the transportation (nearly $ billion, via the national transportation safety board [ntsb] and national highway traffic safety administration [nhtsa]) and nuclear and chemical industries (more than $ billion via the nuclear regulatory commission [nrc] and chemical safety board [csb]) demonstrates that the united states has already recognized the importance of these types of studies. in addition to studies investigating human reliability, applied, laboratory-based studies should also be conducted to gather additional data about failures due to mechanical systems or decontamination protocols. given the recent widespread attention on failures of inactivation protocols ( , ) , learning how these protocols fail will also enhance safety and our ability to ascertain risk. finally, although failure rates for many types of personal protective equipment (ppe) are known, many kinds of data are still lacking. primary research in this area would be straightforward and would additionally improve biosafety risk assessments. although incident recordkeeping and human reliability are the two largest and most urgent areas, other areas of biosafety research have also suffered due to a lack of funding. in our experience visiting laboratories undertaking gain-of-function research, we noted some institutions maintained a strong safety culture that likely played a significant role in reducing the risk of accident in these labs. yet, how to create this culture and the effect it has on overall safety remain understudied. in addition, we noted that some labs put into place some unique best practices, such as maintaining a separate entrance at the designated local clinic for potentially exposed laboratory personnel, limiting public exposure, yet these practices were not widely known or shared within the biosafety community. funding for collection and dissemination of these best practices is sorely needed, would be relatively modest, and would have a nearly immediate return on investment. federal, state, and local governments, educational institutions, and industry participants do commit significant resources to biosafety and biorisk management, through biosafety officers, institutional biosafety committees, the biosafety in microbiological and biomedical laboratories manual ( ) , and a host of risk assessment practices, polices, and reporting requirements. these efforts play a critical role in ensuring that biological research is conducted safely. however, despite these efforts, large gaps in our knowledge on how accidents did and could occur in laboratories still exist, and this fact is surprising and inexcusable, given that an accident in a biological laboratory, while already extremely unlikely under today's safety precautions, could lead to a global infectious disease outbreak that kills more people than all aviation and industrial accidents combined. given the vast gaps in knowledge that exist, a significant return on investment could be expected in terms of reduced biosafety risk in the near term, making this one of the safest research investments the federal government could make. transforming biosafety into a quantitative practice would ensure that our research enterprise can produce the cures and treatments to improve our quality of life without a single accident vitiating millions of hours of toil at the bench. it is time to make that transition a reality. government gain-of-function deliberative process and research funding pause on selected gain-of-function research involving influenza, mers, and sars viruses. us department of health and human services recommended policy guidance for potential pandemic pathogen care and oversight (p co) supporting an estimate of absolute risk risk and benefit analysis of gain of function research, supplemental information for chapter biosquare phase ii supplemental final environmental impact report final revised environmental assessment for the proposed construction and operation of a biosafety level facility at lawrence livermore national laboratory national bio and agro-defense facility, science and technology directorate, us department of homeland security evaluation of risk and protective factors for work-related bite injuries to veterinary technicians certified in minnesota human reliability assessment in biological laboratories a guide to practical human reliability assessment human reliability assessment: theory and practice high-containment laboratories: national strategy for oversight is needed. us government accountability office publication no. gao- - nih guidelines for research involving recombinant or synthetic nucleic acid molecules animal and plant health inspection service, department of agriculture. part . possession, use, and transfer of select agents and toxins the development of the nasa aviation safety reporting system. nasa reference publication . scientific and technical information branch commercial aviation accidents - . a statistical analysis boeing commercial airplanes. . statistical summary of commercial jet airplane accidents: worldwide operations - . aviation safety, boeing commercial airplanes reporting and preventing medical mishaps: lessons from non-medical near miss reporting systems near-miss incident management in the chemical process industry recommendations for the evaluation and oversight of proposed gain-of-function research. national science advisory board for biosecurity hundreds of safety incidents with bioterror germs reported by secretive labs cdc failed to disclose lab incidents with bioterror pathogens to congress summary statement of the asilomar conference on recombinant dna molecules niaid budget data comparisons. national institute of allergy and infectious disease biosafety lapses prompt us cdc to shut labs and launch review review committee report: inadvertent shipment of live bacillus anthracis spores by dod. committee for comprehensive review of dod laboratory procedures, processes, and protocols associated with inactivating bacillus anthracis spores, us department of defense, the pentagon public health service, centers for disease control and prevention, and national institutes of health, us department of health and human services key: cord- - qqrn w authors: lanteri, charlotte; mende, katrin; kortepeter, mark title: emerging infectious diseases and antimicrobial resistance (eidar) date: - - journal: mil med doi: . /milmed/usz sha: doc_id: cord_uid: qqrn w introduction: the infectious disease clinical research program’s (idcrp) emerging infectious diseases and antimicrobial resistance (eidar) research area is a department of defense (dod) clinical research capability that is responsive and adaptive to emerging infectious disease (eid) threats to us military readiness. among active-duty and other military health system (mhs) beneficiaries, eidar research is largely focused on evaluating the incidence, risk factors, and acute- and long-term health effects of military-relevant eids, especially those caused by high-consequence pathogens or are responsible for outbreaks among us military populations. the eidar efforts also address force health protection concerns associated with antimicrobial resistance and antimicrobial stewardship practices within the mhs. methods: the eidar studies utilize the approach of: ( ) preparing for emergent conditions to systematically collect clinical specimens and data and conduct clinical trials to assist the military with a scientifically appropriate response; and ( ) evaluating burden of emergent military-relevant infectious diseases and assessing risks for exposure and development of post-infectious complications and overall impact on military readiness. results: in response to the ebola virus epidemic in west africa, the idcrp partnered with the national institutes of health in developing a multicenter, randomized safety and efficacy study of investigational therapeutics in ebola patients. subsequently, the eidar team developed a protocol to serve as a contingency plan (epicc-eid) to allow clinical research activities to occur during future outbreaks of viral hemorrhagic fever and severe acute respiratory infections among mhs patients. the eidar portfolio recently expanded to include studies to understand exposure risks and impact on military readiness for a diversity of eids, such as seroincidence of non-lyme disease borreliosis and coccidioides fungal infections among high-risk military populations. the team also launched a new prospective study in response to the recent zika epidemic to conduct surveillance for zika and other related viruses among mhs beneficiaries in puerto rico. another new study will prospectively follow u.s. marines via an online health assessment survey to assess long-term health effects following the largest dod shiga toxin-producing escherichia coli outbreak at the u.s. marine corps recruit depot-san diego. in cooperation with the trauma-related infections research area, the eidar research area is also involved with the multidrug-resistant and virulent organisms trauma infections initiative, which is a collaborative effort across dod laboratories to characterize bacterial and fungal isolates infecting combat-related extremity wounds and link lab findings to clinical outcomes. furthermore, the eidar team has developed an antimicrobial resistance and stewardship collaborative clinical research consortium, comprised of infectious disease and pharmacy specialists. conclusions: the eidar research area is responsive to military-relevant infectious disease threats that are also frequently global public health concerns. several new eidar efforts are underway that will provide combatant command surgeons, infectious diseases service chiefs, and other force health protection stakeholders with epidemiological information to mitigate the impact of eids and antimicrobial resistance on the health of u.s. military service members and their dependents. and the u.s. army medical research institute of infectious diseases. in addition, department of defense (dod) overseas labs can leverage opportunities to conduct surveillance for emerging threats, as well as conduct large scale vaccine or therapeutic trials. one significant gap; however, was having a clinical platform in dod medical facilities to conduct multi-center clinical research on potential emerging threats and biodefense. therefore, in , the infectious disease clinical research program (idcrp) leadership decided to add a research area related to biodefense and emerging infectious diseases (bd/ eid). this nascent program has evolved, perhaps more significantly than any other research area since its establishment. in addition to the aforementioned gap in the dod clinical centers, the decision to add this new research area was based on other strategic needs. the military's focus on agents that threaten national security or military personnel on the battlefield may be different than civilian preparedness response efforts for bioterrorism or pandemics. this research area helped align the program with key dod and non-dod stakeholders to enhance the idcrp's ability to respond to new or re-emerging threats and support military clinical capabilities to study biodefense countermeasures. agents causing viral hemorrhagic fevers, including ebola and marburg viruses, have been targets of vaccine and therapeutics development in the dod. viral hemorrhagic fevers have threatened combat, peacekeeping, and training operations for decades in areas such as the balkans (dobrava virus), korea (hantaan virus) and the south pacific (dengue virus) and, as a result, have motivated the development of preventive measures. consequently, the first study in the bd/eid program area was a collaboration between the national institute of allergy and infectious diseases (niaid) vaccine research center and military investigators on the safety and immunogenicity of ebola and marburg dna vaccines, assessed at a wrair collaborative field site at makerere university in kampala, uganda. the results showed that, given separately or together, both investigational dna vaccines, one encoding ebola virus zaire and sudan glycoproteins and one encoding marburg virus glycoprotein, were well-tolerated and elicited antigen-specific humoral and cellular immune responses. these findings contributed to the accelerated development of more potent ebola virus vaccines that encode the same wildtype glycoprotein antigens that were tested during the - ebola virus disease outbreak in west africa. , in , the largest-ever ebola virus outbreak was recognized in the west african countries of guinea, liberia, and sierra leone. the dod responded with providing military members to build ebola treatment units, provide logistics support, and diagnostics teams and assays in-country under operation united assistance. multiple different investigational countermeasures were employed during the outbreak and niaid led a multi-center group to prioritize the potential countermeasures and develop a randomized controlled trial to study the most promising options. idcrp investigators participated in the prioritization effort and the idcrp's team at the walter reed national military medical center served as the only dod site participating in the protocol: a randomized controlled trial of the monoclonal antibody zmapp for treatment of ebola virus disease. the study enrolled patients and demonstrated improved outcomes from ebola virus disease in those receiving zmapp, although the results did not meet pre-specified criteria for statistical significance. as a naturally occurring infection, smallpox was the target of a successful global eradication campaign in the s and vaccination of civilian populations ceased in the s. nevertheless, because of ongoing concerns of bioterrorism, the u.s. military continues to vaccinate at-risk operational forces, some healthcare workers, and service members in korea. during the last decade, over one million individuals received the vaccine, which is made from the vaccinia virus. in addition to conferring protection against smallpox, the vaccinia virus demonstrated promise as a vaccine-delivery system, inducing immunity to pathogens such as hiv. as a result, another early study assessed host immune factors that might attenuate the response to vaccinations. because of its potential use in hiv candidate vaccines, investigators at the idcrp and the military hiv research program, with support from the u.s. military vaccine agency, evaluated the potential impact of pre-existing immunity to vaccinia with the use of vaccinia-vectored vaccines in a cell-based infection model. using longitudinal sera from military personnel who had been vaccinated, the investigators found that serum antibody responses to smallpox vaccination did not persist over extended periods of time (e.g., years post-vaccination), suggesting that prior vaccination for smallpox might not interfere with the effectiveness of novel vaccines using vaccinia virus as a vector. within the idcrp, there is a broad intersection of emerging infections across multiple other idcrp research areas, such as the skin and soft-tissue infections and trauma-related infections. , in particular, research has focused on the epidemiology, risk factors, and prevention of community-associated methicillin-resistant staphylococcus aureus skin and softtissue infections in military personnel, as well as emergent trauma-related infections, such as invasive fungal wound infections. in line with the president's executive order for combating antibiotic-resistant bacteria, idcrp also led the way to identify and assess the public health impact of multidrug-resistant gonococci. other early investigations into eid threats assessed a range of disease agents, such as rickettsia parkeri in the tidewater region of virginia shortly after it was identified as an endemic disease in that region. as the bd/eid program continued to evolve, it was renamed as the emerging infectious disease & antimicrobial resistance (eidar) research area in and continues to grow to encompass a broad clinical research portfolio, covering a diversity of military-relevant eids. infectious disease threats to military forces remain high due to deployment worldwide, frequently to areas with limited medical and public health infrastructure. novel respiratory pathogens, such as mers-cov and avian influenza strains, pose significant risk to military populations. the - ebola outbreak in west africa exposed vulnerabilities and delays in responding to research opportunities, and led to the recognition that the u.s. needs the capability to study infectious disease outbreaks in real time, as well as being included in routine public health response planning. the rapid deployment of large numbers of military personnel into disease endemic areas heightens their risk for acquiring infectious diseases. in these settings, the conditions and circumstances (i.e., crowding and inadequate access to hygiene) facilitate potential for disease transmission to other susceptible individuals. the return of military forces from diseaseendemic areas also imposes a significant disease risk to other military personnel, military dependents, as well as the general public, by creating an avenue for the importation of novel pathogens into the united states and increasing the risk of localized outbreaks and major epidemics. the impact of monitoring for and describing the epidemiology and clinical characteristics of novel respiratory pathogens with pandemic potential among military personnel is necessary to ensure a timely, comprehensive epidemiologic, immunologic, virologic, and clinical characterization of the initial cases of an eid in what may be a mounting epidemic or pandemic. the idcrp has prided itself on responding rapidly to new research opportunities; however, the time it takes to draft and receive regulatory approval for new protocols may lead to missed opportunities during an outbreak, especially for shortlived events. with this in mind, in , the idcrp established the epidemiology, immunology and clinical characteristics of emerging infectious diseases with pandemic potential (epicc-eid) protocol, which was designed to fill critical needs by providing military hospitals with a plan to respond rapidly to public health crises/outbreaks of diseases with severe outcomes with potential to spread to the civilian populations in the united states and abroad. the objectives of this protocol are to characterize the epidemiologic, laboratory, immunologic, and clinical characteristics of infections caused by high-consequence pathogens, such as severe acute respiratory infections or viral hemorrhagic fevers, among individuals presenting for care at mtfs to inform effective patient care. this protocol is intended to reduce the response time to facilitate collaborative research partnerships for interventional therapeutic trials, vaccines, or new diagnostic assays for a novel respiratory pathogen or viral hemorrhagic fever as part of a whole of government approach. the eidar research area directs studies in response to the global health security agenda and the national security strategy requirements for preparedness and response to infectious disease outbreaks. as discussed above, the epicc-eid protocol provides the mhs with a unique capacity for preparedness in facilitating systematic collection of clinical specimens and data at mtfs to support the military with a scientifically appropriate response. in addition, eidar supports studies investigating the disease burden, risk factors, and clinical outcomes associated with emerging and/or reemerging pathogens responsible for causing outbreaks of disease affecting u.s. military populations. recently, zikv emerged as a major public health crisis, with the world health organization in february declaring zikv-related congenital abnormalities a public health emergency of international concern. the u.s. military service members deploying to disease endemic areas where the outbreak was occurring (primarily in south america) or stationed in puerto rico were also at risk for zikv exposure. given limitations on the availability of laboratory testing, u.s. (and dod) policy precludes testing of asymptomatic men for diagnosis of zikv infection. to date, there have been confirmed zikv cases in the mhs, primarily in deployers and travelers to puerto rico and south america. following the zikv outbreaks, there was a recognized need for establishing means for conducting active surveillance of zikv (and related arboviruses, such as dengue and chikungunya) among mhs beneficiaries in a disease-endemic location to better prepare for any subsequent re-emergence of these diseases. to this end, the eidar team, in collaboration with the wrair viral diseases branch, developed a prospective protocol to conduct surveillance of zikv and related arboviral infections among active-duty service members and their dependents presenting with symptoms at the rodriguez army health clinic in puerto rico. this study will allow early detection of outbreaks among mhs beneficiaries on the island, assessment of impact on military readiness, and ready reporting to force health protection officials. chikungunya virus is another mosquito-borne infectious disease threat to deployed u.s. military service members that is caused by an rna virus related to dengue and zikv, and sometimes is misdiagnosed since these viruses share similar clinical signs and common geographic distribution. chikungunya infection causes febrile illness, often characterized by debilitating joint pain and, in some cases, progresses to severe rheumatic disease. while the majority of patients recover fully, some cases are associated with chronic issues of joint pain; eye, neurological and heart complications; and gastrointestinal complaints. an eidar virtual cohort study is underway, involving analysis of data extracted from the disease reporting system internet by collaborators at the navy marine corps public health center epidata center and the mhs data repository, to evaluate short-and longterm health outcomes, disability, and healthcare utilization attributable to chikungunya infection in mhs beneficiaries. in addition to vector-borne tropical infectious diseases, diarrheal disease is another major threat to u.s. military operations worldwide; however, further investigation is merited to determine the potential long-term health consequences of severe gastrointestinal infections. the impact on military medical readiness of diarrheal illness (travelers' diarrhea) encountered during overseas deployments is a well-characterized force health protection issue and is a major focus for prevention and treatment research within the idcrp deployment and travel-related infection research area. [ ] [ ] [ ] in contrast, less is known about the deleterious health consequences associated with outbreaks of enteric diseases among military populations at installations within the continental united states. moreover, mounting evidence reported in the literature, primarily from epidemiological studies among civilian populations impacted by diarrheal disease outbreaks, suggests post-infectious sequelae (medical complications observed following infection) is a growing concern. - shiga toxin-producing escherichia coli (stec) infection, a major cause of foodborne illness in the united states and associated with consuming undercooked beef, is linked to life-threatening complications manifesting in the acute phase of infection as severe hemorrhagic colitis and hemolytic uremic syndrome, as well as lesser known sequelae emerging months to years later. in response to the u.s. military's largest outbreak of stec (nearly cases) which occurred at the u.s. marine corps recruit depot-san diego in the fall of , a new eidar protocol was developed to investigate long-term health impact of stec infection through a -year follow-up online surveybased study assessing clinical outcomes. the overall objective is to determine the incidence of post-infectious sequelae, including functional bowel disorders (irritable bowel syndrome, dyspepsia, constipation), osteoarticular symptoms (reactive arthritis and avascular necrosis), and renovascular disease (hypertension and chronic kidney disease) in marines identified as stec cases during the outbreak relative to nonill matched controls. a series of analytic modeling approaches will be used to develop a risk model health outcomes risk score to determine how comorbidities, demographics, and other factors affect risk for developing post-infectious sequelae. this health outcomes risk score could be a force health protection tool for identifying individuals at greatest risk for long-term complications in military populations affected by future diarrheal illness outbreaks. the eidar portfolio also supports studies among u.s. military populations to identify the exposure risk to pathogens encountered within the continental united states. specifically, there are two ongoing retrospective studies involve laboratory analyses of dod serum repository specimens from service members to evaluate the burden of military-relevant infectious diseases: newly emergent non-borrelia burgdorferi tick-borne borrelia bacterial infections causing lyme disease and related syndromes at u.s. military training installations and coccidioides fungal infection at the high-risk disease endemic naval air station lemoore. findings from these studies will inform if future investigations into protective measures, novel therapeutics, or improved diagnostics are warranted to protect service members from these pathogens. the eidar research area aims to be increasingly responsive to addressing clinical research gaps informative to shaping force health protection policy. recent strategic partnerships with the armed forces health surveillance branch global emerging infections surveillance section and the uniformed services university of the health sciences (usu) center for global health engagement have been critical to aligning eidar research efforts with geographic combatant command surgeons' requirements for addressing the threat of regional infectious diseases affecting military operations. these and other partnerships with key dod stakeholders will continue to advance the operational relevance of eidar work. antimicrobial resistance is a serious health threat worldwide. new forms of antibiotic resistance have emerged over the past decades and can spread rapidly with some pathogens becoming resistant to multiple types and classes of antimicrobials. [ ] [ ] [ ] [ ] [ ] colonization with and infections caused by multidrug-resistant organisms (mdros) appear common and have a significant impact on military populations. [ ] [ ] [ ] [ ] [ ] in addition, mdro infections further complicate the care of wounded warriors, are associated with high morbidity and mortality, and add substantial and avoidable costs to the healthcare system due to prolonged and costlier treatments, longer hospital stays, and additional patient visits. the idcrp has had a long interest in studying antimicrobial resistance in several of its research areas. as a result, eidar research on mdros and infections caused by these organisms overlap with several other idcrp research areas, including trauma-related infections, deployment and travel-related infections, and sexually transmitted infections research areas. , , in alignment with the president's launch of the executive order for combating antibiotic-resistant bacteria, eidar efforts address force health protection concerns associated with antimicrobial resistance (amr) and filling clinical knowledge gaps related to best practices for antimicrobial stewardship programs (asps) within the mhs. one such major effort is the trauma infectious disease outcomes study (tidos) multidrug-resistant and other virulent organisms (mdr/vo) trauma infections initiative, which is a collaborative effort across dod laboratories with expertise in wound microbiology and infections (including usu, brooke army medical center, u.s. army institute of surgical research, wrair, and nmrc). the mdr/vo trauma infections initiative was established to investigate the combat trauma wound microbiology and infections linked to well-characterized clinical data and outcomes to expand our understanding of the complex microbiology inherent within combat wounds. through the tidos mdr/vo initiative, several analyses have been performed to examine microorganisms collected from combat casualties from iraq and afghanistan, assess antimicrobial resistance, and evaluate associations with clinical outcomes. patients colonized or infected with pathogens having in vitro resistance are reported to have more co-morbidities, longer hospital stays prior to infection, and increased exposure to antibiotics. each of these factors can independently influence clinical outcomes regardless of in vitro susceptibilities. therefore, understanding patient comorbidities is important when assessing the impact of in vitro resistance on outcomes, along with the development and prevention of infection. comorbidities may differ in a military population compared to the general public, especially in combat-related trauma patients and, thus, may affect outcomes differently in this population. the impact of in vitro resistance in a military population has not been evaluated. to address this clinical knowledge gap, an eidar retrospective study is investigating association between phenotypic in vitro bacterial resistance, patient comorbidities and outcomes in antimicrobial-resistant bloodstream infections (ar-bsis) in adult dod beneficiaries, utilizing data extracted from the mhs data repository. analyses are underway of microbiology and clinical data from~ , cases to describe the demographics and risk factors of mhs patients who develop ar-bsis. results from this study will help direct mhs resources to more effectively prevent and plan care for these patients. antibiotic use is a crucial factor leading to antibiotic resistance. up to half of all antibiotics prescribed are not needed or are not effective as prescribed, increasing the risk of bacteria becoming resistant to those antibiotics. , antibiotic misuse also contributes to adverse drug reactions in patients and increased healthcare costs. thus, there is an urgent need to ascertain further clinical knowledge on optimal and appropriate antibiotic use to inform policies that form the basis of hospital asps. the eidar team leveraged the idcrp's extensive clinical research network, which includes active-duty investigators located at mtfs worldwide, to develop an amr/asp collaborative clinical research consortium, comprising infectious disease and pharmacy specialists from army, navy, and air force mtfs. to date, the consortium performed a landscape review of asp efforts within the mhs and identified clinical evidence gaps to be addressed by the group through multisite studies. one ongoing effort of the consortium is creating a knowledge, attitudes, and practices survey to identify trends and practices driving antimicrobial prescribing patterns among mhs healthcare providers, with the aim of generating findings informative to making recommendations for improving asp practices and training within the mhs to support optimal practices in managing microbial infections. the eidar research area was established to ensure the idcrp remained nimble and responsive to studying new and emerging disease threats to military populations in a constantly evolving landscape. in addition to the specific efforts within eidar, the eidar team will continue to partner with other idcrp research areas, as well as external collaborators, to achieve the goal of mitigating the impact of military-relevant eids, especially high-consequence pathogens and mdr infections, on military readiness. newer efforts such as the epicc-eid protocol, stec-short and long-term health effects, zikv epidemiology, and amr efforts, will continue to advance the idcrp's military operational relevance. emerging infections: microbial threats to health in the united states emerging infectious diseases in : years after the institute of medicine report safety and immunogenicity of ebola virus and marburg virus glycoprotein dna vaccines assessed separately and concomitantly in healthy ugandan adults: a phase b, randomised, double-blind, placebo-controlled clinical trial efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, clusterrandomised trial (ebola ca suffit!) ebola outbreak in west africa operation united assistance: infectious disease threats to deployed military personnel a randomized, controlled trial of zmapp for ebola virus infection immunization healthcare branch. information paper, smallpox disease and smallpox vaccine: defense health agency humoral immunity to primary smallpox vaccination: impact of childhood versus adult immunization on vaccinia vector vaccine development in military populations executive order -combating antibiotic-resistant bacteria detecting rickettsia parkeri infection from eschar swab specimens research as a part of public health emergency response world health organization. who director-general summarizes the outcome of the emergency committee regarding clusters of microcephaly and guillan-barre syndrome armed forces health surveillance branch a: years in of chikungunya clinical vaccine development: a historical perspective a scoping review of published literature on chikungunya virus diarrhoea during military deployment: current concepts and future directions military importance of diarrhea: lessons from the middle east german outbreak of escherichia coli o :h associated with sprouts chronic sequelae of e. coli o : systematic review and meta-analysis of the proportion of e. coli o cases that develop chronic sequelae the epidemiology, microbiology and clinical impact of shiga toxin-producing escherichia coli in england shiga toxin-producing escherichia coli infection prevention: carbapenem-resistant klebsiella pneumoniae associated with a long-term -care facility -west virginia complete sequence of a novel -kilobase plasmid carrying bla(ndm- ) in a providencia stuartii strain isolated in afghanistan centers for disease control and prevention: notes from the field: hospital outbreak of carbapenem-resistant klebsiella pneumoniae producing new delhi metallo-beta-lactamase centers for disease control and prevention: notes from the field: new delhi metallo-beta-lactamase-producing escherichia coli associated with endoscopic retrograde cholangiopancreatography -illinois carbapenemase-producing enterobacteriaceae bacteriology of war wounds at the time of injury infection-associated clinical outcomes in hospitalized medical evacuees after traumatic injury: trauma infectious disease outcome study multidrug-resistant bacterial colonization of combat-injured personnel at admission to medical centers after evacuation from afghanistan and iraq phenotypic and genotypic changes over time and across facilities of serial colonizing and infecting escherichia coli isolates recovered from injured service members carbapenem-resistant enterobacteriaceae and the correlation between carbapenem and opportunities and obstacles in the prevention of skin and soft-tissue infections among military personnel after the battlefield: infectious complications among wounded warriors in the trauma infectious disease outcomes study biomedical response to neisseria gonorrhoeae and other sexually transmitted infections in the united states military the acute respiratory infection consortium: a multi-site, multi-disciplinary clinical research network in the department of defense fluoroquinolone usage and resistance in the us military health system the commonality of risk factors for nosocomial colonization and infection with antimicrobial-resistant staphylococcus aureus, enterococcus, gram-negative bacilli, clostridium difficile, and candida immune response to traumatic injury: harmony and discordance of immune system homeostatis vital signs: improving antibiotic use among hospitalized patients antimicrobial resistance we are indebted to our dedicated study team of idcrp investigators, clinical research managers, clinical research coordinators, laboratory personnel, and data management staff for their hard work and contributions to the success of our projects. key: cord- - zu fn authors: riley, william t; glasgow, russell e; etheredge, lynn; abernethy, amy p title: rapid, responsive, relevant (r ) research: a call for a rapid learning health research enterprise date: - - journal: clin transl med doi: . / - - - sha: doc_id: cord_uid: zu fn our current health research enterprise is painstakingly slow and cumbersome, and its results seldom translate into practice. the slow pace of health research contributes to findings that are less relevant and potentially even obsolete. to produce more rapid, responsive, and relevant research, we propose approaches that increase relevance via greater stakeholder involvement, speed research via innovative designs, streamline review processes, and create and/or better leverage research infrastructure. broad stakeholder input integrated throughout the research process can both increase relevance and facilitate study procedures. more flexible and rapid research designs should be considered before defaulting to the traditional two-arm randomized controlled trial (rct), but even traditional rcts can be designed for more rapid findings. review processes for grant applications, irb protocols, and manuscript submissions can be better streamlined to minimize delays. research infrastructures such as rapid learning systems and other health information technologies can be leveraged to rapidly evaluate new and existing treatments, and alleviate the extensive recruitment delays common in traditional research. these and other approaches are feasible but require a culture shift among the research community to value not only methodological rigor, but also the pace and relevance of research. despite increasing demands to produce timely and relevant research findings, our traditional research process remains painstakingly slow. randomized efficacy trials take approximately . years from the initiation of enrollment to publication [ ] , and years or longer after adding the time from grant application submission to enrollment initiation. extensive follow-up periods for relevant outcomes such as morbidity/mortality as well as delays in participant recruitment and publication can extend this time period to a decade or longer. during this period, scientific and technological advances will occur that may make the eventual findings less relevant or even obsolete. for illustration, figure shows a few of the salient consumer technologies introduced during a typical seven year clinical trial. these recent advances in consumer technologies are most impactful for mobile and wireless health research, [ ] but many less mainstream scientific and medical technological advances also occur while clinical trials are being conducted. for example, one explanation for the recently reported negative results of the sammpris trial was that stent technologies and surgery procedures had advanced substantially since study initiation [ , ] . this protracted period from concept to publication is further exacerbated by the slow and limited uptake of research findings into practice. balas and boren have estimated that it takes approximately years from concept to evidence implementation for the % of evidence that progresses to implementation [ ] . a recent report from the president's council of advisors on science and technology (pcast) estimated that , treatments are in development and concluded that major upgrades are needed in the research system to evaluate these treatments [ ] . an institute of medicine report on clinical trials states that "recognition is growing that the clinical trials enterprise in the united states faces substantial challenges impeding the efficient and effective conduct of clinical research to support the development of new medicines and evaluate existing therapies [ ] ." clearly, our current research enterprise is too slow, inefficient, and cumbersome to meet the rapidly evolving demand. what are needed are "rapid-learning research systems" that integrate researchers, funders, health systems, practitioners, and community partners asking clinically relevant questions, using efficient and innovative research designs, and leveraging rich, longitudinal data sets from millions of patients. to begin progress toward such a system, we considered and have described approaches to make research more rapid, responsive and relevant (r ), organized into four sections for the purpose of this paper: ) stakeholder engagement, ) design, ) review, and ) infrastructure (see table ). these suggested approaches are viewed as a starting point for a dialogue among the health research community to challenge our current cumbersome research enterprise and to consider these or other approaches to maintain scientific rigor while speeding the process by which more responsive and relevant research findings are produced. broad stakeholder engagement involving patients, providers, health plans, policy makers and other relevant stakeholders may seem counterintuitive as a strategy to speed research, but this time investment has the potential to improve the recruitment and retention of study participants, thus increasing the pace of conducting the study. more importantly, stakeholder engagement increases the likelihood that findings will be relevant to stakeholders and more readily adopted into practice, thereby making the overall research pipeline more efficient. these "evaluability assessments" [ ] or participatory approaches are considered key to facilitating the adoption of research findings by practitioners [ ] . the patient centered outcomes research institute (pcori), for example, is creating public advisory groups and soliciting patient input on specific comparative effectiveness questions that are relevant to practitioners and stakeholders [ ] . an ongoing relationship between researchers, healthcare providers, health plans, and patients is critical to a better, faster research system. clinical trial recruitment is a major problem with about % of us trials failing to meet enrollment goals [ ] . the nih health care system collaboratory (hcsc) offers an important resource for rapid research. like the hmo research network and the va queri program, the hcsc will make available opportunities to conduct large-scale studies within well-organized healthcare delivery systems [ ] . research embedded in organized delivery systems and networks enhances not only research relevance, but also facilitates recruitment, retention, study start-up, operations, data capture, and integration into practice. an accelerated research system can also use information technologies to speed the process of seeking and obtaining stakeholder feedback. consistent with a citizen-scientist model [ ] , a virtual network of various stakeholders can participate and provide feedback throughout the research process using online surveys, virtual meetings, and social media systems [ ] . via innovative technologies, stakeholder feedback can be obtained efficiently to increase research relevance and responsiveness, even for researchers who are not fully integrated into the practice setting or community where their research findings are likely to be translated into practice. traditional study designs and procedures are wellestablished, rigorous, and notoriously slow and costly. this belabored research process typically begins with pilot trials that we posit have limited benefit, are used inappropriately to estimate effect size [ ] , and often prematurely concretize a less than optimal intervention. instead, we recommend replacing the traditional pilot trial with a more flexible iterative intervention testing and optimization approach, analogous to the agile software development process that places a premium on failing early to succeed later [ ] . for example, n-of- trial designs provide intervention development flexibility. with the increasing availability of intensive longitudinal data from wireless sensors and mobile devices, n-of- trials can be rapidly implemented and provide results congruent with a more personalized medicine approach [ ] , and bayesian analyses from a series of such trials [ ] may provide sufficient evidence of generalizability to limit the need for a larger trial. intervention optimization designs such as fractional factorial and sequential multiple assignment research trials (smart) are particularly valuable when the intervention development questions involve combinations or sequences of intervention components [ ] . dynamic system models have also been used to optimize treatments [ ] . some optimization approaches may take more time than the traditional pilot trial, but the pace of the overall research enterprise will be improved by more quickly discarding or modifying interventions that are unlikely to be found effective in larger and more expensive trials. within the traditional rct, researchers have a number of design decisions that can increase efficiency. trials that utilize within-group designs in which participants serve as their own controls can speed the research process by reducing the number of study participants needed to detect outcomes, and can often simplify study procedures as well. the minimal intervention needed for change (minc) standard [ ] provides a standard pragmatic comparison anchor across studies for comparative effectiveness research. the va is adopting a "point of care" randomization that computer-randomizes patients to different treatments, and then uses adaptive algorithms to change allocation of new patients as evidence accumulates [ ] . recent technological advances make it possible to conduct "automated rcts" in which the enrollment, random assignment, intervention delivery, and outcome assessments are fully automated. to fully realize the potential of automated rcts and other rapid learning systems, the nature of and procedures for informed consent need to be resolved. follow-up periods also can be shortened or segmented. results can be analyzed at the point where the maximal benefit of the intervention is hypothesized to occur. longer-term outcomes can be modeled from these results, or one of the investigators can remain blind to conduct the follow-up portion of the study and publish the follow-up results separately. in addition to improving the efficiency of rcts, we also need to consider alternative designs that may be more appropriate to the research question and provide more rapid and relevant answers. a range of within-subject and quasi-experimental designs such as interrupted time series [ ] , stepped wedge [ ] , and regression discontinuity [ ] may have less internal validity than the rct, but offer a number of advantages [ ] . for example, these quasiexperimental approaches facilitated participation of the major minnesota health insurers in the diamond depression treatment program [ ] . these designs may be particularly appropriate for evaluating treatments already adopted in practice. it takes to months from nih grant submission to funding [ ] . if revised and resubmitted, and assuming a six month revision period, it can take two years from initial submission to the award of a revised (a ) nih grant application. during this time, science and technology continue to advance; research partnerships, especially with nonresearch stakeholders, must be maintained; and the research questions may become less relevant or timely. grant review and funding processes could be streamlined in a number of ways. for the recovery act challenge grants [ ] , a flexible, two-stage review process was implemented to reduce to five months the time from receipt to funding. rapid review processes are already used by the nih for time sensitive natural experiments [ , ] . in response to the sars (severe acute respiratory syndrome) outbreak, the canadian institutes of health research developed and issued a funding announcement that resulted in submissions within weeks, and these submissions were reviewed and four approved for funding within days of submission [ ] . these rapid review examples clearly indicate that it is possible to review and fund research applications quickly when necessary, and that such rapid review systems should be considered for a broader range of research, including timely and pressing clinical and public health questions. the grant application review process could facilitate more rapid research not only by reviewing more efficiently, but also by placing a greater premium on more rapid and innovative research designs. despite the addition of innovation as an nih review criteria, a recent study of grant applications revealed that novelty is associated with a . percentile point drop, and that feasibility concerns did not contribute substantially to this "novelty penalty" [ ] . innovative designs, especially those that speed the pace of research relative to traditional designs, should be rewarded, not penalized. institutional review boards (irbs) also should consider streamlining review procedures. slowness of research should be considered a risk, both to study participants who may continue in their assigned treatment even as newer treatments become available, and to the broader public who are delayed getting answers to relevant research questions. revisions to the common rule are anticipated to allow for a more flexible and rapid review process [ ] . online and open access publication practices have greatly reduced the time from acceptance to publication [ ] but could be further facilitated by a better or more incentivized process for acquiring reviewers and obtaining reviews. as green noted, new technologies for publication, systematic reviews, and dissemination of evidence-based guidelines reduce the time from research findings to practitioner adoption, but the publication and dissemination process should continue to be reviewed to further reduce the time lapses between the various stages of the dissemination and implementation process [ ] . improving our research infrastructure has the potential not only to speed the pace of research, but also increase its rigor and relevance. the health system has lagged decades behind other sectors in it implementation [ ] . as a result, health research has been severely constrained by a datapoor environment in which acquiring needed research data is expensive, difficult, and time-consuming. since the rapidlearning health system and learning healthcare system concepts were advanced in [ ] , major investments have been made in databases and learning networks to take advantage of the research potential of electronic health records. it is now possible to conduct some studies in weeks or months instead of years. the fda mini-sentinel system accesses million patient records to generate several studies per week on drug safety questions [ ] . large biobanks are now coming on-line at kaiser-permanente, [ ] the veterans health administration [ ] , the en-code network, [ ] , and the uk biobank [ ] . using these and other patient databases, researchers have been able to assess the unintended effects of treatments [ ] and produce outcome findings comparable to rcts [ ] . large future investments are now being considered that could offer extraordinary opportunities for researchers and a faster, more efficient infrastructure for rapid learning research. the nih director has proposed a new national patient-oriented research system with electronic health records databases, including genomics, for - million patients [ ] , and pcori recently released a funding announcement to support development of the national patient centered clinical research network [ ] . the big data to knowledge (bd k) initiative [ ] also provides the opportunity to leverage these large data sets for rapid research. researchers can accelerate the collective pace of learning with greater attention to reporting comparable data. there have been a number of efforts to encourage the use of common data elements [ ] . standardized outcome data are particularly problematic for patient-reported outcomes. to address this problem, there have been consensus measurement efforts such as phenx [ ] as well as efforts to co-calibrate various patient-reported outcome measures on a single metric [ ] . the national research council report on precision medicine calls for a new science commons and national learning system that will revolutionize biomedical research, clinical care, and public health [ ] . one of the benefits of such a system is that researchers can more readily target drug approval studies to predicted high-response populations, and cut years from the drug research process. gleevec, an anti-cancer drug, was approved in a trial of only patients because nearly all showed benefit [ ] . with targeted therapeutics and research, it took only four years from target discovery to drug approval for the lung cancer drug xalkori [ ] . rapid learning systems of large patient populations appear to provide the infrastructure to rapidly evaluate treatments. we have outlined a number of actions to enhance relevance, streamline design, speed review, and use new research infrastructures to make research more rapid, relevant, and responsive to the st century demands on health research. this transformation to a rapid research learning system will require a concerted effort by research funders, academic institutions, healthcare systems, researchers, and a variety of practice, community, and policy stakeholders to produce a culture change among the health research community. will we continue to use limited funds to support the currently slow and cumbersome research enterprise that produces costly results that may not be relevant or easily translated into practice, or are we willing to pursue alternative approaches that in other research disciplines have produced rapid and relevant improvements? the rationale and opportunities for such a culture change have never been greater or rapid answers more needed. our recommendations to speed research are undoubtedly incomplete, and we invite the research community to contribute additional recommendations to increase the speed and relevance of the research enterprise. this call to streamline and speed the research process is also likely to be met with skepticism, especially among those who fear that methodological rigor might be compromised in a quest for greater efficiency. we believe that the recommendations outlined in this paper can be achieved without compromising scientific rigor, and any efforts to streamline research should be judged based on methodological soundness. we are convinced, however, that the currently dominant health research paradigm is too slow and inefficient to address today's challenges, and that we must produce a more rapid, responsive, and relevant research enterprise. effect of the statistical significance of results on time to completion and publication of randomized efficacy trials advancing the science of mhealth stenting versus aggressive medical therapy for intracranial arterial stenosis interpretations and implications of the prematurely terminated stenting and aggressive medical management for 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health and science research (rc ) time-sensitive obesity policy and program evaluation (r ) innovation and challenges in funding rapid research responses to emerging infectious diseases: lessons learned from the outbreak of severe acute respiratory syndrome the novelty paradox and bias for normal science: evidence from randomized medical grant proposal evaluations human subjects research protections: enhancing protections for research subjects and reducing burden, delay, and ambiguity for investigators what does the marriage of open access and online publication bring? what' s holding back online medical data etheredge lm: a rapid-learning health system a systematic review of validated methods for identifying pancreatitis using administrative data the encode project: encyclopedia of dna elements unintended effects of statins in men and women in england and wales: population-based cohort study using the qresearch database use of primary care electronic medical record database in drug efficacy research on cardiovascular outcomes: comparison of database and randomized controlled trial findings nih common data element research portal the phenx toolkit: get the most from your measures measuring fatigue in persons with multiple sclerosis: creating a crosswalk between the modified fatigue impact scale and the promis fatigue short form delivering value through personalized medicine: an industry perspective rapid, responsive, relevant (r ) research: a call for a rapid learning health research enterprise the authors declare that they have no competing interests. preparation of this manuscript was supported in part by a grant from the robert wood johnson foundation to george washington university to the third author. the funders had no role in this manuscript. the opinions expressed are those of the authors and do not necessarily reflect those of the robert wood johnson foundation or the national cancer institute.authors' contributions wtr led the writing and preparation of this paper. reg, le, and apa all contributed sections to this paper, and revised and edited drafts of the paper. all authors read and approved the final manuscript. convenient online submission rigorous peer review immediate publication on acceptance open access: articles freely available online high visibility within the fi eld retaining the copyright to your article submit your next manuscript at springeropen.com key: cord- -af oq d authors: friedman, henry; ator, nancy; haigwood, nancy; newsome, william; allan, james s.; golos, thaddeus g.; kordower, jeff h.; shade, robert e.; goldberg, michael e.; bailey, matthew r.; bianchi, paul title: the critical role of nonhuman primates in medical research date: - - journal: pathog immun doi: . /pai.v i . sha: doc_id: cord_uid: af oq d the sponsors of this report endorse carefully regulated research with nonhuman primates. this research is essential to learning about the biology, treatment and prevention of diseases and conditions that cause human suffering. research with nonhuman primates (nhps) -monkeys for the most part -has led to critical health advances that have saved or improved millions of human lives. while nhps account for just one-half of one percent of animals in current medical research, it is no exaggeration to say they are essential to our ability to find cures for cancer, aids, alzheimer's, parkinson's, obesity/diabetes, and dozens of other diseases that cause human suffering and death. research with monkeys is critical to increasing our knowledge of how the human brain works and its role in cognitive, motor, and mental illnesses such as alzheimer's, parkinson's, and depression. this research is also fundamental to understanding how to prevent and treat emerging infectious diseases like zika and ebola. nhp research is uncovering critical information about the most common and costly metabolic disorder in the u.s. -type diabetes -as well as the obesity that leads to most cases. without nhp research, we lose our ability to learn better ways to prevent negative pregnancy outcomes, including miscarriage, stillbirth, and premature birth. this research is also helping scientists to uncover information that makes human organ transplants easier and more accessible, literally giving new life to those whose kidneys, hearts, and lungs are failing. news headlines tout medical breakthroughs. breakthrough sounds dramatic, and to someone hearing about how the virus that causes polio is being used to put an aggressive form of brain cancer into remission, it is indeed. but as the scientists involved in that cancer research-and research into every other area of medicine-will tell you, breakthroughs might be dramatic, but they are never sudden. a well thought-out and structured process is behind virtually every medical breakthrough and the discovery process probably took decades or more. every step in the process was essential to the next, from basic research to human clinical trials. monkeys are often involved at the later stage of the processwhat is called translational or applied research. here all of the knowledge accumulated earlier is applied to specific medical questions such as: will this vaccine protect a pregnant woman (and her baby) from zika infection? and is the vaccine likely to be safe? research scientists are like detectives solving mysteries. we want to know why. more importantly, we want to save lives. but monkeys also play a vital role in basic science research that can come decades earlier. basic nhp research in the s helped scientists understand the inner workings of the basal ganglia, the part of the brain that coordinates movement. those early findings led to the "breakthrough" years later in which deep brain stimulation is used to reduce involuntary movements of parkinson's disease. see more breakthroughs linked to nhp research in appendix a. regardless of where it occurs in the scientific discovery process, research with monkeys is highly regulated (see appendix b). scientists use monkeys only when no other research model can provide the required information. while rodents are used extensively and are extremely helpful in answering many basic research questions, their usefulness is limited by differences from primates in their lack of sophisticated brain structures, less developed immune systems and motor skills, and differences in how their metabolism functions, among other traits. to cite an example, rodent brains are very different from human brains. the rodent lacks the prefrontal cortex specialization that is found in monkeys and humans. this difference limits the applicability of rodent studies in relation to studies of injury in the human brain. nhps are also the main animals that allow quick response and research into emerging viruses, like zika. what scientists learn about zika itself, as well as what they learn about the best use of monkeys in zika studies, they will apply to studies of future emergent diseases. and with recent history as a guide (zika, ebola, middle east respiratory syndrome [mers], sars, pandemic flu, etc.), we should expect more infectious disease outbreaks in the near future. modified poliovirus is being tested as a way to help the body's immune system see and destroy glioblastomas, the deadliest type of brain cancer. glioblastomas can double in size every two weeks and can be deadly within months of diagnosis. the new treatment has led to complete remission in two glioblastoma patients and investigations are ongoing. this type of research, called immunotherapy, uses the body's natural defense -the immune system -to destroy cancer cells. the harmless form of poliovirus is injected into glioblastoma tumors where it attaches to the cancer cells. the immune system recognizes the poliovirus as a dangerous invader and attacks -killing it and the cancer along with it. current studies in monkeys are helping to find ways to help wounded soldiers and stroke victims regain their independence after losing limbs or the ability to control them. this is the most promising therapy i've seen in my career. it was years from the earliest research until the first study in humans. monkey research was essential in mapping out how to get the poliovirus through the brain and inside the cancerous tumors. the national institutes of health and the food and drug administration also mandated testing the treatment first in monkeys to be sure the modified poliovirus would be harmless to humans. doctors now are designing research to use this approach in treating many forms of cancer -including breast and prostate cancers -since the same receptor on the glioblastoma tumors that allows the poliovirus to attach itself also is found on virtually every cancerous tumor in humans. scientists are looking for vaccines that can prevent hiv infection and treatments leading to a cure. just years ago medical advances changed the disease from a death sentence into a chronic, manageable disease. drugs that keep the virus in check now give millions of hiv-infected people hope for a long and productive life. drug therapies effectively prevent hiv disease, but scientific advances in hiv are still needed. people with well-treated hiv still face more health problems than those without hiv [ , ] . they age faster, too. doctors estimate people with hiv are at least to years older than their chronological age [ , ] . monkeys are crucial to ongoing hiv research because of the combination of their unique biology among animals and their longevity, which is key in hiv studies that take from months to years to complete. their similar biology helps scientists understand hiv disease, infection routes, the potential for vaccine-induced protection, and even an hiv cure. an experiment reported in early looked at preventing mother-tochild hiv transmission [ ] . after being exposed to simian-human immunodeficiency virus (shiv), which is similar to hiv, infant monkeys with early stage infection were treated with human antibodies to block the infection. all of the monkeys in this experiment had no detectable virus in their blood or any of their tissues at the end of six months of observation. in another experiment reported this year, rhesus macaques infected with another hiv-like virus were treated with standard anti-hiv medications plus an experimental drug that stimulates the immune system [ ] . at the end of the study, days after both medications were stopped, two monkeys showed no detectable virus in their bloodstream. this immune system stimulator was tested earlier in nhps infected with chronic hepatitis b, leading to current research in humans with this potentially deadly infection. [ ] the value of studying monkeys is scientifically proven. hiv is no longer an impending death sentence. several years ago, this young woman was diagnosed with a deadly brain tumor the size of a tennis ball. when conventional treatment didn't work her doctors suggested a new treatment developed with primate research. she agreed. today, she's cancer free. these studies hold promise for protecting babies from hiv infection and for finding a cure for those already infected, but much more research with monkeys will be needed to get there. in human clinical studies, a fundamental question is, "do the potential benefits of this treatment outweigh the potential risks?" this question takes on added meaning when the study is in pregnant women. researchers must not only consider the risks and benefits to the pregnant woman, but also to her developing fetus and ultimately to the child [ ] . but how do researchers even begin to define these risks and benefits before human clinical trials? the answer is research with monkeys, since their fetal and placental development is uniquely similar to humans. researchers are working with macaque monkeys to understand the impact of zika, the latest virus to emerge as a global threat. zika infection in pregnant women can cause microcephaly, a condition where the child is born with a small head due to abnormal brain development. it also appears to cause stillbirth, miscarriages, and fetal growth restriction. these problems all appear to be rooted in how the zika virus affects the developing fetus and the placenta, which nourishes the baby in its mother's womb. the zika virus infects monkeys just as it does humans, and both experience the disease in the same way. researchers can study pregnant monkeys much as an obstetrician follows a woman's pregnancy -they can take blood, monitor fetal development through ultrasounds, and collect amniotic fluid. they can then test vaccines and drugs with the hope of protecting the fetus. no other animal model allows for this entire spectrum of study and application of the findings to pregnant women. more than , people in the u.s. are waiting for organ transplants and of them die every day [ ] . it is all the more tragic, then, when an organ transplant fails. this failure, or rejection, is caused when the recipient's immune system sees the new organ as "foreign" and attacks it. to reduce the chance of organ rejection, transplant patients receive drugs to suppress their immune system. but the drugs come with a risk of toxicity and increase the risk of other problems, including development of cancers and infections resulting from a weakened immune system. research with monkeys is focused on achieving transplant tolerancewhere the body's immune system does not see the new organ as foreign, thus eliminating the need for immunosuppressive drugs. while scientists have already made great strides in kidney transplant tolerance, they understand that tolerance is organ specific, so knowledge about the kidney primate research will help us learn new ways to prevent miscarriage, stillbirth, and premature babies. understanding all brain diseases relies on us understanding how the healthy brain works during normal functioning. may not transfer to the heart, lungs, liver, pancreas/pancreatic islets, or other types of transplants. transplant tolerance also differs by species. in other words, what works in a mouse may not work in a pig, and what works in a pig may not work in a monkey. scientists learned about kidney transplant tolerance by starting with mice and then working up through swine and eventually into monkeys and humans. the same process is underway now for many other types of transplants. how does the brain work? no question could be more important for understanding human behavior and mental health, and for acquiring new information about the triggers in the brain that cause psychiatric, movement and other neurological diseases. the u.s. national institutes of health-supported brain initiative has developed a plan for improving our knowledge in these areas and research with monkeys and other species is critical to its success [ ] . scientists are mapping the activity of the billions of neurons deep inside the brain -the special cells that transmit the signals that drive thinking, mood, movement, and much more. by tracking neuron activity in monkeys while they are performing new tasks, scientists can actually see what parts of the brain are involved in sending the signals that take in, process, and store the newly acquired information. what is unique to -or at least greatly enhanced by -the use of monkeys in this research is the range of cognitive behaviors that can be studied, the amount and precision of the data that can be collected, and the relevance of that data to human behavior and mental activity. seeing what is happening in a healthy monkey brain helps scientists understand what has gone wrong when a human brain is no longer working as it should. this type of research has relevance to parkinson's disease and other movement disorders, all forms of dementia, including alzheimer's, and behavioral and psychiatric problems from alcoholism and attention-deficit disorder to bipolar disorder and autism. you see someone walking haltingly, dragging one leg behind him, or sitting with one arm draped listlessly on a table and immediately know he has had a stroke. scientists learned long ago that it's not the muscles that are at fault; it's the nerve impulses inside the brain that have been affected. alzheimer's and other dementias cost the u.s. $ billion each year [ ] . monkeys have certain traits and characteristics that make them essential and irreplaceable in medical research. they're the "bridge to the clinic." a combination of scientific breakthroughs in neuroscience, computer processing and robotics has led to development of "brain-machine interfaces" -devices that allow humans to interact with their environment with prosthetic arms when they have lost the use of their own. brain-machine interfaces translate signals in the brain into directions to move prosthetic arms. this area of research shows enormous promise for humans who are paralyzed, such as injured veterans, or those with brain damage and paralysis due to stroke. as nhps and humans have similarly developed brains and movements, experiments in monkeys have been vital to moving this field forward both conceptually and technically. nhps are essential to vaccine research. among research animals, they alone can reproduce the entire biological process of the infections being studied. they allow researchers to monitor for information that is vital in understanding human infectious diseases -such as how a virus or bacterium reproduces inside the body, what symptoms it causes, and how the body's immune system responds to attack the invader. among the viruses currently in vaccine research trials is respiratory syncytial virus, or rsv -the most common cause of lower respiratory tract infections in u.s. infants and small children [ ] . there is no treatment for rsv, [ ] which hospitalizes nearly , u.s. children under age every year and sends . million more to the doctor [ ] . vaccine research with monkeys is evaluating the safety of potential rsv vaccines in infants. other viruses under study include ebola and marburg, which can cause extreme bleeding that leads to death; the mosquito-borne dengue and zika viruses, capable of causing massive epidemics; and mers plus the dangerous h and h bird flu strains, all of which have very high death rates. lowering blood pressure is vitally important to individuals and our society. high blood pressure is a major factor in heart disease -the number one killer in the u.s. and the world [ ] . and it's not just heart disease; high blood pressure leads to stroke, kidney damage, memory problems, and many other illnesses [ ] . decades ago, researchers made a breakthrough discovery that longterm blood pressure regulation is nearly identical in humans, baboons, brain-machine interfaces can help paralyzed veterans interact with their environment. the top benefits of our human/monkey research partnerships? safety. efficacy. and greater predictability. scientists recently discovered that baboons share another unique trait with humans -a characteristic in their red blood cells that can lead to salt-sensitivity and an inherited form of hypertension that is particularly difficult to treat. current research is looking for new targets to control this type of high blood pressure. research with monkeys provides another key benefit -lifespan. high blood pressure becomes more common as we age and researchers are able to work with older baboons to gain essential information about the mechanisms driving this age-based increase -vital to the health of our aging population. type diabetes develops in monkeys just as it does in humans, even following the same age patterns, that is to say, more disease as we get older (one-fourth of u.s. seniors have diabetes) [ ] . nhps with diabetes even develop the same complications that are common in humans: eye disease, kidney disease, nerve damage and pain, and blood vessel disease, among others [ ] . nhps and humans have very similar systems that regulate blood sugar. for example, the structure and function of the group of cells in the monkey pancreas (called islets) that produce insulin are very similar to human islets. the islets in mice, rats, pigs, and other animals share some similarities with humans, but there are important differences, making monkeys a critical model for developing treatment and prevention methods, and for testing new therapies for people with diabetes. type diabetes and the u.s. obesity epidemic are linked -obesity is a contributing factor to the condition. more than a third of u.s. adults are obese and another third are overweight [ ] . as with diabetes research, monkeys provide a critically important study model for human obesity. monkeys that are fed a diet similar to the typical american diet respond like humans, gaining weight and later progressing to type diabetes. researchers are examining the role of gastrointestinal proteins called glucagon-like peptides in the development of obesity in bonnet macaques. bonnet macaques are unique among nhps because they have a strong genetic predisposition to obesity. this research is looking for obesity treatments that will be as effective as invasive bariatric surgery, but with far less risk. in conclusion, because nhps are the most readily available models with the greatest psychological and genetic similarities to humans, they play an indispensable role in the process of medical research and development. nonhuman primates are the ideal model for testing new therapies for people with diabetes, including the artificial pancreas, drugs and devices. all of our ability to address diseases in applied research comes from efforts in basic science research. william newsome, phd, neurobiologist studying how • components of blood and plasma discovered. • ability to diagnose and treat typhoid fever. • modern anesthesia. • mumps virus discovered. • treatment of rheumatoid arthritis. • discovery of the rh factor, blood-typing knowledge critical for safe blood transfusions. • development of polio vaccine. • development of antipsychotic medication chlorpromazine and its tranquilizing derivatives. • cancer chemotherapy. • development of yellow fever vaccine. • mapping of the heart's connections to arteries. • development of german measles vaccine. • therapeutic use of cortisone for reducing inflammation and allergy symptoms. • corneal transplants. • development of treatment and prevention of radiation sickness. • development of measles, mumps, and rubella (mmr) vaccine. • discovery of the biochemical cause of depression. • transmissibility of human prion diseases, such as creutzfeldt-jacob disease, discovered. • treatment of leprosy. • procedures to restore blood supply in the brain. • interaction between tumor viruses and genetic material. • understanding of slow viruses, which linger in the nervous system. • understanding of the inner workings of the basal ganglia, the part of the brain that coordinates movement. • discovery of mechanisms of opiate withdrawal and the anti-withdrawal effects of clonidine. • development of cyclosporine and other anti-rejection drugs helpful for organ transplants. • processing of visual information by the brain. • identification of physiological and psychological co-factors in depression, anxiety and phobias. • treatment of malnutrition caused by food aversion following chemotherapy. • treatment of congenital cataracts and "lazy eye" in children. • first animal model for research on parkinson's disease, enabling doctors to more accurately research human parkinson's disease. • heart and lung transplant to treat cardiopulmonary hypertension. • first hepatitis b vaccine. • development of rhesus monkey model for hiv/aids. • addition of taurine to infant formulas. taurine is necessary for normal eye development. • first treatment of naturally diabetic nhps with a hormone-like insulin stimulus that is now in wide use both for diabetes and obesity treatment (glp- agonist). without continued nonhuman primate research, many important translational discoveries in the field of transplantation will never happen. these preclinical investigations are critical to the many patients who are currently waiting for transplantation and the many others who are facing the possibility of organ rejection after transplantation. • estrogen discovered to control an enzyme key to making serotonin, the brain chemical that regulates mood. represents first step to providing effective medications for depression at the end of the menstrual cycle, and postpartum and postmenopausal depression. • demonstration of the effectiveness of early administrationof azt to prevent or treat hiv infection. thanks to this, hiv-infected mothers can give birth to hiv-free babies. • demonstration in monkeys of the high efficacy of the hiv drug tenofovir to prevent or treat infection. • lead toxicity studies help u.s. fight childhood lead exposure. • ongoing development of a one-dose transplant drug to prevent organ rejection. • first controlled study to reveal that even moderate levels of alcohol are dangerous in pregnancy. • breakthroughs in understanding the mechanisms of puberty and disorders of puberty. • primate embryonic stem cells studied extensively for the first time, advancing efforts to better understand reproduction and genetic disorders. • control of intimal hyperplasia, a complication of coronary bypass surgery. • parent to child lung transplants for cystic fibrosis. • nhps shown to naturally develop diabetes, which is the same disease as in humans, thus opening the path to research for new treatments. • naturally regenerative mechanism discovered in the mature nhp brain, spurring new research toward curing alzheimer's and other degenerative brain disorders. • development of anthrax vaccine. • development of life-saving medications for lupus. • gene that boosts dopamine production and strengthens brain cells used to successfully treat monkeys showing symptoms of parkinson's disease. • monkey model developed to study the effects of malaria in pregnant women and their offspring. • nhps are prime model for development of hiv treatments and potential vaccines. • insulin-treated diabetic patients live longer, fuller lives. • the most common and debilitating complications of diabetes can now be studied in nhps. • high blood pressure is treated to prevent heart attack, stroke, and kidney failure. • patients can receive hip replacements and are no longer reliant on wheelchairs. • people with degenerative eye diseases are able to see more clearly. • better medications improve lives of people with severe depression, bipolar disorder, and other psychiatric illnesses. • better pre-and postnatal care protects children. • earlier diagnoses and better treatments help those with polycystic ovary syndrome, endometriosis, and breast cancer. • improved treatments help more men survive prostate cancer. • secondhand smoke shown to affect prenatal, neonatal and child lung development, cognitive function and brain development. • exposure to wildfire smoke adversely affects development of the immune system. • better understanding of the effects of bpa, a chemical found in plastic, on prenatal development improves health of children and adults. for research funded by any public health service entity, such as the national institutes of health (nih) or the national science foundation, the nih: g establishes public health service policy on humane care and use of laboratory animals. [ ] g mandates use of the guide for the care and use of laboratory animals, which is issued by the national academy of science institute for laboratory animal research. this guide addresses day-to-day aspects of caring for laboratory animals. [ ] g mandates that every institution appoints an institutional animal care and use committee (see below). the usda's animal plant and health inspection service enforces the animal welfare act with unannounced compliance inspections of all regulated entities using animals in research, testing or teaching at least yearly [ ] . the association for the assessment and accreditation of laboratory animal care international is an independent non-government accrediting organization. while voluntary, this accreditation includes broader requirements than the regulations. this demonstrates research facilities want to go "above and beyond" in the care of animals [ ] . every institution involved in nonhuman primate research is required by the animal welfare act as well as public health service policy to appoint and empower an institutional animal care and use committee [ ] that reviews and approves the research. scientists must justify their use of primates and also explain why alternative forms of research (for example, studying cells or using computer simulations) are not able to achieve their scientific goals. they must also confirm that their research does not unnecessarily duplicate previous research [ ] . the number of nonhuman primates used in research is less than %. but its impact on human health is enormous. all others geriatric syndromes in older hiv-infected adults the end of aids: hiv infection as a chronic disease methylome-wide analysis of chronic hiv infection reveals five-year increase in biological age and epigenetic targeting of hla acceleration of age-associated methylation patterns in hiv- -infected adults early short-term treatment with neutralizing human monoclonal antibodies halts shiv infection in infant macaques gilead announces data from new preclinical study evaluating an investigational tlr agonist in siv-infected monkeys gs- , an oral agonist of toll-like receptor- , induces prolonged suppression of hepatitis b virus in chronically infected chimpanzees ethical issues related to the inclusion of pregnant women in clinical trials (i) american transplant association. facts and myths national institutes of health. what is the brain initiative? available from alzheimer's disease facts and figures respiratory syncytial virus infection (rsv) american lung association. diagnosing and treating rsv the burden of respiratory syncytial virus infection in young children heart disease and stroke statistics-at-a-glance high blood pressure (hypertension) statistics about diabetes the rhesus monkey (macaca mulatta) manifests all features of human type diabetes national institute of diabetes and digestive and kidney diseases public health service policy on humane care and use of laboratory animals. revised committee for the update of the guide for the care and use of laboratory animals; institute for laboratory animal research. guide for the care and use of laboratory animals animal welfare act inspections. at .usa.gov/ xcaq m american association for the accreditation of laboratory animal care. the aaalac international accreditation program office of research integrity. how to work with your institutional animal care and use committee (iacuc) good laboratory practice for nonclinical laboratory studies the authors wish to acknowledge the following experts who participated in conference calls that served as the basis for the paper's content and/or who provided comments during drafting: key: cord- -upzxscux authors: adeoti, adekunle olatayo; marbus, sierk title: the european respiratory society course on acute respiratory pandemics: how to plan for and manage them date: - - journal: erj open res doi: . / . - sha: doc_id: cord_uid: upzxscux learn about the @erstalk course on acute respiratory pandemics http://ow.ly/xge i acute respiratory infectious diseases are more likely to cause future pandemics, as droplet generation and potential transmission of infectious agents could constitute a major public health threat [ ] . the exposure and associated risk of infections are not limited to the individuals' immediate environment, as some cases could cross boundaries and constitute global health challenges of international concern [ ] . a gap in emergency preparedness in the event of an outbreak was apparent during pandemics like those of severe acute respiratory syndrome coronavirus (cov), influenza virus a (h n ), ebola virus and middle east respiratory syndrome cov [ , [ ] [ ] [ ] . it is apparent that conducting clinical research in response to swiftly emerging disease outbreaks could be challenging and is often delayed [ ] . moreover, transmission of such infections during pandemics may be widespread in healthcare settings, especially with the increased demand on medical personnel and associated morbidity, resulting in a reduction in the available workforce. hence, it is imperative to create awareness among medical personnel, and train them on how to prepare for pandemics and conduct research in order to reduce mortality and morbidity during epidemics. the first european respiratory society (ers) course on acute respiratory pandemics was organised to train and improve participants' knowledge on how to plan for and manage pandemics [ ] . course members shared an interest in infection prevention and control but had different backgrounds, ranging from medical specialists to general practitioners, epidemiologists and researchers. participants from three continents, namely africa, europe and asia, were involved in the -day training programme held in amsterdam, the netherlands, from november to , (figure ). seasoned faculty members were carefully selected based on their research work, skills and experience in the area of outbreak investigation, communications, planning and management of pandemics. emphasis was laid on the practical workshops, which simulated pandemic scenarios with the aim of stimulating course participants to respond appropriately in cases of a pandemic. the aim of this course report is to give a short overview of some of the key lectures, highlights and take-home messages. • creating awareness, training medical personnel and planning an appropriate response at all healthcare levels is essential for pandemic preparedness. • integrating research into pandemic preparedness plans is often forgotten but is crucial for timely clinical research during and after pandemics. • during a pandemic, communication and collaboration between all stakeholders should be optimised for effective outbreak control. on the first day of the course, an overview of the european union seventh framework pandemic preparedness project prepare was given. prepare is a european research framework designed to harmonise large-scale clinical research studies on infectious diseases in order to provide real-time evidence for clinical management and timely healthcare interventions during a pandemic (www.prepare-europe.eu). the three outbreak research modes identified in the prepare programme are preparation, mobilisation and response, thereby differentiating the levels of research preparedness in case of an outbreak. the one health approach, which is a multidisciplinary collaborative approach to solve global and health challenges for humans, animals and the environment was also advocated by p. penttinen of the european centre for disease prevention and control (ecdc) to enable effective control of outbreaks [ ] . a public health preventive stance and outbreak investigation measures were stressed by v. prikazsky from ecdc during an interactive lecture in which he focused on the need for proper epidemiology in the investigation and sampling in case of an outbreak as well as factors responsible for vaccine failure. the key contributors on the second day stressed the importance of adequate communication during epidemics. crucial aspects of communication during an epidemic include finding out who your audience is, getting to know their social and cultural background, and delivering understandable, tailored and specific messages. taking the audience's comments into account, acknowledging risks and modifying your next messages are important for effective communication during an epidemic. the world health organization (who) risk communication course is worth taking as an e-module on the who website to improve communications skills in this setting [ ] . in another lecture, the need to escalate medical provision within the hospital and the value of a checklist for pandemic preparedness was emphasised [ ] . this checklist will help hospitals set up regulations and protocols on essential issues such as communication, surge capacity, logistics, case management, surveillance and scale-up of laboratory services. during outbreaks of severe acute respiratory infections, intensive care units (icus) are often fully occupied; l. derde gave an overview on the icu perspective on conducting research and management in critical cases during a pandemic. special attention was paid to children during pandemics by p. fraaji, as they may be worst hit due to their relatively low immune response to infectious agents. furthermore, j. buchanan stated the vital role played by an effective primary healthcare facility in the event of an outbreak. she also stressed the need for training and retraining of healthcare personnel in the use of personal protective equipment as basic as gloves and facemasks. on the final day of the course, ethical aspects of pandemics, in general and in research, were covered. these dealt with the ethical challenges, especially as regards to balancing social and individual benefits from research in an emergency situation against the potential harms and risks to individuals and the community at large (www.prepare-europe.eu). in addition, study participants may be more vulnerable because of public panic or fear; thus, the principles of autonomy, justice and beneficence may be subordinated to that of non-maleficence. a fast-track review by an ethical committee, which should include local representatives, is necessary to conduct timely research during such periods. the high point of this course was the workshop simulating a pandemic scenario due to a new cov strain. during this workshop lead by g. carson, the knowledge acquired on the ers course was put into practice. the participants were divided into six groups representing stakeholders from every healthcare level: clinicians, laboratory healthcare workers, researchers, government, and local and national public health personnel. each stakeholder had to adapt their strategy and management plan depending on new information given while the scenario evolved. there were live active inserts by skype from who, public health england and the us centers for disease control and prevention as the scenario escalated, to add realism. at the end of the workshop, situational reports were given by each stakeholder, underscoring the need for integrated care, effective communication skills, early research and information management in the event of an outbreak. important take-home messages were the need to improve communication between stakeholders and establish effective collaboration in outbreak control, as well as early efforts to integrate research activities in the event of a pandemic. factors associated with death or hospitalization due to pandemic influenza a(h n ) infection in california infection prevention and control of epidemic-and pandemic-prone acute respiratory infections in health care. geneva, world health organization emerging and reemerging diseases in the world health organization (who) eastern mediterranean region -progress, challenges, and who initiatives clinical review: sars -lessons in disaster management diagnostic preparedness for infectious disease outbreaks talking to the people that really matter about their participation in pandemic clinical research: a qualitative study in four european countries ers training course: acute respiratory pandemics: how to plan and manage mers-coronavirus: from discovery to intervention who checklist for influenza pandemic preparedness planning acknowledgements: we thank the organising committee, a. simonds, p. penttinen, l. sigfrid and g. carson. the course was funded by european union seventh framework research grant prepare (workpackage create). key: cord- - pjdutgg authors: awaisu, ahmed; mukhalalati, banan; mohamed ibrahim, mohamed izham title: research designs and methodologies related to pharmacy practice date: - - journal: encyclopedia of pharmacy practice and clinical pharmacy doi: . /b - - - - . - sha: doc_id: cord_uid: pjdutgg abstract the need for evidence to inform policy and practice in pharmacy is becoming increasingly important. in parallel, clinical pharmacy and practice research is evolving. research evidence should be used to identify new areas for improved health service delivery and rigorously evaluate new services in pharmacy. the generation of such evidence through practice-based research should be predicated on appropriate use of robust and rigorous methodologies. in addition to the quantitative and qualitative approaches used in pharmacy practice research, mixed methods and other novel approaches are increasingly being applied in pharmacy practice research. approaches such as discrete choice experiments, delphi techniques, and simulated client technique are now commonly used in pharmacy practice research. therefore, pharmacy practice researchers need to be competent in the selection, application, and interpretation of these methodological and analytical approaches. this chapter focuses on introducing traditional and novel study designs and methodologies that are particularly pertinent to contemporary clinical pharmacy and practice research. this chapter will introduce the fundamentals and structures of these methodologies, but more details regarding the different approaches may be found within the encyclopedia. the mission of pharmacy profession and the role of pharmacists in healthcare have evolved toward patient-centered care in the last few decades. pharmacists with their expertise in drug therapy and accessibility to the public have unprecedented opportunities to assume increasing responsibility for direct patient care (bond, ) . new cognitive pharmaceutical services and new roles for pharmacists continue to emerge. in the era of evidence-based practice and health services, it is not just adequate to propose those new pharmacy services or new roles without evidence of their benefit (awaisu and alsalimy, ; bond, ) . new pharmacy services and new roles must be proven to be feasible, acceptable, cost-effective, and increase health outcomes. pharmacy practice research provides such evidence and can confirm the value of a new service, inform policy, and result in practice changes (bond, ; chen and hughes, ) . research evidence should be used to identify new areas for improved health service delivery and rigorously evaluate new services. the research used to generate such evidence should be grounded in robust and rigorous methodologies (chen and hughes, ) . traditionally, common quantitative and qualitative methods such as randomized controlled trials, cohort study, case control study, questionnaire-based surveys, and phenomenology using qualitative interviews have been used in pharmacy. however, in recent years, novel and more complex methods are being developed and utilized. pharmacy practice researchers need to know how these old and new methodological approaches should be selected, applied, and interpreted in addressing research problems. various study designs, including, but not limited to experimental, quasi-experimental, observational, qualitative, and mixed method designs, have been used in pharmacy practice research. furthermore, different classification systems (e.g., quantitative vs. qualitative, experimental vs. observational, descriptive vs. analytical study designs) have been used in the literature. the choice of a study design to answer a research question in pharmacy practice research is driven by several factors, including the type of the research question or the research hypothesis, expertise of the investigator, availability of data, and funding opportunities. pharmacy practice researchers need to be competent in the selection, design, application, and interpretation of these methodological and analytical approaches. today, many of the research methods used in pharmacy practice research have been adapted from fields such as sociology, anthropology, psychology, economics, and other disciplines. this paradigm shift has led to a greater emphasis on the appropriate choice of a specific research design or method to answer a specific research question (chen and hughes, ) . consequently, pharmacy practice researchers should place an emphasis on the reliability of the methods selected, the correct interpretation of their findings, the testing of a specific hypothesis, and the internal validity of their data, among other considerations. novice and early career researchers should be familiar and have sound foundation in a variety of methods applied in pharmacy practice research, which will be covered in this chapter and other chapters in this encyclopedia. we do believe that more experienced researchers should focus on certain methods in order to advance research in our discipline. traditionally, core quantitative approaches used in pharmacy practice research include nonexperiments, quasi-experimental designs, and true experimental designs such as prospective randomized controlled intervention trials. nonexperiments also include observational study designs that are often described as pharmacoepidemiologic study designs such as case-control study, cohort study, nested case-control study, and cross-sectional study (etminan, ; etminan and samii, ) . in recent years, conventional qualitative approaches and their philosophical paradigms are increasingly been used in pharmacy. these include the five qualitative approaches to inquiry: narrative research, phenomenology, grounded theory, ethnography, and case study. these qualitative methods are often difficult for pharmacy practice researchers to comprehend, and researchers tend to describe the methods of data collection such as individual interviews and focus group discussions as qualitative methods of inquiry. these data collection methods are briefly described later in this chapter, among others. furthermore, there is an increasing importance on the appropriate selection and use of mixed method approach (hadi et al., ; closs, a, b) , which are often designed and applied wrongly. finally, it is worthwhile to be familiar with novel research methodologies such as discrete choice experiments, delphi techniques, simulated client technique, and nominal group techniques, which fall between quantitative and qualitative approaches, often with no clear differentiation on where they belong. although called "novel" in the context of this chapter, these methods are not new in other relevant disciplines, but new and not commonly used in pharmacy practice research. pharmacy practice researchers begin by conception of a research idea or identifying a research question and defining a hypothesis based on the question. the researcher then selects a study design that will be suitable to answer the research question. the study design should be appropriately selected prior to initiation of any research investigation. selecting an inappropriate study design may potentially undermine the validity of a study in its entirety. investigators are encouraged to critically think about the possible study designs to ensure that the research question is adequately addressed and should be able to adequately justify their choice. these study designs have been variously classified and one common classification system is quantitative vs. qualitative study designs. study designs play a major role in determining the scientific value of research studies. inappropriate choice of a study design is impossible to correct after completion of the study. therefore, thorough planning is required to avoid unconvincing results and invalid conclusions. good understanding of basic study design concepts will aid researchers in conducting robust and rigorous practice-based research. this chapter introduces the structure and the fundamentals of common study designs used in pharmacy practice research and discusses the important considerations for conducting pharmacy practice research in terms of study design, data collection, data analyses, and ethical considerations. various classifications for research designs and methods used in pharmacy practice have been used in the literature. the following are some of the approaches for the classification of research designs: . classification based on time orientation: retrospective vs. prospective designs a. retrospective design-a retrospective study design observes what has happened in the past. it begins and ends in the present. this design involves a major limitation as it looks to collect information about events that occurred in the past. an example of this design is retrospective case-control study. case example: investigators were looking for the association between acute myocardial infarction and smoking status, type of tobacco, amount of smoke, etc. (teo et al., ) . another example of a case-control study from published literature is the study investigating the association between the use of phenylpropanolamine and the risk of hemorrhagic stroke (kernan et al., ) . b. prospective design-a prospective study design begins in the present and progresses forward, collecting data from subjects whose outcomes lie in the future. an example of this design is prospective cohort study. case example: investigators were interested to determine the long-term effectiveness of influenza vaccines in elderly people; they recruited cohorts of vaccinated and unvaccinated community-dwelling elderly (nichol et al., ) . . classification based on study purpose: descriptive vs. analytical designs a. descriptive design-a descriptive study describes a population/sample in terms of distribution of the variables, and frequency of outcomes of interest. unlike analytical studies that include control (comparison) group, descriptive studies do not include a comparison group. descriptive studies include case reports, case series reports, cross-sectional studies, surveillance studies, and ecological studies. case example: a case report was written by a physician who contracted severe acute respiratory syndrome (sars) during an outbreak in hong kong (wu and sung, ) . another example is an ecological study examining diet and sunlight as risks for prostate cancer mortality (colli and colli, case example: a group of investigators carried out a study to establish an association between the use of traditional eye medicines (tem) and corneal ulcers. in this case, both case-control and cohort study designs are applicable. in an example of a case control study, archibugi et al. aimed to investigate the association between aspirin and statin exclusive and combined and pancreatic ductal adenocarcinoma occurrence (archibugi et al., ) . another example of a cohort study is a study carried out by wei et al. in which they investigated whether or not acid-suppression medicines increased the risk of bacterial gastroenteritis (wei et al., ) . . classification based on investigator orientation: experimental vs. quasi-experimental vs. observational designs a. experimental design-in experimental design (also known as interventional design), the investigator performs an intervention and evaluates cause and effect relationships. case examples: investigators conducted a study about the newer versus older antihypertensive agents in african hypertensive patients (noaah) trial (nct ) to compare the efficacy of single-pill combinations of newer versus older antihypertensive agents (i.e., a single-pill combination of newer drugs, not involving a diuretic, with a combination of older drugs including a diuretic) (odili et al., ) . in a crossover design, a group of investigators evaluated the effect of spironolactone on nonresolving central serous chorioretinopathy (bousquet et al., ) . b. quasi-experimental design-the quasi-experimental design is very similar to the true experimental design described above and it involves an intervention. the design has been employed when randomization is inappropriate or impossible, especially when implementing complex interventions. case examples: prashanth et al. aimed to understand if (and how) a package of interventions targeting primary health centers and community participation platforms affect utilization and access to generic medicines for people with noncommunicable diseases using quasi-experimental design approach (prashanth et al., (murphy et al., ) . c. mixed method designs-mixed method design brings together qualitative and quantitative methodologies within a single study to answer or understand a research problem (hadi et al., ) . case examples: shiyanbola et al. combined focus group discussion with a survey tool to investigate patients' perceived value and use of quality measures in evaluating and choosing community pharmacies (shiyanbola and mort, ) . below is a brief description of traditional and novel pharmacoepidemiologic study designs. several examples of pharmacoepidemiologic study designs are provided above. some descriptive studies including case reports, case series, and ecological studies will not be described in this chapter. a. case-control studies-in this design, patients (those who develop the disease or outcome of interest) are identified and control patients (those who do not develop the disease or outcome of interest) are sampled at random from the original cohort that gives rise to the cases (etminan and samii, ; newman et al., ) . the distribution of exposure to certain risk factors between the cases and the controls is then explored, and an odds ratio (or) is calculated. b. cohort studies-this can be described as a study in which a group of exposed subjects and a group of unexposed subjects are followed over time and the incidence of the disease or outcome of interest in the exposed group is compared with that in the unexposed group (etminan and samii, ; hulley et al., ) . c. case-crossover studies-the case-crossover may be considered comparable to a crossover randomized controlled trial in which the patients act as their own control (etminan and samii, ) . pattern of exposure among the cases is compared between event time and control time. the between-patient confounding that occurs in a classic case-control study is circumvented in this design. tubiana et al. evaluated the role of antibiotic prophylaxis and assessed the relation between invasive dental procedures and oral streptococcal infective endocarditis, using a nationwide population-based cohort and a case-crossover study design (tubiana et al., ) . d. case-time control studies-this design is an extension of the case-crossover design, but includes a control group (etminan and samii, ) . a group of researchers assessed medication-related hospitalization. they used the case-time control study design to investigate the associations between high risk medication categories (e.g., antidiabetic agents, diuretics, benzodiazepine hypnotics) and unplanned hospitalizations (lin et al., ) . e. nested case-control studies-in this design, a cohort of individuals is followed during certain time periods until a certain outcome is reached and the analysis is conducted as a case-control study in which cases are matched to only a sample of control subjects (etminan, ) . de jong et al. examined the association between interferon-β (ifn-β) and potential adverse events using population-based health administrative data in canada (de jong et al., ) . f. cross-sectional studies-in this type of study, the investigator measures the outcome of interest and the exposures among the study participants at the same time setia, b) . it provides a snapshot of a situation for a particular period. a wide range of quantitative methods are commonly applied in pharmacy practice research. these methods are widely used in published pharmacy practice literature to explore appropriateness of medicines use, appropriateness and quality of prescribing, and medication safety, through analyzing existing datasets, direct observation, or self-report (green and norris, ) . pharmacy practice research questions also seek to determine the knowledge, behaviors, attitudes, and practices of pharmacists, other healthcare providers, patients, policy-makers, regulators, and the general public. quantitative methods are also used in evaluating the effect of new pharmacy services and interventions to improve medicines use. these practice research projects provide valuable insights about how medicines are used, and how to maximize their benefits and minimize their harmful effects. in the context of this chapter, quantitative study designs will be broadly classified into three: ( ) observational, ( ) experimental and quasi experimental, and ( ) other designs. pharmacoepidemiology is a "relatively new science that explores drug efficacy or toxicity using large observational study designs" (etminan, ; etminan and samii, ) . these study designs explore drug use studies that usually cannot be answered using randomized controlled trials or other experimental designs. in several instances, experimental study designs may not be suitable or feasible; in such circumstances, observational study designs are applied . as the name implies, observational studies involve merely observing the subjects in a noncontrolled setting, without investigator intervention or manipulating other aspects of the study. therefore, observational studies are nonexperimental. the observation of the variables of interest can be prospective, retrospective, or current depending on the type of the observational study. in pharmacoepidemiology and other areas of pharmacy practice, researchers are often interested in measuring the relationships between exposure to a drug and its efficacy, toxicity, or other outcomes of interest using observational study designs. it is worthwhile to note that observational study designs investigate association, but, in most cases, not causation. here, we provide descriptions of some commonly used study designs in pharmacoepidemiology and pharmacy practice research in general. case-control study design is used to determine association between risk factors or exposures and outcomes. it is a useful design to study exposures in rare diseases or diseases that take long time to develop . it investigates exposures in individuals with and those without the outcome of interest. nevertheless, case-control studies can help to identify harmful or beneficial exposures. furthermore, the outcome of interest can be undesirable (e.g., mortality) or desirable (e.g., microbiological cure). as the name suggests, in a case-control study design, there are two groups of subjects: ( ) cases (individuals with the outcome of interest) and ( ) controls (individuals without the outcome of interest) . cases are randomly selected based on prespecified eligibility criteria from a population of interest. appropriate representative controls for the cases selected are then identified. the researchers then retrospectively investigate possible exposures to the risk factor. fig. represents a schematic diagram of a case-control study. case-control studies are relatively inexpensive, less time-consuming to conduct, allow investigation of several possible exposures or associations, and are suitable for rare diseases. selection of the control group is a critical component of case-control studies. case-control studies have several drawbacks: confounding must be controlled, subject to recall, observation, and selection biases. or is the measure of association used for the analysis of case-control studies. this is defined as the odds of exposure to a factor in those with a condition or disease compared with those who do not have the condition or disease. similar to case-control studies, cohort studies determine an association between exposures/factors and development of an outcome of interest. as previously described, a cohort study is a study in which a group of exposed subjects and a group of unexposed subjects are followed over time to measure and compare the rate of a disease or an outcome of interest in both groups (etminan and samii, ; hulley et al., ) . a cohort study can be prospective (most common) or retrospective. while a case-control study begins with patients with and those without the outcome of interest (e.g., diseased and nondiseased patients), a cohort study begins with exposed and unexposed patients (e.g., patients with and those without certain risk factor) setia, a) . in a cohort study, both the exposed and the unexposed subjects are members of a larger cohort in which subjects may enter and exit the cohort at different periods in time (etminan and samii, ; hulley et al., ) . typically, a cohort study should have a defined time zero, which is defined as the time of entry into the cohort (etminan and samii, ) . the cohort (a group of exposed and unexposed subjects, who are free of the outcome at time zero) is followed for a certain period until the outcome of interest occurs. in addition, information or data related to all potential confounders or covariates should also be collected as failure to account for these can bias the results and over-or underestimates the risk estimate. there are two types of cohort studies: retrospective cohort and prospective cohort studies. retrospective cohort study, also known as historical cohort study, begins and ends in the present, while looking backward to collect information about exposure that occurred in the past (fig. ) . historical cohort studies are relatively less time-consuming and less expensive than prospective cohort studies (etminan and samii, ; hulley et al., ; setia, a) . in addition, there is no loss to follow-up and researchers can investigate issues not amenable to intervention study designs. however, these studies are only as good as the data available, the investigator has limited control of confounding variables, and it is prone to recall bias. on the other hand, prospective cohort study, also known as longitudinal cohort study, begins in the present and progresses forward, collecting data from enrolled subjects whose outcomes fall in the future (etminan and samii, ; hulley et al., ; [ ( f i g u r e _ ) t d $ f i g ] setia, a) (fig. ) . prospective cohort studies are easier to plan for data collection, have low recall bias, and the researcher has a better control of confounding factors. on the other hand, it is difficult to study rare conditions; they are more prone to selection bias, more time-consuming, expensive, and loss of subjects to follow-up is common. relative risk (rr) is the measure of association used for the analysis of a cohort study. this is defined as the risk of an event or development of an event relative to exposure (i.e., the risk of subjects developing a condition when exposed to a risk factor compared with subjects who have not been exposed to the risk factor). this is a relatively new design in the field of epidemiology in which the patients act as their own controls (maclure, ) . in this design, there is a case and a control element both of which come from the same subject. in other words, each case serves as its own control. it can be considered equivalent to a crossover rct with a washout period (etminan and samii, ) . pattern of exposure to the risk factor is compared between the event time and the control time (etminan and samii, ) . case-crossover study design is useful to investigate triggers within an individual. for instance, it is applicable when studying a transient exposure or risk factor. [ ( f i g u r e _ ) t d $ f i g ] however, determination of the period of the control and case components is a crucial and challenging aspect of a case-crossover study design. since the patients serve as their own controls, the interindividual variability that is inherent in classic case-control studies is eliminated. this is important in studies involving progressive disease states in which disease severity may differ between patients such as multiple sclerosis. or is estimated using techniques such as mantel-haenszel statistics and logistic regression. cross-sectional studies also known as prevalence studies identify the prevalence or characteristics of a condition in a group of individuals. this design provides a snapshot of the prevalence or the characteristics of the study subjects in a single time point. the study investigator measures the outcomes and the exposures in the study subjects simultaneously (etminan and samii, ; hulley et al., ; setia, b) . hence, cross-sectional studies do not follow up patients to observe outcomes or exposures of interest. data are often collected through surveys. cross-sectional design cannot provide cause and effect relationships between certain exposures and outcomes of interest. in a typical experimental study design, the investigator assigns subjects to the intervention and control/comparison groups in an effort to determine the effects of the intervention . since the investigator has the opportunity to control various aspects of the experiment, this allows the researcher to determine the causal link between exposure to the intervention and outcome of interest. the researcher either randomly or conveniently assigns the subjects to an experimental group and a control group. when the investigator performs randomization, the study is considered a true experiment (see fig. ). on the other hand, if subjects are assigned into groups without randomization, the study is considered a quasi-experiment (refer to fig. ). as with experimental designs, quasi-experimental designs also attempt to demonstrate a causal link between the intervention and the outcome of interest. due to the challenges of conducting a true experimental design, the quasi-experimental study designs have been consistently used in pharmacist intervention research. rcts are considered the gold standard of experimental study designs in pharmacy practice and evidence-based research . the investigator randomly assigns a representative sample of the study population into an experimental group and a control group (fig. ) . randomization in rct is to minimize confounding and selection bias; it enables attainment of similar experimental and control groups, thereby isolating the effect of the intervention. the experimental group receives the treatment or intervention (e.g., a new drug or pharmaceutical care for treatment of a certain disease), while the control group receives a placebo treatment, no treatment, or usual care treatment depending on the objective of the study . these groups are then followed prospectively over time to observe the outcomes of interest that are hypothesized to be affected by the treatment or intervention. the result of the study is considered to have high internal validity if significant changes on the outcome variable occur in the experimental group, but not the control group. the investigator can infer that the treatment or intervention is the most probable cause of the changes observed in the intervention group. the unit of randomization in rcts is usually the patient, but can sometimes be clusters to circumvent the drawbacks of contamination. rcts are very challenging to undertake and pharmacy practice researchers should ensure design of robust experiments, while considering all essential elements and adhering to best practices. for instance, to determine the impact of a cognitive pharmaceutical service, the selection of a representative sample of the population is a prime consideration in an rct. moreover, rcts are expensive, labor-intensive, and highly prone to attrition bias or loss to follow-up. in pharmacy practice research, it is often difficult to comply with the stringent requirements of true experimental designs such as rcts, due to logistic reasons and/or ethical considerations krass, ) . whenever true experimental models are not feasible to be applied in pharmacy practice research, the researcher should endeavor to use a more robust quasi-experimental design. for instance, when randomization is not feasible, the researcher can choose from a range of quasi-experimental designs that are non-randomized and often noncontrolled krass, ) . quasi-experimental studies used in pharmacy literature may be classified into five major categories: ( ) quasi-experimental design without control groups (i.e., one group pre-posttest design); ( ) quasi-experimental design that use control groups with no pretest; ( ) quasi-experimental design that use control groups and pretests (i.e., nonequivalent control group design with dependent pretests and posttests) (see fig. ); ( ) interrupted time series and; ( ) stepped wedge designs (brown and lilford, ; grady et al., ; harris et al., ) . the one group pretest posttest design and the nonequivalent control group design (fig. ) are the most commonly applied quasiexperimental designs in practice-based research literature. these designs have been commonly used to evaluate the effect of pharmacist interventions in medications management in general and specific disease states management. the lack of randomization and/or the lack of control group is a major weakness and a threat to internal validity in quasi-experimental designs . the observed changes could be due to some effects other than the treatment. in addition to the common observational, experimental, and quasi-experimental designs described above, there are other designs that are used in pharmacy. these research methods include, but are not limited to, simulated client technique, discrete choice experiments, and delphi techniques. these methods, which are considered relatively new to pharmacy, are now commonly used in pharmacy practice research. in this chapter, we briefly describe these methods and their application in pharmacy. however, a more detailed description of their components and the nitty gritty of their application in pharmacy practice are available elsewhere within this textbook. the use of simulated client or simulated patient (mystery shopper) method to assess practices or behaviors in pharmacy practice has received much attention in recent times (watson et al., (watson et al., , . "a simulated patient is an individual who is trained to visit a pharmacy (or drug store) to enact a scenario that tests a specific behavior of the pharmacist or pharmacy staff" (watson et al., ) . a review by watson et al. demonstrated the versatility and applicability of this method to pharmacy practice research in both developing and developed countries (watson et al., ) . the investigators also identified some important characteristics that should be taken into consideration in designing studies that use this technique. this method can be used to assess wide range of cognitive pharmacy services including counseling and advice provision, treatment of minor ailments, provision of nonprescription medicines, and public health pharmacy, among other things. this method can be a robust and rigorous method of assessing pharmacy practice if used appropriately (watson et al., ; xu et al., ) . more recent developments have documented that the simulated patient methods have been used to provide formative feedback in addition to assessing practice behavior of pharmacists and their staff (xu et al., ) . in a case example, a group of investigators evaluated qatari pharmacists' prescribing, labeling, dispensing, and counseling practices in response to acute community-acquired gastroenteritis (ibrahim et al., ) . in another example, the investigators documented the state of insomnia management at community pharmacies in pakistan (hussain et al., ) . [ ( f i g u r e _ ) t d $ f i g ] figure quasi experimental study design. evidence in healthcare suggests that understanding consumers' preferences can help policy-makers to design services to match their views and preferences (ryan, ) . traditionally, studies to understand patients' and consumers' preferences for pharmaceutical services used opinion or satisfaction survey instruments. nevertheless, such satisfaction surveys lack the ability to identify the drivers of satisfaction or the relative importance of the different characteristics of the service (vass et al., ) . discrete choice experiments are a novel survey-based method in pharmacy that are predicated on economic theories that allow systematic quantification of preferences to help identify which attributes of a good or service consumers like, the relative value of each attribute, and the balance between the different attributes (naik panvelkar et al., ; ryan, ; vass et al., ) . in-depth description of this method and its essential elements are described in another chapter in the encyclopedia. qualitative research methodology is applied to investigate a problem that has unmeasurable variables, to get a comprehensive understanding of the topic, through discussing it with the involved individuals, and to recognize the natural context in which the investigated issue takes place (creswell, ) . the use of qualitative research methodology is becoming increasingly common across diverse health-related disciplines, including pharmacy practice. this is because of its ability to describe social processes and behaviors associated with patients or healthcare professionals, which strengthen the research impact (mclaughlin et al., ) . therefore, pharmacy researchers and practitioners need to be better oriented to qualitative research methods (behar-horenstein et al., ) . in the following section, interpretative frameworks and philosophical orientations, methodologies, data collection and analysis methods, approaches to ensure rigor, and ethical considerations in qualitative research are briefly discussed (cohen et al., ; creswell, ) . interpretative frameworks are the conceptual structures for comprehension, which form researcher's reasoning and views of truth and knowledge (babbie, ) . different scholars have categorized qualitative research paradigms or interpretative frameworks differently. the following are examples of interpretative framework categories that are used in health science research based on the categorization of creswell ( ): ( ) social constructivism (interpretivism) framework; ( ) post-positivism framework; ( ) transformative, feminist, critical frameworks and disabilities theories; ( ) postmodern frameworks; ( ) pragmatism frameworks. philosophical assumptions are theories and perspectives about ontology, epistemology, axiology, and methodology, which underpin the interpretative frameworks selected by qualitative researchers (cohen et al., ) . as with interpretative framework, there are numerous means to categorize the philosophical assumptions that are folded within interpretative framework. the following are explanations of philosophical assumptions based on the categorization of creswell ( ): . ontological assumptions, which define the nature of reality . epistemological assumptions, which clarify means for knowing reality . axiological assumptions, which explain the role and influence of researcher values . methodological assumptions, which identify approaches to inquiry it is important that a qualitative researcher understands how interpretative frameworks (e.g., social constructivism, postpositivism, and pragmatic interpretative frameworks) are differentiated because of their underpinning philosophical assumptions (i.e., ontological, epistemological, axiological, and methodological assumptions). it is important that qualitative researchers understand the differences between the characteristics of the five qualitative approaches to inquiry, in order to select an approach to inquiry and attain methodological congruence (creswell, ) . the five approaches to qualitative research inquiry are: a. narrative research: describes participants' written and spoken stories about their experiences with a phenomenon being investigated, while considering the chronological connection of the phenomenon's series of events (anderson and kirkpatrick, ; creswell, ; czarniawska, ) . b. phenomenological research: describes the essence of participants' common experiences of a phenomenon, so that the description is a general essence rather than an individual experience (creswell, ; giorgi, ; moustakas, ) . c. grounded theory research: aims to generate a theory grounded in participants' data that conceptually explain a social phenomenon, which could involve social processes, or actions or interactions (creswell, ; strauss and corbin, ; woods et al., ) . d. ethnographic research: involves describing the shared patterns of values, behaviors, and beliefs of culture-sharing participants (creswell, ; harris, ; rosenfeld et al., ). e. case study research: provides an in-depth examination of a real-life contemporary phenomenon that researchers cannot change over time, to illustrate the significance of another general topic (baker, ; creswell, ; de leó n-castañ eda et al., ; mukhalalati, ; yin, ) . . data collection methods data collection tools in qualitative research can be categorized into the following fundamental categories (creswell, ): a. observation b. documents c. individual semi-structured interviews d. focus groups (fgs) e. audio-visual materials f. emails chat rooms, weblogs, social media, and instant messaging. a. topic guides: topic guides guide the discussions in focus groups and individual interviews, and contain open-ended questions and probes, to enable the researcher to understand the complete picture, based on participant views and experiences. they are developed based on the literature review, aim and objectives, research questions, and propositions (kleiber, ) . b. audio recording of fgs and interviews: audio recording of discussions that take place in interviews and fgs is essential for managing and analyzing data, and for increasing the accuracy of data collection and analysis, and ultimately enhancing the dependability and credibility of the research (rosenthal, ; tuckett, ) . c. transcription of fgs and interviews recording: verbatim transcription refers to the word-for-word conversion of oral words from an audio-recorded format into a scripted text format. transcribing data is considered as the first data reduction step because it generates texts that can be examined and rechecked (miles et al., ; grossoehme, ) . . data analysis data analysis comprises several fundamental steps, including reading the transcribed text, arranging data, coding data deductively based on prefigured themes or inductively to produce emergent themes, and then summarizing the codes into themes, and finally presenting the analyzed data as results (cohen et al., ; crabtree and miller, ; pope et al., ) . the most commonly used data analysis methods in health science research are: a. thematic analysis thematic analysis is characterized by identifying, analyzing, and reporting themes that are available in the data (braun and clarke, ; castleberry and nolen, ) . b. content analysis content analysis comprises systematic coding followed by quantification of the analyzed data in a logical and unbiased way (berelson, ; vaismoradi et al., ) . c. discourse analysis discourse analysis emphasizes the core format and the structure of texts to examine the assumptions and concealed aspirations behind discourses (brown and yule, ; gee, ) . qualitative research validation involves ensuring the rigor of the utilized data collection, management, and analysis methods, by utilizing approaches to ensure the quality. in pharmacy practice research, closs ( a, b) argued that quality in qualitative research topic has not been discussed widely in the literature, and therefore closs ( a, b) suggested using several trustworthiness criteria to ensure the rigor of qualitative study. the trustworthiness criteria for ensuring quality in qualitative research (lincoln and guba, ) are: . the trustworthiness criteria a. credibility this criterion aims to ensure that the results are true and increases the possibility that the conclusions are credible (cohen and crabtree, ) . this criterion aims to indicate that the research results are repeatable and consistent, in order to support the conclusions of the research (cohen and crabtree, ) . c. confirmability this criterion aims to confirm the neutrality in interpretation by ensuring that the perspectives of participants, not the bias of researchers, influence the results (krefting, ) . d. transferability this criterion involves identifying the contexts to which the study results can be generalized, and indicating if the study conclusions can be applied in similar setting (yin, ) . reflexivity implies revealing and evaluating the effect and biases that researchers can possibly bring to research process, by explaining the researcher's opinion, feelings, and experience with the phenomenon in question, and explaining the influence of this experience on research methods, findings, and write-ups (creswell, ; krefting, ; lincoln and guba, ) . obtaining an ethical approval from the institutional review board (irb) is required before conducting the qualitative research (creswell, ) . the key ethical issues that need to be considered are: a. informed consent and participant information leaflet informed consent refers to the decision taken by a competent individual to voluntarily participate in a research, after adequately understanding the research. participant information leaflet is usually distributed to participants before they consent to participate in the research to clarify them the voluntary nature of research participation, the aim and objectives of the research, the rights of the respondents and the potential risks and harms, the data collection, management and storage conditions, and the right of participants to withdraw from the research (jefford and moore, ) . b. anonymity and confidentiality the anonymity is usually ensured by not disclosing names of participants and by utilizing a code system to identify them during data collection, management, analysis, and in the writing up of the research. the confidentiality of participants and data is ensured by using a code system to identify participants, and by storing all data in a locked cabinet and a password-protected computer for a specified period of time (creswell, ) . c. power relations in qualitative research settings power imbalance is caused by the fact that participants have the experience about the investigated phenomenon, and researchers need to obtain information about these experiences. the power imbalance is usually associated with interaction between the researcher and participants during recruitment stage, and during data collection, analysis, interpretation, and validation stages. hence, researchers should take suitable measures at each stage to decrease the influence of possible power imbalance, and should enhance trust with participants (karnieli-miller et al., ; yardley, ) . research studies in pharmacy practice usually utilize single-method research designs. however, often these report numerous limitations and may not adequately answer the research question. therefore, the combination of more than one research method to answer certain research questions has become increasingly common in pharmacy practice research (ryan et al., ) . mixed methods research design is now a popular and widely used research paradigm in pharmacy practice research fields (hadi et al., (hadi et al., , closs, a, b; ryan et al., ) . mixed methods research allows the expansion of the scope of research to offset the weaknesses of using either quantitative or qualitative approach alone (creswell et al., ; hadi et al., ; closs, a, b; pluye and hong, ) . typically, qualitative and quantitative data are collected concurrently or sequentially in order to increase the validity and the comprehensiveness of the study findings (creswell et al., ; hadi et al., ; closs, a, b; pluye and hong, ; ryan et al., ) . the mixed method approach provides an expanded understanding of phenomenon under investigation through the comparison between qualitative and quantitative data (hadi et al., ; closs, a, b; pluye and hong, ) . this section provides an overview and application of mixed method research in pharmacy practice. however, considerations in selecting, designing, and analyzing mixed methods research studies as well as the various typologies of mixed methods research are discussed elsewhere. johnson et al. ( ) proposed the following definition for mixed methods research: "the type of research in which a researcher or team of researchers combines elements of qualitative and quantitative research approaches (e.g., use of qualitative and quantitative viewpoints, data collection, analysis, inference techniques) for the broad purpose of breadth and depth of understanding and corroboration." mixed methods design allows the viewpoints of participants to be reflected, enables methodological flexibility, and promotes multidisciplinary teamwork (ryan et al., ) . furthermore, the approach allows a more holistic understanding of the research question. however, its major limitations include: need for wide range of research expertise across the research team members, highly labor-intensive, and the complexity of data integration. scholars believe that it is challenging to provide researchers with a step-by-step guide on how to undertake a mixed methods study and that this is driven by the specific research question (ryan et al., ) . nevertheless, the investigator should precisely determine the type of qualitative and quantitative methods to be employed, the order of data collection to be undertaken, the data collection instruments to be used, and the method of data analysis (ryan et al., ) . this approach encompasses a synthesis of findings from both quantitative and qualitative components, which is achieved through integration of the findings from each approach (hadi et al., ; closs, a, b; pluye and hong, ) . different models or typologies for mixed methods research have been described in the literature. the most common typologies used in pharmacy practice and health services research include: concurrent or convergent parallel design, exploratory sequential design, explanatory sequential design, and the embedded design (hadi et al., ; pluye and hong, ) . scholars believe that there are several factors to consider when selecting the typology or model of mixed methods research to use. these factors include: the order of qualitative and quantitative data collection (concurrent vs. sequential); priority of data (i.e., which type of data has priority between quantitative and qualitative data); purpose of integration of the data (e.g., triangulation); and number of data strands (hadi et al., ; pluye and hong, ) . in mixed methods research, integration of qualitative and quantitative findings is critical, and this research approach does not simply involve the collection of these data (ryan et al., ) . • in the era of evidence-based practice, it is not sufficient to propose new pharmacy services or roles without evidence of their benefit. • new pharmacy services and new roles must be proven to be feasible, acceptable, beneficial, and cost-effective. • practice-based research provides such evidence and can inform policy, confirm the value of the new service, and change practice. • various study designs, including, but not limited to experimental, quasi-experimental, observational, qualitative, and mixedmethods designs, have been used in pharmacy practice research. • pharmacy practice researchers need to be competent in the selection, design, application, and interpretation of these methodological and analytical approaches. • the choice of any study design in pharmacy practice research is driven by the expertise of the investigator, type of research question or hypothesis, data availability, time orientation, ethical issues, and availability of funding. there is a great demand for innovation and quality in pharmacy practice. these can be achieved partly through robust and welldesigned pharmacy practice research. pharmacy students, practitioners, educators, and policy-makers are exposed to a variety of research designs and methods. we need to have the best evidence (e.g., in policy, regulation, practice) for making decisions about the optimal research design that ensures delivering an ultimate pharmacy practice and a quality patient care. pharmacy practice research the canadian pharmacists association defines pharmacy practice research as a component of health services research that focuses on the assessment and evaluation of pharmacy practice (koshman and blais, ) . quantitative research is simply defined as "a research in which things are counted." these things might be people, medicines, opinions, behaviors, etc. "while qualitative research is very useful for describing phenomena in depth (particularly motivations, feelings, understandings), quantitative research can tell us how common and widespread the phenomena are" (green and norris, ) . qualitative research qualitative research within the health sciences has developed as a means to gather an in-depth understanding of human behavior, as well as to find the underlying reasons, attitudes, and motivations that govern such behavior (kaae and traulsen, ) . mixed methods research a mixed methods research typically involves both the components of qualitative and quantitative research approaches embedded within a single study or research program for the broad purpose of breadth and depth of understanding and corroboration (creswell et al., ; johnson et al., ; tashakkori and creswell, ) . observational studies observational studies involve merely observing the study subjects in a noncontrolled setting, without investigator intervention or manipulating other aspects of the study in order to determine an association between an exposure and an outcome of interest. experimental studies in experimental design (also known as interventional design), the investigator performs an intervention and evaluates cause and effect relationships between exposure to the intervention and an outcome of interest . subjects are typically randomly assigned into experimental and control groups. quasi-experimental studies quasi-experimental designs are studies that aim to evaluate interventions, but that do not use randomization and at times noncontrolled (harris et al., ; krass, ) . similar to true experimental studies, quasiexperiments aim to determine causality between an intervention and an outcome of interest. narrative interviewing exclusive and combined use of statins and aspirin and the risk of pancreatic cancer: a case-control study pharmacists' involvement in and attitudes toward pharmacy practice research: a systematic review of the literature the contribution of case study research to knowledge of how to improve quality of care perceptions of pharmacy faculty need for development in educational research the need for pharmacy practice research spironolactone for nonresolving central serous chorioretinopathy: a randomized controlled crossover study using thematic analysis in psychology the stepped wedge trial design: a systematic review thematic analysis of qualitative research data: is it as easy as it sounds? why have a special issue on methods used in clinical pharmacy practice research? societal perspective on access to publicly subsidised medicines: 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influenza vaccine in the community-dwelling elderly progress report on the first sub-saharan africa trial of newer versus older antihypertensive drugs in native black patients combining the power of stories and the power of numbers: mixed methods research and mixed studies reviews qualitative research in health care: analysing qualitative data improving access to medicines for non-communicable diseases in rural india: a mixed methods study protocol using quasi-experimental design interdisciplinary medication decision making by pharmacists in pediatric hospital settings: an ethnographic study qualitative research methods: why, when, and how to conduct interviews and focus groups in pharmacy research mixed methods research in pharmacy practice discrete choice experiments in health care methodology series module : cohort studies methodology series module : cross-sectional studies patients' perceived value of pharmacy quality measures: a mixed-methods study tobacco use and risk of myocardial infarction in countries in the interheart study: a case-control study dental procedures, antibiotic prophylaxis, and endocarditis among people with prosthetic heart valves: nationwide population based cohort and a case-crossover study rigour in qualitative research: complexities and solutions: anthony g tuckett outlines the strategies and operational techniques he used to attain rigour in a qualitative research study through relying on guba and lincoln's trustworthiness criterion. research strategies such as use of personal journals, audio recording and transcript auditing, and operational techniques including triangulation strategies and peer review, are examined content analysis and thematic analysis: implications for conducting a qualitative descriptive study discrete choice experiments of pharmacy services: a systematic review simulated patients in the community pharmacy setting -using simulated patients to measure practice in the community pharmacy setting a systematic review of the use of simulated patients and pharmacy practice research acid-suppression medications and bacterial gastroenteritis: a populationbased cohort study communities of practice and the construction of the professional identities of nurse educators: a review of the literature haemorrhagic-fever-like changes and normal chest radiograph in a doctor with sars a systematic review of simulated-patient methods used in community pharmacy to assess the provision of non-prescription medicines dilemmas in qualitative health research qualitative case study methodology: study design and implementation for novice researchers transforming qualitative information: thematic analysis and code development avoiding common pitfalls in qualitative data collection and transcription the enlightened eye: qualitative inquiry and the enhancement of educational practice research methods and methodologies in education postmenopausal hormone use and secondary prevention of coronary events in the nurses' health study. a prospective, observational study is verbatim transcription of interview data always necessary? qualitative research methods for medical educators holding on to the indispensable medication-a grounded theory on medication use from the perspective of persons with medication overuse headache moral dilemmas of community pharmacists: a narrative study a grounded theory of the process of adherence to oral chemotherapy in hispanic and caucasian children and adolescents with acute lymphoblastic leukemia problems of reliability and validity in ethnographic research improving accuracy of transcripts in qualitative research focus groups as qualitative research the contracted relationship: ensuring protection of anonymity and confidentiality conducting small-scale investigations in educational management. nunkoosing, k., . the problems with interviews learning how to lead through engagement with enquiry based learning as a threshold process: a study of how post-graduate certificate in education healthcare professional students learn to lead data analysis in health-related qualitative research essentials of nursing research: appraising evidence for nursing practice determining counselling communication strategies associated with successful quits in the national health service community pharmacy stop smoking programme in east london: a focused ethnography using recorded consultations pharmacists in general practice: a qualitative interview case study of stakeholders' experiences in a west london gp federation quality of qualitative research in the health sciences: analysis of the common criteria present in assessment guidelines by expert users i did not want to take that medicine": african-americans' reasons for diabetes medication nonadherence and perceived solutions for enhancing adherence understanding the meaning of medications for patients: the medication experience doing qualitative research: a practical handbook informed consent: ethical implications for physiotherapy transparency in transcribing: making visible theoretical bases impacting knowledge construction from open-ended interview records i don't think i'd be frightened if the statins went': a phenomenological qualitative study exploring medicines use in palliative care patients, carers and healthcare professionals key: cord- -bhvemgjv authors: counsell, chelsie w. w.; elmer, franziska; lang, judith c. title: shifting away from the business-as-usual approach to research conferences date: - - journal: biol open doi: . /bio. sha: doc_id: cord_uid: bhvemgjv to combat the climate crisis, we need rapid, unprecedented social change. scientists can play a lead role by signaling to society that we recognize the critical importance of redesigning our business-as-usual approach to research conferences. traditional research conferences have high co( ) emissions as well as significant financial and travel time costs for participants. using available technology, early career scientists chelsie counsell and franziska elmer created a global, virtual, coral reef research conference with live talks, recorded contributions, and networking events. funding from the company of biologists allowed this event to be free, supporting attendance of subscribers and content contributions from participants from diverse backgrounds and career stages. we provide metrics on content viewership and participation in networking activities, note the success of incorporating regionally focused sub-events, and discuss the emergence of a collaborative research project. we highlight the broad accessibility of virtual conferences as well as their increased flexibility in programming, health benefits, and cost savings. our approach to organizing and hosting a global, low-carbon emission research conference is documented. finally, we propose a hybrid approach to future conferences with virtually connected remote (sub-regional or local) hubs. research conferences provide opportunities to share science, network, and reach innovative solutions through synthesized ideas. we discover collaborators, obtain positions, present our research, and feel inspired by plenary talks. conference attendance is considered vital for the growth of scientific fields and for the career trajectory of most scientists. unfortunately, the traditional approach to research conferences, often involving travel over great distances (including intercontinental flights), can have a large co footprint. global coral reef week (gcrw) was designed to rethink this paradigm by organizing a global coral reef research conference with low co emissions and high accessibility. the average amount of co emitted from travel to a research conference has been calculated at ∼ kg per presenter (spinellis and louridas, ) . long-distance travel is particularly problematic: a seat on a one-way intercontinental flight is estimated to release more co ( kg) than the average annual emissions for one person living in britain or ten people living in ghana (klöwer et al., ) . we know the climate crisis is real. its effects occur globally through increased risk of deadly heat waves (mora et al., ) , intensified weather patterns, land degradation, increased food insecurity (ipcc, a) , loss of ice sheets and glaciers, marine heat waves, and ocean acidification (ipcc, b) . coral reef scientists personally experience the high costs of climate change, as our study sites bleach with an increasing frequency in overly warm oceans, such that their continued existence is threatened (ipcc, b) . to reduce the effects of climate change, we need to stop burning fossil fuels (ipcc, a) . the climate crisis is not a technological or scientific problem. the climate crisis is a problem of inadequate willpower, resulting from capitalism and institutionalized definitions of growth and success at all levels, from the global community to the individual (jewell and cherp, ) . scientists cannot save coral reefs through research alone. we can lead by example, through rapid shifts in our collective actions by redesigning our business-as-usual approach to both our professional and personal lives. we should be part of the energy that drives necessary, unprecedented social change. international, in-person coral reef conferences are intended to help study and save coral reefs, but, paradoxically, the long-distance air travel of participants to these events directly contributes to their demise (nevins, ) . gcrw was designed to pilot an approach to research conferences that removes long-distance flights while retaining the ability to network and share science. this dramatically reduces conference co emissions and increases conference accessibility (hiltner, ) . we originally envisioned a collection of inexpensive, virtually-connected, remote meeting 'hubs' organized at local and sub-regional scales. this approach would blend in-person interactions with access to live-streamed plenary talks (including interactive questions and answers) and include the curation of a digital content repository. our vision was enthusiastically received by colleagues, particularly in the caribbean, brazil, hawai'i, the continental us, and parts of europe. from january to march, we confirmed local hosts for remote meeting hubs in locations. when covid- then restricted faceto-face meetings and group gatherings, we pivoted to create an entirely virtual conference. gcrw thus became an opportunity for coral reef scientists to share their research, network with new colleagues, and try the virtual conference experience. when organizing gcrw, we strived to retain the key components of an in-person conference, i.e., research talks, plenary talks, workshops, networking, and other social events. contributed talks were uploaded as recorded presentations to a gcrw conference youtube channel divided into nine topical playlists. our conference website included advice on how to use freely available software to create a digital recording of a presentation and tips for producing engaging science communication videos. the research talks that were contributed from participants across the globe featured reefs in the atlantic, indian, and pacific oceans, as well as the caribbean, mediterranean, and red seas. early career scientists and students submitted over % of the presentations. reef scientists, resource managers, and coral reef enthusiasts submitted the remainder. on the final day of the conference, recorded research presentations had an average of views per video. one month after the end of gcrw, this average had increased to views per video for a total of over , views. for our ten live-streamed plenary talks, we invited a diverse group of speakers who collectively represented seven countries, five of whom were within eight years of their phd (two within two years). plenary talks were divided into five thematic sessions that attendees could join via zoom to participate in questions and answers. all plenary talks were also live-streamed on our youtube channel, and the recordings are digitally archived on our website. an average of attendees watched the plenaries live, with an average additional views on youtube at the end of gcrw. after covid- restrictions forced the shift to an entirely virtual format, the local hosts for three hubs requested virtual support to live-stream regionally focused research talks and discussions. we therefore added live-streamed sessions for brazil (four speakers, hosted by cesar cordeiro), cayo arcas (five speakers, in spanish, hosted by eduardo cuevas), and the mesoamerican reef ( speakers, hosted by healthy reefs for healthy people). on average, people watched these live-stream regional sessions and people viewed each recording. furthermore, cordeiro and his team hosted a -day sub-event in portuguese, encontro recifal brasileiro, with its own youtube channel, website, and social media marketing (box ). as part of our call for abstracts, we welcomed contributions of virtual workshops. we suggested a recorded format with an introduction, guided activities, and a wrap-up. we received two virtual workshops contributions, one focused on designing and implementing creative ways to combat the climate crisis, from chelsie counsell, hawai'i institute of marine biology, which received views by the end of gcrw, and another designed in four sequential episodes from alizee zimmermann, turks and caicos reef fund, showing citizen scientists how to identify the coral species susceptible to stony coral tissue loss disease, which received an average of views per episode. in response to participant requests shared during gcrw, conference organizer franziska elmer partnered with plenary speaker robby thigpen (marine conservation without borders) and participant neus figueras (university of barcelona) to lead a live workshop on ways to strengthen outreach and communication. forty-four conference participants attended this live workshop and the recording had an additional views. for social activities, gcrw included two networking sessions and a trivia event. the networking sessions included rotation through breakout rooms and icebreaker questions for a total of participants. our virtual trivia event, led by a team of research interns from the school for field studies' center for marine resource studies, had registered participants. while casual networking is a challenging element of conferences to recreate virtually, our participants provided positive feedback for these sessions, noting that much of the challenge of inserting oneself into an ongoing in-person conversation was removed. excitingly, enough science and ideas were shared at gcrw to catalyze a new research collaboration. building from live-streamed plenaries and conversations in networking sessions, participant tatiana becker (wageningen university) and plenary speaker phillip box . cesar cordeiro (universidade federal do rio de janeiro) and his team hosted a -day sub-event, encontro recifal brasileiro, in portuguese as part of global coral reef week. the brazilian reef meeting, encontro recifal brasileiro -erebra (portuguese), held as a regionally focused component of gcrw , was the first event dedicated to reef environments in brazil. the lack of such events in the past has made the local audience seek out international events, involving long distance travel at high economic and environmental costs (sarabipour et al., preprint) . traveling is the main cost associated with scientific events for attendees (rowe, ) , especially in a large country like brazil. being part of gcrw provided the erebra audience with access to global coral reef research content and a wider network of collaborators. in return, erebra contributed a live-streamed session focused on technology innovation in reef research to the core gcrw program and access for all gcrw attendees to erebra's regionally focused recorded talks. erebra had registered attendees from all coastal states and five inland states in brazil. the majority of participants were students ( %), followed by non-academic ( %), academic professionals ( %), and coral reef enthusiasts ( %). while erebra's regional program was only available in portuguese, participants were from portugal, chile, méxico, guatemala, the usa, and ireland. from a feedback poll, % of respondents (n= ), rated the event format and content as 'very good' or 'good.' erebra was supported by projeto costão rochoso, projeto conservação recifal, projeto coral vivo, & projeto budiões. dustan (college of charleston) are leading a global effort to study the effects of the return of tourism on reefs when covid- travel restrictions are lifted. to date, gcrw has hosted six meetings, providing digital and administrative support for the collaborators participating in this project. a week before the conference, people had subscribed to gcrw. this number increased to people by the day before gcrw and to people at its end. the incredible jump in email subscriptions for an inaugural research conference not affiliated with any specific program or society demonstrates the ease with which digital content can be shared across social networks (box ). while gcrw was designed to decrease conference co emissions, we quickly recognized a multitude of other benefits of a global virtual research conference. the public, digital archive of content increases access compared to an in-person conference by eliminating scheduling conflicts, enabling rewinding, pausing and re-watching, facilitating content sharing, and providing anyone with internet access an opportunity to learn about current coral reef science. the increased accessibility of digital content is of particular importance for participants who are experiencing personal life events or have certain disabilities that make travel to conferences unfeasible. for example, an attendee of gcrw had just finished cancer treatment, yet was able to join our virtual conference and even to contribute a presentation. conference content in the form of youtube recordings provides access to researchers who are deaf or who have limited understanding of the language through autotranslation subtitles. when contributors choose to keep their videos available online, these benefits of increased access to virtual content continue beyond the end of the conference. broad sharing of science with current or future colleagues, employers, advisors, and students, as well as resource managers, politicians, the media, and community partners is enabled. hosting a research conference on virtual platforms affords additional flexibility for programming and content compared to inperson conferences. without temporal and spatial restrictions on the number of conference rooms, we could accept all topically relevant research contributions. we were able to adjust the thematic sessions, i.e., youtube playlists, to match the interests of conference participants revealed in their contributed talks. although we asked for - or - min presentations, when presenters took an additional minute or ten, the talks could be included in their entirety without disrupting the conference schedule. alternatively, when a subset of contributed abstracts did not result in video submissions, these gaps in content were easily removed from our digital program. no attendees arrived expecting a speaker, only to find an empty room. another powerful aspect of high flexibility in virtual programming is the ability to respond to participant requests in real time. we deliberately did not schedule live events on the last two conference days, which enabled us to organize and host additional programming (i.e., live-streamed workshops and collaborative research-project-development meetings) in response to the expressed interest of our participants. virtual conferences incorporate a variety of health benefits for attendees as a result of minimizing long distance air travel. personal benefits of flying less include reduced exposure to germs and misalignment of the circadian rhythm. jet lag affects gene expression connected with aging processes and immune system functioning (cohen and kantenbacher, ) . chronic jet lag can lead to memory impairment and increase the risk of stroke and heart attack (cohen and kantenbacher, ) . in addition, flying exposes travellers to radiation at exposure levels hundreds of times higher than other travel options and chronic exposure to engine noise at levels within passenger cabins is linked to increased risk of heart disease and hearing loss (cohen and kantenbacher, ) . the health impacts of traveling to conferences can also include social strain from leaving families and stress from postponing other work activities (høyer and naess, ) . the cost benefits of gcrw were enormous. in-person conference organizers with a similar number of speakers to gcrw need to raise around $ , to $ , for non-labor related costs, whereas our non-labor costs were <$ (all estimates in usd). in-person conference attendance can cost participants >$ ; registration for gcrw was free and participation did not require travel or lodging. researchers from diverse social-economic backgrounds attended gcrw, including those who are unable to afford in-person conferences. gcrw cost <$ per presenter and <$ . per attendee, expenses that were covered through the support of a grant, sponsors, and donations. in terms of the time required of the organizers, this virtual experience was similar to in-person conferences. however, the time commitment will likely be reduced building a social media presence is crucial for promoting research conferences. for gcrw, we used three different platforms: instagram ( followers), twitter ( followers), and facebook ( followers). to create content, we built a photo bank by reaching out to scientists and photographers around the globe, and then used canva to design promotional graphics. posts included conference details (abstract deadlines, subscription information, program scheduling), interesting facts about coral reefs, and details on the research of gcrw participants. to increase interest in our educational content as a mode of informal outreach, we used creative hashtags e.g., #weirdfactwednesday, #sharkysunday, and #funfactfriday. our content also included instagram 'takeovers' where invited scientists shared photos and brief summaries of their research through posts on the gcrw account. on all three social media platforms, people interested in gcrw shared and promoted the conference with their networks by re-posting our content. this chain reaction expansion in reach greatly boosted our audience. on facebook, > , individuals saw our posts in their feed from june to july . for future gcrw events as it included concept development, building a website, familiarizing ourselves with the capacity of various virtual platforms, and writing guides to help participants navigate the virtual milieu. to guide others who are interested in organizing research conferences with virtual components, we have documented the key steps required to host a meeting like gcrw along with a timeline that reflects our process. in total, > h were required to organize gcrw. . clarify event-specific details (> months before conference, + h of organizer time). this includes selecting the event name, creating a logo, defining a target audience, scoping potential research themes, selecting event dates, and building a website (we created a wix website linked to a google domainwww.coralreefweek.org). assessing finances and technological support capacity can help guide platform selection: for live-streaming content we used zoom, for sharing recorded videos and creating a digital archive we used youtube, for developing a mailing list to share content links and updates we used wix built-in tools and mailchimp, and for promoting the event on social media we used instagram, twitter, and facebook (box ). . develop core meeting content ( months before conference, + h). invite and confirm participation for plenary speakers. develop plans for social and networking events, include protocols alerting participants how their contact information may be used (e.g., for email updates) or shared (e.g., with fellow participants). strategize effective techniques for virtual workshops and begin conversations with potential workshop hosts. . marketing/outreach to solicit for contributed content and conference attendees (start as soon as possible, + h). we created a google form to collect abstract submissions for contributed talks and workshops. the link to this form and a subscribe option, for people interested in attending the conference without contributing content, were provided on our website. we started marketing the event on email lists for our affiliate research organizations. we contacted individuals acting as local organizers for remote hubs and/or research sessions regarding support needs and programming ideas. we sent newsletter and email templates to local organizers for marketing the event. we used social media accounts to market the conference and abstract submission deadline. . conference administration ( - months before conference, + h). even if content is virtually collected using built-in platform tools, organizers will need to curate the contributions. it is also critical to maintain regular communication and logistical organization with plenary speakers and local partners. . technological support ( our vision for a conference of virtually connected, in-person regional hubs would build on the success of the inaugural grcw. in addition to low co emissions, low cost, and high accessibility, remote hubs would provide additional opportunities for face-toface interactions with local colleagues, educators, students, policy makers, politicians, and the media, greatly strengthening the dissemination of science. each hub could incorporate a day of environmental or societal service to strengthen engagement with local communities. virtual connections across the remote hubs would enable participants to network with global community members. we remain energized by local organizers who volunteered to host remote events in international locations and hope someday to realize this vision. completely free participation for all attendees of gcrw was possible because of the copious volunteer time contributed (in particular from elmer and counsell), a grant from the company of biologists, institutional infrastructure support, donations, sponsorships, and merchandise sales. however, there may be good reasons to charge a small conference fee for future events. attendees and participants may be more actively engaged in paid events to get 'value' from their investment. the funds raised could be used to outsource components of the conference organization process and to provide support for virtual platform software and in-person meeting venues. without prior experience and without everyone having met faceto-face, we saw a way to provide an option for a collective shift in professional behavior, and we worked hard to make our vision a reality. along with many others during this difficult pandemic year, we took quick action to create a successful virtual research experience for our colleagues. we have shown that virtual conferences have many benefits and strong potential for growth. to solve the climate crisis, we must embrace creative new approaches for achieving our personal and professional goals with dramatically reduced co emissions. flying less: personal health and environmental co-benefits writing a new environmental era: moving forward to nature conference tourism: a problem for the environment, as well as for research? sustainable land management, food security, and greenhouse gas fluxes in terrestrial ecosystems summary for policymakers on the political feasibility of climate change mitigation pathways: is it too late to keep warming below . c? wires clim an analysis of ways to decarbonize conference travel after covid- global risk of deadly heat academic jet-setting in a time of climate destabilization: ecological privilege and professional geographic travel the economic cost of attending educational conferences evaluating features of scientific conferences: a call for improvements the carbon footprint of conference papers we are fortunate to have various technological digital tools available to record, screen, and globally share science. we are exceptionally grateful to the work of ken hiltner (university of california at santa barbara) and team who documented their protocol for holding nearly carbon neutral conferences. we thank two colleagues who provided insightful comments on this manuscript. the authors declare no competing or financial interests. the company of biologists, tradewind colours, marine conservation without borders, and donations from colleagues. key: cord- -lw xd m authors: ha, kyoo-man title: integrating the resources of korean disaster management research via the johari window date: - - journal: eval program plann doi: . /j.evalprogplan. . sha: doc_id: cord_uid: lw xd m it is not widely known that quite a few researchers are faced with difficulties in using various resources of disaster management research in korea. the article aims to assess how rigorously the korean field of disaster management research resources has been managed or how it can be improved for the ultimate goal of disaster management. descriptive content analysis has been used as the major methodology by referring to the johari window. in doing so, electronic research resources have been systematically compared with integrated research resources via the perspective of korean-speaking researchers and that of english-speaking researchers. the conclusion is that two researchers have to be integrated with all four research resources (open, blind, hidden, and unknown resources) by implementing assigned responsibilities as well as freely asking questions. ultimately, this will be conducive to reducing down the impacts of disaster in korea. south korea (hereinafter korea) has continually been hit not only by natural disasters but also by man-made emergencies (on that point, this article covers all kinds of disaster). sample disasters include the collapse of sampoong department store in , typhoon maemi in , the sinking of ferry sewol in , the outbreak of middle-east respiratory syndrome (mers) in , gyeongju earthquakes in , pohang earthquakes in , miryang fires in , the chronological outbreak of avian influenza and foot-and-mouth disease, and others (ministry of interior & safety (mois), ). due to the many occurrences of disasters, the people asked the government to take progressive actions, but its policy was incomplete because of many factors, including the lack of disaster awareness, poor level of scientific research, lack of appropriate education, and lack of efficient government efforts. as a policy tool, impact assessment deals with evaluating or assessing the consequences of various environmental or disaster programs (durning, ; morgan, ) . with impact assessment, there is a need to identify the critical impacts of certain programs and then provide alternatives to reduce these impacts. in the field of disaster management research, impact assessment may improve the effectiveness of disaster management. disasters cause not only human loss and economic damages but also psychological impacts to human society. many researchers have also studied such impacts. however, with much more complex issues of disasters or disaster management, several complicated questions remain unanswered (benight & mcfarlane, ) . indeed, more sophisticated and in-depth researches on disaster management are urgently needed. to illustrate, there are some researchers who concentrate on a single resource in a particular topic. other researchers also fail to access research resources in a timely manner. in the case of disaster management, availability of integrated resources and timely access are of the same importance, given the need for organized action and quick response when it comes to curtailing the impact of disasters (alamdar, kalantari, & rajabifard, ; srinivasan et al., ) . despite the availability of diverse research resources in the internet, many researchers continue to encounter difficulties in using and making them more useful in their works (scott & few, ) . in many cases, this is also the experience in the field of korean disaster management. this raises the question: how can the integration of research resources on disaster management be improved to benefit not only korea but also the international community? this article aims to assess the state of integration of korean disaster management research resources. descriptive content analysis was used to compare resources with the viewpoints of korean-speaking researchers and english-speaking researchers using the johari window. electronic research resources refer to materials available in computers or via the internet that may be structured, but not necessarily coherent. integrated research resources are not only available electronically; they are also structured, organized, and maintained in a system that allows them to be searched and used significantly. the key is the need for transformation to be in an integrated setup or platform for both korean-speaking researchers and english-speaking researchers. in , researchers joseph luft and harrington ingham developed the johari window (named after them) (clayton, ) . it is a tool used to help assess self-awareness and understanding, while observing multiple relationships between the self and others. many internationally known researchers have discussed the issue of research in the field of disaster management. at the same time, some researchers have applied the concept of the johari window to their disaster management research (lander & atkinson-grosjean, ) . however, based on the literature review, not many international researchers have made the same efforts to systematically assess the korean disaster management research or the johari window in the korean context. the johari window has four behavioral areas, specifically, open, blind, hidden, and unknown. the use of this technique plays a role in setting up goals, improving critical thinking, studying prejudices, identifying all elements or personal boundaries, and developing relationships. by analyzing the four areas, concerned individuals can improve their knowledge and information through an appropriate assessment (south, ) . research, to include not only research itself but also its development, has played many roles in supporting disaster management. throughout the life cycle of disaster management, namely, disaster prevention/mitigation, preparedness, response, and recovery, research has contributed in mitigating the loss of human lives, economic damages, and psychological impact in the international community. although invention or use of cutting-edge technologies or new theories continues, research has changed how the field of disaster management operates (king, edwards, watling, & hair, ) . surely, research has many resources, depending on an individual's perspective. as an example, disaster management research may have two resources, namely, primary resources and secondary resources (university of maryland university college (umuc), ). primary resources as original materials include raw data, survey and interview results, official documents, and other firsthand accounts. on the other hand, secondary resources as secondary materials include assessments, syntheses, discussions, and others. although disaster management research consists of many sub-areas, each sub-area has its own characteristics (xu, wang, shen, quyang, & tu, ) . for example, research on natural disaster as a sub-area has not adequately predicted disaster victims' psychological change, as the field has not provided enough amount of pre-disaster data or related statistical analysis (green, lowe, & rhodes, ) . although this may depend on the type of natural disaster, research on natural disaster has relied so much on victims' post-disaster data. in general, disaster management research has more noteworthy characteristics, as compared with other academic researches. similar to the thought that a disaster may be efficiently managed by multiple professionals or complicated knowledge, related research is also a sort of multidisciplinary one. also, the contents of disaster management research are specific and thus may be applicable to concrete cases. by using common and basic terminologies, disaster management research allows ordinary citizens to understand the research results. disaster management needs organized and quick action, especially in the case of researching and developing vaccines, such as with mers, ebola, and h ni virus (callaghan, ) . without timely resolution, the adverse impacts of pandemic outbreaks may be serious and damaging. in disaster management, integration does not only concern uniting data resources but also stakeholders, methodologies, disciplines, etc. (ye, browne, grdisa, beyene, & thabane, ) . integration is also about highly coordinated and collaborative networks available to extend services toward effective disaster management. integrated disaster management research was strongly vindicated for the past or years in the international community, mainly because disaster has a complex structure, wherein one or two experts alone may not be enough to easily solve or handle disaster management. however, the majority of integrated research was carried out by european researchers or north american researchers concerning their own topics or fields of interest. surprisingly, even though they maintained their achievement on their own integrated research, some critics raised questions on whether related research was indeed performed or not (gall, nguyen, & cutter, ) . like disaster management research in many countries, the korean field of disaster management research has a wide scope. it is surrounded by unique management styles, cultures, or other environments. furthermore, the korean field includes many international principles on disaster management, considering that it has started to associate itself with the international community. if the korean research field fails to assess or use the networks of all disaster management research resources, it may not smoothly suggest appropriate alternatives not only for decision-makers but also for disaster victims. alternatively, without the help of self-assessment via the johari perspective, the adverse impacts of disasters would not be mitigated in korea. integration of all research resources relative to disaster management is imperative. doing so may help lessen or address the potential ripple effects of disasters. using the johari window as an assessment tool will aid in achieving this integration goal (hills, ). an increasing number of korean researchers have examined disaster management in korea. however, not many korean researchers have studied the issue of disaster management research. furthermore, no rigorous study has ever been attempted to assess or evaluate disaster management research or its resources in terms of the johari window (national disaster management institute (ndmi), ). therefore, the goal of this article has potential value to the study. descriptive content analysis was the methodology used for this study, as it has been considered as one of the most effective tools in analyzing the important features of korean disaster management research resources and evaluating not only the tangible effects of research resources but also their intangible effects (fenriam, ; vo, ) . the procedure of descriptive content analysis includes four steps: defining a research question, identifying information and data, reviewing collected information and data, and documenting a descriptive summary (bengtsson, ) . in short, descriptive content analysis plays a role in assessing the significant characteristics of disaster management research resources by describing the outcomes of disaster management research resources. meanwhile, relevant subjects and texts were identified, collected, and then interpreted for the research. for information and data identification, some terms (e.g., johari window, principles of disaster management, science and technology policy, korean disaster management, etc.) were searched in widely used search engines, such as sciencedirect, ebsco, google.com, oxford university press, korean search engines, etc. moreover, the author performed subjective assessment to qualify the identified materials and resources. primarily, as the johari window was proposed about years ago, not many researchers have discussed related issues in the st century. thus, the author identified or included as many articles or documents as possible on the johari window into the references or resources for analyzed texts. in general, the majority of identified or collected references were labeled as favorites as shown in the results of the article. using impact assessment, this article assesses (or evaluates) how the korean field of disaster management research has been doing with its resources and what the field should do to improve the current situation and establish appropriate alternatives. in the process, the article analyzes all the resources of korean disaster management research using the johari perspective, with the goal of naming a current model and an alternative model on disaster management research resources. similarly, this research has shown a comparative perspective between korean-speaking researchers and english-speaking researchers regarding disaster management research resources. the comparative study leads to identifying many factors that are unique to national disaster management system when reflecting on traditional approaches, such as a single-case study, being too parochial, rigid, or even narrow in their theoretical process (jreisat, ; luft & fakhouri, ) . additionally, traditional approaches have not been based on cross-cultural situation. therefore, this comparative study plays a role in fostering the development of effective planning and programs in the field, particularly by referring to the cross-language perspective. as can be seen in table , the two major stakeholders identified are korean-speaking researchers and english-speaking researchers. these two groups have been directly or indirectly involved in all major resources of korean disaster management research. research resources written in other languages are excluded from the scope of this framework. by cross-checking the two stakeholders, four research resources, including open research resources, blind research resources, hidden research resources, and unknown research resources, are analyzed. descriptive content analysis has been well known for its systematic review of the subject (kim & lennon, ) . similarly, the johari window has thoroughly pursued the systematic aspect of disaster management research resources by referring to open/free resources, blind resources, hidden resources, and unknown resources in that order (newstrom & rubenfeld, ) . the johari window comprehensively includes not only the bright sides of research resources but also their dark sides. considering these aspects, the johari window technique is quite relevant for the methodology used in this article. open research resources include four major english resources, such as international journals, the united nations' official documents, googlescholar.com, and yahoo.com, which are some of the many open research resources. it is worth noting that those four major english resources have been widely used by both english-speaking researchers and korean-speaking researchers. to elaborate, all english-speaking researchers may access such resources and generally understand related contents. also, although only a few korean-speaking researchers can understand the contents from such resources, all korean-speaking researchers have equal access to them. many publications have held a monopoly of their research results. without the internet, it would be more difficult for researchers to communicate or share their research results with their colleagues. through the use of internet, many researchers have overcome these difficulties. with the support of public funds, many researchers have been able to read, copy, print, download, and distribute research results without much restrictions. nevertheless, open research resources are, at times, said to be useless resources to many korean-speaking researchers, mainly because they do not understand the english language very well. furthermore, even though those open resources are electronically suitable to all researchers, even english-speaking researchers still face difficulties in freely accessing such resources because of strict copyright laws (sharma, ) . a good example of blind research resource is the emergency management institute (emi)'s independent study program (isp). various english-speaking researchers have recognized important courses in the program. at the same time, they have used it as their research resource (emergency management institute (emi), ). however, almost all korean-speaking researchers were not able to use this program as their research resource. the emi's program includes many disaster management principles, such as comprehensive emergency management, integrated emergency management system, incident command system, whole community approach, and many others. the international association of emergency managers (iaem) has contributed to spreading the emi's program to many isolated areas while encouraging local emergency managers to take appropriate courses and become certified emergency managers. blind research resources have been frequently ascertained as a sort of "liability" in the field of disaster management research (katsikopoulos & gigerenzer, ) . a liability is something that causes negative problems and, thus, should be efficiently solved. the emi's program has been electronically usable to korean-speaking researchers; however, they have not recognized its existence. some have surely recognized it, but they have not fully understood it due to language barrier. thus, at present, the emi's program is considered a challenge for korean-speaking researchers. hidden research resources include local definitions on disaster management, local community data, and knowledge of indigenous resources in korea. although all korean-speaking researchers have comparatively understood them, many english-speaking researchers are not aware of them. a good example is that "banjae" to korean-speaking researchers means "to manage typhoon accompanied by flood," but it has frequently been translated as "disaster prevention" in english. language does matter in the field of disaster management research (kim, ferrini-mundy, & sfard, ) . when english-speaking researchers work on their mathematical research, they predominantly explain the issue in procedural terms. on the other hand, koreanspeaking researchers respond to the issue via structural ways. when researchers work on the issue either in the english language or in the korean language, they are less prolific than when bilingual researchers resolve the same issue. electronically, hidden research resources have been certainly available to both korean language researchers and english-speaking researchers. however, many english-speaking researchers have not been able to use those resources for their research because of the language barrier or the lack of appropriate understanding of the local culture. in terms of understanding relevant resources written in english, table analytical framework via the johari window. sources: (chapman, ; luft & ingham, korean-speaking researchers are at a disadvantage. on the contrary, english-speaking researchers are at an advantage because they do not face any challenge in understanding the language. unknown resources, as the name suggests, are unfamiliar to many. they remain to be determined or discovered for meaningful use. one example would be the precise differences between korean language research and english language research regarding disaster management. further examples are the applications coded in programming languages and experiments with results that are not yet available to the public and are known only to some scientists. a few researchers have pointed out the existence of unknown research resources in the field of disaster management (ramasesh & browning, ) , such that they have willingly or unwillingly faced unknown research resources while attempting to develop new disaster management products or implement new disaster management processes. despite these unknown research resources, they have paradoxically made their new theories. a superficial form of integration is based on the fact that albeit far too many resources are available in the internet, they remain unknown to many. exactly evaluating the gap between and among researchers and precisely assessing the gap between and among resources are quite a challenge. with the above-mentioned analysis of the four research resources, it is summarized that the current korean model should be named as electronic research resources. although research resources have been located and then directly or indirectly managed under their own circumstance, they have all been just electronically suitable for not only korean-speaking researchers but also english-speaking researchers via the internet. as long as only electronic research resources are used in korea, the field of research will surely lose important knowledge and information on disaster management. in other words, disaster management researchers may not efficiently use knowledge and information from electronic research resources due to the associated challenges. without the appropriate research results as their source, most researchers will not be able to produce better alternatives. therefore, electronic research resources must be changed into integrated research resources. integrated research resources mean that all research resources are not only electronically usable but also substantially used by various researchers without much difficulty. both korean-speaking researchers and english-speaking researchers have to re-evaluate the way they use integrated research resources. it has been known that any change in one research resource may influence the other research resources in many cases (luft, ) . thus, all researchers should initiate their drive within appropriate resources to start realizing appropriate changes. based on the initial change, those researchers need to work together to reach the apex of the targeted model. while thinking that disasters cause not only physical impact but also social impact to the human society, this article has examined critical problems and appropriate alternatives on disaster management in the viewpoint of impact assessment. to illustrate, this article has made efforts to provide theories on research resources, while also trying to mitigate or assess the physical impact and social impact of disasters. in summary, both electronic research resources and integrated research resources have been engaged under the context of impact assessment. fundamentally, both korean-speaking researchers and englishspeaking researchers should be aware of the negative aspects of electronic research resources or the necessity of integrated research resources. up to present, some researchers are not aware how the four resources are being used. in other words, they are not interested in examining the issue and, thus, work for their own research under a limited scope. specifically, without related awareness, they would not be willing to work for change. based on the high level of related awareness, all researchers need to plan the integration of disaster management research resources in advance (chang, wilkinson, brunsdon, seville, & potangaroa, ; jugend, araujo, pimenta, gobbo, & hilletofth, ) . by systematically planning how to overcome the status of superficially integrated resources or how to implement related countermeasures, they may flexibly tackle diverse research issues, as shown in table . while changing to integrated research resources, it is inevitable for the research field to coordinate thorny research issues. without voluntarily coordinating the stark differences among various researchers, the research field will be in a rut and unable to advance. also, it is quite necessary not only for english-speaking researchers but also for korean language researchers to give up their vested interests or privileges, if any, for the ultimate goal of disaster management. considering that disaster management is a public service, the korean governments, such as the ministry of the interior and safety (mois), should play an appropriate role in supporting the integration of research resources (hakaloba, mumba, dambe, & michelo, ) . by officially addressing the changing process via legalization, budget allocation, tax reliefs, international relations, and others, the mois may contribute to the sustainability of resource integration. furthermore, the mois may develop its research management section as a formal international organization and then facilitate it to work for integrated research resources with the full participation of both korean-speaking researchers and english-speaking researchers. in doing so, the mois may create a series of working committees between and among countries. as they approach integrated research resources, all korean-speaking researchers and english-speaking researchers should be allowed to ask questions at anytime (armstrong, ) . many english-speaking researchers have more frequently asked questions on their researches under the western culture than what korean-speaking researchers have performed under the confucian culture. with barriers in freely asking questions, the research field would not be able to sufficiently advance. alternatives toward integrated research resources. sources: (lee & kim, ; westerwick, kleinman, & knobloch-westerwick, ; yoon, ) . appropriate alternatives ① open research resources -all kinds of open research resources should be freely accessible to korean-speaking researchers as well as english-speaking researchers without any restriction, for the goal of distributing research results. ② blind research resources -with the support of iaem, korean-speaking researchers must understand the status of emi's isp as a fundamental research resource. also, the emi (or english-speaking researchers) may add the characteristics on korean disaster management or its research resources into isp. ③ hidden research resources -to reduce the extent of language barrier between korean-speaking researchers and english-speaking researchers, the field of research must operate on diverse networks in multi-languages. ④ unknown research resources -bilingual (english language and korean language) researchers need to work on comparative perspectives between korean language research and english language research or joint international research. k. -m. ha evaluation and program planning ( ) accordingly, all answers need to be composed of appropriate knowledge and information. if those answers do not provide related information or data to those who ask questions or they just keep silent due to political reasons, those questions will just be meaningless. therefore, when there is a question from any researcher, there should be an answer for the goal of integrated research resources. without an answer, the embodiment of integrated research resources would not succeed. although a huge amount of information has been advanced with high speed, almost no one has ever been completely competent in understanding or interpreting them. diverse kinds of information have been produced in incremental pieces. hence, the free flow of information exchange has to be facilitated in the research field before jumping into integrated research resources. basing on the concept of open communication in the research field, various researchers must freely exchange appropriate research knowledge and information with others (little, ) . for the ultimate goal of disaster management, all researchers need to utilize interpersonal communication as well as twoway communication. the use of johari window has been an excellent technique, which makes individuals and groups better grasp their relationship with not only themselves but also others. on the effect of the johari window, it has provided a fair opportunity to see how the field of disaster management research resources has been operated. by assessing how the self and others are doing, the johari window has played a role in positively supporting the field of disaster management research. also, the johari perspective may be considered as a cornerstone in evaluating the level of korean disaster management research, particularly by equally investigating all important areas/aspects. similarly, as the realization of disaster management research is much related to either the success or failure of disaster management, the johari window is regarded as a supervision tool, as well as a selfassessment tool for disaster management research resources (halpern, ) . while comprehensively overseeing the big picture of scientific research, the field of disaster management can efficiently use the johari window in supervising research processes. it is known that the johari perspective has provided new research opportunities for many past researchers (giarratano, savage, barcelona-demendoza, & harville, ) . on this point, appropriate researchers may meet with new research agenda on disaster management research, with the support of this article. in other words, when considering that the johari window has shown both the resilience and vulnerability of disaster management research in korea, related researchers may figure out much more research agenda. ultimately, the general direction of korean disaster management research will be able to free itself from fragmentation. if research resources will remain as only electronically or superficially available, related researches will just be fragmented. however, when the integrated research resources are addressed, related researches will be carried out in a more holistic way (waring, ) . as a result, researches in the future will have a more comprehensive quality. on top of doing impactful assessment, the purpose of this article is to assess how efficiently the korean field of disaster management research resources should be improved to contribute to the ultimate goal of disaster management. considering that both electronic research resources and integrated research resources have been systematically drawn via the johari perspective, this article has been successful in achieving its original goal. the biggest finding is that not only korean-speaking researchers but also english-speaking researchers must exert efforts to change electronic research resources into integrated research resources. for its implementation, all four research resources, namely, open, blind, hidden, and unknown researches, have to carry out their own assignment, such as distributing open research data, approaching the emi's isp as a basic research resource, enhancing diverse networks in multi-languages, and encouraging comparative perspectives and joint international researches. while reflecting on the fact that no rigorous study has ever been attempted yet to analyze the korean field of disaster management research resources via the johari perspective, this article has many advantages as a pioneer study. at the same time, this study has illustrated that both korean-and english-speaking researchers can benefit from using the johari perspective. moreover, it has shown the problems and the alternatives surrounding research resources in the field of disaster management in korea. this article has directly indicated the need for diverse studies in the future. for example, it will be necessary for korean-speaking researchers and english-speaking researchers to further delve into their assigned responsibilities regarding open, blind, hidden, and unknown research resources. in addition, researchers are encouraged to apply the johari perspective to their own case studies. then, they may eventually compare or contrast their results with a korean case or among themselves. the author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. towards multi-agency sensor information 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management and public policy key: cord- - m iozj authors: yang, hyeonchae; jung, woo-sung title: structural efficiency to manipulate public research institution networks date: - - journal: technol forecast soc change doi: . /j.techfore. . . sha: doc_id: cord_uid: m iozj with the rising use of network analysis in the public sector, researchers have recently begun paying more attention to the management of entities from a network perspective. however, guiding elements in a network is difficult because of their complex and dynamic states. in a bid to address the issues involved in achieving network-wide outcomes, our work here sheds new light on quantifying structural efficiency to control inter-organizational networks maintained by public research institutions. in doing so, we draw attention to the set of subordinates suitable as change initiators to influence the entire research profiles of subordinates from three major public research institutions: the government-funded research institutes (gris) in korea, the max-planck-gesellschaft (mpg) in germany, and the national laboratories (nls) in the united states. building networks on research similarities in portfolios, we investigate these networks with respect to their structural efficiency and topological properties. according to our estimation, only less than % of nodes are sufficient to initiate a cascade of changes throughout the network across institutions. the subunits that drive the network exhibit an inclination neither toward retaining a large number of connections nor toward having a long academic history. our findings suggest that this structural efficiency indicator helps assess structural development or improvement plans for networks inside a multiunit public research institution. public research more inclines to distribute its findings than commercialize in contrast to industrial research (geffen and judd, ) . in general, institutes conducting public research are largely government funded and target the public domain (bozeman, ) . because of their national orientation and stable funding source, public research institutes do cutting-edge research at least one academic field through long-term plans (greater than three years) (bozeman, ) . a public research institution often develops as an association of research institutes rather than a single organization. research entities with a public research institution enjoy institutional autonomy in choice of subjects notwithstanding the fact that they are under the same umbrella of governance. naturally, research organizations have different characteristics depending on national circumstances. some public research institutions, such as the max planck gesellschaft (mpg) in germany, are faithful to pure research (philipps, ) , while others have significance within a particular national context: part of the national laboratories (nls) in the united states (us) addresses defense-related technologies (jaffe and lerner, ) , and the government-funded research institutes (gris) in korea attempt to assist in the country's economic development by promoting indigenous public research (mazzoleni and nelson, ; arnold, ; lee, ) . with recent advances in our understanding of network, it is possible to apply novel network knowledge to manage public research institutions in response to internal and external changes. for example, entities in national innovation systems (freeman, ) or the triple helix models (phillips, ; leydesdorff, ) can be external factors affecting research of public research institutions. the notion of national innovation systems provides a framework to explain underlying incentive structures for technological development at a national level and international differences in competence from a network perspective of public and private organizations (patel and pavitt, ) . the triple helix model considers coevolving academic, industry, and government which provokes techno-economic developments of a country (leydesdorff et al., ) . in these systems, public research institutes provide fiscal and technical assistance to other organizations. kondo (kondo, ) pointed out that public research institutes dedicated to transferring technologies to industry by means of consulting, licensing, and spinning off. by doing so, they contribute to promoting integration and coordination within the system (provan and milward, ) . in order to formulate policies and procedures to steer the entire system, system organizers are able to guide public research institutes properly. in this context, control of those key agencies is important to achieving desirable outcomes. moreover, there is a growing need for an efficient implementation throughout public research institutions composed of multiple sub-organizations in order to deal with internal controls (yang and jung, ) . for example, most public research institutions have undergone transformations in recent years due to modernization, imperatives for efficiency, and the promotion of collaboration with the industry (buenstorf, ; cohen et al., ; simpson, ; senker, ) . in unfavorable economic conditions, declining government funding causes the restructuring of research areas (malakoff, ; izsak et al., ) or the government demands more practical outputs from them, such as conducting applied research and setting standards (oecd, ) . in an attempt to harness technology for socio-economic development, governments often prioritize future research through foresight activities (priedhorsky and hill, ) and accordingly assign new academic missions to public research institutions. in particular, developing countries have lately been paying more attention to the technology-driven development model under government supervision (arnold, ) . at that time, controlling every entity enables the institution to fully guide those internal changes but entails great expense. from to , public research institutions engaged in national strategic areas, including exploration of mineral resources, industrial development, and military research and development (r&d) (oecd, ) . after the termination of world war ii, the establishment of public research institutions grew in an effort to advance military technology in many countries. moreover, at that time, public research institutions extended almost all areas with which governments were associated, such as economic and social issues. they continued growing until the s. in the s and s, many countries expressed doubts on their contributions to innovation. however, as deepening the understanding of national innovation systems or the triple helix models, public research institutions started to be seen in a new light. in these models, public research institutions have played an indispensable role in preventing systemic failures, which reduce the overall efficiency of r&d (lundvall, ; sharif, ) due to their relations with external collaborators (klijn and koppenjan, ; mcguire, ) . still, the importance of public research institutions are emphasized in particular for scientific innovation (cabanelas et al., ) . in this regard, a network approach is necessary to efficiently implement transformations throughout sub-organizations, and the academic interest also grows for the effective operation of the network (cabanelas et al., ; jiang, ) . there is, however, a lack of empirical research on managing public research institutions through a network system. hence, in this paper, we conceptualize three major public research institutionsthe mpg, nls in the us, and gris in koreaas networks, identify the sub-organizational network structure of each, and examine its structural efficiency. a collaborative research network is one of the most prevalent inter-organizational configurations (shapiro, ) . however, we deem that topical similarity between research institutes is suitable to represent a relation between them in research interests. most transformations involve changes in research areas, and changes in organizational research topics frequently occur when governments prioritize specific research fields or delegate new roles to institute (wang and hicks, ) . prior studies emphasized the importance of similarity in knowledge content among entities to effectively manage inter-organizational networks as well (tsai, ; hansen, ) . for these reasons, a network here is formed by pairs of subunits having the most similar research profiles. with the addition of temporal dynamics to inter-organizational relations, a chain of networks over time allows the description of the structural evolution of public research institutions. based on revealed networks, we determined the structural efficiency with which network-wide actions can influence entities for finite time periods. no matter the measure puts in place, all members of network need to adopt it to achieve collective actions. in the early stages of change implementation, network organizers select initiators to change among entities. as the change initiators propagate control actions to the remainder of entities, a public research network can be steered in the desired direction like a car. we can derive a minimum number of suitable initiators from a theory of "structural controllability" (yuan et al., ) . in the theory, change initiators refers to injection points of external energy used to steer the network, which are theoretically selected depending on network structure. in this process, structural efficiency is obtained by calculating the share of change initiators in the network: the lower the efficiency value, the smaller the number of entities the network manager is required to handle. therefore, by comparing efficiencies with structural properties over time, we can estimate network characteristics specific to institutions. in this study, we divided institutional research portfolios into six time periods based on scientific output over eighteen years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , and estimated structural efficiencies of research similarity networks. considering structural efficiency, we can observe that networks in all three research institutions can be managed with less than % of sub-organizations, and the values reflect the changes that have occurred in research institutions. each research institution has some suborganizations consistently selected as suitable change initiators over a period of time. our results primarily highlighted young subordinates as appropriate change initiators, which means that information blockades in network might occur unless the selected units are properly managed. moreover, the estimated changes initiators tend to have a lower connectivity in network than the rest of nodes. we expect that our work has implications for decision-making bodies and network managers seeking to an efficient way to influence their intention on a network of public research institutes. the remainder of this paper is structured as follows: in section , we briefly describe the impact of structure on network effectiveness associated with public research institutions based on past research. section is devoted to an explanation of data sources, network construction processes, and the calculation of structural controllability in a network. we discuss the results of our experiments in sections and , and offer our conclusions in section . methods for utilization and development of networks have grown in an attempt to address complex problems that require collective effort. when the purpose of the network is to deliver public services, independent organizations are generally involved in the process, and interdependency between participants facilitates the formation of links (kickert et al., ) . by exchanging knowledge through a network, public research organizations attain a higher level of performance, at the same time, create a greater ability to innovate (morillo et al., ) . goldsmith and eggers ( ) claimed that using a vehicle for networks is favorable to organizations that require flexibility, rapidly changing technology, and diverse skills because actors can exchange goals, information, and resources while interacting with each other. resources usually refer to units of transposable value, such as money, materials, and customers, and information signifies exchangeable units between agencies, such as reports, discussions, and meetings. with regard to exchanged goods between organizations, van de ven (van de ven, ) underlined the importance of information and resources as "the basic elements of activity in organized forms of behavior." in research systems, organizations can take advantage of network participation to have a greater possibility of funding, to broaden their research spectrum, or to reduce the risk of failure (beaver, ) . therefore, networks are beneficial because they can pool resources, permit the mutual exploration of opportunities, and create new knowledge (priedhorsky and hill, ) . however, strategies are needed to coordinate interactions while managing networks because different actors have different goals and preferences concerning a given problem (kickert et al., ; o'mahony and ferraro, ) . the capability of network management is also necessary to promote innovations (pittaway et al., ) , but there remain questions as to how to manage such organizational interactions as beaver (beaver, ) pointed out. orchestrating activities seems unnecessary because of interactions between autonomous organizations, but addressing conflicts keeps agencies cooperative in effort to achieve the goal of the network, thereby facilitating the effective allocation and efficient utilization of network resources. furthermore, a network sometimes needs to be intentionally formed to boost management by governing parties which would be either an external organization or network participant(s) (provan and kenis, ) . public research institutions can be said to be governed by external organizations, considering that different entities are in charge of their administration in general, such as ministries, research councils, and other steering bodies. both the mpg and korean gris are apparently steered by a single entity. the fundamental management policy of the nls in the us also originates in a federal agency, although several laboratories are operated by contract partners. by frequently repeating interactions among actors, networks produce certain outcomes. the performance of a network is evaluated according to whether the network effectively attains its goal. the outcome varies depending on governing strategies, and the course of attainment can be enhanced by taking advantage of structural properties of the network (kickert et al., ; goldsmith and eggers, ) . provan and milward ( ) argued that the assessment of network effectiveness should involve consideration not only of beneficiaries, but also of administrative entities and the participants of the network. nevertheless, the literature on networks has paid more attention to the evaluation of their effectiveness by treating networks as a whole, such that the common goal is primarily involved in network-level accomplishment (provan and milward, ; möller and rajala, ) . there remain difficulties in determining network effectiveness. the problem primarily resides in the impossibility of quantifying the exact network outcome (provan and lemaire, ) . as agranoff ( ) claimed, networks are not always directly related to policy adjustments because some interactions are forged by voluntary information exchange or educational service. in the public research institution, researchers engaged in specialized fields have the opportunity to share ideas across administrative boundaries given that they have the goal and intend to generate public knowledge. outcomes of research networks can be approximated by proxy variables, such as patent and paper citations, innovation counts, new product sales, and productivity growth (council, ) . furthermore, such networks also indirectly affect subsequent movements and policies. thus, network efficiency needs to be measured for various types of networks, by considering factors beyond collaborations. in order to increase network effectiveness, structural efficiency in networks is important: since all entities are connected, damage to one part can cause the collapse of the entire system through a cascade of failures. in this regard, considerable research on networks has focused on deliberately building efficiently manageable networks (cabanelas et al., ; kickert et al., ; van de ven, ; provan and kenis, ) . certain network structures can affect innovation performance by catalyzing knowledge exchange (valero, ) . enemark et al. ( ) argued the importance of the network structure to collective actions via experimental tests that demonstrated structural variations in a network can either improve or degrade network outcomes. however, there is ambiguity in appropriate network structures to achieve effective control. pittaway et al. ( ) suggested that longitudinal network dynamics need to be taken into account when designing network topologies. a network is required to change its members or structures in order to adapt to environmental changes. much of the literature on networks emphasized that instability is an opportunity for transformation (hicklin, ) . although the capability of flexible response is one of the strongest features within a network model, such network dynamics challenge for effectively managing networks. with regard to network size, it is widely known that the greater number of actors involved, the more difficult it becomes for the network to achieve collective cooperation (kickert et al., ) . increasing the number of participants results in more complex network governance because the number of potential interactions also exponentially escalates. however, prior research found that research networks evolved to be more centralized as growing the network (ferligoj et al., ; hanaki et al., ) . the growing patterns of research networks imply that adding an entity does not always increase complexity of network management. theorists rather claimed that the introduction of a new node would improve efficiency to control networks (klijn and koppenjan, ) . centralization captures the extent of inequality with which important nodes are distributed across the network, and is often measured in terms of freeman's centralities (freeman et al., ) . a degree (the number of connections) centralized network is known to readily coordinate across agencies and closely monitor services (provan and milward, ) . in complex networks, a minority of nodes, referred to as hubs, dominates connections while the majority is connected with a small number of points (barabási and albert, ) . research revealed that complex networks were robust against random attacks (albert et al., ) . hubs in research networks were not only empirically impressive in their performance (echols and tsai, ; dhanarag and parkhe, ) , but also easy to access new knowledge developed by other entities (tsai, ) . hanaki, nakajima and ogura (hanaki et al., ) also found r&d collaboration networks evolved toward more centralized structures because organizations prefer to collaborate with reliable partners based on referrals obtained from former partners. however, a high degree of integration is not always desirable. provan and lemaire ( ) proposed that connective intensity between organizations should be appropriately controlled for effective network structure. cabanelas et al. ( ) also found research networks producing high performance featured nodes with low degree centrality. no matter the types of networks that develop out of interactions, the goal achievement is possible only when the relevant information spreads throughout the network to encourage actors to conform. in recent years for public research institutions, the controllability of organizational portfolios has been seen as constitutive of dynamic capabilities, which means the "ability to integrate, build, and reconfigure internal and external competencies to address rapidly changing environments" (teece et al., ; floricel and ibanescu, ) . in this sense, estimating efforts to control entities of public research institutions is related to assessing the feasibility of research reorganization over networks. at the same time, the number of key points in information flow within a network affects burden on the network administration. although earlier work emphasized that selectively activating critical actors is more effective to integration than full activation, the system must secure the capability to exercise influence across agencies (kickert et al., ; provan and lemaire, ) . furthermore, the efficiency with which network structure can be manipulated would be a suitable criterion to evaluate the built structure. this section is devoted to describing methods of network construction based on collected bibliographies and analytical methods. we describe a quantification method for structural efficiency given structure to control the whole network, and explain structural properties to explore their relation with structural efficiency. in the process of efficiency calculation, we extract suitable organizations to initiate transformation. this investigation was conducted in the r ver. . . environment (r core team, ) , and used the following add-on packages for convenience: ggplot (wickham, ) and igraph (csardi and nepusz, ) . we identified research portfolios based on scientific output, and gathered bibliographic data regarding nls, mpg, and gris from the thomson reuters web of knowledge. academic output over eighteen years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) was compiled according to institutional names and abbreviations of authors' affiliations. we only used affiliations in english for this study. subordinate research institutes listed in official websites were considered, and their portfolios were tracked using at least twenty papers for each. all disciplines, which are the constituent elements of a portfolio, need to be identified using the same classification system for ease of institutional comparison. we utilize the university of california-san diego's (ucsd) map of science (borner et al., ) as a journal-level classification system. the map classified documents into subdisciplines belonging to disciplines on the basis of journal titles. naturally, a research portfolio has two levels of classification: discipline and sub-discipline. particularly, a discipline refers to the aggregate level of sub-disciplines in the hierarchical structure in this study. fig. shows an example of disciplinary mapping using sci (sci team, ) in order to analyze the thematic evolution of network over time, we split the portfolios into time intervals. with regard to an adequate duration of assessment period to represent scientific output being measured, abramo et al. ( ) claimed that a three-year period is adequate to assess scientific outputs. by accepting their recommendations, we observed the development of institutional portfolios for six consecutive time slices. as a well-known analytical method, a complex network is suitable for exploring dynamic topology changes (strogatz, ) . here, an inter-organizational network is formed between subordinate institutes building similar research profiles. representing sub-organizations, nodes are connected by a link when two sub-organization have similar research portfolios. in order to measure similarities, we used "inverse frequency factors" for weighting system and "second-order cosine similarities" (garcía et al., ) . the inverse frequency factor borrows from a term discrimination method for text retrieval (salton and yang, ; salton and buckley, ) . the factors weight each subdiscipline in the research portfolio. the weight of sub-discipline m for research institute i is determined by w m;i ¼ f m;i  logð n nm Þ where f denotes the number of articles; and ð n nm Þ implies the inverse frequency factor to file out prevalent research (jones, ) . the logarithmic frequency factor is calculated inversely from the ratio number of subunits n m that publish their achievements in sub-discipline m to the total number n of research institutes. as a result, the set of weights generates a sub-disciplines-by-institute matrix. the similarities between two institutional research portfolios primarily take the cosine measure (salton and mcgill, ; baeza-yates and ribeiro-neto, ) . for the purpose of improving the accuracy of similarity, we applied second-order approaches to the sub-disciplineby-institute matrix. colliander and ahlgren ( ) explained that first-order approaches directly reflect the similarity between only two profiles, whereas second-order similarities determine those between two given portfolios and other institutional portfolios. a large number of studies have confirmed the superior performance of the secondorder approach as well (ahlgren and colliander, ; thijs et al., ) . moreover, to render easier structural analysis and network visualization, we strip weak similarities from the research similarity matrix. using the maximum spanning tree (mst) algorithm (kruskal, ) , we extracted tree like-structures. the mst algorithm ensures that all institutes are connected with maximal similarity, which implies that the institutes are connected through the most relevant links. therefore, a linked pair of institutes indicates greater potential for common intellectual foundations. among various well-known algorithms to detect mst, the backbone of thematic networks is derived by prim's algorithm (prim, ) . in order that the network structure can efficiently elicit the desired response from its elements, a certain amount of energy needs to be injected into the network to change the behavior of actors. thus, the selection of several agencies to initiate changes depending on network structure is inevitable. at the same time, it is important to minimize the number of injection points due to management cost. studies on complex networks consider that nodes can dynamically make decisions or can dynamically change their states by responding to information received through links between nodes. as individual actors, nodes on research networks can be researchers or research institutions, and the nodal states can be represented by individual research interests or disciplinary composition. here, we estimate the capability that controls the behavior of such nodes in complex networks with the minimum involvement of intervention adopting the notion of structural controllability. in recent years, a number of studies have focused on driving networks to a predefined state by combining control theory and network science (liu et al., ; wang et al., ; lombardi and hörnquist, ; gu et al., ) . according to network controllability, if a network system is controllable by imposing external signals on a subset of its nodes, called driver nodes, the system can be effectively driven from any initial state to the desired final state in finite time (kalman, ; lin, ) . thus, network controllability depends on the number and the placement of the control inputs. for this reason, structural efficiency refers to the share of the driver nodes. in this study, agencies found using structural controllability are key locations to steer the entire inter-organizational research network. we applied the structural controllability for undirected networks, introduced by yuan et al. ( ) , to matrix representation of our temporal msts. each temporal matrix g(a) was considered a linear timeinvariant model _ xðtÞ ¼ axðtÞ, where the vector x ∈ ℝ n represents the state of the nodes at time t, a∈ ℝ n×n denotes the research similarity matrix of mst, such that the value a i,j is the portfolio similarity between institutes i and j (a ij = a ji ). the controlled network g(a, b) corresponds to adding m controllers using ordinary differential equations _ xðtÞ ¼ axðtÞ þ buðtÞ, where vector u(t)∈ ℝ m is the controller and b ∈ ℝ n×m is a control matrix. the problem of finding the driver nodes of the system is solved by the exact controllability theory following the popov-belevitch-hautus (pbh) rank condition (hautus, ) . to ensure complete control, the control matrix b should satisfy rank[λ m i n − a, b]=n, where i n is the identity matrix of dimension n, and λ m denotes the maximum geometric multiplicity μ(λ l ) (=n-rank(λ l i n − a)) for the distinct eigenvalues λ l of a. therefore, from a theoretical perspective, changes initiated from the drivers are likely to affect the entire structure. hence, driver institutes are crucial to the functioning of networks for public research institutes. in this paper, we regard the share of drivers in all agencies as an efficiency indicator in that the number of drivers is important for efficient control. network properties have been utilized by a considerable amount of literature in the area to better understand structural features of networks (newman, ; albert and barabasi, ; woo-young and park, ) . in order to understand the relation between efficiency and the inter-organizational research network, we extracted major features across institutions based on some structural properties, such as network size and connectivity. the number of participants represents network size associated with network volume. centrality is one of the most studied indicators in network analysis, and measure the influence of a node in a network using degree centrality (freeman et al., ; borgatti et al., ; freeman, ) . we examine the degree feature of driver nodes. as a nodal attribute, we assign research experience in time periods to nodes to characterize driver nodes. this section contains the major results of our investigation of the structural features of the inter-organizational networks. to form our desired skeletal network, we extracted pairs of academically close institutes based on portfolio similarities among their participants using the construction algorithm of the maximum spanning tree (mst). the results obtained from the backbone networks are related to structural controllability. in order to address the evolution of inter-organizational research, we assessed the structural features of temporal msts. figs. - show tree-like structures of institutions over time. each node represents a sub-organization, and its size is proportional to the total number of documents published. the colors filling the nodes were determined by the discipline in which the institute was found to be most productive. the portfolio similarities between pairs of linked institutes represented the weight on the network, and these weights affected the width of links as well. the descriptive statistics of portfolio similarity summarize and describe the distribution of the skeletal relationships between subordinates, as listed in table . for all institutions, we found that the distributions were biased toward high similarities between research portfolios. for the nls networks in the us, the overall greater averages and smaller standard deviations of portfolio similarities than the other two institutions indicated that most research units were seen as connected, with the smallest difference in their research areas. on the other hand, in case of the gris, the lowest values of average similarity signified that each unit had a distinct research portfolio. the largest standard deviation and the low values of kurtosis for most time periods also showed that their research similarities were the most widely distributed. in order to represent the dynamic characteristics of msts, their structural properties are listed in table . the number of nodes n increased and, accordingly, the number of links increased to n − following the definition of an mst in the context of a connected network. in spite of sparsity of the network, nodes having a relatively large number of links could be found in some institutions, in particular the oak ridge national laboratory (ornl) within the nls, which was connected to approximately a quarter of the other organizations for four time periods, and the korea research institute of standards and science (kriss) and the korea institute of science and technology (kist), which appeared as a maximally connected node in each half of the dataset. however, there was stiff competition among institutes with the maximum number of connections in the mpg. we noted that network density ( /n) could be obtained from the number of nodes, and the transitivity always reduced to zero because mst rules out cycles. we calculated a periodical change of structural efficiency, and then examined the relations between network efficiency and structural properties. following this, we investigated the features of estimated driver nodes in terms of degree and period of appearance. note that although the number of driver nodes is theoretically fixed in a network, there can be multiple sets of drivers (jia and barabasi, ) . we randomly selected a set where multiple driver sets existed. as an indicator of network efficiency obtained from structural controllability, fig. denotes the share of driver nodes over time. according to the graph in the figure, the proportion of drivers varied, but institutions did not have to consider all their agencies for network-wide transformation. less than % of nodes were selected as suitable points at which to inject external information in all three institutions because the maximum value of structural efficiency in the entire datasets was about % at the second period ( ) ( ) ( ) in the gris. in particular, the nls could be influenced with a relatively small share of drivers among the institutions at all times, which the exception of the period - , whereas in the gris, the largest portion of nodes mostly needed to initiate changes. the efficiency fluctuation of the mpg was more stable than other two institutions over time periods. an understating of drivers enables administrators to take preemptive action to prevent information isolation, like the knowledge of the relation between the share of drivers and network efficiency can help plan structural development. the total number of driver appearances for the entire period corresponded to , , and for the nls, the gris, and mpg, respectively but , , and agencies were selected as drivers. this was an evidence for the existence of memory in the drivers. moreover, figs. and capture some features of the drivers. fig. compares the average number of links between drivers and the entire nodes over different periods. despite the common knowledge that nodes possessing large connectivity are influential, our results showed that drivers with low connectivity tended to determine collective agreement on the network. fig. shows the average durations of appearance by institutional drivers. we see that the driver nodes were the ones newly entered to the network based on the average durations. of the institutions, the research units of the nls showed a wide difference between drivers and non-drivers. public research has contributed to major innovations by improving competitiveness among existing industries and developing new ones. as prominent contributors to public research, governments have implemented a variety of support policies and programs for higher efficiency and excellence. among the actors involved in public research, public research institutions aim to disseminate their knowledge, by providing various functions: priority-driven research to address national and academic agendas or blue skies research engaging large-scale research facilities to complement university research (pot and reale, ) . to maintain such diversity, public research institutions seek to coordinate elements with varying specializations and missions in adapting to dynamic technological environments. as a part of the effort, institutions occasionally attempt to restructure research portfolios or modify organizational placements in relation with other research units. in order to assess the development of public research institutions, we examined structural evolution derived from research similarities in the context of networked organizations in this paper. more precisely, this study focused on public research institutions composed of several specialized research units, and extracted a network from similarities between sub-organizational research portfolios over eighteen years. a pair of connected agencies would be most influenced by the same type of exertion on a specific research area. in addition, suborganizations connected to each other can be potential partners to collaborate because they share similar academic backgrounds. for example, the similarity networks of the gris give implications for inter-disciplinary research groups operated by the research council. in the research group, researchers working at different gris seek a solution together to technological difficulties and research similarities can indicate proper gris to resolve the difficulties. moreover, offering the advantage of predictable network controllability, network modeling helps to understand the system's entire dynamics, which could be guided in finite time by controlling the initiators (liu et al., ) . as a result of the modeling, we can measure the efficiency of the network, where network efficiency implied the proportion of elements required as initiators to change the states of the entire agencies. the lower the proportion, the greater the network efficiency because the initiators are injection points for external information. we also revealed the structural properties of estimated initiators. our research here is different from other studies concerning network effectiveness in that it quantitatively estimated the effort required to control an entire inter-organizational network based on its structure. naturally, if we send control signals to every single node, the network is operated with high controllability but involves significant cost. thus, by employing the concept of structural controllability, we can theoretically detect the initial spreaders of information that need to be properly treated. otherwise, they would have produced barriers to the exertion of authority; in extreme cases, the information blockades could have caused network failure (klijn and koppenjan, ) . however, handling these elements incurs extra cost, because of which it is important to build networks with the minimum possible number of initiators to reduce enforcement costs incurred for complete control (egerstedt, ) . common structural features of estimated initiators can direct network management of public research institutions. we generated results to provide a clear idea of how structural efficiency of research network is related to structural properties, such as size and nodal degree. previous work on network governance structures has provided recommendations on how to build and design inter-organizational networks for innovation acceleration. for example, related to the number of participants, it is natural to expect that the share of drivers would also increase owing to a higher risk of insularity in information due to increasing structural complexity. however, our findings suggest not necessarily complying with the idea. each of the institutions considered by us was different in size from others: the mpg was the largest-scale organization, whereas the nls formed the smallest group in terms of number. however, according to our results, the size of the network did not seem to meaningfully affect table network properties of inter-organizational network. time span - - - - - - the proportion of drivers in public research institutions. despite being a medium-sized institution, the networks of the gris were more likely to be inefficient than those of the mpg and the nls. we think this was because an institution more experienced with managing such a union has built more effective structures. even we found that the gris took advantage of the structural reorganization of the network because additions improved their network efficiency. in this regard, kickert et al. ( ) claimed that the introduction of new actors can be a strategy to accomplish a mutual adjustment, since the new institute would cause structural changes within the network. proposition . a subset of nodes positioned in structurally important locations will have ability to steer a whole network of public research institution. our findings indicated that control actions applied to only less than half of the research units can lead to changes of an entire system, and the units repeatedly appear over time. we suspect the reason that public research institutions are designed to be a cost effective and resilient, as do their infrastructure networks. however, as national research structures can be affected by government policies (hossain et al., ) , the network efficiency also changes over time. the drastic fluctuation in the share of drivers would be related to changes in the relevant institution's strategy or operation. for example, the gris underwent a restructuring to remove redundancy, and began operating under the research councils after . we can capture drastic changes at the same time in our results because their structural efficiency significantly increased between the second and third periods between and . the results would imply that the organizational rearrangements in the gris worked well. besides, the research subjects of the nls were revamped in the - period due to several events, i.e. the september attacks and the outbreak of the severe acute respiratory syndrome (sars). since the terrorist attacks of september , , the nls have made greater efforts to reinforce national security by working on nuclear weapons or intelligent detection of potentially dangerous events. moreover, a sudden epidemic of sars accelerated multidisciplinary research in the nls on vaccines, therapeutics, bioinformatics, or bioterrorism. we also find that the structural efficiency of the nls were severely affected during the readjustment period. these changes in portfolio composition would cause temporary disarray in the structure of the networks. on the other hand, the property of stable fluctuations in the mpg would be attributable to internal transitions for scientific advances rather than external impact. the mpg makes an expansion of research topics by mostly spinning off units because each unit has its own research area. proposition . variations in structural efficiency of research networks will reflect structural changes in research composition. another difference between past research and our work here is that degree centralization is not invariably recommendable. policy makers and network scientists have hitherto paid attention to highly connected institutes because hubs are regarded as network facilitators. however, our findings indicated that most key elements were apt to have low degrees. our study focused on revealing the injection points to infuse their nearest neighbors with energy regardless of the amount of energy required, and the nodes impart directions to connected neighbors at a time rather than exuding control forces over their adjacencies simultaneously. obviously, the energy-entering hub can effectively reach agencies within its orbit, but there is a diffusion range. thus, our observations suggest that network-wide influence was dependent upon nodes with a low connectivity. in this context, a network with moderately distributed focal points can be more effective to influence all organizations than a thoroughly concentrated one. furthermore, emergent sub-organizations show a tendency to have greater effect on structural efficiency than sub-organizations with a long research experience. we suspect that this is why a new research institute is often derived from a larger unit in public research institutions, holds low research similarity with other units than its parent, and takes position at the border, beyond any energy ranges. another possible is that that a newly-established research institute has unstable research portfolios, as braam and van den besselaar ( ) pointed out. the instability that a new research institute has in its research areas can increase uncertainty about the consequences of network-wide changes. therefore, network managers may have the need to monitor the degree of acceptance of a network-wide action especially among emerging sub-organizations. this result is also consistent with recent observations, whereby driver nodes in real-world networks tend to be reluctant to link with high-degree nodes (liu et al., ) . proposition . other things being equal, a possibility to control the whole research network will increase when control actions work properly at nodes with a low connectivity and a short research history. we consider the differences in network effectiveness between existing studies and our findings originate from: whether the complete functioning of all elements was considered. previous studies regarding the maximization of network effectiveness implicitly presupposed the complete performance of all entities a priori despite conflicts between participants, but at least network managers need to ensure complete operation of their network. for the full functioning of a network, all elements are required to be within the sphere of influence of the network manager for network-wide control. we deal with a possibility to manage the network behavior in public research institutions by quantifying the effort required to implement maneuvers. in order to avoid control blockades, we showed the importance of the elements, inter alia, with low connectivity and brief experience in academia. this study provided theoretical results for structural controllability assuming some ideal situations, such as that measures were implemented on network skeleton without redundant connectivity, sufficient resources were provided to change the network, all institutes respected the administrator's intention, and there were no conflicts between a pair of connected institutes. the success or failure of such measures can be determined once the processes are completely implemented because a network's dynamic nature in inter-organizational network raises difficulties in coordination. nevertheless, estimating the completion of network-wide objectives is still critical to network planning and design. our theoretical calculations here can assist decision making for structural improvement plans. moreover, the common features of the selected initiators are sufficient to suggest elements significant to attaining a synchronized response across and institutional network to reorganize research portfolio. public research institutions continue to gain prominence in the development national agendas of science and technology. institutions have their own strategies according to their values and interests in research trends to a greater or lesser extent. governments and research councils significantly affect these institutes through polices, programs, funding, and financial support in an effort to better coordinate their research agencies (rammer, ) . therefore, guiding the subunits of these institutes in a network is important to efficiently deliver managerial control. in doing so, administrators should be concerned with improving network structure to enhance its outcomes. however, manipulating network structure is difficult because of complex and dynamic states of the sub-organizations. in this study, we quantified network structural efficiency to maneuver a set of spontaneous elements into network-wide goals by using the theory of structural controllability (yuan et al., ) , and tracked the efficiency of networks of these public research institutions: the gris in korea, the nls in the us, and the mpg. for the relevant calculations, we extracted a hidden network structure from each institution based on similarities between the profiles of their subordinate organizations. the results of structural efficiency enabled the assessment of the operational strategies of each institution for eighteen years. the elements selected by structural controllability implied suitable points to inject external energy for governing networks. revealing the injection points was important to prevent information blockages that hinder collective action. apparently, the greater number of injection points required, the lesser the efficiency of network due to the increased burden of management. our findings indicate that structural efficiencies reflect changes in research interests of an institution. in this sense, research institutions have the necessity to track the structural controllability to assess their structural changes, such as portfolio adjustments on all of sub-organizations. the structural controllability can also provide the suitable spots for an intervention by a network manager (ministries, research councils, or steering bodies) as driver nodes with regard to structural changes. according to our results, the proper intervention points tend to be with a low connectivity as well as young suborganizations. in spite of these implications for managing strategies of interorganizational networks, this study has shortcomings that limit the generalizability of our findings. scientific articles represent only part of an institute's capacity for research. major scientific outputs are classified into two types: scientific articles and patents. depending on the major research types, some institutes concentrate on patents instead of publications. as a result, research portfolios derived from richer data sources than were used would more precisely depict institutional research capacity. another limitation of this study is that network properties other than those considered here, such as network density, clustering coefficient, and betweenness centrality, might affect structural efficiency like. furthermore, our findings raised several questions that suggest directions for future research. these include exploring the range 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resources in rtd establishing 'green regionalism': environmental technology generation across east asia and beyond emergence and development of the national innovation systems concept after the reforms: how have public science research organisations changed? r&d manag exploring complex networks dynamic capabilities and strategic management do second-order similarities provide added-value in a hybrid approach knowledge transfer in intraorganizational networks: effects of network position and absorptive capacity on business unit innovation and performance effective leadership in public organizations: the impact of organizational structure in asian countries on the nature, formation, and maintenance of relations among organizations detecting structural change in university research systems: a case study of british research policy optimizing controllability of complex networks by minimum structural perturbations the network structure of the korean blogosphere a strategic management approach for korean public research institutes based on bibliometric investigation exact controllability of complex networks hyeonchae yang is a ph.d. candidate of the graduate program for technology and innovation management at pohang university of science and technology the authors are grateful to the editors of the journal and the reviewers for their support and work throughout the process. this work is supported by mid-career researcher program through the national research foundation of korea (nrf) grant funded by the ministry of science, ict and future planning ( r a a a ). key: cord- - lfjfvs authors: hanney, stephen r.; kanya, lucy; pokhrel, subhash; jones, teresa h.; boaz, annette title: how to strengthen a health research system: who’s review, whose literature and who is providing leadership? date: - - journal: health res policy syst doi: . /s - - - sha: doc_id: cord_uid: lfjfvs background: health research is important for the achievement of the sustainable development goals. however, there are many challenges facing health research, including securing sufficient funds, building capacity, producing research findings and using both local and global evidence, and avoiding waste. a who initiative addressed these challenges by developing a conceptual framework with four functions to guide the development of national health research systems. despite some progress, more is needed before health research systems can meet their full potential of improving health systems. the who regional office for europe commissioned an evidence synthesis of the systems-level literature. this opinion piece considers its findings before reflecting on the vast additional literature available on the range of specific health research system functions related to the various challenges. finally, it considers who should lead research system strengthening. main text: the evidence synthesis identifies two main approaches for strengthening national health research systems, namely implementing comprehensive and coherent strategies and participation in partnerships. the literature describing these approaches at the systems level also provides data on ways to strengthen each of the four functions of governance, securing financing, capacity-building, and production and use of research. countries effectively implementing strategies include england, ireland and rwanda, whereas west africa experienced effective partnerships. recommended policy approaches for system strengthening are context specific. the vast literature on each function and the ever-growing evidence-base are illustrated by considering papers in just one key journal, health research policy and systems, and analysing the contribution of two national studies. a review of the functions of the iranian system identifies over relevant and mostly national records; an analysis of the creation of the english national institute for health research describes the key leadership role played by the health department. furthermore, who is playing leadership roles in helping coordinate partnerships within and across health research systems that have been attempting to tackle the covid- crisis. conclusions: the evidence synthesis provides a firm basis for decision-making by policy-makers and research leaders looking to strengthen national health research systems within their own national context. it identifies five crucial policy approaches — conducting situation analysis, sustaining a comprehensive strategy, engaging stakeholders, evaluating impacts on health systems, and partnership participation. the vast and ever-growing additional literature could provide further perspectives, including on crucial leadership roles for health ministries. keywords: biomedical research, capacity-building, evidence-based practice, health ministries, health research systems, health services research, policy-making, priority-setting, research utilisation, sustainable development goals, translational medical research background interest in strengthening health research systems has intensified following increasing recognition of the importance of research in achieving key goals such as universal health coverage [ ] and the sustainable development goals (sdgs) [ ] . however, achieving progress in health research faces many challenges, including securing sufficient funds [ ] [ ] [ ] [ ] [ ] [ ] [ ] , building and retaining capacity [ , , [ ] [ ] [ ] [ ] [ ] , producing research findings, and using both local and global evidence [ , [ ] [ ] [ ] [ ] [ ] [ ] . chalmers and glasziou [ ] dramatically highlighted the extent of the challenges facing health research by claiming, in , that even where there was funding and capacity, up to % of all biomedical research was wasted because it asked the wrong questions, was poorly designed, or was either not published or poorly reported, with only about % of studies being published in full. many of these challenges have long been recognised and the adoption of a systems approach advocated. in , the bangkok declaration on health research for development promoted the importance of a systems approach, following consideration of how a health research system could "be integrated with a nation's health development plan" [ ] . it suggested that establishing and strengthening an effective health research system needed coherent and coordinated health research strategies [ ] . national strategies should have specific combinations of various health research system components, tailored to the country's circumstances. the who's knowledge for better health initiative involved further work on these issues [ , ] . the mexico statement on health research, issued in by a ministerial summit, called for nations to take actions to strengthen their national health research systems (nhrss). it was endorsed in by the fifty-eighth world health assembly in a resolution committing its member states to strengthening their nhrss as a pathway to improve their overall health system [ ] . as part of the initiative, pang et al. [ ] developed a conceptual framework to guide the analysis and strengthening of health research systems, including development of a health research strategy. while this can be used for planning, monitoring and evaluation of health research systems, it did not claim to provide a precise blueprint. the framework defined a health research system as "the people, institutions, and activities whose primary purpose in relation to research is to generate high-quality knowledge that can be used to promote, restore, and/or maintain the health status of populations; it should include the mechanisms adopted to encourage the utilization of research" [ ] . the framework indicates the range of constituent components and how they can best be brought together into a coherent system. it identified four main functions for an effective system, namely stewardship, financing, capacity-building (or creating and sustaining resources), and producing and using research [ ] . each function is defined by operational components and consists of one or more of a total of nine such components. since then, progress is evidenced by analyses of developments in individual countries, including the national institute for health research (nihr) in england [ ] [ ] [ ] , and in repeat surveys conducted in various who regions, including africa [ , , ] and the pan-american health organization (paho) [ ] . however, as reported by those surveys and other publications, many challenges remain. for example, in february a new analysis by the who global observatory on health r&d examined health research funding, concluding that "neglected diseases such as those on the who list of neglected tropical diseases remain very neglected in terms of r&d investments" [ ] . nevertheless, there are various initiatives underway, including in who's regional office for europe, which commissioned an evidence synthesis on the topic as part of its action plan to strengthen the use of evidence, information and research for policy-making in the who european region [ ] . the synthesis is published in the who region's health evidence network (hen) report series and consists of a scoping review addressing the question "what is the evidence on policies, interventions and tools for establishing and/or strengthening nhrss and their effectiveness?" [ ] . the evidence synthesis focuses on the systems level and so primarily includes publications taking a systems approach at either the national or multi-national level. not surprisingly, health research policy and systems (harps) is the single largest source of papers included in the hen report. these were papers directly identified in the review's search or papers included in the hen report to illustrate a key point because they had been cited in one of the who reports or other systems-level collations of papers included in the synthesis. while the system level papers did provide considerable data about each function, limited resources to conduct the scoping review meant that we had to exclude papers focusing solely on one specific function of a health research system or on just one field of health research. as acknowledged in the hen report's agenda for further research, there is a large number of publications (papers and grey literature) covering each function [ ] . therefore, reviewing all of these publications would be a major task but some exploration of the extent of the task, and the nature of such literature, could be informative. furthermore, additional papers are continuously emerging, including from the various initiatives that are ongoing or just underway, for example, the european health research network [ ] . the three sections of this paper sequentially address the question of how to strengthen a health research system by: . describing key points and conclusions from who's hen report. . illustrating the nature of the ever-widening literature available on each function, or component, of a health research system by examining two sources in particular. first, the full range of papers published in harps in the months up to february . second, the range of data gathered from publications or interviews that is included in detailed studies of the national health research systems in two countries -iran [ ] and england [ ] ; between them, these two papers also illustrate diverse aspects of the additional material that could be drawn upon. . considering a key question in the analysis of the current and future initiatives, namely who is going to steer the development of health research systems? here, information and insights from the hen about this sometimes-controversial issue, along with wider continuing analysis, are drawn on in the more flexible and speculative way that can be undertaken in an opinion piece compared to a formal evidence synthesis. who's review, whose literature and who is providing leadership? who's review the evidence synthesis described by the hen report [ ] starts by describing the importance of nhrss in helping to achieve universal health coverage [ ] and the sdgs [ ] . it goes on to analyse the challenges facing health research and describes how issues remain unresolved despite the development and application of a systems approach including who's framework for health research systems [ ] . many countries do not have comprehensive national health research policies or strategies that would facilitate the introduction of a systems approach. therefore, challenges remain around two key and overlapping sets of issues. first, how to develop a systems approach to maximise the benefits from the research resources availablethis can be a challenge even in high-income countries with considerable research funding. second, how best to strengthen each specific function and component of a health research system [ ] . the hen identifies two main systems-level approaches to strengthening nhrss. the first is comprehensive and coherent strategies, which can be contained in either policy documents, such as those from the english nihr [ ], the irish health research board (hrb) [ ] and the rwandan ministry of health [ ] , or in specific legislation as in the philippines [ ] . the second systems-level approach involves partnerships and multicountry initiatives, especially with international organisations. two initiatives from the west african health organization (waho) are particularly important examples [ , ] . here, the ministries of health of the west african member countries worked together in a joint initiative covering all the countries and with funding and expertise from a range of partners, including the council on health research for development (cohred), the canadian international research centre, the special programme for research and training in tropical diseases, and the wellcome trust. all who regions have seen multi-country activities by who and/or cohred to strengthen nhrss, including the repeat surveys that identify areas for action [ , , ] . then, broadly using the who framework as the structure [ ] , the hen identifies key points from systems-level literature on each of the four functions and nine components. the components of the stewardship and governance function include defining a vision, ethical review, research priority-setting, and appropriate monitoring and evaluation [ ] . consultation with health system stakeholders should enhance the relevance of the research priorities to the healthcare system, with examples of extensive priority-setting engagement activities sometimes being seen as a key aspect of building the nhrs as in brazil [ ] . evaluating the impact of research on policy and practice should help researchers to focus on achieving such impact and was therefore promoted in the world health report [ ] . securing finance can involve obtaining funding from sources within the country and from external donors or multi-national organisations [ ] . targets for research expenditure, such as the % of national health expenditure set by the commission on health research for development [ ] , can usefully be brought into health research system strategies as in rwanda [ ] . major health research strategies from countries within the european union can highlight the importance of european union funding as in france [ ] , ireland [ ] and malta [ ] . requests for funding can be more effective when linked to other parts of the overall strategy, including identified priorities that need supporting through donor funding [ ] and assessments of the benefits obtained from previous funding such as in england [ ] . capacity-building involves building, strengthening and sustaining the human and physical capacity to conduct, absorb and utilise health research [ ] . in , santoro et al. [ ] identified the generally low levels of research production in countries of the former soviet union and south-eastern europe and made recommendations for the sustained investment in training and career development of researchers, which should go beyond scholarships for training abroad and involve comprehensive strategies to ensure clear career structures. strategies such as that from inserm in france set out comprehensive plans for capacity-building [ ] and strategies in both england and south africa addressed priority gaps identified in the research capacity within the healthcare professions [ , ] . donors can play an important part in building capacity but, recognising the need to avoid donor domination, often do so through partnerships. these can take diverse forms ranging from multi-country initiatives, such as that by waho, which included an initiative focusing on the challenges of postconflict countries but was unable to meet all the needs [ ] , to accounts that focus on the partnership to address a broad range of capacity issues in a single country such as malawi [ ] , to partnerships between individual institutions. examples of the latter can feature particular challengesthe james cook university in australia worked with the atoifi adventist hospital in malaita, the most populous province of the solomon islands, to start establishing health research system capacity on the island using an inclusive, participatory approach [ ] . increasingly, there are also south-south partnerships, for example, an account of the panamanian health research system described how the country's first doctoral programme in biotechnology was established with support from acharya nagarjuna university in india [ ] . the rwandan strategy described plans to tackle the 'brain drain' through making the country an appealing place to conduct health research in terms of job requirements and providing opportunities for career advancement [ ] . the three mutually reinforcing components of the producing and using research function encourage the production of scientifically valid findings that are relevant for users and communicated to them in an effective manner [ ] . major research funding bodies increasingly seek to address the waste issues raised by chalmers and glasziou [ ] by working together in the ensuring value in research (evir) funders' collaboration and development forum. it issued a consensus statement committing the organisations signing it to "require robust research design, conduct and analysis" [ ] . the forum is convened by the english nihr, the netherlands organization for health research and development, and the patient-centered outcomes research institute (united states) with the active support of major research funding organisations from australia, ireland (hrb), italy, sweden and wales, plus the special programme for research and training in tropical diseases [ ] . the first waho intervention also worked to boost research publications, including by creating a regional peerreviewed, multilingual journal [ ] . how research is produced can increase the chance that the evidence will be used in the health system, for example, the english nihr strategy noted that leading medical centres with substantial funding to conduct translational research can act as "early adopters of new insights in technologies, techniques and treatments for improving health" [ ] . fostering the use of research requires specific knowledge translation and management approaches that draw on both locally produced and globally available evidence. various health research strategies promote the role of cochrane, including in england, where a unified knowledge management system to meet the needs of various stakeholders, including patients and their carers, involves funding both cochrane and a review centre focusing on the needs of the national health system [ ]. in ireland, the hrb strategy facilitated evidence-informed decisions through promoting access to the cochrane library and supporting training in conducting high-quality cochrane reviews [ ] . south africa cochrane featured as an important element in the nhrs [ ] . the rwandan strategy stated that "the government of rwanda is committed to using research findings to make evidencebased decisions that will improve health in rwanda" [ ] . it aimed to orientate various functions, including agenda-setting, monitoring and evaluation, and capacitybuilding, towards facilitating this challenging aim. the world health report highlighted various mechanisms that health research systems could adopt, including evipnet (evidence-informed policy network), to promote the use of research [ , ] . the review also considers the effectiveness of approaches to strengthening nhrss. several reviews identified the effectiveness of the comprehensive approach taken by professor dame sally davies in creating the english nhrs [ , , ] . the title of one analysis, 'nihr at : examples, themes, transformation', emphasises that the success of the nihr depended on a range of elements being brought together in one transformation [ , ] . one of the themes was the involvement of patients in decisions about research priorities and processes and, based on this, another recent analysis highlighted england and alberta (canada) as having health research systems that had made important progress [ ] . davies herself reflected on the success of the nihr and stated: "what we envisaged was integrating a health research system into the health care delivery system so that the two would become interdependent and synergistic" [ ] . who's regional office for africa drew on their series of surveys of the performance of countries in building nhrss and analysed the data from the and surveys using the nhrs barometer that they developed to score progress on a range of items linked to the list of nhrs functions [ , ] . in the survey, the rwandan system was identified as the best performing and it, along with the majority of systems, was reported to have further improved in the survey; by then, south africa was reported to have the best performance in africa. the surveys also illustrate how the multi-country approach makes a useful contribution to strengthening nhrss by helping to target action. furthermore, the waho interventions made some progress but, while the evaluations identified the importance of political will and leadership provided by waho's parent organisation of west african states, they also emphasised that building capacity for a whole nhrs is a significant task requiring commitment over the long-term [ , ] . the hen review collated a range of examples of tools for nhrs strengthening. these were identified from the systems level discussions of nhrs strategies and partnerships and/or the major reports calling for nhrs strengthening such as the world health report [ ] . the hen lists these in an annex [ ] . the discussion in the hen draws on the literature that was included to identify five key policies that those responsible for strengthening nhrss could consider [ ] , namely conduct context, or situational, analyses to inform strengthening activities [ , , , , [ ] [ ] [ ] , develop a comprehensive and coherent strategy [ , [ ] [ ] [ ] , engage stakeholders in the development and operation of the strategy [ , , , , , , , , [ ] [ ] [ ] [ ] [ ] , adopt monitoring and evaluation tools that focus on the objectives of the nhrs, including health improvement [ , , , , ] , and develop partnerships [ , , , , ] . examples of the evidence to support or illustrate each policy are given in table . in summary, therefore, this section shows that the who evidence synthesis, published as a hen report [ ] , provides a firm basis for decision-making by policy-makers and research leaders looking to strengthen the health research system in their country. it analyses, in turn, the individual functions and components within a system and identifies a series of tools that can be used for strengthening many of them. finally, this section highlights the five crucial policy approaches that the hen report suggests can be applied as appropriate to the context of the country (table ) . as noted above, the hen was a scoping review and focused on the literature at the systems level rather than on publications (papers and grey literature) related solely to specific functions, types or fields of research [ ] . therefore, there is scope for further work to incorporate an even wider range of publications than the included in the hen review [ ] . the discussion in the hen suggests that further research could usefully take the form of a series of reviews on the extensive literature on each of the nhrs functions or components, which could then be collated [ ] . just two of the many available sources illustrate the nature of the vast literature available on each function, or component, of a health research system and the way the literature on that, and the system level developments, is ever-widening. first, we can examine the papers published in harps, the specialist journal in the field of building nhrss. second, we can focus on two very different but detailed studies of individual nhrssone conducted for a phd thesis to show the year history of the development of all the functions in the iranian health research system [ ] and the other an interview-based study to understand the factors behind the creation of the nihr with its new strategy [ ] . in terms of further reviews of the literature on specific functions or components, harps would probably be a key source. in the summer of , an analysis by the retiring editors of the papers published in the journal from its inception in identified many papers that had been published on each of the functions or components of a health research system [ ] . while this editors' analysis was included in the hen review because it organised its discussion of the papers at the systems level, the individual papers in it were, in general, only included in the hen review if they, too, adopted a systems approach at the national or partnership level, or were also cited in a report such as the world health report [ ] . examples of such papers include viergever et al. on priority-setting [ ] , bates et al. on capacity-building [ ] , and lavis et al. on the support tools for evidence-informed policy-making [ ] . therefore, many additional papers related to specific functions (or fields) could be consulted, in a formal review or otherwise, in any future series of reviews, each with a narrow focus on strengthening a specific function. to further inform this current opinion piece, a quick 'hand-search' was conducted of the papers published in harps in the months since the previous analysis in mid- [ ] . this again identified a wide range of papers on specific components, especially priority-setting, evaluation of research impacts, capacity-building and the translation of research (or knowledge mobilisation). various papers linked the final two points and discussed capacity-building and knowledge translation [ , ] . such a focus is entirely consistent with the aim described by the incoming editors in autumn of bringing "all elements of the research-policy world togethersuch that the research which is done is useful and that it is used" [ ] . in this more recent phase of harps, there have also been important papers on issues related to the policies 'recommended' at the end of the hen and listed above, including the contribution of stakeholder engagement in research [ ] . the more recent papers could sometimes provide useful further tools on specific functions. their narrow focus meant they had not been directly included through the hen search and, further, they had not been included in any of the major reports also used as sources for tools such as the world health report [ ] . in some instances, this was because they were too recent, for example, the isria statement by adam et al. [ ] describing the ten-point guidelines for an effective process of research impact assessment prepared by the international school on research impact assessment (isria). even more recently, the intervention scalability assessment tool, developed by milat et al. [ ] , was proposed for use not only by health policy-makers and practitioners for selecting interventions to scale up but also to help design research to fill evidence gaps. this analysis of the papers from just one journal reinforces the message that there is likely to be a plentiful supply of literature for a future review on any of the main specific components. this message is further reinforced by a more detailed analysis of the papers in harps in the first months of . articles on the main components of a nhrs were supplemented by some important papers on topics that are highly relevant but which feature less frequently in harps. these include a study aimed at reducing the research waste that arises from disproportionate regulation by examining the practices for exempting low-risk research from ethics review in four high-income countries [ ] , the global observatory's paper on research funding described earlier [ ] , a study on the governance of national health research funding institutions [ ] , and one on a more recent topic of growing significancean analysis of attempts to boost gender equality in health research [ ] . additionally, some of the papers on specific components, such as impact evaluation or use of evidence, are extending the analysis. examples include consideration of how research impact assessments are conduct context or situational analyses of current national position to inform strengthening activities cohred, in particular, has developed tools to assist countries in conducting situational analyses as part of wider advice [ ] and this approach was an important element in the waho interventions being successful to the extent that they were [ , , , ] . strategies informed by analyses of their current situation include those for the english nihr [ ] and the irish hrb [ ] develop a comprehensive and coherent nhrs strategy comprehensive and coherent strategies with at least some degree of success (as seen in progress on some or all of the nihr functions) had set out how they intended to take action on the range of health research system functions and components, even if not necessarily explicitly using the who framework [ ] ; examples include the strategies for the english nihr [ ], the irish hrb [ ] , and in the philippines [ ] and rwanda [ ] engage stakeholders in the development and operation of the nhrs strategy strategy documents such as those for the nihr [ ] and hrb [ ] , plus ones in british columbia [ ] , malta [ ] and new zealand [ ] , describe the importance and/or range of stakeholders engaged in developing the strategy. articles describing the approach in south africa [ ] and zambia [ ] also highlighted the importance of wide stakeholder engagement. an analysis of stakeholder engagement in the creation and operation of the nihr identified it as making a key contribution to its success [ ] . there is increasing support for the engagement of stakeholders in setting the priorities for research as well as in research processes and translation [ , , , , ] adopt monitoring and evaluation tools that focus on the objectives of the nhrs, including health system improvement a range of documents, including ones on the nihr [ ] , hrb [ ] and rwandan strategies [ ] , and the world health report [ ] , demonstrate the importance of adopting monitoring and evaluation approaches that include a focus on assessing the impacts of research on health polices/practice and the economy, e.g. through application of the payback framework [ , ] develop/participate in partnerships across regions, bilaterally or within the nhrs examples of progress made by partnerships between countries, sometimes along with international organisations and donors, include the waho interventions [ , , , ] and the work of who regional offices for africa [ , ] implemented in practice within research organisations [ ] and how evidence is used in decision-making in crisis zones [ ] . to illustrate the volume of studies being produced, there has been a flurry of studies, in the first months of alone, on the collaboration and coproduction of health research. the titles include 'building an integrated knowledge translation (ikt) evidence base: colloquium proceedings and research direction' [ ] , 'using a 'rich picture' to facilitate systems thinking in research coproduction' [ ] , 'exploring the evolution of engagement between academic public health researchers and decision-makers: from initiation to dissolution' [ ] , 'research co-design in health: a rapid overview of reviews' [ ] , and 'conceptualising the initiation of researcher and research user partnerships: a metanarrative review' [ ] . finally, another article in may presented a new conceptual model for health research systems to strengthen health inequalities research [ ] . here, we have focused on just one journal, harps, because it was the largest single source of papers in the hen report, which totalled publications (additional publications were included to the in the review to help set the background, provide examples of key tools, etc). however, even with the review's focus on the system level, harps only provided % ( out of ) of the publications; % ( of ) came from other journals and % ( of ) were other types of publication. if the focus was shifted to including papers on specific functions it is highly likely that there would be a higher proportion of papers from other journals. the authors of two single-country papers on the development of the health research system, mansoori [ ] about iran and atkinson et al. [ ] on the creation of the nihr in england, both highlight the importance of context but also claim their findings could have wider application. examining these two papers is also informative because of the differences between the studies, including one being located in a low-or middle-income country, and the other not. mansoori's narrative review of studies addressing the health research system of iran included relevant and mostly national records, categorised using an approach informed by the functions and components of who's nhrs framework [ ] . the papers and grey literature documents included were all available in english or persian, and mostly published in journals other than harps, and illustrate the vast literature available at a global level on the various components of a nhrs. they informed an impressively detailed account of the various nhrs components and the attempts to strengthen them. for example, the account of the development of the national level ethical overview includes a fully documented chronology of the progress over years and some insightful analysis of how the progress was facilitated by the pivotal role of professor bagher larijani, who was a prominent medical practitioner, leading researcher and founder of the medical ethics research centre in iran. he was able to "use the confidence that iranian authorities had in him as an opportunity" [ ] . while mansoori's review was included in the hen review, only a tiny fraction of the available data about iran could be included, primarily in a brief description of the system's effectiveness [ ] . however, the full paper could usefully inform the approach of researchers and/or policy-makers planning a detailed analysis of their own nhrs prior to embarking on exercises to strengthen it, and "[t]he findings emphasized that improvement of hrs functions requires addressing context-specific problems" [ ] . as an illustration, mansoori's review identified a need for "a more systematic, inclusive" approach to research priority-setting [ ] and, in the same stream of research, she co-led just such a priority-setting exercise to help address the knowledge gaps related to achieving both iran's national health policies and the sdgs [ ] . atkinson et al. examined the creation of what might be viewed as the most successful attempt to strengthen a health research system in their paper ''all the stars were aligned'? the origins of england's national institute for health research' [ ] . compared with mansoori, the authors adopted a different but equally detailed approach in their analysis, which was conducted principally through interviews and a witness seminar but also drew on the existing literature and documents [ ] . they showed how the formation of the nihr was led from the department of health by a key group driven by sally davies. they aimed to improve patient care through both the strengthening of evidence-based medicine and through boosting the infrastructure to facilitate pharmaceutical clinical trials that would also meet wider industrial and economic goals. as with mansoori's study, consideration was given to how the full analysis could be informative to any planned detailed study or reforms in any other country. the key observations were similar to the recommendations from the hen report with a focus on stakeholder engagement and building support: "[t]wo measures likely to contribute to political support are to place the greatest emphasis on 'problem' rather than 'investigation' research, and to devote attention to measuring and reporting research 'payback'" [ ] . atkinson et al.'s paper is also a link to the other main source considered here because it was a recent paper published in harps. in summary, if further analysis and research beyond that in the who evidence synthesis [ ] is thought to be relevant in the particular country looking to strengthen its health research system, this opinion piece indicates some of the types of additional sources of information that are available and how they might be organised. the vast literature on each function and the ever-growing evidence base are illustrated by considering papers in just one key journal, harps, and analysing the contribution of two national studies. a review of the functions of the iranian system identifies over relevant, mostly national, records and an analysis of the creation of the english nihr describes the key leadership role played from the health department. who is providing leadership? the above analysis demonstrates that there is no shortage of useful material on which to draw when strengthening health research systems. however, key questions remain as to who might best lead or steer attempts to strengthen such a system. the papers by both mansoori [ ] and atkinson et al. [ ] illustrate that, where a key committed individual has the capacity and opportunity to provide leadership, this can be a vital element in making progress. however, the institutional factors are also crucial. the hen developed the argument that a department or ministry of health will have a particular interest and perhaps experience in promoting research agendas that meet the needs of the healthcare system and in helping to develop mechanisms to use the findings from such research, where appropriate, to inform local policy and practice [ ] . the health ministry or a research council responsible to it played an important role in the various systems identified above as being effective, as was also the case in the waho initiative [ ] . in some cases, as with zambia, more progress was made once the ministry of health elected to play a more important role, sometimes in place of other stakeholders [ ] . examples of the important role that health ministries can play were described in the world health report, including on paraguay: "the support of the minister of health backed by the president of paraguay has been a key factor in the development of a national health research system" [ ] . additionally, naturally enough, the activities of the various who regional offices in boosting nhrss tend to focus on working with the national ministries of health, including work in europe [ ] and by paho [ ] . conversely, several analyses illustrate that progress in strengthening the nhrs might be limited where key parts of the ministry of health, for whatever reason, do not provide support [ , ] . nevertheless, some disadvantages or dangers were identified when the ministry of health plays the leading role. first, in england prior to the creation of the nihr as well as in some other countries, the research funds controlled by the health ministry were sometimes appropriated by other parts of the health system when they were under particular pressure for resources [ ] . similarly, there have been a few reports that health research funding lost out when donor funds that had previously been allocated specifically for health research programmes were replaced by donations of funds to be allocated by the nation's own health system according to its own priorities [ , ] . one way of attempting to mitigate the danger is, as undertaken by the nihr and described by atkinson, by building support for health research through measuring and reporting the payback from research [ , ] . the second danger arises because, traditionally, many researchers argued that the best science came when they had the freedom to identify the key research topics, rather than having priorities set by others [ ] . therefore, they argued, the responsibility for funding and organising health research should be left to organisations that are part of the research system and independent of the health system [ ] . furthermore, despite the growth of interest in coproduction approaches noted above, there have also been recent doubts raised about the assumption that coproduction is always the most appropriate approach [ ] . this issue clearly requires sensitive handling. indeed, atkinson et al. [ ] argue that one of the great successes of the nihr is that this issue has been so skilfully handled by the nihr that external input, or stakeholder engagement, in setting agendas has become widely accepted and the structures created give ministers a sense of ownership without sacrificing scientific independence. the efforts of waho [ , ] and the who regional offices for africa and paho [ , , , ] indicate that partnerships can be helpful. in europe, the who regional office worked with member states to create the european health research network, which is intended to help nations with limited nhrss who wish to make more progress [ ] . partnerships can provide important support and encouragement, but the evidence suggests there must be strong political will somewhere within the political and/ or health systems for a health research system to be fully strengthened. the central asian countries in who's european region seem to provide an illustration of this point. a cohred collaborative initiative successfully resulted in situation analyses being produced in each country and then jointly discussed as the basis for action [ ] , but according to the analysis by santoro et al. [ ] , limited progress seems to have been made in the subsequent years. the importance of partnerships and collaboration in focusing research efforts in an extreme crisis, with a leadership role for the who, has been seen in the race to find treatments for covid- and vaccines against severe acute respiratory syndrome coronavirus (sars-cov- ), which causes the covid- disease [ ] . in many nhrss across the globe, including in the philippines, scientists are coming together to participate in who's solidarity trial, which will test the safety and effectiveness of various possible therapies for treating covid- [ ] . sarah gilbert, leader of oxford university's jenner institute's work on developing one of the leading vaccine candidates explained that cooperation was vital for tackling the crisis: "work is continuing at a very fast pace, and i am in no doubt that we will see an unprecedented spirit of collaboration and cooperation, convened by who, as we move towards a shared global goal of covid- prevention through vaccination" [ ] . a key issue going forward is how such cooperation can be built on in strengthening nhrss into the future. for now, it is recommended that a prospective study be conducted to analyse all that is being done in different nhrss to speed up research during the pandemic, with a view to taking lessons about cooperation, partnerships and other matters into strengthening nhrss in the future [ ] . the who evidence synthesis, published as a hen report [ ] , provides a firm basis for decision-making by policy-makers and research leaders looking to strengthen the health research system in their country. it identifies five crucial policy approaches that can be applied as appropriate to the context of the countryconducting situation analyses, sustaining a comprehensive strategy, engaging stakeholders, evaluating impacts on health policies and practices, and partnership participation. it also analyses, in turn, the individual functions and components within a system and identifies a series of tools that can be used for strengthening many of them. if further analysis and research is thought to be relevant in the particular country looking to strengthen its health research system, this opinion piece indicates some of the types of additional sources of information that are available. the opinion piece also discusses aspects of the sometimes-controversial question of who should lead or steer attempts to strengthen nhrss. again, the context of the particular nation will be crucial in determining the most appropriate course to take, as emphasised by both mansoori [ ] and atkinson et al. [ ] , but at least some involvement of the ministry of health is likely to be beneficial; additionally, sometimes, key individuals can play a crucial leadership role in strengthening the whole system or one component. in countries with a less developed tradition of conducting health research, partnerships with other countries and/ or with international organisations can help lead the progress and learning for all partners. the valuable role that international organisations, such as who, can play in leading partnerships and cooperation to strengthen health research systems is being highlighted during the covid- crisis. overall, therefore, the full who hen report not only provides a detailed analysis of nhrs strengthening, it also provides a structure within which an even wider and ongoing literature can be considered. additionally, it contains a perhaps more nuanced account, on which this paper builds, of some aspects of the literature around the issue of who should provide leadership 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towards fair and effective north-south collaboration: realizing a programme for demand-driven and locally led research bringing health research forward the dark side of coproduction: do the costs outweigh the benefits for health research? strengthening health research systems in central asia: a system mapping and consultative process from covid- research to vaccine application: why might it take months not years and what are the wider lessons who western pacific: philippines. ph solidarity trial for covid- treatments receives green light from ethics review body. press release. who. carving a path towards a covid- vaccine publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions sp, lk and sh planned the original phase of the who evidence synthesis, including the search strategy. lk led the original literature search and contributed article selection and data extraction and analysis. ab and sh planned the second phase of the evidence synthesis. tj led the second phase of the literature search and contributed to the article selection and data extraction. ab contributed to the final version of the health evidence network report. sh led the data extraction and analysis and drafting of the report. sh conducted the additional analysis of the literature and initial drafting for this opinion piece. all authors commented on the opinion piece and approved the final version. key: cord- - i rfpvd authors: norton, alice; de la horra gozalo, arancha; feune de colombi, nicole; alobo, moses; mutheu asego, juliette; al-rawni, zainab; antonio, emilia; parker, james; mwangi, wayne; adhiambo wesonga, colette; marsh, kevin; tufet, marta; piot, peter; lang, trudie title: the remaining unknowns: a mixed methods study of the current and global health research priorities for covid- date: - - journal: bmj glob health doi: . /bmjgh- - sha: doc_id: cord_uid: i rfpvd introduction: in march , the who released a global research roadmap in an effort to coordinate and accelerate the global research response to combat covid- based on deliberations of experts across the world. three months on, the disease and our understanding have both evolved significantly. as we now tackle a pandemic in very different contexts and with increased knowledge, we sought to build on the work of the who to gain a more current and global perspective on these initial priorities. methods: we undertook a mixed methods study seeking the views of the global research community to ( ) assess which of the early who roadmap priorities are still most pressing; ( ) understand whether they are still valid in different settings, regions or countries; and ( ) identify any new emerging priorities. results: thematic analysis of the significant body of combined data shows the who roadmap is globally relevant; however, new important priorities have emerged, in particular, pertinent to low and lower middle-income countries (less resourced countries), where health systems are under significant competing pressures. we also found a shift from prioritising vaccine and therapeutic development towards a focus on assessing the effectiveness, risks, benefits and trust in the variety of public health interventions and measures. our findings also provide insight into temporal nature of these research priorities, highlighting the urgency of research that can only be undertaken within the period of virus transmission, as well as other important research questions but which can be answered outside the transmission period. both types of studies are key to help combat this pandemic but also importantly to ensure we are better prepared for the future. conclusion: we hope these findings will help guide decision-making across the broad research system including the multilateral partners, research funders, public health practitioners, clinicians and civil society. introduction covid- was declared a public health emergency of international concern on january and then a global pandemic on march . the who published their global research roadmap on march , within the context of the situation and what is already known? ► the who produced a roadmap that sets out the research priorities following a meeting in february, just before covid- was declared a pandemic. now, at this point in the evolution of this novel disease across the world, and almost months later, it is important to assess whether these priorities remain and if research teams in all countries across the globe agree that these are the most important questions that need to be tackled within their healthcare setting and communities, both to mitigate this outbreak and to learn for next time. what are the new findings? ► over healthcare workers and researchers contributed to this research and their data tell us that across the globe there has been a shift in priorities and new questions have emerged, particularly from low-resourced settings. for example, there is a strong call for evidence on the relative effectiveness and optimal implementation of public health interventions in varied global settings, for social science studies to guide how to gain public trust and mitigate myths, to understand the impact on already present diseases within communities and to explore the ethics of research within a pandemic. what do the new findings imply? ► the who roadmap is globally relevant; however, our findings also provide insight into the temporal nature of these research priorities, highlighting the urgency of research that can only be undertaken within the period of virus transmission, as well as other important research questions but which can be answered outside the transmission period. both types of studies are key to help combat this pandemic but also importantly to ensure we are better prepared for the future. bmj global health the epicentre of infection at that time. the roadmap was built on deliberations of the global research forum, whereby over participants from different sectors across the world identified three to four immediate research priorities for the following months across each of nine themes. now, in june , we see the evolution of this pandemic at different points across the globe. we know from our previous experience with ebola and other outbreaks that it is essential to embed research into the response to an outbreak, and that there is a finite and unknown window where these questions can be answered. covid- is an unprecedented situation and therefore we must take every opportunity to undertake all the possible research that funding and capabilities allow; and high-quality studies should happen everywhere there are cases in order to maximise the evidence generated and ensure that the resulting data and findings are globally applicable. therefore, it is important to assess now, what are the most key remaining global health questions that need to be addressed, both to ensure this pandemic can be halted and to learn for future outbreaks of this pathogen or another. this research intentionally builds from the who roadmap, with the aim of strengthening the global health research response effort already aligned to this, rather than generating a completely new set of priorities. using broad consultative workshops, we have identified additional considerations beyond the who roadmap scope in order to broaden the current global research priorities at this point in time to tackle the covid- pandemic and to help learn for any future outbreaks. an online multilanguage survey was developed where ranking questions were coupled with open-ended questions. this was based on a previous survey led by the african academy of science (aas) that was undertaken in march to assess how well the who priorities were applicable to africa. here, we worked from the aas survey so we could now assess whether the findings remained relevant across the globe, and if they had changed over time. seventy-three potential priorities ( from the original who document and generated as part the aas survey and consultations) were arranged under the nine topic headings used in the who research roadmap. participants ranked their top three options for both short-term and long-term priorities ( total ranking questions). free text boxes were provided under each of the broad topics, where participants were asked to list any research priority they felt was not included in the options provided. recognising that this survey inherently focused respondents on the existing who priority framework, we expanded our consultation through workshops to enable broader discussions of research priorities. after the survey closed, a virtual workshop was held on the fifth of june to seek wider global comment and discussion on the survey findings and to discuss current priorities and unmet research areas beyond the scope of the existing who priority framework. we conducted further open access workshops with research teams and health workers across the globe, led by the the global health network (tghn) covid- research implementation and knowledge hub between april and june . these workshop meetings were recorded with permission of participants, and comments and questions captured. a thematic content analysis methodology was developed to report the findings of each. here, we applied this to the cumulative data of all workshops to add to the survey data and better address the question: what are the current global research priorities during the covid- pandemic? quantitative data analysis methods responses from the survey were downloaded in excel format, all data were fully anonymised, password protected and access restricted to the study team. descriptive analyses were undertaken within excel to provide a ranking score for each research priority for immediate and longer term, as per the survey. priorities ranked as first were given a score of , those ranked second were given a score of and those ranked third were given a score of . this analysis was conducted within the category headings from the who roadmap and included both the original who priorities and new priorities suggested in the aas report. therefore, these data show us how responders currently rank the priorities set within the who roadmap and the aas report. the data were split for comparison between the global researcher responses and those originating from less-resourced settings. within the less-resourced setting category, we include low and lower middle-income countries as defined by the world bank. the aim of the open-ended survey was to determine whether there are new priorities that were not included in the original who roadmap or the aas survey findings. these written comments were imported into nvivo qualitative data analysis package and we undertook a pragmatic thematic content analysis. analysing the data from the workshops allowed a further open consideration of current research priorities as this step expanded beyond the limitation that the survey had of asking questions within the framework of the who roadmap. following the methodology established after the first workshop, we compiled a dataset by transcribing the spoken and written comments from each workshop. a coding framework was generated through an inductive and then deductive approach, following the same categories used in the survey. the participants in this study were the global health research and healthcare community and the very aim was to give them a voice in the requirement to assess bmj global health whether the right research questions are being tackled in covid- . we made ongoing open calls through social media for contributions to surveys and the workshops were open access on tghn and also on facebook. the research question was set to address prior lack of engagement with the wider, global community, and the design was based on ongoing engagement with this community and our understanding of how to most effectively engage and gain their involvement. the study was entirely open throughout all the steps and the time taken to complete the survey and taking part in the workshops was made clear to participants. in total, individuals completed the online survey and attended the workshops, from across countries, ensuring representation from all of the who regions (african region= ( %); american region= ( %); eastern mediterranean region= ( %); european region= ( %); south east asia region= ( %); western pacific region= ( %)). participants were most commonly employed in academia ( %), hospitals ( %), research organisations ( %) and nongovernment organisations ( %). the survey results (table ) show how priorities were ranked across the immediate and longer term within the who categories. we present these globally, along with a subgroup analysis of less-resourced countries, to understand whether there are differences in priorities for lessresourced countries. the ranking of these priorities broadly indicates what researchers feel to be the most important research areas from the who roadmap at this point within this pandemic. the qualitative data from the survey and the workshops then provide further insight to guide where emphasis should be placed and where completely new priorities are relevant, particularly in low-resourced nations. the qualitative data analysis from the survey, workshops and working groups supported the existing who roadmap and highlights where greater research emphasis is needed at this later point in the pandemic. however, most importantly new broader priorities have also come through from this study (table ) . these data suggest that that original who covid- research roadmap remains broadly globally applicable. here, we also show which research questions require the most emphasis and also that potential new priorities have emerged that were not within the initial roadmap. some newly suggested priorities reflect the progress of the pandemic and acquisition of knowledge as to where the gaps lie; notably research in children, pregnancy, long-term health impacts of the disease and that there is a strong call for research that assesses the effectiveness of public health measures put into place across the globe to reduce transmission of this virus. these were alongside a demand for greater social science research to determine public perception, and better ways to change behaviours and build trust (including a need for social sciences to cross-cut the other more biomedical priorities). we also identified a range of new priorities relating to addressing covid- in lower resource settings, where multiple pressures including ongoing endemic infectious diseases and other comorbidities are competing within the health and policy systems for limited resources. these pressures have led to emphasis on cheaper and field applicable tools and research and health capacity strengthening. the need for further studies to evaluate public health measures and studies on other potential interventions as they arise were ranked highly by the survey respondents and workshop participants. these studies must be undertaken as quickly as possible, in highly varied social contexts, if we are to gain evidence now on just how effective measures such as lockdown, handwashing and social distancing are on reducing transmission and to understand the relative risks and benefits. the need for social science research and mixed methods came through very strongly, with an emphasis on determining how to gain trust and successfully deliver public health messages. this needs evidence-based community engagement strategies; tested and evaluated everywhere. limitations of our approach include the fact that we built the questions to align with the original who broad priority headings, this would have inherently focused the survey respondents around the largely biomedical focus of these priorities and this meant that some headings (eg, the animal human interface) had relatively few suggested priorities, while others (eg, social sciences in the outbreak response) had much larger numbers. we also retained the original order of priorities from the who research roadmap and the aas survey and this may have influenced the ranking given by respondents. the workshops however were open and purposefully invited researchers to make whatever comments they wanted in regard to where current research priorities lie, beyond the scope of the who research roadmap. therefore, taken together, we suggest that these data support the importance of the who research roadmap approach and highlight where funders and researcher should be placing emphasis as well as identifying potential new areas that should be tackled within this pandemic. consideration of both immediate and long-term priorities is important to address this specific pandemic and to better prepare for the future. there are studies that need ongoing transmission, at a high enough rate to answer the question they set. these might be essential for this pandemic, for example, clinical trials to determine the efficacy of drugs or vaccines, or address questions to guide future outbreaks, such as evaluating the effectiveness of public health interventions. other studies do not need circulating virus and could still guide the effort to bmj global health investigate ways of ensuring transparency of information flow and mitigating false information spread by various mechanisms ensure that knowledge is produced according to local, national and regional needs ensure that knowledge is produced according to local, national and regional needs promote the prioritisation of knowledge needs according to epidemic dynamics examine optimal ways of communicating about potential interventions in high-density low socioeconomic status urban settings ppe, personal protective equipment. table existing priorities now requiring greater research emphasis and new priorities not in the who roadmap or aas list (all data from participants working in less-resourced countries apart from those priorities asterisked which originated from participants working in higher income countries) existing priorities now requiring greater research emphasis social sciences in the outbreak response understanding covid- in the contexts of conflict, civil war and refugee situations. examine the effects of the pandemic on the participation of the public in democratic processes. infection prevention and control how to ensure effective social distancing in public spaces and congregate settings post lockdown** the environmental impact of the response to covid- determine the impact of: public health interventions on the environment (including air pollution and carbon dioxide emissions) ► disinfectants and hand sanitisers on the environment. ► large-scale ppe production and disposal. preparing for the next pandemic ensure effective measures including community surveillance and animal screening techniques are in place to rapidly identify emerging zoonotic diseases. evaluation of governmental policies and lessons learnt in preparation for the next pandemic. cross-cutting the use of technology in various aspects of pandemic response. assess effective ways of conducting cross-disciplinary research. all data from low-income countries apart from the three priorities marked as ** which are only from participants from high-income countries. lmics, low-and-middle-income countries; ppe, personal protective equipment. priorities to enable funders and researchers identify gaps and opportunities, and inform future research investments or coordination needs. finally, we want to highlight both the importance of fully involving the global research community in priority settingand the ongoing need to review priorities where knowledge and practice is advancing rapidly. we recognise that these efforts need to be complemented by further research priority scoping work, beyond the global health focus to further strengthen cross-disciplinary efforts. here, we have shown that the global health research community supports the recommendations of the who research roadmap, but that important new priorities have emerged both due to the transition through the pandemic and consideration of differing global epidemiological, health system, policy and research contexts. twitter wayne mwangi @thogoto patient consent for publication not required. ethics approval this research was limited to seeking the views of healthcare professionals and research staff; patients and the wider community were not involved. therefore, this research would be considered 'minimal risk' and does not come under the definition of research involving human subjects. however, this work does still fall with our research methodology and remit for the protocol that is approved by the university of oxford research ethics committee (oxtrec) protocol number oxtrec - . provenance and peer review not commissioned; externally peer reviewed. data availability statement data are available in a public, open access repository. all the data from this study will be openly available on the global health network. open access this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by-nc . ) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by-nc/ . /. author note the results from the survey were shared with the community through the 'research priorities' workshop and the reports from each workshop are being shared on the platform. the wider, cumulative report is being shared online and the release of that will also be widely disseminated. one of our core aims with this research is to make these findings as widely known as possible so that the prioritise that this work highlights translates to studies undertaken by this same community. who director-general's statement on ihr emergency committee on novel coronavirus ( -ncov) who director-general's opening remarks at the media briefing on covid- a coordinated global research roadmap: novel coronavirus geneva figure priority assessment matrix for research within the covid- pandemic. ppe, personal protective equipment ebola: embed research in outbreak response emergent threats: lessons learnt from ebola research and development goals for covid- in africa -the african academy of sciences priority setting exercise malaria and covid- : a rapid determination of unknowns and call for research the uk collaborative on development research. covid- research project tracker by ukcdr & glopid-r, acknowledgements we acknowledge and thank all survey and workshop participants for their contributions.contributors km, ca, wm, jma and ma developed the original survey with input from mt; all the authors then contributed to further developing and delivering this global version with oversight from tl. an and tl guided this analysis along with adlhg, nc, ea with support from za-r and jp. the workshops were delivered by tl and nfdc, with support from za-r and jp. tl led the drafting with an and mt, pp and km were closely involved throughout and contributed to the draft and review. the other authors contributed significantly and equally in conducting the study and analysing the data. the corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. tl is responsible for the overall content as guarantor.funding the global health network is supported by a grant from the bill and melinda gates foundation (https://www. gatesfoundation. org/ grant number: opp ). the covid- knowledge hub is supported by a grant from uk research and innovation (https://www. ukri. org/ grant number: mc_pc_ ). an & mt are employees of ukcdr, which receives funds from beis, dhsc, dfid, ukri and wellcome for its core activities. no other specific funding supported this work. the funders played no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication. all researchers are independent from funders, and all authors, external and internal, had full access to all of the data in the study and can take responsibility for the integrity of the data and the accuracy of the data analysis.patient and public involvement patients and/or the public were involved in the design, or conduct, or reporting or dissemination plans of this research. refer to the methods section for further details. key: cord- - rt wga authors: pattnaik, debidutta; hassan, mohammad kabir; kumar, satish; paul, justin title: trade credit research before and after the global financial crisis of – a bibliometric overview date: - - journal: nan doi: . /j.ribaf. . sha: doc_id: cord_uid: rt wga abstract this study presents an overview of the state-of-the-art in trade credit research by examining publications between and . applying bibliometric and econometric tools, this study presents a comparative analysis of the extant research across the three sub-domains of banking and finance, production and operations, and accounting. findings suggest that the financial emergency in the global market had resulted in a watershed moment in trade credit research. about % of the literature was found to have emerged after the global economic crisis of . a network analysis grouped the trade credit articles into four major and four minor clusters. the banking and financing cluster exhibited the highest growth followed by the production and operation cluster while the perspectives of accounting are yet to gain traction. conversely, reputation of the publishing hub, empirical studies, and the production and operational dimensions of the research positively and significantly influence citations. alongside thorough introspection, the study also provides new areas to direct the course of future research. emerging from the field of banking and finance, research in trade credit has evolved as a multi-disciplinary scientific domain with contributions from business management, industrial engineering, production and operations, finance, economics, etc. (paul & boden, ) . prior reviews have provided partial qualitative and (or) quantitative perspectives of their respective disciplines (see table ), while the need for a holistic overview of the extant literal corpus is unaddressed. in addition, the precise factors contributing to the growth in literature in this fastevolving academic domain lack empirical support. as an example, in one of the recent reviews, pattnaik et al. ( ) provided a comprehensive overview of the literal corpus on trade credit covered in top finance and economics journals between and february, . however, the study misses out the basic multi-disciplinary nature of the research domain. therefore, views from other important disciplines e.g., industrial engineering, production and operations etc. excluded in the former are included in this endeavor. alternatively, while the former evaluates only articles covered in web of science (wos); this work analyzes , works of research available in scopus. in addition, both the studies also differ in their analytical outfits. the primary concern of the first review is on network analysis and therefore exhibits limitations in the descriptive front. however, this endeavor is rich in both descriptive, network, and predictive contents of the multidisciplinary research domain. j o u r n a l p r e -p r o o f clustering exposes their semantic (dis)association. further, we also explore the thematic components and introspect on the extant literature to provide some future research directions. in addition, we also identify a number of factors influencing the quantum of citations, thus indicating a growth of trade credit research. therefore, apart from the de novo researchers, such a detailed introspection may also inform the established academics in the field about theories which require future validation and the study methods they could deploy. moreover, this study contributes significantly to the extant research by asking the following research questions (rqs): rq . what is the state-of-the-art of the research front-in terms of publications, authorship and citation structure, influence, impact, activity, and productivity of its contributing authors and publishing sources-and how does it vary in the pre and post crisis era of ? rq . what is the thematic layout of the research domain? rq . how do the extant literature, its prolific contributors, publishing sources, and highly cited references converge intellectually? rq . what factors significantly contribute to the growth of the research domain? rq . what is the direction for actionable research in future? in the remaining sections, we summarise the research methods, discuss the study outcomes, provide key summaries, and conclude the paper. (insert table about here) trade credit-in form of receivables-is a financing provision by non-financing firms (garcía-teruel & martínez-solano, ) with or without bank intermediation. in the context of a complex and competitive global market, credit supply is inevitable to retain b b relations and impart business growth (chowdhury & lang, ) while financing supply chain is must for obtaining competitive advantage (pirttilä et al., ) . enhanced fluctuations in the level of industrial production backed by disruptive technologies significantly distress trade credit policies both for the upstream and downstream supply chain. the nature of the product, fluctuations in market demand and the level of hostility in the business environment further impact its demand and supply. buyers benefit from the time value of money by increasing their demand for credit supplies while suppliers, on the other hand, register a growth in sales (schwartz, ) . simultaneously, empirical evidences assert the influential role of a number of qualitative indicators such as the national culture (el ghoul and zheng, ) , the cultural background of finance managers (bedendo et al. ) , and level of social trust (levine et al. ) impacting the demand and supply of trade credit. demand for trade credit is also influenced by the level of financial market development and financing constraints. more importantly, trade credit substitutes bank credit during a crisis (bastos & pindado, ) and therefore is a viable source of finance both in the developed and developing world (mcmillan & woodruff, ; afrifa & gyapong, ; li et al., ) . suppliers' credit played a crucial role in the survival of smes during the crisis. empirical evidence substantiates the argument that trade credit reduces the likelihood of financial distress among smes (mcguiness et al. ) . simultaneously, it impacts the production cycles, optimal ordering quantity, level of inventory, firm performance, and industry growth (haley & higgins, j o u r n a l p r e -p r o o f ; barth et al., ; huang, huang, , huang & hsu, ; fisman & love, ; chung & huang, ; chung et al., ; liao, ; allen et al. ). in the chronological progression of the research domain, the intertwined financing and accounting perspectives of trade credit shows signs of separation. few notable thinkers such as brick and fung ( ) proposed the tax theory of trade credit, hypothesising that businesses offer trade credit to obtain tax benefit. other themes explored in this area include revenue management and budgetary participation among others. thus, the literature analysed exhibit three broader academic domains: first, the banking and financing aspects, and second, the production and operational dimensions, and third the accounting perspectives of trade credit. in order to delineate the potential factors contributing to the popularity of trade credit literature in academia this study applies both quantitative and qualitative tools. in the field of scientometrics citations are predominantly considered to be the indicators of quantity versus that of quality (aksnes et al. ). on the basis of citations, hirsch ( ) proposed the "h-index" while egghe ( ) proposed the "g-index". both hirsch and egghe provide authorial performance indicators measuring the quantitative performance of authors. drawing from such prior evidence, we examined citation as a growth factor for a research domain as every new citation is the birth of a new publication. alternatively, it also adds to the influence and impact of the academic source cited, the specific theme cited, and contributes to the intellectual influence of its contributor(s). given the debatable qualitative indications of citations (aksnes et al. ), we present qualitative perspectives i.e. journal's academic reputation, an author's academic reputation, etc. alternatively, ball & tunger ( ) argue that growth in publications indicates a growing research area while acedo et al. ( ) and finardi & burati ( ) associate growth with authorial collaborations. other factors include an increase in global submissions and publications in prestigious academic outlets (merigó et al., ; baker et al. ) , etc. alongside the hypothetical constructs, this study also applies some of the tested factors affecting citations (growth in publications) by drawing upon schwert's work ( ) . by combining all the theoretical perspectives, the conceptual model of our study is proposed in figure . during academic emergency or external threats to industry (environment)-established research domains grow faster. growth of the broader academic domain is also due to the upsurge in research activities of the interrelated disciplines. in such circumstances, published articles in reputed journals positively affect growth. the growing inter-disciplinary variable mediates between the primary established academic domain and the discipline's overall growth. in other words, by drawing wisdom from the (reputed) publications and the established academic domain, the growing inter-disciplinary sub-domain(s) expand(s) the broader horizon of knowledge further. j o u r n a l p r e -p r o o f data analysed in this study was furnished from scopus. it is one of the largest databases (bartol et al., ) of peer-reviewed works in social science (norris & oppenheim, ) , extensively accessed for quantitative analyses (durán-sánchez et al., ; baker et al. ; donthu et al. ) . figure summarises the study design. (insert figure about here) to carry out the investigation, a broad search term was finalised. the term, "trade credit" or "account* receivable*" or "account* payable*", searched in the title, abstract or keywords enabled access to the extant literature in scopus. redundant documents were eliminated using subject filters. literature confined to business management, accounting, economics, econometrics, finance, social sciences, arts and (or) humanities were included. since our focus was accessing the core bibliographic records on trade credit; only those documents classified as articles, reviews, or conference proceedings were included and the rest excluded. further, due to the dynamic nature of the research domain literature published in is omitted. finally, a total of , documents published between and were considered for analyses. the descriptive analysis in this study was conducted manually using microsoft (ms) excel under the four broad categories of: . publication trend. . authorship pattern. . citation structure. . the influence, impact, activity, and productivity indicators (donthu et al. ; baker et al. ) . refer appendix i for definitions of the descriptive variables. j o u r n a l p r e -p r o o f the study explores the thematic factors of the research domain using principal component analysis (pca) with varimax rotation under kaiser normalisation. it was conducted with the help of the statistical packages for the social sciences (spss) software. the core of such an analysis is based on the co-occurrence counts of the keywords indicating themes (callon et al. ; marrone, ) . ponzi ( ) applied a similar technique to explore the intellectual structure among frequently co-occurring authors. to carry out the analysis, prolific themes appearing at least five times in the shortlisted articles are identified. using ms-excel, the co-occurrence matrix of those themes was later processed in spss to obtain the pearson's correlations. finally, we used the correlations' matrix to explore the thematic components. when two distinct works cite one or more common documents, they exhibit intellectual similarities (kessler, ) ; whereas, co-citation is the citation of two or more existing researches in a later article (small, ) . the network analyses in the forms of bibliographic couplings (kessler, ) and co-citations (small, ) unveil the semantic clustering among the citing and cited documents, contributing authors, publishing sources, etc. (merigó et al., ; donthu et al. ; baker et al. ) . the networks in this study are visualized using vosviewer and gephi software (van eck & waltman, ; van eck & waltman, ; bastian et al., ) . both the programs apply two standardized weights for visualising the networks e.g. the total number of links and the total strength of the links. the size of the nodes in a network indicates its relevance; whereas, the j o u r n a l p r e -p r o o f number and size of the interlinking lines represent the strength of the association among the nodes (van eck & waltman, ). we apply the ordinary least square (ols) regression in spss to explore the potential growth factors of the research domain (dragos et al. ) . drawing primarily from previous research (schwert, ) , the following model is proposed: total citations = article length + number of contributing authors + authorship type (sole-authored or co-authored) + publication year (before or after ) + ajg ratings + bibliographic cluster of the article (major or minor) + study method (primary or secondary) + thematic components' score + error (eq. ) the dependent variable, total citations, is defined as the total quantitative growth of the research domain. article length-defined as the number of pages of the article. number of contributing authors-defined as the total number of authors who contributed to the article. thematic score-respective thematic component scores of the articles. dummy variables include, authorship type (sole-authored or co-authored), publication year ( figure shows the yearly growth in publications, its influence (h-index), and the average annual citations of the articles published between and . figure depicts the annual trend of the intelligentsia, while figure distributes the trade credit articles by its number of contributors. it also compares the authorial distribution of the articles before and after . (insert table figure depicts an exponential growth in publications since . as indicated in table , about % (tp: of , ) of the study articles are published after . thus, the majority of the research on trade credit followed the global economic crisis of . such a trend is not surprising as-during the collapsing phase of banking-academia largely proclaimed trade credit as the primary source of alternate finance that sustained the global economy (giannetti et al. ; chor and manova, ) . conversely, as the demand was non-decreasing post , the proposition of the economic order quantity model by teng et al. ( ) attracted further research attention (see table ). not only did academia recognise trade credit as a major source of finance over these years, trade credit also managed to establish itself as one of the predominant factors influencing factors like economic order quantity (eoq), economic production quantity (epq), the shifts in the volume of production, etc. from another perspective, an evolving research domain triggers further research attention by attracting and engaging more scholars (ball & tunger, ) . the notion is evidentially affirmed in figure . the figure depicts an increasing trend in the number of thinkers. , unique researchers have contributed to the domain between and (see table in conclusion, the protective mechanism of trade credit as an alternative source of finance during banking emergency has historically triggered academic attention between and . with the pandemic onset of covid- shutting down operations, we foresee a similar trend in the production and operational area of trade credit research in the near future. table presents the top articles published before and after . (insert table interestingly, the top three highly cited works exhibit convergence in their respective philosophical outlooks. petersen and rajan ( ) predominantly present the banking and financing views of trade credit research while both dechow et al. ( ) and barth et al. ( ) navigate the broader finance and accounting dimensions. however, the model proposed in the note by huang ( ) modified the production and operational dimensions concerning the trade j o u r n a l p r e -p r o o f credit policies of retailers. while earlier notions predominantly argued against the extension of credit period by retailers, huang ( ) proposed that like suppliers, retailers stimulate their demand by extending the credit period to their customers. thus, the highly cited articles, depicting the existing knowledge in the established academic domain, presumably influence their respective research sub-domains. however, a deeper investigation of the highly cited articles reveal that the accounting dimension of trade credit is dormant without any significant contribution in the post-crisis era thereby presenting scope for future research. in the subsequent discussion, we recognize some of the most prolific and influential thinkers in the broader domain. table lists some of the most prolific authors who have contributed at least five works of research between and . (insert table (insert table table enlists the prominent themes of the extant literature on trade credit which have been discussed in at least articles. (insert table table presents the communalities of the thematic items. table shows the loading of the thematic items to their respective components and also presents the reliability of the thematic scale. (insert table about here) (insert table (kessler, ) while small ( ) argued that frequent co-citations of published articles in the subsequent research evidentially confirms intellectual association. unfortunately, the argument of small ( ) may suffer a time lag effect as more recent publications require time to influence academic evidence in the form of citations (marrone, ) . since our study incorporates articles till , we applied bibliometric coupling analysis on the trade credit articles, authors, and publishing sources. however, to expose the prior seminal ideas, we applied co-citation analysis on the cited references as explained by small ( ) . table presents the descriptive analyses of the bibliographic clusters, figure reveals the publication trend of the clusters in the pre and post crisis era, while figure shows the kind of studies that prevailed within the respective bibliographic clusters. (insert table further, as indicated in figure , most of the articles are empirical investigations while the cluster contributes a smaller number of qualitative researches such as conceptual papers, reviews etc., thereby suggesting few methodological gaps for the aspiring contributors to fulfil. of note is that some of the influential themes of the cluster include terms such as firm value, corporate social responsibility, earnings and cash flows, prediction of future cash flows, etc. some of its recent and original publications draw upon transactions cost theory; for eg. the third cluster is the most competitive cluster of the research front occupying the topmost rank in the majority of the indicators reported in table (tp: the supplier-retailer's relation mediate the above proposition. we argue that apart from establishing the credit quality of the new buyers, the incremental increase of credit supply with age may naturally eliminate the risk of free-riding. cluster most of the latest articles published in the cluster work in the area of green supply chains, operational management, optimal ordering quantity, optimal wholesale price, optimal carbon j o u r n a l p r e -p r o o f emissions, etc. as an example, the amalgamative supply chain operations model of dash ( ) addressing the concern of optimal carbon emissions (oce), optimal ordering quantity (ooq) of a capital-constraint retailer, and the optimal wholesale price (owp) for a manufacturer operating in a viable trade credit financing and bank financing market environment proposed and validated that lower levels of carbon emissions fosters win-win outcomes. future empirical validations of such models can sensitise controlled carbon emissive behaviour as it positively impacts profitability. it can be concluded that the bibliographic coupling analysis affirms three major subdomains of trade credit (banking and financing, accounting, and production and operations) gradually expanding to eight specialized sub-domains. the following section visualizes the intellectual epicentres of trade credit research, and the network among the most prolific authors and contributing sources. schwartz ( ) . interestingly, excluding goyal ( ) , aggarwal and jaggi ( ) , and teng ( ) all the remaining articles are closely knitted. it suggests that the operational dimension of trade credit stand out from the remaining perspectives. interestingly, figure also presents three broad groups of intellectuals. however, x. chen and d. yazdanfar stand out in their referencing pattern exhibiting similarities with n. h. shah and l. -y. ouyang, respectively. in figure , the majority of the journals present the financial perspectives of trade credit in the subsequent section we report the outcome of the regression analysis revealing some key variables influencing the total citations of the discipline over the years. j o u r n a l p r e -p r o o f table presents the description of the studied variables, table presents the correlation matrix of the studied variables while table presents the regression coefficients of the variants. (insert table about here) (insert table about here) (insert table although the directions for future research are already presented during the analysis of the bibliographic networks, we summarise some of the key aspects here. while analysing the gaps in research methods, we suggest future research to be more qualitative in nature. researchers should consider providing more conceptual and theoretical models, specialized pathways, survey-based studies etc. in the emerging clusters to fortify the domains. simultaneously, we call for more studies with empirical insights from emerging economies that would educate global academia which has largely observed this phenomenon simultaneously , paul et. al. ( ) presented receivables as an alternative investment. however, the social nature of trade credit is unexplored. given its importance, we argue that trade credit is not only commercial, it also has social implications, especially when the relation is between the large-scale lender to the micro, small, and medium enterprises (msmes). further, drawing from the model of dash ( ), future research could also explore the impact of green trade credit on firm's accounting, market, and social performances. conversely the behavioral dimensions in the research front is unexplored. with regard to the application of theories in this area, we suggest that there are opportunities to carry out researches using theories such as the prospect theory and dynamic capability theory. prospect theory was originally developed by economists kahneman and tversky ( ) . it constitutes one of the first economic theories formulated using experimental methods. prospect describes how individuals asymmetrically assess their loss and gain perspectives relative to their specific situation. they found that individuals are risk-averse when facing gains but risk-lovers when facing losses. accordingly, the prospect theory describes the actual behavior of people. in the original formulation of the theory, the term prospect referred to the predictable results of a lottery. however, the prospect theory can also be applied to the prediction of other forms of behaviors and decisions in areas such as trade credit. similarly, tenets of dynamic capability framework (teece, ) can be applied in trade credit research to analyze how the firm capabilities and trade credit influence firm performance. the state-of-the-art in trade credit literature indicates an evolving growth trend. a growing discipline attracts more researchers from multiple disciplines resulting in synergetic ( ), - . https://doi.org/ . /j. - . .tb .x chung, k.-j., goyal, s. k., & huang, y.-f. ( . the optimal inventory policies under permissible delay in payments depending on the ordering quantity. international journal of production economics, ( ) financial and economic perspectives notes: this table presents some of the former reviews on trade credit. it includes the author(s) of the study, type of study (tos), the study method, study period, number of articles analysed (na) and the primary focus of the study. nd stands for not defined period/articles. this table presents the publication here, iiap = influence, impact, activity, and productivity; tp = total publications; b = number of publications before ; a = number of publications after ; nca = number of contributing authors; ci = collaboration index; sa = number of sole-authored articles; ca = number of co-authored articles; pcp = proportion of cited publications; c/ca = citations per contributing author; ct = first citation threshold i.e. between and cites; ct = second citation threshold i.e. between and cites; h = h-index; g = g-index; nay = number of active years; and pay = productivity per active year. here, iiap = influence, impact, activity, and productivity; tp = total publications; nca = number of contributing authors of those publications; ci = collaboration index; sa = sole-authored articles; ca = co-authored articles; pcp = proportion of cited publication; tc = total citations; c/cp = citations per cited publication; c/ca = citations per contributing author; ct = first citation threshold i.e. between and citations; ct = second citation threshold i.e. between and citations; ct = third citation threshold i.e. above citations; h = h-index; g = g-index; nay = number of active years; and pay = publications per active year; and na = not available. here, sd = standard deviation, n = number of cases, and na = not applicable. the dummies include: authorship type of the trade credit articles i.e. sole-authored or co-authored; publication year of the articles i.e. before or after ; ajg ratings of the publishing sources of the trade credit articles such as *, , , , or ; bibliographic clustering of the trade credit articles i.e. major or minor cluster; and research type of the trade credit articles i.e. primary (empirical) or secondary (review, conceptual, model building, etc.). apart from reporting the standardized coefficients (sc) of the independent variables influencing the dependent (total citations), the table also presents the collinearity statistics (cs) such as tolerance (t) and the variance inflation factors (vif) of the regressors. of note, the r of the model is . with an adjusted r value of . . the regression is significant at % confidence interval (p-value ≤ . ). here, t = tolerance; and vif = variance inflation factor. citations, citation indicators, and research quality: an overview of basic concepts and theories understanding informal financing twenty-five years of the journal of corporate finance: a scientometric analysis bibliometric analysis -a new business 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two-level trade credit application of entity linking to identify research fronts and trends european trade credit use and sme survival interfirm relationships and informal credit in vietnam research in production and operations management: a university-based bibliometric analysis assessing the usefulness of bibliometric indicators for the humanities and the social and behavioural sciences: a comparative study comparing alternatives to the web of science for coverage of the social sciences' literature qualitative research in financial markets, ahead-of-print(ahead-of-print the secret life of uk trade credit supply: setting a new research agenda trade credit: theories and evidence working capital management in the russian automotive industry supply chain scholarly influence in the field of management: a bibliometric analysis of the determinants of university and author impact in the management literature in the past quarter century the intellectual structure and interdisciplinary breadth of knowledge management: a bibliometric study of its early stage of development statistical bibliography or bibliometrics an economic model of trade credit co-citation in the scientific literature: a new measure of the relationship between two documents explicating dynamic capabilities: the nature and microfoundations of (sustainable) enterprise performance. strategic management journal on the economic order quantity under conditions of permissible delay in payments economic order quantity model with trade credit financing for non-decreasing demand citation analysis of ted nelson's works and his influence on hypertext concept software survey: vosviewer, a computer program for bibliometric mapping vosviewer manual using payment behaviour data for credit risk modelling supply chain finance: a systematic literature review and bibliometric analysis financial intermediary development, and industry growth" fisman r., love i. bank credit: evidence from recent financial crises sarria-allende v. in-kind finance: a theory of trade credit bank discrimination in transition economies: ideology, information, or incentives are accruals during initial public offerings opportunistic an eoq model with noninstantaneous receipt and exponentially deteriorating items under two-level trade credit" liao j.-j. multi-delay-in-payments under single-setup-multiple-delivery policy in a global sustainable supply chain chain finance: a systematic literature review and bibliometric analysis international trade during the - crisis: in search of the smoking gun ranking (r) of the articles are based on their respective average citations per year (cpy). here, tc = total citations, and c/ca = citations per contributing author notes: this table presents the pearson's correlation among the potential variables influencing the citations of trade credit articles = type of the trade credit research (empirical or secondary); = empirical research defined as the sum of total publications b defined as the number of publications before a defined as the number of publications after authorship pattern nca number of authors contributing the research article(s). annual increment of authors added to the research domain. ratio between the number of contributing authors to total publications less the number of total publications ( − ). number of articles contributed by a single author. number of articles contributed by multiple authors. the number of articles cited at least once in scopus. ratio between the number of cited publications to the total number of publications. sum of the citations accredited to an article, an author, a journal, a cluster, etc. c/p average citations per publication. average citations per cited publication. average citations per contributing author. citations between and times. citations between and times. ct citations and above. influence, impact, productivity, and activity h h number of publications cited at least h times. gsum of g number of highly cited publications cited at least g times. number of years an article or a theme on trade credit was published or the number of years an academic source &/or a cluster published on trade credit. number of publications in each of the active years. key: cord- -b qn ynk authors: baillie, l.; dyson, h.; simpson, a. title: dual use of biotechnology date: - - journal: encyclopedia of applied ethics doi: . /b - - - - . - sha: doc_id: cord_uid: b qn ynk this article addresses issues that are central to the dual use of biotechnology, such as the public perception of risk and the need for physical containment to prevent the release of potentially dangerous microorganisms. it also examines the public and media perception of the scientists who handle and manipulate these pathogens and discusses the controls that are currently in place to ensure that scientists engaged in defense-related dual-use medical research act in a transparent and ethical manner. finally, the article discusses what can be done by scientists to allay the fears of their fellow citizens. research in the area of life sciences and biotechnology has the potential to bring great benefit to humankind. in a relatively short period of time, the life sciences have evolved from a simple cataloguing exercise of the diver sity of nature to a position in which researchers are adding to that diversity through the construction of modified and potentially novel life-forms. the vast majority of this activity has had a positive impact on the quality of life of at least some of the human race. indeed, the past years have seen major advances in the fields of micro biology and biochemistry, and these have been followed by the emergence of the disciplines of immunology, molecular biology, and genetics. in practical terms, this has resulted in the introduction of sewers and clean water, the development of antibiotics and vaccines capable of eliminating infectious diseases such as smallpox, and the ability to create genetically modified organisms able to synthesize production-scale quantities of human hor mones such as insulin. indeed, on a daily basis biomedical researchers manip ulate microorganisms in an effort to understand how they produce disease and to develop better preventative and therapeutic measures against the infections they cause. the efforts of plant and animal biologists using similar techniques to improve agricultural yields have resulted in the development of disease-resistant crops and transgenic animals. some of these species have transitioned from the confines of the laboratory into mainstream agriculture in countries such as the united states and india. on first disclaimer: any views expressed are those of the authors and do not necessarily represent those of dstl, ministry of defence, or any other uk government department. inspection, these emerging technologies hold enormous potential to improve public health and agriculture, strengthen national economies, and close the develop ment gap between resource-rich and resource-poor countries. however there is also a potential dark side to this benign picture. throughout human history, every major new technology has been used for hostile purposes, and thus it would be naive to believe that the life sciences might not be similarly exploited for destructive purposes by state-sponsored biological warfare programs or by individual terrorist or doomsday groups. research with the potential to be misused for illicit purposes is said to be 'dual use.' simply stated, the techniques needed to engi neer a bioweapon are the same as those needed to pursue legitimate research. there are also concerns that rapidly advancing technological possibilities could enable the creation and production of unforeseen new biological threats with uniquely dangerous but unpredictable characteristics. a key challenge faced by regulatory authorities is the need to balance legitimate public concerns over the mis use of life sciences against the enormous potential that they have to benefit humankind. getting this balance right will be central to ensuring that governmental actions do not impose blanket restrictions and cumbersome rules on scientists that stifle legitimate research and reduce industrial competitiveness while having little impact on real security. it could be argued that any new regulations specific to dual use of biological technologies would be largely inef fective because they would only affect scientists working in government-funded laboratories, who already follow very stringent rules. indeed, even if new regulations were implemented, it is debatable as to how effective they would be. the anthrax attacks in in the united states are thought to have been undertaken by a 'regu lated' lone u.s. government scientist working in a government-controlled facility. does this mean that we need new regulations, or does it suggest that regulations alone are likely to be ineffective? it is also a fallacy to believe that life science research is limited to government-regulated facilities; indeed, the technology has reached a stage at which an individual with a graduate-level education, access to the internet, and a credit card can set up a garage laboratory anywhere in the world. the emergence of organizations such as diybio is a testament to this new movement. this spon taneously formed community of more than individuals is in the process of establishing community laboratory spaces in major cities throughout europe and the united states to enable their members to carry out their own 'hobby research.' how can these free spirits be assisted or regulated to ensure that both their own safety and that of the community in which they live and experi ment remain secure? an approach proposed by a number of advocates has been to encourage life scientists to take the lead in tack ling the issue of dual-use technology. indeed, some have stated that these scientists have a moral obligation to prevent the misapplication of their research because they are believed to be in the best position to understand the potential for misuse. although the validity of this argument is debatable, it is also extremely unlikely that the average research scientist will have more than a hazy comprehension of the factors important in developing an effective bioweapon. this view does, however, point to the need for life scientists to move more to center stage and proactively engage with both the public and the security and regulatory communities to ensure that the control systems that are ultimately adopted are both proportionate and likely to be relevant in the real world. it should not be forgotten that the reason for these control measures stems from a desire to protect the well being of the general public. although it is highly unlikely that they will understand the intricacies of the research, it is important that they support the outcome that the researchers are trying to achieve. indeed, the support and tacit consent of the general public and their elected representatives is essential in the development of propor tionate regulatory systems. unfortunately, scientists in general, and particularly those engaged in defense and industry-funded research, have a poor track record in communicating the impor tance of their research to fellow citizens. this is primarily due to the constraints imposed on them by their parent organizations, but it also flows from a lack of understand ing of science among the media industry generally, and particularly the popular press, which often results in incomplete and inaccurate reporting. as a consequence, this perceived lack of openness has created an atmosphere of suspicion in which conspiracy theorists, the media, and hollywood thrive, routinely conjuring up lurid images of evil scientists working on government-funded frankenstein projects to destroy the world. it is thus perhaps not surprising that public perception of scientists and their motives may not be as positive as it once was. this article addresses issues that are central to this theme, such as the public perception of risk and the need for physical containment to prevent the release of potentially dangerous microorganisms. it also examines the public and media perception of the scientists who handle and manipulate these pathogens and discusses the controls that are currently in place to ensure that scientists engaged in defense-related dual-use medical research act in a transparent and ethical manner. finally, this article discusses what can be done by scientists to allay the fears of their fellow citizens. although microorganisms capable of causing disease are widespread in the environment, medical, technological, and economic advances have, to a large extent, shielded individuals in the developed world from their adverse effects. notable examples include the reduction in the incidence of ( ) puerperal fever and surgical sepsis in the nineteenth century following understanding of the modes of transmission of bacterial infection, ( ) enteric fevers due to improvement in sanitation, and ( ) food poisoning due to better education and food preparation practices. while there are still intermittent outbreaks of food poisoning in the united kingdom, the real concern is that the overuse by the farming industry of powerful antibiotics to promote animal growth could result in the emergence of multi-drug resistant pathogen bacteria making them increasingly difficult to treat. the importance of a society's organizational and tech nological status in mitigating the effects of disease is well demonstrated by the contrasting fortunes of new zealand and haiti following earthquakes in . although the earthquake that hit south island at . local time on september was of a similar magnitude ( . compared to . ) to the one that struck haiti at . on january , the outcomes for the two populations have been remarkably different. there were no fatalities in new zealand (only two people were admitted to hospital in the immediate aftermath), and there have been no epidemics, despite disruption of sewage and water supply systems. this contrasts with a high initial death toll ( ), many injuries ( ), catastrophic disruption of haitian society ( million people made homeless), and an epi demic of cholera in the displaced population. aspects of a society that determine its resilience to major disasters include the general health and education of its popula tion, its technological infrastructure, the state of readiness of its societal organization to respond to the event con cerned, and its political and governance structures. new zealand and haiti appear to be at opposite ends of the spectrum for all of these criteria. a particular infection of a certain severity may have widely different impacts on an individual depending on the person's general health and specific circumstances. thus, an enteric infection in an undernourished child in the medical center of a refugee camp outside port-au-prince could well prove fatal (particularly because the child is likely to be only one of many), whereas a similar infection in a healthy child in christchurch might be overcome with little more than good nursing care from the child's parents at home. perception of the risks of such infection also varies considerably according to a society's recent experience; childhood deaths from enteric infec tions are an accepted fact of life in many poor areas of the world, whereas in richer, technologically advanced areas they are not. typically, as the prevalence of infectious disease decreases over time in a society, concern regarding rare and particularly novel infections increases. thus, fre quent but relatively mild infections (e.g., the common cold) may inflict a significant overall burden on a society in terms of general ill-health, use of health services, and loss of economic activity without arousing much outcry from the general population. in contrast, a rare but severe treatable infection such as methicillin-resistant staphylococcus aureus may cause much consternation in the media but actually inflict a much smaller overall burden on society. novel or emerging infections (e.g., severe acute respiratory syndrome (sars) and swine flu) may have major economic and societal impacts worldwide, with an actual disease burden that is a minute fraction of that caused by well-known diseases such as malaria and tuberculosis. perhaps counterintuitively, the fear engendered by rare diseases in a society appears to be inversely related to the actual disease burden that they impose. this fear will clearly be modified by experience of the disease; thus, sars was rightly to be feared and swine flu less so. by extension, an unknown infection can cause disproportion ate fear in a population, exaggeration by the media, and the risk of overreaction by the authorities. the anthrax letters episode in the united states was an exam ple of a major response to a relatively small overall disease burden ( infected and deaths in a country of million people), but it demonstrates the fact that a society's response to an unexpected human-originated event may have a much greater impact on the society than the event itself. media-dominated, internet-connected, technologically advanced, economically developed areas of the world are therefore more prone to exaggerated responses based on fear of a horrifying unknown than are those areas that are less privileged. the controversies in europe regarding genetically modified (gm) crops (feared in europe for ideological reasons but welcomed by more pragmatic societies in india and the united states for the increased yields they bring) highlight the fact that advanced societies may, for cultural reasons, have different views of the risks associated with certain technologies. although all advanced societies would be expected to have a marked fear of the sequelae of a deliberate release of infectious organisms, those with an already heightened fear of biotechnology might be more prone to extreme reactions. deliberate release of harmful biological material would provoke a number of emotions, including fear of the unknown, the ancient fear of plague or conta gion, anger and fear of malicious human action, anger directed at law enforcement agencies for failing to prevent the event, and anger at politicians for possibly provoking the event. a release of dangerous biological material from a research laboratory would provoke many similar emotions, although anger would be direc ted more at the incompetence of those operating the laboratory and at the relevant authorities for failing to prevent it. so what is the real risk of an accidental or deliberate release of dangerous biological material in the united kingdom? fortunately, escape of infectious material from laboratories is very rare; examples include a small pox outbreak in birmingham in and the foot-and mouth disease outbreak associated with faulty drainage at the animal health facility at pirbright in . the root cause of such accidental releases was a breakdown in containment (the physical control measures put in place to prevent microorganisms escaping to the environment). in fact, containment technology and practices have improved dramatically during the past years, with significant improvements often being identified by analy sis of accidents or near-misses. at the porton down site, which houses both ministry of defence and department of health microbiological containment laboratories, there have been only two cases of laboratory-acquired infec tion; these occurred in the s and both were the basis for considerable improvements in procedures. in the united kingdom, there have been no known deliberate releases of biological material. there have been deliber ate releases of infectious material in other countries -the rarity of such events has probably contributed to their celebrity status. given the extreme rarity of such events, why are they so feared? it is instructive to compare the annual inci dences of certain other commonly accepted events using headline statistics relating to work-related ill-health and accidents in the united kingdom during - : • ill-health: . million people who worked during this period were suffering from an illness (long-standing as well as new cases) that they believed to be caused or made worse by their current or past work. a total of of these instances were new conditions that started during the year. an additional . million former workers (who had last worked more than months previously) were suffering from an illness caused or made worse by their past work. a total of people died from mesothelioma in , and thousands more died from other occupational cancers and diseases. • injuries: workers were killed at work -a rate of . fatalities per workers. a total of other injuries to employees were reported -a rate of per employees. a total of reportable injuries occurred, according to the labour force survey -a rate of per workers. • working days lost: . million days were lost overall ( . days per worker), . million due to work-related ill-health and . million due to workplace injury. thus, real risk is very different from perceived threat, which may sometimes appear greater the rarer the event (and hence the lower the probability of actually experi encing that event). data for england and wales for indicated that the more common avoidable causes of death (e.g., cardiovas cular disease due to smoking and obesity) carried a risk of in compared to a risk of in for spectacular events, such as railway accidents, that generally attract media attention. these risks were calculated retrospec tively from the reported causes of death during that year. the risk of dying in england and wales from infection due to bioterrorism in that year was zero (as it was in ). however, when looking forward into an uncertain future, many more factors than likelihood affect the per ception of threat, and it may be that the very rarity of an event adds to its perceived impact, making it more inter esting to society at large and therefore much discussed in the media. it is clearly the case that if these rare events remained unreported, the public would not dread them so much, but such censorship would not be acceptable in a democratic society, and the media should take a respon sible approach to explaining real risks and suggesting appropriate and proportionate precautions to mitigating them. although past experience suggests that release of danger ous biological material, whether accidental or deliberate, from facilities is extremely rare, it is important that we consider how such an event could occur in the future. the most likely routes of escape are following an accident in a laboratory (hospital, academic, government, or commer cial research) or as a consequence of defective physical containment processes or equipment as occurred at pirbright in when foot-and-mouth virus was released to the outside world. the mainstay of preventing release of dangerous bio logical material rests on principles of biosafety, biocontainment, and biosecurity. biosafety covers the procedures needed to work safely with hazardous organ isms. biocontainment includes the measures (facilities, equipment, and apparatus) within which work on these organisms can be carried out safely without danger of release into the environment. biosecurity is the process of ensuring that the whereabouts of hazardous organisms are known and tracked and that access to them is restricted to appropriately authorized personnel. these principles actually apply more widely to other human activities, including hygienic preparation of food, supply of clean drinking water, safe processing of sewage, sterile procedures in surgery, and safe operation of hospital microbiology laboratories, as well as the more obvious situations of microbiological research laboratories. welldesigned facilities and procedures both facilitate the con duct of good science and minimize the opportunities for accidental misuse. unfortunately, although good engineering can reduce the physical risk of pathogen release, it cannot stop a researcher from deliberately removing material for his or her own use. the motives for such an action could include ideology (extremist apocalyptic, islamist, or ani mal rights philosophies), blackmail by members of an extremist group, disorders of perception (mental illness or desire for revenge against society following some real or imagined disadvantage), or severe disaffection with employers or colleagues. the u.s. anthrax mail attacks represent just such a case, in which anthrax spores alleged to have been deliberately removed from a u.s. government defense research facility by a government scientist were used to carry out indiscriminate attacks against the general population. although extremely rare, this event is likely to have had a major impact on the public perception of scientists engaged in defense-related research and their motivation. scientists have an image problem. the charming and charismatic scientist is not an image that permeates popu lar culture. although it is common for the entertainment industry (and news media should be included in this category) to portray professions such as medicine, law, and journalism as exciting and glamorous, scientists are often depicted as unattractive, reclusive, socially inept white men or foreigners working in dull, unglamorous careers on projects that could destroy the world. indeed, there is evidence that this impression may be imprinted in childhood and once established is difficult to modify. the reasons for this stereotyping are complex but can be broken down into two main areas: a failure to grasp the nature of the scientific process on the part of the public (education) and a failure to present their message in an accessible manner on the part of scientists (communica tion). these tendencies are compounded by an understandable desire on the part of the entertainment industry to produce content that is popular and profitable. the issue of how our children are taught basic science is an area of obvious concern, as highlighted by the observation that approximately % of adult americans do not understand the scientific process and have to depend on others to help them understand the signifi cance and consequences of scientific advances. in the advanced economies, the major source of information is television, whereas the internet (another unregulated environment) is increasingly used to research specific scientific issues. given the importance of these media in 'educating' and shaping public opinion, how good are scientists at ensuring that their message is getting across? it is safe to say that whatever they are doing, it is not having the desired effect. part of this failure is due to the inability or reluctance of practicing scientists to engage with the media in such a way as to convey their story in a form that is understandable by their fellow citizens. a survey com missioned and funded by the wellcome trust found that the majority of scientists believed that the public saw them as detached, poor at public relations, secretive, and uncommunicative. furthermore, they identified a lack of knowledge and/or interest in science within the general public as a major barrier to communicating concepts and ideas. most of those questioned believed that they were insufficiently trained to deal with the media; more impor tantly, the majority of scientists surveyed distrust the role of the mass media in communication of their results. the role of the mainstream media and popular press is primarily to entertain their customers and make money. in that light, it is not surprising that there is a tendency to focus on stories and issues that seize public attention. all journalists know that scares make good stories and fre quently generate a momentum of their own that does not require any facts to keep them moving forward. for example, in recent years we have seen the emergence of numerous scare stories in the media (flesh-eating bacteria, falling sperm counts, chlorofluorocarbons, bovine spongi form encephalopathy, harmful gm foods, etc.), many with little in the way of scientific evidence to support them. it is perhaps not surprising, then, that a climate has been created in which the ordinary person regards scientific developments with suspicion, having the under lying assumption that he or she is being put at risk by reckless scientists operating in an uncontrolled manner in their ivory towers. this perception is not helped by hollywood, which provides a seemingly endless diet of disaster films in which dastardly government scientists are either blowing something up or pursuing genetic experiments in a top secret government laboratory to produce new species that could escape and destroy the world. when was the last time a blockbuster film was released in which a dedicated scientist carried out an experiment that did not involve a chiseled-jawed hero saving the day? a further element that may contribute to the public distrust of science is the rise of pseudoscience, which includes topics such as astrology, alternative medicine, yogic flying, and ufos. indeed, it has been suggested that the entertainment industry (e.g., the popular x-files series) is partially responsible for the large numbers of people who now believe in astrology, esp, alien abduc tions, and other forms of pseudoscience that contribute to the scientific illiteracy of the public. against such a cultural backdrop, it is not surprising that the public has little problem in believing that government scientists employed in defense-related research are not to be trusted. as a consequence, scientists, particularly those engaged in research considered dual use, find themselves in an almost impossible position when trying to explain their research and allay understandable fears harbored by the public. so why are members of the public concerned about research sponsored by the defense community? many nations view research into the development of medical countermeasures (mcms) against biowarfare agents as an essential element of risk reduction. although civilian and military populations are equally susceptible to the same biological agents, the relative risk of exposure differs markedly. thus, although there is considerable common ality in the research priorities of each group, some biological threats, such as anthrax and plague, are cur rently seen as being more relevant to the military. in addition, the nature of the work undertaken by the mili tary and the environment in which it operates are likely to influence how and when mcms are administered. for example, the military may consider immunizing troops with a new vaccine prior to deployment as the most effective means of protecting individuals and ensuring operational effectiveness in a high-risk environment. in contrast, the civilian authorities are more likely to treat with antibiotics after an outbreak has occurred rather than vaccinate large numbers of the public against a disease with a very low likelihood of a deliberate release (albeit very high impact), such as anthrax. thus, differences in the relative risk of exposure of each target population are a major driver of the research undertaken by defense scientists. to develop mcms capable of dealing with biothreat agents such as anthrax, there is inevitably a requirement to handle and manipu late these dangerous pathogens, which in turn generates concerns, rightly or wrongly, about the possibility of their inadvertent release or potential misuse (dual use). indeed, these concerns derive partly from the fact that the government-sponsored organizations currently devel oping defensive mcms were engaged many decades earlier in the development of offensive biological weap ons. although this research was discontinued in the s in the united kingdom, there are still concerns, in some quarters at least, regarding the potential for this type of work to be resurrected. when the public's mistrust of politicians and scientists is added to this mix, it is not difficult to understand why people are willing to believe the worst. indeed, the perceived lack of 'public visibility' of defense research further stimulates the public imagination despite the fact that the results of this research are widely disseminated through peer-reviewed journals and at international conferences. the nature of modern research is such that it is rare to find a project that does not require collaboration with academic and/or industry-based part ners, thus ensuring at least some degree of scientific visibility. in addition, the regular inspection of defense research facilities by national regulatory agencies or under the auspices of international treaties is an attempt to alleviate some concerns. openness, combined with inspection by independent scrutinizers, is an important tool in tackling dual-use concerns. if one accepts that defense-related research is war ranted, then how does one justify the development of a new medical countermeasure costing millions of dollars to protect against an event that may never happen? this is particularly important given that any new mcm must first undergo clinical trials in human volunteers to demonstrate both safety and efficacy. this will require the exposure of healthy individuals to an experimental treatment that carries with it the risk of adverse reactions. given that this is a man-made risk, how can this be justified? fortunately, in countries such as the united kingdom, these decisions are taken out of the hand of the defense scientists. indeed, investigators conducting clinical trials need to justify their study to an independent research ethics committee, which determines if any potential health risks to trial participants are justified. a key element in the committee's deliberations is to determine if there is a real-world justification for the new mcm; thus, the committee represents an important reality check. once the study has received approval, it is subjected to further scrutiny at the national level in the united kingdom by the medicines and healthcare products regulatory agency (mhra). each of these layers of control has the power to stop a clinical trial if it is concerned that an ethical breach has occurred and thus plays a key role in preventing inappropriate research. although regulatory scrutiny is vital to prevent harm to volunteers, a further level of protection is provided by the financial realities of drug development. the cost of bringing new mcms to market is considerable, amount ing to hundreds of millions of dollars, and as a consequence, the engagement of the pharmaceutical industry is essential. drug companies are focused on making money and given the relatively small size of the military market will only invest significantly in the devel opment of mcms that could also be used to protect civilian populations. thus, any mcm derived from mili tary research will undergo intensive public scrutiny on route to being licensed and, as a consequence, will be exposed to intense public and financial scrutiny. even when an mcm has been approved for human use, there are still questions regarding its administration to service personnel. for example, should immunization with biodefense-specific mcms such as the anthrax vac cine be mandatory as it is the case for the u.s. army? this raises issues of military governance and consent to treat ment, which can only be dealt with by the relevant law in each country. if new mcms are being developed, there needs to be consideration of the target population in advance of likely use. it would be wasteful for defense research to develop new drugs that would not be accep table to service personnel and would therefore be effectively unusable. public concern regarding dual-use issues and the ethics of performing defense-related research has led to the instigation of a range of checks and balances in the united kingdom designed to reduce risk to a minimum. the effectiveness of these measures is rightly open to public debate, and it is hoped that future scientists, as well as members of the public, will be encouraged to make a full and active contribution to this debate to ensure that future regulatory decisions are based on evidence rather than driven by popular misconception. biological material with the capacity to cause harm can be found in a range of different institutions (hospital, aca demic, pharmaceutical, and government establishments, both civilian and military). potentially, such organisms could be released into the environment following unfore seen accidents, due to negligence, or by deliberate intent. however, experience to date shows that the actual like lihood of human infection as a result of deliberate or accidental release is vanishingly small, particularly com pared to that of contracting infections naturally or suffering harm from other types of accidents or being the victim of a criminal or terrorist assault of some kind. the disproportionate fear that the threat of such infec tions arouses in the general population reflects a lack of understanding of the nature of risk, hazard, and proba bility, coupled with an understandable tendency of the popular media to exaggerate the impact of rare or ima gined spectacular events. the research undertaken in organizations in which microorganisms can be found has produced results of enormous benefit to human society in terms of improving health outcomes (better sanitation, advances in medicines, and vaccines) and increasing safety and efficiency of food production. future developments in biotechnology hold the promise of major benefits to humanity in such diverse fields as mitigating the impact of climate change, improv ing agricultural yields in poor areas of the world, synthesis of novel materials on an industrial scale (e.g., biofuels), and the discovery of cures for major scourges such as tuberculosis and malaria. how do we balance the enormous potential for good that biotechnology offers against concerns regarding its misuse? in the united kingdom, the vast majority of microbiological research is performed in civilian organi zations, with only a very small fraction being conducted by defense laboratories. research activities in defense and civilian facilities in the united kingdom are carefully regulated by a number of statutory bodies such as the health and safety executive, which monitors studies involving genetically modified organisms, and the home office, which oversees experiments involving animals. clinical trials involving human volunteers are regulated by the mhra and are overseen by research ethics com mittees (which are themselves approved by the uk ethics committee authority). however, more regulations, such as intrusive psychological profiling of staff working in microbiological laboratories or heavy-handed, overbear ing, rigid assessment programs of scientific staff to 'ensure' reliability, are unlikely to further reduce the probability of an already extremely unlikely event. rather, they are more likely to alienate well-motivated staff, thereby sti fling research and the development of products and techniques that could bring major benefits to the united kingdom and humanity as a whole. indeed, fostering a supportive community among well-rewarded and appre ciated scientists and staff would make it much easier to detect early signs of unhappiness, social problems, or the unacceptable behavior of individual researchers. such an approach would also be expected to produce better scien tific outcomes. the role and responsibility of scientists is central to minimizing misuse of technology, and thus it is vital that life scientists are encouraged to take ownership of this problem and in doing so assume a more proactive role in regulating, communicating, and explaining their activities to the wider public. unfortunately, to date, the majority of scientists have demonstrated a marked reluctance to fill this role for the reasons outlined previously. it has been suggested that improved education of scientists, the media, and the public would go some way toward addressing this issue. improving the aware ness of scientists could take many forms, such as the inclusion of teaching material covering biosecurity and dual-use issues into the curriculum of all life science undergraduates and in seminars, conferences, and pub lications dedicated to the subject. scientists, particularly those engaged in areas of research that have the potential for misuse, must be encouraged to communicate the nature of their research as widely as possible to their fellow citizens. the most obvious vehicle through which to achieve this aim is the mass media, which is in a position to be a creative and positive influence in bring ing scientists and the public together around these issues. it has the capability to improve communication and understanding, reducing unwarranted fears and sensa tionalist reactions to imagined threats. how we achieve this utopian dream in the face of the economic realities of a / multimedia society is a question beyond our powers. the committee for skeptical inquiry uk -the health and safety executive les baillie is a professor of microbiology within the welsh school of pharmacy of cardiff university. prior to joining the university in , he was director of the biodefence medical countermeasures department based at the naval medical research center in washington, dc. in this role, he led a multidisciplinary team developing novel therapeutics to combat the threat posed by biothreat agents. this research built on previous experience gained at dstl porton down, where he led a research team working on anthrax. having worked in both government defense and academic laboratories on two conti nents, he has a unique insight into the challenges faced by life scientists engaged in research in this area.dr. hugh dyson is a principal medical officer at dstl porton down. he has previously worked in the national health service and academia, holding posts in renal medicine and pharmacology.dr. andrew simpson is a clinical microbiologist at dstl porton down. he has previously held posts in the national health service and academia, and he worked for many years in thailand at the mahidol oxford tropical medicine research unit. key: cord- -vrqltz s authors: nan title: isar news date: - - journal: antiviral research doi: . /s - ( ) - sha: doc_id: cord_uid: vrqltz s nan as i enter the last six months of my presidency, i'm amazed how fast time has moved. soon we'll convene in la jolla for the th icar, and i'll hand over the reins to josé esté as our next president. we'll also know the results of the election for president-elect, for which we have two excellent candidates in rhonda cardin and johan neyts. after many years of participation and service to isar, it's great to know that we continue to attract such high quality people, who will lead our society forward. the program for the th icar is coming together nicely under the leadership of mark prichard, and we're just entering the abstract submission phase, using our new electronic submission system. i hope that everyone will find the site easy to use, and i look forward to your feedback. the best part of the new submission website is that we can modify many of its features, to provide a much better experience for both abstract submitters and reviewers. as this issue of isar news goes to press, i hope that everyone will have taken a moment to vote for our new officers and board members. for many years, voting has unfortunately remained fairly constant at around % of our membership. this year phil furman and the nominations committee have solicited an excellent slate of candidates, who have all been heavily involved with isar and represent the future of our society. i hope that we'll double the turn-out this year, especially since voting is now easy, using the system set up by our webmaster andrea brancale. the past year has also seen some new developments in isar that i hope will produce lasting benefits. first, raj kalkeri has formed an initial team of isar ambassadors around the world, who are now hard at work soliciting new sponsors and members. the ambassador program utilizes the isar website, facebook and other social media to spread the word about the society and our annual meeting. if you would like to get more involved in isar, i strongly recommend that you join our ambassador program and help bring more people to the meeting in california. we all know scientists from our local geographic areas and countries who would benefit from attending icar, and it will greatly benefit from their new ideas and vision. in another important development, the women in science committee is now soliciting applications for the second wis scholarship program -now proudly known as the chu family foundation women in science scholarships. thanks to generous funding from the chu family, up to five scholarships will be awarded for . applications are due by december st , so please encourage women students and postdocs to submit their applications. unfortunately, one of my goals as president that has not yet materialized is my desire to use the wis program as a model for developing ways to ensure that all young scientists are able to attend icar and contribute to the society. during my last six months in office, i would like to develop a committee of young researchers that would meet at icar and have regular conference calls between meetings, to discuss how the society could promote more participation from graduate students, postdocs and other young investigators. over the years, we've had special sessions at icar featuring talks by our young members, such as the shotgun poster presentations and a fantastic euvirna session at raleigh. we've also held young investigator receptions during the meeting (always one of my favorites!). i hope that many young scientists from around the world will want to get involved in the society through membership on this new committee, and will work with me to promote participation in icar. if you're interested, please email me, so that we can get started! i hope everyone will enjoy this informationpacked issue of isar news, and appreciates the efforts of our publications committee to expand it to four issues per year. it takes plenty of work to pull these issues together in a timely fashion, and i know that the guest editors spend a lot of time sending out reminders for submission of articles. i also hope that our newsletter will begin to feature more news items submitted directly by our membership. please let us know what's going on with you and your research, such as new grants, publications, and products. to summarize: submit abstracts, apply for chu family scholarships and volunteer for the ambassador or young investigator programs! i look forward to seeing everyone in la jolla in april. bob buckheit, isar president the th international conference on antiviral research will be held at the hilton la jolla torrey pines hotel in la jolla, california from sunday, april th through thursday, april st , . this venue will take advantage of the vibrant research community in the san diego area. while the meeting will focus on the latest scientific developments in antiviral research, it will also emphasize the interdisciplinary nature of the field and will foster collaborations by providing dedicated time for networking with colleagues. icar is designed to provide opportunities for virologists, chemists, pharmacologists and clinicians to establish and maintain the close collaborative relationships that are needed for the discovery and development of effective antiviral therapies. it also serves to stimulate innovative thinking on the drug development process, and provides specific events to welcome new scientists to our ranks to help them to establish successful careers. the breadth of viruses discussed and topics covered provide a rich environment to learn how scientists in different fields approach problems common to the development of all antiviral therapies. the conference will begin with a session on drug discovery and development at pm on sunday, april th . rich whitley, a past president of isar, will introduce the new "antiviral drug discovery and development center" funded by niaid/nih. this consortium illustrates how the cooperation of us academic institutions with a not-for-profit research organization and a commercial partner can lead to the rapid development of therapies for emerging infections. this interactive session will feature presentations by maaike everts, mark denison, bob bostwick and rob jordan. the th icar will begin at pm on april th , with presentations by two keynote speakers: ♦ heinz feldmann, m.d., ph.d. (niaid/nih) will speak on "ebola virus: past, present, future", reviewing research from the original discovery of the filoviruses in marburg, germany through the present west african epidemic. he will also provide a look ahead: what research is needed, where do we go from here? ♦ richard h. scheuermann, ph.d. (j. craig venter institute) will speak on: "decoding viral genomics in the next generation era". the availability of whole-genome sequence data, combined with standard representations of virus phenotypic characteristics from large numbers of viral isolates is allowing for extensive genotype-phenotype association studies that go well beyond traditional phylogenetic lineage tracing. his lecture will demonstrate the use of statistical genomics analysis to predict influenza virus evolution in the face of adaptive immunity and to identify novel genetic determinants of disease severity in enterovirus d . the first of two symposia will take place on the morning of monday, april th and will highlight recent developments in the use of structural biology to discover and develop antiviral drugs. the session is being organized by a trio of prusoff young investigator award winners: andrea brancale, bruno canard, and erica ollman saphire. the second symposium, focusing on dna viruses, is being organized by rhonda cardin and graciela andrei and will be held on the morning of wednesday, april th . it will feature recent advances in therapies and will included presentations by thomas lion on adenovirus infections, margaret a. stanley on papillomavirus, david bernstein on herpesvirus vaccines, and timothy kowalik on the evolution of human cytomegalovirus. each year icar features a poster awards competition, and the tradition will continue in . the committee, chaired by kathie seley-radke will review the candidates for awards. in past years, the competition has been intense and the program committee is fortunate to have dedicated members who are willing to serve on this important subcommittee. cash prizes of up to $ will be awarded in the categories of graduate student, postdoctoral fellow and young investigator. awardees will also have the opportunity to present their work in the shotgun presentation session. the prominence of the poster presentations at icar reflects their excellent quality and offers both new and experienced investigators a high-profile venue to present their work. the icar program, the posters presented and many of the posters will be placed on the isar website, so that members can review the material before and after the meeting. the program committee and the society are committed to bringing you the most rewarding scientific experience. in response to feedback from our membership, the committee has worked diligently this year to make changes to the meeting format, keeping the best features of icar, while adding scientific sessions and events which we believe will heighten the experience for all attendees. following the success of the session on women in science at the rome icar, it will be held again in , and will include panel discussions to provide advice to women scientists as their careers progress. the society will also maintain its commitment to the newest isar members by once again sponsoring an interactive career forum, in which attendees meet with established scientists and other professionals in the pharmaceutical, biotech, academic and government sectors to discuss various career options. there is no additional fee for the career forum, but space is limited, so attendees should indicate their interest when registering for the meeting. on thanks to a generous donation from the chu family, as many as five young women with the potential to make significant contributions to the field of antiviral research will receive $ scholarships in . the funds may be used to attend a conference, visit a laboratory, take a course or acquire specialized training. in general, the career development activity should not be one for which the applicant's advisor is already funded. the awards will also include a -year membership in isar and a commemorative certificate. to be eligible, an applicant must be working in an area of antiviral research and either be an undergraduate or graduate student, or have no more than five years of cumulative postdoctoral experience. graduate students and postdocs must be a member of isar at the time of application. each applicant must submit: • a cv and a statement, not exceeding two pages, describing her academic achievements and goals, including an explanation of how the award will help her career. • a letter of support from her research project director, department chair or center director. • if the funds will be used to visit another scientist's laboratory, the candidate must submit a letter from the head of the lab. • if the funds will be used to attend a meeting or take a course, the candidate must provide a description of the activity, including a link to online information. successful candidates will have demonstrated their ability to do independent scientific work, their potential for a high level of scientific endeavor and their leadership skills. for more information, and to apply online for a scholarship, go to http://www.isar-icar.com/?page=wiscda applications must be submitted by december st. winners will be selected by the cff award committee by march st , , and the awards will be presented at the th icar in la jolla. ilane (pronounced "elaine") studied veterinary medicine in her home country of mexico. in her first research project, she joined a group developing a dna vaccine against salmonella infections in chickens, and her thesis won the award for excellence in veterinary public health from paho in . however, when the novel swine-origin h n virus emerged in mexico that year, she switched her focus to influenza diagnostics and epidemiology. working jointly for the ministry of agriculture and the fao, ilane and her colleagues validated new diagnostic tests and screened specimens from hundreds of pig farms. she notes, "i quickly realized that i preferred virology to bacteriology." in , ilane moved to the netherlands to enter a masters program at wageningen university in cellular and molecular biotechnology, focusing on development of next-generation viral vaccines. when it came time for further training, her supervisor drew her attention to the european training network ilane hernández morales on (+)rna virus replication and antiviral drug development (euvirna). she applied for a ph.d. position in the consortium, and in became a euvirna fellow, conducting research on dengue virus in a project that reflects a strong interaction between academic science and the pharmaceutical industry. her day-to-day work is at janssen infection diseases and vaccines, part of j&j belgium, under the supervision of dr. marnix van loock, in collaboration with prof. johan neyts at the rega institute. she is studying the response of primary human mononuclear cells to dengue virus infection, aiming to establish a more relevant in vitro model for antiviral drug testing and to identify potential new antiviral targets, using genome-wide transcriptomic analysis. my primary interest is the study of virus-host interactions. i would like to contribute to the field of antiviral drug development by identifying new targets for prevention or treatment. i'm also strongly committed to bringing new knowledge and research collaborations back to mexico and to learn linking basic research to the "real world." i hope to make a positive impact as an individual -and specifically as a woman scientist -in under-represented groups, developing societies and in science itself. i attended the icar in raleigh, where the emphasis given to connecting with young scientists impressed me. networking was a priority, not only among experienced researchers, but more importantly with young ones. i was very motivated by all the members who invested so much time, effort and "heart" in sessions such as women in science and career discussion and networking. at that time, i already knew andrea brancale, who was my mentor in euvirna. i told him the great joy i felt meeting and learning from all the people attending icar, and asked how i could become part of such a great team. he suggested that i join the isar communications committee and help manage the isar facebook page and twitter media. i could only say "yes!" it was the right moment of inspiration, not just because of the friendly environment, but also to match my long-standing wish to work in science communications. what is your personal experience with social networking and science? i started following virology news by listening to vincent racaniello's podcast, "this week in virology," while on the bus going from antwerp to the j&j lab. it's been extremely valuable -like reading three scientific papers in one hour! also, in the euvirna training program, i attended a workshop on science dissemination and society outreach through social media. i got some "tips and tricks" from professors and experienced teams on blogging, facebook and twitter. together with euvirna fellows, we prepared a blog about viruses and antiviral research, which was reviewed by professor racaniello. he gave us serious feedback that helped me to get some experience in this kind of communication. one of my main activities is to follow science news sources such as science, nature and cdc for items of interest to our members, which i then post on isar's facebook page or send out on twitter. for each item, i include a link to the source and a short comment or paragraph explaining the information. i also contact scientists to ask permission to use figures from their publications. this has been very rewarding, because they typically are willing to share, and they also get to know isar. it's incredible how much networking a simple image can bring. for example, a picture that i posted from the "new attendees" reception at the rome icar reached almost people in less than a week -more than the total attendees! how can isar members make use of our social media sites? first, go to facebook, twitter or linkedin and sign up for an account, then look for isar in the network and like the page (facebook) or follow (on twitter). once you've signed up, then every time isar posts something new, you'll receive a paragraph called a "post" from facebook or a character "tweets" from twitter, and if you're interested, you can follow the link to the complete story. the isar linkedin page is mostly managed by andrea brancale, for sharing information about isar activities. once you have a facebook account, you can post information on the isar page by using the "post" space. you may respond to posts using the "comment" space. once you've signed up for twitter, you'll see the "tweets" automatically in your notification page. you may respond to a new tweet in two ways: "re-tweet," to share it with your followers; and "reply," to post your comments. members can make use of these sites in many ways, such as: • use isar social media to enhance communication with isar members and promote society activities; • network with isar members and with others doing antiviral research; • find reminders for important isar deadlines, such as nominations for the wis award and icar abstract submission; • promote the ambassador program. • find photos from past icars, including the poster sessions and the banquet; • share a link to a recent publication; • exchange information from other webpages and sites, such as news from research groups, job openings, etc. i encourage you to give it a try -there's a big benefit that you can only see once you've tried! the best thing is that social media is flexible and offers different resources for all personalities. other agencies and communities, such as cdc, who, nasa, etc., have had great success using social media. if you would like more information, or have a news item that we could publicize through the isar social network, email me at ilane.hernandezm@gmail.com those of us who follow news of the ebola epidemic are aware of the case of the scottish nurse who was accidentally infected in late while working as a volunteer in sierra leone, was treated at the royal free hospital in london and discharged this past january, but was readmitted to the same hospital in early october, when her ebola virus infection relapsed in the form of acute meningitis (see link below). this recurrent infection is an example of the increasingly recognized ability of ebola virus to persist in anatomical sites that are relatively inaccessible to the immune system, such as the chambers of the eye, the central nervous system and the seminal vesicles. fortunately, it's the only known example to date of a recrudescence involving the central nervous system. another unusual feature of this case is the antiviral medication used to treat the nurse, a new gilead compound, gs- (above) (gilead sciences press release, oct , ). according to isar member robert jordan, who heads the gilead team developing antivirals against respiratory viruses, the parent compound was originally discovered as part of the hepatitis c program, targeting the hcv polymerase, but the strong clinical efficacy of sofosbuvir, especially in combination with ledipasvir, resulted in the molecule being evaluated for other indications, including respiratory viruses such as respiratory syncytial virus (rsv). the story of how gs- moved from in vitro testing against respiratory viruses in california to an ebola patient in a high-security unit in london provides a good example of how a promising drug can be fast-tracked in an emergency. it began in , when robert decided to expand the testing of gilead nucleosides and nucleotides from rsv to two other members of the paramyxovirus family, the highly virulent nipah and hendra viruses, by establishing a collaboration with michael lo in the viral special pathogens branch at the cdc in atlanta. by may , when the ebola epidemic in west africa began to spread, the cdc researchers began testing the gilead compounds against filoviruses and identified a modified ′-cyano-substituted adenine cnucleoside ribose analogue gs- as a novel inhibitor of the west african ebola strain and other ebola species (below). the promising in vitro data led to further collaboration between gilead and usamriid to evaluate gs- in a macaque model of lethal ebola virus disease. a team headed by travis warren tested multiple different treatment doses and regimens, and obtained complete protection when they gave a daily iv dose of mg/kg, beginning on day postinfection. as some of the animals were already viremic when treatment began, the result was very impressive: usamriid science director sina bavari stated in a press release "this is the first example of a small molecule which can be easily prepared and made on a large scale, that shows substantive postexposure protection against ebola virus in nonhuman primates." as a consequence of the successful in vivo testing, gilead filed an ind this past july for the use of gs- to treat ebola virus disease, and phase i testing in healthy human volunteers began in august. when the scottish nurse was hospitalized in early october, sufficient data had been obtained to support compassionate use of the drug. as described by robert and tomas, gs- employs a phosphoramidate prodrug strategy that is similar to sofosbuvir and the recently approved tenofovir alafenamide. the prodrug structure enables the molecule to permeate the cell membrane and rapidly enter the cytoplasm. the ester bond is then cleaved by a cellular hydrolase, followed by chemically catalyzed release of phenol to yield the negatively charged ala-nucleotide conjugate, which is cleaved by an amidase to release the monophosphate nucleotide. cellular kinases then quickly produce the triphosphate molecule, which is incorporated into viral rna, terminating its synthesis. the prodrug strategy thus avoids the slow first phosphorylation kinetics of nucleoside analogues and greatly enhances intracellular concentration of the active triphosphate metabolite. additional in vitro testing at usamriid and at the university of north carolina, chapel hill has found that gs- is also active against the mers coronavirus. it will be interesting to see if gs- will have as great an impact on the treatment of ebola, mers and other severe rna viral infections as sofosbuvir has had on the therapy of chronic hepatitis c. (thanks to travis warren, robert jordan, tomas cihlar and michael lo for information and editing of this article.) http://www.nytimes.com/ / / /world/europe/ new-clues-into-ebola-as-ill-nurseimproves.html?_r= following the success of euvirna (www.euvirna.eu), whose participants received many prizes at icar , a new training network has been funded by the european union: antivirals. its mission is to prepare talented young researchers for leading roles in antiviral drug discovery in european industry or academia, by providing them with a multi-disciplinary, intersectoral training program that may lead to a ph.d. degree. the antivirals partnership includes seven outstanding european academic partners and four industrial partners (see map and logo above). all are leaders in their field of expertise, ensuring state-ofthe-art training, and their skills are highly complementary. three of the academic partners (utrecht university, leiden university medical center, university hospital of heidelberg) specialise in various aspects of molecular virology, while the other four (cardiff university, université d'aix marseille, university of leuven, university of vienna) focus on the identification and development of novel antiviral compounds and strategies. the four industrial partners include a pharmaceutical r&d company specialising in antiviral drug discovery and development (aicuris), another large company specialised in medicinal chemistry (prestwick chemical), a small company that pioneers an exciting novel class of biopharmaceuticals (complix), and a small business that develops antiviral drugs for animals (virovet). a company specialising in training (virology education) will contribute its expertise to the programme. to make a serious impact, the next generation of scientists must do more than good research. the antivirals network will therefore organise training both on relevant research topics, such as virus replication, structural biology and medicinal chemistry, and on a wide range of transferable skills, including teamwork, science communication, dissemination and societal outreach, ethics, biosafety, innovation and entrepreneurship and intellectual property rights. the selected group of young researchers consists of fourteen women and one man from different countries. they were chosen from hundreds of applicants from all over the world, and they excel in enthusiasm, laboratory skills and their potential to become researchers with a broad view and the desire to make a societal impact. they will each work on their own projects, which aim to gain insight into antiviral drug development for a selected group of viruses. in three years, you'll have the opportunity to meet these young interdisciplinary, intersectorial researchers at their final conference, hopefully at isar ! for more information, go to www.antiviralsetn.eu or contact the project manager antivirals-etn@uu.nl. this project has received funding from the european union's horizon research and innovation programme under the marie skłodowska-curie grant agreement . for more than years, scientists at the rega institute have made important contributions to the development and clinical application of antivirals against herpesviruses. that tradition continues with the creation of regavir, the research group for antiviral resistance. graciela andrei and robert snoeck, the program directors, would like to make sure that isar members are aware of what this reference center has to offer. regavir provides clinicians with rapid genotyping and and/or phenotyping of clinical isolates of human cytomegalovirus, herpes simplex virus and , varicella-zoster virus and human herpesvirus . the staff also provide assistance with the interpretation of results and treatment options. since , regavir has performed about , herpesvirus drug-resistance tests, and is now recognized as the belgian reference center for drugresistant dna viruses. thanks to funding from the belgian national cancer plan, an interactive network of hospitals in belgium and abroad has been established and is continuously growing. regavir carries out herpesvirus drug-resistant tests, assists clinicians in the choice of the adapted therapy and promotes scientific interactions in this network. the rationale for the establishment of regavir lies in the increasing importance of antiviral therapy for herpesviruses, which cause significant morbidity and mortality among cancer and leukemia patients, recipients of stem cell and solid organ transplants, neonates, patients with genetic immune deficiencies and hiv+ individuals. these viruses often reactivate and cause persistent infections that require prolonged treatment, increasing the risks for selection of drug resistant mutants. it is remarkable that the antiviralresistance of hsv has remained at a low level (< %) for three decades in immunocompetent individuals, but they are also susceptible to herpesvirus infections in "immune-privileged" sites, such as the eye and the central nervous system; the latter is life-threatening, and requires rapid therapeutic decisions. except for acyclovir, its oral prodrug valacyclovir and famciclovir, current fda-approved drugs for herpesviruses are associated with some toxicity. ganciclovir, its oral prodrug valganciclovir, foscarnet and cidofovir may produce hematologic abnormalities (primarily neutropenia, anaemia, and thrombocytopenia). ganciclovir has also caused long term-reproductive toxicity, and cidofovir and foscarnet are nephrotoxic. alternative antiviral regimens are therefore preferred, to avoid cumulative toxicity and the selection of multidrug-resistant viruses. physicians typically recognize drug-resistant herpesviruses based on signs of infection in patients. regavir aims to support clinical decision-making by providing prompt characterization of the virus. correct use of the few available drugs is necessary to avoid selection of multiple-resistant viruses. simultaneous adjustment of antiviral and immunosuppressive therapy enhances the success of treatment, and also decreases health care costs. for more information, please go to www.regavir.org or contact us at robert.snoeck@rega.kuleuven.be, graciela.andrei@rega.kuleuven regavir@rega.kuleuven.be in march, , antiviral research published two reports of the efficacy of the novel antiviral favipiravir (t- ) against ebola virus infection in ifn receptor-knockout mice. one study, by steve lever's group at the dstl at porton down in the uk showed that aerosol-infected mice were protected by treatment beginning an hour after virus challenge [ ] . the other, from stephan günther's team at the bernhard nocht institute of tropical medicine in hamburg, found that intranasally-infected mice were protected against death when treatment was started as late as days postinfection, when the mice were viremic and showed biochemical evidence of liver injury [ ] . probably because it demonstrated the efficacy of favipiravir late in the course of infection, the paper from the hamburg bsl- lab attracted wide interest, and it has been downloaded some , times, more than any other article in the history of avr. the two simultaneous reports on the anti-ebola activity of favipiravir may have helped to jump-start efforts to deliver the drug to patients in the west african epidemic: an open-label phase ii trial began months later at two treatment centers in guinea, as a collaboration between inserm and médecins sans frontières (see nct on www.clinicaltrials.gov). the first report of antiviral drug testing by the hamburg lab appeared in , when stephan and his colleagues described the utility of rt-pcr in drug screening [ ] . however, antiviral testing has accelerated in the past two years, as postdoc lisa oestereich (below) has taken the lead in evaluating antivirals against several highly pathogenic viruses. she was first author on the study of favipiravir treatment of ebola-infected mice, and in a follow-up paper, she and colleagues in hamburg and in france performed mathematical modeling of the effect of favipiravir therapy on the kinetics of ebola virus replication, using data from the earlier paper [ ] . for her doctoral thesis at the bni, lisa developed a new mouse model of lassa fever, and in a recently published paper, she and coworkers describe the use of that model to demonstrate favipiravir's activity against lassa virus, alone or in combination with ribavirin [ ] . lisa and her colleagues have also examined the efficacy of favipiravir, ribavirin and arbidol in mice infected with crimean-congo hemorrhagic fever virus [ ] . stephan's research in hamburg dates from , when he began a postdoctoral fellowship at the heinrich pette institute, studying hepatitis b virus. the direction of his career underwent a major change in , when he accepted a position as a research group leader at the bni and shifted his focus to tropical medicine and viral hemorrhagic fevers. in , he published his first paper on lassa fever, a report of a case imported into germany [ ] . he has since become a world authority on the disease, as author or co-author of more than articles. in , stephan became head of the department of virology at the bni, director of the bsl- lab and of the who collabor-ating centre. in addition to antiviral drug testing, stephan's group performs routine diagnostic services for more than different tropical and emerging viruses, testing samples from about patients each year, including cases imported into europe. they also routinely carry out isolation and biobanking of lassa, ebola and other bsl- viruses, providing them to academia and companies within the european virus archive (http://www.european-virus-archive.com/). since , the laboratory of virology has had a very successful collaboration with the irrua specialist teaching hospital in nigeria, focusing on the development of rapid diagnostics and research on the pathogenesis and immunology of lassa fever. other collaborative research projects are under way in guinea and ghana. any isar member interested in influenza and ebola virus infections should know about two websites that provide access to a wealth of scientific resources on those diseases. at www.filovir.com and www.influenzavir.com you'll find links to recent news items, scientific publications, official reports, notices of upcoming conferences and other useful information. both sites interface with the worlds of social networking and micro-blogging via twitter, which through manually curated annotation and filtering delivers timely, updated scientific news. in particular, filovir has provided daily coverage of the ebola epidemic in west africa, with tweets of news alerts almost in real time. luca zinzula at the max planck institute, munich the websites are the brainchild of luca zinzula (above), who created them in - , while he was a ph.d. candidate in the lab of enzo tramontano in cagliari, sardinia. the concept of filovir was born when luca found that, despite the existence of numerous virus-related databases on the internet, the information they presented was highly disparate, and it was limited to specific sub-fields and disciplinary areas. especially for ebola and marburg viruses, there was no middle ground between the extreme level of detail of repositories intended for specialists and the superficial (and frequently incorrect) information on non-scientific websites. luca's concept was to bring together information from a wide range of authoritative third-party resources in real time in a single web environment, in which every tool would be readily accessible and navigable. his goal was to create a virtual space where a researcher could find abstracts of the latest publications, pin-point the location of the latest outbreak, retrieve a genome sequence or a protein structure, or hear about upcoming conferences, with everything "just a click away". filovir went live in january , and one year later, influenzavir went online as a twin site focused on influenza viruses. once he had created the two websites, luca realized that he would need help to maintain them, and he was fortunate to obtain the support of two colleagues, massimiliano orsini, a bioinformatician expert in genomic databases, and cristian romagnani, a biologist who has moved to the field of network systems and big data analysis. luca presented the websites in a poster at the th icar in rome. in recognition of his efforts for the benefit of the virology community, he was recognized by antiviral research as the first recipient of an annual award for "most promising antiviral researcher." luca is a native of sardinia. before beginning his scientific studies, he spent several years working as a professional scuba diver, and was frequently involved in rescue and conservation activities for endangered marine species, with a special focus on monitoring infectious disease casualties. after hanging up his fins, he did undergraduate work in biochemistry and virology at the university of cagliari, then continued there for his doctoral research with enzo tramontano. he received his ph.d. from the university of rome tor vergata in for studies characterizing the dsrna-binding activity of the zaire ebolavirus vp protein. since the beginning of , luca has been a postdoctoral fellow in the lab of wolfgang baumeister at the max-planck institute of biochemistry in martinsried, munich, learning cryoelectron micro-scopy and using the technique to further investigate the role of the vp protein in filovirus replication and innate immune antagonism. in addition to his current work with cryo-em, luca's wide-ranging scientific interests include the expression, purification and characterization of recombinant viral proteins, in vitro assays of protein-rna binding and immune evasion by highly pathogenic rna viruses. in , he and enzo published of a remarkably thorough review article in avr on the latter topic: zinzula l, tramontano e. strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit. antiviral res. : - . lieve is an associate professor in the laboratory of virology and chemotherapy at the rega institute, katholieke universiteit leuven, belgium. she is clearly very happy at the university of leuven, as she began her undergraduate studies there in , and has spent her entire career in the rega institute. my passion for virology began during my masters studies, when i did my thesis on the recombinant hepatitis b vaccine. i then by chance was invited for an interview with erik de clercq, and was charmed by his positive and enthusiastic attitude towards young researchers. i immediately decided to do my phd work in his lab. at that time, the acyclic nucleoside phosphonates (anps) had just been discovered, and i was very lucky to be able to work on their preclinical development. my doctoral project focused on the pharmacokinetics and efficacy of antiretroviral anps in animal models, and my direct mentor was jan balzarini. i also had close interactions with the team of antonín holý in prague. in collaboration with gilead, i was the first to demonstrate the in vivo efficacy of the oral prodrug of tenofovir ( ), soon before it became a leading hiv blocker. at the end of my ph.d. period, i got the chance to compile my insights into this successful drug class in a highly cited review article in antiviral chemistry & chemotherapy ( ) . at the start of my postdoctoral period, i left the field of antiretrovirals, but not the anps. having worked with adefovir and tenofovir, it was easy to move to anti-dna virus anps like cidofovir. collaborating with my colleagues graciela andrei, robert snoeck and johan neyts, my new research focused on two dna viruses: adenovirus and hhv- . both are important pathogens, especially for recipients of organ or stem cell transplants, but unfortunately they are very much neglected in antiviral drug development. i also became involved in the hhv- foundation, which aims to keep this neglected virus on the research agenda. for many years, i have been a member of their scientific board. during this "dna virus period", i was assisted by my first two graduate students; our focus was on the virus-host interactions for hhv- and adenovirus, and how this can be combated with novel inhibitors ( , , ) . it was also during this period that i was appointed for academic teaching (i teach virology and immunology). as i just mentioned, i'm on the scientific board of the hhv- foundation, where my main role is to give advice on antiviral therapy. at the moment, however, i'm not performing any drug development for hhv- , since the need for new therapies is not clear. in the clinic, patients are treated with the classical anti-herpetic agents, such as ganciclovir and foscarnet. brincidofovir also appears to be a promising new drug for hhv- infections. after working for several years on dna viruses, i decided to shift my focus to influenza, because it was becoming evident that the current antivirals, which are neuraminidase inhibitors, are not sufficient, and new drugs are clearly needed. from a virological viewpoint, this was a new virus, but from a technical/experimental standpoint, i could rely on expertise i had acquired with other viruses. my group now has two main topics for influenza antivirals: inhibitors of the polymerase complex and inhibitors of viral entry. the first encompasses inhibitors of pa, pb or pb , or a cellular factor associated with the viral polymerase. regarding the influenza pa endonuclease, i am the driving force behind a network of academic researchers with expertise in medicinal chemistry, structural biology, enzymology and virology ( , ) . our cell culture and biochemical data are of high and direct relevance for the development of pa inhibitors, a drug class that several pharmaceutical companies are working on. inhibitors of viral polymerases are a good fit for our lab, because this concept has been a continuous line of research in my career. the second topic includes inhibitors of the hemagglutinin, such as fusion inhibitors ( ), or compounds that interfere with a cellular factor involved in virus entry, such as compounds that inhibit endocytosis or the proteolytic activation of ha ( ). the influenza virus hemagglutinin is an amazing research topic. even though this protein has been studied extensively, there are still knowledge gaps regarding its link to receptor signaling and the biochemical details of its proteolytic cleavage or species adaptation. hence, we use hemagglutinin inhibitors as tools to resolve these basic scientific questions. as for drug development, the hemagglutinin is not an easy target, given its subtypedependent and variable structure. because influenza viruses evolve very rapidly and easily develop drug resistance, i believe that inhibitors of a cellular factor will have high clinical relevance. some of these strategies are explained in our recent review article on influenza virus entry inhibitors ( ) . my influenza team now has seven coworkers. as the pi, i'm responsible for project design, mentoring graduate students, grant applications, writing papers, etc… i also have a network of medicinal chemists, mainly at european universities, with whom i collaborate to design and synthesize inhibitors. this is a very interesting translational aspect of my research. as an expert on anps, i hypothesized that these compounds may act like product inhibitors of hypoxanthine-guanine phosphoribosyltransferase, which is a crucial enzyme in plasmodium parasites and mycobacteria. the validity of the concept was proven in collaboration with the teams of luke guddat in brisbane, australia and the late antonín holý in prague. since our first joint publication in ( ), this has become a totally new concept in antiparasitic drug design. the project has been very successful, with many high-impact papers and citations. my current role in this network is the pharmacological aspect, relating to prodrug design, cell permeability and cytotoxicity. i strongly believe that pharmacologists should look at potentially new applications of inhibitor concepts. for instance, nucleoside analogues have been very successful as anticancer and antiviral drugs, and they can be equally relevant in the fields of antiparasitic or antibacterial therapy. i consider myself a pharmacologist-virologist. i don't do medicinal chemistry, but to reveal the interaction of a molecule with its viral target, i need to understand the sar of inhibitors, so some basic knowledge of chemistry is required. my goals are to find innovative drugs that inhibit viruses and elucidate their mechanism of action. the aim is not only to discover new inhibitors, which may have only short-term clinical relevance, but to use the inhibitors to understand viruses. it's a nice balance between applied and basic research. isar members know andrea (standing, above, with cardiff colleague salvatore ferla) as the winner of the prusoff young investigator award, but not all of us are familiar with the direction of his career, which has increasingly explored the use of computer-based methods to design new antivirals and anticancer drugs. andrea's involvement with antiviral research began in - , when he did an undergraduate project in romano silvestri's lab at the universita "la sapienza" in rome, investigating novel nonnucleoside rt inhibitors. after receiving his degree, he did his doctoral work in chris mcguigan's lab at the welsh school of pharmacy, focusing on the design, synthesis and evaluation of novel antiviral nucleosides. he became a research associate in , and has continued as a staff member. after finishing my undergraduate degree and military service, i wanted to do doctoral research in medicinal chemistry, possibly abroad. i applied to several places in the uk and the us, and chris mcguigan replied right away. it was very exciting to join chris's lab and continue to work in the antiviral field. in particular, i was very happy to have the opportunity to work on nucleoside analogues. difficult chemistry sometimes, but very satisfying when it works as planned! what aspects have you found most satisfying? i always wanted to have a career as an active, independent researcher, and i consider myself very lucky to have achieved it. the best part of my job is the possibility of interacting with a wide variety of collaborators in different fields. it's never boring, and i'm always learning something new. cardiff university, and my school in particular, is a dynamic and stimulating place, always committed to excellence. in particular, it's an ideal place to foster collaborations and develop new ideas: in the time i've been there, seven patents have been filed based on projects i've contributed to, and ten students have received their ph.d. under my supervision. my current group of three postdocs and five graduate students is involved in a variety of collaborative projects, and we receive funding from both the public and the private sector. to a certain extent, chemists like to work in specific fields, especially if they have solid collaborations with biologists, but they're also happy to move in a different direction if they have an indication that their beloved compounds could be interesting in another therapeutic context. from the point of view of computational chemistry, fidelity to a specific research area is even more tenuous, as the methods are "neutral," and can be applied to almost any drug design project [ ] . for example, i've collaborated on projects spanning all the way from designing antivirals against chikungunya virus [ ] to modeling simulations on small peptides for treatment of gastrointestinal diseases. what new approaches are you using in your research? i'm applying several different structure-based methodologies, including molecular docking, homology modeling and molecular dynamics. of course, we also make use of traditional organic synthetic methods and classical medicinal chemistry approaches to design and synthesize novel potential drugs. more recently, i've made a strategic decision to expand my research into the developing area of molecular modeling software. in particular, i've become interested in how researchers relate with the computer and how an interactive interface between the human and the computer would improve the quality of in silico drug design. this has led our group to develop a haptic-driven molecular modeling simulator, which we presented at icar in miami. coming from a medicinal chemistry background, this was a completely new research field for me, but i quickly become fascinated by it. recently, i attended a very interesting faraday discussions meeting on "molecular simulations and visualization." anyone who is interested in understanding where new software and methods are heading should look at the conference papers: http://pubs.rsc.org/en/journals/journalissues/fd#!issue id=fd &type=current&issnprint= - the development of antivirals shares some common ground with the anticancer field, and nucleoside analogues are very important drugs in both fields. in fact, this is not the only common aspect. we are increasingly seeing how some drugs designed to hit target host cell pathways are finding new applications in the antiviral field. some of the clearest examples are cyclin-dependent kinase inhibitors, such as roscovitine, that were developed as anti-cancer agents, but have also shown antiviral activity. efforts to develop new drugs often lead to disappointment, but are sometimes unexpectedly successful. what has been your own experience? disappointment is the most common emotion a researcher faces in his career, so we have to learn to forget failures quickly and use them to move forward with more impetus. i've had several small and some big disappointments in the last few years, but i tend to remember only the successes! potential interaction of a virtual screening hit compound with the bcl- protein, leading to reduced motility of malignant cells, blocking metastasis. at the moment, our most exciting project focuses on the development of an anti-metastatic agent, based on the recently discovered role of the bcl protein in cancer cell motility, especially for mammary cancer (above). it began with a virtual screening simulation just four years ago, but has since become the foundation stone of a spin-off company, tiziana life sciences (http://www.tizianalifesciences.com) which is now listed in the aim section of the london stock exchange. the major focus of your career has been drug discovery for flaviviruses. what are the principal diseases for which antivirals are needed? although vaccines are the best countermeasures against flaviviruses, approved vaccines are currently available only for yellow fever, japanese encephalitis and some tick-borne encephalitis viruses. antiviral drugs are needed for agents lacking approved vaccines, and dengue virus (denv) is the top priority within this category. even for those diseases for which vaccines are available, such as yellow fever, the low vaccination rates in many endemic regions mean that effective therapies are still needed. yes, many lessons can be learned from hcv drug discovery. for example, the pan-genotype coverage and high resistance barrier of sofosbuvir show a clear advantage of the nucleotide analog approach. it would be ideal if a single compound could be identified that inhibits a broad spectrum of flaviviruses. compared to hepatitis c, a chronic disease that is treated for at least weeks, the duration of therapy for dengue or other flaviviral diseases is expected to be no more than a week, so that the toxicity barrier will be much lower. over the past decade, both academia and industry have made effort to develop inhibitors of flaviviruses (especially denv). various approaches have been taken; each approach has proven to have its own challenges (lim et al., ) . nucleoside/nucleotide analogs exhibit the attractive features of broad-spectrum coverage and a high resistance barrier. these advantages have already been demonstrated by an adenosine analog (nitd- ) in both denv cell culture and mouse model (yin et al., ). however, a number of scientific hurdles still need to be addressed. for example, for an anti-denv drug, in what cells and tissues does the virus replicate during the acute phase of infection in humans? how could we engineer compounds to be selectively loaded onto these sites? for those who are interested in the nucleoside/nucleotide approach, please refer to our recent review on this topic (chen et al, ) . protease inhibitors appear to be less promising. in contrast to the hcv protease, for which peptidomimetic drugs have been successful, the two positively-charged amino acid residues located immediately upstream of the flavivirus protease cleavage site make this approach challenging. more effort is needed to develop a denv protease inhibitor. compounds targeting the denv envelope and capsid proteins have also been reported, but the physical/chemical properties of these inhibitors prohibit further development. other types of compounds, such as ns b inhibitors (xie et al., ) , remain to be explored. drugs that target host factors required for viral replication, such as alpha glucosidase inhibitors, or that play a critical role in disease development, such as mast cell stabilizers, are worth investigating. in addition to small-molecule drugs, therapeutic antibodies are being explored for denv and other flaviviruses, and antibodies active across serotypes have recently been reported in cell culture and in mouse models. potent serotype-specific antibodies have also been reported, but this approach will require at least four antibodies, each inhibiting one denv serotype. some of them are expected to enter clinical trials soon. vaccines and antivirals complement each other for disease control and intervention. recent results for front-runner dengue vaccines have shown great promise, despite certain limitations (e.g., weak efficacy against denv serotype for the cyd-tdv vaccine). the ongoing vaccine trials will continue to unravel many questions about dengue diseases and immune response in humans; such information will facilitate antiviral development, especially when antiviral candidates reach clinical trials. i'm really excited to witness and participate in the effort to translate our knowledge about dengue disease to benefit patients. safety and efficacy are the two most important parameters for any therapeutics. for travelers to a dengue-endemic area, prophylactic dosing may be sufficient to prevent disease during a short visit. for people living in endemic areas, it is likely that multiple treatment courses (either prophylactic or therapeutic) will be needed, making drug safety even more important. just as success in developing hcv drugs should benefit dengue antiviral development, success in therapeutic development against denv is expected to facilitate discovery for other flaviviruses. due to similarities among flavivirus proteins, it would be ideal if an inhibitor of denv would cross-inhibit other viruses, even if at a lower efficacy. such inhibitors would then be good starting points for chemical modifications to improve their efficacy against other flaviviruses. this strategy remains to be experimentally investigated. rapid point-of-care diagnostics will be essential, especially for recruitment of dengue patients with early, acute infection for clinical trials. like other acute viral infections, the treatment window for dengue patients is short. the feasibility of such short treatment window can only be experimentally addressed using a safe and efficacious compound in a clinical setting. once efficacy and safety have been established, one could conceive of treating febrile patients in areas with ongoing dengue outbreaks without a confirmed diagnosis. an opportunity that i find especially exciting is to help build partnerships between utmb and leading research institutes in china, especially the wuhan institute of virology of the academy of sciences, which is opening china's first bsl- laboratory. we hope to establish working relationships for the exchange of scientists and collaborative research projects. this year marks the th anniversary of the discovery of the australia antigen, subsequently identified as the hepatitis b virus surface antigen, by baruch blumberg and his colleagues at the national institutes of health and the fox chase cancer center. by providing a specific serum marker of hepatitis b, the discovery led to rapid progress in multiple areas, including the development of diagnostic tests, improved safety of blood transfusion and production of the first hepatitis b vaccine. the ability to detect asymptomatic infections also revealed the huge numbers of people around the world with chronic hepatitis b and its relationship to terminal cirrhosis and hepatocellular carcinoma. this th anniversary is a particularly special event for the hepatitis b foundation and its director, isar member tim block. tim and his wife joan established the foundation in as a public health, advocacy and outreach organization committed to helping people with hepatitis b. in , they created the institute for hepatitis and virus research, as an independent, non-profit organization dedicated to finding therapies "to improve the quality of lives of those affected by hepatitis b and liver cancer." dr. blumberg (below) maintained an office at the institute until his death in . after his passing, the institute was renamed in his honor. the blumberg institute is located within the pennsylvania biotechnology center, a life sciences "incubator" in doylestown, pa, which is home to in addition to hepatitis b, the blumberg institute also has strong programs in antivirals against rna viruses. its filovirus antiviral program has produced novel imino sugars shown to have efficacy in animal models. in , the merck company donated its collection of natural products, including the schering plough legacy, to the institute, which now has one of the most diverse and "druggable" libraries of natural products in the world. institute scientists screen them for antiviral and anticancer leads, and make the collection available to other researchers and institutions. other blumberg institute activities of potential interest to isar members include: --"out and up": established professionals from universities and the pharmaceutical and biotech industry may be offered faculty appointments, funding or space to develop their ideas; --recent college graduates may perform -or -year research fellowships, working with mentors on assigned projects; --graduate students in masters or ph.d. programs may obtain adjunct appointments at the university of pennsylvania, drexel university and other nearby institutions. to mark the th anniversary of the discovery of the australia antigen, the blumberg institute is sponsoring a collection of invited articles in antiviral research. the symposium papers review a range of novel therapies for hepatitis b that are now on the horizon. they can be accessed at http://www.journals.elsevier.com/antiviralresearch/symposia/symposium-hepatitis-b/ isar member bart tarbet reports two new projects at utah state university. the first is a response to recent outbreaks of enterovirus d in the midwestern usa and of ev- in china and other areas: the iar has received a contract from extramural niaid/nih to develop mouse models of enterovirus infections and use them to evaluate therapeutics. bart will direct the study. funding for the first two years will support model development, and if that's successful, additional funds will cover drug testing. the other project is something new for the iar. in october, , bart met dr. emmanuel assana, a professor of veterinary medicine in cameroon in central africa, at the annual meeting of the international society for vaccines. after a discussion of possible research collaborations, they decided to apply for a grant to establish a laboratory in ngaoundéré, cameroon for the surveillance and detection of emerging and re-emerging zoonoses. the government's minister of livestock, fisheries and animal industries has supported the proposal, recommending that the lab be included in the national program for the prevention and fight against emerging and re-emerging zoonoses, part of the usaid program on global health security and emerging pandemic threats. although the award is not yet official, recent word from usaid is that the proposal has been submitted to its implementing partners in one health central and eastern africa. if the grant is approved, bart will help set up the lab and provide training in biosafety and biosecurity, and personnel from cameroon will train in virology and diagnostics in the iar and the utah veterinary diagnostic laboratory. bart would enjoy hearing from isar members who have suggestions for a larger scope of activities for this project, such as the study of infectious disease transmission at the wildlife-livestock interface and environmental drivers of emerging infectious diseases. this year the nominations committee was charged with finding two candidates for the position of president-elect, two for the post of secretary, and six candidates for three positions on the board of directors. they have all given of their time and talents to the society and would make excellent board members. the election was held through the isar website beginning on november th , and has just concluded. we hope that all members voted, and were able to make the difficult choice among this slate of excellent candidates! in , rhonda joined parke-davis pharmaceuticals as a senior scientist, where she supervised the evaluation of anti-herpesvirus compounds in animal models and was also part of the hiv entry inhibitor program. after the pfizer merger, she joined chemocentryx, inc. to work on chemokine therapeutics for diseases and vaccine strategies. in , she joined the faculty of cincinnati children's hospital medical center. her research focuses on murine cytomegalovirus pathogenesis and latency as a model for human cytomegalovirus and is currently nih-funded. since , she has served as co-pi on a nih contract for evaluating novel antivirals and vaccine strategies in cmv and hsv animal models. she joined isar in and has actively attended and presented at icar and has served as a poster judge and herpesvirus co-chair. she is currently a member of the finance, membership, and publications committees and has served on the isar executive board for the past four years. she is also a member of the women in science committee and has led efforts to develop the women in science mentoring program. johan neyts is full professor of virology at the faculty of medicine of the university of leuven (ku leuven) in belgium, where he teaches medical virology at the school of medicine and dentistry. the focus of his laboratory is the development of novel antiviral and vaccination strategies. he is author of ~ peer reviewed papers and holds several patents. sixteen people have obtained their ph.d. degree under his guidance and bachelor or master students have been trained in his laboratory. he is an editor of antiviral research and is on the editorial board of several other journals. johan was co-founder and cso of the ku leuven spin-off okapi sciences nv a biotech company that developed antivirals for veterinary use. he is cso of virovet (www.virovet.com ), a new ku leuven spin-off that is currently being incorporated. research topics covered in his laboratory at the university in leuven include the development of novel antiviral strategies against a number of rna viruses, including flaviviruses (hepatitis c, dengue and others), picornaviruses (entero-and rhinoviruses), alphaviruses (chikungunya and others), paramyxoviruses (rsv and others), rabies, noroviruses and the hepatitis e virus, as well as a novel thermostable dna vaccine technology. johan has attended icars since . in , he received the william prusoff young investigator award. he has also served as a board member and chair of the membership committee. graciela's main scientific activities include the unraveling of the mode of action of novel antivirals, the molecular mechanisms of drug resistance in herpesviruses and poxviruses, the molecular anticancer mechanism of action of nucleotide analogues and the development of organotypic epithelial raft cultures for the study of epitheliotropic viruses. in , she participated in the set-up of the regavir, a translational research platform for typing drug-resistant herpesviruses in immuno-compromised patients who fail antiviral therapy. she has been a member of isar since , and in , she was elected secretary. she is a member of the editorial board of antiviral research and plos one. kara carter has years of experience in virology research and the discovery and development of antiviral agents. following undergraduate research at stanford university, in collaboration with chiron to develop an hsv- -specific diagnostic, she received her ph.d. in virology at university of chicago in the laboratory of dr. bernard roizman, identifying novel genes of hsv- and elucidating the mechanisms of their protein products. she performed postdoctoral studies at harvard university and brigham and women's hospital in the laboratory of dr. elliiott kieff, focusing on ebv-induced transcriptional changes of infected b cells and their effect on transformation. she then moved to praecis pharmaceuticals, where she led drug discovery post-exposure efficacy of oral t- (favipiravir) against inhalational ebola virus infection in a mouse model application of real-time pcr for testing antiviral compounds against lassa virus, sars coronavirus and ebola virus in vitro evaluation of antiviral efficacy of ribavirin, arbidol, and t- (favipiravir) in a mouse model for crimean-congo hemorrhagic fever imported lassa fever in germany: molecular characterization of a new lassa virus strain update on human herpesvirus biology, clinical features, and therapy inhibition of hypoxanthine-guanine phosphoribosyl transferase by acyclic nucleoside phosphonates: a new class of antimalarial therapeutics pmea (adefovir) and related acyclic nucleoside phosphonate analogues: a review of their pharmacology and clinical potential in the treatment of viral infections antiretroviral efficacy and pharmacokinetics of oral bis(isopropyloxy carbonyloxymethyl)- -( -phosphonylmethoxy propyl) adenine in mice antiadenovirus activities of several classes of nucleoside and nucleotide analogues mutational analysis of the binding pockets of the diketo acid inhibitor l- , in the influenza virus pa endonuclease an integrated biological approach to guide the development of metal-chelating inhibitors of influenza virus pa endonuclease antiviral treatment is more effective than smallpox vaccination upon lethal monkeypox virus infection novel inhibitors of influenza virus fusion: structure-activity relationship and interaction with the viral hemagglutinin emerging antiviral strategies to interfere with influenza virus entry the search for nucleoside/nucleotide analog inhibitors of dengue virus ten years of dengue drug discovery: progress and prospects targeting dengue virus ns b protein for drug discovery an adenosine nucleoside inhibitor of dengue virus programs in rsv, influenza, hsv and hcv, focusing on cellular targets.in , kara moved to the genzyme corporation, where she led drug discovery programs in virology, immunology and oncology, as well as supporting the transplant business unit in the evaluation and acquisition of antiviral products to complement their portfolio. she is currently head of antiviral research at sanofi. she is an isar member and has served as a member of the women in science committee. since his appointment as lecturer, andrea's research has focused on the use of computer-aided techniques in the design and discovery of novel antiviral and anticancer compounds. in the antiviral field, his research has focused on the in silico design of rna virus inhibitors, including dengue, wnv, hcv, chikungunya virus and coxsackie virus. he is the chemistry editor for antiviral chemistry and chemotherapy. he has been a member of isar since and was elected a board member in . he has also been a member and chair of the website committee since and the publications committee since . he has been the isar webmaster since , and has implemented several technical developments, including online registration and membership management and enhancement of isar's social profile on facebook, twitter and linkedin.randall lanier is vice president for biology at chimerix, inc. before joining chimerix in , he contributed to the hiv and cancer programs at burroughs wellcome, glaxowellcome and glaxosmithkline, where he supervised a clinical virology/immunology laboratory, led teams for drug discovery and was involved in preclinical and clinical development, product differentiation, post-marketing support and licensing opportunity evaluation.randall has over years of experience in the discovery and development of antivirals, and has focused much of his career on understanding the activity, mechanism, and resistance profiles of nucleoside analogs used for prevention and treatment of viral disease caused by hiv, cmv, adenovirus, and poxviruses. his received his undergraduate degree in biology from new college and ph.d. in cellular and molecular biology from the university of texas health science center in san antonio. professor of chemistry and biochemistry at the university of maryland, baltimore county. she earned her ph.d. in organic chemistry from auburn university.her research involves a synthetic organic/medicinal chemistry approach to nucleoside and heterocyclic drug discovery and development. current projects include the investigation of flexible nucleosides/nucleobases "fleximers" for use against sars, mers-cov, ebola, hcv, hiv and other viruses. she has published over peer-reviewed papers, and is the president of the international society of nucleosides, nucleotides and nucleic acids (is na).kathy has chaired the icar poster awards for a number of years. notably, she initiated a new program for is na, the women's career development scholarships, as well as to obtain funding for the women in science scholarships for isar, both of which have been generously funded by the chu family foundation. she is a member of the acs medicinal chemistry division awards committee and is an associate editor for current protocols in chemical biology. karen's interests have also expanded into other areas of women's health, and she is involved in the continuing development of imquest's programs to identify prevention and treatment agents for hiv, hsv- , hsv- , hpv and various bacterial and fungal organisms. she has been first author or coauthor of more than papers on topical microbicide development. she has assisted with the development of isar programs for drug discovery and development and serves on the women in science committee. bill initially worked as a clinical virologist in support of the approved hbv nucleotide prodrugs adefovir dipivoxil and tenofovir disoproxil fumarate, which included characterization of the first clinical adefovir resistance mutations. following their approval, he focused on hcv, which included leading discovery efforts on ns protease inhibitors and overseeing biological support for other programs, including the ns a inhibitor program that led to ledipasvir. he is currently senior director of biology and heads gilead's viral hepatitis discovery biology group. he has been an isar member since , received the william prusoff young investigator award from the society in and was elected to the isar board of directors in . simon tucker is updating the calendar of future conferences on antiviral therapy, medicinal chemistry and other topics of interest that he posted on the isar website last year. isar members can access the calendar by logging in and downloading the pdf. key: cord- - wuh k g authors: dong, mengying; cao, xiaojun; liang, mingbiao; li, lijuan; liu, guangjian; liang, huiying title: understand research hotspots surrounding covid- and other coronavirus infections using topic modeling date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: wuh k g background: severe acute respiratory syndrome coronavirus (sars-cov- ) is a virus that causes severe respiratory illness in humans, which eventually results in the current outbreak of novel coronavirus disease (covid- ) around the world. the research community is interested to know what are the hotspots in coronavirus (cov) research and how much is known about covid- . this study aimed to evaluate the characteristics of publications involving coronaviruses as well as covid- by using a topic modeling analysis. methods: we extracted all abstracts and retained the most informative words from the covid- open research dataset, which contains all the , pieces of coronavirus related literature published up to march , . using latent dirichlet allocation modeling, we trained an eight-topic model from the corpus. we then analyzed the semantic relationships between topics and compared the topic distribution between covid- and other cov infections. results: eight topics emerged overall: clinical characterization, pathogenesis research, therapeutics research, epidemiological study, virus transmission, vaccines research, virus diagnostics, and viral genomics. it was observed that covid- research puts more emphasis on clinical characterization, epidemiological study, and virus transmission at present. in contrast, topics about diagnostics, therapeutics, vaccines, genomics and pathogenesis only accounted for less than % or even % of all the covid- publications, much lower than those of other cov infections. conclusions: these results identified knowledge gaps in the area of covid- and offered directions for future research. keywords: covid- , coronavirus, topic modeling, hotspots, text mining coronaviruses (covs) are enveloped, positive single-stranded large rna viruses that cause respiratory and intestinal infections in animals and humans ( ) . to date, there have seven human coronaviruses (hcovs) been identified. the alpha-covs hcovs-nl and hcovs- e and the beta-covs hcovs-oc , hcovs-hku only cause mild respiratory illness ( , ) . however, in the past two decades, two novel coronaviruses, severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov), caused severe human diseases in local countries and regions, representing fatality rates of around % and %, respectively ( ) ( ) ( ) . now, the seventh hcov, sars-cov- (formerly -ncov), is causing an outbreak of coronavirus disease all over the world. compared to the earlier epidemics by covs, covid- is highly contagious and has been reported with more than , , confirmed cases as on april , ( ) . given the spread of the new cov and its impacts on human health, governments are facing increasing pressure to stop the covid- pandemic. the white house and a coalition of leading research groups have prepared a covid- open research dataset (cord- ) and issued a call to action to the world's artificial intelligence experts to support the ongoing fight against this infectious disease. the research community has also responded rapidly and substantial literature about this epidemic has emerged. understanding of covid- is evolving rapidly. it is essential to understand the emerging scientific knowledge to coordinate covid- research globally. recently, the who has published a global research roadmap with immediate, mid-term and longer-term priorities to enable the implementation of priority research ( ) . bonilla-aldana dk et al. ( ) and md mahbub hossain mbbs ( ) have performed bibliometric analysis to evaluate the scientific literature on coronavirus infections as well as covid- , basing on indicators such as the number of articles, the productivity of authors, geographic distribution of articles and prominent keywords. however, to the best of our knowledge, there is still no quantitative thematic analysis available to date that focuses on covs. addressing this knowledge gap requires not only elaboration at the text level but also an understanding of semantic structures. top modeling is the most notable approach for analyzing . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint text to identify semantic content ( ) . its objective is to find latent semantic topics within vast and unstructured collections of literature. among several topic modeling algorithms, latent dirichlet allocation (lda) is the most fundamental one and has been shown to be effective at discovering semantic structures and distinct topics from a corpus ( ) ( ) ( ) ( ) ( ) ( ) ( ) . this kind of data-driven analysis can generally shift the information overload burden from researchers to the computer via algorithmic and statistical methods. the purpose of this work was to conduct lda modeling for semantic and quantitative evaluations of the current status of literature on cov infections as well as covid- , identify broad research topics and how these topics interact with one another. more importantly, this work can benefit covid- research coordination by recognizing high priority scientific topics. we found that topics of clinical characterization, epidemiology, and virus transmission are hotspots for covid- at present, while research on pathogenesis, therapeutics, virus diagnostics, vaccines and viral genomics are urgently needed. data used in this study were obtained from cord- , which was prepared by the white house and a coalition of leading research groups in response to the covid- pandemic ( ) . contains all research about covid- , sars-cov- , and other covs (e.g. sars, mers, etc.) up to march , , including over , scholarly articles, from the following sources: ) pubmed's pmc open access corpus; ) a corpus maintained by the who; and ) biorxiv and medrxiv pre-prints. abstracts were firstly exported from cord- and merged into a local repository for further processing. next, , records with an empty or non-english abstract (i.e., not provided in the database) were removed from the corpus. then, duplicates were removed based on the title (stripped of all special characters and spaces) or the digital object identifier (doi, if available). the final corpus contained , abstracts of all coronaviruses-related articles. we further retrieved covid- data by administering the query, "covid" or "covid- " or " -ncov" or "sars-cov- " or "novel coronavirus", and limiting the publication time to . in . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint total, , articles were identified as covid- -related research. this study is a retrospective analysis of accepted and publicly disclosed research, which does not meet the definition of human research under the us department of health and human services protection of human subject regulations. data were not deidentified and are publicly available. there was no intervention, interaction with researchers, or use of private information to perform the analyses in this study. we applied a standard approach for tokenization, lemmatization and part-of-speech tagging to preprocess the biomedical texts. we also removed the following words to get the clean corpus: ) punctuation; ) digits; ) special characters; ) words that appeared fewer than times in the corpus; ) useless words from text collection, including coordinating conjunctions, cardinal numbers, prepositions, pronouns and so on; ) common terms in scientific articles, e.g., 'method' and 'introduction', as well as 'virus', which is thought to appear in most abstracts. we built a topic model using lda ( , ) from the article abstracts within the corpus. lda assumes individual document as random mixtures over latent topics, with topics in turn being probability distributions over multiple vocabularies, and topics being uncorrelated. the gibbs sampling algorithm is used to estimate the topic distribution parameters of the document and the multi-distribution of the vocabulary on each topic. the evaluation index in the statistical language model, coherence score, was used to find the optimal number of topics in the corpus. the coherence score is calculated by the co-occurrence frequency of the words in the sliding window, which increases with the increase of sentence similarity, meaning the higher, the better. although the coherence score can help to decide the most appropriate topic number, such number sometimes can be still large for manually understanding. under this circumstance, manual examination should be performed to evaluate models with different numbers of topics with the aid of expert knowledge to thoroughly understand the corpus. for each model, top words per topic were examined to assess scientific coherence of the words as a set, overlap in topic words across topics, and human understandability. the selected model was used for all subsequent analyses. lda modeling is performed and visualized on the entire corpus via the python language. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint before profiling topics in the corpus, a co-occurrence map of the most frequent words in the abstracts were generated to describe the research hotspots of coronaviruses from a macro-level viewpoint (fig. ) . the top ten co-occurring words were 'infection' (n = ), 'cell' (n = ), 'protein' (n = ), 'diseases' (n = ), 'patient' (n = ), 'human' (n = ), 'respiratory' (n = ), 'response' (n = ), 'gene' (n = ), and 'identify' (n = ). it is obvious that most of these words take 'infection' as the central node, depicting the virus attack event from clinical, macroscopic and microscopic viewpoints. certainly, such map treats each word as a topic and gets the relationships between words simply basing on co-occurrence statistics. its granularity is too small to capture the semantic information of cov research. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint model. increasing the number of topics would make each individual topic more specific and might increase overlap between topics. decreasing the number of topics would result in topics to be more high-level abstract. we assigned a potential theme to each topic by manual examinations based on semantics analysis of representative words in each topic. table shows the most frequent words for each of the eight topics. in most cases, topics were easily recognizable representing specific subjects about the viruses, or the disease, or the public health and so on. the first most dominant topic was enriched for the clinical characterization, with words such as 'infection', 'cause', 'disease', 'severe', 'respiratory', 'acute', 'child' and 'symptom'. representative words of topic include 'cell', 'protein', 'expression', 'bind', 'replication', 'activity' and 'membrane', which usually are . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. it's common that one article discusses more than one topic, i.e., the topics are not isolated but semantic-related. to identify the semantic relationships between topics, we assigned each article two topics with the highest probabilistic proportion, measured the relationships between two topics by using their co-occurrence statistics, and showed them vividly in a direct chord diagram. as shown in fig. , the topic 'clinical characterization' has strong relationships with all the other . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint number of relationships with that same co-occurrence as the dominant and second topics. to evaluate the current state of knowledge on covid- , we compared the topic distributions of , covid- related articles with that of , other covs infections related articles. fig. shows here we applied lda to the abstracts of , covs-related research articles up to march , . results of this exploration present a comprehensive overview and an intellectual structure of the coronavirus research. we identified eight major research topics, analyzed their semantic relationships, and compared the topic distributions between the evolving covid- research and . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. normally, for viruses that broke out earlier, certain topics can be attractive and widely discussed. in the case of topics with high proportions, we consider that they have been elaborated and specified by research community, such as the topics 'clinical characterization', 'pathogenesis research', and 'therapeutics research' in other covs research. while as for the new virus sars-cov- , the research topic distribution is in a state of dynamic change. therefore, comparison between the 'old' and 'new' viruses could clearly highlight the knowledge gaps between them. this work suggests that research needs to be focused on pathogenesis, therapeutics, virus diagnostics, vaccines and viral genomics for covid- , which could then necessitate nationallevel support for ongoing research, if required. pathogenesis research is usually important but time-consuming. we believe such studies will be available in the future. the more important and urgent topic is therapeutics research, which accounts for only . % in covid- research, much lower than . % in other cov infections research. with the number of infection cases increasing rapidly, therapeutics research will be more and more urgently needed. in addition, virus diagnostics, vaccines and viral genomics show small proportions in all cov infections research, but even smaller in the field of covid- . this shows how little we know about covs. it is time . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org / . / . / to translate research findings into effective measures such as a diagnostics, vaccine or effective therapeutic options, as with other priority diseases ( ) . similar to the study of md mahbub hossain mbbs ( ), we did not identify any topic about social, economic, political, psychological, or cultural consequences of covid- as well as other cov infections. the who roadmap has noticed this and taken 'social sciences in the outbreak response' as a priority. the social science research community should act to assess the impact of a major public health crisis like covid- on the complex network of modern societies ( , ) . this present work has several limitations that must be acknowledged to apprehend the findings. first, as a computer model, topic modeling has difficulties with understanding nuances and subtext. we identified eight major topics with frequent appearances, whereas some specific topics such as virus natural history and human-animal interface were buried due to low proportions. future work will attempt to develop an approach to detect and visualize the conceptual sub-domains. second, it is also necessary to be aware that the granularity of terms used to label topics may vary a little for different topics. we have tried our best to keep them at the same level through carefully curating the top words in each topic, as well as reviewing text intention of the corresponding publications. third, the lda model only provides a cross-sectional profile of the coronavirus research, which is methodologically different from academic disciplines . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint such as evidence-based medicine and clinical assessment that synthesizing evidence centered on a specific question. but this study may facilitate these disciplines by informing what is already available and what is urgently required for the covid- research. future research could also study the occurrence of topics over time and analyze links to historic events and virus characteristics, in order to better understand possible temporal patterns. notwithstanding its limitations, this work is the first to thoroughly assess research output of coronavirus research in statistical perspective. the present study provided machine-extracted research topics in various contexts based on massive number of articles over a long period. such a bottom-up approach can complement the existing manual content analysis techniques. it can also help to find the latent semantic relationships between topics and enrich the research landscape. in conclusion, this study specifically shows a holistic picture of the current research in response to the outbreak of covid- . we conducted a topic-based bibliometric study to address two questions "what is the covs research interested in?" and "where are we with our research about covid- ?". an lda-based model was used to identify the hotspots and their difference between covid- and other cov infections in a quantitative way. generally, eight main research areas are identified, i.e., clinical characterization, pathogenesis research, therapeutics research, epidemiological study, virus transmission, vaccines research, virus diagnostics, and viral genomics. the results also indicate that studies in diagnostics, therapeutics, vaccines, viral genomics, and pathogenesis are urgently needed to minimize the impact of the covid- outbreak. as covid- can be considered a recent emerged disease, we characterize a 'snapshot' of this field at an early stage in its development. our findings can potentially aid governments and the research community in understanding recent development of the research field, optimizing research topic decision, and monitoring new scientific or technological activities. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint advances in virus research coronaviruses -drug discovery and therapeutic options origin and evolution of pathogenic coronaviruses severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease molecular pathology of emerging coronavirus infections mers-cov and now the -novel cov: have we investigated enough about coronaviruses? -a bibliometric analysis current status of global research on novel coronavirus disease (covid- ): a bibliometric analysis and knowledge mapping engineering e. topic discovery and evolution in scientific literature based on content and citations international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) the copyright holder for this preprint a hotspots analysis-relation discovery representation model for revealing diabetes mellitus and obesity years of sepsis: using topic modeling to understand historical themes surrounding sepsis analyzing research trends in personal information privacy using topic modeling detecting and predicting the topic change of knowledge-based systems: a topic-based bibliometric analysis from to measuring author research relatedness: a comparison of word-based, topic-based, and author cocitation approaches clustering scientific documents with topic modeling cord- ). . version - - distance metric learning with application to clustering with side-information. advances in neural information processing systems latent dirichlet allocation severe fever with thrombocytopenia syndrome-a bibliometric analysis of an emerging priority disease virus will have huge socioeconomic impact in china. (oxan-db) costly lessons from the middle east respiratory syndrome coronavirus outbreak in korea the authors declare that they have no competing interests. it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity.(which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint key: cord- -w uaet l authors: nayeri, shadi; walshe, margaret; lee, sun-ho; filice, melissa; rho, stella; jeyakumar, ajani; stempak, joanne; smith, michelle i; silverberg, mark s title: conducting translational gastrointestinal research in the era of covid- date: - - journal: j crohns colitis doi: . /ecco-jcc/jjaa sha: doc_id: cord_uid: w uaet l abstract spread of the novel coronavirus sars-cov- has resulted in a global pandemic that is affecting the health and economy of all world health organization [who] regions. clinical and translational research activities have been affected drastically by this global catastrophe. in this document we provide a suggested roadmap for resuming gastrointestinal translational research activities, emphasising physical distancing and use of personal protective equipment. we discuss modes of virus transmission in enclosed environments [including clinical workplaces and laboratories] and potential risks of exposure in the endoscopy environment for research staff. the proposed guidelines should be considered in conjunction with local institutional and government guidelines so that translational research can be resumed as safely as possible. the novel coronavirus sars-cov- [which causes covid- ] remains a major public health threat. this virulent organism has caused the deadliest pandemic since the 'spanish' influenza pandemic of . the virus is transmitted mainly through respiratory droplets. however, the virus is also detectable in the gastrointestinal [gi] tract. a recent study reported the isolation of viral nucleocapsid protein and expression of angiotensinconverting enzyme [ace ] protein [a receptor which facilitates entry of sars-cov- to cells] in the gastric, duodenal, and rectal epithelial cells of patients infected by sars-cov- . , additionally, stool samples from approximately % of covid- patients remain positive for viral rna up to weeks after their respiratory samples test negative. , currently, the viability and infectivity of the virus in faeces is poorly understood. , droplet [> - мm] transmission occurs primarily during close contact [usually within - m] with an infected person who has respiratory symptoms [eg, coughing, sneezing]. however, asymptomatic infected individuals also play a major role in transmission of sars-cov- . exposure to high concentrations of bio-aerosols in relatively closed environments has also been suggested as a route of virus transmission. sars-cov- can also be transmitted through fomites in the immediate environment of an infected person. one study reported detection of the virus from sink and toilet bowl samples taken from the isolation room of a covid- patient. in addition, viable virus particles can be detected on surfaces [such as plastic and stainless steel] for up to h. , globally, government and public health bodies have implemented policies in an attempt to mitigate the spread of sars-cov- . efforts focus primarily on physical distancing, use of phase personal protective equipment [ppe] , and addressing capacity needs of health care systems to deal with the outbreak. this has led to significant curtailment of translational research activities for multiple reasons. first, physical distancing measures have restricted the ability of researchers to work 'on site' and handle samples at the same capacity as before the pandemic. second, the pandemic has resulted in limitation of resources such as access to shared laboratory equipment, ppe, and endoscopy. third, availability and willingness of patients to engage in research has been negatively affected, in part due to drastic reduction in non-urgent clinical activity. we suggest that a phased approach be taken to re-expand non-essential research activities. in this guidance document, we address the roadmap to re-engaging in gi translational research in the era of the covid- pandemic, while keeping researchers and research participants safe. these guidelines were formulated with collaboration across our translational research group, with incorporation of international as well as local institutional recommendations. given the rapidly evolving landscape of the pandemic worldwide, these guidelines should be considered in conjunction with local institutional and government regulations. considering the risk of viral transmission associated with conducting office/laboratory-based research, re-opening of research environments should be performed in stages. potential risks relate to sharing of work space and handling of biospecimens. the following suggestions should be considered in the context of local factors including capacity, ppe availability, and feasibility of monitoring procedures to ensure new safety measures are being followed. all workplaces should be prepared to re-introduce restrictions on research activities in the event of sars-cov- resurgence. we propose that re-expansion of research activities take place across four phases [as outlined in figure ]: phase , preparation; phase , re-start research activities with total staff numbers not to exceed % of on-site capacity; phase , continue to increase staff numbers to maximum - % of on-site capacity; and phase , continue to increase staff numbers to approximately - % of on-site capacity, while maintaining significant sars-cov- restrictions for the foreseeable future. this phased approach will enable researchers to ramp up projects in order of priority. we propose suggested time frames for implementation of each phase, but the decision to progress through phases must factor in local risk assessment based on prevalence of infection in the community. the time frames described should allow for monitoring of adherence to safety measures and detection of outbreaks resulting from increased traffic in the workplace, both of which must be prospectively and actively monitored within each phase. decision makers for advancing through the phases should be designated based on institutional policies. phase should be completed within an estimated -week time frame. the main scope of this stage consists of: ] increasing the number of staff on site while introducing new safety routines to maintain physical distancing; ] provision for increased levels of hygiene [hand, surfaces, and equipment]; ] increasing access to critical supplies when supply lines may already be stretched. we suggest the following phase measures. • access to all research areas should be restricted to research personnel only. all visitors from outside research institutes, including other researchers, service personnel, delivery personnel, and vendor representatives must follow local sars-cov- restrictions for booking appointments. additionally, they must follow screening procedures and wear appropriate ppe. • programme leaders should develop specific plans for resuming work in their laboratories, allowing identification of staff who will work on site. this should take laboratory space, layout, and ventilation into account to allow for physical distancing in all shared areas such as laboratory bays, equipment rooms, tissue culture rooms, offices, and break areas. for common areas we suggest an online calendar for booking equipment and rooms. • re-organisation of workplace layout may be considered to facilitate shared use of space and equipment while maintaining physical distancing. • presence of staff in the workplace should be prospectively recorded to ensure that future contract tracing [if required] is feasible, and to monitor occupancy on site. we suggest web-based sign-in to facilitate this process. • in-person meetings should be limited to maintain the m rule for physical distancing. in addition, face masks should be required for face-to-face meetings in enclosed spaces. • meetings [including in-laboratory meetings and meetings with external groups and collaborators] should take place online wherever possible. • all staff who can work from home should continue to do so; this includes staff coming into the workplace to carry out specific activities but who do not need to remain for the entire day. re-assignment of 'on-site' tasks should also be implemented where feasible, in order to minimise staff numbers in the workplace. • staggered work hours to avoid crowding of work spaces should be considered. • in order to maximise opportunity for staff to work from home, access to relevant resources should be addressed. this may include laptops, analysis software, and remote access to datasets. subsidies for work-related costs incurred to staff as a result of working from home [eg, internet access costs] may be considered. • an updated cleaning schedule for common areas should be executed by housekeeping. cleaning schedules should include wiping down door handles and other highly used surfaces with approved disinfectants. • on-site laboratory staff should regularly wipe down common surfaces/equipment using approved disinfectants or % ethanol. these areas include but are not limited to: • equipment: incubators, fridge and freezer doors, bench tops, biological safety cabinets [bsc], fume hoods, keyboards, microscopes, centrifuges, etc. • office and break areas: tables, chairs, desks, microwaves, coffee pots, etc. • as research programmes restart, staff in different supply centres, research receiving, stabilisation, and glass washing should re-schedule staffing to match research activity. • laboratory managers should ensure availability of supplies for at least - months following re-initiation of research activities. this includes availability of ppe, molecular kits, plasticware, chemicals, and reagents. • research units must take responsibility for acquiring ppe, and remain cognisant of any impact on the availability of ppe for clinical care workers. co-ordination of ppe procurement with allied hospital services may help to mitigate costs through 'bulk buying'. the estimated time frame considered for this phase is - weeks. during phase , we suggest that areas be restricted to a maximum of % occupancy at any one time, though this can be customised based on the overall size of the research group. • as staffing numbers increase at this stage, cleaning logs should be implemented for all laboratory areas. • staff will be responsible for self-monitoring for symptoms of covid- [eg, cough, sore throat, dyspnoea, rhinorrhoea, fever, anosmia]. symptoms and/or close contact with infected individuals should be reported immediately to occupational health and laboratory management. self-isolation should be adopted while awaiting further direction from occupational health. the suggested time frame for this phase is - months. phase is subject to a maximum - % staff occupancy at any given time. • the plan for this stage is to return to research activity based on approvals of local research group work committees. • some dry laboratories can move directly to phase , where physical distancing [ m] can be practised or working remotely is possible. • physical distancing of m should continue to be practised. during this time, the occupancy of areas is suggested to be maintained at - % at any one time depending on how space constraints limit capacity for physical distancing. this phase will persist as long as sars-cov- remains a community health risk. • the research environment will essentially run at full capacity but on-site occupancy may need to remain reduced by up to %. • staff should be encouraged to continue to work from home where possible. translational research relies on in-person involvement of research staff and patients in most circumstances. researchers must remain cognisant at all times of any potential risk posed to research participants and research staff. whereas all persons should consider themselves at risk of covid- , research patients may represent a particularly vulnerable population due to underlying disease processes and/or medical intervention. as always, the option to withdraw from research studies must remain open to participants, whose willingness may be significantly affected by the pandemic. local and institutional guidance is required to resume translational research activities, including patient interactions. these guidelines are intended to assist safe resumption of such activities. • wherever possible, research study participants should be engaged remotely. • study protocols should be adapted in order to minimise in-person patient visits. suitability of phone/video or electronic interaction should be considered. all such adaptations must be subjected to reb approval before implementation with stringent protection of patient privacy and confidentiality. • visits to hospitals and research facilities should be minimised and confined to clinical research areas. • for research relating specifically to sars-cov- infection, in-person contact with patients known to be infected may be necessary. for all such contact, full ppe including n masks or equivalent, long-sleeve gowns, gloves, and goggles or face shields must be worn. fit testing of n masks must be performed before use. • invitation of persons currently infected with sars-cov- from the community into the research environment would cause unnecessary and inappropriate risk of viral transmission. as such, research involving patients with current sars-cov- infection should be limited to inpatients. as outlined above, sars-cov- has been isolated from gi biopsies and stool samples. , it is unclear at this time whether transmission of sars-cov- can occur via handling of biospecimens. no cases have been reported to date, but precautions are required. in keeping with standard laboratory protocols, all specimens should be regarded as potentially infected. additionally, particular consideration is necessary when obtaining biospecimens in the endoscopy environment. the nature of endoscopic procedures poses potential for viral transmission via aerosolisation of viral particles. , the risk of viral transmission to staff from patients during gi endoscopy has not been quantified, but many consider gi endoscopy 'high-risk'. , here, we provide guidance on laboratory biosafety in relation to sample collection, handling, processing, transportation, and storage. • for outpatient blood sample collections, patients should be sent to commercial medical laboratory services or hospital outpatient laboratories if possible. • if in-person blood sampling by research staff is needed, it should be performed in areas where there are minimal additional exposed individuals [ie, dedicated examination rooms], and with adequate ppe. for research staff, gloves and masks should be mandatory. we recommend also using eye protection and gowns. patients should also be wearing masks. • stool and urine sample kits can be couriered to subjects to obtain samples at home. the samples should be couriered back to research staff, if possible. • where patients must return biospecimen samples in person, sample drop-off by the patient and pick-up by the research staff should be sequenced with minimal contact. designated drop-off locations will facilitate these practices. • samples need to be wiped down with disinfectant before placing them in the storage container and transfer bag. • a drop-off bay should be designated. • all surfaces touched by the research staff or specimen containers during drop-off and pick-up must be sanitised. all transfer bags and container bags should be sanitised between uses. • dedicated standard operating procedures should be in place for transfer of samples which may contain live virus to research areas. • standard universal precautions should be followed when handling clinical specimens which potentially contain infectious materials: hand hygiene, use of ppe, ie, laboratory coats or gowns, gloves, and eye protection. • all laboratory processing of samples should be performed based on risk assessment and only by certified technicians following local or institutional guidelines. • processing of all specimens should be performed in certified class bsc [with the exception of virus propagation, for which class bsc is required]. viral inactivation through addition of % detergent or heat treatment is highly recommended and significantly reduces concerns for laboratory handling. - • a sample manifest or tracking log should be maintained. • routine laboratory practices including procedures for decontamination of work surfaces and disposal of laboratory waste should be followed using local safety protocols. • there should be a clear framework of communication between management and research staff such that relevant parties are notified in a timely manner should inadvertent potential exposure to sars-cov- occur. • a contingency plan with a specific protocol must be developed in case of a biosafety incident, ie, exposure to a potentially infected biospecimen. • such incidents should be reported immediately to the appropriate personnel. • spill kits and first aid kits including medical supplies should be prepared at all times. • research staff exposed to a potentially infected biospecimen or infected patient should be self-isolated and be tested for sars-cov- as soon as possible. this should be performed in collaboration with occupational health services. we have proposed guidelines for gradual re-expansion of gi research activities during the sars-cov- pandemic. stage-wise resumption of research activities should be implemented with consideration for ongoing risk assessment, availability of resources such as appropriate ppe, and proper physical distancing measures. considering the risk of exposure in enclosed environments, we propose re-engagement in research activities in four phases: phase , preparation, phase , start-up, phase , ramp-up of research activities; and phase , maintaining and monitoring the safety situation at the new normal. these guidelines address safety precautions in relevant workspaces [including laboratory and endoscopy environments] as well as in specific research activities such as sample collection, handling, and transportation. as the pandemic continues to evolve, vigilance and flexibility must be applied, particularly as risk of future waves of infection fluctuates. accordingly, the guidelines should be interpreted in conjunction with local institutional and government policies. influenza: the mother of all pandemics the epidemiology and pathogenesis 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biosafety guidance related to coronavirus disease clinical trials for inflammatory bowel disease: a global guidance during covid- pandemic. j crohn's colitis endoscopy in inflammatory bowel diseases during the covid- pandemic and post-pandemic period the time sequences of oral and fecal viral shedding of coronavirus disease covid- pandemic: which ibd patients need to be scoped-who gets scoped now, who can wait, and how to resume to normal practice of endoscopy during covid- pandemic: position statements of the asian pacific society for digestive endoscopy peyrin-biroulet l. the day after covid- in ibd: how to go back to 'normal aga institute rapid recommendations for gastrointestinal procedures during the covid- pandemic evaluation of heating and chemical protocols for inactivating sars-cov- sodium lauryl sulfate, a microbicide effective against enveloped and nonenveloped viruses inactivation of the coronavirus that induces severe acute respiratory syndrome, sars-cov we wish to acknowledge the zane cohen center for digestive diseases, lunenfeld-tanenbaum research institute and toronto academic health science network for providing the principles for the guidelines. specifically we would like to thank jim woodgett for his detailed comments and review of the paper. the authors have no financial disclosures or conflicts of interest. key: cord- -o vbuxnw authors: tiberius, victor; schwarzer, hannes; roig-dobón, salvador title: radical innovations: between established knowledge and future research opportunities date: - - journal: nan doi: . /j.jik. . . sha: doc_id: cord_uid: o vbuxnw the fast growing body of radical innovation research is fragmented and difficult to overlook. we provide an overview of the most cited journals, authors, and publications and conduct a bibliographic coupling to structure the literature landscape. we identified the following research clusters: management of radical innovations, organizational learning and knowledge, financial aspects of radical innovation, radical innovation adoption and diffusion, radical industry innovations as challenges for incumbents, and radical innovation in specific industries. based on an in-depth content analysis of these clusters, we identify the following future research opportunities: a systematic compilation of all intra- and extra-organizational management aspects, moderators, and mediators, extending radical innovation research's epistemological basis by adding strategic foresight, further research in individual, group (team), organizational, and inter-organizational capabilities required for radical innovation, a managerial perspective on adoption and diffusion of radical innovations, applying portfolio theory and real options theory to radical innovation research, stronger research efforts on coping strategies for firms faced with competitors' radical innovations, and intensifying both industry-specific and cross-industry research. the smartphone as an example for a radical innovation unites the functions of common mobile phones, photo and video cameras, navigation systems, partially computers, and other devices. additionally, installable apps allow for endless possible uses like instant messaging, fitness tracking, and mobile payment. radical innovations, lacking a clear definition (garcia & calantone, ) , differ from incremental ones in regard to the extend they add new value to customers (souto, ) , their novel knowledge intensity (dewar & dutton, ) , and the required strategies and structures (ettlie, bridges, & o'keefe, ) . firms engage in the development of radical innovations to pursue the opportunity to change or generate new markets, act as (temporary) monopolists, and significantly increase their profits (rubera & kirca, ; slater, mohr, & sengupta, ) . especially due to current digitization trends, there is a high chance that radical innovations will still become more relevant (kraus, roig-tierno, & bouncken, ) . article and citation numbers which are considered to objectively represent research productivity and impact (kücher & feldbauer-durstmüller, ; ramos-rodríguez & ruíz-navarro, ; zupic &Čater, ) . we retrieved our data set of publications on january from the web of science (wos), which is regarded as a comprehensive database of scholarly literature (gaviria-marin, merigó, & baier-fuentes, ; norris & oppenheim, ) . we conducted a title rather than a topic search for 'radical innovation' to ensure that all publications in the data set focus on our topic rather than only deal with it marginally. we reduced the data set by selecting the wos categories business, business finance, economics, management, multidisciplinary sciences, and operations research/management science. we also excluded publications from as the first days of the year would not have resulted in representative numbers in the temporal analysis. one article from which was still in the data set was removed manually. at the end, the data set comprised articles. our bibliometric analyses can be separated into performance analyses focusing on productivity and impact and a bibliographic coupling as a science mapping which searches for research themes within the field (noyons, moed, & luwel, ) . our performance analyses include a temporal analysis of the productivity and impact of radical innovation research which can locate the current state of research in the research lifecycle and answer the question if it is growing, stagnating, or declining. they also include an analysis of the productivity and impact of journals, the productivity and impact of authors, and the impact of the most cited articles. these productivity and impact analyses help to find the most relevant research. science mappings search for links between publications based on citation data. more specifically, we conducted a bibliographic coupling, for which we used vosviewer to visually structure the bibliometric data (van eck & waltman, ; waltman, van eck, & noyons, ) . we clustered the radical innovation literature based on strong links between citing and cited articles which are expected to deal with similar research questions (zupic &Čater, ) . we decided not to conduct a co-citation analysis which examines articles citing older articles (small, ) and therefore is more suitable to capture the historic structure of the research field. rather, we are interested in the structure of the current state of research and therefore employed the less common bibliographic coupling which examines younger publications that jointly cite older publications (kessler, ) . for this analysis, we further reduced the data set consisting of documents to by only including documents that were cited at least once. of them, were connected with , links. the temporal distribution of publications on radical innovations as depicted in fig. shows the development of the number of publications. the development can broadly be separated in three stages. the first period, from to , produced a few, but several seminal publications. during the second period, from to , the number of articles published per year gained momentum as it is more than five times the previous stage on average. the third period, from to , displays the most productive timeline in radical innovation research so far, with an average of publications per year. in , with a total of publications, the most contributions were published so far. the development of citations the focal articles of our data set received is displayed in fig. . also three stages can be identified. period one, from to , displays the timespan with a low average number of citations. during the second period, to , a significant increase in citations was measured. citations passed per year and reached almost . the last period, from to , again displays a significant growth in citations, peaking at , in . since , a constant growth in citations can be measured. the analysis of the most influential journals reveals that the documents in our data set were published in different journals. table shows the top most cited journals in which percent of articles of the data set were published. therefore, these journals are also highly productive. journals that rank highest in average citations per article are the journal of marketing ( citations per article), academy of management review ( citations per article), and strategic management journal ( citations per article). the h-index refers to radical innovation articles only and does not display the overall h-index of the depicted journals. for example, the h-index of held by the journal of product innovation management means that articles on radical innovation published in that journal received at least citations (hirsch, ) . the second and third highest h-index with each belongs to the journal of engineering and technology management and r & d management. the analysis of the most influential authors in the radical innovation field selects the most cited of authors in the sample. in order to give an overview of the most influential ones, table indicates the most cited ones. the ranking is led by chandy who laid focus on larger organizations which struggle to facilitate and deal with radical innovations (chandy & tellis, , sorescu, chandy, & prabhu, ) . tellis co-authored several of chandy's articles and also focused on the dynamics of technological change (sood & tellis, ) . o'connor, who also is the most productive author and has the highest h-index, predominantly added insights into the relationships between radical innovation and market learning (o'connor, ) and the management of radical innovations (mcdermott & o'connor, ) , especially in mature firms (leifer, o'connor, & rice, ) . the h-index, again, regards not the authors' overall publications but only those on radical innovation. the most cited and therefore most influential articles are listed in table . accordingly, the first places are occupied by older seminal articles. dewar and dutton ( ) was the most cited article in the sample. the authors empirically assessed whether there is a need for different theoretical models in the adaption of technological process innovation, regarding radical and incremental innovations. they suggested that, regarding adaption processes to technical process innovations, the investment in human capital in the form of technical specialists appeared to be a major facilitator. ettlie et al. ( ) ranked second in citations. similar to dewar and dutton ( ) , the paper conceptualized the innovation typologies of radical and incremental innovations which today is the oldest and most consolidated innovation typology (klarin, ) . in contrast to dewar and dutton ( ) , ettlie et al. ( ) suggested that centralized decision-making has a strong impact on radical process innovation in organizations. the article ranking third in citations is chandy and tellis ( ) . the paper proposed another view on the classification of radical and incremental innovations based on two dimensions. innovations that had a high degree of novelty and a high degree of customer need fulfillment per currency unit, were considered to be radical innovations, while others ranking low in both dimensions were considered as incremental innovations. in addition, the authors propose the willingness to cannibalize as a strong indicator for organizations' innovation capabilities. chandy and tellis ( ) , ranking fourth in citations, discuss that large incumbent firms rarely produce radical innovations and rely on incremental innovations instead. this is striking considering that they had the resources to invest in radical innovations. additionally, us firms were less likely to be radically innovative than japanese or western european firms because the latter could make use of source: own elaboration. source: own elaboration. less risky government subsidies whereas us firms had to rely on venture capital. the article ranking fifth in citations is henderson ( ) who compares incumbents with new entrants and finds that incumbents underperform when they face new technology as their focus on incremental innovations reduced their capabilities to exploit radical innovations. furthermore, the author connects radical innovations to greater performance features and significant impacts on existing markets. other influential articles that shaped the field of adical innovation cover a variety of subjects including incumbent firm performance (hill & rothaermel, ) , corporate culture (tellis, prabhu, & chandy, ), strategy (mcdermott & o'connor, ) , knowledge (zhou & li, ) , and design and meanings (verganti, ) . table also includes a row with the average annual citations which allow for a fairer comparison of the yearly impacts of publications as this indicator compensates for the amount of time a paper has been accessible. the highest annual citations on average ( . ) are attributed to zhou and li ( ) as well as tellis, prabhu, and chandy ( ) who received an average of . citations per year. zhou and li ( ) explain how firms with different knowledge distributions should act to be able to develop radical product innovations. they argue that firms with wide cross-technological and cross-market knowledge benefit from internal knowledge sharing. on the contrary, they suggest that firms which have in-depth specialist knowledge regarding existing technologies and markets need to expand their knowledge scope by acquiring external market knowledge. tellis, prabhu, and chandy ( ) analyze drivers of radical innovations across nations. their findings imply that corporate culture was the strongest driver of radical innovations. in contrast, drivers usually considered as important such as government regulations or capital accessibility do not seem to be as relevant. the bibliographic coupling analysis led to eight research clusters (fig. ) . the statistical interconnections between publications do not guarantee that they are also dealing with the same topics. in that sense, one cluster did not show a common thread. three clusters were very small, containing only one to three publications. three clusters have a firm focus, two have a market focus, and two small clusters address specific industries. against this background, the clusters can be described as follows. this large cluster comprises publications which deal with various aspects in firms that should be managed to foster radical innovations. several papers address organizational aspects such as organizational structure (o'connor & demartino, ) , teams (alexander & van knippenberg, ) , organizational interconnectedness between radical innovation initiatives and the established organization (kelley, ) , organizational processes (hooge, béjean, & arnoux, ) , organizational culture (mclaughlin, bessant, & smart, ) , institutional work (radaelli, currie, frattini, & lettieri, ) , and trust (brattstrom, lofsten, & richtner, ) . other articles deal with the integration of customers in the radical innovation process (lettl, herstatt, & gemuenden, ) , co-creation (perks, gruber, & edvardsson, ) , and open innovation (kennedy, whiteman, & van den ende, ) . another sub-cluster concerns human resource issues such as human resource management practices (aagaard, ) , leadership (aronson, reilly, & lynn, ) , and employee motivation (pihlajamaa, ) . organizational learning and knowledge: with publications, this cluster is similarly large and can be seen as a specific thematic subset of the prior one. knowledge (zhou & li, ) and capabilities (chang, chang, chi, chen, & deng, ) are regarded as key resources for radical innovations, and organizational learning, the corresponding process of changing the organizational knowledge and capability base, is therefore also considered as highly relevant (sheng & chien, ) . especially interorganizational learning (jean, chiou, & sinkovics, ) and the corresponding absorptive capacity (ritala & hurmelinna-laukkanen, ) are addressed as means to acquire external knowledge (flor, cooper, & oltra, ) and use it commercially. financial aspects of radical innovations: this very small cluster with only two publications also addresses a specific organizational variable-financial resources. it concerns financing constraints and sources (caggese, ) as well as financial consequences (sorescu, chandy, & prabhu, ) of radical innovations. radical innovation adoption and diffusion: this cluster with publications shifts the focus from the firm to the market as it stresses that the breakthrough success of radical innovations does not only rely on firms' innovativeness but also on customers to adopt them (dewar & dutton, ) . for radical innovations to diffuse in the market, market barriers have to be overcome (sandberg & aarikka-stenroos, ) and the right market entry (montaguti, (reinders, frambach, & schoormans, ) have to be pursued. radical industry innovations as challenges for incumbents: this cluster with publications employs a market perspective. here, firms are not seen as drivers but as potential victims of radical innovation. incumbents previously dominating a market might be threatened by competitors or new entrants introducing radical innovations (ansari & krop, ; hill & rothaermel, ) . in face of such crisis, incumbents might react with incompetence and underinvestment (henderson, ) or, proactively, with an imitation strategy (hurmelinna-laukkanen, sainio, & jauhiainen, ) or business model innovation (souto, ) . radical innovations in specific industries: two small clusters with one and three publications specifically address car manufacturers and financial service firms. however, in the other clusters, also several other industries, such as pharmaceuticals (sorescu, chandy, & prabhu, ) etc., are used to generate propositions or test hypotheses regarding radical innovations. the temporal distributions of both publications and citations clearly indicate that the research field is in a stage of growth and that the interest in radical innovation research has strongly increased over the last years. it could have been expected that the financial crisis which also had consequences for the real economy could have had an increasing effect on the research output, either because firms had to cut research and development costs or because generating radical innovations could have been a coping strategy. however, such an effect is not detectable. it is unclear what effect the current covid- crisis might have on the innovation-related research output (kraus, clauß, breier, gast, zardini, & tiberius, ) . we did not find any explanation for the two publication peaks in and . several publications were based on papers presented at conferences. however, none of them specifically focused on radical innovations, and the share of such papers was not higher than usually. the analysis of the most influential journals shows that research on radical innovations is published in a wide variety of journals which focus on different topics. interestingly, the journal of product innovation management, the most productive and most cited journal, and technovation are the only two among the top which specifically focus on innovation. radical innovation research is also published in economic, general business, general management, marketing, operations research, r&d (research and development), strategic management, and technology management journals. this indicates that radical innovation, despite being a narrow topic itself, addresses all aspects in business. the top author analysis shows that productivity and impact do not necessarily run concurrently. for example, chandy is the overall most cited author, but the average number of citations per publication is . , whereas dewar and dutton received , citations with just one article. however, even the least cited author on the top list received an average of . citations per article, again showing that research on radical innovations is regarded as highly relevant. the closer look at the most cited individual publications, not surprisingly, find older articles on the first places. however, dewar and dutton's ( ) article also has a high average citation rate per year. the article not only analyzes whether incremental and radical innovations are adopted differently but also provides an interesting definition of the two innovation types which does not focus on the extent to which the market is disrupted but on the degree of new knowledge that is integrated in the innovation. the publication with the highest average annual citation rate and also the newest in the top is by zhou and li ( ) who also focus on the role of knowledge in the radical innovation process. both high scores demonstrate that the relationship between innovation and knowledge is considered highly relevant. based on the bibliographic coupling and the content analysis within the identified clusters, we propose the radical innovation research framework depicted in fig. . to generate a radical innovation (or enhance the likelihood of its emergence), firms have to implement a favorable setting in their organization and coordinate the radical innovation process (management of radical innovations). as seen in the cluster description, the setting consists of various organizational variables such as structure, processes, culture, and many more. as previous research often analyzes relationships between specific organizational variables and partial radical innovation variables, innovation practitioners lack a systematic overview of all relevant aspects that should be subject to proper management of radical innovation. the scattered insights should be unified by future research. a radical innovation management "checklist" could name the intra-and extra-organizational aspects managers should pay attention to and how to best configure them. this overview would also help to identify research gaps as many organizational variables, such as agility (brand, tiberius, bican, & brem, ) , internal idea contest designs (hober, schaarschmidt, & von korflesch, ) , entrepreneurial orientation (gupta, mortal, & yang, ) , organizational innovation climate (liu, chow, zhang, & huang, ) , organizational justice (akram, lei, haider, & hussain, ) , or organizational wisdom practices (akgün, keskin, & kırç ovalı, ) , as well as human resource variables, such as emotional intelligence (aç ikgök & latham, ), incentives (ritala, vanhala, & järveläinen, ) , individual innovation behavior (strobl, matzler, nketia, & veider, ) , slack time (medase, ) , and top management teams (sperber & linder, ) , and many more have been related to innovation performance but not yet been subject to in-depth analysis regarding specifically radical innovations. it would also be interesting to contrast findings about what radical innovation managers should do and what they really do (maier & brem, ) . at its core, the radical innovation process can be seen as a knowledge processing and learning process which feeds from internal and external sources (organizational learning and knowledge). the role of the customer as a knowledge carrier and especially respondent of his or her own wants and needs has been stressed. external knowledge can also be assimilated through inter-organizational learning from suppliers and other partner firms. more generally, market learning also involves understanding technological trends and competitors' behavior. apart from the knowledge that is directly relevant for the potential radical innovation, research also addresses radical innovation (dynamic) capabilities which can be seen as the applied procedural meta-knowledge or organizational metacognition regarding the radical innovation process. future research might consider extending its epistemological foundation which is mainly based on the concept of "hard" knowledge which can be assimilated and verified or falsified. as radical innovations are absolutely novel rather than based on existing predecessors, it makes sense not only to concentrate on "what is", but also "what could be". apart from knowledge about current customer wants and needs, current structure of competition, and available technologies, strategic foresight (fergnani, ; iden, methlie, & christensen, ; rohrbeck, battistella, & huizingh, ; semke & tiberius, ) , and, more specifically, the use of the scenario technique (tiberius, ; tiberius, siglow, & sendra-garcía, ) , could explore multiple future developments which do not represent factual knowledge but rather mental images. rather than forecasting the most probable future state (cuhls, ) , the occupation with alternative possibilities might not only allow for a better future preparedness (rohrbeck & kum, ) but also increase creativity (rohrbeck & gemünden, ) as an antecedent of (radical) innovation (anderson, potočnik, & zhou, ) . also the conceptualization of capabilities can be enhanced (saunila, ) . whereas current research often refers to the dynamic capabilities approach (verona & ravasi, ) , the capabilities needed for radical innovation should be further specified on the individual, group (team), organizational, and inter-organizational level. the success of radical innovations depends not only on firms' proper management but also on the market. the most ingenious innovation fails when market barriers are too high to overcome or customers do not want to buy it (radical innovation adoption and diffusion). whereas the questions of adoption and diffusion are well researched, explanations of their mechanics often take a market perspective rather than focusing on how to actively foster these processes and increase the likelihood of a radical innovation's market success. future research could convey a managerial perspective more intensely. if a radical innovation can be marketed successfully, the revenues and thus firm performance (helm, mauroner, & pöhlmann, ) will significantly increase. in the ideal case, the firm has generated a new market and acts as a monopolist which would lead to massive growth. the small cluster financial aspects of radical innovations but also publications scattered across other clusters address these financial outcomes of radical innovation. however, they also see financial resources not only as an output but also an input because radical innovations are considered to require sufficient liquid funds. that this cluster is so small and that hardly any other publication with a focus on financial aspects regarding radical innovations exists, is quite surprising and provides several future research opportunities. from a financial perspective, engaging in radical innovation is a highly risky investment. however, also the decision not to engage in radical innovation is a risky endeavor as competitors could come up with a radical innovation which could diminish or even destroy the focal firm's market position. comparing such risks and providing an investment recommendation is financial studies' daily business which is rather unused so far. interestingly, paulson, o'connor, and robeson ( ) recommend evaluating the individual risks in a firm's portfolio of radical innovation projects. however, portfolio theory usually does not focus on high risk investments alone but tries to figure out the optimal diversification of securities regarding risk and return (rubinstein, ) . in that sense, all investment activities of a firm, i.e., also replacement and expansion investments as well as investments in incremental innovations, should be seen as part of a firm's investment portfolio which should be optimally diversified. the portfolio perspective can therefore assist with decision-making regarding whether or not to engage in specific radical innovation projects. another attractive research opportunity is to apply real options strategy to radical innovation research. real options apply the notion of financial options to real investments. a real option can be defined as the right but not the obligation to realize a business opportunity (adner & levinthal, ) . a firm can make a compa- rably small investment, the option price, to secure the possibility to engage in the business opportunity later. if the firm decides not to pursue the opportunity, the option price is lost, but not the whole investment amount that would have been required if the opportunity was implemented right away. this concept which already is applied to r&d projects (huchzermeier & loch, ) , could also prove useful for radical investment projects, especially in an early stage when the firm has several ideas for radical innovations but it would be beneficial to wait until uncertainty diminishes. apart from the main identified variables relevant for radical innovation, the in-depth analysis of the clusters revealed that many publications address indirect variables -mediators and moderators -which also influence the overall success of radical innovation initiatives. building on the idea of a radical innovation management checklist, a broader radical innovation checklist which also includes all variables which cannot be managed directly would prove helpful for practitioners involved in radical innovation initiatives. future research should engage in building such a systematic overview which could be based on our suggested research framework (fig. ) . a firm's performance and even survival is at risk if a competitor or startup succeeds in generating and marketing a radical innovation which might diminish the focal firm's revenue and market share (radical industry innovations as challenges for incumbents). if firms cannot engage in the development of radical innovations due to financial and human resource constraints, they have to find a suitable coping strategy in case a competitor's radical innovation emerges. future research could more intensely and systematically immerse in such coping strategies as they could become imperative with the growing number of radical innovation initiatives. additionally, coping strategies can be seen as alternative strategic options which complement the engagement in radical innovations themselves. the cluster radical innovations in specific industries and further publications across other clusters suggest that radical innovation is relevant in almost all industries. however, most research seems to analyze radical innovation in a specific industry exemplarily, possibly due to access to data, and then generalize the findings. yet, both market structures and attributes of isomorphic firms (populations) might differ significantly from industry to industry. therefore, future research should search for both generalizable findings which are valid across industries and industry specifics which differ in regards to radical innovation. our bibliometric analysis is associated with several (potential) limitations. first, the quality of the analysis depends on the quality of the data set. the bibliometric data derived from the wos might be incomplete and contain errors. due to our conscientious handling of the data, we do not see problematic inadequacies. however, future research could use additional databases. second, bibliometrics are subject to the risk of the so-called matthew effect which addresses the social reality that highly cited publications are only further cited due to their already high citations (garcía-lillo, Úbeda-garcía, & marco-lajara, ). this effect increases over time and can cause that research is wrongly perceived as highly relevant. third, and as already mentioned, clusters derived from science mappings usually do not provide accurately delimited categories as they are not content-but citation-based. the content analysis especially of rather blurry clusters is not as objective as bibliometrics usually suggest but resemble the methodology of qualitative research. due to this subjectivity, other researchers might have labeled clusters differently. fourth, future research opportunities, despite being based on the clustering and an in-depth content analysis, also involve a creative act and therefore have a subjective component. researchers with different knowledge bases and associations might have come up with other suggestions. in this study, we structured the large and fragmented literature on radical innovations using bibliometric analyses. the performance analyses revealed that radical innovation research has been growing fast in both annual publication numbers and citations, especially over the last years, and it can be expected to gain further momentum. articles are published in economic, general business, general management, innovation, marketing, operations research, r&d, strategic management, and technology management journals. the most cited authors are r. k. chandy, g. j. tellis, g. c. o'connor, r. d. dewar, and j. e. dutton. the most cited articles are dewar and dutton ( ) , ettlie et al. ( ) , chandy and tellis ( ) , chandy and tellis ( ) , and henderson ( ) . zhou and li ( ) is the paper with the highest average annual citation rate and also the most recent in the top . the bibliographic coupling identified the following research clusters: management of radical innovations, organizational learning and knowledge, financial aspects of radical innovations, radical innovation adoption and diffusion, radical industry innovations as challenges for incumbents, and radical innovations in specific industries. based on an in-depth content analysis of these clusters, we recommend engaging in the following future research opportunities: a radical innovation management checklist should compile the intra-and extra-organizational phenomena innovation managers should focus on and give recommendations on how to best manage them. the radical innovation process should be conceptualized as an intra-and inter-organizational knowledge processing and learning process. however, radical innovation research should extend its epistemological basis. apart from factual knowledge ("what is") it could also ask: "what could be?" using strategic foresight might enrich the knowledge base and increase creativity necessary for radical innovation. further research in capabilities required for radical innovation should address individual, group (team), organizational, and inter-organizational characteristics. rather than conveying a market view on the adoption and diffusion of radical innovations, future research might more intensely use a managerial perspective. the financial side of radical innovations needs more attention. we recommend applying portfolio theory and real options theory to radical innovation research. building on the radical innovation management checklist, a broader radical innovation checklist could also address moderator and mediator variables and systematically present them to innovation practitioners. stronger research efforts regarding coping strategies for firms which are faced with competitors' radical innovations would help top managers deal with such challenges. industry-specific future research should not try to generalize its findings but elaborate on the industry specifics, whereas cross-industry research should identify knowledge that is applicable to a wide range of markets. facilitating radical front-end innovation through targeted hrm practices: a case study of pharmaceutical and biotech companies the relationship of perceived emotional intelligence with adaptive performance in new product development teams what is not a real option: considering boundaries for the application of real options to business strategy organizational wisdom practices and firm product innovation the impact of organizational justice on employee innovative work behavior: mediating role of knowledge sharing teams in pursuit of radical innovation: a goal orientation perspective innovation and creativity in organizations: a state-of-the-science review, 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the adoption process of radical innovations incremental and radical innovation in coopetition -the role of absorptive capacity and appropriability the role of employee incentives and motivation on organizational innovativeness in different organizational cultures corporate foresight: an emerging field with a rich tradition corporate foresight: its three roles in enhancing the innovation capacity of a firm corporate foresight and its impact on firm performance: a longitudinal analysis firm innovativeness and its performance outcomes: a meta-analytic review and theoretical integration markowitz's "portfolio selection": a fifty-year retrospective what makes it so difficult? a systematic review on barriers to radical innovation innovation capability in smes: a systematic review of the literature corporate foresight and dynamic capabilities: an exploratory study rethinking organizational learning orientation on radical and incremental innovation in high-tech firms radical product innovation capability: literature review, synthesis, and illustrative research propositions co-citation in the scientific literature: a new measure of the relationship between two documents the impact of top management teams on firm innovativeness: a configurational analysis of demographic characteristics, leadership style and team power distribution individual innovation behavior and firm-level exploration and exploitation: how family firms make the most of their managers technological evolution and radical innovation sources and financial consequences of radical innovation: insights from pharmaceuticals business model innovation and business concept innovation as the context of incremental innovation and radical innovation radical innovation across nations: the preeminence of corporate culture scenarios in the strategy process: a framework of affordances and constraints scenarios in business and management: the current stock and research opportunities visualizing bibliometric networks design, meanings, and radical innovation: a metamodel and a research agenda unbundling dynamic capabilities: an exploratory study of continuous product innovation a unified approach to mapping and clustering of bibliometric networks how knowledge affects radical innovation: knowledge base, market knowledge acquisition, and internal knowledge sharing bibliometric methods in management and organization key: cord- -h vigeuy authors: zhang, lin; zhao, wenjing; sun, beibei; huang, ying; glänzel, wolfgang title: how scientific research reacts to international public health emergencies: a global analysis of response patterns date: - - journal: scientometrics doi: . /s - - - sha: doc_id: cord_uid: h vigeuy as of the middle of april , the unprecedented covid- pandemic has claimed more than , lives (https://coronavirus.jhu.edu/map.html). because of its extremely fast spreading, the attention of the global scientific community is now focusing on slowing down, containing and finally stopping the spread of this disease. this requires the concerted action of researchers and practitioners of many related fields, raising, as always in such situations the question, of what kind of research has to be conducted, what are the priorities, how has research to be coordinated and who needs to be involved. in other words, what are the characteristics of the response of the global research community on the challenge? in the present paper, we attempt to characterise, quantify and measure the response of academia to international public health emergencies in a comparative bibliometric study of multiple outbreaks. in addition, we provide a preliminary review of the global research effort regarding the defeat of the covid- pandemic. from our analysis of six infectious disease outbreaks since , including covid- , we find that academia always responded quickly to public health emergencies with a sharp increase in the number of publications immediately following the declaration of an outbreak by the who. in general, countries/regions place emphasis on epidemics in their own region, but europe and north america are also concerned with outbreaks in other, developed and less developed areas through conducting intensive collaborative research with the core countries/regions of the outbreak, such as in the case of ebola in africa. researches in the fields of virology, infectious diseases and immunology are the most active, and we identified two characteristic patterns in global science distinguishing research in europe and america that is more focused on public health from that conducted in china and japan with more emphasis on biomedical research and clinical pharmacy, respectively. universities contribute slightly less than half to the global research output, and the vast majority of research funding originates from the public sector. our findings on how academia responds to emergencies could be beneficial to decision-makers in research and health policy in creating and adjusting anti-epidemic/-pandemic strategies. in april , we are in the midst of the covid- pandemic, the spreading of which does not yet show any sign of success in flattening the infection curve. italy and spain are in their darkest hours, and the impending devastation in the usa looms alarming news breaks each hour. the attention of the global scientific community has turned toward flattening the curve, stopping the spread, treating the infected patients and racing to find a vaccine. however, time is short and, as governments pump billions of dollars into keeping their economies afloat, money is even shorter. this makes an international review of what research is conducted, where and by whom research is done, an insightful exercise and useful source of an expeditious and efficient global research effort. in situations like this, when the global research landscape is changing every day, it would be an unfeasible and even irresponsible attempt to closely follow up research and to keep the review up to date. what may be more valuable is to identify the 'response patterns' of academia in the face of public health emergencies towards controlling and stopping outbreaks. therefore, we aim to achieve two goals in this paper. the first one is to provide insights into how academia responds to the pandemic situation through a comprehensive analysis of over , articles and reviews on five virus outbreaks during the last decades. the exercise comprises the volume of research, geopolitical region, subject field, research sector and funding agency. the second goal is to provide a preliminary review of the global research effort in defeating covid- in the light of the response patterns detected in the first part of the analysis. these insights into the characteristics of research output in the relevant domains could assist policymakers in planning, creating and adjusting scientific strategies for a preferable response to health emergencies. public health emergencies confronting society have by nature strong science and technology components, which means that substantial improvements in health come as a result of significant advances in knowledge and technology (howitt et al. ) . hence today, controlling pandemics rests to the largest extent in the hands of scientific researchersfirst, by understanding the sources and transmission routes of the infection, then through ways to reduce infection rates and finally with the development of treatments, cures and methods of prevention. the most common outlet for scholars to communicate and fight through each of these stages is via academic publications (persson et al. ) . researchers are producing publishable work in record time (chen et al. ; holshue et al. ; li et al. ; lu et al. ) . journals are slashing the typical time lags between peer review and publication, processed within days, what otherwise might have taken months or even years. funding agencies have created special grants supporting immediate research on covid- (medical research council ; national natural science foundation of china ). using bibliometric approaches to analyse scientific output relating to recent public health emergencies helps reveal the specific contributions academia makes to resolve health crises. several studies stand out as good examples of where bibliometrics has provided a useful international view of medical and multidisciplinary research efforts. for example, a global research of fabry's disease conducted by klingelhöfer et al. ( ) identified the need to foster research infrastructure on this rare disease in developing countries with the focus on genetic research. other studies include exploring the research status and directions on several infectious diseases, such as tuberculosis (ramos et al. ) , leishmaniasis (soosaraei et al. ) , and leprosy (khasseh et al. ) . several studies of emergent infectious diseases have been conducted on single outbreaks in specific countries/ regions. those studies relied on research collaboration, publication language and citation impact to map the publication-activity patterns and scholarly impact of research related to the diseases (chiu et al. ; luchs ; nasir and ahmed ; pouris and ho ) . as far as we know, the present paper presents the first comparative bibliometric study of multiple outbreaks in a global context that has ever been conducted. the main research questions addressed in this paper are as follows: . is there any identifiable response from academia to public health emergencies? if so, what are the features of publication dynamics and distribution by country/region and research discipline? . what kinds of research institutions and funding agencies are the main actors in research relating to public health emergencies? . how has academia, and especially chinese academia, reacted to the early stages of the current covid- pandemic? the paper unfolds as follows. the next section provides a brief background on covid- , public health emergencies according to the who, and our strategy for collecting publication data on recent infectious disease outbreaks. in "results: the first group-zika, h n , ebola and sars" section, we present the patterns and participants on the previous outbreaks, followed by the preliminary analysis and discussion on covid- in "preliminary analysis: covid- " section. the last section contains our findings and reflections on this research, plus our intended directions for future work. is an infectious disease caused by a newly discovered coronavirus, which was firstly reported in december . the disease rapidly spread with a continued increasing number of confirmed cases and, by jan , the world health organization (who) had proclaimed covid- to be a public health emergency of international concern (a pheic). a pheic is a formal declaration by the who of "an extraordinary event which is determined to constitute a public health risk to other states through the international spread of disease and to potentially require a coordinated international response". pheics were instituted following the sars epidemic of - as a way of building surveillance and early warning systems for outbreaks, mobilising rapid international responses and containing the spread of infection at the source (hoffman ; who ) . the twenty-first century has seen six pheic declarations since sars: the h n pandemic, the polio declaration, the outbreak of ebola in west africa, the - zika virus epidemic, the - kivu ebola epidemic, and the - coronavirus outbreak. given our aim to map the patterns of academic 'response' to severe emergencies, we deemed all these outbreaks to be primary research subjects, with the exception of polio. this is because pheic warns of polio's resurgence after its near-eradication. hence, including literature on a virus that has been of significant concern to academia for over a century and to the who since its inception would introduce considerable bias into the sample. details of sars and the five subsequent pheics included in the study are provided in table . we subdivided these viruses presented in table into the first four viruses and covid- , and analysed the two groups separately. the analysis of covid- is only preliminary due to the fast-developing situation currently. the document retrieval strategies for the first four viruses, as shown in table , are constructed by referring to entry terms of each specific name of virus/disease in the mesh vocabulary and retrieving rules of wos. our final sample of the first four viruses, drawn from the science citation index expanded (sci-e) & social sciences citation index (ssci) of the clarivate analytics's web of science (wos) core collection, comprised , articles and reviews. note, however, that the number of publications in table will add up to more than total since some publications related to more than one disease. because of the limited number of publications indexed by wos in the initial stage of the outbreak, we expanded our search for publications on covid- beyond wos to conduct a more comprehensive analysis. we retrieved articles and reviews in pubmed, and the source categories of the chinese social sciences citation index (cssci) and chinese science citation database (cscd) in cnki (chinese national knowledge infrastructure), which is the largest continuously updated database of chinese journals in the world. the search strategies are listed in table . data were obtained on april . all retrieved publications were manually checked, and irrelevant publications were removed. the final corpus comprised of publications. our first observation is that very few publications on any of these viruses were published prior to . hence, fig. shows the number of publications by year between and . from all four cases, a clear trend is apparent. immediately following the outbreak, there is a sharp rise in disease-specific publications up to a peak within years after the outbreak, beyond which the number of publications gradually decreases. these patterns reflect a quick and remarkable response by the research community to public health needs. perceptible increase in publication activity. in fact, the number of publications on ebola, which was already in decline since , experienced an even slightly sharper drop. the zika virus first isolated from a rhesus monkey in the ugandan zika forest in , later from aedes africanus mosquitoes in the same forest (nasir and ahmed ) . from the s to the s, rare sporadic cases of human infections were found across africa and asia. research on zika appeared early in our database, but publication numbers remained low until the outbreak in - , which resulted in sharply growing attention with a peak in . the ground zero of each outbreak varies as does the research capability of different countries/regions, both affecting the geographic distribution of research effort. the country/ region assignment of publications has been done on the basis of the author's institutional address as reported in the by-line of the paper. in this study, a full-count assignment scheme has been applied, that is, national publication counts express how many papers the country/region has contributed to. figure shows the geographical distribution of publications for the top countries/regions by the number of publications on the four viruses. the usa, china and the uk are the three countries with the largest number of publications. the usa ranks first in all viruses except sars. within each disease, the distribution patterns across europe and north america are quite similar, i.e., a relatively higher proportion of publications on h n and ebola than for zika and sars. h n got lots of exposure from all ten countries except for brazil, which has devoted most of its effort to zika. in addition, studies on h n are relatively evenly distributed, whereas papers on the other three diseases show significant regional differences. the three countries with the most publications on ebola are the usa, uk and germany, even though both ebola outbreaks had their epicentre in africa. unlike ebola, the country with the most sars-related publications was the one in the heart of the outbreak-china. in general, these ten countries tended to place greater emphasis on diseases that caused epidemics in their own regions, especially sars in china and zika in brazil. countries in europe and north america paid relatively close attention to ebola, which was not an epidemic in their own regions, perhaps partially because of the high fatality rate coupled with early media reports of isolated cases in the uk (cooper ) and the usa (botelho and wilson ) that eventually did not escalate. we further analysed the geographical distributions of publications by year to explore the differences in response speed and continuity of research from various countries/ regions. figure shows the variations of publication distributions with the top countries/regions from to , where the vertical axis refers to the number of publications for each disease. the top countries/regions that contributed to the highest number of publications vary for four diseases, in which the usa, the uk, china, canada, australia and germany are on the four diseases' top list. interestingly, chinese researchers showed a strong and quick response to the sars outbreak in - . however, the research interest declines steadily with the end of the outbreak. in contrast to china, the usa tends to pay more sustained attention to sars. of particular note is that sierra leone is the only african country that appeared on the list of ebola, which is the core area of the outbreak of ebola in western africa (who ). to further investigate joint research efforts by different countries/regions on health emergencies, we generated international collaboration networks among the top twenty countries/regions according to their publication activity of four viruses (see fig. ). the size of a node indicates the total number of publications of the country/region, which corresponds to the first number presented on the node. the second number on each node represents the number of papers published by the country/region as the first authors' address. the thickness of links refers to the strength of collaboration between two countries/regions. it is calculated based on salton's measure, which is defined as the number of joint publications divided by the square root of the product of the number (i.e., the with the largest number of publications in total, the usa plays a significant role in collaboration with other countries/regions regarding all these four diseases. apart from canada and european countries, the usa also collaborated intensively with several african countries on ebola research, such as sierra leone, liberia, guinea etc., which are the main areas of the ebola outbreak. african countries, although particularly contributed to ebola research, in general, do not play a leading role in international collaborations, which is also reflected by the low share of 'first-author' publications. different from the massive joint efforts contributed by the usa and european countries on ebola, sars related research reveals a quite different pattern that european countries had a relatively weak role in this china-centric outbreak. china, as the country with the largest number of publications in sars, focussed more on collaboration with the usa. in this section, we adopt the science overlay maps proposed by rafols et al. ( ) and carley et al. ( ) , and generated using the vosviewer (waltman et al. ) to illustrate those disciplines that proved most relevant for research on these four infectious diseases, shown as fig. . every node presents a wos category, and the size of the node indicates the number of the publications based on a full counting scheme. fig. shows, publications have a relatively centralised distribution around those subject categories, which primarily belong to medical and health-related fields, such as 'infectious diseases', 'health care', 'pharmacology' and 'clinical medicine'. a non-negligible share of papers has also been published outside the core of the life sciences, above all in 'chemistry', 'environmental science' and 'material science'-fields with relevant links to medical research. the structure of the fine-grained picture base on the wos subject categories provided in fig. is strongly influenced by multiple subject assignments, on the one hand, and the different broadness of the journals' scopes underlying this assignment, on the other hand. therefore, we further used the major fields of the ecoom classification scheme (cf. glänzel and schubert ; glänzel et al. ) , which actually forms an aggregation of the wos categories, to produce a more explicit depiction of the subject distribution. the charts in fig. show the annual change of this distribution, where the vertical axis represents the number of publications. 'clinical and experimental medicine i (internal medicine)', 'clinical and experimental medicine ii (non-internal medicine)', 'biology' and 'biosciences' are the four subject fields with the most publications on all four diseases. the remarkable attention given to ebola in 'social science i' (with regional and community health issues) is worth mentioning as it certainly reflects the social perspectives of this epidemic. according to the who ( a), "ebola is rare, but the disease has a high risk of death, killing % to % of those infected". the symptoms of the ebola virus can be sudden and striking, including high fever, fatigue, vomiting and both internal and external bleeding. the high mortality rate and scary symptoms have led to public panic about ebola. furthermore, the generally lower level of economic and technological development in the affected areas of africa in order to gain a more in-depth insight, we combined geographical distributions with the disciplinary one. the above analysis indicated quite similar patterns in the disciplinary distributions of publications in general. in a next step, we subdivided the major fields into the ten most frequent wos subject categories in medical science for a fine-grained exploration of the differences between countries/regions. thus, instead of the bottom-up approach in fig. , we applied a top-down solution to filter out redundancies caused by multi-assignment and journals with 'general scopes' and to focus on the most relevant disciplines. figure illustrates the results, where the ten radial lines depict the discipline distributions of ten countries' publications on the listed fields, and edges with different diameters of the map represent the percentage of publications involved by each country in the listed fields. for example, almost % of publications that australia contributed to was devoted to the category 'infectious diseases'. as shown in fig. , 'virology', 'infectious diseases' and 'immunology' are the top three categories with the largest shares of publications in all countries except for brazil. as the centre of the zika outbreak, brazil devoted the lion's share of its research efforts to this virus as compared with the other three. zika is primarily transmitted by the aedes mosquito, which makes it highly relevant to 'parasitology', 'tropical medicine' and 'infectious diseases'-the top three disciplines in brazilian research. it is particularly noteworthy that brazil also paid relatively close attention to 'public, environmental & occupational health'. china and japan also demonstrated quite different disciplinary interests compared to other countries. 'tropical medicine' is the least studied discipline in china and japan, followed by 'parasitology'. compared with other european and american countries, 'public, environmental & occupational health' also received less attention from these to consolidate our observations, we calculated the cosine similarities between disciplinary distribution in different countries. the results in table confirm our findings that brazil has distinct differences, and china and japan have low similarity with the other countries. although germany is somewhat similar to japan, mainly due to sharing the same top four disciplines and relatively much attention paid to 'pharmacology & pharmacy' (see fig. ). research interests in the usa and canada are the most similar at the cosine level of . . australia, the uk and france form kind of cluster with an average cosine similarity of . . apart from the top three disciplines for these three countries, 'public, environmental & occupational health' also received relatively much attention, above all in the uk. scientific output represents not only the efforts of researchers but also the involvement and support of research institutions and funding agencies. the institution information was cleaned through correcting the clerical errors from authors, merging spelling differences and various expressions in different times of institutions. following the rules of country/ region and subject assignment, the number of publications for each research institution was calculated on the basis of a full counting scheme. as expected, universities play a pivotal role in enabling and conducting relevant research and responding to public health emergencies. as shown in fig. , universities have contributed to more than % of the publications, which is almost equal to the contribution of all other institutional sectors of research combined. table lists the ten institutions with the largest number of publications. universities and governmental research institutes are the most actively involved in responding to health emergencies. the centers for disease control and prevention (cdc) and the national institute of allergy and infectious diseases (niaid) in the usa have notably contributed to improving understanding of different diseases, especially in the context of h n and ebola. furthermore, five of the ten institutions that published research results on ebola are government agencies in the usa and canada, which reflect the concern from the developed countries' government bodies on epidemics in underdeveloped areas. the university of hong kong and the chinese academy of sciences are found as the two most active institutions from china. their focus was more on sars and h n . consistent with the previous analyses, research institutions in brazil showed a distinct tendency toward zika. funding information in scientific publications often lacks standards and is incomplete. completeness and accuracy of funding information in bibliographic databases do practically not allow any profound bibliometric analysis at this level (alvarez-bornstein liu et al. ) . nevertheless, to gain some insight into the funding patterns of research on the epidemics, we also analysed the information obtained from the wos metadata. we found in terms of funding agencies that , papers ( . %) in our dataset acknowledged support from a grant, of which , ( . %) supplied valid information about the funding agency(s). we have analysed these papers in the following. the assignment of publications with multiple funding agencies is based on full-counting method. we subdivided the funding agencies into public and non-public agencies. public agencies mainly refer to governmental funding bodies and institutions, including public universities. the non-public sector includes non-profit, charity foundations, private firms and other non-public. the number of publications with grant information by virus is shown in fig. . h n has the largest number of publications with valid grant information; sars has the smallest (cf. figure ). the sars outbreak lasted only months with a peak of infection even much before, which might explain why there are so few funded publications for this case. this was a very short period to establish grants and parameters, apply for and receive funding, let alone to have sufficient time for doing research and to publish. furthermore, research interest declined with the disappearance of the sars virus. the same seems to hold for funding interest. figure shows the breakdown between public and non-public funding. public funding agencies contributed to more than % of the research output for all four viruses, which demonstrates the large degree of governmental support for research in response to public health emergencies. the public sector dominates the top ten funding agencies. as table indicates, the usa department of health & human services (hhs) ranks first in supporting research in all four cases, within which the national institution of health (nih), as one of the world's leading public medical research and funding organizations, plays a prominent part. the national natural science foundation of china (nsfc) and the european commission (eu) also made notable contributions to supporting research and, like the other countries/regions, public funding agencies in brazil occupied an essential position in responding to zika. as to the non-public sector, all organisations/companies in the top ten list are from europe: glaxosmithkline (gsk) and roche, inc., and the fourth wealthiest charitable foundation in the world, the wellcome trust ( ), which ranked sixth and tenth in supporting ebola and zika research, respectively. from the perspective of countries and world regions, funding agencies in the usa, china, and the uk contributed most to supporting research in response to public health emergencies, as shown in fig. . in all four cases, the usa ranks first. china is second for sars and h n , and the uk ranks third for sars and ebola. of particular note is that brazil ranks second in supporting the research on zika, which is in line with our previous results. other countries in the top five list are japan for h n , germany for sars and canada for ebola. this ongoing - covid- pandemic has spread globally with unprecedented speed, which raised considerable concern from scientists in various fields from all over the world. the sharply increasing number of papers in the beginning stage of covid- substantiates the immediate and decisive response of academia to public health emergencies again. perhaps the most remarkable observation, before we examine the research literature on covid- in detail, is how this pandemic has catalysed such intensive scientific activities, which generated an unprecedented amount of knowledge and allowed research to move faster than during any previous outbreak (kupferschmidt ) . following the retrieval strategies given in table , publications both in international and domestic journals of china were collected on april . the publication data of domestic journals in china was added for two reasons. first, china was one of the first countries on the front line of fighting the pandemic, and, second, china has been acknowledged by the who ( b) as having "rolled out perhaps the most ambitious, agile and aggressive disease containment effort in history" in response to covid- , much of which was informed by research findings. therefore, the studies from china hold a special position that could provide a comprehensive understanding of the efforts made by the whole of chinese academia. figure shows the cumulative number of publications sourced from each of the different databases from the middle of january to early april. the entrez date, i.e., the date that the record was added to pubmed, was adopted here to represent the publication time indexed by pubmed. due to the incompleteness of 'publication date' information in wos, the publication time in wos was acquired through two approaches: ( ) for papers with pubmed id (pmid), matching papers to pubmed and adopting the entrez date as publication time, ( ) for papers without pmid, searching manually and taking the first online published date as publication time. for publications in cnki, publication time was directly obtained from the database. as fig. shows, pubmed dwarfs ckni and wos for the number of publications and growth rate. the low covid- topic coverage in wos is to a large extent a consequence of the update time of document indexing for the database but also due to the coverage of its journal collection. the first study on covid- indexed by cnki emerged on jan , somewhat later than pubmed and wos. however, the number of papers published in domestic journals still reflects strong growth. the keywords provided with each paper show the different focuses of publications across the databases. we chose three as the threshold of the minimum number of keywords' occurrence and merged similar terms to generate better visualizations. figure provides the author-keyword co-occurrence maps using vosviewer for each database. apart from the high-frequency terms and phrases related to the name of the virus (e.g., covid- , sars-cov- ), it is quite apparent that research on other coronavirus diseases, such as "mers" and "sars" is the common interest for both papers in the international and domestic journals. with the spread of covid- around the world, epidemic prediction, control and management strategies have also become the general concern of academia. furthermore, mental health under the crisis time (e.g., anxiety, depression) and preexisting condition and diseases that may complicate the disease course and cause severe cases of covid- (e.g., old age, obesity, diabetes) also attracted attention for scholars from all over the world. publications in international journals reflect the focus on fundamental research on the virus and the development of a vaccine as reflected by keywords like "genome", "vaccine" etc. the susceptibility of the population, public health concerns and the disease's status in different countries (e.g., australia, china) as an epidemic/pandemic also received considerable attention in papers indexed by pubmed. unlike the publications in international databases, those indexed in the cnki focus more on clinical studies and diagnosis and treatment schemes. terms like "clinical trial", "diagnosis and treatment scheme", "clinical features", "remdesivir" occur frequently. as the core of the outbreak from the end of january until early march, china required timely research on treatment schemes to diagnose patients and prevent further spread. in addition, societal issues such as medical support teams from other provinces in china to hubei province and personal protective equipment also received attention from research published in domestic journals. it is worth noting that traditional chinese medicine features prominently in these studies, as evidenced by terms in red clusters, such as "chinese medicine", "chinese herbology", "syndrome differentiation and treatment" and so forth. figure depicts the countries, institutions, subjects and countries of funding agencies with the highest number of publications indexed by wos. we limited this analysis to publications from wos for comparability with the previous analysis. chinese scholars have contributed to more than % of the papers related to covid- , and the majority of the projects have been granted by chinese funding agencies with the national natural science foundation of china (nsfc) at the top of the list. the three institutions with the highest research output are chinese universities. in line with previous epidemics, the field with the most studies is infectious diseases. to gain insight into how covid- is being studied collaboratively from different countries, we used publications indexed by pubmed to observe the international collaborative pattern due to its relatively high volume of publication data at the present stage. figure was generated following the same rules as fig. . as fig. shows, china, as the heart of the outbreak in the initial period, has taken a prominent role in covid- research with the largest number of publications, followed by the usa and the uk. close collaboration is observed between the two leading countries of china and the usa. apart from the uk, italy stands out among other european countries with publications in total. as of april , italy is one of the world's centres of the covid- outbreak with , confirmed cases. chinese researchers have consistently played an important role in contributing to knowledge production on emergent epidemics in previous pheics since , which signals the growing role of chinese scholars in international academia. as covid- continues, a vivid public discussion has arisen in china on whether scientific achievements from china should be prioritized to publish in international or domestic journals. critics of international publication argue that, due to language barrier and possible pay-walls of international journals, necessary information will not be shared with china in a timely manner with the consequence that gaining and applying knowledge of how to treat the disease and control the epidemic would slow down. academia is continuing to adjust its methods to respond outbreaks more efficiently and effectively, which may also result in permanent transformations of scientific and technological systems. research needs to make more specific and more substantial contributions to the health and wellbeing of people, especially during outbreaks. as pallari and lewison ( ) wrote, biomedical research could influence its two main goals: better patient fig. international collaboration pattern of covid- research treatment and prevention of illness, by studying the research base of clinical practice guidelines linked to patient treatment, and stories in the mass media as an expression of healthcare policy. in the context of the covid- disease, this means that domestic publication in chinese could help achieve this goal faster and earlier by reaching the targeted community directly without and paywalls and language barrier that may make relevant literature less accessible to many on the global front line (larivière et al. ) . in addition to formally published papers in scholarly journals, many findings related to covid- have been publicly shared as preprints. preprints have the benefit of accelerating the release of results with a solution that provides fast evidence-based responses, although not peer-reviewed and thus officially unverified (johansson et al. ; chen et al. ). the desire, and perhaps necessity, to publish as preprints have been reflected thoroughly during the covid- outbreak so far. according to kupferschmidt ( ) , the plethora of data has been released daily by preprint servers that did not even exist a decade ago. for example, by early april, two of the largest biomedical preprint servers, biorxiv and medrxiv, had already posted more than papers on covid- (see fig. ). the two servers "are currently getting around ten papers each day on some aspects of the novel coronavirus," says john inglis, head of cold spring harbor laboratory press, which runs both servers (kupferschmidt ) . johansson et al. ( ) found that preprints posted during the ebola and zika outbreaks published novel analyses and new data more than days before the publication of the journal version, which is a substantial acceleration of data dissemination and information sharing. however, despite all benefits of preprint publication, there are also some validity issues. without any peer review or other quality control, there is the risk of disseminating inaccurate results and unvalidated information. a preprint paper (deposited on biorxiv) stating "uncanny" similarities between sars-cov- and hiv has fostered conspiracy theories about genetic engineering and might serve as an example. although the paper was retracted, it still raised a vivid discussion on this issue, leaving people in doubt. notes have been posted on biorxiv and medrxiv for each paper to emphasise that preprints only contain preliminary findings. regardless of whether scholars choose formal publication channels such as a peer-reviewed international or domestic journal or rather publish a preprint, sharing information in a timely manner and open science is of great importance if fig. accumulated number of papers posted on biorxiv and medrxiv [note publication data was acquired from https ://conne ct.biorx iv.org/relat e/conte nt/ (covid- sars-cov- preprints from medrxiv and biorxiv) on april, ] academia is to build the first line of defence against infectious diseases. with increasing the scale and complexity of the scientific study, a global vision and awareness of international collaborations are essential for scholars to enhance their research ability, the quality and usefulness of outputs and for disseminating their findings in high-level journals. for academia, joint efforts should be made to promote the timely and wide dissemination of relevant information, which is critical for saving lives in times of crisis (larivière et al. ). in this work, we have explored the response patterns of academia to six international public health emergencies in terms of the number of publications, geographic region, subject matter, institutional sector and funding agency. in two separate analyses, we compared academia's response to five outbreaks of four viruses-ebola, h n , zika and sars-with research-activity patterns in the early stages of the covid- pandemic. our analysis showed that researchers typically respond quickly to public health emergencies with a sharp increase in the number of publications immediately following a pheic by the who and during each outbreak. countries/regions give greater emphasis to epidemics in their own region. however, europe and north america are also concerned with outbreaks in other, developed and less developed areas through conducting intensive collaborative research with the core countries/regions of the outbreak, such as in the case of ebola in africa. as a contrast, research on sars is primarily conducted by the epicentre of the outbreak-china, with a joint force of usa. the participation of european countries in sars research is relatively low. in terms of the research field, most papers have been published in the highly relevant disciplines 'virology' and 'infectious diseases'. there are also clear indications that european and american countries pay closer attention to the public health aspects of outbreaks, while china places more emphasis on biochemistry & molecular biology, and japan tends to focus on pharmacology. our results also indicate that universities and public funding agencies are the main respondents in global health emergencies. as the core area of the covid- outbreak in the beginning stage, researchers in china are playing a prominent role in producing knowledge and international dissemination of scientific information regarding the virus and the disease. while preprint proved important means of extremely fast response and scholarly communication, the lack of peer-reviewing also raises the risk of spreading inaccurate information. more research on preprint publishing is needed to investigate how preprints might affect the response patterns of academia to health emergencies. outbreaks of epidemics on that scale may also affect or even result in transformations of national health security systems. after the sars outbreak in , the chinese government undertook major programs to strengthen the public health systems (wu and ye ). despite these initiatives, however, the attention and efforts on improving the system are declining over time. some argued that severe shortcomings of public health and security system had been revealed in this outbreak (ding et al. ) . sustained attention from both governments and academia is necessary if treatments, cures and preventative measures have to be developed for diseases caused by these viruses and their possible future mutations. recent public health emergencies have uncovered the disruptive effect of outbreaks on individuals and society, leading many scholars and practitioners to call for increased investments in "global health security" (puyvallée et al. ) . their implementation in system reforms could be an interesting topic for future research. funding acknowledgments in the web of science: completeness and accuracy of collected data thomas eric duncan: first ebola death in u visualization of disciplinary profiles: enhanced science overlay maps the risk of a pandemic with the influenza a (h n ) virus epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study the position of preprint in scholarly communication: a bibliometric and empirical study of arxiv bibliometric analysis of severe acute respiratory syndromerelated research in the beginning stage ebola in the uk: infected nurse pauline cafferkey treated with survivors' blood plasma and experimental drugs ebola death toll in west africa tops . the new york times an interim review of lessons from the novel coronavirus (sars-cov- ) outbreak in china a new classification scheme of science fields and subfields designed for scientometric evaluation purposes the challenges to expand bibliometric studies from periodical literature to monographic literature with a new data source: the book citation index the evolution, etiology and eventualities of the global health security regime first case of novel coronavirus in the united states technologies for global health preprints: an underutilized mechanism to accelerate outbreak science cluster analysis and mapping ofiranian researchers in the field of parasitology: with an emphasis on the co-authorship indicators and h index global research on fabry's disease: demands for a rare disease preprints bring 'firehose' of outbreak data the coronavirus (covid- ) outbreak highlights serious deficiencies in scholarly communication early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia funding information in web of science: an updated overview genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding profile of brazilian scientific production on a/h n pandemic influenza covid- rapid response call a bibliometric analysis of research on zika virus indexed in web of science special project guide of "fundamental research on origin, pathopoiesis and prevention of -ncov how biomedical research can inform both clinicians and the general public research collaboration at nordic universities a bibliometric analysis of research on ebola in science citation index expanded crisis! how emergency preparedness logic changes global health policy science overlay maps: a new tool for research policy and library management a bibliometric analysis of tuberculosis research indexed in pubmed a decade bibliometric analysis of global research on leishmaniasis in web of science database a unified approach to mapping and clustering of bibliometric networks wellcome trust annual report and financial statements international health regulations ebola outbreak in west africa declared a public health emergency of international concern ebola virus disease report of the who-china joint mission on coronavirus disease (covid- ) china's public health practice in years acknowledgements this work is supported by national natural science foundation of china (grant nos. , ), the national social science foundation of china (grant no. vsj ) and the national laboratory center for library and information science in wuhan university.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - hd iibk authors: solbakk, jan helge; bentzen, heidi beate; holm, søren; heggestad, anne kari tolo; hofmann, bjørn; robertsen, annette; alnæs, anne hambro; cox, shereen; pedersen, reidar; bernabe, rose title: back to what? the role of research ethics in pandemic times date: - - journal: med health care philos doi: . /s - - -x sha: doc_id: cord_uid: hd iibk the covid- pandemic creates an unprecedented threatening situation worldwide with an urgent need for critical reflection and new knowledge production, but also a need for imminent action despite prevailing knowledge gaps and multilevel uncertainty. with regard to the role of research ethics in these pandemic times some argue in favor of exceptionalism, others, including the authors of this paper, emphasize the urgent need to remain committed to core ethical principles and fundamental human rights obligations all reflected in research regulations and guidelines carefully crafted over time. in this paper we disentangle some of the arguments put forward in the ongoing debate about covid- human challenge studies (chis) and the concomitant role of health-related research ethics in pandemic times. we suggest it might be helpful to think through a lens differentiating between risk, strict uncertainty and ignorance. we provide some examples of lessons learned by harm done in the name of research in the past and discuss the relevance of this legacy in the current situation. these are uncertain times-for all, wherever one lives, and whatever one aspires to know about the covid- pandemic. at present, there are so many things we do not (yet) know: how it will evolve and spread, when, if ever, it will end, how and why it started, whether infected persons develop permanent immunity, whether safe and effective cures and vaccines will be possible to develop, and last, but not least, what impact the pandemic will have on each and every one; be it individuals, families, societies, nations, regions, or globally. the aim of this paper is to address the ongoing debate about covid- chis and discuss the concomitant role of medical and health-related research ethics in the present situation the word pandemic comes from two ancient greek words (pan 'all' + dēmos 'people'), so literally speaking 'pandemic' means all the people, all the world. where there is an urgent need for developing effective methods of detection, treatment, and prevention to cope with the covid- pandemic and notably in ways that minimize harm and that benefit all human beings. although at present all attention and focus is on the covid- pandemic, it is key not to forget there have been pandemic surprises in the past and that future, unknown pathogens may lead to crises with unprecedented implications. the issues we discuss here are therefore relevant beyond the covid- pandemic; above all they concern how we should navigate (research ethics) in times of great threats to individual, public and population health and under conditions where different forms of uncertainty still prevail. the british medical association's report on biomedical research and human rights states: «research is driven by a desire to understand the causes of disease or dysfunction and find effective methods of prevention and treatment." however, the report continues, "even such humanitarian aims can be risky", in particular under circumstances perceived as extreme or exceptional. in the report, nine risk-factors for abusive research are identified, of which three are of particular relevance in the present context: ( ) the perception of an urgent and overriding scientific need; ( ) the perception of a national necessity or government pressure to conduct research; and ( ) the situation of contingent populations chosen as research subjects. table covid- and three forms of uncertainty (risk, strict uncertainty and ignorance) a goldstein and burstyn ( ) b sethuraman et al. ( ) c mutambudzi et al. ( ) d mcintosch et al. ( ) e hofmann ( ) f mcintosch et al. ( ) g yelin et al. ( ) h european group in ethics in science and technology ( ) i mcintosch et al. ( ) j hofmann ( ) k yelin et al. ( ) l kalil ( ) risk a,b,c,d (known outcomes and known probability distributions) test accuracy (sensitivity, specificity, predictive values) for the various tests in different contexts effects and side effects of new treatments prevalence of disease the risk of healthcare workers versus the risk of non-essential workers testing positive for covid- strict uncertainty e,f,g,h (known outcomes and unknown probability distributions) basic reproduction number (r) case fatality rate/infection fatality rate the precise interval during which an individual with sars-cov- infection can transmit infection the pathogenic effect of the sars-cov- in different age groups the extent to which transmission occurs from a-symptomatic or pre-symptomatic subjects and how much it contributes to the pandemic whether all infected patients mount a protective immune response and how long any protective effect will last reinfection how long sars-cov- can persist on surfaces whether pre-existing immune responses impact the risk or the severity of covid- and whether they will influence sars-cov- vaccine responses long-term sequelae and late-stage consequences of covid- ignorance i,j,k,l (unknown outcomes and unknown probability distributions) in a recent letter to the editor of the american journal of bioethics, stoeklé and hervé state that now is not the right time for ethics reflection, but rather for political action and for "indisputable confidence in medical care staff and scientists, not only in france, but everywhere around the world". in a response to this view, and notably with the opposite title, the respondents state : in times of crisis, like the current pandemic of covid- , the perception that ethical standards can be relaxed due to the urgent need for solutions is growing, according to stoeklé and hervé. for them, ' ethics is only useful if you have the time, and right now, time is exactly what we do not have.' it is a misperception without any doubts. ethics has always preserved its identity as a rationalization of human action. therefore, ethical reflections to take decisions are useful all the time and must be reinforced in times of pandemic. another way of visualising this tension is by differentiating between the epistemological ethos of doing biomedical research, such as developing knowledge and skills for effective diagnostic, treatment and prevention, versus the ethical ethos of the same enterprise; the attempt to protect the interests and wellbeing of patients and healthy individuals involved in such research. ethos is here used in the meaning of 'accepted standards.' in research, two such normative standards or rules of play are used: epistemological rules of play (such as truth, probability, coherence, relevance, fruitfulness, interestingness, and utility), and ethical rules of play (such as autonomy, informed consent, justice, beneficence, non-maleficence, truthfulness, dignity, trust, vulnerability, and solidarity). the tension between these two normative standards is permanent, and one that probably never can be fully resolved. but in times of perceived urgency, the danger is increased tension, or worse, a disregard and violation of epistemological as well as ethical standards, and that shortsighted expediency with questionable results will ensue. in the pages to follow we argue in favour of an ethics of precaution, with particular emphasis on the role of research ethics in particularly challenging situations, such as pandemics. that is, we will analyse and critically assess the epistemological and ethical justification of several research initiatives that have been implemented or are at the planning stage. we claim that in the current situation of a palpable sense of medical and scientific urgency, and of national and global necessity, there should be no room for epistemological or ethical exceptions or shortcuts. on the contrary-and perhaps more than ever-there is a need to conduct biomedical and health-related research in compliance with existing rules of play and fundamental human rights commitments. faced with the "toxic brew of uncertainty" the pandemic has caused, we suggest it would be helpful to differentiate between three different forms of uncertainty: risk, strict uncertainty, and ignorance or non-knowledge. risk represents a form of uncertainty with known potential outcomes, and, where the probability distribution is known. the plethora of uncertainties that the covid- pandemic are causing cannot, however, be addressed within this narrow framework of risk estimation; uncertainty considerations of the two additional kinds mentioned above should also be included. strict or fundamental uncertainty is a form of uncertainty where possible outcomes are known, but the probability distribution is unknown, while ignorance or nonknowledge represents forms of uncertainty where only some possible outcomes are known while the statistical likelihood of each of them is unknown. the relevance of differentiating between these three forms of uncertainty in the present context is visualized in table . in the aftermath of another global disaster, world war ii, several normative initiatives were taken to prevent a similar catastrophe to recur, such as the establishment of the united nations and the development of the universal declaration of human rights. in addition, more robust ethical standards for biomedical research were crafted in order to avoid inhuman research in the future-not the least in crisis-situations. the then first lady of the usa, eleanor roosevelt, served as the first chair of the un commission on human rights, which drafted the universal declaration of human rights ( ). the first paragraph in the declaration's preamble states that "the foundation of freedom, justice, and peace in the world" is the "recognition of the inherent dignity and of the equal and inalienable rights of all members of the human family". of the articles in the declaration article and are of particular relevance for research ethics; article restates the freedom and equality of all human beings in terms of stoeklé and hérve ( ) . hellmann et al. ( ) . outka ( ). wynne ( ) , rørtveit and strand ( ) , nielsen and sørensen ( ) and hofmann ( ). https ://www.ohchr .org/en/udhr/pages /udhri ndex.aspx. dignity and rights, and article emphasizes the right to be protected from torture or cruel, inhuman or degrading treatment or punishment. the essence of these normative statements harkens back to the ethical code of medical research issued by the war crimes tribunal at nuremberg the previous year, in . of the standards laid down in this code, and with which physician-researchers must comply when carrying out experiments on human subjects, standard , in particular, has become highly relevant these days due to pressure from influential medical stakeholders, agencies and bioethicists to permit the conduct of controlled human infection studies (chis), also labeled human challenge trials (hcts), or challenge studies (css) to possibly shorten the development time of vaccines to protect against covid- caused by the sars-cov- virus. in the next paragraph of this paper we will examine and critique in detail four position statements advocating the epistemological and ethical justifiability of conducting covid- chi-studies: p. singer and r.y. chappell's, pandemic ethics: the case for experiments on human volunteers ; the report, key criteria for the ethical acceptability of covid- human challenge studies, issued by a working group set up by who ; the policy forum statement, ethics of controlled human infection to study by shah, miller, darton et al.; and jamrozik and selgelid's statement, in addition, we will consider other recent papers advocating the use of covid- chis studies. in table below we have made a summary of prevalent arguments for and against such studies. standard of the nuremberg code reads: no experiment should be conducted where there is an a priori reason to believe that death or disabling injury will occur; except, perhaps, in those experiments where the experimental physicians also serve as subjects. while repeating (in the first clause of article ) that no one shall be subjected to torture or to cruel, inhumane or degrading treatment or punishment, the legally binding un international covenant on civil and political rights adds a second clause that "in particular, no one shall be subjected without his free consent to medical or scientific experimentation". the prohibition is intended to prevent the recurrence of atrocities such as those that took place during wwii and the first decades after the war. even though this prohibition is implicit in the first clause, the matter was deemed so important as to require a specific and precise provision. during the drafting of the covenant, there was a proposal that compulsory measures might be taken "in the interest of community health", but the proposal was rejected on the grounds that this might lead to abuse. the consent requirement is thus formulated as absolute, without any exceptions. the human rights committee has reaffirmed what is already explicitly mentioned in article ( ), that even in situations of public emergency, no derogation from article is allowed. controlled human infection studies (chis) "are clinical studies that, as part of the protocol, deliberately expose trial participants to an infectious pathogen. these studies are often done in the context of vaccine development, with trial candidates exposed to a pathogen after being immunized with an experimental vaccine". the main advantages of chis compared to large field trials are that they can generate data much faster, they are much less expensive and they do not require thousands of research participants, normally only between and participants. the four position statements on chis referred to above all suggest that suitable candidates for such studies are "young people without underlying medical who ( ). shah et al. ( ) . jamrozik and selgelid ( a callaway ( ) , eyal ( ), eyal et al. ( ) , jamrozik and selgelid ( a, b) , plotkin and caplan ( ) , schaefer et al. ( ) , shah et al. ( ) , , who ( ) and wolemonwu ( ) . . the urgency of the current pandemic gives substantial weight to a challenge study j the endemic argument the probability of dying or developing disability if infected would be smaller in a chi trial k the risks in question do not entail a major net increase in risk (in light of background risks of infection) l participants face a background risk of infection in the community m only people with an especially high baseline risk of getting exposed during or soon after the trial period should be recruited (e.g., people residing in areas with high transmission rates) n the risk and safety argument participation in a covid -chi trial would be less risky than joining a standard efficacy trial for the same vaccine o covid- chis have a much higher risk than the minor risk threshold. p for a live sars-cov- challenge there are deadly risks q currently, we lack sufficient knowledge of sarscov- pathogenesis to inform inclusion and exclusion criteria for a sars-cov- chim r the medical benefit argument the probability of averting death in the event of infection would be substantially better inside a chi trial than outside s the altruistic argument it might seem that anybody volunteering to participate in such a study lacks capacity for rational decision-making. but humans do many important things out of altruism t volunteers who participate in the challenge trials should be motivated to advance human health and wellbeing rather than driven by their economic needs u the social value argument benefits to the subject + benefits to society > risks to the subject v,w given the risks to participants, sars-cov- challenge studies would need to demonstrate very substantial social value before proceeding. arguably, this bar might already be met given the high death toll and severe disruption caused by the pandemic x covid- chis could help prioritize among the almost investigational vaccines and over experimental treatments for covid- currently in development y young healthy adults may not generalize to older individuals and those with comorbidities who would most benefit from effective vaccines z conditions" /"healthy young volunteers" /"young healthy adults". the last part of standard of the nuremberg code deserves particular attention in this context because it rejects high risk experiments with human beings be they young or old, "except perhaps", in cases where physician researchers are willing to conduct such experiments on themselves. the proponents of starting such studies all admit that there remains "significant uncertainty" with regard to the pathogenic effects of sars-cov- in both elderly and young people, and that high or significant risk is involved in such studies with the potential of causing severe harm, or death. even if the recommendations of the nuremberg code of letting researchers themselves serve as participants had been followed strong caution is still warranted for at least six reasons. first, as uncertainty still surrounds the pathogenic effect of the sars-cov- in different age groups, challenge-studies would not be justified because of the lack of robust knowledge and understanding of the short term consequences of the sars-cov- , and because of the almost complete absence of data on the long term effects of the virus. for these reasons it would be very difficult to justify the inclusion even of the most knowledgeable individuals in such studies, i.e. covid- researchers, because even the most knowledgeable are currently deeply ignorant. it is worth noting here that in a document on a eyal ( ) and callaway ( ) b the international alliance for biological standardization ( ), jamrozik and selgelid ( a, b) , menikoff ( ) , eyal et al. ( eyal et al. ( , pp. eyal et al. ( - who ( ) and jamrozik and selgelid ( a) . eyal ( ) , who ( ) . in a recent paper reviewing and synthesizing available evidence on asymptomatic sars-cov- infection, subclinical lung abnormalities was found even among asymptomatic persons (for this see oran ) . this additional form of uncertainty is admitted by jamrozik and selgelid ( a, p. ) . jamrozik and selgelid ( a, p. ) briefly address the question whether "the permissibility of high-risk human challenge studies" would increase if researchers used themselves as research participants, but they warn against this, and notably for the reason that "clinical and research staff might feel pressure to participate". shah et al. ( ) . . challenge-studies for vaccine development adopted in by who's expert committee on biological standardization there is no talk about accepting such studies if the risk is of the size referred to in the who-working group's report of this year; i.e. "high" risk with the potential of causing "severe harm". on the contrary, in the document emphasis is on "minimizing risks to subjects" and reference is made to "situations in which there may be greaterthan-minimal risk", or risks "…considerably greater than minimal", but still manageable as "…e.g. accepting that they [the trial candidates] will develop an acute, but manageable, disease that will resolve but in the meantime may cause considerable morbidity, such as severe diarrhea managed with fluid and electrolyte replacement". however, risks "considerably greater than minimal" are still far from being equal to high risk with the potential of causing severe harm. further down the same paragraph of the who expert committee document the following is added: "however, accepting such risks requires absolutely that the elements of voluntary consent are based on truly being informed". it is, however, difficult to see how it would be possible to comply with this condition when so much is still unknown with regard to both the short and long-term pathogenic effects of the sars-cov- . a valid informed consent is a legally binding absolute human rights requirement. second, if something goes wrong after infecting healthy young volunteers with the virus, the treatment options at present are limited. accepting such studies in relation to the sars-cov- would therefore not be in compliance with two of the core conditions in current ethical frameworks for challenge studies: namely that the pathogen studied does not induce infections that may cause severe harm, or for which there exist no effective treatment. despite this, challenge studies for covid- are proceeding and the recruitment of health volunteers has exceeded , . if something goes wrong, compensation to participants ought to be a major consideration for these trials. in a recent publication, carl elliot argues that although the focus of challenge studies is to recruit healthy volunteers, if the model is based on remunerating these volunteers, the studies may attract economically vulnerable volunteers who view participation as a means to an end. and as stated by ruth macklin : …given standard practice, it is virtually certain that monetary payment-which may be considerable-will serve as an inducement to enroll. the likely result is that a disproportionate number of volunteers would come from lower-income brackets, including many people who lost their jobs because of the pandemic. it is also likely that many volunteers would be members of racial and ethnic minorities, raising a serious question of social justice. economically vulnerable volunteers who become injured or "permanently disabled" may not receive compensation, especially in the us. in contrast to other developed countries, research sponsors in the us are not under any legal obligation to pay for the medical care of research participants who become injured or ill, hence, few do. furthermore, the us national vaccine injury compensation program excludes payment for experimental vaccines. research participants in the us can therefore be left without compensation for injury caused by their participation in a challenge study. third, the technical label 'controlled human infection studies' is unfortunate, because it might give healthy young volunteers and other non-experts the impression of a kind of control that is misleading. fourth, at present one does not know whether controlled infections studies conducted with young and healthy adults (or covid -researchers) will provide results that enhance survival and/or healing-rates of those most affected by sars-cov- , i.e. old fragile persons with comorbidities. hence, there is a significant problem of external validity. fifth, the argument that participants "might benefit from controlled infection and/or vaccination if they become immune to virus" is also undermined by major uncertainty, as openly admitted by the proponents of such studies. sixth, and, perhaps, most important, such studies violate the core ethical principle of human subjects research; i.e. the priority of the individual principle. this principle emerged as a normative response to the medical horrors that had been practiced during wwii in concentration camps in nazi germany and in japan. yet in spite of the universal declaration of human rights and the nuremberg lynch ( ) . cohen ( )-for this, see also guarino and johnson ( ) . elliott ( ) . macklin ( ) . elliott ( ) . ibid. ibid. jamrozik and selgelid ( a) . this concern is also raised by deming et al. ( ) , and by schaefer et al. ( ) . shah et al. ( ) . shah et al. ( ) , who ( , note , p. code, several ethically compromised studies involving vulnerable groups have been conducted after wwii. to underscore this point, and analyze their implications, allow us to describe two such examples. first, the hepatitis studies, conducted from , at willowbrook state school, an institution for mentally disabled children on staten island, new york. more than children, predominantly african-american and puerto-rican, were included in these studies, and a subgroup of almost non-infected children were fed a suspension with the local strains of the hepatitis virus prepared from the stool of six children collected "during the first days of recognized jaundice". written consent from the parents of these children had been obtained by the chief investigator, dr. krugman. but later investigations indicated that the parents' consent might have been based on indirect coercion since volunteering their children to the infection study was allegedly put forward as a condition for admitting the children to care at willowbrook state school. krugman defended the contested infection and immunization studies his whole life with reference to their scientific merits; the "confirmation of two types of hepatitis, a and b, with different infection pathways (oral versus close contact), and the preparation of a "crude vaccine" containing the hepatitis b virus. in a letter to the editor of the lancet in he justified the exposure of "a small number of newly admitted children to the willowbrook strains" of the hepatitis virus with reference to: ( ) inevitability; the children were "bound to be exposed" to the same virus strains "under the natural conditions existing in the institution", ( ) safety; they would be admitted to "a special, well-equipped and well-staffed unit", thus shielding them from exposure to other prevalent infectious diseases in the institution, ( ) immunity; "they were likely to have a subclinical infection followed by immunity", and ( ) informed consent; "only children with parents who gave their informed consent would be included". the second study worth mentioning is the cancer injection study that took place in the early s at the jewish chronic disease hospital in brooklyn, new york. the chief investigator, dr. chester m. southam, had since conducted research on the role of the immune system in protecting against cancer, using two different groups of research participants who were injected with a suspension of foreign cancer cells to study the difference in immunological rejection of the cells between the two groups. the first group was a cohort of patients at memorial hospital in ohio, in total , with different forms of widespread cancer, and the second group consisted of healthy individuals from the ohio state penitentiary ; i.e. a prison in downtown columbus, ohio. these studies documented that healthy individuals rejected the injected cancers cells faster than the cancer patients ( - weeks versus weeks to months). in his presentation of southam's studies john d. arras labeled the second group of research subjects "healthy prison volunteers", and notably, without any reflection whatsoever either about the ethical justifiability of recruiting prisoners for a study that would not benefit them, or whether it is appropriate to consider prisoners to be free to volunteer. on this point southam himself seems to have been, at least, partly aware of the dilemma of recruiting incarcerated individuals. in an interview in science he admitted that, although there was no theoretical likelihood that the injections would produce cancer, he had nonetheless been unwilling to inject himself, or his colleagues, when there was a group of normal volunteers at the ohio penitentiary fully informed about the experiment and its possible risks and nonetheless eager to take part in it: "i would not have hesitated", southam said, "if it had served a useful purpose. but to me it seemed like false heroism, like the old question whether the general should march behind or in front of his troops. i do not regard myself indispensable-if i were not doing this work someone else would be-and i did not regard the experiment as dangerous. but, let's face it, there are relatively few cancer researchers, and it seemed stupid to take even the little risk". southam persuaded the then director of the jewish chronic disease hospital, emmanuel e. mandel, to permit, as a third part of his immune reaction studies, the injection of foreign cancer cells into old patients with other debilitating chronic diseases than cancer. the scientific justification for this study was to get "direct evidence" that it was the cancer disease that caused the delay in rejection of the foreign cancer cells, and not the fact that most of the cancer patients he had studied were elderly, debilitated and with additional chronic diseases. such evidence, he maintained, would be possible to establish by doing the same immune reaction study in a group of elderly patients with other chronic and debilitating diseases than cancer. in the letter southam wrote to mandel on july , , he also discussed whether consent ("written permission") from the patients was warranted, something which he warned against, for two reasons; first, at memorial hospital they considered it a "routine study, much less hazardous than other routine procedures", and second, the only risk related to the use of cancer cells in these injections was the "phobia and ignorance surrounding the word "cancer". in the same letter southam informed mandel that with regard to the prisoners, "signed permits" had been obtained, but this, according to southam, was "because of the law oriented personalities of these men, rather than for any medical reason". in the two doctors were found guilty of fraud, deceit and unprofessional conduct, and they were, in particular, criticized for assuming they were entitled to perform any kind of research without consent as long as the research in question was scientifically justified. two years later, however, southam, was elected president of the american association for cancer research for the period of - . in miller and grady proposed a way of evaluating the ethical justifiability of planned challenge-studies by locating each candidate along a continuum from legitimate studies to clearly unacceptable ones. in the "border zone" between these two extremes they locate studies that are neither indisputably justifiable nor clearly unacceptable. among legitimate ones they count studies for common cold, cholera and malaria, while chi-models for lyme disease or helicobacter pylori are labeled "more controversial". finally, among clearly unacceptable studies they mention two examples; hiv-chi-studies or chi-studies for hepatitis c virus. their arguments for labeling these two chi-models unacceptable were twofold; "non-existent or ineffective" treatment, and "intolerable symptoms and/or the likelihood of serious morbidity or mortality". two additional possible studies deemed unacceptable are referred to in cioms' commentary on guideline of the international ethical guidelines for health-related research involving humans : for example, a study that involves deliberately infecting healthy individuals with anthrax or ebola-both of which pose a very high mortality risk due to the absence of effective treatments-would not be acceptable even if it could result in developing an effective vaccine against these diseases. therefore, researchers, sponsors, and research ethics committees must ensure that the risks are reasonable in light of the social and scientific value of the research, and that the study does not exceed an upper limit of risks to study participants. an additional example-deemed unacceptable at the time of the evaluation-was a zika virus-chi-study. the nih ethical review committee's reasons for deciding against the study were with reference to three kinds of uncertainty; uncertainty of the risk to research participants as well as to third parties (fetus and sexual contacts), uncertainty about the duration of protection needed, and, third, uncertainty of the study's societal value. viewing the studies conducted by drs. krugman and southam in the light of miller and grady's differentiation between legitimate, more controversial and clearly unacceptable challenge-studies, and in view of current ethical standards pertaining to such studies, calls for reflection, not only for historical reasons; they may also be of help in investigating where the arguments in favor of chi-studies in the current context of the covid- -pandemic differ from those of krugman and southam, and where these arguments seem to overlap. when it comes to locating the studies under discussion along the ethical line of decreasing permissibility proposed by miller and grady, southam's immune reaction studies on elderly, debilitated subjects arguably deserve the label ethically unacceptable, in spite of their scientific merits, for at least three reasons: (a) no consent was obtained, (b) a chi-study in healthy volunteers contributed to the development and licencing of the live oral cholera vaccine cvd -hgr study in . for this, see: roestenberg et al. ( , p. ) . in , the first malaria vaccine allegedly achieved through the use of chi-studies gained ema approval. for this, see roestenberg et al. ( , p. ) . for this, see also: european medical agency: https ://www.ema.europ a.eu/en/docum ents/medic ine-outsi de-eu/mosqu irixsumma ry-publi c_en.pdf. for such a study, see: graham et al. ( ) . grady ( , p. ) . ibid., p. . cioms ( , p. ). the international alliance for biological standardization ( , p. ). ibid., p. . for a similar verdict concerning a zika chi-trial, see recommendation in shah et al. ( , p. ) : "whether a zika virus human challenge trial has sufficient social value to proceed depends on the reasons for doing it and whether there are alternative ways to obtain the information. the most compelling rationale for conducting a zika virus human challenge trial, given the risks and uncertainty, would be if field trials were prohibitively difficult to conduct in light of a waning epidemic. this rationale is not currently met, but it could come to pass in the future. another valuable reason to conduct a challenge trial would be to accelerate the development of a vaccine that could prevent congenital zika infection. this rationale must be accompanied with strong evidence that results from a zika virus human challenge trial would be used by stakeholders (e.g., indication from regulatory agencies that finding a correlate would speed up the licensing of a vaccine). the committee did not hear sufficient evidence that this rationale is currently met. finally, using a challenge trial solely to learn about the pathogenesis and natural history of zika infection is unlikely to justify the risk involved given the alternative ways to obtain similar information". southam in his letter to mandel. ibid. the element of deceit involved and (c) the fact that participants did not stand to benefit in any ways from the studies. southam's reluctance against using himself and colleagues as study subjects was based on two considerations: first, exposure to possible risks caused by the injection of foreign cancer cells; second, since neither he nor his colleagues were physically debilitated they were unsuitable candidates for the study. in two of the position statements referred to above reservation is expressed against using researchers as studysubjects in covid- cih-studies, although for a different reason; "clinical and research staff might feel pressure to participate", and "such individuals could feel pressured to participate (thereby undermining the voluntariness of informed consent". krugman's challenge studies at willowbrook are more difficult to locate and label according to miller and grady's linear differentiation, not least because his defense of the studies has several striking similarities with arguments used in favor of conducting chi-studies to speed up the development of efficient vaccines against the sars-cov- virus. first the reference to the studies' scientific merit; the "confirmation of two types of hepatitis, a and b, with different infection pathways, and the preparation of a "crude vaccine" containing the hepatitis b virus. a similar type of argument is used by and shah, miller, darton et al. : for example, chis could clarify dynamics of infection, viral pathogenesis, and risk of vaccine pathogenesis or identify correlates of protection-all of which could inform the development and implementation of vaccines. second, their high social value. in his letter to the editor of the lancet in krugman emphasized the high social value beyond willowbrook of his studies: "it is unnecessary to point out the additional benefit to the world-wide population which have been plagued by an insoluble hepatitis problem for many generations". in support of such arguments, shah, miller, darton et al. argue : sars-cov- chis could have high social value in several ways. for example, they could help prioritize among the almost investigational vaccines and over experimental treatments for covid- cur-rently in development. chis could help identify the most promising agents, which would inform the design of larger trials, guide decisions to scale up manufacturing early, and thereby accelerate product development and implementation. in a similar vein, singer and chappel refer to the high social value, or in their wording-"the broader humanitarian benefits"-of such studies. third, the exposure of a small number of individuals to risk for the sake of benefits to the rest. different versions of this argument are used in the four position statements, and also by the international alliance for biological standardization. fourth, the endemic argument-the children were "bound to be exposed" to the same virus strains "under the natural conditions existing in the institution". two -versions of the same argument read: "the risks in question do not entail a major net increase in risk (in light of background risks of infection", and "participants face a background risk of infection in the community". fifth, the safety-and better care argument. in krugman's wording the study subjects would be admitted to "a special, well-equipped and well-staffed unit", to minimize risks to study personnel, participants should be in inpatient isolation, with contact reduced to the extent possible and robust personal protective equipment provided. and who's working group highlights the importance of "supportive care, including critical care" and "long-term follow-up" as two crucial risk-minimization strategies. sixth, the immunity-argument; krugman argued that the children involved in his studies "were likely to have a subclinical infection followed by immunity" , while who's working group refers to "[i]mmunity induced by experimental vaccines" as a potential benefit of study participants. jamrozik and selgelid combine the safety-/better care argument with the immunity argument : …potential direct benefits of being infected with sars-cov- in the course of human challenge studies would include participants being exposed to less infection-related risk than if they are infected in the community (e.g. because of early diagnosis and medical care) and gaining immunity to future infection in the context of a high background risk. seventh, and last but not least, krugman as well as the authors of the four position statements referred to above all emphasize the importance of informed consent. this comparison between krugman's arguments and the arguments in the four position statements here subject of detailed analysis and in other recent papers advocating the use of covid- chis shows that their overall views are pretty much the same. in fact, the only difference in terms of substance is the use of vulnerable individuals or groups as study-participants which two of the four position statements warn against. singer and chappel do not address this issue, while the authors behind the fourth position statement, jamrozik and selgelid, in a recent paper on cih studies in endemic settings, argue that sometimes it may be justifiable, in fact "ethically important" to include vulnerable populations: "…especially where the results of research in other populations are not likely to be generalisable to the vulnerable populations in question. this is one consideration that sometimes favours conducting (more) hcs in low-middle income countries (lmics)". so where does all this lead us? should we accept the 'neo-krugman'ian' views advocated by proponents of covid- chis, or should we rely on the normative principles that were formulated as a reaction to the kind of studies southam and krugman had conducted? of these principles the priority of the individual principle is the most fundamental. the original formulation of this principle in biomedical research ethics occurred in the first, i.e the -version of the declaration of helsinki as basic principle and reads : every biomedical research project involving human subjects should be preceded by careful assessment of predictable risks in comparison with foreseeable benefits to the subject or to others. concern for the interests of the subject must always prevail over the interests of science and society. this principle has been maintained in all versions of the declaration. in the latest version (wma ) it is included as general principle : while the primary purpose of medical research is to generate new knowledge, this goal can never take precedence over the rights and interests of individual research subjects. whereas the declaration of helsinki is a professional ethics norm, the principle was restated in article of the une-sco universal declaration on bioethics and human rights, which was adopted by all member states of the united nations in october , and notably with reference in the first section of the article to human dignity, human rights and fundamental freedoms: the interests and welfare of the individual should have priority over the sole interest of science or society. in their position statement singer and chappell claim that current research-ethical principles are based on "assumptions developed in calmer times when much less was at stake". this claim is historically wrong, as this is not the first time the world has faced a public health crisis since the development of the core principles of research ethics. we have managed to live through those less calm times without who ( , p. ). for the immunity argument, see also eyal ( , p. ) . jamrozik and selgelid ( a, p. ) . a slightly different version of this argument reads thus: "in short, if researchers conducting challenge trials act as recommended, admittedly, the probability of getting infected would remain larger inside a challenge trial than either outside any trial or in a standard efficacy trial; but the probability of death or disability is likely to be much smaller inside a challenge trial than in these alternative scenarios. overall, a × b could be smaller for any individual inside the challenge trial than either outside any trial or in a standard efficacy trial. what the individual would lose in the probability of averting infection (with that probability rising) she could gain in better protection from death" (eyal , p. ) . krugman ( , p. ) : informed consent by proxy; shah et al. ( , p. ): "robust consent"; jamrozik and selgelid ( a, p. ): "proper" or "adequate consent"; who, , p. : "sars-cov- challenge studies must involve rigorous informed consent". for this, see also bambery et al. ( , pp. - ) , eyal ( , p. ), plotkin and caplan , p. ), schaefer et al. ( ), and wolemonwu ( , p. ) . shah et al. ( , p. ): «sites should be selected for sound scientific reasons while avoiding especially vulnerable populations"; who ( , p. ): "those whose background risk is high as a result of social injustice should be excluded from participation because their inclusion could be considered unethical exploitation (i.e., taking advantage of those who have already been wrongly disadvantaged. any prospective participants who could reasonably be perceived to be vulnerable in other ways that would undermine their consent or put them at greater risk (for example as a result of the mental health strain of inpatient isolation during the study) should also be excluded". jamrozik and selgelid ( b, p. ). https ://www.cirp.org/libra ry/ethic s/helsi nki/. infringing those principles, and we have no particular reason to overstep them now. so to conclude our analysis and critique of controlled human infection studies (chis) with sars-cov- : there is a consistent, historical line of research ethical principle from nuremberg, through the revisions of the helsinki declaration, and the successive un human rights declarations and other normative documents. this principle is that the interests of the individual research participant is paramount in research ethics, i.e. that if there is a conflict between the interests of society (e.g. in speeding up vaccine development) and the interests of the participants (e.g. in not dying or being permanently harmed) then the interests of society has to yield to the interests of the participant. there is no exception for times of crisis, or for instances where societal interests are large. one might argue against this principle, and some have done so, but well-founded and sustainable ethical principles can't be disregarded just because they seem inconvenient at a certain point in time. the concern here is not just an abstract philosophical principle, but a part of the normative core of all existing research ethics processes. if we take this principle seriously it prohibits the conduct of sars-cov- challenge studies at the present time where the challenge virus would be the native virus with full virulence and where there is no rescue treatment yet available. in a policy forum statement in science alex london and jonathan kimmelman, warn against "pandemic research exceptionalism" and against using crises as an excuse for lowering scientific standards. they focus on five epistemological conditions of "informativeness and social value" that research should embody, even in times of emergency: first, importance : trials should address key evidence gaps…as of this writing, more than clinical trials enrolling more than , patients have been registered in north america for testing various hydroxychloroquine regimens for covid- . this massive commitment concentrates resources on nearly identical clinical hypotheses, creates competition for recruitment, and neglects opportunities to test other clinical hypotheses. second, rigorous design. third, analytical integrity: "designs should be pre-specified in protocols, prospectively registered, and analyzed in accordance with pre-specification". fourth, complete reporting: "trials should be reported completely, promptly, and consistently with pre-specified analyses". fifth, and last, feasibility: "studies must have a credible prospect of reaching their recruitment target and being completed within a time frame where the evidence is still actionable". similar warnings against research exceptionalism, and notably with reference to things that went wrong in the past when arguments in favor of this were used, are made by deborah doroshow, scott podolsky and justin barr : the coronavirus disease (covid- ) pandemic has incited remarkable disruption in biomedical research. at academic institutions worldwide, laboratories have been forced to halt all but the most critical activities. clinical trials of novel agents for such diseases as cancer are temporarily suspended, limiting access to potentially life-prolonging medications […] although this boom has already begun to transform our response to the pandemic for the better, medical and scientific responses to past crises suggest that urgency may also result in compromised research quality and ethics, which may in turn jeopardize public faith in government and science, waste precious resources, and lead to the loss of human life. another problem with knowledge-production of the covid- pandemic has been the lowered standards of quality assurance of published research. it has been documented that the peer review process has been rushed ("express" or "opinion based peer review") and so far (october , ) research papers about covid- have been retracted. a stunning example of this is an observational study based on the health records of almost , patients around the world published in the prestigious journal lancet in may , which indicated that hydroxy-chloroquine had a sharply higher risk of death and heart problems compared to those who did not receive the drug, and that hydroxychloroquine did not provide any benefit. on june , this study was retracted by the authors due to doubts about london and kimmelman ( , pp. - ) . ibid., p. . ibid, p. . doroshow et al. ( ) . ioannidis ( ) . retraction watch. retracted coronavirus (covid- ) papers. https ://retra ction watch .com/retra cted-coron aviru s-covid - -paper s/. mehra et al. ( a) . the veracity of the data used and the analyses conducted. on june , preliminary clinical trial results from the recovery study of the university of oxford were broadcasted all over the world suggesting that a commonly used drugdexamethasone-reduced deaths among the sickest covid- patients by a third. hopefully, the published study will prove this claim to be justified, but it is unfortunate when covid- researchers start "doing science by press release" instead of following generally accepted publication procedures. as stated by dr. atul gawande at brigham and women's hospital in boston : typically, researchers extensively detail their work in scientific journal articles. before publication, other scientists take an in-depth look at how the study was designed, who the patients were and whether any potential side effects were uncovered-a process called peer review. it takes time-weeks or months in some cases-for independent, unbiased experts to pore over the manuscripts, looking for any concerns. taking epistemic shortcuts inevitably produces poor evidence and often leads to bad decisions with potentially severe consequences for vulnerable persons. although tempting, we should avoid epistemic shortcuts, as high-quality evidence is needed in exceptional times, as otherwise. when taking chances, we must consider the risks of harm, not only the benefits. one such risk worth mentioning here is that persons selected for testing emergent vaccines become victims of enhanced disease, i.e. presenting worse symptoms from the effects of an unproven vaccine compared to persons catching e.g. the covid- flu through usual paths of contagion. potential covid- vaccine volunteers might e.g. end up with life-threatening complications (such as irreversible and untreatable clogged lungs) whereas, in the current situation most unvaccinated patients display only mild flu-indicators, if infected. as there as yet does not exist any known therapy, the perils these volunteers risk is ethically unacceptable. two examples of enhanced disease precipitated by insufficiently proven vaccines occurred in connection with the inoculation of children against rsv (respiratory syncytial virus in the late s. similarly, from october to january more than million adult citizens in the usa were vaccinated with a swine influenza virus vaccine. during the same period more than vaccinated persons fell ill with a rare neurological illness (guillain-barré syndrome) and of them died. these unexpected events led to immediate cancellation of the vaccination program indicating that the effects of an insufficiently tested vaccine in some cases cause greater harm than benefit. hence, ethical reflection and compliance with epistemological rules of play are needed more than ever. in a paper reflecting on what might be learned from the swine flu vaccination program, sencer and millar warn against politicization of scientific information in a way that is well worth listening to also for today's public health leaders and their political peers : while all decisions related to niip [the national influenza immunization program] had been reached in public sessions (publishing of the initial virus findings in cdc's weekly newsletter, the morbidity and mortality weekly report (mmwr); new york times reporter harold schmeck's coverage of the acip [the advisory committee on immunization practices of the united states public health service] sessions, the president's press conference, and congressional hearings), effective communication from scientifically qualified persons was lacking, and the perception prevailed that the program was motivated by politics rather than science. in retrospect (and to some observers at the time), the president's highly visible convened meeting and subsequent press conference, which included pictures of him being immunized, were mistakes. these instances seemed to underline the suspicion that the program was politically motivated, rather than a public health response to a possible catastrophe. mehra et al. ( b) . university of oxford. dexamethasone reduces death in hospitalised patients with severe respiratory complications of covid- . june , . accessible at: https ://www.ox.ac.uk/news/ - - dexam ethas one-reduc es-death -hospi talis ed-patie nts-sever e-respi rator y-compl icati ons. on october , , who reported that "dexamethasone is the only effective drug for coronavirus". this information is accesible at: https ://www.aa.com.tr/en/lates t-on-coron aviru s-outbr eak/who-dexam ethas one-only-effec tive-drug-for-coron aviru s/ . the expression "doing science by press release" we have borrowed from an interview about the study with dr. george anesi, director of the medical critical care bioresponse team at the hospital of the university of pennsylvania. accessible at: https ://www.nbcne ws.com/healt h/healt h-news/scien ce-press -relea se-docto rs-view-covid - -drug-resul ts-excit ement -n . nbc news. doctors view dexamethasone results on covid- with excitement and skepticism. accessible at: https ://www.nbcne ws.com/healt h/healt h-news/scien ce-press -relea se-docto rs-view-covid - -drug-resul ts-excit ement -n . macklin ( ) . acosta et al. ( ) . langmuir ( , p. ) . sencer and millar ( ) . rounding them should be communicated'. this goal is much better accomplished if the explanations are communicated by those closest to the problem, who can give authoritative scientific information. scientific information coming from a nonscientific political figure is likely to encourage skepticism, not enthusiasm. another function of research ethics is to provide guidance on the "compassionate use" of drugs unapproved for covid- . in the past months we have read about compassionate use access of covid- patients to hydroxychloroquine and remdesivir. by compassionate use (otherwise known as expanded access), we refer to : …a potential pathway for a patient with an immediately life-threatening condition or serious disease or condition to gain access to an investigational medical product (drug, biologic, or medical device) for treatment outside of clinical trials when no comparable or satisfactory alternative therapy options are available. in principle compassionate use must have regulatory oversight and, in some countries such as the us, spain, and italy, research ethics committee approval as well. us regulations as well as those of individual eu member states stipulate the requirements before an investigational drug can be offered to either an individual, a limited group, or a wider population. but what exactly is the role of research ethics? compassionate use is therapy in the sense that the purpose of it is to cure. however, compassionate use is not only therapy. it is, after all, the provision of a drug with yet-to-be determined levels of efficacy and safety. considering the not very impressive success rate of an investigational drug of only % (i.e., from phase to successfully being licensed for market distribution), the risks of compassionate use, especially when doctors cannot be provided with definitive guidance on dosage or exclusion criteria, can actually be worse than the risks of a controlled clinical trial. this being the case, research ethics guidance is imperative for access to investigational drugs via compassionate use. also, faced with a surge of sick patients suffering from a new unknown disease, well-motivated clinicians and investigators all over the world, including drug companies and funding agencies, should adopt "a more integrated approach to learning while doing", and they should join forces, i.e. engage in collaborative efforts so as to increase the "exploitation/exploration trade-offs", and, hopefully, shorten "the period until effective treatments are discovered and implemented". one of the tenets of research ethics is the provision of the benefits of research to the intended patient population. all major international ethics guidelines for research have provisions with different degrees of specifications. the declaration of helsinki article , for example, requires clinical trial "sponsors, researchers, and host country governments" to ensure post-trial provisions to participants who, at the end of the trial, might still need the trial intervention that has been "identified as beneficial in the trial". unesco's universal declaration on bioethics and human rights provides more specificity and directive. article says the following : benefits resulting from any scientific research and its applications should be shared with society as a whole and within the international community, in particular with developing countries. in giving effect to this principle, benefits may take any of the following forms: (a) special and sustainable assistance to, and acknowledgement of, the persons and groups that have taken part in the research; (b) access to quality health care; (c) provision of new diagnostic and therapeutic modalities or products stemming from research; (d) support for health services; (e) access to scientific and technological knowledge; (f) capacity-building facilities for research purposes; (g) other forms of benefit consistent with the principles set out in this declaration. guideline of cioms' international ethical guidelines for health-related research involving humans provides a similar specification in terms of provision of the fruits of research to the population or community where the research was carried out, most especially for research conducted in low-resource settings. then, on guideline , it stipulates the applicability of guideline for research during disaster and disease outbreaks. during the covid- pandemic, we see these principles tested again and again. there were moments filled with high spirits and solidarity. the sequencing of the genome of sars-cov- , as well as the identification of relevant proteins and enzymes, happened at lightning speed because of spontaneous collaboration between universities from different countries. we also saw how public funds were earmarked covid- research and therapy development. , , at the same time, we saw how public funds were used to secure advanced orders of potential covid- vaccines originally researched using public funds and how pharmaceutical companies played hardball with the public for covid- testing kits. there were real reasons to be worried, most especially if pharmaceutical companies continue to be granted free rein in pricing, which usually means paying "a small fortune" for new interventions. to address potential access concerns brought about by patent market exclusivity, costa rica spearheaded what is now the who solidarity call for a covid- technology access pool. specifically, this is a call for "key stakeholders and the global community to voluntarily pool knowledge, intellectual property and data necessary for covid- ". it was this same spirit of solidarity that spurred the sequencing of the genome of the sars-cov- and it is this same spirit of pooling, collaboration, and sharing of benefits that provides hope for timely and equitable access to much needed interventions. research ethics has provided this foundation, as we saw above, and it remains research ethics' task to ensure that covid- drug discovery and development take this course. to date, countries have signed the who solidarity call, which countries such as the us and the uk have yet to become signatories. the role of research ethics in research on and with those most vulnerated by the covid -pandemic: the case of elderly people in nursing homes older people in nursing homes are among the most vulnerable in contemporary society. this has become very clear in several countries during the covid -pandemic. in may a report from bergen (one of the largest cities in norway) revealed that % of the city's covid- deaths stemmed from patients living in nursing homes. and in sweden nursing home residents account for nearly half of deaths linked to while future estimates in england are that more than half of coronavirus-related deaths will affect people living in care homes. research on how to prevent deaths in nursing homes during a pandemic, and ways of minimizing the risk of contagion represent issues of great ethical and health political urgency. there are, however, several research ethical challenges related to the inclusion of nursing home residents in research projects because the pandemic has rendered them victims of additional vulnerability. the more vulnerable 'cioms' stands for council for international organizations of medical sciences. for this, see cioms ( ). ibid. editorial in nature. everyone wins when patents are pooled. nature, ; : . aj impact/europen union ( ). novavax ( ). cnbc ( a). cnbc ( b). dutchnews.nl ( ). lazarus ( ) . hoen ( ) . covid- technology access pool, . https ://www.who.int/ emerg encie s/disea ses/novel -coron aviru s- /globa l-resea rch-onnovel -coron aviru s- -ncov/covid - -techn ology -acces s-pool. ibid. emanuel et al. ( ) . bergens tidende: accessible at: https ://www.bt.no/nyhet er/lokal t/i/ vxd / -av- -doede -var-sykeh jemsb eboer e. bbc news. accessible at: https ://www.bbc.com/news/world -europ e- . the guardian. accessible at: https ://www.thegu ardia n.com/socie ty/ /jun/ /more-than-half-of-engla nds-coron aviru s-relat ed-death s-will-be-peopl e-from-care-homes . the differentiation between vulnerability and being vulnerated is important to distinguish between on the one hand persistent and on the other variable forms of vulnerability (solbakk , pp. - ) . the persistent form of vulnerability we all share, is part of the human condition, while the second form of vulnerability is contextdependent, in the sense that some people because of disease, poverty, lack of freedom etc. are vulnerated, i.e. harmed or wounded. this distinction points to the need for a differentiation between at least two distinct regimes of protection. firstly, a human rights-based regime aimed at protecting persistent or universal vulnerability. this regime requires negative action on the part of the state, in the sense that its responsibility is to guarantee basic liberties by securing a just social order that gives equal protection to the vulnerability of each citizen. these protective measures are, however, in need of being supplemented by additional measures of protection-of affirmative actionto cope with accidental states and situations when human vulnerabil-a research participant is, the greater the risk of causing harm. furthermore, the inherent asymmetric power relation between the researcher and his/her patients fuels powerimbalance, about which we as researchers should be ever more concerned, many older and vulnerable nursing home patients, many of whom either lack the competency to consent, or are verbally deficient. the principle of consent is one of the most basic research principles. the un human rights committee notes that special protection is warranted when including persons not capable of giving valid consent. in some cases a next of kin may consent on behalf of the patient, but since we cannot be sure whether this consent reflects the wishes of the patient him/herself, this poses a problem. we should therefore be aware of both verbal and non-verbal signs and reactions from the participants' signs of discomfort and resistance throughout the research process. here we will argue that researchers need a moral sensitivity, not only to avoid physical harm or risks, but also to avoid psychological and social damage. it is not sufficient to conform to "procedural ethics" at the onset of a researchproject or to gain/secure approval from a research ethical committee. moral sensitivity in research may be even more important during pandemics, when research protocols seem to be "rushed" and misgivings ignored. in addition to moral sensitivity as researchers, we may also argue for a kind of ethical calmness or what guillemin and gillam refer to as "ethical reflexivity" throughout the research process. the arguments presented above suggest that the role of research ethics in pandemic times is exceptionally important, but not only in the sense of deviating from hard won core ethical and epistemological principles in the wake of wwii. on the contrary, perhaps more than ever, it is vital to restate the importance of human dignity, human rights and fundamental freedoms as the normative bedrock on which medical research involving human subjects should rely. there is no alternative pathway for research ethics that is viable; returning to the core values and principles enshrined in the universal declaration of human rights is urgently needed. we therefore share the views expressed by the european group on ethics in science and new technologies in a statement on the covid- pandemic : it is natural in these circumstances of deep uncertainty to focus on immediate action and speed of measures. this must not, however, lead to a continuous suspension of rights and liberties. we therefore call for vigilance about the necessity, evidence, proportionality of any policy and technological intervention that, even temporarily, suspends fundamental rights. consideration needs to be given to the immediate and lasting impacts that such measures have on our societies. funding open access funding provided by university of oslo (incl oslo university hospital). open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. for the scientific merit argument, see also bambery for this argument, see also eyal jamrozik and selgelid performing chi is a way to learn and test, while minimizing the number of subjects for this argument, see also eyal also eyal ( , p. ) highlights the importance of covid- -chi-trial participants having "access to standardof-care life-sustaining treatments accessible at: https ://www. pharm acyti mes.com/news/fda-annou nces-two-drugs -appro ved-forcompa ssion ate-use-in-treat ing-covid - ; and, fda is allowing two drugs to be used for here's what that means publi c-healt h-focus /expan ded-acces s. for this, see also reference is here made to: department of health and human services. annex : pandemic influenza response and preparedness plan brief history and characterization of enhanced respiratory syncytial virus disease aj impact/europen union. . eu may deploy emergency funds in covid- vaccine race optimizing the trade-off between learning and doing in a pandemic the jewish chronic disease case article (prohibition of torture, or other cruel, inhuman or degrading treatment or punishment) ethics, reflexivity, and "ethically important moments" in research. qualitative inquiry statement on european solidarity and the protection of fundamental rights in the covid- pandemic footnote (continued) fallen vulnerability" is a metaphor that has been suggested to designate such situations ethical criteria for human challenge studies in infectious diseases ethics review in compassionate use the medical profession & human rights. handbook for a changing agenda should we infect healthy people with coronavirus? international ethical guidelines for health-related research involving humans trump signs $ . billion emergency 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epidemiology useful bodies: humans in the service of medical science in the twentieth century challenge model for helicobacter pylori infection in human volunteers volunteers sign up to put their lives on the line for a coronavirus vaccine ethics, reflexivity, and "ethically important moments" in research the importance of moral sensitivity when including persons with dementia in qualitative research covid- : think first, act better later protect against market exclusivity in the fight against covid- the first casualty of an epidemic is evidence coronavirus disease : the harms of exaggerated information and non-evidence-based measures covid- human challenge studies: ethical issues. the lancet infectious diseases human infection challenge studies in endemic settings and/or low-income and middle-income countries: key points of ethical consensus and controversy treating covid- -off-label drug use, compassionate use, and randomized clinical trials during pandemics experimentation with human beings. the authority of the investigator, subject, profession, and state in the human experimentation process vulnerability: what kind of principle is it? medicine experiments at the willowbrook state school viral hepatitis, type b (ms- -strain). further observations on natural history and prevention guillain-barré syndrome: the swine influenza virus vaccine incident in the united states of america here's why a covid- vaccine could end up costing you a small fortune against pandemic research exceptionalism the right to withdraw from controlled human infection studies: justifications and avoidance human challenge studies for covid- vaccine: questions about benefits and risks coronavirus disease (covid- ): epidemiology, virology, and prevention hydroxychloroquine or chloroquine with or without macrolide for treatment of covid- : a multinational registry analysis retraction-hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis the regulation of covid- «challenge» studies the ethical challenge of infectioninducing challenge experiments occupation and risk of covid- : prospective cohort study of , uk biobank participants. medrxiv. the preprint server for health sciences how to take non-knowledge seriously, or "the unexpected virtue of ignorance novavax to receive up to $ million funding from cepi for covid- vaccine development and manufacturing prevalence of asymptomatic sars-cov- infection: a narrative review how pandemics seep into literature extraordinary diseases require extraordinary solutions experimental infection of human volunteers. the lancet infectious diseases risiko, usikkerhet og uvitenhet i medisinen [risk, uncertainty and ignorance in medicine) covid- vaccine development: time to consider sars-cov- challenge studies? reflections on the swine flu vaccination program interpreting diagnostic tests for sars-cov- ethical considerations for zika virus human challenge trial ethics of controlled human infection to study covid- the principle of respect for human vulnerability and global bioethics pandemic ethics: the case for experiments on human volunteers covid- : act first, think later the international alliance for biological standardization infectious hepatitis. studies of its natural history and prevention declaration of helsinki human challenge trials for vaccine development: regulatory considerations key criteria for the ethical acceptability of covid- human challenge studies human challenge trials for a covid- vaccine: should we bother about exploitation? uncertainty and environmental learning-reconceiving science in the preventive paradigm long-term consequences of covid- : research need key: cord- - wtyk r authors: sabroe, ian; dockrell, david h.; vogel, stefanie n.; renshaw, stephen a.; whyte, moira k. b.; dower, steven k. title: identifying and hurdling obstacles to translational research date: journal: nat rev immunol doi: . /nri sha: doc_id: cord_uid: wtyk r although there is overwhelming pressure from funding agencies and the general public for scientists to bridge basic and translational studies, the fact remains that there are significant hurdles to overcome in order to achieve this goal. the purpose of this opinion article is to examine the nature of these hurdles and to provide food for thought on the main obstacles that impede this process. it has achieved much, including the sequencing of the entire human genome, but it is already under serious attack for its failure to deliver effective therapies in many areas. this opinion article will provide a subjective view of our understanding of translational research, identify obstacles to its successful development, and propose a series of initiatives to improve the effectiveness of translational research strategies. originating from latin, translation means to 'carry across' . in biomedical research, the goal of translational science is to develop a thorough operational understanding of the human organism in health and disease, with the goal of 'carrying across' this knowledge to alleviate disease and suffering and to improve the quality of human existence. to be translational in medicine we must acquire knowledge from the broad arena of molecular and cellular biology and then apply this knowledge to human disease. the quality of our scientific output (perceived as a change in disease incidence and/or the development of a therapy) is largely dependent on the quality of the input data and the methods for their processing and interpretation, although the process of generating effective translational science is not as linear (that is, from molecules to models to humans) as is often thought. failure to ask the appropriate questions of optimized systems leads to the acquisition of knowledge that might be less relevant than anticipated. further corruption of the process comes as a result of limitations in our models, which are often not fully appreciated (or are simply ignored) at the time of the study. additionally, grant agencies must be sufficiently flexible to allow researchers to follow up on novel observations because many of the most exciting developments arise from unexpected findings. determining what research is intrinsically translational, or has been translated effectively, is therefore surprisingly difficult, and in a healthy global biomedical research environment, translation will continue to mean very different things to different groups. few scientists will see a process through from the conception of a hypothesis to the development of a specific medical therapy. as scientists, we are nonetheless required to measure our performance in terms of our 'translational potential' , particularly when it comes to justifying and generating funding and publications. therefore, in the absence of being able to measure contributions to health directly, we often quantify individual success using surrogate markers (such as publications and their impact, prestige, and funding), which depend on the prevailing concepts of what constitutes importance. however, these markers may be flawed for this purpose. a more global assessment of the output of translational science might consider whether the scientific community has improved specific disease outcomes, quality of life or life expectancy. specific examples of global success might not be as common as we would wish, but increased successes in the areas of organ transplantation or the ability to eradicate diseases such as measles virus highlight our ability to be successful. the improved treatment of many cancers through the combination of good science and high-quality clinical trials of new therapies or combinations of therapies has been strikingly impressive and provides many examples of highly effective translational science , . there remains, however, a lack of available mechanisms by which to relate our individual contributions to the global progress of translational science, and many factors conspire to impede our progress. the translation of basic scientific research faces a myriad of hurdles, both obvious and occult. these revolve around our understanding of the nature of the translational process, the integration of the outputs of different technological approaches to disease, the use of models, access to tissues and appropriate materials, and the need for support in increasingly complex areas such as ethics and bioinformatics. in addition, owing to technological advances, well-meaning safeguards of personal privacy have been imposed in relation to carrying out research in humans, and these have, in fact, greatly impeded progress in translational studies. problems with the models. entirely appropriate restrictions on what research can be done in humans have contributed to the status quo in which the mouse has become an indispensable model for translational biology; however, it is often not possible to predict biological responses in humans accurately based solely on results obtained from animal models [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . within immunity, exposure of mice to their own species-specific commensals and pathogenic organisms might contribute to a species-specific immunological phenotype , that affects the translational relevance. for example, studies of the effects of irak (interleukin- receptor-associated kinase ) deficiency have revealed increased identifying and hurdling obstacles to translational research susceptibility to a greater range of pathogens in mice than is observed in humans who are deficient for irak . where good potential translational concepts are generated from models, determining how to move to the human can be challenging, as highlighted by difficulties in calculating tolerogenic doses of insulin in the prevention of diabetes . in addition, technical limitations, or the dominance of prevailing models, can sometimes limit the scope of in vivo work, as illustrated by studies of airway disease, where studies in mouse models are focused predominantly on the important t helper (t h ) component of asthma but are poor models for the contribution of other mediators. mouse models are also complicated by our limited understanding of the phenotypes of human diseases , . designing perfect models of diseases that we do not fully understand is a tall order, and chronic diseases that involve life-long interactions between the host and the environment present a particularly difficult challenge [ ] [ ] [ ] [ ] . developing truly chronic models of disease is, however, heavily militated against owing to ethical concerns about prolonged suffering. moreover, the typically short-term nature of research funding, where outputs must be deliverable at a reasonable cost and within the time frame of a project grant, hinders the development of chronic models of disease. it is equally clear that work on a single cell line is often poorly predictive of the behaviour of the whole organism. although the use of primary cells from normal tissues can overcome some of these problems, the phenotype of these cells might be altered by their removal from the microenvironmental influences in vivo, and they often show markedly different responses to those of primary cells that are obtained from diseased tissue. therefore, rodent models remain essential and cannot be replaced by in vitro approaches at present, but are an imperfect translational conduit for both biological and practical reasons (such as the difficulty in establishing chronic disease models). other models such as flies and zebrafish (danio rerio) have many advantages for forward genetics and other related studies, but have substantially greater differences to humans than mammalian models. primates are the most compatible mammalian models for immunological research, and have provided invaluable insights into diseases such as hiv, but their broader use is not feasible because of major ethical concerns, as well as other practical and cost-related issues. even these models can have limitations. recently, differential expression of siglecs (sialicacid-binding immunoglobulin-like lectins), which are inhibitory receptors thought to downregulate immune-cell activation, has been noted between apes and humans , and such differences in immune responses between species require careful consideration, as highlighted by the development of a 'cytokine storm' when individuals received an agonistic cd -specific antibody that did not show the same effects in monkeys , . research is being increasingly hindered by bureaucracy. evident at many levels in research management, nowhere is this more of a problem than in the areas of access to human samples and tissues, and in the establishment of clinical collaborations. liaison with industry also provides additional challenges . furthermore, some of the most exciting areas of current research, such as work with embryonic stem cells, are subject to special ethical concerns. even the simplest research involving humans, or archived specimens from humans, is often encumbered by a multistaged, intimidating application process that might dissuade individuals from carrying out the proposed study. overcautious interpretation by ethics committees and regulatory authorities of what is ethical results in restrictive practices that can, for example, prevent re-screening of dna banks for relevant markers and mutations, or apply over-elaborate protection of clinical phenotype data. although it is absolutely clear that robust systems are needed to prevent exploitation of patients or clinical data, it is often unclear how the ethical standards that are applied to the review of proposed studies are developed. apparently arbitrary standards that might be perceived to be more ethical simply because they are more restrictive seem to be ever more on the increase. it is also unethical to excessively hinder research, but this imperative sometimes seems to come second to apparently minor scruples over details such as the methods of recruitment. although the introduction of national standards to ensure timely review of proposals has, in the united kingdom, begun to address these issues, increasingly complex review processes are frustrating and sometimes of questionable ethical value. the area of personal privacy is particularly complex. the majority public view is supportive of the use of some personal data in confidential research . however, although many individuals are less concerned that well-regulated data usage is an invasion of privacy than some ethics committees seem to be, due consideration is needed of the views of the minorities who are more concerned with their privacy. the future potential for apparently confidential genetic research resulting in the identification of individuals through the cross-referencing of dna databases is clearly of concern , . in addition, governments tend to respond to highly public episodes of research offences or clinical misconduct with legislation. this runs the risk of creating additional administrative burdens for the majority of individual scientists who practise ethically and well, without ensuring the prevention of future wilful misdeeds by a rare minority. increasingly, clinical practice is becoming more defensive and less willing to engage in research as a response to a society that is becoming more risk-adverse, litigious and blame-centred when adverse events occur. this generates further barriers to research in humans and requires a frank reappraisal of what experimentation is acceptable in humans or on samples that are derived from humans. additional problems arise in setting research priorities. without effective public input, we run the risk that research priorities become the whim of changing government focuses, which lack the stable, long-term input necessary for meaningful scientific advancement. although charities and private foundations that are centred on specific diseases provide important contributions, their focus is often too narrow to tackle the wider research agenda. this has led to a culture of increasing financial pressures that are governed by short-term policies, which require rapid outputs, and piecemeal funding, stifling longer term, more open-ended research that might produce real translational progress . for example, political pressures to study agents of bioterrorism or emerging infectious diseases such as sars (severe acute respiratory syndrome) can be welcomed and are clearly important; however, more long-term commitment to studying infections of historical global importance is also essential. with respect to developing long-term goals, the national institutes of health translational biomedicine must be seen by scientists and the wider public in the context of a broad-based body of science that has the potential to contribute, in the near or long term, to the advancement of human health. increased financial and political pressures on scientists to maximize directly measurable patient benefit from research carried out over short time frames runs the risk of destroying a healthy science base and should be actively resisted. a continued and robust engagement of the public is mandatory for scientists, because unless the public values the broad sweep of translational biomedical science, funding will inevitably be diverted away from science or applied only to those areas of research that immediately influence patient care. as scientists, we must learn to articulate not only the promise of science, but also the difficulties that are associated with moving an idea along to the product stage, so that unrealistic expectations of perfect medications for every disease in short time frames are not raised. we must also wrestle the debate on the usefulness of in vivo models away from the more extreme ends of the anti-vivisection movement so that a constructive, rather than confrontational, discussion can evolve. equally, we must foster a culture of research in the curricula of medical students, and promote the goals of translational research to basic science undergraduate and graduate students. we must seek to marry the skills of basic scientists with those of clinicians to convey the idea that strong translational research underlies improved health and is inseparable from the provision of good standards of clinical care. improving our use of the models? in many biomedical fields, a series of debates have highlighted deficiencies in our current in vivo models. strain differences and variance in experimental conditions between research groups pose major challenges in comparing the results derived from different studies. a collaborative investment in phenotyping human disease by clinicians and basic scientists is required for developing robust models. there is clearly an opportunity to define the characteristics, behaviour and relevance of model systems, particularly with a view to standardizing optimal strains and protocols. for example, multiple models exist for common diseases such as asthma and rheumatoid arthritis , although harmonizing protocols and strains for particular features of a disease must avoid the loss of sufficient diversity in order to allow the finding of the unexpected. this challenge could be met by interest groups or individuals working in a particular area, by large national funding agencies and/or international funding initiatives, or alternatively by specialist societies. such debates might facilitate the comparison of data between laboratories and between species, and might highlight the components of specific diseases that are ripe for the development of new in vivo models and protocols (for example, there remains a great need to more effectively model the role of the innate immune system in acute and chronic asthma), broadening the number of disease processes or phenotypes that are modelled in pathology. although much work will continue to focus on the mouse, for some diseases comparative or independent studies in other species will continue to be important. beyond the scope of this article, there are issues ahead with respect to subject selection for early clinical trials and the development of individualized treatment regimes that are based on pharmacogenetic and individual disease phenotypes , . figure | developing interactive models. a | in vivo experimentation, perhaps in particular the generation and characterization of knockout mouse strains in experimental models of disease, is often viewed as the gatekeeper between in vitro science and the generation of drug targets that are appropriate for human disease. although central to an effective understanding of immune biology and the role of new candidate drug targets, the predictive value of in vivo experimentation is less than desired, particularly in the context of studies of single knockouts in specific disease models and mouse strains. b | an example of a more holistic network in which multiple lines of evidence allow the refinement of objectives and target relevance in order to increase the chance of successful drug discovery. such approaches reflect the approach of many researchers, but (acknowledging that no branch of science is 'easy') the main difficulties associated with undertaking human-based research run the risk of degrading the quality of data that arise from an integrated scientific approach. the tendency to view translational research as a linear process in which mice are the gateway between basic science and humans does rodent models a great disservice. in particular, the classical route of identifying genes in vitro, followed by generating knockout animals in vivo, has, in general, been poorly predictive of the consequences of targeting specific molecules in humans. advances in medical sciences that have emerged in this manner are vastly outnumbered by those that arise by serendipity or through less predictable routes. a more holistic integration of in vivo models with in vitro science and studies in the human is needed when summarizing the translational relevance of a system, in which in vivo models contribute strongly to an iterative strategy, but are not themselves the final arbiters (fig. ) . the potential to identify medicines for use in human disease by screening less complex biological systems has long been recognized in the pharmaceutical industry, and there is now considerable interest in the use of model organisms such as caenorhabditis elegans and zebrafish in high-throughput screens for new drugs , . it is becoming increasingly evident that these systems can be used to identify therapeutic targets in the immune system . in this way, understanding the biology of pathways and gene products is deferred, and the ability of a compound to intervene in complex biological processes is directly tested, as highlighted in recent studies of calcium-channel antagonists in c. elegans . combining the use of mice and humans in effective strategies. it seems self-evident that research which aspires to influence pathogenesis in humans needs to be carried out on the human system. at the same time, we must also anticipate the potential for doing harm that might accompany any new approaches to treating diseases, which militates against a rapid progression to phase i human trials. nonetheless, the extraordinary difficulties that are associated with carrying out human studies in parallel with animal models has facilitated a culture in which such studies rarely occur, and in which prioritizing research is not driven by the inclusion of such processes. indeed, almost quixotically, work in human tissues and cell lines is sometimes not deemed to be of importance until verified in a mouse with a targeted mutation. bridging this divide requires the scientific community to value more highly the studies that seek to bring mouse and human studies together, and to appreciate that in human studies a lack of phenotype or subtle modifications of processes might be as important as models that generate dramatic phenotypes. from simple co-culture models of normal human tissues and ultimately to the generation of whole organs or representations of whole organs in the laboratory, we are now beginning to produce in vitro human systems that can complement essential work that is currently only achievable in vivo. increasing success with new gene-delivery systems, combined with new technologies such as gene knockdown by rna interference, might allow us to overcome the inability to study humans with targeted gene deficiencies. such models are in their infancy, and cell-culture-based approaches are, in many scientists' views, farther from human biology than techniques that investigate biology in mice. it would seem that both are required, and therefore, a major thrust to develop standardized co-culture models that are based on primary human cells from healthy and diseased subjects would provide a complementary scientific base to our strong expertise in the mouse. our commitment to this development is essential, not only to boost the translation of science to the human, but also to ensure that we honour the principles of reduction, refinement and replacement (minimizing the number of animals that are used and finding alternatives wherever feasible) that are central to all animal experimentation. we are faced with many difficulties in generating appropriate human tissue models, including defining differences in similar cell types that are pathologically relevant (for example, comparing the biology of endothelial cells isolated from the umbilical cord with those isolated from different microvascular beds). we also need to determine the optimal representative culture conditions for primary cells. for example, when should epithelial cells be studied at an air-liquid interface, or when should leukocyte-endothelial interactions be studied under flow? importantly, diseased tissues are often modified by inflammation, genetic traits and epigenetic processes that require further consideration when translating from the biology of health to the biology of disease. nevertheless, we have recently shown that simple co-cultures of primary human cells can in some circumstances replicate inflammatory systems that are observed in vivo in mice , ; such co-culture models are becoming increasingly common. the future ability to study systems that resemble organs or complete tissues, and verifying the work of simple co-culture experiments in such systems, offers a future we should not only embrace, but also be actively working towards at every opportunity. it is routine in clinical practice to combine drugs with similar or differing modes of action when treating disease. there is also increasing appreciation of the roles of cellular and molecular networks in the aetiology of disease , a fact that is implicitly recognized by the need for in vivo models in which complex interactions between tissues and cells can be studied without a need to identify the full panoply of the systems involved (box ). in the context of an inflammatory disease, we have recently proposed that these networks are best described by the terminology of 'contiguous immunity' , whereby, in disease, temporally and spatially contiguous networks comprising multiple processes of innate and adaptive immunity are in continual dialogue and evolution . it is curious, then, that so many studies aim to land a single 'killer blow' on pathological processes, rather than considering the impact of therapeutic combinations. our increasingly deeper understanding of the complexity of the inflammatory process often results in the targeting of downstream components for which there might be redundancy or very specific functionality. the targeting of such systems in combination, although complicated to achieve and less satisfying with respect to the identification of single clear targets, is nonetheless likely to have relevance for successful translation. allowing access to tissues and overcoming ethical anxieties. the diversion of research funding into bureaucracy and the delays in productivity that result from meeting statutory requirements for ethical practice contribute to the erosion of research capacity. research cannot do without governance, but it is disappointing that increasing regulation has not been met by increased support for ethical human-based research, and www.nature.com/reviews/immunol the future ability to study systems that resemble organs or complete tissues … offers a future we should not only embrace, but also be actively working towards at every opportunity. although many processes conspire to make research harder, few exist to make it easier. a simplification of the regulations and national-level funding that provides templates for ethical applications, together with training and support for investigators, would do much to make this easier. because many projects involve international collaborations, simplified standard international regulations would also greatly facilitate progress. many research projects have conceptually similar goals (such as the tackling of inflammation in a tissue), and generic pre-approved protocols requiring modest local adaptation would obviate many problems. in the united kingdom, the proposed establishment of a single health research board to coordinate government-funded health research provides an opportunity to develop programmes that examine the effects of government regulation on research and mechanisms for overcoming the issues discussed earlier. research on human tissues could be enhanced by improved access to normal and diseased specimens. the establishment of central and devolved services whose function would be to source human tissues in an ethical manner, characterize the donors and tissues anonymously, and make these resources freely available to investigators in research-active countries would transform biological research (indeed, some progress is being made here, but there is still much to be done). an emphasis on dialogue with scientists and flexibility with respect to supporting science would be required to underpin access to these resources by researchers. where materials are scarce, such systems could be supported by expertise in cell-line immortalization. the development of the lung tissue research consortium by the national heart, lung and blood institute is an exemplar of a clinical data and tissue bank that offers enormous potential for research in lung disease; in addition, the development of the uk biobank shows it is feasible for human resources to be sourced ethically in ways that allow relatively broad use in medical research according to need. the proposed tissue banks would promote a positive view of human science working in parallel with mouse biology, which also requires commitment and support from the public to make it work. 'omics'. one area in which large-scale biology is excelling is the use of public-access databases (such as those pertaining to gene expression (genomics) or the production of metabolites (metabolomics)) , but we are missing opportunities to further expand these databases. local and national guidelines define the optimal management of many diseases, but it can be argued that the providers of health care are not given appropriate opportunities to engage with the scientists whose work is developing the future treatments for these diseases. scientific initiatives that are associated with national care plans could drive disease phenotyping and tissue collection for tissue, dna, rna, protein and metabolite databanks with good the difficulty in designing in vitro and in vivo experiments to model human disease has inevitably required and generated simplifications of pathology, often leading to relatively linear models of disease (for example, tissue damage, followed by antigen presentation, the generation of immunological memory and autoantibodies, and a resulting autoimmune disease). in reality, pathology is generated by networks that can exhibit substantial plasticity over the course of a disease. these principles are illustrated in the figure. a disease process -pathology -is represented at the centre of a series of simple conceptual networks, components of which are left intentionally blank to avoid attempting to define specific diseases. each component (or node) within the network might have different roles at different times in a disease, or if active at more than one tissue site, might even have different roles simultaneously in a disease process. therefore, the depicted connections are plastic over time and in individual microenvironments. in a cellular network. pathology can be considered in the context of networks of cells that are recruited or resident at inflammatory sites, whose communication through cytokines and other molecules regulates inflammation. in a cytokine network. pathology is driven by the interrelated actions of cytokines, again forming a dynamic plastic network. in a process network. process behaviour (for example, angiogenesis, scarring and leukocyte recruitment) will contribute differently to pathology at specific tissue locations and different times in the disease. as an illustration, wound-healing responses might contribute to the resolution of normal tissue architecture, the development of fibrosis, or the regulation of inflammatory cell recruitment and survival. depending on the nature and duration of the stimulus driving the woundhealing process, and the location in which it is set, multiple resulting pathological phenotypes are feasible. it is the nature of tissues to contain multiple cells and supporting structures that are physically associated, with many more cells that can transit through or become resident. we have proposed that the immunity seen in pathology rarely falls clearly into categories of innate and adaptive immune, or t helper (t h ) and t h responses. rather, pathology is generated by a networked interaction that changes over time. the networked relationships of immune pathology might be better described as 'contiguous immunity', in which multiple processes or networks can be operational and in dialogue in the same space (physically contiguous); equally, processes might be linked together in evolving patterns (temporally contiguous). understanding these networks, and, where necessary, developing new models to elucidate and target them, is essential to effective translational biology. ifn, interferon; il, interleukin. public access. good clinical practice not only should focus on ensuring that existing best practice is reliably reproduced, but also should put equal weight to research and development of practice through translational research. it is not just the priority of scientists to engage with clinicians: the imperative is equally strong that clinicians should engage more with basic scientists. in both cases it is important that such engagement is facilitated and supported by national policy. another major challenge is to take the input of a large amount of descriptive data generated by an 'omics' approach and both interpret and reapply it to a translational problem in a hypothesis-driven manner. as we learn to integrate complex data sets with in vitro cell biology and in vivo models, we might begin to generate virtual phenotypes, allowing conclusions to be drawn on the basis of the relatedness of a series of data sets to the questions asked. in essence, all researchers do this when they read published data, but the process is inherently subjective and formalizing such integrated biology could be more objective and so better inform future experiments. we are now experiencing an unprecedented blossoming in available technologies, unparalleled through history, which makes biomedical science extraordinarily exciting. with this comes an ever greater financial burden and increasingly complicated ethical issues. an increasing focus on the need for effective translation from the use of these resources highlights the many obstacles that impede such progress. we have highlighted these difficulties, and have suggested strategies to rejuvenate and maximize our translational potential. tyrosine kinases as targets for cancer therapy adjuvant chemotherapy for breast cancer - years later pro: mice are a good model of human airway disease modelling the human immune response: can mice be trusted? modeling allergic asthma in mice: pitfalls and opportunities con: mice are not a good model of human airway disease the mouse trap satisfaction (not) guaranteed: re-evaluating the use of animal models of type diabetes modelling infectious disease -time to think outside the box? lost in translation: barriers to implementing clinical immunotherapeutics for autoimmunity distinct mutations in irak- confer hyporesponsiveness to lipopolysaccharide and interleukin- in a patient with recurrent bacterial infections pyogenic bacterial infections in humans with irak- deficiency severe impairment of interleukin- and toll-like receptor signalling in mice lacking irak- phenotypes in asthma: useful guides for therapy, distinct biological processes, or both? evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics loss of siglec expression on t lymphocytes during human evolution differences in immune cell 'brakes' may explain chimp-human split on aids cytokine storm in a phase trial of the anti-cd monoclonal antibody tgn national survey of british public's views on use of identifiable medical data by the national cancer registry confidentiality in genome research no longer de-identified mouse model of airway remodeling: strain differences murine models of asthma rodent models of rheumatoid arthritis concordance among gene-expressionbased predictors for breast cancer a genomic strategy to refine prognosis in early-stage non-small-cell lung cancer a small-molecule screen in c. elegans yields a new calcium channel antagonist in vivo drug discovery in the zebrafish a transgenic zebrafish model of neutrophilic inflammation capturing complex d tissue physiology in vitro cooperative molecular and cellular networks regulate toll-like receptor-dependent inflammatory responses agonists of toll-like receptors and activate airway smooth muscle via mononuclear leukocytes pulmonary perspective: targeting the networks that underpin contiguous immunity in asthma and copd a bioinformatician's view of the metabolome we thank the many scientists whose stimulating conversations have in some measure been represented in these pages. i. the authors declare no competing financial interests. ian sabroe's homepage: http://www.shef.ac.uk/medicine/staff/sabroe.html lung tissue research consortium: http://www.ltrcpublic.com uk biobank: http://www.ukbiobank.ac.uk access to this links box is available online. key: cord- -vre bq authors: groneberg, david a.; braumann, hannah; rolle, stefan; quarcoo, david; klingelhöfer, doris; fischer, axel; nienhaus, albert; brüggmann, dörthe title: needlestick injuries: a density-equalizing mapping and socioeconomic analysis of the global research date: - - journal: int arch occup environ health doi: . /s - - - sha: doc_id: cord_uid: vre bq background: needlestick injuries have caused a deleterious effect on the physical and mental health of millions of health-care workers over the past decades, being responsible for occupational infections with viruses such as hiv or hepatis c. despite this heavy burden of disease, no concise studies have been published on the global research landscape so far. methods: we used the new quality and quantity indices in science platform to analyze global nsi research (n = articles) over the past years using the web of science and parameters such as global versus country-specific research activities, semi-qualitative issues, and socioeconomic figures. results: density-equalizing mapping showed that although a total of n = countries participated in nsi research, large parts of africa and south america were almost invisible regarding global participation in nsi research. average citation rate (cr) analysis indicated a high rate for switzerland (cr = . ), italy (cr = . ), and japan (cr = . ). socioeconomic analysis revealed that the uk had the highest quotient q(gdp) of . nsi-specific publications per bill. us-$ gross domestic product (gdp), followed by south africa (q(gdp) = . ). temporal analysis of hiv versus hepatitis research indicated that nsi-hiv research culminated in the early s, whereas nsi-hepatitis research increased over the observed period from the s until the last decade. conclusion: albeit nsi research activity is generally increasing, the growth is asymmetrical from a global viewpoint. international strategies should be followed that put a focus on nsi in non-industrialized areas of the world. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. background as elegantly stated by recent reviews, the risks of needlestick injuries (nsi) may have fallen over the past three decades, but even in the presence of guidelines, they still occur frequently (king and strony ) . health-care workers have the highest risk of experiencing needlestick injuries, but also other occupations are endangered (king and strony ; mona et al. ) . when injured, the individual clinical, psychological and economic burden can be substantial (cooke and stephens ) . as previously stated, the rates of nsis have been estimated to vary between . and . % in health-care workers, depending on different settings, countries, and methodologies (cooke and stephens ) . hence, research in this area is crucial and knowledge about previous research is important to plan future strategies. since there are no data existing on the international architecture of nsi research available, the present study used the newqis project platform (groneberg-kloft et al. a) to construct the first picture of global nsi research strategies. we aimed to ( ) perform an in-depth analysis of the global nsi research activities and to ( ) visualize a variety of figures related to nsi research by the use of density-equalizing procedures proposed by gastner and newman (gastner and newman ) . to analyze nsi research, we used the new quality and quantity indices in science (newqis) platform termed newqis (groneberg-kloft et al. a, b) , ranging from public and global health issues trost et al. ) to fields such as internal medicine (schoffel et al. ) , surgery (schoffel et al. (schoffel et al. , or gynecology and obstetrics (bruggmann et al. a, b) . since its foundation, about peer-reviewed studies have been published using newqis. as data source, we used the web of science (clarivate) since this database enabled us to calculate various citation parameters ). to cover studies related to occupational needlestick injuries, we constructed a detailed search algorithm, illustrated in fig. . using this term, we performed a topic search in the period - . as in earlier studies, the period after - - was excluded to avoid incomplete data acquisition. as earlier stated, newqis enables to analyze and visualize a wide range of different parameters (i.e., the time of publication, the originating countries, the attributed subject categories, the citations). with these raw data, further parameters such as country-specific h-indices (hi) of the present set of publications were calculated. these hi are not related to single authors, but to the countries. also, country-specific citation rates of nsi-related research of each country were calculated (cr, total country-specific citations numbers per total numbers of country-specific nsi publications) (hirsch ) . in a next step, the numbers of collaborating countries in collaborating articles were also analyzed. absolute research activity is important to assess the overall contribution of a single country toward the global research efforts in the areas of nsi. however, there are vast socioeconomic differences between single countries. therefore, the research activity data should also be related to socioeconomic figures of each country to be able to interpret the relative contributions. we approached this issue by relating nsi research activity to the ( ) total economic power index "gross domestic product" (gdp) per billion us-$, ( ) gdp per capita, and ( ) country population sizes (world factbook (world economic outlook database )). we here used the approach of density-equalizing map projections (demp) established by gastner and newman in (gastner and newman ) . to construct the demps, we introduced several nsi-specific parameters to the algorithm including total numbers of nsi-related publications, country-specific citation rates of nsi-related research and others. as stated earlier, newqis also assesses global collaborative networks. using a methodological approach which was previously described, all affiliations of nsi-related articles were analyzed for their originating countries. then, chart diagrams were generated to visualize the network (mund et al. ). in two separate analyses, we assessed the research activities for the most prominent nsi-related acquired diseases-hiv and hepatitis. in specific, the search term listed in fig. was separated into two different, hiv-and hepatitis-specific terms as listed in online supplement . the metadata was analyzed for chronologic development and countries of research origin. the first publication on nsi was identified for the year , but until , only a moderate activity was present with less than articles being published annually. in the s, an increase was present and beginning of the s, more than annual articles on nsi were published for the first time. in total, articles were identified. more than % of all articles were published after the year . a regression analysis of the investigated research interest over time reveals a high coefficient of determination (r = . ) since (fig. ) . in total, countries published nsi-related articles. the nation with the highest level of nsi research activity was the usa with n = , articles. next, great britain showed an activity of nsi-related articles, followed by canada (n = ), australia (n = ), germany (n = ) and france (n = ). when all data are transferred to a demp algorithm, a world map distorted toward north america and western europe is present. in the eastern hemisphere, japan and australia are prominent. australia is the only country south of the equator with an activity over nsi articlesindicating a strong north-south slope (fig. ) . a similar landscape is present, when the number of nsi publishing institutions/affiliations per country is analyzed (fig. ) : the usa is the leading country with i = different affiliations, followed by great britain (i = ), france (i = ), italy (i = ), germany (i = ), and japan (i = ) (fig. ). the analysis of the crude parameter of the total citation count followed the picture of the total publication activities with only small variations being present: the usa was at the lead position with c = , citations. it was followed by great britain (c = citations), italy (c = ), canada (c = ), japan (c = ), australia (c = ), and germany (c = ) (fig. a) . as a second citation readout, the country-specific hirsch indices (cohi) were assessed for the presently identified nsi-related publications. in this assessment, demp calculations led to a global architecture similar to the total citation count projections with the usa at the lead position with a cohi of publications being at least cited times. the usa was followed by the uk (cohi = ), canada (cohi = ), australia (cohi = ), france and italy (both a cohi of ), germany, japan and switzerland (all a cohi of ) and the netherlands (cohi = ). the only african country with a cohi more than is south africa (cohi = ) (fig. b) . a complete different global architecture was found when the nsi publications were analyzed for the average citation rates with a threshold of at least publications: here, switzerland was ranked first (cr = . ), followed by italy (cr = . ), japan (cr = . ), usa (cr = . ), and spain (cr = . ) (fig. c ). countries with a high level of economic prosperity are able to allocate more funding toward nsi research from a financial point of view. therefore, we also assessed nsi research in relation to socioeconomic figures. we first assessed the correlation between the country-specific number of nsirelated articles and the countries' gross domestic product in bill. us-$ (gdp , table ). when applied for all countries with at least specific articles related to nsi, the uk is ranked highest with a quotient q gdp of . nsispecific publications per bill. us-$ gdp. the uk is followed by south africa with a quotient of q gdp = . , and iran (q gdp = . ). next, pakistan (q gdp = . ), switzerland (q gdp = . ), australia, belgium, canada, and israel follow with a q gdp of . . the usa, turkey and the netherlands have a q gdp of . ), followed by sweden and poland (q gdp = . ), and spain (q bip = . ). in a next step, we assessed the correlation between the country-specific number of nsi-related articles and the countries' gross domestic product per capita (table ): in this ranking with a threshold of at least specific articles, india is ranked first with . nsi-specific articles per gdp/capita, followed by the usa and pakistan. in a final assessment, nsi research was related to population sizes. here, switzerland was ranked first with a calculated number of . nsi-related articles per million of inhabitants, followed by australia, the uk, canada and the usa (table ). in total, there were collaborative articles (n coop ) present. about % were bilateral cooperations (n coop = ). trilateral publications were present in with n coop = cases, followed by collaborations of four countries (n coop = and five countries (n coop = ). there were , us collaboration articles, followed by uk (n coop = ), french (n coop = ), chinese (n coop = ), german (n coop = ), and canadian (n coop = ) collaborations (fig. ) . the leading nation was the usa with a total of collaboration publications. the most prominent collaboration was between us-american and canadian scientists (fig. ) , followed by us-swiss and us-uk collaborations. subject category analysis of published nsi research showed an increase in public, environmental and occupational health-topics related to nsi over the past decades (fig. a) . also, the field of nursing gained research activity in the period of - . when single countries were analyzed for differences in their research focuses, a heterogeneous picture was present (fig. b) . the most apparent difference was found for japan with an emphasis on the field of gastroenterology and hepatology. besides, turkey was the country with the highest proportion of nsi research related to the field of nursing. a temporal analysis and comparison of research activities focused on nsi and hiv or nsi and hepatitis shows that there are major differences present. whereas nsi research, specifically on hiv, culminated in the beginning of the s, nsi-research focused on hepatitis increased over the observed period from the s until the last decade (fig. ) . when a sub-analysis of countries' research activities was performed (table ) , the usa dominated both nsi research on both acquired diseases, followed by the uk. it is noteworthy that south africa also appeared in the top ten most active countries concerning nsi and hiv. by contrast, the only asian country which is present in the overall activity ranking, japan, does not belong to the most active nsi-hiv-focusing countries. however, it is present in the nsi-hepatitis top ten ranking countries. nsis are omnipresent around the globe and endanger the health and life of hundreds of thousands of health-care workers. as stated before, they generate a large amount of indirect, direct, potential, and intangible costs, which why? probably, because nsis are not just a single entity such as a disease or a pathogen, but an accident, resulting from a series of failures, which can be analyzed from different perspectives: determinants, characteristics, distribution, consequences, prevention of accidents, and prevention of consequences. therefore, the present results need to be interpreted with caution. our approach aimed to visualize global research activities in the field of nsi and related areas of science. to approach this aim, metadata were used from the web of science between and and , publications were identified which were specifically related to nsi. interestingly, almost all nsi-related articles ( . %) were published between the years and , indicating only a very minimal activity in the decades before. the reason for this increase after is based upon two findings: observations that (a) hepatitis b (first articles appearing in - ), and then (b) hiv (first documented case in literature in ) could be acquired through a needlestick. one might argue that due to the minimal activities more than three decades ago, the analysis should have been limited to, i.e., the past decades. however, including a time span the usa plays a dominant role in country-specific analyses with the highest number of articles published, the largest number of institutions of higher education as well as the highest modified h-index. furthermore, the usa possesses the most national and international collaborations among all nations. how can these data be interpreted? first of all, it is important to discuss these results with regard to the overall global research performance and general trends. to achieve this, the presently observed increase in nsi research activities needs to be related to the increase in global research activities, in specific, biomedical research activities. it is wrong to take the growth of databases such as the presently used web of science as a precise tool for increase in research activities (since no existing database captures everything) (noorden ). however, it may serve as a proxy (bornmann and mutz ) . by analyzing more than million cited references in million publications from to , bornmann and mutz estimated the growth of research for between to % (bornmann and mutz ) . although only restricted to wos data, our analysis shows for nsi a growth rate which is not constantly increasing, but varying between years, especially in the s to the s. this is an uncommon picture, because usually the growth is more or less linear, as previously demonstrated (noorden ; bornmann and mutz ) . how is the global architecture of nsi research? as stated above, the usa is by far dominating the international efforts in nsi research. is this a common picture-yes, there is a multitude of other bibliometric studies that demonstrated a unique leading role of the usa in almost every area of science. but how is the pattern of leading countries apart from the usa? does this ranking follow any previously known pattern? our ranking with the uk at position , followed by canada (n = ), australia (n = ), germany (n = ) and france (n = ) shows a global architecture that is similar to those of many other fields of biomedicine. but is this pattern specific for research conducted in the area of viral diseases? there are a number of studies that addressed global research activities on infectious diseases so far: both nsirelevant infectious diseases/agents such as hiv or hepatitis virus and other diseases such as ebola or influenza. in general, there are two global patterns present: a pattern for infectious diseases with a large burden of disease in industrialized countries and/or infectious diseases with the possibility of vaccination such as hepatitis (schmidt et al. ) , hpv , or influenza (fricke et al. ) and a pattern for diseases that are more common in non-industrialized or tropical countries such as leishmaniasis (al-mutawakel et al. ) . the present nsi research activity pattern is more or less similar to the ones reported for hiv or hepatitis (schmidt et al. ) . it differs from the patterns of diseases linked to viruses such as yellow fever ) and mers-cov (zyoud ) . here, countries such as brazil and india appear in the top ten ranking most active countries. next to these continental differences, one might also focus future studies upon further variations, i.e., between countries of the european union, which enjoy shared health and safety legislation, and other regions of the northern globe. in this respect, it has been recently demonstrated with regard to psychosocial risks and violence in the workplace that there are important differences between these regions (chirico et al. ) . a similar study set up using databases such as legosh" of the international labour office (ilo) might add important new insights into the understanding of global and regional nsi research differences. we also related overall nsi research figures to socioeconomic features including gdp or population sizes. a limitation of this approach is the constant change in these socioeconomic parameters. however, they are commonly used as proxies to relate research productiveness to socioeconomic features (chong et al. ; wang and zhao ) . apart from the country-specific analysis of research activities, it is also of major importance to analyze the subject areas in which nsi research is performed and published. in contrast to global research activities, e.g., on hepatitis (schmidt et al. ) , or hpv ), we here found for nsi that the field of public, environmental and occupational health gained importance in the past decades. in the final -year period of analysis, this field was among the most active areas of nsi research. this points to the importance of nsi for this area of biomedicine. obviously, nsi cases among health-care workers have also occurred in countries where the hiv epidemic was widespread (i.e., african countries), but they were less likely to be identified due to the ongoing epidemic in the general population, and the lack of specific surveillance at the beginning of the epidemic. in our sub-analysis, south africa was detected to belong to the top ten most active countries concerning hiv-specific nsi research. other countries, such as japan, with a significant prevalence of hcv in the general population, observed cases of hepatitis c linked to nsis after the identification of a specific antibody test to detect seroconversion in the early s, and this is why papers appear in gastroenterology and hepatology journals. this is visualized by our nsi research area analysis in which japan is very prominent in the field of gastroenterology and hepatology. also, in our sub-analysis, japan appeared in the top ten activity ranking of specific nsi-hepatitis research. the different waves of publications are in relationship with the different subjects related to nsis (and countries) as demonstrated here. the evolution of these areas with regard to nsi occurred over time, i.e., pathogens acquired, effects of the first preventive measures (universal precautions, banning of recapping, sharps containers, hepatitis b vaccination), post-exposure prophylaxis against hiv, introduction of safety devices, and impact of laws and regulations supporting preventive measures. we here present data on activity of nsi research in the twentieth and twenty-first centuries. our data clearly show that there is a heavy global asymmetry with the southern globe being underrepresented to a major extent. although the area of public and occupational health-being underpresented concerning research activity in the majority of other medical areas-is omnipresent in nsi research. this study points to the need to foster nsi research activities in non-industrialized countries in which health-care personnel are crucial for public health. substantial contributions to the interpretation of the data. they all have been involved in drafting and revising the manuscript. all authors have read and approved the final manuscript. funding this study was supported by bgw (institution for statutory accident insurance and prevention in the health and welfare services), germany. the bibliometric data are owned by and have been obtained from the web of science database. any researcher with access to the web of science database can obtain the data using the methods described in the paper. readers who do not have access to web of science should contact clarivate analytics to obtain a license. as per clarivate analytics terms of use, the authors may also be able to provide limited access to data, subject to their agreement. conflict of interest the authors declare that they have no conflict of interest. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. scientometric analysis of the world-wide research efforts concerning leishmaniasis growth rates of modern science: a bibliometric analysis based on the number of publications and cited references endometriosis and its global research architecture: an in-depth density-equalizing mapping analysis global architecture of gestational diabetes research: density-equalizing mapping studies and gender analysis human papilloma virus: global research architecture assessed by density-equalizing mapping yellow fever disease: density equalizing mapping and gender analysis of international research output psychosocial risk prevention in a global occupational health perspective. a descriptive analysis global economic burden of schizophrenia: a systematic review clinical, economic, and humanistic burden of needlestick injuries in healthcare workers influenza: a scientometric and density-equalizing analysis diffusion-based method for producing density-equalizing maps silicosis: geographic changes in research: an analysis employing densityequalizing mapping snakebite envenoming-a combined density equalizing mapping and scientometric analysis of the publication history quality and quantity indices in science: use of visualization tools new quality and quantity indices in science (newqis): the study protocol of an international project an index to quantify an individual's scientific research output how much do needlestick injuries cost? a systematic review of the economic evaluations of needlestick and sharps injuries among healthcare personnel a systematic review on occupational hazards, injuries and diseases among police officers worldwide: policy implications for the south african police service global research on smoking and pregnancy-a scientometric and gender analysis pancreatic cancer-critical examination of the global research architecture and recent scientific developments hirschsprung disease: critical evaluation of the global research architecture employing scientometrics and density-equalizing mapping crohn's disease: a critical approach to publication procedures and citation behavior of the global research network statistisches jahrbuch deutschland und internationales immigration: analysis, trends and outlook on the global research activity global scientific output doubles every nine years worldwide research productivity in the field of back pain: a bibliometric analysis world economic outlook databa www global research trends of middle east respiratory syndrome coronavirus: a bibliometric analysis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements open access funding provided by projekt deal. we thank c. scutaru and b. kloft for their pioneering work concerning newqis. this study is part of a md/phd thesis project (hb). we also thank the two peer referees for excellently helping us to improve the quality of the data.author contributions dag, hb, dk, and db have made substantial contributions to the conception and design of the study, acquisition of the data and interpretation. sr, dq, af, and an have made key: cord- -mc pifep authors: rowhani-farid, anisa; allen, michelle; barnett, adrian g. title: what incentives increase data sharing in health and medical research? a systematic review date: - - journal: res integr peer rev doi: . /s - - - sha: doc_id: cord_uid: mc pifep background: the foundation of health and medical research is data. data sharing facilitates the progress of research and strengthens science. data sharing in research is widely discussed in the literature; however, there are seemingly no evidence-based incentives that promote data sharing. methods: a systematic review (registration: . /osf.io/ pz e) of the health and medical research literature was used to uncover any evidence-based incentives, with pre- and post-empirical data that examined data sharing rates. we were also interested in quantifying and classifying the number of opinion pieces on the importance of incentives, the number observational studies that analysed data sharing rates and practices, and strategies aimed at increasing data sharing rates. results: only one incentive (using open data badges) has been tested in health and medical research that examined data sharing rates. the number of opinion pieces (n = ) out-weighed the number of article-testing strategies (n = ), and the number of observational studies exceeded them both (n = ). conclusions: given that data is the foundation of evidence-based health and medical research, it is paradoxical that there is only one evidence-based incentive to promote data sharing. more well-designed studies are needed in order to increase the currently low rates of data sharing. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. research waste: hidden data, irreproducible research the foundation of health and medical research is data-its generation, analysis, re-analysis, verification, and sharing [ ] . data sharing is a key part of the movement towards science that is open, where data is easily accessible, intelligible, reproducible, replicable, and verifiable [ ] . data sharing is defined here as making raw research data available in an open data depository, and includes controlled access where data is made available upon request which may be required due to legal or ethical reasons. despite the wide-scale benefits of data sharing such as addressing global public health emergencies, it is yet to become common research practice. for instance, the severe acute respiratory syndrome (sars) disease was controlled only months after its emergence by a world health organization-coordinated effort based on extensive data sharing [ ] . likewise, the researchers working on the ebola outbreak have recently committed to work openly in outbreaks to honour the memory of their colleagues who died at the forefront of the ebola outbreak, and to ensure that no future epidemic is as devastating [ ] . notwithstanding these benefits, numerous studies have demonstrated low rates of data sharing in health and medical research, with the leading journal the british medical journal (bmj) having a rate as low as . % [ ] and biomedical journal articles % [ ] . there are of course legitimate reasons to withhold data, such as the concern about patient privacy, and the requirement for patient consent for sharing [ ] . with % of the world's spending on health and medical research, an estimated $ billion, wasted every year, it is clear that the scientific community is in crisis, leading to questions about the veracity of scientific knowledge [ ] . data sharing and openness in scientific research should be fundamental to the philosophy of how scientific knowledge is generated. thomas kuhn introduced the concept of paradigm shifts that arise from a scientific crisis. the paradigm shift before us today is from closed, hidden science to open science and data sharing [ ] . sharing scientific data will allow for data verification and re-analysis, and for testing new hypotheses. open data reduces research waste in terms of time, costs, and participant burden, and in turn, strengthens scientific knowledge by ensuring research integrity [ , ] . the many current problems in health and medical research have led to the emergence of a new field, metaresearch, which is concerned with improving research practices [ ] . meta-research has five sub-themes with 'reproducibility' and 'incentives' as two of the themes [ ] . reproducibility is concerned with the verification of research findings, which can be achieved through the sharing of data and methods [ ] . incentives is concerned with rewarding researchers, which includes incentives to share their data and methods [ ] . we were interested in how researchers are incentivised to openly share their raw data, thus combining two sub-themes of meta-research. historically, it has not been common practice for the content of a research article to include access to the raw data from scientific experiments [ ] . this flaw, created by technological limitations among others, has hindered the progress of scientific knowledge [ ] . however, we can no longer blame technology for outdated research practices. there are many data depositories which allow researchers to easily share their data using a citable doi. there have also been many recent policies and frameworks to encourage openness in research [ ] . yet, uptake in health and medicine is low and what is lacking, it appears, are rewards that incentivize researchers to share their data [ ] . incentives are defined here as rewards that are given to researchers if they participate in sharing their raw scientific data [ ] . the queensland university of technology (qut) library staff assisted in developing a rigorous and clearly documented methodology for both the search strategy and the selection of studies. the aim was to minimise bias by documenting the search process and the decisions made to allow the review to be reproduced and updated. the cochrane handbook for systematic reviews was used as a guide for this systematic review: http://handbook.cochrane.org/. the equator network additional file : prisma ( ) checklist [ ] was used to ensure good practice as well as accurate reporting. three systematic review registries (prospero, joanna briggs institute, and cochrane) were checked to ensure our proposed systematic review had not already been done. our systematic review protocol was registered at the open science framework on august (doi.org/ . /osf.io/ pz e). this review considered published journal articles with empirical data that trialed any incentive to increase data sharing in health and medical research. articles must have tested an incentive that could increase data sharing in health and medical research. for the purposes of this review, health and medical research data is defined as any raw data that has been generated through research from a health and medical facility, institute or organisation. incentives are defined here as 'a benefit, reward, or cost that motivates an […] action'. this was based on the definition of incentives in economics, which groups incentives into four categories: financial, moral, natural, and coercive [ ] . the review included any paper with empirical data on sharing that compared an intervention and control, which used a clear research design (including randomised and non-randomised designs). the types of measures included are the percent of datasets shared, or the number of datasets shared, or the relative ratio of data sharing. this review excluded the following, but still classified these excluded papers by field: all editorial and opinion pieces that only discuss strategies to increase data sharing without trialling them. strategies that do not involve incentives, e.g., education seminars, change in a data sharing policy or some other policy, access to data management tools and managers. observational studies that describe data sharing patterns. this search strategy was designed to access published articles through the following steps: ) ((("open science" or "open data" or "data sharing") and (incentive* or motivation* or reward* or barrier*))) ) ) relevant articles that did not appear in the database search but were known to the reviewers were handpicked and extracted into endnote. two reviewers, arf and ma, screened the titles of the articles and based on the inclusion and exclusion criteria, extracted them into endnote. duplicates were removed. the reviewers independently screened the extracted article titles and abstracts based on the inclusion and exclusion criteria and categorised them into five groups: arf read the titles and abstracts of all extracted articles and ma verified her findings by reading a random sample of %. discrepancies between the two reviewers were approximately %, however these were relatively minor and resolved through discussion of the scope of each of the categories. for instance, a research paper outlined the introduction of a data system, one reviewer classified it as an observational study, but after discussion it was agreed that it was a strategy article as its objective was to increase data sharing rates rather than observing data sharing patterns. the two reviewers independently read eligible documents and extracted data sharing incentives in health and medical research. both reviewers were agnostic regarding the types of incentives to look for. the final list of incentives was determined and agreed on by all authors [ ] . individual incentives were grouped into research fields. a qualitative description of each incentive was presented. based on our prior experience of the literature, the research fields and sub-fields for classification were: a. health and medical research i. psychology ii. genetics iii. other (health/medical) b. non-health and medical research i. information technology ii. ecology iii. astronomy iv. other (non-health/medical) the other article-strategies, opinion pieces, and observational studies were also grouped into the same research fields. the database searches found articles, of which met the inclusion criteria based on assessment of titles and abstracts and were exported into endnote. after automatically removing duplicates, articles remained and after manually removing the remainder of the duplicates, articles remained. titles and abstracts were read and categorised based on the above inclusion and exclusion criteria. one study was hand-picked as it met the inclusion criteria, bringing the total number of extracted articles to . after screening titles and abstracts, nine articles were classified under incentives in health and medical research. these articles were then read in full, and one of them was judged as an incentive that satisfied the inclusion criteria. the prisma [ ] flow chart that outlines the journey of the articles from identification to inclusion is in fig. . the categorisation of all articles into the sub-fields and article type is in table . a review of the reference list for the one included intervention was undertaken [ ] . the titles and abstracts of the full reference list of this study ( papers) and those that cited the study ( papers) were read, but none met the inclusion criteria of this systematic review. articles were irrelevant, bringing the total number of screened articles to . the distribution of articles across type of study was similar for both health and medical research and non-health and medical research ( table ) . observational studies were the most common type (n = , n = ), then opinion pieces (n = , n = ), then articles testing strategies (n = , n = ), and articles testing incentives were uncommon (n = , n = ). these articles did not fit the inclusion criteria, but based on the abstracts they were mostly concerned with observing data sharing patterns in the health and medical research community, using quantitative and qualitative methods. the motivation behind these studies was often to identify the barriers and benefits to data sharing in health and medical research. for instance, federer et al. ( ) conducted a survey to investigate the differences in experiences with and perceptions about sharing data, as well as barriers to sharing among clinical and basic science researchers [ ] . these articles also did not fit the inclusion criteria, but based on the abstracts they were opinion and editorial pieces that discussed the importance and benefits of data sharing and also outlined the lack of incentives for researchers to share data. open data and open material badges were created by the center of open science and were tested at the journal psychological science [ ] . [ ] . a limitation of the badge study was that it did not use a randomized parallel group design; notwithstanding, it was the only incentive that was tested in the health and medical research community, with pre-and postincentive empirical data [ ] . the pre-and post-design of the study makes it vulnerable to other policy changes over time, such as a change from a government funding agency like the recent statement on data sharing from the australian national health and medical research council [ ] . however, the kidwell et al. study addressed this concern with contemporary control journals. a limitation of the badge scheme was that even with badges, the accessibility, correctness, usability, and completeness of the shared data and materials was not %, which was attributable to gaps in specifications for earning badges. in late , the center for open science badges committee considered provisions for situations in which the data or materials for which a badge was issued somehow disappear from public view and how adherence to badge specifications can be improved by providing easy procedures for editors/journal staff to validate data and material availability before issuing a badge, and by providing community guidelines for validation and enforcement [ ] . of the non-health/medical incentives, seven were categorised as information technology, and nine as other. upon reading the full text, all the non-health/medical incentives were proposed incentives or strategies as opposed to tested incentives with comparative data. given that the systematic review found only one incentive, we classified the data sharing strategies tested in the health and medical research community. seventy-six articles were classified under 'strategies' and table shows the further classification into categories based on a secondary screening of titles and abstracts. the articles are grouped by whether they presented any data, descriptive, or empirical. the majority, / , of strategies were technological strategies such as the introduction of a data system to manage and store scientific data. seven of the strategies concerned encouraging collaboration among research bodies to increase data sharing. eight were a combination of collaboration across consortia and the introduction of a technological system. three had a data sharing policy as the strategy but did not test the effectiveness of the policy, but two of them reported descriptive data from their experience in implementing the policy. one strategy was an open data campaign. below we give some examples of the strategies used to promote data sharing. two articles discussed an incentive system for human genomic data and data from rare diseases, namely, microattribution and nanopublication-the linkage of data to their contributors. however, the articles only discussed the models and did not present empirical data [ , ] . another article discussed the openfmri project that aims to provide the neuroimaging community with a resource to support open sharing of fmri data [ ] . in , the openfmri database had full datasets from seven different laboratories and in october , the database had datasets openly available (https://openfmri.org/dataset/). the authors identified credit as a barrier towards sharing data and so incorporated attribution into the openfmri website where a dataset is linked to the publication and the list of investigators involved in collecting the data [ ] . an article discussed open source drug discovery and outlined its experience with two projects, the praziquantel (pzg) project and the open source malaria project [ ] . the article did not have pre-and post-strategy data. the authors discussed the constituent elements of an open research approach to drug discovery, such as the introduction of an electronic lab notebook that allows the deposition of all primary data as well as data management and coordination tools that enhances community input [ ] . the article describes the benefits and successes of the open projects and outlines how their uptake needs to be incentivised in the scientific community [ ] . an article discussed the development of the collaboratory for ms d (c-ms d), an integrated knowledge environment that unites structural biologists working in the area of mass spectrometric-based methods for the analysis of tertiary and quaternary macromolecular structures (ms d) [ ] . c-ms d is a web-portal designed to provide collaborators with a shared work environment that integrates data storage and management with data analysis tools [ ] . the goal is not only to provide a common data sharing and archiving system, but also to assist in the building of new collaborations and to spur the development of new tools and technologies [ ] . one article outlined the collaborative efforts of the global alzheimer's association interactive network (gaain) to consolidate the efforts of independent alzheimer's disease data repositories around the world with the goals of revealing more insights into the causes of alzheimer's disease, improving treatments, and designing preventative measures that delay the onset of physical symptoms [ ] . in , they had registered data repositories from around the world with over , subjects using gaain's search interfaces [ ] . the methodology employed by gaain to motivate participants to voluntarily join its federation is by providing incentives: data collected by its data partners are advertised, as well as the identity of the data partners, including their logos and url links, on each gaain search page [ ] . gaiin attributes its success in registering data repositories to date to these incentives which provide opportunities for groups to increase their public visibility while retaining control of their data, making the relationship between gaiin and its partners mutually beneficial [ ] . this study did not have pre-and post-strategy empirical data, but described the importance of incentives in motivating researchers to share their data with others [ ] . an article described how data sharing in computational neuroscience was fostered through a collaborative workshop that brought together experimental and theoretical neuroscientists, computer scientists, legal experts, and governmental observers [ ] . this workshop guided the development of new funding to support data sharing in computational neuroscience, and considered a conceptual framework that would direct the data sharing movement in computational neuroscience [ ] . the workshop also unveiled the impediments to data sharing and outlined the lack of an established mechanism to provide credit for data sharing as a concern [ ] . a recommendation was that dataset usage statistics and other user feedback be used as important measures of credit [ ] . one article addressed the need to facilitate a culture of responsible and effective sharing of cancer genome data through the establishment of the global alliance for genomic health (ga gh) in [ ] . the collaborative body unpacked the challenges with sharing cancer campaign ( ) [ ] genomic data as well as the potential solutions [ ] . the ga gh developed an ethical and legal framework for action with the successful fostering of an international 'coalition of the willing' to deliver a powerful, globally accessible clinic-genomic platform that supports datadriven advances for patients and societies [ ] . an article discussed the efforts of the wellcome trust sanger institute to develop and implement an institutewide data sharing policy [ ] . the article outlined that successful policy implementation depends on working out detailed requirements (guidance), devoting efforts and resources to alleviate disincentives (facilitation), instituting monitoring processes (oversight), and leadership [ ] . the topic of disincentives (facilitation) included concerns about lack of credit [ ] . they propose that cultural barriers to data sharing continue to exist and that it is important to align the reward system to ensure that scientists sharing data are acknowledged/cited and that data sharing is credited in research assessment exercises and grant career reviews [ ] . one intervention was an open data campaign which was included in the review via an open letter in june from the alltrials campaign to the director of the european medicines agency to remove barriers to accessing clinical trial data [ ] . the alltrials campaign is supported by more than , people and organisations worldwide [ ] . this letter contributed to the european medicines agency publishing the clinical reports underpinning market authorization requests for new drugs, which was part of a more proactive policy on transparency that applied to all centralized marketing authorisations submitted after january [ ] . the adoption of this policy was a significant step in ensuring transparency of health and medical research in europe [ ] . this systematic review verified that there are few evidence-based incentives for data sharing in health and medical research. the irony is that we live in an evidence-based world, which is built upon the availability of raw data, but we hardly have any evidence to demonstrate what will motivate researchers to share data. [ ] . it is interesting to note the great number of opinion pieces (n = ) on the importance of developing incentives for researchers, which outnumbered the number of articles that tested strategies to increase data sharing rates (n = ). 'opinion pieces' are mutually exclusive from 'strategies' as the former is concerned with discussing possible strategies and incentives and the latter tests the ideas and strategies and provides evidence of what works or does not work. these strategies included: the introduction of data systems such as electronic laboratory notebooks and databases for data deposition that incorporated a system of credit through data linkage; collaboration across consortia that also introduce data systems that also use data attribution as an incentive; collaboration across consortia through workshops and development of frameworks for data sharing; implementation of data sharing policies; and campaigns to promote data sharing. these strategies discussed the requirement of introducing rewards to increase data sharing rates and the only form of incentive used was via data attribution and advertising on websites. studies that test the effectiveness of attribution and advertising as a form of credit are necessary. in light of the small number of studies, we see a clear need for studies to design and test incentives that would motivate researchers to share data. organisations are promoting the development of incentives to reduce research waste. in late , the cochrane and the reward alliance combined to create the annual cochrane-reward prize for reducing waste in research. the monetary prize is awarded to 'any person or organisation that has tested and implemented strategies to reduce waste in one of the five stages of research production [question selection, study design, research conduct, publication, and reporting] in the area of health'. this prize is an example of an incentive for researchers to design studies or implement policies that reduce research waste; it will be interesting to see the impact of this initiative [ ] . another endeavour in the area of developing incentives and rewards for researchers is the convening in early of a group of leaders from the usa and europe from academia, government, journals, funders, and the press to help develop new models for academic promotion and professional incentives that would promote the highest quality science, organised by the meta-research innovation center at stanford (met-rics). the focus will be on designing practical actions that embody principles that this community has embraced, while also recognizing that the effect of any such policies will need empirical evaluation. while the systematic barriers to widespread data sharing are being addressed through the general shift towards more openness in research, the conversation on data sharing includes an alternative view where users of shared data are called 'research parasites' who 'steal from research productivity' and who are 'taking over' [ , ] . there is also some questioning of whether data sharing is worth the effort [ ] . these points, however, are contrary to the purpose of sharing data, which is to progress science as a body of knowledge and to make the research process more robust and verifiable [ , ] . a limitation of this systematic review is that we did not search the grey literature (materials and research produced by organizations outside of the traditional commercial or academic publishing and distribution channels). this review could be perceived as having a narrow design, given that we anticipated a lack of evidence-based incentives for data sharing in health and medical research, hence making the topic of this systematic review too simple. however, we could not be sure that there were no incentives and the recent paper by lund and colleagues ( ) emphasises the importance of conducting systematic reviews prior to designing interventions in order to avoid adding to the already large issue of research waste [ ] . the current meta-research discourse outlines the numerous benefits of openness in research: verification of research findings, progressing health and medicine, gaining new insights from re-analyses, reducing research waste, increasing research value, and promoting research transparency. however, this systematic review of the literature has uncovered a lack of evidencebased incentives for researchers to share data, which is ironic in an evidence-based world. the open data badge is the only tested incentive that motivated researchers to share data [ ] . this low-cost incentive could be adopted by journals and added to the reward system to promote reproducible and sharable research [ , ] . other incentives like attribution require empirical data. instead of evidence-based incentives, the literature is full of opinion pieces that emphasize the lack of incentives for researchers to share data, outweighing the number of strategies that aim to increase data sharing rates in health and medicine. observational studies that identify data sharing patterns and barriers are also plentiful, and whilst these studies can provide useful background knowledge, they do not provide good evidence of what can be done to increase data sharing. the open knowledge foundation: open data means better science meta-research: evaluation and improvement of research methods and practices perspectives on open science and scientific data sharing:an interdisciplinary workshop data sharing: make outbreak research open access reproducible research practices and transparency across the biomedical literature a systematic review of barriers to data sharing in public health avoidable waste in the production and reporting of research evidence the structure of scientific revolutions increasing value and reducing waste in biomedical research regulation and management open science: many good resolutions, very few incentives, yet. in: incentives and performance: governance of research organizations reproducing statistical results preferred reporting items for systematic reviews and meta-analyses: the prisma statement accessed badges to acknowledge open practices: a simple, low-cost, effective method for increasing transparency biomedical data sharing and reuse: attitudes and practices of clinical and scientific research staff accessed microattribution and nanopublication as means to incentivize the placement of human genome variation data into the public domain speeding up research with the semantic web towards open sharing of task-based fmri data: the openfmri project open source drug discovery -a limited tutorial the collaboratory for ms d: a new cyberinfrastructure for the structural elucidation of biological macromolecules and their assemblies using mass spectrometry-based approaches the global alzheimer's association interactive network data sharing for computational neuroscience facilitating a culture of responsible and effective sharing of cancer genome data developing and implementing an institute-wide data sharing policy open data campaign open letter: european medicines agency should remove barriers to access clinical trial data the cochrane-reward prize for reducing waste in research accessed data sharing data sharing -is the juice worth the squeeze? towards evidence based research assessing value in biomedical research: the pqrst of appraisal and reward northwestern university schizophrenia data and software tool (nusdast). frontiers in neuroinformatics schizconnect: a one-stop web-based resource for large-scale schizophrenia neuroimaging data integration coding rare diseases in health information systems: a tool for visualizing classifications and integrating phenotypic and genetic data universal syntax solutions for the integration, search, and exchange of phenotype and genotype information a web-portal for interactive data exploration, visualization, and hypothesis testing lord: a phenotype-genotype semantically integrated biomedical data tool to support rare disease diagnosis coding in health information systems proteomics fasta archive and reference resource using the wiki paradigm as crowd sourcing environment for bioinformatics protocols disgenet-rdf: harnessing the innovative power of the semantic web to explore the genetic basis of diseases brisk-research-oriented storage kit for biologyrelated data isa-tab-nano: a specification for sharing nanomaterial research data in spreadsheet-based format usage: a web-based approach towards the analysis of sage data. serial analysis of gene expression a universal open-source electronic laboratory notebook grin-global: an international project to develop a global plant genebank information management system padma database: pathogen associated drosophila microarray database all the world's a stage: facilitating discovery science and improved cancer care through the global alliance for genomics and health scens: a system for the mediated sharing of sensitive data the us-mexico border infectious disease surveillance project: establishing binational border surveillance medetect: domain entity annotation in biomedical references using linked open data open science cbs neuroimaging repository: sharing ultra-high-field mr images of the brain race by hearts: using technology to facilitate enjoyable and social workouts using a database/repository structure to facilitate multi-institution utilization management data sharing real-time data streaming for functionally improved ehealth solutions real-time clinical information exchange between ems and the emergency department integration of a mobile-integrated therapy with electronic health records: lessons learned loni mind: metadata in nifti for dwi application of foss g and open data to support polio eradication, vaccine delivery and ebola emergency response in west africa improving hiv surveillance data for public health action in washington, dc: a novel multiorganizational data-sharing method an i b -based, generalizable, open source, self-scaling chronic disease registry next generation cancer data discovery, access, and integration using prizms and nanopublications owling clinical data repositories with the ontology web language design and generation of linked clinical data cubes the current and potential role of satellite remote sensing in the campaign against malaria the ebi rdf platform: linked open data for the life sciences a digital repository with an extensible data model for biobanking and genomic analysis management catalyzer: a novel tool for integrating, managing and publishing heterogeneous bioscience data. concurrency computation pract experience a simple tool for neuroimaging data sharing e-health systems for management of mdr-tb in resource-poor environments: a decade of experience and recommendations for future work the cardiac atlas project-an imaging database for computational modeling and statistical atlases of the heart american college of rheumatology's rheumatology informatics system for effectiveness registry pilot the rheumatology informatics system for effectiveness (rise): enabling data access across disparate sites for quality improvement and research the preprocessed connectomes project: an open science repository of preprocessed data kimosys: a web-based repository of experimental data for kinetic models of biological systems the nih d print exchange: a public resource for bioscientific and biomedical d prints. d printing addit manuf implementation of chemotherapy treatment plans (ctp) in a large comprehensive cancer center (ccc): the key roles of infrastructure and data sharing implementing standards for the interoperability among healthcare providers in the public regionalized healthcare information system of the lombardy region rapid growth in use of personal health records software breakthrough makes data sharing easy. hospital peer review preventing, controlling, and sharing data of arsenicosis in china possibilities and implications of using the icf and other vocabulary standards in electronic health records the functional magnetic resonance imaging data center (fmridc): the challenges and rewards of large-scale databasing of neuroimaging studies one health surveillance -more than a buzz word? xnat central: open sourcing imaging research data flexible specification of data models for neuroscience databases health care provider quality improvement organization medicare data-sharing: a diabetes quality improvement initiative the global alzheimer's association interactive network (gaain) pediatric patients in the track tbi trial-testing common data elements in children rapid learning in practice: validation of an eu population-based prediction model in usa trial data for h&n cancer developing the foundation for syndromic surveillance and health information exchange for yolo county, california. online journal of public health informatics the national academies collection: reports funded by national institutes of health new models of open innovation to rejuvenate the biopharmaceutical ecosystem, a proposal by the acnp liaison committee the preclinical data forum network: a new ecnp initiative to improve data quality and robustness for (preclinical) neuroscience data sharing in neuroimaging research sharing data with physicians helps break down barriers. data strategies & benchmarks : the monthly advisory for health care executives sharing overdose data across state agencies to inform public health strategies: a case study act levels the playing field on healthcare performance the qut librarians assisted in designing the search strategy for this review. no monetary assistance was provided for this systematic review; however, support was provided in kind by the australian centre for health services innovation at the institute of health and biomedical innovation at qut. the datasets generated and analysed during the current study are available at the open science framework repository (doi . /osf.io/dspu ). authors' contributions arf collected and analysed all the data for the study and wrote the manuscript. ma collected the data and analysed ( %) for the study and edited the manuscript. agb provided close student mentorship for this research, which is a part of arf's phd under his primary supervision, and was a major contributor for the writing of this manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. not applicable.ethics approval and consent to participate not applicable. key: cord- -dezauioa authors: johnson, stephanie; parker, michael title: ethical challenges in pathogen sequencing: a systematic scoping review date: - - journal: wellcome open res doi: . /wellcomeopenres. . sha: doc_id: cord_uid: dezauioa background: going forward, the routine implementation of genomic surveillance activities and outbreak investigation is to be expected. we sought to systematically identify the emerging ethical challenges; and to systematically assess the gaps in ethical frameworks or thinking and identify where further work is needed to solve practical challenges. methods: we systematically searched indexed academic literature from pubmed, google scholar, and web of science from to april for peer-reviewed articles that substantively engaged in discussion of ethical issues in the use of pathogen genome sequencing technologies for diagnostic, surveillance and outbreak investigation. results: articles were identified; nine united states, five united kingdom, five the netherlands, three canada, two switzerland, one australia, two south africa, and one italy. eight articles were specifically about the use of sequencing in hiv. eleven were not specific to a particular disease. results were organized into four themes: tensions between public and private interests; difficulties with translation from research to clinical and public health practice; the importance of community trust and support; equity and global partnerships; and the importance of context. conclusion: while pathogen sequencing has the potential to be transformative for public health, there are a number of key ethical issues that must be addressed, particularly around the conditions of use for pathogen sequence data. ethical standards should be informed by public values, and further empirical work investigating stakeholders’ views are required. development in the field should also be under-pinned by a strong commitment to values of justice, in particular global health equity. genetic information derived from pathogens is an increasingly essential input for infectious disease control, public health and research . although routine sequencing of pathogens was, until recently, unthinkable, the centers for disease control (cdc), food and drug administration (fda), state, and global public health laboratories now routinely sequence more than foodborne bacterial isolates a day and more than , influenza virus genomes a year , . in the united kingdom, public health england now engages in routine clinical genomic diagnostics and drug sensitivity testing for mycobacterium tuberculosis . in the research setting, phylogenetic analysis (the study of evolutionary relationships among pathogens) is being used to track and understand factors associated with the spread of infections such as hiv and to monitor the global spread of drug-resistant infections . mobile genomic sequencing technology is also being applied to disease outbreak investigation, most publicly in the case of the ebola outbreak in west africa - . going forward, the routine implementation of genomic surveillance activities and outbreak investigation is to be expected. while the technical developments of sequencing technology are being implemented at a rapid pace, the non-technical aspects of implementing this technology are still being broadly discussed between the different stakeholders involved . the successful implementation of this rapidly developing technology will, for example, require sharing of samples and metadata, interdisciplinary global collaborative partnerships, and will need to offer useful evidence for public health decision-making. importantly, the successful and appropriate response to these challenges will also require the systematic identification, analysis and addressing of a number of complex ethical, legal and social issues. a number of factors will contribute to the types of ethical issues that arise in different instances. these are likely to include characteristics of the disease, the environmental, political and geographical context, existing laws and policies, public attitudes, and cultural differences . in the work reported in this paper, taking these and other ethical issues as our focus, we sought to systematically examine the available literature to: identify the emerging ethical challenges and proposed solutions; and to systematically assess the gaps in ethical frameworks or thinking and identify where further work is needed to solve practical challenges. scoping reviews seek to identify literature relevant to a research objective and may include a variety of research formats and conceptual literature [ ] [ ] [ ] . this study sought to review published literature on ethical aspects of pathogen sequencing. inclusion criteria for the study encompassed a broad range of article types, including empirical studies, news articles, opinion pieces, features, editorials, reports of practice, and theoretical articles. we systematically searched indexed academic literature from pubmed, google scholar, and web of science from to april for peer-reviewed articles that substantively engaged in discussion of ethical issues in the use of pathogen genome sequencing technologies for diagnostic, surveillance and outbreak investigation. the search was then updated in january . the initial search strategies were developed through an iterative process and used a combination of controlled vocabulary (mesh terms) and free text words. an example medline search strategy is provided in table . reference lists of included articles were searched for relevant articles and further database searchers were conducted using the names of researchers commonly publishing in this field. finally, we also reviewed relevant international research and clinical practice guidance for relevant guidelines e.g. website of the world health organization. we sought to maximize the literature included in the review by reviewing guidelines, frameworks, commentaries and original research reviews related to pathogen sequencing. we also included studies on molecular typing where enough accuracy could be included to include transmission tracking, as this was thought to provide useful insights into the ethical challenges pathogen sequencing technologies may pose. we excluded studies considering genomics outside of infectious disease or focusing on host response studies as these were not deemed relevant or specific enough to the topic under investigation. duplicates were removed. sj undertook title and abstract screening to remove obviously irrelevant studies, borderline cases were discussed with mp and a decision reached by consensus. data was then abstracted by sj and cross-checked for accuracy by mp. names of study authors, institutions, journals of publication and results were non-blinded. analysis sj initially inductively analysed all studies, recording the aims and main findings in microsoft excel ( . . . ), and developing descriptive codes to chart the broad themes describing the literature base. similar findings were then grouped according to topic area and a preliminary list of themes was developed in collaboration with mp. both authors engaged in iterative discussions about organization of findings, after which the final themes were decided. sj subsequently re-examined each study and extracted data using a standard format (design, results and recommendations). the search produced articles after duplicates were removed. all articles were initially screened by title and abstract and thirty-nine full text articles were assessed for eligibility. twenty eight articles were included in the final analysis; three ethical guidelines or frameworks - ; seven empirical research studies , - ; eight reviews of the ethics , - ; and ten publications that contained a section on the ethical aspects of pathogen sequencing - . the literature largely originated from the us and other high-income countries (hics, country determined by lead author institution): nine from the united states (us), five from the united kingdom (uk), five from the netherlands, three from canada, two from switzerland, one from australia, and one from italy. only two publications, by the same author, originated from the global south (south africa). eight articles were specifically about the use of sequencing in hiv , , , , , , , . eleven were not specific to a particular disease. table presents a summary of studies. results were organized into four themes: tensions between public and private interests; difficulties with translation from research to clinical and public health practice; the importance of community trust and support; equity and global partnerships; and the importance of context. when considering the implications of collecting, using and sharing pathogen genome sequence data, the interests and rights of individuals were universally acknowledged. in particular, the literature pointed to the importance of considering and/or protecting individual rights to autonomy , , , , and to privacy , , , . many pointed out that the potential of sequencing techniques to detect the origin and routes of transmission of an outbreak may result in negative consequences for the individuals involved . consequences may include stigmatization, penalties, economic risks, problems with interpersonal relationships (e.g. inadvertent disclosure of infidelity), emotional distress and the capacity for discrimination , , , , , , , , [ ] [ ] [ ] . there was also concern that sequencing could lead to serious legal consequences, particularly with regards to the criminalization of hiv transmission , , , , , , , . it was also acknowledged that individuals may have an interest in avoiding the use of information about them for purposes they do not endorse, such as to support anti-gay sentiment or as part of a criminal investigation , . somewere concerned about forced testing either of certain groups such as gay men or healthcare workers , , - . there was acknowledgment of individual professional interests of researchersand practitioners to ownership and use of data , , . sequencing was also seen to carry risks for communities and groups. many authors noted that certain groups can be placed at risk through characterization as high risk or likely to transmit virus (hiv), including geographically defined groups, sexual or gender minorities, or those defined by ethnicity, nationality, or migration status . similarly, data regarding transmission patterns of multidrug-resistant tuberculosis (tb) could be used for discrimination based on ethnicity, and possible challenges to immigration . institutions could be subject to increasing numbers of legal claims, or companies could suffer reputational or economic damage . it was also noted that some communities may be particularly at risk of being exploited by research, especially during emergency outbreak situations , . on the other hand, it was acknowledged that widespread availability and use of sequence data contributes important benefits to the clinical and research communities . in particular, the rapid sharing of data can help identify etiological factors, predict disease spread, evaluate existing and novel treatments, symptomatic care and preventive measures, and guide the deployment of there was strong support for the permissibility of conducting sequencing studies, as long as potential risks were thoughtfully mitigated , , , , . in one empirical study, patients and healthcare workers were asked if the benefits of hiv molecular epidemiology outweigh the risks; all said yes . threequarters of respondents answered with an unqualified, yes, and one quarter gave a positive answer with qualifications, such as 'it's very necessary, just as long as parameters are set in place and they're kept', or 'with proper protections in place, the benefits outweigh the risks' . in another study, expert delphi panelists held that the protection of the public was of overriding importance, but that most of the potential harms could be managed . there were differences across the literature in the priority afforded to different conditions of use, and to the types of risk or amount of risk deemed acceptable. it was broadly agreed that any research should have a favourable risk benefit ratio , , and that maximizing the utility of data must be weighed against concerns over interests of individuals and that policies on data collection and release should seek to align the interests of different parties . there was, however, disagreement as to whether privacy concerns or public interest should take precedence , , and some noted that the balance between the public health benefit and personal privacy risk for individuals whose genetic data (personal or pathogen) are included is difficult to delineate, since neither the true benefit nor the actual risk to participants has been adequately defined , . below we set out the key recommendations from the literature on the conditions of use for pathogen sequence data. box summarises recommendations from the literature for future research focus and study design. it was clear that the release of information of relevance to public health should not be delayed by publication timelines or concerns over academic ownership of data , , . recommendations to address such conflicts of interests included: that medical journals should update their policies to support pre-publication sharing of pathogen sequence data related to outbreaks ; publication disclaimers prohibiting use of sequence data for publication without permission , ; acknowledgment of data sharing contributions and the inclusion of such criteria to the assessment of academic research credit ; establishment of governance structures and dispute resolution mechanisms that can mediate where disagreements arise . much of the literature suggested that traditional methods of de-identification or anonymization of data are insufficient to meet their purpose in the context of pathogen sequencing , , , , , , . existing approaches to minimize the risk of privacy loss to participants are based on de-identification of data by removal of a predefined set of identifiers . however, this has three key limitations in the context of pathogen genomics. first, sharing of corresponding sample metadata (minimally time and place of collection, ideally with demographic, laboratory, and clinical data) is essential to enhance the interpretation and the value of genomic data , therefore removal of key identifiers such as geographic location may severely limit the utility of genomic data . second, removing predefined identifiers may be ineffective at protecting privacy and confidentiality , , , . for example, one study demonstrated how sample collection dates associated with microbiological testing at a large tertiary hospital were highly correlated with patient admission date (protected health information), meaning data is re-identifiable . small study populations may also mean that individuals who are part of a transmission chain may be able to identify others during the course of routine contact tracing (e.g. sexual partners) . third, anonymization of data does little to mitigate potential risk to communities and groups , , . perhaps a consequence there was clear support for re-visioning of existing privacy standards, and for privacy policies specific to the context of sequencing studies. there was debate in the literature around the importance of consent to the use of sequence data and associated meta-data in epidemiological investigation. in the research setting, coltart et al. state that research participants and patients whose samples are being used for phylogenetic analysis should ideally have consented to such use, but suggest that when using data from previous studies, where only broad consent for hiv-related research might have been obtained, waivers of specific consent are allowable when samples are no longer linked to identifiers, or when broad consent for sample collection for research and storage in future studies was given . in the public health and clinical setting, an australian study reported that one of the key differences amongst participants in a modified delphi social and behavioural research into conceptual and normative aspects should be backed up by empirical research . . new inter-disciplinary collaborations including microbiologists, engineers and bioethicists . . as real-time and other intervention strategies that build on hiv phylogenetic information continue to emerge, it will be critical to address questions of efficacy for cluster growth interventions to ensure that the benefits outweigh potential risks. implementation science research may also inform best practices for discussing the meaning and limitations of sequence data and cluster membership with community members and help to identify acceptable and evidence-based approaches that impose the least risk to persons within specific contexts. these might involve partnerships with providers for non-intrusive patient follow-up related to clusters, more detailed consent procedures for future follow-up related to hiv test results or partner services referrals, and specific guidelines and education to mitigate criminalization risks . . communication methods that increase the understanding of phylogenetic studies need to be designed and evaluated. these must emphasise potential harms, thoughtful mitigation of harms to risk groups, processes for monitoring risk, and clear protection procedures to minimise risks . . collaboration between stakeholders is necessary, with an active exchange of experiences and best practices. the first step, should be sought in creating awareness and consensus within sectors on the causal factors of barriers to sharing of sequence data . ethical conduct of studies . need to pre-define exceptional circumstances where un-validated techniques might be used in emergency situations . . to ensure scientific validity, researchers and their associates should be competent to implement the proposed study design. in order to maximize scientific validity, the researchers should ensure that they have all necessary resources, that the community accepts the protocol and that a competent and independent research ethics committee (rec) or institutional review board (irb) reviews and approves the protocol . . the scientific objectives of research should guide the choice of participants and determine the inclusion criteria and appropriate recruitment strategies. it is unethical to use privilege, convenience and/or vulnerability as criteria for selecting participants. exclusion of certain population sub-groups or communities in a research study without appropriate scientific justification is also considered unethical . . risk mitigation strategies must also provide for redress mechanisms in cases of abuse or misuse of phylogenetic data. these strategies might require the establishment of ties with local legal services, organisations working to protect people with hiv, and criminalised or stigmatised populations, to ensure that they have access to the means to protect their rights . study included the necessity for consent before testing and data-linkage. no panelists agreed with the statement "under no conditions should a study be conducted without prior consent", although only ten of thirty agreed that consent is not required under any conditions . in a dutch study, outbreak managers thought intervention without seeking explicit consent of all individuals involved is justified when there is at least be a substantial public health threat, realistic expectation that deploying the techniques will help to mitigate the outbreak, and that source and contact tracing would most likely not be successful without the use of molecular typing techniques . there was a strong commitment to rapid and open data-sharing, particularly in emergency or outbreak situations , , and in such conditions for incentives and safeguards to encourage rapid and unrestricted access to data release , , . the world health organization (who) recommended that in emergency outbreak situations "the first set of sequences providing crucial information on the pathogen, genotype, lineage, and strain(s) causing the outbreak should be generated and shared as rapidly as possible. sharing of corresponding anonymised sample metadata (minimally time and place of collection, ideally with demographic, laboratory, and clinical data) is essential to enhance the interpretation and the value of genomic data" . however, access to data gathered as part of clinical care was seen as ethically more contentious as "publicly accessible databases are not an appropriate storage location for the level of metadata required to enable clinical and epidemiological analysis for the purposes of providing patient and population care" . suggestions were made for a tiered approach to data release, whereby a separate database governed by appropriate public health authorities would collate and store metadata in a location to which access to data could be limited to users with a legitimate clinical or public health need to use it, and data that cannot be released into public domains but is needed by authorised healthcare and public health professionals for service delivery remains within a suitable secured access database . a public survey on tb explored questions related to database access and the potential benefits and risks associated with it. most felt that medical professionals and the research community should have access to such a database; and a significant proportion thought that other agencies, such as the police ( %) and immigration officials ( %), should also have access to the genomic database. experts, however, were clear that they felt transmission data should not be used in litigation; this was partially because it was deemed too unreliable , and also because of the potential for 'abuse' of data , , . overall, there was broad support for further work in defining the conditions for collection use and storage of data and samples , , , , and for policy and legal clarity to aid the ethical implementation of these technologies. this will require more work to carefully assess and understand risks ; research to decide how much individual privacy might be risk in the name of public health ; consideration of alternative strategies required to mitigate this risk, such as suppression of data in the public domain where it may cause serious harm; and adjustments to communication plans . effective phylogenetic work often occurs at the interface between research and public health practice because the same data can be used for both purposes . in this regard, pathogen sequencing was described as 'straddling the boundary between research and clinical use' . the hybrid nature of sequencing activities imposes important ethical challenges. clinical implementation of metagenomics sequencing (un-targeted testing) has the potential to detect unexpected or incidental findings that may include infections with hepatitis or hiv . incidental findings of a different type may occur if non-germline samples (such as faecal samples) are contaminated with germline cells, which could potentially reveal predictive information about developing inherited disease . furthermore, informed consent for phylogenetic studies that are difficult complex and difficult to understand, and in which the benefits and risk may not be fully determined, may also be difficult to achieve . mutenherwa suggests that where sequence data are generated for routine clinical management, its subsequent use for research and surveillance may be underestimated by patients . others suggested that the right to withdraw from research activities-a key indicator of voluntary participation in research-was overlooked by expert stakeholders . understanding and interpreting phylogenetic data requires significant expertise , and presents a challenge to established professional boundaries. expertise in phylogenetic studies creates new obligations for researchers, such as deciding whether or not to participate in forensic investigations and potential prosecutions of individuals , and to consider the down-stream uses and misuses of data . the routine implementation of pathogen sequencing studies may create new responsibilities for clinical microbiologists (related to public health) , and require major changes in culture such that diagnostic interpretation, therapeutic management decisions and antimicrobial treatment regimes are delegated to physicians instead of microbiologists . many noted that there are important reasons to ensure that the public and individuals understand the uses of data collected as part of a sequencing studies, and the potential risks. first, this was seen to have some intrinsic value in that it supports patient autonomy and truth telling is a respected moral virtue , , , . second, truth-telling was seen to be important because it may lead to better outcomes in research and public health practice. this is both because this was deemed to promote trust in research and therefore lead to increased participation, and because it promotes disclosure, which is helpful from a public health perspective. third, promoting understanding of uses and risks of data was also seen as a way of avoiding harm and exploitation of vulnerable individuals and communities, by enhancing understanding of risks that may be specific to that them. in some cases, this was balanced by a number of practical challenges to telling people the truth, such as: risk of fear mongering ; information needed for legal proceedings in public interest ; and the fact that it may be difficult to adequately inform the public and/or ensure full understanding . none-the-less, there was a clear recommendation in the literature to raise public awareness and understanding of these techniques , , , and for early and meaningful community engagement , , , prior to conducting sequencing studies. this was seen as particularly important when working with vulnerable groups or when the risks of participation are high. the notion of justice appeared to be a widely recognized ethical principle in the field . stakeholders pointed to the importance of equitable access to data , and to benefit-sharing obligations , . this included an ethical imperative that outbreak related research and countermeasures, such as diagnostics and vaccines, should be accessible to all affected countries , and towards reciprocal arrangements such that countries that participate in sequencing activities should derive some corresponding local benefit . collaboration between researchers from africa and hics was raised as an important ethical consideration . in one empirical study, interviewees were concerned that african researchers were not meaningfully engaged in the scientific research process in health research in general and phylogenetic research in particular, and that for equitable and mutually beneficial collaborative research partnerships to be realized, local researchers were encouraged to take leading and active roles throughout the research process . this type of collaborative research practice was supported elsewhere in the literature , , . for some, this was to enhance equity as well as to help maximize the utility of data and lead to better public health outcomes. it was argued that local researchers were more likely to understand their health care and research systems and study results were more likely to be easily translated into policy , and that context specific responses to particular outbreaks were likely to be required. recommendations were made to conduct studies exploring the nature of existing collaborative partnerships between researchers from low-and middle-income countries (lmics) and hics to explore team composition and distribution of roles, including contribution to intellectual property . outbreak related research and countermeasures, such as diagnostics and vaccines, must be accessible to all affected countries not only as a legal obligation, but also as an ethical imperative . it was also noted that global and interdisciplinary partnerships are a necessary component of an effective genomic informed response to infectious disease. this was because of the vast range of stakeholders and varying interests involved in control of infectious disease outbreaks, and because issues may resist simple resolution and span multiple jurisdictions . for example, conflict may result from governments wishing to keep an outbreak quiet and/or from the tension between lmics with few resources for generating and using data and the researchers or response teams from better-resourced settings , . ownership of samples and data was seen as an important barrier to global cooperation. the nagoya protocol (np), for example, was developed to facilitate access to genetic resources and the fair and equitable sharing of benefits arising from their utilization . nevertheless, despite the importance of reinforcing sovereignty rights of states over genetic resources in their territory, uncertainties about intellectual property rights and the resulting disputes hamper access to samples , . ribeiro et al. explain that: "the real or perceived possibilities for the commercial valorization of microbial genetic resources (mgr) has enforced their appropriation for further use in research, innovation and product development. the problem for public health surveillance occurs when such appropriation is triggered at initial (upstream) phases of the research and innovation cycle, such as sampling and sequencing of microorganisms, instead of later stages, such as the actual product development (in this case drugs, diagnostics and vaccines). as such, stakeholders are reluctant to share their (intangible) assets even in early phases of the innovation process, decreasing the scope of innovation efforts due to the lack of access to upstream research inputs." the same authors suggest that standardized and simplified sharing agreements , and collaboration between stakeholders with an active exchange of experiences and best practices are required. in general, recommendations were made for a global approach to ethics, policy and legal frameworks , , , , . for example, it was suggested global data sharing arrangements should include "a global data governance or ethical framework, supplemented by local memoranda of understanding that take into account the local context" ; or to investigate how the global alliance for genomics and health (ga gh) framework for responsible data-sharing could be adapted for digital pathogen surveillance . lastly, it was clear that the types of ethical issues likely to arise are in part dependent upon the contexts in which studies are conducted, as well as the nature of the pathogen under study. chiefly, information that may impact on interpersonal relationships was viewed as particularly sensitive and therefore, worthy of additional ethical reflection. examples included: sexually transmitted infections , ; consent requirements to use isolates collected from dead neonates for the purposes of epidemiological research ; and disclosure of family members as the source of infection . it was suggested that the balance of risks to patients and public health benefits is likely to be affected by the characteristics of the pathogen, in terms of likely morbidity and mortality: infectivity; treatability and drug resistance . stakeholders also suggested that the ethical permissibility of sharing data about, particularly with regards to the source of transmission, may be different in professional contexts, where healthcare providers or companies are seen to carry a responsibility to control risk, as opposed to outside of professional contexts where protecting individuals from 'naming and shaming' may be of greater concern . it was also noted that the legal and regulatory structures in which studies are conducted may also influence the implementation and ethics of conducting pathogen sequencing studies. in particular, use of phylogenetic analyses in criminal convictions was raised as an ethical risk , , . although quality assessment of all included materials is desirable in systematic reviews, it was not possible in this case due to the inclusion of a diverse range of research formats and literature, such as commentaries and ethics guidelines. a second limitation of this review is that a large proportion of the literature included related to phylogenetic and hiv specifically ( out of ), meaning that the issues relevant to this context may be over-represented. this review highlights that while pathogen sequencing has the potential to be transformative for public health and clinical practice and to bring about important health benefits, there are a number of key ethical issues that must be addressed. in particular, there was clear support in the literature for innovative and critical thinking around the conditions of use for pathogen sequence data. this includes context specific standards of practice for consent, data collection, use and sharing. these practices should be informed by public values, and further empirical work investigating stakeholders' views are required. this should include experts in pathogen sequencing, patients and the general public, as well as end users such as public health professionals and clinicians. lastly, it is both a scientific and an ethical imperative that development in the field is under-pinned by a strong commitment to values of justice, in particular global health equity. all data underlying the results are available as part of the article and no additional source data are required. this manuscript presents the results of a scoping review of the literature on the topic of the ethical issues in the use of pathogen genome sequencing technologies. the authors make excellent use of the scoping review methodology which they implemented clearly (good use of tables!) and rigorously. there are minor formatting issues and typos: p. under theme : somewere concerned about forced testing either of certain groups such as gay men or healthcare workers , , - . there was acknowledgment of individual professional interests of researchersand practitioners to ownership and use of data. p. under theme : 'that may be specific to that them' the main substantial issue is the substantial focus of the manuscript on the hiv context ( articles out of ) which the authors acknowledge. hiv is a particularly stigmatized, serious condition, with lifelong consequences for patients which is not the case for many other communicable diseases. the problem is that in the past few months, covid- has been a game changer in the field. covid- is both much more contagious but less stigmatizing and, for most people, less dangerous than hiv. considering that the last update to this scoping review was made in january , none of the covid- emerging literature was considered. the result, which is not the fault of the authors, is that the article will only have a limited relevance to the current global pandemic. given the importance of covid to the field and beyond, it could be worth it for the author to take the time to update their research accounting for very recent developments before publishing. otherwise, the publication maybe perceived as already outdated and not garner much attention from readers. a second issue of the manuscript is that while it acknowledges the tension and blurry demarcation between the research and the public health context, it doesn't really provide any solution in this regard. for example, in the context of pathogen genome sequencing for outbreak surveillance during a public health emergency, informed consent is often not required. however, the lack of consent can create issue later for data sharing with the research community. such a scenario is not really discussed in the manuscript. similarly, the impact of the public health vs. research situation on the potential requirement for ethics review is not discussed. perhaps this was not touched upon in the literature, but it is certainly a preoccupation of researchers in the covid context. is the study design appropriate and is the work technically sound? yes are all the source data underlying the results available to ensure full reproducibility? yes are the conclusions drawn adequately supported by the results? yes in the paper in the review appear to be appropriate and informative. some additional concepts that weren't included, possibly because the article presents a review of already published ideas, are the following: the potential for civil legal consequences from publication of genomic data. the article discusses at a few different points the potential for criminal prosecution, but publication of data could also open up participants of a study to civil penalties as well. how ownership rights over microorganisms affect infectious disease control and innovation: a rootcause analysis of barriers to data sharing as experienced by key stakeholders pubmed abstract | publisher full text | free full text societal implications of the internet of pathogens reviewer expertise: ethical, legal and social issues of genomic research, medical law, bioethics. reviewer report july https://doi.org/ . /wellcomeopenres. .r © armstrong g. this is an open access peer review report distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. office of advanced molecular detection, national center for emerging and zoonotic infectious diseases, centers for disease control and prevention, atlanta, ga, usa the article is generally well written, although it would be stronger if it had a more concise, more focused set of recommendations for readers to act on. the focus of the paper is mostly on the use of microbial genomic data for research but also touches a little on the issues around public health use of the data. this reviewer is not an expert in ethics; that said, the ethical concepts brought out ○ the potential for violation of a privacy when cases are rare. for example, early in an outbreak, as is highlighted in the article, there is an urgency to making sequence data from the pathogen in question public. however, it's common early in an outbreak for the media to discover and publish the names of early cases. in that situation (which is relatively common), there's a risk that sequence data made public could be linked to a specific person. ○ one concern that is often under-appreciated is that, under certain circumstances, a researcher or public health agency might be legally compelled to release the identity of someone. in general, public health laws are quite strong in shielding public health data, but that may not be the case with research data. if there is uncertainty about whether such data are protected, any participants in research should be notified. ○ gh ge is mentioned in the manuscript, but there's no mention of pha ge ( https://pha ge.github.io/), which is much more applicable here. ○ issues around the nagoya protocol are addressed, but the tension between gisaid and insdc is not. gisaid provides protections for intellectual property rights that the insdc members do not, and such protections are key for participation of lmics. however, where public funds are used to obtain data, there is a strong argument that the data should be made publicly available (i.e., through insdc) without restriction as long as privacy and confidentiality are not placed at risk. the tension between the two models has been particularly strong with the advent of covid- , and the very assertive push by gisaid to prevent researchers from submitting to insdc or from citing it. ○ some (minor) specific issues that need to be addressed: i would remove the reference to microsoft excel. there's a strong argument for including information about statistical software, but which spreadsheet is used is not particularly relevant.○ "theme : …": there are typos in the first paragraph.○ box , item : this is not a complete, declarative sentence like the others.○ reference appears to be the incorrect reference. also, the quote from it (pages - ) appears to be a quote from a separate document (that should be cited separately, if still available). is the study design appropriate and is the work technically sound? if applicable, is the statistical analysis and its interpretation appropriate? are all the source data underlying the results available to ensure full reproducibility?no source data required competing interests: no competing interests were disclosed.reviewer expertise: pathogen genomics to support public health.i confirm that i have read this submission and believe that i have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. key: cord- -a jc g i authors: shrivastava, paul; stafford smith, mark; o’brien, karen; zsolnai, laszlo title: transforming sustainability science to generate positive social and environmental change globally date: - - journal: one earth doi: . /j.oneear. . . sha: doc_id: cord_uid: a jc g i despite the decades-long efforts of sustainability science and related policy and action programs, humanity has not gotten closer to global sustainability. with its focus on the natural sciences, sustainability science is not able to contribute sufficiently to the global transition to sustainability. this perspective argues for transforming sustainability science into a transdisciplinary enterprise that can generate positive social and environmental change globally. in such transformation, the social sciences, humanities, and the arts can play an important role to address the complex problems of culture, institutions, and human behavior. to realize a truly integrated sustainability science, we need renewed research and public policies that reshape the research ecosystem of universities, funding agencies, science communications, policymaking, and decision making. sustainability science must also engage with society and creatively employ all available sources of knowledge in favor of creating a sustainable earth. global environmental challenges are now manifesting at a planetary scale in the form of climate change, ocean acidification, biodiversity loss, environmental pollution, deforestation, and land-use change, among many others. these characteristics of the anthropocene era have been driven by accelerating socioeconomic trends, including population growth, various measures of consumption and economic growth, urbanization, telecommunications, and other aspects of globalization. the so-called great acceleration, which started in the s, has been compounded by the rapid development and spread of information technologies and telecommunications. decades of international conferences and reports-beginning with the report the limits to growth by the club of rome and the united nations (un) conference on the human development in that same year and evolving to the creation of the sustainable development goals (sdgs) by the un general assembly in -have warned of the challenges and risks associated with current development pathways and have fostered the evolution of sustainability science at the global scale ( figure ). science has played a pivotal role in helping society to identify and understand the scope and scale of planetary environmental changes. it has also helped to guide policies and practices that address sustainability challenges from local to global scales. in recent years, global-scale challenges have become a growing concern. the s saw the development of a number of global-change research programs and various environmental science initiatives, such as the world climate research program, the international geosphere biosphere program, diversitas, the international human dimensions program, and some attempted unification in the earth systems science program. sustainability science emerged from the recognition of a need to extend global-change research to seek solutions, which in turn requires multidisciplinary and interdisciplinary research. this is slowly moving further from the perceived hegemony of the disciplinary expert toward the transdisciplinary co-design and co-production of research. international science coordination and funding approaches have also evolved over the past decades, as promoted by the international science council, the belmont forum, and others. many of the global-change programs merged into future earth in . , in , a unified coordination of the natural and social sciences was advanced by the formation of the international science council through the merger of the international science union and the international social sciences council. this year, the transdisciplinary sustainability research & innovation congress was planned to unite global researchers, industry practitioners, and world leaders to inspire action and promote sustainability transformation. although the event is now postponed because of the coronavirus disease pandemic, its aim of cultivating a space for sustainability scholarship, innovation, collaboration, and action won't be delayed. global-change research reflects a growing demand for research to become more impactful and solution oriented, as spelled out in the future earth vision, which is aimed at a multiscale (from local to planetary) understanding of environmental changes that transcend national boundaries. over the past decade, global-change research has made significant efforts to be more inclusive of science communities from china, russia, africa, and the global south. global-change research has first and foremost promoted a systems approach to environmental problems. this approach has focused on relationships and interactions among the atmosphere, biosphere, hydrosphere, cryosphere, and soils and explores how they are influenced by (and influence) human activities. an understanding of human impacts on earth system processes gave rise to the notion of planetary boundaries, the idea of tipping points, , and the concept of the anthropocene. these scientific metaphors have been useful in articulating the risks of continuing with business as usual and development as usual. it is fair to say that global sustainability science has contributed significantly to a number of global agreements to address the challenges of the anthropocene, most notably the paris agreement and the un agenda , which were signed in . these universal agreements reflect a global consensus to address climate change and strive for sustainable and balanced social and economic development that promotes the well-being of socio-ecological systems. they also provide a powerful statement of intended direction for humanity, as captured in the un's sdgs. they create an imperative for transformative change in response to global challenges. however, such changes have not been forthcoming at the rate and scale required by global agreements. as a consequence, we continue to see increasing concentrations of co in the atmosphere, biodiversity loss, land degradation, and growing inequalities among people and nations. , the great acceleration is generated and sustained by powerful economic mechanisms, including globalization, marketization, and financialization, which are based on the mainstream model of doing business and promoting economic growth. to foster growth and maximize profit, companies and other organizations aggressively appropriate value as much as possible from nature and society and externalize the costs to natural ecosystems and to human communities, including future generations. , the mainstream business model drives competition and consumption and guarantees that human and social activities continue to drive the overuse of ecosystems beyond their carrying capacity toward breaching several planetary boundaries. the current economic paradigm has led us to the point where the functioning of our global life-support system is no longer assured. the global economy and functioning earth system are on a collision course that will ultimately have dire consequences for humans and the biosphere; we need an urgent course correction, and current research paradigms are not meeting this challenge. in this perspective, we argue that in order to generate positive social and environmental changes globally, sustainability science must transform into a transdisciplinary enterprise. our modest goal here is to lay out the special research challenges posed by the emerging anthropocene era and exhort sustainability science to address these challenges impactfully. we focus our suggestions primarily on research policy measures that shape the conduct of science and agency of scientists and only secondarily on institutional policy measures. this is not intended to be a broad public-policy discussion on sustainability research. sustainability science needs deep integration of the social sciences, humanities, and the arts with its dominant natural sciences. the focus should be on complex problems of nature, culture, institutions, and human behavior and the co-creation of knowledge with stakeholders. to do this, sustainability science and its stakeholders will need to better understand the funding dynamics and higher levels of research funding. to make sustainability science impactful, we need to modify the current research ecosystems. sustainability science must engage with society (economic, social, and cultural spheres) to extend many successful small-scale seeds for change. the goal of sustainability science is to create a sustainable earth by creatively deploying all available knowledge of humanity. the evolution of what is now known as sustainability science has been important, but clearly it is not yet enough to play a pivotal role in social transformations needed for human preservation in the face of accelerating changes of the anthropocene. we argue that we need to transform our approach to research itself. a number of known attributes of global change currently create not only are these changes fast, but in most instances they are also increasing non-linearly, as described by the great acceleration. the human capacity to respond to these changes has not kept pace. some of the historical drivers of social change, such as generational turnover, have slowed as a result of increasing longevity. consequently, cultural and value changes that influence how humans relate to the environment have to occur within a generation rather than over generations. researchers themselves are challenged to think differently and engage in interand transdisciplinary research processes. however, it takes time to develop common understanding between disciplines and engage across different stakeholder knowledge epistemologies; therefore, integrative research risks lagging behind the changes it is addressing (table ) . spatial connectivity sustainability policies and practices must be local and context specific, yet they are embedded in larger systems that are increasingly and more rapidly connected. although developments in inter-and transdisciplinary research over the past few decades have delivered much success at local levels in agriculture, development, and other domains, replicating this success across scales and globally is genuinely challenging. transformations of social, economic, and institutional systems must also be negotiated across scales. sustainability science research has not reconciled the gap between global-scale problems and locally based solutions. we still do not know how to scale up solutions in an interconnected world (table ) . social-ecological systems have always been affected by social and cultural norms, vested interests, and power dynamics. however, in today's globalized system, many social norms have been shaped by market-based capitalism and consumption, and the rates and distributed extent of change mean that managing the dynamics of winners and losers is increasingly challenging. for example, oil profits in one part of the world could finance investments that undermine sustainability interventions in another. at a time when there is a need to build engagement across all levels of society, identifying and transforming the nature of power relationships can be difficult. sustainability science has not fully acknowledged or integrated global power dynamics into understandings of change (table ) . selective embeddedness of science in society science is extremely specialized and operates within universities and corporations, increasingly fulfilling the mandates of government and corporate funders. science does not reach wide swathes of global society, particularly the communities where challenges of the anthropocene are concentrated. many sectors, especially the informal sector of the economy, are not benefiting from scientific knowledge. the informal sector accounts for up to % of global output in developed countries and over % in developing countries. according to the international labor organization, two billion women and men make their living in the informal economy, which provides over % of non-agricultural employment in africa, latin america, and asia. in africa, the informal economy could account for %- % of the gross domestic product (gdp) and % of new jobs. even in sectors where science does influence economic and social decisions, it does not always effectively serve broad public interests. science funding for the public good and commons protection is insufficient to address the massive and expanding problems of the anthropocene (table ) . above all, the challenges involve culture, society, behavior, and institutions. yet, the current approach to sustainability science still pays insufficient attention to the social and human dimensions, and holistic integrated research remains the exception rather than the norm. sustainability science is still dominated by the natural sciences, and there is grossly less investment in the social sciences, the humanities, and arts than in the ''hard sciences,'' as well as a general underinvestment in research overall. the uptake of research insights from the social sciences and the arts and humanities has been limited to the findings that fit within the existing paradigm. particularly debilitating is the failure to open up to new perspectives and different types of knowledge in the name of keeping science ''objective'' and apolitical. even where efforts have been made to integrate social, human, and natural sciences in transdisciplinary research, these successes are challenged by scale. in a recent review, norströ m et al. identified four key principles for good transdisciplinary co-production of knowledge: the engagement by research should be context based, pluralistic, goal oriented, and interactive. these principles, which draw on a great body of understanding from past transdisciplinary successes, are all challenged by the attributes of the anthropocene. engagement must be context specific, yet it seems to be required at all levels, including globally. it must be pluralistic, yet the diversity of stakeholders even at the local level is immense, and more so for global processes. goal-oriented science is challenging with diverse actors across scales, and making engagement interactive, with feedback loops that promote reflection and learning, requires new thinking at the global level. systems thinking emphasizes that the most powerful leverage points for system change come through modifying the design and intent of the system. paradigms-or the shared thought patterns that influence the way that systems are perceived and designed-are also recognized as a powerful driver of systems change. understanding how to achieve paradigm shifts that influence the goals, values, and behaviors of systems is not the normal purview of the natural sciences. in fact, alternative paradigms are usually dismissed rather than encouraged within research systems. although global assessment initiatives such as the intergovernmental science-policy platform on biodiversity and ecosystem services (ipbes) recognize and include cultural beliefs, values, worldviews, and indigenous and local knowledge, many perspectives from the social sciences and humanities have not been taken seriously in sustainability science. for example, research within feminist political ecology, decolonization studies, and ''new materialism'' offers new thinking on ethical and political challenges. , to capture the breadth and depth of global challenges, sustainability science needs to support efforts to recognize multiple perspectives and ways of knowing. this includes developing profoundly holistic views on how natural and cultural systems coevolve. interdisciplinary and transdisciplinary research activities in academic institutions are seeking such integration. there have been numerous attempts to encourage transdisciplinary collaboration and team science. in higher-income countries, there is a tradition of collaborative cross-sectoral research, such as the agricultural extension research in the us and that conducted by the cooperative research centres (crcs) in australia. recently, the us national science foundation has encouraged research collaborations across science fields, which it calls ''convergence science,'' by awarding grants for themes such as ''navigating the new arctic.'' global programs such as future earth are developing ''knowledge-action networks'' to promote transdisciplinary research. however, the societal ecosystems in which such initiatives operate do not embrace their logic or provide resources to scale them globally as necessary. current efforts to support integrated and transdisciplinary research do not match the scale of the problem. maintaining the power of conventional, objective science has come at a cost for sustainability science. not only has failure to integrate important insights from the social sciences and environmental humanities limited the perceived ''solution space'' for responding to global challenges, but sustainability science has also failed to engage with the ''how'' of transformative change. it has had limited success in connecting with key stakeholders (policymakers, corporate decision makers, political leaders, and civil society) through a language that they understand. it has also failed to convey not only the sense of urgency underscored by global changes but also the possibilities for responding. currently, research on global environmental change largely speaks to the converted-to those who are influenced by rational arguments and scientific evidence. very little attention has been paid to alternatives that challenge the tenets of conventional science and business as usual. the normative dimension of sustainability science is one area that is often challenging to researchers. the question of which changes are ethical and the ''right ones'' to pursue introduces a normative dimension to science that can also be seen as highly political. however, this is an area where global agreements have provided direction for sustainability in recent years-the sdgs of agenda clearly articulate normative visions for global sustainability in alignment with providing food, water, and energy to the approximately ten billion people expected to live on earth by , just as the paris agreement clearly states an intent to stay below c warming, and . c if possible. although they provide a strong statement of goals and intents, both still contain ambiguities around more detailed interpretations relating to different places, levels of governance, and interests, which together can limit their impact for sustainability. to integrate the diversity of interpretations of particular goals, the research system has to open itself up to a wide range of disciplines and epistemologies. many examples of existing research can contribute to a more integrative discourse within sustainability science. however, it is important to consider these different perspectives together rather than as separate boxes or silos. drawing on an integral framework, figure depicts how sustainability science has been largely confined to the domain of ''systems'' science and how less attention has been paid to behavior, culture, and experience. an integral approach to sustainability science includes a more balanced representation of these other three perspectives. below, we highlight some specific examples of research in these other ''quadrants'' to illustrate the potential to broaden the systems-oriented perspective of sustainability science and deepen our understanding of how to respond to environmental change. behavioral change there is still a tendency within sustainability science to draw on the information-deficit model and assume that once individuals ll open access and organizations realize the risks associated with environmental change, they will take action or at least can be manipulated or nudged in that direction. yet, plenty of research in psychology-including research on the relationship between the intrinsic and extrinsic motivation of human actors, i.e., between motivation that is inherent in the task itself and motivation that relies on external rewards or sanctions -shows that human behavior is more complex than this. the latter can result in the crowding-out effect, where external rewards actually reduce the intrinsic motivation of the actors. a crucial result of this mechanism is that people's environmental commitment declines and more environmentally harmful behavior emerges. , a key message here is that intrinsic motivation is critical to sustainable behavior within rapidly changing systems. research also describes how mechanisms of moral disengagement enable otherwise considerate people to commit transgressive acts without experiencing personal distress or guilt. in other words, moral control can be selectively disengaged from detrimental conduct through psycho-social mechanisms. selectively activated disengagement strategies (such as moral justification, euphemistic labeling, advantageous comparison, displacement of responsibility, diffusion of responsibility, disregard for or distortion of the consequences, dehumanization, and attribution of blame) are at work in harmful environmental practices of corporations and other economic entities. this points to the need for a more critical engagement with the social structures and systems in which sustainability measures are enacted, as emphasized in social practice theory. cultural change embedding sustainability sciences and practices at a global scale will require a new and evolutionary narrative about hu- an integral framework recognizes four perspectives or domains of reality that present different types of knowledge about a subject, in this case sustainability science. global-change research has tended to prioritize the systems domain and put less emphasis on research from the meaning-making, cultural, and behavioral domains. integration does not diminish the systems perspective but rather adds valuable knowledge that can help society meet the sdgs. adapted from wilber. man-environment relationships. research on the power of narratives (language, metaphors, and storytelling) points to the need for new cultural narratives on the journey of humans in the universe across geological time. , the current narrative that humans are a special, privileged species destined to dominate over nature is deeply flawed, and it has contributed to a fascination with techno-fixes, such as geoengineering. cultural changes need to embrace research on the power of the arts, aesthetic inquiry, and the humanities to heal the emotional disconnection between humans and nature, which is at the root of our current environmental crisis. the role of artists and art-based practices in achieving sustainability is increasingly being recognized. dutton refers to the art instinct of humans as having survival value across cultures and time. art is ubiquitous in all human communities globally over most of history. communities able to tell better stories and draw better pictures of natural and predatory hazards were able to avoid annihilation more effectively. the rich human legacy of the arts also allows us to connect cognitive understanding to emotional empathy and embodied learning about sustainability. the experiential dimensions of environmental issues are becoming increasingly pronounced through emotions such as hope, grief, anger, and despair. whether and how we identify and respond to environmental problems are also closely tied to the ways that we perceive the world and our place in it. issues such as climate change will be interpreted differently depending on the beliefs, values, and worldviews held by individual or groups. within psychology, meaning making is defined as ''the process of how people construe, understand, or make sense of life events and experiences.'' research in developmental psychology shows that multiple worldviews coexist today and that these can change over generations and within individual lifetimes. acknowledging both differences and developments in perspective-taking capacities and meaning making can provide insights into why people relate to sustainability issues differently and how to connect with a diversity of worldviews. for example, in a study of meaning making among farmers in el salvador as it related to climate-change adaptation, hochachka found differences in the object of awareness, the number of perspectives taken, the complexity of thought, and the scope of time considered in one's understanding of climate change and adaptation. such findings suggest that it could be time for sustainability science to engage with the diversity and dynamics of meaning making rather than to assume that sense making of global challenges is universal and static. in the three research areas described above, one can already see the ways in which researchers are synthesizing knowledge within and across the ''quadrants'' to study the dynamics of sustainability at a deeper level. for example, understanding behavioral changes involves grasping the processes of motivational crowding, understanding socio-cultural changes involves studying the narratives that underpin social values, and understanding experiential responses to environmental change involves indepth research into meaning making. yet, questions remain on how to forge this integrative research more broadly within sustainability science. the integration of different perspectives and domains of knowledge and methodological approaches to research can be enhanced through awareness and education. yet, disciplinary science has privileged cognitive modes of learning, and as disciplines have fragmented, so have the content domains of learning. in highly scientized fields such as medicine, expertise is being isolated and narrowed into increasingly restricted specializations. education for global-change problems needs to be more holistic and contextually anchored through the incorporation of systems and their interconnectedness with culture and experience. education that embraces an integrative discourse recognizes multiple perspectives and the need for critical thinking, reflection, and experience-based learning that leads to action and a sense of agency. one way of integrating different perspectives is through ''action research,'' which focuses on driving change and doing rather than just reaching understanding. otto scharmer and colleagues at the presencing institute created an actionresearch platform at the intersection of science, consciousness, and social change. their ''theory u'' framework and methodology for leading profound innovation proposes that the quality of the results that people create is a function of the quality of awareness, or consciousness, from which the participants in the system operate. the inner shift, from fighting the old to sensing and presencing an emerging future, is at the core of deep leadership work. people are encouraged to expand their thinking from the head to the heart. it is a shift from an ego-system awareness to an eco-system awareness that cares about the well-being of all, including oneself. the presencing institute has reached over , people in countries to help leaders at any level find new solutions to the disruption that humanity faces. there are a variety of approaches to linking research epistemologies with experiential knowledge systems. the crcs in australia have been a generally successful approach to making public funding contingent on a close working relationship between researchers and stakeholders, the latter of which provide funding input, in a vast array of different topics (https://www. business.gov.au/grants-and-programs/cooperative-research-centres-crc-grants). crc structures increasingly require a formal, even dominant, role for stakeholders in governance; even so, the growing experience suggests that it takes up to years for the diverse partners to really work out a common mission and deliver on the promise of transdisciplinarity to get novel research into action. another promising approach is through greater investment in coordinated, applied, local innovation hubs or sustainable development labs. at community colleges and universities in the us, ''living labs'' are using university operational facilities to create engaged action learning opportunities for students and engaged scholarship opportunities for researchers. examples of integrated and holistic knowledge-creation and action initiatives that embrace more diverse epistemologies are also emerging from social and cultural institutions, as exemplified by the encyclical letter laudato si' by pope francis. the encyclical presents an integral ecology that underscores the human origins of the ecological crisis and proposes fundamental changes in organizing economic life. among the important suggestions put forward by the pope is increased frugality in consumption, and he also acknowledges the intrinsic value of nature. to realize his vision, pope francis initiated the economy of francesco project. this international cooperative platform brings together , young economists, entrepreneurs, and change makers to co-create and put in place a new economic model in the spirit of integral human development. notwithstanding the examples of integrative approaches described above, sustainability science has not been transformed enough to successfully address the mounting grand challenges faced by society. the challenge of evolving sustainability science cannot be just endogenous and isolated. science is an instrument of society, and it operates within a broader socio-economic, cultural, and technological milieu that sets the operating context for research. at present, the external context is characterized by neoliberal capitalism that sets up competition rather than collaboration; devalues or overlooks social, human, and environmental capitals; fails to see systemic effects; and encourages inequality. science needs to rethink its social relevance and social connections to effectively contribute to the grand challenges of the anthropocene. it must play an active role in the concurrent co-evolution of economies and their underlying cultural assumptions in sustainable directions. within that broader context, scientific institutions and business organizations supporting science would need transformation of their core purpose, administrative processes, and reward systems. sciences would need deep unification across epistemic and disciplinary lines and be willing to play a more active, participatory role in the transformations required for global sustainability in the world at large. at a more synoptic level (see figure ), waddell et al. have argued that research needs to support the establishment of a ''transformation system'' in parallel with the system being transformed (see https://transformationsforum.net/). they outline four strategies that can contribute to transformation: doing change, forcing change, directing change, and co-creating change. according to these authors, most transformations involve a mix of all four types, and research can play a different role in each of these strategies. there is research to be done on actual transformations, as well as on transformation processes, practices, and pathways. although systems thinking emphasizes that addressing design and intent are the strongest leverage points, these ll open access are also the hardest to achieve, particularly when the personal sphere of transformation is considered to be ''external'' or irrelevant to systems change. an integration of experience, culture, behavior, and systems within sustainability science recognizes that the system is not ''out there,'' and it highlights the importance of ''being change,'' i.e., embodying the changes that are considered necessary at all levels, right here and now, and critical to the process of creating an equitable and sustainable future. recognizing that people engage with the future differently is also essential for activating transformations to sustainability. the three horizons framework helps to generate agency and future consciousness by recognizing different ways of addressing uncertainty. this approach distinguishes between incremental and transformative change and emphasizes the need for participatory, reflective, and action-based research. research on how to pre-condition the system for transformation through incremental changes also provides valuable insights on how to create transformative change , by drawing, for example, on parts of the australian wine industry that were adapting incrementally as opposed to transformationally. when change is seen as a process, incremental changes offer an opportunity to shape the pathways for transformative change, as depicted on the righthand side of figure , and thereby activate easier systems interventions (left side) in order to pre-condition the system for more transformative change in the harder systems characteristics of structure and intent. in practice, this requires a diversity of the intervention strategies (top of cone), as can be seen in examples such as the german energy transition or marriage equality in the us. then arises the question, how can similar insights be applied to sustainability science itself? the cone of increasingly transformative change: systems interventions with increasing leverage (from bottom to top on left side , ) as the cone opens out have greater potential to cause transformational change and require a larger application of the four strategies for systems change (top plane ); easier but directed incremental changes can contribute to the transformability of the system (right side). , enabling sustainability science action sustainability science is a part of the global system that needs to transform to achieve the sdgs. this means that researchers have to reflect on how science should evolve from the research that is already available on transformations to research that can activate sustainability transformations in the wider world. we note four lessons in this regard, examine incremental changes in research organizations (where a great deal of science is operationalized), and then share some thoughts on how science can drive transformation across society. first, most environmental problems, especially the global ones, are complex, messy, and wicked. too often the problem formulation by researchers is partial and inadequate because they reduce the real-world, multifaceted problems into a single scientific-technical dimension and seek to solve this. instead of providing a precise answer to the wrong question (i.e., committing an ''error of the third kind''), approaches should address the full scope of the problem in question (the scientific-technical, the interpersonal-social, the systemic-ecological, and the existential-spiritual) and then develop some satisficing balance among them. the job of sustainability science is to identify problems and then to produce response options that are substantively adequate and ethically acceptable in broad socio-economic contexts. the same logic can be applied to thinking reflexively about sustainability science itself. second, the growing literature on transitions and transformations in society covers the many dimensions and scales needed for responding to global environmental challenges. [ ] [ ] [ ] much of this research emphasizes small-scale (pilot) experiments as important to demonstrate proof of concepts. it also explores how local success stories can be scaled. we need rapid cross-system learning about the successful ones in a context of changing the institutions and rules (scaling ''up'') and the cultural milieu (scaling ''out'') in which these can be scaled. a growing set of examples and opportunities for scaling can influence how we do sustainability science. we are at a point in time when sustainability science needs to think about how to selectively scale these exemplars massively, including the transformation of science itself. third, as already noted (figure ) , systems thinking highlights how the strongest leverage points for changing a system are in modifying its design and intent. it is usually much easier to fiddle with parameters and perhaps feedbacks. the resilience literature addresses what makes a system more or less ''transformable.'' it highlights how important well-chosen incremental changes can be to pre-conditioning a system to be more likely to transform in the ''right'' direction when a suitable impetus comes along. therefore, a good strategy for transforming sustainability science (as for transforming the world) should integrate well-directed incremental changes with preparedness to transform at the appropriate opportunity. fourth, enabling sustainability science to be impactful could also involve modifying the identity and role of scientists. historically, the role of scientists has been to be ''objective'' observers of phenomenon, scientific data collectors, unbiased analysts, and accurate recorders and reporters of findings. in a world beset with grand challenges, scientists must also become translators of knowledge, communicators to the public, policymakers, implementors of action, advocates of solutions, and co-designers of the future. they must also listen deeply and maintain reflective, open minds as they engage not just with theory but also with actions that generate a sustainable world. noting these points, we turn to asking how sustainability science could become more pre-adapted to transform (bottom of figure ), as well as actually transform (top of figure ), in order to play a more effective participatory role in global transformations. this must be in the context of the challenges of the anthropocene, i.e., that change is rapid, spatially interconnected, and more than ever influenced by global power dynamics as it is increasingly embedded into all societal systems. acknowledging universities as a key operational venue of sustainability sciences, and knowing how difficult it is to change them, we suggest some modest yet potentially transformative incremental changes. progressing incremental changes in research and public policy scientific research is guided by research policies and public policies, both of which progress through incremental changes. research institutions, especially universities, are being challenged to produce impactful research. it is widely held that good transdisciplinarity (convergence science) is a means to greater and more enduring impact, especially in more complex and value-laden problems, such as many of the sustainability challenges discussed here. such transdisciplinary challenges almost inevitably end up also requiring interdisciplinary approaches. , the incremental moves noted above from universities and funders toward promoting more inter-and transdisciplinarity must now be accelerated and empowered in both intent and design. however, there are a number of well-recognized barriers to this-traditional mainstream pressures and incentives (e.g., university tenure based on citation counts and grant dollars or corporate instrumentality of research-delivering products); systemic problems in publication, granting, and training processes (e.g., difficulties in publishing integrated research, funding review processes that favor narrow disciplines, and underresourced student training); and disciplinary research cultures of universities that undermine the commitment to inter-and transdisciplinarity and solving real-world public problems. these barriers need to be systemically addressed. change can be initiated if funders of science articulate more demand for impactful science. funders should also modify the institutional constrains to addressing challenges in the global south, where research funding is woefully inadequate and sustainability problems are also pervasive. universities where researchers are employed should modify tenure and promotion rules and reward systems to actively encourage transdisciplinary, solution-oriented sustainability science. even where inter-and transdisciplinarity are encouraged, we have noted that their co-design and co-production processes can be too slow to meet the rates of change we are experiencing. this could require additional resources to speed up these social processes. many current funding mechanisms and processes do not recognize the need for these additional resources to be effective. however, we must also work harder to recognize where transdisciplinarity is really needed and where it is not. we must obtain better evidence for what works and what doesn't in multistakeholder processes so the resources can be transparently focused where they will have the best effect. a critical impact of science-related public policies occurs via funding priorities. inter-and transdisciplinarity need to be supported by a much greater investment in social science and the humanities if we are to promote an equal partnership across the disciplines. as noted earlier, this investment is miniscule in comparison with that in the natural sciences, even if these also are under-resourced. even if we don't wait for more deep interdisciplinarity, many research areas require greater attention: understanding the deeply social nature of desire and consumption as it expands globally, mapping power dynamics across world consumer demand, exploring how culture change can support global transformations, rethinking the idea of ''progress'' in western cultures, and understanding global social and cultural tipping points and other aspects of mobilizing leverage points for systems change. , in addition to this improved investment, there should be a significant effort to improve holistic conceptual models and research practices that draw on multiple traditions. there is potential for increasing resources via government university and private-sector partnering. , these could help to resolve epistemological conflicts among the natural sciences, social sciences, humanities, and arts. this is important both interdisciplinarily (e.g., through conceptual models that deeply support the indivisibility and universality of sustainable development as framed by the sdgs) and also transdisciplinarily (e.g., through extended engagement and experimentation with traditional knowledge systems and faith-based systems). another key incremental and ''no-regrets'' step is increased training of researchers. this encompasses building the cohort of early-career researchers comfortable with interdisciplinary and transdisciplinary processes. we should also provide researchers with the skills to engage with stakeholders, convene and facilitate multistakeholder meetings, identify community problems that need to be addressed, and communicate results to the public and to policymakers. effective co-evolution of science and society will also need to address joint training of the next generation of researchers and stakeholders so they will be able work together to meet sustainability challenges. last, we can begin to recognize reflexively that sustainability is itself an open-ended social learning process in which we all need to play a part. the notion of ''being change'' implies the need to ''walk the talk.'' sustainability scientists should be models of sustainable behavior yet also draw attention to the norms and systems that are impediments to sustainability. this means engaging in public debates and contributing to critical and reflective conversations about sustainability. drawing on the experiences described in this paper, we should engage our own organizations to practice sustainability, drive toward net-zero carbon emissions and zero waste, and deliver gender equity and better social outcomes to meet the sdgs in our institutions and communities. driving global transformations to sustainability moving toward a transformational agenda for sustainability science implies not only driving the incremental change in the right direction but also being sufficiently active so that transformational interventions can take place rapidly when the opportunity arises. , for this we suggest a number of interlinked approaches. first, building on the incremental efforts already taking place, sustainability science should be pursuing funding and other support for a large network of co-learning innovation hubs in all sorts of contexts. these can be modeled in various ways (e.g., sdgs and cities ). they share the attributes of obtaining close engagement between research and society at a local level in many projects in combination with a strong monitoring, learning, and knowledge-exchange infrastructure that enables success stories to be learned from and scaled rapidly in ways that are sensitive to understanding the contexts in which they will or will not work. this transformation of the ivory-tower model of research not only provides an innovation scaffolding for transformation in society when the opportunity arises but also creates good outcomes at the local level in the meantime. , it also engages a much wider portion of society in discussions and learning about sustainability. second, sustainability science needs to engage in an action research mode with the high societal leverage points in such a way that an opportunity for transformation will lead these levers to be pulled in the ''right'' direction. ethically, these should be in areas where the direction is both normatively justified by the framing of goals such as the sdgs and factually justified by research and analysis. examples already noted include dramatic changes to our economic system away from narrow valuation of gdp to broader human well-being, the removal of structural barriers related to the skewed distribution of wealth and consequent inequalities, and a fundamental rejection of simplistic marketbased capitalism, all of which are underpinned by a deep realignment of values to a more collaborative, reflexive, and kinder view of humanity's stewardship of our planetary home. needless to say, such changes would require supporting changes in all areas of institutional and governance design, but these are, of course, past human inventions themselves. the role of sustainability science (and scientists) here would be as activists, as envisaged in waddell et al.'s ''forcing change'' quadrant. third, in addition to playing this activist role, sustainability science needs to have a greater voice in societal processes, such as international un governance discussions or discourse with global religions and traditional societies. the past attitude of scientists has tended to emphasize an autocratic expert identity. the transformed role must be as a partner among equals in seeking the normatively defined outcomes. global-change research has sought this collaborative role, for example, at the un conference on sustainable development (rio+ ) and through the intergovernmental panel on climate change and ipbes assessment processes, but in the sdg development process it was still relegated to being one among many lobby groups. the un secretary general's science advisory panel is still seen as a set of experts on the outside of the political process, as are most government scientific advisory boards. this is a failing on both sides of the relationship, and it must be worked on without hubris in policy and other domains. fourth and finally, sustainability science needs to be goal oriented to pursue transformation and accept the challenging roles implied by this. in the absence of clear intentionality in the design of these systems, science is dragged along by the dominant socio-economic forces that themselves should be the subject of change. sustainability science should examine and prepare for its role in all the complementary transformation strategies noted by waddell et al. -understanding when to be an activist and when to partner and being ready to distribute these tasks in a credible way. the community of researchers and their co-design partners also need to accept the goal of transforming themselves to help enable societal transformation in order to meet the sdgs and thereby set the direction for all the above points. maintaining transparency and accepting open debate while seeking sufficient consensus to move forward rapidly to achieve global sustainability and human well-being will be the hardest part of the balancing act. it is difficult to reach final conclusions to the broad critique and transformational vision for sustainability science as explored in this paper. we acknowledge that the realization of this vision could take time, yet the current global situation is challenging assumptions about incremental and transformational change. now is the opportunity for sustainability science to engage and coevolve with the underlying socio-economic roots of environmental changes. can the various branches of science afford to remain at epistemological odds with each other? can research that is driven by funding needs and competition continue to serve private corporate interests rather than the public good? research has not sufficiently emphasized how the most pressing environmental problems are caused and conditioned by social, cultural, economic, and business activities based on individualistic, competitive, profit-seeking models of economics and finance. if there has ever been a time to explore paradigm shifts that support an equitable and sustainable world, this is it. in this article, we have offered some suggestions for moving sustainability science forward on its evolutionary path. profoundly, on this path, the interlinked nature of science and society means that an overall societal commitment to sustainability (as articulated in the un agenda ) is a prerequisite for transforming science and society toward sustainability. for this to happen, macroeconomies would need to be designed for human and biosphere well-being rather than for financial economic growth. , cultural values to support this economic transition would be based on a more eco-centric vision of life and organizations. [ ] [ ] [ ] [ ] [ ] [ ] transformations like these require a different, more impactful version of sustainability science. the research system of sustainability science itself needs to evolve rather than just seek to change everything else. this means that sustainability scientists and policymakers have to be more reflexive and challenge their own limiting assumptions related to global change. global sustainability research and practices strive to provide adequate food, water, energy, income, education, resilience, voice, jobs, health, and gender and social equity for all in an ecologically safe operating space for humanity. this will require weaving together models of economic growth with models of ecosystem changes and managing winners and losers and the related changes in power dynamics. it will also require addressing the structural economic barriers that have emerged over the past century. the most important of these is wealth and income inequality. currently, % of wealth is owned by less than % of the world population. two billion people still consume under $ per day and aspire to increase it. growing inequality makes ''economic growth'' ineffective at addressing the needs of vulnerable sections of society by concentrating wealth with the rich. the co-evolution of sustainability science and sustainable societies implies increasing consumption of some and decreasing consumption of others without degrading ecosystems. the research ecosystem of universities, funding agencies, policymakers, and decision makers needs to be modified in other ways. science operations are different in each country, and there is no one-size-fits-all solution. some general actions for consideration include the following: d universities explicitly prioritize impactful research on environmental grand challenges. d universities adjust their research productivity, measurement methods, and reward systems to beyond academic measures (publications, citations, and grant dollars) to include the impact on social and sustainable development metrics. d funding agencies make more stringent and concrete demands that research should have an impact on sustainability (via the sdgs) in communities. d science communication with policymakers and with the public should be made a priority. many global environmental changes are being addressed outside academia through citizen activism, non-governmental organizations, and social movements. sustainability science could be leading some of these activities instead of merely studying or observing them. however, in order to take such leadership, researchers will need to acknowledge that science is not just a neutral objective pursuit of knowledge when it comes to sustainability. by bringing in reflexivity, values, and ethics, they can justify participation in structural and systemic changes that would produce an equitable and thriving world. scientific pursuit of knowledge involves much more than constructing accurate and analytically powerful 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fskqeqtt rigorous sustainability science includes addressing pressing real-world problems, weaving multiple knowledge systems, and striving for transformative change. however, these key attributes of sustainability science often conflict with university structures and established academic work practices, for instance with regard to frequent long-distance travel. such contradictions between key principles of sustainability and everyday practices are experienced by many researchers not only at university level, but also in their individual behaviors. to help resolve this widespread divergence, we present ten principles to foster the sustainability of a research group working in sustainability science, based on our personal experiences and experiments as research group leaders. these principles comprise: ( ) monitor the environmental footprint, ( ) foster learning and innovation, ( ) reduce the environmental footprint, ( ) nurture campus sustainability, ( ) embrace sustainability in private life, ( ) constructively deal with environmental anxiety, ( ) design research projects for sustainability impact, ( ) engage with stakeholders, ( ) capitalize on sustainability teaching, and ( ) recognize biases and limits. applying sustainability principles in everyday research practices can provide important social tipping points that may trigger the spreading of new social norms and behaviors. sustainability science has grown rapidly over the past years. being considered "not yet an autonomous field or discipline" in the early s (clark and dickson , p. ) , sustainability science has now come of age for a while, as demonstrated by numerous journals, conferences, professorships, university departments and faculties, and research programmes (spangenberg ) . to guide society toward sustainability is a most central characteristic of sustainability science (horcea-milcu et al. ) . correspondingly, identifying, conceptualizing, and supporting seeds (raudsepp-hearne et al. ) , leverage points , scenarios (kishita et al. ) , visions (wiek and iwaniec ) , and pathways (luederitz et al. ) towards sustainable development are overarching features of the field (miller et al. ) . rigorous sustainability science comprises, among other attributes, addressing pressing real-world problems (schmidt and pröpper ) , weaving multiple knowledge systems (tengö et al. ) , and striving for transformative change of society globally (díaz et al. ). however, these key attributes of sustainability science often conflict with university structures and established academic work practices (haider et al. ) . for example, flying to international conferences across the world remains a widespread (and sometimes required) practice in academia, food offered in university cafeterias is often unhealthy and not produced sustainably, and university endowments are frequently handled by osamu saito, institute for global environmental strategies, japan. invested in fossil fuel companies (though these phenomena are increasingly questioned). many researchers experience these contradictions between key principles of sustainability and everyday practices not only at university level, but also in their individual behaviors (for instance, when flying back from academic meetings to have more time with their families). such knowledge-action gap can inhibit impactful sustainability science in multiple ways. in particular, researchers can be affected by psychological stress, comprising negative mental and emotional consequences and leading to unclear role expectations and ultimately environmental anxiety (usher et al. ) . also, contradictions between research outcomes and research practices may undermine credibility of sustainability scientists in the public (fiske and dupree ) . surprisingly, sustainability researchers and academics in 'green' research areas do not produce significantly less carbon emissions than their 'non-green' counterpart researchers (wynes et al. ) . many sustainability scientists cope with this cognitive dissonance by suppressing inconsistencies and using justifications such as denial of control or responsibility, comparisons with the behavior of less-environmentally aware persons, and compensation through perceived sustainability benefits of their work (schrems and upham ) . the central argument of this note is that research groups are key drivers for shaping and implementing more reflective and more sustainable behaviors and by that for resolving the widespread knowledge-action gap in sustainability science. therefore, they should receive more attention as nuclei for developing and living innovative sustainability practices. our hope is that best practices implemented in leading sustainability science groups would spread to other groups and be upscaled to other disciplines eventually. we here suggest ten principles to foster the environmental sustainability of research groups working in sustainability science, based on our personal experiences and experiments as research group leaders at european universities. our principles ( fig. ) are grouped into clusters on 'learning about sustainability' ( , ), 'improving sustainability' ( , , , ) , and 'scaling up and spreading the word' ( , , ) , while the final principle ( ) is an overarching one. to run a sustainability science research group in a sustainable manner starts by creating an understanding of the social-ecological impacts of different types of group activities. build a knowledge basis to understand where the group currently stands, define common sustainability targets, and monitor impacts regularly, for example on an annual basis. typically, the largest share of researchers' environmental footprint is caused by professional travel, in particular when carried out by airplane and car (kalmus ). hence, the first step is to calculate the carbon footprint of the group's travel activities. several tools exist to perform this, but one recently developed calculator is targeted particularly to the scientific community: the travel carbon footprint calculator combines seven publicly available flight emission calculators and also considers train emissions (barret ) . it is specifically applicable to calculate the carbon footprint of a large set of trips and can help to identify a meeting place that minimizes emissions. a second step is to pay attention to energy and resource consumption, for example in relation to the use of internet (most notably cloud services), technical equipment, physical printing, and food catering. although exact monitoring of energy and resource use may be impossible, current practices can be listed: what are the internet habits of the group, what cloud services are used, and where is big data analysis done? how energy-efficient are equipment, instruments, and processes being used? to what degree are they being shared? what activities produce the bulk of lab waste? the relation between innovation and sustainability has many facets. on one hand, it is clear that we need to adopt new practices to become more sustainable and that technologies can greatly support us in this. on the other hand, particularly set of ten principles for research groups in sustainability science for learning about sustainability, improving sustainability, and scaling up and spreading the word technological innovation can be even detrimental to sustainability when it leads to rebound effects. it might for instance make a lot of sense to continue to use and repair old printers, even though they are less energy-efficient than a new one promoted as 'green' and 'sustainable'. therefore, it is indispensable to critically assess available options (office infrastructure, networking procedures, etc.) with regard to their actual sustainability impacts and figure out what kind of innovation and learning is needed to become more sustainable. using approaches such as social innovation (mehmood et al. ) or design thinking (fischer ) may offer opportunities to use the knowledge and skills of the research group members as catalysts to co-design innovative sustainability solutions. motivation towards sustainability also comes from following how colleagues perform in this task and from mutual learning. such benchmarking between sustainability science research groups and wider academia can lead to good practices that can be adopted among a research group. learning can be fostered by actively exchanging thoughts with colleagues at other institutions or by following arenas promoting discussion, such as blogs (e.g., academic flying ) or podcasts (e.g., essn ). the covid- pandemic forced academia to remote working modes and pushed us seriously to master various technical platforms for teaching and collaboration. this disruption and the resulting digital leap have created new norms and innovations for working in an unexpectedly rapid manner. after the crisis, it will be essential to consider which of such practices to maintain instead of returning to practices that were more energy-or resource-intensive. although frequent air travel is not a precondition for success in academia (wynes et al. ) , long-distance travel to conferences, workshops, seminars, and field sites is common in sustainability science. several steps can be taken to reduce this footprint. at least in europe, prioritizing railway for mid-range academic travel is feasible. for example, thousands of academics in germany have pledged not to use airplanes for trips of less than km distance. innovative conference styles have potential for substantially lowering the environmental footprint of a conference while keeping the character of a live meeting and lowering barriers to inclusiveness. templates are offered by the 'nearly carbon-neutral' (ncn) conference (hiltner ) or the 'all continents, balanced gender, low carbon transport, diverse backgrounds' (abcd) conference that mixes live-streamed and pre-recorded talks with in-person ones (blackman et al. ) . changing annual live meetings to biannual ones may be another option. such actions can reduce conference travel emissions by up to % (klöwer et al. ) . department seminars and phd defenses can also be moved into online formats, making them accessible to a much larger and wider audience. smaller meetings, such as research group events, faculty search panels, and other committees, can take place in the virtual space as well. in selection processes, committees should remove the number of physical talks given at distant departments from the evaluation criteria. rather, visibility and networks can also be developed through original use of social media, engagement in academic societies, or editorial tasks. due to their generally higher environmental footprints, senior academics bear more responsibility to reduce their footprints than junior ones. reducing the environmental footprint of travel to field sites is more difficult: does it make sense to 'regionalize' empirical sustainability research? to what degree can online surveys and remote sensing replace fieldwork on the ground? can lesser and longer field campaigns replace more frequent and shorter travel to field sites? can stronger reliance on local partners reduce travel? offsetting air travel-related carbon emissions by voluntary compensation schemes (now practiced for instance by some universities in germany) is contested, but the purchase of serious gold standard certificates for unavoidable flight travel is in our view substantially better than flying without compensation. changes in energy use have also potential to reduce a research group's environmental footprint, for example by deploying conscious internet search engines (e.g., ecosia), taking energy-and resources-aware decisions when purchasing lab equipment, or choosing a service provider that openly informs about energy demands. universities are large institutions with high societal impact: they involve diverse actors with connections to numerous societal groups, constitute important economic players not only at local level, and provide key impulses through research and their 'think tank' character particularly in regard to economic and technological development. moreover, they have a high reputation and serve as points of reference and orientation for the public and political decisionmakers alike. more and more universities around the globe are currently setting examples by withdrawing investments in fossil fuel-based companies and suspending research that is connected to these. but typically, university administrations will only get active for sustainability when they perceive pressure to do so. often, a few key persons have been enough to kick off university actions for sustainability. universities can also nicely link research, teaching, and sustainability impact. for example, university of natural resources and life sciences (boku) vienna ( ) established as the first university worldwide its own carbon offsetting scheme with projects in several countries. off-setting academic travel of employees, but also critically assessing the social and ecological complexities of such off-setting in research and teaching is at the heart of this endeavor. a similarly participatory project has been developed with the 'stay grounded-keep connected' strategy of eth zurich ( ). other important tasks for universities to advance sustainability are enhancing energy and water resource efficiency of university campuses, upscaling the generation and use of renewable energy, establishing more sustainable university infrastructure development, and climate-and biodiversityproofing of university greenspace management. many academics have fluidity in terms of separation of work and private lives. although everybody has a right to a private life, a large discrepancy in sustainability goals and actions at work compared to those taken in one's private life can jeopardize credibility (attari et al. ) . being a sustainability scholar means we can spread the message not only at work. we also personally contribute to important social tipping points that may trigger the spreading of new social norms and individual behaviors (centola et al. ). the 'value belief norm' theory (stern ) , a theory of environmental behavior change, suggests that awareness of environmental consequences comes together with responsibility for these consequences. this fosters the personal norm of pro-environmental behavior, both in public and private lives. more importantly, pro-environmental behavior can be linked to wider co-benefits to one's personal health and thus further motivate embracing sustainability (cohen and kantenbacher ). one everyday example is active transportation to work by foot or bicycle with substantial improvements to both personal health and climate indicators. the environmental stewardship literature offers rich examples of the linkages between consciousness, individual and collective environmental action, and personal well-being (bennett et al. ). there are multiple ways of reacting to the current sustainability challenges. given the existential and complex character of the issue, particularly young people struggle with feeling overwhelmed and intimidated (taylor ) . this may set free energy for taking action, but for many, at least at times, causes rather the contrary: they fall into despair, feel depressed, or adopt a fatalistic or cynical attitude (usher et al. ) . it is therefore an important part of a research group's sustainability agenda to create an atmosphere and space in which people can openly exchange on their mental reactions to the current ecological and social crises. considering 'eco-anxiety' as a normal reaction is a good first step towards taking it up constructively (lawton ) and will relief already some pressure. a next step focuses on the development of positive visions of our future and pathways towards them, acting as shared counter-narratives to images of inescapable destruction and despair that are dominating in the current debate. examples such as thousands of landscape-level sustainability initiatives (carmenta et al. ) , 'bright spots' (cinner et al. ) , or 'seeds of good anthropocenes' (raudsepp-hearne et al. ) help us to develop hope and imagine positive futures of a sustainable and good life as something achievable, although it will not come without backlashes and frustrations. while striving to minimize negative environmental impacts, we want to maximize the positive sustainability impact of our research. there are many directions for achieving impact, whether instrumental (triggering changes in practice and policy), conceptual (fostering new understanding), capacity building (training relevant actors), attitudinal/cultural (influencing societal values), or enduring connectivity (fostering follow-on interactions) impacts (reed ) . societal impact can unfold at many spatial scales, from local to global ones, and can often be achieved by very simple means-although a more comprehensive design of projects for sustainability impact may require acquisition of some new skills. one example are the self-made 'innovation leaflets' produced in the agforward project ( ) that proved very influential in upscaling novel agroforestry systems across europe. another possibility is to build impact activities into our formal research plans. for example, phd researchers could be expected to develop at least one impact activity besides the typically three scholarly papers that comprise a dissertation. a next step is writing of a guidance document and best practices collection of the impact activities that are suitable for phd projects. in such contexts, multiple synergies between academic and real-world impact can be created, for example when policy papers are published in scientific journals (see e.g., pe'er et al. ) . but beyond that, we consider it important to make striving for real-world impact an everyday practice. potential opportunities include, among others, engaging local media when carrying out fieldwork; releasing targeted plain-language summaries for any research paper published; and creating and disseminating short video documentaries (see e.g., utu tanzania team ). helpful resources on how to achieve short-and longterm sustainability impacts are reed ( ) and comms-consult ( ). sustainability science has potential to spread the word to diverse groups of stakeholders at the science-society and the science-policy interfaces. a robust knowledge of the interests and needs of the stakeholders that are dependent on and/or influential for one's research is key so that messages can be strategically targeted toward these actors (kusmanoff et al. ) . multiple tools-partly simple, partly sophisticated-exist to identify, group, and understand stakeholders and the relationships among them (reed et al. ). the most straightforward way to engage with stakeholders is integrating them as partners into research projects. we experienced stakeholder organisations such as the european landowners' organisation (elo), the international union for conservation of nature (iucn), or the world business council for sustainable development (wbcsd) as highly effective partners in european research projects. at more local levels, municipalities, regional businesses, associations, and even individuals are important stakeholders to ensure links to practice. the intergovernmental science-policy platform on biodiversity and ecosystem services (ipbes) developed a format for stakeholder participation that can be adapted to multiple domains and levels of governance. stakeholder engagement within ipbes has helped to communicate, disseminate, and implement findings; to co-develop guidelines and measures for biodiversity conservation within member countries; and to create linkages between global policy and local actors (krug et al. ) . similarly, the biodiversa platform offers detailed resources for informing and engaging with stakeholders (durham et al. ) . at best, stakeholder engagement leads to knowledge co-creation and supports transformative changes to sustainability. while many researchers aim for creating leverage for sustainability through research projects and publications, teaching is also a key arena and tool to achieve sustainability. in many countries throughout the world, research-based teaching is a key element of higher education, and correspondingly, a large share of researchers is in close contact with students as part of their daily work. this involves a huge potential: after finishing their study programs, generations of students pursue careers in relevant natural resource management agencies, ngos, or companies at various levels and in diverse fields, thus becoming decision-makers in all societal spheres. spreading the idea of sustainability and making it part of the worldview and commitment of students-not only in sustainability teaching, but in all kinds of classes-is therefore a very powerful, yet relatively easy to accomplish task for researchers. what is necessary for this is establishing a strong and clear link to the global sustainability agenda throughout all kinds of teaching offers, for example organized along the un-sustainable development goals (bowser et al. ) . for this, the manifold experiences, role models, and tools established within education for sustainable development can be drawn upon (mulà et al. ). an essential element is a focus on real-world problems, to be achieved for instance in the course of project work that considers the complexity of the matter, but also opens up pragmatic and solutions-oriented pathways for action. innovative teaching formats for sustainable development, for instance using living labs to engage students with applied sustainability challenges (evans et al. ) , allow for handing over responsibility to students. this can create life-changing experiences to them and promote long-lasting impacts (chawla ). although the potential for promoting sustainability in a research group is vast, there are limitations. in practical terms, any research activity comes with social-ecological trade-offs, ambiguities, and compromises, so that we have to accept that we cannot act % perfect in our lives. for some eminent workshops, conferences, or stakeholder contacts, long-distance air travel may simply be irreplaceable (and useful) . while it may be rewarding to strengthen research activities close to campus, the global south is known to be particularly rich in sustainability challenges and lessons (nagendra ) , and north-south exchange is a fundamental pillar of sustainability science (kates et al. ) . in more fundamental terms, navigating normativity in sustainability science may pose challenges. sustainability science is a value-laden or 'crisis discipline' (chan ) and-in the interest of promoting sustainability-to some degree departs from 'objective' science in that it comprises a facts-based and a normative dimension. most sustainability scientists embrace science-based advocacy for sustainable development (shrivastava et al. ) . however, how to (and how not to) advocate for sustainability in policy and practice is more contested. for getting the facts straight, it is indispensable to strictly consider good scientific practice codes and use rigorous peer-review processes. we can avoid misusing the scientific process also by grounding policy and practice recommendations firmly in our own scientific expertise. peery et al. ( ) provide details on how to avoid activities outside scientific norms that we consider valid for sustainability science. for the normative dimension, we need to make our background and claims explicit and consider the ones of other people, to open up a debate on the many different things sustainability might mean in a specific context, on who should take over which responsibility, and on how to settle the manifold dilemmas and conflicts in sustainability-directed action. in very practical terms, building up a research group that comprises students and scientists of diverse backgrounds and values may be the best premise for delivering good sustainability science to society. in this note, we provide a set of principles for reducing the negative and increasing the positive impacts of a research group. most principles focus on environmental sustainability, while we keep the multiple interactions between social and ecological sustainability dimensions in mind. the solutions that individual research groups and their leaders can contribute may appear small given the magnitude of current sustainability challenges. many relevant decisions may not lie in the hands of a research group. also, there is a debate on the effectiveness of individual action for sustainability (marris ) . however, we argue that reducing a sustainability scientist's environmental footprint is setting an important example, with high potential for multiplication and scaling. this potential can be further increased by speaking up for sustainability in social media and the mass media and by identifying 'allies' in other disciplines and making them change agents. addressing sustainability issues that researchers can influence through their own activities at the level of research groups can be particularly powerful by mediating between individual and-undoubtedly indispensable-collective and institution-level action for sustainability. thus, applying sustainability principles in everyday research practices can provide important social tipping points that may trigger the spreading of new social norms and behaviors, but also policies and economic processes (nyborg et al. ; otto et al. ). funding open access funding provided by projekt deal. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the 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academic air travel has a limited influence on professional success we are grateful for constructive feedback from christian albert, wolfgang schweiger, and one anonymous reviewer. this study contributes to the global land programme (www.glp.earth ) and the programme on ecosystem change and society (www.pecsscien ce.org). key: cord- -j hgzrtm authors: magoon, rohan; ohri, ruchi title: compounded research challenges amid the covid- pandemic date: - - journal: anaesth crit care pain med doi: . /j.accpm. . . sha: doc_id: cord_uid: j hgzrtm nan highest-priority societal goal of mitigating the morbidity and mortality associated with the novel severe acute respiratory syndrome coronavirus (sars-cov- ). despite the concerted endeavours of the scientific powerhouse, the real-time research challenges have considerably intensified amid the pandemic, particularly when the contributions of the scientific fraternity are under a perpetual microscopic scanner given the immensely broadened readership, in view of the understandably high hopes worldwide. with a colossal magnitude of covid-related research being published, it becomes increasingly difficult to discern the "signals" from the "noise", necessitating a continuous reflection upon the relevance, reliability, reproducibility and robustness of the same. the "pendulum-swings" as a result of the premature acceptance of attributes like treatmentefficacy or contraindication, can have potential consequences. to name a few, the dramatic escalation of the hydroxychloroquine requirement in the pandemic times raises possible concerns of shortages for the cohort receiving medication for the u.s. food and drug administration (fda) approved indications, exemplifies the aforementioned fact [ , ] . similarly, the threats surrounding the use of ibuprofen in covid- limited the availability of acetaminophen for patients with a well-known contraindication to ibuprofen [ ] . nevertheless, the research community is not new to such examples (premature embracement of recombinant human activated protein-c, etc.) with these previous experiences only highlighting the need of maintaining a high quality standard of evidence for moving practice forward [ ] [ ] [ ] . in this context, the fda has recently outlined a regulatory document of guidance on the conduct of clinical trials related to the public health emergency of covid- [ ] . in addition to pragmatic guidelines dictating our research efforts backed by a meticulous peer-review, we researchers are also obligated to adhere to j o u r n a l p r e -p r o o f the principles of scientific integrity aiming at the most accurate and objective representation of the study results, despite a condensed time frame in a pandemic situation [ , ] . the concept of scientific-integrity is much more holistic (compared to scientific-misconduct encompassing the fraudulent publication practices, at large) and focuses closely on a firm adherence to the epistemic values in the way a scientist evaluates, discusses and accepts the research results. as an extension of the same, research-related accountability is pivotal while science serves society, wherein a sound comprehension of the scientific research can be augmented by ensuring an enhanced transparency (an improved researchrepresentation quality by sharing methodological intricacies, protocols, patient-registries, appropriate regulatory approval, funding details, data-analysis script, etc.), vetting the involved experimental designs and techniques to minimise the associated biases, which could compound the study representation and interpretation, alike [ , ] . moreover, a second major concern emanates from the "viral" spread of misinformation (misleading information, a major plague to the scientific community and public health), which tends to transpire in a much greater extent during times of disaster, considering the humans' natural tendency to discover resolution in absence of its true existence [ , ] . given the demanding situation, the healthcare fraternity can also be reasonably vulnerable wherein the intrinsic zeal to contribute may potentially blur the spectacle of critical appraisal. however, intentions kept aside, the contagion-effect of the misinformation phenomenon (the concurrent "infodemic", in its own peculiar ways) presents the biggest peril to the ongoing covid- fight [ ] [ ] [ ] , as the popular adage most aptly puts it: "a lie can travel halfway around the world while the truth is still putting on its shoes". the information on the most trending search engines bear testimony to the above mentioned [ ] . in addition, rovetta and bhagavathula, in their analysis of google searches and instragram hashtags pertaining to covid- , reveal that myriad infodemic monikers circulate propagating information that potentially classifies as fake news or misinterpretation, all coming within the purview of misleading information [ ] . appropriate to the above mentioned context, merchant and asch delineate the remedial measures to safeguard the value of science in the social media age of "liking" and "retweeting" [ ] . their exemplary tenets can contribute to combat the menace of misinformation in times of the pandemic. identifying the potential sources of misinformation at the level of social media blogs, news reports and other mass media regulators, the scientific community needs to engage at these sources (in the form of commentaries, interviews, expedited reviews and joint author-journal posts, vetting the dissemination of information) to augment the resultant control on the derived narratives (alongside the preservation of veracity) and to execute every possible opportunity to curtail the dissemination of the infodemic monikers, which tend to accelerate misinformation in one or the other form [ , ] . to conclude, the research fraternity discovers itself under the spotlight, much more than ever before. while every part of the community has their own lessons to learn from the pandemic, the peculiar research-related challenges have intensified as we navigate through this epic pandemic, which presents unique opportunities to learn long-term lessons in the subject of tenuously balancing the "hope" and the '"hype" paralleled with the aim of strengthening the integrity of and the confidence in the medical research enterprise. the covid- pandemic: effects on low-and middle-income countries fact versus science fiction: fighting coronavirus disease requires the wisdom to know the difference pediatric policy council. covid- impact on research, lessons learned from covid- research, implications for pediatric research prowess-shock study group: drotrecogin alfa (activated) in adults with septic shock implications of practice variability: comment precision cardiac anesthesia: welcome aboard! fda guidance on conduct of clinical trials of medical products during covid- public health emergency. guidance for industry, investigators and institutional review boards research integrity is much more than misconduct teaching scientific integrity and research ethics coronavirus: the spread of misinformation the infodemics of covid- amongst healthcare professionals in india misinformation of covid- on the internet: infodemiology study quality of novel coronavirus related health information over the internet: an evaluation study global infodemiology of covid- : analysis of google web searches and instagram hashtags protecting the value of medical science in the age of social media and "fake news key: cord- -qa cjszv authors: termini, christina m.; traver, david title: impact of covid- on early career scientists: an optimistic guide for the future date: - - journal: bmc biol doi: . /s - - - sha: doc_id: cord_uid: qa cjszv nan nationally mandated social distancing efforts have been implemented throughout the world during the covid- pandemic to support the health and safety of the public. in response to the pandemic, research institutions have enacted strict changes to permitted research operations, requiring scientists to abide by social distancing guidelines in the laboratory, facility closures, and ramped down laboratory activities. while scientists at all stages in their careers have been impacted by these changes to the research environment, early career scientists such as postdoctoral fellows and junior faculty are particularly vulnerable during these unconventional times. because early career scientists are in the process of establishing independence during times of restricted research activities, we believe that they are particularly susceptible to the direct and indirect effects of the covid- crisis. while not all researchers will be equally affected by covid- research restrictions, we believe that the impact of such restrictions will be significant and longlasting. in this piece, we discuss how changes enacted over the past months in response to the covid- pandemic have impacted early career scientists that are members of wet laboratories. we will emphasize how changes in research productivity, the job market, and funding will impact early career scientists in the immediate and late phases of their career. meanwhile, we will discuss how continued social distancing and limited domestic and international travel will influence early career scientists and, ultimately, the scientific landscape as a whole. with a reduction of personnel in laboratories and social distancing measures in place, the traditional laboratory dynamic limits collaborative research that relies on multiple team members working simultaneously. furthermore, social distancing reduces the natural exchange of ideas that occurs through informal conversations in the laboratory. depleted of this valuable information sharing channel, junior scientists may not benefit as greatly from the rich training environment offered by a traditional laboratory. to circumvent this loss, we suggest that research advisors sustain scientific creativity and knowledge sharing by holding virtual lab meetings and journal clubs, which can build a sense of community and routine among group members and support research productivity. beyond meetings focused on research, we suggest that advisors also hold informal meetings such as social hours to enable scientists to connect in a casual setting, which will further strengthen the team morale that may have been lost during quarantine. hosting informal meetings can help reduce the stress and anxiety that early career scientists may experience during these uncertain times. we also suggest that early career scientists hold virtual meetings with colleagues and collaborators to update them about their research projects and use this as an opportunity for informal feedback and conceptualization of subsequent studies. as international travel will not be possible in the immediate future, it is necessary for researchers to continue to foster ongoing collaborations using virtual platforms. while virtual interactions may not replace the in-person environment of a laboratory, we are optimistic that digital interactions can still support collaborative research productivity. during times of social distancing, scientists have reduced access to the laboratory resources needed to move their research forward. while scientists who are capable of working remotely may not experience as strong of an interruption to research productivity, those requiring lab space, core facility support, and specialized equipment may be unable to perform necessary experiments to support their research program. to mitigate this, scientists may consider adapting the direction of their research to take advantage of resources that they do have access to. this may range from seeking training in bioinformatics, computational biology, or molecular modeling to learning how to utilize new analysis software platforms. some universities are already hosting online courses and workshops on using bioinformatics tools and learning coding languages. in many cases, it may not be possible to change research direction due to timeline or funding restrictions. we suggest that postdoctoral fellows meet with their research advisors to identify the best mechanisms for maintaining productivity during quarantine and lab distancing, while also taking responsibility for their own productivity. for example, it may be best to use time away from the bench for analyzing data, generating figures, and writing manuscripts. we suggest that postdocs generate specific and achievable goals that they can revisit during subsequent meetings with their advisor. for example, your goals may be to create an outline for a manuscript in preparation, or to generate a specific panel for a figure. by establishing and revising their goals, postdocs can hold themselves accountable for progress and also identify barriers that may exist preventing them from achieving their goals. finally, time away from the bench can be utilized to apply for funding, from postdoctoral fellowships, career transition awards (nih k , k , k mechanisms), or short-term research grants in collaboration with the laboratory head (nih r , r mechanisms). acquisition of funding can thus help bridge the gap before fellows apply for independent positions by providing resources to extend fellowship time. depending on the nature of the project, time away from the bench will have varying impact on researchers. for example, some scientists may have many of their materials frozen and stored such that they can pick up where they left off when the laboratory re-opens. meanwhile, in the event that an experiment was terminated prematurely due to research restrictions, the researcher will lose the time that their experiment was incubating in addition to the time spent away from the bench, which can add up to over a year in some cases. along these lines, it is possible that after re-opening, researchers may have to again ramp down their research programs, leading many to question whether it is appropriate to begin long-term projects. we suggest that researchers think creatively prior to initiating a long-term study to identify concrete stopping points at which a project can be paused or terminated, while still enabling scientists to obtain meaningful data. as such, we believe that the impact of covid- restrictions on the research timeline will be case specific, although all scientists will experience setbacks while working to rebuild their research programs. additionally, the restrictions imposed on researchers may extend the publication timeline by preventing submission or resubmission of manuscripts due to incomplete experiments, which may lead to tenure applicants being viewed as less productive than expected. this may have long-term consequences on tenure decisions for a generation of scientists, and we may see increased departure of the academic workforce if they are held to pre-pandemic standards. to circumvent this change in productivity, many departments have provided junior faculty with an extension in their tenure application timelines. for postdoctoral fellows, first-author publications are a crucial metric for future employment. if fellows are unable to publish as planned, they may be perceived as unproductive by future employers and, thus, a generation of untapped talent be excluded from research landscape. one solution to this problem is to extend the postdoctoral timeline, but contract extension may not be possible for scientists with funding restrictions or for international scientists on strict visa timelines. we suggest that research advisors meet with their fellows to discuss potential strategies to support alternative employment possibilities, such as contract extension or in some cases, promotion to project scientist or research/adjunct professor. additionally, advisors may consider supporting non-traditional publication mechanisms, such as registered reports, depositing the manuscript on a preprint server to bring the study to the attention of the community before publication in a journal, or adapting a larger story to a brief reports format. journals are critical intermediaries in the publication process, and some of them have already adopted measures to support the publication process, such as extending the submission deadlines and working with researchers on revision plans to take into account the challenges of this restricted time. for example, journals of springer nature, elsevier, cell press, embo press, elife, and many others have publicized practices to support the dissemination of research findings during the covid- crisis. keeping the lines of communication open is essential, and we would prompt researchers to be proactive in their discussions with the editorial staff about these challenges. along these same lines, scientific productivity is used as a metric for funding decisions across the world. given the impact of covid- restrictions on research productivity, it is unlikely that the rising generation of scientists will be able to compete for funding resources if scientists are to be held to the pre-pandemic standards. without sufficient funding, early-stage investigators may be unable to continue their research programs as planned, thereby preventing their scholarly contributions from being realized. we suggest that grant reviewers consider how changes induced by covid- may have impacted researchers. more specifically, funding agencies may consider allowing scientists to write an addendum to their application to describe how their research program and institution were impacted by covid- . this may provide researchers with an opportunity to explain that timepoints were omitted from an analysis because they did not have access to the laboratory, or that experiments were performed with an alternative animal strain because researchers were unable to order animals from outside vendors. this would give researchers an opportunity to acknowledge the shortcomings of studies while explaining that certain unconventional choices were made during a time of restricted resources. networking represents a crucial opportunity to support the career development of scientists at all stages in their career and is particularly important for early-stage scientists working to build their scientific community. a majority of networking interactions occur via in-person interactions at conferences, seminars, and other events whether through formal meetings or informal introductions. to comply with social distancing restrictions, many societies have postponed or canceled their scientific events, significantly changing the mechanisms by which networking can occur [ ] . we suggest that researchers can still take advantage of scientific networking at virtual meetings by reaching out to scientists in the audience through follow-up emails. social media involvement using twitter and linkedin also represent powerful mechanisms to promote networking with the international scientific community. additionally, several slack channels have been generated that allow researchers with common interests to engage in discussions at their own liberty, which will help scientists generate new connections with researchers around the world. with reduced in-person interactions, early career scientists also have limited opportunities to promote their science and research vision, which may prevent them from reaching larger audiences to disseminate their results. in fact, however, it seems that virtual seminars or conferences may actually reach a larger audience because scientists do not have to travel to attend and the conference fees are sometimes reduced. however, we do not yet understand how presenting our work in the digital realm may impact situations in which keeping results confidential or without sharing is preferred. as early-stage scientists are at particularly vulnerable stages in our careers, scientists should carefully consider what information to share publicly as participants may have digitized access to sensitive information or novel ideas. however, participation in virtual presentations can also be incredibly beneficial by facilitating collaborations that may not have been forged previously. the job market has been significantly modified by the covid- pandemic, with many open searches being put on hold indefinitely, leaving a generation of fellows unable to transition to the next stage in their career. for postdocs nearing the end of their contracts, it may be necessary to seek employment in non-academic sectors before entering the job market or face unemployment. working outside of academia may equip researchers with new perspectives and expertise, and we are hopeful that employers and funding agencies will recognize this and not penalize fellows looking to re-enter academia. furthermore, in the event that job searches are continuing, most interviews are taking place using virtual platforms, which can be an unfamiliar experience for interviewees to navigate. we strongly suggest that postdoctoral fellows utilize time away from the bench to practice key job skills such as interviewing and presentation skills for seminars and chalk talks using digital platforms such as zoom. by using this time to refine these skillsets, applicants will be better prepared for the interview process when the time comes. additionally, we hope that hiring committees will take into consideration the impact of the pandemic on the publication record of applicants from specific fields to ensure representation is given to fields that have been more drastically affected by laboratory limitations. again, a solution to this problem may be to generate a short narrative to include in the application packet to how restrictions incurred from the covid- pandemic specifically impacted the applicant to ensure that a diverse group of scientists remains in the scientific community. for recently hired junior faculty, university-mandated hiring freezes will likely limit the ability to bring in necessary staff and trainees to support the research vision of your laboratory. however, it may be necessary to communicate your staffing needs to your department chair, who may advocate to decision-makers to enable hiring, which will ultimately support the research of junior faculty. the scientific community as a whole will be even more drastically impacted by the covid- pandemic if early career scientists are not supported. early career researchers represent the next generation of leaders in scientific research, bringing with them unique expertise, creativity, and ideas for the future of biological research. to ensure that a generation of scientists is not lost, we encourage advisors, colleagues, and collaborators to offer help and support. we also encourage early career researchers to lean on their community for support and guidance. whether it be through virtual mentoring sessions, online seminars, or informal meetings between scholars, we believe that discussing our unique experiences and concerns for the future will help scientists persevere and grow during and following the pandemic. covid- impact on early career investigators: a call for action publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations article conceptualization was by c.m.t.; c.m.t. and d.t. wrote and edited the manuscript. the authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- - drl xas authors: farah, i.; lalli, g.; baker, d.; schumacher, a. title: a global omics data sharing and analytics marketplace: case study of a rapid data covid- pandemic response platform. date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: drl xas under public health emergencies, particularly an early epidemic, it is fundamental that genetic and other healthcare data is shared across borders in both a timely and accurate manner before the outbreak of a global pandemic. however, although the covid- pandemic has created a tidal wave of data, most patient data is siloed, not easily accessible, and due to low sample size, largely not actionable. based on the precision medicine platform shivom, a novel and secure data sharing and data analytics marketplace, we developed a versatile pandemic preparedness platform that allows healthcare professionals to rapidly share and analyze genetic data. the platform solves several problems of the global medical and research community, such as siloed data, cross-border data sharing, lack of state-of-the-art analytic tools, gdpr-compliance, and ease-of-use. the platform serves as a central marketplace of 'discoverability'. the platform combines patient genomic & omics data sets, a marketplace for ai & bioinformatics algorithms, new diagnostic tools, and data-sharing capabilities to advance virus epidemiology and biomarker discovery. the bioinformatics marketplace contains some preinstalled covid- pipelines to analyze virus- and host genomes without the need for bioinformatics expertise. the platform will be the quickest way to rapidly gain insight into the association between virus-host interactions and covid- in various populations which can have a significant impact on managing the current pandemic and potential future disease outbreaks. in december , a cluster of severe pneumonia cases epidemiologically linked to an open-air live animal market in the city of wuhan, china , . the sudden spread of this deadly disease, later called covid- , led local health officials to issue an epidemiological alert to the world health organization's (who) and the chinese center for disease control and prevention. quickly, the etiological agent of the unknown disease was found to be a coronavirus, sars-cov- , the same subgenus as the sars virus that caused a deadly global outbreak in . by reconstructing the evolutionary history of sars-cov- , an international research team has discovered that the lineage that gave rise to the virus has been circulating in horseshoe bats for decades and likely includes other viruses with the ability to infect humans . the team concluded that the global health system was too late in responding to the initial sars-cov- outbreak, and that preventing future pandemics will require the implementation of human disease surveillance systems that are able to quickly identify novel pathogens and their interaction with the humans host and respond in real time. the key to successful surveillance is knowing which viruses caused the new outbreak and how the host genome affects severity of disease. in outbreaks of zoonotic pathogens, identification of the infection source is crucial because this may allow health authorities to separate human populations from the pathogen source (e.g. wildlife or domestic animal reservoirs) posing the zoonotic risk . even if stopping an outbreak in its early stages is not possible, data collection, data sharing, and analysis of host<>virus interactions is nevertheless important for containment purposes in other regions of the planet and prevention of future outbreaks. such surveillance measures are easily implemented with the pandemic preparedness platform showcased in this report that is based on the novel data sharing and data analytics marketplace shivom (https://www.shivom.io/). the platform is a proven research ecosystem used by universities, biotech, and bioinformatics organizations to share and analyze omics data and can be used for a variety of use cases; from precision medicine, drug discovery, translational science to building data repositories, and tackling a disease outbreak. in the current coronavirus outbreak, the global research community is mounting a large-scale public health response to understand the pathogen and combat the pandemic. vast amounts of new data are being generated and need to be analyzed in the context of pre-existing data and shared for solutioncentered research approaches to combat the disease and inform public health policies. however, research showed that the vast majority of clinical studies on covid- do not meet proper quality standards due to small sample size . our approach is designed to provide healthcare professionals with an urgently needed platform to find and analyze genetic data, and securely and anonymously share sensitive patient data to fight the disease outbreak. no patient data can be traced back to the data donor (patient or data custodian) because only algorithms can access the raw data. researchers querying the data-hub have no access to the genetic data source, i.e. no raw data (e.g. no sequencing reads can be downloaded). instead, researchers and data analysts can run predefined bioinformatics and ai algorithms on the data. only summary statistics are provided (e.g. gwas, phewas, polygenic risk score analyses). in addition to genetics bioinformatics pipelines, specialized covid- bioinformatics tools are available (e.g., assembly, mapping, and metagenomics pipelines). using these open tools, researchers can quickly understand why some people develop severe illness while others do show only mild symptoms or no symptoms whatsoever. we might hypothesize that some people harbor protective gene variants, or their gene regulation reacts differently when the covid- virus attacks the host. the usability of digital healthcare data is limited when it is trapped in silos limiting its maximum potential value. examples of covid- data depositories include the european commission covid- data portal , the canadian covid genomics network (cancogen), genomics england and the genomicc (genetics of mortality in critical care) consortium , the covid- host genetics initiative , the national covid cohort collaborative (n c) , the covid- genomics uk (cog-uk) consortium , the secure collective covid- research (scor) consortium , the international covid- data research alliance , and the covid human genetic effort consortium , among several others. all of them, while very valuable for fighting the pandemic, present complicated access hurdles, limited data-sharing capabilities, and missing interoperability and analytics capabilities that limit their usefulness. compartmentalized information limits our healthcare system's ability to make large strides in research and development restricting the available benefits that can be generated for the public. this is especially true for omics data. if omics data silos are broken up and can be globally aggregated, we believe that society will realize a gain of value that observes accelerating returns. if individuals and medical centers around the world shared their genomic data more fully, then as a whole this data would be significantly more useful. particularly the role of host genetics in impacting susceptibility and severity of covid- has been studied only in a few small cohorts. expanding the opportunities to find patients somewhere in the world that possess unusual mutations and phenotypes relevant to national efforts, or have sars-cov- resistant mutations, would shift precision medicine to another level. by combining genome sequenced data with health records, researchers and clinicians would have an invaluable resource that can be used to improve patient outcomes, and additionally be used to investigate the causes and treatments of diseases. for big data approaches to thrive, however, historical barriers dividing research groups and institutions need to be broken down and a new era of open, collaborative and data-driven science needs to be ushered in. there is also a cost to using and producing data, and if this cost is shared amongst many stakeholder's new data sources, which outperform older databases significantly, can be built. another obstacle is data generation itself. genomes have to come from somewhere and currently researchers have limited access to them. healthcare organizations and private individuals often have restricted access to their own data and even when they do, they are limited in the ways they can use this data, or for sharing with third parties (e.g. for scientific studies). computer scientists, bioinformaticians, and machine learning researchers are tackling the pandemic the way they know how: compiling datasets and building algorithms to learn from them. but the vast majority of data collected is not accessible to the public, partly because there is no platform that offers anonymized data sharing on a global level, while addressing privacy concerns, and regulatory hurdles. what is needed is a platform that can store data in a secure, anonymized, and impenetrable manner with the goal of preventing malicious attacks or unauthorized access. while ensuring access controls are maintained and satisfied, the database must also be user friendly, publicly accessible, and searchable. in addition, there must be ways to quickly and easily analyze the collected data. these properties make the data in the database usable, meaning it must be easy for use in population health studies, pharmaceutical r&d, and personal genomics. in addition, such a platform must be accessible on a global scale, as well as offer data provenance and auditing features. we believe that for such a platform to be successful, adding new genomic data to the platform must be trivial and accessible to everyone. we aim to provide such a platform by using convergent technologies including genomics, cryptography, distributed ledgers, and artificial intelligence. global pandemics come and go. the tragic impacts of the unusually deadly influenza pandemic in , called the spanish flu, or the bubonic plague that killed as many as one-third of europe's and north africa's population in the th century reminds us that covid- wasn't the first global pandemic, and it is clear that it won't be the last. in contrast to the middle ages, we now have the technological tools and exceptional scientific networks to fight pandemics. already, the covid- pandemic has demonstrated that sharing data can improve clinical outcomes for patients, and that quick access to data literally has life-or-death consequences in healthcare delivery. however, only a minority of healthcare organizations are participating in datasharing efforts. without proper coordination amongst initiatives and agencies, and making the efforts public, these initiatives run the risk of duplicating efforts or missing opportunities, resulting in slower progress and economic inefficiencies and further data silos. comprehensive solutions to problems such as the sars-cov- virus outbreak require genuine international cooperation, not only between governments and the private sector, but also from all levels of our health systems. researchers from all over the world need to be able to act quickly, without waiting for international efforts to slowly coordinate their efforts, to understand early local outbreaks and to start intervention measures without delay. new solutions to tackle the pandemic need also early adopters that want to move away from our archaic data information systems that can hamstring real-time detection of emerging disease threats. many research and innovation actors have reoriented some of their previously funded activities towards covid- , but often with little guidance from policy makers. despite all complications and concerns, the advantages of sharing data quickly and safely can be significant. a lack of coordination at international level on numerous initiatives launched by different institutions to combat covid- resulted in notably reduced cooperation and exchanges of data and results between projects, limited interoperability, as well as lower data quality and interpretation. open data plays a major role in the global response to large-scale outbreaks. for instance, analysis of the ebola outbreak points to the importance of open data, including genomic data to generate learnings about diseases where effective vaccines are lacking or have not been developed. similarly, open data played a major role in the response to the zika outbreak with a commitment from leading national agencies and science organizations to share data. with the shivom platform, it is possible to help the global research community fasttrack discover new preventive measures and optimize logistic solutions. moreover, this data lends itself naturally in predicting and preparing for future, inevitable disease outbreaks. a commitment to open access in genomics research has found widespread backing in science and health policy circles, but data repositories derived from human subjects may have to operate under managed access, to protect privacy, align with participant consent and ensure that the resource can be managed in a sustainable way. data access committees need to be flexible, to cope with changing technology and opportunities and threats from the wider data sharing environment. the spread of sars-cov- has taught us that prevention relies on community engagement and has exposed the fragility in our research relationships. in an ideal scenario, health workers, such as doctors, virologists, data custodians at datarepositories, biobanks and in pharma, as well as students at the university should be able to quickly collect, share, and analyze health data in a global, cross-border manner without complicated, lengthy access-procedures. the ability to share data securely should not be restricted to selected specialists in the field but should be possible for all healthcare workers, as such the access and sharing procedures need to be easy. data collected during a pandemic should not only be accessible, but also analyzable; meaning all stakeholders and the public should have the ability to analyze the data, not only the data custodians or project leads of research consortia. having such an affordable and easy-to-use platform in place, even developing countries are enabled to work together to reduce a pathogens' spread and minimize its impact, despite severe resource constraints. however, our health systems are -more often than not-conservative and innovation averse. what is needed is true leadership in the research community that is able to adopt new solutions, who are ready to tackle the uncertainty a situation like covid- creates. there is no value in competing for data during a global crisis, instead humanity needs to collaborate more than ever before to defeat a common enemy that has taken the world hostage. collective solutions that provide a 'one-stop shop' for the centralization of information on pathogen <> host interactions can help ensure that appropriate conditions for collaborative research and sharing of preliminary research findings and data are in place to reap their full benefits. the provided platform offers such a one-stop shop solution, without being an exclusive storing solution. data shared on the platform can also be collected and shared elsewhere. beyond short-term policy responses to covid- , the shivom platform supports research and innovation that could help tackle future epidemics early, on a national or international scale. the shivom data analytics platform was built on a principle of open science, transparency, and collaboration and has been designed to give researchers a one-stop ecosystem to easily, securely and rapidly share and analyze their omics data, therefore simplifying data acquisition and data analysis processes. no coding proficiency is needed for using the platform, so everyone can use it and get results, minimizing work times. our organization started out with the mission to accelerate data access, easy analysis, and exchange across international and organizational boundaries to promote equal access to healthcare for all. the platform facilitates sharing and analysis of genetic, biomedical, and health-related data in a safe and trusted environment and contains two connected marketplaces, one for omics data and one for bioinformatics/ai pipelines and tools (see fig. ). . cc-by-nc . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint figure : data flow in the shivom platform. the user does not need in-depth bioinformatics expertise and is guided through all necessary steps from data upload to data analytics. basically, data from a variety of sources is uploaded by the data custodian and data permission rules are set. if the data is set to open or monetizable, it will be searchable in the data marketplace. once a dataset of interest is identified and filtered, the user can select a bioinformatic workflow (pipeline), configure user-specific parameters if needed, and then start the analysis. the user can then investigate and share the resulting report and, depending on the workflow, run further downstream analysis. it was shown recently that clinical data collection with rapid sequencing of patients is a valuable tool to investigate suspected health-care associated covid- cases . similarly, the shivom platform can enable quick insight into local outbreaks to identify opportunities to target infection-control interventions to further reduce healthcare associated infections. if widely adopted by researchers and doctors around the globe, this wealth of genomic and healthcare information is a powerful tool to study, predict, diagnose and guide the treatment of covid- . the information from combined epidemiological and genomic investigations can be fed back to the clinical, infection control, and hospital management teams to trigger further investigations and measures to control the pandemic. signing up on the platform is easy and doesn't require complicated access procedures. the idea behind this mechanism is to provide access to clinical and health data and analytics tools for everyone. calls for data sharing have mostly been restricted to publicly funded research, but we and others argue that the distinction between publicly funded and industry-funded research is an artificial and irrelevant one . at the center of precision medicine should be the patient which should override commercial interests and regulatory hurdles. data sharing would lead to tremendous benefits for patients, progress in science, and rational use of healthcare resources based on evidence. as such, the shivom platform stands out for its ease of use from the very first steps; the only requirements are a valid email, preferably institutional, in case the user wants to use advanced features that come with institutional plans. the user needs to set a password, and if required, can utilize an additional step verification procedure to add an extra layer of security. the personal data stored by the platform is protected under gdpr regulations whether it is hosted in the european union or another country on other continents. after the sign-up, users are directed to a settings and profile page that allows to add additional information about the user and organization, research interests and dataset needs, or to switch between light and dark mode to make the user experience more personalized. the personal profile can be changed at any time. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint depending on the account type (e.g. academic group or company), the user can invite team members to the workspace to work on projects together and to share data and bioinformatics pipelines. team members can be easily assigned user roles, i.e. any new user can be upgraded to administrator for the account. team members can be removed any time from the account by the administrator (project lead or manager). with the 'manage plan' tab, the platform can be adapted and scaled to meet the requirements of the users, from single/private users (e.g. students or freelance bioinformaticians), academia and startups, to small and medium enterprises with vast amounts of data and unique requirements as well as pharmaceutical organizations looking for custom solutions. the shivom platform was designed to make the process of uploading and sharing covid- and other genetic data as easy and simple as possible. the reason is that access to the data sharing and analysis features should not be restricted to a few trained experts (data analysts or bioinformaticians) but should be easy for all healthcare professionals. importing records (genetic or metadata files) into the user's workspace is done via the 'my datasets' page. in case of sequencing data, the user will primarily choose to upload variant call format (vcf) files. vcf is a tab delimited text file format, often stored in a compressed manner, that contains meta-information lines, a header, and then data lines each containing information about a position in the genome . the format also has the ability to contain genotype information on samples for each position in the genome (virus or host). vcf is the preferred format on the platform because it is unambiguous, scalable and flexible, allowing extra information to be added to the info field. many millions of variants can be stored in a single vcf file, and most bioinformatics pipelines on the platform use vcf files as input. most ngs pipelines use the fastq file format, the most widely used format, generally delivered from a sequencer. the fastq format is a text-based format for storing both a nucleotide sequence and its corresponding quality scores. fastq.gz is the preferred file format for the standard covid- pipelines preinstalled on the shivom platform. despite vcf being very popular especially in the statistical genetic area, there are also other file formats that can be used on the upload or directly at the point of configuring pipeline parameters. the purpose of these other genetic file formats is to reduce file size -from many vcfs to just three distinct files: a) .bed (plink binary biallelic genotype table), that contains the genotype call at biallelic variants, b) .bim (plink extended map file) which also includes the names of the alleles: (chromosome, snp, cm, base-position, allele , allele ); c) .fam (plink sample information file), is the last of the files and contains all the details with regards to the individuals including, whether there are parents in the datasets, and the sex (male/female). this file will also contain information on the phenotype/traits of the individuals in the cohort. ideally, the sequencing file is accompanied by phenotypic metadata (e.g. is it patient or control data, age, height, comorbidities, etc..), describing the uploaded datasets (see metadata). the metadata file is uploaded as a csv file, a comma-separated values file, which allows data to be saved in a tabular format. csv files can be used with most any spreadsheet program, such as microsoft excel or google spreadsheets. for ease-of-use, a sample template file is provided on the platform. in the next step, the user has the option to save specific data sharing permission settings related to the uploaded dataset to allow for fine-grained data sharing with others. these unique permission settings transform the data analytics platform into a massive data marketplace. providing health information for a data marketplace and sharing it is a key in leveraging the full potential of data in precision medicine and the fight against pandemics. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint the marketplace is a central point of entry for the users in order to find needed data faster and more structured. in addition, interoperability can be created through the marketplace, which supports cross-sectoral applications. unrestricted access to relevant pandemic data in particular will become increasingly important in the future. the user has three options: open -all data designated open will be available for the public to analyze. however, open does not mean that the data is downloadable, this permission setting only allows only for algorithms/pipelines to use the data in the data analytics module. all open data is searchable on the platform. being a data-hub for covid- data, we highly encourage all researchers and data custodians to make their data open/public and accessible for the scientific community, thereby increasing the amount of opensource covid- data currently available and usable. private -private data will be stored in a secure, private environment, and is only available and analyzable by the data owner and team members from the same organization or project and will not show up in data search results. this setting is usually used when data is not supposed to be shared (or only within a consortium), or if the data needs to be analyzed or published before sharing commences. monetizable -by choosing this option, the data owner/custodian authorizes the marketplace to license their information on their behalf following defined terms and conditions. in this context, data consumers can play a dual role by providing data back to the marketplace. data marketplaces make it possible to share and monetize different types of information to create incremental value. by combining information and analytical models and structures to generate incentives for data suppliers, more participants will deliver data to the platform and the precision medicine ecosystem. to monetize pandemic data, making datasets available for researchers at a cost, can make sense if a laboratory is not otherwise able to finance the data collection, e.g. when located in an underfunded developing nation. at any time, the permission settings of data files can be updated. the settings have an audit trail and the platform owner or server host are not able to change the settings, only the data owner/custodian is able to modify the settings. in the next step during data import is to add additional information to the dataset, such a dataset name, a digital object identifier (doi), health status, author or company name, a description of the dataset, or the name of the genotyping or sequencing system used (a list of sequencing platforms is available in a drop down menu). other features will be added in later versions. after requesting the user's digital signature to confirm the operations carried out so far, the data will be uploaded, and the user will be redirected to a page containing a summary of his/her data sets. during the upload, the data is checked for quality metrics and for duplicates (compared to data already existing on the data marketplace) by internal algorithms that prevent monetization of datasets that were shared by others before. genetic data is largely transformed into a common format as part of the data curation process. the platform also starts automated proprietary anti-fraud mechanisms to identify data that was tampered with. . cc-by-nc . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint all data in a user's workspace are easily searchable and can be sorted by therapeutic area (based on collection of biomedical ontologies) and other parameters. generally, the workspace allows for building larger datasets in time, and at any point to share data whenever it is needed, or to revoke access. there are several potential use cases for using the platform as storage space for data. first of all, having a sharing infrastructure will enable healthcare professionals to quickly accumulate and share anonymized data with the public (e.g. sequencing data from covid- patients), which is particularly important during an early disease outbreak. second, the platform is an ideal storage infrastructure for data custodians (e.g. biobanks, direct to consumer genetic companies, or patient support groups) that inherently have large curated patient and volunteer datasets. the data can then be monetized, but more importantly, the datasets can be made available to the wider research and scientific community to make medical breakthroughs. since the basis of the platform is precision medicine, at this point in time, data custodians are encouraged to store their data as vcf files; however, a plethora of other file formats are planned to be added to the platform in time. vcf are particularly useful as they are one of the final file formats of most genomic technologies across both microarray chips and sequencing (genome vcf/ gvcf). they are also the most common file format when it comes to secondary genetic analysis, but equally their smaller file size reduces the cost on storage. third, using the platform as a repository for research data during a peer-review publication process. researchers are increasingly encouraged, or even mandated, to make their research data available, accessible, discoverable and usable. for example, the explicit permission settings in the platform would allow researchers to grant access to the journal's reviewers to their data and analyses, prior to publication. this time-restricted data sharing can be valuable when data should not yet be available to the wider public. fourth, the platform is an ideal data sharing infrastructure for research consortia. groups that are located in different countries can easily share data and result and build larger, statistically more relevant datasets. such a procedure is particularly valuable for pre-competitive consortia, to significantly lower the costs to accumulate valuable datasets, while the in-house research can be kept confidential. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint the shivom platform was developed with a comprehensive security strategy to protect user data privacy and security. the central pillar of the shivom data-hub is patient data privacy and confidentiality. the duty of confidentiality is a medical cornerstone since the hippocratic oath, dating back to the th century bc. on the other hand, the right to privacy is a relatively recent juridical concept . the emergence of genetic databases, direct-to-consumer genetics, and electronic medical record keeping has led to increased interest in analyzing historical patient and control data for research purposes and drug development. such research use of patient data, however, raises concerns about confidentiality and institutional liability . obviously, doctors and other healthcare workers have to make sure that no private and protected information of a covid- patient, such as name, address, phone number, email address, or biometric identifiers are exposed to rd parties. as such, institutional review boards and data marketplaces must balance patient data security with a researcher's ability to explore potentially important clinical, environmental and socioeconomic relationships. in this context, the platform was designed from the beginning to be gdpr compliant and to provide full anonymization of patient records (privacy by design). the advantage of this approach is that personal or confidential data that has been rendered anonymous in such a way that the individual is not or no longer identifiable is no longer considered personal data. as such, by making it impossible to connect personal data to an identifiable person, data controllers (i.e. the data custodian or patient who can upload and manage data) and processors (researchers) are permitted to use, process and publish personal information in just about any way that they choose. of course, for data to be truly anonymized, the anonymization must be irreversible. anonymization will provide opportunities for data controllers to use personal data in more innovative ways for the greater good. full anonymization is achieved with several steps. first of all, no private, identifiable data of patients is stored together with clinical data. second, and most importantly, the platform provides only summary statistics/analytics on the data. that means sharing of data only allows bioinformatics pipelines or machine learning algorithms to touch the data; no raw data (e.g. dna sequencing reads) can be downloaded or viewed by the data user/processor. all data must be stripped of any information that could lead to identification, either directly or indirectly-for instance, by combining data with other available information, such as genealogy databases . however, at this point in time, the consent mechanisms as well as the process of removing private or confidential information from raw data remains with the data custodian, for example the hospital that uploads the covid- related sequences. in this context, it is important to understand that only the data owner/custodian has all the access rights and can manage the data such as granting permissions to use the data. the platform provider has no power to manage the data and only has the possibility to identify duplicate datasets that will automatically be excluded from the data marketplace. other security features on the platform are that no statistical analyses are possible on single individuals, and only vetted algorithms and bioinformatics pipelines are available on the analytics marketplace to avoid that algorithms are deployed to specifically read out raw data from individuals or to cross-link data from individuals to other, de-identifying databases. by harnessing the power of cloud computing the platform enables data analysts and healthcare professionals, with limited or no computational and data analysis training, to analyze and make sense of genomic data and covid- in the fastest and most inexpensive way possible. the genetic data stored with the platform is handled and managed with state-of-the-art technology and processes, using a combination of security layers that keep data and management information (cryptographic keys, permission settings etc.) safe. currently, all the covid- related data is uploaded to amazon web services (aws) s buckets that provide data storage with high durability and availability. like other services, s denies access from most sources by default. the shivom platform requires specific permission to be allowed to perform transactions or computation actions on the bucket. the s storage service allows to devote a bucket to each individual application, group or even an individual user doing work. access to the buckets is regulated via the platform on different, non-aws server(s), and all information related to the access is encrypted using state-of-the-art key management services (kms). in addition, no user/custodian data is stored alongside genetic data and is completely separated and encrypted using different cloud storage solutions. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint one powerful feature of our solutions is the ability to maintain an immutable data-access audit trail via blockchain. this immutability property is crucial in scenarios where auditability is desired, such as in maintaining access logs for sensitive healthcare, genetic and biometric data. in precision medicine, access audit trails for logs provide guarantees that as multiple parties (e.g., researchers, laboratories, hospitals, insurance companies, patients and data custodians) access this data create an immutable record for all queries [ ] [ ] [ ] . since blockchains are distributed, this means that no individual party can change or manipulate logs , . such a feature can help journal reviewers and research collaborators to protect themselves from inflation bias, also known as "p-hacking" or "selective reporting". such bias can be the cause of misreporting or misinterpretation of true effect sizes in published studies and occurs when researchers try out several statistical analyses and/or switch between various data sets and eligibility specifications, and then selectively report only those that produce significant results. all permission settings stored in the system are coded into a blockchain, guaranteeing that nobody can temper with the permission setting provided by the data owner. each data transfer on the platform represents a cryptographic transaction hash. hashing means taking an input string from the platform of any length and giving out an output of a fixed length. in the context of blockchain technology, the transactions are taken as input and run through a hashing algorithm (e.g. a hash function belonging to the keccak family, the same family which the sha- hash functions belong to) which gives an output of a fixed length that is stored immutable on the chain. a transaction is always cryptographically signed by the system. this makes it straightforward to guard access to specific modifications of the database. transactions such as monetization of datasets are encoded as smart contracts on the blockchain. a "smart contract" is simply a piece of code that is running on a distributed ledger, currently ethereum. it's called a "contract" because code that runs on the blockchain can control digital assets and instructions. practically speaking, each contract is an immutable computer program that runs deterministically in the context of a virtual machine as part of the ethereum network protocol-i.e., on the decentralized ethereum world computer. once deployed, the code of a smart contract cannot change. unlike with traditional software, the only way to modify a smart contract is to deploy a new instance, making it immutable. all the contracts used by the shivom platform only run if they are called by a transaction, e.g. when a file is used in a data analytics pipeline. in this case, a smart contract is triggered that can, if the file permissions are set to monetizable, transfer a fee to the electronic wallet of the data owner. the outcome of the execution of the smart contract is the same for everyone who runs it, given the context of the transaction that initiated its execution and the state of the blockchain at the moment of execution. if needed, smart contracts can be put in place to accommodate specific terms needed in more complex research or drug development agreements, e.g. if datasets belong to a consortium or a biobank, or if there are data lock constraints (e.g. an embargo on data release) and usual access procedures are complicated. for example, most biobanks contracts for accessing data are long, arduous, detailed documents with complicated language. they require lawyers and consultants to frame them, to decipher them, and if need be, to defend them. those processes can be made much simpler by applying self-executing, self-enforcing contracts, providing a tremendous opportunity for use in any research field that relies on data to drive transactions. these contracts already possess multiple advantages over traditional arrangements, including accuracy and transparency. the terms and conditions of these contracts are fully visible and accessible to all relevant parties. there is no way to dispute them once the contract is established, which facilitates total transparency of the transaction to all concerned parties. other advantages are speed (execution in almost realtime), security (the highest level of data encryption is used), efficiency, and foremost trust, as the transparent, autonomous, and secure nature of the agreement removes any possibility of manipulation, bias, or error. advances in data capture, diagnostics and sensor technologies are expanding the scope of personalized medicine beyond genotypes, providing new opportunities for developing richer and more dynamic multi-scale models of individual health. collecting, and sharing phenotypic and socioeconomic data from patients and healthy controls, in combination with their genotype and other omics profiles, present new opportunities for mapping and exploring the critical yet poorly characterized "phenome" and "envirome" dimensions of personalized medicine. therefore, in is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint the context of covid- , if available, metadata should be shared next to sequencing information of the patients. such metadata can contain information with regards to the comorbidities, ethnic background, treatment and support that they receive rather than biological or clinical data. currently, metadata is uploaded as a csv file to the platform, which allows almost all phenotypic data to be saved in a tabular format. other inputs may be available in the future, e.g. to parse information directly from a medical record or from wearables. using a simple tabular format also allows it to be source agnostic, e.g. it is not difficult to combine genetic information with patient questionnaires. indeed, patients support groups, direct-toconsumer genetic companies or patient registries are less likely to have biological data types in their repositories and often have only metadata that comes from questionnaires that volunteers have filled. an example of this is the dementia platform uk (dpuk) , a patient registry that comprises thousands of individuals from across dozens independent cohorts -which allows a variety of analysis to be conducted including machine learning and mendelian randomization techniques. the platform is multi-purpose built, which allows for all types of data that is alpha-numerical encoded to be stored. other platforms are engineered specifically for a particular type of cohort, e.g. tailored towards analyzing biobank data. the shivom platform is completely source agnostic and is engineered to allow large cohort datasets to be uploaded regardless of biobanks or cohorts. additionally, the platform will allow for biostatistical (non-genetic), epidemiological and machine learning analysis types to be undertaken. the metadata is structured in a simple and intuitive way in order to allow the user to be able to quickly query the dataset. depending on whether the data being uploaded has accompanying genetics information, the first column of the file will either contain the name of the vcf file or the first metadata attribute. csv files are supported by nearly all data upload interfaces and they are easier to manage if data owners are planning to move data between platforms, export and import it from one interface to another. a template that illustrates how metadata can be organized, including a standard metadata set (e.g. height, smoking status, gender, etc..) is provided on the platform. for covid- datasets (provided as vcf) we encourage data providers to share as many as possible phenotypic, clinical and socioeconomic data, keeping in mind that all data should be anonymized. working with several covid- consortia, we established a guideline of what an ideal dataset should include, keeping in mind that many clinical data are usually not available see supplement for metadata structure): • ethnicity, age, gender, height, weight is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . the platform supports fine-grained search queries across global omics datasets. the idea is to find and analyze genetic and other omics dataset from various global sources in the easiest, fastest, and most efficient way. on the 'discover' page, with just a few simple steps, the user will be able to quickly discover and select the data according to the disease of interest and filter the dataset based on the appropriate metadata for their study or research question. one strength of the platform is that the user can find and combine open-source, in-house, and proprietary datasets in a matter of seconds. the user has the opportunity to perform a free search with advanced search operators (e.g. searching for gene-or disease-name), or can use separate clinical search bars, and therapeutic area, disease or dataset title. the discover window will then be populated with the search results. currently, the search query form supports doid ontology, a comprehensive hierarchical controlled vocabulary for human disease representation. but other standards (efo, icd ) are planned to be added in the future as well. once the data has been selected, it will be possible to further filter it based on the information contained in the associated metadata files, generally gender, diet, country, etc., and also the type of data permission associated. for example, it is possible to select only free data that is not associated with any costs. this fine-grained filtering feature therefore allows to stratify patients based on the information the user is interested in, so that it will be possible to select the most significant data for the analyzes to be conducted. for example, once the database is populated, it should be possible to select covid- patients by ethnicity or smoking status to quickly find confounding factors that may affect the outcome and severity of the disease. once the user has completed the selection of the data that has to go into the data analysis, a simple click on the 'choose pipeline' box will bring the user to the data analytics marketplace for downstream analytics. after selecting datasets for analysis, the user is directed to the data analytics marketplace. the marketplace is designed to provide researchers with or without bioinformatics expertise to perform a wide variety of data analyses and to share in-house pipelines as well as machine learning tools with the scientific community. since the launch of the platform, a variety of standard bioinformatics pipelines/workflows, primarily for secondary analyses, were already added to the marketplace that are free to use, including pipelines to analyze covid- patients. the shivom analytical platform hosts a variety of genomic-based pipelines; from raw assembly and variant calling, to statistical association-based analysis for population-based studies. in time, a vast variety of new data analytics tools and pipelines will be added to the marketplace, including ai analytics features. currently, all provided standard pipelines are built on nextflow technology . nextflow is a domainspecific language that enables rapid pipeline development through the adaptation of existing pipelines written in any scripting language . nextflow technology was chosen for the platform because it is increasingly becoming the standard for pipeline development. its multi-scale containerization makes it possible to bundle entire pipelines, subcomponents and individual tools into their own containers, which is essential for numerical stability. nextflow can use any data structure and outputs are not limited to files but can also include in-memory values and data objects . another key specification of nextflow is its integration with software repositories (including github and bitbucket) and its native support for various cloud systems which provides rapid computation and effective scaling. nextflow is designed specifically for bioinformaticians familiar with programming. however, it was our aim to provide a platform that can be used by inexperienced users and without common line input. as such, the shivom data is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint analytics marketplace was designed to provide an easy to use graphical user interface (gui) where all pipelines can be easily selected and configured, so that no coding experience is required by the user. in addition to the data marketplace, this feature sets the platform apart from other cloud computing tools that use nextflow or other workflow management tools such as toil, snakemake or bpipe. users can easily automate and standardize analyses (e.g. gwas, meta-gwas, genetic correlation, two sample mendelian randomization, polygenic risk score, phewas, colocalization etc..) by selecting all parameters for sets of different analysis activities. this makes it possible to run analyses fully automated, meaning that even new users can start using sophisticated analyses by running a workflow in which all analysis steps are incorporated, i.e. assembled by more experienced bioinformaticians or machine learning experts. findings that cannot be reproduced pose risks to pharmaceutical r&d, causing both delays and significantly higher costs of drug development, and also jeopardize the public trust in science ( ) . by using standard pipelines and allow for rd parties to share their workflows on the platform, and by creating reproducible and shareable pipelines using the public nextflow workflow framework, not only do bioinformaticians have to spend less time reinventing the wheel but also do we get closer to the goal of making science reproducible. the disadvantage of many research consortia, including some covid- consortia, is that they do not open the data to all consortium members to analyze the collected data individually, but only provide data analysis by a central coordinator. then the summary statistics of the analyzed cohorts are fed back to the consortium members. for example, the host genetics initiative agreed on a standardized genome-wide association studies (gwas) pipeline . the shivom platform works differently, and all participants of a research consortium (and other rd parties if permitted) can analyze the datasets independently. to make gwas analyses available to the whole research community, one of the most important preinstalled pipelines on the platform is a standard gwas analysis workflow for data quality control (qc) and basic association testing. genome-wide association studies are used to identify associations between single nucleotide polymorphisms (snps) and phenotypic traits . gwas aims to identify snps of which the allele frequencies vary systematically as a function of phenotypic trait values. identification of trait-associated snps may reveal new insights into the biological mechanisms underlying these phenotypes. gwas are of particular interest to researchers that study virus-host interactions. the sars-cov- virus does not affect everyone in the same way. some groups seem particularly vulnerable to severe covid- , notably those with existing health conditions or with genetic predispositions. variation in susceptibility to infectious disease and its consequences depends on environmental factors, but also genetic differences, highlighting the need for covid- host genetics to engage with questions related to the role of genetic susceptibility factors in creating potential inequalities in the ability to work or access public space, stigma, and inequalities in the quality and scope of data . the genome sequencing of the coronavirus can provide clues regarding how the virus has evolved and variants within the genome. the highlighted platform contains a set of bioinformatics pipelines that allow the interrogation of host and virus genomes. genetic information might enable targeting therapeutic interventions to those more likely to develop severe illness or protecting them from adverse reactions. in addition, information from those less susceptible to infection with sars-cov- may be valuable in identifying potential therapies. the current version of the platform comes with preinstalled covid- specific pipelines covering assembly statistics, alignment statistics, virus variant calling, and metagenomics analysis, and other pipelines that can be used for analyzing covid- patient data such as gwas or metagwas. the is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint research community is encouraged to add more pipelines and machine learning tools to support the fight against the pandemic. all the standard preinstalled covid- pipelines work with fastq sequencing files. the platform works with illumina short reads as well as oxford nanopore long reads. long and short reads differ according to the length of reads produced and the underlying technology used for sequencing. the short-read length ( - bp) limits its capability to resolve complex regions with repetitive or heterozygous sequences, so the longer read lengths (up to kbs) have fundamentally more information. however, these tend to suffer from higher error rates than the short reads. the short reads come in zipped format (.gz) and do not need to be extracted before use; since there are paired-end, each sample will have two files: r and r fastq that need to be included in an analysis. long reads come as single fastq files. each analysis requires a sample metadata sheet, which specifies the fastq.gz files for each of the sample and other associated metadata files. a template metadata file can be accessed on the platform, in which the user can also find the description of all columns and the example values. one of the common analyses done on patient sequences is to identify the pathogens contained in the analyzed sample and to assign taxonomic labels, usually obtained through meta-genomic studies . metagenomics allows researchers to create a picture of a patient's pathogens without the need to isolate and culture individual organisms. the prediction power on covid- also depends on data related to underlying morbidities, including other flu-like illnesses. though metagenomic testing for other viruses can therefore increase the specificity of the gwas studies and other algorithms. the shivom platform comes with a metagenomics analysis pipeline that is based on the nf-core/vipr pipeline, a bioinformatics best-practice analysis pipeline for assembly and intrahost low-frequency variant calling for pathogen samples. the pipeline uses the kraken program metagenomics classification [ ] [ ] [ ] . using exact alignment of k-mers, the pipeline achieves classification significantly quicker to the fastest blast program. the process of running pipelines on the platform are largely the same and do not require in-depth bioinformatics expertise or command line entry. the user is guided through all steps. after the upload of the selected fastq files and associated metadata, the user can choose to add a quality control and then to modify the parameters of the pipeline, if required. after starting the computation, the user can easily monitor the progress of the pipeline and all other tasks in the 'projects' tab (see fig. ). is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint by pressing on a project, the user can see more details about the status of the computation and parameters used. if required, it is possible to download a log file and to monitor the costs of computations. in case a pipeline fails, e.g. in the case there are problems with the raw data or metadata sheet, all processes the pipeline went through are monitored in real time with additional information on the computation's cpu load and memory. in this way it is easy to find the step where a pipeline failed, and the error can be addressed. after the pipelines are finished, the user can monitor the results by clicking the 'results' button. all the pipeline-specific plots are presented in an interactive view and can be exported to a pdf report that contains all the parameters and settings of the analysis for easy results sharing. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint by interrogating the species profile, the presence of sars-cov can be confirmed and the presence of other pathogens that may add to observed phenotypes can be analyzed. the other preinstalled pipelines also work with patient sequencing files: this is a bioinformatics pipeline for sars-cov sequence assembly, by aligning and merging fragments from a longer viral dna sequence in order to reconstruct the original sequence. the raw data, represented by fastq files, a mixture of human and pathogen sequences, are taken as input and then checked for quality. after removal of the adapter sequences, the reads are mapped to the human reference genome (hg ; grch . ). the unaligned, viral sequences are then taken for de novo assembly using the spades program and evaluated using the quast program. the assembled genome files are viewable in programs such as bandage. the contigs are subjected to gene/orf prediction and the resulting sequences are further annotated using prokka. this pipeline produces variant files and an annotation table from the sars-cov viruses present in the covid- patient. unlike going for a de novo assembly, the user can directly align the reads to the available pathogen reference genome and identify the variants presented within the analyzed samples. the resulting variant calls can be used in downstream analyses to identify clues regarding how the virus has evolved and the distribution of variants within the virus strain. the raw data (fastq short reads) from the patient are taken as input and are checked for quality with fastqqc. the reads are then mapped to the hg human reference genome. the unaligned reads are then taken for alignment with the coronavirus (nc_ ) reference genome. the alignments are checked for duplicates and realigned using picard. the variants calling tools (lofreq) are used on these realigned files to get the list of the variants in the patient's viruses. the variants are annotated with snpeff. this pipeline is designed to produce vcf variant files from the covid- patients sequencing reads. the resulting vcf files can be valuable in identifying the variants associated with the coronavirus infection and can be easily exported and used for downstream analyses. the pipeline performs a quality check on the reads and trims the adapters. these trimmed reads are then aligned to the human reference genome (hg ). the alignment files are checked for duplicates, realigned and variants are called. these variants are annotated using the snpeff program. overall, the whole workflow, from data upload to running a covid- related pipeline was designed to be as easy as possible, with only a few steps involved and no command line entry needed, so that is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint researchers and healthcare professional can run analyses of patients sequences and share the files and results with the research community. researchers with other nextflow pipelines related to tackle the covid- pandemic are highly encouraged to share them on the shivom platform. the main goal of this precision-medicine platform is to provide the global research community with an online marketplace for rapid data sharing, managing & analytics capabilities, guidelines, pilot data, and deliver ai insights that can better understand complex diseases, aging & longevity as well as global pandemics at the molecular and epidemiological level. among other use-cases, the provided platform can be used to rapidly study sars-cov- , including analyses of the host response to covid- disease, establish a multi-institutional collaborative datahub for rapid response for current and future pandemics, characterizing potential co-infections, and identifying potential therapeutic targets for preclinical and clinical development. no patient data can be traced back to the data donor (patient or data custodian) because only algorithms can access the raw data. researchers querying the data-hub have no access to the genetic data source, i.e. no raw data (e.g. no sequencing reads can be downloaded). instead, researchers and data analysts can run predefined bioinformatics and ai algorithms on the data. only summary statistics are provided (e.g. gwas, phewas, polygenic risk score analyses). in addition to genetics bioinformatics pipelines, specialized covid- bioinformatics tools are available (e.g., assembly and mapping pipelines). using these free tools, researchers can quickly see why some people develop severe illness while others do show only mild symptoms or no symptoms whatsoever. we might hypothesize that some people harbor protective gene variants, or their gene regulation reacts differently when the covid- virus attacks the host. particularly during an early outbreak, virus-host genetic testing has value in identifying those people who are at high or low risk of serious consequences of coronavirus infection . this information is of value for the development of new therapies, but also for considering how to stratify risk, and identify those who might require more protection from the virus due to their genetic variants. identifying variants associated with increased susceptibility to infection, or with serious respiratory effects, relies on the availability of genetic data from the affected individuals. however, here covid- research encounters challenges associated with the population distribution of genetic data, and the consequent privileging of specific groups in genetic analyses . most data sets used for genome-wide association studies are skewed toward caucasian populations, who account for nearly % of individuals in gwas catalogs . in comparison, those from african and asian ancestry groups are poorly represented. even large-scale genetic analyses may consequently fail to identify informative variants whose frequency differs among populations, either under-or overestimating risk in understudied ethnic subgroups. data from many countries showed that people from black and minority ethnic (bame) groups have been disproportionately affected. people from a bame background make up about % of the uk population but account for a third of virus patients admitted to hospital critical care units. black americans represent around % of the us population but % of those who have contracted the virus. more than % of healthcare professionals who have died in the uk have been from bame backgrounds. similar patterns showing disproportionate numbers of bame virus victims have emerged in the us and other european countries with large minority populations. in the us and uk, the proportion of critically ill black patients is double that of asians, so geography and socioeconomic factors alone are unlikely to explain the stark differences. the key to fighting early disease outbreaks is speed. with the reality that some infections may originate from unknowing carriers, fast patient stratification and identification is even more paramount. it is important to investigate if the severity of the disease may lie in the interaction with the host genome and epigenome. why do some people get severely sick and die while others do not show any symptoms? it could be that some people harbor protective gene variants, or their gene regulation is able to deal with viruses attacking the host. very quick data sharing and analysis ensures that precision medicine is brought to patients and healthy individuals faster, cheaper, and with significantly less severe adverse effects, leveraging information from the interaction between labs, biobanks, clinics, cros, investigators, patients and a variety of other stakeholders. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint as outlined in this paper, we have the technology to make easy data sharing a reality. however, the observation that data sharing technologies are still extremely underutilized and most data custodians are reluctant to share their data, demonstrates that the global healthcare community needs an elemental paradigm shift, a major change in the concepts and practices of how data is shared and utilized, particularly during a pandemic. a paradigm shift, as outlined by the american physicist and philosopher thomas kuhn, requires a fundamental change in the basic concepts and experimental practices of a scientific discipline. kuhn presented his notion of a paradigm shift in his influential book the structure of scientific revolutions. we argue that the world needs such a revolutionary paradigm. researchers use real-world data from laboratories, testing centers, hospitals, academic institutions, payors, wearables, and other data sources such as direct to consumer genetic companies to better understand disease outbreaks and to develop vaccines and medicines quickly. such data is used across the whole spectrum of drug discovery, vaccine development, and basic academic research. however, whoever has ever applied for data access to a major biobank knows that typically, it's costly, time-consuming and nerve wrecking, in the bestcase scenario, to obtain such data. data is often received from a third-party partner that aggregates, anonymizes it, and makes it usable for their research purposes. often, there is a significant lag time between when the data is generated and when it's available for use. for example, when an infected patient is sequenced in a hospital, it can take between weeks and even years until the data surfaces and is available in a public database. but if healthcare systems are trying to control a pathogen outbreak and want to understand more about the affected patients and host-virus interactions, things are changing day by day, we need that insight into the real-time aspect of what's going on. what we need is an ecosystem that, for example, allows a doctor who sees a patient and sends a buccal swab out for sequencing, to get sequencing results, sharing it with the global community and gets in-depth data analytics back to evaluate if a similar host-virus interaction was observed anywhere else on the planet -within hours or less. in the current pandemic, for example, there is a need to understand covid- patient data at the local level, directly from the sources, to get data in real time. omics data from patients, exposed individuals, and healthy control subjects will be a critical tool in all aspects of the response-from helping to design clinical trials, identifying vulnerable groups of the population, to understanding whether covid- patients taking specific medicines are at higher risk of developing severe side-effects. the ability to collect, analyze, and interpret data is fundamental to the management of infectious diseases. to do so, stakeholders need to be incentivized to share their data. sharing data is good for science, not only because transparency enhances trust in science, but because data can be reused, reanalyzed, repurposed, and mined for further insight. sharing data on the shivom platform can increase transparency. the more transparent the contributors are about their data, analytic methods, or algorithms, the more confident they are of their work, and more open to public scrutiny. using the platform can also help researchers and consortia that want to collaborate within a safe space. we need more data aggregators that are committed to supporting an ecosystem of research communities, businesses, and research partners, by sharing data or algorithms in safe and responsible ways. such an open ecosystem approach can yield high dividends for society. in addition, consortia can come together to build patient cohorts, maximizing their research budget. research organizations who share similar goals can join efforts to create insight at scale, creating datasets that meet the needs of multiple stakeholders at lower costs. a successful response to the covid- pandemic requires convincing large numbers of scientists and healthcare organizations to change their data sharing behaviors. although the majority of countries have population studies and build massive digital biobanks, to our knowledge, none of the larger biobanks in the world have a data sharing infrastructure to share data with other countries. the shivom platform provides the only crossborder data marketplace to allow data custodians and biobanks to easily & securely share and monetize anonymized datasets. a massive, decentralized, and crowd-sourced data can reliably be converted to life-saving knowledge if accompanied by expertise, transparency, rigor, and collaboration. overall, there are several obvious advantages using the platform: • making data actionable as data can be analyzed on the bioinformatics platform • breaking down data silos, putting data in a larger, global context • making data easily findable & searchable . cc-by-nc . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint • ability to easily share data with fine-grained permission with clients, collaborators, or the public • improved accessibility by avoiding complicated access procedures, promoting maximum use of research data • advanced interoperability by integrating with applications or workflows for analysis, storage, and processing; e.g. new ai algorithms • better reusability so that data is not stored and forgotten, but reusable in many research projects • increased impact as sharing data will help the global fight against complex and rare diseases • gain reputation for the user's organization by giving researchers the ability to find, use and cite their work data monetization options. usually, for most data custodians such as clinical laboratories, data cleanup procedures and data deposition are not formal responsibilities. these jobs have to be done by some fairly busy people, in an increasingly demanding environment. there is no remuneration for the labs to submit their data; but done out of goodwill. as long as the data collection is funded by public institutions this is no major problem, but private hospitals, small biotech or genetic companies, data sharing can become a challenge. the shivom platform offers the possibility to monetize their datasets. practically, that means if a dataset is monetizable, whenever a bioinformatics pipeline or algorithm touches the dataset, a smart contract is triggered that sends a predefined fee to the data custodian. the pricing can be adjusted depending on the quality, data type and other factors. while open data sharing is encouraged for the publicly funded research community, such monetization options can be a valuable extra resource of funds for organizations that have to worry about sustainability and revenue models. unlike many other data brokers, organizations can upload and monetize data directly without a distributor or middleman. this leads to real-time payouts, higher revenue share rates, and instant upload availability. it allows researchers to upload their own data and get it in front of an audience of other scientists. in a way, it almost acts like a content creation tool, rather than a pure data sharing service. from the data-user perspective (e.g. a pharma or biotech company), getting access to large genetic datasets gets much easier and more affordable as it is not necessary to acquire exclusive licenses that can cost millions of dollars. instead, the data user only has to pay for accessing the data that is actually used, similar to the way people access music on streaming services. in addition, since the data custodian remains full ownership of the data, there is no need for complicated business relationships, nor will their internal business model be challenged. using the shivom platform as the regular data repository can help researchers with their data curation tasks, preventing what was termed 'data curation dept' . researchers can migrate/copy their data to the platform to protect themselves from breaking in-house legacy hardware, e.g. old unmaintained servers. also, digitizing raw data and storing it increases security of the data as many datasets are still stored on decaying physical media (e.g. consents done on paper, metadata stored on cds/dvds, minidisc, memory sticks, external hard drives etc.). if the physical medium decays, then the raw data are lost, making it impossible to reproduce the original research or apply new techniques to analyze the original raw data. loss of data has far-reaching consequences: a recent survey demonstrated that the lack of availability of raw data and data provenance were common factors driving irreproducible research . for many academic labs, the loss of data could lead to a loss of funding because future grant applications would not be able to list their data as a resource. even for normal daily tasks in a typical university laboratory, if data is not lost but requires additional work to locate, for example, after a phd student left the research group, then an additional time and staff cost is involved to find the data and re-learning knowledge that was the domain of the departed team member. particularly in multinational pandemic cohort studies where the recruitment and data collection are so difficult, it is unacceptable that there is such a risk to the hugely valuable resource of historical data through the accrual of data curation debt . joining forces and sharing information at the national level also eases and supports international cooperation initiatives. national coordination of pandemic policy responses can also benefit from joining forces with international research cooperation platforms and initiatives. it is important to note that data stored and shared on the shivom platform does not need to be exclusive to this data marketplace; the data can be hosted in other databases as well. the benefits of data sharing do not end here, other benefits that should incentivize researchers to share their datasets include the chance of getting more . cc-by-nc . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint citations, increase of exposure that may lead to new collaborations, boosting the number of publications, empowering replication, avoiding duplication of experiments, increasing public faith in science, guiding government policy, and many more. also, far from being mere rehashes of old datasets, more and more evidence shows that studies based on analyses of previously published data can achieve just as much impact as original projects and published papers based on shared data are just as likely to appear in high-impact journals, and are just as well-cited, compared with papers presenting original data , . building a massive global data-hub with its wealth of multi-omic information is a powerful tool to study, predict, diagnose and guide the treatment of complex disease and help us to live longer and healthier. also, data sharing should not stop with a successful vaccine rollout. healthcare systems need to monitor closely the characteristics of the most promising vaccine candidates. such monitoring includes understanding the patient's response, dosing regimen, potential efficacy, and side effects. measures to monitor and control the covid- pandemic by the scientific community will be relevant for as long as its risk continues. moving genomics data and other information into routine healthcare management will be critical for integrating precision medicine into health systems. however, data sharing is often hindered by complicated regulatory and ethics frameworks. obviously, there are ethical and legal issues to consider in any work that involves sharing of sensitive personal data. nevertheless, it is obvious that we need to be able to share data to fight global pandemics. the sars-cov virus does not care about jurisdictional boundaries. data from covid- patients enables experts to interpret complex pieces of biological evidence, leading to scientific and medical advances that ultimately improve care for all patients. but these advantages must be balanced against the duty to protect the confidentiality of each individual patient. it is out of the scope of this study to discuss ethics in depth. the shivom platform is built to be operational in all jurisdictions around the world. many regulatory frameworks do already exist that allow to collect and hold anonymized data from patients in the interest of improving patient care or in the public interest. it is now time to go a step further and decouple procedures from regional regulatory frameworks, to make data sharing easy across borders and regulatory frameworks. in this context, the magic word is anonymization. on the outlined data sharing platform, no access to raw data is permitted. only algorithms that provide summary statistics are able to touch the data, guaranteeing that no dataset can be traced back to an individual. this process makes it possible to share important multi-omics information anonymously and securely, whilst still enabling linkage to metadata such as diagnosis, lifestyle factors, treatment and outcome data. the key is that data on the platform is largely protected from reidentification of individuals. re-identification in this context is understood as either identity disclosure or attribute disclosure, that is, as either revealing the identity of a person, thus breaching his or her anonymity, or disclosing personal information, such as susceptibility to a disease, which is a breach of privacy . the shivom platform was designed to make it impossible to disclose information from genetic data not solely on the individual, but also on their relatives and their ethnic heritage, e.g. protecting the identity of siblings or other relatives in the context of forensic investigations. ideally, patients should be involved in such data sharing initiatives. institutions that collect and host data and promote data sharing should explain the benefits of collecting and using patient data to their clients. there is a need for public education on genetics, to take the frightening aspect away, ultimately incentivizing patients and healthy control groups to share their data anonymously for the greater good. one of the cornerstones of the proposed data ecosystem is easy public access to data. if adopted by the research community, researchers seeking to use public data must no longer comb websites and public repositories to find what they need. it is important that every human being has the potential to participate in this process, either on the data provider side or by analyzing datasets. access to the data should not be restricted to certain organizations or researchers with a long publication record. these systems are outdated and archaic. usually, with most databases and biobanks, the bona fide user must apply for access with a corresponding data access control body, provide an institution id such as openid or orcid, and often provide a long publication record. usually, that means they must register from, and be affiliated with, an approved research institute and need to register their organization which means they will need their organization's signing official to is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . participate in the registration process. often, when interested researchers want to access data in public databases, they are asked to submit a lengthy proposal detailing the data they want to use and the rationale for their request. most of the time, projects must involve health-related research that is in the public interest. sometimes the researcher needs to go through an institutional review board (irb) first and even then, all proposals received by the data repository are considered on an individual basis and may have to be evaluated by an internal or external ethics committee. on average, a straightforward application and approval process takes - months; sometimes it has taken up to months . obtaining access to data that are available from international sources adds another layer of complexity, due to international law. most databases require the user then require a standard material transfer agreement (mta) to be signed prior to any data delivery that governs how the researcher can use the data. but even when data access is granted, it is common that datasets are restricted to only those data and participants that the researcher required at that time (e.g. covid- patients only or specific case-control subsets). in addition, most agreements grant access for a limited time only, at the end of which a report summarizing research progress is required; for continued data access, yearly renewal requests are often obligatory. to exemplify the problem, our researchers applied for access to covid- data with four dataproviders (e.g. data repositories, covid- research consortia, and biobanks) in the uk, eu and us to support our covid- research efforts. all four requests were rejected, each for a different reason. one public data repository denied access to their data because their legal terms only allow access for groups working in public institutions but not private/commercial companies. another biobank was concerned about the lack of publications from our research unit that could demonstrate the organization's current activity in health research, keeping in mind that our researchers are highly respected in their field and have a vast list of scientific publications from previous appointments. other objections were that the proposed covid- research was too broad, basically no exploratory studies were permitted, or that our junior researcher had no publication record ( or more papers). one covid- data consortium suggested that our organization shares our genetic data with them but did not allow access to their data sets and only provided access to secondary analyses that their researchers performed on the data. such processes are often undemocratic, against the public interest and simply take too long to provide any benefit under a health crisis. a large majority of biobanks and databases are nonprofit organizations, and most of them operate on a not-for-profit basis. consequently, public funding puts some obligation on data-custodians to enable research with any scientific and social value. against this background, we argue requiring data custodians to prioritize access options and avoid underutilization of data. thus, biobanks ought to make all necessary arrangements that facilitate the best possible utilization of the data sets. as such, interested researchers on our data hub do not require a long publication record, nor do they need to provide a -page proposal, referrals, or a membership to an elite club (e.g. an accredited, approved research institute) to search and analyze data. usability and accessibility needs to be guaranteed for every interested individual, the academic researcher from any elite university as well as the pharma manager, the phd student from kenia, the doctor at a local clinic in wuhan, a student in a remote area of pakistan, or the nurse in a village hospital in brazil. in addition, data should not only be accessible for a specific research purpose but should enable research that is broader in scope and exploratory in nature (i.e. hypothesis-generating). many diseases hide in plain sight -in our genomemaking genomics the most specific and sensitive way to identify disease early and guide both patients and healthcare practitioners to the best course of treatment. yet despite many advances in data analytics and ai, our understanding of the human panome (including epigenome, proteome, metabolome, transcriptome, and so forth) is incomplete and the ways in which we utilize this information is limited and imperfect. clinical diagnosis. having a secure data-hub for fast data dissemination and analytics in the clinical setting will improve routine clinical care. most complex diseases, such as cancer have become increasingly dependent on genetic and genomic information. when biopsy samples are collected from patients and sequenced, there is the need to be able, as quickly as possible, to tell whether any variants detected are associated with the disease or indicate that a patient is likely to respond to a particular drug. at the same time, with every sequence shared, the accuracy of the global diagnostic accuracy improves, not only for the current patient but for all cancer patients around is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint the globe. in addition, linking patients' clinical data with genomic data will help answer research questions relevant to patient health that would otherwise be difficult to tackle. rare diseases. access to genetic data and 'realworld evidence' (rwe) obtained from observational data is crucial to help push research and innovation particularly on rare diseases. the standard approach to clinical trials is challenging, if not impossible for most rare diseases. it is the characteristics of rare diseases that they can only be adequately tackled if there is enough data to make evidence-based decisions, given that the number of patients is so small. to make this happen it is absolutely necessary to break down global data silos. to move things forward, many global initiatives, such as the eu regulation on orphan medicinal products (omps) were introduced specifically to address the challenge of developing medicines that treat patients with rare diseases. however, the initial progress seen since the introduction of these initiatives has tapered off in recent years, with a decline in a number of drug approvals over the past years. in fact, % of rare diseases conditions remain without treatment. one of the main hurdles is the collection and access to data, particularly obtained outside the context of randomized controlled trials and generated during routine clinical practice. gaining the critical mass of data to close data gaps needs global coordination and represents a critical step for driving forward research in the area of orphan drugs. in addition, the described platform can be used to collect valuable datasets to specific themes and therapeutic areas that are not yet available. currently, several pilot data initiatives started to collect research-area specific datasets on the shivom platform: cannabis data-hub. in collaboration with direct to consumer companies, patient support groups and medical professionals, we aim at collecting data on the interaction of individual's genomes and medicinal compounds found in medical and recreational cannabis. cannabis-related research allows governments and pharma researchers to better understand efficacy and potential side effects of hundreds of compounds like cannabinoids and terpenes and to improve legal frameworks for cannabis products. for example, several gene variations are known to affect the user's endocannabinoid system, increasing sensitivity to Δ -tetrahydrocannabinol (thc), potentially posing risk factors for thc-induced psychosis and schizophrenia , . similarly, certain variations in akt can make people's brains function differently when consuming marijuana . about percent of occasional cannabis users develop a physiologic dependence, cravings, or other addiction-related behaviors that can affect everyday life. as a result, many cannabis users often go through various products blindly before their ideal balance between their drug's efficacy and their tolerability is reached. patients and their doctors can learn about pharmacogenetic aspects of cannabis use and be informed on their susceptibilities and can therefore adjust cannabis habits to best fit their genotype. having a comprehensive anonymized genetic database available combined with real world data will pave to way to better pain management as it's been demonstrated that cannabis may be a promising option to replace opiates in the treatment of pain, as well as replacing other drugs such as antiepileptics and antipsychotics . the longevity dataset is an ongoing, concerted effort to provide genetic and epigenetic information on individuals who have attained extreme ages. the remarkable growth in the number of centenarians (aged ≥ ) has garnered significant attention over the past years. consequently, a number of centenarian studies have emerged, ranging in emphasis from demographic to genetic. however, most of these studies are not aimed at breaking down data silos. as such, data will be collected from centenarians, with a subset of people who attained an age of years or higher -so-called "supercentenarians" to yield sufficient numbers to warrant descriptive studies and to find genetic factors associated with exceptional longevity. having enough datasets will improve the search for meaningful subnetworks of the overall omic network that play important roles in the supercentenarians' longevity and will help to better understand epigenetic mechanisms of aging , . a data repository for the scientific publishing process. having data deposited in a public data-hub is of utmost importance, particularly when data needs to be published rapidly, as in a time sensitive pandemic. the case is easily demonstrated by retraction of two covid- studies in may of . the new england journal of medicine published an article that found that angiotensin converting enzyme (ace) inhibitors and angiotensin receptor blockers were not associated with a higher risk of harm in patients with covid- . the lancet is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . published an observational study by the same authors, indicating that hospital patients with covid- treated with hydroxychloroquine / chloroquine were at greater risk of dying and of ventricular arrhythmia than patients not given the drugs . both journals retracted these articles because the authors said they could no longer vouch for the veracity of the primary data sources, raising serious questions about data transparency. the problem was that both studies used data from a healthcare analytics company called surgisphere. after several concerns were raised with respect to the correctness of the data, the study authors announced an independent third-party peer review. however, the data custodian surgisphere refused to transfer the full dataset, claiming it would violate confidentiality requirements and agreements with clients, leading the authors to request the retraction of both studies. this problem could have been avoided by publishing the datasets to a data repository as the described platform in this article. journal reviewers and other researchers could have easily accessed and analyzed the data before problems arose, a method that could easily be a mandatory part of the submission process. the urgency of sharing emerging research and data during a pandemic is obvious. rapid publication and early sharing of results is clearly warranted, but it means that the research community must also be able to access and analyze the data in a timely manner. publishing research data to an open platform with audit features and data provenance increases the chance of reproducibility and to have reliable assurances for the integrity of the raw data. considering that we have the technical capabilities to publish anonymized datasets, journals, in principle, should try to have their authors publicize raw data in a public database or journal site upon the publication of the paper to increase reproducibility of the published results and to increase public trust in science . in this context, sharing raw omics data publicly should be a necessary condition for any master or phd thesis as well as research studies to be considered as scientifically sound, unless the authors have acceptable reasons not to do so (e.g. data contains confidential personal information); keeping in mind that fine-grained permission settings also allow to publish and analyze industrial & proprietary research. overall, by utilizing an easy-accessible data-hub, it is possible to incorporate valuable omics information into routine preventive care, research and longitudinal patient monitoring. in the end, we can leverage the benefits of omics to expand patient access to high-quality care and diagnosis. it is the aim of the authors to further evolve the proposed data sharing and analytics platform to the point that it is a widely used and truly actionable data hub for the global healthcare ecosystem. together with the research community, we are working on adding a variety of other functionalities to make this precision medicine ecosystem more valuable and innovative, particularly in the fields of artificial intelligence, interoperability, pandemic preparedness, and other new research fields. longterm this can include projects to add functionalities for clinical trials, ai marketplaces, iot data, or quantum computing. some of the planned addons in the roadmap for future releases include a cohort browser, improved search engines, many new pipelines and ai tools, as well as fully federated data sharing capability. we plan to add a forum to the platform to help researchers exchange information on datasets and data curation. often, those who want to make their data more open face a bewildering array of options on where and how to share it. providing a space for sharing data and guiding young researchers in their task by sharing necessary expertise in data curation and metadata will help to ensure that the data they plan to share are useful for others. the urgency of tackling covid- has led governments in many countries to launch a number of short-notice and fast-tracked initiatives (e.g. calls for research proposals). without proper coordination amongst ministries and agencies, they run the risk of duplicating efforts or missing opportunities, resulting in slower progress and research inefficiencies. the best way to prevent a further spread of the virus and potential new pandemics is to apply the same principles as we use to prevent catastrophic forest fires: survey aggressively for smaller brush fires and stomp them out immediately. we need to invest now in an integrated data surveillance architecture designed to identify new outbreaks at the very early stages and rapidly invoke highly targeted containment responses. currently, our healthcare system is not set up for this. first, modern molecular diagnostic technologies detect only those infectious agents we already know exist; they come up blank when presented with a novel agent. infections caused by new or unexpected pathogens are not identified . cc-by-nc . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint until there are too many unexplained infections to ignore, which triggers hospitals to send samples to a public health lab that has more sophisticated capabilities. and even when previously unknown infectious agents are finally identified, it takes far too long to develop, validate and distribute tests for the new agent. by then, the window of time to contain an emerging infection will likely be past. using the platform as demonstrated in this article, all those processes can be sped up dramatically. it would enable our healthcare ecosystem to halt the spread of emerging infectious diseases before they reach the pandemic stage. in this new surveillance architecture proposed here, genomic analysis of patients presenting with severe clinical symptoms would be performed up front in hospital labs piggybacked on routine diagnostic testing. researchers can use the data in our data-hub to compare new cases with existing datasets. given national epidemiological data on infection rates; where symptomatic people seek healthcare, a remarkably small number of surveillance sites would be required in order to identify an outbreak of an emerging agent. using genomic sequencing, the novel causative agent could be identified within hours, and the network would instantaneously connect the patients presenting at multiple locations as part of the same incident, providing situational awareness of the scope and scale of the event. using a globally accessible, easy-to-use solution that provides a 'one-stop shop' for the centralization of information on new virus-host interactions can help ensure that appropriate conditions for collaborative research and sharing of preliminary research findings and data are in place to reap their full benefits. beyond short-term policy responses to covid- , incorporating data sharing with the shivom platform into ongoing mission-oriented research and innovation policies could help tackle future pandemics on a national or international scale, for example to enhance datasets informativeness to reveal host infection dynamics for sars-cov- and therefore to improve medical treatment options for covid- patients. in addition, it can help to determine and design optimal targets for therapeutics against sars-cov- . the platform is in alignment with recent guidelines published by the research data alliance (rda) covid- working group . the rda brought together various, global expertise to develop a body of work that comprises how data from multiple disciplines inform response to a pandemic combined with guidelines and recommendations on data sharing under the present covid- circumstances. the rda report offers best practices and advice -around data sharing, software, data governance, and legal and ethical considerationsfor four key research areas: clinical data, omics practices, epidemiology and social sciences. in line with these recommendations, the shivom platform can be used for: • sharing clinical data in a timely and trustworthy manner to maximize the impact of healthcare measures and clinical research during the emergency response • encouraging people to publish their omics data alongside a paper • underlining that epidemiology data underpin early response strategies and public health measures • providing a platform to collect important social and behavioral data (as metadata) in all pandemic studies • evidencing the importance of public data sharing capabilities alongside data analytics • offering a general ecosystem to exploit relevant ethical frameworks relating to the collection, analysis and sharing of data in similar emergency situations at this point in time, the research community should be clear that a second or third wave of the covid- pandemic will hit most countries. in addition, other epidemics and global pandemics will follow; it is just a matter of time. as such, the global research and medical community need to be prepared. when analysis of datasets shared on the shivom platform results in publications, we encourage the researchers to cite this publication to enhance widespread adoption of this data sharing and analytics marketplace. we also encourage sharing data from covid- and other virus outbreaks, including data from people who were exposed to the same pathogen but did not get the disease or possibly were infected but did not get symptoms. . cc-by-nc . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint we developed a next-generation data sharing-& data analytics ecosystem that allows users to easily and rapidly share and analyze omics data in a safe environment and demonstrate its usefulness as a pandemic preparedness platform for the global research community. adopting the platform as a data hub has the clear benefit that it enables individual researchers and healthcare organizations to build data cohorts, making large, or expensive-tocollect, datasets available to all. in this way, data sharing opens new avenues of research and insights into complex disease and infectious disease outbreaks. this is not just true of large-scale data sharing initiatives, even relatively small datasets, for example rare disease patient data, if shared, can contribute to big data and fuel scientific discoveries in unexpected ways. particularly during early disease outbreaks, the patient-level meta-analysis of similar, past outbreaks may reveal numerous novel findings that go well beyond the original purpose of the projects that generated the data. using the platform as demonstrated will enhance opportunities for co-operation and exploitation of results. our data sharing approach is inclusive, decentralized, and transparent and provides ease of access. if adopted, we expect the platform to substantially contribute to the understanding of the variability of pathogen susceptibility, severity, and outcomes in the population and help to prepare for future outbreaks. in providing clinicians and researchers with a platform to share covid- related data, the clinical research community has an improved way to share data and knowledge, removing a major hurdle in the rapid response to a new disease outbreak. as, if, db, and gl are employees of shivom ventures limited. conceived and designed the platform: as, if. analyzed data: if, gl, db. wrote the paper: as. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint a pneumonia outbreak associated with a new coronavirus of probable bat origin early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia evolutionary origins of the sars-cov- sarbecovirus lineage responsible for the covid- pandemic sample sizes in covid- -related 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testing for covid- assembling genomes and mini-metagenomes from highly chimeric reads. in lecture notes in computer science (including subseries lecture notes in artificial intelligence and kraken: a set of tools for quality control and analysis of high-throughput sequence data improved metagenomic analysis with kraken ultrafast metagenomic sequence classification using exact alignments the missing diversity in recognizing, reporting and reducing the data curation debt of cohort studies , scientists lift the lid on reproducibility data sharing and the future of science assessment of the impact of shared brain imaging data on the scientific literature barriers to accessing public cancer genomic data association between abcb c t polymorphism and increased risk of cannabis dependence cannabis consumption and psychosis or schizophrenia development protein kinase b (akt ) genotype mediates sensitivity to cannabis-induced impairments in psychomotor control systematic review efficacy of cannabis-based medicines for pain management: a systematic review and meta-analysis of randomized controlled trials unraveling the complex genetics of living past handbook of epigenetics epigenetics and late-onset alzheimer's disease data transparency: 'nothing has changed since tamiflu drug therapy, and mortality in covid- retracted:hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis no raw data, no science: another possible source of the reproducibility crisis rda covid- working group. rda covid- recommendations and guidelines rda recommendation key: cord- - tapkjb authors: nan title: th escp-nsf international symposium on clinical pharmacy: clinical pharmacy tackling inequalities and access to health care. oslo, norway, – october date: - - journal: int j clin pharm doi: . /s - - - sha: doc_id: cord_uid: tapkjb nan pharmacy, sint maartenskliniek, ubbergen, pharmacy, radboud university medical centre, nijmegen, clinical pharmacy and toxicology, maastricht university medical centre, maastricht, netherlands please specify your abstract type: research abstract background and objective: according to literature adherence to statins ranges from to %. medication adherence is affected by both practical barriers and patient's beliefs about medication. however, physicians also have their beliefs about medication. several studies have shown that these beliefs also impact the decision of patients to agree with a particular treatment or not. as current published interventions on medication adherence (which focus predominantly on patients) are not or just partly effective, physicians' beliefs might be a promising target for interventions to improve adherence. however, there is currently no information available on physician's beliefs about statins and whether these beliefs affect patient's beliefs and adherence. therefore, the objective of this study is to examine whether physicians' beliefs about statins influence the beliefs and adherence of patients using a statin. setting and method: this cross-sectional study was conducted in gp practices and community pharmacies, between september , and march , . physicians' and patients' beliefs about statins were assessed with the beliefs about medicine questionnaire (bmq) specific. patients' adherence on statins was assessed with both the mars- and the morisky- questionnaires. please specify your abstract type: research abstract background and objective: nhs highland and nhs western isles are the most remote and rural health boards in the united kingdom, with high numbers of dispensing medical practices. a pilot is underway in dispensing practices with clinical pharmacists undertaking targeted medication reviews. a previous quantitative service evaluation demonstrated its value, with pharmaceutical care issues identified in almost all patients, the vast majority of which ( . %) were managed by the pharmacist without any need for general practitioner (gp) referral. the objective was to undertake a qualitative exploration of the service. setting and method: all patients and staff involved in the service were invited to participate. a semi-structured interview schedule was developed and piloted. telephone interviews were conducted with all consenting staff and a purposive sample of consenting patients recruited to the point of data saturation. interviews were audiorecorded, transcribed verbatim and analysed thematically. nhs ethics and research and development approvals were obtained. were the most confident with doacs (range from . to . %) please specify your abstract type: research abstract background and objective: patients are at risk of drug-related problems (drps) at transition points during hospitalization. the community pharmacist (cp) is often the first healthcare professional patients visit after discharge. cps lack sufficient information about the patient and so they may be unable to identify problems in medications, which may lead to dispensing the wrong drugs or dosage, and/or giving wrong information. we aim to assess the impact of a complex intervention comprising of medication reconciliation performed at discharge by a hospital pharmacist (hp) with communication between the hp and cp on drps during the days following discharge. setting and method: cluster randomized crossover trial involving medical and surgery care units (each unit corresponding to a cluster) in french hospitals during two consecutive -day periods, randomly assigned as 'experimental'(e) or 'control' c (usual care) periods. during the experimental period, the hp performed a medication reconciliation that was communicated to the patient's cp. main outcome measures: the primary outcome was a composite outcome of any kind of drp (prescription/dispensation, gap or patient) during the days following discharge assessed at day seven post-discharge by phone from patient and cp. the secondary outcomes were /unplanned hospitalizations assessed by phone contact at day after discharge and /the iatrogenic potential exposure scale from to for each patient established by a clinical team. analysis was conducted in intention to treat. results: hospitals corresponding to clusters enrolled patients ( e group v/s c group). no difference was observed on age, sex, autonomy, and number of drugs in home medication at admission and discharge. at day ; ( . %) patients in e group had at least one drp v/s ( . %) in c group (or . ; ic % [ . ; . ] p = . ). intervention was especially efficient for patient discharged from surgery unit (or . ic % [ . ; . ]) and aged less than years (or . ic % [ . ; . ] . although intervention decreased patient exposure to drp with high iatrogenic potential (from . to . % p \ . ), un-planned hospitalizations at day weren't different between groups ( . vs. . % p = . ). conclusion: medication reconciliation associated to communication between hospital and community pharmacists is efficient to decrease patient exposure to drp but not sufficient to decrease un-planed hospitalization. hp-pc : clinical pharmacists bridging health care levels by medication reviews in primary care katherine wendelbo *, , kristine lundereng namsos hospital pharmacy, central norway hospital pharmacy trust, namsos, levanger hospital pharmacy, central norway hospital pharmacy trust, levanger, norway please specify your abstract type: descriptive abstract (for projects) background and objective: nord-trøndelag county is sparsely populated and many inhabitants live far from the hospital. additionally, only half ( of ) of the municipalities have a local pharmacy. traditionally, namsos and levanger hospital pharmacies have performed quality audits of the implementation of drug administration procedures in primary care units. since , a service where clinical pharmacists participate in multidisciplinary medication reviews in municipalities throughout the county has been established. the objective of this poster is to describe the practical approach and design of the service. design: a descriptive report of an implemented clinical pharmacy service in primary care where clinical pharmacists, as part of multidisciplinary teams, perform medication reviews. results: medication reviews are performed on patients admitted to nursing homes and patients in home care, receiving help with handling of their drugs. primary care nurses prioritise patients (by selecting frail elderly with multiple co-morbidities and polypharmacy), usually five patients in each meeting. prior to the review, nurses collect medical information using a checklist including; diagnosis, drug-related symptoms, standard laboratory tests and an updated medication list. the clinical pharmacist receives de-identified medical information by postal mail or e-mail before the meeting. based on this information the pharmacist identifies possible drugrelated problems (drps) and provides recommendations on how to solve them. this is performed in a structured approach according to the integrated medicine management (imm) model. subsequently, the pharmacist visits the municipality and discusses the medication reviews in a multidisciplinary team meeting with nurses and physicians. in addition, the pharmacist gives lectures in a medication related topic (e.g. treatment of insomnia and anxiety, oral anticoagulants and cognitive side effects). following the meeting, the pharmacist reports the drps and suggested interventions to the multidisciplinary team, for further follow-up. during , totally medication reviews were performed in municipalities. in the same period, lectures were given by the clinical pharmacists. conclusion: this clinical pharmacy service enables multidisciplinary medication reviews even in municipalities with limited health professionals and resources. as a part of multidisciplinary teams, the clinical pharmacists contribute with medical competence. camille castel , arnaud de la blanchardière , vincent cattoir , guillaume saint-lorant *, pharmacy, infectious and tropical diseases, microbiology, chu caen, caen, france please specify your abstract type: research abstract background and objective: antimicrobial stewardship have clearly demonstrated their efficiency towards a more adequate use of antibiotics. since , the use of daptomycin, a ''critical last resort antibiotic'' has intensified in our hospital, occasionally outside the scope of its approved indications. this situation has led to the implementation of an antimicrobial stewardship and the drafting of local guidelines. the aim of this study is to analyse the evolution and pertinence of daptomycin prescriptions, after distribution of these guidelines within our institution. setting and method: a monocentric prospective study was conducted between july and november in a -bed university hospital. each daptomycin prescription recorded by pharmacy department was analysed by an infectious diseases specialist in the presence of the prescriber and considering local guidelines and the patient's clinical conditions. main outcome measures: the indicators chosen to determine prescription pertinence were: treatment indication, prescribed dose and other antibiotics associated with the daptomycin prescription. results: daptomycin prescriptions were analysed. observed indications were: sepsis ( %), infective endocarditis ( %), bone and joint infections ( %) and vascular prosthetic infections ( %). identified pathogens were: mrsa ( %), methicillin-resistant coagulase-negative staphylococci ( %), methicillin-sensitive staphylococcus aureus ( %), enterococci ( %) and methicillinsensitive coagulase-negative staphylococci ( %). daptomycin was prescribed as first-line treatment in % of cases. the mean dose was mg/kg/day [ - mg/kg/day] for a mean duration of days [ ; days] . local guidelines were followed in % of cases. daptomycin use was relevant for % of prescriptions. the irrelevant prescriptions triggered the modification or stoppage of antibiotic therapy in % of cases, respectively, generating an % decrease in consumption and an economy of over € for our institution. conclusion: this study shows the efficiency of antimicrobial stewardship in adequately using antibiotics, limitating ecological impacts, improving patient care and decreasing healthcare costs. it also shows that guidelines alone are insufficient to ensure a proper use of antibiotics. without a close prescription follow-up, constant reminders and sustainable evaluations, guidelines only affect a few prescribers. within the context of an ''antimicrobial crisis'', further development of guidelines and antimicrobial stewardship is essential to fight increasing bacterial resistances and requires a close collaboration between all healthcare professionals including pharmacists. interviews were transcribed verbatim and data were analysed using systematic text condensation. results: three major themes were identified: benefits, unrealised potential and criteria and barriers for success. ( ) benefits described by physicians included increased patient safety, increased awareness on drugs, and an ease of workload. drug interaction management was emphasized as one of the clinical pharmacists' most important work tasks, as well as being a resource for collaborating healthcare professions and to the patient himself. ( ) the clinical pharmacists expressed that they had an unrealised potential and could contribute to a greater extent in the multidisciplinary team than they did already. they mentioned education towards physicians and nurses, contribution in treatment decision-making and patient counselling as examples for possible extended work tasks. ( ) as criteria to succeed as a clinical pharmacist, physicians highlighted the importance of oral communication and physical presence on the wards. as barriers for integration in the team, the clinical pharmacists identified the physicians' lack of knowledge about the clinical pharmacists' skills as well as unclear expectations regarding their responsibilities. conclusion: physicians agreed that the clinical pharmacist represent a valuable contribution to the multidisciplinary team, where patient safety and drug interaction management are highlighted as main benefits. clinical pharmacists should to a greater extent educate healthcare professions in drug related topics and provide patient counselling. continuous effort on making the clinical pharmacist a natural part of the multidisciplinary team is crucial for the development of clinical pharmacy. by gathering perceptions from the collaborating professions as well as educating them on what clinical pharmacists can provide, we can develop a multidisciplinary team that enhances patient safety. hp-pc : assessment of dual antiplatelet therapy following acute coronary syndrome using grace and crusade sadeer fhadil * , paul wright, sotiris antoniou please specify your abstract type: descriptive abstract (for projects) background and objective: mortality and morbidity benefits of dual antiplatelet therapy (dapt) following acute coronary syndrome (acs) have been unequivocally demonstrated in a large body of evidence. with the availability of more potent antiplatelet agents, balancing ischemic and bleeding risks to prevent adverse outcomes is an on-going challenge, in particular, recognising that patients with high bleeding risk were excluded from clinical trials. grace and crusade scores stratify risk of mortality and in-hospital major bleeding post acs respectively. these tools should be used to support antiplatelet choice in light of newer more potent agents that equally pose a greater risk of bleeding. design: grace and crusade scores were calculated for patients presenting with acs. clopidogrel was recommended for patients with a high or very high crusade score (greater bleeding risk). ticagrelor was recommended for patients presenting with st-elevation myocardial infarction (stemi) or those with nsteacs with a grace score of intermediate or above (greater ischemic risk) and a crusade score of moderate or less (low bleeding risk). in either case, treatment was at the discretion of the clinician and patients received concomitant aspirin. a registry was collated of risk scores, diagnosis and choice of antiplatelet therapy. results: patients were included in the registry, of which ( %) presented with stemi and ( %) presented with nsteacs. of ( %) patients with a greater ischemic risk received ticagrelor as part of their dapt regime. advanced age, concomitant anticoagulation and those awaiting surgery were the most common reasons for patients with a greater ischemic risk to receive clopidogrel. ( %) had a high or very high crusade score. of these, ( %) received clopidogrel as part of their dapt regime. conclusion: risk stratification was streamlined using the data collection tool and useful to support choice of dapt. european society of cardiology (esc) guidance recommends use of established risk scores for prognosis and bleeding; however evidence to correlate to choice of dapt is lacking. outcome data is currently being reviewed and will provide further evidence to correlate choice of dapt to grace and crusade scores. please specify your abstract type: descriptive abstract (for projects) background and objective: in europe, approximately % of the patients with the human immunodeficiency virus (hiv) infection are co-infected with the hepatitis c virus (hcv). treatment recommendations in hiv/hcv co-infected patients are identical to those in patients with hcv mono-infection. however, potential drug-drug interactions (ddis) between antiretroviral agents and new direct-antiviral agents (daas) imply the need of a careful selection of the hcv treatment regimen. the aim of the present study was to evaluate the need of a change in the antiretroviral therapy (art) due to potential ddis in patients with hiv/hcv co-infection who started treatment for hcv with new daas. we also assessed the effectiveness of hcv treatment weeks after hcv treatment completion. design: we retrospectively registered clinical data about hcv and hiv management: hcv genotype, fibrosis metavir score, initial hcv viral load, hcv treatment and previous art regimen. we recorded the changes in art prior to starting hcv treatment and the reason of this switch (ddi, simplification or duplication of the therapy). results: between february and january , hiv/hcv coinfected patients started hcv treatment with a daas regimen. of them, had advanced liver disease (fibrosis score: f /f ) and were infected with hcv genotype . prior to starting hcv treatment, patients needed a switch in art regimen due to potential ddis with daas. simeprevir and the co-formulation ombitasvir/paritaprevir/ritonavir were the daas most frequently implicated in ddi with protease inhibitors or non-nucleoside reverse transcriptase inhibitors: / and / , respectively. also, we observed some changes of art due to other causes. five switches occurred to adequate the regimen (discontinuation of ritonavir in candidates to take the co-formulation ombitasvir/paritaprevir/ ritonavir or art improvement to decrease pill burden). as for hcv treatment effectiveness, / ( %) patients achieved sustained viral response weeks after therapy completion. conclusion: a large proportion of patients with hiv/hcv co-infection who initiate treatment with daas for hcv need to switch art due to potential interactions that may impact on effectiveness and safety of both treatments. additionally, some changes in art treatment are made to facilitate therapeutic adherence. these results highlight the need of a multidisciplinary approach in which interactions between art and hcv treatments should be carefully assessed. please specify your abstract type: descriptive abstract (for projects) background and objective: the potential impact of polymedication, iatrogenic events and medication error is a serious concern in hospitalized patients. clinical pharmacists can limit these risks by identify high risk. the aim of this study are to identify in six medical units high risk patients by using three predictive scores of rehospitalisation ( ps) , early mortality (charlson) and drug related problems (drp) . design: clinical and therapeutic variables in patients were collected through medical records and prescriptions by clinical pharmacists. scores were calculated during months in six units (internal medicine, n = ; nephrology, n = ; geriatrics, n = ; rheumatology, n = ; cardiology, n = and endocrinology, n = ). the data were analysed by mann and whitney test for the continuous variables and chi square test for the qualitative variables. the coefficient of correlation between the three scores were calculated by a pearson test for normal distribution and by a spearman test for non normal distribution. patients were considered at a high risk for re-hospitalization ( ps [ ) , early mortality (charlson [ ) and iatrogenic events (drp c ) . results: in the general population, the average age was . ± . years old and the sex ratio was . . the average treatment used was . ± . charlson scores were higher in geriatric unit ( . ± . ) follow by medical interne unit ( . ± . ). the ps and drp scores were higher in nephrology unit respectively . ± . and . ± . follow by internal medecine unit . ± . and . ± . . on contrary the rheumatology unit presented the lower level for the three scores. patients were considered at high risk for three scores, % (n = ) in nephrology unit (almost % of unit), % (n = ) in geriatric unit, % (n = ) in internal medicine unit, % (n = ) in cardiology unit, % (n = ) in endocrinology unit and % (n = ) in rheumatology unit. conclusion: knowledge of the variables associated with these predictor scores could help clinical pharmacists to prioritise various medicine units and target those at risk. we identified especially three units at risk: nephrology, geriatric and internal medicine. thanks to these results, clinical pharmacists can rapidly and efficiently target patients who present iatrogenic and/or re-hospitalization risks. design: a retrospective observational analysis was conducted in our hospital, based on medical records of patients presenting atrial fibrillation (af) and treated by doacs from january to may . to identify patients hospitalized due to severe bleeding, we analysed prothrombin complex concentrates (pccs) and activated pccs prescriptions, as well as pharmacovigilance declarations. results: patients were treated with doacs: with rivaroxaban ( . %), with dabigatran ( . %) and with apixaban ( %). fifty-nine ( . %) patients experienced at least one bleeding leading to hospitalization: with rivaroxaban ( . %), with dagibatran ( . %) and with apixaban ( . %). thirty-eight severe bleeding were identified ( . %): occurred with rivaroxaban ( . %), with dabigatran ( . %) and with apixaban ( . %). they included intracranial bleeding ( %) and gastro-intestinal bleeding ( %). seven haemorrhages resulted in hypovolemic shock (dabigatran: , rivaroxaban: , apixaban: ) and of them were fatal (dabigatran: ). rates of bleeding (p = . , v test) and of severe bleeding (p = . , v test) were not statistically different for the three molecules. in case of major haemorrhage, the recommended factor concentrate in our protocols differs between the anticoagulant. with dabigatran, the antidote idarucizumab ( g, intravenously) should be administered, without waiting for plasma concentration results. with rivaroxaban, apixaban or unknown doacs, pcc ( - units/kg) is indicated. in case of pcc failure, activated pcc ( - units/kg) is suggested. pcc, activated pcc or idarucizumab ( ) were used in / patients ( %). in rivaroxaban and apixaban-related haemorrhages, patients received activated pcc: two had a ui/kg dose and one had a ui/kg dose. regarding dabigatran-related bleeding, one patient received pcc instead of idarucizumab. compliance with local recommendations was % ( , p [ . , v test) . pharmacovigilance reports were issued. conclusion: management of doacs-associated severe bleeding in our hospital respects local protocols. it should also be pointed out that patients with life threatening bleeding may benefit from pcc. however, the risk of thrombosis associated with pcc must be weighed against the risk of haemorrhage. since specific antidotes are emerging, like idarucizumab or andexanet alpha, new guidelines for doacs-related haemorrhage are expected. please specify your abstract type: descriptive abstract (for projects) background and objective: this project is part of a prospective quasi experimental proof-of-concept investigation of a clinical pharmacist intervention to reduce drug-related problems among people admitted to a ward in a rural hospital in northern sweden. the aim of this particular study is to explore doctors' and nurses' expectations of having a ward-based pharmacist providing clinical pharmacy services in a rural hospital. design: eighteen face to face semi-structured interviews were conducted with a purposive sample of doctors and nurses working on the ward were the clinical pharmacy service was going to be implemented. semi-structured interviews were digitally recorded, transcribed and analysed using thematic analysis. results: the majority of participants had limited experience or a vague idea of what pharmacists are able to do in a ward. most participants described traditional roles such as inventory, drug distribution and dispensing. most respondents were unaware of the pharmacists' knowledge, skills and competences. for some it was unclear how having a clinical pharmacist in the ward was going to impact on their workload this was particularly important for the nurses. some doctors (mainly experienced) were concerned that having a pharmacist may mean losing or not gaining competence on drugs. for others it was unclear how the pharmacists' will work with patients or what clinical skills they have. however most participants were positive about the implementation of the new service. conclusion: this study provided a rare opportunity to explore the doctors' and nurses expectations of the role of clinical pharmacists before a clinical pharmacy service was implemented. the results showed that the participants' expectations of the clinical pharmacist role were unclear. to successfully implement clinical pharmacy services in a clinical pharmacy ''naïve'' setting; roles, clinical competence and responsibilities should be clearly described. furthermore, it is important to focus on inter professional collaborations between doctors, nurses and pharmacists. practical for the local hospital setting. seven out of experts agreed with pharmacist prescribing for the conditions identified. pharmacists (n = ) were more willing to prescribe antihypertensive and antidiabetic medication ( . %) when compared to oral anticoagulants ( . %). these values are higher than those obtained by vella in . the majority of pharmacists ( . %) recommended that pharmacists should take up further studies to a master or doctorate level degree in a clinical aspect in order to be authorised to prescribe. conclusion: the developed framework for pharmacist prescribing and the guidelines developed for pharmacist prescribing of oral anticoagulants and pharmacotherapy of hypertension and diabetes mellitus were shown to be reliable and were accepted by pharmacists and physicians. please specify your abstract type: research abstract background and objective: valproic acid (vpa) and its derivates and mycophenolate mofetil (mmf) and mycophenolic acid used during pregnancy increase risk of congenital malformation and cognitive impairment. thus, the french national agency for medicines and health products safety (ansm) decided to establish new conditions of prescription and dispensation of drugs containing vpa (may ) and mmf (april ). a signed care agreement and a co-prescription of contraceptives are now mandatory in the drug dispensation for reproductive-age adolescent girls and adult women. this study will describe the impact of these new guidelines on our practice. our objective is to compare the vpa and mmf media coverage and the impact on the prescriptions. setting and method: we compared the mass communication between vpa and mmf on social media, webpages and journal article (public and professional journal) on google and googletrends in the first months around these new rules. we combined different keywords such as ''accord de soins'' and the drug name. in the same time, we collected and analysed vpa and mmf prescribing and dispensing data and compared it to the data for the first months. main outcome measures: results: just before the vpa rule, the vpa was presented in the general press as the new health scandal after benfluorex mediator°w ith google searches in march compared to searches usually per month. simple research combining keywords reveal always more than twice more webpages concerning vpa than mmf. at the same time, a patients association (renaloo for renal failure) wrote to the ansm to contest the new rule with the double contraception and without any consultation of patients association. in our daily practice we also faced some physician reluctant to sign this prescription agreement with patient (too many agreements already asked, decision of ansm without any consultation of learned societies). the care agreements are kept in the patient records, a statement ''care agreement signed'' is reported in the electronic prescription of vpa and mmf. the overall consumption of mmf and vpa increase for respectively the first and months after rule implementation (from + to + %) except for the micropakin mg. the months mmf data will be presented for the final communication. conclusion: the media pressure and the new regulation have an impact on prescription trends. these new prescription and dispensing rules concerned two different contexts: pathology, media coverage, possible drug alternatives. we were faced to some difficulty in implementing the new guidelines, which reveals a certain reluctance of the prescribers or the patients represented by associations. tdmp : vancomycin trough serum concentrations are frequently subtherapeutic in a population of critically ill patients: a prospective observational study please specify your abstract type: descriptive abstract (for projects) background and objective: to design and characterise a framework of international pharmacy standards for pharmaceutical care application on oral anticoagulation for prevention of atrial fibrillation (af) related strokes. design: literature review (including existing international guidelines and quality measures) was conducted to characterise the standards and design an international framework for pharmaceutical practice application on oral anticoagulation for prevention of af-related strokes. expert opinions were sought through a delphi method to reach consensus on the framework domains and standards. results: the framework consisted of twelve overarching standards, which were defined and grouped into four domains as follows; ([personal care package]:-communication with patients, support decision making process, education and counselling, adherence. [medicines optimisation]:-clinical review and therapy optimisation, initiation and control, maintenance, supply and transfer between care settings. [workforce]:-workforce planning, training and development, analysing information; and [governance]:-assurance of service provision) specific to oral anticoagulation in prevention of af-related stroke. each standard was also categorised within dimensions and supporting statements to describe what a quality pharmacy service should deliver. a total of forty-five dimensions and twelve statements were incorporated into the framework. conclusion: a clearly defined framework of international standards was developed as a clinical tool and quality assurance to optimise the delivery of care for oral anticoagulation in prevention of af-related strokes. it will support pharmacists and their teams to develop their professional practice, improve services, and deliver safe and high quality patient care across all pharmacy settings. poster discussion forum i: community pharmacy and public health cp-pc : nurses' and pharmacists' learning experiences from participating in inter professional medication reviews in primary health care: a qualitative study hege t. bell *, , anne gerd granås , ragnhild omli , ingela enmarker , aslak steinsbekk nord university/ntnu, trondheim, hioa, oslo, nord university, namsos, norway, department of nursing, Østersund, sweden, ntnu, trondheim, norway please specify your abstract type: research abstract background and objective: traditionally, drug prescription and follow up have been the sole responsibility of physicians. however, interprofessional medication reviews (imrs) have been developed to prevent drug discrepancies and patient harm. what participating nurses and pharmacists learn from each other during imr is poorly studied. the aim of this study was to investigate nurses' and pharmacists' perceived learning experience after participating in imrs in primary health care for up to years. setting and method: a qualitative study with semi-structured focus group interviews and telephone interviews with nurses and pharmacists with experience from imrs in nursing homes and home based services. the data was analysed thematically by using systematic text condensation. main outcome measures: a qualitative method is useful when looking at objects from the perspective of how they are experienced. results: sixteen nurses and four pharmacists were interviewed. the nurses' perception of the pharmacist changed from being a controller of drug management routines towards being a source of pharmacotherapy knowledge and a discussant partner of appropriate drug therapy in the elderly. the pharmacists became more aware of the nurses' crucial role of providing clinical information about the patient to enable individual advice. increasingly the nurses learned to link the patient's symptoms of effect and side effect to the drugs prescribed. with time both professions jointly spoke of an increased awareness of the benefit of working as a team and the perception of contributing to better and more individual care. conclusion: imrs in primary health care meet some challenges especially concerning how to ensure participation of all three professions and how to get thorough information about the patient. possible solutions might be to use shared communication tools like internet based communication programs and to introduce the patient as a participant at the imrs. please specify your abstract type: research abstract background and objective: international good pharmacy practice guidelines describe how pharmacists should counsel the patients about their medicines, offer additional services where needed, and intervene at drug related problems. daily practice often differs from theory. this study aimed at illustrating the whole process of prescribed medicines dispensing in daily community pharmacy practice. part b of the project focuses on pharmacists' opinions. setting and method: community pharmacies in basel, switzerland, were invited in random order for study participation. one master student in pharmacy performed non-participant observations during day at each included community pharmacy. at dispensing of prescribed medicines, patient data, content of counselling, communication style, and provision of further services (e.g. follow-up offer) were documented on a checklist with predefined themes. interventions were documented systematically. a semi-structured interview on the pharmacists' opinions about the counselling, triggers, facilitators and barriers, and the documentation of interventions was conducted at each community pharmacy. main outcome measures: counselling content at prescription dispensing by numbers and by pharmacists' opinions; barriers, facilitators, and triggers for counselling at prescription dispensing. results: in march and april , of invited community pharmacies participated in the study. out of documented observation periods, encounters were analysed (first prescription: /refill prescription: ). counselling was provided to ( . %) clients with an average of . (± . ) themes per encounter. a total of clients refused counselling. themes most counselled at first and refill prescription dispensing were: drug intake ( / ), dosage ( / ) , and administration ( / ). for the pharmacists (n = ), most important themes to be discussed at first prescription dispensing were indication ( ), administration ( ), and anamnesis ( ); for refill prescription dispensing they were adherence ( ), therapy benefits ( ), and adverse effects ( ). the majority of pharmacists ( ) felt that it was their obligation to ask questions about patients' health during the dispensing of prescription medicines and named trigger (e.g. patient knowledge gap, patient motivation, interactions), but one-third reported difficulties with it. barriers were refusal by patients ( ), communication problems (language, ), lack of medical data ( ) , and lack of time ( ) . conclusion: a discrepancy in counselling content by observation compared to pharmacists' opinions was revealed. this might indicate that pharmacists are aware but hindered by barriers to practice according to good pharmacy practice guidelines. please specify your abstract type: research abstract background and objective: the workforce vision in scotland envisages 'making more and better use of technology …to increase access to services and improve efficiency' across the healthcare interface. services offered by community pharmacy remain limited by lack of shared access to patients' clinical information. in scotland, every patient has a unique identifier, their chi (community health index), which facilitates identification of/searching for patient records. the aim of this research was to explore the experiences of community pharmacists granted clinical portal access to patients' records. setting and method: from april , community pharmacists across nhs tayside (n = ) who had completed technical and information governance training were invited to maintain a portal log of their experiences of using the clinical portal to access patient records. each was asked to record when/why they considered accessing a patient's record and whether their information needs were met. portal logs were subject to independent summative content analysis by two researchers. this study gained ethical approval from robert gordon university. main outcome measures: not applicable. results: clinical portal logs were received from most participating pharmacists (n = / ). two were unavailable (moved to hospital setting; maternity leave). a third had not had occasion to access the clinical portal which he speculated was due to not working at weekends but also raised concerns about gaining patient consent. frequency of seven identified themes provided a partial indication of balance of reasons for usage. firstly (# ), to confirm a patient's prescription (n = ), secondly (# ), for additional information (n = ). less frequently (# ), portal access was to check repeat medications (n = ). other reasons for access were (# ) to check hospital discharge (n = ) followed by (# ) check on multi-compartment appliance aid status (n = ) or (# ) check the emergency care summary (n = ). there were also instances (# ) when portal access was not found to be helpful (n = ) so traditional offline routes were followed. conclusion: preliminary findings indicate mainly positive experiences with no technical issues raised and community pharmacists' information needs largely met. although limited to a small number of pharmacists in only one health board, findings support scottish policy aims, including 'prescription for excellence.' further work is underway around patients' perspectives of community pharmacist access. please specify your abstract type: research abstract background and objective: multicompartment compliance aids (mcas) such as the multidrug punch cards pharmis Ò are used to support patients in the daily management of their medication. solid oral medicines are unpacked from their original packaging and repacked in mcas although controversy exists about the stability of repackaged medicines. different countries published contradictory lists of medicines not recommended for repackaging. we aimed to define and apply criteria able to assess visual alteration of medicines repackaged in mcas. setting and method: eight criteria describing physical alteration of tablets/capsules were retrieved from the who international pharmacopoeia : chipping, swelling, capping, rough surface, cracking, crushing under pressure, mottling, and discoloration. absence of one criteria gives point. a maximum score of points can be obtained. twenty-two critical medicines and three half tablets were repackaged in the multidrug punch cards pharmis Ò and stored at accelerated conditions ( °c/ % rh) for weeks. original blisters of medicines were stored at room temperature as control. each tablet/capsule was visually inspected after , , and days. main outcome measures: score according to eight criteria. results: after weeks, tablets/capsules including of the half tablets showed no visual alteration and were identical to controls. a reduced score ( - points) was given to seven repackaged medicines and one half tablet within weeks: madopar Ò ( ), pravastatin sandoz Ò ( ), carvedilol mepha Ò ( ), plavix Ò ( ), pantoprazol nycomed Ò ( ), adalat Ò cr ( ). swelling and rough surface were the most frequent. chipping and capping were not observed. conclusion: our eight visual criteria are able to detect physical alteration of repackaged solid oral medicines under stress conditions. in absence of reliable data we suggest to apply this simple quality control for repackaged medicines in pharmacy practice. because chemical stability testing is not feasible in practice, pragmatic solutions are sought. further studies are needed for storage at room temperature. cp-pc : drug-related problems and symptom burden in nursing home residents kerstin bitter *, , ulrich jaehde , christina pehe , gabriela heuer , manfred krü ger clinical pharmacy, university of bonn, bonn, aok rheinland/ hamburg, pharmacists' association north rhine, düsseldorf, germany please specify your abstract type: research abstract background and objective: drug-related problems (drp) are common in the elderly due to polymedication. community pharmacies supplying drugs to nursing homes may play an important role in detecting and solving drp in nursing home residents. this project aims to evaluate whether community pharmacists can enhance the medication safety of nursing home residents by solving drp by means of a simple medication review (mr). furthermore, the applicability of a new tool to detect symptoms as potential adverse drug reactions in elderly patients should be tested. setting and method: nursing home residents at the minimum age of years insured by aok rheinland/hamburg and regularly taking at least five drugs per day were invited to participate. pharmacists performed a mr based solely on the patients' medication data, including dosage regimens of the nursing home and self-medication data. the detected and solved drp were counted. additionally, a simple questionnaire (sympel) was distributed to the patients periodically in order to assess their symptom burden. main outcome measures: frequency and type of the patients' drp as well as their symptom burden before and after the mr. results: after testing the feasibility of this intervention in a pilot study, patients were included in the main study so far. in average, the pharmacists identified two drp per patient and reported to the responsible general practitioner (gp) . as in the pilot study, most frequent drp documented by pharmacists were drugdrug-interactions ( %). % of the pharmacists' recommendations were accepted by the gp. % of the patients took at least one drug considered as potentially inadequate in the elderly. out of six different symptoms, patients reported dizziness and bruises most frequently. conclusion: pharmacists can detect many drp in nursing home residents by means of a simple mr. however, the full potential of this service to solve drp can only be exploited if the cooperation with the gps is improved. please specify your abstract type: research abstract background and objective: medicines for rare diseases (rd) are costly and have limited efficacy evidence but they represent around % of all innovative medicines. therefore, countries are facing challenges in providing patient access to them. the purpose of the study was to assess the patient access for slovenia and compare it with other european countries in the last decade. setting and method: the medicines for rd that obtained marketing approval between and via centralised procedure were included in the study based on the orphanet list from january . using the quarterly ims health database, sales data were analysed for slovenia and other european union countries, norway and switzerland. patient access was assessed for each country and comparisons between the countries were made using the three main outcome measures. main outcome measures: the number of medicines for rd available; time to first continuous use after marketing approval; total pharmaceutical expenditure for medicines for rd in euros. results: altogether, medicines for rd were approved between and . complete sales data were available for medicines which were included in the comparison. for germany and the united kingdom the continuous use of ( %) and ( %) was observed, respectively. the following italy, france, denmark and sweden use - % of all the medicines. in slovenia, ( %) medicines for rd were introduced. germany and the united kingdom times to first continuous use were the shortest (median time - months after marketing approval). median times below months were observed for norway, sweden, austria, the netherlands, france and switzerland. other countries were slower in enabling first continuous use (median time from to months). germany, france, switzerland had the largest pharmaceutical expenditure per inhabitant in ( . , . and . euros/inhabitant) while slovenia amounted to . euros/inhabitant. in slovenia more than a half of the medicines for rd approved in europe are used which ranks it in the middle of all the countries in comparison. comparing to the important european pharmaceutical markets like germany, united kingdom, france and italy, slovenia's median time to first continuous use is longer and pharmaceutical expenditure per inhabitant for these medicines is lower. pec : mapping of the dlqi scores to eq- d utility values using ordinal logistic regression and monte carlo simulations: is it plausible? please specify your abstract type: research abstract background and objective: converting dermatology life quality index (dlqi) scores to generic measure data would allow utility calculations and enable cost-effective analysis. this would meet the needs of health technology assessment agencies (htas) such as nice, who preferentially use the general health measure eq- d. the dlqi is a specialty-specific measure unlike the eq- d, a generic measure from which utility values can be derived. often several measures are implemented in studies with increased cost and patient burden. ordinal logistic regression (olr) was used to develop a model to convert dlqi scores to eq- d based utility values for use in economic appraisal of medicines. setting and method: data from patients were randomly divided into estimation and validation sets to fit and test the model. a series of ordinal logistic regressions were fitted in spss v for the five eq- d dimensions based on age, sex and all individual items of the dlqi as predictors. the model produced three estimated probabilities per subject per eq- d domain. using these estimated probabilities, a series of monte carlo (mc) simulations were run for each subject resulting in predicted domain responses. from these, utility values were calculated and compared to actual patient values. main outcome measures: conversion of dlqi scores to eq- d domain data results: there are conceptual overlaps between items of the dlqi and eq- d. the validation data set (which was not included in the creation of the model), demonstrated that the models were highly predictive compared to actual responses, except for minor differences for the pain/discomfort domain. for example, for the eq- d ' mobility' domain, patients answered 'no' (predicted and patients answered 'some or extreme' (predicted ) . we examined the model's latent variables, which also demonstrated high predictability at individual level. for example for the 'usual activities' domain the mean latent variable scores were - . , - . and - . for those responding 'no', 'some' and 'extreme' respectively, showing a clear increase in the scores with response. after excluding subjects with missing variable data there were patients in the estimation set and in the validation set. the model was shown to be highly predictive and repeated simulations demonstrated a stable model. the average predicted utility value for the entire validation set ranged from . to . across the mc simulations compared to the actual average utility value of . . conclusion: using olr, we have developed a method of mapping the disease-specific dlqi onto the eq- d: utility values may then be derived for population data sets, and possibly for individuals. the olr technique could be used to convert data from other disease-specific quality-of-life measures to generic utility data for incorporation into cost-effective analyses, greatly enhancing the potential value of such information. tomi laptoš *, , tanja kersnik levart hospital pharmacy, the division of paediatrics, department of nephrology, university medical centre ljubljana, ljubljana, slovenia please specify your abstract type: descriptive abstract (for projects) background and objective: having to take medication during time in school may present certain discomfort for some children. suboptimal dosing regimens (i.e. dosing more frequent than determined by drug's trough:peak ratio) can be prevented by a clinical pharmacist's overview and therapy optimization. the aim of the study was to review and evaluate dosage regiments in paediatric patients on antihypertensive therapy. dosage regiments are defined as schedule of doses of a therapeutic agent per unit of time and the amount of a medicine to be given at specific time. design: electronic health records (ehr) review, evaluation of actual dosage regiments against regiments recommended in smpcs and clinical database (uptodate Ò ), a consecutive case series study. results: ehrs of patients, admitted to or discharged from the department of nephrology of the division of paediatrics, umcl in with suspected icd- diagnoses from i to i were reviewed. patients were excluded (diagnosis not confirmed or lifestyle-change disease management only). the remaining patients (average age . years (range - )) received daily on average . medications (range - ) in . individual doses (range [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . most frequently used drugs were perindopril (n = ), ramipril (n = ), amlodipine (n = ), bisoprolol (n = ) and doxazosin (n = ). patients received ex tempore oral suspensions. dosage regimen was not optimized in % (n = ) of the patients, among those % (n = ) receiving one medication only and % (n = ) receiving more than one medication where at least one was not optimized. furthermore, % (n = ) patients on stabledosage therapy (no dosage change of either medication in last months with satisfactory clinical outcomes) were eligible for fixeddose combination medication. with optimized dosage regimens patients would receive daily on average . medication (range - ) (- %) in . individual doses (range - ) (- %). the difference was statistically significant ( % ci, p \ . ) in both cases. conclusion: our data show that the majority of the paediatric patients on antihypertensive therapy ( %) received their medication in optimal dosage regimens. however, with an estimated every fifth patient not being on optimal dosage regimen, a multidisciplinary approach is crucial to assure that the individual patient achieves the best clinical, humanistic and economic therapy outcomes. ph : polypharmacy management programmes in the elderly: a case study in greece dimitra gennimata *, , christos kampolis , aggelos vontetsianos , jennifer mcintosh , alpana mair , on behalf of simpathy consortium please specify your abstract type: descriptive abstract (for projects) background and objective: polypharmacy management and medication adherence in the elderly are significant public health issues throughout the european union (eu). simpathy (stimulating innovation management of polypharmacy and adherence in the elderly) is a consortium of organizations representing eight european countries, aiming at stimulating innovation around management of appropriate polypharmacy and adherence, ultimately providing tools for eu policy makers to develop and/or improve, implement and evaluate programs addressing these issues. design: a mixed-methods case study was carried out in greece, to identify policies on the management of polypharmacy and adherence issues in the elderly. a desk review of the polypharmacy and adherence policies at the government, regional and institutional level has been completed. key informant interviews were conducted with policymakers and health professionals responsible for developing and implementing strategies. focus groups consisting of policymakers, clinicians and patients validated the research findings. results: although e-prescription implementation is widespread (& % coverage nationwide) and disease-specific guidelines have been developed, polypharmacy management is only associated with direct economic indicators. no formal policies or programmes are identified. significant contributions are coming from different health professional organizations that have chosen to provide expanded services to their patients, aiming at optimising drug therapy and thus polypharmacy management and medication adherence in the elderly. community pharmacists offer pharmaceutical care to patients, which includes management of prescribed medication, otc remedies, vitamins and supplements and food-drug interactions. hospital pharmacists in some state (public) hospitals review medication for inpatients and out-patients, communicate with prescribers and confirm the ''benefit-no harm'' principle in the prescribed medication (e.g. incompatibilities, side effects, -rights of medication). medical doctors, mostly general practitioners and some specialized ones, usually in primary healthcare settings, keep health records of their patients and have an overview of all administered medication. however, key barriers still remain the lack of coordination of institutions and authorities and overlap of their responsibilities, healthcare workforce and infrastructure shortages and several cultural issues. conclusion: all initiatives to medication management and medicines optimisation are provided without directive from national policies or guidelines. therefore, these activities rely on the goodwill of the health professionals to address pharmacotherapy and therefore polypharmacy management but they are not necessarily representative of what is happening nationwide. a national policy to implement the management of polypharmacy nationwide could mobilise the willingness of health professionals and ensure consistency of care. development and implementation of this policy should build on the grassroots efforts currently underway in this area. ph : clinical profile and treatment discontinuation in a tuberculosis control state programme in brazil: preliminary results from sinan database simone s. bezerra , mara guerreiro *, , , nathany pessoa , maria paula athayde , rodrigo auad , joão josé gomes , josé lamartine soares sobrinho post graduation program in therapeutic innovation, federal university of pernambuco, recife, pernambuco, brazil, centro de investigação interdisciplinar (ciiem), instituto superior de ciências da saúde egas moniz, monte de caparica, please specify your abstract type: research abstract background and objective: challenges remain in tuberculosis (tb) control. discontinuing treatment can leave patients infectious and contributes to the emergence of resistance. this study aimed to describe the clinical profile and cure and discontinuation rates of tb patients enrolled in the pernambuco tuberculosis control programme (pect). setting and method: the study was conducted in three sites in recife, brazil, designated a (one polyclinic plus eight general practice units), b (one hospital for medium-complexity patients) and c (one hospital for high-complexity patients). data were extracted from the notifiable diseases information system (sinan) for all pect outpatients, from / to / (n = ). analysis was performed with the aid of action for excel; there is on-going analysis to further explore differences across sites. ethical approval was granted. main outcome measures: clinical form of the disease, hiv testing, new cases, cure and treatment discontinuation. results: sociodemographic data were available for sites a and b only. most patients were male ( %, n = ), with age raging from to years old ( . %, n = ); most had a low education level ( %, n = ) and low socioeconomic status ( %, n = ). the most common clinical presentation was pulmonary tb. most cases were new ( . %, n = ); recurrence and enrolment after discontinuation were respectively . and . % (n = and n = ). with respect to hiv, . % of patients were seronegative (n = ); about a third ( %; n = ) had not performed hiv test. rates for cure were respectively . % (n = ), . % (n = ) and . % (n = ) in the sites a, b and c. correspondent rates for treatment discontinuation were . %(n = ), . % (n = ), . % (n = ), respectively. tb-related mortality ranged from in site c to . % in site b. conclusion: missed hiv tests may represent undetected tuberculosis/ hiv coinfection. site b presented the highest rates of intrapulmonary plus mixed tb forms, discontinuation of treatment and tb-related mortality; additionally, it had the lowest rate of enrolment after treatment discontinuation. patients co-infected with tb and hiv are firstly referred to this site, which may explain this finding. our findings may help managers allocating resources and assist clinical pharmacists in planning their interventions. findings also suggest the need of more intensive interventions in tb patients, such as pharmaceutical care programmes. please specify your abstract type: research abstract background and objective: pharmacists working in primary health clinics have various roles. pharmaceutical care is one of them. how to provide this service varies across countries and settings. the most optimal way to provide pharmaceutical care is important to define when developing clinical pharmacy services in a new setting such as primary care practices. general practitioners are key stakeholders in this endeavour. the aim of this study was to find the most optimal approach to providing pharmacist-led pharmaceutical care in primary health care clinics in iceland in collaboration with general practitioners. setting and method: action research provided the framework for this research. data was collected from pharmaceutical care interventions with patients, field observations, field notes, and interviews with general practitioners over the period of the study. the study ran from september to june . three separate semi-structured in-depth interviews were conducted with five general practitioners from one primary health care clinic in iceland at different time points throughout the study. pharmacist-led pharmaceutical care was provided to patients (n = ) before and between general practitioners' interviews. the study settings was a primary health care clinic in reykjavik area and the patients' homes. main outcome measures: how to provide pharmaceutical care in collaboration with general practitioners in the icelandic health care environment. results: direct contact between pharmacists and general practitioners over short distances are essential to providing optimal pharmaceutical care services. pharmacist's access to medical records is necessary even though face-to-face communication between pharmacist and patients are most effective in providing pharmaceutical care. pharmacist-led clinical service was deemed most needed in dose dispensing polypharmacy patients. patients require more information about drugs prescribed to them coupled with an accurate drug list with greater detail. conclusion: the most efficient collaboration when pharmacist and general practitioner is obtained when they work side by side at the primary health care clinic. when new services are developed it is vital to identify different requirements of the primary health care clinics to optimize the running of a clinical pharmacist service. ph : accompaniment of patients treated with oral chemotherapy: a survey on patients' experience laure napoly *, , pascal paubel , , sylvie burnel oncorif -regional cancer network, health law and health economics deparment, health law institute, inserm, umr s , paris descartes university, sorbonne paris cité, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: antineoplastic agents taken orally are more and more used in cancer care. these medicines confer autonomy to patients. although, they may cause adverse effects that can lead to treatment adherence issue, unjustified hospitalizations, or premature treatment interruption. proper accompaniment of patient can prevent these issues. in order to define how to organize this accompaniment, we conducted a survey on patients' experience. the objective is to describe patients care pathway and identify their needs. design: a descriptive, qualitative, prospective, survey was conducted using self-administered questionnaires, in hospitals of ile-de-france region. inclusion criteria were: having being treated with oral neoplastic agents during minimum months, age [ , solid tumor, no concomitant iv chemotherapy. collected data were: patients' sociodemographic and clinical profile, their insight about different steps of care pathway (information, treatment delivery, follow up), their behaviours and interactions with healthcare professionals, and their overall opinion. results: patients were recruited in six hospitals. their sociodemographic and clinical characteristics were variable. % were treated with targeted therapy, % with cytotoxic agent, and % with endocrine therapy. patients showed high satisfaction for given information at the beginning of the treatment. principal source of information identified was the oncologist ( %). while the delivery of treatment, % of patients beneficiated of advices from the pharmacist. accompaniment of patients during treatment seemed unequal, with: a frequency of visits with the oncologist ranging from less than - months; % of patients not knowing any telephone number they can call in case of worries or questions about their condition or treatment; % not remembering any particular accompaniment treatment (visits or calls from nurses or doctors for example). for adverse effects management, three principals actors were identified: oncologist ( %), general practitioner ( %) and pharmacist ( %). regarding the use of oral chemotherapy, patient are satisfied with: it's comfort ( %); being more actively involved in their cancer care ( %); and state not having any anxiety taking chemotherapy home ( %) . asked openly about their concerned and needs three major concepts were identified: high concern about adverse effects, positive feedback about maintaining contact with health professionals between cures and difficulties of communication between health professionals. conclusion: there is a high satisfaction regarding oral chemotherapy and health professionals. the oncologist has a primordial place in patient pathway, whereas implication of pharmacist and general practitioner stays variable. adverse effects are a major concern that needs proactive accompaniment. the variability of our results suggests that accompaniment must be flexible and adapted to the needs of each patients. the key to flexibility could be good coordination and communication between healthcare professionals. ph : general beliefs about medicines among independent elderly adults in sweden: data from an rct lina hellström *, , , victoria throfast the pharmaceutical department, kalmar county council, ehealth institute, linnaeus university, kalmar, sweden please specify your abstract type: research abstract background and objective: there is a need to improve prescription and use of medications by the elderly. the objective of a recent rct was to investigate the effects of e-learning about medicines among elderly adults. a positive impact on the primary outcome measure, knowledge about medicines, is reported elsewhere. a secondary outcome measure, general beliefs about medicines, is reported below. setting and method: the study was a randomized controlled trial in elderly people (aged c years). participants were recruited from patient associations and pensionerś associations. participants were randomized to either an intervention group that participated in the e-learning, i.e. internet modules with video and audio, or a control group that did not take part in the e-learning. post-intervention data was collected using paper-based questionnaires, completed within two weeks after agreeing to participate in the study. the general beliefs about medicines questionnaire (bmqgeneral), comprising the subscales necessity, harm and overuse, was used to elicit beliefs. higher scores indicate stronger endorsement of scale constructs (range [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . main outcome measures: bmq-general subscale scores. results: a total of elderly people were included in the study, in the intervention and in the control group. the mean age in the total population was . years and % were women. eleven percent did not use any prescribed drugs while % used more than five prescribed drugs. the patients' scores were very similar in the two groups for all three bmq subscales. the median ''overuse'' score was in the intervention group versus in the control group, the median ''harm'' score was (iqr - ) in both groups and the median ''necessity'' score was in both groups. in the total population the most commonly expressed negative beliefs referred to overuse of drugs. . % of respondents agreed with the statement ''if doctors had more time they would prescribe fewer medicines'', . % stated ''doctors prescribe too many medicines'', and . % stated ''doctors place too much trust in medicines''. a majority of the respondents agreed with the four items on the ''necessity'' scale. for example ''medicines help people to live a better life ( . % agreed)''. conclusion: the studied e-learning intervention was not shown to have any impact on general beliefs about medicines. beliefs about medicines have been associated with a number of background variables which might explain why increased knowledge about medicines alone cannot change such beliefs. in general, respondents in the study had highly positive beliefs about the necessity of medicines. nevertheless, the results indicate that overuse of medicines is regarded as a problem. ph : maf-plus: pharmacists' contributions to provision of financial assistance for medications ian wee * , charlene ong, niron naganathar changi general hospital, singapore, singapore please specify your abstract type: descriptive abstract (for projects) background and objective: the medication assistance fund plus (maf-plus) is a government scheme introduced in singapore in to provide financial assistance to needy patients who meet pre-set criteria based on means testing. unlike previous schemes, maf-plus provides broader discretion to institutions when providing financial assistance. in our institution, pharmacists reviewed patients' case and medication histories, and filed recommendations to a multidisciplinary committee tasked with approving deserving maf-plus applications. the pharmacists' contributions to the committee, and the outcomes of the applications, are presented in this study. design: all maf-plus applications received between st october and st december were reviewed. pharmacists' comments for each application, where provided, were noted, as well as the range of medicines applied for, review approval rates, and cumulative percentage of available funds utilised. the effect of a recent widening of the scheme's scope to include notable high-cost items was also evaluated. results: between and , maf-plus applications were reviewed, of which ( . %) were approved. of the rejected applications, ( . %) were channelled to alternative financial assistance schemes on the recommendation of the pharmacists. the medicines most commonly applied for were intended for the treatment of cardiac ( . %), respiratory ( . %), and psychiatric ( . %) conditions. pharmacists' recommendations also led to a gradual expansion of our institution's list of pre-approved medicines-from in - to by end- . from onwards, pharmacists previewed increasing numbers of applications for high-cost medicines, particularly those for treatment of retroviral disease, hepatitis c, and rare diseases. cumulative utilisation of maf-plus funds (inclusive of annual replenishment) rose from . % in - to . % by end- , representing an average year-on-year growth of . %. conclusion: using a process of judicious previewing of maf-plus applications, and recommendations to the maf-plus committee, pharmacists contributed to a high percentage of patients receiving financial assistance for medications. despite a steep growth in the number of applications received between and , this approach helped to prevent over-extension in fund utilisation. pharmacists will likely be increasingly relied upon due to an anticipated rise in the number of applications for high-cost medicines. please specify your abstract type: research abstract background and objective: adolescents often treat themselves and take medications without parental supervision. lack of experience and knowledge of medicines in this age group frequently leads to inappropriate use of medicines and adverse drug reactions. data about the use of medicines among slovak adolescents and their knowledge of medicines have not been studied yet. setting and method: for our study we used the questionnaire method. the questionnaire contained multidimensional items with closed-ended and open-ended questions, which focused on the characteristics of the adolescentś health status, use of medicines, also in relation to parents and adolescentś knowledge and perception of medicineś risk. we distributed validated questionnaires for adolescents aged from to at secondary schools in all regions of slovakia. response rate was . %. questionnaires were finally analysed. the differences in the distribution of categorical variables between groups were evaluated using the chi square test. sas . . was used as statistical software. main outcome measures: to determine adolescentś knowledge of medicines in terms of efficacy, self-medication, safety of therapy and analyse which medicines are the most frequently used by adolescents. to compare adolescents with chronic disease and healthy ones from the perception of pharmacotherapy point of view. results: in the analysed group . % (n = ) of adolescents are treated for chronic disease. mostly they suffer from allergy ( . %, n = ) and skin diseases ( . %, n = ). adolescents with chronic disease use regularly prescription medicines ( . %, n = , p \ . ) and over the counter medicines ( . %, n = , p \ . ). this group of adolescents better accept the pharmacotherapy with parental supervision ( . %, n = , p = . vs. healthy adolescents) and they believe in effectiveness of prescription medicines ( . %, n = , p = . vs. healthy adolescents). most frequently prescribed medicines were azithromycin, levocetirizine, ofloxacin and over the counter medicines were ibuprofen, paracetamol, ascorbic acid. we found out in all group of adolescents that . % (n = ) prefer self-medication without check-ups, . % (n = ) used drugs in the last months without a prescription, . % (n = ) take over the counter medications independently without the supervision of parents, . % (n = ) buy medicines themselves in the pharmacy, . % (n = ) do not take medications as recommended, . % (n = ) believe that they have enough knowledge of medicines which they take, . % (n = ) resp. . %, (n = ) believe that prescription medicines resp. over the counter medicines are safe. conclusion: questionnaire analysis pointed out that slovak adolescents have not enough knowledge of medicines. the study provides new information in the field of risk perception and adolescentś knowledge of medicines in the slovak republic and highlights the areas that need to be studied in the future in terms of adolescentś education. federal university of pernambuco, state technical school prof. agamenon magalhães, recife, pernambuco, brazil please specify your abstract type: research abstract background and objective: the effectiveness of tuberculosis (tb) control programmes depends critically on patients completing appropriate treatment. this study aimed to outline the cure and discontinuation rates of patients enrolled in the pernambuco tuberculosis control program (pect), based on dispensing data. setting and method: the study was carried out in three sites in recife public health system, brazil, designated a (one polyclinic plus eight general practice units), b (one hospital for medium-complexity patients) and c (one hospital for high-complexity patients). data were collected between - / , through reports from the stock management software for public pharmacies (horus) for pect outpatients. reports corresponded to a total of patients ( , and in sites a, b and c, respectively). horus defines ''cure'' as medicines collection for three, six or nine consecutive months without interruption, depending on the treatment scheme; discontinuation is defined as non-sequential collection of medicines or treatment interruption for two consecutive months or more. patients were assigned an ''undetermined'' status if treatment was ongoing. data were inputted onto an excel spreadsheet and checked for accuracy. quisquared test, fisher's exact test and bootstrap analysis were performed with r statistical computing. ethical approval was granted. main outcome measures: cure and discontinuation rates for pect outpatients. results: demographic data are not available for the sample. rates for cure were respectively . % ( ), . % ( ) and % ( ) in the sites a, b and c, while rates for treatment discontinuation were . % ( ), . % ( ) and % ( ), respectively. discontinuation rates were significantly different among the sites a, b and c (p \ . ). bootstrap analysis showed that overall the proportion of patients with an ''undetermined'' status in each site did not significantly change these differences. conclusion: only site a had an acceptable discontinuation rate, in light of the world health organization recommendations. this deserves attention as default treatment leaves patients infectious for longer, increases the risk of poor outcomes and fosters resistance to antibiotics. pharmacists could use dispensing data to signal tb patients at-risk of discontinuation, and subsequently tailor interventions addressing its causes. site b had the greater number of patients which discontinued treatment. patients co-infected with tb and hiv are firstly referred to this site, which may explain this finding. our findings suggest the need of more intensive interventions in patients co-infected with tb and hiv, such as pharmaceutical care programmes. please specify your abstract type: research abstract background and objective: many efforts are done to organise good quality and safe pharmaceutical care. in general, the involvement of a hospital pharmacist or hospital pharmacy personnel in the process of medication reconciliation results in a reduction of the number of medication discrepancies. however, in case of emergency admissions this topic is still insufficiently studied. the introduction of good medication reconciliation on the emergency department (er) requires firm logistical and organisational efforts. we investigated the effects of a drug reconciliation intervention by pharmacy personnel during emergency admissions in order to identify discrepancies between medication lists taken by er physicians and by pharmacy personnel. setting and method: this observational, comparative, non-randomised intervention study was performed in . we calculated that a population size of patients was sufficient to perform reliable measurements. inclusion criteria: all patients presented at the er and admitted to a hospital ward \ after presentation with usage of one or more prescription drugs. exclusion criteria: age \ years, residency outside the region delftland, inability to undergo an oral interview, absence of a medication list of the public pharmacy (ozislist), decease of the patient during er-stay and patients undergoing surgical procedures. discrepancies between both medication lists taken by an er physician or pharmacy technician were classified in four categories of increasing severity ( = no discrepancy to = clinical relevant discrepancy) using the index of the national coordinating council for medication error reporting and prevention (www.nccmerp.org). discrepancies were categorised by a panel consisting of a pharmacy technician, a (senior) hospital pharmacist and a th year pharmacy student. statistical analysis was carried out with a statistical software package (spss ) using the mann-whitney u test and chi squares test. main outcome measures: during the intervention measurement we analysed the reconciliated medication by comparing the er's physician's list with the list of the pharmacy technician after a medication verification interview. the number of discrepancies were measured and judged by the panel. discrepancies were given a category , , or as defined. results: during the intervention measurement patients were admitted to the er. sixty-five ( ) patients ( . %) met the in-and exclusion criteria. the number of medication discrepancies decreased significantly after intervention of the pharmacy technician by %, from to discrepancies. the average number of discrepancies per patient after intervention decreased by . %, from average . to . discrepancies per patient. conclusion: medication verification by pharmacy personnel in the er reduces the number of medication discrepancies by half. medication lists generated with a standard interview by pharmacy technicians in combination with an ozis-list on admission of patients at the er is more complete and accurate than the current method. hp-pc : discharged patients: a problem for community pharmacists? information transfer, as well as the role, needs, and objectives of pharmacists when they care for recently discharged patients. setting and method: a focus group was conducted with a sample of six community pharmacists from personal contacts to represent different characteristics. the focus group consisted of different questions and the recording was transcribed, fragmented and categorised. based on these results, a nationwide online-survey was created with the following questions: a) responder's characteristics, b) number and origin of prescriptions, c) role fulfilment of the joint-who/fip-guideline on good pharmacy practice, rated with a -point likertscale, d) information items derived from the focus group discussion grouped into four categories and evaluated for their availability and for their usefulness by likert-scales, e) goals for discharge optimisation, f) additional comments. the questionnaire was piloted and translated forward and backward to french and italian by native speakers. it was sent to all managers of pharmacies belonging to the swiss pharmacist's association in summer (n = ). main outcome measures: conclusions from focus group discussion and responses to questions a-f from the nationwide questionnaire. results: the focus group participants ( . ± . years, % female, % employees) emphasised the importance of an expanded information transfer, especially for medication changes, unclear prescriptions, and information about a patient's medication acquisition. they were concerned about their extensive workload of discharge prescriptions, and mentioned treatment continuity as one of their goals. the questionnaire was answered by pharmacists (response rate . %, . ± . years, . % female). there were . % of responders who reported to fulfil their role (to manage a patient's therapy, function b) not satisfyingly. unavailable but essential information were allergies and the specification of off-label use prescription. unavailable although desired information were the reasons for therapy changes, indications, appointments, contact information, or compounding formulations. concerning design and transfer, information should be written in a structured way but no clear preference for a transfer method was found. goals of community pharmacists were: improved treatment continuity, patient safety, and pharmaceutical care. conclusion: swiss community pharmacists rarely receive sufficient information on discharge prescriptions. appropriate pharmaceutical care is therefore impeded. the knowledge and application of the findings enable directed optimisation of discharge. hp-pc : patients attitude for using antipsychotic medication in the norwegian early intervention in psychosis, tips study rafal yeisen *, , stein opjordsmoen , , , inge joa , , jan olav johannessen , , jone bjørnestad on behalf of centre for clinical research in psychosis, psychiatric division, stavanger university hospital, stavanger, norway background and objective: poor drug adherence in patients with psychosis leads to relapse, re-hospitalization, poor outcome and increased consumption of health services. pharmacoclinical studies have demonstrated that the treatment response decreases with each relapse. it is estimated that % of patients suffering from chronic illness are not taking medication as prescribed after months. the purpose of this study is to investigate which experiential factors that potentially might affect adherence with medication in adults with psychotic disorders. setting and method: in a descriptive qualitative sub-study in the ongoing norwegian early intervention in psychosis, tips study, where twenty-first episode patients ( male, female) participated in semi-structured interviews years after inclusion. they were still using or had used antipsychotics during the last years. data were analysed using interpretative phenomenological analysis. main outcome measures: adherence to antipsychotics. results: the data suggested four main themes, reflecting the patients' subjective experiences and their impact on the desire to adhere to antipsychotics: ( ) admission experience as a psychotic's patient; ( ) information from healthcare staff; ( ) limited involvement in decision-making; ( ) attitude to antipsychotics. conclusion: a number of factors had a positive influence on adherence to antipsychotics. pleasant admission/stay experiences, feeling that antipsychotics had therapeutic effects, mild or no side effects, and believing that antipsychotics are necessary and useful, were typical statements. please specify your abstract type: research abstract background and objective: the hospital-to-home transition is a vulnerable stage in a patient's care. patients can experience problems with medication supply, which possibly lead to therapy interruptions. the objectives of this study were to investigate medication supply after discharge, and patients' and physicians' opinions about the current discharge process and possible optimisations. setting and method: a telephone interview was conducted with discharged patients from the surgical and internal medical wards from the cantonal hospital in baden (switzerland). inclusion criteria were: patients c years old, discharged home with a discharge prescription. patients were called between the nd and th day after discharge and a piloted, structured interview was performed, consisting of questions on experiences and optimisations. afterwards, semi-structured interviews were conducted with five physicians from the study hospital. results from patient interviews and the general discharge process were discussed. main outcome measures: proportion of filled prescription, frequency and type of supply problems including therapy interruptions. opinions of physicians and patients on current discharge process and possible optimisations. results: discharged patients were . ± . years old, % female, % from internal medicine, and % regularly visit the same pharmacy. of the interviewed patients, have not filled their prescriptions yet and had their prescription filled when they were called. of these, % of them visited the pharmacy on the day of discharge, but it took up to the th day until all of them received their medication. supply problems were encountered by patients ( %), mainly because of the medication not being in stock in the community pharmacy. only four patients experienced therapy interruptions, which took up to the rd day post-discharge. patients discharged from internal medical wards had more supply problems compared to surgical wards (relative risk = . , p = . ). patients experiencing supply problems had statistically significant more medicines on a daily basis ( . ± . vs. . ± . , p = . ). physicians were surprised about the late prescription filling and worried about the disease outcomes. however, interruptions were interpreted as unfrequent. when asked if, in future, hospitals should transfer prescription to the community pharmacy prior to discharge, % of patients refused and physicians were undecided, mainly because of a questionable benefit. but both groups indicated that giving some bridging supply would be welcome. conclusion: this study showed that patients discharged from a swiss hospital encounter supply problems, but therapy interruptions are seldom. giving some bridging supply was preferred over an early information transfer by patients and physicians. interventions should consider these opinions and focus on internal medicine patients with high number of medication. please specify your abstract type: research abstract background and objective: adherence to secondary prevention evidence-based medical (ebm) therapies for patients with st-segment elevation myocardial infarction (stemi) is essential to reduce long-term rates of major adverse cardiovascular events. current guidelines recommend the long-term use of low-dose aspirin, highintensity statins, angiotensin-converting enzyme inhibitors (acei)/ angiotensin receptor blockers (arb) and beta-blockers (bb), in addition to p y inhibitors for year. we aimed to assess the adherence to secondary prevention ebm therapies from discharge to one-year follow-up among patients with stemi undergoing primary percutaneous coronary intervention (pci) in contemporary practice. setting and method: observational single-centre study including consecutive patients with stemi undergoing primary pci in a tertiary hospital in switzerland over a one-year period. secondary prevention ebm therapies were assessed at discharge and at one-year follow-up. main outcome measures: prescription of key secondary prevention ebm therapies (aspirin, p y inhibitors, statins, acei/arb and bb) from discharge to one-year follow-up after stemi. bb was recommended only for patients with heart failure or left ventricular ejection fraction (lvef) \ %. results: a total of patients were included. ebm drug prescription at discharge was . % for aspirin (n = ), . % for p y receptor inhibitor (n = ), . % for statin (n = ), . % for acei/arb (n = ) and . % for bb (n = , among patients with lvef \ %). ticagrelor ( . %) was the major p y inhibitor prescribed. overall, ebm drugs were missing at discharge, with of these missing drugs having no justification for no-prescription (contraindications, allergy or intolerance). at one-year follow-up (median . months, n = ), aspirin, statins and acei/ arb prescription rates were . % (n = ), . % (n = ) and . % (n = ) respectively. out of patients ( . %) with lvef \ % received a bb. among patients treated with ticagrelor at discharge, ( . %) were receiving ticagrelor at follow-up, whereas ( . %) were switched to another p y inhibitor. among patients who discontinued ticagrelor (n = , . %), duration of dual antiplatelet therapy was months for % (n = ) and discontinued prematurely (\ year) for % (n = ) patients. reasons for ticagrelor early discontinuation or switch were not specified. conclusion: in a real-world cohort of patients with stemi undergoing primary pci, prescription of recommended secondary prevention medications at discharge is excellent. adherence to ebm therapies at year remains high with more than % of patients receiving all ebm drugs. early discontinuation of dual antiplatelet therapy was observed in % of patients, whereas ticagrelor was switched for another p y inhibitor in . % of patients. these observations highlight key opportunities to improve longitudinal use of secondary prevention therapies after stemi in routine clinical practice. although side effects are less common than traditional chemotherapies, certain ones such as pain, fatigue, nausea and vomiting can still be bothersome. in oncology outpatient clinics, side effects are monitored by oncology nurses; however due to high patient turnover and limited numbers of nurses, the assessment of side effects might not be performed adequately. therefore, aim of this study was to determine side effects of immunotherapy and targeted therapy and to compare the severity assessment of side effects by clinical pharmacist and nurses. setting and method: the study was conducted in the hacettepe university oncology hospital outpatient clinic. the patients who have been taking ipilimumab, nivolumab, pembrolizumab, bevacizumab, panitimumab or cetuximab during october -march were included. the assessment of side effects were undertaken by a clinical pharmacist and nurses separately on each visit using the common terminology criteria for adverse events version- toxicity assessment scale. an independent clinical pharmacist compared the side effects' assessments by pharmacist and nurses for analysis. ethical approval was obtained from hacettepe university ethics committee. main outcome measures: to compare the severity of side effects of targeted drug therapies which were assessed by a clinical pharmacist and nurses. results: during the study period visits of patients were evaluated. a total of side effects assessments were recorded. among those assessments ( . %) was assessed in different ranking by nurses and pharmacist. the differences in the number of assessments were mainly seen in criteria related to pain (n = ; ), sensory loss (n = ; ), fatigue (n = ; ), stress (n = ; ), insomnia (n = ; ) which was performed by nurses and pharmacist respectively. other side effects detected only by clinical pharmacist were oedema, cough, gastrointestinal complaints (heartburn, cramp) and sensitivity of odour which require close monitoring and in-depth counselling by clinical pharmacist. conclusion: this study explores the differences in assessment of side effects by pharmacist and nurses in targeted therapies. routine assessment of side effects between chemotherapy cycles might yield to misinterpretation or inadequate assessment due to workload of outpatient clinic. therefore, inter-professional interactions in outpatient clinics might close the communicational gaps and improve patient care. hp-pc : implementation of clinical pharmacy in the acute psychiatric wards: improving quality of medical treatment across health care sectors amila zekovic *, , signe kristensen , lisbeth lund pedersen clinical pharmaceutical services, capital regional pharmacy, head of clinic, mental health services, copenhagen, denmark please specify your abstract type: descriptive abstract (for projects) background and objective: a study from shows that people with a mental disorder had a two-to threefold mortality compared with the general population in denmark. life style diseases are the major reason for the excess mortality, partly due to undertreatment of physical disease and well known side effects from medicines such as obesity, diabetes, and heart disease. in may , a clinical pharmacy service (cps) was implemented in all acute psychiatric wards (apw) in the capital region as a part of a three-year project funded by the danish health authorities. the objective is to illustrate how the implementation of clinical pharmacy in the apw in copenhagen increases the focus on drug related problems, rational pharmacotherapy and side effects, increasing the quality of medical treatment and patient safety across health care sectors. design: data was collected at the apw in copenhagen which consists of three wards and has a total capacity of beds. inclusion criteria were patients to which two or more of the following apply: • c years of age • c drugs • high risk drugs (clozapine, sertindole and opioids) • combination of antipsychotics and benzodiazepines • diagnosed with liver/kidney disease the secondary inclusion criteria were all patients receiving c drugs as a single criterion. to obtain a valid medication history and secure medication reconciliation, the pharmacist interviewed included patients. the patients were also asked about side effects, compliance, and perceived effects of treatment. a medication review was conducted based on the patient interview, screening for interactions in an interaction database, and consideration of biomedical data in order to evaluate if treatments should be adjusted, initiated or discontinued. the pharmacist's input was discussed with the doctor, as inputs are more likely to be considered if they are communicated orally. finally, all inputs were documented in the patient's journal as a pharmacist note. the model for improvement was used as a tool for implementing the cps and is being used continuously for improving the service. results: between may th and june th , patients were screened at admission to the apw, of which . % met the inclusion criteria ( patients). in this period the pharmacist conducted notes, indicating that . % of the included patients were seen by the pharmacist. in april , patients were in average admitted with . drugs and . inconsistencies between the hospital's medication orders and the medications that the patient had been taking. regarding patients who are discharged to community care shortly after admission, the pharmacist note is sent to their general practitioner for follow up. conclusion: overall, the implementation of a cps in the apw has been successful. medication reconciliation ensures that the patient is provided with correct medicine at admission, transfer or discharge. by performing a thorough medication review based on a consultation with the patient, the service contributes to an increase in quality of medical treatment. please specify your abstract type: descriptive abstract (for projects) background and objective: the importance of the role of a clinical pharmacist resident in the operating room during months, in a private hospital belonging to a group devoted to healthcare for over years. the hospital is recognized as a reference centre of excellence of hospital care in portugal. it has inpatient beds, two surgical blocks with rooms and beds in the intensive care unit. the aim of the clinical pharmacist in the operating room is to ensure compliance with good clinical practice, safety and pharmacotherapeutic effectiveness, as well as optimization of drug costs. design: . logistics restructuring of pharmaceutical services and the need of the physical presence of the pharmacist in the operating room. . furthermore, the workstation of the pharmacist is moved to the operating room and the in-depth study of all medicines used in the operating room. . in compliance with the joint commission, definition, optimization and adjustment of drug stocks to the needs of the service itself. in close collaboration with the nursing staff, consumer kits were created for registration of drugs by type of surgery in order to facilitate registration and ensure billing efficiency. control of the analgesic drug's dispensation circuit in hospitalized surgical patients that stay less than h in the hospital. ensure compliance with the project through which the health regulatory authority evaluates several hospitals in the country, creating a national ranking among hospital specialties. . clinical phase: creation of prescription protocols by type of surgical intervention based on national clinical guidelines. validate prescriptions in the intra-surgical block in compliance with antibiotic prophylaxis, antiemetic and thromboembolic, checking deviations in therapy according to good practice. identify pharmacologic hypersensitivities of patients by consulting the clinical process and anaesthesiology records. provide information on drugs, drug efficacy monitoring and adverse drug reactions in risk management platform. check off label use of drugs. results: of a total of interventions, relate to revenue optimization and relate to clinical interventions. there was an increase of approximately % in billing. on what regards to clinical interventions, the majority of them showed deviations from good clinical practice. the physical presence of a clinical pharmacist in the surgical block is essential as the prescription and administration of drugs is carried out simultaneously, allowing immediate therapy validation, in order to increase the safety and efficacy. the pharmacist has the ability to interact with the multidisciplinary team, as well as monitoring the patient's clinical process, the pharmacotherapeutic profile and drug allergies, allowing the detection of any adverse drug reaction on-time. all these interventions are possible in the pre, intra and postoperative phases. results: counselling (av.(±sd) duration: ± min) was performed in patients ( . % female; av.(±sd) age: . (± ) years; av.(±sd) medicines at discharge: . (± . )). in % of patients mrps were intercepted. the five most common mrps (%) were: need for organisational support ( . , e.g. proper prescriptions' writing), therapy-related discussions ( . ), untreated indications ( . ), errors in documentation ( . ), and medicines without an indication ( . ). patients ( . %) classified for study inclusion, of whom ( . %) consented to be followed-up and ( %) provided data. roughly % of patients report having received information about medicines at discharge, of which three-fourths remember being informed by the pharmacist. more then every second patient ( . %) reported having received valuable new information. changes in chronic-use medicines occurred in . , . , and . % of patients at -, -, and -month, respectively. at -month, in . % of patients chronic-use medicines were newly prescribed, in . % discontinued. medical specialists initiated these changes in . % of patients. one out of five patients couldn't recall the reasons for changes in medication. nearly % of patients showed moderate to little medication adherence at -month. it did not significantly change during the follow-up period. conclusion: clinical pharmacists' counselling prevents mrps at the transition from hospital to home. follow-up data show that changes occur in one out of three patients. medication adherence remains stable, but generally needs to be improved. please specify your abstract type: research abstract background and objective: until , prescription analysis was based in our hospital pharmacy. clinical pharmacy has been deployed in care units since . many clinical pharmacy services were developed: medication reconciliation, patient's therapeutic education and counselling, and prescription analysis unit based. the purpose of this study is to assess the impact of the clinical pharmacist as a direct patient-care team member on prescription analysis. setting and method: we collected pharmaceutical interventions (pis) of the first months of and at the same period of the year in the neurology unit when the pharmacist was unit based. we studied and compared type of pis (medication, drug related problem-drp), rate of pis acceptance and clinical impact. focus was made on high alert risk medications and potentially inappropriate medications. . % in versus none in . when prescription analysis was based in the pharmacy unit, % of drp detected by the pharmacist had a potential clinical impact versus % when the pharmacist performed prescription analysis in the care unit (p \ . ). three drp detected in had serious potential harm. results: ward-based prescription analysis allowed detecting five more times drp with a significant more important clinical impact than pharmacy unit based prescription analysis. the clinical pharmacist as a direct patient-care team member is more efficient in detecting serious potential harm. indeed, the pharmacist has a greater knowledge of the patient's clinical condition. nevertheless the global rate of acceptance of pis was greater when the prescription analysis was based in the pharmacy unit even if the difference is not significant. but prescription analysis is more complex when performed in the care unit, taking account adherence of the patient, and potentially inappropriate medications resulting in much higher risk-taking by the ward-based pharmacist. conclusion: this study showed that unit based prescription analysis is the best way to detect drug related problem. it must be competed by medication reconciliation and medication review to improve medication safety process. hp-pc : qt-prolongation in an acute psychiatric setting: fact or fiction? eva jacxsens *, , hans van den ameele , jü rgen de fruyt , yves vandekerckhove , frank vancoillie , veerle grootaert pharmacy, psychiatry, cardiology, az sint-jan brugge-oostende av, bruges, belgium please specify your abstract type: research abstract background and objective: several psychotropic drugs can induce qt-prolongation, which is a well-known risk factor for developing torsade de pointes (tdp) and sudden death. the clinical relevance of this side effect of psychotropic medication remains unclear, especially in patients hospitalized in an acute hospital. to interpret the clinical importance of psychotropic drug induced qt-prolongation, we investigated the prevalence of these electrocardiographic changes. setting and method: a prospective study was conducted on four psychiatric wards in a general hospital: two acute, short-term psychiatric units (asp and asp ), one addiction service unit (asu) and one geriatric-psychiatric ward (gpw). all adult patients admitted between october st and march th on a psychiatric ward were eligible for inclusion. at admission, an ecg (ecg ) was performed and creatinine and potassium levels were measured. a second ecg (ecg ) was performed at least days after the start of a psychotropic drug associated with a risk of qt-prolongation. qtcprolongation was defined as ms for males and ms for females. clinically relevant qtc-prolongation was defined as c ms. statistical analysis (r software) was done as appropriate. main outcome measures: prevalence of psychotropic drug induced qtc-changes and correlating factors. results: patients (mean age years, %female) were enrolled in the analysis. in patients, an ecg was performed. qtc + were prolonged in . %( / ) of females and . %( / ) of males. no clinical relevant prolongation (c ms) was registered. higher qtc intervals were measured in the geriatric population. . %( / ) of all measured qtc were situated between [ c qtc + b ms] in gpw versus . %( / ) in the other units. significant difference in qtc-changes was associated with sex (p = . ). there was no correlation assessed between qtc-prolongation and age, number of psychotropic drugs or a specific single psychotropic drug (p [ . ). conclusion: in this study qtc-prolongation due to psychotropic drugs is less common than previously described. ecg monitoring may be unnecessary in the follow up of patients without risk factors and could reduce hospital and community costs. however, considering the potential harm associated with tdp, qt-prolongation should be avoided. we recommend recording an ecg before the start of a qt-prolonging psychotropic drug in risk patients: patients with a chronic alcohol or drug addiction, a cardiac history, on concomitant therapy with at least two qt-prolonging psychotropic drugs, or geriatric patients ([ years). hp-pc : implementation of medication reconciliation aase m. raddum *, , anne-lise sagen major sykehusapotekene i midt-norge, sjukehusapoteket i Å lesund, avd. volda sjukehus, volda, sykehusapotekene i midt-norge, sjukehusapoteket i Å lesund, Å lesund, norway please specify your abstract type: descriptive abstract (for projects) background and objective: a correct and accurate medication list should accompany patients at transitions in care from one setting to another, including admission to hospital. complete information on drug use is a prerequisite for all hospital treatment, whereas incomplete information represents a potential patient safety risk. medication reconciliation is defined by the world health organization (who) as ''…the formal process in which health care professionals partner with patients to ensure accurate and complete medication information transfer at interfaces of care.'' the objective of this study was to investigate the quality of the medication history obtained for admitted patients. furthermore, measures to improve the quality of medication histories, i.e. implementation of medication reconciliation, were initiated. design: the study included patients admitted to the internal medicine ward. a comprehensive medication history was determined by performing a standardized patient interview and/or by using relevant sources of information. the primary endpoint was discrepancies between the medication history obtained on admission and the one determined prospectively by a clinical pharmacist. the clinical relevance of the discrepancies was not determined, but sorted according to six major categories, such as: medication not in chart, but patient reports using (omission) and medication in chart, but patient reports not using (commission). further on, in order to minimize the risk of discrepancies, it was focused on implementation of medication reconciliation. a campaign was initiated, where a clinical pharmacist held information meetings regarding the medication reconciliation procedure. for the next weeks, the degree of medication reconciliation was recorded. to spur the degree of medication reconciliation, each ward's weekly numbers were published and the ward with the highest degree of medication reconciliation won a prize. results: among the patients included, a total of discrepancies were revealed. in summary, patients had at least one discrepancy in their medication history, resulting in discrepancies in the medication lists of % of the included patients. at the start of the study, the level of medication reconciliation varied among the wards ( - %), while at the end of the study the levels were increased ( - %). conclusion: all the included wards improved their level of medication reconciliation during the study period. however, these new combinations have potential drug-drug and herb-drug interactions which can affect the safety and effectiveness of the treatment. in our clinical practice, the clinical pharmacist provides patient education about direct acting antiviral drugs (daa) based-regimens, promotes medication adherence and manages potential interactions with hcv treatment. the aim of the present study was to determine the prevalence of use of herbal products in the patients on hcv treatment, and to describe the potential hepatotoxicity of the herbal products and their interactions with hcv treatment. design: we included all adult patients on daa treatment for hcv who were dispensed drugs from / / to / / . we retrospectively recorded demographic data (age and gender), clinical data related to hcv infection (hcv genotype, fibrosis stage, daa regimen and treatment outcomes) and type of herbal products consumed. we then assessed the presence of herb-drug interactions and the potential hepatotoxicity of herbal products. results: we obtained data from patients on daa-based treatment for hcv. the prevalence of consumption of herbal products prior to starting the treatment was . % ( / ). the most consumed herbal products were (prevalence [ % among herbal products users): milk thistle, green tea, chamomile, valerian, pennyroyal, boldo and artichoke. we detected four herbal products with potential hepatotoxic effects according to the literature: milk thistle, green tea, pennyroyal and aloe vera. the prevalence of consumption of these hepatotoxic plants among herbal products consumers were, respectively: . , . , . and . %. we detected herb-drug interactions or potential for hepatotoxicity in out of patients who consumed herbal products. the management of these potential interactions consisted of stopping the herbal product before starting the hcv treatment. conclusion: the consumption of herbal products in our hcv patients was frequent. the management of potential interactions was conservative, recommending to stop herbal products. clinical pharmacists have an important role in the counselling, detection and management of potential herb-drug interactions and herbal products-related hepatotoxicity. poster discussion forum iii: hospital pharmacy and pharmaceutical care please specify your abstract type: research abstract background and objective: anaemia is a common comorbidity of chronic kidney disease. intravenous (iv) iron is used when oral iron formulation became insufficient or to reduce the use of erythropoiesis-stimulating agents (esas) in haemodialysis (hd) patients. the lack of generic group for iv iron sucrose (is) preparations leads to a controversial issue about their clinical effectiveness. in this study, we evaluated the effectiveness of original is compared to is similar (iss) in hd patients. setting and method: a retrospective monocentric observational cohort study was conducted from / / to / / , in a stable hd population to compare is and iss. the follow-up periods lasted weeks and were separated by a one-month wash-out period. original is and iss were administered respectively during the first (p ) and the second (p ) periods. the comparisons were performed using the paired student's t test or the paired wilcoxon test for continuous data and the fisher's exact test for categorical data. main outcome measures: the main endpoint was the difference in haemoglobin (hb) levels between p and p per patient. anaemia parameters (serum iron, serum ferritin, transferrin saturation ratio), the number of transfused patients, the doses of iv is and the doses of erythropoiesis stimulating agents (esas) were compared before and after the switch from is to iss, as secondary endpoints. results: a total of patients were included. there was no significant difference in mean hb value between p and p ( . ± . mmol/l versus . ± . mmol/l p = . ). anaemia parameters were significantly different between p and p (mean serum ferritin, serum transferrin and transferrin saturation ratio) with p \ . , except to the mean serum iron. the mean monthly dose of iv iron per patient and the mean dose of esas were respectively in p and in p : . ± . mg versus . ± . mg (p = . ) and . ± . ui/kg/week versus . ± . ui/kg/ week (p = . ). transfusions occurred less frequently in p than in p (p = . ). conclusion: this study showed that iss was as effective as original is regarding hb levels. however anaemia parameters appeared to be in favour of is; the mean dose of esas seemed to be higher after switching from is to iss. these outcomes should be further explored using prospective comparative clinical studies. please specify your abstract type: research abstract background and objective: the pharmacy residents are sometimes up to deliver chemotherapy when they are on night or week-end duty at the hospital. a dispensation's error (delivery of metoject Ò (methotrexate) for intrathecal (it) injection whereas it doesn't have the indication for this use), led us to test the pharmacy residents' knowledges about the it access in order to underscore the points to be improved. the final aim of this work is to secure the pharmaceutical care of the patient h a day, days a week. setting and method: an online and anonymous survey of questions was sent to the residents of our area. it was composed of three parts: specific general information, questions about the chemotherapy specifically (indication, maximum dose and volumes, molecules used), illustrated questions about real situation for the dispensation on duty. the answers were collected over a two weeks period. main outcome measures: we studied the rate of good answers in global and by respondent. results: twenty-five residents answered the survey, among them % never achieved any internship in a centralized unit of reconstitution of chemotherapy (urc). all the levels of internship are represented: st year (n = ), nd year (n = ), rd year (n = ) and th year (n = ). only % know where a medicine is injected intrathecally on the spinal column, % know on which level of the meninges. three residents think that a nurse can inject intrathecally. they also had to select the molecules which can be injected by this access: % answered vincristine, % vinblastine, % bortezomib; despite these three molecules are mortals if they are injected intrathecally. the majority know the indication of the it chemotherapy: prevention and treatment of cancers' meningeal localizations. sixty percent do not know that several molecules can be injected for the same patient in the same time. the maximum dose of methotrexate is known for half of the respondents, but only % for the cytarabine'one. only residents out of know that ml is the maximum volume allowed to be injected for an adult. six residents would have delivered metoject Ò mg in pre syringe filled if a doctor had asked for an it during a night. lastly, there are only two people who know that aracytine Ò (cytarabine) mg must be reconstituted with sodium chloride for it use and not with the provided solvent containing benzylic alcohol. the score of the residents having already done an internship in an urc is . / compared with . / for those who never did. the respective scores per year of internship are . ( st year), . ( nd year), . ( rd year) and . ( th year). conclusion: results and answers have been presented in a meeting and sent to the residents. we initially note many gaps in knowledge. the residents who already worked in an urc and the elders got better results. all the residents could be on duty at the hospital and all must be formed. a second session will be organized in a month to evaluate the formation's impact. it also has been presented to the assistants during an interactive lesson. this formation is essential to guarantee the dispensation of the adequate product and a secured medical care of the patient. please specify your abstract type: descriptive abstract (for projects) background and objective: patient adherence to prescribed medications is crucial for reaching metabolic control goal. to better understand the impact of polypharmacy on medication adherence, we undertook a detailed survey of medication use among patients with endocrinologic diseases. the aim of this study was to determine medication adherence in a cohort of patients with endocrinologic diseases and to test the hypothesis that adherence decreases with increased number of medicines prescribed. design: we conducted structured interviews to determine self-reported adherence of patient on a scale of (high) to (low observance) (srap- ) and a measurement using morisky medication adherence scales . demographic and medication information were collected from medical record. for statistical analysis, mann-whiney u-test for continuous variables, with chi square for categorical variables and kendall test for correlation were used. results: our cohort included patients, % were women and % were diabetic ( % suffering from type diabetes). the mean age was ± years, the average number of medication was . ± . . ( %) patients were not able to estimate their adherence. patients reported srap- scale with an average of . ± . , this estimation was significantly higher than mmas- with an average of . ± . (p \ . ). the proportion of adherence level were identical between srap- and mmas- with respectively and % of high, and % of medium and and % of low adherence. a significand correlation between srap- and mmas- scales (r = . , p \ . ) was found. however no correlation between adherence scale and number of treatment (r = . for mmas- and . for srap- scale) nor number of daily doses (r = . for mmas- and . for srap- scale). on the medications, % presented difficulties with observance. cardiovascular ( . %), diabetes ( . %) and psychiatric ( . %) treatment are the three most involved drug classes in nonadherence. conclusion: in this cohort, patients reported high medication adherence. we highlighted a correlation between srap- and mmas- scales. surprisingly, we didn't find correlation between adherence scales and number of treatment or dose by day. the next step of this work will be the identification of risk factor of nonadherence using logistic regression analysis. hp-pc : the office of access to healthcare: how to optimize secured access to treatments? claire chatron, adeline flatres, claudine hecquard, guillaume saint-lorant * , alexandra muzard pharmacy, chu caen, caen, france please specify your abstract type: research abstract background and objective: office of access to healthcare (oah) is an organization which offers a medical and social coverage to people who can't access to care and to medication because of the absence of social welfare, living conditions, or financial difficulties. medications are free dispensed thanks to retrocession activity in hospitals pharmacies. the aim of this study is to analyse this activity and to improve communication with patients and access to treatments by an adapted pharmaceutical interview. setting and method: this study includes all dispensations of year . in order to get medications in our hospital, a social worker and the patient come at the hospital's pharmacy. one people of retrocession team (four assistants, two externs, two residents and two pharmacists) dispenses necessary drugs to the patient according to hospital drug formulary and operating protocol. a switch or a special order can be purposed if the drug is not available. then, we give the patient a medication management plan (mmp) to explain him how to take his treatment at home. retrocession team filled a quiz about this activity and ways to improve it. main outcome measures: the main topics included in the quiz and evaluated were: dispensation organization, english talking, feeling during the interview and evaluation of the mmp. results: three hundreds and ten patients were admitted in oah . these patients come mainly from the eastern europe and do not speak french in most of cases. social workers, who can help for communication, are not always present because these patients can come during on-call duty. quiz results showed that weak points occurred during the interview: explanation of the mmp, languages barriers, mention '' if needed '' not understood by the patient. explanation of the order for a particular drug was difficult to operate too. mmp were only drafted in french which was not convenient for foreign people. however, modalities of dispensation were well understood by the retrocession team. following quiz results, mmp was translated into english by the retrocession team. mentions '' if needed '', '' number of maximum tablet a day: … '', ''your medication is in order, thank you for coming to look for this treatment back to the hospital on … '', '' … is the same as …, prescribed by your doctor on your medication list '' have been added and translated. results of the study and new mmp will be presented to the pharmaceutical team and to social workers in staff. an index card for ''communication in english with a patient'' has also been drafted. it contains sentences meadow drafted in english. conclusion: access quality health care service is important to achieve health equity and to increase the quality of everyone's life. these documents improve communication with patients and by the way their understanding about their treatment. the use and the impact of these documents on well understanding will be soon evaluated with social workers and patients. hp-pc : improve the medication in an associated to general hospital nursing home luisa alonso * , marta vidal iglesias, lucia gómez carrasco, guillermo goda, laura garcia, laura marin, ana hernandez, alvaro moreno please specify your abstract type: descriptive abstract (for projects) background and objective: in order to improve the medication reconciliation and to implement training programs for the medical team in an associated to general hospital nursing (asnh) home we measured the discrepancies between pharmacy registered treatments (prt) and medical prescriptions (mp), and we analysed potentially inappropriate prescriptions according to ''american geriatrics society beers criteria'' and ''stopp-start criteria. design: retrospective observational study that included patients admitted in the asnh. the ''consensus document on terminology and classification in medication reconciliation'' was considered for discrepancy classification. data collected: discrepancies between mp and prt. in patients from the original group of , we reviewed potentially severe drug interactions, potentially inappropriate mp and drug classes to avoid in older adults and medications to be used with caution in older adults (according to stopp-start and beers ) . all data were registered, measured and analysed in excel Ò . results: patients and a total of mp were reviewed. discrepancies ( . %) were found between the medical order and the prt, those discrepancies included errors of omission in prt ( . %), absence of discontinuation of medication ( . %), incorrect dosage ( . %). potentially moderate to severe interactions: the most frequent drug groups were proton pump inhibitors (ppis) ( . %), benzodiazepines (bzds) ( . %), oral hypoglycemiants ( . %), other groups with frequency over %, oral antihistaminic, statines, low molecular weight heparine (lmwh), laxatives, calcium salts and iron salts. stopp criteria were identified that affected to mp and the distribution was as follows: laxative combinations ( . %), long term ppis ( . %), cns depressants combinations ( . %), long half life bzds combinations ( . %), aspirin incorrect drug strength ( . %) and other groups with lower frequencies, nsaids and prokinetics. start criteria: being all of them by omission of the drug at the time of admission. beers criteria: prescriptions in the ''avoid prescription in adults'' group of which corresponded largely to concomitantly cns depressants and long term use of ppis in no risk patients. conclusion: the difficult working conditions, the excessive workload and the high staff turnover, where doctors have a patient ratio over / , make difficult to update treatments according to patient daily needs. a clear communication problem between the hospital pharmacy and the asnh prescribers exists due to lack of infrastructures, and it has been demonstrated with the high percentage of discrepancy, that implies an important logistic problem (not a safety problem) since the nurse team works directly with the original medical orders. the analysis of prescriptions showed the need for updating the medical knowledge. the high volume of stop and beers criteria and lack of doctors time made impossible the individual acting upon each patient, so short summaries of continuous training related to most frequent problems have been designed. please specify your abstract type: descriptive abstract (for projects) background and objective: our french university hospital is one of the most active centre for liver transplant ( transplants annually). various professionals are involved in the graft patient care and education. much information and education sessions are exempted before and after the transplant. the objective of this work was to realize a short movie for patients ( ) to get them ready for transplant ( ) to give the key messages to support their transplant ( ) to make family understanding the process and to promote the life behaviour changes. design: three members of the pharmaceutical team with nurses-led care coordination and a surgeon wrote the scenario. we requested two directors for days of shooting. we defined the key points for the patient and places to film, and fixed the duration ( - min). the scenario was validated by the chief of the liver transplant unit and nurses-led care coordination. after the days of film shooting, we selected sequences. results: the movie was a succession of six parts. ( ) the movie has been burned onto cds, put on flash-drives and will be uploaded on the internet. because of the international origin of our patients, the video will be subtitled at least in english. the video will be broadcast to hospitals which do not transplant patients and refer them to our hospital. since the medical team was involved in a collaborative project, the making of the video has permitted to strengthen the cohesion. indeed, this work would not succeed if everybody did not express himself. patients understood the interest to testify about their lived experience with the liver transplant, because they wished to have such information when they were waiting for the graft. conclusion: this movie is very useful for patients and families who are looking for information before and after liver transplant. it is a tool to get them into condition patients. this video presents the advantage of being personalized (local and caregivers that the patient will encounter are filmed). furthermore, it maintains a dynamic involvement of the pharmacy (already well established with clinical pharmacy, patient education and medication reconciliation) in the liver transplant unit. the making of the film has been an opportunity to bind the members of the team together, by valuing the work of everyone. the film could be screened if this abstract is selected for an oral communication. please specify your abstract type: descriptive abstract (for projects) background and objective: numerous procedures on medication management at oslo university hospital aim to minimize the risk of medication-related errors. error reports and observations show great variation in the use of these procedures, primarily due to difficulties in their implementation and maintenance. our aim was to assess the effect of a novel teaching strategy, the impala project, on doctors and nurses compliance with the medication management procedures. design: the project was carried out at general medicine wards at oslo university hospital for a period of weeks at each ward. assessment of medication-related error reports yielded the following areas of focus: (i) correct medication prescription, (ii) specification of doses for medications given on an ''as required''-basis, (iii) double control of medication dosing, (iv) correct and documented generic substitution. weekly presentations by pharmacist(s), lasting for a maximum of min, were given to doctors and nurses as part of daily ward routines. this was repeated over weeks. data on medicationmanagement procedure compliance were recorded before the start of the intervention, during and after each intervention period. the results were presented and made available to both leaders and employees throughout the project period both as an incentive to improvement and as a motivation factor for continued effort. results: there was a marked increase in medication-management procedure compliance among the nurses, especially after the second week of intervention. the most marked increase was shown for double control. increase in medication-management procedure compliance was also present among the doctors, but was less prominent. the data presented gave an extra motivational kick according to the participants. the leaders and the employees stated that the impala strategy was easy to follow and gave results without much organizational effort. conclusion: fifteen minutes presentations given by a pharmacist(s) as part of daily ward routines, combined with presentation of results demonstrated considerable improvement in medication-management procedure compliance. please specify your abstract type: research abstract background and objective: high unexpected serum vancomycin concentrations (svcs) were observed in patients without impaired renal function during the therapeutic drug monitoring (tdm) in our pharmacokinetic service. the aim of this study was to analyse the evolution of the svcs and its relationship with the markers of renal function. setting and method: retrospective study conducted at a university hospital with a follow-up period of months. only adult patients having at least two tdm were selected. trough svcs were measured by cmia (architect i- analyser, abbott Ò ) and fitted to a two-compartment model by using bayesian analysis (pks Ò , abbott). clinical and demographic data and daily dose, as well as timings of vancomycin administration and of blood sample collection were accurately recorded. spss Ò , version . was used to compare data from both tdm by student t-test (parametric data) and wilcoxon (nonparametric data). main outcome measures: concentration-to-dose ratio (cdr: trough concentration * /daily dose); glomerular filtration rates (gfr) estimated by cockroft-gault formula; measured and predicted svcs levels. results: adult patients were included (females: %); median age [ - ] years).the first and the second tdm were carried out after . [ . - . ] and [ . - . ] days from the beginning of the treatment, respectively. in the first tdm, no difference was found between the measured concentrations ( . ( . ) lg/ml) and those predicted ( . ( . ) mg/l. however, predictions were less accurate in the second tdm and predicted concentrations were significantly higher svcs ( . ( . ) mg/l vs. . ( . ) mg/ml, p \ . ). the median cdr in the second tdm was significantly higher than that calculated in the first one ( . [ . - . ] l - vs. . [ . - . ] l- ; p \ . ), indicating a lower clearance and a drug accumulation. however, no statistically significant differences in the glomerular filtration rates were found ( [ - ] ml/min vs. ml/min) in the first and second tdm, respectively. conclusion: although the markers of renal function did not change during the treatment, a decrease in vancomycin clearance was observed. the pharmacokinetic model does not accurately predict evolution of the svcs over the treatment. the introduction of covariates such as the length of treatment or the cumulative dose in the pharmacokinetic model could improve its predictive performance. please specify your abstract type: descriptive abstract (for projects) background and objective: genetic polymorphism or major physiological changes have to be considered in patient therapeutic management. clinical pharmacists have a role to evaluate and optimize the appropriateness and effectiveness of patient's medications. we report here the impact of the clinical pharmacist and his collaboration with the clinical pharmacologist in the therapeutic management of a patient suffered from anorexia nervosa, a psychiatric disorder leading to body composition change that may influence drug pharmacokinetics and efficacy. design: case report. results: the patient was a -year old woman hospitalized for chronic pulmonary aspergillosis previously treated by voriconazole, posaconazole and itraconazole. her medical history included anorexia nervosa since with a body mass index of . kg m - , pulmonary tuberculosis in with relapse in , and chronic pulmonary aspergillosis since . at admission, a treatment by oral voriconazole at mg/ h was introduced. the trough concentration of voriconazole at steady state was . mg/l (therapeutic range - mg/l) despite taking drug on empty stomach. although the voriconazole dosage increased in mg/ h, the trough concentration did not increase significantly ( . mg/l). we hypothesized anorexia led to a significant mucosal atrophy and accordingly, a significant decrease in intestinal absorption surface which is a major determinant of the level of drug absorbed. thus, a switch from oral to intravenous route was performed (voriconazole mg/ h). according to subtherapeutic voriconazole concentrations (trough concentration: . mg/l) despite the use of intravenous route, we decided to perform genotyping to look for mutations of cytochromes p a * , c * and c * , particularly implicated in voriconazole metabolism. the presence of an ultrarapid metabolizer genotype ( * allelic variant of the c isoenzyme) in our patient should lead to increase drug dosage from to %. finally, the patient was treated by intravenous voriconazole at mg/kg/ h (i.e., increase by %). the maximum concentration performed h after iv route initiation was at . mg/l, suggesting a better efficacy. conclusion: this case report highlights the potential complexity of therapeutic management in some patients given anatomical and functional changes or genetic polymorphism, which can affect drug efficacy. clinical pharmacists in collaboration with clinical pharmacologists have to be able to help physicians in this type of situations. please specify your abstract type: research abstract background and objective: posaconazole (pcz) is widely used for invasive fungal infections as prophylactic, pre-emptive or curative therapy in lung transplantation. recently, a new formulation of pcz has been available in enteric-coated tablets. this new formulation improves pcz bioavailability, as compared to the oral suspension, which leads to increase pcz plasma trough concentrations (c min ) in haematological patients. no data related to pcz exposure and its effects on tacrolimus (tac), an immunosuppressant with narrow therapeutic index widely used, exists in lung transplantation. we aimed to assess the consequences of the treatment by pcz entericcoated tablets on pcz and tac exposure in lung transplant patients. setting and method: a single-centre retrospective study was conducted among lung transplant patients receiving tac and either enteric-coated pcz or both galenic forms. main outcome measures: pcz and tac exposure were estimated by the measurement of c min . to overcome the influence of dose (d), c min were adjusted on dose (c min /d) for both pcz and tac. a spearman test (nonparametric distribution) was performed to assess the correlation between pcz c min /d and tac c min /d. results: eighteen lung transplant patients (median age [q ; q ] = . [ . ; . ] years; % female) were included between june and march . eight patients received only pcz entericcoated tablets. pcz enteric-coated tablets were associated to an increase in pcz c min /d as compared to oral suspension ( . ± . l - vs. . ± . l - , p \ . ). overall, pcz therapy initiation led to an increase in tac c min /d ( . ± . l - before initiation vs. . ± . l - after initiation, p = . ). tac c min /d was significantly higher with pcz enteric-coated tablets, as compared to pcz oral suspension ( . ± . l - vs. . ± . l - , p \ . ). a weak correlation was observed between pcz c min /d and tac c min /d, independently to pcz galenic form (r = . , p = . with pcz enteric-coated tablets and r = . , p = . with pcz oral suspension). conclusion: this pilot study in lung transplantation confirms the better bioavailability of pcz enteric-coated tablets as compared to oral suspension. our results show a more important increase in tac exposure with pcz enteric-coated tablets compared to pcz oral suspension, suggesting a concentration-dependent cyp a inhibitor effect of pcz. these findings are of interest in clinical practice to monitor transplant patients treated by the new formulation of pcz. further analyses, including the consideration of confounders, will be conducted. please specify your abstract type: descriptive abstract (for projects) background and objective: within months, two patients receiving apixaban developed agranulocytosis. based on temporal and clinical plausibility as well as published literature, the objective was to determine the causal relationship between agranulocytosis and apixaban. design: description of two agranulocytosis cases reported in our hospital. results: first case is an years old male, admitted to the neurology unit (d ) for ischemic stroke. at admission, blood count showed no abnormalities. four days after admission, treatment administered consisted in: dextrose % infusion iv, sodic heparin iv, acetaminophen, atorvastatin, metoprolol. neutrophils count was normal ( . g/l). heparin was stopped at d and replaced with apixaban according to following dose regimen . mg twice a day. at d , patient presented with hyperleukocytosis (neutrophils count g/l) and high crp ( mg/l). thus, a cytobacteriological urine test was performed. at d , patient presented with hypothermia followed by hyperthermia related to acute sepsis. blood count showed agranulocytosis (neutrophils count . g/l). broad spectrum antibiotherapy was started (ceftriaxone and gentamycin). despite treatment, death of patient occurred at d . the suspected cause of death was septic shock added to severe febrile neutropenia. following haemocultures confirmed sepsis (e. coli) possibly originating from urinary tract infection. second patient is a years old male, admitted to the cardiology unit (d ) for bronchopneumopathy associated with tachycardia and atrial fibrillation. a treatment with heparin was immediately started in association with patient usual treatment (bisoprolol, valsartan, rosuvastatin, hydrochlorothiazide and manidipine). in addition, broad spectrum iv antibiotherapy was started with ceftriaxone and spiramycine followed at d by an oral treatment with cefixime and spiramycine until d . heparin was replaced by apixaban at d ( . mg twice daily). antihypertensive treatment was adapted throughout patient's stay. patient presented neutropenia at d (neutrophils count . g/l), followed by agranulocytosis at d (neutrophils count . g/l) when it was decided to replace treatment with apixaban by fluindione. the following day, neutrophils count was about . g/l and patient received filgrastim. a myelogram showed a possible peripheral neutropenia. in the absence of other confounding factors (hiv, hbv, hcv, cmv), an iatrogenic agranulocytosis related to apixaban was suspected. conclusion: causal association with heparin is unlikely as neutropenia is not an adverse drug reaction known included in the smpc of this drug having a well-established safety profile. since the two patients were taking their usual treatment for a significant period of time, a causal relationship is deemed unlikely. temporal and clinical plausibility seem to indicate a possible relationship between agranulocytosis and apixaban. as this medicine has been recently approved, this might explain why no case has been reported in the literature and the absence of agranulocytosis as an adverse drug reaction of apixaban. please specify your abstract type: research abstract background and objective: taste is tightly connected to children's acceptability of medicines. two ways to overcome lack of acceptability are to administer solid formulations which are easier to taste mask and change to better tasting medicines. dicloxacillin is an antibiotic known for its unpalatability, and taste studies suggest that this might jeopardize its adherence. the aim of this study was to explore if prescription data can be used to estimate acceptability of antibiotics among children on a population level using dicloxacillin as an example drug. the research questions were: when comparing dicloxacillin with other antibiotics commonly used in children, ( ) is there a difference in the age of conversion from liquid to solid formulation and ( ) is there a difference in re-prescription rates on day and after the initial prescription? setting and method: we included all initial prescriptions of oral dicloxacillin, phenoxymethylpenicillin, amoxicillin and erythromycin for children - years registered in the norwegian prescription database (norpd) - due to dicloxacillin mixture being discontinued from the norwegian market in . the age of conversion was defined as the age where half of the children were prescribed liquids and the other half prescribed solid formulations. re-prescription rates were defined as re-prescriptions of a different antibiotic or formulation on day and after the initial prescription, divided by the total number of prescriptions. main outcome measures: age of conversion and re-prescription rates of dicloxacillin compared with other common antibiotics. results: the age of conversion for dicloxacillin was . years, compared to years for other common antibiotics. the average represcription rate for dicloxacillin was . % for children - years and . % % for children - years. the highest re-prescription rate of . % was found in -year olds. corresponding numbers were . , . and . % for common antibiotics. conclusion: the lower age of conversion from liquid to solid formulation and higher re-prescription rate of dicloxacillin mixture compared to common antibiotics indicates that prescription data can be used to identify antibiotics with low acceptability for children - years. further studies are needed to investigate if this also holds true for other antibiotics. please specify your abstract type: research abstract background and objective: attention deficit/hyperactivity disorder (adhd) or hyperkinetic disorders (hkf) is among the most common mental disorders in children, and may persist through adolescence into adulthood. pharmacotherapy used for treating the disorders also has potential for misuse/abuse. the aim was to describe the prevalence and magnitude of use of stimulant drugs and atomoxetine, and compare consumption in the nordic countries. setting and method: a descriptive pharmacoepidemiological study from the * million inhabitants of the five nordic countries in the period - . data were collected from national prescription registers, public drug reports and by correspondence with public health institutions. population data were obtained from official statistical databases or by correspondence with public health institutions. main outcome measures: trend over time, comparison between countries, type of pharmaceutical, gender, age, comparability of data. results: the annual consumption has been increasing from to , both in volume and prevalence of use. denmark had the largest increase in volume, from . to . ddd/ inhabitants/day. sweden had the highest increase in prevalence of use over the period, from . to . users/ inhabitants. iceland had the largest consumption of adhd medications in , . ddd/ inhabitants/day. prevalence data was not available for iceland but sweden was highest in prevalence of use among the other countries in : . users/ inhabitants. males aged - years had the largest volume and prevalence of use in , but females' consumption had been increasing faster both in terms of numbers of users (* . faster) and in volume (* faster) than men's consumption. conclusion: variation in consumption is considerable and cannot be explained by diagnostic and prescription guidelines, as these are similar in the five countries. consumption has been increasing fast in the period in all the countries, and faster for women than for men, although men still consume larger volumes than women, and are more frequent users. please specify your abstract type: research abstract background and objective: in and , regulatory bodies in usa (fda) and europe (ema), issued warnings on use of metoclopramide due to an increased risk for serious adverse drug reactions (adr), especially neurological adrs. ema recommended that metoclopramide only should be prescribed for up to days while fda concluded that treatment longer than weeks should be avoided. metoclopramide is commonly used to treat nausea and vomiting in pregnancy (nvp) and deficient breast milk production ( days course). ema did not make any recommendations concerning use during pregnancy and lactation. the objective of this study is to assess the disproportionality of reporting of adr from metoclopramide, with special emphasis on neurological adrs and women in reproductive age. setting and method: data from whos global adr database vigibase Ò for the time period november to may was used. the measure of disproportionality of reporting calculated was the proportional reporting ratio (prr), and % confidence intervals (ci). analyses were performed according to gender and age. time-toonset of adr was calculated. main outcome measures: proportional reporting ratio (prr) results: vigibase contains over million adr reports. metoclopramide is a suspected/interacting drug in of the reports, most common ( %) are neurological adrs. the majority ( %) of the metoclopramide adrs occurred within the first days of use. a total of % of the reports was received the last years ( ) ( ) ( ) ( ) ( ) . the reporting of neurological adrs was higher for metoclopramide than other medications in vigibase. women in reproductive age ( - years) reported higher proportion of neurological adrs (prr = . , % ci . - . , n = ) than women + years (prr = . , % ci . - . , n = ) but a similar proportion as men - years (prr = . , % ci . - . , n = ). conclusion: there is a . to three fold higher proportion of all reports regarding neurological adrs for metoclopramide than for other drugs. patients initiating treatment with metoclopramide should be informed about risks of adrs and that most adrs occur within days, and instructed to contact health care personnel and stop treatment if adrs occur. please specify your abstract type: descriptive abstract (for projects) background and objective: self-induced drug intoxications (sidi) are one of the most frequent reasons of hospitalization in emergency service ( %) with around - / inhabitants and represent around % of admissions in intensive care unit (icu). it is the most frequently used method of suicide attempts (sa) and the leading cause of hospitalization for young people under . the main objective of our study was to analyse, stratify and pharmaceutically map the different sidi identified in our icu. design: this is a prospective study over months, including all icu patients following sidi from june to january . we have collected psychiatric history and previous sa by sidi, usual treatment, state of consciousness, incriminating drugs, drug classes stratified according to the clinical severity score igsii, evolution, transfer in a specialized centre and average cost of stay. results: ninety-two cases were reported, representing % of icu admissions. the average hospital stay was days for an average cost of . €. this amount is low compared with the average cost of all stays gone through the icu for the period ( , €). ninety percent of patients had a psychiatric history and % a previous sa. the usual treatment was involved in % of sidi. half of the patients arrived conscious with an average of severity score igsii of / , being the highest found for a patient who had swallowed simultaneously pregabalin and nitrazepam. clinical severity of these patients is less than that found on average for all patients in the icu in this period ( / ). eighty-seven percent had a favorable evolution. only one death was observed after ingestion of propranolol. fifty-six and a half percent of patients were then hospitalized in a specialized centre. the great family of psychotropic is the most frequent with benzodiazepines %, neuroleptics %, antidepressants . % and antiepileptic . %. the main drugs involved are oxazepam %, alprazolam %, cyamemazine %, bromazepam % and quetiapine %. antihypertensives then arrive and represent % of sidi. the stratification of severity scores does not appear to show significant differences between drug classes, nor between mono or polydrug ingestions. conclusion: sa by drug ingestion are very common and are often linked to risky behaviours. for these epidemiological and economic findings, it is necessary to continue and develop prevention strategies avoiding the appearance of intoxication (primary), limiting the consequences (secondary), and reducing the risk of recurrence (tertiary). please specify your abstract type: research abstract background and objective: interpretation of quality of life scores to render them meaningful to aid clinical decision-making is an ongoing challenge. interventions often result in statistically significant quality of life (qol) improvement, but may not reach the threshold of clinical importance. the minimal clinically important difference (mcid) is the minimal score change of relevance clinically. the aim of this systematic review was to assess the impact on quality of life of topical, systemic and biologic treatments for psoriasis in randomised controlled trials (rcts). setting and method: prisma guidelines were followed. all available articles describing rcts of therapies for psoriasis that included qol measurements published up to november were identified. six databases were examined with search terms. abstracts of articles were reviewed independently by two assessors: a third adjudicator resolved any opinion differences. risk of bias was assessed using the jadad scale. main outcome measures: reporting of the use of qol endpoints and impact of interventions in psoriasis. results: of screened article abstracts, articles were selected for detailed review: trials met the eligibility criteria, describing research on a total of , patients. reports of psoriasis interventions that fulfilled inclusion criteria have gradually increased over time : - = , - = , and - = ( ) to evaluate the relationship between the use of different therapeutic agents and the severity of osa, and ( ) to determine the effects of commonly used medications on continuous positive airway pressure (cpap). setting and method: patient medical records (n = ) of patients, that underwent sleep studies between the years and were collected over an eight-month period from the sleep laboratory department at mater dei hospital using a random sampling technique. data collected included body mass index, gender, age, epworth sleepiness score (ess), drug history, apnoea hypopnoea index (ahi) and cpap therapy prescription. likelihood ratio chi square test, paired samples t-test and multinomial logistic regression were the statistical tests used for data analysis. main outcome measures: assessment of the drug history in response to osa control using the ess and ahi scores. results: one hundred and seventy ( . %) patients of the patients ( males, females) were diagnosed with osa. forty-five ( . %), ( . %) and ( . %) patients suffered from mild, moderate and severe osa respectively. patients had a mean age of years. angiotensin ii receptor antagonists (arbs) (p-value = . ), sulphonylureas (p-value = . ), insulin therapy (p-value = . ) and non-benzodiazepine sedating agents (p-value = . ) were found to be associated with the presence of osa. a decline in the use of the arbs (p-value = . ), angiotensin converting enzyme inhibitors (p-value = . ) and non-benzodiazepine hypnotics (pvalue = . ) was observed over the study year period. reduction in the cpap therapy benefit was detected with the use of histamine (h ) antagonists (p-value = . ), b-adrenergic blocking agents (pvalue = . ) and antiplatelets (p-value = . ). conclusion: it is confirmed that hypertension and diabetes mellitus type ii are the main co-morbidities associated with the presence of osa. reduction in the use of certain therapeutic agents is observed secondary to cpap therapy use. patients using specific drugs have been identified as being at risk of a reduced cpap therapy benefit. please specify your abstract type: research abstract background and objective: people are using increasingly more common of social networks such as facebook, twitter and youtube for different purposes. many people are using these networks with the aim of getting information and knowledge sharing. there are many groups that pharmacist is a member in social networks at turkey. the largest of these groups has , members. pharmacists are shared common problems, information and experiences in these groups. but the accuracy of the information shared on social networks are not always conclusive. the study aim to evaluate the impact of social network information sharing in the knowledge and attitude of pharmacists. setting and method: clinical pharmacy group has been created to share information on facebook. pharmacist joined this clinical pharmacy group. the group was fed by information which include new drugs, fda alerts, adverse event and case report and also drug related problems during the months. pharmacists were assigned in two major groups, group a active pharmacist who becomes a member of our clinical pharmacy group, share and discuss information through the network and group b who is not a member. a knowledge measurement survey (ams) was given to both of them. main outcome measures: acknowledge measurement survey (ams) was developed and the difference in the score was used to evaluate the difference between the two study groups. results: pharmacists participated in the study, . % of the participants were a member of our facebook group and . % of participants were not. . % of the participants have doctoral degree or student, . % have master degree or student, % have bachelor degree from year-pharmacy faculty, . % have bachelor degree from year-pharmacy faculty. the education level distribution between the two groups was not statistically significant. while . % of the ams questions were answered correctly in the member group only . % were answered correctly in the non-member group. conclusion: the study emphasizes the importance of social network in providing the accurate and fastest information for the daily use of the pharmacists, there is a significant difference in knowledge between the pharmacist who join, share and discuss information on the social network and the one who do not join. cp-ce : impacts of a community pharmacy practice experiences on student professionalism yunn-fang ho , , hung-wei lin *, , fang-ju lin , , sheng-ping chang , yen-ming huang graduate institute of clinical pharmacy, school of pharmacy, college of medicine, national taiwan university, taipei, taiwan, r.o.c please specify your abstract type: research abstract background and objective: professionalism is valued globally and pharmacy schools are expected to nurture competent practitioners to better serve the public with humanity attitudes and behaviours. the study aims: ( ) to understand possible differences in professionalism between pharmacy students and potential community pharmacist preceptors, and ( ) to evaluate student changes in professionalism upon completing the community pharmacy practice experiences (cppe) at the end of the third (p ) year. setting and method: a modified chisholm's pharmacy professionalism instrument ( -item, -point likert scale) was administered to p students, pre-cppe and hopefully post-cppe in september, and community pharmacist practitioners who participated in a two-day preceptor training workshop. participants also provided their significance ratings toward ten traits, namely altruism, accountability, excellence, duty, honor and integrity, respect for others, communication, ethics, humanism, and teamwork. main outcome measures: differences or changes in chisholm professionalism scores. results: thirty-two students and fifty pharmacists participated in the survey. honor and integrity ( . ± . ) and communication ( . ± . ) were recognized by students ( . %) and pharmacists ( . %), respectively, as the most significant trait. humanism was rated the lowest in both groups (students, . ± . ; pharmacists, . ± . ). the -item professionalism scores ranged from . ± . (''i do not expect anything in return when i help someone.'') to . ± . (''i am respectful to individuals who have different backgrounds than mine.'') in the student group; whereas . ± . (''i do not expect anything in return when i help someone.'') to . ± . (''it is wrong to cheat to achieve higher rewards (i.e., grades, money).'') in the pharmacist group. in general, pharmacists' professionalism scores were higher and, in certain items, statistically significant differences were achieved. conclusion: professionalism might grow with professional competency and practice experiences as demonstrated by potential pharmacist preceptors. upon completion of cppe, students could probably exhibit gains in professionalism. more investigations are still underway. please specify your abstract type: descriptive abstract (for projects) background and objective: in france, a significant consumption of benzodiazepines (bzd) is observed in prisons. they are widely used during incarceration to treat or prevent anxiety and insomnia. furthermore, it is known that, an important traffic exists with these drugs because of the releasing properties of bzd in case of misuse. based on these observations, the pharmacist has set up a plan to improve the use of bzd in prison. the purpose of the study was to evaluate the impact of these measures after year of implementation. design: in january , we shared with physicians in a meeting to explain our plan for a better use of bzd and to set up new rules of prescription in prison: • regularly reducing the dose to limit drug tolerance • promoting the use of long half-life molecules which allow reducing addiction and misuse • advising sedatives anti-histaminics to treat insomnia • providing information to patients about addictives risks of bzd on the tv channel please specify your abstract type: descriptive abstract (for projects) background and objective: some drug combinations (described in thesaurus of national agency of drug) are contraindicated because they appear to increase the risk of torsade de pointes. the aim of this work is to standardize our pharmacists' intervention and to propose guidelines for doctors and pharmacists, depending on the situation and drugs, to limit these combinations and to reduce this risk at our hospital centre ( beds). design: a prospective survey was realized over a period of months to identify the drug combinations prescribed in medical prescription software, from the national drug agency thesaurus, that might be inducing torsade de pointes. a multidisciplinary staff was then constituted composed of a cardiologist, a geriatrician, a paediatrician, an anaesthesiologist, a psychiatrist and pharmacists to identify the different situations and to establish guidelines. results: from the survey drug combinations were found to be contraindicated due to increased risk of inducing torsade de pointes on a list interventions realized by pharmacists. the work group identified three drugs with a therapeutic alternative: hydroxyzine, domperidone, escitalopram, the other drugs can't be switched because they are vital or have no alternative. the work group decided to maintain hydroxyzine but only on premedication and child anxiety, to eject domperidone from our therapeutic index and substitute it with metoclopramide or metopimazine, to not initialize escitalopram but to keep it if the patient has no have others risk factors associated or no contraindication. if the patient has a contraindication with a risk factor the doctor could prescribe other ssri. in addition, pharmacists alert doctors about the risk of torsade de pointes on medical prescription software if some contraindications are identified. conclusion: the contraindications identified must not be underestimated. this work allows identification of torsadogenic drugs commonly prescribed and provides guidance for doctors and pharmacists regarding drug combinations. the collective decision will be disseminated to sensitize all the doctors in the establishment. some treatments could not be substituted despite the contraindication; these must be retained but with clinical monitoring. conclusion: a substantial proportion of medication waste in the community pharmacy could have been prevented. unused medicines in the community pharmacy are generally of low economic value, making it unlikely that the costs that pharmacies will make with the redispensing of unused medicines will be covered. therefore, other actions to decrease medication waste in the community pharmacy, such as preventing that too much medicines are dispensed, should be considered. please specify your abstract type: research abstract background and objective: flaws in usage technique for inhalationmedicines is common, as much as half of the users may need some correction measures, to get the active substances down to the lungs and provide the intended effect. inadequate compliance, especially for regular-use preventive medications, is common. good guidance in pharmacies enhances correct use of medicines. the new norwegian pharmaceuticals policy (legemiddelmeldingen) from opened up for paid cognitive services, leading to the first such service being implemented in march . the service can contribute to a more correct use of the medicines and, as a consequence, lead to better control of the symptoms for patients with asthma or copd. our objective was to map the variation in pharmacies' handling of an inquiry regarding lack of effect of an inhalation-medicine. the study was done prior to the implementation of the standardized service ''inhalation-guidance'' in norwegian pharmacies. setting and method: simulated patient (mystery shopper) visits in pharmacies in oslo, akershus and buskerud in november/december . the mystery shopper expressed just having started to use an inhaler because of her asthma, but not experiencing effect. structured data collection sheets were used to register the handling immediately after the visit. main outcome measures: scoring of the quality and contents of the information based on the products' patient information leaflets. results: the issue of inhalation-technique was mentioned in of the pharmacies, whereof asked the ''patient'' to show their inhalationtechnique, in order to correct and advice and used an inhaler or demo-inhaler as an aid in the guidance. going through the instructions or watching a video-demonstration with the simulated patient also occurred, or referring the patient to read the instructions and/or watch the video-demonstration on his own. half of the pharmacies discussed the difference between use for preventive treatment of asthma and inhaler that is being used for treatment of attacks. sixty-five pharmacies gave no information about the importance of regular use of the preventive treatment. conclusion: there was considerable variation in how the pharmacies guided, which indicates a potential for improvement. the new guidance-service, implemented in norwegian pharmacies in march , will contribute to better guidance. please specify your abstract type: research abstract background and objective: in portugal, tobacco addiction was responsible for over , deaths in ( % of the total deaths). the community pharmacist's contribution to control this public health problem is insufficiently documented. the aim of this study is to assess the contribution of the community pharmacist for smoking cessation. setting and method: a retrospective and longitudinal study of a convenience sample of patients integrating quit tobacco consultations, as part of a pharmaceutical care programme implemented by an outsourced pharmacist was performed at several community pharmacies. the smokers, aged or over, were invited to join the programme. patients signed an informed consent and were submitted to a comprehensive approach by face-to-face consultations and telephone contacts. richmond and fagerström tests were used to evaluate motivation and nicotine dependence, respectively. the therapeutic plan (pharmacotherapy and behavioural counselling) was personalised to each smoker. the quit rates were evaluated by patient selfreport and confirmed by carbon monoxide measurements. the continuous variables are expressed as mean ± standard error of the mean. main outcome measures: quit rates at , , and months. results: between january and june , smokers joined the programme, dropouts ( . %). the remaining smokers, ( . %) were male, with mean age of . ± . years. on average, each smoker consumed . ± . cigarettes per day. the mean age of initial tobacco use was . ± . years with . ± . years of consumption. about % reported moderate or high motivation and % medium or high dependence. a total of consultations were held and, on average, each patient received . ± . interventions. all smokers received non-pharmacological interventions (e.g. motivational approach) and ( . %) also accepted pharmacological interventions, usually nicotine replacement products. the quit day was achieved by patients ( . %). a month after quit date, patients were abstinent ( . %). the number reduced to after months ( . %), to after months ( . %) and to after year ( . %). these data upgrade and are consistent with our previously published results ( ). the smoking cessation consultation in the scope of a pharmaceutical care programme in community pharmacy seems to effectively contribute to the reduction of tobacco addiction in portugal. cp-pc : patient counselling at dispensing of oral anticancer drugs in european countries from the pharmacists' perspective andreja eberl * , on behalf of epic working group pharmacy, institute of oncology ljubljana, ljubljana, slovenia please specify your abstract type: research abstract background and objective: the number of oral anticancer drugs (oads) available on the market grows constantly. consequently the number of patients, which have to manage the complex treatment with oads at home is increasing. the pharmacists present an important member of healthcare team, since they are dispensing oads to the patients, which need a high quality information at that crucial moment. therefore, our aim was to evaluate pharmacists perceived confidence and needs for specific continuing education in connection to oads dispensing in european countries. setting and method: we used an electronic mailing approach and a standardized online survey to ask practicing pharmacists in european countries about their experience with dispensing of oads. main outcome measures: frequency of patient counselling and fields of counselling, assessment of knowledge and skills. results: the frequency of patient counselling varied widely in participating countries between ''never'' and ''more than %'' at initial fill of an oad. at following refills the frequency of counselling was generally even lower. counselling mostly encompassed directions of use, the proper use of antiemetics and side effects. however many pharmacists stated, that they do not feel comfortable counselling patients of oads ( %) and even more acknowledged that they were uncomfortable with managing patients' side effects ( %). on the other hand only % of pharmacists believed, that they have received adequate knowledge of oads through undergraduate program, continuing education (ce) events and professional practice. many of pharmacists ( %) have not attended any of ce events related to oncology in last years. pharmacists' responses differed little between the countries. conclusion: the proportion of pharmacists who regularly counsel their patients on oads is insufficient in view of importance of the patients' needs to manage their therapy at home. however the pharmacists seems to be aware of their knowledge deficits and educational needs. the field of oads needs better coverage in under-and postgraduate education. the number of ces has to be increased in order to improve the knowledge and skills in the areas of oads counselling. please specify your abstract type: research abstract background and objective: treatment guidelines for diabetes recommend that patients are well-informed about their disease, treatments and treatment goals, e.g. glycosylated haemoglobin (hba c). the objective was to describe diabetes patients' self-monitoring of blood glucose (smbg) and potential need of guidance. please specify your abstract type: descriptive abstract (for projects) background and objective: in , the international pharmaceutical federation collected data of remuneration models for community and hospital pharmacy and identified large variations between remuneration models and highlighted that the focus is largely on products and not on cognitive services. the aim of the study is to map the remuneration models of different pharmacist-led cognitive services in primary care across europe, with a special interest on medication reviews and to update a prior survey by bulajeva (bulajeva a et al. medication review practices in european countries. res social adm pharm ; : - .). the definition of terms is pivotal for such a european survey to avoid results based on pseudoconceptions. hereafter we present the development of the survey and we will present first results from pilot tests. design: pharmacist-led cognitive services were selected based on a previous study by our group and by searching the literature, official government websites, the pcne wiki and arising links. the definitions of the terms of these services were based on searches in the mesh browser, medline and google scholar. additionally, a search in grey literature and in the internet was conducted to find appropriate foundation for the formulation of the definitions. the questionnaire will consist, of a first part about the remuneration of the pharmacist-led cognitive services. the focus is on country-specific differences in remuneration and the different levels of supply across europe. the second part of the survey is about the different types of medication review services with a focus on e.g. the implementation level, addressed issues, eligibility criteria. this survey will have a cross-sectional study design with an online questionnaire specific for invited participants across europe. to achieve the best quality of answers we will send this survey to at least two researchers with references in pharmacy practice, in each european country (purposive sample). the answers from each country will be checked for discrepancies and these potential discrepancies will be solved by a discussion with the responders. results: by the end of the pre-pilot phase, different pharmacist-led cognitive services were identified and the correlating definitions of the terms were developed. conclusion: at the time of submission the pre-pilot phase has been finished and the pilot will start july . please specify your abstract type: research abstract background and objective: medication adherence is one of the key aspects in assuring optimal health outcomes in majority of chronic diseases. the aim of the study was to evaluate copd patients' medication adherence in slovenia and its association with health outcomes. setting and method: patients were recruited by community pharmacists at the time of dispensing medication for copd. medication adherence was evaluated by using morisky medication adherence scale (mmas- ). patients who scored b points, . - . points and points were regarded to have poor, moderate and good adherence, respectively. quality of life was evaluated by saint george's respiratory questionnaire (sgrq) and the impact of disease by copd assessment test (cat). the study was conducted in september and february . the association between potential predictors and copd impact or quality of life was estimated using multiple linear regression in ibm spss statistics version . main outcome measures: medication adherence rate (mmas- ), quality of life (sgrq total score) and impact of disease (cat score). results: of patients, majority were men ( %) with mean age years. in average, patients were prescribed . medicines for copd and . medicines for other diseases. good, moderate and low adherence to copd medication regimens was found in . , . and . % of patients, respectively. mean cat scores and sgrq scores were . (range - ) and . (range - ), respectively. thirtyeight percent of patients experienced an exacerbation in the past year. linear regression showed no statistically significant association between medication adherence and quality of life or copd impact on patient. factors that statistically significantly predicted patients' quality of life were exacerbation in the past year, education level and number of concomitant medicines for other diseases. the latter was found to be the only factor associated with copd impact. conclusion: the study showed half of the copd patients to be optimally adherent to their treatment and only a small proportion of patients not taking their medicines regularly. due to the nature of the disease medication adherence does not seem to play the most important role in assuring optimal health outcomes in copd patients. please specify your abstract type: descriptive abstract (for projects) background and objective: intermediate care units (imcu) are designed to serve patients in need of more advanced medical care than the ordinary nursing home units can provide. the aim of this study was to see; ( ) how medication information follows patients in and out of icmu and nursing home short-termcare units (stcu) ( ) the type and amount of drug related problems (drp), focusing inappropriate drugs, and ( ) if there are differences between the icmu and stcu in drug use and drps. design: patients c years old admitted and submitted at the imcu or stcu in the study period ( weeks) were included. transfer of medication information were evaluated and given a score. the clinical pharmacist provided medication reconciliation upon admission, medication review and monitoring, and presented identified drps and a suggestion for solving the problem, to the multidisciplinary team. inappropriate drugs, identified by screening tools (stopp/norgep), and systematic medication reviews, were recorded. results: patients from imcu and five from stcu were included. a hospital discharge summary including medical history followed mostly all patients. the score of the medication history was . points out of . by submission from either imcu or stcu, the score was . . systematic drug review identified . drp in the imcu and . in the stcu. imcu patients used . drugs, stcu patients . in the icu, % of the identified drps was inappropriate drugs, none in the stcu. the clinical pharmacist in the multidisciplinary team presented % of the identified drps. the doctors agreed in % of the suggestions for solution, and started immediate changes in %. conclusion: a hospital discharge summary followed the patients, but the medical history part needs improvement. although few patients, the results suggest that imcu patients had more complicated medication and more inappropriate drugs than stcu patients did. clinical pharmacist in a multidisciplinary team provides useful contribution to identify, solve and prevent clinical relevant drps, including inappropriate drugs. please specify your abstract type: research abstract background and objective: lack of clinical effects of medication review on health-related quality of life of older people may be due to insufficient focus on health-related complaints. goal attainment scales (gas) are an instrument to formulate specific health-related goals. the objective of this early process-evaluation of the dreamer-study (drug use reconsidered in the elderly using goal attainment scales during medication review) is to investigate if pharmacists are able to formulate gas during a medication review of older people with polypharmacy. setting and method: older patients aged years or older using or more medicines are included in this study. half of the patients were randomized into the intervention group, where they received a medication review. during the patient interview, the pharmacist formulated gas in concordance with the patient. recommendations were made to reach these goals in collaboration with the gp. main outcome measures: number of performed medication reviews, total number of formulated gas and the three most frequent types of gas. results: until now patients have been included in the drea-mer study ( % of the target). half of them ( ) were randomized into the intervention group. by now ( %) of these patients have received a patient interview. goal attainment scales were formulated yet. the number of gas ranged from to per patient. the four most frequent gas were: polypharmacy-reducing the number of medicines ( ), reducing pain ( ), increasing mobility ( ), reducing fatigue ( ). conclusion: gas seem to be a feasible approach during medication review that increased focus on patient's needs and health-related complaints. cp-pc : oral transmucosal fentanyl citrate: a regional survey of dispensing practices in community pharmacy please specify your abstract type: descriptive abstract (for projects) background and objective: oral transmucosal fentanyl citrate (otfc) is an opioid analgesic indicated for management of breakthrough cancer pain in patients with malignancies who are already receiving and who are tolerant to opioid therapy for their underlying persistent cancer pain. otfc are usually use off-label prescription, especially in noncancer patients or patients without opioid maintenance treatment. this practice can expose to iatrogenic risks, lack of efficacy, abuse and addiction. the observatory of drugs, medical devices and therapeutic innovation of upper normandy, conducted a study to assess the knowledge of pharmacists on these medications and assess dispensing practices (pharmaceutical analysis and advice to patients). design: between june and september , two quizzes were sent to the pharmacists and pharmacies in upper normandy: one included questions of knowledge and general practice, the other assess dispensing practices of otfc prescriptions received at the counter, regarding indication, dosage and associated opioid medication. results: of the pharmacists who participate in the survey, % know the all of the oftc specialties, % of them confuse transdermal and transmucosal fentanyl specialties. indication, dosage, titration methods and the main interest of oftc are known by , , and % of them. only % have dispensed oftc more than times over the past months, % never have. they already have dispensed oftc in noncancer patients ( %) or without opioid maintenance treatment ( %). they consider not know enough about these drugs to be able to provide the necessary advice to patient ( %) and would like specific training on oftc ( %). of the analyzed prescriptions, only % are consistent with the marketing authorization: otfc medicines are prescribing in noncancer patient ( %) and/or dosage is higher than four units per day ( %) and/or there is no prescribed opioid maintenance treatment ( %). only two prescriptions have been discussed with the prescriber, and all were approved and dispensed. conclusion: otfc specialties are occasionally dispensed and often misunderstanding by pharmacists. a good knowledge of otfc is necessary to achieve the pharmaceutical analysis and provided appropriate advice to patients, in order to guarantee the good use of these medicines. support tools for dispensation, recalling indication, . the most frequent interventions were drug substitution (n = ), dose adjustment (n = ), and clarification of information (n = ). common services were reconstitution of suspension (n = ), provision in advance for continuing supply (n = ), and follow up offers (n = ). conclusion: the observation of the dispensing process in community pharmacies revealed a broad range of tasks performed by the pharmacy and identified several variables likely to influence the counselling. in addition, pharmacy activities could be pictured by the documentation of pharmaceutical interventions. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is a multidisciplinary process to correct medication errors resulting from miscommunicated information at transitions of care. development of this activity is essential but it is hindered by the time required for its implementation. we must carefully choose which services can develop this activity. as it was recently introduced in cardiac surgery unit, this study aims to evaluate impact of this process to hospital admission (severity of potential harm of medication error intercepted) and to determine the relevance of this activity in this unit. design: prospective study conducted from january to april . the data is recorded in an excel table, filled after each mr. there are five items: patient's age, best possible medication histories (bpmh), implementation period of the mr, inadvertent discrepancies (ids) and clinical impact. to assess the severity of ids, a scoring method was used (doerper et al. ) with the cooperation of surgeon and pharmacist. results: eighty-two patients (mean age ± years old) were included in the study, which represents % of the patients hospitalized in this service. the mean number of drugs per patient was ± . the bpmh were obtained within h to h of admission to hospital. a total of ids were detected, with a mean of . ids per patient. the most frequent type of ids was omission ( %, n = ), error of dose ( %, n = ). the three most common classes involved in ids were hypolipaemic drug (n = ), antidiabetic drugs (n = ) and the drugs for acid related disorders (n = ). the mean of ids per patient ( . ) as well as the percentage of patients affected by a ids ( %) are less important in cardiac surgery than those observed in other services of the institution and in the literature. about clinical impact, % of patients presented with ids considered as minor, % significant and % major. among the major ids, none was evaluated as critical or catastrophic. in our study, this process remains retroactive. conclusion: one of challenge experienced when implementing mr process in hospitals is demonstrating its clinical impact. in order to address this concern, we found that the little ids with a serious clinical impact in this unit. mr is an interesting process to detect drug errors. to optimize our study we will improve our organization in order to be closer to the patient and to strengthen the doctor-pharmacist collaboration. please specify your abstract type: research abstract background and objective: special packaging like multidose drug dispensing (mdd) may optimize medication use in patients with a decreased ability to manage their own medication. however, it remains unclear how a 'decreased ability to manage medication' is defined. the objective of this study is to assess potential medication problems that contribute to a decreased ability to manage medication in patients starting with mdd compared to patients who use manually-dispensed drugs. setting and method: patients starting with mdd (cases) and patients using manually-dispensed drugs (controls) were interviewed in community pharmacies. questions to assess potential medication problems covered three domains; medication adherence ( ), practical management issues ( ) and medication knowledge ( ) . every potential medication problem was scored with one point. cognition was assessed with the mini-cog and frailty with the groningen frailty index (gfi). main outcome measures: mean scores of potential medication problems on the domains medication adherence, practical management issues and medication knowledge. results: patients starting with mdd and patients using manually-dispensed drugs were interviewed. patients starting with mdd scored more potential medication problems on all domains: adherence . versus . , practical management issues . versus . , medication knowledge . versus . . on the three domains together, patients starting with mdd scored . [ . - . ] potential medication problems compared to . [ . - . ] for patients with manuallydispensed drugs. forty-two percent of the patients starting with mdd might be cognitive impaired and % was classified as frail compared to and % respectively of the patients using manually-dispensed drugs. conclusion: patients starting with mdd reported significantly more potential problems on three domains that may contribute to a decreased ability to manage their medication. cp-pc : fifteen key questions to assess patient knowledge on new oral anticoagulants corina metaxas * , valerie wentzky, sonja luginbü hl, kurt e. hersberger, isabelle arnet please specify your abstract type: research abstract background and objective: knowledge on new oral anticoagulants (noacs) is crucial for their safe and effective use. validated tools that assess patient knowledge exist for vitamin k antagonists, but not for noacs. we aimed to identify which questions are relevant for patient knowledge on noacs. setting and method: based on a systematic literature search, questions were compiled for the assessment of noacs knowledge. key questions were selected through three rounds of ranking by an expert panel (four physicians, four pharmacists, four nurses). round (online survey; importance): the questions grouped into the nine educational topics of wofford,adapted for noac (disease, mode of action, risk-benefit, adherence, accessing healthcare professionals, diet/life-style, lab-monitoring, medication interactions, self-care) were to be rated as important/not important and educational topics were to be ranked according to decreasing importance. round (online survey; relevance): the questions were to be ranked according to decreasing relevance. round (focus group): number of questions was reduced by voting. main outcome measures: ranking of educational topics and questions ( = most important/relevant) in march/april . results: experts ranked adherence ( . ± . ) as the most important topic, followed by risk-benefit ( . ± . ), disease ( . ± . ), accessing healthcare professionals ( . ± . ), self-care ( . ± . ), lab-monitoring ( . ± . ), medication interactions ( . ± . ), diet/life-style ( . ± . ) and mode of action ( . ± . ). one question was judged as unimportant by all experts. out of the remaining questions, ( . %) were selected as relevant for basic knowledge, ( . %) were combined into four questions and one new question was generated. a total of key questions remained after the focus group discussion. conclusion: a multiprofessional expert panel was able to select key questions retrieved from literature and ensured content validity. the selected questions will be compiled into a tool to assess patient knowledge on noacs. background and objective: medicines use review (mur) was defined by the slovene chamber of pharmacies in december and an education program was set to assure pharmacists competencies. in june the first pharmacists were certified and implemented the service in the community pharmacies. additionally, an online database was established to collect mur reports and provide feedback on pharmacists' performance. the aim of the study was to evaluate identified drug related problems (drp) as well as pharmacists' interventions from mur documentation. setting and method: a preliminary retrospective analysis of documentation for mur services provided in the first year after implementation was performed. drps were classified using a slovenian drp classification system, which is based on the pcne classification v . [ ] . data were analysed with descriptive statistics measures. main outcome measures: number and type of identified drp and pharmacists' intervention. results: a preliminary analysis was performed on mur cases, performed by certified pharmacists. in total drps were identified: ( . %) manifested and ( . %) potential. patient had on average two drps, however patients had none. main risk factor for potential drps was inappropriate use of medicines. adverse drug events (ades) presented . % of manifested drps; the main risk factor was again inappropriate use. in two cases ades happened due to an allergic reaction. different medicines were the cause of ades; mainly statins resulting in muscles pain and sleeplessness. another frequently manifested drp was insufficient effectiveness of treatment. drug interactions were risk factors in cases of manifested drps, mainly in connection with antidepressants: serotonin syndrome due to escitalopram, bleedings in concurrent use of escitalopram and ginkgo, sleepiness, etc. pharmacist intervened independently in . % of cases; times recommendations were given to physicians. however, in . % of cases the outcome of intervention is unknown. the preliminary results of the first mur cases points to a high number of identified manifested drps. however, the knowledge of intervention outcomes is lacking and therefore more attention has to be put on establishing adequate follow up on this issue. official definition represented harmonisation of several similar activities that have already been performed in slovenian pharmacies and also provided an educational program to assure pharmacists competencies. in may the first pharmacists were certified and implemented the service in the community pharmacies. therefore, the aim of the research was to get an insight into the implementation of mur in slovenia from the perspective of the first community pharmacists that provide the service in practice. setting and method: a focus group with seven community pharmacists, that provide mur in practice, was run in february . guided discussion included three main themes: the development and assurance of competencies, experience with the provision of service in practice and the future of the service. the discussion was voice recorded and analysed with the nvivo . written consent from included participants was obtained. main outcome measures: views, challenges and opportunities for the medicines use review service in slovenia. results: in total themes were identified and organized in three main categories: competencies for quality provision of mur, mur's recognisability and organizational aspects of mur provision. participants emphasized broad knowledge in pharmacotherapy is pharmacists' key competence and advantage in performing mur when compared with other healthcare professions. recognisability of mur among other health care professions as well as participants' work environments is low. hence a comprehensive approach in marketing of the service is needed. positive patient's feedbacks were reported, however persuading patients to attend mur presented a challenge. another barrier was the time to perform mur, which could be overcome by suitable work organization and special time intended for mur. conclusion: participants of the focus panel had positive experience with the development of competencies and implementation of the service in the practice. several challenges were presented connected with the recognition of the service by patients, physicians and health care payer. they strongly believe that continuing professional development forms the base for quality of the service in the future. cp-pc : evaluation of rational antibiotic dispensing in the community pharmacy setting: a simulated patient study betul okuyan * , mehmet ali savan, fikret vehbi izzettin, mesut sancar please specify your abstract type: research abstract background and objective: in the present study, it is aimed to evaluate rationale antibiotic dispensing without prescription in the community pharmacy setting; this will be done by using a simulated patient methods. setting and method: this study was conducted in malatya, located in the east part of turkey. the simulated patient visited the community pharmacies to meet the pharmacist, posing as the husband of a patient with acute uncomplicated rhinosinusitis. the simulated patient was trained regarding the standard information to be provided by the researchers and informed about the privacy of all information that would be gathered during the present study. the sample size was sixty-seven pharmacies, with a confidence interval of % and error of margin of %. the study was conducted over a total of pharmacies. all the pharmacies were listed alphabetically and were randomly selected and allocated random numbers by a computerbased program. main outcome measures: after each community pharmacy was visited, the simulated patient filled the check list which had been drawn up for the purpose of the present study. due to ethical concerns, no audio or video records were used during the study. any suggested medications were not purchased from the community pharmacy. results: of the total community pharmacies that were visited . % of them had female pharmacists and . % were run by male pharmacists. the mean number of questions asked by pharmacists to the simulated patient was . ± . . only eleven pharmacists did not suggest any medication for the simulated patient. however, thirty-two ( . %) pharmacists recommended various medication regimens, including antibiotics. of them, . % referred the simulated patient to a physician. conclusion: in conclusion, it was observed that dispensing antibiotics without prescription was still high, pharmacists did not take comprehensive medical or medication history from patients, and pharmacists provided insufficient medication information to the patient regarding suggested medications at community pharmacy setting. to avoid irrational antibiotic dispensing, it is essential to educate both health care providers and the general population. although dispensing antibiotics without prescription is illegal in some countries, it is necessary to actualize new regulations to avoid antibiotic dispensing without prescription. please specify your abstract type: research abstract background and objective: the medication adherence is an important part of active (as well as passive) attitude of a patient to the disease treatment. it represents the level of keeping the treating procedure as well as the recommendations of doctors, pharmacists and other healthcare professionals. this study deals with the adherence in patients with hypertension. the hypertensive patients are a substantial part of patients, daily visiting the community pharmacy to pick their prescriptions. these patients represent group of patients with typical asymptomatic disease. this means that they do not take the medicines or use them according to their own will. the result of their non-adherence could lead to later complications. the aim of the study was to evaluate the level of adherence and its relation to the clinical outcome-the blood pressure in hypertensive patients. setting and method: the methodology was based on a single anonymous questionnaire survey combined with the blood pressure measuring in a community pharmacy in slovakia. the modified morisky -item medication adherence tool was used in this study. main outcome measures: the results of medication adherence were evaluated as follows: - points = full adherence, points = partial adherence and - = non-adherence. each participant should use at least one antihypertensive agent and fulfil the anonymous questionnaire in the community pharmacy. the pharmacist measured the blood pressure in each participant twice, within the interval of min and used the average value in data sheet. results: the research included hypertensive patients ( . % females and . % males). the results showed that almost % of the respondents were non-adherent to the prescribed pharmacotherapy ( . % of those were males and . % were females). the group of partially adherent patients consisted of . % of the respondents ( . % of those were female). only . % respondents were fully adherent according to modified morisky score ( . % of those were women). fully adherent patients reached an average blood pressure . / . mmhg; partially adherent hypertensive patients recorded an average blood pressure . / . mmhg; and in the non-adherent patients has been observed the average blood pressure . / . mmhg. the results showed an alarming situation, and confirm the published data. non-adherent patients could not goal the good clinical outcomes. this leads to adding of another medications, raising the risk of interactions and adverse drug reactions, complications of undertreated disease, and finally, to pharmacotherapy costs increasing. please specify your abstract type: research abstract background and objective: in psychology, depression is a mental state characterized by feelings of sadness, dejection, inner tension and indecision. in psychiatry, the depression is defined as a severe mental affective disorder which paralyzes clarity of thought, psychomotorics, sleep cycle and raises pessimistic and depressing emotions often lead to pathological changes of personality. during treatment of depression is often needed psychotherapy and pharmacotherapy as well. using of antidepressants requires the sufficient level of medication adherence in patients. non-adherence to antidepressant medication significantly contributes to the undertreatment of depression in primary care populations. the aim of this study was to evaluate the level of medication adherence to antidepressants to better understand the socio-behavioural factors associated with non-adherence. setting and method: the anonymous, face-to-face questionnaire survey was set in the community pharmacy in slovakia. questionnaire obtained questions on socio-behavioural factors and adherence tool-modified -item morisky score (mmmas- ). main outcome measures: respondents were patients ( males, females) using at least one antidepressant. the results were evaluated as follows: points = full adherence, - points = partial adherence and - = non-adherence. results were evaluated in relation to socio-behavioural factors. results: average level of the medication adherence in our group was , which means the line between partial and full adherence. the results showed non-significant higher medication adherence level in males ( ) compared to females ( ). the highest level of medication adherence ( ) has been shown in patients - years old, the lowest average adherence level (non-adherence) was observed in patients up to years old ( ). patient living in the city were more adherent to their medication ( ) compared to patients living in countryside ( ). the highest level of the partial medication adherence has been shown in secondary educated patients ( ). partial adherence level was higher in patients with monthly income over € ( ) compared to non-adherent patients with monthly income up to € ( ). in patients using no other medications, only antidepressant, we have observed the highest partial adherence ( ). conclusion: our survey showed the partial antidepressant medication adherence levels in our study group. poor adherence results in low stabilization of clinical state in patient, in using more types of therapy and in increasing costs. there might be very important role of the community pharmacists and other health care professionals to improve the medication adherence and persistence through counselling and education patients on importance and need of antidepressant medication. ( ) and medication regimen complexity was assessed by using the medication regimen complexity index (mrci) ( ) . five and more medication usage has been defined as polypharmacy. results: a hundred and two elderly subjects ( . ± . ; male) were included in this study. of them, . % had two and more chronic diseases. the most common chronic diseases determined in study population were cardiovascular diseases (especially hypertension), diabetes and hyperlipidaemia. the polypharmacy has been defined in . % of them. the mean of mrci per elderly patient was . ± . . one or more pims use was observed in seventy-four elderly subjects ( . %). of all elderly subjects, . % were dispensed one and more medicines with a potential for drug-disease/ syndrome interaction. pims use was more frequently determined in patients with polypharmacy ( . vs. . %, p \ . ). the total score of mrci was significantly increased with elevated number of pims (r = . , p \ . ). conclusion: this study highlights a significant association between utilization of pims and both polypharmacy and higher total score of mrci in elderly patients. pharmacists could be evaluated utilization of pims in especially elderly patients with used five or more medications and/or higher total score of mrci. please specify your abstract type: research abstract background and objective: nursing home patients with multimorbidity often use multiple drugs simultaneously, which makes these patients more susceptible to adverse drug events. several studies have pointed to a need to increase the quality of prescribing to this population. to achieve this there is a need for reliable information about patients' diagnosis, and what is recorded as the drug's indication in different electronical and handwritten health records. the aim of this study was to examine the registered diagnoses, and indications for drug use in nursing home patients. we also wanted to study the extent to which diagnoses are untreated with drugs, as well as the extent to which drugs have a registered indication for use and a suitable recorded diagnosis. setting and method: data was collected for long-term patients, on average years old, and % females from four nursing homes in tromsø municipality, norway. we retrieved information about patients' diagnoses and indication for drug use from the electronic health record and written drug charts. two pharmacists conducted the linkage between the reported diagnoses and drug use. main outcome measures: percentage of untreated diagnoses and the percentage of drugs with a registered indication for use. results: as considered by the pharmacists, % of the registered diagnoses was untreated with drugs. dementia, gout and osteoporosis were the most commonly untreated diagnoses with, , and %, respectively. in comparison, the indication for use listed on the patients' drug charts was reported for % of the drugs. the drugs with the highest percentage of recorded indications were acetylcysteine (n = ), oxycodone (n = ) and zopiclone (n = ), where , and % had a listed indication, respectively. conclusion: a high percentage of nursing home patients' diagnoses seem to be untreated. however, most drugs that patients received were listed with indication for use in the drug charts. to increase quality of drug prescribing, one should put emphasis on improving the recorded information in electronical health records. cp-pc : personal changes in drug regimen: dangerous for health system? inga urtane, raivis pastars, dace bandere please specify your abstract type: research abstract background and objective: patient compliance is a key factor for a successful treatment and lack of it is the main reason for predicting treatment failure. in multiple researches patient adherence is determined to be as low as %. therefore it is important to identify the reasons of patients not following their drug regimen. objective. to analyse the patient comprehension of their drug regimen depending on the duration of hypertension and received treatment. setting and method: during the period from december to march a quantitative survey was conducted to include respondents who have been diagnosed with arterial hypertension and whose regimen includes at least one fixed dose combination drug. main outcome measures: in an anonymous survey data was collected about their demographic information, co-morbidity, other prescribed medication, intake regime, the average blood pressure during treatment, and patient's assessment of the prescribed therapy. collected data was analysed with spss. results: the study included participants, most of whom ( . %) were women. participants average age was . ± . years and the median arterial hypertension duration was ( ; ) years. the study participants, who sometimes consciously adjusted dosing regimen, observed arterial hypertension for a longer period of time compared to the group, which follows the prescribed regimen according to their doctor's recommendation, respectively, ( ; ) vs. ( ; ); p = . . group of respondents (n = ) receiving c prescription drugs, more often deliberately adjusted treatment regimen compared to respondents (n = ) treated with b prescription drugs, respectively . versus . %; p = . . respondents who deliberately adjusted drug were more often not satisfied with the number of longterm daily use of tablets (n = ) compared to the group (n = ), which had to intake fewer tablets every day, respectively, . versus . %; p = . . conclusion: arterial hypertension duration was associated with more frequent conscious adjustment of therapy without consulting a doctor. more individual prescriptions (c ) and an increase in the number of tablets per day at the same time also increases the risk of patients deliberately changing their dosing regimen. long-term drug users should receive additional attention during pharmaceutical care process to their respective treatment schedule in order to promote proper use of medication. please specify your abstract type: research abstract background and objective: diabetes is a health issue and real burden for in belgians. better adherence to the treatment could potentially reduce complications, decrease morbidity and mortality, and have a beneficial economic impact due to fewer consultations and hospitalizations. setting and method: a one-year program was started in belgian pharmacies to accompany diabetes patients taking dpp- inhibitors and encourage them to be compliant with their treatment. this study concerns of these pharmacies, all part of the same cooperative group. all pharmacists received prior training in motivational techniques and reviewed the bases of diabetes therapy with an e-learning program. materials developed for the patients included brochures on diabetes and its treatment, nutritional advice, physical exercise, foot care and tips and tricks for diabetics. main outcome measures: the impact on pharmacological adherence was measured using mmpr and pdc. two control groups were included: a historical control group and a group of patients that were not included in the project. non-pharmacological adherence was assessed using questionnaires. results: in the subgroup of pharmacies, patients were included in the program. by the end of april , only of them had completed the program; patients came only once to the pharmacy. they either stopped their treatment after one prescription, or were occasional clients. adherence rates were found to be high in all groups ( . - . % of patients with mmpr b %). only for the pdc, a statistically significant difference was measured between the intervention and control group ( . vs. . %; p = . ). no other statistically significant impact was measured (neither pharmacological, nor non-pharmacological). conclusion: adherence was very high in all groups. the underlying reasons still need to be investigated (choice of adherence measure, healthy user effect, etc.). however, both patients and pharmacists were very pleased with this type of program. this new role of the pharmacist will definitely be more developed in the future. please specify your abstract type: research abstract background and objective: oral anticoagulants (oac) have a beneficial effect on the long term survival of patients with atrial fibrillation and venous thromboembolism. however oacs have also side effects such as bleeding, especially when used inappropriately. pharmaceutical care interventions aim to optimize medicines use and improve patient health outcomes. the literature lacks a review on the impact of pharmaceutical care interventions in patients using oac. therefore, we systematically assessed the impact of pharmaceutical care interventions on the effective and safe use of oac compared to usual care. setting and method: a systematic review was performed in pubmed and embase with synonyms/detailed specifications of the terms oral int j clin pharm ( ) . it was motivate for the need to sort the instruments for urm, including professional participation, and on the basis of the clinical management unit, and reduce variability in decisions. the p&t or ''multidisciplinary commission rational use of medicines'' is constituted by people: one hospital medical director (president), head of pharmacy (secretary), and three directors of healthcare centre, three directors of department of specialities, one epidemiology, one hospital pharmacy, one primary care pharmacy and one paediatric. because some of these members are far between them, and normally dose not have too much time, we create an online platform to work, discuss and download all the necessary documents. setting and method: we used the facilities of the andalusian agency for healthcare quality (www.acsa.junta-andalucia.es), and as a base the law of the administrative decision. we have organized a session to discuss methodology with the participation of all members. main outcome measures: number of meeting and number of internal discussion emails. drug or protocol decisions. design of the platform. results: the design platform consists of five tabs: ( ) has the member information, position, telephone and address, ( ) email forum, following a subject line, ( ) a place for meeting requests and then hang up the meeting minutes. ( ) a tool allows you to upload documents to the portal ( ) a search engine. two sessions are schedules and total of mails. we have of members who have never participated online. at this moment we have adopted two decisions. conclusion: it is an online experience of one andalusian p&t committees, the low turnout makes go slower than expected, therefore physical meetings are necessary in this moment. we are working how to get more participation and involve in the project the committee members. please specify your abstract type: research abstract background and objective: liver cirrhosis can have a major impact on drug metabolism, requiring evaluation of drug safety and dosage in individual patients. currently, there are no guidelines on safe prescribing for medications in patients with liver cirrhosis, and these patients have many questions about safety and side effects of medication. the objective of this study is to explore the patient's needs on information about medication. setting and method: qualitative, semi-structured interviews were performed in patients with a (history of) liver cirrhosis. the patients were approached through an item in the newsletter of de dutch association of liver patients. topics in the interview guide were preferences about information about medication, side effects, safety, drug dosage, and how patients preferred to receive this information. interviews were audiotaped and transcribed verbatim. interviews were analysed using thematic content analysis. main outcome measures: the experiences and needs of patients with liver cirrhosis concerning information about medication. results: patients indicated they had received sufficient information about the indication, possible drug-drug interactions and the duration of treatment. they preferred (more) information about how medications work, what adverse drug reactions could be expected and practical aspects concerning intake of medication. informational needs were related to questions 'how to act': patients with more informational needs took a more active role in responsibility for their own medication management. patients needed information to know what to do, e.g. in case of adverse drug reactions or when a dosage was forgotten. the doctor and internet were the preferred sources of information: doctors because of the personal contact and internet because of the accessibility. facilitating factors were 'taking time' in healthcare provider-patient contact and 'everyday language' for texts on the internet and in package leaflets. a combination of verbal information by the healthcare professional and written information was preferred. conclusion: patients with liver cirrhosis need information about medication to take an active role in their drug management. comission for medicines and medical devices, chu de toulouse, toulouse, france please specify your abstract type: descriptive abstract (for projects) background and objective: due to its common use, insulin is often considered as a harmless medication by lots of health professionals while an overdose can lead to dramatic consequences and death. between january and june , in our university hospital, % ( out of ) of the declared adverse drug events have involved insulin: were caused by prescription errors and by administration errors. all were discovered after the medicines have been administered but thankfully none had serious consequences. the british national health service (nhs) and the french medication safety national agency (ansm) made a list of ''never events'': avoidable events which should never happen and misadministration of insulin is among them. the objective was to increase patient safety in the hospital by setting different actions to promote and improve the appropriate use of insulin and warn health professionals about the real dangers of this medicine. design: different actors participated in the implementation of these actions: the commission for medicines and medical devices (which is composed by doctors and pharmacists) directed a group made by physicians and clinical pharmacists from the department of cardiological and metabolic diseases'. results: in addition of the usual analysis of any adverse event linked to medication declared in the hospital, several actions were set up: • a didactic document summarizing all the ''sensitive'' steps during the prescription, stocking, dispensation and administration of insulin has made the front page of the hospital's intranet and was also diffused throughout the establishment. • a chart resuming all the different insulins commercialized in france has also been diffused. it contains their types, durations and onsets of action, conditions of storage and pictures of their packaging. • the computerized protocols involving insulin are going to be reviewed in order to lower their numbers and harmonize their content. • a revision of the list of insulins available at the hospital is in progress to reduce their number and avoid any confusion between the different products. • an evaluation of insulin's computerized prescription practices will be made via a data request. : this topic about insulin shows a greater willingness to secure the medication circuit in the hospital. other action plans such as this one will be set up involving other medications among the never-events list. meanwhile, the commission for medicines and medical devices pursues its actions of promoting the appropriate use of medication. please specify your abstract type: descriptive abstract (for projects) background and objective: one of the hospital pharmacist tasks is to suggest substitutions to ensure conformity of medical prescription with the hospital formulary. indeed, when an eye drop isn't available at the hospital, there is a specific supply circuit which has to remain exceptional: it's ordered directly to the pharmaceutical wholesaler. in this context, ophthalmologists and clinical pharmacists created a table proposing therapeutic equivalencies with eye drops available at the hospital. after approval by the commission for medicines and medical devices, this tool has been diffused within the establishment via the intranet website since the beginning of to the medical and paramedical staff. the purpose of the study is to evaluate professional practices concerning the use of the eye drops' equivalence chart. design: in the study, we compared eye drops' orders made to the pharmaceutical wholesaler before and after the table's diffusion, thus between january and december . for each order, we used the table available at this time to determine if equivalencies could have been proposed or not. if so, we identified the hospital ward and the pharmaceutical specialty. market changes have also been considered. results: we noticed a decreased frequency of eye-drops ordered despite available equivalencies: % in (before the table's diffusion), % in (after its diffusion), % in and % in . prisons units are responsible of % of these orders: they have the lowest rates of substitutions. their most ordered pharmaceutical specialties are ophthalmic glaucoma agents: % ganfort Ò (bimatoprost . mg/ml/timolol . %), % xalacom Ò (latanoprost + timolol . %), % azopt Ò (brinzolamide) for which the authorized substitutions are for the first two specialties: monoprost Ò (latanoprost) + ophtim Ò (timolol . %) and for the third: dorzolamide Ò . conclusion: equivalence table diffusion throughout the hospital has facilitated and improved the prescription and substitution of eyedrops. orders of pharmaceutical specialties despite authorized equivalencies available have declined by half. probably for practical reasons regarding long-term treatments, prison units make less substitutions but an awareness campaign will be carried out to reduce these rates. please specify your abstract type: research abstract background and objective: the patient's education and information is a mean to reduce medicine misuse and it can be performed with support of a leaflet or informative material about medicines. in brazil, there is a lack in regulation about this type of informative material to compounded medicines. the aim of this study was to evaluate the quality and effectiveness of leaflets developed to compounded medicines' users through knowledge's level and medicine treatment adherence. setting and method: analytical and quantitative study; month prospective study through interviews, at time zero (t ) and after days (t ) in a university pharmacy in goiás, brasil; fisher's exact test to measure effectiveness; ethics committee number / . main outcome measures: categorization into high adherence and low adherence by morisky test; categorization into sufficient, regular or insufficient knowledge about medicine prescription; perceptions and suggestions about delivered leaflet in medicine dispensing process. results: of patients ( . % female, mean = . years), . % considered as relevant the leaflet's content, as well . % of them kept it and . % of them read it. suggestions of . % included a desire in increase font size, more emphasis on drug interactions and images. there was a predominance of regular knowledge in both analysed times ( . % e . %), however there was a decrease in high adherence to medicine treatment ( . - . %). among patients who read the leaflet, no statistically significant association was found on these two variables at t and t (p = . and p = . , respectively). knowledge about ''administration schedules'' showed a significant improvement after intervention (p = . ). . % of patients considered that there was no need to obtain more information. conclusion: this study demonstrates the evaluated leaflets had relevance to patients and demonstrate clinical relevance. however was not observed statistically significance. this highlights the need of using different ways to measure the effectiveness of an informative material to promote rational use of medicines and depth studies and stimulation of greater attention from the health professionals to the topic. di : chlormethine gel: effectiveness and tolerance to treat mycosis fungoides françois dugre *, , anne lefebure , sonia martelli , marion pin , eve maubec , philippe arnaud pharmacy, dermatology, bichat-claude bernard hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: to determine the effectiveness and tolerance of chlormethine gel in treating mycosis fungoides. design: mycosis fungoides is the most common form of cutaneous t-cell lymphoma (mf-ctcl). early stages (ia and ib) can be controlled by skin-directed therapies such as chlormethine and carmustine. these drugs which are solutions for injection are usually used for skin application. chlormethine or mechlorethamine gel is an alkylating agent representing an alternative for previously treated patients diagnosed with mf-ctcl, in case of therapeutic failure and intolerance, or in case of chlormethine and carmustine solutions supply disruption. a retrospective observational analysis was conducted based on medical records of all patients treated by chlormethine gel in our hospital from the first of july to the first of september . the following data were collected with an excel table: body surface area or bsa affected by disease, location of the lesions, therapeutic management, effectiveness and treatment tolerance. results: fourteen patients ( women, men, mean age [min ; max ]) were treated with chlormethine gel in our hospital. twelve ( %) were treated three times per week, ( %) once a day. before treatment by chlormethine gel, ( %) patients were treated by dermocorticoids, ( %) by dermocorticoids and phototherapy, and ( %) by bexarotene, all of them stopped their treatment on account of inefficacity. one ( %) patient was treated by carmustine and dermocorticoid, and ( %) by only carmustin, all of them stopped it because of supply disruption. one ( %) patient received it in first line therapy. ten ( %) patients showed a response (partial or complete), one ( %) experienced a stabilization of his disease. before treatment with topical chlormethine, seven patients ( %) had an involved bsa [ % and four of them ( . %) experienced adverse effects. seven patients ( %) had an involved bsa \ % and three of them had ( . %) side effects. a total of seven patients ( %) presented at least one adverse effect. five patients ( %) stopped the treatment on account of adverse effects; two of them ( %) interrupted it temporarily. reported side effects were: irritant dermatitis and erosive toxicity ( ), rash ( ) and telangiectasia ( ) . conclusion: our results indicate that chlormethine gel can be effective to treat mycosis fungoides. however, it involves side effects that seems to be more frequent than those observed with chlormethine solution (used for skin application). indeed, the french national authority for health reports % of adverse effects for chlormethine solution versus % in our study for chlormethine gel. moreover, telangiectasia was never documented with chlormethine. this significant number of side effects of chlormethine gel can be explained by the gel formulation which induces patients to apply more product, especially in patients with plaques affecting more than % of the bsa. it is important to explain to patients to apply a thin film of chlormethine gel to involved skin areas and allow the skin to dry completely. sophie dumas *, , capucine devaux , nathalie le guyader diaconesses croix saint-simon hospital, diaconesses croix saint-simon hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: aprepitant, a neurokinin- receptor antagonist, prevents nausea and vomiting due to high and moderate emetogenic chemotherapy in combination with other antiemetic agents. it induces cytochrome p (cyp) c and moderately inhibits cyp a . drug-drug interaction could occur with intravenous anticancer or antiemetic drugs metabolised by these isoenzymes. it may lead to adverse effects or loss in efficacy. regarding recent international antiemetic guidelines, emergence of new intravenous chemotherapy and lack of bibliographic data, a report on aprepitant interactions is performed in oncology. the aim of this study is to review pharmacokinetic interactions with aprepitant in order to prevent potential toxic effects of intravenous anticancer or antiemetic agents and provide the best patient care. design: anticancer and antiemetic agents metabolised by cyp a and c were identified. pharmacokinetic literature review was performed using medline Ò database and laboratory data. clinical assessment and non-aprepitant pharmacokinetic studies were excluded. a table was established to summarize data. results: ten intravenous anticancer agents used in oncology are identified as cyp a substrates. pharmacokinetic assessments are achieved for docetaxel, cyclophosphamide, vinorelbine, irinotecan and trabectedin. studies dealing with the five other drugs are strictly clinical assessments. among the different pharmacokinetic studies, only trabectedin showed relevant interaction with aprepitant. in this association, aprepitant dose needs to be adjusted. cyp c catalyses the cyclophosphamide activation pathway with minor contribution. however, it would have few repercussions on cyclophosphamide pharmacokinetic. corticosteroids and hydroxytryptamine type ( ht- ) receptor antagonists are also metabolised by cyp a . aprepitant significantly increases corticosteroid plasma concentrations. in this case, corticosteroid dose adjustment should be applied. furthermore, no interaction has been found with ht- receptor antagonist. conclusion: regardless of the emetogenic level of anticancer agents, all drugs have been studied because of theirs potential combinations. two relevant pharmacokinetic interactions have been demonstrated leading to dose adjustment recommendation. corticosteroids doses, in association with aprepitant, should be reduced one-fourth for intravenous form and one half for oral form. aprepitant first dose should be decreased to mg when it is co-administrated with trabectedin. these two results lead us to re-evaluate our prescription practices. please specify your abstract type: research abstract background and objective: nsaids are associated with serious adverse reactions which in turn are responsible for significant risks of morbidity and mortality. the aims of this project is to identify risks involved in nsaid administration including over-usage and significant drug interactions, and to analyse occurrence of side-effects. the trends of nsaid prescribing by physicians and pharmacists are also determined. setting and method: a pharmacy from each electoral district was chosen by stratified sampling. a sample population (n = ) was obtained from pharmacies in malta. data was collected through the completion of questionnaires carried out by the patients. the trends of nsaid prescribing were determined by another questionnaire directed to pharmacists and physicians that was available online. main outcome measures: use of nsaids by patients and prescribing trends. results: back pain (n = ), muscular pain (n = ), headache (n = ) and arthritic pain (n = ) accounted for the most frequent use of nsaids. diclofenac accounted for the most commonly administered nsaid, taken by of the patients, of which use the mg dose. chronic disorders of symptoms experienced by the patients included hypertension (n = ), heartburn (n = ), dyspepsia (n = ), asthma (n = ) and a history of helicobacter pylori infection (n = ). other disorders suffered by single individuals include epilepsy, crohn's disease and renal dysfunction. more than half of the respondents (n = ) admitted to self-prescribing regardless the fact that the majority of nsaids are prescription-only medications. epigastric pain ( . %), stomach ulcers or gi bleeding ( . %) and elevated blood pressure ( . %) were the most common sideeffects that pharmacists and physicians come across. nsaids were frequently found to be co-administered with antihypertensives ( . %) and ssris ( %) regardless of their significant risks of interacting with nsaids. . % of the pharmacists and doctors believe that nsaids are being over-used and . % state that closer monitoring of nsaid adverse effects is necessary. conclusion: the risk involved with nsaid administration due to over-usage and drug interactions is identified, and healthcare professionals are aware of this risk. pierre leduc, antoine lanneluc, christophe gellis * , sylvie poux, dominique plats, regine larnaudie corrèze, ch brive la gaillarde, brive la gaillarde, france please specify your abstract type: research abstract background and objective: proton pump inhibitors (ppi) are widely prescribed in hospital while their long-term use may be responsible of many potentially serious long-term side effects (hypomagnesemia, neutropenia, gastric cancer) and drug interactions (ppi are inhibitors of cyp c ). the objective of this study was to assess the appropriateness of ppi prescriptions in a geriatric department in order to optimize their conditions of prescriptions. setting and method: this prospective study involved patients hospitalized between january and april in a geriatric department. the accordance of the prescriptions with the marketing authorization indications and the french guidelines was analysed. data collection was done using a table excel. main outcome measures: collected information were related to patients (age, sex) and ppi prescriptions (active substance, administration route, dosage, duration of therapy, therapy indication and reassessment of ppi therapy). results: ninety-one patients were included: sr: . , mean age: . years [ ; ]. ppi therapy prevalence over the period was %. the ppi were prescribed in the geriatric department in patients (mostly esomeprazole) whereas patients had ppi therapy (mostly esomeprazole) at the admission, for more than years in patients. oral route was the most frequent one (n = ). ppi were administered once a day and only three ppi were administered in the morning. % of ppi prescriptions were considered unjustified; the indications were prevention of haemorrhage with antiplatelet therapy (n = ), prevention of haemorrhage with corticoid (n = ), prevention of haemorrhage with anti-vitamin k (n = ), dyspeptic disorders (n = ), gastralgy (n = ) and others reasons (n = ). % of ppi prescriptions were considered relevant. the reassessment of ipp therapy (n = ) lead to prescribe another dosage (n = ), to stop therapy (n = ) or no change (n = ). conclusion: the study showed that the majority of ipp prescriptions were not in accordance with french guidelines. limiting the prescription to the indications, reassessing the therapy or respecting the therapy duration should reduce the risk of long term side effects and the economic burden of ppi in a long term use. please specify your abstract type: descriptive abstract (for projects) background and objective: to evaluate the effectiveness and safety of the use of high dose of tigecycline ( mg followed by mg every h) a tertiary care hospital. design: retrospective observational study. period: january to december . inclusion criteria: episodes use of tigecycline ( mg followed by mg every h. exclusion criteria: time less than days treatment. data source: corporate program stories electronic health. results: we identified episodes in patients ( men, mean age: years ( - )). treatment was directed to multidrug-resistant organism infection in cases (seven klebsiella pneumoniae oxa- , two enterobacter cloacae, two enterococcus faecium and one methicillin resistant staphylococcus aureus. in one episode they coincided e. cloacae and e. faecium). in cases had severe sepsis or septic shock (seven abdominal focus, six respiratory focus and one unknown focus). the median number of days of treatment was ( - ). tigecycline was administered as monotherapy in three cases, bitherapy and triple combination therapy in . the antibiotics were associated were: beta-lactam ( ), aminoglycosides ( ), quinolones ( ) colistin (three, two inhaled cases), cotrimoxazole ( ) and vancomycin ( ) . in episodes produced clinical and/or microbiological resolution and antibiotics are rotated by progression picture or lack of improvement, death occurred in three cases and was suspended on suspicion of hepatotoxicity. among the seven episodes of klebsiella pneumoniae oxa- infection there were four pneumonias, three with favourable evolution and one patient died, two bacteraemia, both with resolution clinical and microbiological, and one urinary tract infection resolved. among the episodes in severe/ septic shock were five cures, six cases of antibiotic rotation progression or lack of improvement and three deaths while patients receiving therapy tigecycline. patients showed an adverse effect possibly related to therapy tigecycline: diarrhoea after days of treatment and case of liver toxicity after days of tigecycline and piperacillin-tazobactam which led to their withdrawal. int j clin pharm ( ) : - conclusion: tigecycline has been used in double dose defined in data sheet especially in situations of severe sepsis or septic shock and infection multiresistant microorganisms. the effectiveness is conditioned by the clinical situation patient, being worse in severe/septic shock sepsis. tigecycline high dose was well tolerated and there was only a case of stopping the medication for suspected damage hepatic. di : wikipedia and medicines: who edits medicine articles on the english wikipedia? kristian husvik skancke , kristian svendsen *, department of history, uit -the arctic university of norway, hospital pharmacy of tromsø, tromsø, norway please specify your abstract type: research abstract background and objective: the medical profession and pharmacists are divided on the usability of wikipedia for looking up health information. nevertheless wikipedia is widely used, more than half of us physicians and percent of all medical students use wikipedia as a source of health-related information. there is a potential for incorrect and biased information being added by the pharmaceutical industry. the aim of this project was to examine who edits wikipedia articles on medicines and to investigate whether the pharmaceutical industry edits these articles. setting and method: two different groups of articles has been examined; the top ten bestselling medicines (substances) in the world in and the ten most recently approved medicines on the european market (until december ). the top ten medicines were selected from a consultancy report by evaluatepharma/ep vantage. the ten most recently approved medicines (new substances) were found on the european medicines agency webpage. we queried the english wikipedia on january and information from the edit history and the editors' user information were extracted. unregistered editors were checked using a whois service. for the new medicines all editors were checked, while for the bestselling medicines large edits and initial edits was checked. main outcome measures: edits suspected of being made by the pharmaceutical industry. results: ten bestselling medicines: there are many users editing these articles and/or watching them, limiting the risk of misinformation from the industry. there was no indication that the pharmaceutical industry had edited any of the articles. ten most recent medicines: no article existed for dasabuvir. for the nine other substances there were relatively few editors and watchers. in four out of the nine articles we found evidence of edits from the pharmaceutical industry. these edits, were done by registered editors with very few edits except for the medicine in question and they had made large additions to the articles sometimes even before the medicine was marketed. conclusion: the pharmaceutical industry seems to edit articles about medicines on english wikipedia however we found no evidence of harmful edits and bestselling medicines have many editors monitoring the quality of articles. please specify your abstract type: research abstract background and objective: the pharmaceutical professional service of the monitored dosage systems (mds) tries to improve the adherence of the patients to the treatment. the aim was to analyse the relevance of the repackaging of the most sold medicines in our country being used by patients included in the mds professional service and to determine the information discrepancies according to the source used by the pharmacist. setting and method: cross-sectional descriptive study. community pharmacy and healthcare institutions. all the patients included in the pharmaceutical professional service of mds on june , . data source: patients' records in the professional service, database of medicines ranked by sales in units in our country to december ( medicines), information sources on medicines: ( ) vademecum of medicines and ( ) the centre of drug information of our agency of medicines. main outcome measures: number of institutionalized and ambulatory patients included in the professional service of the mds and demographic characteristics, sum of different repackaged medicines belonging to the studied patients, analysis of the repackaged medicines of major use, number of discrepancies on the repackaging of the medicines according to the information source. results: patients were included in the professional service of the mds. of them were institutionalized (average age: . years, . % men, . % polymedicated defined as using c prescribed chronic medicines) and the remaining were ambulatory (average age: . years, . % women, . % polymedicated). different medicines prescribed in the institutionalized patients were taken into account, of them included in the sales ranking in our country. according to the first source, of medicines were eligible for repackaging, medicines could be repackaged according to the laboratory manufacturer and the remaining ones could not be repackaged. according to the second source, of medicines could be repackaged, and the remaining ones could not. different medicines prescribed in the ambulatory patients were taken into account, of them included in the sales ranking in our country. according to the first source, of medicines could be repackaged, medicines would depend on the laboratory manufacturer and the remaining ones could not be repackaged. according to the second source, of medicines could be repackaged, and the remaining ones must remain in the original package. discrepancies were observed in the information for ( . %) and ( . %) medicines in institutionalized and ambulatory patients, respectively, based on the sources used. conclusion: a considerable number of discrepancies in the information on the relevance of the repackaging of medications in the mds were found between two analysed sources. these findings have already improved the quality of this professional service. it would be necessary to alert the pharmacist of the existence of the above mentioned discrepancies to be able to prevent errors from occurring at the time of repackaging the medicines in the mds and, thus, increasing patient safety. please specify your abstract type: research abstract background and objective: despite the global advances of pharmacy practice and subsequently pharmacy education, students experience insufficient opportunities to practice the activities, tasks and processes essential to deliver pharmaceutical care. objective: to describe the development, implementation, and assessment of a clinical pharmacy practice (cpp) experience course in internal medicine, cardiovascular, respiratory clinics and drug information centre that is newly integrated into pharmacy curriculum at a university in north cyprus. setting and method: a weeks structured pharmacy practice experience was designed for fifth year students. student competence was assessed using formative osces and summative written exams before and after the course, and mapped in eight main cpp competences. the course utilized a wide variety of learning and practical activities including rounds participation, morning case reports, interdisciplinary activities, carrying interventions, role-play, direct patient care, formal case presentations, journal clubs and answering drug queries. competencies tested and strengthened include: taking medication history, response to the symptoms, pharmacotherapy knowledge application, comprehensive patient assessment, data interpretation using evidence-based approach, public health counselling, drug related problems management, patient counselling and communication skills. student perceptions and experience was assessed using semi-structured group interview and a questionnaire. main outcome measures: student scores in osce; student's perceptions. results: student reported that the course met pre-set objectives with substantial learning in different areas of cpp. students scored best in communication skills ( . ± . %), public health promotion ( . ± . %) and patient counselling ( . ± . %) than in resolution of drps ( . ± . %) and pharmacotherapy application ( . ± . %), while they significantly enhanced in di manipulation ( . ± . %) compared to baseline assessment ( . ± . %)(p = . ). conclusion: the course provided a rich experiential learning environment rather than just theoretical knowledge of clinical pharmacy. students well perceived the course structure assessment and knowledge attained. this could be implemented in other faculties of pharmacy through turkey. please specify your abstract type: descriptive abstract (for projects) background and objective: clinical pharmacy and clinical pharmacology have many similar aspects. both areas present professionals who have groundings in drug therapy principles and who aim to optimize the efficacy and safety of therapies for patient's benefits. however, there are clear distinctions. clinical pharmacologists are in general doctors with an additional education in clinical pharmacology. many of these are prescribers of drugs in practice but are in usual connected to academic parts responsible for education and research. they belong to a well-recognized but small sub-specialty of medicine. in contrast, clinical pharmacists are part of a much greater group of professionals working in most hospitals in developed countries. while the former one is restricted and subordinate to distributing the drugs requested by the medical prescribers, the role of the pharmacist has increasingly developed to encircle monitoring outcomes of medicine treatment and report management, patient safety and budgetary responsibilities. pharmacists are currently capable to take on prescribing responsibilities in developed countries and have been actively involved in collaboration in practice of prescribing with doctors. they also take on a great part in education related to rational prescribing that was once thought the area of the clinical pharmacologist. given the difference in size of the two areas there is understandably increasing confusion in the minds of managers in health services as to the continuing role and identity of clinical pharmacology. this may illuminate, in part, the diminishing in numbers and visibility of clinical pharmacologists in certain countries. in fact, some might see the continuous development of clinical pharmacy as a direct danger to the viability and future existence of the specialty of clinical pharmacology. however, clinical pharmacy and clinical pharmacology working synergistically would serve for the well-being of the public. design: . results: . conclusion: . maxime apparuit *, , lea boissinot , ngauv melodie , stephanie charles weber , isabelle lopez , françois chast pharmacy, hopital cochin, pharmacy, hopital hotel-dieu, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: hereditary angioedema (hae) is a rare disease characterized by episodic attacks of swelling which can be life-threatening. treatment for hae involves prophylaxis and management of acute attacks. the objective of this study was to evaluate patients' knowledge of their disease and their treatment. design: a questionnaire about the disease and drug treatment has been implemented. it was distributed to patients through either a pharmacist during patients stay at the hospital, or the french association des malades souffrant d'angioedèmes (amsao). answers were collected by electronic or conventional mail. results: patients completed the questionnaire. the average patients age is . ± . years. all of those interviewed could name their disease. for % of patients, the crisis happened unpredictably but in most cases a triggering factor was described, such as stress ( %), fatigue ( %) or an emotional shock ( %). oedema were located mainly in extremities ( %), abdomen ( %), ent sphere ( %) or face ( %). patients ( %) reported having more than crisis each year (eligible to prophylaxis), among them, patients ( %) said they had no preventive treatment. all patients knew the difference between prophylactic and curative treatment of crisis. among the patients receiving treatment for crisis, were able to define which treatment to be used depending on the intensity and location of the crisis. the majority of patients used icatibant during a crisis, but the most frequently cited prophylaxis treatments were tranexamic acid ( %) and danazol ( %). for injectable drugs to treat acute episodes, icatibant (subcutaneous) and c esterase inhibitor (intravenous) were self-administrated respectively in and % of patients. conclusion: this study showed that patients generally knew their disease and its treatment. however, they are insufficiently informed on drugs to be used according to the clinical situation and especially intravenous self-administration. therefore, it seems necessary to increase pharmacist involvement in patient's information about therapeutic strategy and drugs routes of administration. this for a major objective: an optimal self-care in a skilled patient. please specify your abstract type: descriptive abstract (for projects) background and objective: hospital pharmaceutical educations (hpe) on patients with oral anticoagulant (oa) can improve their overall management by providing skills on proper use. an ambulatory monitoring is necessary to ensure good compliance and understanding of the treatment. our study aimed at the establishment of hpe for patients with oa, the establishment of a hospital-city link in burgundy, and an evaluation of the expectations of ambulatory health professionals (ahp). design: the development of hpe has been performed in our centre for patients with oa and assessed between may and september . in order to ensure continuity in their support, patients then received a binding document to the attending physician, pharmacist and nursing home stating the treatment and acquired skills. a satisfaction survey, with anonymous electronic questionnaire circulated by the representative boards evaluating the expectations of ahp, took place in order to improve and make the programme more attractive. results: two hundred and ninety-one patients could benefit from hpe and came out with an oa. one hundred and forty-three answers were collected: officinal pharmacists and nurses. ninety-seven percent of ahp have judged relevant the following stated security goals: the name of the drug, its use, its risks and to be able to inform all ahp. ''associated pathologies and treatments,'' ''the last coagulation test'' and ''potential factors for non-adherence'' seem necessary for the binding document. more than % of participants found that this action will facilitate the establishment of pharmaceutical anticoagulant educations in cities, the dialogue around the oa with the doctor, patient's compliance and will secure the treatment. conclusion: hpe certainly help patients. its implementation for patients with oa in our hospital has generated a real interest. the addition of an ambulatory link allows continuing at best their support. the questionnaire has also allowed us to know the opinions of ahp involved and some improvements to the binding document may have been done. participants were asked to associate the task to the profession by determining whether each profession had the main responsibility for undertaking the task, a supportive responsibility, or whether they should not be involved at all. data was analysed using spss Ò, version . the chi squared test was used to assess any significant association between categorical variables. main outcome measures: perception of the oncology pharmacist's role by healthcare professionals. results: from a total of completed questionnaires, it was found that for tasks listed as ''patient education and counselling'', % were considered as the pharmacists' main responsibility, whereas % were believed to be supportive roles. main tasks included educating the patient regarding which medication to avoid during their treatment. for tasks listed as ''drug related problems'', and % of tasks were found to include pharmacists as having main and supportive roles respectively. supportive tasks included dose calculation of anti-tumour therapy required per patient. in the ''authorisation of medication'' category pharmacists' main roles carried a total of % and supportive that of % of the total number of tasks. this included ordering anti-tumour medication. further analysis of data revealed that years of experience did not have a significant association with results obtained (p-value = . ); however physicians, pharmacists and allied healthcare professionals were found to involve the pharmacist most extensively (pvalue = . ). conclusion: tasks associated with the pharmacist were representative of the current role they possess within the oncology setting; however this association was limited to professionals having a close working relationship with pharmacists. this may be due to the lack of an established multidisciplinary team approach within this scenario thus limiting the perception of the oncology pharmacist's contribution. an implemented multidisciplinary team may improve communication between the professionals involved and optimises patient care. the aim of the study is to analyse from a qualitative and quantitative point of view the pharmacy resident's activity in pneumology service. setting and method: the study included all the daily prescriptions of three units of pneumology from january to april . pi and data were extracted from the software pharma Ò and collected in a summary excel Ò table: nature of potential errors, nature of the proposals offered by residents, way of transmitting pi, and rate of pis' acceptance. main outcome measures: potential errors are collected by following the validated and standardized criterions of french society of clinical pharmacy. results: over months, lines of prescriptions from patients aged years old (median [ - ]) were evaluated. sex ratio (m/f) was . . one hundred and two medication problems have been found: overdose ( . %), contraindication (ic) ( . %), under dosage ( . %), wrong rhythm of administration ( . %), forgotten treatment ( . %), dose unit error ( . %), antibiotic indication missing ( . %), drug not listed in the hospital formulary ( . %), potassemie unchecked ( . %), dose unadapted to renal function ( . %) or to inr ( . %), treatment not indicated ( . %), wrong administration route ( . %), antibiotic unreevaluated ( . %), redundancy ( . %). the proposals made to the doctors were: stopping treatment ( . %), posology adaptation ( . %), substitution ( . %), dose unit modification ( . %), adding information about the indication ( . %), treatment renewal ( . %), administration modalities changing ( . %), biological monitoring ( . %), therapeutical monitoring ( . %), antibiotic treatment reevaluation ( . %). all pi were made by informatical way. all medicinal classes were found in this study. hydroxyzine, cyamemazine and escitalopram were often found in contraindication errors. they are involved in cardiac disorders with qt extension. pis' acceptance rate was %. conclusion: this study shows the importance of pharmaceutical analysis on the quality of access to healthcare. the statement of pi allows us to identify the most frequent errors, warn and prevent doctors from these potentials errors by proposing solutions. the rate of acceptance is high which means that doctors agree with our proposals. pharmacists' implication in clinical pharmacy activities and their participation to medical rounds will improve this activity and by the way optimization of the management of the patient. please specify your abstract type: descriptive abstract (for projects) background and objective: ppis consumption is largely practiced in europe, because of their excellent tolerance in short time, and their misuse with regard to indications, dosage and treatment duration (in , france was the nd,ppi consumer in eu). the result is drug iatrogenic disease and unjustified expenses in health insurance. objectives: assess the ppis consumption and appreciate conformity according to the latest recommendations for relevant prescriptions of ppis. design: prospective study via an audit (model created internally), every hospitalized patients with a ppis prescription, in two hospitals, on a given day. data collected through the patient's medical record. prescriptions conformity defined, by taking account of indication level ( : approved by the ma (marketing authorization), : non-valid but certified by international publications or learned society, : nonvalid without scientific proofs, : non-indicated), dosage and treatment duration. analysed situations with no conformity (inappropriate dosage despite conform indication, treatment duration unjustified and ppis prescribed in wrong indications ( and ) . results: patients have ppis prescriptions ( male, £ years] among patients ( %). % ppis prescriptions began during the hospitalization. ( %) of the ppis prescriptions are in accordance with the experiment (indication + dosage +treatment duration), as well in community than in hospitals. details: indication level ( . %), indication level -gi bleedings-( . %). of the ppis prescription aren't in accordance. details: treatment duration ( %), dosage ( %), indication level -prevention of iatrogenic bleeding risk without nsaids prescription-( %), indication level ( %). regarding level indications, ppis are always taken with anticoagulant and/or platelet aggregation inhibitors and/or corticoid. conclusion: the part of ppis prescriptions in this study is high. the majority of non-conformity is caused by ppis prescribed with an indication level . the improvement program will involve feeding back ppis' good use, to educate physicians (junior and senior) about the relevant ppis prescription and give advice in complex situations (indication and ). in collaboration with prescribers, shutdown protocol of ppis, prescribed in long term, could be implemented in order to avoid the acid rebound effect after brutal treatment discontinuation. hp-ce : impact of a self-management program on inflammatory bowel disease patient in a university hospital caroline egon * , xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: inflammatory bowel disease (ibd) is a group of chronic inflammatory diseases that affects the colon and the small intestine. crohn's disease and ulcerative colitis are the principal types of ibd and involve severe diarrhoea, pain, fatigue and weight loss. ibd affects young adult with an increasing annual incidence ( . million concerned people in europe). patients with ibd are affected by somatic or psychosocial problems and patient education may contribute to their well-being. since september , individual educational sessions have been set up and since september , collective educational sessions. these sessions have been developed to improve patient's understanding of treatment options and medical adherence. the aim of this study was to demonstrate that a therapeutic education program (tep) could have a significant effect on ibd patient's skills with regards to their disease. design: after individual education sessions with a nurse, a group education session was introduced for outpatients with ibd. the collective session include approximately six to ten patients and is organized in a half day workshops (about disease and treatment) conducted by a multidisciplinary team. the workshops were performed by an education nurse, two hospital gastroenterologists, two hospital pharmacists and a community pharmacist. these sessions were wrapped up by a short satisfaction and knowledge questionnaires. results: in total, ibd outpatients participated to the educational program, patients with crohn's disease and patients with ulcerative colitis ( . % male; median age: ). for the individual educational sessions, two competence questionnaires were performed about anti-tumour necrosis factor alpha (tnfa) therapy: one about general knowledge, another one about self-administration subcutaneous injection. patients completed these questionnaires. for the collective educational session, the competence questionnaire developed consisting of six questions covering few items: disease, symptoms, treatment and complications. patients completed this questionnaire. after the questionnaire, each participant received a summary document about drugs, side effects, therapeutic and medical advice. conclusion: the patient education program contributes to the improvement of self-management skills when it comes to ibd. pharmacists joining medical specialists and nurses provided pharmaceutical care with a positive impact on compliance, which is a determining factor for the success of the treatment and the quality of life in patients living with an ibd. this program will be continued and a new program for teenagers is to be established as well. hp-ce : desensitization study of paclitaxel and carboplatin drug in the ovary tumor protocol in cuf descobertas hospital miguel  . freitas * , daniela brites, ana bota pharmacy, hospital cuf descobertas, lisbon, portugal please specify your abstract type: descriptive abstract (for projects) background and objective: the hospital pharmacy should be an integral part of the multidisciplinary team and implement strategies that meet the patient's needs. pharmacy, in oncological area, is in constant renewal. josé de mello saúde uses paclitaxel/carboplatin protocol as first line in ovarian tumor. although the antineoplastic agents are essential for the treatment of cancer, they can also cause hypersensitivity reactions, which may carry serious consequences. both immunoallergologist and oncologist create a desensitization protocol, which allows the reintroduction of the drug with greater security. the desensitization protocols involves the gradual administration of small quantities of the drug, resulting in a refractory period of the white blood cells (mastocytes) and a lower production of cytokines until the dose has been totally administrated. objective:to evaluate the efficacy of methods used to prevent and treat hypersensitivity reactions of carboplatin and paclitaxel, in order to carry on the treatment. design: a retrospective review of the patient files was performed in the day hospital between and . we included only patients with moderate to severe immediate hypersensitivity reactions (b h) receiving carboplatin and paclitaxel. the desensitization protocol brigham and women's hospital was applied using three solutions with increasing concentrations (dilution : , : and : ) in twelve successive steps for about h. results: in the period - were desensitized five patients with platinum group drugs, carboplatin (n = ) and paclitaxel (n = ) and the total elapsed six desensitization. almost all patients reached the scheduled daily dose, except a patient, which suspended the desensitization program for disease progression. conclusion: the desensitization protocol allowed the successful reintroduction of antineoplastic drugs in patients with a history of hypersensitivity reactions, in order to treat the disease. please specify your abstract type: research abstract background and objective: in the context of harmonization of clinical pharmacy activities within our region, a common medication reconciliation project was developed between two general hospitals. the objectives of this study were to initiate, a common medication reconciliation activity in the two hospitals, to analyse the results, and to communicate to all professionals in the area. setting and method: a working group composed of pharmacists of each hospital was formed to develop analytical documents. a -month prospective study was conducted in two general hospitals: in the first one, in an emergency department, and the other one, in a medicine department. patients included in the study were either elderly and/or had polypharmacy and/or were hospitalized for iatrogenic reason. at int j clin pharm ( ) : - the point of admission and discharge for each patient, the pharmacist has completed a conciliation record, and has detected potential discrepancy. unintentional discrepancies were reviewed and corrected by doctors. at the discharge, medication changes were sent to general practitioner and community pharmacies. a satisfaction survey about this process was sent to healthcare professionals (gp, pharmacist and nurses). a medication reconciliation's workshop was organized for a hundred healthcare professionals in the area. main outcome measures: at the point of admission, the conciliation record included the list of patient's home medication, admission medical orders, and the types of discrepancies. at the discharge, drugs prescribed were compared to admission medical orders. the satisfaction survey included seven questions to assess the process. results: during the study period, patients were included corresponding to prescription lines. reconciliation process required about min per patient. we identified at admission unintentional discrepancies. the most common unintentional discrepancy was the omission of medication ( %). % concerned alimentary tract and metabolism group. at the discharge, no discrepancies were found; the process required min per patient. % of healthcare professionals answered to our satisfaction survey to date. % are satisfied and believe that the process of medication reconciliation secures the patient medicinal treatment. healthcare professionals were present at the medication reconciliation's workshop, indicating an interest in the process. conclusion: in this experience of medication reconciliation, due to unintentional discrepancies observed, we had better implement this activity in the two general hospitals. a pharmacist devoted to this activity will be hire in each hospital. this relevant practice is well accepted by clinician. thus, we will improve communication with gp and community pharmacies. please specify your abstract type: descriptive abstract (for projects) background and objective: the sickle cell disease (scd) is a genetic, chronic disease, paroxystic in its unpredictable and polymorphic acute events. this most frequent genetic illness in the world is a major public health concern in french overseas territories. haute autorité de santé (has) recommendations for the care of scd advocate the development of therapeutic patient education (tpe). in martinique (french west indies), we consider the population of patients with scd in among which are followed in the adults sickle cell centre (ascc). one of the actions carried out by the ascc of our hospital is the tpe. the objective is to set up an original (because specific in the scd) tpe method, which enables the patient to live better with his disease on a daily basis, by teaching him and his family to recognize prematurely certain complications. design: we analysed needs from the outcomes of a national french survey has which one participated martinique and retained the following themes: the red blood cell, the genetic transmission, the main symptoms, the role of the water, the medicinal treatments and the questions of everyday life. we chose the innovative educational tools called the ''malles des savoirs Ò **'', a set of unusual experiments, accessories and models, which, by using a method of active pedagogy ''omca*: observer, manipuler, comprendre, agir ***'', value the learner by offering to him to manipulate and to experiment by himself. results: in , healthcare professionals (doctors, pharmacists, nurses) and a president of patients with scd association followed one week of formation in the omca* method for the animation of six workshops for - teenagers and adults. every ''malle des savoirs Ò **'' contains the necessary material for the animation and a guide of the organizer, including, for every tackled issue, a generic introduction, a presentation of the themes, the index cards of educational animations proposing the activities and one time of synthesis grouping the approaches concepts. the interactive manipulation allows the appropriation of the discoveries become then long-lasting experiences. a final evaluation allows to spot the problems met by learners to understand, to analyse the difficulties and to proceed to the useful adaptations during the next activity. conclusion: this tool, playful and perfectly adapted to the scd, engages, accompanies and helps patients in the construction of their own knowledges to return them actors of their disease. in , we shall estimate the impact of the development of this specific tpe programme of the patient with scd. please specify your abstract type: research abstract background and objective: to investigate the frequencies and clinical relevance of unintentional medication discrepancies, between preadmission medication lists and discharge medication lists, at discharge from hospital. a discrepancy is considered unintentional if there is no documentation explaining the intent of the medication change or if it is unintentional according to the prescribing physician. setting and method: systematic literature review. main outcome measures: frequency of unintentional medication discrepancies per patient and per medication; frequency of clinically relevant medication discrepancies. results: of the patients included - % experienced at least one unintentional medication discrepancy. of the medications used by the patients, - % were involved in unintentional medication discrepancies. of unintentional medication discrepancies found in five studies, - % were clinically relevant. conclusion: the review documented a high frequency of medication discrepancies, of which many were clinically relevant. ensuring sufficient communication of correct and complete medication information in transitions of care is a process which should be better implemented, to enhance patient safety. please specify your abstract type: research abstract background and objective: to investigate the frequency of medication changes not documented in the discharge letter, at discharge from hospital, for both regular, as needed and over-the-counter medications, supplements and herbal remedies (otc). secondary, differences between variables and patients with undocumented medication changes were investigated. setting and method: the patients included were all part of the intervention groups from an intervention study, conducted by one of the authors (tg), from april to december . the best possible discharge medication list was compared against the medication list in the discharge letter and any discrepancy between the two lists was noted, taking into account the text in the discharge summary. main outcome measures: the proportion of patients affected by at least one undocumented medication change at discharge and proportion of medications with undocumented changes. the proportion of patients was compared using a test according to gender, age, number of preadmission/discharge medications and length of hospital stay. results: two hundred patients were included in the study. the proportion of patients experiencing at least one undocumented medication change for the three subgroups: regular medications; as needed; otc, were , and % respectively. the proportion of medications involved in undocumented changes for the three subgroups were , and % respectively. the proportion of patients experiencing undocumented medication changes was significantly higher in patients with more than five regular medications at admission, (p \ . ) and at discharge (p \ . ). in both regular and as needed medications, the proportion of patients experiencing undocumented medication changes was higher in patients hospitalized longer than days (p \ . and p: \ . respectively). for otc, the rate of patients experiencing undocumented medication changes, was higher in females (p: \ . ). conclusion: a high proportion of patients are affected by at least one undocumented medication change and many medications are involved in undocumented changes. correct and complete medication information at admission and discharge may resolve many of these errors, ensuring patent safety at transitions of care. hp-ce : participation in courses at learning and mastery centre and the impact on patients' beliefs about medicines merethe nilsen *, , erik oie , kirsten k viktil diakonhjemmet hospital pharmacy, department of internal medicine, diakonhjemmet hospital, oslo, norway please specify your abstract type: descriptive abstract (for projects) background and objective: patients with chronic diseases are referred to learning and mastery centre (lmc) where the main objective is to support patients to cope with chronic diseases. education about the disease(s) (by a physician) and the medication treatment (by a clinical pharmacist) are important elements of these courses. little is known about how the participation at lmc influences the patients' beliefs about medicines. design: patients c years participating at a days course at lmc regarding acute coronary disease or atrial fibrillation were included in the period september -december . the patients filled out 'beliefs about medicines questionnaire'(bmq) before and immediately after the course, and also months after the course to evaluate their concern (bmq-concern) and necessity (bmq-necessity) of their cardiovascular medications. the bmq scores were dichotomized at scale midpoint (scale - ) to evaluate high and low concern and necessity, and these scores were combined to calculate the 'ambivalence'and 'acceptance', 'sceptical', and 'indifferent'rate to medications, and also the mean scores of the bmq were calculated. results: fifty patients were included, mean age years, % were women, using a mean of . cardiovascular drugs taken regularly. fifty-eight percent of the patients had high concern prior to the course, whereas and % had high concern immediately after and months after the course, respectively. ninety-nine percent of the patients assessed their medication as highly necessary before the course, % immediately after, and % months after the course. the mean score for bmq-necessity was . (sd . ) prior to course and . ( . ) and . ( . ) immediately after and months after the course, respectively. the corresponding scores for bmq-concern were . ( . ), . ( . ), and . ( . ), respectively. the proportions of patients classified to be 'accepting'were , , and % at the three time points, respectively, and the corresponding numbers for patients classified as 'ambivalent'were , , and %, respectively. conclusion: the lmc course had an immediate positive influence on the patients' concern about their medicines and on 'acceptance'. however, the effect seems not to persist over time. a closer follow-up could be discussed. please specify your abstract type: research abstract background and objective: the narrative-based medicine was intended primarily for health care professionals, and the use of narratives can be applied in any settings to better understand the meaning of own profession, to rediscover/strengthen the motivation to work as a team. the italian society of hospital pharmacist (sifo) promotes a qualitative study aimed at getting the real picture of pharmacist's role within the national health system (nhs), the interaction with other health professionals and patients through the narratives of under specialization pharmacists (ui) and pharmacists already working in the nhs (hp). these data can be further investigated to increase the perceived value/role of the pharmacist. setting and method: sifo hps and uis joining the national pharmacy school specialization network were invited to participate. all pharmacists participating to the study were given a semi-structure interview. the methodology was developed within the conceptual framework of the grounded theory (gt) a research methodology that arises in the context of qualitative research. gt is a systematic methodology involving the construction of theory grounded in data systematically gathered and analysed. main outcome measures: analysis of narratives. narratives were analysed according to the classifications of kleinman, frank and launer and robinson together with transitional analysis (ta). results: a total number of narratives were collected ( ups and hps). narratives from both group of participants show the need of strengthening the professional identity already in the early years of the pharmacy curriculum and more effectively during the years of specialization as well as the need of being educated to deliver patientcentred care as members of an interdisciplinary team. conclusion: this is the first step of a study that also includes patient's contribution to the definition of pharmacist's professional identity. hp-ce : impact of pharmaceutical counselling on cancer patients' information desire and treatment satisfaction stephanie wuyts *, , jacques de grève , veerle foulon , hilde collier , pieter-jan cortoos pharmacy, medical oncology, university hospital brussels, brussels, faculty of pharmaceutical sciences, catholic university of leuven, leuven, belgium please specify your abstract type: research abstract background and objective: appropriately educating onco-/haematological patients is a prerequisite to improve patient empowerment, satisfaction and outcomes. objective: to quantify patients' information need and satisfaction on cancer drug therapy and how this can be improved by clinical pharmacist's counselling. additionally, the pharmacist's impact on therapy quality and costs is assessed. setting and method: setting: prospective, randomised study in the ambulatory ( beds) and in-hospital onco-/haematology unit ( beds) in a tertiary hospital. inclusion criteria: adult patients on intravenous or oral cancer therapy, with informed consent. methods: all patients were asked to complete standardised surveys (extent of information desired, eid; patient satisfaction with cancer treatment education, ps-cate and cancer satisfaction of treatment questionnaire, ctsq) on three occasions (at the start of a new therapy, during the second cycle and after months). patients in the intervention group received additional counselling by a clinical pharmacist including medication reconciliation and review. control patients received standard of care (information on drug therapy was provided by the onco-/haematologist, followed by limited administration instructions by nursing staff). main outcome measures: patient information desire and satisfaction on cancer treatment results: patients were included over a period of months (control (n = ); intervention (n = )). no significant differences were found between contact moments or patient groups for eid, ps-cate and ctsq-scores. however, scores for ps-cate on medication side effects were positively correlated with contact moment (r s = . ; p = . ). multiple linear regression analysis showed a similar trend (b = . ; p = . ). patients receiving first-line therapy (b = . ; p \ . ) and ambulatory patients (b = . ; p = . ) were more satisfied on treatment education. the clinical pharmacist documented more drugs than were recorded in the patient file ( vs. . drugs/patient; p \ . ). on average, each patient required two pharmacist's interventions per occasion. intervention acceptance rate on drug related problems was high ( %). during the study, interventions shifted from therapy adjustments towards advice on supportive measures ( st contact: %; rd contact: %). improved medication stock control on the ward led to a savings of € , . conclusion: the clinical pharmacist can play an important role on the onco-/haematological ward, leading to improved drug reconciliation, patient counselling and cost savings. hospitalised patients and patients receiving salvage therapy appear to have higher educational needs, making them possibly overlooked target groups. finally, pharmaceutical counselling should be repeated and primarily focused on side-effect management to have a meaningful impact on patient satisfaction. please specify your abstract type: research abstract background and objective: europe is ahead of the usa and canada on approval, regulatory and marketing aspects of biosimilars. however, there is still uncertainty about interchangeability and substitution of biosimilars. the aim of the study is to assess pharmacists' perceptions about biosimilar interchangeability. setting and method: a cross-sectional study was carried out in june-july . hospital pharmacists from quebec and france were invited to respond to an online survey of nine questions (surveymonkey Ò , palo alto, ca, usa). the survey focuses on pharmacist's exposition to biosimilars (general knowledge, dispensing) and their perceptions about biosimilar interchangeability. a -item likert scale was used to answer to statements based on key issues about biosimilar interchangeability. main outcome measures: levels of agreement on biosimilar interchangeability key issues. results: a total of pharmacists responded ( % in quebec vs. % in france). the global response rate is: % ( % quebec vs. % france) (n = / ). % attended at least to one conference on biosimilars ( vs. %). % had already dispensed biosimilars ( vs. %). more than % of the pharmacists knew that: biosimilars can cause immunogenicity, clinical studies are requested for their approval, automatic substitution is not permitted. % considered that post-marketing surveillance for biosimilars should be reinforced. pharmacists considered that biosimilars are cheaper than the reference product ( vs. %). there was no difference between the level of agreement of french and quebec pharmacists for the statements. pharmacists agree that a list of biosimilar and interchangeable biologic products is necessary ( vs. %), using the international nonproprietary name to prescribe a biological product can create confusion between the reference product and its biosimilar ( vs. %), pharmacists should check if patients already experienced an immunogenic reaction before dispensing a biological product ( vs. %). pharmacists disagree that a biosimilar can be used for all the indications of the reference product ( vs. %). conclusion: perceptions of quebec and french hospital pharmacists about biosimilar interchangeability issues are very similar. this study highlights the need to deal with the lack of clarity of national guidances. clinical studies on biosimilar interchangeability must be conducted in the future to help pharmacists and physicians to take clear-headed decisions. please specify your abstract type: research abstract background and objective: analgesics are essential drugs in hospitals and especially in emergency units. medical and nurse staffs are used to the narcotic status of opioids. for some drugs, a regulatory change to narcotic status can discourage their use. for others, it could limit their access particularly in developing countries; that's why who did not recommend ketamine to be placed under international control (http://www.who.int/medicines/access/controlled-substances/ recommends_against_ick/en/). yet, the french drug agency has recently considered to register drugs containing ketamine as narcotics. the aim of this study was to assess the impact of this possible regulatory change on the pharmaceutical and medical practices in some paediatric french hospitals. setting and method: the survey was conducted in january-february in four parisian paediatric hospitals: four pharmacies, paediatric neurology and anaesthesia departments, intensive care units and pain management services. main outcome measures: pharmacists, clinicians, health managers and nurses were interviewed, using a standardized questionnaire with closed and opened questions, on the drug circuit including ordering, storage, distribution, prescription, administration and destruction. results: all the health professionals (five pharmacists, ten clinicians, five nurses) indicate that the change to narcotic status would not preclude the use of an analgesic drug. they consider that the pharmaceutical aspects (dispensation, storage and transport, etc.) are not limiting, provided that clinical usefulness is demonstrated: short action onset allowing rapid efficacy, short duration of action allowing the replacement by another drugs if needed, and moderate clinical monitoring. change to narcotic status was rather seen as advantageous since allowing better traceability, use and prescription. half of the pharmacies (n = / ) had a computerized register of narcotics and % of care units (n = / ) had a drug staffing in addition to nominative prescriptions, which was used in all care services. the drugs were kept into secured rooms. none of the emergency units (n = / ) had a computerized secured cabinet. conclusion: according to this survey, narcotic status is not a limiting factor for a drug use in paediatric hospitals, when its clinical usefulness is clearly demonstrated. to promote its use, it is important to inform medical and nurse staffs and include it into care protocols. beyond the nominative prescription, implementation staffing is a key step. please specify your abstract type: research abstract background and objective: port-a-cath is an implanted venous access device most commonly used for frequent or continuous chemotherapy administration. however, the procedure and its subsequent maintenance are not free of complications and requires additional intervention by the clinical pharmacist who can provide further patient care to make a positive impact on. to assess the effective provision of appropriate patient counselling offered by a clinical pharmacist on reducing port-a-cath relatedcomplications in cancer patients. setting and method: a controlled prospective observational study carried out on patients newly diagnosed with cancer eligible for chemotherapy administration at the oncology unit. assessment of port-a-cath related-complications were assessed at regular schedule of chemotherapeutic protocols administration. main outcome measures: to assess, reduce and solve port-a-cath related-complications. results: the most significant port-a-cath related complications were skin rash . % (p \ . ) with occurrence in males (n = ) and females (n = ), skin erythema . % with equal occurrence in both genders, followed by skin discharge . % with also equal occurrence in both genders. a high occurrence of skin rash . % occurred among diabetic cancer patients. a significant improvement in port-a-cath related complications after the provision of patient counselling by the clinical pharmacist was observed as skin rash ( . %), skin discharge ( . %), and skin erythema ( . %). conclusion: results of this study pointed out the essential role of clinical pharmacist in argumenting patient care and improving port-a-cath related-complications in cancer patients. please specify your abstract type: research abstract background and objective: polytherapy, frequently used in the elderly, is associated to an increased risk of potential drug-drug interactions (pddis) and adverse drug reactions (adrs). literature demonstrated that medication reconciliation and medication review performed by hospital pharmacists are correlated to drug related problems (drps). aim: to define a structured and feasible model where hospital pharmacists support clinicians identifying drps and promote the safe use of medicines. setting and method: prospective, feasibility study conducted in four internal medicine wards of a hospital in northern italy. inpatients (c years old, treated with c drugs) were consecutively included; the recognition/reconciliation process was performed by pharmacists in order to identify changes between prescription profile at home and during the admission (active principles, dose, administration route). these changes were classified as intentional documented discrepancies (id), not documented (ind), not intentional (ni). prescriptions during the first -hours of hospitalisation were analysed to retrieve drps (ddis, inappropriate medications for elderly, off-label, over/ under dosage, duplications, adrs) then discussed with clinicians. based on literature, referring almost drp in % of patients, a sample size of patients should allow an estimate of drp rate over % (need of intervention) with a % power and a confidence interval of % (software stata version . ). main outcome measures: rate and type of: discrepancies, drps at admission and discharge, pharmacists consultations accepted by clinicians. results: ad interim results are presented. between october/ -february/ , inpatients ( male, . mean age) were included. overall, patients were admitted with drugs used at home and prescribed during the first -hours; pharmacists retrieved discrepancies ( %id, %ind, %ni) and drps, of which % ddis, % off-label, % overdoses, % duplications, % inappropriate drugs, % not notified adrs. the % of drps was known to clinicians and % considered clinically relevant for the patients. please specify your abstract type: research abstract background and objective: hypertension is a major risk factor for cardiovascular morbidity and mortality worldwide, for which management is based on two principal, complementary approacheslifestyle modification and lifelong treatment with antihypertensive medication. adherence to hypertension therapy is a major public health challenge, despite the availability of multiple classes of antihypertensive agents. factors contributing to non-adherence are multifactorial and include intolerances to drugs at standard doses that result in therapy discontinuation. medication intolerance (mi-htn) refers to patients who experience adverse drug reactions (adrs) to at least one antihypertensive medication, without a known immunological mechanism and the need to discontinue them. we sought to determine factors associated with mi-htn and to identify patients' beliefs and concerns about their antihypertensive treatment and medication in general. setting and method: a cross sectional survey consisting of selfreported questionnaires including beliefs about medicines questionnaire (bmq), perceived sensitivity to medication (psm) and quality of life was undertaken in an unselected patients attending a hypertension centre of excellence out-patient clinic based in london. main outcome measures: to determine factors associated with mi-htn and the impact of health beliefs and self-reported perceived sensitivity to medications on mi-htn and bp control. chi squared tests for comparisons between cases/controls and multiple logistic regression analysis were used for statistical analysis. results: participants were included, of which ( %) participants had mi-htn. two-thirds were female (p = . ) with a mean age of ± years (p . ), of whom . % had uncontrolled hypertension (p = . ). calcium channel blockers were the most commonly reported intolerance by drug class followed by diuretics. being female and age [ were statistically associated with a greater likelihood of reporting medicines intolerance (p \ . ). patients who believed that medicines are harmful were [ -times more likely to report mi-htn (p = . ) and -times more likely to have uncontrolled bp ([ / mmhg) (p = . ). patients with high self-perceived sensitivity to medication was -times more prone to mi-htn (p = . ). conclusion: our findings suggests the need for greater focus on behavioural change interventions to both improve patients' perception of the necessity to persist with lifelong antihypertensive medication and allay concerns regarding harmful effects of drugs may help with long term control of hypertension. please specify your abstract type: research abstract background and objective: today, the number of medical problems in heart transplant recipients has increased due to aging and complications common to immunosuppressive drugs. the co-existence or emergence of other disease states such as renal dysfunction, infection, diabetes, obesity, hypertension, hyperlipidaemia, malignancies, and osteoporosis necessitates the use of other medications. the use of these medications in combination with immunosuppressive agents increases the risk of drug-drug interactions. the aim of this study is to identify the frequency and significance of drug-drug interactions for the patients who received cardiac transplantation. setting and method: this retrospective study was conducted at a cardiovascular specialty hospital. all patients who received cardiac transplantation from the same surgery team between and ( years) were included in the study. all data were collected from the medical records of the patients. only the most recent prescription before discharge was analysed for the presence and significance of drug-drug interactions. drug-drug interactions were checked using micromedex(r) interaction checker. main outcome measures: main outcome measures were the frequency and significance of drug-drug interactions. results: a total of patients met the inclusion criteria and prescriptions were analysed. each prescription contained an average of drugs. a total of drug-drug interactions were identified: . % was classified as moderate; . % as major and . % as contraindicated. almost half of all interactions (n = ) included immunosuppressive agents ( . % was classified as moderate; . % as major and . % as contraindicated). conclusion: cardiac transplant recipients were found to have a high number of drug-drug interactions. in order to advise on these interactions which increase with poly-pharmacy, drugs with narrow therapeutic index or drugs that require intensive monitoring, it is recommended to include a transplantation pharmacist in the transplantation team. please specify your abstract type: research abstract background and objective: the aim of our study was to assess the impact of patient education provided by the pharmacist on gylcemic control, medication knowledge level and medication adherence of patients with type diabetes. patients who were diagnosed with type diabetes for at least one-year time and were receiving at least one antidiabetic medication, attending to the outpatient diabetes clinic for the control visit were informed about the study and invited to participate in the study. patients who gave their informed consent were included in the study. setting and method: the setting is a diabetes outpatient clinic of a state hospital. the medication knowledge levels, medication adherence scores, fasting blood glucose levels, hba c levels and blood pressure of the patients were measured before pharmacist's education. after provision of standard information and individualized patient education all these parameters were measured again after monthstime and the impact of the education was assessed. main outcome measures: main outcome measures are change in the clinical parameters (hba c; fasting blood glucose; blood pressure), as well as improvements in medication knowledge and adherence levels. results: the study was conducted on patients who met the inclusion criteria; none of the patients were lost to follow-up. majority ( %) of the patients was female and the mean age was . years. pharmacist intervention resulted in positive outcomes at all clinical parameters. systolic blood pressure decreased by mmhg, while diastolic blood pressure decreased by . mmhg (p \ . ). hba c level decreased by . % (from . to . %; p \ . ) and fasting blood glucose level by . mg/dl (p [ . ). on the other hand, the number of patients reaching the blood pressure goal increased from to ; and those reaching to hba c goal increased from to (p \ . for all). similarly, the medication knowledge level [usual range - ] increased from . to . (p \ . ); and the medication adherence score [usual range - ] increased from . to . (p \ . ). conclusion: it can be concluded that pharmacist's contribution results in positive outcomes in glycaemic control and management of co-morbid conditions of type diabetic patients by improving medication knowledge and adherence levels of the patients. pharmacists should take active role in management of chronic diseases. hp-pc : impact of a pharmaceutical care program on glycemic control, medication knowledge and medication adherence levels of type diabetic patients residing at a nursing home nimet saglam *, , sule apikoglu-rabus , betul okuyan , fikret v. izzettin , nuran yildirim clinical pharmacy department, marmara university faculty of pharmacy, darulaceze nursing home, istanbul, turkey please specify your abstract type: research abstract background and objective: the aim of our study was to assess the impact of pharmaceutical care provided by the pharmacist on glycaemic control, medication knowledge level and medication adherence of patients with type diabetes residing at a nursing home. setting and method: this prospective cohort study was conducted in a state nursing home (darülacaze nursing home) in istanbul, turkey on patients who completed the whole study. all the patients received pharmaceutical care provided by the pharmacist. this pharmaceutical care program was held for months. it consisted of an initial visit, followed by ''care and control'' visits and a final control visit; each visit was held at two-week time intervals. at the initial visit, demographic and general clinical data were collected and medication knowledge and medication adherence levels of the patients were also assessed. pharmaceutical care needs were identified for each patient and recommendations addressing these issues were structured. education regarding the medications of the patients was provided in both verbal and written forms using the standard patient education leaflets prepared by the pharmacist. at each visit pharmaceutical care needs are assessed and pharmaceutical care is tailored accordingly. main outcome measures: main outcome measures are change in the clinical parameters (hba c; fasting blood glucose), as well as improvements in medication knowledge and adherence levels. results: majority ( %) of the patients was male and the mean age was . years. pharmacist intervention resulted in positive outcomes regarding hba c levels. hba c level decreased by . % (from . to . %; p \ . ) and fasting blood glucose level by mg/dl (p [ . ). similarly, the medication knowledge level [usual range - ] increased from . to . (p \ . ); and the medication adherence score [usual range - ] increased from . to . (p \ . ). conclusion: it can be concluded that pharmacist's contribution results in positive outcomes in glycaemic control of type diabetic patients by improving medication knowledge and adherence levels of the patients. pharmacists should take active role in management of type diabetes at the nursing home setting. please specify your abstract type: research abstract background and objective: haemoglobin variability is related to mortality and morbidity in haemodialysis, renal transplantation and pre-dialysis patients. some demographic, haematological and pharmacological variables may affect hb variability. but there are some controversies about the influences of different erythropoiesis stimulating agents (esa).the objective of this study is to determine the influence of different esa on haemoglobin variability in pre-dialysis patients. setting and method: we conducted a prospective observational study with chronic kidney disease patients recruited from outpatients of nephrology department of a tertiary university hospital (from january to june ). exclusion criteria were: stage i and ii, not treated with esa, haemodialysis, peritoneal dialysis, renal transplantation, thalassemia, and deficit of glucose- -phosphate dehydrogenase . main outcome measures: patients included were treated with esa in maintenance phase (stable months prior).hb variability was calculated by standard deviation (sd) and residual standard deviation (residual sd) of hb levels. statistical analysis was performed with spss . (spss inc, chicago). observation period was months and data were recorded from the clinical records. ( ) . %, sofosbuvir/daclatasvir/ribavirin ( ) . %, sofosbuvir/simeprevir ( ) . %, sofosbuvir/ledipasvir ( ) . %, sofosbuvir/ledipasvir/ribavirin ( ) . %, dasabuvir/ombitasvir/paritaprevir/ritonavir ( ) . %, dasabuvir/ombitasvir/pari taprevir/ritonavir/ribavirin ( ) % ombitasvir/paritaprevir/ritonavir/ ribavirin ( ) . %, sofosbuvir/ribavirin ( ) . %. viral load at week was \ iu/ml in patients and at the end of treatment . conclusion: the results of rapid viral response at end of treatment were similar to those obtained in studies published to date. due to its recent access to these treatments it is necessary to continue monitoring these patients to assess virologic sustained response at weeks after end of treatment. please specify your abstract type: research abstract background and objective: fragile patients are considered those vulnerable patients with a certain degree of complexity in their care (polypharmacy, multi-pathological, palliative and/or residents in social and healthcare institutions). to ensure their continuity of care and safety in the use of drugs we applied a medication reconciliation process at admission, transition of care and/or hospital discharge. objective: to analyse the results of the medication reconciliation process of a fragile patient. setting and method: we developed a list of current medication with the following sources of information: medical history, clinical databases and information provided by the patient (interview). clinical case: -year-old woman admitted through emergency department due to severe dyspnoea. no known drug allergy. background: heart failure, chronic hypertension, hypercholesterolemia, hyperthyroidism, hyperuricemia, gouty arthritis, chronic kidney disease and cognitive impairment by alzheimer disease. exploration and complementary tests: echocardiogram and analytical control. clinical judgment: acute decompensated heart failure. acute myocardial infarction. prerenal acute kidney injury. main outcome measures: medication reconciliation made at admission with the detection of discrepancies and deprescribing criteria at hospital discharge. results: fragile patient (high-risk) with medicines as home treatment. patient was hemodynamically stable during the hospital stay. discrepancies were detected between the prescribed medication and the home treatment. discrepancies justified ( ): five by omission of medication (two new clinical situation, two therapeutic exchanges to adapt to the pharmacotherapy guide and one wrong drug) and two beginning of medication. discrepancies unjustified ( ): by omission of medication. to discharge: one antiplatelet therapy was. after the comprehensive review, we made the following recommendations of deprescription: suspend one non-steroidal anti-inflammatory drug-nsaid (by risk of bleeding in association with concomitant antiplatelet and antidepressant therapy) and one benzodiazepine (central nervous system-cns side effects); modify treatment: reduce doses of diuretics (blood pressure lowering effect). pharmacotherapeutic recommendations were accepted. conclusion: detection of discrepancies in the medication reconciliation and deprescription process are effective and safe strategies that allow optimization of pharmacotherapy in fragile patients. the use of drugs such as nsaids (gastrolesive effect), the combination of drugs with cns side effects and hypotensive action (associated with falls) in elderly patients constitute situations of risk that should be reviewed in fragile patients, as an essential part of the clinical evaluation. please specify your abstract type: descriptive abstract (for projects) background and objective: there are no positions for clinical pharmacists at the hospital, so we are dependent on projects to be able to show how pharmacists can contribute in the clinical team. our aim in this project was to introduce pharmaceutical knowledge by implementing medication reconciliation and medication review in different hospital wards. we wanted to show that many patients have discrepancies in their medication lists during hospital stay and that some of the drugs or doses given can cause drug related problems for the patient. our final goal was to get the physicians to be more aware of these issues when treating their patients. design: the method used was based on the two first parts of the integrated medicines management. the pharmacist conducted a standardized drug interview with patients who prior to admission were responsible for their own drugs. for patients who could not be interviewed or were not responsible for administering their own drugs, a current medication list from relevant care level was obtained. the medication lists obtained were compared to the documentation in the patient's drug chart and discrepancies communicated to the physician. during the hospital stay, a medication review and monitoring was also conducted by the pharmacist. results were presented to the patients physician and discussed. results: a total of patients were included and of these % had c discrepancy identified by the process. the most frequent type of discrepancy was the use of a drug that was not registered on admission (omission discrepancies). other discrepancies were wrong dose, dosage or formulation and registration of a drug the patient didn't use. drug-related problems were discovered in % of the patients and the most frequent were use of anticholinergic drugs in elderly, interactions, lack of treatment and monitoring and too high doses regarding kidney function. many of the detected drug related problems results in change in medication, other times the physician addresses the problem to the gp. the physicians were surprised of the high numbers of discrepancies in medication lists and drug related problems discovered. almost all the physicians considered that the pharmacist could be an important part of the treatment team and they wanted the participation of the pharmacist to be permanent. conclusion: the project led to increased awareness of the importance of medication reconciliation and medication review and showed the importance of pharmaceutical knowledge in the treatment team. unfortunately this was not sufficient to create positions for pharmacists in our hospital. new projects will focus on pharmacists teaching interns to improve the reconciliation at admission. please specify your abstract type: descriptive abstract (for projects) background and objective: we aimed to assess the quality of fluoroquinolones (fq) prescriptions at the toulouse university hospital emergency department as part of significant increase in consumption. design: retrospective mono-centric study of fq prescriptions written to adult patients managed at the emergency department (february th, -march th, . a pair consisting of a biologist pharmacist and a clinical pharmacist has analysed them using tools provided by the centre de coordination de lutte contre les infections nosocomiales (cclin). various criteria (pertinence of prescription, choice of antibiotic, dosage, duration of treatment, method of administration…) were faced with the guidelines issued by the société de pathologie infectieuse de langue française (spilf). results: about files were examined, contained fq prescriptions for systemic use. the most frequently prescribed antibiotic was ofloxacin ( %) and the most frequent indications were urinary tract infections ( %). among the prescriptions of fq, the establishment of fq was justified in % of cases and the antibiotic chosen was always the most suitable. nonconformities of dosage and/ or treatment time were found in a quarter of cases. overall, % did comply with guidelines. the prescriptions, due to the particularity of emergency were still permormed probabilistic. however, a reassessment of them was scheduled for two-third of outpatients. conclusion: this study highlights the conformity of less than half of the prescriptions. this demonstrates that there are still actions to ensure the accuracy of fq prescriptions. and it is in this sense that this audit should be registered under the impetus of the committee on anti-infectives. it will raise awareness among doctors in the proper use of this family of antibiotics. please specify your abstract type: descriptive abstract (for projects) background and objective: the number of persons suffering from end-stage renal disease (esrd) is growing worldwide, mainly due to the aging of the population. esrd incidence has been increasing by - % per year for years. it is estimated that worldwide, more than . million patients with established renal failure are being treated with haemodialysis (hd). water for haemodialysis must meet the physicochemical and bacteriological compliance standards defined by the european pharmacopoeia. as a medicine, this water is placed under the responsibility of hospital pharmacists. addressed to hospital pharmacists, this methodology guide will enable them not only to validate controls of haemodialysis water as well as drug prescriptions for dialysis patients, but also to familiarize themselves with the best currently existing dialysis techniques and medical devices. we have tried to simplify and synthesize existing circulars and guidelines so as to render them more readily understandable for the pharmacist in charge of a haemodialysis service, and thereby help to ensure optimally safe treatment of haemodialysis patients. design: the themes developed in this guide are: • a review of the different existing dialysis techniques, • a review of the different sampling points for controls of hd water, • a review of the physicochemical and bacteriological standards of these controls according to the latest recommendations of the european pharmacopeia, and of appropriate conduct for exceeding established thresholds, • a review of the main international recommendations with regard to clinical signs of chronic kidney disease: anaemia, mineral and bone disorders (ckd-mbd), high blood pressure. • a review of the various medical devices used in haemodialysis and haemodiafiltration. results: the recommendations of good practices summarized in this guide are integrated perfectly adapted to the concept of quality assurance and its role in the accreditation process. they are focused on improving patient safety by harmonizing pharmaceutical haemodialysis practices in different dialysis centres. conclusion: these types of recommendations may be transposable to other pharmaceutical fields and/or be used as a training tool for pharmacy students or young pharmacy school graduates. the format of this guide makes it convenient, easy to use every day. it will be revised regularly to ensure the sustainability of quality plans. please specify your abstract type: descriptive abstract (for projects) background and objective: combination antiretroviral therapy (cart) has strongly improved disease control in hiv-infected patients. however, aging and comorbidities are becoming a major problem in this group of patients. most hiv-infected patients are treated with five or more medications, and harms by polypharmacy increase proportionally with number of medications. possible risks include: poor medication adherence and consequently inefficient care, increased risk of drug interactions and adverse events, with prolonged hospitalization. the problem is worsened when patients are of nonnative language and so their comprehension and adherence to drug therapy can be very poor, compromising efficacy. the hivig study is designed to evaluate the impact of the interventions promoted by the clinical pharmacist in the optimization and comprehension to personal drug therapy, favouring compliance, in a cohort of patients, hiv infected with comorbidities like cancer; the cohort includes a high number of non-native italian language individuals. design: hivig is a randomised, parallel groups clinical trial. in april the study protocol was approved by the local ethical committee, aviano. the project is scheduled to start in autumn . main objective: evaluation of the impact of a series of tools-''drug therapy setting interventions'' (dtsis) applied by the clinical pharmacist on a cohort pf hiv-infected patients with comorbidities, afferent for care at cro aviano. the treatment arm will be submitted to dtsis. dtsis interventions (treatment group) consist in: motivational interview, sharing and delivery of printed, explanatory material in the patient's native language, reconciliation of patients medications at hospital admission and at discharge; identification of potential risks due to drug-drug interactions; monitoring of compliance to drug therapy, and finally detection of adverse drug reactions (adrs) occurring in the course of care. the control group will undergo only to scheduled standard medical visits at cro. results: we expect to recruit a total patients for a -months period of follow-up. statistical analysis will be performed by intention-to-treat and by protocol. at cro aviano, the italian cooperative group on aids and tumors (gicat) has studied malignancies in hiv-positive patients since and has a leading role for studies conducted in italy (vaccher, ) conclusion: previous collected data from the previous trial performed at cro aviano (target-vig), showed a positive impact in the optimization of individual drug therapy and in the reporting of adrs. hiv has an enormous impact on life of infected patients and represents a priority issue for the entire community. we consider the method of dtsi, combined with a close monitoring of patients by means of telephonic motivational interviews, the best added value performed by the profile of the clinical pharmacist in optimizing drug therapy and personal awareness about medicines. please specify your abstract type: research abstract background and objective: the world health organization reports that ''one in four people in the world will be affected by mental or neurological disorders at some point in their lives. around million people currently suffer from such conditions, placing mental disorders among the leading causes of ill-health and disability worldwide». to review the evidences published about the roles and the impact of pharmacists in psychiatry. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, mental illness and psychiatry from january st until june th . manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in psychiatry. results: a total of articles were included. described pharmaceutical interventions included patient-pharmacist relationship ( ), medication reconciliation ( ), patient care needs assessment ( ), drug therapy assessment ( ), patient follow-up ( ), interdisciplinary work ( ), knowledge transfer ( ), competencies maintenance ( ). the impact of pharmacists interventions was studied using a total of indicators from which ( %) had outcome measures. of these outcome indicators, ( %) were positive, neutral and negative (knowledge transfer strategy). positive impacts of pharmaceutical interventions were identified in the following areas: morbidity ( ), patient adherence ( ), patients or clinicians satisfaction ( ), side effects management ( ), medication errors prevention ( ), mortality ( ) please specify your abstract type: research abstract background and objective: the world health organization reports that . million people die each year from cancer, an estimated % of all deaths worldwide and that there is a % increase in new cases of cancer expected over the next two decades. to review the evidences published about the roles and the impact of pharmacists in cancer. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, neoplasms from january st until june th . manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions as well as descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in cancer. results: a total of articles were included. described pharmaceutical interventions included patient-pharmacist relationship ( ), patient care needs assessment ( ), drug therapy assessment ( ), drug compounding/dispensing ( ), patient follow-up ( ), interdisciplinary work ( ), knowledge transfer ( ). the impact of pharmacists interventions was studied using a total of indicators from which ( %) had outcome measures. of these outcomes indicators, ( %) were positive, ( %) neutral and ( %) negative. positive impacts of pharmaceutical interventions were identified in the following areas: morbidity ( ), patient adherence ( ), patients or clinicians' satisfaction ( ), side effects management ( ), medication errors prevention ( ), mortality ( ), costs ( ) setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, myocardial infarction, acute coronary syndrome from january st until june th . manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in myocardial infarction. results: a total of articles were included. described pharmaceutical interventions included patient-pharmacist relationship ( ), medication reconciliation ( ), patient care needs assessment ( ), drug therapy assessment ( ), drug compounding/dispensing ( ), patient follow-up ( ), interdisciplinary work ( ), knowledge transfer ( ). the impact of pharmacists interventions was studied using a total of indicators from which ( %) had outcome int j clin pharm ( ) : - measures. of these outcome indicators, ( %) were positive, ( %) neutral and ( %) negative. positive impacts of pharmaceutical interventions were identified in the following areas: morbidity ( ), patient adherence ( ), side effects management ( ), mortality ( ) and others ( ). conclusion: the role and the impact of pharmacists have been studied in myocardial infarction and % of outcome indicators used in these studies show a positive impact of pharmaceutical interventions. pharmacists should pay attention to these evidences to improve their practice, contribute to prevention or insure treatment of patients with potential or found myocardial infarction. hp-pc : impact of pharmaceutical care in vaccination: a review of literature please specify your abstract type: research abstract background and objective: the world health organization reports that ''immunization is the process whereby a person is made immune or resistant to an infectious disease, typically by the administration of a vaccine and a proven tool for controlling and eliminating lifethreatening infectious diseases and is estimated to avert between and million deaths each year. it is one of the most cost-effective health investments, with proven strategies that make it accessible to even the most hard-to-reach and vulnerable populations». to review the evidences published about the roles and the impact of pharmacists in vaccination. setting and method: literature review. a literature search was conducted using pubmed and the following terms: pharmacists, clinical pharmacy, pharmaceutical services, pharmaceutical care, pharmacy, vaccination and immunization from january st until july th . manual search was also conducted using selected articles. the selection of articles was based on abstracts. selected articles were reviewed, analysed and entered in impactpharmacie.org website according a standard operating procedure. relevant key data were extracted for each article including the type and the description of pharmaceutical interventions and descriptive and outcomes indicators with their results. no statistical analysis was conducted. main outcome measures: proportion of outcome indicators associated to pharmaceutical interventions with a positive impact in vaccination. results: a total of articles were included. described pharmaceutical interventions included patient-pharmacist relationship ( ), medication reconciliation ( ), patient care needs assessment ( ), drug therapy assessment ( ), patient follow-up ( ), interdisciplinary work ( ), knowledge transfer ( ), competencies maintenance ( ). the impact of pharmacists interventions was studied using a total of indicators from which ( %) had outcome measures. of these outcome indicators, ( %) were positive, ( %) neutral and negative. positive impacts of pharmaceutical interventions were identified in the following areas: cost ( ), errors ( ), morbidity ( ), patient adherence ( ), patients or clinicians satisfaction ( ) please specify your abstract type: descriptive abstract (for projects) background and objective: evaluating the appropriateness and effectiveness of the patient's medications by analysing prescriptions is pharmacist side work. bedside drug administration and computerised drug administration traceability (cdat) in nursing care plan (ncp) are nurse's one. however, in order to check adherence, efficiency and tolerance of a drug, pharmacist has to ensure that the patient takes the medication appropriately. therefor ncp could be a useful tool. the aim of this study is to evaluate the effectiveness of cdat, and if not, define causes of divergences with real life situation. design: • comparison between unused drugs remained in individual patients' seven daily pill dispensers (considered as not taken) which come back from the evaluated service to the pharmacy, and their cdat status completed by nurses (taken, not taken or no status) • two recorded data, each collecting a three-week period, separated by a period of discussion with nurses: first results presentation, analysis of divergences by taking into account their feedbacks, and actions to raise their awareness about the importance of cdat. • pill dispensers' cdat is correct only if all returned drugs' status in ncp is ''not taken''. results: during the first period (n = pill dispensers), . % of pill dispensers had an incorrect cdat status. on average, . drug per pill dispenser didn't have an appropriate status in ncp (taken or no status). major causes of divergences were the lack of time and insufficient human resources, the fact that they often are interrupted in the middle of this task, a software which isn't ''user-friendly'' and a deficit of information about the issue. corrective actions were implemented, prior to the second recorded data period, targeting human factor of divergences (oral and written reminders about cdat with didactic memorandum on computers). after awareness actions, results (n = ) were . and . respectively. conclusion: efforts about cdat have been done but not enough to observe a significantly improvement in short terms. ncp's level of reliability is not optimal yet and still dependent on nurses' practices. this study allowed us to strengthen the relationship between clinical service and pharmacy, and opens the way for further works particularly through corrective actions targeting material and organizational causes of divergences. please specify your abstract type: descriptive abstract (for projects) background and objective: in , our teaching hospital has participated to a worldwide survey (global point prevalence survey (global-pps)) aimed to explore antimicrobial consumption and resistance in hospitals. because broad spectrum antibiotics have to be followed with the attention of resistance prevention, we focused our analysis on these antibiotics in our hospital (carbapenems, piperacillin/tazobactam and amoxicillin/clavulanic acid). design: the survey was performed by pharmaceutical team (senior and resident) with help of microbiologists and referring physicians. all wards of the hospital were included. hospitalized patients treated with antimicrobial agent (j , j , j a, p ab, a , j ah, p b from the atc classification) prescribed at a.m. on the day of the survey, were involved. from those who were treated by carbapenems, pip/taz or amx ac, following data were collected: age, gender, weight, doses, indications (probabilistic? documented? and if documented microbiological data), and mention of stop/review date of prescription. results: the survey was carried out from april to june in wards. among the patients included, patients ( . %) were treated with antimicrobial agents. patients ( . %) were treated with broad spectrum antibiotics: ( . %) with amx-ac, with pip-tz, with imipenem and with meropenem. the mean age of patients was . ± . and the weight was . ± . kg. their prescriptions were concentrated in three types of wards: ( . %) in icu, ( . %) in medicine, ( . %) in surgery. moreover, we observe that bsa were used to treat ( . %) community acquired infections, ( . %) nosocomial infections, ( . %) used as medical prophylaxis, or surgical prophylaxis (n = , . %). in relation to the type of treatment: were empirical treatment (including prophylaxes) and were targeted treatments ( bacteraemia, joint and bones infections, cardiovascular system infection, urinary tract infections, lower respiratory tract infections, skin and soft tissues infections and others infections). finally, extended spectrum beta-lactamase (esbl) producing enterobacteriaceae and third generation cephalosporin resistant enterobacteriaceae non-esbl producing were targeted by bsa regimen. conclusion: in this survey, use of bsa is globally compliant to french guidelines and we identified no improper prescription: multidrug resistant bacteria infections, several diseases and empirical treatments with limited duration of regimen. this shows that control of the proper use of antibiotics especially those with a broad spectrum is efficient in our hospital and has to be continued. this has been made possible due to a multidisciplinary approach including physicians, bacteriologists and pharmacists. please specify your abstract type: descriptive abstract (for projects) background and objective: in , our teaching hospital has participated to a worldwide survey (global point prevalence survey (global-pps)) aimed to explore antimicrobial consumption and resistance in hospitals. from these results, we observed that sulfamethoxazole/trimethoprim (tmp/smx) was largely prescribed in our hospital. we focused then our analysis on these results with the attention of check of its proper use. design: the survey was performed by pharmaceutical team (senior and resident) with help of microbiologists and referring physicians. all wards of the hospital were included. hospitalized patients treated with antimicrobial agent (j , j , j a, p ab, a , j ah, p b from the atc classification) prescribed at a.m. on the day of the survey, were involved. from those who were treated by tmp/smx, following data were collected: age, gender, weight, doses, and indications (probabilistic? documented? and if documented microbiological data), and mention of stop/review date of prescription. please specify your abstract type: research abstract background and objective: in france, benzodiazepine (bzd) is frequently prescribed in elderly people (ep). long-term efficacy is often questioned, and treatment has to be regularly re-examined, especially in ep. in our geriatric day-hospital for assessment of frailty, a multidisciplinary team evaluates the patients and gives them preventative measures against the loss of autonomy. medication evaluation is part of these measures. the aim of our study was to evaluate the impact of a standardized intervention on the optimization of bzd treatment. setting and method: after a short interview and the delivery of an information booklet about bzd, patients were proposed an optimization of their bzd treatment (dosage reduction, occasional medication, switch to a short half-life bzd, or total discontinuation). patients were followed up monthly by a phone-interview over a -months period. main outcome measures: the main outcome measure was the prevalence of bzd optimized treatments after a months follow-up. results: patients were included. among them, % have been taking a bzd for more than years, and % were prescribed a long half-life bzd, which can be qualified as inappropriate in ep. % of the subjects were frail and % pre-frail according to the fried criteria. at the end of the study, % of the patients had their bzd treatments optimized, including % of total discontinuation. conclusion: in frail or pre-frail elderly population, a standardized intervention can be useful to improve bzd treatment. an extension to this intervention would be the creation of an organisation tasked with routinely monitoring the patients withdrawal over a month period. hba c and weight were significantly reduced by . ± . %, p \ . and . ± . kg, p \ . , respectively; systolic bp ( . ± . mmhg, p \ . ), diastolic bp ( . ± . mmhg p \ . ) and triglycerides ( . ± . mg/dl, p . ).genital-and urinary tract infections were reported by . % patients. any diabetic ketoacidosis case was reported. conclusion: sglt- inhibitors added to other oral antidiabetic drugs or insulin in patients with uncontrolled t dm significantly improved glycaemic control, reduced weight, blood pressure and triglycerides, and was generally well tolerated. in conclusion, sglt- inhibitors, appears to be an important addition to the therapeutic options for the management of type diabetes, particularly when used as add-on therapy. ( ). treatment safety takes part of the decision to undergo bariatric surgery. during multidisciplinary team meetings, the clinical pharmacist must rely on guidelines to limit drug-induced iatrogenesis. this review aims at assessing influence of bariatric surgery on the clinical impact and pk of cardiotropic drugs so as to document pharmacists' notifications. setting and method: literature review on medline- to may -with terms: cardiovascular drugs and bariatric surgery or malabsorption syndrome. related articles were reviewed. main outcome measures: pharmacokinetic or pharmacodynamic data and clinical impact of cardiotropic drugs. results: a total of titles, and abstracts when necessary, were screened for eligibility. after reviewing process, studies were included: nine concerning digoxin, five beta-blockers (bb) and one amiodarone. published studies varied in methodology: five case report, seven case control and three cohort studies. studies reported variations of digoxin plasmatic concentrations among patients versus , suggesting liquid oral form are preferred. no clinical event was notified. more the bb is liposoluble (propranolol), the higher the toxicity is, such as heart rate and blood pressure decreasing, with potential fatal outcomes. a case of amiodarone-induced hyperthyroidism is described after bariatric procedure showing an increase plasma concentration adjusted to weight. conclusion: while the impact on narrow therapeutic range drugs is documented, others cardiotropic drugs may cause serious patient injury justifying their monitoring. therefore, risk must be identified for all patients undergoing bariatric surgery to setting up closely therapeutic monitoring. further studies are still expected to lead to recommendations about posology and treatment withdrawal to improve patient safety. please specify your abstract type: research abstract background and objective: the issue of non-compliance to prescribed medical treatment has been reported to be a crucial problem in psychiatric outpatients. the aims of this study were to assess the extent of non-compliance in a cohort of psychiatric outpatients in malta and to investigate the applicability of using a -day multi-dose pill box in terms of practicality, ease of use and impact on compliance within this patient group. setting and method: the study was conducted at mount carmel hospital, a psychiatric hospital in malta. twenty outpatients were recruited by convenience sampling. the study was divided into two phases. during phase , patient compliance was assessed using the medication adherence rating scale (mars) survey and patients were administered part a of a questionnaire entitled 'assessment of the -day multi dose pill box'. this questionnaire evaluated the patients' opinion regarding the -day multi dose pill box before and after its use. in phase , the chosen patients were given a demonstration on how to use the -day multi dose pill box and the device was given to them to use at home for one week. after one week, part b of the questionnaire was completed and compliance was re-assessed using mars . main outcome measures: evaluation of adherence before and after use of the compliance aid device. results: of the patients recruited, were male and were female. the mean age was years (range - ) and the mean number of daily medications (range - ). upon initial scoring using mars, patients were adherent and patients were nonadherent. a higher adherence was observed in patients taking or more medications daily. ten patients accepted to move on to phase of the study and took the device home to use for one week. out of these patients, felt that the way they take their medication improved following use of the device and out of patients would consider buying the device since they found it practical and easy to use. statistical analysis of mars score before and after use of the device showed no significant improvement in compliance (p [ . ). there was no significant association between level of adherence and type of psychiatric condition (p [ . ). furthermore, results did not indicate increased adherence in patients who have a carer in-charge of their medication administration or in patients using a compliance aid device (p [ . ). conclusion: the use of a compliance aid device in psychiatric patients is challenging due to difficulty in establishing patient communication and motivation. the pharmacist is in a position to identify patients who would benefit from the compliance aid device. adrien borowik * , anne fratta, fabien hernandez pharmacy, ap-hp, armand trousseau paediatric hospital, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: enoxaparin, a low molecular weight heparin, is the most prescribed anticoagulation treatment in paediatric indications. however, the marketing authorization mentions that due to the lack of data, the use of enoxaparin is not recommended to children nor anyone weighing less than kg. thus, expert recommendations described specific dosage for the paediatric use: we aimed to compare these with our hospital practices. ( ), sofosbuvir/ledipasvir ( ), ombitasvir/ paritaprevir/ritonavir ( ), ombitasvir/paritaprevir/ritonavir + dasabuvir ( ), simeprevir + ifn ( ) . twenty-six patients ( %) were treated for weeks ( week pay-back policy). twenty pharmacists' interventions were carried out with an acceptance rate of %. the interventions included treatment adjustments due to drug interactions ( ), inappropriate treatment according to genotype ( ), duration of treatment ( ) and switch to a more cost-effective therapy ( ). seven pharmacists' interventions concerning treatment switch were applied ( %) resulting in a cost saving of € , . . all assessable patients ( ) have a negative serum hcv rna weeks after the end of treatment (svr = %) while patient died during follow-up (due to the disease). conclusion: the hospital pharmacist, as an active member of the multidisciplinary team, has an essential role in guaranteeing optimal care for hcv patients at the best cost. monitoring has also shown to be fundamental to evaluate the real world effectiveness of these drugs approved with surrogate endpoints. hp-pc : are hospital pharmaceutical staff educated on the criticality of thermosensitive drugs? camille castel, guillaume saint-lorant * please specify your abstract type: research abstract background and objective: in the use of thermosensitive drugs, the safety of patient care involves compliance with allowed temperatures. having the right information at time of care is essential. the aim of this study is to assess, within a french university hospital, pharmaceutical staff knowledge on the criticality of thermosensitive drugs and to educate them accordingly, including associated patient risks. setting and method: an assessment of knowledge using a questionnaire was led in january among pharmaceutical staff in a -bed hospital ( pharmacists, pharmacy residents, pharmacy technicians). evaluation criteria were: storage temperature of refrigerated drugs and frozen drugs, thermosensitive drug retention period after removal from the refrigerator, highest risk situation for a thermosensitive drug (t [ °c or t \ °c) and action to be taken during a temperature excursion. main outcome measures: to determine shortcomings in the management of thermosensitive drugs in order to adapt appropriate tools. results: completed questionnaires were collected. collected questionnaires included % from pharmacists (n = ), % from pharmacy residents (n = ) and % from pharmacy technicians (n = ). regulatory variations in storage temperatures of refrigerated and frozen drugs are known in respectively and % of cases. % of pharmaceutical staff are aware of thermosensitive drug retention periods after removal from the refrigerator and % of the highest risk situation for a thermosensitive drug (t \ °c). the measures to adopt during a temperature excursion are understood in % of cases. conclusion: this study highlights the lack of knowledge on the management and criticality of thermosensitive drugs and the lack of information available to pharmaceutical staff. dissemination of data and questionnaire reponses have been beneficial for the pharmacy department and have reduced inequalities in available information among pharmaceutical staff. subsequent to the study, thermosensitive drug management procedures have been revised. the deployment of this questionnaire is continuing via the university hospital intranet in order to train all health professionals in good patient care. please specify your abstract type: research abstract background and objective: temocillin is a beta-lactam antibiotic exclusively active against gram-negative pathogens. its use can avoid that of broad spectrum antibiotics, such as carbapenems, for the treatment of infections due to extended-spectrum beta-lactamase producing enterobacteriaceae. however, the absence of recommendations by learned societies on temocillin use could lead to misuse and the emergence of resistance. the aim of this study is to identify the role of temocillin in a french university hospital arsenal in order to limit ecological risks. setting and method: a retrospective study was conducted in a -bed university hospital. all adult patients having received at least days of treatment between june and april were included. data collected for the study were: age, sex, treatment indication (type of infection, identified pathogen, dosage and treatment duration), previous antibiotics and therapeutic outcomes. main outcome measures: the indicators chosen were: treatment indication, prescribed dose and treatment duration. results: two patients were included. in july , temocillin was used in a year old female as first-line treatment of intraperitoneal haematoma infection due to multiresistant klebsiella pneumoniae. prescribed at a dose of g twice daily by an infectious diseases specialist, treatment was continued at the same dose for up weeks with therapeutic success. in august , temocillin was used in a year old male for the treatment of bacteraemia due to multiresistant enterobacter aerogenes. previously treated by imipenem/cilastatin, temocillin was prescribed as second-line treatment at a dose of g twice daily by an infectious diseases specialist. treatment was continued at the same dose for up weeks with therapeutic success. conclusion: the dissemination of antibiotic resistance among gramnegative enterobacteriaceae continues to be an increasing threat for healthcare worldwide. within this context, temocillin could be an interesting alternative. determining the role of temocillin in a therapeutic arsenal is essential. our hospital considers temocillin as a ''critical antibiotic'' although its use is not exclusively limited to the new drug application. therefore, temocillin prescriptions are monitored permanently by infectious diseases specialists, microbiologists and pharmacists in order to improve the good use of this antibiotic and to optimise patient safety. please specify your abstract type: research abstract background and objective: drinkable solutions are more susceptible to deterioration and can lead to a potential risk for patient care. having the right information at time of care is essential. the aim of this study is to assess nursing staff knowledge in a french university hospital on the management of drinkable solutions to elaborate tools to help health professionals and to enhance equality of information in order to optimise patient care. setting and method: an assessment of practice using a questionnaire was conducted in may among a share of the nursing staff in a int j clin pharm ( ) results: completed questionnaires were collected. % of nursing staff replied that the period-after-opening is the same for all of drinkable solutions. this period is estimated at month in % of cases, weeks in % of cases and days in % of cases. % of nursing staff do not know how to store drinkable solutions after opening. the date of opening or the date of expiry after opening are specified on the medicine bottle in respectively and % of cases. only % of nursing staff have tools pertaining to the management of drinkable solutions. these observations led the pharmacy to create and distribute appropriate tools. storage methods for the drinkable solutions available in our hospital were collected directly from pharmaceutical laboratories. this information has been made available to nursing staff via drug control software (pharma Ò , computer engineering, paris). conclusion: this study highlights the lack of knowledge on the management of drinkable solutions and the lack of information available to nursing staff. in our hospital, the dissemination of appropriate data reduced inequalities in available information between care units. data will soon be integrated within the drug prescription software (mc kesson usv Ò , crossway, san francisco) in order to homogeneously train all health professionals in good patient care. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is a process which allows prevention of iatrogenic injuries during patient's hospitalisation and transfers. since , a clinical pharmacist has been integrated into the orthopaedic surgery care. he has performed mr at patients' admission. the aim of this study was to evaluate the impact of medication reconciliation performed by a clinical pharmacist. design: a prospective monocentric study was conducted on patients admitted in an orthopaedic surgery care (elective or unplanned surgery), during months. the clinical pharmacist established the best possible medication history (bpmh) from at least three sources of information (including patient interview when possible). then, it was compared to the admission medication order (amo) (from anaesthetists when elective or orthopaedists when unplanned). unintended medication discrepancies (umd) detected were discussed with prescribers in order to be corrected. epidemiological data, number and type of umd, therapeutic classes involved and the percentage of corrected umd were collected and their potential clinical impact was assessed. results: in this study, patients were included during months. elective surgeries were concerned in % of the cases. at least one umd was identified in patients ( %) (median age: . years old; male/female ratio: . ). of these, ( %) were older than years old. finally, umd were detected, being . by patient. main therapeutic classes concerned cardiovascular system ( %), nervous system ( %) and digestive system ( %). of the umd detected by mr, there were % of omissions, % of inappropriate dosing and % of renewal prescriptions stopped by the patient. finally, % of umd were corrected. of these umd, % were major errors (i.e. causing potential harm), % were significant errors (i.e. monitoring or intervention potentially required to preclude harm) and % were minor errors (i.e. without potential harm to the patient). conclusion: medication reconciliation process performed by a clinical pharmacist allows detection and correction of umd on half of patients in surgery care particularly on elderly patients. the high proportion of umd can be explained by the multiplicity of actors involved in medication management. health information technology could help to focus mr on patients at high-risk of adverse drug events. please specify your abstract type: research abstract background and objective: darunavir plus ritonavir (drv/r) have shown optimized results in simplification strategies (monotherapy (mt) or dual therapy (dt)) for selected hiv + in randomized clinical trials and real life experience. recent introduction of one pill drv plus cobicistat co-formulation (drv/c) may be particularly suited for both mt/dt allowing once daily administration optimizing dosage and adherence. the objective of our study is to evaluate efficacy and security of drv/c in mt and dt. setting and method: all hiv + adults with antiretroviral change to drv/c in mt/dt at a reference hospital in the northwest of spain were included in this retrospective study. a statistical analysis was performed using the spss v. .software. main outcome measures: epidemiological, clinical, antiretroviral regimen, serum creatinine, lipids and inmunovirological data (rna-hiv and lymphocytes cd ) were compared previous and after change to drv/c. results: hiv treatment-experienced patients have received drv/c in dt ( ) or mt ( ). . % were men with a mean age of years. main risk factors were: . % heterosexual, . % msm, . % injection drug users, . % mother-to-child transmission, . % transfusion in haemophiliac patient and . % unknown. cdc category distribution was . % a, . % b, . % c and . % unknown. overall mean nadir cd counts were . ± . cells/mcl. mean time since drv/c prescription to discontinuation or until analysis was . days [range - ]. . % drv/c mt were prescribed to patients with prior drv/r mt in order to simplify treatment and the mean time of the duration of these prior therapies were . years. in case of dt, . % were prescribed on patients with prior drv/ r + tc with a mean duration of . years. serum creatinine increases ( . vs. . ; p \ . ) and cd decrease ( . vs. . ; p = . ) when patients move to drv/c. no significant change in the other analytical parameters and all patients maintained undetectable. patients discontinue drv/c due to intolerance and inability to swallow in each case. conclusion: this preliminary study concludes that drv/c in mt or dt is efficacy (no viral rebound) and safety. although an increase in creatinine was observed, it would not be considered clinically significant. of note, lymphocytes decreased significantly and it will be important closely monitored to check that maintain effectiveness during the follow up. hp-pc : developing clinical pharmacy in emergency department setting up a medication reconciliation process marion collignon *, , antoine gantier , florent lapacherie , hélène dewaele , laura foucault , anne-laure raso , emmanuel cirot , said laribi , xavier pourrat pharmacy, emergency department, chru tours, tours, france please specify your abstract type: descriptive abstract (for projects) background and objective: in emergency department (ed), if a drug related problem (drp) happens at the patient admission, the risk is the error remains until discharge. one part of drp may be avoided with using medication reconciliation (mr). the objective of this study was to evaluate the feasibility of setting up a medication history (mh) of patients in ed in an acceptable lap of time before they were transferred in another unit or discharged. design: a months prospective study was conducted in ed in a university hospital in france. two junior pharmacists coached by a senior pharmacist, after a months training for mr, were in charge of the data and mh collection. for all patients, we collected age, mh according to number of sources, discrepancies identified, adherence to treatment (according to the social security questionnaire), type of sources. mh were established according to community pharmacies, patients, previous electronic patient files, prescription sheets, patient's family, packs of pills and to the general practitioner (gp). for patients from long term care facilities (ltcf), the mh was established only by communication with the ltcf. then, the current prescription was compared with the home medication regimen. mh and discrepancies (omitting medication, incorrect dose, ambiguous name) were recorded in the electronic patient files to be available during hospitalization. because ed does not have a pharmaceutical review of prescriptions, only major discrepancies were transmitted to physicians. results: we collected mh ( from ltcf), with a sex ratio of . and a medium age of . years old. it represented an average of . mh per day or min per mh. among patients who did not come from ltcf, sources used by pharmacy students were patient's community pharmacy ( , % of cases), patient ( , %), previous electronic patient file ( , %), prescription sheets ( , %), call to gp ( , . %), gp mail ( , . %), patient's family ( , . %), packs of pills ( , . %), community nurse ( , . %). finally, patients ( %) had been hospitalized, others were discharged. we analysed mh for patients: at least one drp occurred for patients ( %). among patients, ( %) had an immediate pharmaceutical intervention because of the risk due to discrepancy. among patients who did not come from ltcf and who could communicate, were good adherent to treatment ( %). conclusion: this study highlights the great interest of the mh by pharmacists at ed, which avoids many drp. the presence of pharmacists in ed contributes to maintain a safe environment for medication and to assist prescribers in the continuity of treatment between home and hospital. spending min by mh, we identify one drp every min. nevertheless, it could be benefit to develop this activity because of the satisfaction of the emergency physicians. currently, mr is the first step to develop clinical pharmacy in the ed. please specify your abstract type: research abstract background and objective: emerging evidence in the literature suggests a high prevalence of suboptimal vitamin d (vitd) and an association between lower serum levels and higher mortality in cancer. the objective of this study was to quantify vitd deficiency in patients after surgery for head and neck cancer, and to determine the effect of one cholecalciferol intramuscular dose. setting and method: intervention study with a follow-up period of months (november -february ) performed on patients followed by the nutrition support unit after surgery for head and neck cancer. demographic and physiopatological data, including admission diagnosis, age, gender, calcium, magnesium and phosphate were collected. nutrition screening by conut index was carried out. a single intramuscular dose of . ui cholecalciferol (vitamine d bon Ò ) was administered to vitd-deficient patients and serum -hidroxy-vitamin d (s ohd) records after the administration, including primary carés records after discharge, were evaluated (reference range - ng/ml). main outcome measures: s ohd (\ ng/ml: deficiency; - ng/ml: insufficiency; c ng/ml: sufficiency). results: data from patients with a mean (sd) age of . ( . ) years were collected (males: %). the admission diagnosis was laryngeal squamosis cell carcinoma (n = ), glottis carcinoma (n = ) and nasopharynx, tongue and skull base cancer (n = ). at baseline, , and patients were considered have high, medium and low risk of malnutrition, respectively. the mean (sd) serum ohd was . ( . ) ng/ml (deficiency: patients; insufficiency: patient). despite the role of vitd in mineral balance, calcium, magnesium and phosphate mean (sd) serum levels were between the normal range . ( . ) mg/dl, . ( . ) mg/dl, and . ( . ) mg/dl, respectively. s ohd records were available week after the administration (mean (sd) = . ( . ) ng/ml). and patients still showed deficiency and insufficiency, respectively. primary care's records from patients were available after discharge ( . , . and . ng/ml). conclusion: poor nutritional status and high prevalence of suboptimal vitd in patients with head and neck cancer were found. a single dose of intramuscular cholecalciferol slowly raises s ohd. follow-up after discharge is essential to evaluate the achievement of the therapeutic objective. setting and method: this is a descriptive retrospective study. it took place in a teaching hospital. antifungal broad spectrum therapies (liposomal amphotericin b, caspofungin, micafungin, posaconazole, voriconazole) used between st january and st december were included. main outcome measures: indications, type of combination and patients specifications were analysed. results: only patients ( . % over all patients receiving antifungal therapy; n = / ) received an antifungal combination therapy during the study period. majority of patients presented risk factors: % of patients had an organ transplant (n = ), % suffered from malignant blood disorders (four acute myeloid leukaemia, two chronic lymphoid leukemia, one non-hodgkin's lymphoma, one hodgkin's lymphoma and two refractory anaemia with excessive blast), % suffered from solid cancer (one lung cancer and one breast cancer) and % suffered from chronic obstructive bronchopneumopathy (n = ). antifungal combination therapy was used against invasive aspergillosis in % of cases (n = ) among which complications such as brain and cardiac impairment were found in % of patients (n = ). the six remaining patients ( %) were co-infected with candidiasis for three patients and mucomycosis for three patients. voriconazole was logically the most used in combination, and just one patient received oral form. it was in majority prescribed with caspofungin ( %, n = ) and intravenous liposomal amphotericin b ( %; n = ). combination including liposomal amphotericin b and caspofungin (n = , %) or posaconazole with liposomal amphotericin b (n = ) were found in our study. five patients deceased during the hospitalization of the fungal infection ( %) which shows the gravity of these cases. majority of patients ([ %) was treated less than days with these combinations. conclusion: this retrospective study shows that patients who received antifungal combination therapy were mostly immunocompromised, co-infected or experienced a severe infection with severity factors. the antifungal combination was in majority initiated because monotherapy failed to cure the patient. all prescriptions were discussed with a mycologist who tried to shorter the combination treatment duration. this multidisciplinary approach is a major key in the process of these type of treatments. please specify your abstract type: research abstract background and objective: because of its broad spectrum and the risk of resistance mutation, delivery of posaconazole is nominative and controlled by hospital pharmacists. the aim of this work was to describe the use and pharmaceutical follow-up of posaconazole tablets over a -months period. setting and method: this is a descriptive retrospective study over a -months period from november to may in a teaching hospital. all patients who received posaconazole tablets were included. main outcome measures: indications and dosage were reported. results: patients were included in the study. posaconazole tablets were used for: fungal invasive infection prophylaxis in case of stem cell transplantation ( %; n = ), fungal invasive infection prophylaxis if a chemotherapy was started to treat a chronic myeloid leukaemia or a myelodysplasic syndrome ( %; n = ); treatment of invasive aspergillosis ( %; n = ); mycetoma ( %; n = ); zygomycosis or mucormycosis while patient had renal impairment ( %; n = ). all of these indications were approved for posaconazole (marketing authorization and local guidelines). only patients ( %) received a loading dose ( milligrams twice a day) as recommended in approval authorization. posaconazole blood levels were monitored by pharmacologists: % of patients (n = ) did not need dosage modulation which shows that variability is not so important. but three patients did not have any assay to monitor posaconazole blood concentration. patient received a loading dose and was switched to intravenous voriconazole after icu transfer. patients needed increase and/or reduction dose to obtain optimal posaconazole blood levels. conclusion: this study describes the use and the follow-up of posaconazole tablets during the first months after its approval in europe. all indications are approved for posaconazole but this analysis shows that pharmacist have to remind the necessity of a loading dose. dosage can be adjusted according to assays results. please specify your abstract type: research abstract background and objective: due to the acute, hectic environment in a fast-paced work-flow emergency department (ed) it is a challenge to verify the correct and updated medication list for the admitted patients. when performing medication reconciliation (mr) in this environment, these challenge has to be taken into account and prioritizing patients for mr could be necessary. the objective of this study was to identify risk factors correlated to clinical relevant medication discrepancies (crmds) among patients admitted to ed, and based on these revealed risk factors, develop a model for prioritizing patients for mr in the fast-paced work-flow at the ed. setting and method: patients continuously included at the ed, diakonhjemmet hospital (dh), oslo, norway. trained pharmacists and emergency nurse conducted mr. patient specific factors and revealed crmds, between hospital admission records and information about prehospital medication use, were recorded. binary linear regression was used to identify risk factors correlated to crmds. the prioritizing model was built using statics and clinical experiences. main outcome measures: what risk factors is correlated to crmds and how precisely do the prioritizing model classify the patients as high-and low-risk patients. results: % of the patients had c crmd. the following were identified as risk factors correlated to crmd and were suitable for inclusion in the prioritizing model; gender (woman), age (c ), c admission to hospital last months, admission causes; surgical, malfunction, cancer. the model correctly classified . % of the patients with crmds as high risk. further, . % of the patients with crmds were classified by the model as low-risk patients (false negatives). the model classified . % of the patients who did not have a crmd as high-risk patients (false positives). conclusion: the prioritizing model developed can be helpful in identifying what patients are at increased risk of having crmds in the fast-paced work-flow at the ed. identifying these patients will result in using the resources available in the ed in the most efficient manner and utilizing the full potential of the mr method. as a consequence of this, patient safety would be increased. hp-pc : intravenous potassium chloride: quick audit of prescribers knowledge and recommendations regarding safe practice and proper usage asmaa damou * , vincent zaugg, martine postaire please specify your abstract type: descriptive abstract (for projects) background and objective: our hospital has established methods that try to ensure the safe use of high alert medications. intravenous potassium chloride (kcl) was the subject of preventive measures: separation of different dosages (kcl . % vials reserved for paediatric services and kcl % vials reserved for adult services); creation of an advice record for doctors and nurses; specific labelling of storage areas; double-check the prescription and administration. the objective of this study was to evaluate the knowledge of the safe use of intravenous kcl by prescribers. design: multiple-choice questions were developed for prescribing recommendations established by our hospital with the collaboration of the doctor who is chairman of the central committee of vigilance and risk associated with care (cvris). a link to the online survey was sent by email to physicians practicing in departments (eight paediatric services and six adult services). the results were extracted and interpreted in excel Ò . results: % of physicians responded to the survey ( medicine residents, hospital doctors). in paediatric services, % of doctors know that only the kcl . % should be used. % know the unit of prescription to be used (mmol/kg or meq/kg), and % know that the maximum recommended infusion rate is . mmol/kg/hour (or mmol/kg/h in recovery unit). in adult services, the recommended maximum rate of infusion ( g/h) is known to all prescribers, but only % know that the concentration of kcl must be less than g/l. % of paediatric doctors say that their kcl prescriptions are checked by a second doctor, but the answers in the same service area are sometimes contradictory. in adult services, only % of physicians say that the prescriptions are double-checked. the information brochure available on the intranet of the hospital is known by % of prescribers. the response rate of physicians to the survey was satisfactory. therefore, the recommendations are rather well known by prescribers, except the value of the maximum concentration of infusion for adults. the results of this audit were returned to the doctors, accompanied by a reminder stating the need to double-check the prescription and the existence of advice records on the website of the hospital. conclusion: this audit is an approach to increase the safety of the use of high alert medications. it will be completed a second time, by an evaluation of prescriptions collected and the storage conditions of potassium chloride in the care units. please specify your abstract type: research abstract background and objective: data listed behind each unit dose of a primary packaging of a pharmaceutical product are essential for a safe identification for the patient. however, the last medical services of the lausanne university hospital where nurses remove the solid form drugs (sfd) from their blisters when they prepare in advance the week container were in the vaud's prisons. the aims of the study were: ( ), quetiapine ( ) and ibuprofen ( ) and were psychotropics ( . %). part . the four data identified as essential: brand name, dosage [mg], batch number, expiration date. the sfd unit doses were classified as green when the blister included four data, yellow with two or three and red with less than two. of the sfd in cupboards, were green ( %), yellow ( %) and red ( %); an infovigilance was sent to each manufacturers. part . potential barriers identified: trays' sizes and space in drug's cupboards; preparation time to cut versus to remove the blisters; risks of self/hetero-aggression with pre-cut blisters; drugs packaged in bulk; multidose liquid medications. using containers larger than is usual was rarely necessary; space in cupboards was sufficient. the preparation time gradually decreased during the study. ingestion or aggression with pre-cut blisters was considered as limited, based on literature and experiences of two others prisons (geneva; lyon). for bulk sfd and multidose liquid drugs: proposals to the pharmacy to store some alternatives blistered sfd; blistering expensive bulked drugs; availability of the entire package delivered to inmates. the pilot phase was initiated in may . conclusion: a majority of inmates takes a drug treatment. half of sfd unit dose is identifiable (trade name and dosage) but an effort from manufacturers would better secure the drug supply chain. the study of the barriers helped to further implement the pilot phase. since early , none of the five prisons medical wards are removing the blisters and no incident was reported. please specify your abstract type: research abstract background and objective: nefopam is a widely used antalgic in hospital. its use is contraindicated in the epileptic patient as it results in lowering the epileptogen threshold and is likely to trigger epileptic seizures. the clinical pharmacist should systematically warn the prescriber against this contraindication when analysing prescriptions. following the onset in our establishment of an epileptic condition in a patient treated with nefopam, who had not been subject to any pharmaceutical intervention (pi), we set about analysing the validation practices regarding this contraindication and possibly implementing actions designed to improve those practices. setting and method: retrospective collection over a period of months of prescriptions for patients hospitalized in hospital beds with clinical pharmacy service (associating med-reconciliation, checking prescription according to medical file and participation to medical rounds): orthopaedic surgery, hepatic-gastro-enterology, general surgery, liver transplant and chest surgery. records of patients with nefopam prescription associated to medication belonging to the therapeutic class of antiepileptics were consulted with a view to finding cases of epilepsy. the pharmaceutical alerts were extracted from the pharmaceutical software. main outcome measures: number of epileptic patients treated with nefopam, number of pharmaceutical interventions issued when prescribing nefopam in epileptic patients. the study focused on , patients. ( . %) of them were prescribed nefopam, and ( . %) of them were prescribed nefopam associated to medication belonging to the therapeutic class of antiepileptics. after analysis of the patients' records has shown that of them were really epileptic. only pi's were effected ( . % of problematic prescriptions), and ( %) of them had an immediate prescription change. . % ( / ) of the patients have a pi in medicine services compared to . ( / ) in surgery services (p \ . ). the results of this study show that % of the contraindications related to the use of nefopam in epileptic patients are not reported to the prescriber. these results will be presented to our pharmacists so they can take them into account. subsequently a new study will be conducted to measure the relevance and efficiency of this program. hélène dewaele * , anne-laure raso, emmanuel cirot, marion collignon, laura foucault, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) has been demonstrated to reduce drug-related problems in inpatients. in our university hospital, mr has been performed for beds for years at the same time as prescriptions review. the aim of this study was to assess the impact of mr on pharmaceutical interventions (pi) during prescriptions review. design: a -month prospective study in orthopaedic surgery, hepato-gastro-enterology, general surgery, liver transplant and chest surgery was conducted. during medication review all pis were collected and those related to mr (rpi) were identified. thereafter for each patient we collected age, type of hospitalization unit (med or surgery) and for pis the drug associated and its acceptance by the medical team. results: during the study patients had a daily prescription review. patients ( %) had at least one drug-related problem. lines of prescriptions were mentioned to have at least one rpi. rpi represent % of drug-related problems. ( %) discrepancies were corrected by prescribers. the age of the patient was significantly different between patients with rpi (mean age: years old) and with pi (mean age: years old; p \ . ). the type of unit did impact the percentage of prescriptions with drug-related problems (medicine: . ; surgery: . ; p \ . ), the rate of corrected pi (medicine: %, surgery: %, p \ . ), but did not impact the rate of corrected rpi (p = . ). in surgery units the rate of corrected rpi ( / ) is significantly higher than corrected pi ( / ; p \ . ). medicines belonging to the four classes of: digestive and metabolism system, blood and blood flow, cardiovascular system, neurological system represent more than % of all the medication concerned by a resolved pi or rpi. the proportion of medicines from the digestive and metabolism class is the only class among those four that is not significantly different between resolved pi and rpi. conclusion: mr highlights a large number of discrepancies in inpatients. a modification of prescriptions due to mr occurs in % of the patients. in surgery units, these rpi are more frequently taken into account than drug-related problem warned by pis. indentifying patients for whom mr has the bigger impact could help us to reinforce our actions. please specify your abstract type: research abstract background and objective: diabetes is very frequently causing cardiovascular complications, thus impairing various systems and organs. therapy for these multiple conditions has to be revised and improved constantly. the aim of this closed retrospective study lead in bucharest emergency clinical hospital was the assessment of some of the diabetes mellitus (dm) complications and the related medication. setting and method: data was collected from cardiology, neurology, gastroenterology, internal medicine wards from bucharest emergency clinical hospital. only patients diagnosed with type dm were included in the study. there were analysed records from patients aged - of whom were men, following the presence, signalling and monitoring of diabetic nephropathy and arteriopathy. main outcome measures: we investigated the relationship between diagnosis and/or biochemical signs of kidney disease (serum urea, serum creatinine levels), diagnosis of arteriopathy, and the drug therapy administered in the respective cases. we also assessed the sex and age distribution of the patients diagnosed with diabetes mellitus and facing at least one of its complications. results: kidney disease, as a dm complication, was present in % of cases, patients aged - , of whom % were men. patients received diuretic treatment, of them being given hydrochlorothiazide, contraindicated in dm because of its hyperglycaemia-inducing effect. of the patients, had high serum urea levels ([ mg/dl), had high levels of serum creatinine ([ . mg/dl), and presented risen levels for both, but only were also diagnosed with kidney disease. patients with kidney disease were given furosemide, known for altering the renal function. circulatory failure was found in % of the patients, aged - and % of subjects, aged - , had both diabetic complications. conclusion: the present study emphasizes the role of the clinical pharmacist in adapting the medication of the diabetic patient, an inappropriate pharmacotherapy worsening dm complications. this is essential especially for elders, where polypathology and polymedication lead to a significant increase of dm complications risk. hp-pc : epileptic seizure after treatment with thiocolchicoside: discussion about a case report valérie dobremez *, , adeline martin-dupray , jacqueline berlioz , pierric giraud pharmacy, neurology, centre hospitalier annecy-genevois, metz-tessy, france please specify your abstract type: descriptive abstract (for projects) background and objective: thiocolchicoside is a semisynthetic derivate of naturally occurring colchicoside, which is largely used in humans as a centrally acting muscle relaxant. this compound also has anti-inflammatory and analgesic effects. the objective of this work is to report a recent case of serious adverse effect of thiocolchicoside occurring in context post traumatic brain damage without sequalae. design: a -year-old woman suffered from headaches and neck pain since days, she was treated with thiocolchicoside. she took mg in the evening and mg the next morning. five generalized tonic-clonic seizures, without recovery of normal consciousness between seizures, have occurred suddenly - min after the second administration. the patient was admitted to intensive care unit in order to control the epileptic seizures. a status epilepticus was diagnosed requiring intravenous drugs with clonazepam, phenobarbital and propofol. the patient was controlled and transferred in neurologic unit in order to complete paraclinical investigations. its main antecedent was a severe head injury at the age of years following a public road accident. the brain scan revealed an old frontal hypodensity. rest of etiological assessment was negative (lumbar puncture, no infectious disease), numbers were normal. the definitive diagnosis was a status epilepticus on post-traumatic sequelae, sensitized by taking a proconvulsant drug. a treatment with levitiracetam was initiated at mg twice a day. outcome was favourable with no recurrence months later, a recommendation was requested to pharmacovigilance. results: the muscle relaxant activity of thiocolchicoside results of an agonist action on glycinergic receptors located primarily in the brain stem and spinal cord. however, thiocolchicoside also acts as an antagonist of the gaba-a receptor (mainly located in the cerebral cortex), this pharmacological action can cause a proconvulsant effect. epilepsy is a very rare adverse effect, only few cases have been reported in literature. the epileptogenic activity of thiocolchicoside occur mainly in patients with a history of epilepsy, acute brain injury or possible blood-brain barrier disruption. the chronology is consistent with the responsibility of the drug as a promoting factor. pharmacovigilance retains after analysing drug causality. conclusion: the case history indicates that thiocolchicoside has a powerful epileptogenic activity. thiocolchicoside can precipitate seizures in predisposed patients, and that its use should be avoided in patients with brain diseases (and therefore lower seizure thresholds) or blood-brain barrier disruption. pharmacists could warn physicians and should verify the absence of notable history before dispensing thiocolchicoside. hp-pc : acute exacerbation generalized myasthenia after red yeast rice use: a case report valérie dobremez *, , amélie serra , déborah grosset-janin , jacqueline berlioz , aymeric dopter , jean-henri ruel pharmacy, neurology, centre hospitalier annecy-genevois, metz-tessy, nutrivigilance, french agency for food, environmental and occupational health and safety, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: many drugs can induce acute exacerbations or reveal myasthenia gravis. self-medication or complementary and alternatives medicines expose patients. the objective of this work is to report a recent case of acute exacerbation of myasthenia gravis because of a dietary supplement use. design: intermittent vertical diplopia and ptosis of the left eye settled in a -year-old man. its main antecedent is hypertension treated with perindopril. the neurovascular origin was ruled out. the electromyogram (emg) found a significant decrement ( %) of a postsynaptic block in the tongue and right orbicularis muscle. acetylcholine receptor-antibodies were positive. myasthenia gravis was diagnosed (osserman score / ) and the patient was treated with pyridostigmine. the identification of carotid atheroma required a treatment with a statin that the patient refused. he preferred a cholesterol lowering dietary supplement, containing red yeast rice. six days later, he was hospitalized for an acute decompensation of myasthenia with bilateral ptosis, oculomotor paresis, drooping head, int j clin pharm ( ) : - chewing trouble and dysphagia (osserman score / ). the patient is treated with high-dose intravenous immunoglobulins then corticosteroids. the dietary supplement is stopped. an opinion was requested to the clinical pharmacist of neurology. the osserman score gradually increases to / . results: red yeast rice contains a range of compounds known as monacolins, of which monacolin k-renamed lovastatin, which was found to be an inhibitor of cholesterol synthesis and the progenitor of the statin family. a literature review has highlighted the responsibility of statins in acute exacerbations or reveal myasthenia gravis occurrences. in this case, the chronology is consistent with the responsibility of red yeast rice. the case was reported to the french system of nutrivigilance, which retained after analysing a probable intrinsic imputability score. conclusion: dietary supplement with red rice yeast are not recommended in case of myasthenia gravis. this is the first case of acute decompensation of myasthenia recorded with red yeast rice in the french system of nutrivigilance. multidisciplinary collaboration (neurologists, clinical pharmacist) has optimized the patient management. fanny durand * , camille lambert, antoine dupuis please specify your abstract type: descriptive abstract (for projects) background and objective: development of computerized prescription highlights the need to harmonize pharmaceutical analysis practices. the aim of this study is to analyse the antibiotics prescriptions in the treatment of urinary tract infections, to develop a pharmaceutical validation tool. design: a prospective observational study was conducted for one week, in care units. pharmacists, interns, and pharmacy students were trained on spilf (french society of infectious pathology) recommendations, on pharmacist's role in the management of urinary tract infections, and on the data collection. all patients with antibiotic prescription for urinary tract infection were included. some data were collected: reason for hospitalization, clinical signs, results of susceptibility testing, risk factors for complications (organic or functional abnormality of urinary tract, male, pregnancy, elderly, severe immunodeficiency, severe renal impairment) and signs of severity (severe sepsis, septic shock, interventional surgical drainage). then, the treatments prescribed to the patient, probabilistic on the one hand and documented on the other hand, were compared to spilf recommendations. finally, during a multidisciplinary meeting (pharmacist, expert in infectious diseases), we selected the relevant pharmacist interventions. results: twenty-three patients were included ( women, men), % had a urinary catheter. . % of prescriptions were concordant with spilf recommendations: probabilistic and documented treatment, and duration. among the non-conforming prescriptions, nine pharmacist interventions have been formulated: four prescriptions did not specify the duration of treatment, one antibiotic was prescribed on an insufficient period, two cases of severe acute pyelonephritis without prescription of aminoglycoside, one prescription was not reassessed according to results of susceptibility testing, one pregnant woman with urinary colonization without clinical signs, was treated before obtaining results of susceptibility testing. three cases of poor management are identified: two cases which treatment began only after results of susceptibility testing (a urinary tract infection linked to care, an acute pyelonephritis with complication risk), and a cystitis treated with nitrofurantoin while the germ was resistant. conclusion: a synthetic tool was created. there are three elements for helping pharmaceutical analysis: the questions to ask oneself facing a prescription of antibiotic for urinary tract infection, a flowchart to identify the recommendation adapted to the case, and finally a summary table showing spilf recommendations. this tool will be distributed and evaluated. hp-pc : off-label use of rituximab in refractory antisynthetase syndrome (as) through a long-time experience in a neuromuscular diesases center lise durand * , carole metz, patrick tilleul, helga junot pharmacy, gh pitié salpêtrière, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: as is an idiopathic autoimmune inflammatory myopathy, characterized by presence of antisynthetase antibodies: anti-jo , anti-pl , anti-pl . patients are usually first treated by corticosteroids (cs) or immunomodulating drugs. rituximab (rtx) has become another option for refractory as, supported by few uncontrolled studies . because of its off-label use, our hospital pharmacy has implemented a controlled drug delivery. this work assesses a -years follow-up of patients treated by rtx and the resulted drug costs. design: patients registered in our database since december who received c injections of rtx to treat as, were analysed to describe their eligibility criteria, conditions of management and the clinical and biological effects of the treatment (creatine kinase (cpk) used as biomarker). patient files were consulted to collect all individual data and pharmaceutical software was used to review deliveries. drug costs were also reckoned based on prices from french health insurance. results: for months, patients (median age (min-max): ( - ), % women) have been treated with rtx for refractory as, the majority with anti-jo antibodies ( ). all patients suffer from muscular and lung affections, particularly interstitial pneumonia. many are also living with arthropathies ( ) or cutaneous disorders ( ). cardiac involvement is seldom ( symptomatic patients). the mean age of diagnostic is . years and the mean treatment period is . years. the common treatment is g at day (d ) and d , then g all months. before rtx treatment, seven patients received c other drugs such as cs ( %), azathioprine ( %), methotrexate or mycophenolate mofetil ( %). prednisone and azathioprine are also prescribed with rtx respectively for and %. treatment is associated with cures of intravenous immunoglobulins for four patients. to date, median number of administrations per patient is ( - ), d and d included. all patients have presented positive effects on both clinical and biological markers, mainly during the first months after treatment induction. wilcoxon tests show a significant difference in cpk level between d and m , also between d and the last known result. today, three complete remissions are specified in patient file; only one hepatitis b virus reactivation is reported. since , budget impact due to drug cost amounts to €. conclusion: whereas the use of rtx is controverted for treatment of all types of myopathy, as could have one of the best response . our cohort shows real clinical results and positive effect on usual biomarker. our experience demonstrates the safe and successful use of repeated administrations in refractory as. however, there is a need for further controlled studies to assess the efficacy/safety of rtx and to define its place in the strategy in view of its cost-effectiveness ratio. the pharmaceutical controlled drug delivery has to be continued to supervise, support and document its proper off-label use. please specify your abstract type: descriptive abstract (for projects) background and objective: as a part of the national patient safety program, the northern norway regional health authority are implementing new procedures for medication reconciliation (mr) in hospitals in the region. the procedure defines that mr is the doctor's responsibility and describes how it should be performed. the aim of this study was to investigate whether the implementation of the procedure reduces medication discrepancies (mds) in the charts at bodø hospital. and . % ( / ) of the patients died before discharge. parenteral nutrition was administered an average of . days ( % ci . - . ), of which . ( % ci . - . ) were with ipn. previous spn had been administered in . % ( / ) of the patients. before beginning ipn, the average triglycerides level was . mg/ dl ( % ci . - . ) but at the end of the ipn it was . mg/ dl ( % ci . - . ), which lead to a mean reduction of . mg/dl ( % ci . - . ; p = . ). regarding to the total amount of lipids provided with parenteral nutrition, with ipn there was a mean reduction of . g ( % ci . - . ; p = . ) comparing to those administered with spn. conclusion: usage of ipn in critically ill patients with htg permits to adjust parenteral nutrition formulations to meet specific nutrition needs, enables to reduce the total amount of lipids administered and, therefore, it allows to significantly decrease triglycerides levels. jennifer a. esteban gonzález * , elisabet nogué pujadas, angels andreu crespo, xavier bonafont pujol, nuria romero pascual please specify your abstract type: descriptive abstract (for projects) background and objective: the incidents involving patient misidentification (pm), or wrong patient medical errors (wpme), are medication errors (me), near-miss or close-call situations which can pose a considerable threat to patient health. pm may be under-reported due to the unawareness of the error or the difficulty of identifying them. the aim of this study is to describe the incidence and categories of wpme in a university hospital. design: observational, retrospective analysis of the voluntary reported wpme in the pharmacy database since march until june . these were classified in prescription, transcription, dispensing, administration and drug system errors. in addition, the national coordinating council for medication error reporting and prevention (nccmerp) taxonomy was used for classifying me according to the severity of the outcome. results: of me registered, of them were wpme ( . %). . % of them were due to prescription errors, which consist on wrong labelled medical orders, intermingled patient prescriptions or patient misidentification in computerized physician order entry (cpoe). the administration errors supposed a . % of the total amount of wpme and dispensing errors were . %. % of wpme were transcription errors, which occurred previously to the implementation of cpoe, and the remaining . % were system errors after cpoe. the wpme reported took place in the hospitalization wards ( . %), pharmacy ( . %), outpatient services ( . %), intensive care unit ( . %) and day-care hospital unit ( . %). . % occurred at working days and . % at the weekends. wpme were notified by pharmacists ( . %), nurses ( . %) and physicians ( . %). referring to the classification according to nccmerp, . % of wpme didn't reach the patient (category b) whereas . % reached the patient but didn't cause harm (category c) and . % required patient monitoring (category d). the remaining wpme ( . %) caused harm to patients and required medical intervention (category e). finally, in int j clin pharm ( ) : - more than half of wpme ( . %), reporters suggested measures to prevent these errors. conclusion: wpme represents near % of total me reported in our hospital. given that more than % reached the patient, safety measures must be implemented to reduce the risk of hazardous events. additionally, further encouragement in notification is necessary in order to improve patient safety. results: two men diagnosed with rrms aggressive evolution were included in the study. age: and . both of them without any treatment by the time they started being treated with alemtuzumab (previously one of the patients had been treated with fingolimod, suspended by inefficiency). the protocol design for the elaboration and control of alemtuzumab in the pharmacy service ensures greater safety and represents a saving strategy. in addition, the development of the protocol in the electronic prescription system (silicon Ò ) facilitates the prescription, proper administration and standardization of treatment among patients. the protocol includes daily alemtuzumab infusion for days and other necessary medications including premedication (metylprednisolone, omeprazole, paracetamol and metoclopramide) and anti-infective prophylaxis (aciclovir). developed adverse effects during infusion were skin erythema, pruritus and fever. it was not necessary to stop the alemtuzumab infusion in any patient. during treatment, one patient developed a severe lymphopenia and upper respiratory tract infection (influenza a). conclusion: the role of the pharmacist is critical at various stages, from the preparation and the administration guidelines, to detection, monitoring and reporting of adverse effects. alemtuzumab is presented as an alternative for those patients who do not respond to standard therapies or who have rapidly evolving severe rrms. because of its mechanism of action it is important to closely monitor patients, with particular emphasis on prophylaxis of possible infections. hp-pc : descriptive analysis of patients receiving oral anticoagulation following acute coronary syndromes sadeer fhadil * , paul wright, sotiris antoniou please specify your abstract type: descriptive abstract (for projects) background and objective: triple therapy with concomitant anticoagulant and dual antiplatelet therapy (dapt) following acute coronary syndrome (acs) increases bleeding risk by % compared to patients on dapt. bleeding post acs increases mortality and reinfarction risk; balancing ischemic and bleeding risks is particularly challenging in this population. european society of cardiology (esc) produced a consensus document, providing guidance for patients presenting with acs requiring concomitant anticoagulation; however optimal duration of triple therapy and safety and efficacy of novel oral anticoagulants (noacs) and more potent antiplatelet agents requires further evidence. design: a registry was collated of patients presenting with acs requiring concomitant anticoagulation. baseline characteristics, bleeding and ischemic risk scores, periprocedural treatment and antiplatelet/anticoagulant choice and duration was recorded and analysed for trends in prescribing. results: patients have been included in the registry between oct and june , of which ( %) were naïve to anticoagulation prior to admission, ( %) were taking warfarin and ( %) were on noacs. atrial fibrillation (af) accounted for ( %) cases, (average chadsvasc score of , hasbled score of ), and ( %) were for lv thrombus. of those naïve to anticoagulation, ( %) were initiated on warfarin and ( %) on a noac (last patients all received noacs). of those on a noac for af, ( %) were dose reduced on triple therapy; apixaban being the most commonly prescribed ( % apixaban, % rivaroxaban, % dabigatran). background and objective: solid oral formulations are more convenient than liquids to manufacture, store and administer for most adults. given this superiority, one would think that children were prompted to use solid formulations when available in an eligible dose. there are indications, however, that the conversion from liquid to solid formulation in children is influenced by characteristics of the liquid medication, rather than the child's ability to swallow solid medications. the aim of this study was therefore to explore if the proportion of oral liquid formulations differed between antibiotics commonly used for upper respiratory tract infections (urti) in hospitalized children. setting and method: we collected the sales data for for the children's department of the five university hospitals in norway. the three most common oral antibiotics used for urti in children were included: penicillin v, amoxicillin and erythromycin. the proportion of oral liquids was calculated by dividing the number of defined daily doses (ddd) of liquids by the total oral ddds for each substance. main outcome measures: the proportion of ddds of oral liquid antibiotics. results: a total of ddds of common oral urti antibiotics were sold in , distributed as % erythromycin % amoxicillin and % penicillin v. amoxicillin had the highest proportion of liquid with %, followed closely by erythromycin at %. in contrast, only % of the ddds sold of oral penicillin v were liquids. conclusion: higher proportions of liquid amoxicillin and erythromycin compared to penicillin v were sold to children's departments in hospitals. there are several limitations regarding the quality of sales data, as we lack information of the administered doses as well as the child's age, gender, infection and specific needs. infections in hospitals often require initial intravenous treatment, and oral switch will often be based on the initial treatment. despite these limitations, the results fit well with earlier findings which indicate that children prefer liquid amoxicillin and erythromycin to penicillin v. hp-pc : proactive medication reconciliation: a preliminary study to identify barriers before its implementation in surgery departments laura foucault * , marion collignon, hélène dewaele, anne laure raso, emmanuel cirot, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: it's well known that medication reconciliation (mr) decreases drug-related problems at patient admission (pa). in surgery departments, for planned hospitalizations, mr is performed - h after the pa (pourrat x and al, ). during this period, some chronic treatments are unintentionally not prescribed to patients. the aim of proactive mr (pmr) is to anticipate the pa by collecting their medication history before their hospitalization. the objective of this study was to identify the barriers preventing pmr implementation in our hospital. design: one week prospective study in digestive and orthopaedic surgery units in a beds' university hospital. the main outcome is to identify which barriers prevent the collection of mr before pa including the evaluation of time required to collect the relevant information, reconcile any discrepancies after the pa and identify the right sources from which to perform the mr. results: eighteen patients with a median age of years old ( - ) were contacted by phone one week before their scheduled surgery. these calls were conducted by pharmacy residents mainly between and p.m. (a more practical time for patients and at the end of pharmacist's routine tasks). an average of . ( - ) calls per patient were conducted. one patient was unreachable by phone. the average duration of the calls was min ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . twelve community pharmacy (cp) were contacted. in all cases, cp have accepted to share information about the patient's prescriptions by phone and sending it by fax during the day. five pharmacists were not contacted because patients had no chronic treatment and consequently no regular cp. on lines of prescriptions, discrepancies between the patient's information and prescriptions were identified and between prescriptions and the anaesthesia records. drug history was reported in the patient's records by pharmacy students on the day of pa in order to be used immediately by prescribers. surgery was cancelled for one patient. conclusion: the first step of an mr is made by a hospital anaesthetist some weeks before hospitalization but we have demonstrated that this step is not able to avert all potential errors. our study highlights that the time necessary to perform an mpr appears to be shorter than for an mr. in fact, it's sometimes difficult to properly interview patients during hospitalization (patient in operating room, drug-induced drowsiness). additionally, a key hurdle is to obtain any necessary modification of the prescriptions by surgeons. pmr can be expected to produce time saving efficiencies given that at pa, prescribers will have their full medication history. this study also allowed us to highlight the good cooperation between patients, cp and the hospital. it is worth noting that efforts were made to accommodate the schedules of a majority of working patients. however, as we would expect pharmacy student to perform the pmr, they will most likely attempt to contact patients during standard working hours which may impact the number of patients they are able to reach. laura foucault * , hélène dewaele, marion collignon, emmanuel cirot, anne laure raso, xavier pourrat please specify your abstract type: descriptive abstract (for projects) background and objective: the french legislation has clearly defined and integrated the therapeutic education of patient (tep) for healthcare professionals. the pharmacist is invited to get involved in tep as a caregiver around the patient. in our study, we are investigating how the pharmacist's role is viewed by patients with chronic diseases that are included in a tep program. design: prospective study on patients included in a tep program (chronic inflammatory bowel diseases, rheumatoid arthritis, ankylosing spondylitis) between september and april . in july , the participants of group sessions (gs) conducted with health professionals, including a pharmacist, were interviewed on the phone. the principal outcome of the interviews was to evaluate how their view of the involved health professional's roles evolved before and after gs; to evaluate if they would consider being followed by their pharmacist for individual sessions (is) in a community pharmacy (cp); and if the information supplied by the pharmacist during gs was understandable. to health care. however, discussions between patients appear to be essential to facilitate their acceptance of a chronic condition. some patients also questioned the cp's skills and knowledge when it comes to their particular disease. nevertheless, . % of patients have found that the vocabulary and documents used by pharmacist during gs was adapted and that the information supplied was very useful. conclusion: this study highlights that although the pharmacist is the drug's specialist, a majority of patients will more likely ask their physician about medication. their participation to the gs hasn't changed their habits even if the pharmacist intervention was relevant and understandable. the fact that the pharmacists took into account the level of health literacy of each participant was an appreciated aspect. cp should be more proactive in their relationship with the patients in order to highlight their skills and the assistance they can provide in a chronic disease. however, it's important to take in consideration that in some cases, patients have lived with their disease since childhood. the role of is is likely to be much more limited than in other situations given their key need is to interact with patients afflicted with the same condition. hp-pc : use and safety of trastuzumab emtansin in her + metastatic breast cancer in a tertiary hospital c. chaguaceda galisteo * , alba manzaneque gordon, héctor josé del río torres, natália creus baró please specify your abstract type: descriptive abstract (for projects) background and objective: novel anti her drugs have changed the management of her + metastatic breast cancer patients. the aim of this study is to describe the use of trastuzumab emtansin (tdm- ) in clinical practice in a tertiary hospital and to evaluate its safety profile. design: we performed a retrospective study of patients who started tdm- between january and december . we recorded demographic data, clinical and treatment variables, number of doses received, reasons for discontinuation, progression-free survival (pfs) and adverse effects (aes). data were obtained from the chemotherapy prescription program and medical records. aes were classified according to the common terminology criteria for adverse events version . of the national cancer institute. results: eleven female patients with a median age of . years [ . - . ] and an ecog ( / ) were included. tdm- was prescribed as a third or further line treatment in / patients and as firstline in one patient who develop disease recurrence within months of completing adjuvant therapy. median number of tdm- cycles was . . all treatments discontinuations were due to disease progression ( / ). pfs was . [ . - . months] (patients that received less than three cycles were excluded (n = )). most frequent aes were plaquetopenia, neutropenia and transaminitis but only grade in three patients (two transaminitis and one neutropenia). conclusion: the lower pfs obtained comparing to the pivotal study ( . vs. . months) could be explained by the later use of tdm- in clinical practice ( / patients received tdm- as third or further line while % in the pivotal study were first or second line). tdm- safety profile was according to the summary product characteristics. few data are currently available regarding the use of tdm- in clinical practice. further data are required to position this drug in clinical practice. please specify your abstract type: descriptive abstract (for projects) background and objective: the hospital pharmacist for their specialized training in the area of medicines, possess a greater responsibility in the detection and reporting of adverse drug reactions (adrs), as well as other problems related to treatment, which may be subject to monitoring and reporting to the regulatory authorities and the respective laboratories. thus, the pharmaceutical services of the cuf infante santo hospital has implemented a pharmacovigilance program, with two main objectives: . optimization of the detection and reporting of problems related to therapy; . implementation of corrective and/or risk minimization measures. the pharmacovigilance program is based on the following methodology: . detection of adrs/problems related to therapy/medical device: the detection can be performed by the pharmacist or other health professional that guides the process to the pharmaceutical services. . information processing by the pharmaceutical services and realization of spontaneous reporting: the notification is performed both for the portuguese regulator (infarmed) as to the appropriate laboratory (if applicable). after evaluation by both entity, the conclusions are communicated to the pharmaceutical services, which has the responsibility to share it with all the other hospital services. . report of the event in the internal risk management platform: when applicable, the pharmaceutical services internally report the adverse event to the hospital's risk management department, leading to an internal evaluation of the current process. . completion of the process and implementation of corrective measures: when the regulatory authority and/or the laboratory sends the report/technical advice about the notification, the pharmaceutical service in partnership with the risk management team perform a reassessment of the whole process. if needed, corrective and/or monitoring measures are implemented. . monitoring of implemented measures: after the implementation of corrective and/or monitoring measures there is a period of evaluation. results: the implementation of this program for the period of year, has led to a total of fourteen spontaneous reports. from all of these notifications, seven were related to quality defect of medicines, four were of adr, one was due to suspected lack of therapeutic efficacy, and lastly, one of the notifications was medication error derived. conclusion: the obtained results, over a year period, by the pharmacovigilance program were satisfactory but the aim of the pharmaceutical services is to consolidate and optimize the same program with a view to achieving better results. the pharmaceutical services will continue to take responsibility for the pharmacovigilance circuit management in this hospital, by promoting a proactive approach to monitoring the safety, quality and efficacy of medicines, which possess the primary objective to patient safety assurance. please specify your abstract type: descriptive abstract (for projects) background and objective: for prematures, parenteral nutrition (pn) is essential for medical care but is complex (specific needs, daily change of intakes…). now, the software logipren Ò , developed by the french society of neonatology, allows the prescription of pn as well as all the childish therapeutics. it is also in link with our production robot (baxa pomp) for individual pn bags. our objective was to integrate this software while optimizing our pharmaceutical validation process. design: the software implementation was lead by a physician/ pharmacist collaboration with several preliminary steps: • identification of pharmaceutical validation settings (pertinence of individual pn vs. industrial bags, parenteral approach, elements…). before the life-sized use of logipren Ò , a base test has been experimented to identify possible difficulties and to realize some correctives actions of the software or our process. results: logipren Ò leads us to a change in our pharmaceutical validation process, by introducing new elements: • the pharmaceutical validation of pn bags is done in collaboration with the physician, during the prescription step. • all the therapeutics are known, which allow the pharmacist to take into consideration all the intakes (micro-nutrients, vitamins…). • remove the transcription step of pn bags in our production software (abacus Ò ) thanks to an interface with our production robot. • less production problems because of the coverage of those pharmaceutical aspects during the prescription. since months, this reorganization helped us to propose pharmaceutical notices for prescriptions: • omissions (remove lipids, levocarnyl Ò , micro-nutrients, electrolytes, remove industrial bags…) • modification (reduce proteins according to urea level, micronutrients and electrolytes posology, duration of lipids infusion…) conclusion: the implementation of logipren Ò enabled us to reorganize of the pharmaceutical validation process with a consolidation of the role of the pharmacist during the prescription step, in the paediatric ward. it had a beneficial aspect by the reduction of the validation and production time, a decreased risk of error (suppression of job interrupts and better communication) and an improved production by the end of transcription step to abacus Ò . furthermore, during our experimentation, we could bring to the software editor new ways to improve it and make it more efficient. % ( . % in ) , difficulties in swallowing/psycho-behavioural distress in . % ( . % in ) , and rejection of oral drug in . % ( . % in ) . physicians and nurses indicate the reason in the medical record in . % of case versus % last year. this year, drug were crushed versus drugs in : % concerned nervous system group (vs. % in ), % concerned cardiovascular system group (vs. % in ) , and % concerned alimentary tract and metabolism group (vs. % in ) . nurses use guideline in % of cases versus . % last year. as the previous year, in % of cases, washing hands before preparation and after administration are met. last year, none of them was wearing mask and gloves during this operation while this year, % was wearing mask and gloves. finally, in the two assessment, for each patient, drugs are systematically crushed together and then mixed with the patient's meal. conclusion: this study shows that crushing drugs is still problematic in our units. however, best practices were observed, such as the indication of the reason of crushing in the medical record, or the consultation of guideline. a new training for nurses will be conducted to create awareness about risks of crushing drug. please specify your abstract type: research abstract background and objective: in invasive candidemia, three echinocandins are indicated: caspofungin, mycafungin and anidulafungin. the aim of this work is to establish which echinocandin to prescribe in a french university hospital, given the scarcity of available clinical data in the literature regarding obese patients. setting and method: in a french uhc with beds, a multidisciplinary working group composed of a microbiologist, an infectious disease specialist and a pharmacist has been set up to analyse the various therapeutic options. main outcome measures: analysis of the literature, pharmacoeconomic study. results: four medications have been identified as possible therapeutic options. their adverse effects are similar and their administration rhythm is the same. according to recommendations by the esmid ( ) and the idsa ( ), the level of evidence for these three echinocandins in initial treatment of candidemia is equivalent. concerning obese patients, no weight limit is mentioned, int j clin pharm ( ) : - despite recommended dosage adjustment. caspofungin must be prescribed at a dose of mg/day for patients weighing over kg. micafungin must be administered at a dose of mg/day regardless of patient weight. in the case of persistence of cultures or if clinical condition does not improve, the dose may be increased to mg/day. anidulafungin, which is not referenced in our establishment, must be prescribed at the same dose regardless of patient weight. from an economic point of view, in our hospital, micafungin at a dose mg/day remains the least costly therapy. however, if its posology is doubled as indicated, caspofungin then becomes the most economic therapy. amphothericin b, an optional treatment, is never the most economically advantageous therapy. conclusion: as a result of this study, the chosen prescribed therapy for obese patients is caspofungin at a dose of mg/day. this work has improved access to healthcare for obese patients. pharmacokinetics and survival data must be collected on the basis of various patient weights in order to predict clinical efficacy. kristin f. heier * , liv czynski please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of this study was to develop a system to prioritize patients for medication reconciliation by pharmacists in the emergency department. it also proved a useful setting for evaluating how other health care professionals perceived the role of the pharmacist performing medical reconciliations within the emergency department. design: the study was located in the Østfold municipal hospital, located in kalnes, norway. pharmacists used a prioritization model to identify ''high-risk patients'' having clinically relevant prehospital medication discrepancies between hospital admission records and the information obtained via medication histories, general physician referrals and nursing homes. pharmacists registered patient information such as age, gender and drug-related problems (drps). seventeen physicians and thirty nurses in the emergency department answered structured questionnaires anonymously. main outcome measures: • number of patients with medication reconciliation performed by a pharmacist. • number of drug-related problems denoted in the electronical journal and presented to the physician. • the overall experience physicians and the nurses had with pharmacists when located in the emergency department. results: pharmacists performed medication reconciliation for patients, identifying drps and potential drps in total. fourteen of the physicians had read the journal notes from pharmacist and found them helpful (n = , %) or greatly beneficial (n = , %). most physicians (n = , %) and nurses (n = , %) reported a good cooperation with the pharmacist in the care of the patients. some of the physicians (n = , %) and most nurses (n = , %) wanted more information about the pharmacists work in the emergency department. the majority of ed staff ( % of physicians and % of nurses) found pharmacist as a good academic resource in the emergency department. conclusion: the physicians reported an improvement regarding the quality in the medication reconciliation made by pharmacists in the emergency department and both physicians and nurses expressed a need that pharmacists work in the emergency department on a more permanent basis. more information in general and especially better communication with nurses regarding the care of the patients are important actions need to optimise collaboration with pharmacist in the emergency department. results: a total of patients were included in the study, median age was years and % were males. they used in average four drugs regularly (range - ). almost three-quarters ( %) of the patients reported high or moderate adherence to all their regularly used drugs (mmas- c (max )). of the patients using oral spasmolytics, % reported high or moderate adherence to these drugs. the majority ( % of the patients) had high perceptions of necessity to their treatment (bmq [ . (max )), and % had a high level of concern (bmq [ . (max )). logistic regression analysis showed that there was no association between adherence and pain, nor between adherence and spasticity. younger age was found to be associated with higher risk of nonadherence. conclusion: even though overall adherence was high, the patients were more concerned to take their medicines compared to other patients with other chronic conditions. further studies are required for understanding adherence and attitudes toward medication in this population, and to help the patients feel safe about their medication regime. please specify your abstract type: descriptive abstract (for projects) background and objective: errors in medication lists often emerge in transition between health care levels, and there is need for strategies to communicate medication information. therefor we aimed to describe reasons why medication discrepancies (md) occurs in the transfer of patients between hospital and primary care service. design: in conjunction to a study based on use of structured medication report at transition from hospital to primary care service, we observed different reasons to why mds occurs. our observations and experiences linked to communication between health care levels is outlined. results: we observed that many md's disclosed at discharge could most likely be attributed to lack of medicines reconciliation at admission to hospital. for instance, several medicines were prescribed in primary care service prior to admission, but not at admission to the hospital. in addition, at admission, some medicines were listed as prescribed medications although not found in the medication lists in primary care service. we also observed that newly started and discontinued medicines were documented in the hospital discharge letter, but not implemented in primary care service. according to health care personnel in primary care service, insufficient communication about the patients' medications at discharge from hospital, led to corrections in the medication lists based on their previous knowledge about the patients. in addition, justified medication changes at discharge from hospital were not always implemented in primary care service due to professional disagreement. some stated that lack of trust was one reason for not always taking changes into account, often based on earlier experience. conclusion: these observations indicated that mds occurred both with and without intent when patients from primary care service were admitted to hospital and returned back due to poor communication. medication errors during hospitalisation and unproven intentional changes may be the consequences. due to this, it is important to improve the communication and confidence between professionals in the hospital and primary care service in order to reduce the number of mds and to enhance patient safety. please specify your abstract type: descriptive abstract (for projects) background and objective: intravenous human immunoglobulins (iv igs), plasma protein products, may cause in patient to a range of adverse side effects (headache, skin rash, kidney failure, thromboembolic event). in the framework of securing medicinal care, an assessment of professional practices has been conducted within our university hospital. the overall goal of this study is to evaluate the process of intravenous administration of human immunoglobulins done by the nurse staff. design: this prospective study has been carried out in three departments of neurology. an observation grid was established on the basis of guidelines on good practices. all in all, criterions have been examined resuming: prerequisites before administration, patient setup, iv igs administration, monitoring, traceability of drug delivery and management of adverse side effects. results: during the course of this investigation, administrations were observed. only % of nurses deliver information about the treatment to their patients before administration and % question patients about previous hypersensitivity reaction. the presence of spontaneous diuresis is verified in % of cases. emergency cart is not reachable in % of all cases. % of nurses ask patients to decline their identity. the use-by date on the bottles is checked in % of cases. at the time of preparation of perfusion, labelling does not mention either patient's name ( %) or date and hour of perfusion ( %). int j clin pharm ( ) : - during perfusion, only % of nurses follow diuresis and % watch rate of administration. hydration is not always kept min after the end of perfusion ( %). patient monitoring varies between min and h after perfusion's end. in % of cases, diuresis is monitored after the end of administration. % of nurses explain to patients side effects that may occur remotely. finally, administration traceability is was conform in % of all cases and in the event of adverse side effects, statement was made in % of cases. conclusion: best compliance scores have been achieved in myology department where patients are fewer than in the two others departments ( vs. and ). a presentation of those results will be given in theses three departments in order to improve patient management and securitization of iv igs administration. this audit will be carried out soon in other departments. please specify your abstract type: descriptive abstract (for projects) background and objective: a new human polyvalent immunoglobulins dose ( g) for intravenous administration is available on our establishment since . in order to secure the administration, this new dosage was initially reserved for the healthcares using administration pumps (being four health-care). the aim of this survey was to evaluate the satisfaction of the nursing staff already user of the new g dose and to estimate the motivation of the nonuser nursing staff by the audit date. design: this satisfaction survey was carried out with the most igiv consumer services (being internal medicine, neurology, cardiology and haematology). the questionnaire was structured in two sections: the first section regarding igiv in general, the second section concerning the new g dose. the survey included multiple choice questions or questions with answers based on a four levels evaluation scale (not satisfied, mildly satisfied, satisfied, and very satisfied). results: the audit was realized on eight health-care, involving nurses. among the interviewed, ( %) have already used the g dosage. in % of cases, users were very satisfied and % were satisfied. the most positive points noted were: gain of time provided ( . % of satisfaction), less manipulation needed ( . % of satisfaction), and reducing of infectious risk ( . %). moreover, the influence of the injection technique on users' satisfaction was further reported. indeed, according to nurses interviewed, the use of an injection pump is safer and improves the job comfort of nursing staff, unlike the injection by gravity (used in % of cases), which seems to slow down the use of this new dosage. in two cases, a positive opinion given by patient was also reported. finally, negatives points noted were related to administration instruments (use of pump or not) and to less flexibility in daily dose regulation. among the % not-user of this new dose, the % showed a strong interest for the product apart from services making the igiv administration by gravity. conclusion: in light of these results, the use of g dose will be spread to other services. the general diffusion of this dosage will provide a gain of time also at the pharmacy, during the unitary delivery and the computer-based administration of every units. a second survey will be soon effected within patients involved in the switch g/ g. the capital region pharmacy, clinic of neurology, rigshospitalet, blegdamsvej, copenhagen, denmark please specify your abstract type: research abstract background and objective: the clinic of neurology, rigshospitalet, copenhagen, denmark experience continuous medicine-related patient safety incidents (psi) related to newly admitted patients and patient transfers between wards. in order to prevent drug related problems (drp), the pharmacists increased their focus on these patients and provided systematic medication reconciliation. thus, the objective of this pilot study was to investigate if the intervention would help identify drug discrepancies (dd) and prevent drp. four wards were included in this study; two neurological, one neuro-anaesthetic (icu), and one neurosurgical ward(s). three wards use electronic medication module (epm), whereas the icu uses critical information system (cis). furthermore, all patients' prescriptions are registered on shared medication record (smr), which provides an overview of prescribed medicine. prescriptions cannot be transferred from smr and epm to cis and vice versa. we suspected that psi resulted from these system incompatibilities. setting and method: patients admitted or transferred from may nd to june rd were included. medication reconciliations using smr, epm and cis were conducted by a pharmacist on weekdays. dd were presented to a physician orally and documented. only dd accepted by physicians led to drug prescribing change. main outcome measures: number of identified dd. results: the study included patients, of which ( %) were newly-admitted. patients ( %) were transferred between wards. of the transferred patients, ( %) were transferred from the icu to other wards and ( %) were transferred from other wards to the icu. of the newly-admitted patients, ( %) were admitted to the icu and ( %) were admitted to other wards. the pharmacists identified dd; dd ( %) in the transferred and dd ( %) in the newly-admitted patients. in the transferred, dd were all related to the icu. in the newly-admitted, dd ( %) was related to the icu and dd ( %) to other wards. of the dds, ( %) were accepted by the physician. an example of a severe dd identified was an omission of prednisolone to a patient admitted to the icu. conclusion: most dd were identified in patients admitted to or transferred from icu, which uses the incompatible system cis. pharmacist systematic medication reconciliation helps identify these dd and prevent drp. please specify your abstract type: research abstract background and objective: antibiotic related drug interactions are more likely in intensive care unit patients due to common polypharmacy and antibiotic usage. the aim of this study is to determine the antibiotic related drug interactions with three different online databases (micromedex-paid, medscape-free and drugs.com-free) and to evaluate these interaction information by clinical pharmacist. setting and method: a retrospective, descriptive study was set up in hacettepe university hospital's intensive care units, between november and december , . patients who use at least one antibiotic were involved in this study. all drugs were assessed by each three databases and only antibiotic drug interactions were evaluated. clinical significance of identified drug interactions were evaluated by clinical pharmacist. main outcome measures: clinical pharmacist's assessment in significance of drug interactions indicated by three online databases. please specify your abstract type: research abstract background and objective: an implementation of clinical pharmacy practice by postgraduate students in intensive care units is a new way of learning in postgraduate education which creates opportunities in multidisciplinary collaboration in clinical pharmacy research, and also has influence on clinicians' routine patient care process. this system in educational program was ongoing in the department of clinical pharmacy since . as a part of this educational program, drug related problems in intensive care units were described and analysed, an influence of clinical pharmacy postgraduate students on patient treatment process was sought. setting and method: a prospective, cross-sectional study was performed between the march-june in hacettepe university hospitals, department of internal diseases intensive care units which consists of beds. three postgraduate pharmacy students from the department of clinical pharmacy, faculty of pharmacy conducted medication reconciliation in order to identify any problems in patients' medical orders. drug related problems (drps) were identified by the students and recommendations for management were approved by a supervisor of clinical pharmacy department before they were directed to physicians for approval. the students were not authorized to undertake any action in patient care process, therefore all required interventions for drp were undertaken by physicians and the acceptance ratio of the interventions were recorded. the pharmaceutical care network europe foundation classification system (v. . ) was used to asses drps. main outcome measures: determination of drps by pharmacists and evaluation of their interventions' acceptance by physicians in intensive care units. results: during the study period, patients were admitted to the intensive care units. each patient's medication orders were evaluated and interventions were recommended by postgraduate students. the number of interventions per patient was . . the acceptability rate of interventions by physicians was . %. in addition, physicians were provided drug information on seven different occasions. recommendations regarding drug therapy were mainly related with treatment effectiveness and adverse reactions. the common causes of drps were requiring dose adjustment due to pharmacokinetic problems ( . %), no therapeutic drug monitoring ( . %), inappropriate timing of administration and/or dosing intervals ( . %), requiring dose adjustment due to deterioration/improvement of diseases ( . %), inappropriate drug selection ( %) and new indication for drug treatment presented ( %). the most common drugs responsible for drps were ranitidin, levothyroxine, allopurinol, pantoprazol, piperacillintazobactam and vancomycin. the study showed that the most common drps was dose-related, therefore close monitorisation of the intensive care unit patients by students in clinical pharmacy postgraduate program can help physicians in terms of detecting, preventing and minimizing drps in order to improve patients' health outcomes. please specify your abstract type: research abstract background and objective: antibiotic stewardship is the process of salvaging important antibiotic agents from becoming ineffective due to bacterial resistance. this is important because throughout the world antibiotics continue to be one of the most important classes of therapeutic agents due to their vital role in saving patient lives. key goals of antimicrobial stewardship are to improve clinical outcomes, prevent antibiotic resistance, promote patient safety, and reduce health care cost. pharmacist are in the frontlines because they perform antibiotic stewardship activities, such as selecting the most optimal antibiotic agent, adjusting drug-dosage, and stopping use of unnecessary antibiotics. as a result of the continuous rise in antibiotic resistance and decline in development of new antibiotics, antibiotic stewardship programs are proving to be indispensable in a health care settings. setting and method: adult and paediatric inpatients receiving antibiotic therapy in the hospital medipol university has been evaluated. patients were selected randomly in the hospital system. patients were evaluated for antibiotic susceptibility results and compliance with antibiotic management guidelines. main outcome measures: to evaluate the antibiotic therapy in patients with culture results and to determine according the treatment guidelines. results: it was observed different pathogens in blood culture results of inpatients out of patients who were treated with antibiotics in hospital. antibiotic susceptibility results for acinetobacter spp, staphylococcus spp, enterococcus spp, pseudomonas aeruginosa, klebsiella spp, e. coli spp, streptococcus spp, corynebacterium spp, streptococcus pneumonia and enterobacter spp are evaluated in the study. klebsiella spp was the most isolated pathogen at total of culture results. most frequently resistance were int j clin pharm ( ) results: a total of ( %) questionnaires were completed. of these, approximately % were answered by hospital nurses, the remaining mainly by physicians ( %) and % ''other''. on the question ''what is your general perception of the benefit of the clinical pharmacy service; for collaborating health professionals? for the patient?'' the total benefit was ranked . and . respectively (scale from (''no benefit'') to (''beneficial to a very large extent''). the open questions: ''what disadvantages/advantages have you experienced by the introduction of clinical pharmacist into multidisciplinary teams?'' received / comments respectively. physical obstacles regarding office space, interference with the decision making process, more time consuming processes and the issue of relying too much upon the advices given was reported as possible disadvantages. respondents answered ''none'' to this question. the comments regarding advantages dealt mainly with general increased patient safety and quality assurance. in addition, advantages as work-load relieve, time saved, collegial support, practical help, and learning interchange between professions, were highlighted. conclusion: health-professionals assessed the clinical pharmacy service as highly beneficial. the advantages outlined were higher patient safety and quality regarding medication, in addition to collegial support, practical help and learning interchange. please specify your abstract type: descriptive abstract (for projects) background and objective: in june , the french health authority, the «has», published an index resuming the recommendations of benzodiazépines (bzd) prescriptions and proposing an approach to stop using it. indeed, it has been established that there is a too high and too long consumption of bzd in france. a study of prescriptions' prevalence has been done in our hospital centre. the aim of this study was to know our situation regarding the use of bzd in order to set up some improvements and take part in their proper use. design: a prospective study has been done on a months period in different services: geriatric, post-op and rehabilitation facilities, endocrinology, internal medicine, pneumology and cardiology services. the data were raised on a given day in each services and recovered thanks to the prescriptions software but also through interviews with the patients and their doctors. it was examined whether there was a bzd prescription (hypnotic or anxiolytic), whether the duration was superior or not to the duration of the amm and whether the prescription was done in our hospital centre. if the prescription was already part of the patient treatment, we looked if it was possible for the patient to stop using it, according to the has criteria. on their discharge, the letters and bzd prescriptions were also analysed and some patients' general practitioner were contacted after their discharge. results: patients (median age years old) were included from november to march . . % ( / ) of the patients had at least one bzd prescription the day we collected the data. we found only one bzd in prescriptions ( . %) and among them . % ( / ) were anxiolytic bzd. among those prescriptions, . % ( / ) already existed before the hospitalization and . % ( / ) were given during the hospitalization ( were prescribed automatically). . % ( / ) of the prescriptions did not respect the legal duration of the amm ( pieces of data were not found). . % ( / ) already exceeded this duration limit. among the patients who already had a bzd treatment before going to hospital, . % ( out of ) could consider stopping their use of bzd. by the end of this study, patients were discharged from hospital, among them . % ( / ) with a prescription of bzd. . % ( / ) of the prescriptions established during the hospitalization had been renewed when the patient came out of the hospital, we managed to contact ten general practitioners (approximately . days after their discharge), nine patients carried on their bzd treatment, among them one patient had reduced his consumption. conclusion: this study is an example of the high proportion of bzd prescriptions in france which the majority doesn't respect the legal length of the amm. the prescriptions of bzd in the hospital are generally systematically renewed by the general practitioners. the patients must be informed about the risks of using those molecules. in order to ameliorate this practice in the hospital, a proper use leaflet, reminding the prescriptions of bzd, has been created and distributed in each services to make people aware. main causes of admission were infections ( %) (respiratory disease ( %) and other ( %)), hepatic disease ( %) and neoplasias complications ( . %). patients died during their admission; due to hepatic disorder, due to neoplasia, and due to infections. conclusion: last diagnosis of hiv or no art treatment are causes of admission. immunovirological situation is related with their adherence but isńt with admissions. coinfection with hcv or hbv or others infections are risk factors for admission. center for psychopharmacology, diakonhjemmet hospital, oslo, norway please specify your abstract type: research abstract background and objective: complex medical history and treatment can potentially cause problems. the objective of this study was to investigate the prevalence of drug-related problems (drps) and medication discrepancies in internal medical patients with complex treatment at hospital admission. further, to investigate to which extent drps were identified as a result of medication reconciliation, and to which extent drps could be associated to the hospitalization. setting and method: patients with at least four regular medicines from two different therapeutic groups were consecutively included at admission to an internal medicine ward at a university hospital in norway in the period . . - . . . pharmacists used the integrated medicines management (imm) model for medication reconciliation and medication reviews at admission. a medication discrepancy was defined as any discrepancy between the recorded medication list at admission and the patient's actual use of medications, as revealed by medication reconciliation. the patients' actual use of medications, medical journal and laboratory results, were used to perform a medication review at admission time and identify drps. the proportion of drps revealed due to medication reconciliation was calculated. moreover, the project group retrospectively assessed possible drp-induced hospitalizations based on clinical history, cause(s) of admission and identified drps. main outcome measures: the main outcome was the median number of drps per patient at admission. the proportion of drps revealed due to medication reconciliation, the proportion of patients with drps possibly associated to the hospitalization, and the median number of medication discrepancies, were included as secondary outcomes. results: patients were included, . % women. median patient age was (range - ) and most of the patients were home-living before admittance ( . %). in total drps were identified at admission, with a median number of (range - ) per patient. drps ( . %) were identified due to medication reconciliation. for patients ( . %) a causal relationship between the hospitalization and the drps was assessed as ''possible''. medication discrepancies were revealed in of the included patients ( . %), with a median number of (range - ) per patient. conclusion: internal medical patients with complex drug regime are frequently exposed to drps and medication discrepancies at hospitalization. medication reconciliation could be essential to identify drps, which is likely a common cause of hospitalization in the studied patient population. hp-pc : assessment of oral anticoagulant prescriptions and pharmaceutical analysis at the hospital by regional audit damien fuss *, , clélia monchablon , anaïs breteau , marie lefebvre-caussin , rémi varin , jean doucet , mikael daouphars , doreya monzat omedit normandie -chu rouen, chu rouen, please specify your abstract type: descriptive abstract (for projects) background and objective: oral anticoagulants (oa) are the most common drug class associated with preventable adverse drug events in hospitalized patients that require optimizing the pharmaceutical analysis (pa) process. in this context, a regional audit was conducted on pa of prescriptions oral of oa. the aim of this study is to provide an overview of the treatment by oa in the hospital by evaluating the consistency of the oa prescriptions compared with national and european guidelines and evaluate the pharmaceutical interventions. design: this study is based on the collection of pa data (demographics, indication, posology, drug interactions, monitoring) as well as the collection of pharmaceutical interventions and discordance int j clin pharm ( ) : - between guidelines recommendations and clinical practice. the inclusion criteria were any patient treated with oa (vitamin k antagonists (vka), non-vitamin k antagonist oral anticoagulants (noacs)). included patients were followed minimum months. the primary outcomes include description of baseline characteristics of patients, the number of inappropriate prescriptions compared to the different clinical recommendations, the number of pharmaceutical interventions, the number of adverse drug reactions (adrs) related to oa use and the assessment of patient monitoring. results: during the -months study period, patients were included in six health institutions. the average age was years ( % of patients over years old) and % of the patients were women. % of patients had renal impairment. % of patients were treated with vka, and % with noacs. it was the first prescription of oa for % of patients ( % with vka; % with noacs). the most common indication was the non-valvular atrial fibrillation ( %). in this indication, % of patients had cha ds -vasc score c , and nearly % had a high risk of bleeding (has-bled score c ). drug interactions were observed, and adrs occurred related to oa. % of patients with an adrs had a has-bled score c . . % of prescriptions were considered inappropriate, including % noacs (no monitoring renal function in % of patients over years initiating treatment, inappropriate posology in %, and % of contraindications). the rate of pharmaceutical interventions was %. nearly % of the prescriptions were already adapted when the pharmacist was starting analysis. conclusion: prescribers are sensitized of the risks on the oa prescriptions, which explained the delay upon pa and low rate of pharmaceutical interventions. however, the high number of inappropriate prescriptions shows the necessity to improve the pa process on these drugs, particularly by actions on therapy initiation and patient monitoring, especially for noacs. for this class, the impossibility of assess the level of anticoagulation by laboratory monitoring requires appropriate initiation and monitoring, especially an assessment of baseline renal function. please specify your abstract type: descriptive abstract (for projects) background and objective: the development of bacterial resistance these last years is a public health major problem in the world and needs to implement actions. in france, the national drug safety agency has defined a list of ''critical antibiotics''. this list includes antibiotics particularly generator of bacterial resistance (amoxicillinclavulanate, cephalosporine, fluroquinolone) and antibiotics called ''last resort'' (antibiotics against gram-positive cocci, car-bapenem…). at our regional level, an evaluation of prescription of these critical antibiotics was proposed to all medical centers. the aim was to evaluate the quality of prescription of these critical antibiotics. design: the regional working group (pharmacists, infectious diseases physicians and biologists) had developed a collection grid including data on patients, antibiotics and four criteria: adequate molecule, compliance with medical prescriptions, duration of antibiotic therapy and reassessment at h. this is a prospective study proposed to all health institutions (public and private), which had to be completed on a given day in all care units and had to be conducted by a team of multi-professional evaluators. the study included a quantitative part (number of patients hospitalized in the audited units, number of patients receiving antibiotics and number of critical antibiotic prescriptions) and a qualitative part (adequate to the four criteria). results: response rate was of %. the study investigated on patients hospitalized in the audited units, including patients ( %) receiving antibiotics. among the patients, % were hospitalized in medical, surgery or obstetrics units we recorded prescriptions of ''critical antibiotics particularly generator of bacterial resistance'' ( % amoxicillin-clavulanate, % ceftriaxone, % fluoroquinolone and % other third-generation cephalosporine) and prescriptions of antibiotics called ''last resort'' ( % carbapenem). the average age of the population was . years (± years). sex ratio was . . % corresponded to curative use and % to prophylactic use. the expertise of infections diseases physician was requested in only % of the cases. the antibiotics were prescribed in majority to treat bronchopulmonary infections ( %), urinary tract infections ( %) and intraabdominal infections ( %). ninety-two percent of the prescriptions had a proper indication. % of the prescriptions complied to the guidelines. the duration of antibiotic therapy was adequate in % of the cases. only % of the prescriptions were correct according to these three criteria. forty-four percent of the prescriptions were reassessed and adapted by the physician. conclusion: this study is original because of its regional dimension and antibiotic analysis. the number of analysed prescriptions was significant with an overall proper prescription in adequate with the guidelines. however, actions must be implemented on duration and reassessment and adjustment of treatment. these results were presented to the participating hospitals. these three points will be reevaluated during a new regional audit. the criterion «no more psychotropic drugs» has been met in . % of assessment. otherwise, or more psychotropic drugs are prescribed in . % of assessment from the point of admission. the criterion «no more a benzodiazepine drug» has been met in . % of assessment. otherwise, more than one benzodiazepine drug is prescribed in . % of assessment from the point of admission. no contra-indication is detected in . % otherwise, a contra-indication between two drugs causing torsade de pointes is detected from the point of admission in this department. no more anticholinergic drug is prescribed in . % of assessment. according to the french criteria, one or more inappropriate drug is prescribe in . % of assessment. the most common inappropriate drug group prescribed was alimentary tract and metabolism drug ( . %) (the hospital at home team needs these class of drug) followed by nervous system ( . %) (prescribed at the point of admission) and by cardiovascular drugs ( . %) (prescribed at the point of admission). finally, the criteria «no more one non-steroidal anti-inflammatory drug» and «no illogical association» have been met in all cases. conclusion: this analysis shows that most of criteria for «assessment of prescription among elderly in a «hospital at home» department have been met. when one has not been met, either the hospital at home team needs the drug prescribed, or this drug have been yet prescribed from the point of admission in this department. this study could be used for the next certification. hp-pc : access to health care: case of autologous serum eye drops batiste martel, fabien lindenberg, camille castel, guillaume saint-lorant * please specify your abstract type: research abstract background and objective: autologous serum eye drops (ase), prepared from patient's serum, are indicated in the treatment of severe dry eye syndrome and defective epithelial healing. its in-hospital preparation within a controlled-atmosphere zone unable it to be dispensed by non-equipped hospital pharmacies. the aim of this study was to implement security measures to allow transport towards distant hospital pharmacies and all patients even those residing far from a regional university hospital (uh). setting and method: this study was conducted in a -bed french university hospital. patient blood samples were taken within the university hospital every weeks. serum was then biologically controlled (negative tests for hiv, hbv, hcv, tpha, vdrl). preparation was conducted days after blood sampling. sterile preparations were then stored at a temperature of - °c. studies showed that eye drops were stable days after being thawed. transport of eye drops to distant hospital pharmacies requires to be conducted under controlled temperature i.e. below °c, to ensure the stability of eye drops. these pharmacies are located close to patient's homes. the entire process was examined by a pharmacy team in order to study and secure each step, transport in particular. main outcome measures: validation of each step of the autologous serum eye drop dispensing process, from sampling to receipt by different hospital pharmacies, transport in particular. results: patients benefitted from the preparation. all patients resided more than kilometres from a uh. a follow-up form was completed to qualify dispatching and to trace each step during transport. a temperature sensor was placed inside the box. the receiving agent was required to stop and control the sensor. a double retrospective control was performed by a pharmaceutical team via the recording of temperature sensors. a second follow-up form was drafted in order to track dispensation reviews, ongoing dispensation and future planning. a patient information booklet was distributed to hospital pharmacies to inform patients about good practice concerning eye drops. conclusion: technological necessities concerning autologous serum eye drop preparation and transport limit access to health care. in this study, the role of the pharmacist consisted in reducing inequalities among patients residing at a distance from the only regional uh. the role of the pharmacist is to ensure absolute quality of preparation between the uh and the patient. hp-pc : computerized medication reconciliation: overview of pharmaceutical software used and support for development of integrated modules julie mocquard, anaïs berthe, elise rochais * , nicolas prévost, jean-claude maupetit, on behalf of centre de ressources régional en conciliation médicamenteuse omedit pays de la loire, nantes, france please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation aims to improve continuity of care for patients. in , a national survey identified barriers for implementation of this activity in france, among which computerized systems were judged unsuitable for hospital practices. in the absence of appropriate hospital information systems (his), medication reconciliation remains a time consuming process implying manual transcriptions, potentially leading to a lack of traceability and medication errors. the objective of the study was to assess the current his used in a french region including the integration of medication reconciliation into the software and to define courses of action to assist this integration. design: an online survey conducted in may was addressed to head pharmacists of the health facilities in the region, giving a total of head pharmacists concerned. it included questions on the software used by the health facility, the development of medication reconciliation and its traceability, formulation of operational requirements to the editors of software and availability of a module integrating medication reconciliation provided by the software. results: seventy-eight pharmacists ( %) participated in the study, with all types of health facilities represented: public hospitals, clinics, home health agencies, haemodialysis structures and after care and rehabilitation facilities. thirty different software were identified in the region. ( %) pharmacists planned to develop medication reconciliation in their health facility and ( %) were already carrying out this activity. within these %, medication reconciliation was conducted on paper only for ( %) of them, while ( %) were using a computerized system (patient file, pharmaceutical software, other) for traceability. the most widely used software in the region contains a module enable for computerized medication reconciliation, and three other editors are currently developing one. no development is scheduled for another three editors nonetheless commonly used in the region. ( %) pharmacists had contact with the editor of the software, and had given thought to the preparation of requirement specifications to the editor to develop an integrated module of medication reconciliation. conclusion: despite the interest attributed to medication reconciliation and despite the need of a fully integrated module of medication reconciliation to his, only a few health facilities of the region possess an appropriate computerized system to develop this activity. this study underlines the approaches already made by pharmacists to editors in order to integrate medication reconciliation to the his. subsequently, retrieving these approaches and writing specifications common to all health facilities is scheduled, in order to assist them in providing a strong incentive for the editors to integrate medication reconciliation to existing his. please specify your abstract type: descriptive abstract (for projects) background and objective: medication reconciliation (mr) is an interactive and multiprofessional process that ensures the continuity of care by integrating the ongoing treatment to the new hospital prescription. this helps securing the patient's care pathway particularly at transition points. the objective is to initiate the mr process in our medical institution with a pilot study in the department of internal medicineemergency downstream to validate a methodology and adapted tools. design: the mr takes place in three steps performed by a pharmacy student: ( ) realization of best possible medication histories (bpmh), combining at least three sources of information and using sources' collection form. this research begins with a patient interview done in pairs with a medical student using an interview guide. ( ) comparison bpmh with the initial hospital prescription in the department (after passing through the emergency department) on the treatment reconciliation form. a status is assigned to each line of drugs and then the differences are identified (stopped, changed or added). these two steps are validated by a pharmacist. ( ) discussion and characterization of observed differences (intentional/unintentional and documented/undocumented) with the senior physician. results: twenty-six mr were performed over weeks in . the mr is performed within days after admission. on average, . information sources per patient were used for the bpmh: mainly drug prescription (dispensed in pharmacies community); analysis of emergency medical records and patient interview. for the patients included, drugs were listed. discrepancies were observed and were studied (status stopped or changed only): one documented intentional discrepancy, undocumented intentional discrepancies and unintentional discrepancies (ud). these uds affected patients ( - medication errors per patient) and corresponded to a non-prescribed drug in % of the cases. vitamins, antihistamines, anti-reflux and proton-pump inhibitors were involved in % of cases; cardiovascular drugs in % and antiinfectious in %. through this pilot study, the methodology was validated: (a) need to have a minimum of three sources to achieve a relevant bpmh and to confirm each information with two sources; (b) need for a dedicated time with trained staffs; (c) development of tools to improve the traceability of information obtained from each source and traceability of medication reconciliation activity. conclusion: the mr establishment in the internal medicine department was helpful in identifying medication errors that have been corrected. it is proposed to archive the treatment reconciliation form in the patient file to contribute to the traceability of information on treatment. this study strengthens the deployment of this method and mr tools to other services of the hospital. alma mulac * please specify your abstract type: research abstract background and objective: clinical pharmacists have an important role in improving healthcare services. there is lack of knowledge of clinical pharmacists' experiences in interprofessional collaboration. our objective was to explore the challenges and barriers experienced by clinical pharmacists in interdisciplinary teams in norway and incorporation of expanded pharmacist roles in hospital settings. setting and method: this qualitative study was conducted using semi-structured interviews. a total of clinical pharmacists from four (government) hospitals were included in the study. the interviews were audio recorded using a digital recorder. the recordings were transcribed verbatim. main outcome measures: challenges and barriers clinical pharmacists experience in interdisciplinary teams in hospital setting. results: the main findings are that the pharmacists' role is little known to other health care professionals, particularly at hospitals with short tradition for clinical pharmacy services. clinical pharmacists have great motivation from being able to influence drug treatment for patients. from the perspective of the participating pharmacists they succeed in interdisciplinary cooperation when their professional knowledge solves the patients' drug-related problems. communicating recommendations to physicians with professional credibility has great importance for the intervention to be implemented. using the theoretical framework of communicating tensions, we argue that the pharmacists in our study use indirect communication to prevent physicians defensiveness to recommendations. lack of education in interprofessional cooperation and communication is apparent in this study. the participants also stated that there should be some form of quality assurance or education requirements before one can work as a clinical pharmacist. conclusion: training in communication for graduates and interprofessional collaboration during the undergraduate pharmacy education, can possibly help pharmacists with integration in interdisciplinary teams. increased attention to teamwork from the hospital leadership is essential for the implementation of interprofessional collaboration in a larger context. please specify your abstract type: descriptive abstract (for projects) background and objective: antifungal therapy in the icu, particularly therapy targeting resistant aspergillosis, mucormycosis and systemic candida, is often of lifesaving importance. posaconazole and voriconazole are the antifungal agents of choice. our aim was to compile a tool that can be used at the icu to address aspergillosis, mucormycosis and systemic candida in an optimal manner. design: female patient, age + , liver transplant, crp [ mg/ l, creatinine [ lmol/l. abdominal x-ray imaging revealed four large abscesses and laboratory analyses confirmed mucormycosis. posaconazole intravenous ( mg one times daily) and liposomal amphotericin b ( mg/kg/day) were initiated. the inflammatory markers remained unchanged days following initiation of therapy with no change in size or number of abscesses and the patient developed sepsis. amphotericin b dose was increased to mg/ kg/day. after week the inflammatory parameters and size of abscesses began to fall. the dosage form of posaconazole was switched from intravenous to mixture. the dose remained the same and within h the crp rose to mg/l. results: pharmacist intervention revealed a missing loading dose of intravenous posaconazole as well as incorrect dosage of the per oral form due to bioavailability variation. posaconazole mixture dose was increased to mg two times daily. through serum concentration analysis of posaconazole was suggested prior to the dose increase. the serum concentration was . mg/l (range [ . - . ) . through serum concentration days later was . mg/l. both crp and abscess size were on the decline. a dosage and tdm pocket card for posaconazole therapy of mucormycosis, aspergillosis and candida was compiled. conclusion: optimal systemic fungal infection therapy is essential, especially in the critically ill. of special importance is tdm and correct dose adjustment when dosage-form changes occur. please specify your abstract type: research abstract background and objective: potentially inappropriate prescriptions and omission of prescription, respectively ip and op, are common issues in the pharmacotherapy, especially in vulnerable population, such as elderly and children. there are many available tools detecting ip and op for geriatrics, however, similar tools are less common in paediatrics. therefore, a first target tool for paediatric population: popi «paediatrics: omission of prescriptions and inappropriate prescriptions» was created and was validated by delphi method in . we aim to evaluate inter-rater reliability between health care professionals, who apply popi. our study also assessed their satisfaction and the accessibility of this tool. setting and method: twenty cases with or without ip or op were selected. these cases were identified in a previous retrospective ip-op prevalence study on . patients. these patients were admitted to the emergency department of a university mother and child hospital, between october and march . one doctor and one pharmacist, who participated in the creation of popi tool, identified ip and op in cases and composed ''standard answers''. these cases were then reviewed independently by eleven clinicians (including generalists, paediatricians, pharmacists, residents, general practitioners), who did not experience this tool before. inter-evaluator agreement was calculated by using the agreement kappa test. the satisfaction of users was also evaluated. main outcome measures: inter-evaluator agreement, the median time of use and the satisfaction of users. results: a high level of agreement of ip and op detection was recorded (ip: k median = . ; op: k median = . ). the easy use of popi was approved by % evaluators. the median time of use was min s per case (quartiles : . - . ) . as a result, there were % of clinicians satisfied with the provided popi and they would like to apply this tool in their daily practice. conclusion: popi demonstrated a good interrater reliability and is easy to use. this strong validation by many specialists prove popi is a reliable tool. it can be applied daily at work in paediatric section by doctors and pharmacists. other multicentre and prospective study should be conducted to evaluate economical and clinical impacts of popi. please specify your abstract type: descriptive abstract (for projects) background and objective: drug dosing during cvvh is challenging due to changes in pharmacokinetic parameters brought about by the patients' deterioration in health and factors associated with the physical process of filtration. this is of particular significance in the icu. in addition, there is the issue of the patients' diuresis or lack of such. this will affect the total clearance (cl total ) of the drug. the dose of antibiotics must therefore be calculated individually taking into account all of the above as well as changes of filtration parameters. our aim was to illustrate how such dosage calculations can be undertaken. design: a -year-old male patient, weight kg, diagnosed with stenotrophomonas maltophilia infection. the trimethoprim/sulfamethoxazole dose was . mg trimethoprim/kg/day every h as specified for anuric patients on cvvh. patient was initially anuric for days after which diuresis was started. the dose was recalculated. results: creatinine clearance (crcl) related to cvvh during the anuric period was calculated accounting for ultrafiltration rate, sieving coefficient, blood-flow, haematocrit concentration and pre-dilution. the value was ml/min. following diuresis on day , remaining kidney function was assessed by measuring urine and serum creatinine. the value for crcl renal ( ml/min) was added to the extracorporeal clearance, and gave a total clearance of ml/min. this warranted dose adjustment of trimethoprim/sulfamethoxazole since this drug requires normal dosage at crcl [ ml/min. conclusion: during cvvh, the presence or absence of diuresis must be taken into consideration when dosing antibiotics. in anuric patients, the cvvh-machine set up constitutes crcl total , but in patients with diuresis, the remaining crcl renal should be added. please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of the study is; to evaluate patients' home (prescribed and non-prescribed) and hospital medication during hospital admission by computing medication regimen complexity index and investigating possible drug-drug interactions. design: patients (aged and older) who applied internal service during months ( days/a week) were included to the study. patients' medical profile were obtained from patients' file. their home medication and hospital medication were calculated with medication regimen complexity index ( ) and checked drug interactions with micromedex drug interaction program. results: a total of from of patients who applied to the internal service (male . %, female . ; the mean age of patients was . ± . .) were included to this study during months. of them, . % had low education level (\ education years), . % had and more chronic diseases of them, % hospitalized last months before this hospital admission. the most prevalent diagnoses documented at admission were kidney disease ( . %), cardiovascular disease ( . %) and cancer ( . %). the mean of patients' home medication number was . ± . and the mean of their mrci scores was . ± . . % in patients hospitalized in the last months. at least one possible drug-drug interactions were found in . % of patients at home medication and in . % of patients at hospital medication, respectively. the mean number of possible drug-drug interactions at patients' home medications was . ± . , while the mean number of possible drug-drug interactions at patients' hospital medications was . ± . . of them, . % had polypharmacy at home medication. the frequency of possible drug-drug interactions and the score of medication complexity index was found high among patients' hospital medications when compared with their home medications. conclusion: the potential role of pharmacist including medication reconciliation and medication review could improve rationale drug use during hospital admission. coronavirus. experts' local committee has approved to use oral ribavirin for the treatment of these respiratory viral infections. we aimed to assess the effectiveness and safety of oral ribavirin as main treatment in respiratory viral infections. setting and method: from may to october , we performed a retrospective monocentric study including patients who received oral ribavirin for non-hcv infections. main outcome measures: viremia negativation was used to determine the response rate to oral ribavirin. specific toxicities (anaemia, cytopenia, liver dysfunction) and renal function were assessed biologically. results: thirty-five immunocompromised patients (f/m: / , age: ) were included. underlying conditions were lung transplant (n = ), heart transplant (n = ), pulmonary fibrosis (n = ) and acute myeloblastic leukaemia (n = ). the median duration between transplantation and infection was . years ( . - . ). nine patients were exclusively infected by rsv, by hpiv ( hpiv- ; hpiv- ; hpiv- ; hpiv- ; non-identified hpiv), by hmpv and by coronavirus. there were six co-infections: rsv/ hpiv- , rsv/coronavirus, hpiv- /hpiv- and hpiv- or /coronavirus ( patients). all the patients were admitted in pulmonary division, except for the patient with heart transplant who was in cardiac intensive care unit. the administered dose was mg tid or mg tid if there was renal insufficiency ( patients). the median duration of the treatment was days . four patients prematurely discontinued the treatment due to severe toxicity or therapeutic change; three didn't respond to the treatment (no data for the last one). four patients were re-treated despite having a virological response to the first cure. one patient treated for a hpiv- /coronavirus coinfection had an hpiv- relapse days after ribavirin discontinuation. concerning the three other patients, they received a second cure to treat a new infection (coronavirus, hpiv- and hmpv, in opposition to hpiv- twice and hpiv- respectively). virological response rate was % ( / for rsv, / for hpiv, / for coronavirus and / for hmpv). two non-negative viremia patients (rsv and hpiv- /coronavirus) received intravenous ribavirin after oral ribavirin therapy. no patient died from viral infection. twelve patients presented specific toxicity: one hepatic cytolysis and cholestasis, eight haemoglobin decrease, two pancytopenia and one mucositis. conclusion: despite the poor number of patient, our study shows that oral ribavirin seems to be efficient to treat hpiv, hmpv and coronavirus in immunocompromised adults. we observed known side effects that could generally be managed. oral ribavirin may thus represent a therapeutic strategy in several respiratory viral infections. please specify your abstract type: descriptive abstract (for projects) background and objective: reconciliation of medicine lists is important to ensure correct medical treatment of patients both in hospital and other healthcare levels. while reconciliation upon admission is part of the normal routine at surgical ward b, molde hospital, there has been less focus on reconciliation at discharge. as such, this study aimed to ensure reconciliation and correct transfer of medical information at discharge. design: medicine lists of all patients discharged from surgical ward b, molde hospital between week and in (n = ) were investigated. the forms were gathered and counted based on the tasks signed for to ensure completed reconciliation and sufficient information given to the patient. the count was performed every - weeks, and the forms in each count was pooled together as one point of measure. the quality of medicine lists in discharge lists was evaluated based on the norwegian patient safety program criteria. medicine lists in discharge lists from week to (n = ) were pooled together and compared to medicine lists in weeks - (n = ). results: the results of reconciliation was divided into the subsections of surgical ward b, and represent the number of completed tasks as signed for in the reconciliation form. the surgical subsection showed a significant increase in patients with pre-checked medicine lists and reconciled medicine lists over the measured time period. similar results were not found in the orthopaedic subsection. as for the quality of medicine lists in the discharge lists, significant improvement was seen in all set criteria, with the exception of ''source'' in the surgical subsection. in the orthopaedic subsection however, no significant improvement was seen in any of the criteria other than ''indication for use''. conclusion: the implementation of reconciling medicine lists at discharge was successful. however, both subsections need to work further to ensure continuation and improvement of the process. furthermore we found varying results in the writing of medicine lists depending on subsection. still, regardless of the individual results of the two subsections there is big room for improvement to ensure that sufficient medical information is included in the discharge papers. please specify your abstract type: descriptive abstract (for projects) background and objective: from july clinical pharmacists began conducting medication histories and reviews (pharmacist notes) at the emergency surgical ward (esw), north zealand hospital (nzh). inclusion criteria are acute patients using c drugs or c risk drug (antidiabetics, anticoagulants, antipsychotics, benzodiazepines, opioids and digoxin). the aim of the service is to identify drugrelated problems and secure correct medication reconciliation between the medicine the patient is admitted with and the medicine in the electronic medication system (ems) in the hospital. the service ensures that the patients' medication follows across healthcare sectors. the objective is to determine if the discrepancies between the medicine the patient is admitted with and the medicine in ems (documented in the pharmacist notes) are used by the physicians. in addition to determine if the pharmacist interventions increase the physicians' acceptance rate of the discrepancies. design: data were collected at the esw at nzh (capacity of beds). data consist of pharmacist notes conducted from august to may . pharmacist notes were compared to the patient record and ems to identify if the pharmacist notes were considered by the physicians. in order to increase physicians' acceptance rate of the discrepancies suggested in the pharmacist notes, interventions were made according to the model for improvement. throughout the period, the focus was on oral delivery of the pharmacist notes. in december the pharmacist optimized the clinical relevance of the discrepancies, by creating and testing a list of products (including vitamins, herbal drugs, glucosamine etc.) which the pharmacist should not intervene on. in december the pharmacist also started to follow up on the pharmacist notes not considered by a physician the previous day to ensure that the physician considered the discrepancies. results: there were identified discrepancies between the patients' actual medication at admission and ems at the hospital in patient records ( . discrepancies per patient). in total discrepancies were accepted by the physicians ( . discrepancies per patient). the physicians' acceptance rate was based on the acceptance of one or more of the discrepancies in the pharmacist note. baseline data were collected from august to november , where out of pharmacist notes were accepted by the physicians resulting in an acceptance rate of . %. from december to may the interventions made by the pharmacist contributed to an increase in acceptance rate to . % ( out of notes accepted). if the pharmacist notes were not delivered orally to the treating physician the acceptance rate was % ( out of notes accepted). conclusion: the pharmacist interventions contributed to an increase in the physicians' acceptance rate of discrepancies from . to . %. a result indicating that the pharmacist notes contributes to an increase the quality of the medication process across sectors. hp-pc : how the centralization of medicines manufacturing enable to generalize the pharmaceutical validation? samantha oses * , soizic vandierdonck, vincent servant, dominique breilh please specify your abstract type: research abstract background and objective: the centralization of the reconstitution of injectable anti-infective drugs enhance to decrease costs and several risks. this minimization of the risks operates at several levels such as i) reduction of the staff exposure and external contamination of preparations during the reconstitution phase (with controlled atmosphere areas, isolators, etc.), ii) improvement in the quality of the management of infective diseases thanks to a pharmaceutical validation systematically performed after the prescription and before the reconstitution phase. the main objective of the study was to describe and quantify pharmaceutical validation on injectable anti-infective drugs prescriptions restored in a pharmaceutical reconstitution unit. setting and method: an observational descriptive study was carried out on each prescription with at least one injectable anti-infective drug that has to be reconstituted before administration. the process was as follows: -prescription by the physician on an electronic prescription software, -pharmaceutical validation and if necessary pharmaceutical intervention (pi) made by phone call, -reconstitution at the pharmacy, -administration to the patient. the pharmaceutical validation methodology followed the french society of clinical pharmacy (sfpc) guidelines ''prescriptions screening and analyses level'' published in the good practices of clinical pharmacy and one resident and one pharmacist were devoted to the activity every day. main outcome measures: the pharmaceutical validation was quantified by the number of pi by patient, which were categorized according to the sfpc guidelines. results: during months, a total of pi were collected. they concerned patients with an average of . pi per patient. among them, . % ( ) concerned paediatric population. antibiotics were involved in . % ( ) then followed by . % ( ) cases ( . % ( ) biological assessment issues, . % ( ) absence of therapeutic drug monitoring (tdm) and . % ( ) the drug hasn't been adapted to the weight), dosage adjustments in . % ( ), information missing concerning the treatment indication in . % ( ) and miscellaneous pi in . % ( ) such as wrong clinical service on the prescription, etc. approval rate of physicians was . % ( ). conclusion: this study has shown that even if prescriptions were secured by electronic prescription software, the pharmaceutical validation remains essential. in that case, the centralization of the reconstitution of injectable anti-infective drugs enabled to generalize this activity on all prescriptions of the hospital. however, the pharmaceutical validation was focused only on anti-infective drugs, that was not fully efficient and must be extended to the whole prescription. it is a priority to develop a comprehensive and exhaustive validation on every medical prescription; however, this activity is highly time consuming and needs larger and more trained staff. hp-pc : the start/stopp criteria as a helping tool to the pharmacists' medication review in the acute admissions unit of the regional hospital in horsens hans pedersen * please specify your abstract type: research abstract background and objective: polypharmacy occurs often increasing the need for patients' medications to be reviewed. the start/ stopp criteria help detects potentially inappropriate prescriptions in older people. in this study we aimed to measure and categorize the different start/stopp criteria found in medication reviews in the acute admissions unit of horsens and the acceptance rate. setting and method: patients admitted to the acute admissions unit were selected based on their age and the number of prescriptions in a period of months. patients years or more which received six or more drugs were included in the study. only patients who later were transferred to another medicine ward were included in the study. the pharmaceutical medicine review was performed by a clinical pharmacist using minimum two different sources; the electronic medical record and medication-lists. the guideline of pharmaceutical medicine review in the hospital pharmacy central denmark region was used as the standard-guideline. in addition, thestart/stopp criteria version was used. main outcome measures: the number of start/stopp criteria found in medication reviews. the different start/stopp criteria were scored equally with one point each. results: patients, males and females, out of , were included. the mean age was years and the patients received in average prescribed drugs. at admission the average number of stopp criteria were . ± . and . ± . for the start criteria. in average, % of the purposed stopp criteria were accepted by the physicians. the most frequently accepted stopp criteria were in the category of drugs that predictably increases the risk of falls in older people. the benzodiazepines where the most common drugs to be discontinued. in the start category, % of the suggested start criteria were accepted, which included: calcium and vitamin d supplement, beta- agonist and bisphosphonate. conclusion: the present study demonstrates that it was possible to integrate the start/stopp criteria as a helping tool in the medication reviews in the acute admissions unit of horsens. the start/stopp criteria were found within the different categories, however only a minor part of the registered start/stopp criteria were accepted by the physician. please specify your abstract type: research abstract background and objective: the objective of this work is to assess prescribing practices of somatostatin analogues in a surgery department, and to analyse the conformity of switching from immediateacting octreotide to the long-acting release (lar) form, in accordance to laboratories' guidelines. setting and method: retrospective observational study. a focus was realized on patients admitted in a digestive surgery unit between january and december , . the patients' medical records were reviewed for clinical features, diagnosis workup and treatment strategies. main outcome measures: medical records for patients with diagnosis of gastro-entero-pancreatic or endocrine tumors who had received injections of lar octreotide during hospitalization were reviewed and the economic impact of prescriptions errors has been evaluated. results: of the evaluated patients, ( %) were hospitalized in surgery digestive unit; mean age at first administration of octreotide was years and % were male. the male and female ratio was . : . reasons for hospitalization were: digestive system neoplasms ( %), fistula ( %), intestinal obstructions ( %) and other pathologies ( %). of the patients treated with octreotide, ( %) received a lar form. only four patients received doses in accordance with guidelines: one at mg/month lar form and three at mg/month lar form, after having respectively been treated by intravenous octreotide at and mcg/day during - days. medical prescriptions of the remaining patients did not comply: all patients received mg/month after an intravenous treatment of mcg/day, instead of mg/month. from a financial perspective, these misuses have led to an additional cost of . euros for the hospital, excluding tax ( mg: . €/unit and mg: €/unit). conclusion: despite the publication of octreotide release form proper use recommendation in our hospital, % of patients of digestive unit are not right treated. a new guideline will be written added by doses of long-acting release and economic data. this work will be transmitted to specialists by clinical pharmacists. hp-pc : pharmaceutical process for intrathecal analgesia in clinical oncology practice vivien pigeon * , guillaume binson, claire grignon, antoine dupuis please specify your abstract type: descriptive abstract (for projects) background and objective: in some cases, patients with cancer pain remain painful despite the use of high dose of intravenous opioids and intrathecal analgesia becomes the ultima recourse to manage acute pain. until , intrathecal syringes were prepared by nurses in the unit care which involve a risk for patients. therefore, the aim of this work is to describe the set-up of the prescription and preparation process with the potential benefits for the safety. design: multidisciplinary concertation took place between pharmacists, physicians and surgical teams and several points were discussed to secure the process: • identification of patients with high level of infection risk; • identification of critical points of the pharmaceutical process; • validation of quality control and drug stability studies regarding drug compounding involving morphine, ropivacaine, baclofen and clonidine, alone or in admixture. results: multidisciplinary concertation lead us to define the most important points to set up the pharmaceutical process for intrathecal analgesia: • chosen patients are cancer patients; • implementation of a prescription software to secure the prescription step; • production of syringes by the pharmacy department implying several criteria: • preparation in controlled atmosphere area; • training of pharmacy technicians; • implementation of quality control and drug stability studies at °c in syringe over h and at °c in pumps over month; • microbiological control and bacterial endotoxin level. the implementation of a pharmaceutical process for intrathecal analgesia gave us the opportunity to reorganize the care of cancer patients tolerant to high dose of opioids. in this process, the pharmacy department plays a major role leading to decrease the risk of infections and errors of dosing. ingrid plessala *, , xavier deviot , thomas sidibe , zohra mostefaï , michèle minvielle , marta wyrtwal , roselyne gervais pharmacy, geriatrics, saint-denis hospital centre, saint-denis, france please specify your abstract type: descriptive abstract (for projects) background and objective: proton pump inhibitors (ppis) are indicated in gastro-oesophageal reflux and peptic ulcer disease. they are widely prescribed, often in off-label indications. the objective of this work was to reassess ppis prescriptions in collaboration with geriatricians. proton pump inhibitors (ppis) are indicated in gastro-oesophageal reflux and peptic ulcer disease. they are widely prescribed, often in off-label indications. the objective of this work was to reassess ppis prescriptions in collaboration with geriatricians. design: prospective study in three geriatric wards. the study included ppis treated patients from these three geriatric wards. dose, indication of the ppi, age, gender and duration of treatment have been recorded for each patient. the relevance of each ppi treatment has been reassessed by a geriatrician, a pharmacist and a junior pharmacist, regarding the indication and the patient's clinical condition. following this re-evaluation, three situations arose: • to maintain ppi at the same dose ( mg or mg) • to maintain ppi but half dose (from mg to mg) • to stop ppi corrective actions have been recorded in patients' files to allow their traceability. results: patients were included in the study. % of ppis prescriptions were off-label, % had no indication mentioned in patient's file and % were conform to the marketing authorization. % of patients have been on ppis medication longer than months, which is the recommended treatment' duration in france, % longer than a year and % longer than years. in collaboration with the geriatricians, ppi prescriptions were maintained for % of patients. we reduced the dose in % of cases. finally, we decided to stop a third of the ppis prescriptions. conclusion: ppis prescriptions are often longer than recommended. this can lead to side effects for patients. in france, lack of new recommendations since may explain this frequent misuse of ppis. there is also a reserve from doctors to stop these treatments, especially with fragile patients. in our case, the relevance of each ppi treatment was re-evaluated in three geriatric wards and we succeeded in shortening and stopping ppis medications in half of the situations. to assess the impact of this action on our geriatricians, a new review of ppis prescriptions relevance is programmed in . ppis prescriptions are often longer than recommended. this can lead to side effects for patients. in france, lack of new recommendations since may explain this frequent misuse of ppis. there is also a reserve from doctors to stop these treatments, especially with fragile patients. in our case, the relevance of each ppi treatment was re-evaluated in three geriatric wards and we succeeded in shortening and stopping ppis medications in half of the situations. to assess the impact of this action on our geriatricians, a new review of ppis prescriptions relevance is programmed in . hp-pc : oral anticoagulants and heparin for children: standardized protocols for prescription, dispensation and administration alexandra liauzu , marie-françoise hurtaud-roux , ronan bonnefoy , caroline farnoux , philippe sachs , theresa kwon , olivier bourdon , sophie ajzenfisz , sonia prot-labarthe *, pharmacy, hématologie clinique, ap-hp hôpital robert-debré, cardiologie, néonatologie, ap-hp hôpital robert debré, réanimation pédiatrique, ap-hp hôpital robert-debré, néphrologie, pharmacy, ap-hp hôpital robert debré, coordonnateur de la gestion des risques associés aux soins/ responsable du système de management de la qualité de la prise en charge médicamenteuse, ap-hp hôpital robert-debré, paris, france please specify your abstract type: research abstract background and objective: high-alert medications (ham) are medications that are associated with a high risk of serious harm if used improperly. we already identified paediatric ham used in our institution to identify safety measures for their use. anticoagulants and heparin were part of these high-alert medications. we aim to write standard protocol of use for low weight heparin and oral anticoagulant used in our mother-child teaching hospital. our secondary objectives were to decrease medication errors, anti-xa and inr unexplained variability and to help nurses to administer the drugs (standard dilution, oral solution available) setting and method: we carried out a literature search on pubmed Ò , on websites of several learned or professional societies and agencies. the results of the literature search were compiled on written protocol and presented to our institute drug safety-steering committee composed of four doctors, two head nurses, two pharmacists, and one risk manager. main outcome measures: not applicable. results: the protocols concerned enoxaparin, tinzaparin, warfarin but we chose to also include protamine. the most difficult issue was to have standardized dilution and protocol for all ages and weight: from premature to adolescents and all units of care (from cardiology to intensive care unit, nephrology and neonatology). we took into account the administration errors we had in our hospital and the preexisting protocol to avoid any drastic error-prone change. the final version of these protocols will be presented on the final communication with web link to upload them. conclusion: for now we did note evaluate the impact of these protocols but a before/after analysis of error reports and users evaluation will be done. however, these protocols can help all health professionals working in paediatric units for benchmarking. hp-pc : does a hospital formulary system impact timely medication administration and quality of inpatient care? anne-valérie putallaz *, , vera jordan-von gunten , pierre-auguste petignat , pierre turini , johnny beney division of pharmacy, institut central des hôpitaux, division of internal medicine, medical coordinator for quality of care and patient safety, hôpital du valais, sion, switzerland please specify your abstract type: research abstract background and objective: the prevalence of drug omissions is often underestimated but their impact can be clinically relevant. we hypothesized that delays in the administration of non-formulary/nonstored drugs could impair the quality of care. the aims of this study were: °to determine the time between the prescription and the administration of the first prescribed dose and, if applicable, to calculate how many doses were omitted. °to analyse the clinical relevance of the identified delays. setting and method: three months retrospective study of electronic records of patients hospitalized on the internal medicine wards of a network of hospitals supplied by a centralized pharmacy. this pharmacy is located in one of the sites; other sites are - km apart. main outcome measures: . for the main hospital site and the three distant sites: • median time between the prescription and the administration of the first prescribed dose • mean number of omitted doses for formulary and non-formulary/ non-stored drugs. . categorization of patient's harm caused by the delays of timecritical drugs, according to the ncc-merp taxonomy of medication errors. results: ' prescriptions were analysed. calculated delays for non-stored/non-formulary drugs were longer than for formulary drugs. however, the median time to administration is less than h for both formulary and non-stored/non-formulary drugs; and more than % of formulary drugs and around % of non-stored/non-formulary drugs were administered within h following their prescription. there was no significant difference in the mean number of omitted doses or in the delays between the site where the centralized pharmacy is located and the other sites, except for one of them. a delay representing . or more omitted doses was found for ( . %) prescriptions. among them, only were considered potentially clinically relevant. none of them caused severe harm to the patients involved. conclusion: in our setting, non-stored/non-formulary drugs take more time to be delivered than formulary drugs, but more than % of formulary drugs and around % of non-stored/non-formulary drugs are administered within h following their prescription. none of the patients who experienced delays underwent severe harm. our study showed that delays also occur for formulary drugs but no systematic cause of omission was identified; further studies should focus on all dose omissions during hospitalization. penelope randuineau *, , roger jeremy , lauriane cornuault , anne lecoeur , franck lemercier , isabelle javerliat , thomas tritz service de pharmacie à usage intérieur, service de chirurgie vasculaire, hôpital ambroise paré, boulogne-billancourt, france please specify your abstract type: descriptive abstract (for projects) background and objective: a french national survey of inpatient adverse events reveals that nearly half of adverse drug events (ade) are preventable. medication errors behind these ade occur mainly during the transition steps of care pathway. in this context, medication reconciliation process has been implemented in our vascular surgery department. the objective of this study is to identify unintentional discrepancies (uid) and assess their potential clinical impact design: a pharmacy resident or a pharmacy student reconciliated patients: aged older than or with at least five chronic treatments at admission or suffering from chronic diseases. patients were considered reconcilable if at least two reliable sources on usual patient's treatment were available. these many sources of data (patient interview, prescription or interview of general practitioner, reference dispensary, drug box …) were compared to the admission prescription during the first h of hospitalization to detect and correct uid. based on gravity scale promoted by the french high authority of health, two pharmacists (a resident and a senior) and a vascular surgeon reviewed every uid in order to define their potential clinical impact. the uids were considered minor if it leads to no consequence for the patient, clinically significant if it leads to essential monitoring, major if it could cause temporary clinical consequences, and critical if it could result in permanent clinical consequences or the involvement of the prognosis. results: between february th and may st , a total of patients have been reconciled. patients were excluded due to a lack of reliable sources. mean age was . years old (± . ) and sex ratio m/f was . . % of the reconciliated patients' admissions were scheduled. the mean number of medication was . (± . ). patients ( %) had at least one uid and the mean uids per patient was . (± . ). the most common types of uids were omission ( %), incorrect dose ( %) and incorrect administration frequency ( %). more than % of these uid presented a potential clinical impact: an adverse effect (high blood pressure, hyperglycaemia) was observed for nine patients and lead to therapeutic optimization and monitoring; uid were considered to have potential clinically significant impact ( %), a potential major impact ( %) and a potential critical impact. conclusion: these results appear consistent with those reported in literature. vascular surgeons have appreciated the approach and would like systematic medication reconciliation before surgery. as a major part of admissions were scheduled, we would like to establish the reconciliation before the patient's hospitalization every time it's possible. this new organization should facilitate the care pathway before surgery and decrease preventable postoperative adverse events. hp-pc : delirium in elderly patients: successful use of melatonin gaëlle jouin , aurélie reiter-schatz , pierre bentzinger , fatem-zohra laalou , bénédicte gourieux *, pharmacy-sterilization, orthopedic's intensive care unit, university hospital of strasbourg, strasbourg, france please specify your abstract type: descriptive abstract (for projects) background and objective: postoperative delirium happens to about one-third of elderly patients and is a major cause of morbidity and mortality. it is reported that haloperidol, an antipsychotic, has been the agent of choice for managing delirium. however, it induces cerebrovascular adverse effects and greater mortality. the hyperactive type of delirium is known to be associated with a low melatonin level and the loss of a normal melatonin secretion rhythm. the postoperative administration of melatonin to elderly could decrease the symptoms of delirium. the purpose of this study was to evaluate melatonin effectiveness in a cohort of patients suffering from postoperative delirium. design: a retrospective study of melatonin prescriptions has been conducted over a months period. medical background, type of surgery, symptoms of delirium, use of antipsychotics and benzodiazepines have been studied in all patients who received melatonin in an orthopaedic surgery unit. length of hospital stay, time between delirium and melatonin administration and the effect of melatonin had been evaluated. results: a total of patients were included: average age was . years ( - ), sex ratio m/f = . twelve patients ( %) were hospitalized because of an infection (prosthesis or osteoarticular). in % of cases (n = ), the prescription of melatonin was started when the patients were hospitalized in our intensive care unit. nine patients ( %) were under chronic treatments like benzodiazepines or antipsychotics. the average length of hospital stay was days ( - ). melatonin was started on an average of days after surgery , and administered at the dose of mg xr in the evening, during an average of days ( - ). cognitive impairments requiring a prescription of melatonin were: confusion ( %, n = ), agitation ( %, n = ), daytime sleepiness ( %, n = ), temporal-spatial disorientation ( %, n = ), nocturnal awakening ( %, n = ), hallucination ( %, n = ), difficult falling asleep ( %, n = ). the average time to recover from confusion was days, agitation days, daytime sleepiness days, temporal-spatial disorientation days, nocturnal awakening days, hallucination days and falling asleep days. melatonin treatment helped stopping benzodiazepines treatment in six patients ( %). conclusion: after administration of melatonin, delirium symptoms were improved for all patients and benzodiazepines treatment stopped for six patients. earlier prescription of melatonin could regulate sleep-wake cycle and reduce the duration and incidence of delirium. please specify your abstract type: research abstract background and objective: denosumab (xgeva Ò ), a fully human monoclonal antibody targeting rankl, which inhibits bone resorption, is indicated to prevent skeletal complications in patients with solid tumors and bone metastases. about % of patients develop hypocalcaemia, a common adverse event that may induce spasms, muscle cramps, paraesthesia, prolonged qt interval, tetany, convulsions… we report the management of ionic supplementation and physicochemical incompatibilities in a case of hypocalcaemia due to denosumab. setting and method: the clinical case was analysed with the pharmacovigilance regional center. main outcome measures: a year old patient, with nodal and bone metastasis in prostate cancer, was treated with denosumab (stopped with the last injection months before, on the th of march). he went to emergency on the th of may with asthenia, anorexia, nausea, diarrhoea, qt prolongation. biological results showed hypocalcaemia (corrected calcaemic = . mmol/l) and hypophosphatemia (phosphorus \ . mmol/l). concomitant calcium and phosphorus intravenous supplementation started with loading doses ( g of calcium and . g of phosphorus) and then a week of following daily intakes: phosphorus ( g iv and . g oral); calcium ( g iv and . g oral). however, low-serum corrected calcium and phosphorus levels persist at . mmol/l and . mmol/l. results: incompatibility between phosphorus and calcium by formation of soluble or not-soluble complexes is described in literature. in our case, calcium and phosphorus were mixed in a same infusion. after a week of supplementation, calcium infusion is continued with increased dose ( g/day) and phosphorus infusion is stopped. phosphorus oral supplementation remains stable ( . g per day); calcium oral supplementation is increased ( . g per day). h between intakes is applied to avoid digestive complexation. h later, corrected calcium levels are normalized at . mmol/l and phosphorus levels are still low. therefore, as hypocalcaemia due to denosumab induced a secondary hyperparathyroidism and thus hypophosphatemia; phosphorus levels are expected to increase subsequently. conclusion: this case report shows that recurrent hypocalcaemia with denosumab is possible few months after administration. supplementation with large amount of calcium is needed and administration methods may impact the effectiveness of supplementation. indeed, it seems that the incompatibility between phosphorus and calcium did not allow an effective supplementation. gunnhild langdal *, , , ida rudberg , lone holst , anne-lise sagen major , central norway hospital pharmacy trust, Å lesund, centre for pharmacy, university of bergen, bergen, møre og romsdal health trust, Å lesund, norway please specify your abstract type: descriptive abstract (for projects) background and objective: drug interactions (dis) can cause side effects and lack of therapeutic effect. the objective of this study was to describe the prevalence of dis at the medical department of Å lesund hospital, and to investigate how dis were managed by clinical pharmacists and physicians. design: at the medical department, Å lesund hospital, clinical pharmacists serve seven out of ten wards, from which patients were included during a five weeks period. the clinical pharmacists selected patients for screening for potential dis (www.interaksjoner.no) as int j clin pharm ( ) : - usual (= pharmacist group). detected dis were classified according to a predetermined classification system, and it was registered whether the physician implemented suggested changes in prescription. for patients not selected by clinical pharmacists (= non-pharmacist group), a pharmacy student performed the search for dis. results: in total patients were admitted. on average, each patient had . dis, and . % of the admitted patients had at least one di. the prevalence of dis was significantly higher among the patients in the pharmacist group compared to the patients in the non-pharmacist group (median@@@ vs. , respectively, p \ . ). the groups differed significantly regarding number of drugs used, age, duration of hospital stay and number of warfarin users. . % of the dis detected in the pharmacist group were discussed with the physician. the remaining . % were considered not necessary to discuss for various reasons e.g. because they were considered not clinically relevant ( %) or already adjusted for in clinical practice ( %). for dis the clinical pharmacist suggested a change in prescription, and of these suggestions ( %) were implemented by the physician. conclusion: just over half of the patients were selected by the clinical pharmacist for screening of dis, and the pharmacist seemingly made a reasonable priority of patients with many drugs, old age, a long hospital stay and users of warfarin. only of dis was discussed with physicians. this indicated that pharmacists do a considerable work in assessing the relevance of dis before discussing with the physicians. it also seemed that changes in prescription suggested by the clinical pharmacist were reasonable. hp-pc : securing the paediatric use of oral chemotherapy: a proactive risk assessment samia mouffak *, , linda an , anne fratta , anne auvrignon , , nadia marquis , karine morand pharmacy, risk management committee, hematology, armand trousseau hospital -aphp, paris, france please specify your abstract type: descriptive abstract (for projects) background and objective: oral chemotherapy is an important part of the therapeutic strategy in childhood cancer or haematological malignancy. it also represents an emerging risk area in oncology practice. several medications errors involving oral chemotherapy were reported in children of our onco-haematology department, fortunately without clinical consequences. nevertheless, the potential severity of such errors led us to implement a failure analysis of the paediatric oncology care pathway in order to identify and prevent potential risks, and secure the paediatric use of oral chemotherapy. design: we conducted a failure modes, effects and criticality analysis (fmeca) which is a proactive risk assessment approach. first, process maps were detailed for each step of the oncology care pathway. it was performed by a multi-disciplinary group composed of physicians, coordinating nurse, hospital pharmacists and pharmacy resident. then, for each step of the medication-use process, the team identified the failure modes, their main causes and effects. finally, participants rated the expected severity, frequency and detectability for each failure mode, assigning a score on a five-point scale. a risk priority number (rpn) was then calculated by multiplying those three indexes. the risks getting a high rpn were categorized as critical risks and have been the object of safety improvements. results: failure modes were identified, including critical risk failure modes. critical failures were related to hospital discharge prescriptions and were about the dispensation of oral chemotherapy by pharmacy assistants. most failures were due to prescriptions heterogeneity, lack of clinical information reported on prescriptions, and lack of training of pharmacy assistants in reading oral chemotherapy prescriptions and in mistake detection. two improvement strategies were implemented. first, physicians' awareness led to the harmonisation of practices and to the standardisation of discharge prescriptions. then, to enhance pharmacy assistants' abilities, an educational program on oral chemotherapy dispensation was planned. conclusion: the implementation of a fmeca has highlighted the most critical risks of oral chemotherapy medication-use process. the awareness of all caregivers and the targeted changes in our practices allowed us to improve the safety of the paediatric oncology care pathway. please specify your abstract type: descriptive abstract (for projects) background and objective: the purpose of this study was to investigate if medication reconciliation and medication review, by using the integrated medicines management (imm) model, were suitable to assure the quality of patients' medical treatment at a gastrointestinal surgical ward. furthermore, to analyse frequency, type, handling and clinical relevance of medication discrepancies (mds) and other medication related problems (mrps). design: patients, above years of age, from two departments at a gastrointestinal surgical ward at a norwegian university hospital were included consecutively. medication reconciliation was performed at admission by a clinical pharmacist. the resulting medication histories were compared with the medications documented in the medical records. mds were detected and categorized. thereafter the clinical pharmacist identified mrps by reviewing the medical records systematically and categorized the revealed mrps. mds and mrps were presented for the physician with proposed solutions. the physician's actions to manage the mrps were registered. later a multidisciplinary team assessed the clinical significance of mds and mrps in a subset of patients. results: a total of patients were included. overall, mds and mrps were identified. at least one md was revealed in % of the included patients, whereas at least one mrp was identified in % of the patients. the most frequent type of mds was omission, whereas mrps most often were related to medications that were considered unnecessary. totally, % of the mds and mrps were discussed with the treating physician. the physicians followed the pharmacist's input in % of the discussed md-cases and % of the mrp-issues. longterm consequences of mds and mrps were considered more serious than short-term consequences for the patients. conclusion: medication reconciliation and medication review revealed, solved and prevented a large number of mds and mrps in this study. the results emphasize that pharmacist involvement, by using the multidisciplinary imm-model, contributed to more correct medical records and furthermore to quality assurance of the patients' medical treatment at a gastrointestinal ward. hp-pc : prevention and management of drug interactions in oncology day-hospital: results from a months study involving drug assessment and pharmaceutical report to oncologist pauline-saraï zeller *, , chloé hugard , céline mongaret , , juliette vella-boucaud , antonin maréchal , olivier bouché , dominique hettler , florian slimano , pharmacy, oncology day hospital, university hospital reims, clinical pharmacy, faculty of pharmacy, reims, france please specify your abstract type: research abstract background and objective: quality during transitions of care is a major concern in drug safety for patients. traditional hospitalization allows to reconciliation medication but there is not possible for dayhospitalization (patient's hospitalization short time and no outpatient medication prescribe by oncologists). however, lack of communication between health professionals may expose patients to drug-drug interactions (ddi). while ddi between oral antineoplastic and other drugs are well known, there is a lack of knowledge about ddi between parenteral antineoplastic (ak) and other drugs. in this pilot study, we aim to investigate prevalence and characteristics of ddi between ak and other drugs in real life and to propose a pharmaceutical report model to enhance patient's drugs safety. setting and method: during months, all new oncologic patients (thoracic and digestive) receiving chemotherapy in day-hospital have been recruited by clinical pharmacist. first it was conducted a patient-clinical pharmacist interview and carried out the best possible medication history (bpmh) by contacting at least three different sources of drug information. then, the bpmh has been confronted with oncologic treatment (including concomitant medications such as like antiemetic) with support at least with two different database of ddi analysis. finally pharmaceutical recommendations in order to manage potential relevant ddi were reviewed with oncologists then reported and inserted in personal health record (phr). main outcome measures: prevalence and description of potential clinically relevant ddi in an ambulatory oncology population. results: from november, to april, n = oncologic patients were included with following characteristics: mean age of . , sex ratio : , majority of oncologic thoracic localization ( %). number of oncologic concomitant medications per patient was . ± . (mean ± standard deviation). patients present an average of . ± . comorbidities (excluding cancer) and . ± . linked medications per patient. pharmaceutical analysis revealed potential clinically relevant ddi ( . ± . per patient): % of them concern antiemetics (ondansetron and aprepitant): pharmaceutical interventions were formulated (including recommendations to adapt chronic treatment) and % of them involved biological monitoring (for renal function, inr, potassemie or magnesemie). conclusion: our pilot study confirms high prevalence of ddi between oncologic and non-oncologic drugs. clinical pharmacy services with bpmh performing and pharmaceutical recommendation appears to be useful to enhance patient' drug safety in oncology dayhospital. we currently are deploying our study in order to convey a pharmaceutical letter to general practitioner and community pharmacist. hp-pc : loading dose of anti-infectives: elaboration of a tool helping pharmaceutical analysis julie soyer *, , cécile sanchez , guillaume beraud , nicolas venisse , pauline lazaro , antoine dupuis pharmacy, infectiology, pharmacokinetics, university of poitiers, poitiers, france please specify your abstract type: descriptive abstract (for projects) background and objective: the recent data on vancomycin and ceftazidime confirm that continuous infusion is the best way of administration of these antibiotics. moreover a loading dose before the administration is required for the antibiotics to prevent from the infratherapeutic period at the start of infusion and limiting the risk of resistance emergence. long half-life antibiotics and antifungals also require a loading dose to be effective. the aim of this study is to analyse the prescriptions of anti-infective requiring a loading dose in order to develop a tool to help pharmaceutical analysis. design: a prospective observational study was carried out during days in units. initially, pharmacists, residents and students were trained (role of the loading dose, drugs concerned). then, all patients with anti-infective requiring loading dose were included. some data were collected: weight patient, creatinine clearance, loading dose or not, dose, administration mode, monitoring of steady state concentrations (vancomycin and ceftazidime) and dose adjustment. the results were analysed and compared to bibliographic research before discussion during a multi-disciplinary meeting (pharmacists, infection control specialist and pharmacokinetic specialist). finally, a list of relevant pharmacist interventions was selected. results: out of the patients, were enrolled for prescription of anti-infective requiring loading dose. twenty-six prescriptions including vancomycin, ceftazidime, the others fluconazole, caspofungine, voriconazole and posaconazole. concerning vancomycin, the loading dose was prescribed in % of case, monitoring of steady state concentrations was performed in % of case and dose adjustment after first dosage was required in % of case. selected pharmacist interventions were: • to favour continuous infusion (excepted paediatric) • to keep loading dose at full dose even in patient with renal failure • to monitor steady state concentrations after the first h in patient with renal failure or obesity • to adapt dosage when the target concentration is not reached concerning ceftazidime, the interventions were: • to recommend continuous infusion: g/ h after loading dose of mg/kg • to monitor steady state concentrations in patient with renal failure a total of interventions (dosage, adaption of posology at the monitoring, patients with renal failure, obese, paediatric patient, administration…) were identified by the group of experts. conclusion: this study allowed creating a recap data sheet for students and hospital pharmacists. the selected interventions will allow the harmonization of practices. these recommendations have been validated by the commission of the anti-infective. finally, this study shows that the pharmacist has a key role in the management of antiinfective requiring loading dose. hp-pc : assessment of potentially inappropriate medications in orthogeriatric patients using the rasp list the detection of inappropriate prescribing. the objective of this study was to investigate if the rasp list (rationalization of home medication by an adjusted stopp list in older patients), an explicit screening method adapted to the belgian context, can be used to reduce the number of potentially inappropriate medications (pims) in orthogeriatric patients. setting and method: single-centre, interventional study conducted at the orthogeriatric department of the uz brussel, a -bed university hospital. the rasp list was first applied by a last year pharmacy student to the admission medication of orthogeriatric patients hospitalised in october . after potential adaptations to the medication by a liaising geriatrician, the rasp list was additionally applied by the same pharmacy student to the discharge medication of these patients. main outcome measures: detection and reduction of the number of pims. results: in total, orthogeriatric patients, from whom an informed consent was obtained, participated in this study. on admission, a total of pims were detected in this population. at discharge, the number of pims decreased to . the median number of pims per patient decreased from (on admission) to (at discharge). this difference was statistically significant (p \ . ; wilcoxon signed rank test). drugs of atc class n (nervous system) were responsible for the highest number of pims. conclusion: pims can be detected and reduced in the hospital using the rasp list. a structured and collaborative medication review between (student) pharmacists and physicians appears a good approach to reduce the number of potentially inadequate drugs. nevertheless, more research is necessary to substantiate this further as well as to assess the clinical impact of the findings. hp-pc : impact of implementing ward based dispensaries across a hospital site on both service delivery and patient care michelle sullivan, paul wright, christopher watson, malcolm smith, sotiris antoniou * please specify your abstract type: descriptive abstract (for projects) background and objective: waiting for medication at discharge is often quoted as a key factor for delaying patients leaving hospital. feedback from service users (patients and healthcare professionals) was for a more patient facing pharmacy service. this led to a phased installation of remote dispensaries on wards within the hospital to supply medicines. this new and innovative service enabled the supply function to be fully co-ordinated on the ward. this model was initially implemented on wards, which coupled with one-stop dispensing meant % of discharges require nothing to be supplied at the point of validation, % of discharge prescriptions meeting key performance indicator of being dispensed and ready within h with average turnaround time of min for a discharge prescription and a reduction in missed doses- . % in september to . % in march . this success prompted further installation of remote dispensaries in all clinical areas on site. design: implementation included; purchasing hardware, pharmacy labellers, locating appropriate computer terminals and stock cupboards. the main pharmacy labelling and stock control system was fully integrated at ward level, enabling the automatic reordering of replacement stock. identification of items and quantities to stock for remote dispensaries was also needed prior to role out. there was a need to scope staffing requirements including the redeploying of roles from a main inpatient pharmacy to patient facing areas. results: over items are supplied at ward level each month via satellite pharmacies for all wards, equating to more than % of the total dispensing workload for the site allowing for pharmacy staff to be redistributed from dispensary to the ward. this offered the benefit of being more patient facing and supporting other initiatives such as patient counselling and medicines reconciliation. the project has impacted the pharmacists as it has enabled them to focus on clinical aspects of service delivery, including attendance of ward rounds as well as supporting a ward team approach with the pharmacy technician. results of missed dose audit from june shows across the site % ( ) wards scored below the national . % target and ( %) wards had no unintentional missed doses. conclusion: ward based dispensing has led to pharmacists and pharmacy technicians being % ward based. as a constant presence on the ward, the team offer consistency within the pharmacy service for patients, nursing and medical staff. impact of pre-discharge planning has been beneficial to nurses, patients and work flow of the pharmacy teams. ward based dispensing has improved supply at discharge as well as promoting a more patient facing pharmacy service that has seen the pharmacy team instilled as integral to service delivery at ward level. kutay demirkan * , nursel surmelioglu, aygin bayraktar-ekincioglu clinical pharmacy, hacettepe university, ankara, turkey please specify your abstract type: research abstract background and objective: hacettepe university hospitals clinical pharmacy unit was established in april . this unit runs its services by clinical pharmacy postgraduate students under the supervision of two qualified clinical pharmacists as part-time and oncall basis, in adults, paediatrics and oncology hospitals. the aim of this study was to identify drug related problems and describe its management strategies in inpatient and outpatient settings by pharmacists in clinical pharmacy postgraduate education program. setting and method: during a total of months study period (period i: february-july , and period ii: november-february ), clinical pharmacy postgraduate students followed patients for - times in a week in different services in hospitals (internal medicine, internal medicine intensive care, infectious diseases, neurology intensive care, paediatric bone marrow transplant/haematology unit, paediatric intensive care, geriatrics and nutrition units) and drug related problems were identified and pharmacists' recommendations were listed. main outcome measures: determination and evaluation of drug related problems by pharmacist in hospital. results: a total of recommendations was provided for patients. those recommendations were classified as alteration or discontinuation of drug treatment ( . %), dose adjustment ( . %), change in drug administration time ( . %), inadequate treatment ( . %), healthcare staff training/consulting ( . %), patient education ( . %) and error/deficit in therapeutic drug monitoring ( . %). a majority of recommendations (n = ) were related with alteration or discontinuation of drug treatment provided mainly in departments of internal medicine (n = , geriatrics (n = ), neurology intensive care (n = ) and infectious diseases service (n = ). the following main reason for pharmacist's recommendation was related with dose adjustment (n = ) which were provided in departments of internal intensive care (n = ), infectious diseases service (n = ), neurology (n = ) and internal medicine (n = ). conclusion: clinical pharmacy practices are being carried out effectively in many services, particularly in internal medicine services, internal medicine intensive care unit and infectious diseases services. a collaborative and bed-side education in postgraduate programs in clinical pharmacy help to increase the knowledge and skills of students in real life circumstances and also maintain safe and effective drug therapy by an involvement of clinical pharmacists in hospital services. hp-pc : development of a tool to help pharmaceutical analysis in patients with hepatic failure barbara troussier *, , eric gautier , astrid bacle , florian charier , christine silvain , pauline lazaro pharmacy, gastroenterology, hepatology and gastroenterology, university hospital of poitiers, france, poitiers, france please specify your abstract type: descriptive abstract (for projects) background and objective: hepatic impairment can cause significant changes in the pharmacokinetics of many medicines. however hospital pharmacists can be helpless in performing pharmaceutical analysis behind the lack of precise guidelines. we need a strategy to first detect accurately patients with hepatic impairment, then lead us in dose adjustments. the objectives of this project were to develop a helping tool for hospital pharmacists in the pharmaceutical analysis of patients with hepatic failure's prescription and to select relevant pharmacist interventions. design: we first planned an investigation of patients with hepatic failure's management, with multidisciplinary experts groups. the study was conducted during one week in post-surgical, gastro-enterology, endocrinology, cardiology, pulmonology, geriatric departments and reanimation care units. a flowchart based on hepatic's biomarkers helped us including patients. criteria used to assess hepatic impairment could be: a stage c child-pugh score, prothrombin score inferior to %, bilirubin superior to micrograms per millilitres of blood without haemolysis, aspartate and alanin aminotransferases superior to three times the high normal value, and presence of a vitamin k antagonist interfering with those results. after a review of each included patient's prescription, we checked the major pharmacokinetic elimination pathway of each prescribed molecule (biliary or renal) and if hepatic biotransformation was expected. we also checked if the molecule could cause hepatic side effects. results: out of patients, patients were included for liver failure ( . %) and for a cholestasis ( . %) mainly in reanimation care units ( . %) and gastro-enterology ( %). among the lines of prescribed medicines, the main pharmacological classes encountered were cardiology, ( %) pain ( %), psychiatry ( %), haemostasis ( %) and antibiotics ( %). at the end of the investigation, the expert group decided on the relevant pharmacist interventions. these were based on dose adjustment of anti-infectious, psychotropic drugs, painkillers, oral anti-diabetics, anti-coagulants and corticosteroids. alternatives are proposed for each class. conclusion: to conduct a better pharmaceutical analysis, steps are necessary. first, any liver failure or cirrhosis must be detected thanks to the patient's biological results and medical record. then the patient's prescription can be analysed in order to highlight drugs that need a dose adjustment in a context of hepatic impairment. finally, the physicist and the pharmacist discuss about dose adjustments or alternatives if presence of contraindication with the drugs prescribed. soon the designed tool will be available to all pharmacists to harmonize clinical pharmacy practices. please specify your abstract type: research abstract background and objective: data regarding adherence rates to oral chemotherapy in lymphoma patients is limited. the aim was to assess pharmacist intervention on adherence to oral chemotherapy in patients suffering from hodgkin's (hl) and non-hodgkin's lymphoma (nhl). setting and method: following ethics approval, hl and nhl patients attending chemotherapy sessions at the medical investigations and treatment (mitu) at mater dei hospital accepted to participate. a questionnaire was compiled to evaluate adherence to oral chemotherapy and to assess pharmacist intervention. the questionnaire was divided into sections (a-c). the same questionnaire was used for both the first interview (t = ) and after weeks (t = ). an additional section (d) was incorporated at t = to evaluate pharmacist intervention. section a consisted of questions regarding patient management of lymphoma. section b incorporated the morisky -item medication adherence scale (mmas- ) to evaluate adherence to oral chemotherapy. a total mmas- score of zero indicates high adherence, a score between and indicates medium adherence and a score between and indicates low adherence. section c consisted of additional questions regarding medication adherence. between t = and t = , pharmacist intervention involved providing each patient with an information leaflet which was developed in this study, an individualised treatment chart and verbal advice. ibm Ò spss version and the wilcoxon signed-rank test were used to assess changes in medication adherence between t = and t = . main outcome measures: evaluation of pharmacist intervention on adherence to oral chemotherapy in patients suffering from hl and nhl. results: out of the patients with hl at t = , 'never' missed a dose, missed a dose 'once in a while' and 'sometimes' missed a dose. for the patients with nhl at t = , 'never' missed a dose, missed a dose 'once in a while' and 'sometimes' missed a dose. the reason for missing a dose was forgetfulness. all nhl and hl patients indicated the haematologist as their source of information about the management of lymphoma. of the nhl patients, scored low adherence and scored medium adherence at t = and after weeks (t = ) all nhl patients who participated scored medium adherence. of the hl patients, scored low adherence and scored medium adherence in the first interview (t = ) and after weeks (t = ) all hl patients who participated scored medium adherence. there was a statistically significant increase (p \ . ) in the number of patients who scored medium medication adherence between t = and t = for both nhl and hl patients. conclusion: this study shows how pharmacist intervention and extended professional services could be implemented in the clinical setting to impact on the management of hl and nhl patients. please specify your abstract type: descriptive abstract (for projects) background and objective: in may , an activity of medication reconciliation was implemented in the gastroenterology service to carry on the optimization of the medication care of patients due to the recent computerization of their prescriptions. design: this project, worked in collaboration with the gastroenterology service has been introduced in two medical committees. this activity gathers pharmacy students, the pharmacist, senior and junior doctors. reconciled patients are selected according to several criteria (advanced age, poly pathological, poly-medicated and those for whom a drug background is difficult to retrieve for the medical team). a minimum of information sources is used for the collection of the drug background. all information are synthetized on a paper, validated by the pharmacist and discussed again with the prescriber. results: on a -month period, patients were reconciled with on average age of . the reconciliation is executed on average . days after the entry in the service. . % of reconciliations are retroactive. the main sources of information used for the collection of the drug background are: in . % of the cases an oral interview with the patient and/or the family; in % of the cases the prescriptions, the hometown pharmacist ( . %) and a medical letter ( . %). . drugs are on average on the hospital prescription, and . % ( / ) of the patients are concerned with at least one non intentional divergence (nid). on average there are . nid/patient and . intentional divergences (id)/patient. the main types of nid are omissions ( . %), drug dose errors ( . %) and errors in administration frequency ( . %). after the detection of nid, the proposed modifications to the prescribers are accepted in more than % of the cases ( / ). the average time of a reconciliation is min. exchanges on the id and nid are made with the junior doctors in . % of the cases. conclusion: some nid are occurring for . % of the reconciled patients. it is therefore necessary to extend this new activity to reconciliation in other services in order to increase the interception of eventual medication mistakes and allow their correction. please specify your abstract type: research abstract background and objective: diuretic therapy is routinely used in the management of congestive heart failure (chf).,compliance with clinical practice guidelines is reported to result in improved outcomes for patients with chf such as reduced exacerbations. the aim was to assess the effect of pharmacist intervention on adherence to diuretic treatment in a hospital and community pharmacy scenario. setting and method: the study was undertaken at karin grech hospital (kgh), a geriatric and rehabilitation hospital, and in one community pharmacy. inclusion criteria for patients recruited from kgh were age over years, suffering from chf and on bumetanide therapy. the validated -item morisky medication adherence scale (mmas- ) was administered to patients on admission (t = ), repeated after two weeks hospital stay (t = ) and again one-month post-discharge (t = ). a total mmas- score of zero indicates high adherence, a score between and indicates medium adherence and a score between and indicates low adherence. in the community setting patients on diuretic therapy were chosen by convenience sampling. the same adherence scale was administered prior to pharmacist intervention (t = ) and one-month after pharmacist intervention (t = ). pharmacist intervention in the community setting involved dissemination of an informative leaflet regarding chf and diuretic therapy developed for the purpose of this study. main outcome measures: impact of pharmacist intervention on adherence to diuretic therapy in chf patients. results: a total of patients were recruited from the hospital setting, of whom were female and were male with a mean age of years (range - years). on admission (t = ), patients scored high adherence, scored medium adherence and scored low adherence to bumetanide therapy. following weeks at the hospital (t = ), the number of patients scoring high adherence increased from to and the number of patients scoring low adherence decreased from to . one-month post-discharge (t = ), patients scoring high adherence decreased from to and patients scoring low adherence increased from to (p \ . ). a total of patients were recruited from the community pharmacy, of whom were female and were male, with a mean age of years (range - years). after pharmacist intervention (t = ), the number of patients scoring high adherence increased from to , while the patients scoring low adherence decreased from to (p [ . ). conclusion: pharmacist intervention in the hospital setting improved adherence to bumetanide therapy. in the community pharmacy setting, there was a slight improvement in the compliance. pharmacist monitoring and patient support is important post-discharge to ensure patient compliance to therapy. conclusion: surveillance of aeds may be followed by combination of data from adverse drug reaction databases and drug utilisation data from prescription databases. focus on reporting adverse reactions is important for pharmacists and clinicians, especially for newly approved drugs. awareness of increased exposure of aeds to new groups of patients followed by data regarding safety aspects is important and contributes to improved pharmacovigilance. please specify your abstract type: research abstract background and objective: the medication review of polymedicated patients is a priority shared among all healthcare professionals. a multidisciplinary approach of these patients is necessary to achieve the best results for their treatment ( ) . the objective was to analyse the rate of acceptance of the recommendations made by the primary care pharmacist (pcp) to the general practitioner (gp) regarding the treatment of polymedicated patients. setting and method: setting: a primary health care centre ( , population). method: a review of the medical records of polymedicated patients (c chronic drugs for c months). the patients' data were collected from january to june from their clinical records. statistical descriptive analysis of data was performed. main outcome measures: drug related problems (drp) for each patient: interactions, contraindications, inadequate dosages, nonindicated drugs, omission of a necessary drug, duplications, medication with low therapeutic effect, and inappropriate medication for patients c years old. treatment alternatives proposed to gp's by pcp were also measured. results: patients were included in the study (average age: . ± . , % women). out of the patients, interventions were laid out to reduce the risks of drp's and to improve the efficiency of treatments. % of patients presented some drp or some intervention to improve the efficiency of their treatment, this mean an average . interventions for patient. the prevalence of intervention proposals were: non-indicated drugs ( %), interventions for improve the efficiency of treatments ( %), interactions ( %), inappropriate medication for patients c years old ( %), contraindicated drugs ( %), duplications ( %), medication with low therapeutic effect ( %), inadequate dosages ( %) and omission of a necessary drug ( %). % of these intervention proposals were accepted by the gp: % of the accepted proposals were carried out and from the remaining , . % led to a prescription from a specialist physician. in % of the cases, the patient did not accept the changes. . % were not carried out due to other issues. the main drug related problem was the prescription of non-indicated drugs and the most involved drug was omeprazole. conclusion: acceptance by gp's to changes proposed by primary care pharmacists was high. a significant number of changes was not accomplished due to the negative response by some patients and led prescriptions from a specialist physician. the gp greatly values the multidisciplinary aid in approaching the complexity of polymedicated patients. background and objective: case-reports provided evidence that influenza infections, particularly severe episodes, may exert neuronal damage in the cns and thereby increase the risk of depression. it was the aim of this study to analyse the association between influenza infections and the risk of developing incident depression. setting and method: we conducted a case-control analysis using the large uk-based primary care database clinical practice research datalink (cprd). this database contains anonymous longitudinal data from primary care. at present, it contains over million person-years of data from some million active patients. the study encompassed , patients below the age of years with an incident major depression diagnosis between and , and we matched each case to one control patient on age, sex, general practice, number of medical encounters, and years of history in the cprd prior to the index date. main outcome measures: major depression diagnosis was identified by read-codes based on icd- codes (f ), with a minimum of three prescriptions for antidepressant drugs recorded after the diagnosis. we calculated relative risk estimates of developing depression in association with previous influenza infections, stratified by the number, timing and severity of such events, and we adjusted for a variety of comorbidities, smoking status, alcohol intake, body mass index, use of oral corticosteroids, and benzodiazepines. results: patients with a previous influenza infection had an increased risk of developing depression (or . , % ci . - . ) compared to patients with no history of influenza infections. a recent influenza infection recorded within - days prior to the index date yielded an adjusted or of . ( % ci . - . ), and an increasing number of previous influenza infections was associated with increasing odds ratios (c recorded influenza infections, adjusted or . , % ci . - . ). we did not see any differences in the relative depression risk associated with influenza with regard to a previous influenza vaccination. conclusion: this study suggests that influenza infections are associated with a moderately increased risk of developing depression. please specify your abstract type: research abstract background and objective: warfarin is known for its interactions with many drugs. elderly patients are particularly sensitive to warfarin interactions. to evaluate the incidence of potential drug interactions when prescribing new drugs to elderly patients on warfarin, a prospective observational study was conducted. setting and method: patients on warfarin older than years were included and monitored for months in community pharmacies in croatia. data regarding new prescribed drugs was obtained from pharmacy records at the moment of dispensing or by patient selfreporting. the potential interacting drugs were identified using the lexicomp Ò lexi-interact online software. only the clinically significant (levels c, d, x of clinical significance as classified by lexicomp Ò lexi-interact online) interactions were included in this analysis. main outcome measures: number of new proscribed drugs, level of interaction with warfarin, mechanism of interactions. results: we included elderly patients with an average age of years. in the follow-up period, new drugs were prescribed to patients ( . %). there were prescriptions of new drugs and ( . %) of those were drugs with a clinically significant interaction with warfarin. there were prescriptions of drugs with level c of interaction ( . %), and ( . %) with level d. there were no drug interactions of level x. in the group with level c the most prescribed drugs were antibiotics with prescriptions: amoxicillin/clavulanate %, clindamycin %, ciprofloxacin %, norfloxacin %, azithromycin %, cefuroxime %, clarithromycin %, doxycycline %. the remaining prescriptions included tramadol with paracetamol %, rosuvastatin %, simvastatin %, fluvastatin %, levothyroxine % and torasemide %. the dominant mechanism of the potential interactions was pharmacokinetic. in the group with level d the most prescribed drugs were nonsteroidal anti-inflammatory drugs with prescriptions-diclofenac %, ibuprofen %, indomethacin %. among other drugs, prescriptions were antibiotic sulfamethoxazole with trimethoprim %, fenofibrate %, miconazole %, and fluconazole %. the dominant mechanism of the potential interactions was pharmacodynamic. conclusion: pharmacists should actively monitor prescribing of new drugs to elderly patients on warfarin in order to reduce the risk of clinically significant drug interactions. please specify your abstract type: research abstract background and objective: explicit criteria of potentially inappropriate medications in the elderly (pims) have been published in the usa, canada, australia and many eu countries. there is a lack of studies describing prevalence of pim use in central and eastern europe. the aim of the eu cost action initiative wg b ( wg b ( - is to evaluate the registration rates and use of pims in central and eastern europe compared to other eu countries participating in this initiative. this abstract describes preliminary findings on different registration rates of pims in different eu countries. setting and method: researchers/members of the eu cost action initiative from the czech republic, serbia, hungary, spain, turkey and portugal were asked to fill in evaluation tables for the list of pims in the period - / . items available in these evaluation tables related to: registration of individual pims on the pharmaceutical market, registered doses, drug forms, availability of pims on prescription or as otc drugs, prescription limits and the most frequently used brand names. data were evaluated using comparative descriptive statistics. main outcome measures: overall prevalence of registered pims in different countries, cross-country differences in availability of individual pims. results: of pims . % were registered in at least participating country. for the czech republic ( . %), turkey ( . %), spain ( . %) and hungary ( . %) overall prevalence rates of registered pims were found to be similar. however, these prevalence rates substantially differed in serbia (low prevalence- . %) and portugal (high prevalence- . %). substantial differences were found also in the lists of individual pims registered in different countries. these lists were similar in spain and portugal compared to the czech republic, hungary, serbia and to turkey. conclusion: although overall prevalence rates of registered pims were similar in the majority of evaluated countries (except serbia and portugal), availability of individual pims was substantially different. our pilot results confirmed that there are substantial geographical/ regional differences in europe in the lists of pims available (in spain and portugal compared to central and eastern europe and compared to turkey). please specify your abstract type: research abstract background and objective: inappropriate prescribing is a common circumstance found in polymedicated patients. screening tools for identifying potentially inappropriate prescription (pip) and pharmacist interventions for evaluating them have been developed to decrease this ( ) . the aim of this study was to evaluate the effectiveness of a pharmacist provided intervention to reduce pips in polymedicated patients. setting and method: the design was a quasi-experimental study focusing on a single group before and after intervention. the study took place from july to december of at three primary care centres ( , population). polymedicated patients were those using c chronic drugs for c months. main outcome measures: reduction in the rate of pip per polymedicated patient (number of pips found divided by the total number of polymedicated patients) before and after intervention, and the influence of the following variables: type of pip (inappropriate medication for patients c years old, medication with low therapeutic effect, duplication of benzodiazepines (bzd) or angiotensinconverting enzyme (ace) inhibitors, combination of anticoagulant and antiplatelet, combination of non-steroidal anti-inflammatory drug (nsaid) with a diuretic and ace inhibitor, nsaid in cardiovascular disease, chronic antipsychotic in dementia, chronic bzd, or chronic nsaid), gender and age of patients with at least one pip, and the main prescribed drugs involved in the pips based on atc classification system of world health organization. results: there were and polymedicated patients before and after intervention, respectively. . % (n = , before) and . % (n = , after) of the total patients had at least one pip. the number of pips was reduced from to , while the rate of pip per polymedicated patient decreased from . to . , achieving the limit established by the regional health authority. . % (before) and . % (after) of patients had more than one pip at the same time, up to pips per patient. before and after intervention, more than half of patients with at least one pip were c years old, and approximately out of were c years old. also before and after intervention, out of patients with chronic nsaid and with bzd duplication were women. out of patients with combination of anticoagulant and antiplatelet were men. the main pips before and after intervention were, respectively: chronic prescription of bzd ( . vs. . % of the total pip), medications with low therapeutic effect ( . vs. . %) and inappropriate medication for patients c years old ( . vs. . %). the main atc group involved in the total of pips was drugs for the nervous system, and the five most prescribed drugs were all bzd (lorazepam being the first). conclusion: pharmacist provided intervention was able to reduce pip in polymedicated patients. gender, age and atc classification of drugs involved were factors in the pips. please specify your abstract type: research abstract background and objective: up to % of women are exposed to selective serotonin reuptake inhibitors (ssris) during pregnancy. information on their effect on birthweight and gestational age remains conflicting. the aim of this sibling-controlled prospective cohort study is to address shared genetic and family-level confounding to investigate the effects of prenatal ssri exposure and maternal depression on birthweight and gestational age. setting and method: we used the norwegian mother and child cohort study (moba) and the medical birth registry of norway (mbrn). our study population consisted of siblings; were prenatally exposed to ssris and were unexposed to any antidepressant medication. random and fixed effects analysis with propensity score adjustment was used to evaluate the effects on birthweight and gestational age. main outcome measures: birth weight. gestational age. results: ssri exposure during two or more trimesters was associated with a decrease in birthweight of g [ % confidence interval (ci) to ] and a decrease in gestational length of . days ( % ci . to . ). neither maternal ssri use in one trimester, lifetime history of major depression nor depressive symptoms during pregnancy were associated with these pregnancy outcomes. conclusion: prenatal exposure to ssris during two or more trimesters may decrease birthweight and gestational length. our results indicate that neither maternal depression nor shared genetics and family environment fully explain this association. please specify your abstract type: research abstract background and objective: the drugs burden index (dbi) is a tool to evaluate the burden of medications with anticholinergic and sedative effects and this exposure has been associated with poorer physical and cognitive function in older people. objectives were; to determine the cumulative burden of anticholinergic and sedative medicines in older adults with intellectual disability (id) using the dbi, to examine the relationship between dbi score with demographics and comorbidity. setting and method: data from wave of the intellectual disability supplement to the irish longitudinal study on ageing (ids-tilda), a nationally representative study of ageing people with id in ireland. dbi scores were calculated for all participants with available medication data (n = ). bivariate associations between dbi and demographic and clinical characteristics were examined with a significance level of . main outcome measures: dbi scores of participants categorised into low ( ), medium ( - ) and high (c ). dbi score categories were related to demographics, cognitive effects and to a modified functional comorbidity index (fci), which is associated with physical function in older adults. results: of participants, . % ( ) had dbi exposure; . % were exposed to any anticholinergic medication, . % to any sedative medication; mean number of dbi medications . (± . ), mean dbi score: . (± . ). ( . %) participants had dbi score , ( . %) - , and ( . %) c . antiepileptics accounted for the greatest contribution to cumulative score ( . %), antipsychotics ( %) and antidepressants ( %). there was no significant association between higher dbi score and sleep difficulties (p = . ). there was a significant age gradient associated with higher dbi score (p = . ) and significant association between higher scores and increased comorbidity scores; mean fci of . in those with dbi c , . in dbi - and . in those with dbi . conclusion: cumulative exposure to sedative and anticholinergic medicines was high in older adults with id. higher dbi scores were associated with higher comorbidity and associated poorer physical function. optimising use of medications with anticholinergic and sedative effects through medicines review by pharmacists as part of multidisciplinary teams using a tool such as the drug burden index may reduce functional decline and improve quality of life among older adults with id. please specify your abstract type: research abstract background and objective: poor adherence to pharmacotherapy may have considerable consequences for the patients' health and for healthcare costs to society. there was observed that diabetes patients have higher risk of later health complications development. it is necessary to be adherent to non-and pharmacological recommendations as well, to improve the clinical outcomes and decrease the cardiovascular risk (cvr). the aim of this study was to evaluate the medication adherence and cvr in group of patients with diabetes, and to find an association between them. setting and method: the methods were based on a questionnaire survey using a modified -item morisky score and score charts ( ). medication adherence and cvr were evaluated in the whole group (n = , males and females, range - years) as well as in subgroups according to age, gender, (no-/ex-) smoking, level of education, residence, number of used medicines, exercises, compliance to the diabetic diet, and total cholesterol levels. the survey was realized in three ambulatory diabetic centres in slovakia. the study has been approved by ethics committee of university hospital bratislava -ruzinov. all participants signed an informed consent. main outcome measures: the results of medication adherence were evaluated as follows: points = full adherence, - points = partial adherence and - = non-adherence. the cvr (estimating -year cardiovascular attack risk) was evaluated according to score charts using data from questionnaire and medical records-gender, age, smoking, total cholesterol levels and blood pressure. the results showed a partial medication adherence in the study group in average ( . ± . ). the average value of cvr in the study group was . %. the highest average medication adherence has been observed in males b years ( . ), with elementary education ( . ), in ex-smokers ( . ), in patients with regular physical activity-at least times a week ( . ), in patients non-adherent to the diabetic diet ( . ), in patients using medications ( . ), and in patients with satisfactory ( . - . mmol/l) total cholesterol levels ( . ). the lowest cvr has been observed in females b years ( . %), in no-smokers ( . %), with elementary education ( . %), in patients with irregular physical activity ( . %), in patients adherent to the diabetic diet ( . ) , in patients using medications ( . %) and in patients with satisfactory ( . - . mmol/l) total cholesterol levels ( . ) . on the other hand, the highest cvr has been observed in males [ years ( . %), smokers ( . %), secondary educated patients ( . %), without any physical activity ( . %), in patients partially adherent to diabetic diet ( . %), using medications ( . %) and, surprisingly, in patients with satisfactory (\ . mmol/l) total cholesterol levels ( . %). conclusion: our survey has showed that medication adherence in our study group has been decreased and cvr has been increased. cvr and adherence to pharmacotherapy in the study group did not correlate with each other. the medication adherence, cvr and their relationships are specific in every patient. please specify your abstract type: research abstract background and objective: studies show that quality of life (qol) of patients with diabetes mellitus can influence medication adherence, satisfactorily improving clinical outcomes and reducing the morbidity and mortality rates and disease progression. this applies even upside down-medication adherence could significant contribute to improving patient qol. the aim of this study was to evaluate the medication adherence in group of patients with diabetes, to evaluate their qol and find a correlation between them. setting and method: the methodology was based on a questionnaire survey using a modified -item morisky score and questionnaire eq- d- l, including visual analogue scale (vas). medication adherence and qol were evaluated in the whole group (n = ) as well as in subgroups according to age, gender, level of education, monthly income, number of used medicines and type antidiabetic treatment. the survey was realized in three ambulatory diabetic centres in slovakia. the study has been approved by ethics committee of university hospital bratislava-ruzinov. main outcome measures: the results of medication adherence were evaluated as follows: points = full adherence, - points = partial adherence and - = non-adherence. the qol in levels of dimensions results were evaluated as follows: the lowest qol in every dimension = point, the highest = points. the highest vas evaluation has been points and every patient should mark number on the scale - to indicate his/her health on current day. results: the results showed a partial medication adherence in the whole group in average ( . ± . ). the average value of the qol in the study group was . and vas . . the highest medication adherence has been observed in males ( . ± . ), patients \ years old ( . ± . ), with primary education ( . ± . ), with monthly income over € ( . ± . ) and in patients using medications ( . ± . ). the highest qol and vas (qol; vas) has been observed in males ( . ; . ), patients \ years old ( . ; . ), university educated ( . ; . ) , with monthly income over € ( . ; . ) . qol has been highest in patients using medications ( . ), vas has been highest in patients using medication ( . ). we have observed the highest level of medication adherence in patients treated with combined therapy-with oral antidiabetic agents and insulin ( . ), the lowest in patients treated with only insulin therapy ( . ). highest qol was recorded in patients treated with oral antidiabetic agents ( . ), and the lowest qol in patients with insulin therapy ( . ). the highest vas has been observed in patients using only oral antidiabetic agents ( . ), the lowest in patients using combined therapy ( . ). conclusion: survey has showed that medication adherence and qol in our study group has been decreased. qol and adherence to pharmacotherapy in the study group did not correlate with each other. the medication adherence, qol and their relationships are specific in every patient. the role of health care professionals should be in education and counselling with patients to improve qol and medication adherence as well. please specify your abstract type: research abstract background and objective: to assess the appropriateness of antibiotic prescriptions used for urinary tract infections (uti) in the elderly. setting and method: we included patients aged years and older, hospitalized in the geriatric department and for whom a urine culture was performed between march and may . a prescription was qualified as inappropriate: when the antibiotic prescribed was not the narrowest compared to the culture result, or when there was a contra-indication, or when the treatment duration was shorter or longer than recommended. prescriptions were consistent with the guidelines when they were identical to those adopted by the french society for infectious diseases in december . main outcome measures: appropriateness of antibiotic prescription (type and duration) results: elderly patients were included (women: . % (n = ), mean age: . years). % of antibiotic choices were appropriate and % of treatment durations were consistent to the guidelines. urinary clinical signs were mentioned in the medical files for . % of the cases (n = ). patients received an empirical antibiotherapy ( . %). . % (n = ) of urine cultures were positive with bacteria, escherichia coli being the most prevalent (n = ). the urine culture results led to a change in antibiotics for . % of the cases. for cystitis, . % of the antibiotics chosen were appropriate (n = ). the main reasons of non-conformity were the lack of deescalation (to amoxicillin or pivmecillinam), and the prescription of ciprofloxacin when the bacteria was in vitro resistant to other fluoroquinolones. the average duration of effective antibiotherapy for cystitis was . days (appropriateness: . % (n = )). for pyelonephritis, . % of the antibiotics chosen were appropriate (n = ). the average duration of effective antibiotic treatment was . days (appropriateness: . % (n = )). . % of the patients had a transurethral catheterization (n = ). another infection was diagnosed for . % of the patients (n = ). conclusion: according to these results, it appears important to reemphasize to the prescribers the guidelines around the uti diagnosis and treatment in order to improve the prescriptions appropriateness in elderly patients. it is particularly necessary to promote the de-escalation of antibiotherapy (with pivmecillinam for example which has recently become available in our hospital) and to insist about the recommended durations of treatment. please specify your abstract type: research abstract background and objective: to measure the use of potentially inappropriate medications (pim) in the general elderly population several criteria lists exist, e.g., beers criteria. last year, a set of explicit criteria for assessing pharmacologically inappropriate medication use in nursing homes was developed; the norwegian general practice-nursing home criteria (norgep-nh). the aim of this study was to investigate the prevalence of pims in nursing home patients using this new assessment tool. furthermore, we studied possible associations between the use of pims and factors like gender, age, geographical area and the number of drugs used. setting and method: cross-sectional study comprising nursing home patients from two geographical different regions in norway; tromsø city (n = ) and lofoten islands (n = ). data was collected from november to january . pims were identified by norgep-nh. we used logistic and poisson regression to examine possible associations between the use of pims and factors like gender, age, geographical area and the number of drugs used. main outcome measures: number of pims per patient, and odds ratios (or) and marginal effects for associations. results: nursing home patient used a mean (sd) of . ( . ) drugs; . ( . ) regularly and . ( . ) as needed. at least % of patients used one pim. concomitant use of three or more psychotropic drugs was the criterion most commonly identified ( %), followed by the use of antidepressant ( %) and hypnotics ( %). an increasing number of regularly used drugs increased the odds of having pims (or: . ), as well as it lead to . more pims per extra drug used. on average, patients c years had . fewer pims than patients \ years. no statistical significant associations were seen between having pims and gender, nor geographical area and the use of as-needed medication. yet, statistical significant differences were identified in some criteria. conclusion: this is the first study that explicit uses norgep-nh. our results confirm that nursing home patients often use potentially inappropriate medications. this is an area where further work is necessary, not to measure the prevalence of pim, but to develop interventions in order to prevent pims from being used. pe : use of pharmacy dispensing data to measure adherence and identify nonadherence with oral hypoglycaemic agents please specify your abstract type: research abstract background and objective: a framework for calculation of adherence for oral hypoglycaemic agents (ohas) based on data from health-insurance claims is available. pharmacy dispensing data aid identification of nonadherent patients in pharmacy practices. however, use of these data for calculation of oha adherence requires additional methodological categories. we examined the impact of different methodological choices on estimation of oha adherence using pharmacy dispensing data. setting and method: a framework for adherence calculation for pharmacy dispensing data was developed from health-insurance claims. a basic scenario was developed from methodological categories. consequences of choices for different parameters within these categories on the scores of the three adherence measures were calculated from dispensing data. main outcome measures: for oha use between july and july , three adherence measures were calculated: ( ) average medication availability (ama); ( ) mean rate of adherent patients with an ama c % (mrap ); ( ) please specify your abstract type: research abstract background and objective: ulcerative colitis (uc) is a chronic inflammatory disease usually affecting young adults and impacting on patient's quality of life. although many biological agents (bas) have been approved for the treatment of moderate-to-severe uc in patients who have responded inadequately to conventional therapy, the selection of bas is controversial due to the lack of head-to-head trials. indirect economic comparisons of these costly drugs are available from national healthcare perspectives that are not the italian ones. therefore, the objective is to evaluate cost-utility of bas for the treatment of refractory moderate-to-severe uc both in italy and in the lombardy region. setting and method: a markov model (considering transition states: remission, clinical response, relapse) was constructed using the software r . . markovchain-package to evaluate incremental cost-utility ratios (icur) of adalimumab, infliximab, infliximab biosimilar, golimumab and vedolizumab treatments of patients over a ten-year time horizon from the perspective of the italian (n) and lombardy region (r) healthcare system. clinical parameters were derived from clinical trials. costs (which have been actualised- . %) were obtained from the national database and regional public tender. utility was expressed as qaly (quality adjusted life years). main outcome measures: icur. results: costs per treatment were different from a n and r perspective (adalimumab - %; infliximab - . %; infliximab biosimilar - . %; golimumab - . %; vedolizumab - %). direct healthcare costs (treatment cost, visits, lab tests, hospital admissions) were calculated over years of treatment per patient: adalimumab (n: € , . , r: € , . , - . %), infliximab (n: € , . , r: € , . , - %), infliximab biosimilar (n: € , . , r: € , . , - . %), golimumab (n: € , . , r: € , . , - . %), vedolizumab (n: € , . , r: € , . , - . %) with associated qaly respectively of . , . , . , . , . . from a n perspective, infliximab biosimilar was dominating compared to all other treatments. the icur of vedolizumab/infliximab biosimilar was € . for years (willingness to pay (wtp) € . /qaly). from a r perspective, adalimumab was dominating compared to all other treatments. the icur of vedolizumab/adalimumab was € , . for years (wtp € , . /qaly). conclusion: national and regional cua produced different results. as regional price discounts can occur, local analyses are needed to estimate the economic impact of therapies to ensure optimal choice. please specify your abstract type: research abstract background and objective: automated dispensing systems (ads) have been implemented to reduce overall medication errors related to picking, preparation and administration of drugs. costs of drug storage between ads and classic dispensing system (cds) had not been yet performed in france. our objective was to assess economic impact of ads compared to cds. setting and method: retrospective quasi experimental study was conducted in university hospitals in , one with ads ( beds, ads) and one with cds ( beds, cds ( ) for ads and ( ) for cds (p \ . ). mean number of costly drug per system was for ads and for cds. the global stock value in the wards was , € in ads and , € in cds representing respectively . and . % of total pharmacy stock value. conclusion: our data demonstrate that despite the same storage capacity, ads allow the storage of more expensive drugs such as innovative drugs fully reimbursed up to national reimbursement prices, due to the lower risk of pilferage. this preliminary study was focused mainly on stock value. subsequently, another study is conducted to evaluate cost of these two drug storage systems, satisfaction of pharmaceutical technicians and nurses and time allowed for systems reloading. please specify your abstract type: descriptive abstract (for projects) background and objective: in france, pharmacists are not entitled to substitute an original biological drug with its biosimilar, due to specific issues of efficiency, safety, and patient monitoring. our hospital referenced a biosimilar of infliximab on january . according to the french medication safety national agency's recommendations, it has been decided that naïve patients would be treated with biosimilars, and changes between specialties would be proscribed. the objective is to compare prescribing practices between infliximab and its biosimilar, year after its introduction. design: a database tracking patients treated with infliximab was set up. data comparing prescribing practices of biosimilar and reference treatment were analysed between june and may . regional and national infliximab consumption between january and february were used to compare the practices of our hospital with other hospitals. the past and future savings were estimated from repayments data of the regional health agency. results: infliximab was administered to patients, of which ( %) were naive. patients were treated with biosimilar (i.e. . % of all patients), of which were naive. in the end, nearly % of naive patients actually received the biosimilar and . % of patients treated with infliximab switched specialties during treatment. in % of cases, biosimilar prescriptions were consistent with the recommendations (vs. % for infliximab). in % of cases the off-label prescriptions of the biosimilar were explained in the patient record (vs. % for infliximab). in february , the share of biosimilars was % in france, % at regional level and % locally. in year, infliximab and its biosimilar's consumption in our hospital have increased by % in quantity and only % in expenditure (+€ m expenditure). negotiating a lower purchase price and costs has enabled the hospital to save € , (vs. € , during the previous year). because of the decline of refund rates, the gains would have been zero without using the biosimilar but € , if it had been prescribed to every naive patient. conclusion: current data from the literature on security and effectiveness of infliximab biosimilars are very reassuring and the french medication safety national agency doesn't exclude the possibility of changing specialties during treatment. in our hospital, there is room to improve the efficiency of treatment with infliximab. feedback on prescribing practices will be given to prescribers and a campaign to widespread prescriptions of biosimilars will be made. the arrival of biosimilars on the market is a real economic opportunity for hospitals, which are increasingly financially constrained in particular by the arrival of therapeutic innovations which are more and more expensive. setting and method: the study used health claims data on prescription ppis from st january to st july obtained from the health insurance institute of slovenia. to assess medicine use and costs before and after trp implementation data were aggregated into four periods: jan-dec , pre-baseline period; jan-dec , baseline period; jan-sept , transition period between announcement and introduction of trp; oct to jul , period after trp enforcement. main outcome measures: medicine costs; defined daily doses (ddds) dispensed per inhabitants per day; market share; herfindahl-hirschaman index (hhi); number of active substance switches; number of exceptions when medicine is fully reimbursed since physicians may choose option ''not to switch medicine'' when adverse consequences are predicted. results: average monthly cost of ppis declined from € , , in pre-baseline period to € , in period after trp introduction although the consumption increased from . to . ddds/ inhabitants/day. cost of ppis decreased the most in baseline period ( %), however trp induced . % cost reduction compared to the transition period. the reference pantoprazole was market leader already in the transition period, but its use increased significantly after trp introduction and represented % of total ppis consumption. manufacturers' market shares were constant before trp, whereas trp caused decrease of the largest market share for %. still, this resulted in the minor market concentration change; hhi was on average . before and . after trp introduction. further, at least one active substance switch was detected in approx. and % of patients before and after trp introduction, respectively. similarly, the proportion of exceptions when medicine was fully reimbursed increased from . % in transition period to . % in period after trp introduction. conclusion: enforcement of trp for ppi contributed to approx. € m annual cost savings. from the payer's perspective the new policy was proven to be effective in reducing pharmaceutical expenditure; however trp also affected physician prescribing pattern and use of ppis. pec : blood coagulation factor: improvements of the supply chain samantha oses * , serri traore, sonia caroline sorli, lea damery, philippe cestac, sylvie pomies, julien tourel please specify your abstract type: descriptive abstract (for projects) background and objective: most of the antihemophilic factor (ahf) must be held by a teaching hospital to face serious bleeding events. to ensure better availability, offsite-stocks at critical points are required (emergency unit, intensive care unit, etc.). however, this management system increases the risk of economic loss and alteration of the quality due to expired products. in this context, we carried out an optimization of the supply and management system of the ahf. to identify critical points of the supply and management system and to implement improvement solutions. design: a multidisciplinary working group belonging to a regional management centre of haemophilia was set up. two lines of improvement were discussed: i) optimization of stocks ii) optimization of the supply system. results: the optimization of stocks has led to the modification of the threshold of the lowest stock (ls) for ahf out of . in % of cases, this stock modification has exceeded %. the overall cost of ls has been reduced by . % ( , €) for the general stock at the central hospital pharmacy (hp) and by . % for offsite-stocks ( , €). the ahf mainly involved in this reduction was fvii mg ( , €), then followed by the strengths of mg and mg ( , € for each). in order to improve the ahf management, several propositions have been implemented: ( ) developing an online, easily accessible and monthly updated spreadsheet that displayed several accurate data such as the shortest expiry date and the storage location. this operative tool is shared between all pharmacists involved in ahf management in order to facilitate a stock rotation and decrease economic losses, ( ) regular reminders to physicians and health care staff concerning the guidelines for inventory management and the importance of checking the drug expiry date, ( ) presentation of the financial results and raising awareness on ahf costs to the medical consultant[ppip ] and ( ) optimizing stock distribution based on consumption on the different hospital sites for better patient care management (pcm). conclusion: this optimization of stocks and improvement of the supply chain have led to a direct cost saving of , €. however, a more accurate assessment has to be performed to quantify the direct and indirect impact on pcm and cost saving. this work has been done in a context of a sharing operative network at a regional level. the aim of such project is to share, to optimize and to improve practices, knowledge, human and medical health resources at a widespread level to enhance the security and quality of health services and to promote cost and time saving. please specify your abstract type: descriptive abstract (for projects) background and objective: the overall pharmaceuticals consumption in hospitals is rising, which has led to an increasing expenditure, challenging health care professionals and threatening patients safety. clinical trials in hospitals have increased over the past few years and currently play an important role, giving access to new investigational medicinal products and also avoiding costs with standard treatments. the objective of this study is to evaluate the savings of centro hospitalar do porto, a central university hospital with beds and currently clinical trials, with patients included in clinical trials between january and may . design: retrospective observational study over months. all the clinical trials ongoing between january and may were analysed and the data was collected based on: pathology and doses established; number of treatments per patient and the medium prices of standard treatments that patients would be receiving if they were not in the clinical trial. results: there were clinical trials ongoing between january and may , but only were selected to be included in this study. the total number of patients included was . the clinical trials selected for this study were conducted in medical specialties: in dermatology, in immunology clinical unit, in hemato oncology, in gastroenterology, in ophtalmology and in neurology. during these months, with all ongoing clinical trials, centro hospitalar do porto was able to save, in medical products, more than million euros. conclusion: during the period of time established, of the clinical trials ongoing, were not selected due to: not including patients or not having an alternative treatment. hospitals and patients can benefit from clinical trials not only financially but also by preserving resources and medication. on centro hospitalar do porto, the pharmacists specialized in clinical trials, as members of the study team, are more and more required to perform specific tasks, their contribution has been increasing over the years and also have become more aware of all the advantages from participating in clinical trials. these savings can be used to provide a better assistance and contribute, in general, to a higher quality health care. please specify your abstract type: descriptive abstract (for projects) background and objective: several studies show a misuse of opioid maintenance treatment (omt) in detention. in fact, buprenorphine (bup) when it's misused, could present the same effects as heroine. in order to reduce misuses, the pharmacist decided to switch all the patients under bup to buprenorphine/naloxone (bup/nlx). bup/ nlx prevents patients from misusing by a withdrawal syndrome when it's issued by another route of administration than sublingual route. in france, bup/nlx is more expensive than bup which may explain why this therapeutic strategy is not often observed. the purpose of this study is to evaluate the extra cost after switching patients from bup to bup/nlx in order to decide if this choice could be maintained. design: to identify our population, we used the administration reports drugs written by nurses. please specify your abstract type: research abstract background and objective: haemophilia b is an x linked genetic disorder characterized by spontaneous or prolonged haemorrhages due to factor ix (fix) deficiency . within the next few years, new treatments are willing to hit the market. among them are recombinant extended half-life products that will reduce by half the number of injections and will potentially improve the patient quality of life. the aim of the study is to describe the development of haemophilia treatments market between and and to forecast the potential impact of these new therapies on the haemophilia market. setting and method: national and french hospitals of paris (aphp) consumption data of fix between and have been studied. new therapies in development or soon to be marketed have been identified. potential benefits and interest in the therapeutic care of these new products were discussed with haemophilia's medical experts. main outcome measures: quantity (ui) and value (euros) of fix aphp and national consumption. results: in , recombinant (rfix) and plasma-derived factors (pfix) were on the french market. the ap-hp's purchases of these factors represent almost million ui and million euros, which comprise % of national fix expenditures. in france and aphp, ambulatory care is a major part of the use of these treatments with nearly % of the fix purchases in . french rfix consumptions are higher than pfix consumptions ( % against %). in the ap-hp hospitals, rfix even account for % of consumptions against % for pfix. both national and ap-hp rfix purchases have steadily increased between and . the added competition arising from new treatments may lead to more competitive market procedures in hospitals and may reduce costs of haemophilia treatments. according to haemophilia doctor, long-acting (la) fix would offer obvious benefits like fewer infusions and presumably fewer bleeds. these treatments will mainly be used in a prophylactic wayin ambulatory care-than in a curative way (such as surgical use). conclusion: the therapeutic extent of these new treatments is still hard to define. the choice of treatment must remain consensual between physicians and patients. please specify your abstract type: descriptive abstract (for projects) background and objective: good practice about medicines imposes to health institutions a close monitoring of prescriptions, especially off-label prescriptions. patient care should take into account clinical profile, respect of guidelines and health expense control. we report here a case highlighting the significant role of the clinical pharmacist in care units to ensure medication good use in a castleman syndrome, a rare disease due to human herpesvirus (hhv- ) and associated with human immunodeficiency virus (hiv) infection. design: case report. results: our patient, a years old man (creatinine clearance rate (crcl): ml/min), was diagnosed with hiv infection in february (cd at ui/l), leading to introduce a therapy by emtricitabine-tenofovir, darunavir, and ritonavir. the evolution was hampered by repeated episodes of acute renal failure (arf; crcl: ml/min) and pancytopenia (hemoglobinemia at . g/dl, leucopoenia at . g/l, and thrombopenia at g/l). because of hhv blood pcr at copies/ml, transient crises with pancytopenia, arf, and hiv infection, a diagnostic of kaposi sarcoma herpesvirus (kics), an atypical castleman syndrome, was retained. given the lake of data in literature for this rare disease, a multidisciplinary team (medical specialists and clinical pharmacists) was gathered to choose an appropriate therapeutic strategy. treatment regimen consisted of: day , intravenous etoposide at mg; day , rituximab at mg/ m ; following one week later by rituximab day and oral etoposide at mg the day after. good communication between medical specialists and pharmacists enables the patient to get an optimal and personal treatment. relaying the information by clinical pharmacists in care units to pharmacists in charge of good practice facilitate the reimbursement. conclusion: clinical pharmacists in care unit help to optimize therapeutic strategies according to their experiences and scientific works. cooperation with physicians is improved, as well as prescriptions follow-up of off-label drugs, and health patients fully respected. quality and relevance of prescriptions are strengthened, with a better control of economic expenses. please specify your abstract type: research abstract background and objective: the maltese government launched the hpv vaccination scheme in and the national healthcare system (nhs) has since provided the cervarix Ò vaccine free of charge to girls aged . the aim of this study was to assess the cost of the administration of hpv vaccines in the healthcare system of malta. this study was based on the scheme provided by the nhs. the number of girls born per year was used to estimate the annual cost for vaccinating year old girls, based on the wholesale price and tender price respectively. the estimated yearly cost using the wholesale price was approximately € , while the average estimated cost based on the tender price was approximately € , . this signifies that cost savings based on the tender price compared to wholesale costs were of approximately € , . the cost for the cohort who completed the three dose schedule using the tender price on average was of € , per year. this result proved to be more than the anticipated cost. a reason for this could be that the number of girls aged increased possibly due to an influx of immigrants. including boys in the vaccination scheme would increase costs by an average of € , per year. conclusion: this study shows that procuring branded vaccines using the tendering process reduces expenditure for the government and the tax payer. wholesale prices were found to be more expensive than tender prices. this proves that the tendering system in malta is a potent system with many advantages for the tax paying public. the impact of the tendering process must therefore, be safeguarded. please specify your abstract type: research abstract background and objective: with the old age, presence of comorbidities, and overcrowding in mass gatherings such as the annual hajj pilgrimage in saudi arabia, there is a high risk of spreading infectious diseases among pilgrims and then within their country of origin. knowledge and application of hygiene principles in such an environment is therefore important to reduce the transmission of infectious diseases. up to date, there have been no studies to evaluate pilgrims' knowledge, attitude and practices toward mers-cov during the annual hajj pilgrimage in order to see whether there is a need for these aspects to be improved. setting and method: a cross-sectional survey study was conducted with a convenience sample of participants. participants were pilgrims, aged over , and able to speak arabic or english. a selfadministered structured questionnaire was distributed during hajj season in mecca. descriptive and multiple linear regression analysis were used in data analysis. main outcome measures: assessing pilgrims' knowledge, attitude and practices regarding mers-cov. results: two hundred and fifty-seven participants completed the study, % of whom were female, and the median (iqr) age was ( . - . ) years. pilgrims had moderately correct knowledge and accurate attitudes towards mers-cov with median scores of (iqr - ) and (iqr: - ) respectively. they were less educated about management ( %), hallmark symptoms ( %), high-risk individuals ( %) and source of coronavirus ( %). almost % of participants showed a negative attitude towards the use of protective measures such as avoiding food prepared under unsanitary conditions and contact with live animals. some participants ( %) were unable to comply with hygiene practices, particularly washing hands with soap and water or disinfectant after sneezing/coughing and wearing a face mask in crowded areas. educational level and employment status were significantly associated with knowledge whereas gender and age were significantly associated with attitude and practices respectively (p \ . ). the correlation between knowledge, attitude and practices was significant (correlation coefficient: . ; p \ . ). better knowledge was found to be a predictor for positive practice. conclusion: these findings aided in the assessment of the adequacy of current pilgrims' educational measures. they will also provide insight when designing future interventions to promote specific messages to improve knowledge, change attitude and improve practice regarding mers-cov. please specify your abstract type: research abstract background and objective: the prevalence of type diabetes significantly increased in the paediatric population, which is affected by obesity worldwide. today, type diabetes accounts for % of all cases of new-onset diabetes in adolescents. preventive health care particularly taking place at community pharmacies may involve risk assessment for the children and the adolescents, early referral for seeking relevant medical care and patient education on healthy lifestyle choices. the aim of the study is to conduct a type diabetes risk assessment program for the kids b years of age of whose parents visited the community pharmacies involved in the study and also to identify the behavioural parameters that might be associated with this risk. setting and method: the study was conducted in community pharmacies. all patients with kids aged b years who visited the study pharmacies during one-week period were informed about the study and invited to participate in the study. patients who gave their informed consent were included in the study. all data were provided by the parents. demographic data, height and weight of the kid, as well as data regarding the behavioural features (eating habits, exercising, time spent in front of a screen, etc.) of both the children and the parents were collected using standardized forms. type diabetes risk test consisted of questions and identified subjects at risk. the parent of the kid who was identified to have risk for type diabetes was referred to a physician for further examination. also, information regarding type diabetes and the importance of preventive measures such as converting to a healthy life-style was provided. main outcome measures: main outcome measures were the percentage of kids identified to be at risk of developing type diabetes and the behavioural parameters associated with type diabetes risk. results: the study involved subjects. of the subjects % were identified to be at risk of type diabetes. more girls than the boys had the risk ( vs. . %). those with type diabetes risk were older, taller, heavier and had higher body mass index. they were spending more time in front of a screen (tv, pc, tablet, smart phone); . % were spending more than h a day. although the kids' eating habits were similar for those with and without risk, the parents' of the kids with risk ate out more frequently, consumed rice, pasta and pastry more frequently. both the kids with risk and their parents exercised more regularly and frequently. conclusion: this study shows that pharmacist have a vital role in identifying children and adolescents at risk for type diabetes; thus at early management of this condition. identifying and addressing the behavioural parameters associated with the risk will be helpful in lifestyle modification interventions. please specify your abstract type: descriptive abstract (for projects) background and objective: analyse and promote the reporting of adverse drug events (ade), to improve the quality and safety of care to be able to control the risks. design: a software is available on the intranet website of the institution, to enable health professionals to report ade. the drug and medical devices commission (comedims) of the hospital, centralizes these statements and always makes a multidisciplinary and overall analysis of the event, using a collection sheet which is based on the pdca model (plan, do, check, act). it proposes the nursing and medical teams axes of improvement. results: in , only ade were reported and analysed by the comedims, including from the paediatric centre ( %), particularly sensitized to this issue. health professionals are divided as follows: healthcare executives ( %), nurses ( %), pharmacists ( %), residential students ( %), doctors ( %) and others ( %). the main impacted steps of the drug circuit are: administration ( %), prescription ( %) and the use or implementation of a sterile medical device ( %). identified causes include related following factors: operational tasks and procedures ( %), health professionals ( %), work environment ( %), organization and management ( %), drugs or associated medical devices ( %). the number of ade reports, taking into account the size of the institution, remains very low. in january , the comedims decided to broadcast a communication campaign to promote ade reporting, on the hospital website via the intranet. three months after the release, this document was viewed times, and the number of reports increased by % compared to the same period in . conclusion: in front of the low number of returns of adverse drug events, and relying on the charter of non-punishment, the come-dims wants to increase health professionals' awareness. in our hospital, where e-learning about drug-related iatrogenesis is already available, the communication campaign with poster and analysis of adverse events seems to be a useful complementary tool to enhance awareness of medication safety concerns. please specify your abstract type: research abstract background and objective: the migration of modern social networks to the internet has facilitated the transition of traditional pharmacy networks online. the ubiquitous nature of social media (some) combined with merging of personal and professional personas have led to organisations publishing guidance on online behaviour and responsible use of social media. the research to date on the use of social media as a support for professional practice in general is limited. as the pharmacy profession evolves to embrace the technologies which underpin core services and mainstream online daily social activities, it is important that research tracks and evaluates its use and impact within the profession. the objective of this research was to explore and describe how and why pharmacists interact with hosted networks on social media. setting and method: two one-hour online hosted micro-blogging twitter chats were held in december via the #weph network. topic guides were developed around 'exploring the use of twitter and wepharmacists' in line with the wenetwork guidelines (#wecommunities), informed by existing literature, discussion with the #weph moderator after review by an expert panel. all research was carried out in accordance with university governance processes and association of internet researchers guidelines. themes were inducted from analysing the textual content of the chats using the topic guide as a framework. the research was approved by the school of pharmacy and life sciences ethics committee. main outcome measures: tweets per chat results: each of the chats had over million impressions with participants representing international pharmacy practice. themes of e-professionalism and online privacy emerged as concerns; however, the benefits included using social media for education, networking, support mechanisms and career development. tweets highlighted personal experiences of 'trolling' (angry, offensive behaviour) and the effect on user interaction with social media. twitter was also recognised as a career development tool and, in particular, collaborative outcomes around mentorship networking early career pharmacists with more experienced colleagues. conclusion: results support the responsible use of social media as a force for inclusion, breaking down geographical barriers in support of pharmacy practice. further research is underway including a systematic review of guidance on the use of social media by registered healthcare professionals. please specify your abstract type: research abstract background and objective: it is estimated that half of the , persons with diabetes in norway have not been diagnosed. with early treatment, life expectancy can be increased and the incidence of longterm complications and health costs reduced. community pharmacies may be able to help uncover undiagnosed diabetes, but being diagnosed with diabetes can lead to strong emotional reactions, and how the diagnosis is given may influence the experience. the aim of this study was to explore how norwegian people living with type diabetes (t d) experienced being diagnosed, and what led up to the diagnosis. in addition, their attitudes towards a planned community pharmacy service to identify undiagnosed t d was investigated. setting and method: three focus group interviews with people with t d were conducted using a semi-structured interview guide. eleven participants were recruited through a course about type diabetes. the interviews were audio-taped and transcribed in modified verbatim form and analysed in accordance with malteruds principles of systematic text condensation. the study was approved by the norwegian data protection authority, and did not require approval from the regional committee for medical and health research ethics. main outcome measures: how people with t d describe their experiences of being diagnosed with t d, how the disease was revealed and reactions towards using community pharmacies to perform risk assessment for t d. results: none of the participants were diagnosed due to their own suspicion of having diabetes. some saw their doctor because of unspecific symptoms such as fatigue and thirst, and were thereafter diagnosed with t d. others were diagnosed through a routine checkup. negative reactions like shock, discontent and denial were commonly used to describe the experience of being diagnosed with t d, but some participants also expressed a more relaxed attitude, especially if they were familiar with the disease through family members. participants expressed a strong wish for more and better information following the diagnosis. ''it's a jungle out there'' was used to describe how difficult they felt it was to find trustworthy and understandable information. they described change of lifestyle, side effects from drug use, and stigma as challenges following the diagnosis. while in general the participants were positive to using community pharmacies to uncover undiagnosed diabetes as this could help reduce the number of people who were undiagnosed, some were sceptical. they questioned whether the pharmacy staff had the necessary competence of the for this type of service, and saw it as the doctor's responsibility. conclusion: more information and support when people are diagnosed with diabetes may lead to that the experience being diagnosed will be more adaptable and that the challenges living with diabetes are reduced. community pharmacies are important healthcare providers, and risk assessment of t d at the pharmacy can be valuable. however, the pharmacies may also be helpful to reduce the information gap. please specify your abstract type: research abstract background and objective: chemotherapy-induced nausea and vomiting (cinv) is a disruptive and unpleasant side effect in chemotherapy patients and is associated with decline in patients' quality of life and decrement in the adherence to effective chemotherapy regimens. setting and method: chemotherapy naive patients were included in this study. consistency with guidelines were assessed according to mascc/esmo . flie questionnaire was administered to patients before chemotherapy, and days after receiving chemotherapy to assess the difference in the quality of life due to chemotherapy administration. main outcome measures: patients were categorized into two groups as consistent with guidelines group (acute (gcga) and delayed (gcgd)) and inconsistent with guidelines group (acute (giga) and delayed (gigd)). flie score differences between the two groups were assessed. results: the median flie score for patients prior to chemotherapy was and a dramatic decline was noticed post chemotherapy (flie score ; p \ . ). the post-chemotherapy score were for nausea and for vomiting ( . , respectively). although the flie score differed significantly between gcgd and gigd (p \ . ), these differences were not significant in gcga and giga. conclusion: the significant drop in flie scores in the study ( pre-to post-chemotherapy) reflected substantial declination in patients' quality of life. the lower postchemotherapy flie score of nausea emphasized the negative impact of nausea, and to a lesser extent vomiting on the patients ability to complete normal daily activities such as enjoying meals and maintaining social activities. although there were no significant differences in flie scores between giga and gcga groups for acute cinv prevention, significant differences were noted between gigd and gcgd (p \ . ). the flie score was lower for gigd patients. this result implied guideline inconsistency associated with high incidence of nausea which negatively affect patient quality of life. as for the degree of compliance with gp, the results are expressed as percentage of compliance compared to the ideal of %. prescription criterion was fulfilled to %: all requirements of pntb were performed using standardized procedure. in what concerns validation, % of pntb prescriptions were validated by a pharmacist. the invalidated prescriptions were made outside opening hours of the pharmacy service, which is open monday to friday from : to : and on weekends and holidays from : to : . % of the dispensations were individualized and not pntb stocks were found in hospital wards. as for preparation, % were supplemented with micronutrients. pntb of kabiven peripheral administration ml are not supplemented in our centre. of the remaining prescriptions central administration, % were supplemented. in all cases, the addition of micronutrients was performed in laminar flow hood in pharmacy service and the corresponding galenic validation was performed. finally, in the process of administration, % of pntb identified with a complete label: name of the patient, medical record number, type of pntb, qualitative and quantitative composition, date of administration and infusion rate. conclusion: use practices of pntb of our centre are far from those recommended by the sefh standards. this initial evaluation will serve for improvement measures that increase the quality of prescribing and safe use of pntb, in order to minimize errors that can occur with the use of this therapeutic modality. please specify your abstract type: research abstract background and objective: methadone maintenance treatment was developed in malta in and is provided to patients by sedqa, the national agency against drug and alcohol abuse. methadone is the most frequently prescribed opioid in opioid substitution treatment and is dispensed through a centralised service through the substance misuse outpatients unit. in , patients were in opioid substitution treatment, of who were on methadone. in , the government introduced a take-home methadone program. the prescribing, purchasing and dispensing of methadone are regulated by subsidiary legislation . . the objectives were to determine whether community pharmacists in malta would be willing to dispense and supervise the consumption of methadone and to investigate the involvement of community pharmacies in the development of a regionalised methadone dispensing service. setting and method: the study was set in community pharmacies. a cross-sectional study, through the use of a questionnaire, was performed to quantitatively analyse whether pharmacists in malta would be willing to dispense methadone. the questionnaire consisted of questions divided into sections, with each section assessing a particular aspect of community pharmacists' attitudes towards methadone dispensing. community pharmacies were then chosen via a systematic sampling procedure. a hard copy of the questionnaire, addressed to the managing pharmacist, along with a cover letter, instructions on how the questionnaire was to be returned, and a prepaid self-addressed envelope was distributed via postage to community pharmacies. an online format of the questionnaire was also circulated to community pharmacists through the pharmacy council. data was analysed using spss version . main outcome measures: community pharmacist's attitudes towards methadone dispensing. results: a total of responses were obtained and a response rate of . % was achieved. eighteen percent of the pharmacists (n = ) who responded to the questionnaire worked in a community pharmacy located in the north of malta, % in the centre, % in the south, % in the southeast and % in gozo. thirty-two percent of community pharmacists were willing to dispense methadone to drug misusers. the number of community pharmacists who are willing to dispense methadone increased to % if they were provided with appropriate education and support. twenty-nine percent of community pharmacists were prepared to handle the duty of supervising the consumption of methadone while % had never learnt about methadone and its clinical application within opioid substitution treatment. conclusion: community pharmacists should be provided with education and training regarding methadone substitution treatment before embarking on a new regionalised methadone dispensing service within community pharmacies. this would allow more community pharmacists to become involved in a new dispensing methadone service. pt : evaluation of regorafenib in patients with colorectal cancer please specify your abstract type: research abstract background and objective: the colorectal cancer is the second more frequent cancer in europe and the third in the world. regorafenib is only approved in adult patients with metastatic colorectal cancer who are previously been treated with available therapies or are not considered suitable candidates to these treatments. regorafenib is an oral anti-tumor drug that blocks the kinases involved in the tumor angiogenesis (vegfr , - , - , tie ), the oncogenesis (kit, ret, raf- , braf, brafv e) and the tumor microenvironment (pdgfr, fgfr).in this study, we are reviewed the reports of the patients with colorectal cancer who are been treated with regorafenib in our hospital and analysed the information in order to evaluate the efficacy and safety of regorafenib. setting and method: descriptive and observational study about the use of regorafenib from april to the present day. the variables studied, obtained from the software applications archinet and diraya, were: sex, age, pathology, location of metastasis, posology and adverse effects of regorafenib, tumor markers (cea y ca . ) before and after the treatment with this drug and the mutational state of kras. main outcome measures: the tumor markers cea and ca . only decreased in the . % of the patients after the regorafenib treatment. results: regorafenib was taken by patients ( %men).the average age of these patients was . ± . years old. the patients took regorafenib to treat: metastatic and non-intervened gastrointestinal stromal tumors (gist) e-iv that progressed with the previous treatment of imatinib and sunitinib ( . % patients), intervened colon adenocarcinoma e-iv ( . % patients), sigma adenocarcinoma e-iv ( . % patients) and unresectable and non-intervened rectal adenocarcinoma e-iv ( . % patients).all patients presented metastasis in different locations on the body: liver ( . % patients), diaphragm ( . % patients), intestine ( . % patients) and lung ( . % patients).the % of the patients started the treatment with mg of regorafenib, administrated once a day for weeks followed by one week without this drug; while the . % of the patients started the treatment with mg. however, the . % had to decrease the initial dose and the . % of the total patients had to get off the treatment because of the development of side effects. the most frequent adverse effects were: hypertension associated with headache, hyperbilirubinemia, elevation of ast and alt, intense asthenia. the . % of the patients presents native kras. the native kras was presented in the % of the patients treated with regorafenib who had an appropriate development of the illness (decrease of cea and ca . ) conclusion: the decrease of cea in the . % of the patients and the high development of side effects reveal that regorafenib has low effectiveness and security in the control of the progression of colorectal cancer. in addition, it is supposed that this drug has better results in native kras patients. however, more studies are necessaries in order to demonstrate the effectiveness of regorafenib in this pathology. pt : evaluation of nintedanib in patients with non-small-cell lung carcinoma (nsclc) please specify your abstract type: research abstract background and objective: the nsclc means a high rate of mortality in developed countries. patients diagnosed with nsclc who debut with advanced or metastatic disease have a median survival of months. one of the innovative drugs approved to improve survival in nsclc is nintedanib: an inhibitor of multiple tyrosine kinases, which can be found in some receptors on the surface of cells involves in the growth and spread of cancer cells (''pdgfr'', ''fgfr'' and ''vegfr''). nintedanib is not yet marketed in spain. hospital pharmacists are responsible for applying this treatment as ''expanded drug'', only after the elaboration of an exhaustive report. in this study, we have reviewed all the reports and classified the information in order to present our clinical practice. the objective of this study is to evaluate the effectiveness and safety of nintedanib in patients with nsclc treated in a tertiary hospital. setting and method: descriptive observational study of the use of nintedanib from november to september . sex, age, body mass index (bmi), pathology, smoking habits, line of treatment, posology and adverse reactions of the treatment with nintedanib and tumor markers (cea an ca . ) before and later the treatment with nintedanib were collected from medical history through archinet informatic application. main outcome measures: the tumor marker cea decreased in % of the patients and ca . no decreased in any patient after nintedanib treatment. results: nintedanib was used in patients ( % men and % smoker).the average age of these patients was years old. the average bmi was kg/m ( - ).all patients received nintedanib together with docetaxel for metastatic nsclc with adenocarcinoma histology and with non-mutated egfr and alk in third line treatments. posology: all patients started the treatment with nintedanib mg/ h from day to day every weeks; but patients had to reduce the initial dose to mg/ h ( patient) and mg/ h ( patient) because of some adverse reactions. the side effects were: asthenia, diarrhoea, alteration of transaminases, muscle pain and cramps, weight loss and mucositis. conclusion: the decrease of cea in % of the patients reveals that nintedanib is effective in controlling nsclc progression which involves an increase of the survival and the quality of life of these patients. however, more studies are required to demonstrate the efficacy of nintedanib in this illness. please specify your abstract type: research abstract background and objective: patients with sore throat symptoms often seek fast, meaningful relief when presenting to their local pharmacy. flurbiprofen is a non-steroidal anti-inflammatory drug, which has been developed as a spray and lozenge to provide targeted relief for the main underlying process responsible for the symptoms of sore throats, inflammation. to study the relief provided by flurbiprofen . mg delivered as a spray or lozenge, we conducted a multicentre, randomised, double-blind, double-dummy, parallel group, activecontrolled, single-dose, non-inferiority study. setting and method: adult patients with acute sore throat were randomly assigned to take one dose of either flurbiprofen . mg spray plus a placebo lozenge, or flurbiprofen . mg lozenge plus placebo spray at sites across russia. main outcome measures: patients rated sore throat relief using the sore throat relief rating scale (strrs; a -point scale, = no relief, = slight relief, = mild relief, = moderate relief, = considerable relief, = almost complete relief, = complete relief) at timed intervals throughout h starting from min post completion of first dosing ( min after administration of the spray, and min after the lozenge had fully dissolved). adverse events (aes) were recorded over h post-dose. results: patients were assessed (n = for spray, n = for lozenge). [ % of patients in either treatment group experienced some relief (a score of [ on the strrs) at min post-dose, which increased to % of patients by h. - % of patients reported 'at least moderate relief', which is a well-recognised measure of a clinically meaningful effect at min post-dose, which increased to - % of patients by h. over the h post-dose, a total of drugrelated aes were reported by patients across both treatments and no severe adverse events were reported. conclusion: flurbiprofen . mg delivered as a lozenge or spray provides fast, clinically meaningful relief from sore throat. pt : analising antiangiogenics prescription in an ophtalmology service after a protocol implementation silvia cornejo-uixeda * , ivan de la vega-zamorano, celia aparicio-rubio, olga carrascosa-piquer, manuel prieto-castello, agustin sanchez-alcaraz pharmacy, hospital universitario de la ribera, alzira, spain please specify your abstract type: descriptive abstract (for projects) background and objective: after some years using antiangiogenics in our hospital, we observed a large variety of use. considering the high cost of these treatments, we proposed ophthalmology service to develop a protocol of use, attending efficiency criteria. in this paper, we analyse the protocol implementation repercussion. design: a protocol of use was designed with the main of unify criteria and to use the most efficient treatment depending on the specific situation on each patient. once it was implemented, we compared two periods, the period after the implementation (january-may ) and the period before of it (january-may ). the protocol designed is the following: the cost for each injection and patient was the following: aflibercept €, bevacizumab €, ranibizumab €. results: in the period, patients were treated with antiangiogenics. ( %) with aflibercept, ( %) with bevacizumab and ( %) with ranibizumab. in the period, patients were treated, ( %) with aflibercept, ( %) with bevacizumab and ( %) with ranibizumab. the consumption of aflibercept decreased a %, bevacizumab consumption increased % an ranibizumab increased a %.we also observed, some patients had more than one diagnostic at the same time. once the protocol was implemented, the percentage of use was the following: % . please specify your abstract type: research abstract background and objective: drug prescribing is the most common medical intervention in the elderly. however, elderly patients are more sensitive to the drug's effects due to pharmacokinetic and pharmacodynamic changes associated with aging. chronic diseases and co-morbidities often require the use of a large number of medications. therefore, when prescribing drugs for the elderly, the choice of suitable drugs, dosage and duration of treatment should be carefully considered as well as clinically significant drug interactions. inappropriate prescribing is often associated with an increased risk of adverse drug reactions, increased morbidity and mortality, and health care costs. the aim of this study was to determine the incidence of potentially inappropriate medications (pim) prescriptions in the elderly (c years) using the original protocol developed by mimica matanovic and vlahovic-palcevski. setting and method: we enrolled patients hospitalized in clinic of internal medicine. data about patients' medications was collected during patient interview taken by the pharmacists on hospital admission. pharmacotherapy was analysed using the original protocol developed by mimica matanovic and vlahovic-palcevski in order to detect pims. main outcome measures: number and type of potentially inappropriate medications, potential clinically significant interactions. results: the average age of patients was years (range - ), and the average number of drugs per respondent was . (range - ). a total of patients ( . %) were taking at least one pim. the most common pim were long-acting benzodiazepines, central antihypertensive moxonidine and non-steroidal anti-inflammatory drugs (nsaids) in patients with hypertension. in the study population, patients ( . %) have taken at least one combination of drugs that could result in a clinically significant interaction. the most common combinations included application of nsaids and antihypertensive drugs or diuretics, concomitant use of multiple medications with effects on the central nervous system and drug combinations that can cause hyperkalaemia. conclusion: this study revealed the high prevalence of inappropriate prescribing. clinical application of this protocol could be an effective method for improving and optimizing drug prescription with the aim to reduce the number of side effects and the morbidity and mortality associated with the drug use in the elderly. please specify your abstract type: research abstract background and objective: to reduce adverse effects of conventional amphotericin b formulation (deoxycholate or d-amb) it can be infused in intralipid Ò (a fat parenteral nutrition), or lipid-based formulations can be used (i.e. amphotericin b lipid complex (ablc), amphotericin b colloidal dispersion (adcd) and liposomal amphotericin b (l-amb)). studies evaluating safety profiles present conflicting results. the aim of our study was to gather evidence on nephrotoxicity rates of d-amb versus lipid-based formulations in immunosuppressed patients susceptible to invasive fungal infection. setting and method: a systematic review, including randomized controlled trials (rcts) that compared the use of d-amb and amphotericin b lipid-based was performed. a search was conducted in pubmed, scopus, web of science and scielo. results were synthetized and meta-analysis was performed using software review manager . . main outcome measures: nephrotoxicity rates. results: eighteen rcts were identified (n = participants). the result from the meta-analysis favours the treatment with the lipidbased amphotericin b formulations (or: . ( . , . ) and presents a low heterogeneity (i = %). about % of patients from lipid-based treatment group presented an increase in serum creatinine of one to two times, which corresponds to stage one or two of acute renal failure (arf). and % presented an increase of tree times in serum creatinine achieving a stage three in arf (severe) which will require dialysis. while in group treated with conventional formulation int j clin pharm ( ) all of these patients, except one whose treatment adherence was inadequate, were cirrhotic ( / ), liver transplanted ( / ) and/ or presented hepatocellular carcinoma ( / ). / patients were coinfected with hiv. / patients ( %) were genotype . the total genotype patients treated with daas (svr /relapsed) were , which means that . % ( / ) of all genotype patients has had a relapse. / patients ( %) were treated with ledispavir/sofosbuvir ( . % of a total of patients (svr /relapsed) treated with this option). % of patients who suffered a relapse were treated with daas sofosbuvir, simeprevir, daclatasvir, previously to the introduction of the newest antivirals (dasabuvir + ombitasvir/ paritaprevir/ritonavir, ledispavir/sofosbuvir), which represents . % of the total of patients treated with the older option. conclusion: relapses rate was . %, slightly lower than reported in other studies. according to the references, these results show that genotype is the one presenting more relapses. all the patients presented a deteriorated performance status, except for one whose treatment adherence was inadequate. patients treated before april , when the newest daas where introduced, showed more relapses. more studies have to be developed in the near future since other daas will appear, the treatment options will be amplified and the number of relapses is expected to decrease. please specify your abstract type: research abstract background and objective: the inappropriate use of antibiotics remains a major issue since it causes bacterial resistance, longer hospital stay and increased mortality. antibiotic prescriptions must be monitored: the clinical pharmacist has a key role in ensuring patient safety and quality of pharmaceutical care. therefore, an antimicrobial stewardship program has been implemented as part of a national project of the italian society of hospital pharmacy (sifo). the objective is to describe the results obtained at the hospital. setting and method: a multidisciplinary antimicrobial management team has been implemented including clinical pharmacists, microbiologists and infectious disease specialists. the pharmacist examines drug charts on a daily basis in the department of medicine and supports clinicians to improve the appropriate use of antibiotics. data from time-points were extracted from medical records and collected in an excel database: t (november -january ) and t (february -april ). main outcome measures: type of infection, antibiotic consumption data, type of isolated pathogens, patient allergies, clostridium difficile infection assessment and adverse drug reactions (adr). results: records were analysed (t -t ), of which contained at least one antibiotic prescription. the most frequent infections were urinary tract ( %), respiratory ( %) and gastro-intestinal ( %). antibiotic therapy was started in . % of cases due to aspecific increase of c-reactive protein (crp). ddds were calculated for each treatment and were grouped by type of infection and setting (empiric vs targeted): ceftriaxone, meropenem and metronidazole were the most widely used antibiotics for empiric therapy. at t , an increase in the use of piperacillin-tazobactam instead of meropenem was observed. the ddd of ceftriaxone for targeted therapies decreased significantly, while an increase was observed for carbapenems, levofloxacin, glycopeptides and, in case of mdr bacteria, tigecycline. three allergies to antibiotics were reported in medical history. there were clostridium difficile infections ( relapses), confirmed by antibiogram. a total of adrs were identified: of these were related to antibiotics. conclusion: antimicrobial stewardship is a fundamental step to optimise antibiotic management, ensure patient safety and improve quality of care. the results obtained so far demonstrate the added value of a multidisciplinary team in controlling bacteria resistance and in the improving the use of antibiotics. please specify your abstract type: descriptive abstract (for projects) background and objective: the aim of this study was to analyse effectiveness and safety of pirfenidone, an anti-inflammatory and antifibrotic agent used for treatment of idiopathic pulmonary fibrosis. design: a retrospective, descriptive, observational study including all patients treated with pirfenidone at the hospital between march and june ( month) was carried out. to identify patients and collect data the outpatient medication dispensation software farhos Ò and the electronic medical record software hcis Ò were used. statistical analysis was carried out using microsoft excel Ò . demographic (age and sex), clinical (forced vital capacity (fvc), diffusing co capacity (dlco) and six-minute walk test (wt m)) and therapeutic (dosage and adverse reactions) variables were collected. results: throughout the study period, a total of patients ( males) started treatment with pirfenidone, with a median age of . years ( - ). during this period patients were excluded for lack of monitoring. the median fvc, dlco, wt m values prior to pirfenidone therapy, were % ( [ %), . % ( [ %) and m ( - m) respectively. all patients met the inclusion criteria of capacity trial according to fvc and wt m; however of them didn't meet the dlco criteria (at least %).'' all patients were monitored every months. the median in fvc percentage change at the end of the study was - % (- % to + %). patients ( %) showed an improvement on fvc during treatment with a median change of %. in the other eight patients fvc value decreased with a median of - %. only one patient would be candidate to discontinue treatment due to a lack of efficacy, according to discontinuation criteria established at the hospital (absolute decrease of c % in fvc during first year of treatment). dlco percentage was measured in patients, with a median change of % (- % to + %). dlco decreased in patients. wt m was monitored in patients, with a median change of - . m (- m to + m). adverse effects related to pirfenidone were gastrointestinal disorders ( / ), increase of hepatic ggt ( / ), and dermatologic toxicity ( / ). six patients ( %) required a dose reduction because of gastrointestinal adverse effects. five patients ( %) discontinued treatment with pirfenidone due to hepatotoxicity ( ), gastrointestinal ( ) and dermatologic effects ( ). one patient died. conclusion: half of the patients improved fvc during the period of the study. the other half, showed a decrease in fvc value which was similar to the median obtained in capacity trial. gastrointestinal disorders were the most frequent adverse effects and cause of discontinuing treatment. treatment monitoring is important to achieve therapeutic benefit and control the adverse effects. the national centre for epilepsy, oslo university hospital, oslo, norway please specify your abstract type: research abstract background and objective: systematic medication reviews in interdisciplinary teams can help to identify potential and actual drugrelated problems (drp). the centre for development of institutional and home care services in oslo, norway, conducted medication reviews for polypharmacy patients with mental disabilities in - , based on a lack of knowledge about drug-related problems in this patient group. the objective was to examine prescribing patterns, frequencies and types of drp in patients with mental disabilities. setting and method: the forms for medication reviews were developed by the national patient safety campaign in norway. the nurse/social educator recruited eligible patients, observed them, and ordered test if needed. the clinical pharmacist (jwa) reviewed the medications to identify drps. the interdisciplinary case conference took place at the different general practitioners' offices being responsible for the individual patients. the general practitioner, the nurse/social educator and the pharmacist were present, and in some cases, also patients took part. main outcome measures: an independent researcher (aqm) collected and analysed the data based on the drp-forms containing information on the prescribed medicines, strength, dose, indication, a description of drp and suggested interventions to resolve them. results: overall, patients with mental disabilities, aged - years, consented to have a medication review. they used on int j clin pharm ( ) : - average medicines (range - ). the team identified drp in of the patients (average . , range - ). overall, % of all drp were resolved. for one-third of the medicines, an action was taken to improve the prescribing. the most commonly medicines were analgesics ( %), antiepileptics ( %) and anxiolytics ( %). the most frequent drps were unnecessary drug choice ( %), side effects ( %) and too low dose ( %). drps were most common in antipsychotics ( %), antidepressants ( %) and anxiolytics ( %). conclusion: patients with intellectual disabilities take more medicines and have many drps compared to other patient groups. they are also more prone to taking combinations of cns-active medicines and therefore more at risk of side effects and drug interactions. pt : protocol feasibility and patient findings when using a dry extract of zingiber officinale roscoe (ginger extract gr ) during pregnancy please specify your abstract type: research abstract background and objective: there is limited information about the use of dry extracts of ginger root. the objectives of this study are ( ) to evaluate the feasibility of a pilot study with a food supplement among pregnant women ( ) to learn what the patient findings are when using the dry extract of ginger during pregnancy. this abstract deals with the intermediate evaluation of a study conceived to investigate the safety of the ginger extract gr during pregnancy. setting and method: a prospective, interventional and real life pilot study with pregnant women between and weeks of gestation and having symptoms of nausea and vomiting or digestive complaints. the included patients can use the ginger extract gr for digestive comfort during pregnancy when needed. during the use, the score of digestive discomfort is noted and the researcher reports adverse events. main outcome measures: ( ) number of included patients as an indicator of feasibility: including a number of patients was taken as a target ( ) analysis (qualitative and quantitative) of the patient diaries, more particularly patient behaviour, wellbeing and impressions. results: within twelve weeks, patients were included with an average age of . years and a median age of ( - ) years. patients used gr : patients were dissatisfied, patients had a neutral opinion and patients were satisfied to very satisfied. one miscarriage occurred at a gestational age of almost weeks (only tablets of gr were used, with no relevant medical history in preceding pregnancies). two patients were hospitalized, of which with hyperemesis gravidarum. one patient complained about heartburn and one patient experienced a bad taste and heartburn. three patients have indicated that they experienced more nausea after taking the tablets. patients experienced no adverse events. the remaining patients were not yet evaluated. of the included patients, six patients decided not to use the product: because their gastrointestinal complaints were not serious enough, because problems of swallowing (using ginger gums instead). one patient was afraid for the negative consequences for her unborn child. the last of the nonusers indicated that she had no confidence in the product. conclusion: conducting a pilot study with the ginger extract gr in case of pregnancy is feasible. the majority of the evaluated patients were satisfied. signing the consent form does not guarantee the intake of the product. pregnant women remain very cautious in the use of unknown products during their pregnancy, even though it concerns a food supplement and not a drug. the severity of symptoms does not give a good indication whether or not and how often the product will be used. please specify your abstract type: descriptive abstract (for projects) background and objective: to analyse effectiveness and safety of ibrutinib, an oral inhibitor of bruton tyrosine kinase, in patients with mantle cell lymphoma (mcl) who have received at least one prior therapy. design: a descriptive observational study was carried out. all patients with relapsed or refractory mcl who started treatment with mg of daily ibrutinib between september and june were included. patients were identified and followed through electronic medical record. demographic and baseline clinical characteristics of patients were collected: age, sex, ecog (eastern cooperative oncology group scale), number and type of prior regimens, simplified mipi status (mantle-cell lymphoma international prognostic index), and disease stage (relapsed or refractory). progression free survival (pfs) and response to treatment were recorded to evaluate effectiveness. adverse effects related to ibrutinib and possible interactions with concomitant medication were documented to measure safety. statistical analysis of the data was carried out using microsoft excel Ò and spss Ò . results: throughout the period of study a total of patients ( males and female) with a mean age of . ± . years started treatment with ibrutinib. the median of previous treatments were ( ) ( ) ( ) ( ) ( ) including first-line treatment with high dose chemotherapy ( %), steam-cell transplantation ( %), rituximab ( %), bortezomib ( %) and lenalidomide ( %). the median ecog value prior to ibrutinib therapy was (range - ). the mipi status was intermediate risk in patients and high risk in , the disease stage was relapsed in % of the patients. partial response was reported in patients. the mean pfs estimated at the end of the study period was months ( % . - . ). adverse effects related to ibrutinib were: fatigue ( %), diarrhoea ( %) leucocytosis ( %) and infections ( %), including upper respiratory and urinary tract infections, sinusitis and pneumonia. one possible interaction between ibrutinib and everolimus was found in a liver transplant patient. close monitoring of everolimus plasmatic levels was recommended. conclusion: the mean pfs estimated in our study was similar to the median obtained in the pivotal phase ii trial. infections were the most frequent adverse effects. concomitant medication to ibrutinib should be checked, as ibrutibib is metabolised by cyp a and interactions may be frequently present. pharmacy, hiv unit, germans trias i pujol hospital, badalona, spain please specify your abstract type: descriptive abstract (for projects) background and objective: dolutegravir (dtg) is one of the preferred options for initial antiretroviral therapy (art) due to its high efficacy, good tolerability and low potential for drug-drug interactions. nevertheless, an unexpectedly high rate of dtg discontinuation (up to %) due to adverse events in the clinical practice has been recently reported. therefore, we aimed at assessing the dtg discontinuation rate and reasons for discontinuation in our hospital. design: single-centre, retrospective study from september to june of patients cohort with art both naive and pretreated. patients who had started dtg-based art containing regimen were identified and the reasons for the discontinuations were analysed. data were collected using the primary care service program and the electronic prescription program. results: out of patients attended by pharmacy department in our hospital, patients ( males, mean age years (range - )) had started a dtg-based art. out of them, patients were art naive and art-experienced. at the moment of starting dtg, mean cd cells were cell/mm (range - ) and hiv- rna load in plasma was detectable in patients. treatment discontinuation was reported in / patients ( . %) with a median treatment time of days (range - ). / patients ( . %) were naïve and / patients ( . %) pre-treated. most of the patients ( ) were in single tablet regimens (str) containing dtg in combination with abacavir and lamivudine, whereas the rest were in combination with other antiretroviral drugs. the main reason for treatment discontinuation was toxicity in / patients ( . %). the rest of the patients discontinued due to other motives (clinical trial inclusion ( / ), treated in another hospital ( / ), exitus ( / ) and others ( / ). reasons for the discontinuation were classified in different side effects: / ( . %) related to central nervous system (cns) (insomnia, psychiatric disorders such as anxiety, nightmares and depression), / ( . %) gastrointestinal effects, / ( . %) headaches, / ( . %) musculoskeletal effects, / ( . %) fatigue, / ( . %) allergy and / ( . %) for other reasons. some patients reported various toxicities at once. conclusion: more than % of patients treated with dtg discontinued by toxicity reasons. it is important to note that half of these patients had cns adverse effects. please specify your abstract type: research abstract background and objective: hcv therapy has been revolutionised recently by the approval of antiviral agents direct-acting (daa) facilitating the treatment of patients coinfected with hiv/hcv. however, potential drug interactions and overlapping toxicities of both treatments represent the major challenges in adapting therapy. to analyse the prescription profile of direct acting antivirals (aad) in patients coinfected with hiv/hcv. setting and method: retrospective observational study from january to january in a specialty hospital. the data were collected from the hospital program of clinical stories, archinet Ò , and the outpatient program farmatools Ò . the results were analysed using the statistical program r-commander. main outcome measures: inclusion criteria: adult patients coinfected with hiv/hcv with undetectable viral load. the following variables were collected: age, gender, hcv genotype, degree of fibrosis, patient type (naïve or pre-treated), baseline cd count, cd levels end of treatment, sustained viral response (svr) and hcv treatment. results: patients, of whom were men, mean age years were included. patients received daclatasvir and sofosbuvir for hcv, patients had genotype a and b respectively, patients genotype and patient genotype . patients had fibrosis f f , . of the patients they had not received previous treatment (naïve) and had failed to treatment. hiv treatment was modified in patients, patients achieved svr. the cv was undetectable to hiv treatment change for all patients. cd levels increased in all patients at the end of treatment for hcv with a median of cells/ul and at the beginning and end respectively. patients received ombitasvir/paritaprevir/ritonavir and dasabuvir, who had a genotype a. these two patients had received previous treatment and had a f and f fibrosis. none of them was modified hiv treatment and only one got svr. cv remained undetectable and cd slightly increased after the treatment. patients received ledipasvir and sofosbuvir, patients had genotype a, patients genotype b and patient genotype . patients had f fibrosis and had f . patients had received previous treatment (naïve). the hiv treatment was modified only in one of the patients, patients achieved svr. cv increase in patients after the treatment while cd followed the trend of increasing. conclusion: the aad that caused fewer changes in the hiv treatment were ombitasvir/paritaprevir/ritonavir and dasabuvir followed by ledipasvir/sofosbuvir. sofosbuvir and daclatasvir present a greater number of interactions with hiv drugs so they behaved to a major change. more patients are needed to assess more accurately the aad leading to a minor modification. please specify your abstract type: research abstract background and objective: the simplification strategies reduce the amount of tablets and the toxicity in order to facilitate adherence in patients with virological suppression. the strategy more studied is monotherapy with a ritonavir-boosted protease inhibitors (pi/r). to analyse the effectiveness of monotherapy with pi/r in pre-treated patients infected with hiv. setting and method: retrospective observational study. selected hiv patients treated with pi/r monotherapy at any time of pharmacotherapeutic history to / / , with at least one clinical and analytical control months before the beginning. data were collected from the medical record archinet Ò and outpatient farmatools Ò program. variables included were age, sex, duration of monotherapy, virological failure, treatment failure, cd % during monotherapy. main outcome measures: inclusion criteria: virological suppression for year prior to the start of monotherapy, no previous ip virological failure, high cd count ([ cell/ml) and a high level of drug adherence. the effectiveness is defined as the percentage of patients without virological failure ( consecutive plasma viral load (vl) [ copies/ml) and without treatment failure (any event causing retirement monotherapy). results: patients with monotherapy, which represent % of patients with antiretroviral therapy (art) at our institution were identified. were excluded ( co-infected with hepatitis virus, with insufficient data and no had more than months included), including patients in the analysis, with a mean age of years and % were men. the median of time monotherapy treatment was . years ( . days), ( . %) patients received darunavir/r and ( . %) lopinavir/r. the effectiveness of monotherapy treatment during the follow up period was % with undetectable pvl at follow-up. the median of cd % over the treatment time was cell/ml ( %). conclusion: the effectiveness of treatment with ip/r monotherapy in our hospital obtained good results. according with our results treatment adherence plays a very important role. this is a current and valid strategy that brings benefits to the patient and to the healthcare system. please specify your abstract type: research abstract background and objective: the access to investigational drugs for patients who are not included in a clinical trial and without authorized therapeutic alternatives is known as compassionate use. the incorporation of the evidence-based medicine in the area of oncohaematology has implied that an important part of clinic therapy validated by evidence that could not be controlled from an administrative point of view. this is due to the continuous and progressive development of investigation and information on cancer treatment and the delay of the administration regulation. the use of drugs in this way is regulated by royal decree / ( / ). the objective of the study is to describe the use of cancer drugs through compassionate use in the last years in a specialty hospital. setting and method: descriptive retrospective study on a specialty hospital. all the applications for a compassionate use drugs were analysed from january until october . the data were obtained from medical records programme diraya Ò and from an excel database of medicines in compassionate use of the pharmacy service. main outcome measures: the following variables were registered: • number of patient clinic history • authorized medicine • authorization date • applicant service results: we recorded requests of cancer drugs in compassionate use during the years of study. oncology was the service that recorded more authorizations with %, followed with gynaecology with . % and finally endocrinology and haematology with . %. drugs of the requests were approved ( %) and unauthorized ( %) in the years of study. the year in which more applications were received was ( . %) and the least requests were received in ( . %), being the year where all requests were authorized. in fewer applications were authorized, %. in the years , and were authorized . , and . %, respectively. a total of different active drugs were received during the study, the most requested bevacizumab ( %) for grade iii oligoastrocytoma, ovarian cancer (monotherapy), metastatic gall bladder cancer and metastatic platinum-resistant ovarian cancer, everolimus ( %) for indications of neuroendocrine carcinoid tumour and metastatic breast cancer, nab-paclitaxel ( %) for invasive lobular carcinoma indications of high-grade and metastatic pancreatic cancer, ipilimumab ( %) for the indication of metastatic melanoma, and regorafenib for indications of colorectal cancer and metastatic gist i pre-treat with imatinib ( %). the solicitude of drugs through compassionate use needs effective commissions of pharmacy and therapeutics, along with the medical management to establish an agile and faster requesting circuit and the consequent use monitoring. please specify your abstract type: research abstract background and objective: to describe the standard procedure for the elaboration and control of a magistral formula (mf) to assess their effectiveness in two patients with cutaneous metastases of malignant melanoma refractory to other treatment. setting and method: medication for compassionate use was requested for two patients of and years with histopathologic diagnosis of cutaneous metastases of malignant melanoma in the left thigh and left heel in which the lack of response to first-line treatments made to be valued to start with adesleukina intralesional therapy. the first week was infiltrated mu ( ml) in lesions less than cm, mu ( ml) in the larger lesions and repeating each week until complete remission of the lesions. in the nd patient we proceed in the same way but the second week was infiltrated mu ( ml). the following week, infiltrated mml, in metastases and we turn to weekly infiltrations. the response was assessed by clinical disappearance of the lesions treated. complete response (cr) is defined as a clinical disappearance of lesions and partial response (pr) greater than % reduction of the lesion diameter. main outcome measures: we performed a literature search (pubmed, trissel, spc) for all studies published to determine the standard procedure for preparing and monitoring the mf (processing, preservation, stability, dose and indication). results: the standard procedure of preparation and quality control was carried out following the rules established in rd / . it was made in a vertical laminar flow cabinet. the aldesleukin vial was reconstituted with . ml api ( mu/ml) and then diluted with . ml of a solution of . % albumin, % glucose as stabilizer, to avoid aggregate formation, preparing ml syringes ( mu/ml). it was obtained a homogeneous and clear solution without precipitate or opalescence appearance. stable days in a refrigerator ( - °c), protected from light. initially patients had approximately a total of injuries. after months of treatment it was obtained a cr of most lesions in the first patient and rp of the second patient injuries. treatment was well tolerated. the side effects presented were only a flu-like syndrome in the second patient. conclusion: intralesional administration aldeslukina has been effective in treating malignant melanoma skin metastases in our patients, allowing the extension of its use in patients with the same involvement refractory to other primary treatments. the results are similar to those of the publications consulted. please specify your abstract type: research abstract background and objective: chronic infection with hepatitis virus c (hcv) affects about million people worldwide and is a leading cause of liver cirrhosis and hepatocellular carcinoma. the new direct acting antivirals against hcv have revolutionized the treatment of this disease. due to the high cost of these drugs it is necessary to assess their use in clinical practice. to evaluate the effectiveness of daclatasvir in combination with sofosbuvir in patients with hcv monoinfected in a specialty hospital. setting and method: retrospective observational study of patients who began treatment with the combination of daclatasvir and sofosbuvir from january to january in a specialty hospital. the data were collected from the hospital program of clinical stories archinet Ò and the outpatient program farmatools Ò . the results were analysed using the statistical program r-commander. main outcome measures: the sustained virologic response (svr) was considered the primary endpoint of the study. as secondary variables were analysed: sex, duration of treatment, naïve patients or pre-treated, degree of fibrosis, hcv genotype, concomitant use with ribavirin, viral load (vl) before treatment and medical service. results: there were included patients of whom were men. baseline characteristics were: patients with genotype , genotype b, with genotype a and genotype . the degree of fibrosis in the study was patients with f , f and to f . among the patients infected with hcv genotype , had not received prior treatment (naïve) and had failed therapy. the duration of the treatment was weeks to patients and weeks for patients. only patients receiving ribavirin of these had genotype and genotype b. from ribavirin patients it was greater the number of patients in whom the treatment duration was weeks ( patients versus with p-value = . ). the digestive service attended to patients while patients were followed by infectious. the median cv was , , iu/ml. svr was achieved in . % of patients with hcv genotype in . % with genotype b and % with genotype and a. after weeks of treatment % of patients achieved svr and % after weeks. only one patient died during treatment. the results are similar to those obtained in clinical trials. svr has not been influenced by hcv subtype, duration of treatment, degree of fibrosis, pre-treatment or by concomitant use of ribavirin. further studies are needed to evaluate the efficacy of this treatment. please specify your abstract type: research abstract background and objective: the safety and efficacy of medications can vary significantly between patients as a result of genetic variability. as genomic screening technologies become more widely available, pharmacists are ideally suited to utilize this tool to optimize medication management. the objective of this study is to evaluate the feasibility of implementing personalized medication services into community pharmacy practice and to assess the number of drug therapy problems identified as a result of pharmacogenomic screening. setting and method: the study was designed as open-label, nonrandomized, and observational. two community pharmacies in toronto, ontario offered pharmacogenomic screening as part of their professional services program. prior to initiation, participating pharmacists received structured, comprehensive training in pharmacogenetics. pharmacists then facilitated voluntary subject enrolment among patients who they believed would benefit from screening and met inclusion criteria. eligible patients received a simple buccal swab followed by dna analysis using pillcheck Ò . pillcheck Ò is a genotyping assay that translates genomic data and generates a personalized, evidence-based, report that provides insight into patients' inherited drug metabolic profile. upon receiving the report, pharmacists invited patients back to the clinic for interpretation of the results. clinically significant drug therapy problems were identified and recommendations for medication optimization were forwarded to the primary care physician. main outcome measures: number of clinically significant drug therapy problems identified by pharmacists as a result of pharmacogenomic testing. results: patients were enrolled in the study. average age was . years and patients were taking a mean of . chronic medications. pharmacists cited the most common reasons for testing as ineffective therapy ( . %), to address an adverse reaction ( . %), and to guide initiation of therapy ( . %). an average of . drug therapy problems were identified per patient. pharmacist recommendations included change in therapy ( . %), dose adjustment ( . %), discontinuation of a drug ( . %), and increased monitoring ( . %). generally, physician feedback was positive but did reveal an opportunity for a broader understanding of the technology. conclusion: these results highlight the readiness of community pharmacists to adopt pharmacogenetic screening into practice and their ability to leverage this novel technology to positively impact medication management. community pharmacists are ideally suited to both offer personalized medication services and interpret genomic results. please specify your abstract type: descriptive abstract (for projects) background and objective: visual impairment is a common geriatric syndrome and glaucoma/miotic eye drops treatment is a frequent therapeutic option. pharmacist's role in medication reconciliation is an effective process for reducing medication errors and supporting safe medication use. we observed that mentioned medication reconciliation was occasionally not performed during hospital stay and could be cause of delirium because of visual impairment. the aim of this study was to evaluate the influence of omission errors of eye drops treatment on incidence of acute confusional state. design: we conducted an observational, descriptive and retrospective study in an orthogeriatric unit with an average of patients with hip fractures per year ( % surgically treated). data collection was performed from june to march . reconciling medications at admission was performed by implementing the tools and resources of the canadian patient safety institute (cpsi). we extracted from our electronic database (filemaker pro Ò ): • demographic patient data (age and gender). • name and posology of the glaucoma/miotic eye drops treatment. • medication reconciliation performed and identification of professional in charge (pharmacist, geriatrician or orthopaedic surgeon) registration during hospital stay. • protocolar management of delirium with tiapride occasional intramuscular administration performed if necessary was also registered to establish the incidence of acute confusional state. results: thirty-two patients ( women and men) were included, median age year-old . in patients, eye drops reconciliation treatment was performed by the pharmacist in of the patients, the geriatrician in cases and the orthopaedic surgeon in . in patients, the mentioned medication reconciliation was not performed (pharmacist absentism). considering the patients on eye drops treatment during hospital stay, ( . %) of them suffer from acute confusional state. on the other hand, among the patients without medication reconciliation, delirium was registered in cases ( . %). concerning ocular topic treatment, . ± . active principles per patient were observed, being the most frequent timolol ( . %), brinzolamide ( . %) and latanoprost ( . %). conclusion: we consider of paramount importance the pharmacist evaluation availability at an orthogeriatric unit, minimizing the impact of acute confusional state during hospital stay by medication reconciliation. please specify your abstract type: descriptive abstract (for projects) background and objective: to report the therapeutic management of haemorrhagic rectocolitis onset in a lung-transplanted patient with mycophenolate-induced diarrhoea. design: case report. results: a -year-old-man lung transplant patient for alpha -antitrypsin deficiency in receiving mycophenolate mofetil, tacrolimus and corticosteroid developed chronic diarrhoea worsened by sigmoid and cecal necrosis in , and treated successfully by sigmoidectomy. severe diarrhoea attributed to mycophenolate mofetil reappeared in april , which motivated a switch to mycophenolate sodium. the absence of clinical improvement in june led to stop mycophenolate sodium and introduce azathioprine at mg/day (absence of mutation for the thiopurine methyl transferase gene). one month later, the patient presented melena, diarrhoea, bloating, nausea, and knee pain, attributed to azathioprine. this latter was stopped and mycophenolate mofetil was rechallenged associated with symptomatic treatment (i.e., diosmectite and loperamide). in january , a colonoscopy, performed in a context of profuse chronic diarrhoea with mucus during months, highlighted haemorrhagic rectocolitis. therefore, the patient initiated sulfazalasine therapy with no clinical improvement, and then high doses of oral corticosteroids. because high-dose of oral corticosteroids was not recommended as a long-term treatment, mercaptopurine was proposed as a new therapeutic option. mercaptopurine has no indication as an immunosuppressive treatment in solid organ post-transplant supportive care. however, as the active metabolite of azathioprin, an immunosuppressive drug widely used in transplantation, mercaptopurine has immunosuppressive functions towards t-lymphocytes. after multiprofessional collaboration between gastroenterology, pneumology and pharmacy specialists, it was decided to stop mycophenolate mofetil and introduce mecaptopurine at . mg/kg/day, as immunosuppressant for haemorrhagic rectocolitis as well as lung transplantation. this unusual lung transplant immunosuppressive therapy, associated with tacrolimus, improved digestive disorders and patient's quality of life. currently, mercaptopurine is biologically and clinically well tolerated. the dosage of blood residual concentrations of purinethol metabolites ( -thioguanine and -methylmercaptopurine) is going to be performed. conclusion: immunosuppressive therapy in solid organ transplantation is a real challenge for patients who have comorbidity onset. despite a lack of data in the literature, a multidisciplinary collaboration based on comprehensive pharmacology skills is essential to choose the best therapeutic option in this type of patients. please specify your abstract type: descriptive abstract (for projects) background and objective: the use of complementary medicines (cm) in oncology is the subject of broad but still controversial interest. a large part of patients with cancer uses cm, including complementary drugs, during their treatment period. indeed, according to different studies, this proportion ranges from to %. importantly, the risk of interaction between cm and anti-cancer drugs is not negligible; hence we need to identify these cm to ensure the security of our patients and the success of their treatment. design: to achieve this purpose, a monocentric retrospective analysis was conducted with collection of data by pharmacy students during medication reconciliation of hospitalized patients from january to june . collected data are patients' characteristics, prevalence of cm use and potential cm-anticancer drug interactions. results: patients were included in the study ( men- women); median age was [ - years]. a total of . % (n = ) were using a least one cm, most frequently homeopathy ( %, n = ) or phytotherapy ( %, n = ); some patients were using a combination of two cm ( %, n = ). cm are mainly used by women in comparison to men ( . % versus . % and p = . , chi square test). for phytotherapy, at least different herbs were described by patients and among them the most frequently used were mistletoe (viscum album), propolis and fireweed (epilobium angustifolium). data analysis showed that % (n = ) of patients were at risk of potential cm-anticancer drug interaction. moreover this risk was increased to % if we considered only patients taking phytotherapy. interactions included pharmacokinetic ( %, n = ), such as altered hepatic metabolism, and pharmacodynamics ones ( %, n = ). conclusion: in conclusion, our work clearly demonstrates that the use of cm by patients is associated with high risk of relevant drug interaction with their anti-cancer treatment. even if further investigations are necessary to clarify the clinical impact of these interactions, the use of cm must be considered during prescribing process. please specify your abstract type: research abstract background and objective: since their reimbursement, the direct oral anticoagulants (doacs) are increasingly used for stroke prevention in atrial fibrillation (af). the objective of this study was to identify the proportion of real life patients with af eligible for doac therapy, based on the inclusion and exclusion criteria used in the clinical studies and based on the officially approved indications as mentioned in the summary of product characteristics (smpc). setting and method: data for this retrospective cross-sectional study was extracted from the uz brussel stroke registry, containing anonymized data of patients with a suspected stroke. characteristics of patients with documented af were compared with the patient characteristics in clinical trials and the approved indications in the smpc. main outcome measures: proportion of real life patients with af eligible for doac therapy. results: data of patients with af was analysed. based on the selection criteria of the clinical trials, significantly less patients were eligible for treatment with rivaroxaban compared to dabigatran etexilate ( . % versus . %; p = . ), but not compared to apixaban ( . %; p = . ). based on the indications and contraindications in the smpc, significantly fewer patients were eligible for apixaban compared to dabigatran etexilate and rivaroxaban ( . % for apixaban, . % for dabigatran etexilate and . % for rivaroxaban; p \ . and p \ . , respectively). significantly more patients were eligible for doac therapy based on the indications and contraindications in the smpc compared to the inclusion and exclusion criteria of the clinical trials ( . % versus . %; p \ . for dabigatran; . % versus . %; p \ . for rivaroxaban and . % versus . %; p \ . for apixaban). conclusion: when taking into account the selection criteria from the pivotal clinical trials with doacs for stroke prevention in af, less than half of real life patients are eligible for therapy with one of the doacs. however, the indications mentioned in the smpcs of these drugs are less strict. please specify your abstract type: research abstract background and objective: idiopathic pulmonary fibrosis (ipf) is a disease in which tissue deep in the lungs becomes thick and stiff, or scarred, over time. the formation of scar tissue is called fibrosis. pirfenidone is an anti-fibrotic and anti-inflammatory agent, thus offers a new hope for treating progressive fibrotic diseases. int j clin pharm ( ) : - our objective is to set a description of idiopathic pulmonary fibrosis patients treated with pirfenidone, as well as the adverse reactions observed. setting and method: descriptive study in which all patients have received pirfenidone. the data were obtained through the dispensing program of outpatient (farmatools) and review of medical records of the hospital database (archinet) and clinical station (diraya). main outcome measures: we have extracted from each patient baseline data, comorbidities, dose received, reported adverse reactions and data about haematology and biochemistry. results: we have a total amount of patients treated with pirfenidone, all diagnosed with idiopathic pulmonary fibrosis, including women and men. the age of patients is between and years, with an average of . years. all patients are ex-smokers and one of them is also ex-alcoholic. concerning concomitant pathologies, patients have diabetes mellitus, have arterial hypertension, and one of them has ischemic heart disease. another has upper gastrointestinal bleeding prior, among others chronic pathologies. pirfenidone dose received was the usual dose in of the patients: days - mg every h, days - mg every h and a maintenance dose of mg every h. in one patient due to its low imc the dose received was smaller ( - days mg every h, days - mg every h and maintenance dose of mg every h). in relation with the adverse effects, digestive discomfort were observed in of the patients, causing the interruption of the treatment in one of them (with prior gastrointestinal bleeding). in the other patient it was relieved by lowering the dose received. also, one patient has experienced photosensitivity. alterations in transaminase levels were observed in patients but that didn't force to discontinue the treatment. no alterations were observed in the blood count. conclusion: treatment with pirfenidone is being generally well tolerated by patients. it has improved their life-quality and reached the objective data of a slowdown in disease progression. currently, the number of patients is no enough to give conclusive information in relation to the drug effectiveness. please specify your abstract type: research abstract background and objective: to describe the total amount of patients treated with a magistral formula of sodium cromoglycate mg without excipients: indications, concomitant therapy and the response to therapy. setting and method: we run a descriptive study in which we included the totality of patients in treatment with a magistral formula of sodium cromoglycate mg without excipients in a tertiary hospital. the data were obtained through paracelso (development of magistral formulas program), as well as with farmatools (dispensation program of outpatient) and the review of medical records from the hospital database (archinet), and diraya clinical station. main outcome measures: from each patient we extracted data relative to sex, age, diagnosis, time in treatment with the formula, dose received, response to therapy, concomitant antihistamines treatments and adverse effects. results: a total of patients in treatment with a magistral formula of sodium cromoglycate mg without excipients were reviewed: women and men with a mean age of . years old (range - years). regarding the indication of the prescription, patients have been diagnosed of indolent systemic mastocytosis and the remaining were diagnosed of mast cell activation syndrome. in all cases, the diagnosis was established by examination of the bone marrow in the mastocytosis studies institute of castilla la mancha (spain). on average, patients took the treatment . months, with a range between months and months. the dose received was mg every h in patients, having to be increased to mg times daily in a case with poor response to the therapy. in the remaining patients, the treatment response has been optimal. in relation to the concomitant anti-allergic treatment received, patients took fexofenadine daily during the study. no cases of adverse effects related to the therapy received have been reported. conclusion: both indolent systemic mastocytosis and mast cell activation syndrome are considered rare diseases, and we should indicate that in spain there are no commercial medicines available of sodium cromoglycate without excipients for its treatment. the treatment with this magistral formula of sodium cromoglycate mg without excipients has been effective and well tolerated in all patients, improving the symptoms associated with their condition as well as their quality of life, and also, assuming a solution to the lack of marketing of the drug currently in spain. please specify your abstract type: research abstract background and objective: to analyse the prescription profile, safety and effectiveness of new therapies available for the treatment of hcv genotype b in a tertiary hospital. setting and method: we run a retrospective observational study in which we included a total amount of patients infected with hcv genotype b treated with the new therapies against hcv from february to december in a tertiary hospital. the data were obtained through the outpatient dispensing program farmatools and the review of the medical records from the hospital database, archinet and prescription hepatitis c portal of the andalusian health service. main outcome measures: from each patient the following information was collected: sex, age, viral genotype (gen.), naive/nonnaive, hiv coinfection, presence of cirrhosis, degree of hepatic fibrosis measured by fibroscan, treatment prescribed and duration, adverse effects, sustained viral response (svr) and the service that made the prescription. results: a totality of patients with hcv gen. b were reviewed which . % of them were men with a mean age of . years (range - years). of the patients were naive and only of them were hiv co-infected, there were a . % of cirrhotic patients. regarding the degree of hepatic fibrosis, patients had grade f , f grade patients, patients grade f and f grade patients. the most commonly therapy prescribed was lepidasvir + sofosbuvir in patients ( without ribavirin and with ribavirin ) using a treatment schedule of weeks in of them. the treatment was discontinued in one case because of the adverse effects, achieving svr in the remaining patients. the combo treatment with paritaprevir/ombitasvir/r + dasabuvir was prescribed in times ( without ribavirin and with ribavirin) choosing only in one of them for a treatment period of weeks. there were no treatment discontinuations and svr was achieved in all patients treated in this way. patients received simeprevir + sofosbuvir for weeks ( without ribavirin and with ribavirin), one patient of the left the treatment due to adverse effects. svr was found in the remaining patients who completed treatment. sofosbuvir + daclatasvir was prescribed to patients, associating ribavirin in only one case. a treatment duration of weeks was used in patients and weeks in the remaining two. one patient failed rvs without any incidences of adverse effects in any case. interferon + ribavirin sofosbuvir + was prescribed to patients in -week regimen which was well tolerated achieving svr. digestivo service treated the % of the total amount of patients. conclusion: new therapies for hcv have been used in all the treated patients and the older drugs have been relegated. about the effectiveness, svr was achieved in . % of patients. regarding the safety, only patients have discontinued the treatment due to adverse effects representing less than % dropout rate of the therapy. please specify your abstract type: descriptive abstract (for projects) background and objective: thanks to pharmacogenetics we can identify and predict different responses to the same drug among different individuals. during these last years we have noted a big increase of dosing guidelines and advices about the use of several drugs due to the influence of different polymorphisms. the aim of this study is to describe and evaluate the use of pharmacogenetics in our hospital from april , when we started our first research about pharmacogenetics, to the actual time, using these information in our daily clinical practice; and indeed quantify the number of different tests and the number of different clinical advices done because of pharmacogenetic information, by different healthcare specialty areas and drugs. design: we reviewed all the pharmacogenetic test requests in our hospital from april to april , noting which health specialty and for which drug was asked the test. polymorphisms were genotyped using taqman Ò genotyping assays technology by independent laboratories to confirm the results. results: from april we were asked for pharmacogenetic tests from different healthcare specialty areas: rheumatology ( . %), infectious diseases ( . %), oncology ( . %), cardiology ( . %), vascular surgery ( . %), neurology ( . %), ophthalmology ( . %); this information was asked about different drugs: clopidogrel ( . %), trastuzumab ( . %), ranibizumab ( . %), azathioprine ( . %) and tocilizumab ( . %). from all the genotypes, ( . %) were done after using the drug (study phase) and ( . %) were done previous to the use of the drug in daily clinical practice to make a ''clinical recommendation''; from these recommendations affected to the prescription of clopidogrel. conclusion: during the last years we could implement the use of pharmacogenetics in the daily clinical practice in our hospital in different healthcare areas affecting drugs and we started research studies previous to its use on the clinical practice for other three different drugs. please specify your abstract type: descriptive abstract (for projects) background and objective: the drug burden index (dbi) is a tool used to quantify the anticholinergic and sedative burden of medication on an individual. it has been independently associated with poor physical and cognitive performance in community-dwelling older people. objectives were: to create an inventory of medications used in ireland with clinically significant anticholinergic and/or sedative activity and to decide upon the minimum daily dose (mdd) for each medication. design: medications with potential anticholinergic and/or sedative burden were identified by literature review and examination of the summary of product characteristics (smpc) for all medications registered in ireland. each medicine was classified as anticholinergic or sedative. drugs with both anticholinergic and sedative properties were classified as primarily anticholinergic. the mdd, a key component of the dbi score calculation, was selected by reference to the irish smpc. other options which were also considered for this value include the defined daily dose (ddd) of a medication, as available from the world health organisation (who), and the mdd as outlined in the british national formulary (bnf). mdds were decided upon regardless of indication as the lowest effective therapeutic dose as specified in the smpc for the medication. the final list of medicines and mdds to be included in the inventory was then defined by consensus of three pharmacists. results: in total, medicines with potential anticholinergic and/or sedative activity were considered for inclusion. a final list of medications was identified by consensus ( anticholinergic, sedative). of these, ( %) were agents which act primarily on the nervous system. the three main therapeutic groups contributing to the inventory of dbi medications were antipsychotics ( medications), antidepressants ( medications) and antiepileptics ( medications). conclusion: creation of an inventory of medications with anticholinergic and/or sedative properties, in combination with the individual mdds, was achieved. this is a useful resource for use in analysis of drug burden in an older population. it could help in both identifying patients who would benefit from medication review as well as analysing population medication data. please specify your abstract type: research abstract background and objective: vancomycin is an antibiotic widely used to treat infections such as bacteraemia, infective endocarditis, osteomyelitis, meningitis and pneumonia. nowadays, optimal trough concentration is stablished between and mg/l to avoid development of resistance or - mg/l to improve penetration in complicated infections. some articles have been published explaining the methodology to calculate an expected trough level in steady state. our aim was to compare the trough serum value estimated by the mathematical method with a two-compartimental bayesian forecasting model. setting and method: observational retrospective study carried out in a tertiary hospital from january to december . non obese adult patients with creatinine clearance (crcl) \ ml/min and who have achieved steady state level were included. vancomycin serum values were measured using a chemiluminescence's immunoassay (cmia) and bayesian analysis was performed with abbottbase pksystem Ò (pks Ò ). the statistical analysis was made with medcalc software Ò . bland-altman plot and passing-bablok regression were used to compare both methods. main outcome measures: sex, age, weight, dose, creatinine, and size were collected from clinical history. serum trough values (cminr) were collected from cmia. trough values were estimated using two methods: mathematical method (cminf) and bayesian calculations (cminb). results: patients were included, with a mean age of (± . ) years. % were male and % female. they received a median dose per h of ( - ) mg. the mean of cminr was . mg/l ( % ci . - . ), cminb . mg/l ( % ci . - . ), cminf . ( % ci . - . ) . correlation coefficients (r) comparing both methods were significantly different: r between cminf and cminr was . ( % ci . - . ), while r between cminb and cminr was higher: . ( % . - . ). bland-alman plot analysis showed both methods cannot be used interchangeably. the regression equations estimated by passing-bablok regression were y = - . + . x and y = - . + . x. conclusion: bayesian method has demonstrated better correlation with real measures than mathematical method. most part of our patients could be underestimated or overestimated using mathematical methods which could cause toxicity or lack of efficacy, so this method is unsuitable for clinical use. bayesian estimation remains the best option for optimal dosing of vancomycin. please specify your abstract type: research abstract background and objective: combination therapy with digoxin and acenocoumarol is common in patients with atrial fibrillation (af). getting optimal concentrations of digoxin leads an appropriate response; taking into account its narrow therapeutic range and all the factors which can affect to its pharmacokinetics. interaction between them has been studied, even though its mechanism is not clear yet. patients who are taking both drugs need higher doses of digoxin; because they get lower concentrations by using the same dosage. the objective of this study was to analyse digoxin concentrations in patients treated with this combination compared to expected concentrations according to population parameters. setting and method: retrospective observational study from december to march performed by pharmacokinetic unit. patients included had chronic treatment with acenocoumarol and digoxin, which determination were realized in the steady state before the next dosage. patients with toxics concentrations of digoxin, or who were suspected nonadherence, were excluded. the plasma digoxin concentrations were determined through the autoanalyzer architect c- Ò (petinia). dosage adjustment was realized by the program abbot pharmacokinetics system (pks). a comparative between the real measured concentrations in patients and estimated concentrations were realized based on population parameters. finally, in order to get optimal concentrations, some dosage changes were proposed based on pharmacokinetic monitoring. data collected: population characteristics (gender, age, weight, and height), analytical data (potassium, urea, creatinine and clearance). main outcome measures: digoxin serum concentrations (optimal range . - ng/ml). results: data from patients, . % women with a mean (sd) age of . ( . ) years were included in the study. at baseline, potassium, urea, creatinine and clearance mean (sd) was . ( . ) mmol/l; . ( . ) mg/dl; . ( . ) mg/dl; . ( . ) ml/min. . % of the patients had lower concentrations than expected according to population parameters. finally, digoxin dosage was increased in . % of patients, it was maintained in . %, and it was decreased in . %. conclusion: digoxin concentrations in patients with af in combination therapy of digoxin and acenocoumarol are lower than would be expected in most cases. it is important monitoring digoxinaemia to achieve optimal concentrations and a good clinical response. further studies are needed to determine the relevance of this interaction in clinical practice. please specify your abstract type: research abstract background and objective: tocilizumab (tcz) is a humanized monoclonal antibody inhibitor of il- receptor, indicated in combination with methotrexate in the treatment of rheumatoid arthritis (ra) in patients with inadequate response or intolerance to prior therapy. interleukin is involved in the pathogenesis of rheumatoid arthritis via its broad effects on immune and inflammatory responses. previous studies have shown that c-allele at the - g[c (rs ) polymorphism is related with a bad response to tocilizumab (according to eular criteria). the aim of our study was to explore the potential role of il- genetic polymorphisms as a predictor of tocilizumab efficacy in rheumatoid arthritis (ra) patients and check this association depending on the genotype. setting and method: the il- (g[c) (rs ) genetic variant was genotyped using predesigned taqman Ò genotyping assays technology and analysed on a viia Ò real-time pcr system. main outcome measures: clinical response was evaluated at , , and months according to the eular criteria. patients were classified as ''responders'' (good and moderate response according to eular criteria) and ''non-responders''. the statistical analysis was performed using spss v. . results: we recruited patients with ra treated with tocilizumab, these were aged . ± . (mean ± sd), ( %) were women. the mean das at baseline was . ± . . of these patients, the il- g[c genetic polymorphism was significantly associated with ''responders'' at months after the baseline (cc vs non-cc p = . , or . , % ci . - . ) but not at (p = . ), (p = . ) and (p = . ) months. conclusion: the il- g[c may be useful as a genetic marker of tocilizumab efficacy at months. other polymorphisms, clinical parameters and other pharmacological treatment during the follow-up may be checked about their influence on the response to tocilizumab. tdmp : daptomycin pk/pd profile in neutropenic cancer patients with beta-lactam-resistant gram-positive infection nancy perrottet *, , frederic tissot , laurent decosterd , thierry buclin , guy prod'hom , christina orasch , oscar marchetti , farshid sadeghipour , , thierry calandra , véronique erard pharmacy service, infectious diseases service, laboratory and division of clinical pharmacology, service of biomedicine, institute of microbiology, lausanne university hospital, lausanne, school of pharmaceutical sciences, university of geneva, university of lausanne, geneva, switzerland please specify your abstract type: research abstract background and objective: the pharmacokinetics (pk) and pharmacodynamics (pd) of many antibiotics are modified in neutropenic patients and few data are available on daptomycin in this population. this prospective study aimed to assess the pk/pd profile of daptomycin in the treatment of neutropenic patients with beta-lactamresistant gram-positive cocci infections. setting and method: this substudy was performed in the context of a prospective pilot study on daptomycin versus vancomycin in adult hemato-oncological patients with febrile neutropenia and proven or suspected infection with methicillin-resistant staphylococci or betalactam-resistant enterococci. patients received daptomycin mg/ kg/day ( mg/kg/day for enterococci) for c days as a -min infusion. main outcome measures: pk analysis using a published non-linear mixed effect model with nonmem Ò , followed by comparison of parameters with values published for healthy subjects. pd analysis based on auc/mic (area under the concentration-time curve/minimal inhibitory concentration). according to eucast, an auc/mic ratio [ is required for bacteriostatic effect against staphylococci and [ for a two-log reduction in bacterial count. for e. faecium, an auc/mic ratio of . has been suggested for bacteriostasis and . for a -log bacterial count reduction. results: model-derived mean auc observed in patients was . ± . mg h/l, maximum concentration (cmax) ± mg/ l, minimal concentration (cmin) . ± . mg/l. clearance was . ± . l/h and volume of distribution at steady sate . ± . l, both values found higher than those reported in healthy subjects. all patients ( / ) with a staphylococcal infection achieved auc/mic values predictive of bacteriostatic effect on staphylococci, and out of values associated with two-log bacterial killing. of note, infection relapse occurred in the only patient with suboptimal daptomycin exposure (auc/mic of ). the pd targets were also reached in the two patients with e. faecium infection. an asymptomatic elevation of creatine phosphokinase was reported in two patients ( u/l and u/l) with cmin of . and . mg/l, respectively. conclusion: daptomycin pk profile in neutropenic cancer patients indicated higher total clearance and volume of distribution, along with lower total exposure, compared to healthy subjects. despite this, standard dosages allowed attainment of pd targets in / patients with a staphylococcal infection (two-log drop) and / with e. faecium infection ( -log drop) . please specify your abstract type: research abstract background and objective: individual clinical response to infliximab can be influenced by their pharmacokinetics and immunogenicity, so therapeutic monitoring of drug levels (tdm) can guide these biologic treatments. the objective was to analyse the suitability of serum infliximab trough levels (sitls) in patients with inflammatory bowel diseases (ibd) receiving dose schemes based only on clinical response. setting and method: prospective and descriptive study of patients with ibd treated with infliximab and under tdm. medical records were reviewed. dose schemes were established according to clinical guidelines ( mg/kg every weeks) and optimized based on an index of clinical response (mayo, pcr…). sitls (therapeutic range - mcg/ml) and anti-drug antibodies (ada) were measured in all of patients by elisa (promonitor Ò ). ada presence was considered as a therapeutic failure indicator. informed voluntary consent was obtained from all patients. main outcome measures: sitls and ada. results: a total of patients, with a median age of years (range ), were included in the analysis. infliximab standard dose according to clinical guidelines were administered to patients: . % showed sitls under the therapeutic range ( . % with ada). in eight patients with maintained good clinical response, dose decrease or interval elongation had been implemented: % of these patients showed sitls below the therapeutic range ( % with ada). it had been necessary to increase the dose or shorten the interval in patients due to inadequate clinical response: . % of these patients with sitls below the therapeutic range ( % with ada). conclusion: optimization based on clinical response of infliximab treatments in patients with ibd is not always an effective strategy, since it leads to a high percentage of patients with sitls below the therapeutic range and adas. tdm together with clinical response should guide the optimization of infliximab treatments. please specify your abstract type: research abstract background and objective: in addition to its anticonvulsive properties, valproate is also used as a mood stabiliser in bipolar disorder and as augmentation treatment of other psychiatric disorders. the unpredictable relationship between dose-plasma valproate concentrations and correlation between concentrations-efficacy suggest therapeutic drug monitoring (tdm) of plasma valproate concentrations might be useful. the aim of our study was to evaluate the rationale of a new protocol for measuring valproate concentrations and the incorporation of a clinical pharmacist in the process of valproate tdm service, compared to pre-existing standard measuring. setting and method: in the retrospective study we analysed the process of measuring plasma valproate concentrations at the department of psychiatry and at the unit for forensic psychiatry of a large teaching hospital in slovenia before the enrolment of a clinical pharmacist. for the prospective study we created a protocol for tdm of valproate in adults based on literature research. the protocol included reference range, sampling time, indications for sampling and schedule of other laboratory tests that have to be monitored during valproate therapy. main outcome measures: percentage of plasma valproate concentrations in reference range (c trough = - mg/l) before/after the enrolment of a clinical pharmacist, percentage of measured valproate c trough . results: in the retrospective study randomly chosen patients with measured plasma valproate concentrations were included ( % male, age ± years, length of hospital stay ± days). plasma valproate concentrations were measured . ± . times per patient, % were in the reference range (other % subtherapeutic), % were drawn at c though , . % were drawn for assessing compliance (nontrough). in the prospective study patients were included ( % male, age ± years, length of hospital stay ± days). plasma valproate concentrations were measured . ± . times per patient, % were in the reference range (other subtherapeutic), % were drawn at c trough , . % were drawn for assessing compliance. conclusion: the inclusion of a clinical pharmacist in valproate tdm service increased the number of valproate plasma concentrations in the reference range by almost % and increased the number of concentrations drawn at c trough , when indicated. including a clinical pharmacist in valproate tdm is beneficial and the new protocol is useful for optimising valproate therapy. concurrent and predictive validity of a self-reported measure of medication adherence the effect of pharmacist-led interventions in optimising prescribing in older adults in primary care: a systematic review aflibercept: . neovascular membranes with visual acuity higher than . . one eye affection severe cardiovascular pathology (severe episodes in the last months) non-responders to other anti-vegf bevacizumab: . diabetic macular edema macular edema secondary to vascular pathology setting and method: a longitudinal study was carried out in primary care centres. participants: patients aged c , under treatment with or more drugs and belonging to primary care areas in different towns. patients should have at least one of the following potential safety problems: (a) concomitant use of a non-steroidal anti-inflammatory drug (nsaid) with an antihypertensive drug, anticoagulant or antithrombotic drug; (b) use of two or more benzodiazepines. two clinical management units (cmu) were randomized per area to be included in the study. thirty patients per cmu were randomized to be enrolled and monitored during months number of adverse effects ( . ; p \ . ) and number of clinical problems ( . ; p \ . ).with each year increase in age ) and a significant rise in physician ( . ; p \ . ) and nurse ( . ; p \ . ) home visits. women compared to men resulted in a significant decrease ) but a significant increase in visits to nurses ( . ; p \ . ), hospital admissions ( . ; p \ . ) and hospital visits ( . ; p \ . ). age, sex and npsp had no significant effect on falls, fractures or cardiovascular events. conclusion: the npsp in elderly patients contributes to an increase in the use of health services and comorbidity. effective interventions should be addressed to general practitioners to reduce inappropriate prescriptions bpa was found in the dialysate ( ng/l) and ls ( ng/l) wherein the concentration of bpa decreases over time to reach ng/l at the end of a session. finally, bpa was present in all tested dialysis at concentrations of up to . ng/dialyzer in the compartment mimicking the blood and to . ng/dialyzer in the dialysate despite prior rinsing with l of . % nacl. conclusion: our study is the first one to show the risk of exposure to bpa and bpa-clx hdf-ol. while assessment of the impact of this exposure in a patient under treatment remains to be done, it is now possible to better master contamination by bpa and its four chlorinated derivatives through better practices (choice of medical devices) and improvement of the overall water treatment process san cecilio university hospital, genomic unit san cecilio university hospital, genomic unit, genyo, centre for genomics and oncological research the aim of this study is to compare the apparition of stroke, acs, cardio-vascular death and the need of surgery in patients after percutaneous transluminal angioplasty (pta) or stroke depending on the presence of cyp c * * polymorphisms. setting and method: retrospective cohort study. we recruited patients treated with clopidogrel after a pta of the lower limb or stroke (without surgery) from to in our hospital. data collected: age, sex, cyp c * (rs ) and cyp c * (rs ) genotypes and the primary end-point: stroke, acs, cv death and surgery of the affected vessel during months after discharge. polymorphisms were genotyped using taqman Ò genotyping assays technology. main outcome measures: we recruited patients with stroke ( . % men; mean age . ) and patients after pta ( . % men; mean age . ) treated with clopidogrel after discharge %) suffered the primary end-point during months after discharge; of these patients had the cyp c * allele. among patients with pta of the lower limb: % of them had the cyp c * allele and no one a cyp c * allele; ( . %) of these patients suffered the primary end-point during months after the discharge and of these had the * allele of the cyp c isoenzyme * allele and treated with clopidogrel have a higher risk of the primary end-point than those patients not carrying it spain please specify your abstract type: research abstract background and objective: the engagement of fcgrs by tnf antagonists could affect to macrophage-mediated clearance of immune-complexes. the aim of our study was to evaluate the potential role of fcgr a (a[c) (rs ) single nucleotide polymorphism (snp) as a predictor of tocilizumab efficacy in rheumatoid arthritis (ra) patients. setting and method: the fcgr a (a[c) (rs ) snp was genotyped using predesigned taqman Ò genotyping assays technology and analysed on a viia Ò real-time pcr system. the statistical analysis was performed using spss v the mean age of the patients was . ± . years and % were women. the mean das at baseline was . ± . . we found no statistically significant association between our end-point and the genetic polymorphisms studied tdmp : therapeutic drug monitoring of infliximab biosimilar and anti-infliximab antibodies in inflammatory diseases patients with dermatological conditions and inflammatory bowel disease being treated with ifx-b ( mg/kg/ weeks after the induction dose) were included. the concentrations of ifx-b and ati-b were quantified by two sandwich-type elisa immunoassays (triturus Ò analyser). main outcome measures: plasma levels of ifx-b and ati-b, clinical response and infusion reactions. the clinical response was assessed according to pathology of each patient (based on specific clinical variables for the pathology into the electronic history) pharmacokinetic results (% assessments): (a) . % no ifx-b detection (c \ . mcg/ml) and positive ati-b (c [ ua/ml) ( assessments/ patient). atis = , y ua/ml. no clinical response (nr) in . % assessments. (b) . % ifx-b and ati-b (c b ua/ml) no detection ( assessments/ patient). nr %. (c) . % ifx-b detection (c [ . mcg/ml) and negative ati-b ( assessments/ patients) weight: ( - ) kg. twenty assessments, . ( - ) assessments per patient, ( - ) ifx-b doses, % concomitant treatment ( / -azathioprine, / -corticosteroid) the incidence of ati-b was low. a correlation was observed between the presence of ati-b and loss of clinical response, as infliximab original. tdmp : serum concentration of non-vitamin k antagonist oral anticoagulants (noacs) in older hip fracture patients ina linnerud , mette i martinsen estimation of t of noacs by t / = ln /kel [kel; elimination constant] using two s-concentration measurements. results: we included patients (median age years, . % women). noac use was detected be serum analysis in patients ( . %; % coherent with mr), while patients ( . %) used warfarin. of the noac users ( . %) had s-concentrations of noacs above the reference range at admission, and five patients ( . %) had s-concentrations within the reference range before surgery. patients using noac had significantly longer median waitingtime for surgery than warfarin-users ( vs h, p = . ). blood transfusions were given to . % of noac-users vs . % of warfarin-users (p = . ). mean estimated t of noacs were , . and . h for dabigatran (n = ), apixaban (n = ) and rivaroxaban (n = ), respectively. conclusion: mr is effective in detecting noac use in older hip fracture patients, but importantly s-concentrations are higher than expected in this population. this might reflect the significantly longer waiting-time for surgery this column is supplied with packing material made of totally porous spherical silica coated with a silicone polymer monolayer containing octadecyl (c ) groups. the mobile phase was composed of . % na po h o (ph . ), acetonitrile, and methanol ( : : , v/v/v), which was degassed in an ultrasonic bath prior to use. the flow rate was . ml/min at ambient temperature and sample detection was carried out at nm. plasma samples were obtained from patients with cml receiving nilotinib treatment. sampling was performed at the steady state. blood samples were collected by venipuncture h after oral administration of nilotinib. plasma was separated by centrifugation at g for min and stored at - °c until analysis. plasma samples ( ll) were then extracted as described above. the same samples were also sent to a commercial laboratory (bml, inc.) for assaying nilotinib concentration by liquid chromatography-tandem mass spectrometry (lc-ms/ms). in addition, we applied this method to tdm of cml patients receiving nilotinib at our hospital. main outcome measures: the calibration curve exhibited linearity over the nilotinib concentration range of - ng/ml at nm, with relative standard deviations (n = ) of . , . , and . % for , , and ng/ml, respectively. the detection limit for nilotinib was ng/ml due to three blank determinations (q = ). in addition, we compared the results with those measured by lc-ms/ ms at bml, inc. (a commercial laboratory). as a result, a strong correlation was observed between the nilotinib concentrations measured by our hplc method and those obtained by lc/ms-ms (r = . , p \ . ). in addition, tdm of nilotinib was performed to six cml patients. there was the case which participated in dosage adjustment of nilotinib in hepatic dysfunction and poor glycaemic control. results: we have developed a simple ultraviolet detection method for the determination of nilotinib, which has high sensitivity and large dynamic range please specify your abstract type: research abstract key: cord- -rz r sj authors: nan title: abstracts for the th annual meeting of the japan neuroscience society (neuroscience ) date: - - journal: neuroscience research doi: . /j.neures. . . sha: doc_id: cord_uid: rz r sj nan the brain basis of conscious experience is one of the great unsolved mysteries of science. how can a material object became aware of the world around it and of its very own awareness? many scholars think this question is unanswerable. new approaches to this age-old mind-body problem have recently been encouraged by the development of pet and mri and the powerful tools of modern neuroscience. we are now witnessing the explosive growth of a new field, called cognitive neuroscience which focuses on behavior and uses classical reaction-time paradigms together with new computer-based technology. a parallel approach is to cross-correlate formal aspects of conscious experience as the brain spontaneously pursues its regular trajectory through the objectively defined states of waking nrem and rem sleep. at the level of the brain it is possible to record pet and mri and by using an animal model to analyse cellular and molecular mechanisms. the advantage of this approach, which i call the conscious state paradigm, is that it, is quantitative, integrative, and holistic (in the rigorous sense of that word). the brain basis of activation (a) input-output gaining (i) and modulation (m) can now be described in relation to the changes in consciousness associated with them. the data can be used to create a three-dimensional model (aim) which describes the brain-mind trajectory in its state space. neural circuits in many parts of the developing nervous system exhibit highly rhythmic episodes of electrical activity. such activity has generally been considered to fine tune connections that are initially made by axons responding to a complex array of molecular guidance cues. however, chick and mouse spinal cords exhibit activity at early stages as motoneurons are still migrating and beginning to extend their axons. modest alterations in the frequency of such episodes produced changes in the expression of several guidance/recognition molecules and caused motor axon pathfinding errors. interestingly the type of pathfinding decision, the binary dorsal-ventral choice or the subsequent pool specific fasciculation and projection of axons to specific muscles, was differentially affected by decreasing or increasing the frequency respectively. thus, activity generated by developing circuits interacts at early stages with the molecular signaling pathways that govern the formation of precise circuits and any drugs that alter this activity could adversely affect circuit formation. supported by nih grant ns . sl - - frontal cortex and higher-order motor control: preferential use of multiple premotor and prefrontal areas dependent on behavioral context jun tanji brain science research center, tamagawa university, machida, tokyo, japan in the cerebral cortex of primates, there exist a number of motor areas rostral to the primary motor area and caudal to the prefrontal cortex. although each area has been defined based on anatomical and physiological criteria, functional roles played by each of them have been the matter of much debate. in fact, in a recent trend of research reports, it is popular to stress commonalities rather than specificities in the use of multiple cortical areas in motor control. for instance, the involvement of the primary motor cortex in cognitive aspects of motor behavior has been an eye-catching subject of recent reports. the involvement of the prefrontal cortex in movement planning has also been inferred. up to a certain extent, it is true that the use of different areas have some factors in common. however, it is a mistake to ignore profound differences in the use of each area, depending on specific aspects of motor behavior. in this lecture, i will describe such differences that could be clarified only when neural activities are examined properly, by studies designed to reveal individual aspects of functional significance of each area. sl - - neuronal functions and molecular motor, kinesin superfamily proteins, kifs: from transport of synaptic proteins and mrnas, to brain wiring, neuronal survival and higher brain function nobutaka hirokawa department of cell biology and anatomy, graduate school of medicine, university of tokyo, japan the intracellular transports are fundamental for neuronal morphogenesis, functioning and survival. to elucidate this mechanism we have identified and characterized kinesin superfamily proteins, kifs, using molecular cell biology, molecular genetics, biophysics, and structural biology. kifs play essential roles on neuronal function and survival by transporting synaptic vesicle precursors (kif a/kif bbeta), nmda type(kif ) and ampa type(kif s) glutamate receptors and mrnas(kif s) such as camkii ␤ mrna and arc mrna with a large rna-transporting protein complex containing at least rna related proteins such as hn rnp-u, staufen, and fmrps. kif a is fundamental for correct brain wiring by suppressing elongation of axon collaterals through depolymerizing microtubules in growth cones. kif suppresses tumorigenesis by transporting ncadherin/beta catenin containing vesicles from golgi to plasma membrane in the neuroepithelium. kif controls the activity-dependent survival of postmitotic neurons by regulating parp- activity in brain development. thus, kifs play significant roles not only on various neuronal functions but also on brain wiring, development and higher brain functions such as memory and learning. tsukahara award - what we can learn from the functional recovery after brain injury tadashi isa national institute for physiological sciences, okazaki, japan it is believed that when a part of a neuronal system is damaged, some of the lost functions can be taken over by residual systems through training. such concept is considered as the basis of neurorehabilitation, however, the mechanism of the "take-over" is not well understood. in this talk, i will present our recent progress in two lines of studies using non-human primate models related to this issue. the first topic is on the recovery of dexterous finger movements after lesion of the lateral corticospinal tract at the cervical spinal cord. after the virtually complete lesion, relatively independent finger movements can be recovered in - months. we have found that bilateral primary motor and ventral premotor cortices are involved at various stages of the recovery process by combining the pet imaging and reversible local inactivation technique. in the second, i will talk about the visuo-motor processing in monkeys with unilateral lesion of the primary visual cortex (v ). after complete ablation of v , the monkeys recover performance of visually guided saccades toward the blind field in months. saccades to the blind field have low sensitivity and less accurate. however, the monkeys can perform surprisingly cognitively demanding tasks in the blind field. i will discuss on our hypothesis on the bottom-up and top-down control of learning during recovery. takuji iwasato riken brain science center (bsi), wako-shi, saitama, japan in the rodent primary somatosensory (barrel) cortex, the configuration of the whiskers on the face is topographically represented as "barrels", discrete modules of layer iv neurons and thalamocortical afferent (tca) terminals. while barrel formation is an important model of the establishment of patterned topographic connections between the sensory periphery and the brain, the molecular mechanisms underlying this process are poorly understood. we developed and applied mouse reverse genetic technologies to examine these molecular mechanisms. we have focused on the nmda-type glutamate receptor (nmdar) and calcium-stimulated adenylyl cyclases, as nmdar-and camp-cascades are central to various types of neuronal plasticity, both in adulthood and during development. series of global and region-specific knockout mice have revealed the roles of these molecules in the patterning of barrel cortex and differentiated the specific mechanisms at presynaptic tca terminals compared to those at postsynaptic cortical neurons. research funds: presto (jst), kakenhi , kakenhi sy - - - distinct roles of two -pass transmembrane cadherins in neurite growth control tadashi uemura , , yasuyuki shima , , keisuke sehara , manabu nakayama , shinya kawaguchi , mikio hoshino , yoichi nabeshima , tomoo hirano , graduate school of biostudies, kyoto university, kyoto, japan; school of science, japan; kazusa dna research center at chiba, japan; graduate school of science, japan; graduate school of medicine, japan; crest, jst, japan drosophila flamingo (fmi) and mammalian celsr - , which are pass transmembrane cadherins, have been considered to mediate the regulation of neuron contact-dependent neurite growth. we show that mammalian -pass transmembrane cadherins celsr and celsr are activated by their homophilic interactions and regulate neurite growth in a distinct manner. both gene-silencing and co-culture assay showed that celsr enhanced neurite growth whereas celsr suppressed it. our result suggested that celsr had a stronger activity as g-protein coupled receptor than celsr did, most likely due to a difference of a single amino acid residue in the transmembrane domain, and this functional difference resulted in distinct effects in neurite growth regulation. thus, neuron-neuron interactions modulate neurite growth differentially through this couple of -pass transmembrane cadherins. masatoshi takeichi riken center for developmental biology, kobe, japan for synapse formation, axons need to recognize their specific partners, and subsequently stabilize their contacts. while a number of cell surface molecules should be involved in such processes, cadherin adhesion molecules play a role. when cadherin activities are blocked, synaptic contacts become destabilized in cultured neurons. this is also the case in vivo; e.g., in the neural retina whose cadherin activities are impaired without perturbing their overall architecture, a certain class of synaptic contacts does not normally form. another series of our study demonstrate that the cadherins cooperate with nectins, a subfamily of ig-domain molecules, for establishment of axon-dendritic contacts: in early hippocampal pyramidal neurons, nectin- is preferentially localized in axons; and nectin- , in both axons and dendrites. we present evidence that the heterophilic binding between axonal nectin- and dendritic nectin- is important for facilitating the axon-dendritic attachment; and cadherins seem to be required to stabilize the nectin-initiated contacts. thus, multiple classes of adhesion molecules work together to ensure the correct linking between axons and dendrites. hitoshi sakano department of biophysics and biochemistry, university of tokyo, tokyo, japan we have studied how the olfactory sensory neurons (osns) expressing the same odorant receptor (or) gene converge their axons to a specific set of glomeruli in the olfactory bulb (ob). retrogradestaining of osn axons indicated that the dorsal/ventral (d/v) arrangement of glomeruli in the ob is correlated with the expression areas of corresponding ors along the dorsomedial/ventrolateral axis in the oe. in contrast, the anterior/posterior (a/p) arrangement of glomeruli appears to be independent of the epithelial locations of osns and more dependent on the expressed ors. it was found that g proteinmediated camp signals regulate the positioning of glomeruli along the a/p axis in the ob. we also found that multiple sets of cell adhesion molecules, e.g., ephrin-as and eph-as, are expressed in a complementary manner, whose transcription levels are uniquely correlated with the expressing or species. we propose that differential levels of repulsive/adhesive interactions of axon termini may regulate the sorting of like-axons during the process of osn projection. research funds: crest, jst, kakenhi sy - - - lamina-restricted guidance of hippocampal mossy fibers hajime fujisawa , fumikazu suto nagoya university, nagoya, japan; institute of genetics, mishima, japan axons from different sources terminate at particular dendritic segments of target neurons in a laminal fashion. one important issue to be addressed is how individual axons are instructed to invade and arborize in particular laminae. projection of hippocampal mossy fibers is one of good experimental models to analyze molecular mechanisms that govern lamina-restricted termination of axons, because the fibers project to the proximal dendritic segment of ca pyramidal cells. we here report the following three mechanisms that provide lamina-restricted projection of mossy fibers. first, a neural repellent sema a is expressed in ca pyramidal cells and principally suppresses invasion of mossy fibers to ca . second, the repulsive signal of sema a is mediated by plexin-a expressing in mossy fibers. third, the repulsive activities of sema a are attenuated by plexin-a in the proximal dendritic segments of ca pyramidal cells, resulting in the segments permissive for mossy fibers to invade. over the course of development, children become increasingly able to control their thoughts and actions (i.e., cognitive control). the term cognitive control is an umbrella term for a set of putative control processes. these control processes may reach adult levels at different rates, depending on the rate of functional development of the specific brain structures involved. the structure most closely associated with cognitive control is prefrontal cortex (pfc). pfc is composed of what are believed to be functionally distinct subregions, including ventrolateral, dorsolateral, rostrolateral, and medial pfc. i will discuss the control processes associated with each of these regions, and how the functionality of these regions differs between school-aged children, adolescents, and young adults. three fmri studies will be presented, focusing on ( ) working memory maintenance and manipulation, ( ) rule representation and task-switching, and ( ) relational reasoning. based on these data, i will discuss some general points about neurodevelopment changes in cognitive control, and outline the approach that our laboratory has taken in our developmental cognitive neuroscientific research. sy - - - towards manipulative neuroscience based on non-invasive brain decoding in atr computational neuroscience laboratories, we proposed several computational models such as cerebellar internal models, mosaic, modular and hierarchical reinforcement-learning model. some of these models can quantitatively reproduce subject behaviors given sensory inputs and reward and action sequences which subjects received and generated. these computational models possess putative information representation such as error signals for internal models, action stimulus dependent reward prediction, and they can be used as explanatory variables in neuroimaging and neurophysiology experiments. we named this approach as computationalmodel-based neuroimaging, as well as computational-model-based neurophysiology. this new approach is very attractive and appealing since this is probably the only method with which we can explore neural representations remote from either sensory or motor interfaces. but, sometimes limitation of mere temporal correlation between the theory and data became so apparent, and we started to develop a new paradigm 'manipulative neuroscience' where physical causality is guaranteed. research funds: nict, karc sy - - - neural mechanisms in williams syndrome-insights from neuroimaging andreas meyer-lindenberg nimh, bethesda, md, usa williams syndrome (ws), a rare disorder caused by hemizygous microdeletion of − genes on chromosome q . , has long intrigued neuroscientists with its unique profile of striking behavioural abnormalities, such as hypersociability, combined with a differential impact on cognitive functions, with some types of abilities only mildly affected while others are severely impaired. ws, thus, raises fundamental questions about the neural mechanisms of social behaviour, the modularity of mind, and brain plasticity in development, and provides a privileged setting to understand genetic influences on complex brain function in a bottom-uph way. recent months have seen dramatic advances in uncovering the functional and structural neural substrates of ws and a beginning understanding of how these are related to dissociable genetic contributions characterized both in special participant populations and animal models. we will review neuroimaging work indicating abnormal function and structure in subsystems of visual processing, long term memory, and emotional regulation and social cognition, and discuss advances in relating them to the underlying molecular biology of this unique syndrome. daisuke yamamoto tohoku university graduate school of life sciences, japan the fruitless locus of drosophila was originally recognized by its mutants, the males of which preferentially court males rather than females. the fruitless primary transcript is subject to sexually dimorphic splicing mediated by transformer, and encodes a group of proteins that are putative transcription factors of the btb-zn finger protein family. the male-specific fruitless protein is expressed in small groups of cns neurons of males, but not of females. fruitless masculinizes these neurons thereby establishing the neural substrates for male-typical behavior. by experiments that label individually the neurons that express fruitless, we have identified a subset of brain interneurons that display marked sexual dimorphism in their number and projection pattern. fruitless supports the development of those neurons with male-specific dendritic fields, which are programmed to die during development in females as a result of the absence of fruitless. thus, the fruitless protein expression can produce a male-specific neural circuit likely used for heterosexual courtship by preventing cell death in identifiable neurons. hitoshi okamoto riken brain science institute, wako, saitama, japan the emotional behavior depends on the evolutionarily most conserved neural circuits. especially the fear behaviors involve the basal telencephalic nuclei such as the amygdala and the nucleus accumbens. thanks to the progress in understanding of the telencephalic development among different species, we can determine the correspondence of the parts between the teleost and mammalian telencephalons. with these in mind, we initiated the characterization of the emotional neural circuits in the zebrafish brain which are amenable to various modern technology. we already reported the asymmetric axonal projection from the left and right habenulae which act as the relay station to conduct the emotional information from the telencephalon to the monoaminergic neurons in the midbrain and the hindbrain. to investigate whether such asymmetric neural circuits cause the laterality for emotional behaviors, we are now in the process of establishing the paradigms for combining the behavioral assay with genetic manipulations to control the activity of the emotional neural circuit in zebrafish. research funds: kakenhi ( ) sy - - - a molecular biological approach for songbirds to study learned vocal communication kazuhiro wada, erich jarvis duke university, usa songbirds possess one of the most accessible neural systems for the study of brain mechanisms of behavior, particularly that for learned vocal communication. however, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene manipulation tools. to overcome this limitation, we generated a resource of full-length cdnas for gene expression analyses and functional gene manipulation in songbirds. we constructed total full-length cdna libraries from brains in different behavioral and developmental conditions. with these cdnas, we created a novel database and k songbird cdna array. we used the arrays to reveal a set of genes regulated by singing behavior. their molecular functions spanned most cellular and molecular categories, including signal transduction, structural, and synaptically released molecules. with the full-length cdnas, we were able to express proteins of singing-regulated genes in targeted brain area, using a lentiviral system. this resource now opens to more thoroughly study molecular neuroethological mechanisms of behavior. research funds: uehara memorial fellowship to k.w. and nih grant to e.j. - - - stepping pattern learning using mice: histochemical identification of activated neuronal circuits takashi kitsukawa graduate school of frontier biosciences, osaka university, osaka, japan identifying brain areas and neuronal circuits activated in a behavior is a critical step in understanding how the brain works in that behavior. also, identifying neuronal types involved in a behavior is a key step toward connecting behavioral approaches with molecular and genetical approaches. an efficient method of clarifying neuronal types activated by behavior is histochemical identification of neuronal types combined with c-fos staining. i would like to introduce our work as an example of using this method. in order to understand the neural processing involved in sequential motor skill learning we built a wheel running system in which a mouse learns sequential stepping patterns. we double-stained brain sections from mice which performed this task with c-fos and a neuronal marker such as enkephalin, substance p or nitric oxide synthase, each of which denotes a particular neuronal type. our results indicate that particular types of stiriatal neurons are activated during this learning, suggesting that cortico-striatal circuits are involved. synaptic plasticity that is dependent on precise timing of spikes between pre-and postsynaptic neurons plays important role in development and plasticity of brain functions. such spike-timingdependent plasticity (stdp) has attracted wide attentions because of its high computational power and physiologically plausible induction. we previously demonstrated that long-term potentiation was closely associated with structural plasticity of dendritic spines. however, how stdp is associated with structural changes has not been elucidated. we here report that paired two-photon uncaging of a caged-glutamate compound at a single spine and postsynaptic spike of whole-cell clamped neuron rapidly induced long-lasting bidirectional structural plasticity of spines in hippocampal ca pyramidal neurons. our results indicate that stdp is intimately associated with bidirectional structural plasticity at the level of single spines. research funds: kakenhi sy - - - role of camkii as a structural protein that stabilizes actin cytoskeleton in dendritic spines kenichi okamoto, radhakrishnan narayanan, yasunori hayashi massachusetts institute of technology, usa actin serves as a major cytoskeleton which maintains spine structure and exists in a equilibrium between f-actin and g-actin. tetanic stimulation causes a persistent shift of actin equilibrium towards f-actin which enlarges dendritic spines. but the mechanisms which maintain these changes remain elusive. we propose that camkii ␤ acts as an actin stabilizing molecule to maintain spine structure. camkii ␤ is not only an abundant f-actin binding protein, it can also make oligomers. we found that camkii ␤ oligomer crosslinks f-actin and stabilizes actin depolymerization kinetics. in spines, camkii ␤ oligomer slows down actin dynamics and camkii ␤ is enriched in spines by actin polymerization. the suppression of endogenous camkii ␤ alters spine shape to filopodia-like structures. these experiments suggest that camkii ␤ plays a role as a major stabilizer of the actin cytoskeleton to maintain spine structure. we also found that camkii ␤ detaches from f-actin in an activity dependent manner. we will discuss how camkii ␤ maintains actin equilibrium in activity dependent dendritic plasticity. ryohei yasuda duke university medical center, usa the small gtpase protein ras plays central roles in calcium signaling important for many forms of synaptic plasticity and regulation of neuronal excitability. using -photon fluorescence lifetime imaging microscopy in combination with a fret-based ras activity sensor, we visualized the activity of ras signaling with high spatiotemporal resolution. our studies indicate that calcium entry due to action potentials causes ras to activate in a supra-linear manner (yasuda et al., ) . furthermore, in response to single spine stimulation using -photon glutamate uncaging, ras activation initially occurs at the stimulated spine, subsequently spreading into its parent dendrites and nearby spines. these results suggest that nonlinear filtering by ras regulators as well as the spatial spreading of ras and ras regulators shape spatiotemporal patterns of ras signaling. hiroshi shibasaki takeda general hospital, kyoto, japan involuntary movements are unintended, generalized or focal, movements of abnormal nature, and include tremor, myoclonus, dystonia, chorea/ballism, athetosis and dyskinesia. myoclonus is characterized by abrupt, shock-like movements caused by brief muscle contraction (positive myoclonus) or abrupt cessation of on-going muscle contraction (negative myoclonus), or their combination. depending on the estimated origin, it is classified into cortical, brain stem, and spinal myoclonus. cortical myoclonus is short in duration ( ms). by back averaging eeg or meg time-locked to spontaneous myoclonus, a cortical activity is demonstrated in the corresponding area of the contralateral primary motor cortex immediately preceding the myoclonus (by ms for hand). it is mediated by fact-conducting corticospinal pathway. cortical myoclonus is often stimulus-sensitive (cortical reflex myoclonus), showing extremely enhanced cortical responses to somatosensory or visual stimulus, and enhanced longloop transcortical reflexes. these findings, together with transcranial magnetic stimulation, suggest increased excitability of sensorimotor cortex in cortical myoclonus. mark hallett ninds, bethesda, md, usa there have been many recent advances in the understanding of the physiology of focal dystonia. three main avenues of research have shown abnormalities in cortical inhibition, sensory processing including sensorimotor integration, and plasticity. this lecture will emphasize the abnormal inhibition. abnormal inhibition appears to be the most direct cause of unwanted muscle contractions that make up both the involuntary spasms and the overflow movements also seen in this condition. a loss of inhibition is seen in spinal and brainstem reflexes, but these changes are likely secondary to cortical abnormalities. cortical inhibition is also diminished as demonstrated most clearly with transcranial magnetic stimulation. gaba content may be decreased as shown with magnetic resonance spectroscopy. a particular type of defective inhibition is surround inhibition, the inhibition that normally operates to sharpen fine skilled movements. studies are now in progress to determine the synaptic mechanisms of surround inhibition and how this becomes abnormal in dystonia. understanding about inhibition in dystonia has led to some new treatments including some non-invasive cortical stimulation methods. research funds: nih intramural program sy - - - basal ganglia-cortical systems reinforcing tonic motor activity in health and disease peter brown sobell department, institute of neurology, uk the synchronisation of neuronal activity in the beta frequency (∼ hz) band has been noted in healthy primates, including humans, at both striatal, putamenal and cortical levels. it is most obvious in the motor cortex during tonic motor activity and is suppressed by voluntary movement. in this talk i will develop the idea that beta band synchronisation in the basal ganglia-cortical loop promotes tonic/postural contraction at the expense of new movements. thus, spontaneous phasic increases in beta activity in healthy subjects can be shown to be associated with a slowing of voluntary movements and a reinforcement of transcortical stretch reflexes. beta synchrony is also greatly exaggerated in untreated parkinson disease, where it may bias against new movement and contribute to bradykinesia and rigidity. excessive dopaminergic stimulation, either during treatment for parkinson disease, or in conditions such as dystonia, may overly suppress beta activity in bg-cortical loops leading to excessive movement. recordings of local field potentials in the basal ganglia of patients with movement disorders will be described that support this schema. research funds: mrc sy - - - coding of reward value of actions and valuebased action selection in the basal ganglia a damage of the nigrostriate dopamine system results in severe impairments of voluntary movements as well as involuntary behavioral states like rigidity, akinesia and tremor as typically observed in parkinson disease. recent studies revealed that long-term potentiation of corticostriatal synaptic transmission occurs dopaminedependent manner, and that neuronal firing related to external stimuli and body movements are modulated by whether the stimuli and movements are associated with reward or not. we recorded striatal neurons of monkeys who chose between left-and right-handle-turns based on the estimated reward probabilities of the actions. during a delay period before the choices, activity of more than one-third of striatal projection neurons was selective to the values of one of the two actions. during handle-turns, another subset of neurons was activated. these results suggest representation of action values in the striatum, which can guide action selection in the basal ganglia circuit. roles of the basal ganglia circuit in voluntary and involuntary action selection will be discussed. in vivo reporter gene imaging is expected to be a powerful tool in gene and cell therapy monitoring. we designed a new pet reporter gene system with f- fluoroestradiol (fes) and human estrogen receptor ligand binding domain (herl), which would work in various tissues with little physiological effect. we have been evaluating its potential in gene therapy monitoring constructing a plasmid co-expressing thymidine phosphorylase (htp), a factor works for revascularization, as therapeutic gene and herl. cos cells transfected with the plasmid expressed the both proteins and, when the plasmid was in vivo electroporated into mouse calf muscle, the electroporated muscle accumulated significantly higher amount of fes that the control side. this system was successfully applied to es cell transplantation monitoring also. inducible herl expression system was stably transfected into mouse es cells and viable es cells could be detected in vivo using fes. these data support the prospect that our in vivo reporter gene system would be useful in gene/cell transplantation therapy monitoring. tetsuya suhara molecular imaging center, national institute of radiological sciences, chiba, japan the molecular imaging using positron emission tomography enables to visualize various brain molecules with radio labeled ligand. neurons and glias express various receptors and transporters and those can be a specific target of the imaging. the functions of those molecules can be examined in various types of pharmacologically or genetically modified animal models. amyloid precursor protein transgenic mice provide the target of in vivo imaging of amyloid protein and glial reaction. because pronounced neuronal death is frequently heralded by microgliosis, in vivo analysis of glial activation in a quantitative manner could be a powerful means for assessing neuroglial degeneration. on the other hand, clinical finding of molecular imaging can also provide important cues for the basic research targets. since there is no ideal animal model for psychiatric disorders, the abnormal dopamine d receptor found in clinical research indicates a possible therapeutic target of negative symptoms of schizophrenia. the bidirectional interaction between basic research and clinical research using the molecular imaging technique can expand our knowledge in brain disease. sumiko mochida department of physiology, tokyo medical university, tokyo, japan in presynaptic terminals, packages of neurotransmitters, synaptic vesicles (svs), are localized at specific sites in different stages by regulation of proteins complexes. the recent outstanding studies have revealed molecular mechanisms of presynaptic structure and function. for svs, new proteins were found and the anatomy of vesicle structure was clarified. readiness for transmitter release, svs are docked and primed at the cytomatrix at the active zone where proteins complex formation is regulated by phoshorylation. new kinase sad- found at the sv and active zone or pka phosphorylates specific proteins. sv exocytosis is triggered by conformational changes in the fusion proteins complex when ca + sensing protein was activated. synchronies of sv fusion are maintained by a ca + sensing protein, synaptotagmin i. after transmitter release svs are recycled: surprisingly, recycled svs are shared between synaptic boutons regulated by cytoskeletal and motor proteins. these new findings suggest fine mechanisms in presynatic terminals that regulates transmitter release. shigeo takamori st century coe program, department of neuorology and neurological science, tokyo medical and dental university, tokyo, japan synaptic vesicles are storage organelles for neurotransmitters that recycle in the presynaptic terminals. to achieve their functions, i.e. neurotransmitter uptake and membrane fusion, they have to be equipped with specified proteins that play essential roles for each process. since decades ago, we have witnessed major advances in our knowledge about the molecular constituents on synaptic vesicles and we've functionally characterized many key players on membrane fusion machinery such as snare proteins and the rab gtpases and on neurotransmitter uptake such as vesicular transporters and vacuolar h + -atpase. however, a detailed picture of a vesicle membrane with all of its constituents is not yet available. in the present study, we have applied a combination of biochemical and biophysical approaches on purified synaptic vesicles from rat brains in order to arrive at a comprehensive and quantitative description of synaptic vesicles. in particular, with a newly developed counting method for synaptic vesicles in solution, we estimated the copy number of each molecule in a single synaptic vesicle. sy - - - sad: a novel kinase implicated in phosphoproteome at the presynaptic active zone toshihisa ohtsuka department of clinical and molecular pathology, faculty of medicine/graduate school of medicine, university of toyama, toyama, japan sad is a serine/threonine kianse, which has been shown to regulate various neuronal functions during development, including clustering synaptic vesicles, maturation of synapses, and axon/dendrite polarization: these have recently been revealed by genetic studies in c. elegans and mice. to test if sad is also involved in synaptic functions at mature neurons such as neurotransmitter release, we have recently isolated and characterized human orthologues of sad. interestingly, sad localizes both on synaptic vesicles and at the presynaptic active zones. moreover, sad, together with prominent active zone proteins cast and bassoon, is tightly associated with the active zone cytomatrix. in accord with its unique localization at the nerve terminals, sad appears to be involved in a late step of neurotransmitter release, via direct phosphorylation of another active zone protein rim . thus, these results suggest that sad regulates not only neural polarization during development but also neurotransmitter release at mature synapses. toshiaki sakisaka , takeshi baba , sumiko mochida , yoshimi takai department of molecular biology and biochemistry, osaka university graduate school of medicine, osaka, japan; depatment of physiology, tokyo medical university, tokyo, japan two types of factors are involved in ca + -dependent neurotransmitter release: the snare system and its regulators. the regulators of the snare system include many factors, such as the rab system, munc- , munc- , doc- , and tomosyn. we have previously reported that tomosyn, a syntaxin- -binding protein, works as a molecular clamp that controls free syntaxin- availability for the formation of the snare complex and thereby regulates synaptic vesicle exocytosis. here we show that pka-catalyzed phosphorylation of tomosyn decreases its binding to syntaxin- , resulting in enhanced the formation of the snare complex. conversely, rock phosphorylates syntaxin- , which increases the affinity of syntaxin- for tomosyn and forms a stable complex with tomosyn, resulting in inhibition of the formation of the snare complex. thus, tomosyn is likely to be an upstream regulator of the snare system, whose activity is regulated via well known signal transduction pathways. tei-ichi nishiki department of physiology, okayama university, okayama, japan a synaptic vesicle membrane protein, synaptotagmin i is thought to be a ca + sensor for neurotransmitter release. however, the physiological contributions of its ca + -binding domains (c a and c b) are still unclear. we have studied the roles of aspartate (asp) residues in the c b ca + -binding sites. in synaptotagmin i deficient neurons, although synchronous release was abolished as previously reported (cell , p. ) , asynchronous release was significantly increased. this defect was completely rescued by expressing wild-type synaptotagmin i, indicating the crucial roles of synaptotagmin i for triggering synchronous release and suppressing asynchronous release. synaptotagmin i with mutations in the second or third asp inhibited synchronous release, but still could suppress asynchronous release. thus, we conclude that synaptotagmin i maintains the synchrony of transmitter release in two ways. ca + binding to the c b is essential for synchronizing release. suppressing of asynchronous release seems not to require ca + binding to the c b because mutation in the second asp inhibits ca + binding, yet still allows the protein to suppress asynchronous release. yukiko goda, kevin j. darcy, kevin staras, lucy m. collinson mrc cell biology unit and lmcb, university college london, uk it has been assumed that vesicle replenishment at central synapses operates autonomously at individual presynaptic terminals. we tested the classical model of a compartmentalized synaptic vesicle cycle using fluorescent styryl dyes in combination with methods of fluorescence recovery after photobleaching and correlative light and electron microscopy in cultured hippocampal neurons. we found that endocytosed, recycling synaptic vesicles travel along axons and incorporate into non-native synapses by an actin-dependent mechanism. these newly-incorporated vesicles underwent exocytosis upon stimulation, demonstrating that they form part of the functional recycling pool at their host synapses. our findings indicate that synaptic vesicle recycling is not confined to individual presynaptic terminals but rather a substantial proportion of synaptic vesicles are shared constitutively between synapses. research funds: mrc, nih and narsad hiroshi kunugi department of mental disorder research, national institute of neuroscience, ncnp, japan neurotrophins such as brain-derived neurotrophic factor (bdnf) and neurotrophin- (nt- ) have been implicated in the phathogenesis of several neuropsychiatric diseases including schizophrenia, mood disorders, and neurodegenerative diseases. in the past decade, we have systematically screened bdnf, nt- and its low-affinity receptor p genes for polymorphisms and their possible association with neuropsychiatric diseases. as a result, three polymorphisms of the bdnf gene (c t in the noncoding region, bdnf-linked polymorphic region, and val met), three polymorphisms of the nt- gene (g- a, microsatellite in intron , and gly- glu) and a missene polymorphism of the p gene (ser leu) have been found to be associated with susceptibility to schizophrenia, bipolar disorder, depressive disorder, or alzheimer's disease, although some contradictive negative results have also been reported. here i summarize these findings, review the relevant literature, and discuss future directions of the promising role of the genetic variations of neurotrophins and p in neuropsychiatric diseases. recently, a single nucleotide polymorphism (val met) in the bdnf gene resulting in a prodomain substitution at position from a valine (val) to methionine (met) has been shown to lead in humans to altered hippocampal size and function, and susceptibility to neuropsychiatric disorders. we have recently determined in vitro that bdnf met aberrantly engages a specific vps protein, sortilin, that is part of a highly specialized sorting machinery that regulate bdnf trafficking to secretory pathways. in order to determine whether these trafficking defects are responsible for impaired hippocampal functioning, we have developed a transgenic knock-in mice containing the genetic variant bdnf (bdnf met/met ). we have determined that there is a regulated secretion defect for bdnf met , as well as altered hippocampal structure and function in these bdnf met mice, in a manner similar to that reported in humans with this variant bdnf. thus, this bdnf met/met mouse may provide an in vivo model system to inform human studies focused on associations of this variant bdnf with clinical disorders. research funds: nih grant# ns sy - - - processing of bdnf and brain disorders masami kojima , aist, osaka, japan; sorst, jst, saitama, japan the fact that pro-and mature neurotrophins elicit opposite effects through p neurotrophin receptor (p ntr) and trks, respectively suggests that proteolytic cleavage of proneurotrophins is an important mechanism that controls the direction of neurotrophin actions. here we examined the effects of two rare single nucleotide polymorphisms (snps); (t/g) and (t/g) of the human bdnf gene, causing amino acid substitution (r m and r l) near the cleavage site. western blot analysis and two-side elisa demonstrated that these snps prevented the cleavage, resulting in secretion of probdnf, but not mature bdnf. these snps did not affect intracellular distribution or mode of secretion of the protein. application of the uncleavable probdnf (probdnfml) elicited apoptosis of cerebellar granule neurons, but inhibited dendritic growth of basal forebrain cholinergic neurons. together, these results reveal structural determinants for the cleavage of probdnf, and demonstrate distinct functions of probdnf for different populations of neurons. we have now analyzed the brain functions of the mice expressing this form of bdnf. sy - - - pleiotropic effects of gdnf in regulation of enteric nervous system development hideki enomoto laboratory for neuronal differentiation and regeneration, riken center for developmental biology, kobe, japan formation of the enteric nervous system (ens) is governed by multiple extracellular signals at a given time during development. inactivation of the gene encoding gdnf, ret or gfr␣ leads to nearly complete absence of enteric neurons during early development. although this finding establishes gdnf as an essential extracellular signal acting at the initial stage in ens development, little is known about whether and how enccs continue to depend on gdnf later in development. we have generated mice in which function of gfr␣ , the high affinity receptor for gdnf, is conditionally inactivated in a time-specific fashion. we will show how gdnf signal influences cell migration, differentiation, proliferation and survival of developing enteric neurons, and discuss the biological significance of the findings in development and regeneration of the nervous system in general. bone marrow stromal cells (mscs) including the primitive pluriopotent mesenchymal stem cells and the multipotent adult progenitor cells, are attractive targets for cell and gene therapy for the range of central nervous system disorders. we present using replicationincompetent hsv- vector that msc population can be efficiently engineered to secrete a series of various cytokines in the large quantities and in long term in vivo to be able to treat the ischemic stroke of the brain potentially. three kinds of gene-transferred mscs, hgf, il ss+fgf- , and vegf were prepared and directly transplanted into the lesioned brain of rat transient middle cerebral artery occlusion model. each growth factor gene-transferred mscs achieved the remarkable amelioration of neurological symptoms and apparent decreasing of infarct volume comparison with native research funds: kakenhi ( ) sy - - - hgf gene therapy for the treatment of spinal cord injury masaya nakamura , akio iwanami , kazuya kitamura , , yoshiaki toyama , hideyuki okano department of ophthalmology surgery, keio university, tokyo, japan; department of physiology, keio university, japan hepatocyte growth factor (hgf) has recently been reported to exhibit neurotrophic activity and to play a role in angiogenesis. in this study, we demonstrated the validity of hgf for treatment of spinal cord injury (sci) in adult rats. first, we analyzed temporal expression of hgf and c-met in the injured spinal cord. hgf-mrna expression was relatively low in the acute phase of sci compared with c-met mrna expression. hypothesizing that hgf is insufficient immediately after sci, we induced sci at th level in adult rat days after injecting herpes vector-mediated gene transfer of hgf (hgf group) or lacz (control) into spinal cord. motor function was evaluated by bbb score. or weeks after injury, histological analyses were performed. there were significant decreases in apoptotic cell number and significant enhancements of angiogenesis and gap +fibers in hgf group compared to the control group. animals of the hgf group showed better functional recovery than the controls. these findings suggest that hgf could have therapeutic effects for sci. hiroshi funakoshi, toshikazu nakamura division of molecular regenerative medicine, osaka university graduate school of medicine, osaka, japan hgf was initially identified and molecularly cloned as a mitogen for primary hepatocytes (nakamura et al., ) . recently, hgf is found to be a novel neurotrophic factor for various types of neurons, such as hippocampal, cerebral cortex, cerebral granular, motor, and sensory neurons. mutant c-met/hgf receptor knock-in mouse reveals that hgf decreases neuronal survival and axonal elongation of several types neurons, including motor, sympathetic and cerebral granular neurons, during development (maina et al., ) . therefore, it is possible to speculate that hgf could play an important role in the retardation or regeneration of neurons in neurodegenerative diseases. here we show the examples of beneficial effects of hgf on model animals of different neural and neurodegenerative diseases, using several delivery methods for hgf including gene therapy approach. we also present the possible application of hgf in modifying the neurogenesis for the disease. references maina et al., . nature neuroscience. nakamura et al., . nature. hiroyuki kato center for clinical medicine and research, international university of health and welfare, nasushiobara, japan we examined stroke patients using fmri at acute/subacute and chronic stages, and visualized areas of brain activation during paretic hand movements. normal hand movement activated the contralateral primary sensorimotor cortex, supplementary motor areas, and ipsilateral cerebellum. at the acute/subacute stages, we observed reductions of these activations and/or addition of activation in ipsilateral cortex or contralateral adjacent cortex during paretic hand movements. at the chronic stages, recovery of activation and/or persistent addition of activation were observed. thus, motor functional recovery was accompanied by restoration of brain activation and/or appearance of additional activation within the motor network of the brain. the findings suggest that cortical motor reorganization as well as recovery from reversible injury plays a role in the restoration of motor function. interestingly, the time period during which reorganization occurred was limited to first - months after stroke, suggesting the presence of a critical period. in the cat, illert et al. ( ) first demonstrated that disynaptic pyramidal excitation in forelimb motoneurons can be mediated via propriospinal neurons located in the c -c segments. in contrast, recently it has been shown that polysynaptic pyramidal epsps are only rarely observed in forelimb motoneurons of macaque monkeys and humans. we reexamined the indirect corticomotoneuronal inputs in the primates, and obtained the following evidence for the pathway. ( ) in the macaque, recordings from forelimb motoneurons showed polysynaptic pyramidal epsps after blockade of glycinergic inhibition by strychnine. moreover, we recently identified c -c propriospinal neurons, which receive pyramidal inputs and project to forelimb motor nuclei. ( ) in human arm motor units, magnetic stimulation of the motor cortex produced multiple peaks at short latency in the post-stimulus time histogram, whose total duration was longer than the corresponding value of a finger muscle. stimulation of the pyramidal tract in the medulla could also produce multiple peaks, though in a lower frequency. functions of the pathway both in physiological and pathological conditions will be discussed. bisphenol-a (bpa) has been extensively evaluated for toxicity in a variety of tests as the most common environmental endocrine disruptors. we previously reported that prenatal and neonatal exposure to bpa potentiated central dopaminergic neurotransmission, resulting in supersensitivity to psychostimulant-induced pharmacological actions. many recent findings have supported the idea that astrocytes, which are a subpopulation of glial cells, play a critical role in neuronal transmission in the central nervous system. we found that in vitro treatment with bpa caused the activation of astrocytes, as detected by a stellate morphology and an increase in levels of gfap. a low concentration of bpa significantly enhanced the ca + responses to dopamine in both neurons and astrocytes. these findings provide evidence that bpa induces dopaminergic changes in neurons and astrocytes. this phenomenon may, at least in part, contribute to the enhancement by bpa of the development of psychological dependence on drugs of abuse. mami yamasaki, yonehiro kanemura the department of neurosurgery, clinical research institute, osaka national hospital, national hospital organization, osaka, japan l cam(l ) is a member of the immunoglobulin superfamily of cell adhesion molecules. x-linked hydrocephalus, masa syndrome and certain forms of x-linked spastic paraplegia are now known to be due to mutations in the gene for l . therefore, these syndromes have been reclassified as l syndrome. we performed a nation-wide l gene analysis and identified li gene mutations in families with l syndrome. all the patients showed developmental delay in various degree. we discussed genotype and phenotype correlations, a striking correlation between the mutation class and the severity of symptoms and molecular basis of severity of developmental delay. the loss of extracellular domain functions like l -mediated cell adhesion and cell migration is considered to be responsible for molecular genesis of ventricular dilatation and disturbance of the functions of cytoplasmic domain would cause symptoms related axon growth in l syndrome. research funds: kakenhi ( ), ( ) sy - - - rett syndrome and developing brain yoshiko nomura segawa neurological clinic for children, japan rett syndrome (rtt) is a neurodevelopmental disorder with mental retardation, autistic feature, and stereotyped hand movements. hypofunction of the brainstem monoaminergic neurons is suggested. pathology showed no degeneration. methyl-cpg-binding protein gene (mecp ) located at xq is the causative gene. types of mutation at different functional domains are correlated to clinical severities. x-inactivation also influences phenotypic variability. mecp was thought as a global transcriptional repressor, but finding of bdnf as a target gene suggest its role in the neuronal activity-dependent gene regulation. genetic heterogeneities have been suggested and the mutation of cyclin dependent kinase-like gene (cdkl ) manifest as atypical rtt. the mutations of mecp are found in other clinical conditions, such as x-linked mental retardation, angelman syndrome, autism, and severe neonatal encephalopathy. thus, the evaluation of rtt gives the clue to study the clinical, pathophysiological, biological and molecular correlation of not only rtt but also other neurodevelopmental disorders. in our previous studies, we have proposed that ros and/or ros-mediated signal play(s) an essential role in -ohda-induced, caspase-dependent apoptosis. in contrast, mpp+-mediated death is not blocked by caspase inhibition and is accompanied by an increase in intracellular free calcium. subsequently, we have demonstrated that mpp+ induces release of cytochrome c but not activation of caspase and proposed that depletion of atp and/or calcium-activated calpain-mediated degradation of procaspase- are responsible for the absence of subsequent activation of caspases. furthermore, we have identified that degradation of several important proteins by activated calpain and proteasome system is linked to mpp+-mediated dopaminergic neuronal death. as such, we have found that one of onconeural proteins seems to play a role as a potential survival factor, degradation of which is involved in mpp+-induced cell death. taken together, we reason that distinct set of proteases activation is involved in experimental models of pd. therefore, novel strategies interfering activation of these proteases may contribute to prevention of dopaminergic neuronal death. satoshi ogawa department of neuroanatimy, kanazawa university of medical school, ishikawa, japan we discuss the role of er-stress in neuronal cell death in snpc by introducing two models. upregulation of pael-receptor in the substantia nigra pars (snpc) of mice induces endoplasmic reticulum (er) stress leading to a decrease in tyrosine hydroxylase and death of dopaminergic neurons. the role of er stress in dopaminergic neuronal vulnerability was highlighted by their enhanced death in mice deficient in the ubiquitin-protein ligase parkin and the er chaperone orp , suggesting parkin dysfunction result in er-stress mediated neuronal cell death. conversely, transgenic rats overexpressing megsin (tg meg), a newly identified serine protease inhibitor (serpin), demonstrated intraneuronal periodic-acid schiff (pas) positive inclusions, which distributed throughout the deeper layers of cerebral cortex, hippocampal ca , and substantia nigrta. enhanced er stress was observed in dopamine neurons in snpc, accompanied with loss of neuronal viability and motor coordination. in both subregions, pas-positive inclusions were also positive with megsin. these data suggest that enhanced er stress causes selective vulnerability in a set of neuronal populations. noradrenaline (na) transmission modulates synaptic excitability and plasticity through distinct receptor subtypes. accumulating evidence has suggested that the central na system modulates consolidation and reconsolidation of long-term emotional memory. here we show that the na system is particularly important for retrieval of reconsolidated emotional memory. the mutation of the gene encoding tyrosine hydroxylase causes a deficit in conditioned taste memory after its reactivation. this memory deficit is restored by pharmacological stimulation of na activity before the test and is also restored by intra-amygdala na stimulation through ␣ or ␤-adrenergic receptors. moreover, intra-amygdala na stimulation in the wild type animals increases their susceptibility to recall reconsolidated memory. our findings indicate that the amygdalar na system, primarily through ␣ and ␤-adrenergic receptors, acts to improve the retrieval of reconsolidated memory trace. shigeru morinobu, shigeto yamamoto, shigeto yamawaki departmnet of psychiatry and neurosciences, hiroshima university, hiroshima, japan as psychophysiological reactivity on exposure to cues resembling an aspect of the trauma is the major symptom in ptsd, it is hypothesized that impaired extinction may be involved in ptsd. rats subjected to single prolonged stress (sps) exhibit the enhanced negative feedback of the hpa axis, exaggerated startle response, and analgesia. thus, sps is a good model of ptsd. we examined whether extinction of fear memory was impaired in sps rats, using the contextual fear conditioning. sps rats exhibited the significant longer freezing during re-exposure to the context - days after the conditioning. furthermore, repeated administration of d-cycloserine markedly inhibited the development of enhanced freezing in sps rats. we measured the levels of nmda receptor subunits (nr , nr a, b, c), glycine transporter , and eaac , by real-time pcr. no significant changes were found in the hippocampus. based on these findings, it is speculated that the increase in other types of glutamate transporters or nmda receptor modification may play a role in impaired extinction in sps rats. ichiro masai masai initiative research unit, riken, wako, japan in human, there are hereditary retinal diseases such as retinitis pigmentosa. to understand these molecular mechanisms, we performed a large-scale mutagenesis using zebrafish as an animal model. here we report two zebrafish mutants, twilight (tli) and eclipse (els), both of which show no normal erg and okr response. in the tli mutant, photoreceptors initially differentiate but degenerate later. electron-microscopic analyses revealed that photoreceptive membranes are severely disorganized in the tli mutants, suggesting that tli is required for the formation of photoreceptive membranes. in the els mutant, photoreceptors seem normal in morphology, suggesting that phototransduction is compromised. we found that the els gene encodes cgmp phosphodiesterase ␣ -subunit (pde c), a component of cone-type pde. since genetic mutations of pde c have not been reported in human, the els mutant provides a good model for studying roles of cone-pde in visual functions. shinichi nakagawa , masatoshi takeichi , , fumi kubo , nakagawa initiative research unit, riken, wako, japan; riken cdb, kobe, japan; department of biostudies, kyoto university, kyoto, japan the marginal region of the optic vesicle contains retinal stem cells that remain undifferentiated and proliferate for a much longer period compared to other progenitor cells in the central retina. we have previously shown that wnt b, a signaling molecule expressed in a region neighboring the stem cell area, functions as a putative stem cell factor that endows undifferentiated retinal cells with the characteristics of the stem cells. interestingly, wnt b inhibits cellular differentiation in the absence of notch activity, a well-known signaling receptor that inhibits neuronal differentiation. wnt b antagonizes proneural gene functions independent of the notch signaling pathway, presumably through unidentified transcriptional repressors. we have isolated several candidate genes that are upregulated upon an activation of the wnt signaling pathway, and some of them are expressed in the stem cell containing region. physiological roles of those genes will be discussed. research funds: kakenhi ( ) sy - - - identification of cell lineage of retinal progenitor cells by cell surface markers sumiko watanabe, hideto koso, shinya satoh department of molecular and developmental biology, university of tokyo, tokyo, japan i would like to discuss about early cellular developmental stages of retina, which we identified by examination of the expression pattern of cell surface markers. we found c-kit and ssea- to be spatiotemporal markers of distinct populations of retinal progenitor cells, and these cells dramatically changed their expression profiles of c-kit and ssea- during development. c-kit-positive cells expressed various immature retina specific genes; and later onset of rhodopsin expression and stronger proliferation activities were observed. c-kit/ssea- double-positive cells showed stronger proliferation activities than ckit single-positive ones. although the number of ssea- -positive cells was augmented by beta-catenin signal, c-kit-positive cells were positively regulated by notch signaling, suggesting that c-kit and ssea- have intrinsically distinct characters. prolonged expression of c-kit by a retrovirus resulted in promotion of proliferation and the appearance of nestin-positive cells in response to scf, suggesting a role for c-kit in retinal development. the retinal photoreceptor cells play a primal and central role in the phototransduction system. they are susceptible to deterioration in human retinal diseases, which lead to severe visual impairment. we have been demonstrated that transcription factors, otx and crx play critical roles in retinal photoreceptor development. while otx is a key molecule for retinal photoreceptor cell fate determination, crx is essential for the terminal differentiation and maturation of photoreceptors. meanwhile, the photoreceptor cell is a highly polarized neuron and also has epithelial characteristics such as adherens junctions. our investigation of a role of apkc, which has been proposed to play a critical role in the establishment of epithelial and neuronal polarity, in differentiating photoreceptors has shown that apkc is required for the formation of outer & inner segments and ribbon synapse. in addition, we also found that photoreceptor polarity formation has important roles in proper retinal lamination. we would like to present our recent analysis of photoreceptor development. research funds: kakenhi ( , , ) raj ladher riken center for developmental biology, kobe, japan the inner ear translates mechanical energy into neural signals that the appropriate centers of the brain can decode into balance or sound information. the inner ear forms from bilateral thickened discs of ectoderm located on either side of the hindbrain, early during development. induction of the inner ear is mediated by localized signals emanating from the paraxial mesoderm. in the chick, the inner ear is induced by localized fgf found in the mesoderm. we find that although fgf can induce the inner ear, it is unable to support differentiation of the inner ear. differentiation, that is the development of the chick inner ear hair cells, is triggered by another family member fgf and is actually inhibited by fgf . for full functionality, the inner ear needs to be integrated into the larger auditory complex, made up of the middle ear, the external ear and the auditory centers in the hindbrain. these components develop from diverse origins but are intimately linked during development. we have been trying to understand how integration occurs and present one model by which this could occur. research funds: center support grant, mext leading projects grant sy - - - how is olfactory receptor-dependent axonal wiring conducted? shou serizawa, kazunari miyamichi, haruki takeuchi, yuya yamagishi, tokiko tsubokawa, hitoshi sakano department of biophysics and biochemistry, crest jst, university of tokyo, tokyo, japan in the olfactory system, termini of primary axon segregate depending on the type of olfactory receptor (or) expressed, forming the olfactory sensory map. to study how the or-dependent axonal wiring is conducted, we analyzed the gene expression profile in the olfactory epithelium of the transgenic mouse in which the majority of olfactory sensory neurons (osns) express a particular or gene. we found that the expression of the immunoglobulin superfamily gene kirrel , encoding homophillic adhesion molecule, is down-regulated in the transgenic mouse compared to the wild type control. the expression level of kirrel in each osn is found to be correlated with the type of or species expressed in the osn. moreover, kirrel promoted fasciculation of osn axon termini in the mosaic gain-of-function experiment. here, we propose that the information of which type of or is expressed in the osn is converted to the expression level of kirrel which determines the adhesiveness of axon termini, contributing to or-dependent segregation of osn axons. in spite of its morphological similarity to the other species in the melanogaster species subgroup, drosophila sechellia has evolved distinctive physiological and behavioral characters adapting to its host plant morinda citrifolia, known as the tahitian noni fruit. the ripe fruit of m. citriforia contains hexianoic acid and octanoic acid, the main components of the odor from the fruit. d. sechellia is attracted to these two fatty acids, while the other species are repelled by them. using inter-species hybrid between d. melanogaster deficiency mutants and d. sechellia, odorant binding protein e was identified as the gene responsible for this behavioral difference among the species. obp e forms a gene cluster with obp d, and these two genes are expressed in the same cells associated with the chemosensory organ. the history of dynamic obp d/e-cluster evolution was revealed by comparison of the genomic sequences of the obp d/e region obtained from species phylogenetically located between d. melanogaster and d. pseudoobscula. sy - - - an approach of dissociating complex traits into fine genetic elements using consomic strains of mouse aki takahashi , , akinori nishi , , toshihiko shiroishi , , tsuyoshi koide , sokendai, kanagawa, japan; national institute of genetics, shizuoka, japan much of the genetic variation that underlies most behavioral traits is complex and is regulated by loci that have quantitative effect on the phenotype. we have previously shown that laboratory strain c bl/ (b ) and wild-derived strain msm/ms have great differences in many behavioral traits. consomic strains were established by natural mating between b and msm, and those strains have the same genetic background as b except for one chromosome from msm. by examining bunch of consomic strains on many behavioral trait, such as spontaneous activity, anxiety-like behavior, pain sensitivity, and social behavior, we were able to map which chromosome have a locus or loci affecting those phenotype. one strain b - msm, which have msm chromosome , showed increased fear responses and riskassessment behavior, and thus it is thought that there is a locus/loci related to the emotionality. to identify the gene in the loci, we have made congenic strains, and successfully narrowed the locus down in the telomeric region. research funds: kakenhi ( . ) sy - - - cloning of the major quantitative trait locus underlying capsaicin resistance in mice capsaicin is the main compound of hot chili peppers, and induces sensations of heat and pain. however, sensitivity to capsaicin differs among individuals. a genetic approach using a mouse model reveals some quantitative trait loci for this sensitivity. capsaicin resistance linked on chromosome (capsq ) is the major locus affecting reduced taste sensation in kjr mice. here we show that intracellular recycling of capsaicin receptor (trpv ) was impaired in kjr neurons in contrast to that of c bl/ j mice. by searching the candidate genes, eh domain-containing four (ehd ), a trp-binding scaffold protein encoding gene was found. ehd binds to c-terminal of trpv . three mutations were found in ehd of kjr, which remarkably diminished the binding, leading to changes in the intracellular distribution of trpv . this study is the first genetic dissection associated with capsaicin/heat resistance in a nature strain and shows a novel binding protein to trpvs. sy - - - comprehensive behavioral analysis of genetically-engineered mice tsuyoshi miyakawa hmro, kyoto university, kyoto, japan one of the major challenges in the life sciences of the post-genome era is to elucidate the functions of the genes at the level of individual animals. final output level of the functions of the genes expressed in the brain is behavior, indicating a need for systematic investigations of the behavioral significance of the genes. in our laboratory, we use a "comprehensive behavioral test battery" for genetically-engineered mice to reveal causal relationships between genes and behaviors. the battery covers broad areas of behaviors, from simple reflexes to highly cognitive functions. so far, we have assessed more than different strains of mutant mice with the battery. surprisingly, more than % of the strains showed at least one significant behavioral phenotype, suggesting that a large part of the genes expressed in the brain may have some functions. representative results for a few strains of mutant mice and the meta-analytic results of the combined data will be presented. also, a potential impact of our approach to "large-scale neuroscience" will be discussed. research funds: kakenhi ( , , , ) , jst bird hiroshi takashima department of neurology and geriatrics, kagoshima university, kagoshima, japan inherited neuropathies are clinically and genetically heterogeneous. at least genes and loci have been associated with charcot-marie-tooth disease (cmt) and related inherited neuropathies. most causes of inherited neuropathy have been discovered by positional cloning technique and in the past two years, the pace of cmt gene discovery has accelerated. these recently discovered cmt causing genes/proteins include those which, although showing unpredictable correlations with the peripheral nervous system, are definitely important for the peripheral nerve. their discovery should pave the way for dramatic progress in the understanding of peripheral nerve biology. on the other hand, genotype-phenotype correlations of these genes are also important in order to understand the pathomechanisms of inherited neuropathy since, based on mutation studies, a large number of genes associated with both the demyelinating and axonal forms of cmt have been identified. to clarify the specific features and molecular mechanisms, we reviewed recent progress in cmt research, especially cmt f caused by prx, and scan caused by tdp . sy - - - gangliosides are important for the maintenance of the nodes of ranvier nobuhiro yuki, keiichiro susuki department of neurology, dokkyo university school of medicine, tochigi, japan gangliosides are abundant in vertebrate nervous system, but the function has yet to be elucidated. some patients with guillain-barre syndrome have autoantibodies to gangliosides such as gm , who show failure of peripheral motor nerve conduction. sensitization of rabbits with gm can produce the disease model. in ventral roots from the paralyzed rabbits, igg and complements deposited on the nodes of ranvier, and sodium channel clusters were disrupted. in ganglioside-deficient mice with disrupted gm /gd synthase gene, motor nerve conduction velocities were reduced in the sciatic nerves. some myelin loops failed to contact the paranodal axolemma, and potassium channels were aberrantly localized at the paranodes. the abnormality became prominent with age. these findings using different models showed that gangliosides are important for the maintenance of the node of ranvier and saltatory conduction along the myelinated nerve fibers. hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan neuropathic pain caused following nerve injury is one of important issues in neuroscience as well as clinics, since its pain pathway is apparently distinct from that in healthy humans and naive experimental animals. this is clearly evidenced by the finding that the tactile information is converted to noxious one in allodynia characterized in neuropathic pain. in our recent paper (nature medicine, ) , we firstly demonstrated that lysophosphatidic acid (lpa) and its receptor (lpa ) activation initiate the neuropathic pain. in this and following studies we proposed that the demyelination of nociceptive fibers reorganizes the nociceptive spinal inputs through sprouting and electrical synapses (ephapses). i will discuss four issues, lpa-induced demyelination of dorsal root fibers using in vivo and ex vivo culture models, the signal transduction of underlying lpa-mediated downregulation of myelin proteins, evidence for sprouting and ephapses following demyelination and the origin of injury-specific lpa production in terms of demyelination and allodynia. research funds: kakenhi ( ) toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan axons of adult central nervous system are capable of only a limited amount of regrowth after injury, and an unfavorable environment plays major roles in the lack of regeneration. some of the axon growth inhibitory effects are associated with myelin. three myelin-derived proteins have been identified to inhibit neurite outgrowth in vitro. these proteins induce activation of rho in some neruons. inhibition of rho or rho-kinase promotes axon regeneration in vivo. these findings establish rho and rho-kinase as key players in inhibiting regeneration of the central nervous system. i will review recent findings regarding the signaling mechanism of axon growth inhibitors. our experiments suggest that several new candidate proteins may be axon growth inhibitors. these proteins activate not only rho/rhokinase but also other signals to inhibit neurite outgrowth from some neurons in vitro. these findings suggest that agents that block the multiple signals elicited by these axon growth inhibitors may provide efficient tools that produce functional regeneration following injuries to the central nervous system. sha mi biogen idec, usa lingo- is a cns-specific protein expressed in both neurons and oligodendrocytes. in neurons, lingo- mediates the inhibition of axonal growth as a component of the ngr /p /lingo- and ngr /troy/lingo- signaling complex. inhibition of endogenous lingo- by soluble lingo- or dominant negative lingo- can reverse the inhibition of neurite outgrowth by myelin components. soluble lingo- treatment significantly improves functional recovery of spinal cord injured rats as determined by bbb scores. soluble lingo- treatment promotes axonal regeneration and reduced axon dieback in the corticospinal tract, rubrospinal tract, and optic nerve. in oligodendrocytes, lingo- mediates the inhibition of differentiation and myelination. loss of lingo- function using dominant negative lingo- , lingo- rnai, or soluble lingo- or lingo- knockout increased oligodendrocyte differentiation and myelination, whereas over-expression of lingo- led to inhibition of oligodendrocyte differentiation and myelination, in vitro and in vivo. the discovery of a significant role for lingo- in neurons and oligodendrocyte biology are an invaluable step for understanding cns axon regeneration and myelination. alex reyes new york university, usa neurons in the auditory cortex exhibit a wide range of firing patterns. to elucidate the cellular properties and circuitry that give rise to these responses, a d sheet of excitatory and inhibitory neurons were reconstructed in vitro using an iteratively-constructed network (icn) modified to contain both feedback and feedforward circuits. a disc of neurons was stimulated and the resultant firing pattern and spread was documented. simultaneous whole-cell recordings were performed from pyramidal and interneurons in a slice preparation of the mouse auditory cortex. a computer simulated the activities of thalamic neurons and calculated the net synaptic conductance that would be generated by their firing. this waveform was converted to current, injected into the recorded neurons via a dynamic clamp circuit, and the resultant firing documented. using the icn method, we reproduced the firing of a realistic network of excitatory and inhibitory neurons. we replicated many of the responses recorded in vivo. morever, the firing patterns of neurons depend substantially on their distance from the stimulus center and on the identity of the local interneurons. research funds: nih dc - a sy - - - neuronal avalanches reveal neuronal wirings of layer / cell assemblies jun-nosuke teramae, tomoki fukai brain science institute, riken, japan how cortical neurons process information crucially depends on how their local circuits are organized. spontaneous synchronous neuronal activity propagating through neocortical slices displays highly diverse, yet repeatable, activity patterns called 'neuronal avalanches'. they obey power-law distributions of the event sizes and lifetimes, presumably reflecting the structure of local cortical networks. however, the explicit network structure underlying the power-law statistics remains unclear. here, we present a neuronal network model of pyramidal and inhibitory neurons that enables stable propagation of avalanche-like spiking activity. we demonstrate a neuronal wiring rule that governs the formation of mutually overlapping cell assemblies during the development of this network. the resultant network comprises a mixture of feedforward chains and recurrent circuits, in which neuronal avalanches are stable if the former structure is predominant. we investigate how the resultant power laws depend on the details of the cell-assembly formation as well as on the inhibitory feedback. research funds: kakenhi ( ) sy - - - spike-timing dependent and homeostatic plasticity from an optimality viewpoint taro toyoizumi , jean-pascal pfister , kazuyuki aihara , , wulfram gerstner department of complexity science and engineering, university of tokyo, japan; school of computer and communication science & bmi, epfl, japan; aihara complexity modelling project, erato, jst, japan maximization of information transmission by a spiking neuron model predicts changes of synaptic connections that depend on timing of pre-and postsynaptic spikes as well as on the postsynaptic membrane potential. under the assumption of poisson firing statistics, the synaptic update rule exhibits all the features of the bienenstock-cooper-munro rule, in particular regimes of synaptic potentiation and depression separated by a sliding threshold. the learning rule is found by maximizing the mutual information between presynaptic and postsynaptic spike trains under the constraint that the postsynaptic firing rate stays close to some target firing rate. an interpretation of the synaptic update rule in terms of homeostatic synaptic processes and spike-timing dependent plasticity is discussed. research funds: grant-in-aid for jsps fellows j and sci. res. on priority areas from mext of japan, and swiss natl. sci. found. - / sy - - - timing computations in the auditory brain stem john rinzel center for neural science and courant institute of mathematical sciences, new york university, usa sound localization involves precise temporal processing by neurons in the auditory brain stem. the first neurons in the auditory pathway to receive input from both ears can distinguish interaural time differences (itds) in the sub-millisecond range. these cells in the mammalian medial superior olive have specialized biophysical features: two dendrites, each receiving input from only one side; very short membrane time constant; specialized ionic channel properties, including a low-voltage activated k+ current, i-klt. this i-klt contributes to phasic firing (one spike in response to a step of current), precise phase-locking, and extremely timing-sensitive coincidence detection. we will describe the temporal feature-selecting properties of mso cells based on biophysical (hh-like) modeling, in vitro electrophysiology and application of concepts from dynamical systems theory and coding theory. neuronal information is often inferred by counting spike numbers over tens to hundreds of milliseconds. however, if relative spike timings at the scale of milliseconds would carry information, neuronal circuits could have large information capacity. in response to various visual inputs, the retina fires spike bursts separated by hundreds of milliseconds of silent periods. onsets and spike numbers of these bursts are highly reproducible. we asked if spike patterns, i.e., combinations of interspike intervals within single bursts, carry information. using the retinas of salamanders and mice, we found that bursts have various spike patterns, which are unique to the preceding inputs. differences in spike patterns at the scale of milliseconds encode differences in the input as long as - ms. when single bursts contain three or more spikes, the multiple interspike intervals combinatorially encode multiple features of the input. this suggests the spike patters are not determined sorely by slowly modulating instantaneous firing rates. we propose that the retina encodes multiple features in hundreds of milliseconds of input into burst spike patterns at the scale of milliseconds. accumulating evidence reveals that the generalized seizure activity can produce regenerative, in addition to degenerative, structural changes in the hippocampus, including the enhancement of progenitor cell division of dentate granule cells. although the regulatory mechanisms underlying such neurogenesis are unknown, we hypothesized that newly generated granule cells may contribute to the reorganization of the hippocampal formation in the early course of seizures, constituting a possible substrate for epileptogenicity. to address this issue, we examined the division of dentate granule cell progenitors in rats after kainic acid administration, or perforant path kindling. the results indicate that initial limbic seizures trigger the enhancement of dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. the degenerative process is not necessary for triggering the upregulation. it is also suggested that newly generated granule cells may play a role in the network reorganization that occurs during epileptogenesis. the molecular basis underlying such neurogenesis will be discussed. keiichi itoi , ikue otaki , saya suzuki , yasunobu yasoshima , kazuto kobayashi laboratory of informational biology, graduate school of informational science, tohoku university, sendai, japan; institute of biomedical science, fukushima medical university, japan in order to examine functional roles of the noradrenergic (na) neurons in the locus coeruleus (lc) we developed a novel method to ablate specifically the na neurons in the lc, and examined the behavioral and stress responses using the animal model. a transgenic mouse line was used in which human interleukin- receptor ␣ subunit (hil- r␣) was expressed under the control of dopamine ␤-hydroxylase gene promoter. anti-hil- r␣ antibody fused to pseudomonas exotoxin was microinjected into bilateral lc of a transgenic mouse stereotaxically to destroy specifically the na neurons. as behavioral paradigms, elevated plus maze and open field test were used. plasma adrenocorticotropin levels were measured following lipopolysaccharide injection intraperitoneally, as an immune stress. thus, the effect of lc ablation how it affects the behavioral and stress responses will be elucidated. - - - integrated circuits controlling the stress response james p. herman department of psychiatry, university of cincinnati, oh, usa the hypothalamo-pituitary-adrenocortical (hpa) axis is a primary stress-response system in all vertebrates. the end-product of hpa activation, glucocorticoids, serve the general function of redirecting bodily resources to meet a real or perceived challenge. however, prolonged glucocorticoid secretion has deleterious effects on metabolism, immune function and behavior, making control of hpa activity a priority for the organism. this control is exerted in large part by limbic structures in the brain. our studies indicate that the amygdala, hippocampus and prefrontal cortex play major roles hpa axis regulation. the amygdala is primarily stress excitatory, whereas the hippocampus has an inhibitory influence on hpa activity. the role of the prefrontal cortex is considerably more complex; its prelimbic region is primarily stress inhibitory, whereas the infralimbic region may participate in stress activation. all of these regions exert their influence via subcortical relays to hypothalamic paraventricular nucleus (pvn) neurons controlling the hpa response, allowing convergence of information from multiple limbic sources prior to the pvn. sy - - - molecular mechanism for the inverse incidence of parkinson's disease and cancer: synuclein as stimulator of tumour differentiation makoto hashimoto department of chemistry and metabolism, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative disease and cancer are major age-associated disorders. however, the pathogenesis of these diseases may be in sharp contrast, since the former is featured by cell death, whereas, the latter is associated with immortalization. in parkinson's disease (pd) research, smoking, the risk factor for a variety of cancers, had been known to reduce the risk of pd. furthermore, epidemiological studies described that the incidence of cancer was reduced in pd patients. recent study provides evidences of the inverse relationship of pd and some cancers at the molecular level. for example, loss of neuroprotection of dj- is causative for familial pd, while increased expression of this molecule stimulates oncogenesis. in this context, we show that proteasomal inhibition by ␣-synuclein, which has been thought as one major pathogenic mechanism for pd, may induce differentiation of cancer cells. thus, unifying approach on the basis of the opposite pathogenic mechanism to neurodegenerative disease and cancer might uncover unexpected findings in both fields. kiyomitsu oyanagi department of neuropathology, tokyo metropolitan institute for neuroscience, tokyo, japan neurodegenerative diseases and malignant tumors develop symptoms usually at middle or old-age in humans. however, it is well known that critical periods of some malignancies are in fetal period, which are ( ) leukemia in patients exposed with atomic bomb during the iind world war, and ( ) brain tumors in rats with ethylnitrosourea administration. as to neurodegenerative diseases, ( ) many genetic/familial diseases show clinical symptoms at the middle or old age. ( ) epidemiological study revealed that emigrants from guam to the main land of usa show relatively high incidence of amyotrophic lateral sclerosis, and the critical period of exposure to some environmental noxiousness was considered to be childhood/adolescence. ( ) relating to parkinson disease, low magnesium intake over generations induced selective degeneration of the dopaminergic neurons in the substantia nigra in rats [oyanagi et al., in press] . these findings indicate that not only certain malignant tumors but also some sporadic neurodegenerative diseases may be induced originally by the insults in embryonic stage/childhood. to understand the role of synuclein, the major component of pathological inclusions, we examine the expression of synuclein in the embryonic mouse cerebral cortex. we found that a-synuclein and b-synuclein were predominantly detected in the subplate neurons, which are known to enter programmed cell death at a postnatal stage. in another line of inquiry, we are interested in a zinc finger protein containing poz domain, rp , which functions as a sequence specific transcriptional repressor and involved in cortical layer formation. when the rp gene is disrupted, apoptosis is enhanced, and a-synuclein, but not b-synclein, is upregulated in the mutant cortex, suggesting that a-synuclein is involved in the cell death. interestingly, in the mutant cortex the expression of s-phase marker, pcna increased, suggesting that rp mutant mice are useful to analyze the relation among neurodegeration, synuclein and cell cycle. minoru saitoe, junjiro horiuchi, daisuke yamazaki tokyo metropolitan institute for neuroscience, tokyo, japan age-related memory impairment (ami) is a striking feature of ageassociated neuronal dysfunction. to identify gene mutations that affect ami, we screened ∼ drosophila lines and found that heterozygous mutants for the pka catalytic subunit (dc /+) exhibit robust suppression of ami without affecting memory at young ages. this result suggests a causal relationship between pka and ami. of particular interest, igf/pi k/akt signaling, which results in decreased gsk activity, has also been shown to ameliorate ami. both pka and gsk phosphorylate the microtubule-associated protein tau, causing tau aggregation and neurodegeneration. while igf signaling suppresses activity of gsk at young ages, declining igf levels during aging may increase gsk activity in aged animals. in support of this idea, we found suppression of ami in flies fed gsk inhibitors. we hypothesize that similar to the mechanisms occurring in neurodegenerative diseases, tau phosphorylation by pka and gsk causes neuronal dysfunction during normal aging. research funds: kakenhi sy - - - molecular mechanism of cancer progression by gamma-synuclein koji okamoto radiobiology division, national cancer center research institute, tokyo, japan synucleins, a family of small proteins consisting of three known members, are implicated in both neurodegenerative disorder and tumorigenesis. ␣synuclein is involved in the formation of pathologically insoluble deposits characteristic of neurodegenerative diseases such as alzheimer disease and parkinson disease, whereas overexpression of ␥synuclein is associated with progression of breast and ovarian cancer. however, the normal cellular function of synucleins remains largely unknown. in order to get an insight into biological function of synucleins, we focus on cancer progression induced by ␥synuclein. we introduced ␥synuclein into breast cancer cells in order to recapitulate malignant transformation of breast cancer. using such cells, the attempt to elucidate the biochemical function of ␥synuclein is underway. the impact of synuclein over-expression, especially on known tumor suppressor pathways such as the p pathway, will be discussed. research funds: kakenhi ryuichi sakai growth factor division, national cancer center research institute, - - tsukiji, chuo-ku, tokyo - , japan numbers of growth factors and their membrane receptors which possess tyrosine kinase activity are involved in proliferation and differentiation of the neural system. shc family docking molecules conduct signals directly downstream of various growth factor receptors as substrates and binding partners of these tyrosine kinases. in the neural systems, two unique shc family molecules, shcb and shcc, are found to be specifically expressed and analysis of mice lacking these proteins revealed that they have redundant functions during mammalian neural development as mediators of ngf/trka signaling. it was recently found that tyrosine phosphorylation of shcc is frequently detected in majority of neuroblastoma cell lines. we showed that hyperphosphorylated shcc detected in some of neuroblastoma cell lines is associated with constitutively activated anaplastic lymphoma kinase (alk) caused by the gene amplification. identification of binding partners of shcc and expression of mutant shcc in several cancer cell lines revealed novel roles of shcc as a regulator of differentiation and proliferation of neuroblastic tumors. research funds: kakenhi sy - - - identification of estrogen receptor target genes and role of their gene products in cancer and nervous system satoshi inoue , department of geriatric medicine, university of tokyo hospital, tokyo, japan; research center for genomic medicine, saitama medical school, saitama, japan estrogen has crucial roles in the cancer growth and in the neural function. here, we have isolated and characterized novel estrogenresponsive genes to clarify the molecular mechanism of the estrogen action in target cells using genomic binding-site cloning (gbsc) method. one of the first identified genes is the estrogen-responsive ring finger protein (efp). efp expression was observed in uterus, mammary gland and certain regions of the brain where er is also expressed and positively regulated by estrogen. we revealed that efp targets proteolysis of - - sigma, a negative cell cycle regulator that causes g arrest and that efp is an essential oncogenic factor in breast cancer growth. on the other hand, another gene identified by gbsc is nr d, an nmda receptor. this gene was regulated by estrogen in the hypothalamus, together with er, pr and efp. these estrogen responsive genes could mediate roles of estrogen action in specific organs, utilizing differential mechanisms as well as sharing common mechanisms. keiji tanaka , hossein esteky , kiani roozbeh , tadashi sugihara , gang wang riken brain science institute, wako, saitama, japan; institute for studies in theoretical physics and mathematics, tehran, iran; kagoshima university, kagoshima, japan individual cells in the monkey inferotemporal cortex, which is the final unimodal stage along the ventral visual pathway, respond to moderately complex features, but not to objects nor to object categories. then, questions arise where and how view-general objects and object categories are represented. a possibility is the representation by a population of inferotemporal cells. to examine it, we recorded responses of inferotemporal cells to object images in a fixation task. we also conducted psychophysical experiments with monkeys to determine conditions for view-invariant object recognition. the results suggest that a population of inferotemporal cells represent object categories and their relational structure, and that the representation is common to nearby views of objects with up to • rotation. research funds: kakenhi alexander thiele , gene stoner , louise s. delicato , mark roberts university of newcastle upon tyne, uk; the salk institute, japan a variety of different roles of synchronized activity for sensation and perception have been proposed, ranging from object binding, through attentional enhancement, to mechanisms of learning. we have employed different paradigms to investigate the role of neural synchrony in visual perception and attentional selection in the awake macaque monkey. using two different tasks and stimulus conditions, well suited to probe the role of feature binding in the motion domain, we found no support for the idea that neuronal synchrony in macaque area mt underlies the binding of an object's component features. recent reports have focused on the role of synchrony in the mediation of attention. we will discuss the role of synchronized activity in primate v while attentional load was varied, and how it could be mediated by cholinergic mechanisms. research funds: hfsp, wellcome trust, bbsrc sy - - - context-dependent changes in noise correlation in mt william newsome, marlene r. cohen stanford university and howard hughes medical institute, usa changes in the correlated firing of a pair of neurons may provide a metric of changes in functional circuitry within the nervous system during ongoing behavior. we studied dynamic changes in functional circuitry by analyzing the noise correlations of simultaneously recorded mt neurons in two behavioral contexts: one that promotes cooperative interactions between the two neurons and another that promotes competitive interactions. we created cooperative or competitive contexts by changing the axis of motion of the discrimination task from trial to trial. we found that identical visual stimuli indeed give rise to differences in noise correlation in the two behavioral contexts. specifically, noise correlations were higher in the cooperative than in the competitive context. this result suggests that mt neurons receive inputs of central origin whose strength changes with the task structure. the changes in correlation appear to reflect differences in how mt neurons are pooled for the purpose of perceptual discrimination, and may derive from higher-level cognitive processes such as feature-based attention. research funds: howard hughes medical institute sy - - - effects of task demands on color processing in area te of the monkey hidehiko komatsu , , kowa koida national institute for physiological sciences, okazaki, japan; sokendai, okazaki, japan color vision has two different functions, namely, categorization and discrimination, and the same color stimulus can be processed according to these two functions depending on task demands. lesion studies suggested that inferior temporal (it) cortex of the monkey plays a key role in color vision, and we have recently found that color selective neurons are concentrated in a small region in area te of it cortex. to study how the color selective responses in this region are affected by the task demands, we trained monkeys a color categorization task and a color discrimination task using the same set of color stimuli, and analyzed how the responses are affected. we found response magnitudes of many neurons changed between two tasks while the color tuning is well reserved. in several extreme cases, large gain change almost completely eliminated the responses in one task. these results suggest that color signals are gated by the top-down signal representing task demands in area te and color channels specific to different tasks are formed at this level of the visual cortex. yoichi sugita neuroscience research institute, aist, tsukuba, japan early visual experience is indispensable to shape the maturation of cortical circuits during development . monocular deprivation in infancy, for instance, leads to an irreversible reduction of visually driven activity in the visual cortex through the deprived eye and a loss of binocular depth perception - . here, i show exposure only to monochromatic illumination in infancy results in the disruption of color perception. infant monkeys were reared for nearly a year in a room where the illumination came from only monochromatic lights. after extensive training, they were able to perform color matching. but, their judgment of color similarity was quite different from that of normal animals. furthermore, they had deficits in color constancy; they could not compensate for the changes in wavelength composition. these results indicate that early visual experience is also indispensable for color perception. research funds: crest sy - - - dendritic growth, spinogenesis and synaptogenesis in response to neurosteroids in the developing purkinje cell kazuyoshi tsutsui , hirotaka sakamoto , katsunori sasahara , hanako shikimi , kazuyoshi ukena , mitsuhiro kawata laboratory of brain science, faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima, japan; department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan new findings over the past decade have established that the brain synthesizes steroids de novo from cholesterol. such steroids synthesized de novo in the brain are called neurosteroids. recently we have identified the purkinje cell as a major site for neurosteroid formation in the brain. this is the first demonstration of de novo neuronal neurosteroidogenesis in the brain. in mammals, the purkinje cell actively synthesizes progesterone and estradiol de novo from cholesterol during neonatal life, when cerebellar cortical formation occurs. subsequently, our recent studies on mammals using the purkinje cell have demonstrated organizing actions of neurosteroids. both progesterone and estradiol promote dendritic growth, spinogenesis and synaptogenesis via each cognate nuclear receptor in purkinje cells. research funds: kakenhi ( and to kt) sy - - - roles of estrogen receptors in the regulation of socio-sexual and emotional behaviors-studies with knockout mice and rnai sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan the gonadal steroid estrogen plays a major role in the regulation not only of female reproductive behavior but also an array of social and emotional behaviors in both sexes, by acting through intracellular estrogen receptors (ers), ligand dependent transcription factors. a series of studies using single and double knockout mice for er-␣ and/or er-␤ genes have revealed that activation of er-␣ and er-␤ differentially regulate a number of behaviors as well as neuroendocrine functions. our studies have suggested a unique role of activation of er-␤ in the hypothalamic and limbic brain areas, dorsal raphe nuclei and locus coeruleus in the regulation of socio-sexual and emotional behaviors. in this talk, our findings from behavioral studies using er-␣ and er-␤ knockout mice along with possible brain mechanisms underlying the behavioral effects will be first overviewed. our most recent studies on brain site-specific manipulation of er gene expression with the use of small interference rna combined with adeno-associated virus will then be presented. research funds: kakenhi ( , ) sy - - - sex steroid receptor function in sexual behavior shigeaki kato , , takashi sato , takahiro matsumoto , imcb, university of tokyo, tokyo, japan; erato, jst, saitama, japan androgen actions are believed to mediate nuclear androgen receptor (ar)-mediated gene regulations. ar is a member of nuclear receptor, and acts as a hormone-induced transcription factor to control of target genes through chromatin remodeling/histone modification. we generated the floxed ar mice to avoid testicular feminization mutant (tfm) abnormalities with infertility, and then crossed with female ar(−/+) heterozygoutes expressing cre to generate ar(−/−) female mice. the ar(−/y) ko males grew healthy with typical features of tfm abnormalities, and genital organs were atrophic with a marked decrease in the serum testosterone level, but with normal estrogen level (kawano et al., ) . no sexual behaviors and reduced aggressive behaviors were seen in ar(−/y) male mice (sato et al., ) . female ar ko mice were normal in sexual behavior but exhibited premature ovarian phenotype (shiina et al., ) . together with these results, the ar function will be discussed in terms of ar function as a transcription factor. references kawano, h., et al., . pnas usa , . sato, t., et al., . pnas, usa , . shiina, h., et al., research funds: probrain sy - - - annexin : a mediator of cell-cell communication in the neuroendocrine system julia buckingham , helen christian , john morris imperial college london, uk; department of human anatomy and genetics, university of oxford, uk annexin (anxa ) plays an important part in mediating the regulatory effects of glucocorticoids (gcs) on neuroendocrine function, particularly within the hpa axis. it is expressed by folliculostellate (fs) cells in the pituitary gland and by ependymal cells and activated glia in the hypothalamus but not by classical secretory cells. gcs act on cells expressing anxa to cause the translocation of the protein to the plasma membrane at points with particular accumulation at points where the cells make contact with endocrine cells. this process is effected via a non-genomic mechanism and is dependent upon phosphorylation, lipidation and a transport protein, possibly abca . the released protein then acts, via cell surface receptors on the endocrine cells to suppress stimulus-evoked peptide release. the nature of the anxa receptor is unclear but, increasing data suggest that members of the formal peptide receptor family may be important in this regard. katsuhiko nishimori , yuki takayanagi , masahide yoshida , yoshiyuki kasahara , masaki kawamata graduate school of agricultural science, tohoku university, sendai, japan; department of physiology, jichi medical university, minamikawachi-machi, japan we examined the behaviors of mice lacking oxtr gene and discovered that oxtr null females displayed impaired nurturing behavior, and their pups showed defect in ultrasonic vocalization, instead, increased locomotor activity by social isolation test. those are implying impaired mother-infant relationship. oxtr null males also showed more aggressive and having social amnesia as well as the phenotype of oxt null mice. in addition, oxtr null mice failed to maintain their body temperature after acute cold exposure. their rectal temperature rapidly dropped in comparison of that of wildtype animals at • c ambient temperature. our studies demonstrate that oxtr plays a critical role in regulating several aspects of social behavior and the other physiological function, and may have important implications for developmental psychiatric disorders, such as autism. research funds: grant-in-aid for scientific research (b) ( ) sy - - - cortical mechanisms mediating visuomotor control of primate grasp roger n. lemon ucl institute of neurology, uk primates demonstrate an exquisite ability to precisely shape their hand when grasping an object. a network of parietal and frontal motor areas is thought to play a key role in this behaviour. our work shows that: hand shape can be unambiguously determined from emg activity of hand and digit muscles. information about grasp is represented by neuronal populations in the ventral premotor cortex (area f ); f activity shows graspspecific discharge soon after an object becomes visible, well in advance of activity in primary motor cortex (m ). local field potential activity in f and m is also tuned to grasp, and there is strong beta coherence between f and m , indicating reciprocal transmission of information. this is also seen in synaptic responses of m neurones to stimulation of f (and vice-versa). single pulse stimulation in f strongly modulates corticospinal outputs from m through corticocortical pathways between these two areas. paired-pulse tms can probe the excitability of these pathways in humans. facilitation of meps is both object and muscle specific and indicates that activity in these pathways is selectively enhanced during object grasp. research funds: wellcome trust, bbsrc sy - - - where tactile signals are ordered in time shigeru kitazawa , department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; crest, japan science and technology agency, saitama, japan how does the brain order successive events? it is generally accepted that the brain can resolve the order of two stimuli that are separated in time by ms. this applies to temporal order judgment of two tactile stimuli, delivered one to each hand, as long as the arms are uncrossed. however, crossing the arms caused misreporting (that is, inverting) of the temporal order. the reversal was not due to simple confusion of hands, because correct judgment was recovered at longer intervals (e.g., . s). when the stimuli were delivered to the tips of sticks held in each hand, the judgment was altered by crossing the sticks without changing the spatial locations of the hands. we recently found that temporal order judgments of tactile stimuli are strongly affected by visual distractors and/or eye movements. the results suggest that tactile stimuli are ordered in time only after they are referred to relevant locations in space, where multiple modalities of sensory signals converge. results from functional imaging support this idea. sy - - - decision making and underlying neural mechanisms-auditory-visual ambiguity solving and preference shinsuke shimojo , biology/cns, california institute of technology, pasadena, ca, usa; jst.erato shimojo implicit brain function project, atsugi, japan we explore mechanisms underlying crossmodal ambiguity solving (passive decision), and preference (active decision). we've employed the illusory flash effect, where a single flash appears to be doubled when accompanied by two sounds. -channel meg was employed, while the observer reported number of flashes. partial directed coherence was applied to see if there was a causal influence by the auditory on the visual cortices. the results indicate a strong causal influence in a-v direction in alpha ( ) ( ) ( ) ( ) ( ) and ranges only in the illusion-reported trials, while stimulus parameters were identical. no such difference was found in v-a direction. for preference, the observer's gaze shifted towards the to-be-chosen stimulus (face) before conscious decision. our fmri study shows activity specific to preference task in the ventral amygdala and the ventromedial prefrontal. while such results enable the same causality analysis, it also raises a question as to what determines active/passive nature of decision. research funds: jst.erato, hfsp sy - - - why look there? insights from spatial neglect and the medial frontal cortex masud husain ucl institute of neurology, uk why do we look where we do? studies in humans show that when we look at a scene, our initial fixation patterns can be predicted to a high degree of accuracy. our eyes go to the most salient locations where local feature contrast is greatest. these findings have led to the concept of a salience map which directs attention and gaze bottomup. in humans, damage to the right posterior parietal cortex often leads to dramatic neglect of the left side of space. recent research has begun to unravel the components of this syndrome, demonstrating several underlying mechanisms. these include a disturbance of the salience representation, a failure to keep track of spatial locations across saccades and difficulty in sustaining attention over time. gaze is directed not only bottom-up by but also top-down by voluntary mechanisms. our recent investigations of human medial frontal regions reveal important roles for the supplementary eye field and the pre-supplementary motor areas in the control of competing eye movement plans and deciding where to look. parietal and medial frontal gaze regions appear to play different, complementary roles in controlling why we look where we do. research funds: wellcome trust ( ) sy - - - recognizing self actions through externalized eyes atsushi iriki , symbolic cognitive development, riken brain science institute, saitama, japan; cognitive neurobiology, tokyo medical and dental university, tokyo, japan we can recognize ourselves and our own actions through the mirror or video images. thus, human can use such apparatus as externalized eyes (sensory tools), while non-human animals can normally use tools as extension of their effectors (motor tools) at most. human fmri studies revealed that the right temporo-parietal junction region and the mesial superior frontal gyrus are involved in perceiving and manipulating the representation of the self actions under different third person perspectives. japanese monkeys could be trained to use a hand-held video camera as a manipulable extension of their eyes only when their own vision was gradually transferred to the distant cues via motor-tools to extend their body images. the emergence of novel cortico-cortical projections between temporo-parietal junction and the intra-parietal cortex was described in monkeys that were trained to use motor tools, therefore, integrate the tool in their own body image. thus, presence of a self-objectification mechanism is suggested for acquisition of sensory tools as externalized eyes to recognize self actions. yoshiyuki kubota division of cerebral circuitry, nips, okazaki, japan gabaergic nonpyramidal cells in the neocortex are composed of several different subtypes. we found that most of gabaergic cell types, including fs basket and somatostatin martinotti cells, that innervate dendritic spines in addition to the somata and dendritic shafts. most postsynaptic spines also received an asymmetrical input, called double innervated (di) spines. to better characterize the other asymmetrical input on the di spines, excitatory presynaptic terminals were stained by immunohistochemistry for two types of vesicular glutamate transporters (vgluts): vglut , existing mostly in cortical pyramidal cells, and vglut , found in thalamocortical fibers. gabaergic inputs were rarely observed in spines innervated by vglut -expressing terminals (n = ), but were found in- % of spines innervated also by vglut -expressing terminals (n = ). symmetrical synapses on di spines were positive for gaba, as shown by postembedding immunohistochemistry. these results indicated that some thalamocortical inputs are likely selectively inhibited at the spine level by gabaergic synapses from cortical nonpyramidal cells. research funds: kakenhi sy - - - gabaergic recruitment of excitation by cortical axo-axonic cells gabor tamas, csaba varga, gabor molnar, szabolcs olah, pal barzo, janos szabadics university of szeged, hungary the axon has the lowest threshold for action potential generation and axons in the cerebral cortex receive input only at the axon initial segment exclusively from axo-axonic cells (aacs), which use the dominant inhibitory neurotransmitter, gamma-aminobutyric acid (gaba). thus, aacs are considered as strategically placed inhibitory neurons controlling cortical information flow. we applied multiple patch clamp recordings in slices of rat and human neocortex and found that single spikes in aacs can trigger action potentials in pyramidal cells and initiate stereotyped series of multiple synaptic events in the cortical network. the excitatory action of aacs is based on a depolarized reversal potential for axonal relative to perisomatic gabaergic inputs as determined in paired perforated patch recordings. powerful axo-axonic depolarization from the resting membrane potential is supported by a ∼ -fold decrease in the potassium-chloride co-transporter (kcc ) expression from somatic to axon initial segment membranes detected by quantitative immunogold labeling. in my talk i will describe the integrative and plasticity properties of thin basal dendrites of cortical pyramidal neurons. these dendrites receive the majority of the cells' synaptic inputs, so determining their integrative and plasticity properties is of prime importance. previous studies have most often reported global linear or sublinear summation in these dendrites. using confocal imaging and dual-site focal synaptic stimulation of identified thin dendrites in rat neocortical pyramidal neurons we show that thin dendrites provide a layer of independent computational "subunits" that sigmoidally modulate their inputs prior to global summation. next i will describe the plasticity rules used by these fine basal dendrites putting a special emphasis on the role of nmda-spike in local synaptic plasticity processes. yumiko yoshimura department of visual neuroscience, research institute environmental medicine, nagoya university, nagoya, japan neocortical circuits contain fine-scale networks of excitatory neurons interconnected precisely. we previously showed that layer / pyramidal cells in visual cortex share common excitatory inputs from layer and from within layer / , when they are directly connected. here, we tested whether inhibitory cells are incorporated into the fine-scale specificity of excitatory connections. we recorded photostimulation-evoked synaptic currents from pairs of layer / cells, consisting of one inhibitory cell and one pyramidal cell in rat visual cortex slices, and measured the extent of common inputs to the pairs based on cross-correlation analysis. fast spiking inhibitory cells shared extensive common excitatory inputs with neighboring pyramids only when the pairs of cells were reciprocally connected. adapting inhibitory cells shared little or no common input with neighboring pyramids, regardless of their direct connectivity. therefore, fine-scale specificity depends on the type of inhibitory cell and on the direct connectivity between neighboring pyramidal-inhibitory cell pairs. research funds: kakenhi ( , ) sy - - - local circuitry of cortical inhibitory neurons edward callaway, takuma mori, xiangmin xu the salk institute, usa we used laser scanning photostimulation to map local input to inhibitory neurons in layer of rat visual cortex and layer / of mouse barrel cortex. mouse studies used transgenic animals with gfp expressed in subsets of inhibitory neurons. in layer , axondescending cells receive excitatory input predominantly from layer / while neurogliaform cells receive stronger input from deeper layers. layer / neurons also receive inputs that vary systematically by cell type. two subtypes of martinotti cells, distinguished by calretinin (cr) expression, also differ in morphology and intrinsic physiology. cr+ martinotti cells receive excitatory input predominantly from layer / , while the cr− martinotti cells also receive strong excitation from layer . irregular-spiking basket cells also receive strong excitatory input from layers / and , but they often have a gap at the top of layer , with little or no input. fast-spiking basket cells and pyramidal cells in mouse barrel cortex receive input indistinguishable from cells in rat visual cortex, with strong input from layers / and , and only weak input from deeper layers. research funds: nih sy - - - physiological genomics of cortical microcircuits sacha b. nelson brandeis university, czech republic cortical microcircuits are comprised of highly diverse neuronal cell types that differ in their morphology, synaptic connectivity and intrinsic electrophysiology. presumably, these phenotypic differences are orchestrated and maintained by unique transcriptional programs. in order to begin to reveal those programs we have recently developed methods for measuring genome-wide gene expression from small numbers ( - ) of fluorescently labeled, hand-sorted neurons. subsets of pyramidal neurons and gabaergic interneurons were labeled genetically with gfp or anatomically with fluorescent microspheres. labeled neurons were characterized electrophysiologically and sorted for expression analysis. the resulting expression profiles revealed highly diverse expression of molecules involved in cell-cell signaling and cell-cell adhesion, as well as transcription factors. based on this diversity of expression we constructed a taxonomic tree using an unsupervised clustering algorithm, that correctly reflects known relationships between cortical cell types. research funds: r ey , mcknight neuroscience of brain disorders award sy - - - axon guidance mediated by localized ca + signals in the growth cone hiroyuki kamiguchi laboratory for neuronal growth mechanisms, riken brain science institute, wako, japan axonal growth cones migrate along the correct paths, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (cams). many guidance cues attract or repel the growth cone via asymmetric ca + signals. its turning direction depends on the occurrence of ca + -induced ca + release (cicr) through the ryanodine receptor type (ryr ). the activity of ryr is controlled by cams via camp and pka. in this way, axon-guiding and cam-derived signals are integrated by ryr , that serves as a key regulator of axon guidance. attractive ca + signals facilitate intracellular membrane transport to the leading front and subsequent vamp -mediated exocytosis on the side with elevated ca + . in contrast, repulsive ca + signals do not trigger such membrane dynamics. growth cone attraction, but not repulsion, is prevented by inhibition of vamp -mediated exocytosis. the results indicate that growth cone attraction is driven by asymmetric membrane dynamics and that growth cone repulsion is driven by different mechanisms, not simply by changing the left/right polarity of the same molecular machinery. sy - - - molecular mechanisms for signaling through plexin-a hitoshi kikutani, atsushi kumanogoh, toshihiko toyofuku research institute for microbial diseases, osaka university, suita, japan sema a acts as a guidance cue for axons through a receptor complex comprising neuropilin- as the ligand-binding subunit and plexin-a as the signal-transducing subunit. the ferm domain-containing gef, farp , associates directly with plexin-a . sema a induces the dissociation of farp from plexin-a , resulting in activation of farp 's rac gef activity, rnd recruitment to plexin-a and down regulation of r-ras. simultaneously, the ferm domain of farp sequesters pipki␥ from talin, thereby inhibiting its kinase activity. these activities are necessary for sema a-mediated repulsion of outgrowing axons. plexin-a also functions as a ligand binding receptor of a transmembrane semaphorin, sema d and contributes to cardiac morphogenesis. sema d exerts a migration-inhibitory activity on cells from the ventricle and a migration-promoting activity on those from the conotruncal segment. plexin-a forms a receptor complex with off-track in the ventricle or with vegf receptor type in the conotruncal segment, which are responsible for the effects of sema d on the respective regions. research funds: crest sy - - - axonal transport elicited by axon guidance molecules yoshio goshima department of molecular pharmacology & neurobiology, yokohama city university graduate school of medicine, yokohama, japan for the wiring of individual neurons together into an orderly network, control by axon guidance molecules of navigation to their targets is a critical event, and molecular components destined for specific subcellular domains of neuron must be targeted correctly. we previously reported that semaphorins a (sema a), a repulsive axon guidance cue, increases the rate of bi-directional axonal transport in dorsal root ganglia (drg) . to address if the individual molecules rides on the sema a-facilitated cargo, we used time-lapse imaging of several egfp-fusion proteins expressed in drg. sema a stimulated the transport of neuropilin- ::egfp, plexin-a ::egfp, and fyn::egfp, which are the components of sema a signaling, but not neuropilin- ::egfp. interestingly, sema a accelerated the anterograde transport of trka::egfp. these facts suggest that sema a selectively facilitates the transport of its own signaling components and that sema a may modulate ngf-sensitivity of neurites by accelerating the transport of trka. kozo kaibuchi department of cell pharmacology, nagoya university graduate school of medicine, japan neurons are one of the most highly polarized cells, comprised of two structurally and functionally distinct parts, axon and dendrites. however, it remains largely unknown how neuronal polarity is established. we previously showed that collapsin response mediator protein- (crmp- ) is enriched in growing axon, and play a crucial role in axon specification. crmp- interacts with tubulin dimers to promote microtubule-assembly, and binds to sra- , an effector of rac to regulate wave-dependent reorganization of actin filaments. crmp- links kinesin- to tubulin dimmers and sra- , and participates in the kinesin- -dependent transport of tubulin dimmers and the sra- /wave complex to growing axons. we also found that the par- /par- complex and the ras/pi -kinase/akt/gsk- b pathway are involved in neuronal polarization. akt appears to phosphorylate gsk- b and inactivates its kinase activity downstream of ras/pi -kinase, thereby increasing non-phosphorylated active crmp- which promotes axon growth. this time, i summarize and discuss functions of these polarity molecules in regulation of neuronal polarity. research funds: grant-in-aid for creative scientific research sy - - - regulation of actin dynamics during neurite initiation and axon guidance frank gertler, adam kwiatkowski, doug rubinson, erik dent, leslie mebane mit, usa nervous system development requires extensive cell migration and elaboration of neurites that become axons and dendrites. axons are guided to their targets by motile growth cones. both whole cell and growth cone migration involve dynamic remodeling of the actin and microtubule cytoskeleton in response to environmental cues. the ena/vasp protein family regulates cell migration and axon guidance. ena/vasp proteins modulate actin remodeling and promote the formation of long, sparsely branched actin networks, such as those found in filopodia. results of recent work on phenotypes arising in mice lacking all three ena/vasp proteins (mena, vasp, evl) will be presented. such animals exhibit a "cobblestone cortex" in which groups of neurons migrate beyond the pial membrane. the mutants also contain little if any cortical axonal fiber tracts. analysis of primary cells from the mutants indicates ena/vasp function is required for neurite initiation. mutant neurons express differentiation markers but form few, if any filopodia and exhibit alterations in actin-microtubule interactions. kimitaka anami department of psychiatry, national center hospital for mental, nervous and muscular disorders, ncnp, tokyo, japan recent years, studies using eeg and fmri in simultaneous manner has become flourished. this is because the feasibility that any eeg events is, in principle, able to be mapped on the mri tomographic view has attracted many researchers. applications of this methodology are to basic eeg researches including event-related potentials and background activities, and as clinical aspect, localization of epileptic foci. and applications of this methodology is not matured yet but still developing. in this presentation, we will introduce our study using this method to epilepsy and to other eeg events. masaya misaki , , takashi abe , , , shigeyuki kan , , satoru miyauchi , national institute of information and communications technology, kobe, japan; japan society for the promotion of science, tokyo, japan; graduate school of biosphere sciences, hiroshima university, higashi-hiroshim, japan; kyushu institute of technology, kitakyushu, japan; crest, japan science and technology agency, tokyo, japan recording fmri and an eeg simultaneously is effective for studying spontaneous brain activities. we used this method to examine the relationship between an eeg rhythm and a bold signal. some studies have hypothesized that the hemodynamic response for a change in power of certain eeg frequency bands, such as alpha waves, is canonical in shape. however, the appropriate response shape for a change in the rhythmic eeg has not yet been determined. we measured the eeg and fmri simultaneously while subjects were in a resting or sleeping state. we applied nonlinear regression analysis using an artificial neural network to detect correlations between the changes in rhythmic eeg waves and fmri signals without a priori hypothesis of the response shape. research funds: crest of jst and grant-in-aid for jsps fellows norihiro sadato, hiroshi toyoda department of cerebral research, national institute for physiological sciences, okazaki, japan previous studies have demonstrated a nonlinear relationship between blood oxygenation level dependent (bold) response and stimulus parameters. however the origin of this nonlinearity still remains unclear. to investigate the origin for the nonlinearity of bold response, we underwent simultaneous measurement of fmri and near infrared spectroscopy (nirs) . temporal dynamics of the responses in oxy-, deoxy-and total hemoglobin concentrations as well as bold signal were simultaneously measured during a visual stimulation with various durations. to quantify the degree of the nonlinearity of responses, we introduced a model using an impulse response function modified with additional nonlinearity scaling. this model was applied to the nirs measures as well as bold responses. the nonlinearity of the response in oxygen extraction fraction (oef) was also estimated from nirs measures. the non-linearity of bold was almost identical to oef across the wide range of nonlinearity of the neuronal responses. and hence we conclude that the non-linearity of bold responses to the neural activity is mainly due to the nonlinear response of oef. the bold-fmri signal is ambiguous regarding the underlying neurophysiology. in our work we attempt a) to better understand the neurophysiological basis of fmri and b) to improve on the information obtained by functional brain imaging by adding additional information, e.g. obtained by electrophysiological measurements. in one series of experiments, we combined transcranial magnetic stimulation with near-infrared imaging in order to clarify how changes in deoxy-hb concentration (the inverse of bold) is related to neuronal inhibition. in another series of experiments, we combine eeg with fmri in order to identify bold correlates of neuronal background rhythms such as alpha rhythm, mu rhythm, etc. in a third series of experiments, we combine fmri with the measurement of high-frequency oscillations in eeg. the latter is an expression of evoked spike burst in the somatosensory cortex, i.e. this kind of measurements adds the information about action potentials to fmri haruhiko akiyama tokyo institute of psychiatry, japan activation of microglia is a part of host defense mechanisms in the brain. microglia remove invading microorganisms as well as cell debris that contains hazardous substances such as lysosomal proteases. brain is particularly vulnerable to the immune and inflammatory attacks and, therefore, has multiple mechanisms that regulate the immune and inflammatory responses more strictly than other organs. nevertheless, many neurodegenerative lesions are associated with activated microglia and low-grade, but sustained, inflammation. neuroinflammation is a term that refers to such conditions. in alzheimer's disease (ad), microglia play a central role for phagocytic removal of amyloid beta-protein (abeta) from the brain. the process is enhanced by complement activation. however, these cellular and humoral responses to abeta may be toxic to neurons in ad. neuroinflammation could be a double-edged sword in the brain. in patients with neurodegenerative diseases, complication of systemic inflammatory diseases, depletion of some neurotransmitters such as catecholamines, and the presence of brain lesions may adjunctly upregulate neuroinflammation, which further accelerates neuronal degeneration. makoto sawada department of brain life science, riem, nagoya university, japan microglia, macrophage-like cells in the cns, are multi-functional cells; they play an important role in removal of dead cells or their remnants by phagocytosis in the cns degeneration as well as are one of important cells in the cns cytokine network. they are thought to be originated from mesoderm, and to be similar cells to other tissue-resident macrophages. activated microglia have been shown to remove potentially deleterious debris and promote tissue repair by secreting neurotrophic factors at the neuronal injury sites, however, they can release potentially cytotoxic substances in vitro, and at least so-called fully activated form of microglia which are observed at the injury site in aids dementia is completely neurotoxic. these suggest that some factor(s) may contribute to change microglial phenotype from protective to toxic, but the detail is not clear. recently we generated hiv-derived nef protein tranduced microglia. they are found to increase both the potential to produce o -and mpo-like peroxidase activity resulting in the neurotoxicity. therefore, the target protein(s) of nef might to be involved in the control of microglial neurotoxicity. there is abundant evidence that extracellular atp have an important role in pain signaling. the focus of attention now is on the possibility that atp receptor of microglia might be involved in neuropathic pain. neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. this type of pain state is generally resistant to currently available treatments. we recently reported that the expression of p x receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly p x receptors produce a reduction of the neuropathic pain behaviour ( . nature , - ) . more recently, we have reported that brain-derived neurotrophic factor (bdnf) released from microglia by the stimulation of p x causes the depolarizing shift in reversal potential of anion in li neurons of rats with nerve injury ( ( . nature , ( - . understanding the key roles of these atp receptors may lead to new strategies for the management of neuropathic pain. research funds: kakenhi ( ) sy - - - pet imaging of microglia using peripheral benzodiazepine ligands richard b. banati university of sydney, australia brain disease often results in significant changes in the functional state of microglia, the brain's resident tissue macrophages. the response is thought to be an important step in the pathophysiology of traumatic, inflammatory, neoplastic and degenerative brain disease. part of the structural and functional plasticity of microglia is the de novo expression of the kda transposor protein or "peripheral benzodiazepine binding site" (pbbs). the pbbs is bound by the isoquinoline pk , which labeled with carbon- can be used for positron emission tomography (pet). using c-(r)-pk pet in inflammatory and neurodegenerative brain disease as well as receptor autoradiography, we have shown that distributed regional pbbs up-regulation correlates with clinical deficit and mirrors the histologically described activation of microglia in the penumbra of focal lesions, but importantly also in the distant, anterograde and retrograde projection areas of the lesioned neural pathway. sy - - - application of simulation of light propagation in tissue to nirs imaging of brain function eiji okada department of electronics and electrical engineering, keio university, japan in nirs imaging, the functional image is obtained from the variation in intensity of detected light caused by concentration change in haemoglobin in cortical tissue. the serious problem of nirs imaging is ambiguity in light propagation in the head caused by the scattering of tissue. this poses results in poor spatial resolution and contrast in the image. the development of simulation model to calculate light propagation in the head to deduce the path length in the brain and the spatial sensitivity profile is very important to improve the nirs imaging. in this study, simulation of light propagation in the head model for nirs imaging is briefly reviewed. the heterogeneity of the tissues in the head, especially low-scattering cerebrospinal fluid (csf), has a strong effect on the light propagation in the brain. the sensitivity to concentration change in haemoglobin in the cortical tissue is improved by the effect of the csf. the simulation of nirs imaging indicates that the intensity and size of activated region in the functional image depend on the relative position of activated region to fibre pairs. yoko hoshi integrated neuroscience research team, tokyo institute of psychiatry, tokyo, japan quantification of near-infrared spectroscopy (nirs) data has been a central issue in the nirs field. over the past years, many approaches to quantification have been tried, and in the case that hb concentration changes are global within the tissue, the quantitative accuracy of time-resolved spectroscopy (trs) and phase-resolved spectroscopy (prs) has been established. when the changes are localized, however, as with functional brain activation, the difficulty of quantification has not yet been fully overcome because elimination of the effects of hemodynamic changes in the extracerebral tissue is also challenging. the temporal profile of detected light intensity measured by trs carries information about depth-dependent attenuation, because light that penetrated into deeper positions in the head is detected later. thus, several time-domain methods to determine absorption changes with depth resolution have been proposed. diffuse optical tomography (dot) is also a potential technique for quantitative detection of focal changes in cerebral hemodynamics. in this symposium, i will outline these approaches. sy - - - brain functional imaging in cerebral ischemic disorders: comparison of nirs and fmri kaoru sakatani department of neurological surgery, nihon university school of medicine, tokyo, japan we compared the evoked cerebral blood oxygenation (cbo) responses measured by nirs and activation maps of bold-fmri in stroke patients with mild and severe (misery perfusion) cerebral ischemia (ci). in the age-matched controls, deoxyhemoglobin concentrations decreased with increases in oxyhemoglobin and total hemoglobin in the primary sensorimotor cortex (psmc) during contralateral motor tasks. the psmc on the non-lesion side exhibits normal cbo response pattern. on the lesion side, the mild ci did not affect the cbo response pattern, but the severe ci caused an increase of deoxyhemoglobin during the task associated with increases of oxyhemoglobin and total hemoglobin. the mild ci caused only slight reduction of the activation volumes of bold imaging on the lesion side; however, the severe ci, caused markedly reduction of the activation volumes on the lesion side. misery perfusion caused marked reductions of activation volumes of bold imaging associated with an increase of deoxyhemoglobin during activation. bold-fmri should be performed, giving consideration to the baseline circulatory conditions. masato fukuda, toru uehara, masahiko mikuni department of psychiatry and human behavior, gunma university graduate school of medicine, gunma, japan near-infrared spectroscopy (nirs) has been increasingly employed in psychiatry researches such as personality ( . neuropsychobiology , ), aging ( . neuroimage , , and psychiatric disorders ("progress in schizophrenia research", nova science publishers, ) . frontal lobe reactivity was investigated using multichannel nirs machines in unipolar depression, bipolar depression, and schizophrenia ( . biol. psychiatry , ; . neuroimage , ) by monitoring changes of oxy-hemoglobin concentration ([oxy-hb]) every . s during a verbal fluency task. the unipolar depression was characterized by smaller [oxy-hb] increase, the bipolar depression by comparable but delayed [oxy-hb] increase, and the schizophrenia by reduced [oxy-hb] increase during the task period followed by [oxy-hb] re-increase during the post-task period, suggesting reduced, preserved but delayed, and inefficient frontal lobe reactivity, respectively. collaborators: itsuro ida, akihiko oshima, makoto ito, tomohiro suto, masaki kameyama, yutaka yamagishi, toshimasa sato, masashi suda sy - - - clinical application of nirs to neurorehabilitation optical imaging using near-infrared spectroscopy (nirs) is suitable for assessing cortical activation during human gait because of its flexibility and portability. in healthy subjects, walking induced increase of oxygenated hemoglobin levels that centered in the medial sensorimotor cortex and supplementary motor area. in hemiparetic patients with stroke, cortical activation was characterized by asymmetrical activation in the sensorimotor cortex and recruitment of the premotor and prefrontal cortex. a longitudinal study revealed that locomotor recovery was associated with improvement of asymmetrical activation in the sensorimotor cortex as well as enhanced activation in the premotor cortex. sensorimotor stimulation by facilitation technique induced enhanced activation in the motor related areas, particularly in the premotor cortex. partial body weight support and speed-dependent exercise decreased sensorimotor activation, suggesting relative shift of locomotor control to the hierarchically lower structures including the spinal cord. thus nirs may contribute to establishing brain-based strategies for neurorehabilitation. research funds: funds for comprehensive research of aging and health sy - - - measurement of language related brain activities during recovery from aphasia eiju watanabe , yumiko muroi , chizuru nakajima department of neurosurgery, jichi medical university, tochigi, japan; department of neurosurgery, tokyo metropolitan police hospital, tokyo mechanism which supports the recovery of language after aphasia is not well understood. it has long been discussed that language related areas including the regions surrounding the language area and compatible cortex on non-dominant side could be candidates which support the recovery. several studies suggest the compensation by these areas using fmri and pet. we used optical topography (ot) to know the participation of these areas during the recovery from the aphasia. we measured aphasics who showed recovery from the aphasia after apoplexy. word generation task was used. in seven cases ot was measured more that twice. seven cases showed the activity on the non-dominant frontal lobe. they all showed activities on the dominant frontal lobe in the follow-up measurements along with the deactivation of non-dominant side. these findings showed dynamic participation of non-dominant frontal lobe during the recovery phase suggesting that the rehabilitation protocol should be changed according to the area activated in each phase. tamami fukushi research institute of science and technology for society (ristex), japan science and technology agency (jst), tokyo, japan recent development of neuroscience has provided remarkable scientific discoveries, as well many newer philosophical, ethical, legal and social issues. for example, the consequences of the progress of non-invasive neuroimaging technologies, such as functional magnetic resonance imaging (fmri) and near infrared spectroscopy (nirs) show the possibility to read the mind of others, which may lead the criminal and commercial applications. brain-machine interface (bmi) technology and pharmacological manipulation of the human brain can cause the unpredictable enhancement of human ability. in advance to the expansion of "applied neuroscience", neuroscientists should consider "what the ethical problem is in the current neuroscience" and "how we learn and interact with the ethics". in this symposium, the panels will talk about the history of neuroethics then provide the ethical aspects of basic research. we will also discuss the future perspective of the neuroethics in japan and world in terms of sharing the problems across neuroscientists, ethicists, mass media, and public. judy illes stanford university, usa akin to genetic testing in the s, the translation of neuroimaging capabilities from the laboratory to the clinical setting has raised ethical questions about how new diagnostic and predictive information will be managed in the absence of effective treatments for certain diseases, about the timing of technology transfer and handling of technology that falls in the regulatory gray zone between research and clinical use, and what impact increasing opportunities for selfreferral to health care will have on patient-physician relationships, medicine, and society overall. potential off-label uses of advanced neuroimaging outside the health care setting -in law, education, employment and even for national security -are already being tested and debated. we will discuss how these issues converge in st century neuroethics, the presence of neuroethics in the international domain, and the critical role of ethics in neuroscience in the future. sy - - - neuroethics from primate's eyes naotaka fujii bsi, laboratory for symbolic cognitive development, japan neurophysiologists working on monkeys have been trying to understanding how their brains are working. the aim of the studies was not merely revealing functions of primate's brain but also trying to extrapolate the findings in primates onto human brain functions. this was true but not really true due to technical limitations which prevented us from expanding the findings in primates directly to the human brain function. however, recent advancement of technology has changed the world. findings in primates can be directly applied to human studies with or without researcher's intention. technologies invented in primate physiology are now readily transferred into human without much discussion. brain machine interface is one example. now, monkey's brains are forcing us to think about social impact of our research from ethical view, which we have not discussed before. as an experienced primate neurophysiologist but with little ethical training, i will discuss 'what is ethically correct primate research' and 'how our scientific contribution has to be made' from very practical and ground level of neuroethics. sy - - - neuroethics of nurturing the brain takao k. hensch critical period mechanisms group, riken brain science institute, japan at no time in life is the brain so easily shaped by experience than in infancy and in early childhood. it is during these critical periods that neural circuits acquire language and other basic brain functions. unraveling mechanisms that limit such dramatic plasticity to early life would pave the way for novel paradigms or therapeutic agents for rehabilitation, recovery from injury or improved learning in adulthood. conversely, a deeper insight into early postnatal brain development will provide valuable inspiration for effective brain-based education methods for our children-perhaps the greatest potential contribution of neuroscience to society. this raises urgent and important ethical questions for our consideration: is there an "ideal" brain we should be nurturing? to what extent can/should drugs be used not merely to correct developmental disorders but also to enhance performance? how do we determine what is good or bad for the maturing brain? research funds: riken bsi sy - - - neuroethics beyond laboratories and across cultures daofen chen national institute of neurological disorders and stroke, usa recent progress in systems and cognitive neuroscience poses new ethical challenges to both investigators and to the funding agencies that support scientific investigations. potential uses of many of these recent advances go beyond their intended medical applications. a growing array of neurotechnologies capable of monitoring or even intervening in human cognition makes it imperative to consider the social, ethical, and legal implications of these scientific advances. while it once might had been possible to conduct research with naive ignorance of its societal implications, this is no longer the case. moreover, we need to be cognizant that modern brain science is profoundly influenced by the distinct cultural and social values held by different sectors of the world population. we will discuss what can be done from the perspective of funding agencies to facilitate intercultural dialogue, foster mutual understanding, and develop a framework and strategies to address emerging neuroethical issues and prioritize our future efforts in neuroscience research. sy - - - bridging neuroscience and public: neuroethics in cultural contexts osamu sakura , interfaculty initiative in information studies, university of tokyo, tokyo, japan; research institute of science and technology for society (ristex), jst, japan to bridge between neurosciences and public society-it should be one of the important aims of neuroethics. for this purpose we need to draw the outline of neuroscience within the cultural context. the method and the result of natural sciences are universal, of course, but its social consequences are highly variable among cultures. although the systematic comparative researches are open to the future, we should discuss how the neurosciences could create the healthy relationship between the public society, especially focusing on the method for public participation and on the previous successful cases. mitsuru kawamura , , akira midorikawa , yoshiki kaneoke , shinichi koyama , masato taira , argye hillis showa university school of medicine, japan; crest, jst, saitama, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan; johns hopkins university, baltimore, usa this symposium aims to provide an opportunity to talk between clinical neuropsychologists and neuroscientists. focusing on the visual system, we will discuss up-to-date studies from neuropsychological and neuroscientific viewpoints. the topics include motion perception in brain-damaged patients, neuroimaging of motion perception, surface and depth perception in brain-damaged patients, and neuroimaging of surface and depth perception, and neuropsychological and neuroimaging studies of visual attention. we will discuss consistency and inconsistency of our findings, and discuss what to do in order to produce synergy between clinical neuropsychology and neuroscience. research funds: kakenhi ( ), crest sy - - - impairment of surface perception in patients with ventral occipital damages shinichi koyama , mitsuru kawamura , akira midorikawa , yoshiki kaneoke , masato taira , argye hillis showa university, tokyo, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan; johns hopkins university, baltimore, usa we examined the perception of faces and objects in two patients with ventral occipital damages, using psychophysical techniques. patient was a -year-old woman with bilateral damage in the fusiform and parahippocampal gyri. although she could recognize pictures of famous people, she frequently failed to decide the races of unfamiliar faces and occasionally failed to see the hollow-face illusion (gregory ) . in addition, her performance in object identification task became poorer when the objects were presented in inverted (negative) pictures. patient was a -year-old men with bilateral damage in the lingual and fusiform gyri. his recognition of faces and objects became poorer when they were presented in inverted pictures. based on the above results, the role of the ventral visual cortex in the perception of faces and objects will be discussed. research funds: grant-in-aid for jsps fellows sy - - - how do pictorial cues influence d information processing in the parietal association cortex? masato taira , arish, nihon university, tokyo, japan; department of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan pictorial cues are one of the most influenced cues for d perception. basically, it is thought that the parietal association cortex processes d visual information by binocular disparity cues and the temporal association cortex processes that by pictorial cues in the concept of two visual information processing systems in the brain. however, recent studies have suggested that there are many crosstalk of d information between these association areas. our recent studies have shown that a group of neurons in the parietal cortex (area cip) is involved in perception of d surface orientation and used both disparity and pictorial cues. functional mri data in human also suggest that pictorial cues, such as attached and cast shadow, are processed in the parietal cortex in d perception. furthermore, human psychophysical data gives us some insights of how the pictorial cues influence d information processing in the parietal association cortex. research funds: kakenhi sy - - - case report of a patient with posterior cortial atrophy who relatively preserved perception of moving objects akira midorikawa national center of neuroscience, national center of neurology and psychiatry, tokyo, japan it has been presented that severe type of bálint syndrome behaves like a blind person; however, it also reported that there are some cases who behave like a blind person but could walk without collision. up to today several cases have been reported, but the underlying mechanism of the phenomenon has not been mentioned. in this report, i will talk about a patient with bálint syndrome due to degenerative disease known as posterior cortical atrophy (pca), who could not only walk around without collision but also play catch very well, nevertheless having blind like behavior. the crucial visual information underlying these phenomenons was assumed to be motion parallax induced by not only objects movement but also self movement. in addition, discrepancy between the patients who could walk and not walk will be discussed. research funds: crest, japan science and technology agency sy - - - neural mechanism underlying visual perception of motion as revealed by non-invasive human study yoshiki kaneoke department of integrative physiology, national institute for physiological sciences magnetoencephalography (meg) measures the neural activity representing the synchronized inputs to the millions of pyramidal neurons in the localized cerebral cortex. thus, it will show us another aspect of the neural activity related to the specific brain function that would not be revealed by the recording of single neuronal activity. our meg studies have revealed several important findings in the human visual motion detection system. first, the response properties for the apparent motions indicate the importance of the human mt/v + for the perception and the existence of the parallel processing for the motion and light blinking. second, the existence of the spatiotemporal filtering mechanism for the perception of motion speed is shown by the various motion stimuli. third, we present the evidence that the spatial integration of the speed without direction information occurs in our visual system. based on the results, scalar fields theory for the spatial integration of motion is proposed to explain various complex motion perception. research funds: kakenhi ( ) sy - - - neural correlates of visual attention argye hillis , mitsuru kawamura , akira midorikawa , yoshiki kaneoke , shinichi koyama , masato taira johns hopkins university, usa; showa university, tokyo, japan; national institute of neuroscience, ncnp, tokyo, japan; national institute for physiological sciences, okazaki, japan; nihon university, tokyo, japan in this paper i report a series of studies of unilateral spatial neglect (usn) in acute stroke, demonstrating a frequent double dissociation between stimulus-centered neglect and viewer-centered neglect, and showing that these types of neglect can be modality-specific. other data reveal that different types of usn are observed when there is hypoperfusion of temporal cortex versus parietal cortex. still other data provide evidence that severity of neglect depends on the volume of hypoperfused tissue in acute stroke, and that reperfusion results in early recovery of neglect. finally, i will review new evidence that right usn is common after left cortical infarcts or hypoperfusion in acute stroke, but the distribution of types of usn is very different from the distribution of types of usn after right hemisphere stroke. takeo kubota, takae hirasawa, kaoru nagai department of epigenetic medicine, university of yamanashi faculty of medicine, chuo, yamanashi, japan how are brains controlled molecularly? this is one of fundamental questions in neuroscience. several lines of evidences have suggested that genes are more strictly controlled in the brain than in other organs. epigenetics is one of such systems to control expression of the genes not based on dna sequence, but based on 'beyond the dna sequence' (chromatin modifications and small rnas). the failure of this system is known to result in neurodevelopmental diseases, such as an autistic rett syndrome. it has recently been demonstrated that the epigenetic system is affected by an environmental stress after birth, and that the system is associated with neural differentiation and cell fate determination and human brain diversity. these findings imply that epigenetics is an important research field to understand mechanisms of neural development and mental diseases. topics from update epigenetic researches in neuroscience will be discussed in this symposium. as one of epigenetic disorders, we introduce studies of rett syndrome (rtt) and its model mouse. rtt is a neurodevelopmental disorder, characterized by mental retardation and peculiar behavior. mutations of the mecp gene, encoding methyl-cpg binding protein , cause rtt. mecp acts as a transcriptional silencer. abnormal expression of some genes due to mecp dysfunction is thought to be the first step of rtt pathophysiology. to understand how mecp mutation makes rtt, we have two approaches that are morphological investigation of brain and discovery of mecp -downstream genes. using mecp -null mice (mecp −/y ), we revealed small number of dendritic spines and premature postsynaptic density at presymptomatic period. premature synaptogenesis may be the initial neuronal changes of rtt. we also found that mecp directly regulates expression of insulin-like growth factor binding protein (igfbp ) gene in human and mouse brains. pathological features of mecp −/y have the similarity of igfbp transgenic mice, which show reduction of early postnatal brain growth. over-expression of igfbp due to lack of mecp may lead to delayed brain maturation. growing evidence suggests that alterations in the epigenetic status such as dna methylation and histone modifications in brain are involved in the behavioral response to environmental factors and the pathogenesis of psychiatric diseases. however, in contrast to mrna profiling, there are few established methods for profiling the genomewide epigenetic status to date. we developed a method for profiling the genome-wide dna methylation pattern using tiling arrays, and focused our analysis on human brain samples derived from psychiatric patients and control subjects. in this symposium, general picture of the genes that are heavily methylated or non-methylated in human brain, and the relationship between dna methylation and mrna expression patterns will be presented. understanding what produces neuronal diversification has been a longstanding challenge for neuroscientists. the recent finding that long interspersed nucleotide elements- or l (line- ) retroelements are active in somatic neuronal progenitor cells provided an additional mechanism for neuronal diversification. together with their mutated relatives, retroelements sequences constitute % of the mammalian genome with l elements alone representing %. the fact that l can retrotranspose in a defined window of neuronal differentiation, changing the genetic information in single neurons in an arbitrary fashion, allows the brain to develop in distinctly different ways. this characteristic of variety and flexibility may contribute to the uniqueness of an individual brain. however, the extent of the impact of l on the neuronal genome is unknown. the characterization of somatic neuronal diversification will not only be relevant for the understanding of brain complexity and neuronal organization in mammals but may also shed light on the differences in cognitive abilities, personality traits and many psychiatric conditions observed in humans. sy - - - notch-induced acquisition of astrocyte differentiation potential of neural stem cells kinichi nakashima , jun kohyma , tetsuya taga , masakazu namihira grad. sch. biol. sci., naist, ikoma, japan; inst. mol. embryol. genet., kumamoto univ., kumamoto, japan neurons and astrocytes are generated from common neural stem/precursor cells, however, neurogenesis precedes astrocytogenesis during brain development. we have previously reported that gfap-positive astrocyte differentiation is dependent on the transcriptional activity of stat . a cpg dinucleotide in the stat recognition sequence within the gfap gene promoter is highly methylated at midgestation which becomes demethylated as the brain develops, enabling stat to induce gfap expression. thus, it is conceivable that the epigenetic modification such as dna methylation of cell type-specific gene promoter controls the switch from neurogenesis to astrocytogenesis in the developing telencephalon. here we report that neurons, which are generated earlier than astrocytes can potentiate neural precursors at midgenstation to differentiate into astrocyte through the activation of notch-signaling. the activated notch-signaling accelerates demethylation of stat binding element in the gfap gene promoter. sy - - - neurogenesis and stem cell supply as therapeutic approach to overcome ischemic stroke masayasu matsumoto department of clinical neuroscience and therapeutics, hiroshima university graduate school of biomedical sciences, japan in order to overcome the brain damage caused by ischemic stroke, several strategies have been so far applied. in the present symposium, i will address the following points to be considered prior to clinical application of neurogenesis and/or stem cell supply to repair the damaged brain function. ( ) which type of brain infarction will be a future target of this therapeutic approach? ( ) which phase of brain infarction (i.e., acute or chronic phase) will be selected as a future timing of therapeutic intervention? ( ) which will be the best way to be applied in the clinical settings, neurogenesis control, stem cell supply or both? the present research status and future directions will be presented and fully discussed. research funds: kakenhi sy - - - gene therapy for cerebral ischemia setsuro ibayashi, hiroaki ooboshi department of medicine and clinical science, graduate school of medical sciences, kyushu university, fukuoka, japan cerebrovascular disease is the leading cause of the disabled people in japan and western countries. gene transfer technique may be applicable to the treatment of serious types of cerebrovascular disease. cerebral blood vessels have been targeted by gene transfer with intravascular or perivascular approaches. several experimental studies have revealed potential usefulness of gene therapy for prevention of vasospasm after subarachnoid hemorrhage. as for cerebral infarction, studies using various brain ischemia models have shown effectiveness of gene transfer in reduction of infarct size and functional recovery. our recent studies of post-ischemic gene transfer have provided promising results in attenuation of ischemic damages by inhibiting apoptosis, inflammation and vascular permeability. approaches to cerebral ischemia using gene transfer for angiogenesis and neurogenesis appear to be novel and promising strategies. thus, gene therapy has a potential for the future therapy against cerebral ischemia. isao date department of neurological surgery, okayama university, okayama, japan cerebral ischemia is one of the neurological disorders that cell transplantation is expected to be applied. in this presentation, the author will summarize our recent basic research and clinical application reported in the literature. it is now possible to make several types of neurotrophic factor secreting cell line by genetic manipulation. in order to prevent immunological reaction and tumor formation, we have been using encapsulated cell grafting technique. we transplanted several types of neurotrophic factor secreting cell line into the middle cerebral artery occlusion model and could confirm the histological and behavioral efficacy. we have also been using adultderived neural stem cells as donor cells because they have merits to make autografitng possible. as donor tissue, neural protection can be expected similar to fetus-derived neural stem cells. the effect of neural protection increases when neurotrophic factor secreting genes such as gdnf were inserted into neural stem cells. cell transplantation is considered a new therapeutic approach for cerebral ischemia and clinical application is expected. we now know that ( ) motor function may recover after minor injury to the primary motor cortex, ( ) this recovery is, at least in part, associated with reorganization of cortical motor representation, ( ) the molecular mechanism for synaptic plasticity and axonal regrowth is being elucidated, and ( ) recent clinical experience revealed that the motor function in patients with spinal cord injury is improved after transplantation of her/his own olfactory mucosa. furthermore, recent neuroimaging techniques can display the cortical functions as we as the specific fiber connections in individual brain. virtually any part of the brain can be approached with the accuracy of millimeters by the current image-guided neurosurgery. those theoretical and technical backgrounds suggest we might be ready for the reconstruction of brain function. nobuyuki nukina laboratory for structural neuropathology, riken brain science institute, japan a major hallmark of the polyglutamine (pq) diseases is the formation of pq inclusions. recently, misfolding has come to be considered one of the primary factors for pq protein aggregation, although, the nature of misfolding is not yet well known. the protein misfolding induced by pq expansion was investigated with our molecular model system using mutant myoglobin which is inserted different size of pq. expanded polyglutamine stretches form intramolecular and intermolecular beta sheets and amyloid fibrils. the surface of the mutant myoglobin with expanded pq was partially unfolded and destabilized. we also investigated the early phase of fibrillization by small-angle x-ray scattering and electron microscopic studies, revealing that the expansion of pq to repeats induced the formation of quasi-aggregate in the earliest stage of the protein fibrillization. this structure could be closely involved in recruitment of various functional proteins into aggregates, leading to the cellular dysfunction that causes pq diseases. furthermore using cellular model system we also studied the aggregates interacting proteins (aips) by analyzing the purified polyglutamine inclusions and the lists of aip including chaperones, proteasome subunits, ubiquitin interacting proteins and others suggest the pathological role of aips in the disease cascades. sy - - - neuronal dysfunctions in dentatorubralpallidoluysian atrophy (drpla) shoji tsuji , toshiya sato , mitsunori yamada department of neurology, the university of tokyo, tokyo, japan; center for bioresource-based researches, japan; department of pathology, brain research institute, niigata university, niigata, japan to investigate molecular mechanisms of neurodegeneration in drpla, a polyglutamine disease caused by expansions of cag repeats of drpla gene, we have established transgenic mice harboring a single copy of the full-length human mutant drpla gene with cag repeats. the q mice exhibited neurological phenotypes similar to juvenile type of drpla characterized by ataxia, myoclonus and epilepsy. electrophysiological studies disclosed age-dependent abnormalities in the globus pallidus and cerebellum. neuropathological studies revealed progressive brain atrophy without obvious neuronal loss and an age-dependent increase in neuronal intranuclear accumulation of mutant proteins with the regional distribution vulnerable to drpla. expression profiling analyses revealed down-regulated genes including camp responsive genes. these results suggest that "neuronal dysfunction", but not the "neuronal cell death", is the essential mechanism of neurodegeneration in drpla. huntington's disease (hd) is caused by an expansion of a cag repeat encoding polyglutamine in the huntingtin protein and involves progressive motor, cognitive and psychiatric symptoms. using a transgenic mouse model of hd, we have shown that environmental factors can dramatically modify the disease process and delay the onset and progression of motor and cognitive symptoms. further, we have attempted to correlate these behavioural findings with changes in gene expression, neuronal morphology, neurogenesis, and cortical plasticity, in an attempt to elucidate cellular and molecular mediators in hd, and understand how gene-environment interactions can modulate these pathogenic pathways. our findings indicate that the modulatory effects of environmental manipulations are mediated by amelioration of specific molecular and cellular deficits, and provide experimental paradigms for the identification of novel therapeutic targets for hd and related brain disorders. sy - - - control of neural organization in the developing cerebral cortex yasuto tanabe mitsubishi kagaku institute of life sciences, tokyo, japan in the developing cerebral cortex, the generation of neurons with distinct identities and patterns of connectivity is controlled by a hierarchical series of cellular interactions that culminate in the laminar organization of distinct cortical areas. over the past three years we have begun to examine cerebral cortical development by focusing on three distinct major neuronal subtypes, namely, cajal-retzius cells, cortical projection neurons, and cortical interneurons. the analyses of these distinct neuronal subtypes allowed us to identify several candidate molecules and cellular interactions that might contribute to the laminar and areal organization of the cerebral cortex. in the first part of my talk, i would like to deal with the issue of ontogeny of cajal-retzius cells, and present the way cajal-retzius cells are generated, migrate and finally distribute in the developing cerebral cortex. then, i would like focus my talk on the issue of the way the acquisition of radial migration and axonal trajectory patterns of distinct cortical projection neurons is controlled during the development of the cerebral cortex. research funds: kakenhi , sy - - - mechanisms of the regulation of neuronal migration and corticogenesis kazunori nakajima , dept. of anat., keio univ. sch. of med., tokyo, japan; inst. of dna med., jikei univ. sch. of med., tokyo, japan mammalian cerebral cortex has a six-layered structure where the neurons are aligned depending on their birth-date. to determine whether the migration from the ventricular zone (vz) to beneath the marginal zone (mz) is essential for neuronal segregation into layers, we investigated whether migrating neurons have different cell aggregation properties in vitro depending on their birth-dates, even before they arrive beneath the mz. we analyzed vz cells and cells from the intermediate zone (imz) mainly composed of migrating cells, and found that the cells had acquired a birth-date-dependent preferential segregation mechanism in a reelin-independent manner. these findings suggest that cortical neurons acquire a birth-date-dependent segregation property (or fate) before their somas reach the mz. in silico experiments of the reaggregation culture supported that this mechanism might indeed contribute to the layer formation in the developing cerebral cortex in concert with other mechanisms such as reelin signaling. kenji shimamura division of morphogenesis, institute of molecular embryology and genetics, kumamoto university, japan neurons of the thalamus originate in restricted regions of the proliferative zone of the diencephalic compartment before settling in their final locations in the nuclei. to investigate cellular and molecular mechanisms underlying nucleus formation, we analyzed the sequence and pattern of expression of specific markers that distinguish the subsets of neuronal precursors during development of the thalamus. we found that a morphogen-like activity of sonic hedgehog (shh) precisely defines positions of neurons with distinct properties, and that some gabaergic interneurons migrate from their birth place to distant nuclei in a highly organized manner. we also provide evidence that shh produced by the zona limitans intrathalamica (zli), which abuts the prethalamus and thalamus, is likely to be a cue for this directed migration. our results suggest that local production of prespecified neurons coupled with distinct migration properties and local guidance cues such as compartment boundaries could be principle elements for the nucleus formation. layers and nuclei are important functional units in the vertebrate cns. neurons in these structures have common physiological and anatomical features. despite their importance, mechanisms for nucleogenesis are poorly understood. we focused on the lower rhombic lip (lrl)-derived precerebellar neurons, and utilized exo utero electroporation with an enhanced yellow fluorescent protein (eyfp) gene, to study the process of nucleogenesis. after the unilateral transfer of eyfp to the lrl of embryonic day . mice, eyfp-labelled neurons migrate tangentially from the lrl in two distinct streams, one toward the ventral metencephalon and the other toward the ventral myelencephalon. the former formed the pontine grey nucleus and reticulotegmental nucleus and the latter the external cuneate nucleus and lateral reticular nucleus. before forming the clusters, the labelled neurons begin to migrate toward the ventricle along the radial fibres, and aggregate as they detach from the fibres. perturbation experiments such as introduction of dominant negative constructs and sirna suggested involvement of several molecules in the migration of these neurons. the brains of fetal alcohol syndrome patients exhibit impaired neuronal migration, but little is known about the mechanisms underlying this abnormality. here we show that ca + signaling and cyclic nucleotide signaling are the central targets of alcohol action in neuronal cell migration. an acute administration of ethanol reduced the frequency of transient ca + elevations in migrating neurons and cgmp levels, and increased camp levels. experimental manipulations of these second messenger pathways, through stimulating ca + and cgmp signaling or inhibiting camp signaling, completely reversed the action of ethanol on neuronal migration in vitro as well as in vivo. each second-messenger has multiple but distinct downstream targets, including camkii, calcineurin, pp , rho gtpase, mapk and pi k. these results demonstrate that the aberrant migration of immature neurons in the fetal brain caused by maternal alcohol consumption may be corrected by controlling the activity of these second-messenger pathways. sy - - - membranes, water and diffusion denis j. le bihan shfj/cea, france among einstein papers is one which unexpectedly gave birth to a powerful method to explore the brain. molecular diffusion was explained by einstein on the basis of the thermal random translational motion of molecules. in the mid s it was shown that water diffusion in the brain could be imaged using mri. a dramatic application of diffusion mri has been brain ischemia, following the discovery that water diffusion drops immediately after the onset of an ischemic event, when brain cells undergo swelling through cytotoxic edema. also, water diffusion is anisotropic in white matter, because axon membranes limit molecular movement perpendicularly to the fibers. this feature can be exploited to map out the orientation in space of the white matter tracks and image brain connections. more recently, it was discovered that diffusion mri could detect transient swelling of activated cortical cells. this represents a significant breakthrough, allowing non invasive access to a fast and direct physiological marker of brain activation. this approach will bridge the gap between invasive optical imaging techniques and current functional neuroimaging approaches in humans, which are based on indirect and remote blood flow changes. sy - - - diffusion tensor fiber tractography using a tesla mr system yukio miki department of diagnostic imaging and nuclear medicine, kyoto university, kyoto, japan diffusion tensor imaging (dti) is an mr imaging technique that is sensitive to orientation of mobility in water molecules. dti reveals two specific characteristics: diffusion anisotropy; and directional distribution of water diffusivity. white matter shows high diffusion anisotropy, because diffusion is faster in parallel to fiber direction than in other directions. dti of the brain can be reconstructed to display d macroscopic fiber tract architecture, in a process known as fiber tractography. with recent advances in actively shielded -t magnets and parallel imaging techniques, high-field mr imaging has become practical in clinical settings. we have demonstrated that depiction of most fiber tracts was improved on -t tractography compared to . t. we have also established an integration of tractography and intraoperative subcortical motor-evoked potential, and demonstrated that diffusion tensor tractography of the corticospinal tract using -t mr was able to provide interactive information on fiber tracts, depicting the course of eloquent fiber tracts during an operation. to test whether mr tractography is reproducible and reliable, we used this technique to assess acute tiny infarcts located in the supratentorial brain. we analyzed the data of patients who presented to our institute with sensorimotor symptoms. there was an excellent correlation between the location of the infarct as assessed by tractography and clinical symptoms. next, we applied the technique to patients with evolving symptoms after admission to hospital. we specifically assessed the change in the tract-infarct relationship over time. the data showed that, in most cases when there was symptomatic progression, the distance between the tract and the infarct border depicted on dwi diminished. finally, we studied whether the use of tractography could help predict a patient's prognosis. to simplify the analysis, we specifically focused on patients with lenticulostriate artery (lsa) infarcts. we analyzed the correlation between the extent of cst involvement within the infarcts and the severity of motor deficits. the data indicated that the tractographic technique could be useful to predict a patient's outcome. sy - - - anatomical and functional tractography: a combined approach with diffusion tractography and corticocortical evoked potential riki matsumoto department of neurology, kyoto university graduate school of medicine, japan recent advances in diffusion-weighted imaging have raised the possibility of in vivo investigations of brain circuitry in humans. the probabilistic tractography provides estimates of the likelihood of a pathway between two brain regions without tensor estimation and thus could trace the fiber pathways beyond regions of low diffusion anisotrophy into the grey matter. however, the results depend merely on anisotropic movement of water molecules and need validation. for presurgical evaluation of epilepsy patients, we developed an in vivo tracking method, cortico-cortical evoked potential, to electrically track the cortico-cortical connections by stimulating a part of the brain through epicortical electrodes and recording the cortical evoked potentials that emanate from a distant region of the cortex via projections. combined with preoperative diffusion analysis, this invasive evaluation provides a unique opportunity to study the cortico-cortical connectivity both functionally and anatomically. results of the combined approach will be presented. parkinson disease (pd) is the second commonest neurodegenerative disorder after alzheimer disease characterized by tremor, rigidity, bradykinesia, and postural instability. pathologically, the most outstanding change is the neurodegeneration of the nigral dopaminergic neurons. although familial forms of pd can be encountered up to % of the patients, the remaining cases are sporadic. it has been postulated that nigral neurodegeneration in pd is induced by the interaction of genetic risk factors and environmental factors. epidemiological studies revealed numbers of environmental factors that are positively correlated with increased risk of pd; such factors include pesticide, herbicides, rural living, well water drinking, metals such as manganese and iron, fuel oil, industrial chemicals, and hydrocarbon solvents. in addition, certain employments were reported to be associated with increased risk of pd; these include steel/alloy industry, wood/pulp plant, farming, carpentry, cleaning, orchard, mining, and welding. these studies suggest importance of environmental factors in the pathogenesis of pd. recent progress in these areas will be discussed. masami ishido national institute for environmental studies, tsukuba, japan there are getting much public concerns about children health since environmental factors such as industrial chemicals cause deficit in developing brains. it has been suggested that they may be incident of attention deficit hyperactivity disorder or autism. epidemiologic studies also suggested that parkinson's disease was found in the peoples who were exposed to pesticides in their childhood. thus, we examined the effects of industrial chemicals, called endocrine disruptors, on rat neurodevelopment. oral administration of an endocrine disruptor ( - mg/kg) into male wistar rats (from days to weeks of age) significantly caused hyperactivity at - weeks old. immunohistochemical analyses of the brain tissues at weeks of age revealed a large reduction of immunoreactivity for tyrosine hydroxylase, but not for glutamic acid decarboxylase, both of which are localized in the substantia nigra, suggesting the specific degeneration by the chemical of dopaminergic neurons. tunel-positive cells were seen in the substantia nigra. thus, environmental insults in early life may be of particular etiologic importance. sy - - - non-thermal effects of mobile phones upon the rat brain leif g. salford, b. persson, j. eberhardt, g. grafstrom, l. malmgren, a. brun dept of neurosurgery and the rausing laboratory, lund university, sweden we have shown that rf electromagnetic fields can cause significant leakage of albumin through the bbb of exposed rats as compared to non-exposed animals. one remarkable observation is that sar values around mw/kg give rise to a more pronounced albumin leakage than higher sar values -all at non-thermal levels. if the reversed situation were at hand, we feel that the risk of cellular telephones, base-stations and other rf emitting sources could be managed by reduction of their emitted energy. the sar value of around mw/kg is exists at a distance of more than one meter away from the mobile phone antenna and at a distance of about - meters from a base station. another remarkable observation in our studies is the fact that a significant (p < . ) neuronal damage is seen in rat brains days after a hour exposure to gsm at sar values , and mw/kg. we have followed up this observation in a study where animals were sacrificed and days respectively after an exposure for hours to gsm mobile phone electromagnetic fields at sar values , , , . and (controls) mw/kg. significant neuronal damage is seen after days and albumin leakage after . our findings may support the hypothesis that albumin leakage into the brain is the cause for the neuronal damage observed after and days. sy - - - the mitochondrial toxin -nitropropionic acid: an environmental toxin to study striatal degeneration in huntington disease emmanuel brouillet neuronal death laboratory, ura cea-cnrs , france huntington disease is a neurodegenerative disorder caused by a mutation in the gene encoding huntingtin. the mechanisms underlying the preferential degeneration of the striatum, the most striking neuropathological change in huntington disease, are unknown. the behavioral and anatomical similarities found between huntington disease and animal models of striatal degeneration using the environmental toxin -nitropropionic acid ( np) support the hypothesis that mitochondrial defects could play a role in huntington disease. we will discuss the mechanisms of np toxicity and show that np and mutated huntingtin have certain mechanisms of toxicity in common. in particular, we show that mutated huntingtin can alter the expression of mitochondrial complex ii, the respiratory chain enzyme specifically inhibited by np. in summary, the np story is a good example showing how the study of environmental toxins can greatly help to elucidate the complex mechanisms underlying chronic neurodegenerative disorders. we recently demonstrated that rats received intrecisternal injection of -ohda or environmental chemicals, such as bisphenol a, nonylphenol, p-octylphenol, diethylhexylphthalate or dibutylphthalate, at days of age showed behavioral hyperactivity at - weeks of age. immunohistochemical studies revealed a deficit in the development of dopamine (da) neurons. adult rats received these chemicals showed degeneration in nigro-striatal da neurons similarly to parkinson's disease. in this study, we investigated the mechanism of -ohda-induced neurotoxicity, using pc cells as an in vitro model system. we observed the generation of reactive oxygen species (ros) and p-quinone via auto-oxidation of -ohda. we also characterized the oxidation of cellular proteins by -ohda and the protective effect of antioxidants such as catalase, glutathione, and n-acetylcysteine with different manner. we will discuss about apoptotic cell death pathway including cytochrome c release and caspase activation induced by -ohda, ros and p-quinone. sy - - - environmental factors in the pathogenesis of alzheimer's disease joanna l. jankowsky california institute of technology, usa epidemiological studies indicate that environmental factors significantly influence the risk of developing alzheimer's dementia. foremost among those factors are education, occupation, and leisure activities. although not universal, most studies have found that individuals with greater education, more challenging occupation, or active leisure hobbies show relative protection against dementia. animal models for alzheimer's disease have recently been used to explore the mechanism of this effect. transgenic mice designed to recapitulate alzheimer's amyloid pathology are protected from functional decline by enriched housing designed to provide cognitive stimulation. both enriched housing and exercise modify the level of amyloid-beta in the brains of transgenic mice, demonstrating that environmental factors can significantly influence brain biochemistry. intriguingly, traditional environmental enrichment can improve cognitive behavior while paradoxically elevating amyloid-beta levels in transgenic mice, suggesting that environmental stimulation may alter amyloid metabolism and cognitive function by competing mechanisms. the efficacy of synaptic inhibition depends on the number of gaba a receptors expressed on the neuron surfaces. in the present study, we have elucidated the role of prip (plc-related inactive protein) in trafficking of the receptors by analyzing prip knockout (ko) mice; the sensitivity to diazepam was reduced as assessed by biochemical, electrophysiological and behavioral analyses of ko mice, suggesting the dysfunction of the ␥ subunit-containing receptors. we then examined the mechanisms by which prip molecule regulates cellsurface expression of ␥ subunit-containing receptors. disruption of the direct interaction between prip and the ␤ subunit of receptors by prip-binding peptide inhibited cell-surface expression of ␥ subunit-containing receptors in gh and hek cells. constitutive internalization of the receptors was also modified by the peptide. collectively, prip molecules are involved in trafficking of ␥ subunit containing gaba a receptors to/from cell-surface membrane. research funds: kakenhi ( ) sy - - - involvement of bdnf in the induction of ltp at visual cortical inhibitory synapses yukio komatsu dept. visual neurosci., res. inst. environ. med., nagoya univ., nagoya, japan high-frequency stimulation (hfs) induces long-term potentiation (ltp) at inhibitory synapses of layer pyramidal cells in rat visual cortex. this ltp requires a postsynaptic ca + rise for induction and spike firing of presynaptic cells for maintenance, although the necessary frequency is low, suggesting that ltp is expressed presynaptically and some information must be sent backwards from the post-to presynaptic cells during induction. in this study, we investigated whether bdnf could act as such retrograde messengers. ltp did not occur when hfs was applied in the presence of k a at nm, inhibiting trk receptor tyrosine kinases selectively at that dose. hfs induced ltp when k a application was started soon after hfs or when k a was loaded into postsynaptic cells. ltp did not occur in the presence of trkb-igg or anti-bdnf antibodies. in cells loaded with bapta, the addition of bdnf to the medium enabled hfs to induce ltp without affecting basal synaptic transmission. these results suggest that bdnf released from postsynaptic cells activates presynaptic trkb, enabling the induction of ltp. research funds: kakenhi ( ) sy - - - autocrine mglur activation in cerebellar purkinje cells regulates gaba-mediated synaptic inhibition trevor smart, ian c. duguid university college london, uk in the cerebellum, retrograde signalling is important for the induction of short-and long-term changes to synaptic inhibition at interneuron-purkinje cell (in-pc) synapses. endocannabinoids, via cb receptors, mediate a short-term decrease in synaptic efficacy, while glutamate, via presynaptic nmda receptors, induces a sustained increase in gaba release. we now report that dendritically released glutamate also acts as an autocrine messenger, activating mglur on pcs to enhance synaptic inhibition via the release of endocannabinoids. this process was triggered by repetitive pc stimulation and blocked by uncoupling the mglur -gq/ transduction pathway as well as being initiated by direct mglur activation during pc depolarisation. glutamate uptake by excitatory amino acid transporters controlled the extent of autocrine mglur activation, whilst basal glutamate levels were unable to enhance endocannabinoid release. our study suggests that autocrine mglur activation provides a powerful homeostatic mechanism to dynamically regulate inhibitory synaptic transmission. sy - - - regulatory mechanism of inhibitory synaptic transmission in the cerebellum shin-ya kawaguchi , , tomoo hirano , dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan at the gabaergic synapses between inhibitory interneurons and a purkinje neuron in the cerebellum, postsynaptic depolarization induces long-term potentiation of transmission efficacy mediated by gaba a receptors (rebound potentiation: rp). the signaling cascades regulating the induction of rp has been clarified. the balance of activities of protein kinases and phosphatases determines whether rp is induced or not. here we show another molecular mechanism involved in the rp induction. using both electrophysiological experiments and computational kinetic simulation of biochemical reactions, we demonstrate how the long-term potentiation of gaba a receptormediated responses is brought about. rp induction was impaired by inhibition of ca + -activated protease calpain or by disturbance of association of gaba a receptor ␥ subunit with gabarap (gaba a receptor associated protein). binding of gabarap to microtubule was also involved in the regulation of rp. our results suggest that structural alteration of gabarap caused by calpain activity is critical for establishment of rp. sy - - - contribution of hebb's "organization of behavior" to the development of brain science masataka watanabe tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan more than years have passed since the publication of "organization of behavior". this book has been one of the most influential books in neuroscience. around the time of the th anniversary of this book, special issues and articles concerned with this book appeared in several journals. his idea, which is a general framework for relating behavior to synaptic organization through the dynamics of neural networks, has stimulated variety of neuroscience researches in relation to, for example, environmental effects on development, naturenurture interaction, memory consolidation and sensory deprivation. however, he also made some mistakes, for example he advocated frontal lobotomy. in this symposium, i will briefly review how influential this book has been on basic and practical neurosciences, and will re-consider the importance and limitation of studying mental processes, such as emotion, memory and thought by exploring brain mechanisms, in reference to the idea of cell assembly, phase sequence and hebb synapse. sy - - - detection of cell assembly in neuroscience experiments and brain-machine interfaces yoshio sakurai , , susumu takahashi department of psychology, kyoto university, kyoto, japan; crest, japan science & technology agency, japan the reality of cell-assembly coding in the working brain depends on how we could detect specific properties of cell assembly from multi-neuronal activities in behaving animals. first in the present paper, we show experimental results indicating some of the properties, i.e., functional overlapping of individual neurons and connection dynamics among multiple neurons, that depend on tasks and events being processed and on the distance among the neurons. second, we demonstrate a newly developed method, brain-machine interface (bmi), to test the reality of cell assembly as neural information and the plastic formation of cell assemblies during learning processes. we introduce our recent bmi system with independent component analysis (ica) and specific multi-electrodes and show some neuronal and behavioral data obtained by the bmi system. hebb postulated that coincident activities of pre-and postsynaptic neurons trigger input-specific plasticity. how relevant is it in protein synthesis-dependent late-phase plasticity (lp)? synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific lp. using live-imaging techniques, we measured entry of vesl- s-egfp into dendritic spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as synaptic tag in many criteria. ve trapping conforms to the hebb's rule in a sense that it required both presynaptic activity and postsynaptic no-pkg pathway, but their coincident time window was far wider (∼h) than that of early-phase plasticity, suggesting an involvement of persistently synchronized rather than transiently coincident activity. no spreading from the activated synapses may persistently prime the postsynaptic tag components at the surrounding synapses, during which brief inputs to these synapses will establish associative and heterosynaptic tags. thus, tagging one synapse would lead surrounding synapses to multiple metaplastic states. tomoki fukai laboratory for neural circuit theory, riken brain science institute, saitama, japan in the cell-assembly hypothesis, cortical neurons are considered to form functional subnetworks depending on a particular demand of information processing. such cell assemblies may be organized through synchronous firing of the constituent neurons and synapses modifiable by hebbian learning. in this talk, i will overview recent in vivo and in vitro experimental findings that provide new evidence for synchronous or precisely timed neuronal activity. i propose a hardwired structure of local cortical networks, "entangled synfire chains", on the basis of the experimental observations of cortical activity. in this model, multiple cell assemblies can be defined by the pattern of neuronal wiring. however, the same experimental findings can lead us to a different type of cortical network models. in this type of models, cortical networks may self-organize to develop a critical dynamical state, which may be useful for realizing a hypothetical "liquid-state machine". i will discuss the characteristic properties of both types of models and the possible implications in cortical computations. research funds: kakenhi ( ) sy - - - impact of hebbian hypothesis on neuroscience keisuke toyama shimadzu institute of basic technology, seikacho, hikaridai, kyoto - , japan hebb has seeded two major concepts in the modern neuroscience, i.e., cell assembly hypothesis for perception, and hebbian synapses to construct that cell assembly. the former concept stimulated extensive searches for the response selectivity extending from the primary visual cortex to the inferotemporal cortex and even to the hippocampal cortex, while the later concept triggered neuroscience studies of the learning and memory in the developing and adult brains. recently, these concepts refreshed the impact with new dressing of 'dynamics'. cell assembly that was originally assumed to be static, became dynamic and opened a new possibility for the neural computation, combined with dynamic hebbian synapses conceptualized as the spike-timing dependent plasticity (stdp). i would like to discuss about speaker's talks in this context. sy - - - tonic gaba a receptor mediated conductances: properties, functions and plasticity alexey semyanov riken brain science institute (bsi), japan communications mediated by non-synaptic receptors are important for information processing in the brain. high affinity extrasynaptic gaba a receptors mediate a persistent "tonic conductance" which reflects their activation by ambient concentrations of gaba. this phenomenon is found in different brain regions, shows cell-type specific differences in magnitude and pharmacology, and changes during brain development. our findings have revealed functional significance of gaba a receptors mediated tonic conductance in the hippocampus. we have shown that it modulates rate-coded information processing by individual neurons, and acts in a cell-type specific manner to regulate the excitability of the local neuronal circuit. the magnitude of the conductance is regulated by efficiency of gaba uptake and membrane potential. gaba a receptor mediated tonic conductance undergoes adaptive plasticity. it is up-regulated in hippocampal pyramidal cells in a model of pilocarpine status epileptics in rats. in mice lacking gad the amount of the tonic inhibition is reduced in ca hippocampal interneurons, while unchanged in pyramidal cells. sy - - - modulatory effects of peri-interneuronal glial cells on neuronal activities in hippocampal ca region yoshihiko yamazaki , yasukazu hozumi , kenya kaneko , satoshi fujii , hiroshi kato dept. of neurophysiol., yamagata univ. sch. of med., yamagata, japan; dept. of anat. & cell biol., yamagata univ. sch. of med., yamagata, japan glial cells, in addition to their supportive roles in the nervous system, make up a functional unit with neurons and have been suggested to play novel roles in neuronal activities. we focused on interneuron/peri-interneuronal glial cell (pg) pairs in the hippocampal ca region and performed dual whole-cell recordings to investigate the modulatory effect of glial cells on neuronal activities. direct depolarization of pg suppressed the excitatory postsynaptic currents in an adjacent interneuron. this suppression was inhibited by adenosine a receptor antagonist. moreover, pg activation modulated the firing pattern of the interneuron. since interneurons in the hippocampus are mainly inhibitory and the terminals of a single interneuron make a large number of synapses on a group of pyramidal cells, direct inhibitory regulation via pg would have marked effects on the information processing of neurons in the ca region. research funds: kakenhi ( ) sy - - - carbachol-induced beta oscillations in rat hippocampal slices kiyohisa natsume, jun arai graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan rat hippocampus has the cholinergic input from medial septum and diagonal band in vivo. the input involves the generation of hippocampal rhythm, theta, gamma rhythm. to mimic the system, we applied carbachol, a cholinergic agent, to rat hippocampal slices. carbachol can induce beta oscillation as well while carbachol can induce theta, and gamma oscillations in the slices. in the present paper, we introduce the beta oscillations. the application of m carbachol induces beta oscillations which occur intermittently with the interval of - s. during the intervals, gamma oscillations are induced. the mean frequency of the oscillations is . ± . hz (mean ± s.e.m.). the oscillations are induced via muscarinic m , , receptors. the frequencies of them are significantly decreased by the application of bicuculline, a gaba a antagonist. they are sensitive to bicuculline, while theta oscillations are not. it is indicated that the character of beta oscillations are different from those of theta oscillations. the neocortex and the hippocampus are connected by way of the entorhinal cortex and the subiculum. to examine ongoing network interactions among these distinct cortices during neocortical slow oscillations ( - hz), we recorded intracellular potentials in single neocortical, entorhinal, subicular, and hippocampal neurons, together with hippocampal field and multi-unit activities in adult anesthetized rats. we have found that ( ) most entorhinal and subicular neurons displayed bimodal active (up) and quiet (down) states of membrane potential, in synchrony with neocortical slow oscillations, ( ) no bimodal up-down transition was present in hippocampal neurons. hippocampal granule cells were directly driven by entorhinal up-state activity, while ca and ca neurons discharged during both up and down states, ( ) gamma and fast (ripple) oscillations were observed in hippocampal ca area irrespective of up-down transition. these observations suggest that entorhinal and subicular regions are "neocortex-like" and hippocampal networks can generate self-organized activity independent of neocortical slow oscillations. the cholinergic neurons in the mesopontine reticular formation (mprf) seem to control sleep-wake cycle and hippocampal activity, because stimulation of the mprf elicits rem sleep and hippocampal theta wave. in this study, we recorded neuronal activity in the mprf and pontine and hippocampal eeg during rem sleep and investigated time-relationship between them. our results are summarized as follows: ( ) most of the mprf neurons were active during rem sleep; ( ) the mprf activity increased over ten seconds before transition from nrem to rem sleep, i.e. from non-theta to theta period; ( ) the theta wave was instantaneously accelerated concomitant with activation of the mprf neurons. these results suggest that cholinergic neuron in the mprf is important in generation and maintenance of rem sleep and theta wave. because hippocampal theta waves are involved in memory consolidation during rem sleep, our findings might help to clarify this mechanism. research funds: kakenhi ( ) sy - - - clock mechanisms of the scn involving in the entrainment to the morning and evening light sato honma, natsuko inagaki, nobuko tokumaru, ken-ichi honma dept. physiology, hokkaido univ. grad. sch. med., sapporo, japan the circadian clock, by entraining to the light-dark cycle of different day length, controls seasonality in biological functions. the mechanism is currently explained by morning and evening oscillators which change their coupling intensity depending on the day-length. by using clock gene expression as a marker of clock functions, we examined the localization and molecular bases of the two oscillators. rats and mice were housed under light-dark (ld) : h and ld : h. clock gene expression patterns in the entire scn were examined by in situ hybridization on the first day of constant darkness. the phase relations of per and per rhythms suggest that light-on resets per rhythms in both light conditions, while per rhythm also relates to the light-off. in cultured scn of transgenic mice expressing luciferase under the control of per promoter, we observed two bioluminescent peaks a day only in the anterior scn from the mice kept in ld : . the finding suggests that two distinct oscillators, which respond to the day-length, reside in the anterior scn. the suprachiasmatic nucleus (scn) is the center of the mammalian circadian clock. tissue transplantation of the scn restores the behavioral circadian rhythm in scn-lesioned mice in spite of the impaired neural connection with the host brain. we have investigated whether grafted scn regulates the circadian oscillator in peripheral organs using the scn-transplanted mice that have a limited time information transmission paths. as a result, the grafted scn restored not only circadian behavior rhythm but also the circadian rhythms of peripheral organs. many of clock genes showed dynamic oscillations with identical phase relationship as shown in intact animals, however, per and per showed low amplitude of oscillation. the findings suggest that diffusible signal molecules released from the transplanted scn entrain the circadian clock in peripheral organs and that they differentially modulate the expression of clock genes. sy - - - genome-wide analysis of adrenal-dependent and independent circadian regulation of mouse hepatic genes norio ishida clock cell biology group, national institute of advanced industrial science and technology (aist), ibaraki, japan recent progress in genome-wide expression analysis has identified hundreds of circadian regulated genes in the suprachiasmatic nucleus as well as in peripheral tissues of mammals. adrenal gland is important for circadian regulation for mammalian peripheral clocks. to identify circadian expressed genes regulated by adrenal glands pathways, we performed dna microarray analysis using hepatic rna from adrenalectomized (adx) and sham-operated mice. we identified genes that fluctuated between day and night in the livers, lost circadian rhythmicity in adx mice. these included the genes for key enzymes of liver metabolic functions such as glucokinase, hmg-coa reductase, and glucose- -phosphatase. the present study showed that the circadian expression of mouse liver genes is governed by core components of the circadian clock such as clock, and the other genes depend on adrenal glands pathway such as glucocorticoids. hitoshi okamura kobe university graduate school of medicine, japan light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. in the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (scn)-sympathetic nervous system. moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamoadenohypophysial axis. the abolishment after scn-lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. the surge of plasma corticosterone after light exposure indicates that environmental light signals are instantly converted to glucocorticoid signals in the blood and csf. the light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment. sy - - - neuronal and hormonal control of peripheral clock function through suprachiasmatic nucleus shigenobu shibata, naomi hayasaka, takashi kudo, tsuyoshi yaita department of pharmacology, school of science and engineering, waseda university, tokyo, japan the clock genes are expressed not only in the suprachiasmatic nucleus (scn) of the hypothalamus where the master clock exists, but also in other brain regions and various peripheral tissues. in the liver and lung, clock genes are abundantly expressed and show clear circadian rhythm. although oscillation of clock genes in the liver and lung is controlled under the circadian clock mechanism in the scn, we do not know the resetting signals on peripheral clock function. communication between the scn and peripheral tissues occurs through various systems involving the sympathetic, nicotinic and glucocorticoide functions. this symposium mainly describes both anatomical and physiological experiments to reveal the sympathetic and glucocorticoid control over peripheral clock function. sy - - - a to z of gene transfer with adenoviral vector-application to neuronal birth date-specific gene transfer using replication-defective adenoviral vectors, we successfully performed 'pulse gene transfer' into progenitor cells in a neuronal birth date-specific manner. when adenoviral vectors were injected into the midbrain ventricle of mouse embryos between embryonic days (e) . to e . , the adenoviral vectors introduced a foreign gene into a specific cohort of birth date-related progenitor cells. this technique allows us to distinguish a cohort of birth date-related progenitor cells from other progenitor cells with different birth dates and to introduce a foreign gene into specific subsets of neurons by performing adenoviral injection at specific times. this adenovirus-meditated gene transfer technique will enable us to examine the properties of each subset of progenitor cells that share the same neuronal birth date. i will explain directions how to use an adenoviral vector and application of an adenoviral vector in my talk. research funds: crest sy - - - live imaging in the specific neuronal cells by the combination of transgenic mice and viral vectors kaori kashiwagi, naoaki saito lab. mol. pharmacol. biosig. res. ctr, kobe univ, kobe, japan live imaging analysis has revealed that each protein kinase c (pkc) subtype shows spatio-temporally distinct targeting in response to various stimuli. we demonstrated that the trans-synaptic stimulation induced translocation of ␥pkc-gfp in cerebellar slices from bitransgenic mice (nse-tta/tetop-␥pkc-gfp) which express ␥pkc-gfp in time and region-specific manner. this translocation was not restricted, but propagated from the distal to the proximal dendrites close to the soma of purkinje cells. in order to gain further insight in to the molecular mechanisms of pkc translocation, we introduced viral vectors to primary cultured purkinje cells. the propagative ␥pkc-gfp translocation was also observed in cultured purkinje cells derived from nse-tta mice. the molecular mechanisms of pkc translocation in purkinje cells were analyzed by live imaging with various kinds of viral vectors. the combination of tg mice and viral vectors is useful to understand the physiological role of pkc in the specific neuronal cells. research funds: kakenhi ( ) sy - - - visualization and manipulation of the signaling systems in the cns using sindbis viral vectors sho kakizawa dept. of pharmacol., grad. sch. of med., the university of tokyo, tokyo, japan virus vectors can efficiently deliver genes to neurons and other cells in the nervous systems in vitro and in vivo. because many viral vectors are in common use, it is important to select the best viral vector for each specific application, and a number of factors must be considered when making a decision. sindbis virus is an enveloped plus-strand rna virus belonging to the alphavirus genus of the togaviridae family. the sindbis viral vector is characterized by its effective, rapid, high-level and preferential transduction of neurons. these facts indicate that the vector is a powerful tool for the robust expression of target genes in the specific population of neurons. in this symposium, we will introduce our recent topics on the synaptic functions in the cerebellar systems revealed by visualization and manipulation of signaling molecules, such as nitric oxide and inositol , , -trisphosphate, in the cerebellar purkinje cells. research funds: grant-in-aid for scientific research on priority areas-molecular brain science-from the ministry of education, culture, sports, science and technology of japan sy - - - rescue of phenotypes of null-mutant mice by virus vector-mediated gene transfer kazuhisa kohda, wataru kakegawa, kyoichi emi, michisuke yuzaki dept. of physiol., keio univ. sch. of med., tokyo, japan even after the completion of genome project in major species, functions of many molecules remain uncharacterized. a transgene-based rescue approach is one of the powerful methods to decipher the mechanisms of actions of an orphan receptor; however, it is quite labor intensive and time consuming. here, we have developed a virus vector-based rescue approach and applied to investigate the mechanisms of action of the orphan glutamate receptor ␦ (glur␦ ) in the cerebellum. by introducing a sindbis virus carrying a wild-type glur␦ into glur␦ -null cerebellum in vivo, we could rescue abnormal phenotypes, such as impaired long-term depression at parallel fiber-purkinje cell synapses. by examining whether a mutant glur␦ lacking a specific domain could similarly rescue the phenotypes, we could evaluate functional importance of the domain. alternatively, by introducing a partial sequence of the gene of interest into wild-type brain and examining its dominant-negative effect, we will be able to identify the region of the gene product that is functionally important. research funds: kakenhi sy - - - gene transfer into in vivo cerebellar purkinje cells by hiv-derived lentiviral vectors hirokazu hirai advanced science research center, kanazawa university, ishikawa, japan cerebellar purkinje cells are key elements regulating motor coordination and motor learning. gene transfer into purkinje cells is an effective approach for the study of cerebellar function and treatment against cerebellar disorders. although adenoviral vectors or sindbis vectors are frequently used for gene delivery into neurons, the former has extremely low affinity for purkinje cells, while the latter causes substantial damage to the infected cells. to achieve effective gene transfer into purkinje cells, we used human immunodeficiency virus (hiv)-derived lentiviral vectors. purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. gene expression was also detected, though less efficiently, in other cortical cells, whereas no transduced cells were observed outside of the cerebellar cortex. these results suggest that hiv-derived lentiviral vectors are useful for the study of gene function in purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect purkinje cells. research funds: jst/presto, kakenhi ( ) sy - - - physiological basis for stereotaxic surgery in basal ganglia atsushi nambu division of system neurophysiology, national institute for physiological sciences, and school of life science, the graduate university for advanced studies, okazaki, japan stereotaxic surgery in movement disorders such as parkinson's disease dramatically improves the symptoms of such diseases. i assume the physiological basis for the treatment is to disrupt abnormally increased information flow through the basal ganglia. i will discuss the pathophysiology of basal ganglia disorders and the effect of stereotaxic surgery in light of the three major pathways in the cortico-basal ganglia loop, i.e., hyperdirect (cortico-stn-gpi), direct (cortico-striato-gpi) and indirect (cortico-striato-gpe-stn-gpi) pathways, that dynamically control the activity of the thalamus and cortex to perform correct motor programs in correct timing. research funds: kakenhi ( ) sy - - - deep brain stimulation of subthalamic nucleus on parkinson's disease fusako yokochi department of neurology, tokyo metropolitan neurological hospital, tokyo, japan parkinson's disease is a progressive and degenerative disease caused by dopamine deficiency attributed to the degeneration of neurons in the substansia nigra. consequently, various symptoms appear, such as cardinal symptoms, those in the advanced stage and those as the side effects of long-term levodopa therapy. many antiparkinsonian drugs have been developed, but all of the symptoms are not improved by these drugs. stereotaxic surgery was started for treating severe tremor and rigidity in the mid- th century. stimulation by chronically implanted electrodes in the brain, that is, deep brain stimulation (dbs), has recently been applied and has been shown to have marked effects on the symptoms resisted to conventional treatments. in particular, dbs of the subthalamic nucleus (stn) is an effective treatment for the symptoms. much basic research on the function of stn has been reported, but stn function is still unclear. clinical outcomes including the side effects of stn dbs, the neural activities recorded from stn and the localization of clinical effects are reported in this paper. sy - - - firing patterns of basal ganglia neurons and effects of deep brain stimulation in parkinson's disease takao hashimoto center for neurological diseases, aizawa hospital, matsumoto, japan high-frequency electrical stimulation of the subthalamic nucleus (stn), internal segment of the globus pallidus (gpi) and thalamus can improve motor signs in patients with parkinson's disease, however, its mechanism of action remains unclear. a leading hypothesis regarding the development of movement disorders of basal ganglia origin suggests that hyperkinetic and hypokinetic disorders occur as a result of changes in the mean discharge rate in the gpi and substantia nigra, which in turn suppress thalamocortical output. on the other hand, altered firing patterns in the basal ganglia have been reported in mptp-induced parkinsonian animals: increases in bursting activity and periodic oscillatory activity in the gpi and stn, and synchronization of gpi, or gpi and striatal neurons. synchronous oscillation in the basal ganglia may break down independent processing in the motor circuit and disrupt signal processing at the cortical level. kaoru takakusaki department of physiology, asahikawa medical college, asahikawa, japan locomotion is composed of volitional and automatic processes. particular attention is required to perform volitional processes such as avoiding obstacles and accurate limb placements during locomotion. however, we are largely unaware of the automatic control processes of rhythmic limb movements, muscle tone and postural reflexes that accompany locomotion. because each process is seriously impaired in parkinsonian patients, the basal ganglia must play a crucial role in integrating the volitional and automatic processes of locomotion. the basal ganglia contribute to the planning and execution of voluntary movements via basal ganglia thalamocortical loops. on the other hand, recent our findings suggest the importance of the direct basal ganglia outflow to the brainstem where fundamental neuronal networks for controlling postural muscle tone and locomotion are located. in this presentation we discuss the role of the basal ganglia in the integration of volitional and automatic movements during locomotion, which has been less understood aspect of gait control. research funds: grant-in-aid for scientific research (c) and priority area (area no. ) sy - - - characteristics of neuronal activity within the globus pallidus interna (gpi) in patients with dystonia yoichi katayama , department of neurological surgery, nihon university school of medicine, tokyo, japan; division of applied system neuroscience, nihon university graduate school of medical science, tokyo, japan dystonia represents disordered muscular tonicity of the trunk and/or extremities, and is often dramatically controlled by chronic gpistimulation in humans, indicating that dystonia is attributable to certain abnormal activity of gpi neurons. little is yet known, however, regarding characteristics of neuronal activity within the gpi underlying dystonia. we analyzed activity of gpi-neurons in patients with dystonia or parkinson's disease, which were recorded during surgery for chronic gpi-stimulation. as compared to gpi neurons in patients with parkinson's disease, gpi neurons in patients with dystonia were distinctive with following three characteristics; firing rate ( . ± . hz, n = ) was low, firing pattern was often composed of irregular pauses and bursts, and many were responsive to body movement with wide receptive fields. these findings suggest that dystonia may be related to unstable movement-related sensory processing within the gpi. it has been considered that dystonia, which is generally characterized by postural abnormalities and involuntary movements including torsion, is caused by dysfunction of the basal ganglia. the purpose of the present work is to clarify the neural mechanisms underlying the onset of dystonia by analyzing the pathophysiology of a model for torsion dystonia, wriggle mouse sagami (wms). the genomic mutation of wms occurs in the gene encoding plasma membrane ca + -atpase isoform that is located on the th chromosome. recent immunohistochemical and electrophysiological investigations on wms have shown that ( ) d -type dopamine receptors are downregulated presynaptically in the striatum, and ( ) a large number of purkinje cells in the cerebellum express tyrosine hydroxylase (the synthesizing enzyme for dopamine) and their excitability is greatly reduced. in this symposium, the possible correlation between these data and dystonic phenotypes will be discussed. kei-ichiro maeda reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan gnrh has been well established to regulate reproduction through two modes of secretion: surge and pulse. the surge mode is female-specific and induces lh surge and then ovulation through positive feedback action of estrogen. the pulse mode tonically activates gonads in both sexes with being negatively regulated by gonadal steroids. environmental cues, such as photoperiod or nutrition, are considered to affect reproductive activity through altering pulse mode of gnrh release. pulse mode seems much more robust than surge, because estrogen can induce a surge under a certain condition when the pulse is suppressed. the following four papers aim to unveil the physiological mechanism underlying two modes of gnrh secretion in various experimental models. sy - - - metastin: a neuropeptide playing a central role in the regulation of ovulatory cycle hiroko tsukamura, kei-ichiro maeda graduate school of bioagricultural sciences, nagoya university, japan estrous cyclicity is regulated by a sequence of neuroendocrine events consisting of hypothalamus-pituitary-gonadal axis. gonadotropinreleasing hormone (gnrh)/luteinizing hormone (lh) surge is induced by positive feedback action of estrogen secreted by mature ovarian follicles. the central mechanism of positive feedback action of estrogen on gnrh/lh secretion, however, is not fully understood yet. metastin was first isolated as a natural ligand for a g-proteincoupled receptor, gpr ( . nature , ) . recent studies reported that a genetic alteration leading to homozygous loss of function of gpr impairs pubertal development in mice and human. we have first demonstrated that endogenous metastin plays a physiological role in inducing ovulation through stimulating gnrh/lh surges to control estrous cyclicity in the female rat ( . endocrinology , ) . the present paper focuses on the role of metastin in regulating gnrh/lh surge based on our recent study in rats and discusses possible mechanism underlying positive feedback action of estrogen. suzanne moenter, catherine christian university of virginia, usa a surge in gonadotropin-releasing hormone (gnrh) secretion is the cns signal that triggers the luteinizing hormone (lh) surge, which causes ovulation. the gnrh surge depends on a switch in estradiol (e) feedback from negative to positive and in rodents on time of day, occurring in the pm. treating ovariectomized (ovx) mice with a constant release e capsule (ovx+e) elicits daily pm lh surges; there is no diurnal change in ovx controls. likewise, extracellular recordings of firing activity of gfp-identified gnrh neurons showed no diurnal changes in cells from ovx mice. in contrast, e increased firing in the pm compared to am. gabaergic neurons form a major input to gnrh neurons, and activation of the gabaa receptor can be excitatory in these cells. whole-cell patch-clamp recordings of synaptic activation of gabaa receptors on gnrh neurons revealed e-dependent decreases in transmission during the am (negative feedback) and increases in transmission near surge onset that persisted for some populations of afferents thru the surge peak. together these data indicate one mechanism by which e induces the gnrh surge is by altering gaba transmission to gnrh neurons. yoshitaka oka grad. sch. sci., univ. of tokyo, tokyo, japan the gonadotropin-releasing hormone (gnrh) peptidergic neuronal systems consist of hypothalamic neuroendocrine and extrahypothalamic neuromodulatory gnrh neurons. here, i introduce our recent studies on the physiological properties of neuroendocrine preoptic (poa) gnrh neurons in comparison with the neuromodulatory terminal nerve (tn) gnrh neurons. to study the both types of gnrh neurons, we use a fish model system in which we can easily identify both of them in intact brain preparations in vitro, which is a great advantage over most other vertebrates. the poa-gnrh neurons had quite different basic electrophysiological membrane properties from those of tn-gnrh neurons and showed alternating active and silent phases of firing activities, in contrast to the regular pacemaker activities of tn-gnrh neurons. now that we have various experimental approaches (electrochemical measurement of gnrh release, ca + imaging after single-cell electroporation, single-cell rt-pcr, double patch clamp recording, etc.) in hand, simultaneous multidisciplinary approaches should be possible to study the physiology of gnrh neurons. research funds: kakenhi sy - - - metabolic regulation of puberty onset using the prepubertal rat model helen i'anson biology department and neuroscience, washington and lee university, usa we hypothesize that glucose availability determines the timing of puberty onset in rats. abdominal fat stores are low in dieted, prepubertal female rats with delayed puberty, suggesting that such rats may be particularly sensitive to dietary fuel changes. such rats are fed a single daily meal and demonstrate decreased blood glucose levels between meals. we hypothesized that such daily hypoglycemic bouts delay onset of puberty during dieting. when glucose supplement was given to prepubertal dieted rats, and they exhibited first estrus with similar timing to previously dieted re-fed rats. conversely, when dieted rats were re-fed ad libitum and simultaneously glucose-deprived, first estrus was delayed. blood glucose levels during glucose-supplementation which induced first estrus, and during glucoprivation and re-feeding which delayed first estrus, were similarly elevated, suggesting that central, rather than peripheral, glucose levels are monitored in the prepubertal animal. in summary, central glucose availability may be an important signal timing puberty onset. research funds: jeffress memorial trust and washington and lee university sy - - - molecular mechanisms of thyroid hormone action in developing brain toshiharu iwasaki, noriyuki koibuchi department of integrative physiology, gunma university graduate school of medicine, maebashi, japan thyroid hormone (th) plays an important role in the developing brain. the action is mainly exerted by controlling gene expression through binding to its specific receptor, th receptors (trs). trs are ligand-regulated transcription factors that are expressed in many organs, including brain. trs bind to target dna sequences known as th-response element (tre). coactivators and corepressors are involved in the tr-mediated gene regulation through histone modification. we characterized the coactivator and the corepressor that were expressed in embryo brain. although precise mechanism of the th action for brain development has not been fully clarified, these cofactors may be involved in these actions. th affects brain development only during a limited period, which is referred as the critical period of th action. recently, it has been speculated that environmental chemicals may cause the abnormal brain development. possible mechanism of action of such chemicals on tr system will be discussed. research funds: kakenhi , sy - - - thyroid hormone and organic anion transporters in brain hiroyuki kusuhara, yuichi sugiyama graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan organic anion transporting polypeptides (oatps/slco) currently consist of isoforms in human and rodents. they are initially identified as part of detoxification system in the body, and involved in the tissue uptake of xenobiotics, especially amphipathic organic anions. some members accept a variety of structurally unrelated compounds as substrate. oatp c is characterized by its unique substrate specificity, highly selective for thyroid hormones, particularly for t and reverse t , but the transport activity of t is quite low. it is predominantly expressed in the brain capillaries and choroid plexus, acting as barrier of central nervous system, where oatp a , the transport activities of t and t are similar, is also expressed. the uptake of t and t by the brain determined using in situ brain perfusion technique was saturable and inhibited by oatps substrates and inhibitors. these two transporters may play a role in regulation of brain levels of t and t . research funds: the advanced and innovational research program in life sciences from the ministry of education, culture, science and technology sy - - - alterations of gene expression profiles in the developing brain by chemicals disrupting thyroid hormonedependent signals takayuki negishi , masaki takahashi , yasuhiro yoshikawa , tomoko tashiro department of chemistry and biological science, aoyama gakuin university, kanagawa, japan; department of biomedical science, the university of tokyo, tokyo, japan there is increasing concern about the possibility that environmental chemicals such as polychlorinated biphenyl (pcb) and its hydroxylated metabolites interfere with normal brain development through acting as thyroid hormone disrupting agents. in this presentation, based on comprehensive dna microarray analysis, we demonstrate alterations in gene expression in the brain of neonatal rats perinatally exposed to -hydroxy- , , , , pentachlorobiphenyl (anti-thyroid hormone-like), as well as in primary cultured rat hippocampal neurons exposed to -hydroxy- , , , , , , -heptachlorobiphenyl (thyroid hormone-like). among genes whose mrna expression levels were affected by these compounds, a number of genes essential for the establishment of synaptic networks were detected, suggesting that long-lasting effects on higher brain functions may result from exposure of the developing brain to these compounds. hydroxylated metabolites of pcbs (oh-pcbs) have chemical structures similar to thyroid hormones (ths). we reported that low doses of two types of oh-pcb inhibited th-dependent extension of purkinje cell dendrites ( . dev. brain res. , ) . koibuchi et al. ( ) clarified that they interfere with th-dependent gene expressions in reporter gene assays. further, takasuga et al. ( ) detected some pcb and oh-pcb congeners in human csf, of which oh-cb is the highest concentration. to determine its effects on developing neurons, we examined oh-cb in rodent cerebellar culture cells. interestingly, oh-cb promoted dendritic development of purkinje cells in the absence of th and increased significantly their survival numbers. these results indicate that oh-pcb congeners may disrupt normal brain development by different mechanisms depending on their chemical structures. we have reported that rat pups exposed to an antithyroid agent, propylthiouraci l (ptu), via maternal milk exhibit hyperactivity, impairment in spatial learning and social interaction, and audiogenic hypersensitivity extending to audiogenic seizures, thus this ptu rat can be a possible candidate of animal model for autism. in ptu rats, the delay in migration of the extragranular cells of cerebellum, and in innervation from inferior olive nuclei to purkinje cells were shown. these neurons transiently expressed serotonin-ir, therefore we treated ssri or -htp to examine the relevance of serotoninergic function. the treatment of -htp but not ssri recovered the delay of cell migration. these effects of serotonin manipulation in ptu rats on the behavioral impairment and the development of cns will be discussed. it is hoped that embryonic stem (es) cells will be used in transplantation therapy for neurological diseases. however, because grafts of neural stem cells derived from es cells may contain residual undifferentiated cells, there would be a risk for teratomas. to reduce this risk, we applied to es cells herpes simplex virus thymidine kinase (hsv-tk) gene and ganciclovir (gcv) treatment. stable mouse and cynomolgus monkey es cell lines expressing hsv-tk were obtained. gcv sensitivity was higher in undifferentiated es cells than in es cell-derived neural stem cells. es cell-derived neurons were resistant to gcv treatment. nude mice with transplants of undifferentiated es cells expressing hsv-tk formed teratomas, but the tumor growth was suppressed after the gcv treatment. suicide gene delivery might increase the safety of the use of es cells in cell replacement therapy. enzymatic degradation of chondroitin sulfate is known to promote axonal regeneration in the central nervous system. the physiological role of chondroitin sulfate up-regulated after injury was examined in the nigrostriatal dopaminergic system which was unilaterally transected and treated with chondroitinase abc. in transected mice, dopaminergic axons did not extend across the lesion. chondroitin sulfate was up-regulated around the lesion and a fibrotic scar containing type iv collagen deposits were developed in the lesion center. in chondroitinase abc-treated mice, numerous dopaminergic axons were regenerated across the lesion. in these animals, chondroitin sulfate immunoreactivity was remarkably decreased and the formation of a fibrotic scar was unexpectedly prevented. these results support our previous supposition that chondroitin sulfate does not act as an obstacle to regenerating axons, but involved in the repair process of the brain injury including the formation of the fibrotic scar (kawano et al., ) . reference kawano et al., . j. neurosci. res. , - . research funds: kakenhi os a- - neurotransmitters that maintain and suppress the tonic firing of the serotonergic neurons in the dorsal raphe during sleep waking cycles yoshimasa koyama , kazumi takahashi , yukihiko kayama cluster of science and technology, fukushima university, fukushima, japan; department of physiology, fukushima medical university, fukushima, japan the present experiment was done to examine, under unanesthetized natural sleep-waking condition, which neural systems were involved to regulate the firing of the serotonergic ( ht) neurons in the dorsal raphe (dr) during sleep waking cycles. using head restrained, unanesthetized rats, single neuronal activity was recorded and each drug was applied iontophoretically or by pressure close to the recording neurons. spontaneous firing of the ht neurons in dr were excited by glutamate and orexin a or b. they were inhibited by noradrenaline. an ␣ receptor agonist (phenylephrine or methoxamine) increased the firing rate during sws or ps, but had no effect when applied during w. in ps-off type dr neurons, cessation of firing during ps was recovered by bicuculline, however in the dr neurons that did not stop firing during ps, bicuculline had almost no effect. os a- - correlation between regional grey matter volume and proficiency increase in second language: a vbm study arihito nauchi , , kazuyoshi hirano , , yukimasa muraishi , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, japan; secondary education school, university of tokyo, japan although neuroimaging studies have contributed to clarify the brain function, the neural basis of individual variation in cognitive abilities such as language still remains unknown. in the present study using voxel-based morphometry (vbm), a whole-brain unbiased technique for detecting regional differences in mr images, we examined the relationship between the proficiency increase in second language (l ) and grey matter volume among students aged - , who received special classroom training in the use of english verbs. in specific regions including the left lateral premotor cortex and the left inferior frontal gyrus (the grammar center), we found a positive correlation between the regional grey matter volume and improved performance for the grammaticality of english sentences. these results suggest an anatomical basis for the language faculty, such that the capacity of a specific region is related to proficiency increases in l . os a- - grammar center activation in honorification judgment of japanese sentences kanako momo , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, japan one linguistic theory proposes that japanese honorification is a syntactic feature, because syntactic agreement is required between subject/object and honorific forms. to investigate whether such syntactic processing is actually realized in the brain, we examined cortical activation using fmri under several types of normal/anomalous judgment for japanese sentences including honorification. when activation in a honorification task was contrasted with that in a spelling task, we observed significant increase in the left including grammar center (ifg) as well as the bilateral cerebellum for all tested participants. moreover, the lower performance group showed greater activation in the left f op/f t and the bilateral cerebellum. these results suggest that syntactic process is required for japanese honorification and that activation in these regions shows modulation according to performance level, even in native language. research funds: crest, jst os a- - top-down modulation for melody-related activity in the right auditory areas: an meg study takuya yasui , , , kimitaka kaga , kuniyoshi l. sakai , dept. of basic science, univ. of tokyo, tokyo, japan; dept. of otolaryngology, univ. of tokyo school of medicine, japan; crest, jst, japan we previously reported right-hemisphere dominance for melody error-induced fields (m ) (neurosci. res. , s ). in a subsequent study, we confirmed that m was independent from mismatch negativity usually induced by oddballs. in the present study, we examined whether m was induced by deviation from a memorized melody. we used four pairs of unfamiliar songs, each pair consisting of an original song and a modified song in which the third note deviated from that of the original. subjects learned these songs and judged whether there were one or two deviations in notes. there was no significant difference in dipole amplitudes between m elicited by the original songs and that by the modified ones. however, while m without the deviation showed no significant effect of lateralization, m with the deviation resulted in significant enhancement in the right hemisphere. these results suggest the existence of memory-induced, i.e., top-down modulation for melody-related activity in the right auditory areas. research funds: crest, jst os a- - cortical plasticity in adulthood for learning phonics rules for english orthography and phonology makiko muto , , kuniyoshi l. sakai , department of basic science, university of tokyo, tokyo, japan; crest, jst, tokyo, japan although matching english orthography with correct pronunciation is difficult for second language learners, learning phonics rules may rapidly improve their performance. in the present fmri study, we tested an english matching task during the course of phonics training in sessions, in which infrequent words were visually shown, while matched/unmatched speech sounds were simultaneously presented. comparing the first half of the sessions with the latter half, the left posterior inferior temporal gyrus (the letter center) and a part of the left lateral premotor cortex (the grammar center) showed activation decreases, when the performance was significantly improved. these results suggest that the plasticity of functional systems involving these critical regions is essential for establishing phonics rules and for forming a new link between orthography and phonology. research funds: crest, jst os a- - hierarchical syntactic processing in the left frontal region: an meg study kazuki iijima , , naoki fukui , kuniyoshi l. sakai , dept. of basic science, univ. of tokyo, komaba, japan; crest, jst; dept. of linguistics, sophia univ., yotsuya, japan previous erp studies have shown word-related activation based on semantic association or context. however, it remains unclear how syntactic information of preceding words is integrated into the ongoing sentence processing. in the present meg study, we measured brain activity during each of four tasks: a syntactic task, a semantic task, a memory task, and an evaluation task. sentence stimuli consisted of one noun phrase and one verb, where the noun phrase had either an objective or nominative case particle. the first peak of the activity for a verb presentation was observed at the left frontal region as early as ms after the onset. in the objective-case condition, this activity was enhanced only for the syntactic task, while in the nominative-case condition no such task-selectivity was observed. these results are consistent with the current linguistic theory (the minimalist program), which holds that a noun phrase with an objective case particle is directly merged with a verb, to form a new hierarchical level. research funds: crest, jst os a- - individual difference of brain activity in medial prefrontal cortex and superior temporal sulcus during social cognition koji jimura, seiki konishi, tomoki asari, junichi chikazoe, yasushi miyashita dept. physiol. univ. tokyo sch. med., japan previous neuroimaging studies have reported brain activity in the medial prefrontal cortex (mpfc) and the superior temporal sulcus (sts) during performance of theory of mind tasks. the present fmri study explored individual difference of the mpfc and sts activity by employing false belief paradigms. the task consists of two sessions, study and test. during the study session, subject studied a brief story in which two characters have false beliefs. then, the subject answered questions about the false belief and the fact that constitutes the false belief during the test session. consistent with previous studies, significant activity was observed in the mpfc and the sts during representing the false belief. the individual differences of the mpfc and the sts activity were correlated with psychodiagnostic indices that represent controlled and automatic idealization, respectively. these results suggest that the two indices represent distinct neural mechanisms participating in social cognition. research funds: grant-in-aid from mext ( ), jsps research fellowship ( ) os a- - brain activity of happy facial recognition in mother-daughter relationship jun shinozaki , nobukatsu sawamoto , toshiya murai , takashi hanakawa , hidenao fukuyama hbrc, kyoto univ. grad. sch. of med. kyoto, japan; neuropsychiatry, kyoto univ. grad. sch. of med. kyoto, japan relationship between parents and children is special, and affective facial recognition between them should evoke specific neural activity not shared by other personal relationships. eleven healthy females participated in this fmri experiment. the subjects saw happy and neutral faces of their own mothers, and newly learned other subjects' mothers during the scan. when happy face recognition was compared with neutral face recognition, the mother-daughter combination induced greater activity than the non-familial combination in the following areas; the lateral prefrontal cortex, anterior cingulate cortex, middle temporal cortex, striatum, and anterior insula. it has been shown that the lateral prefrontal and anterior cingulate cortices are associated with familial facial recognition, whereas the middle temporal cortex is related to happy facial recognition. the activity in the striatum and anterior insula might be related to positive affection and empathy, respectively. os a- - anatomical connections among functionally identified brain regions for sentence processing yukari yamamoto , , atsushi maki , , kuniyoshi l. sakai , advanced research laboratory, hitachi, ltd., tokyo, japan; crest, japan science and technology agency, saitama, japan; dept. of basic science, univ. of tokyo, tokyo, japan we have functionally identified the left dorsal inferior frontal gyrus (ifg), the left lateral premotor cortex, and the triangular/orbital part of the left ifg (f t/f o) as regions associated with sentence and discourse level processing. in the present study, we examined whether there are direct anatomical connections among these regions by using diffusion tensor tractography. f t and f o of the left ifg were chosen as seed areas for fiber tracking. fiber bundles that went through two spherical regions were extracted from the tracking data. the central coordinates of these regions were (− , , ) , (− , , ) , and (− , , − ) in the standard brain, which are associated with syntactic processing (the first and second coordinates) or sentence comprehension (the third coordinate). direct connections among these regions were consistently observed among the subjects. this result suggests a critical network among multiple regions that are associated with sentence processing. os a- - effect of the incongruity controlled by semantic distance on visually evoked magnetic fields nobuyoshi harada , sunao iwaki , mitsuo tonoike aist, osaka, japan; chiba university, chiba, japan visual incongruities of heads changed on animal pictures, which were controlled by the semantic distance of the word of the animal, were investigated on visually evoked magnetic fields. the semantic distance was decided by the numbers of links of the semantic network of the taxonomic layer in a japanese thesaurus. the words for mammalians were grouped into five semantic categories in the thesaurus. the heads of the animals were changed with those from another semantic category (deviant, d = ), and with those from an inner semantic category (middle, d = ), while others were not changed (normal, d = ). peak amplitudes of waveforms of the root mean square values on the components of ms (f ( / ) = . , p = . ) and ms (f ( / ) = . , p = . ) were significantly decreased with increments of the semantic distance in left occipital sensors. the gradient of the decreasing line of the amplitudes of the and ms components indicated the capability of extracting the structure of a typical prototype of the form of the animal. we call this capability, the structural sensitivity for prototype (ssp). ryohei yasuda duke university medical center, usa calcium signaling in dendritic spines is important for many forms of synaptic plasticity. however, the quantitative mechanisms of how calcium elevations are translated into spatial and temporal patterns of biochemical reactions leading to modifications of synaptic strength are unclear. identifying and following the spatiotemporal activation of molecules necessary for synaptic plasticity is crucial for a better understanding of this complex process. to visualize the activity of signaling pathways in neurons deep in brain tissues, we have combined fluorescence lifetime measurements and two-photon microscopy. this technique allowed us to measure spatiotemporal aspects of the activity of signaling proteins including ras gtpase proteins in response to physiologically relevant stimuli with single spine resolution. research funds: burroughs wellcome fund, dana foundation os p- - mechanisms of p y purinoceptor-mediated long-term enhancement of inhibitory transmission examined by multiple-probability fluctuation analysis at cerebellar gabaergic synapses yumie ono , xiaoming zhu , takashi tominaga , fumihito saitow , shiro konishi , waseda-olympus bioscience research institute, singapore, singapore; department of neurophysiology, tokushima bunri university, kagawa, japan; department of pharmacology, nippon medical school, tokyo, japan postsynaptic p y receptor activation by atp enhances ipscs at cerebellar interneuron-purkinje cell (pc) synapses. to investigate the underlying mechanisms, we here employed the non-stationary fluctuation analysis to estimate the number (n) and single channel conductance (i) of gaba a receptors in pcs using whole-cell recordings of evoked ipscs in pcs of rat cerebellar slices before and - min after application of atp. the atp-induced enhancement of the ipsc amplitude was associated with a significant increase in the single channel conductance, but not the number, of gaba a receptors in pcs: i and n after atp treatment were ± . % and ± . % of the controls, respectively. pretreatment with the protein kinase a inhibitor h- , but not the calmodulin kinase ii inhibitor kn- , completely abolished the atp-induced ipsc enhancement. os p- - activin induces long-lasting nmda receptor activation via scaffolding pdz protein arip isao inoue, akira kurisaki, hiromu sugino institute for enzyme research, tokushima university, tokushima, japan calcium entry into the postsynaptic neuron through nmda type glutamate receptors (nmdars) triggers the induction of long-term potentiation (ltp). the ca + permeability of nmdar is regulated by phosphorylation of its tyrosine residues. we report here that activin, a member of the transforming growth factor-b (tgf-b) superfamily, and one of proteins synthesised after ltp, promotes phosphorylation of nmdars and increases the ca + influx through those receptors in primary cultured rat hippocampal neurons. this signal transduction occurs in a functional complex of activin receptors, nmdars, and src family tyrosine kinase, fyn formed on a multimer of postsynaptic scaffolding pdz protein, activin receptor interacting protein (arip ). activin-induced nmdar activation persists more than h, which is complimentary to the transient activation of nmdars by brain derived neurotropic factor (bdnf). our results show that activin is a long-lasting potentiator involved in synaptic plasticity regulatory mechanisms. os p- - roles of cam kinase i in the hippocampal longterm potentiation kohji fukunaga, takashi komori, shigeki moriguchi department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan cam kinase i (camki) family members are highly expressed in the adult rat hippocampus and camki-alpha is predominantly localized in the cytosol. camki activation requires phosphorylation of thr by camkk as an upstream kinase. we here documented a marked increase in camki-alpha-thr phosphorylation following ltp induction in rat hippocampal ca region. like camkii activation following ltp (fukunaga et al., ) , the increased camki-thr phosphorylation remained elevated at least for min after ltp induction. the increased camki-thr phosphorylation was closely associated with prolonged increases in phosphorylation of creb and myosin light chains in the ca region. this is in contrast with transient increases in camkiv and erk phosphorylation. treatment with camkk inhibitor, sto- significantly inhibited both creb and mlc phosphorylation with concomitant reduction of ltp in the ca region. taken together, camki likely mediates the late phase of creb phosphorylation and an increased mlc phosphorylation in the hippocampal ltp. to investigate how the excitatory postsynaptic inputs of the proximal dendrite effect the information processing of synaptic inputs at the distal dendrite, stimulation was applied to induce bap and epsp at the alveus and the proximal dendrite, respectively. the resulting coincidence of magnitude of bap and epsp at the distal dendrite was enhanced when the bap was delivered at a timing ( ms) to induce ltp. furthermore, the magnitude of bap at the distal dendrite was attenuated by the input from the proximal dendrite at a timing ( ms) to induce ltd. these results suggest that the magnitude of bap delivered to the distal dendrite can be amplified or attenuated depending on the relative timing between proximal input and bap. this may be due to an effect on the coding process at the distal dendrite and could support the basis for a novel learning rule in the brain. research funds: kakenhi ( ) os p- - mouse brains deficient in neuronal pdgf receptor-␤ develop normally but are vulnerable to injury yoko ishii, takeshi oya, lianshun zheng, masakiyo sasahara department of pathology, faculty of medicine, university of toyama, japan the platelet-derived growth factors (pdgfs) and pdgf receptors (pdgfrs) are widely expressed in the mammalian cns. here, we developed novel mutant mice in which pdgfr-␤ subunit gene was genetically deleted in the neurons of cns to elucidate the role of pdgfr-␤. our mutant mice reached adulthood without apparent anatomical defects. the cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with the controls. furthermore, this exacerbated lesion formation was suggested to be, at least partly, due to the enhanced excitotoxicity after injury, because nmda-induced lesion formation was also extensively enhanced in the cerebral cortex of the mutants without altered nmda receptor expression. this is the first known report to address the postnatal function of pdgfr-␤ expressed in cns neurons, using genetically engineered mutant. it was clearly demonstrated that pdgfr-␤ expressed in neuron protects cns neurons from cryogenic injury and nmda-induced excitotoxicity. early postnatal days (especially the first three weeks in the rat) are the critical period for newborn hippocampal granule cells (gcs) to dynamically migrate from the dentate hilus and form the gc layer. to investigate the mechanism that regulates newborn gc migration, we developed a new slice coculture system. the hilar parts of entorhino-hippocampal slices prepared from postnatal six-day-old (p ) rats that had received a single brdu injection at p were substituted with the corresponding region of entorhino-hippocampal slices from p rats. after five days in vitro, newborn gcs, detected by brdu and prox , migrated out of the hilar graft and reached the host gc layer. chronic application of picrotoxin, a gaba a receptor antagonist, facilitated the migration of newborn gcs into the gc layer. these results indicate that gaba a receptors regulate the migration of newborn gcs in early postnatal days. os p- - cdk is required for neuroblast migration in the adult mouse brain yuki hirota , , , toshio ohshima , takuji iwasato , ashok b. kulkarni , hideyuki okano , , kazunobu sawamoto , , bridgestone lab. keio univ., tokyo, japan; dept. physiol., keio univ., tokyo, japan; dev. neurobiol. riken, tokyo, japan; behavioral gen. riken, tokyo, japan; nih, bethesda, usa; sorst, jst, saitama, japan neuroblasts generated in the subventricular zone (svz) of the lateral ventricles migrate into the olfactory bulb (ob) through the pathway called rostral migratory stream (rms). molecular mechanisms regulating the directional long-distance migration remain largely unknown. here we studied adult function of cyclin-dependent kinase (cdk ) that has been revealed to play a role in neuronal migration in the embryonic brain. crossing the floxed-cdk mice to emx cre mice resulted in decreased size of ob and abnormal distribution of neuroblasts. svz explants from these mice cultured in matrigel showed decreased migration distance. leading process of neuroblasts infected with cre-encoding retrovirus were found in random orientations and frequently failed to migrate out of the svz compared to control cells. these results indicate that cdk has a cell autonomous function in neuroblast migration in the adult brain. os p- - colocaliztion of neuron markers and glial markers in gabaergic neuron progenitors as revealed by singlecell microarray analysis shigeyuki esumi , wu sheng-xi , yuchio yanagawa , , kunihiko obata , nobuaki tamamaki kumamoto univ., kumamoto, japan; fourth military medical univ., xi'an, people's republic of china; gunma univ., maebashi, japan; sokendai, hayama, japan; riken, wako, japan gabaergic neurons and oligodendroglia share many characters in the murine forebrain. both of the cell types has been reported to originate in the medial ganglionic eminence and migrate to the neocortex. in addition, it is reported that they share several glial markers, such as ng , plp, and cnp at their prematured stages. in order to investigate its nature, we have established a single-cell microarray analysis method. single gfp-positive gabaergic neuron progenitors were corrected from the subventricular zone of the gad -gfp knock-in mouse neocortex at e -p by dissociation and picking. complemental dna from the single cells was amplified by universal pcr amplification and converted into biotin-labeled crna using t rna polymerase. after these procedures, crna sufficient for a microarray analysis was obtained. as the result we found, mbp and s -␤ expression in the gabaergic neuron progenitors. os p- - role of ␤-catenin signaling in regulating proliferation of transit-amplifying cells in the adult mouse subventricular zone kazuhide adachi , , , masanori sakaguchi , toru yamashita , , , yuko fujita , yukiko gotoh , arturo alvarez-buylla , takeshi kawase , hideyuki okano , kazunobu sawamoto , neurosurgery, keio univ. sch. med., tokyo, japan; bridgestone lab. dev. regenerative neurobiol., keio univ. sch. med., tokyo, japan; physiol., keio univ. sch. med., tokyo, japan; inst. mol. cell biosciences, univ. tokyo, tokyo, japan; neurosurgery, ucsf, san francisco, usa; neurol, okayama univ, med, dentistry and pharmaceutical sci, okayama, japan the subventricular zone (svz) continuously produces olfactory bulb neurons in the adult rodent brain. neural stem cells generate migratory neuroblasts via highly proliferative transit-amplifying cells in this region. here, we studied the role of ␤-catenin signaling in the adult mouse svz. ␤-catenin accumulated in the nucleus of only the transitamplifying cells in the svz. activated ␤-catenin signaling promoted the proliferation of transit-amplifying cells, resulting in an increased number of new neurons in the olfactory bulbs. these results suggest that ␤-catenin signaling plays a role in the proliferation of transitamplifying cells in the adult mouse svz. the ciliary marginal zone (cmz) is a region between the neural retina and ciliary epithelium, and contains retinal progenitor cells that give rise to neuron and glia. wnt b is expressed in cmz, and has been shown to control the differentiation of the retinal progenitor cells. we have isolated a novel bmp antagonist, chick tsukushi (c-tsk), which belongs to the small leucine-rich proteoglycan family. in the eye, the expression of c-tsk is observed in the cmz which is similar with that of wnt b. to examine the molecular interactions between c-tsk and wnt b, we co-electroporated them into the optic vesicle at stage - chick embryo and observed the proliferation of the retinal progenitor cells. we found that c-tsk inhibited the wnt b activity that sustains prolonged proliferation of retinal progenitor cells. our result suggests that c-tsk controls the proliferation of retinal progenitor cells interacting with wnt b. to reveal the role of epigenetic gene regulation in neuronal differentiation, we studied subcellular distributions of histone deacetylase (hdac) in developing cortical neurons. an expression vector of gfp-tagged hdac was transfected to dissociated cortical cell cultures as well as cortical neurons in vivo. hdac was primarily localized in nuclear until week in vitro, but was translocated to cytoplasm in the later stages. such translocation was found in a similar time course after birth in vivo. to examine a possibility that neural activity is involved in the translocation, firing activity of cultured neurons was examined using multi-electrode dishes. as a result, spontaneous firing activity was prominent in the late stages when cytoplasmic translocation occurred. however, ttx addition to the culture medium produced the inverse translocation. these results suggest that activity-dependent intracellular localization of hdac contributes to neuronal differentiation in cortical development. research funds: kakenhi ( ) dept. of physiology, fujita health univ. sch. of med., japan we described electrical synapses in alpha retinal ganglion cells (␣-gcs). precise temporal synchronization of spikes is generated from ␣-gcs (hidaka et al., ) . the fraction of open channels in gap junctions were evaluated with techniques of dual patch-clamp, connexin immunocytochemistry, and high-voltage electron microscopy. junction conductance (maximum . ns) was measured. in high-voltage electron microscopy (hitachi m, nips, , gap junctions (average size . m long) were present in contacts. in confocal laser-scanning imaging, connexin localization at contacts counted gap junctions (seven sites in a pair on average). assuming that the density of connexons would be /m and a single channel conductance is ps, the conductance of each junction would be ns. the presence of seven junctions between a pair will lead to estimate a total junction of ns. the measured conductance could allow to estimate a fraction of open channels as . %. the open fraction is small, when we consider whether electronic transmission acts to synchronize the spikes in the intercellular network. the visual system separates different types of information into parallel, anatomically distinct processing streams. despite their significance for visual processing, the molecular mechanism underlying the physiological stream formation is largely unknown, partially because these physiological streams have not been reported in mice. to identify molecular correlates of functionally distinct streams, we fabricated a custom cdna microarray of higher mammal ferrets. we successfully identified molecules whose unique distribution and developmental profiles define the lgn itself, its constituent layers, or identify cells comprising one of the physiological streams in the lgn. using these molecules as temporal and spatial markers, we investigated mechanisms of the physiological stream formation in the ferret lgn. research funds: kakenhi ( ), coe research akira muto, herwig baier department of physiology, university of california san francisco (ucsf), usa the visual system operates over a broad range of luminances. this is accomplished by adjustment of photosensitivity, called light adaptation. to study the molecular mechanisms of light adaptation, we screened for zebrafish mutants that showed compromised optokinetic responses (reflexive eye movements to large field motion) after an abrupt dark-to-light transition. in this experimental paradigm, wildtype fish larvae recover their full optokinetic response within about two minutes after being brought back to light. in a screen of almost genomes, we identified five mutants all of which showed substantially delayed recovery of the okr. positional cloning of one of the loci revealed a mutation in the dna-binding domain of glucocorticoid receptor (gr). gr is known for its role in the stress response, but its function in the visual system is unexplored. we propose that gr is regulating genes essential for light adaptation in the retina. os p- - multisite recording of the signal propagation pattern in the visual cortex makoto osanai, yusuke takeno, satoshi tanaka, tetsuya yagi graduate school of engineering, osaka university, suita, japan recently, the visual prosthesis systems with implanted stimulus electrode in the visual cortex are developed. but the signal propagation pattern induced by electrical stimuli in the visual cortex is not fully investigated. therefore, we studied the signal propagation pattern induced by electrical stimuli in the mouse visual cortex slice, using a channel multielectrode array and a calcium imaging system. in the electrophysiological study, the responses conducted vertically against the layer of the cortex with layer stimuli and propagated horizontally in the layer / . in the calcium imaging study, the area of the higher calcium concentration region spread vertically with layer stimuli. signal propagation was restricted within several tens m around the stimulus electrode by ap + cnqx administration and was completely blocked by ttx administration. administration of bicuculline increased the area of the signal propagation in a dose-dependent manner. we concluded that these restricted patterns of the signal propagation in the visual cortex were due to the inhibitory system. os p- - presence of two phases in the sensitive period of orientation plasticity shigeru tanaka , toshiki tani , kazunori o'hashi , , jerome ribot brain science institute, riken, saitama, japan; graduate school of life science and systems engineering, kyushu institute of technology, kita-kyushu, japan recently we have revealed that orientation-restricted visual experience induces drastic reorganization of orientation maps in the cat visual cortex. in this study, we examined the effect of release from single orientation exposure on once reorganized orientation maps during the sensitive period using intrinsic signal optical imaging. when kittens were returned to the normal visual environment by removing the goggles after weeks of goggle rearing starting around the age of weeks, the over-representation of the exposed orientation was preserved. on the contrary, when the goggle rearing started around the age of weeks and then the animals were returned to the normal visual environment, orientation maps rapidly changed to represent orientations equally. these findings indicate that the sensitive period of orientation plasticity consists of two phases: orientation map reorganization is irreversible in an early phase and reversible in a late phase. os p- - residual visuomotor processing in the animal model of blindsight: comparison with normal, near-threshold vision masatoshi yoshida , , , tadashi isa , , dept. dev. physiol., nat'l inst. physiol. sci., okazaki, japan; sch. life sci., grad. univ. adv. stud., hayama, japan; crest, jst, kawaguchi, japan in two macaque monkeys with unilateral v lesion performing a visually guided saccade task, saccadic parameters were compared between the saccades to the affected hemifield and those to the intact hemifield. the luminance contrast of the target presented in the intact hemifield was reduced so that the detectability was comparable to that in the affected hemifield ( - % correct). in the saccades to the affected hemifield, the curvature of the trajectories was smaller and the deviation of the saccadic end points from the target was larger than those to the intact hemifield. these results suggest that without geniculo-striate pathway, online compensation for the variation of the initial saccadic command is not fully functional, thus leading to inaccurate saccades. we propose that the residual visuomotor processing of monkeys with v lesion is unlike normal, near-threshold vision. research funds: kakenhi , kakenhi and crest, jst os p- - comparison of the angle representation in macaque visual areas v and v minami ito , , hidehiko komatsu , national institute for physiological sciences, okazaki, japan; the graduate university for advanced studies, hayama, japan previously, we have reported that fairly large number of area v neurons has angle selectivity. here, we studied the angle selectivity of area v , which is the major source of inputs to area v . we conducted single-unit recordings from the superficial layer of area v , while animals performed a fixation task. for comparison, we used a similar stimulus set. the stimuli were much larger than the size of the classical receptive fields. area v neurons responded mainly to sharp angles ( • ), straight lines ( • ) or right corners ( • ), but not to intermediate angles ( • or • angle width). this contrasted with area v , where neurons showed a variety of the optimal angle width including intermediate angles. we also observed several v neurons showed fine orientation tuning to short line segments, while weak or no responses were induced by a set of large angle stimuli. we suggest that area v neurons largely contribute to representing line components (lines and line-ends) and to sending such information to area v . os p- - firing rates and dynamic spatiotemporal patterns of ganglion cells both contribute to retinal information processing xin jin, ying-ying zhang, xue liu, hai-qing gong, pei-ji liang department of biomedical engineering, shanghai jiao tong university, shanghai, china population activities of retinal ganglion cells (rgcs) were recorded using a multi-electrode recording system. single unit analysis showed that firing rate of individual neuron was strongly dependent on the luminance intensity of stimulation. however, population activity of ganglion cells usually showed particular spatiotemporal pattern, in response to a specific velocity of the moving bar. differing from single direction-selective ganglion cell (dsgc), which responds to its preferred direction of movement by firing at its maximal rate, population activity of non-direction-selective ganglion cells may encode the motion information in a temporally ranked manner, independent to their individual firing rates. these results suggest that an efficient and economical coding mechanism may be employed by the retina, where the firing rate of individual neurons and spatiotemporal pattern of population neuronal responses could act in parallel to encode different aspects of visual information. yasuto tanaka, satoru miyauchi, masaya misaki brain information group, nict, kobe, japan visual long-range interaction was reported to be limited in space. here, we show the evidence of long-range interaction extending to an order magnitude larger using the right-left symmetrical configuration. two horizontally collinear gabor signals, one defined as probe and the other cue, were presented at the left and right side of the visual field at mirror symmetrical regions. detection threshold of gs probe reduced with cue-probe separations up to • . the facilitation was highly tuned to the symmetrical locus. furthermore, the facilitation was substantially longer at upper visual field than the lower visual field. the reduction was specific to orientation, phase, and horizontal direction, the results indicate long-range mirror symmetrical interaction across vertical meridian, suggesting symmetrical neuronal communication between early visual cortices. the anisotropy between left-right hemifield (symmetry) and upper-lower hemifield (upper-field advantage) signifies hemifield inhomogenity in human vision. os p- - integrity of visuospatial attention in a split brain patient noudoost behrad, seyed reza afraz, maryam vaziri, hossein esteky school of cognitive sciences (scs), iran transfer of visual information between hemispheres is severely impaired following transection of posterior part of the corpus callosum. we investigated whether attentive visual object tracking across vertical meridian of the visual field is possible for a posterior callosotomized patient (md). we asked md to track one bouncing ball among four identical distracters while fixating at the center of the screen. target crossed the vertical midline in half of the trials. her performance in crossed conditions was significantly above chance level. also, we asked her to make decision about horizontal alignment of two balls presented simultaneously in one of three conditions: both in right or left hemifield, or each in one hemifield. in this alignment task md was able to compare location of the two bilaterally presented stimuli well above chance level. our data suggest that inter-hemispheric transfer of position information required for spatial attention is preserved without posterior corpus callosum. pei sun, justin l. gardner, mauro costagli, kenichi ueno, r. allen waggoner, keiji tanaka, kang cheng laboratory for cognitive brain mapping, riken brain science institute, wako-shi, japan although the preference for stimulus orientations in human visual cortex has been inferred indirectly in a few studies using fmri, tuning to particular stimulus orientations has not been directly demonstrated using this technique. in an effort towards revealing orientation selectivity and its spatial arrangement in human v , we have conducted an fmri study with a novel stimulation paradigm and a differential mapping method. we found that responses of the majority of activated voxels were modulated by the grating orientation and individual voxels were sharply tuned to particular orientations. our results provide the first demonstration that orientation selectivity in humans can be directly studied using fmri. os p- - probing the spatial scale of classifier performance with high spatial resolution fmri justin l. gardner , , pei sun , keiji tanaka , david j. heeger , kang cheng department of psychology and center for neural science, new york university, usa; laboratory for cognitive brain mapping, riken brain science institute, japan recently, classifier analysis with conventional resolution fmri has been used to decode the orientation of a grating stimulus from the fmri responses of early visual cortex. it has been proposed that classifier analysis exploits small but robust orientation biases in voxels that are created by local inhomogeneities in the columnar organization. we have examined this proposal by using classifier analysis to decode stimulus orientation using high spatial resolution fmri ( . mm × . mm × mm voxels) in human v . we found that many voxels that are weighted heavily in the classifier analysis and carry similar orientation biases closely follow draining veins that are visible on t *-weighted venograms. we suggest that large draining veins with orientation specific responses, rather than local inhomogeneities in orientation maps, may provide a basis for classifier performance using large voxels. research funds: nrsa fellowship from the nih ( f ey - ) os p- - relationship between horizontal connections and functional structure in macaque anterior inferotemporal cortex (area te) hisashi tanigawa, kathleen s. rockland, manabu tanifuji riken brain science institute, wako, japan we have studied the relationship between horizontal connections and functional structure in te using a combination of optical imaging, unit recording, and anatomical tracing. intrinsic signal imaging was performed in exposed te, under anesthesia, during presentations of visual object stimuli. this resulted in multiple optical spots evoked by each stimulus. in some animals, subsequently, unit recording was carried out at multiple sites within the imaged region. then, an anterograde tracer was injected into one of the spots. both optical imaging and unit recording revealed regions with stimulus preference similar to that at the injection site. however, these regions and the injection site were not always connected by horizontal axons. some regions sharing a preference to particular stimuli were connected, even though they showed different preferences to the other stimuli. these results suggest that horizontal axons can connect regions with different stimulus preferences in te, in contrast to like-to-like connectivity, as understood in early visual cortices. we recorded single cell responses from the inferotemporal cortex of a fixating monkey while visual stimuli with various durations ( - ms; isi = s) were presented. presentation of visual stimuli at all of the tested durations resulted in prolonged responses. the brief presentations evoked multiple phasic responses while the long presentations evoked sustained activities. there was a significant difference in average firing rate of late phase ( - ms) of response to optimal stimulus across presentation durations. but no such differences were found for the first phase ( - ms). in addition, the optimal stimulus evoked significantly different response magnitudes in the first and second phase particularly in the short presentation durations. but the suboptimal stimulus (∼ % of max response) evoked similar response magnitudes in the first and second phase. these results suggest that stimulus selectivity of inferotemporal cells depends on the stimulus presentation duration and the time window that is used to measure the firing rate. os p- - the perceptual learning effect in myopes by the lateral masking procedure keiko mizobe , kazuto terai , osamu hieda , shigeru kinoshita dept. of ophthal., kyoto second red cross hospital, kyoto, japan; dept. of ophthal., kyoto pref. univ. of med., kyoto, japan; baptist eye clinic hospital, kyoto, japan purpose: the study of the visual cortex revealed the lateral masking collinear configuration modulated the neuronal responses and psychophysical studies also showed perceptual learning improved the visual detection. we asked whether perceptual learning could improve the myopic blurred vision, using the new instrument of the lateral masking technology, neurovision. method: nine low myopes were studied. non-corrected digital visual acuities (va) ranged from . to . . the logmar average was . . eight sessions of neurovision treatment were performed to each individual. the estimation was done by comparison of va before and after the treatment. results: four eyes showed more than one octave improvement of va. the logmar average of the four was . , improved from . . the residual five eyes showed less or no improvement. the change of logmar average was from . to . . conclusion: some myopes showed the perceptual learning effects by new treatment, using the lateral masking technology. os p- - the hindbrain neuroepithelial cells exclude the migrating facial motor neurons by expression of planar cell polarity (pcp) genes hironori wada , hideomi tanaka , , satomi nakayama , miki iwasaki , , hitoshi okamoto , laboratory for developmental gene regulation, bsi, riken, japan; crest, jst, japan many neurons migrate tangentially through one cell layer at a specific depth within the brain. in the developing zebrafish hindbrain, the facial (nvii) motor neurons originate in rhombomere (r) and migrate tangentially to r near the pial surface of the hindbrain. in this study, we demonstrate that expression of the planar cell polarity (pcp) genes celsr and frizzled a in neuroepithelial cells maintain the nvii motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. mosaic analyses show that expression of the frizzled a gene in the surrounding neuroepithelial cells prevented the entry of the nvii motor neurons in the neuroepithelial layer. the demonstration of a role for neuroepithelial cells in excluding differentiated neurons from the neuroepithelial layer may provide new insights into the general mechanisms underlying formation of the layered structures in the mammalian brain, such as in the cerebral cortex. os p- - disrupted-in-schizophrenia (disc ) regulates the transport of the nudel/lis complex to axons via direct interaction with kinesin- shinichiro taya, kozo kaibuchi department of cell pharmacology, nagoya university, nagoya, japan disc is a candidate gene for susceptibility to schizophrenia. in a scottish family, the chromosome translocation interrupts the coding sequence of disc . disc is reported to interact with nudel, which forms a complex with lis . although the functional significance of this complex in axon growth and neuronal development has been reported, the transport mechanism of the complex into axons and the functions of disc remain largely unknown. here we report that disc interacted with kinesin- , a motor protein of anterograde axonal transport. kinesin- interacted with the nudel/lis complex through disc , and these molecules accumulated at the distal part of axons. the knockdown of disc by rnai of disc induced the delocalization of nudel and lis from the axons and reduced axonal growth. the knockdown of kinesin- induced the delocalization of disc from the axons. taken together, these results indicate that disc links kinesin- to the nudel/lis complex and regulates its transport as a cargo receptor for axon elongation. research funds: mext os p- - role of a novel collapsin response mediator protein- interacting molecule, synaptotagmin-like protein in hippocampal neuron nariko arimura, saeko kawabata, atsushi hattori, kozo kaibuchi department of cell pharmacology, graduate school of medicine, nagoya university, nagoya, japan during the development, neurons recognize the extracellular signals and extend the axons to proper directions. certain kinds of receptors are transported from the nerve cell body to the axon terminal, and participate in the recognition of extracellular environments. however, the mechanism of controlled recruitment of receptors remains unsolved. here, we report that synaptotagmin-like protein (slp ) can mediate the vesicle transport. slp is known to associate with rab . we found that slp associates with collapsin response mediator protein- (crmp- ), which is a key regulator of axon formation. slp could form the trimeric complex with rab b and crmp- , and also associate with kinesin- through crmp- . slp is accumulated on microtubules at the axonal growth cones, and is co-localized with a receptor of growth factor. these findings suggest that slp functions as a mediator of recruitment of certain receptor depending on crmp- and kinesin- . os p- - absolute quantification of mdr a, mrp , mrp and bcrp proteins at the mouse brain blood barrier by lc-ms/ms junichi kamiie , , yuki katsukura , , sumio ohtsuki , , xiao-kun cai , , tetsuya terasaki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan the abc transporter proteins are thought to limit permeability across the blood-brain barrier as the efflux transporters. however, contribution of each transporter to the bbb function is not clarified. the purpose of this study was to clarify the protein amounts of mdr a, mrp , mrp , and bcrp in the brain capillaries of mouse by newly developed membrane protein quantification method using lc-ms/ms. by this method, the standard curve showed linearity between and fmol, and amino acid sequence of the detected fragment was confirmed by ms/ms spectrum. in the brain capillaries, the protein amounts of mdr a, mrp , bcrp were . , . and . fmol/g, respectively, while it of mrp was under detection limit of standard curve. this quantitative profile suggests that mrp and bcrp function as the efflux transporter at mouse blood-brain barrier as well as mdr a. os p- - dominant expression of claudin- in highly purified brain capillary endothelial cells sumio ohtsuki , , hirofumi yamaguchi , saori sato , tmoko asashima , , tetsuya terasaki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan claudins are major constituents of tight junctions (tjs). the purpose of this study was to clarify the expression levels of each claudin subtype in brain capillary endothelial cells (bcecs), which form the blood-brain barrier. mouse bcecs were highly purified using endothelial surface antigen (pecam- ) and magnetic cell sorting. mrna expression of caludin- - was measured by real-time rt-pcr. claudin- showed the highest mrna expression in the purified mouse bcecs. mrna levels of claudin- and - were . % and . % of that of claudin- . claudin- mrna was concentrated in the purified bcecs, while claudin- and - mrna in the purified bcecs were lower than that in the whole brain. rat claudin- mrna was also concentrated in rat brain capillary fraction, but claudin- mrna did not. these results suggest that claudin- is a dominant tjs protein in bcecs, and expression of claudin- and - , which was reported as tj protein in bcecs, are not restricted in bcecs. os p- - effects of hydrogen peroxide towards gap junction communication in astrocytes and permeability of blood brain barrier f. ahmad , a. pauzi m. yusof , p.d. mourad , m. bainbridge , s. ab ghani universiti sains malaysia; university of washington, seattle, usa; brody school of medicine, east carolina university, nc, usa h o is the main peroxides produced in mammalian cells that consume o . the main source of h o in the brain, produced in large amount, was from the superoxide dismutase catalyzed reaction in mitochondria. therefore, we look into the effects of h o towards the gap junction communication in astrocytes and permeability of blood brain barrier. in this study, by using a h o microsensor, we investigated the level of h o in the brain that altered the permeability of bbb. the microsensor was implanted in the rat's brain and operated amperometrically. we measured h o level from the current generated by the electron transfer at the electrode. we observed a change in permeability when external h o was injected into the brain. fatality occurs when the injected h o exceeds m. these finding showed that the altered paracellular permeability in the presence of h o is caused by a series of events that happen one after another. research funds: short term grants pkimia and pfar-masi os p- - somato-ovarian sympathetic reflex discharges in anesthetized rats sae uchida, fusako kagitani, harumi hotta dept. auton. nerv. syst., tokyo metropol. inst. gerontol., tokyo, japan ovarian sympathetic efferent reflex discharges caused by single electrical shock stimulation of spinal (t - ) afferent nerves or limb (tibial) afferent nerves were studied in urethane anesthetized rats. in central nervous system (cns) intact rats, stimulation of the t - spinal afferent nerve produced early and late a-reflex discharges, and a late c-reflex discharge. after spinalization at the third thoracic level, stimulation of the same spinal afferent nerve produced an a-reflex with the same latency as the early a-reflex in cns-intact rats and an early c-reflex discharge with the similar latency as the late a-reflex in cns-intact rats. on the other hand, stimulation of the tibial afferent nerve produced late a-reflex and c-reflex discharges in cns intact rats, those were not observed after spinalization. it was concluded that ovarian sympathetic aand c-reflex discharges evoked by stimulation of a segmental spinal afferent nerve in cns-intact rats are of spinal and supraspinal origin, and those evoked by tibial nerve stimulation are of supraspinal origin. os p- - responses of renal sympathetic nerve activity and sodium excretion to days sodium loading in rats misa yoshimoto, nozomi iinuma, rie itokawa, eri hayashi, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan in the present study, a month recording of renal sympathetic nerve activity (rsna) in freely moving rats was made to explore the long-term regulation of rsna and sodium excretion. wistar male rats were instrumented chronically with electrodes for the measurements of rsna and electrocardiogram. after the days recovery period, rsna, heart rate and sodium balance were measured over three weeks. animals were allowed to drink four different concentration of sodium chloride solutions ( , , , meq./l nacl) over days. the sodium loading with meq./l nacl suppressed rsna significantly and then it gradually recovered while either meq./l nacl or meq./l nacl loading had no effects on rsna. sodium excretion changed significantly in proportion to the each sodium loading levels. these results indicated that the changes in rsna were not always correlated with the changes in sodium excretion in rats. os p- - cross correlation analysis of respiratoryrelated optical imaging signals yoshitaka oku , haruko masumiya , yasumasa okada dept. physiol., hyogo col. med., nishinomiya, japan; dept. med. keio univ. tsukigase rehab. ctr. shizuoka, japan we aimed to establish an objective method to identify the distribution of respiratory-related regions and the timing when these regions are activated relative to the inspiratory activity from optical imaging signals. optical signals were recorded from the ventral medullary surface of neonatal rats in vitro using a voltage-sensitive dye. cross correlation between integrated c ventral root (c vr) activity and each pixel was calculated after cycle-triggered averaging and detrending. the maximum of cross correlation coefficients and the lag at which the cross correlation became maximal (lagmax) were displayed as d pseudocolor maps. in all preparations, two respiratory-related regions were consistently identified: ( ) a continuous column extending from the para-facial region to the pre-bötzinger complex, and ( ) a region corresponding to the ventral horn. pixels where lagmax were negative (meaning that the activity preceded the c vr activity) tended to be distributed in the para-facial region, and this tendency was more evident when superfusate ph was lowered. os p- - slow afterhyperpolarization determines the firing pattern of action potentials in rat gnrh neurons masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan gonadotropin-releasing hormone (gnrh) neurons play a pivotal role in the hypothalamo-pituitary-gonadal axis. gnrh neurons must be able to continuously fire in response to depolarizing stimuli. for this type of firing, gnrh neurons may have a certain intrinsic property. to address this issue, we investigated the ca + -activated voltage-independent k + currents underlying afterhyperpolarization. dispersed gnrh neurons from adult gnrh-egfp transgenic rats were cultured overnight and used for an electrophysiological experiment with perforated patch clamp configuration. the gnrh neurons showed a slow afterhyperpolarization current (i sahp ). in contrast to previous reports, the i sahp observed in rat gnrh neurons was potently blocked by an sk channel blocker apamin. in current clamp condition, gnrh neurons evoked a train of action potentials to depolarizing current pulse. apamin increased the susceptibility to spike failure. the results indicate that rat gnrh neurons exhibit an apaminsensitive i sahp , which regulates the firing pattern. research funds: kakenhi , os p- - the effect of music to sex hormones of elderly person hajime fukui , kumiko toyoshima , kiyoto kuda , katsuhiko iguchi nara univ. of edu., nara, japan; grad. school of human sciences, osaka univ. japan; nara city medical clinic, japan it has been known that testosterone or estrogen protects nervous system and regulates cell death in a brain. also, it is pointed out that the decline of t and est accelerates depression. therefore the treatment such as hormone replacement therapy (hrt) has been tried to cure depression and alzheimer's disease. however, it has been pointed out that hrt has serious side effects. on the other hand, there are reports that music influences on a steroid hormone. in addition, it is known that music has certain therapeutic gain toward ad and dementia. in this study, from a point of view of the prevention of ad and dementia, we examined the effect of music to sex hormones of normal elderly person. four males and females participated music session and t and est were evaluated. as a result, in female, in the high hormone group, the values decreased after the session, and in the low hormone group, the values increased. from above, there might be possibility that through a steroid hormone music participates in protection and improvement of function on brain. tuberculous meningitis (tbm) is the most common form of chronic infection of the central nervous system. despite the magnitude of the problem, the general diagnostic outlook is discouraging. this study identifies a specific protein marker in csf, which will be useful in early diagnosis of tbm. we have demonstrated the presence of a -kda protein band in csf of % (n = ) of confirmed and % (n = ) of suspected tbm patients out of tbm patients. the -kda protein band was analyzed by lc-ms/ms analysis. in the present study we have identified two mycobacterial proteins rv c (ag a) and rv c (ag b) and one host derived protein as the components of the tbm specific -kda protein. involvement of mitochondrial extrinsic and intrinsic apoptotic pathways in dopaminergic neurodegeneration was tested in rotenone-and mpp + -induced rat models of parkinsonǐs disease (pd). hplc-ec, patch clamp, fluorimetry, immunoblot and rt-pcr were used for measuring neurotransmitters/free radicals, membrane currents, caspases activities, levels of proteins and mrna of mitochondria-linked signaling in brain. we report here a retrograde mode of neuronal death via mitochondrial intrinsic pathway in mpp + -, but an extrinsic mode of cell death in rotenone-induced model. drug screening in these models (l-deprenyl as positive control) indicated that quercetin, coenzyme q , vitamin d and melatonin act via interfering the signaling events in neurons. loss of complex-i and -iv activities and changes in some of the protein subunits in pd postmortem brains were confirmed in pd and control cybrids. results from the present study provide evidences for a direct involvement of mitochondria and are suggestive of existence of both intrinsic and extrinsic apoptotic pathways in dopaminergic neuronal death. os p- - involvement of thioredoxin on the neuroprotective effect of (−)-deprenyl tsugunobu andoh , boon chock , dennis l. murphy , chuang c. chiueh dept. applied pharmacol., univ. toayama, toyama, japan; lab. bioch., nhlbi, nih, md, usa; lab. clin. sci., nimh, nih, md, usa; cent, brain diseases and aging, taipei med. univ., taipei, taiwan the present study investigated whether the induction of thioredoxin (trx) involves in the cytoprotective mechanisms of (−)-deprenyl which is known as the inhibitor of mao-b. after confirming (−)-deprenyl protects against mpp + -induced cytotoxicity in human sh-sy y cells, we observed further that (−)-deprenyl induced trx for protection against oxidative injury caused by mpp+. the induction of trx was blocked by pka inhibitor through a pka-sensitive phosphoactivation of map kinase erk / and the transcription factor c-myc. (−)-deprenyl-induced trx and associated neuroprotection were concomitantly blocked by the antisense against trx mrna in human sh-sy y cells. consistently, trx increased the expression of mnsod and bcl- supporting cell survival. in conclusion, (−)-deprenyl augments the gene induction of trx leading to elevated expression of antioxidative mnsod and antiapoptotic bcl- proteins for protecting against mpp + -induced neurotoxicity. os p- - pgd induces neuronal apoptosis via d- , -pgj tatsurou yagami , noboru okamura , toshiyuki sakaeda facul. health care sci., himeji dokkyo univ., himeji, japan; kobe univ. grad. sch. med., japan prostaglandin d (pgd ) is abundant in the brain, but its neuropathologic role has been unclear. here, we found that pgd induced neuronal apoptosis in rat cortical cultures. however, a pgd receptor blocker did not suppress neurotoxicity of pgd . little pgd receptor was detected, suggesting an involvement of pgd metabolites in the apoptosis. among pgd metabolites, -deoxy- , -prostaglandin j ( d- , -pgj ) caused neuronal apoptosis most potently and rapidly. although d- , -pgj is an endogenous ligand for peroxysome proliferator-activated receptor ␥ (ppar␥), ppar␥ activators did not kill neurons, suggesting that d- , -pgj induces apoptosis independently of ppar␥ activation. we found specific binding sites of [ h] d- , -pgj (jbs) in plasma membranes. there was a close correlation between the neurotoxicity of various eicosanoids and their affinity for jbs. in conclusion, we demonstrated that pgd induced apoptosis via d- , -pgj in rat cortical neurons, and suggested that jbs in the plasma membrane was involved in the d- , -pgj -induced apoptosis. yoshiki iwamoto, daisuke umetsu, shigeru ozaki, naohito terui department of physiology, university of tsukuba, tsukuba, japan stability of a driver's head is crucial for clear vision and consistent, smooth operation of a vehicle. we reported last year that bilateral sternocleidomastoid muscles (scm) of drivers showed a symmetrical increase in activity during forward acceleration of a vehicle. in the present study, we analyzed the relationship between scm activity and vehicle acceleration. emgs of the right and left scm of drivers were recorded during rapid forward acceleration. the time course of the rectified, smoothed emgs did not match that of vehicle acceleration. for a given acceleration, emg was larger when acceleration was increasing than when it was decreasing. we compared emgs and a linear sum of acceleration and its time derivative, jerk. with optimal weights for the two variables and a proper time lag, the linear sum reproduced the emg profile. the optimal weight and lag varied across subjects and vehicles. we suggest that the jerk-related muscle activity may be necessary to quickly restore proper head position after sudden acceleration. grasping is a highly developed movement in primate including human. in contrast to the well-known involvement of cerebral cortex, role of spinal neurons in controlling this behavior has never been examined. here, we show the first direct evidence suggesting the significant contribution of spinal neurons. we trained japanese monkeys to perform the precision grip task, pinching the two springloaded levers with their index finger and thumb, and recorded neural activities through an oval recording chamber implanted over the cervical spinal cord (c to t ). majority of the recorded neurons showed movement-related modulation of firing rate, and the modulation sometimes started before movement onset. spike-triggered averaging of muscle activities revealed some neurons had post-spike effects to hand muscles, suggesting that spinal neurons were capable to generate and modulate muscle force during precision grip. we suggest that primate spinal neurons have a significant role in preparation and execution of grasping movement. research funds: kakenhi os p- - compartmentalization of the cerebellar nuclei: aldolase c expression and the olivonuclear projection pattern izumi sugihara, yoshikazu shinoda dept. systems neurophysiol., tokyo med. & dental univ., tokyo, japan the cerebellar cortex is compartmentalized into more than longitudinal stripes by the aldolase c (=zebrin) expression pattern, which is tightly correlated with the topographic olivocortical projection. however, no equivalent compartmentalization has been known in the cerebellar nuclei. we mapped aldolase c labeling of terminals of purkinje cell axons and anterograde labeling of collaterals of olivocerebellar axons in the rat cerebellar nuclei. the cerebellar nuclei were divided into the caudoventral aldolase c-positive and rostrodorsal negative parts, indicating purkinje cells in the positive and negative stripes in the cortex project to the caudoventral and rostrodorsal parts in the nuclei, respectively. olivonuclear projections showed clear topography within these parts, which was completely congruent with the olivocortical topography. these results clarified the compartmentalization of the cerebellar nuclei and supported that the aldolase c expression is tightly related with the functional organization of the cerebellum. we examined a context dependency of neuronal activity of the pedunculopontine tegmental nucleus (pptn) in monkeys during visually guided saccade tasks. about half of movement-related activities occurred for only the saccades to the saccade target in the task, but they did not occur for the saccades outside the task. on the other hand, for the other half of neurons, movement-related activities occurred for every saccade regardless of the task condition. for visual responses, some neurons responded either the initial fixation point or saccade target, and others responded equally to both stimuli. we further analyzed mutual relationship among modulation timing, preferred direction, effect of reward expectation and this context dependency of the activities, and discussed the visuo-motor processing of pptn. in the reinforcement learning theory, the midbrain dopamine (da) neurons send reward prediction error signal to the striatum. the cholinergic pedunculopontine tegmental nucleus (pptn) is one of the strongest excitatory input sources to da neurons. we hypothesized that pptn may play an important role for relaying necessary components of reward prediction error signals to da neurons. during recording of pptn neurons, we utilized reward predictable visually guided saccade tasks where a shape of fixation point indicated a reward volume. for more than half of the neurons, which showed cue related responses, the cue responses were dependent on association of cue feature and reward size. from another population, we recorded reward related activity. in conclusion, pptn neurons may relay both reward and reward prediction signals, sufficient for computation of reward prediction error. research funds: kakenhi ( ) os p- - timing activity in supplementary eye field during a saccadic eye movement task shogo ohmae , xiaofeng lu , , yusuke uchida , toshimitsu takahashi , , shigeru kitazawa , dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; crest, jst, tokyo, japan to act properly in our daily life, the ability to detect and predict timing of events is always required. how do we deal with timing in the brain? to address this question, we trained two japanese monkeys to perform a visually guided saccadic eye movement task in which the monkeys made saccades to each of targets following a gosignal given at a random timing between and ms after the appearance of the target. we recorded neuronal activity from the supplementary eye field (sef) during the task. we found a group of cells that showed activity related to the length of the delay period from target-on to the go-signal. these cells were classified into two types: ( ) those that showed buildup activity during the delay period until the go-signal, and ( ) those that displayed changed activity after the go-signal in relation to the length of the delay period. the results suggest that sef is involved in timing the onset of the go-signal during the saccadic eye movement task. in reaching, a spatial visuomotor transformation should occur in our brain. we can make the transformation not only when the relationship between visual and motor coordinates is default, but also when a gain for the relationship is changed, for example, in a microsurgery. we trained monkeys to make reaching movements when visuospatially identical targets were presented on a computer display by aligning a cursor that indicated their hand position, while the gain was systematically changed. we recorded and analyzed movement-related neuronal activity in the ventral premotor cortex (pmv) and the primary motor cortex (mi) during reaction time. it was revealed that a majority of the mi neurons and a part of the pmv neurons showed activity changes depending on executed movement direction, amplitude, and velocity, whereas a number of the pmv neurons exhibited activity consistent to the visual location of the targets, but not to motor parameters such as amplitude and velocity. the results indicate that the pmv contributes to gain control of reaching during visuomotor transformation. local oscillatory changes in the human sensorimotor cortex induced by simple motor tasks were investigated using supragyral and intrasulcal surface electrodes which was temporarily implanted for the treatment of intractable deafferentation pain. time frequency spectrogram and coherence between electrodes revealed that, before and after several hundred milliseconds of the motor execution, the coherence in the premotor cortex increased cooperatively between neighboring electrodes but that the coherence in the intrasulcal primary sensorimotor cortex decreased exclusively. this result reflects that the premotor cortex plays a role in motor planning with diffuse network while the primary motor cortex plays a role in selective motor execution with local motor output unit. the human sensorimotor processing may be hierarchical and similar to an artificial neural computer. we have shown that the trigeminal oral nucleus (vor) neurons with the receptive field in the intraoral structures project bilaterally to either the jaw-closing (jc) or jaw-opening (jo) motor nucleus in the cat. it is known that neurons in the somatosensory cortex project to the trigeminal sensory nuclei in the rat. thus, we conducted this study to reveal whether there are vor neurons that receive cortical projections and project to the jc or jo nucleus in the rat. we injected a retrograde tracer, fluorogold (fg), in the vor, and found many retrogradely labeled neurons in the contralateral rostral primary somatosensory cortex (si). thus, we injected an anterograde tracer, biotinylated dextranamine (bda), in the rostral si, and also fg in the jc or jo nucleus in the same animals. we found a considerable number of fg-labeled vor neurons made contact with bda-labeled axon terminals. these results suggest that si neurons control jawreflexes through vor neurons. tsunehiko kohashi, yoichi oda grad. sch. science, nagoya univ., nagoya, japan the mauthner (m) cells, paired large reticulospinal neurons in teleost hindbrain, are known to initiate fast escape from sudden aversive stimuli. to investigate how the fast escape is established during early developmental stages, we examined motor performance of the escape in zebrafish embryos or larvae, and the contribution of mcell activity on the behavior. the rostral portion of the zebrafish, - h post fertilization (hpf), was embedded in agar and the tail flip in response to water pulse applied to the head was examined. thirty hpf embryos, in which m-cell has already received trigeminal nerve innervation and is still extending its axon in the spinal cord, showed tail flips contralateral to the stimulated side with longer latency (> ms) than larvae (> hpf, ms). m-cell activity monitored with confocal ca + imaging during the tail flip (> hpf) tightly correlated with the initiation of fast escape, whereas delayed escapes without m-cell firing appeared in some cases (< %) after hpf. thus, the development of the escape behavior coincided with that of m-cell circuit. junctophilins (jps) expressed in the er/sr interacts with plasma membrane thereby constructing junctional membrane complexes (jmc). we here report that lacking neural jps subtypes exhibit an irregular hindlimb reflex and impaired memory. to define neural mechanism of memory deficit in jp-dko mice, we performed whole-cell patch clamp recording of hippocampal neurons. in wild mice, an obvious afterhyperpolarization (ahp) was observed and its ahp was totally blocked by apamin. by contrast, ahp was absent in the jp-dko mice and was insensitive to apamin treatment. the er ca + release through ryanodine receptors, triggered by glutamate receptor-mediated ca + influx, is essential for the activation of sk channels toward ahp generation in the hippocampal neurons. therefore, jp-mediated jmc formation likely plays an essential role in neural excitability underlying neural plasticity and memory. os p- - distribution of voltage-gated calcium channel ␣ ␦- mrna in mouse central nervous system takeshi houtani, satoru sakuma, masahiko kase, tetsuo sugimoto department of anatomy and brain science, kansai medical university, moriguchi, osaka - , japan the ␣ ␦ subunits are the auxiliary subunit of voltage-gated calcium channels and modulate the biophysical properties of the pore-forming ␣ subunits. these auxiliary subunits are composed of four genetically different molecules, ␣ ␦- to ␣ ␦- . the distributions of ␣ ␦- , - , - mrna have been intensively investigated in the rat central nervous system by in situ hybridization, but that of ␣ ␦- remains to be determined. we cloned ␣ ␦- cdna fragment from mouse brain by rt-pcr and examined the distribution of ␣ ␦- mrna-expressing cells in the mouse central nervous system by in situ hybridization using digoxigenin-labeled crna probe. while the ␣ ␦- mrna was found to be broadly expressed, some neuronal types or sites such as piriform cortex, hippocampal pyramidal cells, paraventricular hypothalamic nucleus, facial nucleus and motor neurons of the ventral horn had intense mrna expression. our results suggest that ␣ ␦- subunit may play an important role in learning and memory, neuroendocrine secretion and somatic motor control. the mushroom bodies of insect brains are essential in associative olfactory learning. here we show that the drosophila larval mushroom body calyx, the dendritic region, is organized in about glomeruli, which we have mapped. individual glomeruli receive specific innervation from second order olfactory neurons. by contrast, they contain dendrites from hundreds of mushroom body neurons (kenyon cells), which show low specificity for individual glomeruli. glomeruli therefore potentially transmit specific sensory inputs to a large fraction of kenyon cells. quantitative analysis of dendritic termini of single larval-born kenyon cells suggests that they arborize in about glomeruli in an apparently random manner. this pattern of connectivity is consistent with a model in which kenyon cell dendrites process olfactory input by a combinatorial mechanism that allows the discrimination of a large number of odors. withdrawn os p- - hypothalamic defense reaction involves purkinje cells in the flocculus folium p via orexin and gaba in anesthetized rabbits, electric stimulation in the hypothalamic defense area either excited or inhibited "simple spike" discharges in purkinje cells located in folium p of the flocculus. iontophoretic application of an orexin antagonist (sb ) depressed the excitation, while bicuculline depressed the inhibition. h or h histamine antagonist had no effect. labeling orexin fibers by immunocytochemistry showed that they were most numerous in folium p as compared with other folia of the flocculus. stimulation of the hypothalamic defense area produced little field potentials in the folium p unlike those evoked by mossy fibers. these observations suggest that the excitation and inhibition are mediated by orexin-containing fibers, which contact purkinje cells directly and also indirectly via other gabaergic neurons. os a- - activity-dependent development of corticispinal synapse in mouse slice co-culture takae ohno, masaki sakurai dept. physiol., teikyo univ. sch. med., tokyo, japan we showed nmda-dependent synapse elimination of corticospinal (cs) tract in vitro in rat. in order to use the genetically modified mice to study the underlying molecular mechanisms of this developmental plasticity, we studied development of cs synapses in c bl/ mice. by recording field epsp (fepsp) along m interval lattice in the spinal gray matter in response to the stimulation of deep cortical layer, we evaluated spatial distribution of synapse formation quantitatively. fepsps were recorded diffusely throughout the spinal gray matter at - div, then the amplitudes of fepsps in the ventral side began to decrease at - div, and dominated in the dorsal area at div. cs axon terminals labeled anterogradely with biocytin distributed diffusely throughout the spinal gray matter at - div but the axons terminals in the ventral area were eliminated until div. this synapse elimination from the ventral side was blocked by apv application from div, indicating that this process is also nmda-dependent. in slice coculture study, we showed that corticospinal (cs) axons grow rapidly and reach the ventral spinal gray until div. the number of those ventral axons is reduced before div. to study the behavior of the cs axons at the single axonal level, we transfected a small number of cortical neurons with eyfp expression vector pcag-eyfp by way of electroporation to visualize them and took the time-lapse images of their axons under the confocal microscope equipped with an on-stage co incubator. some axons showed rapid growth, reaching the ventral most part of the spinal gray matter already at div. some axons had collaterals at the dorsal part and retracted the ventral branch while extending the dorsal branch during - div. some ventral axons showed a fragmented tip during retraction, which was indicative of axonal pruning. these observations provide direct evidence that there are early cs axons that once reach the ventral spinal gray and then retract to stay dorsally. we identified click-iii/camki␥ as a novel brain-enriched isoform of the camk-i family that was lipid-anchored by multiple lipid modifications, prenylation and palmitoylation, resulting in enrichment of click-iii into lipid rafts fractions. in situ hybridization revealed the abundant presence of click-iii transcript throughout the central nervous system in mouse embryos. to test the role of click-iii during early neuritogenesis, a shrna vector specific for click-iii was delivered into dissociated cortical culture. we found that knock-down of click-iii resulted in significant decrease in the number and total length of dendrites. results from introduction of click-iii into a click-iii-null context confirmed this finding. surprisingly, lipid modifications of click-iii seemed to contribute to fully elicit such an effect. we thus uncovered a novel signaling mechanism by which lipid raft insertion and local activation of a camk can be efficiently coupled to actin cytoskeletal signaling during dendritogenesis. os a- - transcription factor control of dendrite arbor ultrastructure adrian moore, reiko amikura, shiho nakao, andrew liu, emi kinameri riken brain science institute, japan the different functions of neurons in a complex nervous system are reflected in a large diversity of dendrite arbor morphologies. the drosophila larva dendritic arbourization (da) neurons consist of four classes (i-iv) with increasing levels of arbor complexity. these diverse arbor shapes develop due to class specific mechanisms of dendrite branching and outgrowth. here we show that these class specific differences in dendrite arbor morphology are controlled by a combinatorial code of transcription factors. we have developed a system to label individual dendrite arbors then subsequently identify them in electron microscopic sections. using this method we illustrate that the dendrites of class i neurons, with a simple arbor, contain a high density parallel array of microtubules; on the other hand class iv neurons, with a complex arbor, contain a low density meshwork of microtubules. we are presently investigating how these differences in ultrastructure are controlled by the transcription factors making up the combinatorial code. os a- - segmental and hox related cues are involved in the establishment of the somatotopy yasunori murakami igbmc, strasbourg, france in the rodent, trigeminal sensory inputs are topographically relayed, and mapped in the somatosensory cortex. little is known about the mechanism underlying the development of the somatotopic organization. by fate mapping of specific rhombomeres (r), we found that principal sensory (prv) neurons derived from r receive predominantly inputs from the maxillary branch of the trigeminal nerve and uniquely contribute to the whisker map. by conditional inactivation, we found that early expression of hoxa in r is required for pathfinding and positioning of trigeminal nerve afferents. at later stages, hoxa expression in prv neurons provides instructive cues for topographic arborization of maxillary axons. moreover, while prv neurons appeared normally specified, loss of hoxa function resulted in selective loss of eph expression, and altered axonal projections from prv to the ventral posterior medial (vpm) nucleus of the thalamus, and absence of a postnatal whisker map at any level of the neuraxis. thus, hoxa dependent cues are required to determine the territory for whisker representation in r and the assembly of a somatosensory circuit. os a- - the second wave of corticospinal innervation after synapse elimination of the first wave tsutomu kamiyama, masaki sakurai dept. physiol, teikyo univ. sch. med., tokyo, japan in the previous study we showed that the rat corticospinal (cs) terminals and synapses were widely distributed at p and those in the venrtolateral (vl) area were eliminated from p to p and that the number of terminals in the dorsomedial (dm) and vl area began to increase again from p and further increased thereafter. in the present study we further studied the subsequent developmental time course of cs terminal distribution. cs axons were anterogradely labeled by injection of biotin dextrane (bda) into the sensorimotor cortex. the number of the terminals began to increase from p , reaching peak around the third postnatal week. labeling of single or a few by microinjection axons revealed that at p some additional cs axon branches appeared within the dorsal column of the target spinal segment and further ramified after entering the gray matter. however, the number of axons did not increase in the brainstem and the upper cervical cord. these suggest that the second wave of innervation is explained mainly by branching of cs axons just before and after entering the spinal gray matter. os a- - proteomics of the growth cone: i. protein profiling of the growth cone the growth cone is a motile tip formed at the developing neuronal processes, and functions for the accurate determination of the axon pathway and the synaptogenesis. in higher organisms, however, the molecular basis of the growth cone is poorly understood for the present, since the information on the protein localization there is insufficient to explain the growth cone functions. proteomics is a powerful strategy for identifying the protein composition in a given cell or a subcellular compartment, and the application of this method to the growth cone should help us solve the above question. we obtained the whole growth cone (gcp) obtained from neonatal rat forebrain and the membrane subfraction of the gcp (gcm), and then those fractions were analyzed using proteomics. we have identified several hundreds of the distinct proteins of these fractions. here, we show the profiling of gcp and gcm, and will discuss the overview of these protein profiles in relation to the growth cone functions. axonal branching is thought to be regulated by not only genetically specified molecules but also neuronal activity. however, the interplay between these two mechanisms remains largely unknown. to study this issue, we analyzed the role of electrical activity in layer-specific thalamocortical (tc) axon branching by using organotypic cocultures. during the second week in vitro, yellow fluorescent protein-labeled tc axons formed branches primarily in the target layer. spontaneous firing was found to increase when branches were formed abundantly. pharmacological blockade of synaptic transmission diminished layer-specific branching considerably. moreover, time-lapse imaging showed that branching was generated dynamically by elimination as well as addition in the target layer and that blockade of synaptic activity reduced this remodeling. these findings suggest that synaptic activity modifies layer-specific tc axon branching by regulating the remodeling process with molecular cues expressed in the target layer. research funds: kakenhi ( ), kakenhi ( ) os a- - the application of navigation-guided repetitive transcranial magnetic stimulation for intractable deafferentation pain naoki tani, yoichi saitoh, haruhiko k., satoru oshino, masayuki hirata, amami katoh, toshiki yoshimine department of physiology, university of osaka, osaka, japan repetitive transcranial magnetic stimulation (rtms) has been applied to control intractable deafferentation pain (dp). but nobody has investigated which cortical area is the most effective target for pain relief. therefore, we stimulated m , s , sma, premotor accurately with a navigation-guided rtms and compared their effects of pain relief. at the same time, rtms ( , , hz, stimulations) was compared in dp patients. the pain relief was evaluated with visual analogue scale. high frequency ( , hz) rtms of m was the only effective stimulation for treating intractable pain in of patients ( %). the pain relief continued for h significantly. we would like to discuss the mechanism of pain relief with high frequency rtms of m . os a- - involvement of atp on nociceptive modulation in rat model of masseter muscle pain yasuo sugiura, noriyuki ozaki, masamichi shinoda department of functional anatomy and neuroscience, nagoya university graduate school of medicine, nagoya, japan we determined the role of p x r on pressure pain and mechanical hyperalgesia in a newly developed rat model of pain in masseter muscle (mm) . the pain in the mm was assessed by the pressure pain threshold (ppt) defined as the amount of pressure required to induce head flinching. the mm injection of ␣,␤-meatp (p x , , / rspecific agonist) significantly enhanced the behavioral response to the pressure. this enhanced response was completely blocked by the co-application of ␣,␤-meatp with ppads (p x , , , , / , / r-specific antagonist). excessive muscular contraction of mm produced by the electrical stimulation significantly decreased the ppt indicating mechanical hyperalgesia of the mm. administration of ppads to the exerted mm produced a complete recovery of decreased ppt. p x rpositive neurons innervating the exerted mm increased in trigeminal ganglia. our results suggest that p x r plays an important role in pressure pain, and mechanical hyperalgesia caused by excessive muscular contraction of mm. the present study was undertaken to investigate the change in the activation of the nociceptive neuronal circuit under a neuropathic pain-like state. here we found sciatic nerve ligation (snl) produced a marked increase in the number of c-fos-positive cells in the periaqueductal gray (pag). using the fluoro-gold (fg) microinjection into the pag, numerous fg-labeled cells were detected in the hypothalamus. in the arcuate nucleus (arc) of the hypothalamus, the immunoreactivity (ir) for an excitatory neuronal maker, fosb was increased, whereas the ␤-endorphin (␤-ep)-ir was decreased days after snl. furthermore, the subpopulations of ␤-ep-positive cells were co-labeled with fosb in the arc. the present data suggest that the hypothalamus can be received by snl-induced concomitant nociceptive signals, leading to continuous activation of neurons projecting to the pag. this phenomenon, in turn, indirectly controls pain transmission in the dorsal horn through the descending antinociceptive pathway. os a- - the cantor-like patterns in rat hippocampal ca pyramidal neurons tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca . this prediction includes an attractor dynamics expected in the associative network, which was proposed by many authors, since marr's theory of simple memory in the hippocampus. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to find cantor-like patterns experimentally from the ca pyramidal neurons. temporally associated and non-associated electrical stimulations were delivered to schaffer collaterals, and membrane potentials were recorded by patch-clamp recording method. in our results, cantor-like patterns were observed in hippocampal ca pyramidal neurons. young songbirds shape their songs using memorized tutor songs and auditory-vocal feedback. we prevented zebra finches from hearing their own vocalizations by exposure to loud noise after days of age, before which they had been reared with song tutors from birth. when the noise stopped at - days of age, the birds sang unstable and noisy song syllables that did not resemble the tutor syllables. the similarity to the tutor syllables steadily increased until the time of song crystallization ( days later). these findings show that the memory of tutor syllables still exists well beyond the normal age of song crystallization (d of age) and that zebra finches can develop songs using the memory well after the normal period of song development. the temporal order of syllables resembled the tutor model only in birds released from the noise before days of age. thus, different schedules and processes may govern the learning of syllable phonology and syntax. in addition to well-characterized areas, a novel adult neurogenic region; the temporal germinal layer (tgl) was identified in rats (takemura, ) . a tracer study revealed that there is an interconnection between the dorsal part of the tgl and the lateral nucleus of the amygdala, suggesting a functional implementation of tgl neurogenesis in amygdala-dependent emotional memory processing. to investigate this possibility, we performed a tgl region-specific low-dose irradiation, which can selectively kill proliferating cells and hence can reduce neurogenesis, using a gamma knife. the tgl-irradiated rats expressed a significantly increased tone-related long-term fear memory, indicating a functional significance of the tgl neurogenesis for aversive memory reduction. we (tsukada and pan, ) systematically examine the functional difference between spatio-temporal learning rule (stlr) proposed by tsukada ( ) and hebbian learning rules in a single-layered neural network, computing their ability to differentiate spatiotemporal sequence. in this paper, we tested physiologically the cooperative plasticity without a postsynaptic spike in the ca hippocampal network. tsuda and kuroda proposed a mathematical model for the cantor coding in the hippocampal ca . they also predicted chaotically transitory dynamic behavior called chaotic itinerancy in the hippocampal ca . this prediction includes an attractor dynamics expected in the associative network, which was proposed by marr and others. the time series of events, which could be output from ca , may be encoded in ca in an efficient way. the proposed cantor coding is effective, because the topology of time series is naturally measured on the cantor set since each element of cantor set represents a single time series. however, our mathematical model is too abstract to describe physiological feature of neurons. then, we have tried to make more realistic model of ca , using -compartment model of neuron, and we found the cantor coding of information of time series in the model ca . it is known that neurons can propagate action potentials with high temporal precision. however, it is unclear how precisely closely neighbouring neurons synchronize and whether they can code information. here we show that sub-millisecond synchronization can code information as well as the discharge rate modulation. we found that closely neighbouring pyramidal neurons in the ca region of the hippocampus synchronize with sub-millisecond precision. the optimal frequency bands for transmitting these synchronizations matched the beta, gamma and fast-ripple oscillations. moreover, we found that the synchronizations were commonly coupled with rate modulations in relation to both internal (retention and comparison) and external (stimulus and motor) events. the synchronization often occurred in relation to stimulus inputs even when rate modulation was clearly absent. therefore, our results suggest that sub-millisecond synchronization plays an important role in propagating information in the hippocampus. the alterations of cerebral motor function by chronic ischemia are poorly understood, since no motor symptoms are noticeable in most of the cases. we evaluated spatial distribution and intensity of eventrelated desynchronization of beta band (beta-erd) evoked in motor area using synthetic aperture magnetometry in patients with chronic ischemia due to diverse vascular occlusive diseases (n = ) and moyamoya disease (n = ). contrary to the normal motor activation, ipsilateral beta-erd was dominant during grasping task of affected hand in patients. this abnormal activation was obscured by self-paced finger tapping requiring more selective hand motor programming. and it was more frequently observed in the atherosclerotic hypoperfusion (with white matter change) than in other pathogenesis. ipsilateral beta-erd may be a new indicator of subclinical functional alteration in motor cortices caused by chronic ischemia. os a- - hypothermia protects against cerebral ischemia by suppressing ␦pkc activation takayoshi shimohata , , heng zhao , gary steinberg department of neurology, brain research institute, niigata university, niigata, japan; department of neurosurgery, stanford university, stanford, usa hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not fully elucidated. ␦pkc is reported to induce apoptosis upon activation. its activity is modulated by phosphorylation, translocation and proteolytic cleavage. we investigated effects of hypothermia on ␦pkc activation using a rat permanent distal mca occlusion model. mild hypothermia ( • c) reduced infarct size by %. western blots indicated that ␦pkc cleavage increased markedly in ischemic core but moderately in penumbra after stroke, which is suppressed by hypothermia (p < . ). p-␦pkc (t ) dephosphorylated after stroke; this effect is blocked by hypothermia. full-length and cleaved form ␦pkc as well as p-␦pkc (s ) translocate from the cytoplasm to the mitochondria and nucleus, which is suppressed by hypothermia. ␦pkc activator suppressed the protective effect of hypothermia. taken together, hypothermia blocks ␦pkc activation after focal ischemia. this effect might contribute to hypothermic neuroprotection. calcium responses in situ following ischemia remain unclear. we sought to determine, in rats, the calcium changes following transient forebrain ischemia. in anesthetized adult rats, -vessle occlusion was induced. fluo- /am was microinjected, and the fiber-coupled confocal microscope [imaging fiber bundle coupled to the microlensattached nipkow-disk scanner (csu- , yokogawa, japan) equipped with × objective lens] was inserted into the brain. -vessle occlusion induced comparable ischemia in both hippocampus and frontal cortex. fluorescence intensity of fluo- increased up to %, and persistently increased up to % during -min reperfusion, indicating the long-lasting ca + increase in the ca region. in contrast, in the frontal cortex, -min ischemia increased fluorescence intensity during ischemia but not reperfusion. in the ca region but not in the frontal cortex, transient forebrain ischemia induces long-lasting increase in ca + in situ. research funds: kakenhi # , # os a- - reevalution of classical view on resident microglia: neutrophils may play more critical roles than resident microglia at acute phase of ischemic and traumatic brain insults hiroaki matsumoto , h. watanabe , y. kumon , t. ohnishi , chi ii , y. imai , j. tanaka dept. neurosurgery, ehime university, japan; dept. molecular and cellular physiology, ehime university, japan resident quiescent microglia (mg) are thought to respond quickly to a variety of pathologic events in the brain, by proliferating and producing a number of bioactive substances including proinflammatory cytokines and nitric oxide (no). in the present study, however, we found that the majority of resident mg died through apoptosis within h after the onset of ischemic and traumatic brain insults. we further noticed that traditional mg markers isolectin b and cd b recognized with ox antibody histochemically stained neutrophils, which were identified by neutrophil-specific elastase, rather than iba + mg or macrophages. accumulation of neutrophils was observed at the very early phase of the insults, while they expressed proinflammatory cytokines and inducible no synthase. iba + amoeboid-shaped mg started to accumulate days after the insults. the data prompted us to reevaluate the roles and the fate of resident mg in the brain. os a- - insulin regulates the hepatic clearance of amyloid ␤ peptide tetsuya terasaki , , chihiro tamaki , sumio ohtsuki , graduate school of pharmaceutical sciences, tohoku university, sendai, japan; sorst, jst, japan the liver is the major organ that eliminates amyloid ␤-peptide (a␤) from the circulation, and we have revealed that low-density lipoprotein receptor-related protein (lrp- ) is a molecule responsible for the hepatic clearance. since epidemiologic investigations suggest the high incidence of alzheimer's disease in diabetes mellitus, the purpose of this study was to clarify the effect of insulin on the hepatic clearance of a␤ . insulin infusion into the rat portal vein increased lrp- expression in plasma membrane fraction of liver, but did not affect the expression in whole lysate. insulin treatment also increased the hepatic uptake of a␤( - ), which reached . -fold greater uptake than non-treated control after min treatment. increase of the hepatic uptake of a␤( - ) by insulin was concentration dependent (ec = pm), and was completely suppressed by rap ( m), an lrp inhibitor. these results suggest that insulin induces translocation of lrp- to the plasma membrane of hepatocytes, leading to increase of a␤ hepatic clearance from the circulation. research funds: sorst, jst os a- - mr images of intra-arterially administered microglia surrounding ␤-amyloid deposit in the rat brain the therapeutic use of microglial cells has recently received some attention for the treatment of alzheimer disease (ad), but few noninvasive techniques exist for monitoring cells. here we present a magnetic resonance imaging (mri) technology to track micrgolia cells injected intra-arterially in a rat model of ad. we labeled microglia expressing gfp with resovist using the hvj-e vector. we administered labeled microglia into the carotid artery of the rats. mri revealed clear signal changes attributable to resovist-containing microglia in a␤-injected areas. this study demonstrates the usefulness of mri for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia as therapeutic tools for ad. extravasation of protease-activated receptor (par) activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of cns disorders, which causes pathophysiological changes in neurons and glial cells. to elucidate the mechanism of thrombin-induced activation of astroglial cells, we used n astrocytomas that show a characteristic retraction of bipolar protrusions after activation of pars with thrombin. the thrombin-induced morphological change of n cells was inhibited by an inhibitor of ip receptors, -aminoethoxydiphenyl borate ( -apb) or an endoplasmic reticulum ca + -atpase inhibitor, cyclopiazonic acid (cpa). in parallel, thrombin-induced mobilization of ca + was inhibited by -apb and cpa. moreover, removal of external ca + accelerated the reversal of thrombin effects. these results suggest that refilling of ca + store by ca + entry play an important role in the cytoskeletal dynamics of astroglial cells. to clarify the occurrence range of neurofibrillary tangles (nft), we reexamined an autopsied alzheimer patient with the onset at age and a -year-clinical course. the brain showed severe atrophy ( g). microscopic examination disclosed that all telencephalic neocortices had nft of more than and sp of more than . all limbic cortices and nuclei had nft of more than and sp of more than . although there was no sp, various numbers of nft were observed in the following structures: claustrum , caudate , globus pallidus , hypothalamus , meynert's nucleus , thalamus , substantia nigra , central gray , locus ceruleus , purkinje cells , posterior root ganglion , adrenal medulla . this study revealed that there exist nft-rich neurons and free neurons. the latter includes purkinje cells and posterior root ganglion cells. considering the pathogenesis of nft, it must be valuable to clarify qualitative/quntitative differences between nft-rich neurons and free neurons. os a- - transcriptional regulation of androgen receptor in aging mouse brain androgen receptor (ar) mediates action of androgen, which is involved in memory, behavior and other brain functions that deteriorate with advancing age. in aging mice brain, ar mrna expression was measured by rt-pcr, ar promoter methylation by southern hybridization, and proteins binding to promoter by emsa. ar mrna level was significantly higher in male than female, and it was downregulated by testosterone, but upregulated by estradiol in adult mice. female mice exhibited higher methylation of ar promoter than males. methylation was increased by testosterone, but decreased by estradiol. furthermore, dnasei accessibility to ar promoter was reduced in males, increased by gonadectomy but reduced by sex steroids in adult male. incubation of brain nuclear extract with plabeled ar promoter yielded three specific complexes. the intensity of these complexes varied with age and sex. these findings show that ar mrna expression and promoter methylation are inversely regulated by sex steroids in the adult mice cerebral cortex. such regulation of ar expression might influence androgen action and consequently brain function during aging. reliability of synaptic transmission depends on the efficiency of transmitter removal from the synaptic cleft, as well as on the release machinery and the postsynaptic response mechanism. it has been shown in various synapses that postsynaptic and glial excitatory amino acid transporters (eaats) contribute to glutamate removal. however, the role of presynaptic eaats remains unclear. using mouse retinal slices, we examined the contribution of eaats at the rod to rod bipolar cell (rbc) synapse. the kinetics of the rbc current evoked by electrical stimulation of rods was slowed by pharmacological blockade of eaats. recordings of the evoked rbc currents from eaat subtype-deficient mice and the eaat-coupled anion current revealed that functional eaats are localized to rod terminals but not to postsynaptic or glial cells. model simulations suggest that rod eaats are densely packed near the release site, and that rods are equipped with an almost self-sufficient glutamate recollecting system. trpv is a thermosensitive trp channel, and activated by body temperature. we found functional-trpv was expressed in soma, dendrites and synapses in the neurons. since trpv was firstly cloned as an osmotically activated channel, we hypothesized trpv might be involved in volume regulation of the spines. therefore, we quantified the spine volume changes by glutamate stimulation, and confirmed trpv expression related to the volume increase of spines. next, we compared the resting membrane potential (rmp) between wild type and trpv -deficient neurons at • c, and found rmp in wild type was more depolarized by approximately mv than rmp in trpv -deficient neurons. we also performed current-injection experiments in both neurons, and found that trpv -deficient neurons required much bigger currents to get their firing. thus, we conclude that trpv is involved in regulation of both neural activity and spine motility in hippocampus. os p- - a system for rapid uncaging in defined patterns and its application hiroshi kojima department of intelligent information systems, tamagawa university, tokyo, japan neurons integrate many sysnaptic signals at dendrite. understanding these information processes is a central topics in experimental and computational neuroscience. the use of focused laser beam for uncaging can provide fine spatial resolution to analysis of neural function. however, most experiments were carried out either at spatial locations or in a very simple scanning patterns. we developed a system for performing uncaging in arbitrary pattern in order to emulate realistic neural activity. our system is capable of patterned photorelease of caged neurotransmitters at locations per ms with submicron resolution. ultraviolet laser light is steered by galvano-mirrors and projected onto the surface of preparations for uncaging the caged chemicals. simultaneously, imaging of neurons are obtained by -photon microscopy and electrophysiological experiments can be done. we briefly report the present system for rapid uncaging and its application to neurophysiological research. os p- - d -like receptors selectively block p/q-type calcium channels to glutamate release onto cholinergic neurons in the rat basal forebrain a number of molecules have been identified in the sensory ganglia including those involved in the signal transmission to the brain. their functions, however, remain largely unknown. we tried to develop a method enabling to inhibit gene expression in the sensory ganglia in vivo by rnai and to evaluate its effect on the synaptic transmission in the brain slices. for this purpose, we selected the nodose ganglion (ng), in which the neurons sending glutamatergic projections to the nucleus tractus solitarii in the brainstem, are located. in anesthetized young wistar rats, synthetic sirna against the genes coding adenosine a receptors (adora ) was introduced to the ng by electroporation. one to five days after sirna delivery, the expression level of adora in the ng decreased by > % of that in the non-treated ng, being not accompanied by a change in mrna level for a a receptors. this technique might be promising in analyzing the function of specific molecules involved in transmitter release regulation at the brain synapses. nmda-receptors are specific constituents of glutamatergic system in brain responsible for molecular mechanisms of recognition and learning. activation of neurons by nmda results in intracellular generation of reactive oxygen species (ros) and reorganization of cell metabolism. exposure of rodent and human lymphocytes with nmda results in ros increase within the cells which is suppressed by nmda antagonists. moreover we have demonstrated by rt-pcr technique and by using anti-nmda-antibodies the expression of nmdareceptors on lymphocyte membranes. in addition, we shown that nmda receptor dependent signal from lymphocyte membrane is transformed into specific intracellular reactions controlling caspase- activity and interferon-␥ synthesis. in the presentation, properties of nmda-receptors and their functional role in immunnocompetent system are discussed. small molecule g-protein arf in combination with phospholipase d (pld) is essential for intracellular trafficking of the proteins from endoplasmic reticulum to golgi apparatus. however, it is recently reported that it also regulate ionic channel activity at the cytoplasmic membrane. to examine possible involvement of arf and subsequent pld in regulation of receptor-induced responses in neurons, we recorded k + -current response to dopamine (da) in the ganglion cells of aplysia under conventional two-electrode voltage clamp. intracellular application of arf blockers such as brefeldin a, exo , and arf n-terminal peptide, markedly suppressed the da-induced response. furthermore, intracellular application of ␣-synuclein, a specific blocker of pld, significantly depressed the k + -current response to da. these results suggest that arf and subsequent pld may regulate the k + -current response induced by da. os p- - p gap, a brain-enriched rhogap, is involved in the nmdar-mediated signaling takanobu nakazawa , toshihiko kuriu , ayako m. watabe , toshiya manabe , shigeo okabe , tadashi yamamoto div. of oncology, inst. med. sci., univ. of tokyo, tokyo, japan; dept. of cell biol., tokyo medical and dental univ., tokyo, japan; div. of neuronal network, inst. med. sci., univ. of tokyo, tokyo, japan nmdar regulates structural plasticity by modulating actin organization within spines. however, the signaling pathways that link nmdar activity to the postsynaptic actin cytoskeleton are poorly understood. we identified a brain-enriched rhogap, p gap, which interacts with the nr b subunit of nmdar. within neurons, p gap was highly concentrated in the postsynaptic density and co-localized with nr b and an actin-binding protein, cortactin. p gap promoted gtp hydrolysis of cdc and rhoa in vitro and in vivo. nmdar stimulation led to de-phosphorylation and redistribution of p gap. when over-expressed in dissociated neuron, p gap suppressed the activities of rho gtpases, which resulted in spine elongation. taken together, the results suggest that p gap is likely to be involved in nmdar activity-dependent actin re-organization in spines. os p- - non-static method to directly quantify the transfer of firing correlation from one neural population to another: fokker-planck method hideyuki cateau riken brain science institute, saitama, japan firings of only a few neurons are too weak to be transmitted safely, to activate other neurons to fire, or to contract muscles. therefore, we implicitly assume that brain function is exerted by macroscopic population of neurons. to characterize how a macroscopic neural population behave, the simulation method provide an indirect approach. many single neuron simulation runs need to be performed first before extracting macroscopic features by statistically averaging. unlike this method, the fokker-planck (fp) method directly evaluates the macroscopic features, thereby giving a clearer insight into function achievable with neuronal population. despite the lasting interests in firing correlation in coding and conveying information, theoretical studies on it have been largely confined to complicated simulation studies. here, we provide a first non-static fp analysis to directly calculate how correlation and population rates are transferred from one population to another and elaborate a dynamical interplay between these macroscopic quantities at work in time. os p- - spatial frequency tuning of disparity-selective neurons in macaque v hironori kumano , seiji tanabe , ichiro fujita grad. sch. of engineering science, osaka univ., osaka, japan; grad. sch. of frontier biosciences, osaka univ., osaka, japan to examine whether convergence across spatial frequency channels contribute to stereoscopic processing, we recorded single neuron activity from area v of awake, fixating monkeys. for each neuron tested, we first measured the spatial frequency tuning with sinusoidal gratings or two-dimensional ( -d) filtered noise images, and then examined the disparity tuning with both correlated and anticorrelated dynamic random-dot stereograms (rdss). neurons with broader spatial frequency tuning had more attenuated disparity tuning for anti-correlated rdss. in a subset of v neurons, we analyzed responses to various combinations of binocular disparity and spatial frequency by using -d filtered noise stereograms. the disparity tuning of most v neurons was consistent across a range of spatial frequencies to which they were sensitive. we suggest that v neurons pool disparity signals across spatial frequency channels to create an unambiguous representation of stereoscopic depth. os p- - predicting the monkey's behavioral choice in a stereoacuity task from neuronal responses in area v hiroshi shiozaki, seiji tanabe, ichiro fujita lab. cognitive neurosci., grad. sch. frontier biosciences, osaka univ., japan many neurons in visual area v of macaque monkeys are selective for binocular disparity. most disparity-selective neurons in v are sensitive to small changes in disparity near zero, suggesting that they might contribute to stereoacuity. however, the role of these neurons in stereoscopic depth discrimination has not been directly addressed. we recorded single unit activity from v while a monkey was engaged in a fine stereoscopic depth discrimination or stereoacuity task. the monkey was trained to report by saccadic eye movement whether the center region of a random-dot stereogram was nearer or farther than its immediate surround. trial-to-trial fluctuation of visual responses of v neurons was correlated with the monkey's subsequent behavioral choice. given the cell's disparity preference, an ideal observer can predict the monkey's upcoming behavioral response from the visual response of v neurons. the results suggest that v neurons are involved in mediating stereoacuity. os p- - the role of disparity energy and binocular matching processes in stereopsis takahiro doi, seiji tanabe, ichiro fujita lab. cognitive neurosci., osaka univ., japan the early visual system computes disparity energy of stereo images. some of the next stages retain this information, while other stages perform further computation to solve the stereo correspondence problem. we addressed how the energy and correspondence computations underlie stereopsis. we asked human subjects to discriminate depth of random-dot stereograms with various amounts of disparity. at each disparity level, we manipulated the proportion of dots with the same luminance contrast between the two eyes by reversing the contrast of some dots in one eye. at small disparities, the proportion of correct choices increased monotonically from chance to perfect as the proportion of the same-contrast dots was increased. at large disparities, the subjects perceived reversed depth when contrastreversed dots dominated, and the proportion of correct choices reached only chance level when the two types of dots were balanced. the results suggest that the correspondence and energy computations underlie fine and coarse stereopsis, respectively. we introduce a novel receptive field (rf) analysis, lsrc, which can reveal various aspects of visual receptive fields that were undetectable previously in a single measurement. the visual stimuli are standard wide-field -d ternary dynamic random noise, generally refreshed every - ms. unlike the conventional reverse correlation which computes a spike-triggered average (sta) of the stimuli themselves, lsrc computes the sta of the spectra of localized regions of the stimuli. both simulations and recordings from cat v /v neurons demonstrate that lsrc is capable of revealing details of complex cell rfs, cross-orientation suppression, variations of orientation tuning within rfs that might lead to shape selectivites. since the stimuli can cover a wide visual field area, and few assumptions are made regarding specific shapes or features in stimuli, lsrc is highly suitable for multi-neuron, multi-area studies spanning retina, v , and especially areas beyond. research funds: mext( ), jsps( ), coe os p- - analysis of center-surround organization of v neurons as a high-order receptive field hiroki tanaka, izumi ohzawa graduate school of frontier biosciences, osaka, japan responses of area (v ) neurons are influenced by stimuli not only in their classical receptive field (rf) center, but also in its surround. such a center-surround organization may be considered as a unified higher-order rf. we have sought to obtain detailed structures of such a rf by harmonic analyses of responses to drifting contrast-modulated sinusoidal gratings that cover both the center and surround regions. of cells analyzed, % showed spatial frequency tuning curves that were well fitted with gaussian. by taking the inverse fourier transform of these curves, spatial center-surround rf was obtained as gabor functions with spatial phases between ± degrees. highly asymmetric structures were observed for cells with strong surround suppression. estimated sizes of center and surround were well correlated with those from size tuning curves. moreover, there was no space-time tilt in the center-surround rf. the results suggest that neurons with surround suppression are capable of coding various spatial forms of higher-order features (figure-ground borders), but are insensitive to motion of such stimuli. os p- - spatial organization of receptive fields of complex cells in the early visual cortex kota sasaki , izumi ohzawa , grad. school of eng. sci., osaka univ., japan; grad. school of frontier biosci., osaka univ., japan little is known about the quantitative internal structure of the receptive fields (rf) of complex cells, although this is crucial for understanding how a complex cell acquires its function by collecting inputs from neurons in the preceding stage. therefore, we have analyzed the relationship between the spatial nd-order interaction kernels and the rf envelopes of complex cells. extracellular single unit recordings were performed in anesthetized and paralyzed adult cats. threevalued (i.e. gray, dark, and bright) dynamic white noise stimulus with × dots was presented over an area to times larger than the rf of a complex cell. for each dot location, a nd-order kernel and its envelope (by hilbert transform) were calculated. the rf envelope of the neuron was determined by summing the envelopes of nd-order kernels at all locations. nd-order kernels had roughly comparable extent as the rf, and contained . subregions on average (n = ). among complex cells, whose rf envelopes were elongated, cells exhibited the horizontal elongation. research funds: mext( ), jsps( ), coe os p- - orientation tuning of neuron in cat lateral geniculate nucleus tomoyuki naito , osamu sadakane , masahiro okamoto , hironobu osaki , hiromichi sato grad. sch. med., osaka univ., osaka, japan; grad. sch. front. biosci., osaka univ., osaka, japan; med. sch., osaka univ., osaka, japan we examined the orientation selectivity of lgn neurons of anesthetized cats and found that although about % lgn neurons showed significantly orientation-biased response to the grating with optimal size and spatial frequency (sf), and that % of lgn neurons exhibited significant orientation selectivity to gratings with diameter larger than its classical receptive field (crf) and sf higher than the optimal for crf response. two stimulus-size tuning curves measured for responses to stimulation with the optimally-or null-orientated grating exhibited profile similar to each other under the optimal sf condition. however, high sf grating caused stronger surround suppression for response to the orthogonally oriented stimulus than that to the optimally orientated stimulus. our results suggested that elliptic crf center produces orientation-biased response of lgn neurons. furthermore, surround suppression of lgn neurons tuned to particular stimulus orientations enhances orientation selectivity of lgn neurons. os p- - temporal dynamics of suppressive receptive field surround in cat v satoshi shimegi, hiroyuki kida, ayako ishikawa, hiroshi sakamoto, hiromichi sato graduate school of medicine, osaka university, toyonaka, japan in the primary visual cortex (v ), a neuronal response to stimulation of the classical receptive field (crf) is suppressively modulated by the stimulus presented at the receptive field surround (srf). using stationary flashes ( ms) of sinusoidal grating with optimal parameters and varying radii as stimuli, we examined the temporal dynamics of the surround suppression in v cells of anesthetized cats. stimulus slightly larger than the crf caused suppression in early response (< ms) but not in middle ( - ms) and late responses ( - ms). as stimulus size was further enlarged, the middle and late responses were remarkably suppressed while the early response was only moderately or weakly suppressed. radius of surround suppressive field progressively expanded in temporal sequence from . deg (early response) to deg (middle response) and . deg (late response). thus, modulation of early response seems to reflect whether stimulus is larger than crf size or not, and late response to reflect how wide area is stimulated. research funds: kakenhi ( ) os p- - spatial-frequency dependent surround suppression in cat v ayako ishikawa , satoshi shimegi , hiroyuki kida , hiromichi sato grad. sch. front. biosci., osaka univ., osaka, japan; grad. sch. med., osaka univ., japan; grad. sch. eng. sci., osaka univ., japan we examined the temporal dynamics of the surround suppression of visual response in terms of spatial-frequency (sf) tuning of neurons in cat v . we used a stationary flash (duration, ms) of a circular sinusoidal grating patch with optimal orientation and sf as crf stimulus, and that of an annulus ( ms) with optimal orientation but varying sf as srf stimulus. first, we stimulated crf and srf simultaneously (stimulus-onset-asynchrony (soa) = ) and analyzed time course of surround suppression. sf tuning of the surround suppression changed along time course of response, and effective sf of surround suppression shifted from the sf lower than that optimal for crf response (c-sf) to that near c-sf. next, changing soa, we examined surround suppression on different temporal phases of crf response. soa-dependency of surround suppression changed according to the temporal phase of response. these results suggest that multiple mechanisms with different sf-and temporal characteristics are involved in the surround suppression. os p- - contrast-dependency of spatial summation property in cat v and lgn masahiro okamoto , tomoyuki naito , osamu sadakane , hiromichi sato grad. sch. front. biosci., osaka univ., japan; grad. sch. med., osaka univ., toyonaka, japan we examined contrast-dependent change in a receptive field (rf) size and strength of surround suppression of neurons in the primary visual cortex (v ) and the lateral geniculate nucleus (lgn) of anesthetized cats. rf structure was modeled by spatial interactions of excitatory and inhibitory gaussians. both in v and lgn, ratio of gaussians (rog) model captured size-tuning curves of responses better than difference of gaussians (dog) model. under the high contrast stimulus condition, the peak of size tuning curve shrank by . and . times in v and lgn, respectively. in lgn, surround suppression was strengthened under high contrast stimulus condition, but in v , the strength of surround suppression did not affected by stimulus contrast on average. we conclude that ) rog model describes the surround suppression better than dog model both in v and lgn, ) under high contrast stimulus condition, there is a reduction of rf size with a shrinking of excitatory gaussian, which is confirmed with rog model. hiroyuki kida , satoshi shimegi , ayako ishikawa , hiroshi sakamoto , hiromichi sato grad. sch. eng. sci., osaka univ., japan; grad. sch. med. sci., osaka univ., japan; grad. sch. front. biosci., osaka univ., japan in the primary visual cortex (v ), neuronal responses to stimulation of the classical receptive field (crf) were suppressed by the presence of stimuli at surround receptive field (srf). we examined whether the suppression varied according to spatial configuration of srf stimuli in v neurons of anesthetized cat. the crf stimulus was a circular patch of sinusoidal grating with optimal stimulus parameter. srf was divided into flanks ( • step), and stationary stimulated with an annulus, oppositely-faced flanks ( -fk) or a flank ( -fk) stimulus. the durations of stimulus presentation were ms for crf and ms for srf stimulation. localized srf stimulation with either -fk or -fk exerted significant suppression on crf responses. according to the analysis of spatiotemporal change in srf effects, there was no particularly suppressive srf area for -fk stimulation throughout the crf response. however, -fk stimulation of end position to crf had strong and long-lasting suppression on responses during - ms after onset. os p- - temporal-frequency dependency of receptive field size and surround suppression in lgn and v osamu sadakane , tomoyuki naito , hironobu osaki , masahiro okamoto , hiromichi sato grad. sch. med., osaka univ., japan; med. sch., osaka univ., japan; grad. sch. front. biosci., osaka univ., osaka, japan spatial summation property of neurons in the primary visual cortex (v ) varies depending on stimulus parameters (e.g., stimulus contrast). in this study, we examined how temporal frequency (tf) of grating stimulus affects size-tuning properties of cat v neurons. our results showed that, when the tf was higher than the optimal, the strength of surround suppression became weak and receptive field size became larger, suggesting that v neurons change their spatial property according to tf in such a way that neurons integrate wide visual field for fast moving stimulus, whereas localized field for slow stimulus. we also tested the effect of changing stimulus size on tf tuning curve. consistent with above-mentioned results, large grating made the peak and the high cut-off of tf-tuning curve higher than those for small grating. in the lateral geniculate nucleus (lgn), we obtained basically similar results to those of v neurons, suggesting that the subcortical tf tuning property contributes to that in v . ryo sasaki, takanori uka department of physiology (i), juntendo university, tokyo, japan a few studies have shown that basic tuning functions in early visual cortex change during visual perceptual learning (schoups et al. ; yang and maunsell ) . the change in neuronal sensitivity in these studies, however, is small compared to the improvement in behavioral sensitivity. here we hypothesized that the read out of information from sensitive neurons was modified by learning. to test this hypothesis, we investigated whether learning modifies neuronal sensitivity or read out of middle temporal (mt) neurons during learning of a depth discrimination task. two monkeys were trained to report the depth of moving dots (near or far), and we recorded from isolated mt neurons during the course of training. the monkeys showed improvement in discrimination thresholds across daily sessions. in contrast, the sensitivity of mt neurons did not change, whereas the correlation between neuronal activity and the monkey's behavioral choice increased during the course of training. these results suggest that plasticity due to perceptual learning occurs within the neural pathway following area mt. we developed an in vivo method to localize the fine tip of a glassinsulated tungsten microelectrode for chronic recording using . t mri. the scan conditions were first optimized by imaging a microelectrode that was sunk into copper sulfate solution. the microelectrode tip was precisely localized up to a resolution of m under particular geometrical scan condition. we then examined the applicability of the method in vivo under this optimized scan condition in the temporal cortices of three monkeys. the microelectrode was penetrated into the dorsal or ventral bank of the superior temporal sulcus and the tip was localized by the high-resolution mri. the accuracy of this method was validated by comparing the localized positions of the microelectrode tips with the corresponding electrolytic lesion marks in histological sections. a transient signal change in diffusion-weighted image of the brain has been detected in human visual cortex. the time course of this signal was ahead of the bold signal and characterized by a steep onset. diffusion-mri thus represents a new exciting mechanism for fmri. in order to increase its efficiency we aimed at defining a diffusion response function (drf) as a counterpart of the hemodynamic response function (hrf). an volume of interest was defined using spm with a boxcar function. gamma-variate functions were used to model the steep onset. the parameters of the drf were estimated by fitting the time-course with the drf convolved with a boxcar. although the magnitude of the signal change (around %) was smaller than that of bold (> %), the temporal profile showed a constant precedence of the diffusion signal by . s. os p- - new insights on normal and pathological brain function from tomographic analysis of magnetoencephalographic signals laboratory for human brain dynamics, brain science institute (bsi), riken, wako-shi, japan tomographic analysis of magnetoencephalography (meg) data combines exceptional temporal resolution with accurate localization, at least for places a few centimeters away from the center of the head [moradi, et al., neuroimage; ioannides et al., cerebral cortex] . this unique capability of probing brain function across the entire cortex and deep brain structures from milliseconds to minutes in the same experiment has already provided new insights about normal [ioannides et al., cerebral cortex;ioannides et al., neuroimage] and pathological [ioannides et al., j. neurosc.] brain function. novel ways of analyzing meg data provide direct measures of regional brain activity over much longer timescales. these new methods are used in ongoing studies to probe the nature of global brain activity in different states of awareness (e.g. different stages of sleep) and explore the relationship between estimates of electrophysiological activity derived from meg with hemodynamic measures of brain activity. os p- - spatial registration of stand-alone fnirs data to mni space ippeita dan, archana singh, masako okamoto national food research institute, japan the registration of functional brain data to the common brain space offers great advantages for inter-modal data integration and sharing. however, this is difficult to achieve in functional near-infrared spectroscopy (fnirs) because fnirs data is primary obtained from the head surface and lacks structural information of the measured brain. therefore, we present a method for probabilistic registration of fnirs data to the standard montreal neurological institute (mni) template through international - system without using the subject's magnetic resonance image (mri). the standard deviation in probabilistic registration thus performed for given head surface points is approximately within cm. this means that if the spatial registration error is within an acceptable tolerance limit, it is possible to perform multisubject fnirs analysis to make inference at the population level and to provide information on positional variability in the population, even when subjects' mris are not available. stochastic perturbation in scale is a basic property of biological systems and generates scale-independent structuration and functional dynamics in spatial and temporal patterns, which can be characterized by fractal dimensionality. it allows a user-independent evaluation and does not rely on subjective evaluation in image assessment. we have used a box-counting algorithm in scale-space segmented images to determine the mass fractal dimension of ventricles in different neurological disorders. three groups of subjects [alzheimer disease (ad), obstructive hydrocephalus (oh) and normal controls] were examined. mass fractal dimension is high for ad ( . ), approaching unity (∼ . ) for oh, and in between for control ( . ). statistical analysis was performed and significant differences were observed for these groups (p < . ). the observations are accounted by a flow dynamics heterogeneity model. the implications are that stochastic structuration and fractal dimension may be useful to track temporal progression of disease and assess therapeutic management. thrombin, a serine protease essential for blood coagulation, also plays an important role in injury associated with intracerebral hemorrhage. in this study, we revealed that mitogen-activated protein kinase (mapk) pathways contribute to thrombin-induced brain injury in two experimental models. firstly, we employed organotypic cortico-striatal slice cultures. application of thrombin to slice cultures resulted in cortical neuronal injury and striatal shrinkage. the cortical neuronal injury was ameliorated by inhibition of extracellular-signal regulated kinase (erk) but not p mapk, while the striatal shrinkage was prevented by both of them. secondly, thrombin was injected into rat striatum. thrombin-induced brain injury determined by immunoreactivity of neuronal marker was reduced by inhibition of erk and p mapk. these results suggest that mapk pathways play important roles in thrombin-induced brain injury and they should be therapeutic targets against neurodegeneration associated with blood-brain barrier destruction. positron emission tomography was used to study brain activations during motor imagery of standing and during performance of standing posture in parkinson's disease (pd). eight pd patients performed mental and motor tasks: ( ) resting, ( ) staring at a standing human object, ( ) thinking of standing, ( ) standing with eyes open, ( ) standing with eyes closed. regional cbf data analyzed by spm were compared with normal counterparts. the cerebellar vermis was more activated during imagination of standing in the pd group than in healthy group. as seen in healthy subjects, standing also activated the primary sensorimotor foot area and cerebellar vermis in pd patients, but the between-group comparison generated greater activations in the vermis and prevuneus in pd. the cerebellar vermis engages in postural balance both in mind and reality, and the precuneus may play a more important role in postural control in pd. os p- - potentiation of nmda receptor-mediated current by metabolic failures through glycine release facilitation in the hypoglossal motoneurons of the rat yu kono , , eiji shigetomi , kiyoharu inoue , fusao kato dept. neurol., jikei univ., sch. med., tokyo, japan; lab. neurophysiol., jikei univ., sch. med., tokyo, japan to elucidate the mechanism underlying the selective vulnerability of motoneurons (mns) to metabolic failures (mfs), we compared the membrane current responses of mns and non-mns to mfs. experiments were performed on neurons in the hypoglossal nucleus (xii) and dorsal motor nucleus of the vagus nerve (dmx) in the young rat brainstem in the presence of ttx. mfs were induced by nacn or oxygen deprivation. in xii neurons, mfs induced large persistent inward currents accompanied by marked increase in strychnine-sensitive synaptic inputs, indicating facilitation of glycine release onto xii neurons. furthermore, nmda receptor-mediated current evoked by exogenous nmda was increased by nacn. in dmx neurons, mfs evoked outward currents without affecting synaptic inputs. these pre-and postsynaptic responses to mfs in mns might play a role in their selective vulnerability in various neurodegenerative diseases including the amyotrophic lateral sclerosis. os p- - effects of mdma on serotonergic neurons in rat organotypic mesencephalic slice culture including the raphe nuclei yuichi suzuki, megumi higuchi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharmaceu. sci., kyoto univ., kyoto, japan , -methylenedioxymethamphetamine (mdma) is a recreational drug of abused which has been shown to increase serotonin ( -ht) release and cause degeneration of -htergic nerve terminals via -ht transporter, although the mechanisms are unclear. in this study, we developed rat organotypic mesencephalic slice culture including the -htergic raphe nuclei, and examined the effects of mdma and methamphetamine (meth) on -ht release and -htergic neurotoxicity. immunohistochemical studies for tryptophan hydroxylase revealed abundant -htergic neurons around the raphe nuclei. treatment with a -htergic neurotoxin , -dihydroxytryptamine dramatically reduced the tissue contents of -ht and its metabolite, which was blocked by a selective -ht reuptake inhibitor. mdma and meth ( . - m) increased -ht release, and reduced the tissue contents of -ht and its metabolite at higher doses. the mesencephalic slice culture including the -htergic raphe nuclei may be useful to examine the mechanisms underlying -htergic neurotoxic effect of mdma in vitro. os p- - studies on drug dependence (rept. ): involvement of platelet-derived growth factor (pdgf) receptor in the morphine-induced rewarding effect masami suzuki, minoru narita, michiko narita, tomoko takeuchi, yasuyuki nagumo, keiichi niikura, tsutomu suzuki dept. of toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan the present study was undertaken to investigate the involvement of platelet-derived growth factor (pdgf) receptor in the morphineinduced rewarding effect in rodents. extensive coexpression of tyrosine hydroxylase with pdgf receptor was apparently observed in the rat ventral tegmental area (vta). the levels of dopamine and its major metabolites in the nucleus accumbens (n.acc.) were markedly increased by the microinjection of pdgf into the rat vta. the morphine-induced rewarding effect was suppressed by intra-vta microinjection of pdgf receptor fc chimera. the increased level of dialysate dopamine produced by morphine in the rat n.acc. was significantly decreased by intra-vta injection of pdgf receptor fc chimera. these findings suggest that the stimulation of -opioid receptors in the vta by morphine leads to the activation of pdgf receptor, which may be directly responsible for the morphine-induced rewarding effect in rodents. os p- - prostaglandin d is a strong mediator of neuroinflammation in genetic demyelinating mouse model prostaglandin (pg) d , an inflammatory mediator, mainly produced by hematopoietic pgd synthase (hpgds). microglial activation and gliosis are commonly observed during the neuroinflammation. in twitcher (galct wi/twi ), a genetic demyelinating mouse model, we found that hpgds expression was upregulated in activated microglia accompanied by the dp receptor induction in hypertrophic astrocytes. using primary culture of glial cells, we demonstrated that activated microglia produced large amount of pgd by hpgds and that astrocytes expressed both dp and dp receptors and were activated by pgd . we found that gliosis and demyelination were well suppressed in hpgds-or dp -null twitcher and twitcher treated with an hpgds-inhibitor. these results suggest that pgd is a key molecule of neuroinflammation involved in the demyelination. research funds: , os p- - on a sodium channel distribution enabling high frequency signal processing go ashida , , kousuke abe , kazuo funabiki grad. sch. medicine, kyoto univ., kyoto, japan; grad. sch. informatics, kyoto univ., kyoto, japan some auditory neurons, such as the owl's nucleus laminaris (nl) cells, can sense very high frequency signals (up to khz). from the theoretical point of view, it seems exceptionally difficult to handle these high frequency signals because the membrane time constant is far longer. first, we discuss a biophysical mechanism of shifting the membrane time constant by connecting the large cell body (soma) with the small node of ranvier. next, we discuss the effect of sodium channel distribution on the impedance function of the membrane. sodium conductance in the soma amplifies low frequency signal components below khz, while that in the node does up to khz. last, as a typical example, we discuss the capability of high frequency signal processing in the owl's nl neuron. some biological evidences indicate that sodium channels in the nl neuron are distributed mainly in the nodes but less in the soma. by using an nl neuron model, we show that a neuron with low somatic sodium conductance and high nodal sodium conductance can achieve fine sensitivity to high frequency signals. interaural time difference (itd) is calulated using axonal delay lines and coincidence detector neurons (nucleus laminaris:nl). however, little is known about the cellular mechanisms of coincidence detection. here, we report the results of in vivo intracellular recordings from the barn owl's nl. we used coaxial glass electrodes in which one (microelectrode) was inserted into a patch-electrode type capillary. the inner sharp electrode was protected by the outer one during penetration of the cerebellum. we isolated nl cells from owls and achieved intracellular recordings in of them, as judged by a sudden dc potential drop and the resting membrane potential (mean rp = ± mv). nl neurons produced small spikes and oscillatory potentials whose waveform closely resembled the superposition of the tones delivered to the two ears (sound analogue psps:sap). the amplitude of saps varied as a function of itd. spike rates changed in linear proportion to the amplitude of sap. we evaluated sound localization ability of vision impaired and sighted persons by using a 'two-sound sources discrimination test' in a semianechoic darkroom. in total, vision impaired ( blind and low vision) and sighted persons participated. the stimuli were pure tone pulses. for each trial, the same single sound pulse was emitted consecutively from a pair of speakers with the same angle either left or right from the midline of the subject. localization ability was assessed whether the subjects are able to discriminate two sound sources or not in each trial. the discriminability of the blind subjects slightly exceeded that of sighted subjects but the difference was not significant. the discriminability of the low vision subjects, on the other hand, was significantly lower than that of blind or sighted. it was suggested that a peculiar 'object perception' of blind persons is not able to measure by means of 'two-sound sources discrimination test.' os p- - autocrine bmp signaling in astroglia sensitizes the glial scarring masahisa yamada , runa araya , naoto kitamura , yuji mishinsa yamada unit, riken bsi, saitama, japan; nihs, nieh, nc, usa bone morphogenetic proteins (bmps) affect growth of glial cells however, contribution of bmps during glial scar formation is unknown. to study the role of bmp signaling in vivo, we disrupted bmpr a, one of the type i receptors for bmps, in a telencephalic neuronal stem cell-specific manner. we found that aberrant architecture of microvessels that led to a failure in maintaining the blood-brainbarrier in the mutant mice. although mutant mice showed inflammation around the cortical microvessels, proliferation of hypertrophic reactive-astrocytes in the mutant mice was attenuated. disruption of astroglial bmpr a expression by cre-adenovirus recapitulates the same phenomena. bmps were upregulated in reactive astrocytes in after brain injury. knocking down of bmpr a by small interfering rna in primary astrocytic culture negatively affected their astrocytic growth injured by scratch, which reinforced the importance of autocrine bmp signaling in astrocytes. this result opens up the understanding of novel mechanisms underlying the autocrine bmp signaling on glial scarring after cns injury. the present study was undertaken to evaluate the functional role of the glial cells in the induction of stress. here, we found that aging mice promoted anxiety-like behaviors as characterized by both the light-dark and elevated plus-maze tests, and they exhibit an increase in astrocytes in the cingulate cortex. a robust increase in gfap-positive astrocytes was noted in the cingulate cortex of nerve-ligated mice that exhibited the anxiety-like behavior. in contrast, iba -positive microglial cells were dramatically increased as compared to that in control mice and some of them were co-localized with brdu-like immunoreactivity in the hippocampus of mice exposed to chronic psychological stress. our results indicate that the increase in astrocyte or microglia in the cingulate cortex or hippocampus may lead to emotional disorders including aggravated anxiety under aging, chronic pain-like state or exposure to chronic psychological stress. withdrawn os p- - fucosylation prevents overshooting of the migration by the vagus motor neuron precursors shigeharu kinoshita , , hideomi tanaka , , sachiko tsuruoka , hironori wada , , hitoshi okamoto , riken bsi, wako, japan; jst crest, kawaguchi, japan; riken rrc, wako, japan the vagus motor nuclei are important as the autonomic center for the maintenance of homeostasis. aberrant positioning of nuclei is implicated in the etiology of the sudden infant death syndrome (sids). therefore, control of precursor cell migration into the right position may be crucially important. the zebrafish embryo has two vagus motor nuclei, the dorso-laterally and medially located nuclei (dmx and mmx). the dmx precursors are born near the floor plate, migrate dorso-laterally and then are accumulated at the defined position. in the towhead mutant embryos, ectopic neurons are distributed between bilateral dmx where precursors aberrantly migrate in dorsal direction and fail to stop at the right position. positional cloning and mrna rescue analysis identified towhead as a gdp-mannose , dehydratase (gmds), a key enzyme for de novo synthesis of a gdp-fucose. as a result, the mutant embryos showed exclusive reduction of fucosylated glycans. our findings represent that fucosylation is responsible for maturation of these neurons. in development of the drosophila visual center, photoreceptor cells extend their axons (r axons) to the lamina ganglion layer and trigger proliferation and differentiation of synaptic partners (lamina neurons) by delivering the inductive signal, hedgehog (hh). this mechanism helps to establish an orderly arrangement of connections between the r axons and lamina neurons, termed a retinotopic map because it results in positioning the lamina neurons in close vicinity to the corresponding r axons. it is found that the bhlh-pas transcription factor single-minded (sim) is induced by hh in the lamina neurons and is required for the association of lamina neurons with r axons. in sim mutant brains, lamina neurons undergo the first step of differentiation but fail to associate with r axons. as a result, lamina neurons are set aside from r axons. the data reveal a novel mechanism for regulation of the interaction between axons and neuronal cell bodies that establishes precise neuronal networks. research funds: kakenhi ( ) os p- - initial molecular steps in synaptogenesis in vivo: trans-synaptic interaction of cell adhesion molecule is involved in postsynaptic assembly of psd -homolog dlg hiroshi kohsaka, etsuko takasu, akinao nose department of physics, university of tokyo, tokyo, japan trans-synaptic interaction via cell adhesion molecules (cam) is essential in constructing synapse structures. although this notion has been supported by various studies in vitro, evidence in vivo has been lacking. here we used live-imaging and genetic analysis to show that a drosophila cam fasciculin (fas ) mediates early interaction between pre-and postsynaptic cells in synaptogenesis in vivo. by visualizing gfp-tagged fas genetically expressed on a muscle, we found fas accumulated at postsynaptic site just after the contact between growth cones and its target muscle. genetic and deletion analysis implied that trans-synaptic interaction with presynaptic fas is crucial for the postsynaptic localization of fas . in addition, postsynaptic localization of a scaffolding protein dlg, psd -homolog, and glutamate receptors was impaired in fas mutants. these results provide the first in vivo evidence that trans-synaptic cell adhesion molecule has a role in inducing the assembly of synapses. gaudilliere brice harvard medical school, usa postsynaptic differentiation of dendrites is an essential step in synapse formation. we report here a requirement for the transcription factor myocyte enhancer factor a (mef a) in the morphogenesis of postsynaptic granule neuron dendritic claws in the cerebellar cortex. a transcriptional repressor form of mef a that is sumoylated at lys promoted dendritic claw differentiation. activity-dependent calcium signaling induced a calcineurin-mediated dephosphorylation of mef a at ser and thereby promoted a switch from sumoylation to acetylation at lys , leading to inhibition of dendritic claw differentiation. our findings define a mechanism underlying postsynaptic differentiation that may modulate activity-dependent synapse development and plasticity in the brain. research funds: ns , ag ps a-a characterization of mrna species that are associated with postsynaptic density fraction by gene chip microarray analysis we previously reported the partial identification by random sequencing of mrna species that are associated with the postsynaptic density (psd) fraction (tian et al., ) . we report here further characterization by gene chip analysis of the psd fraction-associated mrnas, which were prepared in the presence of rnase inhibitor. we confirmed that a large number of mrna species are associated with the psd fraction and found that mrnas encoding various postsynaptic proteins were highly concentrated in the psd fraction. we identified some mrna species that were highly concentrated in the psd fraction. we also constructed a cdna library using the psd fraction-associated mrnas as templates, and identified randomly selected clones by sequencing. our data suggested that the psd fraction-associated mrnas are a very useful resource, in which as yet uncharacterized genes are concentrated. tian et al., . mol. brain res., , - . research funds: kakenhi ( ) ps a-a the distribution of snap- protein is regulated in isofom-specific manner makoto itakura, saori yamamori, kouta takano, masami takahashi department of biochemistry, kitasato university school of medicine, sagamihara, japan two isoforms of snap- derived from exon splicing are expressed in brain. we generated two specific antibodies for snap- a and b, and studied the distribution in rodent brain. there was a sticking difference in expression of snap- a and b during early postnatal period. snap- b was low at the birth and increased remarkably thereafter. by the contrast, snap- a increased transiently and attained a maximum level around seven day after birth. furthermore, there seemed to be a difference in their distributions in plasma membrane, since a substantial amount of snap- b but not snap- a was recovered in raft-enriched fractions of triton x- -treated lp membrane after sucrose density gradient centrifugation. immunohistochemistry demonstrated that snap- b was widely distributed throughout brain, whereas, snap- a was restricted to some particular regions of brain. these results indicate that expression and distribution of snap- protein are regulated differently in isoformspecific manners, and snap- a and snap- b play different functional roles in brain. ps a-a erc(elks/rab ip /cast) regulates syaptic short-term plasticity by recruiting bmunc - to the active zone camkii in the postsynaptic sites is localized as a psd-anchored or a cytoplasmic form. camkii in the two sites is interchangeable by its translocation. translocation and targeting of this kinase to appropriate subcellular compartments are crucial for its physiological function. we have previously suggested that postsynaptic camkii is also localized in lipid raft microdomain ( . mol. brain res. , - ) . in this report, we proved the lipid raft localization of camkii by detergent-treatment and successive sucrose floatation assay of spm or cos cells expressing camkii, and by cholesterol depletion from membrane using mbcd. we also investigated the mechanism and properties of camkii targeting to lipid raft. camkii targeted to lipid raft microdomain possibly through protein-protein interaction. our data suggest that lipid raft microdomain is a major site of camkii distribution, as well as postsynaptic density and cytoplasmic region, at the postsynaptic site. glial glutamate transporters, glast and glt- , are co-localized in processes of bergmann glia wrapping excitatory synapses on purkinje cells (pcs). although glast is expressed six-fold more abundantly than glt- , the decay kinetics of climbing fiber (cf)mediated excitatory postsynaptic currents (cf-epscs) in pcs in glast(-/-) mice are not significantly different from those in wildtype mice. here we attempted to clarify the roles of glial glutamate transporters in cf-pc synapses using glast(-/-) and glt- (-/-) mice, and a novel antagonist of glial glutamate transporters, ( s, s)- -[ -( -methoxybenzoylamino)benzyloxy]aspartate. our results indicate that glial glutamate transporters can retain the fast decay kinetics of cf-epscs in the normal range if a small proportion (approximately %) of functional transporters, glast and/or glt- , is preserved. glutamate is well known as an essential neurotransmitter in nervous system. how glutamate-mediated synaptic transmission is controlled in neural circuit of live animal, however, remains to be poorly understood. we found that the loss-of-function mutations in vglut (vesicular glutamate transporter) encoded by eat- gene led to abnormal sensory behaviors including thermotaxis in c. elegans. thermotaxis defect of eat- mutant was caused by malfunction of both thermosensory neuron afd and its downstream interneuron ria, suggesting that thermal signals from afd or ria to their downstream neurons are transmitted by glutamate through eat- vglut. a mutation in avr- glutamate receptor also led to abnormal thermotaxis. we are trying to investigate whether avr- functions in the downstream neurons of afd or ria, and to identify other glutamate receptors involved in thermotaxis. through the analysis of thermotaxis neural circuit, we are hoping to reveal the mechanisms of glutamate-mediated synaptic transmission at neural circuit level. ps a-a biochemical characterisation of the vesicular glutamate transporter stephan schenck, shigeo takamori department of neurology and neurological science, st century coe program, tokyo medical & dental university, tokyo, japan vesicular glutamate transporters (vgluts) load synaptic vesicles with glutamate, the major excitatory transmitter in the brain, thus making these transporters of outstanding importance for the function of the central nervous system. the three known isoforms of these secondary active transporters have been characterised in terms of tissue distribution, developmental expression patterns and some pharmacological features. while the third isoform constitutes only a minor fraction, vglut and vglut are abundantly expressed in the brain with a complementary distribution pattern and divergences in the expression profile during ontogeny. so far, no clear difference in the function of vglut and vglut has been found. to further characterise the specific properties of the transporters we make use of the vglut -ko mouse which gives us the opportunity to investigate a brain devoid of vglutl. we focus on synaptic vesicle fractions from ko-mice to study the vglut-associated transport biochemically. in recent years, three isoforms of vesicular glutamate transporters (vgluts) have been molecularly identified in mammals. histological investigations have revealed that the distribution of three vglut isoforms in the cns is largely complementary with limited overlap, suggesting that differential expression of vglut isoforms may contribute to functional diversity in glutamatergic synapses. however, functional differences among the isoforms remained poorly understood. to get insights into their isoform-specific property, we searched for interacting protein(s) to the c-terminus of vglut by yeast two-hybrid screening and found endophilin a . as expected for the interacting molecule to vglut , endophilin a was typically localized to vglut -positive synaptic terminals in cultured hippocampal neurons. we are currently investigating physiological significance underlying their direct interaction and co-localization. the aim of this study is to investigate the molecular basis for lactate utilization. hippocampal neuronal culture was continuously superfused with glucose or lactate solution and spontaneous excitatory postsynaptic currents (sepscs) were recorded from a voltage-clamped pyramidal neuron. in lactate solution, amplitude of epscs was decreased in ∼ min, followed by spontaneously recovered after min, while epsc in glucose medium remained unchanged. application of apv+ni in lactate medium, spontaneous recovery was not observed. in neuron cultures, incorporation of c-lactate was gradually increased, which was suppressed by applications of inhibitors for calcium calcium channels or protein kinase c. in glial cell cultures, incorpotation of lactate was initially maintained. increased expression of monocarboxylate transporter (mct) was demonstrated in the lactate medium. results suggested that increased mct expression of neurons may lead to utilization of lactate to sustain synaptic function via calcium-dependent manner. yumei wu , kazuhito tomizawa , shuang liang , iori ohmori , teiichi nishiki , kohji takei , hideki matsui dept. of physiol., okayama univ., okayama, japan; dept. of neurosci., okayama univ., okayama, japan synaptic vesicle endocytosis is regulated by phosphorylation of endocytotic proteins, such as amphiphysin (amph) i and dynamin i. here, we show a novel type of regulation of vesicle endocytosis by proteolysis. in mouse hippocampal slices, amph i was found to be cleaved by a ca + -activated protease, calpain during prolonged depolarization or stimulus trains. the calpain-cleaved n-terminal amph i fragment lost its ability to bind dynamin and inhibited transferrin uptake as overexpressed in cos- cells, indicating that the calpain cleavage of amph i inhibits endocytosis. amph i in hippocampus was also cleaved by calpain in vivo after kainate seizure. although the second administration of kainate caused less severe seizure activity than the first one, this relieved second seizure was not observed in pre-treatment with a calpain inhibitor, allm during the first seizure. thus, the proteolytic activity of calpain could protect neurons from excitotoxicity by inhibiting vesicle recycling. synaptic vesicles (svs) are effectively recycled by endocytosis for continuous synaptic transmission. previously, we have suggested that a high level of synaptic transmission is maintained by recycling of svs through two types of endocytosis operating coordinately ( th this meeting). in the present study, we labeled endocytosed svs at nerve terminals of drosophila with fluorescence dyes, fm - and fm - , and also measured quantatively exocytosis and endocytosis of svs, using these dyes. egfp-labeled cacophony ca + channels and anti hrp stained the active zone and non-active zone at synapse, respectively. imaging analysis revealed that two distinct types of endocytosis of svs occurred at the active zone and the non-active zone of motor nerve terminals. we have previously shown that baclofen, a gaba b receptor agonist, inhibits exocytosis in synapses of mouse hippocampal neurons. syntaxin a is also known to modulate exocytosis. to characterize the molecular mechanisms involved, the inhibitory effects of baclofen in neurons transfected with antisense oligonucleotide to syntaxin a were investigated by patch-clamp recording and counting the number of release sites. transfected neurons showed higher frequency of miniature epscs and stronger inhibition by baclofen than controls, but no change in number of sites. increased exocytosis is thus induced by increases in transmitter release per site, rather than by more sites due to neurite sprouting. these results suggest that gaba b receptor shares part of the mechanism involved in modulation of exocytosis with syntaxin a in mouse hippocampal neurons. we have previously shown a transient localization of tubulin (tub) during synaptic vesicle (sv) cycling in drosophila nerve terminals. the tub localization is detected during sv recycling, while microtuble (mt)-loop is observed throughout sv cycle. in this study, we characterized the two distinct tub localizations and showed their relation with sv pool formation. axonal mts and mt-loops abounded in acetylated (acetyl) tub. the transient localization was either polymerized or depolymerized, and organized by non-acetyl tub. taxol decreased the non-acetyl tub localization but not mt-loops, and inhibited exo/endo cycling pool (ecp) formation. in boutons containing mt-loops, ecp formation was also inhibited. acetyl mt-loops tend to be stable whereas presynaptic non-acetyl tubs are either free dimers or dynamic mts. these results suggest that presynaptic dynamic tub, especially non-acetyl tub, controls ecp formation. presynaptic tub dynamics may regulate functional presynaptic plasticity by controlling sv pools. research funds: grant-in-aid for jsps fellows the mechanism by which pregnenolone sulfate (pregs) enhances synaptic transmission was studied at the rat calyx of held. pregs increased the amplitude of evoked epscs, without affecting that of spontaneous miniature epscs, indicating that the site of its action is presynaptic. pregs facilitated presynaptic voltage-gated ca + channel (vgcc) currents via accelerating their activation kinetics, but had no effect on k + currents, resting conductance, or action potential waveforms. in simultaneous pre-and postsynaptic recordings pregs did not change the relationship between presynaptic ca + influx and epscs, suggesting that exocytotic machinery downstream of ca + influx was not involved in the pregs effect. neither bapta nor gtp␥s loaded into presynaptic terminals blocked the effect of pregs. we conclude that pregs enhances transmitter release via facilitating vgccs by a novel mechanism, which is independent of intracellular ca + or g-proteins. ps a-b spine targeting of endocannabinoid synthesizing enzyme, diacylglycerol lipase-␣ in the cerebellum and hippocampus endocannabinoids are neuromodulator that is released from postsynaptic neurons, acts retrogradely on presynaptic cb cannabinoid receptor, and induce suppression of transmitter release. to understand the retrograde signaling mechanisms, we investigated subcellular localization of a major endocannabinoid biosynthetic enzyme, diacylglycerol lipase-␣ (dagl␣), in the mouse brain. in the cerebellum, dagl␣ was predominantly expressed in somatodendritic membrane of purkinje cells, and highly concentrated at the base of spine neck. however, dagl␣ was excluded from the main body of spine neck and head. in hippocampal pyramidal cells, dagl␣ was selective to spines, but widely distributed within spines. these results indicate that dagl␣ is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two cell types. synprint site of voltage-gated ca + channels interacts with synaptotagmin. however, its physiological role is not entirely clear. here we report that ap- subunit can directly bind with synprint site. this interaction was ca + -dependent, being weaker at concentrations higher than nm. in contrast, the interaction of synaptotagmin with synprint was optimal at m ca + , being weaker at lower or higher concentrations. the binding domain of synprint for ap- and synaptotagmin was indistinguishable, and these proteins competed with each other for the synprint site. to assess physiological role of these interactions, we made a peptide containing synprint site, and loaded it directly into the nerve terminal at the calyx of held. this peptide blocked endocytosis measured with capacitance, and gradually diminished exocytosis upon repetitive presynaptic activations. we conclude that ca + channel synprint site makes ca + -dependent interactions with ap- and synaptotagmin thereby contributing to vesicular endocytosis. ps a-b acl- , an evolutionarily conserved acyltransferase like gene is required for normal synaptic transmission in c. elegans naoko hara, takao inoue, yasukazu takanezawa, hiroyuki arai department of health chemistry, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan it is generally accepted that various phospholipid molecular species are formed by phospholipids acyltransferase reactions. however, the physiological significance and the molecular mechanism of the remodeling are largely unknown. to address these questions, we focused on evolutionarily conserved acyltransferase like genes in c.elegans acl- ˜ , and generated their deletion mutants. the mutants of acl- gene, which is predominantly expressed in neurons and muscles, showed no apparent phenotype. however, the mutants exhibited severe movement abnormalities in fat- mutant background in which long chain polyunsaturated fatty acids are depleted. pharmacological analysis revealed that these mutants showed presynaptic defects in synaptic transmission. these abnormalities were rescued by neuron specific acl- expression, suggesting that certain phospholipid species produced by acl- are involved in maintaining normal synaptic transmission and motility of c.elegans. daisaku yokomaku , hussam jourdi , akiyoshi kakita , tadasato nagano , hitoshi takahashi , nobuyuki takei , hiroyuki nawa dept. mol. neurobiol., brain res. inst., niigata univ., japan; brain resource center, brain res. inst., niigata univ., japan; dept. pathology, brain res. inst., niigata univ., japan scaffolding proteins containing pdz domains interact with synaptic receptors and cytoskeletal components and are therefore implicated in synaptic development and plasticity. little is known, however, about what regulates the expression of the pdz proteins and how the levels of these proteins influence synaptic development. here, we show that ligands for epidermal growth factor (egf) receptors (erbb ) decrease a particular set of pdz proteins and negatively influence synaptic formation or maturation. in neocortical cultures, egf decreased the expression of grip and sap . moreover, egf treatment resulted in a decrease in the frequency of pan-pdzimmunoreactive aggregates on dendritic processes. these findings revealed a novel negative effects of erbb receptor ligands that attenuates the expression of the pdz proteins and inhibits postsynaptic maturation in developing neocortex. takatoshi iijima , eriko miura , keiko matsuda , tetsuro kondo , , masahiko watanabe , michisuke yuzaki dept. physiol., sch. med., keio univ., tokyo, japan; dept. anatomy, hokkaido univ., sch. med., sapporo, japan; mol. neurophysiol., aist, tsukuba, japan cbln is a member of the c q and tumor necrosis factor families predominantly produced in cerebellar granule cells. recently, we have shown that cbln is secreted as a glycoprotein and plays crucial roles in synaptic plasticity and synaptic integrity of purkinje cells. although other members of the cbln family, cbln - , are known to be expressed in the brain, their precise expression patterns and biochemical properties remained unclear. here, we show that each cbln member is expressed in various regions of developing and mature brains. all cbln family members could form both homomeric and heteromeric complexes each other in heterologous cells. like cbln , cbln and cbln were secreted as glycoproteins, whereas cbln was retained in the endoplasmic reticulum. these results suggest that each cbln member is potentially involved in synapse development and plasticity in various brain regions. s-scam is a synaptic membrane-associated protein with pdz domains, a guanylate kinase domain and ww domains. it interacts with various synaptic components including nmda receptor subunits, psd- and neuroligin. as we previously reported, s-scam is recruited to excitatory synapses by ␤-catenin. s-scam forms a ternary complex with neuroligin and psd- . more importantly, s-scam is involved in synaptic accumulation of neuroligin and subsequently affects the localization of psd- at excitatory synapses. in the course of these studies, we observed signals detected by anti-s-scam antibody at inhibitory synapses. we have here examined whether s-scam is indeed localized at inhibitory synapses in hippocampal neurons. we have raised questions which molecules s-scam interacts with at inhibitory synapses and which role s-scam plays in the assembly of inhibitory synapses. eriko fujita, yuko tanabe, takashi momoi division of differentiation and development, department of inherited metabolic disorder, national institute of neuroscience, ncnp, oawahigashi, tokyo, japan igsf /ra (ra ), which is a member of immunoglobulin superfamily having pdz binding domain at c-terminals, has ca +independent homophilic trans-cell adhesion activity. ra participates in synaptic junction and epithelial junctions in various tissues including testis. homozygous null (ra -/-) male is infertile and shows the defective elongating spermatids and fails to mature further. ra interacted with par- being involved in the polarity of epithelial cells via pdz binding domain at c-terminals. par- was colocalized in the cell adherent region of p embryonal teratocarcinoma cells during ra-induced differentiation into epithelial-like cells and mainly localized in the spermatid of ra +/+ testis, whereas it was undetectable in the spermatid of the ra −/− testis. ra and jam-c were localized around the head portion of spermatid and ra deficiency provided the abnormal polarization of the jam-c, which is necessary for the differentiation of round to elongated spermatid. jam-c inhibited the interaction between ra and par- . research funds: izumi kawabata, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan coordinated development of excitatory and inhibitory synapses is critical for both stability and temporal fidelity of neuron network in the hippocampus. however, there have been few analyses on postsynaptic molecular assembly in interneurons during development. to address this question, we examined dynamic properties of psd- clusters in cultured hippocampal interneurons. higher density of dendritic psd- clusters was observed in interneurons at div. at div, this difference was less prominent, mainly due to > -fold increase of psds in excitatory neurons. psd- -gfp imaging revealed lower rate of cluster appearance/disappearance in interneurons at div. the higher rate of cluster turnover in excitatory neurons, together with their higher rate of net cluster increase, may explain the delayed boost of cluster density. photobleaching of psd- -gfp revealed similar kinetics in two neuron types, suggesting additional determinants of cluster dynamics apart from the steady-state assembly rate. possible involvement of other postsynaptic molecules in interneuron psd dynamics is now being investigated. ps a-c two-photon imaging of immature dendritic protrusions and astroglial processes in hippocampal slice cultures hideko nishida, shigeo okabe department of cell biology, tokyo medical and dental university, tokyo, japan several lines of evidences indicate roles of astroglia in synaptogenesis, possibly mediated by either cell adhesion or diffusible factors. however, structural evidences supporting this claim are virtually lacking, mainly due to technical limitations in simultaneous imaging of neuronal and astroglial structures. here we visualized astroglia and pyramidal neurons in hippocampal slice cultures by combining adenovirus-mediated, cre-dependent expression of gfp with electroporation of rhodamine-dextran. two-photon time-lapse imaging of immature dendritic protrusions and astroglial processes in - div slice cultures revealed longer lifetime of dendritic protrusions having experienced astroglial contacts than those without contacts. dendritic protrusions with astroglial contacts also showed higher tendency to form spines. furthermore, expression of mutant rac in astroglial cells induced significantly longer, non-spiny protrusions than control. these findings suggest an involvement of direct astroglia-filopodia contacts in subsequent maturation of dendritic protrusions. taiko imura, fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan application of p x receptor agonists to the neurons in the nucleus of the solitary tract (nts) results in glutamate release facilitation (kato & shigetomi, ; shigetomi & kato, ) . recently accumulated evidence indicates that astrocytes affect the neuronal excitability by releasing gliotransmitters such as atp. this study was performed to determine whether such astrocyte-neuron interaction takes place in the nts. first, we analyzed the spatial localization of these cells by immunohistochemistry. a large number of gfap-positive cells with processes in the close apposition to the neun-positive neurons were found. second, we analyzed the effect on synaptic activity of localized application of atp using laser-based photolysis of caged atp in brainstem slices. uncaging of atp at neuronal dendrites ( s, -micrometer diameter) resulted in an immediate rise in mepsc frequency, in a manner sensitive to p x receptor antagonists. these results provide supports for the possible interaction between astrocytes and neuronal presynaptic terminals. research funds: kakenhi ( ) ps a-c ealy synapsin i accumulation in a granule cell axon at the filopodial attachment site of developing rodent purkinje cell dendrites in vitro isao nagata, junko kimura-kuroda department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan synapse formation between the parallel fibers (pf) and dendrites of purkinje cells (pc) occurs at an early stage in the developing cerebellar cortex of the neonatal rodent. however, the precise spatio-temporal pf-pc interaction has not been elucidated. we have found that growth of pc dendrites was initiated by the attachment of axonal neurite bundles of granule cells orienting at right angles in several types of d-and d-cerebellar cultures. here, we investigated the expression of a synaptic vesicle marker, synapsin i, in granule cell axons by multiple immunofluorescence labelings in these cultures. synapsin i was first expressed at the filopodial attachment site of a pc dendrite as a cluster of faint punctate deposits in a long axon, then they appeared to gather into a slender and finally into a small round deposit. thus, the filopodial attachment of the juvenile pc dendrites to the axons of granule cells may induce rapid formation of presynaptic terminals via local clustering of synaptic vesicles. ps a-c integrative spike dynamics of rat ca neurons: an in situ multineuronal imaging study takuya sasaki, rie kimura, norio matsuki, yuji ikegaya department of pharmacology, university of tokyo, tokyo, japan the brain operates through a coordinated interplay of numerous neurons. our new technique with large-scale optical recordings reveals the diversity of synaptic integration in hundreds of neurons. in hippocampal slices bolus-loaded with calcium fluorophores, we stimulated the schaffer collaterals and monitored the bulk presynaptic activity from the stratum radiatum and individual postsynaptic spikes from the ca stratum pyramidale. single neurons responded to varying synaptic inputs with unreliable spikes, but at the population level, the networks output a linear sum of synaptic inputs. the network activity varied from trial to trial, even though given constant stimuli. this variation emerged through time-varying recruitment of different neuron subsets, which were shaped by correlated background noise. our imaging approach enables linking single-cell behaviors to their communal dynamics, and we discovered that, even in a relatively simple ca circuit, neurons could collectively be engaged in complex information processing. it is assumed based on previous in vitro experiments by other researchers that mglur connects with syntenin at the dendrites and mglur with pick at the axon terminal in on cone bipolar cells. to prove this possibility, we investigated wild-type mouse retinas immunohistochemically and confirmed their co-localized immunopositive labels at the respective places. next, we examined which scaffold protein would connect with mglur that was known to be ectopicly expressed in the dendrites of mglur -deficient on cone bipolar cells. we observed no pick but only syntenin at the mglur -deficient dendrites, and also the syntenin immunopositivity was co-localized with mglur immunopositivity. these findings suggest that mglur connects with syntenin in place of mglur that was knockout from the on cone bipolar dendrites. noriko trpv family is identified as thermosensitive, ca + -permeable channels. trpv , expressed in sensory neurons, is activated by noxious heat above • c, whereas trpv , expressed in keratinocytes, is sensitive to moderate temperatures (> • c). here we examined the role of trpv and in regulation of body temperature (bt) by using infrared laser as a heat stimulus. in wild type mouse, though the laser irradiation which caused the increase in skin temperature up to • c did not induce the change in bt, desensitization of trpv with capsaicin resulted in the increase in bt. on the other hand, in trpv -knockout mouse, moderate thermal radiation (> • c) caused the increase in the bt. the processing of noxious and moderate thermal radiation stimuli may depend on the trpv and respectively. research funds: kakenhi ( ) ps a-c generation and biochemical analysis of a glur␣ knockout mouse hirotsugu azechi , manabu abe , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, niigata, japan; sorst/jst, saitama, japan glur␣ (glur ) is a key subunit of ampa receptors, since it is a critical determinant of their calcium permeability. to clarify the molecular function of glur␣ , we generated a conditional glur␣ knockout mouse using the cre/loxp recombination system. we first established a "floxed" mutant line gra f using c bl/ (b ) es cell line renka. the homozygous floxed mutants showed no significant abnormalities, thus our gra f was used as a target of glur␣ line. by crossing gra f and tlcn-cre that expressed cre in germ line cells, glur␣ null ko mice were produced, but most of them died within days after birth. to overcome the lethality, the glur␣ mutation was transferred onto b / or b /cd- genetic background. subcellular fractionation and quantitative immunoblot showed changes in the amount of ampa receptor subunits. these results indicated a significant role of glur␣ in the distribution of functional ampa receptors in vivo. ps a-c gisp: a novel brain specific protein that binds to the gaba b subunit and promotes its surface expression here we report the identification and characterisation of a novel brain specific kda protein, gaba b r interacting scaffolding protein (gisp), that interacts directly with the gaba b subunit via a coiledcoil domain. gisp coimmunoprecipitates with gaba b and gaba b from rat brain. in cultured hippocampal neurons gisp displays a punctate dendritic distribution and colocalises with gaba b receptors. when co-expressed with gaba b rs gisp increases the amount of gaba b protein and also promotes gaba b surface expression in the heterologous cells. furthermore, gisp increases surface expression of gaba b /gaba b complexes. these results suggest that gisp is involved in the forward trafficking and stabilisation of gaba b rs. thus gisp is an novel gaba b -binding protein potentially involved in the cell surface and/or synaptic targeting of the gaba b rs. three distinct isoforms of vesicular glutamate transporters (vglut - ) have been cloned and shown to exhibit differential distribution patterns in the brain. recent work shows the presence of vgluts in synaptic-like microvesicles (slmvs) of endocrine cells. mammalian pineal melatonin-secreting cells, pinealocytes, contain numerous slmvs which likely accumulate glutamate to inhibit melatonin synthesis. vglut and vglut seem to participate in this glutamate accumulation. in the present study, we found that vglut mrna is also expressed in the adult rat pinealocytes. vglut immunoreactivity (ir) was distributed throughout the pineal gland, and was co-localized with vglut -ir or vglut -ir in many, but not all, processes of pinealocyte. these data indicate that there are some subpopulations of slmvs which differ in the kind of vglut isoforms contained and/or in their combinations, suggesting vglut isoform-dependent sorting of slmvs to pinealocyte processes. kenzi saito , , , kenji nakamura , toshikazu kakizaki , , satoe ebihara , masakazu uematsu , shigeo takamori , minesuke yokoyama , shiro konishi , masayoshi mishina , , jun-ichi miyazaki , kunihiko obata , yuchio yanagawa , gunma univ., maebashi, japan; sokendai, hayama, japan; sorst, kawaguchi, japan; mitsubishi kagaku inst. life sci., machida, japan; kumamoto univ., kumamoto, japan; toyohashi univ. tech., toyohashi, japan; tokyo med. den. univ., tokyo, japan; univ. tokyo, tokyo, japan; osaka univ., suita, japan; riken, wako, japan the vesicular gaba transporter (vgat) loads gaba from neuronal cytoplasm into synaptic vesicles and is selectively expressed in inhibitory neurons containing gaba and/or glycine. to assess the functional role of vgat in development, we have disrupted the gene encoding vgat using cre-loxp system. western-blot analysis showed that vgat protein was absent in the homozygous embryos, indicating that the mutation had generated in a null allele. vgat knockout mice died around birth. all vgat knockout mice displayed cleft palate and omphalocele. our results suggest that vgat plays essential roles in both palate formation and ventral body wall development. research funds: kakenhi ( ) ps a-c postnatal changes in the colocalization of vglut and vglut immunoreactivities at single axon terminals of the mouse neocortex kouichi nakamura , , akiya watakabe , hiroyuki hioki , fumino fujiyama , yasuyo tanaka , tetsuo yamamori , takeshi kaneko , dept. morphol brain sci., grad. sch. med., kyoto univ., japan; crest, jst, japan; div. brain biol., nat. inst. basic biol., okazaki, japan vesicular glutamate transporter (vglut) and vglut accumulate transmitter glutamate into synaptic vesicles. the vgluts show a complementary expression pattern in the brain, but colocalize at single axon terminals in some synapses. here we quantitatively evaluated postnatal changes in the colocalization of vgluts at single axon terminals of the developing mouse neocortex by using a pixel-based correlation coefficient (cc) as an index of the colocalization. the cc was calculated from pixel values for vglut and vglut in each pixel of confocal micrographs of double immunofluorescence-labeled brain sections. in the barrels, the cc showed a prominent increase transiently around p . the cc was higher in area s than areas m and area v throughout postnatal development. our results indicate that the colocalization of vgluts in the neocortex is regulated in an age-, area-and layer-specific manner. gaba b receptors mediate slow and prolonged synaptic inhibition in the brain, and are members of the g protein-coupled receptors. here we have investigated the role of amp-activated protein kinase (ampk), as an endogenous regulator of gaba b receptor function. site-specific mutagenesis identified multiple phosphorylation sites for ampk within the cytoplasmic tails of both gaba b r and r . the activation of ampk regulated stability of gaba b receptors coupling with k + channels. together highlights a novel role for ampk in regulating the functional properties of gaba b receptors, by direct phosphorylation. given the role of ampk as a sensor of cellular stress this potential mechanism may be relevant in regulating the efficacy of synaptic inhibition under anoxic conditions and during periods of high synaptic activity. takao hirai, hiroaki nishio department of molecular pharmacology, faculty of pharmacy and pharmaceutical sciences, fukuyama university, hiroshima, japan serotonin ( -hydroxytryptamine, -ht) is a central neurotransmitter that is widely implicated in the regulation of mood and cognition, and is a peripheral signaling molecule that affects hemostasis, immune function, intestinal physiology, and other systems. there is increasing evidence for contribution of neuronal system to regulation of bone metabolism. this study was thus aimed at elucidation of possible functional expression of serotonergic system in mouse osteoblasts. rt-pcr analysis revealed constitutive expression of mrna for several -ht receptor subtypes, -ht transporter ( -htt) and vesicular monoamine transporter (vmat ) in primary cultured mouse osteoblasts and mc t -e osteoblastic cells. sustained exposure to fluoxetine, a selective -ht reuptake inhibitor, significantly prevented increase in alkaline phosphatase activities and mineralization in mc t -e . these results suggest that serotonergic system may be functionally expressed to regulate mechanisms underlying cellular differentiation and maturation in mouse osteoblasts. junko motohashi department of physiology, keio university school of medicine, tokyo, japan hotfoot mice are spontaneous mutants with ataxic phenotype. most hotfoot alleles identified so far have deletions of one or more exons coding for portions of the n-terminal domain of the ␦ glutamate receptor (glur␦ ). however, because only genomic dna was available for most hotfoot mutants, it was unclear whether truncated forms of glur␦ were actually translated and involved in the ataxic phenotype. here, we report that a newly identified hotfoot mutant, ho j, was caused by a new type of intragenic deletion of the grid gene, which was indeed translated as glur␦ lacking -amino acids in the n-terminus. mutant glur␦ proteins were retained in the soma of purkinje cells and degraded. as a result, ho j mice exhibited a severe motor discoordination on rotarod tests. furthermore, these mice exhibited sustained innervation of purkinje cells by multiple climbing fibers, and impaired long term depression, which is thought to underlie motor learning. these results indicate the importance of the n-terminal domain in glur␦ signaling and cerebellar functions. research funds: kakenhi ( ) ps a-d role of the dry motif in melanin-concentrating hormone receptor in signaling yumiko saito , yoshimi aizaki , mituse nakano , kei maruyama dept. pharamacol., saitama med. sch., saitama, japan; international university of health and welfare, tochigi, japan considerable attention has been focused on the functional importance of the highly conserved dry triplet in class a g protein-coupled receptors (gpcr). here we investigated the role of asp , arg and tyr in the dry of rat melanin-concentrating hormone receptor (mch r). in transfected cells, mutation of asp (d/a) resulted in nonfunctional receptor despite of showing moderate level of cell surface expression and an apparent affinity to mch. d/a mutation occurred with no increase in basal signaling pathway, suggesting no indication for constitutive activity. y/a mutation also yielded a loss of function phenotype that is similar to d/a mutation. mutation of the arg (r/a) showed higher ec value in signaling with a decrease in mch binding, while the level of cell surface expression exhibited only moderate decrease. these data suggest that a function for dry motif different from that widely accepted for class a gpcrs in regulating mch r-mediated signal pathway. in this study we confirmed functional heteromultimerization between a r and p y r electrophysiologically using xenopus oocyte expression system. when a r and p y r were coexpressed, application of non-hydrolyzable atp analogue induced g i/o response, showing formation of functional heteromultimers with a unique phenotype. it was also observed that the heteromultimers can activate g q/ pathway by atp analogue and also g i/o pathway by adenosine analogue, maintaining the features of the original subunits. ps a-d dual signaling via metabotropic glutamate receptor ␣ is regulated by a cytoskeletal protein . g michihiro tateyama , , yoshihiro kubo , department of biophysics and neurology, nips, aichi, japan; sorst, jst, saitama, japan the g protein-coupled metabotropic glutamate receptor ␣ (mglur ) is known to functionally couple to different types of g proteins. recently we have reported that the signaling pathways through mglur are differentially regulated by different types of ligands, glutamate and gd + . on the other hand, several cytoskeletal proteins have been reported to interact with the c-terminal cytoplasmic tail of mglur . these proteins, such as homer and . g, are also known to change the membrane expression of and modulate the function of mglur . here we investigated whether or not these cytoskeletal proteins regulate the multi path signaling of mglur . interestingly, the functional couplings of mglur to gq and gs pathways were altered by co-expression of . g, but not by homer. deletion of the c-terminal tail abolished the effect of . g, indicating that the interaction of . g with the c-terminal tail of mglur regulates the multi path signaling. ps a-d modulation of the eaac -mediated glutamate uptake by the addicsin mutant mitsushi j. ikemoto , , saori akiduki , age dimension research center, aist, ibaraki, japan; graduate school of science, toho university, chiba, japan addicsin is a murine homologue of rat glutamate-transporterassociated protein - (gtrap - ), an inhibitory modulator of neural glutamate-transporter excitatory amino acid carrier (eaac ). it contains two potential pkc phosphorylation motifs at positions - and - . however, its physiological function remains almost unknown. to clarify a significance of these pkc phosphorylation motifs, we investigated eaac -mediated glutamate transport activity in c bu- cells provided with a mifepristone-inducible expression of addicsin (wt), its mutants mutated at serine into alanine (s a) or at serine into alanine (s a). as compared with wt, s a had no inhibitory effect on glutamate transport activity under exposure to nm pma, and had increased glutamate transport activity under normal condition. by contrast, s a had the same glutamate transport activity as that of wt. thus, the eaac -mediated glutamate transport activity may be regulated by a pkc-dependent phosphorylation at serine in addicsin. kaori akashi , manabu abe , toshikazu kakizaki , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, japan; sorst-jst, saitama, japan kainate type glutamate receptors are composed of various combinations of glur␤ - (glur - ) and glur␥ - (ka - ) subunits. although their physiological functions and subunit compositions have been inferred from various studies, they are still not clear. to clarify the functions and subunit dynamics of kainate receptors, we generated glur␤ ko mice from c bl/ es cell line. the glur␤ ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, the amounts of glur␥ and glur␥ proteins were significantly decreased in the crude fraction of ca region of glur␤ ko. furthermore, subcellular localizations of both subunits were also changed in glur␤ ko. these results suggested that native kainate receptors might function as heteromeric channels (glur␤/␥) and the glur␤ subunit might determine subcellular localization of the glur␥ subunits, similar to the roles of nmda receptor glur subunits determining stability and distribution of the glur subunits. ps a-d sema d/plexin-b activates gsk- ␤ via r-ras gap activity, inducing growth cone collapse yuri ito, izumi oinuma, hironori katoh, manabu negishi laboratory of molecular neurobiology, graduate school of biostudies, kyoto university, kyoto, japan plexins are receptors for repulsive axonal guidance molecules semaphorins. we have recently reported that semaphorin d (sema d) receptor plexin-b induces growth cone collapse by functioning as an r-ras gap. here we characterized the downstream signaling of plexin-b -mediated r-ras gap activity, leading to growth cone collapse. sema d suppressed the endogenous r-ras activity in hippocampal neurons, in parallel with dephosphorylation of akt and activation of gsk- ␤. ectopic expression of the constitutively active mutant of akt, myr-akt, or treatment with gsk- ␤ antagonist suppressed the sema d-induced growth cone collapse. the r-ras gap activity was necessary for plexin-b -induced dephosphorylation of akt and gsk- ␤. plexin-a also induced dephosphorylation of akt and gsk- ␤ through its r-ras gap activity. thus, we conclude that plexin-b dephosphorylates akt and gsk- ␤ through r-rasgap activity, inducing growth cone collapse. to find proteins having relations in receptor trafficking, we searched human genome database and selected hepatocyte odd protein shuttling (hops) as a candidate gene. hops had three transmembrane domains, and expressed abundantly on a brain tissue. hops was detected in membranous regions from subcellular fractionation and immunohistochemistry. hops was recruited to membranous structures when overexpressed in cos cells. when expressed in hippocampal cultures, hops enhanced the amplitude of mepsc. from antibody feeding assay, we discovered that hops enhanced the recycling of glur . hops was co-immunoprecipitated with grip (glutamate receptor interacting protein ) when they were co-transfected to hek cells. thus, it was suggested that hops had roles in synaptic transmission enhancement by stabilization of surface glur via grip binding. serine must be taken up into neurons for their survival, because neurons lack serine biosynthetic enzyme. we have recently identified a serine transporter asc- . a neural amino acid transporter snat /ata also transports serine. we investigated their roles as serine transporters by comparing the localization of these serine transporters in the rat brain. the asc- immunoreactivity (asc- -ir) was detected in dendrites and somata of pyramidal neurons. the snat -ir was widely detected in neurons, whose intracellular localization was similar to that of asc- -ir. deferent from asc- -ir, snat -ir was also located in astrocytes and ependymal cells, especially around capillary blood vessels and ventricles. these results suggest the significant contribution of asc- and snat to the neuronal uptake of l-serine. snat might also accumulate l-serine in astrocytes from the extracellular spaces including blood and csf. ps a-d neuronal glutamate transporter eaat controls climbing fiber-mediated presynaptic inhibition of gabaergic transmission at cerebellar interneuron-purkinje cell synapses shin'ichiro satake , si-young song , shiro konishi , keiji imoto natl. inst. physiol. sci. (nips), okazaki, japan; mitsubishi kagaku inst life sci, tokyo, japan; tokushima bunri univ., sanuki, japan through extrasynaptic diffusion and activation of presynaptic ampa receptors in bc terminals. we here examined possible roles of glutamate transporters in this cf action. the eaat /glt- blocker threo- -methylglutamate, but not the glt- blocker dihydrokainate, augmented the cf-induced inhibition. cf stimulation obviously inhibited gabaergic transmission onto pcs in the lobule iii, where eaat expression was low, whereas the cf-induced inhibition was minimal in the lobule x, where eaat was abundant. the results suggest that eaat plays a major role in regulating the concentration of cf transmitters, possibly glutamate, in the route of its extrasynaptic diffusion, and determining the degree of cf-induced inhibition of gaba release from bcs depending on the regional difference of eaat expression in postsynaptic pcs. chitoshi takayama , yoshiro inoue department of molecular neuroanatomy, hokkaido university school of medicine, sapporo, japan gaba mediates inhibitory transmission in the adult central nervous system (cns). in contrast, gaba induces depolarization in the immature cns. this developmental shift from depolarization to hyperpolarization may be caused by decreasing of the intracellular chloride ion concentration regulated by two chloride ion co-transporters, na-k- cl co-transporter (nkcc ) and k-cl co-transporter (kcc ). in this study, we focused on kcc , which lowers the intracellular chloride ion concentration, and examined the developmental localization of the kcc with special reference to the neuronal development in the cerebellum. kcc was negative in the proliferating and migrating neurons. post-migratory neurons, which formed synapses, expressed the kcc . the kcc -protein was localized at the membrane of dendrites and cell bodies, whereas growth cones, axons and terminals were negative. these results suggested that formation of synapses might induce kcc -expression and localization, and gabaergic transmission might shift from excitation to inhibition after synapse formation. akinori nakajima , hisashi mori molecular neuroscience, university of toyama, toyama, japan the actions of many neurotransmitters are mediated by the members of a superfamily of receptors coupled to heterotrimeric guanine nucleotide binding proteins (g-proteins). the dopamine receptors are classified into two categories, d -like and d -like according to their pharmacological properties. the d -like receptors consist of d and d receptor, and are coupled to the adenylyl cyclase activating g proteins (gs). in the present study, we have generated a series of d receptor mutants and examined the effect on the gs coupled receptor signaling. we found that the expression of the third intracellular loop ( i loop) domain of d r fused with egfp effectively reduce camp production mediated by d and d receptors. interestingly, we also identified that the i loop domain of d r interfere with gs coupled beta adrenergic receptor signaling. these results suggest that the third intracellular loop of the d receptor is a primary determinant in its coupling to gs signaling. the activation of phosphatidylinositol-linked d -like dopamine receptor profoundly suppresses the exaitatory transmission in the developing hippocampus yoshinobu noriyama , yoichi ogawa , hiroki yoshino , masayuki yamashita , toshifumi kishimto dept. psychiatr.; dept. physiol i, nara med. univ., kashihara, japan we studied the effect of dopamine (da) on gabaergic and glutamatergic transmission in neonatal rat hippocampus from the early period of synapse formation by whole-cell patch-clamp recordings from ca pyramidal cells. da ( m) profoundly decreased gaba a receptor-mediated postsynaptic currents to % in the first postnatal week, when gaba provides excitatory drive. da also decreased ampa receptor-mediated excitatory post synaptic currents to % in the second postnatal week, when glutamate responses first appear. the da-induced inhibition declined after these periods. the receptor subtype involved in the da-induced inhibition was phosphatidylinositol (pi)-linked d -like receptor, since skf , a selective agonist for pi-linked d -like receptor, clearly mimicked the action of da. these results suggest that the activation of pi-linked d -like receptor profoundly suppresses the excitatory transmission during the early period of synapse formation in the developing hippocampus. ayuka ina , jinko konno , sachine yoshida , hideki ohmomo , hitoshi kawano , fumihiro shutoh , haruo nogami , setsuji hisano graduate sch., comprehensive human sci, univ. tsukuba, tsukuba, japan; tokyo metro inst neurosci, tokyo, japan supporting critical neurobiological roles of glutamate in mouse corticogenesis, we recently reported that cortical cells express vglut or - mrna at early fetal ages. to know roles of fetal vglut in cortical development, we studied expressions of vglut proteins in mouse fetuses by immunohistochemistry. on embryonic day (e ), vglut immunoreactivity (ir) was first detected in the marginal zone (mz), subplate (sp) and intermediate zone (imz). on e , vglut -ir was seen as puncta close to l -ir thalamocortical fiber tracts in the sp and also localized to fiber tracts expressing l or tag -ir in the imz, whereas vglut -ir was first observed in the sp and upper imz where l -ir existed. these results show that vglut ir corticofugal fibers appose to elongating vglut -ir thalamocortical fibers, suggesting that vglut may play a crucial role in glutamatemediated axon guidance to determine thalamic innervation patterns in the developing cortex. ps a-d developmental changes in the mechanism underlying activity-dependent swelling of the hippocampal ca regions michie kon , yoichi avil , hiroshi tsubokawa , dept. of information engineering, tohoku univ., sendai, japan; grad. schl. of information sciences, tohoku univ. to investigate the mechanisms underlying swelling of brain cells in association with neuronal activity, we analyzed interactions between changes in cell volume and synaptic activities in mouse hippocampal slices. swelling of several areas within the ca region were detected as increases in transmittance of near infrared light (irt). field epsps (feps) were recorded simultaneously from the stratum radiatum of ca region. in adult mice, repetitive stimulation of afferent fibers induced transient increases in irt at both somatic and dendritic regions in a frequency-dependent manner, which was temporally associated with feps. application of the bicuculline, a gaba-a receptor antagonist, reduced these optical signals. however, in mice under days old, the optical signals did not follow by high-frequency stimulation of inputs, and were not affected by an application of bicuculline. these results suggested that gaba-dependency in the mechanisms of cell volume regulation developmentally changes in the hippocampal ca region. in the present study, the effects of bilateral injections of glutamatergic agents into the hippocampal ca region on morphine-induced conditioned place preference (cpp) were investigated in rats. subcutaneous administration of different doses of morphine ( . - mg/kg) produced a dose-dependent cpp. using a -day schedule of conditioning, it was found that intra-ca administration of nmda receptor antagonist, mk- ( and g/rat) significantly attenuated the morphine ( . mg/kg)-induced cpp. moreover, nmda receptor agonist, nmda ( . , . and g/rat) significantly potentiated the morphine ( . mg/kg)-induced cpp. these results suggest that the development of morphine-induced cpp may be related to nmda and mk- receptors in that the glutamatergic system can modulate opiate reward. takahiro sonomura , kouichi nakamura , , hiroyuki hioki , masanori uemura , takeshi kaneko , dept. anatomy for oral sciences, grad. sch. med. and dent., kagoshima univ., kagoshima, japan; dept. morphological brain sciense, grad. sch. med., kyoto univ., kyoto, japan; crest, jst the majority of neostriatal neurons are medium-sized projection neurons with spiny dendrites and have so far been classified into three groups: striatonigral neurons producing ppd, and striatopallidal neurons producing ppe, and striatoinnominatal neurons producing pptb. these projection neurons are regulated in part by dopaminergic input from the substantia nigra pars compacta. it has been assumed that d receptor are expressed in striatonigral neurons and d receptor are expressed in striatopallidal neurons. in recent years, molecular cloning work has shown that there are at least five dopamine receptor genes (d , d , d , d , d ). in this study, the double-labeling method combining in situ hybridization and immunocytochemistry revealed how these five dopamine receptor subtypes are distributed among three projection neuron groups. the cerebellar tissue is good model system for the analysis of neuronal development, since dynamic neuronal development such as migration and axonal and dendritic outgrowth after birth. in the present study, we examined the localization of chondroitn sulfate proteoglycans (cspgs) by cspg-specific antibodies and lectins. cspgs are mainly observed at molecular layer in developing cerebellum (p - ) but they scarcely seen at external granular layer. electron microscopic observation demonstrated that phosphacan, one of cspgs, is localized at axonal membrane of parallel fibers. moreover, phosphacan inhibited adhesion and axonal extension of cerebellar granular neurons, while it promoted axonal fasciculation of their aggregated cultures. thus, cspgs, inhibitory molecules for axonal extension, are participated in axonal guidance cue in developing cerebellum. the vasopressin neurons are well knwon to show structural plasticity during chronic physiological stimulation such as salt loading. in the present study, salt loading significantly diminished the levels of chondroitin sulfate proteoglycans (cspgs) in vasopressin neurons. this downregulation is possibly due to proteolysis by tpa, since ( ) tpa immunoreactivity was observed at neurosecretory granules of vasopressin dendrites and terminals, ( ) salt loading increased protein and mrna levels of tpa in the somata and dendrites in the supraoptic nucleus but reduced protein levels of it in the terminals of the neurohypophysis, ( ) depolarizing agent released tpa from isolated neurosecretosomes, ( ) tpa knockout mice revealed lower ability of osmotic homeostasis and vasopressin release. thus, it is probable that tpa is participated in regulating structural plasticity of vasopressin neurons by degrading cspgs. chondroitin sulfate (cs) proteoglycans are essential for neuronal morphogenesis, including neural migration, survival and neurite formation in the developing brain. cs chains are modified by various sulfotransferases generating diverse sulfation patterns, which are assumed to be involved in the selective binding to various proteins such as growth factors. in this study, we analyzed the expression patterns of several cs sulfotransferases in the developing mouse cerebrum. using in situ hybridization analysis, it was revealed that cs sulfotransferase mrnas (u st, galnac - st, d st) were expressed in various types of cells, especially in the ventricular zone, and the cortical plate neurons just below the marginal zone. immunohistochemical analysis with anti-cs antibodies revealed that cs were highly expressed in the ventricular zone and the marginal zone. these results suggest that the cs structural domains generated by these cs sulfotransferases are involved in the regulation of the proliferation of neural progenitor cells and neuronal migration. nobuaki maeda , maki ishii , isao nagata , yumiko shimazaki dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; dept. of brain structure, tokyo metro. inst. for neurosci. chondroitin sulfate (cs) is a long polysaccharide with enormous heterogeneity that binds with various proteins in a structure-dependent manner. previously, we revealed that cs is involved in the morphogenesis of the purkinje cell dendrites. in this study, we analyzed the expression of cs in the postnatally developing cerebellum using monoclonal antibodies that recognize specific structural motifs in cs. among the epitopes recognized by these antibodies, the expression of mo- epitopes, glca( s)␤ - galnac( s) (d unit)containing structures, remarkably increased during development. detailed immunohistochemical analysis indicated that d unit-rich cs was deposited between purkinje cell surface and the processes of bergmann glia. furthermore, it was found that pleiotrophin bound to d unit-rich cs on phosphacan distributed around purkinje cells. these observations suggest that d-type structure in cs is important for the signaling of pleiotrophin, which play roles in purkinje cell-bergmann glia interaction. nobuna fukazawa , mineko kengaku , nobuaki maeda dept. of dev. neurosci., tokyo metro. inst. for neurosci., tokyo, japan; lab. for neuronal cell polarity, riken bsi, wako, japan ptp is a receptor-type protein tyrosine phosphatase, which is synthesized as a chondroitin sulfate proteoglycan that pleiotrophin-ptp signaling regulates the morphogenesis of purkinje cell (pc) dendrites. we previously revealed that ptp associated with delta/notchlike egf-related receptor (dner), which mediates the pc-bergmann glia (bg) interaction and regulates morphological differentiation of these cells. here, we found that ptp was expressed by both pcs and bgs and the expression by pc occurred at relatively late developmental stage. ptp showed patchy distribution in the dendritic shafts of pcs, which partially overlapped with the localization of dner. furthermore, we revealed that multiple tyrosine residues in the cytoplasmic domain of dner were phosphorylated and that these tyrosine phosphorylated residues were efficiently dephosphorylated by the ptp catalytic domain. these results suggested that ptp participate in the pc-bg interaction by regulating tyrosine phosphorylation level of dner. masahiko tanaka , tohru marunouchi division of cell biology, institute for comprehensive medical science, fujita health university, toyoake, aichi, japan cerebellar purkinje cells have the most elaborate dendritic trees among the neurons in the cns. to investigate the cellular and molecular mechanisms of dendrite development of purkinje cells, we cocultured purkinje cells on a coverslip with other cerebellar cells such as granule cells and astrocytes on the cell culture insert of m pore size. when purkinje cells were co-cultured with granule cells, dendrite development of purkinje cells was promoted in comparison with that in control conditions. this co-culture effect was abolished by addition of a glutamate antagonist in the cultures. in contrast, dendrite development of purkinje cells was inhibited when purkinje cells were co-cultured with astrocytes. we propose that (i) glutamate secreted by granule cells and diffused through the porous membrane of the cell culture insert promotes the dendrite development of purkinje cells and (ii) astrocytes inhibit the effect of glutamate through their glutamate transporting activity. heparan sulfate (hs) proteoglycans regulate neural development through the interaction with cell surface proteins and extracellular matrix molecules. an extracellular endosulfatase, sulffp , has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp gene, and examined the motor control. homozygotes appeared to be normal, showing no sign of ataxia. performances of the rotarod and beam-walking tests were normal compared with the control mice. both short-term and long-term adaptations in the optokinetic response were normal, while the gains in optokinetic response and vestibulocular reflex were significantly reduced. heparan sulfate (hs) proteoglycans regulate a number of developmental signaling through interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific sulfation patterns in hs, but the mechanism generating such modifications has not been fully elucidated. here we show that a new class of hs endosulfatases plays an important role in brain development. the mice deficient in either sulffp or sulffp appeared to be normal, while most of the double knockout mice died soon after birth. mutant brains had higher content of -o-sulfated disaccharide units in hs, suggesting a role of sulffps in heparan sulfate remodeling in vivo. the double mutant brains were smaller than the controls and showed some axon guidance defects. these data demonstrate that specific hs modification generated by sulffps is important for normal brain development. recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. here, we examined roles of -hydroxytryptoamine ( -ht), noradrenaline (na) and dopamine (da) in the formation of dendrites and synapses by dissociation culture. embryonic day or rat cerebral cortex was cultured in the presence of -ht, na or da. after days, we analyzed dendrite formation using anti-map antibody. after - days, we analyzed synaptogenesis with anti-psd- , anti-synaptophysin, and anti-map antibodies. the addition of -ht ( - nm), na ( - nm) or da ( - nm) increased dendritic length of pyramidal neurons. -ht ( - nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that dendritic outgrowth may be promoted by -ht a receptor, ␣ a receptor and d receptor, while inhibited by -ht a and ␤receptors. in addition, synaptogenesis was promoted by -ht a and -ht c receptors, whereas inhibited by -ht a receptor. tatsuya mori, tomoe wada, takahiro suzuki, naoyuki inagaki department of cell biology, nara institute of science and technology, nara, japan most neurons have polarized shape consisting of a single long axon and multiple dendrites. several proteins have been implicated in the establishment of neuronal polarity; however, the mechanism for neuronal polarization is not well understood. in this study, with proteomic approach, we identified a novel protein, singar, as one of the proteins which are up-regulated during neuronal polarization of rat cultured hippocampal neuron. singar was expressed specifically in brain and developmentally up-regulated during neuronal polarization in vitro and in vivo. in t cell, singar associated with p and p , the subunits of pi kinase which is considered as one of the key molecules in neuronal polarization. moreover, inhibition of singar by rna interference induced the formation of multiple axon-like neurites. these data suggest that singar ensures the formation and maintenance of neuronal polarity by suppressing the formation of surplus axons. ps a-e lrfn , a neuronal leucine-rich repeatcontaining transmembrane protain can interact with psd- naoko morimura, takashi inoue, kei-ichi katayama, jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan in a variety of organisms, proteins with leucine-rich repeat domain (lrr) function significantly in neural development. lrfn, a neuronal lrr transmembrane family, was expressed in the brain specifically. expression of lrfn was low in embryonic brain, and increased dramatically after birth. in the rat dissociated hippocampal neurons, lrfn protein was detected predominantly at mature dendrites, where it was accumulated at spines and colocalized with psd- , a postsynaptic scaffold protein. we examined the physical interaction between lrfn and psd- by immunocrecipitation and pull-down assay, since lrfn contains class i pdz domain-binding motif at its c-terminal tail. we revealed that lrfn associated with psd- /nmda receptor complex in the brain extracts and lrfn directly bound to psd- via its pdz domain-binding motif. in this study, we suggest that lrfn may play an important role in the regulation of synaptic functions. tsuya taneda, shingo miyata, hiroaki okuda, masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan protein arginine methylation is a common post-translational modification catalyzed by a family of protein arginine n-methyltransferases (prmt - ). among the prmt proteins, the prmt has some characteristic motifs in the n-terminal tract which follows its active methyltransferase site. although little attention has been paid to protein methylation in the nervous system. first of all, we have examined the distribution of the prmt in the rat brain. the prmt was expressed in the cell bodies and dendrites in the hippocampal neurons. further, the ontogenetic analysis revealed the prmt expression increased from the perinatal stages to the adulthood. these findings suggest that the prmt relates to the neural function in the young and adult brain. furthermore in order to study the role of the prmt in the brain, we tried to identify novel interacting proteins with the prmt in rat hippocampal neurons using tandem affinity purification assay coupled with mass spectrometry. ps a-e proteomics of the growth cone: ii. the systematic immunostaining analysis of the growth cone proteins identified by the proteomic research motohiro nozumi , , michihiro igarashi , div mol cell biol, grad. sch. med dent sci; trans-diciplinary res program, niigata univ., niigata, japan proteomics is a powerful method to understand the molecular composition of a given cell or a compartment of the cell. in the accompanying paper, we applied this method to the growth cone from the rat forebrain, and we identified more than several hundred proteins there. although the proteins have been determined using the powerful methods, the localization of each protein in the neuron should be confirmed; thus, we checked the immunostaining in the cultured rat cortical neurons. currently, we have already performed the immunocytochemistry concerning more than identified proteins including cytoskeletal components, signaling molecules, receptors, and cell adhesion molecules. by quantitative analyzing the fluorescent intensity using the digital imaging, we classified the growth cone proteins into several groups. we have found more than twenty proteins specifically localized in the growth cone by this analysis. research funds: kakenhi ; project-promoting grant from niigata univ ps a-e netrin- is involved in the sensory axonal projection toward the spinal cord as a repulsive guidance cue tomoyuki masuda , keisuke watanabe , kazuhiro ikenaka , katsuhiko ono , hiroyuki yaginuma dept. anat., fukushima med univ. sch. of med., fukushima, japan; div neurobiol bioinfo, nat inst physiol sci, aichi, japan in higher vertebrate embryos, the ventral spinal cord exerts chemorepulsion for dorsal root ganglion (drg) axons to orient them toward their targets. netrin- is known to be a chemorepellent for a subset of axons, the role of netrin- for ventral spinal cord-derived repulsion is, however, unknown. by employing culture assays, we report here the involvement of netrin- in this repulsion. in the mouse embryo at e , netrin- is expressed in the floor plate and the dermamyotome, and the netrin- receptor unc c is expressed in drg neurons. we show that hek-cell aggregates secreting netrin- repelled chick e drg axons. moreover, using function-blocking antibody against netrin- , we revealed the fact that netrin- plays an important role in ventral spinal cord-derived repulsion. together, these findings suggest that the ventral spinal cord repels drg axons by secreting netrin- to shape the initial trajectories of drg axons. research funds: grants-in-aid on priority area (c) (mecst to t.m.) hitoshi maeda, masaki sakurai department of physiology, teikyo university school of medicine, tokyo, japan in the previous reports, we showed that in the early development, corticospinal synapses (cs) were formed widely in the spinal gray matter but those in the ventral side were eliminated later in an activity dependent manner. however, the property of postsynaptic cells to cs input is poorly understood. in the present study, we investigated the electrophysiological and morphological properties of the neurons that receive cs synapses in the acute spinal cord slices of neonatal rat. the postsynaptic neurons that were confirmed by the stimulation of the posterior funiculus, where the cs tract is located in rodents, were whole cell patch clamped and labeled by neurobiotin tm . responsive neurons are widely distributed in the p neonates, but the ventral neurons became unresponsive after p . the majority of the ventral neurons are of multipolar type with large somata showing "repetitive" or "phasic" firing patterns; on the other hand, most of dorsal neurons have smaller somata and multipolar branches with "single" or "phasic" patterns. keisuke watanabe , hirohide takebayashi , , kazuhiro ikenaka , katsuhiko ono div. neurobiol. bioinfo., natl. inst. physiol. sci., okazaki, japan; dev stem cell biol. program, ucsf, usa netrin- is a long-range diffusible factor that exerts chemoattractive or chemorepulsive effects on developing axons growing to or away from the neural midline. however, it is not known whether netrin- also exerts chemoattractive effect on ventral-ward migrating dorsal interneurons in the developing spinal cord. to test this hypothesis, we examined dorsal interneuron migration in netrin- −/− background, using olig -lacz knockin allele, which marks most of ventral-ward migrating dorsal interneurons. in the embryonic spinal cord of olig +/lacz ;netrin- −/− mice, ventral migration of olig cells was significantly impaired. furthermore, a netrin receptor, dcc was expressed in olig -positive cells. these results suggest that netrin- exerts chemoattractive effects on ventral-ward migrating dorsal interneurons in vivo. netrin-g and netrin-g are vertebrate-specific membrane-anchored members of the unc- /netrin family that have no affinity to classic netrin receptors and their function is unknown. here we show that netrin-g and netrin-g proteins are selectively distributed on axons of distinct pathways, and each interacts with a specific receptor on target dendrites. netrin-g and netrin-g differentially bind to lrrcontaining proteins, ngl- and a related molecule nag , in vitro. ngl- and nag in the mouse brain are concentrated in distinct dendritic segments, corresponding to lamina-specific termination of axons expressing netrin-g and netrin-g , respectively. furthermore, in netrin-g and netrin-g deficient mice, in which axonal pathfinding is normal, there is selective mislocation of individual receptors within dendrites. together, these results suggest that axonal netrin-g proteins transneuronally regulate the localization of distinct receptors on dendrites, and thereby determine the properties of subdendritic segments. jinhong huang , ryuichi sakai , teiichi furuichi lab. molecular neurogenesis, brain science institute of riken, saitama, japan; division of cell growth factor, national cancer center of japan, tokyo, japan cas is a tyrosine-phosphorylated docking protein that is indispensable for the regulation of actin cytoskeletal organization and cell migration in fibroblasts. the neuronal function of cas, however, is poorly understood. here we report that cas is dominantly enriched in the brain, especially the cerebellum, of postnatal mice. during cerebellar development, cas is highly tyrosine phosphorylated and is concentrated in the neurites and growth cones of granule cells. cas coimmunoprecipitates with src family protein tyrosine kinases, crk, and cell adhesion molecules. the axon extension of granule cells is inhibited by either rna interference knockdown of cas or overexpression of the cas mutant lacking the crk binding motifs. these results demonstrate that cas acts as a key scaffold to link the proteins associated with tyrosine phosphorylation signaling pathways to the granule cell axon elongation. research funds: huang was a postdoctoral fellowship recipient of jsps in in vitro cerebellum-pons-medulla block preparations isolated from neonatal rats on p -p , stimulation of parallel fibers produces excitation of purkinje cells lasting for - ms. this unusually prolonged response is observed in the lateral region of the cerebellum (paraflocculus/flocculus), where purkinje cells develop primary dendrites on p -p . since -agatoxin iva and -conotoxin mviic abolished the prolonged response, we suggest the involvement of p/q type ca channels. immunohistochemical labeling revealed that p/q type ca channels emerged in paraflocculus/flocculus and uvula/nodulus lobules on p and that they then locate in purkinje cells, in cell body on p and in primary dendrites on p -p . the parallel development of p/q type channels, primary dendrites, and the occurrence of prolonged parallel fiber-purkinje cell transmission suggests their causal relationships. naoya ichikawa, yasuo kitagawa, tatsuhiko kadowaki graduate school of bioagricultural sciences, nagoya university, aichi, japan we have recently identified a novel gene, mahya, which is specifically conserved between hymenoptera and deuterostome. mahya encodes a secretory protein with a follistatin-like domain, two immunoglobulin domains, and a c-terminal novel domain. mouse mahya genes (mmahya- and mmahya- ) are expressed in the olfactory bulb, hippocampus, and cerebellum of the adult brain. we have found that mmahya- protein is specifically synthesized in the pre-migratory granule cells and localized at the molecular layer of the postnatal cerebellum. these results suggest that mmahya- is involved in either the migration of granule cells or the dendritic maturation of purkinje cells. we will further report the analysis of the functions of mmahya- for the early cerebellum development. toshitaka morishima , erina fukushi , kazuto kobayashi , naohiro hozumi , sachiko yoshida toyohashi university of technology, toyohashi, japan; honda electronics co. ltd., toyohashi, japan in cerebellar development, granule cells migrate with elongation their axon, called parallel fibers, and form neuronal circuit in molecular layer. although density and thickness of parallel fibers are important information for cerebellar development, few were simple and useful methods. we have proposed a new method for two-dimensional acoustic impedance imaging for developing cerebellar slices. an acoustic impedance microscopy was obtained by mechanically scanning the transducer and the reflection intensity was interpreted into local acoustic impedance of no treated acute slices with no invasion. the developing parallel fibers were clearly observed as the contrast in acoustic impedance, whereas they were cloudy in immature egl from neonatal rat. the reflection from molecular layer enlarged and floated to deep layer, so that its spatial pattern was changed during cerebellar development. this imaging method is believed to be a powerful tool for observation of neuronal development, as neither fixation nor staining is required. tatsuro yamamoto , hideyuki dekimoto , tomiyoshi setsu , masahiko watanabe , mikio hoshino , yo-ichi nabeshima , toshio terashima dept. of anat., kobe univ. grad. sch. of med., kobe, japan; dept. of anat., hokkaido univ. grad. sch. of med., sapporo, japan; dept. of pathol and tumor biol, grad. sch. of med., kyoto univ., kyoto, japan a mutant mouse, cerebelles (cbll), lacks the entire cerebellar cortex but survives into the adult. the responsible gene for this mutation is ptf a, whose expression is lost in this mutant. in the present study, we examined cerebellar afferent and efferent systems of this mutant mouse by neural tracing methods with a combination of immunohistochemistry. the injection of fluoro-gold (fg) into the cbll thalamus resulted in retrograde labeling of neurons in the contralateral cerebellar nuclei. these fg-labeled neurons were glutaminase-positive. after the injection of bda into the cbll lumbar cord, spinocerebellar terminals projecting to the deep cerebellar nuclei were anterogradely labeled in spite of absence of the cerebellar cortex. these findings suggest that afferent and efferent systems of the cerebellar nuclei of the cbll are preserved in spite of absence of the cerebellar cortex. kumiko ishida , tomoko nishiyama , hitoshi tatsumi , masahiro sokabe , department of physiology, nagoya university graduate school of medicine, nagoya, japan; icorp, cell mechanosensing project, japan science and technology corporation sprouting and synaptic reorganization of the mossy fiber (mf) are commonly found in the hippocampus of temporal lobe epilepsy patients. as the muscarinic agonist, pilocarpine, can induce similar morphological changes, hippocampal slices treated with this drug have been widely used as a model of epilepsy. we found that pilocarpine induced a transient retraction and subsequent elongation of the neurites of granule cells in the slice cultures; the retraction was peaked approximately h and the elongation started at approximately h after the drug application. tetrodotoxin strongly inhibited both the retraction and elongation, while the bdnf sequestering protein, trkb/fc, retarded only the elongation. this result suggests that na + channel dependent neuronal excitation and following activitydependent bdnf releases are essential in the biphasic morphological changes induced by pilocarpine in hippocampal slices. rieko muramatsu, yuji ikegaya, maki k. yamada, norio matsuki, ryuta koyama laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, the university of tokyo hippocampal granule cells extend their axons, i.e. the mossy fibers (mfs), from the dentate gyrus (dg) to the area ca . once this oneway projection is disrupted, the mfs retrogradely innervate granule cell dendrites and make excitatory synapses that induce epileptic neural activities in the dg. to clarify the mechanism that regulates normal, anterograde mf projections, we used a co-culture system of hippocampal slices. when a dg slice from a gfp(+) rat was juxtaposed to the ca region of a host hippocampal slice from a wild type rat, the gfp(+) mfs ran through the host ca toward the host dg but failed to invade it even after ten days in vitro. thus the dg seemed to serve as a barrier that blocks retrograde projections of mfs. however, the mfs extended into the dg when forskolin, an activator of adenylate cyclase, was chronically applied. these results suggest that the dg has a mechanism supporting anterograde mf projections to ca , which is regulated by the levels of adenylate cyclase activation. calcitonin gene-related peptide (cgrp) is a amino acid neuropeptide that is widely distributed in central and peripheral nervous systems. cgrp is expressed from early developmental stage in rat brain, suggesting that cgrp may be involved in not only neurotransmission but also neural development. but roles of cgrp in neuronal development of cerebral cortex and hippocampus remain unclear. in the present study, we made dissociation culture of cerebral cortex and hippocampus of embryonic day (e) or e rat. dendritic outgrowth of pyramidal neurons was analyzed after days using anti-map antibody. synapse formation was analyzed after - weeks, using anti-psd- and anti-synaptophysin antibodies. in the presence of cgrp ( - nm), both dendritic length and synaptic density were increased. however, the number of dendritic branching was not affected. these results suggest that cgrp promotes dendritic outgrowth and synapse formation. chisako kanamaru, kazunori suda, kouji senzaki, takashi shiga university of tsukuba, graduate school of comprehensive human sciences, tsukuba, japan recent studies have suggested monoamine affects neural development, but it is unclear which receptor subtypes mediate actions of monoamine. in this study, we examined roles of hydroxytryptoamine ( -ht) and noradrenaline (na) in the formation of dendrites and synapses using dissociation culture of rat hippocampus. embryonic day rat hippocampus was cultured in the presence of -ht or na. after days, we analyzed formation of dendrites using anti-map antibody. after days, we analyzed formation of synapses using anti-psd- , anti-synaptophysin, and anti-map antibodies. the addition of -ht ( nm) or na ( nm) increased dendritic length and number of branches of pyramidal neurons, whereas decreased number of primary dendrites -ht ( - nm) and na ( - nm) also increased the synaptic density. by using receptor agonists and antagonists, it was suggested that ␣ a receptor promotes dendritic outgrowth, while ␤ receptor suppress dendritic outgrowth and branching. in addition, -ht a receptor and ␣ a receptor promote synapse formation. kenji amano down syndrome cell adhesion molecule (dscam) knock-out (ko) mouse died within h after the birth. to investigate possible etiology of the neonatal death, we examined the respiratory activity using whole body plethysmography and the c inspiratory activity using brainstem-spinal cord preparation. the respiratory activity of dscam-ko mice using plethysmography was irregular frequency and small amplitude accompanied with apnea. furthermore, c inspiratory activity also showed irregular frequency and narrow duration of the bursting. we then analyzed spatio-temporal pattern of the respiratory neuronal activity using combination of the voltage-sensitive dye (di- anepeq) and the imaging system (micam ). in dscam-ko mice, the optical signal which precedes c inspiratory activity was depressed. these results suggest that pre-inspiratory neuronal network, which determines respiratory rhythm, does not develop normally in dscam-ko mice and causes lethal respiratory dysfunction. ps a-f hippocampal cells cultured on d collagen substrate secrete a dense extracellular matrix, supporting neuritic outgrowth shantanu sur, thomas launey, masao ito brain sc. inst., riken, japan the brain extracellular matrix (ecm) influences neuronal migration and morphogenesis. we explored how hippocampal cells modify their extracellular environment when seeded onto collagen gel, a major component of the ecm. after weeks in vitro, neurons formed a dense layer, > . mm below the gel surface, with neurite outgrowth toward the surface, within the top gel layer (tgl). initially, we thought that hippocampal cells were penetrating the gel, following partial degradation of the collagen matrix. however, ( ) collagenasespecific inhibitor did not affect cell depth, ( ) limiting gliosis by antimitotics reduced the thickness of the tgl by %, ( ), neither glial nor neuronal cell body were found in the tgl by gfap/map detection, ( ) neurite outgrowth was observed only within this tgl, but not toward the bottom of the gel. to see whether the tgl is the remains of the initial collagen substrate, we embedded fluorescent beads in the collagen gel before cell seeding. the tgl was completely devoid of beads after weeks, suggesting that the tgl is newly formed by ecm material, largely secreted by glial cells. emi kumamaru, tadahiro numakawa, yuki yagasaki, hiroshi kunugi disorder research, national institute of neuroscience, ncnp, tokyo, japan the level of glucocorticoid is regulated through hpa axis, and glucocorticoid itself has a negative feedback effect on hpa axis. however, under the intense stress, the glucocorticoid level is increased, and the high level of it is suggested to induce neuronal damage and to cause the mood disorder. on the other hand, it is possible that the reduction of neuronal function mediated by bdnf is partly related to the cause of the disorder. therefore, in the present study, we investigated the effect of glucocorticoid (dexamethasone, dex) on synaptic maturation and function enhanced by bdnf in early developing hippocampal neurons. we found that bdnf increased the expression of synaptic proteins including glutamate receptor and presynaptic protein, however, pretreatment with dex significantly inhibited the up-regulation of these proteins by bdnf. further, increase in release of glutamate and in intracellular ca + by bdnf was suppressed after dex pretreatment, suggesting that dex inhibits the maturation of synaptic function mediated by bdnf. takashi ueyama , kazuto kujira , tetsuya kawabe , takao ito , yoshihiro tsuruo department of cell biology and anatomy, wakayama medical university, wakayama, japan; department of cardiovascular medicine, wakayama medical university, wakayama, japan in this study, we investigated the effect of castration on the emotional stress response in the brain by comparing the c-fos expression in response to immobilization stress (imo) between castrated rats (cast) and sham-operated rats (sham). increased c-fos immunoreactive cells in response to imo were observed in septum, thalamus, hypothalamus, midbrain, pons and medulla oblongata in accordance with previous findings. in cast compared with sham, the numbers of c-fos-ir cells were significantly lower in the medial parvocellular part of paraventricular hypothalamic nucleus, while they were significantly higher in the supraoptic nucleus and medial amygdaloid nucleus. these data suggest that neuronal activity in these areas is influenced by systemic androgen level. this may underlie the pathophysiology of partial androgen deficiency in aged men (padam). research funds: grant-in-aid for scientific research (c) ( ) ps a-f metabolic and glucagon response of a genetically heat-tolerant rat to ambient heat and cold fujiya furuyama , hitoo nishino , takehiro yahata nagoya city university graduate school of medical sciences, nagoya, japan; nayoro city college, nayoro, japan the inbred fok rat was developed by us using heat selection and inbreeding for generations. fok rats avoided serious multisystem disorders caused by heat stroke and by extreme dehydration. saliva spreads widely over the whole ventral body surface in fok rats. however, no strain difference was not found in vitro in the salivation rate, suggesting exsisting of a negative feedback loop between the central thermoregulation system and evaporation system. on the other hand, body temperature of the fok rats did not decreased in a extream cold environment as those in control rat strain. thermogenesis induced by cold in fok rats was larger than those in control rat strains. the larger increase in thermogenesis was partly attributable to glucagon-induced thermogenesis in brown adipose tissue. blood levels of triglryceride was lower, but polyunsaturated fatty acids were higher in fok rats than those in control rat strains. these changes can be considered to be results of genetically acquired heat-tolerance. oxidative stress is involved in the degeneration of nigrostriatal dopaminergic system in parkinson s disease (pd). vitamin e is a potent antioxidant, and its retention and secretion are regulated by alpha-tocopherol transfer protein (ttp) in brain. dysfunction of ttp has been shown to result in systemic deficiency of vitamin e in human and mice. in the present study, we using the ttp knockout mice, investigated the effect of vitamin e deficiency in pd development by generating mptp mouse model of pd. we confirmed that vitamin e depleted in the brain of ttp knockout mice completely. while the mptp treatment decreased striatal dopamine in the all three ttp genotypic groups, there were no significant differences among them. our results suggest that vitamin e does not play a major protective role in mptp-induced nigrostriatal dopaminergic neurodegeneration in the brain. priyanka dikshit , anand goswami , nobuyuki nukina , nihar ranjan jana national brain research centre, india; laboratory for structural neuropathology, riken brain science institute, - hirosawa, wakoshi, saitama - , japan a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins, often associated with various chaperones and proteasome components. but, how the polyglutamine proteins are ubiquitinated and degraded by the proteasome is not known. here, we demonstrate that the expanded polyglutamine proteins that are misfolded, become ubiquitinated. secondly, we identified chip ubiquitin ligase that is able to target polyglutamine expanded huntingtin and ataxin- for the misfolding-dependent ubiquitination and degradation by the proteasome. the over expression of chip reduces the aggregate formation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when chip is over expressed along with hsc . finally, we show that the expression of chip is increased in the expanded polyglutamine protein expressing cells. hypothalamic-pituitary-adrenal axis is central to the regulation of stress response. for the comprehensive detection of genes responsive to stress, we identified and catalogued the entire partial complementary dna sequences (expressed sequence tags (ests)) from rat hypothalamus. we have identified the total of , ests ( , non-redundant sequences). of them matched known genes of rodents in the genbank databases, but remained unknown. now we classified a full set of hypothalamic ests on the basis of their functional domains. complete profile of them will be presented in the meeting. these ests will also be applied to a cdna microarray for stress experiments. the present study will provide a refined genomic resource for molecular studies of animal models of stress-related disorder. research funds: grants-in-aid from the ministry of health, labor and welfare shinya yanagita, seiichiro amemiya, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, japan our previous study suggests that acute running is one stressor activating corticotropin-releasing hormone (crh) neurons in the hypothalamic paraventricular nucleus (pvn). many studies have reported that several weeks of voluntary running improved stress tolerance during non-exercise stress. it is, thus, possible that housing in cages attached running wheel can alter activation of stress-related neurons during acute running. in this study, we examine the effects of , , or weeks prior wheel running (i.e. housing in the cages attached running wheel) on activation of stress-related neurons, such as pvn, central nucleus of amygdala (cea), locus coeruleus, dorsal raphe, ventral tegmental area (vta), and prefrontal cortex during acute running using immunohistological methods in rats. prior wheel running altered activation of various stress-related neurons during acute running, especially markedly decreased activation of cea, and increased that of vta. these results suggest that prior wheel running influences stress-related neuronal activity during acute running. ps a-f transforming growth factor-␤ in the brain regulates fat metabolism during exercise kazuo inoue, toma ishikawa, wataru mizunoya, tetsuro shibakusa, tohru fushiki division of food science and biotechnology, graduate school of agriculture, kyoto university, kyoto, japan we have previously reported that the concentration of transforming growth factor-␤ (tgf-␤) increases in the cerebrospinal fluid of rats during exercise and that an increase in fat oxidation was observed following intracisternal administration of tgf-␤. these results led us to postulate that tgf-␤ in the brain regulates the enhancement of fatty acid oxidation during exercise. to test this hypothesis, we carried out respiratory gas analysis during exercise while inhibiting the effect of tgf-␤ in the brain using intracisternal administration of anti-tgf-␤ antibody or sb- , an inhibitor of the type tgf-␤ receptor (t␤r ). we found that each reagent blocked the increase in fatty acid oxidation. these results suggest that brain tgf-␤ has a role in enhancing fatty acid oxidation in peripheral tissues during endurance exercise, and this regulation is executed at partly via the t␤r signal transduction system. yoshii takanobu it has been demonstrated that vasopressin (avp) might play a role in anxiety-related behavior. we hypothesized that traumatic stress changes avp activity and avp contribute to the symptom of ptsd. we carried out in situ hybridization (ish) for avp mrna expression and avp immunohistochemistry (ihc) with an experimental paradigm of single prolonged stress (sps) as ptsd model. sd male rats were exposed to sps ( h restraint; min forced-swimming; ether anesthesia) then they were put in untouchable situation for days. avp mrna expression significantly decreased in the son. ihc showed no significant change in avp-ir, but after additive stress (forced swimming min), avp-ir in the son was significantly diminished. we considered that the stress decrease avp synthesis, but has little effect to the storage of avp. mumeko tsuda, takaaki ozawa, aosa fukushi, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan neonatal maternal separation (ms) is known to affect anxiety and fear responses in adult whereas its effect on socio-sexual behaviors is not fully understood. in the present study, we examined the effect of ms on an array of emotional and socio-sexual behaviors in both sexes of c bl/ j mice. pups were separated from mothers daily ( h) on postnatal days through . starting at weeks of age they were tested for ( ) emotionality and anxiety levels in open field (oft), light-dark transition (ldt), and elevated plus maze tests; ( ) responses to social stimuli in social investigation (sit) and social preference tests; and ( ) socio-sexual behaviors in aggressive and sexual behavior tests. overall, there was no apparent effect of ms on behaviors measured in the oft and ldt except for higher levels of exploration in the ms group compared to the non-stressed (ns) group in both sexes. during the sit, social investigation time and general activity in ms females were much lower than those in ns females suggesting ms females may be more fearful to social stimuli. in the present study, we investigated the effect of environmental stress applied during perinatal period on spatial learning activity of mouse evaluated by morris water maze test. mice were exposed to the noise of db (so), or were forced to swim (sw). these manipulations were performed for min once a day at weeks after birth (from postnatal days to ) or weeks after birth (from postnatal day to ). normal mice were left undisturbed (no). the spatial learning activity was tested at the age of weeks. it was found that the spatial learning activity of both so and sw mice manipulated weeks after birth was impaired as compared to no mice. so mice manipulated weeks after birth exhibited the same learning behavior as no mice, while that of sw mice manipulated weeks after birth was impaired. present results indicated that the effect of the environmental stress on the learning activity of the adolescent mice might be dependent on the period of the stress manipulation. kin-ya kubo , yukiko yamada , mitsuo iinuma , yasuo tamura , fumihiko iwaku , kazuko watanabe , minoru onozuka dept. oral anat., asahi univ. sch. dent., japan; dept. ped. dent., asahi univ. sch. dent.; dept. physiol., gifu univ. sch. med., japan; dept. physiol. and neurosci., kanagawa dent. coll., japan recent studies have suggested that occlusal disharmony is related to temporomandibular arthorosis and braxism, which may come from a hypothalamic-pituitary-adrenal (hpa) axis. in addition, aged mice with masticatory dysfunction show deficits in spatial memory, being due to various pathological changes in the hippocampus, suggesting the link between malocclusion induced by abnormal occlusion and hippocampal pathology. in this study, to prove this hypothesis, we examined the effect of this malocclusion on plasma corticosterone levels, the numbers of hippocampal neurons and spatial performance in water maze in samp mice. this treatment age-dependently advanced a decline in spatial memory, an increase in plasma corticosterone levels, and a decrease in neuron density in the hippocampal ca region. the results suggest that abnormal occlusion may progress hippocampal neuron loss via stress, thereby leading to senile deficits in memory. yurie nakamoto, go mugishima, mitsuko sato, masako miwa, mitsunobu yoshii division of psychobiology, tokyo institute of psychiatry, tokyo, japan it has been shown that pbr are increased after acute stress and decreased under chronic stressful conditions. in our previous studies, expression of pbr was significantly correlated with trait anxiety in normal human subjects, which might reflect polymorphism of the pbr gene. in addition, males appeared to have higher pbr densities than females in their prime lives. the present study was designed to analyze these sexual differences in rats. blood samples were obtained from adult male and female slc wistar rats immediately after acute random electrical footshock and also from these animals after chronic social isolation (for weeks after weaning). in naïve, male rats expressed higher densities of platelet pbr than females. chronic social isolation caused a marked increase in platelet pbr in male rats compared to female. the results indicate that pbr responses to environmentally induced stress are much less in female, probably under the influence of estrogen. kanako tambara , yayoi kitamura , junichi tanaka , yukio hattori , yasushi hayashi department of human nutrition, notre dame seishin university, okayama, japan; department of curriculum, teaching and memory, naruto university of education, tokushima, japan we investigated the effects of exogenous putrescine on stressinduced hyperthermia (sih) in male c bl/ j mice after systemic injection of putrescine to clarify the role of brain putrescine in stressful conditions. in addition, we examined the effects of spermidine, spermine, and the anxiolytic diazepam on sih. the rectal temperature of singly housed mice was measured twice at a -min interval, to measure the basal temperature (t ) and stress-enhanced temperature (t ), respectively. the difference ( t = t − t ) gives the sih. in control mice, t was approximately • c. pretreatment with diazepam caused dose-dependent inhibition of the sih. similarly, putrescine reduced t, although it caused a dose-dependent decrease in t . furthermore, spermidine and spermine also lowered t and t at doses lower than that of putrescine. these results suggest that endogenous brain putrescine and other polyamines have an anxiolytic-like effect in stressful conditions. eriko iguchi, yasuhiro tanaka, toshiyuki matsuoka, shuh narumiya department of pharmacology, kyoto university, kyoto, japan prostaglandins (pgs) are synthesized in many organs including the brain. of their synthesis, the rate limiting step depends on cyclooxygenase (cox), which has two subtypes, cox- and cox- . it has been known that, under some stressful conditions, cox- is induced in some neurons and increases pgs production. but the roles of the increased pgs under stress are not fully elucidated. in this study, we restrained mice in small tubes individually for h and subjected them to the elevated plus maze task h later. these mice showed more anxiety. immunohistochemistry showed significant induction of cox- by restraint in some parts of the brain, such as cerebral cortices and amygdala. next, we examined the effect of indomethacin on this stress-induced anxiety. indomethacin is expected to reduce pgs production. mice treated with indomethacin stayed on open arms longer than control mice. these data suggest that pgs synthesized during stress may have anxiety-increasing effect. ps a-g imaging brain and immune association accompanying cognitive appraisal of acute stressor to investigate association between brain and immune systems accompanying cognitive appraisal of an acute stressor, we recorded o-water positron emission tomography, cardiovascular, neuroendocrine, and immune indices, when male subjects conducted a mental arithmetic task in a high controllability (hc) condition and a low controllability (lc) condition. activation in the orbitofrontal (ofc) and medial prefrontal (mpfc) cortices was observed in the lc compared to the hc. furthermore, significant correlations between brain activation and hr, hrv, bp, and nk cells were found commonly in the ofc in the lc, but not in the hc. thus, the ofc is a pivotal region for top-down regulation over immune activity accompanying cognitive appraisal on a stressor. wei zhang , takesi sakurai , yasuitirou fukuda , tomoyuki kuwaki , dept. molec. integ. physiol., chiba univ., japan; dept. pharmacol., univ. tsukuba, japan; dept. autonom. physiol., chiba univ., japan we have previously proposed that orexin plays as a master switch to elicit multiple efferent pathways of the defense response. it is still open question, however, how information of stressor activates the orexinergic neurons. in this study, we examined possible afferent nuclei to activate orexinergic neurons. in urethane-anesthetized mice, a gaba-a receptor antagonist, bicuculline, was microinjected into the amygdala or the bed nucleus of stria terminalis (bnst), of which electrical stimulation induced simultaneous increases in blood pressure, heart rate, and respiration. bicuculline dose-dependently induced cardiorespiratory excitation in both orexin neuron-ablated and wild-type mice. however, dose-response curve was rightward shifted in the former. we conclude that the amygdala and bnst constitute one of the afferent pathways to the orexinergic neurons that involved in the defense response against stressor. in this study, developmental changes of anxiety behavior as well as myelin formation were investigated in male balb/c mice. the early-weaned mice had lower number of entries to the open arms of elevated plus maze at the age of - weeks, indicating persistent higher anxiety. high performance thin layer chromatography analysis was conducted for amygdaloid galactosyl ceramide, which is a typical lipid of myelin. the early-weaned mice had higher levels of galactosyl ceramide at the age of weeks, and an electron microscopic study suggested increased number of myelinated axon and reduced diameter of myelinated axon in the basolateral amyglaloid nucleus. these results suggest that the early weaning induces precocious myelin formation in the amygdale between and weeks of age, which would be related to higher anxiety state in the early-weaned mice. research funds: sasakawa sci. res. grant takefumi kikusui, yuji mori veterinary ethology, university of tokyo, tokyo, japan we previously reported that early-weaned mice developed persistent increase in anxiety as well as aggression. in this study, developmental changes of brain derived neurotrophic factor (bdnf) protein levels were investigated in early-weaned icr mice. the early-weaned male and female mice had lower number of entries to the open arms of elevated plus maze at the age of weeks, and this change was persistently observed in males. concurrently, the early-weaned males showed decrease of bdnf in the prefrontal cortex between and weeks of age, and in the hippocampus at the age of weeks. however, there was no difference of bdnf expression in females. in addition, the early-weaned males, but not females, showed reduced brdu immunoreactivity in the dentate gyrus. these results suggest that the deprivation of mother-infant interaction during the late lactating period augments the anxiety in the adulthood by decreasing the level of bdnf in the pre-limbic system, and that these stress responses are sexually dimorphic, i.e., male is more vulnerable to early weaning stress. research funds: kakenhi # ps a-g a systematic analysis of genetic factors associated with behavioral diversity between msm and c bl/ koide tsuyoshi , , aki takahashi , , toshihiko shiroishi , , akinori nishi mgrl, national institute of genetics, mishima, japan; sokendai, hayama, japan; mammalian genetics lab, nig, mishima, japan in the previous study conducting a multi-phenotype behavioral tests, we observed a great difference of the behavioral phenotype between mouse strains, msm and c bl/ . in order to elucidate a genetic factors underlying the behavioral difference, we analyzed a series of consomic strains which are made by replacing one of the chromosomes with that of msm strain. the behavioral data clearly indicated involvement of multiple genetic factors for each behavioral phenotype. one of the consomic strains, b - cmsm, which carries chromosome of msm, showed extreme behavioral differences from c bl/ . the strain showed lower activity in home cage and novel cage, and showed decreased number of transition in the light dark box test. by conducting analyses of composite interval mapping and a series of sub-consomic strains, we successfully identified genetic loci for the behavioral phenotype. tomoko soga , yu kajiyama , shigenobu shibata , hiroshi kunugi department of mental disorder research, national institute of neuroscience, center of neurology and psychiatry, tokyo, japan; department of electrical engineering and bioscience, waseda university the hypothalamus-pituitary-adrenal (hpa) axis plays an important role in the pathophysiology of depression. alterations of brain derived neuronal factors (bdnf) have been implicated in depression. we examined the effects of synthetic glucocorticoid (dexamethasone; dex) on emotional behavior and gene expression of hpa-related molecules and bdnf in mice. dex treatment for days after birth showed a significant decrease in locomotor activity and a significant rise in the time of immobility during forced swimming test. dex treatment to mature mice resulted in significant decrease in the number of entries into the open arm during elevated plus maze test. there was no change in gene expression of hpa-related molecules in dex-treated group. bdnf gene expression decreased significantly in dex-treated group, which showed behavioral abnormalities. our results lend further support for the involvement of glucocorticoid and bdnf in depression-related behavior. sachiko chikahisa, hiroyoshi sei, atsuko sano, kazuyoshi kitaoka, yusuke morita department of integrative physiology, the university of tokushima graduate school, tokushima, japan music is known to be able to elicit emotional changes including anxiolytic effect. the gonadal steroid hormone estrogen (e ) has been associated with anxiety levels. in this study, we examine whether the effect of music on anxiety is related with ovarian steroid in female mice. behavioral paradigms measuring anxiety (open field, elevated plus maze, dark-light transition and marble burying test) were tested in gonadally intact (sham-operated) and ovariectomized (ovx) female mice treated with placebo (ovx + placebo) or chronic estradiol (ovx + e ) replacement. in three behavioral tests except for open field, sham-operated mice exposed to music showed less anxiety than those exposed to white-noise and silence, while ovx + placebo mice did not show these effects at all. ovx + e mice showed the anxiolytic effect of music only in the marble burying test. these results suggest that exposure to music reduce anxiety levels, and ovarian steroids may be, at least partially, involved in the anxiolytic effects of music observed in female mice. tatsuhiro yasuda free, tokyo, japan strength and periodicity of periodical air pressure ascent around ones' ears induced by others' respiration may impact upon ones' awaken level, i.e. cognition. the air vibration acts upon tympanic membrane and then cochlear receptor stereocilia transforms it to neural signals which are sent via cochlear nucleus to inspiration nucleus in the medulla, and inspiration is induced. simultaneously afferent signals generated by external intercostals contraction are forward to medulla, thalamus and cortical areas. the stimuli with larger strength and periodicity compared to ones body size yields to auditory startle reflex. continuation of this may induce hyper ventilation or tension. if the input may be lasting with smaller strength and periodicity, insufficient diaphragm activity after hypoxia and gasping fade-out may induce afferent signal shortage that shrinks various cortical neural activity. lasting this situation may fall into depression. suitable timing of inspiration inducing may keep good mood, strong motivation and effective cognition. body system is suggested to own inherent observer that detects alerting or safe state so called homunculus. kenichi sasaguri , takero in general, it has been proposed that the mandibular retrusive position resulted from either malocclusion or inadequate occlusal reconstruction is one of the causes of indefinite complaint. we determined whether the malocclusion model influences brain activities by using fmri study. the results indicated that in some of volunteers, significantly bold signals in the hypothalamus and the amygdala, being associated with emotion and/or stress increased during clenching. it is, therefore, suggested that malocclusion influences the whole body through emotional system, thereby causing the indefinite complains. ps a-g synaptic organization between the amygdaloid axon terminals and the parvicellular reticular formationprojecting neurons in the retrorubral field of the rat toshiko tsumori, yi qin, shigefumi yokota, tatsuro oka, yukihiko yasui dept. anat. & morphol. neurosci., shimane univ. sch. med., izumo, japan the retrorubral field (rrf) is known as one of the areas containing numerous dopaminergic neurons in the midbrain. in the present study, we showed that the axon terminals from the central amygdaloid nucleus (ace) made synaptic contacts with non-dopaminergic rrf neurons sending their axons to the parvicellular reticular formation (rfp), where many premotor neurons projecting to the orofacial motor nuclei have been well known to exist. the ace axon terminals, which usually contain small pleomorphic vesicles and occasionally contain both small pleomorphic vesicles and large dense-cored vesicles, formed symmetrical synapses with cell bodies and dendrites of the rfp-projecting rrf neurons. moreover, most of these axon terminals showed glutamic acid decarboxylase immunoreactivity. the present study suggests that the ace exerts inhibitory influences upon the non-dopaminergic rfp-projecting rrf neurons to control orofacial movements closely related to emotional behavior. research funds: kakenhi ( ) ps a-g involvement of nr b tyrosine-phosphorylation in emotional responses mediated at the amygdala mina delawary , takanobu nakazawa , yuji kiyama , toshiya manabe , tadashi yamamoto div. of oncology, ims, univ. of tokyo, tokyo, japan; div. of neuronal network, ims, univ. of tokyo, tokyo, japan nr b is tyrosine-phosphorylated, with tyr- being its major phosphorylation site. to investigate the role of tyr- phosphorylation, we generated mice with a tyr phe knock-in mutation (yf/yf mice). in the elevated plus-maze test, time spent in open arm was reduced in yf/yf mice as compared to that in wild-type mice. similar phenotype was seen in the corticotropin-releasing factor (crf) overexpressing mice. this phenotype of yf/yf mice was canceled by the administration of crf receptor antagonist. as expected, in yf/yf mice, the expression level of crf in the amygdala was increased compared with that in wild-type mice. in the slice of amygdala from wild-type mice, nmda application induced de-phosphorylation of tyr- and up-regulation of crf mrna level. given that crf is important in emotional responses, these data strongly argue that phosphorylation of nr b is involved in the control of emotional responses by regulating crf content. ps a-g increase in anxiety in transgenic mice overexpressing camkii in forebrain previous studies have shown that ␣calcium/calmodulin dependent protein kinase ii (␣camkii) plays important roles in aggressive and fear response in mice. to understand roles of alpha camkii in emotional behaviors, we have generated transgenic mice overexpressing ␣camkii in forebrain. because these mutant mice showed increase in anxiety in open field and elevated zero maze tests, we here examined effects of administration of selective serotonin reuptake inhibitor (ssri) on anxiety-related behavior of these mutant mice. treatment with ssri suppressed anxiety-related behavior of camkii mutant mice, suggesting that camkii mutant mouse is a mouse model of anxiety disorder. to investigate the mechanisms for increase in anxiety led by overexpression of camkii, we next compared the expression profiles between wild and mutant mice using dna micro array. these mutant mice showed abnormal changes in expression levels of genes related to ca + signal transduction in hippocampus. yumiko ikeda, katsunori kobayashi, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan environment is known to influence behavior of animals. however, cellular and synaptic mechanisms underlying behavioral changes by environment remain largely unknown. we examined effects of changes in environment on locomotor activity and mossy fiber (mf) synaptic transmission in hippocampal slices. in mice housed in enriched condition for weeks, locomotor activity and longinterval ( , and ms) paired-pulse facilitation (ppf) at mf synapses were reduced. in contrast, in mice housed in isolated condition for weeks, there was no detectable change in either the total ambulation distance or the magnitude of ppf. we compared properties of the mf synaptic transmission with the locomotor activity in individual mice used in all experiments and found that the magnitude of synaptic potentiation induced by dopamine was negatively correlated with the ambulation distance. our results suggest that the modification of the hippocampal mossy fiber synaptic transmission could be involved in the environmental regulation of locomotor activity. yilong cui , , yosky kataoka , , yasuhisa tamura , yasuyoshi watanabe , , hisao yamada department of anatomy and cell science, kansai medical university, osaka, japan; department of physiology, osaka city university graduate school of medicine, osaka, japan; molecular imaging research program, riken frontier research system, saitama, japan during long-term intracranial self-stimulation (icss; electrical stimulations to the hemi-lateral medial forebrain bundle of rats by their lever pressing behavior at - times/min), inhibition periods (less than times/min) were often observed h after start of icss. we have been demonstrated that the inhibition was not induced by thermal effect on the neural function or by muscular fatigue. furthermore, the inhibition period was significantly decreased by pre-treatment with ns- , a selective cox- inhibitor. these observations indicate that the arachidonic acid cascade is involved in inhibition of long-term icss and would be in weariness or fatigue sensation. male bluegill, lepomis macrochirus, is known to display alternative reproductive tactics. "parental" males defend nests and provide parental care, and "satellites" or "sneakers" are non-nesting, attempting to achieve parasitic fertilizations via sperm competition. in teleost and other non-mammals, arginine vasotocin (avt), the homologue of mammalian avp, is known as an important hypothalamic peptide involved in the alteration of reproductive behavior. behavioral evaluation and immunohistochemical study in preoptic area (poa) were conducted in parental and satellite bluegills to clear the role of avt in teleost reproductive tactics. parentals displayed more aggressive and courtship behavior than satellites and satellite males had significantly more cells than parentals, while the size of avt cells showed no difference between the male morphs. these results suggested that hypothalamic avt might play some part in the central control of reproductive behavior in teleost. ps a-g impulsive choice in domestic chicks: context dependence and dissociation between delay and handling cost toshiya matsushima , naoya aoki , andras csillag biology, hokkaido univ., sapporo, japan; agriculture, nagoya univ., nagoya, japan; anatomy, semmelweis univ., budapest, hungary choice between small/immediate reward and large/delayed reward has been widely used as a behavioral measure of impulsiveness. to study how ecological factors shaped underlying neural processes, we examined week-old chicks in four different tasks with identical economical consequences. in task , chicks chose between small reward (one pellet) delivered immediately and large reward (six pellets) after a delay up to s. in task , chicks chose between small reward located at cm and large reward at − cm, where cues signaled the distance of invisible food. task was similar to the task , except that cues signaled the food quantity. in task , total handling time differed due to lowered food accessibility, while the delay was kept identical. lesion experiments revealed that ventral striatum was specifically involved in choices based on anticipated proximity (but not quantity), whereas arcopallium (association cortex analogue) in choices based on anticipated handling cost. research funds: kakenhi ( , ) ps a-g analysis of the brain regions associated with the dance language of the honeybees taketoshi kiya, takekazu kunieda, takeo kubo dep. biol. sci., univ. tokyo, tokyo, japan social animals have highly developed communicative abilities. the worker honeybees (apis mellifera l.) can transmit location of food sources by the dance language. in spite of the simple structure of the honeybee brain and the stereotyped dance behavior, its neural mechanisms remain totally unknown. previously, we found active brain regions in the dancing workers (dancers) by using a novel immediate early gene, kakusei, as a marker for neural activities and found its prominent expression in the small-type kenyon cells (skcs) of the mushroom bodies. here, we report that kakusei was similarly expressed in the skcs of the foraging workers (foragers), which do not always show the dance behavior. in contrast, the skcspreferential kakusei expression was not observed in the brains of the orienting workers, which were flying to learn the hive location. these results imply that the activities of the skcs in the dancer brain are neither due to dance presentation itself nor sensory inputs during foraging, but complex information processing accompanying the foraging behavior. c. elegans wild type animals are usually attracted to nacl, but show avoidance behaviors after being conditioned with nacl and starvation (food−/nacl+). this behavioral plasticity is not induced under the food−/nacl− or food+/nacl+ conditions. we isolated learning-defective mutants including pe , which had a missense mutation in the casy- gene. several casy- deletion mutants also showed learning defects. casy- has an extensive similarity to human calsyntenin/alcadein, which is a single-pass transmembrane protein with cadherin-like repeats localized to the postsynaptic membrane of cns synapses. alcadein forms a stable tripartite complex with app and x l/mint . however, after dissociation of x l, alcadein is susceptible to cleavage by protease(s). we found that casy- was expressed mainly in neurons and functioned at the adult stage. we are now investigating the localization pattern of the gfp-tagged protein, and whether casy- can also be proteolytically cleaved. ps a-g insulin-like signaling is required for association between temperature and feeding state in c. elegans eiji kodama , atsushi kuhara , akiko mohri , , kotaro kimura , , masatoshi okumura , masahiro tomioka , yuichi iino , ikue mori , div. of biol. sci., nagoya univ., japan; present address: univ. of texas, health sci. cent., usa; present address: natl. inst. of genet., japan; mol. genet. res. lab., univ. of tokyo, japan; inst. for advanced res., nagoya univ., japan c. elegans can associate cultivation temperature with feeding state. mutations in ins- encoding insulin homologue caused defective associative learning, mutations in daf- and age- encoding the homologues of insulin receptor and pi -kinase, respectively, suppressed the defect of ins- , and the mutation in daf- encoding forkhead transcriptional factor caused the learning defect. this suggests that ins- antagonizes daf- insulin-like signaling for associative learning. interestingly, age- animals associate their cultivation temperature with feeding-state quicker than wild type. this defect was rescued by expressing age- in some head interneurons. in addition, the activity of these interneurons were down-regulated by starvation through ins- . we suggest that insulin-like signaling modulates the neuronal activity of interneurons essential for associative learning. ps a-g analysis of ttx- : novel thermotaxis gene conserved among various organisms akiko miyara, akane ohta, yoshifumi okochi, masatoshi okumura, ikue mori laboratory of molecular neurobiology and institute for advanced research, nagoya university, nagoya, japan c. elegans can memorize the food condition in relation to the cultivation temperature and migrate to the cultivation temperature when looking for the food. this response to temperature is called thermotaxis. several neurons and genes required for thermotaxis have been identified, but molecular mechanism of thermotaxis is still poorly understood. the ttx- (nj ) and ttx- (nj ) mutants are obviously defective in thermotaxis and partially defective in chemotaxis. we revealed that ttx- encodes novel protein and is expressed in many neurons and functions in several neurons responsible for the thermotaxis behavior. the predicted protein structure of ttx- is similar to ric- , identified in c. elegans at first and conserved among several species (halevi et al., (halevi et al., , . ric- is thought to be required for the maturation of acetylcoline receptor (halevi et al., ) , so ttx- may play a similar role such as folding, assembly, transmission or anchoring of some kind of membrane protein. ps a-g analysis of aho- mutant that cannot associate cultivation temperature with feeding state in c. elegans nana nishio , akiko mohri , , eiji kodama , atsushi kuhara , mizuho koike , kotaro kimura , , ikue mori , div. of biol. sci., nagoya univ., japan; present address: univ. of texas, health sci. cent., usa; present address: natl. inst. of genet., japan; inst. for advanced res., nagoya univ., japan the nematode c. elegans can associate cultivation temperature with feeding state: well-fed animals migrate to and starved animals avoid from the cultivation temperature on a temperature gradient. to identify genes required for this associative learning, we screened mutants that are defective in starvation-induced cultivation temperature avoidance. we isolated aho- (nj ) mutants that were normal in thermotactic migration after cultivated well-fed state and normal in response to food in locomotion assay (sawin et al., ) , indicating that they are normal in temperature and food recognition and may be defective in the associative learning. aho- gene encoded a predicted hydrolase and the molecular properties have not been characterized yet, although aho- is a highly conserved protein throughout yeast to human. currently, we are trying to dissect the molecular and cellular analysis of aho- gene further. we have demonstrated aversive conditioning in lymnaea using mm sucrose presentation as the appetitive stimulus (cs) and mechanical tactile stimulation to the head as the noxious stimulus (ucs). we measured the feeding response before and after pairing with the aversive stimulus to determine whether learning alters the innate preference for sucrose. we also measured the neuronal activity of b , located in the buccal ganglion. an associative memory, lasting h, was produced with pairings of cs and ucs. the learning was characterized by a shift in the response to the ucs from a whole body withdrawal response to the cessation of feeding behavior. b neuron responded with repetitive impulse discharge regularly as fictive feeding patterns to a sucrose application in naive animals, on the other hand cs application failed to generate regular impulse activity rather it resulted in generation of epsps in the conditioned animal. this can interpret that the conditioning decreased the excitability of b neuron activity thus to decrease the fictive feeding behavior. yasutaka nomura , dai hatakeyama , tetsuro horikoshi , etsuro ito , manabu sakakibara lab. neurobiol. engr, sch. high-tech, tokai univ., numazu, japan; cris, hokkaido univ., sapporo, japan calexcitin, low molecular weight gtp-binding protein is found to be phosphorylated in the visuo-vestibular conditioned hermissenda at the type b photoreceptor. we found positively stained neurons to anti-calexcitin antibody (gift from dr. kuzirian) at the cerebral and pedal ganglion in the circumesophageal nervous system of conditioned lymnaea with two different ways. one was the same conditioning paradigm as hermissenda and the other was taste aversion conditioning. both of these conditioning response is the whole-body withdrawal. no positive neuron was found in naïve animal. neurons in cb cluster and pea cluster showed both positivity to calexcitin and serotonin. this suggested the functional role in conditioning. ken honjo, katsuo furukubo-tokunaga graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan the fruit fly drosophila melanogaster has been utilized as a successful model to study underlying mechanisms of learning and memory. we have established a novel larval olfactory paradigm and found that appetitive and aversive memories are considerably different in their stability whereas both are localized to the mushroom bodies (mbs). we found that larval memory induced by sucrose lasts six times longer than that induced by quinine although the initial learning performances are comparable. by expressing shi ts in larval mbs, we demonstrate that disruption of neural output from mbs abolishes both appetitive and aversive memory indicating that both memories are stored before the mb output synapses. moreover, we show that disruption of either creb or amnesiac functions abolishes appetitive but not aversive memory. thus these data suggest that appetitive and aversive reinforcements stimulate different intracellular and/or intercellular signaling pathways generating distinct memory components in mbs. motomi matsuno , minoru saitoe , , tim tully tokyo metropolitan institute for neuroscience, tokyo, japan; department of biology, tokyo metropolitan university, japan; cold spring harbor laboratory, usa we identified ruslan as a novel memory mutant, and found that it encodes a cell adhesion molecule, klingon (klg). klg belongs to the immunoglobulin superfamily and was originally identified as an essential gene for the development of photoreceptor neurons. we report here that klg is necessary for long-term memory as well as early-phase memory. we show that klg expression is dependent on neural activity and functions as a downstream of both the transcription factor, creb and the cell surface receptor notch, both of which are well known to function in ltm formation. transgenic expression of klg improves memory of a klg mutant. since klg protein localizes along the surface between neuropil and neuropil glia, we propose that klg mediates an interaction between neurons and glia that is required for memory formation. we have investigated the ability of context-dependent olfactory learning in the cockroach, periplaneta americana. we trained one group of cockroaches to associate peppermint odor (conditioned stimulus, cs, p) with sucrose solution (appetitive unconditioned stimulus, us+), and vanilla odor (cs, v) with saline solution (aversive us, us−) under illumination (l), and to associate p with us− and v with us+ in the dark (d). another group received training with opposite stimulus setup (l: v+/p−, d: v−/p+). before training, cockroaches preferred v over p. day after training, the former group significantly preferred p over v under illumination but preferred v over p in the dark, and the latter group displayed the invert odor preference. result of the control experiment excluded the possibilities that conditioning hours of the day or its order was used as cues to disambiguate the meaning of css. thus cockroaches are capable of disambiguating the meaning of cs odors according to the visual context. hidehiro watanabe, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan a century had passed since pavlov reported classical conditioning of salivation in dogs. however, the cellular mechanisms underlying this conditioning remain obscure. in insects, salivation is regulated by salivary neurons of the subesophageal ganglion which innervate the salivary grand. here, we established antennal classical conditioning of salivation and that of activities of salivary neurons in cockroaches, periplaneta americana. in insects, antennae are elaborate sense organ that processes many sensory modalities including odor and taste. we found that responses of salivary neurons to an odor was increased after repetitive pairing of the odor with sucrose or saline solution presented to an antenna, but those to an odor paired with water or tactile stimulus presented to an antenna did not changed. the level of salivation to sucrose-associated odor was significantly greater than that to non-associated odor. these results are the first to suggest the classical conditioning of salivation in non-mammalian species. these results are useful to study neural mechanisms underlying classical conditioning of salivation. research funds: kakenhi ke zhang , jian z. guo , ai k. guo , institute of neuroscience, chinese academy of sciences, china; institute of biophysics, chinese academy of sciences, beijing, china the cooperation of dopamine system and other brain cortices is essential for decision-making in mammal. drosophila can make clearout choice in visual flight simulator when facing conflicting visual cues based on the saliency of the cues previously trained to follow. here we show this behaviour is impaired when the transmission of dopaminergic neurons or mushroom bodies (mb), was genetically silenced by gal /uas-shi ts system, suggesting that this behaviour is mediated by dopaminergic system acting through mb, a structure shown to be densely innervated by dopaminergic fibers. however, the dopaminergic and mb synaptic activities were required only during the early choice period (< min), but not for the sustenance of the chosen flight path. thus the dopaminergic system and mb are specifically devoted to the cognitive function exemplified by the flyǐs choice behaviour and further studies of the circuit in drosophila may help to understand the neural basis of higher cognitive functions. sae unoki, yukihisa matsumoto, makoto mizunami graduate school of life sciences, tohoku university, sendai, japan in mammals, the dopaminergic reward system plays ubiquitous roles in reward learning. previous studies in insects suggested that octopamine (oa) and dopamine (da) mediate various kinds of reward and punishment signals in olfactory learning. however, whether such roles can be generalized to learning of sensory signals other than odors remained unknown. we pharmacologically studied the roles of oa and da in appetitive and aversive forms of visual pattern learning in crickets. crickets injected with oa receptor antagonists exhibited no significant levels of appetitive visual learning, but aversive one was unaffected. the opposite influences were observed by injection of da receptor antagonists. our finding that oa and da participate in reward and punishment conditioning in visual learning, together with results of previous studies in olfactory learning, suggests ubiquitous roles of the octopaminergic reward system and dopaminergic punishment system in insect learning. this suggests conserved roles of aminergic reinforcing systems among different phyla. aiko watanabe, neal a. hessler laboratory for vocal behavior mechanisms, riken brain science institute, saitama, japan in adult songbirds, neural turnover occurs in hvc, a forebrain motor control nucleus. cells labeled by bromodeoxyuridine (brdu), a cell birth marker, appear in the ventricular zone, migrate into hvc, and some of them mature into projection neuron. to assess the role of neurogenesis in adult song plasticity, we deafened adult bengalese finches, whose songs are disorganized and become plastic within the first month after deafening, and then stabilize. deafened birds had more brdu-labeled cells in hvc than control birds within the first month. more tunel-stained apoptotic cells also tended to be seen in hvc of deafened birds. however, number of the brdu-labeled cells decreased months after deafening, when the songs had stabilized. most of the brdu-labeled cells in hvc of deafened birds were immunoreactive for a neuron-specific marker, hu. additionally, amount of singing in deafened birds, which may affect amount of neurogenesis, did not significantly differ from that in control birds. these results suggest that the amount of neurogenesis is related to adult song plasticity. yasko tobari , , kazuo okanoya , , lab. for biolinguistics, riken-bsi, wako, japan; grad. sch. of sci. and tech., chiba university, chiba, japan; presto, jst. kawaguchi, japan a set of brain nuclei controls song production in songbirds. among these nuclei, the robust nucleus of arcopallium (ra) is the telencephalic site of direct projections onto vocal motor neurons and respiratory premotor neurons. the projections of ra to the mudulla included the tracheosyrigeal part of the hypoglossal nucleus (xiits), which innervates the syrinx, the birds , vocal organ, and respiratoryrelated nucleus, retroambigualis (ram) were present in bengalese finches. in this study, we have focused our attention on the descending projections of ra, with a view to the presence of contralateral projections to xiits and ram, using in vivo tract-tracing technique. the results indicated that ipsilateral and contralateral projections of ra to respiratory-vocal nuclei in the brainstem were defined in adult male bengalese finches. birdsong is composed of various song elements that have typical frequency modulation. each element is aligned in own sequential rule. especially in bengalese finches, the sequential rule obeys finite state grammar. it has been focused what neural mechanism enables such a complex sequential rule. in order to learn and maintain their own song, they have auditory neural representation of their own song in the forebrain area hvc. we collectively recorded the activities of hvc neurons driven by all possible element pair stimuli. the results show that most of neurons in hvc respond not only the sequence included in their own song but also the sequence not included. each neuron has typical response distribution toward the whole element sequence. in addition, the distribution property is different among neurons in same individual. taken together, information of the entire song element sequence would be stored in the neural ensemble of these neurons as a population coding. hironobu sakaguchi department of physiology and biological information, dokkyo university, school of medicine, japan avian vocal learning provides a good model for human speech learning. young male songbirds learn to imitate their tutor's song during a specific time in development, which is referred to as a sensitive period. many behavioral studies have shown that vocal learning is affected by a song template and social factors. if a young bird is raised without a tutor's song template (father) and/or social contacts with other birds, including its mother and siblings, it produces an abnormal isolated song, meaning that isolation delays the sensitive period for song learning. here, we investigated for the delayed song learning of socially isolated zebra finches from new tutors. consequently, isolated birds, exposed to new tutors from day , developed the zebra finch-typical song (song syntax), similar to song acquisition in young birds during the sensitive period of song learning. however, they were not able to imitate the syllable phonology from new tutors. the differences between two aspects of song organization suggest that the schedules and processes of the learning of phonology may be different from those of song syntax. ps a-h facilitatory effects of oxytocin on synaptic plasticity in the olfactory bulb and olfactory learning in young rats fumino okutani, jing-ji zhang, guang-zhe huang, hideto kaba department of integrative physiology, kochi medical school, nankoku, japan oxytocin (ot) within the olfactory bulb (ob) has been reported to be important for the induction of maternal behavior and recognition of offspring. the activity of mitral cells, olfactory relay neurons in the ob is inhibited by granule cells via reciprocal dendrodendritic synapses. electrophysiological studies have revealed that ot modulates mitral cell activity by acting on mitral and granule cells. in a classical conditioning paradigm, young rats show aversion to the odor that has been paired with foot shock. our studies have shown that plasticity in the ob is critical for this olfactory learning. pups that received ot infusion into the ob in the presence of citral odor developed an aversion to the odor without shock, suggesting that ot infusion has a facilitatory effect on olfactory learning. using ob slices, long-term potentiation (ltp) was induced in field epsps recorded in the granule cell layer. ot administration also facilitated ltp. these results demonstrate that ot is involved in olfactory learning in young rats. research funds: kakenhi ps a-h the gaba a receptors in the ventral pallidum are involved in the retrieval of conditioned taste aversion in rats tadashi inui, tsuyoshi shimura, takashi yamamoto div. behav. physiol., dept. behav. sci., grad. sch. human sci., osaka univ., japan we examined the effects of microinjections of gaba a receptors antagonist bicuculline into the ventral pallidum (vp) on the retrieval of conditioned taste aversion (cta). in experiment , rats received a pairing of saccharin or quinine hydrochloride (cs) with an i.p. injection of . m lithium chloride (us). after this conditioning, vehicle or bicuculline was bilaterally infused into the vp just before the re-exposure to the cs. the microinjections of bicuculline significantly increased the intake of saccharin cs, but not quinine hydrochloride. in experiment , rats were presented with saccharin as cs via an intraoral cannula. the microinjections of bicuculline significantly increased ingestive responses and decreased aversive responses. these results suggest that the gaba a receptors in the vp play an important role in the expression of ingestive and/or aversive responses to saccharin cs during the retrieval of cta so that the microinjection of bicuculline might increase the intake of saccharin cs. research funds: kakenhi ( ) ps a-h transient blockade, but not genetic deficiency, of c-fos gene expression impairs long-term memory in taste aversion learning roles of c-fos gene expression and its protein product, fos, in conditioned taste aversion (cta) learning were examined using the antisense oligodeoxynucleotide (odn) method in rats and in mice carrying c-fos gene deficiency. infusion of antisense odn (as-odn) directed against c-fos mrna into the parabrachial nucleus (pbn), but not into the amygdala or insular cortex (ic), impaired the acquisition, while infusion of randomized and inverted control odns had no effect. suppression of fos synthesis in the amygdala or ic impaired the retention. retrieval of an acquired cta was not impaired by as-odn infusion into the pbn or amygdala. in contrast, mice carrying c-fos gene deficiency showed normal acquisition and retention. the present results suggest that the fos-mediated signals in the pbn, amygdala or, ic plays key roles in the acquisition and/or consolidation, but not the retrieval, of long-term cta memory. ps a-h gaba receptors in the deep cerebellar nuclei are essential for mouse eyeblink conditioning classical eyeblink conditioning is a useful experimental system to analyze the neuronal substrate underlying learning and memory. the knowledge on the mouse eyeblink conditioning is far less compared with rabbit's. we examined the role of the deep cerebellar nuclei (dcn) during delay eyeblink conditioning in c bl/ mice by using gaba a receptors agonist and antagonist. in the acquisition tests, in which muscimol (msc) or picrotoxin (ptx) was injected from beginning of training, acsf-injected control mice learned this task, but both msc-and ptx-injected mice showed a significant impairment in acquisition of conditioned response (cr). in the retention tests, in which the drug was injected after acquisition of training, cr % in acsf-injected mice were kept over %, while those in the mscand ptx-injected mice decreased to %. these results revealed that gabaa receptors in the dcn play important roles in acquisition and retention of mouse eyeblink conditioning. various forms of synaptic plasticity are found in cerebellar circuits, but their significance in motor leaning remains unknown. in the cerebellum, delphilin is expressed selectively in purkinje cells (pcs) and localized exclusively at parallel fiber (pf) synapses, where it interacts with glutamate receptor ␦ that is essential for long-term depression (ltd) and motor learning. here, we showed that ablation of delphilin proteins facilitated ltd induction at pf-pc synapses and enhanced optokinetic response adaptation without affecting histology. this finding suggests that threshold regulation of ltd at pf-pc synapses is a limiting step for motor learning efficiency. ps a-h post-training cerebellar cortical activities are necessary for transfer of memory trace of motor learning from cortex to nuclei soichi nagao , , takehito okamoto , fumihiro shutoh , lab for motor learning control, riken bsi, saitama, japan; sorst, jst, saitama, japan; dept. anat., grad. univ. tsukuba, ibaraki, japan one-hour optokinetic training induces short-term adaptation of horizontal optokinetic response (hokr) gains in mice. succession of h daily training for week induces long-term adaptation. we recently reported that the memory trace of adaptation of hokr is initially acquired within the cerebellar flocculus through long-term depression (ltd), and later transferred to the vestibular nuclei for consolidation. in order to reveal the neural mechanisms underlying the memory transfer, we reversibly inactivated the neural activities of flocculus bilaterally by local application of muscimol immediately after the end of daily training. mice treated with muscimol showed depressed long-term adaptations, while the short-term adaptations were intact, suggesting that the neural activities of cerebellar cortex in a certain period after training are necessary for the transfer of memory trace from flocculus to vestibular nuclei. research funds: kakenhi ( ) ps a-h modification of gene expression in the cerebellar cortical neurons related with long-term motor learning yuji t. katagiri , , takehito okamoto , shin-ichi nishimura , fumihiro shutoh , , soichi nagao , lab. for motor learning control, riken bsi, saitama, japan; univ of the air, chiba, japan; dept. of anatomy, human comprehensive science, grad. univ. tsukba, japan; sorst, jst, japan we recently reported that the cerebellar ltd plays a crucial role for both acquisition and consolidation of memory trace of long-term motor learning using the adaptation paradigm of mouse horizontal optokinetic eye movements (shutoh et al., ) . in order to listup the molecules involved in the motor learning, we sampled total rna from the cerebellar flocculus of short-and long-term adapted mice, and quantified amounts of gene expression by the microarray methods. we found that the number of genes modulated by longterm motor learning much exceeded that modulated by short-term motor learning, and the number of down-regulated genes were larger than that of up-regulated genes. we furthermore examined the gene expression of purkinje cells by the laser micro-dissection and quantitative rt-pcr methods. ps a-h influence of spatial cues on hippocampal neuronal activity in spatial navigation tasks in mice hippocampal neurons were recorded while mice performing spatial tasks of searching for unpredictable and predictable rewards. the influence of spatial cues, including distal and proximal cues, on the response of hippocampal cells that exhibited place-related activity was examined. place cells predominantly shifted their fields accordingly by changes of visual and auditory distal cues, and fewer cells shifted their fields by changes of proximal cues. these results provide evidence that hippocampal neurons of mice can use flexibly information of spatial cues to represent the environment, and this ability is important for spatial learning. son ho , , t kobayashi , , e hori , , k umeno , , t ono , h nishijo , system emotional science, univ. of toyama, toyama, japan; crest, tokyo, japan we investigated a role of the hippocampal formation (hf) in encoding of a moving object in an open field. rats acquired icss rewards if they moved freely. then, a remote-controlled car was placed inside the open field. the rats could receive icss if it chased and approached the car. of a total of place cells recorded, activity of was significantly modulated by the car speed and/or distance between the car and rat; , and cells displayed distance-dependent, car speeddependent, and distance and car speed-dependent firing, respectively. furthermore, six cells, which did not show the place field in reference to rat position, but showed the place fields in reference to car position. in a control experiment, the same car was introduced, but the rats could receive icss rewards without relation to relative distance between the rat and car. so far, place cells were recorded in this experiment. of these, six and three place cells displayed distancedependent and car speed-dependent firing, respectively. the results suggest that hf encodes not only spatial information of own location, but also that of other moving object in an environment. hisae gemba, kazuko nakao, ryuiti matsuzaki, yusaku amaya department of physiology, kansai medical university, moriguchi, japan cortical field potentials were recorded by electrodes implanted on the surface and at a . - . mm depth in the cortex of monkeys in the process of learning somatosensory-initiated hand movements and then analyzed. it was found that an s-n, d-p potential, at about ms latency from stimulus, in the caudal bank of the left arcuate sulcus (homolog of broca's area) was related to recognition learning (association of stimulus with movement), and that an s-n, d-p potential in the motor and somatosensory cortices, and areas and , contralateral to the operating hand, was related to skill learning (making movements quicker and more appropriate). in visuo-initiated hand movements, the left prefrontal cortex was related to recognition learning; the motor and somatosensory cortices, and area to skill learning, as previously reported. this indicates that motor programming for somatosensory-initiated and visuo-initiated hand movement differs. computational studies of hippocampal function generally assume that ca performs a match-mismatch comparison of memory retrieval with sensory input. here we investigated this comparator model using an ensemble recording during task behaviors in the rat. we employed directional memory-guided alternation and visual cue discrimination tasks for the same animal. after training, the animals tended to predict a next direction according to the alternation paradigm even in the visual cue discrimination task. during this task, we found that some ca neurons showed specific bursts when a predicted event did not occur or along the trajectories of their corrective movements from a wrong cite to a correct cued one. these data suggest that ca plays an important role in the mismatch detecting and correcting process of behavior. n-methyl-d-aspartate (nmda) receptor has high permeability to ca + but is blocked by mg + in a voltage-dependent manner. this property is a molecular basis of nmda receptor-dependent long-term potentiation, which is thought to play a central role in learning and memory. we have generated the genetically engineered mice in which mutated nmda receptors defecting in mg + binding ability are expressed specifically in the granule cells of the dentate gyrus, the entry point to the hippocampal trisynaptic circuit. the mutant mice showed a variety of behavioral abnormalities including hyperactivity, impaired prepulse inhibition. to elucidate the effect of mutation on the information processing in the hippocampus, we recorded the place-related activity from hippocampal ca cells, the output stage of hippocampal circuit. the link between the behavioral anomaly and the hippocampal activity is discussed. mikako sakurai, ko zushida, masayuki sekiguchi, keiji wada department of neurodegenerative diseases, national institute of neuroscience, ncnp, tokyo, japan uch-l is a component of the ubiquitin system. uch-l is expressed at high levels in the hippocampal neurons. however, the functional role of uch-l in synaptic plasticity and behavior is not understood. we examined behavior and synaptic plasticity in gad mouse which is an autosomal recessive spontaneous mutant carrying an intragenic deletion in the gene encoding uchl . gad mice have significantly impaired performance in the open field and one-trial passive avoidance tests. theta burst stimulation (tbs) of shaffer collateral in hippocampal slices from gad mice elicited decremental long-term potentiation (ltp) in the area ca . in contrast, non-decremental ltp was induced in control wild-type mice. the maintenance of tbsinduced ltp in the wild-type mice was impaired by actinomycin d, an inhibitor of transcription, whereas tbs-induced ltp in gad mice was insensitive to actinomycin d. these results suggest that uch-l is a molecule participating in the synaptic plasticity elicited by tbs and the memory function. ps a-i learning stages in rat operant reversal task and cross-correlation between hippocampal and prefrontal local field potential powers yoshinori izaki, tatsuo akema department of physiology, st. marianna university school of medicine, japan to investigate whether the relationship between hippocampus (hip) and prefrontal cortex (pfc) spontaneous local field potentials changes with leaning stages, we analyzed cross-correlation (cc) of these local field potential powers during operant reversal training sessions. rats were trained with initial discrimination task until a stable discriminative performance was achieved (learning stage ). then the rats received the reversal training. learning stages examined were as following: the first training session (stage i), leaning stage for s+ (stage ii) and for s− (stage iii). different changes of the cc in some frequency-band powers with learning stages were observed. the cc in higher gamma-band ( - hz) was strong at stage and changed with leaning stages. particularly, the cc decreased to almost zero at stage ii. these results suggest that functional connection between hip and pfc is reflected in this frequency-band and changes with learning stages. ps a-i longitudinal fiber systems in the dentate gyrus of the rat norio ishizuka, yoshitomo umitsu department of brain structure, tokyo metropolitan institute for neuroscience, tokyo, japan longitudinal fiber systems in the dentate gyrus of the rat were investigated by anterograde labeling method of pha-l and retrograde labeling method with fluorescent dyes. the flattened hippocampal formation allowed sections to be cut perpendicular to the full septotemporal axis of the dentate gyrus. injection of pha-l into the hilar region elucidated that two longitudinal fiber systems existed in the dentate gyrus. the first fiber system gives rise to projections to the superficial portion of the dentate molecular layer, and the longitudinal axonal trajectory of this system ceased within the range of about . mm from the injection level. in the second fiber system, axonal terminations began to appear at the level of mm apart from the injection level and were distributed in further full septotemporal extent of the dentate molecular layer. the axonal arborizations of the second system were found in the deepest portion of the dentate molecular layer immediately above the granular cell layer. in the experiment of fluorescent dye injection, several kinds of cells in the hilus were retrogradely labeled. ryoichi moki, ryang kim, hisahiro umeeda, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan recent studies have shown that when conditioned fear memory is retrieved, fear memory becomes labile and requires gene expressiondependent reconsolidation for the re-storage. in addition, previous our study using conditional creb mutant mice indicated that creb is required for reconsolidation of conditioned fear memory. we also observed protein synthesis-dependent reconsolidation of spatial memory using morris water maze. in this study, to understand the mechanisms of reconsolidation of spatial memory, we examined a role of creb in reconsolidation of spatial memory. using conditional creb mutant mice that enable to induce the inhibition of creb activity in a tamoxifen-dependent manner, we found that inhibition of creb activity leads to disruption of spatial memory after the retrieval. this result indicates that creb is required for reconsolidation of spatial memory. shunsuke hasegawa, hirosi hosoda, satoshi kida department of bioscience, tokyo university of agriculture bhlh-pas transcription factor bmal ubiquitously expresses in brain. bmal functions by forming a heterodimer with either clock or npas , which has been known to play important roles in control of circadian rhythm and memory formation, respectively. to understand roles of bmal in forebrain function, we generated conditional mutant mice that enable to induce the inhibition of bmal function in a forebrain. using a dominant negative mutant of bmal (bmal r a) that forms a heterodimer with clock but loses the binding activity with e-box (hosoda et al., ), we generated transgenic mice expressing this mutant under the control of tetracycline-dependent promoter. these mutant mice were crossed to transgenic lines expressing tetracycline-dependent transcription factors (tta) under the control of alpha camkii promoter. we observed the expression of bmal r a in several double transgenic lines in a tta-dependent manner. behavioral analyses showed that these mutant mice showed an impairment of memory formation, indicating crucial roles of bmal in learning and memory. stress sometimes causes memory deficits. and chewing has been shown to reduce stress. however, the chewing-related mechanism in stress-induced memory deficits is unclear. we thus examined the effects of chewing on spatial memory using morris water maze and fos induction in the hippocampus and amygdala in stressed mice. when mice were exposed to restraint stress, reduction in learning ability and density of fos-positive cells in the dg and the bla was seen, but not in the mice chewing a thin wooden bar during stress exposure. the results suggest involvement of the amygdaloidmechanism by which chewing may prevent the stress-induced impairment of hippocampus-dependent memory. seiichiro amemiya, shinya yanagita, satoko suzuki, ichiro kita graduate school of science, tokyo metropolitan university, tokyo, japan we examined the effect of background noise (bgn) on spatial learning and its neuronal activity related to arousal and stress using maze task and c-fos immunostaining. rats performed maze task under different intensity of bgn ( , , or db; intermittent white noise). db bgn induced significant decreases in number of error and time to goal in maze compared with and db. although bgn increased fos positive acetylcholinergic neurons (fos-chat) in mesopontine tegmentum (mt) regardless of the intensity, fos-chat in basal forebrain (bf) increased intensity-dependently. in locus coeruleus (lc) and cortex, fos positive cell increased intensitydependently. furthermore, db bgn remarkably enhanced fos expression in stress-related nuclei, such as paraventricular nucleus and central nucleus of the amygdala. these results suggest that bgn improve spatial performance by enhancing arousal following activation of cholinergic neurons in mt and bf, and lc neurons. however, higher bgn intensity could evoke over-arousal and stress responses, thereby prevent the maze task. siriporn chattipakorn , , anucha pongpanparadorn , wasana pratchayasakul , anchalee pongchaidacha , nipon chattipakorn fac. dent. cmu, chiang mai, thailand; cert, cmu, chiang mai, thailand current pharmacotherapy of ad is the use of ache inhibitors. previous in vitro study showed that tde inhibited ache activity. this is the first study investigating the effects of tde on cortical ache activity and neuronal activity in in vivo. we used fos immunohistochemistry to determine the neuronal activity and the colorimetric method to investigate cortical ache activity following the single injection of various tde doses. mean fos-positive neurons in cortex were ± , ± and ± in the groups administered , and mg/kg tde, respectively. cortical fos-positive neurons in all three tde-treated groups were greater than those in the control group. percent inhibition of cortical ache activity was . ± . , . ± . and . ± . for , and mg/kg tde, respectively. these ache inhibitory effects were significantly different from the control. these findings suggest that tde could be beneficial as a possible novel therapeutic agent for ad. we showed the effects of a -h tde administration in animals on the inhibition of cortical ache activity and the enhancement of cortical neuronal activity. ache activity in circulation following a -h tde administration was not different compared to the control. this study investigated that the effects of tde on circulating ache activity (cache) in animal models was time-dependent. we used the colorimetric method to investigate cache activity in rats following the single administration of tde at various doses at different time courses ( , and min). percentage inhibition of cache activity following a single tde injection at doses and mg/kg significantly decreased at and min after tde injection, but not at min. cache inhibitory effects among two doses of tde administrated groups at various time courses were not significantly different. these findings suggest that tde may be a short-acting ache inhibitor. donepezil, galanthamine and tacrine are acetylcholinesterase (ache) inhibitors used for treatment of alzheimer's disease. we examined the neuroprotective mechanisms of ache inhibitors against apoptotic glutamate neurotoxicity using cortical neurons. we show that they protect neurons through mechanisms other than ache inhibition. the protective effects are mediated through nicotinic receptors (nachrs). donepezil and galanthamine protect neurons through ␣ and ␣ -nachr and kinases involved in pi k-akt pathway, and increase the levels of phosphorylated akt and bcl- . these results suggest that these ache inhibitors express their neuroprotective effects against glutamate neurotoxicity through nachrs and that donepezil and galanthamine protect neurons through pi k-akt pathway via ␣ and ␣ -nachrs. ps a-i arachidonic acid preserves hippocampal neuron membrane fluidity in senescent rats yasuto kashiyae , yoshiyuki ishikura , shigeaki fujikawa , yoshinobu kiso , manabu sakakibara lab. neurobiol. engr., tokai univ., numazu, japan; inst. health care sci. suntory, shimamoto, japan previous studies indicate that long-term dietary supplementation with arachidonic acid (aa) in -month-old rats (oa) effectively restores performance in a memory task and induction of long-term potentiation in the hippocampus to the level of young control animals (yc). this study examined fluorescent recovery after photobleaching (frap) in yc, old control (oc), and oa neurons in hippocampal slice preparations. three measures: mobile fraction (mf), diffusion constant (d), and time constant (τ), were estimated among yc, oc, and oa. each of these parameters was significantly different between oc and yc, suggesting that membrane fluidity is lower in oc than in yc. in contrast, d and τ were almost comparable in oa and yc, indicating that hippocampal neuronal membranes supplemented with aa were more fluid than those in oc, whereas the fraction of available molecules remained smaller than in yc. long-term administration of aa to senescent rats might help to preserve membrane fluidity and maintain hippocampal plasticity. ps a-i thimet oligopeptidase co-exists in gfap-and cd b-positive glia in rat pc/rsc treated with mk- takeshi kato , mohammad arif , michiyuki yamada , toshiyuki chikuma , md. mahiuddin ahmed lab. natural info. sci., grad. sch. integr. sci., yokohama city univ., yokohama, japan; grad. sch. integr. sci., yokohama city univ., yokohama, japan; dept. hygien. chem., showa pharmaceut. univ., machida, japan; dept. r&d, bioelectro. anal. sci., japan thimet oligopeptidase (ep . ) hydrolyzes not only neuropeptides but also the peptides generated by proteasomes. in the present immunohistochem study we found that mk- activated gfapand cd b-positive glia cells in rat posterior cingulate/retrosplenial cortex (pc/rsc) day after the treatment. mk- also increased ep . and prolyl oligopeptidase. immunohistochem data showed that ep . co-localized with gfap and cd b positive glial cells. since mk- causes schizophrenia-like psychosis and produces neurotoxicity in adult rodent brain, we further examined the pretreated effect of neuroleptics. clozapine co-administration suppressed the increased ep . in the pc/rsc. these data suggest that ep . in the astroglia and microglia cells of rodent brain might in part control positive and/or negative schizophrenia symptoms. ps a-i effect of age and sex steroids on the expression of alzheimer's disease presenilin (ps) and in the mouse brain soumi ghosh, m.k. thakur banaras hindu university, india alzheimerǐs disease is a neurodegenerative disorder characterized by the impairment of cognition and memory. these functions are improved by supplementation of sex steroids. the genes causing lateonset of ad, presenilin (ps) and , code for highly homologous integral membrane proteins. the proteolytic fragments of these proteins are main biological components. we have analysed the effect of age, sex and gonadal hormone supplementation on ps expression at protein level by western blotting. ps shows a significant decrease with aging in both males and females. however, there is no significant variation in expression of ps and ps with sex. gonadectomy also lowers the level of presenilin proteins in old age. ps protein shows increase in expression with gonadal hormone treatment in both ages, but estrogen supplementation to old mice lowers ps level. these modulatory effects of age, sex and gonadal hormones on ps proteins may explain the therapeutic interventions of hormone replacement therapy. research funds: ministry of science and technology, india ps a-i effects of the monomeric, oligomeric and fibrillar beta-amyloid peptides on the proliferation and differentiation of adult neural stem cells from svz dept. of pharmacol., seoul natl. univ., south korea the subventricular zone is the largest neurogenic area of the adult brain. in this region, neural stem cells (nsc) serve to produce newly generated neurons and glia cells throughout adulthood. however, the common neurogenesis of nsc cannot replace neuronal loss in alzheimerǐs disease (ad) induced by amyloid deposits composed mainly of amyloid␤proteins. in vitro, we examined the effects of various form of a␤peptide on the proliferation and differentiation of nsc from svz of -week-old adult mice. in this study, a␤ peptide was prepared three forms of aggregating stage, monomeric, oligomeric and fibrillar a␤ peptide. we found that treatment of nsc with oligomeric form of a␤ peptides remarkably increased the number of neurospheres during proliferation and neurons during differentiation in-vitro. we also found that these neurogenesis was accompanied by morphological change of neuron. the number of secondary and tertiary neurites increased at submicromolar concentrations of oligomeric a␤ peptide without shrinkage of axonal length. in alzheimer's disease (ad) brain, the formation of senile plaque with accumulated microglia is observed. although the role of microglia in ad is not clarified, their involvement in a␤ clearance is noted. high mobility group box protein- (hmgb ) is a non-histone chromosomal protein. here, hmgb was associated with senile plaques and protein level was increased in ad brain. diffuse hmgb immunoreactivity was observed around dying neurons in the kainic acid-and a␤ - (a␤ )-injected rat hippocampi. hmgb was not co-localized with a␤ in transgenic mice which show massive a␤ production without neuronal loss. furthermore, co-injection of hmgb delayed the clearance of a␤ and accelerated neurodegeneration in a␤ -injected rats. these results suggest that hmgb released from dying neurons may inhibit microglial a␤ clearance and enhance the neurotoxicity of a␤. perineuronal nets consisting of chondroitin sulfate proteoglycan (cspg) and hyaluronic acid (ha) are associated with distinct populations in mammalian brain. in the present study, we observed perineuronal net-like structure by rat cortical neurons in dissociated culture using wisteria floribunda lectin, ha binding proteins, and cspgspecific antibodies. this perineuronal net-like structure was observed often at parvalbumin-positive neurons, indicating gabaergic ones. it is well known that perineuornal nets-containing neurons are survive against alzheimer disease in human. to elucidate significance of perineuronal nets in alzheimer disease, we applied beta-amyloid peptide into cultured cortical neurons. perineuronal nets-containing neurons were resistant against beta-amyloid peptide, while negative neurons were often dead. these results indicate that perineuronal nets are participated in protecting neurons from cytotoxic substances such as beta-amyloid. ps a-i x -like protein regulates metabolism of app in the mouse brain yoshitake sano , , tadashi nakaya , shigeyoshi itohara , toshiharu suzuki riken bsi, saitama, japan; hokkaido university, neuroscience, sapporo, japan abnormal metabolism of amyloid beta precursor protein (app) results in the accumulation of beta amyloid (a␤) in the brain, and contributes to the pathogenesis of alzheimer's disease. app has a functional sequence in its cytoplasmic domain, the yenpty motif, which is involved in trafficking, internalization, and metabolism of app. x -like protein (x l) was identified as a molecule that interacts with the motif and regulates app metabolism in cultured cells ( . j. biol. chem. , . j. biol. chem. , ) . there is no evidence, however, that endogenous x l suppresses app metabolism and a␤ generation in vivo. to examine the physiologic role of x l in app metabolism in the brain, we generated x l null mutant mice. the mutant mice developed normally without gross anatomic brain abnormalities. there were increased amounts of cterminal fractions cleaved at the ␤-site and a␤, but the amount of total app was unaltered in the mutant mouse brain. these results suggest that x l suppresses the production of a␤ by inhibiting ␤secretase-induced proteolysis of app. it is still unknown how human's central nervous system (cns) controls its body system to keep the body balanced. this study aims to analyze the characteristics of spectral response of body sway in eyes open and in eyes closed during static upright stance based on a pid control model. in this model, body sway in medial-lateral direction is considered, and the body is simply modelled as a multi-link inverted pendulum system. spectral response analysis showed the gain varied with input frequency and time lag. peaks of the gain were intensively influenced by controller's parameters (kp, kd and ki). parameters identification showed that kd is decreased in eye-closed. by simulation, the spectral responses of the pid model quite agreed with the experimental data. the results proved that the spectral characteristics of body sway is determined by the dynamics of body system and its controller's parameters, suggest the balance-keeping control in cns can be modelled as a pid controller. nuclear dysfunction is a critical element of the pathology of polyglutamine (polyq) diseases. proteome analysis of soluble nuclear proteins in the nuclear matrix of neurons expressing normal or mutant huntingtin or ataxin- protein by d-electrophoresis and tof-mass delineate that mutant at and htt proteins similarly reduce transcriptional factor x and x . immunoprecipitation and pull-down assays support interaction between polyq and factor x and x . immunohistochemistry of hela cells and primary neurons reveal sequestration of factor x and x into inclusion bodies and reduction of them in the nuclear matrix. compensatory expression of factor x and x ameliorates poly-q pathology in htt-/at -expressing neurons and transgenic drosophila. these results suggest that factor x and x are critical regulators of polyglutamine disease pathology and could be a target for developing therapeutics. ps a-j ba - was reduced in rat brains fed with coconut juice n. radenahmad, p. subhadhirasakul psu, thailand young coconut juice (ycj), cocos nucifera (arecaceae), believed to contain phytoestrogen and other sex hormone-like substances, was investigated for its possible beneficial effects on halting dementia in ovariectomized (ovx) rats, a model system for the postmenopausal condition. sixty ovx rats were divided into six groups, rats/group (g). group received e at . g/kg per day; groups and received ycj at ml, and ml/kg day, respectively, once everyday. group received ycj ml/kg plus e at . g/kg day twice a week, all for weeks. the other two were ovx and sham-operated controls. using a chemiluminescent immunoassay, circulating e in group was insignificantly different from the control groups. after rats were sacrificed, brains were removed, fixed and paraffin embedded for ihc staining. using anti-␤-amyloid - antibody, this alzheimer pathology was found in cytoplasm and dendrites, but not in nuclei or axons, of pyramidal cells both in hippocampus and in layer and layer of cerebral cortex. it was found that amyloid deposition in frontal, temporal and hippocampus of rat brains in group was lesser than ovx and control groups. amyloid deposition was correlated with e serum at r = − . . ps a-j correlation between semantic memory and regional gray matter volume of anterior aspect of right temporal lobe in normal elderly subjects. a voxel-based morphometry yasuyuki taki , shigeo kinomura , kazunori sato , shinya uchida , ryoi goto , kentaro inoue , ichiro tsuji , hiroyuki arai , ryuta kawashima , hiroshi fukuda department of radiology and nuclear medicine, institute of development, aging and cancer, tohoku university, sendai, japan; tohoku univ. grad. school of med., sendai, japan; niche, tohoku univ., sendai, japan the purpose of this study was to determine whether there is a correlation between semantic memory and regional gray matter volume in community-dwelling normal elderly people by voxel-based morphometry. we collected brain magnetic resonance images of community-dwelling normal elderly subjects. we performed multiple regression analysis of raw score in the wais-r information subtest, gender, and regional gray matter volume. the volumes of the right superior and middle temporal gyri showed significant positive correlations with raw score in the information subtest. our study indicated that normal elderly individuals show a significant correlation between regional gray matter volume and semantic memory. research funds: (h -kenko- ), (h -choju- , h -choju- ) ps a-j effects of fluoxetine on the cognition of patients with mild cognitive impairments arash mowla, azadeh pani shiraz university of medical sciences, iran recent researches suggest a role for monoaminergic hypofunction in age related cognitive decline. in several studies selective serotonin reuptake inhibitors demonstrated neurogenesis in hippocampus. we studied the effects of fluoxetine on cognition of patients with mild cognitive impairment (mci). fifty-two non-depressed patients with mci were randomly assigned to take fluoxetine or placebo. the patients were administered mini-mental status examination (mmse) and wechsler memory scale iii (wmsiii) pre intervention. twenty-six patients completed the weeks trial. treatment response was defined as a final mmse and wms-iii scores. the patients in the fluoxetine group showed improvement in mmse and immediate and delayed logical memory scores of wms-iii. the placebo group had not significant changes in the cognitive measurements. fluoxetine enhanced memory and cognition in the patients. this was consistent with pervious studies that emphasized on the role of fluoxetine in improving memory and promoting neurogenrsis in the hypocampus. however, this conclusion should be tempered by the small sample size. lisa l. cook , d.g. goodenowe , y. yamazaki , j. flax phenomenome discoveries inc., saskatoon, canada; precisionmed inc., san diego, ca, usa dementia affects about % of the population over the age of and can result from various neuropathological conditions. currently, there is no way to differentiate specific forms of dementia (alzheimer's disease (ad), vascular dementia, etc.) prior to autopsy. pdi has discovered an -metabolite biomarker panel within the serum of patients with ad, non-ad dementia and healthy non-demented controls that can simultaneously differentiate the type of dementia and identify cognitive impairment using a non-targeted metabolomics technology based a fourier transform ion cyclotron resonance mass spectrometry (fticr-ms). the accurate measurement of the metabolite mass is sufficient to elucidate its molecular formula, thereby leading to metabolite identification, explication of biological significance and efficient biomarker validation. the -metabolite biomarker panel could provide a non-invasive method to aid in the diagnosis of specific subtypes of dementia. the development of a high throughput assay for these markers will also be presented. neurons become photosensitive by genetically introducing one of green algae-derived protein, channelrhodopsin- (chr ). in this study, we quantitatively investigated the rapidness of the light-gated current of chr expressed in pc cells using blue led light. the light-gated current consists of two components, inactivating and noninactivating. the magnitude of inactivating component was almost linearly related to the light intensity. the non-inactivating component showed the tendency to saturate at high illumination. we also found that the activation rate is about -fold faster than the inactivating rate, but both are linearly dependent on the light intensity. since the photoactivated current was very rapid in both onset and offset, the neuronal firings were phase-locked to short light pulses in an acute slice of hippocampus. it is suggested that the genetic expression of chr is one of the most ideal photostimulation methods of a genetically identified neuron with defined activity patterns in intact nervous system. yujiro hattori , shigeki ohta , kenji hamada , naofumi yamada-okabe , yonehiro kanemura , hideyuki okano , yutaka kawakami , masahiro toda , neuroimmnology research group, keio univ., tokyo, japan; chugai co. ltd., kanagawa, japan; inst. cli. res., onh, japan; physiology, keio univ., tokyo, japan; cellular signaling, institute for advanced medical research, keio univ., tokyo, japan; neurosurgery, keio univ., tokyo, japan to identify neuron specific genes, we performed two gene profiling techniques, dna microarray and est analysis. in this study, we focused on genes expressed specifically in the normal brain tissues but not in glioma tissues and identified the human kiaa gene which was a homologue of rat synarfgef (po). rt-pcr analysis revealed that the human kiaa homologue was expressed only in adult brain tissue. western blot and immunocytochemical analyses showed the kiaa protein was expressed in adult brain tissues and differentiated neuronal cells but not in fetal brain tissues nor neural stem/progenitor cells. in conclusion, we identified an adult neural-specific gene using the combined gene profiling method and our results suggest the usefulness of this method to identify tissue specific genes. ritsuko the objective of this study was to find the proteins related to sexual differentiation and to elucidate its molecular mechanism. methods: developing hypothalamic and cortical cells from fetuses on embryonic day were dissociated. after the cells were treated with nm estradiol- beta (e ) or ethanol for days, proteins were extracted and labeled with cydyes. two-dimensional difference gel electrophoresis ( d dige) was then performed. the differential protein spots were analyzed by software analysis, subject to in-gel digestion, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof-ms). results: more than spots were detected from d dige. compared with ethanol treatment, e increased the expression of spots in the hypothalamic cells and spots in the cortical cells (p < . , difference > . ). proteomics analysis showed different effects of e for hypothalamic and cortical cells. in order to relate cellular brain structure to function, it is necessary to manipulate neural circuits at the level of individual cell types. genetic methods for neuronal inactivation combined with cell-typespecific promoters will achieve this goal. here, we have developed a genetic method for quickly and reversibly inactivating in vivo mammalian neurons using allatostatin receptor (alstr), which causes neuronal hyperpolarization when treated with peptide ligand allatostatin (al). in rat barrel cortex neurons expressing alstr, al reversibly inactivated neuronal activity evoked by electrical stimulation of the whisker pad. both inactivation and recovery were seen within several minutes. we also confirmed the effectiveness of the al/alstr system in ferret visual cortex, lateral geniculate nucleus (lgn), and monkey lgn. therefore, the al/alstr system will be a powerful tool to investigate neuronal circuits and function. prospective purification of neural stem cells (nsc) through the specific cell surface marker is crucial for functional recovery of the damaged brain. in the last meeting, we showed that living nsc were enriched from mouse whole brain as positive cells for erythro-phytohemagglutinin (e-pha), which binds to complex type asparagine-linked oligosaccharide (n-glycans). in this study, by using facs system, we found that high selective affinity of e-pha binding to the brain cells; e-pha negative cells were neurons, mid-positive cells were nsc, and highly positive cells were endothelial cells, respectively. ligand blot analysis revealed the existence of the e-pha binding proteins different from the known selective nsc markers in e days brain homogenate, suggesting that n-glycosylated proteins could be distinctive markers for nsc. masahiro waza, hiroaki adachi, masahisa katsuno, makoto minamiyama, fumiaki tanaka, manabu doyu, gen sobue department of neurology, nagoya university, nagoya, japan the pathogenic gene product of spinal and bulbar muscular atrophy (sbma) is polyglutamine (polyq)-expanded androgen receptor (ar), which belongs to hsp client protein family. -allylamino- -demethoxygeldanamycin ( -aag) is a new derivative of geldanamycin that shares its important biological activities but shows less toxicity. -aag is now in phase ii as a potential anti-cancer agent because of its ability to selectively degrade several cancer-related client proteins. we examined the efficacy and safety of -aag in a mouse model of sbma. administration of -aag significantly ameliorated polyq-mediated motor neuron degeneration by preferential proteasome degradation of mutant ar. the ability of -aag to preferentially degrade mutant protein would be directly applicable to sbma and other neurodegenerative diseases. modulation of hsp function by -aag has emerged as a candidate of molecular targeted therapy for neurodegenerative diseases. the avian embryo has long been a popular and an excellent model for studying vertebrate development because of its classical manipulative advantages. in the present study, we tried to develop regulated gene transfer by using the tet regulatory system in the chick. the reverse tetracycline-controlled transactivator was expressed under the control of the motor neuron (mn) specific hb promoter. tetracycline responsive elements were used for inducible gfp expression. after these constructs were introduced into neural tube by in ovo electroporation, gfp expression was induced in spinal mns in the presence of doxycycline. approximately - % of mn express gfp very intensely whereas the remaining mns never express gfp, suggesting that, although the transgene is induced in limited numbers of mns, once activated, cells express a large amount of the protein product of the experimental gene. thus, this strategy can be applicable for a variety of experiments that require specially and temporally regulated gene expression in the chicken embryo. ps a-k selective collection of catecholaminergic (ca) neurons in the brain and its application to gene expression analyses hiroaki nakamura , yoshiyuki ishii , kazuto kobayashi , yasufumi sato , keiichi itoi lab. info. biol., grad. sch. info. sci., tohoku univ., sendai, japan; dept. mol. genet., fukushima med. univ., japan; inst. develop., aging, cancer, tohoku univ., japan ca neurons are involved in a wide spectrum of physiological functions in the brain. most ca neurons are localized in the brainstem and hypothalamic regions and typically make clusters of cells, among which the noradrenergic (a , a and a ) and dopaminergic (a , a and a ) neurons predominate. in order to explore functional roles of these neurons, we collected ca neurons selectively using tyrosine-hydroxylase (th)-green fluorescent protein (gfp) transgenic mice in which gfp was expressed under the control of th gene promoter. in fetal mice, most gfp-positive neurons expressed th-immunoreactivity in limited brain regions including the locus coeruleus (lc). therefore, lc-containing region was dissected under fluorescent microscopy, and neurons were dispersed by treating with trypsin, then gfp-positive cells were sorted out by flow-cytometry (facs). rna was extracted from the gfp-positive (th) neurons, reverse-transcribed, and analyzed by pcr. atg b has been shown to play an important role in the processing of lc , a mammalian homologue of yeast atg , but the tissue distribution of atg b remains unknown. to understand the role of atg b in rat tissue cells, we prepared an antibody to atg b and pc cells in which atg b expression was knocked down by rnai. in rnaitreated pc cells where atg b expression was % of that in the wild-type pc cells, the expression of cytosolic lc -i was similar to that in wild-type cells. the knockdown cell lysates, however, suppressed cleavage of prolc to lc -i. moreover, the expression of atg b mrna was high in the cerebellum and olfactory bulb, while its protein was evenly distributed in the brain. immunostaining for atg b was intense in neurons, especially in the cerebellum. these results suggest that atg b plays a major role in the processing of lc , while autophagy is deeply associated with the metabolism in neurons, especially in the cerebellum. ps a-k spatial and time-dependent transneuronal propagation of swine coronavirus (hemagglutinating encephalomyelitis virus, hev) in the rat central nervous system after its hind footpad inoculation transneuronal propagation of hev n strain into the central nervous system was examined after its subcutaneous inoculation ( pfu) in the rat hind footpad. on day post-inoculation (p.i.), antigenpositive neurons were detected in cell groups of the ipsilateral spinal cord of lumber segments. on day p.i., they increased in number, and higher-order transneuronally infected neurons were observed in restricted brain areas that project to the spinal cord. on day p.i., the viral infection became more extensive and complex, and neurological signs appeared from this period. in this model the th day would be critical for the analysis of the long-distance connections. hev can be used as a novel tracing probe, being equivalent to other reported virus probes. hiroyuki hioki , hiroshi kameda , hisashi nakamura , taro okunomiya , koji ohira , kouichi nakamura , , takahiro furuta , takeshi kaneko , dept. of morphol. brain sci., grad. sch. of med., kyoto univ., kyoto, japan; crest, japan vesicular stomatitis virus g-protein (vsv-g) pseudotyped lentiviral vectors are useful vectors for gene transfer into the central nervous system. vsv-g achieves a broad transduction spectrum and lentiviral vectors provide an efficient vehicle to integrate transgenes into dividing and non-dividing cells. thus, vsv-g pseudotyped lentiviral vectors with ubiquitous promoters, such as human cytomegalovirus (hcmv) promoter, infect and express transgenes in neuronal and glial cells. the purpose in this study is to explore neuron-specific promoters and to quantitatively examine their characteristics. at first, we used five kinds of well-known neuron-specific promoters; hsyn , rta , mcamkii, rnse and hpdgf promoters. then, we developed new hybrid promoters by a combination sequence of hcmv enhancer and neuron-specific promoters listed above. all of the new hybrid promoters dramatically improved expression of reporter gene (gfp), but the specificity deteriorated in the rat striatum, thalamus and neocortex. although green fluorescent protein (gfp) is a useful tool to label living neurons, neuronal processes are not completely labeled with gfp. in the present study, we tried to develop dendritic membrane-targeted gfp using non-prolilferative lentivirus vector with human synapsin i promoter. palmitoylation site of gap n-terminal and myristoylation site of fyn n-terminal were first tested for membrane targeting of gfp. myristoylated gfp (myrgfp) was efficiently localized at the plasma membrane of infected neurons, but not palmitoylated gfp. since myrgfp was located at both dendritic and axonal membranes, we further added the putative dendrite-targeting or basolateral targeting signals, such as c-terminals of telencephalin (tlc), fc ␥ ii ␤ receptor (fcr), polymeric immunoglobulin receptor (pigr), and low density lipoprotein receptor (ldlr), to c-terminal of myrgfp, and compared their efficiency on dendrite targeting. recently, we developed recombinant rabies virus vectors which were expected to act as a potential neurotracing tool. the vectors infected neurons specifically from axon terminals and were transported to the downstream neurons trans-synaptically. by using two different recombinant vectors, each of which expresses reporter protein of different kind, we attempted double labeling of a neuron. it was expected that we could detect and visualize the divergence or convergence of a neurocircuit. in the study, we could demonstrate the efficiency of double labeling in vivo. in the present study, we examined the potential of this technique particularly in terms of the quantitative detection of double labeled neurons in complicated neurocircuit. the experiments were performed in the hippocampus and the neighboring cortices of rats. we could show that this technique is also useful for the quantitative analysis of neurons which forms projections to different region of the brain. shuchen lee, lihao ge institute of neurobiology, institute of brain science, fudan university, shanghai, china glycine receptors on bullfrog retinal cone photoreceptors were characterized by immunocytochemical and whole-cell patch clamp techniques. cone terminals were both gly␣ and gly␤ immunoreactive. in freshly dissociated cones, an inward current could be induced while glycine was focally applied to the terminal. the glycine-induced current was strychnine-sensitive and reversed in polarity at a membrane potential, close to the equilibrium potential of chloride ions. these results suggest that glycine, which may be released by glycinergic inplexiform cells, could modulate functions of cone photoreceptors. ␦-catenin has armadillo motifs and a carboxyl terminal type i pdz ligand. in neurons, ␦-catenin is enriched in the postsynaptic density, where it serves as a link between the adherens junction and the post-synaptic protein complex including the nmda and ampa receptors. electrophysiological recordings from ca hippocampal neurons overexpressing ␦-catenin demonstrated that ␦catenin increased the ampa receptor-mediated epsc but had no significant effect on the nmda receptor-medicated epsc. the effect of ␦-catenin on the ampar-epsc was medicated by its pdz ligand. in cos cells, co-transfection of ␦-catenin/grip showed that ␦-catenin regulated the membrane localization of grip through its pdz ligand. co-transfection of ␦-catenin/grip/gulr increased the surface expression of glur in cos cells compared with grip/glur or ␦-catenin/glur transfection. this study points to ␦-catenin as a regulator of glur receptor trafficking. inseon song, kunihiko obata, alexey semyanov bsi, riken, japan gaba a receptor mediated tonic conductance is a major component of membrane conductance which determines the way how neuron integrates incoming synaptic signals as well as input-output characteristics of the cell. we measured density of picrotoxin (gaba a receptor antagonist) sensitive holding current (which reflects gaba a receptor mediated conductance) in interneurons of hippocampal ca area in wild type (wt) and gad knockout (ko) mice. this parameter was twice lower in gad ko mice (wt: . ± . pa/pf, n = ; gad ko: . ± . pa/pf, n = ; p = . ). the total membrane conductance was similar in both types of animals suggesting adaptive compensation. application of gaba ( m) increased tonic current in both type of mice by the same amount. no significant difference in amplitude or frequency of spontaneous ipscs was detected, although their decay time was shorter in gad ko animals (wt: . ± . ms, n = ; gad ko: . ± . ms, n = ; p = . ). the changes in inhibition which we have found may explain previously reported behavioral abnormalities in gad ko. research funds: bsi, riken hiroki mutoh, thomas knopfel lab. for neuronal circuit dynamics, bsi, riken, wako, japan olfactory glomeruli constitute the first stage of central odor processing. yet, their role in integration of odor information is only partially understood. we previously discovered that hz olfactory nerve (on) stimulation induces long-term depression (ltd) in young (p to p ) mice. the present experiments were designed to understand in more detail the molecular mechanisms underlying on ltd. bath application of dhpg, a selective group i mglur agonist, induced on ltd and occluded subsequent hz stimulation-induced ltd. on ltd was not induced by activation of group ii or iii mglur agonists. the dhpg-induced on ltd was mediated by mglur but not by mglur because it was antagonized by the mglur antagonist ly but not by the mglur antagonist mpep. expression of dhpg-induced on ltd was accompanied by an increase in paired-pulse ratio suggesting that on ltd is caused by a decrease of release probability. we propose that mglur is expressed at the on. on ltd may be important for establishment and maintenance of odor maps in the olfactory bulb but may also involve in the regulation of the sensitivity for specific odorants. ps p-a involvement of dopamine system in long-term potentiation of thalamo-prefrontal cortex pathway masatoshi takita , michiko ohtomi cognition and action group, national institute of advanced industrial science and technology (aist), ibaraki, japan; department of biomolecular science, faculty of science, toho university, chiba, japan a mesocortical dopaminergic (da) input to prefrontal cortex (pfc) with the d receptor is necessary for long-term potentiation (ltp) to occur at hippocampal-pfc synapses, which is involved by working memory (wm) in rats. here the da system was investigated in another wm-involved pathway from mediodorsal nucleus of the thalamus (md) to pfc. preliminarily, local perfusion of the d antagonist sch into pfc by using a microdialysis method impaired md-pfc ltp but the d antagonist sulpiride did not. extracellular da levels in the pfc robustly increased after the tetanus of md (by - %). as a result both excitatory synaptic inputs to the pfc involved the wm-related da profile, implying da system enables a contrast-emphasis for cooperative crosstalk among several neuroplasticities in the pfc to selectively store intersectional information of multiple brain areas. neuronal activity is necessary for postnatal maturation of synaptic connections only grossly laid out in the neonatal brain. in sensory cortices, synaptic maturation involves strengthening of sensory-evoked responses and development of receptive field (rf) maps with defined rf size and shape. evoked activity is thought to shape synaptic maturation in sensory cortices by mechanisms of competitive hebbian plasticity. dendritic excitability, mediated by voltage-gated na + channels, is required for active backpropagation of axosomatic action potentials (aps) and initiation of dendritic spikes; backpropagating aps and dendritic spikes enable forms of synaptic hebbian plasticity, such as spike-timing dependent plasticity (stdp). here we examined the role of dendritic excitability in synaptic maturation of layer / pyramidal neurons in the rat somatosensory barrel cortex. in the present study we compared ltp induction in neocortex of captreated and normal rats in present of gaba antagonist, picrotoxin (ptx). the result of present experiment showed that ptx plays an important facilitatory role in the induction of ltp in both normal and cap-treated group. in cap-treated group, in present of ptx, the ltp responses significantly were higher than normal group. we conclude that the enhancement of ltp by ptx can be explained by product of competition between excitatory and inhibitory pathways or synapses. these results suggest that gabaergic system has an important role in synaptic plasticity. also, these results indicated that gabaergic inhibition has been increased in cap-treated group. tohru kurotani, komatsu yukio department of visual neuroscience, research institute of environmental medicine, nagoya university, nagoya - , japan we showed in previous study that somatic inhibitory synapses of neocortical layer pyramidal neurons undergo long-lasting depression and potentiation depending on the intrinsic firing pattern of the cell that mimics slow wave sleep (sws) and arousal states. in the present study, using a minimal stimulation method, we recorded somatic ipscs from layer pyramidal cells in visual cortical slices prepared from rats at sws like state under urethane anesthesia and in those prepared from rats at arousal state. the average amplitude of somatic ipscs recorded in slices from the former group was significantly larger than that recorded in slices from the latter group. the mean rise time, decay time constant of ipscs and the mean input resistance of the cells were not significantly different between these two groups. the present results further confirmed that the somatic inhibition in neocortical layer pyramidal neurons is bidirectionally modified in accordance with behavioral state. corticothalamic fibers (ct), originated from cerebral layer pyramidal cells, make excitatory synapses with both thalamic relay neurons and reticular neurons. since these pyramidal cells abundantly express kainate receptors (kars) mrna, we studied the effect of kainate on the presynaptic function of the two ct synapses in mouse thalamic vb nucleus. bath application of kainate ( nm) depressed ct-epscs and increased the paired pulse ratio in relay neurons. in contrast, kainate at the same concentration facilitated ct-epscs and decreased the paired pulse ratio in reticular neurons. these results suggested that kars differentially regulated release at the two ct synapses. furthermore, high frequency stimulation of ct depressed relay cell synapses but facilitated reticular cell synapses. blocking endogenous kars abolished these effects. because reticular cells are the main source of inhibitory input to relay neurons, we suggested that endogenous kars presynaptically regulate the balance of excitatory and inhibitory inputs to thalamic relay neurons. to examine the involvement of ntr in the regulatory mechanisms for ltp in the amygdala, we utilized ntr -knockout (ko) mice. we performed whole-cell patch-clamp recordings from the pyramidal neurons in the basolateral amygdala (bla), where da-nt neurons project. we found that the bla-ltp, induced by la stimulation, was significantly greater in ntr -ko mice than in wild-type mice. the bla-ltp in ntr -ko mice was attenuated by sulpiride, a d receptor antagonist. these results suggest that d -ntr interaction regulates the extent of ltp in the mouse la-bla synapses. ps p-a facilitation of axonal plasticity in recovery from traumatic brain injury and the role of tnf␣ in mouse model recent studies suggest axonal plasticity as possible mechanism of recovery from brain injury. apart from that, tnf␣, an inflammatory cytokine, has also been suggested to serve neuroprotective roles. the present study evaluated motor function recovery after controlled cortical impact (cci) brain injury, and also the facilitation of plasticity by biotin dextran amine (bda) axonal tracing in tnf␣ko mice and wild type (wt) mice. mice were subjected to left sided cci or served as sham controls, and were evaluated by composite neuroscore and rotarod over -day period. bda was injected in right cerebral cortex to observe new axonal connections. so far, we observed recovery of motor function in wt mice, whereas tnf␣ko mice showed continuous functional deficit. we also observed greater number of new axonal connections in wt mice. our results suggest that tnf␣ is necessary for functional recovery after brain injury, and axonal plasticity may be the mechanism involved. disuse of synaptic activity causes homeostatic adaptation presynaptically and/or postsynaptically. here we show that in hippocampal autaptic cultured neurons tetrodotoxin-induced chronic inactivity increases the fraction of high vesicular release probability pool with the entire readily releasable vesicle pool size remained intact. kinetics of short-term plasticity and unchanged apparent ca + sensitivity indicate that ttx-induced presynaptic modification is unlikely due to an increase in the fusion rate crucial for the ca + at the final fusion step. in addition, analysis of neurons genetically lacked the synaptic vesicle protein synaptotagmin- , and timing-dependent rescues using two different viruses provide a novel conception, namely, vesicle machinery requires prolonged period so that the fast burst vesicle pool orchestrates presynaptic homeostasis system underlying "vesicle mobilization". ps p-a inhibitory modulation of the hippocampal ca transmission and plasticity by glucagon-like peptide- jun-ichiro oka, takashi iwai lab. pharmacol., fac. pharm. sci., tokyo univ. sci., japan glucagon-like peptode- (glp- ) is a proglucagon-derived peptidehormone in the intestine and brain. we reported that glp- (i.c.v.) improved the concussive brain injury-or scopolamine-induced amnesia in mice. however, the mechanisms of glp- effects on hippocampal neurons are unclear. in this study, we investigated the effects of glp- on the synaptic function of neurons in the acute hippocampal slices. hippocampal slices ( m) were prepared from to days wistar rats of both sexes. patch-clamp recordings were made from pyramidal cells of the ca in the whole-cell mode using glass microelectrodes (resistance: - m ). in extracellular recordings, field excitatory postsynaptic potentials (fepsp) were evoked with a bipolar tungsten electrode, placed in the mossy fibers. glp- ( nm- m) inhibited spontaneous excitatory postsynaptic current. glp- ( nm) did not affect fepsp amplitude or the paired-pulse ratio, but attenuated the long-term potentiation. these results suggest that glp- may play an inhibitory role in the dg-ca transmission. ps p-b quantitative imaging of exo-endocytosis at mossy fiber presynaptic terminals of hippocampus by genetically expressed fluorescent probe takuya hkima, rikita araki, toru ishizuka, hiromu yawo dept. of dev. biol. and neurosci., tohoku univ. grad. sch. of life sci., japan both presynaptic and postsynaptic mechanisms are proposed for the synaptic plasticity. however, the presynaptic mechanisms have been analyzed indirectly on the postsynaptic responses. it has been difficult to quantify the exocytosis at the presynaptic terminals, particularly those in vivo or in acute slices. to measure exocytosis directly, we applied the synaptophluorin (sph) method to the individual presynaptic terminals in hippocampal slices of a mouse genetically expressing a conjugate protein of vamp- and superecliptic phluorin selectively in the mossy fiber terminals. the sph fluorescence at individual mossy fiber terminal was increased by nerve stimulation and was followed by its reduction which is blocked by bafilomycin a , a vesicular h+-atpase inhibitor. therefore, the rising phase of sph fluorescence corresponds to exocytosis whereas the decreasing phase to endocytosis and subsequent re-acidification of vesicles. this method would enable us to evaluate the presynaptic contribution to synaptic plasticity. jyoti parkash, gurcharan kaur gndu amritsar, india we have earlier reported that gnrh nerve terminals in the me continue to express high levels of polysialylated form of neural cell adhesion molecule (psa-ncam) in a cyclic fashion and psa-ncam covers both the gnrh axon surfaces and the associated glial cells in the proestrous phase rats indicating that psa plays important role in the neurosecretory activity in hypothalamus. to further establish the functional significance of psa-ncam molecule, we have studied the expression of psa-ncam on gnrh axon terminals and glial cells in the proestrous phase of cycling rats as well as gaba and pbz treated proestrous rats by using dual immunohistofluorescent staining. both gnrh and psa-ncam immunostaining was much higher in proestrous phase rats, whereas, gaba and pbz treatments significantly reduced their expression. the expression of pst has been studied within gnrh cell bodies as well as at their terminals by combining in situ hybridization with immunohistofluorescent in poa and me-arc regions of cycling female rats as well as in gaba and pbz treated proestrous rats. cortical plasticity has important roles in the development of neural circuits in sensory cortices. however, the roles and mechanisms for various types of ltp and ltd are not clear. we investigated supragranular ltp and two types of supragranular ltd in the slices obtained from the rat auditory cortex, and compared their properties. frontal cortical slices were prepared from male wister rats. supragranular field potentials elicited by the stimulation applied to layer vi were recorded. ltp was induced by tetanic stimulation (ts, hz for s) applied to layer vi. ltd was induced by low-frequency stimulation (lfs, hz for s) applied to layer vi. ltd was also induced by ts applied to supragranular layers near the recording site. lfs-induced ltd and ts-induced ltd were completely abolished in the presence of m apv, m bicuculline, but not m mcpg. lfs-induced ltd and ts-induced ltd occluded each other, suggesting that that both types of ltd share cellular and molecular mechanisms. kazuyoshi kawa department of neurophysiology, tohoku university, graduate school of medicine, sendai, japan using slice-patch techniques, synaptic transmission in neurons of the area postrema (ap) of the rat was studied. when mm kcl was applied from a "y tube" to ap neurons (whole-cell clamped at − mv), massive inhibitory postsynaptic currents (ipscs) were induced. most of the evoked ipscs were blocked by bicuculline confirming gabaergic identity. when nicotine ( - m) or capsaicin ( . - m) was applied to ap neurons, robust appearance of ipscs with gabaergic identity was induced. after blocking action potential generation in the slice with tetrodotoxin ( m), nicotine and capsaicin could still induce gabaergic ipscs. interestingly, responses to capsaicin of the synaptic facilitation showed marked desensitization even after min of rigorous washout. it is concluded that nicotinic receptors, as well as capsaicin receptors (presumably, trpv ), are expressed at gabaergic presynaptic terminals in area postrema neurons and play a distinctive role in controlling autonomic neural functions. research funds: grant from the smoking research foundation (japan) takako morimoto-tanifuji , akira komatu , akinao nose dept. phys., univ. tokyo, tokyo, japan; dept. physiol., sch. med., tokyo women's med. univ., tokyo, japan the molecular mechanisms that target neurotransmitter receptors to the postsynaptic membrane and keep them clustered remain unknown. we investigated how the localization of glutamate receptors (glurs) is regulated in neuromuscular junctions (nmjs) of drosophila rd instar larvae. there are mainly two classes of glurs, containing either gluriia or iib. gluriia has a sequence predicted as ca + -permeable site. when camkii was inhibited by the expression of inhibitory peptide, ala, the content of gluriia in synapses was dramatically increased and the mean amplitude of extrajunctional potential (ejp) was enhanced. the expression of constitutively active form of camkii (t d) resulted in decreased gluriia content and enhanced gluriib content. although miniature ejp amplitude was reduced, ejp amplitude was normal in t d expressing larvae, suggesting the existence of some homeostatic mechanisms. taken together, camkii regulates the localization of glurs in a subunitspecific manner and modulates synaptic function in nmjs. ) . notably, neuronal dnrs from dnr * flies did not show mg + blockade, and dnr * flies displayed significant impairment in transcription-dependent long-term memory (ltm) but not in transcription-independent acquisition and short-term memory. we identified salient increases in genes involved in l-ltp formation, e.g. homer, and activin, as well as the increase in genes involved in ltm, e.g. staufen, upon ltm formation. however, such increases were absent in dnr* flies. transcription for ltm is mediated, at least, by transcription factors such as creb, adf- , and notch. we examined how mg + blockade of dnr links to these transcription factors. research funds: kakenhi ps p-b response properties of wind-sensitive giant interneurons in the th-instar nymphs of the cricket tetsuya matsuura , masamichi kanou dept. of welfare eng., iwate univ., morioka, japan; dept. of biology, ehime univ., matsuyama, japan the response properties of four wind-sensitive giant interneurons (gis) - , - , - and - in the th-instar nymphs of the cricket gryllus bimaculatus were investigated. air current was presented to the animal from different directions in the horizontal plane. the intensity-response curves showed that the response magnitudes of gi - increased with stimulus velocity up to mm/s regardless of the stimulus direction. the response magnitudes of gi - reached a plateau at a stimulus velocity of mm/s in most stimulus directions. the response magnitudes of gis - and - increased with stimulus velocity up to mm/s regardless of the stimulus direction. the directional sensitivity curves revealed that the preferential directions of the gis in nymphs were the ipsilateral-side in gi - , the ipsilateralfront and contralateral-rear in gi - , the ipsilateral-rear in gi - and the ipsilateral-front in gi - , designated with respect to the side of the ventral nerve cord containing the axons, which were basically the same with those of adults. yasuyuki ishikawa, sadao shiosaka division of structural cellular biology, nara institute of science and technology, nara, japan long-term potentiation (ltp) is an enhancement of synaptic strength that may contribute to information storage in the mammalian brain. ltp expression can be regulated by previous synaptic activity, a process known as "metaplasticity." we report a novel form of cellwide metaplasticity in hippocampal area ca . serine protease, neuropsin, is involved in the regulation of synaptic plasticity. neuropsin increased the stability of ltp induced later at the same inputs via l-type vdcc. moreover, neuropsin-deficient mice impaired l-ltp induction by l-tbs. our findings have revealed the effects of neuropsin on the conversion of e-ltp to l-ltp. ptp, a form of presynaptic short-term plasticity, is mediated by a transient increase in transmitter release probability caused by tetanic stimulation. although it has been known that ptp is induced by the elevation of presynaptic ca + , the molecular mechanism of ptp is poorly understood. in order to elucidate the specific role of presynaptic trkb receptors in ptp, we analyzed ptp using hippocampal slices from conditionally gene-targeted mice in which the knockout of the trkb gene is restricted to presynaptic sites in the ca region. we found that ptp induced by the -hz tetanus was reduced in mutant mice, and that ptp in control mice was partially reduced by an n-type ca + channel blocker, while ptp in mutant mice was unaltered by the blocker. thus, these data suggest that the n-type ca + channel-dependent component of ptp requires trkb receptor activation. research funds: jsps and mext of japan kiyoshi ohnuma , kunihiko kaneko , , makoto asashima , grad. sch. of arts & sci., univ. of tokyo, tokyo, japan; jst, tokyo, japan measuring fluctuations or population distributions of a system can be used to understand the dynamics of the system. we have used this approach to study intercellular interaction between neuronal cells. here we show that the shape of the population distribution of intracellular ca + concentration ([ca + ] i ) may change because of nonsynaptic communication. we loaded pc cells with a ca + indicator, indo- am, and the [ca + ] i of more than , cells was measured using flowcytometry. the [ca + ] i distribution of unstimulated single cells had a long right tail, suggesting that [ca + ] i is usually low but sometimes becomes high. on the other hand, the distributions of cell clumps and depolarized single cells were bell shaped, suggesting that many ca + -related mechanisms such as channels and pumps were activated by nonsynaptic communication or by depolarization to change the shape into a normal distribution according to the central limit theorem. our results suggest that measuring the distributions is useful in researching intercellular interaction. na x is a sodium channel involved in sensing the sodium level of the body fluid. our recent studies showed that na x is specifically localized to perineuronal lamellate processes of specialized glial cells in the circumventricular organs, the cns organs involved in the sodium reception. however, molecular and cellular mechanisms underlying the sodium reception of the glial cells has not been elucidated. to address this issue, we developed a functional expression system of the channel protein in cultured glial cells, and found that na x enhances glucose uptake and lactate release in an extracellular sodium-dependent manner. these results suggest that na x alters the state of energy metabolism of the glial cells by sensing a physiological increase of the sodium level. the state of inexcitable glial cells thus play a key role for the control of excitable neural cells in the circumventricular organs. we have isolated spinesin/tmprss from human and mouse cns. in mouse cns, four isoforms (types - ) were expressed. subcellular localization analysis revealed that type (full length) spinesin was predominantly localized to the er, golgi apparatus and plasma membrane, whereas type variant was localized to the cytoplasm. furthermore, we performed expression analysis of m-spinesin in some cell lines. nsc and nb a derived from neuronal cell express only type , whereas os and kt- derived from astrocyte express both type and type . interestingly, it was observed that the level of spinesin mrna was increased by a dibutyryl cyclic amp treatment only in os and kt- . we analyzed promoter region of m-spinesin gene, and identified that -flanking region from − to − bp was essential for m-spinesin gene expression. however, this region did not involve camp-dependent regulation of m-spinesin expression. these results indicate that cell-specific expression and regulation of spinesin gene may play multifunctional roles in cns. it has recently been elucidated that l-serine (l-ser) is one of the glia-derived neurotrophic factors in the brain and its biosynthetic enzyme -phosphoglycerate dehydrogenase (phgdh), which is the first committed enzyme of l-ser biosynthesis in the phosphorylation pathway, is selectively expressed in glial cells, but not in neurons. since l-ser seems to be important in retinal functions as well, we investigated in the present study the cellular distribution of phgdh in the mouse retina. phgdh immunoreactivity was detected in müller cell soma in internal nuclear layer, being close to internal plexiform layer. immunopositive profiles were cellular processes surrounding rod spherules and retinal neurons in internal nuclear layer through nerve fiber layer. it was suggested that müller cells contribute in l-ser synthesis and its transportation to neurons in the retina. astrocytes frequently show spontaneous intracellular ca + signals, such as intra-and intercellular ca + waves; however, their physiological roles remain elusive. the overexpression of an ip -hydrolyzing enzyme, ip -phosphatase, suppressed the spontaneous ca + signals in rat hippocampal astrocytes in culture without noticeable effects on their viability. hippocampal neurons were cultured on a monolayer of astrocytes, and their neurite outgrowth was analyzed. the total neurite length and the number of proximal dendrites and branches decreased significantly when neurons were cultured on the monolayer of ca + -signal-deficient astrocytes. moreover, time-lapse imaging revealed that the extension speed of growing neurites was markedly reduced on ca + -signal-deficient astrocytes. these results indicate that spontaneous ca + signals in astrocytes are essential for glial cells to promote neurite outgrowth. katsuyasu sakurai , noriko osumi , tohoku univ. sch. med., japan; crest, jst, japan astrocytes are the most numerous cells in mammalian brain tissues, although factors regulating their structure and function are still poorly understood. we have previously reported that pax transcription factor is expressed in gfap positive cells in the rat hippocampus. in the present study, we first investigated the expression patterns of pax in postnatal mouse brain and found that pax was expressed in almost all astrocytes in the cerebral cortex. to address the role of pax in the astrocytes, we examined the morphology of the astrocytes in the wild type (wt) and pax heterozygote mutant (sey/+) mice at weeks. confocal imaging revealed that arborization and extension of the astrocytes were poor in sey/+ mice as compared with the wt. in primary culture, the astrocytes isolated from sey/sey cortex showed no morphological difference. however, and h after dibutyryl-camp treatment, the majority of the wt astrocytes had undergone the conversion from a polygonal to stellate shape, while sey/sey astrocytes rarely showed this response. these results suggest that pax regulates the morphology of astrocytes, thereby being involved in astroglial functions. we raised mouse monoclonal antibody (mab) dim to study its distribution and function in cell membrane and found not only its preferential reaction with ptdglc on tlc, but also its labeling in rodent cns (yamazaki et al., ) . we previously reported a unique expression of dim ag in developing mouse brain, especially in cell membranes of embryonic radial glia (kinoshita et al., ) . we show here that mab dim also recognizes adult neural stem cells and glial cells at postnatal period. dim , brdu and gfap co-expressed in cells of mouse neurogenic subventricular zone. we discuss a possibility that the dim ag may be expressed in the radial glia/astrocyte lineage cells. the bone morphogenetic protein (bmp) receptors are thought to have a role in neural patterning of early neuronal development. the bmp receptor is widely expressed throughout the central nervous system (cns) including cerebellum. however, the physiological roles of bmp signaling in mature brain remains obscure. to understand bmp function in cns, we generated a transgenic mouse line that conditionally overexpresses bmp signaling through the type i receptor alk (alternatively known as avcri) in a purkinje cell-specific manner using a cre-loxp system. we bred this mouse line with the cre transgenic mouse line of which expression was driven by l promoter. tissue specificity of cre recombination was monitored by a bicistronically expressed egfp following a constitutively active alk cdna. increased bmp signaling was confirmed by ectopic phosphorylation of smad / / (p-smads) in purkinje cells. we will discuss functional changes of the purkinje cells which receive excess amount of bmp signaling through alk . lipopolysaccharide component of the cell wall of certain bacteria is pyrogenic whose administration to spinal cord injured animals was found to inhibit glial scar formation. glial scar being considered as an impediment for axonal growth, it had been proposed in s and s that sub-febrile doses of pyrogen could be considered for spinal cord injury repair research. we tested this ignored hypothesis in paraplegic bonnet monkeys and found that such sub-febrile doses of bacterial pyrogen derived from salmonella typhi was indeed effective in preventing the glial scar formation in short-term and at least prolong the formation of such scar in long term. therefore, pyrogen therapy may be considered as an adjunct to other strategies such as transplantation approaches to treat spinal cord injury. kavita seth, r.k. chaturvedi, s. shukla, a.k. agrawal dev. tox. div., industrial toxicology reserch center, lucknow, india crosstalk between neurons and glial cells (astrocyte and microglia) in neurodegenerative conditions such as parkinson's disease has gained attention of more than supportive interaction. here contribution of glial cells in -ohda induced degeneration of dopaminergic neurons was investigated. glial cultures showed significant loss in cell viability after h ( and %) and h ( and %) exposure to − and − m -ohda respectively. it was accompanied by morphological changes and induction of gfap, s- and ox . -ohda ( − m, h) was found to cause a significant impairment in h glutamic acid uptake ( %) and gsh levels ( %). further neurons (in coculture with -ohda pre exposed glial cells) on exposure to -ohda ( − m), showed loss of th expression and significant neuronal cell death ( %). the results of the present study suggest that -ohda may impair glial cell functioning, which eventually affect neuronal fate making them more vulnerable toward toxic insults. nestin is an embryonic intermediate filament component, which is transiently expressed by the immediate precursors to neurons and glia during brain development. we studied nestin distribution in the olfactory system after injection of diethyldithiocarbamate in adult rats to cause reversible lesion of the olfactory epithelium (oe). the oe presented a near-complete destruction at day after injection, then started to repair at days and returned to the normal levels at weeks. nestin was expressed in olfactory ensheathing cells (oecs) of the olfactory mucosa at ∼ days, but not in those of the olfactory bulb (ob). simultaneously strong expression of nestin was detected in certain population of astrocytes in glomeruli. the reversion of astrocytes in glomeruli to immature phenotype may reflect their involvement in reinnervation of glomeruli. (ng ) is currently considered a marker of multipotent progenitor cells in the brain. in the present study, most iba + cells accumulated in stab wounds and ischemic lesions were found to express ng , of which molecular weight of its core protein was higher by kda than that of ng expressed in contralateral brain region. this was due to the lack of shedding of ng in the brain lesions. we found that iba + cells accumulated in stab wounds and ischemic core lesion, most of which were ng +/pdgfra+. furthermore, some of these cells expressed gfap, nestin, cd and von willebrand factor. ng + mg isolated from stab wounds often formed cell aggregates bearing alkaline phosphatase activity turned into cells with neuroectodermal phenotypes in serumfree culture medium. these variety of antigens expressed by ng + mg in brain lesions may be related to their multipotentiality to regenerate damaged brain tissue. saroj sharma, l.k. singh, b. ray, t.s. roy all india institute of medical sciences, india oculomotor nerve (on) supplies most of the extra-and intraocular muscles. it shows changes with normal ageing, metabolic and degenerative diseases. though there are various studies on the on, no definitive data regarding the morphometry and the fine structure is available. so, in the present study, neural and the connective tissue organization of the extradural part of the on from cadavers were studied. light microscopy revealed multi-fascicular nerve with myelinated fibers of various calibers. small sized myelinated fibers were noted at the junction of the central and the paracentral zone of most of the nerves. using unbiased stereology techniques the size of myelinated fiber axonal areas showed a multi-modal distribution and presented range from < to m . most of the fibers were myelinated and counts produced a mean of , ( , - , ). ultrastructurally, difference in the compactness of arrangement of connective tissue was observed with advancing age. the cell junctions of the perineurial cells and the endoneurial capillaries were observed. myelin thickness ranged from . to . m (from fetal age to years age). during the development of the drosophila visual system, retinal axons project to the optic lobe through the optic stalk. the optic stalk is composed of glial cells and adopts tube-like structure. fak is a non-receptor protein tyrosine kinase involved in many aspects of cell behavior including cell migration through the regulation of actin or microtubule dynamics. in drosophila fak (dfak) mutant animals, the optic stalk was abnormally broadened and retinal axons were defasciculated. cdgapr encodes one of gaps that regulate rho-family gtpases. putative cdgapr mutants showed dfak-like phenotype. since dfak and cdgapr interacted genetically, they are likely to act in the same signaling pathway to regulate cytoskeletal rearrangement via rho-gtpases. tissue specific rescue experiment showed that dfak autonomously acts in the glial cells. our results suggest that dfak and cdgapr regulate glial cell rearrangement to establish precise tubelike structure of the optic stalk and organized retinal axon projection. astrocytes are thought to be active participants in synaptic plasticity in the developing nervous system. spontaneous gabaergic postsynaptic activity is reported to be decreased in small neurons of the caudal nts at the end of the first postnatal week. to investigate whether astrocytes might be involved in this phenomenon, we examined developmental expression of gfap, an astrocytic marker. gfap began to be immunohistochemically expressed in the caudal nts at p - . costaining with calbindin, a marker for a certain type of small neurons, showed that gfap positive processes were thereafter closely apposed to soma of small calbindin neurons. electron microscopy showed that some astrocytic processes were interposed between orphan gabaergic varicosities and soma of small neurons at the specific developmental stages. these findings indicate that astrocytes may participate actively in regulating the postnatal differentiation of local neural network of the caudal nts. hitoshi ozawa, naoyuki yamamoto, nobuhiko sawai, hao-gang xue department of anatomy and neurobiology, nippon medical school, tokyo, japan it is well known that the hypothalamo-pituitary-adrenal (hpa) axis is an important system for responding and mediating the stress. in addition, hippocampus is also an important area for the stress response. in the hippocampus, the expression of glucocorticoid receptor (gr) has been reported in the ca , ca pyramidal neurons and the dentate gyrus neurons. on the other hand, while astroglia around the hippocampus also expresses gr, the morphological and functional changes under different corticosteroid condition have not been well elucidated. in the present study, we investigated morphological changes of astroglia around pyramidal neurons. under the lack of corticosteroids, astroglia showed well developed morphology with the spread fibrous processes, however the changes recovered to the control level with corticosterone replacement. these suggested that the astroglia were directly regulated by glucocorticoids as associated with the changes of hippocampal neurons. ps p-d impact of s b on hippocampal spontaneous activities in anesthetized and epileptic conditions seiichi sakatani , akiko seto-ohshima , shigeyoshi itohara , hajime hirase neuronal circuit mechanisms research group, japan; lab. for behavioral genetics, bsi, riken, wako, japan s b is a calcium binding protein mainly expressed in astrocytes and has a role in synaptic plasticity and learning. in order to assess the physiological roles of s b, we have recorded hippocampal spontaneous activities from urethane anesthetized s b ko and wt mice. typical eeg patterns including theta ( - hz) and sharp wave associated fast ripple ( - hz) oscillations were observed in both populations and these patterns were indistinguishable between the wt and ko. when epileptic activity was induced by kainic acid (i.p.), a difference appeared in ca radiatum, where ictal event was characterized by hyper-synchronous gamma band ( - hz) activity. while both populations developed ictal event within min, mean power during the development was significantly smaller in ko mice. our results suggest that deficiency of s b does not have a profound impact on neural activity in normal conditions. however, when neural activity was raised, activation of s b related pathways could potentially be activated. yoshiko takagishi , erina okabe , xiaoyang sun , sen-ichi oda , yoshiharu murata riem, nagoya univ, nagoya, japan; grad sch bio-agricult sci, nagoya univ, nagoya, japan shambling (shm) is a spontaneous mouse mutation that causes neurological and motor deficits, characterized by ataxia and the hind limb paralysis. we have recently identified the shm gene that encodes caspr, which constitutes paranodal junction of myelinated nerves. to determine whether the mutation alters the node of ranvier, we performed morphological analysis of myelinated nerves in shambling mice. by electron microscopy, we found that paranodal loops were disorganized and septate-like transverse bands were absent in mutant mice. immunohistochemistry revealed that caspr was diffusely located at the paranodal region, though the staining was extremely weak in mutant sciatic nerves. contactin, a component of the paranodal junction, was distributed similar pattern to that of capsr. further, k + channels were mislocalized to the paranode, while na + channels were normally restricted to the node. these findings suggest that the mutation disrupts the paranodal structure and may disturb salutatory conduction of myelinated nerves in shambling mice. ps p-d regulation of hippocampal neurocircuit activity by glutamate transporter glt- noriko koganezawa, shinsuke muraoka, ken-ichiro tsutsui, toshio iijima div. systems neurosci. tohoku univ. grad. school of life science, japan glial cells are now recognized as an essential functional element in synapses. in order to investigate their function, we focused on the activity of glt- , the glutamate transporter which is expressed in the astrocytes of hippocampus, in the rat brain slice preparations. response to an electrical stimulation of the schaffer collaterals was recorded using the optical imaging technique. by combining the application of the glutamate receptor blockers (nbqx, ap ) and the glt- blocker (dhk) with the signal subtraction, we could visualize the activity of glt- as a slow, tonic rise of the optic signal following electrical stimulation. then we evaluated the function of glt- by applying its blocker dhk. an obvious reduction of neural activity was observed in the hippocampal neurocircuit after application of dhk. furthermore, the blocking of glt- function in the ca region was elicited by much lower concentration of dhk than that in the ca region. ps p-d monoclonal antibody rip specifically recognizes , -cyclic nucleotide -phosphodiesterase in oligodendrocytes the antigen recognized with monoclonal antibody (mab)-rip has been used as marker for oligodendrocytes and myelin sheaths. however, the rip-antigen has been unknown yet. to identify the rip-antigen, we performed immunopurification with mab-rip using the differentiated cg- cells lysate. maldi-qit/tof ms n analyses revealed that one of molecules was , -cyclic nucleotide phosphodiesterase (cnp). immunocytochemical and immunohistochemical studies showed that rip-antigen colocalized with cnp in rat cerebellum, cultured rat oligodendrocytes and cg- cells. moreover, the same localization was also observed in rat cnp transfected hek t cells. overall we first demonstrated that the antigen labeled with mab-rip is cnp in oligodendrocytes. the expression of bdnf gene is regulated by four promoters (pi-piv), and is under activity-dependent control. until now, it has been established that bdnf pi is activated by ca + signal via cre. on the other hand, neuron-restrictive silencer cis-element (nrse), located in bdnf pii, represses bdnf gene expression through binding nrsf and recruiting hdac in non-neural cells. here, we found that nrse repressed the activity of bdnf pi in neuron. using rt-pcr and chip assay, the bdnf exon i expression and the histone acetylation of bdnf pi were increased by the administration of ca + signals or hdac inhibitor. in addition, nrsf bound to bdnf pii in neurons but was detached by ca + signals. these results suggest that bdnf pi activity is regulated by creb and nrsf through an alteration of chromatin structure. since creb and nrsf are playing an important role in neuronal differentiation, it is considered that the bdnf pi is deeply involved in the regulation of neurogenesis. singo suzuki , , hisatsugu koshimizu , megumi kashihara , tomoko hara , masami kojima , research institute for cell engineering; sorst, jst, japan brain-derived neurotrophic factor (bdnf) plays a crucial role in synapse development, especially, in the central nervous system (cns) . although this concept is now accepted extensively, the underlying molecular mechanisms are poorly understood. here we show that -day treatment with bdnf leads to a significant increase in cholesterol content in primary neuron. this change was in its dose-dependent manner and blocked by co-application of a cholesterol synthesis inhibitors. to understand the molecular relationship between cholesterol content and synapse development, we estimated the amount of cholesterol and sv proteins in lipid raft fractions prepared from cultured cortical neurons. the results indicated that bdnf treatment increased the amount of cholesterol and sv proteins in lipid rafts, but not in non-rafts fraction. these data suggested a possibility that bdnf regulated synapse development by increasing the amount of cholesterol and sv proteins in synaptic rafts. (p ) is known to be expressed in the cells of the central nervous system, and supposed to be involved in the control of cell proliferation, differentiation and survival. in this study, we found that p expressed in the neural progenitor cells at embryonic days (e ) mice brain. to ascertain the function of p on these cells, we treated the cultured e cells with the selective chemical inhibitor for p (sb ) for days, and determined the number of neural progenitor cells. the inhibitor specifically enhanced the number of neural progenitors compared to the control cells. this result suggests the involvement of p in the proliferation and/or survival of neural progenitor cells in developing mouse brain. hemragul sabit , takashi yamazaki , , takeshi oya , yoko ishii , masakiyo sasahara pathology ii, university of toyama, toyama, japan; oral and maxillofacial surgery, university of toyama, toyama, japan purpose: we had reported the increase of pdgf-b and active src in rat peripheral nerve regeneration. here examined activation of pdgf receptors (pdgfrs) and signals in the peripheral nerve regeneration. method and result: crushed sciatic nerve was removed on to days after injury, and activation of pdgfrs, mapks, akt and p were examined by phosphoprotein purification kit (qiagen) and western blot. expression of pdgfrs increased from to days after injury. p-tyr was highly detected from to days after injury, and activation of pdgfrs also increased during this period. activation of erk and jnk increased up to days after injury and then gradually decreased. activation of akt and p continuously increased from to day after injury. conclusion: pdgfrs and their signals were activated in rat peripheral nerve regeneration. autocrine signal of pdgf may contribute to the regenerative processes, such as proliferation and differentiation of schwann cells and axonal extension. cbln is a cerebellum-specific protein structurally related to the c q and tumor necrosis factor families. recently, we have shown that cbln is secreted from cerebellar granule cells (gc) and controls synaptic structure and plasticity of gc-purkinje cell (pc) synapses. however, because cbln was previously shown to serve as a precursor of a pc-specific peptide cerebellin, it remains unclear whether cbln needs to be processed before it trans-synaptically activates signaling pathways in pc. here, we show that purified recombinant cbln proteins, which formed a hexamer, preferentially bound to spines on pc dendrites. furthermore, cbln mutants that did not form a hexamer lost the binding affinity to pc spines. although cerebellin peptide may also contribute to different aspects of signaling, these results indicate that cbln released from gc directly bind to postsynaptic pc as a hexamer and activates signaling pathways in pc. activity-dependent gene expression in neurons contributes to expressing a variety of neuronal functions including a long-lasting neuronal plasticity. recently, we found that specific kinds of mrna can be stabilized in an activity-dependent manner. to elucidate the mechanisms for activity-dependent mrna stabilization, we have focused on bdnf, which is a member of neurotrophin family and plays an important role in exerting neuronal functions. we constructed firefly luciferase gene fused to -untranslated region (utr) of bdnf mrna to investigate the effect of the utr on the calcium signal-mediated mrna stabilization. in cultured neurons, we found that the degradation of firefly luciferase-bdnf utr mrna induced by the treatment with actinomycin d was prevented by calcium signals evoked via l-type voltage-dependent calcium channels (l-vdccs) and nmda receptors. we are now investigating to identify the cis-regulatory elements involved in the calcium signal-mediated stabilization of bdnf mrna using a series of mutant bdnf utr. recently, it has been established that bdnf and pacap regulate the expression of a group of genes which encode proteins involved in expressing neuronal functions. in this study, we found that the treatment of cultured rat cortical neurons with bdnf or pacap acutely induced the mrna expression of the activityregulated cytoskeleton-associated protein (arc) and homer a, whose products are necessary for the synaptic plasticity. bdnf induced arc mrna expression through the activation of trkb-erk/mapk pathway, whereas pacap induced it partly through the activation of nmda-receptors. using affymetrix genechips, we are now investigating a comprehensive profile of gene expression controlled by bdnf or pacap in cultured rat cortical neurons. ps p-e bmp expression in the adult rat brain bone morphogenetic protein- (bmp ) is a member of the transforming growth factor ␤ (tgf-␤) superfamily and plays important roles in multiple biological event. although bmp expression has been well described in the early development of central nervous system (cns), little information is available for its expression in the adult cns. we, thus, investigated bmp expression in the adult rat cns using immunohistochemistry. bmp is intensely expressed in most neurons and their dendrites. in addition, intense bmp expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. furthermore, we found that astrocytes also express bmp protein. these data indicate that bmp is more widely expressed throughout the adult cns than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that bmp plays pivotal roles also in the adult brain. ps p-e hgf as a target-derived trophic factor for rat nigro-striatal dopaminergic (da) system during post natal development wakana ooya, hiroshi funakoshi, toshikazu nakamura div. molecular regenerative medicine, osaka univ. grad. sch. med., osaka, japan hgf is a novel neurotrophic factor in vitro on da neurons. however, little is known about expression and biological activities of hgf in nigral-striatal system in vivo. here we show that hgf is a targetderived trophic factor for rat da system. real-time rt-pcr revealed that c-met mrna was expressed in substantia nigra (sn) and striatum (str), while hgf mrna was expressed in str but not in sn in programmed cell death period. hgf, c-met, phospho-c-met, th, da transporter immunostaining revealed the presence of concentration gradient of hgf from sn to str and c-met was phosphorylated in da nerve end during early postnatal development. phospho-c-metpositive da neurons decreased at later developmental stage, while it became prominent in oligodendrocytic leanage. hgf application into str increased da neuronal number and neurites and modified oligodendrocyte maturation, while opposite effects were observed by the application of blocking antibody for hgf. therefore, hgf may be a critical trophic factor for nigro-striatal da system development. the neuroprotective effects of g-csf were reported in neurological disease models. in the present study, we examined whether g-csf can protect dopaminergic neurons from mptp-induced cell death in a pd. the mice were intraperitoneal injected with mptp for five consecutive days, g-csf is intraperitoneal administered two days and one day before first mptp injection, and min before each mptp injection. in our results, g-csf significantly prevented mptpinduced loss of th-positive neurons, and increased bcl- protein, decreased bax protein expression. these findings suggest that g-csf has therapeutic potentiality to protect mptp-induced cell death through increasing the level of bcl- expression, decreasing the level of bax expression in c bl/ mice. kazue takahata has been shown to increase the expression of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, and the activity of superoxide dismutase . here, we evaluated the effects of (−)-bpap on the phosphorylation of mitogen-activated protein kinase (mapk) and akt in slice cultures as well as in an in vivo model. (−)-bpap significantly increased phosphorylation levels of mapk, but not those of akt. (−)-bpap attenuated the decrease in nigrostriatal tyrosine hydroxylase immunoreactivity of -methyl- -phenyl- , , , tetrahydropyridine-treated mice. (−)-bpap may exert antiparkinsonian activity through neuroprotective effects on dopaminergic cells in addition to catecholaminergic enhancement in parkinsonian substantia nigra. shyuichi maeda , yoko tohyama , shinichi kohsaka , tadashi kurihara , kazuyuki nakajima , soka university, hachioji, tokyo, japan; dept. of neurochem., national institute of neuroscience, kodaira, tokyo, japan astrocytes (ast) are a cell type that supports cns not only nutritionally but also neurotrophically, by supplying neurotrophic factors (ntf) required in the neuronal survival, maturation and protection. however, the ability of ast to produce/secrete ntf has not been accurately known. thus, in the present study, we investigate the capacity of ast to produce/secrete various ntf in vitro. ast were prepared from the mother culture of neonatal rat brain. the ntf in the conditioned medium (cm) were detected by immunoblotting. the analysis of neurotrophins in the cm revealed that ast produce/secrete ngf, bdnf and nt- , and promote the production of them by stimulation with lipopolysaccharide (lps). furthermore, tgf␤ among tgf␤ family was detected in the cm of ast, and the production was enhanced by stimulation with lps. these profiles of ast were different from those of microglia, suggesting the differential regulation of ntf by glial cells. ritsuko katoh-semba , chiaki nakagawa , masako tsuzuki , motoko matsuda , satoshi ichisaka , yoshio hata inst. dev. res., aichi human ser. ctr., kasugai, japan; div. neurobiol., tottori univ., sch. med., yonago, japan; div. integrative biosci., tottori univ. grad. sch. med., yonago, japan the sleep-awake rhythm is formed during the early period after birth. the rhythm is very important for the functional development of brain. when the rhythm formation is disturbed, autism-like behaviors are often observed. brain-derived neurotrophic factor (bdnf) is known to be one of the factors forming circadian rhythms. we have found increases in levels of bdnf in the entorhinal cortex as well as the visual cortex from adult male rats h after beginning an -h phase advance of the light-dark cycle. here we planned to reveal the effects of the phase advance on neurotrophins in juvenile rats. we first examined circadian changes in the concentrations of bdnf and neurotrophin- (nt- ) in selected brain regions from -day-old male rats and compared to those from adults. the changes in levels of bdnf and nt- were observed in the neocortex and hippocampus. objective: this study was aimed to investigate the possible beneficial effects of granulocyte colony stimulating factor (g-csf) compared to methylprednisolone (mp) in experimental spinal cord injury (sci). materials and methods: (in vivo) adult female sprague-dawley rats had moderate sci ( kdyne, ih injury device) at t / and were assigned to three groups; a (placebo), b (mp treated; mg/kg i.v. immediately after injury), c (g-csf treated; g/kg i.v. for days after injury). animals were assessed with the bbb locomotor rating scale for w post injury and then killed for assessment of tissue sparing around the lesion. result: the behavioural recovery rates of the group c was as good as group b and significantly better than that of group a. morphological assessment showed better tissue sparing in group b and c compared to group a. these results suggest that g-csf is a possible neuroprotective agent in sci. research funds: kakenhi ( ) ps p-e glutamate signals enhance the expression of rnase-l in primary cultured cortical neurons of mice chie sugiyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan mitochondrial dysfunction results from a decline in the mitochondrial rna (mtrna) transcripts and mitochondrial enzyme activity, as well as from mitochondrial dna (mtdna) damage. to evaluate involvement of mtdna expression in glutamate-induced neuronal death, in this study, we examined the effects of glutamate exposure on mtrna level in cultured cortical neurons of mice. cultured neurons ( div) exposed to glutamate for min at m. glutamate exposure led to a decrease in mrna of nd and nd , which are subunits of nadhubiquinone oxidoreductase, before cell death. since mtrnas level is regulated at least in part by rna degradation mediated by rnase-l, we next examined the effect of glutamate on expression of rnase-l. rt-pcr analysis revealed that glutamate was effective in increasing the level of rnase-l mrna at least - h after treatment. the increase in the expression of rnase-l was abolished by the nmda receptor antagonist mk- . these results suggest that the activation of nmda receptor by glutamate reduces mtrna level probably through enhanced expression of rnase-l in cultured cortical neurons. yuka gotoh, kiyokazu ogita dept. pharmacol, setsunan univ, osaka, japan expression of dj- is enhanced by oxidative stresses. although exact functional significance of dj- has still unknown, it is thus proposed that dj- is protective against neural damage under oxidative stresses. in this study, we tested expression of dj- in the hippocampus damaged by trimethyltin (tmt) treatment in mice. tmt was systemically injected into mice to cause neural damage in the dentate gyrus selectively. immunohistochemical analysis indicated that dj- was markedly increased in the molecular layer of the dentate gyrus on days and post tmt injection. on day post tmt injection, enhanced expression of dj- was observed in the stratum lucidum of the ca . in glutathione-depleted mice, tmt was more effective in enhancing expression of dj- , compared with that in untreated mice. furthermore, double staining of dj- and gfap demonstrated that most of cells highly immunoreactive to dj- were co-localized with gfap in the dentate gyrus of tmt-treated animals, but not of untreated animals. these results suggest that dj- is enhanced in the dentate astrocytes activated by tmt treatment. kei higuchi, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan the systemic administration of trimethyltin (tmt) is known to induce granule cell death in the dentate gyrus of mice. we have previously shown that an injection of tmt ( . mg/kg, i.p.) led to significantly reduction of granule cells in the dentate gyrus days later, with visually apparent recovery of the granule cell layer days afterward. in this study, we examined the effects of glucocorticoids on tmt-induced damage in the dentate granule neurons of mice. tmtinduced neuronal cell damage was assessed by the immunohistochemical analysis using an antibody against single-stranded dna. the systemic injection of dexamethasone ( . - mg/kg) led to a significant reduction in neuronal damage induced by tmt in the dentate gyrus. the neuronal damage induced by tmt at the dose of . mg/kg was enhanced by adrenalectomy. dexamethasone was effective in completely preventing this neuronal damage in adrenalectomized animals. taken together, these results suggest that glucocorticoid released from adrenal cortex may be capable of protection against tmt-induced dentate granule cell death in mice. masami ishido national institute for environmental studies, tsukuba, japan melatonin, a secretory product of the pineal gland, has antitumor activities and is involved in the regulation of circadian, seasonal rhythms and in inducing osteoblast differentiation. furthermore, melatonin is reported to be a scavenger of a number of reactive oxygen and reactive nitrogen species both in vitro and in vivo. in this chapter, antioxidant nature of melatonin was demonstrated to prevent the cultured neural cells from apoptosis induced by endocrine disrupting chemicals, maneb. neurotoxicity of maneb ( g/ml) on the pc cells was elicited through apoptotic cell death. activation of caspase- / was associated with this process. a fluorescence rationing technique using mitochondrial dye revealed that maneb altered mitochondrial membrane potential of the neural cells. however, melatonin ( nm) could largely prevent the neural cells from the neural toxicant by inhibition of both caspase- / activation and disruption of the mitochondrial transmembrane potential. thus, melatonin could be a powerful free radical scavenger against manebcaused mitochondrial dysfunction in pc cells. ps p-e kinesin superfamily protein (kif ) regulates activity-dependent neuronal survival by suppressing parp- enzymatic activity ryosuke midorikawa, yosuke takei, nobutaka hirokawa department of cell biology and anatomy, university of tokyo, tokyo, japan in brain development, apoptosis is a physiological process that controls the final numbers of neurons. here we report that the activitydependent prevention of apoptosis in juvenile neurons is regulated by kinesin superfamily protein (kif ), a microtubule-based molecular motor. the c-terminal domain of kif is a module that suppresses the activity of poly (adp-ribose) polymerase- (parp- ), a nuclear enzyme known to maintain cell homeostasis by repairing dna and serving as a transcriptional regulator. when neurons are stimulated by membrane depolarization, calcium signaling mediated by camkii induces dissociation of kif from parp- , resulting in upregulation of parp- activity, which supports neuron survival. after dissociation from parp- , kif enters into the cytoplasm from the nucleus, and moves to the distal part of neurites in a microtubule-dependent manner. we suggested that kif controls the activity-dependent survival of postmitotic neurons by regulating parp- activity in brain development. research funds: ps p-e expression of hsp , apg- , and apg- in the hippocampal neural cells by trimethyltin masanari orita, kiyokazu ogita dept. pharmacol, setsunan univ, osaka., japan we tested changes in expression of high-molecular-weight heat shock proteins (hsps) in the hippocampal dentate gyrus in vivo and in the cultured cortical neurons in vitro after trimethyltin (tmt) treatment, which caused neuronal damage in the dentate gyrus and cultured hippocampal neurons. tmt ( . mg/kg) was systemically injected into mice, and then an immunohistchemical analysis was performed to identify cells immunoreactive to antibodies against hsps, neun, and gfap in coronal sections of hippocampus. tmt was effective in enhancing the expression of hsp , apg- , and apg- in the granule cell layer of the dentate gyrus, but not in ca -ca pyramidal cell layer, h to days later. double staining of neun and these hsps revealed that these hsps expressed by tmt almost co-localized with neun in granule cells of the dentate gyrus. whereas hsp highly expressed in survival neurons in the culture, apg- and apg- highly expressed in damaged neurons with nuclear condensation. taken together, the high-molecular-weight hsps may be involved in neuronal survival and damage caused by tmt. brain irradiation is often performed in patients with brain tumors. however, little has been known about radiosensitivity of neurons, especially in the developmental stages. in this study, we investigated the effect of irradiation on immature neurons with that on mature neurons. primary neuronal cultures were prepared from fetal rat hippocampi at embryonic day . thirty gray of x-irradiations were performed on the cultured cells at or days in vitro (div). then the cells were fixed at h after the irradiation with dapi. at -div, irradiation significantly increased the number of nuclear pyknosis of neurons. in contrast, radiation did not induce any nuclear pyknosis of neurons at -div. this indicates that the radiosensitivity of -div immature neurons is higher than that of -div mature neurons. glutamate receptors are believed to be involved in various neurological disorders via its excitotoxicity. ataxic mice lurcher (lc) are caused by a mutation in the ␦ glutamate receptor (glur␦ ), which shows constitutive channel activities in purkinje cells and leads to the cell death. thus, lc is the first example of neurodegeneration caused by chronic excitotoxicty. interestingly, glur␦ is also suggested to regulate autophagy via its association with beclin. however, it is unclear how excitation caused by constitutive channel activities is related to the autophagic pathway and cell death. here, using heterologous cells in vitro, we show that continuous influx of na + , but not ca + , was necessary and sufficient to induce autophagic cell death. in addition, we found that intracellular atp levels and subsequent activation of map kinase are involved in this process. junko taniguchi a major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions (niis) of the disease proteins that are ubiquitinated and often associated with various transcription factors, chaperones and proteasome components. but, how the expanded polyglutamine proteins or their aggregates elicit a complex pathogenic responses in the neuronal cells are not fully understood. here, we demonstrate that the expression of expanded polyglutamine proteins down-regulates the nf-kb-dependent transcriptional activity. expression of expanded polyglutamine proteins increases the stability and the levels of ikb-a and its phosphorylated form. we have also found that various nf-kb subunits and ikb-a aberrantly interacts with the expanded polyglutamine proteins and associates with their aggregates. finally, we have shown several nf-kb-dependent genes are down-regulated in the expanded polyglutamine protein expressing cells. molecular mechanisms for selective neuronal death in polyglutamine diseases remain to be clarified. by microarray analysis, we compared gene expression profiles in cerebellar granular cells under expression of normal and mutant ataxin- and found a novel gene down-regulated in response to mutant ataxin- in cerebellar granular neurons. we named the novel gene maxcell (mutant ataxin-affected gene in the cerebellum). nothern blot shows that maxcell mrna in human brain is expressed in cerebellum and cerebral cortex. immunohistochemistry with anti-maxcell antibody shows cytoplasmic stains of neurons but not glial cells in mouse brain. confocal microscopy shows that maxell-egfp is colocalized with ribosomal protein s as a ribosomal marker. we are analyzing the function of maxcell protein that might relate to sca molecular pathology. ps p-f permeability transition in mitochondria isolated from cold perfused brain and spinal cord-a detailed comparison of calcium sensitivity the purpose of this study was to compare the sensitivity of isolated brain and spinal cord mitochondria to ca + -induced permeability transition (mpt). the spinal cord is more traumatized than the brain during the extraction because it takes more time. in order to minimize confounding factors, we induced severe hypothermia in animals prior to removal of tissue and isolation of mitochondria. sensitivity to ca + -induced mpt was evaluated in brain and spinal mitochondria in energized and de-energized model of swelling with or without mpt inhibitor, cyclosporin a (csa). the present findings imply that the general features of mpt are similar in brain and spinal cord mitochondria and that mpt may be an important pharmacological target in disorders affecting the spinal cord. the role of cyclophosphamide monohydrate (cp), which is known as an immunosuppression drug, in the central nervous system (cns) has not been elucidated. in the present study, we found that treatment with cp prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, cp exposure induced the activation of both the map kinase (mapk) and pi kinase (pi k) pathways. interestingly the up-regulation of bcl- , a survival promoting molecule was observed after cp treatment. these observations suggest that cp protects cns neurons from neuronal damage through intracellular signaling pathways. the research of cell death mechanisms has rapidly progressed. however, cell death is an inherently difficult process to measure. to investigate the roles of cell death in vivo, we introduced scat probe. scat is an indicator protein for caspase- activation that uses fret between two types of fluorescent protein, ecfp and venus, linked by a peptide containing the caspase- cleavage sequence. using this probe, we could monitor the activation of caspase- at the singleneuron level in culture. we will discuss about neural cell death through the detection of caspase activity. keiichi seko, koichi kawada, chie sugiyama, masanori yoneyama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka, japan trimethyltin chloride (tmt) is a kind of organotin derivates that are known to induce neuronal damage in human and rodent. in this study, we examined tmt-induced neuronal death in mouse primary cultured cortical neurons in vitro and the frontal cortex in vivo of mice. in vivo analysis using mice revealed that injection of tmt ( . mg/kg, i.p.) led to an increase in single-stranded dna-positive cells, as well as in dnase ii-positive cells, in the frontal cortex days later. in cortical neurons, tmt exposure for h led to a marked decrease in the cell viability, as well as to an increase in nuclear condensation and ldh released. tmt exposure was effective in activating dnase ii in the nucleus. in addition, caspases and , but not caspase , were significantly activated by tmt treatment. cytochrome c release was not affected by tmt treatment. the caspase inhibitor zvad-fmk completely prevented tmt-induced neuronal death. these results suggest that tmt-induced neuronal death is involved in caspases and dnase ii activated by mitochondria-independent pathway in cortical neurons. we investigated the pattern of hippocampal damage and the levels of brain polyamines after systemic injections of trimethyltin (tmt) chloride ( . mg/kg, i.p.) in -( w) and -week-old ( w) icr mice. in addition, we measured the brain tin level following tmt injection. tmt induced marked, localized cell death in granule neurons of the dentate gyrus in w mice. by contrast, slight, diffuse neuronal damage was found in the ca and ca subfields and dentate gyrus of w mice. the hippocampal putrescine level was elevated markedly in w mice on tmt administration, whereas a minor putrescine increase was detected in w mice. there was no difference in the brain tin level between these two age groups. these results revealed the age-dependent vulnerability of mice hippocampal neurons to tmt administration, and suggest that massive activation of polyamine metabolism is associated with tmt-induced neurodegeneration. withdrawn ps p-f establishment of memory guided actions of taking food with tweezers in monkeys naoki hirai , toshinori hongo , kimisato naito , shigeto sasaki department of physiology, kyorin university, school of medicine, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo, japan monkeys learned a task of taking food with tweezers (twz) under the visual guidance. the task consisted of sequential actions of looking at twz, grasping it by hand simultaneously shifting gaze to food, bringing twz to food, and picking it up with twz. brief interruption of vision for . - . s during any actions by a liquid crystal shutter disrupted the ongoing actions, indicating that each action needed visual information as guidance. this contrasted with the task of taking directly with hand, which was done without vision. with repeated practice, they developed a mode of using more memory and somatosensory cue as guidance. they directed their gaze to invisible food in advance, and when vision of . s became available, they grasped twz, brought the twz to memorized location of food and grasped the food without vision. these results show that they acquired food taking actions using twz based on memory and somatosensory cues, the latter allowing monkeys to use twz as an extension of the hand. masahiko nishimura, yoshihiko yoshii university of ryukyus, okinawa, japan we have experienced that the patients with arm impairments by brain disorder were difficult to manipulate the tools with the paralyzed arm, and healthy arm. lt. parietal lobe and ifg are commonly recognized tools-semantic neuro-system. however, nobody knows a neural network contributed to suitability for a purpose of toolsmanipulation. we examined an fmri to evaluate the brain activation of tools-manipulation in volunteers. experiment was performed by three tasks, control task is a forearm rotation, task is simulation of tools-manipulation, task is execution of tools-manipulation. we found different brain regions by this experiment. task to investigate the role of synchronous firing in the prefrontal cortex (pfc), we performed cross-corelational analysis of the pfc neurons, while monkeys performed a path-planning task, which required multiple steps of actions to reach goals. first, we analyzed synchrony among pfc neurons during the execution period in comparison with that during the preparatory period. we found that neuronal synchrony was enhanced transiently for each step of movement during the execution period. next, we examined relationship between neuronal synchrony and task-related activities. we found that the relationship between neuronal synchrony and response selectivity of pfc neurons was more distinct during the preparatory period than during the execution period. we would discuss dynamical roles in neuronal synchrony in planning multiple steps of actions. the functional significance of primate medial prefrontal cortex in the selection of action has been unclear. we studied neuronal activity in this region while monkeys were performing a variant of conflict solving task in which visual cues instructed them to push either the left or right. the location of the cue was either compatible (congruent) or incompatible (incongruent) with the target's location. we found a focus of reaching-related neurons in the medial prefrontal cortex rostral to the pre-sma. the activity of neurons in this newly identified area was dependent on conflict. intracortical microstimulation in this area did not evoke eye movements, distinguishing this area from the sef. we found that the local field potential in this area, but not in other areas, differed when congruent and incongruent trials were intermixed, and when only the congruent trials were presented repeatedly, suggesting the involvement of this area in the selection of actions is dependent on the task demand. masaki maruyama, peter fenwick, andreas a. ioannides laboratory for human brain dynamics, riken-brain science institute, saitama, japan we used infrared corneal reflection, sampling at khz, to record simultaneously and independently the • horizontal saccades of each eye for subjects. two paradigms were used, in go-only sessions saccade direction with the cue to move, and in go/no go sessions, saccade execution, direction and move. mutual information (mi) analysis showed the two eyes were most consistently yoked for position than for velocity, but both provided adequate signals. mi showed coupling between the start and end of saccades and the importance of velocity signals in their ballistic nature. surprisingly leftward movement latency was longer to peak-velocity and showed more complex mi interactions. comparing go-only to go/no go saccades, significant differences were longer onset latencies and a higher eye velocity before the end of saccades. a recent meg study using this protocol, found just before and during the saccade the additional go/no go difficulty led to more interaction between left and right brainstem and cerebellum. these could be related to eye velocity changes with the higher cognitive loading. wriggle mouse sagami (wms) has been presented as a mouse model for dystonia, as it is characterized by postural and motor impairments, such as sever tremor, sustained muscle contractions of the limbs, and wriggling of the neck and trunk without coordination. by extracellular unit recordings under awake conditions, we analyzed neuronal activity in the basal ganglia and the cerebellum of this mutant mouse. in the basal ganglia (globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), neither rates nor patterns of spike discharges were significantly different as compared to normal mice. on the other hand, the discharge rate of cerebellar purkinje cells in wms was markedly decreased. these results suggest that the decreased activity of purkinje cells may be responsible for movement disorders in wms. hiromi hirata division of biological science, nagoya university, nagoya, japan wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions at day, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the cns. recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. furthermore, immunostaining with glyr antibody failed to show clusters in beo neurons. these data suggest that clustering defect of glyrs at synapse causes the impairment of glycinergic transmission and abnormal behavior in beo. indeed, mutations in the glycine receptor beta subunit were identified in beo. this is the first direct demonstration that glyr␤ is essential for physiologically relevant clustering of glyrs in vivo. since glycine receptor mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, zebrafish bandoneon mutant should be a useful animal model for this condition. medium-sized spiny projection neurons in the striatum receive inputs from gabaergic and cholinergic interneurons as well as from extrinsic sources, including the cerebral cortex. in the present study, the effect of gabaergic modulation on striatal projection neuron activity was investigated by infusion of the gaba a receptor blocker gabazine in the vicinity of the recorded neurons in monkeys who performed a memory-guided reaching task. the gabazine infusion enhanced the activity of striatal projection neurons in response to both cortical stimulation and task events, while the neuronal activity specific to the task events was decreased. these results suggest that local gabaergic input may play an important role in fine tuning of striatal projection neuron activity. research funds: kakenhi ( ) ps p-f dependence of synchrony in the subthalamic network on temporal characteristics of afferent inputs katsunori kitano, fumito kosuga department of human and computer intelligence, ritsumeikan university, japan the subthalamic nucleus (stn) and the external segment of global pallidus (gpe) constitute the indirect pathway of the basal ganglia and highly modulate the basal ganglia functions. the evidence that the emergence of synchronized oscillatory activity in the network of the two nuclei is relevant to movement disorders such as parkinson's disease shows temporal structures of the neuronal firings play an important role for the functions. among possible underlying mechanisms for the abnormal activity, the characteristic membrane properties of stn neurons is likely to be one of the most crucial origins. to clarify the detailed mechanism, we focus on and investigate the dynamical properties of the neuron theoretically and numerically with the model neuron. we apply the phase reduction method to the dynamics of the neuron to analyze the stability of synchronous activity. in particular, how the stability depends on temporal characteristics of afferent inputs to the neurons as well as the intrinsic membrane properties are investigated. during phasic voluntary movement, electromagnetic oscillatory activities of ∼ hz around the central sulcus show decrement and increment (erd/ers, respectively), that are assumed to reflect the cortical activation and inhibitory/recovery process respectively. we investigated the correlation between personality and these oscillatory changes. from healthy subjects, high and low scorers (n = each) of novelty seeking dimension on the psychometry were selected. magnetic fields were recorded while they performed selfpaced movements of their right index fingers, and frequency analysis was carried through the beta band ( - hz). high ns group showed less amount of erd in the left hemisphere, smaller magnitude, larger latency of ers in the right hemisphere and smaller amount of baseline activity in both hemispheres than low ns group. it was suggested that individuals with high ns trait may have less inhibition after the movement and higher readiness during resting state. despite the emerging methodology of combined fmri and tms, the quantitative relationship between tms intensity and bold signals is poorly understood. eight healthy subjects were scanned on a -t scanner, with an mri-compatible figure-of-eight tms coil attached for eliciting right hand movement. bold measurement was performed with the stepping stone sequence (tr = . s) with online monitoring of meps. the intensity of tms pulses was varied from % to % at a % step (frequency at ∼ . hz). bold signal changes were assessed in the primary motor cortex. a sharp increase in bold signals was observed above % stimulation. bold signals were weakly but significantly correlated with tms intensity adjusted by the resting motor threshold (r = . , p = . ). this finding gives a theoretical background for the application of fmri with tms to cognitive brain regions. ps p-f shift of activation areas induced by hand movement during recovery from post-stroke hemiparesis: an nirs study kotaro takeda , , yukihiro gomi , itsuki imai , nobuaki shimoda , , hiroyuki kato , international university of health and welfare, ohtawara, japan; crest, jst, kawaguchi, japan; nasu neurosurgical center, nasushiobara, japan we investigated the cerebral hemoglobin (hb) changes in hemiparetic stroke patients under voluntary hand grasping task from acute to chronic phases by using near infrared spectroscopy (nirs). fortyfour channels ( channels on each side) were placed on the scalp overlying both sensorimotor cortices, and the cerebral hb changes were observed during four to six cycles of s task and s resting periods while sitting on a chair. the amounts of oxy-hb change were significantly increased in the bilateral sensorimotor areas during hemiparetic hand grasping at the acute phase, though the significant increase was mainly observed in the contralateral sensorimotor area during hemiparetic hand grasping at the chronic phase and during normal hand grasping at all phases. this result suggests that the functional recovery from post-stroke hemiparesis may be attributed to neuronal reorganization of sensorimotor areas via recruiting ipsilateral cortex. research funds: crest, jst todd pataky , , rieko osu , hiroshi imamizu , mitsuo kawato , computational brain project, icorp, jst, japan; atr cns laboratories, japan movement direction encoding in primate single cortical cells has been widely documented. this study was designed to test whether this directional tuning is observable at the voxel level in human fmri. three subjects performed hz isometric force pulses to seven targets separated by • in the shoulder/elbow flexion-extension plane. while in mri, online force feedback was provided by a d strain gauge. twenty repetitions of each condition were performed in s blocks (tr = s). all subjects showed broad activation over the contralateral motor area, and from functional rois an average of voxel time series were extracted. many voxels exhibited continuous cosine-like tuning with movement direction. decoding using linear svm revealed that while correct classification rate was only . % (chance: . %), errors were distributed normally about the target such that . % (chance: . %) of the data was classified correctly to within • . these data demonstrate that non-invasive neuroimaging is sufficiently sensitive to study the problem of coordinate system representation. the behavioral thermoregulation is important for living in various temperature environments. however, the neural mechanism of behavioral thermoregulation is poorly understood. in this study, we aimed to establish a new model to analyze the neural mechanism of behavioral thermoregulation using zebrafish. we investigated whether zebrafish perform behavioral thermoregulation against heating. when water temperature was changed from • c, fish showed repetition of short time swimming in the range . - • c. the frequency of the heat induced escape behavior was increased with temperature dependant. these results suggest that the heat induced escape behavior is a part of behavioral thermoregulation. the heat induced escape behavior was observed stably in - days past fertilization, indicating that the neural mechanism which control behavioral thermoregulation is matured in days. in conclusion, we established an effective new model to analyze behavioral thermoregulation. ps p-f to learn with one limb or two: limited transfer between unimanual and bimanual skills recent studies on neural activity in primary motor cortex of nonhuman primates suggest that unimanual and bimanual movements are controlled by partially overlapping neural processes. here we demonstrate that unimanual and bimanual motor learning also reflect a partially overlapping process. first, motor adaptations to reach with a novel force field applied to a limb could not be fully transferred to the same limb across unimanual to bimanual conditions, and vice versa. second, learning acquired during unimanual reaching could not be fully eliminated by repeated bimanual reaching with no loads, and vice versa. rather, some learning remained intact (but invisible) until the original context was again performed. lastly, two conflicting force fields can be learned simultaneously if they are separately associated with unimanual and bimanual reaching. these results support the view of partially overlapping neuronal processes and illustrate the intimate relationship between neural control and motor learning. research funds: jsps and nserc rieko osu , ken-ich morishige , jun nakanishi , , hiroyuki miyamoto , mitsuo kawato atr computational neuroscience labs, kyoto, japan; kyushu institute of technology, japan; jst-icorp, japan human can execute multiple motor tasks by using the same limbs, which makes human different from industrial robots. recently the optimal feedback control hypothesis has given a significant impact on the motor control community because it produces an optimal behavior for a given task by avoiding offline computation of optimal desired trajectory that would result in suboptimal behavior in the presence of noise. it, however, requires considerable amount of resources and learning to realize multiple tasks on nonlinear system. on the other hand, a desired trajectory enables the brain to share resources with multiple tasks and save learning time by dividing a difficult problem into easier sub-problems of plan and execution. considering the modularity of the brain and viability for nonlinear system, the hierarchical implementation is a better solution for global optimality as a versatile creature. here, we experimentally demonstrate that the hand variance modulation during multiple via-point tasks supports the existence of a desired trajectory. the purpose of this study is to computationally predict arm-reaching movements and posture controls from neuronal activity of premotor (pm) and primary motor area (mi). the activity was collected with single-unit recording method during the animal performing a visually guided arm-reaching task. electromyograms (emgs) and kinematics were also measured. we reconstructed the emgs from the neuronal data using a linear regression model, and then we estimated the kinematics from the reconstructed emgs with an artificial neural network model and proportional derivative controller. as a result, these serial processes allowed us to accurately predict the kinematics during both moving and maintaining her posture from the activity. the advantage of our bmi system is to estimate not only the kinematics but also the muscle tension from the neuronal activity. we have recently reported essential role of the tongue in breastfeeding in the hypoglossal (xii) nerve-injured newborn rats. of particular interest were the findings that the rates of the amounts of milk intake in the unilateral xii nerve-injured p pups of the surviving cases increased greatly between p ( % of the control value) and p ( %), suggesting adaptive tongue movement during development. this study was undertaken to reveal underlying basic mechanisms for such adaptation focusing on neural plasticity allowing effective suckling. after resection of the ipsilateral xii nerve on p , dii, a postmortem neuronal tracer, was applied to the contralateral uninjured xii nerve of p and p pups. dii-labeled neuronal fibers were successfully traced within the tongue and were found to extend over the xii nerve-injured side with gradual increase from p to p . we show evidence for functional neural plasticity that allows effective suckling in the xii nerve-injured newborns with suckling disturbance. previously we reported that decorticated rats showed abnormal righting movements in the air when dropped from the supine position, while the air righting reflex (arr) could be evoked purposefully ( • turn around the body axis) in decerebrated ones. thus, the basal ganglia might send interference signals to the arr center via the midbrain tegmentum. to clarify its functional roles in arr control, we examined arr movements in rats with the midbrain lesioned. wistar, male rats were prepared; after the posterior cerebral cortex was removed by sucking, the superior colliculus and surrounding structures were ablated in various degrees. arr movements were examined post-operative , , and days. in rats with the superior colliculus lesioned extensively on both sides, arr onset were delayed and body turn around the longitudinal axis was weakened, so that either insufficient or no rotation occurred in the air. furthermore, coordination between the body and tail rotations was lost in many cases. the ablated region may relay cortical signals that give a top priority to the arr center. ps p-g role of plateau potentials in feeding system of aplysia kurodai aiko kinugawa , tatsumi nagahama dept. of life sci., grad. sch. of sci. & technol., kobe univ., kobe, japan; fac. phar. sci., toho univ., funabashi, japan rhythmic motor activities seen in the animal behaviors can be generated by specific neural circuits termed the central pattern generator (cpg). in the feeding system of aplysia kurodai, the le neuron we identified produces the long-lasting plateau potentials and may be a cpg element. during the feeding-like responses duration of the depolarization of the follower neurons was shortened by hyperpolarization of the le. in this study we found that the le plateau potentials had refractory periods and they were turned to activation periods by application of large depolarizing currents. and various depolarizing pulses tended to produce the stable plateau potentials with almost constant depolarizing size and duration, suggesting that the le can supply the constant long-lasting depolarizing outputs to the follower neurons even when it receives various length and intensity of excitatory inputs from the presynaptic neurons. the le may be an important cpg element to determine the size and duration of the basic depolarization of many buccal neurons. withdrawn ps p-g neural organizations for vocal control in a social rodent, the deguneural organizations for vocal control in a social rodent, the degu naoko tokimoto , sayaka hihara , kazuo okanoya , atsushi iriki lab for symbolic cognitive development, bsi, riken, japan; lab for biolinguisutics, bsi, riken, japan vocalizations of most animals are innate, the region for the direct control of such sound is known to be localized in the pag. on the other hand, a few animals with the cortico-medullary projection path can learn a new sound. in this research, we investigated about vocal control in pag of social rodent, the degu (octodon degu). it is known that degus have fifteen kinds of vocal repertoires, and that their courtship song has a complex structure. we verified the hypothesis by electrical stimulation of the pag that the neural mechanism of degus that enables complex vocalization differs from that of guinea pigs with simple vocalization. guinea pig is near relation with degu. as a result, in guinea pigs, each sound is controlled in different area of the pag. in degus, however, multiple sounds are controlled by the same area, and the different sound was occasionally evoked by the different kind of stimulation. the sound with the time-series specific to the spontaneous vocalization of degu was not emitted. the effects of a heat-and steam-generating sheet (hsg sheet) on autonomic nerve activity and bowel movement were examined in women suffering irregular defecation, the hsg sheet was applied to the lumbar or abdominal regions, causing the temperature between the sheet and skin to increase to about . • c. application of the hsg sheet to either the lumbar or abdominal region significantly increased the rate of miosis in the pupillary light reflex. as for changes in r-r, the hf increased after application, suggesting that the parasympathetic nerve system had become dominant. bowel movement assessed by electrogastrography increased in amplitude. based on the above findings, we concluded that the application of an hsg sheet to the lumbar or abdominal region may lead to dominant parasympathetic nerve activity and improve gastrointestinal motility. ps p-g prostaglandin e -induced thermogenesis involves a gaba-receptive mechanism in the preoptic area toshimasa osaka national institute of health and nutrition, - - toyama, shinjuku, - , japan unilateral microinjection of pge into the region around the rostroventral wall of the third ventricle (av v) elicited thermogenic, tachycardic, vasoconstrictive, and hyperthermic responses simultaneously in urethane-chloralose anesthetized rats. the magnitude of these responses increased dose-dependently in a range of - pg, except for the vasoconstrictive response. next, the effects of pretreatment with a gaba a receptor antagonist, bicuculline methiodide ( . mm, nl), microinjected into the preoptic area (poa) ipsilateral or contralateral to the pge injection site was examined. this treatment alone had no effect on the o consumption rate and temperatures of colon and skin but elicited a bradycardic response. however, all pge -induced responses were blocked min after the pretreatment with bicuculline, and recovered at ∼ min. pretreatment with vehicle saline had no effect on the pge -induced responses. these results suggest that the gaba-receptive mechanism in the poa is required for the pge -induced thermogenesis. tetsufumi ito , hiroyuki hioki , kouichi nakamura , takeshi kaneko , yoshiaki nojyo dept. anat., univ. of fukui, fukui, japan; dept. of morphological brain sci., kyoto univ., kyoto, japan although gaba-immunoreactive (ir) fibers in the rat superior cervical ganglion (scg) were thought to originate in small cells located in the cervical sympathetic trunk (cst), almost all gaba-ir axon terminals showed markers for sympathetic preganglionic neurons (spns) in our recent report. in this study, we performed series of experiments to confirm the origin of gaba-containing fibers. gad -ir fibers were not found in dorsal roots (drs), but in ventral roots (vrs), stellate ganglion, cst, and scg. gad -positive somata were not found in dr ganglia and cst, but in intermediolateral (iml) nucleus of thoracic spinal cord (tsc). after intraperitoneal injection of fluorogold (fg), to label the entire spns, some fg-ir neurons were also positive for gad . we injected sindbis virus, an anterograde tracer, in iml, and some labeled terminals in scg showed gad -ir. after cutting t -t vrs and drs, almost all gad -ir fibers were abolished in scg. these results indicate that gaba-containing fibers in scg originate from spns in iml of tsc. masato nagahama , ning ma , reiji semba , satoru naruse dept. of anat. ii, mie univ. school of med., tsu, japan; inst. for developmental research, kasugai, japan; dept. of int. med., nagoya univ. graduate school of med., nagoya, japan aquaporin (aqp ) is first found as a water-transporting protein and has been demonstrated in various organs and tissues. in the present study, we have demonstrated the presence of aqp immunoreactivity in a particular neuronal subtype in the enteric nervous system (ens) of the rat ileum. aqp -immunoreactive (ir) neurons simultaneously expressed a neuronal marker huc/d. moderate numbers of aqp -ir neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. aqp -ir neurons can be classified as dogiel type i cells, which have several short processes and a single long process. many aqp -ir fibers were found both in the myenteric and submucosal plexi. many aqp -ir varicose fibers were closely associated with neuronal somata in the ganglia, whereas other aqp -ir fibers penetrated into the muscle layers. these results suggest that aqp -ir neurons probably play a significant role within the ens to control gut functions. research funds: kakenhi ( ) ps p-g abdominal expiratory nerve activity in the decerebrate neonatal rat center for medical sciences, ibaraki prefectural university of health sciences, ibaraki, japan the abdominal expiratory activity was recorded from the iliohypogastric nerve in the decerebrate, vagotomized, paralyzed, ventilated neonatal rat at postnatal days - . the increase in the volume and frequency setting of the artificial ventilator (fio = %, fico = %) failed to make the rat apnea. under this condition, the phrenic nerve showed unstable rhythmic inspiratory bursts, and the tail pinch increased the respiratory frequency. although the iliohypogastric nerve showed expiratory discharges, their amplitudes and shapes were not consistent. when fico was increased, the cycle period was prolonged and the abdominal expiratory activity was enhanced. in many rats, the iliohypogastric nerve showed biphasic discharges that consisted from the pre-and post-inspiratory discharges. the preinspiratory discharge has larger amplitude and shorter duration than the post-inspiratory discharge. since the post-inspiratory discharge was usually small or indistinguishable in the adult rat, the present results suggest that the pattern of abdominal expiratory activity will change during the postnatal development. we investigated the role of gabaergic neurons in the rostral ventrolateral medulla (rvm) in central respiratory control. we used gad -gfp knock-in mice in which we could identify gabaergic (i.e., gfp-positive) neurons in a living condition. we recorded gabaergic neuron activities (n = ) in medullary transverse slices. about % of gabaergic neurons were inspiratory, and all of the remaining neurons were non-respiratory. about % of gabaergic neurons recorded in the superficial rvm were co inhibitory, and all of the remaining neurons were co insensitive. we suggest that gabaergic inhibition in the rvm respiratory neuron network is mediated mainly by inspiratory neurons. gabaergic neurons are also involved in central chemosensitivity. we investigated two groups of people with a different initial level of an emotional tension before intellectual loading (il). first group had the initially increased emotional level, stressed group (sg), and the second group had not, calm group (cg). reaction time (rt) of simple visual sensorymotor reaction and asymmetry of skin potential level (spla) in two facial zones: forehead and nasal were measured. from research groups, subgroups with and mv spla were extracted. thus the distinction in the greater rt gain after il in subgroups with mv forehead spla, in comparison to subgroups mv forehead spla. such law was common for both for sg and for cg. in two subgroups of and mv nasal zone spla in sg the greater rt gain after il was in mv nasal spla subgroup, and for cg in mv subgroup. it was shown, that individual features of il performance are related to spl lateralization. but the low of the relationships of spl asymmetry in nasal zones depends on the level of emotional tension of investigated groups. mitsuko kanamaru, ikuo homma department of physiology, showa university school of medicine, tokyo, japan we have reported that serotonin ( ht) in the dorsomedial medulla oblongata in mice increases tidal volume and minute ventilation via ht receptors. peripheral administration of p-chlorophenylalanine (pcpa) reduces the whole brain ht level. the present study examined whether peripheral pcpa pretreatment affects hypercapnic ventilatory responses in mice. adult male mice (c bl/ n) were pretreated with pcpa ( . g/ ml/kg body weight) or saline intraperitoneally for consecutive days. on the next day, each mouse was placed into a double chamber plethysmograph to obtain respiratory flow curves. one hundred percent o inhalation was changed to stepwise , and % co in o inhalation every min. hypercapniainduced increases in tidal volume and minute ventilation during % co inhalation were reduced by pcpa pretreatment; these results suggest that ht may increase tidal volume in hypercapnic ventilation. saori nishijima, kimio sugaya, minoru miyazato division of urology, department of organ-oriented medicine, faculty of medicine, university of the ryukyus, okinawa, japan we investigated the effect of gosha-jinki-gan on bladder activity and the autonomic nervous system in rats. forty-two female rats were divided into a control diet group and a . % gosha-jinki-gan diet group. after weeks, continuous cystometry with physiological saline or . % acetic acid solution and biochemical analysis were done. the amplitude of bladder contraction with physiological saline was lower in the gosha-jinki-gan diet group than in the control diet group, and plasma dopamine and serotonin levels were also lower in the gosha-jinki-gan diet group. when cystometry was done with . % acetic acid, the interval between bladder contractions was shortened in the both groups. however, the interval and duration of bladder contractions were longer in the gosha-jinki-gan diet group than in the control diet group. therefore, it is suggested that gosha-jinki-gan inhibits bladder activity by maintaining the balance of the sympathetic nervous system and the parasympathetic nervous system at a low level. satoko suzuki, shinya yanagita, seiichiro amemiya, ichiro kita graduate school of science, tokyo metropolitan university, japan we examined the effects of negative air ions (nai) on physiological responses and neuronal activity with c-fos immunohistochemistry. in addition, we investigated the effect of vagotomy to reveal afferent pathways of nai stimulation. we analyzed neuronal activity of the paraventricular nucleus of hypothalamus (pvn), the locus ceruleus (lc), the nucleus ambiggus (na), and the nucleus of solitary tract (nts). nai significantly decreased blood pressure, heart rate, and respiratory rate, and increased hf component which is an index of parasympathetic nervous activity. nai decreased c-fos expression in the pvn and lc, and enhanced in na significantly. after vagotomy, the physiological responses and changes of c-fos expression in pvn, lc, and na was disappeared. furthermore, increase of c-fos expression in nts induced by nai was also disappeared. these results suggest that effect of nai on sympathetic and parasympathetic nervous activity was induced by reducing the activity of the pvn and lc, whereas enhancing the na activity, and that these effects of nai was caused through vagus nerve. yusuf o. cakmak, umit suleyman sehirli school of medicine, university of marmara, turkey previous assessments of the autonomic nerve supply of testis from vagus and brainstem nuclei were conflicting in the literature. we challenged this consensus by using neuronal tracer fluorogold in rats. fluorogold dye solutions was injected unilaterally under the capsule of rat testis. rats were sacrificed by transcardiac perfusion-fixation in the fifth and seventh days after injection. brainstem of the control group, subdiaphragmatic vagotomy group and main group rats were dissected. in the main group the fluoroscent-labelling were dense in area postrema. dorsal vagal nucleus, nucleus of solitary tract and nucleus ambiguous were also labelled. these preliminary data provide an evidence of testicular innervation by vagus nerve. taking into account that brainstem structures could be labelled from the testis, it can be assumed that the areas detected might be involved in the neural control of testicular functions. the results of this study cautioned that innervation of the testis may not be fully explained by innervation from pelvic and paraaortic ganglia. research funds: scientific researches comittee of medical school of marmara university ps p-g differential control of renal and lumbar sympathetic nerve activity during freezing behaviour in conscious rats yoshimi tahara, misa yoshimoto, keiko nagata, kenju miki integrative physiol. grad. sch. humanities and sci. nara-women's univ., nara, japan the present study was designed to examine sympathetic and hemodynamic responses to loud noise exposure, which induced freezing behaviour, in chronically instrumented rats. wistar male rats were instrumented with electrodes for measurements of renal (rsna) and lumbar (lsna) sympathetic nerve activity and catheters for measurements of systemic arterial and central venous pressure. rats were exposed to db white-noise for min. db noise exposure resulted in an immediate and significant increase in rsna while lsna did not change significantly during the exposure in sham-operated (so) rats. there was a significant difference in the response between rsna and lsna during the db noise exposure in so rats. sinoaortic denervation attenuated the magnitude of the increase in rsna while it had no influence on the changes in lsna observed in so rats. these data suggest that arterial baroreceptor significantly contribute to the differential control of rsna and lsna during freezing behaviour in conscious rats. here, we first demonstrated that in both the kolliker-fuse nucleus (kf) and the rostral ventral respiratory group (rvrg) region, phrenic nucleus (phn)-projecting neurons were embedded in the plexus of axons originating from the ventrolateral subnucleus of the nucleus of the solitary tract (vlnst) and that the vlnst axon terminals made synaptic contacts with somata and dendrites of the phnprojecting neurons, using a combined anterograde and retrograde tracing technique. secondly, we indicated that some of the vlnst neurons innervate both the kf and the rvrg by way of axon collaterals, using the double-labeling method. using retrograde tracing combined with in situ hybridization for mrna encoding glutamic acid decarboxylase (gad ), we finally showed that most of the kf/rvrg-projecting vlnst neurons expressed gad mrna. these results suggest that vlnst neurons may exert inhibitory influences upon the phn-projecting kf/rvrg neurons for inspiratory control. we have examined whether the neurons of the dmv have direct synaptic contacts on the myenteric ganglia using wga-hrp. the myenteric ganglia of the stomach were composed of four types of neurons. the average numbers of axosomatic terminals per profile were . on the small neurons, . on the medium-sized neurons, . on the large neurons, and . on the elongated neuron. most of the terminals contained round vesicles and formed asymmetric synaptic contacts on the small, medium-sized and large neurons. about % of the axosomatic terminals on the elongated neurons contained pleomorphic vesicles and formed asymmetric synaptic contacts. when wga-hrp was injected into the dmv, many anterogradely labeled terminals were found around the myenteric neurons. the labeled terminals were large ( . m), and contacted exclusively the somata. most of them contained round vesicles and formed asymmetric synaptic contacts. serial ultrathin sections revealed that almost all neurons in a ganglion received projections from the dmv. ps p-h neuronal mechanisms of respiratory rhythm modulation induced by external k + concentration change in the newborn rat brainstem-spinal cord preparation hiroshi onimaru, ikuo homma dept. physiol., showa univ. school of med., tokyo, japan it has been suggested that two distinct rhythm generators (pfrg-pre-i and pre-bötzinger insp) for respiration in the medulla possess different sensitivity to various neuromodulators. we hypothesize that the dominancy of these rhythm generators to determine basic respiratory rhythm depends on the back ground stimulation level. to verify this hypothesis, we studied neuronal mechanisms of respiratory rhythm modulation induced by external [k + ] change. we recorded membrane potential of pre-i neurons, c nerve and facial nerve activities. addition of mm k + to the standard superfusate decreased burst rate of c activity. addition of or mm k + caused initial inhibition of c burst and subsequent high frequency c burst. the facial nerve burst was depressed. pre-i neuron was depolarized strongly by application of high k + , and the burst activity was disturbed and action potentials were inactivated. results suggest that pfrg-pre-i or pre-bötzinger insp rhythm generator is dominant in low or high back ground stimulation level, respectively. research funds: kakenhi ( ) ps p-h regulation of synaptic transmission in the reticular formation of medulla oblongata by substance p we have examined the response of neurons in the reticular formation near the nucleus ambiguous (na) to the administration of substance p (sp). whole-cell recording was applied to the postsynaptic neurons in coronal slice preparations of medulla oblongata isolated from infant rat. bath application of sp ( m) increased or decreased the frequency of spontaneous activities. several neurons were clamped at − mv and recorded epscs evoked by electrical stimulation to dorsoventral adjacent area from recording neurons. in several neurons, evoked inward epscs were augmented by sp perfusion. i-v curve suggested that voltage dependent current was both augmented and not changed by sp. our previous studies have shown that administration of neurokinin receptor (nk r) antagonist near the na inhibited gastric and respiratory movement in anesthetized rat. these results indicated that sp affect to both post and presynaptic nk r and regulate the transmittance efficiency to generate the output signal of certain autonomic reactions. ps p-h effects of local warming in the back or abdominal region by means of a heat-and steam-generating sheets on physiological response in the low temperature room to investigate effects of local warming of the body on physiological functions as well as subjective feeling, eegs, ecg, respirometer, bis (bispectrum) index, blood pressure (bp), and local skin temperature of the body were monitored while a steaming heat pack was put on the lower lumber or abdominal region of the subjects for h in the cold room. in the control experiment without the heat pack, lf/hf of hr variability (lf/hf-hr) and lf of bp variability (lf-bp) increased, while hf of hr variability (hf-hr) and skin temperature decreased, suggesting elevation of sympathetic nervous activity. in the warming experiment with the heat pack, an increase in lf/hf-hr and/or lf-bp was suppressed and hf-hr increased. we will discuss these autonomic data in relation to subjective unpleasant or pleasant feeling, eeg and bis data. junichi arai, yasuhisa endo, ryouichi yoshimura, huan wang kyoto institute of technology, japan in the ventromedial hypothalamic-lesioned animals, the abnormal cell proliferation in liver and pancreas are thought to be due to the vagus hyperactivity and/or the sympathetic repression. we conducted the co-culture system of several cell lines and demonstrated that the proliferation of hepatocytes and min- cells (a cell line of pancreatic b cells) were stimulated by the administration of carbachol, when they were co-cultured with cell lines of endothelial cells or smooth muscle cells. these effects were also found in the filter-insert coculture system, but never seen in the culture using single cell line. we discuss the possible mechanism of their intracellular signal transduction. research funds: kakenhi to study the correlation between the trans-cranial oxy-and deoxyhemoglobin (hb) dynamics and sbp, we measured hb dynamics (f-nirs ® , omm- , shimadzu corp. japan) over the frontal area and sbp (finapres ® , bp monitor, ohmeda , usa) at the right middle finger from volunteers ( . ± . years). mild thermal stimuli ( ± • or ± • ) were administered every min alternatively to the left hand. some area showed positive correlation between the oxy-hb and sbp, the other showed negative correlation between them. hb dynamics over the frontal area have any correlation to sbp to some extent. so, trans-cranial nirs should be discussed carefully for neural activation. we thank shimadzu corp. for the use of omm- . we examined the effects of color environments on cognitive function in healthy subjects and patients after traumatic head injury using p components and loreta analyses. the examination was performed in color environments of red, green, or black using visual oddball tasks with photographs of a crying baby face as the target stimuli. the p latency in the red environment was significantly shorter in controls than in patients. the p amplitude in the red environment was significantly larger in controls than in patients. loreta analysis demonstrated that the neurological activities in the occipital lobes, left tonsillar nucleus, anterior cingulated gyrus, and brodmann area in the red environment were significantly higher in controls than in patients. hironori nakatani, cees van leeuwen riken brain science institute, saitama, japan some figures, such as rubin's vase/face and the necker cube, have two or more distinct interpretations and are, therefore, called 'ambiguous'. when an ambiguous figure is presented continuously for a period of time, we experience spontaneous switching between the alternative interpretations. as this occurs without any changes in the figures themselves, perceptual switching phenomena are eminently suitable to study how perceptual processes are influenced by the intrinsic dynamics of neural activity. we analyzed eye-movement and eeg during perceptual switching in the necker cube. blink probability showed a peak about ms before the button press responses. we found that only blinks that appeared around the peak time led to a characteristic spatiotemporal pattern of eeg. our results indicate that some, but not all, blinks play an active role in perceptual switching processes. ps p-h neural basis of social cognition investigated by functional near infrared spectroscopy and electroencephalograms recorded from the whole brain tsuneyuki kobayashi , , mikinobu takeuchi , , takahiro omote , naoyuki yosimura , etsuro hori , , kazuo sasaki , taketoshi ono , , hisao nishijo , system emotional science, univ. toyama, toyama, japan; crest, japan science and technology agency, japan; bio-information engineering, univ. toyama, toyama, japan; molcul. & integ. emotional neurosci., univ. toyama, toyama, japan neural basis of social cognition was investigated by functional near infrared spectroscopy (fnirs) and electroencephalograms (eegs). a head cap for recording fnirs and eegs was set on heads of subjects. the probes of the fnirs imaging systems ( channels) and/or electrodes of the eeg system were attached on the heads of the subjects. the subjects were required to perform social cognition tasks to discriminate ( ) human facial stimuli with different gaze directions and ( ) simple animation videos representing social interaction. whole brain hemodynamic images were superimposed on the d reconstructed mri images of the brains. now we are analyzing hemodynamic images and eeg data related to social cognition, and the results indicated some heterogeneity of the cortex in social cognition. hiroshige takeichi , sachiko koyama , ayumu matani , andrzej cichocki riken, wako, japan; hokkaido university, sapporo, japan; university of tokyo, kashiwa, japan to evaluate the level of spoken sentence comprehension objectively and quickly, electroencephalograms (eeg) were recorded from five japanese adults, while they were listening to fifty-second spoken sentences. natural japanese (native) and spanish (foreign) sentences were modulated in amplitude by an eleventh-order m-sequence at hz, and played twice: forward and backward. evoked responses to the modulation were analyzed as follows: ( ) circular cross correlation functions were calculated between the eeg data and the m-sequence for each subject. ( ) the functions were averaged across subjects. ( ) independent component analysis (ica) was applied to the averaged functions and independent eeg components were estimated for each stimulus for each subject. ( ) phase-locked component responses to the modulation were inspected. as a result, two components showed differential responses to the comprehensible forward japanese and the other incomprehensible stimuli. research funds: jst and kakenhi ( ) perceptual rivalry, such as ambiguous figure perception and binocular rivalry, reflects the flexibility of our brain, because it produces fluctuating perception though an unchanging stimulus. in this study, we carried on meg recordings of healthy subjects while they reported perceptual alternation of bistable apparent motion. we investigated power and phase synchronization analyses of meg signals and compared the spatiotemporal patterns during spontaneous perceptual alternation (rivalry condition) with the externally-triggered alternation (replay condition) to extract the inherent dynamics of perceptual alternation. as results, we detected transient anterior-posterior synchronizations in advance of subjects' reports of perceptual alternation in the rivalry condition. these results suggest that these synchronized activities are involved in a higher-order process inducing spontaneous alternations in perceptual rivalry. ps p-h the reflection of category perception of sound in the auditory evoked n m magnetic responses to periodic complex sounds with equivalent acoustic parameters except for different fundamental frequencies (f ) and different spectral envelopes of vocal, instrumental and linear shapes were recorded to clarify the cortical representation of timbre categorization. responses to vocal and instrumental (nonlinear) sounds were localized significantly anterior to linear sound responses. n m source strength for nonlinear sounds was significantly larger than that for linear sounds. n m peak latency only for vocal sounds was not affected by f . these results suggest that perceptual categorization was reflected in n m source strength and location (linear or nonlinear), and in n m latency (vocal or nonvocal). sunao iwaki , hiroko kou , kouichi sutani , mitsuo tonoike national institute of advanced industrial science and technology, osaka, japan; chiba univ., chiba, japan interactions between neural activities detected at multiple brain regions involved in the visual target detection processing were assessed using meg and the causal modeling. meg signals were measured during subjects performing a visual infrequent target detection task. distributed source model was used to infer the dynamic neural activities at the multiple regions and the structural equation modeling (sem) was then used to compare two possible causal models underlying the generation of major event-related components, namely p , related to the target detection. we used akaike information criteria (aic) and goodness-of-fit index (gfi) as measures of the goodness of the models. the results of the comparison of two possible sem models, whose major difference was on the contribution of the activities in the parieto-temporal region to the generation of p components, suggested the involvement of frontal and anterior cingulate cortex in the early p component (p a) and the contribution of the parietal and temporal regions to the later component (p b in our study, we investigate whether or not bilinguals use distinct neural substrates to recognize words in their first and second languages (l and l , respectively). we compared the brain activity of chinese learners of japanese as l with that of japanese natives studied in our previous study. we obtained written informed consent from each subjects. in data analysis, we used spm . while natives showed specifically greater activation in the left middle temporal gyrus than learners, learners showed specifically greater activation in the bilateral parieto-occipital and left occipito-temporal junction than natives. these results indicate that there are distinct neural substrates for word recognition of l and l . neural activations for lexical processes were measured using noun, vowel, and pseudo-character decision tasks with magnetoencephalography (meg) and functional magnetic resonance imaging (fmri) on ten right-handed subjects, and their time courses were analyzed with an fmri-constrained meg-multi-dipole method. the average activations rose at latencies around ms in the occipital gyrus or cuneus (og/cuneus) and ventral occipito-temporal areas (vot), and at latencies around ms in the posterior superior temporal and inferior parietal areas (pst/ipl), anterior temporal area (at), and posterior inferior frontal gyrus (pifg). the differences in activation between tasks are considered to reflect visual-form process in the og/cuneus and r.vot, phonological process in the l.pst/ipl and l.pifg, and semantic process in the l.at. the decay of activation for these areas was found to be well fitted to exponential functions with time constants around ms. the effectiveness of a habituation/dishabituation paradigm for determining the cerebral dominance for language was examined using a . t fmri. healthy right-handed adult volunteers with prior written informed consent were instructed to listen to analysis-synthesized words. after habituated to a single word presented repeatedly, the subject was presented with contrastive words which comprised comparison and habituation words in a pseudo-random order. the two blocks were repeated alternately for times. comparison words were phonemic or intonational derivative of the habituation word, and presented in respective sessions. the results showed that the left auditory cortex responded more to the phonemic contrast, and the right to the intonational contrast, which is in line with other paradigms/techniques for determining cerebral dominance, while the present paradigm demands little effort on the subject. the issue that whether meaning of kanji words is accessed from orthography, or from both orthography and phonology representations is still debated. the present fmri study investigated brain areas underlying the use of orthography and/or phonology in kanji reading by engaging subjects in semantic categorization task with homophone and orthographic similarity effects. fifteen native japanese volunteers participated. stimuli were pairs of definitions and their target words, including correct words and foils. the subjects were asked to decide the correct target words of definitions. the results showed that homophone versus non-homophone foils increased activation of the left fusiform and middle frontal gyri. orthographically similar versus dissimilar foils increased activation of the left middle and inferior frontal gyri. these findings reflected the roles of both orthography and phonology in kanji reading. moreover, homophone versus non-homophone minus orthographically similar versus dissimilar foils revealed activation of the left fusiform gyrus. this might suggest the role of this area in character-to-sound conversion of kanji words. chieko takamiya , mie matsui , , tsuneyuki kobayashi , , hisao nishijo , , michio suzuki , , yasuhiro kawasaki , , masayoshi kurachi , , jun nakazawa , kyo noguchi , hikaru seto neuropsychiatry, univ. toyama, toyama, japan; crest, japan science and technology corporation, japan; psychology, univ. toyama, toyama, japan; system emotional science, univ. toyama, toyama, japan; neuropsychiatry, univ. toyama, toyama, japan; developmental psychology, univ. chiba, chiba, japan; radiology, univ. toyama, toyama, japan an individual has a theory of mind (tom) if he imputes mental states to himself and others. this ability is necessary for our well-rounded social communication. we used functional magnetic resonance imaging (fmri) in ten healthy subjects to study the neural mechanisms underlying tom. we adopted the picture sequencing tasks which demanded inferring mental states to self and others as tom task. as a result, there were significant brain activations in the medial frontal cortex and middle frontal gyrus. these activations coverged with a part of results in previous neuro-imaging studies on tom and social cognitive functions. objective: the purpose of this study was to investigate the neural bases of evaluation of ambiguous facial expression using whole brain functional magnetic resonance imaging (fmri). methods: participants underwent fmri scanning during which they performed a task evaluating facial expression of human (happy or sad). the task consisted of three conditions: ambiguous, middle, and high intensity of facial expression. pictures were chosen from atr facial expression image database. results: subtraction between ambiguous and other conditions revealed the activation of anterior cingulate cortex and prefrontal cortex in evaluation of ambiguous expression. the present results suggest that these area may be involved in evaluation of ambiguously expressed emotions. motoaki sugiura , atsushi sekiguchi , keisuke wakusawa , , yuko sassa , , hyeonjeong jeong , , kaoru horie , , shigeru sato , , ryuta kawashima , miyagi university of education, sendai, japan; niche, tohoku univ., japan; dep. pediatrics, tohoku univ. school of medicine, japan; ristex, jst, japan; gsics, tohoku univ., japan; lbcrc, tohoku univ., japan using an fmri, we examined the cortical mechanisms for risk perception during observation of risky tool usage. normal subjects were presented with a picture of a naturalistic situation involving two actors, in which risks related to a tool and the direction of action were modulated in a two-factorial design. after the fmri, each subject self-evaluated the degree of risk in each picture. main effects of object-and direction-related risks were observed in the left ventromedial prefrontal cortex, and dorsolateral parieto-frontal network, respectively, suggesting that the object-and direction-related risk signals are separately processed in these networks. significant positive correlation between self-evaluated risk and cortical activation was observed in the anterior part of the left superior frontal sulcus, suggesting an involvement of this region in phenomenal risk-perception. in this fmri study, we identified cortical areas where activation during experience of risky situation is correlated with the harm avoidance (ha) scores, subscale of temperament and character inventory (tci). forty-six healthy subjects performed a rule speculation task in risky, normal, and safe situations in fmri. each situation was arranged for subjects to gain , , points or lose , , points, respectively. cortical activation induced by experience of risky situation was estimated. a significant positive correlation with the ha scores, was observed in activated areas in the right anterior insula in risky versus safe comparison. the results suggested that activation in this region predicts the individual difference in behavioral response to risky situation. this finding indicates that the right insula underlies individual difference in response to risky situation. ps p-h brain activation related to the evaluation of absolute and relative value of outcome juri fujiwara , masato taira , , toshio iijima , ken-ichiro tsutsui div. sys. neurosci., tohoku univ. grad. sch. life sci., sendai, japan; arish, nihon university, tokyo, japan; appl. sys. neurosci., nihon univ. grad. sch. med. sci., tokyo, japan one way to evaluate the behavioral outcome is in terms of absolute gain or loss (absolute value), but the evaluation can also be achieved by comparing the outcome with the possible outcomes of unchosen options (relative value). here we attempted to disentangle the brain processes involved in the absolute and relative value evaluation by using event-related fmri. subjects were instructed to compete with a computer to maximize the income in a task, in which they had to choose one option out of two, each of which were associated with either yen or a gain or loss of , , or yen. in each trial, a choice period was followed by a serial presentation of the outcomes of the chosen and unchosen options. we analyzed the brain activity during the presentation of each outcome. the activation changes related to the evaluation of absolute and relative value were observed mainly in the basal ganglia and in the cerebral cortex, respectively. ps p-i neural activation during experience-based reasoning chisato suzuki , , takashi tsukiura , hiroko mochizuki-kawai , yayoi shigemune , , toshio iijima neurosci. res. inst., aist, japan; div. systems neurosci., tohoku univ., japan the aim of this study is to investigate neural activations when reasoning future events based on experienced events. before fmri, subjects encoded two kinds of four-scene comics; the complete version with four scenes and incomplete one without the last scene. after encoding, subjects performed three tasks during fmri. in the first task, subjects chose a last scene associated with the first scene encoded in the incomplete version (memory-based reasoning: mr), whereas in the second task, subjects recognized a last scene encoded in the complete version (memory: m). in the third task, subjects chose a last scene appropriate to the first scene in the new comics (reasoning: r). activations specific to mr was found in a relatively anterior part of the left pfc and right pfc. the common activations between mr and m were identified in the right mtl, whereas a relatively posterior part of the left pfc was activated commonly between mr and r. the findings suggest that the network including bilateral pfc and right mtl may contribute to the experience-based reasoning of future events. to assess neural responses to reciprocal mindreading in socially strained human relationships, we performed an fmri study in healthy subjects who participated in the chicken game. statistical parametric mapping showed that the counterpart effect (human versus computer) activated the anterior paracingulate cortex (pcc) and the posterior superior temporal sulcus (sts). when we analyzed the data to evaluate whether the subjects made aggressive or reconciliatory choices, the posterior sts showed that the counterpart had a reliable effect regardless of risky or safe decisions. in contrast, a significant opponent x selection interaction was revealed in the anterior pcc. it could be inferred that the posterior sts and the anterior pcc play differential roles in mentalizing; the former serves as a general mechanism for mentalizing, while the latter is exclusively involved in socially risky decisions. creativity is the ability to generate new and original ideas. the most of studies of creativity used linguistic tasks which involve multiple aspects oflinguistic information processing in addition to creativity. we used new artistic creativity task such as designing new tools, in which we could quantitatively evaluated the creativity by the originality (os: originality score) of the products. using fmri, we observed bold signal change during designing task in art students (trained) and non-art students (untrained). we observed clear difference between two groups; in the trained highly creative group, the os is correlated with the interhemispheric difference of neural activities of the prefrontal cortex with right hemisphere dominance. in the untrained group we saw no such correlation. thus, our result supports the notion that both right prefrontal dominance and the increase of interhemispheric cooperativity could be the source of the artistic creativity. ps p-i the difference of brain activity elicited by different styles of art hiromi yamamura , yasuyuki kowatari , , shigeru yamane , miyuki yamamoto , comprehensive human sciences, university of tsukuba, tsukuba, japan; system brain science division, aist, tsukuba, japan artworks are categorized according to time and place where they were produced (cultural effects). surrealistic art is one of those categories and it gives uneasy impression to our mind. we investigated brain activity during viewing pictures of different art styles using functional magnetic resonance imaging (fmri). works of several artists who are well-known as representatives of renaissance, impressionism and surrealism were used as stimuli and results were analyzed by spm . while renaissance arts or impressionism arts elicited a similar activation pattern in the occipital and inferior temporal areas, surrealisms showed deactivation in parietal with the activation in the right dorsal prefrontal cortex (ba , ba ). these results suggest that a particular style of artwork may have commonly activated brain regions. research funds: coe(j- ) ps p-i effects of chewing on the activity of the prefrontal cortex in working memory processing: an fmri study in general, it has been proposed that chewing produces holding or enhancing effect on attention. furthermore, recent studies have shown that chewing causes activation of various brain regions, including prefrontal cortex. we therefore examined the influence of chewing on brain activities using fmri. the subjects used were - aged healthy adults, being conducted continuously to two-back task with intermittent gum-chewing. gum without odors and taste component was used to remove effects other than chewing. the results indicated that chewing tended to increase the bold signals in the prefrontal area including the dorsolateral prefrontal cortex during two-back task. this suggests the possibility that chewing may accelerate the process of working memory. research funds: kakenhi , ps p-i the tip-of-the-tongue with an emotional reaction caused by recall of celebrities' names hirohito m. kondo , michio nomura , jun kawaguchi ntt commun. sci. labs., ntt corp., atsugi, japan; dept. psychol., tokai women's univ., kakamigahara, japan; dept. psychol., grad. sch. environ. studies, nagoya univ., nagoya, japan the tip-of-the-tongue (tot) phenomenon is a mental state where you cannot recall something though you have every confidence that you know it. the tot state generates emotional reactions, but it is not clear what neural mechanisms are involved in the awareness of frustration. participants were instructed to recall the full names of celebrities when their faces were presented. event-related fmri analysis demonstrated that the anterior cingulate cortex (acc), anterior insular cortex (aic), inferior frontal cortex, intraparietal sulcus, and fusiform gyrus were activated during the tot state with frustration. activity of the acc and right aic was positively correlated with the degree of frustration in unsuccessful retrieval. roi analysis indicated that the acc and right aic were sensitive to retrieval demands and awareness of frustration, respectively. we suggest that the cinguloinsular circuit regulates the self-monitoring processes during the tot state. noriko kudo , , , yulri nonaka , katsumi mizuno , kazuo okanoya , riken, bsi, biolinguistics, saitama, japan; chiba university, chiba, japan; jsps, japan; department of pediatrics, showa university, tokyo, japan; presto, jst, japan segmentation of speech stream is a prerequisite for language acquisition. language learners use the transitional probability between vocal tokens to segment continuous auditory stream into distinctive words. we consider that the ability for statistical learning is not specific to language, but more general cognitive competence. and we ask whether this ability could be considered as innate. in this study, we measured erps for neonates within days, in order to examine whether neonates can learn transitional probabilities and statistically segment words. four three-tonal-words were presented in random order without intervals during recording of the eeg. as a result, only the first tone of each word evoked a significant positive component in the frontal area. since this potential is not evident during the first session, this is likely to be due to statistical learning. these results suggest that the ability to distinguish words based on statistical information is innately prepared in humans. using near-infrared spectroscopy (nirs), changes in concentration of oxygenated hemoglobin (oxy-hb) in the prefrontal cortex were evaluated while eleven human subjects performed the paintings appreciation task. in this task, subjects were required to appreciate abstract and representational paintings that appear one after another on a computer monitor. subjects were then required to judge the degrees of interest, beauty, and desirability immediately after the appreciation. it was shown that the peak of averaged oxy-hb change was higher while subjects appreciated abstract paintings. average differentiation for each oxy-hb change revealed that the changes while the appreciation of representational paintings were more accelerated than that while the appreciation of representational paintings. these results suggest the different cortical activity dependent on appreciation of abstract and representational paintings. we used meg to investigate the spatiotemporal cortical activities during mental calculations and their modulation by arithmetic complexity. eleven healthy subjects have participated in the study. three conditions were considered: easy: add three ( ) to a two-digits number without carry-over; difficult: stimuli were the same as easy, but with carry-over; nocalc: add zero to the two digits number. probe stimuli were presented s after the presentation of task stimuli (a pair of two-digit and one-digit number), and the subjects were required to respond by lifting the right index or middle finger. root-mean-square values for different meg sensor groups covering entire cortical area were calculated to evaluate local signal power in each condition. increased neural activities in the bilateral frontal/prefrontal and the parietal regions during both calculation conditions were observed in the latencies around - ms. the activities in the bilateral prefrontal and the left parietal areas in the same latencies were found to be complexity-dependent, i.e., increased activities in these regions were observed in difficult condition compared to easy condition. we investigated an effect of auditory feedback on self-produced speech in children with and without autism by measuring the lombard effect. ten children with autism ( : - : ) and agematched typically developing children ( : - : ) were instructed to name pictures of objects aloud in control and masking conditions. in masking, weighted-white noise was continuously delivered through a headset. the subjects' speech responses were recorded from a microphone. in typically developed children, the enhancement (masking/control) in masking was significantly greater (duration = . ± . , loudness = . ± . ) than in the children with autism (duration = . ± . , loudness = . ± . ) (p < . ). the present findings suggest that deficits in speech audio feedback in autistic children and this could be one of the reasons for their delay in speech development. since the mechanism underlying the effect of low power laser irradiation on the soft tissue is still unknown, we examined whether it can influence the muscle contraction as well as its fatigue in the frog (xenopus laevis) gastrocnemius or not. muscle tension continuously induced by a supramaximal stimulus to the sciatic nerve at . /s chronologically attenuated and showed a simple fatigue curve. direct irradiation of laser ( nm, mw) to the muscle surface ( . mm ) significantly delayed its attenuation (p < . ). when the rest period was set between stimulating sessions and the laser irradiation was applied during the rest period, averaged muscle tension during stimulating period for min decreased according to the session sequence. however, comparing with no or cooling application during the rest periods, such laser irradiation case significantly delayed the muscle fatigue (p < . ). it is suggested that laser irradiation has a potential to more activate atp synthesis during as well as after muscle contraction. ps p-i nedl , a novel e ubiquitin ligase for dishevelled- , targets mutant superoxide dismutase- and interacts with p yuanyuan li , , , kou miyazaki , toshinori ozaki , akira nakagawara division of biochemistry, chiba cancer center research institute, chiba, japan; production technology development center, the furukawa electric co., ltd., ichihara, japan; hisamitsu pharmaceutical co., ltd., tokyo, japan we have cloned a novel hect-type e ubiquitin ligase gene termed nedl . previous study has shown that nedl is exclusively expressed in neuronal tissues and its expression level is high in favorable neuroblastomas and undetectable in unfavorable ones. dishevelled- , a regulatory molecule in the wnt signaling pathway, was identified as the physiological target of nedl for uniquitination and proteasome-mediated degradation. on the other hand, nedl bound and ubiquitinated mutant (but not wild-type) sod in a mutant sod type-dependent manner, which is proportionally related with the fals severity. in the present study, we show that nedl physically bound p , and induced apoptosis in a p -dpendent manner. taken together, our results suggest that nedl may play a critical role in neuronal cell death occurring in fals through interacting with mutant sod and p . spinal and bulbar muscular atrophy (sbma) is an inherited motor neuron disease caused by the expansion of polyglutamine tract within the androgen receptor (ar). chip (carboxyl terminus of hsc interacting protein), u-box type e ubiquitin ligase, has been shown to interact with hsp or hsp and ubiquitylates unfolded proteins trapped by molecular chaperones and degrade them. we demonstrated in a neuronal cell model that transient over-expression of chip reduced the monomeric mutant ar more than the wild-type, suggesting that the mutant ar is more sensitive to chip than is the wild-type. we also demonstrated high expression of chip ameliorated motor impairments in the sbma transgenic mouse model. these findings suggest that chip over-expression ameliorates sbma phenotypes in mice by reducing nuclear-localized mutant ar, which probably due to enhanced mutant ar degradation. we performed an electrophysiological study demonstrating inhibition of spontaneous muscle action potentials within a co-culture of rat muscle and spinal cord by exposure to patients with guillain-barré syndrome (gbs) serum, as well as purified igg, from selected patients with gbs. using a whole-cell recording technique, we then investigated the effects of serum and purified igg from patients with gbs on voltage-dependent calcium currents (vdcc) in ngf-differentiated pc cells and cerebellar purkinje cells. serum from selected patients with gbs and purified igg from some serum of patients with gbs inhibited ca + current in both cells. these results suggest that muscle weakness in some patients with gbs might be induced by changes in p/q-type calcium channel function within motor nerve terminals. the aim of the present study was to explore the possible role of cox- inhibitor, rofecoxib in pentylenetetrazol (ptz, mg/kg, i.p.)induced kindling. rofecoxib was administered orally daily min before either ptz or vehicle. seizure severity was measured according to a prevalidated scoring scale. biochemical estimations were performed on the th day of ptz treatment. chronic treatment with rofecoxib ( . and . mg/kg, p.o.) for days showed significant decrease in ptz-induced kindling score. chronic treatment with ptz significantly increased lipid peroxidation, nitrite levels (no levels), and myeloperoxidase levels and decreased the reduced glutathione (gsh) levels in brain homogenate, which was reversed with rofecoxib treatment. research funds: university supportted study ashish dhir, shrinivas kulkarni uips, panjab university, chandigarh, india the objective of the present study was to elucidate the effect of cyclooxygenase inhibitors on pentylenetetrazol (ptz)-induced ( mg/kg) convulsions in mice with possible mechanism of action. various cox-inhibitors were administered min prior to the ptz administration. onset, duration of clonic convulsions and percentage mortality/recovery were recorded. pretreatment with cox-inhibitors aspirin ( and mg/kg, p.o.), naproxen ( and mg/kg, p.o.), nimesulide ( - mg/kg, p.o.) or rofecoxib ( - mg/kg, p.o.) dose dependently showed protection against ptz-induced convulsions. rofecoxib ( mg/kg) or nimesulide ( mg/kg) also enhanced the subprotective effect of diazepam or muscimol showing gabaergic modulation of cox- inhibitors. cox- inhibitors also antagonized the effect of flumazenil ( mg/kg) against ptz-induced convulsions further confirming the gabaergic mechanism. ps p-j cell proliferation after domoic acid-induced neuronal damage in adult rats domoic acid (da) is structurally related to kainic acid, which is a rigid analogue of the putative neurotransmitter l-glutamate that causes neuronal excitation. da-induced convulsions affects limbic structures such as hippocampus and entorhinal cortex. in this study we examined the neuronal damage after intraperitoneal da administration and cell proliferation in the adult rat brain. the most extensive neuronal cell damage was observed in ca subfield as evaluated by he staining, while tunel positive cells were mainly observed in the granular cells of cerebellum and dentate gyrus (dg) of the hippocampus. to elucidate the relations between damage and cell proliferation, we examined bromodeoxyuridine (brdu) labeled cells. brdu labeled cells were detected in dg and the granular cells of cerebellum. the cell proliferation was not associated with damage. ps p-j a-type potassium channel truncation mutation in temporal lobe epilepsy the role of voltage dependent calcium channels on the pentylenetetrazol (ptz) kindling induced learning deficits was investigated in rats. in this study animals were divided into three groups. in the test group verapamile were injected in the hippocampus ( mg/ min). after min kindling was established in rats with ptz. the control animals were the same age and undergone the same treatment in term of acsf injections and post-kindling waiting time as the kindled animals. and in sham group the animals received saline. one month after induction of kindling spatial learning and memory was tested by morris water maze. results showed that intra-hippocampal injection of verapamil significantly decreased spatial learning, suggesting that only working memory impaired but reference memory remain intact. the results with this study suggest that intera-hippcampal injection of verapamil significantly impaired spatial learning in rats. we showed that -oxoguanine ( -oxog) in mitochondrial (mt) dna and cellular rna increased significantly in the ca subregion of the mouse hippocampus after kainate administration. laser scanning confocal microscopy revealed that -oxog accumulated greatly in mtdna of the ca microglia. wild-type and mth -null mice, the latter lacking an ability to hydrolyze -oxo-dgtp and -oxo-gtp to the monophosphates to avoid their misincorporation into dna or rna, exhibited similar degree of the ca neuron loss after kainate administration, however, levels of -oxog accumulated in mtdna and cellular rna in the ca microglia were significantly increased in mth null mice in comparison to wild-type mice. we thus demonstrated that mth efficiently suppresses the accumulation of -oxog in both cellular dna and rna in the hippocampus, especially in microglia, caused by excitotoxicity. ps p-j transcription factor nrf regulates brain response to kainate-induced excitotoxicity yukihiko dan , kosuke kajitani , noriko yutsudo , ken itoh , masayuki yamamoto , yusaku nakabeppu kyushu univ., med. inst. bioreg., div. neurofunc. genomics, japan; univ. tsukuba, grad. sch. comp. hum. sci., japan nf-e related factor (nrf ) is the key transcription factor that serves to transmit the inducer signal to an antioxidant response element (are), a cis-acting element required for gene expression of a battery of proteins acting on anti-oxidative stress and detoxification of electrophiles. since loss of nrf has been reported to increase neuronal death under increased oxidative stress, nrf seems to play a role for neuroprotection. administration of kainite, a potent agonist of an excitatory neurotransmitter glutamate, to rodents produces epileptiform seizures followed by a delayed loss of pyramidal cells in the ca subregion of hippocampus. to unveil the functional significance of nrf in the brain, we compared seizure responses between wild-type and nrf -null mice after systemic kainate administration. we found that nrf -null mice exhibited an increased susceptibility to the kainate-induced seizure, and their loss of the pyramidal cells and gene expression profiles are now under investigation. ps p-j synaptic plasticity and -aminopyridineinduced epileptic discharges in rat hippocampal slices makoto otani, tetsuo furukawa, kiyohisa natsume department of brain science and engineering, kyushu institute of technology, kitakyushu, japan four-aminopyridine ( -ap) at the concentration below . mm suppresses k d channel and induces the epileptic discharges in rat hippocampal slices. in the present study, the involvement of the activation of nmda receptor on the ictogenesis of the -ap induced discharges in ca region was studied. ten m -ap induced the epileptic discharges with the frequency of . ± . hz (mean ± s.e.m.; n = ) and the amplitude of . ± . mv. when ap- , an nmda receptor blocker, was applied to the pre-established epileptic discharges, the frequency and the amplitude of the discharges did not change significantly. on the other hand, when ap- was applied from the ictogenesis period of the discharges, the discharges did not appear. these results suggest that the nmda receptor-dependent synaptic plasticity involves in the ictogenesis of -ap-induced epileptic discharges. chronic exposure of cultured astrocytes to morphine is reported to induce differentiation of the cells. using primary astrocyte cultures, we observed that under thyroid hormone (th) deficient conditions, morphine significantly decreased cell viability. further studies showed that the loss of cell viability was due to apoptosis of the cells. the effect is attenuated by th supplementation to the culture medium. the observed effect of morphine appears to be mediated through the opioid receptor since the opioid antagonist, naloxone, inhibited the decline in cell viability. ni, a specific inhibitor of nnos, completely blocked loss of cell viability suggesting that morphine induced intracellular no production, leads to cell death. studies suggested that no acts through a cgmp independent pathway. the involvement of no induced cgmp independent pathway in morphineinduced apoptosis during th deficiency has been investigated. collectively, the present study demonstrates that morphine mediated cytotoxicity of astrocyte is critically influence by the level of thyroid hormone in cultured medium. ps a-a influence of conductance-input signal and prior activation history on spike generation in rat somatosensory cortical neurons takashi tateno , hugh p.c. robinson engineering science, osaka university, osaka, japan; university of cambridge, uk in the cortex, a profusion of electrophysiological cell types, which form specific synaptic connections, is becoming apparent. a quantitative understanding of the dynamics of different cell types when responding to complex, natural inputs, is an important prerequisite for understanding the cortical network. neurons compute by transforming excitatory and inhibitory synaptic conductance inputs into a spike train output. we have examined the properties of synaptic conductance inputs which are most effective in evoking spikes, by injecting broad-band excitatory and inhibitory conductance inputs, and using spike-triggered reverse correlation and wiener-kernel estimation to calculate the average conductance input trajectory (acit) preceding spikes. the time course of the acit provides a general description of a neuron's response to dynamic conductance stimuli. our analysis showed that the acit reflects both previous stimulus history and previous discharge history, and that the relative influences of these two factors depend on the cell type. amyotrophic lateral sclerosis (als) is a rapidly progressive neuromuscular disease caused by the destruction of motor neurons. our study has investigated the effects of als-csf on voltage-gated calcium p/q-type channel (␣ a) expression in pre synaptic terminals of rat spinal motor neurons. csf from als and non-als (neurological patients) was injected into the -day-old rat pup spinal subarachnoid space at the rate of l/ . min. the rats were sacrificed h after csf injection and spinal cord sections were processed for immunocytochemistry with p/q-type channel ␣ a antibody and also for cytochrome oxidase labeling. als-csf significantly increased p/qtype channel expression compared to csf from non als patients. als-csf significantly decreased cytochrome oxidase activity in the rat spinal motor neurons, which may be a sign of degeneration. it is probable that, toxic factors present in the als patients csf might induce the expression of p/q-type channel observed in pre synaptic terminals synapsing on the spinal motor neurons. ps a-a on the membrane potential profile of ca pyramidal cells recorded with voltage sensitive dye imaging in rat hippocampal slices takashi tominaga , , yoko tominaga dept. neurophysiol., kagawa sch. pharmaceutical sci., tokushima-bunri univ., kagawa, japan; lab. for dynamics of emergent intelligence, riken bsi, hirosawa - , wako, saitama, japan integration of membrane potential response in a single neuron is a basis of neuronal calculation. we have been aiming to visualize this with voltage sensitive dye (vsd). hippocampal slices, with its unique laminar structure, allow us to assign optical signals to particular membrane fractions. but, it has not been clear whether the profile of optical signal could be a measure of membrane potential profile. to solve this, we visualized rather steady membrane potential change caused by perfusion of high potassium medium. a steep peak in optical signal was seen along stratum pyramidale. an application of ttx diminished this peak, and made the optical signal profile flat along the cell. thus, we concluded that the specificity of the vsd is small. with "neuron", by assuming a population nature to the optical signal, the membrane potential profile in a response to stimulation was successfully simulated. ps a-a overexpression of inwardly rectifying k + channel . in hippocampal slice culture masayoshi okada, hiroko matsuda department of physiology, kansai medical university, japan the expressions of mrnas for the inwardly rectifying k + channel (kir) . have been reported in mammalian central nervous system, but regulation of expression or its role in synaptic transmission remains unknown. in our rat hippocampal slice cultures, the endogenous kir current was hardly detected with whole cell recordings in the ca pyramidal neurons. then, egfp and kir . expressing virus vectors were constructed, and infected to the neurons in the slices. the vectors succeed to express the kir current, and the translocation of the fusion protein to the plasma membrane was also observed. furthermore, the overexpression significantly reduced the raise in whole-cell membrane potential evoked by depolarizing current injection, suggesting that kir plays a role of noise-filter for synaptic input in central neurons. takeshi otsuka, mieko morishima, yasuo kawaguchi div. cerebral circuitry & structure, nips, okazaki, japan layer pyramidal cells are heterogeneous in morphological and physiological properties, and project to multiple subcortical areas. although recent studies have addressed anatomical features of pyramidal cells identified projection regions, little is known about intrinsic membrane properties of these subtypes. here, we obtained whole cell recordings from rat frontal layer pyramidal cells that project to the striatum (ccs) or pontine nucleus (cpn), identified by injection of fluorescent retrograde tracer to these regions. firing properties of pyramidal cells had similarity depending on the projection regions. ccs cells showed strong adaptation of successive spike intervals in response to the depolarizing current injection. however, cpn cells exhibited very little spike frequency adaptation during current injection. we also examined synaptic inputs from layer / neurons to these subtypes by single cell stimulation, and detected excitatory inputs in both subtypes. our results suggest that physiological properties of layer pyramidal cells are correlated with their subcortical target. this study aimed to clarify expressional changes in types and of ryanodine receptors (ryr and ryr ) in the cerebellum of a ca + channel ␣ a subunit mutant, rolling mouse nagoya. semi-quantitative rt-pcr revealed altered mrna signal levels of ryr but not ryr in the rmn cerebellum: a less ryr mrna signals than in the control cerebellum. well consistent with the semi-quantitative rt-pcr results, ryr immunostaining in soma and primary dendrites of purkinje cells was less intense in rmn than in control mice. in contrast, ryr immunostaining was detected in cerebellar glomeruli but the staining intensity was not different between rmn and controls. the present study suggests that somatodendritic ryr expression in purkinje cells was decreased in the cerebellum of rmn. this may suffer ryr -mediated ca + release, contributing altered ca + homeostasis in the rmn purkinje cells. ps a-a dopamine-based modulation of lateral amygdala neuron excitability: a possible involvement of potassium current ryo yamamoto, yoshifumi ueta, noubuo kato integrative brain sci. med., kyoto univ., kyoto, japan the amygdala and dopaminergic innervation thereonto are considered to cooperatively regulate emotional states and behaviors. in the present slice experiments, we investigated the effects of dopamine (da) on lateral amygdala (la) neurons by whole cell recordings. application of da depolarized la neurons, reduced the action potential threshold, and induced slow afterdepolarization (sadp). this sadp was induced voltage dependently, and lasted for more than s. d receptor agonists induced the same sadp. previous reports have repeatedly suggested that sadp is triggered by calcium influx. consistently, calcium channel blockers or chelating intracellular calcium inhibited the present da-induced sadp. a membrane conductance decreased at the peak of sadp current (i sadp ). also, i sadp was suppressed by including cesium in the pipette solution. these results suggest that the present da-induced modulation of la neuron excitability may depend on a potassium current that can be masked by calcium influx. toru aonishi , , hiroyoshi miyakawa , masashi inoue , masato okada , tokyo institute of technology, japan; brain science institute, riken, japan; tokyo university of pharmacy and life science, japan; the university of tokyo, japan it has been reported that amplification of ap paired with epsp boosts the induction of ltp. there are two alternative hypotheses of such amplification mechanisms; one is activation of the na channel and other is inactivation of the a-type k channel. which is essential? in this talk, by mathematical analyses and the neuron simulator, we demonstrate that the balance of inward and outward currents, which can be controlled by down/up-regulation of the a-type k channel induces a divergence of the membrane input resistance, i.e. a singularity, and such super-sensitivity is the fundamental mechanism for boosting amplification of ap paired with epsp. the balance of na and a currents is essential for controlling dendritic integration manners. we also show that the down-regulation of the a-type k channel, which modifies the ratio between the inward and outward currents, leads to a drastic change from amplifying ap mode to shunting epsp mode. miharu komai , maya yamazaki , , mika tsujita , manabu abe , rie natsume , , kenji sakimura , department of cellular neurobiology, brain research institute, niigata university, niigata, japan; sorst/jst, saitama, japan we previously reported that stargazin family (␥ , ␥ , ␥ , and ␥ ) not only promoted ampa receptor surface expression but also modulated receptor activity and channel property (yamazaki et al., ) . therefore, we assumed these family proteins were auxiliary subunits of ampa receptors. to prove this hypothesis, we generated ␥ subunit knockout (ko) mice using the cre/loxp recombination system and analyzed their phenotypes. the ␥ subunit ko mice were viable, fertile, and displayed no overt phenotype. on the other hand, on western blot analysis, protein expression levels of ampa receptor subunits were reduced in ko mice compared with those in wild-type at postnatal day , while the reduction was not so significant in adult brain. these results suggested that ␥ might regulate dynamics of ampa receptor subunits during early development. in the cns, neural damages, such as hypoxia, ischemia and degenerating diseases, are often accompanied by disturbances in the ph environments. ambient ph plays as a significant signal for neural functions. microglia (brain phagocytes) express abundant voltagegated proton (hv) channels which have extremely high selectivity for h + and potent h + efflux ability. exposure to na-lactate (ph . ) induced cell acidosis and activation of the hv channels. the channel activation was characterized by increased conductance, facilitation of activation kinetics, prolongation of deactivation kinetics and a shift of the activation voltages to negative potentials. consequently, the hv channel could open more easily over a wide range of the membrane potential during lactic acidosis, and may contribute to a quick relief of the cell acidosis. mari sasaki, masahiro takagi, yasushi okamura okazaki institute for integrative bioscience, aichi, japan here we report a novel four transmembrane protein similar to the voltage sensor domain (vsd) of the voltage-gated channels that exhibits activities of a voltage-gated proton channel. voltage-gated proton channel currents have classically been described in snail neurons and recently in mammalian blood cells. however, the molecular basis underlying this channel has been elusive. here we identify a novel cdna clone named as mouse voltage-sensor domain only protein (mvsop ). cells overexpressing this protein showed depolarization-induced outward currents accompanied by tail currents during repolarization, which reversed at equilibrium potentials for protons. imaging analysis demonstrated that phin recovers rapidly after an acid load in mvsop -transfected cells. mvsop induced currents exhibited two key features of native voltage-gated proton channels: ph-dependent gating and zn + sensitivity. neutralization of a positive charge in the s -like segment caused shift of the voltage-conductance relationship, suggesting that it plays important role in gating. oscillatory extracellular electric fields have been observed in mammalian brains. the electric fields modulate neuronal excitability and synaptic events. to investigate the effect of the oscillatory electric fields on the ca pyramidal neuron, we applied sinusoidal electric fields to the rat hippocampal slice and recorded voltage responses with a voltage sensitive dye (rh ). application of sinusoidal electric fields induced transmembrane voltage oscillations in all the layers of the ca region. in the pyramidal layer, the amplitudes of the responses to the -hz field were the largest. the amplitudes were decreased monotonically when the frequency of the fields became higher. however, in the stratum radiatum, the amplitudes of the responses to the - -hz fields were larger than those to the other frequencies. the frequency preference in the dendritic region may be an underlying mechanism for the synchronization of the membrane potentials among large population of neurons within the theta frequency range. acid sensing ion channels (asic ) have proposed to constitute mechanoreceptors and nociceptors. we examined the localization and characterization of asic -expressing cells in rat central nervous system (e -p ) using immunohistochemical techniques. asic positive fiber first appeared in brain stem and spinal cord at e - stage. asic -expresseing cells appeared in white matter of brain stem and spinal cord at e stage. in early postnatal stages asic expressing cells appeared in corpus callosum, cerebellar medulla and dorsal horn of spinal cord at p stage. these cells were identified as an oligodendroglia by oligodendrocyte specific antibody and immunoelectron microscopy. these results are suggested the hypothesis that the function of asic mediate the myelin formation in the developmental stages of central and peripheral nervous system. masato shino, seiji ozawa, yasuhiko saito department of neurophysiology, gunma university graduate school of medicine, maebashi, gunma, japan nucleus prepositus hypoglossi (nph) is involved in horizontal eye movement. previously, we found nph neurons exhibiting a characteristic firing pattern in response to depolarizing current pulses (fil neurons). fil neurons exhibited a spike train with a long first interspike interval ( st isi) that is attributed to a large, slow hyperpolarization (ahp) after the first spike. in this study, we investigated ionic conductances underlying the long st isi by whole-cell recordings in rat slices. application of m apamin, an sk-type ca + -activated k + (kca) channel blocker, shortened the st isi and decreased the amplitude of the slow ahp. the shortening of the st isi was observed when membrane potentials were depolarized. moreover, application of m mibefradil, a t-type ca + channel blocker, shortened the st isi. these suggest that the firing pattern of fil neurons arises from activation of sk-type kca channels induced by ca + influx through t-type ca + channels. research funds: kakenhi (c) ( ) jafar vatanparast , , mahyar janahmadi , houri sepehri , ali haeri-rohani , ali reza asgari neuroscience research center, shaheed beheshti medical sciences university, tehran, iran; dept. of biology, university of tehran, tehran, iran the roles of the ionic channels and muscarinic receptors in paraoxon (px) induced burst firing in snail neurons were studied using current clamp method. px ( . m, within min) increased the frequency of spikes and shortened ahp. slow waves of depolarization with superimposed bursts were recorded within min. atropine blocked the depolarization shift but not the other effects of px. px was able to reversibly decrease the duration of calcium spikes elicited in a na + free ringer. this effect observed in the presence of atropine and was along with a decrease in the duration of ca + spike ahp and an increase in the spike frequency. the px blockade of ca + channels may decrease the activation of ca + dependent k + channels that underlies ahp. blockade of these channels possibly makes the neurons susceptible for burst induction, while activation of metabotropic muscarinic receptor by px underlies the depolarization shift with associated bursts. dendritic membrane properties are reported to be non-uniformly distributed in a single neuron and the non-uniformity could be important for synaptic integration. however their distribution is still unclear. we estimated distribution of membrane resistance by fitting a compartment model to voltage imaging data of membrane response in hippocampal ca slices to perturbation, such as propagating epsp induced by synaptic inputs and biphasic response to extracellular electric field. by numerical simulations, we found that these imaging data were consistently reproduced if we assume a step function as distribution of membrane resistance. this implies that a steep decrease of membrane resistance exists in distal dendrite of hippocampal ca pyramidal neuron. it is known that cooling-induced desensitization of cold receptors, however, its intracellular mechanism has remained unresolved. in this study, we analyzed molecular mechanism of desensitization of cold/menthol receptors (trpm ). repeated menthol application induced trpm desensitization. this desensitization was depended on extracellular ca + , indicating that involvement with ca + -dependent kinase. pkc activator (pma) desensitized trpm and go (pkc inhibitor) abolished pma-induced trpm desensitization. pma similarly desensitized wild type trpm and mutant trpm , in which serine or threonine residues in some putative pkc phosphorylation sites were replaced by alanine. pma treatment did not induce internalization of trpm . as the basis of cooling-induced desensitization of cold receptors, we conclude that cooling-activated trpm causes pkc to desensitize trpm itself. yosuke sawada , hiroshi hosokawa , kiyoshi matsumura , shigeo kobayashi dept. of int. sci. and tech., grad. sch. of info., kyoto univ., kyoto, japan; dept. of info. sci. and tech., osaka institute of technology, osaka, japan cooling below • c evokes cold pain sensation. however, the molecular basis of the cold pain sensation is still unknown. trpa is activated by pungent compounds stimuli. if trpa responded to cooling to noxious cold range, it could be candidate for evoking cold pain sensation. however, whether trpa is activated by cooling or not is still controversial. here, we investigated that trpa -expressing hek cells responded to noxious cold stimuli. whole-cell recording demonstrated that cooling below threshold evoked inward current. threshold temperature was . ± . • c. in inside-out singlechannel recording, cooling activated trpa directly. single channel conductance was . ± . ps. single channel currents showed inword rectification. in conclusion, trpa is the cooling activated cation channel. yoshiki matsuda, foong yen ang, jinsun yoon, noriko ebisu, satoshi takahashi, shinichi kogure dept. bioengin., soka university, tokyo, japan hyperplarization-activated and cyclic-nucleotide-gated nonselective cation channels (hcn - ) have been demonstrated in the cns. since they contribute to various physiological functions including neuronal pacemaking activity, setting of resting membrane potential and generation of paroxysmal discharge, we examined their expressions as well as functions in the pns using the frog (xenopus laevis) sciatic nerves. western blot analyses for hcn - demonstrated that samples from the nerve and the heart showed an hcn band whereas those from the dorsal part of skin and the gastrocnemius did not, and that immunoreactivities for hcn , hcn and hcn could not be found in those samples. when an hcn channel blocker, zd was applied on the stimulus portion of sciatic nerve and the nerve was elicited at . /s by a duration of ms pulse with supramaximal intensity, the generation of anode-break-excitation rather than cathode-makeexcitation was significantly blocked (p < . ). it is suggested that hcn channels exist in the pns and they contribute to the burst or recurrent discharges. ifenprodil, a clinically used cerebral vasodilator, interacts with several receptors, such as ␣ adrenergic, n-methyl-d-aspartate, serotonin and receptors. however, the molecular mechanisms underlying the various effects of ifenprodil remain to be clarified. here, we show that ifenprodil inhibited g protein-activated inwardly rectifying k + (girk; kir ) channels, which play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate, expressed in xenopus oocytes. in contrast, kir . and kir . channels in other kir channel subfamilies were insensitive to ifenprodil. the girk currents induced by -opioid receptors or ethanol were also attenuated in the presence of ifenprodil. the inhibitory effects of ifenprodil were not observed when ifenprodil was applied intracellularly. our results suggest that inhibition of girk channels by ifenprodil, at submicromolar concentrations or more, may contribute to some of its therapeutic effects and adverse side effects. ps a-b proliferation of rat c glioma cells is controlled by the concentration-sensitive na + channel (na c ) shigeru yoshida, hiroyuki yamaguchi, takashi takeuchi, hokuto tanaka, yoshiyuki morimoto, teruki hagiwara department of life science, kinki university, higashi-osaka, japan the role of na + as a regulator of cell growth was studied using the tumor cell line (c ), which has a large quantity of concentrationsensitive na + channels (na c ; c = concentration). cell proliferation was suppressed when [na + ] o was raised from control ( mm) to or mm. an increase in [na + ] i was revealed by an image processor in c cells loaded with a na + indicator (sbfi), under high [na + ] o conditions. [na + ] i elevation was augmented by ouabain or by bumetanide (na + /k + /cl − cotransporter blocker), while it was decreased when na c expression was inhibited by rnai techniques. the real-time pcr method revealed that the expression level of the immediate early gene egr- , which is involved in cell growth, was concomitantly reduced. it is to be noted that similar alterations in cell growth, egr- level and [na + ] i were induced by a na + ionophore (monensin) without raising [na + ] o . these data indicate that na + enters through na c upon [na + ] o increase, and [na + ] i elevation itself is responsible for these phenomena. hiroshi kuba, takahiro ishii, harunori ohmori dept. physiol., univ. kyoto, kyoto, japan na + channels are concentrated in the axon to generate action potentials. however, little is known about how distribution of na + channels contributes to the activity and function of single neurons. in avian nucleus laminaris (nl), neurons act as coincidence detectors for sound source localization, and are tuned to both characteristic frequency (cf) and interaural time difference (itd) of sounds. we show here in the chick that nl neurons have distinct distribution of na + channels along the axon and optimize the itd sensitivity depending on their cf. neurons of high and middle cf (higher than khz) had small action potentials, and had no na + currents in the somatic membrane, but clustered only in the axon at some distance from the soma ( - m). while, neurons of low cf generated large overshooting spikes, and na + channels were clustered closer to the soma ( m) in the axon. thus, nl neurons had a spike generator on the axon, at a greater distance from the soma with the increase of cf. by computer simulation, these unique distributions of na + channels were found essential to enhance the coincidence detection. research funds: kakenhi ( ) il-sung jang , in-sun choi , eun-ju park , jin-wha cho , man-gee lee , byung-ju choi kyungpook national university, school of dentistry, south korea; kyungpook national university, school of medicine, south korea bisphenol a (bpa), an endocrine disrupter, is contained in cans, polycarbonate bottles and some dental sealants. here we report the effect of bpa on gaba a receptors using a conventional whole-cell patch clamp technique from acutely isolated rat ca pyramidal neurons. bpa itself elicited a postsynaptic current, which is highly sensitive to bicuculline, in a dose-dependent manner. bpa increased postsynaptic currents induced by gaba at lower concentrations (< m), but decreased those induced by gaba at higher concentrations (> m). in addition, bpa decreased both the current amplitude and decay time constant of gabaergic mipscs. finally, mechanisms underlying bpa-induced modulation of gaba a receptors will be discussed. we recently generated nav . -deficient mice and showed that these mutant mice developed epileptic seizures and died prematurely. we have now used these nav . -deficient mice as negative controls to examine nav . distribution in the mouse brain using rna in situ hybridization histochemistry and immunohistochemistry. at low magnification, nav . expression was higher in the thalamus, superior colliculus, inferior colliculus, pons, medulla and cerebellar nuclei relative to other brain regions. contrary to previous studies indicating a somato-dendritic nav . distribution, in the present study, higher magnification analysis revealed that nav . is predominantly distributed to axons in some brain parts. this apparent discrepancy may reflect the lack of specificity of anti-nav . antibodies used in these previous studies, none of which utilized nav . -deficient mice. based on our findings, we propose that nav . might be involved in propagating action potential to presynapses. keiji miura , , masato okada , , , shun-ichi amari department of physics, kyoto university, kyoto, japan; "intelligent cooperation and control", presto, jst, japan; department of complexity science and engineering, university of tokyo, chiba, japan; brain science institute, riken, saitama, japan we considered a gamma distribution of inter-spike intervals as a statistical model for neuronal spike generation. a gamma distribution is a natural extension of poisson process and it can generate spike trains with various irregularities. the model parameters consist of a time-dependent firing rate and a time-independent spiking irregularity. because the environment changes over time, the firing rate varies for each interspike interval. we used a novel method of information geometry to estimate the spiking irregularity whatever the functional form of the firing rate is. our estimator is simple and easily applicable to experimental data. the estimator is useful for characterizing spiking irregularity which varies among neuron types. it may be possible to classify neurons into functional groups according to their spiking irregularities. research funds: grant-in-aid for scientific research (nos. and ) mitsuyo watanabe, yuko ishimaru, taketo nakadai, tomoyuki kanamatsu graduate school of bioengineering, soka university, tokyo, japan we examined the effect of colchicine, inhibitor of axonal flow, on cerebral amino acid metabolism in the rat. the rats were injected with [ - c] glucose intravenously ( g/kg) or h after the intraventricular injection of colchicine ( g/ l) and the amino acid fractions were extracted from the brains at , or min after the glucose injection. the amount of [ - c] glucose in the cerebra was increased, however, the c incorporation into glutamine, glutamate, gaba and aspartate from [ - c] glucose were decreased. only glutamine concentration in all amino acids was increased in the cerebra of the colchicine group, compared to those values in the control group. the microdialysis analysis showed that the amount of gln in the dialysate was increased by three times in the colchicine group compared with the control group. these data may suggest that the glycolysis of glucose is decreased and that the influx of glutamine from blood to brain occurs with neuronal dysfunction induced by colchicine. these results indicate that a ␤ alters the bhlh gene expression in neural stem cells toward cell death. ken kojima, akiko nishida, shinji takebayashi, jyuichi ito department of otolaryngology-head and neck surgery, graduate school of medicine, kyoto university, japan basic helix-loop-helix (bhlh) transcription factors play crucial roles in development of the central and peripheral nervous systems. to visualize expression of hes or hes gene, phes -and phes -egfp transgenic (tg) mice were generated (ohtsuka et al., ) . in each transgenic mouse, a promoter of hes or hes gene drives enhanced green fluorescent protein (egfp) gene. in the inner ear, it is suggested that hes or hes regulate cell division and differentiation of sensory and supporting cell progenitors via notch signaling pathway. by use of immunohistochemical technique, we examined distribution of gfp expressing cells in the inner ear of the transgenic mice from embryonic day (e ) to postnatal day (p ). in the phes -egfp tg mouse inner ear, gfp immunoreactive (gfp-ir) cells were detected from e to p . in the phes -egfp tg mouse inner ear, gfp-ir cells were observed from e . to p . gfp-ir cells in phes -gfp tg mouse are candidates of sensory cell progenitors in mature mammalian inner ear. ohtsuka et al., . mol. cell neurosci. ps a-c expression of zfh- in the developing mouse brain: mrna, antisense rna and protein expression yuriko komine , kenji nakamura , motoya katsuki , tetsuo yamamori national institute for basic biology, okazaki, japan; mitsubishi kagaku institute of life science, machida, japan zfh- is a transcription factor containing three homeodomains and zn fingers and expressed in differentiating neurons. we have reported that the level of zfh- mrna is negatively regulated by antisense transcripts of the zfh- gene. in several types of neurons, including pyramidal cells in the hippocampus and granule cells in the cerebellum, the zfh- antisense rna is expressed prior to the mrna; as the level of the antisense rna gradually decreases, zfh- mrna starts to be expressed. recently, we have raised an antibody against mouse zfh- and examined the expression profile of the zfh- protein. in the most regions of the brain, the protein expression pattern consisted with that of mrna. however, in the several types neurons mentioned above, zfh- protein was not detected even when the zfh- mrna was already expressed. this observation together with other data suggested that the zfh- protein level is regulated by several mechanisms including suppression by the antisense rna and translational control. takashi inoue , maya ota , katsuhiko mikoshiba , jun aruga laboratory for comparative neurogenesis, riken bsi, saitama, japan; laboratory for developmental neurobiology, riken bsi, saitama, japan zic family zinc-finger proteins play various roles in animal development. in mice, five zic genes (zic - ) have been reported. despite their partially overlapping expression profiles, mouse mutants for each zic gene show distinct phenotypes, suggesting the functional redundancy of zic proteins. it is expected that the common and specific roles of mouse zic proteins can be clarified by studying compound mutant mice. in the present study, we characterized zic /zic compound mutant mice. mice carrying homozygous zic mutant allele together with zic null allele showed defects in midline structures, including abnormalities in forebrain and thalamus. especially, the compound mutants showed severe anatomical abnormalities in the dorsal and ventral telencephalon and olfactory system, which are not obvious in either zic -or zic -single mutant. these observations indicate that zic , in cooperation with zic , have an essential role in controlling proliferation and differentiation of the neuronal projenitors in the medial telencephalon. chiaki maruyama, haruo okado department of molecular physiology, tokyo metropolitan institute for neuroscience, japan rp , a novel zinc finger protein containing a poz domain, functions as a sequence specific transcriptional repressor. rp gene disrupted mice show severe abnormalities in brain cortical layer formation, suggesting that rp has a crucial role in cerebral development. to understand the role of this protein in brain development, we examined rp gene expression in mouse embryo and adult brain by in situ hybridization. as a result, we found that rp transcripts are first detected at embryonic day in the neuroepithelium of the spinal cord and telencephalic vesicle. in the day - embryos, rp transcripts are predominantly observed in the preplate region but not in outside the nervous system. at e , rp transcripts were detected throughout the neocortex and hippocampus, but not in the thalamus and striatum. in the cortex, the transcripts were detected primarily in cortical neurons, but not in the marginal zones and ventricular zone. in adult mice, rp is expressed in neocortical and hippocampal neurons and granule cells in the cerebellar cortex toshiki kameyama, fumio matsushita, yuzo kadokawa, tohru marunouchi division of cell biology, fujita health university, toyoake, japan neural zinc finger (nzf) proteins are transcription factors with dnabinding domains of c hc-type zinc finger motifs. using p cells, we demonstrated that nzfs were expressed transiently during neuronal differentiation, and forced expression of nzf cdnas resulted in neuronal differentiation. these results suggest that nzf family have a function regulate neuronal differentiation. to elucidate in vivo functions of nzf family in detail, we generate knockout mice of nzf- and nzf- respectively. nzf- null mice are born alive, but die within min after birth with cyanosis. on the other hand, nzf- null mice are viable, fertile and appear normal. these mice look normal morphologically. then we generate double knockout mice of nzf- and nzf- by intercrossing. double knockout mice have a forelimb posture abnormalities like arthrogryposis multiplex congenita. and we find out that the spinal nerves projecting forelimb and trunk are decrease dramatically in the double knockout mice embryo. , ) . in the present study, to examine the role of runx in the development of drg in more detail, we examined the development of drg neurons in runx -deficient mice from the early embryonic stages to birth, using various markers for subpopulation of drg neurons. in newborn runx −/− mice, parvalbumin-positive drg neurons were greatly reduced in number, whereas calretinin-positive neurons were slightly decreased. similar decreases were observed in embryonic days . and . . shin hisahara , , susumu chiba , hiroyuki matsumoto , yoshiyuki horio department of pharmacology, sapporo medical university, sapporo, japan; department of neurology, sapporo medical university, sapporo, japan in mammalian cns, the function of histone deacetylase sirt is still unclear. recent studies indicated that sirt interacts with nuclear receptor co-repressor (n-cor) and n-cor represses intracellular domain of notch-icd activation of the hes promoter. we performed overexpression of sirt and n-cor in neurosphere by nucleofection, then induced differentiation. we found remarkable promotion for neural differentiation by overexpression of sirt and n-cor in the sirt with n-cor. sirt and n-cor suppressed hes transcription by notch-icd in the luciferase assay. hes transcription was suppressed in overexpression of sirt and n-cor, suggesting that interaction between sirt and n-cor represses hes transcription. consistent with this, chip assays revealed that not only n-cor but also sirt bind to the promoter of hes gene. taken together, these results indicate that sirt and n-cor accelerate neural differentiation of the undifferentiated cells via binding hes promoter site and repressing hes transcription. yasushi maruyama , mitsuhiko kurusu , masataka okabe , katsuo furukubo-tokunaga grad. school life and envir. sci., univ. tsukuba, japan; natl. inst. genetics, mishima, japan; inst. dna medicine, jikei univ. school of medicine, japan during brain development, a large number of neurons are generated by proliferation of neural stem cells. with a characteristic proliferation mode that persists through development, the neuroblasts of drosophila mushroom bodies (mb) provide an attractive model system to study mechanisms of neural stem cell proliferation. here we show that tailless (tll), a member of the orphan nuclear receptor super family, has a crucial function in maintaining cell cycle progression of the mb neuroblasts. mosaic analysis demonstrates that cell autonomous activity of tll is crucial for maintenance of the mb neuroblast cell cycles. moreover, gain-of-function analyses confirm instructive functions of tll in maintaining neuroblast activity. we propose that tll plays pivotal roles in proliferation of the mb neuroblasts and suggest a conserved mechanism of neural stem cell control with the tll/tlx homologs in both drosophila and vertebrate brains. kouji senzaki, masaaki yoshikawa, shigeru ozaki, takashi shiga graduate school of comprehensive human science, university of tsukuba, ibaraki, japan runx family transcription factor is an important component of tgf-␤ and bmp signaling. we reported previously that runx mrna is expressed in the dorsal root ganglion (drg) from the early developmental stages, and that runx regulates axonal projection of trkcexpressing proprioceptive drg neurons (inoue et al., ) . furthermore, we announced previously that runx mrna is expressed in cranial ganglia of v, vii, viii, ix and x in mouse developmental stages. the expression was restricted to subset of neurons in each ganglion. to examine the influence of runx on the differentiation of trigeminal ganglion neurons, we investigated the expression of neurotrophin receptors, calcium binding proteins and neuropeptides in trigeminal ganglia of runx knockout mice using immunohisitochemical staining. we found the decrease of trkc-expressing neurons in trigeminal ganglia of neonatal runx knockout mice, however, we observe little change in the proportions of nuen-expressing neurons. kouko tatsumi , hirohide takebayashi , takayuki manabe , kazuhiro ikenaka , akio wanaka dept. anatomy, nara med. univ., kashihara, nara, japan; division of neurobiology and bioinformatics, nips, nins, okazaki, aichi, japan our previous study with double labeling of brdu and cell lineage markers suggested that a number of astrocytes were differentiated from resident oligodendrocyte progenitor (opcs)-like cells in the injured adult brain. and we found out that these opcs expressed ng proteoglycan and olig at early phase after injury. to directly trace the lineages of these opcs, we employed double transgenic mice that express tamoxifen-sensitive creer under the control of the olig promoter together with rosa-egfp reporter. the gfp positive cells were detected around the injured region, and the almost all of these cells co-expressed gfap at late phase after injury. furthermore, we confirmed that the morphological characteristics of these cells were those of the astrocyte by immunoelectron microscopy. our results clarified that dormant opcs in vivo differentiate into astrocytes in adult injured brain, and suggested that these cells participate in glia scar formation after brain injury. olig is a bhlh transcription factor, essential for oligodendrocytes (ols) and motoneurons differentiation in the spinal cord. however, differentiation of olig lineage cells in the forebrain is largely unknown. here we examined fates of olig expressing cells in the fetal forebrain by tamoxifen (tm)-inducible cre-loxp system. olig -creer knockin mice were mated with reporter mice, and tm was injected at embryonic day (e) . or . , when most of olig + cells are observed in the basal forebrain. the olig + cells at e . gave rise to more neuron than glia that included both ols and astrocytes. majority of neuronal olig lineage cells differentiated into gabaergic neurons, and a lesser number, into cholinergic neurons. the olig + cells at e . generated more glial cells than neurons. these results indicate that olig lineage cells generate three major types of neural cells with a stage dependent manner, and may have multiple functional roles on neural differentiation in the fetal forebrain. mana igarashi , , masato yano , , satoru hayashi , , hirotaka j. okano , , hideyuki okano , dept. physiol., keio univ. sch. med, tokyo, japan; sorst jst, japan the mammalian neuronal hu rna binding protein family is homolog of drosophila elav protein which is essential for differentiation and maintenance of the nervous system. in mammals, neuronal hu expresses in both early postmitotic and mature neurons and has ability to induce neuronal differentiation by binding to the utrs of specific target mrnas. to understand the molecular mechanism of hu induced neuronal differentiation, we purified hub associated complexes. among them, nf family, a double strand rna binding protein which is one of hu associated proteins, is known to bind to utrs of p , p and tau mrna known as hu targets. we generated rabbit polyclonal antibodies against nf and nf , binding partner of nf , respectively. in mouse embryonic brains, we found that nf / expressed highly in postmitotic neurons where neuronal hu proteins are highly distributed. moreover, we found that hu and nf / formed mrnp complexes in mouse brain extracts. we will discuss the role of hu binding partners in neuronal differentiation through post-transcriptional regulation. sachiko the pallium is specified as a homologous field in the vertebrate telencephalon. however, little is known about how species-specific pallial structures are generated during embryogenesis. to address this issue, we compared several neuronal subtypes and their migration patterns in the developing pallium of the mouse and quail. cell tracing analysis revealed that neurons born at the dorsal pallium tangentially migrated in the developing quail telencephalon, as in the mammalian cortex. next we investigated distribution of later-born neurons in the quail telencephalon using laminar specific genes (er and brn ) in the cerebral cortex. in situ hybridization and immunohisitochemical studies indicated that these neuronal markers were expressed in discrete regions of the developing quail telencephalon. our data suggest that early stages of cortex/pallium development are comparable between the mammalian and avian embryos, whereas neuronal specification in later stages is regulated by distinct mechanisms in each species. research funds: kakenhi ( ) ps a-d protein expression in hippocampal cells dissociated and re-cultured from organotypic slice cultures we established a re-cultivation technique of hippocampal cells dissociated from long-term cultured organotypic slices. protein phenotype of the cells was analyzed using immunocytochemical technique. antinestin immunoreactivity was observed in cells with short processes days in the re-cultivation. the anti-nestin immunoreactivity was progressively declined, whereas number of cells expressing anti-␤iii tubulin immunoreactivity increased through the re-cultivation for - weeks. presence of neurons, astrocytes and oligodenderocytes was examined using anti-␤iii tubulin, anti-glial acidic fibrillary protein and rip antibody, respectively. apart from the cells expressing one of the markers, the cells marked with multiple sets of antibodies were observed. these results suggest that protein expression was changed backward in normal differentiation course in hippocampal cells once matured in organotypic slices. we have shown that perineuronal ng + cells are major populations of proliferating cells in the cerebral cortex of rats. in the adult cortex, ng is known as a marker for oligodendrocyte progenitor cells (opcs) that retain ability to proliferate and differentiate into new oligodendrocytes. however, it is still unclear whether all ng + cells in the neocortex are the opcs. we investigated about subtypes of ng + cells found in the perineuronal regions of the cerebral cortex using cell markers. two subtypes of perineuronal ng + cells could be distinguished by the subcellular localization of gst-protein. one is nuclear type, the other is cytoplasmic type. only the nuclear gst-+ cells have the proliferative activity. these data suggest that the nuclear gst-+ /ng + cells in the perineuronal territory are progenitor cells engaging in reproduction of cortical cells. muguruma keiko, su hong-lin, matsuo-takasaki mami, watanabe kiichi, sasai yoshiki neurogenesis and organogenesis group, riken center for developmental biology, kobe, japan in this study, we report in vitro generation of math + cerebellar granule cell precursors and purkinje cells from es cells by using soluble patterning signals. when neural progenitors induced from es cells in a serum-free suspension culture are subsequently treated with bmp and wnt a, a significant proportion of these neural cells become math + . the induced math + cells mitotically active and express markers characteristic of granule cells precursors (pax , zic , and zipro ). after purification by facs and coculture with postnatal cerebellar neurons, es cell-derived math + cells exhibit typical features of neurons of the external granule cells layer, including extensive motility and a t-shaped morphology. interestingly, differentiation of l + /calbindin-d k + neurons (characteristic of purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by fgf rather than by wnt a. this is the first report of in vitro recapitulation of cerebellar neurons by using the es cell system. sachiyo misumi, kim hye-jung, hideki hida, hitoo nishino department of neurophysiology and brain science, nagoya city university graduate school of medical science, nagoya, japan regulation of the cell cycle plays an important role in cell proliferation, differentiation, and apoptosis. we have shown that pretreatment with cell cycle blocker increase the number of neurons from neural stem or progenitor cells (npcs) without influencing apoptosis after differentiation. in this study, we investigate the molecular mechanism of neuronal differentiation by cell cycle arrest. in rt-pcr, the expression of p cip , p kip and p kip mrnas were elevated during differentiation to neuron from npcs. especially, prolonged enhancement of p kip mrna was shown. transfection of p kip into npcs induced activation of neurod promoter and increase of number of ␤tublin iii-positive cells. treatment with deferoxamine to npcs from e . rat midbrain and hb .f cell line did not activate erk and akt phosphorylation during the treatment. date suggest that prolonged p kip elevation is related to enhanced production of neuron from npcs, and that cell cycle regulation in g /s phase did not activate mapk and pi -k signaling. yuichi tanaka , yusuke tozuka , dai muramatsu , kin-ichi nakashima , tatsuhiro hisatsune departement of integrated biosciences, graduate school of frontier sciences, university of tokyo, kashiwa, japan; graduate school of biological sciences, nara institute of science and technology, ikoma, japan we previously reported no definite evidence for in vivo neurogenesis in adult neocortex. however, we also confirmed dividing cells in this area. in this study, we analyzed the characteristics of adult cortical nestin+ cells. in vivo, they belonged to ng + and olig + cells, showed slowly proliferating ability compared to those in adult dentate gyrus. for in vitro analysis, we precisely isolated progenitor cells by percoll gradient procedure. they differentiated into tuj- + or map- + neuronal cells by adding retinoic acid or bdnf. more than % of newborn neurons expressed gabaergic neuronal markers, gaba, gad or calretinin. we also purified nestin-gfp+ cells from nestin-gfp transgenic mice using the facs system, and confirmed their neuronal potential. moreover, integration of a neural bhlh transcription factor neurod significantly promoted this neurogenesis. we demonstrated neurogenic potential of adult cortical nestin+ cells. mie gangi , michiko imanishi , teiko kuroda , masao tachibana , masahiko takada department of psychology, graduate school of humanities & sociology, university of tokyo, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo metropolitan organization for medical research, tokyo, japan a kv subfamily of voltage-gated k + channels is thought to play an important role in high-frequency repetitive firings. it is unknown which subtype of kv channels is expressed in the frog retina where ␥-range oscillatory spikes are evoked presynaptically by light stimulation. we found immunohistochemically that kv . b and kv . were expressed both in the mouse and frog retinas. however, a laminar pattern with two bands in the inner plexiform layer was displayed by kv . in the frog retina and by kv . b in the mouse retina. it has been shown that mammalian cholinergic amacrine cells express kv . b. thus, the differential expression of kv channels may reflect their functional diversity between the frog and mouse retinas. hiroshi jouhou , , kazunori yamamoto , masayuki hara , akinori homma , akimichi kaneko , masahiro yamada tokyo metropolitan univ., hino, tokyo, japan; astellas pharma. inc., osaka, japan; sch. rehabili., seijoh univ., aichi, japan in order to interpret the formation of receptive field surrounds in the retinal neurons, hirasawa and kaneko ( ) proposed a phmediated mechanism to substitute for the gaba-mediated feedback hypothesis from horizontal cells (hcs) to cone photoreceptors. to verify the idea that the depolarized hcs release protons we measured, by a fluorescent ratio imaging technique, the ph of the immediate external surface (ph o ) of hcs isolated from carp or goldfish retina. when hcs stained by -hexadecanoylaminofluorescein, a phsensitive lipophilic dye, were depolarized by application of kainate or by high extracellular k + , ph o acidified. the amount of ph o acidification was monotonically dependent on the amount of depolarization, as much as . ± . ph unit by mm k + . acidification of pho was suppressed by . m bafilomycin a , a specific inhibitor of v-atpase, suggesting that the hc depolarization enhanced an outward proton movement by the outward electrogenic h + pump. ps a-e analysis of spread of activity in the local circuit of superior colliculus by using multi-channel field potential recording system penphimon phongphanphanee, katsuyuki kaneda, tadashi isa national institute for physiological sciences, japan to study how the visual signal is processed in the local circuit of superior colliculus (sc) from the superficial layers (ssc) to the deeper layers (dsc), we analyzed the propagation of excitation following the electrical stimulation of the ssc by using a planar -channel field potential recording system in slice preparations obtained from to days old mice. stimulation at ssc induced negative field potential with short latency and short duration ( - ms) at the recording site in ssc adjacent to the stimulating site. after application of bath containing m bicuculline, the same stimulus induced a large negative field response with long duration ( - ms) that spreads laterally in ssc and ventrally to dsc. these responses disappeared after application of m apv, when only short latency response remained. the results suggest that when gaba a receptormediated inhibition is reduced, visual signal in the ssc propagates to the dsc as large response with long duration and nmda receptors contribute to propagation of the response. osamu hosoya , ken tsutsui , kimiko tsutsui dept. of neurobiol. and neuroanat., okayama univ. grad. sch. of med., dent., and pharm. sci., japan; dept. of genomics and proteomics, okayama univ. grad. sch. of med., dent., and pharm. sci., japan amphiphysin ir (amph ir) is alternatively spliced variants of amphiphysin i which is specifically expressed in retina. amph ir is composed of conserved domains including the n-terminal bar, the central clathrin/ap- binding, and the c-terminal sh domains and the variant specific two novel insertions (a and b). insert a may be a determinant for the retina-specific expression. insert b has no significant homology to known proteins and two shorter transcripts with -truncations in the insert were also expressed. recently, we found that a human retinal pigment epithelia cell line, arpe- , also expresses amph ir. arpe- thus can be a useful tool for investigating the cellular function of amph ir in retina. immunofluorescence analyses with arpe- cells revealed that amph ir occasionally colocalized with mitochondria, raising the possibility that amph ir may participate in structural or functional organization of mitochondria. further characterization of the variant is under investigation. hironori takamura , satoshi ichisaka , chihiro hayashi , hirotoshi maki , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan monocular deprivation (md) induces significant plasticity in the primary visual cortex (v ) during critical period. it was reported that inhibition of extracellular signal-regulated kinase (erk) activity in the visual cortex suppressed the ocular dominance plasticity. if erk is involved in the mechanism of this plasticity, visual deprivation would change the activity of erk in v and such change might be induced only in the critical period. to test this possibility, we examined effects of md on the amount of phosphorylated (activated) erk (perk) in the rat visual cortex. by md, we found a significant decrease in the amount of perk in v receiving deprived eye inputs in both young and adult rats. as to the subcellular localization of erk, we found a significant increase of the nuclear perk only after md in young rats. these results suggest that erk signaling might be regulated by different mechanism between young and adult rats. research funds: kakenhi ( ) ps a-e rapid pre-synaptic weakening by experiencedependent competition in mouse visual cortex nobuko mataga, yoko mizuguchi, takao hensch neuronal circuit development, riken brain science institute, saitama, japan in the binocular zone (bz) of mouse visual cortex, critical period (cp) plasticity is accompanied by a transient loss of spines on pyramidal cell dendrites. to explore a correspondingly rapid and local pre-synaptic refinement by sensory deprivation, excitatory intracortical or thalamocortical axon terminals were visualized in the bz by vesicular glutamate transporters (vglut) and vglut , respectively. a complementary distribution of vglut and vglut was established by postnatal day (p) and both signal intensities increased further by p - (peak cp). the immunoreactivity for vglut decreased around layer iv after brief monocular deprivation ( dmd) during the cp. interestingly, both signals in all layers were lower in the bz contralateral to an eye injected with ttx than in the ipsilateral bz, consistent with the stronger functional plasticity and rapid dendritic refinement as compared to dmd. these results suggest that rapid and local weakening of excitatory inputs corresponds to dendritic spine pruning during experience-dependent competition. reiko meguro, masao norita department of sensory and integrative medicine, niigata university, graduate school of medical and dental sciences, niigata, japan we investigated how the geniculate and the extra-geniculate visual systems reorganize by monocular deprivation at birth. using anterograde/retrograde tracer, biotinylated dextran amine (bda), we made a small injection into the dorsal lateral geniculate nucleus (dlgn) or the lateral posterior nucleus (lp) of the degenerated side of the monocular deprived rat. the geniculate projection terminated mainly in layer iv of area , with a small projection to layer vi of areas and a. cells in layer vi of area projected to dlgn. in addition, cells in layer v of area projected to dlgn, which is not observed in normal rats. in area a, cells in layers v and vi projected to dlgn. the projection from lp terminated mainly in layer iv of a. cells in layers v and vi of area a projected to lp. smaller number of cells in layer v of area also projected to lp. these findings suggest that major parts of visual system developed normally, but some developed cross talk between geniculate and extra-geniculate systems. ps a-e activity dependent plasticity of feedback projection from the primary visual cortex to the dorsal lateral geniculate nucleus miho yoshida , takemasa satoh , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., yonago, japan; div. neurobiol., sch. life sci., fac. med., tottori univ., yonago, japan the projection from the lateral geniculate nucleus (lgn) to the primary visual cortex (v ) shows significant morphological plasticity responding to visual experiences in early life. such experiencedependent plasticity enables the geniculocortical projection to form functionally specific connections. it is not clear whether similar plasticity operates in other neural connections in the visual system. therefore, we tried to investigate the plasticity of feedback projection from v to the lgn. we focused on the density of type metabotropic glutamate receptor ␣ (mglur ␣) in the lgn which locate postsynaptically at synapses of feedback projection. immunohistochemical signal for mglur ␣ in the lgn decreased after elimination of v , showing that this signal reflects density of functional feedback synapses. to explore the activity dependent plasticity, we examined the effect of cortical activity blockade on the mglur ␣ signal in the lgn of young rats. yu morishima , hiroshi sakamoto , takahumi akasaki , yoshio hata div. integrative biosci., tottori univ. grad. sch. med. sci., japan; div. neurobiol., sch. life sci., fac. med., tottori univ., japan monocular deprivation (md) during postnatal development reduces cortical response to the deprived eye and input axons serving the deprived eye retract. however, when md is combined with continuous inactivation of the visual cortex by muscimol, cortical neurons lose their response to the open eye and the open eye axons retract. to clarify mechanisms underlying the two forms of ocular dominance (od) plasticity in different direction, we examined other characteristics of them, ( ) how rapidly the reverse od shift proceeds and ( ) whether the shift is induced only in young animals. we infused the cortex with muscimol in -week-old kittens and in adults. the reverse od shift was observed after days md, but not significant after days md. in adults, od distribution remained unchanged. morphological change of individual input axons was also examined after days md. the reverse od shift might reflect a mechanism of developmental plasticity that has a slower time course than the normal od shift. ps a-e experience-dependent plasticity in the absence of ampa receptor subunits in mouse visual cortex youichi iwai , nafiseh atapour , john renger , john roder , peter seeburg , takao hensch neuronal circuit dev., riken, bsi, wako, japan; mount sinai hospital, toronto, canada; max planck inst. med. res., heidelberg, germany two ampa receptor subunits (glur , ) play prominent roles in hippocampal models of homosynaptic plasticity (ltp/ltd). brief monocular deprivation ( d md) rapidly alters both the phosphorylation state and surface expression of glur in visual cortex. here, we addressed whether these coincidental events are essential for subsequent ocular dominance (od) plasticity. mice lacking glur (ko) displayed little ltd in visual cortical slices, while baseline transmission was normal. they also exhibited normal visual receptive field properties in vivo and shifted responsiveness toward the open eye after d md during a typical critical period. the rate of plasticity appeared somewhat slowed, as d md eventually led to full od shifts. in glur ko mice, even d md robustly activated od plasticity. thus, experience-dependent modification of ampa receptors is not essential for plasticity in vivo, although glur may contribute to the very earliest stages. shigeyoshi higo, nobuaki tamamaki department of morphological neural science, kumamoto university, kumamoto, japan virus-assisted transduction with reporter genes is a useful technique to investigate morphology of neurons in the central nervous system. however, the mechanisms to induce reporter expression in vivo often depend on gene-manipulated mice. since mice are not the best experimental animal for the study of mental disorder, we developed an adenovirus in which gfp expression is driven by dlx promotor and dlx / enhancer. this virus labels gabaergic neurons and oligodendrocyte in the wild-type mouse neocortex and allows us to trace gfp-labeled axons of gabaergic neurons in serial brain sections. we used this virus to investigate gabaergic neurons with long projecting axon branches beyond a functional area. the virus was injected into the stratum oriens of the mouse hippocampal field ca and revealed a nonpyramidal neuron projecting to ca and fimbria. further we shall introduce this virus to the cat brain and investigate axon branches of gabaergic projection neurons in the neocortex. akiko yamashita , takao oishi , motoharu hayashi div. appl. system neurosci., nihon univ. grad. sch. med. sci., tokyo, japan; dept. cell. and mol. biol., primate res. inst., kyoto univ., inuyama, japan gabaergic cells in the cerebral cortex are divided into subgroups: parvalbumin (pv)-, somatostatin (som)-, calretinin (cr)-, and calbindin-containing types. to clarify inhibitory system in primates, we determined coexistence of these molecules and proportions of these subtypes within gabaergic cells in the various cortical areas. pv, som or cr did not coexist with each other in primates as observed in rodents. more than % of gabaergic cells contained pv; showing that pv cells are more abundant in primates than in rodents. proportion of som cells in gabaergic cells was smaller in the primary visual area ( . %) than in other areas, such as the prefrontal ( . %), primary motor ( . %), somatosensory ( . %) and secondary visual areas ( . %), indicating cortical differentiation in gabaergic system of the primate cerebrum. our recent retrograde labeling studies in mice and cats showed that the neocortical areas are connected not only by excitatory neurons but also by gabaergic projection neurons. in order to address the importance of the gabaergic projection neurons in the neocortical information processing, we need to know the branching pattern and postsynaptic elements of the gabaergic projection axons. since more than % of the gabaergic projection neurons showed npy immunoreactivity, we used npy-cre transgenic mouse that express cre in npy neurons and adenovirus that encodes gfp in the downstream of floxed stop to label the gabeergic projection axons. after injection of the adenovirus into deep layers of the npy-cre mouse neocortex and immunoperoxidase staining of gfp in the brain section, we could reveal gabaergic neurons in a golgi-like image with their axons. also this method seemed to allow us to label gabaergic projection neurons retrogradely. koji ikezoe , guy n. elston , , tomofumi oga , hiroshi tamura , , ichiro fujita , osaka univ., japan; univ. queensland, australia; crest, jst, japan layer iii pyramidal cells in adult monkeys exhibit systematic differences in their dendritic morphology among cortical areas. basal dendrites of cells in visual association cortex such as inferior temporal area te spread more extensively and are more branched than those in the primary visual cortex (v ). pyramidal cells in prefrontal cortex, such as area , have even more dendritic branches than those in area te. here, we investigated whether a similar regional difference in the dendritic morphology was present in infant monkeys. we stained individual layer iii pyramidal cells in v (n = ), area te (n = ), and area (n = ) of a -week old monkey (macaca fascicularis) using intracellular dye-injection techniques in lightly fixed tissues. the number of branches and the tangential extent of dendrites was greatest in area , followed by area te, and v . thus, considerable heterogeneity in pyramidal cell structure already exists -weeks after birth. hiroaki matsushita , mahito ohkuma , masami watanabe , ei-ichi miyachi department of physiology, fujita health university, aichi, japan; department of perinatology, institute developmental research, aichi, japan acetylcholine (ach) receptors are believed to be expressed in developmental and regenerative process of retinal neurons. we performed the patch-clamp recording and fura- based calcium imaging in cat retinal ganglion cells (rgcs). under whole cell clamp conditions, transient sodium currents and action potentials were observed in all of normal or axonal regenerated rgcs. however, these currents and spikes were not observed in the % of axotomized rgcs. bath application of m carbachol, an ach receptor agonist, rose [ca + ] i in % of normal rgcs. although the % of rgcs responded to carbachol at days after axotomy, no responsive rgcs appeared during - days. ach responsiveness recovered in axonal regenerated rgcs ( %). since pycnotic cells were observed few days after axotomy, ach may modulate neurotrophic effect in survived rgcs. these results suggest that ach is an important marker for neuronal degeneration and regeneration in cat rgcs. research funds: kakenhi ( to em, to mw) kenichiro miura, masakatsu taki, hiromitsu tabata, kenji kawano dept. integrative brain sci., grad. schl. of med., kyoto univ., kyoto, japan the initiation of smooth pursuit eye movements is facilitated by the bottom-up attention to the target (hashimoto et al., ) . to study the effects of the attention on the processing of second-order motion stimuli, we recorded smooth pursuits in three humans with a dualpurkinje-image eye tracker. the pursuit target, presented on a crt monitor ( hz), was a gaussian patch of texture displayed on a neutral gray background. the gaussian envelope moved at deg/s, while the texture consisting of black and white random-noise pixel blocks remained stationary (drift-balanced stimulus). the number of the frames displaying the target before the motion onset was selected to manipulate the attention to the target, either eight frames ( ms) or only one frame ( ms). the initial tracking responses were larger when the target became visible eight frames before the motion onset. the result suggests that the second motion processing underlying the smooth pursuit initiation is facilitated by the attention to the target. ps a-e motion picture effects on eye movements and blood flow in the frontal area atsuhiko iijima , , tohru kiryu , kazuhiko ukai , takeshiko bando div. integrative physiol., grad. sch. med., niigata univ., niigata, japan; div. inform. sci., grad. sch. & tech., niigata univ., niigata, japan; dept. appl. phys., sch. sci. & tech., waseda univ., tokyo, japan motion pictures taken by rider's view of motocross bike elicited horizontal eye movements coherent to the motion vectors in some subjects, and not coherent in the other subjects, while those taken by passenger's view of roller coaster evoked similar eye movements in all of the subjects. subjects watched the two-dimensional motion pictures in random order. eye movements were measured by a binocular video oculography (newopto), and head movements were measured by a magnetic motion sensor (polhemus). blood flow in the frontal area was simultaneously monitored with a near infrared spectroscopy (hamamatsu). the patterns of eye movements and the blood flow variation during movie presentation changed in relation to motion components of the movie. possible mechanisms of the differences will be discussed. kiyoto matsuura , kenichiro miura , masakatsu taki , hiromitsu tabata , naoko inaba , kenji kawano , frederick a. miles grad. schl. med., kyoto univ., kyoto, japan; lab. sensorimotor res., nei, nih, bethesda, md, usa human ofrs show winner-take-all behavior when elicited by moving grating patterns composed of two sinusoids (sheliga et al., sfn ) . we recorded the ofrs to the motion of vertical grating patterns composed of two sinusoids of spatial frequency f and f, which created a repeating pattern with beat frequency, f, in two monkeys. motion consisted of successive steps ( hz), each one-fourth of the wavelength of the beat, so that with each step the two components shifted one-fourth of their wavelengths and had opposite directions, the f forwards and the f backwards. the contrast of the f was fixed at , , or %, while the contrast of the f was varied from one-fourth to four times the contrast of the f. when the contrast of the f ( f) was less than about half that of the f ( f), the f ( f) dominated initial ofr: winner-take-all. thus, the motion processing underlying the ofr in monkeys, like that in humans, includes nonlinear interactions. masazumi katayama, takahiro fujita department of human and artificial intelligence systems, faculty of engineering, university of fukui, fukuki, japan when executing prehension movement to grasp an object such as a tool, we plans the hand shape and grasping position to grasp a target object. while, goodale proposes the hypothesis that the roles of two visual streams (dorsal and ventral streams) are "vision for action" and "vision for perception", respectively. from the above points of view, we investigated independence of visual estimation and motor execution for grasping position of a target object. in this experiment, grasping positions were measured under the following four conditions: visual estimation without grasping, grasping without lift-up movement, grasping and lift-up movement and visual estimation without grasping. as a result, we found that grasping positions of visual estimation are significantly different from grasping positions of motor execution in the second and third conditions (p < . ). we concluded that grasping positions of visual estimation and motor execution are independent and these results support the goodale's hypothesis. ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst. niigata univ., niigata, japan cortical sensory areas are divided into modality-specific domains such as the visual and auditory cortices, in which sensory neurons are driven by modality-specific inputs. recently, wallace et al. found that multimodal neurons clustered in deep layers are present near the borders between sensory cortices. multimodal properties of these neurons may be explained by three types of inputs: overlapped projections from the thalamus, projections from multi-modal sites, or overlapped horizontal projections from the modality-specific sensory cortices. in this study, we tested the third possibility. we prepared the mouse cortical slices including the visual and auditory cortices. the horizontal activity propagation elicited by local electrical stimulation were visualized using flavoprotein fluorescence imaging. these results indicate that cortical areas between the visual and auditory cortices receive horizontal projections originated in the visual and auditory cortices, suggesting that multimodal horizontal connections are important for the multimodal properties of sensory neurons. jumpei naito , yaoxing chen , yukiko tanada dept. animal sci., teikyo univ. sci. & tech., uenohara, japan; china agricul. univ., beijing, china twenty white-leghorn chicks (p - ) were perfused with % paraformaldehyde through the heart under deep anesthesia of nembutal ( mg/kg bw). two to three small crystals of dii were implanted into the optic tectum, thalamus, or hypothalamus under a dissecting microscope. a total of rgcs were classified into six groups according to the somal area and dendritic field (naito and chen, ). group ic projected dominantly to the tectum. group is and iiis showed high hypothalamic-and thalamic-dominance, respectively. group iic was non-specific in the central projections. group ivc was tectal-dominant. patterns of the dendritic stratification were counted to in tec-rgcs, in tha-rgcs, and in hyo-rgcs. of these stratification patterns, many patterns were common among tec-, tha-, and hyp-rgcs. in contrast, the rgcs that showed a same dendritic pattern were consisted of a single rgc in most of the non-common rgcs, and their dendrites extended mainly to the superficial inner plexiform layer (sublayers - ). yasuro atoji, shouichiro saito laboratory of veterinary anatomy, gifu university, gifu, japan the present study was examined afferent and efferent fiber connections of the intermediate part of the caudal nidopallium (nci) in the pigeon by a tract-tracing method. in the present study we define nci an area which is located lateral to the field l complex and ventral to ncl. following a ctb injection into nci, a large number of neurons was labeled in nci, the mesopallium, and intermediate arcopallium (ai) and in the thalamic posterior dorsointermediate and posterior dorsolateral nuclei. contralateral ai contained a small number of labeled neurons. a few labeled neurons were found in lst. few labeled cells were found in ncl, field l, piriform cortex, or hippocampal formation. following a bda injection into nci, a large number of labeled fibers extended in nci, mesopallium, and ai. lst contained a small number of labeled fibers. few labeled fibers were located in ncv and limbic regions. the diencephalon contained very few labeled fibers. in summary, nci has strong reciprocal connections within nci itself and with the mesopallium and ai, and little connections with the limbic system. hidenori horie , kenji yuda , eiichi okawa , katsuyoshi maruyama , hiroshi uozato , hiroko horie , satomi nakajima , kenkichi tanioka , yuji ohkawa , tomoki matsubara , wolfram tetzlaff advanced res. centr. biological sci., waseda univ., tokyo, japan; technomaster co. ltd., yokohama, japan; kikuna yuda eye clinic, yokohama, japan; healthcare business co., matsushita electric ind. co. ltd., yokohama, japan; dept. ophthalmol. & visual sci., kitasato univ., kanagawa, japan; nhk sci. technical res. labo., tokyo, japan; icord, univ. british columbia, vancouver, canada we describe here a highly effective method to improve visual acuity of children with myopia and adult with presbyopia by repeatedly offering a visual object at variable distances while keeping the apparent retinal projection size of the object constant using a novel electronic device. in our experiments on human subjects, we used an lcd screen that was rhythmically moved between and cm toward and away in a high speed (top speed: mm/s) from the subjects. the device significantly improved visual performances in over % of the school-aged children with myopia and % of adults with presbyopia. hiroyuki miyamoto , toral s. surti , takao k hensch laboratory for neuronal circuit development, riken brain science institute, wako, japan; san francisco, usa competitive plasticity of binocular response following monocular deprivation (md) is prominent in the primary visual cortex (v ) during an early critical period. recently, md has been shown to enhance head-tracking behavior induced by slow rotation of grating stimuli in adult mice and is critically dependent upon the integrity of v . here, we addressed to what extent these two types of plasticity induced by the same md share common mechanisms. adult mice lacking a gaba-synthetic enzyme (gad ko), which do not exhibit ocular dominance (od) plasticity by brief md during the critical period, showed normal optomotor acuity and enhancement with day md. od shifts did not correlate with optomotor enhancement in these mice. finally, early md spanning the entire critical period had no effect on optomotor acuity through the deprived-eye. these observations support the view that adult perceptual learning and classical od plasticity are independent. junya hirokawa , miquel bosch , shuzo sakata , yoshio sakurai , tetsuo yamamori division of brain biology, national institute for basic biology, okazaki, japan; mit, ma, usa; rutgers university, nj, usa; department of psychology, kyoto university, japan the brain is able to integrate information from different sensory sources to enhance behavioral responses. to identify the neuronal populations responsible for multisensory enhancement in rats, we have mapped the activation of neurons during an audiovisual integration paradigm (sakata, et al., ) by the expression of c-fos. a pronounced c-fos upregulation was found in superior colliculus and in layer iv and deep layer of latero-medial secondary visual area (v lm). local injection of gaba agonist muscimol into this region selectively suppressed the behavioral enhancement related to multisensory integration, while no suppression was found by the injection into primary auditory and visual areas. these results suggest a key role of v lm in integration of auditory and visual information to facilitate the behavioral reaction for bimodal stimuli. takashi shinozaki , youichi miyawaki , tsunehiro takeda department of complexity science and engineering, university of tokyo, chiba, japan; mathematical neuroscience, riken bsi, saitama, japan drifting grating patterns with different motion directions independently presented to the two eyes induce two sets of perceptual rivalries: interocular rivalry (left or right eye's image) and motiontype rivalry (pattern or component motion). we studied this double rivalry process based on psychophysical and magnetoencephalography (meg) measurements. pattern-motion percept exclusively arose and persisted for a long duration whereas component-motion percept was soon followed by percept of either of left or right eye's single motion direction. reaction time (rt) measurement showed that the pattern-motion was perceived faster than left or right eye's motion direction. we then compared meg signals among those perceptual conditions and found a meg response of interocular rivalry in the latency range expected from the result of rt measurement. these results suggest that the double rivalry process has a hierarchical structure in which motion-type rivalry is resolved before interocular rivalry. visual stimuli evoke several brain potentials with relatively precise time courses. the role of these brain potentials in visual object categorization is not clear. in this study we recorded event related brain potentials (erp) while subjects participated in a face/non-face categorization task. gray face and non-face natural object images were presented briefly ( ms) followed by a noise mask with pseudo randomly selected stimulus onset asynchrony (soa = - ms). subjects reported presentation of face or non-face images by pushing one of the two assigned keys. we found that the face category discrimination performance significantly declined only in short soa ( and ms) with a larger impact of masking on non-face discrimination. in erp, the peak amplitude and latency of p , n and area under curve of a late positive potential expanding from to ms were correlated with the subjects behavioral performance. the effect of backward masking on early erp components may be due to altering sensory processing of visual stimuli while the effect on late erp potential could be related to its impact on decision making processes. yasushi naruse , ayumu matani , , tomoe hayakawa , , norio fujimaki university of tokyo, kashiwa, japan; nict, kobe, japan; teikyo university, tokyo, japan to study the process of alpha rhythm resetting, we investigated the relationship between visual evoked potential and the seamlessness: how much the phase angle of prestimulus alpha rhythm and the backward-extrapolated phase angle from poststimulus alpha ringing synchronize. alpha ringing is an evoked potential in alpha frequency band around ms in latency. eight clinically normal adult volunteers participated in the experiment, in which the subjects passively viewed a series of flash stimuli with their eyelids closed throughout the experiment. eeg was simultaneously recorded during the experiment. we classified the trials into four subsets owing to the seamlessness, and then averaged the trials in each subset. the result showed that the larger the amount of the alpha rhythm resetting is, the larger the p amplitude becomes. this suggested that a factor of the variability of the p amplitude is the amount of the prestimulus alpha rhythm resetting. research funds: a grant-in-aid from the ministry of education, culture, sports, science and technology (no. ) hitoshi sasaki , takuya ishida , masayoshi todorokihara , junichiro miyachi , tahei kitamura , ryozo aoki dept. physiol. & biosignal., osaka univ. grad. sch. med., suita, japan; dept. phys. & elec., osaka pref. univ. grad. sch. eng., sakai, japan; dept. elec. eng. & elec., col. industri. tech., amagasaki, japan recently it has been shown that noise can improve detection of sensory stimuli in several modalities. here we investigated whether visual contrast detection sensitivity can be improved by adding a certain amount of noise. contrast detection thresholds of a light changing brightness periodically were measured with or without overlapping noise in five normal participants. the contrast detection threshold, measured by using the psychophysical method (up-anddown method), decreased at around the threshold level of the noise intensity. these findings are consistent with our previous findings obtained by using another psychophysical method and confirm that noise can improve signal detection in human visual perception. narumi katsuyama , nobuo usui , izuru nose , , masato taira , department of applied system neuroscience, nihon university school of medical science, tokyo; faculty of human science, bunkyo university, saitama, japan; arish, nihon university, tokyo, japan when an object is moving, perception of its d trajectory in depth can be strongly influenced by the trajectory of its cast shadow. for example, a ball moving in a diagonal trajectory can appear to rise in a frontal plane when the shadow moves along the horizontal trajectory (rising configuration) or to roll in depth when the shadow follows the same trajectory as the ball, while the trajectory of ball is identical. using fmri, we found that several visual areas, including human mt and the posterior sts and the posterior parietal cortex, are activated in the comparison between rising configuration and ball only condition. additional correlation analysis by modifying the slope of the shadow' s trajectory also showed activation in the posterior part of sts and the posterior parietal cortex, including precuneus. these results suggest that cortical areas in the temporal and parietal cortex might be involved in the processing of apparent motion of ball induced by the moving cast shadow. ps a-f local area network in the gerbil's auditory cortex: reversible focal inactivation with infrared laser irradiation akira yamamoto, hiroshi riquimaroux gratuate school of engineering, doshisha university, scnrl, japan this study investigated local area networks in the primary auditory cortex (a ) and the anterior auditory field (aaf) by blocking neural activities with the near-infrared laser irradiation (wave length = nm). in previous in vivo studies, the laser irradiation could focally inactivate neural activities in a few minutes after the irradiation started, while the activities recovered in a few minutes after its cessation. by using this technique, the present study examined corticocotical relationships in the auditory cortex of the mongolian gerbils (meriones unguiculatus). cf (constant frequency) and fm (frequency modulated) tones were presented to anesthetized animals, and neural responses were extracellularly recorded contralaterally to the ear of stimulation. when irradiated aaf area and recorded neural responses from ai, the irradiation changed phasic responses into tonic responses, and vice versa. these results indicate that there are functional connections within ai or aaf, and between ai and aaf. takashi doi, hiroshi riquimaroux department of knowledge and engineering and computer sciences, doshisha university, kyoto, japan in a previous behavioral study, ablation of right auditory cortex (ac) made the discrimination between ascending and descending frequency modulated (fm) tones by mongolian gerbil (meriones unguiculatus) difficult (wetzel et al., ) . this result indicates that some neurons in gerbil's right ac represent the directions of fm sweeps. actually, we could find direction-dependent neurons and these neurons were mainly in anterior auditory field (aaf). in aaf, bfs are gradually shifted along the rostrocaudal direction, and the same bfs are arranged in dorsoventral direction (thomas et al., ) . moreover, aaf has dense synaptic connections within the area (budinger et al., ) . we made network models based on this structure of aaf and could gain similar responses to the actual responses of directiondependent neurons. this result suggests that aaf in gerbil's ac has good structure to process fm tones. research funds: a grant to rcast at doshisha univ. from mext, innovative cluster creation project by mext it has been demonstrated that the auditory space, namely the direction of a sound source, is represented topographically in the mammalian superior colliculus (sc). however, it is unclear as to how this auditory space map of the mammalian sc is formed in the auditory pathway. the present study investigated the topographical representation of auditory space in the external nucleus of the inferior colliculus (icx) of anesthetized gerbils. the icx is the major auditory nucleus that has projections to the sc. the stimuli were -ms noise bursts whose azimuths varied on the horizontal plane in the virtual acoustic space. single-unit responses were recorded from the icx. the majority of units exhibited some degree of spatial selectivity and preference for the azimuth contralateral to the recorded side. for supra-threshold stimulus only, there were topographical gradients of preferred azimuths in the icx. however, the spatial tuning width and preferred azimuth of the units depended markedly on stimulus level. the results indicate that in mammals, the formation of a rigorous auditory space map is incomplete at the icx level. manabu toyoshima , yasuo takeda , yasushi shimoda , kazutada watanabe department of bioengineering, nagaoka university of technology, nagaoka, japan; department of clinical pharmacy and pharmacology, kagoshima university, kagoshima, japan nb- that we isolated and identified is a neural cell recognition molecule belonging to contactin subgroup. we reported previously that nb- expression is prominent in the auditory system. nb- knockout mice exhibit impaired neural function in the auditory system. these findings indicate that nb- is indispensable for the function of auditory system. here we report the detailed analysis of the nb- localization using anti-nb- monoclonal antibody that we produced recently. immunohistochemical analysis of the rat brain showed that nb- was detected not only in all brain regions of the auditory pathway, but also in substantia nigra (sn), caudate putamen (cpu) and fibers projecting from sn to cpu. the nb- immunopositive cells in sn are restricted to gabaergic neurons. since gabaergic neurons play essential roles in the development and function of the auditory system, it is highly likely that nb- regulates the development and/or function of gabaergic neuron in the auditory pathway. reiko nagashima, kiyokazu ogita department of pharmacology, setsunan university faculty of pharmaceutical sciences, osaka, japan sensorineural hearing loss can be caused by a variety of insults, including acoustic trauma. there is compelling evidence that reactive oxygen species (ros) are formed in the cochlea during acoustic stimulation. glutathione (gsh) protects against the hearing loss through scavenging ros generated by noise. in this study, we investigated the changes in expression of gamma-glutamylcysteine synthetase (gcs) gene, which is the rate-limiting enzyme in de novo gsh synthesis, in the cochlea following acoustic stimulation. nuclear extracts were prepared from the cochlea at various time points after acoustic stimulation ( khz octave band, db, h), and then subjected to electrophoretic mobility shift assay to determine activator protein- (ap- ) dna binding. ap- binding was increased - h after the exposure. rt-pcr and immunostaining revealed that noise exposure was effective in elevating the expression of gcs in the cochlea h later. taken together, ap- may participate in the expression of gcs gene in the cochlea after acoustic stimulation. masaharu kudoh, ryuichi hishida, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan multiple formants compose vowels. we have previously reported that bilateral lesions including in the auditory cortex (ac) of rats impaired discrimination learning between synthesized vowel-like sounds with multiple formants, while discrimination between stimuli of a single formant or pure tones was not significantly impaired. in the present study, we determined the responsible auditory fields, which were required for the discrimination leaning between vowel-like sounds. water-deprived rats were trained to discriminate between two sounds including four different formants. licking a spout during presentation of one sound was rewarded with water while the other was not. surprisingly, local lesions in the primary ac or the ventral association cortex had no clear effect on the discrimination learning. in contrast, the dorsal association areas impaired the discrimination learning. these findings indicate that the dorsal auditory association cortex plays a critical role in discrimination learning of vowel-like sounds with multiple formants. hiroaki tsukano, yamato kubota, manavu tohmi, masaharu kudoh, katsuei shibuki department of neurophysiol, brain research institute, niigata university, niigata, japan we used transcranial flavoprotein fluorescence imaging for visualizing cortical responses to missing fundamentals in mice. c bl/ mice were anesthetized with urethane. the skull on the auditory cortex was exposed and covered with liquid paraffin to keep the skull transparent. cortical images of green fluorescence in blue light were recorded by a cooled ccd camera. responses in the auditory cortex elicited by sound stimuli ( - khz for ms) exhibited mirrorsymmetrical tonotopic maps in the primary auditory cortex (ai) and anterior auditory field. the activity patterns in ai elicited by khz were different from those elicited by or khz. however, the areas activated by khz were also activated by the mixture of plus khz but not by that of plus khz, suggesting that cortical responses to missing fundamentals in ai were visualized using flavoprotein fluorescence imaging. hiroko kosaki national priting bureau, tokyo, japan we constructed a functional scheme of macaque auditory by distribution of calcium binding protein, parvalbumin (pv). auditory cortex is consisted of one core (primary cortex), and five surrounding rings, which correspond with secondary, tertiary, quaric, and quintic cortices. parvabumin showed a graduation, that is, inner core is most pv-rich, and outer rings showed the decrease of pv concentration. comparing with pv staining in visual cortex, these six-levels suggested similar hierarchic and reciprocal structure, which are proposed by deyoe and vanessen by analysis of feed-forward and feedback connections. akihisa kimura, tomohiro donishi, keiichiro okamoto, yasuhiko tamai department of physiology, wakayama medical university, wakayama, japan tonotopically comparable subfields of the primary and non-primary auditory areas in the rat cortex have similar topographies in the projection to the medial geniculate body but reverse topographies in the projection to the thalamic reticular nucleus (trn). in the present study, we determined how cortical and thalamic afferents intersect in the trn with regard to tonotopic organization. in light of the fact that a subset of auditory cells in the trn responds to visual or somatosensory stimulus, we also explored the potential sources of cortical and thalamic afferents that would set up polymodal sensory interaction in the trn. small injections of biocytin into the trn, which were made with guidance of electrophysiological recording of auditory response, resulted in retrograde labeling. retrogradely labeled cortical and thalamic cells exhibited distinctive patterns of distribution depending on the injection sites. the results indicate anatomical nodes in the auditory trn that would implement selective relay of auditory and/or polymodal sensory inputs. ps a-f functional connections between the core and belt fields of the guinea-pig auditory cortex observed by optical recording and partial cortical inhibition using muscimol junsei horikawa, daisuke uchiyama, tatsunori matsui, shunji sugimoto department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan guinea pigs were anesthetized with ketamine and responses to puretones in the auditory cortex stained with a voltage-sensitive dye rh were recorded with a photodiode array. after determining the core (ai and dc) and belt fields, ai or dc was inhibited by putting a muscimol-containing agar piece on each field. the inhibition of ai resulted in reduction of responses in the belt fields by - %, whereas the inhibition of dc resulted in reduction only by - %. the reduction by ai inhibition was larger in the anterior and ventral belt fields and that by dc inhibition was larger in the posterior belt fields than in the other fields. further inhibition of dc after the ai inhibition or vice versa resulted in suppression of the responses in all the fields. these results suggest that the responses of the belt fields are elicited mostly via connections from the core to the belt fields and the belt fields receive differential connections from ai and dc. masataka nishimura, hiroyuki kaizo, wen-jie song department of electrical, electronic, and information engineering, graduate school of engineering, osaka university, suita, japan the auditory cortex of many mammals has a core area and surrounding belt regions. in guinea pigs, the primary auditory area, the secondary auditory area, and many belt regions have been reported. however, the activity of the belt regions has not been fully examined. using a high-resolution optical imaging system, we examined cortical responses to tone stimulations in anesthetized guinea pigs. the auditory cortex of six guinea pigs was exposed to the ventral end and stained with the voltage-sensitive dye rh- . a novel field in the ventro-anterior region was identified based on its isolated responses to a pure tone stimulation and the relatively long latency of the responses. the field was located ventro-caudal to the primary auditory area, and was close to the ventral edge of the auditory cortex. we thus named the field as ventro-caudal field (vc). smooth frequency gradient was observed in vc in rostro-caudal direction, with the frequency axis in opposite direction to that of the primary auditory area. yoko kato , , kazuo okanoya laboratory for biolinguistics, riken bsi, wako, saitama; graduate school of humanities, university of chiba, chiba, japan bengalese finches sing complex courtship songs. to sing complex sequences, they require auditory feedback during singing. song nucleus nif has a projection from primary auditory area field l and then it projects to sensory/motor nucleus hvc. moreover, bilateral lesion of nif cause song deterioration on complex sequences (hosino and okanoya, ) . we recorded auditory responses by multiunit activity from nif and field l. auditory responses of nif showed selectivity to bird's own song (bos) than its reversed song (rev). comparing selectivity of nif and field l, nif showed stronger selectivity than field l. however, nif did not show sequence dependent selectivity. these results suggest that nif relays auditory information and enhances bos selectivity. however, we did not observe a direct evidence that nif related to generation of complex sequences. we started to record responses extracellularly from ac neurons of guinea pigs. in general, animals show a stereotyped pattern of behaviors; they have a quiet, almost-motionless period, usually for tens of min. during this period, animals do not appear to sleep but be sensitive to the environmental disturbance. thereafter they usually fall asleep with their eyes closed. during this presleeping period, the best frequency tone was repeatedly presented - times at a fixed interval, through a speaker at - db spl. responses to such repetitive tones are apparently irregular, with the occurrence of spikes in most trials but no spikes in some trials. however, if all the trials are accumulated, there was global phase alternation every a few to tens of seconds. one phase constitutes relatively high rates of spike occurrence, while the other very low rates of spike occurrence. we suppose that, unlike a machine, the brain has a unique mechanism that automatically turns on and off the cortical processing of the redundant sensory stimulus. masashi sakai, sohei chimoto, ling qin, yu sato department of physiology, university of yamanashi, japan a periodic click train produces a continuum of several perceptual qualities: (i) at low repetition rate (< hz), the individual clicks are clearly heard as discrete events so that the entire train produces "rhythm" percept, (ii) at high repetition rate (> hz), the entire train is heard as a single continuous event leading to a strong "pitch" percept, and (iii) in the transition range, the periodicity can still be detected as "roughness". we physiologically explored how those perceptual qualities are represented in the primary auditory cortex in awake cats. we found that distinct population of cells conducted two coding modes: (i) representing low-rate stimuli through stimuluslocking activity (i.e., temporal code) and high-rate stimuli as only onset responses or (ii) exhibiting sustained responses with generating larger amount of discharges at higher repetition rate (i.e., rate code). in addition, pure-tone stimuli elicited onset responses or sustained responses in each of these cell populations, respectively. we will discuss functional consequences and spike evocation mechanisms of each population. atsuhito toyomaki , , , , , hokkaido university, sapporo, japan; hokkaido university, sapporo, japan; sakushin gakuin university, utsunomiya, japan; kobe shoin women's university, kobe, japan; riken, wako, japan; sakushin gakuin university, utsunomiya, japan gaps in a continuous sound play important roles for perception of voiceless consonants (i.e./k/,/p/,/t/) and japanese special mora (sokuon). we recorded auditory evoked responses to short gaps and tones from children ( - years old, n = ) and adults ( - years old, n = ). there were six gap conditions with durations of , , , , and ms embedded in a continuous tone and six tone conditions with the same durations. the frequency of all the tone was hz. the responses elicited by the onset of gaps differed between the children and the adults: the responses in children were significantly larger and more sustained than those in adults for all the durations. in contrast, an n and p complex followed the onset of all the tones in all the subjects. thus development time course of neural process is conceivably different between gaps and tones. ps a-f an fmri study on pitch control of voice using transformed auditory feedback method akira toyomrua , tamaki miyamoto , atsushi terao , sachiko koyama , takashi omori , harumitsu murohashi , shinya kuriki jst, saitama, japan; hokkaido university, sapporo, japan auditory feedback plays an important role in natural speech production. in the present study, we conducted an fmri experiment while subjects performed a transformed auditory feedback (taf) task to delineate the neural mechanism for control of pitch. the subjects were required to vocalize a and to hold the pitch of a feedback voice constant. in taf condition, the pitch was altered suddenly two or three times, whereas in non-taf condition the pitch was not modulated. under the taf condition, auditory feedback control is selectively expected to work more strongly than the non-taf condition. thus, a comparison between these conditions could neatly extract brain regions involved in auditory feedback control of pitch. as a result, right supramarginal gyrus, right frontal lobe (ba ), right anterior insula, left premotor area and right superior temporal gyrus showed greater activation ( subjects, p < . corrected). this result suggests that auditory feedback of pitch is mainly controlled by the right hemisphere. sachiko koyama-takeichi , yuko toyosawa , fumiya takeuchi , michinao matsui , shinya kuriki research institute for elecronic science, hokkaido university, sappro, japan; jst, saitama, japan; hokkaido university, school of medicine, japan; kobe shoin institute for linguistic science, kobe, japan sounds with relatively long duration elicit a sustained component (slow field, sf). in the present study, we recorded cortical magnetic responses elicited by vowels and examined whether sf differs between native and non-native vowels (n = ). four synthesized vowels were used as stimuli (stimulus duration ms). two of the vowels (a, o) are native for japanese and the other vowels (ae, schwa) are not. two inter-stimulus intervals were used ( / ms). for the native vowels, an early sf ( - ms) was larger for the long than for the short interval session in both hemispheres. for the non-native vowels, the early sf was larger for the long than the short interval session only in the right hemisphere. neither an effect of interval nor hemisphere was significant for a late part of sf ( - ms) regardless of stimulus types. research funds: japan science and technology agent (brain sciences and education), kakenhi ( ) ps a-f spatio-temporal representation of frequencymodulated sounds in the auditory cortex revealed by optical imaging shunji sugimoto , , junsei horikawa department of knowledge-based information engineering, toyohashi university of technology, toyohashi, japan; riken brain science institute, wako, japan optical imaging (voltage-sensitive dye, rh ) showed spatiotemporal response patterns for frequency-modulated (fm) sounds in the multiple fields of the guinea pig auditory cortex. an fm sound evoked a strong onset response spreading widely over the cortex, which was followed by a later response moving across the iso-frequency contours in the core fields. the location of the later response was corresponding roughly to the instantaneous frequency input of each fm sweep. on the other hand, a pure tone evoked a wide-spreading onset response followed by strong and long-lasting inhibitory effects. the later response to an fm sound appeared clearly when the frequency of the fm sweep was modulated over a wider range of frequencies, while it was diminished when the sound frequency was less modulated. these results imply that the cortical representation of such a later response contributes to a detection of frequency modulations in sounds. yamato kubota, kuniyuki takahashi, ryuichi hishida, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan mitochondrial flavoprotein fluorescence is intimately coupled with energy metabolism. if the flavoprotein fluorescence is photobleached, energy metabolisms and neural activities can be inactivated. we applied this photo-inactivation technique to demonstrate auditory signal transmission from the anterior auditory field (aaf) to the primary auditory cortex (ai). cortical responses in aaf and ai after sound stimuli ( - khz) were visualized using transcranial flavoprotein fluorescence imaging in mice anesthetized with urethane. after determination of tonotopic maps, the auditory cortex was irradiated with strong blue light derived from a xenon lamp for min, while the surface either aaf or ai was covered with a piece of carbon paper for preventing photo-inactivation. although photoinactivation of ai had almost no effect on the responses in aaf, photo-inactivation of aaf significantly reduced the responses in ai. these results suggest the presence of auditory signal transmission from aaf to ai. kousuke abe , go ashida , , kazuo funabiki graduate school of informatics, kyoto university, kyoto, japan; hmro, faculty of medicine, kyoto university, kyoto, japan sound signals are translated to dispersed sporadic firing of the auditory nerves, and are converged to the third auditory station called the nucleus laminaris (nl) in birds. in vivo intracellular recording from owl's nl cells revealed that sound waveforms are observed in the postsynaptic membrane potentials (sound analogue potential; sap). we simulated synaptic inputs to the owl's nl neurons by recruiting the convergence of phase-locked excitatory inputs. several parameters such as the degree of phase-locking, the number of convergence and the time course of a unitary synaptic input affected the amplitude of sap, the amplitude of dc depolarization and the spectral features of synaptic noise in a complex manner. biophysical mechanisms for recreating sound waveforms by synaptic potentials will be discussed. takashi nihashi , shigenori takebayashi , masahiko bundo , masazumi fujii , toshihiko wakabayashi , jun yoshida , hiroyuki fujisawa , kazunori ando , kazumasa hayasaka department of radiology, national hospital for geriatric medicine, obu, japan; department of neurosurgery, national hospital for geriatric medicine, obu, japan; department of neurosurgery, nagoya university school of medicine, nagoya, japan we identify si, using fmri in a routine scan for the patient who need a surgical approach. the activation of the brain with a tumor is complicated. we considered the pattern of the response. twelve patients were participated in this study. using . tesla mr imager, tactile stimulation was applied to bilateral palm, respectively. the statistical threshold was set for individual. contralateral activation on si was found in out of patients in the affected hemisphere. when region is near central sulcus, the multiple sites were activated. on the other hand, when the tumor is from central sulcus, the activation is simple: contralateral si. this method is useful to decide si in affected hemisphere in a short time. however, there are great inter-individual differences due to the locations of the tumor. takayuki iwano, shinya yamamoto national institute of advanced industrial science and technology (aist), neuroscience research institute, tsukuba, japan to examine how body surface with low spatial resolution is represented in the brain, we conducted a tactile identification task on toes. subjects (n = ) lay on their backs with their eyes closed, and one of their toes was touched with a toothpick. the subjects were required to identify the toe by verbal response. the subjects responded correctly when the great or fifth toe was touched (cf. fein, ) . surprisingly, subjects tended to misidentify the second toe as the third ( . %), and the third toe as the fourth ( . %), while the reverse misidentification rarely occurred (third as second, . %; fourth as third, . %). this unidirectionality suggests that misidentification arises not only from large overlapping receptive fields associated with the toes, but from some additional factors such as a lack of experience with visuotactile integration, which could be used to reshape the toe receptive fields. ps a-g effects of saccades on subjective temporal order of somatosensory signals toshimitsu takahashi , , shunjiro moizumi , ayami okuzumi , humine saito , shigeru kitazawa , department of neurophysiology, juntendo university graduate school of medicine, tokyo, japan; crest, jst, saitama, japan morrone et al. ( ) recently reported that subjective temporal order of two successive visual stimuli was reversed when the stimuli were delivered just prior to the onset of a saccade. in this study, we examined whether saccades affect temporal order judgments of tactile stimuli. right-handed subjects were required to make a visually guided rightward saccade ( • ), and to judge the order of successive tactile stimuli that were delivered one to each hand at various timing relative to the onset of the saccade. with a stimulation interval of ms, subjects generally judged the order correctly as long as the stimuli were delivered after the saccade. however, they often misreported (i.e., inverted) the order when the stimuli were delivered just prior to the onset of the saccade (within ms). the results show that the reversal effect of saccades is multimodal and further suggest that multimodal brain areas are involved in ordering sensory events in time. ps a-g function-directed organization of the postcentral somatosensory cortex representing oral structures takashi toda , , miki taoka , department neuroscience oral physiology, osaka university graduate school of dental sciences, suita, japan; secondrary cognitives neurobiology, tokyo medical & dental university, tokyo, japan; department physiology, toho university school of medicine, tokyo, japan the representation of oral structures in areas b and of four conscious macaque monkeys was studied by recording single-neuron activities. a total of electrode penetrations were made in areas b and . in penetrations, pairs of adjacent neurons along the track had receptive fields (rfs) on continuous oral portions with or without overlapping, or otherwise on the same portion. in the remaining penetrations, however, % of adjacent pairs ( / ) had rfs on discrete but functionally-related sets of oral portions, e.g., the lip and tongue tip, the cheek mucosa and lateral margin of the tongue, the corresponding portions of the upper and lower lips, the corresponding portions of the palate and tongue, etc. we speculated that such an organization in areas b and might be responsible for forming composite rfs of area neurons. those composite rfs often covered discrete but functionally-related oral portions as reported earlier. research funds: kakenhi ( , ) miki taoka , michio tanaka , hisayuki ojima , atsushi iriki secondrary cognitives neurobiology, tokyo medical and dental university, tokyo, japan; laboratory of symbolic cognitive development, riken brain science institute, wako, japan we previously reported neuronal projections from the hand region of the second somatosensroy cortex (sii) to higher motor cortices (vetral premotor cortex etc.) suggesting that sii may be related to motor control of the hand movement. in the present study, we investigated the activities of sii hand neurons during voluntary movements. we recorded neurons from two animals that were active when animals took small pieces of food by hands and put them into the mouth. among them ( % contra-, % bi-and % ipsilateral hand movements), we could determine receptive fields for only neurons ( %). most of activities ( neurons) were related to a certain phase of movements such as reaching, pinching a food piece, and putting it into the mouth. we found neurons showing phasic activities just before/after a certain phase, for example, just before pinching the object, or just after putting it into the mouth. those results suggest that sii hand neurons code the start or end of a certain act of hand. takahiro furuta , , kouichi nakamura , takeshi kaneko department of morphological brain science, graduate school of medicine, kyoto university, kyoto, japan; crulrg, laval university, canada we investigated response properties of whisker-responsive neurons in the nucleus interpolaris (spvi) combining juxtacellular recording and a piezo-stimurator. the spvi is one of the first relay stations in the vibrissal system. rostral part of the spvi sends axons to the posterior thalamic nuclear group, whereas the caudal part of the spvi projects to the ventral lateral part of the ventral posterior medial nucleus. in the rostral part of the spvi, the vast majority of recorded neurons were multi-whisker responsive neurons, which are considered as projection neurons. in the caudal part of the spvi, about a half of neurons were mono-whisker-responsive neurons, which are thought as local circuit neurons. almost all neurons had angular tunings to upward deflection of whiskers in the rostral part of the spvi, while neurons in the caudal part of the spvi exhibited various angular tunings to all directions. these results indicate that the spvi could be divided into two sectors by response properties. seiji komagata , , shanlin chen , hiroki kitaura , masaharu kudoh , minoru shibata , katsuei shibuki department of neurophysiology, brain research institute, niigata university; department plastic surgery, school of medicine, niigata university, japan we visualized neural responses in the mouse somatosensory cortex using transcranial flavoprotein fluorescence imaging. mice were anaesthetized with urethane ( . g/kg, i.p.), and somatosensory responses were elicited by vibratory brush stimulation ( hz for s) applied to the left plantar forepaw. changes in green fluorescence in blue light were observed in the right somatosensory cortex. immediately after cutting the left median and ulnar nerves, the somatosensory responses were almost completely abolished. however, the responses appeared again within a few hours after the partial denervation, and almost complete recovery was observed a few weeks after the partial denervation. the recovered responses were eliminated by cutting the remaining radial and muscle cutaneous nerves. the rapid recovery of the responses observed in the present study may explain the mechanisms for allodynia and cortical plasticity in the somatotopic maps. shin-ya nakamura, takaaki narumi, ken-ichiro tsutsui, toshio iijima division system neuroscience, tohoku university graduate school of life science, sendai, japan in the rat somatosensory pathway, information received with a whisker is conveyed to the barrel cortex via trigeminal and thalamic nucleus mainly by two parallel pathways, the lemniscal and paralemniscal. the former includes the nucleus of trigeminal prv and thalamic vpm, which are known to contain neurons selective to the direction of whisker stimulation, and the latter includes trigeminal spvi and thalamic pom. in this study, we examined the specific involvement of the lemniscal pathway to the discriminative perception of whisker stimulus direction. rats were trained to perform a single-whisker directional discrimination task, and the task performance was evaluated before and after the selective electrolytic lesion or muscimol inactivation of each trigeminal and thalamic nucleus. the lesion or inactivation of prv or vpm significantly impaired the task performance, whereas those of spvi or pom did not. this result suggests the specific involvement of the lemniscal pathway in the single-whisker directional discrimination. kumiko yokouchi , nanae fukushima , tetsuhiro fukuyama , akira kakegawa , tetsuji moriizumi department of anatomy, shinshu university school of medicine, matsumoto, japan; department of pediatrics, shinshu university school of medicine, matsumoto, japan to know sensory cues for suckling behavior, rat pups of the suckling period received unilateral or bilateral resection of the infraorbital (io), mental (m) or lingual (l) nerves responsible for sensation of the upper and lower lips, and the tongue. for comparison, unilateral or bilateral removal of the olfactory bulb was done in these pups. the control, unilaterally io or m nerve-injured, and unilaterally bulbectomized pups showed successful suckling by their access to the mother's nipple, oral contact to it and long-lasting sucking. the bilaterally bulbectomized pups could not have access to the nipple. the bilaterally io nerve-injured pups could have access to the nipple with no oral contact, while the bilaterally m nerve-injured pups showed successful suckling. suckling behavior of the bilaterally l nerve-injured pups was characterized by frequent oral contacts for a very short duration, resulting in ineffective milk-intake. the results show fundamental roles of olfaction and sensation of the upper lip and the tongue in suckling. takahiro kawashima , takeshi kawano , hidekuni takao , , , kazuaki sawada , , , hidekazu kaneko , , makoto ishida , , department electrical & electronic engineering, toyohashi university of technology, aichi, japan; issrc, toyohashi university of technology, aichi, japan; aist, hsbe, ibaraki, japan; jst, crest, japan a si microprobe array (probe: au-coated recording site at the tip, m in diameter, m in length; array: -m pitch) to record neuronal activities has been developed by using selective si probe growth technique. to examine electrical properties of the array, single motor unit action potentials evoked by the electrical stimulation of the sciatic nerve of a rat were recorded in the left tibialis anterior muscle. signal-to-noise ratio of observed signals decreased with probe impedance, suggesting that lower impedance is better for recording small action potentials. however, lower impedance makes more difficult to record local signals, because signals observed at probes with low impedance were highly correlated (r = . ). to record local signals, it is necessary to decrease the area of the recording site of each probe at the expense of an increase in the impedance. research funds: kakenhi ( ), the st century coe program "intelligent human sensing" ps a-g a microelectrode positioning system for longterm extracellular recording of multiple neuronal activities hidekazu kaneko , hiroshi tamura , shinya s. suzuki , takahiro kawashima aist, hsbe, ibaraki, japan; laboratory of cognitive neuroscience, graduate school of frontal bioscience, osaka university, osaka, japan; department electrical & electronic engineering, toyohashi university of technology, aichi, japan a novel microelectrode-positioning system was devised that tracks a target neuron by countermoving a microelectrode against uncontrollable movements of brain tissue. the system automatically adjusted the position of a seven-core microelectrode such that the amplitude of each spike of a target neuron at the tip was the same as that at a lateral recording site (differential mode). the differential mode was compared with a conventional (peak-search) mode in which the spike amplitude of a target neuron at the tip was continually maximized. the differential mode was more stable to forced electrode movements and more sensitive to small displacements than the peak-search mode. furthermore, the differential mode enabled stable recording of not only spikes of a target neuron but also those of non-target neurons for over h. seiji matsuda , takehiro terashita , tetsuya shimokawa , kyoujy miyawaki , yuji miguchi , takuya doihara , jie chen , shuang-yan gao , chun-yu li , min wang , zhong wang , bing xue , naoto kobayashi , , kazuhiro shigemoto department anatomy, ehime university of medicine, ehime, japan; medical education c, ehime university of medicine, ehime, japan; department of hygiene, ehime university of medicine, ehime, japan this study shows the phylogenetic development of cajal's initial glomeruli (ig) and dogiel's pericellular nests (pcns) in the dorsal root ganglion of the healthy adult frog, chick, rat, and rabbit. the three-dimensional architecture of the neurons was observed in ganglia by scanning electron microscopy, after removal of the connective tissue. the proportion of neurons having ig or pcns increased with increasing phylogenetic complexity in the species examined here. in the chicks, the stem processes were longer and sometimes tortuous. typical ig were observed not in frogs or chicks, but in rats and rabbits. dogiel's pcns also have been observed in the drg of rats and rabbits. the nerve fibers in the pcns were less than . m in diameter and had some varicosities. some pcns contain tyrosine hydroxylase-positive nerve fibers and varicosities. masayo okumura, eiji kondo matsumoto dental university, institute for oral science, shiojiri, japan we established a rat nerve injury model using axotomy of the inferior alveolar nerve (ian), and investigated its effect on gene expression in the trigeminal ganglion. microarray analysis three days after surgery showed the up-regulation of some genes which are regulated by transcription factor stat , whereas other genes known to be regulated by stat were not detected. stat is expressed in many tissues and plays various roles. however, there have been few reports about the role of stat in the peripheral nervous system, despite its welldocumented activation in the central nervous system after injury or stress. the aim of this study is to elucidate the role of stat in gene expression in the trigeminal ganglion after ian injury. at various time points, we analyzed and investigated changes of gene expression which are known to be influenced by stat and stat phosphorylation, which indicates transcriptional activity, as well as cell types in which the genes and stat are expressed. these results should help us understand injury-induced change mechanisms of the peripheral nerve. hirofumi hashimoto , susumu hyodo , makoto kawasaki , minori shibata , takeshi saito , hiroaki fujihara , takashi higuchi , yoshio takei , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; laboratory of physiology, department of marine bioscience, ocean research institute, university of tokyo, japan; department of integrative physiology, university of fukui, japan adrenomedullin (am ) (identical to intermedin) belongs to the super family of am. centrally administered am and am activated oxytocin (oxt)-secreting neurons and increased plasma oxt level in rats. in the present study, we examined the effects of central administration of am on oxt-secreting neurons and sympathetic outflow in comparison with that of am in conscious rats. effects of central administration of am was stronger than those of am and the effects of am on oxt secreting neurons could not be blocked completely by pretreatment with cgrp or/and am receptor antagonists. these data suggested that am would have unknown receptor except cgrp and am receptor. arata oh-nishi , makoto saji , taku uchida , sen-ichi furudate , nobuyuki suzuki division of brain science, kitasato university, graduate school of medical science, kanagawa, japan; division of reproduction and fetal development, kitasato university, graduate school of medical science, kanagawa, japan the mechanism whereby neonatal hypothyroidism impairs cognitive function has not been well studied. in this respect, nmda receptors are thought to be crucially involved in cognitive and memory function. we have examined the effect of neonatal hypothyroidism and hyperthyroidism on the nmda receptor function, using rats treated with methylmercaptoimidazole (mmi), which specifically blocks the biosynthesis of thyroid hormone and mmi-treated rats injected with thyroxine, respectively. dose-response curves indicated that the sensitivity to nmda of the nmda receptors was significantly reduced in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. concomitant with this observation, western blot analysis showed that the nmda receptor subunit nr expression significantly decreased in the hippocampus of the hyperthyroid rats, compared to that of normal and the hypothyroid rats. our lab demonstrated that estradiol is endogenously synthesized within hippocampal neurons in the adult male rat (pnas, ) . here we report that the density and morphology of spines of pyramidal neurons in ca region are rapidly altered by treatments with nm estradiol and bisphenol a (xenoestrogen). hippocampal slices are incubated with estradiol or bisphenol a for h, and then neurons were injected iontophoretically with lucifer yellow. three-dimensional imaging of neurons is performed by confocal laser microscopy, and the analysis of individual spines is performed by neurolucida software. the results showed that in ca , both estradiol and bisphenol a induce a significant increase in the total spine density, especially the density of thin spine. synaptic plasticity of hippocampal neurons is demonstrated to be rapidly modulated by estrogen and xenoestrogen. we investigated the effects of stress on enhanced green fluorescent protein (egfp) expression in the arginine vasopressin (avp)-egfp transgenic (tg) rats. after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide egfp fluorescences were increased in the parvocellular division of the paraventricular nucleus and the external layer of the median eminence. this tg rat is a convenient tool to study dynamic changes of avp expression in the hypothalamus under stressful condition. chitose orikasa, yasuhiko kondo, yasuo sakuma department of physiology, nippon medical school, tokyo, japan we report here a sex difference expression of somatostain mrna within the sexually dimorphic nucleus of the preoptic area (sdn-poa), the volume of which depends on gonadal hormones during the ontogeny. in infant rats aged day - , the volume of somatostain mrna-positive region within the poa was significantly larger in males than in females and overlapped the sdn-poa in both sexes. the sdn-poa visualized by nissl staining in adjacent sections agreed precisely with the extent of somatostain mrna-positive cellular distribution. orchidectomy of males neonates and estrogen treatment of female pups reverse brain phenotypes when examines on day . the staining of somatostain mrna in individual neurons was diminished when examined on day or , albeit that the sex difference of the volume of somatostain mrna-positive region persisted thoughtout the observed period. somatostatin may play a role in the establishment of the sdn-poa, which lacks classic nuclear receptor for estrogen. ps a-g short chain sugar acid, -buten- -olide, activates oxytocin-secreting neurons in the hypothalamus of rats makoto kawasaki , tatsushi onaka , hirofumi hashimoto , hiroaki fujihara , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; department of physiology, jichi medical school, tochigi, japan -buten- -olide ( -b o), an endogenous sugar acid, which may be involved in the regulation of feeding. we examined the effects of -b o on the hypothalamo-neurohypophyseal system in rats. the plasma oxytocin (oxt) levels were significantly increased at - min after intraperitoneal (i.p.) administration of -b o ( mg/kg), whereas plasma arginine vasopressin (avp) levels did not change. dual immunostaining revealed that fos-like immunoreactivity (li) was predominantly observed in oxt-secreting neurons in the paraventricular and the supraoptic nuclei min after i.p. administration of -b o. in addition, many fos-li neurons were also observed in the nucleus of the tractus solitarius (nts) after i.p. administration of -b o. these results suggest that peripherally administered high dose of -b o activates oxt-secreting neurons in the hypothalamus through the activation of the nts neurons. ps a-g estrogen receptor ␣ gene promoter activity is a marker for the sexually dimorphic nucleus of the preoptic area tomohiro hamada, yasuo sakuma department of physiology, nippon medical school, tokyo, japan the volume of the sexually dimorphic nucleus in the preoptic area (sdn-poa) is two to four times larger in male rat than in female, however function of this nucleus has not well known. in contrast, estrogen causes the sexually dimorphism by acting in perinatal periods. recently, transgenic rats expressing enhanced green fluorescent protein (egfp) under the control of an estrogen receptor (er) ␣ promoter were generated to tag er␣-positive neurons in the brain. in the present study, we examined gfp expression could be used a marker for the sdn-poa. gfp labeled cells were distributed in the core of sdn-poa of male and female transgenic rats and in the majority of these cells included er␣, immunohistochemically. both area and number of gfp expressed cells in the sdn-poa were larger in male than in female, however, female gfp cells in the sdn-poa showed concentrated distribution than male. these results suggest that gfp labeled cells in sdn-poa could be useful marker to make clear the function of the sdn-poa. recent studies on gonadal steroids imply that testosterone and estradiol are involved in learning and memory with modification of excitatory synapses in the hippocampus. although previous in vivo studies have demonstrated that these steroids increase the number of dendritic spines in neurons, it is still unclear whether each steroid has a direct effect on the modulation of the spatio-temporal patterns of dendritic morphogenesis. in the present study, we investigated steroid-induced morphological changes using cultured hippocampal neurons derived from neonatal or embryonic mice. the neurons were transfected with venus-actin. time-lapse images were taken by laser scanning confocal microscope during steroid treatment. testosterone but not estradiol increased the number of spines/filopodia of the dendrites within h. these results obtained from in vitro studies suggested that testosterone affects dendritic morphogenesis of hippocampal neurons in short term. tetsuya kimoto , , shinpei higo , , yasushi hojo , , kouhei nakajima , , hironori nakanishi , , hirotaka ishii , , suguru kawato , department of biophysics & life science, university of tokyo, tokyo, japan; crest, jst, japan hippocampus is one of the main target of sex steroids (androgen and estrogen) and stress steroids (corticosteroids). neuronal signal transmission in the hippocampus is modulated acutely by these steroids, and we recently demonstrated that the hippocampus of the adult male rat contained enzymes required for the synthesis of these steroids. however, the full diagram of hippocampal neurosteroid synthesis has not been obtained yet. in the present study, we therefore investigated the synthesis of sex steroids and corticosteroids in the hippocampus of adult male rats, by monitoring the metabolism of tritiated steroids with hplc system. ps a-g gaba depolarizes gnrh neurons isolated from adult gnrh-egfp transgenic rats chengzhu yin, nobuyuki tanaka, masakatsu kato, yasuo sakuma nippon medical school, department of physiology, tokyo, japan gnrh neurons are essential in the reproductive neuroendocrine system. in regulation of gnrh neurons, gaba may be one of the major players, especially in relation to gnrh/lh surge. we, therefore, performed a cell-physiological analysis of gaba action on rat gnrh neurons. cells were dispersed from adult gnrh-egfp transgenic rats and cultured overnight. gnrh neurons, were applied to the perforated patch-clamp configuration with gramicidin d. gaba evoked cl − conductance, which was almost completely blocked by either picrotoxin or biccuculin. the reversal potential of the response was ranged from − to - mv in identified gnrh neurons in both sexes. there was no difference in the reversal potential among the stages of estrous cycle. in unidentified neurons, however, the reversal potential was more negative than - mv and most of them were ∼− mv. in conclusion, gnrh neurons isolated from adult rats express gabaa receptor and its reversal potential is more positive than the resting potential. although the neural activation in the subfornical organ (sfo) by angiotensin ii (angii) is widely regarded for the increments of angii-induced water intake and vasopressin release, galanin (gal) have been reported to inhibit them. therefore, gal may inhibit neural activity of angii-sensitive sfo neurons. rt-pcr analysis demonstrated existences of all mrnas of gal receptor subtypes, galr , galr and galr , in the sfo. in extracellular recording on sfo slice preparation, gal dose-dependently led to inhibition of neural activity. all gal sensitive neurons showed excitatory response by angii. galr selective agonist m induced inhibitory responses, as well as gal. in patch-clamp recordings, gal induced outward current in some neurons. these results suggest that gal inhibits neural activity in sfo neurons through, at least partially, outward current following activation of galr . hiroaki fujihara , tomoki fujio , david murphy , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; molecular neuroendocrinology research group, the henry wellcome laboratories for integrative neuroscience and endocrinology, university of bristol, bristol, uk we have generated transgenic (tg) rats expressing an arginine vasopressin (avp)-enhanced green fluorescent protein (egfp) fusion gene. in this study, we investigated the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight after drinking % saline for days in , , and months old tg rats. in and months, there were no difference between tg rats and tg(−) rats about the amount of drinking and food intake, the urinary output, the urine osmotic pressure, the urine sodium concentration and body weight under normal condition and salt loading. in aged tg rats ( and months old), there were no obvious changes in water balance. these results suggest that the expression of avp-egfp transgene does not disturb body fluid homeostasis in tg rats. ps a-h prolactin-releasing peptide is a potent mediator of stress response in the brain through the hypothalamic paraventricular nucleus takashi mera , hiroaki fujihara , hirofumi hashimoto , makoto kawasaki , tatsushi onaka , takakazu oka , sadatoshi tsuji , yoichi ueta department of neurology (division of psychosomatic medicine), school of medicine, university of occupational and environmental health, japan; department of physiology, school of medicine, university of occupational and environmental health, japan; department of physiology, jichi medical school we examined the effects of restraint stress (rts), nociceptive stimulus and acute inflammatory stress on the prolactin-releasing peptide (prrp) gene expression in the hypothalamus and brainstem. moreover, we examined the effects of pretreatment with an anti-prrp antibody on nociceptive stimulus-induced c-fos gene expression in the hypothalamic paraventricular nucleus (pvn). rts, nociceptive stimulus and acute inflammatory stress upregulated the prrp gene expression in the brainstem. pretreatment with anti-prrp antibody significantly attenuated nociceptive stimulus-induced c-fos gene expression in the pvn. these results suggested that prrp is a potent and important mediator of stress response in the brain through the hypothalamic pvn. taieb bousejin , afsaneh eliassi , nasser naghdi , ali ghanbari ghsemi; pastor institute, tehran, iran the purpose of this study was to consider the role of the ventromedial hypothalamus (vmh) d receptors on histamine-induced gastric acid secretion (gas). the animals were anasthetized and guide cannulas were implanted unilaterally above ( . mm) vmh. animals were anasthetized and two polyethylene tubes were introduced into the stomach through esophagus and pylorododenal junction. iv infusion of histamine in sham grup induced marked increase in gas with a peak response that started from min up to the end of experiments ( min). at the peak acid response, the vmh microinjection skf ( . , ) significantly reduced the amount of gas (p < . ). there was no any effect by microinjected sch ( . ) into the vmh. injecting skf into the vmh, min after sch , had no effect on gas in compare with control. but, the acid suppressant effect of skf was completely removed by peripheral injection of sch (p < . ). our results show that the vmh d dopamine receptors have regulatory mechanisms of gas by interaction with h receptors through an inhibitory neural pathways. zhilin song, celia d. sladek department of physiology, uchsc, aurora, co, usa although prior studies demonstrated expression of p x purinoceptors in supraoptic neurons (son) and indicated their importance in atp stimulated vasopressin release, in studies monitoring the effect of atp on intracellular ca ++ ([ca ++ ] i ), we have obtained evidence that p y purinoceptors (p y r) are important in the response to atp. atp stimulated [ca ++ ] i increase was maintained in ca ++ -free medium and reduced by pretreatment with thapsigargin to deplete [ca ++ ] i stores. p y r agonists increased [ca ++ ] i in son, with p y r agonist being the most effective. the possibility that p y r mediates atp induced [ca ++ ] i increase in son was further evaluated using a p y r antagonist, mrs . atp stimulated increase in [ca ++ ] i was greatly attenuated by mrs ( m) in mm ca ++ medium. in ca ++ -free medium, there was no significant response to atp in the presence of mrs . furthermore, combined treatment with mrs and ppads ( m, a p x r antagonist) also abolished the [ca ++ ] i response to atp. these results demonstrated that p y r mediates a large portion of the [ca ++ ] i response to atp challenge in son. ps a-h analysis of the ontogenic expression of enzymes for brain neurosteroids in the male rat hippocampus hirotaka ishii , , yasuhiro sonoki , , aizo furukawa , , yasushi hojo , tetsuya kimoto , , suguru kawato , department of biophysics and life science, university of tokyo, tokyo, japan; crest, jst, japan; kurihama national hospital, japan brain neurosteroids are steroids synthesized endogenously in the brain. our recent studies have demonstrated that the adult male rat hippocampus is equipped with a complete machinery for the synthesis of androgen and estrogen. to define the physiological role of brain neurosteroids in the hippocampal development and function, detailed information about the expression profiles of enzymes for brain neurosteroids in the hippocampus is essential. this study have comprehensively investigated the temporal patterns of enzymes for brain neurosteroids in the male rat hippocampus from postnatal day (pd ) to the adult stage using rt-pcr/southern blotting. enzymes required for the synthesis of estradiol from cholesterol were expressed form pd to pd with a higher level than in the adulthood. these results indicate that the rat hippocampus synthesizes estradiol more vigorously during the postnatal stage than in the adulthood, which may play an important role in the hippocampal development and function. hideo mukai , , gen murakami , shirou kominami , john h morrison , william g.m janssen , tetsuya kimoto , , suguru kawato , department of biophysics and life sciences, graduate school of arts and sciences, the university of tokyo, meguro, tokyo - , japan; crest project, jst, japan; faculty of integrated arts and sciences, hiroshima university, higashi-hiroshima , japan; kastor neurobiology of aging laboratories, fishberg research center for neurobiology, usa estrogens elicit rapid non-genomic effects on the synaptic transmission, and spinogenesis in the hippocampus. however, the existence of estrogen receptor alpha (er␣) still remains elusive. with highly purified antibody rc- , mass spectrometric analysis identified er␣ in the hippocampus and immunohistochemistry showed er␣ localization in principal neurons of ca , ca , and granule cells in dentate gyrus. further, western blot revealed that er␣ is contained in psd fraction, confirming the observation with immunoelectron microscopy. these results imply that the synaptic er␣ mediates the effects of estrogen in hippocampal neurons. ken takumi gnrh neuron is the key modulator of reproductive systems, directly regulating the synthesis and secretion of gonadotropins from anterior pituitary gland. gnrh neurons have been reported to be contacted by various neuronal systems, suggesting that the biosynthesis and release of gnrh is controlled by a complex of excitatory and inhibitory inputs. however, anatomical studies which quantified the direct input on gnrh neuron are few. in this study, we quantitatively analysed glutamatergic and gabaergic input onto gnrh neurons of the rhesus monkey by immunofluorescence method and confocal laser scanning microscopy; the close appositions between gnrh neuron and axon terminals immunoreactive for either vgluts or vgat were counted and the densities of the appositions on the dendrites and soma were calculated. sabine gouraud , song t. yao , jing qiu , julian fr paton , david murphy university of bristol, hw-line, uk; department of physiology, bristol heart institute, university of bristol, united kingdom the neuropeptide hormone vasopressin (vp) is produced in the magnocellular neurons of the hypothalamic supraoptic (son) and paraventricular (pvn) nuclei and stored in the posterior pituitary (pp). dehydration evokes an increased expression of the vp gene in magnocellular neurons and a massive release of vp from the pp in the circulation to promote the water conservation at the kidney level. in parallel, a functional remodelling of the hypothalamo-neurohypophyseal system (hns) is observed but poorly understood. we investigated this activity dependent plasticity of the hns using proteomic ( d fluorescence difference gel electrophoresis (dige)) combined with maldi mass spectrometry approaches to identify proteins that change in abundance in the son and the pp from days dehydrated rats. a truncated form of prosaas, a granin-like neuroendocrine peptide precursor known as a potent inhibitor of the prohormone convertase , has been found decreased in the pp and increased in the son. ichiro nishimura, masakatsu kato, yasuo sakuma department of physiology, nippon medical school, tokyo, japan function of gonadotropin-releasing hormone (gnrh) neurons is regulated by gonadal steroid estrogen. however, the precise mechanism of estrogen action upon these cells has not been clarified. we investigated a direct action of estrogen on the regulation of potassium current in gnrh neuronal cell line gt - . delayed rectifier potassium current (i k ) and large-conductance calcium-activated potassium (bk) current were recorded by patch clamp configuration in gt - cells cultured in dmem supplemented with % fbs for days. bk current was increased by addition of ␤-estradiol (e ) in culture medium in a physiological concentration range. this action of e was blocked by ici- , , a potent estrogen receptor (er) antagonist. we further examined whether e acted through er ␣ or er ␤ by using selective agonists ppt and dpn, respectively. the dpn augmented the bk currents similar to the effect of e but ppt had no effect. e had no effect on the i k . these results indicate that e increases the bk current by activating er␤ without affecting the i k . research funds: kakenhi , ps a-h myelin protein zero is one of the components of the detergent-resistant membrane microdomain fraction derived from rat pituitary katsutoshi taguchi , haruko kumanogoh , shun nakamura , seiji miyata , shohei maekawa department of biosystems science, kobe-university, kobe, japan; division of biochemistry and cellular biology, national institute of neuroscience, ncnp, tokyo, japan; department of applied biology, kyoto institute of technology, kyoto, japan a membrane microdomain enriched in cholesterol and glycosphingolipids was found to contain many signal transducing and cell adhesion molecules. here, we studied the components of the membrane microdomain fraction derived from rat pituitary, and found specific enrichment of several proteins in this fraction. one of them, kda protein, was identified as myelin protein zero (p ) from mass analysis and this result was confirmed by western blotting that a specific antibody to kda band reacted to an authentic p prepared from rat sciatic nerve myelin. p is a type i transmembrane glycoprotein and a member of the immunoglobulin superfamily. the expression of p has been believed to be restricted to the peripheral myelin in mammals. our result, however, indicates that p expresses more widely and participates in cell communications. mari ogiue-ikeda, norio takata, suguru kawato department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan ␤-estradiol (e ) has a rapid effect on synaptic transmission. recently, we found that hippocampal neurons synthesize e (hojo et al., ) , and express estrogen receptor ␣ (er␣) at synapses. endocrine disrupters are representative estrogenic industrial compounds. while their disrupting effects on reproductive organs are well documented, their effects in the central nervous system are almost unknown. in this study, we investigated the effects of e and endocrine disrupters (des, bpa, np, op and tbt) on nmda-induced ltd in the rat hippocampal ca , ca and dg with a custom made multi-electrode measuring system (med ). ltd was enhanced by e dose-dependently in ca , ca and dg. des, bpa, np, op and tbt had similar or different effects on ltd dose-dependently. our results suggest that estradiol and endocrine disrupters rapidly modulate synaptic plasticity in the hippocampus and that the action of endocrine disrupters can be quantitatively analyzed by measuring the modulation of ltd of the hippocampal neurons. we examined the effects of chronic salt loading on the hypothalamic expressions of the green fluorescent protein (gfp), arginine vasopressin (avp) and oxytocin (oxt) genes and body fluid balance in avp-enhanced (e) gfp transgenic rats. chronic salt loading caused marked increase of the egfp fluorescence in the hypothalamoneurohypophyseal system in transgenic rats. there were no differences of the avp and oxt gene expressions in the hypothalamus, plasma avp and oxt levels and water balance between nontransgenic and transgenic rats under normal condition and after salt loading. humoral responses to chronic salt loading were maintained in avp-egfp transgenic rats. takeshi saito , takushi x. watanabe , tomoko urabe , hirofumi hashimoto , hiroaki fujihara , yukio hirata , yoichi ueta department of physiology, school of medicine, university of occupational and environmental health, kitakyushu, japan; peptide institute, inc., osaka, japan; department of clinical and molecular endocrinology, tokyo medical and dental university, tokyo, japan salusin-␤ was newly discovered as a bioactive endogenous peptide. low concentration of salusin-␤ stimulates the secretion of arginine vasopressin (avp) from perifused rat hypophysis. salusin-␤ coexists with avp, but not oxytocin, in the rat magnocellular supraoptic (son) and paraventricular nuclei (pvn). to further investigate the physiological role of salusin-␤ in body fluid homeostasis, we examined the effects of salt loding for days on salusin-␤-like immunoreactivity (li) in the son and pvn of rats by immunohistochemistry. the marked increase of salusin-␤-li in the son and pvn were observed in the salt loaded rats. the result suggests that salusin-␤ may play a role of body fluid balance by regulating avp release. naoyuki yamamoto , hao-gang xue , yuji ishikawa , yoshitaka oka , hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan; national institute of radiological sciences, chiba, japan; department of biological sciences, graduate school of science, the university of tokyo, tokyo, japan the terminal nerve gnrh (gonadotropin-releasing hormone) system, an extrahypothalamic peptidergic system, is thought to modulate neural circuitries involved in the control of motivational status for certain behaviors in teleosts. the major afferent source to the gnrh neurons is a midbrain nucleus, the nucleus tegmento-terminalis in percomorph teleosts, while a comparable nucleus appears to be missing in cyprinids teleosts. here, we examined the presence of such an afferent pathway in medaka oryzias latipes. injections of a dii crystal into the cluster of gnrh neurons resulted in labeled cells in the midbrain tegmentum, and injections to the midbrain tegmentum resulted in labeled terminals close to the gnrh neurons. these results suggest that the afferent pathway to the gnrh neurons is a character shared by "advanced" teleosts like medaka and percomorphs. takeshi yamazaki , eiji munetsuna , asuka kamogawa , suguru kawato , shiro kominami graduate school of integrated arts science, hiroshima university of higashihiroshima, japan; graduate school of arts and science, tokyou university of tokyo, japan tributyltin, tbt, an endocrine disruptor, induced increases in estradiol content in rat hippocampal slice culture. to analyze molecular mechanism of stimulation of estrogen synthesis, we determined mrna contents of estrogen biosynthetic enzymes and activity of p arom in the hippocampus. method: the cultured hippocampus slices from days male rat were treated with - nm of tbt for h. after the treatment, total rna was extracted and the levels of mrna of estrogen synthetic enzymes were quantified by real-time rt-pcr. activity of p arom was determined by quantification of [ h]estradiol from [ h]testosterone. result: forty-eight hours treatment of hippocampal slices with nm tbt induced increases in mrna contents of p arom, and with nm tbt induced that of ␤-hsd. estradiol content was increased by the treatment with nm tbt, but not affected by nm tbt. tbt may modulate estradiol synthesis by alteration of expression of p arom. the medial preoptic area (mpoa) is an important neural site for regulation and maintenance of sleep. studies have indicated that gabaergic neurons and terminals at the mpoa are active during sleep. present study was carried out to elucidate the contribution of gaba-a receptor at the mpoa in sleep-wakefulness (sw) in male wistar rats. the sw was assessed by chronically implanted electrodes for eeg, eog, and emg. a bilateral guide cannula was also implanted for drug injection into the mpoa. after recovery, three baseline sleep recordings were taken for h on different days. bicuculline methoiodide (gaba-a receptor antagonist) at a dose of , , and ng in nl was injected bilaterally into the mpoa in different groups of rats and their sw was studied for subsequent h. the ng dose of bicuculline methoiodide had minimum effect whereas and ng produced arousal. maximal wakefulness was observed at dose of ng with no further increase in wakefulness at higher dose of ng. the results suggest the involvement of gaba-a receptors at the mpoa in sw. yoshiaki isobe , hiroyuki tsuda department of neuro-physiology and brain science, nagoya city university, graduate school of medical sciences, nagoya, japan; department of molecular toxicology, nagoya city university graduate school of medical sciences, japan locomotor activity in rodents shows free-running circadian rhythms even under the constant light. constant light exerts a promoting effect on hepatic carcinogenesis. after the partial hepatectomy, hepatic cell proliferation is regulated by circadian rhythm information (via wee ). to know the relation of proliferating factor (cell cycle) with circadian rhythmicity, locomotor activity against a diethylnitrosamine (den), widely used to initiate the hepatic neoplastic foci, is analyzed in preliminary. den was injected (i.p., mg/kg) on rats during the free-running condition under the constant dim light (dd) and constant light (ll). the effects of den were gentle under the dd. however, under the ll, phase delay accompanying the elongation of circadian period () was observed. decrement of an amount of activity in h after the den administration was obvious under the ll compared with that under the dd. this study was designed to investigate the central regulating system of hypothermia during maintenance phase of hibernation. although intracerebroventricular (icv) injection of naloxone (non-selective opioid receptor antagonist) and naloxonazine, ( antagonist) were effective, naltrindole (␦ antagonist) and nor-bni ( antagonist) did not interrupt the hibernation. the increment of c-fos expression was observed in arcuate nucleus (arc) at h after from hibernation onset compared with before hibernation. in addition, a localized ␤-endorphin-like immunoreactivity (␤-end ir) was observed in neuronal perikarya in arc at h after from hibernation onset. although ␤-end ir in arc got weak, the ␤-end ir of nerve fibers in preoptic nucleus (pon) got strong with progression of hibernation. these results suggest that the ␤-endorphin was transported to pon from arc by axonal flow and then played an important role in maintenance of hypothermia via -opioid receptors in hibernation. wei-min qu, zhi-li huang, naomi eguchi, yoshihiro urade, osamu hayaishi department of molecular and behavioural biology, osaka bioscience institute, osaka, japan prostaglandin (pg) d is a potent somnogenic substance, and isomerized from pgh through the action of pgd synthase (pgds). pgds has two distinct types, the lipocalin-type pgds (l-pgds) and hematopoietic pgds (h-pgds). selenium compounds have been reported to decrease sleep by inhibiting pgds in rats. to clarify what type of pgds inhibition is involved in sleep reduction by selenium or whether selenium intoxication decreases sleep, we intraperitoneally injected secl into l-pgds and h-pgds knockout (ko), and their wild-type (wt) mice. in wt mice, secl decreased rapid eye movement (rem) and non-rem sleep for h after injection and, concomitantly, increased wakefulness. similar results were observed in h-pgds ko mice. in contrast, l-pgds ko mice did not exhibit any significant changes in sleep-wake profiles after secl administrations. these findings indicate that pgd plays an essential role in the maintenance of the sleep state under physiological conditions, and l-pgds is a key enzyme for the production of pgd involved in sleep-wake regulation. under baseline conditions, h r ko mice showed essentially identical sleep-wakefulness cycles to those of wild-type (wt) mice but with fewer incidents of brief awakening (< s epoch), prolonged duration of non-rapid eye movement (nrem) sleep episodes, a decrease in the number of state transitions between nrem sleep and wakefulness, and a shorter latency for initiating nrem sleep after an intraperitoneal injection of saline. the h r antagonist pyrilamine mimicked these effects in wt mice. these results indicate that h r is involved in the regulation of behavioral state transitions from nrem sleep to wakefulness (huang et al., ) . ps a-h dissociation of responsibility in firing activity to dim light between the optic nerve and the suprachiasmatic nucleus neuron of mice koichi fujimura, ai fukushima, takahiro nakamura, toshihiro jogamoto, kazuyuki shinohara division of neurobiology & behaviour, nagasaki university, graduate school of biomedical science, nagasaki, japan the involvement of light response in the optic nerve to the firing activity of suprachiasmatic nucleus (scn) neuron was investigated by extracellular single unit recordings from the optic chiasma and the scn in mice. recordings were carried out during the early night in a light:dark cycle, and the illuminations were applied to a contralateral retina with a high-power led (λ = nm). the scn neurons responded to the light in intensities above photons/cm /s and were activated maximally at around photons/cm /s, they were about . log units less sensitive than optic fibers with high sensitivity. a sustained illumination in the intensity range between suprathreshold for the optic fibers and subthreshold for the scn neuron did not suppress the subsequent light response in the scn neurons, except in a few neurons. these results suggest that the most of the light responsive scn neurons are driven by any inputs independent of the high sensitive optic fibers. masayuki ikeda, tomoyoshi kojiya department of biology, faculty of science, toyama university, japan the hypothalamic suprachiasmatic nucleus (scn) has a pivotal role in the mammalian circadian clock. scn neurons generate circadian rhythms in action potential firings and neurotransmitter releases, and the core oscillation is thought to be driven by clock gene transcription-translation feedback loops. we have found robust circadian rhythms in the cytoplasmic concentration of ca + in scn neurons. since cytosolic ca + regulates diverse cellular systems, we have hypothesized that the cytosolic ca + rhythms may mediate the cellular output from the clock gene oscillations. here, to address the clock gene functions on the ca + rhythms, mouse bmal and its dominant negative sequence (mbmf r ) are transfected into the organotypic culture of scn with a yellow cameleon ca + sensor by the gene gun. the results demonstrated that over-expression of bmal or mbmf r significantly inhibited the circadian ca + rhythms and thus we concluded that the native bmal rhythm is essential for cellular output processes of the murine clock system. ps a-h the activation of ␣ adrenergic receptor increases the frequency of carbachol-induced ␤ oscillation in rat hippocampal slices masafumi nakano, jun arai, kiyohisa natsume kyushu institute of technology, kyushu, japan recently it is found that locus ceruleus (lc) activation suppresses ␤ rhythm in hippocampus in vivo. noradrenergic fibers derived from lc project to hippocampus. carbachol, a cholinergic agent, can induce ␤ oscillation in rat hippocampal slices like ␤ rhythm in vivo. in the present study, the effect of epinephrine on the generation of carbachol-induced ␤ oscillation in ca region of rat hippocampal slices. carbachol ( m) induced ␤ oscillation with the frequency and the amplitude of . ± . hz, and . ± . mv, respectively (mean ± s.e.m.; n = ). epinephrine ( m) significantly increased the frequency of . ± . hz (**p < . ), not change the amplitude. clonidine ( m), an ␣ receptor agonist, alone significantly increased the frequency at the concentration of m (*p < . ). yohimbine, an ␣ receptor antagonist, suppressed the oscillation. these results suggest that the application of adrenaline will increase the frequency of hippocampal ␤ rhythm via ␣ receptor. attractor dynamics of recurrent neural network are believed to play an important role in information processing in the brain. we recorded transient activities of two neuron groups by two tetrodes apart . mm from each other in the hippocampal ca region in vitro and applied micro-iontophoresis of glutamic-acid near the tetrodes to activate the neurons selectively. it was found that number of spikes during twosite (pairing) stimuli is fewer than the total number of spikes during the single-site stimuli, suggesting synaptic interaction in the network. peri-stimulus time histogram (psth) of ensemble as well as individual neuronal activity in response to the single-site stimuli applied far from the recording site composed of transient (latency - ms), oscillatory ( - hz) and sustained responses. following the pairing stimuli, the psth showed change in transient response properties ( / slices). these results suggest the pairing stimuli would change attractor dynamics of the neural network in the ca region. ryozo aoki , hiroshi wake , hitoshi sasaki , kiyokazu agata dept. physiol. & biosignal. osaka univ. grad. sch. med., suita, japan; dept. elec. eng. & elec. col. industri. tech., amagasaki, japan; dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan by insertion of a stainless-steel monopolar electrode to the head of planarian, continuous waveform of electrical potential could be first observed in microvolts. the frequency spectrum showed an almost monotonously decreasing distribution likely as /f, ranging from − to + hz. during the eeg recordings the planarian was kept still by cooling in several degrees. when it was cooled down to lower temperatures the amplitude of eeg was suppressed, and by warming again restored with spikes provably due to motions. this eeg active state continued beyond min after the electrode insertion but the amplitude gradually decreased, and became natural noise at the time up to min. by observing the sample it turns out the sticked head was degraded. strong photo stimulation suppressed this eeg signals and recovered after over min. however little response to light pulse stimuli was observed on the eeg spectrum. mariko uchida , hiroki sato , , naoki tanaka , atsushi maki , japan science and technology agency, crest, saitama, japan; advanced research laboratory, hitachi, ltd., saitama, japan previous studies about electroencephalography (eeg) described that alpha-wave power (the frequency band from to hz) decreases and the sleep spindle power (from to hz) increases in falling asleep. the purpose of this study is to analyze the crosscorrelation between the eeg power changes (eegpc) of each band and the cortical hemoglobin concentration changes (hbcc) during sleep. we measured optical topography (ot) and eeg simultaneously. the hbcc was measured at eighty-eight positions covering whole head of subject by ot probes. five females and eight males participated in this measurement. the results showed the high correlation between eegpc and hbcc at the location of dorsolateral prefrontal area, both in the period of (i) dominance of alpha-wave and (ii) dominance of sleep spindle. the time lag from eegpc to hbcc was from to s in (i), and from to s in (ii). we examine these differences between (i) and (ii) in detail. carnitine deficiency disturbs fatty acid oxidation under the fasting condition (fc). we show herein that nocturnal locomotor activity (la) was reduced under fc and recovered to normal by carnitine injection in jvs −/− mice, a model of systemic carnitine deficiency. as judged from eeg/emg profiles, jvs +/+ mice showed prolonged wakefulness under fc, but jvs −/− mice revealed disruption of the prolonged wakefulness with a high frequency of non-rem sleep. as the orexinergic arousal system plays an important role in la, we determined orexin neuronal activity in the fasted mice. fasted jvs −/− mice had fewer c-fos + orexin neurons in their lateral hypothalamus and a reduced orexin-a content in their csf, suggesting that the fasted jvs −/− mice exhibited reduced la and fragmented of wakefulness due to suppressed orexin neuronal activity. juhyon kim , kazuki nakajima , yutaka oomura , kazuo sasaki div. of bio-information eng., univ. of toyama, toyama, japan; dept. of integrat. physiol., kyushu univ., fukuoka, japan novel peptide, orexin, identified in the lateral hypothalamus (lh) participates in the regulation of sleep-wakefulness. orexin-containing neurons in the lh project to the pedunculopontine tegmental nucleus (ppt). the ppt is one of brain sites which control sleepwakefulness. thus, we examined effects of orexin on ppt neurons electrophysiologically using brain slice preparations in rats. applications of orexin depolarized the membrane potential of ppt neurons dose-dependently, and the depolarization was associated with the increase in membrane resistance. when extracellular k + concentration was increased, the magnitude of the depolarization significantly decreased. when extracellular na + was replaced by n-methyl-dglucamine, the magnitude of the depolarization also decreased significantly. these results suggest that the ionic mechanism for orexininduced depolarization includes k + channel, non-selective cation channel and/or na + /ca + exchanger, and that orexin participates in the regulation of sleep-wakefulness via the excitatory effect on ppt neurons. ben-shiang deng , wei zhang , akira nakamura , masashi yanagisawa , yasuichiro fukuda , tomoyuki kuwaki , dept. molec. integ. physiol., chiba univ., japan; dept. autonom. physiol., chiba univ., japan; dept. molec. genet., univ. texas, usa we examined whether the respiratory chemoreceptor reflex in prepro-orexin knockout mice (ko) was blunted or not, and if so, whether supplementation of orexin restored the abnormality. we also studied whether pharmacological blockade of orexin in the wildtype mice (wt) resulted in a similar abnormality. ventilation was recorded by whole body plethysmography before and after intracerebroventricular injection of orexin-a, -b, sb- (an orexin receptor antagonist), or vehicle. data were examined for only awake periods because sleeping distorts the chemoreflex. hypercapnic ventilatory responses but not hypoxic responses were attenuated in ko. similar abnormality was reproduced in wt treated with sb- . icv injection of orexin partially restored the hypercapnic chemoreflex in ko. our findings suggest that orexin plays a crucial role for co -sensitivity at least during waking periods. research funds: kakenhi , junko hara , taizo matsuki , katsutoshi goto , masashi yanagisawa , takeshi sakurai , department of pharmacology, basic medical science (coe), university of tsukuba, ibaraki, japan; yanagisawa orphan receptor project, erato, jst, tokyo, japan; howard hughes medical institute and department of molecular genetics, university of texas, dallas, texas, usa when the production of inflammatory cytokines is stimulated by acute inflammatory, the nonrem-sleep amount of animals increases. this is possibly due to changes in the biological activity of the tnfalpha system. besides their important function in sleep regulation during acute immune response, cytokines also seem to be involved in physiological sleep regulation. orexins (hypocretins) are recently identified neuropeptides that are derived from a common precursor peptide. recent studies suggest that specific degeneration of orexincontaining neurons occurs in brains of human narcolepsy patients, suggesting critical roles of these neurons in the regulation of vigilance states. here, we examined the effects of inflammatory cytokines on the activity of orexin neurons, by means of patch-clamp recording. these effects might also possibly be involved in the pathophysiology of narcolepsy. ps a-i prenatal exposure to bisphenol a enhances avoidance response to predator odor and impairs sexual differentiation of olfactory response of medial amygdala neurons tetsuya fujimoto , kazuhiko kubo , shuji aou dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; dept. otorhinolaryngol., chidoribashi hospital, fukuoka, japan prenatal exposure to bisphenol a (bpa) impairs the sexual differentiation of exploratory behavior and enhances depressive behavior (fujimoto et al. ) . in this study, the effects of bpa on general motor activity and avoidance response to predator odor and olfactory responses in medial amygdala neurons were examined. the smell of fox predominantly suppressed locomotor activity and enhanced avoidance response by bpa. in the electrophysiological study, male medial amygdala neurons showed selective excitatory responses to predator odors. this type of neurons did not respond to plant odors. in contrast female amygdala neurons did not show such selectivity. the sex difference in this neuronal response pattern was attenuated by bpa exposure. these findings suggest that bpa impairs sexual differentiation of medial amygdala neurons which affect emotional responses to the olfactory cues of predators. research funds: grants-in-aid for scientific research (no. , s.a.) shuji aou , tetsuya fujimoto , yumi ichihara , kimiya narikiyo , toru ishidao , hajime hori , yukiko fueta dept. brain sci. eng., kyushu inst. technol., kitakyushu, japan; dept. environm. manage., sch. health sci., univ. occup. environm. health, kitakyushu, japan -bromopropane ( -bp), an ozone-depleting substance replacement, has neurotoxicity and exhibited reproductive toxicity in adult animals. in this study, we investigated the effects of prenatal exposure to -bp on sexual differentiation of reproductive and non-reproductive behaviors. pregnant rats were exposed to ppm of -bp during prenatal period. the open-field test, lashley iii maze test and sexual behavior were evaluated at adult age. -bp significantly reduced the locomotor activity and the number of entries into the center area in female rats but not in males in the open-field test. in sexual behavior, the number of ear wiggles, an index of proceptive behavior, was decreased and the rejection score was increased in female rats. these results suggest that -bp is the potential candidate of endocrine disruptors which affect brain development. ( ) ps a-i changes in hippocampal excitability of rats prenatally exposed to -bromopropane yukiko fueta , toru ishidao , susumu ueno , yasuhiro yoshida , hajime hori department of environmental management, school of health sciences; department of pharmacology; department of immunology, school of medicine, university of occupational and environmental health, kitakyushu, japan inhalation exposure to -bromopropane ( -bp), a substitute for ozone depleting compounds, alters the function of gabaergic system in the hippocampus of adult male rats. but the neurotoxcitiy induced by prenatal exposure has not been well investigated. in this study pregnant rats were exposed to -bp ( ppm) during gestational day - ( h/day), and the hippocampal excitability in pregnant rats and their offspring was examined. basic excitability was enhanced and disinhibition was observed in the hippocampus of pregnant rats. offspring, however, exhibited an enhancement of averaged s/r curve of ps in the ca at the pnd - . conversely, s/r curves of fepsp as well as ps in the ca were inhibited at the age of - weeks. our results suggest that -bp causes hyperexcitability in pregnant rats, and disrupts basic excitability in the ca of the offspring during development. research funds: grant-in-aid for exploratory research ( ) ps a-i effects of endocrine disrupting chemical bisphenol a on the development of mouse cerebral cortex keiko nakamura , , kyoko itoh , takeshi yaoi , tohru sugimoto , shinji fushiki dept. pathol. appl. neurobiol., kyoto pref. univ. med, kyoto, japan; dept. pediatr., kyoto pref. univ. med, kyoto, japan bisphenol a (bpa), a widely distributed xenoestrogen, has been shown to disrupt thyroid hormone function. we have thus studied whether prenatal exposure to low-doses of bpa affects morphology and the expression of thyroid hormone-dependent genes in murine fetal neocortex. pregnant mice were injected subcutaneously g/kg of bpa daily from embryonic day (e ). control animals were injected vehicle alone. for evaluating cell proliferation, neuronal differentiation and migration, bromodeoxyuridine (brdu) was given to pregnant mice and processed for immunohistochemistry. the total rna was extracted from embryonic telencephalons at different embryonic period. brdu-labeled cells were decreased in the ventricular zone at e . and e . , whereas those cells increased in the cortical plate at e . , as compared with control mice. some of the genes associated with neurogenesis and thyroid hormone function were upregulated in bpa-treated group. research funds: jsps grant keiko ikemoto , teruko uwano , hisao nishijo , taketoshi ono , masayuki ito , ikuko nagatsu , katsuji nishi , shin-ichi niwa dept. neuropsychiat., fukushima med. univ. sch. med.; toyama med. pharm. univ.; faculty med., mie univ., mie, japan; fujita health univ. sch. med., toyoake, japan; dept. leg med., shiga univ. med. sci., japan we examined the effect of maternal repeated cold stress (rcs) on development of catecholamine neurons of offsprings using by tyrosine hydroxylase (th) immunohistochemistry. rcs was loaded to pregnant rats between day and after fertilization. pups were perfused at postnatal day . in the frontal cortex, the number of largesized (more than m in diameter) th-immunoreactive (-ir) varicosities was significantly smaller in prenatally rcs rats than controls. in the locus coeruleus of prenatally rcs rats, th immunoreactivity was less than that of controls. in the medullary c /a catecholaminergic field, the size of th-ir neurons was smaller and the quantity of thir fibers were less in prenatally rcs rats, although there were not significant differences. it was suggested that prenatal rcs impaired development of catecholaminergic neurons, especially noradrenergic neurons of neonates. ps a-i developmental exposure to pentachlorophenol affects thyroid hormone responsive gene in the brain but not stress response maiko kawaguchi , , , kaori morohoshi , , rie yanagisawa , erina saita , gen watanabe , , masatoshi morita , kazuyoshi taya , , hirohisa takano , , toshiyuki himi , , hideki imai , dept. toxicol and pharmacol., facul. pharmacy, musashino univ., tokyo, japan; res. inst. pharmaceut. sci., musashino univ., tokyo, japan; nation. inst. for environ. stud., ibaraki, japan; grad. sch. environ. sci., univ. tsukuba, ibaraki, japan; wildlife rescue veterinarian associ., tokyo, japan; facul. agriculture, tokyo univ. agriculture & technol., tokyo, japan; the united grad. sch. veterinary sci., gifu univ., gifu, japan; div. environ. health sci., dep. social med., facul. med., miyazaki univ., miyazaki, japan antiseptic pentachlorophenol (pcp) treatment to rats affects thyroid hormone (th) system, which is essential for normal development of central nervous system. in this study, we show the exposure to pcp during gestation and lactation suppressed plasma th level, and induces gene expression of neurogranin and th receptor ␤, which play a role in neural formation. the present data suggest that pcp may affect central nervous system development, though stress response was not affected by pcp exposure. ps a-i the effect of psychological stress during pregnancy on the open-filed behavior, the forced swim test, the fos expression in the brain, and the level of plasma corticosterone in offspring rat hiroshi abe, noriko hidaka, kei odagiri, yuko watanabe, yasushi ishida dept. of psychiatry, miyazaki med. coll., univ. of miyazaki, japan one group (psy) was born from the dams which observed, during their pregnancy, that another rat was exposed to the foot-shock stress in a communication box. the other group (c) was born from the dams not exposed to such stress. psy, comparing to c, showed decreased activities in the open-field test and prolonged immobility time in the forced swim test. on the other hand, there were no significant differences between the number of fos immunopositive cells in various regions of the brain in two groups before and after the foot-shock. however, plasma corticosterone was elevated in psy compared with c. these results suggest that the prenatal psychological stress might enhance reactivity to novel environment and depressive behavior induced by forced swim, and chronically elevated level of corticosterone might be involved in this neurobiological substrate. akane nakasato, yasushi nakatani, yoshinari seki, hideho arita department of physiology, toho university school of medicine, tokyo, japan to evaluate roles of da and serotonergic ( -ht) systems in stressinduced anxiety, we measured brain da and -ht levels before, during and after a forced swimming test (fst) in autistic model of the rat. the model rat was made by exposing a pregnant rat to valproic acid (vpa). our previous study demonstrated that the autistic model exhibited abnormality of -ht system and behavioral impairments related to autism. in the present experiment, we gave a prolonged fst for min in the model rat, which frequently experienced to be drowned after the immobility time during fst. brain da and -ht levels were measured from samples collected from the prefrontal cortex (pfc). we found a gradual and steady increase in pfc da level during fst, although -ht level showed only transient augmentation. behavioral alteration after fst was characterized by an increased appetite during light phase (sleep) of circadian cycle. we suggest that the feeding abnormality may be caused by the stress-induced anxiety mediated by mesocortical da system. shigeo masaki, eiko aoki, satoshi yonezawa, atsuo nakayama dept. embryology, inst. developmental res., kasugai, aichi, japan neuroligin (nl - ) is a family of neuronal cell-surface proteins to be involved in intercellular junctional formation and signalling. recently, several studies have implicated nl and nl in autistic disorders. nls have a relative identical structure (∼ %); nl and nl localize in the glutamatergic excitatory, and inhibitory synapses, respectively, while nl seems express in the olfactory glia, but nl distribution is unknown. here we have generated antibody against human nl , and explored its distribution in the post mortem human tissues. in the central and peripheral nervous system, nl was expressed exclusively in the neurons, and was especially abundant in particular subsets of neurons, including neurons producing nonapeptides. nl was observed in paraneurons and some endocrine cells outside the cns. these results suggested that nl is important for neuroendocrine function. nl cdna was transfected to in neuroblastoma. formed spine-like structures on the cells expressing nl were rough and thicker than those of nl or nl transformants. it suggested the unique activity of nl for synapse formation. motopsin is a serine protease secreted from pyramidal neurons of cerebral cortex and hippocampus. recently, the truncation of motopsin gene has been reported to cause non-syndromic mental retardation. however, the underlying mechanisms are yet to be elucidated. we report here that the knockout (ko) mice deficient in the expression of motopsin exhibit morphological abnormality. golgi-cox staining revealed that spine density on both apical and basal dendrites of hippocampal ca pyramidal neurons in the ko mice significantly decreased than in the wild type mice. similarly, spine density tended to decrease at cingulate cortex of the ko mice than wild type. our results suggest that motopsin affects dendritic spine formation and/or stabilization. mental retardation is a frequent disorder affecting - % of the population. recently the truncation of motopsin/neurotrypsin gene has been identified in algerian family in which four out of eight children affected by a severe impairment of cognitive functions with an iq below . here we report that knockout (ko) mice lacking motopsin gene mildly impaired water maze performance and social behavior. the ko mice significantly delayed the latency to the platform area on a probe test of hidden version of morris water maze although they showed the similar performance to wild-type mice during training session. in a social memory test, the ko mice showed significant elongation of sniffing time to an intruder, despite of normal performance of social memory. our results suggest that the ko mice provide insights into the molecular mechanisms important for development of cognitive functions. natsue yoshimura , daisuke horiuchi , tomoyuki miyashita , minoru saitoe , hitoshi okazawa department of neuropathology, medical research institute, tokyo medical and dental university, tokyo, japan; tokyo metropolitan institute for neuroscience, tokyo, japan polyglutamine tract binding protein- (pqbp- ) was originally isolated as one of the candidates for polyglutamine disease related protein. recently, several groups has reported about pqbp- disease that pqbp- mutant causes x-linked mental retardation (xlmr). to investigate the function of pqbp- in xlmr pathology, we produced two kinds of flies, human pqbp- overexpression flies (hpqbp flies) and drosophila pqbp- knock-down flies (dpqi flies), and examined olfactory learning and memory to analyze their memory consolidation process from short-term memory (stm) to long-term memory (ltm). the hpqbp flies showed memory impairment in ltm. in current study, we analyze memory abilities of the dpqi flies to observe detailed function of pqbp- in memory formation. seiji hayashizaki, masahiko takada tokyo metropolitan institute for neuroscience, usa when two alternatives are available in instrumental behavior, animalǐs behavior is biased toward responding on one lever with which each behavioral response results in delayed large reward delivery, and against responding on the other lever with which each response results in immediate but small reward delivery. this has been used as an index of impulsive behavior and is known to be susceptible to lesions of brain structures such as the basolateral amygdala (bla) and the nucleus accumbens (na). it has been shown that the bla and na are involved in maintaining reward seeking behavior with a secondary reward when a secondary reinforcer is available. thus, a question arises as to how the behavioral response on the delayed lever is maintained through functions exerted by these structures when no secondary reinforcer is available. to this end, we implanted cannulae bilaterally and electrodes into the bla and na to identify neuronal substances and activities involved in the mediation of 'putative secondary reward' without secondary reinforcer. xue-zhi sun , sentaro takahashi , yoshihisa kubota , rui zhang , chun cui , yoshihiro fukui natl. inst. radiol. sci., chiba, japan; sch. med. tokushima univ., tokushima, japan heavy ion irradiation has the feature to administer a large radiation dose in the vicinity of the endpoint in the beam range, and its irradiation system and biophysical characteristics are different from ordinary irradiation instruments like x-or gamma-rays. using this special feature, heavy ion irradiation has been applied for cancer treatment. the safety and efficacy of heavy ion irradiator have been demonstrated to a great extent. for instance, brain tumors treated by heavy-ion beams became smaller or disappearance. however, fundamental research related to such clinical phenotypes and their underlying mechanisms are little known. in order to clarify characteristic effects of heavy ion irradiation on the brain, we developed an experimental system for irradiating a restricted region of the rat brain using heavy ion beams. the characteristics of the heavy ion beams, histological, behavioral and elemental changes were studied in the rat following heavy ion irradiation. yukio imamura department of psychiatry, university of ottawa, on, canada nmdars contain two nr subunits paired with two nr subunits. nr and nr (a-d) subunits harbor the glycine and glutamate binding sites, respectively. nmdars are localized in both synaptic and extra-synaptic areas, but they are found at higher density within the synapse. after the peak of synaptogenesis, the nr /nr a complex, characterized by rapid offset kinetics, dominates at the synapse, while the nr /nr b complex, characterized by slow kinetics, predominates in the extra-synaptic area. the activation of extrasynaptic nmdars by glutamate escaping from the synaptic cleft during episodes of high synaptic activity suggests that they may have a different role. using whole-cell voltage-clamp recordings from ca pyramidal neurons from mice (at weeks of age), we found that following induction of ischemia, ifenprodil, a selective nmdar-nr b antagonist, reduced the inward current of the isolated nmdar at extra-synaptic site while it had less effect at the synaptic nmdar. the molecular mechanisms involved are currently under investigation and these new data will be also presented at the meeting. in the present study, we observed expression and changes of mineralocorticoid receptor (mr) and glucocorticoid receptor (gr) in the gerbil hippocampal ca region after ischemia. in blood, corticosterone levels increased biphasically at min and h after ischemia, and thereafter its levels decreased. in the sham group, mr and gr immunoreactivities were weakly detected in the ca region. by days after ischemia, mr and gr were not significantly altered in the ca region. from days after ischemia, mr and gr immunoreactivities were detected in astrocytes and microglia in the ca region, and at days after ischemia. the specific distribution of corticosteroid receptors in glia may be associated with the differences of mr and gr functions against ischemic damage. the present study was investigated the effects of early treadmill training after cerebral infarction in rats. we determined whether treadmill exercise changes cellular expression of caspase- and midkine in the mca area. stroke was induced by a -min mca occlusion using an intraluminal filament. rats were exercised for min each every day on a treadmill. brain damage in ischemic rats was evaluated by infarct volume. exercised and non-exercised rat brains were processed for immunocytochemistry to quantify the areas of caspase-and mkimmunoreactive calls. no significant differences in infarct volume were found between rats trained with treadmill and non-exercised controls. cellular expressions of mk were significantly increased in striatum (glia) of the exercised rats. treadmill exercise was shown to suppress the decrease in caspase- expression in the penumbra. the present study showed the exercise after cerebral infarction might have important implication for post-ischemic recovery. ps a-j reversed astrocytic glutamate transporter glt- crucial to the ca + paradox-like insult-induced neuronal death in neuron/astrocyte co-cultures tatsuro kosugi, koichi kawahara, takeshi yamada, motoki tanaka lab. of cellular cybernetics, graduate school of information science and technology, hokkaido univ., sapporo, japan "ca + paradox" is the phenomenon whereby the intracellular concentration of ca + paradoxically increases during reperfusion with normal ca + -containing media after brief exposure to a low ca + solution. the present study aims to characterize the ca + paradoxinduced cell injury in neuron/astrocyte co-cultures. prior exposure of the cultures to a low ca + solution for min significantly injured only neurons after reperfusion with a normal ca + medium for h, but astrocytes remained intact. after the onset of reperfusion, the intracellular concentration of na + in astrocytes increased significantly during the reperfusion episode, resulting in a reversal of the operation of the astrocytic glt- . the present findings suggested that ca + paradox-induced accumulation of na + in astrocytes was involved in the reperfusion-induced excitotoxic neuronal injury resulting from the reversed operation of astrocytic glt- during the reperfusion episode. common genetic mutation in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (cadasil) has been associated with missense mutations of notch concerning cysteine residues within the extracellular amino-terminal region. we report new mutations of two japanese cadasil families, which did not directly involve a cysteine residue. exons of the notch were amplified by pcr and subsequently analysed for dhplc and direct sequence. the first patient carried the missense mutation c t, which results in pro ser. the second patient carried the missense mutation c g, which results in arg pro. new mutations had not changed the number of cysteine residues, but coding the extracellular amino-terminal region of the notch receptor which may involve an alteration in the ligand binding or putative dimerisation properties. ps a-j mci - , a radical scavenger, protected cortical neurons from cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol kinase madinyet niyaz , tadahiro numakawa , yoshinori matsuki , emi kumamaru , yuki yagazaki , harumi kitazawa , hiroshi kunugi , motoshige kudo pathology department of tokyo medical university, tokyo, japan; department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan the role of mci - , a radical scavenger, in the central nervous system (cns) has not been fully elucidated. in the present study, we found that treatment with mci - prevented the cultured cortical neurons from cell death induced by serum deprivation. furthermore, we found that mci - exposure induced the activation of both the map kinase (mapk) and pi kinase (pi k) pathways and that the mci - -dependent survival effect was blocked by the inhibitors, u (an mapk pathway inhibitor) or ly (a pi k pathway inhibitor). these results suggested that mci - exerts a protective effect on cns neurons via enhancing survival-signaling pathways in addition to a role such as a radical scavenger. osamu tokumaru , noriko yoshimura , tetsuro sakamoto , takaaki kitano , naoko nisimaru , isao yokoi dept. physiol., sch. med., oita univ., japan; med. edu. ctr., sch. med., oita. univ., japan protective effects of ethyl pyruvate (ep) on energy metabolism of rat brain exposed to ischemia were investigated by p-nuclear magnetic resonance ( • c). brain slices were incubated in standard artificial cerebrospinal fluid (acsf) with mm ep (ep- ), acsf replaced by acsf with mm ep after ischemia (ep- ), or acsf only (control). the brain slices were exposed to ischemia by stopping the perfusion for h. high-energy phosphate, creatine phosphate (pcr) and ␥-atp, levels were measured. decrease in pcr level was not different among the three groups when exposed to ischemia. but increase in pcr level after the reperfusion was significantly larger in ep- than in control (p < . ). these results indicate that ep is effective in the reperfusion period and is more protective when administered before ischemic exposure. the importance of timing of administration of ep in clinical use was suggested. research funds: grant-in-aid for scientific research (c) # from mext to t.k. hideaki tamai , kuniko shimazaki , norimasa seo department of anesthesiology and critical care medicine, jichi medical university, graduate school, tochigi, japan; department of physiology, jichi medical university, tochigi, japan we investigated the effects of acupuncture on cell proliferation in the dentate gyrus (dg) and the lateral ventricle (lv) of adult rats. in this study, acupuncture was performed at the acupoints neiguan (pc ), yintang (ex-hn ) and sanyinjiao (sp ), which have been used for the enhancement of conscious and functional recovery in stroke patients. eight weeks old male wistar rats were used in the experiment. through -bromo- ,-deoxyuridine (brdu) immunohistochemistry, a significant increase in cell proliferation in the dg of the acupunctured group was observed. however, the cell proliferation in the lv was not affected with the acupoints pc , ex-hn and sp . the present findings indicate that the sensitivity on cell proliferation in the dg by acupuncture stimulation is higher than in the lv. yukio ago , keiko takahashi , shigeo nakamura , akemichi baba , toshio matsuda laboratory of medicinal pharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan; laboratory of molecular neuropharmacology, graduate school of pharmaceutical sciences, osaka university, osaka, japan this study examined the effect of isolation rearing on anxiety-related behavior of mice in the staircase test, an animal model of anxiety. the staircase test consisted of placing an experimentally naive mouse in an enclosed staircase with five steps. in group-reared mice, an anxiolytic diazepam increased the number of steps climbed to the top step of the staircase, but did not affect the frequency of rearing behavior. the anxiogenic drug ␤-cca increased the number of rearing, but did not affect the number of steps climbed. on the other hand, methamphetamine increased the number of steps climbed to the second step. in these circumstances, isolation-reared mice showed an increase in the numbers of steps climbed to the top step and rearing in the staircase. these findings suggest that isolation rearing increases in exploratory and anxiety-like behaviors in mice. tomonori fujiwara , tatsuya mishima , takefumi kofuji , kimio akagawa department of cell physiology, kyorin university school of medicine, mitaka, tokyo, japan; radio isotope laboratory, kyorin university school of medincine, mitaka, tokyo, japan hpc- /syntaxin a is believed to regulate the exocytosis of synaptic vesicles. in order to examine the neurophysiological function in vivo, we have produced hpc- /syntaxin a knock-out mice. surprisingly, the null mutant mice revealed normal development and basal synaptic transmission in cultured hippocampal neurons appeared to be normal. however, in conditioned fear memory test, consolidation of the memory was impaired in homozygous mutant mice but not in heterozygote. however, once memory consolidation was acquired, the extinction process was disturbed in homozygote. we further examined latent inhibition of cued fear memory (li) to access behavioral property. interestingly, li was suppressed both in heterozygous and homozygous mutant mice unlike the case of conventional conditioned fear memory test. implication of these behavioral abnormalities in hpc- /syntaxin a knock-out mouse will be discussed. research funds: kakenhi ( ) ps a-k effects of local administration of the gaba agonists into the hippocampus ca area on active avoidance learning and serotonergic systems in the administration area in rats satoko hatakenaka , hiroko miyakubo , junichi tanaka , yasushi hayashi , yukio hattori , masahiko nomura department of curriculum, teaching and memory, naruto university of education, tokushima, japan; department of human nutrition, notre dame seishin university, okayama, japan; department of physiology, saitama medical school, saitama, japan in fischer male rats, bilateral injections of the ␥-aminobutyric acid (gaba) a agonist muscimol into the ca area slightly decreased the avoidance rate in an active avoidance task. similar injections of the gaba b agonist baclofen enhanced the avoidance rate. there are significant differences between the muscimol-and baclofen-treated groups in the avoidance rate, implying that gaba a and gaba b receptors have the opposite action on the performance of avoidance learning. perfusion with muscimol through the microdialysis probe decreased the serotonin metabolite -hydroxytryptamine ( -hiaa) concentration in the ca area, whereas baclofen perfusion had no effect, suggesting that the gabaergic system may exert to inhibit the serotonin release in the ca area through gaba a receptors. sawako arai, taku nagai, kenji takahashi, hiroyuki kamei, kazuhiro takuma, kiyofumi yamada lab. neuropsychopharmacol, kanazawa univ., kanazawa, japan we performed immunohistochemical c-fos mapping after a prepulse inhibition (ppi) test of the startle reflex in mice. startle stimulus increased the number of c-fos-positive cells in the somatosensory cortex, nucleus accumbens shell and the caudal pontine reticular nucleus (pnc), while prepulse trials without startle stimulus increased c-fos expression in the lateral globus pallidus (lgp). in mice subjected to startle stimulus with prepulses, most of the startle stimulus-induced c-fos expression was diminished but c-fos expression remained in the lgp. prepulse-induced c-fos expression in the lgp was colocalized with gad- . fluoro-gold infusion into the pnc and the pedunculopontine tegmental nucleus (pptg) retrogradely labeled neurons in the pptg and lgp, respectively. microinjections of phaclofen, but not picrotoxin, into the pptg impaired ppi of the startle reflex. these results suggest that gabaergic neurons in the lgp which project to the pptg play a crucial role through the activation of gaba b receptors in the ppi of the startle reflex. shiho kitaoka , sho koyasu , akinori nishi , tomoyuki furuyashiki , toshiyuki matsuoka , shuh narumiya department of pharmacology, university of kyoto, kyoto, japan; department of physiology, university of kurume, kurume, japan prostaglandins e (pge ) exert their actions in various organs through specific receptor, ep to . the previous study suggests that ep modulates da system. to investigate the roles of ep in da system, we examined ep ko mice with behavioral sensitization induced by cocaine. the administration of cocaine elevated da concentration in the nucleus accumbens up to ∼ % in both wild-type and ep ko mice. however, increase of locomotor activity in ep ko mice was significantly lower than that in wild-type mice. because locomotor activity is closely related to dopamine d receptor (d r) signaling, we tested the density of d r and d r signaling with phosphorylation of darpp- . there were no differences in d r binding. d r signaling was significantly attenuated in the striatal slices from ep ko mice. the effect of d r agonist on locomotor activity was also attenuated in ep ko mice. these results indicate that pge has enhancing effects on locomotor activity via ep by potentiating the d r signaling. central serotonin ( -ht) function has been implicated in impulsivity. the present study examined rats with -ht depletion by parachloroamphetamine (pca) in simple and reversal go/no-go visual discrimination tasks, and analyzed the relationships between learning performance and focal concentrations of -ht and its metabolites ( -hiaa) in the brain. for both tasks, significant negative correlations between learning performance and -ht and -hiaa concentrations were observed in the medial prefrontal cortex and nucleus accumbens. in contrast, for reversal task only, significant correlations between learning performance and -ht and -hiaa concentrations were observed in the orbitofrontal cortex and amygdala. these data suggest the regional difference of -ht roles on selective indices of impulsivity. yuki sato , , , tatsushi onaka , norimasa seo , eiji kobayashi dept. anesthesiol., jichi med. univ., tochigi, japan; dept. physiol., jichi med. univ., tochigi, japan; div. organ replacement research, center for mol. med., jichi med. univ., tochigi, japan cyclosporine is widely used for preventing allograft rejection. however, in a considerable number of transplant recipients, cyclosporine causes neuropsychological side effects such as confusion, depression, and anxiety. cyclosporine inhibits calcineurin activity and forebrain-specific calcineurin knockout mice exhibit deficits in social behaviour. it is thus possible that cyclosporine causes psychological side effects via disturbing social interactions. here, we examined effects of cyclosporine upon anxiety and social behaviour in mice. calcineurin did not significantly change percent entries into open arms and time spent on open arms in the elevated plus maze test. on the other hand, in the social interaction test in home cage, cyclosporine increased the number of particles in home cage, an index of social activity. all these data suggest that impaired social interaction is a cause of psychological side effects of cyclosporine. to investigate the distribution of functionally activated vestibularrelated brainstem neurons during postnatal development, ombined immuno-/hybridization histochemistry of c-fos expression was performed in sprague-dawley rats (p - ; adult). conscious animals were subjected to rotational or translational stimulus which activates hair cells of the horizontal semicircular canals or utricle, respectively. neuronal activation within brainstem nuclei was defined by the expression of c-fos. labyrinthectomized controls and normal stationary controls showed only a few sporadically scattered fos-expressing neurons. with rotational stimulation that comprised cycles of constant angular acceleration and deceleration, fos-labeled neurons were observed by p in the vestibular nucleus and downstream relay stations of vestibular pathways, such as the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc, ioa, ioc, iok). a later maturation time was evidenced for the utricular system. fos-labeled neurons were only identifiable in the vestibular nucleus by p ; in the prepositus hypoglossal nucleus and inferior olive (subnuclei dmcc and io␤) by p . within the vestibular nucleus of p - rats, neurons activated by canal or utricular inputs were intermingled throughout its rostro-caudal length. in p and adult rats, neurons activated by canal or utricular inputs were intermingled in localized regions of the medial and spinal vestibular nuclei. however, neurons in the rostral half of spinal vestibular nucleus were activated only by utricular inputs. taken together, we have demonstrated that canal-and otolithrelated brainstem neurons that encode rotational and translational movements in the horizontal plane are histologically segregated and exhibit different developmental time frame. to determine whether perineuronal nets (pn) within the vestibular nuclei contribute to plasticity of central connectivity, we studied the presentation of pn within the vestibular nuclei during development (rats, p to adult) and after unilateral labyrinthectomy (ul) in the adult. histochemistry with the lectin wisteria floribunda agglutinin was used to map pn about neun-immunopositive neurons within the vestibular nuclei. in normal postnatal rats, pn was detectable by p in the vestibular nucleus as fuzziness about neuronal cell bodies. from p onwards, the fuzzy pn progressively consolidated into a network organization. the fuzziness was no longer observable after p . during postnatal development, the number of neurons showing pn increased with age, reaching the adult level by p . with ul, the pn network on the lesioned side remained compact until days post-lesion when the fuzziness reminiscent of that in early postnatal rats became evident. by days after ul, the pn of some neurons resumed the network pattern as was observed in normal adult rats. this phenomenon was found in the pn of the remaining neurons by days after ul. the pn on the labyrinth-intact side showed the compact network of uninjured age-matched rats. taken together, our findings indicate pn changes that suggest possible correlation with vestibular nuclear neuronal function both during postnatal development of normal rats as well as in adult rats following destruction of the ipsilateral inner ear. minori ueda, takayuki suzuki, hiroyoshi miyakawa laboratory of cellular neurobiology, tokyo university of pharmacy and life science, tokyo, japan dynamics of transmitters in the synaptic cleft depends on many processes such as transmitter release, uptake and diffusion. to better understand these processes, we analyzed ampar-and nmdarmediated epscs and synaptically induced transporter currents (stcs) elicited with high-frequency stimuli. recordings were made from pyramidal cells and astrocytes in the ca region of rat hippocampal slices, hz/ pulse tetanic stimulations were delivered to schaffer-collaterals, and the evoked currents during the course of tetanic stimulation were isolated. the decay time course of the last isolated stc during the tetanic stimulation was not significantly different from that of the first. while the amplitude of the ampar-mediated epscs showed significant decay in the presence of cyclothiazide, there was no marked decay of the amplitude of the nmdar-mediated epscs. these findings imply that synaptic fatigue and saturation of glutamate transporters do not take place during the course of high-frequency stimulation at hz. ikuko yao , hiroshi takagi , hiroshi ageta , tomoaki kahyo , ken hatanaka , kaoru inokuchi , mitsutoshi setou , mitsubishi kagaku institute of life sciences, tokyo, japan; university of tokyo, tokyo, japan; okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan we identified and characterized a novel ubiquitin ligase named scrapper. scrapper is an f-box protein which has leucine rich repeat and c-terminal membrane localization sequence, highly expressed in neurons throughout the brain. to investigate the physiological role of scrapper in the neuron, we recorded mepscs from the neuron over-expressed the egfp-tagged full-length scrapper construct or truncated form of scrapper constructs. they exhibited a strong suppression or enhancement in the frequency of mepscs while showing a non-significant change in mepsc amplitude, rise, and decay time compared with neurons expressing egfp. the passive membrane properties of neurons such as membrane resistance (rm), series resistance (rs), and membrane capacitance (cm) were not statistically different from those of control. these data suggests a presynaptic effect of scrapper protein. ps p-a presynaptic membrane potential-dependent regulatory mechanism of transmitter release tetsuya hori, tomoyuki takahashi department of neurophysiology, university of tokyo graduate school of medicine, tokyo, japan in simultaneous pre-and postsynaptic recordings at the calyx of held, we addressed the mechanism underlying presynaptic membrane potential-dependent changes of transmitter release. a weak sustained depolarization (e.g., mv, s) of calyceal nerve terminal potentiated epscs despite that it diminished presynaptic action potential (a.p.) amplitude. as we further depolarized the terminal epscs became eventually depressed concomitantly with a marked reduction in the a.p. amplitude. when presynaptic ca + currents (i pca ), induced by an a.p.-waveform command pulse, were used to evoke epscs, a weak sustained depolarization enhanced i pca and epscs in parallel. this epsc facilitation was robust at the calyx of held both in rats and mice, but was almost absent in p/q-type ca + channel knockout mice. we conclude that the p/q-type specific ca + channel facilitation plays an essential role in the facilitation of transmitter release following presynaptic depolarization. hiroshi takagi , koji ikegami , ken hatanaka , , , yoko fujiwara-tsukamoto , mineo matsumoto , ikuko yao , mitsutoshi setou , , mitsubishi kagaku institute of life sciences, japan; presto, japan; school of pharmaceutical sciences, the university of tokyo, japan; okazaki institute for integrative bioscience, japan a variety of post-translational modifications to the exposed cterminal tails of tubulin, such as detyrosination/tyrosination, polyglycylation and polyglutamylation would play a crucial role in the neuron. however, evidence for the implication of these modifications in regulating the translocation of channels and receptors is currently unavailable. of the modifications, polyglutamylation is highly abundant in the mammalian brain, thus, this modification might account for the translocation of channels and receptors in the mammalian brain. in the rosa (−/−) mouse, which shows a gross loss of polyglutamylated ␣-tubulin, transient a-type currents were largely suppressed in hippocampal pyramidal neurons in vitro. we provide herein, using rosa mice, the evidence for the implication of ␣tubulin polyglutamylation in the regulation mediated a-type k current. satoshi kawasaki , shingo kimura , reiko fujita , shuji watanabe , kazuhiko sasaki dept. of physiol., sch. of med., iwate medical univ., morioka, japan; dept. of chem., sch. of lib. arts & sci., iwate medical univ., morioka, japan application of dopamine (da) induces a slow na + -current response in the identified neurons of aplysia ganglia under voltage clamp. this type of response is produced by the activation of trimeric g-protein sensitive to cholera toxin (ctx) as previously reported. the na +current response to da was gradually and irreversibly depressed after intracellular injection of clostridium difficile toxin b, which is known to inactivate all types of rho family g-proteins. intracellular application of clostridium botulinum exoenzyme c , a specific toxin to rhoa-c, also depressed the da-induced response irreversibly. furthermore, the da-induced current response was significantly depressed by gap domain of p rhogap applied intracellulary. in contrast, gef domain of rhogef dbs had a tendency to increase the response. these results suggest that the da-induced na + -current response may be regulated by the activation of rho family g-protein. the ␦ glutamate receptor (␦ r) plays a crucial role in cerebellar functions. although ␦ r has a putative channel pore domain, and ␦ r displayed ca + -permeable channel activities in lurcher mutant mice, it has been unclear whether wild-type ␦ r functions as a channel. here we introduced a ␦ r transgene, which had a mutation (gln arg) in the putative channel pore conserved in ca + -permeable glutamate receptors, into ␦ −/− mice. surprisingly, a mutant ␦ r transgene, as well as a wild-type transgene, rescued all abnormal phenotypes of ␦ −/− mice, such as ataxia and loss of long-term depression. these results indicate that ca + influx through ␦ r is not required for its function in the cerebellum in vivo, and that wild-type ␦ r may not function as a ca + -permeable ion channel. research funds: kakenhi ( ) and takeda science foundation ps p-a distribution of tarp - on hippocampal neurons and its key role in synaptic and extrasynaptic expression for ampa receptors masahiro fukaya , mika tsujita , maya yamazaki , etsuko kushiya , manabu abe , kaori akashi , masanobu kano , haruyuki kamiya , kenji sakimura , masahiko watanabe department of anatomy, hokkaido university school of medicine, sapporo, japan; department of cellular neurobiology, brain research institute, niigata, japan; department of cellular neuroscience, graduate school of medical science, osaka university, suita, japan; department of molecular anatomy, hokkaido university school of medicine, sapporo, japan the - is one of four transmembrane ampar regulatory proteins (tarps). pre-and post-embeding immunogold visualized - on excitatory synaptic and extrasynaptic membrane. in - -ko mice, ampars were reduced in hippocampal homogenates ( % of control) and psd fraction ( %). immunogold labeling also exhibited reduction of extrasynpatic ( %) and synaptic ( %) ampars in ca pyramidal cells. the reduction of extrasynaptic receptors was particularly severe on dendrites ( %) and spines ( %). ampar-mediated responses were reduced at ca synapses ( %). therefore, - is the major auxiliary subunit of hippocampal ampars. etsuko tarusawa , yugo fukazawa , elek molnar , masahiko watanabe , ryuichi shigemoto , div. cerebral structure, nips, okazaki, japan; mrc, univ. of bristol, bristol, uk; hokkaido univ., sapporo, japan; sorst, jst, kawaguchi, japan relay cells in the dorsal lateral geniculate nucleus receive two types of glutamatergic inputs; retinogeniculate (rg) and corticogeniculate (cg) synapses. it has been shown that the synaptic transmission at both rg and cg synapses is mediated via ampa and nmda receptors. however, how ampa and nmda receptors are expressed in these two types of synapses have not been elucidated. we examined the expression pattern of ampa and nmda receptors in rg and cg synapses using sds-digested freeze-fracture replica labeling (sds-frl). the sds-frl revealed that synaptic size of individual rg synapses was significantly smaller than that of cg synapses. rg synapses expressed . to times higher density of ampa receptors than cg synapses. on the other hand, cg synapses expressed . to times more nmda receptors than rg synapses. these results indicate differential effects on the relay cell by the retino-and cortico-geniculate inputs through ampa and nmda receptors. katsuyuki kaneda , , , hitoshi kita dept. of anat. & neurobiol., univ. of tennessee, memphis, tn, usa; japan society for promotion of science, tokyo, japan; dept. of developmental physiology, nips, okazaki, japan to investigate the properties of synaptically induced slow responses in globus pallidus (gp) neurons, whole-cell recordings were performed using rat brain slice preparations. repetitive stimulation of the gp and internal capsule induced mixed fast epsps/ipsps followed by a slow ipsp (sipsp), and a long-lasting slow depolarization (sdepo). bath application of nbqx, cpp, and gabazine blocked the mixed epsps/ipsps. the gaba b receptor antagonist cgp abolished the sipsp. an mglur antagonist, but not an mglur antagonist, partially blocked the sdepo. in addition, cgp enlarged the amplitude of fast ipscs, but not of epscs, that were evoked during the repetitive stimulation, suggesting an involvement of presynaptic gaba b receptors in gaba release. these results indicate that synaptically released gaba and glutamate can evoke gaba b receptor-and mglur -mediated responses in the gp. contribution of these responses to the control of gp activity will be discussed. research funds: nih and the jsps ps p-a essential contribution of glutamate to gaba depolarization involved in hippocampal seizure-like activity yoko tsukamoto , yoshikazu isomura , , michiko imanishi , tomoki fukai , masahiko takada system neurosci., tokyo met. inst. neurosci., tokyo, japan; neural circuit theory, riken bsi, saitama, japan we have previously shown that neuronal synchronization is achieved by excitatory gabaergic and glutamatergic inputs during a hippocampal seizure-like afterdischarge. however, it still remains unclear how the gaba response is converted from inhibitory to excitatory in the process of afterdischarge induction. here we traced the time-course of amplitude and reversal potential of gabaergic transmission in pyramidal cells and interneurons entraining the afterdischarge, and examined influence of glutamate on the conversion of gaba response. the gaba reversal potential in pyramidal cells rose to spike-threshold levels for > s after the induction. gaba amediated cl-influx lasted for . s, and then glutamate enhanced the conversion effectively in a gaba a -independent manner, which was dependent on an extracellular k increase. coapplication of gaba and glutamate caused a similar oscillatory activity. the results show gaba and glutamate may cooperatively induce as well as maintain seizure-like activity. michiko nakamura , yuko sekino , , toshiya manabe , division of neuronal network, department of basic medical sciences, institute of medical science, university of tokyo, tokyo, japan; crest, jst, japan profound activity-dependent facilitation of synaptic transmission at hippocampal mossy fiber synapses is a unique and functionally important property. in the present study, we found that this synaptic strengthening was partially mediated by presynaptic gaba a receptor activation during the developmental period (p < ), using electrophysiological methods and optical imaging. in immature animals (p ), fiber volley amplitudes were activity-dependently increased during short-train stimulation of mossy fibers. this fiber volley facilitation was significantly decreased by either inhibition of gaba a receptors or suppression of gaba release from interneurons. these results suggest that gaba released from inhibitory interneurons and gaba a receptors on mossy fibers contribute to activity-dependent facilitation of the excitatory synaptic transmission during development. takuya nishimaki, il-sung jang, jyunichi nabekura dep. dev. physiol., nips, sokendai, okazaki, crest, jst, japan lateral superior olive (lso) is the first auditory center. during the early postnatal period, the inhibitory synaptic inputs to lso neurons from medial nucleus of the trapezoid body (mntb) change from predominantly gabaergic to glycinergic. we focused on metabotropic gaba b receptor (gaba b r) as the key molecule of difference between gaba and glycine. in immature lso neurons postsynaptic gaba b r could activate k + channels, but this effect ceased by the third postnatal week. baclofen, a gaba b r agonist, reduced ipsc amplitude at mntb-lso synapses in neonate (<p ) with a significant change in the paired-pulse ratio. the presynaptic effect of baclofen was gradually decreased with development (p - ). in situ hybridization and immunohistochemistry experiment revealed gaba b r expression in mntb neurons was also gradually decreased. the property of mntb-lso synapses in gaba b r knock out mouse remained immature at p - compared with control mouse. thus, gaba b receptor seems to play an important role in development of synaptic property. pyramidal cells in the hippocampal ca area receive gabaergic input from interneurons, which make synapses on the soma, dendrites and the axon initial segment (ais). here, we used the sdsdigested freeze-fracture replica labeling method to visualize the cell surface distribution of the ␣ , ␣ , and ␤ / subunits quantitatively on distinct subcellular compartments of pyramidal cells. labelings for these subunits were accumulated over clusters of intramembrane particles (imp) on the protoplasmic face (p-face) of the plasma membrane, indicating that many imp clusters of a certain size represent gabaergic synapses. the synaptic labelling densities for gaba-a receptor subunits were not significantly different on the tested subcellular compartments. double labelling experiments showed that the majority of synapses contains ␣ , ␣ , and ␤ / subunits. in the cerebellar cortex, the effects of ␣-adrenoceptor activation on inhibitory synaptic transmissions have not yet been fully understood. therefore, we investigated the effects of the ␣ -or ␣ -adrenoceptor agonist on firing rates of presynaptic interneurons (ins). presynaptic cell-attached recordings showed that spontaneous activity of gabaergic ins was enhanced by the ␣ -adrenoceptor agonist phenylephrine, while reduced by the ␣ -adrenoceptor agonist clonidine. immunohistochemical studies showed that ␣ -adrenoceptors were expressed in the molecular layer and the purkinje cell (pc) layer, while ␣ -adrenoceptors were observed in cell bodies of pcs and ins. these results suggest that noradrenaline enhances inhibitory neurotransmitter release via ␣ -adrenoceptors, which were expressed in presynaptic terminals and somatodendritic domains, whereas suppresses the excitability of ins via ␣ -adrenoceptors, which were located in the presynaptic in soma. thus, cerebellar ␣-adrenoceptors play roles in a presynaptic dual modulation of gabaergic inputs from ins to pcs. research funds: kakenhi ( ) ps p-b involvement of basal pkc and erk / activities in constitutiveinternalization of ampa receptors in cerebellar purkinje cells tetsuya tatsukawa , takahiko chimura , azumi matsumoto , kazuhiko yamaguchi lab. for motor learning control, riken, bsi, japan; lab. for memory & learning, japan ampa receptor (ampar) internalization provides a mechanism for cerebellar ltd, which is the underlying basis of motor learning and motor coordination. however, the relationship between cerebellar ltd and constitutive ampar internalization in parallel fiber (pf)-purkinje cell (pc) synapses remains unclarified. in the present study, we demonstrated that tetanus toxin (tetx, a blocker of exocytosis mediated by vamp) infusion into pcs caused the rundown of pf-epsc amplitude through the constitutive ampar internalization, and then addressed question whether intracellular signals involved in cerebellar ltd induction modify the constitutive ampar internalization at pf-pc synapses using electrophysiological and immunocytochemical techniques. our results suggest that the regulation of actin polymerization is involved in the surface expression of ampars and the surface expressing ampars are constitutively internalized depending on both basal pkc and mek-erk / activities at pf-pc synapses. taisuke miyazaki , kohichi tanaka , masahiko watanabe department of anatomy, school of medicine, hokkaido university, sapporo, japan; department of molecular neuroscience, tokyo medical and dental university, tokyo, japan glutamate released to the synaptic cleft has to be removed by glutamate transporter. in the cerebellum, glutamate transporter glast is richly expressed in bergmann glial cells (bg) that enwrap synapses, dendrites and somata of purkinje cells (pcs). in this study, anatomical analysis was applied to glast-deficient mice. in the mutant mice, lamellate processes from bg fibers became atrophic, resulting in incomplete sealing of synaptic cleft. furthermore, the distribution of climbing fiber (cf) terminals was decreased proximally. by anterogradely labeling, multiple cf innervation was demonstrated to be formed by ectopic innervation to and from nearby proximal dendrites, particularly at their crossing point. these results suggest that glast is essential for complete ensheathment of pc synapses and for the establishment of cf mono-innervation by suppressing local ectopic innervation between nearby pc dendrites. miwako yamasaki , , kouichi hashimoto , taisuke miyazaki , masahiko watanabe , masanobu kano dept. of anatomy, grad. sch. of med., hokkaido univ., sapporo, japan; dept. of cellular neuroscience, grad. sch. of med., osaka univ., suita, japan the ␥ isoform of protein kinase c (pkc␥) is highly expressed in cerebellar purkinje cells (pcs), and its deficiency impairs the late phase of climbing fiber (cf) synapse elimination. in the present study, we analyzed multiple cf innervation in pkc␥-deficient mouse by immunohistochemistry combined with anterograde tracer labeling of cfs. in general, cf innervation regressed proximally in the pkc␥-deficient mouse. in some mutant pcs, a considerable part of the proximal dendrites lost association with cf terminals. in the majority of multiply innervated pcs, single main cf innervated broad region of proximal dendrites, whereas surplus cfs innervated the somata and basal portion of the proximal dendrites. moreover, immunoelectron microscopy revealed synaptic contact of cf terminals to somatic spines. these results suggest that pkc␥ is crucial for strengthening innervation by a main cf and elimination of surplus cfs from proximal somatodendritc compartments of pcs. keiji imoto , , takashi kodama , , ryuichi shigemoto , , , yugo fukazawa , , yasuo mori division of neural signaling, nips, okazaki, japan; school of life science, graduate university for advanced studies (sokendai), okazaki, japan; division of cerebral structure, nips, okazaki, japan; sorst, japan science and technology agency, kawaguchi, japan; department of synthetic chemistry and biological chemistry, kyoto university, kyoto, japan a murine ataxic mutant, rocker, has a point mutation in the ca v . (p/q-type) ␣ subunit gene and shows the mildest phenotype among the series of ca v . mutants. although functional defects of the mutant channel were relatively mild, we found that synaptic transmission in parallel fiber-purkinje cell synapses was considerably impaired. we demonstrated that this impairment was mainly attributable to postsynaptic changes, which included reduction of the number of ampa receptors in psd, whereas the presynaptic function remained normal. we also found the dendrites of purkinje cells showed poor arborization in rocker mice. these lines of evidence indicate that ca v . exerts its strong influence on postsynaptic maturation and dendritic structure. bai-chuang shyu, chia-ming lee, wei-chih chang institute of biomedical sciences, academia sinica, taipei, taiwan roc the aim of the present study was to investigate synaptic transmission and organization of thalamus evoked activities in the anterior cingulate cortex (acc) using a novel mouse brain slice preparation protocol. the mono-and poly synaptic responses in the acc were excited by direct thalamic activation. the synaptic transmission involves both ampa and nmda glutamate receptors. several thalamus-evoked responses were identified: the early negative component (n ) emerged in layer vi near cingulum. subsequently a positive component (p) appeared in layer vi. the second negative component (n ) became apparent in layers ii/iii and v. then the activities spread downward to layer vi and developed as n component in a medial-to-lateral direction. we confirmed that functional thalamocingular activities were preserved in our newly developed brain slice preparation. the thalamus-evoked responses were activated and progressed along a lateral-medial-lateral trajectory loop in the acc and this process was modulated extensively by glutamatergic synapses. ps p-b target-dependent extracellular ca-dependence of the short-term plasticity of the solitary complex synapses in the rat lab. neurophysiol., dept. neurosci., jikei univ. sch. med., tokyo, japan the discharge frequency-dependence of the transmission efficiency at the synapses from the vagal primary afferents to the second-order neurons in the solitary complex (sc) forms the primary filter of the visceral information. such frequency filtering of the synaptic transmissions from the solitary tract (ts) to neurons in the sc depends on their short-term plasticity as evidenced by strong correlation between frequency-dependence and the paired-pulse ratio (ppr). here we analyzed the mechanism underlying distinct pprs among distinct types of synapses. in synapses from ts to the neurons in nucleus of the solitary tract, which show prominent low-pass filtering, ppr significantly increased from . ± . to . ± . (n = ; mean ± s.d.) by reducing [ca + ] o from to . mm, whereas that in low-pass synapses from ts to the neurons in dorsal motor nucleus of the vagus was affected only slightly. these results suggest that the synaptic frequency filtering in the sc is under distinct control depending on the function of the postsynaptic target systems. ps p-b response of spinal monosynaptic reflex to high frequency stimulation in newborn rat the susceptibility of synaptic transmission to the stimulation frequency is a characteristic feature of immature animals, and has been attributed to the incompleteness of transmitter release mechanisms. in an isolated spinal cord preparation of newborn rat, monosynaptic reflexes (msrs) evoked by dorsal root stimulation, were mediated by both nmda and non-nmda glutamate receptors. msrs were constant in amplitude at / s, and were depressed completely by cnqx. when stimulus rate was increased to /s, msr amplitudes were greatly reduced initially, and recovered later. this recovery of msr was eliminated by apv. non-nmda component of msrs was eliminated completely at /s, but appeared in constant amplitudes in the presence of cyclothiazide. from these results, non-nmda glutamate receptor mediated almost most of msrs at / s, but not at /s due to desensitization. in contrast, nmda glutamate receptor does not mediate msrs, but activated at /sec, in place of non-nmda receptor. in conclusion, incompleteness of transmitter release mechanisms could not be solely attributed to the feature of immature synaptic transmission. kenichi ono , keisuke shiba , ken nakazawa , ichiro shimoyama department of otolaryngology, chiba university, chiba, japan; department of otolaryngology, kimitsu central hospital, chiba, japan; department of integrative neurophysiology, chiba university, chiba, japan; section for human neurophysiology, research center for frontier medical engineering, chiba university, chiba, japan we determined synaptic source of the respiratory-related activity of laryngeal motoneurons (lms) using spike-triggered averaging of lm membrane potentials triggered by spikes of medullary respiratory neurons in decerebrate, paralyzed cats. in inspiratory (i) lms, monosynaptic epsps were evoked by spikes of i neurons with augmenting firing patterns, and monosynaptic ipsps were evoked by spikes of expiratory (e) neurons with decrementing firing patterns and of i neurons with decrementing firing patterns. in elms, monosynaptic ipsps were evoked by spikes of i neurons with decrementing firing patterns and of e neurons with augmenting firing patterns. we conclude that various synaptic inputs from respiratory neurons contribute to shaping the respiratory-related trajectory of lm membrane potentials. ps p-b in-vivo gene transfer of exogenous protein to the primary afferent neurons chiaki yamada , , eiji shigetomi , , fusao kato lab. neurophysiol., jikei univ. sch. med., tokyo, japan; dept. basic biol. sci., kyoritsu univ. of pharm., tokyo, japan; jsps, japan in order to understand the mechanism how sensory information is transmitted to the brain, it is indispensable to identify the functional roles of already-identified molecules related to synaptic transmission between the afferent fibers and the central second-order neurons. here we developed a method for efficient in-vivo gene transfer into the neurons in the sensory ganglia and evaluated expression of their gene products. in the anesthetized young wistar rats, we injected pcaggs-egfp plasmid vector (through courtesy of drs. j. miyazaki and k. nakajima) into the nodose ganglion (ng) at the neck and transferred it by electroporation. two days after transfection, the ng was extirpated and fixed to observe egfp signals. a large portion of somata in the ng as well as the centrally and peripherally projecting afferent fibers expressed high levels of egfp with few damaged cells. this technique might enable to analyze the function of specific molecules in the transmitter release from the sensory afferent termini in the brain. research funds: kakenhi ( , ) ps p-b laminar sources of synaptic input to layer neurons in rat visual cortex takuma mori, edward m. callaway salk institute, usa layer of the mammalian neocortex consists of interneurons, including late-spiking neurogliaform cells and non-late-spiking axondescending cells. very little is known about connectivity of these cells with cells in other cortical layers. we examined the laminar sources of local excitatory and inhibitory functional synaptic inputs onto individual layer neurons of rat visual cortex, using scanning laser photostimulation. we find that axon descending cells get excitatory synaptic inputs primarily from layer / . neurogliaform cells receive more excitatory inputs from layers and . the dominant inhibitory synaptic inputs onto both types of cells originate from layer / . these neurons also get inhibitory inputs from layers and - . the presence of convergent excitatory and inhibitory synaptic inputs from multiple cortical layers suggests that the computations in layer are highly integrative. takako nishi , tsukasa gotow , shiro nakagawa ins. nat. sci., senshu univ., kawasaki, japan; dept. neurol., kagoshima univ. grad. sch. med.-den. sci., kagoshima, japan four extra-ocular photoreceptors, the photoresponsive neurons, a-p- , es- , ip- and ip- which respond directly to light, exist in the abdominal ganglion of the mollusc onchidium. a-p- /es- of these neurons respond to light with a depolarizing receptor potential, whereas light hyperpolarizes the other ip- /ip- . the present study was conducted to examine the functional significance of the above ip- /ip- , having a peak sensitivity at nm light. the body wall of the animal transmitted enough nm to hyperpolarize ip- /ip- in the ganglion covered by the body wall. ip- /ip- were coupled with weak electrical synapses and involved in a spontaneous beating or bursting spike activity. ip- /ip- were not coupled to a-p- /es- one another. finally, ip- /ip- , the first order photosensory cells were also the second order interneurons or motoneurons relaying various sensory inputs and innervating the pneumostome and viscera. ps p-c cellular mechanism of interaural level difference calculation in the auditory brainstem of the chicken tatsuo sato, iwao fukui, harunori ohmori department of physiology, faculty of medicine, kyoto university interaural level difference (ild) and timing difference (itd) are the major two cues to localize the sound source, and are processed separately. although the details of itd processing are well investigated, not much is known about ild. in the barn owl, it is proposed that ild is calculated in lldp (posterior part of the dorsal lateral lemniscal nucleus). we confirmed already that in vivo lldp calculates ild in the chicken as well. in this work using the patch clamp recording with the brainstem slice preparation, we found that lldp neurons fire tonically under the regulation of two k + channels activated at low and high voltage, and receive both excitatory and inhibitory inputs representing the contralateral and the ipsilateral sound intensity, respectively. we are intending to clarify the cellular mechanism of ild calculation as the interaction of both excitatory and inhibitory synaptic inputs. to understand the functional organization of the cns, it is essential to know how sensory information is processed within the cns. we have been approaching this topic by following the ontogenetic patterning of neural circuit formation related to the cranial and spinal sensory inputs using optical imaging. in this study, we surveyed developmental organization of neural networks related to the olfactory nerve in the embryonic chick forebrain. stimulation applied to the olfactory nerve elicited epsp-related optical signals in the olfactory bulb from the -day old embryonic stage. the epsp was mediated by glutamate, and nmda-and non-nmda-receptor components were identified. in more developed stages, in addition to the responses in the olfactory bulb, another response area was discriminated within the cerebrum, which seemed to correspond to the higher-ordered nucleus of the olfactory pathway. the results suggest that the olfactory pathway is functionally generated at early stages of development when neural networks related to other visceral and general somatic sensory inputs are also in the process of developing. yoko momose-sato, katsushige sato dept. physiol., tokyo med. dent. univ. sch. med., tokyo, japan in an accompanying study, using a voltage-sensitive dye recording technique, we examined the developmental organization of the olfactory pathway in the embryonic chick forebrain, and showed that functional synaptic transmission in the olfactory bulb was expressed at around e . it is known that odor stimuli elicit oscillatory events in the olfactory bulb in various species. we found that oscillatory activity was also generated in the chick olfactory bulb during embryogenesis. at early stages of development (e -e ), postsynaptic responserelated optical signals evoked by olfactory nerve stimulation exhibited a simple monophasic waveform that lasted a few seconds. as development proceeded, the pattern of the optical signal became complicated, and oscillatory activity was observed in a later phase of the postsynaptic response. the oscillation was restricted to the olfactory bulb, and this spatial pattern was different from that of the propagating wave activity termed the depolarization wave. we examined spatio-temporal patterns of the oscillatory activity in different stages, and studied its developmental dynamics. akiyoshi shimada , nyberg tobias , nahoko kasai , yuriko furukawa , keiichi torimitsu , , kunihiko obata , yuchio yanagawa , , tadaharu tsumoto , ntt basic research laboratories, ntt corporation, kanagawa, japan; sorst, jst, saitama, japan; neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; department of genetic and behavioral neuroscience, graduate school of medicine, gunma university, gunma, japan in the immature stage of cerebral cortex, gaba a receptor activation depolarizes rather than hyperpolarizes the membrane potential of neurons. cortical neurons of glutamic acid decarboxylase -green fluorescence protein (gad -gfp) knock-in mice at postnatal day - were cultured on a microelectrode array. spontaneous firing of cortical neurons appeared within the first days in vitro (div). when a gaba a receptor antagonist, bicuculline, was added, four types of modulation of firing pattern were observed until at least div: increase, decrease, disappearance and no change. these results and an additional analysis suggest that a portion of the spontaneous activity emerged through an activation of gaba a receptors. ps p-c pathway specific signal modulation by purinergic receptors in the parallel processing system makoto kaneda , toshiyuki ishii , , yasuhide shigematsu , toshihiko hosoya , yukio shimoda dept. physiol., keio univ. sch. med., tokyo, japan; bsi, riken, wako, japan; mri, tokyo womens' medical univ., tokyo, japan; dept. biomolecular science, univ. toho, funabashi, japan the retina divides visual information into on-and off signals, and processes them with independent subsets of neurons. so far no difference between these pathways has been found for neurotransmitter functions. we have previously found that p x -purinoceptors are selectively present on the off-type, but not on the on-type cholinergic amacrine cells of the mouse retina. we therefore asked if atp has different functions in the two pathways. as expected, only the off-type, but not the on-type, cholinergic amacrine cells responded to atp. this response was mediated by the p x -purinoceptors. furthermore, a p x-purinergic receptor antagonist activated the tonic phase firing of the off ganglion cells, but not of the on cells. thus atp has off-pathway specific modulatory functions mediated by p x-purinergic receptors. bhupesh mehta, gulnaz begum, preeti g. joshi department of biophysics, national institute of mental health and neuro science, bangalore, india cultured hippocampal neurons in network exhibit synchronized oscillations of cytosolic ca + . these oscillations are an inherent property of various cell types and are essential for the maintenance and development of neuronal plasticity, and for normal brain functioning. the synchronized ca + oscillations are primarily controlled by glutamate receptor activation, but may be modulated by a variety of other factors. these spontaneous oscillations are inhibited by activation of purinergic receptors. we observed a dose dependent inhibition of the oscillatory activity by purinergic receptor agonists atp and utp, but an enhanced oscillatory pattern was observed when the action of mesatp was observed. the inhibition of mesatp response by p y receptor specific antagonist mrs indicates that different subtypes of purinergic receptors play different role in the regulation of synchronized ca + oscillations. data will be presented on the p receptor mediated regulation of synchronized ca + oscillations in cultured hippocampal neurons. mahomi kuroiwa, akinori nishi department of pharmacology, kurume university school of medicine, kurume, japan presence of additional d -like dopamine receptors that selectively couple to gq/phospholipase c (plc) has been proposed. in recent studies, skf was shown to selectively stimulate plc-linked d -like dopamine receptors. darpp- is a phosphoprotein that regulates the efficacy of d receptor signaling. activation of d receptors that couple to golf/pka signaling leads to darpp- phosphorylation at thr . we investigated the effect of skf on darpp- phosphorylation at thr by using mouse neostriatal slices. treatment of neostriatal slices with skf increased thr phosphorylation. the effect of skf was enhanced by a plc inhibitor, u . in contrast, the effect of skf was partially blocked by a d receptor antagonist, sch , and the sch -insensitive increase in thr phosphorylation was completely abolished by an adenosine a a receptor antagonist, zm . thus, analysis of darpp- phosphorylation revealed that skf activates at least three signaling cascades in the neostriatum: ( ) plc, ( ) d receptor/golf/pka, ( ) adenosine a a receptors. ps p-c thiamylal may more effectively reduce neuronal excitability in cerebral cortex than that in hippocampus naoshi fujiwara division of medical technology, niigata university school of health sciences, japan effects of thiamylal (a barbital derivative anesthetic) on excitation propagation in the cerebral cortex and hippocampus were analyzed using membrane potential imaging. cortical and hippocampal slices of the c bl mouse were dyed with a voltage-sensitive dye rh . in cortical slices, electrical single-pulse stimulation to the layer v evoked transient depolarization in the vicinity of stimulated site, and then this excitatory response propagated to the layers ii-iii and widely expanded in these layers. the excitation propagation in the layers ii-iii was significantly inhibited by thiamylal ( mol/l). in hippocampal slices, excitation propagation elicited by the same stimulation to the ca stratum radiatum remained without significant changes in the presence of thiamylal at the same dose. on the other hand, an ampa/kainate receptor antagonist cnqx ( mol/l) inhibited excitation propagation in both cortical and hippocampal slices. the results suggest that thiamylal more effectively reduce neuronal excitation in cortex than that in hippocampus. research funds: grant-in-aid from jsps no. ps p-c brain-derived neurotrophic factor regulating ampa receptor translocation to postsynaptic density through ip r and trpc calcium signaling hiroko nakata, shun nakamura national institute of neuroscience, tokyo, japan the change in the number of postsynaptic ampa-type glutamatergic receptors (ampars) by neuronal activity is implicated in the excitatory synaptic plasticity. here, we showed that bdnf induced ampar translocation to postsynaptic site of the dissociated cortical pyramidal neurons. using calcium imaging, we located the spines that were activated by bdnf. with thus identified spines, we analyzed the ampar subunit glur localization to postsynaptic density (psd) by immunostaining. we found that bdnf increased the ratio of surface glur at psd within min. bdnf selectively translocated ampar, but not nmdar. the essential signaling was ca + transients evoked by the activation of trkb/plc␥ pathway. both the intracellular ca + rise, that is, ca + release from ip r and ca + influx through storeoperated cation channel trpc contributed to the increase of the surface expression of glur at psd. these results suggest an important role of bdnf in the regulation of ampar trafficking by spatial and temporal ca + signaling regulation. research funds: health sciences research grant of nano- hiroyuki konishi , kazuhiko namikawa , , keiji shikata , yuji kobatake , taro tachibana , hiroshi kiyama dept. anat. & neurobiol., osaka city univ., grad. sch. med., osaka, japan; dept. anat., asahikawa med. col., hokkaido, japan; dept. bioeng., osaka city univ., grad. sch. eng., osaka, japan activation of akt-mediated signaling pathways is crucial for injured neurons to survive and regenerate. in this study, we attempted to identify novel substrates for akt by using an antibody, which recognized phosphorylated consensus motif by akt. using pc cells, which overexpressed constitutively active akt, we screened protein spots that exhibited increased immunostaining by the antibody, and consequently identified the neuronal intermediate filament protein, peripherin. using some mutant forms of the recombinant proteins, the phosphorylation site was determined in vitro. we then generated an antibody against phosphorylated peripherin, and demonstrated that peripherin was phosphorylated not only in akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. these results suggest that peripherin is a novel substrate for akt in vivo and that its phosphorylation may play a role in motor nerve regeneration. the truncated trkb receptor, t , is involved in the control of cell morphology via the regulation of rho proteins via rho gdi . however, it is unclear whether t regulates the downstream of rho signaling and the actin cytoskeleton. in this study, we investigated this question using c rat glioma cells, which express t endogenously. rho gdi was dissociated from t in a bdnf-dependent manner, which also causes decreases in the activities of rho-signaling molecules such as rhoa, rock, pak and erk. moreover, bdnf resulted in the disappearance of stress fibers in the cells treated with lpa, an activator of rhoa, and in morphological changes in cells. furthermore, a competitive assay with cfp fusion proteins of t -specific sequences reduced the effects of bdnf. these results suggest that t regulates the rho signaling pathways and the actin cytoskeleton. anjana kalita eukaryotic gene expression laboratory, national institute of immunology, new delhi, india jak and stat are activated in response to many cytokines and growth factors. this pathway has been primarily studied in non-neuronal origin cells. it has been shown that stat activated cellular transformation, occurs through transcription regulation of specific genes like c-myc, cyclin d , bcl . the present study was undertaken to identify target genes of jak/stat pathway in response to tnf-alpha & igf- in neuronal origin cells. we also looked at the mechanism of activation of these target genes. we saw that jak/stat pathway was involved in upregulation of cyclind in response to igf- both at protein & rna levels but no change was observed in c-myc, bcl levels. interestingly, mapk-erk, which is also known to activate cd in other systems, had no effect on the cd level in neuronal cells. on the other hand, pi kinase not only activated jak/stat pathway but also affected on cyclind level. thus it appears that jak/stat is activated by pi k, which in turn upregulates cyclind level. pi k also activated erk without changing cd level, thus we show that jak/stat pathway is important for survival of neurons, activated by pi k. cd is a one of the major downstream targets of jak/stat. hiroko inoue , haruhisa kawasaki , ritsuko fujii , tohru yoshioka , kazunori sango , toshihiko kadoya , hidenori horie sch. of sci. and eng., waseda univ., tokyo, japan; advanced res. center for biol. sci., waseda univ., tokyo, japan; tokyo metropolitan inst. for neurosci., tokyo, japan; pharmaceut. res. lab. kirin brewery co., ltd., gumma, japan oxidized galectin- (gal- /ox) stimulates macrophages to promote axonal regeneration in peripheral nerves after nerve injury. but, the mechanism how gal- /ox stimulates macrophages remains to be clarified. in the present study with rt-pcr and western blotting, we searched for the molecules contributing to the mechanism. rt-pcr analysis with cultured rat peritoneal macrophages revealed that the application of gal- /ox enhanced mrna expression of nerve regeneration-related factors such as il- , il- ␤. however, those of gdnf, ngf, bdnf, nt- , nt- , igf-i, and igf-ii scarcely detected in control condition were not changed by the application of gal- /ox. since il - , il- , and lif can promote axonal regeneration in the in vitro axonal regeneration model, the up-regulation of these cytokines by the treatment with gal- /ox may enhance nerve regeneration after nerve injury. kennichi katou, taku iwamoto, satoshi kida dep. of bioscience, tokyo univ. of agriculture, japan ␣camkii has been known to play essential roles in synaptic plasticity. in response to increase in ca + concentration, ␣camkii is activated by the interaction with ca + /cam. prolonged increase in ca + level leads to further activation of ␣camkii through autophosphorylation at t . to understand the molecular dynamics of ␣camkii activation in vivo, we have tried to visualize and monitor the ␣camkii activity in various types of cells using fret. in hela cells, increase in ca + level induces continuous interaction between ␣camkii and cam and conformational change of ␣camkii, indicating that the conformational change of ␣camkii mainly reflects the interaction with cam. in sh-sy y cells, the increase in ca + level induces only transient interaction between ␣camkii and cam but the conformational change of ␣camkii is maintained following the termination of the interaction with cam, suggesting that conformational change of ␣camkii is induced by the interaction with cam and then maintained by autophosphorylation. thus, mechanisms for regulation of ␣camkii activation is cell-type dependent. akifumi kamata, yusuke takeuchi, kohji fukunaga dept. pharmacol., grad. sch. pharmaceu. sci., tohoku univ., sendai, japan ca + /calmodulin-dependent protein kinase ii (camkii) is highly expressed in brain including dopaminergic neurons, however, the role of camkii in the dopaminergic neurons remains to be unclear. camkii consist of four isoforms, ␣, ␤, ␥ and ␦, and differential isoforms are implicated in the different neural functions. we have examined the isoforms of camkii expressed in rat substantia nigra (sn). rt-pcr and immunoblot analyses revealed that the ␥ and ␦ isoform mrnas including ␦ isoform, a nuclear isoform of camkii, were predominantly expressed in sn. the immunohistochemical study also confirmed the preferential localization of the ␥ and ␦ isoforms in the sn dopaminergic neurons, and immunoreactivity against anti-camkii␦ - antibody was detected in both nucleus and cytoplasm. furthermore, we defined expression of bdnf mrnas having the exon ii and iv in sn. taken together with our previous observation, the camkii␦ isoform was involved in the expression of bdnf in sn dopaminergic neurons. ps p-d the role of ca + /calmodulin-dependent protein kinase ii in neuronal functions revealed by inactivated camkii␣ knock-in mouse yoko yamagata , , hiroyuki sakagami , keiji imoto , , kunihiko obata , yuchio yanagawa okazaki, japan; sokendai, okazaki, japan; tohoku univ., sendai, japan; riken, wako, japan; gunma univ., maebashi, japan ca + /calmodulin-dependent protein kinase ii (camkii) is one of the major protein kinase in the central nervous system and proposed to play important roles in various neuronal functions. we engineered knock-in mice with the inactivated ␣ subunit of camkii by replacing k with r to study functional significance of camkii activity in intact animals. as expected, camkii activity was selectively reduced, while camkii subunit protein levels were comparable to those of wild type controls in homozygous mutants. in situ hybridization revealed normal expression patterns of camkii subunit mrnas, including dendritic localization of camkii␣ mrna. further analyses of these mice will be presented in the meeting. research funds: a grant-in-aid for scientific research from japan society for the promotion of science (kakenhi , # ) ps p-d characterization of ca + -dependent membrane binding proteins of rat brain shohei maekawa , katsutoshi taguchi , haruko kumanogoh , shun nakamura division of biosystems science, grad. sch. of sci/tech, kobe-u kobe - , japan; division of biochemistry and cellular biology, national inst. neurosci. kodaira, tokyo - , japan ca + -dependent membrane binding proteins were purified through a ca + -dependent membrane binding and egta-induced membrane dissociation protocol from a crude triton solubilized membrane fraction. molecular characterization of these ca + -dependent membrane binding proteins through lc-ms/ms identified annexin vii, annexin xi, and copin isoforms, in addition to annexin vi and neurocalcin␣. since the membrane fraction contains phosphatidylethanolamine (pe), cholesterol, and phosphatidylcholine (pc), the membrane binding activity of annexin vi, a major protein in this fraction, was studied using artificial liposomes. annexin vi showed a pe-dependent, but not cholesterol-dependent liposome binding activity. immunostaining of annexin vi in cultured neurons showed a punctuate staining of neuronal cell membrane and this spots increased during maturation. these results suggest the participation of annexin vi in the membrane cycling at the presynaptic region. ps p-d generation of dominant negative mutants of transcription factor crest takuzou simo, kenichi kato, hiroshi hosoda, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan transcription factor calcium-responsive transactivator (crest) has been shown to be required for dendritic growth of neuron. previous studies have shown that crest contains two functional domains; multifunctional domain (mfd; aa ∼ ) required for transactivation, nuclear localization and homodimerization and c-terminal transactivation domain (ctd; aa ∼ ). to further understand the function of crest on brain function, we tried to generate the dominant negative mutant of crest to use mouse genetics study. indeed, we generated fusion proteins of gal -dbd with mutants of crest; crest c ( ∼ ) lacking c-terminal transactivation domain, mfd and mfd-nls that is a fusion protein of mfd with nuclear localization signal (nls). from the experiments using gal one hybrid system, we found that mfd-nls shows the strong dominant negative effects on transcriptional activation by crest. ps p-d characterization of iq-arfgef, a novel exchange factor for arf , in mouse brain hiroyuki sakagami, hisatake kondo department of cell biology, tohoku university graduate school of medicine, sendai, japan adp-ribosylation factor (arf ) regulates cytoskeletal dynamics and membrane trafficking that are critical processes for synaptic plasticity. to understand its neuronal functions, we have focused on guanine nucleotide exchange factors (gef) for arf . in this study, we determined the entire sequence of a partially identified cdna (mkiaa ) encoding a novel sec domain. the deduced protein contained coiled coil, iq, sec , plekstrin homology domains. interestingly, it contained a type i pdz domain-binding motif at its cterminal tail. arf pull-down assay demonstrated its ability to active arf more selectively than arf . thus, we named this protein as iq-arfgef. in situ hybridization analysis demonstrated the preferential expression of iq-arfgef in the forebrain and cerebellar granule cells. furthermore, iq-arfgef mrna was localized not only in the hippocampal neuronal layers but also in their dendritic fields. our present findings suggested that iq-arfgef may play important roles in dendrites through activation of arf and interaction with various pdz proteins. eriko miura , masahiro fukaya , tokiharu sato , kazushi sugihara , masahide asano , katsuji yoshioka , masahiko watanabe dept. anat., hokkaido univ. sch. med., sapporo, japan; dept. mol. cell. biol., cancer res. inst., kanazawa univ., kanazawa, japan; adv. sci. res. center, kanazawa univ., kanazawa, japan the c-jun n-terminal kinase (jnk) is one of the major mitogenactivated protein kinases (mapks), and jsap (or jip ) is a jnkassociated scaffold that controls the specificity and efficiency of jnk signaling cascades. here we studied its expression and distribution in mouse brains. jsap mrna was expressed in developing and adult brains, showing spatial patterns similar to jnk ∼ mrnas. in embryos, jsap immunolabeling was intense for progenitor cells in the ventricular zone and external granular layer, and for neurons and glial cells differentiating in the mantle zone. in adults, jsap was distributed in spines, dendrites, perikarya, and axons of various neurons, and often associated with ser and cell membrane. this wide spatiotemporal expression suggests its fundamental role in mediating external stimuli to jnk mapk pathway and other intracellular machineries. yasukazu hozumi, kaoru goto department of anatomy and cell biology, yamagata university school of medicine, japan diacylglycerol kinase (dgk) is involved in intracellular signal transduction as a regulator of a second messenger, diacylglycerol, and consists of several isozymes. to address the functional implications of dgk isozymes in the brain, we have raised specific antibodies against the isozymes. by immunoblot analysis, we found that the immunoreactive bands for dgk␤, -␥ and -were detected in the light membrane/microsome enriched fraction of rat brains, suggesting that these isozymes localize to the internal membrane system. immunohistochemical examination on rat brain revealed that dgk␤ was detected as dot-like structure in the cell membrane, and dgk␥ as round-shaped structure in the juxtanuclear region of neurons. dgk was detected in the perinuclear region and in the dendrites of purkinje cells. these data show that dgk isozymes localize differentially to the internal membrane system in neurons, suggesting that dgk isozymes play unique roles in distinct internal membrane system. kotaro hattori , shigeo uchino , tomoko isosaka , , shinichi kohsaka , takeshi yagi , shigeki yuasa dept. ultrastractural res., nat. inst. neurosci., ncnp, kodaira, japan; dep. neurochem., nat. inst. neurosci., ncnp, kodaira, japan; kokoro biology group, fbs, osaka univ., suita, japan recently, we have found that fyn-mediated tyrosine-phosphorylation of nmda-r subunits is required for haloperidol-induced catalepsy. haloperidol induced catalepsy in the control mice, but such response was significantly reduced in fyn-deficient mice. fyn activation and enhanced tyrosine-phosphorylation of the nmda-r nr b subunit were induced after haloperidol injection to the control mice, but both responses were significantly reduced in fyn-deficient mice. both nr b phosphorylation and the nmda-induced calcium responses increased after dopamine d -r blockade in cultured striatal neurons of the control, but not in fyn-deficient neurons. accordingly, d -r antagonist activates striatal fyn and the subsequent tyrosine phosphorylation of nr b alters striatal neuronal activity, thereby inducing catalepsy. we also report further proteomics analysis on this molecular pathway. hiroaki okuda, shingo miyata, tsuya taneda, atsushi yamaguchi, shinsuke matsuzaki, natsuko kumamoto, masaya tohyama department of anatomy & neuroscience, graduate school of medicine, osaka university, osaka, japan the schizophrenia is a well-known debilitating psychiatric disorder. recent studies focused on genetic contributions to schizophrenia and have revealed several susceptibility genes, including dysbindin (dtnbp : dystrobrevin binding protein ) at p . . however it is difficult to explain the relationship between the onset of the schizophrenia and only single genetic factor. since, there is an important another factor, an environmental factor, for the schizophrenia. although little attention has been paid to the importance of both factors for the schizophrenia in the nervous system. therefore, to investigate the mechanism of the onset of the schizophrenia, firstly we performed a yeast two-hybrid screening, using the n-terminal region of dys-bindin as a bait. as a result, we detected several transcriptional factors. thus, we further examine that these interactions regulate the expression level of particular genes in the neurons. ryota adachi , naoko oda , ryuzo shingai , st coe program, iwate university, morioka, japan; department of welfare engineering, faculty of engineering, iwate university, morioka, japan the response to environmental stimuli is important to live for organisms. the soil nematode caenorhabditis elegans responds well to chemical and thermal cues known as chemotaxis and thermotaxis behavior. c. elegans is attracted to some ions, amino acids and alcohol that are byproducts of bacteria, and migrates to the temperature of cultivation with food on a temperature gradient. the sensory neurons detected these stimuli are in the head region. the neural network and genes required in each neuron are well known. however, it is under investigation about the integration of chemical and thermal stimuli. to understand the mechanism of this integration, we investigated the chemotaxis behavior under each temperature using the worms cultivated under each temperature. wild type worms cultivated at • c showed decreased chemotaxis response to water-soluble chemicals and alcohol. alteration of assay temperature did not affect response to cl − but that to na + . to discuss more detail, we used thermotaxis abnormal mutants for chemotaxis assay. takashi takekawa , masaki nomura , , toshio aoyagi , tomoki fukai riken brain science institute, saitama, japan; graduate school of informatics, kyoto university, kyoto, japan; crest, japan science and technology agency, kawaguchi, japan the neuron model proposed by izhikevich can exhibit a variety of firing patterns of cortical neurons and gives an efficient mean to conduct large-scale network simulations. however, simplified models may lose essential properties of real neurons, and hence their properties must be compared with those of more realistic models. in fact, our previous studies have shown that two neuron models with almost identical membrane potential profiles sometimes have quite different phase responses and synchronization properties. here, we study the phase responses of the izhikevich model showing fast rhythmic bursting and demonstrate that it exhibits synchronization properties similar to those shown by complex conductance based models. furthermore, its synchronization properties are consistent with those of realistic models in other firing patterns. therefore, we may conclude that the model with tuned parameters is suitable for simulating the dynamic behavior of large-scale networks. honghai cui, hirotaka sakamoto, mitsuhiro kawata department of anatomy and neurobiology, kyoto prefectural university of medicine, kyoto, japan the amygdala plays a critical role in stress responses, especially for modulation of anxiety and fear. in the present study, we investigated the effects on the neuronal morphology for severe stress in the amygdala by using single prolonged stress (sps) paradigm as ptsd (post-traumatic stress disorder) model. rats were perfused days after sps, and intracellular injections of lucifer yellow were carried out in neurons of central (cea) and basolateral amygdala (bla). at the cellular level, we observed a significant increase of dendritic arborization in bla pyramidal neurons but no effect on the cea neurons. these results suggest that sps-induced structural plasticity of the bla provides a cellular substrate for affective disorders that are triggered in ptsd. takahiko kawasaki , , tatsumi hirata division of brain function, national institute of genetics, sok-endai, mishima, japan; prest, kawaguchi, japan in macrosmatic mammals such as mice, olfactory information is detected by two distinct sensory organs, the olfactory epithelium and the vomeronasal organ, and their signals are integrated into the main olfactory bulb (mob) and the accessory olfactory bulb (aob), respectively. the secondary projections from the mob and aob first run parallel in the lateral olfactory tract and eventually diverge into the discrete targets in the telencephalon. although recent studies have revealed general principles in the primary projections, it has been largely unknown how the secondary projections from the olfactory bulbs are constructed. therefore, we investigated the secondary olfactory projections in mutant mouse lines for several axon guidance molecules and their receptors using a specific dye labeling technique, especially focusing on the distinctive pathways of mob and aob neurons. ps p-d an essential role for click-iii/camki␥ in regulation of neurite extension natsumi ishihara, sayaka takemoto-kimura, hiroyuki okuno, haruhiko bito dept. of neurochem., univ. of tokyo, tokyo, japan click-iii/camki␥, a membrane-anchored camk, is a novel member of the camki family that acts downstream of camkk. in order to uncover the biological function of this kinase, we first examined its subcellular distribution and found that tagged click-iii was concentrated at the tips of filopodia-like processes in cultured hippocampal neurons, in close vicinity of actin cytoskeleton. phenotypic studies in pc- cells expressing various mutants of camkk and click-iii revealed that depolarization-induced activation of camkk-click-iii signaling is sufficient to robustly induce neuritogenesis. interestingly, click-iii localization in membrane fractions was regulated via sequential lipidification in an activity-dependent manner. furthermore, lipidification mutants of click-iii were impaired in their ability to facilitate activity-dependent neuritogenesis. our results thus indicate a novel role of click-iii in activity-dependent neurite formation and extension. takuro maruyama , masahiro matsuura , nobuhiko yamamoto grad. sch. of frontier biosci., osaka univ., suita japan; riso kagaku corp., tsukuba japan during development, thalamocortical (tc) axons invade into the cortex and stop growing in layer . to study tc axonal targeting, we examined the effect of the membrane proteins that are expressed in the developing cortex. the dorsal thalamus dissected from embryonic rat brain was dissociated and plated on the dishes that were coated with preclustered fc-tagged extracellular domains of several candidate molecules. after four days in vitro, axonal growth was enhanced on the substrata with low concentrations of ephrin-a , sema a or kit ligand, while a growth-inhibitory effect was found in higher concentrations. a tendency for tc axons to prefer or avoid the region containing these proteins was observed in a new culture method, in which purified proteins were printed on filter membranes in a real laminar size. these results suggest that ephrin-a , sema a and kit ligand may contribute to regulating tc axonal growth in the developing cortex. research funds: kakenhi , kakenhi ps p-e expression pattern of the guidance molecules and their receptors during gnrh neuron migration from the nose to the ventral forebrain shizuko murakami , katsuhiko ono dept. anat., juntendo univ. sch. of med., tokyo, japan; div. of neurobiol. and bioinfo., nat. ins. for physiol. sci., okazaki, japan using in situ hybridization, we examined the expression pattern of attractive and repulsive guidance molecules and their receptors in the migration pathway of gnrh neurons of chick embryos. netrin- expression was seen in the ventral hypothalamus, whereas was absent in the nasal region and the rostral forebrain. a few gnrh neurons expressed netrin- receptor neogenin. slit- was expressed in the forebrain, but its receptor robo- was not expressed in gnrh neurons. semaphorin (sema) a expression was detected in the olfactory-forebrain region. within the forebrain, sema a was absent in the restricted region of the medial forebrain where gnrh neurons migrated in association with a subset of olfactory fibers. at the end of this pathway, sema a expression was observed in the caudal forebrain, and then gnrh neurons changed their course ventrally. many gnrh neurons expressed sema a receptor neuropilin- , suggesting that sema a may act as a repellent during the migration of gnrh neurons. kazuto fujishima , , qing-hua jin , junko kurisu , tomoo hirano , mineko kengaku riken, bsi, lab. for neural cell polarity, saitama, japan; department of biophysics, graduate school of science, kyoto university, kyoto, japan during development, cortical pyramidal neurons elaborate their dendrites in correct pattern and orientation in response to various environmental factors such as neurotrophins or guidance molecules. dendrite morphology is also regulated by cell-cell contacts with neighboring cells. recent studies have implicated notch signal as a candidate for such a contact-dependent regulator of dendrite patterning in postmitotic cortical neurons. we previously showed that delta/notch-like egf-related receptor (dner), neuron-specific transmembrane protein, acts as a notch ligand and mediates neuron-glia interaction during cerebellar development (eiraku et al., ) . dner is expressed in the dendrite of developing cortical pyramidal neurons. here, we analyzed the role of dner in the development of pyramidal neuron dendrites. we quantitatively compared the dendritic morphology of cortical pyramidal neurons in wild-type and dner knock out mice. reelin signal controls neuronal migration in the developing brain. because the binding of reelin to apoer /vldlr induces dab phosphorylation, dab is a critical component of reelin signal. recent studies suggested that reelin signal is involved not only in neuronal migration but in other aspects of neural development. we investigated a possible function of the reelin signal in the filopodia and neurite formation. administration of reelin protein increased number of filopodia in a lesser extent than bdnf treatment. pp suppressed effects of reelin and bdnf on filopodia formation, suggesting that both signal pathways involve src kinase function. in spite of these similarities of reelin and bdnf functions in the filopodia formation, bdnf did not phosphorylate dab . reelin treatment of the neurons derived from dab deficient mice did not induce significant increase in the number of filopodia. these results suggest that the filopodia and neurite formation involves the reelin-dab signal pathways. hidenobu mizuno , , tomoo hirano , , yoshiaki tagawa , dept. biophys., kyoto univ. grad. sch. sci., kyoto, japan; crest, jst, kawaguchi, japan the left and right hemispheres are interconnected via the corpus callosum. in mouse visual cortex, callosal axons from one hemisphere make dense projections to a restricted region at the area / border of the other hemisphere, where they terminate in layers / and . we used in utero electroporation-mediated gene transfer of gfp to assess distribution of callosal projections during development. gfp-labeled callosal axons arrived at the / border and started to invade the cortical plate around p . they generated exuberant axonal arborizations by p then underwent remodeling to form the adult pattern by p . neonatal enucleation did not affect the development, suggesting no apparent role of retinal activity. exogenous expression of a potassium channel kir . in callosal projection neurons, a manipulation known to reduce neuronal firing, attenuated axonal arborizations in layers / . area-specific targeting to the / border was not affected. results suggest that certain stages of callosal axon development require neuronal activity in cortex. ps p-e identification of a novel tetraspan membrane protein, nplp, that is up-regulated after the facial nerve axotomy chihiro akazawa, yoh sasaki, masato hoshi, yasuko nakamura, shinichi kohsaka department of neurochemistry, national institute of neuroscience, ncnp, japan facial nerve axotomy of adult rats, with its extensive ability to regenerate, provides us of a useful model in which to study the molecular mechanisms of nerve regeneration. to identify genes up-regulated during regeneration processes, we constructed a subtractive library enriched for cdnas expressed in the injured facial nucleus. we identified a novel putative tetraspan protein designated as neuronal pmp like protein (nplp) that has a distant homology to pmp /claudin family proteins. the mrna expression of nplp was detected on the cell bodies of neurons meanwhile mrna of pmp resided on the cell bodies of schwann cells. the nplp mrna level significantly increased in motor neurons of axotomized facial nerve, hypoglossal nerve or sciatic nerve. nplp has a large extracellular domain that may interact with other membrane molecules. overexpression of nplp showed the microspike formation in pc cells. our results suggest that nplp plays an important role in the regeneration of injured motor neurons. shinsuke shibata , shin-ichi sakakibara , hirotaka j okano , hideyuki okano , dept. physiol., keio univ., sch. med., japan; dept. histol. neurobiol., dokkyo med. univ., japan ; crest-jst, japan musashi (msi) is an evolutionarily conserved rna-binding protein. drosophila-msi regulates asymmetric cell division in neural development. in mammalian cns we identified two members, msi and msi , which are co-expressed in neural precursor cells including neural stem cells, suggesting msi proteins regulate the immaturity of cns stem cells. to reveal the roles of msi family in vivo, we generated msi and msi deficient mice. msi −/− mice frequently died of obstructive hydrocephalus with aberrant cell proliferation of cerebral aqueduct. msi −/− mice survive to adult show poor weight gain, low spontaneous movement and temperature insensitivity, with the malformation of pns. msi −/− msi −/− mice die in a few hours after birth with severe defects in the brain, including malformation of hippocampal dentate gyrus. in vitro and in vivo analysis show msi family proteins control the expression of several downstream target molecules post-transcriptionally, and play important roles cooperatively in mammalian cns and pns development. youhei koshimizu , satoshi yamagoe , kazuo suzuki , michiko ohtomi department of biomolecular science, graduate school of science, toho university, chiba, japan; department of bioactive molecules, national institute of infections diseases, tokyo, japan we have previously suggested that lect (leukocyte cell-derived chemotaxin ) is expressed in neurons and may play a role in the regulation of both dendritic extension and morphology of astrocytes in the mouse brain. however, the mechanisms of these regulations have not yet been clarified. in this study, we carried out multi-immunostaining against lect and tau or map in neuronal cell cultures and in tissues of adult mouse brain to examine the functional sites of lect in neurons. at the early stages in cultured neurons, lect was remarkably localized in the vicinity of membrane of soma, and was detected around root and end of neurites. in tissues of adult mouse brain, lect was localized to soma, axons and dendrites in neurons. these results suggest the possibility that lect influences the extension of dendrites and axons from the early stage of neuritic development. furthermore, it is thought that lect may regulate the dendritic and axonal morphology in mature neurons. mitsuru noda , , takahiro moriya , hideyuki terazono , suguru kudo , masahiro yamaguchi , kenji yasuda , izumi nagata , kazuyuki shinohara div. neurobiol. & behav., nagasaki univ. grad. sch. biomed. sci., nagasaki, japan; dpt. neurosurg. radiol. nagasaki univ. nagasaki, japan; dpt. life sci., grad. sch. arts & sci., univ. tokyo, tokyo, japan; dpt. physiol., grad. sch. med., univ. tokyo, tokyo, japan; special division for human life technology, national institute of advanced industrial science and technology (aist), japan neural stem cells (nscs) are defined as self-renewing, multipotent progenitor cells that give rise to neurons, astrocytes and oligodendrocytes. using single-cell-based on-chip cell-cultivation system, we analyzed the process of neural differentiation and examined the effects of bdnf. the individual nscs from e . nestin-promoter gfp transgenic mice were placed into pairs of agar microchambers connected by microchannels and were differentiated in the presence or absence of bdnf. we observed that a part of nscs exhibited neuron-like morphology and extended some neurites. the application of bdnf increased the rate of neurite outgrowth. thus we addressed the process of neural differentiation in a single-cell-based level and demonstrated the effect of bdnf. ayumi kyuka, ryoichi yoshimura, yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan in order to investigate the relationship between neurite outgrowth and target cells, ng cells were co-cultured with vascular smooth muscle cells (sm- ). sm- cells promoted the neurite outgrowth, but repelled the contact of their growth cones. the dna microarray analysis indicated that neuropilin- mrna was specifically up regulated in ng cells. but the western blotting analysis did not show the significant increase of neuropilin- . sema a, a candidate of ligands for neuropilin- , was found in ng cells rather than in sm- cells. the immunocytochemical analysis revealed that neuropilin- was increased in the surface of ng cells co-cultured with sm- cells or cultured with the conditioned medium of sm- cells. these results suggest that neuropilin- may be transferred from cytoplasmic pool to surface of ng cells by some soluble factors released by sm- cells. we will discuss about kinetics of neuropilin and chemorepellents. yoshikuni edagawa , j. nakanishi , h. hamada , y. yokomachi , k. yamaguchi , n. takeda inst. biomed. eng., waseda univ., tokyo, japan; riken, japan; kanagawa univ., japan we show a novel method for controlling neurite outgrowth with a photochemical micropatterning technology, which is based on the photoreactive molecule switching its character by irradiation of uv light. this molecule formed a self-assembled monolayer on a glass coverslip, which was used for cell-culture surface. for the neurite outgrowth, pc cell was recruited as a model of neuron. by irradiating uv light at small spots on the cell-culture surface, single cells were specifically attached to the corresponding spots with intermediated ecm. to draw narrow paths for neurite outgrowth, new regions were irradiated in adjacent to the cell bodies attached on the spots. ngf stimulated each pc cell to extend a neurite along the narrow path: the elongation required both photo-activated path and ngf. the successive irradiation in front of developing neurite achieved bending or branching of the neurite. these results indicate that the timing and direction of neurite outgrowth can be controlled with this functional culture system. tetsuhiro kakimoto, hironori katoh, manabu negishi lab. of molecular neurobiology, grad. sch. of biostudies, kyoto univ., kyoto, japan n-wasp is important for actin polymerization and is strongly expressed in brain. toca- was shown to be required for cdc to activate n-wasp in vitro, although its physiological role is unknown. here we studied neural function of toca- . toca- is strongly expressed in developing brain. knockdown of toca- in pc cells significantly enhances neurite elongation. consistently, overexpression of toca- suppresses neurite elongation through its amino terminus including the f-bar/efc domain, which induces plasma membrane invagination. in addition, knockdown of n-wasp also enhances neurite elongation in pc cells, in clear contrast to the previous report using dominant negative mutants. on the other hand, knockdown of toca- in rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of n-wasp enhances both axon elongation and branching. these results suggest that a vesicle trafficking regulating protein toca- regulates different aspects of neuronal morphology from n-wasp. research funds: kakenhi ( ) ps p-e pragmin, a novel effector of rnd gtpase, stimulates rhoa activity and regulates neurite outgrowth hironori katoh, hiroko tanaka, manabu negishi kyoto university, japan the rho family small gtpase rnd is specifically expressed in brain. although rnd and rnd display an antagonistic action for rhoa, signaling pathways of rnd are not well known. here we have performed a yeast two-hybrid screen using rnd as bait and identified a novel rnd -effector protein, pragmin (pragma of rnd ). pragmin is predominantly expressed in neurons, including cortical and hippocampal neurons. in in vitro and in vivo binding assays, pragmin specifically bound to rnd among the rho family gtpases in a gtp-dependent manner. rnd -bound pragmin significantly stimulated rhoa activity and induced cell contraction through rhoa-and rho-kinase-dependent pathway in hela cells. in pc cells, expression of pragmin inhibited the ngf-induced neurite outgrowth in response to rnd , and knockdown of pragmin by a pragmin-specific small interfering rna enhanced the neurite elongation. therefore, these results suggest that rnd controls neurite outgrowth by regulating rhoa activity through pragmin. ps p-e regulation of growth cone motility by collapsin response mediator protein- (crmp- ) masumi iketani , yanai shigeki , fumio nakamura , yoshio goshima , , kohtaro takei , dept. of mol. pharmacol. & neurobiol., yokohama city univ. grad. sch. of med., yokohama, japan; crest, japan sci. & tech. agency, kawaguchi, japan nerve growth cone located at a distal tip of a neurite is known to be the site governing axonal growth and guidance. collapsin response mediator protein- (crmp- ) is enriched in the distal part of axon and participated in axonal formation and growth as well as growth cone collapse by semaphorin signaling. the local functions of crmp- within growth cones, however, remains unclear. chromophoreassisted laser inactivation (cali) that can inactivate selectively a target protein with high spatial and temporal resolutions. acute localized loss of function of crmp- in the entire growth cone region resulted in temporal growth arrest of neurite in chick dorsal ganglion neurons. asymmetric inactivation of crmp- in a half region of growth cone caused growth cones to turn temporally toward the inactivated region. these findings suggest that a spatial distribution of crmp- activity within the growth cone can regulate growth cone motility and direct neurite outgrowth. research funds: crest, jst ps p-e eph receptors are negatively controlled by protein tyrosine phosphatase receptor type o takafumi shintani , , masaru ihara , , hiraki sakuta , , hiroo takahashi , , ikuko watakabe , masaharu noda , division of molecular neurobiology, national institute for basic biology, okazaki, japan; crest, jst, kawaguchi, japan eph receptors are involved in numerous events such as cell movements, axonal guidance, the formation of synapses, and the maintenance of synaptic plasticity. eph receptors are activated by autophosphorylation of tyrosine residues upon the binding of their ligands, ephrins, however, the protein tyrosine phosphatases (ptps) responsible for the negative regulation of eph receptors have not been elucidated. here, we identified protein tyrosine phosphatase receptor type o (ptpro) as a specific ptp that efficiently dephosphorylates both epha and ephb receptors as substrates. using the retinotectal projection system, we show that ptpro controls the sensitivity of retinal axons to ephrins, and thereby plays a crucial role in the establishment of topographic projections. ps p-e neogenin acts as a displacement factor that releases rho from rho-gdi katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan repulsive gudiance molecule (rgma) is a potent inhibitor of axon regeneration in the adult central nervous system. rgma inhibits mammalian cns neurite outgrowth by a mechanism dependent on activation of the rho/rho-kinase pathway. here we show that direct interaction of the rho gdp dissociation inhibitor (rho-gdi) with neogenin which is a receptor of rgma initiates the activation of rhoa, and this interaction between neogenin and rho-gdi is strengthened by rgma. we also found that neogenin facilitates the release of prenylated rhoa from rho-gdi. thus, understanding of the molecular mechanisms of rgma may be useful for developing methods to overcome the inhibitory signals. nobuyuki fukushima, masumi nakashima, ryou tokunaga, ryutaro moriyama department of life science, kinki university, higashiosaka, japan lysophosphatidic acid is an extracellular signaling lipid that regulates neurite morphology in neuronal cells through g protein-coupled lpa receptors, including lpa and lpa . here we examine how lpa , third lpa receptor subtype, is involved in neuronal development by using pheochromocytoma (pc ) cells. pc cells expressed low levels of endogenous lpa mrna. when pc cells were infected with retrovirus expressing lpa and treated with nerve growth factor under serum-free conditions for days, neurite formation was observed in % of total infected cells, which was similar to that in control virus-infected pc cells. however, the mean length of neurites was increased in pc cells expressing lpa , compared with that in control pc cells. interestingly, multiple neurite sprouts were frequently observed in lpa-treated lpa -expressing cells. indeed, the numbers of neurites and neurite branching points per cell were increased in these cells. these results indicated that lpa was involved in neuritogenesis and branch formation. masao sakurai , tomoko aoki , kyoko ishikawa , shingo yoshikawa , john b. thomas , chihiro hama cdb, riken, hyogo, japan; salk institute, usa in drosophila, odor information is represented as a combination of activated glomeruli in the antennal lobe (al). each glomerulus is formed by specific synaptic connections between olfactory receptor neurons (orns) and projection neurons (pns). in an attempt to address the question of how these glomeruli are stereotypically organized during development, we identified wnt as a regulatory factor for glomerular patterning. in the wnt mutant, several glomeruli were shifted to ectopic positions, and this phenotype was partially rescued by wnt expression in orns. previous studies in embryos have shown that wnt repels the axons expressing derailed (drl). in the als of drl mutants, however, some glomeruli were located at incorrect positions, with the pattern different from that in the wnt mutant. this and other genetic data suggest that drl is an antagonist for wnt signaling. we propose that wnt signal transmitted from orns to pns provide one of mechanisms for glomerular patterning in drosophila. ps p-e a homeodomain transcription factor, homothorax, controls precise dendritic and axonal targeting of the antennal lobe projection neurons in the drosophila brain mai ando, yoko totani, katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan the precise neuronal connectivity in the nervous system depends on specific axonal and dendritic targeting of individual neurons. temporal and spatial regulation of gene expression by transcription factors is though to play seminal functions in determining wiring specificity. we are interested in the identification of the genes that control specification and connectivity of antennal lobe projection neurons, which convey olfactory information from the primary olfactory center to the higher order structures such as mushroom bodies and lateral horns. here, we show that homothorax (hth), a homeodomain protein, is expressed in most of the projection neurons. marcm mosaic analyses showed profound targeting defects in both dendritic and axonal projection patterns. currently, we are looking for genes that are either downstream or work cooperatively with hth. based on these data, we will discuss the functional roles of hth in the specification of antennal lobe projection neurons. ps p-e differential microarray analysis of mushroom body transcripts using chemical ablation ayako ino , masatomo kobayasi , taiki nakajima , lydia michaut , indrayani ghangrekar , kuniaki takahashi , ryu ueda , kaoru saigo , walter j. gehring , katsuo furukubo-tokunaga grad. sch. life envir. sci., univ. tsukuba, japan; biozentrum, univ. of basel, switzerland; genet. strains res. ctr., natl. inst. genet.; dept. biophys. biochem. grad. sch. sci., univ. tokyo, japan mushroom bodies (mbs) are the centers for olfactory associative learning in the drosophila brain. we have surveyed mb transcripts using a drosophila whole-genome microarray and identified , genes that exhibited reduced expression levels with pharmacological mb ablation. among the identified genes, we have further selected genes that exhibited a most consistent reduction of expression levels in the ablated brains. in situ hybridization analyses confirmed preferential mb expression patterns for the majority of the identified genes. moreover, we also demonstrate that rnai of many of the identified genes causes mild to strong mb defects. these results provide novel and genome-wide insights into the repertoire of the genes that control differentiation and function of the mb neurons in brain development and plasticity. mikiko inaki , yoshie suzuki , hiroyuki aburatani , akinao nose dept. phys., univ. tokyo, tokyo, japan; rcast, univ. tokyo, tokyo, japan in drosophila neuromuscular junction, the axons of individual motoneurons precisely recognize and project to specific muscle cells. to understand the molecular logic of target specificity, we tried to identify systematically target recognition molecules expressed on individual muscles. we focused on two neighboring muscles, and , which are innervated by distinct motoneurons, and searched for genes that are differentially expressed between them by using wholegenome microarrays. by comparing gene expression data from the two muscles, we identified ∼ genes with more than two-fold difference in expression level. for candidate genes out of ( %), the differential expression was confirmed by in situ hybridization and/or quantitative pcr. we focused our functional analyses on those encoding transmembrane or secreted proteins, with confirmed differential expression pattern. ectopic expression and/or knockdown of some of them altered the target specificity of muscles or , implicating them in neuronal target recognition. makoto aoki, hiroshi segawa, mayumi naito, hitoshi okamoto laboratory for developmental gene regulation, brain science institute, riken, saitama, japan the sensory neurons have two axons, the central and peripheral axon. previously, we have demonstrated that peripheral axons of sensory neurons are completely abolished in the zebrafish embryos overexpressing the lim domains of islet- . to find the molecules which regulate the formation of peripheral axons of sensory neurons in the islet- signaling cascade, we have created cdna library from trigeminal sensory neurons of zebrafish larvae. most of clones were known genes which are implicated in signal transduction, transcription and cytoskeltal formation, but several clones show no similarity with any known genes. most of them show cns or sensory neuron-specific expression patterns and the expression level of these genes were affected by the overexpression of the lim domains of islet- . loss of function and gain of function studies of these clones, whose function are unknown, showed that these clones might be involved in the development of peripheral axons or the formation of neural circuitry. hiroshi yamamoto, kiyokazu agata development of biophysics, graduate school of science, kyoto university, kyoto, japan highly conserved netrins are known as bi-functional guidance molecules, attracting some axons and repelling others. they act through receptors of the deleted in colorecal cancer (dcc) and unc receptor families. freshwater planarians can proliferate by fission and decapitation, the anterior piece regenerates a tail, and the posterior piece regenerates a head. the planarian central nerve system (cns) can be used as a model for studying neural regeneration and understanding how we can rebuild nervous system after injury. we have identified eight netrin related genes from planarians, dugesiajaponica, including four different netrin homologues and three unc homologuesand one dcc homologue. we analyzed expression patterns of eight genes in both intact and regenerating planarians, and then conducted rnai experiments to investigating their functions in the process of regeneration of visual neurons. here we will summarize these results and speculate their molecular actions during eye regeneration in planarians. ps p-e analysis of the significance and mechanism of neurite elimination using c. elegans as a model yu hayashi , takaaki hirotsu , eriko kage , hideaki takeuchi , hirofumi kunitomo , yuichi iino , takeo kubo dept. biol. sci., grad. sch. sci., univ. tokyo, tokyo, japan; dept. biol., fac. sci., kyushu univ. grad. sch., japan; mol. genet. res. lab., univ. tokyo, japan during brain development, a large amount of neural connections once formed undergo elimination, the mechanism and physiological significance of which are poorly understood. we previously showed that elimination of neurites occurs in the nematode c. elegans and that a novel transcription factor mbr- is involved in this process. in the present study, we attempted to clarify the significance of this phenomenon. the mbr- mutant is defective in odor adaptation, and we therefore hypothesized that neurite elimination is critical for maturation of the olfactory circuit. by executing cell-specific rescue experiments, we suggest that mbr- functions in a set of interneurons involved in olfactory processing. we are now examining whether neurite elimination occurs in these neurons using gfp reporters. we expect that our research provides a useful model for genetic dissection of neurite elimination. taro asakura , ken-ichi ogura , yoshio goshima , dept. of mol. pharmacol. and neurobiol., yokohama city univ., grad. sch. of med., yokohama, japan; crest, japan sci. and tech. agency, kawaguchi, japan netrin is an evolutionary conserved axon guidance molecule. in c. elegans, netrin/unc- is secreted from ventral cells, and attracts some axons ventrally and repels dorsally. one of the neurons guided by netrin/unc- is the hsn neuron. the hsn neuron of c. elegans has interesting axon guidance. netrin/unc- is required for the first ventral growth of the hsn neurons. however, what molecules participate in the axon guidance is largely unknown. in this study, we found that the vulval precursor cells strongly expressed netrin/unc- during the axonal guidance of the hsn neurons. silencing of netrin/unc- only at the vulval precursor cells resulted in abnormal axon guidance of hsn. in addition, expression of netrin/unc- only at the vulval precursor cells in unc- null mutants partially restored the hsn guidance defects. these results suggest that netrin/unc- expressed in vulval precursor cells plays essential roles on axon guidance of the hsn neuron. tomomi sato, takanori hamaoka, hidenori aizawa, hitoshi okamoto brain science institute, riken, saitama, japan the optic tectum is a visual center in vertebrates. it receives topographically ordered visual input from the retina in the superficial layers and then sends motor outputs from the deeper layers to the premotor reticulospinal system in the hindbrain. although it is well known how the retinal axons project to the tectum, it has not yet been well understood how the tectal axons project to the hindbrain. here, we established a stable transgenic line tg(brn a-hsp :gfp) in zebrafish to visualize the retinotectal and the tectobulbar projections. to explore the question, we developed an efficient single-neuron labeling system in combination with cre/loxp and gal /uas systems. using the system, we found that the tectum projected to each hindbrain segment with a two-dimensionally ordered pattern. in addition, we showed that ephrinb a was involved in the patterned projection from the posterior tectum to the hindbrain segment rhombomere . these results suggest a neuroanatomical and a molecular basis for the motor map in the tectum. research funds: grant-in-aid for young scientists (b) masahiko taniguchi , yoshinori mikami , tomoyuki masuda , tomoyuki yoshida , naoto matsuda , masayoshi mishina , takao shimizu department of biochemistry, cancer research institute, sapporo medical university, sapporo, japan; department of molecular neurobiology and pharmacology, graduate school of medicine, university of tokyo, japan; department of anatomy, fukushima medical university, japan; dapartment of biochemistry and molecular biology, faculty of medicine, university of tokyo, japan semaphorin gene family contains a large number of secreted or transmembrane proteins, and some of them function as the repulsive and attractive cues of axon guidance during development. here we report the identification of zebrafish semaphorin d (sema d). the orf of sema d is bp ( aa). zebrafish sema d protein showed a . % identity with mouse sema d protein. to clarify the expression pattern of sema d, in situ hybridization was performed. in the embryos, sema d is expressed in the lens and hindbrain. we also found that sema d has the repulsive activity. these results indicate that sema d repels specific types of the axons and functions in the nervous system development. research funds: kakenhi on priority areas from the mext ( ) mayumi yamada , shohei maekawa , seiji miyata department of applied biology, kyoto institute of technology, kyoto, japan; division of bioscience, graduate school of science and technology, kobe university, kobe, japan obcam belongs to the immunoglobulin superfamily cell adhesion molecule (cam) and was synapse-specific cam in cortical and hippocampal neurons in vivo. however, it is uncertain how obcam is involved in synaptic functions. in this study, we showed that obcam was highly concentrated on spines of cultured mature neurons. the inhibition of obcam function with its specific antibody resulted in a decrease in the number of presynaptic terminals and postsynaptic spines. the suppression of obcam mrna expression with its specific antisense oligonucleotide also resulted in impairment of synapse formation. in contrast, overexpression of obcam augmented the formation of synapses. moreover, obcam was internalized via a raft-dependent pathway and the internalization was promoted by increased neuronal activity. these results suggest that obcam, a spine-specific cam, is a critical modulator of synaptogenesis and its surface expression is dynamically regulated in response to neuronal activity. ps p-f semaphorin d inhibits ␤ integrin activity through the r-rasgap activity of plexin-b izumi oinuma, hironori katoh, manabu negishi lab. mol. neurobiol., grad. sch. biost., kyoto univ., kyoto, japan plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. we have recently reported that the semaphorin d (sema d) receptor, plexin-b functions as a gap for r-ras, a member of ras family gtpases implicated in regulation of integrin activity and cell migration. here we characterized the role of r-ras downstream of sema d/plexin-b in cell migration. activation of plexin-b by sema d suppressed the ecm-dependent r-ras activation, r-ras-mediated pi -k activation, and ␤ integrin activation through its r-ras gap domain, leading to inhibition of cell migration. in addition, inactivation of r-ras by overexpression of the r-ras-specific gap or knockdown of r-ras by rna interference was sufficient for suppressing ␤ integrin activation and cell migration in response to the ecm stimulation. thus, we conclude that r-ras activity is critical for ecm-mediated ␤ integrin activation and cell migration, and that inactivation of r-ras by sema d/plexin-b -mediated r-ras gap activity control cell migration by modulating the activity of ␤ integrins. shun hamada , emi fukuda , , shota katori , , takeshi yagi , dept. nutrition health sci., fukuoka women's univ., fukuoka, japan; grad. sch. frontier biosci., osaka univ., osaka japan, crest, jst, japan cnr/protocadherin (pcdh)␣ is a subfamily of the clustered pcdhs that comprise > cadherin-related molecules and are encoded by three gene clusters (␣, ␤ and ␥). the molecular features and synaptic localization of the clustered pcdhs have raised the possibility that they are synaptic recognition molecules. we have demonstrated that overexpressed pcdh␣ family proteins alone in several cell lines are rarely transported into the plasma membrane. furthermore, we found that a stretch of about fifty amino acids located at the c-terminus of pcdh␣s interfered the trafficking to the cell surface. in the present study, we compared the transport properties of a series of the cytoplasmic region truncation mutants and found that truncation mutants lacking or more c-terminal residues were detectable at the cellular surface suggesting a role for lysine-rich motif in the c-terminus of pcdh␣s in the intracellular retention. mdga is a novel cell surface glycoprotein similar to ig-containing cell adhesion molecules (igcams) with functions in migration and process outgrowth. mdga is expressed by layer / neurons throughout the neocortex at p mice, but is absent in adults. between e . and late p , stages that span the generation and radial migration of layer / neurons, mdga is expressed in patterns consistent with its expression by migrating layer / neurons, suggesting a role for mdga in controlling their migration and settling in the superficial cortical plate. we performed loss-of-function studies using rna interference (rnai) with in utero electroporation into the lateral ventricle at e . to transfect progenitors of superficial layer neurons. we found that an rnai suppressing mdga protein blocks proper migration of superficial layer neurons to the superficial cortical layer. we conclude that mdga acts cell autonomously to control the migration of superficial layer cortical neurons. in various pathological conditions, activated microglia mediate immune responses to injured cns neurons. however, it is not clear whether and how activated microglia affect neurons via direct contacts. this study aimed at examining whether direct contacts between microglia and hippocampal neurons increase following cns injury and whether telencephalin (tlcn), a dendrite specific adhesion molecule, which potentially binds to immune cells, mediates the direct contact. hippocampal neurons were damaged by local injection of excitotoxin, kainic acid (ka). compared to control animals, ka-injected mice showed higher density of contacts between activated microglia and dendrites of ca pyramidal neurons. contacts with longer interface appeared in ka-injected mice. these results suggest the importance of direct contacts for the immune response of microglia to injured neurons. similar contact formation was also observed in tlcn-deficient mice, indicating that the direct contacts are mediated by other molecules than tlcn. kilon is belonging to immunoglobulin superfamily of cell adhesion molecules and contains three igg-like domains. western analysis revealed that the expression levels of kilon is low at early neuronal culture and increased with progress of culture days. immunocytochemical observation showed that kilon was localized at elongating axon and growth cones but not at dendrites on days in vitro (div), while kilon was observed at synapses, mainly at presynaptic terminals on div. similar tendency was observed in kilon immunohistochemistry of brain sections in vivo. kilon was observed at axonal fibers of the cerebral cortex on postnatal day , but it was seen at synapses in adult brains. these results suggest that kilon is axonal cell adhesion molecule to control axonal guidance and/or extension. ps p-f analysis of mice that show abnormal expression of neuroglycan c, a central nervous system-specific transmembrane proteoglycan sachiko aono , yoshiyuki kuroda , fumiko matsui , yoshihito tokita , keiko nakanishi , michiru ida , masahito ikawa , masaru okabe , katsuhiko ono , atsuhiko oohira institute for developmental research, aichi human service ctr., kasugai, japan; research institute for microbial diseases, osaka university, suita, japan; national institute for physiological sciences, okazaki, japan neuroglycan c (ngc) is a membrane-spanning chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. to study the role of ngc in the brain, we produced two strains of ngc-mutant mouse by gene-targeting; a mouse strain with no ngcexpression and a strain with low expression (knockdown mice). both mice were viable and fertile. they did not show obvious abnormalities in gross brain anatomy. to examine their behavioral phenotype precisely, the ngc-knockdown mice were subjected to several kinds of behavioral tests sequentially. they displayed obvious abnormalities in morris water maze and passive avoidance tests, suggesting that ngc is involved in learning and memory. we are now carrying out the same experiments using the ngc-knockout mice. research funds: kakenhi ( ) ps p-f phosphorylation of extracellular signal-regulated kinase in aged rats with acute face inflammation koichi iwata , tatsuhisa watanabe , ikuko suzuki , junichi kitagawa , akiko ogawa , kenro kanda , kazunao kuramoto dept. of physiol., sch. of dent., nihon univ., tokyo, japan; dept. of oral and maxillofacial surgery, sch. of dent., nihon univ., tokyo, japan; dept. of oral diagnosis, sch. of dent, nihon univ., tokyo, japan; shinjuku vocational school of acupuncture, moxibustion and judo therapy, tokyo, japan; division of research animal center, tokyo metropolitan institute of gerontology, tokyo, japan the capsaicin-induced perk expression was studied in the aged rats ( - months) following noxious face stimulation. a large number of perk-li cells were expressed in the superficial laminae of the trigminal spinal nucleus in adult and aged rats following subcutaneous capsainsin injection into the whisker pad region. the larger number of perk-li cells was expressed in adult rats than aged rats following intravenous administration of naloxone before capsaicin treatment. the present results suggest that the descending modulation system was impaired in the aged rats, resulting in the abnormal pain sensation advancing age. hirokazu katsura , koichi obata , masafumi sakagami , koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; department of otorhinolaryngology, hyogo college of medicine, hyogo, japan recent studies demonstrated that the activation of extracellular signal-regulated protein kinase (erk) / and p mitogen-activated protein kinase (mapk) in dorsal root ganglion (drg) neurons contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk (also known as big mapk or bmk ) is activated in the drg and participate in pain-related behaviors in the complete freund's adjuvant (cfa) model. peripheral inflammation induced an increase in the phosphorylation of erk , mainly in tyrosine kinase a-containing small-to-medium-diameter drg neurons at days and after cfa injection. furthermore, time course of phosphorylated-erk level in the drg matched the emergence of cfa-induced pain hypersensitivity. our data suggest that activation of erk in drg neurons may contribute to the development of inflammatory pain. ps p-f activation of erk in drg neurons contributes to acute pain toshiyuki mizushima , , koichi obata , takashi mashimo , koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, hyogo, japan; department of anesthesiology, osaka univ. recently, we have reported that phosphorylation of extracellular signal-regulated protein kinase (erk) / and p mitogen-activated protein kinase (mapk) occurred in primary sensory neurons in response to natural noxious stimulation of the peripheral tissue, i.e., activity-dependent activation of erk and p in dorsal root ganglion (drg) neurons. however, there has been no study examining erk (also known as big mapk or bmk ) activation in drg neurons after noxious stimulation of normal tissue. here, we report intensity-dependent erk phosphorylation in drg neurons by painful stimulation. noxious stimulation induced phosphorylated-erk in small-to-medium diameter sensory neurons with a peak at min after stimulation. furthermore, we found a stimulus intensitydependent increase in the number of activated neurons. our data suggest that activation of erk in drg neurons may contribute to acute pain induced by noxious stimulation. koichi obata, koichi noguchi department of anatomy and neuroscience, hyogo college of medicine, japan there is compelling evidence indicating that the activation of extracellular signal-regulated protein kinase (erk) / and p mitogenactivated protein kinase (mapk) in the dorsal root ganglion (drg) and spinal cord contributes to the development of inflammatory and neuropathic pain. in the present study, we examined whether the newest member of the mapk family of proteins, erk (also known as big mapk or bmk ) is activated in the drg and spinal cord and participate in pain-related behaviors in the l spinal nerve ligation (snl) model. l snl induced an increase in the phosphorylation of erk not only in the injured l drg, but also in the spared l drg at day after surgery. furthermore, l snl induced a striking increase in erk phosphorylation in glial cells in the ipsilateral dorsal horn. our data suggest that activation of erk in the drg and spinal cord may contribute to the development of neuropathic pain. atsushi sakai , minoru asada , naoki seno , hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan; pharmaceutical research center, kyowa hakko kogyo co., shizuoka, japan glial cell line-derived neurotrophic factor (gdnf) has been known to alleviate the neuropathic pain. however, the mechanisms of gdnfinduced analgesia remain almost unclear. gdnf binds to gfr␣- , which forms receptor complex and signals intracellularly through ret. recently, neural cell adhesion molecule (ncam) has been found to be an alternative signal-transducing receptor for gdnf. here, we report that ncam is involved in gdnf-induced analgesia in a rat model of the neuropathic pain. ncam mrna expression was decreased in the ipsilateral dorsal horn of the spinal cord after the nerve injury, but gdnf treatment returned its expression to the normal level. treatment with ncam antisense oligodeoxynucleotide blocked the analgesic effect of gdnf without affecting ret phosphorylation. these results suggest that activation of ncam signaling may provide a new strategy to relieve intractable chronic pain. ps p-f interleukin- ␤ enhanced the excitability of trigeminal root ganglion neurons via activation of satellite glia following inflammation mamoru takeda , jun kadoi , msanori nasu , masayuki takahashi , shigeji matsumoto dep. physiol and res. cent. for odont. nippon dent. univ., japan the present study was investigated whether activation of satellite glial cells modulates the excitability of trigeminal root ganglion (trg) neuronal activity via the il- ␤ paracrine mechanism following inflammation. two days after cfa into the whisker pad area, the mean number of trg neurons that were encircled by glial fibrillary acidic protein and il- ␤-immunoreative satellite cells were significantly increased compared with those in the control. fg labeling was used to identify the trg neurons innervating the site of inflammation. in the fg-labeled small trg neurons, the occurrence of il- ␤ induced depolarization in inflamed rats was larger than that in control rats. il- ␤ application significantly increased the firing rate evoked by depolarizing pulses in the inflamed neurons compared with the control neurons. these results suggest that activation of trg satellite glial cells modulates the excitability of trg neuronal activity via the il- ␤ paracrine mechanism following peripheral inflammation. junichi kitagawa , mamoru takeda , jun kadoi , yoshiyuki tsuboi , shigeji matsumoto , koichi iwata dept. of physiol., sch. of dent., nihon univ., tokyo, japan; dept. of physiol, sch. of dent. at tokyo, japan, nippon dental univ., tokyo, japan the present study was designed to elucidate an involvement of the primary afferent neurons in the trigeminal neuropathic pain using the rats model with chronic constriction nerve injury of the infraorbital nerve (ion-cci). the mechanical escape threshold was significantly lower in ion-cci rats at day after ion treatment and the threshold decrement was lasting more than day . single unit activities of ion were recorded from the ion-cci rats. the firing frequency was significantly higher in a␦ fibers in ion-cci rats as compared with naive at day - after ion-cci. whole cell patch clamp recording was performed from the middle trg neurons. ik and ia currents were significantly smaller and ih current was larger in ion-cci rats than that of naive rats. the present results suggest that ik, ia and ih currents are involved in abnormal firing of trg neurons in the rats with ion-cci, resulting in neuropathic pain in trigeminal region following peripheral nerve injury. hisako urai, munehiro uda, katsuya kami graduate schools of sport and exercise science, osaka university of health and sport sciences, osaka, japan mechanical hyperalgesia of skeletal muscles has been known to occur following intense eccentric contraction such as downhill running (dhr). the present study examined the number of c-fos-positive neurons in spinal dorsal horn to determine peak of mechanical hyperalgesia following dhr in rats. furthermore, we investigated whether glial cells are activated in dorsal horn with excitation of secondary afferent neurons (san). rats performed an intermittent bout of dhr for min. at , , , and h post-dhr, the rats were applied a weight on the right triceps surae muscle. immunohistochemical staining for c-fos and gfap on spinal cords was performed by freefloating abc method. the number of c-fos-positive neurons detected in superficial dorsal horn were increased at h, peaked at h and then decreased. intense gfap immunoreactivities were also detected at and h post-dhr. these results suggest that dhr generates mechanical hyperalgesia by increasing responsiveness of san, and moreover astrocytes may regulate excitability of san. katsuya kami, hisako urai, munehiro uda department of health science, osaka university of health and sport sciences, osaka, japan a production of inflammatory cytokines is increased in injured skeletal muscles. the present study examined relationship between production of inflammatory cytokines in skeletal muscles and fospositive neurons in spinal dorsal horn following downhill running in rats. the rats performed the downhill treadmill running for min at m/min. after the running, rats were applied the weight on the gastrocnemius muscles for min, and then spinal cord and soleus muscles were removed from the rats. productions of il- beta, il- and tnf-alpha in soleus muscles and expression of fos protein in dorsal horn were examined using immunohistochemical approach. at h post-running, number of fos-positive neurons was increased, peaked at h and then decreased to control level at h post-running. vigorous inflammatory reactions with necrotic myofibers in soleus muscles were observed at days post-running. these results indicated that increased numbers of fos-positive neurons in dorsal horn are induced prior to vigorous inflammation of skeletal muscles. shinichi sugiyo , yusuke sakai , aya masawaki , takashi shimoda , masayuki moritani , motohide takemura dept. oral anatomy and neurobiology, osaka university grad. sch. of dentistry, osaka, japan; dept. of fixed prosthodontics, osaka university grad. sch. of dentistry, osaka, japan; dept. of dental anesthesiology, osaka university grad. sch. of dentistry, osaka, japan diabetes mellitus is among the most common causes of painful peripheral neuropathy, worldwide. we examined if there exist the diabetic rat (dm)-specific difference in nociceptive behavioral and c-fos immunoreactivity (ir) by formalin test. injection of formalin into the upper lip weeks before streptozotocin injection induced biphasic specific pain related behavior (prb) for min. first phase was greater in dm than in the control rat (ctrl). in dm, second phase was much greater than ctrl. c-fos ir in the trigeminal caudal nucleus was also greater in dm than in ctrl. these results indicate that dm induced greater prb and c-fos expression following formalin injection into the rat upper lip. yasuko kozaki, satoshi hurune, fukushi kambe, hisao seo, kazue mizumura res. inst. environ. med., nagoya university, nagoya, japan we have reported that prostaglandin ep receptor (ep r) activation attenuates the desensitization of bradykinin (bk)-induced increase of intracellular calcium ([ca + ] i ) in a ptx-sensitive manner in cho cells expressing canine ep r and mouse bk b receptor (b r). in this study, we examined the involvement of protein kinase a (pka) in the desensitization of the bk response. when bk ( nm) was applied twice with a -min interval to the cells expressing b r, the second [ca + ] i increase by bk was markedly attenuated. however, the pretreatment with a specific inhibitor of pka, h- ( m) restored the second response. to further confirm camp increase by bk, the expression of a camp responsive reporter gene was examined. bk ( pm) treatment for h significantly increased the reporter gene expression. it is likely that bk increases the level of intracellular camp, and thus activates pka, resulting in the desensitization of the bk response. these results suggest that the desensitization of bkinduced increase in [ca + ] i was at least in part mediated by pka. ps p-f contribution of peripheral ht a or ht receptors to fos expression in the trigeminal spinal nucleus (vsp) produced by the masseter muscle injury of rats keiichiro okamoto, akihisa kimura, tomohiro donishi, yasuhiko tamai dept. physiology, wakayama med univ., japan we have recently reported that orofacial nocifensive behavior evoked by the masseter muscle (mm) injury is attenuated by blocking peripheral ht a or ht /r in male rats with tmj inflammation. here we tested if these two ht/r subtypes contribute to fos responses in vsp after mm injury. formalin injection into mm produced fos-like immunoreactivity (li) in several areas of vsp and c . fos-li was distributed mainly in the ventrolateral trigeminal subnucleus interpolaris/caudalis transition (vl-vi/vc) and vc/c transition regions. the number of fos-li induced by mm injury was increased in these areas in cfa-evoked tmj-inflamed rats for days compared to naive rats. we tested if local ht a or ht /r antagonist affects fos expression in both groups. the number of fos-li in the vc/c but not vl-vi/vc region was reduced when drugs were injected locally prior to formalin injection in tmj-inflamed rats. these data suggest that peripheral ht a and ht /rs play critical roles in mediating mm nociception during tmj inflammation. keiko abe, hidemasa furue, kohei kga, go kato, toshiharu yasaka, akihiro tamae, toshihiko katafuchi, megumu yoshimura department of integrative physiology, graduate school of medical sciences, kyushu university, japan we examined the postsynaptic effects of -ht on substantia gelatinosa (sg) neurons in slice preparations of rat spinal cord and their relationship to the morphological features. in ∼ % of sg neurons examined, -ht induced an outward current. the outward current was mimicked and suppressed by a -ht a agonist and -ht a antagonist, respectively. in ∼ % of sg neurons, -ht evoked an inward current which was mimicked by a -ht agonist. the outward current was observed mostly in excitatory neurons such as vertical cell, while the inward current was induced in an inhibitory neuron, islet cell. these findings suggest that -ht inhibits excitatory neurons and excites inhibitory neurons in the sg through activation of -ht a and -ht receptors, respectively. the reciprocal postsynaptic actions of -ht on sg neurons in addition to presynaptic inhibitory effects on primary afferents might play an important role in descending control of nociceptive transmission by -ht. we examined effects of levobupivacaine, ropivacaine, bupivacaine and r-bupivacaine on epscs in substantia gelatinosa (sg) neurons of the spinal dorsal horn evoked by dorsal root stimulation, and on action potentials in dorsal root ganglion neurons generated by the dorsal root stimulation. in sg neurons, levobupivacaine reversibly suppressed the amplitude of monosynaptic a␦ and c fiber-evoked epscs. however, a␤ fiber-evoked epscs were slightly inhibited in amplitude. on the other hand, bupivacaine equally suppressed those three fiber-evoked epscs. in drg neurons, ic of bupivacaine and r-bupivacaine were almost equal on a␤, a␦ and c neurons. however, ic of levobupivacaine and ropivacaine on a␦ and c neurons were lower than that on a␤ neurons. the present results suggest that pure s (−) enantiomers especially levobupivacaine effectively inhibits noxious transmission to the spinal dorsal horn by the blockade of ap conduction through a␦ and c fibers. ps p-g nitric oxide-dependent long-term potentiation revealed by real time imaging of nitric oxide production and neuronal excitation in spinal dorsal horn hiroshi ikeda, kei kusudo, kazuyuki murase dept. human & artificial intelligence systems, univ. fukui, fukui, japan no plays an important role in the induction of long-term potentiation (ltp) in spinal dorsal horn, which is believed to underlie hyperalgesia and allodynia. in this study, to elucidate the relationship of no to ltp, we measured the spatiotemporal distribution of no signal with the no-sensitive dye, and neuronal excitation with the voltagesensitive dye, in rat spinal cord slices. in superficial dorsal horn, neuronal excitation evoked by dorsal root stimulation was potentiated for more than h after low-frequency conditioning stimulation (lfs). in the same slices that exhibited ltp, no was produced and distributed in the superficial dorsal horn during lfs. ltp and production of no were inhibited in the presence of no synthase inhibitors and an inhibitor of heme oxygenase, the synthetic enzyme for carbon monoxide (co). research funds: kakenhi to hi ( ) and km ( ) and grants from novartis foundation and promotion of science and sumitomo foundation to hi tao liu, tsugumi fujita, akiko koga, masafumi kosugi, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the effect of a pla activator melittin on inhibitory transmission in the substantia gelatinosa (sg; lamina ii of rexed), we applied the blind whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. in about % of neurons examined, melittin ( m) superfused for min gradually increased the frequency and amplitude of spontaneous glycinergic inhibitory postsynaptic currents which were recorded at mv in the presence of bicuculline. this action was visible about min after the beginning of its superfusion and subsided within min after washout. these melittin actions were reduced in extent by a pla inhibitor -bromophenacryl bromide, while being unaffected by tetrodotoxin, and also by inhibitors of cyclooxygenase (cox) and lipooxygenase (lox). it is concluded that pla activation pre-and postsynaptically enhances glycinergic transmission in sg neurons, possibly not through metabolites of cox and lox; this action would contribute to a modulation of nociceptive transmission. research funds: kakenhi ( ) ps p-g presynaptic p y receptor-mediated enhancement of inhibitory synaptic transmission in the rat spinal dorsal horn terumasa nakatsuka, shugo koga, tsugumi fujita, tao liu, masafumi kosugi, eiichi kumamoto department of physiology, faculty of medicine, saga university, saga, japan using whole-cell patch-clamp recordings, we examined whether the activation of p y receptors can modulate synaptic transmission in dorsal horn (dh) neurons of adult rat spinal cord slices. bath applied -methylthio adp ( mesadp, m), a p y receptor agonist, did not change excitatory transmission, but clearly increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (ipscs) in about % of dh neurons recorded. miniature ipsc in the presence of ttx was increased in frequency by mesadp with no change in the amplitude. the mesadp-induced increase in miniature ipsc frequency was attenuated in extent by mrs ( m), a selective p y receptor antagonist. these results indicate that the activation of presynaptic p y receptors enhances inhibitory but not excitatory synaptic transmission in a subpopulation of dh neurons. thus, spinal p y receptors can be involved in an inhibitory effect on pain transmission. research funds: kakenhi ( ), the japanese health sciences foundation (kh ) ps p-g presynaptic enhancement by proteinaseactivated receptor- agonist peptide of glutamatergic excitatory transmission in rat substantia gelatinosa neurons tsugumi fujita, terumasa nakatsuka, akiko koga, tao liu, masafumi kosugi, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan we have previously reported that proteinase-activated receptor (par)- but not par- agonist (each m) enhances glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons. the present study examined a detail of the par- mediated enhancement by applying the whole-cell patch-clamp technique to sg neurons in adult rat spinal cord slices. par- agonist (sfllrn, m) reversibly increased the frequency of spontaneous epsc without a change in the amplitude and also in holding current at - mv. this facilitatory action was resistant to tetrodotoxin, and was not seen in the presence of par- antagonist (yfllrnp, m). these results indicate that the activation of par- s existing in nerve terminals in the sg results in an increase in the spontaneous release of l-glutamate from there. it is suggested that par- activation in glutamatergic neuron terminals in the sg may be involved in the modulation of nociceptive transmission from the periphery. research funds: kakenhi ( ) ps p-g effect of tramadol metabolite m on glutamatergic excitatory transmission in rat spinal dorsal horn neurons akiko koga, tsugumi fujita, tao liu, terumasa nakatsuka, eiichi kumamoto dept. physiol., facult. med., saga univ., saga, japan in order to know the antinociceptive effect of tramadol, we examined the effect of m , which is one of its metabolites, at mm on glutamatergic excitatory transmission in substantia gelatinosa (sg) neurons of an adult rat spinal cord slice by using the whole-cell patchclamp technique. bath-applied m reduced the frequency but not amplitude of spontaneous excitatory postsynaptic currents (epscs) at − mv. this action was not seen in the presence of a -opioid receptor antagonist ctap ( m). m also reduced the peak amplitudes of epscs which were monosynaptically evoked at − mv by stimulating primary-afferent a␦-and/or c-fibers in a spinal cord slice with an attached dorsal root. we conclude that m inhibits the quantal release of l-glutamate from nerve terminals in the sg through the activation of -opioid receptors; this action is not distinct in extent between primary-afferent a␦-fiber and c-fiber transmission. this effect of m would give a cellular basis for the antinociceptive effect of systemically-administered tramadol. narihito iwashita, natsu koyama department of physiology, shiga university of medical science, otsu, japan in our previous study, subcutaneous injection of glutamate into the human forearm evoked pain and produced skin temperature increase around the injection site. these results suggest peripheral glutamate receptors contribute to nociceptive signaling and neurogenic inflammation. in order to further investigate which subtype of glutamate receptors is involved in neurogenic inflammation, effect of nmda receptor antagonist mk- or non-nmda receptor antagonist cnqx was evaluated in hindpaws of pentobarbital-anesthetized rats. attenuation of skin temperature increase induced by simultaneous mk- injection with glutamate was larger than that of skin temperature increase induced by simultaneous cnqx injection with glutamate at the same concentration. on the other hand, inhibition of paw edema formation by cnqx was stronger than by mk- . these data demonstrate that peripheral nmda receptor predominantly contributes to vasodilatation, while peripheral ampa/ka receptor predominantly contributes to increase of vascular permeability in glutamate-induced neurogenic inflammation. ps p-g studies on pain control system (rept. ): changes in phosphorylation of nr b-contained nmda receptor in the spinal cord obtained from rats with painful neuropathy following chronic ethanol consumption kan miyoshi, minoru narita, michiko narita, tsutomu suzuki dept. toxicol., hoshi univ. sch. pharm. pharmaceut. sci., tokyo, japan chronic ethanol consumption produces a painful peripheral neuropathy. mechanical hyperalgesia was clearly observed during ethanol consumption and even after ethanol withdrawal in rats, and it lasted for weeks. under these conditions, the immunoreactivities of phosphorylated-ser- nr b (p-ser- nr b) subunit and phosphorylated-conventional protein kinase c (p-cpkc) were significantly increased in the spinal cord following chronic ethanol consumption, whereas p-tyr- nr b subunit immunoreactivity was not changed in this region. the hyperalgesia induced by chronic ethanol consumption was significantly attenuated by repeated i.p. injection of ifenprodil, a selective nr b-containing nmda receptor antagonist. these findings provide evidence for a substantial role of the phosphorylation of cpkc-dependent nr b-contained nmda receptor in the development or/and maintenance of ethanoldependent neuropathic pain-like state in rats. ps p-g prolonged depression of nociceptive response in the prefrontal cortex with high frequency stimulation of the amygdala yumi izawa, yoriko kawakami dept. physiol. tokyo women's medical university, tokyo, japan high frequency stimuli (hfs, hz, a, s) delivered to the basolateral nucleus of the amygdala (bl) induced prolonged depression of the nociceptive specific response in the prefrontal cortex (pfc). we examined the receptor mechanism underlying this depression of pfc neuron activity. extracellular neural activities, induced by nociceptive stimulation applied peripherally, were recorded in the rat pfc. inhibitory effects of hfs delivered to the bl on nociceptive responses were blocked by specific antagonists of a metabotropic glutamate receptor (mglur) or nmda receptor microinjected locally into the pfc. dopamine depletion, produced by -ohda injected into the substantia nigra, also reduced the inhibitory effects of hfs. the mglur and dopamine receptor mediated prolonged depressions of nociceptive responses were induced by hfs of the amygdala. our results suggest that emotional condition modulates pain sensation. ps p-g the nav . sodium channel pathologically reconfigures the thalamic pain amplifier-generator after spinal cord injury bryan c. hains, stephen g. waxman yale university school of medicine, usa spinal cord injury (sci) induces pain-related phenomena associated with the aberrant expression of nav . , a rapidly repriming voltage-gated sodium channel. in this study we hypothesized that, following sci, neurons in the thalamus undergo similar electrophysiological changes linked to nav . . four weeks post-sci, nav . protein was upregulated within thalamic neurons, where unit recordings revealed increased spontaneous discharge, afterdischarge, hyperresponsiveness to innocuous and noxious peripheral stimuli, expansion of peripheral rfs, and bursting. these properties persisted after interruption of ascending spinal barrage. lumbar intrathecal administration of specific antisense oligodeoxynucleotides against nav . caused a significant reduction in nav . expression and reversed electrophysiological alterations. these results show, for the first time, a change in sodium channel expression within neurons in the thalamus after injury to the spinal cord, and suggest that these changes contribute to altered processing of somatosensory information after sci. tomoki fukuda , hiroyuki ichikawa , ryuji terayama , tomosada sugimoto department of oral maxillofacial rehabilitation, okayama university, okayama, japan; department of oral function and anatomy, okayama university, okayama, japan ib -sap is a neurotoxin designed for targeting primary nociceptors with ib binding sites on the cell surface. however, the exact cell spectrum that is affected by the toxin has not been thoroughly investigated. we, therefore, unilaterally injected ib -sap ( . l of . % solution for each ganglion) directly into the th and th lumbar (l and ) dorsal root ganglia (drgs). three weeks later, the rats were killed and drg sections were stained for ib -binding. after counterstain, the cell body size of neurons were measured. ib -sap reduced the total number of drg neurons in l and ganglia combined by % ( ± on untreated side versus ± on treated side). small neurons (< m ) were reduced by % whereas large ones (≥ m ) were not affected. ib -binding neurons were mostly small (≥ %) and were reduced by %. the number of small neurons, that were not stained for ib -binding, increased by % ( ± versus ± ). schuichi koizumi , kaoru nasu-tada , makoto tsuda , emiko kunifusa , , kazuhide inoue div. pharmacol., natl. inst. hlth. sci., tokyo, japan; dept. mol. system pharmacol., grad. sch. pharmaceut. sci., kyushu univ., fukuoka, japan although microglial p x receptor, a key molecule for the mechanical allodynia, is increased after peripheral nerve injury, the molecular mechanisms underlying its upregulation remain unknown. here, we describe the influence of fibronectin on p x receptor expression in microglia. microglia that were cultured on fibronectin-coated dishes showed a marked increase in p x receptor expression. western blot examination of the spinal cord from rat with spinal nerve injury indicated that fibronectin was upregulated on the ipsilateral side. interestingly, intrathecal injection of atp-stimulated microglia revealed that microglia cultured on fibronectin-coated dishes was more effective in the induction of allodynia than microglia cultured on control dishes. taken together, our results suggest that spinal fibronectin is elevated after the peripheral nerve injury and it may be involved in the upregulation of the p x receptor in microglia, leading to neuropathic pain. research funds: mf , kakenhi ( ) ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. of biomed. sci., nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root (dr), which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. ). however it remained whether lpa has direct actions on myelinated schwann cells (sc). in the present study we examined the direct effects of lpa on dr fibers in ex vivo culture system. scanning electron microscopy (sem) study revealed that lpa caused a swelling and disruption of myelinated fibers at h. in transmission em analysis, the addition of lpa caused a disruption of myelin sheath of a␦-and a␤-fibers. on the other hand, it was found that c-fibers were separated to each other by scs in naive fibers. following the addition of lpa, c-fibers showed direct contacts and some of them were uncovered. all these effects were also observed either with or without dr ganglion. thus, it is suggested that lpa has direct actions on myelinated and unmyelinated scs to cause demyelination of a-fibers and to uncover c-fibers. research funds: kakenhi ps p-g lysophosphatidic acid (lpa) down-regulates myelin associated proteins in cultured dorsal root fibers norikazu kiguchi, ryousuke fujita, hiroshi ueda div. mol. pharmacol. & neurosci., nagasaki univ. grad. sch. biomed. sci. nagasaki, japan we have reported that intrathecally administered lpa or endogenous lpa generated upon sciatic nerve injury causes demyelination of dorsal root, which is supposed to be one of key molecular mechanisms underlying neuropathic pain (nat. med. ). these treatments also caused a decrease in myelin protein and their gene expression levels. here we report the biochemical evidence underlying this demyelination in cultured fibers. the addition of lpa at mm decreased the protein levels of myelin basic protein (mbp) at h. this action was significantly inhibited by botulinum neurotoxin/c (bont/c ). on the other hand, lpa also caused a decrease in gene expression of various myelin proteins, such as mbp, pmp , mag, p in cultured fibers. the maximal decrease was observed all at as early as h after the addition of lpa. bont/c and y abolished the lpainduced down-regulation of mbp gene. all these findings suggest that the down-regulation of gene expression of myelin proteins is through rhoa-rock pathway underlying lpa-induced demyelination. neuropathic pain arise from peripheral never injury. the purpose of this study was to explore behavioral characteristics and investigate the involvement of nmda receptors and opioid receptors in the behavioural responses following spared nerve injury (sni). the hind paw withdrawal threshold to cold-and mechano allodynia and heatyperalgesia were tested at and , , , , days after operation. pre-emptive co-administration of mk- and morphine were tested. sni produces mechanical and cold allodynia and heat hyperalgesia. co-injection of morphine and mk- decreased cold-and mechanoallodynia, but had slightly effect on heat-hyperalgesia. the present data demonstrate that the sni procedure result in severe changes in behavioral responses in whether hyperalgesia or allodynia. coadministration of both drugs seems to be more effective to alleviate induced neuropathic pain. satoshi deyama , , naomi akiyama , mikie hirata , takayuki nakagawa , shuji kaneko , masabumi minami dept. of pharmacol., grad. sch. of pharm. sci., hokkaido univ., sapporo, japan; dept. of mol. pharmacol., grad. sch. of pharm. sci., kyoto univ., kyoto, japan the bed nucleus of the stria terminalis (bst) is involved in the regulation of negative affective states such as anxiety and fear. in this study, we examined the role of the noradrenergic (na) transmission within the bst in the negative affective component of pain in rats. we found that excitotoxic lesion of the bst attenuated intraperitoneal acetic acid-or intraplantar formalin-induced conditioned place aversion (cpa) without reducing nociceptive behaviors. we showed that na release within the bst was significantly elevated by these noxious stimuli. intra-bst injection of a ␤-adrenoceptor antagonist timolol significantly suppressed these noxious stimuli-induced cpa without affecting nociceptive behaviors. these results suggest that visceral and somatic noxious stimuli-induced na release within the bst contributes to the negative affective, but not sensory, component of pain. noriyuki ozaki, mariko kawai, yasuo sugiura department of functional anatomy and neuroscience, nagoya university, graduate school of medicine, nagoya, japan neonatal maternal separation induces visceral hyperalgesia in colon. this study compares the effects of maternal separation on response sensitivity to gastric and colorectal distension in long-evans rats. maternal separation was performed for h per day between postnatal day and . visceral sensitivities were assessed in stomach and colon at weeks of age by visceromotor responses induced by either gastric or colorectal distension. somatic pain sensitivities were also assessed by von frey filaments and radiant heat. in contrast to the response to colorectal distension, maternal separation induced decreased response to gastric distension, especially in male rats. no difference was found between control and separated rats in somatic pain sensitivities. these results indicate that maternal separation differentially modulates visceral pain sensation in stomach and colon. research funds: grant-in-aid for scientific research ps p-g change by aging in muscular mechanical hyperalgesia after lenghtening contraction k. mizumura, t. taguchi, t. matsuda, t. nasu res. inst. environ. med., nagoya univ., nagoya, japan our previous experiments have shown that the mechanical threshold of the edl muscle underwent lengthening contraction (lec) lowered to days after exercise in rats ( w old). c-fos expression in the superficial dorsal horn increased in l spinal segment when the edl muscle was compressed days after exercise. from these results we have concluded that the muscle became hyperalgesic after lec. in the present experiment, we examined whether this hyperalgesia after lec changes along aging. male sd rats , ( - ) and ( - ) w old were used. the basal mechanical threshold (randall-selitto method) of edl muscle tended to be higher in w old rats, but not significant. after lec, the threshold started to decrease day after lec in all three age groups. it returned to the pre-lec level days after lec in and w old rats only. recovery of w old rats delayed up to days after lec. increased c-fos expression in the superficial dorsal horn was observed in l as well as in l in w old rats. these results suggest that hyperalgesia occurs in larger areas and lasts longer in aged animals. tong liu, hong p. wei, chun y. yuan, ai k. guo institute of neuroscience, chinese academy of sciences, china drosophila can display complex courtship behavior. male-male courtship behavior shown in some fly mutants, but here we report for the first time that the male-male courtship behavior can be induced by disturbance of dopamine level. to up-regulate dopamine level, uas-th/th-gal males were used, which showed high level of dopamine and performed active male-male courtship behavior. this behavior was attenuated by decreasing dopamine level either through drug breeding or genetic method. the increased courtship behavior in uas-th/th-gal males is specific to male partners, because the males courted females normally. to down-regulate dopamine level, pale ts , th temperature sensitive mutant was used. when raised at restrictive temperature, pale ts showed obvious attraction to wild type males. our study shows that the high level or low level of dopamine can induce male-male courtship behavior in active or passive manner. athushi yokoyama , masaharu akita kanagawa life-science res., japan; kamkura, kanagawa, japan we have developed the screening system for drug and chemical compounds of food by the used of ratwhole embryo culture. the advantages of whole embryo culture are to examine the direct effects of l-calnitin (lcal) on embryo and also to find the non-teratogenic agent (d-calnitin:dcal). as the testing agent, lcal was examined in this study using the rat embryo cultured from day to of gestation. in treated embryos of lcal, the embryonic heart beat, the crown-rump length, the embryo weight and the total embryonic somites were not decreased. on the other hand, the malformation (the defects of neural tube) and the short size of head length were observed in the embryos cultured with lcal. in treated embryos of d-calnitin (dcal), there parameter was not decreased. the observed malformation of lcal was not observed in the embryos cultured with dcal. these results might be due to the differences between lcal and dcal in the embryo toxicity. yoshihisa uenoyama, kenji takase, junya hirata, hiroko tsukamura, kei-ichiro maeda laboratory of reproductive science, nagoya university, nagoya, japan the mechanisms underlying the pubertal increase in gonadotropinreleasing hormone (gnrh) secretion are poorly understood. recently, metastin was found to stimulate gnrh secretion and mutations of its receptor are associated with lack of puberty. effect of immunoneutralization of endogenous metastin in the brain on the onset of puberty was examined to clarify the physiological significance of metastin in timing the puberty. when wistar-imamichi strain female rats received an infusion of anti-metastin antibody into the third ventricle during days - of age, they did not show the first estrus before days of age with mean age of . ± . day. in contrast, most of normal mouse igg-treated controls showed the first estrus by days of age with mean age of . ± . day. the age of vaginal opening was also delayed in the anti-metastin-treated rats. thus, the present study demonstrates that the puberty onset was delayed by immunoneutralizing central metastin. central metastin may be involved in timing the onset of puberty in female rats. kenji takase, yoshihisa uenoyama, shunji yamada, hiroko tsukamura, kei-ichiro maeda lab. of reproductive science, nagoya university, aichi, japan metastin has been considered to be involved in triggering pulsatile gonadotropin-releasing hormone (gnrh)/luteinizimg hormone (lh) secretion to time the onset of puberty. the present study aimed to determine expression of metastin, a novel kiss- gene product, in the rat brain during peripubertal period. wistar-imamichi strain female rat shows vaginal opening on around days of age (d ), and first estrus on around d . brain tissues were obtained on d , , , and . kiss- mrna expression in the arcuate nucleus-median eminence region (arc-me) and anteroventral periventricular nucleus (avpv) increased significantly from d to and was kept at a high level thereafter. gpr mrna expression in the medial preoptic area increased significantly from d to . metastin-immunoreactive cells were not found on d but were apparent in the arc-me on d onward. these results indicate that metastin expression increases in the arc-me and avpv before vaginal opening, suggesting that metastin triggers the onset of gnrh/lh secretion in female rats. toshiyuki saito , sei-etsu fujiwara , kenjiro konno , takashi yamaguchi , tetsu nemoto , etsuko kasuya , ryosuke sakumoto anim. neurophysiol. lab., natl. inst. agrobiol. sci., tsukuba, japan; inst. exp. anim. res., fac. med., gunma univ., maebashi, japan; grad. sch. sci. & eng., yamagata univ., yonezawa, japan; sch. health sci., fac. med., kanazawa univ., kanazawa, japan in the present study, we recorded and examined local field potentials (lfps) in the hippocampus of piglets performing an operant task by a radio-telemetry system. under halothane anesthesia, a pair of tungsten electrodes was implanted into the hippocampus and fixed on the surface of the skull with a transmitter using dental cement. after recovery from surgical procedures, the piglets were moved to a training cage. in the lfps, spike-shaped waves were frequently found just before the piglets pushed a switch with their noses. these waves may represent some of the hippocampal neural activities associated with switch manipulation for getting a food reward. yasuo osawa department of bioscience, tokyo university of agriculture, tokyo, japan memory extinction is an inhibitory learning rather than forgetting or erase of conditioned memory. from the view of treatment of phobia and post traumatic stress disease (ptsd) caused by fear memory, it is important to find the drugs to facilitate extinction of fear memory. importantly, previous studies using pavlovian fear conditioning have shown that d-cycloserine, a nmda receptor agonist, facilitates memory extinction. in this study, to examine whether d-cycloserine is applicable for the treatment with another type of fear memory, we investigated effects of d-cycloserine on extinction of aversive memory in mice. indeed, we performed conditioned taste aversion (cta) task, where the ingestion of a novel taste is paired with transient sickness. our results indicated the injection of d-cycloserine before but not after the re-exposure to cs facilitates extinction of cta. ps p-g hippocampal neural responses during a conditional delayed stimulus-response task in awake mice nobuhide kitabayashi , teruko uwano , , anh tran , , eturou hori , , taketoshi ono , , hisao nishijo system emotional science, univ. of toyama, japan; integrative neurosci, molecular and integrative emotional neurosci., univ. of toyama, japan; crest, japan to investigate a hippocampal (hf) involvement in the representation of temporal sequence in mice, neural responses were recorded during performance of a conditional delayed stimulus-response association task. a trial was initiated by one of two different conditioned tones. after a s delay, two serial reinforcements with an intervening delay was presented; aversive air puff-delay-tube protrusion to evoke licking sucrose solution and the opposite order of the same reinforcements. of hf neurons, responded to the tones, the reinforcements, and during the delay. some neurons responded to a presentation of a sensory stimulus, and other responded differentially during the delay depending on the reinforcement sequence. the results suggest a crucial role of the hf in representation of serial events in episodic memory in mice as well as in rats and primates. further studies will be conducted using genetic modified-mice to clarify the neural substrate in episodic memory. naoko inoue , atsu aiba , kaoru inokuchi mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; grad. sch. med., kobe univ., kobe, japan vesl- s/homer- a and vesl- l/homer- c are splicing isoforms encoded by the vesl- gene. vesl proteins bind and regulate mglur / , ip receptor, ryanodine receptor, and trp channel at the postsynapse. the expression of vesl- s is upregulated by tetanic stimulation that elicits l-ltp. vesl- s is thought to play a critical role in the conversion from short-term to long-term memory (ltm). in this study we generated vesl- s gene-targeting mice (ko) and examined whether vesl- s plays a role in the ltm formation. analysis with the contextual fear conditioning revealed a defective in consolidation process, reconsolidation process, and remote memory formation in ko. ko further showed an enhanced freezing decrement within a test session, indicating faster within-session extinction. in contrast, consolidation process of the extinction was normal in ko. these results demonstrate that the vesl- s protein plays critical roles in various processes of the ltm formation. we now examine the signaling pathways important for ltm formation that are altered in ko. hiroshi ageta , r. migishima , s. kida , k. inokuchi , mitsubishi kagaku inst. life sci (mitils), japan, tokyo univ. agricul., japan; crest, jst, japan memory process consists of at least four distinct phases, acquisition, consolidation, maintenance, and retrieval. activin ␤a mrna increases following l-ltp induction in the hippocampus, suggesting that activin plays a role in the memory formation. here, we generated activin and follistatin (antagonizes activin function) transgenic mice in which the transgene expression was tightly regulated by dox in a forebrain-specific manner (tet off system). transgene expression was turned off or on within d by (+/−) dox. contextual fear conditioning with these mice revealed that activin function is required during maintenance phase of fear memory for one week retention. furthermore, activin plays a role in the re-consolidation process. thus, fear memory that was once acquired and consolidated tightly could be "erased" by inhibiting the activin function during maintenance phase. these mice are useful for the study of ptsd. ps p-h sex differences in the effects of chronic estrogen treatment on fear conditioning in c bl/ j mice takaaki ozawa, mumeko tsuda, sonoko ogawa kansei, behavioral and brain sciences, university of tsukuba, tsukuba, japan it has been suggested that estrogen may play a role in the regulation of learning and cognitive functions. although most of previous studies have focused on elucidating facilitatory effects of estrogen on learning in females, estrogen is also known to affect various behaviors in males. in the present study, we investigated the effects of different doses of estrogen on fear conditioning (fc) learning in both sexes of mice. gonadectomized c bl/ j mice were implanted with a silastic capsule containing , , or g of estradiol benzoate. since it is possible that estrogen may indirectly modify learning by affecting general activity, emotionality and anxiety levels, we tested the mice in open-field and light dark transition paradigms prior to fc. mice were then conditioned for fear responses (freezing) to tone stimulus and tested for both contextual and cued fc responses. we found that estrogen facilitated both types of fc learning in females, whereas it inhibited them in males especially at a higher dose, with a small effect on emotional behaviors. ps p-h analysis of brain regions activated during memory consolidation in passive avoidance task zhang yue department of bioscience, tokyo university of agriculture, tokyo, japan short-term memory (stm) is labile. to generate long-term memory (ltm), stm is stabilized through a process known as memory consolidation. importantly, previous studies have shown that memory consolidation requires the function of transcription factor creb whose activation induces c-fos expression. in this study, we tried to understand molecular mechanisms of consolidation of passive avoidance memory that has been known to be amygdala and hipocampusdependent. indeed, we investigated brain regions that are activated following the learning by analyzing the expression level of c-fos using immunocytochemistry. consistent with previous study, we observed increase in c-fos expression in amygdala and hippocampus. more interestingly, we also found this increase in prefrontal cortex, indicating that prefrontal cortex plays critical roles in memory consolidation in light-dark passive avoidance task. hiroshi nomura, norio matsuki laboratory of chemical pharmacology, graduate school of pharmaceutical sciences, university of tokyo, tokyo, japan we have demonstrated the effect of ethanol on reactivated fear memory for the first time, using contextual fear conditioning. rats were conditioned with mild footshock, reexposed to the training context, immediately injected with ethanol or saline, and finally tested h after reexposure. ethanol-treated groups expressed longer freezing and the effect lasted for weeks. reactivation was necessary for the effect. the injection of ethanol itself did not induce a fearful response. as memory retrieval triggers memory extinction and reconsolidation, we investigated whether extinction process is involved in this ethanol effect. increasing retrieval time did not enhance freezing by ethanol, suggesting that ethanol had no effect on memory extinction. post-reactivation injections of anisomycin revealed that retrieval triggered reconsolidation. moreover, picrotoxin inhibited the memory enhancement by ethanol. these studies demonstrate that ethanol enhances reactivated contextual fear memories via activation of gaba a receptors. ps p-h analyses of brain regions activated in reconsolidation and extinction phases of contextual fear memory nori mamiya, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan the retrieval of conditioned fear memory by conditioned stimulus (cs) initiates two processes; reconsolidation or extinction. we previously found that the change in memory stability after retrieval (reconsolidation) associates with memory extinction. to understand the regulatory mechanisms of memory stability after the retrieval at the anatomical level, we here investigated the brain regions that are activated in reconsolidation and extinction phases. we measured the levels of phospho-creb inducing changes in neural plasticity following the re-exposure to cs. short re-exposure to cs inducing reconsolidation increased in phospho-creb in amygdala and hippocampus. in contrast, longer re-exposure inducing extinction increased in phospho-creb in amygdala and prefrontal cortex. these results indicate that distinct brain areas are activated in response to short or long re-exposure to cs and suggests that amygdala plays crucial roles in the interaction between reconsolidation and extinction. ps p-h analysis of molecular mechanism for the destabilization of retrieved contextual fear memory akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan reconsolidation acts to stabilize, whereas extinction tends to weaken the expression of the original memory. to understand the mechanisms for the regulation of memory stability after the retrieval, we have investigated the relationship between reconsolidation and extinction using contextual fear conditioning. we previously found that memory extinction is associated with regulation of fear memory stability, indicating the interaction between memory reconsolidation and extinction phases. in this study, we compared molecular signatures of reconoslidation and extinction using mice. pharmacological experiments using antagonists for cannabinoid receptor (cb ) and l-type voltage-gated calcium channels (lvgccs) indicated that both cb and lvgccs are required for memory extinction but not consolidation and reconsolidation. more interestingly, blockade of either cb or lvgccs function prevents the disruption of the original memory by protein synthesis inhibition. these results suggest that cb and lvgccs are required for not only memory extinction but also the destabilization of reactivated memory. hotaka fukushima, akinobu suzuki, satoshi kida department of bioscience, tokyo university of agriculture, tokyo, japan previous our studies using contextual fear conditioning revealed three distinct time-dependent phases following memory retrieval: stable, reconsolidation, extinction phases. to understand the nature of memory processing following retrieval, we examined the effects of reexposure on memory reconsolidation and extinction using light-dark passive avoidance task. this task is thought to allow us to discriminate between reconsolidation and extinction phases at the time point when mice enter dark box from light box. brief re-exposure to light box did not affect the stability of fear memory (stable phase). further extending re-exposure to light box triggered the requirement of protein synthesis for re-storage of fear memory (reconsolidation phase). in contrast, entry from light into dark box initiated extinction of fear memory (extinction phase). additionally, using pharmacological blockade of cb and lvgccs, we also found that cb is required for only memory extinction but that lvgccs are required for memory extinction and reconsolidation. wakoto matsuda , takahiro furuta , kouichi nakamura , , takeshi kaneko , ps p-h difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatum fumino fujiyama , tomo unzai , kouichi nakamura , , sakashi nomura , takeshi kaneko , department of morphological brain science, kyoto university, kyoto, japan; department of physical therapy, kyoto university, kyoto, japan; crest, japan the striatum, which has patch/matrix compartments, receives glutamatergic inputs from cortex and thalamus. in the present study, the differences in synaptology of these inputs between both compartments were examined. axon terminals positive for vesicular glutamate transporter (vglut) , thalamostriatal inputs, were less dense in patch region, whereas vglut -positive corticostriatal inputs were evenly distributed. quantitative analysis revealed % of vglut positive synapses in patch region were formed with spines, whereas % in matrix region were made with dendritic shafts. in contrast, the targets of vglut -positive inputs were mainly spines in both regions. moreover, vglut -positive axospinous synapses in patch region were larger than vglut -positive ones. the present observation suggests that thalamostriatal connection is more plastic in patch region. research funds: kakenhi ( , , , ( ), ) ps p-h single cell tracing of thalamostriatal projection neurons with reference to patch and matrix compartments of rat striatum tomo unzai , fumino fujiyama , takeshi kaneko , department of morphological brain science, university of kyoto, kyoto, japan; crest, japan the striatum consists of patch and matrix compartments, and receives glutamatergic inputs mainly from the cerebral cortex and thalamus. thalamic intralaminar nuclei are known to project exclusively to matrix compartment. on the other hand, it has not been clarified which thalamic nuclei project to patch compartment. in the present study, we combined single cell tracing with immunohistochemistry for mu opioid receptor, which is specifically expressed by patch neurons, to reveal the distribution of thalamostriatal axon terminals in relation to striatal compartments. recombinant sindbis virus expressing membrane-targeted green fluorescent protein (palgfp) was injected into the rat thalamus. a single neuron in the thalamic paraventricular nucleus extensively projected to the striatum and preferentially to patch compartment compared with matrix compartment. the axons were also distributed in the thalamic reticular nucleus, accumbens nucleus, amygdala, and cerebral cortex. research funds: kakenhi ( , , , ( ) , ) ps p-h lesion of the nucleus accumbens dopamine system shortens the lever pressing interresponse time and delays the response initiation in mice yuji tsutsui , kayo nishizawa , nobuyuki kai , kazuto kobayashi , dept. of psychology, fukushima univ., japan; dept. mol. genet., fukushima medi. univ., japan; crest, jst, kawaguchi, japan dopamine transmission is thought to be important for rodents to perform operant behaviors such as lever pressing. the lever pressing experiment was conducted to examine the effects of -ohda injections into the nucleus accumbens (acb) in c bl/ j mice. all mice were trained to press the lever for a food pellet using a fixed ratio (fr ) schedule. the mice were injected with ascorbate vehicle or -ohda into the acb, and then tested post-surgically using the fr schedule again. the -ohda-injected mice showed the acceleration of response speed, which was revealed by the shortening of interresponse time between each of the five lever pressings, and the suppression of the initiation of the response to the next step. this suppression of initiation was revealed by the increase of time from the last presentation of food to the next initiation. these results suggest that the acb dopamine system is important for the initiation and control of the operant behaviors in rodents. hideshi shibata laboratory of veterinary anatomy, tokyo university of agriculture and technology, fuchu, tokyo, japan retrosplenial area is one of the important structures for spatial memory and behavior in the rat. to understand more fully the functional roles played by area , it is essential to clarify the neural circuitry subserving these functions. in the present study, we analyzed the organization of frontal cortical projections to area in the rat, using retrograde transport of cholera toxin b subunit (ctb). ctb injections into area d retrogradely labeled cells in the orbital cortex and the caudal parts of the anterior cingulate and primary and secondary motor cortices. ctb injections into area c labeled cells in similar cortical regions, except for the orbital cortex. ctb injections involving areas a and b labeled cells in the caudal part of the anterior cingulate cortex. the results show that the orbital, anterior cingulate, and primary and secondary motor cortices have a different pattern of projections to each subdivision of area , suggesting different functional roles played by each subdivision of area in spatial memory and behavior. eiichi jodo , yoshiaki suzuki , tadahiro katayama , ken-yo hoshino , yukihiko kayama dep. of physiol., fukushima med. univ., fukushima, japan; dep. of neuropsy., fukushima med. univ., japan it has been shown previously that the dopaminergic neurons in the ventral tegmental area (vta) selectively respond to a stimulus repeatedly paired with reward stimuli in a classical conditioning paradigm. since the vta receives dense projection from the medial prefrontal cortex (mpfc), such response selectivity of vta neurons may in part be produced by inputs from the mpfc. however, few studies have compared the firing pattern between these two regions. our present experiment was designed to make such a comparison in freely moving rats. two different tones were sequentially presented, one of which (target, %) was paired with intracranial simulation of the reward area. the unit activity was recorded from the mpfc and/or the vta. pfc and vta neurons exhibited phasic excitation with the peak latency of about . s to both tones, while only the target tone induced sustained activation of firing activity lasting until presentation of the reward. masato inoue, akichika mikami primate research institute, kyoto university, inuyama, aichi, japan to investigate the neuronal mechanism in the ventrolateral prefrontal cortex (vlpfc) and inferotemporal cortex (it) for holding information for object and their order of presentation, we examined single neuronal activities in the vlpfc and it while monkeys were performing a serial probe reproduction task. in the task, two sequentially presented objects were memorized and then a target object was selected from memorized objects based on a color stimulus. in % out of vlpfc neurons, the delay-period activity showed objectselectivity and order-selectivity. in only % out of it neurons, the delay-period activity showed object-selectivity and order-selectivity. the starting time of the order-selective activity was earlier in the vlpfc. these results suggest that the vlpfc plays a role in holding information for object and their order of presentation and the it receives information for object and their order of presentation from the vlpfc. masao yukie, yasutaka oosawa department of behavioral physiology, tokyo metropolitan institute for neuroscience, fuchu, japan relational memory theory (eichenbaum et al., ) has been proposed from evidence that the hippocampal damage in rats impairs learning of transverse patterning task (a+ versus b−; b+ versus c−; c+ versus a−). very recent monkey study (alvarado et al., ) demonstrated that lesion of the hippocampus produced a significant impairment in that task and supported such a theory. in our study, however, ischemic damage of the hippocampus has not impaired learning of such a transverse patterning task (yukie et al., ) . in the present study, we examined effects of lesion of the monkey perirhinal cortex on transverse patterning task using two sets of d visual stimuli presented in a wgta. our three monkeys with perirhinal lesions failed to attain a learning criterion within a training limit of sessions in phase , although they learned easily the four problems in phases and . our results suggest that the perirhinal cortex, but not the hippocampus, is important for learning of transverse patterning task, that is, for formation of relational memory. yasuko sugase-miyamoto , noriyuki higo , munetaka shidara neuroscience research inst., aist, tsukuba, japan; grad. sch. of tsukuba univ., tsukuba, japan a recent dopamine d receptor study using antisense cdna showed that d receptor in rhinal cortex is crucial for learning associations between visual stimuli and reward schedules. neuronal responses in the perirhinal cortex differentiate the visual cues only when the cues are associated with the schedule states, while those in area te are related to physical attributes of the cue independently of the schedule states. to investigate the cellular substrate for d mediated associative learning, we examined monkey temporal lobe immunohistochemically with a d receptor antipeptide antiserum. d receptor immunoreactivity was observed in the pyramidal cells in layers ii-vi of the rhinal cortex and area te. the signal was mainly observed in cell bodies, and also in both apical and basal dendrites for some cells. the signal in layers v-vi was stronger in area of the perirhinal cortex than in area teav. the differential localization between area and te suggests the differential roles of the two areas in associative learning process. by using axonal transport of fast blue, diamidino yellow and tritiated amino acids, we determined the afferent and efferent connections of the retrosplenial cortex (rsp) in the macaque monkey. the rsp receives heavy projections from the subiculum, presubiculum and the caudal entorhinal areas (ec-ecl), and projects back to the presubiculum and the ec-ecl. the supracallosal portion of the rsp has connections primarily with the caudal half of the subiculum and presubiculum, as well as the lateral zone of the ec-ecl. the caudoventral portion of the rsp is, in contrast, mainly connected with the rostral half of the subiculum and presubiculum as well as the medial zone of the ec-ecl. the two portions of the rsp, thus, have access to different portions of the medial temporal lobe. these results indicate that there are two distinct neural systems in the retrosplenial-medial temporal network. hideko nakano , natsuko yoshida , kiyohisa natsume kyushu kyoritsu university, fukuoka, japan; kyushu institute of technology, fukuoka, japan; kyushu institute of technology, fukuoka, japan eeg activity was examined in english rhythm acquisition of japanese students who learn english as a foreign language (efl). we measured theta, alpha and beta rhythms of five subjects while they were reading aloud the materials and listening to the audio-recording, using eight electrodes attached to their skulls. the result shows that the increase of theta power at f and f was the highest and suggests that the theta rhythm at f and f may have a relationship to the process of english rhythm acquisition. moreover we found the highest increase in theta power when the subjects began to orally reproduce every line of the rhythm materials. this finding was observed in three right handlers except a left handler and a right handler who had just returned after -month english study experience in australia. these results suggest that the change of theta power at frontal areas may be more closely related to the japanese efl learners' english rhythm acquisition. research funds: kakenhi ( ) ps p-h neural correlates of music retrieval: an eventrelated fmri study using sparse temporal sampling takamitsu watanabe , sho yagishita , hideyuki kikyo , department of physiology, the university of tokyo school of medicine, tokyo, japan; department of molecular neuroimaging, national institute of radiological sciences, chiba, japan we investigated neural correlates of music memory using eventrelated functional magnetic resonance imaging and sparse temporal sampling technique with originally composed musical materials. written informed consent was obtained from all the subjects in accordance with the declaration of helsinki, and the experimental procedure was approved by the institutional review board of the university of tokyo school of medicine. a . t scanner system was used (te = ms; tr = s; acquisition time = . s). we demonstrated that the right hippocampus, bilateral lateral temporal cortices, left prefrontal cortex and left precuneus are involved in music retrieval. in addition, performance-based analysis suggested that the right hippocampus is associated with the accuracy of music memory. in this fmri study, we determined the neural correlates of the intellectual excitement. sentences describing facts in natural and human science were visually presented, and subjects judged whether they know the fact or not. after the fmri, each subject self-evaluates subjective "intellectual excitement" of each sentence. positive correlation with the self-evaluated intellectual excitement for known facts and novel facts were analyzed. significant correlation between cortical activation and self-evaluated intellectual excitement for novel facts was observed in the left and the right parahippocampal gyrus and for known facts was in the left orbital part of inferior frontal gyrus. it suggests the cortical areas related to self-evaluated intellectual excitement are different between getting of novel knowledge and recognition of existing knowledge. hyeonjeong jeong , motoaki sugiura , yuko sassa , keisuke wakusawa , , kaoru horie , , shigeru sato , , ryuta kawashima , gsics, tohoku university, sendai, japan; niche, tohoku university, japan; miyagi university of education, sendai, japan; department of pediatrics, school of medicine, tohoku university, japan; the lbc research center, tohoku university, japan a foreign language word is learned and retrieved either in daily situations (situation) or written text (text), and memory transfer is required when the learning and retrieval modes are different. in this experiment, normal japanese subjects learned korean words in the situation and text modes in video clips. during a subsequent fmri session, subjects were presented with the learned words in different movie clips; half of the learned words was presented in the same mode as in the learning session (match), and the rest was presented in a different mode (mismatch). comparison of the mismatch with match condition revealed significant activation in the orbital part of the left inferior frontal gyrus. the results suggest that this area plays a role in the memory transfer of foreign language words when the learning and retrieval modes are different. georgina e. cruz , christie l. sahley , kenneth j. muller physio. & biophys., univ. of miami, miami, fl, usa; biol. sci., purdue univ., west lafayette, in, usa in some animals much is known at the level of single synapses about mechanisms underlying behavioral sensitization, but in no system is the involvement of interactions at the network level well understood. the s-cell network of the medicinal leech is a chain of electrically coupled interneurons spanning the nerve cord with distributed sensory input and motor output and is crucial for sensitization of reflex shortening. its firing increases with sensitization although few additional s-cells initiate impulses during the reflex. we tested the hypothesis that the initial burst of impulses from the s-cell in the stimulated segment suppresses initiations in adjacent segments. hyperpolarizing the central s-cell to reduce its firing during skin stimulation markedly increased the number of initiations in adjacent s-cells, which corroborated the limited expansion of initiation sites seen in the behaving animal. a computational model of s-cell refractoriness further supported the idea of interaction among s-cells during sensitization. research funds: nih, u.s.a. ps p-h sensory/motor modules regulating the development of peer social relationship mamiko koshiba , , shun nakamura jst, crest, japan; ncnp social intelligence is indispensable for animal's survival and could have evolved to language capability. further, as a recent problem in japan, 'fewer children' supposedly causes the more tight interaction of child-parent, reciprocally the less between siblings or friends. in order to study the genetic and epigenetic development of peer social relationship after birth, we controlled peer interaction through limiting a particular sensory/motor modality as social deprivation and examined the effect on the active attachment behavior of domestic chick to conspecific mates. the chick has a merit of being precocial and unique in higher animal with no need of parent-care. comparing to the chicks reared as a group, the isolated chicks didn't develop their active attachment to peers. meanwhile, the behavior study with the chicks deprived not sensory/motor function itself, but only social interaction in auditory, visual, olfactory or tactile system, suggested that vocal communication at least must play a key role for the development. dna-chip study along the different social context brought candidates of social genes. shogo sakata , minoru hattori department of behavioral sciences, hiroshima university, higashi-hiroshima, japan; graduate school of biosphere science, hiroshima university, higashi-hiroshima, japan peak interval (pi) -s procedure is a very good method to investigate for timing. six male wistar rats were trained for five days a week in pi -s procedure over days. the -s bin of lever press responses on probe trials showed a clear peak point. the temporal distributions had the peak time of regression curve fitting with the gaussian function. the peak time corresponded to near the -s with reinforcement durations. then nicotine was administrated to the rat by intraperitoneal injection before daily pi -s session. results showed that the peak time in the nicotine administration was slightly leftward shift compared to the saline injection. however the pattern of temporal distribution of responses was not changed by the nicotine treatment as well as control condition. it suggests that the nicotine administration affects on the time perception that was reflected by the peak durations of responses. ps p-i the effect of random practice schedule on arbitrary stimulus-response association learning satoshi tanaka , , ritz oshio , , norihiro sadato , , manabu honda , nips, okazaki, japan; jsps, tokyo, japan; nagoya univ., nagoya, japan; ristex, jst, tokyo, japan; sorst, jst, tokyo, japan; ncnp, tokyo, japan previous studies suggest that randomly ordered practice facilitates retention and transfer of motor skills compared to blocked or regularly ordered practices. it remains unclear, however, whether the advantageous effects of random practice can be expended to cognitive skill learning in humans. we examined the simultaneous learning of multiple arbitrary stimulus-response (s-r) associations under three different practice schedules: blocked, random and regularly ordered. behavioral data indicate that subjects performing the random practice showed better performance of the retention and transfer of learning compared to those performing the blocked or regularly ordered practice. the present result indicates that random practice schedule is effective also for s-r association learning, which are considered as a bridge between motor control and cognitive control. ps p-i sports rats show increased level of bdnf in the cerebellum, possibly learning and memorizing well masaki morishima, sayuri hara, yutaka nakaya dept. nutrition and metabolism, univ. of tokushima, japan previously, we reported that the activation of hippocampal norepinephrine neurotransmission following a decrease in monoamine oxidase a was observed in sports, a novel hyper-running rat on wheel. this study assessed whether sports show increased bdnf levels and better learning and memory. compared to control, both protein and mrna levels of bdnf in cerebellum were significantly elevated in sports even without wheel running, and slightly increased in hippocampus. in the cerebellum of sports, trkb/pi k pathway was activated, whereas mapk pathway was activated in the hippocampus. locomotor activity assessed by the open field test showed that the sports were significantly more active in center coat than control. in the passive avoidance test, sports did not enter a dark area at next time indicating that sports showed better passive avoidance learning. these results suggest that bdnf signaling of sports were activated from trkb to mapk and pi k in the hippocampus and cerebellum, respectively, and that these signaling pathways might play an important role in learning and memory. research funds: kakenhi ( ) ps p-i selective manipulation of working memory through d and d receptors: computer simulation shoji tanaka, hiroki yata dept. of electrical & electronics eng., tokyo, japan though a number of experimental results suggest that working memory processes are controlled by the dopaminergic system, its mechanism is still unclear. to elucidate the mechanism, we have constructed a model of the prefrontal cortical neural circuit for working memory. the neurons in the model are leaky integrate-and-fire model with ampa, nmda, gaba, and leak conductances and have dopamine d and d receptors. the computer simulation with this model shows that d receptor activation mainly affect working memory activity itself, while d receptor activation affect the termination of working memory, being consistent with the experimental result. the simulation also mimics the hyper-and hypo-dopaminergic states. under such conditions, like schizophrenia, simulated pharmacological treatments using agonists and antagonists of d and d receptors indicate efficacies of some these treatments for the restoration of working memory. in conclusion, this kind of simulation shows how dopamine controls working memory by using the synergism of the actions of dopamine, glutamate, and gaba. kozo sugioka, tomiyoshi setsu, tatsuro yamamoto, toshio terashima div. anat. & dev. neurobiol., dept. neurosci., kobe univ. grad. sch. med., kobe, japan we examined activity and habituation in rats with experimentallyinduced abnormal morphogenesis of the hippocampus. pregnant rat (jcl:wistar) was injected with saline or mg/kg mam on the th day of gestation. the activity of male and female offspring was measured for each h light and dark period, and the habituation to the visual stimulation was observed by measuring the activity with every min interval for h under min dark/light alternative schedule during weaning and adult periods. activity was measured using infra-red sensor in a home-cage placed in the experimental room. the mam-treated rat showed hyperactivity for dark-period during both weaning and adult periods, and showed retarded habituation during weaning period. sex difference of behavioral alteration was evident during adult period in both groups. these behavioral disorders were discussed in relation to the mam-treated rat showing abnormal hippocampus (disruption of the ca pyramidal layer and ectopic neuron mass). ps p-i long-lasting tagging of functionally activated neurons in the mouse brain naoki matsuo, leon reijmers, mark mayford the scripps research institute, la jolla, ca, usa immediate-early genes (iegs) have been widely used as activity markers for mapping neurons involved in specific animal behaviors including learning and memory. however, conventional ieg approaches that use immunohistochemistry or in situ hybridization allow to detect neurons only shortly after their activation and does not enable genetic manipulations. here we have developed transgenic mice that allow selective and long-lasting tagging of neurons that were activated in a given brain region at a given time point. the mice consist of two components; c-fos promoter driven tetracycline-controlled transactivator (tta) and teto promoter regulated feedback loop. when strong neuronal activity occurs in the absence of tetracycline analogs such as doxycycline (dox), c-fos promoter driven tta initiates the teto-linked expression of mutant tta (tta*) that is not inhibited by dox. this teto-linked gene expression is then maintained indefinitely by feedback activation via the tta* even in the presence of dox. using this system, we have examined the expression of bicistronic teto promoter driven tau-lacz and egfp-glur . hamid gholamipour , shirin babri , khameneh saied department of physiology, university of tabriz, tabriz, iran; university of tabriz, iran; university of tabriz, iran diabetes mellitus is one of the most prevalent diseases in the world. because hippocampus is an important area for memory formation, the present study is scheduled to investigate the effect of insulin injection in ca region of hippocampus on memory formation. fifty male rats were divided into five groups. ( ) control ( ) sham operation ( ) test ( ) diabetic/saline ( ) diabetic/insulin. groups and were made diabetic by treatment with stz ( mg/kg, i.p.). in all but the control group, two canula were stereotaxically implanted in ca region of hippocampus. learning was tested and compared between groups through passive avoidance test. results showed that in the test group the latency increased as compared to control and sham groups (p < . ). compared to sham group diabetic/insulin group showed increased latency (p < . ) but no significant difference was found between diabetic/saline and diabetic/insulin groups. in conclusion, according to the results obtained in this study, insulin facilitates memory in intact rats but not in diabetic sex differences in hippocampus-dependent memory formation are well documented, but the mechanisms are poorly understood. the ca + /calmodulin (cam) kinase cascade regulates gene transcription in the hippocampus, which is required for long-term memory (ltm) formation. we hypothesized that sex differences in transcriptional regulation may account for the sexual dimorphisms in memory formation. we tested this idea by studying the role of cam kinase kinases (camkks). using mouse molecular genetics we found that camkk␤ is required for spatial, but not contextual ltm. consistent with the impaired spatial memory formation, camkk␤ null mutants lacked spatial training-induced creb activation and had impaired late ltp. in contrast to camkk␤, camkk␣ is required for contextual, but not for spatial ltm. furthermore, female camkk mutants had normal spatial and contextual ltm. thus, we show that there are malespecific mechanisms to regulate gene transcription that may explain sex differences in hippocampus-dependent memory formation. akshay anand, sudesh prabhakar, monika bhatia, c.p. das department of neurology, post graduate institute of medical education and research, chandigarh, india background: parkinson's disease has a prevalence rate of per , in india. we studied the park polymorphism in north indian population and parkin expression in early parkinson's disease (n = ) and sporadic parkinson's disease (n = ). methods: pcr, sscp, rflp and direct sequencing analysis were used to screen mutations. results: our results revealed homozygous exonic mutations in exon- , and in early pd and exon- and in sporadic pd, heterozygous mutations in exon and in five early pd and one sporadic pd patient. frequency of s/n polymorphism was significantly high suggesting that exchange of serine to asparagine at position of protein affects the secondary structure or hydrophobicity of the protein resulting in pathogenicity. our facs analysis of these samples indicates reduced parkin expression correlating with severity of mutations. conclusions: we conclude that high frequency of parkin mutations in pd population in india affect parkin expression resulting in pd. wanida tripanichkul , kittisak sripanichkulchai , david finkelstein faculty of medicine, srinakharinwirot university, thailand; faculty of medicine, khon kaen university, thailand; university of melbourne, australia emerging data suggests beneficial effect of estrogen for parkinson's disease (pd), yet the exact mechanisms implicated remain obscured. activated glia observed in mptp mouse model and in pd may participate in the cascade of deleterious events that ultimately leads to dopaminergic nigral neuronal death. estrogen can modify glial expression of inflammatory mediator, such as cytokines and chemokines implicated in neurodegeneration. to determine whether estrogen-elicited neuroprotection in pd is mediated through glia, adult male c bl/ mice were pretreated with beta-estradiol (e ), injected with mptp on the day and brains were collected on day . e pretreatment decreased nigral neuronal loss and diminished striatal fibers deficit induced by mptp. the neuroprotective effect of e was coincident with an attenuation of a glial response within the snpc and striatum. these findings propose that e neuroprotection in mptp mouse model may mediate through reactive glia inhibition. ps p-i effect of angiotensin-converting enzyme inhibitor perindopril in mptp-treated mice; immunohistochemistry and in vivo electron spin resonance (esr) study rumiko kurosaki , fumihiko yoshino , masaichi chang-il lee dept. of food sci. and nutrition, showa women's univ., tokyo, japan; clin. care and med. div. of pharmaco., kanagawa dental college, japan we investigated the effects of perindopril on the dopaminergic system and the oxidative stress in mice after mptp treatment. administration of perindopril showed dose-dependent neuroprotective effects against striatal dopamine and its metabolites depletion after mptp treatment. we have reported that th, gfap, pv, nnos and cu, zn sod positive cells in the substantia nigra was changed after mptp treatment in our immunohistochemical study. the administration of perindopril significantly attenuated mptp induced changes of these immunopositive nigral cells. we could measure increased oxidative stress in the brain of mptp and perindopril treatment mice using by in vivo esr technique. our results provide further evidence that the ace inhibitor perindopril may offer a novel therapeutic strategy for parkinsonǐs disease. research funds: kakenhi ( ) ps p-i recruitment of calbindin into substantia nigra dopamine neurons suppresses the onset of parkinsonian motor signs shigehiro miyachi , kaori sawada , haruo okado , atsushi nambu , masahiko takada tokyo met. inst. neurosci., fuchu, tokyo, japan; natl. inst. for physiological sci., okazaki, japan there is a consensus that dopaminergic neurons in the substantia nigra that express calbindin, a calcium-binding protein, are selectively invulnerable to parkinsonian insults. based on this notion, an attempt was made to test the hypothesis that parkinsonism may be suppressed by recruitment of calbindin into a subpopulation of nigral dopamine neurons that does not normally contain calbindin. an adenoviral vector expressing calbindin was injected unilaterally into the striatum of macaque monkeys, to let calbindin express in the dopaminergic neurons via retrograde transport. two to three weeks later, the parkinsonism-inducing drug mptp was systemically administered several times. parkinsonian motor signs, such as muscular rigidity and flexed posture, appeared only on the side ipsilateral to the calbindin recruitment when cumulative doses of mptp exceeded threshold for their bilateral onset. toru yasuda, hideki mochizuki, yoshikuni mizuno department of neurology, juntendo university school of medicine, tokyo, japan using the serotype- raav vectors, we have recently reported the protective effect of parkin on the ␣-synuclein (␣s)-induced nigral dopaminergic neurodegeneration in a rat model. here we investigated the neuronal specificity of ␣s toxicity and the effect of parkin co-expression in a primate model. another serotype (type- ) of raav (raav ) carrying αs cdna (raav -␣s), and a cocktail of raav -␣s and raav carrying parkin cdna were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral gabaergic and nigrostriatal dopaminergic neurons. the injection of raav -␣s alone caused a decrease of th-immunoreactivity in the striatum, while there was no effect on gabaergic neurons. in the presence of overexpressed parkin, ␣s seemed to be less accumulated and/or phosphorylated at ser residue in gabaergic neurons. these suggest that the ␣s toxicity is not expressed in non-dopaminergic neurons but the ␣s-ablating effect of parkin is exerted in all neurons introduced in primates. tomokazu oshima, yohsuke narabayashi narabayashi memorial laboratory of neurology, neurological clinic, tokyo, japan rigidity in aged parkinsonians is often intractable against surgeries. we investigated how their rigidity scored by updrs was related with ␤-band local field potentials (␤-waves) of the surgical targets in thalamic ventrolateral nucleus (vl) and posteroventral pallidal internal segment (pvp). forty patients aged - s gave informed consent for thalamotomy and/or pallidotomy. we divided the patients into groups with rigidity of . - (i), . - (ii), . - (iii), and . - (iv). the ␤waves were rated with total periods (%) of - hz wavelets in -s sample records. rigidity was re-scored after the surgeries. the vl ␤-waves were rated - % in groups i-iii with a slightly increasing tendency for increasing rigidity, but declined to about % in group iv. the pvp ␤-waves were - %, but with a decreasing tendency for increasing rigidity. the surgeries alleviated rigidity in all the groups, but were least effective in group iv with least vl and pvp ␤-waves. the results suggest that the pathology of aged parkinsonian rigidity develops beyond the pallido-thalamic pathway. yoshihisa tachibana , , hirokazu iwamuro , , masahiko takada , atsushi nambu , div. syst. neurophysiol., natl. inst. physiol. sci., okazaki, japan; sokendai; dept. neurosurg., univ. tokyo, tokyo, japan; dept. syst. neurosci., tokyo met. inst. neurosci., fuchu, tokyo, japan to approach a new therapy for parkinson's disease, extracellular unit recordings combined with microinjections of glutamate-related drugs were performed in the external and internal segments of the globus pallidus (gpe/gpi) of mptp-treated parkinsonian monkeys (macaca cyclopus). compared with the normal state, spontaneous oscillatory discharges were so often observed in the gpe/gpi and the subthalamic nucleus (stn) of the parkinsonian monkeys. microinjections of ionotropic glutamate receptor antagonists into the vicinity of recorded gpe/gpi neurons reduced their abnormal oscillations. these results suggest that glutamatergic excitatory input from the stn contributes to the oscillatory activity of gpe/gpi neurons, and that intrapallidal injections of ionotropic glutamate receptor antagonists may ameliorate some of parkinsonian symptoms. one of the pathological features of parkinsonǐs disease (pd) is loss of dopaminergic neurons in the substantia nigra pars comapacta (snpc). and it has been known that ␣-synuclein is involved in the neuronal loss. during the dopaminergic neuronal loss, activated microglia were centered in snpc. we hypothesize that ␣-synuclein may play a role in microglial activation to migrate to the pathological regions and to perform the neuronal cytotoxicity. we demonstrated that ␣-synuclein induced the cd expression on microglia and also enhanced the mt -mmp expression to shed off cd at the cell surface and degrade surrounding ecm to open the migratory way. a t mutant ␣-synuclein showed greater level of cd shedding and cell migration. extracellular treated ␣-synuclein also increased cd and mt -mmp expressions dose-dependently. among the multiple signaling pathways, erk pathway was involved in ␣-synuclein induced cell migration. these induced cell migration were also confirmed in human pd patients. research funds: national creative research initiative grant ( grant ( - ps p-j serotonergic fibers are involved in the conversion of l-dopa to dopamine in the striatum and the substantia nigra pars reticulata of parkinsonian model rats ryohachi arai , hiromasa yamada , yoshinari aimi , ikuko nagatsu department of anatomy, shiga university of medical science, otsu, japan; fujita health university school of medicine, japan dopaminergic neurons in the substantia nigra pars compacta (snc) project their axons to the striatum (st) and their dendrites to the substantia nigra pars reticulata (snr). dopamine released from these axons and dendrites is important in the regulation of motor activity. in parkinson's disease, dopaminergic neurons in the snc degenerate. l-dopa is the most effective drug for this disease. we hypothesize that, in parkinson's disease, a part of administered l-dopa is converted to dopamine in serotonergic fibers of the st and snr. here we produced parkinsonian model rats by the unilateral injection of hydroxydopamine into the snc, and found that serotonergic fibers in the st and snr were immunohistochemically positive for dopamine after l-dopa administration in the rats. therefore, it is possible that serotonergic neurons may be involved in the therapeutic effects of l-dopa for parkinson's disease. in mptp-induced pd monkey, reactive microglia are observed around neurons in nigra several years after mptp treatment and may be related to the progression of pd. to evaluate if reactive microglia in striatum and/or nigra of mptp-induced pd mice are present for a long time after mptp administration, like pd monkey. iba -and tb distribution in microglia were immunohistochemically investigated at h and days after twice mptp-treatments (one treatment comprised of intraperitoneal injections of mg/kg mptp at h interval) to c bl/ and balb/c at months (mo) interval. the recognizable change of iba -and tb -distibution in microglia of both mice strains was observed even mo after the first treatment. the twice mptp treatments tended to aggravate the symptoms in both mice strains, compared with once treatment. these results suggest that reactive microglia are present for a long time after the treatment by mptp and must play a role in the chronic progression of pd. ps p-j activated microglia affect the nigro-striatal dopamine neurons differently in neonatal and aged mice treated with mptp hirohide sawada , ryohei hishida , yoko hirata , kenji ono , hiromi suzuki , shin-ichi muramatsu , imaharu nakano , kunihiro tsuchida , toshiharu nagatsu , , makoto sawada school of medicine, fujita health university, aichi, japan; division of neurology, jichi medical university, japan; department of biomolecular science, gifu university, japan; research institute of environmental medicine, nagoya university, japan microglia play an important role in inflammatory process of parkinson's disease. we examined the effects between neonatal and aged microglia activated with lps on the nigro-striatal dopamine (da) neurons in mice treated with mptp. by mptp administration to neonatal mice, the number of da neurons in the substantia nigra was significantly decreased, whereas that in mice treated with lps and mptp was recovered. on the contrary, the number of da neurons of the week-old mice treated with mptp was significantly decreased with lps treatment. these results suggest that activated microglia in neonatal mice have neurotrophic potential, in contrast to the neurotoxic effect in aged mice. hyposmia is one of the most characteristic symptoms of parkinson's disease (pd). it may occur even before the motor symptoms start. in the olfactory bulb (ob), dopaminergic cells were present at glomerular layer. furthermore, it has been reported that ob contains neural stem cells. thus, ob has attracted attention because of its unique regenerative potential. in the present study, we established isolation of neurosphere forming cells (nsfcs) derived from adult mice ob, and examined proliferation potential in ob after dopaminergic neuronal loss induced by mptp, a selective toxin for dopaminergic neurons, utilized frequently as pd model. the number of neurospheres derived from adult ob was not decreased with mptp administration, rather significantly increased. we also evaluated nsfcs differentiation into neural subtypes. the isolation of neural stem cells has helped to establish the cellular basis of neurogenesis and the exciting potential for transplant-mediated treatment of degenerative cns disease like pd. ps p-j phosphorylation of erp in adult rat brain with neonatal -ohda treatment qinghua li, yasuyoshi watanabe department of physiology, osaka city university graduate school of medicine, osaka, japan dopaminergic neuron degeneration occurs in sporadic parkinson's disease (pd), but the mechanism of sporadic pd is not clarified. we prepared neonatal dopamine depleted rats, by i.c.v. injection of -ohda at (p ) and days after birth, to investigate the mechanism of dopaminergic neuron degeneration. at p , tyrosine hydroxylase (th) immunostaining cells were significantly reduced in the substantia nigra, and th immunostaining fibers were significantly reduced in the striatum, thus this model mimics the selective dopaminergic neuron degeneraion in sporadic pd. by two-dimensional electrophoresis we found that a certain protein was phosphorylated in the -ohda lesioned rats at p , and it was identified as disulfide-isomerase a precursor (erp ) a kind of molecular chaperone of the endoplasmic reticulum (er) by maldi-tof ms. the result suggests that the phosphorylation of erp may have the key function to induce dopaminergic neuron degeneration and somehow relates to the pathogenesis of sporadic pd. katsunori nishi department of neurology, tokyo metropolitan institute for neuroscience, tokyo, japan regrowth of survived dopaminergic (da) neurons after the administration of psi, a potent proteasome inhibitor, was examined in vitro. dissociated cell co-culture was prepared from embryonic rat mesencephalon and striatum. psi ( or nm, h) was applied to cultures at days in vitro and succeeding changes of da neurons were investigated up to days. more than % of da neurons reduced in number after the administration of psi, and a few truncated da neurons, devoid of neurites, being observed. non-da neurons were less severely affected at these concentrations of psi. regrowth of da neurites was observed approximately weeks after the administration of psi and continued during the observation period. in most of the regrowing da neurons, one of the processes extended far longer than the rest, suggesting that severely injured neurons retain the capacity to reextend axons. regrowth was less remarkable in mesencephalic culture lacking striatum indicating that target cells are necessary for this effect. in conclusion, psi-damaged da neuron has strong regrowth potential in vitro. ps p-j specific expression of proapoptotic factor pag on motor neurons in spinal cords of l-dopatreated parkinsonian models ikuko miyazaki, masako shimizu, francisco j. diaz-corrales, maria f. esraba-alba, masato asanuma dept. of brain sci., okayama univ. grad. sch. of med., dent. and pharmaceut. sci., japan we previously identified a proapoptotic gene, p -activated gene (pag ), as a dopa-induced gene in the striatum of l-dopa-treated parkinsonian models, which increased p expression to promote apoptosis by its nuclear translocation. last year, we also reported specific induction of pag in the internal capsule of l-dopatreated and constitutive expression in the smi- -immunopositive motor neurons in the pontine nucleus and motor nuclei of trigeminal nerve and facial nerve. in the present study, we examined distribution of pag in the spinal cords of l-dopa-injected parkinsonian rats by immunohistochemistry. l-dopa treatment showed inducing tendency of pag expression on the motor neurons in the anterior corn and lateral corticospinal tract of spinal cords. the expression of pag in the motor nuclei of cranial nerves and its induction in the spinal cords suggests its possible involvement in motor dysfunction such as dyskinesia. ps p-j an approach to the generation of ar-jp mouse model: crossbreeding of pael-r transgenic mice with parkin knockout mice hua-qin wang , , yuzuru imai , haruhisa inoue , , ayane kataoka , sachiko iita , nobuyuki nukina , ryosuke takahashi , neurology, university of kyoto, kyoto, japan; bsi, riken, saitama, japan since loss of parkin e activity appears to be causal of ar-jp, accumulation of potentially toxic parkin substrates should result in degeneration of da neurons. however, parkin knockout mice show no different da neuronal loss even at old ages, presumably due to relative short lifespan of mice. pael-r is one of the best characterized parkin substrates. we generated pael-r transgenic mice and crossbred it with parkin knockout mice. pael-r transgenic mice showed modest alterations in dopamine metabolism and behavioral deficits without displaying obvious dopaminergic neuronal loss at the age of one year. however, when pael-r transgenic mice were crossbred with parkin knockout mice, the da neuronal loss was induced in a pael-r gene dosage-dependant manner. these results strongly support that pael-r accumulation substantially contributes to dopaminergic neurodegeneration in ar-jp. parkinson's disease, a common motor disorder, is caused by a degeneration of dopaminergic neurons in the substantia nigra. after dopamine denervation, an over-activity of glutamatergic pathways has been found and that is implicated in the neuropathology of parkinson's disease. previous study (lai et al., ) have found that application of an antisense oligodeoxynucleotide specific for nr have successfully knockdown the expression of nr gene expression in the striatum of -hydroxydopamine-lesioned rats. in the present study, modulation of gene expression of nr was re-addressed using a small interfering rna (sirna) specific for nr . in pc cells, reductions of nr proteins after a single application of nr sirna were found by western blot experiments. and after one single application of nr sirna in the striatum of the lesioned rats, a significant reduction in apomorphine-induced rotation was found. slight reductions in the levels of nr immunofluorescence were found in the striatum after the sirna treatments. lai et al., . neurochem. int. , - . research funds: faculty research grant, frg/ - /ii- , hong kong baptist university ps p-j homocysteine and parkinson's disease: effects of acute intranigral administration on dopaminergic system g. chandra, k.p. mohanakumar indian institute of chemical biology, kolkata, india homocysteine (hcy) is implicated in a number of geriatric multisystem disorders and patients with hyperhomocysteinemia exhibit profound neuropsychological abnormalities. parkinsonǐs disease (pd) patients receiving long-term l-dopa therapy are reported to have elevated plasma hcy levels. we studied whether hcy is neurotoxic to the nigrostriatal dopamine (da)-ergic system in sd rats. animals infused unilaterally in substantia nigra pers compacta (snpc) with hcy ( . - mol in l) showed dose dependent loss of da and its metabolites, in the ipsilateral striatum on th day. animals with mol hcy exhibited significant motor disabilities and spontaneous and da-ergic drug-induced turning behaviors. in these animals a clear loss of neurons was visible in snpc, which were shown to be daergic by tyrosine hydroxylase immunoreactivity. intra-raphe infusion of hcy did not alter the neurotransmitter levels in the serotonergic perikarya or terminals. these results indicate the toxic potential of hcy to the da-ergic system and suggest that chronic l-dopa therapy in pd patients may further deteriorate the disease. ps p-j ubiquitin proteasome system was impaired by the aggregate formation of mutant ␥pkc found in sca takahiro seki , takayuki shimahara , naoko adachi , naoaki saito , norio sakai dept. mol. pharmacol. neurosci., grad. sch. biomed. sci., hiroshima univ., hiroshima, japan; lab. mol. pharmacol., biosig. res. ctr., kobe univ., kobe, japan we have previously demonstrated that several mutant protein kinase c gamma (␥pkc), found in several families of spinocerebellar ataxia type (sca ), are susceptible to cytoplasmic aggregation and cause cell death in cho cells, indicating that this property is involved in the etiology of sca . however, the relationship between the aggregate formation of mutant ␥pkc and cell death remains unclear. accumulating evidences indicate that the impairment of the ubiquitin proteasome system (ups) is related to the pathogenesis of many neurodegenerative disorders. therefore, we examined whether the aggregate formation of mutant ␥pkc affects ups function. the immunoreactivities for ubiquitin and proteasome were intensely accumulated in the aggregates of mutant ␥pkc. decreased proteasome activities were also observed in cells having aggregated mutant ␥pkc. these results indicate that the aggregation of mutant ␥pkc exert cytotoxic effect via the impairment of ups. it is well known that oxidant stress is involved in many pathologic conditions including brain ischemia and neurodegenerative diseases. recently, however, another type of stress, endoplasmic reticulum (er) stress has also been reported to be associated with such diseases. er stress is characterized by accumulation of unfolded proteins in the er that is caused by inhibition of protein modification, disturbance of ca + homeostasis or oxygen deprivation. we recently reported that targeting disruption of herp, a novel er stress-related gene, caused f cells vulnerable to er stress. using these cells, we developed a screening system for molecules that suppress er stress. approximately compounds have been screened, and we found some molecules that protect human neuroblastoma cells against er stress and oxidative stress. we speculate that this system could provide novel therapeutic targets to the er stress and oxidative stressrelated diseases. toshiyuki araki , yo sasaki department of peripheral nervous system research, national institute of neuroscience, ncnp, tokyo, japan; washington university school of medicine, st. louis, missouri, usa axonal degeneration which is observed in a variety of neuropathological conditions or physical damage to axons is a self-destructive program that is independent from programmed cell death. we previously reported that increased nicotinamide adenine dinucleotide (nad) production by the overexpression of nicotinamide mononucleotide adenylyltransferase (nmnat ) or exogenously applied nad can protect neurites from degeneration caused by mechanical or neurotoxic injury of neuronal cells. the mammalian nad biosynthesis is mediated by at least different kinds of enzymes and each enzyme converts different substrate to nad or its precursors. here we investigated whether overexpression of these enzymes or exogenous application of nad precursors protects neurites from degeneration through increased supply of nad. cocaine is considered to affect spine morphology and the composition of postsynaptic density (psd) of medium spiny neurons in nucleus accumbens (nac). we examined the accumulation of several proteins altered by cocaine challenge after withdrawal of repeated cocaine administration in psd fraction of rat nac at different time points. total psd protein yield was decreased at min, but next increased at h and returned to basal at h after cocaine challenge. actin showed a similar pattern but was maintained at high level at h. both psd- and glur were increased between h and h like actin. by contrast, some proteins such as drebrin were decreased after the peak at h. interestingly, the s proteasome subunit demonstrated a dramatic upregulation at h. these data suggest that the composition of psd proteins is regulated by proteasome activity as well as actin cycling. it is possible that some proteins may be removed from psd by proteasome following transient requirement for organizing psd in the nac of chronic cocaine-administrated animals. ps p-k effects of mdma and -meo-dipt on serotonin transporter and dopamine transporter yosuke yamauchi, takaya izumi, takayuki nakagawa, shuji kaneko dept. mol. pharmacol., grad. sch. pharm. sci., kyoto univ., kyoto, japan by two electrode voltage-clamp recordings from xenopus oocytes heterologously expressing serotonin transporter (sert) or dopamine transporter (dat), the effects of two addictive agents, , methylenedioxymethamphetamine (mdma) and -methoxy-n,ndiisopropyltryptamine ( -meo-dipt), on sert and dat were examined. as previously reported, mdma ( . - m) dose-dependently induced transport-associated, inward current response in the sertexpressing cells. interestingly, mdma-induced current response was also observed in dat-expressing cells. on the other hand, -meo-dipt ( . - nm) evoked an outward current response in sertexpressing cells similarly to that of selective -ht reuptake inhibitors. no current response was observed when -meo-dipt was applied to dat-expressing cells. these results suggest that mdma is transported not only by sert but also by dat, and that -meo-dipt suppresses the spontaneous transport activity of sert. junichi kitanaka , nobue kitanaka , tomohiro tatsuta , , yoshio morita , motohiko takemura department of pharmacology, hyogo college of medicine, nishinomiya, japan; department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan we examined the effects of pretreatment with clorgyline on morphine-induced behavioral changes and antinociception. a single administration of morphine ( mg/kg, i.p.) to male icr mice induced a hyperlocomotion. the anova analysis revealed statistical significance of a morphine effect (hyperlocomotion) and of a clorgyline pretreatment x morphine interaction effect (inhibition), but not of an effect of clorgyline pretreatment. clorgyline pretreatment itself did not affect the spontaneous locomotion. clorgyline at a dose of . mg/kg but not other doses tested significantly potentiated morphine-induced antinociception evaluated by tail flick but not hot plate test. clorgyline at the doses of and mg/kg significantly inhibited dopamine and serotonin metabolism. these results suggest that clorgyline showed its inhibitory effect on morphine-induced hyperlocomotion, but not antinociception, through mao inhibition. recent studies in our laboratory have shown that methamphetamine (meth)-induced hyperlocomotion and behavioral sensitization in mice were inhibited by clorgyline, an irreversible monoamine oxidase inhibitor. in this presentation, the effect of clorgyline pretreatment on meth reward was assessed by conditioned place preference (cpp) paradigm, using an apparatus developed with supermex ® sensors. although intact male icr mice showed a significant cpp for meth ( . mg/kg, i.p.), pretreatment with subchronic clorgyline ( . - mg/kg, s.c.) did not affect the magnitude of cpp. pretreatment with clorgyline significantly decreased apparent dopamine and serotonin turnovers in the striatum in a dose-dependent manner. these results indicated that clorgyline pretreatment did not influence meth reward in mice. of lobeline pretreatment on methamphetamine-induced stereotypy and monoamine metabolism in mice motohiko takemura , nobue kitanaka , tomohiro tatsuta , , yoshio morita , junichi kitanaka department of pharmacology, hyogo college of medicine, nishinomiya, japan; department of neuropsychiatry, hyogo college of medicine, nishinomiya, japan the effects of lobeline, an alkaloid constituent of indian tobacco, on methamphetamine (meth)-induced stereotypy and monoamine metabolism were investigated in male icr mice. pretreatment with lobeline ( . - mg/kg, i.p.) min prior to drug challenge significantly decreased an intensity of stereotypies and increased its latency to onset in a dose-dependent manner. in saline challenge groups, doses of lobeline examined did not affect the spontaneous locomotion nor induce any stereotyped behaviors. the range of lobeline doses examined except mg/kg did not affect apparent monoamine turnovers in the brain regions including striatum min after drug challenge. these results suggested that the inhibitory effect of lobeline ( . - mg/kg) on meth-induced stereotypy did not attribute to the change in the brain monoamine metabolism. kazuto sakoori, niall murphy riken bsi, wako-shi, japan previously we showed that endogenous nociceptin suppresses drug reward. here, we examined the effect of blockade of nop receptors on methamphetamine (meth) induced behavioral sensitization in order to understand the role of endogenous nociceptin in the chronic response to addictive drugs. first, nop receptor ko and wt mice were treated with mg/kg meth and locomotor activity measured daily for days. wt mice showed gradually increasing sensitivity to meth with repeated treatment of meth, whereas nop receptor knockout mice did not. next, nmol ufp- (a nop receptor antagonist) and mg/kg meth were co-administrated to mice and locomotor activity measured daily for eight days. ufp- strongly suppressed locomotor activity. thus, it was unclear if ufp- suppressed behavioral sensitization to meth during chronic drug treatment. however, when challenged with meth after four or more days without treatment, ufp- co-administrated mice showed a lower locomotor response. these results suggest that endogenous nociceptin facilitates the plastic changes induced by chronic treatment with addictive drugs. the influence of olanzapine (a d dopamine receptor antagonist) on the morphine-induced conditioned place preference (cpp) in male and female mice was investigated in the present study. subcutaneous (s.c.) injection of morphine ( - mg/kg, three drug sessions) induced place preference both in male and female mice. intraperitoneal (i.p.) administration of olanzapine ( . - mg/kg) induced place aversion (cpa) in female mice but not in male mice. administration of olanzapine ( , . and mg/kg, i.p.) reduced both the acquisition and expression of morphine-induced cpp in male and female mice. however, olanzapine ( mg/kg, i.p.) caused more than % mortality in female but not male mice. the effects of olanzapine were reversed by l-arginine ( mg/kg, i.p.) pre-administration. in conclusion, it seems that olanzapine reduced morphine effects in part via a nitric oxide (no) mechanism. feed-forward associative learning (ffal) theory of cerebellar motor learning proposed by the author presumes that higher motor centers have place-coding systems and the same systems are shared by the cerebellum. when a new motor learning proceeds with respect to a certain movement, previous learning results of the movement will turn out to be modified or erased. ffal theory presumes that transferred memory from the cerebellar cortex to nucleus will serve as the maintenance of the previous learning. from this line, many aspects of saccadic adaptation are successfully demonstrated by computer simulation based on the theory. another theoretical issue is the credit assignment problem of motor error. a motor error is generally an integrated result of maladjusted multiple learning elements, and is to be decomposed to each element credit. this problem naively leads to an idea of a dual redundant system for movement, one for execution and the other for error decomposition. ffal theory naturally and simply resolves the credit assignment problem and demonstrates a computer simulation of motor learning of multi joint movement system, using the place-coding hypothesis. ps p-d regulation of camp responsive element binding protein to stress in rat amygdala and hippocampal formation the department of anatomy and histology, shanghai medical school, fudan university, china amygdala (am) and hippocampal formation (hf) are important structures relating with emotional learning and memory. transcription factor, camp-responsive element binding protein (creb) in am and hf plays important roles in memory modulating processes. creb is a nuclear protein and is wldely accepted as prototypical stimulusinducible transcription factor. creb is activated in response to a vast array of physiological stimuli and then becames phosphorylated creb (pcreb). neurophysiological and neuropharmacological studies said that creb may regulate gene transcription and protein synthesis to maintain the long term and sustaining changing of synaptic efficiency during the long-term process of synaptic plasticity. but we cannot tell exactly via what kind of neurons in am and hf creb regulate these processes. we used the animal model, forced swimming (fs) as emotional stimuli and the experiment methods such as, immunocytochemistry, western-blotting with anti-pcreb antibody. the distributing profiles and changing rules of pcreb immunoreactive nuclei in amygdala and hippocampal formation of both control and experiment groups were investigated. the neuronal types of pcreb immunoreactive nuclei were analyzed by double-labelling immunocytochemistry with anti-pcreb, anti-glu and anti-pv antibodies. the results were: ( ) the number of pcreb immunoreactive neuclei and total amount of pcreb in the subnuclei of rat amygdala, dentate gyrus (dg) and cornu ammonis (ca ) were increased after fs. the rule of this kind of changing was of region-and time-specific. ( ) pcreb immunoreactive neuclei were expressed in glutamate immunoreactive neurons and were devoid in interneurons. these results suggested that pcreb in limbic system regulated the fs process and the regulation was finished via exciting neurons, glutamate neurons. hideto takahashi , , tomoaki shirao dept of neurobiol & behav.; ercgsm, gunma univ. grad. sch. of med., maebashi, japan dendritic spines are developmentally-regulated and activitydependent polymorphic structures based on actin cytoskeleton. drebrin is a spine-rich actin-binding protein regulating spine morphogenesis during development. here we find that chronic blockade of ampa receptors (ampar) inhibits synaptic drebrin clustering during development of hippocampal neurons, but not that of nmdar. further, the analysis of fluorescence recovery after photobleaching for egfp-drebrin a reveals that only . ± . % of drebrin in the spine is stable, with a turnover time of . ± . min. blockade of ampar by m cnqx reduces the population of stable drebrin ( . ± . %), and has no effect on a turnover time. on the other hand, blockade of nmdar by m ap has no effect on the population of stable drebrin, whereas shortens a turnover time ( . ± . min). these data suggest that ampar activities increase the binding capacity of drebrin in spines, and therefore promote drebrin clustering at spine synapses. instead, nmdar activities regulate spine-shaft shuttling of drebrin. itsuko nihonmatsu , yoshito saitoh , kaoru inokuchi , mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; crest, jst, tokyo, japan dendritic protein synthesis requires dendritic localization of mrnas in neurons. however, ultrastructural localization of these mrnas have not been well described. here we employed in situ electronmicroscopic technique to examine the precise localization of ␣camkii mrna in dendrites. ␣camkii mrna was located at the specific sites of dendritic shafts of pyramidal neurons, close to the spines, rather than in a diffused manner. we observed an increase in the ␣camkii mrna signals at the synaptic layer undergone l-ltp in the hippocampal dentate gyrus in unanesthetized freely moving rats. the increase was transient and returned to the basal level at h. the alteration in the ␣ camkii mrna localization in dendrites may reflect a functional change in the translational apparatus along with synaptic plasticity. reiko okubo-suzuki , , daisuke okada , kaoru inokuchi , , mitsubishi kagaku inst. life sci. (mitils), tokyo, japan; yokohama natl. univ. environment information sci., kanagawa, japan; crest, jst, japan late-phase long-term potentiation (l-ltp) depends on de novo protein synthesis. synaptopodin (synpo), an f-actin-associated protein, increases in the activated synapses following l-ltp induction. spine volume and f-actin content in the spines also increase during l-ltp. to reveal the roles synpo plays in the regulation of spine volume and f-actin content, we examined synpo-egfp (se) localization and spine volume in the hippocampal neurons using time-lapse confocal imaging techniques. se-overexpression did not alter spine volume, but the amount of se in spines positively correlated with the spine volume. pharmacological activation of the nmda receptors increased both spine volume and synpo content in spines. furthermore, experiments with ptk cells indicated that synpo stabilizes f-actin. these results suggest that synpo synthesized in soma and transported into the activated spines following l-ltp induction stabilizes spine f-actin that may lead to the maintenance of increased spine volume. mineo matsumoto , mitsutoshi setou , , kaoru inokuchi laboratory for molecular gerontology, mitils, japan; laboratory for nano-structure physiology, nips, japan subcellular localization of rna is an efficient way to localize proteins to a specific region of a cell. a requirement for dendritic rna localization and subsequent local translation has been demonstrated in several forms of experience-dependent synaptic plasticity. in spite of several attempts to identify these rnas, the population of rna species present in dendrites as a whole has not been well described. here we show the results of microarray analyses with rnas isolated from rna granule or synaptosome fractions prepared from the rat brain. these analyses revealed the complex nature of the dendritic rna population, which included rnas that were not expected to be in the dendrites. neural activity caused by an electroconvulsive shock triggered a redistribution of the dendritic transcriptome towards the synaptosome, a translationally active region. our results suggest that the redistribution of dendritic rnas is one of the mechanisms regulating local translation in response to synaptic inputs. ps a-a an activity that traps vesl- s protein into spines serves as synaptic tag synaptic tagging hypothesis explains how new proteins reach the activated synapses to establish input-specific late-phase plasticity, but it has not yet been substantiated. original idea of synaptic tagging is supposed to regulate protein entry into synaptic region including spines. using live-imaging techniques, we measured entry of vesl- s-egfp into spines (ve trapping) of rat hippocampal neurons in culture, and found that ve trapping activity serves as the synaptic tag in many criteria. ve trapping required synergistic activation of postsynaptic no-pkg pathway and an activity abolished by ttx at m, but not nm. because nm ttx is supposed to suppress na channels only postsynaptically, we concluded that ve trapping is a hebbian-like process that requires both pre-and postsynaptic activities. however, their coincidence time window was far wider (hrs) than that of early-phase plasticity, suggesting a requirement of persistently synchronized, rather than transiently coincident, activities, and a possibility of metaplastic states for late-phase plasticity. ps a-a acute effects of dehydroepiandrosterone sulfate (dheas) on the synaptic transmission and plasticity in rat hippocampal slices yuxia xu , ling chen , masahiro sokabe , , dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; dept. physiol., nanjing med. univ., nanjing, china; sorst cell mechanosening, jst, nagoya, japan; dept. mol. physiol., nips, okazaki, japan the neurosteroid dehydroepiandrosterone sulfate (dheas) is known to improve memory and learning in mammals. recently we report that chronic administration of dheas facilitates the induction of ltp in the rat hippocampus. to elucidate the underlying synaptic mechanism of the dheas effects, we examined in this study the acute effects of dheas on the synaptic transmission and plasticity at the ca region in rat hippocampal slices. an application of . dheas for min to the slice augmented instantly the epsp, which was terminated within min. however, even h after the drug application, a subthreshold tetanus could induce ltp without alteration of ppf. this facilitating effect of dheas on ltp induction was blocked by a coapplication of a nmda receptor antagonist with dheas for min, suggesting that the dheas effect involves a sustained modulation of the postsynaptic signaling mediated by nmda receptor. xiaoniu dai , ling chen , masahiro sokabe , , dept. physiol., nagoya univ., grad. sch. med., nagoya, japan; dept. physiol., nanjing med. univ., nanjing, china; sorst cell mechanosensing, jst, nagoya, japan; dept. mol. physiol., nips, okazaki, japan to know whether ␤-estradiol (e ) can protect ca neurons from functional deficit due to ischemia, adult male wistar rats were subjected four-vessel occlusion ( vo) for min, and the effect of e against this ischemic injury was examined. the electrophysiological properties of ca -ca synapses were examined by a real-time optical recording method days after ischemia. the ischemic brain showed a decreased synaptic transmission and an impairment of ltp induction but no alteration in paired-pulse facilitation. administration of e ( mg/kg) h before vo was able to protect ca neurons from these ischemic synaptic dysfunctions. the estrogen receptor-␣ selective agonist ppt ( mg/kg) produced a similar protective effect, but the estrogen receptor-␤ agonist dpn ( mg/kg) did not. above results suggest that e can protect neurons not only from cell death but also from functional damages caused by cerebral ischemia. ps a-a non-genomic rapid effects of estradiol on hippocampal synapses: multi-electrode dish analysis kohei nakajima , mari ogiue-ikeda , yuki oishi , suguru kawato , department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo at komaba, tokyo, japan; department of physics, university of tokyo, tokyo, japan estradiol has a non-genomic, rapid effect on synaptic transmission, which is manifested within seconds to minutes. recently, hippocampal neurons were shown to synthesize estradiol de novo, and to express estrogen receptor ␣ (er␣) at synapses. although these results imply that estradiol rapidly modulates synaptic plasticity through synaptic er␣, there are few electrophysiological evidence about it. here we investigated effects of estradiol on ltd by using wild type, er␣ hetero and er␤ hetero mouse hippocampal slices with a multi-electrode dish (med, panasonic). med enabled us to measure epsps in ca , ca , and dentate gyrus simultaneously. hippocampal slices were perfused with estradiol before nmda-induced ltd. we found that estradiol enhanced ltd both in wild type and er␤ hetero mouse, but not in er␣ hetero mouse. our data suggested non-genomic rapid action of estradiol through synaptic er␣. withdrawn ps a-a morphological changes of dendritic spines mediated by glucocorticoid receptor (gr) in rat hippocampus yoshimasa komatsuzaki , gen murakami , , tetsuya kimoto , , suguru kawato , college of humanities and sciences, nihon university, tokyo, japan; department of biophysics and life sciences, university of tokyo, tokyo, japan; crest, jst, japan modulation of hippocampal synaptic plasticity by glucocorticoids has been attracting much attention, due to its importance in stress responses. dendritic spines are essential for memory storage processes. here we investigated the effect of dexamethasone (dex), a specific agonist of glucocorticoid receptor (gr), on density and morphology of dendritic spines in adult male rat hippocampus by imaging of lucifer yellow-injected spines in slices. the application of nm dex induced rapid modulation of the density and morphology of dendritic spines in ca pyramidal neurons within h. the total spine density increased from . spines/m to . spines/m. dex significantly increased the density of thin and mushroom type spines, however only a slight increase was observed for stubby and filopodium type spines. because the presence of m cycloheximide, an inhibitor of protein synthesis, did not suppress the dex effect, these responses are probably non-genomic. hideki tamura , yuji ikegaya , sadao shiosaka division of structural cell biology, naist, nara, japan; laboratory of chemical pharmacology, university of tokyo, tokyo, japan the capacity of activity-dependent synaptic modification is essential in processing and storing information, yet little is known about how synaptic plasticity alters the input-output (i-o) conversion efficiency at the synapses. in the adult mouse hippocampus in vivo, we carefully compared the i-o relationship, in terms of presynaptic activity levels versus postsynaptic potentials, before and after the induction of synaptic plasticity and found that synaptic plasticity led synapses to respond more robustly to inputs, that is, synaptic gain was increased as a function of synaptic activity with an expansive, power-law nonlinearity, i.e., conforming to the so-called gamma curve. in extreme cases, long-term potentiation (ltp) and depression (ltd) coexist in the same synaptic pathway with ltp dominating over ltd at higher levels of presynaptic activity. these findings predict a novel function of synaptic plasticity, i.e., a contrast-enhancing filtering of neural information through a gamma correction-like process. research funds: st century coe research ps a-a actin organizations within single dendritic spines in ca pyramidal neurons studies with two-photon photoactivation naoki honkura, masanori matsuzaki, haruo kasai center for disease biology and integrative medicine, faculty of medicine, the university of tokyo, japan the major cytoskeleton of dendritic spines is filamentous actin (factin). we have here investigated sub-spine actin organizations using two-photon photoactivation of pa-gfp fused with ␤-actin in rat ca pyramidal neurons. we found segregated and discontinuous organizations of two pools of f-actin, dynamic and stable pools, which turned over with time constants of . min and min, respectively. fractions of the stable f-actin pool were greater in larger spines, therefore, the entire f-actin pool was more stable in larger spines. we succeeded in visualizing a retrograde flow of f-actin in the dynamic pool from the apex to the base of spine, and found that both the speeds ( . - . um/min) and lengths ( . - . um) of the f-actin flow were greater in spines with larger head volumes. moreover, spine heads rapidly shrank when actin polymerization was blocked by latrunculin a, suggesting that the rate of actin polymerization in each spine actively and continuously determines the volume of spine head via the length of f-actin. tomoharu nakamori , katsushige sato , kohichi tanaka , hiroko hamazaki mol. neurosci., tmdu, tokyo, japan; physiology, tmdu, tokyo, japan the visual wulst (vw) in the thalamofugal pathway in chicks is known to have a critical role in the visual learning. to understand the function of the vw in the learning process of imprinting, we investigated the neuronal activity of vw region in chick brain. the slice stained with a voltage-sensitive dye was prepared for a multiple-site optical recording. when chicks were reared in quasi-dark condition, the extent and amplitude of response induced by electrical stimulation were different between at or days post-hatching (p or p ), and at p . this corresponds to behavioral data showing that chicks have high ability of visual learning in imprinting behavior until p , but they lose this ability at p . in addition, the light-exposed chick showed larger optical response than the dark-rearing one. the optical response in the vw was partly inhibited by the glutamate-and gaba-receptor antagonists. these results suggest that the glutamatergic as well as gabaergic neurons are active in the area including vw and that the neuronal activity of vw affects the learning ability for imprinting. withdrawn ps a-b effect of estrogen on hippocampus in male and female mice takanori sugawara , shinji hayashi , victoria luine graduated school of integrated sience, yokohama city university, yokohama, japan; department of psychology, hunter college, city university of new york, new york, usa we examined structural difference in the hippocampal neurons with golgi stain among the male, the female and the female treated with estrogen neonatally. the mice were gonadectomized and received ␤-estradiol (e ) or oil-vehicle injections at adult before golgi impregnation. spine densities m of apical dendrites of the pyramidal neurons in the hippocampus ca region were calculated with categorization into three shapes, i.e., mushroom type with large head, thin type and filopodia-like type. as a result, only in the female not estrogen treated neonatally, the mushroom type and total spine densities were increased but the thin type spine density was decreased by e treatment in adult. the present results indicate that estrogen given at adult induces an enlargement of spine to mushroom type and generates new spines only in the female mice not treated with estrogen neonatally. thus, dendritic spine formation seems sexually dimorphic and depends on the sex steroid environment during the neonatal period. jun-ichi goto , , takafumi inoue , , akinori kuruma , katsuhiko mikoshiba , , lab. developmental neurobiology, brain science inst., riken, saitama, japan; div. molecular neurobiology, inst. medical science, univ. tokyo, tokyo, japan; calcium oscillation project, icorp-sorst, jst, tokyo, japan changes in synaptic efficacy at the parallel fiber (pf)-purkinje cell (pc) synapse are postulated to be a cellular basis for motor learning. although long-term efficacy changes lasting more than an hour at this synapse, i.e., long-term potentiation and depression, have been extensively studied, relatively short lasting synaptic efficacy changes, namely short-term potentiation (stp) lasting for tens of minutes, have not been discussed to date. here we report that this synapse shows an apparent stp reliably by a periodic burst pattern of homo synaptic stimulation. this stp is presynaptically expressed, since it accompanies with a reduced paired-pulse facilitation and is resistant to postsynaptic ca + reduction by bapta injection or in p/q-type ca channel knockout cerebella. this novel type of synaptic plasticity at the pf-pc synapse would be a clue for understanding the presynaptic mechanisms of plasticity at this synapse. aya ishida, wataru kakegawa, michisuke yuzaki department of physiology, keio university, tokyo, japan mitogen-activated protein kinase (mapk) cascade is thought to be essential for the synaptic plasticity and learning. in the hippocampus, three different mapk subfamilies, including extracellular signalregulated kinase (erk), p mapk and c-jun nh -terminal protein kinase (jnk), have been shown to selectively regulate different forms of synaptic plasticity -long-term potentiation (ltp), longterm depression (ltd), and depotentiation after ltp, respectively. although erk was previously shown to play a role in cerebellar ltd in cultured purkinje cells, the role of mapks has not been systemically studied. here, we examined the effect of specific inhibitors of three different mapks on ltd by patch-clamp recordings from cerebellar slices. we found that u , a specific inhibitor for erk activation, significantly inhibited ltd induction, whereas sb and sp , antagonists for p mapk and jnk, respectively, had no effect. therefore, unlike hippocampal ltd, cerebellar ltd was dependent on erk, suggesting involvement of different intracellular downstream pathways. ps a-b regulation of ampa receptor trafficking by aaa atpases in cerebellar purkinje cells: are nsf and vcp playing complementary or antagonistic roles? thomas launey , chou-chi li , yumiko motoyama , junko yamaoka , masao ito riken brain sci. inst., japan; national cancer institute, nih, ma, usa the number of postsynaptic ampa receptors (ampar) is regulated by interactions with multiple protein complexes, throughout its synthesis, maturation, transport, synaptic insertion and degradation. aaa atpases influence several of these stages, the most extensively studied being nsf's contribution to ampar trafficking. in cerebellar purkinje cell (pc), we show that valosin containing protein (vcp), an atpase with high homology to nsf, is bound to ampa receptors in pc's dendritic compartment. following glur co-ip from molecular layer, vcp was detected by ms/ms and by monoclonal anti-vcp. pull-down assay showed a direct interaction between vcp and glur c-term domain, requiring vcp n-term domain and both the nsf and pdz binding domains of glur . glur phospho-ser promotes vcp complex dissociation, suggesting a relation with synaptic plasticity. further, pep m-related peptides, thought to interfere specifically with nsf-regulated ampar trafficking, also blocked the glur -vcp interaction. yuichi kitagawa , , shin-ya kawaguchi , , tomoo hirano , dept. biophys., grad. sch. sci., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan at inhibitory synapses on a cerebellar purkinje neuron (pn), postsynaptic depolarization induces long-lasting potentiation of the gaba a receptor (gaba a r) responsiveness (rebound potentiation: rp). previous studies have clarified the molecular mechanism regulating rp induction. whether rp is induced or not is determined by the balance of activities of protein kinases (camkii and pka) and phosphatases (pp- and calcineurin). to understand the complex behavior of biochemical reactions systematically, a kinetic simulation model to analyze the behaviors of signaling network was developed. computer simulation reproduced the bistable states of gaba a r phospholyration according to stimulation patterns, which apparently corresponded to whether rp was induced or not. we further studied the systematic property of the molecular network, and obtained several experimental predictions. these possibilities were evaluated by experiments such as immunocytochemistry using cultured pns. ps a-b long-term depression of synaptic transmission in a songbird motor nucleus essential for song learning yuki haruta, yachun huang, neal hessler vocal behavior mechanisms riken brain science institute, japan in order to fully understand the neural basis of song learning, it is critical to characterize forms of synaptic plasticity that could be involved in this process. we previously reported that, in synapses of the song motor nucleus ra, participation of postsynaptic nmda receptor nr b subunits and presynaptic transmitter release both decrease from young birds to adults. here, we tested whether synaptic function could be modified in a similar way by acute stimulation. after pairing slight postsynaptic depolarization with presynaptic stimulation, ltd was reliably induced at both hvc and lman inputs in juvenile birds from to days old. this depression required activation of postsynaptic nmda receptors, and was expressed by decreased transmitter release, which required activation of cannabinoid receptors. no ltd could be induced in normal birds over days old, when song learning is nearly complete, but ltd remained possible in birds over days old who had been isolated from song tutors, and thus retained the capacity for learning. ps a-b involvement of ca + -permeable ampar in the repetitive-ltp induced synaptic enhancement (rise) yukiko ueno, keiko tominaga-yoshino, akihiko ogura graduate school of frontier biosciences, university of osaka, osaka, japan we showed previously that exposures to glu of cultured rat hippocampal slices at h intervals produced a long-lasting enhancement in synaptic strength accompanied by synaptogenesis (rise). we examined here whether the conversion of ampar subunits occurred during the development of rise. immunochemical staining for ampar subunits, glur and glur , showed that the number of glur -positive puncta increased transiently after the repeated glu exposures, whereas the number of glur -positive puncta increased gradually and persistently. jstx (a ca + -permeable ampar blocker) suppressed fepsp amplitude recorded at ca -ca synapses by - % in the period corresponding to the transient increase of glur -positive puncta. this transient increase should represent the delivery of ca + -permeable (glur -lacking/glur -including) ampar to synaptic sites. furthermore, jstx application at that period blocked the rise production. these results suggest that the transient delivery of ca + -permeable ampar to synaptic sites is involved in the rise production. yoshihiro egashira, tsunehiro tanaka, yuji kamikubo, yo shinoda, keiko tominaga-yoshino, akihiko ogura osaka univ. grad. sch. frontier biosciences, toyonaka - , japan long-lasting synaptic plasticity, the cellular basis of long-term memory, is assumed to be associated with protein synthesis. using cultured rat hippocampal slices, we previously found that a long-lasting synaptic enhancement coupled with an increase in the number of synaptic structures was established after inductions of ltp, not after its single induction. this synaptic enhancement required protein synthesis for its establishment. we recently found an apparently mirror-image phenomenon; inductions of ltd led to a long-lasting synaptic decrement coupled with a decrease in the numbers of synaptic structures. to know whether this synaptic decrement also requires protein synthesis, we induced ltd times ( h intervals) by applications of dhpg (a type i mglur agonist), during or after which anisomycin (a protein translation blocker) was applied. we found that anisomycin did not block the induction of ltd but blocked the establishment of the long-lasting synaptic decrement. haruo mizutani, tetsuya hori, tomoyuki takahashi department of neurophysiology, graduate school of medicine university of tokyo, tokyo, japan bath-application of -ht ( m) attenuated the amplitude of evoked epscs and facilitated paired-pulse ratio without affecting the miniature epsc amplitude, suggesting that its site of action is presynaptic. the -ht b receptor agonist cp mimicked the presynaptic inhibitory effect of -ht. -ht b receptor antagonist nas- reversed the -ht inhibitory effect, indicating that the -ht induced inhibitory effect occurs by mediating -ht b receptors. the presynaptic inhibitory effect of -ht became weaker as animals matured. in whole-cell recordings from calyceal presynaptic terminals, -ht attenuated voltage-dependent calcium currents, but had no effect on potassium currents. this -ht effect was characterized with a marked desensitization, but sustained under the fast calcium chelating agents, bapta. these results suggest that -ht, upon activating -ht b receptors, inhibits presynaptic calcium channels thereby inhibiting transmitter release and induces receptor desensitization by calcium influx at the immature calyceal synapse. takako ohno-shosaku , masato ano , yuki hashimotodani , tadasato nagano , masanobu kano dept. impair. study, grad. sch. med. sci., kanazawa univ., kanazawa, japan; dept. neurophysiol., grad. sch. med., osaka univ., osaka, japan; dept. cell. neurosci., grad. sch. med., osaka univ., osaka, japan retrograde endocannabinoid signal contributes to activitydependent modulation of synaptic transmissions in various brain regions. endocannabinoid release is triggered by depolarizationinduced elevation of intracellular calcium level or activation of gq-coupled receptors. here we report that nmda receptors can also contribute to generation of endocannabinoid signal. inhibitory postsynaptic currents (ipscs) were recorded in cultured hippocampal neurons prepared from newborn rats. application of nmda induced a transient suppression of cannabinoid-sensitive ipscs but not cannabinoid-insensitive ipscs. the nmda-induced suppression of ipsc was blocked by a cannabinoid receptor antagonist. these results indicate that activation of nmda receptors induces the endocannabinoid release, and suppresses the inhibitory synaptic transmission through activation of presynaptic cannabinoid receptors. the most caudal region of the rat spinal cord, the conus medullaris has a simple anatomical feature, which lacks ventral as well as dorsal root fibers and somatic motor neurons in the ventral horn. a small number of neurons distribute around the central canal, and some of them are nitric oxide synthase (nos) positive. a dense distribution of nerve fibers immunoreactive to cgrp, sp, and npy was found in dorsal part of the conus medullaris similarly to that of other spinal cord levels. in addition, enk-, -ht-, and th-immunoreactive varicose fibers were richly distributed throughout the sectional plane. to analyze this unique structure may provide valuable information on the basic neural cytoarchitecture and fiber connections of the spinal cord, particularly for the intraspinal circuitry. for this purpose, we made an electron microscopic study using nadph-diaphorase histochemistry combined with immunohistochemistry for neuronal markers. adenosine has been known to be a neuro-modulator in the nervous systems and four types of adenosine receptor are identified (a , a a, a b and a ). adenosine a and a receptors have been reported to inhibit high-threshold ca channel currents in neurons. to investigate the interaction between adenosine a and a receptors in rat striatum neurons in culture, l-type ca channel currents were recorded by whole-cell clamp method before and after administration of a agonist (cpa) and a agonist ( -cl-ib-meca). ca currents were decreased after administration of low concentration of cpa and -cl-ib-meca as reported previously. although ca currents were decreased by -cl-ib-meca in the presence of cpa, ca currents applied with cpa were not decreased on cells in the presence of -cl-ib-meca. at administration of cpa and -cl-ib-meca on cells simultaneously, ca currents were not decreased. these results suggested that adenosine a receptor may inhibit adenosine a receptor throughout a intracellular pathway in neurons. ps a-c influence of extracellular gaba and taurine to gaba a receptor-mediated actions in radially migrating cortical plate cells with identified by in utero electroporation t. furukawa , j. yamada , k. inoue , y. yanagawa , a. fukuda , dept. physiol., hamamatsu univ. sch. med., japan; dept. biol. info. process, grad. sch. elec. sci. & tech., shizuoka univ., hamamatsu, japan; dept. developmental and integrative neurosci., gunma univ. sch. med., gunma, japan it is well known that role of gaba a -r mediated actions is important for early cns development. the radially migrating cells may affected by the actions. gaba content in the brain of gad -gfp knock-in mouse decrease compared with the wild type mice. therefore, we investigate the influence of the circumferential gaba concentration to radially migrating cells. furthermore, as it was known that gaba a -r is affected by taurine, the influence of taurine to radially migrating cells was also investigated. there was no significant difference in distribution of radially migrating cells that was labeled by means of electroporation. evoked gaba a -r mediated currents of labeled cells had dose-dependent manner and had no differences among genotypes. therefore, we have examined the influence of circumferential taurine to gaba a -r mediate actions. takashi hayakawa , hiroyuki hioki , kouichi nakamura , , hisashi nakamura , takeshi kaneko , dept. morphol. brain sci., grad. sch. med., kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan we previously reported that almost all vesicular glutamate transporter (vglut )-immunoreactive (ir) cells were also gabair in neocortex and choline acetyl transferase (chat)-ir in caudate-putamen in rat. although, in dorsal and median raphe nuclei, many vglut -positive cells showed immunoreactivity for -hydroxytryptamine ( ht), a significant proportion ( . %) of vglut -postive cells was ht-negative. in this study, triple immunofluorescence staining was performed for vglut , ht and one of the following proteins: neuronal nuclear antigen (neun), glial fibrillary acidic protein (gfap), glutamic acid decarboxylase (gad ) and tyrosine hydroxylase (th). our results showed that all of the vglut -positive/ ht-negative cells were immunoreactive for neun but not for gfap. furthermore, we found that these vglut positive/ ht-negative neurons didn't show any immunoreactivities for gad nor th, and thus it is indicated that there is a group of exclusively glutamatergic vglut -positive neurons in these nuclei. research funds: kakenhi , , ps a-c cortico-striatal and fast-spiking cell activity in the rat frontal cortex during cortical oscillations in vivo: modulation by serotonin m victoria puig , mika ushimaru , yoshiyuki kubota , akiya watakabe , tetsuo yamamori , yuchio yanagawa , yasuo kawaguchi div. cerebral circuitry, nips, okazaki, japan; div. brain biology, nibb, okazaki, japan; dept. genetic and behavioral neurosci., gunma univ. graduate school of med., japan we studied how cortico-striatal (cs) and fast-spiking (fs) cells are modulated by slow-wave-sleep (sws) oscillations and by serotonin ( -ht). cs and fs cells were recorded simultaneously with the electrocorticogram in the secondary motor area of anesthetized rats that expressed a gfp in gabaergic interneurons. fs displayed a highsuccess excitation to striatal stimulation, suggesting a control of cs over fs. during sws, both cs and fs fired during the up-states though with different patterns. the stimulation of the dorsal raphe promoted longer up-states. moreover, % of the cs were inhibited by -ht through -ht a r and % were excited through -ht a r. however, % of the fs cells were inhibited and % excited. these results show that cs cells are more inhibited by -ht than fs. the expression of -htr was confirmed by in situ hybridization. research funds: jsps pe and ryohei tomioka, kathleen rockland laboratory for cortical organization and systematics, riken brain science institute, saitama, japan in small mammals, gabaergic neurons have been shown to contribute to ipsi-and contralateral cortical projections. here, we report in monkey as well that some gabaergic neurons send long-distance projections. identification was partly based on golgi-like labeling of the dendritic tree, achieved by injecting adenovirus as a retrograde tracer in areas v , teo, or tep. aspiny or sparsely spinous nonpyramidal neurons were clearly visualized in the white matter or, less frequently, in cortical gray matter, in mainly layer but also in layer and/or . in each of the cases, about - gabaergiclike neurons were scored, with a preferential location anterior to the injection sites. in addition to their characteristic dendritic morphology, the neurons were identified as positive for gabaergic neuronal markers; namely, gad , somatostatin, or nos. thus, we conclude that gabaergic projection neurons are phylogenetically conserved; but more work is needed to determine ( ) their other features, ( ) possible species variability, ( ) their functional significance. supported by riken bsi. withdrawn ps a-c regional, cell type, and layer-specific differences in cholinergic modulation of neocortical neurons allan gulledge , , susanna b. park , greg j. stuart , yasuo kawaguchi national institute for physiological sciences, japan; div. neurosci., jcsmr, australian national university, canberra, australia we examined cholinergic modulation of pyramidal and nonpyramidal neurons in neocortical areas (prefrontal, somatosensory, and visual cortex). transient ach exposure ( m) inhibited layer pyramidal neurons in all areas via activation of an sk-type potassium conductance. pyramidal neurons in layers / were generally less responsive to ach, but ach inhibited layer cells in visual cortex. prefrontal layer pyramidal neurons were more responsive to ach than were layer cells in other areas of cortex. fast spiking (fs) nonpyramidal neurons were completely non-responsive to ach, even at very high concentrations ( mm). on the contrary, ach generated fast, nicotinic receptor-mediated responses in % of non-fs interneurons ( of cells). laminar or regional differences in ach responses were not observed in nonpyramidal neurons. these data suggest that ach may act to inhibit the output of cortical projection neurons while preserving information processing in superficial neurons. toshikazu kakizaki , , kenzi saito , , yuchio yanagawa , department of genetic and behavioral neuroscience, gunma university graduate school of medicine, maebashi, japan; sorst, jst, kawaguchi, japan; sokendai, hayama, japan a major inhibitory neurotransmitter gaba is synthesized by glutamate decarboxylase (gad), and is accumulated into synaptic vesicles by vesicular gaba transporter (vgat). another inhibitory neurotransmitter glycine could be transported into synaptic vesicles by vgat, and be co-released with gaba. several molecules related to gabaergic or glycinergic neurotransmission are expressed in nonneural tissues, suggesting that gabaergic and glycinergic systems exert their activities outside the cns. vgat-deficient mice die in the perinatal period, and display omphalocele, defect in ventral body wall closure, suggesting that gaba and/or glycine are involved in body wall formation. to further investigate whether gaba is essential for the ventral body wall formation or not, we have been examining how the body wall developed in the gad -deficient mouse fetus. ps a-c gaba mediated glutamate release from developing cerebellar cortex and ca sensitivity sachiko yoshida, miyuki ohshita, masakazu uematsu, shoichiro hirano, shinya tanaka, naohiro hozumi toyohashi university of technology, toyohashi, japan gaba (␥-amino butyric acid) and glutamate are known to play important roles as modulators in the survival and development of cerebellar neurons. during cerebellar development, gaba-mediated responses, gaba excitations, become depolarized inducing an increase in intracellular calcium concentrations, and are thought to have important trophic effects. many observations of gaba excitations using cultured cells have been reported, whereas few using acute slices. we recently reported the spatial nature of glutamate and gaba releases from acute slice with an enzyme-linked assay system and ccd imaging technology. in the present study, we evolved this measurement system to allow observations of spontaneous or gaba-mediated glutamate release from developing postnatal acute cerebellar slices. glutamate was released spontaneously, but gaba-mediated glutamate release appeared from postnatal to day in egl. its release, especially from premigratory zone, was inhibited by ni + , but cd + couldn't. we suggest that gaba excitation induces granule cell migration. ps a-c gabaergic fiber in the rat trigeminal motor nucleus reorganized following masseter nerve transection hiroyuki hayashi , hiroaki wake , junichi nabekura , osamu takahashi department of histology, kanagawa dental college, yokosuka, japan; national institute of physiological science, okazaki, japan it has been reported that gabaergic nerve terminals are seen in the trigeminal motor nucleus (vm) of the rat, and that there are primary afferent inputs from the muscle spindle of masticatory muscles to the vm cell bodies. we recently found that the number of these gabaergic fibers projecting to vm is markedly reduced in postnatal development. in this study, to elucidate the possibility that the re-arrangement of gabaergic circuits could be reproduced after neuronal injury, we examined the effect of axonal injury of the masseter axon on the gabaergic circuits in the vm. two to eight weeks after unilateral surgical transection of the masseter nerve of rats, gabalike immunoreactive (gaba-ir) varicosities were examined using immunofruorescence technique. the significant increase in number of gaba-ir varicosities were seen after eight weeks of the operation. this result suggest that gabaergic inputs may play one of important role for reorganization of afferent inputs in the vm. akiko arata , kunihiko obata , jonathan davies , mark bellingham , peter g. noakes lab. for memory & learning, riken-bsi, wako, japan; obata res. unit, riken-bsi, wako, japan; sch. biomed. sci., univ. queensland, queensland, , australia during embryonic development, approximately half of the motoneurons (mns) undergo programmed cell death. this process depends also on glycinergic and/or gabaergic synaptic activity, as suggested by increased mn number in gephyrin-deficient mice (banks et al., ) . we investigated the involvement of gaba alone in the mn death using gad -deficient mice, in which cerebral gaba is reduced to less than % of the wild-type. mn numbers at embryonic day (e) were counted by the method of banks et al. brainstemupper spinal cord blocks were prepared from e embryos and subjected to electrical recording from the c and c ventral roots and also gaba measurement. in gad -deficient embryos, increase in number of brachial mns ( %) and decrease in both spontaneous discharges in the c , c roots and gaba content (less than %) were observed, compared with those of the wild-type littermates. gaba might control cell death in developing network. abolghasem esmaeili, joe lynch, pankaj sah queensland brain institute, the university of queensland, australia the amygdala has key role in processing emotional information. distribution of gaba a receptor subunits is crucial for understanding physiology and pharmacology properties of these receptors in the amygdala. we examined the pharmacology of gaba a receptors by expressing different subunit combinations in hek cells and comparing the pharmacology with specific gabaergic inputs in the amygdala. dmcm blocked the actions of gaba at expressed ␣ ␤ ␥ and ␣ ␤ ␥ combinations ( % reduction) but had no effect at ␣ ␤ ␥ or ␣ ␤ ␥ . in slice recordings dmcm blocked ipscs by % in the lateral amygdala and had variable effects in the central amygdala. diazepam and zolpidem enhanced ipscs in the lateral whereas the response in the central amygdala was either reduction or enhancement. real time pcr and western blotting revealed differences in the distribution of gaba a receptor subunits between the lateral and central amygdala. we conclude that in the lateral amygdala all inputs have ␥ subunits whereas in the central amygdala some inputs contain ␥ while others contain ␥ subunits. masayuki kobayashi department of pharmacology, nihon university school of dentistry, tokyo, japan noradrenergic agonists have different effects on the excitatory neural transmission according to their subtypes in rat cerebral cortex. the present study aimed to explore what kind of second messengers and the precise site of synaptic membrane, pre-or postsynaptic, is involved in these noradrenergic modulation. the suppressive effect by activation of ␣ -adrenoceptors was mediated by protein kinase c, and excitatory effect by activation of ␤-adrenoceptors was mediated by camp/protein kinase a cascade. phenylephrine suppressed inward currents evoked by puff application of glutamate, and it decreased mepsc amplitude and increased mipsc frequency. isoproterenol increased mepsc frequency and decreased mipsc amplitude. gaba-induced postsynaptic currents were suppressed by isoproterenol. these results suggest that phenylephrine may decrease postsynaptic currents through glutamate receptors and increase the release probability of gaba from presynaptic terminals. on the other hand, isoproterenol may facilitate glutamate release and suppress gaba a receptor-mediated postsynaptic currents. ps a-d hydrogen sulfide modulates synaptic transmission in rat hippocampal neurons mamiko tsugane , takashi iwai , yasuo nagai , junichiro oka , hideo kimura dept. mol. genetics, nat'l. inst. neurosci., ncnp, tokyo, japan; lab. pharmacol., fac. pharm. sci., tokyo univ. sci., chiba, japan hydrogen sulfide (h s), which is a well-known toxic gas and facilitates the induction of hippocampal long-term potentiation, has been proposed as a neuromodulator in the brain. the aim of this study is to understand the mechanism of regulation on synaptic transmission by h s. we examined the effect of h s on spontaneous excitatory postsynaptic currents (sepsc) as well as paired-pulse facilitations using both whole-cell and field potential recordings from rat hippocampal slices. sodium sulfide (na s), a donor of h s, reduced the amplitude of field excitatory postsynaptic potentials and increased the ratio of paired-pulse facilitation. the frequency and the amplitude of sepsc were initially reduced by na s then gradually increased, while the inward currents elicited by glutamate were not significantly suppressed by na s. these observations suggest that h s may modulate glutamatergic synaptic transmission by suppressing the release of a transmitter. several studies show that activation of locus coeruleus (lc) play an important role in the symptoms of opiate withdrawal. in this study the effects of lc inactivation on self-administration of morphine and on morphine withdrawal syndrome in rats has been investigated. male rats were anaesthetized and implanted with silastic catheters inserted in to the right jugular vein. after days animals were fitted and the external end of the catheter was connected with a syringedriven pump, then were placed in the self-administration apparatus. lc was inactivated by ( l) lidocaein ( %) min before training. animals were allowed to self administer morphine ( mg/kg per inf.) ten consecutive daily -h session. during all morphine self administration session lever pressing was measured. our results show that: ( ) lc inactivation produced a significant decrease in the initiation of morphine self administration during all session. after the last test session morphine withdrawal symptom signs (mws) precipitated by naloxone were measured. ( ) most of mws were decreased by lc inactivation in comparison with morphine group. these results suggest that extracellular atp plays a dual role in astrocytic ca + wave propagation with activation of distinct purinergic receptors in the hippocampus of the rats. the electrophysiological analysis of the rescue effect of ␤ estradiol from glucocorticoid activity yuki oishi , suguru kawato department of physics, graduate school of science, university of tokyo, tokyo, japan; graduate school of arts and sciences, university of tokyo, tokyo, japan it is well known that stress reduces several activity of brain. especially, hippocampus is the largest target of stress. these phenomena are caused by glucocorticoids which are synthesized at adrenal when suffering stress. on the other hand, ␤ estradiol is one of the neuro protective factors and rescues neural death caused by several neurotoxins, such as ␤-amyloid, glutamate, glucocorticoids. in this study, we focused attention on the acute effects of steroid hormones and researched the effects of glucocorticoids and estradiol on rat hippocampal long term potentiation (ltp), which is the index of learning and memory. the results was that corticosterone (glucocorticoid of rat) acutely reduced ltp via glucocorticoid receptor. ␤ estradiol rescued this reduction via estrogen receptor ␣ and ␤. so we found that ␤ estradiol affected not only neuro protection but synaptic protection from stress-induced suppression of synaptic transmission acutely. ps a-d the hypothalamic neuropeptide y neuron system of rats after long-term, high-dose dexamethasone treatment jinko konno, ayuka ina, sachine yoshida, hideki ohmomo, fumihiro shutoh, setsuji hisano lab. neuroendocrinol., graduate sch. comprehensive human sci., univ. tsukuba, ibaraki, japan effects of dexamethasone (dex) on hypothalamic neuropeptide y (npy) expression were evaluated with semi-quantitative in situ hybridization and immunohistochemistry. adult male wistar rats received an injection of dex ( . mg/ g b.w., sc) or sesame oil (vehicle control) everyday for - days. the two and intact rats (intact control) were decapitated, and the hypothalamus was dissected out, fixed and cut into paraffin sections. npy-immunoreactive axonal varicosities in the external zone of the median eminence were apparently more frequent in the dex-treated rat than in controls. npy hybridization signals in the arcuate nucleus were significantly higher in the treated-rat than in controls. no difference was found between both control animals. these results indicate stimulatory effects of dex on hypothalamic npy production and suggest enhanced npy influences on pituitary function. akiko shingo, idumi yamashita, shozo kito lab. of neuroscience, hyogo university, hyogo, japan we examined estrogen-like actions of isoflavones in the cerebral cortex and hippocampus on the basis of our previous data that estradiol induces igf- mrna expression, upregulates estrogen receptors and facilitates ere binding in these brain areas. materials are ovxed and non-ovxed rats. each group of rats were divided into the following groups. a: rats fed with phytoestrogen-free control diet, b: rats fed with diet with soy bean-derived estrogen and c: rats fed with control diet combined with chronic intraperitoneal injections of minimum dose of ␤-estradiol. after feeding, rats were sacrificed to remove the cerebral cortex and hippocampus. expressions of mrnas of igf- , estrogen receptors ␣ and ␤, and ere binding were analysed. as the results, it was revealed that isoflavones induced increased expression of mrnas of igf- and estrogen receptors in both ovxed and non-ovxed rats. difference between estrogen receptor ␣ and ␤ in responses to isoflavones were analysed. isoflavones feeding increased ere binding as much as chronic injections of estrogen did in the ovxed rats. research funds: kampo science foundation, japan ps a-d mechanism of central metabolic control by tgf-beta in the rat brain: using the rat with depletion of hypothalamic noradrenaline teppei fujikawa, kazuo inoue, tohru fushiki division of food science and biotechnology, graduate school of agriculture, university of kyoto, kyoto, japan we have previously reported that activated transforming growth factor-beta (tgf-beta) increase in the rat brain during exercise. intracranial administration of tgf-beta induced an increase in fat oxidation, free fatty acid and keton body in the blood. these results suggest that activated tgf-beta in the rat brain participates in metabolic control of peripheral tissue by cns. it is, however, not known how tgf-beta increases in specifically fat oxidation. many investigations suggest that hypothalamus is essential for central metabolic control. in addition, some reports suggest that noradrenergic system in the hypothalamus may play important role for fat oxidation. in this study we measured concentration of extracellular noradrenaline (na) in the hypothalamus by using microdialysis after injection of tgf-beta. then, we measured respiratory exchange ratio and serum samples, after administration of tgf-beta in the rat with depletion of hypothalamic na by injection of -hydroxydopamine. ps a-d the effect of brain-derived neurotrophic factor (bdnf) on neuropeptide y (npy) neurons in the mouse corpus callosum: an examination using organotypic brain slice culture ryoichi yoshimura, kazuto ito, yasuhisa endo department of applied biology, faculty of textile science, kyoto institute of technology, japan the morphology of neuropeptide y (npy) neurons existing in the corpus callosum (cc) and the effects of brain-derived neurotrophic factor (bdnf) on the npy neurons were examined by using organotypic slice culture system. bdnf treatment significantly increased the number of the npy-immunopositive cell bodies and fibers in cc assessed with immunocytochemistry. electron microscopy demonstrated that the npy immunoreactivities were mainly localized in the regions associated with accumulating synaptic or cored vesicles in cc nerve fibers. the sectional area of npy-positive fibers was larger in the bdnf-treated culture than in the control culture. the number of nerve fibers adjacent to the npy-positive fibers was also larger in the bdnf-treated culture than the control. these results suggest that npy may play a key role in the neuronal regeneration, and bdnf takes part in the development of npy neuron fibers as well as the increase of the number of npy neurons in cc. reiji semba , kimi watanabe , munekazu komada institute for developmental research, aichi human service center, aichi, japan; graduate school of medicine, kyoto university, kyoto, japan d-serine is hypothesized to be a glia-derived neurotransmitter activating the nmda receptor because d-serine was reported to be formed and localized exclusively in astrocytes. however, we reported strong immunoreactivity of d-serine in some axons. to reveal which cells are producing d-serine in the brain, an in situ hybridization study of serine racemase, the enzyme producing d-serine from l-serine, was performed. using antibodies against neun, a neuronal marker, gfap, an astrocyte marker, and cnpase, an oligodendrocyte marker, type of the cells containing the mrna was examined. coincidentally with our immunohistochemical study of d-serine, strong signals for serine racemase mrna were found in some neurons while weak signals were found in astrocytes. present results suggest that d-serine will be a neurotransmitter activating the nmda receptors produced in a specific type of neurons. takatoshi hikida , , asif k mustafa , kenji hashimoto , kumiko fujii , , kazuhisa maeda , , hiroshi ujike , richard l. huganir , solomon h. snyder , akira sawa dept. of systems biology, obi, suita, japan; depts of neurosci. & psychiat, johns hopkins univ. med., baltimore, maryland, usa; chiba univ. forensic mental health, chiba, japan; dept. of psychiat, shiga univ. med. sci., shiga, japan; div. of neuropsychiat, tottori univ., yonago, japan; dept. of neuropsychiat, okayama univ., okayama, japan accumulating evidence from both genetic and clinical studies suggests a critical role of d-serine in schizophrenia (sz). we identified and characterized pick as a protein interactor of the d-serine synthesizing enzyme, serine racemase (sr). d-serine levels in the hippocampus and frontal cortex of pick knockout mice were significantly lower than those of their wildtype littermates at age of p , but not in adults, suggesting regulation of pick on sr at developing stage. in case-control association study, we observed an association of the pick gene with sz, which is more prominent in disorganized sz. our findings suggest that pick contributes to sr activity, d-serine production, and nmda neurotransmission in the pathophysiology of sz. ps a-d epileptiform activity is inhibited by taurine which can activate glycine and gaba a receptors in immature rat hippocampus akihito okabe , , werner kilb , ileana l. hanganu , taizhe qian , daiichiro nakahara , atsuo fukuda , heiko j. luhmann dept. of physiol., hamamatsu, japan; inst. of physiol., mainz, germany; dept. of psychol., hamamatsu, japan many studies indicate that the underlying mechanism of epileptic seizures differ between children and adults. the depolarizing gabaergic responses in immature neurons may contribute to higher epilepsy susceptibility. to investigate whether taurine, a neurotransmitter found in high concentrations in the immature cns, modulates epileptiform activity in immature hippocampus, we performed field-potential recordings in neonatal rat hippocampal ca region of an intact preparation. mm taurine blocked epileptiform activity induced by mg + free acsf and m -ap. this taurine effect was prevented by the glycinergic antagonist strychnine and the gaba a antagonist gabazine. inhibition of taurine uptake by ges also suppressed epileptiform activity in strychnine and gabazine sensitive manner. these results suggest that taurine mediates an inhibition in immature hippocampus via glycine and gaba a receptors that suppresses epileptiform activity. ps a-d responses of pge in undifferentiated and differentiated ng - cells kayoko matsushima , takashi imanishi , akinori kawaguchi , tetsuyuki wada , shigeru yoshida , seiji ichida school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan; school of sci. & eng., kinki univ., osaka, japan; school of pharm. sci., kinki univ., osaka, japan our previous findings showed that -ht-and bk-induced [ca + ] i increases were enlarged in differentiated ng - cells. for the next stage, we investigated the effect of pge , an inflammatory mediator for -ht and bk, on the cells. ng - cells were loaded with fura- /am, and the change in [ca + ] i was monitored by an image processor. the results showed: ( ) pge -induced response was decreased when ng - cells were differentiated by bt camp, ( ) − m ah and sc irreversibly inhibited pge -induced response by about % and %, respectively, while − m ah and sulprostone had no effect, and ( ) pge -induced response was abolished under ca +free conditions in about % of both ng - cells. these results indicate that the response to pge , via ep and ep receptors, significantly decreased during differentiation. mitsumasa murano, fumihito saitow, hidenori suzuki department of pharmacology, nippon medical school, tokyo, japan the most of cerebellar outputs are generated as a result of synaptic interaction in the deep cerebellar nuclei (dcn) and by the electrical membrane properties of dcn neurons themselves. this study aimed at examining mechanisms underlying the serotonergic modulations of both the gabaergic transmission at the purkinje-to-nuclear cell synapses and the membrane properties of dcn neurons using cerebellar slices prepared from -to -day-old rats. bath application of serotonin ( -ht) decreased the amplitude of stimulation-evoked ipscs in dcn neurons in a dose-dependent manner. furthermore, slow inward currents ware observed in dcn neurons during -ht application. under the current-clamp recording, -ht markedly depolarized and increased action potential discharges of dcn neurons. taken together, these results suggest that -ht facilitates the voluntary activity in dcn neurons by both pre-and post-synaptic mechanisms. ps a-d searching for endogenous ligands of trace amine receptors in mammals ( ) akira komatsu , airi yamaguchi , noriko makikusa , osamu koizumi dept. physiol., tokyo women's med. univ., sch. med., tokyo, japan; neurosci. lab., fukuoka women's univ. fukuoka, japan trace amine receptors were discovered in mammals, but their endogenous ligands have not yet been found. to search for them, we developed a new method to make antibodies against monoamines for immunohistochemistry (ihc). monoamines, phenylethylamine (pea), tyramine (ta) and histamine (ha), were conjugated to a hemocyanine, klh, using an imidoester cross-linker, dimethyl suberimidate (dms). rabbits were immunized by the conjugated macromolecule. the obtained antibodies were assayed by elisa and competitive elisa technique to check their antibody titer and specificity respectively. the antibodies recognized specifically the monoamine-dms part within the complex. for ihc, the rat brain was perfused by % dms, post-fixed by % formaldehyde and then frozen-sectioned. the antibody against ha revealed the immunoreactive neurons in the hypothalamus, showing that this method is effective to demonstrate the presence and localization of monoamines. the antibodies against pea and ta failed to reveal immunoreactive neurons in the rat brain. ps a-d effects of mg + on neural activity of cultured cortical neurons of the rat and mouse yuriko furukawa , , nahoko kasai , akiyoshi shimada , keiichi torimitsu , , kunihiko obata , yuchio yanagawa , , tadaharu tsumoto , ntt basic research laboratories, kanagawa, japan; sorst/jst, saitama, japan; neuronal circuit mechanisms research group, brain science institute, riken, saitama, japan; dept. of genetic and behavioral neurosci., grad. sch. of med., gunma university, gunma, japan it is well known that mg + plays an important role not only in energy metabolism, but also in neural information processing. however, the mechanism of such a role in cns is not well understood. previously we reported that neural activity and the intracellular ca + concentration are largely affected by mg + removal in cultured cortical neurons of the rat. transient glutamate release was also detected. in the present study, we investigated effects of the mg + removal on neural activity in cultured cortical and hippocampal neurons. in particular, we measured the intra-and extracellular mg + concentration and their actions on neural activity using a mg + indicator, kmg- -am together with fluo -am. we observed different effects of the mg + removal on gabaergic and non-gabaergic neurons by using gad -gfp knock-in mice. research funds: jst/sorst ps a-e transient zinc-positive terminations in the developing rat somatosensory cortical system noritaka ichinohe, daniel potapov, kathleen s. rockland lab. for cortical organization and systematics, bsi, riken, usa synaptic zinc (zn) is a neuromodulator used by a subset of nonthalamic glutamatergic connections, and associated with both experiencedependent and developmental plasticity. during development, transiently high levels of synaptic zn occur in both sensory and nonsensory cortical areas. by injecting the retrograde tracer sodium selenite into barrel cortex, we demonstrated a transient subset of zn + thalamocortical neurons from p -p . zn + cortical neurons were also labeled, intrinsic and extrinsic, from p . unlike in the adult, these were in layer , instead of layers , , and . at p , neurons occurred in layers , , and and, in some areas, layer . at p , zn + neurons first appeared in layer ; and at p , there is the adult lamination. as whisking and exploratory behavior commences in the second postnatal week, these transient zn + terminations may play a role in experience-dependent adjustments in cortical circuitry. research funds: bsi, riken and kakenhi no. ps a-e systematic comparison of the structure of the serotonin immunoreactive neurons between insect species masaaki iwano , , ryohei kanzaki , kei ito , center for bioinform., imcb, univ. of tokyo, tokyo, japan; dept. of mechano-inform., grad. sch. of inform, sci. and tech., univ. of tokyo, tokyo, japan; bird, jst, saitama, japan in the vertebrate central nervous system, the distribution of the serotonin immunoreactive neurons (sirns) is known to be preserved remarkably during evolution. systematic comparison of the invertebrate sirns has not been performed, on the other hand. in the current study we analyzed the morphology of the sirns in the brains of holoand hemi-metabolous insects including flies, bees, moths, beetles, crickets, dragonflies and cicadas. in spite of the large variation in the size and cell numbers of the brain, the number and distribution of the sirns were highly consistent between species. for example, we observed either one or two pairs of bilateral sirns with similar morphology that connect specific subregions of the lateral accessory lobe, a candidate pattern generator of the zigzag locomotion of the insect. variation was greater in the antennal lobe, the insect primary olfactory center, where sirns project either ipsil-or contra-laterally depending on the species. maki kagohashi , , taizo nakazato , shigeru kitazawa neurol, juntendo univ., tokyo, japan; physiol, juntendo univ., tokyo, japan in vivo voltammetry has been used for measuring neurotransmitter releases in the brain of behaving rats (e.g. nakazato, ) . however, task freedom was restricted by cables connecting the head and the measurement system. to overcome the difficulty we developed a wireless voltammetry system and examined its sensitivity in vitro (kagohashi et al., jns ). the system consisted of a wireless transmitter with a potentiostat and a signal receiver. in the present study, we reduced the size and weight and measured dopamine (da) currents in vivo with the wireless system mounted on the back of the rat. a single-step voltage pulse ( to mv for da; to mv for ht) was applied at hz through a carbon electrode that was chronically implanted in the striatum. after administration of l-dopa, da currents showed a gradual increase in good agreement with the data measured with conventional systems. the present wireless system would be applicable to measurement of neurotransmitters in various situations (e.g. social interaction). research funds: scientific research on priority areas (mobiligence) hiroyuki yamazaki, tomoaki shirao department of neurobiology and behavior, gunma university graduate school of medicine, maebashi, japan dendritic spines are multiple functional units that receive most of excitatory inputs in central nervous system. in the purpose of finding a novel molecule that is involved in regulation of dendritic spines, we have done a screening of a novel drebrin binding protein. yeast twohybrid system was conducted with drebrin as bait, and a novel drebrin binding protein was isolated. in neurons, this protein was localized primarily in nucleus and dendritic spines. hence, we named it spikar for its unique intracellular localization in spine and karyoplasm. we studied the role of spikar in spine formation. hippocampal neurons were transfected with shrna expression vector for spikar at several developmental stages. in early stage, spikar knock down (kd) did not affect the density of dendritic protrusions that were mostly filopodia. in contrast, spikar kd reduced spine density at the stage of synapse formation. these results suggest that spikar plays a role in the formation of dendritic spines, without affecting the filopodia formation. ps a-e time-lapse analysis of the translocation of drebrin-actin complex from dendritic spines to dendritic shafts by glutamate stimulation toshiyuki mizui , , yuko sekino , , tomoaki sirao dept. of neurobiol. & behav., gunma univ. grad. sch. of med., maebashi, japan; div. of neural network, inst. med. sci. univ. of tokyo, tokyo; crest, jst, kawaguchi, japan; jsps, japan we have shown that nmda receptor activation induced translocation of drebrin, with retaining its binding to f-actin, from dendritic spines to their parent dendrites. in the present study, we analyzed the time course of gfp-tagged drebrin a (gfp-da) dynamics after glutamate receptor activation. we prepared primary hippocampal cultured neurons, transfected them with gfp-drebrin a expression vector using microinjection methods at days in vitro (div), and analyzed the dynamic localization of gfp-da at div. glutamate stimulation started gfp-da translocating within s and completed in min. after washout of glutamate, gfp-da gradually re-accumulated in the spine, and the fluorescence intensity of gfp-da is fully recovered in min. these data suggest that translocation mechanism of drebrin from spines to shafts is different from that from shafts to spines. research funds: grant-in-aid for jsps fellows ps a-e distribution of the srf co-activator mal in developing mouse brain mitsuru ishikawa , jun shiota , hiroyuki tsutsumishita , hiroyuki sakagami , masaaki tsuda , akiko tabuchi dept. biol. chem., fac. pharm. sci., univ. toyama, toyama, japan; dept. cell biol., tohoku univ., grad. sch. medicine, sendai, japan the srf co-activator mal (megakaryocytic acute leukemia) plays an important role in controlling srf-dependent gene, whose expression is regulated by rearrangement of actin cytoskeleton. recent studies with conditional deletion of srf gene demonstrated that srf was required for inducing genes such as egr- , c-fos,␤-actin but also for neuronal migration and plasticity. in this study, we investigated the expression of mal in developing mouse brain and the role of mal for dendritic morphology. the in situ hybridization analysis revealed that mal mrna was highly and developmentally expressed in hippocampus and broadly expressed in cortex, olfactory bulb. staining of mal displayed cytoplasmic localization at cell bodies and apical dendrites. furthermore, dominant negative mal mutants and rnai led to a reduction of dendritic number, as well as a decrease of srf transcription. these findings indicate that mal is involved in the formation or the stability of dendrites. research funds: kakenhi ( ) to a.t. shoko shimizu , shinsuke matsuzaki , tsuyoshi hattori , ko miyoshi , masaya tohyama department of anatomy and neuroscience, graduate school of medicine, osaka university, japan; department of brain science, graduate school of medicine and dentistry, okayama university, japan disrupted-in-schizophrenia (disc ) was identified as a novel gene disrupted by a ( ; ) (q . ;q . ) translocation segregating with schizophrenia and affective disorders in a scottish family. kendrin was identified as a protein which interacts with disc at centrosome and residues - of disc (kendrin-binding region: kbr) were essential for the interaction with kendrin. in this study, we show that c-terminal of disc downstream of kbr is indispensable structure for kbr to interact with kendrin and also essential for disc to target to the centrosome. furthermore, we have shown that inhibition of the disc -kendrin interaction perturbs the tubulin network formation. these results suggest that the c-terminal region of the disc is important to the disc -kendrin interaction and that a truncated form of disc lacking the c-terminal downstream of the translocation breakpoint might affect the microtubule organization. tatsuro kumada, yasuhiko nakanishi, atsushi fukuda department of physiology, hamamatsu university school of medicine migratory cells exhibit dynamic morphological changes in the cell soma and process in both normal developmental program and tumor growth. the morphological changes in the cells are correlated with the rate of cell migration and ion transfer such as ca + or cl − . although the highly invasive migration of glioblastoma in the brain is known to be influenced by a variety of ion channels, there were a little evidence about the relationships among the morphological changes and ion homeostasis. to clarify it, we have developed a glioma cell culture system for the simultaneous observation of the cell movement and ca + and cl − imaging. we found that the relatively low density a glioma cells actively moved on the substrate. the movement has the correlation with intracellular ca + oscillation in the cells. the relationship between cell movement and intracellular ion levels is further studied. ps a-e involvement of ca + influx in the unpolarized non-vesicular release of fgf- hayato matsunaga, hiroshi ueda division of molecular pharmacology and neuroscience, nagasaki university graduate school of biomedical sciences, nagasaki, japan little is known of molecular basis mechanisms for the er-golgiindependent or non-vesicular release of fgf- lacking a conventional signal peptide sequence. we found that fgf- is co-released with s a , a ca + binding protein from cultured rat astrocytes upon the serum-deprivation stress. here, we report that fgf- is co-released with s a from the axon and dendrites in cultured rat hippocampal neurons upon depolarization stimulation, but serum-deprivation stress leads to release, which is seen in neurites as well as in soma. the interaction between fgf- and s a required ca + . the overexpression of s a - mutant lacking an ability of interaction with fgf- inhibited the their release, suggesting that s a is a cargo molecule. the release of fgf- upon either stimulation was abolished by voltage-dependent n-type ca + channel blocker. these findings suggest that ca + influx may be involved in the unpolarized non-vesicular release of fgf- . in neuron, intracellular calcium involves a large number of physiological phenomena, including cell migration, differentiation, and neurite outgrowth. pc cell is a useful model of neural differentiation and neurite outgrowth, and recently we demonstrated that -ht has an effect on neurite outgrowth via the increase in intracellular calcium concentration ([ca + ] i ) in pc cells. however, it is unclear how [ca + ] i regulates neurite outgrowth via actin cytoskeleton. in this study, we investigated effects of [ca + ] i on actin dynamics in pc cells transfected with yfp-actin. filopodial growth speed and actin retrograde flow were increased by treatment with calcium ionophore, a . treatment with calcineurin inhibitors decreased the filopodial growth speed, while treatment with camk inhibitor did not. these effects could contribute to -ht induced enhancement of neurite elongation. the actin cytoskeleton is a complex protein network that not only provides cellular structure but is fundamental for cellular dynamics. on stimulation of pc cells by ngf, proteins that directly interact with f-actin such as actinin rapidly translocate to the f-actin-rich cytoskeleton. clp is a pdz-lim protein which was originally identified as an actinin-interacting protein in skeletal muscles. here, we show that clp is endogenously expressed in pc cells and plays an important role in actin dynamics during ngf-induced neurite outgrowth. immunofluorescent studies showed that clp is accumulated in irregular cell surface and membrane extrusion soon after ngf-stimulation, where colocalized with actin filaments. we next performed rnai experiments to explore the role of clp in actin dynamics in growth cones and found that knockdown of clp expression lead to the suppression of ngf-mediated neurite outgrowth. in addition, we revealed using clp deletion mutants that both of pdz and lim domains are necessary for the proper function of clp . ps a-e screening of genes expressed preferentially in migrating gabaergic neurons of developing cerebral cortex toshiya kimura , tsuyoshi kobayashi , yuchio yanagawa , kunihiko obata , fujio murakami , grad. sch. of frontier biosci., osaka univ., osaka, japan; grad. sch. of medicine, gunma univ., maebashi, japan; bsi, riken, wako, japan; sorst, jst, japan neuronal migration plays a critical role in constructing brain architecture organization. however, molecular mechanisms underlying this process still remain elusive. in an attempt to identify molecules that regulate the motility of migrating neurons, we focused on migrating cortical interneurons, and performed subtractive hybridization, differential screening and in situ hybridization. subtraction was done between the embryonic and postnatal interneurons, because they robustly migrate prenatally but not postnatally. among the clones tested, two genes, neuronatin and seizure related gene (sez- ) attracted our attention. they were expressed in the subventricular zone of the embryonic cortex, implicating that these molecules are expressed in interneuron subpopulations. postnatally, mrna signals were hardly detectable. these results raise the possibility that they are expressed preferentially in subpopulations migrating cortical interneurons. yan zhu , , tomoko matsumoto , , sakae mikami , takashi nagasawa , fujio murakami , grad. sch. of frontier biosci., osaka univ., japan; sorst, jst, japan; inst for frontier med. sci., kyoto univ., japan long distance neuronal migration takes place typically along the tangential plane of the developing neural tube. the migratory behaviour and the underlying molecular mechanisms of tangential migration are poorly understood. we address these issues using the hindbrain precerebellar system as model system. precerebellar neurons, born dorsally in the lower rhombic lip, migrate in close association with the pial membrane (except inferior olive neurons) ventrally or rostroventrally. we therefore studied the role of pia-secreted chemokine sdf- and its receptor cxcr in the precerebellar migration. we show that cxcr is expressed in the migrating precerebellar neurons, and its expression is down-regulated towards the end of migration. in cxcr and sdf- knock out mice, migrating precerebellar neurons are less confined to the pial surface. more strikingly, the rostrally-directed migration of pontine precerebellar neurons is severely disrupted, leading to a caudalized ectopic pontine-like cluster. ps a-e involvement of an immunoglobulin superfamily molecule, neph /mkirre in the migration of precerebellar neurons kazuhiko nishida , , kazuhide nakayama , saori yoshimura , , fujio murakami , grad. sch. of frontier biosci., osaka univ., osaka, japan; sorst, jst, saitama, japan neural cell migration plays a crucial role in central nervous system development. in this study, we analyze the involvement of neph family transmembrane proteins of the immunoglobulin superfamily in the migration of precerebellar neurons (pcns). postmitotic pcns derived from the rhombic lip in the hindbrain first migrate tangentially along the pial surface, followed by radial migration to settle at their final positions (kawauchi, d., taniguchi, h., watanabe, h., saito, t., and murakami, f., development, in press ). in situ hybridization analysis showed that among neph family members including neph , neph /mkirre, and neph , only neph /mkirre was strongly expressed in pcns. expression of neph /mkirre was detected from e . when pcns migrate tangentially. the expression level became weaker at p , when pcns stop the radial migration, raising the possibility that neph /mkirre might be involved in the migration of pcns. we are currently analyzing the function of neph /mkirre in the migration of pcns. hiroki umeshima , , toshio ohshima , tomoo hirano , mineko kengaku lab. for neural cell polarity, riken bsi, wako, japan; department of biophysics, kyoto university, kyoto, japan; lab. for developmental neurobiology, riken, bsi, wako, japan during lamination of the cerebellar cortex, granule cells exit their final mitiosis at the external granular layer and migrate to the internal granular layer. we analyzed the molecular mechanisms regulating migration of granule cells. using an in vivo electroporation system followed by time-lapse confocal microscopy of a slice culture, we found a dominant negative form of cdk (cdk -dn) disrupted the morphology of granule cells during radial migration. recently, centrosome positioning is thought to be one of the important factors for neuronal migration. double-labeling of the centrosome and the whole-cell images by transfecting centrin -gfp and rfp enabled us to record dynamic movement of the centrosome during radial migration. we found that the motion kinetics of the centrosome was disrupted by cdk -dn. based on these results, we will discuss the role of centrosome during neuronal migration. keisuke ito , , takahiko kawasaki , , tatsumi hirata , division of brain function, national institute of genetics, mishima, shizuoka, japan; department of genetics, school of bioscience, sokendai newly generated neurons migrate through proper pathways toward their own targets, where they are integrated into specific neuronal circuits. we have analyzed a unique tangential migratory stream of early-generated cortical neurons designated as lot cells, and performed pharmacological perturbations to characterize the intracellular mechanism of the migration. among various drugs, we found that a protein kinase inhibitor, k a has the most interesting effect on the lot cell migration. during the normal migration, leading processes and cell bodies of lot cells move forward in a coordinated manner, but k a blocks the migratory movement of cell bodies without inhibiting the extension of leading processes. we also found that k a has a similar effect on cerebellar granule cells. these phenomena are quite intriguing because the drug seemed to switch the neurons from "whole cell migration" to "neurite extension" mode. we are now analyzing possible targets of k a, aiming for dissection of these phenomena. the conserved ser/thr kinase unc functions with unc- to regulate axonal transport in drosophila hiroaki mochizuki , hirofumi toda , , emiko suzuki , joseph gindhart , toshifumi tomoda , katsuo furukubo-tokunaga grad. school life and envir. sci., univ. tsukuba, tsukuba, japan; gene net. lab., natl. inst. genet. mishima; beckman res. inst., city of hope, ca, usa; dep. biol., univ. richmond, va, usa neural network develops through regulated guidance of axons and interconnection among them. despite intensive researches in the past years, genetic mechanisms of axonal development still remain unclear. we have identified the drosophila homolog of unc , which encodes a ser/thr kinase and is required for axonal formation in c. elegans and mouse. we found that unc is essential for neural development in drosophila. loss of function of drosophila unc results in reduced locomotion and axonal transport defects reminiscent of the phenotypes observed in kinesin mutants. we also found that unc genetically interacts with unc- , an evolutionarily conserved cytoplasmic protein that binds to kinesin heavy chain. in unc mutants, unc- was separated from synaptotagmin vesicles. these results suggest that unc coordinates kinesin-cargo interaction via unc- to regulate dynamic axonal transport. ps a-e change in microtubule polarity during the conversion of dendrites into axons kensuke hayashi , daisuke takahashi life science inst. sophia university, tokyo, japan; waseda university, tokyo, japan axons and dendrites of neurons differ in the polarity of their microtubules. the mechanism for the difference, however, is not well understood. we found previously that dendrites convert into axons in cultured neurons isolated from rat cerebral cortex. in this study, we examined whether microtubule polarity changes during the conversion. in dendrites of neurons before culture, microtubule polarity was nonuniform. after h of culture, we found that most of microtubules in the original dendrites had their plus ends oriented distal. this indicates that microtubules with their minus-ends distal disappeared during the culture. microtubule movement along actin filaments is a candidate for this mechanism among several types of microtubule movement reported in neuronal processes so far. however, the change of microtubule polarity within dendrites was observed even in the presence of actin polymerization inhibitors. our results suggest a rearrangement of microtubules by a yet-unreported movement in neuronal processes. research funds: kakenhi ( ) and kakenhi on priority areas ( ) ps a-e generation and analysis of region-specific rac -deficient mice hidetoshi kassai , masahiro fukaya , eriko miura , mizuho sakahara , masahiko watanabe , , atsu aiba div. cell biol., kobe univ. grad. sch. med., kobe, japan; div. physiol. sci., hokkaido univ. grad. sch. med., japan rac is a member of the rho family of small gtpases, and assumed to be involved in regulation of neuronal development through actin cytoskeletal reorganization. nevertheless, physiological role of rac in the cns is poorly understood because of the embryonic lethality of rac knockout mice. in this study, we generated and analyzed region-specific rac -deficient mice (emx -rac ko mice) by the cre-loxp system, in which a promoter for emx homeobox gene induces expression of cre recombinase exclusively in the dorsal telencephalon, including cerebral cortex, hippocampus and olfactory bulb. emx -rac ko mice showed partially abnormal layering of cerebral cortex, indicating impaired migration of neuronal cells during cortical development. furthermore, emx -rac ko mice lacked corpus callosum and anterior commissure, both of which connect the left and right cerebral hemispheres. these results suggest that rac regulates neuronal cell migration and axonal growth in cerebral cortex. previously we reported overexpression of map b containing nterminal amino acids promoted neuronal death. to reveal the mechanism of map b n-terminal induced neuronal death, we searched for the proteins that interact with n-terminal of map b by two-hybrid system. alpha-tubulin was found to interact with map b n-terminal and their in vitro interaction was proved with pull-down assay. the interaction of tubulin and map b n-terminal has not yet been reported. beta-tubulin was also found to interact with map b n-terminal. when ␤ tubulin was divided in fragment at between amino acid and , there was no interaction between ␤ tubulin fragments and map b n-terminal. interaction needs the continuous region over aa and . there were much proportion of round formed cos cells in n-terminal containing map b transfected cells than in n-terminal lacking map b transfected cells. there might be some interference in interaction between map b and tubulin in cells express map b containing n-terminal. otone endo , , masaaki mizuno , yasukazu kajita , jun yoshida department of neurosurgery, ja kainan hospital, aichi, japan; department of neurosurgery, nagoya university, nagoya, japan; department of molecular neurosurgery, nagoya university, nagoya, japan primate es cells have rather different character from rodent ones, but it is inevitable to elucidate mechanism for stable culture, purification and induction into object-oriented differentiation, because human es cells might show wide similarity to cynomolgus ones. we refined the way of large scale culture maintaining totipotency without contacting feeder cells indispensable for primate es cells. our super selective induction method for dopaminergic neurons is also refined, and induced neurons transplanted in vivo which survive without forming tumor such as teratoma for long period, are evaluated not only immunohistologically but eletrophysiologically and ethologically suggesting its enough stability, activity, ability to make neural network system and potentiality to improve clinical symptom of parkinsonism. differentiation of other types of neurons and development of fully functional neural network must be established. shigeki ohta , masae yaguchi , yumi matsuzaki , yoshiaki toyama , yutaka kawakami , hideyuki okano , masahiro toda , neuroimmunology research group, keio univ., tokyo, japan; physiology, keio univ., tokyo, japan; orthopaedic surgery, keio univ., tokyo, japan; institute for advanced medical research, keio univ., tokyo, japan; neurosurgery, keio univ., tokyo, japan we have shown that mouse dendritic cells (dcs) have the ability to induce the proliferation and survival of neural stem cells/progenitor cells (nspcs) in vitro. implantation of dcs into injured mouse spinal cord could improve the motor function through activation of endogenous nspcs in vivo. in this study, to identify an effective dc subtype for the treatment of spinal cord injury (sci), we analyzed the effects of different mouse dc subtypes on the proliferation of nspcs in vitro. among mouse splenic cd c + dcs, cd ␣ + dcs increased the number of neurospheres most effectively in vitro. furthermore, a significant functional recovery after mouse sci was induced by implantation of cd ␣ + dcs compared to cd c + dcs. these results suggest that cd ␣ + dcs can be an effective subtype of mouse dcs for the treatment of sci. ps a-e von hippel-lindau protein regulate the neurogenesis in skin-derived precursor cells atsuhiko kubo , hiroshi kanno , takaakira yokoyama , shuichi nakano , naoki sugimoto , nahoko kobayashi , tetsuhiko yoshida , isao yamamoto dept. of neurosurgery, yokohama city university graduate school of medicine, yokohama, japan; fiber and dept. of chemistry, konan university, kobe, japan; toagosei co., ltd. corporate research laboratory, nagoya, japan skin-derived precursors (skps), multipotent somatic stem cells, are preferred cell source for autologus cns cell replacement therapy. they are proliferated by the mitogens of egf and bfgf. to investigate the effects of von hippel-lidau (vhl) protein in the neural cell fate commitment, skps were inoculated with hsv vector expressing vhl protein. skps showed promotion of neurogenesis and inhibition of gliogenesis. to detect the intrinsic factors that control lineage commitment, vhl peptides fused with the protein transduction domain (ptd) were synthesized. the ptd-vhl peptides showed rapid cell internalization in nearly %, and peptide with the elongin c binding site (residues - ) showed a high ability of inducing neuronal differentiation by interacting with jak/stat pathway. these findings are important in its application to the cns cell grafting. ps a-e the effect of pueraria mirifica on erk / and s- following sciatic nerve injury in rats pornpen chaiworakul, supin chompoopong department of anatomy, mahidol university, bangkok, thailand to investigate the effects of pueraria mirifica (pm) compared with genistein (g) and estrogen (e ) on the expression of erk / and s- following sciatic nerve crush and transection in rats. protein levels of perk / and s- in distal segments of nerve at day were determined by western blot analysis. it was demonstrated that pm and g treatments, similar to e , caused a significant decrease in the expression of perk / levels in both nerve crush and transection injuries. however, transected nerves showed high and sustained levels of erk / phosphorylation. following treatments, levels of s- were significantly decreased both in crushed and transected nerves with respect to control group at p < . . this estrogenic effect was blocked by ici , . because of their structural similarity to e , pm may have therapeutic potential in nerve injuries which as previously reported to enhance sfi following sciatic nerve crush in rats after day . this study suggested that pm as well as g could enhance nerve regeneration like e by interfering with the injury-induced erk signaling pathway. yasuhiro kato , takafumi suzuki , kunihiko mabuchi department of advanced interdisciplinary studies, graduate school of engineering, the university of tokyo, japan; department of information physics and computing, graduate school of information science and technology, the university of tokyo, japan mems technologies have been established to fabricate a multichannel neural probe for interfacing with the nervous system. there is, however, no suitable probe for long-term neural recording and stimulation. one main reason is the death of brain tissues damaged by the probe insertion and implantation. thus, a new skeleton-like multichannel flexible neural probe coated with hybrid biodegradable polymer was fabricated. the skeleton-like probe was designed to minimize the volume of the flexible probe and buffer injurious micromotion between the probe and the tissues in a post-implantation. the probe was coated with mixed polyethylene glycol and microspheres with nerve growth factor (ngf) to improve the stiffness for the probe insertion, and deliver ngf for an optimal period to promote regrowth of damaged neural tissues around the probe. damage-induced neuronal endopeptidase (dine) is a newly identified nerve regeneration-associated molecule. it encodes neuronspecific membrane-spanning metalloprotease and belongs to nep/ece family which degrades/processes neuropeptides. although the precise mechanism of dine including substrate is still unclear, dine seems to play a protective role in damaged neurons. the most marked property of dine is a striking response to various kinds of nerve injury in both central nervous system and peripheral nervous system. to clarify the transcriptional regulation of dine after nerve injury, we analyzed untranslated region of dine gene. previously, we found that lif treatment and ngf deprivation additively increased dine mrna. in this study, promoter analysis showed that dine promoter activity was cooperatively up-regulated by atf- and stat , which were induced after nerve injury and activated at the downstream of lif treatment and ngf deprivation. this combination of transcription factors may be pivotal to promote gene expression, which is responsible for nerve regeneration. tomohiro miyashita, takekazu kubo, masashi fujitani, katsuhiko hata, toshihide yamashita department of neurobiology, graduate school of medicine, chiba university, chiba, japan wnt proteins are known as those concerning with formation of central nervous system. we tested whether they play a role in inhibition of axon regeneration after spinal cord injury. cerebral granule neurons from p - wistar rats were cultured. wnt proteins were added into the culture medium. twenty-four hours after culture, neurite length of each neuron was measured. immunohistochemistry was done employing anti-wnts antibody and anti-ryk (wnt receptor) antibody. anti-ryk antibody was injected continuously for two weeks into the subarachnoid space of contused rat spinal cord. locomotor behaviour was evaluated up to six weeks after injury. immunohistochemistry showed that several wnt proteins and ryk were upregulated after spinal cord injury. wnt proteins inhibited neurite outgrowth of cultured cerebral granule neurons. and this effect was abolished by y , a rho-kinase inhibitor, and anti-ryk antibody. suppression of wnt proteins may promote axon regeneration and improve locomotor behaviour after spinal cord injury. akihito takeda, richard goris, kengo funakoshi department of neuroanatomy, yokohama city university graduate school of medicine, yokohama, japan in contrast to mammals, spontaneous nerve regeneration after lesion of the spinal cord occurs in fishes. we examined tissue remodeling and axon regeneration after spinal hemisection in the goldfish. in the lesioned spinal cord, neurogenesis reached the maximum level days after the hemisection. glial cells positive for glial fibrillary acid protein (gfap) temporarily increased at the lesion site one day after. many gfap positive cells expressed somatostatin. serotonin ( ht) positive cells increased in number progressively from day to weeks after. six weeks after, the regenerated axons with glial fibers invaded fibrotic scar centered about the lesion site, and ht cells surrounded the axons and glia. thus, ht may promote these neural elements to invade the fibrotic scar. six weeks after the hemisection, projections from locomotion center in midbrain to spinal motoneurons were restored, and swimming ability was also recovered. these results suggest that the goldfish have ability to reestablish correct projections after the spinal injury. masao koda , yukio someya , ryo kadota , chikato mannoji , tomohiro miyashita , atsushi murata , masashi yamazaki department of orthopaedic surgery, togane hospital, department of ortopaedic surgery, graduate school of medicine, chiba university, japan; division of rehabilitation medicine, chiba university hospital, japan objective: anoikis is a type of apoptosis due to the detatchment from the extracellular matrix. preparation of graft cells for cell therapy includes dissociation of cultured cells, which may cause anoikis. here we tested the effect of bdnf for anoikis of schwann cell. methods: (in vitro) schwann cells were cultured from sciatic nerves of neonatal rats. schwann cells were transferred to suspension culture. bdnf was added into the culture medium. cell death was detected h after suspension culture. (in vivo) schwann cells were transplanted with or without bdnf treatment into contused rat spinal cord. immunohistochemistry was performed to detect survival of grafted cells. the olfactory bulb and its caudal extension are unique forebrain regions with the residence of neural stem cells and the ability of persistent neurogenesis. however, evidence for active functional involvement of neural stem cells is still very limited. this study was undertaken to know whether or not newly generated neurons are integrated in olfactory neuronal circuits in the neonatally bulbectomized rats that had been proved to show olfactory discriminative abilities. for this purpose, retroviral vector, a very useful tool to trace neural stem cells, was applied to the anterior part of the subventricular zone of the rats of which olfactory bulbs had been unilaterally ablated at the neonatal stage. we will show cell dynamics of newly generated neurons in the neonatally bulbectomized olfactory nervous system, with special reference to their neuronal circuits. koichi kawada, masanori yonayama, kiyokazu ogita dept. pharmacol., setsunan univ., osaka the subventricular zone (svz) contains undifferentiated cells, which proliferate and generate the olfactory bulb (ob) interneurons. throughout life, these cells leave the svz and migrate to the ob via the rostal migratory stream, where they differentiate. we have shown that trimethyltin (tmt) causes neuronal damage in the hippocampal dentate gyrus. in this study, we examined neuronal degeneration and regeneration in the ob after tmt treatment in mice. ddy mice were given tmt ( . mg/kg) to prepare slices for an immunohistochemical analysis using antibodies against single-stranded dna (ssdna), -bromo- -deoxyuridine- -monophosphate (brdu), neuronal nuclei (neun) and nestin. positive cells immunoreactive to ssdna markedly increased in the ob on days after tmt treatment. positive cells immunoreactive to brdu markedly increased in the ob on days after tmt treatment. double staining of brdu and neun in the ob revealed that almost brdu was not incorporated into mature neurons on day after the treatment. these results suggest possible enhancement of neurogenesis in the ob following tmt treatment. ps a-f early migration of human umbilical cord blood neural stem cells transplanted into rat brain miroslaw janowski , hanna kozlowska , marcin jurga , aleksandra habich , elzbieta wanacka , barbara lukomska, krystyna domanska-janik department of neurorepair, medical research center, warsaw, poland many neurological disorders result from progressive cell loss or rapid cell damage. as stem cell technology appeared there is an arising hope for cell replacement therapy and definitive cure. recently, in our laboratory human umbilical cord blood neural stem cell line (hucb nsc) was established. the aim of the study was to analyze the migratory potential of hucb nsc transplanted into intact rat brain. hucb nsc transfected with gfp gene was stereotactically transplanted (tx) into intact brain of csa immunosuppressed adult wistar rats. cell detection was performed h, h, h and days after transplantation using abs anti gfp, hla class i and numa. analysis of rat brains revealed viable gfp positive hucb nsc cells migrating from tx site and dispersed through the host brain tissue , , and days after grafting. immunohistochemical studies confirmed that these cells were of human origin: hla class i or numa. in future we plan to study their lesion directed migratory potential. heparan sulfate proteoglycans (hspgs) are considered to play roles in cns development, such as axonal guidance. however, little is known about the function of hspgs during nerve regeneration. in this study, we examined the expression of ext , one of the enzymes for heparan sulfate biosynthesis, after hypoglossal nerve injury. the upregulation of ext mrna was detected using in situ hybridization in injured hypoglossal motoneurons, and heparan sulfate glycosaminoglycan was also upregulated in the injured hypoglossal nucleus. we also examined the expression of mrna for hspg core protein. the mrnas for glypican- and syndecan- were upregulated in injured motoneurons. these results indicate that the synthesis of hspg is upregulated in injured motoneuron and hspg might be involved in nerve regeneration. masami watanabe , hiroe sagawa , masahiro ichikawa , yoshihito tokita dept. perinatol., int. dev. res., kasugai, japan; dept. ophthalmol., nagoya univ. sch. med., nagoya, japan; dept. neurosurg., nagoya univ. sch. med., nagoya, japan we examined whether rho/rock inhibitor, y , can make injured rgc axons regenerate into the crushed optic nerve (opn) of cats. methods: culture; retinal pieces were cultured in dmem for d. after fixation, the neurites were stained with anti-tuj antibody to obtain number and length of tuj neurites. crush; after an intravitreal injection of drug, the left opn was crushed with thread. on day , wga-hrp was injected into the vitreous. sections of opn were reacted for hrp with tmb reaction. masanori yoneyama, kiyokazu ogita dept. pharmacol, setsunan univ., osaka, japan in this study, we evaluated the effects of glutathione depletion on proliferative activity in neural progenitor cells of -days-old embryonic mice. neural progenitor cells were prepared from the hippocampus of -days-old embryonic mice by culturing in dmem/f medium for days in vitro (div). marked round spheres were formed from cells adhered to each other under the culture conditions in the presence of bfgf and egf, and then subsequently proliferated to form large neurospheres in proportion to the duration of cultivation. to evaluate the effects of glutathione depletion on proliferation in the neural progenitor cells, buthionine sulfoximine (bso) were exposed into cultured neural progenitor cells for a period of - div. treatment with bso resulted in a marked reduction in endogenous glutathione in the cells. mtt assay revealed that the deletion of glutathione led to a marked decrease in surviving neurospheres cultured for - div. these results suggest that glutathione would positively regulate proliferative activity and/or survival in neural progenitor cells of murine hippocampus. michio hashimoto, eisuke kawakita, masanori katakura, osamu shido dept. of environ. physiol., sch. of med., shimane univ., japan docosahexaenoic acid (dha), one of the main lipids in brain, plays crucial roles in the development and function of brain neurons. we examined the effect of dha on neuronal differentiation of neural stem cells (nscs) in vitro and in vivo. nscs obtained from rat embryos were propagated as neurospheres and cultured with or without dha for days. dha increased the number of tuj (+) neurons compared with the control, and the newborn neurons in the dha group were morphologically more mature than in the control. dha decreased the incorporation ratio of brdu, the mitotic division marker, during the first h period. thus, dha promotes the differentiation of nscs into neurons by promoting cell cycle exit. furthermore, dietary administration of dha significantly increased the number of brdu(+)/neun(+) newborn neurons in the granule cell layer of the dentate gyrus in adult rats. these results demonstrate that dha effectively promotes neurogenesis both in vitro and in vivo, suggesting that it has the new property of modulating hippocampal function regulated by neurogenesis. research funds: kakenhi ( ) ps a-f interaction among cues for visual depth motion perception tomokazu shimizu, akitoshi hanazawa kyushu institute of technology, fukuoka, japan when an object surface approaches or leaves us, we perceive visual depth motion. cues for this motion are change in binocular disparity, change in spatial frequency and optical flow. we investigated interactions among these cues by using visual stimuli in which the cues provides opposite depth motion direction. for the stimulus without optical flow component, spatial frequency was changed continuously by presenting uncorrelated random dot patterns filtered by different band-pass filters. when binocular disparity and spatial frequency was oppositely changed, subjects perceived depth motion corresponding to the change in binocular disparity or special frequency. for the stimulus with optical flow component, a random dot pattern filtered by a band-pass filter was expanded or contracted. when binocular disparity and the other two cues were oppositely changed, subjects perceived depth motion corresponding to the change in the other two cues. when depth motion was perceived from binocular disparity, stimulus image was perceived as changing its size. when from the other cues, depth perception from binocular disparity was suppressed. research funds: coe-j kazuyuki takahashi, akitoshi hanazawa kyushu institute of technology, japan in phenomena such as biological motion and structure from motion, a global structure is perceived by an integration of local motion signals. to clarify the fundamental mechanism of this motion integration process, we psychophysically examined the influence of directional motion coherency on motion grouping. moving dots were presented in three apertures that were aligned horizontally. before presenting these stimuli, subjects were instructed to detect a dot moving in a direction among noise dots presented in the central aperture. the dots moving in the same as or different from the instructed direction were presented in the side apertures. the performance of the subjects was the best when the dots in the side apertures moved in the same direction as the instructed one. the performance kept high when the directional difference was up to ± • and declined as the difference increased. the high performance would be due to the grouping of the dots presented in the central and side apertures that have the same or similar motion direction. the underlying motion grouping mechanism was suggested to integrate motion signals that have a certain directional variation. ps a-f effect of spatial context on structure-frommotion perception koshi makino, akitoshi hanazawa kyushu institute of technology, japan when viewing an orthographic projection of dots on the surface of a rotating cylinder, one perceives a transparent rotating d cylinder. this phenomenon is called structure-from-motion (sfm). the direction of the rotation is ambiguous. we investigated the influence of spatial context on the perceived direction of the rotation. three spatially separated stimuli were horizontally aligned. subjects reported in which direction the central random-dot sfm cylinder rotated. they perceived the same direction of rotation as the side stimuli when the side stimuli were corotating cylinders whose direction of rotation was disambiguated by binocular disparity. this effect was strong when the stimuli consisted of a small number of dots, and was attenuated as the number of dots increased. the perception was also influenced by translational motion stimuli that had front and back planes comprising oppositely moving random-dots whose depth was specified by binocular disparity. these results suggest that the neural mechanism determining the rotation direction of bistable sfm is strongly influenced by the d structure of surrounding stimuli defined by binocular disparity. ps a-f rotational motion aftereffect in positive direction for -dimensional random-dot pattern masako ono, akitoshi hanazawa kyushu institute of technology, kitakyushu, japan when viewing a unidirectionally moving pattern followed by a stationary pattern, we will see the stationary pattern moving in the direction opposite to the preceding movement. this phenomenon is well known as motion aftereffect (mae). this mae can be perceived for -dimensional motion such as rotating cylinders. we found that adaptation to the rotation of a stereoscopic random-dot cylinder generate mae like phenomenon in the same positive direction as the rotation of the adaptation stimulus (positive mae). this positive mae was strong when cylindrical random-dot was used as a stationary test stimulus. this aftereffect could not be perceived for uniformly distributed non-cylindrical random-dot. although ordinary mae declined in a few seconds, this positive mae remained for a few minutes. this is a new phenomenon that is different from known dimensional mae. this finding suggests that the visual system has a mechanism that detect -dimensional rotation direction specifically, and this mechanism has a property that gives a bias to the perception of stereoscopic rotation direction in an adapted direction. research funds: coe-j takanori uka, ryo sasaki department of physiology , juntendo university school of medicine, tokyo, japan crowding refers to a subjectǐs difficulty in identifying a target in the presence of distracters. as a first attempt towards identifying the neural mechanism of crowding, we investigated perceptual crowding using a random-dot kinematogram. human subjects were required to report the direction of moving dots within a center patch ( deg) of a center/surround display presented degrees to the left of fixation, and to ignore the dots in the surround. motion coherence of the dots in the center patch, as well as surround size varied randomly across trials. motion coherence of the surround was always percent. for each of subjects, we calculated direction discrimination thresholds (at % correct) at each surround size. consistent with crowding, thresholds increased when surround size was . and degrees, compared to those with no surround. surprisingly, however, thresholds decreased when surround size was and degrees, relative to degrees. our results show that the spatial resolution of motion direction discrimination improves when the area we have to ignore exceeds a defined size. ps a-f generation of receptive fields in higher visual areas based on v columnar structure: a model study yoshitaka toyoda, yoshiyuki shimizu, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan neurons in higher cortical areas of the visual pathway, such as v and v , respond to stimuli with complex shapes. how do these neurons integrate signals from v ? in particular, does the well-known columnar organization of v play a role in determining the shape selectivity of higher-order neurons? to explore these questions, we devised a feed-forward hierarchical model. in our model, higherorder neurons sum the activities of v neurons linearly according to a neural receptive field (nrf), a weighting function defined over the cortical surface. the manner a nrf sums over multiple columns determines its shape selectivity. since there is no physiological data regarding possible forms for nrf, we have tested simple functional prototypes, gaussians and gabor functions. reponses of these model neurons are examined using non-cartesian gratings and other stimuli, and compared to published physiological data. about % of model neurons exhibit responses similar to those of v and v neurons. odd-symmetric gabor nrfs tend to generate more of these neurons. taihei ninomiya, takahisa m. sanada, izumi ohzawa graduate school of frontier biosciences, osaka university, osaka, japan when images with different spatial frequencies (sfs) are projected onto the two retinae, a -d surface slant is perceived (blakemore, ) . the relationship between the binocular receptive fields (brfs) and sf tuning properties indicate that the early cortical neurons can signal slant-in-depth (sanada and ohzawa ) . however, their measurements of brf were conducted in the spatial domain, and the sf tunings were tested monocularly. in this study, interactions are examined directly in the sf domain between grating stimuli presented to the left and right eyes. frequency-domain brfs were measured by a reverse correlation technique. both binocular and monocular sf profiles were obtained by this method. we predicted binocular sf (bsf) maps from monocular sf profiles, and compared the prediction and the actual bsf maps to assess the binocular interactions. with this method, neural response properties which previous studies couldn't access were revealed. research funds: mext( ), jsps( ), coe ps a-f consistency of simple cell receptive fields: space and spatial frequency domain measurements yuka tabuchi , kota sasaki , izumi ohzawa , grad. school of frontier biosci., osaka univ., japan; grad. school of eng. sci., osaka univ., japan frequency-domain subspace reverse correlation and -d spacedomain dynamic dense noise have become increasingly popular for mapping receptive fields (rf) of early visual cortical neurons. however, it is not known whether results from these methods are mutually consistent. to examine this issue, we compared an rf in the space domain measured by -d noise stimuli and an rf reconstructed from the response in the spatial frequency (sf) domain measured by flash grating stimuli of various orientation (or), sf and spatial phase presented in rapid succession. we fitted these two rfs by gabor functions, and examined the consistency of their parameters. all parameters including sf, or, spatial phase, and size of the rf agreed well when an expansive nonlinearity is considered for each cell. the optimal sf obtained in the space domain increased over time to the same extent as that obtained in the sf domain. therefore, responses of a simple cell can be encapsulated in a concise framework of a linear filter followed by expansive nonlinearity. research funds: mext( ), jsps( ), coe ps a-f firing statistics and stimulus selectivity of inferior temporal cortical neurons in the monkey shunta tate , , hiroshi tamura , , ichiro fujita , graduate school of frontier biosciences, osaka university, osaka, japan; jsps, japan; crest, jst, japan inferior temporal (it) cortical cells are selective for visual shape, and vary in their spontaneous firing pattern among them. cluster analysis indicated that it cells were classified into five groups based on inter-spike interval (isi) histograms of their spontaneous firing. the first two groups showed a single peak at a long or a short isi in isi histograms. the other three had multiple peaks, whose positions and relative heights varied among the groups. principal component analysis and other analyses of visual responses showed that the five groups differed in their stimulus selectivity for a predetermined set of visual stimuli. stimulus selectivity was sharper in the single-peak groups than in the multiple-peak groups. one of the single-peak groups was modulated by natural images more strongly than the other groups. the results suggest that cells with different firing patterns carry different aspects of visual information, and may perform different functions in the coding of visual object images. supported by jsps and crest. research funds: kakenhi - ps a-f spatial-frequency dependency of receptive field size and surround suppression in lgn and v hironobu osaki , tomoyuki naito , osamu sadakane , masahiro okamoto , hiromichi sato , med. sch., osaka univ.; grad. sch. med., osaka univ.; grad. sch. front. biosci., osaka univ., osaka, japan in the primary visual cortex (v ), neurons change their responses depending on stimulus parameters such as orientation, size, spatial frequency (sf). we investigated how sf of stimulus affects on stimulus-size tuning property of responses of neurons in v (n = ) and lateral geniculate nucleus (lgn) (n = ) in anesthetized cats. first, we found that v neurons exhibited shifts of their sf tuning from high to low according to a change in stimulus size from small to large. second, we measured stimulus-area summation curve of responses and found that a higher sf stimulus caused a reduction of the receptive field (rf) size and an increase of the surround suppression. similar results were obtained for lgn neurons implying that the relationship between sf and area summation properties observed in v has its origin in lgn. these results suggest that the sf tuning of rf surround is broader than that of rf center and this center-surround mechanism reduces redundancy in visual information processing. hiroyuki nakamura , akichika mikami , kazuo itoh department of morphological neuroscience, gifu university graduate school of medicine, gifu, japan; department of behavioral and brain sciences, section of neurophysiology, primate research institute, kyoto university, inuyama, japan an extrastriate visual area v a is considered to be involved in the dorsal stream visual areas, however, its connections are not understood. to demonstrate the cortico-cortical connections of v a, we injected a bi-directional tracer biotinylated dextran amine into the v a. our results indicated that the v a has connections with the occipital, parietal and temporal cortices. the v a may thus be involved in the visual information processing of both the dorsal and the ventral stream visual areas. in addition to these connections, we found that v a has commissural connections with the v , the v a, the parieto-occipital area, the dorsal parietal area, and the ventral intraparietal area, and receives commissural projections from the dorsal and ventral aspect of secondary visual area v . these commissural connections may convey ipsilateral visual information near the vertical meridian representations. ps a-g activity of neurons in the isthmo-optic nucleus and its relationship with head movements hiroshi ohno, hiroyuki uchiyama department of information and computer science, faculty of engineering, kagoshima university, kagoshima, japan retinopetal neurons in the isthmo-optic nucleus (ion) send their axons to the contralateral retina in birds. the centrifugal visual projection is thought to be involved in attentional modulation of retinal output. we recorded activity of neurons in the ion in awake, headunrestrained japanese quails using an implanted electrode assembly. head movements were videotaped with a high-speed video camera ( fps), and were also monitored with a d or d accelerometer. we found two distinct types of activity pattern: phasic and tonic. the majority of neurons in the ion discharge in a phasic manner. phasic and tonic cells are also different one from another in relation to head movements. phasic cells show phasic elevation of activity - ms after end of head movements, while tonic cells show tonic suppression during head movements. we will discuss the activity profiles of neurons in the ion in terms of their possible role in visually guided behaviors. ps a-g timing of face specificity in fusiform gyrus responses to stimuli in different parts of the visual field yuka okazaki , , arman abrahamyan , catherine stevens , andreas a. ioannides , brain science institute, riken, saitama, japan; graduate school of life science and systems engineering, kyushu institute of technology, fukuoka, japan; school of psychology, university of western sydney, sydney, australia neuroimaging techniques have demonstrated the preferential responses to faces in the fusiform gyrus (fug). event related potential (erp) and magnetoencephalography (meg) studies have shown that such the responses specificity to faces occurs approximately ms (n ) after stimulus onset by comparing with the other objects. in the present study, we examined whether these and earlier fug activities, which have been already identified by our team (within ms), were selective for face. we achieved this by analyzing meg data elicited by static human faces, hands and shoes stimuli placed in fovea and four quadrants. we found robust statistically significant activities for faces in fug about ms after stimulus onset which depended on the stimulus location in the visual field. narihisa matsumoto , shoutaro akaho , kenji fujikumi , yasuko sugase-miyamoto , masato okada aist, ibaraki, japan; ism, tokyo, japan; university of tokyo, chiba, japan to understand the temporal aspects of information encoded at a population level in the inferior-temporal (it) cortex, we applied a cluster analysis method to the responses of neurons. each response was recorded while one of the visual stimuli that consisted of geometric shapes and faces of humans and monkeys was presented. population activity vectors of neurons for visual stimuli were clustered by a mixture of gaussian model. we estimated the number of clusters by using variational bayes algorithm. we assumed that the probability of the number of clusters depended on the one at one time step before. in the early period, the population vectors formed three clusters corresponding to global categories (human versus monkey versus shape). in the subsequent period, each cluster expanded to form sub-clusters corresponding to detailed categories. moreover, the number of clusters changed smoothly over time. these results suggest that the responses of it neurons represent different levels of categorical signals separated along the time axis. ps a-g relationship between color and shape selectivity in area teo of the monkey masaharu yasuda , , hidehiko komatsu , national institute for physiological science, okazaki, japan; sokendai, okazkaki, japan visual objects typically consist of multiple features such as color, shape, texture etc. it is reported that neurons selective for these object features exist in the inferior temporal (it) cortex of the monkey and some of them are selective for more than one of these features. however, little is known about the relationship between the selectivity for different features. last year, we have reported that there exist many neurons in the posterior part of it cortex (area teo) that are selective for both color and shape. to study the relationship between the color selectivity and shape selectivity, we tested the responses of each neuron using all combinations of the sets of colors and shapes, and conducted svd (singular value decomposition) analysis. we found that some teo neurons exhibited selectivities for color and shape that were independent (separable) each other, whereas in some other neurons they were not independent (nonseparable). these results suggest a possibility that color and shape informations interact at cellular level in this area. ps a-g neural correlates of stimulus shape detection in monkey inferior temporal cortex taijiro doi lab. cogn., neurosci., osaka univ., japan we searched for a neural "correlate" of conscious perception of shape by recording neuronal activities from inferior temporal (it) cortex while a monkey performed a -choice shape detection task. the monkey was required to judge whether or not a sample stimulus was presented immediately after a forward masking stimulus. when there was, the monkey was required to select the stimulus identical to the sample from three targets, two shapes and one small dot. trial-totrial variation of firing rates of many it neurons correlated with the monkey's seen versus not-seen choices. the mean choice probability (cp) of it neurons was . , a value significantly larger than the chance level. neurons with stronger visual responses exhibited larger cps. we also searched for temporal firing patterns within the spike train from a single neuron or across - simultaneously recorded neurons, but failed to find any temporal structure related to the monkey's behavioral choice. the results indicate a link between the firing rates of it neurons with conscious perception of stimulus shape. research funds: mext grant ( ) ps a-g behavioral visual performance of the zebrafish mutant, eclipse yuko nishiwaki , atsuko komori , tomonori manabe , toshihiko hosoya , hiroshi sagara , emiko suzuki , hitoshi okamoto , ichiro masai masai initiative research unit, riken, wako, japan; riken bsi, wako, japan; ims, university of tokyo, minato-ku, japan eclipse was identified as a visual zebrafish mutant that does not show both electroretinogram and optokinetic response. in the last meeting, we reported that the els gene encodes the ␣ subunit of cgmp phosphodiesterase (pde c), which functions in phototransduction in cone photoreceptors. since genetic mutations of pde c have not been reported in human patients of hereditary eye diseases, the els mutant is a good model for studying physiological roles of pde c. here we investigated whether the structural integrity of photoreceptors and visual sensitivity are affected in the els mutants. our electron-microscopic analyses revealed that photoreceptors do not undergo degeneration and are maintained in the els mutant until day-post-fertilization. however, we found that visual response to the contrast is slightly affected in larvae heterozygous for the els mutation. these data suggest that the level of pde c activity is important for the sensitivity of vision. ps a-g localisation of two markers of oxidative phosphorylation in the ageing human retina: an immunohistochemical study tapas nag, shashi wadhwa aiims, india the enzymes of oxidative phosphorylation are known to be affected by reactive oxygen species, which cause mutations in them, leading to reduced energy production. we examined the distribution of two markers of oxidative phosphorylation (nadh-ubiquinol oxidoreductase and cytochrome c oxidase) in the human retina at different ages. eyeballs of donors (age: - years) were fixed in paraformaldehyde, frozen retinal sections from macular to midperipheral regions cut and immunolabelled for nadh-ubiquinol oxidoreductase (complex i) and cytochrome c oxidase (complex iv; molecular probe, usa). complex i-immunoreactivity (ir) was moderately present in photoreceptors, outer plexiform layer and few ganglion cells from to years of age, and showed a decline and lack of ir in older retinas ( - years). complex iv-ir was intensely present in most ganglion cells, outer plexiform layer and photoreceptors from to years of age, and absent at years of age. thus, complex i and iv-ir decline with age, with the former showing an earlier reduction in its ir. the data signify a reduced mitochondrial activity in the retina with ageing. research funds: aiims ps a-g temporal characteristics of neural activity related to target detection during visual search tomoe hayakawa , norio fujimaki , toshihide imaruoka nict, kobe, japan; kit, kanazawa, japan meg and fmri experiments were conducted during the orientation singleton search task, and moment magnitudes of dipoles were estimated with an fmri-constrained meg-multi-dipole method to obtain differences between target-present and -absent conditions in each brain region for the whole time course. activity around the cas consisted of a prominent and a subsequent smaller but still obvious peak ( , ms); the first peak showed no difference between conditions while the second peak was significantly larger in the target-present. activity around the pfug had a prominent peak and subsequent small activity ( , ms), whereas the target's presence or not had no influence on either activity. the activity of the right intraparietal sulcus (ips) was significantly larger than that for the left ips at latencies around ms irrespective of the target's presence or not. the results demonstrate that neural activities of multiple regions had different temporal characteristics and the later activity around the cas was related to the target segregation from its surroundings. kaoru amano , , derek arnold , alan johnston , tsunehiro takeda univ. tokyo, chiba, japan; ntt cs lab., kanagawa, japan; univ. sydney, sydney, australia; ucl, london, uk when a moving border defined by small luminance changes (or by color changes) is shown in close proximity to moving borders defined by large changes in luminance, the low contrast border can appear to jitter at a characteristic frequency -a phenomenon we refer to as misc (arnold & johnston, ) . in order to reveal the neurophysiological substrates of this illusion, brain activities measured using magnetoenceohalography (meg) were compared with the perceived rate of illusory jitter measured psychophysically. the result showed that the perceived rate was around hz and matched with the alpha frequency of meg. as hz meg responses were enhanced in the presence of illusory jitter relative to the presence of isoluminant motion and physical hz jitter, we believe that the activity is related to illusory jitter generation rather than to jitter perception or to isoluminant motion per se. these results support our hypothesis that misc is generated within cortex by the dynamic characteristics of a cortical feedback circuit rather than by any physical stimulus properties. ps a-g the internal structure and the visual neuron projection patterns of the ventrolateral protocerebrum (vlpr) in the drosophila central brain kazunori shinomiya , , kei ito , , center for bioinform., imcb, univ. of tokyo, tokyo, japan; dept. comput. biol., grad. sch. frontier sci., univ. of tokyo, kashiwa, japan; bird, jst visual information processing in the insect brain has so far been analyzed mainly within the optic lobe. many visual pathways are known to project from the optic lobe to a central brain area called the ventrolateral protocerebrum (vlpr). the vlpr is therefore expected to be one of the major higher-order visual centers. the neural circuits in this area, however, remain essentially unknown. our study is to reveal the detailed internal structure of the vlpr, for the first time, using the drosophila brain as a model system. we have identified discrete glomerulus-like structures (gls) in the vlpr, among which at least five are innervated by the visual projection neurons from the optic lobe. we analyzed the detailed internal structure of these gls by visualizing single cells in each visual pathway using the combination of the gal enhancer-trap and the flp-out systems, and revealed the directionality of each pathway by specifically labeling the pre-and post-synaptic terminals. ps a-g dynamic reorganization of orientation maps in a late phase of the sensitive period kazunori o'hashi , , toshiki tani , shigeru tanaka , graduate school of life science & systems engineering, kyushu institute of technology, japan; laboratory for visual neurocomputing, brain science institute, riken, japan we have found that there are two phases in the sensitive period of orientation plasticity: an early irreversible phase and a late reversible phase. in this study, we attempted to elucidate how orientation maps are reorganized in the late reversible phase, performing intrinsic signal optical imaging several times from the same kittens. we observed the over-representation of the exposed orientation even one day after the onset of goggle rearing around the age of weeks. we also found that when the goggles were removed after or weeks of goggle rearing, drastically reorganized orientation maps returned to regular orientation maps that had been established before goggle rearing. these results suggest that once established orientation maps in an early phase serve as template maps to which later rapidly reorganized orientation maps are restored by the release of single orientation exposure. manavu tohmi, seij komagata, yamato kubota, masaharu kudoh, katsuei shibuki department of neurophysiology, brain research institute, niigata university, niigata, japan fourier analysis of intrinsic signals produced by periodic visual stimuli has been applied for constructing retinotopic maps (kalatsky and stryker, ) . in the present study, we used fourier analysis of flavoprotein fluorescence signals for constructing retinotopic maps in the mouse visual cortex. periodic bar stimuli that moved across the visual fields produced periodic fluorescence signals in the visual cortex of anesthetized mice. the fourier components of the signals locked with the periodic stimuli were calculated in each pixel regarding the magnitude and phase. retinotopic maps were constructed based on these components. vascular artifacts could be removed when the stimulus frequency was higher than . hz, since fluorescence signals but not vascular responses could follow up to these frequencies. combination of flavoprotein fluorescence imaging and fourier analysis is a powerful tool for investigating high-resolution retinotopic maps with short acquisition time in the mouse visual cortex. yoshitake kohei, manavu tohmi, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst, niigata univ., nigata, japan we have reported that ocular dominance plasticity induced by monocular deprivation can be visualized in mice using transcranial flavoprotein fluorescence imaging. another condition for producing ocular dominance plasticity is strabismus, which causes an increase in the proportion of monocular cells in the visual cortex. however, this possibility has not been tested in mice, mainly because surgical operations for producing large and stable shifts in eye position are difficult in mice. in the present study, we designed a new prism goggle for mice. this goggle was attached on the skull of mice during the critical period. the neural responses in the visual cortex of these mice were investigated using transcranial flavoprotein fluorescence imaging. preliminary experiments suggested that the responses in the monocular zone of the visual cortex were not affected in the strabismic mice. however, binocular interaction, which was additive in the binocular zone of normal mice, turned to be more repulsive in the strabismic mice. ps a-g retinotopy-based morphing of brain activity hiroshi ban, hiroki yamamoto, jun saiki graduate school of human & environmental studies, kyoto university, kyoto, japan the topographic visual field map is a fundamental property of the primate early visual cortex. we propose a new method to represent and sample topographic activities in the space of visual field by extending our previous study (maeda et al., . neurosci. res.) . the procedure was as follows. first, eccentricity and visual angle representations were measured for each subject using standard phase-encoding stimuli. second, individual cortical surfaces were reconstructed. third, the transformation between the position in the visual field and that on the cortical surface was established. finally, by using this transformation, brain activities were sampled and then displayed as an image spanning visual field dimensions, each pixel of which represents the activity of neurons representing a given position in the visual field. this retinotopy-based morphing is useful to analyze brain activity related to spatial and form vision and is more reasonable to integrate individual data than normalizing methods based on stereotaxic coordinates and anatomical structures. masahiro yamada , yasuhiro enami , hiroshi jouhou , takehiko saito , kaj djupsund tokyo metropol. univ., hino, tokyo; astellas pharma. inc., osaka, japan; suny upstate med. univ., center for vision and ophthal., ny, usa; univ. kuopio, dept. neurobiol., kuopio, finland on-off type amacrine cells are intensely connected with each other by gap junctions (gjs), forming a syncytium with a wide receptive field. we studied effects of external ph (ph ) on the control of cell functions. photoresponses of the cells were recorded intracellularly. slits of light stimuli simplified the estimation of the current flow in the cellular network into a one-dimensional problem. by lowering ph only . units from the baseline of . , we found a remarkable reduction of the conduction velocity by - %, an increase of the length constant and a hyperpolarisation of the resting potential. based on our theoretical model, combined with measurements of conduction velocity and length constants of the receptive field, we could estimate both gj and plasmamembrane conductances of the cell. thus, we suggest that protons could contribute to the reduction of conductances, especially at the plasmamembrane but also at gjs. ps a-g analysis of the band-pass filtering of the retinal rod by the ionic current model it is known that the rod network behaves like a band-pass filter. it was found that the time to peak of the response was shorter in rods further away from a slit of light. the band-pass filtering behavior has been attributed to an inductance element, i h , or i k(ca) . however, biophysical mechanism underlying the band-pass filter is not fully understood. to analyze the functional roles of ionic currents in the band-pass properties of rods, a model of the rod network was developed. the model incorporates much of the known parameters in rods, i.e., the phototransduction cascade, ionic currents (i ca , i kv , i k(ca) , i h , i cl(ca) ), calcium system and gap junctions between rods. in simulation, the band-pass properties of the rod was analyzed. it was found that single rod itself behaves as a band-pass filter. the mechanism underlying the band-pass filter was examined by changing model parameters. the result suggests that i k(ca) , i cl(ca) and i h are responsible for the bandpass filtering. research funds: kakenhi ( ) ps a-g stimulus selectivity and correlated spontaneous activity of distant neurons in monkey inferior temporal cortex go uchida, mitsuhiro fukuda, manabu tanifuji bsi, riken, wako, japan in inferior temporal (it) cortices of anesthetized macaque monkeys, we have previously shown that spontaneous spike activities (sas) of % ( of ) of neuron pairs (inter-neuronal distance > m) are significantly correlated. in the present study, to investigate how the correlated sas relate to functional structure in it cortex, we measured stimulus selectivity for each neuron of the pairs and explored similarity of stimulus selectivity by calculating correlation coefficients of responses to visual stimuli. this analysis revealed that the pairs with correlated sas tended to show more similar selectivity than the pairs lacking correlated sas. in addition, model analysis showed that in % ( / ) of the pairs the correlation of sas reflect synchronous transition between two activity states: periods with high and low mean firing rates. these results suggest that a network underlying the synchronous state transition provides circuitry that functionally connects distant it neurons showing similar stimulus selectivity. toshiyuki ishii , , toshihiko hosoya bsi, riken, japan; dept. biomolecular science, toho univ., japan understanding the significance of single spikes can be of critical importance in the analysis of neuronal information coding. it is often assumed that the firing rate is the sole carrier of information. however, if fine temporal patterns of spikes would carry information, the system could have large encoding efficiency. the vertebrate retinal ganglion cells fire burst spikes, separated by hundreds of milliseconds of silent periods. here we show that temporal patterns of spikes within these bursts carry visual information. when three or more spikes are fired, the multiple interspike intervals encode the input in a cooperative, non-redundant manner. this suggests that the spike patterns are not sorely determined by slowly modulating instantaneous firing rates. we also found that millisecond-scale structures in the spike patterns encode light intensity waveforms over ms. we propose that the retina compresses hundreds of milliseconds of light sequences into spike patterns at the scale of milliseconds. kazuhiro shimonomura, takayuki kushima, tetsuya yagi osaka university, osaka, japan purpose of this study is to design a neuromorphic hardware model that emulates fundamental architecture and function in the primary visual cortex (v ). we have constructed a binocular vision system consisting of two silicon retinas and simple cell chips and fpga circuits. the silicon retina has a concentric center-surround laplacian-gaussian-like receptive field. the output image of the silicon retina is transferred to the simple cell chips. the simple cell chip aggregates analog pixel outputs of the silicon retina to generate an orientationselective response similar to the simple cell response in v . this architecture mimics the feed-forward model proposed by hubel and wiesel, and computes physically a two-dimensional gabor-like receptive field. the fpga circuits compute complex cell responses based on the disparity energy model. the system can emulate the neural image of the binocular complex cells responding to natural scene in real-time and is useful to verify computational models of v neurons. masayoshi tsuruoka , masako maeda , bunsho hayashi , ikuko nagasawa , tomio inoue dept. physiol. showa univ. sch. dent. tokyo, japan; dept. anestesiol. showa, univ. sch. dent. tokyo, japan the present study investigated the involvement of ventral root looping afferent fibers in visceromotor function. under halothane anesthesia, the t -l dorsal roots were cut bilaterally to eliminate thoracolumbar influences. an electromyogram (emg) of the external abdominal oblique muscle evoked by colorectal distention was measured. colorectal distention ( mmhg) was produced by inflating a balloon inside the descending colon and rectum. emg activity evoked by colorectal distention significantly increased when the colon was inflamed with mustard oil ( %, ml). the increased emg activity significantly reduced following bilateral l -s ventral rhizotomies. a baseline emg did not significantly alter when the l -s ventral roots were cut bilaterally prior to inflammation. following the development of inflammation, there was less of an increase in emg activities. these results suggest that looping afferent fibers in the ventral root are involved in visceromotor function during colon inflammation. ps a-g hypnotic modulation of the cerebral processing of human visceral sensation using positron emission tomography using positron emission tomography (pet), we examined cerebral processing to visceral perception during neutral, hyperalgesic or analgesic suggestion with standard hypnosis. activation within right dorsolateral prefrontal cortex (dlpfc) and right inferior parietal cortex (ba ) was significantly greater (p < . , uncorrected) during rectal distention with analgesic suggestion than with neutral suggestion. on the other hand, activation within right medial frontal cortex (mpfc) was significantly greater (p < . , uncorrected) during rectal distention with hyperalgesic suggestion than with neutral suggestion. this is the first evidence with pet for a modulation of cerebral processing during visceral stimulation by hypnotic suggestion. these results suggest a role of dlpfc and mpfc in the cognitive control of the interoception. the participation of bladder receptors sensitive to cold temperature has been proposed in overactive bladder for decades. bladder cooling reflex (bcr) which consists of immediate sense of urgency and detrusor contraction in response to ice water infusion may be a neuropathic cause of detrusor overactivity (do). recently, urothelial cells display a number of properties similar to sensory neurons and have many sensors including gene for transient receptor potential (trp). we detected cold sensitive receptor trpm in the urothelial cell by immunofluorescence in an animal model for boo. intravesical administration of trpm agonist (l-menthol: . - mm) in freely moving rats, increased the micturition pressure (mp) in either normal (n = ) or boo rat (n = ). the micturition interval (mi) did not change in normal rat, but decreased in boo that have do. the results suggest that bcr is enhanced in boo by increasing trpm on the urothelium cell of the urinary bladder. ps a-g caudate projection from the vagal responsive site in the thalamic parafascicular nucleus in monkeys shin-ichi ito , a.d. craig dept. physiol, shimane univ. sch. med., izumo, japan; atkinson res. lab., barrow neurol inst, phoenix, usa we investigated efferent projections to the forebrain, from the vagal afferent activation focus in the thalamic lateral parafascicular nucleus (pf) (ito & craig, j neurophysiol ) . evoked potentials were mapped in the right thalamus from stimulation of the left cervical vagus nerve, and fluorescent dextrans were iontophoretically injected at the response focus. the injection sites were all located in the ventrolateral part of caudal pf, lateral to the habenulointerpeduncular tract, medial to the basal ventromedial nucleus, and ventromedial to the centre median. labeled terminals were found in the caudate nucleus (cd) in all cases. terminal patches extended longitudinally in the head of cd, concentrated in its ventral aspect. dense terminal patches also occurred throughout the tail of cd. these results suggest that visceral information modulates the portion of the striatum that has been implicated in cognitive function, and they implicate the caudate nucleus in the control of heart rate and respiration. research funds: nih grant ns ps a-g ascending general visceral sensory pathways to the telencephalon via the medial inferior lobe in a percomorph teleost, tilapia masami yoshimoto, naoyuki yamamoto, chun-ying yang, hironobu ito, hitoshi ozawa department of anatomy and neurobiology, nippon medical school, tokyo, japan general visceral sense is relayed to the telencephalon via thalamic and hypothalamic centers in mammals and birds. in teleosts, an ascending connection that corresponds to the thalamo-telencephalic pathway is present. however, it remained unclear whether or not a hypothalamo-telencephalic pathway exists in teleosts. the medial inferior lobe (mil), which corresponds to part of the hypothalamus of other vertebrates, is known to receive general visceral sensory inputs from the rhombencephalon in a percomorph teleost tilapia. hence, telencephalic connections of the mil were studied in this study. tracer injection experiments into the mil revealed that this hypothalamic zone projects to the preoptic area, the ventral telencephalon (i.e., vs, vd, and vv), and the dorsal telencephalon (i.e., dm, rdc, and dl). these findings suggest that the mil corresponds to hypothalamic relay zones in mammals (e.g. ventromedial hypothalamic nucleus). tatsushi onaka, yuki takayanagi department of physiology, jichi medical university, tochigi, japan administration of prolactin releasing peptide (prrp) decreases food intake. we have previously shown that an icv injection of anti-prrp antibodies increases food intake. neurones producing prrp are activated after peripheral administration of cholecystokinin octapeptide, a satiety factor. it is thus possible that prrp may mediate satiety signals in the brain. here we examined effects of anti-prrp antibodies upon total amounts of food intake and meal patterns. an icv injection of anti-prrp antibodies increased the total amounts of food intake and amounts of food intake during a meal but did not significantly change meal frequency. these data suggest that prrp may play an important role in the short-term control of food intake and are consistent with a hypothesis that prrp is a satiety signal within the brain. research funds: grant-in-aid for scientific research (c) ps a-h fasting induced long-chain fatty acid receptor gpr expression in the anterior pituitary of mouse ryutaro moriyama, shingo imoto, shinya shano, nobuyuki fukushima department of life science, kinki university, higashiosaka, japan g-protein-coupled receptor (gpr ) is known as a receptor for unsaturated long-chain fatty acids. the present study investigated the effect of h fasting on gpr expression in several regions of male mouse by real-time quantitative pcr, in situ hybridization and immunohistochemical method. gpr mrna expression was highly observed in the anterior pituitary, lung, colon, rectum, skeletal muscle, adipose tissue and testis in normal fed animals. h fasting induced gpr mrna expression increase in the anterior pituitary, lung and rectum. in the anterior pituitary, gpr -like immunoreactive cells were only observed in fasting animals. these results suggest that long-chin fatty acid regulates endocrine function in the anterior pituitary via gpr at least fasting period. ps a-h ketone body sensing cells in the lower brain stem to regulate food intake and reproductive functions kinuyo iwata, mika kinoshita, hiroaki sato, hiroko tsukamura, keiichiro maeda laboratory of reproductive science, graduate school of bioagricultural sciences, nagoya university, nagoya, japan ketone bodies are used for energy in the brain under malnutrition, such as prolonged fasting. we have previously revealed that hydroxybutylate ( hb), one of ketone bodies, sensed by the ependymocytes lining the fourth ventricular walls ( v) in the rat brain to regulate reproductive functions and feeding behavior. the present study was aims to determine if the ependymocytes located on the wall of v respond to the change in hb. change in the intracellular calcium concentration ([ca + ] i ) in vitro was measured in dispersed ependymocytes taken from the v in rats. the present results showed that the [ca + ] i increased in response to hb, but the increase was blocked by ␣-cyano- -hydroxycinnamic acid, which is a monocarboxylate transporter (mct ) inhibitor. immunohistochemistry showed that mct -immunoreactivities were located on the v ependymocytes. these results indicate that the ependymocytes may sense hb through a mct -dependent mechanism. research funds: kakenhi ps a-h comparison of hypothalamic histamine release by leptin in normal mice and high fat diet-induced obese mice tomoko ishizuka, kouta hatano, atsushi yamatodani dept. med. sci. and technol, grad. sch. allied hlth sci., fac med., osaka univ., osaka, japan leptin is a satiety factor which is produced by the white adipose tissue. peripheral administration of leptin decreases body weight and food intake acting on the hypothalamus. circulating concentration of leptin is in proportion to body fat mass, however, in obese humans, elevated concentrations of endogenous leptin cannot prevent the accumulation of the adipose tissue. we previously reported that leptin decreases food intake via the activation of the histaminergic system. in the present study, the effect of leptin on hypothalamic histamine release was compared in normal and high fat diet-induced obese (dio) mice. leptin ( . mg/kg, ip) reduced food intake in normal mice but not in dio mice, suggesting that dio mice have resistance for exogenous leptin like obese humans. the same dose of leptin increased hypothalamic histamine release in normal mice, while it had no effect in dio mice. these results suggest that the lack of the activation of the histaminergic system partly contributes to obesity in leptin-resistant dio mice. tomoya kitayama, yuri onitsuka, katsuya morita, toshihiro dohi department of dental pharmacology, hiroshima university, hiroshima, japan parkinson disease (pd) is neurodegenerative disorder of the substantia nigra accompanied by depletion of dopamine levels. symptoms of pd include disorder of aspiration and mastication, and dysphagia. in this study, rats injected with -hydroxydopamine ( -ohda) resulted in an extension of feeding time and a marked increase in the amount of feed powder on cage floor after clump feeding at weeks after -ohda without affect on number of neuron in solitary tract. these rats were transplanted with neural progenitor cells at mm; anteroposterior, + mm; lateral and − and − mm; dorsoventral from bregma at weeks after -ohda injection. the treatment shortened feeding time and decreased the leavings on the cage floor, as well as achieving decrease of neuronal death in substantia nigra. however, neural progenitor cells were not detected in substantia nigra. these results suggest that transplantation of neural progenitor cells may better -ohda-induced eating disorders via protection of neurons. research funds: grant-in-aid for young scientists b most tools used by nonhuman animals are extension of their effectors (motor-tools), while humans can use a kind of tools as substitute for their sensory organs (sensory-tools). to understand biological bases of using such tools, we trained japanese monkeys to use a tool as an extension of the eyes, and analyzed its learning processes to proceed as follows: ( ) retrieving the food with a rake (a motortool), ( ) retrieving the hidden food with a mirror-attached rake, ( ) using the reflected image of the food on a mirror separated from the rake, placed stationally beyond hidden food, ( ) moving a mirror hung along the rail by hand to find the food, ( ) using a rake with a small camera mounted inside, with which the monkeys searched for the food using the live video image captured by the camera on the monitor. finally, they could use a hand-held camera (a sensory-tool) as a manipulable extension of their eyes. thus, acquisition of using the externalized eyes can be achieved by gradual transfer of their own vision to the distant visual cues via motor-tools to extend their body image. kaori sawada , , shigehiro miyachi , michiko imanishi , masato taira , masahiko takada div. applied system neurosci., nihon univ. sch. med., tokyo, japan; dept. system neurosci., tokyo metropol. inst. neurosci., tokyo, japan to investigate the outflow of information from the temporal lobe to the prefrontal cortex, we injected rabies virus into three prefrontal regions: medial area ( m), dorsal area ( d), and ventral area ( v). the retrograde transsynaptic labeling was examined in the temporal lobe cortex days after prefrontal injections when the second-order neurons were labeled. the labeled neurons were observed in the lateral and medial aspects of the temporal lobe. in the lateral temporal lobe, neuronal labeling from m, d, and v was arranged topographically in and around the superior temporal sulcus. the labeing in the medial temporal cortex was also topographically arranged, such that m, v, and d receive multisynaptic projections from the entorhinal cortex, area , and both, respectively. these results suggest that there are parallel streams of information flow from the temporal lobe to the prefrontal cortex. research funds: crest, japan science and technology agency ps a-h new neural activities of reward anticipation and task errors h. ogawa , h. ifuku , t. nakamura , s. hirata kumamoto kinoh hosp, kumamoto, japan; fac educ, kumamoto univ., kumamoto, japan; nat kikuchi hosp, kumamoto, japan; dept. psych, kumamoto univ. hosp, kumamoto, japan neural activities at reward phase were recorded from the primary (pgc: areas g, & - ) and higher-order (hgc: prco & ofc) gustatory cortices of a monkey engaged in a taste discrimination go/nogo task. a lever had to be pressed after led onset when nacl was delivered, but not to water delivery. reward was given ca s after led offset at correct trials. relations between cues and responses were reversed. of reward-related neurons found, . % showed on type responses and the rest usual expectation responses. three types of on responses were noticed; c-type (n = ) only at correct trial, i-type (n = ) at around possible reward onset only at incorrect trials, and c-i type (n = ) at both. two classes of the c-i type were found; class i increased discharges at correct trials but decreased them at incorrect, but class ii increased them at both. all types were found in both cortices, but most class i were found in pgc and most class ii in hgc. i-type and class ii c-i type may represent error signals and reward anticipation. hiroaki ishida, masahiko inase, akira murata department of physiology, school of medicine, kinki university, japan in the macaque monkey, the ventral intraparietal area (area vip) integrated visual-tactile information in the body centered reference frame. the receptive fields of these neurons mapped on the same body parts in each sensory modality, so this area contributes to own body representation often referred to as body image. recent psychological studies implied that shared body representation of self and other might be required in the brain for social interaction. this means other¸s body image is mapped on own body image in the same neuron. in our experiments, we studied visual-tactile receptive field of the bimodal neuron in vip, then recorded activity during observing the experimenter being touched. some of neurons that had receptive fields anchored on the monkey¸s body showed visual response while the experimenter was being touched on corresponding body parts. the results suggested that bimodal neurons in vip may be related to matching mechanism between own body image and others, then we discussed that this area may contribute to the human social ability such as imitation. daichi hirai , takayuki hosokawa , masato inoue , akichika mikami section of brain sciences, primate research institute, kyoto university, inuyama, japan; department of psychology, tokyo metropolitan institute for neuroscience, tokyo, japan amygdala is involved in stimulus-reinforcement association learning, and have neural responses related to prediction of rewarding and aversive outcomes. however, it remains unclear whether representation of reinforcement value in the amygdala depends on other available outcomes in a given trial block. to elucidate how rewarding and aversive infomation are coded in the amygdala, we recorded single neuronal activity in monkey amygdala during delayed color matching task. we compared the neural responses to cue that rewarding outcome in two different stimulus-outcome conditions; one included electrical stimulus as aversive outcome, and the other included only rewarding outcomes. we found amygdala neurons to code the relative preference of available outcomes in a given trial block. ps a-h neuronal correlates of expectation-evaluation based on previous and ongoing contextual memories in the monkey prefrontal cortex kyoko matsuda, toshiyuki sawaguchi lab. cogn neurobiol, hokkaido univ. grad. sch. med., sapporo, japan to expect future events based on the ongoing context and to evaluate it are important for flexible control of goal-directed behavior. to examine a possible involvement of the lateral prefrontal cortex (lpfc) in such functions, we recorded neuronal activity from the lpfc of monkeys that performed an oculomotor task. in this task, the target of a saccade was indicated by combinations of successively presented two cues; symmetrically allocated two objects (cue ), and centrally allocated one of the objects presented in cue (cue ). the frequency of which object was presented as cue , i.e., task context, was manipulated across blocks. we focused on cue period and found that a subset of neurons showed object preference depending on current task context (cc type) or previous task context (pc type). cc type and pc type activities may be neuronal correlates of expectation-evaluation based on current and previous contexts, respectively. thus, neuronal processes for expectation-evaluation based on previous and ongoing "contextual memories" may progress in the lpfc. ps a-h anterior insular cortex neurons in monkey are activated when reward might be delivered, such as occurs in gambling takashi mizuhiki , barry j. richmond , munetaka shidara , grad. sch. of tsukuba univ., ibaraki, japan; neurosci. ri., aist, tsukuba, japan; lab. neuropsychol., nimh, bethesda, usa the human insular cortex has attracted interest because it is activated during risk-taking or decision-making tasks in fmri studies. to identify related neuronal signals, we recorded single insular neurons while two monkeys worked in a reward schedule task in conditions: ( ) a cue is picked at random so it is uncertain whether a correctly performed trial will be rewarded [uncertain condition], ( ) a cue indicates whether the current trial will be rewarded or not [certain condition]. in the uncertain condition / neurons responded in all trials. in the certain condition / neurons responded in the rewarded trials only. of these showed significant differences in firing rate between in the first trials after reward and other trials. these insular neuron responses seem related to reward expectancy and recent reward delivery. these neuronal responses might underlie the activation identified in imaging studies during gambling and decision-making tasks. research funds: kakenhi (priority areas ), aist masamichi sakagami , , kosuke sawa , xiaochuan pan , bsrc, tamagawa university, tokyo, japan; senshu university, kanagawa, japan; presto, jst, japan reward prediction behavior based on integration of associative information was investigated. monkeys were trained to perform a sequential association task with symmetric reward by symbolic delayed matching-to-sample procedure. at first, they learned two sequences of stimuli: a -b -c and a -b -c . after monkeys could acquire the sequences, new pairs of stimuli (i.e., d and d , e and e , etc) were introduced to associated with b or b (d -b , d -b , etc). the asymmetric reward rule was instructed by pairing c (c or c ) with the reward. after this instruction, reward predictive behavior was tested by using trained sequences and new stimuli. monkeys could show reward predictive behavior for not only a and a , which were associated with c and c in trained sequences, but also new pairs of stimuli, which were not directly associated either with c or reward. these results suggested that monkeys could use reward predicting information by integration of association among trained sequences, c-reward association, and new stimuli. research funds: kakenhi ( ), hsfp, presto, jst ps a-h reward predicting activity of prefrontal neuron based on group of stimuli xiaochuan pan , , kosuke sawa , , masamichi sakagami , bsrc, research institute, tamagawa university, japan; presto, jst, japan; department of psychology, senshu university, japan ability to anticipate a reward based on grouped events is important for guiding appropriate behavior. the main purpose of this study is to examine the pfc neuronal mechanism involved in predicting reward using learned associations among groups of stimuli. monkeys performed a sequential association task with symmetric reward. at first, they learned two sequences of stimuli: a -b -c and a -b -c . the asymmetric reward rule was instructed by pairing c (c or c ) with the reward block by block. monkeys were also trained with two different orders of stimuli (b-c-a and c-a-b). out of neurons from the lateral pfc, % showed reward-related activity in the first cue period. and one third of them (sr type) predicted reward only when a preferred stimulus was presented as a first cue. interestingly, the preference was not based on visual properties of stimulus, but on stimulus-group. the results suggest that about % of lateral prefrontal neurons predict reward based on stimulus-groups that were formed through the associative learning. attention evoked by novel stimuli is important for behavioral adaptation to new environment. however, it remains unknown whether the novelty is processed in a specific region of the prefrontal cortex. we trained two monkeys on a pavlovian conditioning task interleaved with an instrumental conditioning task and recorded cell activity from the lateral and medial prefrontal cortex (lpfc and mpfc). in a block of the pavlovian task (pv block), a visual stimulus (cs) was paired with a liquid reward and the trial repeated times. in a following block of the instrumental task, the monkey searched a correct action to obtain the cs as positive feedback. the cs was alternated every pv blocks. in many lpfc cells, responses to the cs were enhanced immediately after the change of cs, while such enhancement was less popular in mpfc. this result suggests that lpfc more contributes to coding of stimulus-novelty than does mpfc. when an outcome of action is uncertain, a top-down attention is directed to the coming outcome. to clarify the neural mechanisms, we trained two monkeys on a task with secondary reinforcers and recorded single cell activity of the medial and lateral prefrontal cortex (mpfc and lpfc). in a pavlovian block (pv block), a visual stimulus was paired with a liquid reward. in a following instrumental block (inst block), the monkey searched a correct action based on the visual feedback. the same visual stimulus as the one presented in the preceding pv block followed a correct action, whereas another visual stimulus followed a wrong action. when the monkey made more than consecutive correct trials, a new pv block started. both mpfc and lpfc cells gradually increased their firing toward the visual feedback when the outcome was uncertain, while the onset of the activity was significantly earlier in mpfc than in lpfc. these results suggest that the top-down attention first occurs in mpfc and propagates to lpfc in individual trials. ps a-h neuronal activity in the presupplementary motor area during a bimanual sequential motor task toshi nakajima , hajime mushiake , jun tanji department of physiology, tohoku university school of medicine, sendai, japan; brain science research center, tamagawa university, machida, japan to investigate the involvement of the pre-supplementary motor area (pre-sma) in organizing bimanual sequential movements, we recorded neuronal activity while a monkey was performing a motor task consisting of pronation or supination of either arm, with an intervening delay. in this report, we focus on neuronal activity during a period when the monkey was preparing to start the -sequence movements in a memorized order. we made regression analysis of neuronal activity in this period. we found that neuronal activity in the pre-sma rarely reflected muscle activity. instead, we found neuronal activity representing forthcoming actions such as supination, regardless of the arm to be used. we also found neuronal activity that reflected the second movement in a preparatory period before the execution of the first movement. we would demonstrate typical examples of pre-sma neurons and discuss their functional implications. ps a-h neuronal activity in the putamen and cm thalamus during response bias and its complementary process yukiko hori, takafumi minamimoto, minoru kimura dept. of physiol., kyoto prefect univ. med., japan we showed previously that cm thalamus participates specifically in complementary process to response bias (minamimoto et al. ) . to study the roles of the putamen and cm in response bias and it complementary processes, we recorded activity of cm and putamen projection neurons from two macaque monkeys performing asymmetrically rewarded go-nogo button press task. instruction of go or nogo activated cm neurons (n = ) preferentially when the instruction was associated with small reward. the instructions activated groups of putamen neurons preferring small reward-(n = ), large reward-action (n = ) and both types of action (n = ). onset latencies of these putamen neurons and rts in large-reward-go trials were shorter than those in small-reward-go trials by - and - ms, respectively. putamen neuron activation lead that of cm neurons by - ms. these results suggested that the putamen plays a major roles in both response bias and its complementary process while cm participates in the complementary process in concert with the putamen. research funds: kakenhi ( ) ps a-h encoding expected total rewards and their errors through a series of action choices by dopamine neurons naoyuki matsumoto, kazuki enomoto, minoru kimura dept. physiol. kyoto pref univ. med., japan to examine how dopamine (da) neurons represent reward expectation and its error through a series of action choices, we recorded activity of da neurons in two japanese monkeys making trial-anderror and repetition choices to find a correct, rewarding target among three alternatives. there are trials of first (t ), second (t ) and third (t ) choices with reward probabilities of about , and %, respectively. monkeys got reward after they hit a correct target, and got one more time by choosing the same target in the next trial (r , %). most da neurons ( / ) responded to the start cue of each trial and reinforcer beep after the choices. magnitude of the start cue responses progressively increased from t to t and to t trials, then decreased in r trial. in another task with two repetition trials (r and r , %), magnitude of start-cue responses decreased gradually from t to r and to r trials. thus, the start cue responses may reflect expected total rewards through a series of action choices for a goal, while reinforcer beep responses may reflect their errors. research funds: kakenhi ( ) ps a-h striatal neuron activity during decisions and action selections for probabilistic, scheduled rewards hiroshi yamada , , hitoshi inokawa , minoru kimura dept. of physiol. kyoto prefect univ. med., kyoto, japan; jsps, japan to study roles of the striatum in decision and selection of actions for probabilistic, multiple rewards, we recorded striatal projection neurons from a monkey. after depressing a start button, the monkey chose of target buttons with correct rates at st, nd, rd and repetition trials of , , and %, respectively. correct choices were followed by reward water. neuronal firing rates at starting each trial were related either to expected reward probability or to schedule states to obtain reward twice ( / ) rather than to upcoming choice of target ( / ). during the target choice, another subset of neurons showed firings selective to choosing particular target ( / ) rather than to expected reward probability ( / ). after the target choices, another group of neurons fired related to expected reward ( / ) rather than to chosen action ( / ). our results suggested that striatal neurons encode expected reward probability, schedule states to obtain multiple rewards and choice of actions during decision and action choices for a goal. research funds: kakenhi ( ), jsps fellows ps a-h encoding of reinforcement after rewardbased action selection by tonically active neurons in the striatum hitoshi inokawa, hiroshi yamada, minoru kimura department of physiology, kyoto pref. univ. of med., japan to study the signals encoded by tonically active neurons (tans) in the striatum, presumed cholinergic interneurons, reward-based decision and action selection, activity of tans was recorded from the putamen and caudate nucleus of a japanese monkey. after depressing a start button, the monkey chose of target buttons at average correct rates of (first), (second), (third) and % (repetition choices). correct and incorrect choices were followed by high-tone beep, reward water and low-tone beep, respectively. about a half of tans ( / ) responded differentially to the high and low tone beep respectively. number of responsive tans and magnitudes of the responses to high-tone beep was highest at the first choices, then, decreased gradually at second, third and repetition choices. these results suggested that the tans may encode reinforcement after reward-based action choices which is modified by reward expectation errors and motivation. research funds: kakenhi ( ) ps a-i representation of value of action, action and its outcome in sub-populations of striate neurons y. ueda , k. samejima , k. doya , m. kimura dept. physiol., kyoto pref. univ. med.; brain sci. res. center, tamagawa univ.; irp, oist to know the mechanisms of reward-based action selection in the basal ganglia, we recorded activity of striatal projection neurons of two macaque monkeys performing a free choice task with probabilistic reward. after a s delay, monkeys chose between left-and right-handle turn, followed by water reward at probability of , or %. a linear regression of neuronal discharge rates showed: neurons encoded reward values of either action during delay period before go signal, with most ( %) of them not having the action value signal in other task epochs. another subset of neurons encoded action signal selectively during action selection after go signal (n = ), while other neurons encoded presence or absence of reward at reinforcer epoch after the action selection. neurons encoding action values were in more anterior part of putamen than the neurons encoding actions. these findings suggested that sub-populations of striate neurons process action values and selection of actions during rewardbased decision and action selection. research funds: kakenhi ( ) ps a-i delay period activity of the monkey striatum in duration discrimination task atsushi chiba, ken-ichi oshio, masahiko inase dept. physiol., kinki univ. sch. med., osaka sayama, japan neuronal activity was recorded from the striatum of a monkey during a duration discrimination task. two visual cues (a blue or red square) were presented consecutively followed by delay periods, and the subject then chose the cue presented for the longer duration. durations of both cues, order of cue duration (long-short or short-long), and order of cue color (blue-red or red-blue) were randomized on a trial-by-trial basis. striatal neurons phasically responded during the first cue (c ), first delay (d ), second cue (c ), second delay (d ), and response periods. activity during the d and d periods was analyzed in this study. firing rates during the d period linearly depended on c durations. on the other hand, d period activity depended on trial types (ls and sl), but not on the variety of c durations in each trial type. our results suggest that striatal neurons encode, in the delay periods, not only temporal information with monotonic dependence on cue durations to prepare a comparison to a forthcoming cue duration, but also encode discrimination results between two cue durations. research funds: kakenhi ( ) ps a-i neuronal activities in the anterior inferior temporal cortex of monkeys during an asymmetrical pair association task based on facial identity satoshi eifuku , ryoi tamura , teruko uwano , taketoshi ono dept. integrative neurosci., univ. toyama, toyama, japan; dept. molecular integrative emotional neuroscience, univ. toyama, toyama, japan to elucidate neuronal basis of face memory, neuronal activities in the area teav of monkeys were recorded during a pair association paradigm that involves recognition of facial identity (i-apa task). in the i-apa task, monkeys were required to memorize paired associates of patterns and facial identity. each association has a particular direction, either the 'face to pattern' direction in which a cue stimulus which is a face is associated with a test stimulus which is a pattern, or the 'pattern to face' direction in which a cue stimulus which is a pattern is associated with a test stimulus which is a face. during the i-apa task, neuronal responses to a particular paired associate were identified. many of these neurons showed asymmetrical activities during the delay periods which were dominant in the 'face to pattern' trials. this asymmetrical delay activity are indicative of the crucial role of the teav area in face memory. research funds: kakenhi ( ) ps a-i reflexive social attention elicited by biological motion in monkeys and humans yoshiya mori , mikio inagaki , wu lisa , taijiro doi , eishi hirasaki , hiroo kumakura , ichiro fujita osaka univ., japan; massachusetts institute of technology, usa determining where another individual is attending and preparing for his/her upcoming action is crucial for members of a social group. here we report that the walking direction of another individual elicits a reflexive shift of visuospatial attention in monkeys and humans. we examined how the reaction time to peripheral visual targets was affected by a prior, brief presentation of a walking biological motion (bm) stimulus. during the task, subjects responded to a target point after the disappearance of the bm stimulus and fixation point. the walking direction of the bm stimulus was not predictive of the target direction, and was irrelevant for performing the task. we found that the reaction times in congruent trials, where the walking direction of the bm stimulus and the direction of the target appearance were the same, were significantly shorter than those of incongruent trials. we believe the attention mechanisms driven by bm may be part of the intentionality inference system. research funds: grants from and takeda science foundation ps a-i response properties of posterior parietal neurons during a multidimensional visual search task tadashi ogawa, hidehiko komatsu natl. inst. physiol. sci., aichi, japan the posterior parietal cortex (ppc) is thought to be one of crucial areas to direct spatial attention toward the target in visual search. visual sensory information (e.g. stimulus features) might be integrated in ppc to form a saliency map that controls spatial attention. to examine this hypothesis, we recorded the neural activity from the lateral intraparietal (lip) and a areas of monkeys performing a multidimensional visual search task. the monkeys had to make a saccade to either shape or color singletons in a stimulus array depending on the instructed search dimension. ppc neurons increased their activity when the receptive field stimulus became the target. some neurons showed target enhancement depending on the stimulus condition (singleton type and stimulus features), whereas others exhibited it irrespective of the stimulus condition. the mixed existence of these two distinct types of activities suggests that ppc is one of critical stages that integrate feature-dependent signals to produce featureindependent signals identifying the target location toward which spatial attention should be directed. monkeys utilize visual information in social communication. to elucidate visual function to categorize sexes, ( ) performance of visually guided sex discrimination task and ( ) neuronal activity during the task in orbitofrontal cortex (obf), the region could be related to sex recognition and vision processes, were investigated. monkeys were trained to discriminate the sex of a monkey shown in a picture that was presented on the display. the monkeys pressed the right bar for pictures of males and the left for females to get water reward. as a result, the monkeys were able to discriminate the sexes of monkeys shown in pictures. extracellular recordings of neurons in obf during the task showed that some cells responded to the pictures in a sexspecific manner. the present results suggest that visual information alone sufficiently contribute to discriminate sex in monkeys. obf could be involved in visual categorization of sex. research funds: kakenhi (a) ( ) (sa) and coe program in kit from the mext ps a-i activities of bursting neurons during color discrimination task in the monkey prefrontal cortex naoki ishikawa , satoshi katai , masanori saruwatari , masato inoue , akichika mikami section of brain sciences, primate research institute, kyoto university, inuyama, japan; third department of internal medicine, shinshu university, school of medicine, matsumoto, japan the neurons in the prefrontal cortex of monkeys are involved in the behavioral control of saccadic eye movements. on the other hand, cerebral cortex consists of different types of neurons. in this study, we trained macaque monkeys to perform a delayed matching to sample task with saccadic eye movement. and we classified neurons whether they had burst episode or not, and then classified bursting neurons into fast spiking (fs), fast rhythmic bursting (frb), and intrinsic bursting (ib) neurons (katai et al. neuro ) . most of bursting neurons activated during the target presentation or during the saccade period were selective to the target location or saccade direction. these results suggest that the bursting neurons have the significant role in the target selection and decision-making of the eye movement toward the specific direction. atsushi matsumoto , tetsuya iidaka department of psychology, nagoya university, nagoya, japan; department of psychiatry, nagoya university, nagoya, japan several studies indicated that gamma band activity (gba: - hz) reflects the process to form mental representation of objects or information. we investigated whether the gba is observed during subliminal visual word processing as well as supraliminal word processing. gba were observed both in masked and unmasked condition. at the - ms time window, gba was significantly higher in the word condition compared to the nonword condition in the unmasked condition. similarly, in the masked condition, gba of the word condition was significantly higher than that of the nonword condition at that time window. these results indicate that the unconscious lexical processing was reflected in the gba at that time window. furthermore, at the - ms time window, gba induced by word was significantly higher than that induced by nonword. this effect was not observed in the masked condition. in addition we found the significant semantic priming effect, indicating that the information of briefly presented words was processed unconsciously. wakayo yamashita, junichi hayashi, tomoki murakami, gang wang department of bioengineering, kagoshima university, kagoshima, japan the purpose of this study was to investigate the dependency of view association learning on the separation of the views. each stimulus set included images ( objects × views). novel objects were generated by deforming a prototype in four directions. for deg-interval object sets, views were obtained by rotating each object with the interval of deg, deg-interval set and deg-interval set were with deg and deg interval respectively. task performances were evaluated while the subjects performed an object matching task, in which the subjects had to recognize one object from others regardless of the viewpoint. the performance across deg separated views was significantly higher in the trials with deg-interval sets than those with deg-interval sets. similarly, the difference was also found in the performances across deg separated views between those with deg-interval sets and deg-interval sets. the results suggest that the exposure of interpolated views significantly improved the association learning of the views. ps a-i brain regional activity during attention task ( the kana pick-out test, treated as inspecting higher brain function, has been proposed to be suitable for screening dementia, which is widely used among public health nurses in japan. however, few fmri studies while demonstrating the test have reported. we therefore assessed the effect of brain regional activity with computerized kana pick-out test projected on the screen with clicking a mouse button to pick kana out under fmri running. executing the test resulted in significant increases in bold signals in right prefrontal area, bilateral hippocampus and broca's area. the results indicate the existence of the attention pathway from and/or to prefrontal area as association mechanisms for execution of kana pick-out test, suggesting that this test is useful in screening dementia. ps a-i obsessive compulsive symptoms in middle school students and its association with tic disorder, body dysmorphic disorder and trichotilomnia in shiraz, iran, ashkan mowla, arash mowla shiraz university of medical sciences, iran aim: the aim of this study is to evaluate ocd symptoms, tic disorder, body dismorphic disorder (bdd) and trichotilomnia (ttm) among middle school students of shiraz, iran. methods: middle school students were selected in a cluster random sampling from the four educational regions of shiraz, iran.persion standardized moci was used to assess obsessional symptoms. for evaluating bdd, tic disorder and ttm symptoms, a semi-structured interview was done according to dsm-iv-tr criteria. results: students with more obsessional symptoms were more girls and demonstrated more positive family history.they were more likely to be from lower socioeconomic class and with lower school average. they also showed more association with body dysmorphic disorder and tic disorder. conclusion: girls especially those from lower socioeconomic class demonstrated more obsessional symptoms. this study, like pervious ones, confirmed bdd symptoms and tics to be more in individuals with ocd symptoms. it was seen that ocd symptoms would affect school performance. ps a-i sirna-induced nr knockdown causes hypofunction of nmda-r and cognitive deficit m. saji , , t. utida , a. ohnishi , k. noda , m. ogata , h. akita , n. suzuki , physiol, health sci. sch. kitasato univ., sagamihara, japan; brain sci., graduate sch. kitasato univ., sagamihara, japan blockade of nmda-r by antagonists causes psychomimetic effects, suggesting involvement of nmda-r dysfunction in mental disorders like schizophrenia. however, the relationship between mental disorders and molecular abnormality has not been cleared. to identify the role of nmda-r in brain function, we performed sirnainduced knockdown of nmda-nr using hvj-envelope vectors. we confirmed that marked down-regulation ( %) of nr expression occurred only in the hippocampus among various brain regions - days after intra-ventricular injection of sirna-vector complex. in the hippocampal slice from rats with the nr knockdown, the nr down-regulation prevented depressive effects of nmda on fepsps, while the treatment did not affect ltp or ltd. in rats with the nr knockdown, the nr down-regulation caused disruption of prepulse inhibition, while the same treatment did not affect locomotor activity. these results suggest that hypofunction of hippocampal nmda-r by sirna-treatment causes a deficit of cognition. ken hatanaka , , hiroshi ageta , ikuko yao , kaoru inokuchi , yutaka kirino , mitsutoshi setou , graduate school of pharmaceutical sciences, the university of tokyo, tokyo, japan; mitsubishi kagaku institute for life sciences, tokyo, japan; okazaki institute for integrative bioscience, national institute for physiological sciences, okazaki, japan schizophrenia is a severe psychiatric disorder that characterized by psychotic symptoms in particular delusions and hallucinations, reduced interest and drive, altered emotional reactivity and disorganized behavior. to know the molecular mechanisms of the disease, we screened altered gene expression on the brain of schizophrenic patients by using microarray analysis, and found that the expression of ubl mrna was significantly decreased in the enthorinal cortex, whose size is known to be reduced in some schizophrenic patients. ubl is a highly conserved protein, which has a ubiquitin-like domain (ubl domain) and caax motif which is a membrane localization signal. we found that ubl mrna was expressed in the hippocampus, and purkingie cells of the cerebellum. the putative molecular function of ubl wil be discussed. research funds: grant-in-aid for young scientists (b), presto ps a-i decreased interneurons in the pax mutant mouse limbic system hasumi haba , tadashi nomura , yoshinobu hara , , noriko osumi , div. dev. neurosci., ctaar, tohoku univ. sch. med., sendai, japan; crest, jst, japan core features of schizophrenia are impairments in certain cognitive functions such as working memory, in which a number of brain regions in the corticolimbic system are involved. recent studies have revealed abnormality in distribution of interneurons in these regions. we have previously found that pax heterozygous mutant rats show behavioral abnormalities including impairment in fearconditioned memory and sensorimotor gating. in the present study, we thus analyzed distribution of interneurons in several regions of pax heterozygous mutant mouse (sey/+) brain. we focused on three subpopulations of interneurons: parvalbumin (pv)-, calretinin-, and somatostatin-posive interneurons. immunohistochemical studies indicated marked decrease in pv-positive interneurons in two brain regions of sey/+ mice, i.e., the olfactory bulb and the amygdala. reduced number of pv-positive interneurons was observed in the sey/+ amygdala at weeks, but not at weeks. our results suggest that age-dependent decrease of pv-positive interneurons might underlie behavioral abnormalities in sey/+ mice. schizophrenia is a complex genetic disorder, characterized by multiple susceptibility genes. dysbindin (dtnbp ) is a susceptibility gene for schizophrenia. genetic evidence for the association between the disorder and the dysbindin gene has repeatedly been reported in various populations world wide. recently, decreased expression levels of dysbindin mrna and protein have been reported in postmortem brain in patients with schizophrenia. thus, we performed behavioral analysis in sandy mouse, which has a deletion in dysbindin gene and expresses no protein. sandy mouse showed decreased locomotor activity and time in the center in the open field test. and an acute treatment of atypical antipsychotic, olanzapine ( . mg/kg, i.p.), improved the decrease in time in the center. moreover, subtle behavioral abnormality was observed in elevated plus maze test and social interaction test in sandy mouse. our results suggest that dysbindin might be involved in anxiety-related behavior in novel environment. research funds: , ps a-i gene expression analysis of dysbindin mrna in peripheral blood in schizophrenia sachie chiba , , satoko hattori , hiroaki hori , tetsuo nakabayashi , hiroshi kunugi , ryota hashimoto department of mental disorder research, national institute of neuroscience; tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan; musashi hospital, ncnp, kodaira, japan although many efforts have been spent to discover a biological marker of schizophrenia, no biological marker has been established. as genetic evidence suggested that dysbindin (dtnbp ) is a susceptibility gene for schizophrenia, we measured dysbindin mrna expression level in peripheral blood samples of patients with schizophrenia and age-sex matched healthy controls by a quantitative real time rt-pcr method. we quantified the expression levels of two major dysbindin transcripts among several known splicing variants. no significant difference in the expression levels of examined dysbindin transcripts was observed between control and schizophrenia. further examination measuring other dysbindin transcripts should be warranted to find a biological marker for schizophrenia. research funds: , ps a-i genetic variation in dysbindin influences memory and general cognitive ability ryota hashimoto , hiroko noguchi , hiroaki hori , tetsuo nakabayashi , satoko hattori , sachie chiba , seiichi harada , osamu saito , hiroshi kunugi department of mental disorder research, national institute of neuroscience, national center of neurology and psychiatry, kodaira, japan; musashi hospital, ncnp, kodaira, japan; tokyo university of agriculture and technology department of biotechnology and life science, koganei, japan dysbindin (dtnbp ) is a susceptibility gene for schizophrenia, a neuropsychiatric disorder characterized by cognitive dysfunction. we examined the possible association between genetic variants in the dysbindin gene and memory and iq in healthy volunteers and patients with schizophrenia. individuals who did not carry a protective haplotype had lower performance in several memory domains wms-r, although this haplotype did not affect iq measured by wais-r. a risk independent polymorphism for schizophrenia influences both memory and iq in the opposite direction. these data suggest that dysbindin gene may have impact on the cognitive function such as memory and iq and that memory might be an intermediate phenotype of dysbindin on risk for schizophrenia. research funds: , ps a-i detection of f-dopa signal in brainstem monoaminergic nuclei in schizophrenia yuri kitamura , nicola bright , toshio yanagida , masatoshi takeda , paul grasby department of physiology, osaka university, japan; department of psychiatry, osaka university, japan; cyclotron unit, imperial college, hammersmith hospital, uk we used f-dopa pet to investigate presynaptic dopamine dysfunction in schizophrenic patients. the object of this study was to test that a schizophrenic cohort would show elevated aadc activity in the substantia nigra, midbrain raphe and locus coeruleus compared to normal controls. all subjects and f-dopa scans were obtained from a database of scans published in mcgowan et al. , archives general psychiatry. the schizophrenic patients all met dsm-iv criteria on medication and healthy volunteers were compared. we attempted to improve the quality of the f-dopa signal by implementing a fbf-realignment movement correction method. significant increases in f-dopa uptake were found in the striatum, substantia nigra and raphe nuclei of schizophrenic patients (p > . ). our result suggests that an elevated presynaptic dopamine function is present in dopaminergic neurons that innervate striatal areas associated with enhanced dopamine activity in schizophrenia. in this study, we analyzed the p component of the visual eventrelated potential in patients with schizophrenia and healthy controls, and also performed loreta analysis. the ethics committee of kurume university approved this study. the p amplitude for the crying face was significantly smaller in patients than in controls. in controls, the p amplitude was significantly larger for the crying face than for the laughing face, while in patients, there was no significant difference in the p amplitude between the faces. loreta analysis demonstrated that there were significant differences in the activity in brodmann area between the faces in controls, while in patients, there was no significant activity difference between the faces. stimulation with crying face induced higher activities in the and right areas in controls than in the patients. these results indicated that the cognitive function was influenced by affective stimulus. ps a-j inappropriate input produces schizophrenialike working memory deficits in a simulated neural circuit kensuke nomura , shoji tanaka , koki yamashita , motoichiro kato , haruo kashima department of neuropsychiatry, school of medicine, keio university; department of electrical and electronics engineering, sophia university a number of studies indicate that the prefrontal cortex (pfc) is intrinsically linked to working memory (wm) and that dopamine critically modulates wm activity. according to the hypothesis proposed by goldman-rakic and her colleagues, we constructed an electrophysiological circuit model for wm which represents eight directions. the computer simulation with this model shows that the working memory activity is dampened by cue-irrelevant inputs and greater noise inputs lose the directional selectivity of the representation. a lot of studies suggested that increase of noise was related to schizophrenia, especially in wm disturbance. our study indicates that noise inputs cause wm impairment in patients with schizophrenia and that working memory performance is not always positively correlated with the neuronal activity of the pfc. ps a-j pericentrin is localized to the base of neuronal primary cilia in the developing cerebral cortex ko miyoshi, ikuko miyazaki, masato asanuma department of brain science, okayama university, okayama, japan we previously identified pericentrin, a mammalian centrosomal protein, as a binding partner of the product of disc , a candidate gene for schizophrenia. in this study, we analyzed in vivo expression of pericentrin in the mouse embryo. in the developing cerebral cortex, pericentrin mrna was highly expressed in migrating cells of the intermediate zone, though proliferating neuroepithelial cells and mature neurons revealed a low expression level of pericentrin. the pericentrin protein was shown to be localized to the base of primary cilia in the pre-plate of the developing cerebral cortex, in agreement with a recent study demonstrating the involvement of pericentrin in primary cilia formation. specific subtypes of receptors such as -ht are known to be localized to the plasma membrane of neuronal primary cilia in certain regions of the brain, and then our results raise the possibility that pericentrin dysfunction may result in perturbed chemosensory function of neuronal primary cilia and increased vulnerability to psychiatric disorders. dysregulation of gr has been thought to play an important role in the pathophysiology of mood disorders. two isoforms of human gr-alpha and -beta arise from alternative splicing of the pre-mrna primary transcripts. previously, we evaluated these two isoforms mrna level in the peripheral white blood cells of the patients with mood disorders. we found that the reduced gr-alpha mrna level in the patients with both bipolar and major depressive disorders, while gr-beta mrna level was not altered. these results suggest that dysregulation of alternative splicing play an important role in the pathophysiology of mood disorders. to test this, we evaluated mrna level of alternative splicing-related sr protein family, which regulate alternative splicing in several genes including gr, in the peripheral white blood cells of the patients with mood disorders. we did not find any differences in of the sr protein mrnas level in the patients compared to healthy controls and now, we are examining other sr family mrnas level. ps a-j alteration of neocortical long-term depression following electroconvulsive shock yoshifumi ueta , ryo yamamoto , shigeki sugiura , kaoru inokuchi , nobuo kato dept. integrat. brain sci., grad. sch. med., kyoto univ.; nara med. univ.; mitsubishi kagaku inst. life sci. electroconvulsive therapy is useful in treating drug-resistant depressive disorders, though its mechanism remains unclear. there have been a few reports that studied effects of electroconvulsive shock (ecs) on long-term potentiation. however, its effects on long-term depression (ltd) have not been investigated to date. the present experiments examined roles of ecs in inducing ltd at a variety of corticocortical synapses in rat cortex slices by using whole-cell patch clamp. following ecs, ltd magnitude at layer ii/iii-to-vi pyramidal cell synapses was significantly reduced in comparison to no-ecs subjects. as described in recent microarray studies, homer a/vesl- s was identified as one of the most up-regulated molecules after ecs. we therefore injected homer a protein by diffusion from patch pipettes. homer a injection, as well as with ecs treatments, reduced ltd magnitude only at layer ii/iii-to-vi pyramidal cell synapses, implicating that homer a may be a biological mediator of ecs effects. masanori kasai , nozomi miyagi , norio kawashiro , daisuke torizuka dept. of chem. & biosci., faculty of sci., kagoshima univ., kagoshima, japan; sanko shokuhin co., ltd., tokyo, japan it is well known that zinc is an essential mineral necessary for a multitude of body functions, including acuity of taste. to know a change of serum level in adjuvant-induced inflammation, we measured a zinc level in serum from male lewis rats received a suspension of complete freund's adjuvant ( . mg), injected intradermally into the tail. body weight, food intake and water intake were also measured. all rats showed signs of systemic inflammation (weight loss, hind paw swelling, nodules around eyes and penis) after the th day. the rats were sacrificed to measure the serum mineral contents (zn, na, cl, p, ca, k, mg) on the nd, th, th, st, th and th days. the serum zinc level was decreased on all of the measurement and the average of serum zinc ( . ± . g/dl, n = ) on the the day was significantly lower than that in intact rats ( . ± . g/dl, n = ). this decrease of zinc was correlated with weight loss but not hind paw swelling. other minerals did not show any significant changes throughout the measurement period. ps a-j molecular cloning of a novel candidate for ethanol-responsive genes, yy ap-related protein (yarp), in rat brain in order to elucidate the molecular mechanisms of etoh action on the cns, we investigated changes in gene expression in the adult rat brain after chronic etoh treatment. by means of cdna subtraction, we identified a candidate for etoh-responsive genes in the hippocampus. cdna cloning and sequence analysis revealed that this gene encodes a novel homolog of yy ap (yy -associated protein) and is well conserved in rats and humans. homology search for functional domains predicted that the yarp polypeptide contains nlss', a dnabinding motif, and a chromatin decondensation domain, as well as yy -binding and transactivation domains previously demonstrated in yy ap. in the brain, neurons such as hippocampal pyramidal cells were stained by in situ hybridization, and co-expression of yarp and yy genes was demonstrated in the same neurons. analogous to yy ap as a co-activator of transcription factor yy , it is postulated that yarp can regulate cerebral gene expression in response to etoh treatment. ps a-j excitotoxic degeneration of hypothalamic orexin neurons: involvement of nr b-containing nmda receptors and rescue by gaba a receptor stimulation hiroshi katsuki, shinsuke kurosu, toshiaki kume, akinori akaike department of pharmacology, graduate school of pharmaceutical sciences, kyoto university, kyoto, japan selective degeneration of orexin neurons, a pathological hallmark of narcolepy, is in part reproduced in hypothalamic slice cultures by application of quinolinic acid (qa), an endogenous nmda receptor agonist. we report here that nr b-selective nmda antagonists ifenprodil ( and m) and ro - ( . and m) markedly inhibited degeneration of orexin neurons induced by h application of nmda ( m) or qa ( . mm). we also show that stimulation of gaba a receptors by muscimol ( and m) or isoguvacine ( and m) potently inhibited qa cytotoxicity. in addition, the protective effect of gaba ( m) plus a gaba uptake blocker nipecotic acid ( mm) was abolished by a gaba a antagonist picrotoxin ( m). norepinephrine and serotonin did not provide a neuroprotective effect. thus, gabaergic inhibition may be decisive on survival of orexin neurons under excitotoxic stimuli mediated by nr b-containing nmda receptors. yoshika kurokawa, shinji tsukahara, hidekazu fujimaki national institute for environmental studies, tsukuba, japan to evaluate neurotoxicological influence of volatile organic chemicals (vocs), such as toluene, on hippocampal function, we attempted to develop an in vivo optical imaging technique for the hippocampus of mice with or without receiving voc inhalation. we dissected out the cerebral cortex in mice anesthetized with pentobarbital in order to prepare an optical window for monitoring the dorsal surface of the hippocampus, and stained the hippocampus with voltage-sensitive dye (rh ). we then monitored optical signals responding to electrical single-pulse stimulation to the parahippocampal region or hippocampal formation with a time resolution of ms. we also examined optical signals in the hippocampus during toluene inhalation. as a result, neural excitation of the superficial layer was observed in the hippocampal formation after electrical stimulation. on the other hand, acute perinasal exposure of toluene gas did not alter any signal pattern in the hippocampal formation. we will discuss the usefulness of this technique for examination of the neurotoxicological influence of vocs. ps a-j a simple method for fabricating electrodes array for multichannel neural recording -investigation of the alignment of the array and the measurement system-noriyuki taniguchi , osamu fukayama , takashi sato , takafumi suzuki , kunihiko mabuchi , dept. biomed. eng., univ. tokyo, tokyo, japan; dept. info. physi. comp., univ. tokyo, tokyo, japan various types of electrodes have been developed for use as brain-machine interface (bmi) to record signals from neurons. electrode arrays can be purchased from vendors. however, economic considerations and the adjustment of the array alignment for experimental design still make it worthwhile to develop fabrication methods inhouse. thus we developed a low-cost multichannel microwire array electrodes for recording from the cerebral cortex of conscious rats. the electrodes were able to align for the experimental paradigms. the effectiveness of the arrangement of the array as a bmi device was investigated. the electrodes were implanted in the primary motor cortex of wistar rats. we used a wheel-formed rat exercising kit to measure the walking speed of a rat. the neural signal of the rat and the rotating speed of the wheels were simultaneously recorded. and we evaluated the estimation of the walking speed by multiple electrodes with different alignments. ps a-j on-chip electrophysiological measurement of artificially constructed single-cell based neuronal networks ikurou suzuki , yasuhiko jimbo , kenji yasuda department of life sciences, graduate school of arts and sciences, university of tokyo, tokyo, japan; department of precision engineering, graduate school of engineering, university of tokyo, tokyo, japan we have developed a single-cell-based on-chip um-diameter multielectrode arrays with an agarose microchambers (amc) for topographical control of the network patterns of living neurons. this system enables flexible and precise control of the cell positions and the pattern of connections through photo-thermal etching. and sampling rates of measurement are khz in ch electrodes simultaneously. using this system, we formed a single-cell-based neural network pattern of rat hippocampal cells within the amc array and controlled the growth direction of axon/dendrite selectively using photo-thermal etching methods during cultivation, and recorded the spontaneous firings and evoked responses. moreover, we identified propagation along patterned neural network and found the effects of tetanic stimulation within this neural network. in the meeting we will present the results in detail and will discuss the potential of our method. yuichi yamashita , tetsu okumura , kazuo okanoya , jun tani lab. for behavior & dynamic cognition, riken-bsi, japan; lab. for biolinguistics, riken-bsi, japan how the brain generates and learns temporal sequences is a fundamental issue in neuroscience. the production of birdsongs, a process which involves complex learned sequences, provides researchers with a good biological model to study this phenomenon. bengalese finches (bf) learn highly complex songs that have grammatical structure. the underlying neural mechanisms that allow the birds to learn these songs are however not fully understood. to address this issue, we developed a neural network model of bf's songs that might explain how different regions of the brain work together. to test the model, we also conducted empirical experiments on the brains of bf. the model shows that complex grammatical songs can be replicated by simple interactions between deterministic dynamics of a recurrent neural network and random noise. moreover, comparison between the model and the empirical data on real birds shows similar trends. this work is a part of an integrated research project combining model simulations and empirical study. please see also the empirical component of this project as reported by okumura. ps a-k local administrations of muscimol into the nif alter song grammar of the bengalese finches (bf) tetsu okumura , yuichi yamashita , kazuo okanoya , jun tani behav & dynamic cognition, riken-bsi, saitama, japan; biolinguistics, riken-bsi, japan songs of passerines are learned behavior which used by males to attract females. their songs consist of several song notes, and these notes are produced in a fixed temporal order. among the passerines, however, bfs sing complex song which follows finite state syntax. the song control system of bf consists of a set of discrete nuclei including the hvc and nif. previous study showed that nif lesioned bfs sung simpler songs, with less phrases to phrases branching. therefore, nif-hvc connection may play important role in generating song grammar. in this study, we perfused nif with muscimol via microdialysis probes as a perturbation on nif-hvc system. following a local perfusion, song grammar was modified. some of chunks in their grammar were disappeared and introductorily notesǐ duration was elongated. nif is also known as one of auditory relay nucleus to hvc. part of the effects is possibly caused by disruption of auditory feedback. we also developed a neural network model of nif-hvc system. please refer yamashitaǐs poster for details of this model. the reason for the emergence of reward expectancy neurons suggested by a model using reinforcement learning and an artificial neural network katsunari shibata , shinya ishii , munetaka shidara dept. of e&e engineering, oita univ., oita, japan; grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan in the experiment of multi-trial schedule task to obtain a reward, reward expectancy neurons, which respond only in the non-reward trials prior to the reward trial, have been observed in the anterior cingulate cortex of monkeys. it is difficult to explain directly by reinforcement learning why they do not respond in the reward trial. here, we interprets that such neurons emerge as an intermediate representation to generate appropriate value and actions in reinforcement learning by simulation analysis using a model that consists of an artificial recurrent neural network trained by reinforcement learning. the simulation result suggests that the reward expectancy neurons emerge to realize smooth temporal increase of the state value by complementing the neurons that respond only in the reward trial. [ ] s. ishii, et al., "a model to explain the emergence of reward expectancy neurons using reinforcement learning and neural network", neurocomputing, behavior is adjusted by outcomes of actions. to examine the neural mechanisms of the behavioral adjustment, we recorded single cell activity of the medial prefrontal cortex (mpfc) of two monkeys performing a behavioral adjustment task. the monkey searched a correct action (left or right lever press) on the basis of the two kinds of visual feedback, one (cs+) paired with a liquid reward and the other (cs−) that did not appear in a preceding pavlovian conditioning. cs+ followed a correct action and cs− followed a wrong action. when the monkey made more than consecutive correct trials, a new block of pavlovian conditioning started. we calculated the prediction errors provided by cs+ and cs− on the basis of a reinforcement learning model of action selection. we found that the neuronal activity corresponds to the prediction error of value of the selected action. this result suggests that mpfc contributes to behavioral adjustment by providing prediction errors of action values. makoto miyazaki , shinya yamamoto , sunao uchida , shigeru kitazawa , faculty of hum sci., waseda univ., tokorozawa, japan; neurosci. res. inst, aist, tsukuba, japan; faculty of sport sci., waseda univ., tokorozawa, japan; dept. of neurophysiol, juntendo univ. grad. sch. med., tokyo, japan; crest, jst, saitama, japan our judgment of temporal order of two sensory signals is not always fixed but subject to changes due to prior experiences, such as repeated exposure to a constant stimulus sequence. to date, such perceptual changes occurred so that signals in the order of the most frequent sequence are judged as simultaneous. in this study, we examined temporal order judgment of two tactile stimuli, delivered one to each hand, using stimulation intervals sampled from biased gaussian distributions (mean = ± ms, s.d. = ms). previous studies predict that the point of simultaneity would be shifted toward the peak of the gaussian, i.e. toward the most frequent interval. however, the point of simultaneity was shifted away from the peak by about ms. our results disagree with the previous studies, but conforms to a contrasting prediction from a bayesian integration theory. research funds: kakenhi ( ) ps a-k single measurement of oxy-and deoxyhemoglobin for a functional near infra-red spectroscopy ichiro shimoyama , fumiko sato , ken nakazawa , kenichi ono chiba university, japan; field of home economics, faculty of education, chiba univ., japan; department of integrative neurophysiology, graduate school of med. chiba univ., japan to study single dynamics for oxy-and deoxy-hemoglobin to a single task, we measured near infra-red spectroscopy (omm- , shi-madzu) over the frontal area ( channels) for volunteers ( - y). thirty tasks were presented visually every s, the subjects were asked to think about the question immediately following the sentences and asked not to think moreover if the question was difficult (e.g., how to cook curried rice? or how to fold paper into a turtle? etc). a comprehension-test was done just after the record. easy/difficult serial tasks were selected, and the oxy-and deoxy-hemoglobin differences between tasks were calculated to obtain correlation coefficients between the oxy-and deoxy-hemoglobin. grand averaged correlation coefficient was − . +/− . between the dynamics of the oxy-and deoxy-hemoglobin. the correlation should be considered in discussing neural activation for nirs. we thank shimadzu corp. for providing the nir station. kazuya ishibashi , , kosuke hamaguchi , masato okada , , department of complexity science and engineering, graduate school of frontier sciences, university of tokyo, kashiwa, japan; jst, japan; riken bsi, wako, japan a synfire chain is one of the networks which generate stable synchronous pulse packets. although the networks with a single stable synfire state is intensively analyzed by using several neuron models, the networks with several stable synfire states have not yet been investigated so thoroughly. by using leaky integrate-and-fire neuron model we construct a layered associative feedforward network embedded with several memory patterns. we analyse the network dynamics with the fokker-planck equation. first, we analyze the activity of the network when we activated one memory pattern of the first layer. we show that the layered associative network has stable synfire state. second, we investigate the activity when we activated different memory patterns. then we observe several characteristic phenomena, which are not observed in the conventional homogenous synfire chain. we will report the details of those phenomena. research funds: kakenhi ( ) and ( ) ps a-k auditory erps can be identified as corresponding stimuli by classifier with naive bayes method akitoshi ogawa , , sachiko koyama , , takashi omori , takashi morotomi research institue for electronic science, hokkaido university, sapporo, japan; japan science and technology agency, saitama, japan; graduate school of information science and technology, hokkaido university, sapporo, japan; sakushin gakuin university, utsunomiya, japan in an attempt to reversely estimate the input stimulus from measured erps, we developed computational classifier using naive bayes method. correct classification rates could be index values of the erp characteristic. in this study, we applied the classifier to identify auditory erps (n = ). the erps were elicited by tones ( hz) with different durations ( , , , , , ms) and gaps ( , , , , , ms) embedded in a continuous pure tone ( hz). to confirm the generality of the method, we used leave-one-out cross validation. erps of each subject were identified by the classifier which was constructed from the others' erps. as a result, the correct rates for and ms were high both for the tones ( ms, %; ms, %) and the gaps ( ms, %; ms, %). ps a-k determination of channel parameters for construction of a neural model of caenorhabditis elegans kazumi sakaa, akane andoh, taro ogurusu laboratory of bioscience, faculty of engineering, iwate university, iwate, morioka, japan caenorhabditis elegans (c. elegans) is one of the most suitable model animal for investigation of the relationship between the connection and the function of the neural network because its connection was revealed with the electronmicroscopy. on the other hand, it has been difficult to build a precise model neuron because the neuronal electrophysiological data of c. elegans has not been sufficient. we have been developing a precise neural model by extracting parameters required for model of voltage dependent channels from the electrophysiological data by the genetic algorithm with a neural simulator genesis and parallel genesis. using these simulation softwares, not only the optimum parameter set was determined for each channel but also the ratio of the conductances of several channles were determined. we report validity of obtained parameters and the possibility of the existence of unknown channel. supported by grant from jsps. ps a-k theoretical consideration about nmda current change and its effect on synaptic plasticity shigeru kubota, tatsuo kitajima department of bio-system engineering, yamagata university, yonezawa, japan it is well known that nmdar plays an important role in learning and memory. several experiments have shown that the property of nmdar epsc can change within a few weeks after birth, leading to the shortening of its decay time course. since the calcium current through nmdar is involved in ltp and ltd induction, it is possible that such change can work as the modulation of the plasticity rule or higher-order plasticity. here we show by the biophysical compartmental model that the alteration of nmdar property can modulate the calcium influx into the spine, which finally switches plasticity rule. we also show that this type of plasticity switch can promote synaptic competition and separate postnatal synapses rapidly into two groups of either strong or week ones. our results suggest that changing nmdar time course is very useful for the developing animals in order to promote fast and stable formation of the polysynaptic circuit. manish kumar jain department of psychiatry, r.d. gardi medical college, india introduction: i want to inform you regarding the some of challenges coming across my practice with the person with the psychiatric disorder in social rehabilitation like education and training, work and employment, family, groups, social, sexual, environmental and regional, coordination with the other health group and care giver, insurance problems, medical, physical, occipital vocational, languages problems mostly how to give oppurtinies with in the society and many more to be come in future. method: i keep the records with me since i join the medical college and my during practice but this is really challenging to calm down for question with their relatives and care givers. results: it is always to see the experience of the other people including self help groups in this regards and most challenging with near by perfect action and required more interaction with the rehabilitation groups because some are social problems in psychiatric disorder. conclusions: there is big challenge in the for social rehabilitation for the persons with psychiatric disorder as multifactor involvement s are there in this groups with early intervention and long term rehabilitation so that we can produced many working induals with in the society among the person with psychiatric disorder the more interaction among the society and care giver working in this field as well as neuroseiencents working in this field so that we will able to achieve almost complete social rehabilitation as till today we are not able to achieve social rehabilitation up to % till now. hepatic encephalopathy (he) refers to acute neuropsychiatric changes accompanying fulminant hepatic failure (fhf). in the present study we investigated changes in lipid composition of membranes isolated from cerebral cortex of rats treated with thioacetamide (taa), a hepatotoxin which induces fhf and thereon he. estimation of phospholipid fatty acid content in cerebral cortex membranes from taa treated rats revealed a decrease in monounsaturated fatty acid namely oleic acid and the poly unsaturated fatty acids ␥-linolenic acid, decosa hexanoic acid and arachidonic acid compared to controls. assesment of membrane fluidity with pyrene, , -diphenyl- , , -hexatriene, and -[ (trimethylammonio)phenyl]- -phenyl- , , -hexatriene revealed a decrease in annular membrane fluidity while the global fluidity was unaffected. the level of thiobarbituric acid reactive species-marker for lipid peroxidation also increased in membranes from taa treated rats indicating prevalence of oxidative stress. results from the present study demonstrate gross alterations in cerebral cortical membrane fatty acid composition and fluidity during taa induced he and their possible implications in the pathogenesis of this condition are also discussed. nagatoki kinoshita, shigenobu yonemura cellular morphogenesis, cdb, riken, kobe, japan rho-gtpases are well known as regulators of cytoskeletal reorganization and many cellular morphogenetic movements. however, little is known about their distributions and their physiological functions in vertebrates. immunohistology of chick embryos revealed apical accumulation of rho, rac and cdc in neural plate cells, especially in bending hinge points. after neural tube closure, the apical accumulation decreased. coordinately, activities of rho-gtpases and myosin ii in neural plate cells were higher during neurulation than after neural tube closure. inhibitions of actin filament formation, myosin ii-mediated contraction or rho-associated kinase activity affected neural tube formation. inhibition of rho activity induced the disruption of its apical accumulation and the defects of neural tube formation. these results suggest that rho-gtpases in an active form accumulate in the apical surface of neural plate cells and play important roles in neurulation. furthermore, we are screening regulators and effecters of rho-gtpases transiently expressed in neural plate cells during neurulation. setsuko sahara, dennis dm o'leary mnl-o, the salk institute, usa gradients of morphogens are postulated to establish the initial patterning of the mammalian forebrain, but little is known about their downstream targets and the mechanisms of patterning. here we report mouse buttonhead homogoues, the sp gene family, as candidates of downstream of those morphogens: sp expression correlates with wnts/bmps in the cortical hem, sp with fgfs in the cop, and sp with shh in the ventral midline and mge. by using in utero electroporation, we show that sp regulates anterior-posterior patterning of the cortex into areas by controlling distinct fgfs that having opposing effects. sp and fgf exhibit reciprocal induction, indicating that sp is a positive feedback regulator of fgf . surprisingly, though, ectopic expression of both sp and its dominant active form shift cortical areas in the opposite manner to fgf , suggesting that sp activates additional targets that overcome fgf function. our results indicate that fgf is an additional target of sp , showing effect on patterning similar to sp . these findings indicate that sp balance the proper cortical arealization through fgf and fgf . research funds: nihr ns ps p-c fyn-fak signal transduction is involved in the radial migration of late-generated neocortical neurons eiko nakahira , kotaro hattori , takeshi yagi , shigeki yuasa dept. ultrastructural res., nat. inst. neurosci., ncnp, tokyo, japan; kokoro biology group, fbs, osaka univ., suita, japan fyn tyrosine kinase posphorylates focal adhesion kinase (fak) that is involved in cell migration. taking into account the defective formation of neocortical layers ii-iii in fyn-deficient mice, fyn-fak signal transduction might be involved in the control of the migration of neocortical neurons. accordingly, we analyzed the neuronal migration in the mutant neocortex and compared the phenotypes to the changes induced by fak gene-knock down by foreign gene transfer by means of in utero electroporation. late-generated neocortical neurons exhibited defective radial migration in the mutant and this defect was rescued by the transfer of fyn-expression vector to the neocortical primordium. fyn and fak were colocalized in the migratory neurons, and fak sirna transfer into neocortical primordium induced migration defect similar to that in fyn deficiency. these findings strongly suggest that the coordination of fyn and fak is essential for the radial migration of late-generated neocortical neurons. noriyo ishibashi , kazuko keino-masu , tatsuyuki ohto , satoshi kunita , satoru takahashi , masayuki masu dept. of mol. neurobiol., grad. sch. of comprehensive human sci., univ. of tsukuba, tsukuba, japan; laboratory animal resource center, univ. of tsukuba, tsukuba, japan heparan sulfate (hs) proteoglycans regulate developmental patterning through the interactions with cell surface proteins and extracellular matrix molecules. these interactions are mediated by the specific hs structures generated by sulfation and epimerization. a recently identified extracellular sulfatase, sulffp , has been implicated in the regulation of growth factor/morphogen signaling through hs remodeling in vitro, but its physiological roles remain unknown. here we generated knockout mice lacking the sulffp gene, and examined the brain development. a previous study showed that the brain-specific disruption of the ext gene, which encode a hs synthesizing enzyme, led to severe brain defects including hypoplasia of the cerebral cortex and cerebellum. in this study, we thus examined the morphological changes of the cerebellum in the neonatal and adult sulffp -deficient mice. heparan sulfate (hs) proteoglycans play a crucial role in mediating important signaling by wnt, hedgehog and fgf. recently, novel sulfatases, sulffp /sulfatase- and sulffp /sulfatase- , which have hs -o-endosulfatase activity have been isolated. since these sulffps are detected in the extracellular space, sulffps are thought to regulate cell surface signaling through hs remodeling. in order to examine the function of sulffp genes in zebrafish, we isolated zebrafish sulffp and sulffp . here we report the isolation and the characterization of the third homologue, sulffp . sulffp has about % and % overall amino acid homology with sulffp and sulffp , respectively. at h postfertilization, sulffp is expressed in the ventral region of spinal cord, whereas sulffp is expressed only in the floor plate and sulffp is expressed in the lateral floor plate and ventral regions of spinal cord. detailed expression patterns of sulffp will be presented. masahiko ajiro, kenichi arai, mika maeda-sato, masuo obinata, wataru shoji dept. of cell biology, idac tohoku univ., japan collapsin response mediator proteins (crmps) are cytosolic proteins involved in neuronal differentiation and axonal guidance. a member of this family, crmp was shown to mediate the repulsive effect of sema a on axons. crmps appear to play more complex roles in axonal differentiation, elongation and branching during development. since less is known about their in vivo function, we studied their roles during development using transparent zebrafish embryos. at early axogenesis stage, zebrafish crmps are expressed in specific patterns. in trigeminal sensory ganglia, crmp , , , and are highly expressed. knocking down of these gene results in disorganization of the ganglia, separating into several clusters. however, their axonal patterns including direction, extension, and branching appears normal. same defects were observed in the knockdown of neuropilins, receptor component for class semaphorins. these results suggest that crmps may functionin keeping trigeminal neurons as a ganglia by mediating semaphorin-neuropilin signals. ps p-c developmental origin of diencephalic sensory relay nucley in teleosts y. ishikawa , n. yamamoto , m. yoshimoto , t. yasuda , k. maruyama , t. kage , h. takeda , h. ito nat. inst. rad. sci., chiba, japan; nippon med. sch., japan; tokyo univ., japan we propose a novel interpretation of the embryonic origin of cells of diencephalic sensory relay nuclei in teleosts, based on our studies in the medaka embryonic brain. it has been proposed that the relay system in teleosts is unique among vertebrates. teleost relay nuclei, the preglomerular complex (pg), have been assumed to originate from the basal plate (posterior tuberculum, pt) of the diencephalon, whereas relay nuclei in mammals are derived from the alar plate. our results show, however, that many pax -or dlx -positive cells migrate laterally and ventrocaudally from the diencephalic alar plate to the basal plate during development. massive clusters of the migrated alar cells become localize in the mantle layer lateral to the pt neuroepithelium, from which the pg appear to differentiate. we therefore consider most neurons in the pg are be of alar, not basal origin. thus, the teleost pg can be regarded as migrated alar nuclei. the organization of the diencephalic sensory relay system may have been conserved across vertebrates. hideyuki dekimoto, yoshihiro oomiya, satoshi kikkawa, toshio terashima, yu katsuyama department anatomy and developental neurobiology, kobe university graduate school of medicine laminaiton is one of features unique to the brain of vertebrates. to understand the evolution of layer formation in the vertebrate brains, we are studying genes which exhibit layer-specific expression. since one of ets family transcription factors, er is expressed specifically in the layer v of the mouse neocortex, we selected this gene for the purpose of our study. here we cloned zebrafish er homologue (zfer ), and found that the amino acid seuqence of the putative protein is highly conserved throughout the entire length. expression of zfer was observed in multiple sites of developing brain. the expression disappears sequentially in some sites, whereas it persisted in other sites until adult stage. er expressing sites in the brain was basically conserved between mouse and zebrafish, whereas expression pattern in each site (i.e. telencephalon, tectum) was different. based on these observations, evolution of the gene expression in the brain lamination will be discussed. hiroyuki koizumi, teruyuki tanaka, joseph g. gleeson university of california, san diego, usa doublecortin (dcx), encoding a microtubule-associated protein, is critical for neuronal migration, as mutations result in x-linked lissencephaly in hemizygous males and subcortical band heterotopia in heterozygous females, whereas in mouse, rnai-mediated knockdown but not germline knockout shows abnormal positioning of cortical neurons. dclk (doublecortin-like kinase) is one of the homologous genes of dcx, encodes for protein with an n-terminus that is % identical to dcx, but also additional c-terminal protein kinase domain. here, we report that the dclk functions in a partially redundant pathway with dcx in the formation of axonal projections across the midline and migration of cortical neurons in mouse. dosagedependent genetic effects were observed in both interhemispheric connectivity and migration of cortically and subcortically derived neurons. rnai-mediated knockdown of either gene results in similar migration defects. these results indicate the dcx microtubuleassociated protein family is required for proper neuronal migration and axonal wiring. hiraki sakuta , , hiroo takahashi , , takafumi shintani , , kazuma etani , masaharu noda , div. of mol. neurobiol., nibb, okazaki, japan; crest, jst, japan in the developing chick retina, the expression of bmp is relieved by that of bmp at around e with a change from a dorsal high to dorsotemporal high pattern, complementary to that of ventroptin, a bmp antagonist. we previously demonstrated that misexpression of ventroptin altered the retinotectal projection along both the dv and ap axes. here, we show that topographic molecules along the dv axis, together with ephrina , are expressed in a double-gradient fashion from e on like ventroptin and bmp . when bmp expression is manipulated by using the gene-specific knockdown and the reagent-inducible gene expression techniques, the expression patterns of these double-gradient molecules are all changed. moreover, in the bmp knockdown and ephrina -misexpressing embryos, the retinotectal projection is altered along the two axes. the expressional switching from bmp to bmp thus appears to play a key role in retinal patterning and consequently in topographic retinotectal projection, by changing the direction of the dv axis toward the posterior side during retinal development. noriyuki morita, teiichi furuichi lab. for molecular neurogenesis, riken-bsi, wako, japan the mammalian cerebellum is anteroposteriorly and mediolaterally compartmentalized at the level of neuroanatomy and also at the level of gene expression. to elucidate the molecular mechanisms underlying the establishment and the maintenance of functional cerebellar compartment, genes responsible for mouse cereballar development transcriptome were examined for patterned expression in cerebellum by whole-mount in situ hybridization. not a few known and novel genes were found to be expressed in parasagittal band pattern in the embryonic mouse cerebellum, which could be categorized as "early-onset-genes". parasagittally expressed genes were classified in comparison with the band pattern of en , wnt b and pcp /l gene expression in declival vermal lobule, to investigate the correlation between spatial expression profiles and transcriptional regulatory elements. our accumulating data suggest that not only patterning genes like engrailed and wnts, also genes related in later events in neural development such as synaptogenesis are expressed as earlyonset-genes. yasufumi tanaka, tomiyoshi setsu, hideyuki dekimoto, yu katsuyama, toshio terashima kobe university graduate school of medicine, japan the nissl staining of the brains of the adult reeler and normal mice showed that the size of the pontine nuclei (pn) was reduced in the reeler compared with the normal counterpart. the injections of dii and di- asp into the left and right hemicerebellum, respectively, resulted in that only a few pn neurons were doubly labeled in the control, but in the reeler most of pn neurons were doubly labeled. the placements of solutions of dii and di- asp into the left and right cerebellar peduncles of paraformaldehyde-fixed brains resulted in that dii-labeled or di- asp-labeled pontocerebellar fibers made a fascicular formation in the cerebellum of the normal mouse, but such a fascicular formation was not recognized in the reeler and labeled terminals of mossy fibers were randomly arranged along the course of the pontocerebellar projection. reelin mrna and reelin were both expressed in the pn of the normal mice. these data elucidate that the reelin may play a key role in fasciculuation and collateral formation of pontocerebellar projections in addition to cell positioning or migration of pn neurons. kudoh suguru , , takahisa taguchi aist, ikeda, japan; presto, jst the spatiotemporal patterns of spontaneous action potential were analyzed, using the multi-site recording system for extracellular potentials of neurons and the living neuronal network cultured on a -dimensional electrode array. the map of functional connections between neurons revealed that each culture contained some hublike neurons and the distribution of the number of functionalconnections approximated a power-law distribution. we confirmed that the spatiotemporal pattern of spontaneous action potentials became more complex pattern along with developmental stage, and the constant pattern of stimulation promote this developmental change. in addition, the spatiotemporal pattern and the functional connections between neurons were drastically re-organized by real-time feedback stimulation. these results strongly suggest that the network structure of the cultured hippocampal neurons is neither stable nor random, but is functionally dynamic and is suitable for certain types of information processing. research funds: presto, jst ps p-d laterality of the human cerebral hemisphere taiko kitamura, jinzo yamada department of anatomy, tokyo medical university, tokyo, japan it has been reported that some functional predominance is located in the right or left hemisphere of the human brain. especially, the speech center and the center related to thought and emotion are located in the left and in the right hemisphere, respectively. in this study, the laterality between the right and the left human hemisphere was investigated macro-anatomically. we measured the weight, the medial-lateral width (m-l), the anterior-posterior lenght (a-p), and the width of the medial surface in the right and the left human hemisphere using in anatomical practice for medical students. the weight of each hemisphere was roughly equal. the m-l was wider in the right side than the left side. the a-p was longer and the width of the medial surface was larger in the left side than in the right side. because of the longer a-p and the larger width of the medial surface in the left hemisphere, it appeared that the left hemisphere overspreads the medial-dorsal marginal surface of the right hemisphere by the naked eye. such overspreading suspects that the left hemisphere develops earlier and faster than the right hemisphere. ps p-d synchrony-induced transition behaviors organized under spike-timing dependent plasticity for retrieving the memorized patterns takaaki aoki , toshio aoyagi department of physics, kyoto university, kyoto, japan; graduate school of informatics, kyoto university, kyoto, japan temporally correlated spikes, such as spike synchrony, have been observed in relation to behaviors or cognitions. however, it is unclear how the neurons read out the incoming spike synchronization in the dynamical behavior of network. in this modeling study, considering a network of excitatory and inhibitory neurons organized under spiketiming dependent plasticity, we present a type of network model in which incoming spike synchrony causes a transition between learned activity patterns in the order they were experienced in the learning process. furthermore, using appropriate training patterns, this network exhibits a context-dependent transition, in which the network switches to multiple patterns from a single pattern depending on the temporal structure of neuronal activity at the onset of incoming spike synchrony. this ability of the network may provide one of mechanisms by which a neuronal system can be trained to carry out tasks in a context-dependent manner. shozo kito, maiko kitagawa, akiko shingo lab. of neurosci., hyogo univ., kakogawa japan in our previous studies, we showed that a part of nicotine's beneficial effects on hippocampal and cortical neurons were due to increased igf- mrna expressions. nevertheless, the situation may be somewhat different as far as nicotine's effects on the neuronal progenitor cell, which is still on the way of differentiation are concerned. to clarify this problem, nicotine was intraperitoneally injected into weekold wistar strain rats in several doses followed by successive injections of brdu for the next days. then rats were sacrificed and vertical sections of the hippocampus formation were offered for double immunohistochemical staining of brdu/psa-ncam, brdu/neun or brdu/gfap. as the results, numbers of both brdu(+)/psa-ncam(+) cells and brdu(+)/neun(+) cells were much decreased nicotine-dose dependently. on the other hand, as much as mg/kg was needed for nicotine to exert its effect on the number of brdu(+)/gfap(+) cells. these results reveal that nicotine inhibits neurogenesis and plasticity in the hippocampus of adult rats. ps p-d the establishment of the organotypic slice culture of postnatal rat forebrain involving egfp-labeled neural progenitors kaoru sato , james e. goldman division of pharmacology, national institute of health sciences, tokyo, japan; department of pathology, columbia university, new york, usa after injecting egfp-encoding retrovirus into p rat svz, sagital sections of forebrain were made at p and cultured for days. the migration pattern of the egfp-labeled neural progenitors in the cultured slices is almost same as that at the corresponding age. the expression patterns of the glial differentiation-markers were also in accordance with those at the corresponding age. when slices were cultured with anti-␣ integrin antibody, the migration of the neural progenitors inside svz was significantly enhanced along the rostrocaudal extent. these results suggest that the organotypic slice culture of postnatal rat forebrain is an efficient experimental system for pharmacological studies about migration and differentiation of neural progenitors. radial glia is involved in the contact guidance of neuronal migration and also the neuronal and astroglial precursors. to make clearer the role of radial glia, we developed a method for the selective ablation of a subset of radial glia. it has been reported that tenascin-c (tn-c) is one of the markers for radial glia. accordingly, diphtheria toxin (dt)gene and enhanced green fluorescence protein (egfp)-gene both driven by tn-c gene promoter were co-transferred into the ventricular zone cells of the mouse neocortical primordium by means of in utero electroporation. the numbers of egfp-labeled cells in that tn-c gene promoter and subsequently dt gene are activated selectively decreased by this approach. using this method, the examination of radial glial morphology and neuronal migration following selective ablation is in progress. takayuki manabe, kouko tatsumi, eri makinodan, manabu makinodan, takahira yamauchi department of nd anatomy, nara medical university, kashihara, nara, japan it has been well documented that neurogenesis persists at the subventricular zone and the subgranular layer of the dentate gyrus in the adult mammalian brain. in the adult mice, we demonstrated that cells around a cryo-injured cortical lesion had a proliferative activity (labeled with brdu in vivo) and formed neurosphere-like aggregates in the sphere-forming culture condition. significantly lager number of spheres was observed in the culture from the injured hemisphere, which excluded the neurogenic regions (i.e. the svz and hippocampus), than those cultured from the control (contralateral and intact) hemisphere. furthermore, the sphere-forming cells differentiated to neuronal-and glial-marker positive cells in vitro. these results suggest that the cells forming sphere-like aggregates in vitro may function as a kind of progenitor cells in the injured brain. if this is a case, it would be tempting to transplant these sphere-forming cells to cure brain injury or disease. further characterization of the cells is underway. ps p-d localization of neurotrophin receptors trka in pc cells: d reconstruction analysis of membrane proteins tomoki nishida , hiroshi jinnai , tatuo arii , akio takaoka , ryoichi yoshimura , yasuhisa endo department of applied biology, kyoto institute of technology, kyoto, japan; department of polymer science and engineering, kyoto institute of technology, kyoto, japan; national institute for physiological sciences, myodaiji, okazaki, japan; osaka university, mihogaoka, ibaraki, osaka, japan it was previously reported that trka (ngf receptor) was associated with caveolae, small invaginations on the cell membrane, but its subcellular localization is not clarified in detail. we performed immunocytochemistry of trka and caveolin- in pc cells, analyzed by high-voltage electron microscopy, and reconstructed d structure of their subcellular distribution by imod. our results indicated that localization of caveolin- , known as an integral membrane protein of caveolae, was never found in the invagination structure in pc cells, but trka and caveolin- immunoreactivities were mainly found as a mesh-like structure in the cytoplasmic matrix. kensuke shiomi, kazuko keino-masu, masayuki masu department of molecular neurobiology, graduate school of comprehensive human sciences, university of tsukuba, tsukuba, japan the wnt signaling plays important roles in cell growth, differentiation, polarity formation, and neural development. previously we identified ccd , a third-type of the dix domain-possessing protein, as a positive regulator of the wnt/␤-catenin pathway. ccd mrna was mainly detected in the neural crest derivatives and differentiated neurons in mouse embryos, suggesting the importance of ccd in the wnt-mediated neuronal development. there are three subtypes of mouse ccd gene products, ccd a, ccd b and ccd c, which are generated by different promotor usage. mouse ccd a as well as zebrafish ccd a has a calponin homology domain which can mediate the interaction with the actin cytoskelton. we found that in the ccdtransfected hela cells, only the type a ccd proteins co-localized with the actin filament. in order to examine the function of the type a ccd proteins, we are now doing overexpression and functional blocking experiments using zebrafish embryos and cell culture. research funds: kakenhi ( , ) ps p-d analysis of a role of r-spondin on proliferation of the cortical neuroepithelium yumiko hatanaka , masahiro yamaguchi , fujio murakami , masayuki masu grad. school of comprehensive human sci., univ. of tsukuba, japan; grad. school of med., univ. of tokyo; grad. school of frontier biosci., osaka univ r-spondin (rspo ) is a secreted activator of wnt/␤-catenin signaling (kazanskaya et al. ) . rspo is expressed in the developing medial cerebral wall and transgenic mice expressing rspo in the entire neuroepithelium show enlarged lateral ventricle with a slight increase of brain size (hatanaka et al. ) . since wnt a has a role for expansion of caudomedial cortical progenitor cells (lee et al. ) , these findings lead us to the idea that rspo may synergistically promote proliferation of cortical neuroepithlial cells together with wnt a. to clarify their role on proliferation of cortical neuroepithelial cells, we first introduced a ␤-catenin/tcf reporter gene into these cells of embryonic day . mouse. an application of wnt a on these cells increased level of the reporter expression, and an addition of rspo further increased its level. we are now monitoring incorporation of brdu in neuroepithelial cells to know whether wnt a and rspo directly promote their proliferation. tae sun kim, hideki hida, tomoko narita, sachiyo misumi, hitoo nisino department of neurophysiology & brain science, nagoya city university graduate school medical sciences, nagoya, japan to investigate whether physiological low oxygen during development and cytokines expressed in the dopamine (da)-depleted striatum increase the number of da neurons from es-derived neural progenitor cells (npcs), npcs were treated with cytokine cocktail (il- ␤, il- , lif, gdnf) or lowered o ( . %), followed by tyrosine hydroxylase (th) immunostaining. low oxygen increased total number of th (+) cells ( . -fold) as compared to normal o . cytokine cocktail significantly increased th (+) cells ( . -fold) compared to nontreated control. treatment of lif and il- ␤ to npcs exhibited major contribution in the effect of cytokine cocktail. data suggest that physiologically relevant low oxygen in development and cytokines and trophic factors that were enhanced in da-depleted striatum cause in the increase of daergic neurons from es-derived npcs. ps p-d structural basis for reelin signaling: determination of receptor-binding site and its three-dimensional structure norihisa yasui , terukazu nogi , mitsuharu hattori , kenji iwasaki , , junichi takagi research center for structural and functional proteomics, inst for protein res., osaka univ., suita, japan; dept. of biomed. sci., grad. sch. of pharm. sci., nagoya city univ., nagoya, japan; core research for evolution and technology (crest) a large secreted glycoprotein reelin acts on target neurons through its receptors (apoer and vldlr), resulting in tyrosine phosphorylation of dab . in the present study, we have carried out structural and functional studies on the reelin signaling. first, we determined the structure of a single reelin repeat by x-ray crystallography. it had a horseshoe-like globular structure with some similarities to carbohydrate binding modules from many enzymes. moreover, electron micrographic d reconstruction of four-domain reelin fragment (i.e. r - ) revealed an elongated rod-like structure. next we determined minimum active unit within reelin. a fragment containing both the fifth and sixth reelin repeats (r - ) was capable of binding to the receptor (apoer ), and was also able to induce tyrosine phospholylation of dab in primary neuronal culture. ps p-d effects of astrocyte-derived factor and cell-cell communication on uni-directional differentiation from mouse embryonic stem cells into neural cells embryonic stem (es) cells uni-directly differentiate into neurons via neuroectoderm and neural stem cells by neural stem sphere (nss) method. cultured with astrocyte-derived factor, colonies of es cells give rise to nsss. we analyzed structure and gene expression of cell spheres formed under various culture conditions, in order to elucidate mechanisms of the uni-directional differentiation into neurons. quantitative real-time rt-pcr analysis demonstrated that the neuronal differentiation did not occur in the cell spheres. these results suggest that astrocyte-derived factor and cell-cell communication are necessary for the differentiation. we have previously established es cell differentiation system, by which we can derive neurospheres containing neural stem/progenitor cells (ns/pcs) with the identity of early caudal neural tube. taking advantage of this culture system, we have recently found conditioned medium of a stromal cell line (cmsc) has the activity to support the formation of neurospheres. this activity was more prominent when cultured at low cell density than when cultured at high cell density, suggesting that it supports the survival of ns/pcs. moreover, rt-pcr analysis of regional identities of the cmsc treated neurospheres revealed elevated expression of pax and pax compared with those of untreated neurospheres, indicating that cmsc promotes dorsalization of ns/pcs or selective proliferation of dorsal ns/pcs. elucidation of underlying mechanisms may provide important tools to derive early ns/pcs which can generate variety of projection neurons and be applicable to regenerative medicine. research funds: sorst jst ps p-d neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells neudesin expressed in adult mouse brain encodes a secreted signal with neurotrophic activity in neurons (j neurosci res : , ) . most neurotrophic factors are involved in neural cell proliferation and/or differentiation. however, the role of neudesin in neural development remains to be elucidated. neudesin mrna was expressed in the neural precursor cells before the appearance of neurons. therefore, roles of neudesin in neural development were examined using the neural precursor cells. neudesin significantly promoted neuronal differentiation. in addition, neudesin transiently promoted neural cell proliferation early in the developmental process. the differentiation was mediated though activation of the pka and pi- k pathways. in contrast, the proliferation was mediated through the mapk and pka pathways. the expression profile and activity indicate that neudesin plays unique roles in neural development. ps p-d fabp is required for maintenance of neural stem/progenitor cells in the postnatal hippocampus motoko maekawa , miho matsumata , , yuji owada , shigeki yuasa , noriko osumi , natl. inst. of neurosci., ncnp, tokyo, japan; tohoku univ. sch. of med., sendai, japan; crest, jst pax transcription factor is a key player for brain patterning and embryonic neurogenesis, and also expressed in the postnatal brain. we have previously shown that pax is necessary for keeping neural stem/progenitor cells in the hippocampus. in this study we have focused on a fatty-acid binding protein fabp , a downstream of pax , regulating maintenance of embryonic neural stem/progenitor cells (arai et al., ) . fabp was expressed in neural stem/progenitor cells in the hippocampal dentate gyrus (dg). % of fabp -expressing cells co-expressed gfap (a marker for early progenitors), and % of them co-expressed psa-ncam (a marker for late progenitors). fabp expression was also overlapped with pax , and expression of fabp was down-regulated in the dg of pax deficient rats and mice. finally, brdu-labeling analysis revealed decreased cell proliferation in the dg of fabp knockout mice. taking all together, it is concluded that fabp is required for maintenance of neural stem/progenitor cells in dg. ps p-d involvement of the psa-ncam expressing cells in early development of the vascular system of the forebrain momoko miyakawa, tatsunori seki department of anatomy, juntendo university school of medicine, tokyo, japan early development of the vascular system of the forebrain were studied in the chick embryo. staining of vascular endothelial cells by fitctomato lectin and immunohistochemical staining of the surrounding cells were performed on the same cryostat sections of embryos of embryonic day - . sections were examined under a confocal laser scanning microscope. capillaries were found in the lateral pallium and seemed to grow from psa-ncam-positive outer zone to negative inner zone of the pallium. psa-ncam is thought to be expressed in the immature neurons. the rims of capillaries were immunoreactive with psa-ncam in both zones. immunoreaction of doublecortin (neuronal marker) and punctate immunostaining of laminin also were observed on rims of capillaries. by immuno-electron-microscopy it appeared that the endothelium were covered with very thin processes of cells of which outer surface was immunoreactive with psa-ncam. psa-ncam expressing cells may be involved in the development of the vascular system of the forebrain by supporting or guiding the growing capillaries. masaharu kotani , , shiki okamoto , masato imada , kouichi itoh , atsushi irie , hitoshi sakuraba , hideo kubo department of molecular biologu, ohu univ., koriyama, japan; dept. deve. physiol., natl. inst. physiol. sci., okazaki, japan; dept. anatomy, nihon univ. shl. med., tokyo, japan; dept. mol. pharma., univ. tokushima bunri, sanuki, japan; dept. biochem. cell res., tokyo metro. inst. med. sci., tokyo, japan; department of clin. genet, tokyo metro. inst. med. sci., tokyo, japan; dept. med. biol, tokyo metro. inst. med. sci., tokyo, japan as randam- shows the highest expression level with the proliferating stage of neural stem cells (nscs), it is thought that the isolation of nscs based on the expression level of randam- is possible. in the present, we show that the isolated randam- high+ cells enrich nscs. the randam- high+ cells had the characteristics as the highly self-renewal capability and potential for multilineage differentiation into neural cells. in contrast, almost all of the randam- low+/− cells exhibited not only the extremely low self-renewability but the differentiation capability restricted to neurons. the results demonstrate that randam- is a usefule marker for the isolation of nscs by facs. yasuharu takamori , yasuhisa tamura , , yosky kataoka , , yilong cui , , hisao yamada department of anatomy and cell science, kansai medical university, osaka, japan; department of physiology, osaka city university graduate school of medicine, osaka, japan; morecular imaging reserch program, riken frs, saitama, japan lamins are major structural proteins of nuclear envelope. three lamin subtypes, a/c, b and b are mainly present in mammalian somatic cells. to investigate the pattern of lamin expression during neuronal differentiation, we immunohistochemically analyzed the existence of lamins in two neurogenic regions of rat brain; subgranular zone of dentate gyrus and subventricular zone, with confocal microscopy. gfap-positive primary progenitor cells possess lamin a/c (++), b (++), b (++), psa-ncam-positive subsequent progenitor cells possess lamin a/c (−), b (+++), b (+), and mature neurons possess lamin a/c (++), b (+), b (+++), in both neurogenic regions. these observation showed that the composition of lamin subtypes was distinct in particular differentiation stages during adult neurogenesis. yusuke tozuka , yuichi tanaka , tatsuhiro hisatsune department of integrated biosciences, university of tokyo, chiba, japan recent work has shown that nestin + neural progenitor cells exist in the adult brain, and suggested that neural activity itself could act directly on these progenitor cells. it has been unclear, however, how do adult progenitor cells sense activity signals from surrounding neural circuit. in the hippocampus where new neurons are continuously produced throughout life, nestin + adult progenitor cells received gabaergic inputs. the gabaergic activity depolarized these progenitor cells, and then promoted their neuronal differentiations. although neuronal production does not readily occur in the adult neocortex, nestin + neural progenitor cells exist in this area too. interestingly, these progenitor cells also received excitatory gabaergic inputs. this gabaergic inputs inhibited their cell proliferations. from these results, we here propose that adult progenitor cells are a direct target of gabaergic neuronal networks, and that this networkto-progenitor cell interaction influences progenitors development by regulating their cell proliferations and/or neuronal differentiations. ps p-e new migration pattern in the postnatal neurogenesis of the dentate gyrus takashi namba , , hideo namiki , tatsunori seki dept. of anat, juntendo univ. sch. of med., tokyo, japan; integrative biosci. and biomed. eng, sch. of sci. and eng, waseda univ., tokyo, japan in the hippocampus, granule cells continue to be generated from embryonic to adult stages. the early postnatal neurogenesis is a transitional state between the embryonic and adult neurogenesis. previously, we have suggested that the postnatal hilus contains astrocytic neural progenitors that divide and differentiate into neuroblasts, and that finally the neuroblasts settle in the granule cell layer (gcl). however, the questions remain how astrocytic progenitors divide and differentiate into neurons, and how the neuroblasts migrate to the gcl. to observe them, we developed a time-lapse imaging system. retrovirus-gfp was injected into the rat hippocampus at p . three days after the injection, the hippocampal slices were prepared for the time-lapse imaging. the present data show that neuroblasts migrate from the hilus to the gcl, changing the direction of their movement. this is inconsistent with the previous report suggesting simple radial migration (rickmann, et al., ) . the dividing pattern is currently under investigation. akiya watakabe , noritaka ichinohe , sonoko ohsawa , tsutomu hashikawa , kathleen s. rockland , tetsuo yamamori div. of brain biol, nibb, okazaki, japan; lab. for cortical organization and systematics, bsi, riken, wako, japan, lab. for neural architecture, bsi, riken, wako, japan by using gene expression profiles, we have tried to classify layer neurons in several areas of monkey neocortex. we previously reported that nurr , ctgf and sema e mrnas are specifically expressed in subsets of layer neurons. we further show here that cholecystokinin (cck) mrna is expressed in a subset of excitatory neurons in layer . by double ish, layer neurons in monkeys are roughly divisible into cck(+) and sema e(+) subgroups. each subgroup was further subdivided by other markers. tracer experiments showed that cck and sema e mrna expression correlate well with corticocortical and corticothalamic connectivity, respectively, but the correlation was only partial. from this, we infer that subtypes defined by gene expression may not directly correspond to classical neuronal types. the implication of our findings will be discussed in terms of constancy of laminar structure across areas and species. research funds: kakenhi ps p-e rbp-j regulates the cortical laminar formation kenji tanigaki , kazue muraki , norio yamamoto , tasuku honjo shiga medical center, research institute, shiga, japan; department of medical chemistry, kyoto university, kyoto, japan precise patterns of cell cycle exit and migration of neural progenitors are crucial for the formation of cortical layer structure. to examine involvement of notch-rbp-j signaling in the cortex laminar formation, we deleted rbp-j from neural progenitors in anatomically restricted areas by in vivo electroporation of cre-expressing plasmids. such studies revealed that rbp-j deficiency caused transformation of glutamatergic pyramidal neurons in layer ii/iii to layer iv neurons with concomitant loss of astrocytes. the loss of rbp-j accelerated neuronal differentiation and changed their laminar fates. in addition, time-lapse studies indicated the migration defect of rbp-j-deficient neurons. the results showed that notch-rbp-j signaling regulates migration of differentiated neurons as well as the timing of the cell cycle exit of neuronal progenitors to determine the laminar and cellular fates of neural progenitors. ps p-e search for the genes that define mammalian cortical progenitor cells using single-cell gene expression profiles ayano kawaguchi , tomoko ikawa , yuya kasukawa , hironori ueda , , kazuki kurimoto , michinori saitou , fumio matsuzaki , lab. for asymmetric cell division, cdb, riken, kobe, japan; functional genomics subunit, cdb, riken, kobe, japan; lab. for systems biology, cdb, riken, kobe, japan; lab. for mammalian germ cell biology, cdb, riken, kobe, japan; crest, jst, japan in the mammalian brain, cellular heterogeneity of the progenitor cells has largely hindered the molecular analysis of neuronal diversity. to overcome this problem, we randomly picked individual vz/svz cells of mouse embryos, and constructed cdnas from each of them by global pcr amplification method. we could classify these "single cell derived cdnas" into several groups retrospectively based on the expression of marker genes, including cell cycle related genes, transcription factors, and regional marker genes. samples that showed typical marker gene expression pattern of the groups were applied for genechip analysis. the obtained data were confirmed by quantitative pcr and in situ hybridization. by this strategy, we identified nine genes that were specifically expressed in the svz progenitor cells. research funds: kakenhi ( ) ryosuke tatsuno , tomoaki sai , , masahiro otsu , kuniko akama , takashi nakayama , tosifusa toda grad. sch. of sci. and tech., chiba univ., chiba, japan; lab. regener neurosci., tokyo metropol. univ. fac. health sci., tokyo, japan; dept. orthop. surg., jikei univ. sch. med., tokyo. japan; dept. biochem., yokohama city univ. sch. med., yokohama, japan; proteomics collab. res., tokyo metropol. inst. of gerontol., tokyo, japan embryonic stem (es) cells possess pluripotency and self-renewal. however, the proteomic analysis of neural stem cells and neurons differentiated in vitro from es cells has not so proceeded yet. we investigated the expression levels of proteins during in vitro differentiation of mouse es cells into neurons via neural stem cells by neural stem sphere (nss) method, using -d gel electrophoresis and maldi-tof ms. we identified vimentin, creatine kinase, atp synthase beta subunit, and some proteins with no annotation in murine brain the database, which were up-regulated in neural stem cells, and down-regulated in es cells and neurons. these results suggest that the neural stem cells have characteristic protein expression profile. ps p-e identification of se , a novel gene expressed in the nural progenitor cells shin-ichi sakakibara, kazuhiko nakadate, shiichi ueda department of histology and neurobiology, dokkyo university school of medicine, tochigi, japan identification of the genes regulating neural progenitor or neural stem cell functions is critical to understand the mechanisms of the adult neurogenesis and neurodegenerative disease. we compared the gene expression profile of proliferating neural stem cell cultures with those of differentiated cells. a subtractive library was constructed by using the suppression subtractive hybridization and the differential screening was performed. among two thousand of the differentially expressed subtracted clones, we identified genes that significantly upregulated in neural stem cell culture. these included several novel genes, in addition to the known genes involving in the cell cycle and signal transduction. in situ hybridization and the developmental northern analysis demonstrated that these mrnas were enriched in the germinal neuroepithelium, embryonic ventricular zone and the postnatal subventricular zone surrounding the lateral ventricles. we further analyzed the expression pattern of the novel gene se in developing and matured cns. teiichi furuichi , akira sto , , yukiko sekine , noriyuki morita , tetsushi sadakata , satoshi shoji , jin-hong huang , toshio kojima laboratory for molecular neurogenesis, riken brain science institute, japan; comparative systems biology team, riken genome sciences center, yokohama - , japan mouse cerebellum develops through a series of cytogenetic and morphogenetic events that are genetically coded within the first three weeks of life. we have extensively investigated the spatio-temporal gene expression profiles during the postnatal development of mouse cerebellum by differential display, rt-pcr, genechip, cdna microarray, and in situ hybridization. we have informatively systematized all the profiles in an online neuroinformatics database cdt-db (http://www.cdtdb.brain.riken.jp) with various search functions. we have demonstrated that the postnatal development of mouse cerebellum is genetically programmed by thousands of genes that exhibit differential expression patterns in time and space. further studies on a scale that includes the underlying expression of all genes and more detailed studies on their transcriptional regulation will shed light on the genetic basis for cerebellar development. miwako ozaki , makoto mizuno , kazuhisa sakai , yoshimoto kiyohara , kazuhiko yamaguchi , tsutomu hashikawa , hiroyuki nawa institute of biomedical engineering, waseda university, tokyo, japan; department of molecular neurobiology, brain research institute, niigata university, niigata, japan; laboratory for memory and learning, bsi, riken, saitama, japan; laboratory for neural architecture, bsi, riken, saitama, japan neuregulin (nrg), a neurotrophic factor, involved in the development, differentiation and repair of the nervous system, regulates the activation of ion channels and neurotransmitter receptors. in order to examine the molecular mechanism on the relationships between network, synapse formations and higher orders functions, we prepared ig-nrg knock out mice (nrg type i and iv were disrupted). the mutant mice showed motor disco-ordination and abnormality of synaptic structure in related areas in cerebellar nuclei and cortex. in addition, the number of vesicles in presynaptic neurons decreased in their synapses. the study on cerebellum that is very clear in the network input information would give some suggestions to the relationship between synaptic functions and behaviors. ps p-e psd- protein expression in rat oromaxillofacial motoneurons during postnatal development kohji ishihama , , satoshi wakisaka , shiho honma , akira ito , , kei azuma , , mikihiko kogo department of oral anatomy and developmental biology, osaka university graduate school of dentistry, osaka, japan; first department of oral and maxillofacial surgery, osaka university graduate school of dentistry, osaka, japan postsynaptic density (psd), which is composed of diverse proteins, involved in synaptic structure, neurotransmission and signal transduction. psd- implicates in formation and maturation of excitatory synapses. psd- regulates the localization of the nmda receptor by means of binding with nr . rhythmical oro-maxillofacial activities, such as suckling and chewing, are generated in the brainstem, and we showed that nmda receptors played critical role for the rhythm and pattern generation and signal transmission around the trigeminal motor nucleus during prenatal and early postnatal development. here we examined the temporal distributions of psd- protein using with immunohistochemical study, in developing rat brainstem from suckling to mature chewing stage. there was early emergence of psd- expression in the interneurons located at medial of the trigeminal motor nucleus. masami miura, masao masuda, toshihiko aosaki neural circuits dynamics research group, tokyo metropolitan institute of gerontology, japan the striatum, an input stage of the basal ganglia, contributes to habit formation as well as motor functions. recent studies suggest that striatal interneurons play an important role in processing of cortical input. we investigated the synaptic connections between interneurons using paired whole-cell recordings and immunohistochemical techniques. we found that fast-spiking (fs) interneurons sent gabaergic inhibitory input to cholinergic interneurons, which were gaba a receptor-mediated and suppressed by gaba b receptor agonist skf . in turn, cholinergic interneurons sent cholinergic excitatory input to fs interneurons. because the excitatory postsypnatic potentials (psps) were blocked by hexamethonium and dihydro-␤-erythroidine, the psps were nicotinic acetylcholine receptor-mediated. these results suggest that gabaergic interneurons and cholinergic interneurons mutually influence their excitability and might modulate the activity of striatal local circuits. ps p-e ocular following responses (ofrs) to a brief background motin are modulated in relation to preparation for upcoming pursuit hiromitsu tabata, kenichiro miura, kenji kawano dept. integ brain sci., grad. schl of med., kyoto univ., kyoto, japan recently, our group reported that the ocular responses to a brief perturbation of a small target during fixation increased when subjects (humans, monkeys) were preparing for upcoming smooth pursuit eye movements (spems) rather than preparing for saccades or stationary fixation. here, we report that the increase in ocular responses based on the anticipation of spems was also observed in monkeys when a large-field visual stimulus (background) was moved briefly prior to pursuit. the result indicates that the visual region where the gain of the visuomotor transmission increased is not limited to a small region near the target but spreads to a larger field. in other words, the anticipation of upcoming spems could affect the generation of ofrs. furthermore, directionally biased ocular responses to the brief background motion were observed when the animals repeatedly performed spems toward one direction, implying that the prediction of the upcoming spem direction might cause the directional asymmetry of the visuomotor transmission gain. ps p-e comprehensive characterization of motor neurons related with locomotory central pattern generator in the earthworm by imaging toshinobu shimoi , kenji mizutani , hiroto ogawa , kohji hotta , kotaro oka ctr. for biosci. and info, keio univ., yokohama, japan; neuro, karolinska inst, stockholm, sweden; bio, saitama med. sch., saitama, japan in this study, we comprehensively identified and characterized motor neurons concerning with locomotory central pattern generator (cpg) in the earthworm by calcium imaging as multiple recording. the candidates of motor neurons were stained with dextran conjugated calcium indicators using retrograde labeling from projection nerves. we obtained the responses of up to cell bodies of motor neurons and sensory neurons on the ventral surface of the segmental ganglion ( % or less for all neurons on the ventral surface). we analyzed the activity patterns of the candidates of motor neurons using pattern matching method comparing between calcium responses or between calcium responses and locomotory motor pattern. as a result, we detected motor neurons as pairs of neurons having strong synchrony to each other neuron or to motor pattern. these results were great progress to identify motor neurons related with locomotory cpg in the earthworm. ps p-e three dimensional ( d) pursuit eye movement signals in cerebellar dorsal vermis takuya nitta, teppei akao, sergei kurkin, kikuro fukushima department of physiology, hokkaido university school of medicine, sapporo, japan for pursuit of a target moving in d space, signals for frontal and vergence-pursuit must be synthesized. studies in our laboratory have demonstrated that d pursuit signals are generated in the frontal eye fields, and also present in cerebellar floccular region. however, the majority of floccular purkinje (p-) cells discharged after onset of vergence-pursuit. cerebellar dorsal vermis is another cerebellar area for frontal pursuit. to examine whether d pursuit signals are present in this area, we examined simple-spike discharge of vermal pursuit p-cells in monkeys. of a total of p-cells that were examined during both frontal and vergence-pursuit, % discharged for both, % only for vergence, and % only for frontal pursuit. these results indicate that most of vermal pursuit p-cells discharged for vergence and that about half of them had d pursuit signals. majority ( %) of these p-cells discharged before onset of vergence eye movements with the typical lead time of ms, suggesting their involvement in the initiation of vergence-pursuit. research funds: kakenhi ( ) ps p-e information processing in fef-rnrtp pathway for smooth pursuit seiji ono, michael j. mustari division of sensory-motor systems, yerkes national primate research center, emory university, atlanta ga, usa the frontal eye field (fef) cortex is known to play a role in smooth pursuit (sp). this role is supported by fef projections to the rostral nucleus reticularis tegmenti pontis (rnrtp) which projects heavily to the vermis. using multiple linear-regression modeling, we have shown that sp neurons in rnrtp were biased towards eye acceleration. however, the functional characteristics of sp related fef neurons that project to rnrtp have never been described. therefore, we used micro-electrical stimulation to deliver single pulses in rnrtp to antidromically activate fef neurons. the majority of sp related fef neurons that we identified as projecting to rnrtp were most sensitive to eye acceleration and much less sensitive to eye velocity. the neurons in fef-rnrtp pathway carry signals that could play a primary role in sp initiation. our antidromic studies may help address a fundamental question regarding whether basilar pontine nuclei integrate signals from multiple cortical areas or mostly relay signals with little transformation to cerebellum. research funds: nih grants ey , rr aya takemura , yumi murata , , kenji kawano , neurosci. res. insti, aist, tsukuba, japan; dept. integ brain sci., grad. sch. med., kyoto univ., japan; grad. sch. compreh hum sci., univ. tsukuba, japan previous studies in monkeys suggest that the medial superior temporal (mst) area is involved in visual motion processing. to understand the role of the mst in optokinetic nystagmus (okn) and afternystagmus (okan), we examined the effects of bilateral chemical lesions in the mst in two monkeys. when each monkey was injected with ibotenic acid ( mg/ml, - l total), the initial rapid rise in okn was reduced. consequently, it took longer for the eye velocity to reach a steady state (i.e., an eye velocity close to the stimulus velocity). by contrast, the steady state okn was not affected and the okan persisted. the initial amplitude and falling time constant of the okan increased. the results suggest that the mst is part of the direct pathway for the initial rapid rise in the okn, but is not involved in the velocity storage mechanism for the steady state okn and okan. smooth pursuit is performed by coordination of eye and head movements. we have reported that the majority of fef pursuit neurons in monkeys with their head free to rotate about a vertical axis were modulated not only during eye-and gaze-pursuit but also head-pursuit to a moving reward feeder while the monkeys fixated an earth-stationary spot without gaze movement. to examine the origin of head-pursuit modulation, we moved the reward feeder in a ramp trajectory at • /s with random intervals. the majority of pursuit neurons discharged before the onset of head movements with the mean lead time of ms. discharge modulation during head-pursuit and passive whole body rotation was not correlated in most neurons. these results suggest that proprioceptive neck inputs or vestibular inputs are not the main origin of head-pursuit modulation. rather, our results suggest that the main origin reflects pursuit commands. ps p-e the local feedback loop of the saccadic system: an analysis of the eye movements induced by pdb stimulation rikako kato department of developmental physiology, national institute for physiological sciences, okazaki, japan saccadic amplitude are controlled by a comparator that calculates dynamic motor error. some models place the comparator in the superior colliculus while others assign this role to the reticular formation. to decide between the two hypotheses one would need to stimulate pathways in between their putative comparators. we stimulated collicular axons descending in the pdb. our data demonstrate that electrical stimulation of the pdb evokes saccades and they always terminate before the end of the stimulus train. the characteristics of evoked saccades are comparable to those spontaneously generated by the cat. our data clearly demonstrate that the feedback path of the local loop of the saccadic system closes downstream of the superior colliculus. katsuo fujiwara , kenji kunita , kaoru maeda , takeo kiyota department of human movement and health, graduate school of medical science, kanazawa university, kanazawa, japan; institute for health and sport sciences, osaka city university, osaka, japan we investigated changes in visual evoked potential (vep) during postural adaptation process while subjects maintaining standing posture on an oscillation floor with periodic vision shut. the subjects were undergraduate students. a shutter goggle was used as a vep stimulator which was opened periodically for ms with -ms intervals. the oscillation trial ( . -hz frequency and . -cm amplitude) ( - s) was repeated times. postural steadiness was evaluated by mean fluctuation speed of the center of foot pressure. the mean speed decreased as trial was repeated, and reached a plateau before the th trial. a significant correlation was shown between th- st trial differences in mean speed and vep amplitude (r = . ). this indicates that the role of visual information is different among subjects with various adaptation processes of postural control. ps p-e primary motor cortex contributes to generating manual following response toshitaka kimura , naoki saijo , hiroaki gomi , ntt cs labs, kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan a large-field visual motion during arm movements induces a shortlatency, involuntary arm response called as manual following response (mfr). the mfr exhibits similar features to the ocular following response (ofr) elicited by the similar visual stimulus, with respect to the stimulus-response directional characteristics and the spatiotemporal frequency tuning property. this suggests that computational mechanism is shared for both responses. however, the neural basis of the mfr motor command generation remains unclear, while ofr is known to be generated subcortically. here we show, by using transcranial magnetic (tms) and electrical (tes) stimulation over the primary motor cortex (m ), that ( ) an emg response evoked by tms was facilitated during mfr, while that by tes was not, and ( ) intracortical inhibition within m assessed by paired-pulses tms was reduced during mfr. these results suggest that mfr is generated through activity of interneuronal networks within m . such cortical mechanisms for mfr generation are distinct from the subcortical processes for ofr generation. naoki saijo , hiroaki gomi , ntt cs labs., kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan when a visual target is suddenly shifted during a reaching movement, we can quickly adjust the arm movement. however, the computational mechanism to generate quick adjustment is still unclear. here we investigated this mechanism from the viewpoint of visuomotor coordinate transformation. we observed the hand responses to the target shifts in radial directions applied during reaching. the data show that the direction of the initial phase ( - ms) of hand response acceleration was slightly biased from the corresponding target shift direction, whereas the direction of the late phase ( - ms) was little biased. additionally, when we use a target shift having less-motion energy, the response latency greatly increased and the directional bias significantly decreased. these results suggest that the on-line reaching adjustment would be generated by two different mechanisms: a reflexive controller which is induced by visual motion with short latency and generates spatially inaccurate response, and voluntary controller which generates spatially accurate response with long latency. ps p-e spatial relationship between gaze and reaching-target modulates manual following response naotoshi abekawa , hiroaki gomi , ntt cs labs., kanagawa, japan; erato shimojo implicit brain function proj, jst, saitama, japan to explore the functional mechanism of the manual following response (mfr) induced by a large-field visual motion during arm movement, we examine its modulation caused by the spatial relationships between gaze, target, and background. on a large vertical screen placed in front of the subject, full field checker pattern, two markers (upper and lower), and a gray mask around one of the markers, were displayed. in the first condition, subjects kept watching the upper marker, and pointed the upper (congruent) or lower (incongruent) marker instructed before every reaching. the checker pattern suddenly moved either rightward or leftward brief after reaching start. in the second condition, subjects did the same task with watching the lower marker. in both conditions, the mfr amplitude was significantly grater in the congruent condition than in the incongruent condition, whereas the mask location did not significantly affect the mfr amplitude. this suggests that the spatial relationship between gaze and target is important in modulating mfr. misako komatsu, eizo miyashita dept. compu. intelligence & systems sci., tokyo tech., yokohama, japan when a subject performed pointing to a remembered target under eyes fixated, we have reported that endpoints tended to sift closer to the fixation point. moreover, we have noted that the greater the distance between a target and the fixation point, the larger the errors. the result was consistent even when the position of the fixation point was changed. the above tendency was considered to occur in eye-or gaze-centered coordinates. it is open question, however, if the brain correctly compensates the difference of the relative position of eyes to the head? to answer this question, we investigated the dependency of the endpoint errors on the positions of a monitor and the fixation point. the subjects, sitting in front of the monitor, were asked to point a remembered target as accurately as possible using a computer mouse. all the results were consistent with the previous ones regardless of the position of monitor or the fixation point. these results suggest either the eye-position doesn't affect how we recognize the target position, or the brain correctly compensates the eye-position with a fixed head position. ps p-f influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot tetsuro muraoka , takashi obu , kazuyuki kanosue asmew, waseda university, saitama, japan; graduate school of human sciences, waseda university, saitama, japan; faculty of sports sciences, waseda university, saitama, japan the aim of this study was to investigate the influence of the coupling of muscle activity on rhythmic movements of ipsilateral hand and foot. the subjects (n = ) were supine, and their hand was prone. they performed cyclical flexion-extension coordinations of the hand and foot in the iso-(iso) or opposite-(oppo) directions, and those with an elastic load against wrist flexion (el-iso and el-oppo) at . , . , and . hz. over % success rate was observed in all tasks except oppo ( - %). the in-phase muscle activity of wrist and foot muscles was obserbed in all tasks except oppo. it was suggested that the in-phase muscle activity might be an important factor in a coordinated movement of ipsilateral hand and foot. research funds: the special coordination funds for promoting science and technology, mext, japan ps p-f simultaneous muscle activity stabilizes the coordinated movement of ipsilateral hand and foot takashi obu , tetsuro muraoka , kazuyuki kanosue , , faculty of human sciences, waseda university, saitama, japan; faculty of sport sciences, waseda university, saitama, japan; asmew, waseda university, saitama, japan in human, voluntary opposite-directional movement (antiphase) of ipsilateral hand and foot is more difficult than iso-directional movement (inphase). the purpose of the present study was to investigate the influence of the coupling of muscle activity on these movements. eight normal subjects lay in supine position with hand prone and their foot was forcedly moved by a dynamometer cyclically at , . , and . hz. they were asked to perform tasks, concentric/eccentric contraction of ankle dorsiflexors with in-phase/antiphase wrist extension/flexion. all tasks were performed successfully. muscle activity of hand flexors was observed in concentric-antiphase and eccentric-inphase tasks, indicating simultaneous muscle activity of hand and foot. it may be suggested that simultaneous muscle activity would make the movement easier regardless of the direction of movement. ps p-f activities of erector spinae muscles during jaw clenching in man kayoko yasunaga , , tadachika yabushita , kazuo toda , kunimichi soma orthodontic science, tokyo med. & dent. univ., tokyo, japan; div. integrative sensory physiology, nagasaki univ., nagasaki, japan recent studies focused the functional relationships between the masticatory and the posture system. the hypothesis of our present study is an existence of functional connections between the masticatory system and the spinal muscles which maintain the posture. therefore, we investigated the effect of the maximum jaw clenching on the spinal muscle activities. bipolar needle electrodes were inserted into erector spinae muscles to record the motor unit activities when the sitting subjects relaxed and performed maximal jaw clenching. as a result, the instantaneous frequencies of the spinal muscles decreased with clenching, compared with relaxed jaw position. our results suggested that there were some relationship between spinal muscle activities and jaw clenching. the effects of bipedal walking on the central nervous systems-influence of bipedal walking on the spinal reflex-naomi wada , sachiko motoyama , futoshi mori , shigemi mori department of veterinary physiology, yamaguchi university, yamaguchi, japan; national institute for physiological science, okazaki, japan the one of the biggest questions in the vertebrate evolution is how human got the highly developed brain. many investigators suggest that upright posture and bipedal walking caused remarkable development of brain and produced the human being. the purpose of our experiments is to show the influences of bipedal habits on central nervous systems. we have established the bipedal walking model using rats (rbm) by amputation of forelimbs and training of upright posture and bipedal walking. after training of upright posture and bipedal walking for - weeks, rats got abilities of the stable upright posture and bipedal walking with symmetrical hindlimb movements between left and right side. in the present experiments, we studied about the effects of bipedal habits on the lumbar spinal reflex. the results of out experiments showed that bipedal habits inhibit the spinal reflex pathways. ps p-f neuronal activity in primary motor cortex during quadrupedal locomotion of the japanese monkey katsumi nakajima , futoshi mori , akira murata , masahiko inase dept. of physiol., kinki univ. schl. of med., osakasayama, japan; dept. of vet. physiol., facult. of agr., yamaguchi univ., yamaguchi, japan to elucidate cortical mechanisms related to the control of primate locomotion, we recorded neuronal activity in m of the monkey walking quadrupedally on the treadmill. tungsten microelectrodes were inserted into m hindlimb region using a custom-made micromanipulator. we found that all neurons recorded in m modulated their discharge phasically time-locked to the step cycle or increased their discharge frequency tonically during simple locomotion. the neuron exhibiting phasic modulation peaked once or twice per step. the peak activity occurred at widely different times during the step cycle in different recorded neurons. as the treadmill speed increased, most of recorded neurons increased their discharge frequency. all these results suggest that m output in monkeys directly and/or indirectly acts on spinal circuitries generating a basic pattern of rhythmic activity during simple locomotion in a manner different from that in subprimates. research funds: kakenhi ( ) ps p-f activity of putaminal neurons receiving inputs from motor cortical areas in behaving monkeys sayuki takara , , nobuhiko hatanaka , , masahiko takada , atsushi nambu , school of life science, the graduate university for advanced studies, japan; division of system neurophysiology, national institute for physiological sciences, japan; tokyo metropolitan institute for neuroscience, japan the putaminal (put) neurons receive motor cortical inputs and change their activity in relation to movements. to investigate how these inputs contribute to put neuron activity in behaving monkeys, extracellular unit activity was recorded from identified put neurons during the performance of a memory-guided reaching task. based on orthodromic spikes evoked by cortical stimulation, individual put neurons were defined in terms of whether they receive input from the primary motor cortex (mi), the supplementary motor area (sma), or both. the results showed that mi-recipient neuron activity was responsive to the movement, while sma-recipient neuron activity was responsive to the cue stimuli and/or the delay period. the activity of neurons receiving convergent inputs was related to both the movement and the delay period. we previously reported that electrical stimulation of cerebrofugal fibers induced short latency facilitation and succeeding suppression on phrenic activities, while train pulse stimulation of caudal raphe nuclei (raphe magnus, rm, and raphe pallidus, rp) induced suppression or facilitation on respiratory neural activities in cats and rats. in this study, in order to analyze the cerebral and raphe projections to the respiratory neuron network, we examined the effects of stimulation of cerebrofugal fibers and caudal raphe nuclei on activities of ventral respiratory group neurons (vrgs) in the medulla and upper cervical inspiratory neurons (ucins). animals were anesthetized, immobilized and artificially ventilated. stimulation of cerebral peduncle (cp) induced short latency facilitation and succeeding suppression on activities of ucins. stimulation of rm or cp evoked inhibitory postsynaptic potentials in the caudal vrgs. these results suggest that rm and cerebral cortex directly inhibit main respiratory output neurons in vrg. ken muramatsu , sei-ichi sasaki , yuichiro cho , kenji sato anatomy and physiological science, tokyo medical and dental university, tokyo, japan; department of physiology, ibaraki prefectural university of health sciences, ibaraki, japan distribution of average diameters of external anal sphincter (eas) motoneurons and peripheral motor fibers were examined in cats. to identify eas motoneurons, horseradish peroxidase was applied to the central cut end of the anal branches of the pudendal nerve. eas motoneurons were found in the onuf's nucleus of s and s spinal levels. to examine size of peripheral motor fibers, ganglionectomy was performed onl -s spinal segments which contain afferent fibers of eas muscles. after weeks survival period, anal branches of the pudendal nerve was examined. histograms of the distribution of average diameters of cell body and motor fiber shows unimodal distri bution. also, distribution of muscle spindles of eas muscle were examined by serially sectioning the distal colon and staining with mayer's haemotoxylin and eosin. no muscle spindles were found. these results suggest that eas muscle is controlled without gamma loop. mariko miura, yoshiki iwamoto, kaoru yoshida neurophysiol., univ. tsukuba, tsukuba, japan saccade accuracy is ensured by an adaptation mechanism. the speed and magnitude of adaptation vary greatly across experiments even for the same subject. one factor that might cause this variability is adaptation history. the present study aims to clarify whether preceding adaptation influences subsequent adaptation over several days. gain decrease adaptation was induced in a monkey by stepping the target backward during saccades. adaptation experiments were repeated for consecutive days. we compared adaptation in day and that in day . the gain decrease for the first saccades in day ( . ± . ) was larger than that in day ( . ± . ) (p = . , n = , paired-t test). the rate of adaptation in day ( . ± . × − /sac) was higher than that in day ( . ± . × − /sac) (p = . ). the overall gain change ( saccades) in day ( . ± . ) was larger than that in day ( . ± . ) (p = . ). thus, both the speed and magnitude of adaptation were increased by preceding adaptation. the present study suggests that the memory of saccadic adaptation is retained for days and facilitates following adaptation. research funds: kakenhi ( ) ps p-f asymmetry of the anticipatory convergence eye movement haruo toda, takehiko bando div. integr. physiol., grad. sch. med. sci., niigata univ., niigata, japan typically, convergence eye movement is known as symmetric adduction of the both eyes. but asymmetrical convergence also found in the natural condition. these asymmetrical convergence may reflect asymmetries of central control of convergence eye movement. the lateral suprasylvian (ls) areas are extrastriate cortices which receive visual information from v . the ls has contralateral dominant receptive fields and convergence eye movements evoked from the long latency regions were asymmetrical. cats (n = ) were trained to start convergence by an alarm signal (buzzer sound or combination of buzz and blinking of led), preceding target movement by s. after training, ocular convergence was elicited by the alarm signal before target movement (predictive open-loop convergence) in % of trials. in three cats, we used training with obliquely approaching target. after training, asymmetrical anticipatory eye movements were observed. based on these findings, related ls neuronal activities and results from lesion study, we will discuss the role of ls in asymmetry of anticipatory and visually-evoked convergence eye movement. yusuke uchida , xiaofeng lu , , shogo ohmae , toshimitsu takahashi , , shigeru kitazawa , dept. of neurophysiol., juntendo univ. grad. sch. of med., tokyo, japan; crest, jst, tokyo, japan we examined reward related neural activity in the supplementary eye field (sef). for this purpose, two monkeys were rewarded after each visually guided saccade from a central fixation point to one of targets that were arranged in a radial pattern. a target appeared while the monkeys were fixating on the central point, and the monkeys made a saccade to the target when the fixation point disappeared and held on the target until the target turned off. reward was delivered during or after target-hold period. we found that many sef cells became active during the period of reward delivery (r-cell). more than half of r-cells showed enhancement of the neural discharge in the specific target directions but not other directions in which the same amount of reward was given (rd-cell). interestingly, most of rd-cells displayed activity with the clear directional tuning. these results demonstrate reward dependent activity specific to spatial direction in the sef, and further suggest that sef cells provide reinforcement mechanism. research funds: kakenhi ps p-f frontal pursuit area is involved in the retinalslip dependent adaptation of monkey post-saccadic pursuit eye velocity hiromasa kitazawa , soichi nagao , lab. for motor learning control, riken bsi, saitama, japan; sorst, jst, saitama, japan smooth pursuit is under learning control by several brain areas including cerebrum and cerebellum. smooth pursuit velocity is modifiable by repetition of target velocity for a brief period at its onsets. role of cerebellar vermis and hemisphere in the adaptive control of smooth pursuit is suggested by lesion experiments, but the role of frontal pursuit area (fpa) is not known. to reveal possible involvement of fpa in the adaptation of smooth pursuit, we identifying fpa by unit recording and microstimulation, and reversibly inactivated it by local injection of muscimol. we found that inactivation of fpa not only reduced of the velocities of pursuit in the ipsi-and contra-versive directions to the inactivated fpa, but also appreciably depressed its adaptation, suggesting that fpa is involved in the adaptation of smooth pursuit. shinji matsutani department of functional morphology, kitasato university school of nursing, kanagawa, japan distribution of terminals on individual centrifugal axons in the main olfactory bulb was studied using an anterograde tracer to elucidate function of the centrifugal system. the tracer was injected into olfactory cortical areas, and individual labeled axons were traced from serial sections. as already reported in the last meeting, the centrifugal axons had multiple terminals with discrete locations. distribution of these terminals was examined in reconstructed maps in which localization of the terminals was projected onto a sagittal plain. in most axons, the terminals were clustered to form a patch that was stretched in a rostrocaudal direction. it was also common that patches belonging to the same axon were found in distant locations and in both sides of the single bulb. while most of the terminals were seen in the granule cell layer, those located in the glomerular layer and in the external plexiform layer were found following injections into the anterior olfactory nucleus. the centrifugal fibers may couple the activity of discrete and distant subsets of bulbar neurons. ps p-f projection targets of the drosophila taste receptor neurons in the primary gustatory center of the brain takaaki miyazaki , , kei ito , , dept. of comput. biol., grad. sch. of frontier sci., univ. of tokyo, japan; center for bioinform., imcb, univ. of tokyo, japan; bird, jst, japan in order to figure out the way of information processing linking gustatory stimulus and taste-associated behavior, systematic knowledge about the underlying neural networks is required. drosophila melanogaster is an attractive model organism for this task, thanks to its relatively simple brain structure and a wide variety of molecular and genetic tools available. gustatory sensory neurons in the labellum of the mouth project their axons via the labial nerve to the suboesophageal ganglion (sog) of the brain. to understand the entire neural circuits of these first-order neurons in the primary gustatory center, we searched for the gal enhancer-trap strains that visualize specific neural fibers in the sog and the labial nerve. screening , strains, we identified about candidate lines. the projection targets of the labeled neurons were classified into seven areas. the terminals of the already identified sensory neurons appear to fall into specific subsets of these areas. research funds: bird, jst ps p-f immunoreactivity and voltage-gated channels of mouse taste bud cells kennji kimura , yoshitaka ohtubo , takashi kumazawa , kiyonori yoshii graduate school of life science and systems engineering, kyushu institute of technology, kitakyushu, japan; department of applied chemistry, saitama institute of technology, fukaya, japan mammalian taste buds comprise four heterogeneous cell types, type i to iv, and their collaboration seems to generate taste sensation. we investigated the electrophysiological properties of these cell types except type iv with taste buds preserved in mouse lingual epithelia. type i cells elicited smaller ttx-sensitive, tea-sensitive, and teainsensitive currents in magnitude than other cell types. type ii cells elicited a smaller tea-sensitive current and a larger tea-insensitive current than type iii cells. these results suggest that type ii and iii cells elicit action potentials with different ionic mechanisms, and that the difference results from the functional differences of these cell types. research funds: kakenhi ( ) and the st coe program (center # ) granted by mext of japan ps p-f inositol monophosphatase maintains synapse localization and regulates behavior in the mature nervous system of c. elegans yoshinori tanizawa , atsushi kuhara , hitoshi inada , eiji kodama , takafumi mizuno , ikue mori , lab. of mol. neurobiol., nagoya univ., japan; institute for advanced research, nagoya univ., japan inositol monophosphatase (impase) is suggested to be relevant to bipolar disorder. although lithium is believed to exert therapeutic effect by inhibiting impase in patients, the mechanism underlying lithium therapy is largely unknown. here we show that the loss of impase causes defects in behavior and localization of synapses in c. elegans. mutations in ttx- gene encoding impase exhibit defective thermotaxis behavior, which is attributable to the loss of impase activity in the most essential integrative interneuron ria in the nervous system. the ttx- mutations also cause mislocalization of synaptic proteins in ria. both behavioral and synaptic defects in ttx- mutants were rescued by expression of impase at adult stage and inositol application, and were mimicked by lithium application in wild type animals. these results suggest that impase is required in the mature nervous system for maintaining synapses of the central interneurons in order for animals to behave properly. research funds: kakenhi ps p-f postnatal alterations in expression of vesicular glutamate transporters in the main olfactory bulb (ob) of rats h ohmomo, f shutoh, a. ina, s. yoshida, h. nogami, s. hisano lab. neuroendocr., graduate sch., univ. tsukuba, tsukuba, japan olfactory information is conveyed to the brain by transmission from primary olfactory neurons to mitral or tufted cells. however, little is known about development of these ob glutamatergic neurons in early postnatal life. vesicular glutamate transporters (vglut) have been used as the best histological markers to identify glutamatergic neurons. we here studied expressions of two vglut isoforms (vglut and - ) during rat ob development from postnatal day (p ) to p by in situ hybridization and immunohistochemistry. at p vglut immunoreactivity (ir) was detected in all layers except the olfactory nerve layer, and thereafter its localization expanded and intensity increased. vglut mrna signals were detectable in the mitral cell layer from p to p . in contrast, vglut ir was prominent in the glomerulus at all days examined, and only at p and p in mitral cells. despite mitral vglut ir disappeared at p , the mrna signals were still detectable. these results suggest that glutametergic neurons in the rat ob continue to develop even after birth. ps p-f v r genes multiplied in amphibian and expressed in the main olfactory system atsuko date-ito , , masumi ichikawa , yuji mori , kimiko hagino-yamagishi tokyo metrop. inst. med. sci., tokyo, japan; the univ. of tokyo, tokyo, japan, tokyo metrop. inst. neurosci., tokyo, japan in rodent, v r gene family is expressed specifically in the vomeronasal organ (vno) and is thought to be responsible for pheromone reception. however, teleost fishes lacking for the vno have a single v r gene, which is expressed in the olfactory epithelium (oe). to examine when the v rs function as pheromone receptors in the course of evolution, we analyzed the amphibian xenopus tropicalis genome, and identified v r sequences. these v rs were not expressed in the vno, but most of them were expressed in the oe of the middle cavity, which is considered for reception of water-soluble odorants. from these results, we speculate that the amphibian v rs get a chance to receive diverse odorants such as pheromones by gene multiplication and sequence diversification. our results raise the possibility that pheromonal information is transmitted via the main olfactory system. ps p-f analyses of ligand binding sites and snps on sweet taste receptor system in human noriatsu shigemura, a.a. islam, yuki nakamura, shinya shirosaki, yuzo ninomiya sect. oral neurosci., grad. sch. dent science, kyushu univ., japan recent studies have shown that t r /t r heterodimer plays a role as a sweet taste receptor. but, mice lacking t r showed diminished but not abolished behavioral and nerve responses to sugars, suggesting t r -independent sweetener binding site also exist in mice. in this study, to predict binding sites on t r /t r and/or other sweet receptor in human, we measured sensitivity thresholds to various sweet compounds and examined the qualitative similarities. we also used gymnemic acid and ␥-cyclodextrin, which selectively inhibits sweet responses and reduces the inhibitory action of it. the ten sweet compounds were classified into five groups [( ) sucrose, glcose, fructose, ( ) saccharin, aspartame, acesulfame-k, glycine, ( ) d-phenylalanine, ( ) d-tryptophan, ( ) l-proline]. in sequencing analysis, four and two snps with amino acid substitution were revealed in t r and t r , respectively. these results suggest that there may be at least five binding sites in human sweet receptor system. the individual differences in sweet sensitivities may be due to these snps. keiko yasumatsu , sachiko saito , yuko murata , ding ming , tatsu kobayakawa , robert f. margolskee , yuzo ninomiya sect. oral neurosci., grad. sch. dent. sci., kyushu univ., fukuoka, japan; saito sachiko taste and smell research institution, ibaraki, japan; national res. institute of fisheries sci., kanagawa, japan; dept. of physiol. & biophys., mount sinai sch. med., new york, usa; national institute of advanced industrial science and technology, ibaraka, japan the effect of unsaturated fatty acids on taste responses was examined by measuring perceived taste intensity in human, behavioral short-term lick responses and electrophysiological taste responses recorded from the chorda tympani and glossopharyngeal nerves in mice. the results showed that dha and other polyunsaturated fatty acids inhibit responses to bitter taste compounds without affecting other taste stimuli. we also found fatty-acid inhibition on bitter responses in an in vitro g-protein activation assay using bovine taste membrane, but lack of the bitter taste inhibition in ggustducin ko mice. these results suggest that fatty acids specifically inhibit responses to bitter stimuli by suppression of activation of t r receptors which coupled with ggustducin. ps p-f newborn infant body odor attenuates their mother's postpartum moods shota nishitani , mayumi kokuryo , tsunetake miyamura , kazuyuki shinohara div. neurobiol. & behav., nagasaki university, japan; obstet. & gynecol. of miyamura hospital, japan mothers are attracted to the body odor of newborn infants, but little is known about its reason. in the present study, we examined whether the body odor of newborn infants exert effects on moods in postpartum mothers. the body odors of newborn infants were collected from their undershirts. postpartum mothers were exposed to odors of a part of the undershirt with control odors, their own infant body odors or other infant body odors. we used the poms to assess the effects of infant body odors on postpartum moods. this study was approved by the ethics committee of nagasaki university. the infant body odors significantly increased hedonics and friendliness scores, and significantly decreased anxiety, depression and fatigue scores, whether infant odors may be originated from their own infants or other infants. these results suggest that body odors of newborn infants attract their mothers because they have calming effects on postpartum mothers. research funds: japan science and technology agency (jst), research institute of science and technology for society (ristex) ps p-f human prefrontal activity in taste encoding: an fnirs study masako okamoto , mari matsunami , haruka dan , tomoko kohata , kaoru kohyama , ippeita dan national food research institute, tsukuba, japan; nippon suisan kaisha, ltd., japan taste remains one of the least-explored human senses. using multichannel functional near-infrared spectroscopy (fnirs), we examined the lateral prefrontal cortex (lpfc) of healthy volunteers (n = ) while they tasted and encoded the quaternary taste mixtures. the contrast between the cortical activation under encoding conditions and that under control conditions without memory requirement revealed activation in the bilateral ventro-lpfc and the right posterior portion of the lpfc. the activation pattern, which was in line with those that have been associated with intentional encoding of non-verbal materials of other senses, supported an amodal role of lpfc in intentional encoding, at least at a macro structural level. this study also demonstrates that, by using fnirs, lpfc functions on taste can be examined with experimental paradigms comparable to those used for other senses. recently, we performed simultaneous respiration and electroencephalographic recordings during odor stimulation. we sought to identify changes in respiratory pattern, inspiratory phase-locked alpha oscillation (i-␣) and location of dipoles estimated from the potentials. electroencephalographic dipole tracing identified the location of dipoles from the i-␣ in the limbic area and the cortex; the entorhinal cortex, hippocampus, amygdala, premotor area and orbitofrontal cortex. in this study, we compared the respiratory pattern during odor stimulations, i-␣, dipole localizations without habituation with those with habituation of odors. onset of inspiration was used as a trigger for averaging, and potentials were averaged before and after the habituation period. habituation of odor caused to return to the normal respiratory pattern, decrease of amplitudes of ␣, and entorhinal cortex, hippocampus, amygdala were less active. akio tsuboi, takaaki miyazaki, takeshi imai dept. of biophys. & biochem., univ. of tokyo, tokyo, japan vertebrate odorant receptor (or) genes are divided phylogenetically into two distinct classes, the fish-like class i and the terrestrialspecific class ii. in the present study, we systematically analyzed mouse class i or genes ( subfamilies) to elucidate the expression profiles in the olfactory epithelium (oe) and the projection sites of their olfactory sensory neurons (osns) in the olfactory bulb (ob). in situ hybridization (ish) revealed that most class i or genes ( subfamilies) were expressed in the dorso-medial zone (zone ) of the oe. furthermore, there appeared to be no significant differences in the distributions of osns expressing class i genes within zone . these results indicate that there is a clear boundary between zone and non-zone areas in the oe. some class i ors are known to possess ligand specificity for aliphatic acids, aldehydes and alcohols. our ish analysis has revealed that osns expressing the class i ors in zone tend to converge their axons on a cluster of glomeruli in an antero-dorsal domain that is assumed to be involved in responses to the aliphatic compounds on the ob. research funds: kakenhi ( ) ps p-g taste response characteristics of putative interneurons in the rat gustatory cortex tatsuko yokota, kunihiro eguchi, katsunari hiraba department of physiology, school of dentistry, aichi-gakuin university, nagoya, japan previous studies have indicated that the extracellular spike waveforms and discharge rate properties of cortical neurons differed between pyramidal cells and interneurons, the latter tending to have narrower spike-widths and higher discharge rates. taste-sensitive neurons in the rat gustatory cortex were classified according to ( ) best-taste profiles and ( ) spike-widths which were found to form a bimodal distribution (narrow and broad). narrow-spike neurons had a significantly larger response to nacl than broad-spike neurons, but no differences were found to other tastants. the proportion of narrow-spike neurons in the n-best neurons was higher than that in the h or nh-best neurons. these results indicate that putative interneurons may play an important role in the coding of salt taste information. research funds: kakenhi ( ) of japan to t.y. yuki sato, nobuhiko miyasaka, yoshihiro yoshihara laboratory for neurobiology of synapse, riken bsi, wako, japan in the fish olfactory system, individual olfactory sensory neurons (osns) are thought to express only one or at most a few different odorant receptors (ors) from the large or family consisting of ∼ members. here, we investigated the mechanisms underlying or gene choice by using transgenic zebrafish that carried a modified bac containing a zebrafish or gene cluster. replacement of the or coding regions in the bac transgene with reporter genes allowed the reporters to be expressed in a small population of osns in the transgenic fish. in situ hybridization analysis using or-specific probes revealed that or genes expressed in reporter-positive cells were mostly restricted within the same or subfamilies to which the replaced ors belonged. additionally, the reporter-expressing osns projected their axons to a topographically fixed cluster of glomeruli in the olfactory bulb. these findings suggest the hierarchical regulation of or gene choice, whereby an individual osn may express one or gene from a limited subpopulation that is chosen from the entire repertoire in advance. research funds: kakenhi ( ) ps p-g identification of perisomatic-targeting granule cells in the mouse olfactory bulb hiromi naritsuka , kazuhisa sakai , tsutomu hashikawa , kensaku mori , masahiro yamaguchi dep. physiol. grad. sch. med., univ. of tokyo, tokyo, japan; laboratory for neural architecture, bsi, riken, saitama, japan in the olfactory bulb (ob), odor information is processed by the local circuit that includes inhibitory interneurons. granule cells (gcs) are major interneurons in the ob, but their diversity is not well understood. in the ob of adult transgenic mice expressing gfp under the control of nestin gene regulatory regions, we observed gcs with strong gfp expression (referred to as type s cells). their dendrites branched and formed spines within the granule cell layer, internal plexiform layer and mitral cell layer but did not reach the external plexiform layer, where typical gcs make synapses with dendrites of mitral and tufted cells. type s cells had huge protrusions at their dendritic ends, which formed contact with mitral cell somata. electron microscopic analysis revealed the existence of reciprocal synapses between type s cell protrusions and mitral cell somata. characteristic morphology of perisomatic-targeting gcs indicates that they have functions distinct from typical gcs in the ob. keiko moriya-ito, kentaroh endoh, yuuki ishimatsu, masumi ichikawa department of neuroscience basic technology, tokyo metropolitan institute for neuroscience, fuchu, tokyo, japan a coculture system of accessory olfactory bulb (aob) neurons and vomeronasal neurons was established for studying the functional roles of aob neurons in pheromonal signal processing. in this study, the effect of vomeronasal neurons on the development of aob neurons was examined in a coculture system. the densities of dendritic spines were lower in the coculture than in single culture. the ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. the volume of spine head was larger in the coculture than in single culture. by electron microscopic observation, the synapses on dendritic shafts were decreased and the synapses on dendritic spines were increased in the coculture. the synapses between aob neurons and vomeronasal neurons were recognized in the coculture. these observations suggest that synapse formation of aob neurons is modified by synaptic contact with vomeronasal neurons. ps p-g nacl induced responses of mouse fungiform taste cells: existence of amiloride sensitive and insensitive taste cells ryusuke yoshida, tadahiro ohkuri, keiko yasumatsu, noriatsu shigemura, yuzo ninomiya sect. of oral neurosci., grad. sch. of dental sci., kyushu univ., fukuoka, japan previous electrophysiological studies showed that the chorda tympani nerve contains two types of nacl-responsive fibers, amiloride sensitive (n-type) and insensitive (e-or h-type) fibers, suggesting the existence of amiloride sensitive and insensitive taste receptor cells in fungiform papillae. in this study, we examined nacl responses of mouse fungiform taste cells in isolated taste bud and amiloride sensitivity of them. some taste cells respond to apical restricted nacl stimulation with increase in firing frequency and their responses were concentration dependent. amiloride mixed with apical nacl solution inhibited nacl responses in some taste cells [amiloride sensitive (as) cells] but not in others [amiloride insensitive (ai) cells]. ai cells responded to other electrolytes such as kcl and hcl. these results suggest the existence of at least two types of nacl sensitive cells, as and ai cells. n-or e-type fiber may selectively innervate as or ai cells respectively. research funds: kakenhi ( ), kakenhi ( ) ps p-g integration of olfactory and oral sensory input in the rat insular cortex hideki kashiwadani, kensaku mori department of physiology, university of tokyo, tokyo, japan axonal connections between olfactory cortex and insular cortex suggest that insular cortex integrates olfactory information and information originated from the oral cavity (taste, tactile, temperature). however cellular mechanisms underlying the integration of multimodality are poorly understood yet. in this study, we examined single-unit spike responses of insular cortical neurons to odor stimulation and intraoral water stimulation in urethane-anesthetized rat. we found that more than % of recorded neurons in the insular cortex responded to odors. about half of the odor-responsive neurons were activated by intraoral water stimulation, indicating the convergence of olfactory and oral sensory information onto individual neurons in the insular cortex. when odor stimulation and intraoral water stimulation were simultaneously applied, some neurons showed spike responses larger than the responses evoked by each stimulus. the integration of olfactory and oral sensory information in the insular cortex might contribute to form the flavor sensation. research funds: kakenhi ( ) ps p-g odor combination selectivity of the rat piriform cortex neurons ikue yoshida, kensaku mori dept. physiol. grad. sch. med., univ. of tokyo, tokyo, japan olfactory cortex is thought to integrate signals from different odorant receptors to form the olfactory image of objects. however, the manner of integration at the level of individual cortical neurons is not well understood yet. using single-unit recording method, we examined the response selectivity of individual neurons in a dorsocaudal part of the anterior piriform cortex (apc) to classes of odorous compounds, each class being present in odors from many different vegetables and fruits. individual neurons typically responded to more than classes of odorants. each neuron was uniquely tuned to a specific combination of odorant classes, and different neurons typically showed different odor combination selectivity. single-unit responses to odor mixtures showed mixture facilitation and mixture suppression. these results suggest that individual neurons in the apc can be characterized by the odor combination selectivity and that the apc neurons may integrate signals from different odorant classes. research funds: kakenhi ( gs ) ps p-g odor-driven activity in the anterior piriform cortex of an in vitro isolated whole brain with the olfactory epithelium takahiro ishikawa , takaaki sato , akira shimizu , ken-ichiro tsutsui , toshio iijima div. of systems neuroscience, grad. sch. of life sciences, univ. of tohoku, sendai, japan; res. inst. for cell engineering, aist, amagasaki, japan to examine the neural mechanisms underlying odor-induced response in the anterior piriform cortex (apc), we analyzed odorinduced local field potential (lfp) and multiunit activity in an in vitro preparation, isolated guinea-pig whole brain with the olfactory epithelium. in apc, odor-induced lfps consisted of a phasic initial component followed by a fast oscillatory activity in the beta range ( hz). by comparison a result of current source-density analysis with unit activity data, we confirmed that the initial component of odor-induced response has a characteristic temporal pattern, generated by a relatively weak direct afferent input, followed by an intracortical associative response, which was associated with a phasic inhibition. the beta oscillation might be generated by the repetition of these network activities. these electrophysiological data were consistent with the results of previous studies that used slice or anesthetized in vivo preparations. ps p-g chemotaxis of c. elegans to concentration gradient of an attractant superimposed on a uniformly distributed attractant lin lin, hiroyuki oikawa, miyako sasaki, tokumitsu wakabayashi, ryuzo shingai department of welfare engineering, iwate university, morioka, japan to investigate the informational interaction between pathways from different sensory inputs to the behavior in the nervous system of c. elegans, chemotaxis toward the concentration gradient of an attractant spotted on a uniformly distributed another attractant was investigated. lysine and chloride ions are water soluble chemoattractants. when m lysine was spotted on ammonium chloride background, . - . m and . m background did not influence lysine chemotaxis, while . m background augmented and . - . m background suppressed the chemotaxis. in contrast, when . m ammonium chloride was spotted on the lysine background, the background did not alter or suppressed the chemotaxis. interaction between informational pathways from different sensory inputs could be seen also in the presentation of an odorant spotted on chemoattractant background, and vice versa. ps p-g glutamate receptors are regulated by the ras-mapk pathway in neural circuit-dependent odor adaptation in c. elegans takaaki hirotsu , , , takeshi ishihara , eisuke nishida , yuichi iino dept. biol., fac. sci., kyushu univ., japan; mol. genet. res. lab., univ. of tokyo, japan; grad. sch. biostudies., kyoto univ., japan c. elegans shows a decrease in chemotaxis to odorants after exposure to the odorant for min. this plasticity, called early adaptation, requires aiy interneurons, which receive synaptic inputs from olfactory neurons, indicating that early adaptation depends on neural circuit. the ras-mapk pathway is activated by odorant exposure in aiy and plays essential roles for early adaptation. the function of glr- , a non-nmda type glutamate receptor, in aiy is also important for early adaptation. glr- appears to localize at postsynaptic sites in aiy. this localization was changed by odorant exposure in early adaptation. mutation of the ras-mapk pathway impaired localization of glr- . in vitro kinase analyses revealed the possibility that mapk directly phosphorylates glr- . these results suggest that the ras-mapk pathway controls odor adaptation by directly regulating glr- localization in aiy neurons. kohei ueno , yoshiaki kidokoro dept. behav. sci., grad. sch. med., gunma univ., maebashi, japan; inst. mol. cel. reg., gunma univ., maebashi, japan sodium chloride (nacl) is the major substance that induces nacl taste. in rodents, some strains prefer nacl solutions (∼ %), but others do not or even avoid them. although it is reported that the difference is based on the genetic background, the molecular information involved in the difference is not known. in the th ns annual meeting, we have shown that nacl preference in several wild-type strains of drosophila melanogaster is variable and p-element insertion in a single gene suppressed nacl preference. here, we carried out the sequencing analysis and found eight single-nucleotide polymorphisms (snps) in the gene. moreover, we found that one of the snps was correlated with nacl preference among wild-type strains. we generated transgenic flies and rescued the low preference phenotype of p-element insertion strain using the gal /uas system. finally, we examined the expression pattern of the gene and found the gene is expressed in taste organs. taken together, we suggest that the gene is a novel nacl receptor gene. ps p-g spatial and temporal organization of odor representation by moth antennal lobe output neurons shigehiro namiki , graduate school of life and environmental sciences, university of tsukuba, ibaraki, japan; department of mechano-informatics, graduate school of information science and technology, university of tokyo, tokyo, japan the antennal lobe (al) is the first relay station for olfactory information in the insect brain and is the anatomical equivalent of the mammalian olfactory bulb. both systems have common structures called glomeruli, functional units of olfactory processing. odor-evoked spatial and temporal patterns by an array of glomeruli are both important in olfactory coding. but the details of olfactory coding mechanisms are still unclear. we confirmed that projection neurons (pns, al output cells) innervating the same glomerulus had similar olfactory responses in the silkmoth. by pooling data from many pns that innervate identified glomeruli i reconstructed odor representations. i found that olfactory information is encoded by distributed spatiotemporal activity of a pn population and that there are no clear correlation between the similarity of slow temporal patterns of pns and spatial distances of innervating glomeruli. research funds: brain ps p-g medial nucleus amygdala neurons have morphologically and electrophysiologically heterogeneous properties makoto yokosuka , yoshinori sahara , shinichiro horie , masumi ichikawa , shun nakamura st. marianna univ. schl. med., kawasaki, japan; ntl. inst. neurosci., ncnp, tokyo, japan; tokyo metropol. inst. neurosci., tokyo, japan we characterize the electrophysiological and morphological properties of the medial nucleus amygdala (mea) neurons using whole-cell recordings in mice slice preparations. most mea neurons showed either tonic-bursting or adapting burst of action potentials to deporalizing currents. biocytin labeling showed that mea neurons possessed bipolar to multipolar cell bodies and dendritic fields covering projection areas from the accessory olfactory bulb. norepinephrine increased the frequency of spontaneous ipscs in some neurons, while serotonin increased spontaneous epscs in others. morphologically and physiologically heterogeneous mea neurons seem likely to produce multiplex outputs of many instinct behaviors. hideyuki matsumoto, kensaku mori department of physiology, graduate school of medicine, university of tokyo, tokyo, japan olfactory sensation sometimes lasts even after odorant stimulation has ceased. neuronal mechanisms for the olfactory afterimage are not well understood yet. single unit recordings from mitral/tufted cells in the mouse olfactory bulb (ob) showed that some neurons continued to discharge for more than s even after the cessation of odorant stimulation. the induction of the sustained spike discharge depended on the intensity of odorant stimulation, and showed an allor-none behavior. spike discharges during the sustained discharge mode phase-locked to the respiration cycle and the phase-locking pattern during the sustained discharge mode differed from that during odor stimulation. these results suggest that neuronal mechanism in the ob may be responsible for the induction of the post-stimulus sustained discharges. the respiratory-phase-locked sustained discharges were recorded from juxta-glomerular cells. this implies that neuronal interactions within the glomeruli are involved in the induction of the sustained spike activity of mitral/tufted cells. ps p-g synaptic transmission shows state-dependent change in the urethane-anesthetized rat olfactory bulb yusuke tsuno, hideki kashiwadani, kensaku mori department of physiology, graduate school of medicine, the university of tokyo, tokyo, japan olfactory cortex (oc) shows a state-dependent sensory gating that is controlled under the modulatory inputs from the basal forebrain and brainstem. since the olfactory bulb (ob) receives the modulatory inputs heavily, neuronal activity in the ob might change in a state-dependent manner. in the present study, we demonstrate a clear state-dependent change in the magnitude of the transmission of granule-to-mitral dendrodendritic inhibitory synapses and olfactory cortex-to-granule excitatory synapses. transmission of granule-tomitral synapses and olfactory cortex-to-granule synapses was facilitated during slow-wave state and suppressed during fast-wave state. in addition, we observed synchronous slow oscillations (about hz) in the granule cell layer of the ob, layer iii of the oc, and the occipital cortex. thus the ob shows state-dependent synaptic modulation and presumably receives top-down periodic signals from the cortex. research funds: kakenhi ( ) ps p-g rem sleep deprivation decreases na-k atpase phosphorylation gitanjali das, birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india it has been hypothesized that "one of the functions of rem sleep is to maintain brain excitability" rem sleep deprivation increases noradrenaline in the brain that increases the na-k atpase activity causing increased brain excitability. however, the molecular mechanism of such increased na-k atpase activity was unknown; although it was known that dephosphorylated state is the active form of na-k atpase. rats were rem sleep deprived by flower-pot method; large platform and recovery from lost rem sleep were carried out as controls. at the end of experiment, brains were quickly removed by cervical dislocation and synaptosomes prepared, which were used for western blotting against phosphoserine and phosphothreonine antibodies as well as for na-k atpase activity. after rem sleep deprivation the activity increased, while the level of phosphorylated form of na-k atpase decreased in the same sample. this confirms our hypothesis that rem sleep deprivation induced increased activity is due to dephosphorylation of na-k atpase. research funds: icmr (govt. of india) and upoe (govt of india) takeshi fujii , , ken yoshikawa , yuki takatori , koichiro kawashima dept. of pharmacol., fac. of pharmaceut sci., doshisha women's coll., japan; dept. of pharmacol., kyoritsu univ. of pharmacy, japan stimulation of muscarinic (machr) and nicotinic (nachr) receptors with respective agonists induces ca + signals in t cells. in the present study, using rna interference approach, we investigated roles of machr and nachr subtypes in ca + signals in ccrf-cem (cem) cells, a human t cell line, as a model of t cells. cem cells express m , m , m and m machr subtypes, and ␣ , ␣ , ␣ , ␣ , ␣ , ␣ and ␤ nachr subunits. transfection of anti-m , anti-m and anti-␣ small interfering rna (sirna) significantly down-regulated respective mrna expression, while no changes were observed in gene expression of other machr subtypes or nachr subunits. ca + signals evoked by oxotremorine-m, a non-selective machr agonist, were reduced by anti-m or anti-m sirna. ca + signals evoked by nicotine were reduced by anti-␣ sirna. these findings indicate that m , m machr and ␣ nachr subtypes play major roles in ca + signals to acetylcholine in t cells, and suggest that these receptors are involved in regulation of immune function. research funds: kakenhi ( ) ps p-g is "seronegative" mg explained by autoantibodies to musk? kazuhiro shigemoto , sachiho kubo , seiji matsuda , naoki maruyama dept. of preventive medicine, ehime univ. schl. of med., ehime, japan; dept. of mol. path., tokyo metro inst. for gerontology, tokyo, japan; dept. of integrated basic medical science, ehime univ. schl. of med., ehime, japan muscle-specific kinase (musk) is critical for the synaptic clustering of nicotinic acetylcholine receptors (achr). musk is activated by agrin, which is released from motoneurons, and induces achr clustering at the postsynaptic membrane. although autoantibodies against the ectodomain of musk have been found in a proportion of patients with generalized myasthenia gravis (mg), it is unclear whether musk autoantibodies are the causative agent of generalized mg. in the present study, rabbits immunized with musk ectodomain protein manifested mg-like muscle weakness with a reduction of achr clustering at the nmj. the autoantibodies activated musk and blocked achr clustering induced by agrin or by mediators that do not activate musk. thus, musk autoantibodies rigorously inhibit achr clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of mg. (shigemoto et al., j. clinical investigation, ) research funds: kakenhi ( ) ps p-g dynamic changes in the thalamo-cortical system associated with thalamic neurodegeneration shin-ichi kyuhou, hisae gemba department of physiology, kansai medical university, japan in purkinje cell degeneration (pcd) mice, degenerating thalamic neurons were found morphologically in the particular thalamic nuclei including the ventral medial geniculate nucleus around postnatal day . electrophysiologically, auditory evoked potentials in the primary auditory cortex began to decrease gradually in amplitude from postnatal day . analysis of spontaneous cortical field potentials by fast fourier transform, revealed that high frequency oscillation (hfo) of around hz appeared prominently in the auditory cortex. local injection of kynurenic acid, a glutamate receptor blocker, into the thalamus suppressed the hfo in the auditory cortex, indicating that the thalamus is involved in the generation of the hfo. the real time polymerase chain reaction analysis demonstrated the upregulation of the mrna of nmda receptors in the auditory cortex. these results suggested dynamic changes occurred in the thalamo-cortical system after thalamic neurodegeneration in pcd mice. research funds: grant c from kansai medical university ps p-h unusually folded sod species sequester specific motor molecules and inhibit the axonal transport of their cargos minako tateno , yumiko simazaki , fuminori saitoh , ryosuke takahashi , toshiyuki araki national institute of neuroscience (ncnp), tokyo, japan; dept. of neurology, kyoto university, kyoto, japan misfolding of mutant sod protein is thought to be responsible for the selective loss of motoneurons in sod -related familial amyotrophic lateral sclerosis (als), although the molecular mechanisms underlying the toxicity of such unusually folded sod species are not yet clarified. since we have detected accumulation of unusual sod species in motoneuronal axons from g a sod -tg mice, we fractionated the ventral white matter of spinal cords to isolate the unusual sod species. immunoprecipitation analyses revealed specific interaction of unusual sod species with certain kinds of motor molecules. moreover, the axonal transport of cargos mediated by those molecules was found to be significantly reduced in symptomatic mutant sod -tg compared with wt sod -tg mice. these data strongly suggest that the toxic property of unusual sod proteins is partially ascribable to the transport inhibition of specific cargos. research funds: grant-in-aid for scientific research c ( ) ps p-h relationship between the amount of the cathepsin d expression and the symptomatic manifestation of neuronal ceroid-lipofuscinosis in a mouse model masahiro shibata, masato koike, yasuo uchiyama department of cell biology and neuroscience, osaka university graduate school of medicine, japan mice deficient in cathepsin d (cd), a representative lysosomal aspartic proteinase, have been shown to be an excellent model of neuronal ceroid-lipofuscinosis (ncl). here we report that the phenotype of mice in which cd is partially expressed is decided depending on the amount of the protein expression of cd. the proteolytic activity and protein expression of cd in the mutant mice were approximately % of those in the wild-type mice, while the growth of the mice appeared intact until postnatal day . the mice started to show ncl symptoms on p , and their life span was prolonged for one to three days, compared to that of the cd-null mice. the protein expression of cd in the heterozygous mice was approximately half of that in the wildtype mice and the mice showed no pathological finding. these results indicate that a threshold of the cd expression required for the manifestation of ncl symptoms in the mice may be present in the range from % to % of that in the wild-type mice. research funds: kakenhi ( ) ps p-h neuronal toxicity of expanded polyglutamine depends on intracellular distribution among cells with similar expression levels mamoru satoh, atsuyoshi shimada, noriko kawamura, yoichi chiba, yuko saitoh, hiromi keino, masanori hosokawa dept. pathol., inst. develop. res., aichi human service center, aichi, japan we previously reported that expanded polyglutamine (polyq) tracts induced cellular toxicity of neuro a cells in the form of massive cytoplasmic aggregates but not of intranuclear inclusion. however, we did not rule out the possibility that such toxicity depends on the level of intracellular expression of polyq. in this report, we compared the toxicity of polyq among cells expressing polyq tracts with a variety of intracellular distribution but at similar expression levels. damages were most remarkable in cells with cytoplasmic massive aggregate in terms of shrunken cellular and nuclear sizes. cells with cytoplasmic homogeneous distribution, cytoplasmic punctate distribution and intranuclear inclusion of polyq tracts were relatively spared. these data suggest that the severity of cell damages depends on the type of intracellular distribution of polyq tracts in cells expressing polyq tracts at similar level. ayumi takamura , katsumi higaki , junichiro matsuda , yoshiyuki suzuki , eiji nanba division of functional genomics, research center for bioscience and technology, tottori university, tottori, japan; national institute of biomedical innovation, osaka, japan; clinical research center, international university of health and welfare, tochigi, japan g m -gangliodisosis is an autosomal recessive lipid storage neurodegenerative disorder. due to a deficiency of lysosomal ␤-galactosidase, excessive lysosomal accumulation of gm is observed in patients and animal model brains. however pathogenesisi of this disease is still unclear. since gm is known to be a major sialoglycolipid constituent of plasma membrane (pm) in neuron, we examined the analysis of brain of mouse model. cerebellar granule cells from this mouse showed gm accumulation of lysosome and pm and the membrane fluidity was also reduced. gm -bound phosphorylated trka was markedly decreased in cultured neuron and brain tissues. subsequent plc␥, known as a downstream signal of trka, was also impaired. these results suggest that dysfunction of neurotrophin signaling may cause the onset of neurodegeneration in g m -gangliosidosis. katsuya inoue , , katsuaki endo , takamitsu fujikawa , seijyun fukuda , tatsuo nakamura department of physical therapy, university of aino, osaka, japan; institute for frontier medical science, kyoto university, kyoto, japan regeneration of spinal cord injury is an important thema in rehabilitation science as well as basic one. the experiment was designed to reveal the process after spinal cord injury by asphyxia. to establish the animal model of spinal cord injury produced by asphyxia, we used adult cats with aorta occulusion under deep pentabarbital anesthesia. twenty minutes after occulusion electrical reflex activity of spinal cord disappeared. after min occulusion, irreversible functional changes were observed, long term depression of reflex activities and disorders of motorsensory function. we also traced time course of electrical and functional changes after min occulusion. ps p-h development of a rodent behavioral model to study the direct interactions of reward and learning adam weitemier, niall p. murphy riken brain science institute, japan cognitive and reward processes often occur simultaneously, and perhaps interdependently. learning is a necessary condition in many experimental models aimed at assessing the rewarding value of a given stimulus. conversely, reward is often used as an experimental tool to engage mnemonic processes in studies aimed at investigating learning and memory. recent studies have demonstrated shared neurobiology between memory and reward. a direct behavioral interaction between reward and memory has never been studied. cognitive impairments observed in psychiatric conditions of dysregulated reward, such as drug abuse and depression, make this issue important, particularly in light of ongoing efforts to investigate higher brain functions. we are developing a rodent behavioral model with which to directly assess the influence of reward processes on learning and memory. we will introduce our recent progress with this new model, including two variations of the procedure designed to study the influence of reward on memory acquisition and memory recall. tetsuya ando , yuya kawanaka , minoru saito , hiroaki mochizuki , ken honjo , hirofumi toda , , toshifumi tomoda , akira sawa , katsuo furukubo-tokunaga grad. school of life & envir. sci., univ. tsukuba, japan; molecular physiol., tokyo metropolitan inst. neurosci. tokyo, japan; beckman res. inst., city of hope. california, usa; dept. of psych. & neurosci. johns hopkins univ. school of medicine. baltimore, usa the disrupted-in-schizophrenia- (disc ) gene, originally identified at the breakpoint of a chromosome ( ; ) (q . ; q . ) translocation in a scottish schizophrenia pedigree, is a promising candidate gene for schizophrenia and affective disorder. however, cellular and molecular mechanisms underlying cognitive impairments are yet to be elucidated. to address disc functions in vivo, we expressed disc in drosophila and examined developmental and behavioral phenotypes. overexpression of disc resulted in marked suppression of olfactory associative learning in flies whereas it caused no symptoms of neural degeneration even in aged animals. we anticipate that the drosophila system will serve as a novel model system amenable to a variety of genetic manipulations for the study of schizophrenia. ps p-h effect of hypothermia on discrepancy between memory learning ability and anatomical brain damages in rats with neonatal hypoxic ischemic encephalopathy yuji miyatake , ayumi kamo , kenji minato , hitoshi haruna , hiritsugu fukuda , yuji murata , takayoshi hosono department of bomedical engineering, osaka electro-communication university, japan; graduate school of medicine, osaka university, japan we investigated the effect of brain hypothermia on neonatal hypoxic ischemic encephalopathy (hie) in hie-model rats using olton t-maze and anatomy. the common carotid artery of of -day-old rats was ligated and cut under anesthesia. after the operation the rats were put in a box containing % oxygen at • c for min. after the insult, of the rats were put in a box at • c for h (hypothermia, h-group). the other rats were returned to their mother without hypothermia (normothermia, n-group). sham operations were performed on three rats (s-group). eight weeks after the operation, their learning and memory ability was assessed by olton t-maze, and no statistical difference was observed in either the working or reference memory in the three groups although the anatomical brain size in the n-group was significantly smaller than in the h-group and s-group. withdrawn ps p-h tau hyperphosphorylation in ts cje, a partial trisomy mouse model for down syndrome ebrahim abdul , a. shimohata , w. yu , m. yamaguchi , m. murayama , d. chui , t. akagi , t. takeuchi , k. amano , h.s. karthik , t. hashikawa , h. sago , c.j. epstein , a. takashima , k. yamakawa research scientist; lab. for neural arch.; lab. for alzheimers disease; div. of fetal med. ncchd; ucsf, usa although down syndrome (ds) or trisomy is the most common genetic cause of mental retardation, its neuropathology remains unclear. ts cje, a ds mouse model partially trisomic for chromosome , shows learning and behavioral abnormalities mimicking ds mental retardation. the trisomic segment, corresponding to parts of human chromosome q , has about genes. importantly, sod and app, which may contribute to the ds phenotype, are excluded from the ts cje trisomic segment. here we report that ts cje brains show hyperphosphorylation of tau in the absence of nft formation, as well as increased gsk ␤ and jnk/sapk activities without alterations in a␤pp metabolism. our results suggest that genes on the trisomic ts cje segment other than app and sod can cause hyperphosphorylation of tau, which in turn may be critical in the pathogenesis of ds mental retardation. research funds: kakenhi number: ps p-h increased oxidative stress and mitochondrial dysfunction in ts cje, a down syndrome mouse model atsushi shimohata , ebrahim a. s. , m. yamaguchi , w. yu , h. sago , c.j. epstein , k. yamakawa lab. for neurogenetics, riken-bsi, japan; div. of fetal med. ncchd, japan; dept. pediatrics, ucsf, usa down's syndrome (ds), caused by chromosome (hsa ) trisomy, is the most common genetic cause of mental retardation and affects every major organ in the body. ts cje is one of a number of segmentally trisomic ds mouse models, and is triplicated for a region of mouse chromosome extending from sod to znf , containing genes syntenic with hsa . since these mice show learning and behavioral abnormalities mimicking ds mental retardation, ts cjespecific trisomic segment genes may be involved in the ds phenotype. in the present study, we observed increased levels of reactive oxygen species (ros), mitochondrial function impairment in primary cultured astrocytes and hippocampal neurons, and increased cabonylated proteins in ts cje brains. collectively, our results implicate dosage imbalanced genes other than sod and app in both ros generation and mitochondrial dysfunction, which in turn possibly contribute to the ts cje ds mental retardation-like phenotype. ps p-h polyinosinic-polycytidylic acid injection in early pregnancy causes the hypomyelination in the hippocampus, but not in the cortex manabu makinodan , , kouko tatsumi , takayuki manabe , takahira yamauchi , , eri makinodan , juro shimoda , toshifumi kishimoto , akio wanaka department of psychiatry, nara medical university, kashihara, japan; department of nd anatomy, nara medical university, kashihara, japan polyinosinic-polycytidylic acid (poly i:c) elicits maternal immune response similar to anti-viral ones. recent studies demonstrated that poly i:c injection into pregnant mice resulted in behavioral changes including deficits in prepulse inhibition in the offspring, rendering this system an animal model of schizophrenia. in the present study, we observed such behavioral abnormalities reproducibly in the experimental group born from poly i:c-injected mice, but not in the control group born from pbs-injected mice. they showed decreased myelination in the hippocampus at juvenile period with unaltered number of oligodendrocytes. on the other hand, myelination in the cerebral cortex did not significantly differ between the experimental and control mice. the hypomyelinaton in the hippocampus at the juvenile period may be a possible cause for the behavioral changes in later periods. joanna doumanis, ritsuko kazama, adrian moore, nobuyuki nukina riken brain science institute, japan the fruitfly drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. to model the polyglutamine expansion disease, huntington disease (hd), we have established stable, inducible cell lines expressing n-terminal truncated huntingtin fused to egfp with an expanded ( q) polyglutamine repeat in a drosophila larval central nervous system-derived cell line. induction of expression results in the formation of protein aggregates, characteristic of hd. utilising rnai, we have carried out a high-throughput screen for modifiers of aggregate formation in these cells. genes, encompassing around % of the drosophila genome, were screened, resulting in the identification of candidates that either suppress or enhance aggregation. most candidates identified have mammalian orthologues, validating the use of drosophila to screen for genes relevant to human disease. we established in vivo models of hd by expressing polyq-egfp in the drosophila nervous system and are further characterising selected candidates in our model. the rodent model of harmaline-induced tremor has been used as an animal model of essential tremor. the present study investigated effects of harmaline on olivocerebellar systems of mice and rats. systemic administration of harmaline produced generalized tremors in both types of rodents. immunohistochemical studies revealed significant degeneration of purkinje cells that was associated with activated microgliosis in the cerebellar cortex, following administration of harmaline in rats but not in mice. however, in mice but not rats, microgliosis was induced following administration of harmaline in the inferior olivary nucleus (ion). numbers of neurons in the mouse ion did not decrease, suggesting the possibility that microgliosis in ion might not be a simple neurotoxic effect. presumably, differences in sensitivity of purkinje cells between rats and mice may be related to differences in functional alterations in their respective olivocerebellar systems induced by harmaline. recognition of these species-specific differences is an important consideration for experimental analysis of the rodent model of tremors. ps p-h analysis of ␣-synuclein expression in young mouse model of multiple system atrophy kimiko nakayama, yasuyo suzuki, ikuru yazawa laboratory of research resources, national institute for longevity sciences, aichi, japan multiple system atrophy (msa) is a sporadic neurodegenerative disease that affects oligodendrocytes and neurons in human central nervous system. glial cytoplasmic inclusions (gcis) are diagnostics of msa. gcis are shown to be abnormal accumulation of filamentous ␣-synuclein. yazawa et al. ( ) generated a transgenic (tg) mice overexpressing human wild-type ␣-synuclein in oligodendrocytes under the control of the , ,-cyclic nucleotide -phosphodiesterase (cnp) promoter. tg mouse study demonstrated that formation of gci-like ␣-synuclein inclusions in the oligodendrocyte leads directly to neuronal degeneration, as shown by motor impairment and novel accumulation of mouse ␣-synuclein in neuron. to elucidate the mechanisms of neurodegeneration in tg mice, we prepared primary cultures of neurons and glial cells from tg mice. the cells are examined the effects of ␣-synuclein accumulation. ps p-h dysregulation of sodium channel ␤ subunit by expanded polyglutamine in huntington disease transgenic mice fumitaka oyama, haruko miyazaki, kazumasa okamura, yoko machida, kurosawa masaru, takashi sakurai, nobuyuki nukina laboratory for structural neuropathology, riken bsi, wako-shi, japan sodium channel ␤ (␤ ) is a very recently identified auxiliary subunit of the voltage gated-sodium channels. we have identified ␤ as an est that was significantly downregulated in the striatum of hd model mice and found that reduction in ␤ started at a presymptomatic stage of the hd model mice. in contrast, spinal cord neurons, which generate only negligible levels of expanded polyq aggregates, maintained normal levels of ␤ expression even at the symptomatic stage. expanded polyq with nls expression suppressed the promoter activity of ␤ gene in pc cells. forskolin, an activator of the camp/pka pathway, did not affect b promoter activity, indicating that ␤ is not camp-responsive gene. these findings strongly suggest that sodium channel ␤ subunit is a novel molecule, which is an upstream non-camp-responsive gene in hd pathogenesis. ps p-h repeat length-and age-dependent changes in behavioral phenotypes of drpla transgenic mice harboring a single copy of a full-length human drpla gene kazushi suzuki , yuji takahashi , jun goto , mutsuo oyake , toshiya sato , shoji tsuji department of neurology, the university of tokyo, tokyo, japan; department of neurology, brain research institute, niigata university, niigata, japan; center for bioresource-based research, brain research institute, niigata university, niigata, japan we carried out detailed analyses of the behavioral phenotypes of drpla transgenic mice carrying an expanded cag repeat of (q ), (q ), (q ), or (q ). in the accelerating rotarod ( w), the latencies of q , q , q and q were %, %, % and %, respectively. in the open field, moving distances of q , q , and q were decreased to %, %, and %, respectively, while that of q was increased to %. home cage activity was decreased depending on the repeat length. the q mice, however, showed increased ratios of the activity during the light time to that during the total day at weeks ( %) and weeks ( %), suggesting that drpla mice display not only impaired motor coordination, but also changes in emotional behavior, and disrupted night and day activity patterns. ps p-h the mice lacking schnurri- show multiple behavioral abnormalities related to psychiatric disorders keizo takao , nobuyuki yamasaki , keiko toyama , tsuyoshi takagi , shunsuke ishii , tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan; riken, tsukuba, japan schnurri- (shn- ) is a zinc finger transcription factor, a mouse homologue of human hiv-ep , that binds to nuclear factor kappa b-binding site in the hiv long terminal repeat. shn- is known to play important roles in the mammalian immune systems. however, the role of shn- in the central nervous system (cns) is still unknown. to investigate the functional significance of shn- in mammalian brain, we analyzed the shn- knockout (ko) mice using a comprehensive behavioral test battery. shn- ko mice were dramatically hyperactive under novel environment and in their home cage. they also showed increased acoustic startle response and impaired prepulse inhibition, indicating their impairment in sensorimotor gating. anxiety-like behavior and depression-like behavior were also significantly reduced in shn- mice. our results demonstrate a critical role of shn- in cns and suggest that shn- ko mice may serve as an animal model of psychiatric disorders. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive brain-behavior phenotyping of camkii␣ heterozygous knockout mice nobuyuki yamasaki, koichi tanda, keiko toyama, yasuyuki fukui, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan ca + /calmodulin-dependent protein kinase ii (camkii) is a ubiquitous serine/threonine protein kinase that is abundant in brain as a major constituent of the postsynaptic density and critically involved in synaptic plasticity, learning and memory. several behavioral abnormalities of camkii␣ mutant mice were reported, but systematic assessments of behaviors of camkii␣ mutant mice have not been conducted. to analyze the behavioral effects of camkii␣ deficiency, we subjected camkii␣ heterozygous knockout mice to a comprehensive behavioral test battery. the mutant mice showed hyperactivity, decreased anxiety, decreased depression-related behavior, increased offensiveness, selective spatial working memory deficit, and dramatic periodic change of locomotor activity in home cage. to identify the mechanism underlying these behavioral abnormalities, gene expression analysis was conducted. the potential involvement of camkii␣ in pathogenesis/pathophysiology of psychiatric disorders will be discussed. research funds: kakenhi ( , , , ) , jst bird ps p-h effects of various factors on the results of a comprehensive behavioral test battery for genetically engineered mice: a factor analytic study hiroshi ougino, nobuyuki yamasaki, koichi tanda, keiko toyama, keizo takao, tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan we have been using a behavioral test battery to reveal unknown phenotypes of genetically engineered mice. for the adequate experimental design and interpretation of data, it is essential to know experimental variables which may potentially influence results, and various kinds of factors which underlie many indices measured in the tests. in this study, we investigated the effects of background strains (c bl/ j, c bl/ n, c bl/ c, svev, balb/c), body weight, age at test, and start time of test on the results of each test, by analyzing data of more than mice (, including wild type and mutant mice from strains of genetically engineered mice), which had been tested in our laboratory. also, we conducted factor analyses of a large set of data to examine the relationship between behavioral indices. the potential implications of our findings for the improvement of the behavioral test battery will be discussed. calcium-and calmodulin-dependent protein kinase iv (camkiv) is a protein kinase that activates the transcription factor, camp responseelement binding protein (creb). camkiv has been hypothesized to play a significant role in synaptic plasticity and in learning and memory. however, functions of camkiv in a variety of behaviors, e.g., motor function, nociception, fear, anxiety, depression, learning and so on, have not yet been fully elucidated. to gain more insight into behavioral significance of camkiv, we subjected camkiv−/− mice to a battery of behavioral tests. camkiv−/− mice did not display any deficit in spatial reference memory and working memory tests, but had mild performance deficit in fear conditioning tests. these results indicated selective and specific involvement of camkiv in regulating emotional behavior. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive behaivoral analysis of ryanodine receptor type knockout mouse suzuko ohsako , koichi tanda , , nobuyuki yamasaki , keiko toyama , hiroshi takeshima , tsuyoshi miyakawa kyoto university graduate school of medicine, kyoto, japan; dep. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan; dep. of biochem. and mol biol., tohoku univ. graduate school of medicine, miyagi, japan ca + signaling is essential for the regulation of neuronal processes including synaptic transmission and transmitter release. ryanodine receptors (ryrs) are family of intracellular calcium channels and mediate calcium-induced calcium release from the endoplasmic reticulum. ryr is highly expressed in the hippocampus, caudate putamen, and thalamus. to investigate the behavioral effects of ryr deficiency, we subjected ryr knockoout mice to a battery of behavioral tests. ryr knockout mice exhibited hyperactivity and abnormal behavior in social interaction test, while they did not show any deficit in motor function, depression, attention, and working memory tests. these results suggest a role of ryr in regulating general locomotor activity and social behavior. research funds: kakenhi ( , , , ) , jst bird ps p-h comprehensive behavioral analysis of neuronal nitric oxide synthase knockout mouse keiko toyama , koichi tanda , , nobuyuki yamasaki , tsuyoshi miyakawa hmro, kyoto university graduate school of medicine, kyoto, japan; dept. of pediatrics, kyoto prefectural univ. of medicine, kyoto, japan nitric oxide (no) plays several important roles in the brain, including in regulation of synaptic signaling and plasticity. no is synthesized from the amino acid l-arginine by the enzyme nitric oxide synthase (nos). in neurons, no is produced by neuronal nitric oxide synthase (nnos), representing one of three nos isoforms expressed in most tissues. to elucidate function of nnos/no in a variety of behaviors, e.g., activity, motor function, nociception, attention, anxiety, depression, social interaction, learning and so on, we subjected nnos knockout mice to a battery of behavioral tests. nnos knockout mice exhibited increased locomotor activity and decreased depressionrelated behavior. furthermore, they displayed increased social contacts in novel environment and homecage. these results indicate that nnos/no is involved in regulation of their behaviors. research funds: kakenhi ( , , , ) , jst bird ps p-h primate model of attention-deficit/hyperactivity-disorders (adhd) shintaro funahashi , keiko shimizu grad. sch. human and environmental std, kyoto univ., kyoto, japan; primate res. inst., kyoto univ., inuyama, japan adhd is one of the prevalent childhood psychiatric disorders. children with adhd show hyperactive behavior and attention problems, suggesting prefrontal (pfc) contribution to adhd. adhd is also known as dopamine (da) related dysfunctions, because methylphenidate is the most effective drug for the treatment of adhd. pfc is the cortical area where the strongest da innervation is observed. injection of da-related drugs to pfc produces behavioral deficits in cognitive tasks. these suggest that da-related dysfunction in pfc could be a candidate of biological causes of adhd. to prove this notion, we injected -ohda into bilateral pfc to destroy da innervation in infant monkeys and examined whether these monkeys exhibited hyperactivity. -ohda injected monkeys showed significant increase of spontaneous activity in test cages. oral administration of methylphenidate reduced spontaneous activity in -ohda injected monkeys. these results suggest that monkeys injected -ohda into pfc are good candidates of the primate model of adhd. research funds: kakenhi ( ) ps p-i training-induced recovery of precision grip after primary motor cortex damage in the adult monkey yumi murata , , , noriyuki higo , , takao oishi , , , akiko yamashita , keiji matsuda , motoharu hayashi neurosci. res. inst, aist, tsukuba, japan; grad. sch. compreh. hum. sci., univ. of tsukuba, tsukuba, japan; crest, jst, kawaguchi, japan; dept. cell mol. biol., primate res. inst., kyoto univ., inuyama, japan; div. applied sys. neurosci., nihon univ. sch. med., tokyo, japan in the present study, we compared the motor recovery between monkeys that received daily training and that did not receive any training after lesion of the primary motor cortex (mi), in order to investigate the effects of postlesion training on motor recovery. we derived a hand representation map in mi, and ibotenic acid was then injected to destroy the digit region, which resulted in hand paralysis. after one or two months of postlesion training, skilled use of the affected hand including a precision grip was recovered. untrained monkeys also became able to grasp objects with their affected hand, but they couldn't use a precision grip. this suggests that recovery of precision grip requires postlesion training. research funds: a grant-in-aid for scientific research on priority areas from mext ( ) mouse mutants with behavioral abnormality are indispensable tools to elucidate molecular pathways underlying behavior. in order to develop numbers of novel behavioral mutants, we have been carrying out dominant behavioral screening in potential mouse mutants that was randomly induced point mutations by a chemical mutagen enu (n-ethyl-n-nitrosourea). we screened about , g animals (dba/ j × enu-treated c bl/ j) for home-cage activity, open-field activity, and passive avoidance response, and obtained lines of dominant behavioral mutants. by linkage analysis, the causative genes were mapped in of mutant lines. hyperactivity was predominant phenotype, and of mutants showed hyperactivity in home-cage and/or open-field. we will report the recent results of initial characterization and the progress of fine mapping in these enuinduced mutants. ps p-i ubiquitin signal in neurons of cathepsin ddeficient mouse brains with special reference to the autophagic process masato koike, masahiro shibata, yasuo uchiyama dept. of cell biol. and neurosci., osaka univ. grad. sch. of med., suita, japan we have shown that autophagy contributes to the accumulation of vacuolar structures in neurons obtained from cd−/− and cb−/−cl−/− mice, murine models for neuronal ceroid lipofuscinoses (ncls) (koike et al., ) . until recently, it remains unknown what signaling is essential for autophagosome formation. interestingly, in the conditional atg -knock-out mice where autophagy is absent specifically in the liver, numerous ubiquitinated aggregates are detected in the cytosol of hepatocytes (komatsu et al., ) , suggesting that protein ubiquitination may serve as a signal to the autophagic process. we therefore examined the immunohisto/cytochemical localization of ubiquitin and lc , and found that in our ncl model mice, positive signals for ubiquitin and lc were co-localized on the membranes of granular structures in the neuronal perikarya. these results suggest that protein ubiquitination may be involved in signaling for autophagosome formation in ncls. research funds: grant-in-aid for young scientists (b)( ) and creative scientific research ( gs ) ps p-i activation of medial prefrontal cortex neurons by systemic phencyclidine is primarily mediated via ampa/kainate glutamate receptors tadahiro katayama , eiichi jodo , yoshiaki suzuki , ken-yo hoshino , yukihiko kayama dept. of physiology, fukushima medical university, fukushima, japan; dept. of neuropsychiatry, fukushima medical university, fukushima, japan it has been shown that tonic activation of the medial prefrontal cortex (mpfc) plays a pivotal role in development of behavioral abnormalities induced by systemic phencyclidine (pcp). however, receptors mediating such activation are not clearly specified, though several studies indicate the increase of extracellular acetylcholine, dopamine, and glutamate in the mpfc. here, we examined effects of local application of those antagonists on increased firing activity of mpfc neurons by systemic pcp in anesthetized rats. after tonic activation of mpfc neurons by pcp had been established, cnqx, sch , mecamylamine or scopolamine was locally applied with iontophoresis or gas pressure on the recorded neuron. cnqx reduced pcp-induced augmentation of firing activity to the baseline level, while others gave little changes. these results suggest that pcpinduced activation of mpfc neurons be mediated primarily via ampa/kainate receptors. ps p-i increased depressiveness and decreased sensitivity to antidepressants in calcium/calmodulin-dependent protein kinase iv (camkiv)-knockout mice jiro kasahara , hiroyuki sakagami , hisatake kondoh , kohji fukunaga department of pharmacology, graduate school of pharmaceutical sciences, tohoku university, sendai, japan; department of histology, graduate school of medicine, tohoku university, sendai, japan calcium/calmodulin-dependent protein kinase iv (camkiv) is expressed abundantly in the nuclei of neurons and thought to regulate ca-dependent gene expressions mediated by the transcriptional factors such as creb. recently, we found that chronic treatments of the rats with antidepressants increased camkiv activity and creb phosphorylation in the prefrontal cortex, suggesting the importance of camkiv in the effects of antidepressants. this result led us to perform the behavioral assessments of depressiveness and the sensitivity to antidepressants in camkiv-knockout mice by some experimental paradigms. from the experiments, the increased depressiveness and decreased sensitivity to antidepressants were observed in the mice, suggesting the importance of camkiv for the regulation of depressiveness and the effects of antidepressants. ps p-i severity of audiogenic seizures is influenced by multiple factors in vlgr -mutated mice hideshi yagi , , makoto sato , division of cell biology and neuroscience, department of morphological and functional sciences, faculty of medical sciences, university of fukui, fukui, japan; research and education program for life science, university of fukui, fukui, japan epilepsy is a highly prevailed disorder and reports are accumulating that demonstrate that single gene mutation causes such disorders. we made vlgr -mutated mice and found that they showed high susceptibility to audiogenic seizure, one of the reflex seizures provoked by loud noise. to evaluate whether the genetic backgrounds influence on phenotype of the audiogenic seizure in our mice, we made c bl/ backcrossed vlgr -mutated mice and /svs backcrossed vlgr -mutated mice. these two backcrossed lines showed different susceptible periods and severity of audiogenic seizure from the original line. furthermore, phenotype of audiogenic seizure was altered by restraining mice from free moving while being exposed to loud noise. these observations suggest that genetic factors and environmental factors may modify the phenotype of seizures and our vlgr -mutated mice are good model of reflex epilepsies that are evoked by multifactors. ps p-i reduction in the density of parvalbumin-positive cells in the medial frontal cortex of rats behaviorally sensitized to methamphetamine tomoko kadota , ken kadota , department of bioenvironmental medicine, university of chiba, chiba, japan; chiba institute of psychiatry, chiba, japan our previous study demonstrated that the development of behavioral sensitization of rats to methamphetamine (map) corresponded in time with the progress of neurotoxic changes induced in the medial prefrontal cortex (mfc). the present study further examined morpholological changes of rats that were administered a daily dose of mg/kg of map i.p. for days (d d ) and then withdrawn from the drug for days (wd wd ). the regimen reduced the densities of parvalbumin positive cells (pac); these were probably gabaergic cells and distributed in the strata covering layers ii, iii and v in the anterior cingulate cortex (cg ) and mfc. the decrease in the density of pac was first observed in cg and then in mfc. the reduction began on d and advanced to higher levels on d and subsequently wd . these findings suggest that the behavioral sensitization regimen leads to the deterioration of inhibitory processes in the neural circuits in cg and mfc, particularly in layers ii and iii. ps p-i up-regulation of ␤ -adrenergic receptor immunoreactivity in astrocytes in the spinal cord after dorsal rhizotomy teruyoshi kondo, yoshihiro ishibashi, kei-ichiro nakamura department of anatomy, division of microscopic and developmental anatomy, kurume university school of medicine, kurume, japan stimulation of ␤ -adrenergic receptor (␤ -ar) induces astroglial proliferation and activation after brain injury, but little is known concerning the potential role of adrenergic receptors in the spinal cord. present study demonstrated that rhizotomy induced a marked and prolonged up-regulation of ␤ -ar-immunoreactivity (ir) in the regions of the dorsal root entry zone and dorsal funiculus containing the central processes of the injured primary sensory neurons. ␤ -arimmunoreactive cells coexpressed gfap-ir and were positive for nestin which is characteristic of reactive astrocytes. a population of ␤ -ar-immunoreactive cells were labeled with ki- , a marker of cell proliferation, indicating some of them went into cell mitotic state. interestingly, a major population of ␤ -ar-immunoreactive cells also exhibited fgf- -ir. these findings suggest that ␤ -ar may play important roles in astrocytic activation and neuroprotection associated with induction of synthesis of growth factor such as fgf- . ps p-i effects of lateral fluid percussion injury (fpi) on the optical signals in dentate gyrus of the rat brain slice preparations shin yamashita , norihiro muraoka , hiroshi hasuo , takashi akasu , minoru shigemori dept. of physiology, kurume univ. sch. of med., kurume, japan; dept. of neurosurgery, kurume univ. sch. of med., kurume, japan we investigated the effects of experimental traumatic brain injury on the neuronal function in dentate gyrus (dg) using optical recording techniques with voltage-sensitive dye (rh ). horizontal hippocampal slices were obtained from the control and the fpi rats (one week after the single moderate impact). electrical stimulation of perforant path (pp) produced the optical signal spread in the molecular layer of dg. temporal change in the optical signal, obtained from an area on the propagation pathway, had two peaks (fast and slow peaks). increment of stimulus intensity ( - v) increased the amplitude of both fast and slow peaks. the intensity for producing the maximal response was - v. the amplitude of slow peak in fpi group was about % larger than that in control group, while the amplitudes of fast peak were not different in the two groups. these data suggest that the excitatory pp synapse onto granule cells of dg is facilitated after fpi. ps p-i comparative study of neural activities in mouse hippocampal slices by flavoprotein autofluorescence and ca + imaging chikafusa bessho, yasuharu mitsushima, ryo matsumoto department of physics, kyoto sangyo university, kyoto, japan recently k. shibuki et al. have succeeded in flavoprotein autofluorescence imaging of neural activities in the rat brain. we examined neural activities in mouse brain (hippocampal) slices by the modified method and ca + imaging. the slices ( m) were prepared from the block in an ice cold acsf medium using microslicer and incubated for h in the oxygenated medium at room temperature. a slice was placed on a recording chamber perfused with the medium at a flow rate of ml/min. green autofluorescence (> nm) of the slices illuminated by blue light ( - nm) was observed by an inverted microscope. images of the autofluorescence were recorded using a calcium imaging system. ca + imaging was also performed in the slices. slices were incubated in acsf medium containing m of fluo / am for h at • c. the ca + image was recorded with an excitation wavelength of - nmand an emission wave length of > nm. the autofluorescence and ca + responses wereobserved in slices perfused with l-glutamate ( mm). takuya hayashi , hiroshi sato , shinichi abe , takashi hanakawa , hiroshi watabe , hidenao fukuyama , babak aldekani , hidehiro iida department of investigative radiology, national cardiovascular center research institute, osaka, japan; human brain research center, kyoto university, kyoto, japan; nathan kline institute for psychiatric research, ny, usa we show connectivity pattern between cortex and striatum in macaque and human by using the non-invasive method of diffusionweighted magnetic resonance imaging (dwi). in macaque, the dwibased striatal connectivity of brodmann's area corresponded to that revealed by the tracer (mncl ) tractography. the dwi-based connectivity pattern also isolated a part of the ventral striatum corresponding to the histochemically-specific 'shell' region in both human and macaque. in addition, we confirmed the species-homology in intra-striatal topography of cortical connection by quantitatively analyzing the connectivity; however, we found that human striatum was more intensively connected to prefrontal cortex and less connected to extra-frontal cortices. these results suggest that human striatum has a dominant and specific role in processing prefrontal information. research funds: h -kokoro- ps p-i optical analysis of synaptic transmission by a fluorescent glutamate probe shigeyuki namiki, hirokazu sakamoto, sho iinuma, kenzo hirose department of cell physiology, nagoya university graduate school of medicine, nagoya, japan glutamate is an essential excitatory neurotransmitter in the central nervous systems. for optical analysis of glutamatergic synaptic transmission, we have developed a fluorescent glutamate probe called eos. by imaging with eos, we successfully detected the synaptically released glutamate following axon firings in cultured hippocampal neurons; the spatial distribution of the glutamate release was non-uniforml along dendrites. we also succeeded in monitoring the phorbol ester-induced potentiation of the glutamate release. furthermore, we found spontaneous and stochastic glutamate release which was confined to small regions. neither application of tetrodotoxin nor removal of extracellular calcium blocked the release. high concentrations of sucrose increased the frequency of the release. these features are reminiscent of those of miniature epsc in electrophysiological recordings and thus suggest that the spontaneous release is quantal vesicular release. in conclusion, our probe directly visualizes the presynaptic release. shingo miyata , , yasutake mori , , tsuya taneda , , hiroaki okuda , , masaya tohyama , department of anatomy and neuroscience, graduate school of medicine, osaka university, osaka, japan; st coe program, tokyo, japan local protein synthesis in neuronal dendrites is one of the mechanisms that may mediate a rapid and synapse-specific mobilization of proteins from the resident mrnas. a great deal of effort has been made in analyzing the dynamic state of protein synthesis in the living cells, chiefly by quantifying protein level. however, the protein level cannot mirror the spatio-temporal alteration of translation, because it cannot be affected only by protein synthesis but also by other factors like degradation. therefore, it is problematic to visualize the dynamic state of translation by the present methods. to solve the problem, we applied fret (fluorescence resonance energy transfer) technique to in situ detection of the assembly and disassembly cycle among a pair of translation initiation factors (eifs), thereby showing that bdnf and ephrin could potentiate local protein synthesis in the dendrites of hippocampal neurons. ps p-i a model selection of glm applied to fmri data using aic jobu watanabe , fumikazu miwakeichi , andreas galka , , ryuta kawashima , tohru ozaki , sunao uchida , institute for biomedical engineering, consolidated research institute for advanced science and medical care, waseda university, japan; department of medical system engineering, faculty of engineering, chiba university, chiba, japan; institute for statistical mathematics, tokyo, japan; institute of experimental and applied physics, university of kiel, keil, germany; new industry creation hatchery center, tohoku university, sendai, japan; faculty of sport sciences, waseda university, tokorozawa, japan in the general linear model (glm) that is widely used in analyses of functional neuroimaging data, several combinations of explanatory variables are possible. the akaike information criterion was applied as a basis of comparison and selection among several glms that analyze block-designed functional magnetic resonance imaging (fmri) data. the glms with/without a resting condition, head motion covariates, time derivatives and dispersion of hemodynamic response function were compared. we demonstrate that a combination of these explanatory variables can effectively improve the model and that aic is a useful tool for model selection in fmri studies. ryuzo shingai, katsunori hoshi, tokumitsu wakabayashi department of welfare engineering, iwateuniversity, morioka, japan to investigate the relationship between the behavior and function of the nervous system of caenorhabditis elegans, quantitative analysis of behavior that indirectly represents the internal states of the worm is necessary. we devised an automated analysis system of c. elegans locomotion. the system is well suited for detecting four locomotion states: forward or backward movement, curl and rest. the system was applied to a phenotype that when a worm is transferred from a seeded plate to a bacteria-free plate, the worm shows frequent backing and short duration of forward movement for - min and then a gradual increase in the duration of forward movement. accuracy of the state identification for wild type and several mutants was sufficiently high, indicating the system is robust in studies of locomotion. ps p-j flavoprotein fluorescence responses elicited by thalamic stimulation in slices obtained from the mouse barrel cortex daiki kamatani, ryuichi hishida, masaharu kudoh, katsuei shibuki dept. neurophysiol., brain res. inst., niigata univ., niigata - , japan we have reported that whisker trimming induced activity-dependent changes in the barrel cortex of rat cortical slices using flavoprotein fluorescence imaging. however, contribution of thalamo-cortical afferents in this plasticity was not clear, since specific stimulation of thalamo-cortical afferents was not possible in the coronal cortical slices obtained from rats. in the present study, we used the mouse cortical slices that kept thalamocortical connections to the barrel cortex intact. the cortical activities in layer iv were observed as fluorescence responses after thalamic stimulation. the magnitude of the fluorescence responses was increased as the amplitude of cortical field potentials was increased. these cortical responses were suppressed by antagonists of glutamate receptors such as cnqx and apv, and almost completely abolished in the presence of cnqx plus apv. in preliminary experiments, we confirmed that whisker trimming induced activity dependent changes in the barrel cortex of mice. ps p-j effects of implicit emotional processes on encoding-related activations of episodic memory: an eventrelated fmri study yayoi shigemune , , takashi tsukiura , hiroko mochizuki-kawai , chisato suzuki , , toshio iijima neurosci. res. inst., aist, tsukuba, japan; div. systems neurosci., tohoku univ. grad. sch. life sci., sendai, japan in this study, we investigated the effects of implicit emotional processes on encoding-related activations of episodic memory using fmri. nineteen healthy right-handed male participated in this study. we prepared emotional pictures with three kinds of emotional valence (negative: nega, neutral: neu and positive: posi) and line drawings for encoding. in the fmri scanning, subjects memorized line drawings, which were presented after the emotional pictures. after the scanning, subjects were presented with the names of line drawings, and were required to judge whether or not line drawings with the names were learned. we found significant activations of the right anterior cingulate gyrus specifically in the nega condition, the right lingual gyrus in the neu condition and the right amygdala and pulvinar in the posi condition. these results suggest that encodingrelated activations of episodic memory may be modulated by the implicit primer with emotional valence. ps p-j different neural correlates of stimulus-actiondependent and stimulus-dependent reward predictions revealed by fmri masahiko haruno , kenji kansaku , yu aramaki , mitsuo kawato atr cns, kyoto, japan; institute of physiology, okozoki, japan efficient decision making requires multiple reward predictions in switching different contexts and learning. we conducted a human fmri experiment (n = ) to examine stimulus-action-dependent and stimulus-dependent reward predictions. in condition a, each of two fractal figures specifies a monetary reward associated with a button push (left or right). if the button is pressed correctly, or yen is provided with a probability of . , but only with . if pressed wrongly. the key difference in condition b is that a fractal determines the reward but not the action. subjects had learned the two conditions fully before scanning. at the fractal onset, the putamen, lateral ventral and medial dorsal prefrontal cortices showed stronger activity correlated with the predicted reward (p < . ) in a, while it was more prominent in the caudate, dorsolateral prefrontal cortex and cerebellum in b. the striatum also showed a similar difference correlated with the reward prediction error at reward feedback, suggesting the different neural substrates for different reward predictions. research funds: nict ps p-j brain networks for communicative speech production: feeling inference and speech content production yuko sassa , , motoaki sugiura , hyeonjeong jeong , , keisuke wakusawa , , kaoru horie , shigeru sato , ryuta kawashima niche, tohoku university, sendai, japan; ristex, jst, tokyo, japan; miyagi university of education, sendai, japan; gsics, tohoku university, sendai, japan; department of pediatrics, tohoku university, sendai, japan; the lbc research center, tohoku university, japan communicative speech production often accompany inference of the targetperson's feeling. in this fmri study, we segregated the brain networks forthe feeling inference and speech content production processes incommunicative speech production. during presentation of a picture showingan actor's utterance in a balloon, normal subjects covertly talked to theactor (speech), inferred feeling (feeling), or described the action (des). greater activation in the contrasts speech-feeling was observed in themedial prefrontal cortex, and that in the contrast feeling-des wasobserved in the right superior temporal sulcus extending to the temporalpole. the results suggest that these two regions play roles in the speechcontent production and feeling inference, respectively. research funds: the st coe program ps p-j the construction of a brain-computer interface using the brain activity measured by near infrared spectroscopy takafumi miyoshi , yasuhisa fujibayashi , yoshiharu yonekura , tatsuya asai department of human and intelligence systems, university of fukui, fukui, japan; biomedical imaging research center, university of fukui, fukui, japan people with severe motor disabilities can increase the quality of life if they can communicate with the external world. a brain-computer interface using brain activity is one of the ways to provide such communication without depending on muscular controls. brain activity was measured non-invasively by multi-channel near infrared spectroscopy (nirs) during various motor tasks from healthy subjects. these spatial brain activities were fed to neural networks, and pattern learning was carried out by matching the tasks and the brain activities. we propose that nirs signals may be used to construct a brain-computer interface. ps p-j imaging of brain activity by near infrared spectroscopy in response to various sounds tatsuya asai , kuniyoshi shinya , tetsuo araki , masahiro kusakabe , yasuhisa fujibayashi , yoshiharu yonekura department of nuclear power and energy safety engineering, university of fukui, fukui, japan; department of human and intelligence systems, university of fukui, fukui, japan; biomedical imaging research center, university of fukui, fukui, japan brain activity can be monitored non-invasively by near infrared spectroscopy (nirs). in the present study, we measured changes in cerebral hemoglobin concentrations during a listening task using multi-channel nirs from healthy right-handed subjects, and hemispheric dominance for various sounds including verbal sounds was assessed. we have found asymmetrical brain activity when subjects listened to sounds with their left or right ear. these results suggest that hemispheric sound dominance may exist in addition to language dominance in healthy humans. kazuo kitamura , , winfried denk , michael hausser department of cellular neuroscience, graduate school of medicine, osaka university, osaka, japan; university college london, london, uk; max-plank institute, heidelberg, germany we describe a new approach for making targeted patch-clamp recordings from single neurons in vivo visualized using two-photon microscopy. the method involves using a patch electrode to perfuse the extracellular space surrounding the neuron of interest with a fluorescent dye, thus allowing the neuron to be visualized as a negative image and identified on the basis of its somatodendritic structure. the same electrode can then be placed on the neuron under visual control to allow gigaseal formation. we demonstrate the reliability and versatility of the method using recordings from principal neurons and interneurons in mouse and rat barrel cortex and cerebellum. we also show that the method can be used for in vivo juxtacellular labelling in identified cell types. this approach thus offers the prospect of targeted recording and labelling of single neurons in the intact native mammalian brain without the need to pre-label neuronal populations. research funds: wellcome trust, gatsby foundation, jsps and uehara foundation ps p-k analysis on viability of gabaergic neurons in cerebral cortical slices of adult mice yasuyo tanaka , yasuhiro tanaka , takeshi kaneko , dept. of morphological brain science, kyoto univ., kyoto, japan; crest, jst, kawaguchi, japan whole cell clamp recording and intracellular staining in adult brain slices are technically difficult because of their low viability. we analyzed the effect of slice cutting and incubation conditions on viability of cortical gabaergic neurons, using gad -gfp knock-in mice. we considered gfp positive cells as having survived. we observed more gfp-positive cells in the slices when nacl in cutting solution was replaced with n-methyl-d-glucamine (nmdg) chloride, choline chloride or sucrose. however, the viability was lower after h incubation in nmdg-based solution than in nacl-based solution. cutting at • c did not reduce the number of gfp-positive cells, but decreased gfp fluorescence in single neurons as compared with cutting at • c. the viability after h incubation was better kept at • c than at or • c. we thus recommend that slices be cut at • c in na-free solution, and incubated at • c in nacl-based solution. we thank dr yanagawa for his generous gift of knock-in mice. research funds: kakenhi ( , , ) ps p-k contribution of reduced and oxidized glutathione to signals detected by magnetic resonance spectroscopy as indicators of local brain redox state takumi satoh , yoshichika yoshioka faculty of engineering, iwate university, morioka, japan; iwate medical university, takizawa, japan we evaluated gsh signals by the mega-press (a frequencyselective refocusing technique) signals assessed by magnetic resonance spectroscopy (mrs). gsh gave a single positive signal ( . ppm) by mega-press. in contrast, gssg gave a multiplet of reversed signals ( . , . , and . ppm). a phantom solution mimicking the normal condition (gsh:gssg = : ) gave a single positive peak. gsh was prominent and gssg signals were minimal. thus, the signals originated from gsh, not from gssg. in the phantom solution (creatine: gsh: aspartate: gaba = : : : ), the creatine signal overshadowed the other signals. through mega-press, a single peak of gsh stood out over other signals. in vivo, the brains of healthy volunteers gave similar signals as the in vitro phantom solution, indicating that the signal originated from gsh. the estimated concentration of gsh in the human brain was . mm. in conclusion, mega-press allowed us to assess gsh levels in vivo non-invasionally. hiroshi kadota, hirofumi sekiguchi, yasoichi nakajima, yutaka kohno, makoto miyazaki department of sensory and communicative disorders, research institute, national rehabilitation center for persons with disabilities, tokorozawa, japan we investigated the brain regions related to the inhibition of habitual responses by using functional mri. we used the rock-paper-scissors game as an example of a familiar habitual behavior. it is considered that making positive attempts to lose when presented with the gesture of a rock, paper, or scissors is associated with the inhibition of habitual responses. in this study, the subjects were randomly assigned to one of the following two groups: the "win group" and the "lose group." a comparison between these groups showed that the lose group displayed activation of multiple cortical areas in the brain. with regard to the prefrontal cortex, the comparison revealed a higher activation in the left middle frontal gyrus (brodmann area ) and the right superior and middle frontal gyri (brodmann area ) in the lose group. these findings suggest that these regions play a role in the inhibition of habitual responses. ps p-k cortical commissural connection in macaque and human callosum using diffusion mri rishu piao , takuya hayashi , hiroshi sato , shinichi abe , , takashi hanakawa , hidenao fukuyama , hidehiro iida national cardiovascular center, osaka, japan; human brain research center, kyoto university, kyoto, japan we investigated the cortical commissural connection in human and macaque using the non-invasive diffusion-weighted magnetic resonance imaging (dwi). we used the probabilistic algorithm to track connection paths between a pair of the left and right homologous in subcortical areas. in macaque, the classification of callosum based on the highest interhemispheric connections paralleled with the results of tracer studies. however, the territory corresponding to the interfrontal connectivity extended more posteriorly than suggested in the tracer studies. the human interhemispheric connectivity showed similar topography in callosum as in the current macaque study, except that the connectivity territory of the frontal areas extended more posteriorly than in macaque. this study revealed that the commissural connectivity of the two species has a common intra-callosal topography. ps p-k optimal resolution of eeg/meg source imaging by spatial filtering wan xiaohong , niche, department of qutantum science and energy engineering, tohoku university, sendai, japan, niche, tohoku university, sendai, japan nowadays, electro-and magnetoencephalography (eeg/meg) is the sole invasive technique which is able to directly measure the human brain neural cortical dynamics. although we are well aware that it is impossible to accurately estimate the -d neural cortical activity using the -d eeg/meg surface potential topography, the upper limit of these techniques is not well described. during the past decades, various inverse approaches based on different criteria have been proposed, from the single dipole or multiple dipoles to the distributed current dipoles. however, it is difficult to systematically evaluate their efficiencies due to the different criteria and regularizations adopted in these methods. in this paper, we ask the question whether there exists an optimal approach based on a systematical criterion. this motivation firstly seems to be conflicted with the primary knowledge that there is no unique solution for the bioelectromagnetic inverse problem. essentially, here we are trying to find an optimal inverse solution that is closest to the real current distribution. ps a-c sensitivity of serotonin synthesis to synthesis inhibitor gtp cyclohydrolase i in senescence-accelerated mouse-prone inbred strain (samp ) nobuyuki karasawa , kazuko watanabe , keiki yamada seijoh universitry, tokai, japan, dept. physio., sch. med., gifu univ., gifu, japan, dept. anat., sch. health sci., fujita health univ., toyoake, japan to study the relationship between aging and levels of monoaminergic neurons, , -diamino- -hydroxypyrimidine (dahp), an inhibitor of monoamine synthesis, was intraperitoneally administered to senescence-accelerated mouse-prone (samp ) mice. time course of immunoreactive intensity for serotonergic ( -ht) neurons in the dorsal raphe nucleus, which were stained using laboratory-raised serotonin-specific antibody, was quantitatively evaluated using an image analysis system. results showed that -ht neruons are not highly sensitive to a synthesis inhibitor common to both catecholaminergic and -ht neurons. katsuya yamada , , , yoshihiro matsumura , takashi miki , makoto wakui ␣-smooth muscle actin + arterioles were fewer in kir . (−/−) barrel cortex than in wild-type one. in addition, whisker stimulation-induced increase in local cerebral blood flow was much smaller in kir . (−/−) barrel cortex than in wild-type one for short ( s, hz) but not long ( s) stimulation, suggesting crtical involvement of thin arterioles in a short-time neuro ps a-h learning to use sensory-tools by japanese monkeys yumiko yamazaki , hiromi namba support by public health research foundation (japan). acknowledgement supported by hkrgc. we wish to thank professor miyashita for valuable advice. ps p-h mechanisms for processing of intellectual excitement kazuhiko yanai, hongmei dai dept. pharmacol tohoku grad. univ. sch. med., sendai, japanthe aim of this study was to investigate the role of histamine h receptor (h r) in cognition in physiological and pathological conditions by using h r mutant (h −/−) mice. in normal condition, several behavioral studies indicated h −/− mice show impaired object recognition and spatial memory, improved conditioned fear memory. moreover, hippocampal long-term potentiation was reduced in h −/− mice. these results indicate h receptor is involved in memory process for which the frontal cortex, amygdala and hippocampus interact. in pathological condition, both h −/− and control mice were subjected to social isolation, an animal model of schizophrenia. social isolation impaired locomotion, prepulse inhibition of startle response and water maze performance in control mice, but not in h −/− mice. mutation of h receptor decreases isolation-induced hyperactivity of cortical dopaminergic neurons. these findings indicate blockage of h r attenuates social isolation-induced behavioral changes. in conclusion, blockage of h r impairs cognition in normal conditions, whereas h r blocking inversely improves cognition in disease models of schizophrenia.research funds: kakenhi ( ; ) ps a-h -ht a receptor gene polymorphism modulates activation in the human ventrolateral frontal lobe during go/no-go task michio nomura , , hirohito-m. kondo , makio kashino , department of psychology, tokai women's university, kakamigahara, japan; ntt communication science laboratories, ntt corporation, atsugi, japan; shimojo implicit brain function project, erato, jst, kawaguchi, japan impulsive behavior has been suggested to be due to a dysfunction of -ht neurotransmission. we examined whether this -ht a receptor gene polymorphism is involved in impulsive aggression by evaluating a reward-punishment go/no-go task using fmri. participants were required to learn to respond to active stimuli and inhibit their response to passive stimuli both under the reward-only (r) condition and the punishment-only (p) condition. the r condition, compared with the p condition caused right prefrontal activation mainly seen in ventrolateral regions. it has been reported that the possible involvement of the -ht a receptor gene polymorphism in impulsive behavior (nomura et al., ) , together with the present findings, this observation indicates the involvement of -ht a receptor gene polymorphisms in ventrolateral frontal lobe.research funds: shimojo implicit brain function project, erato, jst ps a-h role of cortical thin arterioles in neurovascular coupling; analyses of kir . -containing atpsensitive potassium channel-deficient mice ps p-g rem sleep deprivation increases serum ceruloplasmin level manoj jaiswal , chinmay k. mukhopadhyay , birendra n. mallick school of life sciences, jawaharlal nehru university, new delhi, india; special centre of molecular medicine, jawaharlal nehru university, new delhi, india rapid eye movement sleep (rems) is present across higher species and is essential for life. its loss predisposes one to several pathophysiological conditions. continuous loss of rems leads to several diseases and extreme loss may be fatal. rems loss is reported to increase metabolism and food intake though associated with hypothermia. hence, we proposed that the rems deprivation would affect acute phase response protein. in this study rats were rems deprived by platform method. free moving normal, large platform and recovery from rems loss were used as controls. blood was collected from the same rat before and after experimental as well as control periods. level of serum cruloplasmin, a positive acute phase response protein, was detected using western-blot analysis. the results showed that rems deprivation increased the serum ceruloplasmin level suggesting that the rems deprivation triggers an acute phase response at least in rats.research funds: council of scientific and industrial research, india and dst, india the indirect cytopathic effect in hiv- and the direct infection of hsv- are critical in their pathogenesis. we established murine neurosphere and evaluated with cocultivation of hiv- jrfl-infected macrophages or with hsv- . the generation of primary neurospheres did not suppressed by hiv- -infection or by hsv- infection at no more that moi . in the secondary neurospheres, cd + neural stem cells were intact in these infections, although beta- -tubulin + cells were decreased in hiv- infection and intact in hsv- -infection. in the differentiation assay, neun + nfp + neurons in hiv- -infection and gfap + s + astrocytes in hsv- infection were significantly decreased. the migration capacity of the neurosphere cells was suppressed in hiv- infection and in hsv- infection. we conclude that neural stem cells in vitro are resistant to cytopathic effect by hiv- and hsv- infection and their differentiation capacities are different in these infections. our assay will be one of the significant methods in neurovirological research.research funds: kakenhi grant-in-aid for young scientists(b) ps p-g effects of attraction to favorite opposite gender on nervous, endocrine, and immune systems masahiro matsunaga , , taeko yamauchi , toshihiro konagaya , hideki ohira department of psychology, nagoya university, japan; department of internal medicine, aichi medical university school of medicine, japan everybody can "fall in love". thus everybody knows that attraction to favorite opposite gender invokes positive feelings and often makes us energetic. to investigate effects of this positive emotion on the biological systems, we recorded various parameters, namely mood states, heart rate, skin conductance level (scl), serum levels of several hormones, and proportions of t cells and natural killer (nk) cells in the lymphocytes simultaneously when subjects viewed the video films of their favorite opposite genders. when the subjects were evoked their attraction to favorite opposite gender, they became more vigorous and felt better. as for the biological systems, scl and the proportion of nk cells in the lymphocytes significantly increased. these results suggest the possibility that attraction to favorite opposite gender may have a role in activating nk cell-related innate immune system by means of the activation of scl-related sympathetic nervous system. hiroko ikeshima-kataoka , shen jin-song , saburo saito , shigeki yuasa dept. mol. immunol., inst. dna med., jikei. univ. sch. med., tokyo., japan; dept. gene ther., inst. dna med., jikei univ. sch. med., tokyo, japan; dept. ultrastruc. res., natl. inst. neurosci., ncnp, tokyo, japanto investigate the role of tenascin (tn)-expressing astrocytes played in the injured brain, we analyzed tn knockout (tn/ko) mouse. we have previously reported that tn is one of the essential molecules for proliferation of the primary culture of astrocytes. from injured mouse brain model with stab wound, gfap expression was down regulated sharply at earlier stages in tn/ko mouse than in the wt mouse. some of the inflammatory cytokines are known to be expressed in injured cns, and also those receptors are expressed in the primary culture of astrocytes. to evaluate immune responses in the cns, some of the inflammatory cytokine production was determined in the lesioned mouse brain compared with tn/ko and wt mouse. from rt-pcr method, tn seemed to have the possible roles for some of the cytokine prodution at the cns lesion sites. we are currently investigating the function of tenascin for the cytokine production around the lesion site. aiko hori , tomoko yamamoto , kiyoshi matsumura , hiroshi hosokawa , shigeo kobayashi dept. of intelligence science and technology, grad. sch. of informatics, kyoto university, kyoto, japan; dept. of information science and technology, osaka institute of technology, osaka, japan intracerebroventricular (i.c.v.) injection of arachidonic acid (aa) evokes fever. this response has been thought to occur simply because aa is converted to prostaglandin e (pge ), the final mediator of fever. however, our recent study suggested that aa might not only be the precursor of pge but also induce an enzyme cyclooxygenase- (cox- ) that catalyses aa to form prostaglandins. we here examined in rats whether i.c.v. injection of aa induces cox- , and whether cox- is involved in aa-induced fever. two hours after i.c.v. injection of aa, cox- was expressed in the perinuclear region of brain endothelial cells. aa-induced fever was partly suppressed with a cox- specific inhibitor, ns- . these results indicate that aa itself or its metabolites induces cox- that accelerates the formation of pge from aa, and, hence, enhances fever. mitsunari abe , tatsuya mima , shinichi urayama , toshihiko aso , nobukatsu sawamoto , hidenao fukuyama human brain research center, kyoto university graduate school of medicine, japan; nano-medicine merger education unit, kyoto university, japanrepetitive transcranial magnetic stimulation (rtms) can induce lasting changes in the cortical excitability. however, its cellular mechanism remains unknown. diffusion weighted imaging (dwi) is a useful tool for measuring microscopic states of the brain tissue by probing water diffusion.we examined changes of dwi following rtms to further understand its effects. four healthy volunteers received rtms at . hz ( min; % of the rest motor threshold) applied over the left primary motor area (m ). we scanned sets of dwi (before, and min, min and min after rtms) using -t mr scanner, and calculated apparent diffusion coefficient (adc). in out of subjects, the adc decreased (mean . × − /mm s − ) in the left m just after the rtms, which recovered at min. it is possible that the rtms-induced change of adc might occur as the cellular response. further examination is needed for confirming this point. vahe poghosyan, andreas a. ioannides laboratory for human brain dynamics, brain science institute riken, wako-shi, japanretinotopic areas v and v a in macaques occupy almost the entire extend of the anterior bank of parieto-occipital sulcus (pos). v a located more dorsal has a larger receptive field size then v . both areas lack a foveal magnification. we used meg to record brain activity while human subjects were viewing stimuli presented at two different eccentricities in each quadrant of visual fields. to verify the reliability of results, for each subject, the experiment was repeated on three different days. tomographic analysis of meg signal, in each subject, identified highly reproducible activations throughout visual cortex in accord with the known organization. two new areas along the pos with a similar retinotopy to that of macaques v and v a were identified. in the ventral one, activations in response to each stimulus were spatially separated. the foveal magnification was much reduced compared to v in both areas and in the more dorsal area activations elicited by stimuli in the same quadrant could not be separated. given the above finding we suggest these areas as possible homologues of macaques v and v a.ps p-i measurement of magnetic evoked field of ratmeasurement of magnetic evoked field of rat using micro squid naohiro tsuyuguchi department of neurosurgery, osaka city university graduate school of medicine, japanthe study of neural activity in rodents would be enhanced by the stimulation of neuronal function in vivo. magnetoencephalography (meg) is used to study brain function in humans, but the limited resolution and sensitivity of conventional instruments have precluded the use of meg to study neuronal function in rodents. we demonstrate that micro meg developed for use with small animals, can be used to detect assess neuronal activity in conscious rodent brain. we used a micro -channel magnetometer consisting of a × matrix of superconducting quantum interference device (squid) with its integrated base of . × . mm to measure the visual evoked magnetic field (vef) and auditory evoked field (aef) of rats. we obtained the vef wave with - ms peak by the white led flashing stimulation and the aef wave with - ms peak by the tone and burst stimulation. this study demonstrate that micro meg can be used for serial assessment of neuronal function of individual, live animals with a minimal degree of invasiveness, has the potential for use in the study of brain function and plasticity. kentaroh takagaki, michael t. lippert, jian-young wu department of physiology and biophysics, georgetown university, washington, dc, usa voltage-sensitive dye (vsd) imaging is used to study visually evoked responses in rat visual cortex, in single trials without averaging. the signal is small, and a diode array with an effective dynamic range of bits was used, along with a "blue dye" (rh ) with small heartbeat artifact. a subtraction algorithm was used to further remove heartbeat artifact in the data. with the combination of the array, the blue dye and the algorithm, we were able to visualize sensory evoked wave activity with a high signal-to-noise ratio in single trials. the signals were . - . % of the resting fluorescence intensity. spatiotemporally, the evoked response manifested as propagating waves in the visual areas. there were large trial-to-trial variations in the propagating velocity and directions of the waves. the evoked wave apparently interacted with spontaneous waves in the cortex, and varied greatly according to anesthetic regimen. visualizing evoked waves may contribute to the understanding of cortical dynamics underlying sensory processing. masahito nemoto , yoko hoshi , chie sato , susumu terakawa tokyo institute of psychiatry, tokyo, japan; photon medical research centre, hamamatsu university school of medicine, hamamatsu, japanwe investigated interhemispheric interactions and neurovascular coupling by simultaneous recordings of neuronal and hemodynamic signals in rats. bilateral somatosensory cortices were activated with a stimulus time lag between test stimuli (electrical pulses to contralateral hindpaw) and conditioning stimuli (to a homologous somatosensory region of the contralateral hemisphere). we measured electrophysiological signals (local field potentials and multiunit activity) and optical intrinsic signals ( and nm, indicators of cbv and oxygenation), and analyzed the dependence of the signals on the time lag. the results showed that both neuronal and hemodynamic signals were suppressed around -ms time lag. average and trial-by-trial correlation analyses suggested that the hemodynamic signals reflected a balance of neuronal excitation and inhibition via callosal connections. we can infer some parts of underlying neural interactions by imaging of the hemodynamic signals. ps p-j multiple-site optical detection of spontaneous activity in the rat sensorimotor cortex akihiko hirota, shin-ichi ito department of physiology, shimane university school of medicine, izumo, japan multiple-site optical recording provides a powerful tool for the cerebral cortical neurophysiology, but its application has largely been restricted to reproducible, stimulus-evoked activation. we have developed the recording system with longer continuous recording capacity and larger signal-to-noise ratio to detect spontaneous activity in a single sweep. we applied this system to the sensorimotor cortex of rats anesthetized with a mixture of urethane and ␣-chloralose. the hindlimb region was exposed and stained with rh , a voltage sensitive dye. optical records, after compensation for pulsation artifacts, contained deflections time-locked to the high amplitude transient waves, characteristic to ␣-chloralose anesthesia, recorded with a wire electrode placed in the optically sampling area. as the transient in the electrocorticogram fluctuated, the optical signal also varied. this signal was distributed over a broad region, whose latency, amplitude or shape varied systematically within the region, probably reflecting the regional differences in the transient activity. yuko tanaka, r. allen waggoner, kenichi ueno, keiji tanaka, kang cheng bsi, riken, saitama, japanobjective: in this fmri study, we attempted to identify the brain regions involved in the process completing objects with degraded image information.method: fourteen healthy subjects were studied using a t mri scanner while performing a matching-to-sample task with three task conditions. the main condition required the subject to judge in a -s trial if a trial-unique, degraded animal image matched a contour image. in the comparison condition, we reversed the order presenting intact and degraded images (id epoch).results: comparing images acquired in di epochs with those acquired in id epochs, significant activation was found in the left parietooccipital cortex spanning the cuneus (ba ), superior occipital gyrus around the parieto-occipital sulcus (ba ) and superior parietal lobule (ba ). other activated foci include the left anterior cingulated cortex, left dorsal frontal gyrus and right middle frontal gyrus.conclusion: these results indicate that the parieto-occipital cortex is critically involved in the object completion with degraded images.ps p-j influence of task difficulty during meter inspection: an fmri study naoki miura , , makoto takahashi , jobu watanabe , , shinya uchida , , shigeru sato , kaoru horie , masaharu kitamura , toshio wakabayashi , katsuki nakamura , , ryuta kawashima crest, jst, kawaguchi, japan; niche, tohoku univ. sendai, japan; graduate school of engineering, tohoku univ. sendai, japan; bme institute, waseda univ., tokyo, japan; idac, tohoku univ., sendai, japan; lbc research center, tohoku univ., sendai, japan; department of animal models for human disease, ncnp, tokyo, japanthe purpose of the study was to analyze the cognitive process of a subject facing a human-machine interface (hmi) using fmri. we compared brain activation during meter inspection tasks with different task difficulty. during the meter inspection tasks, the subjects were instructed to inspect the three meters, and to press the button, if the subject found abnormal state. the task difficulty was devised by controlling the rate of change for the value to be displayed. in the right occipitotemporal area and the left cerebellar posterior lobule, activation during analog meter inspection was greater when the task difficulty was higher case. the results suggest that these regions are related to attention and perception of visual appearance of hmi.ps p-j neural connectivity among brain areas related to language function shinya uchida , , , naoki miura , , jobu watanabe , shigeo kinomura , kazunori sato , yasuyuki taki , kentaro inoue , ryoi goto , ai fukushima , kaoru horie , shigeru sato , katsuki nakamura , hiroshi fukuda , ryuta kawashima department of nuclear medicine and radiology, idac, tohoku university, sendai, japan; niche, tohoku university, sendai, japan; national institute of neuroscience, ncnp, kodaira, japan; japan science and technology agency, kawaguchi, japan; bme institute, asmew, waseda university, tokyo, japan; graduate school of international cultural studies, tohoku university, sendai, japanthe present study examined the neural connectivity of languagerelated regions using functional mri (fmri) and diffusion tensor imaging tractography (dtt). twenty subjects were participated. functional region of interest (roi) in the left inferior frontal gyrus (lifg) defined by fmri during speech production task was used as a seed point for dtt. in more than % of subjects, tracts between the roi and the left thalamus (lth) were estimated. post hoc fmri analysis showed activation in the lth during speech production tasks. therefore, cortical connectivity between the lifg and lth may have certain functional roles in speech production. keisuke wakusawa , , motoaki sugiura , yuko sassa , , hyeonjeong jeong , , kaoru horie , shigeru sato , hiroyuki yokoyama , kazuie inuma , ryuta kawashima niche, tohoku univ., sendai, japan; department of pediatrics, tohoku univ. graduate school of medicine, sendai, japan; miyagi univ. of education, sendai, japan; ristex, jst, tohoku univ., sendai, japan; gsics, tohoku univ., sendai, japan; lbc rc, tohoku univ., sendai, japanthis study examines the cortical mechanisms of comprehension of implicit social meanings such as irony and metaphor. healthy subjects judged whether the utterance in a picture such as irony, metaphor, or control expressions was situationally appropriate (s), or literally correct (l). greater activation during s than l task was analyzed to identify the activation for implicit meanings and neural responses to irony or metaphor were analyzed. the left medial prefrontal cortex showed higher activity during the s than l task. the medial orbitofrontal cortex and the right temporal pole showed responses selective to the irony; the responses in the former were observed during s task only, while the latter in both tasks. no selective response to metaphor was observed. keiichi onoda , yasumasa okamoto , kazuhiro shishida , akiko kinoshita , shigeru toki , kazutaka ueda , hidehisa yamashita , shigeto yamawaki department of psychiatry and neuroscience, hiroshima university, hiroshima, japan; training and research center for clinical psychology, hiroshima university, higashi-hiroshima, japan anticipation of emotional events may affect perceptual and cognitive processes when the events actually happen. we studied the effects of anticipation of positive and negative affective images on neural processes estimated with meg and event-related fmri. participants were presented emotionally positive or negative images with cue stimuli. the cue stimulus indicated the emotional valence of the image which followed a few seconds later. in meg study, visual evoked field (vef) was smaller for the anticipatable negative image than the anticipatable positive image. this result suggests that when the presentation of a negative image is anticipated before the event, neural processing for the image is depressed compared to when a positive image is anticipated. furthermore, we report the difference of brain activation between anticipation of positive images and that of negative images in event-related fmri study. makoto wada , , , kenji yoshimi , , noriyuki higo , yong-ri ren , hideki mochizuki , yoshikuni mizuno , shigeru kitazawa , , dept. of physiol, juntendo univ. schl. of medicine, tokyo, japan; dept. of neurol., juntendo univ. schl. of medicine, tokyo, japan; crest, jst, tokyo, japan; neurosci. res. inst., aist, tsukuba, japanwe developed a new method for comparing immunopositive cell densities across groups of animals and creating statistical parametric maps on standardized sections. as an example, we compared iba- positive glial cell densities in rats with and without unilateral injection of mpp+. immunopositive cell density map was automatically created in each animal over a coronal section in the midbrain (bregma − . mm). after the map was normalized to a template section, positive cell densities of the two groups were compared in each pixel and a statistical parameter was mapped on each pixel. we were able to detect significant increases of microglias in the side of the injection not only in the substantia nigra pars compacta but also in the white matters. the new method was proven to be useful for detecting significant changes of cell densities over the entire area of immunostained sections. key: cord- -ilhr iu authors: nan title: isev abstract book date: - - journal: nan doi: . / . . sha: doc_id: cord_uid: ilhr iu nan introduction: primary tumours secrete large amounts of extracellular vesicles (evs), which play critical roles in preparing distant sites for a pre-metastatic niche formation, thereby promoting metastasis and even determining metastatic organotropism. whether biogenesis, secretion rates and organotropism of evs are linked remains unknown. we have recently shown that ral gtpases control evs secretion in nematodes as well as in mouse mammary tumour cells (hyenne et al. jcb ) . since both rala and ralb are overexpressed or over-activated in various human cancers, we aimed to investigate the mechanisms by which these two gtpases control evs secretion and to determine how this affects metastatic progression, with a focus on breast cancer. methods: we used t mouse mammary carcinoma cells knocked down for either rala or ralb and determined their ability to induce orthotopic tumours and metastasis in a syngeneic mouse model. in vitro, we investigated ev secretion mechanisms using confocal and electron microscopy (em). evs were isolated either by uc or sec and characterized by nta, em, rna sequencing and mass spectrometry. the function of evs was assessed using a transwell assay. finally, we tracked the organotropism of fluorescently labelled evs and their capacity to induce pre-metastatic niches in mice. results: we show that rala and ralb promote lung metastasis of breast cancer cells in mice without affecting their invasive behaviours. we found that rala and ralb control the biogenesis of exosomes, by acting on the formation of multi-vesicular bodies though the phospholipase pld . as a consequence, knock down of rala or ralb reduces the levels of secreted evs and modifies their rna and protein contents. these differences alter the pro-tumoural function of evs, as demonstrated with an in vitro permeability test. importantly, we show in vivo that evs from rala or ralb depleted cells have a decreased lung organotropism and, as a consequence, are less efficient in priming lung metastasis. finally, we show that high expression of rala or ralb is associated with a bad prognosis in human breast cancer patients. summary/conclusion: altogether, our study identifies ral gtpases as central molecules linking the mechanisms of evs secretion, their dissemination and their capacity to promote metastasis. nuclear proteins are recruited into tumour-derived extracellular vesicles upon expression of tetraspanin tspan introduction: tetraspanin tspan is a transmembrane protein that exhibits a unique expression pattern, being overexpressed in many cancer types, but undetectable in most healthy tissues. although there is increasing evidence of an effect of tspan in invasion, metastasis, and regulation of extracellular vesicle cargo, the molecular mechanisms of tspan are yet not fully understood. methods: to study the function of tspan , we have established a fibrosarcoma model consisting of the parental cell line (ht ) and its derivatives expressing tspan (ht -tspan ) either fused with different fluorescent tags or tag-free. life imaging, sted and storm microscopy were used to determine the intracellular localization of tspan . co-immunoprecipitation from nuclei lysates was performed to detect direct and indirect interacting partners of tspan . small evs were purified from cell-conditioned media using sec and subjected to mass spectroscopy and ngs for a comprehensive comparative analysis of the proteome and transcriptome of the evs. results: the results of the proteome analysis showed a strong effect in the protein cargo of evs upon tspan expression. remarkably, among of the most regulated targets, several histones and ribosomal proteins were enriched in the evs derived from ht -tspan cells. in line with this finding, life imaging and super-resolution microscopy revealed that, while a majority of the intracellular tspan is located on the cell membrane or intracellular membranes, -as it is known for other tetraspanins-, a portion of tspan is located on the nuclear envelope. in fact, several histones co-immunoprecipitated with tspan , indicating their interaction. summary/conclusion: our data show that the expression of tspan in the tumour cells greatly impacts ev cargo. moreover, localization of tspan on the nuclear envelope, together with the enhanced recruitment of nuclear and ribosomal proteins to the evs, suggests a new mechanism of action of tspan . introduction: extracellular vesicles (evs) modulate tissue development, regeneration and disease through the transfer of proteins, nucleic acids and lipids between cells. currently, the mechanism of cytosolic delivery of ev cargo is largely unknown. here, we unravel how evs release their cargo in recipient cells. methods: evs were isolated from gfp-cd and cd -rfp expressing hek t cells by ultracentrifugation. gfp-cd and cd -rfp evs were added to hek t cells stably expressing anti-gfp fluobody and fluorescently tagged galectin- , respectively. clem and fluorescence microscopy were employed to visualize fluorescent markers in recipient cells. bafilomycin a and u a were used to inhibit endosomal acidification and cholesterol export from lysosomes, respectively. results: fluorescent galectin- which binds to betagalactosides present at the luminal side of endosomes was used to detect endosomal permeabilization. the absence of galectin- recruitment to endosomes in presence of cd -rfp evs showed that endosome permeabilization is not the mechanism behind ev cargo release. gfp-cd ev addition to cells expressing anti-gfp fluobodies resulted in the formation of fluobody punctae, reflecting cytosolic exposure of ev cargo. subsequent clem of the fluobody punctae revealed endosomes as the underlying cellular compartments from where cargo release takes place. neutralization of endosomal ph and accumulation of endosomal cholesterol blocked cargo release, showing that ev cargo release is dependent on endosomal ph and cholesterol level. summary/conclusion: we show that genetically encoded cytosolic probes and clem offer an excellent approach to study both the mechanism and efficiency of ev cargo release in cells. we provide experimental evidence that ev cargo release occurs from endosomes. funding: the research was supported by dutch technology foundation ttw and netherlands organization for scientific research nwo, de cock-hadders stichting, and erasmus mundus namaste scholarship. healthy humans. to determine cut-off values for diagnoses, reference intervals of evs in plasma are needed. to establish such reference intervals, ( ) a significant number of healthy donors should be included, ( ) the presence of non-ev particles, residual platelets, lipoproteins, and haemolysis should be quantified, ( ) flow cytometry signals should be in si units. the long-term aim of this study is to determine reference intervals of ev concentrations in human plasma within known dynamic ranges of the detectors. methods: ( ) to establish a clinical reference, we collected blood from healthy volunteers and prepared platelet-free plasma. ( ) we performed quality control measurements including residual platelet count, serum index, and lipid spectrum. ( ) we measured all samples by flow cytometry (apogee a -micro) and used custom software (matlab r b) to automate calibration of all signals and data processing. scatter signals were calibrated in comparable units of scattering crosssection (nm ) and diameter (nm). fluorescence signals were calibrated in units of molecules of equivalent soluble fluorophores (mesf). results: the quality controls showed that most residual platelet concentrations ranged from ^ to ^ per ml except for one outlier, while the serum index and lipid spectrum were normally distributed. preliminary results of the first donors analysed, show that within the ev size range of - , nm, the median concentration of cd + evs is . • ^ per ml (apc> mesf), cd p+ evs is . • ^ per ml (pe> mesf), cd a+ evs is . • ^ per ml (pe> mesf), and cd + evs is . • ^ per ml (apc> mesf). summary/conclusion: we have developed reliable procedures for establishing reference intervals of ev concentrations, within a well-defined size and fluorescence intensity range, in human plasma by flow cytometry. we are currently applying these procedures to samples to obtain, for the first time, ev reference intervals for human plasma. funding: pol, e. van der is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . introduction: the use of extracellular vesicles for diagnostic and therapeutic applications has seen a major interest increase in recent years because of their capacity to exchange components such as nucleic acids, lipids and proteins between cells. isolation of a pure population of evs is the first step in studying their physiological functions since contamination of ev preparations with non-ev proteins can lead to incorrect conclusions about their biological activities. we have developed a new method termed tangential flow for analyte capture (tfac) using ultrathin nanomembranes to purify extracellular vesicles from pure, highly complex biological fluids such as blood plasma, resulting in a new method for extracellular vesicle purification. methods: the tff microfluidic devices are assembled through a layer stack process using patterned polydimethylsiloxane (pdms) sheets with the membranes sandwiched between top and bottom channels. undiluted plasma was tested in both normal flow filtration (nff) and tangential flow filtration (tff) modes on ultrathin nanomembranes. we have utilized a pore patterning technique called nanosphere lithography (nsl) that uses close-packing of nanoscale beads to pattern pores in an ultrathin membrane. results: nff of undiluted plasma resulted in a protein cake of~ μm on the membrane, which prevented further transport across the membrane and evs were buried in the formed cake that were impossible to identify. however, tfac as a modified version of tff, led to capturing cd positive evs on the pores of the membrane with little evidence of protein fouling. nsl allows us to fabricate nanopockets (bowls with a single pore at the base) with various diameter, depth and pore diameter. using nsl, we further utilize nanopocket membranes to purify ev samples in tfac devices. this nanomanufacturing technology will allow us to pattern nanopockets with various diameter, depth and pore diameter which increases the efficiency of capturing of evs. furthermore, nanopockets can be modified and coated by specific ev markers to capture different subpopulation of evs based on size and affinity and further allows identifying the phenotypic subsets of evs by combining both size and affinity-based techniques. summary/conclusion: we have developed a method for the capture and release of nanoparticles such as evs called tfac using ultrathin nanomembranes. nsl technology can be applied to fabricate nanopockets with different physical and biochemical properties. utilizing nanopocket membranes in tfac system will allow us to separate different subpopulations of evs based on size and affinity. funding: this project was supported in part by the national science foundation (iip ) to j.l.m and t.r.g., department of defence (ca ) to j. l.m., and the national institutes of health (r gm ) to t.r.g. the addition of a size exclusion chromatography step to various urinary extracellular vesicle concentrating methods reveals differences in the small rna profile introduction: urinary extracellular vesicles (evs) and their rna cargo are a novel source of biomarkers for various diseases, however non-vesicular rna (e.g. associated with proteins) is also present within urine. this study aimed to identify the optimal method for isolating and enriching evs from human urine prior to small rna analysis. methods: three ev concentration methods, ultracentrifugation (uc); a precipitation-based kit (pk); and ultrafiltration (uf), were compared using ml aliquots of pooled healthy volunteer urine. evs were then separated from protein contaminants by size-exclusion chromatography (sec). presence of evs was confirmed by transmission electron microscopy and western blotting, and evs were quantified using nanoparticle tracking analysis (nta). small rna content of concentrated urine and fractions obtained by sec (evs and proteins) were evaluated with the agilent bioanalyzer small rna chip. results: ev recovery following sec of concentrated samples was - %, however particle: protein ratio (indicating ev purity) was approximately x greater after sec, regardless of the concentrating method used. uf+sec yielded the highest number of evs (per ml of urine) compared with pk+sec and uc+sec. small rna analysis from uf-concentrated urine (prior to sec treatment) identified peaks at nucleotides (nt) and nt. following sec, rna analysis indicated that ev fractions contained mostly small rna of~ nt, whereas the protein factions contained small rna of nt in size (consistent with mirnas). summary/conclusion: uf+sec provided the best balance between ev recovery (per ml urine) and particle: protein ratio. these data indicate that most of the nt sized rnas, presumably mirnas, are not within evs in urine. ev preparations obtained after uc, pk and sec (regardless of concentrating method) contain pre-dominantly~ nt sized small rna. these data outline the importance of removing non-vesicular proteins and rna from urine ev preparations prior to small rna analysis. funding: this research has been funded by petplan charitable trust. the use of rev for the optimization of ev separation and characterization by af introduction: the reproducibility of extracellular vesicle (ev) research has been hampered by the infinite number of separation and measurement techniques and the lack of appropriate reference materials (van deun et al., nat methods, ) . recombinant extracellular vesicles (rev) were developed as a biological reference material to overcome these limitations (geeurickx et al. nat comm ) . since rev have ev-like physical an biochemical characteristics and as they are trackable and distinguishable from sample ev they can be used as a spike-in material for data normalization and method development, and as a quality control. we used rev to optimize ev separation by asymmetrical flow field-flow fractionation (af ). methods: an af long channel column with a frit inlet driven by the eclipse system (wyatt) was coupled to a uv detector (shimadzu), mals dawn helios-ii (wyatt) and fluorescent detector (agilent). a spacer of µm and a regenerated cellulose membrane of kda were used. pbs supplemented with . % nan was used as a running buffer. light scatter profiles and uv profiles were analysed as well as the fluorescent emission spectrum as the rev are gfp positive. fractions were collected and analysed by nanoparticle tracking analysis (nta) and western blot. we also estimated the repeatability and reproducibility of the af technique by light scatter and fluorescence profiles as well as the recovery efficiency by nta. results: in a first step * ^ rev isolated from conditioned medium by a velocity gradient were injected in the af system to optimize the ev characterization protocol. later concentrated conditioned medium was spiked with * ^ rev and injected in the af column to optimize ev separation from non-ev contaminants. the most optimal separation protocol was obtained by varying detector and cross-flow settings. this protocol shows elution of monodisperse particles at each time point and size distribution estimations by af correspond to size determination by nta and electron microscopy. summary/conclusion: we were able to optimize the af protocol for characterization of ev by af as well as for separation of ev from crude conditioned medium samples by using rev. we demonstrate that rev are suitable for method development and that af has high potential as an ev separation technique. comparative evaluation of ev isolation methods for ev subpopulation analysis in human urine, plasma and cell culture media liang dong, richard zieren, kengo horie, sarah amend and kenneth pienta the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: extracellular vesicles (evs) are membrane-enclosed particles of variable sizes that are released by any cell types to the extracellular space and are identified in all body fluids. a shortcoming in ev research is the lack of standardized isolation protocol for various sample types, resulting in heterogeneous outcomes in downstream analyses. in this study, we compared the ev isolation purity and efficiency among ultracentrifugation (uc), precipitation, sizeexclusion chromatography (sec) and a microfluidic tangential flow filtration device (exodisc) in human plasma, urine and cell culture media (ccm). methods: all evs were isolated by different isolation methods and characterized per misev guidelines. single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence and nanoflow (nfcm) were used for single particle analysis. results: in ccm, total particle yield of exodisc was about times higher than those of the rest three methods. size distribution differed per sample, but the ranges were comparable between the different isolation methods. the total protein amount of sec, precipitation and exodisc were similar which were - times higher than that of uc. uc had the highest particle-toprotein ratio followed by exodisc. precipitation and sec had low ratios. when loading ug of total protein for western blot, cd , cd , cd and flot could only be detected in uc and exodisc samples, but not precipitation or sec. sp-iris and nfcm demonstrated consistent purity findings. in urine, total particle yields of exodisc and sec were about times higher than those of the rest two methods. the total protein amount of precipitation was times higher than exodisc and sec, times higher than uc. sec had the highest particle-to-protein ratio followed by uc and exodisc. precipitation had low ratios. in plasma, total particle yields of exodisc and precipitation were times higher than those of the rest two methods. and so were the total protein amount. sec had the highest particle-to-protein ratio followed by uc. exodisc and precipitation had low ratios. western blot, sp-iris and nfcm demonstrated consistent purity findings in urine and plasma. to evaluate particle capture efficiency, we spiked a known number of density-gradient uc purified evs to each method and the recovery rate of uc, precipitation, exodisc and sec was . %, %, . % and %, respectively. summary/conclusion: the order of ev isolation purity in ccm is uc, exodisc, sec and precipitation. in urine it's sec, exodisc, uc and precipitation. and in plasma, this order is sec, uc, exodisc and precipitation. exodisc and sec have similar high isolation efficiency followed by precipitation. uc has low efficiency for ev capture. a capillary-channelled polymer (c-cp) fibre spin-down tip approach for the isolation and biomarker characterization of extracellular vesicles of ovarian cancer origin kaylan d. kelsey, rhonda r. powell, terri f. bruce and r. kenneth marcus clemson university, clemson, usa introduction: extracellular vesicle (evs) profiling has shown promise for disease detection through less invasive sampling (liquid biopsies). current diagnostic tools for ovarian cancer are invasive or only semiinformative. thus, use of evs could prove useful in early disease detection. demonstrated is a hydrophobic interaction chromatography (hic)-based capillarychannelled polymer (c-cp) fibre tip spin-down process for the isolation of ovarian cancer evs for use in diagnostics. methods: polyester c-cp fibre micropipette tips are employed in the isolation of evs from biological matrices including cell culture media, urine, and blood plasma in a spin-down solid-phase extraction (spe) approach. evs were isolated from standards of healthy urine origin and from skov cells (human ovary adenocarcinoma). the c-cp fibre isolation method (taking less than mins and μl sample volumes) preserves the morphology and functionality of evs as confirmed by sem, tem, and confocal fluorescence microscopy. results: the dynamic binding capacity of ev standards on a cm pet c-cp fibre tip was found to be~ e particles ( %). the release of evs was confirmed using dot blot analysis for cd , cd , and cd tetraspanin proteins. immobilized evs were subjected to immunolabeling to allow the positive identification of a profile of ovarian cancer biomarker proteins (her , cd , egfr, epcam, ca ). summary/conclusion: this new ev isolation method introduces a simple capture mode, allowing for direct immuno-characterization and imaging on the fibre surface. this offers a unique and cost-effective opportunity for clinical analyses related to early detection and diagnosis of ovarian cancers (and others). the longterm goal is the creation of a rapid ev isolation and biomarker detection platform. funding: support from the national science foundation, eppley foundation for scientific research, gibson foundation, prisma health system and itor biorepository are gratefully acknowledged. development and optimization of purification method of exosomes by tangential flow filtration and ion-exchange chromatography approach tek lamichhane, ali navaei, sandeep choudhary, yonatan levinson and senthil ramaswamy cell & gene therapy r&d, lonza inc, rockville, usa introduction: extracellular vesicles (evs) such as exosomes have significant therapeutic potential, however, translation of ev-based therapies has been slowed down because of the biomanufacturing challenges. the isolation of evs, especially exosomes, is inherently challenging due to their small size, and heterogeneity in the mixture. the current isolation methods either have low recovery rate, aggregation, damaging the structure, time consuming or co-precipitation of contaminants. specially, it is difficult to process larger sized samples by centrifugation-based or immunoaffinity based methods because of the time and cost associated with these methods. methods: to overcome these roadblocks, we developed and optimized alternative purification techniques to isolate evs with higher purity and yield by using tangential flow filtration (tff) coupled with ion-exchange chromatography. we used bioreactor platform to produce evs from serum-free medium using bm-msc and hek s cells. bm-mscs were cultured on stirred tank bioreactors using microcarriers which provide a high surface area to volume ratio for the optimal cell growth and evs production. impellers were used to enhance mixing and maintain homogeneous culture conditions that can be easily monitored and controlled. results: depth filtration was applied for clarification of conditioned medium. we screened different types of filters during depth filtration for the best recovery of evs. tff membranes with different pore sizes were used to optimize the purity and yield of evs. because of the negatively charged nature of evs, anion exchange chromatography was chosen to capture and separate tff purified vesicles by their surface charge characteristics. we compared monolith based and membrane-based anion exchange columns to remove contaminants and purify exosomal fractions. the purity, size and presence of exosomal markers in isolated evs at each step of purification was evaluated by f-nta, nano-fcm and tetraspanins based elisa kits. summary/conclusion: in summary, our optimized methods improved the speed of isolation and purity of evs to the clinical grade. the production and isolation methods of exosomes that we developed here will be easily expandable to support large-scale and cgmp compatible bio-manufacturing in the future. use of an alternating current electrokinetic microelectrode chip to positively identify oncology, neurology, and infectious disease samples through plasma extracellular vesicle analysis juan pablo hinestrosa, jean lewis, david searson, orlando perrera, alfred kinana, heath balcer and rajaram krishnan biological dynamics, inc., san diego, usa introduction: cancer, neurological, and infectious diseases are leading causes of death, with early detection needed to improve outcomes. extracellular vesicles (evs) in the blood contain disease biomarkers, but current methods do not allow rapid analysis, and are often limited to one biomarker type. methods: we developed methods using alternating current electrokinetics (ace) to isolate evs from bloodbased samples and analyse the evs in situ with downstream assays for protein and nucleic acid biomarkers. we investigated if we could identify tuberculosis (tb) donor samples, protein and nucleic acid biomarkers in evs derived from cancer cell lines, and alzheimer's disease (ad) protein biomarker levels. results: ev isolation was confirmed by positive identification of the proteins cd , cd , and cd and measurement of ev mrnas using a direct rt-ddpcr assay. different disease models were analysed following method development. tb was used as a model for infectious disease, with tb positive and tb negative samples isolated on ace chips and analysed for levels of lipoarabinomannan and ag . using a cut-off above the negatives, the auc of roc curves were . and . , respectively. for oncology, cancer cell lines were cultured and evs isolated from supernatants were spiked into human plasma for analysis. levels of pd-l or glypican- on evs were able to be measured following ace capture. additionally, dna and rna mutations known to be present in the cell lines were able to be detected using ngs and qrt-pcr, respectively. using ad samples as a neurological disease model, tau and phospho-tau t (p-tau t ) in human donor plasma were detected. in ad and healthy donor samples, p-tau t signal increased % in diseased versus healthy donors. summary/conclusion: ace chips are an innovative ev isolation and analysis platform that allow rapid disease sample detection in a wide range of studies with high sensitivity and specificity. introduction: colorectal cancer (crc) is one of the most frequent causes of cancer-related death. in the majority of crc patients, mutation in the apc gene is among the first genetic events. it leads to uncontrolled activation of the wnt pathway, and thus, to adenoma formation. some of these adenomas may then further progress to crc with the accumulation of other mutations. the d organoids maintain the cellular and genetic heterogeneity of in vivo tissues and haves proved to be so far the best ex vivo model of human cancers. here we analysed the ev-based communication between cancer cells and fibroblasts by i) identifying factors that substantially increase ev release from intestinal cancer cells and ii) by determining cargo components of evs that enhance tumour cell proliferation. methods: we used commercially available and patientderived fibroblasts and crc organoids. the medical research council of hungary approved all experiments with human samples and informed consent was obtained from patients. evs were studied by using antibody-coated beads, trps, nta, tem and western-blotting. we introduced apc mutation into wild type murine small intestinal organoids by crispr-cas . results: we found that in crc patient-derived organoid cultures, small evs were preferentially secreted. we observed that apc mutation and the accumulation of the extracellular matrix component collagen critically enhanced ev secretion in intestinal organoids. furthermore, we showed that amphiregulin, present on fibroblast-derived ev, contributed to the maintenance of the intestinal stem cell pool and to cell proliferation in epidermal growth factor-dependent crc organoids. summary/conclusion: by proving the key role of mutations, collagen deposition and ev-bound amphiregulin in the release intensity and functions of the evs, we identified novel mechanisms in the progression if crc. funding: this work was funded by otka-nn , by the national competitiveness and excellence program nvkp_ - (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). prostate cancer-derived evs induce a pro-inflammatory phenotype in the stroma blandine f. victor a , dolores di vizio b , andrew chin c , tatyana vagner b , javier mariscal b , mandana zandian a , catherine grasso a , roberta gottlieb a and helen goodridge a a cedars-sinai medical center, los angeles, usa; b cedars-sinai medical center, west hollywood, usa; c cedars-sinai, los angeles, usa introduction: since % of patients with metastatic prostate cancer (pc) develop bone metastasis, identifying the mechanism that drives this process is essential. most ev research has been focused on the role of exosomes in mediating the pre-metastatic niche formation. however, most of these studies do not separate exosomes from large evs. our preliminary studies have demonstrated that a subclass of evs known as large oncosomes (lo) can reprogram prostate fibroblasts, at the primary tumour site, promoting angiogenesis and enhancing the migration and invasion of pc cells in vitro and tumour growth in vivo. the bone marrow is the initial site of entry into the bone microenvironment for disseminating tumour cells (dtcs) and is a rich source of nutrients that houses various cells types including bone marrow derived mesenchymal stem cells (bm-msc) and immune cells such as neutrophils, which have been implicated in metastasis. here we investigate the role of lo in reprogramming bm-mscs and driving bone metastasis in pc. methods: differential centrifugation, density gradient centrifugation, trps, rna sequencing, qpcr, migration assay, invasion assay, chemotaxis assay. results: we report that pc-derived evs induce distinct gene expressions changes in bm-mscs. rna-seq analysis identified inflammatory and immune regulating cytokines as top differentially expressed genes (deg) in bm-msc. moreover, lo induced a more potent response in bm-msc in comparison to exo and to non-treated controls. the genes enriched in lo treated bm-msc were associated with tumour cell motility. in agreement with the gene expression data, lo-treated bm-msc attracted migration and invasion of significantly more pc cells than exo -treated bm-mscs. in addition, the top deg expressed in ev treated bm-msc were identified as potent neutrophil chemoattractant proteins. in line with the rnaseq findings, the lotreated bm-msc demonstrated enhanced chemotaxis of neutrophils towards them in comparison with exo or vehicle-treated bm-msc. finally, we show that the observed differences in bm-msc's response to lo and exo may be mediated by distinct molecular pathways. summary/conclusion: the results from this study provide novel insight into how tumour derived evs alter the bone marrow microenvironment and how they may drive bone metastasis in prostate cancer. the αvβ integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis introduction: prostate cancer (prca) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sevs). sevs isolated from prca cell media, express the epithelial-specific αvβ integrin, a surface receptor for fibronectin and vitronectin. the αvβ integrin is not detectable in healthy prostate tissues but is highly expressed in prca. in this study, we hypothesized that αvβ in cancer sevs plays a crucial role in angiogenesis. methods: the sevs isolated from prca cell media were characterized by nanoparticle tracking analysis, iodixanol density gradients and expression of sev markers. the αvβ -negative endothelial cells (hmec ) were incubated with αvβ -positive sevs from prca cells to evaluate the transfer of αvβ by immunoblotting (ib) and facs. the effect of αvβ -positive sevs on motility, tube formation and angiogenic signalling were assessed by boyden chamber, angiogenesis assays and ib in hmec . results: we demonstrate for the first time that the αvβ is de novo expressed on endothelial cell surface by sevmediated protein transfer. prca cell-derived αvβ -positive sevs, significantly promote the motility and the formation of nodes, junctions and tubules by hmec . mechanistically, we demonstrate that hmec treatment with sevs from pc cells that endogenously express αvβ , decreases pstat (y ), a negative regulator of angiogenesis, while upregulating survivin, an inducer of angiogenesis. hmec treatment with sevs isolated from pc cells harbouring crispr/cas -mediated downregulation of β , or shrna-mediated downregulation of β , results in increased levels of pstat (y ). this sev treatment also results in a decrease of survivin in sevs and hmec . summary/conclusion: overall, our findings show that αvβ in prostate cancer sevs regulates a novel proangiogenic signalling pathway. funding: this study was supported by nci r - (lrl); p - (lrl and dca). introduction: advanced prostate cancer (pca) is asso-introduction: extracellular vesicles (evs) are secreted from cells, and carry bioactive proteins and rna cargoes. increasing numbers of studies have identified key roles for exosomes in driving aggressive tumour behaviours, including metastasis. however, the detailed mechanisms and responsible factors in the ev cargo are still unclear. recently, immune system has been considered as an important factor in establishing and maintaining metastasis. our goal is to identify the role of head and neck squamous cell carcinoma (hnscc) derived small evs (sevs) in tumour metastasis from the study analysing the effects of sevs on metastasis and tumour immunity. methods: sevs were collected from the conditioned media of hnsccs and purified through cushioned density gradient ultracentrifugation. an orthotopic mouse model was used for the assessment of tumour angiogenesis and metastasis. moc (inflammationinducing rarely metastasizing murine hnscc line) and moc (highly metastasizing murine hnscc line) were used for this study. moc and moc cells were transplanted into mice tongues orthotopically, and moc /moc derived sevs or pbs were injected into the tumour twice in a week. two weeks after tumour transplantation, mice were sacrificed and tumours were sectioned for pathological analysis and facs analysis. in facs analysis, the number and species of tumour-infiltrated immune cells were measured. results: injection of sevs from moc into moc tumours suppressed frequency of lymph node metastasis. on the other hand, injection of sevs from moc into moc tumours didn't promote metastasis. cd positive t-cell distribution in moc tumour was significantly changed by moc sev injection. t-cell deprivation treatment using anti-cd antibody increased the frequency of metastasis in moc -sev treated moc tumours. from the result of proteomics analysis on moc and moc sevs, immune-regulated proteins and metastasis-suppressing proteins were observed in moc sevs. summary/conclusion: we find that low aggressive hnscc sevs affect metastasis of highly metastasized hnscc, and also find that changing immune cell distribution may be related to the result. this mechanism and finding contributes to understanding the possible role of hnscc sevs on metastasis as well as on the tumour immune microenvironment. funding: this work was supported by the nih under award numbers r ca and r ca to aw. desmoglein enhances squamous cell carcinoma tumour development through extracellular vesicles in an il- /mir- a-dependent mechanism introduction: the cadherin dsg is a stem cell marker that is upregulated in many different cancers, including sccs, and its expression correlates with poor prognosis. dsg activates mitogenic signalling and plays a key role in cell proliferation, migration, and survival. we recently showed that dsg enhances ev release, but the mechanism by which these evs modulate tumorigenesis is not fully understood. methods: we established scc cell lines stably expressing wildtype dsg or a palmitoylation deficient mutant, dsg cacs. evs were isolated by sequential ultracentrifugation, iodixanol gradient separation, or qev izon column, and analysed by nta and bca. tumour xenografts were established by subcutaneous injection of cells in scid mice and monitored up to weeks. cytokine profiling was determined by antibody array. mirna expression was analysed by rnaseq and confirmed by qpcr. results: dsg enhanced ev release by % and promoted a~fivefold increase in tumour size in xenograft models. tumour growth was increased when control cells were treated with a single µg dose of evs. loss of palmitoylation, which altered membrane trafficking of dsg , reduced ev release (~ %) as well as tumour development. plasma evs from xenograft mice reflected in vitro particle counts from scc cell lines. a cytokine array analysis was performed revealing that dsg -evs were enriched with pro-inflammatory cytokines including il- , a potent chemotactic and angiogenic factor. most importantly, il- was surface-bound on evs. furthermore, rnaseq revealed mir- a, a negative regulator of il- , to be significantly downregulated in response to dsg . treatment with mir- a mimic or mir- a inhibitor decreased or increased, il- expression in scc cells, respectively. summary/conclusion: in summary, dsg plays a key role in scc tumour development by increasing ev biogenesis and downregulating mir- a, which in turn upregulates il- synthesis and release which can promote invasion, angiogenesis and metastasis. funding: nih r introduction: stem-and progenitor cell transplantation therapy holds great promise for regenerating damaged heart tissue. several lines of evidence suggest that its efficacy is mainly caused by secreted extracellular vesicles (evs). indeed, cardiac progenitor cell (cpc)-derived evs have been shown to protect the myocardium against ischaemia/reperfusion injury in several preclinical models. however, the underlying mechanisms for cpc-ev-mediated cardioprotection remain elusive. here, we utilized the proteomic composition of cpc-evs released during different culture conditions, to unravel protein-mediated effects of cpc-evs on the endothelium. methods: cpcs were stimulated with calcium ionophore (ca ion-evs) or vehicle (control-evs) for hours and evs were isolated from serum-free conditioned medium using size exclusion chromatography. ev concentration and size was assessed using nta. evs were functionally characterized based on endothelial cell activation by western blotting and an endothelial cell scratch assay. the proteomic composition of both ev conditions was profiled using mass spectrometry. cpc-ev knockouts for specific proteins were generated using crispr/cas technology. results: we found enhanced phosphorylation of erk / and akt in endothelial cells and increased wound closure after stimulation with control-evs, but not after stimulation with ca ion-evs. proteomic analysis identified a total of ev-associated proteins, with proteins uniquely expressed in control-evs. another proteins were revealed as candidate proteins, based on their relative enrichment in control-evs compared with ca ion-evs. go analysis demonstrated that differentially expressed proteins were involved in vascular endothelial growth factor signalling, extracellular matrix organization and angiogenesis. to investigate the involvement of the individual candidate proteins on endothelial cell activation, knockout evs of multiple proteins were generated and functionally characterized. summary/conclusion: a specific set of ev proteins is identified that may be functionally responsible for the activation of endothelial cells upon exposure to cpc-evs. generating knockout evs for each of these proteins will help to investigate their individual roles. this may lead to a better mechanistic understanding of the use of cpc-evs as therapeutics for cardiac repair. funding: erc- -cog- evicare grant. hypoxia enhances the therapeutic potential of human cd + stem cell exosomes in ischaemic hindlimb repair ischaemic cardiovascular disease. we have previously shown that human cd + cell-derived exosomes (cd exo) improve perfusion and function of the ischaemic tissues. hypoxia is shown to modulate the secretion and content of exosomes in both cardiovascular and cancer research. therefore, we hypothesized that hypoxia can modulate the content and regenerative efficacy of human cd exo. methods: cd exo were isolated from primary human cd + stem cells cultured under hypoxia ( . % o , or normoxia ( % o , n-cd exo) using density gradient ultracentrifugation. cd exo size was measured using trps, nta, and dls and surface protein expression was determined using imaging flow cytometry. function of cd exo was assessed using cell viability, migration and matrigel tube formation assays in vitro and a mouse hind limb ischaemia model (hli) in vivo. protein content of hypoxic or normoxic cd exo was evaluated via lc-ms/ms and -d -dige followed by lc-ms/ms. results: we did not observe any significant differences in size or in quantity of exosomes secreted from h-or n-cd cells. both h-and n-cd exo expressed cd , cd and cd surface markers. interestingly, h-cd exo significantly improved cell viability, migration and tube formation of huvecs in vitro compared to n-cd exo. in the same line, h-cd exo also significantly improved perfusion (ratio: . ± . v . ± . ) and prevented ischaemic limb amputation ( % v . %) as compared to n-exo (p < . ; n = - ) in a murine (balbc nude) model of hind limb ischaemia. flow cytometry and confocal microscopy indicated that h-exo was uptaken by endothelial cells in the ischaemic limb. remarkably, we detected several proteins (including a fragment of hemopexin) and mirnas (mir- ) that could be responsible for the proangiogenic and beneficial function of h-cd exo. we have also demonstrated that removal of surface proteins diminished the pro-angiogenic function of cd exo. summary/conclusion: hypoxia enhanced the proangiogenic and regenerative potential of cd exo, and thus, may represent a more efficient clinical strategy for cd exo therapy. our research is clinically important to improve therapeutic angiogenesis in diabetic and cardiovascular patients with compromised stem cell populations. hyun-ji park a , jessica r. hoffman b and michael davis b a emory university, decatur, usa; b emory university, atlanta, usa introduction: exosomes, a subset of membrane nanovesicles, transfer cellular information by passing proteins and nucleic acids between cells. exosomes have been implicated as the mechanistic unit in stem cell therapy, as inhibition of exosome synthesis abrogates the effects of cell therapy following cardiac injury. more importantly, increasing evidence indicates that mirnas (mirs) within exosomes serve as important signalling molecules to regulate inflammation, recruit stem cells, and repair diseased tissue. among exosomal mirs, mir- and − are known to decrease angiogenesis, cell migration, and increase inflammation in various types of cells. here, we investigated the inhibition of these negative mirs as a means to improve the reparative capacity of c-kit+ progenitor cell (cpcs) exosomes. methods: cpcs were isolated from three paediatric patients using magnetic-bead sorting. ʹ-o-methylated rna duplexes inhibited mir- and − expressions in cpcs. exosomes (inhexos) were isolated from mirinhibited cpc conditioned medium. mir expression in exosomes and cpc was quantified by qrt-pcr. migration and proliferation of mesenchymal stem cells (mscs) were assessed two days post-exosome treatment. for inflammation analysis, thp cells with/without tnfα exposure were treated with exosomes and the expression of il- , − , and − was quantified by qrt-pcr. finally, the angiogenic potential of inhexos was tested by tube formation of cardiac endothelial cells. results: inhibitor treatment of cpcs decreased exosomal mir- and − expression. treatment with inhexos enhanced msc migration and proliferation compared with normal cpc exosome (norexo). moreover, inhexos showed promising results for immune regulation, as tnfα-induced inflammation was decreased in thp exposed to inhexos for h. however, tube formation capacity is slightly decreased (~ %) by inhexo compared to norexo. summary/conclusion: exosomes from mir- and − -depleted cpcs may be a promising strategy for the treatment of various cardiac diseases, as they enhanced stem cell recruitment and proliferation, and regulated inflammation and angiogenesis. while other studies focus on boosting the reparative potential of exosomes by increasing positive mir and mrna cargo, the inhibition of negative mir in exosomes could be an overlooked strategy for the treatment of cardiac disease. endo-lysosomes as an alternative intracellular location for ev cargo delivery with disease relevance introduction: extracellular vesicles (ev) are lipidbilayer nanovesicles that carry macromolecules and act as paracrine vectors for cell-to-cell communication. the processes regulating ev biogenesis are largely known, whereas how ev cargo is delivered to recipient cells remains poorly understood. a simple mechanism proposed is direct ev fusion with the cell membrane that liberate cargo into the cytosol. in this study, we observed that cargo release occurs also at an alternative intracellular location and that this acquires a disease relevance. methods: ev were isolated by serial centrifugation and characterized. for uptake studies, ev were traced by labelling donor cells with a lipophilic dye or by overexpressing gfp-cd . uptake was assessed by cytofluorimetry or by live confocal imaging. co-localization studies were performed with ectopic marker expression or by immune staining. protein-protein interaction was analysed by bi-molecular fluorescence complementation (bifc). prion-like transmission was studied using a pro-fibrillogenic tau fragment in donor cells and full-length tau in recipient cells. for quantification of subcellular localization, an automated algorithm based on machine learning was developed. lysosomal stress was monitored by nuclear translocation of tfe and lysotracker staining. antibodies directed against pathogenic epitopes of tau were employed to assess prionlike transmission. results: ev were taken up by recipient cells through an endocytic process and accumulated in endo-lysosomes (el). when cells were exposed to ev carrying a profibrillogenic tau, recipient cells accumulated tau within el by an autophagic process. direct interaction of ev-tau and cellular tau in el favoured the appearance of pathological epitopes. cells displaying this condition showed an increased el stress and cytotoxicity. summary/conclusion: in this study, for the first time we report that el represent a critical subcellular location where transcellular prion-like transmission mediated by ev of a neurodegeneration-associated protein occurs. thus, the degradative pathway most likely involved in the recycle of ev and endogenous proteins is highjacked in disease. these findings represent a novel mechanism for ev acting as vector for transcellular propagation of tau, which opens up new therapeutic interventions trying to halt the disease. funding: supported by gelu foundation. anti-human fab fragment of cd antibody prevents the endocytosis of melanoma and colon cancer-derived extracellular membrane vesicles and nuclear transfer of their cargos introduction: interfering with the mechanisms regulating intercellular communication mediated by extracellular membrane vesicles (evs) may find relevance especially in oncology where cancer cell-derived evs have an implication in the malignant transformation of tumour microenvironment. our laboratories recently demonstrated a novel intracellular pathway in which a fraction of endocytosed ev-associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of rab + late endosomes entering into the nucleoplasmic reticulum. here, we have investigated the effect of a monovalent fab antibody against the tetraspanin cd (referred hereafter as cd fab), on the internalization of evs and nuclear transfer of their cargo proteins. chair: david r f. carter -oxford brookes university chair: neta regev-rudzi -weizmann institute of science methods: to monitor the intracellular transport of ev-associated proteins, we used bioengineered fluorescent evs containing cd -gfp fusion protein from femx-i melanoma, sw colorectal cancer and bone marrow-derived mesenchymal stromal cells (msc) as donors and the same cell types as recipients. evs were enriched by differential centrifugation from h serum-free conditioned media and characterized by zetaview nanoparticle tracking analysis, zetapotential and immunoblotting. cd fab was prepared from h hybridoma cells using the pierce fab purification kit. results: we previously demonstrated that silencing cd both in evs and recipient cells strongly decreased the endocytosis of evs and abolished the nuclear transfer of their cargos. here we show that cd fab significantly reduced the cellular uptake of cd -gfp+ evs and the nuclear transfer of their proteins in melanoma, colorectal cancer and msc used as receptor cells in a dose-dependent manner. the effect on the nuclear transfer is probably a direct consequence of the endocytosis inhibition of evs. in contrast, the divalent, intact cd antibody stimulated both events. summary/conclusion: the effect of cd fab appears independent of the used ev-donor cell types or receptor cells, probably due to the widespread expression of cd both at plasma membrane and ev surface. in conclusion, by impeding intercellular communication in the tumour microenvironment, cd fab-mediated inhibition of ev uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti-cancer therapeutic strategies. a bright, versatile reporter for multivesicular body trafficking and exosome secretion and uptake bong hwan sung, ariana von lersner, jorje guerrero, evan krystofiak, david inmann, roxanne pelletier, andries zijlstra, suzanne ponik and alissa weaver vanderbilt university, nashville, usa introduction: live imaging of exosomes is one of the required tools to understand the function of exosomes. our previous live-cell reporter, phluorin-cd allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. however, there were some caveats to its use, including dim fluorescence and the inability to make cell lines that stably express the protein. methods: a stabilizing mutation, m r is incorporated in the phluorin moiety and now exhibits stable expression in cells and superior monitoring of exosome secretion. a dual-tag reporter was created by incorporating a further ph-insensitive red fluorescent protein, mscarlet to the c-terminus of phluo_m r-cd . cancer cells stably expressing the constructs were imaged using a variety of microscopy techniques in vitro as well as in vivo. purified small evs labelled with phuo_m r-cd were imaged using immunogold transmission electron microscopy (tem) and quantitated for the half-life in the blood circulation using flow cytometry. results: phluo_m r-cd and phluo_m r-cd -mscarlet are exclusively detected in exosomeenrich small ev preparations. immunogold tem visualizes the phluo_m r tag is located on the surface of small evs. live cell imaging reveals phluo_m r-cd -positive puncta left behind migrating cells suggesting the deposition consists of exosomes. those puncta and trails are not only positive for exosome markers such as cd , alix, and tsg but also correspond to small evs observed by a scanning electron microscopy. the dual-tag reporter allows visualization of the exosome lifecycle, including multivesicular body (mvb) trafficking, mvb fusion, exosome uptake and endosome acidification. summary/conclusion: using phluo_m r-cd construct, we demonstrate superior visualization of exosome secretion in multiple contexts and a role of exosomes in promoting leader-follower behaviour in collective migration by observing that exosomes are secreted at the front of migrating cells and left behind in exosome trails. the dual-tag reporter allows visualization of the entire exosome lifecycle. we anticipate that these reporters will be broadly useful to investigate regulation and functions of exosome secretion and uptake in diverse physiological conditions. funding: r gm , r ca , u ca - s , r ca . uncovering novel genes regulating ev-mediated functional rna transfer using a crispr/cas -based reporter system introduction: extracellular vesicles (evs) play a pivotal role in intercellular communication through functional transfer of bioactive cargo, including rna molecules. despite increasing interest in ev-mediated rna transfer, our understanding of the pathways and mechanisms regulating ev-mediated rna delivery and processing is limited due to a lack of suitable readout systems. we recently developed a novel crispr/cas -based reporter system that allows study of ev-mediated rna transfer at single-cell resolution. here, we further validate this system by studying the role of known targets involved in ev uptake and intracellular membrane trafficking, and subsequently employ this system to uncover various novel genes that play a regulatory role in functional rna transfer. methods: we employed a novel crispr/cas -based stoplight reporter system, in which egfp expression is activated upon functional delivery of targeting single guide rnas (sgrnas) stably expressed by donor cells. intercellular functional rna transfer was assessed by measuring egfp expression in acceptor cells using fluorescence microscopy and flow cytometry after direct co-culture, transwell co-culture, and upon addition of isolated evs. potential roles of various genes in intercellular rna transfer were assessed by rnaimediated target knockdown in acceptor cells, prior to co-culture experiments. rnai knockdown was confirmed by qpcr analysis. results: a significant activation of egfp expression was observed in acceptor cells after direct co-culture and transwell co-culture with donor cells expressing sgrnas, as well as after addition of evs from cells expressing sgrnas. reporter activation was substantially decreased after knockdown of multiple targets involved in ev uptake through endocytosis and/or intracellular membrane trafficking. based on these results, a potential role of various novel genes in intercellular rna transfer was studied in acceptor cells. these experiments uncovered various novel targets involved in ecm binding, endocytosis, intracellular membrane trafficking, as well as various rho gtpase interactors. summary/conclusion: we previously demonstrated a crispr/cas -based reporter system that allows the study of functional delivery of small non-coding rnas with single-cell resolution. here, we show that this novel approach allows the study of specific genetic targets and pathways in ev-mediated functional rna delivery, and unravel the regulatory pathways that dictate the underlying processes. quantitative characterization of extracellular vesicle uptake and content delivery within mammalians cells gregory lavieu a , emeline bonsergent b , eleonora grisard c and clotilde théry d introduction: extracellular vesicles (evs), including exosomes, are thought to mediate intercellular communication through the transfer of biomolecules from donor to acceptor cells. occurrence of ev-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. methods: we developed a cell-based assay in which evs containing luciferase-tagged cytosolic cargo are loaded on unlabelled acceptor cells. measurement of luciferase activity associated with acceptor cells revealed ev uptake efficacy. additional cell fractionation procedure that separates membranes from cytosol revealed the occurrence of ev-content release within the cytosol of acceptor cells. results: results from dose-responses, kinetics, and temperature-block experiments suggest that ev-uptake is limited ( % spontaneous rate at h), does not depend on bona-fide ev-receptor, at least for the tested acceptor hela cells. yet, further characterization of this limited ev-uptake, through cell fractionation that separates membranes from cytosol, revealed the occurrence of ev-content release within the cytosol of acceptor cells. cytosolic release is inhibited by bafilomycin-a and overexpression of ifitm proteins, which prevent virus content delivery. summary/conclusion: our results show that ev-content release requires endosomal acidification and suggest the involvement of membrane fusion. funding: anr -ce - - and arc pja and pga rf . introduction: glioblastoma is a highly malignant brain tumour with a poor prognosis. its ability to develop therapeutic resistance result in devastating clinical outcomes. to solve the intractable problem, we need highly sensitive diagnostics that can detect the molecular changes during treatments. extracellular vesicles (evs) can be a potential biomarker to monitor treatments and the host cell ev mapping can better reflect molecular changes in the tumour immune microenvironment. we have developed a droplet-based single ev protein sequencing platform that overcomes limitations of current bulk measurement technologies, which make it difficult to discover a rare ev population in the presence of high background. methods: we multiplex protein measurements to profile hundreds of proteins at a time by using an antibody-dna conjugate and sequencing. we barcode each ev in droplets and make amplicons that are comprised of both ev barcodes and antibody barcodes for sequencing. barcoded antibodies are made using tco-tetrazine click reaction and evs are labelled with these barcoded antibodies. the labelled evs are encapsulated into droplets with barcoded beads that serve as a template for ev barcodes. we then perform extension to make amplicons that contain both ev barcodes and antibody barcodes for sequencing. results: we successfully fabricated barcoded beads using a split-pool approach and validated by observing a fluorescence decrease of the sybr green after dna strand denaturation. we used a -channel droplet maker to encapsulate barcoded beads, single ev, and master mix into droplets. close packing of barcoded beads allowed > % encapsulation into droplets. both droplet and tube-based methods achieved a similar high amplification efficiency (ct < for evs). we confirmed the amplicon size by running a gel, which showed the right amplicon size (~ bp) from the droplet and tube prepared samples and no signal from the negative control. summary/conclusion: the droplet-based single ev profiling platform has the ability to identify rare immune ev subtypes in the peripheral blood, which would otherwise be impossible to detect due to the copresence of abundant normal evs. this cutting-edge technique has the potential to revolutionize treatment monitoring of high-cost immunotherapies, avoid unnecessary toxicities, and enhance personalized medicine capabilities. funding: schmidt science fellows, in partnership with the rhodes trust po ca , ro ca , r ca quantbio graduate student award at harvard university. introduction: in this study, we compared four orthogonal technologies for sizing, counting, and phenotyping of evs. the platforms were: single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence, nanofcm nanoflow (nf), nanoparticle tracking analysis (nta) with fluorescence, and microfluidic resistive pulse sensing (mrps) . results from these platforms were compared with results from standard ev characterization techniques such as transmission electron microscopy (tem) and western blot (wb). methods: human t lymphocyte h (high cd , low cd ) and promonocytic u (low cd , high cd ) cells were chosen for their distinct tetraspanin profiles without abnormalities that might result from genetic manipulation. evs were isolated from culture conditioned medium (ccm) by differential ultracentrifugation (duc) and size exclusion chromatography (sec) and characterized per misev guidelines. synthetic particles (silica and polystyrene spheres) with known concentrations and mixed size distributions were also tested. results: particle counts from nf and mrps were consistent, while nta detected approximately one order of magnitude lower for ccm derived evs, but not for synthetic particles. sp-iris events could not be used to estimate particle concentrations. for sizing, nf, mrps, and sp-iris returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of nm. nta detected a population of particles with a mode diameter above nm. additionally, sp-iris, nf, and mrps were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. finally, for tetraspanin phenotyping, the sp-iris platform in fluorescence mode and nf were able to detect at least two markers on the same particle. summary/conclusion: based on the results of the study, we can draw conclusions about existing singleparticle analysis capabilities that may be useful for ev biomarker development and mechanistic studies. funding: this project is funded by mh and ug ca . importance to ev organotropism. yet, most techniques rely on bulk characterization, or are severely restricted by the diffraction limit. the exoview r (nanoview biosciences) combines interferometry, immunocapture, and immunofluorescence, introduced as an alternative technique to multiplex protein detection on single evs below the limit of diffraction. here, we use this technique to characterize tetraspanin multiplexing on evs and to identify spatial patterning of tetraspanins using steric hindrance of antibodies (abs). methods: evs were isolated from conditioned media from skov- cell culture or human serum. evs were incubated overnight on chips to allow immunocapture by anti-cd , anti-cd , or anti-cd . chips were then incubated with three fluorescent abs against the same epitopes and imaged on the exoview r . following concentration optimization, evs were tested after preincubating with carboxy-fluorescein diacetate succinimidyl ester (cfse) or fluorescent abs against tetraspanins. results: using different concentrations of evs, binding curves could be fit to characterize binding kinetics of abs. maximum concentration of evs could be identified that minimized fluorescent overlap. bright-field interferometry (detection limit~ nm) distinguished x fewer bound evs than fluorescent detection, while pre-labelling evs with cfse produced x more detectable evs than immunofluorescence. interestingly, evs captured by one tetraspanin did not necessarily show high fluorescent detection of the same tetraspanin. upon pre-incubating evs with a single ab, vastly different expression profiles were identified, indicating significant steric hindrance between abs. furthermore, pre-incubating evs with anti-cd ab significantly decreased detection of cd with less impact on cd . this discrepancy indicated possible spatial patterning of tetraspanins with cd and cd closely colocalizing on the ev surface. summary/conclusion: this combination of interferometry, immunocapture, and immunofluorescence produces unique information about size distribution of evs and single ev protein profile. this data corroborates that evs have distinct subpopulations of tetraspanins and indicates that tetraspanins may be spatially patterned. regulation of liver homoeostasis, regeneration and diseases by mesenchymal stem cell-derived apoptotic extracellular vesicles university of pennsylvania, philadelphia, usa introduction: billions of cells undergo apoptosis and produce apoptotic extracellular vesicles (apopevs) each day, whereas the roles of apopevs in regulating the organismal health and disease remain poorly understood. mesenchymal stem cells (mscs) emerge as critical contributors to tissue homoeostasis, while mscs suffer from apoptosis in regenerative transplantation. in this study, we investigated the function and mechanisms of msc-derived apopevs in regulating the organismal homoeostasis. methods: fas mutant (fasmut) and caspase knockout (casp -/-) mice were applied for apoptotic and apopev deficiency. mouse bone marrow mscs were cultured and apoptosis was induced by staurosporine (sts). msc-derived apopevs were collected by serial centrifuges and were infused into mouse circulation via caudal vein. tracing of apopevs were performed by radioisotope or fluorescent labelling. liver homoeostasis was evaluated at the histological and functional aspects. liver regeneration was induced by partial hepatectomy (phx). acetaminophen (apap) was used to establish acute liver drug injury. high-fat diet (hfd) was used to establish type diabetes (t d) and non-alcoholic fatty liver disease (nafld). results: after systemic injection, msc-derived apopevs migrate to liver and can be uptaken by liver macrophages and hepatocytes. fasmut and casp -/mice develop hepatomegaly with structural disorders, which particularly reveals hepatocyte polyploidization. furthermore, fasmut and casp -/-mice demonstrate liver glucose and lipid metabolic disorders. importantly, msc-derived apopev infusion significantly rescues structural and metabolic dysfunction in fasmut and casp -/-mice. mechanistically, apopevs use the soluble n-ethylmaleimide-sensitive fusion protein attachment protein receptor (snare) protein for interactions with recipient organelles thus transferring signalling molecules. moreover, msc-derived apopev infusion promotes liver regeneration after phx, prevents apap-induced liver injury, and ameliorates nafld in t d. summary/conclusion: msc-derived apopevs serve as crucial regulators of liver homoeostasis, regeneration and diseases. these findings indicate potential significant roles of apopevs in maintaining the organismal health and in developing therapeutics for diseases. (msc-sevs) mediate osteochondral regeneration in rats. however, the therapeutic effects of these msc-sevs/exosomes in restoring the mechanical competence of the repaired cartilage for joint function in a clinically relevant animal model remain to be addressed. to investigate this, we compared the structural and mechanical properties of the repaired cartilage in a rabbit model after intraarticular administration of msc-sevs and hyaluronic acid (ha) with that of ha alone, which is widely used as visco-supplementation. methods: bilateral osteochondral defects were surgically created on rabbits. immediately after surgery and at days and post-surgery, rabbits received ml injections of µg msc-sevs and ha in both knees, and rabbits received -ml injections of ha in both knees. at and weeks, macroscopic evaluation, histological scoring and compressive testing at different points on the repaired cartilage were performed. results: defects treated with msc-sev/ha showed improvements with time in macroscopic and histological scores and mechanical properties than defects treated with ha alone. in contrast, ha treated defects showed some repair at weeks, but this was not sustained, as evidenced by significant deterioration of histological scores and a plateau in mechanical properties from to weeks. by weeks, the msc-sev/ha repaired tissues demonstrated significantly better macroscopic score ( . vs . ; p < . ) and histological score ( . vs . ; p < . ). mechanical strength as measured by the young's modulus was significantly higher in the msc-sev/ha repaired cartilage than that in ha repaired tissues [defect centre ( . vs . mpa; p = . ) and overall periphery ( . vs . mpa; p = . ], and approximated that of the adjacent native cartilage. summary/conclusion: our findings demonstrated that msc-sevs and ha not only improved tissue morphology of the repaired cartilage but also promoted functional mechanical competence. this study establishes a clinically translatable protocol for use of msc-sevs for cartilage repair. introduction: mesenchymal stromal/stem cell (msc)exosome (mex) treatment has shown considerable promise in experimental models of bronchopulmonary dysplasia (bpd) and pulmonary hypertension (ph). mechanisms by which mex afford their beneficial effects remain incompletely understood and here, we embark into investigating them through assessment of mex biodistribution and impact on immune cell heterogeneity. methods: newborn fvb mice were exposed to hyperoxia (hyrx, % o ) at birth and returned to room air at postnatal day (pn) . mice received a bolus mex dose at pn . adoptive transfer studies were used to determine the role of mex-educated myeloid cells in vivo. mice were harvested at pn , , , or to characterize mex biodistribution and for assessment of pulmonary parameters. results: mex therapy effectively ameliorated core features of hyrx-induced neonatal lung injury, improving alveolar simplification, pulmonary fibrosis, vascular remodelling and blood vessel loss. exercise capacity testing and assessment of ph showed functional improvements following mex therapy. biodistribution studies demonstrated that mex localize in the lung, where they interact with lung monocytes/macrophages. whole lung mass cytometry (cytof) revealed that mex treatment promotes a pro-homoeostatic shift in lung immune cell apportion, replenishing the early hyrx-induced depletion in pulmonary cd + immune cells, restoring alveolar monocyte and macrophage populations and suppressing cellular inflammation. ex vivo and in vivo analysis showed that mex promotes a "pro-resolving" ccr -monocyte phenotype. notably, adoptive transfer of mex-educated bone marrow-derived myeloid cells (bmdmy), but not naïve bmdmy, restored alveolar architecture, blunted fibrosis, improved vascular remodelling and pulmonary blood vessel loss. summary/conclusion: mex treatment ameliorates core features of experimental bpd, restoring lung architecture, decreasing pulmonary fibrosis and vascular muscularization, ameliorating ph and improving exercise capacity. the beneficial actions of mex are associated with modulation of immune cell phenotypes, arising from mex-monocyte interaction. furthermore, adoptive transfer of mex-educated bmdmy rescued, at least in part, alveolar architecture, reduce fibrosis, improve vascular remodelling and pulmonary blood vessel loss. funding: this work was supported in part by an american thoracic society foundation grant (grw); the little giraffe foundation (grw); charles h. hood foundation major grants initiative (sk), nih r hl (sk) and united therapeutics research grant (sk and sam). immunomodulatory small extracellular vesicles derived from mesenchymal stem cells: a potential cell-free therapy for acute and chronic pulmonary vascular diseases introduction: vascular inflammation plays a critical role in acute respiratory distress syndrome (ards) and pulmonary arterial hypertension (pah). despite decades of research, there is no curative therapy for either condition. mesenchymal stem cells (mscs) have shown preclinical efficacy, mediated by release of extracellular vesicles. hence, msc-derived small extracellular vesicles (sevs) can harness the benefits of mscs with advantages in cost and safety. this study aims to evaluate the immunomodulatory effects of sevs in preclinical ards and pah. methods: msc-sevs were characterized by nanoparticle tracking analysis, electron microscopy and western blot. live fluorescence imaging measured in vitro and in vivo distribution of sevs. using a lipopolysaccharide (lps)-induced mouse model of acute lung injury (ali), a time course study of inflammatory response guided endpoint analyses. cell count and cytokines were measured in bronchoalveolar lavage fluid (balf) and histological lung injury was assessed. in ali mice, saline, mscs, msc conditioned media or sevs were administered . h post-lps. using a monocrotaline (mct)-induced rat model of pah, animals received saline or sevs at day . haemodynamic changes and right ventricular hypertrophy were evaluated at weeks. results: msc-sevs were nm in size with cd / expression. pkh -labelled sevs were taken up by endothelial cells. in the ali time course study, cell count and il b in balf peaked at h post-lps, whereas il peaked at h. histology showed significant intra-alveolar cell infiltrate at h. msc conditioned media attenuated il b in balf, whereas a trend towards reductions in il b and cell count were seen from delivery of mscs and sevs. using fluorescence imaging, lung accumulation of dir-labelled sevs was highest when administered h post-lps as compared to h, h or h. for pah rats, sevs reduced right ventricular systolic pressure ( . ± . mmhg) as compared to control ( . ± . mmhg; p = . ), whereas no changes were observed for right ventricular remodelling. summary/conclusion: these findings demonstrate the potential of msc-sevs to be used as a cell-free immunomodulatory therapy for acute and chronic lung vascular diseases. additional live and ex-vivo biodistribution studies will determine optimal timing of sev administration, tissue distribution and clearance in both ali and pah. changes in extracellular vesicle protein cargo after pro-inflammatory priming of umbilical cord mesenchymal stem cells (ucmscs) have been shown to suppress inflammatory responses in studies of autoimmune diseases. these therapeutic effects can be attributed to paracrine signalling, by which extracellular vesicles (evs) are one of the essential components. this study looks at how the culture conditions of ucmscs affects the type of evs they secrete. it also aims to identify an ev population with an anti-inflammatory potential for the treatment of autoimmune diseases. methods: ucmscs were isolated and culture expanded in a quantum® cell expansion system, then grown at %o , %o and primed with a pro-inflammatory cocktail. evs were isolated from ucmsc conditioned media by differential ultracentrifugation using a sucrose cushion and characterised by transmission electron microscopy and nanoparticle tracking analysis. ev markers were analysed using a europium-based immunoassay, macsplex exosome detection kit and immunoblotting. a proximity-based extension assay was used to identify inflammatory proteins in the evs. results: there was no difference in evs cultured at %o , %o or with pro-inflammatory conditions when analysed for size and morphology. all evs displayed the tetraspanin markers (cd / / ) and internal proteins (alix, hsp ). evs from primed cells showed a > twofold increase of cc chemokines and a > sixfold increase in cxcl and csf- . protein cargo did not differ in evs from %o and %o . summary/conclusion: this study showed that proinflammatory culture conditions alter ev protein cargo, evidenced by the increased production of chemotactic and angiogenesis associated proteins. upcoming rnaseq analysis will show if ucmsc culture conditions also affect mirna expression in evs. ongoing functional studies will determine how changes in ev cargo correlates with changes in t-cell proliferation and polarisation. funding: this work is fund by the orthopaedic institute ltd, keele university and the rjah orthopaedic hospital charity. alzheimer's disease biomarkers in plasma extracellular vesicles of neuronal origin correlate with brain pathology in mice introduction: multiple studies have shown that neuronal-derived extracellular vesicles (ndes) in blood contain alzheimer's disease (ad) biomarkers, especially tau. however, the convergent validity of tau in blood ndes in relation to brain pathology is yet to be determined. to address this, we measured total and phosphorylated tau levels in matched nde and brain tissue samples from ad mouse models. methods: we collected the cortex, hippocampus and plasma of xtg-ad, xtg-ad, and wild type (total of mice; female, male; age: mean = . , sd = . , - months) . plasma samples were collected retro-orbitally for weeks and at euthanasia via heart puncture. ndes from the pooled serial blood collections (nde ) and the single endpoint (nde ) were immunocaptured by targeting the neuronal marker l cam. we measured human total tau and pthr -tau (p-tau) in ndes and cortex and hippocampus homogenates using a luminex multiarray. results: overall, there were strong positive correlations for both total tau and p-tau between ndes and brain tissues across mice types. total tau in ndes showed positive correlations with levels in the cortex and hippocampus (r = . and . , p < . , cortex vs nde and nde ; r = . , p = . , hippocampus vs nde ; r = . , p = . , hippocampus vs nde ). levels of p-tau in nde showed positive correlations with levels in the cortex (r = . , p = . ) and hippocampus (r = . , p = . ); however correlations were not observed for nde (r = . , p = . vs cortex; r = . , p = . vs hippocampus). summary/conclusion: tau levels in circulating ndes reflect levels in cortex and hippocampus across ad model mice, supporting their convergent validity as "liquid biopsy" biomarkers for ad. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. exosomal ceramide mediates neurotoxicity of amyloid beta (aβ) in alzheimer's disease. ahmed elsherbini a , simone crivelli b , alexander kirov c , michael dinkins d , zhihui zhu a , haiyan qin a , sanjib karki a , priyanka tripathi a and erhard bieberich a a university of kentucky, lexington, usa; b university of kentucky, lexington, usa; c augusta university, augusta, usa; d augusta university, augusta, usa introduction: amyloid beta is a pathologic hallmark of alzheimer's disease (ad), however, the mechanism of aβ neurotoxicity is not fully understood. it has been reported that exosomes associate with aβ, but it is not clear how this association affects aβ neurotoxicity. methods: here we utilized several techniques to isolate exosomes from the sera of wild type (wt) and ad transgenic mouse model. ( xfad) as well as alzheimer's patients and healthy controls. we used exoquick, exoeasy, sequential ultracentrifugation, and size exclusion chromatography. particles' size and number were characterized by nanoparticle tracking analysis (zetaview). results: we report that the sphingolipid ceramide mediates neurotoxicity of aβ. we show that sera from ad transgenic mouse model ( xfad) and ad patients, but not the wt or healthy controls, contain a subpopulation of astrocyte-derived exosomes that are enriched with ceramide and are prone to aggregation (termed astrosomes) as confirmed by nanoparticle tracking and cluster analyses. when taken up by introduction: multiple sclerosis is the most common chronic inflammatory demyelinating disease of the central nervous system, affecting more than million people worldwide. ms is a multifactorial, immunemediated disease caused by complex genetic and environmental interactions. in recent years, extracellular vesicles (evs) have been described as powerful mediators of the modulation of biological processes (e.g. inflammatory and immune response) following environmental exposures such as particulate matter (pm), and have been described altered in ms. we characterized evs in patients with ms and healthy subjects matched for age and gender and evaluated the effects of pm exposure on ev release patients with ms compared with controls. methods: evs isolated from blood samples were characterized by nanotracking analysis and by flow cytometry after labelling with the following markers: cd + (monocyte), cd + (platelet), cd + (neutrophil), cd + (t-reg), and cd + (endothelium). pm and pm . concentrations at the residency of each subject were obtained from the regional air quality monitoring network. results: we observed decreased concentrations of cd + (p < . ), cd + (p < . ), cd + (p < . ), cd + (p < . ), and cd + (p < . ) in patients compared with controls. in cases, pm was inversely associated with cd + evs (pm . , β = − . ; p < . ), cd + evs (pm . β = − . ; p < . ), and cd + evs (pm β = − . ; p < . ; pm . , β = − . ; p < . ). on the contrary, in controls pm was positively associated with cd + evs (pm β = . ; p < . ; pm . , β = . ; p < . ). summary/conclusion: our findings showed a different composition of blood-derived ev subpopulations in patients compared with controls. moreover, we observed that patients and controls react differently to pm exposure in terms of blood-derived ev release, suggesting the involvement of this mechanism in the modulation of both inflammatory and immune responses, and thus in ms pathogenesis. plasma neuronal and astrocyte-derived exosomes serve as biomarkers of neurodegeneration and systemic bioenergetic effects in male cynomolgus monkeys self-administrating oxycodone ashish kumar a , yixin su a , david soto-pantoja a , jingyun lee a , ravi singh a , cristina furdui a , michael nader b and gagan deep a a wake forest baptist medical center, winston-salem, usa; b wake forest baptist medical center, winston-salem, usa introduction: opioid use disorder (oud) is currently a health emergency in the usa affecting millions of people. oud is a complex issue requiring a multipronged strategy. at the biological level, there is an urgent need to understand the dynamic molecular changes and adverse effects associated with opioid addiction. here, we aimed at identifying the biosignature of brain cells-derived exosomes associated with opioid addiction in a non-human primate (nhp) model of oud in which cynomolgus monkeys perform cognitive tasks and self-administer (sa) intravenous oxycodone daily. we also characterized the systemic adverse effects of the brain cells-derived exosomes from drug-naïve and oxycodone sa monkeys. methods: we isolated total exosomes (te) by ultracentrifugation and exoquick methods from the plasma of male monkeys self-administrating oxycodone for years and naive monkeys. subsequently, from the te population, we isolated neuron-derived exosomes (nde) and astrocytes-derived exosomes (ade) using surface biomarkers l cam (l cell adhesion molecule) and glast (glutamate aspartate transporter), respectively. this novel method involved streptavidin coated magnetic beads and photo-cleavable (pc) biotin, providing us biologically intact exosomes useful for co-culture studies. we characterized the exosomes by nanoparticle tracking analyses (nta), western blotting, flow cytometry, immunogold labelling, transmission electron microscopy (tem), elisas and mass spectrometry. respirometric profiling in cardiac myoblasts and monocytes following exosomes treatment was performed by seahorse xf. results: the quality of isolated exosomes (te, nde, and ade) was confirmed by nta (size distribution and concentration), western blotting (e.g. cd ) and tem (size and shape). nta did not show any significant difference in exosomes size and concentration (number per ml) between control and oxycodone sa groups. flow cytometry (e.g. l cam and glast) and immunogold labelling (cd , cd and l cam) confirmed the purity of nde and ade isolated from te. proteomics analyses of te, nde and ade identified several unique proteins present in exosomes from the oxycodone sa group. interestingly, we observed significantly higher expression of neurodegeneration markers neurofilament light protein (nfl) and alpha-synuclein in nde and ade of oxycodone sa group compared to controls. furthermore, te treatment of h c cardiac myoblasts and raw . monocytes significantly compromised their mitochondrial metabolism (basal and maximum respiratory capacity). summary/conclusion: these results suggest the utility of plasma exosomes as biomarkers for better understanding of the neurodegenerative and systemic effects of oxycodone addiction. funding: da , da . vesicles released during mycobacterium tuberculosis infection: immunomodulatory (glyco)lipids and role in host-pathogen interactions emilie layre, pierre boyer and jerome nigou cnrs-université paul sabatier, toulouse, france introduction: the tuberculosis disease remains one of the top causes of death worldwide. mycobacterium tuberculosis (mtb) has evolved strategies to evade immune responses and to persist within the hostile intracellular environment of alveolar macrophages. the current lack of efficient anti-tuberculosis strategies is largely due to our incomplete understanding of the host-pathogen interactions of mtb infection. vesicles released by the bacillus itself (bacterial membrane vesicles, bmv) and by infected cells (host extracellular vesicles, hev) have immunomodulatory properties in vitro and when administered to animals. if vesicles likely play key role in host-pathogen interactions of the tuberculosis infection, their content in bacterial factors, their uptake, trafficking and interaction with host cells receptors remain incompletely deciphered. methods: bmv and hev have been purified by combining differential centrifugation, density gradient and exclusion chromatography. after characterization by microscopy, nanosight and western blot, their content in bacterial (glyco)lipids has been characterized by the use of high sensitivity mass spectrometry-based lipidomic approach. bmv have been tested for their capacity to activate reporter cell lines of pattern recognition receptors. in addition, fluorescent-labelled bmv have been used to study their uptake by host cells thank to super-resolution microscopy. results: we have undertaken to characterize the content, the trafficking and interaction with pattern recognition receptors of bmv and hev released during infection by mycobacteria of variable virulence. we have importantly optimized the purification of bmv showing that lipoproteins aggregates are co-purified with vesicles on density gradient. sfc-ms lipidomic analyses allowed the characterization of the repertoire of immunomodulatory bacterial lipids released by bmv and hev, which excluded a continuum between these two release pathways. preliminary, assays have shown that these vesicles are capable to interact with different pattern recognition receptors including tlr and lectins. finally, we have been able to visualize fluorescent-labelled vesicles uptake by macrophages using superresolution microscopy. summary/conclusion: during m. tuberculosis infection, the bacillus as well as infected cells release vesicles that harbour different content in immunomodulatory bacterial (glyco)lipids, including strain-specific lipids. these vesicles likely play important role in host-pathogen interactions by modulating immune response beyond the infected cells, in part through their interaction with different pattern recognition receptors. funding: fondation pour la recherche medicale, fondation fonroga. introduction: conventional diagnoses of mycobacterium tuberculosis (mtb) rely on quantifying bacteria in sputum samples, which make it incapable of measuring the body's total bacterial load and diagnosing patients that have difficulty producing sputumsuch as children and those that are hiv-positive. nanoscale ( - nm) outer membrane vesicles (omvs), which are shed from their bacterial cells of origin and circulate in the bloodstream, have been found to contain rich molecular information from their mother cells. despite the diagnostic potential, their nanoscale size in the presence of high background has complicated the use of these promising biomarkers for clinical diagnosis of tuberculosis. chair: amy buck -the university of edinburgh chair: cherie blenkiron -the university of auckland methods: here we report two complementary approaches to systematically discover and clinically detect mtb-derived omvs using protein and rna biomarkers. first, we employ a digital droplet elisa on whole, unprocessed samples to detect and quantify the presence of these omvs using surface protein markers. second, we have developed a platform to specifically enrich for mtb-derived omvs using our previously developed magnetic nanopore platform, wherein millions of nanofluidic devices are operated in parallel, increasing throughput relative to a single nanofluidic device by a million-fold. using this approach, we identify rnas that are specifically enriched in mtb-derived omvs and can be used to identify tb strain, infectious activity, and total body burden. results: using these platforms, we enriched for mtbderived omvs from plasma and profiled their cargo, both proteins and rna. we first determined a panel of protein biomarkers for multiplexed detection of omvs through a digital droplet sandwich elisa. we then tested our protein markers on spiked plasma samples as models for clinical tb samples. simultaneously, we performed rna sequencing and discovered a panel of rna biomarkers that are preferentially enriched in omvs. we picked ten of the most highly-expressed rna biomarkers and also tested for them on spiked plasma samples using our magnetic nanopore platform. summary/conclusion: these results demonstrate the capability of omv biomarkers in the development of novel liquid biopsy based mtb diagnostics. building on this work, we are working with clinical collaborators to test our assays on clinical samples from philadelphia and west africa. funding: nih ot . introduction: a dearth of knowledge exists regarding the molecular mechanisms by which host exosomes regulate immune response to infections caused by gram-negative pathogens. to address this gap in knowledge, our laboratory has been using two wellestablished model organisms; yersinia pestis (yp), and burkholderia pseudomallei (bp). yp and bp cause the emerging human diseases plague and melioidosis respectively. currently, no licenced vaccines or highly effective therapeutics are available for either disease. methods: ex were purified from naïve u monocytes (exu) and infected u (exi) by serial centrifugation followed by sucrose density gradient purification, and characterized by tem, zetaview nanoparticle tracking, and exosome markers (cd , tsg , . immune responses of naïve u cells and response mechanisms were analysed following treatment with equivalent amounts of exi or exu (as control). these included macrophage differentiation assays, multiplex measurements of inflammatory cytokines, bacterial clearance assays, quantitative protein microarray analysis of host signalling proteins, sirna knockdown of exi-induced cytokines in recipient cells, and mass spectrometry (ms) analysis of exi contents. for all assays, at least four biological replicates were performed. results: exi induce monocyte differentiation to macrophages and dramatic release of il- , il- and il- cytokines, effects that are also seen when monocytes are infected with the bacteria. the exi also induce a substantial increase in the capacity of the recipient monocytes to clear bacteria in an il- -dependent manner. specific host signalling molecules are strongly modulated by the exi, including p , jak and alk for exi-yp, influencing the observed phenotypes. ms analysis showed lack of lps in exi-yp and demonstrated the presence of specific bacterial proteins that have antigenic properties. summary/conclusion: we have identified some of the molecular mechanisms by which exi assist the host in clearing infection. exi prime distant naïve monocytes through modulation of distinct pathways such as p to mount immune responses similar to when they become infected. these include differentiation to macrophages and migration to infection site for increased il- -dependent bacterial clearance. introduction: recruitment of monocytes to sites of infection is important in restricting growth and invasion of various microorganisms such as pathogenic fungi c. albicans. beside complement supported phagocytosis and extracellular trap formation, human monocytes secrete extracellular vesicles which are crucial in cellular communication in physiology and pathophysiology as they transfer proteins, lipid, and nucleic acids. the current study attempts to shed light on immune evasion mechanisms by c. albicans via extracellular vesicles. methods: human monocytes were isolated by magnetic beads technique and extracellular vesicles were isolated using polymer precipitation or ultracentrifugation or size exclusion chromatography. vesicles were characterized by elisa, lc/ms-based proteomics, confocal laser scanning microscopy (clsm) as well as electron-and dynamic light scattering microscopy, etc. crispr-cas based genome editing was performed to knockout cd b in human monocytic thp- cells. effect of isolated vesicles were determined by using proximity ligation assay (pla), elisa, western blot, next generation rna sequencing, qpcr, immunohistochemistry, etc. results: here we show for the first time that human blood derived monocytes alone and in a whole blood model strongly induced and released extracellular vesicles in response to the pathogenic fungus c. albicans. one induced population carried the anti-inflammatory cytokine tgfβ- . release of these vesicles is triggered by binding of soluble β-glucan from c. albicans to the cr receptor on monocytes as demonstrated by crispr-cas based cr genome editing in thp- cells, and by using cr knock out mice. isolated tgf-β -transporting vesicles reduced the inflammatory response in human m macrophages and in a whole blood model. the anti-inflammatory effect by tgf-β transporting vesicles is investigated in detail and results in inhibition of il- β gene transcription. summary/conclusion: showing that human apoptotic bodies similarly induced tgf-β -transporting vesicles from human monocytes we hypothesize that c. albicans hijacks this new cr -dependent anti-inflammatory vesicle pathway for immune escape. funding: this work was supported by the "deutsche forschungsgemeinschaft" transregio funginet projects c , c , c and z . introduction: to date, most research involving extracellular rnas has focused in rnas encapsulated inside extracellular vesicles (evs) or in total unfractionated biofluids. it is known that exrnas also exist outside vesicles or in lipoprotein particles. however, nonvesicular exrnas remain widely uncharacterized despite being a feasible source of contaminants in ev preparations. our interest in nonvesicular exrnas arises from the observation that some small rnas, such as specific trna-derived fragments, have much higher relative representation in this extracellular fraction. at least in part, this enrichment seems to be a consequence of their differential extracellular stability. methods: to get a representative picture of the whole set of rnas released to the extracellular nonvesicular space by cultured human cells, we inhibited extracellular degradation by adding recombinant ribonuclease inhibitor to the cell-conditioned media and studied the kinetics of rna release and degradation. high-resolution iodixanol gradients were used to separate evs from extracellular rnps or vesicle-free rna. the conversion rate between parental ncrnas and their fragments was studied by high-throughput sequencing and northern blot. results: the inhibition of extracellular rnase activity revealed the presence of full-length trnas and ribosomes in the extracellular space of a variety of malignant and non-malignant cell lines. extracellular ribosomes co-isolate with evs purified by ultracentrifugation or size-exclusion chromatography, but not with evs purified by density gradients.these ncrnas are substrates of extracellular rnases, demonstrating an extracellular biogenesis route for the formation of ncrna-derived fragments, some of which achieve remarkable stability and can be detected in biofluids. we also highlight the immunoregulatory potential of purified rna-containing extracellular complexes. summary/conclusion: in conclusion, ribonuclease inhibition dramatically shapes extracellular rna profiles and uncovers a population of extracellular ribosomes, trnas and other coding and noncoding rnas which exists outside evs. although these rnas are prone to degradation, some of their fragments can accumulate in cell culture media and in biofluids. this dynamic view of exrnas impacts our understanding of rna secretion mechanisms and may offer a window to new molecules with biomarker potential. in contrast, evs confer an rnase-protected environment and contain more full-length ncrnas (trnas, yrnas, sl rnas, rrnas depending on vesicle size) than their fragments. introduction: cd is a ubiquitously expressed membrane protein that functions as a receptor for thrombospondin- and the counter receptor for signal regulatory protein-α in phagocytes. high expression of cd is associated with a poor prognosis for some cancers. conversely, cd blocking agents are in clinical trials for enhancing innate and adaptive antitumor immunity in cancer patients. these studies suggest utility of cd as a diagnostic and prognostic biomarker and as a therapeutic target. cd is also expressed on extracellular vesicles (evs), and we reported that cd expression identifies a distinct population of evs from those that express the traditional ev markers cd or mhc . cd -, cd -and mhc -enriched vesicles contain distinct small rna populations (pmid: ), and these differ in rna content from evs that lack any of these markers. the mechanisms by which cd directly or indirectly regulates which rnas are packaged into ev remain unknown. methods: to elucidate the mechanism by which cd regulates ev rna composition and function, we performed global mirna microarray analysis between evs produced by wild type and cd -deficient t cells. results were further validated using real-time pcr and rna-immunoprecipitation. interactions between cd and exportin- /ran complex was identified by mass spectrometry and confirmed by using co-immunoprecipitation, subcellular localization, flow cytometry, and confocal and electron microscopy. results: ev released from cd -deficient human t cells and in cd -/-mouse plasma were enriched in ʹ- -methylguanosine-capped micrornas and mrnas that depend on the exportin- /rangtp pathway. knockdown of cd in wild type cells or thrombospondin- treatment correspondingly enhanced levels of capped-rnas released in ev and re-expressing cd in null cells decreased their levels. mass spectrometry and co-immunoprecipitation identified specific interactions of cd with components of the exportin- / ran nuclear export complex and its known cargos and between the cd cytoplasmic adapter ubiquilin- and the exportin- /ran complex. interaction of cd with exportin- was inhibited by leptomycin b, which inactivates exportin- and increased levels of cap-dependent rnas in ev released from wild type but not cd -deficient cells. summary/conclusion: these findings indicate that cd -dependent thrombospondin- signalling regulates cytoplasmic levels of cap-dependent rnas in t cells at least in part through ubiquilin- -and gtpdependent physical interactions with the exportin- / ran transport complex, which regulate levels of specific pre-mirnas and mrnas available for sorting into evs. funding: this work was supported by the intramural research program of the nih/national cancer institute (zia sc ). role of membrane protein palmitoylation in extracellular vesicle biogenesis in squamous cell carcinoma introduction: desmoglein (dsg ), is a palmitoylated cadherin that is involved in cell-cell adhesion. interestingly, dsg promotes mitogenic cell signalling and is upregulated in many cancers, including scc, contributing to poor prognosis and survivability. we recently demonstrated that dsg promotes ev release, but the mechanism by which dsg enhances ev biogenesis and role of palmitoylation is poorly understood. methods: pharmacological drug inhibitors -bromopalmitate, gw , and bafilomycin a were used. stable scc cell lines were established by retrovirus infection expressing gfp, wild type dsg /gfp, or palmitoylation deficient dsg cacs/gfp. evs were isolated by sequential ultracentrifugation and iodixanol gradient separation and analysed by nta. proteins associated with the endocytic pathway were analysed by immunofluorescence and imaged by confocal microscopy or immunoblotting and signals were quantitated using imagestudio. results: here we demonstrate that the effect of dsg on ev release was reduced by the palmitoylation inhibitor -bromopalmitate. furthermore, mutations that prevented palmitoylation (dsg cacs) dramatically abrogated ev release by targeting of un-palmitoylated dsg to the lysosomes for degradation. dsg increased expression and subcellular localization of flot , a membrane lipid raft protein critical for membrane invagination. dsg also altered membrane localization of several early (eps and eea ), but not late (rab , rab , and hrs), endocytic pathway proteins. loss of palmitoylation in the dsg cacs mutants abrogated these effects. finally, dsg -induced ev release was abrogated by the sphingomyelinase inhibitor gw or augmented by the v-atpase inhibitor bafilomycin a . summary/conclusion: the combined results of the drug treatments and functional mutations of dsg suggest that dsg plays a critical role in ev biogenesis by modulating proteins involved in early endosome sorting and is dependent on post-translational palmitoylation. introduction: the translation initiation factor eif e ( e) is an oncogenic protein that is upregulated in % of cancers including a subgroup of acute myeloid leukaemia (aml) patients. eif e regulates post-transcriptional rna processing including the nuclear export and/or translation of mrna transcripts. in particular, it selectively increases the expression of genes that have a prominent role in cancer progression such as myc, cyclin d , and mcl . furthermore, our lab pioneered studies demonstrating that a subset of ehigh aml patients is clinically responsive to treatment with a e inhibitor (ribavirin) indicating the importance of e in aml progression and its relevance as a therapeutic target. we investigated an as yet unexplored perspective of e-whether the oncogenic role of e is in part mediated by its function as a master regulator of vesiculation. methods: to assess mrna export and identify ebound mrna targets that correspond to vesiculationrelated genes and associated cargo, we used cellular fractionation and rna immunoprecipitation techniques. to determine whether e regulates the number of extracellular vesicles (evs) released as well as their protein and rna cargo we used nanoparticle tracking analysis (nta) as well as mass spectrometry, antibody microarrays, and rna sequencing technologies. results: eif e upregulates cellular protein levels of the vesiculation marker cd by increasing its nuclear export. in addition to increased cellular expression, cd , cd , cd , and flotillin- proteins are elevated in evs released from e-high cells. this is also associated with an increased release of vesicles that are - nm in size. currently, we have validated the upregulation of several receptors and cytosolic proteins in evs isolated from e-overexpressing cells that function in cell growth, migration, invasion, and stemness. the most abundant rnas in our ev preparations are micrornas (mirs) and we have confirmed downregulation of several of these. summary/conclusion: our work shows that e reprograms the vesiculation of cancer cells changing the release and cargo of evs. this may impact cellular communication and tumour biology, which we are currently addressing in functional studies. we hope that these studies will highlight novel therapeutic strategies for aml patients. intranasal administration of neural stem cells-derived extracellular vesicles promotes neurogenesis and reduces neuroinflammation and amyloid plaques in a mouse model of alzheimer's disease introduction: cognitive and memory impairments worsen with time in alzheimer's disease (ad), likely due to a progressive loss of hippocampal neurogenesis, and escalation of neuroinflammation. these changes are also accompanied by increased deposition of amyloid plaques in the brain. methods: in this study, using the xfad mouse model, we examined the efficacy of extracellular vesicles (evs) shed from the rat subventricular zone neural stem cells (svz-nscs) for disease modification. we first purified evs from the rat svz-nsc cultures through ion-exchange chromatography and then administered intranasally to -months old xfad mice (~ billion/week for two weeks). two months later, the functional effects of ev treatment were quantified through a series of behavioural tests, and animals were euthanized for quantification of hippocampal neurogenesis, oxidative stress, neuroinflammation, and amyloid plaque deposition. results: in comparison to ad mice receiving vehicle, ad mice receiving nsc-evs displayed improved cognitive function to discern minor changes in the environment in an object location test, better spatial recognition memory in an object-in-place test, and improved pattern separation ability in a pattern separation test. besides, ev-treated ad mice displayed no anhedonia in a sucrose preference test. analyses of neurogenesis using the birth-dating marker ʹ-bromodeoxyuridine and the newly born neuron marker doublecortin revealed maintenance of a higher level of hippocampal neurogenesis in ad mice receiving evs, in comparison to vehicle-treated ad mice. moreover, analyses of brain tissues from ev-treated ad mice revealed decreased concentrations of oxidative stress markers malondialdehyde and protein carbonyls and elevated levels of antioxidants catalase and superoxide dismutase. also, the concentration of proinflammatory cytokines tumour necrosis factor-alpha and interleukin- beta and the extent of amyloid plaques were significantly reduced in ev treated ad mice. immunohistochemical analysis showed reduced hypertrophy of astrocytes. summary/conclusion: intranasal administration of nsc-derived evs restrains the deterioration of cognitive and mood dysfunction of ad by maintaining higher levels of neurogenesis and curtailing the progression of neuroinflammation. funding: supported by a grant from the national institute of neurological disorders and stroke ( r ns - to a.k.s.) ot . the case of mesenchymal stromal cells, opening new perspectives in the use of ipsc in regenerative medicine. the aim of this study is to evaluate the potential of ipsc-ev in the treatment of kidney disease. methods: the ipsc were generated from skin fibroblast after informed consent of healthy donors (cytotune®-ips . sendai reprogramming kit -protocol: clementino fraga filho uh . . . / . ). the ev were isolated from ipsc supernatants (cultured h in mtesr- medium) by ultracentrifugation ( , g for h at °c). characterization of ipsc-ev was performed using zetaview, tem, exoview™ tetraspanin kit and macsplex exosome kit. for in vitro injury, renal epithelial cells were cultured under hypoxia ( % o ). for in vivo injury, male wistar rats were submitted to bilateral renal arterial clamping ( min) followed by reperfusion without or with injection of ipsc-ev (protocol approval: federal university of rio de janeiro - / ). kidney damage was assessed by histological and immunohistochemistry analyses (pcna, tunel and ed- ). modulation of rna levels was assessed by rt profiler pcr array. results: the results show that ipsc-ev reduce renal cell death, tissue damage, macrophage infiltration, promote mitochondria protection and ameliorate renal function. the ipsc-ev mechanism of action is related to the regulation of key genes known to prevent damage caused by oxidative stress like gstk , sod , sod , txn and txnrd . characterization of ipsc-ev showed that ipsc-ev can carry important molecules that can support renal recovery as epcam and prominin- . summary/conclusion: ipsc-ev presents renoprotective properties, acting on different aspects of aki. this presents a new relevant application of ipsc as a source of ev for therapeutic purpose in kidney diseases. the hospital for sick children, toronto, canada introduction: incomplete lung development, also known as pulmonary hypoplasia (ph), is a recognized cause of neonatal death. we have previously shown that experimental ph can be rescued by the administration of extracellular vesicles derived from amniotic fluid stem cells (afsc-evs) through an rna-mediated mechanism. this effect was not observed with evs derived from mesenchymal stromal cells (msc-evs) . the aim of this study was to ) evaluate which rna species were responsible for ph rescue, and ) to define the mechanism behind this effect. methods: evs were isolated and characterized from conditioned medium of rat afscs and rat mscs (control group) using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd , hsp , flo- , and tsg (western). to identify the mediators of afsc-evs, we used deseq (fdr< . ) to differentially analyse rna from: a) asfc-ev and msc-ev cargo, isolated with seramir and sequenced with nextseq. b) lung epithelial cells from rat ph lungs treated with vehicle or afsc-evs. epithelial cell rna was isolated with mirvana and sequenced with nextseq. we correlated afsc-ev cargo mirna with validated mrna targets that were downregulated after ev conditioning in lung epithelial cells. results: of the rna species contained in asfc-ev and msc-ev cargo, mirnas were the most proportionally different between the two ev populations. afsc-evs were enriched for mirnas that are critical for lung development, such as mir ~ and their paralogues that control lung branching morphogenesis. afsc-ev administration to ph lung cells significantly downregulated genes, which formed mirna-mrna reported interactions. summary/conclusion: afsc-evs contain many rna species in their cargo, but mirnas are the main effectors of their ability to rescue underdeveloped foetal lungs. we have identified for the first time that afsc-ev biological effect on underdeveloped foetal lungs is in part due to the release of mir ~ cluster. funding: cihr-sickkids foundation grant. bottom-up assembly of fully-synthetic extracellular vesicles oskar staufer a , franziska dietrich a , jochen hernandez a , martin schröter a , sebastian fabritz b , heike böhm a , ilia platzman a and joachim spatz a a max planck institute for medical research, department for cellular biophysics, jahnstraße , heidelberg, germany, heidelberg, germany; b department for chemical biology, max planck institute for medical research, jahnstraße , heidelberg, germany, germany, germany introduction: extracellular vesicles (evs) are considered as key elements for future therapeutic and diagnostic procedures. however, despite enormous research efforts to understand their physiological relevance and several greatly successful clinical trials, evs are currently not authorized for clinical routines by american or european regulation and approval agencies. this is especially because therapeutic evs are produced or isolated from cell cultures or biofluids, both of which are subjected to batch-to-batch variations and ill-defined contaminations. therefore, complementary technologies that produce evs as reproducible and defined as state of the art nanotherapeutics, would revolutionize the application of evs in clinical settings and provide the scientific community with a holistic understanding of ev-mediated signalling processes. in our study, we achieve de novo bottom-up assembly of fully synthetic evs (fsevs) that comprise identical physiological and therapeutic functionalities to natural evs. methods: we applied droplet-based microfluidic synthesis to sequentially amalgamate synthetic lipids, proteins and nucleic acids into defined vesicles that display analogous therapeutic capabilities to natural evs. fsevs were characterized by electron and confocal microscopy, dynamic light scattering and mass spectrometry and tested on organotypic models or in vivo. results: using previously described evs as "naturegiven" blueprints, we assembled several fsevs in their exact molecular composition. in particular, we produced wound-healing promoting evs composed of several exosomal proteins, lipids and micrornas and showed that their therapeutic performance on human skin wounds is equivalent to that of natural evs. besides their high molecular complexity, being composed of dozens of different molecular building-blocks, the presented fsevs are completely defined on a quantitative level. based on this, we achieved a stoichiometric understanding of cell-vesicle interactions. summary/conclusion: by applying bottom-up synthesis of fsevs for quantitative studies on ev signalling, we not only provide innovative and safe compounds for ev-therapeutics but also a vastly new perspective on the application spectrum of extracellular vesicles in fundamental research. introduction: small extracellular vesicles (sevs) contain functional molecules from their cell of origin and can enter recipient cells for intercellular communication. ifnβ has been shown to induce some lncrnas to regulate host immune response and play a major role in the positive regulation of the activity of natural killer (nk) cells. here, we aim to clarify whether ifnβ induced sevs can regulate the cytotoxicity of nk cells by transferring specific lncrnas into nk cells. methods: evs were purified from a with/without ifnβ treatment by serial centrifugation followed by sucrose density gradient purification. elisa assay were performed to demonstrate the cytotoxicity of nk cells. qpcr and western blot were used to verify the expression of nkp . results: surprisingly, ifnβ induced sevs can strengthen the cytotoxicity of nk cells. through human transcriptome array (hta) we found the expression levels of lncrnas were significantly changed within sevs isolated from a cells following ifnβ treatment. additionally we found a specific sev cargo, linc-epha - , acted as a competing endogenous rna (cerna) for hsa-mir- which subsequently up-regulate the natural cytotoxicity receptor (nkp ) expression. furthermore, we verified over-expression of linc-epha - significantly enhance the cytotoxicity of nk cells against zika virus-infected a cells. summary/conclusion: our results demonstrated that ifnβ-induced linc-epha - wrapped in sevs can regulate the cytotoxicity of nk cells. our study provides a novel link between type i ifn and nk cells, which are two major players for the host innate immunity against pathogen infections. introduction: hiv-infected t cells release simultaneously viral particles and small extracellular vesicles (sevs) including mvb-derived exosomes and plasma membrane-derived evs. sevs and hiv share many physical and chemical characteristics, which makes their separation difficult. although several approaches have been used to obtain sevs free of virus they leave a majority of sevs within hiv preparations. for this reason, the function of sevs during hiv infection remains unclear. methods: we have developed a novel un-biased proteomic profiling approach to identify specific markers of the virus or sev subtypes released by a human t lymphoma cell line. our approach was to combine differential centrifugation of medium/small evs contained in the ccm with quantitative mass spectrometry to generate protein abundance profiles across the different sub-fractions. we generated an interactive database to define groups of proteins with similar profiles, suggesting their release in the same evs. results: we thus identified different categories of evs, which bear different surface proteins, e.g. different combinations of t cell surface markers, integrins or tetraspanins. in evs released by infected cells, we identified cellular proteins behaving like hiv proteins, and several that changed behaviour after infection, either moving towards or away from the hiv cluster. we identified two cell-derived proteins that are included in the viral particles and one that is specific of non-viral sevs that are modified by infection, and analysed their respective roles in controlling ev composition or virus infectivity. summary/conclusion: our approach presents a powerful tool for identification of common cargoes of given ev subtypes, and could be now used to identify modifications of ev composition in any given physiological or pathological situation. the encephalomyocarditis virus leader modulates autophagic pathways to promote the release of virions inside extracellular vesicles introduction: recent data indicate that naked viruses belonging to the picornaviridae family can be released from host cells via enclosure in extracellular vesicles (ev). ev cloak virus particles in a host-derived "envelope" and can thereby affect antiviral immune responses and disease severity. a better understanding of the formation and function of ev-enclosed viruses is therefore required. previously, we showed the presence of the autophagosome marker lc in ev isolates from encephalomyocarditis virus (emcv) infected cells, suggesting the involvement of a secretory autophagy pathway in ev-mediated virus release. however, little is known about the viral and host factors that regulate this process. here, we have assessed the role of the emcv leader, a viral protein that is dispensable for replication but is required for symptomatic disease. methods: cells were infected with wildtype virus or a mutant carrying an inactive leader. ev produced during the infection were isolated using differential ultracentrifugation and density gradient purification. ev were characterized by high resolution flow cytometry and their infectivity determined using end-point dilution assay. in addition, the fate of autophagosomes in infected cells was monitored using a reporter assay for autophagosome-lysosome fusion and analysis of the secretion of autophagosomal proteins. results: inactivation of the emcv leader strongly reduced the release of ev-enclosed virus. whereas autophagosomes are typically degraded, we show this is blocked by the leader. instead, autophagosomes fuse with the plasma membrane, as indicated by the secretion of autophagy marker lc during infection with wildtype but not the mutant virus. pharmacological reactivation of degradative autophagy in infected cells resulted in a strong reduction in the release of ev and ev-enclosed virus. similarly, the reduction in evenclosed virus release in the absence of the leader could be partially reversed by drugs that promote the secretion of autophagosomes. summary/conclusion: our data supports a role for secretory autophagy in the release of viruses in ev, a pathway that is regulated by the emcv leader. these findings highlight an unconventional route for ev formation that intersects with autophagosomal compartments and contributes to viral pathogenesis. introduction: zika virus (zikv) causes a public health emergency of international concern because of its correlation with microcephaly. during viral infection, the innate immune response quickly to produce some endogenous functional molecules which can prevent viral invasion or replication. extracellular vesicles (evs) contain molecules from their cell of origin under virus infection and can enter recipient cells for intercellular communication. here, we aim to clarify whether zikv induced evs can regulate viral pathogenicity by transferring specific rna. methods: evs were purified from a with/without zikv infection by serial centrifugation followed by sucrose density gradient purification. human transcriptome array (hta) was used to found rna expression within evs. flow cytometry was used to determine cell cycles. zikv replication was assayed by qpcr and western blot. flow cytometry was used to determine cell cycles. results: through hta we found the defensin alpha b (defa b) expression was significantly increased within evs isolated from zikv infected a cells. additionally, we found that the extracellular defa b but not the intracellular defa b exerts anti-zikv effect mainly before entry step. surprisingly, up-regulate defa b can retard cell cycles of host cell. we verified defa b could bind with the origin recognition complex (orc ) which is required to start dna replication during the cell cycle. furthermore, up-regulate defa b decreased the orc level in nuclear. interestingly, evs with defa b can internalize into recipient cells and inhibit their cell cycles. summary/conclusion: together, our results demonstrated that zikv infection can induce defa b wrapped in evs, and defa b not only exerts anti-zikv effect but also regulate cell cycles which may affect neurodevelopment. our study provides a novel viewpoint that defa b act as first-line anti-viral molecules during zikv infection also correlate with neurodevelopment by retarding cell cycles. extracellular vesicles mediate bacterial-immune cell interactions during respiratory viral-bacterial co-infections sidney w. lane a , matthew hendricks b and jennifer bomberger a a university of pittsburgh, pittsburgh, usa; b university of washington, seattle, usa introduction: respiratory infections are a major cause of morbidity and mortality worldwide and host-derived extracellular vesicles (evs) play important roles in mediating these infections. during respiratory infection, evs are shown to have a modulatory effect: promoting or suppressing infection dependent on the pathogen and cell type. in the age of next-generation sequencing, we now appreciate that many respiratory infections are polymicrobial in nature, with viral-bacterial co-infections correlating with worse disease outcomes. epidemiological studies correlate acute viral infections with the increased likelihood and severity of both acute and chronic secondary bacterial infections; however, the exact mechanisms of these interactions remain poorly understood. evs have been understudied in the context of respiratory viral-bacterial coinfections; thus, their role in mediating these infections is relatively unknown. unpublished data from the lab shows that in airway epithelial cells (aecs), viral infection induces the release of evs that associate with pseudomonas aeruginosa (pa) and promote biofilm growth. here, we aim to expand upon these findings and determine how aec evs mediate pa-immune cell interactions during respiratory viral-bacterial co-infection. methods: to determine how exposure to evs impacts pa-immune cell interactions, evs were isolated from the apical secretions of aecs and co-cultured with pa. ev-treated pa was then co-cultured with macrophages to evaluate ev impact on pa uptake and survival. results: in preliminary experiments using control evs, we observed that evs associate with pa. interestingly, during co-culture with macrophages, ev-treated pa are more susceptible to phagocytosis in comparison to non-treated pa. however, after hours of co-culture with macrophages, ev-treated pa are able to survive and replicate, while nontreated pa are effectively controlled by the macrophages. summary/conclusion: these findings suggest that while pa-ev association promotes pa uptake, it may ultimately enhance pa immune evasion and survival. ongoing experiments in the lab are evaluating the mechanism of pa-ev association and how evs from virus-infected aecs affect the phenotypes observed with control evs. notably, this is one of few reports of a mammalian ev influencing the pathogenesis of a bacterium; thus, results from these experiments will define the function of aec evs in regulating bacterial-immune cell interactions during respiratory co-infections. using machine learning with neuronal ev target proteins and clinical data to predict cognitive impairment in hiv infection lynn pulliam a , michael liston b , bing sun c and jared narvid d a university of california, san francisco, san francisco, usa; b veteran affairs, san francisco, usa; c ncire, san francisco, usa; d ucsf, san francisco, usa introduction: objective biomarkers are needed to assess and predict neuronal function and cognitive impairment. in people ageing with chronic infections, such as hiv, determining the mechanism of impairment will be important when therapies are available. methods: sixty plasma samples from hiv-infected people were obtained from nih-sponsored aids banks. clinical and epidemiological data were collected. all underwent neuropsychological testing and were considered impaired. neuronal extracellular vesicles (nevs) were isolated from plasma and assayed for high-mobility group box (hmgb ), neurofilament (nf-l) and phosphorylated tau- (p-tau) proteins. results: using different algorithms, support vector machines (svm) performed the best with an area under the curve (auc) value of . ± . . using different combinations of clinical data and the nev protein targets, selected clinical data and hmgb best predicted cognitive impairment (auc = . ). the most important features included cd count, hmgb , nf-l and education. summary/conclusion: specific clinical features plus nev hmgb , an inflammatory marker, were the best predictors of cognitive impairment. previous published data showed nev p-tau- elevated in alzheimer's disease and in this study p-tau had no importance in assessing hiv-associated cognitive impairment. nev target discovery can be improved to better identify neuronal damage, possibly to differentiate other neurodegenerative diseases and hopefully recovery after therapies are identified. in recent years, we have been able to separate and characterize extracellular vesicles (evs) from several different viruses including hiv- , htlv- , rift valley fever virus and ebolavirus. however, to date it is not clear whether there is a timing difference between ev and virus release from infected cells. methods: ev isolation by nanoparticle capture and differential centrifugation, ev quantification by nanoparticle tracking analysis, western blot, rt-qpcr, virus rescue assay. results: we have attempted to address the kinetics of ev and virus release from multiple-infected cells using serum starvation experiments from infected ( %) cells. these infected cells were initially put in g quiescent stage using serum starvation. both supernatants and cell pellets were collected postinduction release ( % fbs + pma/pha) at , , , , and hours and examined for the presence of ev, autophagy and viral proteins as well as viral rna expression. results from supernatants of uninfected cells showed a peak of tetraspanin proteins (cd , cd , and cd ) at hours and a gradual decrease of all ev associated proteins by hours. however, the ev from hiv- infected cells showed all three tetraspanins present at hours and expression gradually increased up to hours. when compared to htlv- infected cells, the three tetraspanin proteins peaked at hours and expression continued to decrease up to hours. htlv- infected cells also showed a unique pattern of cd expression. autophagy associated proteins (lc a, lc b and p ) from uninfected cells and htlv- infected cells plateaued at hours, whereas in hiv- infected cells their expression continued to increase and peaked at hours. hiv- viral proteins (p , gp , nef) expression was present at hours and continued to increase and peaked at hours. htlv- proteins (p and gp / ) peaked at hours and gradually decreased overtime. hiv- and htlv- rna gene expression analysis was performed, and data correlated with viral protein expression. additionally, evs release was quantified and showed significant increase of ev concentration overtime in both uninfected and infected samples. finally, experiments of infectivity from -and hour supernatants were performed on three naive cells. hiv- supernatant -hour sample was found not to be infectious. however, hiv- was successfully rescued from -hour sample. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = ). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine distinct uev surface markers of cd +/cd +/cd + vesicles in a multiplexed bead-based manner including respective controls. the accuri c (bd) was utilized for data acquisition. for further misev -compliant characterization, cd +/cd +/cd + uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd , all other surface markers could be identified. the most abundant markers were cd and cd , which were detected in % of samples, followed by cd / ( %), cd ( %), cd and cd ( %, each). cd ( %), cd , cd ( %), cd e ( %) and cd showed similar relative median fluorescent intensities (rmfi), while cd yielded significantly higher (p = . ) and all other markers significantly lower rmfi (p < . ). tem and f-nta revealed cup-shaped vesicles ( ± nm) with . ± . e + particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg , cd , cd and cd without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a ,esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = ) aged - yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a -colour panel, which included annexin v (for the majority of circulating evs), cd (for platelet-derived evs) and cd (for endothelial-derived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and -yr cvd risk detected by qrisk . results: ev numbers, as determined by nta, were strongly associated with bmi (r = . , p < . ), blood pressure (systolic bp: r = . , p = . ; diastolic bp: r = . , p < . ) and plasma triacylglycerol levels (r = . , p < . ). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = . , p = . ). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining . % of the variance for total ev numbers. an additional . % of the variance in total ev numbers was predicted by diastolic bp. ancova of the -yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with -yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd + t cells were isolated from secondary lymphoid organs of foxp -rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th , th , and th ), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, -day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th , th , th ) showed either no change in proliferation (th ) or decrease in proliferation (th , th ), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < . ). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th ), il and il (th ), or il a and il f (th ). in contrast, exposure of tregs to cdc-evs resulted in~ -fold increase in expression of il , a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il- in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il- . this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t hl . prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from patients treated with prostanoids (study group; n = , ± years, % female) and patients not treated with prostanoids (control group; n = , ± years, % female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; . mm), adenosine diphosphate (adp; . µm) and thrombin receptor-activating peptide (trap; µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd + and cd p+; pevs), leukocytes (cd +, levs) and endothelial cells (cd +, eevs) were measured in plateletdepleted plasma by flow cytometry (a -micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = . ) and lower concentrations of pevs and levs (p ≤ . ). furthermore, thrombus formation was delayed (p ≤ . ) and thrombus size was decreased (p = . ) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd + pevs (p = . ). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ . ) and the concentrations of pevs (p ≤ . ). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ . ). introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from cf children (< years of age). for nanoflow cytometry, evs were stained with di- -anepps, and with epcam, cd b and cd (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. results: the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = . , spearman's rho = . ). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = . , rho = . ). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv a ), emory pediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: individuals were enrolled into our study, subdivided into a training (volunteer n = , pneumonia n = , sepsis n = ) and testing cohort (volunteer n = , pneumonia n = , sepsis n = ). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq ) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative realtime pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed significantly regulated mirnas in pneumonia compared to volunteers, and mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: . , pneumonia: . , sepsis: . ). mir- a- p (log fc = . , padj = . e- ) and mir- - p (log fc = . , padj = . e- ) differentiated between pneumonia and volunteers and mir- (log fc = − . , padj = . e- ) between pneumonia and sepsis. expression levels of mir- a- p and mir- were related to disease severity. mir- - p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was . %. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study, healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd and cd were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and mirnas (mir- a-p, mir- - p, mir- a, mir- b- p, mir- - p, mir- - p, mir- - p, mir- - p, let- g- p) , were evaluated by rt-qpcr. after the optimization of the methodology, healthy adults subjects and patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd and cd . however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna- a showed a significant increased (p = . ) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir- a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf -active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds joseph kuhn a , absara hassan b , sonali sharma b , jennifer kwong b , montaha rahman b , salma adam b , jasmine lee b , alvaro villarreal ponce b and piul rabbani b a nyu langone health, new york, usa; b nyu langone health, new york, usa introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid -related factor (nrf ) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf -active exosomes, mscs were incubated with potent nrf activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express > % positive msc markers (cd , cd , cd , and cd ) and < % express negative markers (cd , cd , cd , cd , or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd , cd and tsg . nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of . ± . nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf -active human mscs increase exosome secretion by %, compared to nrf -baseline mscs (p < . ). both nrf -baseline and nrf -active exosome treatment significantly reduced closure time to . and days respectively, compared to . days for vehicle-treated wounds (p < . ). this reduction eliminated the delay in closure time compared to wounds of c /b mice. nrf -active exosome treatment of db/db wounds reduced closure time by a further . days compared to untreated c /b wounds. at day , exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd + vessels compared to vehicle-treated wounds. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist is a key mediator of tumour cell metastasis.. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist . methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc -ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc -ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc -ml lines stably overexpressing or deficient in twist . results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc -ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc -ml overexpressing twist showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. funding: american heart association of symposium introduction: as researchers continue to explore the therapeutic potentials of extracellular vesicles (evs) for the treatment of many diseases, there is a growing unmet need for real-time in vivo monitoring of these therapeutic evs after they are injected into a subject to understand their safety, targeting, and effectiveness. while current optical imaging solutions like bioluminescence and fluorescence are useful for ev tracking studies in animal models, there is limited utility in clinical applications. here we present a novel ev tracking solution utilizing clinically applicable mri technology. methods: to generate trackable evs, cells were labelled with a clinically applicable novel magnetic agent. evs secreted by the labelled neural stem cells and amniotic fluid stem cells (afscs) were isolated by differential ultracentrifugation. the viability and morphology of labelled-cells were evaluated, and the in vitro mr properties of their derived evs were analysed by magnetometer. a proof of concept in vivo biodistribution study was conducted by injecting labelled evs into wt and alport mice (a model of chronic kidney disease) via retro-orbital and intra-cardiac routes and tracking them via mri at min and hr postinjection. results: the magnetic label did not affect the physiological characteristics of the cells. the mr detectability of labelled-evs was confirmed by in vitro/ex vivo mri phantoms. mri studies showed that homing of afsc evs to the kidney injected intra-cardiacally into alport mice were more efficient versus the retro-orbital route, and prussian blue staining of kidney sections confirmed the mr findings. introduction: a central question in ev biology is the fate of circulating ev. this can be evaluated by developing non-invasive ev bioimaging techniques in mice in order to benefit from transgenic and knock-out models. recent reports described ev biodistribution in vivo using optical (fluorescence) and nuclear imaging. but the physicochemical properties of the probes impact ev integrity, labelling efficiency, background signals and observation timecourse. methods: we developed the radiolabeling of red blood cells (rbc) and ev with [ f]fluorodeoxyglucose ( f-fdg). we used rbc-derived ev in their native, intact form, without pre-experimental processing (no centrifugation or filtration). we tracked f-fdg in vivo by pet-scan, within seconds of ev, rbc or free f-fdg injection, and during their dissemination in blood and recruitment by organs over one hour. ev and rbc biodistribution were confronted to the kinetics of free f-fdg. results: we collected images of the biodistribution of rbc, and rbc-derived ev. nuclear imaging was well suited for accurate studies of ev organotropism, with high sensitivity, excellent signal-to-noise ratio, very low signal absorption by tissues and an inherent quantitative tomographic nature. ev-specific signals were mostly accumulated within minutes of injection (tail vein), in the spleen and liver, with a small part in the bone marrow (femurs). signals in other compartments were largely transient and linked to tissue perfusion and blood volume. we selected the most drastic control conditions to secure a correct interpretation of the data. this made kidneys, hearts and brains unavailable for analysis. hence the new approach came with limitations, but we describe how "free" f-fdg signals can be used to draw sound conclusions for ev. summary/conclusion: we propose that three types of compartments coexist in control mice at rest: active ev-capturing organs with high capacity and specificity including the spleen, and to a lesser degree the bone marrow; passive ev-retaining organs with high capacity, including the liver; and ev-neutral organs where transient signals only mirror tissue perfusion. we also report how ev biodistribution patterns are altered in ageing animals, as an example. we hope that this novel, non-invasive, quantitative, dynamic wholebody imaging approach will help characterize native cell-derived ev and help set standards for the reproducibility of ev bioimaging in mice. funding: frm grant "biface", inserm copoc, cnrs. introduction: extracellular vesicles (ev) are important mediators of intercellular communication; however, basic principles of ev biogenesis and loading remain largely unknown. a limited repertoire of tools has thus far made these processes challenging to research. the development of an ev-transfer reporter in a genetically tractable organism such as drosophila has allowed us to study mechanisms of cargo loading in vivo and has provided us with a platform to explore fundamental aspects of ev biology. methods: we have developed a bioinformatic pipeline to analyse the properties embedded in the ʹutr of mrnas enriched in evs released by drosophila cells. in parallel, we have adapted a cre-loxp system for use in fruit flies that appears to be proficient to reveal the exchange of bioactive molecules between secretory and recipient cells. results: taking advantage of computational methods, we uncovered sequence motifs that preferentially appear in combinations along the ʹutr. these sequence motifs occur within characteristic secondary structures, in a way that is more variable and motif dependent than previously reported. identified motifs also show similarities to known binding sites for rna binding proteins; a feature potentially important for ev-loading. in parallel, we developed a drosophila in vivo system to detect cell communication in complex tissues and between different cell types. using this system, we studied the biological significance of specific sequence motifs and identified their ability to modulate mrna ev-transfer in a context dependent and evolutionarily conserved manner. summary/conclusion: in summary, we have developed a novel tool to study cell communication in complex tissues, and shown its effectiveness to study principles of ev biogenesis and loading. beyond improving our understanding of ev biology and providing a novel tool to the scientific community, we hope this knowledge will pave the way to harnessing evs as a means of remotely manipulating cell communication in many biological contexts. introduction: the idea of cross-kingdom, species and inter-individual transfer of bioactive compounds via extracellular vesicles (evs) is a recent avenue. however, the bioactivity and bioavailability of these dietary compounds upon consumption is highly debated. it has been proposed that evs from diet can absorbed by consuming organisms, be bioavailable in various organs and exert phenotypic changes. milk is the most vastly consumed beverage and is an abundant source of evs that may act as signalosomes. whether these milk-derived evs can serve as cross-species messengers and have a biological effect on host organism has been poorly understood. methods: bovine milk-derived evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. the evs were characterised by tem, nta, quantitative proteomics and rna-seq. evs were orally administered to various mice models of colorectal, breast and pancreatic cancer. primary tumour burden was monitored, and the rate of metastases was measured by imaging and qpcr. immune cells were analysed by facs. mechanistic insights were obtained using quantitative proteomics, confocal microscopy and biochemical experiments. results: we demonstrated that upon oral administration, bovine milk-derived evs were able to survive the harsh degrading conditions of the gut and be bioavailable in peripheral tissues. interestingly, oral administration of milk-derived evs reduced the primary tumour burden in various cancer models and attenuated cancer cachexia. intriguingly, despite the reduction in primary tumour growth, milk-derived evs accelerated metastasis in breast and pancreatic cancer mice models. timing of ev administration was critical as oral administration after resection of the primary tumour reversed the pro-metastatic effects of milkderived evs in breast cancer. biochemical and quantitative proteomics analysis highlighted the induction of epithelial-to-mesenchymal transition and senescence upon treatment with milk-derived evs. summary/conclusion: taken together, we were able to demonstrate the capacity of bovine milk-derived evs in mediating cross-species communication and regulating cancer progression in a context-dependent manner. bacterial membrane vesicles (mvs)a bacterial innate defence system against viral infection xiaomei yan, qian niu and ye tian xiamen university, xiamen, china (people's republic) introduction: in order to survive the constant onslaught of phage, bacteria have evolved diverse defence mechanisms that act at every stage of the phage life cycle. it has been suggested that bacterial membrane vesicles (mvs) may play a key role in innate bacterial defence against phage infection by acting as a decoy to prevent phage adsorption. nearly a decade has passed since mvs were first proposed as a decoy, but details of how bacteria utilize mvs to defend against phages remain poorly understood. here we use the laboratory-built nano-flow cytometer (nfcm) to reveal details of the interaction between mvs and phages at the single-particle level, and to provide new insights into innate defence mechanisms of mvs. methods: s. typhimurium was used as the model system. differential ultracentrifugation and density gradient centrifugation were used to isolate and purify mvs and bacteriophage p . cryo-tem was used to determine the morphologies of mvs and phage p . the purity of mv isolates was validated by measuring the particle concentration before and after triton x- treatment. monodisperse silica nanoparticles were used as the size reference standards to measure the size distribution of mvs via single-particle light scattering detection. the purity of phage p was verified by concurrent detection of side scatter and fluorescence signals of single phages upon nucleic acid staining by syto . results: by incubating mvs and af -labelled p , the number of phages adsorbed on single mvs were accurately quantified. we found that s. typhimurium and mvs it secretes express different affinity for phage p attachment. the binding ability of p to mvs is greater than that of bacteria. we confirmed that p can inject their nucleic acids into mvs, and these nucleic acids can be degraded by non-specific nucleases inside mvs for the first time. besides, by labelling the nucleic acids of mvs with syto , we were able to distinguish three different subpopulations of mvs. summary/conclusion: taking advantage of the superior sensitivity of nfcm in single-particle analysis, we developed a novel approach to the characterization of the interaction between mvs and phages. our study revealed that bacteria produce mvs as bait to attract viral adsorption and nucleic acids injection. funding: this research was supported by the national natural science foundation of china (grants , and ). introduction: the development of evs for therapeutic applications requires an in-depth understanding of their in vivo biodistribution and pharmacokinetic profile. in this study, we have made a comprehensive comparison of nuclear, fluorescent, and bioluminescent imaging technologies to identify the most suitable in vivo ev tracking method. methods: evs were purified from expi f cell supernatant by differential centrifugation followed by iodixanol density gradient separation and further characterized following misev guidelines. engineered expi f cells were used to generate evs carrying mcherry or nanoluc (nluc) proteins. the membrane of naïve ev was labelled with indium (in )-dtpa or xenolight dir post-ev isolation. ct tumour-bearing balb/c mice were intravenously dosed with × evs followed by imaging at h, h and h using spec/ct and ivis systems. tissue distribution and blood circulation profile of evs were analysed from ex vivo samples up to h post-injection. results: xenolight dir and (in )-dtpa were the most suitable ev labels for live whole-body animal imaging, ex vivo organ imaging, and tissue lysate quantification. nluc was appropriate for ex vivo imaging and tissue lysates quantification, but suboptimal for live imaging with limited sensitivity. mcherry evs were found not suitable for in vivo tracking studies due to high background signal fluorescence. ex vivo organ quantification of in -dtpa and dir showed that naïve evs mainly accumulate in liver, followed by spleen, kidneys, and lungs at h post-dose, with less than % ev exposure to the tumours. interestingly, nluc-evs accumulated mainly in the lungs, regardless of the small size of the particles injected and the absence of aggregation. blood circulation profile of in -dtpa and nluc evs showed rapid clearance of vesicles from circulation, with % of injected dose detected in blood after min and less than % after h. summary/conclusion: radionuclide imaging is an excellent technology to detect evs in vivo and ex vivo with high resolution and sensitivity but requires advanced infrastructure for radiolabeling. the optical methods have limited tissue penetration and sensitivity but can be improved with the right selection of the dye. these results contribute to the understanding of the biodistribution and pharmacokinetics of evs and are highly relevant to exploiting their potential for targeted delivery to diseased tissues in vivo. symposium introduction: new methods for quantifying extracellular vesicles (evs) in complex biofluids are critically needed. we report the development of a new technology combining size exclusion chromatography (sec), a commonly used ev purification technique, with fluorescence detection of specifically labelled evs (flu-sec). methods: flu-sec was validated using red blood cell derived evs (revs). size and concentration measurements were performed by microfluidic resistive pulse sensing (mrps) using the ncs instrument (spectradyne llc, usa). pe-cd a (anti-glycophorin a) and alexa -wga (wheat germ agglutinin) were used to label revs. flu-sec experiments were performed on a liquid chromatography system using a tricorn / glass column filled with sepharose cl- b gel (ge healthcare). results: a log-normal size distribution was obtained for revs with a mean diameter of . ± . nm and standard deviation of . ± . nm. the concentration of revs measured by mrps was . * e particles/ ml. the fluorescence chromatograms of the rev samples labelled with pe-cd a and with alexa -wga show the typical features of the separation of evs from soluble proteins with sec and enables the determination of the labelling efficiency of the markers. the linear range for quantification of evs in our experiments spans over two orders of magnitude ranging from e particles/ml to e particles/ml. the lod depends on the type of the label. in our experiments the lowest lod was e particles/ml for alexa -wga. summary/conclusion: the results indicate that flu-sec is a quantitative technique with very good linearity over a wide range of concentrations, though the limit of detection depends largely on the employed label (sci. rep. , , ) . moreover, the ratio of ev-bound and free-antibody molecules can be also determined by flu-sec, which can be used to calculate the labelling efficiency of the used marker. funding: this work was supported by the national research, development and innovation office (hungary) under grant numbers pd and nvkp_ - - - . zv was supported by the janos bolyai research fellowship. the conan assay: purity grade and concentration of ev microlitre formulations by colloidal nanoplasmonics. (evs). control over such properties is constantly experienced by researchers to be critical for ev proper manipulation, engineering and translation. however, the need for characterization methods that strike the balance between robustness, working volume, cost and accessibility remains unmet. methods: the colorimetric nanoplasmonic (conan) assay we developed consists of a solution of gold nanoparticles (aunps) into which - μl of the ev formulation is added. the solution turns blue if the formulation is pure, while stays red if soluble exogenous single and aggregated proteins (saps) are present. the colour shift is visible by the naked eye and can be quantified by conventional uv-vis spectroscopy, providing a quantitative index of purity and an estimation the ev molar concentration (particle number). results: the assay specifically targets saps, and not the ev-related proteins, with a detection limit < ng/μl (an order of magnitude higher resolution than the bradford protein assay). for pure solutions, the assay also allows for determining the ev number, as the colour shift is linearly dependent to the aunp/ev molar ratio. instead, it automatically reports if the solution bears sap contaminants, thus avoiding counting artefacts. experiments, conducted on ev separated from milk and ascaris suum culture medium, are repeatable, with an error below %. summary/conclusion: conan proves to be robust and reliable, while displaying appealing performances in terms of cost (inexpensive reagents, run by standard microplate reader), working volumes ( - μl) and time (the procedure takes less than one hour). the ability to assign a quantitative purity grade is, up to date, a unique peculiarity of this assay. finally, the assay is potentially extendable to all classes of natural and artificial lipid micro-and nanoparticles. funding: evfoundry project, horizon -future and emerging technologies (h -fetopen), id: . marina cretich a , roberto frigerio b , alessandro strada b , greta bergamaschi b , marcella chiari c and alessandro gori c a consiglio nazionale delle ricerche (cnr), istituto di scienze e tecnologie chimiche (scitec), milano, italy; b consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy; c consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy introduction: small extracellular vesicles (sev) present fairly distinctive lipid membrane features in the extracellular environment. these include high curvature, lipid packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sev membrane could be then considered as a "universal" marker, alternative or complementary to traditional characteristic surface-associated proteins. here we introduce the use of membrane sensing peptides as new, highly efficient ligands to directly integrate sev capturing and analysis on a microarray platform. methods: we designed and synthesized membranesensing peptide ligands as molecular baits for small ev and we demonstrate their use in a microarray platform as valuable alternative/complement to antibodies. evs from blood serum and plasma were isolated by ultracentrifugation, characterized by tem, nta, wb. samples were analysed by label-free, single particle counting and sizing on peptide microarrays coupled to fluorescence co-localization immune staining with fluorescent anti-cd /anti-cd /anti-cd antibodies. results: peptide microarrays were realized using a click-chemistry strategy for optimal peptide surface orientation and used to analyse evs from human blood. membrane sensing peptides showed a capturing capacity higher than anti-tetraspanin antibodies. in addition to purified vesicles, peptide ligands were tested with pure serum showing capacity to capture evs even from complex samples. in order to get insights into the ev-peptide binding mechanism and verify whether it is directly mediated by the lipid membrane, trypsin-treated evs were captured on peptide microarrays demonstrating that binding is not directly mediated by surface associated proteins. summary/conclusion: we introduced the use of membrane sensing peptides as a novel class of molecular ligands for integrated sev isolation and analysis, reporting for the first time on peptide microarrays for extracellular vesicles. given their affinity to the membrane of small ev, these molecules can serve as general baits, enabling vesicles capturing unbiased by differential surface protein expression. these new class of molecular probes may be integrated with the use of protein markers towards improved small ev isolation and characterization. compared to proteins and antibodies, peptides are characterized by low cost of preparation, remarkable stability and ease of chemical manipulation, offering virtually unlimited possibilities for experimental design. we anticipate that this new class of ligands, may greatly enrich the molecular toolbox for ev analysis. funding: hydrogex (regione lombardia&fondazione cariplo, grant n. - ) and index (european union's horizon research and innovation programme under grant agreement n° ) projects are acknowledged for partial financial support. high-resolution size-based profiling and morphological analysis of extracellular vesicles by scanning electron microscopy sara cavallaro a , federico pevere b , petra hååg c , kristina viktorsson c , rolf lewensohn c , jan linnros a and apurba dev b a kth royal institute of technology, stockholm, sweden; b uppsala university, uppsala, sweden; c karolinska institute, stockholm, sweden introduction: extracellular vesicles (evs) have been found to mediate intercellular communication in physiological and pathological processes. nevertheless, the understanding of evs bio-functionality remains elusive, mainly because of their high heterogeneity in molecular content, but also in size ( - nm) . therefore, accurate size measurements of evs are highly desired, particularly for exploiting their full diagnostic/therapeutic potential. currently available techniques, such as nanoparticle tracking analysis (nta), cannot accurately measure evs smaller than nm and are not capable to distinguish them from protein aggregates. on the contrary, electron microscopy (em) techniques allow high-resolution size-profiling and morphological analysis of evs over their whole size range. however, their low throughput combined with several long preparatory steps have prevented em from being routinely used for ev size profiling. methods: we shall present a method improvement in throughput and reproducibility of ev size-analysis by scanning em (sem). the technique is based on covalent ev capture onto a silicon wafer, using the protocol reported by cavallaro et al. up to the glutaraldehyde step. after immobilization, critical point drying (cpd) is performed to dehydrate evs before sem, while preserving their shapes. results: sem images, showing the comparison in densities of evs prepared by covalent and non-covalent coupling to substrate, indicated a good capture efficiency of our covalent protocol. the size distribution analysis showed good agreement between nta and sem for evs > nm. for smaller evs, sem is more sensitive than nta, thus more suitable to check the purity of ev-isolation techniques. last, atomic-force microscopy (afm) measurements was also used to validate our measurements. introduction: extracellular vesicles (evs) are membrane vesicles secreted into extracellular space, by almost all cellular populations, playing a major role in cell-to-cell communication. it has been already demonstrated that changes in luminal or surface protein cargos of these vesicles, may reflect the status of producing cells. for this reason, evs are considered as potential biomarkers in several types of diseases ranging from cancer diagnosis to heart rejection. periostin (postn) is a matricellular protein associated with evs, and its level is considered a possible biomarker, which indicate malignancy and poor clinical outcome in different types of cancer. here we extensively characterize the presence of postn associated on evs, showing how different isolation methods can drastically affect the amount of postn content in extracellular vesicles fraction. methods: serial ultracentrifugation steps or size exclusion chromatography were used to isolate evs from primary culture of cardiac progenitor cells. evs were characterize, according to misev guidelines, by western blot, nta, facs and cryotem analysis methods. postn amount, associated with evs, was analyses by western blot and elisa. furthermore, functional tests were performed on h c cardiomyoblast cell line, treated with the same amount of evs from different isolation methods; cells response were analysed by western blot. results: evs, from both the isolation methods, showed tsg , syntenin , cd positivity while grp was absent. nta showed no differences, in terms of amount and size of particles. by facs analysis evs resulted enriched with cd , cd and cd . cryotem showed a similar morphology in the two preparations with presence of protein contaminant in the ultracentrifuge pellet. in vitro, h c treated with evs showed activation of pfak after ʹ of treatment, this induction was . times higher in cells treated with evs isolated with ultracentrifuge compared to evs isolated with sec, confirming a drastic effect of postn protein contamination. furthermore, by phospholipase-c treatment, we found that postn is bound to evs surface through a gpi anchor. summary/conclusion: these results suggest that selection of a proper isolation method is critically relevant in evs studies, in particular when protein analysis is considered. different isolation methods dramatically influence protein amount in extracellular vesicles and consequentially their function. furthermore, in this study we show for the first time, that postn is actually bound to evs surface and not carried in their lumen as previously believed. members of the y-rna family have been detected in ev from various cell types and are among the most abundant non-coding rna types in plasma. we previously showed that shuttling of full-length y-rna into ev is modulated by tlr-activation of ev-producing immune cells. this suggested that y-rnas may have potential as biomarker for immune-related diseases. methods: we separated rna-containing structures in plasma based on differences in size, density, and resistance to protease/rnase treatment. using rt-qpcr, we quantified full-length y-rna subtypes (y , y , y ) in ev from various blood-related cell types cultured with or without lps-stimulation. inflammationinduced changes in y-rna were assessed in plasma samples from a human endotoxemia study. results: full-length y-rna in plasma was mainly found in ev (early sec-fractions, density . - . g/ml). in contrast, specific mirnas were either enriched in lpp (e.g. mir- ), in both ev and lpp (e.g. mir- and mir- ), or in ev (e.g. mir- ). evenclosed full-length y-rna was resistant to enzymatic degradation, while lpp-bound mirnas were degradation sensitive. we discovered that ev released by different blood cell types varied in y-rna subtype ratios. these ratios remained stable upon lps-stimulation of the ev-producing cells. in endotoxemia plasma samples, the neutrophil-specific y /y ratios and pbmcspecific y /y ratios changed significantly during systemic inflammation. importantly, the plasma y-rna ratios strongly correlated with the number and type of circulating immune cells during the inflammation process. summary/conclusion: cell type specific "y-rna signatures" in plasma ev can be determined without prior ev-enrichment, and may be further explored as biomarkers to diagnose inflammatory responses or other immune-related diseases. mining public ev small rna-seq data with mirev -insights into potential reference transcripts and abundant mirnas recently, extracellular membrane vesicle (mv) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells. s. aureus produce membrane-bound, spherical, nano-sized, mvs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. the present study sought to examine the nature of the association between nucleic acids and mvs produced by s. aureus. we also sought to analyse the immunostimulatory potential of mvassociated rna and dna, and to evaluate receptormediated recognition of mv-associated rna and dna molecules by innate immune cells. methods: by following a stringent purification protocol, we characterized the rna and dna content of mvs produced by actively growing s. aureus. nuclease protection assays were performed to determine whether mv-associated nucleic acids are protected from degradation. we assessed the immunomodulatory potential of mv-associated rna and dna by treating cultured mouse macrophages with mvs and measuring the induction of interferon-β mrna using qpcr. introduction: urinary extracellular vesicle (uev) transcriptome could potentially reflect the kidney gene expression profile and serve as virtual/liquid biopsy. in order to explore this possibility, we performed mrna sequencing of uevs from individuals with type diabetes to assess whether it can capture a "kidney enriched genes" expression signature that could lead to novel biomarker discovery for diabetic kidney disease. methods: the study included type diabetic individuals ( normoalbuminuric, microalbuminuric and macroalbuminuric). urine samples were collected either overnight (n = ) or during -hours (n = ) and uevs were isolated from - ml of urine by differential centrifugation. the evs quality was ensured by electron microscopy (em), western blotting and ev-rnasprofiling with the bioanalyzer. isolated rnas were subjected to rna sequencing after cdna library preparation (ultra-low amount protocols) using hiseq (illumina) pair-end protocol. the association between kidney specific gene expression levels (> fold higher compared to other tissues, n = ) and degree of albuminuria or glomerular filtration rate was explored. results: isolated ev quality appeared good by em and western blotting. rna quantity and quality were sufficient for sequencing of all samples with > million pair end reads. we detected on average expression of , genes. principal component analysis (pc) of the expression of all genes did not reveal any systematic batch differences between the overnight and -hour urine collections. comprehensive look-up of kidney-enriched genes revealed expression of > % (total ) of these genes in urine evs with high expression of five kidney-specific genes (slc a , slc a , nphs , aqp and slc a ). pc analysis combining the impact of kidney-enriched genes revealed that most macroalbuminuric patients clustered together along the pc axis, and the axis also correlated with the albumin-to-creatinine ratio (p = . ) explaining % of the variance (p = . ) in the whole data set. the pc axis also showed correlation with hba c (p = . ), but not with diabetes duration, bmi, age and egfr. introduction: due to their safety profile, tissue tropism and long-term transgene expression, adeno-associated viruses (aavs) have become the vector of choice for human gene therapy. however, pre-existing neutralizing antibodies (nabs) to many aav serotypes pose a critical challenge for the translation of gene therapies to clinic. here, we describe the use of exosomal aavs (eaav) as a robust cardiac gene delivery system that enhance transduction efficiency while shielding from pre-existing humoral immunity to the viral capsid. methods: we developed an ultracentrifugation-based purification strategy to obtain eaav specimens from aav-producing hek- t cells, and used electron microscopy-based visualization, confocal microscopybased colocalization studies, qpcr, immunoblotting, dynamic lights scattering, exoview technology and protease assays to characterize eaav morphology, contents and mechanism of action. we then evaluated efficiency of heart targeting for eaav or eaav and standard aav or aav encoding for egfp, mcherry or firefly luciferase in different human cell lines in vitro, in black mouse and in passive immunity nude mouse model in vivo using flow cytometry, confocal microscopy, langendorff perfusion system and methods: hlhs patients (n = ) after glenn procedure and swine (n = ) after pab were given rv injections of allogeneic/xenogeneic mscs. donor-specific, hla-i+, exosomes were isolated from plasma. in swine, exosomes were collected and rv fractional area change (fac) was measured post-msc-injection. in the elpis patients, exosomes were collected and outcome measurements (fac, stroke volume (sv), rv mass) were recorded and -months post-injection. exosomal mrna, microrna (mirna), and proteins were quantified and partial least squares regression (plsr) reduced the dimensionality of the datasets to build a swine model, upon which elpis outcome predictions were made. results: multiomics analysis of swine exosome cargo revealed mirna to be the largest contributor to overall variance. in swine and elpis patients, mirnas were similarly expressed ( %, fold-change< ). plsr reduced the dimensionality of the swine mirna dataset to mirnas with the highest weighted coefficients for changes in fac. pathway analysis of mirna targets revealed links to smooth muscle cell proliferation and cardiac chamber development. importantly, the swine mirna plsr model predicted elpis patient improvements in fac, sv, and rv mass with strong correlation (r > . ). summary/conclusion: these findings support the use of: ( ) swine pab model for rv failure in hlhs, ( ) circulating donor-specific msc-exosomal mirna as a novel, non-invasive biomarker of patient outcomes, and ( ) introduction: evs have been shown promising potential as a drug delivery vehicle, especially nucleic acid therapeutics. however, the overall short of specificity to target cancer cells has led to low therapeutic efficacy and potential toxicity. rna nanotechnology is the bottom-up self-assembly of nanometre-scale rna architectures. we previously discovered a stable phi prna three-way junction ( wj) motif and used it to construct multivalent rna nanoparticles with high chemical and thermodynamic stability. the resulting arrow-shape rna nanoparticles are homogenous, uniform in size and shape, and can harbour different functionalities while retaining their tertiary folding and independent functionalities both in vitro and in vivo. this flexible platform using rna nanotechnology to achieve tumour-specific targeting has been demonstrated over the last decade. here we introduce a strategy to take advantage of both evs and rna nanotechnology to develop a versatile platform for efficient target-specific delivery of sirnas for cancer treatment. methods: we design membrane-anchoring arrowtail wj rna nanoparticles to display tumour targeting ligand (psma rna aptamer or egfr rna aptamer or folate) on birc sirnas loaded evs (fig. ). nanoparticles were characterized by nanoparticles tracking analysis (nta), transmission electron microscopy (tem), dynamic light scattering (dls) and atomic force microscopy (afm). evs were produced by hollowfiber bioreactor and purify by tangential flow filtration (tff) follow by ultracentrifugation. cell binding were evaluated by flowcytometry and confocal microscopy and gene knockdown effect were assay by quantity reverse transcription-pcr (qrt-pcr). formulated evs were introduced to tumour (prostate, triple negative breast cancer, colon pdx) xenograft mice by tail-vein injection and evaluate in vivo tumour inhibition. results: ) we found the orientation of arrow-shaped rna can be used to control ligand display on evs membranes for specific cell targeting. ) by placing membrane-anchoring cholesterol at the tail of the arrow results in display of rna aptamer or folate on the outer surface of the evs and enhance cancer cell binding and uptake. ) taking advantage of the rna ligand for specific targeting and evs for efficient cytosolic delivery, the resulting ligand-displaying evs or plant derived evs-like nanovesicles were capable of specific delivery of sirna to cells, and efficiently blocked tumour growth in three cancer models. summary/conclusion: we developed an rna-evs based nanoparticles platform and shown the flexibility for different cancer type treatment. related publications: pi f et.al. nature nanotechnology. , : . li z et.al. sci rep. introduction: extracellular vesicles (evs) contain plasma membrane surface markers that provide insights into their cell source. until now, our understanding of the circulating ev-biome has been limited by the lack of celland size-specific ev quantitation methods. we have developed and validated a multiplex nanoscale flow cytometry approach to image cell-and size-specific ev populations using a novel human "ev-lyoplate" with differently coloured monoclonal antibodies per well in a well plate format (n = separate antibodies with isotype, stained pbs, unstained plasma, and quant-beads controls per plate). we hypothesized that platelet poor plasma samples from patients diagnosed with pancreatic cancer would have significantly different ev-biome profiles than screen negative study subjects. methods: study subjects were enrolled and sampled before clinically scheduled endoscopic ultrasoundguided biopsy (eus-fna) procedures to screen patients with symptoms of pancreatic duct obstruction who later had at least two years of clinical follow up, including surgical resection in cases of pancreatic neoplasia (n = ) or at least one follow up clinic visit to confirm resolution of symptoms. blood samples were uniformly collected, processed, and banked per isev recommended guidelines. uniform machine (facsymphony) settings to standardize light scatter and fluorescence detection were based on commercially available beads (eg. megamix). samples were coded and randomized for testing and results were reported as the mean cell-and size-specific ev events/ul of plasma. results: clinical outcomes confirmed cases of cancer and screen negative controls. principle component analysis suggested that a number of different celland size-specific evs were significantly more common in the cancer cases (adjusted p-value < . , with aucs > . ), including epcam+/cd + events likely from cancer cells and cd +/cd p+/cd + microvesicles from platelets, among others. summary/conclusion: in this proof of principle study employing an ev-lyoplate design and nanoscale flow cytometry, we could reliably discriminate the ev-biomes in patients with cancer from negative controls. ongoing studies will determine whether these discriminators will be validated in larger cohorts and provide at least noninferior predictive value compared with the current gold standard clinical testing assay (eus-fna introduction: small cell lung cancer (sclc) is an aggressive tumour type, usually metastatic at diagnostic leading to poor overall survival. interestingly, sclc tumours are composed by distinct subpopulations of cells that cooperate as an ecosystem to drive tumour survival. since the subtype of sclc may have prognostic significance, the aim of this study was to identify surface marker proteins as biomarkers of sclc. methods: a linear discriminant analysis (lda) model, implemented in python via sci-kit learn, was used to choose the best markers for distinguishing subtypes. this analysis was based on rna-seq data from a previous study. in order to identify ev-based biomarkers that would identify sclc evs and not normal evs, we excluded from this analysis proteins without a verified transmembrane domain and proteins associated with evs expected to be present in white and red blood cells, and endothelial cells (according to exocarta and vesiclepedia databases). we also prioritized proteins that could be pan markers for sclc and that might have prognostic significance. to validate our findings, we performed western blotting and flow cytometry in sclc cell lines from different subtypes. results: our rna analysis indicated that the best surface markers to distinguish sclc subtypes were ceacam , fam a, lrfn , epha . immunoblot analysis validated ceacam and epha but not fam a or lrfn . we also found that ncam , a commonly used sclc marker, only marks some of the subtypes. for further analysis, we chose proteins with antibodies validated for flow cytometry as our chosen biomarker platform. flow cytometry analysis of cd is suitable as a pan-sclc marker. however, the expression of non-ne cell lines was decreased compared to rna-seq data. summary/conclusion: protein analysis of ceacam and epha corresponded to rna-seq data. ncam was not detected as a pan marker for all sclc subtypes. however, we could see cd expression in all sclc subtypes, indicating it may be a useful pan marker for sclc. future studies will be performed to validate the expression of other surface markers in cells, purified evs, and plasma of sclc patients. funding: nih u ca and nih u ca . leukobiopsyexploiting extracellular vesicle-mediated leukocyte sequestration of cancer-specific signatures introduction: in cancer, extracellular vesicles (evs) act as a unique exit mechanism for mutant and oncogenic macromolecules (proteins, rna and dna) en route from malignant cells to blood . while this process has inspired major liquid biopsy efforts, the biology of circulating evs that carry oncogenic mutations (oncosomes) is still poorly characterized. it is also unclear what part (if any) of the tumour-related cell free dna (ctdna) , , a major liquid biopsy analyte, is linked to circulating evs and what is their fate, receptacles and biological activity. methods: we employed as series of cancer cell lines carrying mutations in major oncogenes (hras, her , egfrviii). ev-dna was analysed by digital droplet pcr (ddpcr), along with nuclear anomalies in donor cells (dapi, electron microscopy) and transfer of dna to recipient cells of endothelial (huvec, mmbec), astrocytic (nha) or myeloid (hl ) origin. blood underwent fractionation into red blood cells (rbc), white blood cells (wbc), platelets (plt), evs ( , g ultracentrifugation) and soluble plasma (sup) . results: hras-mediated cellular transformation (in ras- cells) triggers profound changes in the structure of nuclear chromatin, which is driven into the cytoplasm and released as cargo of evs. oncogenic dna is detectable in blood fractions of tumour bearing mice. while evs, ctdna and plts contain intermediate levels of mutant dna, rbcs contain only traces of this material. the highest hras copy number per ml of blood is found in wbcs (monocytes and neutrophils), which contain more cancer dna/cell than liver, spleen and bone marrow. depletion of neutrophils using anti-ly g antibody results in an increase in ev-and ctdna-associated mutant dna in blood, suggesting the role of these cells in regulating the circulating levels of cancer cell-derived particles. uptake of dna-containing evs impacts the phenotype of myeloid cells, which adopt thrombo-inflammatory properties. these cells also retain cancer-specific transcripts and other cargo. finally, normal astrocytes treated with oncogenic evs also exhibit phenotypic changes and signs of genomic instability including formation of micronuclei. summary/conclusion: we propose that the process of leukocyte sequestration of circulating particles containing tumour-related nucleic acids renders these cells potentially usable as a novel liquid biopsy platform (leukobiopsy) in cancer. introduction: early diagnosis of colorectal cancer (crc) and precancerous adenoma patients is of vital importance. previously we profiled small extracellular vesicles (sevs) derived mirnas isolated from plasma, proposed a new promising biomarker category of crc patients. here we further gave a full landscape of circulating sevs derived rnas to explore and evaluate sevs based rna biomarkers for early detection of both crc and adenoma patients. methods: plasma sevs were isolated from participants, including early-stage crc patients, adenoma patients, and normal controls (nc), and characterized according to misv guideline. the total sevs derived rna expression profile of all participants was investigated by next-generation sequencing (ngs). weighted gene coexpression network analysis (wgcna) was performed to categorize differentially expressed rnas, and t-distributed stochastic neighbour embedding (tsne) was adopted to distinguish crc, adenoma, from nc samples with the top-ranked genes in wgcna modules. rt-qpcr validation was performed in a cohort of additional participants. results: a total of rna species (including mirnas, mrnas, and lncrnas) were found differentially expressed between plasma sevs in crc and nc participants. additionally, rna species were differentially expressed between plasma sevs in adenoma and nc participants. rna species were differentially expressed between plasma sevs in crc and adenoma participants. wgcna categorized all rnas into modules, which exhibited different expression trends during the carcinogenesis of crc. a -gene combined tsne model consists of the top genes in each module could perfectly classify crc, adenoma, and nc samples. a -gene combined tsne model consists of the top gene in each module could roughly distinguish crc and adenoma from nc, with only sample misclassified. rt-qpcr assays also confirmed the potential classification ability of those genes in another validation cohort of participants. introduction: although the concept of systematic "liquid biopsy" using bodily fluids is simple and elegant, the path of clinical reality has been challenging. recently, numerous tissue-specific biomarkers have been discovered in evs derived from blood, urine, cerebrospinal fluid, cell culture media, and a variety of other fluids. however, tracing the lineage of evs to their tissue of origin remains challenging due to their minute amount of cargo and unavailability of matching biopsied tissue and bodily fluids from the same patient. we recently demonstrated in three separate publications (dogra et. al; smith et. al; murillo et. al) , a new device (nanodld) for ev isolations, it's comparison with current technologies, bioengineered vesicles, and a detailed study of rna types present in small/ large vesicles, lipoproteins, and ago protein in different biofluids. in the present study, we aim to investigate the lineage of prostate derived evs in biofluids. methods: using our chip technology, we have isolated exosomes from prostate cancer cell lines and patient tissue, blood and urine samples. after exosome isolation, small rna libraries were prepared, and sequencing is carried out at icahn school of medicine and new york genome center using illumine sequencer hiseq . our nanofluidic pillar array is manufactured in an sio mask using optical contact lithography and deep ultraviolet lithography. results: our study revealed i) rna markers, which are exclusive to their prostate tissue of origin and are secreted in evs; ii) approximately - % of prostate tissue-specific rna were discovered in evs; iii) over % ( of rna) of literature curated prostatespecific rna signatures were detectable in serum and urine evs from pca patients; iv) evs contained over - % of noncoding rna ( - % was mirna), while tissue predominantly yielded rrna (> %); v) finally, gene set analyses generated that over % of evs rna were enriched for signalling pathways, yielding mirna-associated, non-canonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively monitor prostate tissue-specific biomarkers, identify tumour-specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. summary/conclusion: in summary, we have investigated patient matched tissue, serum, and urine derived evs in prostate cancer. we present a set of prostatic rna in evs, which are enriched in noncanonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively track prostatic biomarkers, identify tumour specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. a multi-model, liquid biopsy approach for diagnosing and staging pancreatic adenocarcinoma introduction: pancreatic ductal adenocarcinoma (pdac) is the third largest contributor to cancerrelated death in the usa. since there is not yet a feasible technology to diagnose pdac early in the disease, % of patients are diagnosed at an advanced stage. moreover, for patients with confirmed pdac, standard imaging method has low sensitivity to detect early metastatic disease, which complicates the selection of therapy. to address these challenges, there has been great interest in developing minimally-invasive, extracellular vesicle (ev) based blood tests for pdac. to this end, we have integrated measurements of tumour derivedev rna cargo with circulating proteins and cell free dna (cfdna), and use machine learning algorithms to distill this multiplexed diagnostic to . diagnose pdac patients from healthy and disease controls and . distinguish pdac patients with distance sites of metastasis to guide their treatment. we make use of our lab's magnetic nanopore isolation technique to specifically enrich for tumour derived evs directly from patient plasma. methods: we have developed a high throughput nanofluidic sorting platform, which immunomagnetically isolates individual evs from plasma using magnetic nanostructures. however, our architectures is uniquely designed for massive parallelization allowing high throughput, robust processing of ml of plasma in minutes. we performed sequencing on a discovery set of patients and controls (n = ). subsequently, we trained our panel of biomarkers using a training set of n = . finally, we validated the performance of our platform using an independent blinded test set of n = . results: the results of a blinded test set achieved an accuracy = % and an auc = . on binary classification of pdac patients versus those that were healthy or disease controls. in addition, we achieved an auc = . and accuracy = % with sensitivity of % and specificity of % on detecting occult metastasist. summary/conclusion: we developed a highly sensitive pancreatic cancer diagnostics by combining our nanomagnetic isolation platform for tumourderived ev isolation, rna sequencing, and machine learning. we isolated tumour-derived evs and profiled their rna cargo, combined with cfdna and ca - for pancreatic cancer diagnosis. the predictive panels successfully distinguished non-cancer patients from pdac patients, and nodistant metastasis patients(m ) from distant metastasis patients(m ) for appropriate treatment. the resulting auc and accuracy from the independent blinded test set outperformed any individual biomarker, showing both the benefits and the robustness of combining multiple orthogonal biomarkers for pdac diagnosis. introduction: both hypertension and diabetes exhibit significant molecular changes to the vasculature that are associated with increased cardiovascular risk. here we examined the protein composition of large evs (l-evs) isolated from the plasma of hypertensive, diabetic and healthy mice to identify common and diseasespecific molecular changes. methods: we examined circulating l-evs isolated from transgenic mice expressing active human renin in the liver (ttrhren, a model of hypertension), ove type diabetic mice, and their wild-type (wt) littermates. at weeks of age mice were sacrificed and blood samples were obtained by cardiac puncture. l-evs were isolated from platelet-free plasma via differential centrifugation and protein content was assessed via mass spectrometry (ms). results: ttrhren mice exhibited increased blood pressure compared with ove mice or their wt littermates. ( . ± . vs . ± . [ove ] vs. . ± . mmhg [wt], p < . ). ms identified independent proteins with at least peptides per protein. of these, proteins were found in all groups studied, were exclusive to wt mice, were exclusive to ove mice and were exclusive to ttrhren mice. in addition, proteins were observed with > . fold change (fc) compared to wild-type mice, and proteins were reduced by > %. amongst the top ten differentially expressed proteins, fibrinogen was upregulated in both ove and ttrhren mice compared with wild-type controls. similarly trem-like transcript , sarcoplasmic/endoplasmic reticulum calcium atpase and junction plakoglobin were all downregulated in both ove mice and ttrhren mice suggesting molecular changes common to both conditions. conversely, arginase was up-regulated in diabetic, but not hypertensive mice while carboxypeptidase was upregulated in hypertensive but not diabetic mice. summary/conclusion: taken together, these results show that the protein composition of circulating l-evs is altered in diabetes and hypertension and that both common and disease-specific changes may be detected. further analysis of these changes may lead to the identification of novel pathways associated with the pathogenesis of vascular injury in hypertension and diabetes. funding: this study was supported by grants (to db) from the canadian institutes of health research, an ontario early researcher award, and the canada foundation for innovation. understanding the role of endothelial cell-derived apoptotic bodies in inflammatory signalling and cell clearance in an atherosclerosis model of inflammation. introduction: apoptotic bodies (apobds) are a class of large (~ - um) evs formed during apoptotic cell disassembly, that are becoming increasingly recognized as potential mediators of intercellular communication, e.g. via the transfer of proteins and other cargoes to target cells. during the inflammatory vascular disease atherosclerosis, endothelial cell (ec) apoptosis contributes to loss of barrier function and promotes the formation of plaques in regions of ec damage. although, experimentally, ecs generate an abundance of apobds, a specific role for ec-derived apobds (ec-apobds) in the progression of atherosclerosis remains poorly defined. methods: in the present study, a detailed in vitro characterization of ec disassembly was performed via flow cytometry, confocal live cell imaging and cytokine profiling, followed by function analyses of ec-apobds using a murine in vivo model of dead cell clearance. results: characterization of ec disassembly revealed that apobd formation in ecs is regulated by rho-associated, coiled-coil-containing protein kinase (rock ), a process that can be pharmacologically inhibited using a rock- inhibitor, thereby providing tools for functional in vivo studies. the specific cargo and role in clearance of ec-apobds were then investigated. profiling of ec-apobds was performed via cytokine antibody array to reveal that ec-apobds generated under inflammatory conditions contain high levels of pro-inflammatory cytokines including mcp- and il- , suggesting a potential role for ec-apobds in the propagation of inflammation during vascular disease. furthermore, the ability of ec-apobds to be cleared from the vasculature via phagocytosis was investigated, revealing that ec-apobds can travel to distal organs to undergo clearance. summary/conclusion: these findings provide important insights into the potential functions of ec-apobds generated under both non-inflammatory and inflammatory conditions and may contribute to future studies involving the therapeutic targeting of ec disassembly for the treatment of atherosclerosis. funding: this work was supported by grants from the national health & medical research council of australia (gnt , gnt ) adipose mesenchymal stromal cell derived evs foster cardio-renal protection in the doca-salt hypertensive rat model introduction: cardio-renal syndromes (crs) are disorders of the heart and kidneys whereby "acute or chronic dysfunction in one organ may induce dysfunction of the other". stem cell-derived extracellular vesicles (evs) mediates the protection of the kidney from development of chronic kidney disease (ckd). we here investigated the potential of adipose-mesenchymal stromal cells derived evs (asc-evs) as therapeutic tools for the treatment of crs. methods: adult wistar rats were uninephrectomized and treated with a high-na+ diet and deoxycorticosterone-acetate (doca-salt) for -weeks ( / ; a / - - ). evs were isolated by ultracentrifugation method. ev dimension, concentration and surface markers were characterized by nta, cytofluorimetric analysis and transmission electron microscopy. to characterize the role of evs in crs, doca-salt rats were injected weekly with asc-evs. systolic blood pressure was measured by the tail-cuff method. plasma creatinine and urinary protein excretion were determined by colorimetric assays and microalbuminuria by immune turbidimetric assay. qrt-pcr and western blot were conducted to evaluate fibrosis and inflammatory-related genes and proteins in the kidney and heart of doca-salt rats. immunohistochemistry was used to confirm matrix accumulation (a-sma) and immune infiltrate (cd + cells). results: multiple administration of asc-evs in doca-salt rats induced a protective effect on the kidney, by reducing tubular and vascular damage. kidney function was also conserved by ev treatment as detected by the normal glomerular filtration rate and the absence of proteinuria with respect to doca-salt untreated rats. ev administration significantly decreases the pro-inflammatory molecules mcp- and pai and reduce the recruitment of macrophages in the kidney. the mitigation of the inflammatory response by asc-ev infusion consequentially affected the development of fibrosis, as detected by the decrease in collagens (col a , col a ) and fibronectin (fn) expression in respect to doca-salt animals. asc-evs were able to act in multiple organs, preventing fibrosis and inflammation also in the heart, therefore alleviating blood pressure rise during the -weeks of treatment in doca-salt rats. summary/conclusion: our results indicate that asc-ev administrations in hypertensive-induced ckd rats promote protection from renal damage, reduction of the inflammatory response and prevention of interstitial fibrosis in the kidney. asc-evs are also able to protect the cardiac tissue and to control blood pressure increase, displaying complex and multiorgan beneficial effects. introduction: alveolar macrophages (ams) tonically secrete extracellular vesicles (evs) containing suppressor of cytokine signalling (socs ) protein. uptake of socs -containing evs by alveolar epithelial cells is critical for restraint of cytokine-induced janus kinasesignal transducer and activator of transcription (jak-stat ) signalling to promote homoeostasis in the distal lung. at steady state, ams exhibit suppressed glycolytic activity, a metabolic phenotype that promotes homoeostatic function. whether this glycolytic restraint is critical for am secretion of socs is unknown. in fact, to our knowledge, metabolic control over release of any ev cargo has never been explored in any cellular context. methods: immortalized mouse ams (mh-s) were treated with various doses of -deoxy-d-glucose ( -dg) and oligomycin, inhibitors of glycolysis and oxidative phosphorylation, respectively. primary rat ams collected by lung lavage were treated with an aqueous extract of cigarette smoke (cse) with or without -dg. metabolic activity was measured by seahorse assay, evs were quantified by nanoparticle tracking analysis, and vesicular (> -kda) socs secretion was determined by western blot of conditioned medium. additionally, ams collected from wild-type (wt) and lsl-krasg d mice bearing lung tumours weeks after intrapulmonary ad-cre were cultured ex vivo in the presence or absence of -dg. vesicular (> -kda) socs secretion was measured by elisa. results: in a dose-dependent manner, oligomycin inhibited, whereas -dg enhanced, socs and ev release by mh-s cells. treatment of rat ams with cse ( %) attenuated secretion of socs , an effect that coincided with increases in glycolytic activity, and co-treatment of ams with -dg abrogated the inhibitory effect of cse on socs release. finally, ams collected from lsl-krasg d mice exhibited a deficiency in socs secretion relative to wt ams, an effect that was reversible by overnight culture in the presence of -dg. summary/conclusion: in tandem, our data generated using in vitro and in vivo approaches demonstrate that am secretion of vesicular socs is down-regulated by glycolysis. we speculate that metabolic control over release of ev cargoes is a phenomenon of broad biologic relevance within and outside of the lung. introduction: bacterial extracellular vesicles (ev) are described to play roles in defence and resistance, pathogenesis and stress responses. cyanobacteria pioneered oxygenic photosynthesis, and are the ancestors of modern chloroplasts. we previously described that by deleting the gene encoding tolc (Δtolc) in the model cyanobacterium synechocystis sp pcc (s ), a key player in protein-mediated secretion systems, a hyper-vesiculating phenotype could be obtained. the goal of this work was to understand why Δtolc hyper-vesiculates. methods: isobaric tag for relative and absolute quantitation (itraq) was used for quantitative proteomic analyses of total cell extracts. ev were isolated as follows: cells were separated from the extracellular medium (em) by centrifugation ( g, min) and filtration ( . µm pore-size filters). cell-free em was concentrated using centrifugal filters (mwco of kda), and later ultracentrifuged for h at g. the final ev fraction was suspended in growth medium. ev characterization was performed using tem, dls, nanosight, and by the detection and quantification of lps (lipopolysaccharides). detection of specific proteins in ev was carried out by western blot. copper (cu) levels were quantified by atomic absorption spectrometry (aas). results: a large-scale quantitative proteomic analysis was performed, resulting in the identification of several metal-related proteins with differential regulation in s Δtolc. both wild-type (wt) and Δtolc cells were then challenged with different metals. compared to the wt, Δtolc showed impaired growth only when exposed to cu, a co-factor for several proteins with roles in primary metabolism. the intracellular cu levels were quantified and Δtolc accumulates threefold more cu than wt cells. we then asked whether the hyper-vesiculating phenotype observed could be linked to the stress induced by cu accumulation. in ev isolated from Δtolc we detected the metallochaperone copm, a periplasmic cu-binding protein involved in cu-resistance mechanisms in s . in addition, cu could also be detected in isolated Δtolc-ev. in addition, more ev were detected when s wt cells were challenged with cu, in a cu-concentration dependent manner. summary/conclusion: these results support the idea that bacterial ev represent an alternative cu-secretion mechanism to deal with cu-induced stress. funding: fct phd grant sfrh/bd/ / ; feder-compete -poci-fct project: poci- - -feder- . juan wang and maureen barr rutgers university, human genetics institute of nj, piscataway, usa introduction: extracellular vesicles (evs) function in intercellular communication. despite their physiological importance and biomedical relevance, knowledge of ev fundamental biology is not well understood, in part due to a lack of tractable animal systems. our analysis of environmentally-released c. elegans ciliary evs provides strong evidence that nematodes package cargo in evs that mediate inter-organismal communication, in analogy to intercellular signalling in mammals. we predict that conserved mechanisms underlie ev cargo sorting, biogenesis and signalling. cilia act as cell towers to both receive extracellular signals and to send information via ciliary evs. ciliary defects result in human ciliopathies including autosomal dominant polycystic kidney disease (adpkd). adpkd is a life-threatening disease that affects / and is caused by mutations in pkd and pkd , which encode polycystin- and − . in c. elegans and humans, the polycystins are architecturally similar, act in the same genetic pathway, function in a sensory capacity, localize to cilia, and are shed in evs, suggesting ancient conservation. moreover, ciliary ev biogenesis and shedding is an evolutionary conserved process from algae to worms to humans. by studying how cilia make and receive evs, we aim to uncover fundamental principles of how cells communicate using evs. methods: to study ciliary ev cargo sorting and biogenesis, we use genetically-encoded fluorescent-tagged ev cargo and superresolution zeiss airyscan confocal microscopy in living animals. results: we find that cargoes are sorted into distinct populations. in cilia, kinesin- motors and kinesin- klp- /kif transport different ev cargoes to the ciliary tip and generate an ev cargo enrichment zone. from here, evs are shed and released into environment in a spatially and temporally regulated manner. ciliary ev biogenesis and release is regulated by mechanical pressure and ph. our work revealsat the single cell levelthat different evs are made in response to environmental stimuli, which may be important for ev signalling properties. summary/conclusion: cells exploit the spatiallyrestricted cilium and its sophisticated transport system to generate distinct populations of ciliary evs. how these ciliary ev communicate cellular messages awaits decoding. introduction: we recently demonstrated that recycling endosomes marked by rab a generate exosome subtypes distinct in cargos and functions from late endosomes, which we collectively term rab -exosomes. these exosomes are preferentially released from cancer cells in response to metabolic stress and promote adaptive changes in a xenograft model. here we use comparative ev proteomics in hct colorectal and hela cervical cancer cell lines to identify rab -exosome signature proteins and screen for functional effects. methods: we analysed ev preparations by mass spectrometry using tandem mass tag® labelling to identify changes in ev protein cargo in response to glutamine depletion. candidate genes were subsequently knocked down in drosophila secondary cells, which permit visualisation of rab -exosome biogenesis using fluorescence microscopy, and in human cancer cell lines. results: we show that accessory escrt-iii proteins, chmp , chmp and ist , are enriched on glutamine-depletion-induced evs and play a selective and conserved role in generating rab -exosomes. they are, however, not required to traffic ubiquitinated cargos into late endosomes and lysosomes. escrt- components, thought to regulate trafficking of ubiquitinated cargos into intraluminal vesicles, are also required to make rab -exosomes. in flies the escrt- , hrs, localises to the limiting membrane of rab -endosomes. comparative proteomics reveals other proteins enriched in rab -exosomes, which also appear to be needed to mediate this novel exosome formation mechanism. summary/conclusion: we conclude that rab -exosome subtypes are formed via a distinct mechanism requiring accessory escrt-iii components, suggesting a route to selectively target these exosomes. introduction: the tumour microenvironment consists of a complex network of host cells embedded within extracellular matrix. communication between these cellular compartments is critical for tumour progression and exosomes have emerged as important regulators of intercellular communication. while a number of studies have implicated exosomes in cancer progression, mechanisms controlling exosome transfer are not well understood. we developed three-dimensional ( d) culture models to evaluate the role of cues provided by the extracellular matrix in exosome release and uptake. methods: exosomes were isolated from cells in two-and three-dimensional culture via ultracentrifugation and characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. exosomes were labelled with fluorescent lipophilic dyes and uptake in recipient cells quantified by flow cytometry. results: cells cultured in d display decreased exosome release and increased uptake compared to d cultured cells. exosome release in d culture was inhibited with the exosome release inhibitors brefeldin a and gw , but was not significantly altered by knockout of rab b. in addition, disruption of polarity signals provided by d culture did not impact exosome release or uptake in d, but induction of oncogenic hras increased both secretion and uptake of exosomes through activation of pi k signalling. summary/conclusion: release and uptake of exosomes is altered in d environments. these studies help provide insight into exosome production and uptake in vivo and have potential implications for therapeutically targeting exosome release and the development of exosome based therapeutic delivery vehicles. introduction: previous studies in our lab found that expression of r w-fibulin- induces rpe to undergo emt. the purpose of current study was to characterize the extracellular vesicles (evs) in rpe cells expressing wt-fibulin- versus rpe cells expressing r w-fibulin- and investigate the effects of these evs on rpe cell differentiation. methods: arpe- cells were infected with lentivirus with luciferase-tagged wild-type (wt)-fibulin- or luciferase-tagged r w-fibulin- . evs were isolated from the media of arpe- cells by conventional ultracentrifugation or density gradient ultracentrifugation. transmission electron microscopy (tem) and cryogenic electron microscopy (cryo-em) were performed to study the morphology of the evs. the amount and size distribution of evs were analysed by nanosight tracking analysis (nta). ev protein concentrations were quantified using the dctm protein assay (bio-rad). ev cargo were analysed by unbiased proteomics using lc-ms/ms with subsequent pathway analysis (advaita). migration ability was evaluated in arpe- cells with or without the exposure of evs by conducting scratch assays. results: morphologically, tem imaging showed concave-appearing vesicles and cryo-em imaging showed spherical vesicles with two subpopulations of evs: a small group with diameters around nm and a large group with diameters around nm. moreover, tem and cryo-em showed an increased amount of small evs (~ nm) in the mutant group compared to the wt group. this result was further confirmed by nta showing that, in the mutant group, the particle size distributions were smaller than the wt evs. no significant differences were shown in ev protein concentrations per particle between wt and mutant groups. our previous data suggest that the expression of r w-fibulin- causes rpe cells to undergo emt as evidenced by upregulated emt drivers and an increased migration ability. proteomic studies showed that evs derived from arpe- cells overexpressing wt-fibulin- contain critical members of sonic hedgehog signalling (shh) and ciliary tip components, whereas evs derived from rpe cells overexpressing r w-fibulin- contain emt mediators, indicating that ev cargo reflects the phenotypic status of their parental cells. ev transplant studies showed that exposing native rpe cells to mutant rpe cell-derived evs containing emt drivers, including tgf-β-induced protein (tgfbi), vim, and smad , leads to an enhanced migration ability of rpe cells in a dosedependent manner. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms ( d static tissue culture flask vs d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in d t-flasks and d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both d and d culture systems. introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies methods: pca patients and age-matched healthy controls (hc) plasma (n = in each group) were used to isolate sevs with different isolation methods including commercial exoquick ultra kit, qev and qev size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd and cd commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev . furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods summary/conclusion: qev method demonstrated better performance in isolating relatively pure sevs from human plasma; qev has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev but with the highest non-sev protein contaminations. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd , cd and cd , it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: ) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and ) the assessment of subpopulation target enrichment relative to the population average by leveraging tim as an unbiased, lipidbased ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasisassociated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr (her +), t d and mcf- (er+pr+), bt and mda-mb- (triple negative) breast cancer cell lines, as well as five mda-mb- -derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her was broadly detected in her + skbr evs. interestingly, her -t d and mcf- evs also expressed her where it was highly enriched in its epcam+ subpopulations. itg α , β and β were only found in triple negative and organotropic evs with itg β and β differentially enriched based on the organotropism. the population average of mda-mb- and lung-tropic evs had high expression of itg β , where subpopulations of cd + evs showed positive enrichment while cd + and cd + evs showed negative enrichment. itg α , β and β were absent in the bone-tropic cd + ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb- evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. op . = pf . heparan sulphate proteoglycans are required for ev-mediated delivery of multiple growth factors sara veiga, alex shephard, alex cocks, aled clayton and jason webber cardiff university, cardiff, uk introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified targets that bind to hs-gag chains, and also different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of ev- introduction: methamphetamine (ma) and related amphetamine compounds, which are potent psychostimulants, are among the most commonly used illicit drugs. neuroimaging studies have revealed that chronic ma abuse can indeed cause neurodegenerative changes in the brains of human ma abusers including prominent microglial activation throughout the brain. it is still unclear how chronic inflammation caused by ma abuse leads to long-term damage to the brain. with this in mind, we are particularly interested in studying the role of extracellular vesicles (evs) in eliciting chronic inflammation in ma exposed brains. in the present study, we focus on the role of a mirna, mir- a- p (mir- a) in chronic ma exposure. here, we present novel data that shows for the first time how chronic ma impacts not only the biogenesis but also the ev associated mirna cargo thereby affecting the overall health of the neurons and glial cells in the brain. methods: -density gradient centrifugation for isolation of brain-derived vesicles -characterization of bdes by western blotting, nanoparticle tracking analysis and transmission electron microscopy -quantitative rt-pcr -digital droplet pcr -confocal imaging of dendritic spines and synapses results: in the present study, we show from both in vivo and in vitro studies that chronic methamphetamine (ma) treatment alters ev biogenesis and microrna (mirna) cargo. brain-derived evs (bde) isolated from frontal grey tissue of rhesus macaques that were administered ma in a chronic regimen revealed a significant increase in both number and size. further analysis revealed increase in biogenesis genes and increased levels of mirna, mir- a- p (mir- a). in situ hybridization of the frontal brain area revealed that mir- a was exclusively expressed in microglia and neurons. further, in vitro studies revealed that ev associated mir- a elicited not only neuronal damage but also was able to activate microglia to release pro-inflammatory cytokines thereby inducing a chronic inflammatory cycle. finally, we show that an anti-inflammatory drug was able to rescue inflammation, mir- a levels and synaptodendritic injury. summary/conclusion: in summary, our results present for the first time show that chronic ma exposure in the brain affects ev biogenesis and mirna expression. we further confirm that mir- a can serve as potential marker to diagnose synaptic deficits for chronic ma addiction in humans. finally, we reveal that anti-inflammatory drug could rescue the ev biogenesis and reduces the secretion of mir- a, thereby rescues synaptodendritic injury. our data further supports the use of the anti-inflammatory drugs as therapeutic interventions for ma addiction. funding: nida funding # r da blood-borne and brain-derived ectosomes/microparticles in morphineinduced anti-nociceptive tolerance deepa ruhela, veena bhopale, ming yang, kevin yu, eric weintraub, aaron greenblatt and stephen r. thom university of maryland school of medicine, baltimore, usa introduction: opioid pain treatment is impeded because chronic administration decreases analgesia, a condition called tolerance that prompts dose escalation contributing to morbidity and mortality. inflammatory interleukin (il)- β is required for tolerance development, so we hypothesized that pro-inflammatory extracellular vesicles (evs) play a role. methods: evs with opioid administration were assayed in mice and humans. annexin v-positive, . - µm diameter microparticles (mps) were assessed by flow cytometry in murine and human blood and in murine deep cervical lymph nodes that drain brain glymphatics. blood-borne exosomes (< nm) were assayed by tunable-resistance pulse sensing (trps). anti-nociceptive tolerance following morphine administration to mice was assessed by speed of tail removal from warm water. results: repetitive morphine dosing of mice to induce anti-nociceptive tolerance increased blood-borne mps by eightfold, and by tenfold in cervical lymph nodes. mps expressed proteins specific to neutrophils, microglia, astrocytes, neurons and oligodendrocytes. il- β content of mps increased -fold. administration of an il- β antagonist to mice diminished blood and glymphatic mps elevations and abrogated tolerance induction. intravenous polyethylene glycol telomer b that lyses mps and intraperitoneal methylnaltrexone that binds peripheral opioid-mu receptors and myeloid differentiation factor- to inhibit toll-like receptors, inhibited mps elevations and tolerance. neutropenic mice did not develop anti-nociceptive tolerance, elevations of blood-borne mps or cervical node mps expressing microglial proteins. elevations of blood-borne exosomes were not identified based on trps analysis. patients entering treatment for opioid use disorder exhibited similar mps elevations as do tolerant mice. summary/conclusion: neutrophil-derived mps containing il- β are required for morphine-induced antinociceptive tolerance. funding: this project was supported by grant n - - - from the office of naval research and an unrestricted grant from the national foundation of emergency medicine. evs are a conveyor of toxic dipeptide repeat proteins in c orf als/ ftd models thomas jefferson university, philadelphia, usa introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterized by loss of motor neurons. in als, motor symptoms initiate focally and then progress gradually, distal from the initial focus. abnormal forms of als-associated proteins are physically exchanged between neuronal cells. pathogenic als proteins like sod , fus and tdp are transmitted between cells by assisted mechanisms, mainly extracellular vesicles (evs), spreading toxicity and misfolding of native proteins within the recipient cells. an intronic g c aberrant nucleotide repeat expansion in c orf gene is the most common genetic cause of als. translation of this expanded region occurs by a process called repeat associated non-aug (ran) translation that produces five dipeptide repeats proteins (dprs), polyga, polygp, polygr, polypa and polyga. polyga, polygr and polypr are associated with toxicity in neurons. in this work we study the recruitment of these aberrant proteins into extracellular vesicles (evs) and the potential role of these evs in spreading toxicity between cells of the central nervous system. methods: to isolate the evs from cell culture media we isolated by ultracentrifugation the larger vesicles at , xg and the smaller evs at , xg. number, size and fluorescence of the vesicles were analysed by fluorescent nanotrack analysis (f-nta) and by cytoflex. the protein content of the vesicles was analysed by western blot (wb). to evaluate the potential toxicity of the evs, a transwell system (tw) was employed. neuron viability was assessed using live imaging techniques. results: nsc were transfected with reporter constructs expressing dprs tagged with gfp protein. by f-nta, cytoflex and wb analysis we assessed that all the five dprs were loaded in both the large and the small vesicles isolated from cell culture medium. by tw, nsc transfected with the dprs were put in contact with primary cortical neurons (cns) transfected with synapsin driven td-tomato for live imaging purposes. we observed that polygr+ nsc were able to cause a significant decrease in cns viability. we also observed that polygr+ evs associated toxicity was directly dependent on polygr length. this effect was reverted reducing the number of polygr+ evs treating nsc with gw . to understand the downstream effect of polygr+ evs in recipient cells we studied tdp mislocalization, ran-translation and activation of the integrated stress response, finding a dysregulation of all these potentially toxic pathways in neurons treated with polygr+ vesicles. summary/conclusion: concluding, dprs are actively secreted in evs and polygr+ vesicles cause the activation of toxic mechanisms in the recipient cells, possibly contributing to the spreading of als introduction: pregnancy is the a condition that profoundly mitigates symptoms of multiple sclerosis (ms) a complex disease characterized by immune dysfunction and neurodegeneration affecting . million people worldwide. serum exosomes, released by specific cells during pregnancy, modulate the immune and central nervous system function and contribute to pregnancy-associated suppression of experimental autoimmune encephalomyelitis (eae), an induced preclinical model of ms. extracellular vesicles (evs) are the new means for communication among cells. the aim of our study was to characterize the ability of human amniotic fluid stem cells-derived evs (hasc-evs) to antigen presenting cell function thus correcting immune dysfunction in eae. methods: amniotic fluids were obtained from human - -week pregnant women. hasc-evs were collected by ultra-centrifugation. evs were characterized for their specific proteins, lipids and nucleic acids expression. the ability of evs to modulate immune responses was performed in vitro, testing the ability of evs to induce a tolerogenic phenotype in mouse bone marrow derived dendritic cells, and in vivo for their potential to suppress eae, induced by immunization c /b female mice with mog - peptide. results: we found that hasc-evs expressed high levels of galectin- and promoted a significant increase of the immunoregulatory enzyme indoleamine , dioxygenase- enzyme in dcs. moreover in in vivo experiments administration of hasc-evs significantly reduced disease severity in eae. such effect was associated with reduced neurological deficits and suppression of pathogenic t helper (th ) cells, and increased percentage of regulatory t cells (treg-foxp +) cells. summary/conclusion: our findings unravel immunoregulatory effects of evs secreted by hascs. evs may represent a novel cell-free immune regulatory and regenerative therapeutic approach that can potentially mitigate immune dysfunction and promote remyelination. association of neuronal-derived extracellular vesicles cargo with cognitive decline in late middle life introduction: alzheimer's disease (ad) is characterized by a long preclinical stage during which phosphorylated tau pathology spreads in the brain leading to clinical symptoms. pathogenic tau spreads, in part, via extracellular vesicles (evs). we and others have demonstrated that tau cargoes of neuronal-derived evs (nevs) from blood can serve as biomarkers for ad. we aimed to examine whether nev tau cargo can predict cognitive decline in late middle age by leveraging samples from participants in the wisconsin registry for alzheimer's prevention (wrap) study. methods: we blindly immunoprecipitated nevs using antibody against neuronal l cell adhesion molecule (l cam) from serum samples of wrap participants who were cognitively unimpaired at baseline (mean age . ± . years old; . % females; . % apoe carriers), of whom half subsequently developed cognitive decline. we measured phosphorylated (p and p ) and total tau in nevs using electrochemiluminescence assays. we used linear regression models to identify differences between cognitive status groups including age, sex apoe status and the cognitive status*age interaction in the model. results: at baseline, we found trends for higher p -(p = . ) and p -tau (p = . ) levels in future decliners compared to stable participants. further, there were significant cognitive status*age interactions for ptau (p < . ), total tau (p < . ) and ptau (p < . ) with higher levels with increasing age in future decliners summary/conclusion: nev tau cargo differs between late middle-aged individuals at risk for ad with and without future cognitively decline even before decline occurs, presumably due to subclinical spread of tau pathology. further nev biomarker development may allow preclinical ad diagnosis. introduction: in the brain, circulating extracellular vesicles (evs) in the cerebrospinal fluid (csf) contain a variety of signalling factors, including proteins, enzymes, and rna transcripts. while evs have been implicated in many cell-to-cell signalling contexts, the vast majority of these studies are based on findings derived from cell culture conditions. thus, the ability to identify cell typespecific ev release from cellular subpopulations within the brain represents a critical barrier in the field. methods: to address this knowledge gap, we utilized a novel transgenic mouse model to determine the release of cell-type specific evs. here we report the exomap- mouse, which is designed to express an exosomal green fluorescent protein in response to expression of cre recombinase. specifically, the exomap- transgene was inserted at the mouse h locus and consists of (i) a broadly expressed cag promoter/enhancer, (ii) a floxed orf encoding mts-tdtomato, (iii) an orf encoding the exosomal protein acyltya fused to mneongreen (mng), and (iv) a ʹ utr containing the wpre element and polyadenylation signal from the bovine growth hormone gene. results: intracranial ventricular injections of the viral vector aav-ttr-cre, which drives cre recombinase expression from the choroid plexus-specific promotor of the transthyretin gene, leads to acyltya-mng expression in the choroid plexus. moreover, we observed that these mice released mneongreen-positive evs into the cerebrospinal fluid and also visualized the vesicles in the blood. furthermore, these mice displayed an accumulation of acyltya-mng fluorescence in the medial habenula. summary/conclusion: the results indicate that choroid plexus-derived evs are trafficked to the csf and the medial habenula, and more generally, that the exomap- mouse can be used to follow the trafficking of tissuespecific evs into biofluids and between tissues in vivo. introduction: large-scale colorectal cancer (crc) sequencing studies have shown that % of all tumours had at least one mutation in proteins implicated in the wnt signalling pathway. mutations in β-catenin have often been associated with the constitutive activation of wnt signalling pathway and has been established as a major driver of crc. one of the proposed mechanisms of activating wnt signalling involves extracellular vesicles (evs) as cellular couriers to transfer wnt ligands from one cell to another. however, the association of oncogenic mutant β-catenin with evs has not been studied. subpopulations of cancer cells with different mutational loads and behavioural variations lead to intra-tumour heterogeneity methods: integrative proteogenomic analysis showed the secretion of mutant β-catenin via evs. evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. silac-based quantitative proteomics analysis, immunofluorescence, biochemical analysis, qpcr and xenograft models were employed to unveiling the role of evs carrying mutant βcatenin. results: an integrative proteogenomic analysis identified the presence of mutated β-catenin in evs secreted by colorectal cancer (crc) cells. follow up experiments established that evs released from lim crc cells stimulated wnt signalling pathway in the recipient cells with wild type β-catenin. silac-based quantitative proteomics analysis confirmed the transfer of mutant β-catenin to the nucleus of the recipient (rko crc) cells. in vivo tracking of dir labelled evs in mouse implanted with rko crc cells revealed its bio distribution, confirmed the activation of wnt signalling pathway in tumour cells and increased the tumour burden. introduction: there has been a significant increase in incidence of human papillomavirus (hpv ) driven oropharyngeal cancer (opc) in developed countries. there is evidence that hpv alters the molecular cargo of exosomes released by opc. emerging evidence suggests that hpv integration within the human genome is associated with both genomic and transcriptomic alterations. consistent with previous studies, the genomic viral-cellular junctions were identified using dips-pcr method in ( %) saliva samples collected from hpv -driven opc. methods: morphology and molecular features of exosomes derived from three different saliva sampling methods: unstimulated saliva; acid-stimulated saliva; and salivary oral rinses were examined using transmission electron microscopy (tem), nanoparticle tracking (nta) and western blot analysis. hpv- dna detection in salivary exosome was determined by using qpcr method. proteome profile of salivary exosomes derived from both cancer-free controls and hpv -driven opc patients was characterized using liquid chromatography-electrospray ionization-tandem mass spectrometry (lc-ms/ms). results: we demonstrate that unstimulated saliva had greater abundance of exosomes when compared to the other sampling methods. three common exosome markers (cd , cd and cd ) were higher in unstimulated saliva. only salivary exosomes derived from hpv-driven opc patients had a detectable level of hpv- dna. the proteomic signature of salivary exosome was significantly (p < . ) different between cancer-free controls and hpv-driven opc. we found elevated protein abundance of five main glycolytic enzymes (i.e. phosphoglycerate kinase (pgk ), glyceraldehye- -phosphate dehydrogenase (gapdh), aldolase (aldoa) and lactate dehydrogenase a (ldha) in salivary exosomes derived from opc patients, suggesting a functional role of salivary exosome in the reciprocal interplay between hpv-driven opc and glucose metabolism. summary/conclusion: our data suggest that the development of a low-cost non-invasive saliva-based test using both salivary exosomal dna and protein may offer an opportunity to detect hpv-driven opc, that may be clinically useful in managing these patients. continuous in vivo release of mast cell derived extracellular vesicles from an implanted device spreads pro-inflammatory response in mice introduction: mast cells are important players of the immune system and they secrete a wide range of mediators during bacterial infections. mast cells are also able to release extracellular vesicles (evs). here, we report that mast cells communicate with each other in vivo by evs. methods: we isolated bone marrow-derived and peritoneal mast cells from gfp-transgenic and wild type mice. evs were separated from the conditioned media of these cells cultured in the presence or absence of lipopolysaccharide (lps). evs were characterised according to the misev guidelines by flow cytometry, electron and fluorescent microscopy, trps, the spv lipid and the bca protein assays. separated ev-s were cultured with naïve mast cells, and tumour necrosis factor (tnf)-α production was tested by elisa and intracellular flow cytometry. gfp+ mast cells were seeded in diffusion chambers which were implanted into the peritoneal cavities of mice enabling us to investigate the continuous in vivo release of evs. uptake of gfp+ evs and tnf-α expression of peritoneal mast cells were tested by flow cytometry and fluorescent microscopy. results: here, we showed that bacterial lps-sensing mast cells release evs that in turn, induce tnf-α expression in resting mcs in vitro. moreover, we confirmed that evs are transmitted to other peritoneal mast cells in vivo spreading the pro-inflammatory response by inducing tnf-α secretion in peritoneal mast cells. summary/conclusion: ev communication between members of the mast cell network, play an important role in spreading and escalating pro-inflammatory responses to immune stimuli. our data may provide an explanation how the relatively rare tissue resident mast cells can play key roles in diseases such as autoimmune arthritis. the ability of small extracellular vesicles (sevs) to reprogramme cancer cells is known. integrins, receptors for extracellular matrix proteins, are major players in mediating sev functions. previously, we have reported that the αvβ integrin is detected in sevs of prostate adenocarcinoma (prca) cells and transferred into recipient cells in a paracrine fashion; however, its role and expression have never been explored in the most aggressive forms of prca, such as neuroendocrine prca (neprca). neprca does not express androgen receptor (ar) but does express neuron-specific proteins, such as aurora kinase a, synaptophysin and neuron specific enolase, that activate pro-tumorigenic pathways independently from the ar. methods: we isolated sevs from prca c - b cells using iodixanol density gradients and characterized them by immunoblotting and exoview. the experiments were performed in vivo by injecting subcutaneously, in nude mice, du cells treated with sevs expressing or lacking the αvβ integrin, and in vitro, by testing anchorage-independent growth of different cell lines treated with the same sevs. discarded human tissues from prca metastasis were analysed by immunohistochemistry (ihc). results: we demonstrate that a single treatment of prca cells with sevs significantly stimulates tumour growth and anchorage-independent growth. moreover, we show that one treatment with sevs, shed from c - b cells that express αvβ , but not from the control cells that lack αvβ , induces differentiation of prca cells towards a neuroendocrine phenotype and downregulates ar. finally, our ihc analysis shows coexpression of αvβ integrin and synaptophysin in neprca metastatic lesions. summary/conclusion: in conclusion, our current study shows, for the first time, that αvβ integrin expression in donor cells generates sevs that reprogramme recipient cells towards an aggressive tumour phenotype. funding: this study was supported by nci-p - , r - to lrl. introduction: exosomes are small extracellular vesicles (sevs) that carry a variety of cargoes and have been shown to promote tumour cell motility and metastasis. cell motility is influenced by dynamic formation and stability of filopodia: actin-rich protrusions that extend from the leading edge and perform directional sensing. filopodia regulators such as fascin are upregulated in multiple epithelial cancers and can promote invasive phenotypes. however, how filopodia are induced and controlled by extracellular factors is poorly understood. here, we describe a role for sevs in regulating filopodia formation and tumour cell motility. we utilized b f melanoma cells and ht fibrosarcoma cells for fixed-and live-cell imaging to quantify filopodia numbers and dynamics in control and exosome-deplete conditions. itraq proteomics was used to identify sev protein cargoes that contribute to filopodia formation. in vivo experiments were performed using a chick embryo model for metastasis. results: inhibition of exosome secretion in cancer cell lines, via rab a or hrs knockdown, led to decreased filopodia numbers. specificity to sevs was demonstrated by rescue experiments in which purified sevs but not large evs rescued the filopodia phenotypes of exosome-inhibited cells. live imaging of hrs-kd cells revealed that exosome secretion regulates formation and stability of filopodia. proteomics data and molecular validation experiments identified the tgf-beta coreceptor endoglin as a key sev cargo regulating filopodia formation, cancer cell motility, and metastasis. summary/conclusion: in this study, we identified exosomal endoglin as a regulator of filopodia formation and in vivo metastasis. these data are relevant to cancer as endoglin expression is altered in many cancers. in addition, endoglin is the disease gene for hereditary haemorrhagic telangiectasia, and may influence angiogenesis. overall, our data implicate sev-carried endoglin as a key cargo regulating filopodia. astrocyte-derived ev-mediated blood-brain barrier disruption shilpa buch, ke liao, susmita sil, fang niu and guoku hu university of nebraska medical center, omaha, usa introduction: the breach of the blood-brain barrier (bbb), resulting in ensuing neuroinflammation, is a key feature of hiv-associated neurological disorders (hands). while combination antiretroviral therapy (cart) has successfully suppressed peripheral viraemia, cytotoxicity associated with the presence of viral tat protein in tissues such as the brain, remains a significant concern. our previous study has demonstrated that hiv- tat can induce disruption of bbb by downregulation of tight junction (tj) proteins in human brain microvascular endothelial cells (hbmecs) and that this is regulated by the autophagic pathways. methods: evs were isolated from hiv tat-stimulated mouse/human primary astrocytes using the standard differential ultracentrifugation method and characterized by transmission electron microscopy, nanosight & western blot analyses. among the various mirs dysregulated in hiv tat -stimulated astrocyte ev cargo, mir- was found to be upregulated by realtime pcr. confocal microscopy identified uptake of astrocytic evs by hbmecs. functional assessment of astrocytic ev uptake by hbmecs involved cell permeability using transepithelial electrical resistance as well as trans-well endothelial cell monolayer permeability assays. results: hiv- protein tat-mediated induction of micrornas (mirs) in astrocyte-derived extracellular vesicles (adevs) regulated the permeability of bbb by targeting the expression of tj proteins in the hbmecs. exposure of hbmecs to tat-adevs resulted in down-regulation of the tight junction protein claudin , resulting in increased endothelial cell monolayer paracellular permeability. microarray data of tat-adevs demonstrated upregulation of several mirs compared to that of controls, among which upregulated mir- was identified to target the tj proteins using ingenuity pathways analysis. increased expression of mir- was validated in tat exposed astrocytes and tat-adevs. adevs loaded with mir- oligos showed similar effects as that observed with tat-adevs in inducing permeability in hbmecs. increased expression of mir- with downregulation of claudin- was also recapitulated in microvessels isolated from the brains of doxycycline-inducible hiv- tat transgenic mice (itat) mice and in lysates isolated from the frontal cortices of siv+ macaques/hiv+ autopsied brains. summary/conclusion: our findings demonstrated that tat-adevs containing mir- as an important mediator underlying tat-mediated disruption of the bbb. introduction: endogenous exosomes and related extracellular vesicles (evs) are potent nanoparticles released by all cells tested to date. the exploitation of their unique scaffolding for engineering next-generation drug delivery systems represents a major area of academic and commercial interest. the lag in exploiting this potential is in part due to our inability to measured extent and efficiency of modification, e.g., composition and drug loading. here we report a robust pipeline of optical tweezing combined with raman spectroscopy to molecularly characterize engineered evs and quantitatively assess extent of drug loading at single particle resolution. methods: evs derived from cell culture and isolated by ultracentrifugation were fused with synthetic liposomes to create engineered evs (eevs). these eevs were formed via well-established vesicle fusion techniques, namely ( ) mechanical extrusion, ( ) freeze-thawing, or ( ) probe-tip sonication. prior to formation, calcein was encapsulated in the liposomes and used as a surrogate for soluble drug loading. laser trapping raman spectroscopy (ltrs) was used to optically trap single evs, before and after synthetic manipulation. raman spectral analysis was used to assess trapped eevs compared to pure standards to quantify ratiometric variation in chemical composition. results: raman laser trapping experiments confirmed that each formation method results in largely varying ( ) extent of fusion between evs and synthetic calceinloaded liposomes, ( ) efficiency of calcein loading, and ( ) particle size. we could also quantify the molar amounts of liposome vs. ev molecules for single particles, revealing a great amount of variation from particle to particle. functional membrane proteins we left intact to varying degree across fusion methods. summary/conclusion: given the rising importance of analytical tools able to characterize extent of molecular loading for engineered evs, we believe this technology will be very useful, thus warrants further investigation for eev characterization across a variety of clinical applications. funding: randy carney, phd was supported by a research scholar grant, rsg- - - -cdd, from the american cancer society. extracellular vesicles containing host restrictive factor ifitm inhibited zika virus infection of foetuses in pregnant mice through trans-placenta delivery allen z. wu nanjing university, nanjing, china (people's republic) introduction: zika virus (zikv) infection can lead to neurological complications and foetal defects, and has attracted global public health concerns. effective treatment for zikv infection remains elusive and a preventative vaccine is not available yet. therapeutics for foetus need to overcome blood brain barriers to reach placenta and require higher safety standard. methods: in the present study, we engineered mammalian extracellular vesicles (evs) to deliver a host restrictive factor, interferon-induced transmembrane protein (ifitm ), for the treatment of zikv infection. results: our results demonstrated that the engineered ifitm -containing evs (ifitm -exos) were overall safe to the animals and suppressed zikv viraemia by log s in the pregnant mice. moreover, the engineered evs effectively delivered ifitm protein across placental barrier and suppressed overall zikv viraemia in the foetuses to the basal level with significant reduction of viraemia in key foetal organs as measured by q-pcr. mechanistic study showed that ifitm was delivered to the endosomes/lysosomes where it inhibits viral entry to the host cells. summary/conclusion: our study demonstrates that exosomes can act as a cross placenta drug delivery vehicle to foetus and ifitm , an endogenous restriction factor that is highly expressed in placenta, is a potential treatment for zikv infection during pregnancy. introduction: extracellular vesicles (ev) are natural and abundant nanoparticles capable of transferring complex molecules between neighbouring and distant cell types. translational research efforts have focused on co-opting this communication mechanism to deliver exogenous payloads to treat a variety of diseases. important strategies to maximize the therapeutic potential of evs include payload loading, functionalization of the ev surface with pharmacologically active proteins, and delivery to target cells of interest. methods: through comparative proteomic analysis (lc/ms) of purified evs, we identified several highly enriched and ev-specific proteins, including a transmembrane glycoprotein (ptgfrn) belonging to the immunoglobulin superfamily. leveraging ptgfrn as a scaffold for surface display, we generated evs with functional targeting ligands, including single domain antibodies (sdabs), single chain variable fragments (scfvs), single chain fabs (scfabs), and receptor ligands, on the surface to direct ev uptake to cell types of interest. biological activity of these engineered evs was assessed in an array of in vitro and in vivo assays and compared to untargeted controls. results: we engineered evs displaying anti-clec a scfabs to target conventional type dendritic cells (cdc s), anti-cd scfabs to target t cells, and cd ligand to target b cells. in mice, systemic administration of anti-clec a evs resulted in a % increase in the percentage of cdc cells that take up evs over controls. anti-cd evs resulted in both an increase in the percentage of ev positive t cells ( . and -fold for cd + and cd +) and the number of evs per cell ( and -fold for cd + and cd +) in the blood. furthermore, in primary mouse dendritic cells, anti-clec a evs loaded with sting agonist achieved a fold greater pathway induction compared to untargeted controls. preliminary in vivo data suggest that anti-clec a evs reduce the required sting agonist dose -fold to achieve efficacy and induce anti-tumour responses, compared to control evs. summary/conclusion: these results demonstrate the potential of our ev engineering platform to generate novel ev therapeutics targeted to cell types of interest for pharmacologic payload delivery. a novel method for the delivery of cell-free therapy to foetuses with congenital anomalies: a proof of principle study lina antounians, louise montalva, gabriele raffler, maria sole gaffi and augusto zani the hospital for sick children, toronto, canada introduction: antenatal cell-based therapies are currently considered invasive for the foetus. a promising cell-free strategy that holds great regenerative potential for several organs is the administration of stem cell derived evs, whose cargo contains bioactive molecules that epigenetically regulate target cells. herein, we aimed to ) assess the ability of evs to reach foetal organs when administered to the mother intravenously or intra-amniotically; ) compare these administration routes on normal foetuses and foetuses with a congenital anomaly. methods: evs were isolated from rat amniotic fluid stem cell conditioned medium using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd , hsp , flo- , and tsg (western). we injected rat dams with evs stained by exoglow™-vivo or saline (control) via maternal tail vein (iv) or intra-amniotically (ia) at e . . ia and iv injections were performed on dams carrying normal foetuses or foetuses exposed to nitrofen to induce congenital diaphragmatic hernia. after h, dams and pups were sacrificed. d high-sensitivity optical reconstructions of whole foetuses or micro-dissected foetal organs were imaged using the ivis® spectrum imaging system. ev fluorescence signal was compared between normal (n = ) and nitrofen-exposed (n = ) foetuses. results: both iv and ia injection routes were successful in delivering evs to foetal organs. no fluorescent signal was detected in saline only control. ia injections yielded higher signal than iv, and evs reached more organs with ia than iv injections. ia injected evs were detected in the lungs, gastrointestinal, and urinary tract of normal and nitrofen-exposed foetuses. nitrofen exposed foetuses had higher signal than normal foetuses. summary/conclusion: this proof of concept study shows that antenatal administration of stem cell evs is feasible with different routes. although maternally administered evs cross the placenta, ia injection is more effective at reaching foetal organs. further studies are underway to reproduce these findings in experimental models of various congenital anomalies. funding: cihr-sickkids foundation grant os . introduction: safe, efficient and specific nano-delivery systems are essential to the current cosmetic, nutraceutical and therapeutic medicine sectors. the ability to optimise the bioavailability, stability, and targeted cellular uptake of bioactive molecules while mitigating toxicity, immunogenicity and off-target/side effects is of the utmost priority. ves us is a european project, which aims to develop an innovative platform for the efficient production of extracellular vesicles (evs) from microalgae, which constitute a promising renewable bioresource (www.ves us.eu). here we present characteristics of evs from several microalgal lineages, which offer the opportunity for a potentially developing a new and scalable tailor-made biogenic nanotechnology. methods: we cultivated a number of ev-producing microalgal species and developed protocols for ev isolation both at laboratory (differential ultracentrifugation) and pilot scales (tangential flow filtration). the physico-chemical characterization of microalgal evs was carried out according to the minimal information for studies of extracellular vesicles (misev- guidelines): biochemical methods to verify the presence of specific ev-biomarkers, tuned for microalgal evs; dynamic light scattering (dls) and nanoparticle tracking analysis (nta) to assess the particles number and size distribution; electronic scanning microscopy (sem), atomic force microscopy (afm), and cryo transmission electron microscopy (cryo-tem) for imaging analyses; bilayer-specific fluorescence staining (f-nta) to test the purity of ev preparation. results: we identified microalgae as a novel natural source of evs that could constitute a cost-effective and sustainable way of mass-producing them. we screened strains of microalgae and generated an "ev identity card" for each, which contained a variety of ev features relating to their biophysical, biochemical and biological characteristics in line with the misev- . our approach will next focus on the scalable production, surface functionalization and bio-engineering of selected microalgal evs. at the same time, their bioactivity will be explored using both in vitro and in vivo biological models. summary/conclusion: the ves us consortium is investigating the potential of microalgae as novel ev bioresources. this research will attempt to bioengineer novel naturally-derived nanocarriers, microalgal evs, suitable for the development of future cosmetics, nutraceutical or therapeutic formulations. funding: this project has received funding from the european union's horizon research and innovation programme under grant agreement no . sequence-specific rna trafficking to extracellular vesicles is conserved across cell types several sequences have been identified that act as a zipcode for preferential rna targeting into ev (evtropic) or for retention in parental cells (cell-tropic) . in this work, we aimed to compare the ev-tropic capacity of specific rna sequence motifs in promoting loading into ev, across different cell models representing the main cell types found in the body. methods: immune, epithelial and mesenchymal cell lines were transiently transfected with xenogeneic c. elegans micrornas (mirnas) containing ev-tropic or cell-tropic sequences and grown in culture. ev were isolated from the supernatant by differential (ultra)centrifugation. rna was extracted from both cell pellets and isolated ev fraction, and target mirnas were quantified by digital droplet pcr. distribution of cargo mirna across cells and ev was also analysed for chimeras of ev-and cell-tropic sequences. results: the mirnas containing an ev-tropic sequence were highly enriched on the ev fraction, with - , higher levels than in parental cells. contrarily, cell-tropic mirnas were only - times higher in ev. no significant differences were observed in the ev loading efficiency for the various ev-tropic motifs tested. mutations in the ev-sorting motif resulted in reduced ev loading. ev-tropic sequences consistently promoted mirna loading into ev across all the cell models evaluated, suggesting conserved biological mechanisms. summary/conclusion: we showed that rna loading into ev is dependent on the presence of defined evtropic rna motifs, and that sorting mechanisms are conserved across the major cell types tested. the highest loading efficiencies resulted in . mirna copies per particle on average, suggesting a limited scope for ev-tropic motifs for therapeutic rna loading into ev. funding: as, os and eli are fellows of the astrazeneca postdoc programme. introduction: coordinated activity between pancreatic islet cells is critical for the regulation of glucose homoeostasis. chronic exposure to diabetogenic factors such as pro-inflammatory cytokines, perturb islet cell crosstalk and β-cell function in diabetes. extracellular vesicles (evs) derived from cytokine-exposed β-cells modulate physiological and pathological responses to β-cell stress. however, the mechanisms governing this process remain largely unknown. we set out to test the hypothesis that β-cell failure in diabetes is mediated in part through β-cell autocrine release of pro-inflammatory evs which promote inflammation and inhibit βcell function. methods: pro-inflammatory cytokine-exposed evs (cytoevs) were generated using conditioned media from mouse min β-cell line treated with diabetogenic cytokines (tnfα, il- β, ifnγ, h). evs were also isolated from human type diabetic (t dm) and lean non-diabetics (lnd) plasma. gw (n-smase inhibitor) was used in the presence of cytokines to determine the effect of reduced ev concentrations on the restoration of β-cell function. proteomic and rna-seq analysis was conducted on min β-cell cytoev (vs. control ev) and cytoev treated mouse islets, respectively. results: assessment of ev concentrations from cytoev and human t dm plasma revealed a~twofold increase (p < . , vs. control (ctl) and lnd ev). immunofluorescence staining of cd and cd expression was significantly elevated in human t dm pancreas (p < . , vs. lnd). while acute inhibition of ev formation with gw ( µm) showed significant restoration in β-cell function (glucose stimulated insulin secretion assay, gsis) in cytokine-exposed mouse and human islets (~ and fold vs. cytokines alone, p < . ). moreover, functional assessment of mouse islets exposed to cytoev ( h) resulted in suppression of gsis (~ %, vs. untreated, p < . ). identification of cytoev content through proteomic analysis revealed a significant upregulation of the chemokine, cxcl (~ fold vs. ctlev) and rna-seq analysis of cytoev treated mouse islets depicted a marked upregulation of transcripts associated with cxcl -cxcr signalling (p < . ) and downstream pathways (e.g. nfκb; p = . and jak/stat; p = . ). furthermore, inhibition of cytoev (gw ) with cytokines markedly decreased cxcl (~ %) and cxcr receptor (~ %) expression in min β-cells. summary/conclusion: these data suggests that cytokines elevate cxcl expression in β-cell ev to enhance inflammation-induced diabetes. this is mediated through ev-autocrine release of cxcl consequently activating cxcr signalling and downstream pathways to impair β-cell function in diabetes. synergy between -lipoxygenase and secreted pla promotes inflammation by formation of tlr agonists from extracellular vesicles introduction: damage associated molecular patterns (damps) are endogenous ligands that induce innate immune response, thus promoting sterile inflammation. during oxidative stress, stress-derived evs (stressevs) were found to activate toll-like receptor (tlr ), but the activating ligands were not fully determined. additionally, several enzymes, among them -lipoxygenase ( -lo) and secreted phospholipase a (spla ) are induced during inflammation and were suggested to promote damp formation. methods: stressevs were produced from hek cells exposed to um a and isolated with ultracentrifugation. : lysopi was oxidized for min with -lo. additionally, synevs were prepared from phospholipids (pls), oxidized with -lo and hydrolysed with spla . activity was measured by qpcr and elisa on wt and tlr -ko macrophages. -lo oxidized : lysopi was analysed by mass spectrometry. spla activity was measured in synovial fluid from rheumatoid and gout patients using fluorometric assay. k/bxn serum transfer induced arthritis model on wt and tlr ko mice (c bl/ mice) with spla -iia injection was used (approval no. u - / / by mkgp of slovenia). results: stressevs released after oxidative stress were found to activate tlr with a gene profile different from bacterial lipopolysaccharide (lps). stressevs, -lo oxidized synevs, but only -lo oxidized lysopls activated cytokine expression through tlr /md- . hydroxy, hydroperoxy and keto products of : lysopi oxidation were determined by ms and they activated the same gene pattern as stressevs. furthermore, spla activity, which we detected in the synovial fluid from patients, promoted formation of tlr agonists after -lo oxidation. injection of spla -iia into mice promoted k/bxn serum induced arthritis in tlr -dependent manner. summary/conclusion: both -lo and spla are induced during inflammation, therefore these results imply the role of oxidized lysopls in stressevs in promoting sterile inflammation through tlr signalling. the formation of tlr agonists is enzyme driven so it provides an opportunity for therapy without compromising innate immunity against pathogens. funding: h -msca-itn project tollerant (grant no. ), slovenian research agency (project no. j - to mmk, research core no. p - to rj). monocytes traffic extracellular vesicles to damaged muscle and adopt a novel immunophenotype to support muscle regeneration russell g. rogers, akbarshakh akhmerov, weixin liu, lizbeth sanchez and eduardo marbán smidt heart institute, cedars-sinai medical center, los angeles, usa introduction: extracellular vesicles (evs) are secreted membrane vesicles that carry bioactive molecules such as mirnas, mrnas, proteins, and lipids to modify recipient cell behaviour. we recently demonstrated evs secreted by cardiosphere-derived cells (cdc-evs) augment endogenous muscle regeneration in mdx mice, a model of duchenne muscular dystrophy, when delivered intravenously. in parallel, macrophages preferentially accumulate surrounding small regenerating myofibers in cdc-ev treated mdx muscle. however, it is currently unclear how intravenous cdc-evs home to dystrophic muscle and exert their therapeutic bioactivity. methods: fluorescently-labelled and unlabelled cdc-evs were infused into the contralateral femoral vein of wild-type mice with unilateral muscle injury induced by bacl . injured and uninjured muscles were dissected h following infusion and subjected to optical imaging, immunohistochemistry, and confocal microscopy. this experiment was repeated using clodronate liposomes to deplete endogenous monocytes/macrophages. next, rna-seq was preformed on bone marrow-derived m , m , and cdc-ev (mcdc-ev) polarized macrophages from mdx mice. conditioned media (cm) from these macrophages were tested in an in vitro model of myogenesis. lastly, small rna-seq was performed on evs secreted by m , m , and mcdc-ev macrophages. results: when delivered intravenously, cdc-evs naturally home to injured, but not uninjured, skeletal muscle. cdc-evs were detected in the interstitium adjacent to non-muscle cells, macrophages, and within surviving myofibers. after depletion of monocytes/ macrophages by clodronate liposomes, the presence of cdc-evs in the injured muscle was attenuated. bioinformatic analyses indicate cdc-evs confer a novel immunophenotype to mdx macrophages with features of both m and m . indeed, mcdc-ev cm promotes myoblast proliferation and supports myogenic differentiation. interestingly, mcdc-ev evs have a unique mirna signature and contain several mirnas with known roles in myogenesis. summary/conclusion: these data indicate circulating monocytes traffic cdc-evs to damaged muscle where they adopt a novel immunophenotype to support muscle regeneration. we propose mcdc-ev macrophages mediate their pleiotropic effects via paracrine factors, possibly including evs. introduction: microglia, the immunocompetent cells of the cns, play an important role in maintaining cellular homoeostasis in the cns. these cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. the contribution of microglial-derived extracellular vesicles (m-evs) to the maintenance of cns homoeostasis is unclear. in addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to m-evs is scarce. methods: here, we analysed the effects of m-evs produced in vitro by either tnfα-activated or non-stimulated microglia bv cells. we showed that m-evs are internalized by both mouse bv and human c microglia and that the uptake of m-evs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. consistently, exposure of microglia to m-evs increased the protein expression of the autophagy marker, lc b-ii, and promoted autophagic flux in live cells. to elucidate the biological activities occurring at the transcriptional level in c microglia exposed to m-evs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted rna sequencing. results: inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia exposed to m-evs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. summary/conclusion: we demonstrate that in vitro produced microglial evs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homoeostasis. funding: this work was financed by hasselt university and by efro through the interreg v grensregio vlaanderen nederland project trans tech diagnostics. evaluation of plasma extracellular vesicles as biomarkers for longevity xin zhang a and virginia kraus b a laboratory medicine center, nanfang hospital, southern medical university, guangzhou, guangdong, , p. r. china, guangzhou, china (people's republic); b division of rheumatology, duke molecular physiology institute, duke university school of medicine, durham, usa introduction: extracellular vesicles (evs) have emerged as key indicators and effectors of ageing. although plasma concentrations of evs decline with age, the ev biomarkers associated with ageing and longevity are not fully understood. recently, our group found an age-related decline of plasma evs associated with immune cells during normal human ageing. our study aims to evaluate the association of plasma evs with longevity. methods: plasma samples were selected from the established populations for epidemiologic studies of the elderly study subjects (n = ): half dying within years (short-lived group) and half surviving ≥ years (long-lived group) after the blood draw; all matched for age (median age . ± . years, range - ), gender ( % female), and race ( % white/ % black). the samples were acquired under donor consent and irb approval of duke university. evs were separated from the plasma samples, and profiled based on the surface markers of haematopoietic stem cells (hscs), mesenchymal stem cells, immune cells, skeletal muscles, cardiac muscles and adipocytes (cd , cd , cd , cd , cd , cd , cd , cd , cd , cd , cd , cd a, cd a, cd , cd , hla-abc, hla-g, hla-drdpdq, cd , cd , cd , m cadherin, ryr , ryr , fabp , dlk ). the percentages of evs expressing each tested molecule were determined using a high-resolution multicolour bd lsr fortessa x- flow cytometer as we recently reported. graphpad prism . software was used for statistical analysis. results: we found significantly increased percentages of cd +, hla-abc+, cd + and cd a+ large evs ( - nm) in the long-lived compared to the short-lived group. none of the tested surface marker expressing medium ( - nm) or small (< nm) evs showed differential percentages between the shortand long-lived groups. summary/conclusion: evs carry surface markers from their parent cells. cd is expressed by hscs and immune cells. cd regulates homing of human cord blood cd + hscs, and delivers a potent cd independent costimulatory signal to activate t cells. hla-abc, the key human immunogen, is expressed by nucleated cells and platelets. cd is expressed by hscs, immune cells and epithelial cells, and cd + plasma evs declined with age in healthy people. cd a is expressed by hscs, megakaryocytes and platelets, and is functionally relevant for hsc maintenance and haematopoietic homoeostasis. our preliminary data suggest that hscs and immune cell associated plasma evs (cd +, hla-abc+, cd +, cd a+ large evs) inform on health status related to longevity. introduction: it is anticipated that stem/progenitor cells-derived extracellular vesicles (spc-evs) will rapidly progress towards clinical studies, and the development of reproducible, efficient, scalable and costeffective process for their production is expected to boost the therapeutic applications of evs-based products. in addition, the use of defined serum-/xenogeneic(xeno)-free culture medium formulations could result in substantial improvements for spc-evs production in terms of reproducibility, stability and quality, while ensuring the approval of regulatory agencies. the main goal of this work is to develop a full-controlled manufacturing platform for the spc-evs production. methods: human mesenchymal stromal cells (msc) were expanded in a xeno-free microcarrier-based bioreactor culture system operating in fed-batch feeding mode and after days the conditioned medium was collected. different methods for spc-ev isolation/purification from the msc-derived conditioned medium, including chromatography were compared and the the quality of the final product obtained was characterized by different methods according to misev, including nanoparticle tracking analysis, lipidomics and western blot. moreover fourier-transform infrared (ftir) spectroscopy was evaluated in terms of its implementation as a standard technique for the identification and characterization of evs. results: after days of msc expansion under dynamic conditions, we collected . l of conditioned medium with approximately . million evs/msc. a combination of a pretreatment with a nuclease for the digestion of dna/chromatin with a purification using strong anion exchange chromatography led to the best results so far in terms of evs isolation. of notice, by ftir spectroscopy, it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture conditions tested. summary/conclusion: the platform established herein could be applied to the production of wellcharacterized spc-evs targeting their biomedical use in different settings (e.g. as drug delivery systems), as well as evs from other parental cells lines (i.e. dendritic cells) in therapeutic settings as cancer. ultrasensitive protein detection for quantification of extracellular vesicles in human biofluids enables comparison of isolation techniques dmitry ter-ovanesyan, maia norman, wendy trieu, roey lazarovits, george church and david walt wyss institute, boston, usa introduction: extracellular vesicles (evs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. one of the main challenges in studying evs and using them in diagnostics is a lack of suitable methods to quantify evs that are sensitive enough and can differentiate evs from similarly sized lipoproteins and protein aggregates. we propose using ultrasensitive single molecule array (simoa) assays to quantify evs by immunoisolating and detecting ev transmembrane proteins in microwell arrays. we developed single molecule array (simoa) assays using the quanterix hd-x analyser for the quantification of evs using the tetraspanins cd , cd , and cd . simoa allows for the detection of single proteins using arrays of femtoliter wells, turning elisa into a digital immunoassay. we then used these assays, together with an additional assay for albumin, to compare commonly used ev isolation methods from plasma and cerebrospinal fluid (csf): ultracentrifugation, precipitation (exoquick), and size exclusion chromatography (sec) using the izon qev columns. we further used these assays to rapidly optimize and improve sec by comparing different sec resins and column dimensions in both plasma and csf. results: in comparing our simoa assays to traditional elisa with the same antibodies, we found that the simoa assays were more than times more sensitive, detecting the tetraspanins in samples where the proteins were undetectable by elisa. given the high dynamic range and high-throughput capabilities of simoa, we were able to comprehensively compare relative ev yields and ev purity for different isolation methods of evs from plasma and csf. we provide average tetraspanin and albumin levels to directly compare the methods. we also tested different sec resins and provide data for custom sec columns that outperform izon qev and allow for fine tuning of different ratios of evs to albumin. summary/conclusion: our results highlight the utility of quantifying evs using ultrasensitive simoa assays for tetraspanins. we were able to rapidly simoa to rapidly evaluate different ev isolation methods in csf and plasma. in general, the experimental framework we present could be easily applied to evaluate new ev isolation methods, or applied to any other biological fluid. thus, we think simoa is a powerful new tool for relative ev quantitation. introduction: the protein profile of extracellular vesicle (ev) subpopulations has been shown to contain valuable disease information, notably in cancer. currently, techniques aiming to find ev proteins that associate together mainly focus on transmembrane proteins, while methods that also probe cytosolic proteins generally resort to a combination of affinity capture, elution, and lysis, which limits throughput. to allow the high-throughput analysis of both membrane and cytosolic ev proteins, we optimized a total extracellular vesicle antibody microarray (tevam) incorporating fixation and heat-induced epitope retrieval (hier), then leveraged it to perform combinatorial protein profiling of evs from colorectal cancer (crc) cell lines ht and sw . methods: arrays of iggs targeting surface protein markers were incubated overnight with evs purified from cancer cell line supernatants. hier optimization was carried out through variation of buffer contents, presence or absence of prior permeabilization, as well as incubation time and temperature, for a total of conditions. a evs, previously profiled with other methods, were used as a model during the optimization. cytosolic protein hsp and membrane marker egfr, both with high expression in a evs, were probed and the results used to compare hier conditions. following hier treatment, protein targets were detected through incubation with primary antibodies and fluorescent secondary antibodies or streptavidin. the resulting optimized tevam workflow was used to phenotype ht and sw evs through probing of trios of surface ( ) and internal ( ) protein targets. results: the selected tevam protocol successfully maximized hsp signal while minimally affecting egfr detection, enabling simultaneous analysis of surface and internal proteins. profiles of more than combinations, featuring integrins, claudins, cytokines, and other key actors of cancer-relevant pathways, were obtained for ht and sw evs, revealing coexpression patterns that highlight the biomolecular heterogeneity both within and between crc cell line evs. summary/conclusion: using tevam, intra-and extravesicular proteins can be detected simultaneously in evs immobilized based on surface protein content, yielding extensive combinatorial protein profiles with significance for health and biomarker research. characterization of evs using orthogonal techniques identifies discrete ev populations from a mouse dendritic cell line bryce killingsworth a , timothy traynor b , joshua a. welsh c , aleksandra dakic a , jason savage a , kevin camphausen d , kenneth aldape a and jennifer jones a a laboratory of pathology, national cancer institute, national institutes of health, bethesda, usa; b laboratory of pathology, national cancer institute, national institutes of health, gaithersburg, usa; c laboratory of pathology, national cancer institute, national institute of health, bethesda, usa; d radiation oncology branch, national cancer institute, national institutes of health, bethesda, usa introduction: extracellular vesicles (evs) have the potential to serve as valuable biomarkers for patient response to cancer therapy. however, development of robust ev-based clinical assays relies on knowledge of ev concentration and diameter distribution. many different methods exist to measure the size and concentration of evs, and each method exhibits strengths and limitations. it is important to use orthogonal methods for determination of these important properties of ev preparations. here, we use dendritic cellderived evs to demonstrate that some ev analysis methods can give a biased interpretation of both diameter and concentration. through comparison, we highlight why orthogonal assays are essential in providing measurement reliability. methods: dc . mouse dendritic cells were cultured in flasks containing a total of . l of ev-depleted media ( % fbs, centrifuged hr. x , g.) when cells reached % confluency, conditioned media was collected, depleted of debris with two min. x , g spins, and concentrated down to~ ml using a pall jumbosep kda mwco filter. the ev concentrate was purified from protein using an izon qev- column, with ml fractions collected. the protein content of the ev-containing fractions was analysed by a , pierce bca, and bioanalyzer. the diameter distribution of the evs was determined by nanoparticle tracking analysis (nta), resistive pulse sensing (rps), flow cytometry (fcm), and electron microscopy (em.) concentration was compared using nta, rps, and fcm. evs were further analysed by protein mass spectrometry and rna sequencing. results: we have identified two distinct populations of evs with our dc . preparation, one highly abundant population with a power-law distribution, whose peak diameter is below nm, and a second, less abundant population with a peak diameter at approximately nm. these two distinct populations and their relative concentration were not detectable with all analysis techniques. based on cross-platform measurements, these populations appear to have distinct compositions that warrant further investigation. summary/conclusion: the use of orthogonal methods allowed the detection of two discrete populations of evs which was not possible on some platforms and would have resulted in a biased perspective of the sample composition. this work has highlighted the need for orthogonal measurements to be conducted by pairing techniques that do not have the same biases. introduction: extracellular vesicles (evs) are nanosized vesicles shed by all cells that serve vital roles in cell-to-cell communication. tumour-associated ev subpopulations vary in molecular content (lipids, proteins, nucleic acids, small molecules), enabling minimally invasive spectroscopic analysis for a wide variety of cancers. here, we use surface-enhanced raman spectroscopy (sers) in combination with a novel plasmonic substrate for global chemical composition analysis of cancerous and non-cancerous populations of evs to determine distinguishing surface characteristics. methods: evs were isolated from ovarian cancer (ovca) patient serum samples by differential ultracentrifugation. a new hybrid nanoplasmonic scaffold comprised of a microscale biosilicate diatoms embedded with silver nanoparticles (agnps) was used for sers measurements. the substrate was incubated with cysteamine to positively-charge the agnps (responsible for the sers enhancement) so that evs could attach (evs are naturally anionic). in a typical experiment, μl of~ particles/ml evs per sample were incubated with the porous substrate surface, which was inverted on a glass cover slip for raman interrogation. principle component analysis (pca) was used to compare the spectra and determine distinguishing characteristics between populations from tumour and non-tumour sources. we also trypsinized evs before sers analysis to see the extent of influence the surface molecules play in localizing the evs to the agnp "hot spots." results: a total of clinical samples ( ovca and non-malignant control) were tested in combination with ovca skov- cell line evs. simple pca was able to separate clinical samples according to disease subtype and major peaks were identified to provide chemical content analysis. each sample exhibited inherent heterogeneity but clustered together in a distinguishable way from the others. summary/conclusion: despite innate heterogeneity within single samples (i.e., evs isolated from a single patient sample), evs isolated from clinical samples could be easily distinguished from each other using our hybrid sers substrate, with minimal sample processing, a label-free approach, and only a few microlitres of sample. our study using this novel plasmonic material demonstrates its potential for use as a component in next-generation diagnostic platforms. introduction: single-particle analysis is critical for understanding extracellular vesicle (ev) heterogeneity. yet such techniques remain technically challenging due to low detection sensitivity and presence of variable amounts of "contaminants," including lipoproteins. the high degree of structural similarity between evs and lipoproteins in size, density, and chemical composition, results in their co-isolation using any of the standard ev isolation techniques. here we introduce laser trapping raman spectroscopy (ltrs) as a wellsuited, label-free, and non-destructive tool to distinguish evs from various lipoprotein species at single particle resolution. methods: ev samples were isolated from skov- cell culture supernatant by differential ultracentrifugation and their raman spectra measured. as the most abundant lipoproteins in ev isolations from human biofluids are sub-micron low density lipoprotein (ldl), very low density lipoprotein (vldl), and high density lipoprotein (hdl) particles, these were purchased as pure components and also measured by ltrs. ldl and vldl were then spiked-in to isolated evs to mimic "contaminated" post-isolation ev samples. raman spectra were analysed by principal component analysis (pca) using a custom matlab script. results: ldl and vldl have been observed to adhere to ev surfaces in vitro after standard isolation techniques. we could readily distinguish pure vldl, ldl, and hdl standards according to their raman spectra. pca revealed distinction of skov- evs from both ldl and vldl. pca also differentiated skov- evs incubated with ldl from skov- evs incubated with vldl. extent of ldl and vldl adherence to evs could be observed and quantified. summary/conclusion: through raman and pca, classes of lipoprotein and evs can be identified and quantified when co-incubated. ltrs is a quantitative single-ev analysis technique that can be used to differentiate between lipoprotein classes and evs when incubated together. this technique allows for analysis of evs where standard isolation methods fall short. introduction: extracellular vesicles (evs) are endogenous membrane-derived vesicles that shuttle lipids, proteins or nucleic acids between glia and neurons, thereby promoting neuronal survival and plasticity in the cns and contributing to neurodegenerative conditions. although evs hold great potential as cns theranostic nanocarriers, the specific molecular factors that regulate neuronal ev uptake and release are currently unknown. methods: we used a combination of patch-clamp electrophysiology and ph-sensitive dye imaging to examine stimulus-evoked ev release in individual neurons in real time. results: whereas spontaneous electrical activity and the application of a high-frequency stimulus (hfs) induced a slow and prolonged fusion of multivesicular bodies (mvbs) with the plasma membrane (pm) in a subset of cells, the neurotrophic factor bfgf (basic fibroblast growth factor) greatly increased the rate of stimulusevoked mvb-pm fusion events and, consequently, the abundance of evs in the culture medium. proteomic analysis of neuronal evs demonstrated bfgf to increase the abundance of the v-snare vesicle-associated membrane protein (vamp , cellubrevin) on evs. conversely, knocking-down vamp in cultured neurons attenuated the effect of bfgf on ev release. introduction: heat shock proteins (hsps) function as chaperones under both normal and pathologic conditions. as chaperones they assist in protein folding, in holding protein complexes for current or future activation, and in degradation of senescent proteins for recycling of components and display for immune surveillance. during stressful situations, hsp quantities and/or activities increase as cells and tissues seek protection from insults. these insults can result in the cell surface display of hsps, which can then lead to the surface display of hsps on extracellular vesicles (evs). hsps present on the cell surface or in the extracellular space are regarded as "danger signals" in an ancient biologic paradigm. hsp-accessorized evs may act as "danger boli", carrying not only the hsps, but hundreds of components of the stressed parental cell, capable of prompting differential responses depending on the status of the recipient cell. methods: clarified/filtered plasma from patients suffering from neurologic maladies (cancer, brain injury, multiple sclerosis) was incubated with peptides designed to bind hsps. the evs congeal under these conditions and are pelleted (microfuge) and washed with increasing-stringency buffers. we lysed the evs and subjected them to metabolomic analyses (focused on lipids) or assayed them on phosphokinase arrays. results: we show that evs from the blood of patients suffering from brain tumours, or from tbi, or from ms, possess distinct metabolomes compared to blood evs from healthy donors. we found hundreds of differentially-expressed lipids amongst the patients vs the healthy donors. the levels of annotation and identification for these compounds ranges from level (low, no matches in databases) to level (high, annotation matches to known database components). in addition, we found differences in phosphorylated kinases as cargo in these evs between patients with matched primary vs recurrent gliomas, and among tbi/stroke patients compared to healthy donors. summary/conclusion: hsp-accessorized evs present different metabolomic and phosphokinase content which may serve as biomarkers in a "liquid biopsy" setting, but may also play roles in the pathobiology of neurologic diseases. introduction: methamphetamine (ma) has deleterious effects to both peripheral organs and the central nervous system. the rewarding properties and addictive potential of ma are correlated with increased synaptic dopamine availability following alterations in dopamine and vesicular monoamine transporter function. in rodents, ma alters brain mirna expression and the mirna content of serum extracellular vesicles (ev). here we examined plasma evs isolated from human subjects actively using ma (ma-act) for size, concentration, protein markers, and mirna content. methods: plasma samples from ma-act, and controls (ctl) were obtained from the methamphetamine abuse research center. plasma evs were evaluated by vesicle flow cytometry (vfc) for size, concentration, and surface protein markers. vfc antibodies included markers for a pool of tetraspanins (cd , cd , and cd ), platelet evs (cd ), pro-coagulant evs (annv), and red blood cell evs (cd ). next plasma ev isolated by size exclusion chromatography were analysed by qpcr on taqman® array human microrna a + b card set v . . fold change was calculated by ΔΔcq between ma-act and ctl for mirna expressed in ≥ % of samples in at least group. we identified the top % of ranked mirna by fstatistic; of these, the mirna of interest for ma-act were identified by at least a (i) . fold change in expression, (ii) area under the receiver operating characteristic curve of . , and (iii) glass's Δ of . for mirna of interest correlations to additional ma variables were conducted, along with ingenuity pathway analysis of predicted gene targets. tobacco use was controlled for. results: vfc data show that the size (~ nm) and concentration (~ . x particles/ml) of all plasma evs is comparable between ma-act and ctl groups. in addition, the plasma evs primarily consist of tetraspa-nin+, annv+, or cd + evs, and to a much lesser extent cd + evs. of the mirna expressed in ma-act and/or ctl plasma evs, there were mirna that have at least a . fold increase or decrease in ma-act. mirna were identified to be of interest in ma-act based on fold change, effect size and diagnostic potential, compared to ctl. further, of the mirna correlate with a ma associated variable, including frequency of use and age of first use. together the mirna best cluster subjects based on ma-act status, not tobacco use. finally, the predicted gene targets of the mirna are associated with canonical pathways linked to ma. summary/conclusion: ev mirna expression in ma-act subjects was unique to ctl participants, suggesting that ma may affect ev communication among cells. the differential mirna expression also implicates a role for evs in behavioural and physiological effects specific to ma, and suggests that there may be changes in expression of mirna that are relevant to specific drugs of addiction, as well as to a spectrum of drug-mediated addiction disorders. bone marrow-derived extracellular vesicles may alter the ageing phenotype of murine haematopoietic stem cells. sicheng wen a , jill kreiling b , mark dooner c , elaine papa c , michael del tatto c , yan cheng c , mandy pereira c , peter quesenberry c and laura r. goldberg d in natural ageing of haematopoietic stem cells (hscs) is unclear. we tested the hypothesis that bone marrowderived evs (bm-evs) can modulate the ageing hsc phenotype. methods: we flushed bone marrow from old ( - month old) and young ( - -week old) c /bl mice and collected bm-evs by differential centrifugation ( × g for min, supernatant collected and centrifuged , × g for hour, bm-ev pellet collected and quantified by nanoparticle tracking analysis). we injected old mice with x ^ young bm-evs via tail vein, daily x days. control mice were injected with age-matched bm-evs or vehicle alone. we euthanized the mice one month post-injection, harvested whole bone marrow (wbm) and highly purified hscs (lineage negative/c-kit+/sca- +/cd +; lsk-slam) and tested stem cell function in competitive bone marrow transplants ( - recipients/group). results: at months post-transplant, wbm from old mice exposed to young bm-evs exhibited a statistically significant decrease in engraftment when compared to wbm exposed to age-matched bm-evs (percent average donor chimerism ± sem: % ± % (young evs) vs. % ± % (old evs)). for lsk-slam from old mice, we observed a trend towards decreased engraftment when exposed to young bm-evs and a trend towards increased engraftment potential when exposed to old bm-evs (percent average donor chimerism ± sem: % ± % (young evs), % ± % (old evs), % ± % (vehicle)). these findings are consistent with our previous data showing that, in contrast to highly purified hscs which develop impaired stem cell function with ageing, total un-separated old wbm actually has increased engraftment capacity when compared to young wbm. of note, we found that the classic myeloid skewing by old lsk-slam was partially reversed by exposure to both young and old bm-evs. finally, consistent with the known increase in highly purified hscs with age, our preliminary data showed that old mice exposed to young bm-evs had an approximately -fold decrease in the number of lsk-slam cells in marrow, indicating that bm-evs may influence agerelated changes in hsc number. summary/conclusion: these preliminary data suggest bm-evs may play a role in modulating hsc ageing phenotypes, potentially altering engraftment capacity, lineage fate, and lsk-slam population size. future studies delineating the molecular mechanisms underlying these ev-mediated effects could provide key insights into normal haematopoietic ageing. funding: this work was supported by the nih grants p gm , dk - a and by the savit foundation. oral with poster introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr. results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom . mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the biogenesis of evs is neutral sphingomyelinase (nsmase ), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles. methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-( -( -( , -dimethoxyphenyl)- , -dimethylimidazo [ , -b] pyridazin- -yl) pyrrolidin- -yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated xfad mice with mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay. results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting % of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human post-mortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of mdd subjects and hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed. results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd . tem images showed the typical cup-shaped morphology with sizes mostly between and nm. preliminary sequencing results revealed that mir- a- p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir- a- p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. op . = ps . combining nanomagnetic isolation and artificial intelligence to guide the treatment of traumatic brain injury zijian yang a , kryshawna beard b , david meaney b , danielle sandsmark b , ramon diaz-arrastia a and david issadore c a university of pennsylvania, philadelphia, usa; b university of pennsylvania, philadelphia, usa; c department of bioengineering, university of pennsylvania, philadelphia, usa introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores ( nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the protein biomarkers (tau, uchl- , nfl, gfap, il , il , and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl were elevated in mtbi patients compared to controls (p < . ), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il and tnf-with tau from glur + evs showed % accuracy with % sensitivity and % specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. l cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd , cd and cd ) as well as l cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l cam appears. we also immunocapture l cam from csf and plasma and perform western blots for the internal and external domains of l cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l cam in plasma is likely not coming from the brain. this data call into question the utility of l cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in -month-old wt mice, (n = /groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for % of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and non-amplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy y cells with n-myc amplified sk-n-be cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. op . = ps . introduction: quantification and characterization of single extracellular vesicles (sevs) based on surface markers can aid in dissecting the heterogeneous landscape of ev subpopulations. we and others have demonstrated the potential of imaging flow cytometry (ifc) to perform sev characterization. we recently showed release of protoporphyrin (ppix) positive sevs by -aminolevulinic acid ( -ala) dosed glioma cells, in vitro and in vivo. rickfels et al. also used ifc to demonstrate the enrichment of cd +/cd + evs in the plasma of glioma patients. herein, we performed in vitro studies to characterize ev subfractions using -ala as well as ev and cns specific surface markers. methods: we use ifc to characterize evs released by glioma using -ala, fluorescently labelled ev (cfda-se, cd ) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd ) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that % are tenascin c positive, . % are egfrviii positive and . % are -ala positive. there was only a minor overlap (< %) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs ( . -fold), tumour specific evs ( . -fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: f. graminearum (fgr) and f. oxysporum f. sp. vasinfectum (fov) are severe fungal pathogens of cereals and cotton, respectively. fgr and fov cause economic losses and threaten food and fibre supplies worldwide. understanding host-pathogen interactions is crucial for developing new strategies for disease control. we are determining whether extracellular vesicles (evs) have a role in the interaction between fungal pathogens and their host plant. methods: we isolated evs from fgr and fov by sizeexclusion chromatography and characterized them by nta and tem. evs from fgr and fov are between - nm and have morphology similar to evs reported for other fungi. we performed label-free quantitative proteomics to describe the protein cargo of evs from fgr and fov, including a comparative study of evs from fov grown on different media: czapek dox (cd) and saboraud's dextrose broth (sdb). results: a total of proteins were detected in fgr evs and, according to prediction software effectorp, . % of these were potential effectors. similarly, % of ev proteins do not contain signal peptide indicating that packaging into evs is a novel mechanism of secretion for these proteins. notable fgr ev proteins include lipases, proteases and synthases for toxins and chitin. fov produced evs in similar quantities in both growth media tested, but ev protein cargo differed between them. there was a % overlap in proteins identified in the cd and the sbd ev proteins. in general, ev proteins were involved in metabolism, cell wall architecture and oxidoreduction, with . % and . % of potential effectors, respectively. polyketide and toxin synthases, proteases and effectors were present in both types of fov evs. summary/conclusion: this new fungal ev isolation method was rapid, yielded high-quality evs, and did not submit particles to high centrifugal forces. our data revealed that both fgr and fov produce evs enriched with proteins that could alter host immune responses or facilitate fungal infection. furthermore, the protein composition of fov evs was dependant on culture conditions. this supports a potential role for fungal evs in disease progression in plants and provides the foundations to pursue the role of evs in plant-fungal interactions with the potential to identify new targets for disease control. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll receptor (tlr ) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il- and il- , which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells. objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow fieldflow fractionation (af ) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp- ) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk- epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with to nm (ev ) and another with to nm (ev ). ev induced more tnf-alpha, il- , ip- and ccl than ev . it was also more effective in promoting t. cruzi infection in epithelial cells. summary/conclusion: t. cruzi released two ev populations that affects differently host cells. identification of these evs composition might help to better understand the role of evs in the modulation of t. cruzi infection funding: fapesp, cnpq and capes op . = ps . commensal bacterial extracellular vesicles act as carriers for norovirus sutonuka bhar, melissa jones, annalise galbraith and mariola edelmann university of florida, gainesville, usa introduction: human norovirus (hunov) are one of the most common causes of gastroenteritis and, along with inducing morbidity and mortality by diarrhoea, have a massive economic impact resulting in approximately usd billion each year in healthcare costs and missed worker productivity. development of anti-viral therapies for hunov has been hampered by the lack of robust in vitro cultivation systems. several cell types support viral replication but only produce modest amounts of virus due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays. noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr. isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and . um filtration. co-purification of norovirus with the evs was checked. ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems. detection of bacterial extracellular vesicles in blood from healthy volunteers kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of . µg/ml of plasma, as geometric means ≥ . µg/ml were nearly equal. hevs were detected at µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f x electron microscope and measured between - nm, and hevs were between - nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: new zealand (nz) has a population of just . million people with a remote geographical location in the pacific ocean. its unique culture, food-based industries and ethnic population make nz an invaluable place for extracellular vesicle research into all areas. however, as for many places in the world, standardization of methodologies, training and access to appropriate equipment is challenging. methods: the hub for extracellular vesicle investigations (hevi) is a virtual research centre established in with three-year seed funding from a university of auckland strategic research initiatives fund. two staff members are employed to support training, education and optimization of methods. the hevi is guided by a governance group providing valuable input from australasian experts in evs. results: since the hevi has organized research symposia, hands-on training days, hosted international students as well as providing one-on-one training for individuals from universities and institutes across nz. training is provided on multiple isolation and characterization methods and tailored to individuals access to essential equipment without bias towards individual manufacturers or techniques. travel funding has supported people to attend conferences and workshops for the purposes of education, networking and research dissemination. the hevi also provides support for project design with grants awarded to hevi members and a number of manuscripts in submission for publication. the embo practical course "extracellular vesicles: from biology to biomedical applications" is organized each year by a group of researchers active in the ev field in collaboration with the embl advanced training center in heidelberg. the course focuses on training phd students and postdoctoral researchers who enter or are already active in the field of ev research. given the large number of methods and protocols that is being used by researchers in the ev field, the organizers aim to provide practical guidance to new researchers and teach them appropriate skills. methods: participants obtain theoretical knowledge and hands-on experience on different ev purification and characterization techniques, such as fluorescent labelling, density gradient centrifugation, size exclusion chromatography, electron microscopy, flow cytometry and nanoparticle tracking analysis and on databases like ev-track and funrich. in addition, the organizers and invited lecturers from several different research areas explain which strategies are used to understand the role of ev in biomedical applications and give an overview of the current state of the field of ev research. results: the course therefore covers a broad range of topics important in the ev field, including heterogeneity in ev subpopulations, mechanisms of ev cargo selection, ev biogenesis, pre-analytical variables, therapeutic and diagnostic use of ev, and in vivo functions of ev. group discussions are facilitated and stimulated via assignments to analyse data obtained during the practicals and to critically evaluate literature. participants also have the opportunity to present their own research during poster presentations and ask for feedback from organizers and invited lecturers. summary/conclusion: among the participants selected for the course, a large geographical distribution is reached, including researchers from the newer eu member states and from outside of europe, to ensure a broad geographical distribution of the knowledge gained during this course. introduction: on october , we organized the st lugano exoday, first initiative in the southern switzerland to bring together resident researchers and european experts in the field of extracellular vesicles (evs). the workshop, centred on "technical challenges of extracellular vesicle research" aimed to highlight technical requirements and advances in the evs area, focusing on isolation, characterization and tracking. methods: the workshop started with a lecture by dr. cecilia lässer, from the university of gothenburg. the rest of the workshop was divided in two working groups (wg), each introduced by a keynote lecture followed by presentations by young researchers and a round-table discussion. wg , introduced by dr. mercedes tkach, from the institute curie in paris, focused on recent advances on evs characterization and isolation. wg was centred on evs tracking and introduced by dr. frédérik verweij, from the institute of psychiatry and neuroscience of paris. results: dr. lässer opened the workshop with a comprehensive review and introduced recent developments in the evs field. the first wg discussed different isolation methods, focusing on ultracentrifugation, size exclusion chromatography and immunoprecipitation-based techniques. supported by the keynote speakers, the participants agreed that the best approach to optimize the isolation process consists in the combination of different techniques. wg shared insights about new strategies to visualize and tracking evs, focusing on how to improve the routinely approaches used, defining optimal criteria for evs labelling and imaging. all the participants had an in-depth overview on the requirements and the state-of-the-art techniques currently in use for the isolation, characterization and tracking of evs. summary/conclusion: the transferable knowledge acquired during the workshop ensures participants to remain up-to-date with the advances in the field of evs. as our ultimate goal is to create a competence centre in southern switzerland around the field of evs, the workshop was an invaluable opportunity to intensify collaborations between resident laboratories and broaden the scientific exchange with laboratories of the experts hosted during the event. given the success of this first workshop we are already working to prepare the second edition and make the event a recurring appointment. funding: supported by the swiss national science foundation the role of core facilities and emerging technologies in maximizing rigour and reproducibility of ev quantification and characterization and following misev guidelines rachel derita a and andrew hoffman b a thomas jefferson university, philadelphia, usa; b university of pennsylvania school of veterinary medicine, philadelphia, usa introduction: it remains very clear in the field of extracellular vesicle (ev) research that the rapid rate of increase in publications and expansion of interdisciplinary clinical ev interest has created the need for increased standardization and access to the appropriate technologies to uphold these standards. as the first core facility in the usa with the sole intention of creating a space where users can both isolate and characterize evs, we provide a central location for the facilitation of ev research via access to multiple technologies (both established and emerging) such as resistive pulse sensing, nanoparticle tracking analysis, ultracentrifugation, high-performance liquid chromatography, flow cytometric analysis of evs and additional immune or fluorescence-based ev characteri zation techniques. methods: we surveyed a group of leading scientific investigators and researchers in varying stages of their scientific careers in the mid-atlantic region of the us. the survey data demonstrate applications of greatest current and future interest to be employed in a shared lab resource. results: the current demand is highest for isolation services, ultracentrifugation and nta, with a gradually increasing demand for immunophenotying analyses such as the exoview chip array, fluorescent nta and flow cytometry. we additionally present strategies and data-based examples of how shared resource facilities can facilitate multifactorial and rigorous ev characterization in accordance with misev guidelines, and encourage collaboration among ev researchers. summary/conclusion: in order to answer the larger remaining questions in the ev field such as the isolation of specific ev subsets, ev tracking between cells and the use of evs for biomarker discovery and drug delivery, it is essential that shared resource facilities interact not only with investigators, but with each other to integrate the necessary resources to progress. programme to assess the rigour and reproducibility of extracellular vesicle-derived analytes for cancer detection national cancer institute, rockville, usa introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par - , is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par - / , successfully funded r and r grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, ); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, ). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, ). summary/conclusion: drs. sudhir srivastava and matthew young are the program directors for the par which began accepting applications on january . this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute introduction: early detection of cancer as well as monitoring cancer treatment are important to improve cancer care. diagnostics for cancer are mainly based on tissue biopsies and re-biopsy during treatment is challenging. moreover, current diagnostics are expensive, time-consuming and have low-throughput. therefore, liquid biopsies are expected to bring the next breakthrough in cancer diagnostics. in liquid biopsies tumour-secreted material is isolated from body fluids and subsequent analyses thereof allow for non-invasive diagnostics. one type of tumour-secreted materials are extracellular vesicles (evs), which are schedded from tumour cells. evs are surrounded by a lipid bilayer, whichs composition resembles the plasma membrane of their parental cell. as many tumours are driven by over-expression or upregulation of transmembrane proteins e.g. growth factor receptors, detection of the later on evs holds promise for early tumour detection and treatment monitoring. methods: for the immuno-pcr evs were first affinitycaptured on magnetic beads, allowing immobilization of purified evs as well as evs secreted into cell culture medium or spiked into plasma. afterwards each sample was divided and affibody-dna-conjugates directed against different targets were added. affibodies are small affinity proteins, which often are developed as high affinity binders for tumour imaging, making them suitable probes in the presented assay. after washing, the bead-ev-affibody-dna-complexes were analysed for the immobilized dna-amount via qpcr. results: via the presented immuno-pcr evs secreted from the non-small cell lung cancer cell line h as well as the ovarian cancer cell line skov were analysed. the immuno-pcr method allowed the detection of the tumour-associated membrane receptors epidermal growth factor receptor (egfr), receptor tyrosineprotein kinase erbb /her and insulin-like growth factor receptor (igf r). different levels of membrane receptors depending on the ev source and concentration were detected. summary/conclusion: the presented immuno-pcr showed to be a comparably fast and robust method for detection of tumour-associated membrane receptors on evs derived from cancer cell lines with medium through-put and is currently further developed into a method for liquid biopsy for non-small cell lung cancer patients. introduction: introduction. evs produced by cells can originate from different cellular compartments and evs in complex biofluids may originate from many different cell types. traditional biochemical analysis, which reports on the total composition of all evs in a sample can't adequately resolve this heterogeneity. single vesicle analysis methods can, if they have the necessary specificity, sensitivity and speed. flow cytometry (fc) is capable of rapid and quantitative analysis of individual particles, but conventional fc-based assays lack the specificity and sensitivity to measure individual evs. assays that combine sensitive instruments with ev-selective sample staining can measure individual evs with accuracy and precision. to better understand the nature and origins of ev diversity, we used single vesicle fc (vfc) to quantitatively measure vesicle number, size, and surface cargo expression on individual evs. methods: methods. evs in culture supernatants ( t, a , u , thp- , sh-sy y) were used neat or enriched by standard methods including differential centrifugation or ultrafiltration. evs from platelets (plt) and red blood cells (rbc) were induced by ionophore treatment of washed cells, and measured in diluted supernatant. evs were stained with a membrane-selective dye and fluorescence-labelled antibodies using a commercial vfc assay kit (cellarcus biosciences), measured using a commercial flow cytometer (cytoflexs, beckman coulter), and data analysed using fcs express v (de novo software). vesicle size, fluorescence intensity, and antibody binding were calibrated using appropriate vesicle and beadbased standards and essential controls performed as recommended by the miflowcyt-ev reporting guidelines. results: results. to assess the compositional heterogeneity of evs, we first characterized the expression of tetraspanins (tss; cd , cd , cd , cd , cd , cd , cd ) on evs released from cultured cell line and primary cell cultures. we find quantitative differences in the expression of ts on evs from different cell types that generally reflected the expression on the cell of origin, with most ev types expressing detectable amounts at least one of the common ts molecules (cd , cd or cd ) but generally not all three. in evs from some cell types, ts expression was uniform across the ev population (cd on evs), but evs from other cell types differentially expressed tss, with some evs expressing no detectable ts (rbc evs). intracellular cargo labelled genetically using fluorescent proteins (egfp or mneongreen) or fluorogenic enzyme substrates (cfse) were measured in individual evs and revealed distinctive associations between ev surface and internal cargo. summary/conclusion: conclusions. high resolution measurement of cargo on/in individual evs can help interpret ev heterogeneity in terms of cell of origin, signals carried, and effects on target cells. integrated omics reveal conserved and divergent modulation of cardiovascular disease by tissue-entrapped extracellular vesicles introduction: fewer than % of patients develop both vascular and valvular calcification, implying differential pathogenesis. while circulating extracellular vesicles (evs) act as biomarkers of cardiovascular diseases, tissue-entrapped evs are implicated in early mineralization but their contents and function are unstudied. we developed an innovative method to isolate and study evs from fibrocalcific tissue and investigated entrapped ev cargoes in human cardiovascular diseases. methods: human carotid artery endarterectomies and stenotic aortic valves were obtained from donors under irb-approved informed consent. tissues underwent enzymatic digestion, ultracentrifugation, and a -fraction optiprep density gradient. global proteomics was performed on intact tissue, each optiprep fraction, and ev-enriched pooled fractions; the latter also underwent mirna-seq. fractionated samples were also studied by cd immunogold electron microscopy (tem) and nanoparticle tracking analysis (nta). high confidence mir targets were predicted by targetscan, pathway analyses utilized the biocarta/kegg/reactome databases, and protein-protein interaction networks were built in string. results: vesicle-associated pathways were increased . x (p < . ; / vesicle-related top go terms) in proteins common to intact arteries and valves (n = , ). proteomics found ev markers to be highly enriched in the four least-dense optiprep fractions of arteries and valves, while extracellular matrix and mitochondria were predominant in denser fractions, as confirmed by tem/nta. proteomics and mirna-seq of tissue evs quantified , proteins and mir cargoes linked to , target genes. pathway networks of proteins and mir targets common to artery and valve tissue evs revealed a shared regulation of rho gtpase and mapk intracellular signalling cascades. proteins and mirs were significantly altered between artery and valve evs (q < . ); multi-omics integration found that evs differentially modulated cellular contraction and p mediated transcriptional regulation in vascular and valvular tissue. summary/conclusion: our findings delineate a novel strategy for studying tissue-entrapped ev protein and mir cargoes and identify critical roles that tissue-resident evs play in mediating cardiovascular disease. funding: this study was supported by a research grant from kowa company (ma) and nih grants r hl , r hl and r hl (ea). mir- a regulates exogenous cd expression on proliferation, invasion, migration and angiogenesis of gastric cancer zhengzhou university, zhengzhou, china (people's republic) introduction: to investigate the possible mechanism of mir- a regulating the expression of exosome cd on proliferation, invasion, migration and angiogenesis of gastric cancer, and to study the application value of cd in the early diagnosis and prognosis of gastric cancer. methods: the gastric cancer cell line mgc- was used as the research object. the exosomes were extracted from the culture supernatant of mgc- by exosome extraction kit. the extracted exosomes were identified by transmission electron microscopy and western blotting. the expression of cd in exosomes was detected by elisa. the expression of cd in exosomes and cd in whole blood and serum were detected by western blot. they were randomly divided into blank group (mock) and mir- a lentivirus experimental group (mir- a group). the lentivirus control group (mir- a/con) was transfected into cells. qrt-pcr was used to verify the status of mir- a after transfection; western-blot was used to detect the expression of cd and downstream erk / , akt and m tor proteins; mtt assay, cell colony formation assay, transwell migration assay for cell proliferation, invasion, and migration. a nude mouse xenograft model was constructed to observe the growth of transplanted tumours,microvessel density (mvd) was detected by immunofluorescence, and distant metastasis was recorded. results: the expression of cd in exosomes was detected by elisa and western blot. the expressions of cd , akt, erk / and m tor in mir- a group were significantly lower than those in mir- a/con and mock groups. cd protein is positively correlated with downstream protein levels.the growth rate and cell invasion ability of mir- a group were significantly lower than those of mir- a/con group and mock group. the weight of the nude mice in the mock group and the mir- a/con group decreased, while the weight loss in the mir- a group was not significant. the tumours in the mir- a/con group and the mock group showed invasive growth accompanied by abundant microvessels, while the mir- a group had smaller tumour volume and uniform cell distribution. only a small amount of microangiogenesis was observed, and no obvious necrotic area was observed. summary/conclusion: mir- a affects the proliferation, invasion, migration and angiogenesis of gastric cancer mediated by akt/erk/m tor signalling pathway by regulating the expression of exosome cd . streamlined detection and quantification of plasma extracellular vesicles and their protein cargo by high-performance nanoscale flow cytometry and label-free mass spectrometry introduction: nanoscale flow cytometry (fc) and mass spectrometry (ms) are useful for profiling ev surface proteins and performing bulk ev proteomics, respectively. this study sought to develop pre-analytical and analytical pipelines for ev protein profiling that are applicable to clinical studies. methods: to optimize plasma ev detection and quantification by fc, modifications of instrument settings and serial dilutions of platelet-free plasma (pfp) and antibodies were tested for improved separation of signal from noise and reduction of event coincidence and swarming. the high-performance flow cytometry (hpfc) platform was used to assess the effect of time ( , , , , , , or hrs) between blood draw (into acd, nacit, edta or heparin) and blood processing, on ex-vivo release of evs from blood cells. label-free ms was used to examine the intensity and breadth of identified proteins in plasma evs purified using several density and size separation methods, either manually or automated, along with various buffer conditions. results: ev event aborts were minimized at a pfp dilution, prior to staining, of : and by using a narrow cytometer window extension. target ev signals were distinct from noise and were triton x- labile. the most significant changes in plasma evs were associated with platelet-derived fractions, use of heparin and > -hour delay before blood processing. yet, platelet ev numbers did not significantly change for up to hrs in citrated and edta plasma. higher overall coverage of known ev proteins and a fivefold increase in number of uniquely identified proteins were observed in ms profiling of evs prepared by a combination of ultracentrifugation (uc) and manual size-exclusion chromatography (sec) compared to preparation by fplc on capto core /superose resins. uc/sec was better than direct sec at reducing contamination by excipient plasma proteins. column buffers with trehalose increased ev protein recovery while adding protease inhibitors had minimal effect. summary/conclusion: with our optimized hpfc protocol, we established that blood ev numbers remain stable for up to hrs in acd or edta and that uc+sec with trehalose-containing buffer result in high canonical ev protein recovery. we are applying these workflows to investigate cancer-associated changes in plasma ev protein cargo. the value of exosomes as a potential biomarker for devil facial tumour disease. university of tasmania, hobart, australia introduction: the tasmanian devil (sarcophilus harrisii), the largest living carnivorous marsupial is endangered because of two transmissible cancers: devil facial tumour disease (dftd) one and two. current efforts to manage dftd are hindered by the lack of a preclinical diagnostic test for dftd. detecting dftd infection is only possible once tumours are noticed, too late to stop dftd progression. a preclinal test could tell us about unknown components of dftd pathogenesis, such as latent period and host-tumour dynamics. exosomes are extracellular vesicles released by most types of cells under both physiological and pathological conditions. exosomes have utility as diagnosis and prognosis biomarkers in a range of diseases, including cancers. the aim of this study is to investigate exosomes-based approaches towards a preclinical and progression biomarker for dftd and in tasmanian devils. methods: exosomes were isolated from three different dftd- , dftd- and devil fibroblast cell lines by sizeexclusion chromatography. likewise, exosomes were isolated from plasma of healthy and diseased devils. to determine the size and morphology of exosomes, samples were imaged with transmission electron microscopy. exosomes isolated from cell lines and devil plasma were analysed with mass spectrometry to characterise proteins and determine their differential expression between the cell origins, and healthy and diseased animals. results: this study identified the presence of myelin proteins in exosomes from dftd cells relative to fibroblasts, which are diagnostic of dftd. additionally, we found that exosomes derived from dftd- abundantly express the inhibitory checkpoint molecule cd relative to exosomes from dftd- cells and devil fibroblasts, indicating a potential candidate for a differential diagnosis between tumours. moreover, exosomes from dftd cells present a greater amount of proteins related with metastasis in comparison with fibroblast exosomes, such as integrins. finally, we report the protein expression profile of exosomes from healthy and diseased devils, showing clear differences between them and the presence of immunosuppressive and metastasis proteins in animals in late stages of the disease. summary/conclusion: dftd-exosomes may provide a non-invasive diagnosis tool to detect early stages of dftd in tasmanian devils to facilitate the prevention of the disease. furthermore, dftd-exosomes may have utility as a prognosis biomarker, determining late stages of the disease using a simple a blood test, which would facilitate monitoring of wild populations. this project will provide long-term benefits for the future of the devils and encourage exosome-based solutions for other future wildlife disease outbreaks. introduction: despite the increased understanding of evs, from involvement in disease pathophysiology to therapeutic delivery, improved molecular tools to track biodistribution are largely lacking. current approaches used for ev labelling lacks sensitivity and specificity. here, we have explored bioluminescent labelling of evs to achieve a highly sensitive system for absolute in vivo quantification and tracking of exogenous evs at low cost and in a high throughput manner. methods: ev-producing cells were genetically engineered to express various tetraspanin-luciferase fusion proteins. evs purified by uf-sec from these cells were characterized by nta, multiplex bead-based array, tem and wb, followed by luciferase assay to determine the labelling efficiency. for in vitro applications cell lysate from treated cells or the conditioned medium were subjected to luciferase assay. for in vivo applications two different methodologies were applied to determine biodistribution; either by non-invasive real time in vivo imaging using ivis or by luciferase assay on harvested tissues for absolute quantification of injected evs. results: we initially performed a systematic comparison of five different luciferases for endogenous labelling of evs and identified nanoluc and thermoluc as lead candidates. we applied this technology to monitor in vitro cellular uptake and observed cell type differences in cellular uptake of engineered evs. in addition, we also observed an effect of different culturing conditions on exocytosis kinetics. for in vivo application, we applied the nanoluc labelling strategy to determine the pharmacokinetics and effect of different routes of injection on ev distribution. our results indicated a rapid uptake profile of administered evs in different tissues with liver, spleen, and lungs being the primary recipients. we also observed similar results upon tracking in vivo biodistribution in real time immediately after administration. finally, we show how different subpopulations of evs differ in their in vivo biodistribution. summary/conclusion: overall, nanoluc and thermoluc labelling of evs holds great potential for various in vivo and in vitro applications. in addition, it can enable the simultaneous detection of different subpopulations of evs in vivo, which may aid in our understanding of different sub-populations and their behaviour in vivo. apart from monitoring therapeutic evs, with one simple modification this platform offers great potential for tracking tumour derived evs both in vivo and in vitro and thus could aid in the development of anti-tumour therapies. biofunctional peptide-modified extracellular vesicles for cell targeting, macropinocytosis induction, and effective intracellular delivery ikuhiko nakase department of biological science, graduate school of science, osaka prefecture university, sakai-shi, japan introduction: [introduction] in our research group, developing therapeutic techniques based on extracellular vesicles (exosomes, evs) by effective usage of peptide chemistry to deliver therapeutic/diagnostic molecules into targeted cells has been focused. in this presentation, modification techniques using biofunctional peptides such as arginine-rich cell-penetrating peptides [ ] , artificial coiled-coil peptides with receptor targeting [ ] , and cell-penetrating sc peptides [ ] derived from cationic antimicrobial protein, cap for cancer targeting with macropinocytosis induction, on the ev membranes will be introduced. i will also show effects of lyophilization of the peptidemodified evs on their biological activity [ ] . methods: [methods] cd (ev marker)-gfpfusion protein expressed evs were used for cellular evs uptake assessments. all biofunctional peptides were synthesized by fmoc solid-phase method. results: [results] macropinocytosis with actin reorganization has been shown to be crucial for cellular ev uptake [ ] . we developed the methods for modification of arginine-rich cpps or sc peptides on ev membranes using chemical linker techniques, and for example, arginine-rich cpps modification can induce proteoglycan-clustering (e.g. syndecan- ) and macropinocytosis signal transduction [ ] . the artificial leucine zipper peptide-modified evs recognize the peptide-tagged epidermal growth factor receptor (egfr) on targeted cells, leading to macropinocytotic cellular ev uptake [ ] . in addition, lyophilization is a useful technique for long term storage, however, we found that lyophilization negatively affected biological functions of encapsulated proteins in the evs after their cellular uptake [ ] . summary/conclusion: [conclusion] these techniques and findings will contribute to development for the ev-based intracellular delivery systems. reference: [ ] sci. rep. , ( ), [ ] chem. commun. , ( ), [ ] chrmmedchem. , ( ) [ ] anticancer res. , ( ), [ ] sci. rep. , ( ) os . multi-compartmented microvesicles: novel extracellular secretory organelles that release exosomes and extracellular vesicles introduction: extracellular vesicles (ev) bud from the plasma membrane (pm) as microvesicles (mv) or arise from the fusion of multivesicular bodies (mvb) with the pm to release intralumenal vesicles (ilv) as exosomes. the variety of bioactive molecules carried by ev imparts diverse functionality to ev in intercellular signalling. the biogenesis and extracellular release of these specialized messenger organelles is not well understood. to investigate, we studied endothelial cells that line the inside of blood vessels, known to release ev that support angiogenesis. methods: cultured human umbilical vein endothelial cells (huvec) were examined by thin-section electron microscopy (em), serial sectioning and immunogold labelling to study the structure and composition ev release sites. to obtain optimal views of cellular ultrastructure, cells were preserved by fast-freezing and a freeze-substitution. results: a potential release site was identified in em thin sections as a discrete domain, up to several microns long, on the otherwise smooth huvec pm, where numerous bulbous membrane protrusions with thin necks were clustered. the cytoplasm in these protrusions was enriched with mvb and other vesicles and appeared to be on the verge of pinching off to release multi-compartmented mv (mcmv). consistent with this notion, in the neighbouring extracellular space, a plethora of mcmv of - nm with ultrastructural features matching the bulbous protrusions were observed, supporting the concept that mcmv bud from the release site. serial sections confirmed that these extracellular mcmv were independent of cells and not linked by nanotubes or other processes. remarkably, fusion of mvb with the mcmv membrane was directly observed, presumably caught in the act of releasing ilv (exosomes) from the mcmv. immunogold labelling for ev markers is being used to identify proteins enriched at release sites and on released mcmv. summary/conclusion: in summary, ) mcmv bud from localized sites on the endothelial pm, ) mcmv contain mvb, and ) fusion of mvb to mcmv to release exosomes occurs extracellularly. mcmv can now be evaluated as a potential source of exosome and ev release that occurs after budding from the cell of origin, adding new layers of regulation to when, where and how ev are assembled and released. funding: this work was supported by the division of intramural research of the nih. one size does not fit all: overcoming barriers to successful discovery and scaled manufacturing of therapeutic extracellular vesicles jieun lee a , wei guo b , hal sternberg c , mike west d and dana larocca d a stem cell team, seoul, republic of korea; b university of pennsylvania, philadelphia, usa; c agex therapeutics inc, alameda, usa; d agex therapeutics inc., alameda, usa introduction: extracellular vesicles have tremendous intrinsic therapeutic potential. however, the limited availability of production cell lines presents a barrier to scaled ev production and novel ev discovery. indeed, ev sources have been largely confined to a handful of cell types with the vast majority consisting of msc evs. to overcome this limitation, we developed a diverse library of hundreds of clonally pure and scalable progenitor cell lines that provides an alternative resource for ev drug discovery and production. methods: we harnessed the capacity of human pluripotent stem cells (hpsc) to differentiate into virtually any cell type by subjecting hpsc to a wide variety of media and culture conditions to maximize the diversity of partially differentiated cells. the resulting heterogeneous "candidate cultures" were plated at clonal density and further selected for self renewing and scalable clones. transcriptomic analysis indicated > distinct progenitor lines. cell fate potentials were mapped by screening for cell type specific marker expression in various differentiation conditions. evs were produced using cgmp methods (tff and sec) and characterized by nta, trps, surface marker analysis, rna and protein content. bioactivity assays included proliferation, migration, vascular tube network formation, senolysis, and oxidative stress. results: the progenitor library contained > distinct lines with diverse lineage fates including various types of bone, cartilage, muscle,and fat cells, as well as all blood vessel cell types. the lines displayed much longer replicative lifespans ( - pd) than primary cell lines like msc. clonal purity minimized phenotypic drift resulting in maintenance of cell identity, genome integrity, differentiation capacity and bioactive ev production over extended culture. evs were highly diverse in their rna and protein cargo and bioactivity displaying various degrees of migratory, proliferative, angiogenic and senolytic activity. library screening identified evs with higher angiogenic potency than primary adult stem cell evs. summary/conclusion: we demonstrated scalable and stable production of bioactive evs from a large progenitor cell library. library screening resulted in discovery of novel angiogenic and senolytic evs having diverse rna and protein cargo. we are currently creating a corresponding library of progenitor cell evs to accelerate discovery of novel evs and their production cell lines. funding: the initial establishment of the cell library was funded in part by grants from the california institute of regenerative medicine and national institutes of health. introduction: besides extreme potential in biomedical applications, extracellular vesicles (evs) are also promising candidates to expand biophysical understanding of membrane active biomolecules. their complex bilayer composition allows the better understanding of adsorbed proteins and protein coronas as well, which sets of macromolecules will likely be key for advanced ev targeted delivery. considering cargo, membrane active peptides are interesting as these can be both drugs to be delivered, but can also facilitate cargo insertion through lipid bilayers. however, at present very little is understood regarding interactions between the peptides and the ev lipid bilayer, and between peptides and membrane associated proteins on evs. methods: we have recently demonstrated, that ev membrane adsorbed proteins and their interactions can be studied by techniques such as polarized light spectroscopy, microfluidic resistive pulse sensing measurements and freeze-fraction transmission electron microscopy [ ] . furthermore, initially we studied several peptides with known antimicrobial properties and found that these strongly interact with the ev surface proteins, resulting in efficient removal of some from the lipid bilayer [ ] . results: here we present investigation of further evpeptide interactions also focusing on anticancer peptides, which may be promising drug candidates for targeted delivery. these studies allowed to gain insight to novel functions of several peptides, such as melittin, magainin, buforin, lasioglossin, temporin, but also provide a more detailed understanding on how ev protein coronas, or ev bilayers are affected, to such extent that they cannot exert their potential function as delivery systems. summary/conclusion: the above interactions are expected to be interesting both for applicability, i.e. for selecting suitable compounds for ev processing, and also for curiosity-driven understanding of peptide functions, and ev-biomolecule interactions. based on these we promote that peptide -ev interactions will receive increased focus in ev-engineering. introduction: our late-breaking finding is the identification of a non-coding rna (ncrna) in extracellular vesicles (evs) from neuronal cells that is a natural antisense transcript for the dbh gene and associated with epigenetic changes and gene silencing. dna methylation in neurons is involved in memory and neurological disorders (science ( )). earlier work found that during chronic brain infection with toxoplasma gondii induced a decrease in norepinephrine levels and expression of the host dbh gene; and the decrease is correlated with behaviours linked to noradrenergic signalling (infect immun. ( ); infect immun. ( )). dbh catalyzes the production of norepinephrine from dopamine in noradrenergic neurons. we found that evs from infected cultures suppress transcription of the dbh gene and hypermethylation of the gene in noradrenergic cells in vitro. in this study, we identify a ncrna in the evs from infected neuronal cells. methods: neuronal cells were induced by infection with toxoplasma gondii and evs purified on sucrose gradients. evs were characterised by electron microscopy and used to treat rat and human neuronal cells and expression levels of dbh mrna and nascent dbh gene transcription were measured. induced evs were injected into the locus coeruleus of rats and dbh gene expression was monitored. rna purified from evs was screened for natural antisense transcripts (nats) by strand-specific rt-pcr. results: we found that evs purified from infected neuronal cultures induced transcriptional gene silencing (tgs) and dna methylation of dbh in recipient neuronal cells. the induced evs down-regulated dbh gene expression > -fold and induced dna hypermethylation of the dbh gene. this could be induced in the brains of recipient rats by intracerebral injection of evs. using a panel of strand-specific primers, antisense transcripts for the dbh gene were identified in infected cells. this permitted us to examine the rna in purified evs and identify a lncrna in evs selective for evs from infected cultures. summary/conclusion: this is the first study to find a specific neurotransmitter antisense lncrna in evs associated with transcriptional gene silencing and epigenetic changes in the gene. this represents a different type of neuron-to-neuron signalling than the classic chemical and electrical neurotransmission. the findings will enhance our understanding of neurological disorders (ie. schizophrenia, epilepsy, drug addiction) and how memory works. human cd + t regulatory-derived extracellular vesicles and associated micrornas: role in cell-to-cell communication and involvement in the loss of immune tolerance during multiple sclerosis introduction: an impairment of immune tolerance is a determining factor in multiple sclerosis (ms) and dysregulation of cd + t regulatory (treg) cell function is believed to be a major pathogenic factor. micrornas (mirnas) released by treg cells in association with extracellular vesicles (evs) have been shown to participate in the block of pathological immune responses by inhibiting the growth and cytokine production of cd + t conventional (tconv) cells, but the molecular mechanism is still poorly characterized. aim of the present work was to evaluate whether treg cell-derived ev-associated mirna signature is dysregulated in ms and whether this defect may play a role in the development of autoimmunity. methods: human treg cells isolated from blood of naïve to treatment relapsing-remitting ms patients and healthy controls were in vitro stimulated and released evs were isolated by size exclusion chromatography and characterized by nanoparticle tracking analysis, electron microscopy and flow cytometry. evassociated mirnas were quantified by traditional rt-qpcr and droplet digital pcr for absolute quantification. the actual ev-mediated passage of rna molecules from cell to cell was followed through rnaspecific fluorescent staining and treg-derived ev effect on tconv cell transcriptome was evaluated by rnaseq. results: in healthy conditions, the treatment of tconv cells with treg-derived evs was shown to cause the specific repression of genes involved in the proteasome-dependent proteolytic process, known to be crucial for t cell activation. in ms, treg-derived evs may have lost this capability as a direct consequence of a significantly decreased expression of mir- - p, able to target key factors of the proteasome system. summary/conclusion: our results unveil a novel molecular mechanism for treg-mediated maintenance of self-tolerance based on ev-associated mir- - p and its potential alteration in human autoimmunity. funding: fondazione italiana sclerosi multipla, fism, # /r/ and # /r/ revealing the proteome of brain derived extracellular vesicles isolated from human amyotrophic lateral sclerosis post-mortem tissues. introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterised by the deposition of misfolded proteins in the motor cortex and motor neurons. although a multitude of als-associated proteins have been identified, few have been associated with extracellular vesicle (ev) trafficking, a form of inter-cellular communication. additionally, the role of evs in als is undetermined, specifically in relation to pathogenic stress granule formation, a response to cellular stress involving aggregation of non-coding rnas and their rna binding proteins. therefore, this study aimed to determine the proteome of brain derived small extracellular vesicles (bdevs) isolated from als subjects and identify novel alsassociated deregulated proteins and their potential contributions to pathogenic pathways in als. methods: bdevs were isolated from human post-mortem als (n = ) and control (n = ) motor cortex brain tissues through an ultracentrifugation protocol (vella et al., ) . following thorough characterisation, bdevs that successfully met the minimum criteria required by the international society for extracellular vesicles were classified as evs. the bdevs subsequently underwent mass spectrometry analysis on the thermo scientific q-exactive hf with ultimate rslcnano. proteins identified to be statistically significant differentially expressed then underwent validation by western blotting. results: a panel of statistically significant differentially packaged proteins were identified in the als bdevs. this included several up-regulated rna binding proteins and a down-regulated cell adhesion molecule; dhx , stau and vcam , respectively. pathway analysis revealed that the bdevs were enriched in proteins associated with stress granule dynamics, exosomal and lysosomal pathways. summary/conclusion: the identification of the rna binding proteins in the als bdevs suggests there may be a relationship between als-associated stress granules and als bdev packaging. the packaging of stress granule associated rna binding proteins into als bdevs may be an attempt by the cells to compensate for lysosomal dysfunction caused by stress granule accumulation, a feature of als. thus, these results highlight a potentially novel role for evs in the pathogenesis of als for long-term cultivation . the whole cultivation process of tissue preparation, cultivation, and cryopreservation has been established using strict serum-free conditions under a good manufacturing practice. long-term-cultivated hmnpcs retained stemness and hmnpcs have excellent differentiation efficiency into dopaminergic neurons. hmnpcs reversed impaired motor function in a rodent model of parkinson's disease (pd). based on the promising results in animal experiments, the clinical trial is under way (nct ). multiple-system atrophy (msa) is one of fatal neurodegenerative diseases with a combination of progressive autonomic nervous system disorders, parkinson's syndrome, and cerebellar pyramid syndrome. there are three types of msa such as msa-a, msa-c, and msa-p. in case of a msa-p type, it is difficult to diagnose due to the similarity of symptoms with parkinson's disease (pd). methods: in vitro and in vivo animal msa model were established and rotational behavioural was performed. npc cells were isolated and cultured based on moon et al. mirna sequencing (bgi) was performed and several bioinformatics analyses were done. results: based on the finding that hmnpcs exhibited therapeutic effects on pd, we hypothesize that hmnpcs will have a therapeutic effect on msa-p, where sympotoms are largely common with pd. as expected, transplanted hmnpcs survived, integrated, and differentiated in to dopamine neurons in the host brain, consequently leading to the functional recovery in the msa-p model. to further investigate the therapeutic key factors of hmnpcs in msa-p, mirna sequencing of the extracellular vesicles (evs) secreted from hmnpcs was performed. we found that mir- a highly expressed in the npc-derived evs is one of key regulators of inflammatory response via nfkb pathway. we further experimentally demonstrated that mir- a had anti-inflammatory effect on cells of msa-p condition such that the level of cx cl expression and its receptor, cx cr were both decreased in the msa-p modelled cells and in severe inflammatory environment in msa brain. summary/conclusion: our study first showed that mir- a in hmnpcs-evs is one of key therapeutic factors for the recovery of brain damage through immuno-modulation in msa-p. introduction: oxidative insults are known to be involved in the pathophysiology of alzheimer's disease (ad). we have previously demonstrated that some blood-based redox-signature were associated to the cognitive scores in mild cognitive impairment patients and in ad (perrotte et al., ) . the aim of this study was ( ) to evidence the presence of some oxidative markers in circulating extracellular vesicles (evs), and ( ) to compare to their plasma levels. methods: plasma samples from healthy, mci and ad patients were from the memory clinic of sherbrooke (québec, canada). ad patients were stratified in three groups (moderate, mild and severe) according to the mmse and moca scores. total plasma extracellular vesicles (pevs) were isolated from plasma with the total exosome isolation reagent (invitrogen™ by life technologies inc.). pevs were then characterized by electronic microscopy, nta, dls and western blot. antioxidants apolipoprotein j, d (apo j, apod), the glyoxalase- and protein carbonyls were determined by western blot. results: in pevs, we found that apo d levels were higher in mci patients but not in ad patients. protein carbonyls levels were higher later, in pevs from moderate and severe ad while apo j levels were not different in pevs from the five groups of patients. in plasma, the pattern of apo j and apo d was different. the levels of apo d was not different in the five groups of patients while apo j levels were elevated in mci and in all ad groups. protein carbonyls were higher earlier from mild ad group, earlier than in pevs. the levels of the detoxifying enzyme glyoxalase- were higher in pevs than in plasma and were significantly decreased in early ad as compared to control subjects and mci summary/conclusion: these results demonstrate a differential regulation of redox homoeostasis in plasma and in pevs from ad patients. funding: acknowledgements: this work was supported by the chaire louise & andré on alzheimer's disease, foundation armand-frappier (cr) and cihr grant (tf). carlos j. nogueras-ortiz a , pavan bhargava b , sol kim b , francheska delgado-peraza a , peter calabresi b and dimitrios kapogiannis a a laboratory of clinical investigation, national institutes of ageing, baltimore, usa; b department of neurology, johns hopkins university school of medicine, baltimore, usa introduction: multiple sclerosis (ms) is a neurological disorder characterized by white matter demyelination and extensive synaptic pathology. recent studies have shown synaptic loss in the grey matter of ms brains in the absence of demyelinating lesions which could account for disease progression independent of demyelinating episodes. opsonization of synapses with complement components is a mechanism by which phagocytic cells normally prune synapses, but, when occurring in excess, it may underlie pathologic synapse loss. we sought to identify blood-borne biomarkers of hypothesized complement-mediated synaptic loss in ms using circulating neuronal-enriched and astrocytic-enriched extracellular vesicles (nevs and aevs). methods: nevs and aevs were immunocaptured in parallel from the plasma of ms patients ( with relapsing remitting, with progressive ms) and healthy controls, targeting the neuronal-specific marker l cam and the astrocyte-specific marker glast, respectively. we measured the protein levels of preand post-synaptic proteins synaptopodin and synaptophysin in nevs using elisas and multiple complement cascade components (c q, c , c b/ic b, c , c , c a, c , factor b, factor h) in aevs using a luminex array. results: synaptopodin and synaptophysin protein levels in nevs of ms patients compared to controls were markedly reduced ( . -fold; p < . for both), whereas multiple complement components in ms aevs were markedly increased (c q: . -fold change; c : . -fold change; c b/ic b: twofold change; c : . -fold change; c a: . -fold change; factor: . -fold change; p < . ); differences were not observed in total circulating evs or neat plasma. strikingly, we found the nev-associated synaptopodin/synaptophysin and the aev-associated complement levels to be negatively correlated in people with ms (synaptopodin vs: c q, r = − . and p < . ; c , r = − . and p < . ; factor h, r = − . and p < . /synaptophysin vs: c q, r = − . and p < . ; c , r = − . and p < . ; factor h, r = − . and p < . ), but not in controls. summary/conclusion: circulating evs provide markers of synaptic loss and complement activation in ms and suggest a link between astrocytic complement production and synaptic decline. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. methylglyoxal and glyoxal affect the protein cargoes in neuronal-derived extracellular vesicles introduction: advanced glycation end-products (ages) and their receptor rages are known to be involved in the pathogenesis of alzheimer's disease (ad). methylglyoxal (mg) or glyoxal (go) are the precursors of ages and particularly n-( -carboxymethyl)-l-lysine (cml), the most abundant ages. mg induced tau hyperphosphorylation and causes hippocampal damage and memory impairment in mice. the aim of our study was to analyse the effects of mg and go on the neuroprotective, neurotrophic factors, inflammatory and neurodegenerative markers in the human cell line sk-n-sh and their release into the neuronal derived-evs. methods: briefly, sk-n-sh cells were incubated in fbs free media with mg and go ( . mm) for hours. neuronal derived-evs (nevs) from culture media were isolated as previously described (haddad et al. ). nevs were characterized by electronic microscopy, nta and by western blot. cellular and nevs concentrations of bdnf, prgn, nse, app, mmp , angptl- , lcn , ptx , s b, rage, dj- and alpha synuclein were determined by a luminex assay from r&d systems, inc. aβ - , aβ - , ptau t and total tau levels were measured also with luminex assay from emd millipore corp. results: we found that both ages precursors, at non toxic concentration, reduced the neuronal levels of nse with no effect on bdnf, ptrx- , lcn- , dj- , on neurodegenerative markers and on cml. go decreased the levels of prgn, app, angpl- while the expressions of mmp- and angpl- were, respectively lower and higher in the presence of mg. mg and go greatly reduced the release of lcn- by neuronal cells in nevs. bdnf and prgn in nevs were reduced in the presence of go. both mg and go did not modify the release of nse, app, mmp , agntl- , ptx- , dj- , aβ, ptau and cml in nevs. summary/conclusion: our data demonstrated that mg and go differently affect the content of some protein cargoes in nevs and suggest that targeting mg and go may be a promising therapeutic strategy to prevent neurodegeneration. introduction: peripherally circulating brain-derived extracellular vesicles (evs) and their encapsulated rnas may serve as biomarkers for hiv-associated neurocognitive disorders (hand). however, rates of cigarette smoking are significantly higher in hiv+ individuals than the general population, and smoking can modulate the expression of these markers. to better understand how cigarette smoke might modulate rna expression and ev release, we examined several cnsderived cell lines, representing astrocytes (u mg), microglia (sv ), and oligodendrocytes (hog). methods: cigarette smoke extract (cse) was prepared by bubbling through culture medium using a standardized and published method. all cell types were exposed to either % or % cse for hours. cell viability was assessed by musetm cell analyser, and evs were isolated from culture conditioned media (ccm) by size exclusion chromatography. the void (fractions - ), ev ( - ), and protein ( - ) enriched fractions were pooled and concentrated. evs were characterized by transmission electron microscopy (tem), microfluidic resistive pulse sensing, and western blotting. total rna was isolated from cells and circular rna (circrna) expression was assessed with a circrna microarray. results: in response to cse exposure, cell viability was only slightly reduced for all cell types. tem images validate the presence of vesicles in the ev fractions, and their absence in the void and protein fractions. spectradyne particle counts indicated cse exposure substantially increased the ccm particle count in the ev fraction when compared with control. the presence of expected ev markers (cd , cd , and tsg ) in the ev fractions, and their absence in the void and protein fractions was observed via western blot. intracellular circrna expression was significantly altered in all three cell lines. summary/conclusion: cns cells display physiologic responses to cse that include vesiculation pathways and significant alterations in circrna expression. we are now studying the effects of cse exposure on circrna expression in released evs. funding: this work is supported by da , da , and ai . a method for exosomal rna extraction from paired human brain and blood specimens emily n. moya a , lillian wilkins a , esther cheng a , lisa linares b , brian kopell b , navneet dogra c , bojan losic a and alexander charney a a icahn school of medicine at mount sinai, new york, usa; b mount sinai hospital, new york, usa; c department of genetics and genomic sciences, department of pathology, icahn school of medicine, mount sinai, new york, usa introduction: diagnosis and treatment of neuropsychiatric disorders has made little progress in the last half-century likely in large part due to the absence of a scalable technique to profile the complex biological activity of the brain in a living person. exosomes are nanovesicles - nm in size that mediate intercellular communication and contain proteins, lipids, and nucleic acids. it has been shown that brain derived exosomes can be found in peripheral blood, but determining whether peripheral exosomes truly reflect ongoing brain processes has to date not been possible due to the absence of paired living brain and blood specimens. here, we present a novel method for paired sampling of the dorsolateral prefrontal cortex (dlpfc) and peripheral blood from living human subjects for exosomal rna profiling. methods: informed consent, approved by the irb at the icahn school of medicine at mount sinai, was obtained for patients undergoing deep brain stimulation (dbs). paired brain and blood specimens were collected from patients at two deep brain stimulation (dbs) electrode implantation procedures: left hemisphere followed by right hemisphere (total of samples). we developed protocols to profile rna from exosomes of brain tissue extracellular matrix (ecm) and peripheral blood. exosomes were isolated via our in-house protocol using ultracentrifugation. rna was then extracted from the exosomes using the qiagen mirneasy mini kit protocol. quality control (qc) was performed to determine whether rna obtained was sufficient for next-generation sequencing. results: we demonstrate the safety of a novel procedure to sample the brain in living human subjects. bioanalyzer traces and qc data show a mean total rna of . ng (range . - . ng) and no samples fell below the threshold required for library preparation and sequencing ( pg) determined by inhouse optimization on the smart-seq v ultra low input kit. summary/conclusion: to our knowledge, we have performed the first study to sample pairs of dlpfc and blood from living human subjects for exosomal rna for subsequent next-generation sequencing. ongoing analyses by our group promise to establish peripheral exosomal rna transcripts reflective of brain activity. this non-invasive approach to probing neurobiology in the living human brain may facilitate the development of exosome-based diagnostics for neuropsychiatric disorders. introduction: the relationship between obesity and dementia is complex. while obesity in middle age triples the risk of dementia years later, many patients with alzheimer's disease (ad) are cachectic, and a decline in adiposity portends progression of dementia. this suggests adipose-derived factors are important to nervous system homoeostasis. we previously showed that adipocyte-derived small extracellular vesicles (ad-sevs) induce pathologies critical to developing obesity-related diseases and may provide a mechanistic link between adiposity and dementia. we hypothesized that altered expression of ad-sev micrornas involved in neurodegenerative pathways is associated with more severe cognitive impairment methods: we studied serum and cerebrospinal fluid (csf) from participants with ad and non-ad controls. ad-sevs were isolated from samples by precipitation and immunoselection. ad-sev microrna expression was profiled in both biofluids and compared. results: serum and csf microrna expression correlated strongly (r = . ). in serum, micrornas were differentially expressed by a fold change ≥| . | in the ad and control groups (p ≤ . ) and micrornas were differentially expressed in csf. using ingenuity pathways analysis, we identified mrnas expressed in nervous system tissue that are targeted by the differentially expressed micrornas. the mrnas map to diseases and functions; neuronal cell death, neurodegeneration, and neuronal growth and developmental pathways are highly represented. of the differentially expressed micrornas in serum, were moderately correlated with participants' score on the mini-mental state exam, a test of cognitive function (rs = | . - . |). as validation, rencell cx cortical derived neuronal stem cells had decreased doubling time when exposed to ad-sevs from obese adipose tissue in vitro. summary/conclusion: these findings support our hypothesis that altered expression of circulating ad-sev micrornas are involved in neurodegenerative pathways associated with cognitive impairment. these findings support using serum ad-sevs as a surrogate for csf ad-sevs. functional validation is underway to define the connection between ad-sevs and ad. understanding the link between obesity and ad is crucial as the population ages and the global obesity epidemic grows. funding: supported by uw adrc (nih:p ag ) expression of extracellular vesicles after acute traumatic brain injury: an exploratory flow-cytometry study introduction: coagulation derangements related to disseminated intravascular coagulation (dic) are common after tbi and contribute to secondary neural injury. extracellular vesicles (evs) are released from all cell types, including platelets, endothelium, and lymphocytes, which are responsible for dic. we hypothesized that specialized flow cytometry techniques could identify a unique ev signature of dic in acute tbi. methods: using a modified flow cytometry instrument for detection of small particles, fluorescence panels were created to assess for evs from endothelial cells (cd , cd ), platelets (cd , cd p, cd a, cd b), and erythrocytes (cd ) as well as brain biomarkers (s b, uchl- , gfap, tau and nse) and t-lymphocytes (cd , cd , cd , cd ). samples were prepared in trucount tubes to determine volume and treated with triton to confirm presence of evs. results: / study patients and / controls were male. % of study patients presented with a glasgow coma scale of . in the hypercoagulability panel, of the subsets with statistically significant differential expression, involved s b+ and were elevated in patients. platelet-derived cd a evs and uch-l evs were significantly elevated in controls in ev subsets identified in the brain-specific panel. finally, cd +/ + evs, derived from t-cells and identified in the endothelial/t cell panel, are significantly lower in patients suggesting cns recruitment. summary/conclusion: endothelial and platelet/erythrocyte evs may be elevated early after tbi. s bcarrying evs are significantly elevated in circulation of tbi patients; if reproducible, this signature profile may be informative for diagnosis and risk stratification. further study is warranted to evaluate whether this expression correlates with secondary microvascular brain injury. funding: intramural award from the university of pennsylvania enrichment of mir- a in cns extracellular vesicles following impairment of the blood brain barrier nasser nassiri koopaei a , ekram-ahmed chowdhury b , lais da silva a , jinmai jiang a , behnam noorani b , ulrich bickel b and thomas d. schmittgen a a university of florida, gainesville, usa; b texas tech university, amarilo, usa introduction: extracellular rnas (exrnas) are present in essentially all biofluids and include all types of rna including mirna. to enhance their stability outside of the cell, exrnas are bound within ribonucleoprotein complexes or packaged into extracellular vesicles (evs). the blood brain barrier (bbb) is a dynamic interface between the systemic circulation and the cns and is responsible for maintaining a stable extracellular environment for cns cells. the intent of this study was to determine if evs and their contents are transferred from the peripheral circulation to the cns under conditions of an impaired bbb. methods: the bbb of mice was disrupted by hyperosmolar mannitol injections. to validate that the bbb has been disrupted with mannitol, intravenously-dosed [ c]-sucrose was increased in the forebrain by fold with mannitol compared to sham treated mice. evs were isolated from the forebrain, hindbrain and spinal cord following gentle tissue lysis and differential ultracentrifugation. evs were validated by nta, tem and western blotting. mir- a, a mirna that is highly abundant in erythrocytes, was measured in the evs by qpcr. results: qpcr showed that mir- a in cns tissue evs increased with mannitol treatment in the forebrain, hindbrain and spinal cord by -, . -and twofold respectively. qpcr analysis of mrna from reported mir- a target genes showed reduced target gene expression with mannitol. summary/conclusion: we demonstrate that evs containing mir- a, a highly abundant mirna present within erythrocytes and erythrocyte evs, is enhanced in the cns upon bbb disruption. astrocyte-derived extracellular vesicles in morphine tolerance guoku hu, rong ma, naseer kutchy, yuetong zhao, susmita sil and shilpa buch university of nebraska medical center, omaha, usa introduction: opiates, such as morphine are used extensively in the clinical setting owing to their beneficial effects. paradoxically, however, the prolonged use of morphine often results in the development of tolerance, drug addiction, and ultimately leading to various comorbidities associated with drug abuse. although great efforts have been made, at present there is no treatment. the sonic hedgehog (shh) plays a key role in brain development, and brain cells fine-tuning processes such as their proliferation, patterning, and fate specification recent findings have demonstrated that inhibition of the shh signalling prevents morphine tolerance in rodent models. we thus hypothesize that extracellular vesicles (evs) derived from morphine exposed astrocytes and their cargo such as shh are critical for the development of morphine tolerance. methods: mice were received either saline or chronic morphine injection with escalating doses of morphine for days (subcutaneously; mg/kg, day , mg/kg days - , and mg/kg days [ ] [ ] . the development of tolerance was assessed by measuring the tail-flick latency using tail flick analgesia metre (le , harvard apparatus). evs were isolated using either differential ultracentrifugation from astrocyte conditioned media or gradient ultracentrifugation from brain tissues. western blotting and qpcr were performed to determine the expression/activation of shh signalling pathway components. results: our data showed that the levels of shh protein were upregulated in morphine exposed astrocytederived extracellular vesicles (morphine-adevs). furthermore, shh containing morphine-adevs activated shh signalling in astrocytes. our in vivo study further demonstrated the upregulation of shh, as well as the activation of shh signalling, in astrocytes of morphine-administered mice. summary/conclusion: these findings thus demonstrated an autocrine mechanism for shh pathway activation in astrocytes associated with morphine tolerance. these findings could pave the way for the development of shh signalling pathway targeted strategies in the prevention and treatment for substance use disorders. biophotonics-based platforms for the evaluation of circulating extracellular vesicles as biomarkers of neurodegeneration in alzheimer's disease silvia picciolini a , cristiano carlomagno a , alice gualerzi a , monia cabinio a , francesca baglio a and marzi bedoni b a irccs fondazione don carlo gnocchi, milan, italy; b irccs fondazione don carlo gnocchi, milano, italy introduction: in the search for novel and non-invasive biomarkers of alzheimer's disease (ad), both circulating brain-derived extracellular vesicles (evs) and whole serum represent a valuable integration of the currently used classification system. to face the technological challenge of evs and serum analysis, we propose the use of biophotonics techniques as reliable, sensitive, fast and label free methods, potentially useful in tailoring pharmacological and rehabilitation treatments. methods: circulating evs, isolated by sec, and serum samples were collected from healthy subjects (hc) and ad patients. all subjects were asked to complete montreal cognitive assessment scale and mri examination. surface plasmon resonance (spr) was performed in order to detect evs coming from neurons, astrocytes, oligodendrocytes and microglia and to characterize each of them for the amount of ganglioside m (gm ), aβ and tspo expressed on their surface. serum analysis was performed using a raman microscope through the surface enhanced raman spectroscopy (sers) effect by mixing serum with ag nanoparticles. the pearson's correlation index was used to assess the linear correlation between spri data and clinical, mri data and data obtained from multivariate analysis (mva) of sers spectra. results: the spr analysis of evs showed that the selected bioactive molecules are differently loaded on neural ev populations and that their amount is increased on total evs in ad patients compared to hc. we observed a significant correlation between mva data from sers and the presence of aβ on neuronal and microglial evs and of tspo on neural evs, measured with the spr array. summary/conclusion: thanks to our methodological innovation we have verified the potentiality of evs as ad biomarkers, correlating biophotonics blood-based analysis with clinical data. this platform could provide a powerful tool for the evaluation of ad neurodegeneration. funding: the study was supported by the italian ministry of health (ricerca corrente - to irccs fondazione don carlo gnocchi). raman profiling of extracellular vesicles as new blood-based biomarker for brain disorders: focus on parkinson's disease introduction: extracellular vesicles (evs) play a pivotal role in brain homoeostasis and intercellular communication in both physiological and pathological conditions. in parkinson's disease (pd), evs are key players in the transfer of α-synuclein, with blood evs reported to undergo proteomic modifications. nonetheless, the detection and characterization of the ev cargo is technologically challenging, limiting the use of evs as biomarkers so far. herein, we propose raman spectroscopy for the label-free, bulk characterization of blood evs in pd patients. methods: evs were isolated by sec and ultracentrifugation from the serum of healthy subjects (hc) and pd patients. in all patients, the severity of pd was evaluated with the unified parkinson's disease rating scale (updrs) part iii and with hoehn and yahr scores (hy). after proper ev characterization following misev guidelines, raman analysis was performed. the raman microspectroscope was used with a nm laser in the spectral ranges - cm- and - cm- . data from hc and pd patients were compared by multivariate statistical analysis (pca-lda). results: the raman analysis of evs highlighted differences in the biochemical profile of the two groups, with the main variations in the spectral regions related to proteins, lipids and saccharides. a preliminary estimate of the accuracy of raman profiling of blood evs for pd diagnosis was obtained, demonstrating an accuracy of %. even more interestingly, we demonstrated the correlation between the raman spectra and the clinical scales (updrs and hy) used to stratify pd patients. summary/conclusion: in conclusion, the biochemical signature of blood evs can be detected by raman spectroscopy in pd patients and the ev spectral modifications can be related to their clinical status. these data suggest the possibility to use the raman profile of circulating evs as a biomarker for brain disorders, complementary to other specific molecular markers. funding: the study was supported by the italian ministry of health (ricerca corrente to irccs fondazione don carlo gnocchi) impact of circulating extracellular vesicles on brain functions and behaviours introduction: peripheral immune alterations have been described in psychiatric disorders such as schizophrenia, depression, and autistic spectrum disorders. in addition, behavioural changes have been observed in various immunodeficient animal models. however, the mechanisms by which peripheral immune system influences brain development and function are not well understood. in this study, we explored the mechanisms by which circulating extracellular vesicles (evs) mediate immune-brain communication and influence mouse behaviours. methods: mice deficient for rag or rag gene (rag ko mice) were used as a model to study the effects of loss of adaptive immune cells (t and b cells) on brain cellular phenotypes and behaviours. circulating evs were collected from their sera and analysed by using electron microscopy, nanoparticle tracking assay, and western blotting. brain cellular phenotypes were assessed by immunofluorescent staining and gene expression analysis. behavioural phenotypes of rag ko and wt mice were examined in social interaction test. in vivo transfer of evs was performed to see its effects on behavioural alterations of rag ko mice. results: rag ko mice displayed social behavioural deficits, accompanying by enhance c-fos immunoreactivity and altered microglia morphology in the medial prefrontal cortex (mpfc). circulating evs were also affected in these mice and lacked the expression of markers for t cells. a set of micrornas (mirnas) in circulating evs were diminished in rag ko mice. in vivo transfer of circulating evs rescues the social behavioural deficits of rag ko mice and ameliorate the c-fos immunoreactivities in mpfc of rag ko mice. summary/conclusion: our data showed that circulating ev profiles were altered in mice lacking adaptive immune cells and, accordingly, showing social behavioural deficits. notably, our in vivo experiments suggest that circulating evs may contribute to social behaviours. further study will provide a novel biological insight into the mechanisms underlying peripheral-to-brain immune communication via evs. introduction: the involvement of neuroinflammation on ageing process is widely recognized. extracellular vesicles (evs), such as exosomes, are able to cross the blood-brain barrier and were related to neuroinflammation. in this context, evs have been considered a potential mechanism of spreading molecules, including micrornas (mirnas) that can promote mrna degradation or inhibit translation of their targets. our aim was to investigate the mirna profile of circulating total evs during ageing process and their impact on canonical pathways. methods: the local ethics committee (comissão de Ética no uso de animais -ufrgs; n ) approved all animal procedures and experimental conditions. plasma was obtained from wistar rats ( and months-old) and total evs were isolated. ev microrna isolation and microarray expression analysis was performed to determine the predicted regulation of targeted mrnas. results: the analysis of global microrna expression revealed differentially expressed micrornas (p < . ; fold change of ≥ | . |); mirnas were up-regulated and were down-regulated in circulating total evs from aged animals compared to youngadult ones. a conservative filter was applied on ingenuity pathway analysis (ipa) and only experimentally validated and highly conserved predicted mrna targets were used. ipa showed that neuroinflammation signalling is ranked among the top canonical pathway impacted by differentially expressed micrornas and is upregulated in aged animals (p < . ; z-score: . ). the differentially expressed mirnas impacted molecules in the neuroinflammation pathway. interestingly, the ion channel grin b is predicted to be up regulated and is a target of many evs mirnas; in accordance with our results grin b was previously related to neurodegenerative diseases. moreover, let- a- p is predicted to be downregulated and target all the molecules of the neuroinflammation signalling pathway. previous studies have correlated let- a- p and neurodegenerative diseases. summary/conclusion: our data suggest that circulating total evs cargo, specifically mirnas, are altered by ageing and impact neuroinflammation pathway, suggesting the involvement evs mirna on ageinginduced susceptibility of neurodegenerative diseases. introduction: bidirectional cell-cell communication via paracrine mechanisms is critical for wound healing. a new paradigm involving exosome-borne distinctive repertoire of cargo such as mirnas has emerged as a predominant mechanism of cellular communication at the site of injury. unlike other shedding vesicles of similar size, exosomes selectively package mirna by sumoylation of heterogeneous nuclear ribonucleoprotein (hnrnp). methods: keratinocyte-derived exosomes (exoκ) were genetically labelled with fluorescent reporter (gfp) using tissue nanotransfection. purified, gfp-labelled exoκ were isolated from dorsal murine skin and wound-edge tissue by differential ultracentrifugation followed by affinity selection using magnetic beads. distributions of intact exosome were analysed using a prototype jarrold-geometry charge-detection mass spectrometer to directly measure differences in particle mass and charge distributions. complementary ms and ion mobility spectrometry (ims)-ms experiments have been used to characterize surface glycans and glycopeptides. to selectively inhibit mirna packaging within the exoκ in vivo, ph-responsive targeted sirna functionalized lipid nanocarriers (tlnκ) were designed using materials that have prior history of fda approval for human use. results: an increase in mass/charge ratio with glycan binding sites on the surface of wound-edge exoκ were observed compared to dorsal skin exoκ. wound-edge exoκ were selectively taken up by the macrophages in the granulation tissue (n = ). keratinocyte targeting sirnahnrnp functionalized lipid nanocarriers (tlnκ) were designed with encapsulation efficiency of . %. application of tlnκ encapsulating sirna of hnrnp (tlnκ/si-hnrnp) to murine dorsal woundedge significantly inhibited the expression of hnrnp by % in epidermis compared to control (tlnκ/sicontrol)(n = ). moreover, mice treated with tlnκ/si-hnrnp showed impaired barrier function, with significant presence of macrophage in granulation tissue at day , suggesting impaired conversion of macrophage in the granulation tissue. summary/conclusion: this work provides a novel insight wherein exosomes of keratinocyte lineage are recognized as a major contributor that directs macrophage conversion in granulation tissue for wound healing. multifaced effects of milk-exosome (mi-exo) as modulator of scar-free wound healing gna ahn, hyo-won yoon, yang-hoon kim and ji-young ahn chungbuk national university, cheong-ju, republic of korea introduction: recently, milk exosome (mi-exo) has been focused particularly on the possibility of oral distribution for therapeutic agents. however, studies related to the cosmeceutical effects associated with mi-exo are fairly limited. the purpose of this study is to suggest the anti-oxidant and antiinflammatory effect of mi-exo and possibility that can be induced by scar free healing by micro rna in mi-exo. methods: the characteristics of the extracted mi-exo were verified by size measurement, morphological characteristics through cryo-em and western blot. for antioxidant experiments, an abts assay was performed. next, mrna expression through four major cytokines (tnfα, il- , cox- , inos) was used to evaluate anti-inflammatory effects. finally, cell migration assay was performed to confirm the effect of scar-free healing and the detection of mir- b in mi-exo and vegf mrna expression confirmed. results: mi-exo using % acetic acid extraction showed the highest yield. the average size of the exosomes is approximately nm, confirmed the typical double membrane vesicle. as a result of antioxidant experiments, it was confirmed that the treatment of exosomes of ^ particles showed about % antioxidant activity. when ^ particles were treated, rna expression of cytokines showed about times more inhibitory effect than control. elisa test results also confirmed that the concentration-dependent decrease. the activation of the raw cell less proceeded as the treated mi-exo increased. the cell scratch assay cells did not close the cells as the number of milk exosomes increased (wound closing % of ^ particle = . %). and mir- b in milk exosomes was detected at ct value = . summary/conclusion: the antioxidant and antiinflammatory effects of mi-exo showed the greatest efficacy when ^ particles were treated. in addition, it induced to scar free healing rather than wound healing. mi-exo has great potential as a superior natural material in the future cosmeceutical field. extracellular vesicles in human milk expose tissue factor and promote coagulation introduction: tissue factor (tf), a transmembrane protein, initiates coagulation by binding and activating coagulation factor vii (fvii). tf is associated with extracellular vesicles (evs) in saliva and urine, but it is unknown whether also human milk (hm) contains evs exposing coagulant tf. methods: hm was collected from six healthy nursing women with informed consent. evs were isolated by ultracentrifugation and size exclusion chromatography (sec). the presence of tf antigen exposing evs was studied by western blot, flow cytometry, cryo-electron microscopy (cryo-em), and surface plasmon resonance imaging (spri). the ability of tf exposing evs to trigger coagulation was investigated with a plasma fibrin generation test (fgt), performed in the absence or presence of antibodies against tf or fvii(a). results: addition of hm to plasma shortened the plasma clotting time, even when hm was highly diluted. after ultracentrifugation of hm, both tf antigen and tf activity were detected in the ev-containing pellet. after sec, tf antigen and tf activity were present in the ev-containing fractions and . the presence of tf-exposing evs in these sec fractions was confirmed by western blot (cd , cd and tf), flow cytometry, spri, and fgt. in addition, the presence of evs in hm was confirmed by cryo-em. scalable isolation of evs from different probiotic strains with potential as cosmetic ingredients laura soriano-romaní, joaquin espí and begoña ruiz ainia, paterna, spain introduction: extracellular vesicles (evs) are increasing their application in a number of fields. recently, it has been shown that skin health may be affected not only by commensal skin bacteria, but also by the evs that they secrete. however, because most of the efforts have been directed to the characterization and evaluation of evs, the scaling up of the production process remains a bottleneck at the industrial level. in this work, the goal was to evaluate the potential applications of evs produced by different probiotic strains commonly used in the cosmetic field, considering the economic and technical viability of the process. methods: to meet our goal, a standardized workflow was defined to isolate evs from probiotic strains such as lactobacillus and bifidobacterium species, that have demonstrated cutaneous immuno-regulatory effects. the different bacterial strains were produced under standard culture conditions. to isolate the secreted bacterial evs, different chromatographic techniques were performed starting from clarified growth medium. then, evs were evaluated in vitro for a number of biological effects related with skin health. results: the ev yields obtained after downstream processing were calculated for each strain and isolation technique by means of nanoparticle tracking analysis (nta) and total protein content. moreover, evs were visualized by electron microscopy. the in vitro evaluation of isolated evs was based on changes in the expression of five biomarkers related with anti-ageing, anti-inflammatory and whitening effects using distinct skin cell types to identify possible cosmetic claims that could be associated to each probiotic source. summary/conclusion: the potential of evs obtained from probiotic strains as cosmetic active cell-free ingredients was preliminarily assessed with this work, where the process yield and cosmetic function were evaluated. however, additional experiments will be needed in order to increase and optimize the productivity of each step of the ev manufacturing process. acerola derived exosome-like nanovesicles enhances the repair of ultraviolet b-induced dna damage in cultured skin fibroblasts tomohiro umezu, masakatsu takanashi, yoshiki murakami and masahiko kuroda tokyo medical university, shinjyuku, japan introduction: acerola (melpighia emarginata dc.) is a fruit is known to contain not only high amounts of ascorbic acid but also various nutritional components such as carotenoids and polyphenols. previous reports showed the acerola juices are able to confer protection against ultraviolet radiation b (uvb), to improve barrier function of skin. uvb is the main cause of dna damage in epidermal cells, generating several types of pro-mutagenic lesions, like cyclobutene prymidine dimers (cpds) and prymidine ( - ) prymidinone photoproducts ( - pps): if not repaired, this dna damage leads to skin cancer. in this study, we investigated the biological property of the acerola derived exosome-like nanovesicles (adens), aiming to clarify the involvement of adens in repair of uv-induced dna damage. methods: normal human dermal fibroblasts (nhdfs) were purchased from lonza inc. the exosome-like nanovesicles were isolated from acerola juices using exoeasy maxi kit (qiagen). the morphology and size distribution of adens were checked by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta, nanosight lm , malvern). nhdfs were exposed to uvb ( mj/cm ) with pre-or post-adens. effect of uvb was assessed by examining cell viability, cell morphology, and dna damage levels through biochemical assays, microscopy and protein expression studies. results: purified adens were compatible with nta or tem for assessing the nanovesicle size range and concentration ( - nm). when nhdfs were added with adens and incubated at °c for h, there was no effect of adens on cell proliferation of nhdfs. we found that adens treatment to uvb exposed nhdfs significantly reduced cpds and - pps dna adduct formation. present results showed that aden treatment prevented uvb induced dna damage in nhdfs. summary/conclusion: we confirm that adens have the effect of repairing dna damage caused by uvb. these results provide that adens can be a new source to protect human skin from uv-induced skin cancer. introduction: introduction: despite the development of a variety of therapies, complex wounds resulting from disease, surgical intervention, or trauma remain a major source of morbidity. extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) have been shown to improve wound healing, especially via enhanced wound angiogenesis. however, despite their clearly established potential, evs have limitations that may limit clinical relevancy, such as low potency. hypothesis: increased expression of pro-angiogenic lncrna hotair within msc evs enhances their proangiogenic effects and thus their wound healing properties. methods: methods: hotair was overexpressed in human dermal microvascular endothelial cells (hdmecs) to determine any molecular or functional pro-angiogenic effects. anti-angiogenic mirnas and angiogenic mrna levels were quantified by rt-qpcr. effects of hotair on proliferation of hdmecs was also determined. hotair was then loaded into msc evs by delivering a cmv-based hotair plasmid to mscs for endogenous loading via a concentration gradient. evs were collected by differential centrifugation. hotair content within evs was confirmed by gel electrophoresis and rt-qpcr. effects on migration of hdmecs by hotair-loaded msc evs were determined using a scratch assay. results: results: overexpression of hotair decreased mir- c and mir- , while increasing vegf and hif- a. hdmec proliferation was also increased in hdmecs overexpressing hotair (p < . ). hotair was visually confirmed in hotair-loaded msc evs by gel electrophoresis, but was undetectable in unmodified msc evs. rt-qpcr confirmed a -fold increase of hotair compared to control msc evs. hdmecs showed a more statistically significant rate of gap closure when treated with hotair-loaded evs (p < . ) than compared to control msc evs (p < . ). summary/conclusion: summary: loading lncrna hotair into msc evs is achievable by a concentration gradient-dependent method and offers potential to enhance the angiogenic properties of msc evs. nanomaterial labelling of exosomes for cell biology introduction: exosomes are vesicles secreted by many, if not all, cell types and have been known about for decades. among larger micro vesicles that are produced directly from the cell membrane, the small ( - nm), exosomes are similar in size to a virus surrounded by a lipid bilayer. we and others have demonstrated that exosomes contain proteins, lipids, rna, and dna, making them promising materials for diagnosing and treating diseases, including many cancers such as brain cancer. in addition, exosomes from neurons and glial cells represent a novel type of intercellular communication. however, their size makes them hard to track with traditional fluorescence microscopy. to address this, we developed photothermal microscopy (ptm), which uses gold nanomaterial labelling to track exosomes' interaction with and effect on cells/tissue. methods: exosomes secreted by tumour cells and general exosomes found in the blood were isolated using differential ultracentrifugation or a commercially available kit (invitrogen). next, the exosomes were characterized by (tem), (nta), and western blotting to determine shape, size, morphology and the protein profile in the exosomal membrane. after characterization, the exosomes were labelled with gold nanoparticles via sonication. next, the samples were washed, and the exosomes were labelled with fluorescence dye to stain the membrane. after staining and labelling, the exosomes were added to u cells in culture and incubated for h. they were then fixed by % paraformldehyde and imaged by ptm. results: ptm found that exosome-cell interactions are exosome-type dependent, as u cells took up exosomes from other u cells but not human serum exosomes. this suggests that exosome uptake is a selective process and depends on the source of the donor cells. summary/conclusion: exosomes can be labelled with gold nanoparticles via sonication then successfully tracked by ptm to study the effect of exosome source on exosome-cell interactions and communication. cells incubated with u exosomes took the vesicles up rapidly, while cells incubated with serum exosomes had little uptake. ptm will help us design selective exosome-based strategies to treat different conditions, including brain cancer and cns damage. funding: nsf epscor riii award . loading of goat´s whey extracellular vesicles with spiked microrna and curcumin as an strategy for developing new nanocarriers for acellular therapies introduction: extracellular vesicles (ev) are involved in cell signalling and are present in a variety of cell secretions such as milk, from which enormous amount of ev can be purified, thus milk is an attractive raw material for scaling up ev production for therapeutic, cosmetic or other uses. here we isolated evs from the whey fraction of goat´s milk and demonstrated that such evs can be loaded with molecules like polyphenols and mirna. methods: to achieve this, milk was collected from lactating goats and fractionated by acidification and centrifugation into whey and caseins. evs were purified from the former fraction by serial centrifugation and precipitation with commercial kit (total isolation/ thermo fisher) and characterized by electron transmission microscopy (tem), western blot to identify surface markers and measurement of size through nanotracking analysis. once isolated, evs were loaded with different concentration of a spiked synthetic mir or with the polyphenol curcumin. mirna or curcumin were co-incubated over night with evs at oc, precipitated and purified as described above, with an additional washing and precipitation for curcumin. concentration of mirna uploaded by evs was quantified using mir specific qpcr. curcumin was measured using a spectrophotometer at nm. results: evs isolated from whey had an average size of nm, were positive for hsp , cd and alix. in tem, evs were identified with their natural conformation and corresponding size to exosomes. qpcr showed a significant difference of expression of mir in relation to control (loaded with shame) and the negative control (p < . ). curcumin presence was also confirmed after washing and precipitacion. summary/conclusion: in conclusion, milk evs and exosomes can be loaded with mirna and a polyphenol and can be used as alternative nanocarrier for acellular therapies. introduction: extracellular vesicles (evs) are cellderived lipid membrane nanoparticles that serve as messengers of intercellular communication, transferring bioactive molecules to recipient cells. evs have a natural therapeutic potential with high flexibility and biosafety for employing natural and synthetic biomolecules as therapeutic delivery vehicles. considering the importance of evs, their isolation methods are still a bottleneck. to get insights into the tissue-specific cargo in vivo for complete exploitation of evs as therapeutic, biomarker and diagnostic tools, ev purification methods are critical. the aim of the study was brought about to develop an efficient ev purification method both in vitro and in vivo and to further investigate function of evs in cellular senescence. methods: to isolate tissue-specific evs in vivo we developed recombinant evs by genetically fusing snorkel-tag to the cd . the snorkel-tag enables on-column protease treatment for purifying evs which does not rely on traditional immunoaffinity purification protocols using low ph or high salts solutions. results: we systematically evaluated the purification of evs harbouring snorkel-tag by employing different methodologies. our findings suggest that evs harbouring snorkel-tag indeed can be purified at high purity without altering ev biophysical properties. furthermore, we expressed cd -snorkel-tag under p ink a promoter and were able to purify evs derived from senescent cells. summary/conclusion: finally, we are developing an in vivo model with cd -snorkel-tag under p ink a promoter. this will provide us detail insights into the ev cargo secreted from senescent derived cells, by purifying evs harbouring snorkel-tag under pathophysiological conditions, allowing us to develop biomarkers and therapeutic tools. summarized, we have here developed novel tool for studying content and function of evs in the context of ageing and disease. this tool will now pave the way for studying the molecular mechanisms underlying these ev functions in vivo. funding: this work was funded by the austrian science fund phd program biotopebiomolecular technolgy of proteins (w ). engineering exosomes with gata- jie xu, christian paul, yi-gang wang and meifeng xu university of cincinnati, cincinnati, usa introduction: exosomes, are small vesicles ( - nm) secreted from cells that can transport and deliver of their components such as lipids, proteins, dna, mrna, and mirna to target cells. gata- , a cardiac transcription factor, has been shown to regulate differentiation, proliferation, and survival of a wide range of cell types. delivering gata- protein into ischaemic tissues may be one of the most straightforward approaches to improve cardiac function following myocardial infarction. here, exosomes were engineered with gata- by infusing gata- with exosome targeting peptide. methods: the open reading frame of mouse gata- cdna was ligated to xpack lentivirus vector (xpack-gata- ) and plvx-ef -ires-pouro lentivirus vector (plvx-gata- ), respectively. hek cells were transduced by lentivirus, then exosomes were isolated from conditioned medium of hek cells using ultracentrifugation. exosomes were identified using transmission electronic microscope (tem), and the expression of gata- was semi-quantified using western blot. the internalization of exosomes was tracked via treating bend cells with exosomes pre-labelled with pkh . introduction: chinese hamster ovary (cho) cells have dominated as the mammalian cell host for the manufacture of humanized biologics, in part owing to their genomic plasticity and robust growth in suspension culture. there is great interest surrounding the use of extracellular vesicles (evs) as novel therapeutics owing to their capacity to deliver bioactive molecules. however, much remains unknown about the mechanisms involved in ev cargo loading, limiting their development as novel biologics. to this end, we have engineered cho cells to stably express constructs enabling loading of gfp into evs. methods: tetraspanins are established markers of ev identity. accordingly, cd was selected as a tethering point to generate evs with gfp cargo and constructs were generated via golden gate assembly. cho cells were stably transfected by electroporation and expression was verified with fluorescence microscopy and western blotting. growth in batch culture was monitored to establish maximum viable cell densities for ev harvest and recovered evs were characterized by nanoparticle tracking analysis (nta). finally, uptake of gfp-evs was studied using time-lapse fluorescence imaging in co-culture experiments. results: strong localization of cd -gfp was observed at the cell membrane and blotting confirmed intact tetraspanin fusion present at the expected molecular weight. additionally, cells were confirmed to retain high gfp expression post-cryopreservation. stable cell pools were able to reach viable densities greater than million cells/ml in batch culture and nta allowed for detection of gfp cargo even prior to ev isolation. evmediated transfer of functional gfp to recipient cells was found to occur over a period of hours. introduction: extracellular vesicles (evs) are considered promising for therapeutic applications. evs resemble the cell membrane, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. despite the promising potential of evs for therapeutic applications, robust manufacturing processes that would increase the scalability and consistency of ev production are still lacking. methods: in this work, evs were produced by mesenchymal stromal cells (msc), isolated from different human tissue sources (bone marrow, umbilical cord matrix and adipose tissue). msc were selected as these cells allow for a scalable production of evs, while displaying low immunogenicity. a vertical-wheel™ bioreactor system was implemented for the production of msc-derived evs and compared with traditional static systems. the obtained ev products were characterized by nanoparticle tracking analysis, atomic force microscopy, zeta potential and western blot. results: the bioreactor system allowed to obtain evs at higher concentration and productivity, as well as more homogeneous size distribution profiles, when compared to traditional static culture systems. functional studies were performed using breast cancer and lung cancer cell lines. proliferation assays allowed to determine the dose-response profiles of these cell lines when exposed to msc-derived evs. a bell-shaped profile was observed for most cases, since raising the ev concentration lead to increased cell proliferation until a certain point ( - µg/ml), after which cell proliferation was attenuated with increasing ev concentrations. summary/conclusion: the bioreactor culture system allowed a substantial improvement in the production of msc-derived evs, while the obtained dose-response profiles will be valuable to determine the most appropriate ev concentrations for anticancer drug delivery. overall, we demonstrate that this culture system is able to robustly manufacture human msc-derived evs in a scalable manner towards the development of novel therapeutic products such as anticancer drug delivery systems. biodistribution and cellular location of inhaled exosomes and liposomes in the lung introduction: increasing evidence reveals the potential role of extracellular vesicles, such as exosomes and liposomes, in lung regenerative medicine for the treatment of lung diseases. encapsulation and delivery of potential rna and microrna targets into liposomes and exosomes are attractive drug delivery methods, but remain difficult to deliver to the pulmonary parenchyma to reach target lung cell types. here, we demonstrate effective delivery and cellular uptake of exosomes and liposomes to the pulmonary parenchyma via inhalation treatment in a murine model of idiopathic pulmonary fibrosis. methods: human lung stem cells (lscs) were generated and expanded from healthy whole lung donors. lsc-exosomes were purified via ultrafiltration and diilabelled using vybrant☐ labelling solution according to the manufacturer's instructions. dsred-labelled liposomes were generated using lipofectamine™ rnaimax transfection reagent and block-it™ alexa fluor™ red fluorescent control according to the manufacturer's instructions. lsc-exosomes and liposomes were delivered via nebulization to cd mice with bleomycin-induced pulmonary fibrosis. exosome and liposome delivery and biodistribution were visualized -and -hours post-treatment through histological analysis. the study was approved by the institutional animal care and use committee of north carolina state university and complied with all national and state ethical standards. results: exosome and liposome delivery to the pulmonary parenchyma was confirmed by the presence of dii and dsred fluorescence in lung histological sections that penetrated the mucus-lined respiratory epithelium. more exosomes and liposomes surpass mucus-lined surfaces -hours post-treatment compared to -hours post-treatment. fluorescent colocalization of exosomes and liposomes with alveolar type i cells, alveolar type ii cells, basal lung cells, and cd + macrophages was observed through immunohistochemistry analysis. more exosomes and liposomes colocalize with these cell types -hours post-treatment compared to -hours post-treatment. summary/conclusion: lsc-exosomes and liposomes penetrate the mucus-lined respiratory epithelium and reach the pulmonary parenchyma through inhalation treatment. lsc-exosomes and liposomes are uptaken by alveolar epithelial cells, basal cells, and interstitial macrophages with improved biodistribution -hours post-treatment. funding: this study was supported by the nc state chancellor's innovation fund. transfection reagent artefact accounts for some reports of extracellular vesicle function codiak biosciences, cambridge, usa introduction: extracellular vesicle (ev) functions are frequently investigated by transiently transfecting cells with plasmid dna to produce evs modified with protein(s) or nucleic acid(s) of interest. however, evs and the dna-complexes used to transduce cells are physically similar, raising the possibility that they may co-purify during differential ultracentrifugation, the most common ev isolation procedure. activities attributed to evs may therefore be due to contaminating dna -transfection reagent complex. methods: ev producing cells were transiently transfected with plasmid dna encoding gene-editing or split enzymes fused to ev-targeting protein sequences. differential and density gradient ultracentrifugation were used to purify evs from cell culture supernatant or dna lipoplexes from cell-free culture media. protein expression and localization to evs was confirmed by western blot. cell lines stably expressing fluorescent or luminescent reporters were used to assess functional enzyme delivery in recipient reporter cells. results: reporter cells treated with ultracentrifuge pellet material (ucp) from media of transiently transfected cells showed robust and reproducible signal, however fractionating the ucp with an iodixanol density gradient revealed that reporter activity was associated with high-density fractions that were depleted in evs. ucp isolated from identical transfection conditions, but lacking cells (and exosomes), showed identical biological activity levels and distribution in iodixanol gradients, suggesting that the activity was due to contaminating transfection reagent complexes and not evs. serial media changes on ev producing cells post-transfection did not significantly reduce ucp activity on reporter cells. treatment with nucleases did not digest complexed dna, did not significantly reduce dna levels in the ucp as measured by qpcr, and did not decrease activity in reporter cells treated with ucp from either transfected cells or no-cell controls. summary/conclusion: we find that dna-transfection reagent complexes are not separated from evs using differential ultracentrifugation and that common approaches to remove such complexes, including media exchanges and nuclease treatment, are ineffective. due to the pernicious nature of the dna-complex in these cellular assays, it is likely that some reports of ev function are likely artefacts produced by contaminating dna-complexes. we find that density gradient centrifugation can effectively separate evs and dnacomplexes, highlighting the importance of validating elimination of contaminating transfection reagent complexes when using transient transfection to interrogate ev function. chair: suresh mathivanan -la trobe university cancer stem cell-derived exosomes: potential biomarkers for early diagnosis and prognosis in pancreatic cancer introduction: pancreatic cancer (paca) is the most deadly manlignancy, due to late daignosis and early metastatic spread, which prohibits surgery. it is urgently for relaible, early detection. research shows that tumour-derived exosomes, which had been present in the blood in the early stage of tumour formation and before metastasis, is the vanguard forces of tumour formation and metastasis; cancer stem cell-derived exosomes (csc-exos) has stronger migration ability, so the detection of blood csc-exos for early diagnosis and monitoring of progress for paca has great research potential and the value of application. methods: protein markers were selected according to expression in exosomes of paca cell line culture supernatants, but not healthy donors' serum-exosomes. according to these preselections, serum-exosomes were tested by flow cytometry for the pancreatic cancer stem cell marker cd v and tspan . results: the majority ( %) of patients with paca and patients with nonpa-malignancies reacted with anti-cd v and anti-tspan . serum-exosomes of healthy donors' and patients with non-malignant diseases were not reactive. recovery was tumour grading and staging independent including early stages. introduction: chronic traumatic encephalopathy (cte) is a tauopathy that affects individuals with a history of mild repetitive brain injury frequently seen in contact sports. initial neuropathologic change of cte include perivascular deposition of phosphorylated tau (p-tau) in cortical neurons and, in later stages, the formation of neurofibrillary tangles in neurons throughout the brain. extracellular vesicles (ev) are known to carry neuropathogenic molecules in neurodegenerative disease and able to cross the blood brain barrier. we therefore examined the protein composition of ev separated from cerebrospinal fluid (csf) and plasma in former national football league (nfl) players with cognitive dysfunction, and an agematched control group with no history of contact sports. methods: evs were separated from csf and plasma from former nfl players (n = , ) and controls (n = , ) by affinity separation method or size exclusion chromatography, respectively. the ev protein profiling was characterized by simoa for tau and ptau and mass spectrometry. the protein data was analysed for ev enrichment, differentially expressed proteins, pathway analysis and correlation with cognitive function, head impact and tau/p-tau levels by biostatistics and bioinformatics. results: the level of total tau and p-tau in csf evs was not significantly changed, but significantly elevated in plasma evs from former nfl players. the proteins were commonly identified between the paired plasma-csf from the same patients, but there was no significant correlation with disease status. collagen alpha- (vi) chain (col a ), − (vi) chain (col a ) and reelin (reln) were differentially expressed in former nfl players' plasma evs. a combination of these proteins in plasma ev can distinguish former nfl players from controls with % accuracy by machine learning. summary/conclusion: the interacting plasma-csf ev proteomes provide an original resource to ev biomarker development for neurodegenerative disease, and col a , reln and col a in plasma evs can be potential biomarker for monitoring the cte development. density-based fractionation of urine to unravel the proteome landscape of extracellular vesicles in prostate cancer introduction: current diagnostic tests are unable to discriminate indolent from aggressive prostate cancer (pca), leading to overdiagnosis and overtreatment, and an intense interest in biomarkers to improve clinical decision making. urine is considered an ideal proximal fluid for biomarker identification in pca due to its direct contact with the urogenital system. the discovery and translation of extracellular vesicle (ev) content into pca biomarkers remains challenging due to the difficulty of obtaining urinary ev (uev) with high specificity. methods: we developed a step-by-step protocol to separate uev by orthogonal implementation of ultrafiltration and bottom-up density gradient centrifugation (bu-odg). we implemented complementary particle and protein measurements to identify uev (lower density) and protein rich fractions (higher density) and assess the performance of bu-odg (specificity, efficiency and reproducibility). using mass spectrometry-based proteomics we interrogated uev and protein rich fractions from matched urine and radical prostatectomy tissue samples from pca patients (n = ), and urine from men with pca prior to (n = ) and after local treatment (n = ), benign prostatic hyperplasia (n = ) and other urological cancers (n = ). results: bu-odg separated uev from soluble proteins and tamm-horsfall protein (thp) complexes with high specificity and reproducibility, outperforming differential ultracentrifugation, exoquick and size-exclusion chromatography. comparison of the uev proteome from men with benign or malignant prostate disease, allowed us to expand the known human uev proteome and identify a pca specific uev proteome not uncovered by the analysis of the protein rich fraction. proteomic analysis of ev separated from prostate tumour interstitial fluid and matched uev confirmed pca specificity of the uev proteome. analysis of the uev proteome from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uev reflecting their respective cancer tissues of origin. summary/conclusion: we identified hundreds of previously undetected proteins in uev of pca patients and developed a powerful toolbox to map uev and protein rich fractions, ultimately supporting biomarker discovery for urological cancers. immunoglobulin a coating of faeces-derived bacterial vesicles as a marker of inflammatory bowel disease in humans nader kameli a , frank stassen b , heike becker c , john penders c , daisy jonkers d and paul savelkoul b introduction: iga is the most abundant antibody in mucosal secretions and plays a crucial role in maintaining the balance between the host and the gastrointestinal microbiome. recent studies suggested that pronounced iga coating is especially prominent among inflammatory commensals which drive intestinal disease. membrane vesicles (mvs, nano-sized particles released by bacteria) have also been found to interact with the host and modulate development and function of the immune system. however, their interaction with iga has not been studied yet. here we developed a method to isolate and characterize the mvs from faecal samples and checked for possible differences in iga coating patterns of mvs in health and disease. methods: mvs were isolated by using a combination of ultrafiltration and size exclusion chromatography from faecal samples of healthy controls (hc), patients with active crohn disease (cd) and cd patients in a remissive state. quantification and verification have been done with tunable resistive pulse sensing (trpsbased analysis) bead-based flow-cytometer (bbfc) and transmission electron microscope (tem). mvs were selected with specific antibodies for capturing (gram +: lta, gram-: ompa) followed by pe-conjugated anti-human iga antibodies as detection. results: we could successfully isolate * - * particles/ml from mg of faeces. bbfc in combination with trps provide a valuable method for (semi-)quantitative measurements of mixed populations. intriguingly, remarkable differences were found between iga coating mvs derived from healthy controls and active and remissive cd patients as mvs derived from healthy controls were significantly more coated compare to both cd patient groups. in details, for selected g-ve derived mvs: % of the total population of mvs derived from hc were coated, % from remissive cd patients, and < % of active cd patients; and for selected g+ ve derived mvs: % of the total population of mvs derived from hc were coated, % from remissive cd patients, and % of active cd patients. (data are represented as the mean). summary/conclusion: here we demonstrate for the first time that mv isolated from the faecal samples are also coated with iga, and surprisingly mvs from healthy volunteers were more densely coated than mvs from diseased patients. the possible consequence of this difference remains to be determined in future studies. monitoring altered tetraspanin and psma expression in prostate cancer derived extracellular vesicles via advanced image flow cytometry (isx) lukas w. prause a , christopher millan b , natalie hensky c , tullio sulser c and daniel eberli c a universityhospital zurich, zurich, switzerland; b university of zurich hospital, schlieren, switzerland; c university of zurich hospital, zurich, switzerland introduction: new diagnostic and therapeutic options for patients with prostate cancer are urgently needed. prostate-specific membrane antigen (psma)-based imaging and therapy are increasingly used for prostate cancer management. unfortunately, as a membrane protein, psma is not found as a soluble protein in the blood and therefore has limited utility as a diagnostic biomarker. however, psma has reportedly been observed as a cargo protein of prostate cancer-derived extracellular vesicles (evs). we demonstrate altered psma expression on evs derived from prostate cancer cell cultures (c - , lncap) in response to novel next-generation androgen receptor inhibitor (enzalutamide), a standard chemotherapy agent (docetaxel), a novel experimental nonsteroidal antiandrogen (epi- ) that binds covalently to the n-terminal domain of the androgen receptor and dihydrotestosterone (dht). additionally, evs were isolated from the plasma of prostate cancer patients who participated in the prococ biobank campaign at the usz. plasma was taken and stored from patients both pre-and post-prostatectomy. results: transmission electron microscopy, nanoparticle tracking analysis and simple western (wes) analysis show stable size distribution and amount of evs produced by treated and non-treated cells. using advanced image-based flow cytometry, altered tatraspanin and psma expression could be detected in evs isolated from cell culture supernatants of lncap and c - prostate cancer cells following their treatment. summary/conclusion: measuring psma expression on extracellular vesicles might pave the way to use image flow cytometry of evs to develop a blood based diagnostic test for prostate cancer patients with a wide range of possible applications including: ) monitoring response to therapy and, ) early indications of potential relapse. funding: vontobel fondation. proteomic profiling of human neural cells derived extracellular vesicles to identify human brain cell-type specific markers introduction: alzheimer's disease (ad) is a common neurodegenerative brain disease which affects appropriately million patients worldwide. one of the major challenges in ad is to develop reliable biomarkers for early diagnosis and disease-modifying therapies, especially before the clinical symptoms. extracellular vesicles (evs) carry cargos of proteins, lipids and nucleic acids. there was no comprehensive characterization of evs isolated from specific brain cell types, which may be useful for cell type-specific biomarkers. the purpose of this study is to isolate evs from human induced pluripotent stem cell (ipsc)derived brain cells for proteomic profiling and characterization of cell type-specific molecules. methods: human ipscs-derived neurons, microglia and primary cultured astrocytes were differentiated in ev-depleted media. the evs were isolated by differential centrifugation combined with size exclusion chromatography, followed by characterization using nanoparticle tracking analysis and mass spectrometry. the proteomic data were subjected to bioinformatics analysis results: we identified proteins from neuronderived ev (nde), proteins from microgliaderived ev (mde) and proteins from astrocytederived ev (ade) by proteomics. gene ontology analysis indicated that most of these proteins are associated with evs. furthermore, , and proteins are present individually in ndes, mdes and ades. among them, high levels of atp a and syt in ndes, itgam and cd a in mdes, and eaat and gfap in ades were found, all of which are typically and highly expressed in the original cells. summary/conclusion: our results provide us the potential candidates for cell-type specific ev markers, which will be helpful to develop non-invasive tools to enrich ev originating from specific brain cells and may lead to the development of new biomarkers for neurodegenerative disorders. ) are a tremendous resource for extracellular vesicle (ev) research, but they are heavily focussed on mammalian evs, i.e. evs from humans and laboratory animals, where protein cargoes are well characterised, and a wide selection of antibodies are commercially available. protein markers can be used to identify and define the types of mammalian ev and to determine the presence of any contaminants that might confound functional studies. similar resources are not as readily available for bacterial evs as these are not as well characterised, commercially available antibodies are much less abundant and immunological variation between different bacterial species (and there are trillion bacterial species on planet earth!) means that each species, strain, or group of related species may require different antibodies. methods: to identify quality markers for bacterial evs, we have characterised the proteome of cells, crude evs (ultracentrifuge pellet from cell free culture supernatant) and size exclusion chromatography or density gradient centrifugation purified evs from two different (pathogenic vs probiotic) strains of escherichia coli grown under two different environmental conditions, and one strain of mycobacterium marinum grown in one medium. results: our results identify a selection of proteins enriched in purified ev preparations, and proteins that are depleted after purification steps. summary/conclusion: our results allow the identification of potential markers for ev purity and non-ev contaminants, but also highlight the variability in bacterial ev preparations and suggest potential targets that can be used to investigate the heterogeneity of bacterial ev populations. introduction: recent findings indicate an increase in mid-life mortality rates in the usa and persistent, significant race-related health disparities exemplified by differential mortality rates. this suggests that exploring new molecular markers that may be linked to mortality could provide novel insights into factors that are driving mortality rates. accumulating data suggests that extracellular vesicles (evs) circulating in blood may be potential biomarkers of age-related disease. evs are nano-sized membranous vesicles that bear molecular cargo and mediate intercellular communication between different cells and tissues. little is known about whether ev characteristics differ by race or whether evs are associated with clinically relevant mortality risk factors. methods: in this cross-sectional study, plasma evs were isolated from middle-aged african american (aa) and white males and females. results: we report no significant differences in ev size or concentration with race or sex. there were significantly higher ev levels of phospho-p , total p , cleaved caspase , erk / and phospho-akt in whites compared to aas. higher ev levels of phospho-igf- r were found in females compared to males. we examined ev characteristics and protein cargo in the context of well-established clinical mortality risk factors. ev concentration was significantly, and positively, associated with several mortality markers including, high-sensitivity c-reactive protein (hscrp), homoeostatic model assessment of insulin resistance (homa-ir), alkaline phosphatase, pulse pressure, body mass index, and waist circumference. the relationship of ev concentration and cargo with mortality markers differs by race. summary/conclusion: our data show that ev-associated proteins can differ by race and sex and are associated with mortality risk factors. this study provides insight into the characterization of evs in middle-aged aas and whites, which may aid in the development of ev-based diagnostics. funding: this study was supported by the national institute on ageing intramural research program of the national institutes of health. repurposing specialised cell-free dna blood collection tubes for extracellular vesicle isolation introduction: liquid biopsies offer a minimally invasive approach to patient disease diagnosis and monitoring. however, many plasma processing protocols have been designed with a single biomarker in mind. here we investigate whether specialised dna blood stabiliser tubes could be repurposed for the analysis of extracellular vesicles (evs). methods: peripheral blood (n = ) was collected into k -edta, roche or streck cell-free dna (cfdna) blood collection tubes and processed using sequential centrifugation immediately or after storage for days. microev were collected from platelet poor plasma by , g centrifugation and nanoevs isolated using size exclusion chromatography. particle size and counts were assessed by nanoparticle tracking analysis, protein by bca assay and dot blotting for blood cell surface proteins. results: major variations in micro and nanoevs were seen with delayed time to processing. nanoev counts did not change with processing delay or tube collection type but the associated protein amount increased, indicative of cell lysis or activation. the protein was predominantly derived from from platelets (cd ) and red blood cells (cd a). the increase in associated protein was seen more in the k -edta and streck tubes indicating that the roche tubes may offer improved cell stability. conversely, microevs increased in both quantity and protein content with delay to processing indicative of both lysis and cell activation, irrespective of tube type. epithelial cell surface marker epcam abundance remained the same across conditions in both micro and nanoevs demonstrating that epcam+ evs were stable. summary/conclusion: specialised cfdna collection tubes can be repurposed for micro and nanoev analysis, however simple counting or using protein quantity as a surrogate of ev number may be confounded by pre-analytical processing. the evs would be suitable for disease selective ev subtype analysis if the molecular target of interest is not present in blood cells. introduction: nutrigenomics and nutrigenetics have been defined as the effect of nutrients on gene expression and genetic variation on dietary response, respectively. here, we propose the isolation and characterization of exosomes from donors carrying different alleles of hla-dqa and hla-dqb , to investigate their involvement in coeliac disease (cd) management. methods: a chilean population (n = ) was investigated for snps mutations in hla class ii alleles associated to cd predisposition (as well as other mutations related to other food intolerances), using the genochip food technology. exosomes have been isolated from donors' serum by ultracentrifugation and characterized by sds-page, western blotting (cd and cd ), and transmission electron microscopy. exosomes were also studied for their interleukins (il- and il- ra) content. results: among the studied population, % present at least one of the alleles leading to cd development and % carry alleles encoding for αand β-chains heterodimers associated with very high risk to develop cd. in parallel, isolated exosomes from donors with low to extremely high risk for cd showed high il- ra content ( . ± . to . ± . ), as the persons were not following any treatment. however, values of il- ra decrease in exosomes isolated form persons receiving treatment for cd. a relationship between exosomes' content and genetic susceptibility for cd has been observed, which may suggest their possible use as biomarkers for cd as the diagnostic of this disease is still a big issue. summary/conclusion: until this point of this underway project, we demonstrate the existence of a relationship between the exosomes' content in il- ra and genetic susceptibility for cd. furthermore, the genetic predisposition to cd could also modulate the gut colonization process, another important player in intestinal homoeostasis. in the next step, extracellular vesicles from gut microbiota will be isolated and analysed to determine their role in cd management. nasibeh karimi a , razieh dalir fardouei a , jan lötvall a and cecilia lässer b a krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, göteborg, sweden; b krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, gothenburg, sweden introduction: the ability to isolate extracellular vesicles (evs) from blood is vital in the development of evs as disease biomarkers. both serum and plasma can be used but few studies have compared them in terms of amount and type of evs. we have previously developed a method to isolate evs from plasma with minimal contamination of lipoprotein particles (karimi et al ) . the aim of this study was to compare the presence of different subpopulations of evs in plasma and serum. methods: blood was collected from healthy subjects, from which plasma and serum were isolated. evs were isolated using a combination of density cushion and size exclusion chromatography (sec) (protocol ) or a combination of density cushion and density gradient (protocol ) or immune-capturing (anti-cd , anti-cd and anti-cd beads) (protocol ). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, electron microscopy (em), exoview, flow cytometry and mass spectrometry (lc-ms/ms). results: as determined by nta and protein measurement more evs could be isolated from plasma with protocol and the majority of the vesicles were cd / cd a positive as determined with exoview and western blot. additionally, flow cytometry and western blot showed that more cd /cd a positive evs where also identified with protocol . furthermore, western blot showed increased amount of cd a in plasma samples in protocol . when labelled evs were spiked in freshly collected blood, no difference in recovery was seen for plasma and serum. summary/conclusion: this study shows that a larger amount of evs could be isolated from plasma compared to serum when three different isolation methods were used. firstly, this suggests that more evs are present in plasma. secondly, it suggests that these vesicles are probably released by platelets and that evs are not trapped in the clot during serum formation. future studies are needed to answer how this affects the use of blood-derived evs as biomarkers from serum and plasma. tumour-derived extracellular vesicles contain distinct integrin proteins stephanie n. hurwitz a and david g. meckes b a university of pennsylvania, philadelphia, usa; b florida state university, tallahassee, usa introduction: cargo profiling, including proteomic analyses, of tumour cell-derived extracellular vesicles (evs) may provide ripe opportunities for further understanding cancer growth, drug resistance, and metastatic behaviour. accumulating data suggest that cancer-derived evs contain membrane-bound integrin proteins which may aid in cell detachment, migration, and homing to future metastatic niches. we have previously published an extensive proteomic profile of secreted vesicles from the nci- panel of human cancer cells. methods: here, we further examine the distinct integrin components in these cancer-derived evs, and additionally profile evs released from benign epithelial cells by liquid chromatography and tandem mass spectrometry for comparison. results: we demonstrate the enrichment of integrin receptors in cancer evs compared to vesicles secreted from benign epithelial cells. total ev integrin levels, including the quantity of integrins α , αv, and β correlate with tumour stage across a variety of epithelial cancer cells. in particular, integrin α also largely reflects breast and ovarian progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of certain primary tumours. other integrins including α , αl, and β are enriched in vesicles derived from leukaemia cells, and may provide a means to distinguish haematopoietic cell-derived evs. summary/conclusion: this study provides preliminary evidence of the value of vesicle-associated integrin proteins in detecting the presence of cancer cells and prediction of tumour stage. differential expression and selective packaging of integrins into evs may contribute to further understanding the development and progression of tumour growth and metastasis across a variety of cancer types. effect of nicotine and menthol on cytochrome p and antioxidant enzymes in rat plasma-derived extracellular vesicles introduction: tobacco products such as e-cigarettes pose potential adverse health effects caused by direct exposure to aerosolized nicotine, flavorants such as menthol, and other particulates. here, we aimed to study the hypothesis that whether nicotine and menthol modulate nicotine-metabolizing cytochrome p a (cyp a ), antioxidant enzymes (aoes), sod and catalase in plasma extracellular vesicles (evs). modulation of these enzymes would eventually lead to nicotine-induced toxicity and hiv- pathogenesis via evs-based cell-cell interactions. methods: we isolated and characterized evs from rat plasma before and after nicotine self-administration (nic) with audiovisual cue (av) and menthol and characterized using ev markers according to the isev guidelines. protein associated with cyp a , sod , and catalase were quantified by western blot. results: we measured size, total protein, and acetylcholine esterase activity of evs and found no significance difference in these characteristics before and after nic. to investigate the effect av, menthol alone or in combination in the absence and presence of nic, first we evaluated the expression of ev markers cd and cd . the results showed menthol and av together increased the levels of cd (p ≤ . ), the marker of small vesicles, in the presence of nic. the nic with menthol and av showed a pattern of increased levels of small vesicle but could not reach to significance. next, we demonstrated that the nic with av increased the level of sod (p ≤ . ), which showed a pattern of increased levels of catalase and cypa , though statistically non-significant. the expression of nicotine receptor did not change under any conditions used. the results showed an increased level of cyp a (p ≤ . ), sod (p ≤ . ), and catalase (p ≤ . ) in plasma evs in the menthol-nic group compared to menthol group only. nic group with a combined av and menthol, showed further increase in the levels of cyp a (p ≤ . ), and catalase (p ≤ . ). further analysis of plasma evs on inflammatory cytokines/chemokines in these groups, and the effect of plasma evs on nicotine-induced toxicity and hiv pathogenesis are underway. summary/conclusion: nicotine administration increased, though not statistically significant, the levels of circulatory evs. moreover, the study provided evidence that nicotine in the presence of menthol, av, and/or menthol+av increased nicotine-metabolizing cyp a in all the groups and aoes in specific groups. funding: we thank the national institute on drug abuse (grant #da , da- ) for supporting our work. introduction: biomarker discovery in breast cancer (bc) is a clinical need for therapeutics and non-invasive diagnostics. tumour exosomes are involved in premetastatic niche formation and drug resistance and represent a source of non-invasive biomarkers. the identification of tumour exosomal biomarkers provides, not only, the possibility to discriminate patient groups also potential targets to control cancer progression that could be exploited to develop innovate bc therapeutic strategies. methods: we have performed a comparative differenti. al proteomic profile of four bc cell lines and their derived-exosomes, representative of the most relevant bc subtypes in clinic to search non-invasive biomarker candidates. then, we have carried on two bioinformatics approaches: ) protein association network analysis interaction (string) and ) pathway inference analysis (hipathia), to characterize the functional profiling for each bc subtype. results: we have found differentially-expressed proteins, in both cells and exosomes, that include indicators of invasion, metastasis, angiogenesis and drug resistance. exosome proteome profile reflects their different bc cell origin suggesting potential indicators of bc subtype. further, bioinformatics analysis reveals a differential role of exosomes in bc signalling pathways in recipient cells, according to their protein cargo and cell origin. summary/conclusion: our results show a set of cells and exosome proteins that highly discriminate bc subtypes and may significantly contribute to further studies for the design of bc biomarker predictor to stratify bc patients and the development of novel therapeutic strategies. funding: a set of potential biomarkers to discriminate breast cancer subtypes. circulatory evs as potential biomarkers of hiv-drug abuse interactions and neurological dysfunction in hiv-infected subjects and alcohol/ tobacco users sunitha kodidela a , kelli gerth a , namita sinha a , asit kumar b , prashant kumar a and santosh kumar a a uthsc, memphis, usa; b university of tennessee health science center, memphis, usa introduction: abuse of alcohol and tobacco can exacerbate hiv pathogenesis and its associated complications. further, the diagnosis of neurocognitive disorders associated with hiv infection and drug abuse using csf or neuroimaging are invasive or expensive methods, respectively. therefore, extracellular vesicles (evs) can serve as reliable non-invasive markers due to their bidirectional transport of cargo from the brain to the systemic circulation. hence, we aimed to study the specific evs proteins, which are altered in both hiv and drug abusers to identify a physiological marker to indicate the immune status and neuronal dysfunction of hiv-positive drug abusers. methods: evs were isolated from plasma of the following subjects: a) healthy b) hiv c) alcohol drinkers d) cigarette smokers e) hiv+alcohol drinkers f) hiv +cigarette smokers. quantitative proteomic profiling of evs was performed by mass spectrometry and potential ev proteins associated with neuronal dysfunction were quantified by westernblot. results: the evs were characterized according to the isev guidelines. a total of proteins were detected in evs of all the study groups. comparison of proteins among all the study groups revealed that hemopexin was significantly altered in hiv+drinkers compared to drinkers and hiv subjects. further, our study is the first to show properdin expression in plasma evs, which was decreased in hiv+smokers and hiv+drinkers compared to hiv patients. though we couldn't identify the few other cns-specific proteins, g-fap and l -cam, associated with neuronal dysfunction in plasma evs by mass spectrometry, we could detect those by westernblot. the protein expression of gfap (p < . ) was significantly enhanced in plasma evs obtained from hiv-positive subjects and drinkers compared to healthy subjects, suggesting enhanced activation of astrocytes in those subjects. the l cam expression was found to be significantly elevated in smokers (p < . ). both gfap and l cam levels were not further elevated in hiv+smokers compared to hiv+nonsubstance users. summary/conclusion: the present findings suggest that hemopexin, and properdin show potential as markers for hiv-drug abuse interactions. further, astrocytic and neuronal-specific markers (gfap and l cam) can be packaged in evs and circulate in plasma, which is further elevated in the presence of hiv infection, alcohol, and/or tobacco and thus may represent as potential biomarkers for neurological dysfunction in those subjects. funding: we thank the national institute on drug abuse (da ) for supporting our work. . electrochemical detection of mirna- - p introduction: micrornas (mirnas) are small, single-stranded, non-coding rna species that regulate gene expression post-transcriptionally, and are transported by extracellular vesicles (evs). they play an essential role in biological processes, such as development, cell proliferation, apoptosis, stress response and tumorigenesis. thus, mirnas are considered relevant biomarkers in health. more particularly, mirna- - p is expressed in neurons after traumatic brain injury, being expectably transported to peripheral fluids by brain evs that cross the blood-brain barrier. the main goal of this work is to develop an electrochemical biosensor for the detection of mirna- - p in serum. methods: overall, the experimental assembly of the biosensor was made in three stages. the first one consisted in the electrodeposition of aunps, the second one in the incubation of anti-mirna - p on the carbon screen-s printed electrodes and the final stage in the incubation of mercaptosuccinic acid for blocking unspecific bindings. the probe was hybridized with the target mirna - p by a consecutive incubation of several standard solutions. each modification was evaluated with cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis) and square wave voltammetry (swv). the electrochemical behaviour of the biosensor was followed in all steps by monitoring the electron transfer features of a standard redox system. the redox probe selected for this purpose was [fe (cn) ] -/[fe(cn) ] -. results: the results indicated that the electrodeposition of gold was more effective for − . v for s and could lead to better signals upon anti-mirna- - p hybridization. summary/conclusion: in general, the experiments showed increasing charged transfer resistance upon the incubation of higher concentrations of mirna- - p. in these experiments, ev concentration is a critical variable that must be carefully controlled to ensure scientific rigour and reproducibility: without controlling for concentration (dose), experimental outcomes will exhibit excess variability that could mask important biological discoveries. in this study, three orthogonal methods are compared for accuracy in ev quantification: microfluidic resistive pulse sensing (mrps) and nanoparticle tracking analysis (nta) were compared to each other and relative to the gold standard method, transmission electron microscopy (tem). the ability of nta to accurately measure particle concentration is shown to depend on the polydispersity of the sample itself. results validate the accuracy of mrps and emphasize the importance of using orthogonal techniques to quantify evs. methods: reference urinary vesicles were prepared and analysed with the three methods and the relative concentration accuracy of nta and mrps were compared as a function of particle size. the hypothesis that nta concentration accuracy was impeded by sample polydispersity was tested using polystyrene bead mixtures having a range of polydispersity. a theoretical argument based on fundamental physics explains the experimental observations. results: tem and mrps measurements of the evs were in excellent agreement and showed a broad, polydisperse particle size distribution with no peak on the measured size range ( nm - nm diameter). nta differed significantly from tem and mrps by reporting a steep decrease in measured concentration below about nm that resulted in a peak in the reported particle size distribution. bead measurements confirmed the hypothesis to be tested: sample polydispersity significantly affects the ability of the nta method to accurately measure concentration, even for particles as large as nm diameter. summary/conclusion: these experiments validate mrps as an accurate method for quantifying evs and highlight the importance of using orthogonal measurement methods in accordance with misev guidelines. clinically relevant synthetic reference materials to standardize concentration measurements of extracellular vesicles: state-of-the-art and future prospects introduction: there is an unmet need to standardize concentration measurements of extracellular vesicles (evs). flow cytometry is the clinically most applicable method, but the currently available reference materials for calibration are insufficient. for example, the refractive index (ri) between standard particles and evs substantially differs, whereas concentration and fluorescence calibration particles are too bright. the goal of this study is to ascertain the most desired properties of reference materials to standardise ev measurements. methods: an online survey was prepared within the meves ii project to measure the desired size, concentration range, optical properties, choice of fluorochromes, and stability of synthetic ev reference materials for flow cytometry (fcm) measurements. besides the desired properties of ev reference particles, also the available instrumentation was assessed in the survey, which was sent to the members of the stakeholder committee of metves ii project and members of the ev flow cytometry working group. results: the most desired size, concentration, and ri range for ev reference particles is nm to nm, e to e /ml, and . - . , respectively. based on mie-theory evaluation of the sensitivity of the available instruments, none of the respondents would be able to detect nm particles with ri = . with their current instruments. regarding fluorescence intensity, the most desired range according to the responses is from molecules of equivalent soluble fluorochromes (mesf) to mesf. considering the sizes of evs and fluorescent labels, the maximal mesf that can be obtained for ev reference particles with nm diameter and high molecular mass fluorescent dyes is in the range of several hundreds. typical antigen densities on evs fall below copies per ev with nm diameter, i.e. mesf values above are probably not physiologically relevant in this size range. summary/conclusion: a part of the desired properties of ev reference materials precludes either their physical feasibility of production or their detection at most currently available fcms, meaning that the intended reference materials will be future-proofed. funding: this work was supported under hlt metves ii project by the european metrology programme for innovation and research (empir). the empir initiative is co-funded by the european union's horizon research and innovation programme and the empir participating states. comparison of production and activity of amniotic fluid stem cell extracellular vesicles from d hollow fibre bioreactor and d culture. culture conditions may affect ev composition and potency. here we compare production, potency, identity and therapeutic potential of evs collected from cells grown in culture dish ( d) versus hfbr ( d). methods: human clonal afsc were derived from patient-consented amniotic fluids. x e hafsc were seeded in d ( cm ), and . x e hafsc on a small kd mwco hfbr (fibercell-c d, cm ) with fibronectin coating; both cultured in chang medium with % of es-fbs, starved for hr and then evs collected. the effect of harvest frequency was tested ( hrs, hr, hrs, wk). d-evs and d-evs were compared by nanosight, potency assay (by wb), identity (by exoview analysis) and therapeutic effect (in vivo in an animal model of kidney disease, alport syndrome). results: d production was~ . x e ev/ml/ hrs while d was~ . x e ev/ml (first four hrs) and . x e ev/ml (two days of hourly harvests). very little difference in ev concentration and very similar size distribution (~ nm) were observed during harvest intervals; possibly indicating either significant ev re-uptake or inhibition of ev secretion dependent upon free ev in the supernatant. d-evs trapped vegf (an in vitro established potency assay) as efficiently as d-evs, and expressed cd , cd , cd , cd , cd and vegfr as d-evs. summary/conclusion: d-evs had comparable properties and bio-activity to d-evs, but the hfbr produced x more evs. hfbr cell culture conditions for hafsc still need optimization, however an available . m cartridge provides a x scale up potential. the hfbr, a cgmp closed system, can produce sufficient numbers of ev to support pre-clinical and clinical applications with at least similar properties to evs produced by conventional d methods. funding: -intramural funding -intramural ev core pilot funding demonstration of high gain mode in combination with imaging flow cytometry for improved ev analysis luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. in recent years, the importance of evs has become apparent, as they are key mediators of intercellular communication. however, quantifying and characterizing evs in a reproducible and reliable manner is challenging due to their small sizeexosomes range from to nm in diameter. it is well-known that flow cytometers were originally designed to measure and detect cells, and due to the quantitative power flow cytometry offers, there has been a push to quantify and characterize evs using flow cytometric methods. however, these systems have not been designed to measure objects smaller than a cell. methods: here, we describe the use of high gain mode on the amnis® imagestream® imaging flow cytometer to address the challenges of measuring small particles. in this new high gain mode, the charge-coupled device (ccd)-camera is manually adjusted to higher gain settings, increasing the signal obtained from the ev. object thresholds and masking have also been adjusted to better identify and detect small particles. results: preliminary results using murine leukaemia virus-sfgfp reference particles have shown up to a fivefold increase in the number of gfp-positive objects collected in high gain mode, when compared to standard gain on the imagestream system. summary/conclusion: in this study, we demonstrate improved small particle detection, including evs, using this new high gain mode on the imagestream imaging flow cytometer. distance-controlled accelerated catalysed hairpin dna circuit for multiple and sensitive detection of exosomes-associated mirnas introduction: sensitive and simultaneous monitoring of multiplexed exosome-associated rnas is of great value for early cancer diagnosis remains a challenge. methods: here, we report a simple, multiple and sensitive exosomes-associated multiplex mirnas detection method that uses distance-controlled accelerated catalysed hairpin dna circuit (chdc) system without any complex operation or enzymatic amplification. the distance-controlled accelerated chdc can directly enter the plasma exosomes to generate fluorescent signal quantitatively by specifically targeting mirnas without any transfection means. results: we show that distance-controlled accelerated chdc strategy with signal amplification capability could selectively and sensitively identify low level rnas in serum evs, distinguishing patients with early-and late-stage breast cancer from healthy donors and patients with benign breast disease. summary/conclusion: this simple, accurate, sensitive, and cost-effective liquid biopsy by the distance-controlled accelerated chdc method is potent to be developed as a non-invasive breast cancer diagnostic assay for clinical applications. impact of isolation methods on biophysical heterogeneity of single extracellular vesicles university of california los angeles, ca, los angeles, usa introduction: current biophysical analysis of extracellular vesicles (evs) typically encompasses particle density and size distribution determinations using various techniques. however, variabilities in ev isolation methods and the structural complexity of these biological-nanoparticles (sub- nm) necessitate more rigorous nanoscale biophysical characterization of single evs to facilitate more reliable and comparable evbased assays. methods: combining atomic force microscopy (afm), super-resolution optical and conventional particle sizing light scatter and microfluidic techniques, we compared the unique sub-nanometre scale biophysical properties of breast cancer cell-derived ev isolates obtained using different isolation methods. results: afm and dstorm particle size distributions showed coherent unimodal and bimodal particle size populations in centrifugation and immune-affinity isolates respectively. more importantly, afm imaging revealed striking differences in nanoscale morphology, surface undulations, and vesicle-to-non-vesicle ratios among ev isolates from different isolation methods. our findings demonstrate the effectiveness of orthogonal high-resolution biophysical characteristics of single evs, not discernable via particle size distributions and counts alone. summary/conclusion: the identified nanoscale biophysical characteristics of ev isolates represent a strategic and complementary framework to resolve differences in the heterogeneity and purity of evs from introduction: extracellular vesicle (ev) concentrations measured by flow cytometry are incomparable. to improve comparability, the metves ii consortium is developing traceable reference materials and procedures, which require validation by test samples. in previous interlaboratory comparison studies, however, a main source of variation was introduced by pre-analytical variables and measurement artefacts introduced by test samples. to minimize variation introduced by test samples, our aim is to develop off-the-shelf biological test samples containing pre-labelled evs. methods: human urine and plasma were collected from healthy donors. evs were labelled with lactadherin-fitc, isolated by size-exclusion chromatography to remove free dye and minimize swarm detection, and mixed with dimethyl sulphoxide (dmso), exocap or trehalose, frozen in liquid nitrogen and stored at − °c . after thawing, ev concentrations were measured by a calibrated flow cytometer (apogee a -micro). results: compared to the ev concentrations measured in fresh plasma and urine, the concentrations decreased % in plasma (p = . ; mean of the cryopreservation agents) and % in urine (p = . ) after one day of storage. after months of cryopreservation, the concentration of plasma evs decreased % (dmso and exocap) and . % (trehalose) compared to one day of storage, whereas the concentration of urine evs decreased % (exocap) and % (dmso and trehalose). summary/conclusion: we have developed ready-touse, pre-labelled human evs that are stable up to months and dedicated for use in interlaboratory comparison studies. to further increase stability, other cryopreservation agents will be tested. our biological test samples will be key to validate the new reference materials and procedures developed by metves ii in . funding: this project has received funding from the empir program co-financed by the participating states and from the european union's horizon research and innovation program. understanding intracellular fate of ev-delivered content introduction: despite much work performed on evaluating the potential effects of extracellular vesicles (evs), the functional uptake of their cargo is still controversial. this project aimed to demonstrate that ev content (protein and mrna) is protected and can be subsequently transferred with functional activity into recipient cells, while also developing a tool to assess and quantify functional ev uptake. methods: fusion proteins used were mitochondrial localized coxviii-cfp-nanoluc(cox) and nuclear localized h b-rfp-nanoluc(h b). results: hek t cell-derived evs protected cox proteins from proteinase k digestion while demonstrating significantly improved efficiency of uptake when compared to free protein, as measured by bioluminescence that was still detectable in recipient cells hrs post-ev-exposure. to confirm functional uptake, recipient cells exposed to evs containing h b for hrs were imaged and some recipient cells manifested fluorescent red nuclei. to demonstrate the presence of functional mrna within evs, producer cells were transfected for such a duration as not to have detectable levels of protein in the evs while still containing detectable levels of mrna (qpcr) even after rnasea treatment. transfer of these evs to hela cells showed an increase in expression of h b which was blocked by cyclohexamide, confirming translation of the mrna ( . kb). to determine if recycling of ev delivered proteins occurs, recipient hela cells were exposed to evs containing cox for hrs. all extracellular evs were removed and cells were trypsinized ( . % for min) to remove any non-internalized cox protein. hrs later, evs (cd + and cd +) released from cells contained cox suggesting recycling of protein or possibly recycling of entire evs. lastly, an assay was developed to measure functional ev uptake. nanoluc protein was split in two and fused to mturquoise (n ) or mscarlet-i( c). expression of each fragment alone exhibited non-detectable levels of luminescence while expressing both together had a significantly increased signal. delivery of either fragment within an ev to a cell expressing the corresponding fragment worked as confirmation and quantification of ev uptake (hek , u , hela cells). summary/conclusion: this study robustly demonstrates ev delivery of functional mrna and protein to cells, while also establishing a simple assay to quantify and validate functional ev uptake. theoretical model of ev losses due to adsorption on the tube walls. application for immunomagnetic detection of the vesicles introduction: short-term storage of unfrozen samples of vesicles, mainly at °c, overnight or during a couple of days is rather common laboratory practice. however, it was found to lead to significant losses of vesicle concentration supposedly due to adsorption on the walls of the tube. the present work develops a theoretical model intended to describe the vesicle adsorption process. the experimental validation of the model was made using method of immunomagnetic precipitation. methods: the theoretical model considers the "diffusion-limited" case of vesicles storage. the maximal adsorption capacity of the surface of contact between the tube and the solution is given as the number of vesicles in hexagonally packed monolayer. for experiment, the vesicles were purified from ht cell culture supernatant by differential centrifugation, aliquoted and kept at − c. further the aliquots were consequently unfrozen, and placed into the tubes with different surface treatment and kept at + c. the kinetics of vesicles loss was measured by anti cd immunomagnetic capturing followed by cd , epcam and cd staining and flow cytometry. results: the model allows the estimation of the adsorption-associated losses as dependent on initial vesicles concentration, volume of the solution, tube geometry, the storage temperature and duration case of quiet vesicles storage (without mixing) and also accounts an expected effect of active agitation of the solution (ev-beads complexes formation). theoretical calculations were illustrated by analysis of ev at different storage conditions and during reaction of immunomagnetic precipitation of the vesicles. summary/conclusion: it was demonstrated that application of tubes surface treatment allows increasing sensitivity of immunomagnetic precipitation method to x ^ for cd +, x ^ for epcam+ and x ^ for cd + vesicles. introduction: it is now largely accepted that the intestinal microbiota plays a key role in intestinal bowel diseases (ibd). an imbalance in the composition and diversity of the intestinal microbiota (i.e. dysbiosis) of patients has been repeatedly pointed out by several teams. there are also indications that extracellular vesicles produced by bacteria and exosomes produced by epithelial cells might be increased in this family of diseases. methods: in order to differentiate healthy and ibd faecal samples on the basis of their vesicle profiles, we want to develop a means to enumerate rapidly particles in faecal samples, based on interferometric microscopy. the videodrop technology, developed by myriade, relies on the creation of single beam interferences between two signals from the same light path by nanoparticles such as small vesicles. it will permit to compare on large scales the viral load of healthy subjects and ibd patients. results: this fast and easy-to-use device was compared to the nta on several types of eukaryotic and prokaryotic vesicles and our preliminary results are encouraging. introduction: small extracellular vesicles (sevs) produced by mesenchymal stromal cells (msc-sevs) may be useful in cell-free therapies for immunomodulation and tissue regeneration. methods: to characterize msc-sevs produced ex vivo, human bone marrow mscs were cultured in mesencult-acf plus (macfp), an ev-free and animal component-free culture medium for days and spent medium collected to isolate sevs by ultracentrifugation (uc). analyses of sevs were performed by nanoparticle-tracking analysis (nta), western blot (wb), and human umbilical vein endothelial cell (huvec) tube formation assay. results: analysis of fresh uncultured macfp by uc, nta and wb for cd , cd , and cd confirmed the absence of sevs. msc-sevs isolated from spent macfp by uc ranged from - nm in size and were positive for cd , cd , and cd proteins. these sevs could be stored at − °c for > months in solution or lyophilized with minimal loss based on nta and wb analysis. the msc-sevs contained the msc-associated micrornas let a, mir , and mir a as per qpcr analysis. the biological function of ex vivo isolated msc-sevs was assessed using a human umbilical vein endothelial cell (huvec) tube formation assay. huvecs treated with msc-sevs generated tubes as early as h after seeding, which were not observed in control huvec cultures until h. moreover, the number of branch points present in such tube structures was >fourfold higher in huvec cultures (n = ) supplemented with msc-sevs versus control, with the former lasting > h and the latter lasting < h in culture. direct comparison of the performance of macfp medium to media containing non-depleted or ev-depleted foetal bovine serum demonstrated that only mscs cultured in macfp (n = ) were able to expand robustly with a doubling time of . , . and . days in these media, respectively. lastly, methods for isolating sevs using newly developed easysep-ev™ magnetic separation kits and size exclusion columns will be presented. summary/conclusion: taken together, these data demonstrate that msc-sevs can be produced in high yield in macfp medium and that these possess similar physical, phenotypic and functional characteristics as sevs in vivo. funding: this work was privately funded by stemcell technologies inc. introduction: extracellular vesicles (evs) are heterogeneous group of small vesicular structures released by different types of cells, including stem cells (scs). as recent studies demonstrate that they may enclose bioactive content and transfer it into the target cells, growing interest is placed on the utilization of evs in the field of biomedical research. however, there is still lack of standardized methods of evs characterization. as an example, typical flow cytometry-based protocols, commonly used for cells phenotyping, may be inadequate for the characterization of evs as particles with size close to the detection limit of conventional cytometers. thus, the aim of this study was to optimize and compare the use of different flow cytometry platforms for the multiparameter analysis of evs isolated from different types of scs populations. methods: ev samples were obtained by ultracentrifugation of conditioned media collected from selected scs types, including human induced pluripotent scs (ips) and mesenchymal scs (mscs). next, several high resolution flow cytometry systems: cytoflex, apogee (a and a micro-plus) and image stream mk ii were employed to compare their sensitivity and resolution, as well as influence of "swarm" effect. furthermore, we examined evs phenotype, including expression of tetraspanins and other surface markers. results: our results have revealed that tested flow cytometry systems may be utilized for the phenotypic characterization of evs secreted by scs populations. however, the conventional staining and gating strategy protocols have to be thoroughly optimized. additionally, depending on a type of tested cytometer, we have demonstrated the difference in a "swarm" effect and its influence on obtained results regarding evs phenotype. finally, imaging flow cytometry platform was also employed to visualize evs on the single particle level. summary/conclusion: in conclusion, we have demonstrated that tested high-resolution flow cytometry platforms are convenient methods for the multiparameter characterization of evs produced by different types of scs populations. however, careful selection of particular measurement parameters should be performed depending on a type of employed system. funding: this study was funded by ncbr grant strategmed iii (strategmed / / / ncbr/ ) to ezs. evaluation of atcc's exosomes from cell culture supernatant as reference standards in research and development. introduction: exosomes are subcellular particles - nm in size released from cells through a fusion of multicellular bodies with the plasma membrane. exosomes are stable carriers of cell-free cargo in the form of dna, rna, and proteins, thereby making them an attractive candidate for diagnostic and therapeutic applications. however, isolating a consistent population of exosomes can be challenging and there is an unmet need for highly characterized exosomes for use as reference standards in extracellular vesicle research (ev). methods: exosomes were isolated from cell culture supernatants of different atcc cell lines including stem cells and cancer cell lines representing the most prevalent cancer types -prostate, colorectal, breast, lung, cervical and glioblastoma, using tangential flow filtration (tff). these exosomes underwent sterility and mycoplasma tests as a part of their quality control. the morphology and size distribution of these exosomes were evaluated through multiple strategies including nanoparticle tracking analysis (nta), asymmetrical flow field-flow fractionation (af ), cryo-electron microscopy (cryo-em) and spectra dynetm particle analyser. exosome surface markers were also analysed through multiple strategies such as electro chemiluminescent elisa, flow cytometry and western blotting. also, stem cell exosomes and cancer exosomes were further evaluated for functionality through in vitro functional assays including migration assay, angiogenesis and anchorage independent growth assay. results: our optimized tff method resulted in high yields of > × exosomes/ml and average protein equivalent of more than mg/ml. more than % of the exosomes population had an average size distribution of - nm and median size of nm confirmed through a number of different size distribution instruments. although cell line dependent, we were able to obtain similar expression levels of different cell surface markers including tetraspanins (cd , cd , cd ) when evaluated through different methods. our functional data demonstrated stem cell exosomes were functionally active in promoting cell migration and tubule formation. additionally, cancer cell exosomes were found to promote a malignant phenotype in an anchorage independent growth assay. summary/conclusion: collectively, we demonstrated our ability to reproducibly manufacture production-scale batches of exosomes from multiple different cell types. our purified exosomes are of high yield, meet well-established quality control specifications, and are robust in maintaining size distribution, surface marker expression, and functionality in vitro. therefore, they can serve as ideal reference materials that can support different ev-based research applications. exo-cise: extracellular vesicles enriched from plasma post-exercise promotes myogenesis and neurogenesis bianca paris a , yaomeng liu a , vicente pagalday-vergara a , julie davies b , priya samuel a , ayman abu seer a , johnny collett a , laura gathercole a , ken howells a , karl j. morten b , zhidao xia a , daniel anthony b , david r f. carter a , helen dawes a and ryan c. pink a a oxford brookes university, oxford, uk; b university of oxford, oxford, uk introduction: physical activity brings about a widespread physiological response and elicits the beneficial adaptation of several tissues and organs. furthermore, regular participation in physical activity reduces the risk of developing major non-communicable diseases such as cardiovascular disease, diabetes, cancer, osteoporosis, and dementia. two important processes known to occur following physical activity are myogenesis and neurogenesis; both of which involve the activation and proliferation of specialised tissue-resident stem cells. the molecular mechanisms regulating these processes following exercise are poorly understood to date. here, we investigated the contribution of extracellular vesicles, which are released into the circulation after exercise, to benefit adult myogenesis and neurogenesis. methods: small extracellular vesicles were enriched from the blood of healthy participants before and following maximum and moderate intensity exercise. differentiation and proliferation using a range of methods was measured following vesicle treatment onto primary myoblasts and neuronal primary exvivo stem cells. activation of key cellular pathways were measured. results: we show significant proliferation and differentiation changes of both stem cell types. this is independent of extraction method, extracellular vesicle depleted fractions and is interestingly conserved across mammalian species. remarkably, we see an age-related effect. summary/conclusion: this advocates that short single bouts of exercise may promote myogenesis and neurogenesis via systemic signalling of extracellular vesicles which opens an interesting field in endogenous ev therapies. show promise as a cell-based therapy for retinal degeneration. while clinical trials are ongoing, the potential of extracellular vesicles (evs) as biomarkers for monitoring eye health and disease is not well studied. this study characterized the ev surface profile and cargo of hipsc-rpe to offer a baseline assessment in normal and disease conditions. moreover, we evaluated the importance of pnpla , a gene involved in membrane integrity and when mutated causes retinal degeneration, in ev biogenesis and secretion. methods: evs were isolated from serum-free culture medium of hips-rpe and identified with nanoparticle tracking analysis, transmission electron microscopy, and immunoblot analysis of exosomal markers, including alix, tsg , and cd . surface marker detection and proteomic profiling were completed using an ev surface marker kit and mass spectrometry, respectively. small interfering rna targeting pnpla was used to knockdown the expression in hipsc-rpe and evs were characterized. results: nanoparticle tracking analysis confirmed the presence of both microvesicles (> nm) and exosomes (< nm) by size distribution and the concentration of evs ( x particles/ml) from rpe. tem displayed typical morphological characteristics of evs. the presence of known ev markers, alix, tsg , and cd was confirmed via immunoblot and flow cytometry. surveillance of ev surface markers revealed enrichment of epithelial markers (cd ) and stem cell markers (cd / ) that depict donor cell origin and functional proteins including integrin-binding (cd ) and tgf-beta receptors (cd ). in addition, proteomic analysis revealed regulators of inflammation and rpe function, including hemopexin, clusterin, complement factor i, and pigment epithelium-derived factor. furthermore, reduction in pnpla expression reduced vesicle secretion and vesicle size compared to non-targeting controls. introduction: vascular endothelial growth factor (vegf) is a potent angiogenic factor and was first described as an essential growth factor for vascular endothelial cells. vegf plays a role in normal physiological functions such as bone formation, haematopoiesis, wound healing, and development. mesenchymal stem cell (msc) was found to secretes potential growth factors such as vegf when cultured in vitro. however there are some beliefs that foetal bovine serum (fbs) which usually used as serum in cell culture content vegf. methods: msc seeded in in -well plate in with concentration of , cell/well. cells were incubated for hours and fasted for another hours using only dmem. cells were treated with complete medium consist of dmem and % fbs. culture medium were collected after , , and hours after treatment. cell were culture in ºc dan % co . vegf concentration was detected using elisa technique. results: vegf concentration was not found in fbs which do not contact with msc. an increasing of vegf concentration in time-dependent manner was shown when culture medium was used in msc cell culture in normoxic condition. the result of vegf concentration when culture , , and hours were . pg/ml, . pg/ml, and . pg/ml, respectively. the mechanism of msc release growth factor is still under investigated. however, the classic growth factors and cytokines serves paracrine control molecules which were important in regenerative medicine. vegf was found to be an important molecules in angiogenesis process and determine the fate of cells. summary/conclusion: msc secreted vegf and concentration increased in time-dependent manner. isolation and characterization of exosomes from canine stem cells introduction: unlike induced disease models using laboratory animals, naturally occurring disease models display pathophysiologic attributes that are more similar to human diseases. unfortunately these models are underutilized in translational regenerative medicine research. this is partly due to the slow development of species-specific experimental therapeutics to investigate comparative efficacy. thus, we set out to isolate and characterize exosomes from canine adipose-derived mesenchymal stem cells (cad-msc) to use as a comparative therapeutic in dogs. to accomplish this, we optimized an isolation and purification strategy and characterized their molecular properties. methods: exosomes were isolated by sequential centrifugation and subsequent ultrafiltration. the proteome was characterized by tandem mass tag (tmt) mass spectrometry and the mirna cargo was identified using a canine specific pcr array with subsequent target and enrichment analysis using targetscan and the panther platform, respectively. also, nanoparticle tracking analysis and transmission electron microscopy were used to determine exosome size and structure. to investigate bioactivity, we measured the ability of exosomes to inhibit collagen production in an in vitro model of fibrosis. results: exosomes were purified by ultrafiltration using a kda cut-off. proteomic analysis by tmt mass spectrometry identified unique proteins. % of the exocarta top were identified from this list. additionally, we identified the mirna cargo within exosomes and found highly expressed mirnas. enrichment analysis identified multiple pathways of probable regulation including angiogenesis (fold enrichment = . ; p < . ) and transforming growth factor-beta (tgfb) signalling (fold enrichment = . ; p < . ). exosome size was quantified to be . ± . nm with a modal average of nm. lastly, in the presence of exosomes, tgfb stimulated fibroblasts deposited . % less collagen than vehicle controls (p = . ). summary/conclusion: in summary, cad-mscs exosomes display structural and functional features comparable to stem cell derived exosomes from other species. use of these exosomes in naturally occurring disease canine models may provide superior predictive value for human clinical trials. funding: support provided by the ccah, school of veterinary medicine, uc davis. mesenchymal stem cells-derived exosomes promote in vitro the progression of triple negative breast cancer cells introduction: mesenchymal stem cells (mscs) are multipotent stromal cells and have been described as key regulators of different aspects of tumour physiology. in tumour pathogenesis, mscs can integrate the tumour microenvironment after recruitment and are able to interact with cancer cells to promote tumour modifications by affecting epithelial-tomesenchymal transition (emt). it was revealed that exosomes derived from mscs are critical players in the tumour niche. exosomes are a novel way of cellto-cell communication and play crucial roles in the majority of pathways that contribute and affect response to therapy, cell-adhesion molecules and the progression of tumour cells. because of the known importance of this communication we decided to investigate the implication of mscs with triple negative breast cancer (tnbc) cell lines as well as exosomal profiles between the experimental conditions. methods: the interactions of mscs with triple negative breast cancer cell lines (mda-mb- and hs t) was performed by coculturing mscs (or tnbc cell lines) with exosomes derived from tnbc cell lines (or mscs). physical characterization of isolated exosomes was performed followed by their molecular investigations. cell proliferation was detected by mtt assay and migration was analysed by wound healing assay using d cultures. moreover, we also used d culture to assess the exosomes uptake and to observe their capability of internalization into a d structure. the alterations in expression level of some transcripts (mrnas and mirnas) and protein profile were investigated by qrt-pcr, western blot and immunofluorescence staining. results: we found that mscs-derived exosomes are actively incorporated by triple negative breast cancer cell lines ( d culture). in coculture, in tnbc cells the expression level of mesenchymal markers and emt markers (e-cadherin, vimentin) at mrna and at protein levels, as well as mirna-derived exosomes targeting mesenchymal genes were significantly affected. using bioinformatics tools, we highlighted the important biological processes which were activated by promoting tumour modifications. in addition, using d culture we provided a comprehensive understanding regarding exosomes internalization in d structures, which closely mimics in vivo conditions, compared to d culture. summary/conclusion: in this work, we focus on the investigation of mscs-derived exosomes in order to highlight their implication in several biological processes, including tumour proliferation and progression of triple negative breast cancer cells. all these alterations affect the response to therapy and should be considered for developing efficient therapeutic strategies. natural killer cell-derived extracellular vesicles have a potent anti-leukaemic effect and selectively target the cancer stem cell subpopulation introduction: natural killer (nk) cells of the immune system recognize and kill tumour cells. extracellular vesicles (evs) secreted from nk cells are capable of killing tumour cells independent of the cell to cell contact required for nk cell activation. cancer is a leading cause of death, primarily due to metastasis and recurrence. cancer stem cells (csc) within tumours are resistant to chemotherapy and immune attack, and cause metastasis and relapse. identification of the cancer types killed by nk evs is limited, and the effect of nk evs on cscs has not been described. here we determine whether nk-derived evs kill a myeloid leukaemia cell line and its csc subpopulation. methods: nk evs were isolated from our nk cell line, nk . , derived from normal human lymphocytes. nk . evs were characterized by immunoblotting, proteomics, and next generation rna sequencing. human k leukaemia cells were treated with nk . evs in vitro and analysed for proliferation and markers of cell death. results: nk . evs contain ev-associated proteins alix, cd , hsp , and tsg , nk effector molecules perforin, granzymes a and b, granulysin and nklam/ rnf b, an e ubiquitin ligase required for maximal nk cytotoxicity, and tumour suppressor mir- . nk . ev treatment of k significantly decreased its expression of proliferation markers cd and ki , and increased the frequency of apoptotic and necrotic cells, paralleled by elevated levels of active caspases − and − . non-tumorigenic cells were unaffected by nk ev treatment. most notably, nk . ev treatment significantly reduced the frequency of k cells highly expressing aldh, a csc marker. summary/conclusion: nk . -derived evs have a robust anti-tumour effect on k myeloid leukaemia cells and selectively target the csc population, suggesting they may circumvent the evasion and resistance mechanisms used by cscs. nk . evs therefore have introduction: due to their potential as a key bioactive agent in regenerative medicine applications, mscderived extracellular vesicles (msc-evs) are increasingly being investigated as a clinical therapy. manufacturing that generates enough evs for product development and clinical doses is currently a limitation in the field and clearly a scalable manufacturing solution will be necessary for successful translation. moreover, a complementary approach that increases the ev productivity, i.e. the number of evs produced per cell, could further help to accelerate the development of msc-evs as a therapy. methods: we developed a process that leverages a series of new cell culture reagents to couple to our established cell-media system for scalable manufacturing of msc-evs. briefly, human bone marrow-or umbilical cord-derived mscs were rapidly expanded under xeno-free conditions (i.e. > x expansion within days). cultures were then switched to our proprietary ev collection medium and evs were harvested for up to three additional days. at the end of culture, the evs in the conditioned media were concentrated using a tangential flow filtration (tff) system. to increase the productivity of mscs, two medium supplements were developed that increased ev yield by either increasing the number of evs generated per cell in a shortened culture process or increasing the number of collected evs by lengthening the ev collection culture period. results: this scalable msc-ev manufacturing method was implemented in both d flask and d bioreactor culture and generated over , particles per cell in d and over , particles per cell in d. with the addition of a medium supplement to increase evs produced per cell, the ev productivity was increased > x after hrs. alternatively, ev productivity was also increased > x by addition of the medium supplement that extended ev collection culture period. summary/conclusion: msc-ev success in clinical translation will be reliant on a manufacturing method that can scalably and reliably generate large amounts of evs. these results present one such solution. furthermore, increasing ev productivity, for instance by medium supplements that increase evs per cell or lengthen culture times could further address the limitation of generating the evs required for development and translation of clinical therapies. simplifying scalable msc ev production in a microcarrier-based bioreactor system divya patel, josephine lembong, katrina adlerz, jon rowley and taby ahsan roosterbio inc, frederick, usa introduction: the growpt ing numbers of msc-ev clinical applications drives the need for a scalable msc-ev production platform. while most msc-evs are generated while cells are attached to tissue culture plastic, such d cultures cannot be scaled up to meet the yields necessary for commercialization of ev-based therapeutics. we have shown that d bioreactors can be used to generate msc-evs and that paradigm can be scaled directly in terms of yield from the to l scales. the technical expertise of seeding cells onto microcarriers for expansion in bioreactors, however, requires technical expertise not available to all those in the ev field. therefore, our goal here is to simplify and expedite the ev collection process in bioreactors by cryopreserving cells on microcarriers, such that end users can merely thaw and then collect msc-evs. methods: mscs were expanded in d and then seeded on three different microcarriers and cultured in a bioreactor for days. when confluent, cells on microcarriers were cryopreserved. to evaluate the microcarriers and the cryopreservation protocol, the cells-microcarriers were thawed, cultured in a bioreactor in growth media for hours, then in ev collection media for additional days. cell recovery and ev production upon thaw was evaluated and compared to ev collection from fresh, non-cryopreserved cells. results: total cell counts hrs post thaw were comparable to those before cryopreservation and to fresh samples prior to ev collection. following -day ev collection, concentration of particles collected from cryopreserved cells on microcarriers were similar to those collected from the fresh cells ( e particles/ml). this process was validated for two different microcarriers using two separate cryopreservation solutions. summary/conclusion: our results show that cryopreserved hmscs on microcarriers can support ev collection in a d bioreactor process with a particle yield that is comparable to those collected from fresh cells. this cryopreserved product can simplify ev production, reducing cost and time by removing process steps associated with the hmsc expansion, with in a paradigm suitable for scale-up. the whitening, anti-wrinkle, and wound-healing effects of extracellular vesicles from orbicularis oculi muscle-derived stem cells. introduction: skeletal muscle-derived stem cells possess potent therapeutic activities in the treatment of muscle-related disorders. in our study, we tried to isolate and characterize orbicularis oculi muscle (orm)-derived stem cells (orm-scs) from the discarded human tissues which were obtained from the ocular surgery-subjected patients. we also prepared the natural extracellular vesicles (evs) from the cultured orm-scs and assessed the their therapeutic actitities including the skin whitening, anti-wrinkle, and wound healing effects. methods: we isolated the orm-scs from the patients subjected to ocular surgery and characterized the orm-scs by analysing cell morphology, proliferation, expression levels of the cell surface and stemness-associated markers, and tri-lineage differentiation and colony-forming capacities, confirming the stemness properties of the orm-scs. then, we prepared the natural evs from the orm-scs via the centrifugation and filtration of the media supernatants and their therapeutic activity was investigated. results: the isolated orm-scs showed spindle-like morphology and positive expression of cd , cd , and cd , but they were negative in expression of cd and cd . the orm-scs showed the capacity of osteogenic, adipogenic, and chondrogenic differentiations. the evs from orm-scs (orm-sc-evs) possessed the apparent inhibitory effect on the melanin synthesis in b f cells by blocking the tyrosinase activity, although orm-sc-evs treatment did not dramatically change the expression level of melanogenesisrelated genes, such as microphthalmia-associated transcription factors (mitf), tyrosinase (tyr), tyrosinaserelated protein (tyrp- ), and tyrp- . in addition, we confirmed that orm-sc-evs could stimulate skin cell migration and increase the expression level of antiwrinkle related genes and wound-healing properties. summary/conclusion: this study revealed the stem cell property of orm-scs and the whitening, antiwrinkle, and wound healing effects of orm-sc-evs, suggesting that orm-scs and orm-sc-evs can be successfully used for stem cell-based ev therapy and cosmetics, by regulation the melanogenesis, wrinkle, and wound. funding: this work was supported by grants from the national research foundation (nrf) funded by the korean government ( m a h ). use of stem cell extracellular vesicles as a holistic approach towards cns repair introduction: neurological diseases and disorders are leading causes of death and disability worldwide. many of these pathologies are associated with high levels of neuroinflammation and irreparable tissue damage. we have previously shown that extracellular vesicles (evs) from infected cells contain viral by products (noncoding rnas and proteins) and that these evs can exert deleterious effects on recipient cells - . therefore, in the context of neurotrophic viruses evs may contribute to or perpetuate processes relating to neuroinflammation and neurodegeneration. due to their multipotent properties, stem cells have broad applications for tissue repair; additionally, stem cells have been shown to possess both immunomodulatory and neuroprotective properties. in recent years it has been well-established that stem cell evs play a critical role in the functionality associated with stem cells. the diverse biological cargo contained within these vesicles are proposed to mediate their effects and, to date, the reparative and regenerative effects of stem cell evs have been demonstrated in a wide range of cell types. while a high potential for their therapeutic use exists, there is a gap of knowledge surrounding their characterization, mechanisms of action, and how they may regulate cells of the central nervous system (cns). methods: we have isolated and recovered high yields of evs from large scale cultures of both induced pluripotent stem cells (ipscs) and mesenchymal stem cells (mscs) using tangential flow filtration. our ev characterization includes both phenotypic (size, tetraspanin expression) and biochemical assays. ev functionality has also been assessed in vitro utilizing several cellbased assays related to cellular viability, migration, angiogenesis, and immunomodulation in both healthy and damaged recipient cells with relevance to the cns. results: our data suggests that evs from different sources of stem cells display unique phenotypes, exhibit differential association with various cytokines, proteins, and long non-coding rnas, and have the ability to significantly enhance processes that are critical for cellular repair . lastly, utilizing an ipsc-derived neurosphere model, we have observed a robust uptake of stem cell evs and have found that these evs are able to effectively penetrate these d structures. summary/conclusion: collectively, these results highlight the "holistic" properties of stem cell evs by demonstrating their ability to partially reverse or reduce damage in various cell types. funding: this work was supported by national institutes of health (nih) grants ai , ai , ai - , ai , and ns to fk and r ca and r ar to lal. the effect of cell culture media on extracellular vesicle secretion from mesenchymal stem cells and human pluripotent stem cell-derived neurons introduction: cell culture media and its supplements are known to affect the secretion and isolation of extracellular vesicles (evs) from cell cultures. identification of these effects is crucial especially when planning to use evs as therapeutic agents. here, we investigated the effect of cell culture media on ev yield from human mesenchymal stem cells (mscs) and human pluripotent stem cell (hpsc)derived neurons. methods: evs were collected from cell-conditioned media (ccm) and no cell control (ncc) media using size-exclusion chromatography (sec). mscs were cultured in dmem/f :neurobasal medium or in opti-mem reduced serum medium, both supplemented with exosome-depleted foetal bovine serum (fbs). the ev yield from hpsc-derived neurons was compared at two maturation time points (day and ), in dmem/f :neurobasal or in opti-mem, with and without -hour kcl stimulation. sec fractions were analysed by nanoparticle tracking analysis (nta), protein concentration assay and blinded transmission electron microscopy (tem). results: ccm samples had a clear peak of evs in sec fractions - , which was not detected with ncc. interestingly, a second population of evs eluted in sec fractions - in both ccm and ncc, indicating presence of evs in exosome-depleted fbs. moreover, this second population differed largely between used media batches. culture medium had no significant effect on msc ev yield (dmem: . e+ particles/ ml, opti-mem: . e+ particles/ml). with neuronal cultures, no significant differences in ev yield were found between culture media or cell maturation time points. in contrast to earlier findings, -hour stimulation of neurons by kcl resulted in significantly smaller ev yield compared to non-stimulated controls (stimulated: . e+ particles/ml, non-stimulated: . e+ particles/ml, p < . ). summary/conclusion: our results indicate that exosome depleted-media are not entirely devoid of vesicles, which can cause bias in downstream analyses. however, sec is a good method to separate cellsecreted evs from the contaminating medium-derived evs. culture medium did not affect the number of evs secreted by mscs or neurons; instead, we observed larger differences between media batches. this data emphasizes the importance of analysing the ncc as negative control in all cell culture experiments. mouse mesoangioblast stem cell extracellular vesicles are able to influence macrophage cell activity maria magdalena barreca a and fabiana geraci b a dept stebicef university of palermo, palermo, italy, palermo, italy; b dept stebicef, university of palermo, italy, palermo, italy introduction: it is largely demonstrated that stem cells release extracellular vesicles (evs) that are able to modify target cell behaviour. interestingly, there is a bidirectional signalling exchange between stem cell evs and damaged cells. moreover, it is well known that macrophages, could also play a role in wound repair and tissue regeneration. it was also demonstrated that stem cell evs are involved in immune cell regulation. for this reason, today takes hold the idea that evs could replace stem cells in regenerative medicine. the aim of our work was to evaluate if evs released by mouse mesoangioblast stem cells (a ) could have a role in immune cell regulation. specifically, we have investigated the possible a ev effect on murine macrophages (raw . ) in terms of cell proliferation, migration and phagocytic ability, and cytokines/chemokine release. methods: a evs were collected from conditioned milieu by ultracentrifugation. raw . cell proliferation with or without a evs was evaluated via cfse assay. scratch test was performed to assay their migration ability. to study raw . cell phagocytosis they were treated with μm beads. finally, cytokine array was used to monitor their secretion after ev treatment. results: we have found that a evs inhibited macrophage proliferation as proved by a proliferation index significantly reduced after ev treatment. simultaneously, we have noticed that evs increases raw . migration ability. furthermore, a evs are able to increase macrophage phagocytic activity. as it is known that hsp is involved in for macrophagic activity increase and a evs express hsp on their surface, we performed phagocytosis assays assay by blocking the protein or its receptor tlr , tlr and cd . our data demonstrated that a evs increase phagocytosis through hsp and its receptors. we have also proved that a evs modify the expression pattern of cytokines/chemokines released in the extracellular milieu by raw . cells. in particular, we observed an increase in anti inflammatory cytokines, and a decrease in some inflammatory ones, suggesting that evs could polarize macrophages towards an anti inflammatory m phenotype. summary/conclusion: in conclusions, our data show that a evs influence macrophage activity and additional studies could provide a new insight into understanding the underlying potential of evs in tissue regeneration. and ) . in a healthy kidney, the polycystins localize to renal cilia. mutations that abrogate ciliary localization of pkd (yet preserve its channel function) also cause cysts. besides cilia, pkd is also found in other subcellular locations including extracellular vesicles (evs) of human urine. how dysfunction of pkd trafficking and localization leads to the kidney pathology remains unknown. pkd is evolutionarily conserved across all members of eumetazoa. in c. elegans, pkd- is exclusively expressed in ciliated male-specific neurons, where it is trafficked to cilia and evs. gfp-tagged pkd- -containing evs play a signalling role in inter-organismal communication between animals. conservation of polycystin- cellular localization between worm and human suggests that their network of molecular interactions may also be conserved. we propose that pkd- plays distinct roles in cilia versus ciliary evs. methods: to understand the role of evs in c. elegans inter-organismal signalling, we aim to identify the pkd- -associated ev proteome, transcriptome, and metabolome. we established a pipeline for fluorescent labelling and tracking specific ev cargoes in a living animal using super-resolution microscopy. we used fluorescence of the pkd- carrying evs to optimize biochemical procedures for their enrichment. results: our initial analysis revealed two populations of pkd- -carrying evs that differ in their densities: . - . versus . g/ml. we are currently characterizing these two distinct populations using transmission electron microscopy and refining our enrichment procedure for protein identification by mass spectrometry, sequencing of their rna cargoes and metabolome analysis. summary/conclusion: what function human pkd plays within the cilia and within the urinary evs is not well understood. identification of molecular mediators of c. elegans pkd- ev signalling will inform on the interactome of human pkd and its function in cilia versus evs. introduction: ectosomes play roles in many physiological and pathophysiological processes, and their precise is dependent on molecular cargo and parent cell type. a single cell can release distinct subpopulations of evs enriched with different molecular cargo, which adds complexity to elucidating cargo sorting and biogenesis mechanisms. in the nematode c. elegans, ectosomes bud from sensory neuron cilia and are released into the environment to modulate animal behaviour. methods: c. elegans is genetically tractable and optically transparent, allowing for live imaging of fluorescently tagged ev cargo. we express all tagged cargo at endogenous levels, adding physiological relevancy. results: we discovered that the calcium homoeostasis modulator ion channel clhm- localizes to cilia of ev-releasing neurons and observed gfp-tagged clhm- in ciliary evs. using super resolution microscopy, we imaged evs released from animals coexpressing tdtomato-tagged clhm- and gfp-tagged pkd- (another vesicle cargo) in the same neurons. while the two proteins colocalize in the cilia, clhm- ::tdtomato and pkd- ::gfp rarely colocalize in evs. this indicates that separate subpopulations of evs are being released from the same neurons. to determine how the clhm- subpopulation is formed, we are investigating candidate genes. anoh- , a homolog of the ca + scramblase tmem f, localizes to neuron cilia and induces phosphatidylserine exposure on the outer membrane leaflet. in anoh- mutants, the number of clhm- ::gfp evs released is significantly decreased but the number of pkd- ::gfp evs does not significantly change. in addition, i am using facs to isolate clhm- and pkd- containing evs and analysing the respective proteomes with lc-ms/ms. summary/conclusion: we are elucidating mechanisms that give rise to distinct subpopulations of ciliary evs in c. elegans and defining cargoes being enriched in these ev subpopulations to gain insight into ev cargo sorting and biogenesis mechanisms in ciliated neurons. ceramide accumulation induces exosome secretion through lysosomal protein laptm b kohei yuyama, hui sun and yasuyuki igarashi hokkaido university, sapporo, japan introduction: exosomes, a type of extracellular vesicles originated from multivesicular bodies (mvb), are important carriers of cellular molecules and have critical roles in intracellular communication in both health and disease. ceramides (cer) are implicated in biogenesis of exosome, however the molecular machinery that mediates exosome secretion remains obscure. lysosome-associated protein transmembrane- b (laptm b) is a lysosome/late endosome-resident transmembrane protein, which has been reported to bind cer. we demonstrate here that laptm b is involved in the exosome secretion, which are induced by exogenous cer treatment or lysosomal ceramidase inhibition in cultured neuronal cells. methods: neuroblastoma sh-sy y cells were treated with cer (porcine brain-derived cer or synthetic d : /c : ~c : cer) for h. exosomes were isolated from the culture supernatants by sequential centrifugation and their amounts were measured using ps capture exosome elisa kit. to analyse mvb transport, mvb and recycling endosomes are visualized with gfp-cd and rab immunostaining, respectively. results: we found that exogenous treatment of cer, especially those with c and c fatty acids, resulted in a marked increase in exosome secretion. in addition, lysosomal cer accumulation induced by acid ceramidase inhibition also accelerated exosome production. knockdown of laptm b significantly prevented the ceramide-dependent exosome release. in addition, we showed that these cer loading promoted colocalization of cd -positive mvb with rab -positive recycling endosomes, further demonstrated that laptm b knockdown cancelled the cer-dependent increase of the colocalization. summary/conclusion: these data suggest that lysosomal cer binds to laptm b and promote the transport of mvb to plasma membrane, resulting in an increase of exosome secretion in neuronal cells. chloroquine-mediated lysosomal inhibition alters composition and function of cancer-derived extracellular vesicles jing xu a , kevin yang a , shane colborne a , elham hosseini-beheshti b , gregg morin a , emma guns b and sharon m. gorski a a bc cancer, vancouver, canada; b the vancouver prostate centre, vancouver, canada introduction: small extracellular vesicles (sev) are signalling entities released by many types of eukaryotic cells. sev are of special interest in cancer due to their reported roles in modulating the cancer microenvironment and facilitating cancer cell invasion. macroautophagy (hereafter autophagy) is a catabolic process well-known for the recycling of cytosolic cargos through lysosome-mediated degradation. in this study, we profiled the changes in sev content and function under lysosome inhibition and investigated the involvement of autophagy machinery in sev content. methods: chloroquine (cq) was used to inhibit lysosomal degradation and autophagy turnover in triplenegative breast cancer (tnbc) cell lines. sev were collected via precipitation after pre-clearing and concentration of conditioned media. western blotting, nanosight and transmission electron microscopy were used to profile sev. quantitative mass spectrometry was used to characterize cq-induced changes in the sev proteome. antibody-conjugated magnetic beads were used in immunoprecipitation of sev. results: cq treatment did not substantially alter the physical properties of tnbc-derived sev. however, cq treatment altered the sev proteome and growth effects of sev on normal and endothelial recipient cells. cq treatment induced co-localization of mammalian atg proteins with endolysosomal markers in the cytoplasm, which coincided with an enrichment of atg s and their adaptor proteins in sev. cq-induced enrichment of atg s in sev required lipidation, and occured preferentially in one subset of sev. summary/conclusion: our study reveals changes in the content and function of cancer cell-derived sev in response to perturbation of intracellular trafficking pathways, demonstrates the flexibility and heterogeneity of sev composition, and has implications for cq efficacy in therapeutic settings. introduction: introduction: argonaute (ago ) is the essential component of the rna-induced silencing complex (risc) that binds mirnas and promotes mrna degradation. extracellular vesicle (ev)-carried mirnas have been shown to influence gene expression and functional phenotypes in recipient cells. many investigators have found ago in evs and it is postulated that ago is a major transporter of mirnas into small evs (sevs), such as exosomes. others have reported extracellular ago that is non-vesicular. we set out to evaluate the effect of growth factor signalling and serum contamination on the detection of ago in sevs. methods: methods: wildtype kras colorectal cancer cells, dks , were conditioned with different culture media (serum-free dmem, dmem supplemented with ev-depleted fbs, and opti-mem). evs were purified from conditioned media by cushion-density gradient ultracentrifugation. western blot analysis of dks total cell lysates, large evs and density gradient fractions was performed, probing for ago and ev marker proteins. the size and concentration of the evs were determined by particle metrix analysis. results: results: in all conditions, we found the highest abundance of sevs in fractions and , as assessed by western blot analysis. ago was detected in the same fractions as sevs in both the serum-free dmem and opti-mem conditions, although the levels of ago was higher in the serum-free dmem fractions compared to that of opti-mem. in contrast, ago was present in both vesicular and non-vesicular fractions in the dmem supplemented with ev-depleted fbs condition. no significant differences were observed in the size and number of evs collected in the three conditioning methods. summary/conclusion: summary/conclusion: the presence or absence of ago in evs has been controversial. multiple factors may affect the ability to detect vesicular ago , including serum and growth factors in the conditioned media that may provide sources of extravesicular ago and also regulate the trafficking of ago into vesicles. introduction: cancer-associated glycosphingolipids have been utilized as tumour markers and targets of cancer therapy. we have investigated roles of gangliosides in cancers, and clarified that cancerassociated gangliosides enhance malignant properties of cells by forming complexes with membrane molecules in lipid rafts. in this study, we analysed contents of gangliosides and membrane molecules on extracellular vesicles (ecvs) secreted from melanoma cell lines. methods: melanoma cell lines with various ganglioside patterns were used for isolation of ecvs. gangliosidemodified melanomas with genetic engineering were also used. genetic modification was done by cdnas of ganglioside synthase genes. ecvs were collected by ultra-centrifugation, or by tim -beads. contents in ecvs were analysed by immunoblotting or flow cytometry. roles of lipid rafts in the generation and secretion of ecvs were analysed by treating cells with mm methyl β-cyclodextrin. results: using melanoma cell lines, ecvs were isolated by ultra-centrifugation, and their sizes were analysed by nanosight. all samples showed uniform sizes between and nm. protein amounts in ecvs were measured, showing heterogeneous levels at ~ μg/ ml. then, gangliosides expressed on ecvs from these cell lines were analysed using tim beads and flow cytometry. gd and gd were detected on ecvs almost proportionally with expression levels of those gangliosides on the cell surface. then, immunoblotting was performed to analyse integrin levels in ecvs from transfectant cells expressing high levels of gd , showing increased levels of integrins in ecvs from gd + cells compared with those from gd -cell lines. integrin levels in cell lysates from these cells (gd + and gd cells) were almost equivalent. treatment of a gd -expressing melanoma cell line by mm methyl β-cyclodextrin resulted in marked reduction of secreted ecvs and amounts of tsg in them. summary/conclusion: ganglioside expression patterns on melanoma cells were well reflected in the expression of gangliosides on ecvs. these results as well as increased levels of integrins in ecvs from gd + cells suggest that gangliosides and lipid rafts are involved in the generation and secretion of ecvs. introduction: hypoxia, or low oxygen tension, is a common feature associated with tumour growth and is known to regulate tumour cell function, especially through rewiring of cell metabolism. however, how hypoxia influences tumour cell interactions with surrounding cells is not fully elucidated. we sought to evaluate how hypoxia alters metabolite and metabolism-associated mirna packaging in exosomes. methods: exosomes were isolated from t breast cancer cells cultured in normoxia ( % o ) and hypoxia ( % o ) via ultracentrifugation, optiprep gradients, and size exclusion chromatography. exosomes were further characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. metabolite and mirna profiling was performed on exosomes and exosome-producing cells in normoxia and hypoxia. results: secretion of exosomes was increased under hypoxic conditions. metabolite profiling revealed alterations in metabolites specific to exosomes derived from hypoxic cells. profiling of exosomal mirna showed packaging of metabolism-related mirna into exosomes derived from hypoxic cells. summary/conclusion: hypoxia alters the metabolite and mirna profiles of cancer cells, with selective packaging of these molecules into exosomes. we identified metabolites and mirna that are depleted and enriched in exosomes compared to cells. these studies identify hypoxia-associated shifts in exosome cargo, providing insight into exosome cargo packaging with potential implications for understanding how cancer cell-derived exosomes regulate recipient cell function. lysosomotropic agents prompts the release of extracellular vesicles carrying autophagy-associated markers: evidence of a general mechanism of secretion driven by lysosomal impairment introduction: drug-induced lysosomal storage disorders (lsds) are due to the transient intracellular accumulation, mostly of phospholipids, into multilamellar inclusion bodies within late endosomal/lysosomal compartment. they represent a major side-effect for many drugs of several pharmacological categories. most lsds inducers are cationic amphiphilic drug (cad), but the molecular mechanisms leading to accumulation of undigested substrates are unknown. extracellular vesicles (evs) have been implicated in cell waste disposal, but it is unclear whether they might be involved in extracellular release of undigested substrates. methods: to investigate this aspect, we developed hek cells stably expressing the fluorescent fusion proteins egfp-cd and mcherry-cd , separated evs by differential ultracentrifugation and quantified by evassociated fluorescence and nta particle count. results: evs released by these models upon treatment with drugs inducing the accumulation of phospholipids (amiodarone) or glycosaminoglycans (tilorone), showed the release of fluorescent medium/large evs ( k fraction) and small evs ( k fraction), whose size and distribution were similar to the same vesicles released by control cells, but enhanced the recovery of medium/large evs and to a lower extent of small evs, analysis of evs associated markers revealed a dosedependent increase of autophagy-associated markers in medium/large and small evs. similar results were obtained when autophagic flux was impaired by drugs raising lysosomal ph by different mechanisms, such as chloroquine and bafilomycin, but not when autophagic flux was stimulated by drugs such as curcumin or overexpression of the endosomal/lysosomal regulator tfeb. summary/conclusion: overall results show that impairment of autophagic flux, either by indigested substrates or higher lysosomal ph, is associated with an increased release evs enriched in autophagy markers, compatible with autophagomes and/or amphisomes, unravelling a connection with secretory autophagy. tomofumi yamamoto a , yusuke yamawaki b , yutaka hattori c and takahiro ochiya a a tokyo medical university, shinjuku-ku, japan; b national cancer center research institute, chuo-ku, japan; c keio university faculty of pharmacy, minato-ku, japan introduction: multiple myeloma (mm) is a haematological tumour. last decade, the prognosis of mm has improved by the development of therapeutic drugs; however, mm cells acquire drug resistance by longterm exposure of these therapeutic drugs. one of the possible explanations of drug resistance is that cells with drug resistance transmit information to other mm cells and their microenvironmental cells. although the elucidation of the mechanism of drug resistance in mm have been desired, it remains poorly understood. methods: in order to understand the mechanism of drug resistance in mm, lenalidomide resistant cell lines were established by long-term exposure of low concentration of lenalidomide. drug resistance was assessed by mts assay and caspase assay. the amount of ev was measured by exoscreen, which is ultra-sensitive detection method of evs by measuring surface protein of evs, such as, cd and cd (yoshioka et al., nat commun., ) . to identify the genes which involved in drug resistance, rna sequence among the drug-resistant cell lines and their parental cell lines was performed. results: firstly, characterization of these cells was confirmed. we found that all of the lenalidomide resistant cell lines secreted more evs than their parental cell lines. in addition to this, the size of ev derived from resistant cells are smaller than those of parental cells. next, we collected evs from resistant cells and parental cells by using ultracentrifugation, and added them to parental cells in the presence of lethal dose of lenalidomide. compared with ev derived from parental cell lines, the evs derived from lenalidomide resistant cell lines increased a number of living parental cells. these results suggested that the evs derived from lenalidomide resistant cells can affect the lenalidomide sensitive cells. as a result of rna sequence, several genes highly expressed in resistant cell line we found, which associated with lysosome pathway. among them, attenuating the sort and lamp genes could significantly reduce the ev secretion in mm cells, leading to enhance the lenalidomide sensitivity. summary/conclusion: our results showed that ev secretion via sort or lamp could induce the drug resistance in mm. study on biological stimulate mechanism of stem cell-derived exosome generation by nanoparticles introduction: mesenchymal stem cells (mscs) are pluripotent stromal cells known to release extracellular vesicles (evs) containing various growth factors and antioxidants that can positively affect surrounding cells. nanoscale msc-derived evs, such as exosomes, have been developed as bio-stable nano-type materials, but had low yield and were difficult to quantify. we hypothesized that the mechanism of nanoparticleenhanced exosome production would stimulate intracellular molecules. the aim of this study was to elucidate the molecular mechanisms of exosome generation by comparing the internalization of surface-modified positively charged nanoparticles and exosome generation from mscs. methods: mesenchymal stem cells (mscs) were cultured in mem-alpha with % fbs and × antibiotics. the positively charged nanoparticles were synthesized by poly-lactide-co-glicolide (plga) and polyethylenimine (pei) with cy . for tracking nanoparticles. all of the exosome image were identified using an electron microscope. additionally, it was confirmed the internalization of the nanoparticles by if. the primary antibodies used were anti-eea , anti-rab and anti-gm . in order to prove the development of exosomes, rt-pcr using autophagy-related mrna was performed. real-time rt-pcr was performed using the applied biosystems sequence detection system . lastly, mirna from msc-derived exosome analysed automatically in the affymetrix data extraction protocol using the provided affymetrix genechip® command console® software (agcc). all statistical testing and visualization of differentially expressed genes was conducted using r statistical language . . results: we determined that rab , located in the mvb and autolysosomal membrane, was increased upon exosome expression and was associated with autophagosome formation. these results suggested that nanoparticles migrated to lysosomes during treatment; however, intracellular exosome-forming factors were stimulated during endosomal maturation simultaneously. summary/conclusion: therefore, msc-derived exosome research using nanoparticles is useful for increasing exosome yield and the discovery of nanoparticleinduced genetic factors. theoretical description of formation of extracellular vesicles by budding of membrane introduction: understanding mechanisms of extracellular vesicles (evs) formation is of utmost importance for their effective use in science, medicine and technology. in particular, the discovery of universal mechanisms explaining the phenomena taking place in vesiculation appears to be crucial and highly warranted. mammalian erythrocytes and giant phospholipid vesicles have been largely used as model systems to study principles of membrane budding and vesiculation. the mechanisms conveniently studied in these simple systems are then generalized to other types of biological membranes. we present a theoretical description of membrane budding and compare the theoretically obtained shapes with the observed ones. methods: in accordance with the fluid crystal mosaic model, membrane is considered as composed of constituents (inclusions) subjected to the local curvature field created by surrounding constituents. constituents can attain different in-plane orientations in the membrane which correspond to different energies. the thermal motion oposes the complete orientational ordering. the single-constituent energy expresses a mismatch of the curvature of the membrane at the position of the constituent and the intrinsic principal curvatures of the constituent and inplane orientation of their principal axes. the free energy of the whole membrane is obtained by summing up (integration) the contributions of the constituents and using methods of statistical physics, and minimized by using numerical methods. results: to outline the principle of (outward and inward) budding, respective sequences of shapes corresponding to a formation of one (outward and inward) spherical bud were calculated by minimization of the free energy. also the corresponding shapes observed in evs (imaged by electron microscopy) and in erythrocytes and giant phospholipid vesicles (imaged by optical microscopy) are shown. it can be seen that theoretically calculated shapes and experimentally observed ones agree well over up to orders of magnitude (the order of the size of giant phospholipid vesicles is between and micro metres, in erythrocytes it is about micro metres and in evs it is about nanometres). summary/conclusion: budding of the membrane is an universal mechanism in formation of external and internal vesicles. introduction: the release of extracellular vesicles (evs) from cells is important for many cellular mechanisms both in normal physiology and in disease. arrdc (arrestin domain containing protein ) is an adaptor protein known to facilitate the ubiquitination of target substrates by nedd family ubiquitin ligases. it also traffics cargo to extracellular vesicles. previous studies show the involvement of arrdc in the trafficking of the divalent metal ion transporter dmt to evs in a ubiquitin-dependent manner, and we aimed to further understand this mechanism. methods: we performed mass spectrometry to identify ubiquitinated lysine residues in arrdc . we then generated arrdc wt and lysine mutant clones and expressed these in cells to determine the effect on ev biogenesis and protein trafficking. results: mass spectrometry data identified potential ubiquitinated lysine residues. out of these, lysine appeared to be the most important for arrdc function. arrdc k r mutation caused a decrease in the number of ev released by the cell compared to arrdc wt, and a reduction in trafficking of dmt to evs. furthermore, we also observed a decrease in dmt activity and an increase in its intracellular degradation in the presence of arrdc k r. k also appeared to be ubiquitinated with k polyubiquitin chains by the ubiquitin ligase smurf . summary/conclusion: our data suggests that k polyubiquitin chains are the signal for arrdc mediated ev biogenesis and protein trafficking, and loss of this signal causes cargo to be rerouted to intracellular degradation mechanisms. chair: tanina arab -department of molecular and comparative pathobiology, johns hopkins university school of medicine a d-printed model to represent the structure and nature of extracellular vesicles, for public engagement and education events. christian burton a , sara veiga a , jason webber a , kate milward a , muireann ni bhaoighill a , lauren evans a , andreia de almeida b , rachel j. errington a and aled clayton a a cardiff university, cardiff, uk; b cardiff university, research associate, uk introduction: explaining the field of extracellular vesicles to the lay public and young audiences can often be challenging. whilst diagrams and images of evs may be helpful, conveying clearly the shape and composition of an ev by these means is not always a success. whilst many members of the audience may be familiar with concepts of cells and related structures, others will find such discussions very abstract and challenging. in order to aid interactions with lay audiences we embarked on the design of a physical hand-held plastic model, representing a typical ev. incorporating flexibility in the design allowing the community to adapt it to showcase their own research. the second goal was to ensure manufacturability using widely available dprinting technologies. methods: the basic model design was conceived by dr c. burton, and iteratively developed using solidworks, , then exported for use in any cad environment (stl format). a model showing a halved ev hemisphere, with a visible lipid-bilayer was developed. attachable rings allow trans-membrane-molecules to be represented, current designs include mhc class-i, hspgs, integrins, tetraspanins and supported by handouts accompanying the models. intraluminal cargo is included via removeable "pegs", and examples representing rna or simple globular proteins, and a template has been created. results: the design is free and open source, and available to the community at: https://www.thingiverse. com/thing: . instructions for d printing are available from the uk extracellular vesicle society website; https://www.ukev.org.uk/public-engagementmaterials/. models have been produced using entrylevel d printers and trialled at engagement events with good early responses. summary/conclusion: the authors hope the community will use and develop this d-model design and that the approach provides an additional and helpful tool for educating audiences about the complexities and roles of evs in biology and disease. centrifugal filtration-sec is promising for extracellular vesicle isolation from d and d her + breast epithelial cell lines introduction: despite recent developments in breast cancer therapy, there is still need for a more targeted approach. extracellular vesicles (evs), endogenous nanovesicles released from human cells, are an attractive choice as nanodrug carriers due to their size, stability and their unique targeting specificity. the aim of this study was to determine if centrifugal filtration (cf) combined with size exclusion chromatography (cf-sec) would be useful for ev isolation from two epithelial breast cell lines d and d her +, representing the tissue of interest, and the amount of cell culture needed to get measurable ev concentrations. methods: cell culture media (without serum) from the immortalized breast epithelial cell lines d and d her + was concentrated with centrifugal filtration (cf) followed by isolation with size-exclusion chromatography (sec) using hiprep / sephacryl s- column run with Äkta start ( nm), min runs. each fraction ( - ml) was collected with fraction collector. dulbecco's particle free pbs was used as mobile phase. the resulting particles were analysed with nanoparticle tracking analysis (nta, nanosight ns , camera gain , static mode, capture time sec), western blotting (wb), microbca and transmission electron microscopy (tem, samples fixed with % formaldehyse and stained with % uranyl acetate, run at kv). results: although sec did not show any prevalent peaks from early eluting regions previously shown to contain extracellular vesicles, these fractions (f -f , - min) were collected from d her + cell culture medium. interestingly, both nta and tem suggest that f and f contained evs as the isolated particles measured and nm, respectively and tem revealed spherical particles - nm in diameter. wb was unable to detect the ev associated protein alix (but was present in the whole cell lysate). soluble proteins and protein aggregates eluted late in the sec chromatogram ( min), with protein analysis (microbca), tem and wb confirming their presence. summary/conclusion: cf-sec is a promising method for ev isolation for pharmaceutical applications, but further work is needed to optimize the isolation process using Äkta start for these cell lines. customer stories from the ev core of university of helsinki introduction: the ev core, world's first ev-dedicated technology platform established in , is a joint venture of two extracellular vesicle (ev) research laboratories at university of helsinki. as an academic research/service facility, the ev core provides infrastructure, state-of-the-art and emerging ev-technologies for research groups, hospitals, companies and authorities in the ev-field. the ev core provides ev isolation, purification and characterization services and offers contacts to downstream analyses in other core facilities based on optimized ev protocols. here, we present and discuss the customer experiences and prospects with the aim to further develop ev core services. methods: our most wanted services are nanoparticle tracking analysis, electron microscopy, ev isolation and rna isolation and consultation. currently, the key down-stream analysis methods are (mi)rna sequencing, metabolomics, flow cytometry and functional assays. results: we present the stories from our customers starting with their research questions and need for the ev expertise/consultation and equipment. next, we show how the projects advanced and what types of ev core -derived or other downstream services helped them to achieve their aims. in the end, we will acknowledge the customers experience and current status of their research. summary/conclusion: narratives of customer stories are an effective starting point for fruitful discussions about the current status and next developments in the young ev service field. recent isev workshops: open, reproducible and standardized ev research (ghent, ) and evs in immunology (buenos aires, ) introduction: since its founding in , isev has sought to further extracellular vesicle research in various ways including scientific meetings. these events encompass annual meetings as well as smaller, topically focused workshops, with the first isev workshop (on rna and evs) organized in new york city in october, . in december, , the workshop "open, reproducible, and standardized ev research" was held in ghent, belgium. in march, , the workshop, "evs in immunology" was held in buenos aires, argentina, with a preceding education day. methods: the international organizing committees of the and isev workshops prepared scientific programs around key themes of ev rigour and standardization (ghent, belgium, workshop) and evs in immunology (buenos aires, argentina, workshop). abstract and application submissions were invited. applications were reviewed and ranked by panels of ev experts for each event, and participants were invited. results: the and workshops assembled a total of more than individuals for talks and discussions around the themes of rigour and standardization and evs in immunology. the buenos aires workshop was preceded by an education day, coordinated by the isev executive committees for education and science and meetings. during these two workshops, poster presentations were permitted for the first time, affording additional presentation and interaction opportunities. the rigour and standardization workshop also featured real-time discussant polling to facilitate discussion. summary/conclusion: isev workshops such as those addressing rigour and standardization (ghent, ) and evs in immunology (buenos aires, ) continue to provide opportunities for focused discussion of small groups of experts on key topics in the field. often followed by published products, isev workshops help to lead and coordinate progress in ev science. for future isev workshops, educational activities may again expand the reach of each event, while poster sessions and app-driven real-time responses should be considered for enhanced interactions and participant canvassing. ev journal club: exchanging pizza for a worldwide audience during covid- kenneth w. witwer johns hopkins university school of medicine, baltimore, usa introduction: a monthly journal club focused on extracellular vesicle science was established at johns hopkins university in , featuring lunch and presentations by academic and industry participants. when covid- prevented in-person meetings beginning in march, , the journal club was converted to a virtual, weekly format on the popular online meeting app zoom. the journal club has persisted despite initial problems with online vandalism. most sessions are also made public on a youtube channel, https:// www.youtube.com/c/extracellularvesicleclub. methods: weekly ev club sessions are arranged by the host. most focus on a specific manuscript related to evs, but some weeks feature presentations of published or soon-to-be-published research by the presenting authors. sessions are advertised one week to several days in advance on social media platforms such as linkedin, twitter, and facebook, asking interested parties to sign up to join a mailing list via surveymonkey. the log-in information is then sent to the mailing list. upon clicking the link, participants are placed in a virtual waiting room for vetting by the host and volunteers. after admission, all parties but the host and presenter are muted to avoid distractions. questions and comments may be placed in a chat box. contributions are monitored and compiled by the host and volunteers to build a question-and-answer session at the end of the presentation. recorded sessions-with or without editing as needed-are placed on the youtube channel for additional access. results: despite initial problems with online vandalism known as "zoombombing," the journal club has continued weekly during the covid- shutdown in the host country (us). an audience of between and individuals is typical. participants typically ask more questions than can be answered in a one-hour time frame. the online format also allows for debate-style events and polling of the audience. summary/conclusion: this ev journal club is an example of how online tools can be used to facilitate international scientific interactions. further development of such formats could provide alternative approaches for isev activities in the science, education, and communication areas. the study aim is to assess whether the exposure to pm and pm , , chosen as paradigmatic environmental stressors, could modify the composition of nasal microbiota (nm) and extracellular vesicle (ev signalling network, showing a role in allergic ar exacerbation). methods: nm analysis were performed on v -v s rrna gene regions amplified from upper-airway tracts of ar cases and healthy individual controls to perform nm analyses. ev size, concentration and cellular origin for each subject were assessed by nanoparticle tracking analysis (nta) and flow-cytometry (fc). information on daily pm and pm , concentrations at the municipality of residence in the days preceding nasal sampling (i.e. day − to day − ) was assigned to each subject by arcgis software. multivariable and logistic analyses were applied on nm, nta and fc outcomes. results: when taxonomy composition was considered, in controls actinobacteria ( . %) was the most represented, followed by firmicutes ( . %) and proteobacteria ( . %) while in cases proteobacteria were . %, actinobacteria were . % and firmicutes were . %. cases showed a higher concentration of all the investigated ev types, derived from platelets (cd +), activated endothelium (cd e+), monocytes (cd +), eosinophils (cd +), neutrophils (cd +), mastocytes (cd c+), epithelial cells (epcam+), gram+ bacteria (lipoteichoic acid+), gram-bacteria (lps+). the effect was greatest in the case of mastocytes evs which were increased . fold in cases versus controls (p < . ). evs were modified by pm exposure at several time lags. in particular, a negative association between pm and eosinophil evs was observed (beta = − , ; pvalue = , ). as we clustered subjects according to their nm, we observed this variable was a strong effect modifier of the association between pm exposure and ev release. summary/conclusion: our findings start to provide an insight on the effect of air pollution on evs, taking into account the effect of nm, in patients with ar. further research is necessary to disentangle the mechanism exerted by inhaled pollutants in modulating evs and nm, and therefore ar exacerbation. funding: gsk investigator sponsered study aryl hydrocarbon receptor activation induces the expression of specific microrrnas in th cells that are release into extracellular vesicules and associated with arthritis introduction: in rheumatoid arthritis (ra), an autoimmune disorder characterized by a chronic sinovial inflammation, smoking is a major risk factor contributing to disease progression, and poor response to therapy. th cell is actively involved in worsening smooking-associates inflammation mediated by aryl hydrocarbon receptor (ahr), a cytoplasmic transcription factor involved in xenobiotic metabolism. both, ahr and th cells, has important implications during ra development. considering that cigarette smoke is a potent epigenetic modifier, we hypothesized that ahr activation, by cigarette components, would transcribe specific micrornas in th cells as a molecular mechanism to exacerbate inflammation in arthritis. methods: microrna expression was evaluated by largescale approach or real-time pcr. c /bl and ahr null mice were submitted to arthritis experimental models and exposed or not to cigarrete smoke (ethical committee approved / ). extracellular vesicles (evs) were isolated by ultracentrifugation, and characterized by western blot and nanosight. rankl-induced osteoclasts (ocs) differentiation in vitro was stained for trap. inhibition of mirnas were performed using anti-mirs transfection. results: we identified a specific group of mirnas induced in th cells after ahr activation. during arthritis progression, the micrornas are expressed and increases after exposure to cigarette smoke. in the absence of ahr their levels were drastically reduced. interestingly, we found that these micrornas are released by th cells into evs, and are able to promote osteoclastogenesis. ocs differentiation in vitro increases in the presence of th -derived evs, and this process is reduced in the absence of micrornas. summary/conclusion: microrna-mediated gene regulation plays crucial roles in the immune system functions, and their abnormal expression is highly correlated with the pathogenesis of ra. evs are known to function in cell-to-cell communication and are able to transmit their contents and cause changes in the target cell. our findings demonstrate a new molecular mechanism by which cigarette smoke could aggravate inflammation in arthritis; through the activation of ahr receptor in th cells, inducing the transcription of specific micrornas that are released into evs, and act as pro-inflammatory mediators. introduction: chagas disease (cd) is caused by the flagellated protozoan t. cruzi. trypomastigote forms are capable of releasing extracellular vesicles (evs) that contain the major surface molecules of the parasite. the parasite has a complex life cycle that leads to it a rapid adaptation in the environmental changes in the hosts. however, the effects of stress on on evs release are not completely understood. objetive: we evaluated the release of evs by trypomastigotes incubated under different stress conditions and the immunomodulatory role of these evs in pre-activated bone marrow-derived macrophages (bmdm). methods: nanoparticle tracking analysis (nta) and scanning electron microscopy (sem) showed an increase in evs releasing by trypomastigotes at °c under acidic conditions, evs released was affected and triggered amastigogenesis process. results: treatment with sodium azide (nan ) also caused changes in the release of evs regarding size and concentration. nitrosative stress caused by sodium nitrite (in culture medium mildly acidic, ph . ; in this condition nano releases nitric oxide) stimulated an increase in production of evs by t. cruzi. when the parasites were treated with nm s-nitrosoglutathione (snog), we observed a reduction in size and concentration of vesiculate material by trypomastigotes. at a higher snog concentration ( µm), the concentration of the vesiculate material increased. t. cruzi-derived evs exposed to stress conditions increased the expression of inos, arg , il- and il- genes in ifn-γ and lps pre-activated bmms. summary/conclusion: results suggest that the viability and/or integrity of the parasite are necessary for the evs releasing. in those in vitro conditions they triggered a proinflammatory response in host cells. this may be a strategy developed by the parasite to favour its establishment in the host. funding: fapesp, cnpq, capes and fapemig ppm-x / . immuno-toxicological evaluation of human mesenchymal stem cell- introduction: mesenchymal stem cells (mscs) have been widely used to the field of autoimmune diseases or tissue regeneration therapy. recently, many research groups have reported that mscs showed their ability via secreted paracrine mediators including extracellular vesicles (evs) rather than cell-to-cell contact. mscs mainly exist on bone marrow, peripheral blood, umbilical cord and adipose and can mostly secrete evs. it has emerged that evs alone are responsible for the therapeutic effect of mscs in plenty of animal diseases models. hence, msc-derived evs may be used as an alternative msc-based therapy in regenerative medicine. methods: as part of safety programme for human therapeutics, we performed immunotoxicological assessment of evs obtained from human mscs (hevs) in mice and human peripheral blood mononuclear cells (hpbmcs). firstly, mice were treated intravenously with a negative control, a positive control (lps; . mg/kg), or low-dose ( x e paticles/head) and high-dose ( x e paticles/ head) of hevs every other day for days and then analysed lymphocyte subsets from collected spleen by facs. next, we treated the evs on hpbmcs for days with low conc. ( x e particles/ml), high conc. ( x e particles/ml), pma/ionomycin as a cell activator or cpt ( μm) as an apoptotic inducer. annexin v/pi and csfe were analysed by facs. results: as a result, splenic nk cells and b cells were slightly increased about ~ % in hevs-treated mice, without biological significance, compared with a positive control (lps) as an immunogenicity inducer. and there were no effects on serum levels of inflammatory cytokines in mice. in addition, hevs had no cytotoxic effect on hpbmcs at both low and high conc. under the culture medium with evs-depleted fbs, the hevs appeared minimal anti-apoptotic effect on hpbmcs. for the cfse assay, the hevs showed slight proliferation on hpbmcs and pbmc activation induced by pma/ionomycin. summary/conclusion: in conclusion, the hevs have little immuno-toxicological effects in mice and hpbmcs. further detailed studies to elucidate immunological response of hevs for development of human therapeutics are needed. funding: this research was supported by a grant ( mfds ) from ministry of food and drug safety. investigation of immune response to mesenchymal stem cell-derived extracellular vesicles in the cancer setting introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) are thought to be a fingerprint of the secreting cell and therefore may retain the cancer targeting and immune privilege of mscs. thus msc-evs hold immense potential as tumour-targeted therapeutics for breast cancer. the aim of this study was to determine whether msc-ev administration in tumour bearing immunocompetent animals would initiate an immune response. methods: evs were isolated from conditioned media of both human and murine bone marrow-derived mscs through sequential differential centrifugation, microfiltration and ultracentrifugation. evs were characterized by nanoparticle tracking analysis (nta), western blot and transmission electron microscopy (tem). x ( ) human or murine msc-evs were administered intravenously into t breast tumour bearing balb/c mice (n = ) and healthy controls (n = ). tumour tissue, draining lymph node and spleen were then harvested, dissociated and flow cytometry performed targeting markers associated with a range of immune cells including t-cells, macrophages and natural killer (nk) cells. results: evs were successfully isolated from murine and human mscs with the appropriate size of small evs (sevs: - nm) and morphology including a lipid bilayer observed by tem. evs expressed tetraspanins cd , cd , cd ; cytosolic protein tsg and were negative for calnexin. ev concentrations ranged from . x ( ) - . x ( )/ml. in order to study a range of immune cell populations two antibody panels were created using complimentary fluorescent dyes. the proportion of t-cells (cd +, cd +, cd +), neutrophils (gr- +, ly- c+), dendritic cells (cd c+), macrophages (cd b+, mhci+, mhcii+), nk cells (cd +) and b cells (cd +) remained stable in the tumour, draining lymph node and spleen of all tumour-bearing animals that received either human or murine msc-evs, with no significant change observed in any category. summary/conclusion: the data presented supports the hypothesis that msc-evs retain the immune privilege of the secretory cell, with human cell-derived evs illiciting no immune response in mice. this is encouraging and reinforces the potential for use of msc-evs in the therapeutic setting. introduction: mycobacterium avium (m. avium) is a slow growth rate non-tuberculous mycobacterium (ntm). m. avium infection is a severe global health problem. but the mechanisms of pathogenicity of m. avium are poorly understood. outer membrane vesicles (omvs) that traverse the cell wall and contain a varied bioactive components inculding dna, rna, protein and toxins. previous studies have suggested that these omvs are produced in vitro and during animal infection, but the role of omvs secretion during the interaction of m. avium with host cells remains unknown. methods: in this study, m. avium were grown in middlebrook h medium (m h ) supplemented with % (v/v) oadc enrichment and . % (v/v) glycero. m. avium omvs were isolated by ultracentrifugation method. characterization of omvs by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). the raw . murine macrophages were incubated with the m. avium omvs to analyse inflammatory response and production of nitric oxide (no) and reactive oxygen species (ros) of macrophage. results: in this study, we demonstrate by fluorescence microscopy that murine macrophages can phagocytosis omvs produced by m. avium. incubation of m. avium omvs with murine macrophages resulted in increased levels of extracellular tumour necrosis factor alpha (tnf-α), interleukin- β (il- β), terleukin- (il- ) and interleukin- (il- ). meanwhile omvs stimulated macrophages produce no and ros. introduction: hospital associated venous thromboembolism (ha-vte) in paediatric patients is the second most common contributor to harm in hospitalized children. platelet-endothelial interactions are integral to the formation of vte, especially in inflammatory conditions such as sepsis. small extracellular vesicles (sevs) have the ability to reprogramme target cell phenotypes via their microrna contents and are known to contribute to vte formation. we hypothesize that sepsis alters platelet-derived sev micrornas capable of net upregulation of vascular endothelial procoagulant and downregulation of anticoagulant pathways. methods: using a precipitation solution and size exclusion chromatography, we isolated sevs from platelet poor plasma of children admitted to the paediatric intensive care unit for sepsis and from healthy controls. we positively selected platelet-derived sevs using immunomagnetic isolation for cd b platelet antigen and confirmed selection using flow cytometry. microrna was profiled using affymetrix genechip mirna . array. results: microrna from sepsis patients (median age . years; iqr: . - and % female) with a median psofa score of (iqr: . - ) and from healthy controls (median age years; iqr: . - . and % female) was isolated and compared. in septic vs. healthy patients mirnas were differentially expressed (false discovery rate (fdr)< . ; fold change ≥| . |) affecting mrna pathways. in septic children, pathways affecting chemotaxis and cell movement of leukocytes were predicted to be activated with z-scores ≥ . summary/conclusion: we developed a method to successfully isolate platelet-derived sevs. sepsis alters the platelet-derived sev microrna profile in paediatric patients with sepsis. these micrornas are predicted to target chemotaxis and cell movement pathways, important contributors in the formation of ha-vte. further analysis into specifically targeted pathways should be conducted as a potential target for the prevention of ha-vte in sepsis. introduction: sjögren´s syndrome (ss) is a systemic autoimmune disease that mainly affects salivary and lacrimal glands. mechanisms of ss pathogenesis are poorly understood. it is thought that inflammation leads to destruction of exocrine glands, however the triggers of autoimmunity and the mechanisms by which inflammation drives immunopathology are not characterized. our work identifies t cell-exosomederived mir- - p as a pathogenic driver of immunopathology in ss. micrornas (mirnas) are endogenous small noncoding rna molecules that regulate the expression of target genes through translational repression of mrnas. through transcriptomic profiling studies our group had previously documented a significant upregulation of mir- - p in patient ss tissues and in serum exosomes. methods: structured search for target genes of mir- - p involved in salivary gland (sg) physiology was performed with mirdip . serca b, ryr and ac were selected for further validation and functional analysis. binding of the mirna was confirmed by luciferase reporter assays in hsg cell lines and human-derived primary epithelial cells. the mrna and protein levels of serca b, ryr and ac were determined by qpcr and western blot, respectively. to investigate the cell-specific distribution of mir- - p in relation to the expression levels of serca b, ryr , and ac , a double fluorescent in situ hybridization was performed. ca + signalling and camp levels were measured using fluorescent sensor. to isolate exosomes, the t cell medium and serum of ss-patients and healthy volunteers (hv) were collected. results: we show that mir- - p is over-expression in the sgs of ss-patients. next, we demonstrated that mir- - p is contained in exosomes in serum of sspatients significantly more than serum of hv. we also show that activated t cells secrete exosomes containing mir- - p which transfer into glandular cells and affecting intracellular ca + signalling, camp production and protein production by mir- - p targets (serca b, ryr and ac ). summary/conclusion: this study provides evidence for a functional role of the mir- - p in ss pathogenesis and promotes the concept that t cell-activation directly may impair epithelial cell function through secretion of mi-rna containing exosomes. treg-derived il -coated extracellular vesicles promote infectious tolerance (p ) subunits, yet the forms that il assumes and its role in peripheral tolerance, remain elusive. methods: we induce cba-specific, il -producing t regulatory (treg) cells in tregebi wt c bl/ reporter mice, and identify il producers by expression of ebi tdtom gene reporter, plus ebi and p proteins. results: curiously, both subunits of il were displayed on the surface of tolerogen-specific foxp + and foxp neg (itr ) t cells. furthermore, il producers, although rare, secrete ebi and p on extracellular vesicles (ev) targeting a -to -fold higher number of t and b lymphocytes, causing them to acquire surface il . this surface il is absent when ev/exosome production was inhibited, or if ebi is genetically deleted in treg cells. summary/conclusion: the unique ability of ev to coat bystander lymphocytes with il , promoting exhaustion in, and secondary suppression by, non-treg cells, identifies a novel mechanism of infectious tolerance. funding: nih grants r -ai - (to w.j.b.), r ca and p ca (to d.a.a.v.) and the university of wisconsin carbone cancer center support grant p ca . unique formulated dual targeting antigen specific and delivered mirna- gene regulating exosomes acting at the immune synapse to induce apc-derived secondary suppressive exosomes introduction: an exosome-apc circuit we uncovered may be applicable beyond skin immunity we study in mice. methods: high antigen dose tolerized cd + t cells make suppressive antigen-specific exosomes due to chosen surface antibody light chains that enable targeting antigen presenting cells (apc) antigen-specifically for delivery of also chosen inhibitory mirna- to mediate specific functional gene alterations. results: both antigen and gene specificity aspects are lent to naïve but activated exosomes by simple in vitro incubations alone. for mechanism, these primary exosomes bind antigen peptides in mhc on apc that in turn make secondary suppressive exosomes that act peptide/mhc-specifically on the effector t cells at the immune synapse. they transfer another mirna for strong prolonged inhibition of active delayed-type hypersensitivity (dth) for days even, when the primary mirna- -pos exosomes are administered orally at the height of the in vivo response, in a physiological dose. summary/conclusion: it is shown possible to induce therapeutic exosomes with ag targeting of choice due to placed ab on the surface and that also target specific gene functions of acceptor cells due to carriage of a selected mirna. this dual ag and gene-specific therapy has applications in treatment of cancer, autoimmunity and allergies. introduction: previously, our group characterized distinct populations of extracellular vesicle (ev) released from neutrophilic granulocytes: ev formed spontaneously (sev) and upon activation with opsonized particles (aev). the aev differs in protein cargo and its ability to inhibit bacterial growth. we described that mac- integrin (cr receptor) plays key role in the aev production and extracellular calcium supply is crucial in this signalization. in the present work, our aim was to investigate whether mac- activation or casignalling on their own are sufficient for the initiation of the aev biogeneis. methods: we isolated neutrophil derived evs from peripheral human blood and murine bone marrow by two-step centrifugation and filtration. we tested the effect of ca-ionophore and examined the ev production on c bi coated surface and in soluble form. we quantified the vesicles by flow cytometry and determined their protein content by bradford assay. we examined their antibacterial effect in parallel with optical density-based measurement and our flow cytometry based method. results: on c bi coated surface, we observed an increased ev production, and these evs possessed antibacterial capacity. however, in soluble condition, c bi did not induce further ev production, and these evs did not show any antibacterial property. we found that ca-ionophore initiated ev formation, but these ev did not show antibacterial effect. we observed ev production increase after ca-ionofore treatment both in the presence and in the absence of extracellular ca. the ca-ionophore slightly increased the opsonized particle induced ev production, but did not potentiate their antibacterial capacity. summary/conclusion: mac- activation is not just crucial, but sufficient in initiation of the aev biogenesis. clustering of this receptor is required. while the ca-signal is crucial, it is not sufficient in the generation of aevs. extracellular vesicles and their microrna cargo in retinal health and degeneration: mediators of homoeostasis, and immune modulation yvette s. m. wooff, adrian cioanca, riemke aggio-bruce, joshua chu-tan, ulrike schumann and riccardo natoli the australian national university, canberra, australia introduction: photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases such as age-related macular degeneration (amd). however, the molecular mechanisms regulating these biological processes are largely unknown. extracellular vesicles (ev) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. evs, including exosomes, encapsulate and transfer microrna (mirna) to recipient cells and in this way can modulate the environment of recipient cells. dysregulation of evs however is correlated to a loss of cellular homoeostasis and increased inflammation. in this work we investigated the role of isolated retinal small-medium sized ev (s-mev) in the regulation of homoeostasis and immune modulation in both the healthy and degenerating retina. methods: isolated s-mev from healthy and degenerative (photo-oxidative damaged) mouse retinas were characterized using dynamic light scattering, transmission electron microscopy and western blot, and quantified using nanotracking analysis. small rna-seq was used to characterize the mirna cargo of retinal s-mev isolated from healthy and degenerating retinas. finally, the effect of exosome inhibition on s-mev-mediated immune modulation was investigated using systemic daily administration of exosome inhibitor gw and analysed by in situ hybridization of s-mev-abundant mirna. electroretinography and immunohistochemistry were performed to assess functional and morphological changes to the retina as a result of exosome depletion. results: our results demonstrated an inverse correlation between s-mev concentration and photoreceptor survival, with decreased s-mev numbers following retinal degeneration. small rna-seq revealed that s-mevs contained uniquely enriched mirnas, however no differential composition in s-mev mirna cargo following photo-oxidative damage was observed. exosome inhibition using gw exacerbated photoreceptor degeneration, with reduced retinal function and increased levels of inflammation and cell death seen following photo-oxidative damage. further, reduced translocation of the photoreceptor-derived s-mev was demonstrated following exosome-inhibition in photo-oxidative damaged mice. summary/conclusion: taken together, we propose that retinal s-mev and their mirna cargo play an essential role in maintaining retinal homoeostasis through immune-modulation, and have the potential to be targeted using gene therapy for retinal degenerative diseases. impacts of agricultural dust exposure on human lung-resident mesenchymal stromal/stem cells and their extracellular vesicles introduction: agricultural dust is considered a high-risk occupational hazard by the cdc, with impacts reaching throughout the communities surrounding these industries, leading to increased incidence of respiratory illness and disease among individuals within this occupation and these communities. lung-resident mesenchymal stromal/stem cells (lr-msc) have an important role in maintaining homoeostasis in the lung, and mediating pro-and anti-inflammatory effects, particularly during exposure to inhaled irritants, like agricultural dust. one way in which these lr-msc promote lung homoeostasis is through the release of extracellular vesicles (ev), with a variety of cargo that elicit changes among target cells. we hypothesize that exposure to agricultural dust modifies the quantity and cargo of ev released by lr-msc to promote lung tissue homoeostasis. methods: primary human lung-resident mesenchymal stromal cells were exposed to extracts of dusts collected from swine confinement facilities (de) for or hrs and the media from these exposures were collected and enriched for ev by opti-prep density gradient ultracentrifugation. the quantity of these ev were assessed by nanoparticle tracking analysis. additionally, cytokine and chemokine release by lr-msc were analysed by enzyme-linked immuno assays. results: as assessed at hr following treatment, deexposed lr-msc released pro-inflammatory cytokines, il- and il- , with il- release reaching statistical significance at . %, . %, and % de concentrations (p = . , < . , and < . respectively) and il- trending a similar dose response but only statistically significant at % de (p = < . ). de exposure of lr-msc also induced changes in the lr-msc-derived ev populations when compared to vehicle control, where lr-msc released significantly more ev in the and % iodixanol fractions (p = < . and . , respectively) at hr following de treatment. alternatively, there were significantly less ev in the and % density fractions in the media of deexposed lr-msc versus vehicle control. summary/conclusion: following exposure to agricultural dusts, lr-msc-derived ev populations more likely consist of exosomes and ectosomes, which play an important role in promoting lung tissue homoeostasis during exposure-related pulmonary inflammation. introduction: during analyses of single extracellular vesicles (evs) by flow cytometry (fcm), particles below the detection limit may exceed the trigger threshold, which is called swarm detection and generates false-positive counts. serial dilutions are recommended to find the minimal dilution for which swarm detection is absent. however, because particle concentrations in plasma vary, the optimal dilution differs > -fold between donors, but it is unfeasible to do serial dilutions for each clinical sample. therefore, our aims are to ( ) develop a faster method to avoid swarm detection, and ( ) increase the number of detected evs per second. methods: we measured serial dilutions of cd stained evs in platelet free plasma (pfp), with and without spiking of fitc beads, by fcm (apogee a -micro). we triggered either on side scatter or fluorescence. results: for scatter triggering with our fcm, swarm detection consistently occurred for plasma samples exceeding a (total particle) count rate of , - , events/s. the cd + evs concentration scaled linearly over . orders of magnitude of the dilution and most donors required > -fold dilution to avoid swarm detection, thereby reducing cd + ev counts. for fluorescence triggering, the cd + evs concentration scaled linearly over > orders of magnitude of the dilution. for all donors, swarm detection was absent after -fold dilution (relative to pure plasma). the count rates of cd + evs were - -fold higher compared to scatter triggering. the spiked fitc beads confirmed that the median signals remained constant. summary/conclusion: we have developed two clinically applicable ways to avoid swarm detection. for scatter triggering, the count rate provides direct feedback on the presence of swarm detection in plasma samples. for fluorescence triggering, swarm detection was absent for all plasma samples diluted ≥ -fold and compared to scatter triggering, count rates of cd + evs were - fold higher, thereby improving statistical significance. funding: edwin van der pol is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . benchmarking flow cytometric analysis of nanoparticles: a cross-platform study for single extracellular vesicle detection introduction: despite flow cytometry being widely used to analyse cells in suspension, most commercial instruments lack sensitivity when measuring nanoparticles (nps) and extracellular vesicles (evs). furthermore, the use of appropriate reference materials (rms) for calibration and quality control are essential to compare results acquired with different instruments. to work towards successful clinical applications for ev biomarker profiling, benchmarking studies including state-of-the-art flow cytometers are required. we here investigated the ability of three different flow cytometers to detect nps and evs. methods: the instrument sensitivity of light scattering detection was evaluated by using synthetic nps of different sizes and refractive indices. fluorescent calibration was investigated by using molecules of equivalent soluble fluorophores (mesf) beads. biological recombinant evs (revs) were used to validate the detection and quantification of fluorescent evs in a side-by-side cross-platform study using an n nanoflow analyser (nanofcm), an optimized bd influx and a cytoflex lx. results: we found that when light scatter based detection was used, the nanofcm detected the smallest non-fluorescent nps, the bd influx was able to provide reliable fsc information from the smallest detected nps and the cytoflex performance was greatly improved by the use of violet-ssc. biological revs showed that the nanofcm could clearly resolve fluorescent evs while the bd influx and cytoflex were unable to fully resolve revs from background, although fluorescence threshold improved detection. in addition, our findings revealed that different concentrations are required to ensure single ev detection in these platforms. summary/conclusion: we identified several strengths and limitations for each platform with respect to single ev analysis. furthermore, our results showed that proper calibration and rms are of utmost importance to ensure reliable interpretation of ev flow cytometric data. caution when using membrane dyes for sequential extracellular vesicle analysis diana pham, michael wong, desmond pink and john lewis nanostics, edmonton, canada introduction: confirmation that particles detected by microflow cytometry are actually extracellular vesicles (evs), or at least membranous in composition, can be achieved through a variety of methods. positively staining particles with a membrane dye strongly suggest that the particle contains a membrane; loss of stain (or detection) after detergent solubilization of the membrane-dyed particles provides even stronger evidence that the particles were evs. it is important to recognize that the labelling protocol provided by the membrane dye manufacturer may not be ideal for all types of evcontaining biological samples, such as blood, urine, semen etc.). removal of excess dye from stained evs is very difficult and can be impractical depending on the nature of the experiment. however, this means that the potential for excess dye to contaminate subsequent sampling is high. therefore, it is important to determine optimal working concentrations and labelling conditions when using membrane dyes for ev detection to understand properties that may impact your analyses. methods: to assess the utility of membrane dyes, titration curves were generated to determine the optimal working concentrations of membrane dyes for ev detection in conditioned media and human serum samples. once the optimal concentration was determined the potential of dye carry-over from sample to sample during microflow cytometry detection was evaluated by tracking dye positive (dye+) particles in phosphate buffered saline (pbs) blanks and matched, unlabelled, sample replicates. results: we found that optimal concentration of any membrane dye is dependent on sample type. even with the inclusion of system washes to prevent sample carryover, there was carryover of low amounts of dye+ particles into sequentially analysed pbs blanks. if unstained samples were analysed following a stained sample, excess dye (or at least dye+ events) appeared in the data. a sample concentration effect was also seen; samples of lower concentrations were more susceptible to dye carryover. summary/conclusion: when using membrane dyes to stain evs in biological samples, especially if an autosampler is employed to run a series of tests, it is critical to determine the optimal concentration of dye for each type of sample, as excess dye can carry over to the next sample in the queue. in addition, determining the necessary steps to clean any excess dye following each sample run will improve the accuracy of ev detection and analyses. funding: nanostics alberta innovates alberta cancer foundation correlation between size and protein expression of single exosomes by combined atomic force and fluorescence microscopy introduction: there are no universal markers of extracellular vesicles, but often they are identified by the presence of tetraspanins in their membrane. based on this, products have been developed to precipitate or quantify evs by acting upon cd , cd , and cd . however, evs also carry proteins from their parent cells, and capturing evs based their presence allows for a more complete understanding of vesicle heterogeneity from a single cell type, and for evs derived from specific tissues to be enriched from other biofluids in support of biomarker assessment. for example, evs derived from the brain could be captured from the general population of serum evs for better assessment of cargo associated with proteinopathy. the goal of this study was to identify specific antibodies to capture and label evs bearing the neural markers cd , snap , α-synuclein, tau, and ncam. methods: the targets were overexpressed in hek t cells through transient transfection of plasmids (origene). media was conditioned for - hours, and then centrifuged to remove cell debris. cell lysates and concentrated conditioned media (cm) were analysed by western blot. unpurified cm, or cm after performing size exclusion chromatography (sec, izon), were analysed in the exoview r system. diluted cm was incubated on custom antibody microarray chips overnight. then the chips were labelled with a cocktail of labelled antibodies, washed and imaged. vesicles were counted, sized, and phenotyped. next, commercially available pooled human csf was analysed in a similar fashion to determine their abundance in a relevant biofluid. results: multiple antibody clones were tested in different combinations for capture and labelling for the five different neuronal enriched proteins of interest, and optimal combinations were identified. some markers were identified on particles > nm in size that were negative for tetraspanins, while others colocalized with tetraspanins. through comparing permeabilized and intact evs with and without sec to remove non-vesicular proteins, we found that tau could be on the vesicle surface, within the vesicle, and free in solution. summary/conclusion: the exoview platform can be customized to enable the detection of proteins of interest and to determine whether they are on the ev surface, intravesicular, or non-ev associated. methods: forty non-smoking male and female subjects ( - y) at moderate risk for cvd were recruited for the study. evs from platelet-free plasma (pfp) were isolated using size exclusion chromatography (sec). the concentration and size distribution of evs were measured by nanoparticle tracking analysis (nta) and flow cytometry (fcm). three ev markers, including annexin v for the circulating phosphatidylserinepositive (ps+) evs, cd for platelet-derived evs and cd for endothelial-derived evs were used for phenotyping. in addition, coagulation and fibrinolysis were assessed using a thrombodynamics analyser (hemacore). platelet aggregation to determine platelet function was assessed by a high-throughput platelet function assay with a wide range of concentrations of agonists, including adenine diphosphate (adp), collagen-related peptides (crp-xl), epinephrine, thrombin receptor activating peptide (trap- ) and u . the association between thrombogenic risk markers for cvd and ev numbers was tested by pearson's correlation coefficient and linear regression model using the statistical program, spss. results: circulating ev concentration with threshold of nm, measured by nta, were positively associated with coagulation-related risk markers, including rate of clot growth (r = . ; p = . ) and clot size at min (r = . ; p = . ). ps+ evs derived from endothelial cells, determined by fcm, were negatively associated with lysis onset time (r = − . ; p = . ), whereas they were found positively correlated with lysis progression (r = . ; p = . ). both mean and mode size of cevs, detected by nta, were significantly correlated with u -induced platelet aggregation (r = − . ; p = . , r = − . ; p = . , respectively). summary/conclusion: in subjects at moderate risk for cvd, cev numbers were positively related to rate of clot growth and clot size and size of cevs was negatively related to platelet activity. higher numbers of endothelial cell-derived ps+ cevs were associated with lower rates of fibrinolysis. this suggests that cevs promote clot growth and reduce fibrinolysis, and may therefore be an indicator for greater risk of cvd. beyond stem cells: extracellular vesicles from human induced pluripotent stem cells (hipsc) and hipsc-cardiomyocytes as therapeutic approaches for heart failure introduction: heart failure is caused by a variety of underlying diseases, the most common being myocardial infarction. initially regarded as an alternative to pharmacological approaches, stem cell transplantation has failed to demonstrate clinically meaningful results. instead, it has become increasingly apparent that the therapeutic effects of transplanted cells are largely mediated by their secretome, while mounting evidence suggests extracellular vesicles (evs) play a major role in cardiac repair. within this framework, evs from human induced pluripotent stem cells (hipsc) and hipsc-derived cardiomyocytes (hipsc-cm), hold a tremendous potential to treat cardiovascular disease. we isolated evs from conditioned culture media at key stages of the hipsc-cm differentiation and maturation processes, i.e. from hipsc (hipsc-ev), cardiac progenitors (cpc-ev), immature (cmi-ev) and mature (cmm-ev) cardiomyocytes, with the aim of studying their potential role as therapeutics, and whether their effectiveness was influenced by the state of their parent cell. methods: hipsc were differentiated into cardiomyocytes in a d culture approach, using the protocols developed by our group. ev isolation was performed on an iodixanol density gradient, and the evs were characterized in terms of particle size and particle size distribution, presence of ev-specific markers, and imaging through transmission electron microscopy. functional studies were performed using human umbilical vein endothelial cells (huvecs) to evaluate evuptake, cell migration and angiogenesis. results: evs from all hipsc and cardiac derivatives presented a typical cup-shaped morphology and expressed cd and cd . ev yield varied along differentiation, with a minimum for cpc and a maximum for cmi. pkh -labelled evs were uptake by huvecs, and colocalized with calnexin, a protein from the endoplasmic reticulum. wound healing assays showed an increased cell migration in huvecs treated with cardiomyocyte-derived evs, in comparison with control evs isolated from foetal bovine serum. summary/conclusion: our findings suggest a different ev secretion profile along cm differentiation and maturation, with preliminary assays showing ev functionality. ongoing work aims at elucidating the possible differences in function and cargo amongst these types of evs. endothelial cells differentially load and secrete extracellular vesiclederived micrornas into apical and basolateral compartments this may play a role in microcalcification in calcific aortic valve disease (cavd), but this is poorly understood. annexin a is thought to be a marker of membrane-derived evs, but because it can be found on the cytoplasmic or extracellular side of the plasma membrane, its localization within or on the surface of evs is unclear. the goal of this study was to determine whether annexin a is found on the surface of evs in two cell lines relevant to cavd, and develop an assay that can be used to determine whether this changes under pathogenic conditions. methods: evs were isolated by differential ultracentrifugation from the conditioned medium (cm) of smooth muscle cells (smc) and valvular interstitial cells (vic). total protein in the cell lysates and ev pellets was analysed by western blot. evs from cells treated with control sirna or anxa -sirna were enumerated and phenotyped using the exoview r platform. evs with surface expression of cd , cd , cd , and annexin a were captured using a customized antibody microarray chip. then evs were labelled with fluorescent antibodies to assess ev number, size, and colocalization of ev proteins. the knockdown of annexin a allowed us to assess the specificity of the selected annexin a antibody. results: the ev fraction was positive for cd , and lacked markers of other vesicle types. western blot on the ev pellet and supernatant in ± edta indicated that there is annexin a both on the surface of and within the evs. using the antibody microarray chips, numerous annexin a + evs were captured on the annexin a spots from the control cm, and there was a marked decrease in capture and labelling from anxa -sirna treated cells. under both conditions, vesicles were also captured on tetraspanin probes, with the greatest number captured on cd , then cd and cd . there was a significant population of annexin a + evs that was negative for tetraspanins. summary/conclusion: annexin a is found on the surface of evs. the assay developed in collaboration with nanoview biosciences is well suited for assessing the number and phenotype of annexin a + evs derived from smc and vic cell lines, which could provide a useful method for understanding ev populations in cavd patient cell lines. funding: this work was supported by hl and hl . possibility of exosomal micrornas associated with chronic limb-threatening ischaemia, the end stage of atherosclerosis, as a promising biomarker introduction: chronic limb-threatening ischaemia (clti), the end stage of peripheral artery disease (pad), has poor prognosis and is attributed to lifestyle disease. with increasing of atherosclerotic disease all over the world, establishment of biomarker for should play a pivotal role for early detection and preventing aggravation of the disease. the aim of this study is to explore the possibility of liquid biopsy for atherosclerotic disease by analysis of clti-associated exosomal micrornas. methods: clti due to pad was diagnosed by anklebrachial blood pressure index, skin perfusion pressure (< mmhg) and angiography. ten preoperative clti patients and control patients without pad were analysed (all patients with diabetes and % of patients had end-stage renal failure [esrd] ). to identify biomarkers associated with clti, exosomes were extracted from patient's serum after ultracentrifugation and total rna including small rna was isolated from the exosomes. the expression profile of exosomal micrornas associated with clti were evaluated using a next generation sequencing. results: forty-three exosomal mirnas associated with clti were identified. intriguingly, these mirnas were clearly categorized with esrd, which was well known as end-stage of life-style disease: these were stratified into micrornas for esrd patients and micrornas for non-esrd patients. since esrd is the most important factor significantly related to patient's prognosis in clti, exosomal micrornas reflected patient's comorbidity onto the expression profile. summary/conclusion: a portion of the expression profile of exosomal micrornas associated with clti was identified. exosomal microrna could be a biomarker to stratify patient's condition along with their comorbidities and is very promising for individualized diagnosis in atherosclerotic diseases with risk diversity. postoperative plasma exosomal mir- and mir- a signature in patients with left ventricular reverse remodelling after surgical mitral valve repair underwent implantation of a prosthetic mitral ring. lv remodelling was assessed by cardiac magnetic resonance imaging and pexos were isolated by optimized ultracentrifugation before surgery (t ) and six months after surgery (t ). isolated pexos were quantified by nanoparticle tracking analysis and mir- , mir- , mir- a, and mir- a were measured by rt-qpcr. the same analysis was performed on healthy subjects with normal cardiac function (n = ). local ethical committee approved the study (emigrate study, approval n° ) and informed consent was obtained from all patients. results: pexos levels at t were lower (− %, p = . ) in patients with worst postoperative lv function, while they were higher at t (+ %, p = . ) in patients with reversed lv remodelling after surgery. at t , the increase in pexos levels was associated to decreased heart mass index (− %, p = . ) and higher levels of exosomal mir- (+ %, p = . ) and mir- a (+ %, p = . ) were detected in patients with improved lv function. summary/conclusion: higher postoperative levels of pexos delivering mir- and a depict lv reverse remodelling after surgical mitral valve repair. monitoring of exosomal micrornas cargo might predict postoperative outcome in patients with mr. expression of lipocalin- (lcn ) in circulating extracellular vesicles (evs) and femoral plaque-derived evs of peripheral arterial disease patients. introduction: clinically, the drug resistance situation of acinetobacter baumannii is becoming increasingly serious, and its drug resistance has become a difficult problem for nosocomial infection and clinical treatment. in view of the relatively slow development of antibacterial drugs, exploring the resistance mechanism of acinetobacter baumannii is of great significance to improve bacterial resistance and help clinical treatment. studies have shown that outer membrane vesicles (omvs) can transmit resistance genes to mediate the spread of drug resistance, and recent studies have confirmed that high expression of efflux pumps play an important role in the multidrug resistance of a. baumannii. in this study, we want to explore whether the outer membrane vesicles of acinetobacter baumannii can transfer the efflux pump related substances. methods: first, ultracentrifugation and density gradient centrifugation were used to extract the omvs of acinetobacter baumannii antimicrobial-sensitive strains (atcc ) and antimicrobial-resistant strains. then, nanoparticle tracking analysis (nta) technology was used to analyse the particle size and distribution range of omvs. transmission electron microscopy (tem) was used to identify their morphology and structure. bradford method was used to determine the protein concentration of omvs. next, the omvs of antimicrobial-resistant strains were incubated with the antimicrobial-sensitive strains and then the drug susceptibility test was done to determine whether omvs of antimicrobial-resistant strains could transmit antimicrobial-resistance information to the antimicrobial-sensitive strains. finally, pcr, qpcr and mass spectrometry were used to determine whether the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. results: nanoparticle tracking analysis (nta) detected the concentration and size distribution of omvs of acinetobacter baumannii strains. it showed that the extracted omvs have a relatively uniform particle size and a size between - nm. tem showed that omvs had a typical vesicle structure. omvs coculture experiments showed that omvs of the antimicrobial-resistant strains can indeed pass resistance to the antimicrobial-sensitive strains. and the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. summary/conclusion: omvs of the antimicrobialresistant strains can indeed pass resistance to the antimicrobial-sensitive strains. the cause of acquiring antimicrobial resistance in sensitive strains may be caused by resistant strains passing efflux pump-related genes or proteins to sensitive strains. characterization of melanocytic extracellular vesicles during ageing of the choroid kelly coutant a , léo piquet a , nathan schoonjans b , philippe gros-louis a , julie bérubé c , stéphanie proulx a , alain r. brisson d and solange landreville a a université laval, quebec city, canada; b université de lille, lille, france; c centre de recherche du chu de québec-université laval, quebec city, canada; d université de bordeaux, bordeaux, france introduction: the choroid is located at the backside of the light-sensitive retina and is highly vascularized. it contains pigmented melanocytes, and their melanin protects them against oxidative stress. since ageing reduces the number of melanosomes in melanocytes and generates a stiffer extracellular environment, our hypothesis is that surrounding choroidal cells and the retinal pigment epithelium (rpe) are subject to more oxidative stress-related damages. this study aimed to characterize evs released by human choroidal melanocytes in the context of intercellular cooperation during ocular ageing. methods: melanocytic evs were recovered from the conditioned culture medium of young/old melanocytes grown on hydrogels of varying stiffness ( . - kpa) by differential centrifugation. the concentration and size distribution of melanocytic evs were determined by high-sensitivity flow cytometry. cryo-transmission electron microscopy combined with receptor-specific gold labelling were used to reveal their morphology, size and phenotype. the relative abundance of surface markers was evaluated with the exo-check exosome antibody array. the uptake of fluorescent melanocytic evs by the rpe and choroidal endothelial cells was assessed by confocal microscopy. results: choroidal melanocytes released evs positive for annexin- and the tetraspanin cd . young melanocytes produced more annexin- positive evs and evs larger than nm compared to older donors. the stromal stiffness impacted the concentration and size of melanocytic evs. we confirmed the uptake of melanocytic evs by endothelial and rpe cells. summary/conclusion: evs from choroidal melanocytes are internalized by surrounding endothelial cells and rpe. age-related stressors modify the phenotype of melanocytic evs. the identification of melanocytic factors that can protect retina/choroid cells from oxidative stress-induced cell death could lead to more efficient therapy for patients suffering from dry agerelated macular degeneration. introduction: owing to their proposed biocompatibility and ability to cross biological barriers, evs represent an attractive therapeutic delivery platform. however, evs are eminently heterogeneous. a better understanding of ev heterogeneity and its origins will allow for improved design of ev-based therapeutics. ev heterogeneity is mainly studied by focusing on distinct ev subpopulations. other sources of heterogeneity, such as heterogeneity within ev secreting cells themselves, have been investigated in lesser detail. in this study, we assessed the phenotypic drift of cell derived evs to explore the origins of ev heterogeneity and its potential impact. methods: three independent samples of two mda-mb- breast cancer cell sub-clones were cultured for six weeks. evs were harvested weekly and analysed using the macsplex exosome flow cytometry kit. at two time points the proteome of evs was analysed by lc-ms/ms mass spectrometry with subsequent gene ontology and reactome pathway analysis. results: the expression of over proteins was deregulated in evs derived from the two different cell clones. many de-regulated proteins were associated with biological processes predicted to affect potential ev toxicity (platelet activation, neutrophil degranulation, blood coagulation) and ev biological activity (antigen presentation, inflammation, tgf-beta/ mtor/wnt signalling). more surprisingly, within only two weeks, over ev proteins, many associated with immune modulation, apoptosis, interleukins, cytokines and cell signalling pathways (including those affecting t-cell/b-cell receptors) were de-regulated between the two ev isolation time points. summary/conclusion: results suggest that temporal changes can be observed in the ev proteome (potentially by clonal drift, epigenetic changes or cellular genomic instability) over short time periods. these changes could cause significant differences in biological effects and delivery capabilities between evs harvested from the same cells at different time points and conditions. in vivo tracking and biodistribution analysis of mesenchymal stem cellderived extracellular vesicles in a radiation injury murine model introduction: recent studies indicated that extracellular vesicles (evs) play key roles in intercellular communication and have great potential for clinical application. understanding the biodistribution of evs is therefore essential. our previous works have shown the ability of mesenchymal stem cell (msc)derived evs to protect haematopoietic cells from radiation damage. in this study, we evaluated the biodistribution of msc-evs in a radiated mouse model. methods: human msc-evs were harvested by ultracentrifugation and labelled with did lipid dye. the reliability of the labelling evs was confirmed by sucrose gradient fractionation analysis. the distribution of evs in radiation-exposed mice after ev intravenous administration were evaluated by fluorescence molecular tomography and further confirmed by flow cytometry and confocal microscopy analysis. results: we observed that did labelled msc-evs appeared highest in liver and spleen, lower in bone marrow in tibias, femurs, and spine, and were undetectable in heart, kidney and lung. we found the significantly increased msc-ev accumulation in spleen and bone marrow post-radiation appeared with an increase of uptake of msc-ev by cd b+ and f / + cells, but not b + cells, compared to those organs from non-irradiated mice. however, there was a predominant ev accumulation in lung and less accumulation in spleen and liver; in mice infused with human lung fibroblast cell derived evs (lfc-evs) and there was no significant lfc-evs accumulation change in the spleen or liver after radiation. we further found that increasing levels of irradiation caused a selective increase in vesicle homing to marrow and spleen. this accumulation of msc-evs at the site of injured bone marrow could be detected as early as hour after msc-ev injection and was not significantly different between and hrs. post-msc-ev injection. summary/conclusion: this study indicated the specific accumulation of ms-evs at the site of injury of haematopoietic tissue in radiation injury mice. funding: this work was supported by the nih grants uh tr , uh tr - s , p gm , and t hl . linking fat to colorectal cancer: extracellular vesicle crosstalk sheffield hallam university, sheffield, uk introduction: colorectal cancer is the third most common cancer worldwide, and fourth leading cause of malignancy related mortality. understanding the mechanisms of its growth and metastasis is key to elucidating new therapeutic targets and developing treatments in the clinical setting. epidemiological evidence indicates an increased risk of cancer in obese patients, pointing to bidirectional communication between colon and adipose cells. extracellular vesicles (evs) are small membrane enclosed packages released by cells, capable of transporting bioactive cargo from donor to recipient cells and inducing phenotypic changes. adipocytes are a key component of the tumour microenvironment and interactions between adipose tissue and tumour cells may be important in the growth and metastasis of cancer. in this study, we investigate the effects of colorectal cancer evs on adipocytes in vitro, and potential induction of dedifferentiation to a more fibroblastic, pro-inflammatory phenotype. methods: evs were isolated from sw and ht human colorectal cancer cell lines by differential ultracentrifugation and mature adipocytes generated by differentiation of the sgbs human pre-adipocyte cell line. adipocytes were treated with evs and their lipid content measured by oil red o to determine loss of lipids. inflammatory cytokine profile was measured by elisa to assess any increase in pro-inflammatory behaviour, and expression of late adipogenesis markers were determined by western blot. results: ev treatment was shown to reduce lipid accumulation in adipocytes, with up to % reduction in lipids observed at the µg/ml dose. treatment was also shown to reduce the expression of late adipogenesis markers, and increase secreted levels of proinflammatory cytokines il- and il- by over fold and fold respectively. these results provide evidence for colorectal cancer derived ev involvement in the dedifferentiation observed in cancer associated adipocytes in vivo, displaying an altered phenotype, releasing lipid energy stores to fuel tumour growth and increasing pro-inflammatory signalling. summary/conclusion: studies have shown colorectal cancer evs may be involved in signalling which induces functional changes in cells within the tumour microenvironment. our work indicates that ev mediated dedifferentiation of resident adipocytes may potentially contribute to a microenvironment favouring cancer cell growth and metastasis. further work aims to elucidate the specific ev cargo which mediates these effects. introduction: ageing is a major risk factor for many human diseases. it is a complex process that progressively compromises most of the biological functions of the organisms, resulting in an increased susceptibility to disease and death. senescence is a cellular phenotype characterized by a stable cell cycle arrest. senescent cells are accumulated in the body during ageing. it contributes to develop age-related diseases and cancer. the alteration in intercellular communication with age has been demonstrated to be due to senescent cells developing a phenomenon denominated senescenceassociated secretory phenotype (sasp). exosomes are small extracellular vesicles (sev) ( - nm) of endocytic origin whereas microvesicles are formed by shedding of the plasma membrane. they contain nucleic acids, proteins and lipid that generally reflect the status of the parental cell and can influence the behaviour of neighbouring cells. methods: in this study, we demonstrated that the small extracellular vesicles (sev) contribute for transmitting paracrine senescence to proliferative cells firstly, we evaluated the presence of exosome-like particles in the sev from senescent cells by detection of exosome markers (alix, tsg and cd ), transmission electronic microscopy (tem) and nanoparticle tracking analysis (nta). to determine that sev from senescent cells are mediators of the paracrine senescence, we performed functional assays using cre-loxp reporter system and high-throughput results: besides, we confirmed at a single-cell level that the proliferative cells internalizing sev from senescent cells activate senescence process using the cre-reporter system. sev protein analysis from senescent cells by mass spectrometry (ms) and validation of top candidates using a functional sirna screen identify interferon induced transmembrane protein (ifitm ), a component of non-canonical interferon (ifn) pathway, as partially responsible for transmitting senescence to proliferative cells. summary/conclusion: in conclusion, we found that sev are regulators of paracrine senescence and ifitm contained in senescent sev has an important role in the intercellular communication mediated through sev during cellular senescence . bin wu a , lei guan a , ye xu a , likang chin a , ting li a , youhai chen a , gordon mills b , jinqi ren a , ravi radhakrishnan a , rebecca wells a and wei guo a a university of pennsylvania, philadelphia, usa; b oregon health & science university, portland, usa introduction: extracellular matrix (ecm) remodelling and stiffening are associated with solid tumour progression. stiff ecm promotes cell proliferation, epithelial-to-mesenchymal transition (emt), metastasis and chemoresistance. hepatocellular carcinoma (hcc) appears frequently in patients with liver cirrhosis or fibrosis while the mechanism remains unclear. exosomes have been determined to serve as messengers to mediate intercellular communication and influence the extracellular. tumour-derived exosomes have been shown to influence tumour progression, metastasis, drug resistance, angiogenesis and immune regulation. thus, determining whether exosomes provide a mechanism by which stiff matrix modulates tumour microenvironment for tumour progression opens a new way to understand cirrhosis and oncogenesis. here we identified the molecular mechanism of matrix stiffening induced exosome secretion and showed the different effect of exosomes induced by soft or stiff matrix on tumorigenesis. methods: huh cells were cultured on acrylamide gels with the stiffness was modulated to pa (soft) or k pa (stiff). the exosomes in conditioned media were collected and analysed by nanoparticle trafficking analysis (nta) and immunoblotting. protein expression level in cells was screened by reverse phase protein array (rppa). inhibitor or shrna were used to inhibit target proteins function. in vitro phosphorylation and gef assay were used to verify rabin phosphorylation and activation. exosomes from cells on soft or stiff matrix were injected into mice to study their effect on tumour growth. results: ( ) stiff matrix promoted exosomes secretion. ( ) akt was activated by stiff matrix and was required for exosome secretion. summary/conclusion: matrix stiffening promotes exosome secretion via akt-rabin -rab pathway, contributing to tumorigenesis. tridimensional fibroblast culture revealed a novel exososome-dependent extracellular matrix secretion mechanism vincent clément a , bastien paré b , cassandra goulet a , thiéry de serres-bérard a , stéphane bolduc a , françois berthod a and françois gros-louis a a université laval, québec, canada; b norgen biotek corp., thorold, canada introduction: the extracellular matrix (ecm) is constituted of a variety of proteins and polysaccharides that are secreted locally and assembled into a thick d meshwork to provide biophysical and biochemical support to the surrounding cells, and regulate numerous cellular functions such as adhesion, migration and proliferation. dysregulation of ecm components or aberrant ecm remodelling can lead to various pathologies, as well as to play important roles in wound healing. although ecm secretion pathways are still largely unknown, the current paradigm is that ecmassociated proteins are synthesized in the endoplasmic reticulum and transported via the endosomes to the golgi apparatus en route to the cell surface and released by exocytosis. methods: to study ecm secretion pathway, we used dimensional ( d) cultured fibroblasts. this culture method technique has been used widely to generate tissue-engineered self-assembled stromal tissues, free of exogenous materials, and rely on long-term supplementation of sodium ascorbate into the culture medium. non-cancerous fibroblasts, grown in conventional two-dimensional ( d) cellular cultures, are known to be a poor source of secreted exosomes when compared to cancerous fibroblasts. results: here, we provide evidence that non-cancerous dermal fibroblasts can secrete high amounts of exosomes, containing different ecm proteins, when cultivated in a d fashion. we also demonstrated that dermal fibroblast-derived exosomes had the capacity to travel from one cell to another, induce cellular migration and promote wound healing. summary/conclusion: altogether, these findings reveal a novel exosome-dependent ecm deposition mechanism and suggest that the use of d-fibroblast cellular culture may emerge as an innovative approach in precision medicine to better study the role of patient-derived exosomes and ecm proteins in the establishment of cellular microenvironment in health and disease. anthony yan-tang. wu a , charles lai, yun-chieh sung b , steven t. chou c , vanessa guo c , jasper c. chien c , john j. ko c , alan l. yang c , ju-chen chuang c , hsi-chien huang b , syuan wu c , meng-ru ho d , maria ericsson e , wan-wan lin f , koji ueda g , yunching chen h , chantal hoi yin cheung i and hsueh-fen juan j introduction: bionanoparticles including extracellular vesicles and exomeres (collectively termed evs), have been shown to play significant roles in diseases and therapeutic applications. however, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a limited suitable method. methods: we developed a bioluminescence resonance energy transfer (bret)-based reporter, palmgret, to enable pan-bionanoparticle labelling ranging from exomeres (< nm) to small (< nm) and medium and large (> nm) evs and larger evs (> nm). results: palmgret emits robust, sustained signals and allows the visualization, tracking and quantification of bionanoparticles from whole-animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, bret, and fluorescence. using palmgret, we show that evs released by lung metastatic hepatocellular carcinoma (hcc) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. ev proteomics identified hcc-ev lung tropic protein candidates associated with cancer progression, in which slco a and clic expression on non-tropic evs conferred lung-tropism, while cd gave spleen tropism. our results further demonstrate that redirected lung tropism decreases ev distribution to the liver, whereas the spleen tropism significantly reduces over time delivery to most major organs distribution including the liver and kidney. summary/conclusion: we established a multimodal and multi-resolution palmbret method to enable pan-bionanoparticle labelling and imaging and therefore quantification in live cells, whole animals, and preserved tissues. the method can resolve the intricate spatiotemporal dynamics of evs. palmgret revealed that evs derived from lung metastatic hcc are lung tropic, and the tropism can be conferred to non-lungtropic ev- t by decorating evs with identified hcc-ev membrane proteins. importantly, the enhanced ev delivery to tropic organs also significantly alters its distribution to other major organs. our findings suggest that the dynamics of ev biodistribution and targeted design should be investigated at the organ systems level in ev biology and therapeutic developments, respectively. tracking mesenchymal stem cell-derived extracellular vesicles (evs) in a in vivo cancer model introduction: small extracellular vesicles (sevs) are nanoparticles ( - mn) encircled by a phospholipid bilayer, derived from the endocytic pathway and released by all cells. sevs have an inherent role in cell communication and deliver cargo to target cells. mesenchymal stem cells (mscs) and have a natural ability to home to tumours and metastases while avoiding the host immune response. it is hypothesised that msc derived sevs (msc-sevs) also possess tumourhoming and immune-evading capacities therefore could provide a novel targeted delivery vehicle for treatment of cancer. it is imperative to elucidate msc-sevs migratory itinerary in vivo to support translation to the clinical setting. methods: this study aimed to image the interaction of labelled msc-sevs with cancer cells in real time in vivo. sevs were isolated from wildtype mscs and mscs with stably expressing red fluorescent protein (rfp) (via lentivirus) by the combined techniques of differential centrifugation, microfiltration and ultracentrifugation. isolated sevs were extensively characterised by transmission electron microscopy (tem), nanoparticle tracking analysis and western blot. nod scid gamma (nsg) mice with dorsal skinfold window chamber (dsfwc) were injected with either mda-mb- luciferase (luc) expressing cells or ht- -luc cells. bioluminescence imaging was performed to confirm tumour formation. a dose of x ^ msc-rfp-sevs was directly added to the window chamber and rfp expression detected using a microscope with rfp filter attachments. x ^ evs were incubated with the radionuclide, technetium- m tagged duramycin ( mtc-dur) for minutes at room temperature. excess radiolabel was removed using exosome spin column (invitrogen™). the mtc-dur-sevs were then added directly to the window chamber and charged particle imaging carried out. results: hours post-administration; the rfp signal was localised at the tumour site. radiolabelled sev signal could be detected minutes and hours after administration. msc-sevs were successfully detected at the tumour site following direct administration using two different tagging and imaging approaches. summary/conclusion: this promising preliminary data supports the potential of this approach for tracking msc-sev migration in vivo. future studies will investigate systemic tracking of msc-sev migration. vaughn garcia ; aejez sayeed ; rachel derita ; shiv ram krishn ; peter a. introduction: tumor-derived small extracellular vesicles (sevs) have emerged recently as mediators of tumorigenesis. however, the role of sevs in response to irradiation, a widely used therapy in prostate cancer, is not fully understood. methods: our study involved the tramp mouse model of prostate cancer. we used plasma sevs isolated using differential ultra-centrifugation and further isolated using iodixanol gradient fractionation. we also used nanoparticle tracking analysis (nta) to analyze sevs. mouse pelvises were irradiated using gy, for consecutive days. results: we first observed that upon pelvic irradiation of tramp mice, the levels of the signaling oncogene c-src are reduced in plasma-derived sevs, while the average size of sevs is increased from - nms to - nms. furthermore, we show that the sevs from irradiated cells lose the ability to stimulate anchorage independent growth and migration of recipient cancer cells. additionally, sevs from irradiated mice increase the amount of dna damage in recipient cancer cells. summary/conclusion: overall, our data show that irradiation of tramp mice (and prostate cancer cells) significantly reduces the pro-metastatic and pro-anchorage-independent growth potential of sevs when tested on human cells. changes to the composition and behavior of a cancer cell sev population via radiation therapy offers promise for future therapeutic approaches for prostate cancer. introduction: there are emerging physiological and pathological functions of extracellular vesicles (evs) in neurodegenerative diseases including alzheimer's disease (ad). brain derived-evs contain pathogenic proteins, such as tau, amyloid beta (aβ), which have been reported to contribute to cell-to-cell propagation in those diseases. investigation of the brain-derived ev cargo, therefore, is important to further understand the mechanisms of progression in neurodegenerative diseases. we developed the ev separation method from unfixed frozen mouse and human brain tissues and assessed the protein composition. methods: to establish the ev separation method, we separated evs from frozen mouse brain tissue using sucrose density gradient ultracentrifugation (sg-uc) or size exclusion chromatography to compare the results from the particle number, morphology and protein profiling by nta, tem and mass spectrometry. evs were then separated from cortical grey matter of ad (n = ) and control (n = ) by sg-uc. tau and aβ in the evs were measured by immunoassay. differentially expressed ev proteins were observed by quantitative proteomics employing machine learning. results: the separated evs were enriched in ev molecules and devoid of contaminant proteins by sg-uc, showing our method was successful. the levels of ps tau and aβ - were significantly increased in ad evs. annexin a (anxa ), neurosecretory protein vgf, neuronal membrane glycoprotein m -a (gpm a), and alpha-centractin (actz) were differentially expressed in ad evs. a combination of these proteins were confirmed to predict ad with the % accuracy by machine learning. summary/conclusion: these data suggest our method were suitable for the separation of brain-derived evs and ev anxa , vgf, gpm a and actz can be potential biomarkers for monitoring the progression of ad. edta stabilizes the concentrations of extracellular vesicles during blood collection introduction: to establish reliable biorepositories for research on extracellular vesicles (evs) as disease biomarkers, the release of evs during blood collection and handling must be avoided. currently, citrate is recommended as the anticoagulant for blood ev research, but citrate does not inhibit the release of evs from activated platelets. the release of platelet-derived evs excludes pneumatic tube transport and makes assays time dependent, thereby limiting clinical compatibility. therefore, we aim to stabilize the release of platelet ev concentrations. methods: blood samples were collected from healthy individuals and subjected to common circumstances known to induce platelet activation. blood was (i) incubated with or without thrombin receptor-activating peptide (trap; n = ), a potent platelet activator, (ii) send to the lab by a routine blood transport (pneumatic tube system; n = ), and (iii) stored at room temperature or at °c for hours (n = ). the concentrations of evs from platelets (cd +), activated platelets (p-selectin+), erythrocytes (cd a+), and leukocytes (cd +) were determined by flow cytometry (apogee a -micro). results: following activation by trap, concentrations of platelet-derived and activated platelet-derived evs increased . -fold and . -fold in citrate-anticoagulated blood, compared to . -fold and . -fold in edta-anticoagulated blood (edta vs citrate: p = . and p = . , respectively). preliminary data show that during pneumatic tube transport and routine sample handling, both platelet-and activated platelet-derived evs were more stable in edta compared to citrate. the concentrations of evs from erythrocytes and leukocytes were unaffected under all studied conditions. summary/conclusion: to conclude, edta stabilizes platelet ev concentrations during and after blood collection, which would facilitate pneumatic tube transport, enhance reliability and thereby improves the establishment of reliable biorepositories for ev research. introduction: cancer-cell secreted extracellular vesicles, called exosomes, are an emerging biomarker for cancer liquid biopsy. profiling of cancer-associated exosomes usually required lengthy, and multi-step procedures; therefore simple and easy-setup sensing methods are urgently needed for diagnosing cancer in a timely manner. chirality, the foundational property of all biomolecules, including exosomal proteins, can be utilized for exosome detection and differentiation using recent advances in chiral nanostructures. we found that microfluidic sensors can be successfully implemented for successful detection of cancer-associated exosomes taking advantage of unusually high circular dichroism (cd) of chiral gold nanoparticles (aunps). circular dichroism-based exosome (cdexo) detection utilizes chiroplasmonic enhancement of cd signatures of cancer-associated exosomes. we first synthesized donut-shaped aunps conjugated with l-cysteine and immobilized the aunps on a glass slide using a layer-by-layer assembly. the aunps on slide glass were surface functionalized by the standard biotin-avidin reaction after mua treatment. biotinylated annexin v marker, targeting phosphatidylserine (ps) expression on cancer-associated exosomes, was conjugated to the aunp surface. μl of exosome samples from cancer cells (a and h ) or normal cells (mrc ) were injected into the pdms microfluidic device and incubated for minutes. the cd signal before and after exosome exposure was monitored, compared, and systematically analysed as a rapid technique for the detection of exosomes with high sensitivity. results: we showed that the cdexo signals from cancer exosomes showed . folds absolute cd peak value change and . folds shift, respectively, compared to that of healthy exosomes. importantly, the cdexo sensing method takes less than mins in terms of total scanning time and requires minimal sample volumes. from the preclinical studies using blood samples from cancer patients and healthy donors, we found that cancer patients show stronger band shift and signal change comparing to that of healthy donors, implying our platform could be used for cancer diagnosis. summary/conclusion: this new versatile and sensitive method based on chiroplasmonic exosome detection paves the way to profiling disease-associated exosomes in a timely manner for minimal volumes of liquid biopsies. ev classification and fractionation strategy using surface charge labelling takanori ichiki a , hiroaki takehara a , hirofumi shiono b and hiromi kuramochi a a the university of tokyo, bunkyo, japan; b innovation center of nanomedicine, bunkyo, japan introduction: the development of new classification technology is required based on the evaluation of physicochemical properties of exosome surfaces and the diversity of constituent molecules. in this presentation, we present the electric charge activated exosome sorting platform comprising microfluidic device technology and electric charge labelling technique. methods: the single nanoparticle analysis platform, which has been developed by our research group, images rayleigh scattered light (elastically scattered light) obtained by irradiating nanoparticles with convergent laser light and provides information of individual particles by image processing. the method that utilizes electrokinetic phenomena, unlike the method using fluorescent labels, measures the properties of the particle surface without serious difficulty in principle even if the particle size is on the order of tens nanometres, and further enables to perform fractionation. since the number of particles usually handled in exosome research or its envisioned application is enormous, it is not realistic to take an approach such as a cell sorter in which particles are sequentially manipulated one by one following the measurement results of individual particles. results: particles receive attraction or repulsion by an external field according to the charge density on the surface, so there is no need to control the external force, and it is possible to design a device that can autonomously fractionate particles according to the difference in zeta potential. summary/conclusion: in conclusion, we have proposed and demonstrated the new concept of electric charge activated ev sorter. funding: this research was partially supported by the center of innovation program (coi stream) from the japan science and technology agency. high throughput exosome analysis by using reversible microfluidic electrochemical sensor system introduction: exosome is one of the important extracellular vesicles (evs) released from parental cells and it contains various types of molecular cargos from its original cell including proteins, messenger rna (mrna), and micro rna (mirnas) [ ] . the exosomes have recently emerged as biomarkers for early stage cancer detection because the number of exosomes originated from cancerous cells are significantly higher than those from normal cells [ ] . since many different types of exosomes exist in the whole blood, it is necessary to isolate and detect disease-specific exosomes. for this reason, the isolation and the detection of exosomes is an important research issue and has been studied by many groups. however, limitations such as low throughput and low recovery still make it difficult to use exosomes in diagnostics and therapeutics. methods: in this study, we developed an integrated microfluidic electrochemical biosensor to extract plasma from whole blood and subsequently detect cancer related exosomes in a continuous manner. this consists of two parts. the first part is a channel for extracting plasma containing exosomes from whole blood, and the second part is a channel combined with an electrochemical sensor for multiple detection of various exosomes in the extracted plasma. previously, a multi-orifice flow fractionation (moff) channel that consists of a series of expansion and contraction structures has been developed in our group. in this channel, the blood cells are moved to sides of channels by hydrodynamic forces and then are eliminated to outlets. at this time, the plasma is moved to the electrochemical sensor part, the exosomes in the plasma are captured to the electrodes immobilized with the specific antibodies and are quantified the amount of cancer-related exosomes. results: using this chip, blood cells were eliminated from the whole blood with over % of separation efficiency at µl/min flow rate and exosomes were collected continuously with high recovery (~ %). in order to quantify various types of exosomes, a labelfree electrochemical biosensor with electrochemical impedance spectroscopy (eis) was used for the continuous detection of exosomes. the limit of detection was x ^ exosomes/ml. summary/conclusion: the developed device is an integrated device capable of separating exosomes from whole blood with high purity and quantitating exosomes through the electrochemical sensor in a continuous manner. , , ) . the development of highthroughput techniques capable of simultaneously monitoring physical and biochemical properties of evs would significantly simplify and accelerate the characterization process. in this context, microfluidic technology is emerging as an attractive platform. here, we present a microfluidic device based on the combination of diffusion sizing and multi-wavelength fluorescence detection to simultaneously provide information on ev size, concentration and composition. methods: the diffusion of evs in the microfluidic channel provides information on their size distribution, and four different staining protocols with high signalto-noise ratios track different ev native molecules. evs are separated from unbound fluorophores directly during the microfluidic analysis, therefore avoiding the need for sample pretreatments and allowing to operate the device as a single-step immunoassay. results: the microfluidic device coupled with complementary staining techniques allows to individually detect and size particle populations with different ev components such as lipids, primary amines and the ev marker cd . we demonstrate that this approach can probe the abundance of ev-specific markers and impurities such as lipoproteins with high throughput and low sample consumption. summary/conclusion: we present a microfluidic technique capable of characterizing and quantifying evs at low costs, in a time-scale of minutes and requiring only up to µl of non-pretreated sample. this method is an important complementary tool to the current array of biophysical methods for ev characterization, in particular for high-throughput screening applications. funding: h -eu. . . -fet open programme via the grant agreement . immunomagnetic isolation of specific subpopulations of exosomes for liquid biopsy via nano-architected porous materials introduction: exosomes offer the potential to reveal significant biological information in many areas of clinical importance by virtue of their rna contents and protein surface markers. this abstract reports the fabrication of a device for high throughput targeted immunomagnetic capture of exosomes via the use of highly-ordered nano-architected porous metal lattice materials. methods: we have invented a fabrication technique to precisely make millions of nanoscale exosome sorting devices that can operate on unprocessed plasma. each nanoscale device can precisely sort targeted exosomes from background vesicles but is too slow for practical use individually. however, the operation of millions of these devices in parallel preserves the precision of nanoscale sorting while also enabling high throughput and robust use on raw plasma samples. the metal lattice within which these devices are contained is assembled via metal electroplating onto a selfassembled polystyrene bead lattice with face-centred cubic (fcc) symmetry with nanometre pores. the devices feature a conformally-coated layer of nickel-iron with gold passivation atop a base layer of nickel, resulting in a lattice of millions of nanoscale pores capable of magnetic sorting of exosomes tagged via surface-marker-based immunomagnetic labelling with magnetic nanoparticles. results: compared to our previous work on immunomagnetic exosome capture via commercial track-etched membranes (tempo), this device offers superior capture due to increased surface pore density (> x) and three-dimensional pore density (> x) alongside lower required sample volume due to decreased noncapturing volume in the device. finite-element analysis simulations show that strong magnetophoretic traps emerge at the pore boundaries in this structure between higher-permeability metals such as nickeliron permalloy and the lower-permeability sample fluid in the device. preliminary experimental data shows that this device can isolate iron nanoparticles in solution with > x enrichment from input and x capture efficacy versus tempo. summary/conclusion: current methods of exosome isolation such as ultracentrifugation and column chromatography all suffer from low throughput and limited yield. the application of inverse opal materials towards exosome capture offers the potential for isolation of specific exosome populations from very low clinical sample volumes or sparse biological signals. micropatterned growth surface topography affects extracellular vesicle production colin l. hisey a , james hearn b , yohanes nursalim a , vanessa chang a , cherie blenkiron a and lawrence w. chamley a a the university of auckland, auckland, new zealand; b university of auckland, grafton, new zealand introduction: extracellular vesicles are micro and nanoscale packages released by all cells and play an important role in cell-to-cell communication by shuttling biomolecules to nearby and distant cells. however, producing enough evs for many in vitro studies using conventional tissue culture techniques can be challenging, and despite the success of some bioreactors in increasing ev-production, it is still unknown how many independent culture conditions like growth surface topography can alter the production and content of evs. methods: standard mm petri dishes were patterned with µm tall polystyrene microtracks spaced by , and µm across a mm area using standard microfabrication techniques including photolithography, soft lithography and microtransfer printing. the micropatterns were characterized with sem and profilometry, then activated with oxygen plasma and uv sterilized. mdamb cells were seeded onto patterned and smooth (control) dishes and grown in serum-free media for the final hours of culture. evs were isolated using sequential ultracentrifugation of conditioned media and characterized using nta, tem and western blot. cell morphology was imaged using immunocytochemistry and single cell migration was characterized using time-lapse microscopy and manual single cell tracking in fiji. results: we demonstrate the simple and repeatable fabrication of microtracks across a large surface area in order to culture cells on topographically patterned growth surfaces. furthermore, we show that the µm spacing produced significantly more evs than other patterns as well as the highest cell aspect ratio and average single cell migration speed (p < . ). summary/conclusion: these findings have implications in both biomanufacturing of evs and potentially in enhancing the biomimicry of evs produced in vitro. however, further experimentation to assess the differences in cargo on patterned growth surface topographies compared to conventional methods is still required. funding: this project was funded by the maurice and phyllis paykel trust. using miscroscale thermophoresis and surface plasmon resonance to measure the interactions of extracellular vesicles mst is a quick method, easy to handle, has a low sample consumption, has no limitation on molecule size, and enables measurements in solution, either in various buffers or complex biological liquids. these properties make mst an interesting tool for research of extracellular vesicles (evs); therefore, our aim is to apply this method to evs. methods: evs were isolated from jurkat cell line by differential centrifugation. microscale thermophoresis (mst) and surface plasmon resonance (spr) were used to analyse the interaction between antibody and evs. results: we have demonstrated that interactions of evs with antibodies could be analysed by mst. however, the tiny glass capillaries for sample mounting represent a challenge due to adhesion of evs to their surface. we have tested commercial capillaries as well as prepared capillaries in house coated by liposomes or bovine serum albumin. the interactions between evs and antibodies were confirmed by surface plasmon resonance (spr), which is an established method for studying the interactions of evs. introduction: the isolation of extracellular vesicles (evs) from cell culture supernatants and complex body fluids, such as blood and urine, is of high importance for ev research as well as for future medical applications in diagnostics and therapy. nevertheless, it is still challenging to reach the desired recovery, purity and specificity due to many manual and time intensive sample preparation steps. conventional centrifugation for ev isolation or sample preparation prior to affinity-based separation methods can damage evs and cells, leading to misinterpretation of results or inactive evs. alternative field flow fractionation methods employing acoustic fields are highly promising, but so far limited to laboratory usage, based on a complex (moulding) fabrication and/or hardly reproducible. here, we present an innovative surface acoustic wave (saw)-based acoustofluidic device for gentle sorting of cells and particles. methods: our device consists of interdigital transducers patterned on a piezoelectric substrate generating saw propagating on the substrate surface. upon interaction of saw with our on-chip structured, fluid-loaden microchannels, an acoustic pressure field is developed across the fluid wherein particles are suspended. this pressure field can be employed to simply manipulate cells and particles based on their intrinsic properties, such as size, density and compressibility in continuous flow. the device is manufactured using precise and low-cost microtechnological methods and is suitable for reproducible mass fabrication. results: we demonstrated the separation of blood components, i.e. the sorting of erythrocytes and thrombocytes. furthermore, we could also show results on thrombocyte activation indicating a gentle separation without damaging these shear-sensitive cells, as well as first results on plasma separation from whole blood samples and nanoparticle sorting. summary/conclusion: our unique acoustofluidic sorting technology for complex suspensions has the potential to overcome the need for time-effective, cheap and gentle separation of evs. funding: this work was supported by efre infrapro project "champ: chip-based acoustofluidic medtech platform". nanophotonic platform for cancer-associated exosomal microrna detection introduction: exosomes have an important role in intercellular communication at physiological and pathological processes. their cargo includes micrornas (mirs), single-stranded non-coding rnas, involved in alterations on recipient cells, such as development of tumourous phenotype and metastasis. more particularly, mir- excels due to its association with several cancers. determining exosomal mirs as cancer indicators demands selective and accurate methods, which are not currently available or entail high costs. colorimetric photonic-based assays are a promising label-free alternative, which dismisses complex apparatus for signal reading since biorecognition is detected by colour change. moreover, the clinical and economic systems have also been demanding a decrease on the green footprint of biosensors, requirement fulfilled with naturally derived biomaterials. methods: herein, the biosensor is constructed on a biopolymer matrix to meet the requirements of an eco-friendly disposable device, and it is based on a photonic structure obtained by imprinting a nanopattern on the polymer surface. then, the surface is functionalized with the complementary oligonucleotide sequence of mir- as sensing probe. a labelfree detection is thus envisioned and the sensor performance is evaluated by changes in the optical properties when the target is present. results: the combination of biological materials conducted to a biosensor support with great flexibility and low water permeability, allowing easy surface functionalization. the self-reporting ability of the photonicbased sensor enables high intensity colours detected by naked eye. summary/conclusion: the alliance with the high selectivity of oligonucleotide hybridization is expected to offer great exosomal mir- recognition ability and an optimistic perspective for utilization in clinical setups. funding: the authors acknowledge the financial support from the european commission/h , through mindgap/fet-open/ga project. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = ). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine distinct uev surface markers of cd +/cd +/cd + vesicles in a multiplexed bead-based manner including respective controls. the accuri c (bd) was utilized for data acquisition. for further misev -compliant characterization, cd +/cd +/cd + uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd , all other surface markers could be identified. the most abundant markers were cd and cd , which were detected in % of samples, followed by cd / ( %), cd ( %), cd and cd ( %, each). cd ( %), cd , cd ( %), cd e ( %) and cd showed similar relative median fluorescent intensities (rmfi), while cd yielded significantly higher (p = . ) and all other markers significantly lower rmfi (p < . ). tem and f-nta revealed cup-shaped vesicles ( ± nm) with . ± . e + particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg , cd , cd and cd without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a , esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = ) aged - yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a colour panel, which included annexin v (for the majority of circulating evs), cd (for plateletderived evs) and cd (for endothelialderived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and -yr cvd risk detected by qrisk . results: ev numbers, as determined by nta, were strongly associated with bmi (r = . , p < . ), blood pressure (systolic bp: r = . , p = . ; diastolic bp: r = . , p < . ) and plasma triacylglycerol levels (r = . , p < . ). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = . , p = . ). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining . % of the variance for total ev numbers. an additional . % of the variance in total ev numbers was predicted by diastolic bp. ancova of the -yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with -yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd + t cells were isolated from secondary lymphoid organs of foxp -rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th , th , and th ), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, -day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th , th , th ) showed either no change in proliferation (th ) or decrease in proliferation (th , th ), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < . ). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th ), il and il (th ), or il a and il f (th ). in contrast, exposure of tregs to cdc-evs resulted in~ -fold increase in expression of il , a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il- in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il- . this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t hl prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from patients treated with prostanoids (study group; n = , ± years, % female) and patients not treated with prostanoids (control group; n = , ± years, % female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; . mm), adenosine diphosphate (adp; . µm) and thrombin receptor-activating peptide (trap; µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd + and cd p+; pevs), leukocytes (cd +, levs) and endothelial cells (cd +, eevs) were measured in plateletdepleted plasma by flow cytometry (a -micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = . ) and lower concentrations of pevs and levs (p ≤ . ). furthermore, thrombus formation was delayed (p ≤ . ) and thrombus size was decreased (p = . ) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd + pevs (p = . ). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ . ) and the concentrations of pevs (p ≤ . ). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ . ). summary/conclusion: patients with pah treated with prostanoids have increased risk of bleeding both due to impaired platelet aggregation, ev release and thrombus formation, compared to patients not treated with prostanoids. antiplatelet effect of prostanoids varies: whereas epoprostenol decreases the release of pevs, treprostinil and iloprost impair platelet aggregation. funding: ag is supported by the national science centre, research project preludium / /n/ nz / . evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . nanoflow cytometry identifies an imbalance of epithelium-and neutrophil-derived extracellular vesicles in the airway environment of paediatric cystic fibrosis patients brian dobosh, vincent giacalone, milton brown, lucas silva, lokesh guglani and rabindra tirouvanziam emory university, atlanta, usa introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from cf children (< years of age). for nanoflow cytometry, evs were stained with di- -anepps, and with epcam, cd b and cd (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = . , spearman's rho = . ). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = . , rho = . ). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv a ), emory paediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: individuals were enrolled into our study, subdivided into a training (volunteer n = , pneumonia n = , sepsis n = ) and testing cohort (volunteer n = , pneumonia n = , sepsis n = ). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq ) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative real-time pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed significantly regulated mirnas in pneumonia compared to volunteers, and mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: . , pneumonia: . , sepsis: . ). mir- a- p (log fc = . , padj = . e- ) and mir- - p (log fc = . , padj = . e- ) differentiated between pneumonia and volunteers and mir- (log fc = − . , padj = . e- ) between pneumonia and sepsis. expression levels of mir- a- p and mir- were related to disease severity. mir- - p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was . %. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study, healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd and cd were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and mirnas (mir- a-p, mir- - p, mir- a, mir- b- p, mir- - p, mir- - p, mir- - p, mir- - p, let- g- p), were evaluated by rt-qpcr. after the optimization of the methodology, healthy adults subjects and patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd and cd . however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna- a showed a significant increased (p = . ) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir- a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf -active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid -related factor (nrf ) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf -active exosomes, mscs were incubated with potent nrf activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express > % positive msc markers (cd , cd , cd , and cd ) and < % express negative markers (cd , cd , cd , cd , or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd , cd and tsg . nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of . ± . nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf -active human mscs increase exosome secretion by %, compared to nrf -baseline mscs (p < . ). both nrf -baseline and nrf -active exosome treatment significantly reduced closure time to . and days respectively, compared to . days for vehicle-treated wounds (p < . ). this reduction eliminated the delay in closure time compared to wounds of c /b mice. nrf -active exosome treatment of db/db wounds reduced closure time by a further . days compared to untreated c /b wounds. at day , exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd + vessels compared to vehicle-treated wounds. summary/conclusion: enhancing nrf function in mscs multiplies exosome yield. our results demonstrate exosome-based therapies hold tremendous promise and warrant further investigation for rapid translation. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist is a key mediator of tumour cell metastasis. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist . methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc -ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc -ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc -ml lines stably overexpressing or deficient in twist . results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc -ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc -ml overexpressing twist showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. introduction: exercise is associated with various health benefits, including the prevention and management of obesity and cardiometabolic risk factors. however, a strong heterogeneity in the adaptive response to exercise training exists. differential response to exercise training might be mediated by myokines (proteins, nucleic acids, metabolites) that can be released directly into the systemic circulation, or packaged within extracellular vesicles (evs). the objective of this study was to evaluate if changes in evs after acute aerobic exercise (ae) were associated with the responders phenotype following -week resistance exercise (re) training. methods: this is a secondary analysis of plasma samples from the exit trial (clinical trial # ). eleven sedentary obese youth ( . ± . years, bmi ≥ th percentile underwent an acute bout of ae ( % heart rate reserve, min). blood was collected before [time (at) − , min], during [at , , min], and after [ min (at ), min (at )] exercise. afterwards, youth participated in -week re programme, and were categorized into responders or non-responders (nr) based on changes in insulin sensitivity (above or below percentile). primary outcome: evs were isolated using size exclusion chromatography (izon®) at baseline (at ), immediately after ae (at ) and after recovery (at ). ev protein concentration, size, and zeta potential were analysed in a single-blind fashion. results: responders had larger evs (~ . nm) as opposed to nrs (~ . nm) at at (p < . ) and this pattern was maintained at at and at , though not significant (p = . ). nrs displayed differential ev size distribution (peaks at nm or nm), while ev distribution was highest at nm in responders. no difference in average zeta potential or total ev protein yield was observed between groups. an increase in ev yield with exercise time and recovery was observed in both groups. summary/conclusion: our preliminary data suggest that ev size is significantly increased after an acute bout of ae in obese youth responders. further research to delineate the role of evs as predictors of exercise adaptation is warranted. funding: funded by dream and research manitoba. using dual-fluorescent reporter mice to track tissue-specific extracellular vesicles andrea estrada, gabriella hehn, zackary valenti, christopher allen, nicole kruh-garcia and dan s. lark colorado state university, fort collins, usa introduction: extracellular vesicles (evs) from tissues like skeletal muscle (skm) and adipose tissue (at) have been implicated in human disease but are understudied. skm is likely a major player in ev biology as it accounts for~ % of total body mass. tools to define cellular ev origin are needed because tissues like skm are comprised of a variety of cell types. here, we describe our ongoing efforts using the dual fluorescent mg/mt mouse as a tool to analyse skm-myocyte derived evs. methods: wild-type (wt) and mg/mt mice were used for these studies. mg/mt mouse cells express membrane-tagged red (mt) or green (mg) fluorescent protein in the absence or presence of cre, respectively. we made skm myocyte mg expressing mice using a mouse expressing cre on the human skeletal actin promoter. blood was collected via cardiac puncture and platelet-free plasma was obtained via centrifugation. plasma evs were isolated using exoquick, exoquick-tc or size exclusion chromatography. skm and at were dissected into~ mm chunks, placed in serum-free dmem and incubated for hours. tissuederived evs were isolated using exoquick-tc. ev abundance was determined with a horiba viewsizer. individual evs were analysed with a cytek aurora spectral flow cytometer. settings were optimized using polystyrene beads and spectral unmixing was performed to allow detection of mg and mt. results: in wt mice, skm releases > times more evs than adipose tissue per unit of mass (p < . using paired student's t-test). since skm is also a major component of total body mass, these data further emphasize the importance of skm-derived evs. skmderived evs from wt mice were not fluorescent (< . % of events). evs from mg/mt mouse skm overwhelmingly expressed mg (> % of events) with negligible (< %) expression of mt. at-derived evs robustly expressed mt but lacked mg. summary/conclusion: these data provide "proof-ofprinciple" that mg and mt are readily incorporated into evs secreted ex vivo. surprisingly however, plasma evs from mg/mt mice expressed very little mg (~ %) or mt (~ %). this observation was confirmed with three separate isolation techniques. we are currently exploring possibilities to explain this finding, including: ) modification of evs post-secretion, ) clearance of fluorescent evs by the liver or ) that evs secreted from tissues remain predominantly in the interstitial space. funding: this work was supported by an innovative project award from the american heart association ( ipa ) to dsl. endothelial cd delivery of fa loaded extracellular vesicles is critical for thermogenesis. introduction: membrane cd facilitates tissue fatty acid (fa) uptake. we recently found that endothelial cell (ec) cd controls muscle and adipose tissue fa uptake, and influences the tissue's metabolic phenotype. the mechanism for cd -facilitated fa uptake is unknown. here we examined the role of ec cd in thermogenesis and in fa delivery to brown fat tissue. methods: adult male mice were housed individually, restricted from food during acute ( hr) cold exposure ( °c) with core temperature monitored every minutes. after hours, animals were sacrificed and samples collected for analysis. for cellular studies, human microvascular (lonza) or primary murine microvascular ec were used. for primary cells, crude cell pellets from lung homogenates were purified using mouse-cd magnetic beads (miltenyi). for microscopy studies, alkyne fa (cayman) was added to cells and to enable visualization of internalized fas, click chemistry (invitrogen) used to label alkyne-fa with alexa . for radioactive studies, primary lung ec were serum starved for hrs and incubated overnight with h-oleic acid bound to fa-free bsa ( : ratio). media was collected, clarified by centrifugation to remove microvesicles and debris. small extracellular vesicles (sevs) were isolated from clarified media using total exosome isolation reagent (invitrogen) and counted for radioactivity. results: basal core body temperatures are similar in mice lacking ec cd (eccd -/-) compared to controls (cd fl/fl). however, during cold exposure at °c , eccd -/-are unable to maintain body temperature (p < . ). plasma free fa are higher in cold exposed eccd -/-indicating fa clearance by brown fat is impaired. mitochondrial function and expression of thermogenic and mitochondrial genes in brown fat from eccd -/-and cd fl/fl mice were similar. these data suggested that endothelial delivery of fas is necessary for thermogenic maintenance of body temperature. to examine fa handling by ecs we used alkyne fas to visualize the process. we found that fas are transferred by microvascular ec through caveolae-mediated transcytosis involving src signalling and cav- phosphorylation. the internalized cav- and cd positive vesicles containing fas are released as sevs. to determine the dependence of cd on this process, we treated primary microvascular ec with radiolabeled fa and found that sevs secreted by cd -/-cells contain less labelled-fa (p = . ). summary/conclusion: endothelial delivery of fa is critical for thermogenesis. our working model for the mechanism of fa uptake by brown adipose tissue is the following: endothelial cells transfer the fa through caveolae-mediated transcytosis and secrete small extracellular vesicles (sevs) that help deliver fas to brown adipocytes. funding: this work is supported by nih grants dk and dk . introduction: diet-induced obesity modifies intestinal permeability leading to bacteria infiltration and to a decrease in the number of immune cells protecting mucosa. as orange consumption is beneficial for human health and used in preventive medicine, we determined whether orange juice-derived nanovesicles (onv) might be recommended as nutritional strategies for the treatment of intestinal complications associated with obesity. methods: onv isolated from fresh orange juices were characterized by lipidomic, metabolomic, microscopy, nta and for their stability during digestion. intestinal barrier (ib = caco- cells+ht- cells differentiated with oleic acid) were treated with onv and co-cultured with adipocytes to monitor ib fat absorption and release. obesity was induced in mice fed for weeks with a high-fat high-sucrose diet (hfhs mice vs standart chow diet mice). then half of the hfhs mice were gavaged with micrograms/day for weeks. results: onv did not modify high-fat high-sucrose diet-induced obesity and insulin resistance but reversed diet-induced gut modifications. six hours post-gavage, onv accumulated preferentially in jejunum involved in lipid absorption. in jejunum, and no other intestinal region, onv increased villi size, restored immune response and decreased barrier permeability in hfhsd mice. in addition, onv-treated mice had increased expressions of acat , angptl and dgat , but a decreased expression of fabp , fatp , mtp vs hfhsd animals, which indicated that fat absorption, tg synthesis and chylomicron release were strongly reduced. similarly to other plant-derived nanovesicles, these results were likely associated with onv lipid and metabolite compositions (strong enrichment in bioactive phospholipids: pe, pa, pc, pi and leucine) as onv did not resist to harsh digestive conditions in vitro and were poorly incorporated in enterocytes. as the effects of onv on the decrease in tg content and epithelial cell growth were also observed in vitro, gut microbiota unlikely participate to these effects. summary/conclusion: onv are important bioactive compounds of orange juice and for the first time we demonstrated that they can modulate lipid metabolism in the intestinal barrier associated with morphological changes. interestingly onv treatment targets mtp and angptl mrnas, therapeutic intestinal targets to reduce plasma lipids and for attenuating inflammation in gastrointestinal diseases. therefore, onv might be used to reduce the development of dyslipidemia-associated diseases and to restore intestinal functions in obese patients. funding: olga triballat institut; benjamin delessert institut, inrae institut. association, structure, and function of fibronectin in extracellular vesicles from hepatocytes xinlei li, ruju chen, sherri kemper and david brigstock nationwide children's hospital, columbus, usa introduction: we have shown that extracellular vesicles from normal hepatocytes have anti-fibrogenic activity and that they preferentially bind to hepatic stellate cells (hscs, the principal fibrosis-causing cell in the liver) and hepatocytes. in this study, our goal was to determine the molecular nature of the ev components involved in cell binding. fibronectin (fn ) is a key component of extracellular matrix, functioning in processes including cell adhesion, differentiation, and wound healing. two types of fn are present in vertebrates, of which the soluble plasma fn is derived principally from hepatocytes, while cell-associated fn is produced by numerous cell types. here we describe a novel function of plasma fn in facilitating binding of hepatocyte evs to target cells. methods: differential ultracentrifugation was used to collect evs released by parental mouse aml hepatocytes, fn ko aml cells in which fn was ablated using crispr-cas , primary human or mouse hepatocytes, or human hepg cells, or from human or mouse serum. evs were characterized by nanosight tracking analysis (nta), western blot, iodixanol gradient ultracentrifugation, and mass spectrometry. the binding efficiency of pkh -labelled evs from parental (ev-hep) or fn ko (ev-hepfn ko) aml cells was analysed in hepatocytes or hscs. swiss webster mice were injected with ccl for five weeks to induce liver fibrosis, with some mice also receiving i. p. administration of ev-hep or ev-hepfn ko over the last two weeks, followed by determination of hepatic fibrogenic genes by qrt-pcr. results: ev-hep or ev-hepfn ko were - nm in diameter and positive for common ev markers (cd , cd , flotillin- ). mass spectrometry showed that fn was the most abundant protein in ev-hep and comprised principally the plasma form. the abundant presence of ev fn was verified by western blot and co-immunoprecipitation with anti-cd or antiflotillin- . western blot showed that fn was also abundant in evs from primary human or mouse hepatocytes, hepg cells, and human or mouse serum. fn and ev-hep co-sedimented at a density of~ . g/ml. ev-hepfn ko yield and size-range were similar to those of ev-hep, suggesting that ev biogenesis is fn -independent. as compared to ev-hep, the binding of ev-hepfn ko to target cells was highly reduced whereas ev binding was independent of fn expression by the target cells themselves. both ev-hepfn ko and ev-hep were anti-fibrogenic in vivo but only ev-hep attenuated collagen ⍺ expression in mouse hscs in vitro. summary/conclusion: fn is abundantly associated with hepatocyte evs and facilitates ev binding to target hepatocytes or hscs. additional studies are needed to clarify the functional role of fn in mediating ev-hep anti-fibrogenic actions in vitro or in vivo. elevated glucose increases soluble and aggregated forms of human islet amyloid polypeptide in islet-derived extracellular vesicles -implications in type diabetes and islet transplantation introduction: type diabetes (t d) is characterized by reduced beta cell mass and function. islet amyloid, formed by aggregation of human islet amyloid polypeptide (hiapp), contributes to progressive beta cell loss in t d. amyloid also forms in human islets during pre-transplant culture and following transplantation in patients with type diabetes (t d) which is associated with graft failure. the cellular mechanisms underlying islet amyloid formation are still unclear. in this study, we examined the potential role of islet-derived extracellular vesicles (ev) in the clearance of soluble and aggregated (pro)iapp species from beta cells and amyloid formation. methods: human islets isolated from cadaveric pancreatic donors (n = donors) and wild-type or hiappexpressing (hiapp+) transgenic mouse islets (n = / group) were cultured in normal ( . mm) or elevated ( . mm) glucose to form amyloid. ev (exosomes) were isolated from culture medium using classical centrifugation and ultracentrifugation. purified ev were analysed by nanoparticle tracking analysis. western blot analysis and double immunogold transmission electron microscopy were performed to verify the presence of ev markers as well as (pro)hiapp species and oligomers (aggregates). results: human islets formed amyloid during culture with elevated glucose which was associated with progressive beta cell apoptosis. (pro)iapp species were detectable in ev released from human islets cultured in normal and elevated glucose. the latter markedly increased (pro)iapp content in islet-derived ev. interestingly, hiapp aggregates (oligomers) were present in the majority of ev released from human islets cultured in elevated glucose but were not detectable in islets cultured with normal glucose. similarly, ev released from hiapp+ mouse islets which formed amyloid during culture had higher (pro)iapp content compared to wild-type islet-derived ev. moreover, hiapp oligomers were present in ev derived from hiapp+ islets but not wt islets. summary/conclusion: in summary, our data show that (pro)iapp species are present in islet-derived ev and that elevated glucose increases (pro)hiapp and its aggregates in ev released from islets. islet-derived ev may play a key role in the process of amyloid formation in t d and human islet grafts. funding: university of manitoba research grants program (urgp). on. contraction, but not glycolysis, regulates the size of skeletal muscle evs secreted ex vivo. colorado state university, fort collins, usa introduction: skeletal muscle (skm) is a metabolically active tissue and accounts for~ % of total human body mass. acute exercise increases secretion of extracellular vesicles (evs), but the mechanisms responsible are unknown. muscle contraction increases the demand for atp which requires intercellular communication in order to adapt. we hypothesized that this "metabolic stress" during contraction increases skm ev secretion. methods: we tested our hypothesis using an ex vivo ev secretion assay. all studies were approved by the colorado state university institutional animal care and use committee. vastus medialis muscle (skm) from male c bl/ j mice (n = ) or female mt/mg mice (n = ) was cut into~ mg pieces and added to well plates (~ mg/well) filled with ml of serum-free dmem and placed in a cell culture incubator at c for hours. skm from male mice was treated with -deoxyglucose ( -dg) ( . nm - mm) to induce metabolic stress via inhibition of glycolysis. skm from female mice was treated with um of blebbistatin (bleb), a contraction inhibitor. after incubation, skm mass was measured and conditioned media was centrifuged ( , x g for min) to remove cell debris. evs were isolated using exoquick-tc. nta was performed on isolated evs using a horiba viewsizer . ev secretion was normalized to tissue mass and culture media volume then reported as ([particle]/ml/mg tissue). statistical comparisons for -dg experiments were made using a repeated measures -way anova. bleb experiments were analysed using a paired student's t-test. results: there was a trend towards greater ev abundance (p = . ) as a function of -dg treatment, but no effect on ev diameter (p = . ). bleb treatment did not alter ev abundance (p = . ), but significantly reduced ev mean diameter (p = . ; % decrease; dmso: . ± . vs. bleb: . ± . ). summary/conclusion: contrary to our hypothesis, inhibition of glycolysis with -dg did not stimulate skm ev secretion. however, bleb did appear to promote the release of small evs and/or inhibit secretion of larger evs. ongoing efforts are focused on testing other metabolic stressors and defining how blebbistatin promotes small ev secretion. funding: american heart association grant to dsl (ipa ). introduction: extracellular vesicles (evs, exosomes) are nanovesicles ( - nm) secreted from various types of cells. because of vesicular encapsulation of mirnas and enzymes, the evs play crucial roles in cell-to-cell communication by delivering these functional molecules to other cells [ ] . on the other hand, the evs are highly expected as next generation therapeutic tools due to pharmaceutical advantages such as controlled immunogenicity, effective usage of cell-tocell communication routes, artificial modification and encapsulation of functional molecules. however, cellular targeting and uptake efficacy of the evs are insufficient to be utilized as therapeutic tools [ , ] . in this study, we newly developed evs decorated with cellpenetrating sc or (sc ) peptides, which are derived from the c-terminal domain of the cationic antimicrobial protein, cap , because the peptides can be efficiently internalized by breast cancer cells. [ , ] . methods: all peptides were prepared by fmoc-solid phase synthesis. secreted evs from cd -gfp stably expressing hela cells were isolated by ultracentrifugation. cellular uptake of evs was analysed using a flow cytometer and a confocal laser microscope. encapsulation of saponin in the ev was conducted by electroporation. results: sc peptide is known as one of cell-penetrating peptides, and branched structure of sc peptides, (sc ) , further enhances the cellular uptake [ ] . in this research, we examined the effects of the peptide modification on cellular ev uptake, and modification of the sc or (sc ) peptides on ev membranes was conducted via stearyl moiety. as our results, increased macropinocytotic cellular uptake by modification of the peptides was successfully attained. especially, the modification of (sc ) peptides showed higher cellular uptake and macropinocytosis induction efficacy than that of sc peptides. in addition, anticancer protein, saporin toxin-encapsulated evs modified with the (sc ) peptides significantly enhanced their biological activity with dependency of glycosaminoglycan expression on targeted cells. summary/conclusion: the cell-penetrating (sc ) peptide-modified evs shows high abilities to be effectively internalized by cells and are applicable for intracellular delivery of therapeutic molecules. this study is expected to contribute to development of intracellular delivery techniques based on evs. [ introduction: rna therapeutics possess high potential which is yet to be realised, largely due to difficulties involved in delivery to the cytoplasm of target cells. extracellular vesicles (evs) possess numerous features that may help overcome this hurdle and have emerged as a promising rna delivery vehicle candidate. despite extensive research into the engineering of evs for rna delivery, little is known about how their intrinsic rna delivery efficiency compares to current synthetic rna delivery systems. using a novel crispr/cas based rna transfer fluorescent stoplight reporter system, we here compared the delivery efficiency of evs to state-of-the-art dlin-mc -dma lipid nanoparticles (lnps). methods: evs were isolated from mda-mb- cells expressing either a targeting or non-targeting control sgrna and applied to hek t stoplight+ reporter cells. lnps containing targeting sgrna were titrated onto hek t stoplight+ reporter cells to determine the minimum effective dose. lnp and ev particles were characterized using nanoparticle tracking analysis, dynamic light scattering and zeta potential analysis. sgrna copy number was determined using rt-qpcr. results: evs were ± nm in diameter as measured by dls and possessed a negative surface charge of − . ± . mv. rt-qpcr and nta analysis indicated that sgrna ev loading was low, with only in . e ± . e evs containing a single sgrna copy. nevertheless, evs containing targeting sgrna induced significant reporter activation while evs containing non-targeting sgrna did not. lnps were ± . nm in diameter and possessed a neutral charge. these particles also induced significant reporter activation when loaded with targeting sgrna. when delivered via evs, only between to sgrna copies per cell were required to induce statistically significant reporter activation. in contrast, the minimal effective sgrna dose when delivered by lnps was considerably higher at approximately e copies per cell. summary/conclusion: mda-mb- evs deliver rna in a highly efficient manner and are functional at sgrna concentrations several orders of magnitude lower than those required for lnp mediated delivery. this underlines the potential of evs as rna delivery vehicles and highlights the need to study the mechanisms by which evs achieve their efficiency in order for improved development of rna therapeutics. the role of circulating extracellular vesicles in patients with chronic chagas disease introduction: chagas disease is a neglected tropical disease (ntd) caused by the flagellated protozoan trypanosoma cruzi. it is a major public health problem in latin america, and it is now expanding over the globe through immigration of infected individuals. eukaryotic cells release extracellular vesicles (evs) that circulate in body fluids and have an important roles in intercellular communication, both in physiological and pathological conditions. objectives. our study proposes to characterize and to compare the circulating evs isolated from plasma of the chronic chagas disease (ccd) patients with healthy individuals (controls). methods: peripheral blood was collected from patients and controls in the presence of edta and evs enriched from plasma by differential ultracentrifugation. the obtained evs were characterized and quantified by nanoparticle tracking analysis (nta) and added to human thp- cells. after h, the cell supernatants were analysed by elisa for the presence of cytokines. results: lower amounts of evs were obtained from ccd patients in comparison with control individuals. however, the same amount of evs of ccd were more capable of inducing cytokines such as ifn-gamma and il- in relation to controls. summary/conclusion: although less evs are present in the blood of ccd, these evs induce high inflammatory reactions on macrophages suggesting a possible role of these evs in the establishment of chronic disease. funding: supported by fapesp, cnpq and capes. extracellular vesiclesa trojan horse for therapeutic agent delivery introduction: extracellular vesicles (evs) may prove to be one of the optimal payload carriers for therapeutic agents. while they travel through the extracellular space, the ev's lipid membrane layer shields their luminal cargo from deleterious external factors. when autologous evs are used to protect this therapeutic cargo, little immunogenic effects are expected compared to viral vectors and artificial structures, such as liposomes. their usage is potentially manifold, and they are ubiquitously present in all body tissues and fluids. the key is to develop a manageable ev loading agent for adoptive transfer therapies. methods: to exploit the unique properties of evs, highly positively charged proteins were used to load them with multiple biomolecules, such as a cas protein or dicer substrate dsrna as a functional payload and to improve their apparently inadequate natural ability to deposit cargo into the cytoplasm of recipient cells. results: highly positively charged proteins can associate with and/or diffuse through a phospholipid bilayer (thompson et al. ) . when these kinds of charged proteins are mixed with isolated evs in vitro, they are loaded into the evs. the positive charge of the protein has the advantage that it can associate with negatively charged agents, such as rna species, and aids the associated molecule to also incorporate into the ev. moreover, the positive charge of the protein helps with cargo delivery, and thus overcoming the bottleneck of the ev's cargo to escape the endosome post-uptake in a recipient cell. self-quenching fluorescent lipid dyes demonstrated that discharge of the highly positive ev cargo into the cytoplasm is concomitant with lipid mixing between the membrane of evs and the membrane of the recipient cell. when egfp-expressing microglia were exposed to evs loaded with a dicer substrate dsrna able to silence egfp via the positively charged protein, the uptake of dicer substrate dsrna was concomitant with a decrease in egfp expression in the microglia. a similar result was achieved when evs were loaded with cas protein conjugated to the highly positively charged protein. post-uptake of these cas -loaded evs, microglia expressing anti-egfp sgrna (single guide rna) lead to decreased egfp expression. summary/conclusion: our ev delivery technology has the capability of delivering multiple biomolecules, such as protein and rna cargo and demonstrates postuptake of the ev functionality of the ev delivered cargo in the recipient cell. hybrid extracellular vesiclesbiomimetic tool for drug delivery to repair endothelial cell dysfunction introduction: traditional drug delivery systems (dds) are usually based on liposomes, micelles or dendrimers. unfortunately, many dds cause side effects including organ toxicity and/or unexpected immune response. in living organisms, extracellular vesicles (evs) are responsible for delivering biologically active molecules to distant cells. in vitro loading of therapeutic compounds into evs is still not effective and needs developing new strategies. for these reasons we aimed to design hybrid extracellular vesicles (hevs) with high loading capacity for dds. methods: for hev synthesis, we used human endothelial derived evs. using freeze/thawing method we fused them with liposomes composed of cholesterol and one of the three lipids: dopc, sphingomyelin or phosphatidylserine. to confirm membrane fusion, we applied a spectroscopy ruler -fret (förster resonance energy transfer) and cryotem imaging technique. we characterized hevs using nta (for size distribution evaluation), dls (zeta potential) and western blot (for detection of evs markers). we evaluated loading efficiency using calcein as a model drug. additionally, we performed cytotoxicity tests. results: in the cryotem imaging, pure and homogenous hev population with a diameter of ± nm was detected. additionally, we observed changes in zeta potential and in size distribution after fusion. fret measurements showed increased fusion efficiency with the increasing number of freeze/thawing cycles and dependence on a lipid-to-protein ratio in evs. additionally, hev had higher loading efficiency than liposomes and sole evs and that their internalization by endothelial cells did not cause a cytotoxic effect. summary/conclusion: based on cryo-tem and fret, we confirmed that our fusion method of hybrid evs is effective and can be applied as a delivery platform for dds to endothelial cells. response to a range of stressors. the functional activity of these evs in recipient cells may, in part, be driven by changes to their biological cargoes. however, the molecular details of the underlying ev biogenesis and loading processes, and how this may vary in different conditions, is poorly understood. methods: we first studied the effect of oxidative stress on the functional activity of evs in recipient cells using cell viability and mitochondrial membrane potential assays in drosophila s r+ cells. we then carried out total rna sequencing of ev and cellular rna under three stress conditions and compared results to existing data in mouse cells. further to this we have used a bioinformatic pipeline to identify sequence motifs enriched in evs under stress. results: functional assays indicated changes to cell viability and mitochondrial membrane potential in recipient cells, which were donor cell-stress dependent. subsequent characterisation of rna showed an enrichment of ribosomal rna in evs relative to cells, but no significant changes to other biotypes. comparative analysis has also uncovered a set of genes enriched in evs under oxidative stress, and a further subset whose enrichment may be evolutionarily conserved in mouse. we also identified potential ev-loading motifs which may assist in rna loading specifically under stress. summary/conclusion: we have shown that evs derived from oxidatively stressed cells show dose-dependent differences in rna cargo and identified potential sequence motifs that may have a role in its loading. we are now validating the biological significance of these findings by combining different in vivo approaches in drosophila. this will enable us to gain insights into the basic mechanisms which govern ev loading in different contexts, and ultimately the molecular mechanisms underlying ev-mediated intercellular communication. ishai luz a , bibek bhatta a , kanaga sabapathy b and tomer cooks c a ben-gurion university of the negev, beer-sheva, israel; b national cancer centre, singapore, singapore, singapore; c ben-gurion university, beer sheva, israel introduction: mutations in tp are considered one of the most frequent genetic alterations in human cancer. besides the abrogation of the wild-type (wt) p -mediated tumour suppression, a distinct set of missense mutations was reported to endow mutant p proteins with novel activities termed gain-of-function (gof). even though mutations in tp are typically thought to arise in the tumour cells rather than in the stroma, the non-cell-autonomous effects of these mutants over the tumour microenvironment are poorly understood. in the presented studies, focusing on colon cancer as well as on lung cancer microbiome, we investigated intercellular interactions mediated by exosomes and outer membrane vesicles (omvs) in the context of cancers harbouring mutant p . methods: p results: in the colon, tumour cells harbouring mutp were found to exert a non-cell-autonomous effect over macrophages. when exposed to tumour cells harbouring mutp , monocytes became polarized towards a distinguished subset of macrophages characterized by tams-related markers. the mutant p affected tam were characterized as tnf-αlow/il- high, over expressing cd- and cd , with decreased phagocytic ability and increased invasion and matrix degradation potency. investigating the exosomal transfer from mutp tumour cells to macrophages, revealed a mutp -specific mirs signature led by mir- promoting the tam phenotype and creating an invasive front together with tumour cells. mir- was also found to be the top mutp -associated mir in a cohort of human colorectal resected tumours. separately, in two lung cancer cohorts, we identified a signature of microbiome members associated with p mutations. acidovorax temperans, a gram negative bacterium, was found to be abundant in tumours of patients with mutant p . we found a significant increase in tumour volume in animals inoculated with acidovorax temperans as compared to sham treated animals, and increased lung weight as a percent of total body weight. these preliminary data indicate that acidovorax temperans contributes to lung tumorigenesis in the presence of activated k-ras and mutant p . omvs shed by acidovorax temperans promoted inflammatory signalling in lung carcinoma cells and elevated cd expression on tumour cells and sirpα levels on macrophages. summary/conclusion: altogether, these findings are consistent with a microenvironmental role for specific "hot-spot" gof p mutants tightening the interaction between the tumour cell and the immune compartment in colon cancer. in both colon and lung cancer, mutant p facilitates cellular interactions within the tumour microenvironmets mediated by vesicles. funding: intramural funding from the national cancer institute, national institutes of health. lori zacharoff and mohamed el-naggar university of southern california, los angeles, usa introduction: the metal respiring bacterium s. oneidensis creates outer membrane extensions and outer membrane vesicles that are sculpted by the novel bar domain protein bdpa. these vesicles and extensions incorporate mutliheme cytochromes involved in extracellular electron transfer to metals and electrodes. however, the physiological relevance of incorporating these cytochromes into the higher order d architecture of a vesicle or extension is unknown. given that bar domains serve as a protein sorting mechanism in eukaryotes, we investigated the pathway crosstalk between bdpa and outer membrane multiheme cytochromes as means to understand the physiological significance of membrane architecture. methods: o this end, vesicle morphology and content was measured using dry weights, dynamic light scattering, fluorescence microscopy and comparative proteomics from wild type s. oneidensis and deletion strains. results: cells lacking bdpa make large amorphous vesicles that are dense with protein. in contrast, a strain lacking outer membrane cytochromes recruits less total protein into smaller vesicles. proteomics to show that both bdpa and multiheme cytochromes are involved in recruiting other proteins to outer membrane vesicles and have a reciprocal relationship. summary/conclusion: in the absence of bdpa, protein crowding has to become the main driving force of vesiculation and bdpa is essential for efficient incorporation of cytochromes. however, multiheme cytochromes are not only vesicular cargo, but are also important for shaping and loading vesicles. both of these situations make it clear that vesicles play a role in increasing the respiratory surface area of s. oneidensis cells. moving forward, we hope to be able to control bdpa and cytochrome levels for selective recruitment of technologically relevant payloads. introduction: fascioliasis caused by fasciola hepatica represents a major economic loss and clinical burden in cattle farming worldwide. extracellular vesicles (evs) contain pathogen-derived molecules that represent novel biomarkers of disease. in the present study, we have identified potential new biomarkers of f. hepatica infection in evs present in sera of infected cattle. methods: parasites and sera were obtained from local abattoirs (valencia, spain, and medellin, colombia, respectively). sera from infected and from healthy animals. parasites were cultured, and evs obtained by sizeexclusion chromatography (sec) and characterized by nta, tem and proteomic profiling. recombinant proteins from f. hepatica evs (enolase and fh . tegumentary protein) were produced, and coupled to magnetic beads. measurement of bovine igg antibodies was performed using luminex bead array technology. results: a total of proteins were identified associated with evs as shown by the presence of typical ev-markers (tsg , alix, cd ). two parasite proteins, enolase and the fh . tegumentary protein were produced as recombinant proteins and used for detection of cattle igg employing luminex bead array technology. interestingly, significant differences were found in the fluorescence values of both recombinant proteins allowing discrimination between sera from infected and non-infected cattle. the use of the fh . protein generated a highly significant difference between the two groups (p value = . ); as did enolase (p value was . ). summary/conclusion: this study demonstrates the usefulness of ev proteins as new biomarkers for early diagnosis of helminth infections using multiplex assays, a technology that may also be applied to other parasite ev molecules. life stage-specific glycosylation of schistosome-derived extracellular vesicles introduction: glycans play an essential role in pathogen-host interactions. larvae and adult worms from schistosoma mansoni release distinct subsets of glycoconjugates as excretory/secretory (es) products. extracellular vesicles (evs) are also among the es products. we recently found that schistosomuladerived evs are glycosylated and bind human dendritic cells via c-type lectin receptor (clr) dc-sign, leading to increased il- and il- release. here we investigated the glycosylation profile of evs released by s. mansoni adult worms, compared this to schistosomula evs, and addressed how this may affect parasite-host interactions via clrs. methods: evs from cultured s. mansoni parasites were obtained by ultracentrifugation and purified with iodixanol density gradients. isolated evs were analysed by nta and cryo em. n-glycan and lipid glycan content was determined by mass spectrometry. density gradient fractions with evs were loaded onto sds-page gels followed by western blot (wb) analysis using anti-glycan monoclonal antibodies (mabs). results: cryo em showed that adult worm evs lacked the long thin filaments that are characteristic for schistosomula evs. additionally, in contrast to schistosomula evs, glycolipids could not be detected in the adult worm evs. mass spectrometry analysis showed that the most abundant n-glycans in the adult worm evs contained galnacβ - glcnac (lacdinac, ldn) motifs, which correspond to previously published overall glycan profiles of this specific life stage. other differences in ev glycosylation between the two life stages were observed by wb using anti-glycan mabs: adult worm evs showed a paucimannosidic glycan motif whereas in the schistosomula evs galβ - (fucα - ) glcnac (lewis x) was detected in line with previous ms analysis. introduction: phloem plays a central role in plant function, as it is the responsible for the translocation of photoassimilates from source-to sink-organs, and a long-distance route for signals distribution. due to the sap high nutrient content, sieve elements are primary target for plant pathogens and pests. in this work we aimed to isolate and characterize extracellular vesicles (evs) from cucumis melo phloem sap, derived from plant either exposed or not to the melon aphid, aphis gossypii (hemiptera: aphididae). methods: phloem exudates from -week-old melon plants, either uninfested or infested with adults of a. gossypii (n = , replicates each), were collected by cutting the stem with a sterile razor blade between first and second expanded leaf from the top. evs were isolated by size exclusion chromatography, and analysed by nanoparticle tracking analysis (nta) and transmission electron microscopy. evs proteome was determined by quantitative mass spectrometry. results: evs from phloem sap were successfully isolated in every condition. no significant differences were detected among distinct samples, neither in particle concentration and size by nta, nor in protein concentration. most importantly, a total of different proteins were identified in phloem sap evs, including present in exosome databases (exocarta). on top of that, differentially expressed proteins were identified in evs derived from aphid infested or uninfested plants (p value < . ). summary/conclusion: understanding how plants trigger their defences against pests and pathogens is important to develop new control measures. the characterization of several proteins in evs from the phloem sap provide valuable information on long distance signalling in plants. moreover, as plants lack an immune system comparable to animals, the different protein content in phloem sap evs after exposure to aphids could indicate their important role in delivering inducible defences against invading pests and pathogens. extracellular vesicles from nematode species heligmosomoides bakeri and trichuris muris contain distinct small rnas that could enable niche specificity in the host introduction: gastrointestinal nematodes are extremely prevalent parasites that infect most animals and % of human population. their success as parasites is attributed to their ability to secrete diverse molecules that modulate the host immune system. extracellular vesicles (evs) are one of the immune modulatory compounds they release that directly modulate host cells. our goal is to understand how the small rna (srna) cargo underpins ev function, using a comparative analysis of ev cargo from diverse nematode species. methods: we first compared how different ev isolation methodologies (ultracentrifuge (uc), size fractionation, sucrose gradient floatation) effect the small rnas detected in h. bakeri evs using different library preparation kits (cleantag, truseq), with or without polyphosphatase treatment. we then compared this to small rna libraries from t. muris evs using comparable methods, uc ev purification, with or without polyphosphatase treatment and using the cleantag library preparation kit. results: evs from both species contained mirnas, however the mirna gene familes in h. bakeri and t. muris evs are distinct. the mirna content detected in ev samples collected by different purification protocols is robust. the largest difference in detected mirnas was found when comparing different library preparation kits. although both h. bakeri evs and t. muris evs were dominated by srnas derived from intergenic or repetitive elements in the parasite genomes, only in h. bakeri evs were these secondary sirnas. summary/conclusion: h. bakeri and t. muris evs contain distinct small rna cargos, which may underpin their ability to colonise different host niches, and/or modulate the host immune system differently. t. muris evs do not contain secondary sirnas, in contrast to h. bakeri, however they are dominated by srnas derived from intergenic or repetitive regions. comparative analysis of helminth evs could help pinpoint the srnas involved in cross-species communication. please provide any keywords if applicable.: nematode, cross-species communication, small rna introduction: extracellular vesicles (evs) are secreted from various cells including cancer cells and known to contain protein and small rnas including mirna isoforms (isomirs). therefore we also focused on isomirs including other small non-coding rnas for biomarker discovery. although liquid biopsies using small rnas are promising biomarkers for early detection of cancer, current approaches to detecting and analysing mirnas in the blood are still inadequate. artificial intelligence (ai) data analysis may provide better algorisms for diagnosing cancer. methods: small rnas were isolated from serum or purified evs using a mirneasy mini kit (qiagen) and quantified by using the ion s ™ next-generation sequencing system. (thermo fisher scientific). evs were purified using total exosome isolation reagent (invitrogen™). ai data analysis was performed using jmp® genomics and datarobot enterprise ai platform. results: three small rnas, isomir of mir- - p, mir- a- p, and trf-lys (ttt) were significantly upregulated in breast cancer patients compared with the healthy cancer-free individual. the combination algorithm using these three small rnas allows for a more accurate diagnosis of the area under the curve (auc) . . to test the possibilities that these small rnas are derived from cancer cells, we isolated evs from the serum and performed ngs analysis to profiled serum small rnas in evs. interestingly we found that two small rnas, mir- - p and mir- a- p, also high in breast cancer evs, indicating that these small rnas were expected to be derived from cancer cells. in oesophagus cancer, we also performed ngs analysis and identified twenty-four mir/isomirs candidates for diagnostic biomarkers. a multiple regression model selected mir- a- p and two isomirs (mir- - p and mir- - p) . the auc of the panel index was . . we also performed ai data analysis and discovered the novel algorisms that can diagnose breast and oesophagus cancer more accurately. summary/conclusion: we demonstrated combinations of circulating non-coding rnas containing evs potentially useful for the detection of early-stage breast and oesophagus cancers. in addition, the datarobot enterprise ai platform enables us to the more accurate diagnosis of cancers at the early stage. identification of novel ev-associated mirnas as toxic biomarkers in mouse introduction: recent findings reveal that extracellular vesicles (evs), secreted from cells, are circulating in the blood. evs are classified into exosomes ( - nm), microvesicles ( - , nm) and apoptotic bodies ( - , nm). evs contain mrnas, micrornas, and dnas and have the ability to transfer them from cell to cell. recently, especially in humans, the diagnostic accuracy of tumour cell type-specific evs as biomarkers is more than %. in addition, micrornas contained in the evs are being identified as specific biomarkers in blood for chemical-induced inflammation and organ damage. therefore, micrornas contained in the evs released into the blood from tissues and organs in response to adverse events such as chemical substances and medicine are expected to be useful as novel biomarkers for toxicity assessment. in this study, we aimed to identify target organs by comprehensive analysis of ev rnas in the blood of mice after chemical exposure to establish a highly sensitive "next generation type" toxicity test for chemical substances and medicine using ev rna in blood as a biomarker. methods: all animal studies were conducted in accordance with the helsinki declaration and the guidelines approved by the animal care committee of the national institute of health sciences. c bl/ j male mice ( weeks) were orally dosed with ccl (vehicle, , mg/kg). serum were separated from blood after , , and hours after ccl administration. the serum was centrifuged at , x g to remove cellular debris and subsequently ultracentrifuged , x g. the pellet is resuspended in pbs and ultracentrifuged , x g again. the comprehensive small rna-seq of collected evs were performed according to the manufacture's protocols. results: we succeeded in isolating more than novel small rnas, which could be used as novel highly sensitive biomarkers for hepatotoxicity due to carbon tetrachloride (ccl : mg/kg & mg/ kg). well known hepatotoxicity biomarkers, mirna- and mirna- were upregulated more than -fold in the administration of mg/kg ccl , but not responded in the administration of mg/kg ccl . summary/conclusion: these results suggest that mir- and mir- are mainly released from liver to blood directly only in the administration of mg/kg ccl , while novel more sensitive hepatotoxicity biomarkers which responded in the administration of both mg/kg and mg/kg ccl should be included in the ev. our novel biomarkers will accelerate a rapid evaluation of chemical substances and medicine in nonclinical safety evaluation. introduction: advancements in sequencing technologies have allowed analysis of the genomic landscape of cancer using circulating cell-free(cf) dna. however, cfdna does not originate only from tumour cells. we recently demonstrated that most of the dna circulating in plasma of cancer patients is associated with large evs (l-evs), and that l-ev-associated dna reflects genomic aberrations of the cells from which l-evs arise. since l-evs are specifically released by tumour cells, we explore their potential to report cancer-specific genomic alterations in patient plasma and compare it to cfdna. methods: differential ultracentrifugation, tunable resistive pulse sensing, qubit dsdna high sensitivity assay, capillary electrophoresis, whole exome sequencing ( - x), targeted sequencing (qiaseqtm), flow cytometry. results: we show here that l-evs in the size range of > micrometre are present exclusively in plasma obtained from cancer patients and absent in plasma from healthy donors. in agreement with this finding, double-stranded(ds) dna is detected only in l-ev fractions of patient plasma and not in those obtained from healthy donor plasma using the same protocol. we also demonstrate that the fragments of dsdna associated with circulating l-ev are larger in comparison with cfdna (> , bp versus~ bp). a large-scale analysis of l-ev dna obtained from plasma of patients with metastatic castration-resistant prostate cancer (mcrpc) as well as with non-small cell lung cancer (nsclc) demonstrates that dna associated with circulating l-evs reports cancer-specific genomic alterations in both types of cancer. we further investigate if l-evassociated dna is intra-or extravesicular and demonstrate that it is present in both forms. we finally compare the purity of the tumour signal in intravesicular l-ev dna, total l-ev dna, and cfdna obtained from patient plasma. summary/conclusion: our results demonstrate that circulating l-evs contain high quality, large molecular weight dna that contains cancer-specific genomic alterations, supporting the use of l-evs as a source of tumour-derived dna in plasma. introduction: epidermal growth factor receptor (egfr) mutation driven lung adenocarcinoma (ac) represents a unique subgroup that lends itself to treatment with oral egfr tyrosine kinase inhibitors. current methods that are used to detect these mutations (e.g. l r or the resistance mutation t m) involve invasive tumour biopsies or blood circulating tumour dna (ctdna) and cell free dna (cfdna). the sensitivity of blood ctdna and cfdna is limited by the frequency of genomic alterations in the egfr gene; additionally, ctdna does not reflect changes in the egfr protein, against which novel therapies are in development. there remains a need to develop bloodbased biomarkers that can circumvent these disadvantages and replace the more standard, invasive tumour biopsies. we propose the study of exosomes for treatment monitoring as well as to identify egfr resistance related genomic and proteomic changes. methods: we enrolled patients with metastatic lung ac: with egfr mutations and without (control). from the patients with egfr mutant lung ac, we processed blood samples through the patients' treatment course, using ultracentrifugation to isolate exosomes. we then used both droplet digital pcr (ddpcr) to test exosomal rna (exorna) for the mutation of interest and western blots to test protein resulting from exon deletion or l r mutations. results: from patients with egfr exon deletion mutations, we detected identical mutations in exorna from / samples. exorna based mutational load increased and mirrored clinical progression in patients. three patients whose cancer remained stable demonstrated a decrease in their exorna. one patient had blood drawn only at points and was therefore not plotted. exorna from patients with l r and t m mutations demonstrated the corresponding mutations; however, exorna did not mirror their disease course. we also demonstrated mutant egfr protein presence in exosomes from patients. finally, we tested cfdna for egfr mutations from four matched samples using ddpcr. we detected matched mutations in exosomes in all four, while cfdna mutations were only detected in / patients. summary/conclusion: in summary, we detected egfr mutations in / exosome samples isolated from metastatic lung ac. our results set the stage for optimization of exorna methods and inform future experiments relating to exosomal cargo in patients with egfr mutant lung ac. identification of plasma-derived, ev-based biomarkers for glioblastoma introduction: glioblastoma multiforme (gbm) is the most malignant and aggressive primary brain cancer in adults, with an incidence of . per , people. currently, diagnosis is only performed via histopathological investigation of a tissue sample from a gbm lesion, complemented with molecular diagnostics for identification of select biomarkers. mri is the standard of care for follow-up and monitoring of treatment response. therefore, development of a "liquid biopsy" to obtain disease-relevant information from patient's body fluids is highly desirable. methods: we present the results from a clinical study in which extracellular vesicle (ev)-derived mrnas and long non-coding rnas were profiled from the plasma of gbm patients and control individuals. we obtained plasma from patients at the time of initial diagnosis, and matched controls by sex and age. ev-associated rna was isolated from - ml plasma and rna-seq was performed using our proprietary pipeline. sequencing data was analysed for differential gene expression. results: we observed mrnas as differentially abundant between gbm and control samples, with mrnas enriched in gbm samples and mrnas enriched in control samples (p < . ). correlation based on differentially abundant mrnas separated gbm and control samples into two unique populations. eight differentially expressed mrnas were previously identified as part of the mesenchymal gbm subtype. these data, while preliminary, provide a potential basis for the further development of a noninvasive gbm gene panel test. summary/conclusion: we have identified a novel rna signature for gbm from plasma derived evs, which differs from previously identified biomarkers isolated from tissue. further work will refine this signature to enable detection, characterization, and patient monitoring for gbm with minimally invasive techniques. introduction: sjogren's syndrome (ss) is a systemic autoimmune disease in which inflammation progressively damages the moisture producing glands of the afflicted. million americans are estimated to be suffering from the disease, % of which are women with an average age of . overlapping symptoms with other health conditions and co-morbidities make ss particularly difficult to diagnose, with average time to diagnosis of years. saliva exosomal rna profiling has been primarily focused on small rnas and has been limited thus far due to the large contribution of sequencing reads from the oral microbiome. a noninvasive saliva exosomal rna (exorna) based test capable of diagnosis would be highly desirable. methods: we began by first developing a novel long rna-seq workflow to selectively enrich and profile human exosomal mrnas and long non-coding rnas (lncrnas) from saliva. we then profiled salivary exorna obtained from ss patients and healthy matched controls. finally, we performed differential gene expression analysis to obtain an exorna signature for ss. results: rna-seq data analysis demonstrated highly efficient enrichment of human transcriptome, with over % of reads mapping to the transcriptome. further rna biotype analysis showed over % of transcriptome reads mapped to protein coding genes and lncrnas. we detected over , mrnas and approx. lncrnas. differential expression analysis (dex) of ss vs. healthy control exorna identified upregulated genes, including mrnas and lncrnas (p < . ). genes were found to be downregulated in ss, including mrnas and lncrnas. gene ontology analysis of dex genes revealed enrichment of genes involved in various immune system related pathways. most importantly, principal component analysis (pca) resulted in clear separation of ss patients from healthy controls. summary/conclusion: our optimized rna-seq workflow enables saliva-based liquid biopsy for biomarker discovery. the gene signature identified in this ongoing study could potentially provide a non-invasive molecular means of diagnosing sjogren's syndrome. introduction: increasing embryo implantation rates has become one of the greatest challenges in assisted reproduction techniques. usually an endometrial biopsy is done to identify a receptive endometrium, which prevents embryo transfer in the same cycle, as it is detrimental for the implantation. the implantation is a complex process, which requires a synchrony between the development of the embryo and the endometrium, but also, an adequate embryo-endometrial cross talk. the presence of extracellular vesicles (evs) as mediators of this communication has been describe in the endometrial fluid. therefore, we hypothesize that the molecular analysis of the content of the evs and companion molecules from endometrial fluid could be a non-invasive method to recognize an implantative endometrium and consequently improve the implantation rates. methods: the objective is to define a simple, sensitive and reproducible non-invasive ev-based method that allow the quick identification of an implantative endometrium by means of mirna analysis. for the establishment of a robust methodology for analysing evs from endometrial fluid in clinical settings, where the sample is limited and no sophisticated equipment is available, five different methodologies were compared in triplicate. two of them consisted in the direct extraction of rna while in the other three, before the rna extraction an enrichment of evs was done. smallrnaseq was performed to determine the most efficient method. once the best method was selected, it was applied in a set of real samples with different implantation outcome. the content of mirnas (mainly associated with evs) of endometrial fluid samples from women in whom the implantation was successful (n = ) and unsuccessful (n = ) were analysed. results: our results show that the protocols with a previous enrichment step of evs obtained a higher mirna expression. the results obtained from the differential analysis of the set of samples with different implantation outcome are being analysed and it is expected that the results will be available by the time this communication is presented. summary/conclusion: this work demonstrates that it is possible to obtain and analyse evs and evs-associated mirnas from a small volume of endometrial fluid samples, which allows the use of ev-mirnas as a low-invasive biomarkers for the detection of an implantative endometrium. funding: jip is supported by a predoctoral grant from the basque government. small rna cargo of evs is affected by hormone treatment in prostate cancer introduction: small rnas are recently reported as a regulator for prostate cancer progression to castration-resistant disease. our previous work has shown that evs protein cargo is affected by male steroid hormone, dihydrotestosterone (dht). in this study, we assess the small rna cargo of evs in response to androgen manipulations. methods: androgen receptor-positive lncaps are grown in css medium to deplete the androgens. media were then replaced with vesicle-depleted css medium ± nm dihydrotestosterone (dht) ± µm enzalutamide (enz) for h. evs were isolated using sequential ultracentrifugation ( g for min, , g for min, , g for h), washed once in pbs. protein and rna were collected from both parent cells and conditioned medium to allow direct comparison between s-evs cargo and cells. small rna ngs libraries were prepared using the illumina's truseq small rna library prep kit and single-end sequenced at a read length of nucleotides (nt). fastq library files were processed using a custom-designed pipeline. adapters were removed using the cutadapt tool, trimmed reads were mapped with high stringency against ribosomal sequences using bowtie . snorna and trna fragments were identified using the flaimapper software. remaining reads were mapped against the human genome hg using bowtie . results: we found that the presence or absence of androgens does not significantly change the amount of total rna in small evs (s-evs). however, hormone stimulation altered the small rna content of s-evs, in parallel with our previous published data on ev protein cargo. dht increased the abundance of snorna in cells, while a reduction of snornas was observed in the s-evs fraction. interestingly, dht induced the formation of cell filopodia that are not inhibited by androgen inhibitor enzalutamide. pathway analysis indicates the p mediated regulation driven by mirnas found in s-evs upon exposure to dht. the expression profile of snorna and trna fragments in dht treated cells resembles results from clinical prostate cancer specimens. summary/conclusion: our findings show that androgen manipulation alters both s-ev derived protein and rna cargo. changes in the s-ev rna profile due to treatment with androgens are not identical to small rna profiles in parental cells, indicating a specific sorting mechanism of s-ev small rna upon androgen manipulation. further, dht induces the formation of cell filopodia irrespective of enzalutamide, suggesting cargo selection of s-evs. we conclude that small rna ev cargo can be utilised to as prostate cancer biomarkers in androgen targeted treatments. introduction: cancer immunotherapy, such as pd-l blockade, is a method to eliminate cancer cells. ectopic expression of pd-l , on the surface of tumour cells, has been associated with tumour persistence and as an important predictor of therapy response. a test that, specifically and accurately, detects pd-l is critically important in order to identify patients that would benefit from these treatments. emerging evidence has shown that extracellular vesicles (ev) can carry immune checkpoint molecules, such as pd-l , and whose expression have been correlated with tumour immunity response. with a multitude of commercially available antibodies identifying appropriate clones and associated assay is important in order to standardize the diagnostic modality used. methods: pd-l expressing cancer cell lines were used to generate evs. pd-l -myc vector was transfected to generate an overexpression system. exoview® sensors containing different anti-pd-l clones were generated. samples (cell derived and plasma) were incubated on chips to allow the antibody to bind the antigen on the ev. after incubation, chips were immunolabeled with fluorescently labelled antibodies against pdl- or ev associated markers. exoview r reader was used to enumerate the evs captured on the sensor surface and analyse the expression of pdl- on single vesicle through fluorescence imaging. immunoprecipitation and mass spectrometry (ip/ms) were employed as an orthogonal method to verify the specificity of the assay. results: to study the detection efficiency of the antibodies, engineered pd-l -myc evs were used. under these circumstances, all the tested antibodies were able to capture evs. when testing endogenous pd-l positive evs from different cancer cell lines, only . and clones consistently bound to evs. in addition, evs derived from plasma demonstrated to be positive for pd-l , however, only clone . was able to immobilize these evs. the results suggested that clone . could be a potential pd-l antibody to detect pd-l positive evs originating from various sources. to confirm these results, and assure the specificity of the antibody targeted ip/ms was employed. summary/conclusion: in combination with the exoview platform, anti-pd-l antibodies can be screened and potentially used to generate a non-invasive ev-specific assay that could detect this protein in patients. differences in extracellular vesicle protein cargo is dependent on head and neck squamous cell carcinoma cell of origin university of michigan, ann arbour, usa introduction: head and neck squamous cell carcinoma (hnscc) is the sixth most common, eighth most fatal cancer worldwide and includes cancers of the oropharynx, larynx, hypopharynx, and oral cavity. in , there were over , new cases and , deaths estimated in the usa alone. despite recent advances in treatment, including radiation, chemotherapy, surgery, concurrent chemoradiation, and immunotherapy, many tumours develop resistance and progress. patients develop metastases or tumours recur locally or regionally; the -year overall survival rate for hnscc is only - %. factors that contribute to poor survival for patients with hnscc include late stage diagnosis, lack of reliable markers for early stage detection, high level of biologic heterogeneity, and local recurrence and distant metastases after treatment. methods: this study used representative hpv-positive and hpv-negative hnscc cell lines, one hpvtransformed cell line. and two non-cancer oral keratinocyte cell lines. evs were isolated using differential ultracentrifugation and peg precipitation/ultracentrifugation. evs were characterized by tem, nta, and wes protein analysis for reported ev markers. ev and whole cell lysates were assessed by lc-ms/ms analysis using the tandem mass tag- plex kit. cluster analysis was performed on the fold-change peptide spectrum matches (psm) for the evs from the hnscc lines compared to the evs from the normal keratinocyte line (noksi). protein was measured using a capillarybased electrophoresis instrument. results: cd and annexinv were detected in all of the ev lysates tested, while calnexin was detected in all of the whole cell lysates and none of the ev samples tested. selected proteins stat , hla-a, tenascin, e-cadherin, β catenin, cytokeratin , epha , and cd , and hpv-related markers p , p , rb, cyclin d , and egfr were tested using the wes platform. evs from hpv-positive cell lines showed higher protein levels compared to evs from hpv-negative cell lines in stat , hla-a, and tenascin. only kert demonstrated lower protein levels in evs from hpvnegative cell lines. of the common hpv-associated hnscc markers: egfr, p , rb, cyclin d and p , only egfr was positive in any the evs tested. the remaining proteins queried, e-cadherin, β catenin, epha and cd showed varying protein levels in evs from both hpv positive and hpv-negative cell lines. summary/conclusion: our findings suggest that these proteins may be potential hnscc ev markers that may be ) selectively included in ev cargo for export from the cell as a strategy for metastasis, tumour cell survival, or modification of tumour microenvironment, or ) representative of originating cell composition, which may be developed for diagnostic or prognostic use in clinical liquid biopsy applications. validation of antibodies on western blot for extracellular vesicles from biological human samples and cancer cell conditioned media the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: one of the major challenges in extracellular vesicles (evs) research is to prove the particles that are isolated are true evs, rather than other co-isolated contaminants, like lipoproteins. isev recommends using multiple assays to characterize evs. this study aims to validate the positive and negative protein markers for extracellular vesicles from plasma, urine and prostate cancer cell conditioned media (ccm). methods: membrane and cytosolic fractions of mcf cells served as positive and negative controls for all antibodies validated. evs were isolated from plasma of healthy volunteers, urine of healthy volunteers and ccm of pc- cells using differential ultracentrifugation. eight protein markers were assessed: positive markers cd , cd , cd , flotillin (flot ), alix and tumour susceptibility gene (tsg ), negative marker calnexin (canx), and contaminant markers apo-a for plasma and thp for urine. tetraspanins are small transmembrane proteins expressed in evs. flot is membrane protein that forms microdomains in the plasma. alix and tsg , an accessory protein of the endosomal sorting complex required for transport, are involved in the biogenesis of evs. they are positive markers for evs. canx is in the membrane of the endoplasmic reticulum. apolipoprotein-a (apo-a ) is the protein components of lipoproteins, therefore it is marker of contamination for plasma ev. tamm-horsfall protein (thp) is contamination marker for urine ev, because it is most abundant protein in human urine. results: all antibodies were validated in the correct positive and negative control, thus confirmed as usable and reliable antibodies for western blot. in plasma ev, cd , cd , cd and flot were positive and canx and apo-a were negative. in urine evs, cd , cd , flot- , alix and tsg were positive and canx and thp were negative. in ccm evs, cd , cd , flot , alix and tsg were positive and canx was negative. summary/conclusion: we confirmed a high degree of ev purity from sample types: urine, plasma, and ccm. of particular importance, we confirmed that evs isolated from biologic patient samples, plasma and urine, had low contamination. future work will use these methods to confirm purity of ev samples prior to addition analysis, such as examining ev cargo and biologic significance. proteomic study of mesenchymal stem cells derived exosomes modified using mir. introduction: the project we are working on is to modify the immunogenic profile of human cmms from the umbilical cord stroma through its stable transfection with anti-mir- - p, and therefore of the exosomes that these cells generate, for use in free-cell therapy to treat inflammatory process. methods: evs released from a primary culture of human umbilical cord mesenchymal stem cells and from primary culture of human umbilical cord mesenchymal stem cells mir -/-modified through stable lentiviral transfection were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (tem) and measured by nanoparticles tracking analysis (nta). protein extraction from evs was made using ripa buffer and after checking protein integrity the total ev proteins. we performed a shotgun proteomic study using a tmt ( -plex) label of the total mir -/-exosomes protein comparing it with normal exosomes. after labelling the ltq-orbitrap platform of proteored was needed for fraction injections and data acquisition. proteome discoverer . (thermo) was used for protein processing and quantification. results: a total of . proteins were identified at least with a unique peptide and we have able to establish the proteomic profile of mir -/-exosomes against normal exosomes. we found out several protein modulated by mir and related to inflammation. summary/conclusion: we have able to establish the proteomic profile of mir -/-exosomes against normal exosomes focusing on proteins involving inflammation process. all those results seem indicate that exosomes could be modified, which could be used as an anti-inflammatory free-cell therapy. funding: proteored concept test project grant. a novel extracellular vesicle isolation method used to discover urine liver disease biomarkers introduction: hepatocellular carcinoma (hcc) is the th most common cancer worldwide and the rd most common cause of cancer death; additionally, its incidence is increasing. while outcomes for early hcc are superior to those for late stage disease, early detection of hcc remains a challenge. current guidelines have suboptimal sensitivity and specificity. in this pilot study, we hypothesize that urine extracellular vesicles (evs) may identify candidate biomarkers towards the development of an inexpensive, widely accessible screening assay for the early detection of hcc. methods: urine samples from healthy subjects, subjects with cirrhosis, and subjects with cirrhosis plus hcc were collected and processed using ymatrix columns to isolate ev-associated protein and mirna. protein was analysed using a tandem mass tag method on a thermo scientific orbitrap fusion mass spectrometer with comet/paws and edger processing. mirna was analysed using a targeted firefly microarray from abcam. differential expression and predictive modelling for the presence of hcc and cirrhosis was performed to identify candidate mirna and protein biomarkers. results: for mirna, samples were eligible for analysis after low expression filtering. we used pair-wise ratios of cancer-associated mirnas by gradient boosting of decision trees to develop a predictive model for hcc. our best model had a sensitivity and specificity of . and . respectively using mirnas to distinguish hcc from cirrhosis. all samples were eligible for protein analysis. based on differential expression and biologic relevance, we identified protein candidate biomarkers. interestingly, we found liver-selective proteins and known hcc/cirrhosis plasma/tissue markers, demonstrating proof-of-concept for the method. summary/conclusion: urine extracellular vesicles contain liver-selective proteins and known liver disease serum biomarkers as well as novel mirna and protein biomarkers that are significantly up-regulated in disease samples. the described candidate biomolecules may be easily accessible biomarkers with which to develop a sensitive and specific universal screening diagnostic for the early detection of cirrhosis and hcc. introduction: the peptidergic g-protein coupled receptors (gpcrs) are cell-signalling transmembrane proteins, which in their native form comprise of seven segments embedded in the cell membrane. this structural advancement is believed to be maintained in extracellular vesicles (evs). in autoimmune diseases, the presence of autoantibodies towards gpcrs is not uncommon, and to detect plasma autoantibodies, evs carrying gpcr will be used as template in a novel microarray screening tool. methods: purified evs from hek cells were printed on different types of surfaces; polymer coated glass slides and hydrophilic and hydrophobic plastic well plates. five different print buffers were tested in a multiplex assays. spots containing evs were stained with biotinylated antibodies (cd , cd , cd , adrβ , hsp , epcam and flotilin- ) followed by binding of cy -labelled streptavidin and visualized microarray scanner. results: the outcome of these experiments was promising, as some of the chosen printing buffers showed increased tendencies to bind evs. the ev presence was verified with a panel of markers known to be present on small evs. in addition, the ev content of the adrenergic beta- receptor (adrβ -receptor), which is a gpcr of interest in autoimmune diseases, was verified in some of the experimental setups. summary/conclusion: the approach of using evs as template in a screening tool possesses the potential to easily screen for autoimmune illness markers in diagnostic purposes. using the microarray technology allows the screening to be multivariate, specific and highly sensitive. circadian variation of extracellular vesicles secreted in urine: analysis of time point collection and normalization strategy. introduction: urinary extracellular vesicles (uevs) are an ideal source of biomarkers for kidney and urogenital diseases. despite the great deal of interest generated by uevs, little is known about its collection time and normalization approach. the majority of the studies on uevs focus on spot urine collection based on the assumption that it accurately reflects the renal function, although time point of collection is not standardized. therefore the practice to collect spot urine does not allow for calculating and standardizing accurately the uev excretion rate which may vary during the day. in addition, no research has been carried out yet to show the quantitative and qualitative difference of uevs between spot urine and h collections.the aim of this study is to compare uevs excreted in all single voids during a hour collection period and compare it with hour collection performed. methods: uevs were enriched by differential centrifugation and electron microscopy, western blot, nanoparticle tracking analysis, tuneable resistive pulse sensing and imaging flow cytometry were used to quantify uevs and associated markers variation during the hour. creatinine, urine osmolality and particle concentration were used to normalize the assessed analytes. results: electron microscopy showed a heterogeneous population of evs and western blot confirmed the presence of ev markers (tsg , alix and cd ). rna was extracted by a column-based method (mirna extraction kit qiagen) and cel- mirna was spiked in each sample. a multiparametric detection of nephron markers podocalyxin, aquaporin- and uevs pan tetraspanins (cd + dc + cd ) was performed utilizing imaging flow cytometry. whereas the uev composition did not change across the hours analysis, the quantity of uevs and related markers fluctuated during the day depending on the hydration and excretion rate.the results of a hour urine collection reflected the average results of all single voids over a hr period. creatinine and particle count normalization failed to normalize "outliers". summary/conclusion: this study represents the very first report which compares single void urine versus hour uev analysis. we concluded that the hour collection is the preferred choice for a robust and rigorous assessment of uevs and its associated markers. porcine body fluids differ in small extracellular vesicle counts: comparison of blood plasma, seminal plasma and cerebrospinal fluid as vesicle sources for proteomic analyses helena kupcova skalnikova a , jakub cervenka b , jaromir novak a , karolina turnovcova c , bozena levinska a , jana juhasova a , stefan juhas a and petr vodicka a a institute of animal physiology and genetics, czech academy of sciences, libechov, czech republic; b institute of animal physiology and genetics cas, v. v. i. libechov, libechov, czech republic; c analysis tools were used to identify in silico biological pathways and functions governed by detected mirnas. expression of putative targets of selected mirnas was tested using qpcr after in vitro delivery of uterine evs to ptr cells. results: careful characterisation confirmed that uterine lumen is enriched with a diverse population of evs caring mirnas. interestingly, out of detected mirnas showed difference in abundance between tested days of pregnancy and half of them was exclusively detected on d . identified mirnas were characterized as potent regulators of cellular development, growth, proliferation, and movement, in addition to their involvement in organismal and embryonic development. the expression of genes identified as a possible mirna targets was tested after evs delivery to ptr cells in vitro. both down-(e.g., ptger ) and up-regulated (e.g., lifr) genes were found (p < . ); involved in the same molecular and cellular functions enriched by detected mirnas. methods: evs were harvested from wild type and arrdc -/-epididymal cells using differential ultracentrifugation, then characterised using nanoparticle tracking analysis and transmission electron microscopy. sperm motility was measured using computer assisted sperm analysis and imagej. fertilisation capacity was measured using the following assays: capacitation-associated tyrosine phosphorylation, calcium ionophore induced acrosome reaction, zona pellucida binding assay and in vitro fertilization with time-lapse imaging of embryo development. immunohistochemistry was also used to visualise two pronuclei formation and blastocyst morphology. arrdc -/-sperm was supplemented with wild type evs in the above assays to assess whether they could restore function. results: sperm from arrdc -/-mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as evidenced by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and production of two-cell embryos. we observed a significant reduction in ev production by arrdc -/-epididymal epithelial cells, and addition of wild type evs to arrdc -/-sperm dampens the acrosome reaction and restores zona pellucida binding. introduction: gestational diabetes (gdm) is among the most common pregnancy complications. despite treatment, up to % of pregnancies complicated by gdm result in infants being born large-for-gestational-age (lga). this not only causes problems at birth but predisposes offspring to developing cardio-metabolic disease in adulthood. there are no treatments for lga as the cause is unclear, although it is associated with altered placental vascular development. micrornas (mirnas) regulate placental development; they are produced within cells but can be released into the circulation inside evs, which in turn can be transported into target cells and tissues to influence cellular processes. we aimed to characterise circulating evs in pregnancies complicated by gdm-lga and determine if ev-derived mirnas have the potential to influence placental development. methods: maternal serum and plasma samples were collected from women with pregnancies complicated by gdm at - weeks gestation; placental tissue was collected at delivery and birth outcomes recorded. serum and plasma evs were isolated and characterised by electron microscopy (shape), nanoparticle tracking analysis (nta; size/concentration), and western blotting (ev-enriched proteins). mirna qpcr arrays were performed on evs. mirnas were quantified in placental tissue via qpcr. results: em and western blotting confirmed isolation of evs and nta revealed no significant difference in size/ concentration in gdm-lga pregnancies (n = ) compared to gdm-aga (n = ; p > . ). several ev mirnas were altered in maternal circulation in gdm-lga compared to gdm-aga (n = /group; >twofoldchange; p < . ), including four skeletal muscle-specific "myomirs": mir- - p, mir- a- p, mir- b, and mir- a- p (all increased). all four myomirs were present in placenta but only mir- - p was significantly altered in gdm-lga compared to gdm-aga (n = - /group; p < . ). summary/conclusion: ev-bound myomirs could have predictive value for aberrant foetal growth in cases of gdm. mir- - p regulates vascular development in other systems, so we propose that mir- - p contributes to lga by influencing placental vascular development, however further work is required to establish this. introduction: seminal plasma is particularly rich in extra cellular vesicles. myelinosomes are membranous organelles described throughout the seminiferous epithelium of the testis but never reported in semen. the aim of this study was to look for the presence of myelinosome vesicles in human seminal plasma. methods: because of the viscosity of seminal gel and its water-holding capacity, classical transmission electron microscopy does not seem to be an optimal technique to reveal the presence of myelinosomes in this fluid. cryo-electron microscopy is a technique that allows visualization of nanosized structures without prior fixation or addition of heavy metals for contrast. the sample is therefore visualized as close to its native state as possible. using standard myelinosome preparation from tm sertoli cells, we first analysed the appearance of "standard" native myelinosomes by cryo em and then compared it with the vesicles from human seminal plasma samples. results: we have specified by cry-em the morphological aspect of "standard" myelinosomes isolated from the culture media of tm sertoli cells. the vesicles with the same morphological appearance were revealed in human seminal plasma specimens. summary/conclusion: myelinosomes are membranous organelles found in the seminiferous epithelium of the testis and secreted by the somatic sertoli cells in the lumen of the seminiferous tubules.the preparations from human seminal plasma contains a population of large ev (average diameter nm) whose morphological appearance resemble those of myelinosomes. defining the specific biomarkers and functionalities of myelinosomes in human seminal plasma are the concerns to be addressed in our further research. introduction: more than one million patients worldwide suffer from tuberous sclerosis complex (tsc) and have mutations in either tsc or tsc genes. together, the tsc proteins regulate mtorc activity. all tsc patient post-mortem samples exhibit renal disease and % of patients with tsc experience a premature loss of renal function. mouse and human studies are incongruity with the second somatic hit mechanism of disease, because of the low percentage of cystic cells exhibiting loss of tsc expression. we posited that the loss of a tsc protein expression may alter extracellular vesicle (ev) biology and contribute to disease. methods: we used crispr/cas to disrupt the tsc gene in mouse inner medullary collecting duct (mimcd) cells, and isolated evs using gel filtration from the isogenic cell lines. we characterized the evs using tunable resistive pulse sensing (trps), dynamic light scattering (dls), transition electron microscopy (tem), and wester blot analysis. we further performed mass spectroscopy on the ev proteins. results: loss of the tsc gene in mimcd cells induced a greater than three-fold increase in ev production compared to the same cells having an intact tsc axis. electron microscopy confirmed the purity and spherical shape of evs. both trps and dls demonstrated that the isolated evs possessed a heterogenous size distribution. approximately % of the evs were in the - nm size range. western blot analysis using proteins isolated from the evs revealed the cellular proteins alix and tsg , the transmembrane proteins cd , cd and cd , and the primary cilia-related hedgehog signalling-related proteins arl b. proteomic analysis of evs identified a significant difference between the tsc -intact and tsc -deleted cells that correlated well with the increased production. summary/conclusion: evs may be involved in tissue homoeostasis and cause disease by overproduction and altered protein content. the evs released by renal cyst epithelia in tsc complex may serve as a tool to discover the mechanism of tsc cystogenesis and in developing potential therapeutic strategies. introduction: we have shown that evs derived from amniotic fluid stem cells (afsc) of mouse origin present therapeutic effect in an animal model of chronic kidney disease, alport syndrome (as). in light of clinical translation, we isolated afsc-evs of human origin, characterized their cargo and evaluated thier therapeutic effect in vivo. methods: human clonal afsc were derived from amniotic fluid collected after volunteer donors provided consent. evs were obtained from afsc and identity and purity were assessed by rna-seq and proteomics. potency of hafsc-evs was evaluated by performing in vivo studies. ev biodistribution was evaluated by mri and therapeutic effect by measuring renal function and mice life-span. bulk rna-seq was performed on glomeruli obtained from injected and non-injected mice to identify potential ev regulating targets. results: proteomic profiling identified intact proteins and rna-seq data identified , mirs in hafsc-evs. hafsc-ev "fingerprint" was assessed by performing go analysis on the most highly expressed proteins and mirs. the results identified pathways involved in tissue homoeostasis such as mtor pathway, tgfβ and vegf pathways. when injected in vivo into as mice, biodistribution studies showed that hafsc-evs localized in the kidney, corrected proteinuria. no side effects (including teratoma) were noted in the treated mice. rna-seq of glomeruli obtained from treated as mice showed similar gene expression patterns to wilt type mice, by cluster analysis. our data indicated that hevs highly modulated pathways involved in collagen and matrix deposition remodelling, in addition to downstream targets of vegf, fgf, tnf, angiotensin and preserved glomerular cells structure and function. summary/conclusion: our protocol for hevs derivation is reproducible and allows derivation of ev lots with the same identity (specific cargo of proteins and mirs) and potency (present therapeutic effect in as). hafsc-evs modulated signalling pathways that are central to maintaining glomerular homoeostasis and preserved glomeruli structure with improved kidney function. this suggests the possibility of using hafsc-evs as a new therapeutic option for treating renal failure in humans. introduction: recent studies have shown that stem cell-derived extracellular vesicles (msc-ev) therapy improves renal outcomes in models of acute ad chronic renal disease. however, to better investigate the molecular mechanisms of ev-induced regeneration, and to define new ev sources, devices that mimic d organ architecture and flow conditions are needed. the aim of our work is to evaluate the regenerative potential of naïve and engineered ev in a millifluidic in vitro d model of glomerular damage in continuous perfusion. methods: methods: we set a millifluidic in vitro d model of glomerular filtration, a three-layers structure composed by human podocytes and glomerular endothelial cells, and, in between, of a basement membrane of collagen type iv. the barrier thus formed is set up inside a bioreactor, in a closed milli-fluidic circuit in which fluid flows continuously at a certain flow rate. we reproduced different pathological conditions and tested the localization and effect of evs in a dynamic system. : results: we obtained a standardized protocol and an adequate configuration of the milli-fluidic circuit subject to continuous reperfusion. renal damage was induced by doxorubicin or by hypoxia-reperfusion injury. we evaluated uptake, cargo transfer and effect of naïve and mirna engineered msc-evs or of klotho engineered ineffective evs administered into the dynamic co-culture system. evs were able to pass through the system and to deliver to podocytes proregenerative factors, promoting survival and limiting permeability. introduction: worldwide, renal cell carcinoma (rcc) is th most common cancer in men and th most common in women. new biomarkers are needed to aid rcc-diagnosis, provide prognostic information, and to predict response to modern targeted therapies. extracellular vesicles (evs) are an emerging source of cancer biomarkers because all cells, including cancer cells, secrete evs into biofluids as blood and urine. however, benign cells contribute to ev populations isolated from blood and urine reducing the diseasespecificity. we have developed a protocol for ev isolation directly from human rcc tissue that can increase tumour-specificity of biomarkers. methods: we obtained technical and biological replicates from normal kidney tissue and clear cell rcc tissue. serum-free media was incubated with the specimens. a combination of differential centrifugation, filtration, and ultracentrifugation was used for ev isolation. evs were quantitated using two methods, allowing for comparison between nanosight ns and nanofcm. tem was used to determine presence of intact vesicles in the ev samples. presence of ev introduction: urothelial carcinoma (uc) is a malignant cancer that affects the urothelial cells, representing % of all bladder tumours. at diagnosis % of bladder cancers are non-muscle invasive tumours. importantly, upon transurethral resection of the bladder tumour, nearly - % of these patients will experience disease relapse and - % will progress to muscle invasive tumour, requiring thereby, a rigorous and expensive follow-up. currently, this is performed through the frequent use of highly invasive cystoscopy and the low sensitivity urine cytology. thus, innovative liquid biopsy-based biomarkers that circumvent these drawbacks are highly desirable for improved uc clinical management. here, we aim to implement a protocol for the isolation and characterization of extracellular vesicles (evs) from uc patients' urine samples. methods: a two-step protocol involving ultracentrifugation (uct) and by size-exclusion chromatography (sec) was optimized for urine samples. the isolated urine-derived evs from uc patients were then characterized according to their size, concentration (nta), morphology (tem), protein amount (lowry method), presence of ev-associated and disease-associated protein markers (western blot). results: isolated urinary evs from uc patients had a size ranging from nm to nm with characteristic ev morphology, express ev-associated markers as cd and hsp and were negative for cell debris markers. the recovery yield and purity of isolated evs following each isolation technique was characterized. upon uct, sec was required to deplete most of the ev-associated thp and albumin protein contaminants. some disease-associated protein markers were highly enriched in isolated urinary evs compared to crude urine. summary/conclusion: taken together, these results indicate that a two-step ev isolation protocol was properly implemented and validated in uc patients' urine samples. notably, several ev-associated disease biomarkers were detected in the urine of uc patients. this ev-based liquid biopsy might provide the means for real-time monitoring of residual disease and relapse in uc patients. introduction: glioblastoma multiforme (gbm) is a very aggressive type of brain tumour. different gbm molecular subtypes (proneural, mesenchymal and classical) often co-coexist within the same tumour, with the mesenchymal subtype driving the tumour progression. recently, our lab demonstrated that the cargo of extracellular vesicles (evs) could mirror the molecular background of the gbm cells from which they were derived. altogether, we believe that gbm cell-derived evs can be directly involved in the expansion of the mesenchymal signature in tumours, thus supporting gbm aggressiveness. methods: non-mesenchymal (t & u ) gbm cells were "primed" using evs derived from mesenchymallike (u & ln ) gbm cells. ev-primed gbm cells were then co-cultured with their non-primed counterparts to determine whether the mesenchymal signature can "spread" from cell to cell via evs. effect on cell proliferation, migration and invasion (in hyaluronic acid hydrogels) was assessed following ev treatment and co-culture. the expression of mesenchymal gbm markers was measured by western blotting. further mass spectrometry analysis of cell and ev content was undertaken to describe potential underlying mechanisms. results: co-culture with ev-primed gbm cells significantly increased proliferation and hydrogel invasiveness of non-mesenchymal cells. interestingly, the stimulating effect of co-culture was even stronger on the proliferation of ev-primed gbm cells. moreover, further proteomic analysis revealed that expression of mesenchymal gbm markers such as cd was increased in non-mesenchymal cells following coculture. summary/conclusion: our data suggest that evs from mesenchymal gbm cells can be uptaken by gbm cells from different subtypes, thus stimulating tumour progression. overall, we think the present study provides with new insights for the understanding of gbm recurrence and the development of potential therapeutic strategies. introduction: triple-negative breast cancer (tnbc) is the most aggressive form of breast cancer. previously we reported that the heterogenous population of evs released from tnbc cells promotes the growth and aggression of recipient cells. here we investigated if, by using compounds proposed to inhibit ev release i.e. calpeptin and y (to block those budding at cell membrane) and gw and manumycin a (to block evs from mvbs), we could reduce the associated transmission of aggressive phenotype. methods: evs were separated from medium conditioned by tnbc cell line hs ts(i) , using a discontinuous optiprep density gradient, after the cells were treatment for hrs with the compounds listed above. evs (pooled fractions - with a density range of . - . g/ml) were characterised by nta, bca, lipid assay, immunoblot, tem and flow cytometry. to investigate the functional effects of the evs released, proliferation and migration assays were performed on hs t and mda-mb- cells using the ev to cell ratios of × evs/ x cells, × evs/ x cells, × evs/ x cells to evaluate doseresponse. ev-track id ev (score of %). results: gw significantly (p = . ) decreased ev release from hs ts(i) cells. manumycin a and a combination of calpeptin and y (combo) decreased ev release, but significance was not reached. conversely, calpeptin and y actually increased ev release; but not significantly. of the reduced numbers of evs released following gw treatment, hla-dr+ evs were significantly (p = . ) enriched. none of the evs analysed significantly changed hs t or mda-mb- growth rates. however, evs from cells treated with calpeptin (p = . ), gw (p = . ), manumycin a (p = . ) and combo (p = . ) caused significant reduction in mda-mb- migration compared to the effects of evs from untreated cells. similarly, ev from cells treated with gw (p = . ), and combo (p = . ) caused significant reduction in hs t migration. summary/conclusion: while gw was the only compound that caused a significant decrease in quantities of ev released, the evs that continued to be released following treatment with gw or calpeptin and y significantly reduced migration of both recipient cell lines. funding: phd funding: tcd scholarship and carrick therapeutics ltd extracellular vesicles from highly metastatic lung cancer cells induce barrier impairment, permeability, and epithelial-to-mesenchymal plasticity in a -day mature bronchial epithelium purdue university, west lafayette, usa introduction: epithelial-to-mesenchymal (emt) transition plays an integral role in cancer metastasis, which is responsible for as much as % of cancer mortality. cancer exosomes induce emt in bronchial epithelial cells, however, the epithelial cells inhibit emt when allowed to form a mature epithelial barrier with apicalbasal polarity. it is not known if cancer-derived extracellular vesicles (evs) can induce emt and more importantly, barrier disruption in a mature epithelium. here, we show that evs from a highly metastatic lung cancer cell line (calu ) are) are not only sufficient to induce emt in non-tumorigenic bronchail epithelial cells (beas- b), but are also capable of disrupting a -day mature bronchial epithelial barrier by significantly reducing teer, inducing sixfold increase in permeability and complete loss of e-cadherin at cellcell tight junctions. methods: beas- b and calu evs were characterized using electron microscopy, nanosight and western blotting for exosome-specific features. for permeability studies, beas- b cells were cultured in transwell for days to establish an intact epitheliumconfirmed by measuring teer (trans-epithelial electrical resistance). intact beas- b monolayers were treated with calu evs at , and μg/ml for hrs, and barrier intactness and permeability were evaluated by measuring teer, apical-basolateral translocation of dextran beads and confocal imaging of tight junctions (e-cadherin). for emt experiments, beas- b cells treated with calu evs at and μg/ml were evaluated for ecadherin and vimentin levels by qrt-pcr and western blot after hrs. results: beas- b and calu evs were enriched in - nm size range, and cd and cd were enriched in the ev fraction in contrast to the cell lysate and vice versa for gp . calu evs significantly impaired day mature beas- b monolayer's barrier properties, which at the highest dose caused % reduction in teer from . ± . to . ± . Ω.cm (n = ). this was further confirmed by~sixfold increase in dextran beads' apical-basolateral translocation in min ( . ± ng/ml in control vs . ± ng/ml in treated) (n = ) and complete loss of e-cadherin expression at cell-cell tight junctions (n = ). at the transcript level, calu evs induced significant downregulation of e-cadherin by % and upregulation of vimentin (mesenchymal marker) twofold (n = ) in beas- b cells, indicating transition into mesenchymal phenotype. summary/conclusion: we demonstrated the involvement of evs derived from highly metastatic lung cancer cells in inducing emt in bronchial epithelial cells and epithelial barrier disruptionthe initial stage of the intravasation process. grp plays a crucial role in the extracellular vesicle-promoted radioresistance of irradiated head and neck cancer cells introduction: small evs released from irradiated head and neck squamous cell carcinoma (hnscc) cells increase resistance of recipient hnscc cells to radiation in vitro. we have identified the glucose-regulated protein (grp ), a chaperone protein of the hsp family which is involved in cellular stress responses and associated with worse survival in head and neck cancer patients, as an essential component of the ev-mediated radioresistance. methods: small evs were isolated from conditioned medium from irradiated and non-irradiated bhy hnscc cells by combined microfiltration ( . µm) and differential ultracentrifugation. grp surface expression was measured by proteomic analysis, immunoblotting and bead-facs. radiation resistance of bhy cells was determined by a clonogenic survival assay. results: increased grp was identified on the surface of evs from irradiated cells. the increase in ev grp correlated with increased grp expression at the donor cell surface. the grp content of recipient cells also increased upon transfer of evs from irradiated, but not non-irradiated cells, ultimately leading to enhanced cell survival. to check a potential role of elevated grp in radiation resistance we overexpressed grp . here the modest ( x) overexpression of grp was sufficient to confer an enhanced radioresistant phenotype to the bhy cells. a correlation between grp -dependent increase of radioresistance and activation of the akt pathway is yet to be determined. summary/conclusion: our results suggest a pivotal role for ev-transferred grp in modulating the radiation response of recipient hnscc cells. radiation directly increases the cellular and vesicular grp levels, and subsequent ev-mediated transfer leads to enhanced grp levels and radioresistance in recipient cells. this study provides new mechanistic insights into the effects of evs in radiation response and elucidates an interesting target protein and novel strategies for the improvement of radiotherapy. d modelling of ev release in progressing prostate cancer introduction: the modelling of cancer progression should be capable to translate acquired knowledge of cell behaviour to the real human body conditions. however, the extracellular vesicles (evs) isolated from d cell models are commonly exploited in research. taking into account the specificity of the prostate cancer (pc) environment, and a strong need of early diagnosis of castrate-resistance by prostate cancer (crpc) patients, we suggest in-depth profiling of different ev subtypes isolated from d culture as a new tool to model the progressing pc. methods: cells from hormone-resistant prostate carcinoma -rv line were cultured in d and d conditions, using d coseedistm. acd plasma controlled for haemolysis and remaining platelets was taken from patients with pc and crpc. the fractions of ev subtypes from cell culture and plasma were obtained by differential centrifugation (dc) followed by iodixanol density gradient purification. each of the fractions was measured by nanoparticle tracking analysis (nta), tunable resistive pulse sensing (trps) followed by elisa. for that, cd and cd were used as ev markers, apob and apoa for lipoprotein contaminants control, and cd , cd and psma as tissue-specific biomarkers for determination of fractions containing evs of different origin. ev-contained fractions were subjected to next generation sequencing (ngs). results: in d conditions, the -rv cells produce up to -times higher ev number than in d. size and density distribution of evs derived from d cultures but not of d resembled plasma evs. size distribution and biomarker expression among different ev subtypes allowed distinguishing between pc and cprcderived samples, indicating a potential to translate these results into clinics for early cprc detection. summary/conclusion: this work demonstrates a new approach to study the secretome of a progressing pc under d conditions. the profiles of ev subtypes produced by cancer cells growing in a d spatial architecture resemble the profiles of plasma evs and can serve a useful tool for the establishment of new biomarkers. introduction: renal cell carcinoma (rcc) is the most common primary renal neoplasm, with over , cases in the us alone each year. early detection of rcc leads to consistently better patient outcomes, and extracellular vesicles (evs) isolated from patient samples may prove to be a valuable clinical tool in the future. evs are abundant in blood and urine and show a large amount of heterogeneity but are difficult to analyse due to their small size and difficulty in isolation. here, we employ a multiparametric analysis of ev surface markers to identify a set of markers that may prove clinically relevant in future studies. methods: rcc cell lines vok , vok , and vok were cultured in flasks containing ml of ev-depleted media ( % fbs, centrifuged hr x , g). when cells reached~ % confluency, the conditioned media was collected and spun at , g for mins two times to deplete any remaining debris, leaving~ ml of media. this media was concentrated to a final volume of~ ml using a pall jumbosep kda mwco filter. this concentrate was purified from protein by using an izon qev- column, collecting ml fractions. protein content of each fraction was analysed using a absorbance while concentration and diameter distribution were determined through nanoparticle tracking analysis (nta). pooled samples made of the three most concentrated fractions were concentrated to a final volume of~ µl using the pall microsep kda filter and then used for analysis in the miltenyi macsplex exosome kit. flow cytometric data were generated by the cytoflex s and analysed using flowjo and mpapass software. these positive signals were verified through bead-only controls and titrations. results: the mpapass software allowed for heatmap generation, data reduction, clustering and visualization of expression patterns. of the detection antibodies used across capture beads, cd , cd , cd , beta- microglobulin, and cd were found to be prevalent in these rcc evs. these markers were found to be co-expressed particularly with cd , cd , and cd . summary/conclusion: the use of multiplex analysis allowed for detection of five distinctive surface markers found to be prevalent in evs collected from rcc cell lines. these results demonstrate the utility of multiplex analysis and mpapass software for identifying potential markers of interest and provide proteins that are worth exploring further. the next steps to this work will be developing custom multiplex arrays that tailor capture and detection of evs specifically for rcc pathology. low molecular weight protein tyrosine phosphatase (lmwptp) carried by colorectal cancer cells-derived extracellular vesicles as a player in tumour-educated human fibroblast university of campinas -unicamp, campinas, brazil introduction: extracellular vesicles (evs) are doublemembrane-bound nanovesicles released by cells playing a key role as mediators of intercellular communication. low molecular weight protein tyrosine phosphatase (lmwptp) is upregulated in several cancers type, including colorectal cancer (crc), and it has been correlated with aggressiveness, chemoresistance and poor prognostic. methods: the aim of this study was to determine whether crc cells release lmwptp-enriched-evs and influence tumour microenvironment-associated cells as a representative tumour education. crc cells, hct and ht , were cultured in serum-free medium for hours. conditioned medium was concentrated by ultrafiltration (mwco kda) and evs were isolated by total exosome isolation reagent (invitrogen). evs were characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and western blotting (wb). lmwptp levels were analysed by wb and sandwich-elisa. to evaluate tumour education, hff- fibroblasts were used as recipient cells. the uptake of evs (pkh fluorescently labelled evs), proliferation (viability) and migration (wound healing assay) were analysed in a co-culture model of crc-derived evs and hff- . results: nta showed a higher concentration of evs released by ht . hct and ht evs displayed a mean diameter around nm and a cup-shaped morphology. isolated evs were positive for evs-markers cd and tsg and negative for gm a non-evs marker. ht lineage as well as derived-evs are lmwptp-enriched in comparison to hct cells and evs. upon incubation, fluorescently hct and ht derived evs were internalized into hff- cells in a perinuclear region. evs derived from both cells increased the viability and proliferation of hff- cells. intriguingly, evs derived from ht promoted cell migration. summary/conclusion: in conclusion, for the first time, we showed that lmwptp can be carried by evs derived from crc cells and lmwptp-enriched-evs can modulate biological aspects of hff- fibroblast. overall, our findings point lmwptp out as important player in tumour-educated fibroblast. exosomal mir- a inhibition by vincristine and prednisone in paediatric acute lymphoblastic leukaemia. introduction: vincristine and prednisone are standard agents in treatment of paediatric acute lymphocytic leukaemia (p-all). mechanistically, vincristine induces apoptosis by blocking microtubules formation, while prednisone binds to cytoplasmic receptors and inhibits dna synthesis, both of which lead to apoptosis. the effect of these agents on exosomal micro-rna expression and its functional regulation is not yet investigated. elevated levels of mir- a in circulating exosomes (nanoparticles) has been shown to lead to progression in several cancers, including all. we have previously shown that leukaemia-derived exosomes induce leukaemia cell proliferation via up-regulating of mir- a expression and silencing of exosomal mir- a reverses this exosomeinduced cell proliferating effect. the objective is to investigate the effect of vincristine and prednisone on exosomal mi-r a expression in all. methods: jm , sup-b , and nalm- leukaemic cell lines were treated in vitro with vincristine ( . to . µm) and prednisone ( . to . µm) in exo-free medium and apoptosis was measured by mts assay. total rna of exposed cell lines was isolated and cdna was prepared for mir- a analysis. expression of mir- a was analysed by q-pcr. exosomes from conditioned medium of exposed cell lines were isolated by ultracentrifugation method. purity and particle size of exosomes were confirmed by western blot and nanoparticle tracking analysis (nta) assay respectively. total exosomal rna was isolated from exosomes (exo-rna) by trizol method. synthesis of cdna was carried out with the miscript ii rt kit (qiagen). results: vincristine and prednisone promote apoptosis in leukaemia cell lines (jm and sup-b ) in a dosedependent manner. both cellular and exosomal mir- a expression was down-regulated by vincristine and prednisone exposure in all three leukaemia cell lines (jm , sup-b , and nalm- ). these observations demonstrate that cellular mir- a down regulation in the parental cells is stable and can be transferred to exosomes, confirming the concept that exosomes are the fingerprint of parent cells. summary/conclusion: our data suggest that the vincristine and prednisone anti-proliferative effect in p-all maybe induced by another yet unexplored pathway, that suppresses mir- a at a cellular and exosomal level in p-all, resulting in apoptosis. funding: this project is supported by the dimartino family foundation. secreted extracellular vesicles from renal cell carcinoma cells anatoliy samoylenko, artem zhyvolozhnyi, eslam abdelrady, naveed ahmad, genevieve bart and seppo vainio oulu university, oulu, finland introduction: clear cell renal cell carcinoma (ccrcc) represents the most common form of kidney cancer and is among the most lethal of all genitourinary cancers. despite surgery and medication therapy, most patients with metastatic ccrcc have a poor prognosis. intratumoural hypoxia is a key factor involved in renal cancer progression and it is known to promote secretion of evs by many types of tumour cells. methods: rcc-derived renca cells, embryonic kidney derived ub cells, and primary mouse hepatocytes were used in the study. evs were purified from cell culture media by gradient ultracentrifugation, sequential ultracentrifugation and exo-spin™ columns. before ev isolation cells were kept for h either under normoxia or hypoxia ( % oxygen). evs were analysed by transmission electron microscopy with negative staining and immunolabeling, by nanoparticle tracking analysis (nta) and western blotting. cells proliferation and viability were assayed by live cell imaging using incucyte zoom (essen bioscience), cell metabolic activity by seahorse xf analyser (agilent), rna expression by qpcr and ddpcr. proteins were identified by ultra-performance liquid chromatography-mass spectrometry (uplc-ms). rna libraries were made using nebnext small rna library prep kit, and sequenced on nextseq (illumina). results: we showed that hypoxia induced production of evs by rcc cells, and characterized differences in protein and rna content of evs generated by renca cells cultured under normoxic and hypoxic conditions. we also showed that rcc-produced vesicles modify key features of tumorigenesis (gene expression, metabolic activity, motility, and growth) of target cells. these data were obtained by using two target cell types: model mouse kidney cells and primary mouse hepatocytes, which represent typical site of rcc metastasis with an exceptionally poor prognosis. we proposed that a possible mechanism of ev action in rcc is related to changes in caveolin- function. we also tracked renca-derived evs in a chick embryo model and in a novel kidney organoid co-culture assay developed by our group (xu et al., ) . summary/conclusion: hypoxia may influence tumorigenic properties of rcc by changing rates of production and composition of evs. funding: the study was supported by finnish cancer foundation grants. exosomes synthesizing her mirna and engineered to adhere to her on tumour cells surface exhibit enhanced anti-tumour activity introduction: exosomes are small extracellular vesicles averaging - nm in diameter. they serve as a means of intercellular communication. typically they consist of structural proteins as well as selected proteins, mirnas, mrnas, and long noncoding rnas. thus in an earlier report this laboratory designed a mirna targeting a major herpes simplex virus regulatory protein. as predicted by the nucleotide packaging signal the mirnas were packed in exosomes and on exposure to infected cells significantly reduced virus yields. her (human epidermal growth factor receptor ) plays an important role in the neoplasia of some breast cancers. the protein is exhibited on the cell surface and is the target of therapeutic antibodies. methods: firstly, we report on the construction of a mirna targeting the synthesis of her both in cells constitutively expressing her and in cells transfected with a plasmid encoding her . secondly, we report that the mirna targeting the synthesis of her reduced the viability of her positive cancer cells both in cell culture and in implanted tumours. lastly, we enhanced the anti-tumour activity of the exosomes by binding to the exosome surface a ligand with affinity for the her on the surface of tumour cells. the -mir-her exosomes package with mirna designed to block her synthesis and deliver to cells. these exosomes kill cancer cells dependent on her for survival but have no effect on cells lacking her or which were engineered to have her but do not depend on it for survival. the -mir-xs-her exosomes carry in addition a peptide which enables the exosome to adhere her on the surface of the cancer cells. in consequence, these exosomes preferentially enter and kill cells exhibiting her on their surface. the exosomes with -mir-xs-her are significantly more effective in shrinking the size of her -positive tumours implanted in mice than the -mir-her exosomes. summary/conclusion: our studies indicate that exosomes carrying mirna against her have no effect on her negative cells it was nevertheless desirable to increase the uptake of exosomes carrying the her mirnas by her -positive tumour cells. to this end we modified the exosomes to exhibit on their surface a peptide that bound the exosomes to the her on the surface of cancer cells. in consequence, we significantly enhanced the uptake of exosomes carrying the mirnas directed against her by her positive cells. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd , shenzhen science and innovation commission project grants jcyj , jcyj to shenzhen international institute for biomedical research. systematic characterization of ovarian cancer-derived exosomes unveil mirnas interfering with cd + t cell activation introduction: cd + tumour-infiltrating lymphocytes (til) have been widely reported to correlate with cancer patient survival, including ovarian cancer. even with the presence of tils, immunotherapy has limited success in ovarian cancer. understanding the interaction between cd + til and tumour cells is thus important. our hypothesis is that tumour-derived exosomes are released and taken up by cd + til such that specific mirnas contained within modulate physiological processes that inhibit cd + t cell activation. we aim to identify mirnas carried in tumour-derived exosomes that inhibit cd + t cell activation in ovarian cancer. methods: we purified exosomes from nine ovarian cancer cell lines and stocked in high concentration. interferon-gamma (ifn-gamma expression screening was performed after days of co-incubation of tumour derived exosomes, cd + t cells, and activators in conditioned medium. cell counts and viability were tested by trypan blue staining at day and day . rna-seq for exosomes were generated to identify mirnas critical in differentiation effects on cd + t cell activations. microrna target matching uncovered target mrnas while enriched pathway analysis predicted potential signalling pathways involved. results: our ifn-gamma screening results indicated the exosomes exhibit different behaviours in interfering cd + t cell activation owing to different donors. exosomes derived from peo. and ovca cells have consistent polarized results in ifn-gamma expression. exosomes derived from peo. remained a low ifn-gamma expression and from ovca stayed at relatively high level. small rnas profiling analysis between the two cell lines identified mirnas (p < . ), and mirnas have been reported with validated targeting information, and out of have targets involved in immune signalling. mrna targets were uncovered by target matching. cmap search identified complex connections among mrnas with the top enriched pathways actively involved in cell cycle and immune related behaviours. summary/conclusion: our ifn-gamma screening identified crucial mirnas in ovarian cancer exosomes interfering cd + t cell activation. computational modelling on both experimental and public multiomics datasets predicted promising signalling pathways of tumour-immune crosstalk for functional validation. irradiation of breast cancer cells alters the quality of dna cargo in the exosomes that they produce sheila spada, paul zumbo, doron betel, tuo zhang, nils-petter rudqvist and sandra demaria weill cornell medicine, new york, usa introduction: irradiation of breast cancer cells with an immunogenic dose ( gyx ) leads to accumulation of cytosolic dna that is sensed by cgas leading to interferon type i (ifn-i) signalling via cgas/sting pathway [ ] [ ] [ ] . we previously showed that tumour-derived exosomes (tex) secreted by irradiated ( gyx ) (rt-tex) but not untreated (ut-tex) tsa carcinoma cells carry dna that stimulates the production of ifn-i in recipient dendritic cells (dc) via the cgas/ sting pathway [ ] . moreover, mice vaccination using rt-tex, but not ut-tex, elicited anti-tumour immune response inhibiting tumour growth [ ] . here, we hypothesized that the differential ability of rt-tex and ut-tex to activate ifn-i in recipient dcs is due to qualitative differences in dna cargo of rt-tex compare to ut-tex. methods: the length of dna purified from tex and from the cytosolic fraction of tsa cells was measured by agilent bioanalyzer. the dna cargo of tex was analysed by whole-genome sequencing (wgs) and whole-genome bisulphite sequencing. the percentage of methylation of total dna in tsa cells was quantified by -methyl cytosine dna elisa kit. results: dna fragments with size between and bp were enriched in rt-tex compared to ut-tex, as well as in the cytosolic fraction of irradiated compared to mock-treated tsa cells. wgs revealed that the entire genome was represented in tex dna cargo, regardless of rt. more than % of tex dna was of nuclear origin, but mitochondrial dna was increased in rt-tex. interestingly, we found that rt decreases the level of methylation in both exosomal and total dna in tsa cells compared to the controls. summary/conclusion: these data support the hypothesis that immunogenic rt alters some characteristics of the exosomal dna cargo, mirroring molecular changes occurring in parent irradiated breast cancer cells. the enrichment in dna fragments of - bp in rt-tex is intriguing considering that cgas is optimally activated by dna in this length range [ ] . we are currently investigating which features of the cargo dna that differ between ut-tex and rt-tex may explain the differential ability to induce ifn-i pathway activation in recipient dcs. the identification of a dna signature associated with the ability of tex to activate the cgas/sting pathway could provide a circulating biomarker of the rt-driven immunogenic tumour response. introduction: triple negative breast cancer (tnbc) is among the most difficult cancer subtypes to treat and continues to cause a high number of cancer-related deaths annually. extracellular vesicles (evs) transfer cell type-specific cargo and have important implications in disease initiation, therapy and outcome. upon treatment of cancer cells with low-dose chemotherapy, released evs are able to transfer phenotypic traits to other cancer cells. new treatment strategies for tnbc, like inhibitors of the er stress pathway (ire ) might impact on ev biogenesis, cargo delivery and response of cells in the cancer microenvironment. our aim is to identify immune modulatory alterations in breast cancer cells and cancer derived evs upon treatment with inhibitors of the er stress pathway. methods: human tnbc cell lines were treated with ire inhibitor mkc and cells were analysed for immune modulatory surface markers, like hla-i, b -h molecules and different integrins. mitochondrial and lysosomal activities were investigated by the use of a mito-and lysotracker and analysed by imagestream (isx) technology. extracellular vesicles were isolated from cell culture supernatants by sequential centrifugation, quantified by nanoparticle tracking (nta) and characterized by exosome bead array. single ev analysis of total cell free supernatants and of isolated evs was performed by isx and marker positive evs were quantified for absolute fluorescence signals and total amount by objectives/ml. ev uptake into t cells was investigated by the use of different ev labelling strategies. results: several immune relevant surface markers (hla-i and cd ) are downmodulated by ire inhibition across different cell lines. cell surface expressed cd and b -h show cell line specific downmodulation profiles upon ire inhibitor treatment. other immunomodulatory marker such as b -h and b -h , integrin cd , cell adhesion-promoting cd and stemness/metastasis marker (cd and ssea) are unaltered on ire treated breast cancer cells. cancer cell derived evs were tetraspanin positive (cd , cd , cd ), similar in number and showed differential expression of immune markers upon ire treatment. mitochondrial and lysosomal activities were unaltered under ire inhibition, whereas cell proliferation was diminished. no breast cancer-derived ev uptake of externally labelled evs into healthy t cells could be detected. summary/conclusion: ongoing analyses focus on the multicolour analysis of multiple markers on single evs by imaging flow cytometry and on the functional impact of cancer derived evs on t cells delivered by ev receptor binding. funding: dagmar quandt is supported by the sfi (cÚram research centre, /rc/ ), the european regional development fund and the dr. werner jackstädt-stiftung. chair: uta erdbrügger -university of virginia chair: larry harshyne -thomas jefferson university comparison of three isolation protocols to search extracellular vesicles signature in sickle cell disease patients introduction: sickle cell disease (scd) is an inherited disorder characterized by chronic haemolysis and continuous activation of different cell types. extracellular vesicles (evs) were described to be at increased levels in scd patient's plasma compared to healthy subjects and were associated with several clinical manifestations such as leg ulcers and stroke. scd patient's plasma has increased concentrations of haem, free-hb and other proteins and lipoproteins as chronic haemolysis consequence. here, we report the comparison of three mostly used isolation protocols to search ev signature in scd patient's plasma by flow cytometry. methods: blood samples were obtained from scd patients (n = ) following wisgrill et al., ( ) protocol. three different ev isolation protocols were used: differential centrifugation (dc), ultracentrifugation (uc) and size-exclusion chromatography (sec). lactadherin and calcein-am were used to detect phosphatidylserine (ps)+ vesicles and membrane integrity, respectively. platelet-derived evs (pevs), endothelialderived evs (eevs), leucocyte-derived evs (levs) and monocyte-derived evs (mevs) were quantified. silica beads were used to define evs gate and samples were acquired in the cytoflex cytometer platform. results: the quantification of pevs in uc, dc and sec samples was, respectively, x , , x and , x events/ml mean, eevs was , x , × and , x events/ml mean, levs was x , × and , x events/ml mean and mevs , x , , x and , x events/ml mean. uc samples demonstrated a higher concentration of evs, which could be more useful to functional studies than dc and sec, however, it took more time to separate than dc. dc was the fastest method to separate evs from plasma, being useful to study large patients cohorts, but showed the smallest overall number of evs. sec also demonstrated high capability to detect evs in plasma and the possibility of obtaining a purer sample, although it is the most expensive and time-consuming method among all tested. all evs populations were detected in the three protocols tested. summary/conclusion: in summary, all protocols tested were efficiently to detect evs in scd patient's plasma and the definition of the best protocol may vary based on the research aim and time and budget available. funding: fapesp / - . gabrielle lapping-carr, joanna gemel, yifan mao and eric beyer university of chicago, chicago, usa introduction: aberrant cell-cell interactions involving the endothelium are central to the pathophysiology of sickle cell disease (scd), including acute chest syndrome (acs), a deadly and unpredictable complication. we previously demonstrated that the plasma of scd patients contains increased circulating small extracellular vesicles (evs) compared to controls and that those vesicles can disrupt endothelial integrity in vitro by affecting adherens junctions and ve-cadherin. the current study was designed to examine the effects of those evs on other cellular junctions including tight (zonula occludens , zo- ) and gap junctions (con-nexin , cx ) and to test the hypothesis that the junctions would be more severely affected by evs isolated from patients during an episode of acs than by ones isolated from the same patient at baseline. methods: we identified subjects with scd in our biobank who had plasma isolated at baseline and at the beginning of an admission for acs. evs were isolated from platelet free plasma using established methodologies. to determine the effects on endothelium, cultures of human microvascular endothelial cells were treated with evs for h and studied by immunofluorescence, immunoblotting and rt-qpcr. gap junction-mediated intercellular communication was assessed following microinjection of lucifer yellow and neurobiotin. results: the distribution and abundance of zo- at the plasma membrane were minimally affected by scd evs. while baseline evs did not affect the distribution of cx , evs isolated during an episode of acs caused loss of cx from the plasma membrane. the integrated intensity of cx membrane staining was decreased bỹ % following treatment with acs evs. cx protein decreased on average by %, cx mrna levels by % and neurobiotin transfer by - % in cells treated with acs evs, compared to baseline evs. summary/conclusion: circulating evs in scd affect multiple components of endothelial junctions. gap junctions composed of cx are the most sensitive of the cellcell junctions, since their abundance and function are reduced by acs evs even when the endothelial monolayer appears intact. cx -mediated intercellular communication may be an early and sensitive event in the endothelial disturbance caused by evs in scd patients. funding: nih ul tr , comer hospital rbc race funds, ted mullin fund. the effects of platelet concentrate storage time on extracellular vesicle interactions associated with fibrin clot formation in-vitro jamie nash a , christine saunders b , amanda davies a and philip james a a cardiff metropolitan university, cardiff, uk; b welsh blood service, velindre university nhs trust, cardiff, uk introduction: platelet concentrates (pcs) have been utilised for decades to prevent bleeding in thrombocytopenic patients and to stop active bleeding. the storage of pcs however is a logistical challenge due to the limited day shelf life under standard conditions. during storage, platelets undergo a number of mechanical and biochemical changes contributing to the short shelf life of a pc. these changes are collectively known as the platelet storage lesion. platelet extracellular vesicles (pevs) are known to increase throughout pc storage, due to an increase in platelet activation. as pevs have previously been shown to be pro-coagulant and increase in annexin v binding over pc storage. the aim was to investigate the effect of pc storage time on extracellular vesicle interactions on fibrin clot formation. methods: pcs were sampled on alternate days up to days of storage and centrifuged to achieve acellular plasma. the plasma was subjected to ultracentrifugation ( , xg) to pellet evs. the size and concentration of evs was assessed using nanoparticle tracking analysis software, followed by a western blot to confirm evs were of platelet origin. the pevs were added at a fixed number to a control pooled plasma sample with added thrombin and tissue plasminogen activator. the time to clot and % lysis time were recorded by using the turbidometry of the plasma over time. results: evs isolated from the pc were confirmed to be of platelet origin by western blot using cd as a marker of platelet origin and cd as an ev marker. pevs caused a significant increase effect on the fibrin clot formation (p < . ) when compared to the control plasma. pevs also had a significant effect (p < . ) on the fibrinolysis time, extending the time taken to lyse the clot. characterization of mirna from serum derived exosomes in a mouse tibia fracture model of introduction: complex regional pain syndrome (crps) is a debilitating chronic disease that occurs after trauma to the periphery and is intimately associated with nerve injury. its presentation is often described as an injury that is disproportional to the inciting event and manifests neuropathic pain, systemic inflammation, and immune dysregulation. owing in part to our poor understanding of disease aetiology, current treatments for crps are insufficient and as a disease of exclusion there is a lack of quantitative diagnostic markers. exosomes are small extracellular vesicles (sevs) - nm in size which provide a means of cellular communication through their cargo molecules (protein, mirna, mrna, lipids) , and have demonstrated promise in uncovering mechanisms of disease manifestation and identifying potential diagnostic markers. we have shown previously that crps patients have differential expression of several mirnas in serum derived sevs as compared to healthy controls, but little is known on how this compares to the established mouse tibia fracture model of crps. methods: mice undergoing fracture were anesthetized and subjected to a unilateral tibia fracture followed by casting of the injured limb. after confirming the establishment of pain hypersensitivity, serum samples were collected from fracture model and control mice three weeks post-injury. sevs were isolated by differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna-seq analysis is being performed to identify differentially expressed mirnas. results: nanoparticle tracking analysis showed no significant difference in the number or size of sevs present in the serum from the fracture model and control mice. rna-seq is ongoing and differential mirna expression in sevs from fracture model will be compared to control samples. comparative studies identifying mirnas that are common between crps patients and the rodent model will facilitate the development of correlational outcomes between preclinical and human studies. summary/conclusion: identification of similarities and differences between crps patients and animal models will aid in directing future studies at clinically relevant aspects of crps aetiology and identifying potential diagnostic markers for crps patients. extracellular vesicle-based liquid biopsy in acute myeloid leukaemia: a reliable source of residual disease biomarkers? introduction: acute myeloid leukaemia (aml) is an haematopoietic stem cell disorder with a poor -year survival rate. monitoring of measurable residual disease (mrd) in aml patients receiving chemotherapeutic treatment is useful to assess therapy response and predict relapse. indeed, many different leukaemia associated immunophenotypic protein markers (laips) are presently useful to detect mrd. nevertheless, their analysis currently requires invasive bone marrow aspirates, thus severely hindering real-time monitoring of the disease. therefore, alternative peripheral blood-based methods are highly desirable for an easy, real-time and costeffective monitoring of aml progression. this work aims was to assess the feasibility of a peripheral blood ev-based liquid biopsy method for aml disease monitoring, based on the detection of laips with a known negative impact on the prognosis of aml. methods: the profile of evs isolated from paired samples from aml patients' blood plasma collected at diagnosis, complete remission (and some at relapse) was compared and correlated with clinical data. for that, a size-exclusion chromatography (sec) method was optimized to isolate the circulating evs from the blood plasma. the evs of the paired aml patients' blood samples were then characterized according to their size (dls/nta), morphology (tem), proteinto-lipid ratio (lowry/sulpho phosphovanillin assay), surface charge (zeta-sizer) and protein cargo (western blot). results: sec allowed the isolation of size-resolved plasmaderived evs from the peripheral blood of aml patients. isolated evs had a size ranging from nm to nm with an intact morphology, expressing ev-associated markers such as hsp , cd , cd and cd . size-resolved evs also had a differential expression of mitofilin, actinin- , syntenin- and annexin-xi proteins. several laips were detected in the isolated evs and their relative abundance changed throughout the stage of the disease. summary/conclusion: our preliminary data shows that aml patients' circulating evs carry relevant immunophenotypic protein markers, which might predict aml clinical outcome. introduction: cell plasticity regulated by the balance between the epithelial-to-mesenchymal transition (emt) and met is critical in the metastatic cascade. extracellular vesicles (evs) may play an important role in this balance by shuttling molecular cargos into recipient cells. this study aims to evaluate the feasibility of profiling mrnas of parental prostate cancer (pca) cells with different phenotypes and their daughter evs using the nanostring low rna input ncounter assay. methods: pc -epi and pc -emt cell lines representing epithelial and mesenchymal phenotype, respectively, were generated from original pc cell line. the cell culture supernatant was first pre-cleared for any dead cells and debris by centrifugation at × g for min. without disturbing the pellet, the supernatant was then transferred to a fresh ultracentrifuge tube and centrifuged at , × g for min at °c. the remaining supernatant was then centrifuged to isolate the evs at , × g for min at °c. the evs pellet was further washed in × pbs followed by a second centrifugation at , × g for min at °c. the final evs pellet was resuspended in × pbs for subsequent characterization (transmission electron microscopy, nanoparticle tracking analysis and western blot) and ncounter assays. the total rna of cells and their daughter evs were assayed by the ncounter pancancer progression panel to determine expression of selected mrnas. the nanostring ncounter low rna input kit with the multiplex gene primer pool was used for the pre-amplification of mrna and overnight hybridization with the pancancer progression panel. each sample type was submitted to the assay in biological triplicate. results: when comparing all samples, eisen cluster analysis separated all the cells and all evs into two groups, regardless of their phenotypes. in subgroup analysis, the expression patterns between pc -epi and pc -emt cells were significantly different. clec b, kdr, crip , il ra , cc d b were significantly upregulated in pc -emt cells, while cxcl , epcam, esrp , tgfb , cdh , s a , ovol were significantly downregulated in pc -emt cells. the expression patterns between pc -epi and pc -emt evs were also significantly different. tbx , cav , col a , slc a , myc, itgb , timp , camk b, ptgds, p h , itgb , vim, stat were all significantly downregulated in pc -emt cell derived evs. summary/conclusion: the nanostring low rna input ncounter assay can provide reliable mrna expression profiling of evs. the mrna expression patterns are very different between cells and their daughter evs. both cells and evs with different phenotypes have different gene expressions. cancer cell-derived evs containing alphav beta integrin regulate cd , il- and il- levels in peripheral blood mononuclear cells introduction: extracellular vesicles (evs) mediate communication in the tumour microenvironment and play an important role in cancer progression. previously, we have shown the enrichment of alphav beta integrin in small extracellular vesicles (sevs) isolated by differential ultracentrifugation and iodixanol density gradient from pc prostate cancer cells. we have also shown in the past that alphav beta -positive sevs induce peripheral blood mononuclear cell (pbmc) polarization by increasing the expression of pro-tumorigenic m markers, such as cd and cd . finally, we have demonstrated that down-regulation of alphav beta integrin up-regulates the stat -interferon stimulated genes (isgs) pathway in cancer cells and in sevs released by them. methods: in order to investigate whether prostate cancer cell-derived vesicular stat has a causal effect in pbmc polarization, we down-regulated alphav beta and stat in prostate cancer cells derived sevs using sirna as well as crispr-cas strategies. the sevs isolated from these cells were used to analyse m polarization by measuring the levels of cd in pbmc. the results show that sevs lacking alphav beta inhibit cd levels in pbmc in a stat -independent manner. analysis of cytokines released by pbmc upon incubation with sevs lacking alphav beta , show that pbmc selectively up-regulate the levels of il- and il- , which are predominantly anti-tumorigenic cytokines. in contrast, sevs lacking alphav beta do not upregulate pro-angiogenic cytokines, such as vegf. summary/conclusion: these findings suggest that cancer cell-derived sevs containing alphav beta integrin promote a pro-tumorigenic pbmc phenotype in the tumour microenvironment by regulating cd , il- and il- levels. introduction: the recognition of donor-mhc molecules by recipient t cells triggers the immune response leading to rejection of allografts. our recent studies have documented the presence of high numbers of recipient apcs displaying donor-mhc molecules (cross-dressed) on their surface in the lymphoid organs of mice after skin, heart or pancreatic islet transplantation. in addition, we have reported that acquisition of allogeneic mhc molecules by host apcs (mhc crossdressing) is mediated by donor-derived extracellular vesicles (evs) trafficking through blood and lymphatic vessels (marino et al. science immunology, ) . in the present study, we investigated the ability of allogeneic evs and allo-mhc-cross-dressed cells to initiate a t cell alloresponse in vitro and in vivo. methods: evs were isolated (using differential centrifugation) from balb/c bone marrow derived dendritic cells (bmdcs). these evs were used to cross-dress b splenocytes in vitro. the transfer of donor mhc class i and ii on b cells was analysed by imaging flow cytometry. next, t cells from b mice were cultured in vitro with either allogeneic bmdc-derived balb/c evs or b spleen cells crossdressed with allogeneic balb/c mhc. alternatively, × balb/c or b bm derived evs or × balb/c bm cells were injected iv to b mice. in both cases, the t cell response was assessed by activation markers detection, infg production and cell proliferation. results: apcs cross-dressed with allogeneic mhc molecules can trigger a pro-inflammatory direct alloresponse by t cells in vitro and in vivo. on the other hand, allogeneic evs alone were only able to induce early t cell activation but not proliferation in vitro. furthermore, injection of mice with allogeneic evs alone could induce some but suboptimal alloresponse in vivo and only when administered with complete freund's adjuvant. summary/conclusion: blocking donor evs release and subsequent recipient apc cross-dressing may represent a promising target to selectively inhibit anti-donor t cell inflammatory responses thus achieving long-term allograft survival. funding: r dk . antifungal antibiotic activity of outer membrane vesicles from adherent lysobacter enzymogenes c against therapeutic and biocontrol targets. rutgers university, new brunswick, usa introduction: lysobacter enzymogenes is a predatory gram negative bacterial species being studied for biocontrol activity against fungi. planktonic l. enzymogenes c produces outer membrane vesicles (omv) harbouring small molecule antifungal antibiotics (meers et al. ) . we show here that the more biologically relevant surface-associated c exerts remote antifungal activity via omv as well. the results have important consequences regarding the natural mechanism of biocontrol of fungal pathogens by c as well as isolation and delivery of therapeutically relevant antifungal compounds. methods: omv were isolated from scraped adherent c culture on agar by similar methods to meers et al . omv were stained in some cases with fluorogenic syto dna stain for microscopic observation. fungal growth was monitored via turbidity readings in liquid culture or photomicrographs on agar. c was also grown on polycarbonate filter membranes with defined pore sizes to monitor growth of fungal cells on the opposite side. vesicles were also labelled with an amine-reactive probe alexa- and washed x by sedimentation. binding of labelled omv to fungal cells was observed by epifluorescence microscopy. results: syto -stained vesicles from surface-adherent c were similar to previously observed~ nm vesicles (meers et al., ) . the isolated vesicles inhibited growth of saccharomyces cerevisiae or candida albicans in liquid cultures at similar potency and were active against the filamentous species fusarium subglutinans grown on agar or maize leaves. c cultures grown on filters with nm pore size but not nm were able to inhibit the hyphal growth of f. subglutinans on the opposite side. similarly c on filters with a nm pore size were able to inhibit growth of c. albicans. observation of fluorescently-labelled c omv after interaction with c. albicans showed binding specifically to hyphae or pseudohyphae and for f. subglutinans to the growing hyphal tips. summary/conclusion: the omv of c specifically bind and inhibit the growth of fungal hyphae of various species without direct c cell contact. these data elucidate mechanisms of biocontrol and suggest strategies for production of therapeutic antifungal antibiotics. meers et al. elucidating the cellular uptake and tissue distribution mechanism of cell derived vesicles, a novel therapeutic carrier hui-chong lau a , jae young kim a , jin-hee park a , jun-sik yoon a , min jung kang a and seung wook oh b a mdimune inc, seoul, republic of korea; b mdimune inc, seattle, usa introduction: cell derived vesicles (cdvs) are emerging as a novel therapeutic carrier. one of the crucial factors in the development and therapeutic applications of cdvs is to understand the precise mechanism by which vesicles find and enter the target cells. in this study, we aim to investigate the uptake mechanism of cdvs produced from natural killer (nk) cells using a manufacturing process established at mdimune inc. both in vitro uptake assay and in vivo distribution analysis were performed to provide precise insights into how cdv exert its effect at the cellular level. methods: nk cells were mainly used to produce cdvs. breast cancer cells, bt , and human and rodent endothelial cells, with a varying degree of icam- expression, were used to determine the effect of lfa- expressed on the surface of nk-cdvs in cellular uptake using facs and confocal imaging analysis. next, various inhibitors for uptake pathways, such as phagocytosis, dynamin dependent endocytosis, and receptor mediated endocytosis, were used to understand the underlying mechanism of cellular uptake of cdvs. biodistribution profile of cdvs were characterized using both normal and tumour xenograft models by ivis imaging. results: using a recently established manufacturing process, we demonstrate that nk-cdvs can efficiently enter the target cells. this study also shows that the cellular uptake depends on the molecular interaction between icam- and lfa- . in vivo distribution profile of nk-cdvs are also assessed using various tumour models. furthermore, we present a cellular uptake mechanism involved in the entrance of cdvs into the target cells. summary/conclusion: this study demonstrates that the cdvs produced at the manufacturing scale can be easily taken up by cells via specific cellular pathways. this finding will facilitate the development of more efficient therapeutics for cancer and other debilitating diseases. myofibroblasts-derived microvesicles increase dermal fibroblasts collagen production through plgf- syrine arif, sebastien larochelle and véronique j. moulin chu de québec -université laval, loex, québec, canada introduction: a proper wound healing of the skin involves angiogenesis, extracellular matrix (ecm) remodelling and re-epithelialization. these three mechanisms require well-organized interactions between different cell populations. a key role in this context is played by myofibroblasts (wmyo), a cell population mainly differentiated from dermal fibroblasts. these cells contract wound edges and synthesize new ecm. we previously showed that myofibroblasts predominantly produces microvesicles (mvs) and can favour angiogenesis. however, proteomic analysis of mvs from our previous studies indicated some molecules that can potentially be implicated in ecm remodelling. in this study, we evaluated whether myofibroblasts-derived mvs could affect dermal fibroblasts who are highly responsible for ecm regulation. methods: mvs were isolated by differential centrifugation of medium collected from wmyo cells. number and size of mvs were characterized by transmission electron microscopy and nanosizer. multiplex assays of cytokines were evaluated in mvs samples, wmyo and mvs-depleted medium. to examine the interaction of mvs with fibroblasts, we evaluated the uptake of mvs isolated from wmyo transduced with a fluorescent protein. we then treated fibroblasts cultures with mvs or a selected cytokine for days and evaluated collagen production. lastly, we neutralized the selected cytokine in mvs samples before evaluating collagen production. results: plgf- was the cytokine detected in mvs samples in large amount ( . ± . pg/µg proteins in mvs). fibroblasts treated with mvs or plgf- significantly stimulated pro-collagen i level production with a fold change of . ± . and . ± . . moreover, the neutralization of plgf- present in mvs significantly inhibited the production of pro-collagen i by dermal fibroblasts. summary/conclusion: our results indicated that mvs influence fibroblasts pro-collagen production through plgf- signalling. funding: this work was supported by natural sciences and engineering research council of canada (nserc) (rgpin - ); les fonds de recherche du québec-santé (frqs) via the research centre funding grant; the quebec cell and tissue and gene therapy network-thécell (a thematic network supported by frqs). structural insights on fusion mechanisms of extracellular vesicles with model plasma membranes introduction: extracellular vesicles (evs) represent a potent intercellular communication system. while their functional biological properties are more and more investigated, the biophysical aspects of their interaction with recipient cells are often overlooked. small size ( to a few hundred nanometres in diameter) of evs and their heterogeneous origin still pose a great challenge for their isolation, quantification and biophysical/biochemical characterization. in particular the complex network of interactions between differently classified evs and recipient cells remains to be further revealed. here we deeply investigate the fusion mechanism between evs and a model plasma membrane system by an interplay of different structural/morphological techniques to get a molecular description of the interaction helping to clarify the role of different membrane compartments on the evs uptake mechanism. standardized protocols and good manufacturing practice conditions were employed to derive highly stable vesicles of defined size and reproducible molecular profiles from umbilical cord multipotent mesenchymal stem (stromal) cells. after a thorough biophysical and biochemical characterization of evs non-contact liquid imaging atomic force microscopy (afm) and, in parallel, neutron reflectometry (nr), as well as small angle neutron scattering (sans) experiments were performed on evs to determine their interaction with model plasma membranes in the form both of supported lipid bilayers and suspended unilamellar vesicles of variably complex composition. results: we observed that evs tend to fuse with the model membranes with a preferential interaction with the external layer of the fluid membrane. moreover we revealed a stronger interaction with the liquid ordered domains, strengthening the hypothesis of a critical role of lipid rafts in fusion mechanisms. summary/conclusion: our results on the analysis of the interaction of evs with artificial lipid membranes could provide insights on the internalization mechanisms of evs. the approach shown here can be further extended to convey incremental complexity, adding glycolipid and membrane proteins to the model lipid bilayers. this approach combined with data on the specific biological function of each ev subpopulation as retrieved by standard functional assays, will turn useful to select the crucial molecular aspects of evs internalization by cells. introduction: platelet-derived extracellular vesicles (pev) are the most abundant circulating extracellular vesicle (ev) and exhibit platelet-like properties, hence the original term "platelet dust". direct phenotyping of ev surface markers within biofluids is challenging often requiring time-intensive purification steps that can significantly alter resultant ev population characteristics. the exoview™ (nanoview biosciences) specifically captures ev sub-populations and was used to characterise the ev content of platelet free plasma (pfp) and a potential novel haemostatic agent designed for the treatment of severe trauma and haemorrhage, platelet enhanced plasma (pep). methods: freeze-thaw cycling of platelet rich plasma/ expired platelet concentrates was followed by centrifugation to remove platelet remnants and yeilded pep. pfp controls were prepared by double centrifugation ( g for minuntes followed by , g for minutes). rotational thromboelastometry (rotem) and calibrated automated thrombography (cat) were used to assess ev driven haemostasis and thrombin generation. a dilutional and hypothermic model of coagulopathy was designed to assess pep. ev capture arrays comprised of anti-cd , anti-cd , anti-cd and anti-cd were used (exoview™, nanoview biosciences). captured vesicles underewent interferometric imaging and were quantified, sized and further probed with fluorescent tetraspanin markers, annexin-v and intravesicular markers. results: pep is highly procoagulant, exhibits enhanced thrombin generation and can restore haemostasis in a dilutional model of coagulopatic whole blood. pep can be generated from expired platelet concentrates, potentially allowing for upscalable production. the predominant vesicle population were pev with a large cd /cd population that contained a smaller subpopulation of phosphatidyserine positive procoagulant vesicles. pfp as expected has a much lower number of pev and a cd positive ev population. summary/conclusion: pep is a unique resuscitation fluid containing high pev levels for the potential treatment of severe trauma and haemorrhage. exoview measurements can be performed in unpurified plasma and may be useful for measuring circulating ev in health and disease. funding: defence and security accelerator, dstl therapeutic effect of exosomes in mice model of autism daniel offen a , reut horev a , nisim perets a , ehud marom b , uri danon b and yona gefen b a tel aviv university, tel aviv, israel; b stem cell medicine ltd., jerusalem, israel introduction: during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. we have previously shown that intranasal administration of msc-exo, cross the bbb and significantly ameliorate autistic-like behavioural phenotype in btbr and shank animal models of autism, representing a potential therapeutic strategy to reduce symptoms of autism spectrum disorder (asd). our objective is to study the mechanism of action and the cellular pathways in which the msc-exo activate their target, we performed rna sequencing analysis of primary neurons isolated from shank mice treated with msc-exo. methods: primary neuronal cell cultures were prepared from newborn shank homozygotes mice model of autism. cultures were treated with msc-exo ( ^ particles/ul), isolated from human adipocytes, followed by rna sequencing. the alterations in gene expression between the treated and intact neurons were analysed for gene ontology and pathways and were also compared to proteomics analysis of the msc-exo in order to find regulatory proteins that may lead to these differences. results: bioinformatic analysis revealed several up-regulators proteins that might be responsible for the increase in anti-inflammatory and protective factors seen in the mice neurons treated with msc-exo. one of them is bdnf which is known as an essential growth factor responsible for neuroprotection and neurogenesis. importantly, no difference in the genetic expression of cancer-related genes was identified following msc-exo treatment indicating for their safety. summary/conclusion: our data suggest that adipocytederived msc-exo carry therapeutic potential in asd via alternation in gene-expression related mainly to immuno-modulation, reduce neuroinflammation and increase neuroprotection and neurogenesis. the beneficial effects of the exosomes treatment in mice models is being translated into a novel, easy to administer, a therapeutic strategy to reduce the symptoms of asd. introduction: autologous blood-derived products gain increasing focus in regenerative medicine, especially in orthopaedics and osteoarthritis therapy. this disease is characterised by cartilage degradation and inflammation among other symptoms, which are targeted by conventional therapies, but genuine cartilage regeneration is rarely achieved. citrate-anticoagulated platelet rich plasma (cprp) is often clinically applied to stimulate soft and hard tissue healing. recently, cell-free alternatives to cprp including hyperacute serum (hypact™ serum) have been developed. cprp and hypact™ serum contain specific profiles of growth factors, however, they also contain extracellular vesicles (evs) that harbour signal molecules including mirna. methods: evs were enriched by ultracentrifugation (uc) followed by size exclusion chromatography (sec) to obtain purified evs. particle size and concentration of each fraction was measured by nanoparticle tracking analysis (nta). fractions with the highest amount of particles were pooled and concentrated via uc, before mirna expression was assessed via screening with a panel of mirna-specific primer pairs by rt-qpcr. presence of evs was confirmed by cryoelectron microscopy. results: the ev concentration tended to be lower in hypact™ serum than in cprp as determined via nta. similarly, lower diversity of mirna species was found in hypact™ serum than cprp evs. around % of detected mirnas were found in both blood products, whereas only % of mirnas were shared between evs from cprp and hypact™ serum. while mirnas such as mir- were consistently depleted in evs compared to the corresponding blood product, others like mir- a were in enriched in hypact™ evs, but not cprp evs, indicating release of specific mirnas via evs in response to clotting. summary/conclusion: although the purification resulted in high loss of evs, we identified specific mirnas enriched in evs from cprp and hypact™ serum. their functional spectrum with respect to osteoarthritis therapy focuses on inhibition of inflammation, inhibition of tissue remodelling via matrix degrading enzymes as well as preventing senescence. this renders blood product derived evs as interesting candidates for in vitro and in vivo testing with respect to cartilage regeneration. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst -f- / - . protective role of shiitake mushroom-derived exosome-like nanoparticles in d-galactosamine and lipopolysaccharide-induced acute liver injury in mice baolong liu, xingyi chen and jiujiu yu university of nebraska lincoln, lincoln, usa introduction: fulminant hepatic failure (fhf) is a rare, life-threatening liver disease with poor prognosis. new therapeutic interventions are urgently needed to treat this disease. administration of d-galactosamine (galn) and a low dose of lipopolysaccharide (lps) triggers acute liver damage in mice, which simulates many clinical features of fhf in humans and therefore is widely used to investigate the molecular mechanisms and potential therapeutic interventions of fhf. recently, suppression of the nucleotide binding domain and leucine rich repeat related (nlr) family, pyrin domain containing (nlrp ) inflammasome was shown to alleviate the severity of lps/galn-induced liver injury in animal models. therefore, the goal of this study was to identify food-derived exosome-like nanoparticles (elns) with anti-nlrp inflammasome function to potentially control fhf. methods: seven commonly consumed mushrooms were used to extract elns, which were examined for anti-nlrp inflammasome activities in primary macrophages. results: it was found that these mushrooms contained elns composed of biomolecules including rnas, proteins, and lipids. among these mushroom-derived elns, only shiitake mushroom-derived elns (s-elns) strongly inhibited nlrp inflammasome activation by blocking the inflammasome assembly. this inhibitory effect was specific for the nlrp inflammasome because s-elns had no impact on activation of the absent in melanoma (aim ) inflammasome. s-elns also inhibited the secretion of interleukin (il)- and both protein and mrna levels of the il b gene in macrophages. remarkably, pre-treatment of s-elns protected mice from lps/galn-induced acute liver injury. summary/conclusion: therefore, s-elns, identified as potent inhibitors of the nlrp inflammasome, represent a new class of agents with the potential to combat fhf. approaches to assess clinically available exosomes' quality and safety introduction: recent adverse events resultant from an exosome product use in a nebraska clinic, highlight the importance of assuring product quality and safety standards. an often-overlooked safety risk is ancillary reagents remaining within a finished product. when processes to obtain exosomes utilize cow proteins such as fbs or bovine sera albumin, failure to adequately remove these can result in significant adverse allergic reactions. we evaluated different exosome products to test the hypothesis that purity of some products may not be consistent with actual product quality and safety profiles claimed. methods: three different exosome products (manufacturer a, b, and c) were prepared per their instructions for use. sample source identity was blinded from assaying scientists. an independent cro service was used to conduct the experiments to ensure unbiased assay execution and data collection. exosome suspensions were sampled undiluted for bovine protein content using commercially available bovine secretome protein arrays from ray biotech. a total of different proteins found in bovine serum were quantified. results: six of proteins were not detected in any sample. of array antibodies were found to cross react with human antigens. of the bovine proteins that were acceptable for analysis, manufacturers a, b, and c exosomes contained of proteins, of proteins, and of proteins, respectively. concentrations of individual bovine proteins ranged from . to , . ng/ml. summary/conclusion: these results indicate manufactures a and b are selling potentially dangerous products. the successful implementation of exosome products into the clinic requires equivalent demonstrations of safety and quality. this requires adopting strict quality standards and safety testing during their production. physicians must require safety data prior to clinical use. engineering pro-healing ev cargo using a closed-system bioreactor. introduction: chronic wounds, including diabetic ulcers and pressure ulcers, are difficult and expensive to treat. while tissue engineering approaches have largely failed as a viable treatment for chronic wounds, we hypothesize that stem cell-derived extracellular vesicles (evs) may provide several unique advantages. zenbio, inc has developed a methodology to generate commercial-scale stem cell-derived exosomes using a closedsystem hollow fibre bioreactor capable of continuous ev production. additionally, we have shown that by manipulating the cellular environment, we can improve the pro-healing capacity of the evs.this technology leverages the complex healing capabilities of stem cells without the obstacles of replicating cells. methods: we have demonstrated that a mild heat shock resulted in evs enriched for stress-response proteins and increased pro-healing activities in vitro. we extended this innovative approach to include stimulating adipose stem cells with combinations of heat shock and growth factors to generate differential extracellular vesicle packaging that enhances pro-healing activity. to monitor reproducibility across lots and batches, we rigorously characterized tuned evs for particle size and number as well as surface marker and cargo composition. results: our results using tuned evs showed efficacy using cellular models of inflammation, motility, vascularization, collagen production and metalloprotease activity. we utilized an established murine model of pressure ulcers to assess the in vivo efficacy of the tuned evs. these studies showed a single injection into the wound site activated a more rapid wound closure, increased collagen deposition and reduced dermal thickness compared to saline control. summary/conclusion: these data strongly support our hypothesis that evs may be selectively modified to improve their wound healing activity by modulating the culture or tissue microenvironment. future studies will use chronic wound models to determine optimal dosing and routes of administration. introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) can reduce inflammation, promote healing and improve organ function thereby providing a potential "cell-free" therapy. prior to clinical translation, there is a critical need to synthesize existing preclinical evidence supporting their efficacy. this systematic review provides the most comprehensive evidence map of methods, safety and efficacy for msc-ev research to date. methods: medline and embase were systematically searched for in vivo interventional studies using msc-evs. two reviewers extracted data for: ) methodology, ) study design, ) intervention details and ) efficacy/ adverse events. results: after screening articles, studies met our eligibility criteria. msc-evs were used to treat a variety of diseases including renal ( %), neurological ( %) and cardiac ( %) conditions. benefits were described in % of studies across all organ systems and adverse effects were seen in only three studies; two showing tumour growth. however, several key methodological concerns were evident. based on size criteria for ev subtypes (exosomes/small evs~ - nm, microvesicles~ - nm) only % of studies used appropriate nomenclature. ultracentrifugation ( %) and isolation kits ( %) were the most common isolation methods despite marked differences in yield and purity. evs were inconsistently dosed by protein ( %), particle number ( %) or cell count ( %), hindering inter-study comparisons. two-thirds of studies used xenogeneic evs suggesting immunocompatibility. techniques to determine size, protein markers and morphology was highly heterogeneous, and only and studies met the characterization standards recommended in the misev and guidelines, respectively. finally, % of studies did not incorporate randomization which represents a high risk for bias and only a quarter performed biodistribution studies. summary/conclusion: this systematic review reveals extensive heterogeneity in methods and intervention details for animal studies of msc-evs. nonetheless, nearly all studies showed significant benefits in a wide range of distinct conditions. the knowledge gaps we identified highlight important opportunities for improving preclinical design and the need for more standardized approaches in this growing field of ev therapeutics. msc-exosomes as next generation therapeutics for atopic dermatitis exocobio inc, seoul, republic of korea introduction: atopic dermatitis (ad) is a systemic inflammatory disease with unknown cause. recent approval of a targeted therapy, dupilumab, opens new era of ad management. however, current therapeutic options for ad are only targeting inflammation, a component of ad vicious cycle including itching and barrier disruption. human mesenchymal stem cells (mscs) have been highlighted as a novel therapy for suppressing allergic progress of ad in clinical studies. unfortunately, phase iii clinical study of human umbilical cord blood mscs for ad was failed with unknown reason. previously, our group reported that exosomes derived from human adipose tissue-derived mscs (asc-exosomes) alleviated the pathological symptoms in a murine ad model with concomitant reduction of inflammation. methods: our group has further investigated the therapeutic effects of human asc-exosomes in an alternative murine ad model with skin barrier defects. large scale isolation of asc-exosomes was performed by tangential flow filtration and isolated asc-exosomes were characterized according to the recommendation by the isev. the protein and lipid cargo were also analysed. results: we found that asc-exosomes induced restoration of skin barrier by inducing de novo lipid synthesis and reduced the levels of multiple inflammatory cytokines. in addition, asc-exosomes suppressed the expression of itching-causing cytokines. transcriptomic analysis of ad skin lesions revealed that asc-exosomes reversed the abnormal expression of genes functioning in skin barrier function, lipid metabolism, and cell cycle. summary/conclusion: taken together, asc-exosomes could be a promising cell-free therapeutic option for the treatment of ad, which affecting inflammation, skin barrier function, and itching. cell derived vesicles: unravelling the science of novel vesicles with therapeutic promises introduction: cell derived vesicles (cdvs) are nanosized vesicles produced by serially extruding cells through small pores. a growing number of studies have implicated their therapeutic potentials, with superior yield compared to other extracellular vesicles (evs). however, two key objectives remain to be accomplished to demonstrate the utility of cdvs in clinical applications. first, a manufacturing process has to be developed to allow a large-scale production of cdvs. next, these novel vesicles need to be thoroughly characterized at multiple levels. methods: manufacturing-scale extruders were developed to allow extrusion of large volume of cell suspension in a single process. cdvs with approximately - nm in diameter were obtained by a serial extrusion. crude samples were then purified using the tangential flow filtration method to further remove cellular impurities. finally, physical and biochemical characteristics of purified cdvs were analysed using dls, nta, cryo-em, and facs analysis. additionally, cdvs were subject to multi-omics profiling to comprehend our understanding in molecular contents of cdvs. both mesenchymal stem cells (mscs) and natural killer (nk) cells were used for this study. results: in this study, we first demonstrate that the large-scale extruder efficiently produce cdvs with consistent quality at the scale that are compatible for clinical applications. surface marker and membrane composition analyses show that the cdvs are primarily formed using plasma membrane of source cells, with characteristic cellular markers enriched on the surface. comprehensive profiling of molecular components reveals the unique properties of cdvs as well as the underlying mechanism of formation of cdvs. summary/conclusion: recently, we have established a manufacturing process to enable clinical applications of cdvs. this study also highlights key molecular features of cdvs that can be harnessed to offer a powerful tool for regenerative and anticancer medicine. antifibrotic properties of extracellular vesicles derived from human induced pluripotent stem cells introduction: fibrosis is a pathological condition resulting from abnormal healing of various tissues. it is triggered by activation of fibroblasts and their subsequent transition to myofibroblast. in consequence, excessive deposition of extracellular matrix proteins leads to impaired organ function. to revert this process, we employed extracellular vesicles (evs) derived from human induced pluripotent stem cells (hipscs). as a model system, we used human cardiac fibroblasts (hcfs), since heart fibrosis constitutes a serious socioeconomic problem worldwide. methods: we isolated evs from conditioned media from three hipsc lines using ultrafiltration combined with size exclusion chromatography methods. next, we analysed the evs by nanosight, transmission electron microscopy, mass spectrometry and western blot methods. finally, we treated tgf-b-stimulated hcfs with hipsc-evs and evaluated expression of fibrosisrelated genes using real-time qpcr, western blot and fluorescence microscopy. results: we detected anti-fibrotic properties of hipsc-evs exerted on hcfs pre-stimulated with tgf-b. the evs significantly decreased the expression levels of acta , fn, tnc, snai , col a and reduced the number of myofibroblasts. the canonical profibrotic tgf-b-dependent smad / pathway was significantly attenuated in response to ev-treatment. summary/conclusion: in this study we demonstrated strong anti-fibrotic function of hipsc-evs. our findings can further be exploited for future medical applications to treat fibrotic diseases, such as heart fibrosis. funding: this work was supported by the project sonata : umo- / /d/nz / from the national science centre of poland to sbw. induced pluripotent stem cells-derived extracellular vesicles ameliorates d-galactosamine and lipopolysaccharide induced acute liver failure tianjin third central hospital affiliated to nankai university, tianjin, china (people's republic) introduction: liver failure is among the most causes of death in patients with liver disease. promoting liver regeneration will help patients with liver failure recover on their own. extracellular vesicles (evs) can released by induced pluripotent stem cells (ipscs) through paracrine effects and play a pivotal role in inter-cellular communication in the treatment of disease. in this study, we investigated whether the ipscs-evs have therapeutic effects on acute liver failure. methods: the ipscs-evs were isolated by ultracentrifugation and identified using nanoparticle tracking analysis, transmission electron microscopy and western blotting. the isolated ipscs-evs were administrated d-galactosamine-injured heprg cells in vitro and tail intravenously injected into d-galactosamine and lipopolysaccharide induced acute liver failure model mice in vivo, respectively. the anti-apoptosis role and potential mechanism were evaluated using flow cytometry and immunofluorescence staining. and alanine transaminase (alt) and aspartate transaminase (ast) in serum, h&e staining and tunel staining were explored the effect of ipscs-evs on liver injured and liver function. finally, high throughput sequencing of small rnas was performed to investigate mirna expression profiles in ipscs-evs and ipscs. results: the ipscs-evs that were all - nm, doublelayered and oval or round cellular vesicles and expressed the marker proteins cd , tsg and hsp . in vitro, the ipscs-evs treatment inhibited heprg apoptosis induced with d-galactosamine in a time-and dosedependent manner and promote the proliferation of hepatic stem cells. in vivo results showed that ipscs-evs significantly alleviated liver failure, improved liver function and prolonged the survival period. tunel assay showed that ipscs-evs suppress apoptosis of hepatocytes. moreover, mirna expression profiles analysis found that mir - a cluster and mir - cluster were enriched in ipscs-evs and ipscs. summary/conclusion: these findings indicated that ipscs-evs could ameliorate d-galactosamine and lipopolysaccharide induced acute liver failure to attenuate hepatocyte apoptosis, which will be benefit for therapy of liver disease in the future. msc-derived extracellular vesicles promote human cartilage regeneration by control of autophagy introduction: osteoarthritis (oa) is a rheumatic disease leading to chronic pain and disability with no effective treatment available. recently, allogeneic human mesenchymal stromal/stem cells (msc) entered clinical trials as a novel therapy for oa. increasing evidence suggests that therapeutic efficacy of msc depends on paracrine signalling. here we investigated the role of bone marrow msc-derived extracellular vesicles (bmmsc-evs), an important component of msc secretome, in cartilage repair. methods: to test the effect of bmmsc-evs on oa cartilage inflammation the tnf-alpha-stimulated human oa chondrocytes were treated with bmmsc-evs and inflammatory gene expression was measured by qrt-pcr after h. to access the impact of bmmsc-evs on cartilage regeneration the bmmsc-evs were added to the regeneration cultures of oa chondrocytes, which were analysed after weeks for glycosaminoglycan content by dmmb and qrt-pcr. paraffin sections of the regenerated tissue were stained for proteoglycans (safranin-o) and type ii collagen (immunostaining). results: we show that bmmsc-evs promote cartilage regeneration in vitro. treatment of oa chondrocytes with bmmsc-evs induces production of proteoglycans and type ii collagen and promotes proliferation of these cells. msc-evs also inhibit the adverse effects of inflammatory mediators on cartilage homoeostasis. our data show that bmmsc-evs downregulate tnfalpha induced expression of pro-inflammatory cox- , pro-inflammatory interleukins and collagenase activity in oa chondrocytes. the anti-inflammatory effect of bmmsc-evs involves the inhibition of nfκb signalling, activation of which is an important component of oa pathology. autophagy, a cellular homoeostatic mechanism for the removal of dysfunctional cellular organelles and macromolecules, is essential to maintaining chondrocytes survival and differentiation. the expression of autophagy regulators is reduced in osteoarthritic joints, which is also accompanied by increased chondrocyte apoptosis. our preliminary data indicate that bmmsc-evs carry mrna of natural autophagy inducers and promote autophagy in oa chondrocytes. therefore, we hypothesize that msc-evs exert their beneficial effects on cartilage regeneration by restoring the expression of autophagy regulators. summary/conclusion: in summary, our findings indicate that bmmsc-evs have ability to promote oa cartilage repair by reducing the inflammatory response and stimulation of oa chondrocytes to produce extracellular matrix, the essential processes for restoring and maintaining cartilage homoeostasis. thus, msc-evs hold great promise as a novel therapeutic for cartilage regeneration and osteoarthritis. large-scale preparations of small extracellular vesicles from conditioned media of mesenchymal stromal cells modulate therapeutic impacts on a newly established graft-versus-host-disease model in batch dependent manners introduction: extracellular vesicles (evs) harvested from supernatants of humane adult bone marrow-derived mesenchymal stem/stromal cells (mscs) can suppress acute inflammatory cues in a variety of different diseases, including graft-versus-host disease (gvhd) and ischaemic stroke. furthermore, they can promote regeneration of affected tissues. following a successful clinical treatment attempt of a steroid refractory gvhd patient, we intend to optimize msc-ev production strategies for further clinical applications. as we observed functional differences of independent msc-ev preparations in vitro, we aimed to adopt an in vivo gvhd model for the more advanced functional testing of different msc-ev preparations. methods: to this end we set up a bone marrow transplantation mouse model in which endogenous bone marrow was myeloablated by ionizing irradiation (iir). gvhd was induced by the transplantation of major histocompatibility mismatched allogeneic spleen-derived murine t cells. if not treated otherwise, myeloablated mice developed severe gvhd symptoms. results: the gvhd symptoms were effectively suppressed, when msc-ev preparations were applied at consecutive days, which exerted immune modulatory effects in a mixed-lymphocyte reaction assay. msc-ev preparations lacking in vitro immune modulating activities, however, hardly improved the symptoms of the gvhd mice. thus, our results demonstrate that not all msc-ev preparations harvested from adult bone marrow-derived mscs contain the same therapeutic potential. summary/conclusion: thus, successful transplantation of msc-evs into the clinics requires a platform allowing identification of msc-ev preparations with sufficient therapeutic, most probably immune modulating activities. funding: this research was funded by sevrit leitmarkt lifescience.nrw ls- - - g. introduction: malnutrition impacts approximately million children worldwide and is linked to % of global mortality in children below the age of five. severe acute malnutrition (sam) is associated with intestinal barrier breakdown and epithelial atrophy. extracellular vesicles including exosomes (evs; - nm) can travel to distant target cells through biofluids including milk. since milk-derived evs are known to induce intestinal stem cell proliferation, this study aimed to examine their potential efficacy in improving malnutrition-induced atrophy of intestinal mucosa and barrier dysfunction. methods: mice were fed either a control ( %) or a low protein ( %) diet for days to induce malnutrition. from day to , they received either bovine milk evs enriched using differential ultracentrifugation and sucrose gradient purification or control gavage and were sacrificed on day , hours after a fluorescein isothiocyanate (fitc) dose. tissue and blood were collected for histological and epithelial barrier function analyses. results: mice fed low protein diet developed intestinal villus atrophy and barrier dysfunction. despite continued low protein diet feeding, milk ev administration improved intestinal permeability, intestinal architecture and cellular proliferation. summary/conclusion: our results suggest that evs enriched from milk should be further explored as a valuable adjuvant therapy to standard clinical management of malnourished children with high risk of morbidity and mortality. funding: cb was generously awarded a catalyst grant from the centre for global child health at the hospital for sick children to support this work. the impact of spheroids culture on mesenchymal stem cells and ev production introduction: mesenchymal stem/stromal cells (mscs) are now widely believed as bio-factories releasing bioactive products responsible for their therapeutic effect, i.e. cytokines, chemokines, and extracellular vesicles (evs). mscs are highly sensitive to physical stimuli from their surrounding microenvironment and can change their characteristics in response to their environment. the application of d spheroids cell culture allows mscs to adapt to their cellular niche environment which, in turn, influences their paracrine signalling activity. we aim to determine how d and d culture microenvironments can modulate the ev production and investigate their anti-fibrotic activity. methods: for d culture, bone marrow-derived mscs were cultured on standard tissue culture plastic. for d culture, mscs were aggregated into spheroids using non-adherent -well plates and cultured with addition of . % methylcellulose. to collect conditioned media, both d and d mscs were cultured using serum free medium for days. evs were isolated by serial ultracentrifugation and were characterised on exoview platform which allows simultaneous detection of particle size and expression of cd /cd /cd . cell lysates were collected for mirna isolation and qrt-pcr was performed to analyse expression of candidate mirnas. to model the progress of lung fibrosis, human lung fibroblasts (hlfs) were cultured with tgf-β to induce fibroblast activation, subsequently exposed to d and d evs, and collagen production was measured. further, d and d msc-evs were added into human lung mscs isolated from healthy and ipf patients and cell proliferation was assessed using mts assay. results: d and d msc-evs have similar ev characteristics in terms of particle size and ev tetraspanin markers expression. exoview analysis showed expressions of cd /cd /cd and average particle diameters of < nm. on a cellular level, we identified a panel of anti/pro-fibrotic mirnas which are differentially expressed in d and d mscs. d and d msc-evs have similar anti-fibrotic activity shown by their ability to reduce collagen deposition in hlf cultures. both d and d msc-evs could promote cell proliferation on ipf lung mscs but no overall effect on healthy lung mscs. summary/conclusion: this concept of engineering the cellular microenvironment to promote ev production is as yet untouched and we foresee that in d cultures, we can culture mscs for longer timeframe and therefore maximising the overall ev production process. the outcome presents future potential for d culture of msc to increase the efficiency and feasibility of scalable ev production. outer membrane vesicles from photobacterium damselae subsp. piscicida: characterization and antigenic potential introduction: photobacterium damselae piscicida (phdp) is a gram-negative bacterium that causes a septicaemia in > fish species worldwide. it represents a major drawback for aquaculture, whose importance has been sharply growing as a food supplier. given the phdp massive mortality and widespread antibiotic resistance, an effective vaccine is highly needed. extracellular products (ecps) have an essential role in phdp virulence, containing important antigens. however, the ecps' identity remain undisclosed. in our efforts to dissect their composition, we found that they contain high amounts of outer membrane vesicles (omvs). these particles are potent weapons for bacteria and are being explored in the field of vaccinology, since omvs present antigens in native conformations and are strongly immunogenic, without requiring adjuvants. this potential associated to the urgent need for an anti-phdp vaccine prompted us to isolate and characterize the omvs shed by phdp. methods: in order to harvest high amounts of pure phdp omvs, a reproducible optimized protocol was developed: the bacteria-free supernatant from a phdp overnight culture is concentrated, dialysed and ultracentrifuged to collect the omvs. results: analysis of the obtained omvs preparations by transmission electronic microscopy and dynamic light scattering indicate that the main population of vesicles has sizes around - nm. proteomic analysis of the vesicles revealed the presence of the apoptogenic ab toxin aip that is known to play a major role in phdp virulence, a putative pore-forming toxin, a putative adhesin/invasin and several outer membrane proteins (omps), including a kda omp, predicted to be involved in iron acquisition, and other omps ( - kda), with an ompa-like structure that may act as adhesins. moreover, preliminary in vivo studies suggest that some of those proteins may have important roles for virulence, since injection of knock-out strains in sea bass induced a decreased mortality comparing to the wt strains. summary/conclusion: our findings suggest that omvs are a promising vaccine candidate and we are currently studying their biological activities and determining the antigenic potential of the identified proteins. introduction: whole body exposure to high doses of ionizing radiation (ir) can potentially be lethal if radiation injury is not diagnosed and treated expeditiously. when considering a non-invasive approach for the identification of biomarkers of ir exposure, we and others have studied molecules in plasma, serum, saliva, and urine. however, these matrices can potentially have significant background noise, obscuring potential biomarkers of biological importance. extracellular vesicles (evs) are fast becoming a platform for biomarker discovery in radiation research as well as in other pathologies. however, no groups have investigated the use of metabolomics to analyse evs derived from urine in the context of ir exposure. furthermore, the dominant protocols for ev isolation from urine require a large (up to ml) amount of starting volume, which may not be available for many studies. the aim of this study was to optimize ev isolation from rat urine and assess radiation-induced alterations in urine ev number and metabolic content. methods: as a proof of concept, we compared and optimized several ev isolation methods on small volumes of urine from male wag/rijcmcr rats exposed to gy or gy x-rays to the whole body except the hind leg. starting with either µl or µl of urine, we isolated evs using ultracentrifugation (uc) with filtration, size exclusion chromatography (sec), and a proprietary bead-based isolation method developed by a rd party provider. ev samples were characterized using nanoparticle tracking analysis. metabolomics profiles were measured using lc-qtof-ms. results: we found that sec resulted in the highest yield of evs from as little as µl of urine, while uc was the poorest performing. lc-qtof-ms analysis revealed that sec and uc had the most consistent identification of features, whereas the bead-based method contained artefacts likely as a result of the extraction method. we next used sec to isolate evs from a larger cohort of rats exposed to ir and analysed with ms. ev metabolic content will eb related to differences in survival and organ function between sham and irradiated groups. summary/conclusion: we conclude that sec is the preferred method for isolating evs from small volumes of urine for broad-based mass spectrometric analysis, and that the ev metabolome may be a sensitive and specific early indicator of radiation injury. introduction: there is growing evidence that contents (including rna and proteins) of exosomes may serve as biomarkers for early diagnosis and prognostic prediction of cancers. here we aim to identify potential protein markers for oesophageal cancer. methods: using our newly developed label-free exosome automated preparation system (leaps), exosomes were isolated from ml culture medium of various oesophageal cancer cells with different differentiation profiles and different sources of metastasis. exosomes from µl plasma of cancer patients at different clinical stages or with/without relapse and healthy controls were also prepared by leaps. matrix-assisted laser desorption ionization time-of-flight mass spectrometry (maldi-tof ms) was employed to directly analyse exosomes. protein identities of exosomal fingerprint peaks were tentatively assigned by correlation with top-down and bottom-up proteomics. results: start from ml culture medium or µl plasma, high-quality exosomes rapidly isolated by leaps are sufficient for maldi-tof mass spectrometry. it seemed that poorly differentiated cells showed more exosome release. maldi-tof ms fingerprints of exosomes in cells is cell line specific. ms profiles from poorly differentiated cells showed more peaks than that from highly differentiated cells. fingerprints also allowed classification of cancer cell lines through software mathematical analysis. we identified different numbers of significantly differentially expressed peaks in exosomes of various cancer cells. fingerprints of exosomes derived from the poorly differentiated cells showed more elevated peaks. top four peaks ( , m/z, , m/z, , m/z, , m/z) were commonly down-regulated in exosomes of most cancer cells. top four protein peaks ( , m/z, , m/z, , m/z, , m/z) that might be correlated to the differentiation profile of cancer cells were also identified. maldi-tof ms detection of exosomes in the plasma and clarifying identities of potential biomarker peaks will be done in the future. summary/conclusion: the combination of leaps and maldi-tof mass spectrometry provides a fast and high-throughput tool for exosomal marker discovery. potential biomarker identified in exosomes derived from oesophageal cancer cells or from plasma of cancer patients by this tool might be useful in cancer diagnosis and prognosis. fraction-based proteomic profiling of serum extracellular vesicles derived from cervical cancer patients introduction: current evidence indicates that extracellular vesicles (evs) can release from most of cell types and affect adjacent or distant cells by circulating in all bodily fluids. proteomic analysis of evs from clinical samples is complicated by the low abundance of ev proteins relative to highly abundant circulating proteins. size exclusion chromatography (sec) has been overcome as a method to deplete protein contaminants and enrich evs. methods: we collected serum of healthy women and cervical cancer patients with stage i-iii and then counted concentration and size distribution of the evs using nanoparticle tracking analysis (nta). differential ultracentrifugation combined with sec was used to isolate and purify evs from contaminant proteins. isolated evs were investigated their characteristic based on morphology using transmission electron microscope (tem) and on expression of cd , cd , cd protein markers using western blot analysis. fraction no. - of isolated evs in among sample groups were profiled by nano-liquid chromatography tandem mass spectrometry (nanolc-ms/ms) analysis. results: nta shows that the concentration of evs is increased in patients compared with healthy women. proteome profiles of evs isolated by sec were compared in each fraction. moreover, we detected molecular evidence for fraction-specific molecular pathways in connection with cancer progression and complied a set of protein signatures that closely reflect the associated clinical pathophysiology. summary/conclusion: these unique features in each fraction among sample groups would be the informative considering in order to select for further analysis as in vitro. introduction: recently, diagnostic biomarkers from exosomes by proteomic analysis have been reported, but it is required to optimize the isolation protocol to screen out more effective biomarkers. for serum-originated exosomes, it has been also reported to isolate them selectively, however, it is observed that a different method resulted in different protein profiles in -d gel electrophoresis. methods: we isolated exosomes by two discrete methods, using ultracentrifugation and magnetic separation. before ultracentrifugation and magnetic separation, precipitation using polymer materials was perforemd. the isolation of exosomes by these two methods followed by comparison of their size, total vesicle number, morphology, and protein markers. to identify protein biomarkers, proteomic analyis using -d gel electrophoresis was performed. results: both methods induced enrichment of exosome-specific proteins, but protein profiles in each exosome fraction was totally different. the protein profiles showd that the magnetic seperation following a polymer-based precipitation step was more efficient to screen out candidate biomarkers, which showed nearly protein profiles originated from exosomes. summary/conclusion: in our study, magnetic separation of exosomes from serum fraction was optimized for -d gel electrophoresis to observe identifiable biomarkers. an extracellular small rna-seq data processing pipeline optimized for high-performance computing chenghao zhu and angela zivkovic department of nutrition, uc davis, davis, usa introduction: a variety of rna species is found in extracellular biofluids such as blood, bile, and urine, carried by extracellular particles including extracellular vesicles (evs) and lipoproteins (e.g high density lipoproteins (hdls)). the extracellular rna (exrna) carried by evs and hdls is of great interest for two reasons: ) the exrna within different carriers could be diagnostic of the state of the tissues from which the particles originate, and ) exrna has been shown to affect gene expression in target cells. although the origin and functions of exrnas remain largely unknown, there is growing interest in exrna research for the development of diagnostics and new therapeutic targets. small rna sequencing is widely used to estimate the abundance of exrnas in biofluid samples. here we present a data processing pipeline for extracellular small rna sequencing. sequencing data are pre-processed through quality control, and then aligned to the endogenous genome to obtain the gene counts for various rna biotypes, including microrna, trna, rrna, piwi-interacting rna, long non-coding rna (lncrna) and protein coding rna. it also aligns sequencing reads to exogenous databases, including the ribosomal rna sequence database silva, and all sequenced bacteria genomes available on ensembl, to estimate the abundance of exogenous genes. results: we analysed a publicly available small rna-seq dataset of hdl from three systemic lupus erythematosus (sle) patients and three healthy controls using this pipeline. the mirna hsd-mir- , lncrna al . and ac . were elevated in sle patients compared to controls. exogenous rna reads mapped to bacteroidetes were also elevated in sle patients. summary/conclusion: our pipeline is able to process exrna sequencing data and estimate the abundance of major exrna species, as well as exogenous rna taxonomy. the pipeline is optimized for the job scheduler slurm, and can therefore utilize the full computational power of high-performance computers. the pipeline is publicly available on github (www.github. com/zhuchcn/excernapipeline). introduction: ibd is a chronic hyperinflammatory disorder that severely compromises the intestines. the aetiology of ibd is poorly understood. however, it has been associated with a dysregulation of the immune system and gut microbiota and with genetic and environmental factors. cumulative evidence indicates that evs play an essential role in modulating immune responses. recent research suggests that evs derived from dendritic cells, saliva and intestinal epithelial cells may be involved in the progression of ibd inflammation. however, little is known about the contribution of immune cells-derived evs with this pathology. the goal of this study is to shed light on the contribution of pbmc-derived evs on ibd pathogenesis. here we characterized and compared the composition of evs derived from pbmcs of ibd patients and healthy control. evs were isolated by differential centrifugations from the supernatant of pbmc activated with cd -cd beads for days in serum-free media. size and concentration were analysed using a nano sight instrument, while the presence of known evs markers (cd , cd , hsp ) was analysed by immunoblotting. whole evs proteome was performed by ms/ms and functional-enrichment analysis was done using funrich with uniprot database. results: proteomics analyses identified a total of proteins in the four groups. of those, ( . %) were present in both the ibd patients and control. this group of protein was composed of several ras-related proteins, eukaryotic initiation factors, granzyme, cd , tubulin, and serpins among others. patients' evs shared proteins in common such as proteasome subunit beta type- , t cell receptor beta, and the amine oxidase containing copper . interestingly, each patient sample had a unique group of proteins. among these are myeloperoxidase, neutrophil elastase, proteasome subunit alpha type- , and signalling lymphocytic activation molecule (slamf ). summary/conclusion: these preliminary studies show that the ev composition from pbmcs of ibd patients is specific and differs from a healthy control. this exclusive composition has the potential to be used as a biomarker for diagnostics and progression of the disease, and it could also provide new insights into our understanding of the cellular pathways involved in the pathogenesis of ibd. the studies were performed with corresponding irb approvals. proteomic analysis of exosomes isolated using precipitation and columnbased approaches introduction: exosomes are a subtype of small extracellular vesicles (evs) involved in various physiological and pathological processes with huge potential as biomarker resources or as therapeutic tools. although several exosome isolation approaches are available, complementary studies focusing on optimizing the methods for human bloodderived exosomes isolation and method-specific comparative exosomal proteomic profiles will be of clinical value. methods: blood-derived evs were isolated through precipitation-and column-based methods and characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. serumderived exosomal proteomes were analysed by mass spectrometry (ms). the resulting proteomes were then overlapped with the proteomes obtained from exosome-related databases, to determine the % of similar content. in addition, bioinformatic analysis, including gene ontology (go) was carried out. results: both methodologies tested isolated particles with the expected morphology and size range, although the column-based method isolated a higher number of particles. about % of the exosomal proteins identified through ms overlapped with the proteomes extracted from the databases. go terms were similar for the proteomes isolated from the column-and precipitationbased methodologies. the top go terms identified for molecular function were ion binding, peptidase activity and enzyme regulator activity and for biological process were immune system process, transport and response to stress. further, partial least square analysis revealed a clear segregation of proteomes obtained by the distint methodologies and complementary statistical analysis revealed the proteins differently expressed. summary/conclusion: no major differences were found in the top biological processes and molecular function based on go analysis. nonetheless, the two approaches result in different evs yields and significant proteome differences were identified. characterization of distinct methods for blood-derived exosomes isolation can be useful in the context of evs potential in disease diagnostics/therapeutics. introduction: we and others are developing biomarkers for neurodegenerative diseases using neuronalenriched evs immunocaptured from a suspension of total plasma evs. here we assess how the isolation method for total evs affects the yield, purity and enrichment of neuronal evs. methods: for n = subjects, total evs were isolated by ev precipitation solution (exq), ev precipitation solution plus bipartite resin columns (exu) and size exclusion chromatography (qev) from . , . and . ml plasma, respectively. then, neuronal-enriched evs were immunoprecipitated using anti-l cam antibody. in total and l cam evs, we measured particle concentration by nanoparticle tracking analysis, protein concentration, and novel multiplex electrochemiluminescence immunoassays for tetraspanins cd , cd and cd on intact evs. results: for total evs, yield followed the order of exq > qev > exu, assessed by particle (p < . ) and protein concentrations (p < . ). l cam evs immunocaptured after exq showed -fold higher particle (p < . ) and fivefold higher protein (p < . ) concentrations compared to l cam evs after exu, and -fold higher particle (p < . ) and -fold higher protein (p < . ) concentration compared to l cam evs after qev. l cam evs after ev precipitation (exq) showed , and -fold higher cd , cd , and cd concentrations (p < . ) compared to l cam evs after exu, and , and -fold higher cd , cd , and cd concentrations (p < . ) compared to l cam evs after qev. l cam evs following different methods had equal purity assessed by ratios of particle/protein concentrations (p = ns), and tetraspanin/particle concentrations (p = ns). summary/conclusion: l cam ev immunocapture preceded by exq exceeded the yield of immunocapture preceded by exu or qev. recovered l cam evs showed equal purity by particle/protein and tetraspanin/particle metrics. neuronal enrichment results will be available by the time of isev. immunoprecipitation following exq, often considered impure, purifies final isolates as effectively as more onerous methods typically considered purer. balancing sensitivity, purity and scalability is essential for implementation of blood biomarkers in the clinical setting and may be achieved by combining techniques. funding: this research was supported in part by the intramural research program of the nih, national institute on aging. characterisation of breath exosomes: towards non-invasive diagnosis deanna ayupova a , renee goreham b and paul teesdale-spittle c a school of chemical and physcial sciences, victoria univeristy of wellington, wellington, new zealand; b university of newcastle, newcastle, australia; c school of biological sciences, victoria univeristy of wellington, wellington, new zealand introduction: breath-derived exosomes present new potential for non-invasive diagnosis of lung cancer. however, breath-derived exosomes have not been well characterized and methodology for their purification has not been optimised. in order to exploit their potential for diagnosis, it is first necessary to develop methods that reproducibly provide high quality pure exosomes from breath. in this study, we optimise methods for their isolation and characterise them in comparison to exosomes derived from cell culture models. methods: in order to characterize exosomes from exhaled breath condensate (ebc) it was first necessary to optimize methods for isolation of pure, intact, and high quality exosomes. to this end, isolation methods were optimised on cell-derived exosomes and then applied to ebc, yielding high quality exosomes from size exclusion chromatography (sec). ebc exosomes were compared with those from a and wi-cells using dls, tem, and cryo-sem. an immunoblotting-grid technique was used to validate the presence of exosome-specific markers cd and cd . protein content of exosomes were quantified and compared. results: sec-based isolation was more effective at isolation of pure and intact exosomes than ultracentrifugation, with the highest purity exosomes obtained in the middle fractions of the exosome-containing eluate. exosomes from ebc had a size range ( - nm), protein content ( - ug/ml) and molecular markers typical of cell-derived exosomes. summary/conclusion: breath-derived exosomes isolated through size exclusion chromatography are sufficiently pure for diagnostic purposes and are phenotypically similar to exosomes derived from other sources. we foresee their use in non-invasive diagnostics for lung cancer as an important future application. ligand-based exosome affinity purification (leap) is a rapid and reproducible method for the enrichment of functional evs introduction: platelet-derived extracellular vesicles (pevs) represent the next generation of therapeutic biologics as they enable a more refined and targeted approach when compared to crude blood derivatives currently used for treating diseases such as cancer, thrombocytopenia and chronic wounds. however, development of an ev-based therapeutic is hindered by the lack of a scalable, validated and reproducible purification process. in this study, pevs were isolated from activated platelet concentrates and purified using exopharm's ligand-based exosome affinity purification (leap) technology to produce a functionally active ev therapeutic. methods: platelet concentrates (n = ) were obtained from the australian red cross blood service and were activated by exopharm's proprietary process. activation was verified by measuring cd p using flow cytometry. the resulting platelet releasate ( ml) was subjected to leap purification to isolate pevs. for characterization, protein concentration was determined by a bicinchoninic acid assay, microfluidic resistive pulse sensing (mrps) was used to perform a particle count and transmission electron microscopy (tem) enabled visualization of ev morphology. key ev markers were detected using mass spectrometry (ms) and western blots. to confirm biological activity, human dermal fibroblasts were subjected to serum starvation for hours before treatment with pevs ( µg/ml). cell growth was recorded by the real-time xcelligence system and differences in proliferation were statistically analysed using a one-way anova. results: mrps and tem both revealed isolated pevs to be - nm in size. the final product was positive for platelet markers (cd , cd p) and key ev markers (tsg , alix, cd ). treatment with purified pevs significantly increased proliferation in serumstarved fibroblasts over hours. summary/conclusion: exopharm's leap technology is a rapid and reproducible purification process which produces pevs that adhere to misev guidelines and are functionally active. funding: all funding was through exopharm ltd (asx:ex ) a novel but simple method to obtain purified exosomes by one-step ultracentrifugation introduction: exosomes are extracellular vesicles (evs) that are derived from endosome membrane. they are usually - nm in diameter, actively secreted in most living cells. originally, exosomes were thought to act as cellular garbage disposals. recent studies showed that exosomes not only can serve as biomarkers for diagnosis, but also can be used as an ideal delivery vehicle for drugs in therapeutics. exosomes are natural carrier for mrna, mirna, sirna, protein, dna and peptide for long distance intercellular communication. isolation of exosomes is challenging due to their small size and heterogeneity. traditional differential ultracentrifugation method is still the gold standard for exosome purification. to further explore the potentials of exosomes being as the therapeutic delivery vehicle or diagnostic reagent, it is an essential step to purify them in high quality at high yield. methods: here, we report a novel method to obtain intact shape, high-quality and high purity exosomes with one-step ultracentrifugation by using "exojuice". results: data of nanoparticle tracking analysis (nta) and western blotting showed "exojuice" can yield exosomes with a simpler method to obtain higher purity exosomes in comparison to previous method of cushion ultracentrifugation using optiprep. summary/conclusion: our method can be used to purify exosomes from cell culture medium, serum, urine, saliva, and other biofluids. a straightforward device to extract apoplastic fluid from succulent fruits for higher purity of extracellular vesicles introduction: edible plants are emerging as a sustainable source for extracellular vesicle (ev)-based drug delivery vehicles. however, current isolation methods (e.g. grinding or squeezing) may cause destruction of plants' biostructures, and in turn leads to unwanted effects in downstream applications and complicates the study of nanovesiclescell. therefore, we designed a simple device that allows the extraction of apoplastic fluid (af) from succulent fruits, facilitating ev isolation as well as effective downstream applications. methods: an inner filter tube was designed to extract af with a determined membrane pore size. af was collected by low-speed centrifugation method and then filtered to eliminate the impurities from the cytoplasm and damaged cells. minced juice (mj) was homogenized by a blender and then centrifuged to remove large fragments. subsequently, the differential centrifugation method was employed to extract evs from af and mj. fourier-transform infrared spectroscopy (ftir), nanoparticle tracking analysis (nta), and transmission electron microscopy (tem) were performed to discriminate af, mj and their evs. results: the "spectroscopic" protein-to-lipid (p/l) ratio of af ( . ± . ) is significantly lower than that in mj ( . ± . ), showing the higher lipid contents in af, which may result from the loss of lipids in mj obtained from grinding or juicing methods. similarly, ftir showed the difference in p/l ratio between af and its evs ( . ± . and . ± . , respectively). nta showed the sharper peak and smaller vesicle size in the following order: mj ( . ± . nm), af ( ± . nm), af-derived evs collected at , × g and , × g ( . ± . nm and . ± . nm, respectively). furthermore, tem study indicated that the collected evs exhibited a typical lipid bilayer of extracellular nanovesicles. summary/conclusion: by using a reusable filter device, we successfully isolated af from succulent fruits, paving the way to collect plant evs without an interference of significant biodestruction or damaged cells, hence improving the purity of evs and facilitating downstream applications. moreover, this method is straightforward, reproductive, and can be potentially used in a large-scale production. method to simultaneously capture multiple classes of intact extracellular rna carriers including extracellular vesicles and lipoprotein particles introduction: extracellular particles including extracellular vesicles (evs), lipoproteins, and free proteins are carriers of extracellular rna (exrna), which has been shown to regulate cellular function. because these particles have different physiological origins, they have different rna signatures, so the first step to understanding the biology of exrna is to isolate individual particle fractions with high purity and efficiency. current methods for isolating evs are optimized for increased yields and purity of ev fractions but typically require multiple millilitres of starting plasma and do not capture the other exrna carrier particle types. methods that can capture evs from low starting plasma volumes and can also capture other exrna carriers simultaneously are needed for analysing samples from previously conducted large cohort studies, biorepositories, and in populations where sample volume is limiting. methods: we have developed a method adapted from lipoprotein isolation that requires only µl of starting plasma, and uses brief ultracentrifugation (uc) followed by fast protein liquid chromatography (fplc) to capture classes of purified exrna carriers including evs, ldl, hdl, lipidated albumin, proteins, and vldl/chylomicrons. we have validated successful capture of evs by microfluidic resistive pulse sensing (mrps, spectradyne), transmission electron microscopy (tem), and single particle interferometric reflectance imaging system (sp-iris; exoview) with optional fluorescence. results: we have observed . × particles per ml from a ml fplc fraction of evs measured from , events by mrps, confirming that evs are being captured by this method. there were also . × particles/ml and . × particles/ml in the two subsequent ml fractions that are known to contain lipoprotein particles, though these were measured from , events each. by tem we confirmed these observations that evs are eluting before lipoprotein particles with some evs eluting later in fractions containing lipoproteins. summary/conclusion: these results confirm the efficacy of the method in isolating multiple exrna carrier fractions simultaneously from a single ul plasma sample, making it amenable for the analysis of exrna in samples from large cohort studies, biorepositories, and vulnerable populations such as the elderly and young children. funding: nih/nia r ag ; nih ug ca - optimizing the isolation of placental mesenchymal stromal cell-derived extracellular vesicles in a d bioreactor system leora goldbloom-helzner a and aijun wang baaaaa a uc davis, davis, usa; b uc davis medical center, sacramento, usa introduction: extracellular vesicles (evs) derived from placental mesenchymal stromal cells (pmscs) have the potential to provide neuroprotection at sites of injury. however, a rate limiting step in ev research is the low yield, high technical time, and high cost of current isolation procedures. to address this inefficiency, we cultured pmscs in a unique bioreactor system to increase the absolute yield of evs per ml of media and per cell. future studies will determine if this system can improve pmsc ev yield without altering the demonstrated neuroprotective properties of pmsc-evs. methods: pmscs were cultured in the bioreactor for ten weeks. ev-conditioned media was collected weekly and evs were isolated through differential centrifugation. nanoparticle tracking analysis (nta) measured ev size and concentration. western blots tested for normal ev markers (cd , cd , and cd , calnexin(-)) and enzyme-linked immunosorbent assays (elisa) measured levels of characteristic growth factors in conditioned media including vascular endothelial growth factor (vegf), brain-derived neurotrophic factor (bdnf), and hepatocyte growth factor (hgf). results: evs remained consistent until week eight, after which a decrease in both ev size and concentration was seen. western blots revealed normal positive expressions of cd , cd , and cd and negative expressions of calnexin. levels of vegf, bndf, and hgf were comparable after weeks. cost analysis revealed an overall increase in ev yield for shorter labour time and lower material cost. summary/conclusion: this initial study uses a bioreactor system for a unique source of cells and has brought us closer to optimizing pmsc ev isolation protocols for increased yield, lower cost and time commitment, and maintained sample purity. preliminary data suggests the ev phenotype and cell secretome are consistent with those present in current culture settings. future experiments will assess the preserved neuroprotective properties of the pmsc evs. a novel method for isolating extracellular vesicles from cell culture media and plasma using polyethylenimine introduction: due to their ability to transport dna, rna, and protein cargoes between cells, extracellular vesicles (evs) are becoming popular for biomarker discovery as well as for therapeutic delivery. here we describe the development of a novel precipitation method for the isolation of evs from cell culture media and plasma that is based on polyethylenimine (pei), an inexpensive, water-soluble, and biocompatible cationic polymer. pei is a group of hydrophilic cationic polymers that are synthesized as either linear or branched forms of varying molecular masses ( , to , da) and are widely used in the biomedical field as a coating and transfection agent. methods: linear and branched pei of varying molecular weights (mw) were tested for their ability to precipitate evs from either conditioned culture media (ccm) or human plasma. isolated evs were characterized by western blotting and nanoparticle tracking analysis (nta). the small rna profile of evs isolated using pei from human plasma was analysed by ngs and ev-specific mirnas were confirmed by digital droplet pcr (ddpcr). mass spectrometry (ms) was used to analyse the proteome of pei-captured evs from plasma. hek cells producing gfp+ evs were used to optimize conditions for release of evs from both linear and branched pei by fluorescent spectrophotometry and flow cytometry measure-ments. results: linear and branched pei were both able to precipitate evs as determined by western blotting for ev protein markers; however, branched pei with mw > , da and linear pei with mw > , da were more efficient for ev precipitation than lower mw forms. despite its known ability to bind nucleic acids pei was unable to capture cell-free dna from plasma, although rna and in particular ev-associated mirnas such as mir- - p were recovered. ms revealed that pei enriches extracellular exosome proteins from plasma. evs captured from ccm by pei could be released from the complex using heparin or high salt conditions. summary/conclusion: pei has an unexpected preference for associating with evs compared to nucleic acids in complex biological samples and has a hitherto unrecognized application for ev precipitation. introduction: there is ongoing debate about which is the most appropriate method for isolation of evs, with most labs using some combination of differential ultracentrifugation (uc), size-exclusion chromatography (sec), and/or density gradient ultracentrifugation (dg). here we applied a surface-enhanced raman spectroscopy (sers) analysis platform to compare chemical composition of the isolate from each method against lipoprotein standards to assess the relative purity of the ev preps. methods: - ml of plasma was separated from whole blood collected from head and neck cancer patients. each sample was split into batches and evs were isolated by either uc, sec, or dg. following isolation, samples were incubated on commercial sers substrates and raman spectra were collected. lipoprotein standards were purchased and also measured for comparison. using principle component analysis (pca), spectra were analysed for chemical variability. results: sers analysis of sec, uc, and dg isolated evs were chemically distinguishable using simple pca. the chemical changes could in large part be attributed to fitting the differences in spectra to lipoprotein standards. we found that uc isolated populations clustered with the high-density lipoproteins (hdl), sec populations with the low-and very low-density lipoproteins (ldl, vldl), and dg populations were more variable, but mainly clustered together with the highdensity-lipoproteins (hdl). summary/conclusion: this set of experiments matches our expectation that various lipoprotein would contaminate ev preps according to their relative size and density distributions. no single isolation method could separate pure ev samples. this study also illustrates the utility of label-free sers analysis for rapid chemical characterization of evs. bioreactors: lessons to develop an extracellular vesicle factory vanessa chang, priscila dauros-singorenko, lawrence w. chamley, colin l. the university of auckland, auckland, new zealand introduction: high density mammalian cell culture systems (bioreactors) provide valuable advantage for large scale production of secreted products such as extracellular vesicles (ev). however, optimisation of design selection, handling and operational costs can be quite challenging. here we provide our experience with a celline bioreactor system. methods: cultures of adherent cell lines were established in celline ad bioreactors and propagated for up to weeks. media was changed twice weekly and cells shed into serum-free conditioned medium were counted and assessed for viability. nanoevs were isolated by sequential centrifugation ( g - , g - , g) and size exclusion chromatography (sec). nanoevs were characterised in their protein (bca) and particle (nanoparticle tracking analysis) amount, ev markers (western blotting) and morphology (transmission electron microscopy, tem). results: the viability of shed cells varied between cell lines and through time, suggesting a changing dynamic during reactor establishment and continuous growth phases, that was specific to each cell line. hdfa, bt and bt consistently shed mainly dead cells ( - %), as opposed to mcf and mda-mb- which predominantly shed live cells. sec fractionation of nanoevs identified a dominate ev-rich peak and significant quantities of smaller proteins, highlighting the need for further purification. nanoev yields from each - day culture averaged - × particles, representative of yields obtained from cells grown in to conventional t tissue culture flasks. ev markers and tem confirmed the protein profiles and morphology of evs obtained from bioreactors. summary/conclusion: high density bioreactor cultures offer a physiologically relevant, cost and space efficient approach to produce significant amounts of evs, providing sufficient material for numerous experimental uses. in our hands, with careful twice weekly management, they can be propagated for up to weeks without significant changes to the evs. introduction: extracellular vesicles (evs) have potential applications for clinical theranostics. ultracentri-fugation is most commonly adopted to the evs isolation, which is recommended as a gold standard method. however, ultracentrifugation is time-consuming and expensive equipment requirement, resulting in the coisolation of contaminants such as protein aggregates. therefore, our aim is to develop a rapid and efficient platform to isolate heterogonous evs based on the insertion of lipid molecules into the evs membrane to avoid co-isolation of non-membranous protein particles. methods: herein, a defected nanoscale functional metal organic framework (mof) was constructed as an efficient platform for evs isolation. typically, one single-stranded dna was designed and modified with a phosphate group at the ʹ-end and cholesterol at the ʹ-end to form a capture dna named phosphate−dna−cholesterol (pdc). the phosphate group forms a strong covalent bond with the designed defeated site of zr (iv) in mof uio- -nh and the cholesterol inserts into the phospholipid bilayer to capture evs without non-membranous particles contamination. the formed mof−phosphate−dna −cholesterol−evs (mof@pdc@evs) system was further treated with dnase i for dna hydrolysis to give high pure evs. results: a rapid and efficient isolation platform of evs based on a defected mof functionalized with phosphate-dna-cholesterol (mof@pdc) has been constructed successfully. compared with ultracentrifugation, mof@pdc platform promises to isolate size heterogeneous evs i) without non-membranous particles contamination, maintaining evs intact membrane structure, protein components, and biological functions; ii) with the ability to capture evs with % isolation efficiency; iii) makes evs isolation process simple and fast, which could be finished in minutes without requirement of the expensive equipment. summary/conclusion: in conclusion, this rapid and efficient platform is suitable for isolation evs from biological fluid for downstream protein analysis. this work opens a new perspective in mof-based separation researches and may shed light on further studies towards evs isolation. introduction: incorporation of pharmacologically active molecules on the surface or the lumen of extracellular vesicles (evs) is an important strategy for maximizing the therapeutic potential of evs. genetic engineering of producer cells by introducing dna through random or site-specific integration are promising strategies for creating engineered evs. longterm stability with consistent transgene expression in the ev producer cells and therapeutic potency of resulting engineered evs are crucial for biomanufacturing. we present a comprehensive study to investigate stability of transgene expression and potency of two potential therapeutic engineered evs derived from stably selected pools transfected by either random integration (ri) or site-specific integration (ssi). methods: producer cells were engineered to make evs displaying interleukin (il ) or interferon gamma (ifng) by ri or nuclease-mediated ssi into aavs locus. following puromycin (puro) selection, longterm cellular stability and transgene expression without selective pressure was investigated. evs were generated from stable cell pools at , , and months post-thaw and purified by density gradient ultracentrifugation. purified evs were biochemically characterized by nta, bca, western blot, and cholesterol quantitation. transgene expression and biological activity of evs displaying il and ifng were assessed by alphalisa and in vitro reporter assays. results: transfection by ssi resulted in faster recovery in puro selection compared to ri. all stable cell pools, regardless of integration method, resulted in comparable cell culture performance, ev yield, and lipid and protein content at all time points tested. the engineered evs also demonstrated long-term stability of il and ifng transgene expression and in vitro activity from both integration strategies. summary/conclusion: both methods for generating stable cell lines were comparable in terms of cell stability, transgene expression, ev titre and potency, with ssi having the advantage of speed, allowing for more rapid iteration cycle times. thus, both methods are suitable for the precision engineering of therapeutic evs. this work demonstrates feasibility to manufacture therapeutic engineered evs from stable cells from either integration strategy for clinical development. transport of outer membrane vesicles as a model therapeutic delivery system in pathogenic and commensal bacteria introduction: outer membrane vesicles (omvs) in gram-negative bacteria have been shown to be important carriers of biomolecules, including toxins and other virulence factors, peptidoglycan, and nucleic acids. it has been shown that omvs play an important role in the delivery of these biomolecules to host cells and bacterial cells. while many thorough studies have explored omv delivery to host cells, few studies have explored the mechanisms of delivery of omvs to bacterial cells. our goal was to study the delivery of omvs to other bacterial cells. specifically, we were studying the oral pathogen aggregatibacter actinomycetemcomitans (a.a.), a gram-negative organism associated with localized aggressive periodontitis, to study the process by which vesicles from this organism communicate with other bacterial cells. overall, we want to understand the roles specific surface components of omvs play in the transport of these omvs to other bacterial cells. methods: we studied omvs from two strains of a.a.: jp , a highly pathogenic strain, and , a natural commensal strain. af -labelled omvs were incubated with fresh bacterial cultures. association of the omvs with the bacterial cells was quantified using flow cytometry. to examine the role of surface-associated dna in this process, dna was digested with dnase, and the amount of surface-bound dna was quantified with the membrane impermeable dna stain, toto- . results: using flow cytometry, we observed jp omvs were delivered to , cells, and at a lesser amount to jp cells. alternatively, , omvs associated readily with jp cells, more than to , cells. this suggests that the delivery of omvs to bacterial cells may be a targeted delivery mechanism. furthermore, we hypothesized surface-associated dna may play a role in this interaction. we next digested the surface-associated dna on the omvs with dnase, and observed a decrease in association between the omvs and bacterial cells. this supports our hypothesis that dna on the surface of the omvs plays a role in association. current experiments are investigating this interaction in more detail. summary/conclusion: we have demonstrated that omvs are selectively delivered to bacterial cells, and surface-associated dna plays a role in this process. we propose to investigate this process to further understand omvs delivery to bacterial cells. funding: r de & r de . utilizing a gaucher's disease cell line for the evaluation of a novel exosome-based replacement therapy annie k. brown a , jiayi zhang b , brendan lawler b and biao lu b a santa clara university, san jose, usa; b santa clara university, santa clara, usa introduction: engineered nano-scale exosomes have great potential as new and targeted delivery vehicles for the treatment of gaucher's disease, the most common lysosomal storage disease. recently, we have reported the design, production, and isolation of exosomes loaded with lysosomal β-glucocerebrosidase (gba). people suffering from gaucher's disease do not have functional gba, which results in toxic build-up of undegraded substrates within the cell. methods: to evaluate the efficacy of this exosomebased therapy, a human gaucher's disease model is required. here, we have utilized near-haploid human cells (hap ) modified via crispr-cas to model gaucher's disease in vitro. these cells contain a bp insertion in the th exon of the gba gene, resulting in non-functional gba. pcr, enzyme activity assays, and flow cytometry have been employed to confirm the diseased genotype and phenotype. results: characterization of gba-knock out cells shows a total loss of gba enzyme activity. further characterization demonstrates a normal growth rate but an increased number of lysosomes, indicating a diseased phenotype. summary/conclusion: the utilization of a human gba-knock out cell line will enable the evaluation of the efficacy of our engineered exosomes. disease models will be an important resource for the evaluation of new biologic therapeutics, including exosomes. funding: we would like to acknowledge the santa clara university school of engineering for their support. thrxosomes: a novel exosomes based theranostic for lung cancer introduction: chemotherapy is the first-line of treatment for lung cancer. however, inefficient bio-distribution and reduced accumulation of drugs in the tumour results in treatment failure. therefore, improved drug delivery and diagnostic systems are warranted. herein, we propose a novel theranostic system "thrxosomes" where exosomes are loaded with super paramagnetic iron nanoparticles (spions) conjugated to an anticancer drug via a phresponsive linker for controlled release. we hypothesize that thrxosomes will exert profound anticancer tumour activity that can be concurrently be monitored by magnetic resonance imaging (mri). methods: thrxosomes were produced by combining normal human lung fibroblast (mrc ) cell-derived exosomes with spions conjugated to and anti-cancer drug (chemodrug or mirna) via a ph cleavable linker. the physical and biological properties of thrxosomes were determined using transmission electronic microscopy (tem), nanotracker-analysis (nta), inductively coupled plasma mass spectrometry (icpms), western blotting, cell viability, and mri. results: exosomes used in preparing thrxosomes were nm in size with a typical lipid bilayer structure, and were positive for cd , cd , flotillin and negative for annexin a confirming presence and purity of exosomes. charge analysis, tem, and icmps data showed successful loading of spion-drug conjugate. biological studies showed selective and enhanced drug release under acidic condition (ph . ) compared to drug release at ph . . cell uptake and viability studies demonstrated increased uptake and killing of thrxosome-treated human a lung cancer cells compared to mrc- cells. in vivo studies demonstrated accumulation and detection of spions by mri in in-situ tumours of a tumour-bearing mice. summary/conclusion: our study demonstrates thrxosomes will produce profound anticancer activity in lung cancer that is measurable by mri. exosome-modified nanoparticles as an alternative delivery system for small rnas in cancer therapy petro zhupanyn a , alexander ewe b , thomas büch c and achim aigner a a independent division for clinical pharmacology at rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany; b dr., leipzig, germany; c rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany introduction: gene knockdown by rna interference (rnai) is an alternative, non-invasive method for inhibiting proliferation or promoting apoptosis in tumour cells. this technique allows the specific targeting of key signalling proteins or mutated genes. most of the available transfection compounds suffer from rather profound cytotoxicity in vitro. the aim of our study was to establish a novel targeted small nucleic acid delivery system to the cells, with good cellular biocompatibility and applicability for in vivo studies. for this aim, we used native, cell own vesicles-exosomes. since exosomes are known to transport peptides and different rnas between cells and tissues, these unique, small extracellular vesicles (ev) may also be useful as transport vehicles for therapeutic sirna. methods: as detected by multiple cell surface protein expression analysis, exosomes carry specific surface expression markers, allowing the cellular uptake by the most of tissues. we established an ev purification protocol from tumour cell culture supernatants and a strategy for the efficient ev loading with our test sirnas or antimirs. here we used the combination of polyethylenimine (pei)-complexation of the rnas with ultrasound treatment for their loading into the evs. our ev-modified, ultrasound-treated nanoparticles were tested in vitro by measuring knockdown efficacies in luciferase reporter cell lines or by rt-qpcr gene expression analysis. results: more efficient cellular sirna uptake was observed upon ev-modification of our pei/rna nanoparticles, accompanied by efficient inhibition of gene expression. biological efficacies were retained also after storage for several days at room temperature. the monitoring of the ev-based particles by facs revealed a different time resolution of cellular uptake and nucleic acid release compared to the classically formulated peinanoparticles. in an in vivo therapy study in tumour xenograft-bearing mice, high biocompatibility, significant biological knock-down and tumour inhibition were observed after injection of anti-survivin sirnas formulated in our ecv-modified pei nanoparticles. summary/conclusion: our data demonstrate the usability of ecv-modified nanoparticles as efficient delivery system for small rnas in cancer therapy. microglial extracellular vesicles as therapeutic vector for neuroinflammation giulia marostica a , annamaria finardi b and roberto furlan a a san raffaele scientific institute, milan, italy; b san raffaele scientific institute, milan, italy introduction: microglia is considered an eligible target against the progressive multiple sclerosis (ms), but currently available therapies do not allow its efficient targeting. as many cell types, microglia communicate with the neighbouring cells through a complex system of extracellular vesicles (evs) exchange. recently my group described that microglia derived-evs, engineered to encapsulate il , are taken up by microglia itself, mediating a phenotype switch to a protective phenotype. in vivo studies suggest that these evs can ameliorate established neuroinflammation, thus making them a promising drug-delivery tool to target cns in ms. my project focuses on understanding the mechanism of action and the signalling pathway of evs delivery and to exploit this knowledge to specifically deliver different potential therapeutic molecules. for this purpose, we decided to characterize the evs through trps technology. methods: a murine microglia cell line (bv ) was engineered to stably overproduce endogenous il . this cell line was cultured in exosome-depleted rpmi and stimulated with pma( mg/ml) for min. evs isolation was carried out by collecting supernatant and subjecting it to consequential centrifugation of g, min, rt and g, min, °c. the resulting supernatant was filtered ( µm) and ultracentrifuged at , g for h at °c. the evs pellet was re-suspended in ice-cold pbs. the evs analysis with trps shows two populations of evs, one with a mean diameter of - nm and a broad zeta potential ranging from − mv to − mv, while the second population has a mean diameter of - nm and a zeta potential of − /- mv. this difference can be consistent with the different pathway formation of exosomes and microvesicles. we demonstrated in vivo the strong phenotypic change induced by our evs to resting microglia in a dose-and time-dependent effect. then, impairing the physiological procedure of the endosome acidification, the effect of our evs on recipient cells is higher. thus, suggesting an endocytic pathway for the internalization of the vesicles. we further demonstrate with gradient ultracentrifugation the capability of our formulation to vehicle endogenous il inside the vesicles. even if some protein is co-purified in the procedure, we know that the half-life of this cytokine is too short to elicit a strong in vivo response. consequently, we assume that the anti-inflammatory effect of our evs in vivo is a result of the il internalized in our formulation. summary/conclusion: these data help us understand more in detail the process of internalization and phenotype change mediated by these evs. our next goals are to discriminate between different internalization pathways and further validate the efficacy of our therapy on the eae mouse model. targeting il- rα on tumour-derived endothelial cells blunts metastatic spread of triple negative breast cancer via extracellular vesicle reprogramming introduction: the lack of an approved targeted therapy and the early onset of metastasis highlight the need for new treatments for triple-negative breast cancer (tnbc) patients. interleukin- acts as an autocrine factor for tumour-endothelial-cells (tec), and exerts pro-angiogenic paracrine action via extracellular vesicles (nevs). il- rα blockade on tec changes tec-ev (anti-il- r-evs) microrna cargo and promotes the regression of established tumour vessels. as tec are the doorway for "drug" entry into tumours, we have aimed to assess whether il- r blockade on tec impacts tumour progression via their unique ev cargo. methods: human tnbc samples, mda-mb- , mda-mb- and mcf cell lines were evaluated for the expression of il- rα. nevs and anti-il- r-evs were characterized by electron-microscopy, macsplex-exosome-kit and western blot. proliferation, migration, apoptosis and sphere formation were evaluated. scid mice were used for in vivo experiments. results: we noticed that, besides tec and inflammatory cells, tumour cells from . % of the human tnbc samples expressed il- rα. mda-mb- and mda-mb- , but not mda cells, expressed il- rα. in vitro, nevs provide survival and migratory signals, while anti-il- r-evs promoted apoptosis as well as reduced cell viability and migration of human tnbc cell lines. in vivo anti-il- r-ev treatment induced vessel regression in established tumours formed of mda-mb- cells and almost abolished the spread of liver and lung metastasis. moreover, decreased β-catenin and twist were found in tumours from animals treated with anti-il- r-evs. in addition, anti-il- r-evs reduced lung metastasis that was generated via the intravenous injection of mda-mb- cells. nevs that were depleted of mir- - p (antago-mir- - p-evs) were effective as anti-il- r-evs in down-regulating twist and reducing lung-vessel density and metastatic lesions in vivo. summary/conclusion: overall, these data provide the first evidence that il- rα is highly expressed in tnbc cells, tec and inflammatory cells, and that il- rα blockade on tec impacts tumour progression. introduction: high-grade serous ovarian cancer (hgsoc) is the deadliest gynaecologic cancer. its lethality is explained for late diagnosis at advanced stages and frequent recurrences despite achieving complete response with standard therapy. most of recurrences occurs at abdominal cavity with multiple metastasis. therefore, identifying key determinants of metastatic process remains as priority to find better therapies. current evidence assigns a central role of the exosomes in conditioning the metastatic niche in epithelial cancers. recently, we demonstrated that statins reduce metastasis in hgsoc in preclinical models. here, we decided to study the effects of statins on hgsoc-derived exosomes and its capability to condition the metastatic niche. methods: exosomes were isolated from heya ovarian cancer cell line and primary tissue cultures established from advanced-stage hgsocs (with signed informed consent and irb approval) by differential ultracentrifugation and quantified by nanoparticle tracking analysis (nta). enriched-cancer initiating cells (cic) spheroids were established from heya cells by using stem-selecting conditions. the paracrine effect of exosomes on cic migration/invasion was studied using either d migration or boyden chamber invasion assays. previous to exosome isolation, heya cells were treated with simvastatin ( um, h) or solvent and proteins involved in exosome biogenesis/uptake (alix, tsg ), its trafficking (rab a, rab a) and in conditioning the metastatic niche (emmprin) were measured by immunoblotting. results: exosomes isolated from heya cells or hgsocs enhance the metastatic potential of heya established spheroids in d migration or boyden chamber invasion assays. upon simvastatin treatment, we observed a significant reduction in migration/invasion induced by equivalent number of exosomes in heya -derived cics. under same treatment, we observed a significant decrease in protein levels of alix and tsg and an increase in the inactive forms of rab a and rab a in heya cells. we also observed a decrease in emmprin levels in heya -derived exosomes. summary/conclusion: here, we demonstrated a paracrine effect of hgsoc-derived exosomes that favour the metastasis process. in addition, we demonstrated that simvastatin reduces metastasis induced by cancerderived exosomes. such an effect is partially explained by changes in the expression of proteins involved in exosome biogenesis/uptake, its endocytic trafficking and in the content of proteins conditioning the metastatic niche. thus, simvastatin arises as potential therapeutic target to improve outcomes in this disease. funding: this research was supported by fondecyt granted to mauricio a. cuello label-free optical imaging and characterization of cancer-associated extracellular vesicles in tissues introduction: cancer-associated extracellular vesicles (evs) visualized in the tumour microenvironment have been identified as a potential biomarker for cancer-related tissue changes. analyses of evs have traditionally been performed in cells or isolated evs, with no temporal or spatial information that could be critically important for elucidating their roles in carcinogenesis. since the unperturbed distribution and organization of evs in the tumour microenvironment is associated with their cellular function and can potentially serve as a diagnostic and prognostic biomarker, there is a strong need for visualizing evs in freshly isolated tissue specimens. currently, only fluorescent labelling methods enable visualization and tracking of evs. we used a custom label-free multimodal multiphoton optical imaging system to detect and characterize evs and classify them using their optical signatures both in isolated tissues and in situ tumours. methods: label-free multimodal multiphoton imaging was used to provide simultaneous, co-registered structural and functional images of evs in untreated samples. heterogeneous populations of evs could be identified from their unique optical signatures. results: the intrinsic metabolic and structural properties of evs enabled reliable visualization and optical characterization of evs from cell cultures and in situ imaging of tumour-bearing rats. unique optical signatures were then used for identification of cancer-related evs in tissues from human breast cancer patients, and their density was found to be highly correlated with clinical diagnosis. in the current study, evs were isolated from urine of tumour-bearing dogs, and urine and serum from breast cancer patients. analysis of ev content showed higher concentration of nad(p)h in evs isolated from cancer subjects, than from healthy subjects, which reflects the reprogramming of cellular metabolism in carcinogenesis. summary/conclusion: these results suggest a potential label-free optical methodology to detect and characterize evs by their optical signatures, which can be utilized as possible diagnostic and prognostic biomarkers for cancer. funding: this research was conducted under protocols approved by the iacuc and irb at the university of illinois and carle foundation hospital, and supported by funding from nih. novel potential anticancer therapies based on interference with nuclear entry of cancer cell-derived extracellular vesicle components in recipient cells introduction: the intercellular communication mediated by extracellular vesicles (evs) in the tumour microenvironment plays an important role in tumour progression. experimental evidence indicates that evs derived from highly metastatic cells influence the behaviour of less aggressive cancer cells. we have previously described a novel intracellular pathway where a fraction of endocytosed ev-associated proteins and nucleic acids is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into nucleoplasmic reticulum (nr). here, we better characterize this pathway and report that it is required for the induction of an aggressive behaviour induced by evs released from highly metastatic sw colon cancer cells in isogenic primary cancer cells. methods: super resolution-structured illumination microscopy and magnetic-based co-immunoisolation studies were employed to identify the protein components of the nuclear pathway and to monitor the entry of ev-containing late endosomes into the nucleoplasmic reticulum. human sw carcinoma cells expressing er-gfp and rab -rfp were exposed to evs from sw cells and then live imaged. results: we have previously reported that the tripartite protein complex, containing vap-a, orp and rab orchestrates the localization of ev-carrying late endosomes into nr. we now report that silencing of orp or vap-a, but not its homologue vap-b, reverses the pro-metastatic changes induced by evs isolated from metastatic cells on their non-metastatic counterpart, including transition to an ameboid phenotype, cell rounding and blebbing. moreover, we found that certain nuclear pore complex proteins and importin-beta are co-immunoisolated with orp , vap-a and rab suggesting the formation of a large protein complex at the entry of nuclear pores. summary/conclusion: interfering with the mechanisms regulating this novel intracellular pathway may find therapeutic applications particularly in ev field and oncology. educated osteoblasts regulate breast cancer proliferation via small extracellular vesicles thomas jefferson university, philadelphia, usa introduction: breast cancer commonly traffics to bone, where breast cancer cells (bccs) can survive undetected for years before metastatic outgrowth. in bone, bccs interact with surrounding stromal cells, including osteoblasts (obs), to shape the metastatic niche. our lab discovered there are at least two subpopulations of obs in the bone-tumour niche, based on protein marker expression. one group, "educated osteoblasts" (eos) have engaged in crosstalk with bccs whereas another group, naïve obs, have not. we have novel evidence that eos regulate bcc proliferation. the purpose of this study was to determine if extracellular vesicles (evs) produced by eos play a role in regulating bcc proliferation. we hypothesized evs produced by eos would decrease bcc proliferation. methods: eo-derived small evs from culture media were isolated via ultracentrifugation and characterized evs for size, protein marker expression, and density floatation to validate the purity of ev samples. the functionality of eo-derived evs on bcc proliferation was examined using edu and checkpoint proteins p and p . bcc protection from chemotherapy induced cell death was also examined. results: we found that evs produced by eos, but not naïve obs, decreased both triple negative and erpositive bcc proliferation in a concentration dependent manner. furthermore, using an edu assay, we found that exposure to eo-derived evs induces bccs to undergo cell cycle arrest. interestingly, the cell cycle arrest was reversible and bcc proliferation was restored upon removal of eo-derived evs. in addition, exposure to eo-derived evs leads to increases in bcc expression of the g checkpoint proteins, p and p . we next wanted to investigate proliferative signalling pathways that may be deregulated in bccs following exposure to eo-derived evs. we found that eoderived evs reduce bcc levels of erk / . because our data indicate eo-derived evs induce sustained cell cycle arrest in bccs, we desired to know if eo-derived evs protected bccs from chemotherapy-induced cell death. we found that bccs exposed to eo-derived evs and the chemotherapy drug, doxorubicin, have decreased cell death compared to bccs exposed only to doxorubicin. summary/conclusion: altogether, our data suggest eos play a crucial role in bone-tumour microenvironment by regulating bcc proliferation. funding: supported by nih r ca and commonwealth of pennsylvania -department of health sap for kmb. phosphorylation of tyrosine in annexin a is essential for its association with exosomes and for imparting invasive and proliferative capacity to other cells priyanka prakash desai a , pankaj chaudhary b , xiangle sun b and jamboor vishwanatha a a unt health science center at fort worth, fort worth, usa; b unt health science center, fort worth, usa introduction: triple negative breast cancer (tnbc) accounts for %- % of all breast cancer cases. the lack of targeted-based therapies highlights the importance of studying tnbc. elevated levels of annexin a (anxa ), a ca+ -dependent phospholipid binding protein, has been correlated with worse overall survival in tnbc patients. our previous data implicate that exosomal anxa is involved in creating a pre-metastatic niche and facilitating metastasis in tnbc. moreover, n-terminal phosphorylation of tyrosine (tyr) in anxa has been implicated in regulating several anxa activities in cancer progression. here, we demonstrated that n-terminal phosphorylation of anxa at tyr is important for its association with exosomes which imparts invasive and proliferative phenotype to other cells. hence, dissecting the regulatory pathway will be critical for verifying the value of anxa as a therapeutic, diagnostic or prognostic marker in tnbc. methods: pn -egfp plasmids expressing the constitutive phosphomimetic (anxa -y e) and non-phosphomimetic mutant (anxa -y f) gene expressing mutation at tyr site were overexpressed in mda-mb- tnbc cells. mutant cells were experimentally validated for anxa specific functions like migration, invasion and proliferation. exosomes were isolated from the mutant phosphomimetic (exo-anxa -y e-gfp) and non-phosphomimetic (exo-anxa -y f-gfp) cells and were analysed for exosomal surface expression of anxa by immunoprecipitation and flowcytometry. cal- breast adenocarcinoma epithelial cells were treated with exo-anxa -y e-gfp and exo-anxa -y f-gfp to analyse the rate of invasion and proliferation by transwell invasion and proliferation assay, respectively. transfer of exosomal anxa in cal- was studied using immunofluorescence and its implications on signalling pathways were studied by western blot. results: mda-mb- phosphomimetic tnbc mutant cells showed increased migratory, invasive and proliferative capacity compared to non-phosphomimetic tnbc mutant cells. exo-anxa -y e-gfp had elevated surface anxa expression compared to exo-anxa -y f-gfp. cal- cells treated with exo-anxa -y e-gfp showed high migratory, invasive and proliferative characteristics, with a higher expression of p-anxa (tyr ), p-src(tyr ) and p-paxillin(tyr ) compared to exo-anxa -y f-gfp treated cells. summary/conclusion: n-terminal phosphorylation of tyr in anxa in mda-mb- tnbc cells (phosphomimetic mutant cells) is essential for its association with exosomes and for conferring increased invasive and proliferative capacity to other breast cancer cells. funding: the above study is funded by national institute of health ro ca and nimhd's u md to dr.j.k.vishwanatha. a novel method for epithelial-derived extracellular vesicle isolation and enrichment in patients with advanced prostate cancer arpit rao, helene barcelo and bharat thyagarajan university of minnesota, minneapolis, usa introduction: evaluation of changes in prostate cancer biology is difficult due to presence of lymph nodal or bony metastatic disease in a majority of patients. a number of liquid biopsy assays have shown clinical utility in prostate cancer, but are limited by low sensitivity (e.g. circulating tumour cells-based assays) or inability to perform transcriptome sequencing (cellfree dna-based assays). epithelial-derived extracellular vesicles (epi-ev)-based assays are uniquely positioned overcome both these limitations as evs are abundantly secreted into the blood and have rnacargo that mirrors the cell of origin. however, a reliable method to enrich for epi-evs is currently lacking. methods: plasma was isolated from the peripheral blood collected from patients with metastatic prostate cancer enrolled in an institutional biobanking study before initiation of systemic antineoplastic therapy. evs were isolated from µl of plasma using a commercially available qev size exclusion column (izon inc.). without subjecting the evs to any physical stressors such as centrifugation, cd magnetic beads were used to fractionate the evs into cd + (platelet derived) and cd -(non-platelet derived) fractions. multiparameter flow cytometry was used to evaluate evs that expressed cd and epcam and were negative for calnexin. nanotracking analysis (nta) was used to quantify both total ev and cd + and cd fractions in all patient samples. the average ± standard deviation of total evs obtained from the patients was . x ^ ± . x ^ evs/ml of plasma (coefficient of variation [cv] : %) while the average and standard deviation of cd -evs was . x ^ ± . x ^ (cv: %). the cd -ev fraction represented a variable amount of the total evs in prostate cancer patients ranging from . % to . %. multiparameter flow cytometry showed that over % of total evs were cd + and calnexin-, suggesting an endosomal origin for a vast majority of the evs in these plasma samples. however, the proportion of evs expressing epcam (marker of epi-evs) was higher among the cd -fraction ( % - %) as compared to the cd + fraction ( . % - %). summary/conclusion: our novel method was able to isolate and enrich the epi-ev from the plasma of advanced prostate cancer patients. correlation between clinical characteristics and ev quantity is being evaluated to identify the reason(s) for large variations in cd -ev fraction. future studies are planned to use our method in improving the sensitivity of ev-based assays and increase the rna yield to facilitate transcriptome sequencing. funding: this work was funded by grants from randy shaver community and research fund, minnetonka, mn. exosomes drive medulloblastoma metastasis in a mmp and emmprin dependent manner introduction: recurrent/metastatic medulloblastoma (mb) is a devastating disease with an abysmal prognosis of less than % -year survival. the secretion of extracellular vesicles (evs) has emerged as a pivotal mediator for communication in the tumour microenvironment during metastasis. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the blood-brain-barrier. matrix metalloproteinases (mmps) are enzymes secreted by tumour cells that can potentiate their dissemination by modification of the extracellular matrix. we hypothesise that exosomal mmp and its inducer emmprin could enhance metastasis of mb. methods: proliferation, invasion and migration assays were used to evaluate the phenotypic behaviour of primary cell lines pre-treated with metastatic tumour cell-derived exosomes. gelatin zymography and western blotting were performed to confirm mmp functional activity in cell lines and exosomes. nanoscale flow cytometry was used to measure surface exosomal emmprin levels. exosomal mmp and emmprin were modulated at the rna level. results: number of exosomes is directly related to the migratory behaviour of parental mb cell lines (p < . ). notably, functional exosomal mmp and emmprin levels also correlate with this. furthermore, exosomes from metastatic cell lines conferred enhanced migration and invasion on their matched isogenic primary (non-metastatic) cell line pair bỹ . -fold (p < . ). exosomes from metastatic cell lines also conferred increased migration on poorly migratory foetal neuronal stem cells. summary/conclusion: together this data suggests that exosomal mmp and emmprin may promote medulloblastoma metastasis and supports analysis of exosomal mmp and emmprin levels in patient cerebral spinal fluid samples. introduction: exosomes secreted from cancer cells harbour the potential to regulate intracellular signalling and promote metastasis. wherein, metastasis suppressor genes (msgs) play a pivotal role in regulating such signalling cascades. however, the regulation gets hampered due to low expression of msgs under metastatic conditions. nm -h , product of first identified metastasis suppressor gene nme , is significantly downregulated under metastatic conditions. nm -h serves as a regulator of small gtpases. several evidences have highlighted an involvement of small gtpases (such as rab , rab and rab ) in the biogenesis of exosomes. in addition, bacterial homolog of nm has been shown to interact with rab and rab . however, experimental evidence supporting a relationship between exosomes and nm -h is lacking. our current focus is to deduce the relationship between exosomes and msgs. methods: breast cancer cell lines were used to assess the effect of exosomes isolated from highly metastatic cells (mda-mb- cells) on lower/non metastatic cells (mcf- cells). nme was overexpressed in mda-mb- cells and subsequently used to isolate exosome fractions. equivalent amount of isolated exosome fractions from mda-mb- cells and mda-mb- /nme cells were utilized to access their effect on migration and difference in exosome markers. results: we observed an enrichment of nm -h in the exosomes isolated from mda-mb- cells upon overexpression of nme . proteinase k protection assay confirmed the packaging of nm -h inside the exosomes isolated from mda-mb- /nme cells and excluded the possibility of membrane association of nm -h . additionally, overexpression of nm -h led to a significant reduction in the ability of mda-mb- exosomes to stimulate movement of mcf- cells as confirmed by wound healing assays. our data also highlights a clear reduction in the protein levels of exosome markers such as cd , cd and alix in the exosome fraction isolated from mda-mb- /nme cells as compared to mda-mb- cells. interestingly, rab a, a protein involved in the endosome-lysosome fusion was also present in lower amount in the exosomes isolated from nm -h overexpressing cells. summary/conclusion: our data highlights an antimigratory effect of nm -h via exosomes. these findings support a regulatory role of nm -h in the packaging or release of exosomes in highly metastatic breast cancer cells, and further suggest that metastasis suppressor proteins may be involved in the regulation or packaging of exosomes. additional studies will be required to decipher the downstream signalling of nm -h which affects the biogenesis of exosomes as well as to assess the effect of nm -h overexpression on the content of exosomes. these insights could help us delineate the complex exosome biogenesis pathway and provide new potential drug targets for exosome regulation. introduction: exosomes (exs) are emerging as novel players in the beneficial effects induced by exercise on vascular diseases. our recent study has revealed that moderate exercise enhances the function of circulating endothelial progenitor cell-derived exosomes (cepc-exs) on protecting endothelial cells against hypoxia injury. in this study, we aimed to investigate whether exercise-regulated cepc-exs contribute to the beneficial effects of exercise on ischaemic stroke (is). methods: c bl/ mice performed moderate treadmill exercise ( m/min, -wks) before is induced by middle cerebral artery occlusion surgery. acute injury was evaluated at day by determining neurologic deficit, infarct volume, cell apoptosis in the penumbra and neurologic recovery was assessed by determining angiogenesis/neurogenesis, sensorimotor functions at day . the correlations of cepc-exs and their carried mir- with neurological parameters were analysed. the underlying mechanism of the effects of cepc-exs isolated from exercised mice was explored in a hypoxia neuron model. cellular mir- level, apoptosis, axon growth ability and gene expressions (cas- , bdnf and akt) were measured. results: ) exercised mice had a smaller infarct volume on day , which was associated with decreased cell apoptosis and cleaved cas- level, and a higher microvessel density than those in control; ) the elevated cepc-ex level positively correlated with tepc-exs in ischaemic brain of exercised mice on day . the upregulated mir- level positively correlated with the numbers of tepc-exs in ischaemic brain; ) the numbers of cepc-exs and their carried mir- level negatively correlated with the infarct volume, cell apoptosis and positively correlated with the microvessel density in the peri-infarct area on day ; ) exercised mice had decreased infarct volume, increased microvessel density, promoted angiogenesis/neurogenesis and improved sensorimotor functions on day , accompanying with upregulated levels of bdnf, p-trkb/trkb and p-akt/akt; ) cepc-exs of exercised mice protected neurons against hypoxia-induced apoptosis and compromised axon growth ability which were blocked by mir- and pi k inhibitors. summary/conclusion: our data suggest that the protective effects of moderate exercise intervention on the brain against mcao-induced ischaemic injury are ascribed to cepc-exs and their carried mir- . funding: this work was supported by american heart association ( post ) and nih ( r ns ). syndecan- regulates alveolar type epithelial cell senescence mediating through extracellular vesicles during lung fibroproliferation tanyalak parimon a , changfu yao a , adam aziz a , stephanie bora a , marilia zuttion zuttion a , dianhu jiang a , melanie koenigshoff b , cory hogaboam a , paul nobel a , barry stripp a and peter chen a a cedars-sinai medical center, los angeles, usa; b university of colorado, denver, usa introduction: alveolar type epithelial cell (at ) senescence is implicated in the pathogenesis of lung fibrosis, a progressive fatal condition. syndecan- , a heparan sulphate proteoglycan, is overexpressed by at cells of human idiopathic pulmonary fibrosis (ipf) and bleomycin-injured wt mice and the overexpression of syndecan- is profibrotic. moreover, syndecan- deficient (sdc -/-) mice had less lung fibrosis after bleomycin injury. we reported that extracellular vesicles (evs) in bronchoalveolar lavage (bal) of bleomycin-injured wt mice augmented lung fibrosis whereas the sdc -/--bal-evs attenuated the process. moreover, wt-bal-evs expressed lower level of anti-fibrotic mirnas (mir- b- p, − - p, − - p, and − - p) compared to the sdc -/-bal-evs. these mirnas targeted genes in the cellular senescence pathway indicating that syndecan- altered microrna profiles in the bal-evs to promote cellular senescence during lung fibrogenesis. we investigate how syndecan- regulates at senescence through evs. methods: bleomycin was intratracheally given into wt and sdc -/-mice. at day , lungs were processed for single-cell rna sequencing (scrnaseq) and western blot (wb). evs were isolated using ultrafiltration centrifugation method. human (a ) and mouse (mle- ) lung epithelial cell lines were used for in vitro experiments. results: scrnaseq analysis indicated while bleomycin stimulated an overexpression of cellular senescencespecific genes on at cells of wt mice, these genes were significantly downregulated on sdc -/-at cells. senescence proteins, p and p , were also less expressed in the lungs of sdc -/-than of the wt mice by wb. to determine the functional effects of evs in bal, a cells were treated with human ipf or control lung wash-evs and evaluated for beta-galactosidase activity. we found that ipf-evs markedly increased beta-galactosidase enzymatic activity. corroborating with these data, bleomycin-injured bal-wt-evs also significantly upregulated senescence marker, p , by wb on mle cells whereas sdc -/--bal-evs inhibited p expression. summary/conclusion: our data indicate that syndecan- regulates lung fibrosis through the senescence signalling pathway on at cells. furthermore, syndecan- controls at senescence mediating through extracellular vesicles in the bal. lastly, the most likely cargo molecules mediating this process are micrornas. immortalized cardiosphere-derived cell ev-associated pirna, imev-pi, protects against ischaemic injury in the heart alessandra ciullo, ahmed ibrahim, liang li, chang li, weixin liu and eduardo marbán smidt heart institute, cedars sinai medical center, los angeles, usa introduction: cardiosphere-derived cells (cdcs) are a population of heart-derived progenitors with demonstrated therapeutic efficacy in preclinical and clinical settings. cdcs function by secreting extracellular vesicles (evs), lipid-bilayer nanoparticles laden with bioactive molecules. recently our group developed a strategy for immortalizing cdcs (imcdc) that retains their therapeutic potential and enhances cdc function indirectly through their secreted evs. imcdc show a different rna content(mirna, mrna, rrna, trna and pirna) compared to primary cdc. in particular, we focus on piwi rnas (pirnas), small rnas bound by piwi proteins, important regulators of both the epigenome and transcriptome. we seek to explore the role of a pirna highly enriched in imcdc-evs (imev-pi). methods: evs are prepared by conditioning cells for hrs in serum-free basal media, in hypoxic culture. after hrs conditioned medium is cleared of cellular debris and evs isolated using ultrafiltration by centrifugation (ufc). fractions were analysed in terms of particle size, number, and concentration and pirna content. in vitro, bone marrow derived-macrophages (bmdm) were exposed to imcdc-ev, imev-pi and control and transcriptomic profile and potentially activated pathways were assessed. in vivo, - week-old wistar-kyoto female rats received ^ imcdc-ev, imev-pi, scramble or vehicle intracoronary minutes after ischaemiareperfusion(i/r). cardiac troponin i levels, scar size and monocytes were assessed at and hrs. results: by small-rna sequencing analysis we found that pirnas are enriched in both cdc-ev and imcdc-ev. imcdc show a different pirna composition compared to primary cdc. imev-pi was identified as one of the most highly-expressed non-coding rnas (the number of reads were x higher in imcdc-ev compared to cdc-ev). in vitro, imexo-pi-conditioned bmdm exhibit a different transcriptomic profile compared with control, with upregulation of pathways involved in the inflammatory response, cell death, and cell-to cell signalling. in vivo, imev-pi is cardioprotective, as shown by reduced scar size and lower cardiac troponin levels compared to vehicleand scramble-injected animals at hrs post i/r. imev-pi only minimally alters neutrophil counts profile in blood but it alters monocytes profile with a decreased number at hrs and an increase at hrs. summary/conclusion: we posit that imev-pi is a key determinant of imcdc-ev therapeutic efficacy. our results indicate that target cells may be macrophages/ monocytes, given that imev-pi exposure modifies their composition and mrna profile both in vitro and in vivo. introduction: extracellular vesicles (exosomes, evs) are cell membrane particles ( - nm) secreted by virtually cells. during intercellular communication in the body, secreted evs play crucial roles by carrying functional biomolecules (e.g., micrornas and enzymes) into other cells to affect cellular function, including disease progression and tissue regenerations. literature previously reported that the macropinocytosis pathway contributes greatly to the efficient cellular uptake of evs. the activation of growth factor receptors, such as epidermal growth factor receptor (egfr), induces macropinocytosis. in this study, we demonstrated the effects of evs on demal papilla and hair follicle regeneration. methods: identification of distinct nanoparticles and subsets of extracellular vesicles from umbilical cord blood stem cell by asymmetric flow field-flow fractionation. results: the effects of evs from umbilical cord blood stem cell on the propagation of demal papilla and hair follicle regeneration were observed. summary/conclusion: the enhancement of extracellular vesicles from umbilicalcord blood stem cell the propagation of demal papilla and hair follicle regeneration were observed and confirmed. mechanisms of host resistance to plasma membrane damage induced by pneumolysin attack introduction: bacterial pore-forming toxins (pfts) are major virulence factors produced by pathogens. pfts target host plasma membrane (pm) and create transmembrane pores, which allow uncontrolled flux of ions and small molecules across the pm disrupting cellular homoeostasis. to survive, cells display poorly understood repair mechanisms to recover the cell homoeostasis. several mechanisms were proposed to participate in cell recovery: exocytosis of cortical lysosomes; endocytosis of pfts pores; pm blebbing and shedding. methods: we used increasing concentrations of purified ply to intoxicate cells. pm permeability was assessed by flow cytometry using propidium iodide dye. cytoskeleton rearrangements were investigated by confocal immunofluorescence microscopy. extracellular vesicles released during pm repair were isolated by high-speed centrifugation and characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and mass spectrometry/ liquid chromatography analysis. results: ply triggers a complete reorganization of the actomyosin cytoskeleton inducing the formation of cortical actomyosin bundles at sites of pm remodelling. these structures assemble upon loss of pm integrity and disassemble as pm recovers. we detected the release of microvesicles during the recovery of pm integrity. vesicle population is heterogeneous with sizes ranging from to nm, with the majority of them measuring - nm. vesicle proteomic analysis revealed that they contain ply, suggesting they participate in pore removal, proteins involved in vesicle trafficking, pm repair and exosome biogenesis. summary/conclusion: our data demonstrate that cells are able to recover from the damage induced by sublytic concentrations of ply. actomyosin cytoskeleton undergo massive changes with the assembly of cortical bundles possibly at sites of pm damage. we showed that cells produced extracellular vesicles during the process of repair. we are now focusing on understanding the biogenesis of those vesicles and its importance during the process of repair. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms ( d static tissue culture flask vs d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in d t-flasks and d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both d and d culture systems. msc-ev were characterized according to misev guidelines, using techniques as nta, protein and lipid quantification, western blot, imaging and fourier-transform infrared spectroscopy (ftir). the results indicate that msc-ev derived from different sources/donors have similar size distribution, however, ev yields tend to be higher for the d culture system. of notice, several spectral regions were identified by ftir, enabling the detection of differences in the biomolecules present in msc-ev, msc-conditioned media and cells produced under different conditions. summary/conclusion: in summary, this study contributes to the establishment of a scalable process for msc-ev production. evaluation of three different isolation methods for small extracellular vesicles from human plasma in prostate cancer diagnosis introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies. methods: pca patients and age-matched healthy controls (hc) plasma (n = in each group) were used to isolate sevs with different isolation methods including commercial exoquick ultra kit, qev and qev size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd and cd commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination. results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev . furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods. summary/conclusion: qev method demonstrated better performance in isolating relatively pure sevs from human plasma; qev has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev but with the highest non-sev protein contaminations. people can choose higher sev content or higher sev purity according to the downstream analysis. moreover, sevs may also be used for treatment monitoring, as recent studies suggested that the expression levels of certain markers may change during therapy, reflecting tumour response. for cancer diagnostics and therapeutic purposes in clinical settings, it is important to have a device which allows multiplexed measurements, in order to scan a large number of markers simultaneously and compare the expression levels of different patients, or same patients at different treatment stages, in a time efficient manner. methods: herein, we propose a multiplexed platform for label-free detection and surface protein profiling of sevs. the technique is based on the electrokinetic phenomena of streaming current and zeta potential (\zeta*) and measures the\zeta* change upon sev binding on functionalized microcapillary surfaces. for the purpose, we used sevs derived from lung cancer cells. in its current form, the platform can measure up to channels simultaneously, however, it can be further expanded. results: having demonstrated that our electrokinetic sensor successfully detects sevs in a specific way, we tested its ability to measure the expression level of membrane proteins. the analysis showed that it could detect differences in the expressions of egfr on sevs, with a sensitivity of %. we then extended the platform for multiplexed analysis, by connecting and measuring four capillaries, functionalized with different capture probes, simultaneously. for the purpose, we targeted specific tumour markers, i.e. egfr, and exosomal tetraspanin family proteins, such as cd and cd . the results showed successful multiplexed ev detection. summary/conclusion: being the sensor suitable for multiplexed sev detection, we shall present our investigation on a set of pleural effusion samples collected from a cohort of lung-cancer patients with different genetic makeup. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd , cd and cd , it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: ) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and ) the assessment of subpopulation target enrichment relative to the population average by leveraging tim as an unbiased, lipid-based ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasis-associated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr (her +), t d and mcf- (er+pr+), bt and mda-mb- (triple negative) breast cancer cell lines, as well as five mda-mb- -derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her was broadly detected in her + skbr evs. interestingly, her -t d and mcf- evs also expressed her where it was highly enriched in its epcam+ subpopulations. itg α , β and β were only found in triple negative and organotropic evs with itg β and β differentially enriched based on the organotropism. the population average of mda-mb- and lung-tropic evs had high expression of itg β , where subpopulations of cd + evs showed positive enrichment while cd + and cd + evs showed negative enrichment. itg α , β and β were absent in the bone-tropic cd + ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb- evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified targets that bind to hs-gag chains, and also different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of ev-associated hspgs will result in attenuation of ev induction of a tumour-supporting fibroblast phenotype. introduction: ovarian cancer (oc) is the fifth leading cause of cancer-related death in women, partly due to difficulty in early diagnosis. extracellular vesicles (evs) show promise for use in early diagnostics of oc. here, evs from cervical mucus (cm) of ovarian cancer patients were used for discovery of oc biomarkers for diagnostics. machine learning was used to mine ev mirna data to develop an oc biomarker panel (validation via the cancer genome atlas). examination of the mirna targets reveal that the panel is a sufficiently accurate predictor of oc. methods: evs from the cm of patients ( highgrade serous, low-grade, benign) were isolated for small rna-sequencing. the top differentially expressed mirnas were used in a random forest and "voom" (variance modelling at the observational level) model. unsupervised approaches were used and then vetted against patient symptomology data. a tcga ovarian cancer dataset (n = ) was used for validation. results: an oc biomarker panel of micrornas (voom: . % accuracy; random forest: % accuracy) was generated. the panel consists of members from the mir- family and the mir- family, among others. the mirna targets are associated with molecular functions and pathways specific in oc progression. summary/conclusion: our method has identified ev mirna biomarkers that may be crucial for early, noninvasive detection of oc. data science has been used to develop a feedback system integrating biochemical experiments, smaller datasets, and previously available data to identify and verify a biomarker panel for oc diagnostics. introduction: liver disease has become a significant cause of morbidity and mortality among hiv patients. alcohol exposure can further exacerbate liver damage by activating hepatic stellate cells (hscs), leading to hepatic fibrosis or cirrhosis, often seen at all levels of alcohol exposure among people with hiv. due to the potentiating effects of alcohol on hiv-induced hepatocytes (hep) damage, as well as the effect of ethanol in hsc-mediated extracellular remodelling, it is imperative to understand the interplay of hep and hscs. here, we focus on the exosomes released by hiv-and ethanol exposed hep and how these exosomes modulate the functional behaviour of hscs. methods: human hepatocyte huh . cyp e [hepatoma cells stably transfected with cyp e designated as rlw cells] were infected with hiv in the presence or absence of alcohol metabolite, acetaldehyde using the acetaldehyde-generating system (ags). the conditioned medium was collected from groups of cells: untreated, hiv-, ags-and hiv+ags. quantification of exosomes number and size were evaluated with zetaview or nanosight and further characterized for exosome markers following the guideline from minimal information for studies of evs (misev ). the human hepatic stellate lx- cell line was exposed to hepatocyte-derived exosomes and assessed for the activation using pro-inflammatory markers il- β, il- , tnfα, and fibrotic markers acta , and timp using quantitative pcr. we also analysed exosome mirna content in primary human hepatocytes (phh), which potentially regulates the function of recipient cells by "programming" their inflammation/fibrosis status. the network analysis for mrna and mirna were carried out using gene ontology consortium, and mirror . and david bioinformatics resources . . results: ags treatment further enhanced the release of hiv-induced exosome from hepatocytes. size distribution assessed by zeta view or nanosight revealed that approximately - % of particles distributed in the range of to nm, with a peak at~ nm. enriched expression of hiv protein p was observed in fractions f -f . western blotting of hepatocytederived exosome demonstrated positivity for exosome-enriched proteins alix, tsg and cd specifically in f -f fractions and negative for endoplasmic reticulum protein calnexin. the uptake of hepatocytederived exosomes by hscs was apparent as demonstrated by immunofluorescence. the internalization of hepatocyte-exosome induced activation of hscs as evidenced by increased expression of pro-inflammatory il- β, il- , tnfα markers in the latter cells. summary/conclusion: we conclude that ags treatment in hiv-infected hepatocytes potentiates the release of exosomes, which, following uptake by the hscs, leads to their activation. funding: this work is supported by nih- r aa - a . antimicrobial peptide ll- induces neutrophil-derived extracellular vesicles with antibacterial potential and protects murine sepsis yumi kumagai, taisuke murakami, kyoko kuwahara and isao nagaoka juntendo university, bunkyo-ku, japan introduction: extracellular vesicles (evs) released from immune cells or other host cells upon microbial infection modulate the immune response and thereby regulate the infection. sepsis is a life-threatening multiple organ dysfunction caused by systemic dysregulated inflammatory response to infection. nevertheless, numerous therapeutic trails concerning immune dysfunction have still been disappointing outcomes. we have previously shown that ll- , a human cathelicidin antimicrobial peptide, improves the survival of caecal ligation and puncture (clp) septic mice. here, we investigated the induction of ev release by ll- and functions of ll- -induced evs in murine sepsis. methods: evs were isolated from peritoneal exudates of clp mice and the supernatant of ll- -stimulated mouse bone marrow neutrophils by differential centrifugation or size exclusion chromatography. isolated evs were analysed by flow cytometry, western blotting, and nano particle analysis. neutrophil-derived evs were injected into clp mice to assess the protective function of evs against septic mice. the antibacterial activity of evs was evaluated by incubating with escherichia coli. results: in clp mice, ll- augmented the level of evs. evs from ll- -injected clp mice contained higher amounts of neutrophil-derived antibacterial proteins (lactoferrin and cramp, cathelicidin-related antimicrobial peptide) and exhibited higher antibacterial activity compared to evs from pbs-injected clp mice. furthermore, ll- stimulated mouse bone marrow neutrophils to release evs with antibacterial potential, and administration of the ll- -induced evs reduced the bacterial load and improved the survival of clp mice. summary/conclusion: ll- induces the release of antimicrobial evs from neutrophils in clp mice, thereby reducing the bacterial load and protecting mice from lethal septic condition. identification of mirna profiles of serum exosomes in active tuberculosis introduction: tuberculosis (tb) has exceeded hiv as the most lethal infectious disease globally for two consecutive years, mainly due to difficulties in achieving early and definitive diagnosis, and timely treatment. exosomes carrying rna, particularly mirna, have demonstrated their functional and diagnostic potential in diseases including tb. however, few published studies have explored whether exosomal mirnas could be used for diagnosis of tb. thus, more systematic and comprehensive study of exosomal mirnas with regard to their potential as non-invasive tb biomarkers is still urgently needed. methods: we searched the gene expression omnibus database for datasets published before december , and performed meta-analysis on available exosomal mirna profile data for healthy control (hc) and active tb clinical specimens . reprocessing next generation sequencing data under uniform parameters and utilizing state-of-the-art bioinformatics analysis. results: we identified many distinct up-regulated and down-regulated differentially expressed exosomal mirna across multiple studies, and further screened the top , which might provide a potential panel for differentiation of hc and tb. we classified all differentially expressed mirnas into six expression patterns and identified two persistently up-regulated mirna (hsa-mir- - p, and hsa-mir- - p) as potential markers during tb progression. moreover, the differential expressed exosomal genes that we screened from the datasets were consistent with the genes overlapped with predicted mrna targets of differentially expressed mirna. pathway and function analysis further demonstrated down-regulated signalling pathways/immune response and up-regulated metabolism and apoptosis/necrosis. introduction: trypanosoma cruzi is a protozoan parasite that causes chagas disease, a relevant source of morbidity in latin america, which has spread to many countries as result of immigration of the people from endemic areas. many studies have been showed that trypomastigote forms of t. cruzi release extracellular vesicles (ev) that increase parasite infection. objectives. here, we aim to test if previous immunization with evs in adjuvant can generate a protective immune response by decreasing the effects of evs in experimental chagas disease. methods: female balb/c mice were immunized by intra peritoneal (ip) administration with × or evs isolated from trypomastigotes forms, with aluminium hydroxide adjuvant (aloh). injections were administered intravenous in doses during days ( days interval). after immunization, mice were infected intra-peritoneally with trypomastigotes forms. parasitaemia was quantified by counting motile parasites in fresh blood sample drawn from lateral tail veins. mortality and weight were analysed during the infection. in control group, the mice were immunized with aioh. results: the immunization with evs with aloh decreased the blood parasitaemia and the animals survived, while all animals died in the group aloh alone. the animals immunized with evs had an increase of f / + cd b+ and cd /cd expression in cells isolated from the peritoneum. summary/conclusion: these results indicate that t. cruzi ev antigens can induce an immune response that controls the development and establishment of the experimental chagas disease. introduction: acinetobacter baumannii (ab) is a nosocomial pathogen, of major concern due to its multidrug resistance (mdr) and the recent appearance of hyper-virulent strains in the clinical setting. the world health organization included ab as a critical priority pathogen for the development of novel antibiotics. ab pathogenesis is associated with a multitude of potential virulence factors (vf) that remain poorly characterized. there is growing evidence that outer membrane vesicles (omv) are used as vehicles to transport bacterial proteins that contribute to set up the conditions for the infections. in the present work we studied the physiopathology of mdr ab. we focused on the contribution of non-characterized outer membrane proteins (omps) associated to omvs, with special focus on lipoproteins (lp). methods: we conducted a bioinformatic prediction using available datasets to construct a list of omv-associated omps putatively acting as vf in ab . seven genes were selected and the corresponding mutants were obtained from manoil lab collection. physiological analyses of the mutants were performed, and the involvement of the selected proteins in ab pathogenesis was evaluated by adherence, invasion, and cytotoxicity assays on human lung cells a . results: biochemical analysis indicated similar growth rates in rich media, as well as similar levels of omv production for all the mutants as compared to wt. also, no differences in susceptibility to chaotropic agents were observed, indicating no alteration of the om function as a general permeability barrier. all mutants similarly reduced a cell viability, but to a lesser extent than the wt. moreover, three of them exhibited less adhesion and invasion compared to the wt, and omv isolated from these mutants displayed variable levels of cytotoxicity. summary/conclusion: these results suggest roles for the mutant gene products in ab pathogenesis and contribute to the better understanding of ab virulence mechanisms, revealing novel possible targets for therapeutic development. funding: agencia nacional de promoción científica y tecnológica (anpcyt, pict - ) medicine, nanfang hospital, southern medical university, guangzhou, , china, guangzhou, china (people's republic); d zhujiang hospital, southern medical university, guangzhou, china, guangzhou, china (people's republic) introduction: talaromyces marneffei (t. marneffei) grows as a mycelial form in the environment but multiplies rapidly as a yeast form in the host and within macrophages. the yeast can cause disseminated and progressive infections or lethal talaromycosis. but the mechanisms of pathogenicity of t. marneffei are poorly understood. fungal extracellular vesicles (evs) have previously been shown to transmit a proinflammatory message to macrophages. however, the characteristics and effects of t. marneffei evs on the progress of infection have not yet been investigated. methods: in this study, evs of t. marneffei yeasts were isolated by ultracentrifugation method. evs were detected and confirmed by electron microscopy and nanoparticle tracking analysis (nta). the raw . murine macrophages were incubated with the t. marneffei vesicles to observe the changes of macrophage morphology and function, especially in inflammatory response. the proteins, dnas, rnas of t. marneffei vesicles were respectively removed with protease, dnase and rnase. all treated evs were used to incubate with murine macrophages observe the effect on macrophages in inflammatory response. results: we observed that evs secreted by t. marneffei have a typical spherical shape with a diameter of to nm. t. marneffei evs were internalized by raw . murine macrophages and promoted the production of no and proinflammatory cytokine by macrophages in a dose-dependent manner. t. marneffei evs stimulate macrophages to generate reactive oxygen species (ros). addition of t. marneffei evs to macrophages also promoted transcription of the m -polarization marker cd and diminish that of the m markers cd . incubation of t. marneffei vesicles with murine macrophages resulted in increased levels of extracellular interleukin- β(il- β), interleukin- (il- ) and interleukin- (il- ). the proinflammatory effect of vesicles was weakened when the proteins of the vesicles were destroyed. in contrast, no similar changes were observed in degraded dna and rna. summary/conclusion: our results indicate that the extracellular vesicles of t. marneffei can stimulate macrophage towards to m polarization phenotype and promote proinflammatory function. plasma-derived extracellular vesicles as potential biomarkers in chronic chagas disease patients introduction: chagas disease (cd), caused by the parasite trypanosoma cruzi (t. cruzi), is a neglected tropical disease affecting about million people worldwide. currently, one of the main clinical problems is the lack of effective biomarkers for therapeutic response and disease prognosis during chronic infections. in that context, extracellular vesicles (evs) are raising attention as novel, minimally invasive, and inexpensive method for diagnostic and screening of diseases, as well as a new source to identify new biomarkers. the main objective of this study is to use evs derived from biological fluids of chronic cd patients for identifying novel biomarkers, specifically in the context of therapeutic response and disease prognosis. methods: plasma, saliva and urine from a cohort of chronic cd patients are being collected before and at the end of benznidazole treatment. as negative controls, healthy donors have been also included. the purification and characterization of the evs was performed by size exclusion chromatography, followed by nanoparticle tracking analysis, bead-based flow cytometry assay and transmission electron microscopy. a proteomic analysis of the evs was also performed. results: proteins associated with evs secreted by infective t. cruzi have been previously identified in cell culture, but never in human samples. our results, based on the analysis of a single heart-transplanted patient with chronic cd, showed the presence of t. cruzi and human proteins specifically associated with plasma-derived evs. noticeably, several human and parasite proteins identified in evs obtained from plasma samples, were present or upregulated before chemotherapy and were absent or downregulated following treatment. currently, proteomics analyses are being performed with higher numbers of cd plasma samples. summary/conclusion: to the best of our knowledge, this is the first proteomic profiling of plasma-derived evs from a heart-transplanted patient with chronic cd. these results thus open the possibility of using evs from biological fluids as a tool for the identification of new biomarker candidates in chronic cd. these biomarkers are essential for assessing disease introduction: eukaryotic cells communicate with one another through multiple pathways. an established route of communication between eukaryotic cells is via the production of a range of different membrane bound signalling "packages", called extracellular vesicles (evs). evs are produced by all domains of life and carry proteins, nucleic acid (rna and dna), and other biological material, travelling between cells and around the body to deliver a range of chemical messages. bacteria can also produce evs that communicate with each other to coordinate population behaviour, as well as with eukaryotic cells to stimulate host defence or induce tolerance. here i investigate the poorly explored axis where evs are the vehicle for communication between eukaryotic cells and bacteria. methods: as a first step, i have isolated evs from tissue cultured eukaryotic cells grown in advanced rpmi media with minimal ev-depleted fbs. nanoevs were isolated from spent culture media using sequential centrifugation ( , × g, , × g) and concentration ( kda filter) before purifying using size exclusion chromatography columns. nanoev-rich fractions were pooled based on particle (nanoparticle tracking analysis) and protein quantity data. nanoevs were characterised by electron microscopy and expression of exosomal markers. eukaryotic nanoevs were then characterised in their effect upon the growth of escherichia coli as a model bacterium, also grown in tissue culture media to mimic relevant in vivo conditions. results: further experiments with increased dosages are required to determine the effect of human evs on bacteria. summary/conclusion: our work will investigate whether human evs communicate with the resident and pathogenic microbiota, while examining the mechanisms behind this communication. escherichia coli pathogenic bacteria commensal bacteria hydrogen sulphide (h s) derived extracellular vesicles: a potential protective role in response to respiratory syncytial virus (rsv) infection methods: evs were isolated from untreated (control evs) and gyy treated (gyy-evs) a cells, a human alveolar type ii-like epithelial cell line. evs were purified using a two-step enrichment procedure. evs were characterized using particle sizing (size and concentration) and western blot for the ev markers. electron microscopy and immunofluorescence staining were used to investigate presence of multivesicular bodies (mvbs), evs precursors, in both groups. recipient a cells were cultured for hours in the presence or absence of control-or gyy-evs, then infected with rsv for hours. viral titres by plaque assay were measured in recipient infected a cells. results: we confirmed the presence and purity of our evs. we found that gyy reduced the particles number of evs, but did not change ev size. a cells treated with gyy showed an accumulation of mvbs/lysosomes-like structures, as well as an increase in cd expression, a mvbs marker, compared to untreated cells. recipient a cells treated with gyy-evs showed lower viral replication than control ev-treated cells in response to rsv infection. we are currently investigating the potential mechanism for this observation and characterizing the rna cargo composition of gyy-evs. summary/conclusion: no vaccine or effective treatment is currently available for rsv. cellular pretreatment with gyy-evs reduced the rsv replication in airway epithelial recipient cells, suggesting that h s could exert its antiviral activity in the context of rsv infection potentially through modulation of ev composition. therefore, gyy-evs could represent a future novel pharmacological approach for ameliorating virus-induced lung disease. effects of extracellular vesicle-mediated transmission on reoviridae infection results: taken together, these data suggest that multiple particles of reovirus and rotavirus egress in large, virus-modulated evs, and that transmission in evs increases segment complementation compared to transmission as free particles. summary/conclusion: these discoveries may be broadly applicable to viruses that travel in evs and will contribute to general principles of virus transmission and diversification. continued studies will illuminate the specific cellular pathways reovirus and rotavirus utilize for successful egress. these pathways may prove to be critical targets for the improvement of vaccines and oncolytic therapy. multiparameter flow cytometry analysis of the human spleen and its interaction with plasma-derived evs from plasmodium vivax patients introduction: the spleen is a secondary lymph organ that filters blood and elicits immune responses against blood-borne pathogens, such as malaria parasites. extracellular vesicles (evs) are membrane-bound particles involved in intercellular communication. evs play several roles in malaria ranging from modulation of immune responses to induction of vascular alterations. here, we report the first integrated characterization of human spleen cells using multiparameter flow cytometry (mfc) describing subpopulations of splenic leukocytes and red blood cells (rbcs), and studied their interaction with plasma-derived evs from p. vivax patients (pvevs). methods: human spleens were obtained from organ transplantation donors. myeloid, lymphoid, erythroid and haematopoietic stem cells (hscs) were immunophenotyped by mfc. t cells, dendritic cells (dcs) and rbcs were enriched by density centrifugation and immunomagnetic isolation. pvevs and healthy donors evs (hevs) were purified by size-exclusion chromatography (sec) and characterized by bead-based flow cytometry. enriched evs were labelled with fluorescent lipophilic dyes and incubated with total splenocytes or enriched populations. evs-cells interaction was assessed by flow cytometry. results: human spleen immunophenotyping showed that cd + cells included b ( %), cd + t ( %), cd + t ( %), nk ( %) and nkt ( %) lymphocytes. myeloid cells comprised neutrophils ( %), monocytes ( %) and dcs ( . %). erythrocytes represented % whereas, unexpectedly, reticulocytes were . % of total cells. in addition, we also detected hscs, which accounted for . %. sec separated evs from the bulk of soluble plasma proteins as shown by the enrichment of cd , cd l and cd markers. interaction studies showed an increased proportion of t cells (cd + -fold and cd + -fold), monocytes ( . -fold) , b cells ( . -fold) and erythrocytes (threefold) interacting with pvevs as compared to hevs. summary/conclusion: the integrated cellular analysis of the human spleen and the methodology employed here allowed in vitro interaction studies of human spleen cells and evs. a larger proportion of monocytes, t and b lymphocytes as well as erythrocytes was found to interact with pvevs compared to hevs. future functional studies of these interactions can unveil pathophysiological processes involving the spleen in vivax malaria. neuroblastoma-secreted exosomes carrying mir- promote osteogenic differentiation of bone marrow mesenchymal stromal cells introduction: bone marrow (bm) is the major target organ for neuroblastoma (nb) metastasis and its involvement is associated with poor outcome. yet, the mechanism by which nb cells invade bm is largely unknown. tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (mscs) have been recognized as a fundamental part of the associated tumour stroma. here, we explore the potential role of nb-derived exosomes in induction of a pro-osteogenic phenotype on bm-mscs. introduction: extracellular vesicles (evs) are nanosized particles delimited by a lipid bilayer which transfer functional molecular cargos from the cells of origin to target cells. this intercellular crosstalk controls both physiological and pathological conditions. given their presence in body fluids and their characteristics, these nanocarriers might be potentially used in diagnostics and/or therapy. breast cancer is the most frequently diagnosed malignancy and ranks as the leading cause of cancer mortality in women worldwide; the triple negative breast cancer, in particular, is the most aggressive subtype with a poor prognosis. since it is recognized that cell stiffness of cancer cells play a crucial role during the metastatic spreading, we set ourselves the goal of clarify the effects and the activity of small-evs (i.e. with a diameter below nm) in metastatic breast cancer, with a special attention on their correlation with the biomechanical properties of cells. methods: functional assays were performed on the non-invasive mcf breast cancer cell line, before and after the cellular uptake of small-evs originating from the invasive mda-mb- triple negative breast cancer cell line. the mechanical properties (cell stiffness, cytoskeleton organization and focal adhesions) of mcf cells were investigated before and after the vesicle uptake. results: the uptake of small-evs derived from mda-mb- significantly reduces the young's modulus values of mcf cell line making them more invasive. moreover actin and focal adhesion variations were observed in mcf cells before and after small-ev's uptake, suggesting a molecular rearrangement inside mcf cells upon uptake. summary/conclusion: our results evidence that small-evs play a key role in altering biomechanical properties of target cells and underline their relevance in cell-cell crosstalk. our approach is very promising to identify new molecular mechanisms through which evs perform their oncogenic function. stratification of angiogenic or non-angiogenic lesions in colorectal cancer liver metastases patients using extracellular vesicle mirna introduction: colorectal carcinoma (crc) is the second leading cause of cancer death in the western world. over % of the crc patients develop liver metastasis (lm) and % will die from metastatic disease. in the current clinical setting, liver resection provides the only possible cure, but only % of crclm patients are resectable. the combination of angiogenic inhibitors with chemotherapy is used to downsize crclm with the goal of converting unresectable patients to resectable ones. however, only - % of these patients can be successfully converted to a resectable state. we have no way of identifying those crclm patients that would respond/benefit to the addition of anti-angiogenic therapies (e.g. bevacizumab: bev)). proper stratification of patients into angiogenic inhibitor responders and non-responders will permit a proper assessment of the efficacy of angiogenic inhibitors. crclm forms distinct histopathological growth patterns (hgp): angiogenic (desmoplastic) and nonangiogenic (replacement) hgp. we demonstrated that crclm patients with predominant angiogenic lesions receiving bev plus chemotherapy have a more than double -year overall survival compared to patients with non-angiogenic lesions. therefore, nonangiogenic lesions do not respond to angiogenic inhibitors. our study focuses on stratifying angiogenic vs non-angiogenic lesions of crclm through extracellular vesicle mirnas. we are using two approaches in the selection of mirnas to target: . text mining of published ev mirna from crclm patients; and . differentially expressed mirnas present in tumour tissue from both lesion types, we have obtained by sequencing - patients. these two strategies will generate a list of mirnas that we will target using qpcr on plasma-derived ev mirna from the patients used in approach , where we have classified the lesions in the patients. preliminary data on patients will be presented. methods: ev isolation was performed using the gold standard centrifugation method. rnaseq and qpcr are used to generate the expression profile for angiogenic vs non-angiogenic type of crclm. results: the research is under progress. summary/conclusion: the research is under progress. the introduction: it is known that bone metastasis causes a reduction in the quality of life of cancer patients due to fractures and nerve compression. therefore, it is important to elucidate the mechanism of bone metastasis and develop new treatments. metastatic bone tumours occur at particularly high rates in cancers of the prostate, breast, and lung. in this study, we focused on extracellular vesicles (evs) in bone metastasis, and investigated that the role of evs derived from cancer cells in osteolysis. methods: the prostate, breast, and lung cancer cellderived evs were added to osteoclast precursors with rankls. the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase (trap) stain and by measuring the expression level of osteoclast markers using by qrt-pcr. a proteome analysis (lc-ms/ms) and sirna approaches were used to identify molecules which are responsible for promotion of osteoclast differentiation in the prostate cancer cellderived evs. to investigate whether the molecules are suitable for the detection of bone metastasis in serum evs, we isolated evs from serum of prostate cancer patients, and analysed the protein level of the molecules by western blot analysis. results: we found that the prostate cancer and lung cancer-derived evs significantly promoted the rankl-stimulated osteoclast differentiation. our analysis revealed that cub domain-containing protein (cdcp ), which is a membrane protein on the prostate cancer cell-derived evs, was responsible for promotion of osteoclast differentiation. moreover, cdcp was markedly detected in the evs-derived from serum of prostate cancer patients who had bone metastasis than that of normal subjects. we also found that cdcp exits on the breast and lung cancer cell-derived evs. summary/conclusion: we showed that the evsderived from bone metastatic tumours have a role in activation of osteoclastogenesis. moreover, we revealed that cdcp in the evs is responsible for promoting of osteoclast differentiation. these evs could be the novel diagnostic and therapeutic target for bone metastasis. increased expression of chemokine receptor cxcr in non-invasive colorectal cancer cells after incorporation of platelet-derived extracellular vesicles. introduction: blood platelets and platelet-derived extracellular vesicles (p-evs) play a crucial role in tumour growth and metastasis. p-evs, also referred to as platelet microparticles, are recognized as a carrier for proteins and nucleic acids that control cell-to-cell communication, mediate the formation of metastatic niches and affect tumour invasion and metastasis. among the other factors, p-evs contain the chemokine receptor cxcr , known as a co-receptor for hiv entry but also regarded as important in cancer development due to the importance of cxcr /cxcl signalling. overexpression of cxcr was reported in various, especially in invasive cancers, including colorectal cancer (crc). crc, the third most commonly diagnosed cancer, is usually diagnosed at the late stage and patient's death is mainly related to metastasis. increased levels of cxcr has been reported as a poor prognostic factor for survival of crc patients and its blocking has been suggested as therapeutic approach. the aim of this study was to analyse the effect of p-evs on the levels of cxcr in crc cells on various epithelial-to-mesenchymal transition stage. methods: we used crc cell lines ht and sw , which represent distant invasive potential and different phenotypes, epithelial and strongly mesenchymal, respectively. p-evs were isolated from outdated concentrates of human blood platelets after activation by thrombin in the presence of calcium ions, by subsequent centrifugation and ultracentrifugation. the p-evs were labelled using pkh fluorescent dye to visualize their uptake into cell lines by confocal microscopy. we also quantified the levels of cxcr in ht and sw by western blot analysis. the effect of p-evs uptake on the migration of crc cells was studied by "wound healing" method. results: we found that the levels of cxcr in crc lines used in the study were correlated with their emt stage. we show here that p-evs released by activated platelets were incorporated into both ht and sw cell lines. the expression of cxcr in ht was increased after the uptake of p-evs. additionally we observed that migration rate of ht cells with incorporated p-evs was elevated as compared to control cells. summary/conclusion: we posit that circulating p-evs can be incorporated into yet not invasive crc cells to significantly increase the level of cxcr receptors and that may lead to the their more invasive characteristics. introduction: for cancer therapy it is important to identify markers and key processes induced during cancer progression. one of them is epithelialmesenchymal transition (emt) which is associated with cell acquisition of invasiveness, stem cell characteristics and resistance to apoptosis and therapy. also the extracellular vesicles (evs) released from tumour cells, which can be taken up by cells constituting pre-metastatic niches, can alter cancer progression by promoting cells' reprogramming. our group has recently reported that snail transcription factor, a key factor of emt, when overexpressed in crc ht cells, drives their early emt and alters the expression of microrna (mirs). in the present study we analysed the mirs profile of evs released from those cells. methods: evs from three ht clones stably overexpressing snail and from control ht -pcdna were isolated by differential centrifugation and ultracentrifugation of conditioned media after h of culturing in serum-free medium. total rna was isolated and nextgeneration sequencing (ngs) analysis of the mirnas was performed followed by gene ontology ( introduction: prostate cancer (pca) is the most common malignant tumour in male urinary system and osteoblastic bone metastasis is the most observed metastasis in prostate cancer patients. it has been demonstrated that circulating micrornas contained in extracellular vesicles are potential early biomarkers and therapy targets for many diseases. however, the potential role of micrornas in prostate cancer bone metastasis, is not yet to be fully explored. methods: after isolation and purification evs using ultracentrifugation from conditioned media of bone metastatic co-opting prostate cancer cells and normal cells, total rna was extracted. subsequent to library preparation and small rna-seq, differential gene expression analysis was performed. data were filtered by mean mirna expression of ≥ reads, two fold up or down regulation between . − . and adjusted pvalue ≤ . . the uptake of pca-sevs was performed. three candidate mirnas (has-mir- c- p; has-mir- ; has-mir- - p) were internalized and osteoblast differentiation were detected by qpcr, histochemical staining and protein activity detection. results: total reads of mirnas in bone metastatic co-opting pca-evs exceeded significantly than that in normal evs (p < . ), indicating that mirnas delivered by pca cells play critical role in pca bone metastasis. pca-cm enhanced osteoblast differentiation and can be reversed by gw . the uptake of pca-evs by mc t -e was efficient. the high expression of the three candidate mirnas in pca-evs was verified by qpcr. all the three candidate mirnas promoted osteogenesis, verified by mrna expression of osteoblastic markers (alp, ocn, runx , osx), alp activity, alp staining and aliza red s staining. summary/conclusion: these findings suggest that mirna cargos in pca-evs play a pivotal role in the development of osteoblastic bone metastasis of pca, which can be potential early biomarkers and therapy targets for prostate cancer bone metastasis. funding: this work was supported by grants from the national natural science foundation of china ( ); xijing hospital science and technology foundation project (xjzt ptk ). introduction: retinoblastoma (rb) is the most common intraocular cancer of childhood. despite recent advances in conservative treatment have greatly improved the visual outcome, local tumour control remain difficult in presence of massive vitreous seeding. thus, the identification of new biomarkers is crucial to design more effective therapeutic approaches. traditional biopsy has long been considered unsafe in rb, due to the risk of extraocular spread. exosomes, nano-sized vesicles containing nucleic acids and proteins, represent an interesting alternative to detect tumour-associated biomarkers. the aim of this study was to determine the protein signature of exosomes derived from rb tumours (rbt) and vitreous seeding (rbvs) primary cell lines. methods: exosomes from rbt (hsjd-rbt , hsjd-rbt , hsjd-rbt , hsjd-rbt ) and rbvs (hsjd-rbvs , hsjd-rbvs , hsjd-rbvs ) cell lines were isolated by high speed ultracentrifugation. vesicles number and size were confirmed by nanosight and scanning electron microscopy. protein content was analysed by bicinchonic-acid assay and high resolution mass spectrometry. results: a total of proteins were identified. among these, and were expressed in exosomes rbt and one rbvs group respectively. gene enrichment analysis of exclusively and differentially expressed proteins and network analysis identified identified in rbvs exosomes upregulated proteins specifically related to invasion and metastasis such as proteins involved in extracellular matrix (ecm) remodelling and interaction, resistance to anoikis and metabolism/catabolism of glucose and aminoacids. summary/conclusion: in conclusion, in this study, we isolated exosomes from rb primary tumour and vitreous seeding cell lines and characterized their content with a proteomic approach. this is the first evidence describing a proteomic exosome signature specifically associated with vitreous seeding in rb. this characterization may represent a starting point for future analyses that allow defining exosomal markers as promising diagnostic and potential prognostic markers in rb as well as therapeutic targets. activation of hepatic stellate cells by extracellular vesicles released by uveal melanoma cells introduction: uveal melanoma (um) is the main intraocular tumour in adults, and is particularly resistant to treatments when disseminated to the liver. our hypothesis is that extracellular vesicles (evs) released by the primary tumour are priming the liver stroma for metastatic cell colonization by activating hepatic stellate cells (hstecs). this study aimed to characterize evs from um cells, and to determine their interactions with liver cells. methods: evs were isolated from cell lines derived from ocular tumours and liver metastases by differential centrifugation. their concentration/diameter range were determined by high-sensitivity flow cytometry. cryo-tem combined with receptor-specific gold labelling was used to reveal the morphology/size of melanomic evs. the presence of melanoma and ev markers was assessed by western blotting. the internalization of fluorescent melanomic evs in hstecs and their subsequent activation were assessed by confocal imaging using alpha-smooth muscle actin (alpha-sma) and phalloidin stainings. ev impact on invasion was measured with a tumour spheroid model embedded in extracellular matrix. melanomic evs were inoculated into the retro-orbital sinus of immunodeficient mice to study their selective organ distribution. results: melanomic evs were positive for annexin- , tetraspanins, as well as some melanoma markers. stellate cells with internalized melanomic evs expressed more alpha-sma, reflecting their activation. adding evs on tumour spheroids increased the invasion process. melanomic evs were localized into different murine organs, but mainly into the liver, as observed by in vivo fluorescent imaging. introduction: exosomes are being tested for their use as therapeutic agents in degenerative and chronic diseases. however, the optimal source of exosomes is currently under investigation. amniotic fluid (af) is a naturally-rich source of exosomes that is easily obtained for use in regenerative medicine. organicell flow™ is a minimally-manipulated, acellular product derived from human af and consist of over cytokines/chemokines as well as exosomes derived from the amniotic membrane and surrounding tissues. we characterized the exosome fraction of our product to elucidate the protein cargo of af exosomes and demonstrate the therapeutic potential as a novel regenerative therapy. methods: the exosome fraction of our product was analysed using nanosight nanoparticle imaging and macsplex exosome surface marker array analysis. exosomes were precipitated using size-exclusion filtration followed by ultracentrifugation from independent products (in triplicate) and subjected to protein lysis and preparation for mass spectrometry analysis using the easy nlc and q exactive instruments. tune (version . ) and xcalibur (version . ) was used to collect data while proteome discoverer (version . ) was used to analyse data. protein expression lists were created by merging the sample replicates together and commonly expressed proteins were determined using vinny . vin diagram analysis. webgestalt tool kit classification system was used to identify top protein function and pathway hits. results: organicell flow™ contain a mean concentration of . x ^ particles/ml (n = ) with a mean mode size of . nm (n = ). surface marker analysis confirms the presence of exosome associated proteins cd , cd , and cd in addition to a high expression of cd (n = ). the completed analysis revealed commonly detected proteins across products. the top molecular functions of identified proteins included protein-binding, ion-binding, and nucleic acid-binding with enzymes, transcription regulators, and transporter proteins representing the most abundant protein groups. pathway enrichment analysis revealed top hits for integrin, pdgf, and p pathways. a deeper dive into the enzyme category of the protein cargo further demonstrates the presence of proteins that promote dna repair such as dna polymerase (beta and lambda), telomerase reverse transcriptase, and brca . summary/conclusion: organicell flow™ characterization demonstrates the therapeutic potential of afderived exosomes. proteomic analysis revealed protein cargo that may regulate various growth factor and cellcycle associated pathways. furthermore, the presence of dna damage response proteins suggests a possible mechanism for induction of cellular repair. generation of car-t and γδt cell-derived exosomes for future cell free immunotherapies γδt cells are a subset of t cells with dual innate and adaptive qualities. this duality provides various advantages over their more studied and used counterpart, αβt cells. in the present study, we sought to compare the immunotherapeutic potential of car-t cell and γδt cell-derived exosomes as novel cell-free based alternatives. methods: cd -targeting car-t cells were obtained following the isolation, expansion and transduction of αβt cells using a lentiviral vector bearing the car construct. γδt cells were isolated and expanded from peripheral blood mononuclear cells (pbmcs) following innate or adaptive stimulation. exosomes from both cell sources were isolated after a -day culture in serum-free media using ultracentrifugation-based methods. exosomes were characterized by nanoparticle tracking analysis (determination of size) and western blot assays (detection of the appropriate surface markers). nalm- (b cell precursor leukaemia) cells were used as target cells for assessment of exosome cytotoxic/ killing function. car-t cell and γδt cell-derived exosomes were incubated at particles/target cell for -hours. total viable cell counts were assessed via imaging-based cytometry (nc- ) utilizing acridine orange and dapi staining. results: exosomes derived from γδt cells activated via innate mechanisms showed significant killing of nalm- as compared to exosomes from non-activated or adaptively activated γδt cells. in comparison, car-t cell-derived exosomes showed minor killing capabilities of the target cells. summary/conclusion: here, we report for the first time that exosomes derived from cd car-t cells and innately activated-γδt cells show/exert inhibitory action on nalm- cells. further studies are currently underway to identify the underlying mechanism(s) responsible. introduction: age-related cognitive dysfunction is associated with increased oxidative stress, low-level chronic neuroinflammation, and waned hippocampal neurogenesis in the brain. from this perspective, biologics capable of modulating oxidative stress and neuroinflammation, and stimulating neural stem cell activity in the brain might be useful as anti-ageing interventions. methods: we investigated the efficacy of intranasal administration of extracellular vesicles (evs) generated from cultures of rat subventricular zone neural stem cells (svz-nscs) in the middle-aged mice to alleviate cognitive and mood dysfunction, increased oxidative stress, neuroinflammation, and neurogenesis decline in old age. mice were treated intranasally with nsc-evs once weekly for three weeks ( billion per administration) starting from . months of age. a month later, the animals were examined for cognitive, memory, and mood function using multiple behavioural tests, and brain tissues were examined for oxidative stress, neuroinflammation, and neurogenesis. results: object-based tests revealed that aged animals receiving vehicle displayed cognitive impairments for discerning minor changes in the environment as well as for distinguishing similar but not identical experiences. these animals also exhibited spatial memory dysfunction and anhedonia. in contrast, aged animals receiving nsc-evs showed improved cognitive and mood function. biochemical analyses of brain tissues revealed that nsc-ev treatment normalized elevated concentrations of oxidative stress markers malondialdehyde and protein carbonyls and the proinflammatory cytokine interleukin- beta. moreover, nsc-ev treatment stimulated increased production of antiinflammatory protein interleukin- and the antioxidant superoxide dismutase. immunohistochemical analysis revealed modulation of neuroinflammation typified by reduced activity of reactive astrocytes and activated microglia and improved hippocampal neurogenesis. summary/conclusion: the results suggest that the intranasal administration of nsc-evs is a promising approach for maintaining better cognitive and mood function in ageing through modulation of oxidative stress, neuroinflammation, and neurogenesis. funding: supported by a grant from the national institute of neurological disorders and stroke ( r ns - to a.k.s.) chemically modified myocytes-derived evs for the treatment of cardiac fibrosis. marta prieto-vila a , asao muranaka a and takahiro ochiya b a tokyo medical university, tokyo, japan; b tokyo medical university, shinjuku-ku, japan introduction: myocardial fibrosis is a disorder that may occur after cardiac injure due to a malfunction of the cardiac remodelling. fibroblasts resident in myocardium are erroneously activated causing an excessive accumulation of extracellular matrix, which decreases cardiac function and eventually, leads to death. it is known that cardiomyocytes communicate with the surrounding cells such as fibroblast and endothelial cells by extracellular vesicles (evs). the loss of this communication is thought to play a central role in cardiac fibrosis. therefore, cardiomyocytes-derived evs may be a promising a cell-free system for the treatment of fibrosis inhibition. methods: a novel culture medium was stablished to improve the expansion of primary cardiac myocytes. this was tested using two commercially available primary myocytes cell lines. evs were collected by serial ultracentrifuges, and their effect on fibrosis was tested. for that, prior to any treatment, and to mimic fibrosis, primary cardiac fibroblast were activated overnight with tgfβ. results: by the use of a defined conjunct of chemicals, mature cardiomyocytes culture was highly improved to ensure a high collection of evs. terminal differentiation markers, as well as senesce apparition was delayed in comparison to predetermined culture medium. interestingly, those primary cells secreted a rather large amount of evs, which expressed common evs membrane marker. tgfβ-treated cardiac fibroblasts were co-cultured with myocytes showing a decrease of fibroblast activation markers both at mrna and protein levels. similar results were found when activated fibroblast were treated with evs. summary/conclusion: our findings indicate that the use of evs derived from chemically modified myocytes is a promising treatment for ischaemic myocardial fibrosis. however, further molecular experiments have to be done to identify the molecules within evs responsible for the inactivation of fibroblast. evaluation of osteoinductive and anti-inflammatory properties of spinederived exosomes renaud sicard a , tania del rivero b , jonathan messer c , shabnam namin c and timothy ganey c a vivex, biologics, inc., miami, usa; b vivex, biologics, inc., miami, usa; c vivex, biologics, inc., miami, usa introduction: over the last decades, mesenchymal stem cell-derived exosomes have been shown to play a crucial role in a myriad of cell function such as extracellular matrix synthesis, proliferation, differentiation or cell migration. biological sources of exosome (heterogeneous or homogeneous cell population, serum, urine etc.) have a direct influence on the content of their cargo and their therapeutic application and potential. in this study, we evaluated exosomes excreted from cadaveric spine-derived cells. we hypothesized that exosomes derived from a bone source such as the spine, will drive the osteogenic differentiation of progenitor cells. we also investigated their effects on inflammation in nucleus pulposus cells using an in-vitro assay. methods: after their isolation and characterization, exosomes derived from cadaveric human spines were assayed for osteoinductive properties. a c c myoblast cell line was treated with different concentrations of exosomes and expression of alkaline phosphatase was measured after days incubation. treatment with bmp- was used as positive control. anti-inflammatory properties were assessed by incubating tnf-treated nucleus pulposus cells with exosomes for days. qpcr analysis of mrna expression of inflammatory cytokines (il- , il -beta, il- ) metalloproteinases (mmp and adamts ), and apoptotic genes (bax, bcl ) was used to determine the effects of exosomes on inflammation. results: spine-derived exosomes positively expressed the exosome flow cytometry markers tested (cd , cd and cd ). the mean number of exosomes per microgram of protein was . ± . x indicating a relatively high purity. osteoinductive (oi) testing was performed using different concentrations of exosomes. the oi index of treatment of c c cells with bmp- , x , x , x , × or × exosomes alone was . , . , . , . , . and . respectively. anti-inflammatory properties of exosome are currently being assessed and will be presented at the time of the poster presentation. summary/conclusion: administering exosomes alone or in combination with an exogenous scaffold has the potential to repair injured tissue and to restore bone function. the clinical significance of this application is aimed to promote the patients' bone healing process and provide a cell-free therapeutic platform that is safe and effective. administration of human mesenchymal stem cell derived extracellular vesicles modulates the abnormal plasticity of newly born neurons and neuroinflammation in a rat model of status epilepticus maheedhar kodali a , daniel gitai b , dong ki kim a , mariam atobiloye a , bing shuai c , sahithi attaluri c , raghavendra upadhya c , leelavathi n madhu a , olagide w. castro a , darwin j. prockop a and ashok k. shetty c decline in the percentage of newly born neurons displaying basal dendrites. besides, ev treated animals displayed higher percentages of resting microglia (ramified microglia), reduced percentages of activated microglia (microglia expressing iba- and cd ), in comparison to animals receiving vehicle after se. interestingly, diminished abnormal plasticity of newly born neurons was accompanied by the preservation of interneurons positive for reelin; a protein believed to guide newly born neurons to their correct locations. summary/conclusion: the results suggest that even a low dose in administration of msc-derived evs after se can limit neurons loss, dampen the abnormal plasticity of newly born neurons, and modulate the activation of microglia. introduction: autism spectrum disorders (asd) are neurodevelopmental disorders characterized by three core symptoms that include social interaction deficits, cognitive inflexibility, and communication disorders. they have been steadily increasing in children over the past several years, with no effective treatment. two percent of all asd patients are suffering from a disorder caused by a mutation in the shank gene. shank is an important synaptic protein, disruption of this gene directly leads to cognitive and motor impairments. during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. here we test three different autistic mice models. btbr as a multifactorial mice model of autism and two different shank mutated mice. the first is a complete deletion of exon ( q . ) and the second is a specific insertion mutation of guanine to position in the gene (insg ) that leads to stop codon. methods: exosomes were isolated using differential centrifugation protocol and characterized using the misev guideline recommendations. each animal received an intranasal administration of ul containing exosomes/µl. for intravenous administration, the same number of exosomes, were used, injected in µl. results: all three animal models showed significant improvement in their autistic behavioural phenotypes following intranasal administration. the improvement seems to be dose-dependent and was better achieved via intranasal vs intravenous administration. biodistribution of msc-exo showed accumulation in the brain within hours, yet the reduction of the signal was observed in the kidneys, heart and lungs. summary/conclusion: our data suggest that exosomes derived from adipose msc, carry a therapeutic potential in asd, via non-invasive intranasal administration in three different mice models. these data further emphasize our potential therapeutic strategy to reduce symptoms of autism in clinical trials. funding: stem cell medicine ltd. israel. equine tendon injury treatment by evs: an in vitro study introduction: current treatment options for tendinopathies (chronic, painful tendon disorders), are not able to restore the functional properties of native tendons. hence, new treatment options are sought. the efficacy of mesenchymal stem cells (mscs) therapies, which combined with a rehabilitation programme including controlled exercise is the current gold standard in equine tendon treatment, has been shown to be largely due to the cells´paracrine activity. the aim of this study was therefore to evaluate the effect of bone marrow msc derived autologous and allogeneic conditioned medium (cm, full secretome) and their extracellular vesicles (evs) on "tendon healing" in vitro. methods: to compare the "therapeutic" effect of msc derived evs and cm, a standardized scratch assay (wound healing assay) was performed. cm from equine tenocytes, ev depleted medium and medium with or without fcs served as controls. tendons and bone marrow aspirates were obtained from three horses ( , and years) which were euthanized for reasons unrelated to this study. mscs were isolated by ficoll density gradient centrifugation and tenocytes were obtained by migration from tendon explants. for cm and ev production, cells were cultured in ev depleted medium. evs were harvested by a stepwise ultracentrifugation approach and characterized by nanoparticle tracking analysis (nta), western blot (cd , cd ) and transmission-electron microscopy (tem). results: western blot, nta and tem confirmed successful isolation of evs from equine mscs. the strongest positive effect on wound healing (fastest gap closure) was achieved by msc-cm (p < . ). the gap closure achieved with msc-evs was slower than with msc-cm (p < . ) but faster than with cm of tenocytes (p < . ). donor specific differences in wound healing capability were shown for both autologous and allogeneic application. summary/conclusion: treatment with msc-cm resulted in significantly faster wound healing of adult tenocytes in vitro than msc-evs or tenocyte-cm. mscs donor age shows a significant effect on gap closure following autologous but not allogeneic administration. ev-enriched secretome fraction from gmp-compatible, scalable, human ipsc-derived cardiac progenitors improve heart function in chronic heart failure mice introduction: we have shown that research-use-only grade (res) human ipsc-derived cardiac progenitors (cpcres) can produce a secretome whose small-evenriched fraction (svf) can treat chronic heart failure (chf) in mice. gmp-compatible, scalable processes for a cpc-derived svf suitable for human therapeutic use is needed. methods: ipsc-derived cpc were produced and cultured using gmp-compatible, scalable processes (cpctx). media without cells were "cultured" in parallel for "virgin media" controls (mv). cpcres were cultured as previously described. as a proof of concept, svfs were isolated from conditioned media by ultracentrifugation: cpctx-ev, cpcres-ev and mv. particle size distributions/concentrations (nanoparticle tracking analysis), protein levels (bsa), and the presence of cd- (elisa) were determined. in vitro activity was assessed by huvec scratch wound healing assay, and by rat and human cardiomyocyte (cm) survival assays. c bl/ mice in chf received echoguided myocardial injection of pbs vehicle control ( ul, n = ), cpctx-ev ( ul, n = ), or cpcres-ev ( ul, n = ). change in cardiac function was assessed by echocardiography. results: cpctx-ev particle sizes were polydisperse (mode~ nm) at a concentration of~ . e particles/ml (~ , particles/cell) and~ . mu cd /ug protein. cpctx-ev increased wound healing, human cm survival, and rat cm survival in vitro by . x, . x, and x, respectively over mv controls. in chf mice, significantly less cpctx-ev mice, and less cpcres-ev mice had severely progressive heart failure (left ventricular end systolic volume, lvesv, increased > %) than pbs control mice (pbs vs cpctx-ev, p < . ; pbs vs cpcres-ev, p < . ), and the average ejection fraction of the pbs group deteriorated . x more than the cpctx-ev group (− % vs − . %, respectively; ns). summary/conclusion: we have a process for cpc differentiation and production of conditioned media suitable for use in human clinical trials from which can be made an svf with the potential to treat chf, possibly through re-vascularization or preservation of cm viability. introduction: exosomes are nanoscale vesicles that mediate cell-to-cell communication via exchanging molecular cargo. mesenchymal stem cell (mscs) modification towards an osteogenic path can occur by uptake of exosomes from other cells. it is less clear whether vesicle placement in the absence of cells will facilitate site-specific delivery through acellular transfer of osteogenic activity. an electrospun fleece was combined with bone marrow-derived exosomes in the absence of cells to evaluate osteoinductive potential that might be thermo-stable and be used in a biologically neutral collagen carrier. comparisons were made of standard laboratory assay of osteoinductivity (oi), and in vivo expression in a mouse calvarial defect model. methods: electrospun type-i collagen was prepared with and without hydroxyapatite (ha) (spinplant gmbh, leipzig) as a foundation base for application of the bone marrow-derived exosomes. individual discs of the collagen enhanced scaffolds ( -mm) were prepared and placed in a mouse calvarial skull defect. animals were followed for and weeks. exosomes were isolated from qualified cadaveric human spines by differential ultracentrifugation. microscopic observation, quantitative assessment of oi with an alkaline phosphatase assay, and flow cytometry were used to evaluate the composition, the hybrid nature of the addition to the nano-collagen fibres. a fluorescent protein reporter transgenic mouse model expressing osteocalcin, type-i collagen, phex, and sp (osterix) was evaluated at and weeks to determine bone formation across the defect. results: alp activity on the scaffold with ha demonstrated an approximate tenfold increase to that of the collagen scaffold alone. while a dose-dependent effect, with higher doses of exosomes resulting in a greater amount of alkaline phosphatase expression, expression that exceeded that of the ng bmp- control. dose escalation from . , , and e resulted in similar increases in expression that was statistically greater with the combination of the fleece with the exosome component. bone formation in the mouse calvaium did not demonstrate gap closure at or at weeks, but did demonstrate enhanced osteoclastivity and robust bone remodelling at the margins of the defect. summary/conclusion: bone marrow-derived exosomes dried into an electrospun fibrillar collagen demonstrated in vitro osteoinductive potential that might provide site-specific placement that could enhance biologic potential. with the capacity for ambient temperature storage, the provision of site-specific placement becomes a technical consideration. placement of the human tissue derived exosomes in a transgenic mouse calvarial defect model did not demonstrate bridging bone across the defect. exosomes loaded with pten-interfering rna enables functional recovery in rats after complete spinal cord transection daniel offen a , nisim perets a , shaowei guo b , oshra betzer c , rachela popovtzer c and shulamit levenberg b a tel aviv university, tel aviv, israel; b technion, haifa, israel, haifa, israel; c bar ilan university, israel, ramt gan, israel introduction: complete spinal cord transection is a debilitating disease that usually leads to permanent functional impairments, with various complications and limited spontaneous recovery. the current investigation of molecular mechanisms controlling axon regeneration, (e.g., signalling networks and environmental cues), led to new strategies to enhance axonal regeneration. we have previously shown that intranasal administration of mesenchymal stem cells derived exosomes (msc-exo), cross the blood-brain barrier and significantly ameliorate motor and behavioural phenotype in several animal models of neurotrauma and neuropsychiatric disorders. methods: msc-exo were isolated from human bone marrow and were loaded with phosphatase and tensin homolog small interfering rna (pten-sirna). the exosomes were given intranasally to rats two hours after complete spinal cord transaction. eight weeks later we followed the motor function and histology and electrophysiology study was performed in order to reveal the connectivity and the biochemical changes in the treated rats. results: we demonstrate that intranasal (in) administrations of msc-derived exosomes could penetrate the blood-brain barrier, home selectively to spinal cord lesion via chemotaxis, and integrated in neurons within the lesion. furthermore, in rats with complete spinal cord transection, msc-exo loaded with pten-sirna silenced pten protein expression in the lesion and promoted robust axonal regeneration and angiogenesis, companied with decreased astrogliosis and microgliosis. moreover, the intranasal treatment partially restored electrophysiological and structural integrity, and most importantly, enabled the remarkable functional recovery and significant improvement in their movements. summary/conclusion: this rapid, non-invasive, approach, using cell-free nano-swimmers carrying molecules to target pathophysiological mechanisms suggest novel strategy for clinical translation to spinal cord injury and beyond. a novel umbilical cord derived wharton's jelly formulation for regenerative medicine applications introduction: musculoskeletal injuries have traditionally been treated with activity-modification, physical therapy, pharmacological agents and surgical procedures. these modalities have limitations, as well as potential side-effects. over the last decade, there has been an increased interest in the use of biologics for regenerative medicine applications (rma), including umbilical cord (uc) derived wharton's jelly (wj). despite this increase, there is insufficient literature assessing the amount of growth factors, cytokines, hyaluronic acid (ha) and extracellular vesicles (ev) including exosomes in these products. the purpose of this study was to develop a novel wj formulation and evaluate the presence of growth factors, cytokines, ha and ev including exosomes. methods: wj was isolated from human-uc obtained from consenting c-section donors and formulated into an injectable form. randomly selected samples from different batches were analysed for sterility testing and quantified for presence of growth factors, cytokines, ha and particles in ev size range. the results showed all samples passed the sterility test. growth factors including igfbp , , , and , tgfα, pdgf-aa were detected. expression of several immunomodulatory cytokines, rantes, il- r, il- , were also detected. expression of pro-inflammatory cytokines mcsfr, mip- a; anti-inflammatory cytokines tnf-ri, tnf-rii, il- ra; and homoeostatic cytokines timp- and timp- were observed. cytokines associated with wound-healing, icam- , g-csf, gdf- , and regenerative properties, gh were also expressed. high concentrations of ha were observed. particles in the ev size range ( - nm) were detected and were enclosed by the membrane, indicative of true ev. summary/conclusion: our results confirmed the presence of numerous growth factors, cytokines, ha and ev in the wj formulation. more studies are underway to confirm the presence of exosomes in detected ev using exosome-specific markers. we believe the presence of multiple factors within one wj formulation may play a role in reducing inflammation, pain and augment healing of musculoskeletal injuries. this offers a potential expanded use for rma. funding: this study was funded by biointegrate llc, new york, ny, usa. collagen sponge loaded with mesenchymal stem cell-derived small extracellular vesicles promote robust bone regeneration shang jiunn chuah a , chee weng yong a , jacob ren jie chew a , ruenn chai lai b , yi ann cheow a , raymond chung wen wong a , asher ah tong lim a , sai kiang lim c and wei seong toh d introduction: mesenchymal stem cell (msc) therapy has demonstrated effective bone regeneration in clinical studies. however, the therapeutic efficacy of mscs have been attributed to the secretion of extracellular vesicles (evs), particularly - nm small evs (sevs). here, we investigate the efficacy of msc-sevs loaded in collagen sponge in the regeneration of critical-sized calvarial defects in immunocompetent rats. methods: sevs were isolated from conditioned medium of human mscs and stored at − c. calvarial defects of -mm diameter were surgically created on thirty-two -week-old male sprague-dawley rats. these rats were then randomly assigned to groups (n = rats/group): defects treated with collagen sponge containing μg of sevs in μl saline (cs/sevs) and defects treated with control collagen sponge containing an equivalent volume of saline (cs/control). at and -week post-surgery, the calvarial bone samples was harvested for analyses by micro-computed tomography (micro-ct), histology, immunohistochemistry and histomorphometry. results: at -week post-surgery, micro-ct analysis showed little bone formation at the defect site in both cs/sevs and cs/control groups. no statistical differences were observed in micro-ct and histology scores in both groups. interestingly, cs/sevs group showed significantly higher osteocalcin (ocn)+ area of . ± . % than that of cs/control group ( . ± . %; p = . ). cd + microvessels at sizes ≤ µm and > µm in cs/sevs group ( . ± . and . ± . microvessels/hpf) were also significantly higher than that of cs/control ( . ± . and . ± . microvessels/hpf; p = . and p = . respectively). by weeks, cs/sevs group displayed enhanced new bone formation that completely bridged the calvaria defect. in contrast, rats in cs/control showed limited bone formation. consequently, cs/ sevs group displayed a micro-ct score of . ± . which was significantly better than that of cs/control group ( . ± . ; p = . ). cs/sevs group also exhibited >twofold increase in bone volume, and improved bone quality with higher trabecular thickness and number, and smaller separation (p < . ), compared to cs/control group. consistently, cs/sevs group displayed a significantly better histology score of . ± . than that of cs/control ( . ± . ; p = . ). moreover, cs/sevs group showed significantly higher ocn+ area of . ± . % than that of cs/control group ( . ± . %; p = . ). summary/conclusion: this study demonstrates that single-stage implantation of collagen sponge loaded with ready-to-use msc sevs can promote robust bone regeneration in a rat calvarial defect model. funding: national university of singapore, r , national medical research council singapore, r . immunomodulatory potential of extracellular vesicles derived from mesenchymal stromal cells introduction: extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) are promising new agents in regenerative medicine and immunotherapy. considering that independent msc-ev preparations might differ in their therapeutic function, we have set up a functional assay allowing testing for the potential immunomodulatory properties of independent msc-ev preparations. methods: human peripheral blood-derived mononuclear cells (pbmcs) were pooled from up to different healthy donors warranting high allogeneic cross-reactivity, even following an optimized freezing and thawing procedure. after thawing, mixed pbmcs were cultured for days in the absence or presence of msc-evs. thereafter, cell morphologies were documented, supernatants were harvested for cytokines quantification and cells were phenotypically characterized by flow cytometry. by analysing the expression of a collection of different lineage and activation markers, we selected a panel of antigens apparently being regulated by msc-ev preparations considered to be therapeutically active. results: we observed that in the presence of active msc-ev preparations more cd + (monocytes) are recovered from the mlr assay than in corresponding control samples. focusing on t cells, we learned that active msc-ev preparations reduced the content of cd and cd t cells expressing activation markers like cd and cd . summary/conclusion: the mlr assay allows elaborated functional testing of immunomodulatory activities of given msc-ev preparations. currently, we are comparing the immune modulatory capabilities of evs derived from distinct sources and optimize the marker panel to distinguish discrete immune cell subtypes such as different cd cell types, i.e. th , th , th and tregs. extracellular vesicles in platelet-rich plasma: dependency on sample processing zala jan a , saba battelino b , darja božič c , matej hočevar d , ales iglič e , marko jeran c , manca pajnič a , ljubiša pađen a , domen vozel f and veronika kralj-iglič a introduction: platelet-rich plasma (prp) proved effective in regenerative medicine. numerous protocols for its preparation and application are available in the published literature. prp possesses important immune, haemostasis and regenerative factors, however, the mechanisms of their action are yet poorly understood. extracellular vesicles (evs) could be one of the important factors that would contribute to the beneficial effects of preparations. this study was performed as a part of a registered randomised controlled clinical trial (nr: nct ). prp was used to treat chronic middle ear inflammations. here we present the results of prp analyses from blood samples of volunteers with no record of disease. methods: plasma obtained from ml of blood was depleted of erythrocytes and enriched with other particles by repetitive centrifugation of samples. flow cytometry (fcm) was employed to monitor particle contents (cells and smaller particles) throughout the sample processing. the platelet gate was divided into two parts: intact platelets and smaller particles. identity and morphology of particles in the preparations were examined by scanning electron microscopy (sem). standard laboratory tests of blood were performed. results: sem images revealed the presence of heterogeneous population of particles in the preparation of prp, most of which were activated and partially fragmented platelets. the population of smaller particles measured with fcm, was identified as evs. the erythrocyte sedimentation rate was statistically significantly correlated to the volume of plasma obtained in the initial centrifugation step (r = , , p < , ) and to the concentration of evs (r = , ; p < , ). time from sample collection to the preparation of prp was negatively correlated with the concentration of platelets in prp and positively with the concentration of evs (r = , , p < , ). platelet concentration in preparation samples was found to depend on the concentration of platelets in the blood and parameters of sample processing connected with larger centrifugal and shear forces on the samples during centrifugation. these include: sample volume, the size and shape of the centrifuge tube and the distance of the sample from the rotor axis. summary/conclusion: evs are gradually forming upon activation and degradation of cells in the sample throughout the sample processing. optimal processing may importantly contribute to the healing properties of preparation. funding: authors acknowledge support from the european union's horizon research and innovation program under grant agreement no. (ves us project) and slovenian research agency (arrs, grants p - , p - , j - ). satellite cell-derived extracellular vesicles as a therapeutic for mitochondrial dysfunction in duchenne muscular dystrophy duchenne muscular dystrophy (dmd). sc-derived extracellular vesicles (sc-evs) may unlock the therapeutic potential of scs by overcoming these limitations. to investigate their therapeutic potential, we assessed the ability of sc-evs to reverse mitochondrial dysfunction, a key pathological feature of dmd, in oxidatively-damaged c c and primary dmd myotubes. methods: scs from c mice were isolated and cultured. evs were isolated from the supernatant of scs via polyethylene glycol precipitation and characterized using nanoparticle tracking analysis. the ability of sc-evs to deliver protein cargo to c c myotubes, and the localization of the cargo once delivered, were analysed using fluorescence microscopy. to examine sc-ev potential to restore the function of damaged mitochondria, c c myotubes were treated with µm h o for h followed by treatment with . x sc-evs for h. separately, cultured dystrophic myotubes were treated with . × evs every h for h. in both sets of experiments, maximal oxygen consumption rate (max ocr) was measured via seahorse xf cell mito stress test. where appropriate, a t-test was performed to test for statistical significance (p < . ). results: based on estimated cell number and ev quantification, each sc released approximately . × ± . x evs/day. evs delivered protein cargo into myotubes within h. fluorescent labelling of intracellular mitochondria showed co-localization of delivered protein and mitochondria. incubation of myotubes with h o resulted in a % decline in max ocr relative to untreated myotubes. subsequent treatment with sc-evs resulted in a % increase in max ocr. treatment of undamaged myotubes with sc-evs had no effect on max ocr. primary dmd myotubes treated with sc-evs showed a % increase in max ocr relative to untreated dmd myotubes. summary/conclusion: sc-evs rapidly deliver proteins into myotubes, much of which co-localizes with mitochondria, and reverses mitochondria dysfunction in oxidatively-damaged and dystrophic myotubes. introduction: flow cytometry has been used extensively for analysis of ev particles stained with fluorescent antibodies directed to the known cell surface markers. quantitation of the surface markers in terms of the number of molecules or the number of antibodies bound per specific marker has remained one of the largest challenges in the ev research field. changes in instrument setup as well as changes in fluorescent antibodies from different vendors, all impact the relative mfi values for the same ev sample. in this work we report a standardization method of quantitating extra-cellular vesicle surface markers with mesf liposomes. methods: liposomes labelled with fitc fluorescent dye were prepared with a bd proprietary technology. dynamic light scattering analysis was used for size determination of the liposomes. bd facsaria™ fusion system, modified with a small particle side scatter module (sp ssc), was used for analysis of the labelled liposomes by flow cytometry. results: we created a set of nm fitc-modified liposomes of various fluorescent intensities with a known number of fitc molecules incorporated in each liposome intensity. the mfi values of each liposome population (intensity) had a linear relationship to the amount of fitc used for labelling the liposome nanoparticles, suggesting that no self-quenching of fitc fluorescence had occurred. the number for the fitc fluorophores for each liposome intensity was expressed in the units of molecules of equivalents soluble fluorochrome (mesf). a plot of mesf vs. the fluorescent intensity of the liposomes (mfi values) obtained from flow cytometry analysis provided a calibration curve, from which the fluorescent intensity (mfi value) of a stained ev sample can be converted to the number of fluorophores bound (mesf value) to the surface of the ev particles. summary/conclusion: by this approach, the mfi values of stained ev particles are converted to standardized mesf values that are independent of instrument variation, resulting in further improvement of inter-laboratory standardization. furthermore, utilization of liposomes with similar size and refractive index to ev particles simplifies the data evaluation and improves the accuracy of ev surface marker quantitation by flow cytometry. currently, other fluorescent dyes are being explored to expand the utility of mesf liposomes with other fluorescent colours. measuring cholesterol as a high-throughput method for quantifying extracellular vesicles introduction: the extracellular vesicle (ev) field currently lacks a high-throughput method for accurately quantifying evs in solution. ev quantification has traditionally relied on nanoparticle tracking analysis (nta), which is time intensive and indiscriminately counts non-ev particles, such as membrane fragments and protein aggregates. we have rigorously assessed two commercially available methods for measuring cholesterol, a major lipid component of the ev lipid bilayer, and evaluated the utility of these assays to quantify evs in minimally processed samples. methods: the amplex® red cholesterol assay and cedex bio ht were used to quantify cholesterol in ev samples via enzymatic oxidation, with dynamic ranges of - , ng/ml and - µl/ml, respectively. samples throughout various stages of purification were analysed, from clarified cell culture medium to highly purified evs separated on an iodixanol gradient. we evaluated several pre-processing methods, to remove non-ev cholesterol content prior to analysis. results: the amplex® and cedex bio ht assays were found to perform comparably for quantifying cholesterol in purified evs (r = . ). importantly, cholesterol quantification on purified ev samples, ranging from e to e particles/ml, correlated well with nta measurements (r = . ). both µm filtration or an additional , rcf centrifugation step following clarification removed cholesterol associated with cellular debris or other non-ev sources, allowing for accurate quantification of conditioned medium samples or ultracentrifugation pellets (ucp) instead of needing to rigorously purify samples with an iodixanol density gradient. summary/conclusion: cholesterol quantitation can be used to accurately estimate ev concentration, allowing for rapid characterization of samples from clarified cell culture supernatant to highly purified evs. this highthroughput analytical capability may enable more comprehensive assessment of methods to boost ev yield through mass screening of cell culture conditions. optimization of nanoparticle tracking analysis of extracellular vesicles isolated from plasma and bronchopulmonary lavage fluid of patients with non-small cell lung cancer introduction: recent studies show that tumourderived extracellular vesicles (evs) greatly influence the tumour microenvironment and impact the therapy. in non-small cell lung cancer (nsclc), bronchopulmonary lavage fluid (balf) appears to be a good source of tumour-derived evs, providing more accurate information about the tumour microenvironment than evs from plasma. so far there is a lack of accurate and standardized methods for ev quantification. fluorescence nanoparticle tracking analysis (fl-nta) is an emerging method of ev-analysis, allowing discrimination of evs and exosomes from impurities. here we perform an optimization of the fl-nta method to compare evs from plasma and balf of nsclc patients and healthy controls (nc). methods: evs were isolated using homemade sizeexclusion chromatography (sec) columns (plasma) and ultrafiltration or differential ultracentrifugation (balf). nta was performed using zetaview pmx (particle metrix) after ev-staining with membrane dyes or fluorescence-labelled antibodies against typical ev-marker (cd , cd , cd ). results: nta scatter measurements showed a higher total particle concentration in plasma than in balf. however, membrane-specific staining showed a much greater purity of ev-preparations from balf, where nearly % of the particles detected in scatter mode showed positive membrane-staining. in contrast, only around - % of particles in the plasma ev-preparations were positive for the membrane dyes. fluorescence-staining for ev surface marker requires further optimization to obtain reproducible results. summary/conclusion: classical nta using only the scatter mode fails to discriminate between evs, lipoproteins and protein aggregates. for ev-analysis from complex biofluids like plasma, fla-nta and staining for specific ev marker is necessary to receive reliable data. balf seems to be a better source of tumourderived evs than plasma, since the obtained ev-preparations show a higher purity. improving conditions for fluorescence-staining and nta measurement of evs from plasma and balf of nsclc patients will provide an additional method for quantifying and phenotyping of evs. introduction: the exoviewer platform currently enables the user to capture extracellular vesicles (ev) by means of surface antigen-specific antibodies (e.g. targeting tetraspanins), making possible the enumeration of individual particles using single-particle interferometric reflectance imaging sensor (sp-iris, interferometric) imaging as well as fluorescence. currently, through interferometric imaging particles smaller than nm cannot be detected, while fluorescently stained ev smaller than nm can be well resolved. further, it is conceivable that small ev contain antigen numbers in the single digits, making antigen-specific immunostaining a challenge. to further characterize ev populations of different sizes and surface marker composition, it would be highly advantageous to target the vesicular nature of the detected particles linked to a fluorescence readout. methods: the goal of this project is to detect ev with a probe that is ubiquitously distributed across the surface (or lumen) of the vesicle. small ( - nm) ev present fairly distinctive lipid membrane features in the extracellular environment, turning the ev membrane into a "universal" marker, and as such may serve as an alternative marker that is complementary to canonical ev surface markers. results: here we present data on successfully staining ev with the membrane dye di- -anepps (di- ) and the luminal dye calcein-am. we demonstrate that ev from different sources can be efficiently stained with either dye, allowing the quantitative characterization of ev in an unbiased manner using exoviewer's fluorescence mode. while both dyes certainly have their own unique strengths, they exhibit the wanted linear correlation of ev staining versus concentration. further, both dyes are compatible with subsequent immunostaining applications, allowing the user to target specific surface or luminal markers (di- ). summary/conclusion: while a large-panel screening featuring other powerful dyes is continuously ongoing, the current data support the notion of providing the experimenter with a reference for total particle count and at the same time fully exploring the larger dynamic range of the fluorescence mode. moreover, the universal probe will enable the user to correlate intensity and particle size measurements, thereby significantly improving the exoviewer platform and its applications. membrane labelling is essential for the identification and quantification of extracellular vesicles via facs introduction: extracellular vesicle (ev) research is challenged by the lack of standard protocols to identify and distinguish between exosomes and ectosomes being released via exocytosis or plasma membrane shedding, respectively. analysis of small ev populations requires high-resolution technology and can be further improved using fluorescent labels such as carboxyfluorescein diacetate succinimidyl ester (cfse). at the inner leaflet of the plasma membrane, cfse is cleaved enzymatically resulting in covalent binding of the dye. in this study we optimized the conditions for membrane labelling of evs and their subsequent detection by flow cytometry to obtain a maximum yield of intact evs. methods: using sequential centrifugation, we separated ev subpopulations from supernatants of colo pancreas carcinoma cells based on size and mass. after , x g centrifugation, we reconstituted evs from the pellet. we used cfse for ev detection and analysed the expression of tetraspanins by facs to confirm the lipid bilayer structure. furthermore, we determined size distribution of evs by nanoparticle tracking analysis (nta) and electron microscopy. detecting evs as cfse+ events, we quantified our samples and investigated the impact of threshold adjustment on ev quantification. results: after high speed centrifugation of cell free supernatants, we identified cfse+ events as evs, which appeared as round structures under the microscope, and ranged from to nm in size. interestingly, tetraspanin markers cd and cd were detectable only on a subpopulation of purified evs, suggesting heterogeneity of our preparations. for sufficient labelling of evs, minimal temperature variations and short incubation times correlated with ev stability. of note, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of labelled evs and hence, is central for data comparability. summary/conclusion: protocol standardization is of major importance for the use of evs as diagnostic markers in liquid biopsies. funding: this project has been supported in part by annelise-asmussen foundation, luebeck (grant ), leo pharma germany (grant ). surface plasmon field-enhanced fluorescence spectroscopy (spfs) system for quantitative and qualitative extracellular vesicles total evaluation without any sample pretreatment introduction: the function of extracellular vesicle (ev) is interested in the immunology and oncology fields as a key transmitter for cellular communication. however, the conventional ev evaluation methods are required complicated evs preconcentration from the sample, its leads ev analysis uncertainty. in this study, we applied the spfs highly sensitive automated system for quantitative and qualitative ev evaluation without any sample pre-concentration and preparation step. methods: spfs automated system and plastic disposable sensor had been developed by konica minolta corporation in house. anti-membrane protein (cd , cd , cd ) antibody was chemically bonded on hydrophilic polymer which was immobilized through the gold thin film on the spfs sensor. the concentration of standard ev materials was evaluated by the qnano system before using. ev detection without preconcentrating was achieved by sandwich immunoassay step in microchannel round-trip flow reaction (tat min) with the spfs system, and elisa was adapted as a conventional standard method. after spfs highly sensitive fluorescent measurements step, extracted and detected ev were effectively recovered by using the recovery buffer reaction. results: the ev sensitivity performance between spfs and elisa clearly showed a significant difference, and the lod of spfs ( . particles/μl) method was estimated times superior to the lod of conventional elisa ( , particles/μl). the spfs calibration curve showed a wide dynamic range at least over logs as an additional specificity. spfs method also showed fine results in the dilution linearity test with high reproducibility under the serum/plasma sample condition. the data for recovery test of ev expected us that highly accurate measurement can be guaranteed under the condition of dilution about times or less even in the whole blood sample. after the spfs measurement, extracted ev on the spfs sensor chip could be effectively recovered and could be analysed nucleic acid which contains micro rna. summary/conclusion: spfs system might have great potential for quantitative and qualitative ev evaluation. our strategy with spfs system for ev proteomic and genomic profiling will be possible for applying to ev quality control as well as a novel biomarker development. identification of a novel compound that inhibits small ev secretion and tumour progression by a sensitive elisa screening. yunfei ma a , takeshi yoshida a , duc tuan nguyen a , kazutaka matoba b , katsuhiko kida b , taito nishino b and rikinari hanayama c a kanazawa university, kanazawa, japan; b nissan chemical corporation, tokyo, japan; c wpi nano life science institute, kanazawa university, kanazawa, japan introduction: small evs from tumour cells are known to promote tumour progression, therefore, it is expected to develop drugs that regulate small ev secretion, which can be used in clinical applications. methods: to identify such regulators, we first developed a sensitive elisa system for the quantification of small ev secretion using a high-affinity ev binding protein tim . by using this elisa system, we screened for small compounds that promote or inhibit small ev secretion using a drug-repositioning compound library (about , compounds). results: as a result, we identified eight promoters and two inhibitors, including compound a, which significantly reduced small ev secretion from various cell types without affecting cell growth. we further investigated the effects of compound a on a mouse model of osteosarcoma and found that compound a suppressed tumour progression efficiently. summary/conclusion: these data suggest that compound a would be useful not only for the characterization of small ev function but also for the clinical therapy against tumour progression, by inhibiting small ev secretion. introduction: for many years it was believed that several proteins such as cd , cd and flotillin- were unique for exosomes, however recent studies have shown that several of these markers also can be present in other subpopulations of evs (kowal et al pnas ) . furthermore, few markers have been identified as uniquely present in microvesicles. the aim of this study was to in depth compare the proteome of microvesicles and exosomes. methods: mda-mb- -luc-d h , -d h ln and -bmd a were cultured in ev-depleted media. microvesicles ( , x g, min) and exosomes ( , x g . h) were isolated using a combination of differential ultracentrifugation and a density cushion (~ . g/ml). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, and electron microscopy (em). quantitative mass spectrometry (tmt-lc-ms/ms) was used to identify differently enriched proteins in microvesicles and exosomes (n = x cell lines). results: in total proteins were quantified, with being quantified in all samples. in total and proteins were significantly upregulated in exosomes and microvesicles, respectively. go terms associated with the proteins significantly upregulated in exosomes were "extracellular exosome" and "plasma membrane", while the microvesicle proteome was associated with "membrane" and mitochondrion". in exosomes tetraspanins, annexins, escrt and rab proteins were significantly upregulated. in contrast, proteins that were upregulated in microvesicles were involved in protein translocation into the mitochondrial membrane (timm and tomm proteins), in cytokinesis, and in micos complex. however, flotillin- was not differently expressed in the ev subtypes. summary/conclusion: this study identifies several proteins to be differently enriched in exosomes and microvesicles. several of the proteins suggest recently by kowal and colleagues, such as adam and mitofilin could be validated. additionally several novel proteins could be identified. identifying markers separating microvesicles and exosomes is of high importance for the ev field and future studies will have to validate them also in other cells to determine if they are generic. introduction: the cellular elements composing the lining of brain ventricles have drawn much attention from neuroscientists, especially the role of subependymal cells in neurogenesis, but the role of ependymal cells in brain function and disease is still neglected. our objective is to study the morphological aspects of rat brain ventricles and the ependymal cells as analysed by transmission and field emission scanning microscopy in normal or ischaemic rats. methods: for this purpose, male wistar rats were submitted to minutes of global brain ischaemia and divided into two groups: a) sham-operated animals and b) saline-treated ischaemic animals. all animals were allowed to survive for seven days. all procedures were approved by the ethics committee of the federal university of são paulo ( / ). transmission and scanning electron microscopic analysis of lateral brain ventricles were done in buffered , % glutaraldehyde/ %formaldehyde perfused brains. cerebrospinal fluid was collected for nta analysis. results: the morphological characterization of brain ventricle revealed a slight rarefaction of ciliary tufts of animals submitted to ischaemia when compared to normal animals. field emission electron microscopy revealed the secretion of vesicles by the ependymal cilia in the lateral ventricle. size and concentration of particles in the cerebrospinal fluid was confirmed by nta and transmission electron microscopy. summary/conclusion: our results are unprecedented and bring innovative potential regarding the role of extracellular vesicles in both the physiology and pathogenesis of the nervous system. these data may also contribute to the development of new technologies for diagnosis and therapy of chronic degenerative diseases. introduction: the function of mitochondria relies on precise and effective quality controls. neurons have high metabolic demands and employ multiple mechanisms to ensure functional mitochondria. we investigated mitochondrial vesiclesa less understood quality control mechanism for mitochondriaand assessed the effect of cellular stress. methods: we surveyed mitochondrial vesicles in rat and planaria brains with electron microscopy. we quantified these vesicles with serial-section electron microscopy (fib-sem). we also conducted confocal microscopy with airyscan analysis of cultured neurons expressing fluorescently tagged mitochondrial markers. results: electron microscopy showed the ultrastructure of various types of mitochondrial vesicles. serial-section electron microscopy revealed the d ultrastructure of mitochondrial vesicles and their prevalence in neurons. confocal microscopic analysis showed increased numbers of mitochondrial vesicles in neurons under mild stress. summary/conclusion: our findings provide direct structural evidence for mitochondrial vesicles in neurons and their abundance in response to neuronal stress. their detection in the extracellular compartment (evidence for which is expected to be presented by the time of isev) may allow for development of biomarkers for mitochondrial health, with relevance to numerous pathologic conditions. from endosomes, might be involved in the impairment of rna, specific feature of als disease. combining high-resolution flow cytometry and surface marker analysis using an automated platform to study extracellular vesicle in cerebrospinal fluid unity health toronto, toronto, canada introduction: there is growing enthusiasm that extracellular vesicles (evs) carry the potential for a variety of applications in medicine. as biomarkers, evs may aid clinicians in the evaluation of diagnoses, disease progression, or even response to therapy. however, proper characterization of the amount, size, and phenotype of evs in a given sample remains challenging due to their sub-micrometre size and heterogeneity. over the last years, technologies, including high-sensitivity flow cytometry and automated platforms that simultaneously assess ev amount, size, and phenotype, have matured, providing new opportunities to study evs for future clinical applications. using such technologies to analyse cerebrospinal fluid (csf), which is in direct contact with the brain and spinal cord, may yield valuable insights into neurological disease processes. while there is often uncertainty about the exact source of evs in a biological sample, cd has emerged as a surface marker that suggests a neuronal origin. methods: csf samples that had been stored at - degrees celsius for advanced biomarker studies were analysed using two distinct approaches. a becton, dickinson and company (bd) aria iii flow cytometer was converted into using violet side scatter (ssc) for improved detection of evs with instead of nm ssc. for the combined analysis of amount, size, and phenotype, samples were analysed with the nanoview bio r platform. phenotype analysis included probing for the classic tetraspanins associated with exosomes (cd , cd , cd ) and the neural cell adhesion molecule l (cd ). results: flow of csf samples showed similar vesicle counts in control vs. disease and an increase of counts in later disease stages when neurodegeneration is thought to be more prominent. all csf samples showed some binding to classic exosomal markers (cd , cd , cd ). the sample taken at the latest time point showed relatively high vesicle counts, overall larger vesicle size, and abundant cd binding. interestingly, the cd positive evs were not positive for any of the classic exosomal markers (cd , cd , and cd ). summary/conclusion: this data supports the notion that analysing the amount, size, and surface markers of evs in csf can reveal intriguing dynamics in such basic ev characteristics over time and suggests important differences between ev populations in different disease stages. while previous studies indicated that cd could identify an ev to be of neuronal origin, it remains to be determined whether such specific surface markers will emerge as clinically relevant tools to support the evaluation of people affected by neurological diseases. a distinct microrna signature in plasma derived small extracellular vesicles of different neurodegenerative diseases introduction: exploring identifying robust biomarkers is essential for early diagnosis of neurodegenerative diseases. blood stream transports large (levs) and small extracellular vesicles (sevs), which are extracellular vesicles of different sizes and biological functions that are transported in blood. aim of our study was to investigate mrna/mirna signatures in plasma derived levs and sevs of amyotrophic lateral sclerosis (als), alzheimer's disease (ad), parkinson's disease (pdpd), fronto-temporal dementia (ftd) and alzheimer's disease (ad) patients. methods: levs and sevs were isolated from plasma of patients and healthy volunteers (ctr) by ultracentrifugation and rna was extracted. whole transcriptome and mirna libraries were prepared with truseq stranded total rna kit and truseq small rna library kit (illumina). results: our data suggested that the rna cargo in levs and sevs varies among different diseases. mirna analysis in sevs provided the most informative disease specific signatures, while whole transcriptome analysis did not show any specific signature. als was characterized by a small but specific group of circulating mirnas. mirnas profiling revealed that pd and ftd can be subgrouped in two classes while ad appears to be a homogeneous disease population. furthermore, mirnas profiling show the presence of overlaps in the signatures between the analysed diseases. mirna profiling in levs is similar to that observed in sevs, although in levs the overall differences between diseases are less marked. summary/conclusion: in this study we have demonstrated that mirnas are the most interesting subpopulation of transcripts transported by plasma derived sevs since they discriminate a disease from the other and they can provide a signature for each neurodegenerative diseases. may be linked with apoe genotype, we investigated the possible effect of apoe genotype on brain-derived evs (bdevs) and their protein and rna molecular cargo. methods: cortical brain tissues of ad patients with different apoe genotypes [ε /ε (n = ), ε /ε ( ), ε /ε ( ), ε /ε ( )] and non-ad controls (n = ) were obtained. bdevs were separated by size exclusion chromatography plus ultracentrifugation (uc) and characterized per misev . proteins were analysed by mass spectrometry. after protein identification, data were normalized using the cyclicloess method and analysed by principal component analysis (pca). nested factorial design highlighted differentially expressed proteins. rna from bdevs was extracted by mirneasy mini kit. small rna libraries were constructed using the ion total rna-seq kit and sequenced on the ion torrent s ™ using ion™ chips. reads were aligned to human reference transcriptomes using bowtie. differential gene expression was quantified by edger and limma. results: among proteins dysregulated in ad bd-sevs, several have reported roles in ad, e.g., microtubule-associated protein tau and peroxiredoxin- . regarding apoe genotypes, proteins were differentially expressed between ε carriers (ε /ε and ε /ε ) with non ε carriers (ε /ε and ε /ε ). however, ev markers did not differ by apoe genotype. in contrast to protein cargo of bdevs, the overall small rna expression pattern was similar among ad patients with different apoe alleles and non-ad patients. only a few mirnas showed different abundance level between ε /ε and ε /ε groups, or between ad and non-ad groups. summary/conclusion: bdevs carry proteins and mirnas related to ad development and apoe genotypes. further verification of protein and rna expression in brain and plasma derived evs may reveal mechanisms of ev function in neuroinflammation and develop biomarkers for ad disease. funding: this project was funded by mh . efficient pathology spread by extracellular vesicles from human brain tissues in mouse brain and tissue cultured neurons: transmission and propagation to gabaergic neurons however, whether human brain-derived evs induce tau pathology has not yet been characterized in the mouse brain. here, we assess the mechanisms of disease spread after intrahippocampal injection of human brainderived evs into the aged mouse model. methods: ev-enriched fractions were isolated from unfixed frozen human brain samples from ad, prodromal ad (pad), control (ctrl) cases, and tau knockout (tko) mouse brains. isolated evs containing pg of human total tau were sterotaxically injected into the right outer molecular layer of the dentate gyrus of months-old c bl/ female mice. . months after the injection, hippocampal slices were prepared for whole-cell patch clamp recordings of ca pyramidal neurons were undertakent. hippocampi were analysed with immunohistochemistry using phosphorylated-tau (p-tau) epitopes including at . evs were examined for protein composition by protein mass-spectroscopy, the neuronal uptake in vitro, and structural analysis by the atomic force microscopy (afm). results: semiquantitative brain-wide immunohistochemistry of p-tau revealed that inoculation of ad or pad-evs induced tau propagation throughout the hippocampus, including the dentate gyrus, ca and ca subregions. at was localized primarily in gad + gabaergic neurons in pad and ad evs groups, accompanied with reduced amplitude of inhibitory postsynaptic currents and excitatory-inhibitory ratio in amplitube of postsynaptic currents in ca pyramidal neurons in pad evs. afm analysis showed higher density of tau oligomers in both ad and pad evs while only ad evs showed significantly higher neuronal uptake compared to ctrl evs. finally, proteomic analysis showed that ad evs are enriched in disease and glia-related molecules compared to ctrl evs, which may contribute to their enhanced neuronal uptake. summary/conclusion: intracranial injection of ad or pad evs induced p-tau accumulation primarily in gabaergic neurons throughout the hippocampus, resulted in higher uptake by neurons, and tau oligomer conformation, indicating of their pathogenic potency as seeding factors. gabaergic neuronal dysfunction in the hippocampal neuronal circuitry reported in early ad brains could be attributed to specific ev mediated tau propagation in this cell type, a phenomenon meriting further investigation and validation. funding: nih rf ag , nih r ag , nih r ag , cure alzheimer's fund, brightfocus foundation, curepsp, coins for alzheimer's research trust introduction: extracellular vesicles (evs) are released by cells of the central nervous system as a result of injury, including mild traumatic brain injury (mtbi). since mtbi may alter circulating levels of evs, this study aimed to investigate differences in circulating ev numbers between contact sport athletes with and without acute mtbi. methods: circulating evs containing cd (cd + ev), cd (cd + ev), and neural cell adhesion molecule (l cam+ev) were analysed in young, male athletes with or without mtbi ( - yo, n = per group). sodium citrate-treated blood samples were obtained from athletes with mtbi within -hours of injury and from control athletes free of mtbi for one year. athletes were best matched for age and history of prior mtbi. samples were double-centrifuged to obtain platelet-poor plasma and stored at − °c until analysed. quantification of evs was performed using a spectral flow cytometer. the study was approved by temple university's irb, and all athletes provided written informed consent. results: mann-whitney u tests showed that population percentages of small size ( - nm) cd + ev, cd + ev and l cam+evs were significantly higher in mtbi athletes (mean rank: . , . , . ) than controls (mean rank: . , . , . ) (u = . , p = . ; u = . , p > . ; u = . , p > . , respectively). population percentages of large size ( - nm) cd + ev, cd + ev and l cam+evs were also significantly higher in mtbi athletes (mean rank: . , . , . ) than controls (mean rank: . , . , . ) (u = . , p = . ; u = . , p > . ; u = . , p > . , respectively). there were no significant differences between percentages of evs associated with blood brain barrier function (cd + ev) or platelets (cd a+ev) among mtbi athletes or controls. introduction: parkinson's disease (pd) is characterized by clinical heterogeneity, different rates of progression and absence of definitive biomarkers. extracellular vesicles (evs) are easily isolated from plasma and play a central role in intercellular communication which is highly relevant for inflammatory processes implicated in protein misfolding-related neurodegenerative disorders. thus, we characterized distinctive plasmatic ev subpopulations of pd and atypical parkinsonisms (ap) patients, with the aim to identify candidate biomarkers among evs surface membraneproteins. methods: plasmatic evs were collected from pd, matched healthy controls (hc), ap with multiple system atrophy (msa) and ap with tauopathies (ap-tau). evs were quantified by nanoparticle tracking analysis. the expression of ev-surface markers, related to inflammatory and immune cells, were measured by macsplex and correlated to clinical scales. a diagnostic model based on ev markers expression was built via supervised machine learning algorithms and validated in an external cohort ( pd, hc, msa, ap-tau). the cantonal ethics committee approved the study protocol. all enrolled subjects gave written informed consent. results: pd showed the highest ev concentration compared to others groups. pd and msa displayed a greater pool of overexpressed immune markers compared to ap-tau. ev antigens correlate to cognitive impairment and disease gravity in pd and msa. the roc curve analysis of a compound ev marker showed optimal diagnostic performance for pd (auc . ; sensitivity . %, specificity . %) and msa (auc . ; sensi-tivity %,specificity . %)andgoodaccuracyforap-tau (auc . ; sensitivity . %, specificity . %). a diagnostic model based on ev markers expression, cor-rectlyclassified . %ofpatientswithreliablediagnostic performance after validation in an external cohort ( % of accuracy). summary/conclusion: this analysis of multiple immune surface markers of circulating evs in pd and ap well captured the clinical heterogeneity of pd and showed optimal diagnostic performance. furtherly it suggests a different immune dysregulation in pd and msa vs. ap-tau, to be confirmed by functional analysis in experimental models of disease. funding: supported by abreoc. separation and characterization of extracellular vesicles from human cerebrospinal fluid introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off , ), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions ( + ) after the sec void volume as evaluated by detection of cd and cd markers (immunoblotting) and annexin a (peptide mapping by nanolc-ms/ms). there, nanoparticles around nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between and nm, with average diameter ± nm. cd was visualized by immunocytochemistry at the surface of ev around nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin- binding protein (lgals bp or k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between and nm; some of those nanoparticles had ring-like appearance. occasionally k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from k nanoparticles to investigate their individual physiological relevance and biomarker potential. introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off , ), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions ( + ) after the sec void volume as evaluated by detection of cd and cd markers (immunoblotting) and annexin a (peptide mapping by nanolc-ms/ms). there, nanoparticles around nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between and nm, with average diameter ± nm. cd was visualized by immunocytochemistry at the surface of ev around nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin- binding protein (lgals bp or k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between and nm; some of those nanoparticles had ring-like appearance. occasionally k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from k nanoparticles to investigate their individual physiological relevance and biomarker potential. release of extracellular vesicles from platelets requires platelet-platelet interaction aleksandra gąsecka a , naomi c. buntsma b , sytske talsma c , krzysztof j. filipiak d , rienk nieuwland e and edwin van der pol f introduction: arterial thrombosis is a major and global cause of human death and disability, but a biomarker for early-diagnosis of thrombosis is absent. platelet activation and aggregation are the first steps of plateletrich thrombus formation, but their relative contribution to platelet extracellular vesicles (pevs) release is unknown. methods: to study the relation between pev release and platelet interaction (aggregation), citrate-anticoagulated whole blood (wb) from healthy donors was diluted , , , and -fold and activated by μm thrombin-receptor activating peptide (trap). in addition, undiluted wb and -fold diluted wb, which totally blocked pev release, were activated with various trap concentrations. concentrations of pevs (cd + and cd +, cd p + > nm) and activated platelets (cd +, cd p+ > nm) were measured by flow cytometry (apogee a -micro). platelet aggregation was assessed using impedance aggregometry. results: a -fold dilution of wb blocked both aggregation and the release of pevs. compared to baseline, activation of undiluted wb with trap increased the concentrations of cd + . -fold and cd +-cd p + pevs . -fold. the concentration of cd + (r = . ) and cd +-cd p+ (r = . ) pevs as well as platelet aggregation (r = . ) scaled inversely (reciprocal) with the dilution of wb. further, we found a linear correlation between the % of activated platelets and the concentration of cd + (r = . ) and cd +, cd p+ (r = . ) pevs in undiluted wb, which was absent in -fold diluted blood (r < . ). summary/conclusion: the absence of aggregation and pev release upon platelet activation in -fold diluted blood shows that aggregation directly depends on the distance between platelets, which is confirmed by the reciprocal relationship between pev release and blood dilution. because pevs are only released when platelet activation is followed by aggregation, pevs are a potential early biomarker of thrombosis. funding: ag is supported by the national science centre, research programme preludium / / n/nz / . evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . age-dependent alteration in concentration and size distribution of extracellular vesicles in plasma of normotensive and hypertensive rats kosuke otani, muneyoshi okada and hideyuki yamawaki laboratory of veterinary pharmacology, school of veterinary medicine, kitasato university, towada, japan introduction: spontaneously hypertensive rats (shr) are the most widely used animal model of human essential hypertension. we previously reported that plasma small extracellular vesicles (sevs) in shr regulate systolic blood pressure, however, the mechanism has not been clarified. in the present study, we compared the concentration and size distribution of plasma evs (sevs and large evs) from young and aged normotensive wistar kyoto rats (wky) and shr. methods: heparin-anticoagulated plasma was collected from male wky and shr at ~ -(young) and -(aged) week-old. large evs were isolated from the plasma by centrifugation ( x g). sevs were isolated by ultracentrifugation ( , x g) following precipitation with polyethylene-glycol. the concentration and size distribution of sevs and large evs were measured by a tunable resistive pulse sensing analysis. results: there was no significant difference in the total concentration of plasma sevs between wky and shr or between young and aged rats. the mean diameter of plasma sevs from aged rats was larger than that from young rats in both wky and shr. also, the number of particles with a diameter of smaller than nm in plasma sevs from aged rats was lower than that from young rats. the concentration of plasma large evs from aged rats was higher than that from young rats in both wky and shr. there was no significant difference in the size distribution of plasma large evs between wky and shr or between young and aged rats. summary/conclusion: the present results for the first time demonstrate that the concentration of plasma large-sized evs is increased by ageing, while there is no difference in the concertation and size distribution of evs between wky and shr. further research is required to clarify the cause of age-dependent alternation in plasma ev size distribution and its physiological meaning. microrna profiling of circulating extracellular vesicles is involved with susceptibility to age-related diseases: relevance to cardiovascular signalling in ageing process ionara rodrigues siqueira a , laura cechinel b , rachael batabyal c and robert freishtat c a universidade federal do rio grande do sul (ufrgs), porto alegre, brazil; b universidade federal do rio grande do sul, porto alegre, brazil; c children's national hospital, washington, usa introduction: ageing represents a central risk factor for several diseases, such as cardiovascular diseases. our hypothesis is that extracellular vesicles (evs) can be potential mechanism of spreading molecules, such as micrornas, involved with susceptibility to chronic age-related diseases and geriatric syndromes. in this context, the role of micrornas in age-induced detrimental changes in the cardiovascular system has been suggested. although evs can protect micrornas from endogenous rnases and internalization of these vesicles into cells is involved with cell communication, delivering micrornas even to distant tissues, the relationships between evs micrornas profile and chronic age-related diseases has not been evaluated. our aim was to investigate the microrna profile of circulating evs during ageing process and their downstream signalling pathways. methods: the ethics committee (ceua -comissão de Ética no uso de animais -ufrgs; nr. , ) approved all animal procedures and experimental conditions. male wistar rats of -and -month-old were used, and plasma was obtained from the trunk blood. evs were isolated with exoquick following the manufacturer's instructions. microrna was isolated from evs and then amplified. microrna was labelled using the flashtag biotin hsr rna labelling kit and profiled on affymetrix genechip microrna . arrays. ingenuity pathway analysis (ipa) was used to identify pathways regulated by significantly altered micrornas. results: microarray analysis revealed micrornas. of these micrornas, were differentially expressed between aged and young-adult animals, micrornas were significantly upregulated and were downregulated in aged animals compared to young adult (p < . ; fold change of | . |). a conservative filter was applied on ipa and only experimentally validated and highly conserved predicted mrna targets for each microrna was used. ipa analysis showed that cardiac hypertrophic signalling is ranked as highly predicted targets for these differentially expressed micrornas (p < . ). moreover, ipa demonstrated that this canonical pathway is upregulated in aged animals when compared to young adult. in addition to cardiac hypertrophic signalling, other relevant cardiovascular canonical pathways, such as endothelin- signalling and intrinsic prothrombin activation pathway have predicted targets. summary/conclusion: our results showed for the first time that micrornas profile in circulating evs has a potential role to drive heart senescence and consequent cardiac diseases which represents the leading cause of death. introduction: introduction: the vascular endothelium and smooth muscle form adjacent cellular layers that comprise part of the vascular wall. here, we examined the extent to which extracellular vesicles (evs) vesicles participate in endothelial-vascular smooth muscle cell communication. methods: methods: evs were collected from rat aortic endothelial and smooth muscle cell serumfree media by ultracentrifugation. vesicle morphology, size and concentration were evaluated by transmission electron microscopy and nanoparticle tracking analysis. endothelial cell and vascular smooth muscle cell cultures were subjected to various concentrations of evs for various times. functional assays were performed. results: results: western blot as well as shot gun proteomic analyses revealed sets of proteins common to both endothelial-and smooth muscle-derived ev as well as proteins unique to each vascular cell type. functionally, endothelial-derived evs stimulated vascular cell adhesion molecule- (vcam- ) expression and enhanced leukocyte adhesion in vascular smooth muscle cells while smooth muscle evs did not elicit similar effects in endothelial cells. evs from endothelial cells also induced protein synthesis and senescenceassociated β galactosidase activity in vascular smooth muscle cells. proteomic analysis of vascular smooth muscle cells following exposure to endothelial cellderived evs revealed upregulation of several proteins including pro-inflammatory molecules, high-mobility group box (hmgb) and hmgb . pharmacological blockade of hmgb and hmgb and sirna depletion of hmgb in smooth muscle cells attenuated nfkb (p ) phosphorylation and nuclear translocation, vcam- expression and leukocyte adhesion induced by endothelial cell evs. summary/conclusion: conclusions: these data suggest that endothelial cell-derived evs can enhance signalling pathways that induce a pro inflammatory in vascular smooth muscle cells. introduction: graft patency is one of the major determinants of long-term outcome following coronary artery bypass graft surgery (cabg). biomarkers, if indicative of the underlying pathophysiological mechanisms, would suggest strategies to limit graft failure. many studies have generated compelling data on the sensitivity of mvs as biomarkers of cardiovascular disease progression and events. the mv usefulness in cabg has been tested only in a study that highlighted their importance in surgical haemostasis. no information is so far available on the association between the amount or pattern of circulating mvs and cabg outcome. we aimed to evaluate whether mv pre-operative signature could predict mid-term graft failure. methods: this was a nested case-control substudy of the coronary bypass grafting: factors related to late events and graft patency (cage) study that enrolled patients undergoing elective cabg. of these, underwent coronary computed tomography angiography months post-surgery showing % graft occlusion. flow cytometry mv analysis was performed in patients ( /group with occluded [cases] and patent [controls] grafts) on plasma samples collected the day before surgery and at follow-up. results: before surgery, cases had two-fold (p = . ) and four-fold (p = . ) more activated platelet-derived and tf+ mvs, respectively than controls. the mv thrombin generation capacity was also significantly greater (p < . ). this mv signature predicted graft occlusion (auc of . [ %ci: , - , ], p = . ). by using a mv-score ( - ), the or for re-occlusion for a score above was . ( % ci . - . , p < . ). summary/conclusion: the pre-operative signature of mvs is an independent predictor of mid-term graft occlusion in cabg patients and a cumulative mvscore stratifies patient's risk. since the mv signature mirrors platelet activation, patients with a high mvscore would benefit from a personalized antiplatelet therapy. exosomes from engineered immortalized human heart cells improve ventricular function and attenuate fibrosis in mice with arrhythmogenic cardiomyopathy yen-nien lin, lizbeth sanchez, rui zhang, thassio ricardo ribeiro mesquita, chang li, ahmed ibrahim, eduardo marbán and eugenio cingolani heart institude, cedars sinai medical center, los angeles, usa introduction: arrhythmogenic cardiomyopathy (ac) is characterized by progressive loss of cardiomyocytes and fibrofatty tissue replacement. currently, there is no effective treatment for this disease. exosomes (imexos) secreted by heart stromal cells, engineered to be immortal and overexpressing β-catenin, exert anti-inflammatory and anti-fibrotic effects and improve ventricular function in models of ischaemic injury (ibrahim et al., nature bme ). methods: to investigate the effectiveness of imexos in a murine model of ac, four-week old homozygous dsg knockout (dsgko) mice and wild type (wt, age-and strain-matched) mice were compared. dsgko mice were randomized to receive weekly imexos or vehicle via intravenous injection for weeks. neonatal rat ventricular myocyte (nrvm) proliferation and apoptotic assays were performed to explore potential effects of exosomes. results: biodistribution studies of dir-labelled imexos revealed some cardiac uptake, along with strong signals in spleen. at weeks, dsgko mice which had received intravenous imexos showed improved cardiac function (echocardiographic ejection fraction ± vs ± % in vehicle mice, p = . ), with an underlying attenuation in myocardial fibrosis by histology. electrophysiology test showed shorter qrs duration ( . ± . ms imexo vs . ± . ms vehicle, p = . ) and effective refractory period. programmed ventricular stimulation showed dsgko mice which had received imexos were remarkably less prone to ventricular tachycardia induction ( . ± % vs . ± % in vehicle, p = . ). in vitro study showed nrvm exposed to imexos for days exhibited higher brdu expression relative to vehicle group, and less annexin-v expression after oxidative stress induced by -minute illumination with nm uv. summary/conclusion: intravenous administration of imexos improved cardiac function, reduced cardiac fibrosis, and suppressed arrhythmogenesis in ac. our findings motivate clinical testing of imexos in ac, an orphan disease with great unmet medical need. funding: nih r hl (to em) cardiac-derived extracellular vesicles contribute to communication between heart and brain in chronic heart failure (chf) and target nrf / are signalling changhai tian a , lie gao b and irving zucker b a department of cellular and integrative physiology, university of nebraska medical center, omaha, usa; b department of cellular and integrative physiology, university of nebraska medical center, omaha, usa introduction: mirnas regulate the translation of proteins that are involved in redox homoeostasis in the heart and brain. intra-and/or inter-organ communication takes place by multiple mechanisms including extracellular vesicular (ev) transport. our previous studies suggested that cardiac derived mirna-enriched evs contribute to the dysregulation of nrf /antioxidant enzyme (are) signalling in the myocardium via intercellular cross-talk, and result in the decreased nrf /are signalling in the sympatho-regulatory areas of the brain in chf. however, it is unclear if cardiac derived evs circulate to the central nervous system evoking sympatho-excitation by disrupting central redox homoeostasis. methods: cardiac-specific membrane gfp+ mice were generated to track the brain distribution of cardiac evs in rats with chf (coronary ligation). the isolation and characterization of evs were carried out by differential ultracentrifugation, tem, nanosight, western blotting, and qrt-pcr. transfection, labelling, and microinjection of evs into the rostral ventrolateral medulla (rvlm) were performed. results: nrf protein was reduced in the rvlm of chf rats consistent with an upregulation of nrf -targeting mirnas. nrf -targeting mirnas were enriched in cardiac and circulating evs of chf rats. nrf -targeting and cardiac-specific mirnas were abundant in brain-derived evs. circulating evs were taken up by neurons in sympatho-regulatory areas of the brain. mirna-enriched evs from chf animals increased sympathetic tone which was prevented by a cocktail of nrf -targeting mirna inhibitors. summary/conclusion: myocardial infarction-induced mirna-enriched evs mediate the inter-organ crosstalk between heart and brain in the oxidative regulation of sympathetic outflow through targeting the nrf / are signalling pathway. these findings suggest that cardiac-derived ev mirnas targeting nrf /are signalling may act as an endocrine signalling mediator of chf that has potential as a novel therapeutic target. introduction: a fine-tuned communication between cardiac cells is vital to maintain myocardial integrity and contractility. not only an impairment of gap junction (gj)-mediated intercellular communication, but also defects in ev-mediated communication have been associated with ischaemic heart disease, a major causative factor of heart failure. we have previously shown that cx , the main ventricular gj protein, assembles into channels at the evs surface, mediating the release of vesicle content into target cells.the main objective of this work was to characterize the signals underlying protein sorting into extracellular vesicles (evs) in a human pathophysiological context, using connexin (cx ) as a model substrate. methods: animal models of ischaemia/reperfusion (i/ r) injury by ligation of the left anterior descending coronary artery, ex vivo and in vitro ischaemia models and human patients were used to investigate the secretion of ev-cx . results: release of cx was downregulated in circulating vesicles from i/r-injured mice and patients with st-segment elevation myocardial infarction, as well as in intracardiac and cardiomyocyte-derived evs. additionally, we show that ubiquitin signalled the release of cx in basal conditions but appeared to be dispensable during ischaemia. depletion of the autophagy adaptor p partially restored the secretion of cx , suggesting an interplay between ischaemiainduced cx degradation and secretion. summary/conclusion: overall, we demonstrated that ischaemia impairs the sorting of cx into evs, which may ultimately affect long-distance communication. through the identification of the underlying molecular mechanisms and players, these results pave the way towards the development of innovative diagnostic and therapeutic strategies for cardiovascular disorders. introduction: remote ischaemic conditioning is a cardioprotective intervention which protects the heart against ischaemia/reperfusion injury. transient activation of toll-like receptor (tlr ) and its downstream regulators (tnfα and il- ) have been implicated in cardioprotective interventions. extracellular vesicles (evs) play a role in cardioprotection through the activation of the tlrs. however, since isolation of evs in high amounts with suitable purity from blood is a challenge, our aim was to develop a cellular model system from which tlr-inducing, cardioprotective evs can be isolated in a reproducible manner. methods: ev release from hek cells was induced by calcium-ionophore a . evs were characterized, cytoprotection by evs against simulated ischaemia/ reperfusion injury and its mechanism were investigated in h c and ac cell lines. results: a induction of hek cell induced ev release and the isolates contained mostly large evs. evs decreased cytotoxicity and apoptosis due to h ischaemia followed by h reperfusion in h c and ac cells in a dose-dependent manner. evs activated tlr and its downstream signalling pathway in h c and ac cells as well as the expression of cytoprotective haem oxigenase (ho- ) in h c cells. summary/conclusion: a -induced evs exert cytoprotection in h c and ac cells by inducing tlr signalling and ho expression. therefore, evs released via calcium-ionophore treatment may serve as a basis of an efficient carpdioprotective therapy. introduction: biliary strictures may be benign or malignant. the major malignant causes of biliary stricture are a primary cholangiocarcinoma (cca) or pancreatic ductal adenocarcinoma (pdac). there is ongoing debate about adequate diagnostics in biliary strictures of unknown aetiology. micrornas (mirnas) are small non-coding rnas important in tumourigenesis. mirna have been found to be enriched in exosomes, small membrane-bound extracellular vesicles (ev) of endocytic origin, which is a novel pathway for intercellular signalling within the tumour microenvironment and have been implicated in loco-regional pre-metastatic niche formation. this project aims to investigate circulating-free and ev mirnas as biomarkers that can aid diagnosis in patients with a biliary stricture. we will ( ) isolate and characterise evs in plasma and bile from patients with benign and malignant biliary strictures (i.e. pancreaticobiliary cancers); and ( ) identify differentially expressed circulating-free and ev mirnas in plasma and bile suitable for detecting malignancy. methods: sample size (n = ) was calculated for a study power of % and α error of % for the ability of extracellular mirnas to discriminate benign from malignant biliary strictures. prospective matched plasma and bile samples will be collected from patients with benign (n = ) and malignant (n = ) biliary strictures undergoing endoscopic retrograde cholangiopancreatography (ercp). evs will be isolated from the biofluids by ultracentrifugation and/or size exclusion chromatography and then characterised (tem, nta and immunoblotting). circulating-free and ev-associated mirnas will be profiled using small rna sequencing. extracellular mirna "signatures" will then be validated by rt-qpcr, and diagnostic accuracy confirmed (sensitivity, specificity, auc). results: evs derived from patient samples have been characterised using nta, western blotting and tem. sec derived evs appear to be more well-defined than uc evs with marker positivity for cd , cd and cd . ongoing work will be focused on rna profiles of evs from both malignant and benign cohorts. summary/conclusion: there is currently no effective method to differentiate benign from malignant biliary strictures. novel plasma and bile circulating-free and ev-associated mirna biomarkers may improve the speed and accuracy of diagnosis, resulting in considerable patient benefits. furthermore, as little is known about the ev-associated function of these tumours, candidate ev-mirnas could be taken from "bedside to bench" and their function further investigated using in vivo, vitro and silico models. introduction: urine is a source of extracellular rna (exrna) biomarkers that can be obtained non-invasively throughout pregnancy. several studies have profiled extracellular mirnas in biofluids during pregnancy, but few have profiled extracellular mrnas (ex-mrnas) in urine. objective: to optimize methods for ex-mrna isolation and rna-seq library preparation from urine of healthy pregnant and non-pregnant females. methods: rna was isolated from pooled non-pregnant urine using kits based on ev precipitation (mircury exosome kit for csf/urine, seramir), ev affinity purification (exorneasy), and protein precipitation (mirneasy serum/plasma advanced). next, long (> nt) and short rnas were isolated from ev enriched urine of pregnant (n = ) and non-pregnant (n = ) individuals using the mircury kit followed by the mirneasy micro kit. rna-seq libraries were prepared using the smart-seq v ultra low input rna (oligo(dt) priming) and the smarter stranded total rna-seq kit v -pico input (random priming) methods (takara). preliminary data were obtained using the illumina miseq, and aligned using star v. . . .a. results: overall, rna isolation using mircury followed by the smart-seq v library preparation kit yielded the highest % of mapped reads: % in pooled non-pregnant, % in individual non-pregnant, and % in individual pregnant urine. for rna extracted using the mircury kit, the smart-seq v libraries had higher % of mapped mrna reads compared to pico libraries (p < . , t-test). in contrast for mirneasy advanced it was reversed ( % vs %). summary/conclusion: early results from low-depth sequencing show the highest mrna mapping rates for mircury followed by the smart-seq v kit. high-depth sequencing data are now being generated, which will enable us to perform detailed comparisons of different rna species from the rna profiles obtained using different library preparations and rna isolation methods from urine of pregnant and non-pregnant subjects. funding: this study was funded by nih k hd - , nih u hl , and a ucsd igm-illumina mini-grant. il- mutein-induced changes of exosomal mirna cargo in a humanized mouse model emily lurier, erik sampson, patrick halvey, mike cianci and katalin kis-toth pandion therapeutics, cambridge, usa introduction: regulatory t cells (tregs) are key contributors to immune homoeostasis. decreased number and/or function of these cells are frequent features of many autoimmune diseases linked to the development of tissue inflammation. while interleukin- (il- ) is essential for pan t cell proliferation and performance, low dose il- treatment has been shown to preferentially affect tregs and is being evaluated as an intervention in autoimmune diseases. pt is a novel il- mutein fc fusion molecule (il- m) designed to selectively engage with tregs. using a humanized nod-scid il rn-null (nsg) mouse model we have shown that pt expanded tregs without significant effects on other immune cells. we have also shown that tregs from pt -dosed humanized mice exhibit increased expression of foxp and cd , and demethylation of foxp and ctla- genes, suggesting enhanced function and stability. in the current study we investigated the mirna content of plasma exosomes isolated from pt -or vehicle-treated mice in order to identify treg specific mirnas from the il- m treated animals. methods: cd + haematopoietic stem cell humanized nsg mice were dosed once subcutaneously with pt or vehicle. plasma samples from mice were collected at day and exosome isolation was conducted using the exoquick method. small rna was extracted and quantified using the bioanalyzer small rna assay. an illumina nextseq instrument was used for library preparation and sequencing with bp single end reads at an approximate depth of - million reads per sample. raw sequences were mapped to human genome grch and analysed via a pipeline provided by the university of california santa cruz. results: rna within the exosomes from vehicle and il- m-treated groups was mostly comprised of mirna and trna. plasma was pooled from animals per treatment group and differential expression was determined using a twofold change cut-off. we found that pt treatment actively altered the mirna content of plasma exosomes, compared to exosomes from vehicle-treated mice. many of the differentially expressed mirnas are involved in immunoregulation. summary/conclusion: plasma exosomes from pt treated humanized mice encapsulated treatment-specific mirnas which can potentially be used as systemic biomarkers of treg expansion and function. identification of potential biomarkers in microglial specific exosomes isolated from prion-infected serum introduction: transmissible spongiform encephalopathies (tse) are neurodegenerative disorders caused by the misfolding of the cellular prion protein (prpc) to the beta-sheet rich abnormal prion protein (prpsc). prpsc aggregates in the brain and causes amyloid plaques, neuronal loss, spongiform degeneration and microglial activation. currently, definitive diagnosis of tse diseases is only confirmed post-mortem thus a diagnostic test in accessible body fluid is of interest. exosomes are a good resource for biomarker discovery since they cross the blood-brain barrier easily and contain protein, lipids and nucleic acids from the cells of origin. the goal of this study was to look at biomarkers from brain-originating exosomes (specifically microglia) isolated in the serum of prion-infected animals. methods: westerns and nanoparticle tracking analysis (nta) were used to look at the composition of microglial-specific exosomes. as proof of principle, exosomes were isolated from a microglial cell line (bv cells). a cd antibody was labelled with a fluorophore and binding to exosomes was visualized via nta. exosomes were isolated from serum of both prioninfected and mock-infected mice throughout disease course. a macrophage specific antibody (f / ) was bound to beads which were used to isolate exosomes which includes those of microglial origin. microrna was extracted from these exosomes and next-generation sequencing (ngs) was performed using the illumina platform. clc genomics workbench was used for bioinformatics analysis. results: microglial and macrophage proteins (tmem and iba ) were identified in exosomes isolated from bv cells and prion-infected mouse serum. macrophage exosomes were isolated via a novel antibody-bead based system. results of the ngs analysis of the microrna isolated from these exosomes indicated a series of mirna that could differentiate between control and infected samples as well as age-specific markers. summary/conclusion: to our knowledge, this is the first time microglial-specific exosomes have been isolated from prion-infected serum from early and end stage disease. the results of this analysis could facilitate the diagnosis of prion disease in easily-accessible biofluids pre-mortem. comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease introduction: urinary extracellular vesicles (uevs) are emerging as a source for early biomarkers of kidney damage, holding the potential to replace the conventional invasive techniques including kidney biopsy. several methods are available for uev isolation. our aim was to compare different workflows and isolation by hydrostatic filtration dialysis (hfd), ultracentrifugation (uc) and a kit based isolation method for their subsequent use in mirna-seq and rna-seq for biomarker discovery in diabetic kidney disease. methods: type diabetic patients (t d) with macroalbuminuria and normoalbuminuric healthy controls were included in the study. sample collection and all experiments were performed in accordance with the declaration of helsinki. evs were isolated from - ml of h urine collections by uc, hfd, or a commercially available kit (purification based on spin column chromatography, urine exosome purification and rna isolation midi kit, norgen biotech, canada) each with different established urine clarification steps. quality control of the evs was performed with negative staining em, nta and western blotting. isolated rnas were profiled with bioanalyzer pico kit and subjected to mirna and mrna sequencing. for rna-seq, cdna library was prepared using smart-seq v ultra low input rna kit for sequencing (takara bio, japan). rna-seq was performed using hiseq (illumina). mirna-seq library was prepared using qiaseq mirna library kit (qiagen, germany). mirna-seq was performed on the illumina hiseq platform (illumina). results: our data showed that uev yield, morphology and size distribution were closely similar in hfd and uc preparations, while lower yields were obtained using the kit. by western blot, ev markers were detectable in samples isolated by hfd and uc but not readily in samples isolated with the kit. tamm-horsfall protein was detected in all the samples and albumin levels appeared higher in hfd and kit isolated samples relative to uc samples. the number of paired-end reads for rna-seq in hfd and uc samples (in both > m) were closely similar. instead, rna reads were lower than m for the kit samples. for mirna-seq, the number of reads as well as the molecular biotype distribution were similar for the three methods. by principal component analysis of the rna-seq data, we observed that hfd and uc grouped together showing similarities. however, for mirna-seq data such similarities were not obvious. this suggests that the three different workflows and isolation principles may enrich different mirna-rich uev preparation components. summary/conclusion: our transcriptomics data shows that hfd and uc are suitable methods to isolate uevs for mirna-seq and rna-seq. the kit based method appears better suited for mirna-seq. introduction: exosomes contain a variety of biomolecules including dna. knowledge of cfdna distribution and localization in bioliquid is important for understanding both biological function of cfdna and exosomes. some publications state that a large proportion of plasma cfdna is localized in exosomes. to quantify cfdna content in free vs. exosomal form in human plasma, urine, and saliva, we employed subx technology, which allows affinity capture dna via phosphates groups of the polynucleotide chain and exosomes via membrane surface phosphate moiety clusters. subx is a proprietary compound that can simultaneously bind to both cfdna and exosomes in bioliquids, thus allowing precipitation of the [subx-dna/ subx-exosomes] complexes without ultracentrifugation. methods: detection of subx-dna and exosomes binding was done by measurement of particle sizes using zetasizer nano zs and nanosight ns . the samples were processed with the subx exo-dna isolation kit following the standard protocols. dna, protein and lipid concentrations were measured by fluorescent assays using qubit fluorometer. results: subx efficiently and selectively captures and co-precipitates cfdna and exosomes directly from bioliquids. exosomes are easily extracted from the pellet in exosome reconstitution buffer (erb), followed by subsequent isolation of tightly bound cfdna from the subx pellet. erb does not extract dna form the [subx -dna] pellet and thus does not contaminate reconstituted exosomes with cfdna. thus, we separate two distinct types of extracellular materialintact exosomes and purified cfdna in a single protocol from the same sample. over % of dna in plasma and urine exist as a free circulating pool, while in saliva up to % is associated with exosomes. thus, cfdna distribution is probably bioliquid-specific and must be evaluated by methods that eliminate cfdna-outer exosomal membrane aggregation. summary/conclusion: subx technology is suitable for simultaneous isolation of both cfdna and exosomes from the same bioliquid sample. subx separates cfdna fragments non-specifically attached to the outer lipid layers of the exosome membrane from the true intra-exosomal cfdna. in contrast, salting-out peg technique is associated with aggregation of macromolecules and vesicles and thus leads to overestimation of exosome-associated polymers content, including cfdna. tracing extrachromosomal dna inheritance patterns in glioblastoma using crispr eunhee yi, amit gujar, hoon kim, albert cheng and roel verhaak jackson laboratory for genomic medicine, farmington, usa introduction: glioblastoma multiforme (gbm) is the most lethal brain tumour; it is characterized by poor response to standard post-resection radiation and cytotoxic therapy, resulting in a dismal prognosis with a five-year survival rate of %. recurrence after therapy for gbm is unavoidable. there are substantial differences among the cells of gbm tumours in the abundance and types of genetic material. this heterogeneity likely is the major cause of therapy failure, the development of treatment resistance, and ultimately recurrence. a recent study has suggested that the amount of a particular type of dnaextrachromosomal dna (ecdna)differs substantially among different gbm tumours, and differs within a given gbm tumour over time. despite the speculation that ecdna is a key factor of tumour heterogeneity, how ecdna is propagated and distributed amongand how it behaves withincancer cells is completely unknown. methods: to address this gap in knowledge, this study focused on developing a novel cytogenetic crisprbased tool that enables visualization and tracking ecdna behaviour in live gbm cells. results: we found breakpoint sequences resulting from genome rearrangements during ecdna formation by performing computational analysis from whole genome sequencing data. and each breakpoint was regarded as a unique target sequence for ecdnaspecific labelling. the uniqueness of each breakpoint was validated by breakpoint-pcr (bp-pcr). furthermore, the location and the amount of each breakpoint were observed by breakpoint-fish (bp-fish) analysis in gbm cells. summary/conclusion: this results will be strong evidence to make ecdna-specific crispr system in further research. tracing ecdna dynamics will provide new insight into the impact of ecdna on cancer evolution. introduction: small extracellular vesicles (sevs) are - nm vesicles that mediate intercellular communication by transferring rna and proteins to the recipient cells. these cargo molecules are selectively sorted into sevs and mirror the physiological state of the donor cells. given that sevs can cross the bloodbrain barrier and their composition can change in neurological disorders, there is an increasing interest in elucidating the molecular signatures of sevs in circulation as disease biomarkers. however, circulating sevs are derived from multiple cellular sources and determining their source is challenging. information on sev composition can be beneficial in predicting whether these sevs are released predominantly from central nervous system cells. we hypothesized that differentially expressed mirnas between neuronal sevs and astrocytic sevs could be used as cell-typespecific signatures. methods: small extracellular vesicles were isolated from cell culture media of postnatal mouse primary neurons and astrocytes using differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna from neurons, astrocytes, and their respective sevs were used for transcriptome and small rna sequencing. results: we observed that only a subset of cellular mirnas was packaged into sevs; differential expression of specific mirnas between sevs and their corresponding cells suggest that cells employ special mechanisms to sort mirnas into sevs. these mechanisms could be celltype specific since neuronal sevs showed a different mirna profile compared to astrocytic sevs. exomotifs, the short sequence motifs that control the loading of rna into sevs, were present in differentially expressed mirnas. we also observed that five rnabinding proteins, which are associated with passive or active rna sorting into sevs, were differentially expressed between neuronal and astrocytic cells. summary/conclusion: mirna signatures of sevs from neurons and astrocytes could be beneficial in determining if these cell types contribute to the alterations of sev composition in circulation in neurological disorders. cell-type-specific selectivity in rna loading might be attributed to the differential expression of rna-binding proteins. introduction: analytes present in the extracellular fraction of bodily fluids (ex. blood, urine) have utility as a tool for uncovering the molecular landscape of tumours and hold great potential for discovery of individualized cancer medicine. urine, being noninvasive as a sample type, has an obvious advantage over blood when used for liquid biopsy purposes. however, potential for microbial proliferation and the labile nature of host cells and extracellular vesicles (evs) at the point of sample collection/transport to the lab drives the need for stabilization of urine samples. development of such sample stabilization opens up capability for the detection of various biomarkers present in the extracellular fraction to be used in liquid biopsy. this is of particular concern as studies around urinary analytes for cancer diagnosis, progression and therapeutic effect are rapidly expanding in cohort sizes. multi-site collections and at-clinic collections are increasingly prohibitive for large scale recruitment and also lead to variability in the time between collection and processing. methods: in this study, we have analysed two commercially available ev extraction kits and compared them with ultracentrifugation technique for size, concentration and specificity of the isolated evs from human urine samples with and without our proprietary preservation solution using nanoparticle tracking analysis and western blot analysis for exosomal membrane markers. ev rna contents in various urine fractions (first morning first void, random first void and midstream) were compared using rt-qpcr assay to provide better understanding of the collection techniques and fractionations that are ideal for ev research work. results: in our current work, we have bench-marked human urine collection and ev extraction in order to provide recommendations in standardization of sample acquisition and processing for urinary ev studies. we have utilized these standardization in order to develop a novel and efficient sample stabilization principle for preservation of evs and ev rna in urine samples during an ambient temperature hold. summary/conclusion: taken together, we have established a framework for evaluating technologies and techniques in the ev sample processing space, which can be utilized by other research groups. vn -isolated plasma extracellular vesicles improve tumour mutation detection by next-generation sequencing compared to cell-free dna and correlate with tissue biopsy of nsclc patients introduction: liquid biopsy is a minimally-invasive diagnostic method that detects circulating biomarkers and has the potential to improve access to molecular profiling for nsclc patients when tissue biopsy material is unavailable or insufficient. although isolation of cell-free dna (cfdna) from plasma is the standard liquid biopsy method for detecting dna mutations in cancer patients, the sensitivity can be highly variable. vn is a amino acid peptide with an affinity for heat shock proteins that are exposed on the surface of extracellular vesicles (evs); peptide-ev aggregates readily sediment using a benchtop centrifuge and therefore the vn peptide provides a rapid, clinically-amenable procedure for ev isolation. in this study, we determine whether isolation of evs from nsclc patient plasma improves the sensitivity of single nucleotide variants (snvs) detection compared to cfdna and correlate genetic changes observed by liquid biopsy with tumour ffpe tissue biopsy. methods: blood was collected from stage iii/iv nsclc patients with informed consent in either edta or cell-free dna bct® collection tubes and plasma was harvested within minutes. total nucleic acid (tna) was extracted from either vn -isolated evs from edta plasma or directly from plasma collected in edta or cell-free dna bct® tubes (cfdna). snvs were detected by next-generation sequencing (ngs) results: vn isolation of evs from plasma resulted in higher recovery of dna than cfdna isolation. the snvs detected in both ev-dna and cfdna correlated well with those reported in matched ffpe tumour tissue using ngs, including % specificity for egfr mutations. no improvement in snv detection was observed using cell-free dna bct® collection tubes compared to edta tubes. isolation of evs with the vn peptide prior to sequencing improved a number of ngs parameters including library yield, total reads, median read coverage and molecular coverage, resulting in improved sensitivity of snv detection. summary/conclusion: in summary, our research demonstrates that vn -based ev isolation is useful for molecular profiling of nsclc patients for whom tissue biopsy is not an option, thereby improving access to molecular profiling and targeted therapies. funding: atlantic canada opportunities agency novel markers for neuroendocrine prostate cancer divya bhagirath a , michael liston b , theresa akoto a and sharanjot saini a a augusta university, augusta, usa; b veteran affairs, san francisco, usa introduction: prostate cancer (pca) is fuelled by androgens and androgen receptor (ar) signalling. therefore, ablation of ar signalling by androgen deprivation therapy (adt) is the goal of first-line therapy that results in cancer regression initially. however, two to three years post-adt, the disease develops into castration-resistant prostate cancer (crpc). as a second-line of therapy, next generation of ar pathway inhibitors (api) such as enzalutamide (enz) are used that are effective initially followed by emergence of drug resistance. a subset of api-resistant tumours emerges to an ar independent state via undergoing a trans-differentiation to neuroendocrine lineage, a process referred to as neuroendocrine differentiation (ned). due to lack of ar signalling, these pca variants, referred to as neuroendocrine prostate cancer (nepc), are impervious to anti-androgen therapy and constitute an aggressive variant of advanced crpc with poor prognosis. currently, there is a lack of effective molecular biomarkers for predicting api therapy resistance and emergence of therapy-induced ned. methods: exosomes/evs were isolated from sera of a patient cohort with/without ned. the study was conducted in accordance with ethical guidelines of us common rule and was approved by the institutional committee on human research. written informed consent was obtained from all patients. following extensive characterization of evs by electron microscopy, nanosight tracking analyses and western blotting of exosomal markers, small rna sequencing was carried out on illumina hiseq platform to identify differentially expressed transcripts. machine learning algorithms were applied to clinical sequencing data to train a "mirna classifier". further, we probed the proteomic profile of exosomes isolated from nepc cellular model nci-h and enzalutamide resistant crpc cell lines by mass spectrometry. results: we identified that transition from crpc-adenocarcinomas to neuroendocrine states is associated with significant ev-mirna dysregulation, with a specific dysregulation in certain mirna families. with the application of machine learning algorithm, we identified an ev-based "molecular classifier" that can robustly stratify crpc-ne tumours from crpc-adenocarcinomas. proteomic analyses identified novel nepc-specific, glycosylated proteins that can be exploited for nepc diagnosis. summary/conclusion: our data suggest that ev mirna and protein profile can predict neuroendocrine differentiation in advanced castration-resistant prostate cancer patients. exosomal mrna in diagnosis strategy for hepatocellular carcinoma aleksandr abramov, alisa petkevich, vadim pospelov and pavel ogurtsov peoples' friendship university of russia (rudn university), moscow, russia introduction: exosomal cargo is informative source illustrating the genetic events happening in cells, what can be especially advantageous in case of cancer development for disease progression or treatment effectiveness monitoring. methods: plasma samples of hepatocellular carcinoma (hcc) patients, plasma samples of patients with liver cirrhosis - on the hepatitis c virus (hcv) background, healthy donors' plasma samples. exosomes were isolated with ultracentrifugation, western blot (cd , cd ) was performed. total mrna was isolated with exosomal rna isolation kit, norgen biotec corp. sequencing was carried out on a minion sequencer. housekeeping genes (gapdh, b m, actb, tuba a). detected mutations were confirmed by real-time pcr with specific highly sensitive lna probes. results: significant changes in expression levels were identified for genes in hcc and liver cirrhosis groups (increasing up to x compared to control samples and decreasing up to no detected expression). in out of patients with hcc mutant burden was significant increased compared to mutant burden in groups with cirrhotic samples. in out of patients with hcc increased expression for mrna line- was identified compared to cirrhotic patients. summary/conclusion: exosomal mrna expression levels may serve as a prognostic and diagnostic marker for patients with liver cirrhosis caused by hcv for hcc risk development. funding: research is supported with federal funds " - " circulating extracellular vesicle signatures in small cell lung cancer michela saviana a , giulia romano a , giovanni nigita b , robin toft a , patricia le a , kai wang c , mario acunzo a and patrick nana-sinkam a a virginia commonwealth university, richmond, usa; b the ohio state university, columbus, usa; c institute for systems biology, seattle, usa introduction: lung cancer is the leading cause of cancer deaths worldwide and classified primarily as either non-small cell lung cancer (nsclc) or small cell lung cancer (sclc). compared to nsclc, sclc has a faster growth rate, earlier widespread metastasis, and shorter overall survival. the early diagnosis of sclc and the development of novel therapeutics have proven challenging. thus, progression and recurrence rates remain high. non-invasive methods for cancer detection are increasingly being used to inform clinical decision making. extracellular vesicles (evs) have recently emerged as potential carriers of genetic contents such as micrornas (mirs) to induce reprogramming of components of the microenvironment in cancer initiation and progression. moreover, extracellular mirs expression profiles have been shown to have signatures related to tumour classification, diagnosis, and progression. methods: we selected a cohort of patients divided into groups: high-risk smokers, adenocarcinomas, squamous carcinomas, and sclc. we extracted total circulating ev and plasma rna from plasma ( patients in total) and rna from plasma in a separate group ( patients in total). utilizing both next-generation sequencing (ngs) and nanostring platforms, we analysed for global microrna (mirs) expression patterns. candidate mirs were then validated by qrt-pcr. results: we identified several deregulated mirs in both evs and plasma of sclc patients compared to the other groups. for evs, we validated mir- - p as a significant biomarker for the late stage of sclc compared to controls. in the case of plasma, we validated the upregulation of mir- in sclc compared to controls. summary/conclusion: our results indicate that a potential combination of plasma (mir- ) and ev-based (mir- - p) mirs be valuable biomarkers for sclc detection and serve as a basis for a non-invasive sclc classifier. funding: virginia commonwealth university, doim -nih/nci introduction: the isolation of evs from milk is technically challenging due to the complexity of milk. currently used separation procedures allow for the removal of milk fat globules and cells (by low speed centrifugation of fresh milk), removal (by acidification), or disruption (by addition of edta) of casein micelles. using these protocols the integrity, composition and targeting of bovine milk evs has been evaluated and has led to believe that milk evs might withstand these conditions. however, the effects on functionality of milk evs (i.e. immunomodulatory properties) after processing and isolation have not been studied. therefore, we have set up an in vitro culture system using a human t cell line that allows for the rapid screening of milk ev functionality. methods: fresh bovine milk was defatted and cells were removed after x , g centrifugation, followed by differential centrifugation at , g and , g. this milk was either subjected to acidification with hcl, or edta was added, or the milk supernatant remained untouched. top down optiprep density gradient separation followed by sec was used to further purify evs. these highly purified milk evs were added to human jurkat t cells, which were simultaneously stimulated using anti-cd and anti-cd antibodies. after h t cell activation was measured by il- cytokine production. results: precipitation or disruption of casein micelles allowed for the substantial removal of proteins during isolation compared to directly isolated evs, which aids in the purification of milk evs. in vitro analysis revealed that in the presence of directly isolated, or edta isolated milk evs, jurkat cells were suppressed in their activation as measured by il- production. remarkably, evs isolated from hcl-acidified milk were impaired in their suppressive capacity to inhibit il- production. summary/conclusion: although casein removal from bovine milk greatly improves purity of isolated milk evs, the detrimental effects on ev functionality should be considered. interestingly, evs exposed to acidic conditions lost their ability to modulate t cell activation, which is in contrast with the general believe that milk evs could withstand the gastro-intestinal tract. funding: this work is funded by the european union's horizon framework programme under the grant fetopen- evfoundry. optimising methods for separation and characterisation of extracellular vesicles from skim milk and infant milk formula introduction: infant milk formula (imf) is intended to impart nutrition to infants, similar to breast milk. however, although industrial imf production involves harsh treatment, potential consequences on extracellular vesicles (ev) in imf are not yet established. this study aimed to optimise methods for separating evs from imf and skim milk (sm) and to characterise the evs in accordance with misev . methods: sm and imf were either not treated (nt) or treated with acetic (aa) or hcl acid (isoelectric precipitation, ip), to remove caseins. samples were then subjected to differential ultracentrifugation (duc) or gradient ultracentrifugation using iodixanol solution (guc). for duc, ml samples were centrifuged at k g, k g, k g, k g and k g sequentially for min each and pellets re-suspended in ml pbs. preparation of agarose microspheres for high-efficient separation of extracellular vesicles cheng-tai chen, chien-an chen, carolyn yen and nien-tzu chou industrial technology research institute, chutung, taiwan (republic of china) introduction: size exclusion chromatography (sec) is becoming a widely used technique for separating of extracellular vesicles. various commercially available products were launched on the market, however, their separation efficiencies were not fully disclosed. herein, novel porous agarose microspheres with the tunable diameter and pore size were synthesized by emulsion reaction. the performance was evaluated and compared with commercial products. the modified sec column packing materials were shown to exhibit advantages for rapid, high-recovery and high-purity separation of extracellular vesicles from cell culture-conditioned medium and human plasma. methods: the homemade sec column was packed by gravity flow. μl of the sample was loaded and the pbs buffer was used as eluent. factions were collected and analysed by cd /cd sandwich elisa assay and by micro bca assay for determining respectively extracellular vesicles and total protein content. results: agarose microspheres were prepared by emulsification. the particle size can be controlled by the types and concentrations of surfactants. the product was collected by desired screen meshes and used as packing materials of the sec column. our results showed that the extracellular vesicles were clearly separated from proteins. more than . % of proteins were removed while the recovery of extracellular vesicles was close to %, which is much higher than % of the commercial product. the total separation time was less than min. summary/conclusion: we have established an approach for generating spherical agarose microspheres as packing materials of homemade sec columns, which are capable of separating extracellular vesicles from complex samples with high efficiency. further validations with additional samples are currently ongoing. immunomagnetic sequential ultrafiltration (isuf) platform for enrichment and purification of extracellular vesicles from large and small volumes of biofluid eduardo reategui, jingjing zhang, luong t. h. nguyen, richard hickey, nicole walters and andre f. palmer the ohio state university, columbus, usa introduction: evs derived from tumour cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. however, compromises have to be made when using a particular technology/methodology for the isolation of evs. currently, there is a trade-off between sample volume and specificity in ev isolation technologies that limits quantitative molecular analysis of ev contents, ultimately impacting the utility of evs in cancer diagnostics. here, we present an approach called immunomagnetic sequential ultrafiltration (isuf). our platform combines ultrafiltration and immunoaffinity separation. using isuf, we demonstrate that small or large volumes of biofluid can be processed (~ µl or > ml) while concomitantly removing . % contaminating proteins. we also processed serum from breast cancer patients enabling the characterization of different tumour and immune biomarkers on the isolated evs. methods: human samples were collected under an approved irb. size distribution and concentration of evs were measured using a tunable resistive pulse sensing (trps) method. ev proteins and rnas were extracted and quantified using a bca protein assay and uv spectroscopy. isuf and other ev isolation methods were compared for ev concentration, protein, and rna quantity. results: ml of cell culture media (ccm), . ml serum, and ml urine samples were processed with the isuf platform and recovered in µl. for all cases, evs were enriched with recovery efficiency greater than %. the processing time for a ml sample was min with over % of purity. we compared ev concentration and purity isolate from . ml serum using isuf and other commercially available methods, isuf demonstrated superior performance on isolating evs at high concentrations and purities. analysis of total rna amounts in the isolated evs using different methods was corresponding to higher ev recovery efficiency of isuf. we also compared protein and rna levels of evs enriched with isuf present in urine and serum samples from the same donors (n = ), and we found that for the same number of evs, the ev rna concentration from both biofluids showed no significant difference. finally, we have processed serum samples from metastatic breast cancer patients and demonstrated that their isolated evs have expression levels of her , cd and mir biomarkers at significantly higher levels than healthy controls. summary/conclusion: the isuf platform can be scale-down or -up to work with small or large volumes of biofluids for the isolation of evs. using the isuf platform with clinical samples shows the potential of our platform to be used for cancer diagnosis or monitoring treatment response. funding: national institutes of health (nih) grants ug tr (e.r.); r hl , r hl , and r eb (afp). challenges in exosomes isolation from primary biological samples derived from multiple myeloma patients introduction: multiple myeloma (mm) remains incurable despite advances in its treatment and research progress on the crosstalk between mm and surrounding host cells. exosomes are important regulators of the cellular niche. their importance for diagnostic and therapeutic applications has been proven in many cancers. in this context we hypothesized that a better understanding of the molecular role and features of mm-derived exosomes would provide a basis for their use for both risk stratification and as predictive biomarkers of response to anti-mm drugs already in use in clinical settings, given the optimization and validity of their isolation/purification method. methods: exosomes were isolated from human mm cell lines (hmcls) supernatants and peripheral blood plasma (pbpl) isolated from healthy donors, mm and mgus (monoclonal gammopathy of undetermined significance) patients. both fresh and frozen samples were tested. we evaluated commercially-available kits, density-based separation and ultracentrifugation. results: higher purity and recovery, evaluated by western blotting, nanoparticle tracking analysis and electron microscopy, were observed for supernatant density-based purification and for pbpl resin-based isolation. exploring the function of mm-derived exosomes, we observed an increase in proliferation of the immortalized stromal cell (sc) line hs treated with exosomes when compared to untreated cells, and a higher increase in proliferation of scs treated with mm-exosomes when compared to exosomes derived from normal and mgus pbpl samples. summary/conclusion: the method of isolation represents a critical step in the study of exosomes as many factors can affect the purity, yield and downstream application. our data demonstrated that density and resin-based isolation methods provided functional mm-derived exosomes with proliferative effects on scs. altogether our findings may serve as a guide to choose exosome isolation methods for mm studies. further optimization steps, including albumin-depletion from plasma samples and use/type of serum in cell cultures, should be taken into consideration when planning proteomics and genomics as downstream applications. funding: australian government rtp and monash departmental scholarship. a rigorous method for exosome isolation from post-mortem eyes introduction: in order to determine and validate the tissue-specific content of extracellular vesicles (evs) in biofluids, robust ev isolation methods from tissues must be developed. however, to date very few rigorous methods to isolate or enrich for intact evs from tissues have been reported. we present a comprehensive exosome isolation method with a sufficient level of characterization to unequivocally demonstrate true ev identity from ex vivo eyes. methods: iodixanol (optiprep) buoyant density gradient ultracentrifugation (dguc), cushioned dguc (c-dguc), and our newly developed c-dguc immunocapture (c-dguc-ip) method were used to compare yield and enrichment of exosomes isolated from porcine eyes between to hours post-mortem. yield was assessed by nanoparticle tracking analysis (nta) and immunoblotting for exosomal markers along with total protein quantitation. enrichment was assessed by comparison of exosomal markers, ocular-specific markers and known contaminant markers, plus in-depth proteomic mass spectrometry analyses. results: high enrichment of posterior eyecup small evs (sev) were achieved by dguc and c-dguc, with c-dguc resulting in an eightfold increase in yield by nta and two to fivefold increases of exosomal protein markers such as syntenin- and cd by immuno-blotting compared to dguc. interestingly, in-depth proteomic analyses revealed that a majority of these sevs with densities of . - . g/ml isolated by dguc and c-dguc were likely of endoplasmic reticulum (er) and golgi origin, suggesting er-to-golgi transport vesicles resulting from post-mortem tissue cell rupture. in order to enrich further for sevs (including exosomes) we subjected sevs isolated by c-dguc to anti-cd immunocapture. the resulting sev proteome was enriched . -to -fold for bona fide sev and exosome markers compared to c-dguc. summary/conclusion: the c-dguc method provides an enhanced yield and purity of sevs and exosomes from ex vivo eye tissue. however, to avoid significant contamination with er and golgi-derived vesicles from postmortem eyes, a final ev-specific immunocapture step is required to achieve sufficient purity for subsequent analyses. our highly rigorous method paves the way for identification and validation of ocular-derived exosomes in blood and their potential use as eye disease biomarkers. characterization of the extraction of extracellular vesicles using a lab-ona-disc filtration system introduction: personalized treatment for cancer is a promising way to face the multiplicity of the disease, to increase the efficacy of drugs and to decrease their toxicity. as part of this strategy, liquid biopsy explores a new non-invasive approach to diagnose cancer, guide treatment and monitor its efficacy. extracellular vesicles (evs) are nanometric lipid bilayers micelles with high potential as biomarkers. they are involved in the transfer of information (proteins, rna and dna) between cells. evs include a broad spectrum of particle sizes, from the tens to thousands of nanometres. the isolation of evs from complex matrices is the first step of any protocol and is particularly important for the reproducibility and fidelity of the results presented, as it could bring bias in further analysis. in order to explore the heterogeneity of evs, a full characterization (physical and biological) of the extracted evs is needed. we evaluate and compare evs purification methods, including ultracentrifugation, sizeexclusion chromatography (sec) column and an emerging microfluidic technology: labspinner filtration labon-a-disc device isolating evs between two filters of and nm. methods: a cell supernatant was used as a model matrix. we compared three methods of extraction of evs: ultracentrifugation with two cycles of h at , g at degrees celsius (rotor type ti, beckman floor ultracentrifuge optima l k), qev size exclusion chromatography columns from izon (qevoriginal/ nm) and lab-on-a-disc filtration system (labspinner, exodisc c). evs characterization was conducted with nta (nanosightns ), trps (izon), nanodrop (spectrometernd ), tem (fei tecnai kv) and custom micro-immuno-assay. results: in this study, we characterize a filtration system made of two serial filters of nm and nm pores for isolation of evs. compared to ultracentrifugation and chromatography columns, yield of extraction is up to times higher and the size of the extracted particles is smaller. tem imaging was used for assessment of the quality of the extracted evs. however, albumin concentration measurement tends to show that the purity of the solution is decreased. the immuno-labelling analysis shows that the proteomic signature of the extracted evs differs according to the extraction methods. the new filtration technology seems to give us access to a broader range of evs compared to standard methods. summary/conclusion: in this study, we characterized purification methods including lab-on-a-disc filtration, and were able to demonstrate an increase of the concentration of evs by a factor of , a decrease of the size of the accessible extracted particles and access to new proteomic signatures. funding: we acknowledge the support of génome québec and action marie skłodowska-curie. effects of sample processing on isolation of extracellular vesicles from blood plasma by centrifugation darja božič a , matej hočevar b , veno kononenko c , marko jeran a , urška Štibler d , immacolata fiume e , manca pajnič f , ljubiša pađen f , ksenija kogej g , damjana drobne c , ales iglič h , gabriella pocsfalvi i , veronika kralj-iglič f and darja bozic j introduction: the isolation of extracellular vesicles (ev) from body fluids is still controversial and the poor understanding of vesicle stability and effects of sample processing is probably one of the core issues preventing the breakthrough of this field into applicative practices. methods: we performed an in-depth study of sample changes in blood, blood plasma and samples throughout the increasing speed of centrifugation, considering the number, size, contents and shape of particles in the isolates. flow cytometry, light scattering, mass spectrometry and scanning electron microscopy were employed to reveal the properties of material in the samples. results: the particles of size about - nm with characteristic topology of membrane vesicles without internal structure were observed by the scanning electron microscope only in ev isolates prepared from fresh blood sample. inspection of the tube surface in which the isolation took place suggests that those particles are likely formed from activated platelets tearing at the tube wall due to the centrifugal pull. the isolates prepared from frozen blood plasma prepared by centrifugation with different forces contained different amounts of particles with similar protein contents, predominated by highly abundant human plasma proteins, including albumins and immunoglobulins. some lipoprotein clearance and fibronectin precipitation were however observed through increased speed and time of centrifugation. summary/conclusion: the results of this study [ ] contribute to the understanding of stability and dynamics of membrane particles. the reported evidence provides the support for viewing ev isolates as a product, shaped by uniqueness of the starting samples and the thermal and mechanical stress applied upon processing. we believe this kind of insights strengthen our ability of reading the story of evs. introduction: apoptosis is a form of programmed cell death with diverse roles in the tumour microenvironment and emerging data show that, besides its role in tumour suppression, it can also promote oncogenic proliferation. highly aggressive tumours such as burkitt lymphoma (bl) show high levels of apoptosis, which has a diagnostic and prognostic value for classifying and staging the disease. we hypothesize that amongst other elements, extracellular vesicles (ev) are key mediators of apoptotic cell-derived tumour microenvironment signals. here, we report on ev released in vitro by apoptotic bl cells (apo-ev) in relation to their potential use as cancer biomarkers. methods: basic physical properties of apo-ev such as structure, size distribution, surface charge and membrane fluidity are discussed using cryo electron microscopy (em) and tomography, nanoparticle tracking analysis, dynamic light scattering and fluorescence anisotropy respectively. for phenotypic analysis we apply immunocapture and flow cytometry, immunogold labelling on transmission em, fluorescence microscopy and quantitative pcr. in addition, we study the interaction of apo-ev with blood components such as platelets, leucocytes and red cells, in order to understand their effects in the circulation and therefore their potential for analysis in blood samples. results: looking at the differences between apo-and non-apo-ev, apo-ev have larger diameter, while structurally are not different. however, we have identified distinct apo-ev markers such as active caspase and histones, or dna and small non-coding rna-y. there is also strong interaction of ev with platelets and leucocytes but not with red cells, indicating potential routes of transfer of ev cargo in the circulation. summary/conclusion: it is concluded that for the characterization of the heterogenous ev populations, combination of multiple techniques is often required, and also, understanding the strengths and limitations of each method is essential for choosing the appropriate set of analytical tools. finally, we consider that monitoring free circulating apo-ev or blood cells with which they have interacted is a promising approach to improve cancer diagnosis, prognosis and evaluation of therapeutic response. casting a small netrin: functional roles of a novel surface factor on stroma-derived extracellular vesicles in pancreatic cancer kristopher s. raghavan a , ralph francescone b , janusz franco-barraza b and edna cuckierman b a drexel university; fox chase cancer center, philadelphia, usa; b fox chase cancer center, philadelphia, usa introduction: pancreatic ductal adenocarcinoma (pdac) is a devastating disease driven and supported by changes in its microenvironment, or stroma. here we dissect the intercellular communication that exists between the primary stromal component, cancer-associated fibroblasts (cafs) and pdac. pdac communicates with its microenvironment, in part, through the exchange of specific types of extracellular vesicles (evs). specifically, we focus on the mechanism by which caf-secreted evs support pdac survival, with an additional goal to identify biomarkers suitable to generate a future "liquid biopsy" test for early pdac detection and prognosis. methods: evs are isolated from patient-derived pdac-associated fibroblasts via differential ultracentrifugation and validated by isev standards. human pdac cell lines used as recipient cells are treated with caf-evs to assess their role in supporting pdac survival. recombinant proteins, neutralizing peptides, and non-functional mutant proteins are used to block ev interaction with target cells. results: we observe sub-types of caf-evs containing unique surface receptors. one ev sub-population of interest contains a novel surface protein (nsp) expressed on the plasma membrane of pancreatic cafs, but not their healthy counterparts. further, pdac cells up-regulate nsp's lone binding partner, suggesting a role for these factors in pdac-selective ev uptake. functional assays designed to test pdac viability suggest these nsp(+)-evs protect pdac cells from programmed cell death as a result of physiological stress. this ev-mediated survival benefit can also be inhibited by blocking the interaction of nsp and its binding partner, suggesting the engagement of these two factors is necessary for cafs to support pdac via evs. pursuing our biomarker goal we confirm stromal nsp expression increases during early panin stages prior to tumour development, and we are currently seeking to validate nsp(+)-evs in blood of pdac patients. summary/conclusion: this research shines light on a novel mechanism of tumour-stroma communication that may be crucial for cancer progression during early disease stages and a potential target for disrupting the supportive role of the tumour microenvironment. additionally, we describe a sub-population of nsp (+)-evs that have the potential to serve as biomarkers for identifying pdac development. exosomes carry distinct mirnas that drive medulloblastoma progression introduction: extracellular vesicles (evs) represent an ideal source of functional biomarkers due to their role in intercellular communication and their ability to protect cargo, including rna, from degradation. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the bloodbrain-barrier. here we characterised the rna of exosomes isolated from medulloblastoma cell lines, with the aim of investigating exosomal rna cargo as potential functional biomarkers for medulloblastoma. methods: exosomes derived from a panel of matched (original tumour and metastasis) medulloblastoma cell lines were isolated and characterised by nanosight, electron microscopy, western blotting and nanoscale flow cytometry. exosomal mirna and mrna from our matched cell lines and foetal neuronal stem cells, which were used as a normal control, were analysed by rna-sequencing technology. results: based on hierarchical clustering, malignant derived exosomes were distinctly separated from normal control exosomes. mirna profiling revealed several established oncomirs identified in our malignant derived exosomes compared to control samples. using interaction pathway analysis, we identified that our malignant exosomes carry numerous mirnas implicated in migration, proliferation, cellular adhesion and tumour growth. several previously identified oncomirs were also identified to be present at higher levels in metastatic exosomes compared to primary and normal, including hsa-mir- - p and hsa-mir- a- p. summary/conclusion: this study shows that exosomes from mb cells carry a distinct mirna cargo which could enhance medulloblastoma progression. the use of circulating exosomes as markers of metastatic disease could be an innovative and powerful noninvasive tool. introduction: inflammatory changes in the bone marrow (bm) and suppression of haematopoietic stem and progenitor cell (hspc) function during acute myeloid leukaemia (aml) significantly contribute to patient morbidity and mortality. our laboratory has previously shown that aml-derived extracellular vesicle (ev-aml) trafficking confers a state of enforced quiescence and leads to lasting dna damage in hspcs. here we explore the underlying cause. specifically, we hypothesize that ev-aml incite inflammatory regulators as potential mediators of dna damage. methods: as a validated model of aml, we utilized the murine tib cell line as a source of ev-aml. ev-previous work has indicated that mirnas, notably mir- a and mir- , play a critical role in scc tumour development. evs are membrane-bound vesicles involved in cell-cell communication carrying actively sorted cargo, protected from degradation. the potential pathways these vesicular mirnas modulate and the implication they have on cancer biology is under active investigation. we have previously shown that the cadherin dsg , a stem cell marker, modulates ev release. dsg is upregulated in a number of cancers, including scc, and correlates with poor prognosis. here we aim to elucidate the impact of ev-associated mirnas in sccs by bioinformatic analysis. methods: scc cells stably expressing dsg were generated and evs isolated by sequential ultracentrifugation. total cellular and ev rna was isolated by mirneasy, analysed using rnaseq and identified by grch alignment. results were confirmed by qpcr. altered pathways based on targets were identified using mirnet and kegg pathway analysis. potential cancerassociated cytokine targets were confirmed by antibody array. results: rnaseq revealed cellular and ev mirnas that were differentially expressed in response to dsg with overlapping. the highest altered mirnas were validated by qpcr. kegg pathway analysis determined that these mirnas have the highest number of shared targets in cancer, cell cycle, and p signalling pathways. interestingly, mir- was upregulated while mir- a was dramatically downregulated in evs. targets of mir- a, icam- , il- , and il- , cytokines critical for cancer progression were upregulated. summary/conclusion: these results suggest that the mirna content of evs is tightly regulated. by altering the mirna profile, dsg contributes to the pathogenicity of these evs by increasing levels of cytokines important for cancer stem cell renewal and metastasis. in addition, these mirnas may serve as non-invasive diagnostic markers for sccs. funding: nih r cancer cells grown in d release distinct extracellular vesicles during tumour growth and invasion jens c. luoto, sara bengs, leila coelho rato, lea sistonen and eva henriksson Åbo akademi university, turku, finland introduction: cancer cells secrete extracellular vesicles (evs) that affect tumour progression. the characteristics of evs produced during tumour growth and invasion are however poorly understood. in this study, we identify the composition and characteristics of evs produced by noninvasive and invasive tumours and correlate these characteristics with the invasive status of the tumour. for that purpose, we established a protocol for isolating evs from extracellular matrix (ecm)-based three-dimensional ( d) cancer cell cultures. methods: human prostate cancer pc cells were grown in d cultures using ecm-based hydrogel, in standard d culture conditions and in bioreactor. evs were isolated from these cultures with differential and density gradient centrifugation. the isolated evs were characterized with nanoparticle tracking analyses, electron microscopy, immunoblotting and mass spectrometry (ms). results: our results demonstrate that d ecm-based hydrogel cell cultures secrete evs that can be isolated from both the conditioned media and the hydrogel. the invasive d cultivated pc organoids were found to secrete large amounts of evs compared to the non-invasive organoids. interestingly, our ms results revealed that non-invasive and invasive organoids secrete evs with partially distinct protein cargo. summary/conclusion: we have established a novel protocol for ev production in a d cell culture system utilizing ecm-based hydrogel, in which invasive tumour growth can be mimicked. our method allows the specific isolation and characterization of evs derived from different stages of d culture, such as non-invasive and invasive organoids. importantly, we found that tumour-derived evs change in composition during the tumour progression. taken together, our method can be used to define the distinct ev characteristics involved in cancer invasion. we previously showed extracellular vesicles (evs) to be causally involved in transmitting drug resistance. this study aimed to evaluate compounds proposed to reduce/block ev release. specifically, we selected calpeptin and y (proposed to inhibit evs budding from the cell membrane) and manumycin a and gw (proposed to inhibit evs deriving from mvbs). associated effects on -and consequences of-ev release were then investigated. methods: suitable compounds concentrations that were non-toxic to cells were first selected by performing cytotoxicity assay and flow cytometry (fc). conditioned medium (cm) was collected from docetaxel-resistant pc (pc rd) cells after h incubation in dfbs-medium with or without the compounds. evs were separated from tangential flow filtration concentrated cm using optiprep density gradient. . - . g/ml fractions were then pooled and washed. evs were characterised using nta, immunoblot, tem and lipid assay and fc. influences on growth and migration, of evs continuing to be released (at x evs/ x cells, x evs/ x cells), were evaluated on recipient du and rv cells. evtrack id ev , score % results: calpeptin and y , alone and in combination, did not significantly affect quantities of evs released. however, gw significantly (p < . ) increased quantities of released evs, of a larger size; very high protein to lipid ratio; and carrying grp compared to control evs (p < . ). this effect was reverted when gw was combined with manumycin a (p < . ). following all compounds treatments, x evs/ x cells inhibited rv proliferation (p < . ), while at x evs/ x cells only evs from manumycin a (p < . ) and y (p < . ) treated cells reduce rv proliferation. evs following gw treatment significantly (p < . ) inhibited du migration compared to bulk non-treated control and compared to the effect obtained using the entire pool of evs (p < . ). summary/conclusion: while none of the proposed inhibitors significantly reduced ev release, the resulting evs were less potent in transmitting aggressive behaviour, such as proliferation and migration, to receiving cell lines. patient-derived organoids represent a novel tool to study the effect of intra-tumoral heterogeneity on ev release in non-small cell lung cancer introduction: lung adenocarcinoma (luad) is the leading cause of cancer-related death with a low -year survival. although the importance of intra-tumoral cellular heterogeneity of solid tumors in the clinical outcome and treatment is emerging, proper models to study its effects on ev release and cargo in human tissues still lack. the d organoid technology maintains the cellular and genetic heterogeneity of in vivo tissues and has proved to be so far the best ex vivo model of human cancers. by using patient-derived and mouse organoids we set out i) to compare the ev release from normal and tumor tissues and ii) to follow changes in ev secretion when the relative ratio of tumor cell subpopulations is shifted. methods: we used mouse and luad patient-derived normal and tumor organoids. the medical research council of hungary approved our experiments with human samples and informed consent was obtained from patients. evs were detected by antibody-coated beads, nta and tem. intra-organoid heterogeneity was proved by immunostaining and rt-qpcr. results: we provide evidence that both mouse and human normal organoids contain all the bronchiolar cell types. interestingly, luad organoids selected for tp mutation contained not only ki + proliferating cells, but differentiated cell types as well. furthermore, all the lung organoid cultures produced evs and this was shifted to the smaller size range. interestingly however, when modifying the proportion of organoid cell types, we observed an increased ev release when more ki + proliferating cells were present both in normal and in luad samples. summary/conclusion: our data show that patientderived lung organoids represent a novel model to study the role of intra-tissue heterogeneity in ev functions in the humans, leading to improved diagnosis. funding: this work was funded by the national competitiveness and excellence program nvkp_ - (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). exosome mediate heart-adipocyte communication after myocardial ischaemia/reperfusion and impairs adipocyte endocrine function yajing wang, lu gan, dina xie, wayne lau, theodore christopher, bernard lopez and xinliang ma thomas jefferson university, philadelphia, usa introduction: by incompletely understood mechanisms, mi patients sustain systemic metabolic disorder. adipocytes are an important cellular type regulating energy homoeostasis. the impact of mi upon adipocyte function remains unknown. exosomes (exo) are critical vehicles mediating organ-organ communication. however, whether and how exo may mediate post-mi cardiomyocyte/ adipocyte communication have not been previously investigated. methods: adult male mice were subjected to mi/r. serum exo were isolated hours after r and incubated with t l cells for hours. the effects of exo upon adipocyte function were determined. results: compared to control, mi/r exo significantly altered the expression of genes known to be important in adipocyte function. go analysis revealed that genes associated with endoplasmic reticulum (er) function and adipocyte endocrine function are the primary two pathways altered by mi/r exo. venn analysis identified mi-rnas as cardiac-enriched, adipocyte-poor, and er function-related mirnas. rt-qpcr confirmed the mir- a/ a/ - family members are the most markedly increased mi-rnas in mi/r exo. incubation of t l cells with mi-r a mimic significantly downregulated edem , dsba-l, and pparn, and upregulated perk and chop. conversely, mi-r a inhibitor significantly decreased the impact of mi/r exo upon er function genes. additional studies demonstrated edem and pparγ (two critical molecules maintaining er function and adipocyte endocrine function) to be direct targets of mi-r a. one of the most significant endocrine molecules of adipocyte origin, adiponectin is regulated by pparn at the transcriptional level and by dsba-l at the post-translational level. we next determined whether mi/r exo may affect adiponectin expression/ assembly. incubation of t l cells with mi/r exo significantly inhibited total and high molecular weight adiponectin expression, an effect blocked by mir a mimic. finally, in vivo administration of gw (exo biogenesis inhibitor) or mir a inhibitor attenuated adipocyte er dysfunction and restored plasma adiponectin level in mi/r animals. summary/conclusion: we demonstrate for the first time that mi/r causes significant adipocyte er and endocrine dysfunction by exo mediated cardiomyocyte/adipocyte communication via mir- a/ a/ - . funding: nih and american diabetes association pancreatic cancer cell extracellular vesicles drastically alter the behaviour of recipient normal pancreas cells charles p. hinzman a , yaoxiang li a , meth jayatilake a , jose trevino b , partha banerjee a and amrita cheema a a georgetown university medical center, washington, usa; b university of florida health science center, gainesville, usa introduction: pancreatic cancer (paca) is predicted to become the rd leading cause of cancer-related deaths by . patients diagnosed with pancreatic ductal adenocarcinoma (pdac) have a -year survival ratẽ %. detection of pre-neoplastic lesions can potentially improve survival. however, there is currently no screening test for early stage detection. importantly, paca tumours are % non-tumorigenic cells. a better understanding of early paca oncogenesis is needed. cancer cells shed extracellular vesicles (evs) that are internalized by neighbouring and distant cells to induce a myriad of cancer progression events. we hypothesize that in early paca oncogenesis, evs mediate a behavioural change in surrounding normal cells, leading to the formation of this unique stroma. the purpose of this study was to develop a model to examine the phenotypic changes undergone by normal human pancreas cells when they are exposed to paca cell evs. methods: evs were isolated using differential ultracentrifugation with filtration from established (panc- , sw- , capan- and miapaca- ) and patientderived xenograft (ppcl- and ppcl- ) paca cell lines. cells were grown using ev-depleted fbs. ev isolations were validated and quantified using transmission electron microscopy, quantitative elisa, immunoblot and nanoparticle tracking analysis. normal pancreas cells (htert-hpne and hpde-h c ) were co-cultured with cancer cell evs for - hours. metabolic activity was measured using a mito stress test on a seahorse xfe extracellular flux analyser. results: we discovered that normal cells undergo vast behavioural transformations, including significant morphological changes, increased proliferation and an uncharacteristic invasive capability, when co-cultured with paca cell evs. these responses were ev dose dependent. further, paca cell evs metabolically reprogrammed normal cells, causing a bioenergetic switch, from a quiescent, aerobic profile to a highly energetic and glycolytic profile. summary/conclusion: our results indicate that paca cell evs confer enormous transformational properties to normal human pancreas cells in vitro. we hypothesize that evs impart distinct transformational properties to normal cells in vivo and this influence could unveil novel mechanisms regulating cancer onset and progression. these signals may be detectable before progression of early-stage paca to pdac, leading to the development of assays for earlier diagnosis in patients. further studies are underway to identify the biochemical mediators of these changes. plasma extracellular vesicles-mirnas released by hypoxic cells are associated to pro-tumorigenic and immunosuppressive microenvironment in lung cancer introduction: extracellular vesicles (ev) containing specific subset of functional biomolecules, such as micrornas (mirnas) are released by all cell types. it has been widely demonstrated that ev-mirnas from cancer cells can manipulate the tumour microenvironment modulating the gene expression of recipient cells. we previously identified a three levels risk classifier (msc) based on plasma-mirnas associated with lung cancer development and prognosis. the aim of this study was to investigate the potential role of ev- mirnas as mediators of pro-tumorigenic features. methods: evs were isolated from plasma of heavysmoker individuals with high (mscpos) or low (mscneg) risk of lung cancer by differential centrifugation method. purity of evs isolated was confirmed by sizing using nta and tem analysis and expression of ev-enriched proteins. mirna levels were analysed by dpcr. in vitro and in vivo analyses were used to assess the biological effect of plasma evs on different recipient cells. results: levels of mirnas in evs correlated with determination of whole plasma ( % of correlation with msc classifier). mscpos-evs stimulated d and d proliferation of non-tumorigenic epithelial cells through c-myc transfer into recipient cells. furthermore, mscpos-evs increased the ability of huvec to form tubular structures compared to mscneg-evs. in vivo co-injection of mscpos-evstreated huvec with a lung cancer cells resulted in an increase of tumour growth compared to mscneg-evs-treated huvec. mir- modulation in evs with mirna mimics or in recipient cells using mirna inhibitors demonstrated that this mirna is implicated in the autocrine proangiogenic modulation of huvec phenotype. mscpos-evs induced m polarization of macrophages both in vitro and in vivo. we demonstrated using synthetic oligonucleotides that mir- is responsible for the immunosuppressive modulation of these cells. regarding the potential origin of evs in mscpos individuals, we observed that hypoxia stimulated the secretion of evs containing c-myc from fibroblasts, mir- -evs from endothelial cells and mir- -evs from granulocytes. summary/conclusion: these data show that plasma evs of high-risk individuals display pro-tumorigenic features, as documented by their ability to induce a pro-angiogenic and immunosuppressive microenvironment suggesting an involvement of evs in lung cancer development. exploration of ev-associated metastatic targets on melanoma cells introduction: cancer cells secrete evs that may harbour metastatic proteins. previous studies have demonstrated the decrease of cd tetraspanin expression in melanoma cells are correlated with enhancing its metastatic potential. however, other proteins, such as cd , cd , met and nrp which are overexpressed in melanoma cells, are also associated with the spread of cancer. in this study, we sought to investigate the expression of metastatic proteins in evs derived from murine melanoma b f lineage. methods: b f cells were cultured in dmem supplemented with % fbs and antibiotics. the cells supernatant were harvested each hours, filtered through . µm and ultracentrifuged at x g for min at ºc to pellet evs. next, size and concentration was determined using nanoparticle tracking analyses technique, and the morphology of evs were analysed by negative-staining transmission electron microscopy (tem). the ev's surface protein were characterized by flow cytometry and protein content was profiled by mass spectrometry. results: our flow cytometry results have shown the presence of tetraspanins markers cd , cd and cd on vesicle´s surface. in addition, our assay demonstrated a diminished expression of cd molecule in comparison to cd and cd . we have profiled melanoma-evs by mass spectrometry, identifying the presence of proteins that may be associated to metastasis, such as cd , cd , met and nrp . summary/conclusion: these preliminary results are consistent with the literature and suggest that melanoma-derived evs harbour proteins, which may contribute to promote tumour metastasis. in our next step, we plan to generate b f lineages harbouring shrna vectors, in order to knockdown gene expression of cd , cd , cd , met and nrp to investigate the metastatic potential in vitro, in comparison to parental cells. we also may use syngeneic mice models to explore metastatic potential of genetically modified b f -derived cells. introduction: overexpression of her occurs in % of breast cancers and confers aggressive behaviour and poorer prognosis. thankfully, a number of drugs such as neratinib have been developed to target her , potentially providing substantial benefit for many patients. nevertheless, it is estimated that up to % of patients with her -overexpressing tumours do not gain benefit, as a result of innate or acquired drug-resistance. this study aimed to investigate if nano-sized membrane-surrounded extracellular vesicles (evs) released from drug-sensitive and drug-resistant cells reflect the her status of their cells of origin and thus have potential as minimallyinvasive biomarkers. methods: evs were isolated from conditioned media (cm) of her -positive cell lines (hcc and skbr ) and their neratinib-resistant counterparts (hcc -nr and skbr -nr) that we developed in our laboratory. evs from cm of a triple-negative breast cancer (tnbc) cell line variant, hs ts(i) , were evaluated as negative control for her . in brief, cm was centrifuged at g, for min x to get rid of any cells. the supernatant was then centrifuged at , g for h at ºc to collect evs. the resulting evs were washed in pbs, centrifuged as before, and resuspended in μl pbs. ev quantities were estimated by nanoparticle tracking analysis (nts). ev lysates were characterised by immunoblots, for established positive and negative ev markers. particle concentration as well as size distribution of evs were measured using the zetaview (particle metrix, germany). surface proteins on single evs were analysed by highresolution flow cytometry (fc), using an amnis imagestreamx mark ii. all data was submitted to ev-track (ev-track id: ev ). results: neratinib-resistant cell line variants were found to have reduced her protein expression compared to their respective drug-sensitive counterparts. neratinib-resistant cell line variants released fewer evs, when normalised per number of secreting cells, compared to their-drug sensitive counterparts. furthermore, evs from drug-sensitive cells carried her , while those from drug-resistant cells lacked her (similar to the evs from the tnbc cells); reflecting the her status of their cells of origin. summary/conclusion: this study indicates that a reduction in her protein expression is a mechanism by which cancer cells manifest resistance to her -targeted drugs (i.e. by making fewer her receptors available on the cells surface to accommodate the drugs activity). furthermore, ev-carried her seems to reflect the her status of their cells of origin. this suggest that analysis of her on evs in the peripheral circulation may help predict response to her -targeted drugs. thus, analysis of evs in appropriate cohorts of patients' specimens is warranted. introduction: rab a, a small gtpase involved in exosome biogenesis by regulating mve docking at the plasma membrane, and its expression level highly correlated with ohsv replication ability in vitro. oncolytic viruses is a newly promising therapeutic agent for cancer treatment. however, more than % of tumours naturally showed highly resistant to oncolytic viruses for unknown reasons. uncovering the underlying mechanisms of resistance to ohsv can offer potential therapeutic targets to enhance ohsv activity to kill tumour cells. in addition, it will give new insights into the identification of therapeutic biomarkers, which can be used to predict patient response to ohsv in clinical. methods: deep-sequencing data showed that lower expression level of rab a is present in ohsv resistant tumour cells compared to that in sensitive tumour cells. then an ohsv resistant mc tumour cell line was established by repeated injections with ohsv in mc tumour-bear mouse model. lastly, it was verified that ohsv resistance is associated with a downregulation of rab a and overexpression of rab a can rescue ohsv replication. results: ) the lower expression level of rab a is shown in ohsv resistant tumour cells compared to that in sensitive tumour cells shows in deep-sequencing data. ) furthermore, we established an ohsv resistant mc tumour cell line by repeated injections with ohsv in mc tumour-bear mouse model. similarly, in ohsv resistant mc cell line, rab a expression was lower than parental mc cells. and the release of exosomes and virus was decreased in ohsv resistant mc cell line. these results were confirmed by rab a sirna knockdown. ) we verified that in ohsv naturally resistant human cancer cell lines, ohsv resistance is associated with a downregulation of rab a and overexpression of rab a can rescue ohsv replication. summary/conclusion: downregulation of rab a expression restricts the efficiency of ohsv replication and the spreading ability of the released progeny virus which also provide rab a as a potential target and biomarker for ohsv cancer therapy. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd , shenzhen science and innovation commission project grants jcyj , jcyj to shenzhen international institute for biomedical research. inactivation of emilin- by proteolysis and secretion in extracellular vesicles favours melanoma progression and metastasis introduction: studies have demonstrated that melanoma-derived extracellular vesicles (evs) home in distal organs and sentinel lymph nodes favouring metastasis. although lymph node metastases are themselves rarely life threatening, they could be considered as one of the first step of metastasis in many cancer types. therefore, defining the mechanisms involved in lymph node metastasis and pre-metastatic niche formation in lymph nodes could bring the clue to block the metastatic process from the beginning. methods: we have characterized secreted exosomes from a panel of mouse melanoma models representative of low metastatic potential (b -f ), high metastatic potential (b -f ) and lymph node metastasis (b -f r ). we analysed the rna expression in cells and protein content of exosomes derived from mouse melanoma lymph node metastatic models by rna sequencing and mass spectrometry respectively. we validated expression by western-blot, qpcr and immunofluorescence. to define the mechanism of emilin- secretion cells were treated with the ev secretion inhibitor (non-competitive inhibitor of sphingomyelinase), gw . cell viability and cell cycle assays with an overexpression model (b -f -emilin- ) were also performed. in vivo experiments based on subcutaneous and intrafootpad injection for studying the role of this protein during melanoma progression were performed to define the relevance of our findings in vivo. human paraffin samples were analysed for emilin- expression by immunohistochemistry. results: we found a signature of over-expressed genes and hyper-secreted proteins in exosomes related to lymph node metastasis in the b mouse melanoma model. among them, we found that emilin- , a protein with an important function in lymph node physiology, was hyper-secreted in exosomes. interestingly, we found that emilin- is degraded and secreted in exosomes as a mechanism favouring metastasis. further, we found that emilin- has a tumour suppressive-like role regulating negatively cell migration. importantly, our in vivo studies demonstrate that emilin- overexpression reduced primary tumour growth and metastasis in mouse melanoma models. analysis in human melanoma samples showed that cells expressing high levels of emilin- are reduced in metastatic lesions. summary/conclusion: overall, our analysis suggests that the inactivation of emilin- by proteolysis and secretion in exosomes reduce its intrinsic tumour suppressive activities in melanoma favouring tumour progression and metastasis. funding: this work was supported by grants from mineco (saf r), asociación española contra el cáncer, fundación de investigación oncológica fero and mineco-severo ochoa predoctoral program. introduction: the amplification of erbb gene and the consequent overexpression of the encoded protein her have an important role in breast cancer classification at diagnosis and subsequent treatment decision with the anti-her monoclonal antibody trastuzumab. fish and ihc have been used so far to detect erbb gene amplification and her protein overexpression respectively in tissue biopsies. in this context, a major goal for liquid biopsies is to take advantage of the information carried by circulating tumourderived materials (such as extracellular vesicles (evs) and cell free dna (cfdna)) to noninvasively detect erbb gene status in the blood. however, the isolation of diverse tumour-derived materials from a single aliquot of patients' plasma and the accurate detection of cancer biomarkers is still challenging. methods: by adopting a recently published nickelbased evs isolation (nbi) protocol that allows for recovery of cfdna after evs isolation, we generated a high-sensitivity molecular assay to accurately detect erbb amplification and consequent her overexpression on a limited volume of plasma ( . ml) collected from breast cancer patients (stage i, ii and iii) at diagnosis. results: ) we detected erbb amplifications by droplet digital pcr (ddpcr) on the cfdna isolated from the plasma of erbb positive patients; ) we confirmed her overexpression on a subset of patients by setting up an antibody-based affinity reaction designed to detect her protein on the surface of the isolated evs; ) we succeeded in the quantification of her transcripts enclosed within evs by performing ddpcr in samples of patients showing a range of circulating tumourderived material. the specificity and sensitivity of these novel methodological assays were tested on a cohort of healthy individuals (n = ) and on a cohort of her positive (n = ) or her negative breast cancer patients (tnbc; n = ). summary/conclusion: here we report a pilot study on a novel multimodal method for erbb detection from a minimal amount of plasma. this approach integrates information from cfdna with evs-derived rna and proteins analysis. this proof of concept may ultimately translate into relevant clinical applications for disease diagnosis as well as for therapy selection and monitoring of disease progression. introduction: the biological and medical importance of exosomes recognized over the last decade has given rise to a crucial need for the discrimination between true evs and co-purified nanoparticles, such as lipoproteins, protein aggregates or debris. additionally, it is imperative to develop methods to identify and characterize ev sub-populations. considering ev biology and the reliability of labelled biomolecules, we developed both exogenous and endogenous labelling protocols, taking advantage of different dye properties which can target a multitude of compartments. this approach reveals key aspects of ev structure and integrity. methods: nanosized evs/exosomes were purified by size exclusion chromatography (sec) from model cell lines and human plasma. diverse dyes were orthogonally evaluated through different single particle and bulk analysis technologies, such as high-resolution cytometry, nanoparticle tracking analysis and plate fluorimeter. concomitant profiling of specific ev subpopulations was optimized using antibodies (abs) against tetraspanins and cell type specific targets and assessed by single particle analysis and elisa. to assess specificity of labelling protocols we used specific controls such as recombinant rfp expressing vesicles and purified lipoproteins. results: our ev staining protocols allowed for high labelling efficiency and unprecedent ev discrimination, quantification and characterization by combining single particle analysis and bulk measurements in simple matrices (saline buffers) and in complex biofluids (i.e. plasma). different approaches have diverse and complementary advantages (costs, capacity, sensitivity, informative readout) for implementation in research and diagnostic development flows, directly feeding in-house r&d and qc pipeline. summary/conclusion: overall, fluorescent evs are versatile and valuable tracers that can be applied in the optimization of pre-analytical and purification protocols, selection of target biomarkers and diagnostic assay calibration and validation. funding: endevor (por-fse - ) region tuscany and exosomics r&d program. subpopulations of tissue-derived extracellular vesiclesmethodological evaluation for vesicle size measurement introduction: introduction: subpopulations of extracellular vesicle (evs) are commonly classified by their different size, however, the ev size cut off is still under discussion. the aim of the study was to evaluate size range of six ev subpopulations using three methods: electron microscopy (em), nanoparticle tracking analysis (nta) and exoview™. methods: methods: ev subpopulations were isolated from melanoma tissues by a centrifugation based protocol recently established in our lab. large and small evs (levs and sevs) were isolated with differential ultracentrifugation and these were further separated into low and high-density fractions (ld and hd). size of ev subpopulations was then evaluated by: em introduction: small-extracellular vesicles have an important role in cell metabolism and cell-to-cell communication. moreover, sevs when secreted from cells are capable to act as mediators of various neurological diseases. however, sevs show heterogeneity and this may impact their functions. therefore, to characterize sevs at a single-particle level is important to better detail the associated biological activity. in this scenario, we innovatively propose the structured illumination microscopy (sim) as a technique able to complement the non-optical methods (transmission electron microscopy, tem) to analyse single sevs and their markers. methods: human plasma sevs were separated from healthy cognitive control (ctrl), mild cognitive impairment (mci) and demented subjects. the sevscontaining pellet was resuspended in % paraformaldehyde or % glutaraldehyde solutions. for sim, sevsenriched preparations were washed with the blocking solution and incubated with the primary antibody (l cam). the secondary fluolabelled antibody was then added. between the steps with the blocking solution, the primary and secondary antibodies, two ultracentrifuge steps were performed. the image acquisition was done on a nikon sim system with a x oil immersion objective. sevs were imaged with a d-sim acquisition protocol. tem was performed on mesh formvar copper-coated grids. sevs-enriched preparations were incubated first with the blocking solution and then, immunogold-labelled for cd . samples were counterstained with uranyl acetate and observed under a jeol ex electron microscope. data were recorded with a morada digital camera system. participation to the present study was approved by relevant local ethics committee of mendrisio and lugano hospital and written informed consents were obtained from subjects. results: for sim methodology, only vesicles in the range from to nm were detected, as the final resolution achieved was nm. instead, for tem, sevs under nm were identified. none of these methods provided relevant information about possible difference in morphology of the ctrl-, mci or demented subjects-derived sevs. summary/conclusion: even if both methods identified cd or l cam-positive vesicles, sim resolution and the complexity of the protocol represent some disadvantages respect to tem, that may be the first choice screening technique for evs analysis to be then completed by sim for particular tasks. fabrication of nanopore structures via conformal metal-film deposition for ev sensing kwanjung kim a , seung-min han b , soo-hyun kim c and sung-wook nam d luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. these evsreleased under normal physiological conditions as well as in the pathogenesis of neurological, vascular, haematological, and autoimmune diseaseshave been shown to transfer biological molecules such as protein and rna between cells, potentially transmitting signals. to understand more about these signalling mechanisms, there is a need for detecting and quantifying evs with cargo protein and rna in a reproducible and reliable manner. however, this has been challenging due to the small size of evs (ranging from to nm in diameter), and the lack of specific staining reagents. methods: here, we utilize the amnis® cellstream® flow cytometer, which enables high-throughput flow cytometry with increased sensitivity for detecting small particles. we demonstrate that a charge-coupled device (ccd)-based, time-delay-integration image capturing system can be used to detect and quantify evs and their cargo labelled with exoglow™-protein or exoglow™-rna. results: in this study, we show flow cytometry data quantifying ev samples that have been labelled with cargo markers for proteins and rna. the ev cargo contents along with the appropriate control samples will be shown. summary/conclusion: the ccd based detection of the cellstream flow cytometer has the sensitivity to quantify evs and their cargo. single ev imaging reveals novel ev biomarkers and dna cargo siobhan m. king a , ricardo bastos a and andras miklosi b a oni, oxford, uk; b oni (oxford nanoimaging ltd), oxford, uk introduction: extracellular vesicles (evs) are cellderived membrane-bound particles that range in size from - nm and carry active molecules such as dna, rna and proteins. upon secretion, evs can execute many biological functions such as initiating intracellular communication or regulating immune responses. depending on their origin evs have different characteristics and cargo, making them attractive candidates for early diagnostic and therapeutic applications. however due to their small size and heterogeneity, direct visualization and characterization of the surface markers expressed remains a challenge since these vesicles are below the resolution limit of standard light microscopy. methods: here, we describe a method that provides size analysis of single-evs, which falls below the diffraction limit of light. this was done with purified evs, immunostained using fluorescently labelled primary antibodies raised against ev surface markers (cd , cd , cd ), specific cargo such as dna and probed for tissue specific cargo. characterization of the molecular content and structural properties of surfaceimmobilized evs was performed using single-molecule localisation microscopy (smlm) on the nanoimager platform. results: multicolour smlm was used to detect up to three ev biomarkers showing successful characterization of the molecular signature for different ev subpopulations. the distribution of novel components on urinary evs were visualized for the first time using this approach. in addition, smlm revealed the presence of dna on both the surface and also as a cargo inside evs isolated from tumour cell culture media, which was validated using complementary biochemical characterization. summary/conclusion: smlm is a powerful technique for single-ev analysis and characterization. visualization of single-urinary evs enabled accurate sizing and further insights into novel components expressed on the subpopulation's membrane surface. together, the data demonstrates that the quantitative abilities of smlm can significantly enhance our understanding of evs, as structure, phenotypes, and cargoes can now be successfully resolved. introduction: working skeletal muscle is a common site for injury due to unaccustomed exercise with or without underlying pathology. direct analysis of skm injury requires invasive tissue biopsies. circulating extracellular vesicles (evs) are abundant in blood and have been shown to be enriched in microrna; profiles of which may reflect the state of tissues. evs may therefore serve as a non-invasive indicator of muscle injury and regenerative processes in vivo. methods: two consecutive bouts of muscle-damaging exercise (plyometric jumping and downhill running) were performed by healthy male volunteers. serum creatine kinase (ck) and plasma evs were analysed at baseline, and hr post-exercise. perceived muscle pain (pmp) was assessed at and hr post-exercise. large evs were isolated using a g centrifugation step, and small evs were isolated using qev columns. ev-enriched isolates were visualized using tem, and size and numbers were quantified using nta. based on nta results the highest particle fractions ( - ) were pooled for rna analysis. qpcr was done on plasma, large evs and small evs. a group of muscle and immune cell-important mirs were analysed by means of normalization to an exogenous control. results: ck and pmp increased post-exercise, providing evidence for muscle damage. tem revealed an abundant and heterogeneous pool of evs. a concomitant abundance of evs was seen with nta (mean = . x particles/ml plasma). mean ev diameters were ± nm across all timepoints. no change in ev size nor number was seen over time, however, mir- decreased at hr when compared to hr in the small ev isolate only. plasma displayed an immediate increase in myomirs- and − at hr, which returned to baseline at hr. in contrast, myomirs- b and remained elevated over the hr period. myomir- b and , as well as immune-mirs, did not change in evs or plasma as a result of the intervention. summary/conclusion: the decrease in mir- in small evs at hr is consistent with previous data. no decrease in mir- in large evs suggests specific packaging and hence a specific response to the muscle damage in small evs. more changes occurred in plasma myomirs suggesting less specific passive leakage into circulation from damaged cell membranes. funding: south african national research foundation pulsed electromagnetic fields potentiate the paracrine function of mesenchymal stem cells for cartilage regeneration yingnan wu a , dinesh parate a , eng hin lee a , zheng yang a and alfredo franco-obregón b a national university of singpaore tissue engineering program, yll school of medicine., national university of singapore, singapore, singapore; b biolonic currents electromagnetic pulsing systems laboratory, biceps, national university of singapore, singapore, singapore introduction: the mesenchymal stem cell (msc) secretome, via the combined actions of its plethora of biologically active factors, is capable of orchestrating the regenerative responses of numerous tissues by both eliciting and amplifying biological responses within recipient cells. mscs are "environmentally-responsive" to local microenvironmental cues and biophysical perturbations, influencing their differentiation as well as secretion of bioactive factors. we have previously shown that exposures of mscs to pulsed electromagnetic fields (pemfs) enhanced msc chondrogenesis. here, we investigate the influence of pemf exposure over the paracrine activity of mscs and its significance to cartilage regeneration. also, the subsequent extracellular vesicles analysis and isolation are processed for the understanding of how the pemfs affect stem cell evs and consequent differentiation induction. methods: conditioned medium (cm) was generated from mscs subjected to either d or d culturing platforms, with or without pemf exposure. the paracrine effects of cm over chondrocytes and msc chondrogenesis, migration and proliferation, as well as the inflammatory status and induced apoptosis in chondrocytes and mscs was assessed. the cms which have significant effects during chondrogenesis will be analysed by protein and mirna studies. results: we show that the benefits of magnetic field stimulation over msc-derived chondrogenesis can be partly ascribed to its ability to modulate the msc secretome. mscs cultured on either d or d platforms displayed distinct magnetic sensitivities, whereby mscs grown in d or d platforms responded most favourably to pemf exposure at mt and mt amplitudes, respectively. ten minutes of pemf exposure was sufficient to substantially augment the chondrogenic potential of msc-derived cm generated from either platform. furthermore, pemf-induced cm was capable of enhancing the migration of chondrocytes and mscs as well as mitigating cellular inflammation and apoptosis. the cms protein results in the significant promotion chondrogenesis condition showed an increase in proliferation and anti-inflammatory cytokines. summary/conclusion: the findings reported here demonstrate that pemf-stimulation is capable of modulating the paracrine function of mscs for the enhancement and re-establishment of cartilage regeneration in states of cellular stress. the pemf-induced modulation of the msc-derived paracrine function for directed biological responses in recipient cells or tissues has broad clinical and practical ramifications with high translational value across numerous clinical application. effects of extracellular vesicles from blood derivatives on osteoarthritic chondrocytes within an inflammation model introduction: the degenerative disease osteoarthritis (oa) is one of the leading causes of disability especially of elderly people. besides various treatment options depending on the severity of the cartilage degradation, the application of blood derived products such as platelet rich plasma (prp) are getting more and more popular in clinical practice due to its high concentration of platelets and the perceived high growth factor levels. drawbacks of using prp include high donor variability, discrepancies among preparation protocols and the presence of cells (platelets, leukocytes) which can evoke cellular processes, especially inflammation, when injected into the diseased tissue. one possibility is to isolate only extracellular vesicles (evs) from blood derivatives to overcome these problems. in the current study the effects of evs isolated from blood derivatives on oa chondrocytes within an inflammation model was investigated. methods: cd positive primary monocytes were isolated from citrate anticoagulated whole blood by magnetic bead sorting. monocytes were differentiated into resting m macrophages and activated into m macrophages according to published protocols. elisa measurements verified successful differentiation and activation as il β and tnfα levels increased. as control, thp monocytes were used. patient-derived oa chondrocytes were grown in well plates and co-cultivated with activated m macrophages which were seeded into thincerts and added to the wells representing the inflammation model. furthermore, cells were treated for hours with media containing fcs, ev depleted fcs or evs isolated from prp or hypact serum. results: successful differentiation and activation of monocytes (thp and primary monocytes) into m macrophages was demonstrated by elevated levels of the inflammatory cytokines il β and tnfα. within the inflammation model (co-culture of oa chondrocytes with m macrophages), addition of evs isolated from prp or hypact serum resulted in decreased secretion levels of il β and tnfα compared to media supplemented with either fcs or ev depleted fcs. summary/conclusion: taken together, evs from blood derived products might be chondroprotective and anti-inflammatory mediators which protect cartilage from being degraded during oa. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst -f- / - . α, (oh) d regulates growth cartilage matrix vesicle micrornas niels asmussen, michael mcclure, zhao lin, zvi schwartz and barbara boyan virginia commonwealth university, richmond, usa introduction: matrix vesicles (mvs) are small ( - nm in diameter) lipid bound extracellular organelles isolated from calcifying tissues including the growth zone (gc) of growth plate cartilage. α, (oh) vitamin d ( α, ) is a regulator of gc chondrocytes and the mvs they produce. these mvs are key players in the mineralization process and are selectively enriched with enzymes and growth factors. we found that mvs are also selectively enriched with micrornas (mir), including mir- , mir- and mir- . the aim of this study was to determine the regulatory role of α, in the packaging of mirna in mvs by gc cells. methods: gc cells were isolated by enzymatic digestion from costochondral gc cartilage harvested from wk-old male sprague dawley rats (iacuc approved). confluent fourth passage gc cell cultures were treated with - m α, or vehicle for h. media were removed, cell monolayers digested with trypsin and cells and mvs isolated by differential ultracentrifugation. rna was precipitated from cells and mvs. small rnaseq data were trimmed, aligned and counted before undergoing differential expression analysis. experimental groups had an n = per variable. significant differences (p < . ) were determined using r v . . . results: α, treatment altered expression of mv mirs compared to control mvs, whereas cell mirnas were differentially expressed. . % of significantly up or down regulated mir found in mvs overlapped between α, and vehicle groups with the remaining being uniquely differentially expressed. α, increased mv mir- and decreased mir- - p two mirs known to regulate osteoblast proliferation ( increases, decreases). summary/conclusion: α, regulates gc chondrocyte and mv behaviour and this study demonstrates that it also impacts the mir packaging within mvs. mir discovered in mvs have been demonstrated to impact chondrocyte behaviour and the present study indicates that α, regulates the growth plate through mir delivered by mvs. introduction: increasing evidence has proposed extracellular vesicles (evs) as mediators of many of the therapeutic features of mesenchymal stromal cells (msc) that have been widely studied in clinical trials over the last years. these evs have been recognized as nanocarriers of important biological information, which play a central role in cell-to-cell communication. in this context, evs can be used as an alternative to a cell-based therapy, with reduced risks. the present work aimed to evaluate the impact of different culture conditions on the msc-derived evs molecular composition through fourier-transform infrared (ftir) spectroscopy. methods: evs derived from msc from different sources, expanded in two different culture media ((xenogeneic -free (xf) vs serum-containing medium (fbs)) were characterized by ftir spectroscopy, a highly sensitive, fast and high throughput technique. moreover, principal component analysis (pca) of preprocessed ftir spectra of purified evs was conducted, enabling the evaluation of the replica variance of the evs chemical fingerprint in a reduced dimensionality space. for that, different pre-processing methods were studied as baseline correction, standard normal variation and first and second derivative. results: evs secreted by mscs cultured with serumcontaining medium presented a more homogenous chemical fingerprint than evs obtained with xf medium. the regression vector of the pca enabled to identified relevant spectral bands that enabled the separation of samples in the score-plot of the previous analysis. ratios between these spectral bands were determined, since these attenuate artefacts due to cell quantity and baseline distortions underneath each band. statistically inference analysis of the ratios of spectral bands were conducted, by comparing the equality of the means of the populations using appropriate hypothesis tests and considering the significance level of %. it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture medium used for msc expansion and the msc donor. summary/conclusion: this work is a step forward into understanding how different culture conditions affect msc-derived evs characteristics. funding: fundação para a ciência e tecnologia (ptdc/equ-equ/ / , uidb/ / ). performance qualification for microflow cytometers: understanding technical limitations to improve your research desmond pink a , michael wong a , diana pham a , renjith pillai a , leanne stifanyk b , sylvia koch b , rebecca hiebert a , oliver kenyon c and john lewis a a nanostics, edmonton, canada; b dynalife, edmonton, canada; c apogeeflow systems, hemel hempstead, uk introduction: as microflow cytometry and other techniques mature as validated modalities for analysing extracellular vesicles (ev), there has been a concerted effort to improve reproducibility . in order for this reproducibility to occur there has to be a critical understanding of advantages and limitations for each technology. for microflow cytometry, several instruments are available to analyse evs. each platform has different limitations as well as advantages over other platforms. to provide the optimal data for your specific research, it is critical to understand the limitations of your platform. to accurately define these limitations, a performance qualification (pq) of your instrument should be undertaken. methods: an apogee a platform was used in these experiments. experiments were designed with expected ranges and cut-offs for acceptance criteria.initial tests included autosampling of a well plate with either single or double aspiration, single sample reproducibility and linearity proportional to flow rate. other experiments designed to show machine performance included minimal time to achieve valid data, sample volume required for double aspiration, determination of coincidence; detection sensitivity using a spiked sample; flow rate stability for extended periods ( - minutes) . tests should also be performed to determine carryover at a range of sample concentrations. if present, the means to remove contaminating samples should be determined. any performance tests should be applicable to any instrument in the field. results: auto-sampling helped demonstrate consistent data; reproducibility of total events and biomarkers was - % c.v. detected bead concentrations were linear with flow rates between . and . ul/min. double well aspirations provided similar data with aspirations between - ul. valid data was achieved for a low abundant target (~ - events/ul) after only s, < %c.v. detection sensitivity was determined to be~ / , . carryover ranges were determined in the presence of nominal unstained serum. an optimal number of machine washes was determined. some membrane stains, such as cell mask and cfse require much more rigorous cleaning to remove stain carryover. summary/conclusion: to improve data reproducibility, performance qualification of any instrument is key. operational limitations help define optimal performance parameters of any technology. understanding the types of experiments to perform for your particular type of characterization technology depends on the requirements you set for your research. a good performance test should be applicable to any related instrument in the field. funding: funding provided by nanostics, the alberta cancer foundation, and alberta innovates. introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par - , is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par - / , successfully funded r and r grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, ); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, ). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, ). summary/conclusion: drs. sudhir srivastava and matthew young are the programme directors for the par which began accepting applications on january . this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute. introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores ( nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the protein biomarkers (tau, uchl- , nfl, gfap, il , il , and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl were elevated in mtbi patients compared to controls (p < . ), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il and tnf-with tau from glur + evs showed % accuracy with % sensitivity and % specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. ps . = op . l cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma maia norman, dmitry ter-ovanesyan, wendy trieu and david walt wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd , cd and cd ) as well as l cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l cam appears. we also immunocapture l cam from csf and plasma and perform western blots for the internal and external domains of l cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l cam in plasma is likely not coming from the brain. this data call into question the utility of l cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in -month-old wt mice, (n = /groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for % of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and nonamplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy y cells with n-myc amplified sk-n-be cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. ps . = op . results: murine ctl evs were broadly divided into two populations that were eluted at low salt (l-s: . m- . m nacl) and high salt (h-s: . m- . m nacl) concentrations. l-s ctl evs were abundant in late endosome-related proteins, integrins, rabs, and effective mirnas, indicating exosome characteristics, and had biological activity for preventing tumour metastasis after depletion of tumoural mesenchymal cell populations by intratumoral administration (see seo et al., nat. commun. : , ) . contrary, h-s ctl evs were rich in dna, core histones, ribosomal proteins, cytoskeleton proteins, and housekeeping proteins, considering microvesicles and apoptotic bodies, and easily phagocytosed by a kupffer cell line (kup : kitani et al., results immunol. : - . ). in addition, there were noticeable differences between ls and h-s ctl evs in the negative zeta potential width and membrane glycan structure. summary/conclusion: thus, ion exchange can be an optimal mass fractionation method for discriminating bioactive exosomes from cargos for nucleic acids in evs. funding: cryotem was conducted in nara institute of science and technology (naist), supported by nanotechnology platform program (synthesis of molecules and materials: # ) of the ministry of education, culture, sports, science and technology (mext). this work was supported by grants from the japan agency for medical research and development (translational research network program (nagoya univ. seeds a )) and the japan science and technology agency (crest [jpmjcr h ]). clic is essential for breast cancer metastatic competence and predicts disease outcome introduction: metastatic breast cancer is a consequence of complex interactions between cancer cells and the host. clic , a member of a conserved gene family in the glutathione-s-transferase superfamily, mediates crosstalk between tumour and host in breast cancer. tcga and metabric data indicated that elevated clic expression was associated with breast cancers from young women, those with poor prognosis, and those with early stage metastatic disease. methods: since bulk tumour analysis does not distinguish between cancer and host stromal cells, we used genetic modifications of established syngeneic breast cancer mouse models to evaluate the contributions of clic in the host or tumour cells to develop metastases. results: experimentally, the essential clic host contributions for metastatic competence were related to circulating levels of pro-metastatic soluble factors, neoangiogenesis, tumour cell attachment to lung tissue, myofibroblast differentiation, and leukocyte migration. clic was detected as cargo in circulating extracellular vesicles (evs) from breast cancer patients. similarly, circulating evs from tumour-bearing mice have abundant clic in comparison to those from mice bearing tumours that lack clic . tumour cells released evs that induced myofibroblast conversion of wildtype but not clic ablated lung fibroblasts. summary/conclusion: these results illuminate clic expression as a prognostic marker for breast cancer patients, and experimentally, clic is a critical host factor for metastatic competence and potential target within host tissues for anti-metastatic therapy. funding: this work was supported by the intramural program of the national cancer institute under project zia bc . the application of flow cytometry in an ev-based liquid biopsy for the detection of cancer multidrug resistance in myeloma gabriele de rubis a , krishna sunkara a , sabna rajeev krishnan and mary bebawy b a laboratory of cancer cell biology and therapeutics, discipline of pharmacy, graduate school of health, the university of technology sydney, australia, sydney, australia; b the university of technology sydney, sydney, australia introduction: multiple myeloma (mm) is an incurable cancer of bone-marrow plasma cells. it is characterized by unpredictable and highly variable therapeutic response and poor survival, attributed to the development of multidrug resistance (mdr) to chemotherapy. presently, no clinical procedures allow for a continuous, minimally invasive monitoring of mdr. we identified unique extracellular vesicle (ev) populations in the blood of myeloma patients, which serve as biomarkers of disease evolution and mdr to combination chemotherapy. we describe approaches used to optimise the use of flow cytometry (fcm) for ev summary/conclusion: although further investigation is required, our results potentially promise an effective and inexpensive priming agent (i.e., ethanol) for the production of anti-inflammatory msc-evs. this, combined with the significant increase in yield via d dynamic culture, presents practical solutions to both ev manufacturing scalability and potency issues. donor source affects potency of mesenchymal stem cell-derived extracellular vesicles introduction: mesenchymal stem cell (msc) therapies have been heavily investigated for their utility in applications such as wound healing and regenerative medicine due to their angiogenic, immunomodulatory and anti-apoptotic effects. recently, msc-derived extracellular vesicles (evs) have been implicated as primary effectors in msc-based therapies via protein and nucleic acid cargo transfer to patient cells. msc evs represent a superior alternative to msc-based therapies, as they lack the ability to replicate and are much smaller in size, circumventing related safety concerns such as immunogenicity, teratoma formation and blood vessel occlusion. however, a key drawback with msc therapies in general is their variable therapeutic potency, which is dependent on donor source. as a cell derived therapeutic, this crucial limitation is hypothesized to exist in msc evs as well. here, we demonstrate the varying bioactivities of isolated msc evs from differing donors and tissue sources. methods: six separate msc lines were obtained from different donors, with three msc lines derived from donor adipose tissue, and the other three from the bone marrow of separate individuals. evs were isolated from each msc line at passage via differential centrifugation and ultrafiltration. these isolated msc evs were then characterized for size/concentration via nanoparticle tracking analysis, and ev markers (tsg , alix, cd ) via western blot. pro-vascularization capacities of msc evs were determined by a gap closure assay using human umbilical cord vein endothelial cells (huvecs). results: characterization of msc evs revealed similar sizes and ev marker expression across donor groups, frontiers in chemistry, submitted funding: this work was funded by the momentum programme (lp - ), by the national competitiveness and excellence program catalan institution for research and advanced studies (icrea) proteomic profiling of retinoblastoma-derived exosomes reveals potential biomarkers of vitreous seeding angel montero carcaboso g , andrea petretto b , franco locatelli a and angela di giannatale a a department of paediatric haematology/oncology and cell and gene therapy, irccs, ospedale pediatrico bambino gesù ps : separation and concentration a laboratory of clinical biophysics, faculty of health sciences ps : diverse ev biomarkers chair: pia siljander -faculty of biological and environmental sciences urinary evs were isolated using low vacuum filtration method followed by ultracentrifugation. raman spectra of urinary evs were recorded using a renishaw invia raman spectrometer. data analysis was performed using principal component analysis (pca) and hierarchical cluster analysis (hca). the size distribution and morphology of evs were analysed by transmission electron microscopy and nanoparticle tracking analysis methods. results: average raman spectra obtained for urinary evs from studied groups showed differences in intensities of specific bands in the region of - cm- . we found significant correlations between mean area under curve (auc) calculated for raman bands (phenylalanine, dna, proteins, lipids and amide i) and selected clinical parameters such as: egfr, serum creatinine, glucose, urine creatinine. chemometric methods showed spectral pattern responsible for separation between studied groups. nta measurements visualized evs with size of . ± . nm. summary/conclusion: our results showed that characteristic raman spectra of urinary evs are promising candidates for new, non-invasive biomarkers for dkd isolation of circulating extracellular vesicles and cfdna allows for erbb detection in a single aliquot of breast cancer patients plasma michela notarangelo a , mattia barbareschi b , antonella ferro c , orazio caffo c , vito d'agostino a and francesca demichelis d a department of cellular results: results: tissue-derived large and small evs showed difference in size (mean nm vs nm) when examined by em, whereas nta and exoview™ were unable to show a clear difference between the populations (nta: mean . nm vs nm nta can only detected vesicles above nm and exoview™ only measures vesicles between - nm. of the three different methods, em analysis of single vesicles visualized in a significant number of micrographs was the only one able to distinguish ev subpopulations by size. funding: funding: swedish research council knut och alice wallenberg foundation imaging of human plasma-derived small-extracellular vesicles using transmission electron microscopy and structured illumination microscopy mitovesicles: a new extracellular vesicle of mitochondrial origin altered in ageing and neurodegeneration alldred b , chris goulbourne b , hediye erdjument-bromage d , monika pawlik b , mitsuo saito e , mariko saito f , stephen d. ginsberg b an in vitro and in vivo perspective on the role of erythrocyte-derived extracellular vesicles in parkinson's disease pathology frédéric calon c , Éric boilard f and francesca cicchetti b a centre de recherche du chu de québec and faculté de médecine, département de psychiatrie & neurosciences département de microbiologie-infectiologie et d'immunologie evidences on microalgal extracellular vesicles: a morphological assessment antonella bongiovanni i , ales iglič j and veronika kralj-iglič j a laboratory of clinical biophysics, faculty of health sciences a faculty of dentistry, national university of singapore, singapore, singapore, singapore; b institute of medical biology, agency for science, technology and research, singapore, singapore, singapore; c exosome of cancer-associated fibroblast induce anti-cancer drug-resistance of nsclc so-young kim a and yeon-ju lee b a chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea; b chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea introduction: the understanding of interaction mechanisms between cancer cells and the tumour microenvironment (tme) is crucial for developing therapies that can arrest tumour progression and metastasis. cafs are the major constituent of the tme in many cancers. recent studies indicate that exosomes harbour the potential to regulate proliferation, survival and immune status in recipient cells. most of the current studies are focused on cancer cell secreted exosomes; and little is known about cafderived exosomes and their influence on cancer cells. methods: nsclc cell lines (pc gr) and mrc (normal fibroblast cells) were grown in culture with exosome-free fbs. cutured media was filtrated by tangential flow filtration systems. exosomes in supernatant were isolated with the exoquick-tc™ system. considering the important role of cell extrinsic factors on cell growth and survival, we assessed whether factors contained in the mpa exosome could affect proliferation and survival of recipient cancer cells. cells were then treated with μm osimertinib or pbs for days prior to cell quantification of live cells.to investigate mechanisms of resistance to osimertinib mediated by ma or mpa-exosome in nsclc cell lines, we test cell viability by crystal violet assay in trametinib or osimertinib treated after pretreated ma or mpa-exosome, pc gr during days. we will investigate how mpa-exsomes activate erk signalling pathway in pc gr cells to induce antitumor effects by western blot. results: mpa exosome increased proliferation of pc gr cells by more than % compared to control pbs. pc gr cells grown in mpa-exosome and subsequently treated with osimertinib showed a significant increase in cell survival compared to pc gr cells grown in ma-exosome. osimertinib is used to treat egfr-mutant non-small cell lung cancer (nsclc) with tyrosine kinase inhibitor resistance mediated by the egfr t m mutation.these data show that "mrc -pc gr-crosstalk factors" affect proliferation and adaptive drug resistance of cancer cells. mpa-exosome mediates erk signalling activation and attenuated after treatment of um osimertinib. summary/conclusion: cafs support cancer growth and invasion. co-cultured nsclc with mrc lung fibroblast increased cell viability and exosomal mir- through the tgf-ß pathway in treatment osimertinib. exosomal mir- up-regulation in cocultured nsclc with mrc- induced drug resistance to drug-induced apoptosis. thus, exosomal mir- expression in co-cultured nsclc with mrc may support drug tolerance persister cells. introduction: neural stem cell (nsc) therapy has shown promise for brain repair after injury or disease mostly through bystander effects. nevertheless, the translation of nscs derived from human induced pluripotent stem cells (hipscs) to the clinic remain constrained due to safety issues, which include immunogenic risks, tumorigenesis potential, and incomplete differentiation. a way to avoid these issues is by using extracellular vesicles (evs) generated from nscs, as nsc-evs likely have similar neuroprotective properties as nscs and are amenable for non-invasive administration as an autologous or allogeneic off-the-shelf product. however, this would require reliable purification and characterization processes, and testing of evs for composition and biological properties. methods: we generated evs from hipsc-derived nscs using a combination of ion-exchange chromatography (iex) and size-exclusion chromatography (sec) and investigated their composition through small rna sequencing and proteomics. we also performed in vitro and in vivo experiments to determine their biological and functional properties. results: iex and sec facilitated purification of hipsc-nsc evs nearly to homogeneity, which expressed ev markers such as cd , cd , cd , and alix with a mean size of nm. small rna sequencing revealed enrichment of mirnas related to different neuroprotective signalling pathways and diverse metabolic functions consistent with their role in cell-cell communication. the proteomic analysis identified > , proteins, including ev markers and many other proteins involved in central nervous system function and cellular processes. the evs also displayed antiinflammatory activity in an in vitro mouse macrophage assay. intranasal (in) administration of nsc-evs resulted in their rapid incorporation by neurons, microglia, and astrocytes in virtually all regions of the brain. functionally, in administration of nsc-evs reduced inflammatory activity in the brain in a model of status epilepticus, and increased hippocampal neurogenesis in the adult brain. summary/conclusion: biologically active evs with antiinflammatory and neurogenic properties could be purified and harvested from hipsc-nscs. such evs also contain many mirnas and proteins that are of interest for brain repair after injury or disease. funding: supported by a grant from the national institute of neurological disorders and stroke ( r ns - to a.k.s.) introduction: extracellular vesicles (evs) generated from human bone marrow-derived mesenchymal stem cells (hmscs) display anti-inflammatory and neuroprotective properties. our recent study has shown that intranasally (in) administered hmsc-evs incorporate into significant percentages of neurons and microglia in virtually all regions of the intact as well as the injured forebrain within hours (kodali et al., int j mol sci, ) . in this study, using a rat model, we investigated the efficacy of a low dose of hmsc-evs administered intranasally for alleviating the abnormal plasticity of newly born neurons and the activation of microglia after se. methods: approximately billion evs were dispensed bilaterally into both nostrils of young f rats that experienced two hours of kainate-induced se. animals were euthanized seven days after se, and brain tissue sections were processed for immunohistochemical staining of neun (a neuronal marker), dcx (a marker of newly born neurons), iba- (a microglial marker), and parvalbumin (pv) and reelin (markers of subclasses of interneurons). in addition, activated microglia were quantified using iba- and cd dual immunofluorescence. results: in administration of evs reduced the seinduced loss of pyramidal neurons in the hippocampal ca subfield. also, ev administration after se maintained higher levels of pv+ interneurons in the dentate gyrus. furthermore, ev treatment after se modulated abnormal neurogenesis, which was evidenced by a the role of small extracellular vesicles in chronic neuropathic pain zhucheng lin a , renee jean-toussaint b , yuzhen tian b , ahmet sacan a and seena ajit b a drexel university, philadelphia, usa; b drexel university college of medicine, philadelphia, usa introduction: chronic pain is the most prevalent, disabling, and expensive public health condition in the usa. exosomes are - nm extracellular vesicles that can transport rnas, proteins, and lipid mediators to recipient cells via circulation. exosomes can be beneficial or harmful depending on their source and contents. we hypothesized that the composition of small extracellular vesicles (sevs) can be altered following nerve injury and these alterations can provide insight into how the body responds to neuropathic pain. methods: to characterize changes following nerve injury, small extracellular vesicles (sevs) were purified by ultracentrifugation from mouse serum four weeks after spared nerve injury (sni) or sham surgery. mirna profiling and proteomics analysis using tandem mass spectrometry were performed to determine differential expression of mirnas and protein cargo respectively. for in vivo studies, sevs were administrated intrathecally into the mouse lumbar region. animals were evaluated for mechanical and thermal hypersensitivity over days after injection. results: our mirna profiling showed a distinct mirna signature in sni model compared to sham control. proteomics analysis detected gene products. of these, were unique to sni model. neuropathic pain can induce the activation of the complement cascade and we observed significant upregulation of complement component a (c a) in sevs from sni model. intercellular adhesion molecule (icam- ), required for the leukocyte recruitment, adhesion and homing of exosomes was also upregulated in sevs from sni model compared to sham control. administration of sevs from sni model increased paw withdraw threshold in naïve recipient mice and inflammatory pain model, indicating a protective role for sevs in attenuating chronic pain. summary/conclusion: our preliminary studies suggest a critical role for sevs cargo in regulating pain. additional studies are ongoing to determine the functional significance of alterations in sevs composition using mouse models of pain. introduction: amyotrophic lateral sclerosis (als) is a progressive adult-onset neurodegenerative disease caused by selective motor neurons (mns) death. the rapid disease progression strongly suggests that cell-tocell spreading of noxious factors could take place in als pathogenesis. extracellular vesicles could potentially spread the disease. in this study, we characterized large (levs) and small extracellular vesicles (sevs) isolated from plasma of sporadic als patients and healthy controls and determined their different composition in order to understand their neuroprotective or neurotoxic role in als pathogenesis. methods: levs and sevs were isolated from plasma of als patients and healthy volunteers by differential centrifugation and characterized by nanosight ns . cd , cd , cd , cd a and annexin v were used for flow cytometry. sod , tdp , fus protein level was investigated by western blot. for raman spectroscopy, evs were dried on top of a caf slide and raman spectra were acquired using a nm laser line. mirna libraries were prepared by truseq small rna library kit (illumina). results: the mean size both for levs and for sevs resulted increased in als patients compared to controls. levs derived from als patients were enriched in sod- , tdp- and fus proteins compared to ctrls. sevs showed a distinct spectral pattern from levs. in addition, levs of als patients were richer in lipids and had less intense bands relative to aromatic aminoacids compared to healthy controls. we also found a great presence of leukocyte derived levs (lmvs) in als patients compared to ad patients and healthy donors and significant correlation with the progression rate of the disease. on the other hand, mirna and rna whole transcriptome sequencing identified a specific signature of mirnas in plasma derived sevs of als patients compared to a group of healthy controls and three neurological groups of control. summary/conclusion: these data may suggest that levs derived from als patients, enriched in lipids and toxic proteins, might play a role in prion-like propagation and immunity of als disease, while sevs, deriving ps . introduction: dendritic spines are actin-rich structures at the postsynaptic sites of most excitatory synapses in the central nervous system. they are highly important structures for higher brain functions such as learning and memory. several live imaging studies have shown that long, thin, actinrich protrusions called dendritic filopodia are precursors of dendritic spines in hippocampal and cortical neurons. so far, many intracellular factors that regulate filopodia formation have been identified. however, extracellular mechanisms of filopodia formation are largely unknown. also, detailed molecular mechanisms by which astrocyte secreted factors regulate synaptogenesis are not well understood. small extracellular vesicles (sevs)/exosomes have potential to regulate filopodia, spine and synapse formation in autocrine or paracrine manner due to their unique cargo composition. here, we examine role of exosomes in filopodia, spine and synapse formation. methods: primary rat hippocampal and cortical neurons were transiently transfected with the multivesicular body (mvb) docking regulator gfp-rab b or with shrnas against the exosome secretion and biogenesis regulators rab b and hrs. transfected neurons were immunostained for synaptic proteins and analysed for filopodia at day in vitro (div) or spines at div . for rescue experiments, exosomes were isolated using differential ultracentrifugation method from conditioned media of div cortical neurons or primary astrocytes and characterized for their size, common protein markers and morphology. results: here, we find that mvb docking factor gfp-rab b localizes to both the tips and bases of actin-rich filopodia and spines in primary neurons. furthermore, genetic regulation of exosome secretion by overexpression or knockdown of rab b or hrs leads to respective increases or decreases in the number of filopodia, spines and synapses. the defects of exosome-inhibited neurons in filopodia density are rescued by add-back of neuronal exosomes. additionally, treatment of primary neurons with exosomes isolated from primary astrocyte cultures leads to enhanced spine and synapse formation. summary/conclusion: these results indicate that autocrine and paracrine communication via exosomes are a key part of the process of neuronal filopodia, spine and synapse formation. effects of apolipoprotein e genotype on protein and small rna profiles of brain tissue-derived extracellular vesicles of alzheimer's disease patients introduction: multiple sclerosis (ms) is the most frequent chronic inflammatory disease of the young adult central nervous system. nevertheless, the pathogenesis remains largely unknown. it is therefore relevant to better characterise in cerebrospinal fluid (csf), which irrigates the brain, novel bioactive compounds whose dysregulation could be involved in ms pathology. the concentration of extracellular vesicles (evs) has been already found affected in ms patient fluids but the content in bioactive molecules, particularly the micrornas (mirnas), remains barely investigated. the mirna are short oligonucleotides that are major posttranscriptional regulators and we previously showed the dysregulation of specific mirnas in csf of ms patients. evs can potentiate mirna effects by allowing remote action through the shuttling within biological fluids such as csf while providing a protection from circulating rnase. nevertheless, csf remains a challenging fluid to analyse due to limited access, low volume and presence of lipoproteins (other putative mirna carrier) that can be co-isolated with evs. methods: we performed a comparative analysis of ev isolation from csf by size-exclusion chromatography (sec), density-gradient ultracentrifugation, ultrafiltration or chemical precipitation (chemp) to determine the optimal technique(s) to enrich ev. results: sec applied on csf of control patients showed optimal ev purification with sufficient evs from . ml of csf for downstream ev characterization. furthermore, we were able to isolate mirnas from csf and determined their enrichment in evs by rnase-sensitivity treatments. finally, we have combined chemp and sec to enable a fast and largescale isolation of evs from > ml of csf, which successfully provided an increase in particles detected by nanoparticle tracking analysis. we are currently characterising the particles to confirm that they are purified evs, cleared from contaminants. summary/conclusion: this work opens perspective to analyse evs from ms patients and to determine whether mirnas participates in ms pathogenesis through their transit in evs. funding: fondation louvain, charcot foundation. differences in circulating number of extracellular vesicles between contact sport athletes with and without acute mtbi: a pilot study meghan rath a , jacqueline sayoc a , soo-young choi a , karlee burns b , aja corchado c , jane mcdevitt b , jingwie wu d , ryan tierney b , michael selzer e , xiaoxuan fan f and joon-young park a for bottom-up guc, increasing iodixanol gradients with . ml of samples were centrifuged at k g for h. fractions were then pooled based on densities ( . - . g/ml). bca and sds-page were used to analyse total protein; nanoparticle tracking (nta) and transmission electron microscopy (tem) for ev presence; and immunoblotting and imaging flow cytometry (ifcm) to evaluate ev specific markers. (ev-track id: ev ). results: immunoblotting showed absence of actinin from all samples, while cd and tsg were detected for all samples; apart from imf_ip. nt_samples were not analysed reliably by nta and ifcm, due to the high concentration of casein micelles present (~ ^ /ml in milk) that otherwise would be co-counted with evs. as expected, following ip, which most efficiently removed casein micelles, bca showed that samples had lowest total protein. this was confirmed by sds-page. thus, most effects were then focused on the ip casein-depleted samples. ifcm indicated that, post-guc, sm_ip evs had significantly (p < . ) more cd -positive particles/ml of milk vs all other guc and kduc samples. while there were no significant differences in sizes of ev separated from sm or imf, directly comparing the ip pre-treated samples, sm had significantly (p < . ) higher quantities of evs when compared to imf. additionally, tem indicated that evs separated from sm by guc were intact with limited background debris, whereas those separated from sm by duc and all imf evs were not. summary/conclusion: in conclusion, regardless of the method used, imf has fewer intact evs compared to sm. also, to obtain purest sm evs, ip followed by guc separation is optimal. introduction: extracellular vesicles (evs) exist as subpopulations with heterogeneous content. the surface heterogeneity of evs may reflect differences in functionality between ev subpopulations, as interactions with recipient cells may differ between ev subpopulations with different surface profiles. however, it is currently challenging to study functional differences between ev subpopulations due to the lack of suitable techniques to purify intact evs based on their surface signature. here, we showcase a novel capture-and-release platform to enrich intact ev subpopulations by their surface profile and compare their characteristics. methods: mda-mb- and skov- cell-derived evs were isolated using size exclusion chromatography. ev subpopulations were enriched based on surface markers cd , cd , cd or phosphatidylserine (ps) using a novel magnetic bead-based capture-and-release platform. obtained evs were characterized by transmission electron microscopy (tem), nanoparticle tracking analysis (nta) and western blotting. evs were fluorescently labelled using pkh and celltracker deep red (ctdr) and their uptake by recipient cells was examined using flow cytometry. results: western blot analysis showed that ev subpopulations enriched for the selected tetraspanins and ps were successfully isolated using a novel capture-andrelease platform. interestingly, evs isolated based on ps exposure (ps+) lacked most canonical ev markers. all ev subpopulations showed intact, cup-shaped morphology when analysed by tem, but contained less protein contaminants compared to the initial ev isolate. ps+ evs were slightly larger than other ev subpopulations when analysed by tem and nta. to test the capacity of ev subpopulations to interact with recipient cells, evs were labelled with pkh and ctdr prior to subpopulation fractionation. after fractionation, ps+ evs showed a significantly higher ctdr/pkh ratio than other ev subpopulations as determined by fluorescence spectroscopy, suggesting higher esterase activity of ps+ evs compared to other tested subpopulations. furthermore, mda-mb- derived evs isolated based on cd and cd expression were taken up more efficiently by hmec- and mda-mb- cells than evs isolated based on presence of cd or ps. summary/conclusion: using a novel technology to isolate ev subpopulations based on their surface profile, we here show that composition and cellular uptake efficiency differs between ev subpopulations. theoretically, this technology is applicable to any surface marker of interest, allowing its use to further establish ev surface-functionality relationships and enrich evs with desirable characteristics for therapeutic purposes. funding: this work was supported by a veni grant (no. ) of the dutch research council (nwo). aml were harvested from tib cells cultured in evfree medium using serial ultracentrifugation. hspc (ksl; lin-sca + ckit+) clonogenicity and inflammatory responses were assessed using colony-forming unit (cfu) assay and real-time polymerase-chain reaction, respectively. ifn-alpha receptor (ifnar ) expression and intracellular reactive oxygen species (ros) levels were assessed by flow cytometry. dna damage were assessed by quantifying nuclear γ-h ax using immunofluorescent microscopy. results: similar to evs derived from aml patients, tib ev-aml elicited double-stranded breaks in hspcs, and actively suppressed hspc clonogenicity. transcriptional profiling revealed that exposure to ev-aml induced the upregulation of several inflammatory mediators in hspcs, including isg , il- , ifnα, ch h. inflammatory signalling triggered by ev-aml did not depend on ifnα signalling as evident from suppression of clonogenicity in ifnar -null hspcs as well as the lack of evs-induced stat phosphorylation or ifnar downregulation. instead, we found increased levels of ros following ev-aml exposure. summary/conclusion: our findings support a model whereby ev-aml inflammatory signalling and oxidative stress lead to dna damage in hspcs. introduction: basic leucine zipper atf-like transcription factor (batf ) is implicated in inflammatory response and anti-tumour effects. although the tumour suppressive function of batf has been reported, its extracellular role in maintaining a non-supportive cancer microenvironment has not been explored. methods: in this study, we established gbm orthotopic and subcutaneous tumour models in nude and balb/c mice and flow cytometry analysis determined the batf inhibitory effects of mdscs recruiting. we used transwell assay to determine batf -positive evs (evs-batf ) inhibitory of the chemotaxis of myeloid-derived suppressor cells (mdscs) in vitro. in addition, exo-counter detection during the development of the gbm-batf model to demonstrate evs-batf crosstalk with distant tissues. amd blocking in tumour model confirms that evs-batf dominated by the sdf- a/cxcr signalling pathway. in addition, exo-counter detection of evs in pairs of gliomas in different stages proposes plasma-evsbatf (plevs-batf ) as a prognostic marker. results: we found that tumour-derived evs-batf regulate crosstalk between glioma cells and tumour microenvironment by inhibiting mdscs recruitment. evs-batf can be detected in plasma and bone marrow of glioma-bearing mice, this provides direct evidence that glioma-derived evs can communicate with distant site by crossing blood-brain barrier. besides, evs-batf injection significantly reduced sdf- α expression in the tumour tissues. after blocking sdf- α signalling by amd , the inhibitory effects of batf overexpression on mdscs recruitment were rescued. evs-batf inhibit mdscs recruiting and secreting mmp , mmp , and vegfa which promote gbm progression. strikingly, exo-counter detection of evs in pairs of gliomas in different stages reveals that the number of plevs-batf can distinguish stage iii-iv glioma from stage i-ii glioma and healthy donors. summary/conclusion: our results suggest that evs-batf may be an effective circulating biomarker associated with glioma progression. of note, we are the first to determine the regulatory role of evs-batf in regulating tumour microenvironment and propose plevs-batf as a prognostic marker predicting glioma progression and candidate target for gbm therapy. introduction: electrofluidics is an emerging technology of combining electronics and nanofluidics. one important device in electrofluidics is an ion transistor in which the ionic current through a nanopore is regulated by gate voltage bias. here, we suggest a fabrication method of nanopore by introducing focused ion beam (fib) and atomic layer deposition (ald) to sense extracellular vesicle (ev) via metal electrode structures. methods: we deposited nm-thick silicon-nitrite layers on both sides of silicon wafer by low-pressure chemical vapour deposition (lpcvd). we fabricated rectangular patterns by photolithography followed by reactive ion etching (rie) on the backside of the wafer. anisotropic silicon etching by koh was performed. the front side of the chip was patterned by photolithography followed by ti/au deposition for the fabrication of electrode structures. we drilled ~ nm pores in the si n membrane by fib. by the ald process, we deposited highly-conformal metal film, either platinum (pt) or ruthenium (ru) to shrink nanopores by a self limiting process. results: we expect that the ion current through the nanopore is efficiently controlled by the gate-surrounding structures. the nanopore ion transistor can be used to count the number of evs. summary/conclusion: we suggest a fabrication method of nanopore ion transistors by introducing focused ion beam (fib) and atomic layer deposition (ald). this device will be applicable for single ev sensing. introduction: extracellular vesicles (evs) are key players in cell-cell communication and increasing evidence has shown that evs function in cancer by promoting cancer cell motility and metastasis. analysing tumour-derived evs in biofluids is attractive because it would be a novel approach to a non-invasive liquid biopsy. unfortunately, evs are highly heterogeneous. they vary greatly in size, lipid composition, and cargo and are difficult to distinguish from other small particles in complex biofluids. we have developed a novel flow cytometry method to generate a distinct ev fingerprint to profile biological specimens. methods: evs from cell culture media (purified and unpurified) and biological fluids (plasma and urine) were detected by flow cytometry using features on individual evs produced by intrinsic (cd -phluorin) and extrinsic (lipophilic dye, di- -anepps, and antibodies) fluorescent labels. ev subpopulations were visualized with dimensional reduction (t-sne and umap) of - features that defined the vesicle size, shape, and fluorescent emission spectra associated with the fluorescent marker. unsupervised density based clustering (hdbscan) in conjunction with supervised machine learning (xgboost) was subsequently used to define subpopulations. we refer to this method as "ev fingerprinting". results: ev fingerprinting was successfully used to detect evs in complex biological specimens and trace their differential enrichment through conventional purification methods. evs were readily distinguished from protein complexes, lipoproteins and non-lipid particles. calibration with externally validated purified ev, as well as size, lipid, and fluorescence standards enabled ev fingerprinting as a rigorous and reproducible method for resolving heterogenous ev samples. ev fingerprinting applied to conditioned medium from tumour cells and biological fluids from cancer patients reveals unique ev profiles generated by cancer, further supporting the potential of ev fingerprinting as a liquid biopsy. summary/conclusion: our single-ev analysis approach characterizes whole ev populations in complex biological fluids without the need for purification, reducing time intensive purification protocols and subsequent sample loss, permitting efficient analysis of liquid biopsy samples. detection and quantification of extracellular vesicles with cargo protein and rna using the amnis® cellstream® flow cytometer introduction: the particle size distribution (psd) of extracellular vesicles (evs) is commonly measured by tunable resistive pulse sensing (trps) and nanoparticle tracking analysis (nta). both trps and nta have limitations that hamper the accurate measurement of the psd of evs, specifically in the size range from to nm. an alternative technique for measuring the psd of evs is micro-fluidic resistive pulse sensing (mrps). because a standard operating procedure (sop) for characterizing evs by mrps is absent, we aim to establish a reliable sop to ensure reproducible psd measurements of evs by mrps. methods: measurements (n = ) of red-blood cell, prostate cancer cell line supernatant, and human urine and plasma evs were acquired in × s acquisitions. two microfluidic cartridges were used to study a dynamic range of - nm. samples were diluted into phosphate buffered saline with different concentrations of tween or bsa. because the excess of particles affects the detection limit, serial dilutions were performed to find the optimal dilution for each sample. data were evaluated using data viewer software. results: the optimal dilution was determined for each sample by maximizing the particle rate and minimizing the measurement time while preserving a robust detection limit of or nm. moreover, we developed a procedure to optimize the peak filter settings of data viewer by fitting data to normal distributions and identifying threshold values for signal-to-noise ratio, symmetry, and transit time within % confidence. summary/conclusion: we recommend to use . % w/ v bsa in dpbs as sample diluent, because tween affects evs as confirmed by flow cytometry. by using orthogonal techniques and well-characterized biological test samples, we developed and validated a sop for ev detection by mrps, thereby making mrps a valuable tool for ev researchers. real-time measurements of extracellular vesicles binding kinetics achieved through interferometric imaging in a multiplexed microarray modality introduction: extracellular vesicles are very promising diagnostic biomarkers. as a matter of fact, the properties of these biological nanoparticles depend on the health conditions of each individual. however, experiments that involve evs phenotyping are time consuming, due to h-or overnight incubations. in order to get accurate results, maximizing binding efficiency is a necessity; that normally involves ensuring the saturation of the capture reaction, which can result in an unnecessarily long incubation time. with the ability of labelfree kinetic binding measurements using interferometric reflectance sensing in a microfluidic chamber, we perform an optimization of the incubation time in different flow conditions, while demonstrating a new way of multiplexing for real-time evs specific capture and detection.methods: all the real-time binding measurements were performed with the interferometric reflectance imaging sensor (iris). iris chips were first coated with an organic polymer (mcp- ), which provides an active surface for probe immobilization. then, antibodies against cd , cd , cd markers were spotted at different densities in a microarray modality. the chips were then encapsulated with a glass window to form a microfluidic chamber that allows for imaging the sensor surface. samples of hek-derived extracellular vesicles were flowed across the sensor surface in the iris system and real-time images were acquired. incubation was performed at different flow rates, and in static and stopflow modalities. results: in this work, we focus on the specific capture of evs under different flow conditions to achieve an optimization of the incubation time. indeed, through the acquisition of real time binding data, we are able to precisely monitor the equilibrium point of the capture reaction. in this configuration of iris, low magnification optics allow for simultaneous detection of binding on hundreds of capture ligand spots. therefore, surface probes (surface density and specificity) as well as assay conditions can be optimized. we report on the optimization of antibodies against cd , cd , and cd markers. since the sensor chips are identical to the single-particle detection assays developed by nanoview biosciences, the optimization of binding assays will directly impact the phenotyping of individual exosomes. summary/conclusion: our method proved to be very efficient in optimizing the most crucial aspects concerning evs captureflow conditions, incubation time, surface density and sample concentration. introduction: diabetes is a life treating diseases extending its impairing influence on more than billion of people around the world within upcoming years. the most harmful complication generating high treatment and social costs is diabetic nephropathy, which develops in about % of patients suffering diabetes. still we do not have an effective and direct prognostic biomarker to diagnose renal complications in the primary stage of renal disease. methods: extracellular vesicles were concentrated from diabetic patients' urine and washed to perform spectral analysis: fourier transform infrared spectroscopy (ftir), based on the molecular absorption of electromagnetic radiation in the infrared region of the spectrum in a range from cm- to cm- and raman spectroscopy (rs) as a technique based on inelastic scattering of monochromatic light. both techniques provide information on the chemical structure of compounds by identifying functional groups with high molecular specificity. results: average spectral signature obtained for evs from urine samples of patients in the different stage of kidney damage allowed distinguishing specific bands, representative for amide (i/ii), lipids, cholesterol and nucleic acids. spectral parameters correlated with a clinical stage and a commonly used indicator of renal function (creatinine) in diabetic patients. summary/conclusion: infrared and raman spectroscopy are promising tools to diagnose and monitor renal function in diabetes. introduction: several existing bioanalytical strategies for purifying and characterizing exosomes have allowed for fundamental progress to be made. mixtures of evs can be enriched for exosomes by techniques such as ultracentrifugation and size-exclusion chromatography. but, these processes require large amounts of material that are often difficult to obtain and many different types of particles have similar sizes and densities. it is likely that unique subfractions within enriched samples exist, particularly in complex biological matrices such as blood, urine or milk which remain difficult to characterize and isolate with existing analytical technologies. methods: bovine milk exosomes were isolated via differential ultracentrifugation and resolubilized in mm ammonium acetate. these data were recorded using charge detection mass spectrometry (cdms). in cdms, individual particles are reflected back and forth through an electrostatic ion trap where they pass through a sensitive charge detector. each time a trapped particle enters and exits the detector, its charge (z) and mass-to-charge (m/z) ratio is measured. mass distributions are generated by multiplying the m/z values by the charge measured for each ion and binning the resulting masses.results: the masses of particles in a bovine milk extracellular vesicle (ev) preparation enriched for exosomes were directly determined for the first time by cdms. particle masses and charges span a wide range from m~ to~ mda and z~ to~ e and are highly dependent upon the conditions used to extract and isolate the evs. in total, , particles were detected from eight cdms measurements. a simple two-dimensional gaussian model suggests that eight unique subpopulations of particles may be resolvable based on charge and mass. complementary em and proteomics analyses confirm that samples are enriched for exosomes. particles associated with the s , s , and s families that are centred at~ . ,~ . , and~ . mda, respectively, appear too small to be ascribed to exosomes. the remaining , ( %) particles detected by cdms are within the mass range expected for exosomes. while cdms measurements are at an early stage of development, this approach appears to provide a new physical basis for separating and characterizing ev particles. summary/conclusion: this work describes a novel biophysical approach for measuring and characterizing the masses and charges of the extremely heterogenous population of exosomes and other extracellular particles enriched in bovine milk. as new sample preparation methods, aimed at purifying specific types of exosomes from different cell lines, tissues, and other body fluids continue to evolve, rapid and sensitive cdms measurements of the physical properties of mass and charge may become an important means of assessing the efficacy of different protocols. funding: nih (r gm - ). bab is supported by indiana university quantitative chemical biology fellowship (t gm ). in situ detection of exosomal microrna- b by fusion with liposomeencapsulated nanomotor introduction: breast cancer is the most common cause of cancer-associated death in women and has raised global health concerns. early diagnosis and treatment are crucial to improve the prognosis and survival rate of breast cancer patients. liquid biopsy is expected to provide a strategy for early diagnosis of breast cancer. exosomes have been regarded as novel liquid biopsy biomarkers due to their stable cargo of rnas, lipids, and proteins from their origin cells. exosomal micro (mi)rnas have recently been recognized as promising indicators of cancer occurrence and progression. however, most of the reported exosomal mirna detection methods require the lysis or extraction process, which increases the possibility of sample loss. in situ detection strategies avoid interference from body fluid. in this study, we developed a gold nanomotor fluorescence platform based on liposome fusion for breast cancer exosomal mirna in situ detection. the exosomal mirna detection platform was constructed using a gold nanomotor (detector) and liposomes (carrier). the dnazyme amplification sequences which could be especially triggered by mirna- b were identified by sds-page before modified on gold nano-motor and the capacity of the nanomotor was assessed using synthetic target sequence, breast cancer cell mda-mb- , mirna- b-encapsulated anionic liposomes, and mirna- b-expressing exosomes. three kinds of liposomes were synthesized, characterized, and assessed for loading ability. membrane fusion effect was evaluated by confocal laser scanning microscopy (clsm) and nanoflow cytometry. the performance of this method to discriminate between breast cancer patients and healthy individuals was investigated. results: the chosen dnazyme amplification sequences transformed "locked" status to "cleavable" status on target addition, releasing a fluorescence signal. the modified gold nanomotor showed a ten times higher fluorescence signal in the presence of mirna- b than the background and no noticeable fluorescence changes from a single-base-mismatch sequence. moreover, among the three different liposomes, cationic liposomes exhibited great stability, high loading efficiency, and excellent membrane fusion effect. furthermore, the fluorescent experiments confirmed that cationic liposomes could load and transfer the nanomotors into exosomes for mirna- b detection. finally, we were able to distinguish breast cancer patients and healthy individuals by sensing exosomal mirna- b directly from plasma samples without exosome isolation. summary/conclusion: a separation-free and sensitive assay based on dnazyme amplification technique and membrane fusion effect was established for breast cancer-derived exosomal mirna- b detection, which could be a promising tool for the liquid biopsy of breast cancer. isolation of exosomes by membrane affinity column increases non-exosomal rna recovery in comparison to differential ultracentrifugation introduction: exosome-based liquid biopsy is a potential aid in the diagnosis and prognosis of cancer patients. however, in order to incorporate exosomes into clinical routine, there is a need to compare different isolation methods. here we analysed the impact, in exosomal rna yield, of two intermediate recovery/ intermediate specificity methods: differential ultracentrifugation (ucd) and a membrane-affinity column (mac) kit. although mac has a faster performance which is more suitable to the clinic, we found that ucd results in a higher recovery of exosomes and less contaminating non-exosomal rna.methods: exosomes were enriched by mac and ucd from identical volumes of human plasma ( , xg, min/ . ) m filtration/ , xg, h)(n = ) and lymphoma conditioned medium( xg, min/ xg, min/ xg, min/ , xg . h/ , xg, h) (n = ). all exosomes were characterized by nanoparticle tracking analysis (nta), immunoblotting of cd /cd /flotilin/alix and electron microscopy (tem). exosome pellets were pre-treated with proteinase k ( mg/ml/ °c/ min) and rnase a ( mg/ ml/ °c/ min) before phenol-chloroform/glycogen rna extraction. rna yield was measured by both fluorometer and bioanalyzer.results: isolation of exosomes by ucd, in both plasma and medium, resulted in a higher yield in comparison to mac. this was shown by an augmented intensity of marker bands in the ucd samples (p = . , n = ) as well as by an increased number of exosomes in tem.in contrast, mac final exosomal fraction (from both plasma and medium), resulted in a -fold and fold increase in rna, respectively, in comparison to ucd when measured by fluorometer. this was confirmed by bioanalyzer. introduction: there is a need for better techniques for characterizing ev populations. we developed a sensitive multiplexed electrochemiluminescence (ecl)based assay format to characterize evs in cell-conditioned medium (ccm) and human biofluids. here we use the format to analyse ev samples for the presence of ev surface proteins, and to identify changes in ev phenotype associated with different cell lines, purification methods and growth conditions. methods: multiplex plates were prepared on msd's u-plex® platform with antibodies for putative evsurface proteins. each well displayed an array of nine specific capture antibodies and a negative control antibody. evs from samples were captured on the arrays and then detected with a cocktail of anti-tetraspanin antibodies (cd , cd and cd ) conjugated to an ecl label. three distinct cell types were grown at two sites, msd and atcc. resulting ccm were each purified by four common methods: tangential flow filtration, peg-based precipitation, size-exclusion chromatography and centrifugal ultrafiltration. all samples were also assayed without purification.results: fifty-five of the surface markers were detected on intact evs from at least one evaluated cell type. datasets were analysed using correlation matrices, hierarchical clustering, and machine learning. for each cell type, when comparing unpurified ccm grown at different sites or evs prepared by different purification methods, we typically observed correlations above . , indicating that the purification methods did not introduce bias to ev phenotypes, and that the assay format can provide robust phenotypic information without any purification of evs. two unsupervised clustering analyseshierarchical clustering and t-distributed stochastic neighbour embeddingboth generated wellseparated clusters for each of the cell types, regardless of purification method or source. summary/conclusion: we developed multiplex ev surface marker assays and demonstrated their use for multimarker ev phenotyping. this flexible format enables rapid assay development for new ev subpopulations with or without sample purification. these results also demonstrate ev surface marker phenotyping via multiplex ecl assays may be used to distinguish ev populations from various cell types, and characterize bias introduced by purification. detection of misev recommended ev protein-markers using automated western blotting method for isolation of evs and a simple western blotting platform for automated protein separation and immunodetection of misev-recommended proteins.methods: total evs were isolated by affinity-membrane spin columns from pre-filtered . - ml plasma or - ml urine, respectively. intact vesicles were eluted and the ev-depleted biofluid fraction was collected from the flow-through. a small fraction ( μl) was analysed by a simple western blot workflow providing automated capillary electrophoresis-based protein separation and immunodetection, characterizing each fraction for presence or absence of misevrecommended proteins.results: a range of specific antibodies were identified and the ev fractions were shown to be enriched in evproteins, whereas contaminating non-ev proteins were significantly reduced. isolation of evs was necessary to allow detection of the low abundant ev protein markers, whereas non-ev proteins were readily detectable both in the neat biofluids and in the ev-depleted flowthrough. we characterized the effect of washing on the purity of ev isolates and defined the dynamic range of the workflow using titrations of input volume of both plasma and urine ev isolations. summary/conclusion: simple western blotting protocols were established for quality control of isolated evs in accordance with misev-guidelines. evs isolated using affinity-membrane spin columns were shown to be enriched in ev markers and depleted for non-ev proteins. al-pha beads: a library of extracellular vesicle-associated metalloproteinase biosensors (adams) and a disintegrin and metalloproteinase with thrombospondin motifs (adamtss) are highly promising cancer biomarker candidates that have complex roles in cancer pathogenesis and metastasis. importantly, within the context of lung cancer, the detection of adam proteolytic activity might be more informative than the level of adam protein.therefore, the development of low-cost metalloproteinase biosensors could serve as useful biomarker research tools. methods: to this end, we developed advanced proteolytic detector polyhydroxyalkanoates (al-pha) beadsa library of biodegradable, biopolymer-based protease biosensors. broadly, these biosensors utilise phac-reporter fusion proteins that are bound to microbially manufactured bioplastic beads. these phac-fusions also incorporate specific protease cleavage sites. in the presence of a specific protease, reporter proteins are cleaved off of the al-pha beadsresulting in a loss of bead fluorescence that can be measured using flow cytometry. these biosensors were assayed using either metalloproteinases, conditioned media or evs from in vitro cancer models.results: human metalloproteinase recognition motifs were identified in the literature and a total of different al-pha bead biosensors were designed. a control, tev-specific biosensor detected . introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr.results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom . mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the isev abstract book biogenesis of evs is neutral sphingomyelinase (nsmase ), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles.methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-( -( -( , -dimethoxyphenyl)- , -dimethylimidazo [ , -b] pyridazin- -yl) pyrrolidin- -yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated xfad mice with mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay.results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting % of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human postmortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of mdd subjects and hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed.results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd . tem images showed the typical cup-shaped morphology with sizes mostly between and nm. preliminary sequencing results revealed that mir- a- p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir- a- p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. methods: we use ifc to characterize evs released by glioma using -ala, fluorescently labelled ev (cfda-se, cd ) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd ) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that % are tenascin c positive, . % are egfrviii positive and . % are -ala positive. there was only a minor overlap (< %) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs ( . -fold), tumour specific evs ( . -fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll receptor (tlr ) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il- and il- , which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells.objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow field-flow fractionation (af ) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp- ) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk- epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with to nm (ev ) and another with to nm (ev ). ev induced more tnf-alpha, il- , ip- and ccl than ev . it was also more effective in promoting t. cruzi infection in epithelial cells. due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays.noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr.isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and . um filtration. co-purification of norovirus with the evs was checked.ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems.ps . = op . kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of . µg/ml of plasma, as geometric means ≥ . µg/ml were nearly equal. hevs were detected at µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f x electron microscope and measured between - nm, and hevs were between - nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: nasopharyngeal carcinoma (npc) is characterized by a large presence of regulatory t cells (tregs) and the production of tumour-derived exosomes with immunosuppressive properties. our team showed that npc-derived exosomes favour the suppressive activity and recruitment of human tregs via ccl chemokine, thus contributing to npc immune escape (mrizak et al., jnci, ) . more recently, our team has shown that npc-exosomes could induce tregs by altering the maturation of dendritic cells (dcs) and promoting tolerogenic dendritic cells (tdcs) (renaud et al., herpas congress ). our main objectives in this study are (i) to define and compare the metabolic status of mature dendritic cells (mdcs), control tdcs and tdcs generated in the presence of npc-exosomes (exocnptdc) and (ii) to evaluate the chemoattractive potential of npc-exosomes on exocnptdcs, and notably to investigate the involvement of ccl in this recruitment. methods: dcs are generated from human monocytes in the presence or absence of npc-exosomes. the maturation status of dcs was evaluated at a phenotypic level by studying the expression of maturation markers using flow cytometry and at a functional level by analysing cytokines secretion using elisa. this cytokine analyse has been performed in both conditions, on treated dcs and during co-culture assays of autologous cd t lymphocytes with treated dcs. in a second step, a mitochondrial metabolic and glycolytic study was performed using the seahorse technology (ocr and ecar measurement). finally, the chemoattractive potential of npc-derived exosomes on the different induced dcs was analysed (i) using boyden chamber chemoattraction assays or real-time videomicroscopy (chemotaxis µslide ibidi) and (ii) using rt-qpcr analysis of the receptor expression of ccl (ccr ).results: npc-exosomes alter dc maturation, which gives rise to tolerogenic dcs that favour the induction of tregs. in addition, the metabolic analysis of dcs seems to put foward a specific metabolic signature of the tdcs induced by npc-exosomes. and finally, chemoattraction assay suggests that npc-exosomes preferentially attract tdcs and exocnptdcs in a ccl dependant manner. summary/conclusion: taken together our results should allow us to characterize the major role of npc tumour exosomes on the maturation and the recruitment of dc and so identify them as anti-tumoural therapeutic targets. cytotoxic t lymphocyte ev that prevents tumour metastasis by collapse of tumoural mesenchymal stroma is classified into exosome, but not microvesicle or apoptotic body.naohiro seo a , junko nakamura a , tsuguhiro kaneda a , takanori ichiki b , asako shimoda c , kazunari akiyoshi c and hiroshi shiku a a mie university graduate school of medicine, mie, japan; b the university of tokyo, bunkyo, japan; c kyoto university, kyoto, japanintroduction: recently, instead of ultracentrifugation, development of new preparation protocol is demanded for research of reliable bioactivity and drug discovery of extracellular vesicles (evs). in this study, we propose a novel method for large scale preparation of highperformance extracellular vesicles focusing on membrane negative charge. methods: murine cytotoxic t lymphocyte (ctl) evs in supernatant were concentrated more than times at over % purity without leaking by kda mwco ultrafiltration, and subjected to ion exchange deae column chromatography after replacing with pbs. after ion exchange, evs were characterized by bca assay, nta assay, cryotem observation, proteome analysis, dna content measurement, mirna microarray analysis, zeta potential measurement, lectin array analysis, and target cell analysis.biomarker detection and analysis and detail strategies for cross-platform analytical validation. methods: we conducted a cross-platform analysis using two commercially available flow cytometers designed for ev detection. scatter resolution, enumeration accuracy and precision were determined across both platforms by analysing submicron silica beads (apogeemix, - nm) of known concentration.we detected large evs, as established by reference size beads, electron microscopy, expression of phosphatidylserine and the presence of integral membrane proteins of cell of origin. we analysed evs isolated from plasma by high-speed centrifugation ( , g) as well performing analysis by direct plasma labelling followed by validation by detergent lysis of vesicular constituents. a clinical operating range was defined which ensures linearity and avoids swarm detection. we observed comparable scatter resolution, enumeration accuracy (error ≤ %) and precision (cv ≤ %) across both platforms used. we defined two ev size gates: a "latex" gate ( to nm polystyrene latex beads), and a "silica" gate ( to nm silica beads) for evs at the lower end of our size range of interest. to improve detection sensitivity, we identified common contributors to signal noise and applied workflow strategies to minimize these. finally, we identified linear ranges which avoid swarm detection, and which ensures reproducible ev counts (cv < %) across both instruments. summary/conclusion: we present an optimised, standardised and cross-platform reproducible working protocol which supports the use of fcm in an ev-based liquid biopsy application. funding: the project is funded by spark oceania and uts innovation commercialisation seed fund scheme to mb. metabolomic profiling of serum and exosomes isolated from head and neck cancer patients after radiotherapy introduction: cancer radiotherapy (rt) induces the response of the whole body that could be detected at the blood level. searching for new molecular signatures which could correlate with treatment response in cancer patients is of particular importance. radiation-induced changes in proteome and transcriptome of serum have been widely described. however, metabolomic changes in serum, exosomes and other classes of small extracellular vesicles (ev) of cancer patients after rt have not been given as much attention. metabolomics of serum and ev of cancer patients could provide a valuable insight into the response of both tumour and whole organism to the treatment. the aim of the study was to compare serum and ev metabolomic profiles in head and neck cancer (hnc) patients before and after rt. methods: serum samples from hnc patients were taken before (a) and after (b) rt. healthy volunteers were used as a control group (c). ev were isolated from ml of serum using size-exclusion chromatography (sec). selected sec fractions were subjected to extraction of metabolites. a mixture of meoh/h o was used for extraction of metabolites from serum and ev samples. samples were analysed by gas chromatography-mass spectrometry (gc-ms).the study protocol adhered to the tenets of the declaration of helsinki and was approved by the bioethical committee of the maria skłodowska-curie national research institute of oncology, branch gliwice, poland (permit nr. do/dgp/ / / / / /g). results: an untargeted gc-ms-based approach allowed the detection of metabolites in serum samples and exosomal small molecules, of which joint. the identified compounds included amino acids, fatty acids, carboxylic acids, sugars, and others. there were metabolites which levels discriminated compared groups (a,b,c) of serum samples and compounds that discriminate the ev isolated from hnc serum before and after rt from hc. summary/conclusion: rt caused significant changes in levels of serum and ev metabolites witch are involved in amino acid metabolism, lipids metabolism, energy metabolism and oxidative stress response. capable of contributing to intercellular communication and metastasis. numerous studies have focused on elucidating their role in cancer progression. we recently showed that sevs isolated from pancreatic cancer cells can function as an initiator in malignant cell transformation. here, using a mass spectrometry (ms)-based proteomics approach, we analysed the differences in the protein cargo of sevs secreted from normal pancreatic and cancer cells to better understand their biological characteristics. methods: sevs were isolated from human pancreatic cancer cell lines (capan- , mia paca- , and panc- ) and normal pancreatic epithelial cells (hpde) using a combined ultrafiltration-ultracentrifugation method coupled with a sucrose density gradient purification. proteomic profiling of sevs was carried out using an lc-ms/ms method. protein identification from resulting ms/ms spectra was conducted using proteome database search software followed by gene ontology (go) enrichment and reactome pathway analysis.results: a total of , unique proteins were identified confidently across the combined samples. the proteins present in all four sev types ( , proteins) consist of general housekeeping proteins. proteins were uniquely found in all cancer sevs but not in the normal hpde sevs. this group contains an enrichment of proteins that function in the endosomal compartment of cells responsible for vesicle formation and secretion and suggest their important role in driving the increased production of sevs from cancer cells relative to normal cells. moreover, this group includes a set of proteins that have been implicated in malignant cell transformation, consistent with our previous work showing that each of the cancer sevs analysed here could initiate malignant transformation of nih/ t cells. conversely, there were proteins uniquely found in normal hpde sevs. this group includes a number of immune response proteins that are not found in any of the pancreatic cancer cell sevs. summary/conclusion: the differences in the proteomes of cancer and normal sevs may be indicative of their varying roles in cell transformation and helpful in delineating the types of evs that are being produced. in addition, these differences point towards their potential value as cancer biomarkers. proteomic profile of tumour-derived exosomes in plasma of melanoma patients introduction: in the past years, extracellular vesicles (evs) have attracted considerable interest due to their ability to provide valuable diagnostic information from liquid biopsies. the high abundance in all bodily fluids and their cargo stability confers evs the potential as a powerful tool to not only obtain novel biomarkers from inaccessible tissues, therapy response and monitoring, but also to reduce infection risks of conventional highly invasive biopsies. virtually all cells continuously release vesicles into the extracellular environment, diverse in size, content and features depending on the biogenesis, origin and function. this heterogeneity adds a layer of complexity when attempting to isolate and characterize tissue-specific vesicles. methods: hence, we aimed to use a immunomagnetic capture approach for prostate-derived evs from cell culture supernatants, with further investigation into human plasma and urine samples. analysis was performed by nanoparticle tracking analysis, western blotting and electron microscopy. additionally, an in-house spotted antibody microarray is in development. here, we intend to detect different ev sub-populations based on their surface markers. results: isolated immunocaptured ev populations based on the classical ev marker cd show an increased signal for the luminal protein tsg . ev populations targeting the tissue-specific marker prostate specific membrane antigen (psma), were found positive for tsg in a lower extent indicating a subpopulation of evs. the microarray uses less than µl of sample (concentrated cell culture supernatant, human plasma, urine) and leads to a faster characterization within h for ev surface marker as compared to western blot. summary/conclusion: immunomagnetic isolation might be a promising approach for liquid biopsy and thereby the microarray could be valuable to identify potential capture targets. the current design for different surface marker from samples simultaneously could be easily extended for sample size and surface profiling allowing for a more economical way to multiplex samples. paving the way for implementing a feasible and reliable technique for assessing urinary extracellular vesicles as biomarkers for bladder cancer in clinical practice introduction: extracellular vesicles (ev) in urine have been proposed as biomarkers for bladder cancer (bc). however, at present there are no standardized methods for ev isolation or urine sampling. our goal was to evaluate the ev isolation performance between different methods, the effect of the sampling time and the importance of urinary creatinine (ucr) normalization. methods: two urine samples of ml were collected from patients with non muscle-invasive bc: one from the first micturition and another from any time of the day. twenty ml were used for ucr measurement and ml were used for ev isolation by either precipitation with polyethylene glycol (peg), concentration by filtration (uf, centricon plus- , k, millipore), sepharose size exclusion column (sec), or combinations of these methods. additionally, the effect of protease inhibitors (pi) and dtt treatment after collection or during processing was analysed. size and number of particles were evaluated by nanosight and the presence of exosomal markers was evaluated by western blot. results: among the methods evaluated, uf + sec showed the best performance retrieving the highest number of particles in the range of - nm, and the highest protein expression of exosomal proteins. uf alone showed the highest concentration of ev, but with a tendency to isolate larger particles. particle concentration was positively correlated with ucr, reflecting the importance of ucr normalization before journal of extracellular vesicles comparing between patients. finally, no differences in the performance according to the time of collection, nor in the use of pi or dtt were observed. summary/conclusion: uf + sec gave the highest ev yield and was not affected by the time of urine collection. the use of pi and dtt can be avoided, and normalization to ucr should be considered when implementing this technique for assessing evs as biomarkers for bc in clinical practice. funding: pida . the introduction: human tumours, including pancreatic ductal adenocarcinoma (pdac), often harbour a subpopulation of cancer cells with extra centrosomes. we found, that these cells secrete an increased number of small extracellular vesicles (sevs), within the - nm size range. sevs play a role in cancer signalling and progression and are widely studied for their diagnostic potential. we aim to understand the role of sevs secreted by cells with extra centrosomes in shaping pdac-associated stroma, particularly fibrosis. methods: to study the sev mediated changes in the pdac microenvironment, we purified sevs through serial ultracentrifugation and size exclusion chromatography, characterised the content through silac-based proteomics, and assessed phenotypic changes in pancreatic stellate cells (pscs) and extracellular matrix (ecm) production through immunofluorescence staining. results: our data indicates, that the sevs secreted by cells with extra centrosomes are exosomes due to their endocytic origin, and we found, that they can activate pscs, key mediators of fibrosis in pdac. indeed, we observed an increased level of collagen i produced by pscs activated by sevs from cells with extra centrosomes as compared to cells without extra centrosomes. interestingly, we found, that psc activation through sevs is not mediated by tgf-β, assessed by the level of nuclear smad accumulation downstream of tgfβ activation, suggesting a novel mechanism of pscs activation. summary/conclusion: pdac cells with extra centrosomes contribute to a novel type of psc reprogramming, which could alter their ecm deposition and contribute to the extensive fibrosis observed in pdac. we are currently characterising the signalling pathways associated with sev mediated psc activation and how it impacts padc progression to better understand the role of centrosome amplification in the cancer-stromal crosstalk. exosomal carboxypeptidase e confers and cpe-shrna loaded exosomes inhibit growth and invasion of hepatocellular carcinoma cells. methods: exosomes were isolated from the culture media of high metastic hcc h cells and incubated with low metastatic hcc l cells. in other experiments, cpe-shna loaded exosomes from hek cells were incubated with hcc h cells. the recipient cells were analysed for proliferation using mtt assay, colony formation, and matrigel invasion. results: analysis of exosomes derived from hcc h cells revealed cpe-wt mrna and protein. exosomes released from hcc h cells were able to enhance proliferation and invasion of hcc l cells. when cpe expression was suppressed in the hcc h cells before exosome isolation, the exosomes had no effect on proliferation and invasion. these data demonstrate the ability of exosomes to confer growth and invasion in hcc cells and the role of exosomal cpe in driving the process.previously it was shown that down-regulation of cpe expression by shrna can reverse tumour growth and metastasis in an hcc mouse model. we therefore loaded cpe-shrna into exosomes by infecting hek (human embryonic kidney) cells with adenovirus carrying cpe-shrna-gfp. these modified isev abstract book exosomes were used to transfer cpe-shrna to hcc h cells, resulting in significant reduction in proliferation and colony-forming ability of these cells. cpe-shrna loaded exosomes were found to down-regulate the expression of cyclin d and c-myc, two genes with high relavance to tumour growth and metastasis. summary/conclusion: our results demonstrate the ability of exosomal cpe to enhance proliferation and invasion in low metastatic hcc cells and the potential to use shrna loaded exosomes to target cpe as a therapeutic strategy to treat liver cancer.funding: intramural program of the eunice kennedy shriver national institute of child health and human development, and national cancer institute, national institutes of health, bethesda, md. . stress hormones promote prostate cancer aggressiveness through modulation of mir- - p expression and exosome release north carolina central university, durham, usa introduction: despite proactive screening and steady declines in mortality, prostate cancer (pca) remains one of the most prevalent cancers among men. evidence suggests that chronic activation of stress signalling pathways can result in an altered mirnas transcriptome and affect exosomal content and release. here, we study the interaction between leptin and mir- - p expression, previously shown to be downregulated in pca patients. in addition, explored the effect of stress hormones cortisol and leptin on exosomal release and content from pca cells.methods: we utilized normal prostate cell line rwpe- , and pca cells pc , lncap and mda-pca- b. proliferation of cells treated with leptin in the presence or absence of mir- - p mimic or negative control was assessed by mtt, colony formation, wound healing, and expression of targets affected by mir- - p was assessed by western blotting. moreover, exosomes were isolated via differential centrifugation from pca cells treated with leptin or cortisol and exosome number was determined by nanotracking analysis. exosome content was determined by western blotting and proteomic analysis by mass spectrometry.results: we observed that leptin significantly decreased expression of mir- - p in rwpe- cells.co-treatment with mir- - p mimic and leptin abrogated these effects in a cell dependent manner. we also observed that co-treatment with leptin affected mir- - p target jag and other molecules involved in epithelial to mesenchymal transition. in parallel, we demonstrated that cortisol increases exosome secretion particularly in pc cell exosomes with a . -fold increase at nm cortisol compared to untreated. western blotting revealed the presence of gr in exosomes particularly at nm cortisol. summary/conclusion: understanding epigenetic regulation through mirnas and exosomes may be the key to understand stress hormone influence in pca progression. these findings suggest that stress hormones effectively affect mir- - p expression and exosomal release and signalling.introduction: extracellular vesicles (evs) are promising drug delivery vehicles for therapeutic microrna (mirna). for the loading of exogenous cargo, researchers broadly seek to either manipulate the evs directly or the cell that produce them. electroporation, sonication, and direct ev transfection are common methods that work by physical disruption or irreversible chemical addition, which may irreparably damage the molecules intended for therapy. on the other hand, transfection into the producer cells is a simple option that does not imperil ev integrity.methods: there are multiple factors that contribute to ev loading efficiency, including transfection reagent used, timing, and dosage. thus, we sought to establish a basic protocol and improve understanding of the underlying dynamics involved in a basic system consisting of hek t cells and mir- a- p mimic.results: in this work, we examined how different reagents lead to variable ev loading. then we looked at variable dosages, specifically the relationship between rna amount added to reagent, amount present in cell, and amount exported to evs. summary/conclusion: these results will help future studies produce evs with exogenously loaded small rna, and suggest future optimizations. funding: national institutes of health. r and t (host pathogen interactions at university of maryland). we report a single ev trapping method via aptamermediated assembly between au nanoparticle (aunp) and au superlattice template. we propose a chip-based ev trapping technique based on semiconductor processes. methods: we introduce aptamer coated au nanoparticle (aunp) and au superlattices as a template to capture evs. first, we fabricated poly(methyl methacrylate) (pmma) hole pattern on au-coated si substrates by using electron beam lithography (ebl). we designed nm-diameter hole patterns to capture one ev in each hole. to connect the aunp and the au superlattice template, we used an aptamer molecule as a linker strand. also, to capture individual evs, the aptamer molecule is designed to have a hairpin structure to specifically bind to cd , a protein marker of ev. we modified ʹ-terminal and ʹ-terminal of the cd aptamer with thiol group for the formation of self-assembly monolayer (sam) on both aunp and au superlattice surface. results: first, we coat the cd aptamer on the surface of aunp. afterwards, we load the aptamer-coated aunp into au superlattice template. ev solution is specifically bound to cd aptamer. after washing step, each ev is expected to locate within a single hole due to the size confinement of the hole. to separate the evs from the aptamer, we use restriction enzyme, bamhi, to recognize specific dna sequence and cleave them. summary/conclusion: in this report, we propose a aunp -linked au superlattice chip by aptamer molecules for trapping evs. we selected cd aptamer for specifically binding with cd in evs. in addition, we designed cd aptamer as a linker strand to connect introduction: a hallmark of platelet activation is the release of internal granules as extracellular vesicles/ microparticles. thrombolux is a dynamic-light-scattering-based (dls) instrument that was developed for use in clinical setting to check for platelet activation before transfusion. compared to traditional dls, the thrombolux requires no cleaning (single-use capillary) and requires very little sample ( µl). hence the thrombolux may be a useful instrument beyond platelet pack test in blood transfusion laboratory. we have evaluated its use as an in-process monitoring tool for industrial ev manufacturing, for both quantifying cells (input) and evs (output). methods: the thrombolux was used to test the activation status of expired platelet packs (donated by arcbs for research purpose). the readout was compared with platelet swirling test and flow cytometry data (surface marker). furthermore, the thrombolux was also tested for process development and ev manufacturing monitoring purposes at different stages of the process for its ability to rapidly obtain particle presence and size information on evs. time to result was also compared between different particle analysis methods. results: the thrombolux was a better predictor of platelet packs variability compared to the traditional platelet swirling method. however, we did not observe a strong correlation between the activation status and the flow cytometry-based activation marker data. the thrombolux was able to provide a useful estimation of particle presence and sizing of evs in-process.results are obtained rapidly, within minutes, with minimal sample prep. summary/conclusion: although we did not observe a significant direct correlation between flow cytometry activation data and the % microparticles (within a small sample size), the thrombolux has shown potential to become a useful tool for in-process monitoring for ev manufacturing and other ev research, in particular through its speed and ease of use. funding: all funding was through exopharm ltd (asx:ex ). secreted introduction: a major manufacturing challenge related to exosome bioprocessing is that of robust and scalable purification. as efforts to translate exosomes into clinics grows, the more important the design of quality systems which can reproducibly purify the product becomes. the current gold-standard, ultracentrifugation, was adopted from the viral vaccine industry, but remains imperfect in terms of scale up and manufacturing due to labour and time intensive process requirements. in order to follow the preferential adoption of more standard bioprocesses, as previously achieved by the viral vaccine industry, we show the development of two monolith chromatography steps which can be used to purify exosomes from a clinically relevant, allogeneic stem cell product (ctx e ). methods: t-flask expansion of ctx e cells was performed to yield batches of - l of conditioned medium. the medium was subsequently clarified by benchtop centrifugation, and concentrated into a crude concentrate by tangential flow filtration [tff], using a combination of . µm dead-end filtration prior to concentration in a kda hollow-fibre tff system. tff retentate was loaded onto ml hic or aex monoliths, for further purification. potency was assessed by a fibroblast wound healing assay in vitro. results: exosome presence was verified in the tff material by detection of cd and cd . exosomes recovered in this manner could achieve full wound closure in vitro over hours, when dosed at µg. further purification by monolith chromatography showed high levels of reduction of albumin, detected by western blot, as well as heightened ratios of particles to both total protein, and total dna. the results indicate that neither aex nor hic steps cause detrimental loss to product function, either alone or in combination with one another. introduction: custom-made platelet pellet lysate (ppl) and heat-treated ppl (hppl) exert strong neuroprotective effects of neurotoxin-exposed dopaminergic luhmes neuronal cell culture. this effect is significantly enhanced using hppl, which was also highly protective of th-expressing neurons in mice parkinson's disease (pd) model. introduction: there is a critical unmet medical need for new therapies to treat age-related diseases including cardiovascular diseases such as stroke. exosome derived from stem cells have shown intrinsic therapeutic potential in a variety of animal models of ischaemic diseases. we have identified scalable exosome production cell lines (purestem) as a source of angiogenic exosomes and are aiming to generate good manufacturing practice (gmp) grade therapeutic exosomes that can effectively mediate angiogenesis and tissue regeneration. we are developing exosome production and purification protocols that combine methods of tangential filtration flow (tff) and size exclusion chromatography (sec). the particle number and size were measured by both tunable resistive pulse sensing (trps) as well as nanoparticle tracking analysis (nta) for comparison. exosomes were characterized by detection of exosome surface markers and absence of cellular markers. purity was assessed by measuring particles per ug of total protein content. the angiogenic activity of purestem-exosomes was assessed using live-cell imaging to measure endothelial wound-healing and tube formation assays. we further investigated the molecular cargo of purestem-exosomes by screening mirnas targets, rna-seq analysis, and mass spectrometry analysis.results: the isolated purestem-exosomes using our developed protocols were highly purified, resulting purity in the range of e - e particles/ug. we selected angiogenic exosome-producing cell lines from our purestem library by screening for functional activity and characterizing their molecular cargo. we found that purestem progenitor-derived exosomes showed higher angiogenic potency than primary mesenchymal stem cell (msc)-derived exosomes. furthermore, angiogenic micrornas such as mir- were enriched in purestem-exosomes from certain producer cell lines. summary/conclusion: these data demonstrate the potential for using purestem lines as a highly scalable source of therapeutic exosomes. we were able to obtain highly pure exosomes that retain their angiogenic activity. we anticipate that purestem-exosomes will be a valuable resource for developing ev therapies for stroke and other ischaemic diseases. we have developed purification methodologies aimed at achieving a robust and scalable exosome production compatible with gmp for clinical grade purestem-exosomes. these developments have great potential as therapeutic agents for future preclinical in animal model of stroke and clinical trials. neuronal introduction: the hallmark of parkinson's disease (pd) is a-synuclein accumulation, predominantly in dopaminergic neurons, causing neurodegeneration. pd is also associated with insulin resistance, a condition characterized by phosphorylated insulin receptor substrate- (irs- ). besides motor symptoms, some pd patients develop mild cognitive impairment (pd-mci) or dementia (pd-d). given the importance for prognosis, there is an urgent need to develop biomarkers for distinguishing pd with normal cognition (pd-n) from pd-mci/d. neuronal-origin extracellular vesicles (nevs) contain cell signalling and pathogenic proteins (including a-synuclein), which may serve as biomarkers for alzheimer's disease, pd and other dementias.methods: from . ml of plasma from pd-n, pd-mci, and pd-d patients, we immunocaptured nevs using anti-l cam antibody. then, irs- pser and irs- ptyr and a-synuclein were measured in nevs using electrochemiluminescence immunoassays.results: a-synuclein was lower in pd-mci and pd-d compared to pd-n (p < . ) and significantly decreased with increasing motor symptom severity measured by mds-updrs iii score (p = . ). irs- pser was lower in pd-d than in pd-n. irs- ptyr significantly decreased with increasing mds-updrs iii score (p < . ). no biomarker was associated with disease duration. summary/conclusion: pd patients with cognitive impairment exhibited lower nev levels of a-synuclein than cognitively intact pd patients, whereas a-synuclein and irs- ptyr were inversely associated with pd motor symptom severity. additional biomarkers and measurements will be available by the time of isev. plasma nevs is a valuable tool for discovering biomarkers in pd and investigating aspects of disease progression. introduction: despite decades-long advancement in transplant medicine, there is a necessity for personalized approach regarding early kidney allograft injury recognition and immunosuppression therapy towards improved transplant outcomes. biopsy, a gold standard for assessment of kidney allograft injury, cannot be serially used for the diagnosis of subclinical injury due to it's invasiveness and possible sampling errors. instead, urine is easily obtainable and bearing extracellular vesicles (evs), potential carriers of pathological signals related to kidney injury. our aim was to set up a urinary ev (uev) isolation protocol that would allow consistent and reliable identification of their characteristics and cargo. methods: second morning urine sample ( ml) was collected from patients and processed within hours. oxalate precipitation, ph and dilution variability, uromodulin polymerization and high protein content were taken into account. isolated evs were defined by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). uev specific proteins and mirnas were analysed by western blot and qpcr, respectively. results: the optimal protocol relied on low speed urine centrifugation ( . x g, rt) for cell removal and storage at − °c prior to further analyses. after urine thawing at rt, added edta averted cryoprecipitate and uromodulin polymer formation, while concentrated pbs neutralized the ph. filtration through . µm pores was used for large particle removal, while centrifugal kda membrane units (amicon®, milipore) served for sample concentration followed by particle separation on sizeexclusion chromatography (sec; qevoriginal, izon q). protein vacant sec fractions (as rated at a ) were pooled and concentrated to a volume of µl. tem micrographs revealed high sample purity and cup-shaped morphology of uevs. as per nta results, the average mean size of evs was , nm with concentration range of × particles/ml of starting urine. uevs were positive for the tested marker proteins hsc , flotillin, tubulin, gadph and cd . qpcr verified mirna presence in uevs, with ct for mir let- i at . summary/conclusion: we successfully isolated pure uevs. the set up protocol will be used to assess uevs as non-invasive biomarkers of allograft injury in kidney transplant recipients. astrocyte-derived extracellular vesicles regulate dendritic spine formation and neuronal network connectivity introduction: recent advancements in the biology of extracellular vesicles have begun to implicate glial released microvesicles as mediators of glia to neuron communication, suggesting that alterations in the release and/or composition of astrocyte microvesicles could impact neuronal function. methods: astrocytes were allowed to constitutively release extracellular vesicles (adev-cr), or stimulated with atp (adev-atp). adevs were isolated by ultracentrifugation followed by proteomic analysis. we developed a normative whole transcriptome database using primary neurons exposed to adev-cr, and identified changes in neuronal gene expression produced by exposure of neurons to adev-atp. we identified a number of pathways associated with the biological response of synapse, spine and neurite outgrowth that were regulated by adev-atp. the molecular cargo of adev-atp responsible for regulating synaptic functions in neurons were characterized by biochemical, molecular, and functional assays. results: adev-atp enhanced the maturation of dendritic spines and produced functional enhancements in neuronal activity and network connectivity. the mechanism for this effect involved the delivery of integrin- and epha that were enriched in adev-atp. integrin- facilitated binding of adevs to the neuronal surface, and epha -receptor signalled through ephrin to the tyrosine kinase erbb / that regulated the phosphorylation and activation of trkb without increasing expression of the natural ligands bdnf or ntf . this direct activation of trkb increased the expression of the synaptic scaffolding proteins disc , arc, and cplx to promote the maturation of dendritic spines. this increase in mature dendritic spines was associated with increased neuronal activity and network connectivity demonstrating a functional strengthening of synapses. summary/conclusion: these data identify a molecular mechanism whereby modifications in adev protein cargo produced by the stimulation of astrocytes with atp regulates synaptic maturation through activation of trkb in a manner independent of growth factors. stephanie kronstadt and steven m. jay university of maryland, college park, college park, usa introduction: mesenchymal stem cell extracellular vesicles (msc-evs) have been shown to have an immunosuppressive effect in both autoimmune and inflammatory disorders. despite this, clinical translation of ev therapies is hindered by potentially low potency in vivo and the lack of a scalable biomanufacturing process. cell culture parameters are critical in modulating both yield and bioactivity of evs. thus, we hypothesized that the combination of chemical priming and d dynamic culture would enhance the yield and potency of immunosuppressive msc-evs. methods: bone marrow-derived mscs cultured in flasks were chemically primed using ethanol or curcumin. mscs were also cultured using a d-printed scaffold-perfusion bioreactor using a flow rate of ml/min. anti-inflammatory effects were assessed following application of msc-evs to lipopolysaccharide (lps)-stimulated murine macrophages. subsequent inhibition of the production of the pro-inflammatory cytokine il- , quantified using an elisa, was used to characterize evs as anti-inflammatory. in addition, both chemical priming and the bioreactor will be simultaneously utilized to potentially uncover any synergistic effects on ev immunomodulation abilities. nanoparticle tracking analysis (nta) was used to assess ev size and concentration while protein mass was measured via a bca assay. results: preliminary data suggests that priming mscs with µm ethanol for hours prior to ev collection results in a strong inhibition of il- production in stimulated murine macrophages. nta revealed that msc-ev yield increased by about two orders of magnitude in the bioreactor ( . e ± . e ) when compared with flasks ( . e ± . e ). protein measurements also indicated that ev production in the bioreactor (~ µg) was much greater compared with production in the flasks (~ µg). additionally, average protein content per ev was reduced in the bioreactor when compared with flask evs. regardless of tissue source. furthermore, comparison of adipose tissue-derived (ad) msc evs from three donors indicates varying pro-vascularization bioactivity between those donors evaluated in vitro via gap closure assay. similar results were observed for the bone marrow-derived (bm) msc ev donor groups. summary/conclusion: this work highlights the need for screening of donor derived-mscs before use for therapeutic ev production. additionally, standardized criteria for msc donor selection are needed before isolated msc evs can be used as a large-scale, repeatable therapeutic treatment. analysis of extracellular vesicle populations from malaria-infected erythrocytes by field-flow fractionation reveal distinct sub-sets alicia rojas a , paula abou-karam a , anna rivkin a , yael fridmann-sirkis b , yifat ofir-birin c and neta regev-rudzi c a department of biochemical sciences, weizmann institute of sciences, rehovot, israel, rehovot, israel; b wis, rehovot, israel; c weizmann institute of science, rehovot, israel introduction: malaria is one the most devastating infectious disease in the world and plasmodium falciparum (pf) represents the deadliest species. this parasite invades human red blood cells (rbcs) and releases extracellular vesicles (evs) carrying dna, rna and protein cargo components which are involved in the pathogenesis of the disease. recently, it has been shown in mammalian systems that evs are subdivided into different subpopulations, each with a distinct biological function. however, it is still unknown whether pfinfected rbcs (pf-evs) release different ev subpopulations with distinct cargo. methods: we isolated evs from pf-infected and uninfected rbcs, pf-evs or ui-evs, respectively, using differential centrifugation. the ev pellet was subjected to field flow fractionation (fff). the different subpopulations were collected, concentrated with size-exclusion filters and evaluated by nanoparticle tracking analysis. additionally, the presence of ev markers (sr and hsp ) were examined by western blot analysis. results: the fff analysis showed four particle subpopulations derived from the pf-evs and five in the ui-evs. the first three subpopulations were similar in their detection signals in both samples, but the fourth subpopulation was consistently higher in ui-evs than in pf-evs. moreover, hsp was detected in subpopulations and of both pf-evs and ui-evs, whereas sr only in subpopulation . isev abstract book summary/conclusion: pf-ev and ui-ev have similar separation profiles and proteins markers in their subpopulations, consistent with the fact that both samples are derived from host rbcs. additional data regarding the dna and rna cargo, as well as microscopic observations of the pf-ev and ui-ev subpopulations is necessary. this will clarify how malaria parasites sort their components into evs and which fractions are associated to immune evasion and pathogenesis. we have established a small size laboratory production of the microalgae culture in order to harvest the extracellular vesicles (evs) for pharmaceutical and medical uses. in this work we report on globular particles in the isolates from media of microalgae of two types, that we recognize as evs. we observed changes in their production at different temperatures and conditions. methods: samples were fixed by various combinations of aldehyde fixatives and/or osmium tetroxide. they were dehydrated in a graded series of ethanol, hexamethyldisilazane, and air dried. they were au/pd coated for inspection with scanning electron microscopes (sem) crossbeam fib-sem gemini ii (zeiss, germany) and jsm- f field emission scanning electron microscope (jeol ltd., tokyo, japan). results: microalgae were incubated overnight at °c and °c in growth medium and in growth medium supplemented with detergent. the samples obtained from the microalgae culture contained particles that we recognized as extracellular vesicles, however, these particles do not correspond to characteristic shapes of membrane enclosed entities without internal structure. increased temperature and/or presence of surfactant (triton x- and sodium dodecyl sulphate) stimulated formation of evs of different shapes and sizes. the isolates of these samples were rich with evs. in the presence of surfactant, the cell-walls detached from the cell and collapsed upon dehydration. this was documented by sem. summary/conclusion: focused ion beam technique revealed complex internal structure of the algae. it seems from the shapes of the observed structures that the particles deposited on the surface of the microalgae do not derive from budding of the membrane surface, but are instead shed by the cells from the cell interior upon the rupture of the cell wall. key: cord- -uj fe y authors: nan title: scientific abstracts date: - - journal: reprod sci doi: . / sha: doc_id: cord_uid: uj fe y nan by reduced placental oxygenation, hypoxia-induced oxidative stress is a predominant mechanism. we investigated the effect of hypoxic pregnancy, with and without antioxidant treatment, on placental and maternal circulatory indices of oxidative stress in rats. methods: on pregnancy day , wistar rats were randomised into: normoxia ( % o litters), hypoxia ( % o litters) and hypoxia + vit c ( % o + mg. ml - vit c in water, litters). on day , dams were anaesthetised, maternal blood taken, pups measured and weighed, placentae weighed and frozen. only placentae from two male pups from any one litter were investigated. blood was processed for ascorbate, urate, l-cysteine and glutathione (gsh) measurement. placental protein was analysed for heat shock protein (hsp ; western). results: hypoxia + vit c did not affect maternal food or water intake. vit c elevated maternal ascorbate by % of baseline; a similar increment to human trials (poston et al. ) . hypoxia elevated placental hsp and maternal plasma urate and l-cysteine, but decreased gsh. vit c in hypoxic pregnancies prevented all stimulated effects, but the reduction in gsh persisted. hypoxic pups had a reduced ponderal index and elevated head diameter: body weight ratio; effects also prevented by vit c. fetal oxygen uptake in normal and gdm pregnancies. emanuela taricco, tatjana radaelli, veronica cozzi, gabriele rossi, danila puglia, giorgio pardi, irene cetin. department of obstetrics gynecology "l. mangiagalli", irccs policlinico, mangiagalli e regina elena, milan, italy. background. diabetes in pregnancy has been associated with alterations of fetal growth probably due to increased nutrient availability and placental transport. fetal hypoxia and acidemia have been reported in pregestational diabetic pregnancies with poor glicemic control but this is still uncertain in well controlled patients. the role of placental function and the relationship between maternal and fetal circulation is crucial for efficient exchanges of oxygen and nutrients. since umbilical blood flow can be obtained by us in utero, we studied normal and gdm pregnancies in order to evaluate fetal oxygen uptake. methods. normal (n) and gdm pregnancies were studied at term, at the time of elective caesarean section. umbilical vein volume flow (qumb) was measured by us before caesarean section and blood samples from umbilical vein (uv) and artery (ua) were obtained. blood gases and acid-base balance were evaluated. results. average fetal weights were similar in both groups (n= ± ; gdm= ± g) while placental weights were significantly different (n= ± ; gdm= ± g). n and gdm pregnancies showed similar values of qumb and qumb/kg of fetal weight. (qumb: . ± . in n and . ± . ml/min in gdm; qumb/kg: . ± . in n and . ± . ml/min/kg in gdm). in fetuses from gdm pregnancies a significant reduction in o sat, o cont and po and a significant increased lactate conc was found in both uv and ua compared to n (table ) . o umb uptake (n= . ± . ; gdm= . ± . mmo/l/min) and o umb uptake/kg (n= . ± . ; gdm= . ± . mmo/l/ min/kg) were significantly lower in gdm compared to n fetuses. conclusions. our data indicate that fetuses from gdm pregnancies show a significant reduction in oxygen supply despite a normal blood flow/kg of fetal weight. these data may suggest that a good maternal metabolic control is not sufficient to ensure normal placental oxygen supply and/or utilization by the gdm fetus. background: synthetic glucocorticoids (sgc) are given to mothers at risk of preterm delivery to promote fetal lung maturation. evidence is emerging indicating long-term effects of such treatment on endocrine function and behavior in offspring. however, virtually nothing is known concerning potential transgenerational influences on growth, endocrine function and behaviour. we hypothesize that repeated treatment of grandmothers (f ) with sgc will alter hypothalamic-pituitary-adrenal (hpa) function in f offspring with no manipulation of the f pregnancy. methods: pregnant guinea pigs (f ) were subcutaneously injected with betamethasone (beta; mg/kg) or vehicle (veh) on gestational days / , / / . adult f female offspring from each group were mated with control males. hpa function was assessed in adult f offspring by non-invasive measurement of salivary cortisol concentrations: ) under basal conditions, ) during and following exposure to psychological stress (high frequency strobe light) or psychological/physical stress (forced swim) and, ) following dexamethasone suppression. results: there was no effect of beta (f ) on bodyweight from birth to adulthood in f offspring. basal salivary cortisol in the betaf females was lower than in the vehf group in the morning but not the afternoon; there were no differences in male f offspring. in contrast, both male and female betaf failed to mount adrenocortical responses to psychological stress compared to vehf offspring that mounted robust responses (p< . ). swim stress induced robust adrenocortical responses in all groups (p< . ), however, the response was consistently lower in betaf offspring. beta exposure also led to a significant difference (p< . ) in the cortisol response to dexamethasone suppression in female (f ) offspring, with a similar trend in males. conclusion: prenatal exposure to sgc (f ) causes transgenerational programming of adrenocortical function in adult f offspring. grand maternal exposure to beta results reduced basal adrenocortical activity in betaf female offspring, and caused stress hypo-responsiveness in both males and females. dexamethasone suppression tests indicate altered central glucocorticoid feedback. these findings have important ramifications for the management of human preterm labor. support: canadian institutes of health research. in the uterine and umbilical vasculatures, we hypothesized that their remodeling would also be blunted in pregnant enos -/-mice, leading to an elevated vascular resistance and decreased blood flow to the placenta contributing to fetal growth restriction. methods: utero-and umbilical-placental blood velocity waveforms and umbilical arterial diameters were measured using mhz ultrasound biomicroscopy in control (c bl/ j) and enos -/-mice at . days of pregnancy (n= mothers). spiral artery and fetal capillary morphologies, and uterine arterial diameters were evaluated from vascular corrosion casts. tissues were collected for hydroxyprobe- and actin immunohistochemistry to identify hypoxic and smooth muscle regions. we calculated resistance index ((s-d)/s) from systolic (s) and diastolic (d) velocities, and blood flow from mean velocity and vessel area. results: calculated uterine blood flow normalized to the weight of the uterus and its contents was % lower (p< . ) in pregnant enos -/-mothers due to large reductions in uterine artery diameter (- %, p< . ) and mean velocity (- %, p< . ). uterine arterial resistance index was % higher (p< . ), and the spiral arteries were less coiled and contained more smooth muscle actin in enos -/-mice than controls. more intense hypoxic immunoreactivity was detected in the spongiotrophoblast and trophoblast giant cell layers of the junctional zone of enos -/-placentas, whereas fainter staining was only detected in the spongiotrophoblast cell layer in controls. in the umbilical circulation, flow normalized to fetal weight was not significantly changed although the resistance index was slightly elevated ( %, p< . ) and capillary lobule length was reduced by (- %, p< . ). fetal organs showed increased hypoxic immunoreactivity suggesting reduced organ oxygen delivery in enos -/-fetuses. conclusions: results suggest that enos plays an important role in uterine and spiral artery remodeling and in augmenting utero-placental blood flow during pregnancy. it also appears to enhance oxygen delivery to the placenta and fetus. enos may contribute to normal fetal growth by these mechanisms. integrin methods: hpmcs isolated from term placentas were assessed for their phenotype markers, mutilineage capacity, and the expression of integrin molecules. the hpmcs were induced to endothelial cell differentiation in the presence of endothelial cell growth medium with % of fcs and ng/ml vegf for to days. the angiogenesis ability of these cells was demonstrated by using an in vitro angiogenesis kit and in vivo chick chorioallantoic membrane assay from ten-day-old embryos. blocking antibodies specific to integrin , associated with gdm. this study was adequately powered to detect association in the caucasian and asian group. the absence of association suggests that gdm and t dm may have more divergent molecular pathophysiology than previously suspected. background: uterine endometrium has a unique cycle of physiological angiogenesis. in mice, endothelial progenitor cells (epc) contribute to endometrial angiogenesis being incorporated after oestradiol administration. objective: to determine whether circulating © epc number and function vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: ten healthy, nulliparous, pre-menopausal, non-smoking women (mean age years) with regular menses ( - days) were studied. venous blood was collected during menstrual, follicular, periovulatory and luteal phases of a single cycle (days - , - , - , - ) . cepcs, serum oestradiol (e) and progesterone (p) were measured at each phase. cepcs were quantified by phenotype using flow cytometry (leukocytes co-expressing cd , kdr and cd ) and function by the epc-colony forming unit (cfu) assay. epc-cfus were stained for endothelial markers including uptake of acetylated low-density lipoprotein, binding of lectin (ulex europaeus) and endoglin. results: luteal p was > nmol/l in all women. cepc numbers increased in the menstrual and follicular phases being -fold higher in the follicular compared to periovulatory phase (p< . ). there was no significant variation in cepc function over the menstrual cycle. there was no correlation between serum e or p levels and cepc number or function. conclusion: cepcs number but not function (epc-cfu assay) vary during the menstrual cycle with numbers increasing during the menstrual and follicular phase and falling in the periovulatory and luteal phase of the menstrual cycle. this may represent mobilisation of cepcs from the bone marrow and subsequent incorporation into the endometrium. neither function nor cepc number correlate with serum p or e. our previous studies demonstrated that tissue factor (tf), which binds factor vii to act as a potent pro-coagulant and angiogenic factor, is over-expressed in endometriotic lesions. thus, we determined whether tf could serve as a target for the elimination of pre-established ectopic human endometrial growth in a mouse model. icon is composed of a mutated factor vii (fvii) domain targeting tf and an igg fc (fvii/igg fc) effector domain that activates antibody-dependent immune responses against tf bearing endothelial cells. methods: athymic, ovariectomized and estrogen-treated mice received intraperitoneal (i.p.) injections of . mg of proliferative phase human endometrial tissue derived from normal (disease-free) women. twelve days after inoculation to establish lesions, icon protein ( ug) was delivered i.p. once a week for weeks. after sacrifice, animals were subject to gross inspection. residual endometriotic tissue was formalin fixed, paraffin embedded and immunostained for von willebrand's factor (vwf) and tf. results: compared to control mice, treatment with ug of icon abolished all lesions in of mice and reduced both size ( . to . mm) and number of lesions ( . to per diseased mouse) in the remaining mice. moreover, residual lesions from icon treated mice were atrophic and displayed significant reductions in vessel areas of % +/- % (p= . , mean +/-sem, n= ) as determined by vwf immunostaining. no hemorrhagic or thrombotic sequelae were observed in icon treated mice. conclusions: unlike other treatments that target developing angiogenesis, icon can target both developing and established human endometriotic lesions in athymic mice. the gross and microscopic vessel analysis suggests that icon directly or indirectly destroys endometriotic vessels. thus, icon presents a novel, non-toxic therapy for endometriosis. compared to the miscarriage and top groups. ihc for crisp demonstrated increased secretion of crisp in the glandular epithelium and expression in the leucocytes of the tubal uterine decidua. conclusions: there are differences in decidual gene expression in tubal compared to iu pregnancies. we believe that potential biomarkers of tubal pregnancy can be discovered by focusing on secreted proteins associated with uterine decidualization. one of these proteins, crisp , is significantly increased in decidua of tubal ectopic pregnancies and we are currently investigating its expression pattern in sera from women with tubal compared to iu pregnancies. progesterone and hoxa regulate gaba-a pi receptor expression, membrane translocation and activation. homayoun sadeghi, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale school of medicine, new haven, ct, usa. objective the expression of the gaba-a pi receptor has been previously described in the human endometrium in both luminal epithelium and stroma. it is upregulated during stromal decidualization in the rat and in the implantation window of human endometrium. the gaba receptor is modulated by progesterone metabolites, with the resultant opening of the receptor ion channels which allow the water flux necessary for trophoblast attachment. here we identified regulators of pi subunit receptor gene expression and activity. the well-differentiated human endometrial adenocarcinoma cell line (ishikawa) and human endometrial stromal cells (hesc) were transfected with hoxa , treated with progesterone, or treated with vehicle or empty plasmid controls. gaba-a pi receptor mrna upregulation was evaluated by real time rt-pcr. protein expression was evaluated using immunohistochemistry. results gaba-a pi receptor mrna expression was increased with either progesterone treatment ( %, p= . ) or hoxa transfection ( %, p= . ). coadministration of progesterone along with increased hoxa transfection had no additive effect on the expression of gaba-a pi receptor mrna (p= . ). gaba-a pi receptor protein expression was similarly increased by each treatment. either hoxa or progesterone independently caused translocation of the gaba receptor from the cytoplasm to the cell membrane in ishikawa cells. conclusion gaba-a pi receptor expression is increased in the human luminal epithelium and stroma in the window of implantation. activation of the pi subunit leads to opening of ion channels, likely allowing flux of water into the epithelial cells and out of the uterine lumen. progesterone and hoxa each increase both pi subunit receptor expression and membrane translocation. the lack of additive effect suggests progesterone induced pi subunit receptor expression is likely mediated indirectly through progesterone's regulation of hoxa expression. finally, after receptor expression and translocation, progesterone mediated gaba receptor ion channel activation mediates water resorption necessary for implantation. cancer. suzy davies, donghai dai, kimberly k leslie. obstetrics and gynecology, university of new mexico, albuquerque, nm, usa. objectives: endometrial cancer is the most frequent gynecologic cancer in women. it affects an estimated , women in the us every year, and long term outcomes for patients with advanced stage or recurrent disease are poor. targeted molecular therapy against the vascular endothelial growth factor (vegf) and its receptors constitute a new therapeutic option for patients that is now under study by the gynecologic oncology group in a phase trial, gog e (now in second stage accrual). the goal of our work was to assess the potential effectiveness of vegf/vegfr blockade in preclinical endometrial cancer models. methods: these studies employed two agents, bevacizumab (avastin, a vegfa blocking antibody) and vandetanib (zactima, a tyrosine kinase inhibitor of egfr and vegfr ). ic experiments were performed on endometrial cancer cells in culture using four established cell line models. xenografted athymic mice were also employed to test the ability of compounds to inhibit tumor growth in vivo. tumors were isolated from controls and treated animals, mrna was extracted, and affymetrix gene expression arrays were performed to determine the genes consistently modulated by treatment. results: compared to vandetanib with an ic of . m, bevacizumab showed little activity on cell proliferation in vitro, and cell numbers were not reduced by % using concentrations up to . m. however, bevacizumab demonstrated robust activity in the athymic mouse model, resulting in a significant decrease in tumor formation and growth compared to vehicle treated animals when dosed bi-weekly in a concentration of . mg/mouse ip. tumors from this model demonstrated that eighteen genes were consistently up or down regulated in the presence of bevacizumab. among the regulated transcripts was microrna , which was significantly down-regulated. microrna is an anti-apoptotic factor, and its inhibition by bevacizumab predicts for increased expression of the tumor suppressor pten, decreased cell proliferation, and a reduced capacity for metastasis. conclusions: these studies confirm that the vegf pathway is a good target for new therapies against endometrial cancer. blocking this pathway not only inhibits angiogenesis, but also results in changes in gene expression that enhance apoptosis and reduce cellular proliferation and tumor invasion. we collected fibroid and adjacent normal tissues following hysterectomy with patient consent and institutional irbs. to date, data points for each gene from arrays have been collected from patients. the fluorescence readings were log-transformed and normalized with the robust quartile normalization method. quality control of the normalized data was performed to remove arrays that deviated from twice the inter-quartile range calculated from the array signals. results: the custom microarray profiling identified genes that were differentially expressed between normal and fibroid tissues (p < . ; fdr< . ). among them, genes encode receptor tks, genes encode tk ligands, and genes encode cell cycle and apoptosis proteins. clustering analysis of the mean log ratios of these genes has led to the division of the patients into two major groups. thirty four ( %) belong to a group characterized by the significant downregulation of the cyr (ccn ) gene in fibroid tissues. the cyr -down group can be further divided into two sub-groups based on the expression of another tk ligand, efn a. other receptor differentially expressed tk did not segregate with the three defined sub-groups. finally, there was no sub-group segregation based on age of the patient, menstrual phase, or weight of the fibroid. conclusion: our results have demonstrated that tyrosine kinases and their ligands are uniquely differentially expressed between normal and fibroid tissues. unique tyrosine kinase ligands in our population were cyr and efn a, and these markers created three molecular classification groups based on their differential expression. these results support the hypothesis that tyrosine kinase ligands are involved in fibroid growth, and may offer targets for a strategy to avoid hysterectomy. microvascular perfusion sonographic imaging to detect early stage ovarian cancer. joanie mayer hope, arthur c fleischer, brian day, stephanie v blank, bhavana pothuri, robert wallach, john p curtin, david a fishman. gynecologic oncology, new york university school of medicine, new york, ny, usa; radiology, vanderbilt university medical center, nashville, tn, usa. objective: epithelial ovarian cancer (eoc) is the th leading cause of death in us women due to the inability to detect early stage disease. recent sonographic developments involving harmonics, pulse inversion, and the use of contrast agents justify the hope that depiction of aberrant tumor microvascularity associated with early disease can occur. this study utilizes these new techniques to assess the unique microvascularity associated with early stage eoc. methods: we used pulse inversion harmonic microvascular imaging (mvi) technology to depict differences between benign and malignant ovarian lesions. contrast enhanced harmonic transvaginal (tv) sonography was performed using the philips iu scanner after intravenous injection of g of definity (bristol-myer-squibb). split screen real-time images were acquired displaying conventional sonographic views adjacent to harmonic, low mechanical index images. morphologic features (thickened wall, papillary excrescence, calcifications) and aberrant vascularity were noted. q-lab quantification of wash-in, peak enhancement, and wash-out times as well as area-under-thecurve (corresponding to microvessel perfusion) were compared using student's t-tests. results: to date, contrast enhancement patterns of ovaries have been analyzed, benign and malignant. of the malignancies ( fallopian tube, ovarian: stage i, stage iii), / women were correctly identified using conventional tv imaging and others were identified using mvi / . all benign lesions were correctly identified by mvi / while tv detected normals ( false negatives). the lesions detected as malignant by mvi were -stage i fallopian tube and -stage i eoc. contrast enhancement kinetics of malignant lesions demonstrated similar wash-in ( . ± . vs . ± . , p = . ), greater peak enhancement ( . ± . vs . ± . , p < . ), longer wash-out ( . ± . vs . ± . (p < . ), and greater perfusion ( . ± . vs . ± , p < . ) when compared to benign lesions. conclusion: contrast enhancement patterns are significantly different in benign vs. malignant ovarian masses. this technique has clear potential in differentiating benign from malignant lesions and for detecting occult stage i disease. identification and characterization of mir- a as regulator of ikk expression and its function in ovarian cancer cells. rui chen, ayesha b alvero, thomas rutherford, gil mor. department of obstetrics and gynecology, yale university school of medicine, new haven, ct, usa. introduction: the proinflammatory environment associated with tumor growth and chemoresistance is produced by both immune cells and cancer cells. the nf-b pathway plays a critical role mediating the capacity of cancer cells to produce pro-inflammatory cytokines. recently, we described a group of epithelial ovarian cancer (eoc) cells characterized by ikk expression as the main factor promoting nf-b activation and cytokine production. in this study we evaluated the regulation of ikk in eoc cells. we describe the identification and characterization of mir- a as a regulator of ikk expression, thus indirectly of nf-b activity and function. materials and methods: human eoc cell lines were established from malignant ovarian cancer ascites. protein expression were determined by western blotting. ikk mrna was measured by rt-pcr. cytokines were profiled by the luminex system. ikk transfection was done with roche fugene transfection reagent. mirna microarray was done with invitrogen ncode multi-species mirna microarray kit. mir- a qrt-pcr was performed with invitrogen ncode sybr greener mirna qrt-pcr analysis kit. mirna transfection was done with ambion siport neofx agent. the ikk '-utr luciferase reporter plasmid was established based on ambion pmir-report mirna expression reporter vector. results: ikk expression was associated with nf-b cyclic activity and the ability of type i eoc cells (but not type ii) to produce inflammatory cytokines. transfection of ikk into type ii eoc cells reversed their phenotype. mirna microarray identified mirnas differentially expressed in type i versus type ii cells, one of which, mir- a, had putative binding sites in the '-utr of ikk mrna. mir- a introduction into type i cells inhibited ikk expression, and direct inhibition through ikk 's '-utr was confirmed by luciferase assay. conclusion: we describe for the first time the identification of ikk as a potential key switch between chemo-resistant and chemo-sensitive phenotypes, by regulating nf-b activity in eoc cells. furthermore, we identified mir- a as a direct regulator of ikk expression. ikk expression may represent an adaptational stage in tumor progression allowing cancer cells to create their own inflammatory environment. these findings may provide novel molecular targets and potential markers for individual therapy selection. regulation of cd in the rat uterus by nitric oxide and the involvement of p kinase pathway. uma yallampalli, rebakah elkins, pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cd is expressed in many cell types including uterine cells and has been shown to play an important role in protecting against compliment attack. we have previously shown in endometrial cell lines that cd levels were down regulated by nitric oxide (no) . in this study we extend our previous observations to determine if no down regulates cd in rat uterine tissues and assess its mechanisms of action. methods. non pregnant rats ( g; b wt) were bilaterally ovariectomized under ketamine anesthesia. groups of ovariectomized rats were implanted with alzet mini pumps to deliver nitro-l-arginine melthylester (l-name) at or /mg/rat/day in saline or saline alone. uteri were obtained from these rats at or hours after infusion. in another set of experiments uteri were obtained from ovariectomized rats and cut in to small pieces and incubated in vitro with either l-name ( mm), diethylenetriamine-no mm) or worthmanin ( . m) in mem without phenol red for hours. uterine tissues were homogenized in trizol and mrna levels for cd were measured using rt-pcr and expressed as a ratio to s. results. results show that in vivo treatment with l-name to ovariectomized rats caused elevations in uterine cd mrna levels in a time and dose dependent manner with maximal responses seen with mg l-name and at hours. in vitro studies show that deta-no suppressed cd mrna levels in the rat uterus. both l-name and pi kinase inhibitor, worthmanin caused increases in cd levels in these tissues and the effects of worthmanin are reversed by deta-no. these results suggest that cd levels in the rat uterus are down regulated by no and are upregulated when no synthesis is inhibited by l-name. further, pi kinase appears to be involved in cd regulation in the rat uterus and no donor appears to modulate this response. lung. lakeitha r foster, daniel b hardy, carole r mendelson. dept of pediatrics; depts of biochemistry and ob/gyn, ut southwestern medical center, dallas, tx, usa. during % of human pregnancy, the maternal uterus is maintained in a state of almost complete quiescence by elevated circulating levels of progesterone (p ). we previously observed that p acting through the progesterone receptor (pr) serves an anti-inflammatory role and inhibits uterine contractility by antagonizing nuclear factor b activation and cyclooxygenase- (cox- ) expression (hardy et al., ) . our previous findings also suggest that the fetus provides an important signal for the initiation of labor near term through augmented secretion of the major lung surfactant protein, sp-a, into amniotic fluid (condon et al., ) . secreted sp-a, in turn, activates fetal macrophages which migrate to the maternal uterus where they release cytokines and promote an inflammatory response, leading to labor. in the present study, we tested the hypothesis that maternal p also maintains uterine quiescence by inhibiting sp-a production by the fetal lung. age matched icr mice were injected s.c. once daily either with sesame oil (control) or p ( mg/ml) from to days post-coitum (dpc). as expected, treatment with p delayed parturition by - h. this also was associated with a decrease in uterine cox- mrna expression, as compared to the vehicle-injected controls. interestingly, maternal p treatment caused a marked decrease in the levels of immunoreactive sp-a protein secreted by the fetal lungs into in amniotic fluid at dpc. furthermore, sp-a protein and mrna levels were reduced in the fetal lungs of p -injected mothers, as compared to controls. this was associated with an inhibitory effect of p treatment on cox- protein and mrna levels in the fetal lungs. these findings were of interest, since cox- expression is markedly upregulated during differentiation of human fetal lung (hfl) explants in culture (hardy et al., ) and endogenous and exogenous prostaglandins increase sp-a expression in hfl (acarregui et al., ) . collectively, these findings suggest that maternal p treatment prevents increased uterine contractility, in part, by inhibiting inflammatory response pathways within the fetal lung. this, in turn, blocks the developmental induction of sp-a expression and its secretion into amniotic fluid. in this manner, maternal p inhibits an important fetal signal leading to labor. supported by nih p hd ; nih r hl . recent evidence suggests that leukocytes infiltrate uterine tissues at the time of parturition implicating inflammation as a key mechanism of human labor. ccl- is a pro-inflammatory cytokine that may contribute to the development of inflammatory reaction in the myometrium. previously we showed upregulation of rat ccl- gene expression in myometrium during term and ru -induced preterm labor. also ccl- was elevated specifically in the gravid horn of unilaterally pregnant rats suggesting that mechanical strain imposed by the growing fetus controls its expression in the myometrium. the objective of this study was to investigate the role of mechanical stretch as a possible regulator of myometrial leukocyte infiltration and ccl- as a mediator of this stretch response. we also studied the effect of progesterone (p ) on the myometrial secretion of ccl- . methods. we used primary culture of rat myometrium smooth muscle cells (smcs) to study in vitro ccl- gene and protein induction by static mechanical stretch. ccl- gene expression analysis was performed by real-time rt-pcr and immunoreactive (ir) protein content was measured by elisa assay. we used primary rat monocytes to access whether stretch-induced ccl- production by myometrial smcs resulted in enhanced monocyte chemotactic activity. results. myometrial cells were stretched for - hours and the supernatants collected. analysis of media conditioned by primary myometrial smcs revealed that static mechanical stretch ( % elongation for hours) caused a significant accumulation in ir ccl- which was repressed by pretreatment with p ( um). the rise in ccl- protein levels was preceded by a transient increase on ccl- mrna. the migration of primary rat monocytes in response to conditioned medium from stretched myometrial smcs was much greater than that of conditioned medium from control non-stretched cells. co-incubation with a neutralizing antibody to ccl- significantly reduced the chemotaxis of monocytes in response to the stretch-conditioned medium. conclusion: uterine smcs play an active role in uterine inflammation by producing chemokines and promoting the chemotaxis of immune cells into the myometrium. the blockade of this effect by p offers a potential explanation for the therapeutic actions of this hormone in the prevention of preterm birth. membranes during human labor. nardhy gomez-lopez, , guadalupe estrada-gutierrez, lourdes vadillo-perez, felipe vadillo-ortega. direction of research, instituto nacional de perinatologia, mexico city, df, mexico; escuela nacional de ciencias biologicas, ipn, mexico city, df, mexico. introduction. leukocytes arriving to the choriodecidua (chd) during labor are capable to secrete cytokines and matrix metalloproteinases that may play a role in the fetal membranes (fm) extracellular matrix degradation. objective. the aim of this work was to identify changes in the leukocyte subpopulations in the chd and fm during human labor. methods: fm were obtained from two groups of women: ) term without labor (n= ) and ) term with spontaneous labor (n= ). chd cells were isolated and analyzed by flow cytometry. explants of fm were embedded in paraffin and analyzed by confocal microscopy. in both techniques, cd , cd , cd , cd and cd subpopulations of leukocytes were identified. intracellular mmp- was also identified in these cells. results: major changes in leukocytes subpopulations during labor involved a higher amount of cd +, cd+ and cd + cells, both in the chd and inside the fm. mmp- was associated to cd + cells. cd + exhibited a more widespread localization in the fm and cd + cells were localized in the contact with the trophoblast layer during labor. conclusions: leukocyte populations changes both in the chd and fm during labor and are characterized by arrival and infiltration of specific subpopulation of lymphocytes and monocytes. nk cells are enriched in mmp- , which may be related to a role in extracellular matrix degradation leading to the rupture of fetal membranes. women with a history of a pregnancy complicated by preeclampsia or intrauterine growth restriction (iugr) have an increased risk of future cardiovascular disease. excessive weight, particularly abdominal fat mass, is associated with cardiovascular morbidity and mortality. objectives: the aim of this study was to investigate differences in body composition and fat distribution between women with a history of preeclampsia or iugr and uncomplicated pregnancies. methods: from a genetically isolated population in the southwest of the netherlands, non-pregnant women with a history of preeclampsia (n= ), iugr (n= ) and uncomplicated pregnancies (n= ) were recruited at a mean follow up time of . years after pregnancy. body composition and fat distribution were assessed by dual energy-x-ray absorptiometry (dxa) and anthropometric measurements. results: women with a history of preeclampsia compared to controls had higher mean total-, fat-and lean mass (p < . ) as well as higher mean indices of body mass, fat mass and lean mass (p< . ). no significant differences were found for these variables between women with a history of iugr and controls. women with a history of preeclampsia had higher waist circumferences and waistto-hip ratios (p< . ) as well as excess of android fat mass and increased android-to-fat ratios (p< . ). women after pregnancies complicated by iugr had higher waist-to-hip ratios (p < . ). after controlling for body mass index, both women with a history of preeclampsia or iugr had higher waist circumferences (p < . ) and waist-to-hip ratios (p < . ) as well as smaller hip circumferences (p < . ). conclusion: despite differences in body mass index, both women with a history of preeclampsia and women after pregnancies complicated by iugr have a metabolically adverse fat distribution, marked by an excess of fat deposition in the abdominal region relatively to the hip region. these findings may explain, at least partly, their increased cardiovascular risk. women with a history of preeclampsia. marc ea spaanderman, timo h ekhart, robert aardenburg, louis lh peeters. obstetrics and gynecology, radboud university medical center, nijmegen, netherlands; obstetrics and gynecology, university medical center maastricht, maastricht, netherlands. background: a history of preeclampsia is associated with persistent short-term alterations in circulatory function and remote cardiovascular disease. in this study we tested the hypothesis at least years after preeclamptic pregnancy renal and central hemodynamic function is impaired as compared to women with uncomplicated pregnancy. methods: in formerly preeclamptic women (pe) and healthy parous controls (control) who were normotensive at weeks post-partum follow up, we assessed at least years after delivery blood pressure (mmhg), cardiac output (co, doppler ultrasonography, l/min) and effective renal plasma flow (erpf, pah clearance, ml/min/ . m ) after which we calculated total peripheral vascular resistance (. dyne.s/cm ) and renal vascular resistance (. dyne.s/cm ). data were analyzed parametrically (p< . ). results: age and bmi were comparable between groups. blood pressure was higher in formerly pe. moreover, % of formerly pe women and % of control were hypertensive (p< . ). although cardiac out was comparable between groups, total peripheral and renal vascular resistance were about % higher and erpf % lower in formerly pe women as compared to control. conclusion: at least years after gestational hypertensive disease, women who were normotensive at direct follow up have impaired renal and central hemodynamic function and developed more often chronic hypertension. long-term follow up may also be warranted in apparently healthy formerly pe women who are normotensive at post-partum follow up. circulatory function in formerly preeclamptic women and healthy parous controls co erpf tpvr rvr formerly pe . ± . ± * ± * ± * control . ± . ± ± ± * = p< . pregnancy increases blood-brain barrier permeability coefficient (l p ) to lucifer yellow: role of estrogen. marchien j wiegman, , marilyn j cipolla. neurology, ob/gyn and pharmacology, university of vermont, burlington, vt, usa; ob/gyn, university medical center groningen, groningen, netherlands. background: eclampsia is similar to posterior reversible encephalopathy syndrome in which an acute rise in blood pressure causes breakthrough of autoregulation, blood-brain barrier (bbb) disruption, and cerebral edema formation. we previously showed that late-pregnant (lp) animals developed cerebral edema during breakthrough, a response that was absent in nonpregnant (np) animals. in the current study we hypothesized that pregnancy predisposes the brain to edema during acute hypertension by enhanced bbb permeability. we further hypothesized that the underlying effect of pregnancy on the bbb permeability is due to elevated estrogen levels. methods: permeability coefficients (l p ) to lucifer yellow (ly), a polar compound that does not pass through tight junctions, were compared in posterior cerebral arteries (pca) from groups of sprague dawley rats: np (n= ), lp (d ; n= ), ovariectomized and implanted with -estradiol ( . mg, -day release) and estriol ( . mg, -day release) pellets for days (ovx+e; n= ), and ovariectomized and implanted with placebo pellets for days (ovx; n= ). pcas were isolated, pressurized in an arteriograph, and perfused with . mg/ml ly in saline. concentration changes of ly outside the vessel wall were determined at pressures from - mmhg. the slope of the pressure vs. permeability curve is the rate of flux, or l p for ly. results: l p for ly was significantly increased in pcas from lp and ovx animals vs. np (p< . ; figure ). estrogen was protective of the bbb only in ovariectomized animals, decreasing l p % in ovx+e vs. ovx. however, pregnancy did not afford protection and had a l p that was % greater than np. conclusions: pregnancy significantly increases bbb permeability to ly, an effect that may predispose the brain to edema formation during acute hypertension. these data also show that estrogen modulates l p in ovariectomized animals differentially than pregnancy, suggesting that the increased bbb permeability in pregnancy is caused by a mechanism other than elevated estrogen levels. in both pre-and early pregnancy, the sympathoinhibitory response to volume expansion is blunted in formerly preeclamptic women with low plasma volume. ineke krabbendam, marc ea spaanderman, dorette a courtar, robert aardenburg, ben j janssen, fred k lotgering, louis lh peeters. obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; obstetrics and gynecology, university hospital maastricht, maastricht, netherlands; pharmacology and toxicology, university of maastricht, maastricht, netherlands. background: the circulation of formerly preeclamptic women with a low plasma volume (lpv) is characterized by sympathetic dominance. these women respond to a new pregnancy with an aberrant rise in atrial natriuretic peptide (anp) and a times higher chance to develop recurrent gestational hypertensive disease compared to their counterparts with normal plasma volume (npv). anp has sympathicomimetic capacity. we postulate that the sympathetic overdrive in lpv-women is associated with a reduced venous capacitance. to this end, we compared the response to volume expansion (ve) in women with lpv and npv, both before and in pregnancy. method: in non-pregnant normotensive formerly preeclamptic women, we measured pv (hsa i indicator dilution method) at least months post partum. we intravenously infused ml of iso-oncotic fluid over minutes. during the infusion, we recorded changes in heart rate (hr, bpm), blood pressure (bp, mmhg), cardiac output (co, l/min), sympathetic activity (lfsys, mmhg , low frequency component of spontaneous fluctuations in systolic bp, portapress) and anp (nmol/l). eight women became pregnant within year and were evaluated at weeks gestation. changes in circulatory and autonomic function between and within groups were analyzed non-parametrically (p< . ). results: before pregnancy, ve leads to comparable changes in hr, bp and co in women with lpv ( / ) and npv ( / ). in npv, lfsys decreased %, but only % in lpv (p< . ). anp remained unaltered in npv, but increased in lpv. in the pregnant group, women had lpv and had npv. in both groups, pregnancy did not alter the response to ve. conclusion: irrespective of pregnancy, the sympathoinhibitory response to ve is diminished in lpv. these data suggest that in these women ve leads to venous overfill, giving rise to anp-release and consequently sympathetic activation, flattening the normal baroreceptor-mediated sympathoinhibitory response. we speculate that this mechanism contribute to circulatory maladaptation to pregnancy, sympathetic dominance and subsequent gestational hypertensive disease. in both pe and ht, serum sflt- was increased, and plgf reduced at all gestations (p< . ). seng levels were also increased in pe. after weeks (but not before) antihypertensive treatment was associated with a significant fall in serum sflt- and seng, in pe only. the concentrations of both sflt- and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p= . ). only sflt- was significantly reduced in the placenta in women who received antihypertensive therapy. conclusion in pe, antihypertensive therapy after weeks' gestation is associated with a significant fall in serum sflt- and seng, and in placental sflt- . these findings raise the possibility that these drugs may have an effect on the pathophysiology of pe other than their known antihypertensive action. the synergistic effect of soluble vegf receptor pre-treatment and small doses of tnf-on endothelial cells. tereza cindrova-davies, debbie a sanders, olivera spasic-boskovic, graham j burton, d stephen charnock-jones. dept of pdn, university of cambridge, united kingdom; dept of obstetrics and gynaecology, university of cambridge, united kingdom. introduction: preeclampsia is marked by an enhanced endothelial inflammatory response manifested by maternal endothelial activation. soluble fms-like tyrosine kinase- (sflt- , svegf-r ), a naturally occurring circulating antagonist of vegf-a and plgf, is one of the secreted factors implicated in the pathogenesis of preeclampsia. in women who develop preeclampsia, sflt rises sharply, preceding the onset of the clinical disease. the aim of this study was to examine the effect of a combined treatment with recombinant sflt- and tnf-on the activation of human umbilical cord endothelial cells (huvec) by examining leukocyte adhesion, and the expression of icam, vcam, endothelin and vwf. methods: huvec were seeded, grown overnight and pre-treated with recombinant sflt ( - ng/ml) in a basic % fbs-dmem medium for hr. on day , low doses of tnf-( . ng/ml) were applied to pre-treated cells for hr. antagonism of the vegf-a action was mimicked at the protein level by pre-incubating huvec with anti-flt antibody ( - g/ml), anti-kdr antibody ( g/ml), anti-vegf antibody ( g/ml), or vegf receptor inhibitor su ( m). at the rna level, the effect of sflt was mimicked by sirna transfection of huvec with siflt or sikdr. at the end of each experiment cells were either harvested for western blotting, fixed in % pfa for immunofluorescence or incubated with labelled hl leukocyte cells, followed by fluorescent detection of adhesion. results: pre-incubation of huvec with sflt and subsequent treatment with low doses of tnf-increased the adhesion of hl leukocytes and increased icam- , vcam- , endothelin and vwf, compared to tnf-treatment alone. similar results were obtained when cells were pre-treated with su , anti-flt, anti-kdr or anti-vegf. transfection knock-down of flt or kdr gene also significantly increased leukocyte adhesion when small doses of tnfwere added. conclusions: pre-incubation with recombinant sflt, anti-flt, anti-kdr, anti-vegf, su or knocking-down flt or kdr transcripts all antagonised the autocrine actions of vegf and/or plgf. this predisposes huvecs to be more sensitive to the effect of tnf-. our study shows that sflt and tnfcombine to induce an enhanced synergistic effect, activating endothelial cells. maternal obesity is associated with increased production of inflammatory cytokines and risk of poor perinatal outcome. inflammatory cytokines can stimulate production of reactive oxygen (ros) and nitrogen (rns) species, which can covalently modify protein function. placental oxidative and nitrative stress are increased in pathological pregnancies and associated with altered placental function. objectives: determine the effect of increasing body mass index (bmi) on placental nitrative stress, measured by the expression and localization of nitrated (nitrotyrosine) and oxidized proteins. methods: placental tissue was collected at term ( - wks) from lean kg/m ), overweight ( - . kg/m ) and obese ( - kg/m ) patients (n= or /group). tissue was sectioned for immunostaining with nitrotyrosine antibody. protein samples were either dot blotted onto nitrocellulose membrane, probed with nitrotyrosine ab, and nitrated proteins detected using ecl, or derivatized using , -dinitrophenylhydrazine (dnph) (oxyblot® kit), separated on sds-page, probed with anti-dnph ab ( : ) and oxidized proteins detected using ecl. protein band intensity was measured by densitometry. oxidized proteins were selected for maldi mass spectrometry analysis. results: nitrotyrosine residues were immunolocalized primarily in the fetal capillary endothelial cells and the villous stroma, but were almost absent in the syncytiotrophoblast. by dot blot, nitrotyrosine expression differed across the three groups (p< . , anova) with expression in tissue from obese women being significantly increased compared to lean (p< . ) and overweight (p< . , tukey test). several oxidized proteins were detected with significantly greater expression seen in the lean versus overweight groups (p< . , mann-whitney u). one oxidized protein was identified by maldi-ms with four peptide matches and . % coverage as -beta hydroxysteroid dehydrogenase ( bhsd). discussion: with increasing bmi, an increase in nitrative stress appears to occur in parallel with a decrease in oxidative stress. oxidative stress is apparently reduced as ros are consumed by the interaction with rns to give nitrative stress. bhsd is involved in the biosynthesis of steroid hormones and glucocorticoids. its activity and hence steroid metabolism in the placenta may be regulated by oxidation with implications for fetal development. background teenagers are susceptible to delivering small-for-gestationalage (sga) infants. previous studies implicate continued maternal growth as a causal factor . growing adolescent sheep have reduced fetal birthweight due to impaired placental development and nutrient transfer . we hypothesized that placental function is impaired in human teenage pregnancy if there is maternal growth. methods placentas were collected from teenagers ( - years) and adults. activity of the amino acid transporter, system a, was quantified by the sodium-dependent uptake of c-methylaminoisobutyric acid into placental fragments. teenagers were defined as growing (> mm increase in kneeheight per days) or non-growing. system a activity was analysed in relation to individualised birthweight centile and maternal growth. results placental system a activity was significantly lower in teenage compared to adult pregnancies (p< . ). this was unrelated to birthweight; teenagers who delivered infants appropriate-for-gestational age (aga) had significantly lower placental system a activity compared to aga infants delivered to adults (p< . ). growing teenagers did not deliver lower birthweight infants than non-growing teenagers (median birthweight g and g respectively). furthermore, system a activity in placentas from growing teenagers was significantly elevated compared to that in non-growing teenagers (p< . ), and was similar to placental activity in adults. conclusions system a activity was reduced in placentas from teenagers compared to adults. this suggests that inherently lower placental function predisposes teenagers to sga, but that other factors (e.g. adequate nutrition) can compensate in those teenagers delivering aga infants. in contrast to our hypothesis, placental system a was elevated in growing teenagers and mimicked that of adults. this may be related to a hormonal-milieu in growing teenagers that is conducive to fetal growth, in part through stimulating placental transport. homocysteine inhibition of system a amino acid transport activity in human placenta. eleni tsitsiou, susan l greenwood, colin p sibley, stephen w d'souza, jocelyn d glazier. maternal and fetal health research group, st. mary's hospital, university of manchester, manchester, united kingdom. background: elevated plasma levels of the amino acid homocysteine (hcy) during pregnancy are associated with vascular-related complications and adverse neonatal outcomes including a reduced birthweight. fetal and maternal plasma hcy concentrations are positively correlated suggesting placental transport of hcy may be an important determinant of fetal plasma hcy. the mechanisms involved in placental hcy transport are uncharacterised. evidence that the system a amino acid transporter, which transports neutral amino acids in a na + -dependent manner, is important in promoting fetal growth and that a reduced system a activity is associated with intrauterine growth restriction (iugr), led to our hypothesis that system a provides one mechanism for placental hcy transport. this hypothesis was tested by measuring the ability of hcy to inhibit system a activity in isolated microvillous plasma membrane (mvm) vesicles and placental fragments. materials and methods: mvm vesicles and placental fragments were isolated from placentas of normal pregnancies at term. system a activity was measured at initial rate ( s and min respectively) as na + -dependent cmethylaminoisobutyric acid (meaib) uptake into mvm vesicles ( . mm) or fragments ( . mm) in the absence (control) or presence of l-hcy and dl-hcy or model substrates. results: mm l-hcy (custom-synthesised) and dl-hcy (commercial source) significantly (p< . ) inhibited na + -dependent c-meaib uptake into mvm vesicles compared to control; comparable in magnitude to other model substrates (meaib, l-ala, l-ser, l-met; n= , kruskal-wallis with dunn's multiple comparison test). l-hcy, l-met and meaib ( . - mm) caused a dose-dependent inhibition of na + -dependent c-meaib uptake into mvm with ec values (in mm; mean ± se) of . ± . , . ± . , and . ± . respectively, n= ). na + -dependent c-meaib uptake into fragments was reduced substantially in the presence of mm l-hcy or dl-hcy causing a ± and ± % reduction (mean ± sd, n= - ) of control respectively. conclusion: these observations suggest that hcy is a relatively high affinity substrate for system a in the human placenta. we speculate that inhibition of placental amino acid uptake by hcy could impact on fetal growth and development. supported by the mrc and action research endowment fund. glucose regulates placental mtor activity and glucose deprivation down-regulates placental system l activity in an mtor-dependent manner. sara roos, theresa l powell, thomas jansson. department of physiology, institute of neuroscience and physiology, gothenburg, sweden; department of obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. placental amino acid transporters are down-regulated in intrauterine growth restriction (iugr). we have previously shown that mammalian target of rapamycin (mtor) regulates placental system l transporter activity and that placental mtor activity is decreased in iugr. however, the upstream regulators of placental mtor are unknown. in iugr, fetal hypoglycemia and reduced maternal glucose levels are common and the placenta may therefore be exposed to low glucose levels. hypothesis: we hypothesized that glucose availability regulates placental amino acid transporter activity mediated by changes in mtor signaling. methods: cytotrophoblast cells were isolated and cultured until syncytialization at hours. cells were cultured for an additional hours in culture media containing . mm, . mm, or mm glucose (control), which corresponds to standard culture media. at hrs, the activity of the mtor signaling pathway was assessed by measuring the protein expression of s k phosphorylated at thr- , the primary site of mtor phosphorylation. in another set of cells, system l activity was assessed by measuring the bchinhibitable uptake of h-leucine. results: as compared to control, phospho-thr- -s k expression was reduced by % in cells incubated in . mm results: fgr pregnancies were delivered earlier ( ± . v ± . w; p< . ), weighed less ( . ± . v . ± . kg; p< . ) and had higher s/d ratios ( . ± . v . ± . ; p< . ). eaa concentrations were similar between groups, but there were differences (non-parametric testing; p< . ) in transport rates between groups with his crossing considerably faster and ile crossing slower in fgr . the table shows fetal vein/maternal (fv/m) ratios standardized to the leu fv/m ratio for all eaa for both groups. amino acids fell into groups for fgr pregnancies: ) his (ratio . ), ) leu, phe, met (ratios ), and ) val, thr, ile, trp, lys with intermediate ratios ( . ns conclusion: this is the st study to compare the relative rates of in vivo placental transport for all eaa between normal and fgr human pregnancies showing striking differences in the transport rates of two eaa. in the absence of eaa concentration differences between groups, the higher his transport rate in fgr suggests higher utilization by the placenta. vasculature of women who received exogenous estrogen compared to those who received placebo. the purpose of this investigation was to identify the gene expression in response to the estrogen, equilin, as the major component of conjugated equine estrogen and genistein, a phytoestrogen in human coronary artery endothelial cells. human coronary artery endothelial cells from a -year old female were used. cells were treated with estradiol [e ], equilin [eq] ( . nm) or genistein [gen] ( micromolar) for hours. focused oligomicroarrays for cardiovascular disease and endothelial cell function were used to study gene expression. rt-pcr was used to confirm the transcription of genes analyzed in oligoarrays. vascular adhesion molecules and genes involved in the inflammatory response were most affected with the three estrogen sources. significant reduction of integrin alphae was seen with all three . , . and . for e , eq and gen respectively. similarly nfkb was also reduced . , . and . fold compared to controls. significant reduction of ccl was demonstrated with eq ( . fold) and gen ( . fold). tnfreceptor a and b were only significantly decreased by gen. vcam- which is regulated by proatherogenic factors and upregulated mainly at atherosclerosis -prone sites, was significantly reduced ( . -fold) with genistein, and a slight reduction was seen with e and no change was observed with eq. our data support the clinical findings that estrogen has favorable effects on the genes involved in atherosclerotic disease. while e has been shown to be slightly more effective than equilin in the modulation of gene expression, genistein was significantly more effective in the favorable expression of genes involved in particularly the inflammatory response. olga n lekontseva, sandra t davidge. physiology and obstetrics/gynecology, university of alberta, edmonton, ab, canada. introduction: the prevalence of cardiovascular disease in women dramatically rises in the postmenopausal period. although deficiency of estrogen has been implicated in the pathophysiology of systemic vascular dysfunction, the effects of estrogen on vasculature are complex and not completely understood. we have previously shown that estrogen exerts a beneficial effect on the aging vascular system by reducing circulating levels of the inflammatory cytokine tnf . tnf is a known regulator of matrix metalloproteinases (mmps), proteolytic enzymes that may modulate vascular tone through cleavage of vasoactive peptides such as big endothelin- (et- ). the role of estrogen in this pathway is unknown. we tested the hypothesis that in aging/estrogen deficiency, tnf -induced mmp activity mediates greater vasoconstriction, in part, through the et- pathway. we further hypothesized that estrogen replacement reduces vascular sensitivity to the constriction by preventing mmp activation. methods: aged ( month old) female sprague dawley rats were ovariectomized and treated with either placebo [ovx] , -estradiol [ovx+e ], or the tnf inhibitor etanercept [ovx+etan] . after four weeks, resistance mesenteric arteries were isolated and studied on the pressure arteriograph. concentrationresponse to exogenous big in the absence or presence of mmp inhibitor (gm , μm) was assessed in the vessels. results: treatment of "menopausal" [ovx] rats with either estrogen or the tnf inhibitor reduced sensitivity of arteries to big et- (ec = . ± . μm in ovx versus . ± . μm in ovx+e or . ± . μm in ovx+etan groups; p< . ). although mmp inhibition attenuated maximal constriction in all of the arteries, there was a significantly greater (p< . ) role of mmps in big et- -induced vasoconstriction in the ovx+e group (reduction in max constriction= . ± . %) compared to ovx ( . ± . %) and ovx+etan groups ( . ± . %). conclusions: both estrogen and tnf inhibition reduced big et- vasoconstriction. however, contrary to our hypothesis, tnf is not contributing to mmp modulation of et- vasoconstriction. interestingly, our study demonstrates a novel role for estrogen to increase mmp contribution to big et- vasoactivity with the net effect being less vasoconstrictive. understanding this unique pathway of regulation by estrogen in the aged vasculature will allow for development of new therapeutic options for women. mo, usa. introduction: the etiology of pelvic organ prolapse is multifactorial, with both inherited and acquired components. the molecular mechanisms of prolapse have not been established yet. we have previously shown that lysyl oxidase (lox) expression is suppressed in uterosacral ligaments of women with pelvic organ prolapse. it has also been shown that lox is a tumor suppressor gene inactivated by methylation in human gastric cancers. hypothesis: the aim of this study was to analyze the dna sequence of the promoter region of the lysyl oxidase gene in tissues from women with pelvic organ prolapse and identify whether methylation is present. our hypothesis is that the promoters of the lox gene in women with pelvic organ prolapse have significantly more methylation sites than women without prolapse. materials and methods: genomic dna was isolated from the uterosacral ligaments of eight women with pelvic organ prolapse and women without prolapse (controls). genomic dna samples were treated with ez dna methylation kit (zemo research, orange, ca). the lox gene promoter region of - to + was amplified by pcr and then cloned into pcr . -topo (invitrogen, carlsbad, ca) and transformed into an e. coli dh a strain. amplified plasmid dna samples containing the lox gene promoter region from each woman were sequenced and methylated cpg islands were identified by sequence comparison. results: a total of methylated cpg sites were found in the patient group with pelvic organ prolapse while only methylated cpg site was found in the non-prolapse control group. conclusion: these findings suggest that methylation in the promoter region suppresses lox gene expression in women with pelvic organ prolapse. background: reports from case-control and cohort studies have suggested an inverse association between lactation and breast cancer risk, but findings have been inconsistent. methods: we conducted a prospective observational cohort study of , parous women participating in the nurses' health study ii from to . our primary outcome was incident premenopausal breast cancer. results: during the study period, cases of premenopausal breast cancer were diagnosed during , person-years of follow-up. women who had ever breastfed had a % lower incidence of premenopausal breast cancer ( % confidence interval [ci] - %) compared with women who never breastfed, adjusting for parity, age at first birth, year of first birth, height, body mass index (bmi), bmi at age , family history, personal history of benign breast disease, participant birth weight, preterm birth, age at menarche, oral contraceptive use, physical activity, and alcohol consumption. no trend was observed with duration of lactation (p= . ). the association between ever-breastfeeding and premenopausal breast cancer was modified by use of medication to suppress lactation (p= . ); in analyses restricted to women who had never used suppressive medication, ever-breastfeeding was associated with a % ( %ci - %) reduction in incident disease. the association between lactation and premenopausal breast cancer was further modified by family history of breast cancer (p= . ). among women who had never used suppressive medication and reported a family history, those who had breastfed had a % lower covariate-adjusted risk of premenopausal breast cancer ( % ci - %) than women who had never breastfed. among women without a family history, ever-breastfeeding was not associated with breast cancer incidence (hazard ratio . , % ci . - . ). among women who had ever breastfed, we observed no association between breast cancer risk and duration of lactation amenorrhea or exclusive breastfeeding. conclusion: in a large, prospective cohort study, ever-breastfeeding was inversely associated with risk for premenopausal breast cancer. at the durations observed in our cohort, we observed no trend with duration of breastfeeding or lactation amenorrhea. the inverse association with ever-breastfeeding was stronger among women with a family history of breast cancer. regulatory and nkt cells at the maternal-fetal interface. thomas f mcelrath, rachael a clark. brigham women's hospital, boston, ma, usa; harvard skin disease research center, brigham women's hospital, boston, ma, usa. introduction: pregnancy represents an immunologically challenging event requiring maternal tolerance of the fetal semi-allograft. an increase in decidual cd + cd + t cells has been documented but it is unclear if these represent foxp + t cells (tregs) . the possibility also exists that other cd + lineages with potential regulatory function exist within the decidua parientalis. we examined if cd + cd + cells are true foxp + tregs and evaluated the frequency of other t cell subsets with possible regulatory potential. methods: we extracted t cells from term deciduas of planned cesarean deliveries. cells were stained with directly conjugated monoclonal antibodies and were analyzed on a six color flow cytometer. results: we found that only a subset (median %) of cd + t cells were true foxp + regulatory t cells. from donors, foxp + tregs accounted for a median of . % of all cd + cells. these cells were memory cd ro + t cells lacking ccr , and l-selectin but expressing cd , ctla- , gitr, and hla-dr. additionally we found that a median % of cd + t cells expressed the nkt marker cd . these cells were a mixture of cd and cd -cd -t cells, with variable numbers of cd t cells, suggesting they do not represent merely recently activated t cells. there was enrichment for t cells with nkt markers after culture on hela cells expressing cd d, suggesting that these cell represent true nkt cells. nkt cells were of the non-classical type, with a diverse t cell repertoire ( . % iv jq) and also expressed cd , hla class ii, cd ro but were ccr and l-selectin low. comments: we find that only a minority of cd -expressing t cell in the decidua are true foxp + tregs. because much of the work on treg in pregnancy has used cd as a treg marker, this suggests additional studies are needed to confirm the role of these cells in pregnancy. we find that a novel population of non-classical nkt cells exists in the human decidua. nkt cells can either promote or antagonize tolerance, depending on the immunologic context. the large number of these cells in the decidua suggests they may play a role equal or exceeding that of regulatory t cells. prolapse. marsha k guess, kathleen a connell, richard bercik, lloyd g cantley. obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa; internal medicine/neprhology, yale university school of medicine, new haven, ct, usa. objectives: thy- is a cell surface glycoprotien expressed in human fibroblasts, neurons, hematopoetic stems cells and endothelial cells. thy- expression affects fibroblast proliferation and migration, cell-cell, as well as, cell-matrix interactions. moreover, thy- expression has been shown to play a critical role in fibroblast dedifferentiation into myofibroblasts, as well as in extracellular matrix (ecm) production and fibrosis. women with pelvic organ prolapse (pop) have alterations in vaginal ecm protein expression and metabolism, as well as decreases in smooth muscle fractional content. in the current study, we evaluated thy- as well as the smooth muscle markers alpha-smooth muscle actin (asma) and desmin expression in women with pelvic organ prolapse compared to women with normal pelvic support (controls). methods: anterior apical vaginal wall specimens from women with pop and controls were collected at the time of hysterectomy. messenger rna and protein expression of thy- , asma and desmin were evaluated using semiquantitative rt-pcr, real-time pcr and western blot analysis. gapdh and beta actin were used as internal controls. results: rt-pcr demonstrated the presence of thy- , asma and desmin in vaginal tissue from women with pop and controls. further, thy- mrna expression was downregulated % in women with pop compared to controls (p = . ). a parallel decrease in thy- protein was seen in women with pop compared to controls (p= . ). although a % and a % decease were seen in asma and desmin mrna, these differences were not statistically significant. similarly, no differences were seen in asma and desmin protein expression. conclusion: we demonstrate that there is significantly less thy- expression in vaginal tissue from women with pop compared to controls. differential expression of thy- in prolapsed vaginal tissues suggests that thy- may have a functional role in mediating ecm metabolism in the female genitourinary tract. hur expression is altered in ectopic endometrium. s karipcin, t altun, ua kayisli, e seli. ob gyn, yale u., new haven, ct, usa. introduction: cytokines and growth factors contribute to cyclic turnover of the normal endomterium and to the pathogenesis of endometriosis. cytokine and growth factor messenger rnas (mrnas) undergo rapid turnover that is primarily mediated by au-rich elements (are) that consist of multiple stretches of adenylate and uridylate residues located in the ' untranslated region ( '-utr) of their mrnas. hur is a ubiquitously expressed rna-binding protein that stabilizes are containing mrnas and prolongs their expression. we hypothesized that hur may play a role in the regulation of cytokine expression during normal menstrual cycle and in endometriosis. methods: tissue sections obtained from normal (n= ) and ectopic (n= ) endometrium were immunostained for hur. staining intensity was evaluated by hscore and grouped according to menstrual cycle phase. statistical analysis was done with one-way anova. cultured stromal cells isolated from normal endometrium were treated with vehicle, estradiol (e ; - m), or progesterone (p, - m) for and h, and hur expression was determined using western analysis and normalized to ß-actin. results: hur immunostaining was nuclear in endometrial cells. hur immunoreactivity was significantly lower in the early proliferative and late secretory phases ( . ± . and . ± . , respectively), compared to the mid-late proliferative ( . ± . ) and early-mid secretory phases ( . ± . )(p< . ). moreover, hur expression was significantly lower in ectopic endometrial cells when compared to normal endometrium in midlate proliferative and early and mid-secretory phases (p< . ). progesterone suppressed hur levels significantly in cultured endometrial stromal cells at both and h compared to control (p< . ) while estrogen did not cause a significant change. discussion: decreased hur levels in the late secretory and early proliferative phases are likely to contribute to degradation of cytokines and result in lower cytokine levels observed mid-cycle. late secretory decrease in hur levels may be mediated by progesterone as suggested by in vitro findings. in ectopic endometrium, persistent low expression of hur compared to normal endometrium most probably results from elevated cytokine levels associated with endometriosis. the effect of lower hur expression in ectopic endometrium on other are-containing transcripts, and on the pathogenesis of endometriosis remains to be elucidated. tissue. hong zhao, joy innes, scott reierstad, mehmet bertan yilmaz, serdar e bulun. department of obstetrics and gynecology, northwestern university, chicago, il, usa. background: aromatase is the key enzyme for estrogen biosythesis, and is encoded by the cyp a gene. thus far, only three unique untranslated first exons associated with distinct promoters in the mouse cyp a gene were described (brain-, ovary and testis-specific exon i). however, it remains unknown whether aromatase is expressed in other mouse tissues via previously unknown tissue-specific promoters activating new exon is. methods: real-time pcr was used to examine the aromatase expression levels in various c bl mouse tissues. '-rapid amplification of cdna ends ( '-race) was used to determine the transcriptional start sites of cyp a transcripts. promoter activity was measured using serial deletion mutants of dna fused to the luciferase reporter gene. results: real-time pcr results showed that aromatase was expressed in male gonadal fat and the expression level is lower than that in testis. the adipose tissue-specific untranslated exon i of cyp a transcript was isolated using '-race and this novel gonadal fat-specific exon i of cyp a mrna did not show sequence similarities to previously reported ones. this new adiposespecific exon i was mapped to kb upstream of the translation start site in the coding exon ii. the genomic region upstream of the adipose-specific exon i was cloned into luciferase plasmids. transfection of murine t -l cells with these plasmids showed that promoter activity was conferred by the sequence located at - to - bp upstream of the transcriptional start site. dexamethasone significantly induced activity of the adipose-specific promoter region. conclusion: taken together, our results suggest that a novel cyp a transcript is regulated by a tissue-specific promoter in male murine gonadal fat. these sacrificed; reproductive and selected other organs were removed, weighed and evaluated histologically by an observer blinded to treatment. results: the table below shows that tcc augmented effects of tp on weights of accessory sex organs. histological assessment revealed that tcc induced greater glandular distention with more secretions compared to the effects of tp alone; furthermore, mitotic figures were seen only in prostates from rats exposed to tp+tcc. conclusions: tcc is a newly identified endocrine disruptor with unique and novel actions resulting in potentiation of androgen effects on sex organs. these observations underscore possible environmental risks related to exposure to tcc. background: blastocyst implantation is dependent on the differentiation of human endometrial stromal cells (hesc) into decidual cells. transforming growth factor family members have well defined roles in cell differentiation and proliferation. activin a, a tgf superfamily member, enhances hesc decidualization and localizes to decidualized cells in human endometrium. other tgf superfamily members, including bmp , bmp , bmp , gdf , gdf (myostatin), gdf and nodal, may also be present in decidual cells and therefore may also play a role during this important process. this study aimed to determine whether activin is the major family member driving decidualization or whether other family members contribute to the process. methods: broad ranging activin inhibitors (activin-m a and sb ) that effect receptor-ligand interactions of other tgf superfamily members were used in hesc decidualization. protein localization was examined in secretory phase endometrium and first trimester decidua by immunohistochemistry and mrna expression was examined in an ex vivo model. the secretion of candidate proteins was measured during hesc decidualization and certain recombinant proteins added during decidualization to examine their effect. results: m a ( nm) and sb ( m) significantly reduced decidualization ( % and % respectively) demonstrating that activin and possibly other tgf family members are involved in decidualization in vitro. bmp , gdf and tgf protein were detected in decidual cells of mid-late secretory endometrium and first trimester decidua whilst all ligands except nodal, were expressed by hesc. both bmp and tgf secretion increased during hesc decidualization and administration of both these proteins significantly enhanced decidualization in vitro. conclusions: these data support a role for activin a, bmp and tgf in hesc decidualization. this is important as the elucidation of factors involved during decidualization will aid in better understanding implantation and fertility. abnormal chromatin remodeling in diabetic murine oocytes. laura lawrence, ann ratchford, cybill esguerra, qiang wang, kelle moley. ob/ gyn, washington university, st. louis, mo, usa. background: diabetic women experience increased miscarriages and adverse pregnancy outcomes. previous studies suggest adverse diabetic outcomes may occur earlier than the preimplantation period, particularly during oogenesis. we hypothesize that diabetes affects chromatin remodeling and chromosomal condensation in murine oocytes. methods: mii oocytes from diabetic and control mice were fixed with % pfa, permeabilized with . % triton x- for min, and immunostained against a-tubulin and the nucleus for hr at rt. images were obtained with laser confocal scanning microscope. protein expression levels of chromatin with dimethyl h k modifications were measured in nondiabetic and diabetic denuded murine oocytes at hours post pmsg ( hour) and at six hours post hcg ( hour) via western immunoblots. mature nondiabetic denuded oocytes were fixed in %pfa and permeabilized with . % triton x- . they were stained via immunohistochemistry against histone protein h at lysine (h k me ) and heterochromatin. results: immunohistochemistry reveals that diabetic mii oocytes have aberrant spindle formations and metaphase chromosome alignment. approximately / ( %) diabetic oocytes examined had abnormal spindles and metaphase alignments compared with only about / normal oocytes ( . %). western blot demonstrated times higher expression of dimethylated chromatin in diabetic oocytes at time compared with nondiabetic oocytes. at time hours, diabetic oocytes had significantly fewer h k modifications than the controls. when staining mature murine nondiabetic oocytes for dimethylation of h k me by immunohistochemistry, we demonstrated h k me expression in a condensed heterochromatin ring surrounding the nucleolus, consistent with transcriptional silencing. conclusions: diabetic mii oocytes have a significant increase in abnormal spindle formation and metaphase chromosome alignment. they also have increased dimethylation compared with normal oocytes at a time point when they should be transcriptionally active, storing maternal mrna in preparation for the silencing period. in addition, after hcg injection to trigger maturation and gene silencing, the diabetic oocytes had decreased dimethylated chromatin changes. our findings suggest that diabetic oocytes may be exiting the transcriptionally active period prematurely and may ultimately experience decreased, partial, and incomplete gene silencing. and xenopus epab genes and proteins were performed. expression of human epab and pabpc mrna was tested in ten different somatic tissues, testes, and ovaries by rt-pcr. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. epab and pabpc expression in human prophase i (pi) and metaphase ii (mii) oocytes, -cell embryos and blastocysts was evaluated using quantitative real time pcr. results: human epab is a aa protein with % identity and % similarity to mouse epab and contains rna recognition motifs and a pabp domain. human epab mrna is detected in ovaries and to a lesser extent in testes and several somatic tissues including kidney, liver, and muscle. similar to its mouse orthologue, human epab mrna is expressed in pi and mii oocytes, but not in -cell embryos or blastocysts. pabpc mrna is ubiquitously present in all tissues as well as -cell, and blastocyst stage embryos. however, its levels are significantly lower than that of epab in oocytes. conclusions: in this study we report the identification of human epab. similar to that observed in xenopus and mouse, human epab is the predominant poly(a) binding protein in oocytes and it is replaced by pabpc following zga, which occurs at -to -cell stage in human. our findings suggest that the unique translational regulatory pathways that control gene expression during oogenesis and early embryo development may be common between model organisms and humans. objective: c-jun nh -terminal kinase (jnk), a member of mitogen-activated protein (map) kinase family, is involved in cell proliferation, differentiation, and survival. fsh is required for granulosa cell proliferation and antral follicle growth but its mechanism of action in preantral stages is not well defined. we previously showed that pharmacological inhibition of jnk pathway halts invitro growth of murine preantral follicles in serum free media supplemented with fsh ( miu/ml). sigcs (spontaneously immortalized rat granulosa cell line) have characteristics similar to preantral granulosa cells and hence they were used in this study to determine whether the jnk pathway plays a key role in preantral granulosa cell proliferation. our specific aims were to determine whether: a) fsh activates jnk pathway in granulosa cells; b) the inhibition of jnk pathway blocks cell cycle progression. material and methods: sigcs were treated with miu/ml recombinant fsh in serum free media two days after serum starvation. activation of jnk pathway was analyzed with if and wb using phospho-jnk and phospho-cjun expression, respectively. fsh receptor expression (fshr) was analyzed with if. the inhibition of jnk pathway on cell cycle progression was analyzed by facs using a jnk inhibitor sp . results: fshr protein was expressed in sigc indicating that they can respond to fsh (fig a) . likewise by if, phospho-jnk expression was significantly increased in sigc hour post fsh exposure (fig- a) . similarly on wb, phospho-cjun expression increased as early as min after fsh exposure and peaked at hrs. cjun phosphorylation was abolished hr after treatment with sp ( mm) (fig b) . facs analysis showed that the inhibition of jnk by sp resulted in cell cycle arrest at g /m transition in a dose dependent fashion (fig c) . conclusion: these results strongly suggest that the proliferative effect of fsh on immature granulosa cells is mediated through the activation of jnk pathway. this is the first experimental observation implicating jnk signaling in granulosa cell cycle control. (nichd - ). the induction of {alpha}-hydroxylase (cyp ) expression in granulosa cells. satin s patel, victor e beshay, william e rainey, bruce r carr. reproductive endocrinology and infertility, university of texas at southwestern medical center at dallas, dallas, tx, usa; physiology, medical college of georgia, augusta, ga, usa. according to the traditional two-cell two-gonadotropin hypothesis of the ovary, androgen production arises exclusively from theca cells. the granulosa cells, in turn, utilize androstenedione, which is aromatized eventually to estradiol. studies involving immunohistochemical analysis of normal ovaries have shown that granulosa cells express significantly higher levels of the activator protein- (ap- ) transcription factor, cfos compared to theca cells, where cfos expression is virtually absent. we hypothesize that cfos functions to inhibit the expression of cyp in granulosa cells, thereby suppressing androgen production. hence, the inhibition of cfos activity might result in cyp expression in the granulosa cell. our objective was to define the role of cfos, in the regulation of cyp expression in granulosa cells. transformed human luteinized granulosa (hgl ) cells were utilized for all experiments. hgl cells were cultured in monolayer for h. cells were treated for h with and without pd (pd), a mapkk inhibitor, which also blocks cfos expression. rna was isolated and real time rt-pcr was performed for cyp . cfos rna interference experiments were carried out using rnaifect, cfos smartpool sirna and scrambled sirna for h. rna was isolated and rt-pcr was also performed for cyp . immunochistochemical studies were performed on normal ovaries, staining for cfos and cyp . treatment of hgl cells with the mapkk inhibitor pd for h, resulted in a -fold increase in cyp mrna expression compared to basal conditions. in cfos gene silenced cells, cyp mrna expression also increased by -fold compared to control sirna conditions. immunohistochemical staining for cfos and cyp showed significant staining of cfos in the granulosa cell layer, but absent staining for cyp . conversely, the theca cell layer did not stain for cfos, but staining was evident for cyp . these results suggest that the ap- transcription factor, cfos, may play a role in the inhibition of cyp expression in granulosa cells. this may provide an explanation for the lack of cyp expression in granulosa cells. the g /m stages of the granulosa cell cycle. as clip- has been identified as an mtor substrate, we hypothesized that its function at the mitotic spindle would be positively regulated by mtor during the late g and m phases of the cell cycle in granulosa cells. during periods of stress (e.g., mtor inhibition), mtor would fail to phosphorylate clip- , leading to spindle checkpoint failure and follicle undergrowth. objectives: the expression of clip- and mtor were evaluated. computational analysis of potential clip- phosphorylation sites and comparison with residues on known mtor targets were performed. clip- threonine and serine were chosen and evaluated as bona fide mtor phosphorylation sites. a preliminary assessment of the effects of mtor inhibition upon clip- function was performed. methods: for protein expression analyses, western blots, immunostaining of tissues and primary granulosa cells in culture were performed. computational analysis of potential clip- phosphorylation sites was followed by in vitro assessment of mtor kinase activity upon clip- and a peptide substrate. results: clip- was expressed in the ovarian stroma, blood vessels (including the endothelial cells of both arteries and veins), granulosa cells, and in the oocytes of primordial and growing follicles. overlapping expression was found between clip- , mtor, and the mtor cofactors raptor and rictor in granulosa cells. this expression was conserved between the mouse and the human. evaluation of clip- phosphorylation supported thr as a bona fide mtor target. conclusions: clip- was supported as an mtor substrate protein during granulosa cell mitosis. the mechanism of mtor action during granulosa cell growth and survival is likely to include the phosphorylation of clip- and subsequent positive regulation of mitotic spindle function. the effect of a selective oxytocin antagonist (barusiban) in threatened preterm labour: a randomised, double-blind, placebo-controlled trial. steven thornton, thomas m goodwin, gorm greisen, morten hedegaard, joan-carles arce. warwick medical school, university of warwick, coventry, united kingdom; maternal-fetal medicine, university of southern california, los angeles, usa; dept of neonatology, rigshospitalet, copenhagen, denmark; dept of obstetrics, rigshospitalet, copenhagen, denmark; clinical research development, ferring pharmaceuticals, copenhagen, denmark. objective: a mixed oxytocin/vasopressin v a antagonist, atosiban, has been shown to reduce uterine contractions in placebo-controlled clinical trials and is useful in the management of preterm labour. the objective of this study was to determine the effect of a selective oxytocin antagonist, barusiban, in delaying delivery and reducing uterine contractions in women with threatened preterm labour at a late gestational age and relatively high risk of delivery. methods: this was a randomised, double-blind, placebo-controlled multicentre study in countries. a total of women between + and + weeks gestation, and with uterine contractions of sec duration during min, cervical length mm, and cervical dilatation > and < cm were randomised to receive a single intravenous bolus dose of either barusiban . mg, mg, mg, mg or placebo. rescue tocolytics were prohibited. the primary end-point was percentage of women who did not deliver within h. uterine contractions were monitored by cardiotocography. obstetrical and neonatal outcomes were determined. results: there were no significant differences between the placebo and any barusiban group in percentage of women who did not deliver within h ( % in the placebo group and % to % in the barusiban groups). there was no dose-effect relationship nor an effect at or h. none of the barusiban groups were associated with a significant reduction in number of uterine contractions compared to placebo at any time point up to h post-dosing. postpartum blood loss and time to established lactation were not significantly increased with barusiban. barusiban was well tolerated and was not associated with safety concerns for the women, fetus or neonates. conclusion: a single intravenous bolus of a selective oxytocin antagonist, barusiban (dose range . - mg), did not delay delivery or reduce uterine contractions compared to placebo in women with preterm labour at late gestational age and with short cervical length. the results contrast those of the mixed oxytocin/vasopressin v a antagonist, atosiban. prolonged delivery intervals in triplet gestations. tracy a manuck, heather l mertz, leah passmore, david c merrill. obstetrics and gynecology, wake forest university health sciences, winston-salem, nc, usa. objective: delayed interval delivery is one management strategy for previable preterm labor affecting multiple gestations. prior reports of asynchronous deliveries have examined twins and higher-order multiples as a group. this study was conducted to analyze the unique situation of asynchronous triplet deliveries. study design: cases of asynchronous triplet deliveries resulting in an ongoing twin gestation were ascertained through medline. data were abstracted and combined with two similar previously unpublished cases. patients were grouped by management with and without rescue cerclage. variables compared included use of tocolytics, antibiotic administration, gestational age at delivery of each fetus, interdelivery interval, delivery mode, birthweights, and short and long term outcomes. chi-square or t-test analyses were used where appropriate. results: fifty-one cases of asynchronous triplet deliveries met inclusion criteria and were analyzed. twenty-three patients ( . %) underwent placement of a rescue cerclage following delivery of the first infant. these patients delivered the first fetus at a significantly earlier gestational age as compared to those patients without a cerclage ( . +/- . weeks vs. . +/- . weeks, p= . ). patients with a rescue cerclage had a significantly longer prolongation of the remaining twin gestation ( . +/- . days vs. . +/- . days, p= . ). no significant differences in use of tocolytics or antibiotics, gestational age at delivery of triplets "b" and "c," mode of delivery, short term outcome (alive at hours), or long term outcome (alive at discharge) were noted, despite delivery of triplet "a" at a significantly younger gestational age. conclusion: rescue cerclage, particularly when placed following previable delivery of a first triplet, may significantly prolong the delivery interval for the remaining twin gestation. mean pregnancy prolongation (days). objective the aim of our study was to evaluate the role of amnioinfusion in pregnancies complicated by pprom. we studied singleton pregnancies with pprom at < weeks gestation. all patients were managed conservatively with bed-rest, prophilactic antibiotics, tocolytics and steroids. only patients without vaginal bleeding and/or contractions were included: patients showed an amniotic fluid pocket (afp) persistently cm and did not undergo amnioinfusion (group b) whereas had a maximum afp < cm and were offered amnioinfusion to restore an adequate amount of amniotic fluid (group a). in patients of group a amnioinfusion was successful (afp cm for hours following the procedure: group a ) whilst in it was unsuccessful (afp< for hours: group a ) and repeated. results were analized with the student t test for unpaired samples and with the when appropriate. p values < . were considered significant. results the group where amnioinfusion was not successful (group a ) showed the worst outcome (see table) . there were intrauterine deaths, all in this group. pulmonary hypoplasia was present in / ( . %) newborns (both survived and deceased) newborns, / in group a . no maternal complications were recorded. conclusions our data confirm that a conservative-active management with amnioinfusion can be considered a reasonable option in women with pprom. in our series it was effective in preventing both neonatal death and pulmonary hypoplasia. group a (infusion successful) background. synthetic progestogens are effective in reducing the risk of spontaneous preterm birth in high risk singleton, but not multiple, pregnancy. we hypothesized that myometrial stretch may inhibit the response of human myometrium to progestogens. methods. myometrial strips obtained with written consent at the time of term planned cesarean section were studied using a modification of the method of young and zhang (jsgi ; : - ) . strips were maintained in individual tubes in tissue culture media in an incubator for a period of three days. the effect of prolonged stretch was assessed by comparing strips connected to a . g weight with those connected to a . g weight. the effect of prolonged exposure to progestogen was studied by adding medroxyprogesterone acetate (mpa, nm or nm). following the day incubation, myometrial strips were transferred to an organ bath containing kreb's solution. all were placed under g tension and responses obtained to mm potassium then oxytocin. contractility was expressed as the ratio of the maximum response to potassium or oxytocin to the wet weight of the tissue (units = g.tension per gram), summarized as the mean (sem) and compared using student's paired t test. prolonged stretch increased the maximum response to both potassium ( . g weight = . [ . ]; . g weight = . [ . ], n= , p= . ) and oxytocin ( . g weight = . [ . ]; . g weight = . [ . ], n= , p= . ). in strips with a . g weight, incubation in mpa for three days reduced the maximum response to potassium (vehicle = . [ . ]; mpa = . [ . ], n= , p= . ) and there was a trend towards a reduced maximum response to oxytocin (vehicle = . [ . ]; mpa = . [ . ], n= , p= . ). in strips with a . g weight, incubation in mpa for three days had no effect on either the maximum response to potassium . prolonged stretch increases human myometrial contractility in vitro. . prolonged exposure to a progestogen inhibits the contractility of human myometrium but this effect is blocked by prolonged stretch. these properties of human myometrium may explain the failure of oh progesterone caproate to reduce the incidence of spontaneous preterm birth in multiple gestations. sangeeta jain, william l maner, janet l brandon, gary dv hankins, robert e garfield. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine if transabdominal uterine electromyography (emg) correlates with parturition factors such as measurement-to-delivery time (mtdt), cervical dilation (cd), cervical effacement (ce), and station (s) for preterm labor patients with and without tocolysis. materials and methods: pregnant preterm labor women were included. uterine electromyography (emg) was measured for minutes. cd, ce, and s were assessed at or near the time of uterine emg measurement. the power density spectrum peak frequency (pdspf) was calculated on emg. patients were grouped (g : tocolysis, n= ; g : no tocolysis, n= ). pearson-product-moment test was used for correlation. significant differences were sought between groups using student-t test. p< . significant. results: there was a significantly higher uterine emg activity (pdspf: . ± . vs. . ± . ), but no difference in cd ( . ± . vs. . ± . ), for patients delivering within days of emg recording compared to those who delivered later, regardless of tocolysis. there was no apparent difference in uterine emg in tocolytic vs. non-tocolytic patients, regardless of mtdt (table ) . conclusions: uterine emg activity is significantly greater in patients in preterm labor who delivered within days of measurement, making it a viable alternative diagnostic parameter for assessing the state of parturition. tocolytics may not affect uterine emg, but this should be further verified with larger studies. supported by grant nih r -hd . objective: calcium sensitizers are a novel class of drugs with unique molecular and phsiological actions. levosimendan, the best characterized of these compounds and is used in the treatment of acute and chronic heart failure. levosimendan can exert an inotropic effect via sensitization of myofilaments to calcium. it also exerts a relaxant effect on vascular smooth muscle through the opening of atp-dependent potassium channels and has been shown to be a potent inhibitor of human uterine contractions in vitro. for these reasons we investigated the effects of levosimendan on uterine contractions, both spontaneous and agonist induced, in the presence of glibenclamide, a k-atp channel blocker. method: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to glibenclmide ( mmol) followed by cumulative additions of levosimendan in the concentration range of nmol/l to mmol/l. control experiments were performed simultaneously. results: levosimendan exerted an inhibitory effect on spontaneous and oxytocin induced contractions in human m yometrium in vitro, in comparison to control experiments. the effect of levosimendan was significantly antagonized by glibenclamide with the mean maximal inhibition seen due to levosimendan greatly reduced (n= , p< . ). conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on human uterine contractility in vitro. this action was antagonized by glibanclamide and this study demonstrates that the effect of levosimendan on uterine smooth muscle is mediated at least in part through the k-atp channel. introduction: the determination of the beginning and ending points for "bursts" of electrical activity occurring during uterine contractions is sometimes difficult. if bursts cannot be discerned, the preferred burst-by-burst analysis cannot be performed. one solution to is to analyze any given electrical recording in its entirety. but this approach has often lead to meaningless results when traditional analytic methods are applied. chaos analysis, using lyapunov exponents, may provide an answer. materials and methods: term patients were included in the analysis: were in labor (group ), while were non-labor (group ). minute recordings were analyzed using "lyapunov exponent." for each pair of subsequent trajectories in phase space, only the most positive lyapunov exponent was calculated. the mean largest exponent was found by averaging over all of the trajectories in the recording. the lyapunov exponent is given in units of bits per data sample. thus a value of + means that the separation of nearby orbits doubles on the average in the time interval between data samples. the mean largest exponent was found for each patient recording. these values were compared using t-test (p < . considered significant). results: the mean and sd of the lyapunov exponent for all the patients was . ± . . moreover, the lyapunov exponent calculated for each patient was positive. comparing lyapunov exponents of the two groups showed a statistically low value (low chaos) for the laboring group, compared to the non-laboring group (figure) . conclusions: there is a chaotic component associated with uterine emg traces, since small but non-negative lyapunov exponents were found in all the traces observed. the lyapunov exponent indicated significantly lower chaotic behavior in the whole emg traces of patients who were in labor than found in those who were not in labor, implying that this measure could be a good diagnostic parameter for labor, possibly eliminating the need for tedious burst-by-burst analysis. supported by grant nih r -hd . comparing uterine emg to tocodynamometer for monitoring contractions. robert e garfield, lynette b mackay, sangeeta jain, william l maner. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine whether uterine electromyography (emg) plots contractions similarly to tocodynamometer (toco). study design: pregnant term labor patients were recorded using both uterine emg and toco simultaneously. uterine emg signals were sampled at hz and band-pass filtered in the . to . hz range. root-mean-square (rms) function was calculated from the uterine emg signals in order to produce a "toco-like" trace from the original emg trace. emg-generated rms contraction plots were then compared to toco contraction plots using the following criteria: contractions were assigned a marker value of " ." in-between contraction periods were assigned a " ." from these marker values, contraction rates were established. correlation was found between the contraction rates of rms and toco. temporal overlap of contractions plotted by the two methods was used to find overall percent agreement (opa), positive percent agreement (ppa), and negative percent agreement (npa). these parameters were corrected for within-patient variation using a bootstrap method. results: uterine rms contraction plots were seen to correspond with toco contraction plots (fig. ) . corrected opa, ppa, and npa were high at . %, . %, and . %, respectively. there was a large, statistically significant correlation between uterine emg and toco contraction frequency (fig. ) . conclusions: the similarity between toco and uterine emg contraction plots (specifically, using rms to convert) will allow emg to be used interchangeably with toco in the clinic. supported by grant nih r -hd . introduction -this is a secondary analysis of women participating in a center randomized placebo controlled trial (rct) evaluating the impact of -ohpc in preterm birth (ptb) prevention among women with twins. objective -to evaluate the relationship between plasma -ohpc concentrations and gestational age (ga) at delivery in women with twins receiving weekly injections of -ohpc. methods -women with twins were randomized between - weeks to receive weekly im injections of either -ohpc ( mg) or placebo until weeks. after a minimum of five consecutive injections had been administered to assure steady state concentrations a plasma sample was collected between - weeks. the sample drawn just prior to the next scheduled injection was analyzed for -ohpc by hplc-ms in a blinded manner. the lower limit of quantification of -ohpc was ng/ml. we conducted univariate analyses to assess the association of -ohpc concentration and ga at delivery. we also conducted a proportional hazards model to evaluate the time from sample draw to spontaneous delivery (censoring indicated preterm deliveries), and a logistic regression to evaluate ptb< weeks; in both analyses we adjusted for bmi, race and ga at sample draw. results - women assigned to -ohpc were included; all received all of their scheduled injections. the concentration of -ohpc was significantly higher in women delivering < weeks compared with those women delivering > weeks (p= . , table) . concentration of -ohpc was significantly correlated with ga at delivery (r = - . , p= . ). each unit increase of -ohpc was associated with a % increased odds of delivering < weeks (odds ratio . , % ci, . - . , p= . ) and a % increase in hazard of spontaneous delivery (hazard ratio . , % ci, . - . , p= . ) after adjusting for confounders. gestational age at delivery mean (sd) -ng/ml < weeks (n= ) . ( . ) > weeks (n= ) . ( . ) conclusion plasma -ohpc concentrations after weekly injections were inversely related to ga at delivery in women with twins. since -ohpc induces its own metabolism it is possible that higher concentrations during initial treatment are associated with lower plasma concentrations and reduced efficacy in later pregnancy . clearly more studies are needed. objective: in many non-human species, maternal circulating progesterone levels fall prior to delivery, leading to the theory that in humans progesterone withdrawal occurs on a local and/or functional level. our objective was to characterize maternal and fetal progesterone in human preterm and term labor. methods: women between . and . weeks' gestation (cases) or term controls ( - weeks) with either labor with intact membranes or premature rupture of the membranes prior to labor (prom) were enrolled in a prospective case-control study. progesterone was measured by immulite assay in maternal serum collected upon enrollment and again within minutes after delivery and in umbilical cord serum obtained at delivery. maternal progesterone treatment was not used in any subjects. results: cases and controls were studied (see table for comparisons). controls p value ga at enrollment, weeks . ± . . ± . < . interval to delivery, days (median, range) ( - ) ( - . ) < . maternal progesterone at enrollment, ng/ml ± ± < . maternal progesterone after delivery, ng/ml ± ± . cord progesterone, ng/ml ± ± . among cases, fetal but not maternal progesterone was significantly lower in preterm labor with intact membranes ( ± ng/ml, n = ), as compared to prom ( ± , n = ), p< . . this difference increased further when cases of clinical chorioamnionitis were excluded. conclusions: serum progesterone in laboring patients prior to delivery is higher at term than in the preterm period, which may be attributable to increased placental mass in late pregnancy. this disparity disappears shortly after delivery of the fetus and placenta. fetal progesterone levels are several-fold higher than peripartum maternal levels. preterm labor with intact membranes is associated with diminished fetal progesterone, a phenomenon unrelated to clinical infection. these findings suggest the possibility of fetally regulated progesterone withdrawal as a mechanism underlying preterm labor with intact membranes. [snps] and ptb. however, many of these studies are inconclusive and non reproducible. the challenge of identifying robust associations between genetic variation and either susceptibility or protection from ptb is enormous. a systematic review of literature followed by metaanalysis was performed to understand true associations between snps and ptb. methods: for systematic review, articles were chosen based on medline and embase searches ( ( -april and relevant articles were chosen based on stringent inclusion criteria. primarily, studies reporting genetic associations between snps in maternal dna in singleton pregnancies and spontaneous ptb were included. other criteria included, but not limited to, provided genetic data in a complete enough format so that it could be evaluated in meta-analysis and defined the clinical outcome clearly. meta-analysis was performed wherever > replication data sets were available results: a total of abstracts were reviewed and were selected for full text review. data were extracted from articles. over associations were reported between snps on various candidate genes and ptb; however only had replication dataset. meta-analysis documented significant association between pon a g (odds ratio [or]= . ( %ci . - . ), pon (rs# )(or= . ; ci- . - . ), tnfrsf - a/g (fas) (or= . ; ci- . - . ) and ptb. two snps pon s c (or= . ; ci- . - . ) and ifn gamma (rs ; or- . ; ci- . - . ) documented protective effect. conclusions: systematic review concludes significant heterogeneities leading to lack of reproducible data in genetic association studies of ptb. heterogeneities are contributed predominantly by lack of adequate power, poor phenotype selection, and population admixture. the functional relevance of the risk and protective alleles needs to be verified. jignesh parvadia, mounira habli, jeff livingstone, william polzin, foong lim, timothy crombleholme. pediatric and thoracic surgery, cincinnati children's hospital medical center, cincinnati, oh, usa; obstetric and gynecology, university of cincinnati, cincinnati, oh, usa; obstetric and gynecology, good samaritan hospital, cincinnati, oh, usa. objective little is known about the response of ttts to treatment either by amnioreduction or selective fetospopic laser in triplet pregnancy, particularly the survival of the bystander fetuses. in order to define the response of triplet pregnancies to treatment for ttts we reviewed our experience with higher order multifetal gestations complicated by ttts. study design retrospective chart review of patients diagnosed with in high order gestation from - was performed. results among cases of ttts / ( . %) patients with high order gestations were identified (n= ) with a mean ga at diagnosis of . ± . weeks. pregnancies ( . %) were dichorionic triamniotic and ( . %) were monochorionic triamniotic. cincinnati modification of quinterro staging was utilized to characterize recipient cardiomyopathy as mild (stage iiia, n= ), moderate (stage iiib, n= ) and severe (stage iiic or iv, n= ) categories. / ( . %) were treated with amnioreduction alone (ar), / ( . %) with selective fetoscopic laser photocoagulation (sflp) alone, / ( . %) with ar followed by sflp and / ( . %) with ar followed by intrafetal radio frequency ablation (rfa). / ( . %) patient had a cervical cerclage. / ( . %) patients were treated but remain undelivered. mean ga at delivery was . ± . weeks. overall survival was / ( . %) with bystander survival was / ( %), donor survival / ( . %), recipient survival was / ( . %). conclusion triplet pregnancies treated for ttts have % survival rate for bystander fetuses and have . % survival rates for donor and recipients comparable to twins treated for ttts. ga at diagnosis . ± . weeks cincinnati modification of quinterro (i), (ii), (iii), (iiia), (iiib), (iv) ga at delivery . ± . weeks live birth -donor / ( . %)* # -recipient / ( . %)*# -bystander / ( %) # birth weight -donor to assess the effect of breast feeding (bf) on perinatal outcome in relation to maternal antenatal methadone dose. study design a retrospective chart review study of methadone dependent mother and infant pairs. patients were categorized into groups based on maternal dose at time of delivery: group : dose mg, group : dose - mg, group : dose > mg. the finnegan's scoring system was used to monitor neonatal abstinence syndrome(nas). treatment for nas was initiated if there were scores of . neonatal outcome data included:% nicu admission, % of babies discharged(d/c) at time of maternal d/c, % nas, % treated for nas and total hospital stay. data were analyzed by t-test and fisher's exact test. maternal characteristics between the groups were similar. regardless of maternal methadone dose, bf infants have shorter hospital stays and higher rates of d/c at time of maternal d/c, lower incidence of nas and fewer nicu admission (table) . in all three groups, breast feeding did not impact the severity of nas as reflected in finnegan's score(fs). (table) conclusion regardless of maternal methadone dose, breast feeding improves perinatal objective: to evaluate the effects of preventive collagen plugging of the fetoscopic access port at the time of balloon removal on pregnancy outcome in fetoscopic endoluminal tracheal occlusion pregnancies. study design: fifty-one pregnancies involving fetuses with severe congenital diaphragmatic hernia (cdh) were studied. all patients underwent feto between - weeks gestational age (ga) and fetoscopic balloon removal around weeks ga. at the time of balloon removal, a purified dried collagen plug was inserted through the fetoscopic access port in consecutive pregnancies but not in the first pregnancies considered as controls. all patients underwent post-plugging ultrasound and magnetic resonance imaging studies to evaluate for membrane dehiscence, amniotic fluid volume and fetal well being. ga at delivery, incidence of premature rupture of the membranes (pprom), bleeding at port retrieval and adverse fetal effects were compared in both groups. results: mean (sd) ga at feto [ . ( . ) vs. . ( . ) weeks; p= ns] and balloon removal [ . ( . ) vs. . ( . ) weeks; p= ns] was similar in the treatment and in the control group. incidence of pprom following the second fetoscopy was / in the study group compared to / in the control group (p< . ). mean (sd) ga at delivery was . ( . ) weeks in the study group, compared to . ( . ) in the control group (p= . ). bleeding from the trocar insertion site occurred in cases in both groups, but clinically significant bleeding occurred only in one of the controls. membrane dehiscence was noted in patient in the treatment group compared to in the control group (p=ns). conclusion: preventive collagen plugging of the fetal membrane defect created by the fetoscopic access resulted in a significant reduction in pprom rates and a trend towards increased ga at birth without adverse fetal effects in feto pregnancies. wider application of this technique should be considered, but needs evaluation in larger, randomized trials. hydrops fetalis is an uncommon fetal condition characterised by the abnormal accumulation of fluid in two or more body cavities, traditionally associated with a poor prognosis. the relative rarity of this presentation has meant that published case series have consisted of small numbers. a retrospective review of case notes of all cases managed at kemh between and was performed. in western australia, kemh is the only tertiary maternity hospital incorporating a maternal-fetal medicine unit. cases were obtained from the mfm database. in the period to there was a total of pregnancies affected by hydrops (incidence . per births). the average maternal age was years. in cases a fetal abnormality had occurred in a previous pregnancy. the median gestational age at diagnosis was weeks (range - weeks). in just over half ( %) of cases, the diagnosis was confirmed prior to delivery. a post-mortem was performed on all but of the babies not born alive. edema was present in at least cavities in over half of cases (n= ). chromosomal anomalies included trisomy , trisomy and turners syndrome. in all cases of infection, parvovirus b was implicated. cardiac arrythmias included svt and atrial flutter. cases classified as other included alpha thalassemia and syndromic disorders. in cases an interruption of pregnancy was performed at a mean gestational age of weeks. of those who did not elect to terminate the pregnancy, there were fetal deaths in utero, live borns with neonatal death. for the live borns, the median gestational at delivery was . weeks (range to . weeks). the causes of hydrops in live birth cases included cardiac arrythmia (n= ), infection (n= ), chromosomal abnormality (n= ), unknown (n= ) and other (n= (dmv) have been noted to play a role in the development of hemorrhagic and periventricular leukomalacic lesions in premature babies. since deep vein drainage system is relatively more prominent in the developmental brain than adult brain, we investigated if dmv anomalies could be associated with clastic lesions in-utero. methods two senior neuroradiologists reviewed fetal brain exam performed between and , seeking for unequivocal anomalies in dmv, such as periventricular venular engorgement. all mr scanning is performed at . tesla, using surface abdominal coils and single-shot fast spin-echo t -weighted - mm thick sections, with . - . mm in planar spatial resolution. we found cases with dmv anomalies (tab. ). most of the dmv engorgement is located at frontal lobes level. from this limited preliminary series it appears that dmv involvement plays a role in the development of periventricular leukomalacia and periventricular hemorrhagic necrosis. the observation that these lesions are mostly located at frontal level may suggest that some of the term neonates carrying sequelae of atypically located leukomalacia (i.e. deep frontal lobe) might have developed these lesions in-utero. it is of interest to notice that most of our cases were related to heart failure. therefore, central venous hypertension affecting immature deep cerebral venous system has to be taken into account. in our center, patients with an estimated risk for chromosomal abnormalities at term greater that in after st trimester combined screening test (ft) are offered non-directive genetic counseling. the aim of this study was to evaluate the responses of women younger than attending this genetic counseling session. material and methods: data from patients referred for a positive ft from september , to july , was retrieved from our database. information concerning women younger than years of age at the estimated date of delivery was extracted and tabulated. results: during the study period women had genetic counseling for positive ft. thirteen patients were excluded from further analysis ( had incomplete clinical documentation and had spontaneous miscarriages prior to weeks gestation). four hundred and twenty-five patients were older than and were younger than at the estimated date of delivery. table depicts summary statistics for studied variables in this younger group of women. conclusions: overall this data suggests that approximately % of this younger group of women opted for chorionic villous sampling (cvs), % for amniocentesis and more than % declined prenatal genetic testing. moreover, this data also suggests that: ) these women opted for cvs when the ft risk (mean = in ) almost doubled the cvs procedure related risk quoted at % and, ) when the ft risk is between in and in almost half ( out of ) declined not only the st trimester cvs but also the nd trimester amniocentesis. we believe that understanding our patient population is important to optimize both the efficiency and efficacy of the alternative prenatal screening programs. acceptance for prenatal genetic testing after a positive first trimester combined screening test in women of less than to determine the optimal diagnostic test using prenatal ultrasonography and mri for predicting pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. methods: relevant papers were identified by searching medline ( medline ( - , embase ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and the cochrane library ( issue ). in addition, the specialist literature on the topic and reference lists were hand searched for relevant articles. studies were selected if they examined diagnostic tests for the prenatal prediction of pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. the primary outcome measure was perinatal survival. study selection, quality assessment and data abstraction were performed independently and in duplicate by separate observers. results: of a total number of articles (published studies), there were eighteen studies that fulfilled the entry criteria. six examined entirely unique heterogeneous parameters. of the remainder, all examined the ultrasound measurement of lung to head ratios (lhr) at a 'cut-off of greater than or less than the thresholds of . , . , . , . . in addition, the presence of liver in the fetal thorax was included (if present) as a co-variable (liver "up"). a lhr > . compared to < . provided the strongest association with perinatal survival (peto or . , % ci . - . ). the finding of "liver up"in the fetal thorax had a negative association with survival (peto or . , % ci . - . ). only three studies provided data for lhr in conjunction with the presence of liver in the fetal thorax (peto or survival for lhr > . compared to < . was or . , % ci . - . ). data was also available for liver up and lhr > . which had a peto or of . ( % ci . - . ). discussion: the data supports the view that lhr may be a useful prognostic indicator of perinatal survival in fetuses with congenital diaphragmatic hernia. however, heterogeneity still exists regarding the timing of ultrasound measurement and the use of mri. the majority of studies have small sample sizes. objective: to estimate, in fetal anaemia, the diagnostic value of fetal ultrasonography and doppler blood flow in the evaluation of fetal anaemia methods: literature from to was identified using medline and embase, the cochrane library and relevant specialist register of the cochrane collaboration, and by checking reference lists of known primary studies and review articles. studies were selected if the accuracy of the fetal ultrasound parameters or doppler studies of blood flow in the fetal vessels was estimated compared with a reference standard. diagnostic tests evaluated were ultrasound measurement of the fetal spleen and liver length and doppler studies from the umbilical vein and middle cerebral artery. data from the selected studies were abstracted as x tables comparing the diagnostic test result with the reference standard. results were pooled where appropriate. diagnostic accuracy was expressed as sensitivity and specificity. twenty-nine primary studies were identified containing suitable data. twentyone of these gave data on middle cerebral artery doppler peak systolic velocity (mca-psv) and could be pooled in the meta-analysis giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) for cases in detecting severe anaemia. four studies gave data on spleen perimeter and it was possible to pool three of these giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) for cases. three studies had data for liver length measurements and two were pooled. the sensitivity was . ( . - . ) and the specificity was . ( . - . ) but only cases were used in the analysis. there were three studies on umbilical vein maximum velocity doppler studies and all were suitable for meta-analysis giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) with cases analysed. two studies gave data on middle cerebral artery time-averaged mean velocity score giving cases. the sensitivity was . ( . - . ) and specificity was . ( . - . ). discussion: middle cerebral artery peak systolic velocity dopplers remain the gold standard for non-invasive screening of fetal anaemia. middle cerebral artery time-averaged mean velocity scores require further investigation. other tests perform poorly when diagnostic accuracy is assessed. cochrane review of treatment in twin to twin transfusion syndrome. twin-twin transfusion syndrome is a condition affecting monochorionic twin pregnancies associated with a high risk of perinatal mortality and morbidity. the objective of this review was to evaluate the impact (maternal,fetal and pediatric)of treatment modalities in twin-twin transfusion syndrome. register and cochrane controlled trials register. we also searched conference proceedings and made personal contact with experts active in the area of the review. randomised and quasi-randomised studies of amnioreduction versus laser coagulation, septostomy versus laser coagulation or septostomy versus amnioreduction. eligibility was assessed by one reviewer. study authors were contacted for additional information. two studies were included. this review shows that laser coagulation of anastomotic vessels results in less fetal (rr . ; % ci . , . ) and neonatal deaths (rr . ; % ci . , . ) than amnioreduction ( figure ). there is no difference in perinatal outcome between amnioreduction and septostomy. more babies in the laser arm are alive without neurological abnormality at six months of age (developmental delay at < years rr . , % ci . , . )( figure ). conclusions: endoscopic laser coagulation of anastomotic vessels should be considered in the treatment of all stages of twin twin transfusion syndrome to improve perinatal outcome. further research on the effect of treatment on milder forms of twin twin transfusion syndrome (quintero stage and ) are required. (produced in collaration with the cochrane collaboration, uk). (cvs) concluded that although the risks of pregnancy loss are relatively low, lack of adequate controls tends to underestimate the true added risk of prenatal invasive procedures (obstet gynecol. ; ( ): - ). the west midlands is a large region within the uk containing approximately % of the total uk population. the congenital anomaly register for this region is able to monitor pregnancy outcomes with accurate deminator data. results: there were first trimester cvs performed, by ten operators, in the west midlands (uk) in . this equates to . procedures per births. significantly higher rates were noted in areas of high socioeconomic status. the median number of procedures performed per operator was (range - ). all operators were performing other invasive tests such as amniocentesis or cordocentesis,etc. the most common indication for cvs was: i) fetal anomaly on dating scan ( . %) ii) abnormal (> in ) risk on combined first trimester screening ( . %) iii) molecular genetic diagnosis ( . %) and iv) maternal request ( . %). using a combination of first trimester scanning and cvs, % had abnormal karyotype/structural anomaly.the corrected loss rate (background and procedure-related) following cvs in normally formed, singleton pregnancies was . % ( thci . - . %) up to days postnatal (perinatal loss) and . % ( thci . - . %) with days of procedure. the proportion of cvs in which an adequate sample was not obtained was . % ( th ci . - . %). conclusions: this epidemiological study using accurate demoninator data provide interesting statistics relating to the uptake and prenatal risks of first trimester cvs. with the increasing prevalence of obesity in the last two decades, we have seen a tremendous increase in bariatric procedures in reproductive aged women. malnourishment and vitamin deficiency are common complications after gastric bypass which may impact on fetal development. we present the case of a yo who underwent a duodenal switch procedure in . she was discovered to be pregnant during evaluation for persistent malnutrition in . multiple prenatal ultrasounds were performed; the first at weeks gestation was unremarkable. the week sono revealed a male fetus with shortened femurs and humeri bilaterally, nasal bridge hypoplasia, macroglossia, poorly defined hands, and possible clubbed feet. amniocentesis revealed a normal karyotype. d ultrasound redemonstrated the abnormal facial findings. a fetal echocardiogram was normal. the lagging long bone measurements continued to worsen, ultimately with femurs weeks behind. the fetal thoracic circumference was two standard deviations below the mean, giving rise to concern for pulmonary sequelae. growth restriction was noted at the wk sonogram. delivery by cesarean section was at / weeks secondary to nonreasuring fetal status. the birthweight was gm; apgars were , , and . postnatal radiographs confirmed antenatal ultrasonographic findings and demonstrated evidence of epiphyseal stippling. the infant remained intubated until weeks of life, after which it died secondary to respiratory complications associated with pulmonary hypoplasia. gene mapping studies have not found any point mutations on the recessive gene as an etiology of this disorder. rhizomelic chondrodysplasia punctata refers to a heterogeneous group of bone dysplasias with a familiar radiographic phenotype involving punctate calcifications and epiphyseal stippling. the etiology of this may be secondary to chromosomal abnormalities, mendelian gene disorders, or teratogens, notably warfarin. this case may be explained by vitamin k deficiency of the embryo due to maternal malabsorption after bariatric surgery. the maternal vit k level was <. ng/dl at time of delivery(normal > . ). the teratogenic effects of vitamin k deficiency in this instance highlight the need for strict counseling and screening for vitamin deficiency in those women undergoing bariatric surgery since previous obesity-related anovulation is reversed as patients lose weight, resulting in unexpected pregnancies and potentially preventable fetal abnormalities. term preeclampsia: any risk for the neonate? sindhu k srinivas, jamie bastek, christina m andrela, emmanuelle pare, michal a elovitz. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: preeclampsia continues to be a major contributor to maternal morbidity and mortality worldwide. preeclampsia at term is not associated with the same risk of neonatal morbidty and mortality as preterm preeclampsia . however, neonatal outcomes in term women with preeclampsia have not been adequately studied. we sought to compare short term neonatal outcomes in term infants born to women with and without preeclampsia. methods: this study was part of a large case control study. women with preeclampsia (n= ) and term controls (n= ) were prospectively identified. infants with congenital anomalies were excluded. hospital length of stay (los), admission or transfer to the nicu, and use of mechanical ventilation or cpap within first week of life were assessed. associations between neonatal outcomes and preeclampsia were evaluated using chi-square analysis and wilcoxon rank sum test. significant confounders were controlled for using multivariable logistic regression. results: discharge day of life was significantly different between neonates born to women with preeclampsia (median = ; mean = . ) versus those born to women with uncomplicated term deliveries (median = ; mean = . , p< . ). this difference persisted even when neonates with iugr and those born to diabetic mothers were excluded (p< . ). term infants born to women with preeclampsia have a higher odds of being admitted or transferred to the nicu (aor= . [ . - . ], p= . ) after controlling for iugr, delivery mode, race, and gestational age at delivery. these infants also have a higher odds of mechanical ventilation (aor= [ . - . ], p= . ) and cpap use (aor= . [ . - . ], p= . ) after controlling for the same confounders. there was no difference in ivh or nec between the two groups. conclusion: neonates born to women with preeclampsia have differences in short-term morbidity when compared to neonates born to women without preeclampsia, despite being born at term. whether this increase in neonatal morbidity is attributable to medications used in preeclampsia, such as magnesium sulfate, is unclear. these findings may have implications for patient counseling as well as hospital resource allocation. further investigation correlating these findings with long-term morbidity is warranted. could confined placental mosaicism account for adverse perinatal outcomes in ivf pregnancies? benoit c jacod, gh schuring-blom, kd lichtenbelt, jse laven, d van opstal, mjc eijkemans, nick s macklon. reproductive medicine gynaecology, university medical centre, utrecht, netherlands; clinical genetics, university medical centre, utrecht, netherlands; obstetrics gynaecology, erasmus medical centre, rotterdam, netherlands; clinical genetics, erasmus medical centre, rotterdam, netherlands. background: ivf singletons have poorer perinatal outcomes than singletons from spontaneous conceptions. this may be due to the influence of ovarian stimulation on the chromosomal constitution of the embryos which could be translated into localized chromosomal anomalies in the placenta. aim: to compare the incidence of confined placental mosaicism (cpm) in ivf/icsi pregnancies and spontaneous conceptions. methods: multicentre retrospective analysis of karyotype results obtained by chorionic villus sampling (cvs) performed because of advanced maternal age ( years at weeks of gestation) in the netherlands between and . results: from a total of pregnancies, cvs results were analysed: in the ivf/icsi group and in the control group. the mean age of women in both groups was . years (mean difference - . , % ci - . - . ). foetal karyotype was missing in cases of possible cpm, all in the control group. when taking into account missing data, the incidence of cpm was lower in the ivf-icsi group than in the control group, . % vs. . % (odds ratio . , % ci . - . ) whereas the incidence of foetal chromosomal anomalies was increased . % vs. . % (odds ratio . , % ci . - . ) although both differences are not significant. conclusions: the incidence of confined placental mosaicism is not increased in ivf/icsi pregnancies compared to spontaneous conceptions. cpm probably does not account for the adverse perinatal outcomes following ivf/icsi. fetal rh d, cc and ee genotyping using fetal dna from maternal blood is not impaired by the presence of maternal alloimmunization. chad a grotegut, stacey l jeronis, john p gaughan, enrique hernandez, ossie geifman-holtzman. obstetrics and gynecology, duke university, durham, nc, usa; obstetrics, gynecology and reproductive sciences, temple university, philadelphia, pa, usa; biostatistics, temple university, philadelphia, pa, usa. this study was conducted to assess the impact of maternal alloimmunization on the accuracy of fetal rh d, cc and ee genotyping from maternal blood. we performed a literature search of english-written articles describing fetal rh d, cc and ee determination from maternal blood (am j obstet gynecol ; : - ) . using this database, we determined the accuracy of rh d, cc and ee genotyping in the presence of maternal alloimmunization. for each subgroup, % confidence intervals for a proportion were calculated and compared between groups. we found english-written publications reporting non-invasive rhd genotyping and reporting non-invasive rh ce genotyping from maternal blood. fourteen ( . %) of the rh d articles and three ( %) of the rh ce articles provided accuracy results in the setting of alloimmunization. the accuracy results are reported as follows: . % of the samples were determined correctly in the presence of alloimmunization.* this accuracy was significantly lower than the accuracy reported for all rh cc samples. when only studies utilizing free fetal dna for rh cc genotyping were used (vs fetal cells), fetal cc genotype was determined correctly in / ( %, % ci . , ), which was similar to the overall success rate for rh cc determination. overall, there were no differences in the success of rh d, cc or ee determination in the setting of alloimmunization compared to the overall accuracy seen when free fetal dna was used. the presence of maternal alloimmunization does not reduce the accuracy of fetal rh d, cc or ee non-invasive genotyping from maternal blood utilizing free fetal dna. further research into structure and rearrangements of the rh d, cc and ee genes may further improve diagnostic accuracy of free fetal dna from maternal blood. fetal dna, most likely of trophoblast origin, is present in both the plasma of pregnant women and provides a potential basis for non-invasive fetal diagnostic tests. however, fetal dna in maternal plasma is highly contaminated with maternal dna, and this contamination is the main technical challenge in trying to accomplish non-invasive detection of fetal chromosome abnormalities. existing methods for the selective amplification of fetal dna have generally relied on specific sequence differences between the mother and fetus. as an alternative, we have developed a method for selective amplification of fetal dna that makes use of observation that trophoblast dna is globally hypomethylated in comparison with dna from other sources. in this method, a dna mixture is first digested with a methylation sensitive restriction enzyme and then amplified by linker-mediated pcr. after an initial amplification, a second isothermal rolling-circle amplification is performed. this procedure results in the differential amplification of short, relatively hypomethylated fragments. after amplification, the resulting "representations" are comparatively hybridized to a microarray consisting of oligonucleotides that correspond to restriction fragments generated by the initial digest. copy number differences are then detected through statistical analysis of array addresses that show significant amplification. to test the feasibility of this method for detecting aneuploidy, we have prepared mixtures of peripheral blood dna and first trimester trophoblast dna from either normal or from samples with trisomy and trisomy . we present data showing that aneuploidy can be detected even when % of the starting dna sample was derived from a euploid source and only % was from an aneuploid trophoblast sample. two different approaches to data analysis are presented. one relies on prior analyses of trophoblast methylation and the second is independent of any prior knowledge or analysis. both methods provide similar ability to detect aneuploidy. future work will focus on testing whether this approach can be used for non-invasive prenatal diagnosis. chromatin immunoprecipitation and emsa analysis of nf-b and c/ ebp synergism on the otr promoter. shirin khanjani, yun s lee, vasso terzidou, mark r johnson, phillip r bennett. institute of reproductive developmental biology, imperial college, london, united kingdom. we have shown, the transient transfections of the transcription factors nf-bp and c/ebp leads to a synergistic increase in otr promoter activity in human myocytes. this effect is mediated through a bp region of the promoter between - to - from tss. we now report that this sequence binds both nf-bp and c/ebp in vitro and chip studies show binding of both transcription factors to be increased by il- in vivo. materials and methods: emsa studies were performed using a bp oligonocleotide sequence (- to - ) , containing the bp region responsible for the synergistic activation of the otr promoter and nuclear extracts from primary human myocytes treated with il- ng/ml for hours. for chip analysis, dna protein complexes were crosslinked and antibodies recognizing p , c/ebp and h (positive control) and igg (negative control) were used for immunoprecipitation. primers were designed to amplify the region - to - , which includes the bp response sequence. to further confirm the interaction between nf-bp with c/ebp a xnf-b consensus/luc reporter construct was cotransfected with an expression vector for either nf-bp or c/ebp . results: specific nf-bp and c/ebp binding was seen in the emsa study. preincubation with antibodies to nf-bp and c/ebp led, not to supershift, but to elimination of dna binding for both nf-kb p and c/ebp . chip analysis confirmed increased in vivo binding of nf-b p and c/ebp to this region of the otr promoter following stimulation with il- . transfection of the nf-b/luc reporter construct with an expression vector for c/ebp caused a significant reduction in the basal promoter activity suggesting that c/ebp is binding to nf-kb. this interaction was further confirmed using a tf-tf interaction array. conclusion: these data support the role of nf-b and c/ebp in regulation of otr. the bp region contains a c/ebp but not a nf-b dna binding site suggesting that c/ebp primarily binds to this part of the otr promoter but also interacts with nf-b. the emsa data shows that the bp region binds both nf-b and c/ebp . the loss of the supershift observed in previous emsa studies suggests that the antibodies inhibit the interaction between c/ebp and nf-b, therefore inhibiting dna binding. chip analysis supports the concept that il- leads to binding of nf-b and c/ebp to the bp region. regulation of pro-labour genes by c/ebp, nf-b and ap- in human uterine myocytes. suren r sooranna, shirin khanjani, yun s lee, phillip r bennett, mark r johnson. imperial college parturition research group, imperial college, london, united kingdom; irdb, imperial college, london. introduction: the transcription factors c/ebp (lap), nf-b (p ) and ap- (c-fos and c-jun) are implicated in inflammatory processes such as parturition. the promoter regions of the pro-labour genes il- , pghs- and otr contain putative transcription factor-binding sites for these transcription factors. our aim was to determine the effect of transfecting these transcription factors either alone or in combination into uterine myocytes and to determine their effects on the expression of pro-labour genes including il- , pghs- , otr, connexin- and fp. methods: primary cultures of human uterine myocytes (n= ) were grown from myometrial samples obtained at the time of elective lscs. cells were cultured in well plates to % confluence at which point expression constructs for c/ ebp (lap), nf-b(p ), ap- (c-fos and c-jun) were transfected either alone or in different combinations. the empty expression vector psg was used as a filler construct. cells were cultured for and h after which culture medium was collected for elisa and cells frozen at - °c prior to rna extraction. copy numbers of il- , pghs- , otr, fp, connexin- and gapdh were measured by qpcr using a rotor-genetm (corbett research). results: h post transfection with c/ebp (lap), nf-b (p ), c-fos and c-jun alone or in combination showed no significant changes in pghs - , otr and connexin- expression. over expression of p alone or together with either c-fos or c-jun increased fp expression by , and % respectively (p= . ). nf-bp consistently increased il- expression either alone (by %; p= . ) or in combination with lap, c-fos or c-jun (by , and % respectively; n= ; p= , ). rel a, lap, c-fos and c-jun together also increased il- expression (by %; p= . ) and a small but significant increase was seen with a combination c-fos and c-jun (by %; p= . ). the changes observed in il- expression were reflected in the medium il- concentration at h post transfection. in the presence of rela and c-fos il- increased from . ± . to . ± . pg/ml of culture medium (mean ± sem; n= ). conclusions: nf-b (p ) consistently increased fp and il- expression in human myometrium. the data suggest that pghs- activation has greater dependence upon other transcription factor(s) in addition to p . true identity of myometrial pr-c: fact or fiction? yun s lee, suren r soorana, mark r johnson, jan brosens, phillip r bennett. imperial college parturition research group, hammersmith campus, london, united kingdom. progesterone is thought to be central to maintenance of pregnancy. multiple progesterone receptor (pr) isoforms underlie complex and diverse biological action of progestins. previously two human pr isoforms have been identified: pr-b and pr-a. pr-a is n-terminally truncated form of pr-b (initiation site methionine ). in some cells pr-a inhibits pr-b. it has been proposed that increased expression of pr-a in myometrium underlies a 'functional progesterone withdrawal'. the breast cancer cell t d contains a kda progestin-specific binding protein that is not found in pr negative cells. it was proposed that there is a downstream methionine (met ) which serves as a translation initiation site for the generation of a pr isoform of kda. based on such findings condon et al (mol endo ) have focused on the possible role of kda pr-c in human parturition. they found that expression of "pr-c" using pr antibody (sc- santa cruz, sc) is increased in upper segment myometrium with labour and that overexpression of this protein inhibits pr-b function. we cloned the same human pr cdna into psg expression vector. in vivo translation produced a protein of only kda. furthermore overexpression of pr-c did not significantly decrease pr-b activity in human myocytes. we examined other downstream initiation sites, which may produce a kda protein. we constructed potential pr isoforms in psg vector with initiation sites at met and . in vivo translation produced proteins of approximately and kda respectively. to determine the effect of pr isoforms on pr-b function, myocytes were co-transfected with pr-a, pr-c , pr-c and pr-c with constant amounts of pr-b. the progesterone dependent pre/luc was used as reporter. unlike pr-c both pr-c and pr-c significantly inhibited ligand dependent pr-b mediated transcriptional activity. we found in western analysis that the antibodies pgr- (nc) and the sc- (sc) detected both endogenous and overexpressed pr-a and pr-b but none of the pr-c isoforms. the sc- antibody detected only pr-a and pr-b very poorly. our data suggests that the sc- antibody would not detect any pr-c protein and that none of the commercially available antibodies in the uk do so. great care needs to be taken when over-expressing pr isoforms to ensure that proteins are of the expected size. if pr-c does exist in vivo then the kda but not the kda isoform might inhibit pr-b function. tnf receptor antibody (tnf ri ab), nf-b inhibitor (nf-b activation inhibitor) and erk inhibitor (u ) (p< . ), but not by tnf receptor antibody (tnf rii ab), p mapk inhibitor (sb ) and jnk inhibitor (sp ). by western blot analysis, we found that the protein level of tnf receptor associate factor (traf ) was higher than that of tnf receptor associate factor (traf ) (traf >traf ) in untreated ct cells. however, after tnf treatment for h to h, traf protein level was increased, but traf protein level was reduced (traf >traf ). the increase of traf and decrease of traf were blocked by tnf ri ab, but not by tnf rii ab. we also found that tnf rapidly (within - min) and significantly increased phosphorylation of ikk , erk / and jnk / / and the phosphorylation of these protein kinases by tnf was reduced significantly by tnf ri ab, but not by tnf rii ab. moreover, we found that the changes of increased traf and decreased traf in ct cells (traf >traf ) resulted in a dramatic deficiency in phosphorylation of the above protein kinases induced by tnf compared with the normal ct cells (traf >traf ). nuclear localization of nf-b p in tnf treated cells was increased compared to untreated controls. conclusion: we have demonstrated for the first time that tnf stimulates mmp- production in ct cells through tnf ri-trafs-ikk /erk-nf-b signaling pathways, but not through the jnk/p -ap- pathway. these studies reveal steps within this pathway as possible therapeutic targets to inhibit mmp- expression potentially attenuating tnf -induced degradation of extracellular matrix and pre-term rupture of the fetal membranes. objective: women with preterm birth are at elevated risk of cardiovascular disease, but mechanisms that might relate these conditions are not understood. we hypothesized that women with spontaneous preterm vs. term births may have early gestation evidence of activation of the fibrinolytic cascade, as measured by the thrombin-antithrombin iii (tat) complex. we also tested if this relation may be associated with inflammation. methods: tat was measured in plasma collected < weeks gestation (mean . weeks, sd . ) among women without chronic medical conditions, preeclampsia or growth restriction who delivered singleton liveborn infants. inflammation was assessed by c-reactive protein (crp) measured in serum from the same samples. women with spontaneous preterm birth (sptb) < weeks (n= ) and -< weeks (n= ) were compared to women with term births >= weeks (n= ). high tat was defined as > . ng/ml (highest quartile among women with term births) and high crp was defined as >= ug/ml. multinomial logistic regression was utilized to relate elevated tat and inflammation to risk of sptb subtypes. results: women with sptb were more likely to have tat concentrations in the highest quartile compared to women with term births (< weeks, . %; -< weeks, . %; >= weeks . %, p< . ). women with high tat concentrations had a . -fold ( % ci: . - . ) increased risk for sptb < weeks, after adjustment for body mass index, race, age and gestational age at sampling. there was no relation between high tat and sptb -< weeks (or . , % ci . - . ). additional adjustment for elevated crp (>= ug/ ml) did not effect the estimates associated with tat, and elevated crp was independently related to risk for both sptb subtypes (< weeks, or . [ . - . ]; -< weeks, or . [ . - . ]). thus, women with high tat and elevated crp appeared to be at particularly elevated risk of sptb < weeks (or . , % ci . - . ). conclusions: the thrombin-antithrombin iii complex was elevated early in gestation among women with sptb < weeks, perhaps secondary to microvascular injury. this effect was independent of inflammation, suggesting that the elevated fibrinolytic cascade may function independently from inflammation among women with sptb. plasma cortico-releasing hormone and cortisol concentrations and psychological stress among pregnant women. katherine p himes, hyagriv n simhan. obstetrics and gynecology, university of pittsburgh medical center, magee womens hospital, pittsburgh, pa, usa. objective: many studies have found an association between psychological stress and preterm birth. we sought to determine if women with greater psychological stress during pregnancy had higher concentrations of plasma cortico-releasing hormone (crh) or cortisol. study design: this is a secondary analysis of a multicenter case-control study, nested within an observational cohort. of , participants, plasma crh and cortisol concentrations at and weeks gestation were available in controls who delivered after weeks and cases who delivered before weeks. the abbreviated scale for the assessment of psychosocial status in pregnancy (asaps) was available for all women. concentrations of crh and cortisol were compared between women above and below the lowest quartile score on the asaps among cases and controls. the same analysis was done for the portion of the scale related to psychological stress. concentrations of crh and cortisol and psychological stress were also compared between black and non-black cases and controls. univariate analysis was performed with kruskal wallis or chi-square. results: there was no difference in crh or cortisol concentrations at or weeks among women above or below the lowest quartile on the asaps (controls:p= . - . cases:p= . - . ). greater psychological stress was not associated with higher concentrations of crh or cortisol at or weeks(controls:p= . - . cases:p= . - . ). crh concentrations were not different between blacks and non-blacks. among both cases and controls, cortisol concentrations at and weeks were lower in black women than non-black women (controls:p< . cases:p< . ). the median cortisol concentration among control black women was . g/ml at weeks compared to . g/ml among non-black women and . g/ml compared to . g/ml at weeks. black women reported less psychological stress than non-black women (p= . ) conclusion: we found no relationship between psychological stress and plasma crh or cortisol. furthermore, while stress is hypothesized to play a role in the racial disparity of preterm birth, black women reported less psychological stress and had lower cortisol concentrations than non-black women. improved assessments of psychological stress and additional biomarkers involved in the stress response may broaden our understanding of how stress contributes to preterm birth. expression, tissular traffic and activation of mmp- in human fetal membranes during labor. rodrigo vega-sanchez, arturo flores, marisol castillo, nardhy gomez, felipe vadillo-ortega. direction of research, instituto nacional de perinatologia, mexico city, df, mexico. introduction. rupture of the fetal membranes (fm) during human labor occurs as a consequence of extracellular matrix degradation. this process is controlled by increased secretion and activity of matrix metalloproteinases, particularly mmp- .several evidences suggest that mmp- is mainly produced by infiltrating choriodecidual leukocytes that could arrive from placental circulation. characterization of the synthesis, transport and activation of mmp- within the fm is critical to understand the process of membrane rupture during human labor. objectives. expression and secretion of mmp- in placental leukocytes, trafficking of the enzyme through the fm and one possible mechanism for its activation were analyzed. methods. leukocytes were isolated from placental blood of women after active labor. maternal leukocytes were used as controls. cells were cultured for h. relative expression of mmp- by rt-pcr and enzyme secretion by elisa and zymography were followed at different times. to analyze the traffic of mmp- through the fm, fluorescein-conjugated prommp- was added to the choriodecidual side of the fm in an in vitro system that allows the separation of amnion and chorion. labeled mmp- was localized at distinct times by confocal microscopy. the protease responsible for the activation of mmp- was identified using neutralizing antibodies and specific inhibitors. results. no difference in the relative expression of mmp- in leukocytes throughout the culture was found. however, secretion of the enzyme significantly increased since h (p< . ). experiments using labeled mmp- , repeatedly showed that after h in culture, the enzyme was mainly localized within the amniotic epithelium. specific inhibition of mmp- significantly decreased the activation of pro-mmp- . conclusions. our results demonstrate that the increased secretion of mmp- by placental leukocytes is not associated to increased gene expression, suggesting that homing of a specific leukocyte subpopulation to the choriodecidua is occurring during labor. mmp- can be trafficked from the choriodecidua to the amnion, suggesting a transmembranal pathway that may regulate the tissular localization of the enzyme to the area of the fm with high content of connective tissue. once secreted by the placental leukocytes, activation of mmp- depends mainly on mmp- , which seems to be derived from the same leukocytes. objective: preterm labour is a major problem in terms of perinatal morbidity and mortality. the histone-deacetylase inhibitor (hdaci), trichostatin a (tsa) has been shown to have an inhibitory effect on myometrial contractility. the aim of this study was to evaluate the effect of the hdaci's suberic bishydroxymate (sbha) and valproic acid (vpa) on human uterine contractions and hence their potential role as tocolytic agents. methods: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of sbha in the concentration range of nmol/l to mmol/l and vpa ( nmol/l- mmol/l). control experiments were run simultaneously. results: sbha and vpa exerted a potent and cumulative inhibitory effect on spontaneous and oxytocin-induced contractions, compared to control strips. the mean maximal inhibition (mmi) values for sbha were . % for spontaneous contractions (n= ; p< . ), and . % for oxytocin-induced contractions (n= ; p< . ). the mmi values for vpa were . % for spontaneous contractions (n= ; p< . ), and . % for oxytocin-induced contractions (n= ; p< . ). conclusion: these results raise the possibility that hdaci's may have tocolytic potential, in addition to their current clinical indications. the inhibitory effect observed may be linked to the ability of hdac inhibitors to induce the expression of genes involved in the maintenance of myometrial quiescence via epigenetic mechanisms but may potentially also involve non-epigenetic pathways. progestin suppresses thrombin-enhanced interleukin- expression in term decidual cells: implications for abruption-induced preterm delivery. edward kuczynski, lynn f buchwalder, frederick schatz, charles j lockwood. obstetrics/gynecology reprod. sciences, yale university school of medicine, new haven, ct, usa. background and objective: decidual hemorrhage (abruption) promotes binding of factor vii to decidual cell (dc)-expressed tissue factor to generate thrombin. thrombin in turn induces several biological effects leading to preterm delivery via activation of cell surface protease-activated receptors (pars). interleukin- (il- ) is a pleiotropic proinflammatory cytokine induced by pars. this study assessed the separate and interactive effects of thrombin and medroxyprogesterone acetate (mpa) on il- expression in term dcs. methods: term decidua from stripped fetal membranes were isolated and the dcs were purified on a percoll gradient, grown to confluence and passaged until leukocyte-free. confluent dcs were primed in - m estradiol (e ) of e + - m mpa for days, then incubated in a defined medium (dm) with corresponding steroids ± thrombin. after hours, il- levels in conditioned dm were measured by elisa and western blotting. in parallel -hour incubations, il- mrna levels were assessed by quantitative rt-pcr and normalized to -actin mrna. results: secreted il- levels were similar in cultures maintained in e alone ( . ± . ) and e + mpa ( . ± . pg/ml/ug protein; mean ± sem; n = ). the addition of thrombin ( . u/ml) enhanced secreted il- levels by . ± . fold (p< . ) in incubations with e and by . ± . -fold (p< . ) in incubations with e + mpa. the inhibitory effect of mpa was statistically significant (p< . ). in confluent dcs incubated with e + mpa, exogenous thrombin ( . - . u/ml) elicited a concentration-dependent increase in secreted il- levels. hirudin acted as a pure thrombin antagonist, exerting no agonist effects alone, but counteracting thrombin-enhanced il- secretion. western blotting confirmed the elisa results. quantitative rt-pcr confirmed that il- m-rna levels corresponded to protein changes. conclusion: thrombin enhances il- mrna and protein expression in term dcs and progestin blunts these effects. since thrombin-generating abruption is closely associated with preterm delivery, anti-inflammatory effects induced by progestin inhibition of dc-derived il- may contribute to the protection against ptd, and may explain the reported protective effects of administration of -oh-progesterone in recent clinical trials. introduction: catechol-o-methyltransferase (comt) catalyzes the methylation of the phenolic hydroxyl groups in a variety of catechols. during estrogen metabolism, this enzyme converts the catechol estrogen, -hydroxyestrogen ( ohe ), to -ethoxyestrogen ( meohe ). the comt substrate, -ohe , can exhibit an anti-estrogenic effect in multiple biologic assays while the methoxyestrogen ( -meoe ) can exhibit an estrogenic effect. the biologic activities of these estrogen metabolites ( ohe meoe) depend upon their concentrations and tissue type. since comt activity ultimately controls levels of these metabolites, it appears to be a key factor in regulating the cellular estrogenic milieu. we have recently reported that amnion layers of human fetal membranes from laboring women exhibited folds higher comt mrna expression when compared to non-laboring women (wentz et. al. sgi ) . objective: to investigate the impact of comt inhibition on prostaglandin e (pge ) production by human amniotic membrane explants. study design: explants consisting of -cm circular sections of the amnion layer (obtained from term pregnant women who underwent elective repeat cesarean section) were prepared and placed in tissue culture explants media at ºc. after a -hour incubation, explants were treated with the selective comt inhibitor ro - , at and m concentrations. the incubation media was harvested after and -hour intervals. the levels of prostaglandin e (pge ) in the media were measured by sensitive elisa and were normalized against total protein concentration. results: ro - comt inhibitor induced major reductions in pge production in media collected from amnion explants of human fetal membranes. in the group treated with m of ro - for hours there was %± % reduction of pge after hours compared to untreated control (p< . ). in the amnion explants treated with m of ro - , there was %± % after hours and %± % after hours of treatment compared to untreated control (p< . ). conclusions: this finding indicates that comt activity in the amnion layer of human fetal membranes affects pge production. by facilitating a pro-estrogenic milieu in human fetal membranes in late gestation, increased comt activity may indirectly increases production of factors associated with labor such as pge . hypothesis: an extensive remodeling of the human cervical connective tissue takes place throughout pregnancy with a decrease in the total concentration of collagen and proteoglycans due to an altered higher metabolic turnover. we hypothesize that the profound changes in proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. we also hypothesize that proteoglycan production in cervical fibroblasts from preterm partal women are simmilar to the production in fibroblasts from partal women. method: cervical biopsies were obtained from non-pregnant women, women during elective cesarean section, woman after spontaneous parturition and after a preterm vaginal delivery. by explant technique fibroblasts were cultured from the biopsies. produced proteoglycans were metabolically labeled with s during hours and then purified by ion-exchange chromatography and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. results: the total proteoglycan production decreases with approximately % in partal and preterm partal cell cultures. the reduction of proteoglycans in preterm partal and partal cell cultures is significant compared to non pregnant fibroblast cultures. the distribution of biglycan and perlecan are similar in partal and preterm partal cells. biglycan are significantly reduced by around % and perlecan are significantly increased by around % compared to non pregnant cultures. preterm partal cervical fibroblasts secreates significantly more heparan sulfate proteoglycans compared to non pregnant cultures. the changes in total proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. both partal and preterm partal cell cultures differ in their proteoglycan production compared to their non pregnant counterpart, suggesting a role for proteoglycans in cervical ripening. the role of the oxytocin/oxytocin receptor system is not well defined in human amnion. previous studies in rabbit amnion have demonstrated an up-regulation of oxytocin receptors in the end of pregnancy and have shown that there is a large increase in the ability of ot to stimulate pge production. we and others have previously shown a role for nf-b in otr regulation. in whole genome array analysis of human amnion we found that otr was the gene with the second highest increase associated with activation of nf-b (the highest being cox- ). the present work was directed towards further understanding of otr expression and function in human amnion at term. we have shown that pge release by pre-labour primary human amnion cells is significantly increased after oxytocin treatment for hrs (ot: - m; n= ; triplicate samples; fold increase p< . ). the expression of otr in labour (+) and labour (-) primary amnion cells was measured with real time rt-pcr. we found a significantly higher level of expression in the labour (+) cells (n= ; duplicate samples; p< . ). western blot analysis confirmed the upregulation of otr in labouring amnion. treatment with il- b resulted in a significant upregulation of otr which peaked with a fold induction after hours (p< . ). il- caused a fold increase in otr mrna levels in labour (-) cells, bringing the expression level up to that found in labour (+) cells. our findings provide further evidence for a role of otr in human amnion. expression of otr in human amnion is significantly increased after the onset of labour. term non laboured amnion can be stimulated with il- to increase otr expression to levels of laboured amnion. one of the functions of otr in amnion cells is stimulation of prostaglandin synthesis. expression of antioxidant defence proteins in human myometrium before and after the onset of labour. vasso terzidou, mandeep s kandola, shirin khanjani, jan j brosens, phillip r bennett. parturition reserach group, irdb, imperial college, london, united kingdom. background oxidative stress is a result of an imbalance between the production of reactive oxygen species (ros) and the antioxidant defence mechanisms present in biological systems. parturition and infection-induced preterm labour resemble inflammatory processes that are linked to the production of ros including super-oxide (o -), hydrogen peroxide (h o ) and peroxynitrite. low concentrations of ros can act as second messengers in the regulation of several cellular functions. in an attempt to maintain redox homeostasis cells are equipped with machineries to both produce and scavenge ros. enzymatic scavengers include superoxide dismutase (sod), glutathione peroxidise and catalase. sod is the first enzymatic step in the defence system against oxidative stress. nuclear factor-kappa b (nf-b), a transcription factor family classically associated with inflammation, is activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. labour is associated with an increase in nf-b activity in human myometrium and in fetal membranes. nf-b is known to regulate a range of genes associated with the onset of labour. in a study using affymetrix whole genome arrays analysis nf-b overexpression was associated with increased expression of sod- ( . fold). to determine changes in ros scavenging potential upon onset of labour, lower segment myometrial biopsies were taken at term before and after the onset of labour (n= ; each group). cdna was extracted from the tissues and real time rt-pcr was performed for sod- , serum/glucocorticoidinduced protein kinase- (sgk- ) and the dna repair enzyme gadd a. we found that labour onset is associated with a two fold increase in the levels of expression of each. oxidative damage at the fetomaternal interphase has been extensively studied in the placenta as part of investigations for first trimester pregnancy losses, iugr and pre-eclampsia. the role of ros is less well defined in human decidua and the underlying myometrium. our results suggest a role for oxidative stress and redox homeostasis in the maintenance of myometrial quiescene during pregnancy and onset of labour. plasma anandamide levels increase during labour induction and appear to delay labour progression. vijianitha nallendran, anthony h taylor, patricia mw lam, stephen c bell, david j taylor, justin c konje. cancer studies and molecular medicine, university of leicester, leicester, united kingdom. background: the evidence for a role of the endocannabinoid, anandamide (aea) in labour is conflicting. we previously showed elevated aea plasma levels in labouring women whilst another group showed that activation of functional receptors for anandamide actually inhibited uterotonin-induced contractions through an adenylate cyclase pathway . the aim of this study was to explore further the relationship between labour and plasma aea levels. methods: plasma aea levels in women undergoing induction of labour for various indications at term, were measured by a sensitive isotope dilution method using hplc-ms/ms. each volunteer had an assessment of her cervix prior to the start of the induction when the first sample for aea was collected. once active labour was established (cervix cm dilated; contractions every - minutes), a second sample was collected. results: seventeen ( %) of the subjects were multigravida. the inductions were for postdates(n= ); decreased fetal movements(n= ), fetal growth restriction(n= ), symphysis pubis dysfunction(n= ), diabetes mellitus(n= ), pre-eclampsia(n= ), fetal cardiac complication(n= ), spontaneous rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes(n= ). plasma aea levels increased significantly once labour was established (figure) and demonstrated in ( %) of the cases. the median (interquartile range) plasma aea level increased from . nm ( . - . ) at induction to . nm ( . - . ); *p= . ; mann-whitney u-test) at active labour. there was a positive correlation between plasma anandamide levels taken before induction and the time taken for the women to enter into active labour (r = . , p= . pearson correlation). plasma aea levels were higher in labouring women compared to non-labouring women after the induction of labour confirming our previous observations and suggesting a direct role for this endocannabinoid in labour. further studies are required to elucidate this role. nuclear factor-kappa b (nf-b) is a transcription factor family classically associated with inflammation, activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. as a cytokineinducible transcription factor it plays a key role in the expression of a variety of genes involved in inflammatory responses and cell survival. nf-b dna binding and transcriptional activity plays an important role in labour associated gene expression in myometrium. in this study we examined the range of genes regulated by nf-b in myometrium using transient transfections and wholegenome array analysis. myometrial cells were extracted from myometrial biopsies taken at the time of elective caesarean sections at term. transient transfections of primary myocytes were performed using expression vectors for nf-b p .the amount of dna was constant and psg was used as a control construct. cdna was made from myocytes transfected with either nf-b or psg . affymetrix genechip u microarray was performed (n= , each group). we found that genes were significantly differentially expressed between control and overexpressed nf-b samples. twenty eight of these genes were upregulated with nf-b and were down regulated. several chemokines and cytokines were identified in the upregulated group. interleukin demonstrated the highest, fold, induction in the upregulated group, followed by tumor necrosis factor, a-induced protein (tnfaip ), chemokine ligand (ccl ), chemokine ligand (ccl ), pentaxin related gene (ptx ), interleukin (il ) and superoxide dismutase (sod- ). nine genes were present in the nf-b group that were absent in the control group. these included chemokine ligand (ccl ), chemokine ligand (ccl ), chemokine ligand (cxcl ) and il . ingenuity pathway analysis demonstrated that immune response, inflammatory, cell growth and proliferation and cell death were the main pathways involved. standardisation experiments have been performed, and the microarray results were confirmed with real time rt-pcr in several candidate genes. our results provide further support in the role of nf-b in human labour and suggest its direct link in upregulation of inflammatory genes, cytokines and chemokines, consistent with the imflammatory nature of the biochemistry of labour. inflammation is widely accepted to be a key feature of human labor. secretory leukocyte protease inhibitor (slpi), an innate immune molecule, has been shown to be an antimicrobial and anti-inflammatory protein. the aims of this study were to verify its expression and localization in human myometrium methods: specimens were obtained at time of cesarean delivery with or without labor. expression and localization of slpi was detected using immunohistochemistry. slpi expression pattern relative to nf-b p subunit was compared between not in labor and in labor subjects, between different tissue sections as well as in in vitro model systems including myometrial explants, uterine smooth muscle cells (usmc) and ishikawa endometrial adenocarcinoma cells. slpi was predominantly localized to the nuclei of myocytes. the observed nuclear immunoreactivity of myocytes was increased during the labor relative to not in labor, paralleled with p nuclear translocation. the nuclear pattern of slpi is specific to myometrium since slpi immunostaining was present exclusively in the cytoplasm of all other tissues examined, including amnion, chorion, decidua and endometrium. slpi staining was also positive in macrophages, indicated by co-localization of slpi with cd positive cells. treatment with il- or tnf-induced nuclear translocation of p in myometrium explants and usmc, but not in ishikawa cells. in human myometrium, slpi is predominantly localized in the nuclei of myocytes and in macrophages. the nuclear expression pattern of slpi is myometrium-specific and increased following the onset of labor and correlated with nf-b activation. further understanding of its physiological significance may suggest new strategies aimed at preventing preterm birth. application of a new proteomic technology on amniotic fluid in prom. sara consonni, niccolo bosso, marianna andreani, agnese pizzardi, fulvio magni, anna locatelli. obstetrics and gynaecology, university of milano-bicocca, monza, italy; experimental medicine, university of milano-bicocca, monza, italy. objective: mass spectrometry (ms) is the obligatory tool for proteomics studies. biological samples must be purified before ms analysis due to the matrix complexity. a recent approach combines active surface prepurification with maldi-tof (matrix assisted laser desorption) analysis. clinprot technology provides the prepurification of the sample through the use of magnetic beads (mb) with activated surface. this technique can be carried out by robot in an automated way on a large number of samples. a unique example of the use of mb before maldi-tof analysis to determine proteomic profiles of amniotic fluid (af) is reported for rapid detection of fetal aneuploidies (wang ) . the objective of our study was to verify the applicability of clinprot prepurification before maldi-tof analysis on amniotic fluid collected noninvasively in premature rupture of membranes (prom). methods: we sampled af from vaginal posterior fornix of women with preterm prom (group , n= ) and term prom (group , n= ). samples were prepared with mb and analyzed with ms maldi-tof in order to generate proteomic profiles. results: it was possible to generate average proteomic profiles in the two study groups and the observed profiles were different. samples of af non invasively collected in prom can be analyzed by ms maldi-tof after preparation with mb. this technique allows to retain part of the eluted sample for characterization of protein peaks of interest. due to the less laborious characteristics of this method in comparison with techniques based on bidimensional electrophoresis its application can be useful in clinical proteomics. progestins accentuate the maternal but not fetal inflammatory response of women with intra-amniotic inflammation. sonya abdel-razeq, irina a buhimschi, michael cackovic, guoyang luo, antonette dulay, victor rosenberg, mert bahtiyar, errol norwitz, edmund funai, catalin buhimschi. ob./gyn. reprod.sci., yale university, new haven, ct, usa. introduction: data from animal research suggests that progestins have a marked pro-inflammatory capacity. recent studies support the administration of -hydroxyprogesterone caproate in women at risk for preterm birth. we sought to determine the impact of progestins during gestation on the extent of maternal and fetal inflammatory responses in pregnant women with intraamniotic inflammation. methods: amniotic fluid, placenta and cord blood were obtained from women who delivered preterm (median[range], ga: [ - ] wks). an amniocentesis was done to rule out infection. women exposed to progestins (n= ) within one week prior to amniocentesis were matched to controls (crl) by age, parity, history of preterm birth ga, membrane status and interval to delivery. proteomic profiling of amniotic fluid [mass restricted (mr) score] identified the presence or absence of intra-amniotic inflammation. an mr score of or confirmed intra-amniotic inflammation. amniotic fluid and umbilical cord interleukin- (il- ) levels were measured by elisa. histological chorioamnionitis was graded based on recognized criteria. results: overall, women and their fetuses exposed to progestin did not exhibit an increased inflammatory response (table) . however, sub-analysis restricted to women with mr or (n= ) showed that in the context of intraamniotic inflammation, progestins were associated with significantly elevated amniotic fluid il- levels compared to unexposed women (progestins (n= ): vs. crl (n= ): [ . - ] ng/ml, p= . ). these relationships were maintained after correction for steroid and antibiotic exposure. such significance was not found for amniotic fluid glucose, ldh, wbc count or cord blood il- . conclusion: our results suggest that progestins may amplify the maternal, but not the fetal inflammatory response of women with intraamniotic inflammation. objective: recently progestins have been shown to reduce the incidence of recurrent preterm labor in women. progestins have long been known to inhibit preterm labor in some species including rats and are also known to delay term labor. nitric oxide (no) donors, including ng, inhibit uterine contractility and have been used as tocolytics. the aim of this study was to examine the inhibitory effects of r on preterm labor in rats induced with a progesterone antagonist (onapristone, zk) with and without ng. materials and methods: charles river s-d timed pregnant rats (n= /group) were treated with zk ( mg/rat, s.c.) alone or vehicle (controls) on day of gestation. other groups of rats were treated with zk in combination with various doses of r ( . , or mg/rat s.c) with and without ng ( mg s.c. pellet) from days to of gestation. all rats were sacrificed on day of gestation and the number of fetuses and implantation sites were counted to determine the preterm delivery rate. one way anova was used for statistical analysis. p< . was considered significantly different. results: rats treated with zk alone delivered all their fetuses prematurely compared with controls (p< . ) treated with vehicle only (ca. % fetuses delivered). ng treatment alone did not affect the delivery rate (p> . ) compared to controls. similarly zk + ng did not reduce the preterm delivery rate compared to zk alone (p> . ). however r dose dependently reduced (p< . at all doses) the number of fetuses delivered prematurely in response to zk and the premature delivery rate was further reduced when treatment included the combination of r plus ng (p< . ). conclusions: zk effectively induces premature delivery. premature delivery produced by zk can be effectively reduced with a r , a progestin known to bind with high affinity to nuclear progesterone receptors. ng by itself, at the dosage used, does not reduce the prematurity rate caused by zk. however, r and ng act synergistically to reduce the preterm delivery rate. this study indicates that combinations of a progestin with an no donor may be an effective treatment for preterm labor and delivery. monkeys. pl grigsby, jp rasanen, , dw sadowsky, m bertolino, m carbonatto, e gillio tos, s canali, j lacy, a chollet, mj novy. reprod sci, oregon primate res ctr, beaverton, or; ob/gyn, oregon health sci univ, or, usa; rbm, merck serono, italy; merck serono, switzerland. objective: to investigate the pharmacokinetics (pk) and pharmacodynamics (pd) of as , a novel orally active non-peptide oxytocin (ot) antagonist in rhesus monkeys. study design: dose finding and pk/pd studies were done in chronically instrumented non-pregnant (n= ) and pregnant ( - dga, term d; n= ) monkeys. maternal and amniotic fluid compartments were serially sampled during dosing and washout. treatment phases included: ot infusion control, ot infusion + as , and ot rescue therapy. ot infusions ( - mu/kg/hr, iv) were given to determine the lowest dose required to produce stable, sub-maximal uterine contractions in each animal. as was administered p.o. at , , mg/kg and the effects on inhibition of uterine activity (hca, mmhg.sec/hr) were compared. to verify antagonist reversibility, objectives: infection such as chorioamnionitis is thought to be the cause of premature rupture of the membrane and induce uterine contractions leading to preterm delivery. however, the mechanism for enhancement of uterine contractility is not well understood. we have reported that non-selective cation channels (nsccs) regulate pacemaker potentials to generate rhythmical contractions and should be targets of magnesium ions used for tocolysis. the purpose of this study is to investigate the changes of the expression of nsccs during normal pregnancy and the effect of inflammation in preterm. methods; atp receptors (p x) and transient receptor potential canonical (trpc) channels were examined as uterine nsccs. the mrna was extracted from the rat myometrium of non-pregnant and pregnant rats at days , , and . the expression of each subtype of p x and trpc channels was measured by real time rt-pcr (abi ) with taqman probes (abi). as an inflammatory model lipopolysaccharide (lps; . mg/kg) was injected into intraperitoneal cavity at day and the tissue was sampled after six hours. results; p x and p x were determined to be dominant subtypes of p x channel. the expression of p x was increased by % at day , compared with day and that of p x was enhanced by %. on the other hands, trpc and trpc were detected dominantly. the expression of trpc was increased three times in the late stages of gestation. however, trpc was suppressed by %. in the lps treated rat myometrium cox- mrna expression was measured to be fold higher than that of the control rat, showing inflammatory effects in the myometrium. in this model the expressions of p x , p x and trpc were enhanced by . , . and . times, but trpc was not changed. the mrna expression of p x , p x and trpc channels in rat myometrium was increased in the late stages of pregnancy. these channels are suggested to be concerned with onset of labor. in the inflammatory model the expression of these channels was accelerated dramatically and these values were much higher than those in normal pregnancy. this finding supposed inflammation may enhance some types of nsccs to accelerate uterine contractility and induce preterm delivery. anandamide ( to determine the mechanism of rho protein activation during oxytocin and carbachol induced contraction, freshly prepared myometrial strips in krebs henseleit buffer were treated with oxytocin ( nm) and carbachol ( μm) under isometric and tension free conditions. control strips were exposed to buffer only. treated myometrial strips were solubilised and separated into membrane and cytosolic extracts and equal aliquots were immunoblotted with rhoa and rhob antibodies. rhoa translocated to the membrane after oxytocin and carbachol stimulation under both isometric and tension free conditions (p< . ). there were no significant changes in rhob membrane to cytosol ratios relative to control. agonist induced contraction in human myometrium is associated with rhoa but not rhob membrane translocation. during pregnancy components of the intracellular camp signalling pathway show increased gene expression resulting in the maintenance of myometrial quiescence until term where a substantial decrease in expression of these genes is observed. protein kinase a regulatory subunit riia (rii ) is upregulated at both the mrna and protein levels in the human myometrium during pregnancy. this particular subunit is membrane-bound and by directing phosphorylation to myometrial cytoskeletal proteins may affect contractile machinery thus playing a role in maintaining uterine relaxation. acetylation of histones promotes a favourable chromatin environment for transcriptional activity of many genes. this process is largely inhibited by histone deacetylases (hdacs), whereby its activity leads to transcriptional repression. hdacs can be recruited to the promoter region of a gene by other transcription factors such as sp proteins. since the rii promoter contains three sp - consensus binding sequences in its proximal part, we investigated whether this gene is a target for transcriptional regulation by hdacs. using dna precipitation assays we found that sp , and as well as hdac and form complexes with biotin-labelled fragments relating to sp - elements in the promoter region of rii . additionally, treatment of myometrial primary cell cultures with hdac inhibitor trichostatin a (tsa), or with the methyltransferase inhibitor -azac resulted in increased mrna and proteins levels. further studies with full and truncated luciferase constructs of the promoter region of the rii gene in transiently transfected myometrial cells confirmed that all three sp - elements are involved in the transcriptional regulation of the gene. this process involves hdacs, as h treatment with tsa significantly increased the luciferase signal. changes in the binding of sp proteins and hdacs to the promoter after tsa and azac treatment were investigated employing chromatin immunoprecipitation assays. alterations in the methylation status of the promoter after treatment were examined by bisulfite modification and dna sequencing. together, this study highlights the importance of chromatin modifications in the maintenance of uterine quiescence during pregnancy as well as identifying a potential mechanistic target for drugs that may reduce the incidence of preterm labour. objective: progesterone maintains pregnancy by promoting myometrial quiescence. typically progesterone effects are thought to be mediated through the classic genomic pathway. there is evidence, however, that progesterone also acts via a non-genomic pathway by interacting with specific membrane progesterone receptors (mprs) and in particular progesterone receptor membrane component - (pgrmc- ). the role of non-genomic progesterone actions in human pregnancy and parturition is not clearly understood. the goal of this study was to measure the extent of mpr expression in biopsy specimens of human myometrium obtained at cesarean delivery, and to determine whether expression changes with advancing gestation or the onset of labor. study design: lower uterine segment myometrial biopsies were obtained at the time of delivery from consenting women who were at term and not in labor (n= ), preterm and not in labor (n= ) and term and in labor (n= ). protein extracts were prepared and subjected to polyacrylamide gel electrophoresis and immunoblot analyses for pgrmc- and gapdh. abundance of pgrmc- protein relative to gapdh was determined by digital densitometry. we also performed immunohistochemistry (ihc) to determine the cellular localization of pgrmc- in the human pregnancy myometrium. results: pgrmc- protein was identified in each biopsy specimen. there was a -fold increase in pgrmc- protein in term compared with preterm biopsies (relative to gapdh p< . ). the relative level of pgrmc- protein was not different between biopsy specimens from laboring and non-laboring women at term (compared to gapdh, p= . ). pgrmc- immunoreactivity was localized to granular cytoplasmic staining. conclusion: this is the first description of the presence of pgrmc- protein in the human myometrium during pregnancy. its presence suggests that progesterone may influence contractility non-genomically via these receptors. the functional significance of the gestational age associated two fold increase in pgrmc- is unclear. changes in expression of this receptor during pregnancy may be important for the hormonal control of parturition and can be the focus of future studies. (ruddock et. al., abstract smfm, ) . the aim was to examine the mechanism of p inhibition. methods: uterine tissues from women (n= ) at term with cesarean section, were suspended in organ chambers and exposed to various agents or solvents. contractility was registered, stored, analyzed and compared before and after addition of agents or kcl. tissues were treated with p alone ( - to - m) or p bound to bovine serum albumin (bsa/p , - to - m p ), a progestin with low affinity to mpr (r , - - - m), or a non-sex steroid (cholesterol, - to - m). other tissues were pretreated with selective inhibitors of adenylate cyclase (sq , - m), guanylate cylase (odq, - m), phosphodiesterase (rolipram, - m), nitric oxide (no) synthases (l-name, - m) or a nuclear p receptor antagonist (mifepristone, mif, - m), followed by p . data were analyzed by anova for statistical differences (p< . ). results: p rapidly (< hour), effectively ( %) and dose-dependently inhibits spontaneous and kclinduced contractility (ed of < - m incubation of strips with zd- , at concentrations up to m for one hour prior to the experiment, led to no significant reduction in the total work done. however, incubation of strips with l- with a concentration of nm for one hour prior to the experiment, led to a statistically significant reduction in the total work done after one and a half hours. furthermore, when increasing concentrations of pge ( - to - ) were added to l- ( nm) pre-treated strips, total work done per contraction was comparable to that of non-treated controls. similar effects were not observed in zd- ( nm) pre-treated strips. since the reduction in contractility caused by l- was greater than that caused by zd- , it is likely that the stimulating effect of pge acts predominantly via ep receptors. taken together with our previous data showing an increase in ep at labour, this data shows that targetting an ep receptor may be a useful strategy in managaing pre-term labour. recently a truncated kda isoform of the er-has been described in human endothelial and testicular cells. we describe the presence of this kda erisoform in pregnant and immortalized non-pregnant human myometrial cells. methods: myometrial tissue obtained from non-laboring pregnant women undergoing cesarean section at term (n= ) was dissected prior to being finely minced. a portion of the tissue was placed in pbs and sonicated, while the remainder of the tissue was dissociated with collagenase prior to filtration and placement on culture plates. cells were then cultured in mem w/ % fetal bovine serum (fbs) until confluence. cultured cells were then dissociated with . % typsin/edta, subsequently re-plated and grown to confluence. immunohistochemistry directed toward smooth muscle protein was used to verify myometrial phenotype.) protein was extracted and quantified. western blot was performed with mouse monoclonal anti-er-receptor antibody (santa cruz biotechnology) to the f- domain of the human er-receptor. an immortalized non-pregnant human myometrial cell line provided by ann word, htert, was cultured and isolated as described above. results: htert cells expressed both the truncated ( kda) and the full length ( kda) er-isoforms. fresh and cultured myometrial cells from non-labored term pregnant patients also expressed both isoforms of er-. subsequent subcultures of myometrial tissue continued to express the kda erisoform, yet the expression of the kda isoform was lost. a representative blot is below. conclusions: we demonstrate, for the first time, the presence of a kda erisoform in cultured and non-cultured pregnant and cultured nonpregnant human myometrial tissue. discovery of this isoform of er-in myometrial tissue could provide insight into the molecular mechanisms involved in parturition. the action of prostaglandin f (pgf ), a potent uterotonic stimulant that is associated with labour at term and preterm, is mediated by its receptor, ptgfr. myometrial ptgfr mrna levels fall during pregnancy and this likely plays a role in uterine quiescence. however, the mechanisms by which this occurs are poorly understood. we previously reported that pgf downregulates fp mrna expression in cultured human myometrial ultr cells in a protein kinase c (pkc) dependent manner. in addition to the downregulation of mrna levels, receptor desensitization may also represent another mechanism of decreasing ptgfr activity because ligand binding to g protein coupled receptors often results in receptor internalization. we therefore hypothesized that pgf treatment of ultr cells also results in pkc dependent ptgfr internalization. methods: near confluent cultured human myometrial ultr cells were treated +/- - m or - m pgf for , , or hr. cells were fixed with formaldehyde and visualized for localization of ptgfr by immunofluorescence. to examine the potential involvement of pkc in the process, ultr cells were treated +/- - m pgf and +/- m myristoylated pkc inhibitor ( - ) and examined for ptgfr cellular localization as described above. results: pgf treatment resulted in a dose dependent decrease in ptgfr membrane signal at , , and h. this decrease was dependent on pkc as cotreatment with the myristoylated pkc inhibitor ( - ) prevented the pgf induced decrease in membrane ptgfr at h treatment. there was no visible effect of the pkc inhibitor on ptgfr membrane signal on its own. conclusion: we conclude that pgf decreases membrane levels of ptgfr protein in human myometrial ultr cells in a pkc dependent manner. these results suggest that pkc may be required for both the pgf induced internalization and desensitization of ptgfr protein and downregulation of ptgfr mrna in human myometrial ultr cells. therefore pkc may play a crucial role in downregulating ptgfr expression and activity and maintaining uterine quiescence during pregnancy. rhoa is a small gtpase that acts as a molecular switch to control a variety of signalling pathways in smooth muscle, including contractility. it is thought that increases in rhoa-gtp levels facilitates phosphorylation of target proteins such as cpi- , promoting contractility at pre-term and term labour in humans. however, in situations of acute or chronic hypoxia in the uterus, it is important that myometrial contractility and subsequent labour is not facilitated prematurely. given the importance of rhoa to a cell's response to hypoxia in other cell types, it was hypothesised that rhoa plays a central role in the mechanism controlling smooth muscle contraction in the uterus too. following acute hypoxia ( . % o ) for one to six hours, rhoa mrna, total protein and activation (rhoa-gtp) levels were analysed, using semi-quantitative pcrs and western blot, and compared to normoxic non-pregnant human uterine smooth muscle control cells. next, we investigated whether reduced oxygen conditions affected oxytocin induced activation of rhoa, following a two hour treatment of nm oxytocin. firstly, our results demonstrate that the rhoa itself is significantly activated under low oxygen conditions, resulting in phosphorylation of myosin phosphatase, myosin light chain and cofilin, three proteins known to be central in contraction and actin filament organisation. secondly, hypoxia significantly reduced the coupling of oxytocin to rhoa activation under the conditions examined. we observed a significantly reduced level of rhoa expression and activation which correlated with an increase in the level of another rhogtpase protein, rhoe. we propose that rhoa inactivation occurs through a rhoe-mediated mechanism, suggesting a balance in the activity of these two antagonistic rhogtpases in oxytocin-induced hypoxic human uterine smooth muscle cells. these results provide a possible explanation for the reduced coupling of oxytocin as a stimulant of myometrial contractions during slowly progressing labours. oxytocin-induced release of calcium ions (ca + ) from sarcoplasmic reticulum (sr) and sensitisation of contractile proteins to ca + have been suggested to mediate the oxytocin-induced potentiation of myometrial contractions. objective: we investigated the effects of oxytocin in the presence of nifedipine, a known inhibitor of the l-type calcium channel (ltcc). method: samples of myometrium were obtained from women undergoing term caesarean section with the approval of the local ethics committee. a standard organ bath system (ad instruments, uk) was employed to analyse contractile activity. stable spontaneous contractions were recorded for - minutes before addition of nifedipine. results: in agreement with our previous findings, application of oxytocin to spontaneously active strips produced a two-component effect: a transient tetanus-like contraction, followed by prolonged augmentation of phasic contractions. nifedipine ( um) rapidly abolishes spontaneous contractions, subsequent addition of nm oxytocin produced an initial, transient rise in force, approxiamately % compared to oxytocin alone, followed by high frequency oscillations in > % of strips. calcium-free solutions were used to confirm that oscillations were due to ca + entry. disabling the sr store using thapsigargin ( um) had no effect on oscillations, confirming the sr not to be involved. the t-type calcium channel blocker, mibefradil ( um ) showed no inhibition of oscillations. an ip receptor and store-operated calcium channel inhibitor, -aminoethyldiphenylborate ( -apb) um also had no effect on oxytocin-induced oscillations. the store-operated calcium channel inhibitor skf- ( um) showed partial inhibition of oscillations. conclusions: based on these results, we propose that the most likely mechanism of ca + entry producing oxytocin-induced oscillations in the presence of nifedipine is the transient receptor channel-c (trpc) channel, known to be present in the human myometrium. further work needs to be completed to clarify this further. identification of stim and orai in human myometrium. a new paradigm in store-operated calcium signaling. evonne c chin-smith, mark r johnson, rachel m tribe. division of reproduction and endocrinology, king's college london, london, united kingdom; department of maternal fetal medicine, imperial college school of medicine, chelsea and wesminster hospital, london, united kingdom. background: recent reports have suggested that two novel proteins, stim and orai, are involved in the regulation of store-operated calcium entry. we have previously reported that members of the trpc family, putative basal and store operated calcium entry channels, are present in human myometrium and regulated by labour associated stimuli il- beta and mechanical stretch. although stim and orai isoforms have been reported in other smooth muscle cell types, there are no published reports of stim and orai expression in human myometrium. the aim of this study was to identify mrna expression of stim , stim , orai and orai in human myometrium. methods: human myometrial biopsies were obtained from women undergoing elective caesarean section at term (prior to labour) with informed written consent and institutional ethics committee approval. whole myometrial tissue was either snap frozen and stored at - o c or used for cell culture. rna was extracted from whole tissue (n= ), primary cultured myometrial cells (n= ) and passaged (p ) myometrial cells (n= ) and stim , stim , orai and orai mrna expression was assesed by quantitative real-time pcr. results: all four genes were expressed in whole myometrial tissue and cells. stim and stim mrna expression in cultured myometrial smooth muscle cells (primary and passaged) was significantly reduced compared to myometrial tissue expression (p< . ). however, there was no significant difference in either orai or orai expression in whole tissue versus cultured myometrial smooth muscle cells. conclusion: to our knowledge this is the first report of stim / and orai / mrna expression in human myometrium. these genes may contribute to the regulation of calcium signalling in human myometrium, but the functional significance of their expression remains to be determined. funded by the bbsrc. obstetrics and gynecology, national university of ireland, galway, galway, ireland. objective: the ability of uterine smooth muscle cells to stimulate collagen contraction has been well established as an in vitro model of myometrial contractility. devost and zingg ( ) reported that the contractility of human myometrial cell lines layered onto collagen matrices was increased by oxytocin while another group described the stimulation of human uterine smooth muscle cell contractility, cultured within collagen lattices, with endothelin- (dallot et al., ) . our study investigated the response of human primary uterine smooth muscle cells cultured within collagen lattices, to various compounds, including the non-specific depolarizing agent potassium chloride (kcl), the inflammatory cytokine tnf , the rock- inhibitor y- , oxytocin, and oxytocin plus its clinically used antagonist, atosiban. methods: human primary uterine smooth muscle cells (utsmc) (lonza) were maintained in dmem high glucose media. cells ( , per well) passage - , were embedded in . mg/ml collagen, in . ml aliquots, in dmem-f media on well plates (invitrogen) (dallot et al., ) . effects on contraction were studied by monitoring changes in gel area (alpha innotech imager, image j software (nih)). statistical significance was determined by the student t test. results: the utsmcs displayed basal contraction of the collagen gels while the non-contractile cell line hek cells, did not. kcl ( nm) stimulated an % increase in contractility (n= , p= . ) while nm tnf resulted in an . % increase (n= , p= . ), in comparison to unstimulated cells embedded in gel. the rock inhibitor y- ( m) inhibited contractility, with a . % decrease in collagen gel contractility (n= , p= . ). a % increase (n= , p= . ) in utsmc embedded collagen contractility was observed with nm oxytocin, which was antagonized by atosiban ( m) (n= ). conclusion: this study highlights the importance of the development and optimisation of a reproducible human in vitro myometrial contractility model, to evaluate the effect of various known labor-associated, and also unknown compounds. this should aid in our understanding of the many complex biochemical pathways involved in myometrial contractility at labor, and ultimately contribute to the prevention of preterm labor. changes in intra-mural myometrial blood flow during spontaneous labour using d power doppler angiography (pda). nw jones, , nj raine-fenning, h mousa, mj taggart, k jayaprakasan, gj bugg. nottingham university hospitals nhs trust, united kingdom; nottingham university, united kingdom; university of newcastle upon tyne, united kingdom. methods d pda was used to measure the percentage (%) change in the vascularization index (vi), the flow index (fi) and the vascularization flow index (vfi) in a volume of myometrium at the uterine fundus of nulliparous women at term, in the first stage of uncomplicated spontaneous labour. d data sets were obtained during a single cycle of uterine relaxation (r ), contraction and subsequent relaxation (r ). measurements were made independantly by two authors (nwj and gjb) using vocal® (ge kretz) and the mean value was used for analysis. the results from each woman are presented as a % change of r . data is presented as medians [interquartile range (iqr)] and analysed using non-parametric tests. the median volume (cm ) of interest for r was . cm ( . - . cm ), for the contraction was . (fig. ) . the % change for all three indices between r and r was not significantly different. however, there was a significant difference between the contraction and r (p< . ). the mean intra-class correlation coefficient and % confidence interval (ci) of vi, fi and vfi for the authors (nwj and gjb) were . ( . - . ), . ( . - . ) and . ( . - . ), indicative of good inter-observer reliability. conclusion d pda is a useful and reliable tool in the assessment of changes in intramural myometrial blood flow, which was found to reduce significantly during a contraction but increase again during the following uterine relaxation. . raine-fenning nj, et al. the inter-observer reliability of d pda acquisition within the female pelvis. ultrasound obstet gynecol. ; : - . the role of pgf on myometrial contractility; studied with a selective fp antagonist. shankari arulkumaran, andre chollet, phillip r bennet. imperial college parturition research group, institute of reproductive and developmental biology, hammersmith hospital campus, london, united kingdom; merck serono, geneva, switzerland. objectives: human myometrial strips established in culture will usually begin contracting after one hour. we have previously shown that stretch upregulates prostaglandin synthesis and have therefore hypothesized that spontaneous contractions occur because of stretch-related prostaglandin synthesis. methods: experiments were performed using x mm human pre-labour, lower uterine segment myometrial strips in a dmt myograph ms in oxygenated kreb's solution, with adi powerlab software. spontaneous contractions required stretch force and initial experiments determined that the maximum number of strips attaining spontaneous contractions was greatest at a force of - g. a novel antagonist to pgf (fpa) was studied in this model. the fpa has a ki of nm for fp and is - fold selective for fp compared with other prostanoid receptors. results: addition of pge and pgf after the commencement of spontaneous contractions caused a statistically significant increase in the total work done by the strips. the effect of pge was being greater than that of pgf . pre-incubation of the baths with a novel and selective fp antagonist (fpa) with concentrations up to m ( - ) did not affect the total work done by spontaneous contractions compared to non-treated controls. however, increasing concentrations of the fpa ( - to - ) decreased the total work done by -fold on strips treated with pgf beforehand in comparison the pgf -treated strips alone. conclusion: these data suggest that stretch and synthesis of prostaglandins is essential for spontaneous contractility in human myometrial strips. since fpa is able to block pgf induced but not spontaneous contractions, it is likely that pgf does not play a role in spontaneous myometrial contractility in vitro, although it may do so in vivo. because other factors may combine to increase contractility, the combination of an fp antagonist with other inhibitors may therefore, be a more effective strategy in reducing pre-term deliveries. objectives: brain natriuretic peptide (bnp) is synthesized in fetal membranes and inhibits oxytocin-induced contraction of preterm human myometrium. we hypothesized that bnp may be a paracrine mediator of human myometrial quiescence. we showed bnp content is higher in membranes from preterm pregnancies absent labor and significantly decreased with idiopathic preterm labor. while bnp activates natriuretic peptides receptors a (npr-a), b (npr-b), and its clearance receptor (npr-c), we have shown bnp does not inhibit myometrial contraction via npr-a or npr-b. herein, we test in part the hypothesis that bnp inhibits myometrial contractions by activating npr-c by quantitating npr-c in human myometrium at different gestations and labor status. methods: myometrial samples were obtained at the time of cesarean section after informed consent from groups of patients: preterm not in labor (pt-nl), preterm in labor (pt-l), term not in labor (t-nl) and term in labor (t-l). myometrial samples were obtained from women - weeks' gestation, and term between and weeks. mrna for npr-a, b and c were semiquantitated by realtime pcr (normalized by s mrna), and npr-c protein by western blot. results: natriuretic peptide receptors a, b and c mrnas were identified in all groups. while there were no differences in mrna levels among groups, npr-c protein was increased in samples from term laboring women. conclusion: since bnp inhibits the contraction of human preterm but not term myometrium independent of npr-a and npr-b, we have previously speculated that bnp activates another "unknown" receptor. herein we find that myometrial npr-c protein but not mrna increases during human labor at term. the increase in npr-c at term could compete with the unknown quiescent receptor to functionally reduce the availability of bnp and thus permit or promote myometrial activation/contraction. (supported by a grant from chilean government fondecyt ). objective: bnp is synthesized within human chorion and amnion and inhibits oxytocin-induced contraction of human myometrium. bnp activates guanylate cyclase (gc) natriuretic peptides receptors a (npr-a) and b (npr-b). bnp also stimulates the clearance receptor (npr-c) whose action is not mediated by gc. the intracellular pathway of bnp/npr-c inhibition is not known. we determined that bnp does not inhibit myometrial contraction via npr-a or -b. we hypothesized that bnp inhibits myometrium by npr-c activation. we test aspects of our hypothesis by determining whether bnp/npr-c pathway inhibits myometrial contractions via myometrial nos pathway. methods: bnp and canp (specific npr-c agonist) were added to primary human myometrial cell cultures and enos/inos activity/expression determined. cell cultures (n= ) were prepared from myometrial samples of nonlaboring, term pregnant women. after confluence ( d), the cells were incubated ( h) with nm bnp and/or canp. nos activity was measured ( l-citruline assay) and transcription/translation semi quantitated (realtime pcr and western blotting). results: bnp had no effect on either nos expression or activity. canp significantly reduced inos but not enos mrna level. canp did not alter the protein level of nos but significantly reduced nos activity. co-incubation with bnp and canp reduced both mrna levels (p< . ) and significantly decreased enos, but not inos, protein without change in overall nos activity. within the female pelvis. ultrasound obstet gynecol. ; : - . conclusion: the activation of npr-c by canp reduces nos activity and expression. the same effect was not observed by bnp. the difference may be explained because bnp (but not canp) activates all natriuretic peptide receptors, and they may have opposite actions on nos pathway. we conclude unlikely bnp inhibits human myometrial contraction by npr-c activation via the nos pathway. ( introduction: ultr is a retroviral immortalized human uterine smooth muscle cell line which we use as a model for uterine hypertrophy studies. however, this cell line has limited usage due to a reduced rate of cell division and eventually replicative senescence in culture. one of the mechanisms of human somatic cellular senescence is un-compensated shortening of telomeres, the specialized dna structures located at the ends of eukaryotic chromosomes. introduction of human telomere reverse transcriptase (htert) has been shown to induce telomerase activity and telomere elongation, and extend life-span of normal human cells. objective: to recover ultr cell division without altering the phenotypic characteristics of smooth muscle cells by introducing htert, therefore improving ultr cell line. methods: ultr cells were transfected with a modified htert expression vector at a relatively early stage (passage ) in the presence of selective antibiotic. ultr cell growth rate, with (ultr-ht) or without transfection, was determined by plating cells in multiple plates at a fixed density and counting cell numbers after days. single cell clones of stably transfected cells were further isolated by plating in well plates. expression of htert, smooth muscle specific genes (smc-sgs), and target genes was identified by rt-pcr of total rna extracted from ultr and ultr-ht cells. results: rt-pcr demonstrated successful introduction of htert into ultr cells. the growth rate of ultr-ht cells was increased and cell morphology was improved (free of the typical aneupoid appearance). at passage , ultr-ht cells grew . fold (p< . ) and . fold (p< . ) faster than ultr cells at passages and , respectively. currently single cell clones have been selected. ultr-ht cells express a set of smc-sgs, including -actin, caldesmon, calponin, myosin heavy chain, sm , and smoothelin, confirming that the smooth muscle phenotype is preserved. a panel of genes involved in angiotensin ii signalling, including angiotensin ii receptors (at / ), nox family (nox , , and duox ), nox-associated genes (p phox, p phox, p phox, and rac / ), was also preserved. conclusion: this is the first report of rescuing uterine smooth muscle cell replicative senescence via activation of telomerase. we have established a human uterine smooth muscle cell line which will provide an improved in vitro model for studying human myometrium. at term, myometrium is characterized by spontaneous contractions which vary in the amplitude, frequency and duration depending on specie and hormonal status. repetitive depolarization of plasma membrane followed by transient elevation in [ca + ] i is thought to underlie the contractions. however, the detailed pathways which regulate the spontaneous contractility remain unclear. objective: this study was designed to elucidate the effect of protein kinase c (pkc) on the spontaneous contractions and [ca + ] i transients in the myometria from term pregnant mice and women. methods: the human samples were obtained from women undergoing cesarean section with the approval from the institutional review board committee while mice myometria were dissected from the days pregnant mice sacrificed according to the animal study protocol. the isometric force and [ca + ] i were measured simultaneously in fura- loaded myometrial strips using spectrofluorometer equipped with force transducer. results: the pkc activator phorbol , -dibutyrate (pdbu) applied at - m first stimulated the amplitude of spontaneous contractions in mice and human myometria followed by their inhibition. under the pdbu treatment the frequency of the contractions was first increased and then decreased in both species. at the same time, pdbu didn't initially increase the amplitude of [ca + ] i transients but attenuated it over time. however, the frequency of the transients was first increased and later decreased upon the pdbu exposure. in addition, pkc activation with pdbu resulted in the elevation of the uterine basal tone without corresponding changes in the basal level of [ca + ] i in human myometrium. in mice myometrium, on the contrary, pkc activation didn't result in an increase of the muscle tone and the basal level of [ca + ] i . conclusions: we propose that pkc causes bi-phasic effect on uterine spontaneous contraction in mice and human first to potentiate the amplitudes and the frequencies and later decreases them. the amplitude of [ca + ] i was not initially potentiated by pdbu suggestive of the dissociation of the contractile and [ca + ] i response. the stimulatory effect of pdbu on the basal muscle tone in human myometrium and the absence of the effect in mouse suggest different mode of pkc action on uterine contraction in human and mice. introduction: mechanical stretch of uterine myocytes is detected through integrins on the cell surface which form part of the focal adhesion complex, this signals through mapk, ultimately leading to the up-regulation of various pro-labour genes including pghs- and il- . on integrin activation fak is recruited to the focal adhesion complex and activated by autophosphorylation at tyr- , creating a src binding site. this promotes further fak phosphorylation at tyr- , and , enhancing fak catalytic activity. however fak can also act as a scaffold protein and its exact role in the expression of pro-labour genes is uncertain. in this study we used inhibitors for the kinase activity of fak and mek / in order to test the affect of fak kinase activity on expression on pghs- mrna. methods: primary human myometrial cell cultures were grown from myometrial biopsies taken from women undergoing elective caesarean section. cells were plated onto -well flexible bottom plates coated in type i collagen. cells were subjected to % static stretch for up to minutes. cells were also incubated with either the rho kinase inhibitor y or the mek / inhibitor u prior to being stretched. western blots were performed using antibodies to fak phospho- , fak phospho- and erk / phospho- / , -actin was used as a loading control. rna was also extracted and levels of pghs- measured using q-pcr. results: stretch increased levels of phosphorylation at both tyr- and tyr- with the greatest increases occurring at and minutes. however the increase at tyr- appeared to be greater than at tyr- . incubation with the rho kinase inhibitor reduced phosphorylation of fak- , however this did not affect phosphorylation of erk / or stretch induced up regulation of pghs- mrna. in contrast incubation with the mek / inhibitor reduced erk / phosphorylation and expression of pghs- mrna, whilst also reducing fak phosphorylation (n= ; p= . ). conclusions: fak has previously been shown to be important in activation of the stretch induced mapk cascade and pghs- expression. however these data suggest that while stretch causes fak phosphorylation fak kinase activity is not essential to pghs- expression. this suggests fak may be acting as a protein scaffold. . our aim was to examine the effects of both natural progesterone and hp on spontaneous myometrial contractions. myometrial biopsies were taken with informed consent and ethics approval from non-labouring women at elective caesarean section weeks gestation. strips of myometrium mm long , mm wide were cut and suspended under a resting tension of mn in organ baths of krebs gassed with % o / % co within hours of collection. progesterone or hp were added in a cumulative manner at -minute intervals. changes in amplitude were recorded. results were compared using anova. following equilibration for hours, myometrial strips contracted in a rhythmic manner (amplitude . ± . mn, n= pairs). progesterone ( nm- m) produced a concentration dependent inhibitory effect on myometrial contractions (fig ), which was greater than that of vehicle (p< . ). maximum inhibition measured . ± . % and . ± . % for progesterone and vehicle, respectively. hp exerted an inhibitory effect, this was not significantly different from the vehicle (p> . ). progesterone exerts an inhibitory effect on myometrial contractility in vitro. this is apparent within minutes suggesting a nongenomic action. our data are in agreement with some reports in the literature but conflict with others[ ]. this acute inhibition of myometrial contractility may contribute to the mechanism by which progesterone prevents preterm birth. in contrast, we were unable to demonstrate an inhibitory effect of hp on contractility despite its demonstrated ability to reduce the incidence of preterm delivery [ ] . our data suggest that natural progesterone may be a more effective tocolytic agent in the acute setting than hp. obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; pathology, ramathibodi hospital, mahidol university, bangkok, thailand; obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: chemokines has been shown to play an important role in regulating uterine function. recent evidence demonstrates that monocyte chemotactic protein (mcp) level in amniotic fluid increases during spontaneous labor. the aim of this study was to examine the expression of mcp in human myometrium. methods: myometrial biopsies were taken from nonpregnant women undergoing hysterectomy and term pregnant women undergoing cesarean section followed written consent and local ethics committee approval. elective cesarean section was performed before the onset of labor while emegency section was done after the onset of labor. immunolocalization (n = each) was performed on paraffin sections by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp . reverse transcriptionpolymerase chain reaction (n = each) using gene specific primer against mcp and mcp receptor was performed to identify mcp messenger(m) rna in human myometrium . results: immunohistochemical findings demonstrated mcp in human myometrial cells from nonpregnant, term pregnant women before and after the onset of labor. mcp was labelled on plasma membrane and cytoplasm of myocytes from these three groups of women. similarly, mcp and mcp receptor mrna were found in nonpregnant and term pregnant women before and after the onset of labor. in this prospective study a group of fetuses was consecutively enrolled. one ultrasound examination was performed to each patient within days from delivery. we considered fetal macrosomia a birthweight g and big babies a birthweight g. cut-off points for identifying the best value of fetal abdominal circumference for fetal macrosomia prediction were chosen by receiving operator characteristics' curve (roc) analysis. using the best cut-off indicated by roc analysis, specificity and sensitivity were calculated. mean gestational age at delivery was + weeks ( + sd). neonates weighted less than g, had a weight range between and g and weighted more than g. the fetal ac measurement was the selected criterium to evaluate the risk of fetal macrosomia. to identify macrosomic fetuses the roc curve analysis identified a cut off of ac > mm which allowed to select fetuses. analysing this population we found true negative cases, false negatives, false positives and true positives, with a sensitivity of . % and a specificity of %. to detect big babies the roc curve analysis identified a cut off of ac > mm (n. fetuses), reaching a sensitivity of % and a specificity of %, without false negative cases and with false positives ( true positives, true negatives). the cases that weighted between and g, were included in the cases considered false positives by this cut-off. conclusions. these results clearly indicated that ultrasounds alone can not be used to manage a pregnancy suspected for fetal macrosomia or big babies. in fact, to avoid shoulder dystocia and all other complications strictly related to macrosomic fetuses, clinicians should perform a large number of useless elective cesarean sections. . pathway analysis of differentially expressed genes (pa; z-score . ) showed up-regulation of axon guidance, folate biosynthesis, nitrogen metabolism and down-regulation of steroid biosynthesis, insulin signaling, oxidative phosphorylation, tgf-beta signaling, and ubiquinone biosynthesis pathways in cm vs cf. comparison of mnr vs c in f showed genes up-and down-regulated (n= , ; p< . ). pa showed up-regulation steroid biosynthesis, fatty acid metabolism, glycolysis/gluconeogenesis, phosphatidylinositol signaling, ketone body metabolism, and ubiquinone biosynthesis pathways and down-regulation of bile acid biosynthesis, cell adhesion molecules, dna polymerase, notch signaling and ti diabetes mellitus pathways in mnr vs c. comparison of mnr vs c in m showed genes up-and down-regulated (n= , ; p< . ). pa showed up-regulation of jak-stat signaling, autophagy, renin-angiotensin system (ras), and ubiquinone biosynthesis pathways and down-regulation of apoptosis, basal transcription, folate biosynthesis, nitrogen metabolism, protein export, and snare interaction pathways in mnr vs c. only the ubiquinone biosynthesis pathway up-regulation of mnr vs. c was common to both m and f. conclusions: ta and pa demonstrate sex-specific transcriptome expression in fetal kidneys at . g. in addition, these results show sexspecificity in response to mnr. we have seen similar effects of mnr on gene expression for autophagy, apoptosis, ras, ubiquinone and cell adhesion pathways at . g, suggesting these pathways may contribute to persistent affects of mnr. finally, we postulate that stress in utero may contribute to sex differences in risk of hypertension in adult life. leptin and neuropeptied y protein expression paradoxally increased in gestationall food restricted dams. louiza belkacemi, chun-hung chen, andrea jelks, michael g ross, mina desai. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: placental insufficiency is associated with marked increase in placental leptin production. this results in a rise in maternal leptin levels that serves as an early index of placental dysfunction. further increased placental leptin and suppressed neuropeptide y (npy) are associated with preeclampsia. leptin, an anorexigenic hormone and npy, an orexigenic peptide regulate food intake. importantly, leptin also serves as a placental and fetal growth factor. we have shown that maternal food restriction (mfr) results in intrauterine growth ... sf ()to:!)) df(,..)l) ...._ bf(jtolo) ... -m bf(,..lj) =- ( . ) <(u.)) ( . ) )( . )"" ' ~. ) !( . )' d.'cbom< ( ) f( ))""" s( l) background teenagers are more likely to deliver small-for-gestational age (sga) infants than adults, even after adjustment for socioeconomic factors , . previous studies of mostly black and hispanic subjects in the usa have suggested that maternal growth may contribute to reduced infant birthweight, due to preferential nutrient partitioning to the mother . the impact of maternal growth on birthweight and nutrient partitioning in pregnant teenagers in the uk has not been examined. methods skeletal growth (change in knee-height from st to rd trimester), weight gain and skinfold thicknesses were measured in pregnant teenagers (n= , % non-white) in london and manchester. key mediators of nutrient partitioning and metabolism: insulin-like growth factor(igf)- , igf binding protein(bp)- and leptin, were measured in maternal plasma ( weeks gestation). results maternal growth (defined as increase in knee-height > mm/ days) was detected in % of pregnant teenagers. this growth was not associated with sga birth; in fact these mothers were more likely to deliver large-forgestational age (lga) infants (p< . ). maternal weight gain and fat accrual at peripheral and central sites were greater in growers (p< . ). these parameters correlated positively with maternal igf- and leptin but negatively with the igf inhibitor, igfbp- (p< . for all). subjects delivering sga infants gained significantly less weight (p< . ) and had lower igf- levels (p< . ) than those delivering non-sga infants. conclusion maternal growth in teenage pregnancy was not associated with reduced birthweight. indeed the increased weight gain and fat accrual observed in growing teenagers may protect against sga birth and promote fetal growth. igf- and leptin promote fetal growth, primarily through effects on maternal metabolism and nutrient partitioning to the fetoplacental unit. these data suggest that higher maternal igf- and leptin in growing teenagers may provide an anabolic drive for both maternal and fetal growth. background. umbilical oxygen uptake (o umb uptake) has been estimated in human pregnancies only in acute experiments at the time of caesarean section. the recently developed possibility to measure umbilical blood flow by ultrasound in utero, prompted us to study normal and iugr pregnancies in order to evaluate fetal oxygen uptake utilizing the fick principle, i.e. uptake equals umbilical blood flow times (a-v) differences. methods. thirty-six iugr pregnancies were studied at the time of elective caesarean section and compared to twenty-one controls (c) (gestational age: c= . ± . and iugr= . ± . wks). an ultrasound examination was performed within hours from the caesarean section in all the recruited patients. umbilical vein absolute volume flow (qumb) was measured as the result between umbilical vein area and the time-averaged peak velocity * . . blood samples from umbilical vein (uv) and artery (ua) were obtained after the delivery and blood gases and acid-base balance were evaluated. umbilical oxygen uptake was calculated as o umb uptake = qumb*(uv-ua) o content. results. as expected, average fetal and placental weights were significantly different in the studied groups ( ± and ± g in iugr vs ± and ± g in n) . iugr pregnancies showed a significant reduction in qumb ( . ± . vs . ± . ml/min; p< . ) but no differences in the qumb/kg of fetal weight. iugr fetuses showed a significant reduction in o sat, o cont and po in both uv and ua compared to n. (uv-ua)o content ( . ± . vs . ± . mmol/l; p< . ) and o umb uptake/kg ( . ± . vs . ± . mmol/min/kg; p< . ) were significantly reduced in iugr. conclusions. we here report an evaluation of fetal oxygen uptake that proved surprisingly similar to the values reported in chronically catheterized animals. however, iugr fetuses showed a significant reduction in both blood and oxygen supply: this latter was reduced more that % on a per kg basis. iugr fetuses therefore utilize less oxygen than normally grown fetuses. circulating levels of vitamin d and il- in pregnancies with iugr. calvin j hobel, chander p arora, adegoke adeniji, priya arora, susan e jackman, olga miadel, baldjyan lilit. ob-gyn, cedars-sinai medical center, burns ans allen research institute, los angeles, ca, usa; university of california los angeles, los angeles, ca, usa. background: any condition resulting in under exposure to sunlight, including the use of sun block or poor nutrition may result in insufficiency ( . - nmol/l) or even deficiency of vitamin d (< . nmol/l).vitamin d regulates placental development and function. vitamin d deficiency has been linked to increased risk of serious chronic and inflammatory diseases. objective: to determine if the circulating levels of vitamin d and interleukin - (il- ) in maternal plasma correlates to pregnancies resulting in fetus with intrauterine growth restriction (iugr). hypothesis: the metabolism of vitamin d initiates the biochemical cascade of events leading to the expression of il- and the inflammatory response in iugr births. study design: in a behavior in pregnancy study, plasma samples at all three time points were analyzed in a cohort of women for (oh)d using elisa. the samples were also analyzed for il- at three stages of pregnancy: t ( - weeks), t ( - weeks) and t ( - weeks). iugr was defined as birth weight below the tenth percentile for gestational age. none of the iugr cases had spontaneous preterm birth. results: iugr was diagnosed in of women with available samples from a behavior in pregnancy study (bips) . out of these subjects, were selected as matched case controls. circulating levels of vitamin d ( (oh)d) were significantly lower in iugr cases at each visit (p<. ). the levels indicated deficient ( . ± . nmol/l) vitamin d in iugr group at t but sufficient vitamin d levels ( . ± . nmol/l) in controls. subsequent visits also showed lower levels in the iugr cases compared to the control group (t : ± . nmol/l vs ± . nmol /l; t : ± . nmol/l vs ± . nmol /l). at all three time intervals, significantly (p<. ) higher levels of il- were associated with the iugr cases ( pg/ml, pg/ml and pg/ml respectively) as compared to the controls ( pg/ml, pg/ml and pg/ml respectively). conclusions: vitamin d deficiency or even insufficiency may be an unrecognized cause of iugr. it is possible that a primary non-infectious inflammatory process is activated by vitamin d deficiency. combined assessment of vitamin d deficiency and il- expression during different stages of pregnancy may facilitate the resognition of the risk of developing iugr. response to global % maternal nutrient restriction (mnr). nathan drever, thomas j mcdonald, peter w nathanielsz, cun li. obstetrics and gynecology, university of texas health science center at san antonio, san antonio, tx, usa. background: igf-ii is a major growth factor in the developing pancreas and studies in the human fetus demonstrate that the peptide localizes to b cells of the islets (j endocrinology : ). rodent studies show decreased fetal pancreatic growth and igf-ii and ins abundance and increased apoptosis with mnr (j endocrinology : ). we previously demonstrated a fall in most components of the placental and fetal baboon liver igf systems with mnr. here we have evaluated fetal pancreatic igf-ii and ins changes in response to mnr. methods: pregnant baboons were fed ad lib (ctr, n= ) or % of wt adjusted ctr diet (mnr, n= ) from . gestation (g) and fetuses were recovered at c-section under general anesthesia at . (n= ; ctr and mnr) and . g (n= ; ctr and mnr). igf-ii and ins expression were determined by immunohistochemistry (ihc) and quantified by image analysis for fraction (area immunostained/area of the field x %) and density. data are expressed as mean + sem; ctr data are expressed] first; comparison made with two tailed t-test. results: at . g there was no difference in igf-ii or insulin fraction or density between groups. at . g, igf-ii fraction ( . ± . vs . ± . ,p< . ) and density ( . x ± . x vs . x ± . x , p< . ) and insulin fraction ( . ± . vs . ± . , p= . ) were reduced. conclusion: moderate mnr decreases abundance of fetal pancreatic igf-ii and ins at . g. in the fetal baboon and supports the extant evidence for impaired pancreatic development with mnr seen in rodents. maintenance of liver growth in the hypoxic growth restricted fetal sheep: a role for intrahepatic glut ? sheridan gentili, janna l morrison, i caroline mcmillen. sansom institute, unisa, adelaide, south australia, australia. objective: we have previously demonstrated that there is a differential tissue response to chronic placental and fetal growth restriction. growth of fetal tissues such as the brain and the adrenal are consistently spared in the face of chronic substrate restriction, whilst we have demonstrated that the growth of the fetal liver may be either maintained or reduced. it is unclear whether the growth response of the fetal liver to hypoxia and hypoglycemia are determined by intrahepatic metabolic adaptations. hypothesis: we hypothesize that there will be a differential profile of hepatic expression of glut , hsd , the gluconeogenic and glycolytic enzymes pepck and g pdh and the transcription factors pgc and ppar in animals in which liver growth is maintained or reduced. methods: carunclectomy was performed in non-pregnant ewes to induce placental restriction (pr). vascular catheters were inserted in pr and control (c) fetuses at - d and arterial blood samples were collected for blood gas analysis. mean gestation po < mmhg was defined as hypoxic (h; normoxia, n). post mortem was performed at - d. hepatic mrna expression of glut , hsd , pepck, g pdh, pgc and ppar was determined using qrt-pcr. results: four experimental groups were defined by fetal po and liver growth (c-n, pr-n, pr-h and pr-h-reduced liver growth). fetal weight correlated with mean gestational po (r = . , y= . x+ . , p< . ). liver weight was significantly lower in a cohort of pr-h fetuses (c, . ± . ; pr-n . ± . ; pr-h . ± . ; pr-h-rlg . ± . g:kg; p< . ). glut expression was highest in those pr-h fetuses in which liver growth was maintained, whilst the expression of hsd , pgc , ppar and pepck was highest in the pr-h fetuses in which liver growth was reduced (p< . ). conclusions: in the pr-h fetuses in which liver growth was reduced, the increase in hsd expression may be associated with an increase in hepatic exposure to cortisol and an associated increase in pepck, pgc and ppar . interestingly the compensatory increase in hepatic glut expression did not occur in fetuses in which liver growth was reduced. predicting the trajectory of fetal growth. racine n edwards-silva, jeffrey gornbein, calvin j hobel. obstetrics gynecology, los angeles, ca, usa; biomathematics, david geffen school of medicine at university of california, los angeles, ca, usa; obstetrics gynecology, david geffen school of medicine at university of california, los angeles, ca, usa. objective: to evaluate twelve potential predictors of fetal growth trajectory defined as the rate of fetal weight change over time. study design: a longitudinal prospective study of singleton fetal growth trajectory. the twelve potential predictors considered were: fetal gender, gestational age, parity, race, bmi, age, weight at st clinical exam, cumulative weight gain at each exam, smoking, and alcohol use. estimated fetal weight was computed using the hadlock formula and sonographic fetal biometric parameters at - weeks, - weeks, and - weeks. the rate of change in fetal weight was defined as x (fetal wt at exam j -fetal wt at exam i )(j > i)/ (gestational age at exam j -gestational age at exam i ). bivariate statistical analysis included the non-parametric spearman rank correlation and wilcoxon rank sum test. all factors were assessed multivariately using multiple linear regression. results: there were multi-ethnic women included in the study, after were excluded. they underwent a total of , exams. fetal gender (p= . ), maternal weight at st exam (p= . ), and cumulative maternal weight gain at exam (p < . ) were significant predictors of fetal growth trajectory. in this model, male fetuses had an average rate of fetal weight change of . grams per days higher than females. the rate of fetal weight change increased by an average of . grams per days for each lb increase in maternal weight at the st exam. the fetal growth rate increased . grams per days for each lb of cumulative weight gain at the rd exam. conclusions: in this study, maternal weight at the st exam and cumulative maternal weight gain were the significant determinants of fetal growth trajectory. adequate initial maternal weight and cumulative gestational weight gains probably ensure sufficient nourishment for normal placental growth, uteroplacental blood flow, and fetal nutrient uptake. this supports the emphasis on periconceptual nutritional counseling for all pregnant women. the implication of this study is that similar to fetal programming of adult diseases, there is nutritional programming of fetal growth trajectory. high risk patients. other predictors include the known risk factors of age < and > , single, tobacco/alcohol use, and african american race. interestingly, hispanic and native american patients have a lower lbw rate compared to other groups. introduction: catecholamines released by the sympathetic nervous system and adrenal medulla act via b-ars to regulate glucose and insulin function in liver, pancreas, adipose and muscle tissue. b-ar knock out mice show increased fat mass and glucose intolerance (asenslo et al.,diabetes, ) . mnr animal models have increased sympathetic activity. we, therefore, evaluated effects of mnr on baboon fetal liver b -ar. methods: baboons were fed as ad lib controls (ctr) or % of wt adjusted ctr diet (mnr) from . gestation(g) with fetuses retrieved at c-section under general anesthesia at . or . g. protein expression determined by immunohistochemistry for b -ar in the central liver lobule was quantified by image anaysis and expressed as fraction = area immuno-stained/area of the field x %. all data are expressed as mean + sem with ctr data presented first. liver glycogen expression was determined by the periodic accid schiiff (pas) method. comparisions were made with student's t-test with alpha level set at . . results: fetal body and liver wts were not changed by mnr at either age. pas stained liver glycogen at . g = . ± . vs. . ± . %, p< . . b -ar fraction was lower following mnr at . g and at . g (p< . ; fig ) conclusions: mnr decreased b -ar over % at . g and % at . g. decreased fetal liver b -ar in mnr alters glucose metabolisam and may result in reduced lipolysis predisposing to fatty liver. infection with noncytopathic bovine viral diarrhea virus (ncpbvdv) during early bovine pregnancy (< d gestation) results in fetal immunotolerance, persistent infection (pi) and intrauterine growth restriction (iugr). in contrast, infection after the development of adaptive immune competence (> d gestation or postnatal) results in a transient infection (ti). we have previously reported an iugr in pi fetuses presenting as decreased body weight and ponderal index. a growth defect can be the result of many factors, including placental insufficiency and nutrient restriction, however it was hypothesized that the iugr seen in bvdv pi fetuses may be an immunopathological effect caused by the persisting virus. our two part experimental design examined the relationship between bvdv and its pi host. in experiment (exp) , blood cell mrna was collected from pi steers (n= ; confirmed by virus isolation), or uninfected control steers (n= ) and used to identify differentially expressed genes using microarray (affymetrix) and qtrt-pcr approaches. in exp , bvdv naïve pregnant heifers (n= per group) were not infected (control) or infected with ncpbvdv on d. or d. of pregnancy creating pi and ti fetuses, respectively. fetuses were collected by c-section on d. ; infection was confirmed by elisa and qtrt-pcr. histology of placental tissue revealed no placentitis or pathology, and glucose and lactate levels in fetal serum were normal. microarray analysis revealed genes that were differentially regulated in pi vs. controls (p< . , > . fold). qtrt-pcr of steer and fetal blood revealed a significant upregulation of activators and products of the antiviral type-i interferon (ifn-i) pathway. as ifn-i can act as a growthsuppressive cytokine, a long-term upregulation may contribute to the iugr seen in persistent bvdv infection and in other viral infections observed during pregnancy. nricg - from the csrees. patients with a short cervix are at an increased risk for spontaneous preterm delivery. therefore, it is possible that women with a short cervix during pregnancy are also at risk to deliver an sga neonate. this study was conducted to address this question. study design: patients > weeks of gestation were prospectively enrolled into an observational study ( / to / ). transvaginal sonographic examinations were performed every weeks until delivery. the shortest cervical length between - weeks was used for analysis. sga was defined as less than the tenth percentile of birth weight. results: asymptomatic patients were studied. . % of patients delivered an sga neonate ( / ). the median cervical length was mm ( to mm); patients had a cervical length < mm. of these, % ( / ) delivered an sga neonate. similarly, % ( / ) patients with a cervical length < mm had an sga neonate. no relationship was found between a short cervix and sga (short cervix was defined as either < mm and < mm). the frequency of sga was not significantly different between women with a short cervix and those with a long cervix [ % ( / ) vs. % ( / ); p= . ]. newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced background folate is an essential micronutrient for cellular growth. recommendations on periconceptional folic acid use are mainly focussed on prevention of neural tube defects, despite growing evidence that folic acid use may have positive effects on birth weight. objective to examine associations between folic acid use, intrauterine fetal growth and birth weight. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods information on folic acid use was obtained by questionnaires and categorized into three groups: ) preconception start of folic acid use; ) start of folic acid use in first ten weeks of gestation; ) no folic acid use at all. fetal growth measurements included head circumference, abdominal circumference and femur length measured in mid-and late pregnancy, i.e., gestational age - and > weeks, respectively, and birth weight. fetal weight in mid-and late pregnancy was estimated using the haddock method. results data from , pregnant women were available. overall, folic acid use was positively associated with fetal growth. preconceptional folic acid use resulted in an increased growth of grams ( %ci . - . , p< . ) per week from late pregnancy to birth, compared to no folic acid use. similarly, start of folic acid use in the first ten weeks of gestation resulted in an increased growth of grams ( %ci . - . , p= . ) per week from late pregnancy to birth. both preconceptional folic acid use and folic acid use started in the first ten weeks of gestation resulted in higher birth weights of grams ( %ci - ) and grams ( %ci - ), respectively, compared to no folic acid use. a tendency was found for an increased risk of birth weight less than grams when folic acid was not started preconceptionally (or . , %ci . - . ). conclusion periconceptional folic acid use is significantly associated with increased fetal growth resulting in a higher birth weight. ductus venosus isovolumetric relaxation in severely premature growth-restricted fetuses. jason l picconi, katherine drennan, farhan hanif, michael kruger, giancarlo mari. obstetrics and gynecology, wayne state university/dmc, detroit, mi, usa. objective: ductus venosus (dv) doppler waveforms are characterized by two periods in which blood velocity decreases. the first represents the isovolumetric relaxation (ir) at the end of ventricular systole and the second represents atrial contraction at the end of ventricular diastole (a). ductus venosus reversed flow (dvrf) occurring at the time of the a-wave is considered a risk factor for intrauterine fetal demise (iufd). we have previously reported that absent or reversed a-wave flow can be present for weeks before iufd occurs or delivery is performed for non-reassuring fetal testing. the guiding hypothesis for this study is that decreased flow at the time of ir in combination with absent or reversed a-wave flow allows a more accurate prediction of fetal outcome than a-wave absent or reversed flow alone. material and methods: ductus venosus doppler was serially studied in severely premature iugr fetuses (estimated fetal weight < th percentile and umbilical artery pulsatility index > th percentile) from diagnosis until demise or delivery. ductus venosus waveforms were assessed quantitatively for peak systolic velocity (psv), isovolumetric relaxation velocity (irv), and end diastolic velocity (edv). the psv/irv + edv were compared to fetal and neonatal outcome. a kruskal-wallis one way anova, mann whitney u post-hoc test, and a roc, were used for statistical analysis. a p < . was considered statistically significant. results: all fetuses were delivered at < weeks. six cases resulted in iufd, five cases resulted in neonatal demise (nd), and six cases resulted in neonatal survival (ns) at the time of discharge from the hospital. the psv/irv+edv correlated better than a-wave reversal of flow with perinatal outcome. a psv/irv+edv score less than - . resulted in iufd, whereas a score greater than - . resulted in live birth. live births segregated based on estimated gestational age, where those fetuses at less than weeks resulted in nd and those fetuses at or greater than weeks resulted in ns. all results were statistically significant. conclusions: the isovolumetric relaxation velocity is a novel doppler parameter in the assessment of severely premature iugr fetuses. these data indicate that assessment of irv should be considered part of the evaluation of severely iugr fetuses. background: fetal growth restriction has been linked to an increased incidence of chronic hypertension, which may be the result of extracellular matrix changes (ecm) within the vascular tree. in previous studies, ecm changes were observed in the umbilical arteries of preterm growth restricted infants. objective: to determine if there are alterations in collagen subtypes within the umbilical cords from growth restricted fetal rat pups after a period of maternal nutrient restriction. methods: timed pregnant sprague-dawley rats were fed either a % food restricted diet (mfr; n= ) or were fed ad libidum (control; n= ) from d until d of gestation. litter size, fetal weights and placental weights were then noted and umbilical cords from randomly selected pups in each litter were snap frozen. gene expression for collagens i, iii, xiv and decorin was evaluated by real-time rt-pcr with normalization to the gadph housekeeping gene. data were analyzed from fitting general linear regression models with estimation based on the quasi-likelihood estimation (generalized estimating equations) to account for clustering of responses within litters. data are shown as cycles to amplification (ct), which is inversely proportional to mrna levels. results: no difference in median litter size was detected. however, fetal and placental weights in the mfr group were significantly less than those from control dams. no significant differences in umbilical cord gene expression for collagens i, iii, xiv or decorin were detected between mfr and control pups. conclusions: maternal food restriction does not result in any detectable alterations in collagen or decorin expression within the intact umbilical cord, despite causing significant reductions in fetal growth. control (n= ) p-value litter size (median, range) ( , ) ( , ) . fetal weight (mean ± sd) g . ± . . ± . < . placental weight (mean ± sd) g . ± . . ± . . collagen i (mean ± sem) ct . ± . . we have previously demonstrated that the restriction of placental and fetal growth results in fetal hypoxia and fetal brain sparing suggesting a redistribution of cardiac output. furthermore, placentally restricted (pr) hypoxic fetuses are more dependent on their sympathetic nervous system for the maintenance of blood pressure during late gestation. nerve growth factor (ngf) plays a significant role in sympathetic innervation. hypothesis: we hypothesize that the expression of ngf will be higher in the aorta and femoral artery of the pr hypoxic compared to the control fetus. method: carunclectomy was performed in non-pregnant ewes to induce pr. vascular catheters were inserted in pr and control (c) fetuses at - d and arterial blood samples were collected for blood gas analysis. all pr fetuses introduction elevated sflt- levels have been shown to be a feature of pre-eclampsia and is considered to play a significant role in the pathogenesis of the condition. the role of angiogenic factors in fetal growth restriction has not been as well established. this study evaluated the levels of circulating vascular endothelial growth factor (vegf) and its soluble receptor sflt- , in normal pregnancies and isolated placental vascular disease without evidence of pre-eclampsia. method maternal peripheral venous samples were collected antenatally from two groups of pregnant women between - weeks of gestation. group a: uncomplicated normal pregnancies (n = ) and group b: pregnancies complicated by isolated placental vascular disease( n = ) as defined by birth weight less than th centile for gestation and umbilical artery doppler s:d ratio above the th centile for gestation, with no evidence of maternal preeclampsia or pre-existing hypertension. plasma vegf and sflt- levels were measured using standard eliza techniques. comparison between groups were performed by using one way analysis of variance. the maternal plasma sflt- levels (figure ) in group a: normal pregnancies increased with gestation (p < . ). the sflt- levels in group b: isolated placental vascular disease were significantly higher throughout all gestations (p = . ) and did not show a significant variation with gestation ( p = . ). the plasma vegf levels were below the detectable levels of the assay in all samples except normal pregnancies. the significantly increased maternal plasma sflt- levels in established isolated placental vascular disease without evidence of pre-eclampsia suggest a disease of placental origin. the dysregulation of angiogenic factors may be part of a repair and regeneration process in the placenta. objectives: production of -reduced neurosteroids is critical for reducing fetal vulnerability to stressors in pregnancy and inhibition of -reductases ( r) increase acute hypoxia-induced apoptosis in the fetal brain (yawno et al neurosci : . we have developed a model of placental insufficiency that results in fetal growth restriction (gr) in the guinea pig (palliser et al repro sci # ). the aim of the present study was to determine the effects of suppression of -reduced steroid synthesis on apoptosis in vulnerable regions of the fetal brain and on neurosteroid synthetic enzymes in pregnancies compromised by chronic placental insufficiency. methods: placental insufficiency was induced in guinea pig dams by surgical ablation of uterine artery branches at mid gestation (term d). sham operated or gr dams received finasteride (a r inhibitor; mg/kg/day) during late gestation ( d until term). activated caspase- , a marker of apoptotic cell death, was measured by immunohistochemistry and steroidogenic enzymes, r and cytochrome p side chain cleavage (p scc) were measured by real time pcr and western blotting in fetuses ( d) and neonates h after birth. results: placental insufficiency significantly reduced fetal body and organ weight by % whilst sparing brain weight. the number of activated caspase- positive cells was significantly increased in the fetal hippocampus of gr fetuses and further increased in the cortex of gr fetuses receiving finasteride. the neonatal brain also exhibited changes in caspase- activation following gr and finasteride treatment. the fetal adrenal and the placenta responded to the compromise with increased expression of p scc mrna and r protein, respectively. conclusion: the combination of placental insufficiency and suppressed neurosteroid system leads to markedly increased apoptotic cell death in the fetal brain which continues to affect the neonatal brain. the fetus responds to these conditions by increasing steroid synthetic enzyme expression in the placenta and adrenal glands suggestive of a possible neuroprotective feedback process. to study the effect of smoking by mothers on the fetal and neonatal brain using non-invasive magnetoencephalography technique (meg). materials and methods: using fetal magnetoencephalography, cortical auditory evoked responses (aer) were measured from fetuses ranging from to weeks gestational age for a total of recordings. measurements were taken from mothers with a history of smoking (sm) and from mothers with no smoking history (ns). after delivery, five sm and five ns newborns had meg aer measurements twice for a total of recordings. aer was quantified by cross-correlation analysis and its significance was assessed by boot-strap technique. results: aers were detectable in out of fetal recordings in the ns group and of in the sm group. the neonatal response rate was % for each group. the latencies were divided into three components: c ( - ms), c ( - ms) and c ( - ms). in both fetuses and neonates as well, there was a statistically significant difference (p< . ) between the two groups in the c -component and the sm group showed faster aer compared to the lr group. conclusion: meg technique provides a non-invasive approach to study the effects of smoking on developing fetal and neonatal brain. the observed decrease in the latency of fetuses and neonates of the smoking mothers could indicate a hypersensitive cortical response to auditory tone. adenosine (ado) modulates metabolism in adult mammals through multiple mechanisms that involve ado a and a a receptors. objective: this study was designed to test the hypothesis that ado a and a a receptors participate in fetal metabolic homeostasis. methods: experiments were performed in chronically catheterized fetal sheep (> . term). intravascular infusion for h of dpcpx (a receptor antagonist) or zm (zm, a a receptor antagonist) was performed alone or in concert with ado administration. the highly selective ado receptor antagonists were also infused in fetuses in which hypoxia was induced for h by having the ewe breathe a hypoxic gas mixture (fio = . ). data were analyzed by two-way repeated measures of anova. results: blockade of ado a receptors (n= ) increased significantly (p < . ) fetal concentrations of glucose [control (c): . ± . (se)]; experiment (e): . ± . mg/dl] and lactate (c: . ± . ; e: . ± mg/dl) without significantly altering insulin levels and arterial blood gases or ph. antagonism of ado a a receptors (n= ) did not affect plasma levels of glucose, lactate, or insulin. intravenous infusion of ado (n= ), which did not alter pao or paco , increased concentrations of glucose (c: . ± . ; e: . ± . mg/dl) and lactate (c: . ± . ; e: . ± . ). zm (n= ), but not dpcpx (n= ), abolished ado-induced rise in glucose and lactate concentrations. isocapnic hypoxia (pao torr), which increases ( - fold) fetal plasma ado levels to those similar to ado infusion, decreased arterial ph (c: . ± . ; e: . ± . ), and increased fetal levels of glucose (c: . ± . ; e: . ± . mg/dl) and lactate (c: . ± . ; e: . ± . mg/dl) without altering changing insulin concentrations. these effects of hypoxia were not altered by dpcpx or zm. conclusions: ) a receptors modulate plasma levels of glucose and lactate in normoxic fetuses; ) a a receptors mediate the adoinduced rise in plasma glucose and lactate; and ) a and a a receptors are not significant modulators of hypoxia-induced changes in plasma levels of glucose and lactate. supported by usphs hd- . objective: to investigate the dynamic changes of the relationship between leptin, adiponectin and resistin in maternal and fetal circulation during pregnancy and in the early post-natal period. materials and methods: thirty pregnant women with uncomplicated singleton pregnancy delivered at term. maternal and fetal/neonatal venous blood samples were obtained at delivery and at hours from birth. leptin, adiponectin and resistin were measured by specific elisa assays. neonatal anthropometric measurements, glucose metabolism and lipid profile, blood pressure information were obtained. statistical analysis was performed by anova followed by student t test or duncan's test whenever appropriate. correlations were calculated by using the pearson coefficient. results: adipokines concentration at birth and at h from birth was showed in table. multivariate regression analysis showed that fetal leptin levels were positively associated with female gender and adiponectin levels, but not with anthropometric characteristics. fetal leptin and adiponectin levels were not correlated with maternal concentration, whereas fetal and maternal resistin levels were. fetal and maternal resistin concentration was positively associated with gestational age and birth weight and fetal resistin levels correlated negatively with lipid profile. after birth leptin concentration in maternal and neonatal circulation decreased dramatically and the correlation between leptin and adiponectin levels was lost. a positive correlation between resistin and leptin concentration in neonatal circulation was found at h from birth. conclusions: in contrast to adult life a positive correlation between leptin and adiponectin is present in fetal life. placental secretion of leptin, but not of adiponectin and resistin, contributes significantly to maternal and fetal circulating levels. significant changes in the relationship among adipokines occurred immediately after birth and may affect growth and development in early post-natal period. adipokines concentration in maternal and fetal circulation ) antagonism has been shown to normalize placental perfusion and fetal growth in several rat models of fetal growth restriction. however, direct administration of et a antagonists to newborn rats within hours of delivery has been consistently associated with neonatal demise due to failure of the ductus arteriosus to close, raising concerns about the safety of their use late in pregnancy. perinatal exposure to et a antagonists (maternal administration in late gestation) and its impact on rat pup survival and oxygen saturation has not been investigated. objective: to determine the impact of a maternally administered et a antagonist on oxygen saturation in newborn and -day-old rat pups. methods: timed pregnant sprague-dawley rats were treated with fr ( mg/kg/day; et a antagonist) or . % nahco vehicle, by subcutaneous osmotic pump connected to an intravenous catheter, from gestational day (term= days) through parturition. all five pregnant rats in each group delivered spontaneously and nursed their pups through postpartum day . oxygen saturation of each rat pup was measured by pulse oximeter on postpartum days and . results are presented as means ± se. newborn -day neonate vehicle . ± . . ± . eta antagonist . ± . . ± . there were no statistically significant differences between the treatment groups. maternal administration of an et a antagonist from gestational day through parturition, at a dose sufficient to ameliorate fetal growth restriction, has no adverse impact on oxygen saturation in neonatal rat pups. helen l torrance, jan b derks, martijn a oudijk, avnesh s thakor, tereza cindrova-davies, frank van bel, gerard ha visser, graham j burton, dino a giussani. perinatal center, university medical center utrecht, netherlands; department of physiology, development neuroscience, university of cambridge, united kingdom. introduction: the management of perinatal asphyxia remains a major concerns in obstetrics. umbilical cord compressions (ucc) induce fetal asphyxia and ischaemia-reperfusion (i/r). i/r increases reactive oxygen species, for instance via activation of the xanthine oxidase (xo) pathway, which may promote oxidative stress in the fetal circulation. while treatment with allopurinol of asphyxic human neonates reduced free radicals and improved cardiovascular status, treatment started postnatally was deemed too late to prevent oxidative damage (benders et al. arch dis child : , ) . consequently, in complicated pregnancy, recommendations to treat the fetus via the mother, rather than the neonate, with allopurinol are being entertained today. we investigated the effects of maternal allopurinol treatment on indices of oxidative stress in the fetal heart following repeated ucc in sheep. methods: at . of gestation, surgically instrumented sheep fetuses were submitted to i/r ( x min repeated ucc) under maternal allopurinol (n= ) or saline vehicle (n= ) infusion. fetal hearts were collected h after i/r and snap frozen for measurement of (anti)oxidant proteins by western blot. hearts from uninstrumented fetal sheep at . gestation served as controls. statistical comparisons were made using one-way anova. results: i/r episodes led to increased expression of cox- , enos, hsp and decreased expression of sod and gluthatione peroxidase (gpx) in the fetal heart, findings consistent with cardiac oxidative stress. maternal treatment with allopurinol ameliorated these effects (fig. objectives: hypoxic-ischemic fetal brain injury (hie) is a major cause of neonatal death and morbidity. evidence suggests that the brain cell injury associated with chronic hpx (in contrast to an acute ischemic reperfusion injury) is a complex process reflecting a series of adaptive intracellular events that are duration and gestational age dependent. we applied advanced proteomic tools as a next step toward ascertaining a more complete understanding of the impact of hpx on the fetal brain proteome. methods: time-mated guinea pigs were housed in a chamber beginning on day for d, breathing either room air (nmx), or . % or % o ( % o hpx or . % o hpx). on day (term), the fetal brains were removed and preserved for study. total protein was extracted, and the proteome first characterized by d gel electrophoresis. the density of the resulting protein spots were acquired and analyzed using the gs densitometer and pdquest software. identified spots of interest were trypsin digested and subject to maldi mass spectrometry using the proteomics analyzer mass spectrometer for peptide mapping or sequencing. the hpx-induced protein spots were identified based on a minimum of a x change from nmx. results: hpx had a clear effect on the fetal brain proteome. superoxide dismutase (sod), heat shock protein (hsp ), actin (actg ), interleukin- (il- ), and glutamine synthetase (gs) were each up regulated, while cofilin- , brain-type creatine kinase (bb-ck), and - gtp binding protein were each down regulated. all protein changes were proportional to the hpx ( % o hpx vs nmx, % o hpx vs . % o hpx, p< . ). conclusions: this initial application of proteomic techniques confirms that fetal brain damage secondary to chronic hpx (in contrast to acute hpx) is a complex processes characterized by fetal adaptations mediated by multiple protein activations and inactivations. while sod, il- , and gs have each been previously investigated, the potential roles of actg , bb-ck, beta- gtp binding protein, and cofilin- in the fetal response to hpx and the associated brain damage are unclear and represent strong candidates for future investigation. acknowlegement: this study was supported by grants from the phs (r hl - , cpw) and cdc grant (u dp - , cpw). intermittent umbilical cord occlusion occurs in % of human pregnancies.given that elastogenesis within the vascular wall is in part mediated by hemodynamic conditions during development, the blood pressure response to acute hypoxic insults such as cord occlusion may alter arterial composition. elastin content of a central and peripheral artery and blood pressure responses in fetal sheep exposed to varying degrees of cord occlusion were determined. methods: over a day period, near term fetal sheep received total umbilical cord occlusion (uco) lasting min/ hour (mild group; n= ), min/hour (moderate group; n= ), min/hour (severe group; n = ) or no occlusion (control group; n= ). fetal arterial blood samples were drawn min prior to and at the end of cord occlusions. mean arterial pressure (map) was monitored continuously. the carotid and superior mesenteric arteries were excised and a colorimetric assay (biocolor) performed for determination of elastin content. results are presented as mean ± sem. results:umbilical cord occlusions produced decreases in fetal arterial oxygen pressure and oxygen saturation that were progressively more pronounced across mild, moderate and severe uco groups (p < . ). lactate concentration rose during occlusions in the moderate and severe groups, but not the mild group (p < . ).elastin content of the superior mesenteric artery did not differ between the experimental groups and the control group. elastin content of the carotid artery and map response elastin content (μg/mg tissue) max ∆ in map duration of rise in map (min) control . ± . . ± . -mild . ± . . ± . ** . ± . moderate . ± . . ± . ** † . ± . † severe . ± . * . ± . ** † † . ± . † † * p < . ; ** p < . : experimental groups compared to control † p < . ; † † p < . : compared to mild group the max in map was positively correlated with elastin content (r = . , p < . ). conclusions:the transient rise in blood pressure and preferential blood flow to the brain that occur in response to acute hypoxemia during severe intermittent umbilical cord occlusion induce an increase in elastin synthesis in the carotid artery.this may give rise to adaptive programming of postnatal central arterial compliance. electrocortical activity during repetitive umbilical cord occlusions (uco) with worsening acidemia in the ovine fetus near term. martin g frasch, roy mansano, michael g ross, robert gagnon, bryan s richardson. obgyn, chri, univ of western ontario, london, canada; obgyn, harbor-ucla med ctr, torrance, ca. objective: uterine contractions during labour can restrict umbilical blood flow compromising fetal oxygenation and leading to adverse neonatal outcome including newborn encephalopathy/subsequent cerebral palsy. while electronic fetal heart rate (fhr) monitoring is widely used for assessment during labour, abnormal fhr patterns as used clinically have a poor positive predictive value for concerning/significant acidosis at birth. there is limited study of fetal electrocortical activity (ecog) in animal models with induced hypoxia/ acidemia as might be seen during labour. thus, we aimed to induce repetitive ucos in fetal sheep leading to worsening acidemia to determine the predictive value of ecog activity for fetal compromise. methods: near-term fetal sheep (n= ) underwent chronic preparation with artery catheters, ecg/ecog electrodes and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) ucos with each series lasting h or until fetal arterial ph decreased to < . . fetal arterial blood samples for blood gases and ph were taken at selected time points during the baseline, ucos, and recovery periods. arterial blood pressure (abp) and fhr were continuously monitored and spectral edge frequency (sef) was calculated from ecog. correlation of sef with fhr change was calculated for time intervals using sef maxima and fhr minima during uco series. data are presented as means±sem. results: repetitive ucos led to development of a marked acidosis (ph . ± . to . ± . , p< . ). ± min prior to fetal ph drop < . , the sef of ecog began to increase abruptly during each fhr deceleration from ± hz up to ± hz (p< . ) and was correlated to both fhr change and a decrease in fetal abp during each fhr deceleration at this time (p< . ). conclusion: our findings suggest that in the animal model studied ( ) fetal ecog activity is impaired with progressive acidemia accompanied by fhr decelerations and pathological abp decreases; ( ) there is a consistent temporal relationship between the occurrence of ecog alterations and the subsequent critical drop of ph < . . these findings could contribute to improvement in the clinical ability to predict fetal compromise during labour using ecog/fhr monitoring. background: the ductus arteriosus (da) plays a pivotal role in fetal development and circulation. during gestation, patency of the fetal da is maintained by nitric oxide (no) and prostaglandins (pg). no and pgs are downstream effectors of estrogen (e ) and progesterone (p ). however, the roles of e and p in da regulation have not been studied. objective: we hypothesized that: e and p have opposite effects in the da; that rising e and falling p levels help trigger da closure via specific hormone receptors; and that no and/or pgs mediate da responses to e and p . methods: expression of er , er , pra, prb, and the putative membrane receptors mpr-, -, -, pgrmc- , - , and serbp- was examined by rt-pcr and qpcr on days , , (term), and p . the effects of e and p ( - - - m) on the d fetal da were examined in a cannulated microvessel myography system. e and p effects were also studied in the presence of receptor antagonists (ici , ru ) and no and pg inhibitors (l-name, indomethacin). results: er and pr receptors were expressed at low levels; pgrmc- expression was stronger than other membrane prs, and increased with advancing gestation. under fetal o conditions, e induced rapid, concentrationdependent dilation at - - - m (n= ); p induced progressive vasodilation at all doses (n= ). e and p -induced dilation occurred within - seconds of exposure. while e -dilated das did not respond to ici, ici-pretreated das failed to dilate to the same dose of e (n= ) suggesting antagonism of e effects. the vasodilatory effects of e were partially inhibited by pretreatment with l-name. p -dilated das did not respond to ru , whereas ru pretreated das showed a small, significant response to p (n= ), suggesting partial antagonism or signaling via alternative, ru -independent pathways. pre-treatment with indocin did not block the vasodilatory effects of p . conclusions: contrary to our expectation, both e and p have dilating effects on the fetal da, via no, pgs and non-no, non-pg pathways. expression studies and the rapid response of ex vivo das are consistent with non-genomic actions via membrane receptors. hormone shifts in parturition may have longterm effects on da preparation for postnatal closure, but strategies to maintain fetal da patency or treat newborns with pda will require better understanding of this process. background. for the fetus, arterial blood gases are critical in the regulation of cerebral blood flow (cbf) and cerebral oxygenation. however, the relation of cbf, cortical tissue po (tpo ), and electrocorticographic (ecog) activity to arterial o tension (pao ) are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that in the near-term fetus, acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with ecog state. methods. by use of a laser doppler flowmeter with a fluorescent tissue o probe, and with fluorescent-labeled microspheres, and with ecog electrodes, in near-term fetal sheep (n = ) we measured laser doppler cbf (ld-cbf), tpo , ecog (root mean square (rms) voltage with high voltage low frequency, hvlf versus low voltage high frequency, lvhf) and spectral edge frequency- % (sef ) in response to min moderate isocapnic hypoxia. results. ld-cbf, cerebral o delivery, tpo , and several other variables correlated highly with ecog state. in the normoxic control fetus, in association with a shift from hvlf to lvhf ecog activity, tpo decreased briefly to ± from a control value of ± torr; however, as ld-cbf increased ± %, and sef increased to ± from ± %, tpo returned to near normal value. with acute hypoxia (pao = ± torr) when in the lvhf state ld-cbf increased only ± %, as opposed to a ± % increase when in hvlf ecog state. with this degree of hypoxia, tpo decreased to ± torr, sef remained at ± %, and cerebral metabolic rate for o (cmro ) decreased ± % (p< . ). conclusions. for the near-term fetus, normoxia with changes in ecog state was associated with brief periods of decrease in tpo , which were restored quickly by increased ld-cbf. in contrast, acute hypoxia was associated with a significant depression of cortical tpo , cmro , and ecog state, with increased ld-cbf failing to restore cortical tpo . thus, we reject the null hypothesis that in such fetuses, hypoxia demonstrates no compromise in cerebral oxygenation. (supported by usphs hd- ). releasing hormone and arginine vasopressin in the ovine fetus. charles a ducsay, kanchan m kaushal, malgorzata mlynarczyk, kimberly hyatt, dean a myers. ctr. for perinatal biol., loma linda univ., loma linda, ca; ob/gyn, univ. oklahoma hlth. sci. ctr., oklahoma city, ok. background: long term hypoxia (lth) profoundly affects the hypothalamicpituitary-adrenal axis of the fetal sheep. we previously showed that lth causes augmented corticotrope function. the present study was designed to test the hypothesis that lth enhances sensitivity to the acth secretagogues; cortiocotrophic releasing hormone (crh) and arginine vasopressin (avp) resulting in increased anterior pituitary corticotrope secretion of acth. methods: pregnant ewes were maintained at high altitude ( , m) from day to - of gestation (dg), when they were returned to the lab and a maternal tracheal catheter was implanted. maternal po was maintained at a level comparable to that observed at altitude ( mmhg) by nitrogen infusion. on dg, lth (n = ) and age-matched, normoxic control (n = ) fetuses were implantated with vascular catheters. each fetus received a min infusion of either saline vehicle, ng/kg of ovine crh or ng/kg of avp (estimated body weight/min) in a randomized order over consecutive days ( - dg). blood samples were collected at min (baseline prior to infusion), , , and min following the start of the infusion and analyzed for acth, as well as the acth precursors pro-opiomelanocortin and the major processing intermediate kda proacth. results: vehicle had no effect on any of the measured parameters. with crh infusion, acth (pg/ml) increased in both groups over the course of the study. however, peak concentrations (at min) were significantly higher in the lth group compared to control ( ± vs. ± , respectively; p< . ). acth precursor secretion (pm) was greater in lth fetuses compared to controls during the experiment (p< . ). in response to avp, peak acth concentrations were also higher in the lth fetuses compared to control ( ± vs. ± respectively; p< . ), however peak levels were reached at between and min after start of infusion with levels in both groups returning to pre-infusion values. a similar pattern was observed with precursor levels ( . ± . vs. . ± . , p< . , lth vs. control). conclusions: lth significantly increases pituitary sensitivity to both crh and avp. this enhanced sensitivity may be mechanism of our previously observed enhanced corticotrope function. (supported my nih grants hd and hd ). martin g frasch, roy mansano, michael g ross, robert gagnon, bryan s richardson. ob/gyn, chri, university of western ontario, london, canada; ob/gyn, harbor-ucla med. ctr., torrance, ca. objective: repetitive uco leading to worsening fetal acidosis with fetal heart rate (fhr) decelerations (fhr dec ) are accompanied by pathological decreases of fetal arterial blood pressure (bp). we hypothesized this bp change may be caused by the bjr, a vagally mediated reflex with bradycardia and hypotension to reduce cardiac workload, via stimulation of cardiac chemoreceptors during systemic acidemia. methods: ten near-term fetal sheep ( ± dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. after a control period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) uco each lasting h or until ph decreased to < . . fetal arterial blood samples were taken at selected time points during the control and uco periods. bp and fhr were continuously monitored. individual fhr nadir during each fhr dec and accompanying bp change ( bp = [bp at the time of a fhr nadir] -[bp at baseline preceding a uco series]) were determined during all uco. data are presented as means±sem. results: control period ph ( . ± . ), fhr ( ± bpm) and bp ( ± mmhg) were within the physiological range. average depth of fhr dec for all uco was ± bpm and increased with higher lactate concentrations (r = . , p < . ) and lower ph (r = . , p = . ). bp during all uco demonstrated an initial hypertensive response to fhr dec , which decreased with lower ph (r = . , p < . ). bp increased ± mmhg (p < . ) during mild uco (ph to . ± . ), ± mmhg during moderate uco (ph to . ± . , p < . ) and ± mmhg during severe uco (ph to . ± . , p < . ). conclusion: these results suggest that bjr, a short-acting, ph dependent depressor reflex, blunts the physiologic hypertensive response to cord occlusion insults leading to worsening acidemia as might be seen in the human fetus during labour with repetitive variable fhr dec . the failure to increase bp may prevent optimal blood flow distribution responses necessary for preservation of vital organs. role of nos and pde in placental dysfunction following fetal bypass. mitali basu, r scott baker, christopher t lam, kenneth e clark, pirooz eghtesady. cardiothoracic surgery, cincinnati children's hospital, cincinnati, oh, usa; ob/gyn, university of cincinnati, cincinnati, oh, usa. introduction rising placental vascular resistance following fetal cardiopulmonary bypass (bypass) remains the achilles' heel of fetal cardiac surgery. we have previously shown in real-time that nitric oxide (no) production rises during bypass and falls post-bypass, while cgmp levels rise throughout. using immunohistochemical and western analysis of placenta, we examined the involvement no pathway components in this placental vascular pathophysiology. ovine fetuses at - days gestation were placed on bypass for minutes and followed post-bypass for hours. placental samples were collected immediately prior to bypass and at and min post-bypass (n= ) and compared to a group of similarly instrumented controls, (n= ). placental enos, inos and pde protein expression was measured using standard methods and relative expression normalized to beta-actin. statistical analysis utilized students t-test, and anova for trend and group-wise analysis, (significance at p= . ). pre-bypass protein expression did not differ between groups. pde protein levels and phosphorylated pde- expression were both elevated min post bypass, and reduced min post bypass compared to sham, (p= . ). enos levels in the bypass group increased linearly from pre-bypass to min postbypass (p< . ), and were also elevated compared with shams (p< . ), while shams had declined significantly by min post bypass, (p< . ). similarly, phosphorylated enos expression in the bypass group increased linearly from pre-bypass to min post-bypass, (p= . ), while shams trended towards decline by min post-bypass. simultaneously, placental inos expression remained stable within groups, but was lower in the bypass group at and min post-bypass, (p< . ). the preceding data correlated with observed immunohistological changes in the same placental cotyledons. fetal bypass leads to significant increases in placental protein levels of pde , phosphorylated pde- , enos and ostensibly phosphorylated enos. increased pde expression may be a response to increased no and the generated cgmp. this data suggests a compensatory upregulation of pde and enos that eventually fails, leading to increasing placental vascular resistance and subsequent lethal placental dysfunction. angiotensin receptors (rat-ii) in neuronal nuclei of the brainstem have been implicated in integration of baroreceptor responses. in near term fetal sheep, we have previously shown that days of mild chronic hypoxemia increases heart rate (hr) and blood pressure (bp). the aim of the present study was to investigate the effects of chronic mild hypoxemia on the expression of rat-ii in the medulla oblongata and on baroreflex control of the circulation. methods: at days of gestational age, pregnant sheep were submitted to days of hypoxemia ( % of fetal arterial po ; at day fetus ± vs ± . ; mother ± vs ± mmhg). hr and arterial bp were continuously recorded from mother and fetus in control (n= ) and hypoxemic (n= ) animals. baroreflex sensitivity (brs) as well as hr and diastolic bp variability were analyzed (nevrokard software). brainstem was collected and rat-ii expression in the nucleus tractus solitarius (nts) and dorsal motor nucleus of the vagus (dmnx) was measured by autoradiography using i-sarthran labeling. data are shown as mean±sem and were analyzed by t-test. results: hypoxemia induced a greater total binding of i-sarthran in nts and dmnx resulting from an increased expression of at receptors. in the time-domain analysis of brs hypoxemia induces lower baroreflex sensitivity in the fetus (brs in ms/mmhg; . ± . vs. . ± . , p< . ). in the frequency domain, hypoxemia changes the relative power of low frequencies (lf: . - . hz) and high frequencies (hf: . - . hz) of rri and dbp with an increased value of the lf/hf ratio (rri lf/hf . ± . vs ± . , p< . ; dbp lf/hf . ± . vs . ± . , p< . ). also the alpha index for baroreflex sensitivity is decreased in hypoxemic animals (alpha lf . ± vs . ± . , p< . ; alpha hf . ± . vs . ± . , p< . ). no differences were observed in maternal variables. conclusions: our results suggest a link between a prenatal insult, alterations in cns receptors and functional alterations of baroreflex responses. a higher sympathetic outflow, suggested by a greater lf/hf ratio, and impaired reflex gain, both possibly mediated by increases in nts rat , may have potential long-term consequences for the development of hypertension. hd ;hd ;hl . objective : we evaluated velocity profiles, relative wall distension rate (rwdr), and wall shear rate (wsr) of fetal descending aorta (fda) in uncomplicated singleton gestations. study design: ninety seven uncomplicated singleton fetuses were studied throughout gestation using multigate spectral doppler analysis (msda) working with gasp software. this consists of a personal computer (pc) add-on board including a single high-speed digital signal processor. the analysis of echo-signals backscattered from range cells located along the axis of the interrogating ultrasound (us) beam. post-processing was accomplished using gasp software. statistical analysis consisted of spearman correlation and chi-square test. results: velocity profiles, wall distension, wall shear rate were obtained from fetal descending aorta throughout gestation establishing gestational age specific norms. wdr[%] is highly correlated with gestational age in appropriate growth fetuses ( . ± . , rs= . p< . ) with linear regression with standardized coefficient of . (p< . ). in contrast, wsr ( ± ) is unchanged during the first , second and third trimesters (p= . ).conclusion: we speculate that the relative wdr changes observed during gestation, in normally grown fetuses, may be secondary to adaptive vascular and autonomic responses and the evolving composition of the vessel wall, particularly with respect to elastin. conversely, the mean wsr for the study group was independent and constant throughout the gestation. these findings suggest that there is an increase in the diameter of the fetal aorta, which provides adaptation to the progressive flow demands, while preserving other key hemodynamic parameters. in this study, we have established normative values of rwdr and wsr, which are new rheological parameters that may be useful in distinguishing normal and pathologic hemodynamic states. objective: placental dysfunction is a key barrier to successful fetal cardiopulmonary bypass (cpb) for repair of congenital heart defects in utero. endothelial cells regulate vascular tone during fetal cpb through interactions of vasodilation by nitric oxide (no) and endothelin- (et- )-mediated vasoconstriction. the objective was to determine the time during fetal cpb when endothelial cell-mediated changes occur. methods: human umbilical vein endothelial cells (huvec) were cultured in media containing % serum collected from ovine fetuses (n= ) that underwent min of cpb, then were maintained for min. serum was collected before cpb, from pump prime before initiation of cpb, min on cpb, or and min after fetal cpb. control cells were cultured in normal fetal serum. cells were harvested and hr after addition of fetal serum. no production was measured in real time with an electrochemical detection system (inno-t, harvard apparatus). et- was measured in the culture media by elisa. results: no production by huvec after and hr was stimulated above control levels by fetal serum collected during and up to min after fetal cpb (p< . ). serum collected from fetuses that were surgically instrumented, but not yet subjected to cpb, decreased no levels below controls (p<. ). stimulation of et- after and hr of huvec culture peaked with serum collected at min after fetal cpb (p<. compared with control), but was elevated above control levels at each collection time point (p<. , table ). conclusions: fetal cpb releases serum proteins that elevate endothelial cell no and et- production during and for at least min after cpb. although the specific regulatory proteins remain to be identified, the no and et- pathways share circulating mediators and participate in a feedback loop to modulate vascular tone. to test the hypothesis that hypoxia-induced upregulation of no is linked to cardiac inos expression, a selective inos inhibitor, l-nil (l-n -( -iminoethyl)-lysine), was administered in vivo to fetal guinea pigs and no levels measured in fetal hearts. methods: pregnant guinea pigs were exposed to either normal room air (normoxia; %o ) or . %o (hypoxia) in a hypoxic chamber for days prior to term (term= d). l-nil was administered to pregnant normoxic and hypoxic guinea pigs via their drinking water at a dose of - mg/kg/d for days. at d gestation, pregnant sows were anesthetized and near-term fetuses removed via hysterotomy. the fetal hearts were excised, weighed, and normalized to their respective fetal body weights. left cardiac ventricles were obtained and frozen in liquid n and stored at - o c until ready for analysis. the effect of total no product (no and no -, nox) of left ventricles of fetuses exposed to normoxia (n= ), hypoxia (n= ) and hypoxia plus l-nil (n= ) was quantified by a commercial fluorometric no assay kit. results: intrauterine hypoxia significantly reduced fetal body weight by % and increased placenta/fetal body wt by % as expected for hypoxic stress. hypoxia induced a slight increase in heart/fetal body wt by %. fetal cardiac nox levels (pmoles/mg) were increased by hypoxia ( . + . ) by . fold compared to normoxic controls ( . + . ). l-nil significantly decreased (p< . ) nox levels in hypoxic hearts by % ( . + . vs . + . ; hypoxic vs hypoxic+l-nil, respectively). conclusion: l-nil inhibits inos-derived no generation in the hypoxic fetal guinea pig heart. since previous study in our lab showed a significant increase in inos expression but a decrease in enos and no change in nnos expression, we hypothesize that hypoxia upregulates cardiac no generation via the inos pathway. further study is needed to identify the important role of cardiac inos in the adaptive response of fetal hearts to chronic intrauterine hypoxia. objective: fetal asphyxia-mediated metabolic acidosis results in a ph decrease and an increase of lactate and base deficit in blood (bd blood ) and extracellular fluid (bd ecf ). it is not clear whether bd blood and bd ecf are similarly altered with worsening fetal acidosis. in the present study we sought to study the dynamic relations of bd blood and bd ecf to lactate in the ovine fetus subjected to repetitive uco with worsening acidemia. methods: ten near-term fetal sheep ( ± dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) uco with each series lasting h or until fetal arterial ph decreased to < . . fetal arterial blood was sampled at baseline, immediately before and during the first mild, moderate and severe uco and at min intervals during the moderate and severe uco. bd gap (bd blood -bd ecf ) was studied in relation to lactate. presented as means±sem. results: lactate correlated to bd blood (r= . , p< . ). bd ecf correlated strongly with bd blood (r= , p< . ). bd ecf increased by . ± . mmol/l more than bd blood from baseline to ph nadir, with bd gap therefore decreasing with increasing lactic acidemia ( fig. ) . lactate, bd blood , bd ecf and bd gap correlated to ph (r= . , r= . , r= . and r= , respectively, all p< . ) and ph could therefore be predicted with any of the four acid-base parameters. conclusion: the increases of bd blood and bd ecf and the decrease of bd gap with increasing lactic acidemia suggest a relatively more rapid accumulation of [h+] in ecf during uco of increasing severity. this may be because the metabolic build-up of acidosis occurs primarily in the tissues and the endothelial permeability for [h+] is impeded during increasing acidosis with atp depletion thus decreasing [h+] movement from ecf to plasma. previous studies have shown that intraperitoneal transplantation of human umbilical cord blood (hucb)-derived mononuclear cells led to the specific 'homing' of these cells to a hypoxic-ischemic brain lesion in perinatal rats. motor deficits resulting from the lesion were alleviated upon transplantation. thus, the presence of hucb cells at the lesion site seems to be a major prerequisite for their potential beneficial effect. however, the mechanisms of cell 'homing' are still unclear. in this study, we focused on elucidating mechanisms underlying the specific migration of hucb-derived mononuclear cells to the brain lesion. one possibility to induce cell 'homing' are chemotactic signals present at the lesion site. the cxc chemokine stromal derived factor- (sdf- ), which was previously shown to be a potent chemoattractant for directed migration of other stem and progenitor cells, is a putative candidate in our lesion paradigm. therefore we investigated the spatial and temporal expression of sdf- in brain hemispheres with or without hypoxic-ischemic lesion. sdf- expression was substantially increased at the lesion site during the investigated period of fourteen days after the insult. furthermore, hla-positive hucb cells were mainly detected in sdf- expressing brain regions and we were able to show that these cells express the sdf- receptor cxcr on their surface. the functional implication of sdf- in directing hucb cell migration was determined by application of neutralizing sdf- antibodies in vivo, resulting in a reduced number of hucb-derived mononuclear cells at the lesion site. with these functional effects, together with the observed timing and location of its expression, the involvement of the chemokine sdf- in hucb cell 'homing' seems conceivable. novel pathways in inflammation-induced fetal brain injury. michal a elovitz, jinghua chai. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. intro: while survival of extremely preterm infants continues to improve, the number of children with cognitive impairment and/or cerebral palsy is increasing. if preterm birth (ptb) cannot be prevented, then strategies to identify and treat fetal brain injury in the setting of a ptb must be investigated. these studies were performed to explore novel pathways involved in fetal brain injury in the setting of inflammation-induced ptb. methods: cd- mice on e receive intrauterine injection of lipopolysaccharide (lps) or saline. hours after injection, fetal brains from the left upper horn were harvested. fetal brains were removed from each dam with dams per treatment group. separate rna samples were prepared and used for microarray (ma) analysis. all protocols were conducted as described in the affymetrix genechip expression analysis technical manual in the ma core facility using the moe av chip. data analysis was performed using significance analysis for microarray (sam). the brain samples from the same dam were considered as dependent samples. pathway analysis and functional annotation clustering tools were used with david. validations studies using qpcr with fetal brain samples (n= - per group) were performed. results: while there was significant differences in gene expression between lps and saline exposed fetal brains, variability existed even between pups from the same dam. genes were significantly differentially expressed between lps and saline brains (p< . ). with a p value of < . , genes were differentially expressed. pathway analysis revealed significant involvement of ) the cadeherin, cadherin-like, cell fraction and calcium binding (enrichment score . ) and ) ribosomal processing, rna metabolism, pyrimidine metabolism (enrichment score . ). specifically, genes involved in neurogenesis, synaptic function, and neuronal and glial metabolism were most differentially regulated. qpcr confirmed observed fold changes in / genes analyzed. inflammatory pathways were not differentially regulated. conclusions: current theories regarding fetal brain injury in ptb focus on activation of inflammatory processes as essential events. this data suggests that long-term neurological injury in a ptb may be secondary to altered neuronal function, metabolism and/or communication. disruptions in these pathways should be explored as key mechanisms to adverse neurological outcomes in preterm neonates and should be targets for future investigations. regulation of microglial activation in the developing murine brain. mariya hristova, virginia zbarsky, daniel cuthill, adam wallace, donald peebles, gennadij raivich. institute for women's health, university college london, london, united kingdom. introduction: the aim of this study was to assess the process of microglial differentiation in developing white matter, an area of the brain that is particularly vulnerable to damage pre-myelination (approx weeks gestation). microglia form a distinctive non-neuronal component. although related to peripheral macrophages they undergo highly specific processes of regional maturation and differentiation inside the brain with a slimming of the cell body, development of very elaborate crenulated arborised branches and downregulation of most macrophage activation markers. this process is relatively rapid in most grey matter brain regions, but is retarded in and around the subcortical white matter (swm) giving rise to the phagocytic fountains of microglia (fom). methods:we examined the process of deactivation and morphological differentiation in the cortex and swm of mice - days after birth (p -p ) using confocal microscopy for monoclonal antibodies against alpha and beta integrin subunits and the costimulatory factor b . , colocalised with standard microglial marker iba . results: strikingly, only the fom macrophages, but not cortical microglia, strongly expressed typical activation markers alpha- , alpha- , alpha-m, alpha-x, beta- and b . . the data for alpha-x are shown as an example in the figure; cortical microglia are shown in the grey bars and swm in black. fom activation was maximal at p , decreased linearly over p and p and disappeared at p . this process followed the presence of ingested phagocytic material but correlated only moderately with ramification, demonstrated by non-ramified but inactive p cortical microglia and formation of stubby processes in p fom. conclusion: these data describe strong and selective biochemical activation of fom phagocytes in p -p swm, roughly equivalent to early rd trimester human foetal development. this presence of highly active phagocytes in the neighbourhood of vulnerable wm could play an important role in the genesis of axonal damage in the foetus and premature neonate. , pp. - ) . mbp is expressed in mature oligodendrocytes (jakovcevski and zecevic, glia ) . although myelination in the fetal sheep brain, a model extensively used to evaluate fetal development, has been described using conventional staining techniques (barlow, j comp neurol ) the use of specific mbp staining allows a more precise determination of the onset of myelination (antonow-schlorke et al., reprod sci . , , a) . aim: to use mbp expression to determine the trajectory of development of different white matter tracts of fetal sheep brain. methods: the ontogenetic profile of mbp expression was estimated in healthy fetuses at . (n= ), . (n= ), . (n= ), . (n= ), . (n= ), . (n= ) and . (n= ) of gestation (term days). after brain fixation and embedding in wax, sections at the level of the optic chiasm were stained with a monoclonal anti-mbp antibody using the abc-technique. immunohistochemical distribution of mbp was morphometrically assessed in the dorsal internal capsule and cortical white matter tracts, i.e. the lateral centrum semiovale, superficial white matter and median corpus callosum using an image analysis system (scion image . , nih, usa). results: mbp expression of various fetal white matter structures started at different time points; initially in the internal capsule at . of gestation followed by the cortical white matter structures at . of gestation. cortical myelination advanced from the cortical deep white matter via superficial white matter to the corpus callosum reflecting different rates of progression. conclusions: the onset of mbp expression estimated here explicitly antedates previous observations in sheep (barlow, ) the way the embryonic heart functions before cardiac morphogenesis is completed is still subject of many studies. to gain more insight into the early cardiac structure-function relationship, doppler blood flow velocity waveforms at four different locations in the embryonic chicken heart during cardiovascular development were assessed. we collected waveforms using high frequency ultrasound biomicroscopy with a -mhz transducer at hh stages , and which are comparable to humans at to weeks of gestation. waveforms were obtained at the inflow tract, the primitive left ventricle, the primitive right ventricle, and at the outflow tract in ten different embryos per stage. by exploring the time relation between the waveforms, cardiac cycle events were outlined. our results demonstrate that stage and location dependent, intracardiac blood flow velocity waveforms can be obtained in the chicken embryo which reflect stage dependent pumping mechanisms. the blood flow profiles assessed at the four locations in the embryonic heart demonstrated a developmental related increase in velocity. in the primitive ventricle the passive filling wave decreased, whereas the active filling wave increased resulting in a decreased p to a ratio in the course of time. high frequency derived cardiac blood flow velocity characteristics support previous findings that the embryonic heart functions like a suction pump at early development and transforms towards another pumping mechanism at later developmental stages. these findings are of importance for the interpretation of human first trimester cardiac velocimetry studies. figure a . changes in the perimeter of the thymus with gestational age by fetal gender are depicted in figure b . background. for the fetus, the roles of arterial blood gases are recognized to be critical in the regulation of cerebral blood flow (cbf) and cerebral metabolic rate for oxygen (cmro ), cortical tissue po (tpo ), and electrocorticographic (ecog) activity. in addition, metabolites such as adenosine (ado) are important in this regard. nonetheless, the relation of adenosine and its metabolites to these indices of cerebral oxygenation are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with adenosine. the most common cause of perinatal brain injury is chronic hypoxia/ischemia. the mechanisms are complex and poorly understood. applying proteomics tools, we identified a set of hypoxia-related proteins in the fetal guinea pig (gp) brain (companion abstract). one protein, cofilin- , which regulates the rapid cycling of actin assembly and disassembly, was dramatically altered by chronic brain hpx. herein, we test the hypothesis that confilin- modifications during hpx contributes to brain damage via apoptosis and is an example of an adaptive response that becomes maladaptive. methods: time-mated gps were housed in a chamber from d to d (term) with either room air (nmx) or . % or % o . fetal brains were removed and sections prepared for double staining specific to bax and dephosporylated cofilin- . total protein was isolated and equal amounts loaded on a sds gel, separated by electrophoresis, and transfered to pvdf membranes probed with primary antibodies to dephosphorylated and phosphorylated cofilin- , bax and bcl-xl, and incubated with second antibody. protein signals were detected, quantified by densitometry, and normalized to -actin. total rna was isolated, reverse-transcribed, and quantified by real-time pcr using sybr green i labeling. expression was calculated by the ct method using s for control. melt analysis was performed to confirm specificity of the amplification, and efficiency determined by the slope of the standard curve. the mitogen activated protein kinase (mapk) signalling pathway is upregulated in perinatal hypoxic-ischaemic brain. the role of the extracellular signal-regulated kinase (erk) cascade, a pivotal component of mapk signalling, remains unclear with reported actions ranging from mitogenic and trophic effects to a neuronal death-promoting role. the aim of this study was to assess the role of neuronal erk activity using selective neuronal inhibition in a perinatal model of hypoxic-ischemic brain injury. methods: we explored the effects of selective neuronal inhibition of erk activation using transgenic mice expressing dominant-negative (dn) mek , the upstream activator of erk / , which was under the control of the pan-neuronal talpha alpha-tubulin promoter. p-erk immunoreactivity was quantified following a hypoxic inschemic (hi) insult in p c bl/ mice, caused by unilateral occlusion of the carotid artery, followed by hypoxia with % oxygen for mins at °c. the volume of surviving brain on the affected hemisphere was expressed as a % relative to the control side. results: expression of the mek dn was associated with a reduction in erk / activation following hypoxia ischaemia, as assessed by p-erk immunoreativity. compared with the wild type, littermate controls, mek dn animals exhibited significantly decreased volume of forebrain damage brain following unilateral, min hi insult (*p< . , **p< . , anova). similar protective effects of mek dn were observed in cerebral cortex, hippocampus, thalamus and striatum when compared to wild type littermate controls and correlated with a significant reduction in microglial activation in all brain areas. conclusion: overall, these results suggest that neuronal mek and its downstream signals have an important death-inducing role in this model of perinatal brain injury and could serve as potential targets for therapeutic intervention. oup, ) . however, whether melatonin protects placento-fetal development during hypoxic or undernourished pregnancy is unknown. we investigated in rats the effects on placental and fetal growth of maternal treatment with melatonin in hypoxic and undernourished pregnancy. methods: on pregnancy day , wistar rats were divided: control ( % o ) + melatonin ( g.ml - drinking water), hypoxia ( % o ) + melatonin, and undernutrition ( % reduction in food intake) + melatonin (n= per group). on day , dams were anaesthetised, the pups, placentae and fetal brain were weighed. results: relative to controls (fetal weight: . ± . g; brain/body weight ratio: . ± . mg/g; n= ), both hypoxic ( . ± . g; . ± . mg/g, n= ) and undernourished pregnancy ( . ± . g; . ± . mg/g, n= ) promoted asymmetric iugr (p< . ), without affecting placental weight. melatonin treatment had no effect on iugr, but it decreased placental weight in normoxic ( . + . , n= ), hypoxic ( . ± . , n= ) and undernourished ( . ± . , n= ) pregnancies relative to untreated controls ( . ± . , n= ; p< . ). when fetal body weight was expressed relative to placental weight, a measure of placental efficiency, melatonin prevented the fall in the ratio in undernourished, but not hypoxic pregnancies (fig. objective: midkine (mk) is a -kda heparin-binding growth factor with various functions including cell proliferation, migration, differentiation and angiogenesis. mk expression is strictly regulated in temporal sequence; it is highly expressed during midgestation. we recently studied mk expression in the midgestation human fetus, and showed that the highest mk expression were observed in the adrenal gland, brain, lung and kidney. in the present study, we investigated the profile of mk in human amniotic fluid (af) and amnion (am). methods: amniotic fluid and amniotic membranes were collected at diagnostic amniocentesis, preterm no labor, and term no labor. mk protein levels were analysed by western blot. expression of transcripts encoding mk and putative mk receptors were examined by rt-pcr and real-time quantitative rt-pcr (qpcr). results: ) western blot analysis demonstrated abundant mk protein in the human am; mk levels were higher at midgestation than at term ( -fold: wks vs. wks). ) tissue transglutaminase known to polymerize mk was abundant in af. ) qpcr revealed that mk mrna was not expressed in am whereas it was highly expressed in the fetal adrenal, kidney and lung (positive controls). ) among the receptors implicated in mk signaling, lowdensity lipoprotein receptor-related protein and syndecan- were expressed in am while protein-tyrosine phosphatase, anaplastic lymphoma kinase, and syndecan- were not. conclusions: abundant mk protein in the midgestation af is likely to be derived from the fetus. mk in af may play a role in feto-amniotic communications and/or development of fetal organs exposed to af. short term hfix expression. we hypothesized that long term hfix expression could be achieved in fetal sheep using an aav vector, without stimulating an immune response to the transgenic hfix protein. we injected aav hfix vector ( - x p/kg) into the peritoneal cavity of fetal sheep under ultrasound guidance in early (n = ) or late (n = ) gestation. fetal blood was retrieved by ultrasound guided sampling from the intra-hepatic umbilical vein. fetal and lamb blood was tested for hfix expression using elisa, antibody responses using functional assays, and for liver damage up to a year after birth. vector spread was detected in maternal and fetal tissues by quantitative pcr analysis. results: the highest level hfix was detected days after late ip injection ( % and % normal human levels). early gestation ip injection gave . % and . % at and days after injection. hfix levels dropped rapidly correlating with the increase in size of the fetal liver and lamb. however, hfix was detectable at a low levels ( . %) year after birth in early and months after birth in late gestation injected lambs. up to year after birth, liver function tests and bile acid levels were normal, showing no evidence of liver pathology. no functional antibodies to the hfix protein or aav vector were detectable. high vector levels were detected in the fetal liver, and other peritoneal organs; no vector was present in fetal gonadal tissue. conclusion: hfix expression is detectable up to year after delivery of aav vector to the fetal sheep using a clinically applicable method. this is the first study to show long term hfix expression after fetal gene therapy in a large animal. further work will include testing for immune tolerance to exogenous hfix protein in these animals. objective: placental estrogens play a pivotal role in the endocrine control of pregnancy and may be involved in the key changes occuring during parturition. it has been established that crh interacting with crh receptor has a positive effect on estrogen production throughout pregnancy. urocortin (ucn ), a novel peptide of the crh family binding exclusively crh receptor , is expressed by human placenta and the aim of the present study was to evaluate the influence of ucn on estrogen biosynthesis in cultured trophoblast cells. methods: cultured term placental cells were treated with various concentrations of ucn in the presence of the estrogens precursors dehydroepiandrosterone sulphate (dhea-s), androstenedione or testosterone. estradiol secretion was measured in the culture medium using a specific elisa, p arom mrna and protein expression were evaluated by real time pcr and western blot analysis respectively. results: the addition of ucn , in presence of dheas significantly increased e levels at , and hours treatment, while in presence of androstenedione and testosterone an increase in e secretion was detected only at , and hours (at different ucn doses). both p arom mrna and protein were up-regulated in presence of each estrogen precursor and the addition of ucn caused a synergistic increase. anti-sauvagine (a crh type receptor antagonist) resulted in a significantly attenuated ucn effects on e secretion and on p arom mrna and protein expression. conclusions: the present study supports a possible role of ucn on placental e biosynthesis: e secretion and p arom transcript and protein expression were significantly increased after ucn treatment. in conclusion, the crf family may play a major role on placental steroidogenesis, stimulating dheas secretion in fetal adrenals by crh and controlling placental estrogen biosynthesis through ucn . a possible influence on the mechanisms of parturition may be hypothesized. increased expression of kiss- gene in preterm placenta. letizia galleri, michela torricelli, chiara voltolini, fernando m reis, felice petraglia. department of pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. introduction placenta expresses a large number of peptides involved in delivery. neuropeptides, cytokines, are expressed and secreted by human placenta at the time of preterm delivery. human placenta is a major source of kiss- , a -amino-acid peptide encoded by a putative metastasis suppressor gene. kiss- acts on its placental g protein-coupled receptors (kiss- r); this peptide stimulates release of oxytocin in rats, the most potent known uterine stimulant, suggesting a possible role of kiss- in the mechanisms of labor. aim of study the aim of this study was to evaluate placental expression of kiss- at preterm labor. material and methods placental tissue and plasma samples (both maternal and fetal) were collected at term in the absence of labor (tnl), at term spontaneous vaginal delivery (tl), and at preterm labor (ptl). changes in placental mrna expression were determined by real-time quantitative rt-pcr analysis. kiss- protein levels were measured by specific immunoenzymatic assay (elisa). results placental kiss- mrna expression was significantly higher (p< . ) in ptl than in tnl and in tl. however, maternal and fetal plasma kiss- levels did not differ among tnl, tl, and ptl samples. conclusion the present study showed that placental kiss- mrna expression is increased in preterm delivery. further studies are needed to better understand the role of kiss- on cascade leading term and preterm labor. placental angiogenesis is essential for placental development, which occurs under a hypoxic environment ( - % o ) during normal pregnancy. it has been reported that in transformed human dermal microvascular endothelial cells, hypoxia activates erk / , which stabilizes hypoxia-inducible transcription factor- (hif- ). however, signaling mechanisms governing placental angiogenesis under hypoxia is largely unknown. herein, we tested whether hypoxia affected fgf -and vegf-stimulated cell proliferation partly via activating erk / and stabilizing hif- protein levels in human placental artery endothelial (hpae) and transformed human placental microvascular endothelial (hpme) cells. methods: cells cultured under normoxia ( % o ) or hypoxia ( % o ) for days were treated with fgf and vegf. after days, the number of cells was determined. activation of erk / and levels of hif- protein were determined by western analysis. results: under normoxia, fgf and vegf stimulated (p < . ) cell proliferation and induced ( min, p < . ) erk / phosphorylation in hpae and hpme cells. hypoxia promoted (p < . ) fgf -and vegf-stimulated cell proliferation in hpae cells, whereas hypoxia blocked (p < . ) such actions in hpme cells. hypoxia enhanced fgf -, but not vegf-induced erk / phosphorylation in hpae cells. in contrast, hypoxia promoted (p < . ) vegf-, but not fgf -induced erk / phosphorylation in hpme cells. hypoxia increased (p < . ) hif- levels in the hpae cells: first detected at . hr and maintained up to hr. hpme cells had high basal levels of hif- ( folds; p < . ) compared with hpae cells. hypoxia did not alter hif- levels up to hr and decreased (p < . ) hif- levels at hr in hpme cells, conclusions: in hpae cells, hypoxia enhances fgf -and vegf-stimulated cell proliferation possibly partially via promoting activation of different protein kinases. this stimulatory effect is associated with increased hif- protein levels. however, inhibition by hypoxia on fgf -and vegf-stimulated hpme cell proliferation is associated with relatively high hif- levels in the first hr and decreased hif- levels at hr. thus, these data suggest that different signaling mechanisms are involved in hypoxia-modulated growth factor-induced placental angiogenesis in which an increase in hif- levels plays a critical role. objective: corticotrophin releasing factor (crf) is a well established neurohormone involved in the initiation of the stress response. we recently documented that rat placenta is analogous to human placenta in the expression of crf-mrna and protein, and that placental crf release may contribute to in utero meconium passage. time-of-day variation in crf expression is a highly recognized phenomenon at the brain cellular sites of crf synthesis. we sought to determine whether crf expression in rat placenta is subject to time-of-day variation. method: time-dated pregnant rats were obtained on day of gestation (term= days), housed in under h- h light-dark conditions (lights on ). lab chow and water were continuously available. on day , pregnant rats were quickly anesthetized by exposure to isoflurane, abdomen opened and fetuses and placenta exteriorized either at - hr (zeitgeiber time, zt ) or - hr (zt ). individual placentas were processed either for rna extraction or immunohistochemical investigation. the levels of crf-mrna were assessed by pcr using rat specific pcr primers. pcr bands were subsequently cloned and sequenced. bouin's solution fixed paraffin sections of placenta were subjected to crf immunohistochemistry with antibodies specific to rat species and intensity of immunostaining was analyzed using image pro-plus software and expressed in arbitrary units (au). results: one specific pcr band of bp size was consistently identifiable in placenta harvested at zt but not at zt . nucleotide sequence of the pcr band confirmed its identity as crf-mrna. intensity of crf-immunostaining was significantly greater at zt in giant trophoblast (gt) cells (gt-crf: zt = . ± . au, zt = . ± . au, p= . ) but not in spongiotrophoblast cells (stc) (stc-crf: zt = . ± . au, zt = . ± . au, p=ns) or labyrinth cells (lbc) (lbc-crf: zt = . ± . , zt = . ± . au, p=ns) . conclusion: similar to adult brain, rat placenta expresses crf mrna and protein. time-of-day variation of crf expression originally seen in central nervous system neurons is also identifiable in giant trophoblasts cells at zt . these findings suggest that stress-mediated placental crf release, and potentially fetal meconium passage, may be dependent upon time-of-day. objectve: transplacental water flow is essential for the provision and maintenance of fetal body water and amniotic fluid. water flow across membranes is regulated by aquaporin (aqp) water channels in many tissues. thus aqp , expressed in the placenta, is a candidate to regulate maternal to fetal fluid exchange. maternal beta-mimetics have been hypothesized to augment fetal growth by increasing the availability of nutrients and perhaps water. as camp acts as a second messenger to increase expression of selected aqps in other tissues, we sought to determine if betamimetics acting through camp could modulate placental aqp , and potentially influence placental water transfer. methods: trophoblastic cell cultures were established in first trimester-derived extravillous htr- /svneo cells and term placenta-like trophoblast carcinoma cell line jeg- . cultures were treated with sp-camp, a membrane-permeable and phosphodiesterase resistant camp, and forskolin, an adenylate cyclase stimulator, in doses of . , and m for hrs (aqp mrna expression) and hrs (aqp protein expression). for time course experiments, cells were incubated with μm of either sp-camp or forskolin for , and hrs at °c, % co . after cell harvest, mrna and protein expression were assayed using real time pcr and western blotting. results: sp-camp and forskolin increased aqp mrna expression in both cell lines after hrs (p< . ) in a dose-dependent manner. protein expression paralleled the increase seen in the mrna. m of sp-camp and forskolin stimulated aqp mrna expression after hrs in htr- /svneo cells and after hrs in jeg- cells (p< . ). m of either sp-camp or forskolin stimulated aqp protein expression in both cell lines after hrs (p< . ) and the expression remained high at hrs. conclusion: aqp gene expression in trophoblast cells is up-regulated by camp agonists. these results suggest that maternal beta-adrenergic agonists or antagonists may modulate maternal-fetal water flux via modulation of aqp water channels. rat placenta expresses corticotrophin releasing factor-binding protein and mrna. jayaraman lakshmanan, thomas r magee, bindu cherian, sharon k sugano, hanalise huff, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor-binding protein (crf-bp), a kda secreted glycoprotein, was originally isolated from human plasma. it binds crf and urocortin i with an equal or greater affinity than does the crf receptor. crf-bp expression has been reported in target tissues such as brain and placenta. based on its ability to inhibit crf actions in vitro, it is speculated to function as a "gate keeper" for crf-initiated stress responses. we recently documented expression of crf and urocortin- in rat placenta. in the present study we sought to establish the expression of crf-bp in rat placenta by immunohistochemical and pcr-analyses. methods: placental tissues (n= ) collected from sprague-dawley pregnant rats on day of gestation (term= ) were either fixed in % paraformaldehyde solution (ph . ) and paraffin embedded or processed for total rna extraction. paraffin sections of five micron thickness were subjected to immunohistochemical analysis with goat polyclonal antibodies to human crf-bp precursor (sc- santa cruz biotechnology, ca) by avidinbiotin-peroxidase complex technique. the structure of cloned human crf-bp precursor exhibit significant amino acid sequence homology among all species studied. immunoreactivities on the sections were quantified using image pro . software and staining intensity (od/area) expressed as arbitrary units (au). all values are expressed as mean±sem. for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, clone into plasmid vector and dna sequenced. results: the crf-bp antibody elicited strong positive staining in decidua, giant trophoblasts, spongiotrophoblasts and labyrinth cells. the results of image analyses revealed (au): decidua: . ± . , giant trophoblast cells: . ± . , spongiotrophoblasts: . ± . , fetal membranes: . ± . . pcr analyses identified a single bp band, consistent with crf-bp. conclusion: our study establishes for the first time that rat placenta is analogous to humans in that both express crf-bp mrna and protein. immunohistochemical findings reveal that crf-bp protein expression occurs at multiple sites within the placenta. expression of per clock protein in rat placenta: an internal timer for placental functions. jayaraman lakshmanan, reuben lakshmanan, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor (crf) expression time-of-day variation occurs in giant trophoblast cells on the maternal side of the rat placenta. this temporal change in crf expression pattern is very similar to that of central nervous system neurons, suggesting that placental cells may use a similar mechanism to read time of the day. the self-sustaining rhythm generating capacity of the suprachiasmatic nuclei is believed to be derived from cell-autonomous, transcriptional feed-back loops dependent on a number of canonical clock genes. in the present study we sought to determine whether rat placenta expresses period gene (per ), one of the clock/cycle related genes. methods: time-dated pregnant sprague-dawley rats were received on day of gestation and housed in a controlled environment ( - h lights on) with free assess to food and water. placentas collected on days and of gestation (term= days) between and . am were fixed in % paraformaldehyde and paraffin embedded. for each gestational ages a total of placentas from different pregnant rats were used. paraffin sections ( sections per placenta) were subjected to immunohistochemical analysis with antibody to per ( : , santa cruz biotechnology, ca) by abc technique and , 'diaminobenzidine as a chromagen. sections were examined under microscope, imunostaining quantified by image pro . software and expressed in arbitrary units (au). data are presented as mean ± sem. statistical significance was analyzed by anova with a p value < . as significant. conclusion: the present results indicate that rat placental cells, devoid of any neural innervations, express per clock protein, similar to central nervous system neurons. based on the differences in the relative intensities between trophoblasts and labyrinth cells on day of gestation, we conclude that fetalmaternal interactions in per regulation disappear at term (or at the time of birth.). our findings imply that per may function as internal physiological modulator in placenta. to determine the mechanism(s) underlying placental time-of-day crf variation, we examined the effect of maternal administration of betamethasone (a synthetic glucocorticoid) on nuclear gc receptor expression in placental cells. method: time-dated pregnant rats (n= ) on day of gestation were given a single subcutaneous injection (at : am) of betamethasone ( μg/kg body weight) while control pregnant rats (n= ) received saline. dams were exposed to isoflurane anesthesia at : am, and placentas harvested, fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analysis with gc-receptor antibody (sc- , santa cruz biotechnology), with immunoreactive material identified by abc technique using , ' diaminobenzidine as a chromagen. percentages of gc-nuclear receptor (gc-nr) positive cells and their intensities (od/area) were quantified using image pro . plus software. all values are expressed as mean ± sem. statistical analysis was by anova with p< . considered significant. results: in placentas of saline exposed pregnant rats, isolated labyrinth (lb) cells (< %) were positive to gc-nr. no positive staining for gc-nrs was seen either in giant trophoblasts (gt) or in spongiotrophoblasts (st). betamethasone administration was associated with a significant and marked increase in gc-nr staining in all three placental cell types (p< . ). among the cells, gt ( ± %) demonstrated a greater percentage of cells expressing gc-nr than did st ( ± %) or (lb= ± %) (p< . ), though there was similar immunoreactive intensities (gt: . ± . , st: . ± . , lb: ± . au; p=ns) conclusion: our findings indicate that all placental cells respond to gc with upregulation of gc-nr with an enhanced response among gt cells. these results suggest that endogenous or exogenous maternal stress-induced gc exposure may influence signaling responses within both maternal and fetal placental compartments. ovine placenta at very-preterm gestation expresses corticotrophin releasing factor (crf), crf-binding protein (crf-bp) and glucocorticoid receptors (gr). jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: in humans, the placenta is the major source of maternal and fetal plasma crf. based on its critical functions, crf is considered as a "placental clock" of parturition. we recently reported that ovine fetal as well as maternal plasma contain measurable amounts of crf at near-term but not at very preterm gestation. we interpret the absence of crf in plasma at very preterm gestation is either due to lack of placental crf expression and/or placental crf release. here, we examined the expression status of crf, crf-bp (a known specific binding protein for crf and a known regulator of crf functions in human placenta) and gr (a known positive regulator of crf expression in human placenta) in ovine placenta collected at very-preterm gestation. method: placenta harvested from time-dated pregnant ewes on ± days gestation were fixed in bouins's solution and processed for paraffin embedding. paraffin sections were subjected to immunohistochemical analysis with polyclonal antibodies specific to ovine crf( : - : , phoenix pharmaceuticals, ca,) crf binding protein ( : - : , sc ) and gr ( : - : , sc: , santacruz biotechnology, ca). immunoreactive material on the sections were identified as brown staining by abc technique using diaminobenzidine as chormagen. immunoreactive material was quantified by image analysis using image pro . plus software and the immuoreactive intensity (od/area) expressed as arbitrary units (au). results: strong positive staining for crf ( . ± . au), crf-bp ( . ± . au) and nuclear-gr ( . ± . au) were noticed in syncytiotrophoblast cells in all placental sections obtained from pregnant ewes at days gestation. control sections exhibited no positive staining. conclusion: our findings indicated that ovine placenta at very-preterm gestation expresses crf, crf-bp and gr. these results suggest that absence of measurable crf in maternal and fetal plasma at very-preterm gestation is not due to lack of placental crf expression but rather due to the absence of regulated crf secretory mechanisms. rat placenta expresses urocortin i protein and mrna. thomas r magee, michael g ross, john d richard, sharon k sugano, jayaraman lakshmanan. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: urocortin i (ucn- ), a amino acid neuropeptide belongs to corticotrophin releasing factor (crf) stress hormone family. in humans, the placenta and several gestational tissues have been reported to express ucn- protein and ucn- mrna. a number of published studies indicate that ucn- is similar to crf in expression pattern and biological functions. we recently reported that rat placenta is a site of crf protein and crf mrna expression. in the present study we sought to determine whether rat placenta expresses ucn- protein and ucn- mrna. methods: placenta (n= ) collected from pregnant rats at day gestation were either fixed in bouin's solution and processed for paraffin embedding or placed in rna later preservative and frozen for rna extraction. for immunohistochemical localization, five micron thickness paraffin sections were cut and immunostained with rabbit polyclonal antibodies to ucn- ( : to : , sigma) by standard abc technique. control sections were incubated with omission of ucn- antibody. immunoreactive materials on the sections were identified using , ' daminobenzidine as chromagen. immunostaining intensity (od/area) was quantified by image-pro plus software and expressed in arbitrary units (au). for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, cloned into a plasmid vector and dna sequenced. all values are given as mean ± sem. results: ucn- polyclonal antibody elicited strong immunostaining in placental trophoblast cells with variable intensity. the pattern of immunostaining is as follows: giant trophoblast cells: . ± . au, spongiotrophoblast cells: . ± . au and labyrinth cells: . ± . au. the relative intensity in labyrinth cells was significantly lower than the other two cell types (p< . ). pcr analyses revealed the presence of a single band of bp, consistent with ucn- mrna. conclusion: similar to human placenta, rat placenta expresses ucn- protein and ucn- mrna, with most expression localized to the maternal side trophoblast cells. these results support our hypothesis that rat placenta can be used as a model to understand the role of this peptide in feto-maternal stress. the role of the nuclear hormone receptors fxr, pxr and car in placental bile acid homeostasis in normal and pathological pregnancy. victoria geenes, peter dixon, selina raguz, jenny chambers, kishore bhakoo, catherine williamson. imperial college, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder characterised by raised maternal serum bile acid levels and associated with adverse fetal outcome. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from an accumulation of bile acids in the fetal circulation. bile acids are the toxic end products of hepatic cholesterol metabolism and are synthesised from weeks gestation. in common with other waste products, accumulation in the fetal compartment is prevented by excretion across the placenta into the maternal compartment. however, studies of maternal and cord serum from normal and oc pregnancies have suggested that bidirectional transfer of bile acids is possible. hepatic bile acid transport and metabolism is regulated by members of the nuclear hormone receptor family, namely fxr, pxr and car, but the mechanisms for regulating placental transfer are unknown. objectives: this study used placenta from normal and cholestatic pregnancies to investigate the expression of genes involved in hepatic bile acid homeostasis. methods: villous trophoblast samples from oc and normal pregnancies (np), and human livers were collected and preserved in rnalater. explant cultures were prepared from a further np placentas and cultured for days at % oxygen. on day they were treated with chenodeoxycholic acid, lithocholic acid or vehicle for hours prior to fixing in rnalater. total rna was extracted using trizol, and reverse transcribed to cdna. quantitative real-time pcr was performed using sybr green. target gene mrna abundance was calculated from a standard curve and normalised to l . results: the expression of fxr, pxr and car, the nuclear hormone receptors responsible for hepatic bile acid homeostasis and several fxr target genes (shp, mdr and bsep) was found to be very low in np, and unaffected by the presence of maternal cholestasis. furthermore, the expression of these genes could not be induced by bile acid treatment in an in vitro model. here we have shown that the nuclear hormone receptors (fxr, pxr and car) involved in hepatic bile acid homeostasis are expressed at very low levels in normal and pathological pregnancy and are thus likely to be of limited importance in placental bile acid transfer. a placental phenotype for obstetric cholestasis. victoria geenes, jenny chambers, jo wyatt-ashmead, kishore bhakoo, catherine williamson. imperial college, london, united kingdom; hammersmith hospital, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder that affects . % of pregnant women in the uk. biochemically, oc is characterised by liver dysfunction with elevated maternal serum bile acids (sba), and clinically by an increased risk of fetal complications including fetal distress, meconium staining of the amniotic fluid, preterm labour and sudden intrauterine death. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from the toxic effects of bile acids, which can cause vasoconstriction in the placenta and fetal cardiac dysrrhythmias. furthermore, in a rodent model of oc, bile acids cause oxidative stress in the placenta and a reduction in litter size. these changes are absent in animals treated with ursodeoxycholic acid (udca), a drug used to treat oc. however human data are lacking. objectives: this study used whole placenta and explant cultures to investigate the effects of bile acids on human placental architecture and to establish whether udca protects the placenta against bile acid induced damage. methods: villous trophoblast samples were collected from oc and normal pregnancies (np) and fixed in formalin. explant cultures were prepared from a further np placentas, and cultured for days at % oxygen. on day a subset were treated with udca overnight, and on day the explants were treated with taurocholic acid, taurochenodeoxycholic acid or vehicle for hours prior to fixing. m sections were stained with haematoxyllin and eosin. slides were reviewed by a perinatal pathologist and syncytial knots (sk) counted. results: oc tissues showed marked alterations in morphology, including congestion of the terminal villi, and loosening of the stroma and fibrotic change of the membranes of the stem villi. there were significantly more sk the oc samples (mann-whitney u test p= . ). furthermore, sk were increased in explants treated with bile acids, but not those treated with udca prior to the addition of bile acids. conclusions: here we describe several morphological abnormalities of the placenta associated with maternal cholestasis. these changes were confirmed using a placental explant model, which also showed that udca protects the placenta against bile acid induced damage. in summary, this study indicates that udca is likely to protect the fetus in oc. the placenta has complex metabolic and endocrine activities and is essential for the growth and survival of the fetus in utero. ultrasound is the most sensitive and less invasive method to evaluate placental size, morphology and function. the three-dimensional approach allows to calculate placental volume in the i and ii trimester of pregnancy. pregnancies from assisted reproduction technologies (art) show an increased rate of pathologies potentially related to placental insufficiency such as intrauterine growth restriction and preterm delivery. aim. to determine placental volume in art pregnancies in a cross sectional study in the first trimester of gestation. design. using three-dimensional ultrasound machine (ge ) placental volume measurement from art pregnancies (n= ; - wks) were compared with data from normal controls (n= ) matched for gestational age. data were analysed with software stata . results. mean placental volume was . ml (sd± . ) in art pregnancies and . ml (sd± . ) in normal controls. mean difference resulted in . ml (- - . ) and was statistically different (p= . ). multiple linear regression analysis showed a statistically significant interaction (p= . ) between gestational age and case status, i.e. differences in placental volume increase significantly with advancing gestational age between cases and normal controls. mean gestational age at birth was not essentially different between the two subsets ( . weeks ± . ) however a statistically significantly lower mean fetal birthweight was found in art pregnancies: g (sd± ) vs .(sd± . ) (p= . ). conclusions. placental volume is slightly decreased in art pregnancies. measurements of placenta volume by d ultrasound may play a role in identifying the degree of placental growth early in gestation in a risk population. in vitro effects of adenovirus-mediated gene transfer of insulin like growth factor- (ad-igf- ) in trophoblast cells. mounira habli, datis alae, foong yen lim, jignesh parvadia, timothy crombleholme. obstetrics and gynecology; pediatric surgery, university of cincinnati/children's hospital, usa. objective:recently, ad-igf- corrected both fetal and placental weights in rat model of fetal growth restriction (fgr) . to elucidate the mechanism of action of ad-igf- ;we examined the effect of ad-igf- on trophoblast proliferation, differentiation and invasion. methods: rcho- trophoblast cell line derived from rat choricarcinoma was used. ad-igf- and galactosidase transgene (ad-lacz) were given at moi of : and : in all the experiments.transduction efficiency was assessed hr after infection with ad-lacz by galactosidase enzyme activity. the invasiveness of rcho- was measured by using bdmatrigel invasion chamber kit. rcho- proliferation cell density was assessed by crystal violet assay. morphologic analysis of rcho- differentiation in response to treatment (differentiated cells are multinucleated giant cells) was assessed byimunocytochemistry staining using placental lactogen- antibodies(specific hormone produced by giant cells). results: % efficiency of gene transfer of ad-lacz to rcho- cells was observed at both moi's.there was no significant difference in proliferation between treated control group regardless of the dose at and hrs. ad-igf- induced morphologic differentiation of trophoblast cells compared to control (fig ) at hrs.ad-igf- induced higher rate invasion in a dose dependent fashion as compared to control(ad-lacz) group( % at moi vs . % in control p< . )(fig ). conclusion: ad-igf- gene transfer induces differentiation and invasiveness of trophoblast cells. these may be the mechansim of correction of fgr in rat model of placental insufficiency. placental gene transfer may be an effective treatment strategy for placental insufficiency. types of human placenta. martina dieber-rotheneder, ursula hiden, gernot desoye, mila cervar-zivkovic. department of obstetrics and gynaecology, medical university graz, graz, austria. background and hypothesis: endothelin- is a polypeptide with a wide range of functions. in the placenta it acts as a potent regulator of vasotonus on endothelial and smooth muscle cells, whereas on the trophoblast it regulates cell proliferation, invasion and apoptosis. endothelin- action is differently signaled through two endothelin receptor (etr) subtypes, etr-a and etr-b. several alternative splice variants of etr were identified. here we hypothesize that the etr-a splicing varies with gestational age and is different in trophoblast vs endothelial cells of the human term placenta. methods: mrna of placental tissue from first trimester (pregnancy terminations, missed abortions) and term of gestation, first trimester and term trophoblasts, arterial and venous placental endothelial cells as well as cell lines representing first trimester trophoblast (ach p) and term placental endothelial cells (hpec) were analyzed for full length etr-a and known as well as unknown splice variants by sqrt-pcr using primers spanning the exons - . the predominant dna bands in agarose gel were excised and sequenced. results: all tissues and cells expressed full length etr-a. in all tissues an additional -deletion variant was identified which was not found in the isolated cells. trophoblasts expressed another yet unidentified splice variant. the endothelial cells expressed a -deletion variant and a novel splice variant, which was identified as partial -partial deletion. this novel splice variant was found regardless of the arterial or venous origin of the cells as well as in the cell line (sv -transformed). in tissues und trophoblast no difference in splicing was found between first trimester and term. conclusion: gestational age does not alter splicing of endothelin receptor-a. splicing is different between trophoblasts and endothelial cells. the endothelial cells contain a novel splice variant. the differential splicing may allow maternal and fetal endothelin- to induce different effects on the two major cell types of the placenta. (supported by grant , jubilee fund, austrian national bank, vienna). adrenomedullin production and secretion by human trophoblast cells are regulated by glucocorticoids and hypoxia. francesca ciardo, katia pacioni, emanuela marinoni, giovanna corona, massimo moscarini, alfredo patella, romolo di iorio. department of gynecology, perinatology and child health, university "la sapienza", rome, italy; department of obstetetrics and gynecology, university of ferrara, ferrara, italy. objectives: adrenomedullin (am) is produced by intrauterine tissues and is involved in the regulation of implantation, placental hemodynamics and endocrine function. circulating am is increased in pregnancy complications such as preeclampsia and intrauterine growth retardation. we investigated whether am output by human trophoblast cells is regulated by hypoxia and/or glucocorticoids. study design: trophoblast cells obtained by human placentas at term (n= ) were cultured in presence or absence of hypoxia ( % o ) and treated with or without betamethasone at the dose of - , - , - m. media and cells were collected at h and at , and h from syncytiotrophoblast cultures. am was measured in cultured media by specific ria kit. protein expression in trophoblast cells was evaluated with immunohistochemistry and western blot. results: hypoxia stimulated am output and protein expression by cytotrophoblast and syncytiotrophoblast cells. betamethasone induced an increase in am production and secretion in a time-and dose-dependent manner (figure). effects of hypoxia were partially reversed by betamethasone in a dose and time-dependent fashion. conclusions: am production in trophoblast cells is up-regulated by hypoxia and glucocorticoids, independently. increased am levels in pregnancy complications characterized by placental insufficiency might derived by an increase in am placental secretion stimulated directly by hypoxia or indirectly by an increase in fetal cortisol levels. preeclampsia complicates - % of pregnancies, and while associated with significant maternal and fetal morbidity and mortality, the mechanisms responsible for preeclampsia are not completely understood. recent advances suggest there are imbalances of pro-and antiangiogenic factors, present from the time of implantation affecting vascular responsiveness of the fetal placental unit and maternal vasculature. in this study, we investigate vasomotor responsiveness of placental arteries from normal and preeclamptic (pet) pregnancies using an in vitro muscle bath contractility assay. transverse rings of placental arteries were cut and equilibrated in the muscle bath containing a bicarbonate buffer aerated with % o / % co at °c. viability was demonstrated by contraction to mm kcl prior to all experiments. cumulative logarithmic dose dependent responses to four different contractile agents: pgf , serotonin, carbochol, and norepinephrine were compared. placental arteries precontracted with pgf at half maximal concentration were relaxed with three vasorelaxants: sodium nitroprusside (snp), forskolin (fsk) and lactate (lct). agonist studies revealed contraction to both pgf and serotonin but not to carbochol or norepinephrine. there were no statistically significant differences between the responses of normal and pet arteries. both normal and pet arteries demonstrated heightened responsiveness to sodium nitroprusside compared with forskolin and lactate. this study reveals that the placental vessels are more sensitive to sodium nitroprusside, that mediates relaxation through nitric oxide -cyclic gmp signal transduction pathway, than forskolin that induces relaxation through the cyclic amp pathway. cancer complicates approximately one in , pregnancies. depending on the type of cancer and trimester, patients can terminate the pregnancy or choose treatment such as chemotherapy. since there is limited knowledge on the safety of chemotherapy on fetal tissues in utero, clinicians cannot efficiently analyze the risks of particular anticancer agents when deciding on a course of therapy. since carboplatin is among the most common anticancer agents used for treatment of cancer during pregnancy the primary objective of this study was to evaluate if carboplatin will readily cross to placental barrier and determine total potential platinum fetal exposure. this project utilizes an ex vivo placenta perfusion model to determine the concentration of carboplatin that crosses the human placental barrier. placentas are obtained within minutes of spontaneous delivery and re-perfused. two carboplatin concentrations were selected of ng/ml and ng/ml to represent clinically relevant maternal plasma concentrations. antipyrine was used as the internal control. serial samples were collected every minutes for total minutes in open-open model then experiment repeated with closed circuit model. antipyrine concentrations were determined by hplc methods. platinum concentrations in media samples were determined with a validated atomic absorption assay. a cell culture-based approach will be used to determine whether cell cycle or apoptotic proteins are altered (p , bax, bcl- , aif, and pro-caspase- ) using western blotting as a detection method. a total of two placentas have been completed at the low carboplatin concentration and one at the high concentration. the mean carboplatin clearance was . +/- . ml/min and . +/- . ml/min at the low and high carboplatin concentrations, respectively. an estimated % increase in the transport fraction was observed in the high concentration experiments compared to the low concentrations model. fetal carboplatin exposure ranged from to ng/ml. the placental perfusion experiments conducted at carboplatin and ng/ml indicate that carboplatin crosses the placenta through simple diffusion. the toxicology assays are ongoing to determine potential effect on fetal tissues. preterm delivery represents one of the predominant causes of perinatal mortality and morbidity, and is one of the most unpredictable gestational disturbances. urocortin is a peptide expressed by trophoblast and gestational tissues (amnion and chorion), whose maternal levels correlate with gestational length, since they are increased at preterm delivery and decreased in post-term pregnancy, when compared to term pregnancy. the addiction of urocortin enhances contractility of human myometrial strips, suggesting a possible role on uterine contractility. high maternal urocortin levels in threatened preterm delivery correlates with the timing of delivery, suggesting a possible role in the predictivity pf preterm delivery. in the present study urocortin concentrations in amniotic fluid collected at mid gestation for amniocentesis were correlated with gestational age at delivery. a case-control study with amniotic fluid obtained from healthy women undergoing amniocentesis for genetic indications was performed; urocortin concentrations were measured by a specific elisa. amniotic fluid urocortin concentrations resulted significantly lower (p= . ) in women delivering preterm (n= ; ucn= . ± . pg/ml) (m ± se) than in those delivering at term (n= ; ucn= . ± . pg/ml). in conclusion, the present preliminary data showed that amniotic fluid urocortin concentrations at mid gestation may represent predictive marker of preterm delivery. human placentas throughout pregnancy. annunziata mastrogiacomo, elisabetta federico, francesca caprio, maria teresa schettino, gabriele coppola, antonio de luca, luigi cobellis. department of obstetrics and gynecology, second university of naples, naples, italy; department of medicine and public health, section of anatomy, second university of naples, nales, italy. hypothesis apoptosis is intimately involved in placental homeostasis, growth and remodeling and the apoptotic rates increase progressively during normal pregnancy as part of normal placental development. moreover, apoptosis increases in pregnancies complicated by some pathologies such as preeclampsia, fetal growth restriction, diabetes. in the present study, we describe differences in the expression of pro-apoptotic protein bax, in first trimester voluntary termination of pregnancy, first trimester abortion (reserved abortion), caesarean birth, spontaneous birth, preeclampsia and diabetes. material and methods human placental samples were obtained with informed consent from patients undergoing surgery such as first trimester voluntary termination of pregnancy (n= ), first trimester abortion (miscarriage) (n= ), delivery section (n= ), spontaneous birth (n= ), preeclampsia (n= ) and diabetes (n= ). the gestation period ranged from to weeks. the specimens were immediately fixed in formalin for immunohistochemistry. we first observed a strong increase of bax expression in the cytotrophoblast, stroma, endothelial cells and decidua of placentas of the first trimester abortion compared to the low/moderate bax immunopositivity in all the placental compartments during the first trimester voluntary termination of pregnancy. secondly, we showed a more intense immunopositivity for bax in the third trimester spontaneous birth respect to the third trimester caesarean birth. thirdly, we observed an increase of bax expression in preeclamptic placentas compared to the normal full-term placentas. on the contrary, we observed a moderate bax expression in diabetic placentas only slightly decreased compared to the normal full-term placentas. our results seem to suggest that deregulation of apoptotic turnover may lead to placental dysfunction and pathologies. objectives: maternal endothelial activation in preeclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. the application of proteomic technologies such as mass spectrometry promises the reward of identifying and characterising these factors. using a placental explantconditioned culture media model system we have developed a proteomics workflow to obtain relative quantification of proteins released into placental explant culture media. methods: term villous explants were cultured in serum-free conditions and exposed to differing oxygen concentrations ( % %) to mimic physiological and non-physiological intervillous o tensions. the d media was concentrated, immunodepleted to remove fibrinogen (using beckman igy- columns) and proteins labelled using an itraq (applied biosystems) kit following the manufacturer's protocol. labelled peptides were fractionated by strong cation exchange and then analysed using lc-maldi on a maldi-tof/tof (applied biosystems) mass spectrometer. data was analysed using protein pilot . (applied biosystems). results: when matched pooled (n= ) media samples were subjected to our proteomics workflow over proteins were identified with a calculated false positive identification rate of < %. of these proteins a total of display a statistically significant difference in protein levels between the % % o samples (p=< . ). from a list of proteins of interest we selected proteins for validation using elisa/western blotting to measure their relative abundance in both pooled and individual explant media samples. interleukin is an example of a low-abundance differentially expressed protein identified in our proteomic workflow and subsequently validated by elisa; il- (pg/ml) in % o : . , range . to . ; % o : . , range . to . ; values are median with interquartile range. *** p< . , mann-whitney test (n= ) conclusions: we demonstrate a successful reduction to practice of a relative quantitative proteomics strategy applied to placental explant-conditioned culture media. having optimised and validated this proteomic workflow we will apply the same methods to compare proteins released by normal and preeclamptic placentas. a proteomics screen of human placental microvillous syncytiotrophoblast (stb) revealed the expression of dysferlin (dysf), a membrane repair protein associated with certain muscular dystrophies (vandré et al., ) . a second ferlin protein, myoferlin (myof), was also discovered which has not yet been characterized in the placenta. in human c c myoblasts, dysf and myof are reciprocally expressed as a function of differentiation into multinucleate syncytial myotubes, with dysf predominating in differentiated cells. we hypothesized that dysf and myof would show a similar reciprocal expression pattern in human trophoblasts as a function of syncytialization. methods term placentas from uncomplicated pregnancies were obtained with informed consent (n= ) and either fixed for immunofluorescence (if) labeling or flash frozen for subsequent immunoblotting (ib). human trophoblastic (bewo, jar, and jeg- ) and other cell lines (mrc- , hl- , huvec) were cultured in the absence or presence of μm forskolin or solvent control for - days. dysf and myof expression was examined by rt-pcr, ib, and if. results ib validated proteomics data showing myof expression in term placenta. by if, myof labeling was predominant in apical and basal stb plasma membranes. myof was expressed in trophoblastic cell lines (bewo, jar, and jeg- ), cultured endothelium (huvec), and a fibroblast cell line (mrc- ), but was not detected in a leukemic cell line (hl- ). dysf was constitutively expressed in jar, but minimally expressed in unstimulated bewo. following forskolin-induced syncytialization, we observed a time-dependent increase in dysf expression in bewo concomitant with increasing syncytin- levels. by if, dysf expression was restricted to bewo cells in syncytial structures. in contrast, myof expression was robust in mononuclear bewo cells and not down-regulated over the course of days of differentiation. conclusions two ferlin-family genes are expressed in trophoblasts. fusion-competent bewo cells behave similarly to cultured myoblasts and ctbs with regard to dysf expression, which is restricted to syncytializing cells. myof, in contrast, is expressed constitutively in bewo and other models. while both proteins are likely to function in stb plasma membrane repair (e.g., following syncytial sprouting), the relative contributions of each to this process awaits clarification. features of chronic placental insufficiency are significantly more common in small for gestational age placentas from infants with intrauterine growth retardation. emily king, violetta kolesnikova, carri tillotson, jean-marie guise, terry morgan. pathology; center for biostatistics; obstetrics and gynecology, ohsu, portland, or, usa. background: there is a strong association between intrauterine growth restriction (iugr) and small for gestational age (sga) placentas (heinonen et al. placenta ( ), : - ) . the cause is likely multifactorial, but we hypothesize that chronic uteroplacental insufficiency may play a role. our objective was to test whether sga placentas from human neonates with iugr show significantly more pathologic features of placental insufficiency compared to controls. design: we performed a retrospective review of consecutive singleton placentas submitted to ohsu pathology ( - ), excluding elective terminations and spontaneous abortions before weeks gestation. clinical records were reviewed and pregnancy outcomes recorded, including: ) neonatal sex; ) weight (iugr calculated by routine methods), ) trimmed weight of the placenta (sga calculated by routine methods), ) gross placental infarctions, ) gestational age at delivery, and ) maternal features (e.g. race, gravida). controls were defined as cases within the series without sga or iugr (e.g. submitted for meconium, infection, etc). histologic sections were scored by two pathologists while blinded to clinical diagnoses as positive or negative for features of placental insufficiency, including accelerated villous maturation (avm), chorangiosis, and microscopic infarctions. significant associations were tested by analysis and logistic regression for multiple variables. results: similar to prior reports, we observed a strong association between iugr and sga placentas ( . ; p < . ). this relationship was independent of maternal race, fetal sex, and parity, although it was more common in primigravidas. avm and placental infarctions were significantly more frequent in sga placentas with superimposed iugr. controls ( introduction: messenger rna expression peripheral blood cells (pbc) has been recently used as biomarkers of environmental exposures (ionizing radiation or tobacco), physiological conditions (stress) and diseases (hypertension, neurological disorders). pbc evaluation is a useful diagnostic tool in an era of individualized medicine. since there is an urgent need for non-invasive methods for determination of fetal (f) and placental (p) function, this study was designed to evaluate the genes differently and commonly expressed in p tissue and leukocytes in maternal (m) and f circulation.material and methods. p (n= ), f (n= ) and m blood (n= ) were obtained during cesarean section in pregnant baboons at term. total rna from a buffy coat pellet was isolated using a modified procedure of the qiagen rneasy mini kit (qiagen). anti-sense rna (arna) was synthesized and purified using the illumina rna amplification kit (ambion, usa). hybridization of arna to illumina sentrix human whole genome (wg- )beadchips and subsequent washing, blocking and detection was performed using illumina's beadchip ´ protocol. samples were scanned on the illumina beadarrayer gx reader using illumina beadscan image data acquisition software (ver. . . . ). differential gene expression data analysis was performed using illumina beadstudio software (ver. . . . ). results. the detection level of gene transcripts using illumina methodology with control human rna was - at p< . . gene transcripts were detected in f blood and in maternal blood. transcripts were uniquely expressed in fetal blood. transcripts were found in m, but not in f leukocytes. the number of gene transcripts expressed in p tissue was and of these genes were not expressed in m leukocytes. conclusion. despite white blood cells trafficking through the p barrier there is a set of unique genes expressed only in p or in the m or f circulation. the application of these genes as the biomarkers of p barrier function still need to be evaluated. objective: to evaluate if a relationship exists between duration of placental exposure to meconium in vivo and histologic evidence of severity and extent of meconium uptake by macrophages. study design: from a cohort of / ( %) consecutive singleton liveborn infants delivered at term with thick meconium-stained fluid, ( %) had placental histologic examination performed, and in / the timing of meconium appearance after membrane rupture was documented. placental histologic examination quantitatively evaluated the intensity of meconium uptake by resident macrophages based on the number of macrophages/field, and the extent of uptake based on histologic location, graded in a score to . results: mean interval between meconium appearance and delivery was . ± . min (range - ). after exclusion of cases in which severe placental inflammation interfered with analysis, meconium uptake by macrophages was documented in / cases at the level of amniochorionic membranes, in / cases at the placenta, and in / cases at the umbilical cord. there was no correlation between the interval meconium appearance-to-delivery in relation to presence of meconium in the membranes (p= . ), in the placenta (p= . ), in the cord (p= . ), or score of severity of meconium uptake (p= . ). the results did not change after correcting for gestational age, oligohydramnios, presence of placental acute inflammatory or vascular lesions. conclusion: there is no relationship between duration of placental exposure to meconium and the extent and intensity of its uptake by macrophages in cases with exposure up to . hours. transfer of bisphenol a across the human placenta. biju balakrishnan, , kimiora henare, , eric thorstensen, , murray d mitchell. , the liggins institute, university of auckland; national research centre for growth and development, auckland, new zealand. introduction: there are growing concerns over the effects of developmental exposure to the xenoestrogen bisphenol a (bpa). animal studies have shown that bpa is transferred through the placenta and can cause deleterious effects to the fetus. the presence of bpa in feto-placental tissues in humans has also been reported. however, a detailed study of the time-course of bpa transfer across the human placenta has not been performed. the aim of this research was to study the transfer of bpa in ex-vivo perfused human placental tissues. methods: a dual recirculating single cotyledon perfusion was used to monitor the placental transfer of bpa. bpa ( ng/ml), antipyrine (ap) ( μg/ml), and fitc dextran (fitc-dx) ( . μg/ml) were added to the maternal perfusate, and perfusion was continued for hours. perfusate samples were collected from both reservoirs at timed intervals and analysed. bpa, ap, and fitc-dx were determined by hplc with fluorescent detection, hplc with uv detection, and spectrofluorometry respectively. the viability and metabolic activity of the placentae were assessed by measuring -hcg (elisa), glucose utilization, and lactate production (autoanalyzer). fetal pressure, ph, flow rates and fluid shifts were monitored continuously. results: the biochemical validation parameters (glucose consumption, lactate production and -hcg secretion) indicated that the placental tissue was metabolically active and viable throughout the -hour perfusion period. the physical parameters observed (fetal pressure < mmhg, ph range . - . ) were in concordance with other published works for placental perfusion. membrane integrity was confirmed by fluid shifts from either circuit of < ml/hr, and by < % materno-fetal transfer of fitc-dx. an observed ap transfer of - % further validated our model. bpa first appeared in the fetal compartment within minutes of perfusion and reached a peak of about - % of maternal concentration within hours of perfusion. this figure is likely to be an underestimate since it does not include conjugated bpas. conclusion: this first study of bpa transfer in ex-vivo perfused human placental tissue shows that our model can serve as a useful tool to study the transfer kinetics and metabolism of bpa in human term placentae. we found that bpa rapidly crosses the human placenta at environmentally-relevant doses, with potentially harmful effects on the human fetus. angiogenic growth factor secretion by uterine natural killer cells in co-culture with extravillous trophoblast. gendie e lash, katsu naruse, , barbara a innes, stephen c robson, judith n bulmer. institue of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom; obstetrics and gynaecology, nara medical university, nara, japan. objectives. uterine natural killer (unk) and extravillous trophoblast (evt) cells have been proposed to play roles in remodeling of uterine spiral arteries through secretion of various angiogenic growth factors. we have previously demonstrated that unk cells are a significant source of angiogenic growth factors within the decidua, many of which alter with gestational age. however whether the secretion of these proteins is altered by interactions of unk cells with evt is unclear. hypothesis. unk cell angiogenic growth factor secretion is regulated by evt in early pregnant decidua. methods. placental and decidual samples were collected from women undergoing termination of pregnancy with written informed consent ( - and - weeks gestation, n= each group). . × cd + unk cells were positively selected from decidua and co-cultured with evt ( . × ) or cytotrophoblast (ctb; . × ) purified from the same placenta for h in direct or indirect contact (n= each group). angiogenin, ang , pdgf-bb, fgf-basic, timp , icam and vegf-a were measured by fast quant ® angiogenesis multiplex assay system, and vegf-c, ang and plgf by elisa. results of unk co-culture with evt or ctb (negative control) at each gestational age were analyzed with wilcoxon rank test. in addition, the effect of direct and indirect co-culture of unk cells with evt at each gestational age was compared with mann whitney u test. results. at - weeks gestation unk secretion of ang (p= . ), icam (p= . ) and plgf (p= . ) was increased in the presence of evt compared with ctb. no differences were observed at - weeks gestation. in addition, at - weeks gestation unk secretion of ang (p= . ), vegf-c (p= . ) and ang (p= . ) was increased in direct co-culture with evt compared with indirect co-culture. at - weeks gestation unk secretion of timp- (p= . ) was reduced in direct co-culture with evt compared with indirect co-culture. conclusions. unk cell secretion of several key angiogenic growth factors was altered by direct culture with evt. these data suggest that a membrane bound molecule (such as hla-g) mediates this modulation of unk cell activity. abnormalities in placentation and impaired placental circulation can lead to fetal growth restriction. -d ultrasound can be used to evaluate this through the quantification of the power doppler signal that may be expressed as a percentage of colour voxels within a user-defined volume (vi: vascularisation index). we aimed to test the hypothesis that increased fetoplacental blood flow correlates with an increased vi, using the in vitro dual perfusion model of the human placental lobule. three term lobules were dually perfused through both circulations with earles bicarbonate buffer (ebb), and supplemented on the fetal-side only with adult erythrocytes, prepared to a % haematocrit. following initial equilibration perfusion at normal flow values, fetal-side flow was varied between and ml/min, whilst maternal-side flow was held at ml/min. images were obtained with a 'voluson i' ultrasound machine and a neonatal transducer (pulse repetition frequency = . hz, wall motion filter = low , and gain = . ). three -d datasets were acquired at each flow rate from each placental lobule and these were measured in triplicate using vocal. a sphere was centred on a visibly perfused cotyledon along the chorionic-decidual axis, with a diameter corresponding to placental thickness. linear regression analysis was used to assess the relationship between the total fetal-side flow and mean vi. the mean vi showed a high degree of correlation with total fetal-side flow for each lobule ( figure ) suggesting increased vascular perfusion and the inclusion of perfused vessels that cross the detection threshold with increased flow. this data provides qualifying information for translation to a clinical application, where early gestational fetoplacental blood flow will be assessed to predict the onset of fetal growth restriction. anthony n imudia, brian a kilburn, anelia petkova, samuel s edwin, roberto romero, d randall armant. objective survival of first trimester cytotrophoblast cells depends on their upregulation of heparin-binding egf-like growth factor (hbegf), which is downregulated in placental tissues diagnosed with preeclampsia. we have examined the expression and cytoprotective activity of hbegf in term villous explants subjected to hypoxic stress in vitro. non-pathological placentas were collected by cesarean section at term (n= ). chorionic villous explants were prepared and cultured at either % or % o and treated with the hbegf antagonist crm or recombinant hbegf. paraffin sections were assayed for trophoblast cell death by the tunel assay, proliferation by immunohistochemical labeling of nuclear ki and hbegf expression by semi-quantitative immunohistochemistry. data were compared using anova and the student-newman-keuls posthoc test. results crm ( mg/ml) increased trophoblast cell death after culturing villous explants h at % o (p< . ), but only slightly affected proliferative capacity. culture at % o increased trophoblast cell death % above explants incubated at % o (p< . ). trophoblast cell proliferation decreased after h in explants cultured at either % or % o (p< . ). exogenous hbegf ( nm) prevented the elevation of cell death during hypoxia (p< . ) and maintained nuclear ki expression at %, but not %, o . contrary to first trimester trophoblast, hbegf was not upregulated by hypoxia in term trophoblast. the failure of term trophoblast to elevate hbegf expression in response to hypoxia could contribute to their decreased survival at low o compared to early gestation. endogenous hbegf signaling appears to facilitate survival of term trophoblast during villous explants culture. exogenous hbegf supplementation prevented cell death due to hypoxia and maintained trophoblast proliferation rates under in vitro conditions. therefore, hbegf, which is downregulated in preeclampsia, could have significant impact on trophoblast survival during late gestation. supported by the intramural research program of nichd. conspicuous meconium-laden macrophages in the chorionic plate are an independent predictor of clinically significant fetal distress. violetta kolesnikova, emily king, carrie tillotson, jean-marie guise, terry morgan. pathology; center for biostatistics; obstetrics and gynecology, ohsu, portland, or, usa. background: meconium is a common indication for placental examination, present in approximately % of placentas routinely submitted to pathologists (beebe et al. obstet gynecol ; : - ) . prolonged meconium exposure leads to accumulation of meconium-laden macrophages in the chorionic plate (estimated to require at least hours). our objective was to test whether prolonged meconium exposure is associated with clinically significant fetal distress. design: retrospective review of consecutive singleton placentas submitted to ohsu pathology ( - ) was performed, excluding abortions before weeks gestation, and placentas without representative sections of the chorionic plate. clinical records were reviewed and pregnancy outcomes recorded, including: ) evidence of fetal distress prompting c-section (non-reassuring fetal heart rate), ) gestational age, ) maternal diagnosis, ) apgar scores, and ) neonatal length of hospital stay. routine histologic sections were independently scored by two pathologists while blinded to clinical diagnoses and outcomes. cases were scored as positive or negative for: ) diffusely conspicuous meconium-laden macrophages in the chorionic plate (at least / hpf), ) chorioamnionitis, and ) features of placental insufficiency. significant associations were tested by the mann-whitney u test for paired comparisons, x analysis, and logistic regression for multiple variables. results: conspicuous meconium staining of the chorionic plate was common ( / , %) and was significantly more frequent in c-sections performed for fetal distress ( / cases, %) (x . ; p-value < . ). logistic regression modeling showed that this association was independent of chorioamnionitis and features of placental insufficiency. there was no association between meconium and neonatal outcome, including no difference in apgar scores or length of hospital stay. conclusions: our data support the hypothesis that meconium-laden macrophages in the chorionic plate are associated with fetal distress prompting c-section. whether meconium is the cause or consequence of this distress is uncertain. however, given the acute time frame between clinical diagnosis and c-section, we suspect prolonged meconium exposure may be a significant cause of non-reassuring fetal heart rate changes. apoptotic index in normal and intrauterine growth-restricted rat placentas. elissa scotland, tri nguyen, s chiang, radmila runic. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: intra-uterine stress caused by maternal food-restriction may have adverse fetal and placental effects. we sought to assess the effect of maternal food restriction during pregnancy on placental apoptosis. methods: rat placentas were analyzed from control dams fed ad libitum and dams % food-restricted from day of gestation. placentas were harvested at embryonic days and (n= ). placentas were fixed in % pfa and two methods of analysis were utilized to determine the proportion of apoptotic to non-apoptotic cells within maternal food restricted and control rat placentas: immunohistochemistry (ih) using fas-ligand and in situ tunel (terminal deoxynucleotidyl transferase biotin-dutp nick end labeling). tunel: after rehydration, slides were subjected to tdt enzyme. dab was used to visualize brown apoptotic nuclei. dna-se treated slide was used as a positive control. ih: primary rabbit polyclonal fas-ligand antibody was used at : dilution, after which secondary antibody linked to peroxidase and dab staining was performed. results: food-restricted placentas demonstrated . % relative apoptotic index when compared to . % in the control group. the iugr iod (integrated optical density) per unit area in the placental membrane was higher than in the control group. fasl demonstrates an iod of . + . in food restricted placentas as compared to . + . per μm surface area in controls. apoptosis was seen in the amnion as well as trophoblast (syncytialtrophoblasts and some cytotrophoblasts). conclusion: the increased apoptotic index in maternal food restricted placentas suggests that the accompanying iugr may be a result of both maternal/fetal nutrient restriction and increased fetal stress. this study found that maternal food restriction during pregnancy affects placental apoptosis. there is more apoptosis in the iugr placental bed at e than at e , with the reverse being true for the placental membrane. more research is required to statistically validate the data. the suggested next step is to evaluate fas and fas-l and to address possible mechanisms of placental apoptosis. cord. jayaraman lakshmanan, avish arora, lilit baldjyan, sharon k sugano, olga miadel, adegoke adeniji, michael g ross, calvin j hobel. ob-gyn, harbor-ucla medical center, torrance, ca, usa; ob-gyn, cedars-sinai medical center, los angeles, ca, usa. background: crf-bp is a kda protein, that specifically binds corticotrophin releasing factor (crf). the structure of cloned crf cdnas in all species examined predicts that the precursor is larger than the kda in size and contains one n-glycosylation site. marked reductions in plasma crf-bp levels seen in pregnant women prior to both preterm and term delivery led to the notion that crf-bp is a "gatekeeper" of crf responses. recently we identified crf-bp expression in term human umbilical cord (uc) by immunohistochemical analyses. objective: to characterized the expression of crf-bp by immunohistochemical and biochemical analyses in human uc. methods: freshly obtained human preterm ( to < weeks, n= ) umbilical cord ( pieces of mm thickness taken at - cm intervals close to placenta) were fixed in bouin's solution and paraffin embedded. also, pieces of ucs were dissected, arteries and vein separated, weighed and frozen at - c. for immunohistochemical localization, uc sections were subjected to immunostaining with polyclonal antibodies to human crf-bp precursors. for western blot analyses, whole uc as well as isolated arteries and vein were homogenized in a buffer containing detergents and protease inhibitors. the homogenate supernatant proteins were subjected to western analyses by standard protocol. immunoreactive protein bands were identified by chemiluminescent reagent. results: both goat and rabbit crf-bp polyclonal antibodies elicited weak to moderate positive staining in uc epithelial layers, vascular musculature and barely endothelial cells. they identified a strong kda band in whole uc as well as in isolated artery and venous preparations. in addition minor immunoreactive protein bands of , , kda in size were noticed in all three preparations. conclusion: we conclude that preterm uc expresses crf-bp. we postulate that the kda major band is either glycosylated crf-bp precursor or glycosylated kda mature protein. the low molecular weight immunoreactive proteins likely represent proteolytically processed, glycosylated crf-bp precursor or a proetolytically processed mature da crf-bp. uc obtained at delivery could be useful as a tool to understand the critical functions of crf-bp in feto-placental unit. objective: adenosine, known to be released from inflammatory sites and tisse ischemia, has many important biologic roles. four specific adenosine receptors have been cloned to date, termed a , a a, a b, and a . recently our study has shown that increased a receptor in the trophoblast of preeclamptic pregnancy was noted and non-vascular and trophoblast-mediated a receptor may play an important role in the pathogenesis of preeclampsia. there are evidences of impaired trophoblast invasion related to matrix metalloproteinase (mmp) in preeclampsia and the relationship between adenosine receptor and mmp in other fields. the objective of this study is to evaluate the effect of mmp expression by adenosine a receptor in preeclamptic villous explants at different oxygen conditions. methods: placental villous explants from normal (n= ) and preeclamptic (n= ) pregnancies were cultured at high ( %) and low ( %) oxygen levels for days. explants were analyzed for mmp- /- and timp- /- by rt-pcr and western blot. preeclamptic villous explants in hypoxic culture condition were treated with a receptor agonist, cl-ib-meca and a receptor antagonist, mre. mmp- / - expression was determined in a time-and dose-dependent manner by rt-pcr, western blot. also mmp- /- activity was evaluated by zymogram assay. results: there were significantly increased a receptor intensity and reduced mmp- /- and timp- /- expression at low oxygen level in normal and preeclamptic villous explants. interestingly, in preeclamptic villous explants, after high oxygen culture mmp- /- and timp- /- expression were recovered to almost same level compared to those in normal villous explants. treatment of preeclamptic villous explants with cl-ib-meca in low oxygen level resulted in a time-and dose-dependent enhanced expression of mmp- /- . this cl-ib-meca-induced expression of mmp- /- was inhibited by pretreatment with mre. conclusion: to our knowledge, this study is the first to evaluate modulation of mmp secretion by adenosine a receptor in preeclamptic villous explant. our results provide evidence for the existence of functional adenosine a receptors in the trophoblast and suggest that adenosine a receptor will be investigated as a therapeutic target in preeclampsia. murray d mitchell, timothy a sato, anderson wang, jeffrey a keelan, anna p ponnampalam, michelle glass. liggins institute; department of pharmacology and clinical pharmacology, university of auckland, auckland, new zealand. introduction: it is well established that prostaglandins play critical roles in multiple aspects of pregnancy and that the fetal membranes are an important site of intrauterine prostaglandin production. endocannabinoids have been implicated in the maintenance of pregnancy and parturition in women and are a source of arachidonic acid which is a substrate for the production of prostaglandins. the aim of the present study was to determine the effects of endocannabinoids on the production of prostaglandins in extraplacental membranes -amnion, chorion and decidua. methods: explants of term amnion and choriodecidua were established and treated with endogenous endocannabinoids -arachidonoyl glycerol ( ag) and anandamide (aea) and with the synthetic cannabinoid cp , , to determine the ability of these substances to modulate prostaglandin e (pge ) production. pge was measured by radioimmunoassay. the explants were also treated with cp , in the presence of either sr a (a selective antagonist of the cannabinoid receptor cb ) or ns (a cox- inhibitor), to determine whether any observed stimulation of pge production was mediated through cox- activity and/or the cb receptor. cox- , cox- , cpla and pgdh protein levels were measured by western blotting. results: all three cannabinoids caused a significant increase in pge production in amnion but not in choriodecidua. however, separated fetal (chorion) explants responded to cannabinoid treatment in a similar manner to amnion, whereas maternal (decidual) explants did not. the enhanced pge production caused by cp , was abrogated by co-treatment with either sr a or ns , illustrating that the cannabinoid action on prostaglandin production in fetal membranes is mediated by cb agonism and cox- . preliminary data from western blotting show that cannabinoid treatment results in the up-regulation of cox- expression. however, there was no change in cox- expression and no evidence either for up-regulation of cpla or for down-regulation of pgdh expression. conclusion: this study demonstrates a potential role for endocannabinoids in the modulation of prostaglandin production in late human pregnancy, with potentially important implications for the timing and progression of term and preterm labour and membrane rupture. inactivation of vegf receptor- , but not vegfr- or vegfr- , during the peri-implantation period prevents normal pregnancy development in the rodent through disruption of uterine angiogenesis. nataki c douglas, hongyan tang, raul gomez, bronislaw pytowski, daniel j hicklin, jan kitajewski, mark v sauer, ralf c zimmermann. division of reproductive endocrinology and infertility, columbia university, new york, ny, usa; imclone systems, inc., new york. objective: vegf is involved in the regulation of uterine angiogenesis and implantation in both rodents and non-human primates. vegfr- , r- , and r- are expressed in the uterine decidua and are involved in the regulation of vessel formation in many systems. to determine if these receptors have a functional role in the regulation of post-implantation angiogenesis and pregnancy development, we examined the effects of blocking vegfr- , r- , and r- function. design: prospective animal laboratory material and methods: to avoid effects of vegf receptor neutralization on ovarian function, we utilized a progesterone replaced, ovariectomized mouse model. vegf receptor blocking antibodies were administered on ed . , prior to embryonic expression of these receptors. embryonic development was evaluated on ed . , blood vessel density and apoptosis on ed . , and cellular proliferation on ed . (n= per time point in each group). anova with bonferroni correction was used to compare sample means. results: see tables. conclusions: neutralization of vegfr- and vegfr- , but not vegfr- resulted in a significant reduction in cellular proliferation and decidual angiogenesis. vegfr- mediates decidual angiogenesis, but is not required for normal pregnancy development. in contrast, an intact vegf/vegfr- pathway is required for the decidual angiogenesis that mediates early pregnancy development. effect of vegfr neutralization on ed . control anti r- anti r- anti r- no. of implantation sites . +/- . +/- . . +/- . . +/- . hoxa encodes a transcription factor required for endometrial receptivity and embryo implantation. our objective was to identify and to characterize those molecular markers regulated by hoxa in multiple cellular model-systems. using microarray technologies, we identified putative hoxa target genes involved in early implantation. liposome-mediated transfection delivering either empty vector or the same plasmid constitutively expressing hoxa was introduced into newly impregnated mice during laparotomy or layered onto cultured human endometrial stromal-cells (hescs). rna products from these in vivo and in vitro transfections were used to identify targets and to validate the microarray screen employing semi-quantitative real-time pcr (qrt-pcr). we identified statistically-significant genes regulated by hoxa overexpression of which genes were down-regulated greater than -fold when compared to controls. cellular ontogenies of differentially-expressed genes include: cell adhesion molecules, signal transduction factors as well as metabolic regulators. furthermore, we identified the -phosphoglycerate dehydrogenase gene, (pgdh) whose products are regulated by hoxa during implantation in both murine model systems in and cell culture. this genes codes for an enzyme critical to de novo l-serine biosynthesis via a phosphorylation-dependent pathway. microarray analysis demonstrated a fold expression decrease when hoxa is overexpressed. this diminution in pgdh expression was noted in the validation experiments using qrt-pcr and corroborated in hesc cells where the mrna levels decreased to % when compared to controls. the repression of pgdh during the implantation window may represent a conservation of activity as secretory-phase protein synthesis may be suppressed in order to promote cellular differentiation and resultant implantation. these regulatory relationships identified in mouse implantation likely function to enhance uterine receptivity and may have a role in human implantation. objective: hoxa is expressed in endometrium, where it is regulated by sex steroids and is necessary for endometrial receptivity and implantation. hoxa is also expressed in leukocytes. here we hypothesized that hoxa would be regulated by sex steroids in both cell types. we further hypothesized a correlation between expression of hoxa in peripheral blood cell (pbcs) and endometrium in both mice and in humans. methods: real-time pcr was used to determine differential expression of hoxa mrna in u cells, a monomyelocytic cell line, in response to increasing concentrations of estradiol. to determine if hoxa is expressed in leukocytes in vivo, peripheral leukocyte hoxa mrna expression was measured over sequential estrus cycles in mature cd nulliparous mice and correlated to vaginal smear-cytology. additionally, peripheral leukocytes were isolated either from pregnant or normally-cycling women to assess hoxa mrna expression. results: there was a direct, dose-responsive correlation between exposure to increasing estradiol and hoxa mrna expression levels in u cells. in a murine model, we demonstrated that hoxa mrna expression-levels varies throughout the estrus cycle with a marked increase in expression following vaginal plug detection. the nadir of hoxa expression is prior to proestrus and increased up to -fold during the receptive phase. this increased expression continues throughout gestation. the heightened expression in murine leukocytederived hoxa mrna also is demonstrated across species. our preliminary data suggests that the greatest fold-increase of expression occurs during the window of implantation of the secretory phase in normal, cycling women. this level was sustained in pregnancy. there appears to be a trend with the highest levels of expression associated with viable gestations. women with attenuated expression profiles had non-viable gestations. conclusions: hoxa expression is regulated by sex steroids in both leukocytes and endometrium. the temporal pattern of peripheral hoxa transcript expression demonstrated in mice and humans mimics the differential rna expression documented within the uterus. leukocyte hoxa expression during the reproductive cycle in mice and humans is a marker of endometrial receptivity. peripheral leukocyte expression of hoxa mrna may correlate with implantation success. carolien m boomsma, annemieke kavelaars, marinus jc eijkemans, gijs teklenburg, bart cjm fauser, cobi heijnen, nick s macklon. reproductive medicine and gynaecology and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands. the purpose of this study is to assess the association between the intra-uterine cytokine expression profile at the time of embryo transfer and successful embryo implantation following ivf/ icsi treatment. materials and methods women undergoing ivf/ icsi underwent endometrial secretion aspiration prior to embryo transfer. known soluble mediators of implantation were measured using a multiplex immunoassay, namely il ß, il , il , il , il , il , il , il , tnf , vegf, ifn , eotaxin, mcp- , ip- , dkk- and hbegf. mif was determined using an elisa. the total protein concentration was measured for normalization purposes. data were log transformed to obtain normal distribution. multivariable logistic regression analysis with a backward elimination procedure (p< . ) was used, potential confounders (age, blood contamination, embryo quality) were included in a forward stepwise model. ten mediators of the analysed were detectable in - % of the samples. il was detectable in % of samples, dkk- %, il- %, il- %, il- % and hbegf was detectable in % of samples. ifn-was not detectable in any of the samples. multivariable logistic regression showed only logmif concentrations to have a significant correlation with achieving clinical pregnancy (p = . ). higher mif concentrations were correlated with a higher chance of conceiving. endometrial secretion analysis represents a novel means of assessing the intra-uterine milieu encountered by the embryo and offers new perspectives in the study of endometrial receptivity. in this large prospective study assessing an array of cytokines, mif was found to be significantly correlated with pregnancy. mif, macrophage migration inhibitory factor, is a cytokine with numerous proinflammatory, immunomodulatory, angiogenic and tissue remodelling properties. mif induces the synthesis and secretion of matrix metalloproteinases by endometrial cells, which may contribute to embryo invasion. its expression is particularly increased during the secretory phase, suggesting a role in reproductive processes. analysis of aspirated endometrial secretions offers a direct clinical test of endometrial receptivity which can be applied during treatment cycles without disrupting implantation. endometrial receptivity and secretory differentiation require progesterone (p). it has been hypothesized that low p levels result in delayed endometrial differentiation and infertility due to reduced receptivity. objective: test the effects of low luteal p on histologic and molecular markers of differentiation and function. methods: normal cycling women (n= ) were treated with daily leuprolide ( . mg/d) beginning in the midluteal phase and continuing through the protocol. after menses, subjects received transdermal estradiol (e, . mg/d) for days. after day of e, subjects also received daily i.m. injections of p, randomized to mg/d (sub-physiological) or mg/d (physiological). endometrial biopsy was performed after days of combined e and p treatment. additional untreated women had biopsies performed days after spontaneous lh surge. endometrial histologic dating was performed by two individuals according to the criteria of noyes et al. mrna levels were assessed using real-time rt-pcr. results: mean(s.d.) of peak and trough p serum concentrations in the mg/d p group were . ( . ) and . ( . ) ng/dl, respectively, while those in the mg/d group were . ( . ) and . ( . ) ng/dl. there were no differences between treatment groups for histologic dating; the mean(s.d.) histologic date was . ( . ), . ( . ), and . ( . ) for the mg, mg, and spontaneous cycle groups, respectively. there also were no differences among the three groups in mrna levels of ten functional markers (er , pr, integrin subunit, osteopontin, cyr , egr- , fkb , c-fos, and cd ), although variability of gene expression was greater in those who received mg/d p than in those who received mg/d p. there was also no correlation between serum progesterone level and gene expression or histologic date. conclusions: sub-physiological levels of progesterone, in the range seen in ovulatory women, do not induce detectable changes in expression of marker genes or histological dating, although low p levels were associated with greater variability of gene expression. these data suggest that abnormalities in endometrial histologic development and function likely result from intrinsic abnormalities rather than from low levels of p secretion. (supported by unc nova carta fund and nih u hd- ). endometrial ip attracts trophectoderm through cxcr interaction. simcha yagel, caryn greenfield, hen sela, jacob hanna, irit manaster, orna singer, ronit haimov-kochman, shira natanson-yaron, diana prus, benjamin reubinoff, debra s goldman-wohl, ofer mandelboim. obstetrics and gynecology, hadassah-hebrew university medical centers, jerusalem, israel; lautenberg center for immunology, jerusalem, israel; human embryonic stem cell research center, jerusalem, israel. introduction: implantation is initiated in part by attraction of the blastocyst to the endometrium lining the uterus. we hypothesized that this process is partly accomplished by chemokines expressed by the endometrium interacting with chemokine receptors on the blastocyst, suggesting that they play an important role in implantation. materials and methods: chemokine receptors were characterized in jeg cells, placental villi, primary trophoblast cell culture, trophectoderm cells derived from human es cells, blastocyst trophectoderm, and st trimester placental tissue sections. expression of chemokines was tested in decidua, endometrium, and ishikawa and hec- cell lines. immunohistochemistry, intracellular staining, elisa, facs analysis, and rt-pcr were employed to characterize chemokine receptor and ligand expression. functional testing was performed using transwell migration assays and in a nude mouse model using a matrigel gel plug cell attraction assay followed by facs analysis. results: trophoblasts demonstrated expression of various chemokine receptors, most prominently cxcr and cxcr . immunohistochemistry of trophoblast from placental villi plated on matrigel expressed cxcr and cxcr as well as hla-g. noteworthy is that trophectoderm cells derived from hes cells treated with bmp- and jeg cells, and blastocyst trophectoderm expressed principally cxcr . elisa and immunohistochemistry showed that decidua and endometrium expressed chemokines ip and il . migration assays demonstrated that ip significantly attracted various trophoblast and trophectoderm cells in vitro, and in the mouse model in vivo. taken together these results demonstrate the interaction between trophoblasts and endometrial cells is mediated by cxcr and cxcr , and il and ip . these interactions are important in the attraction of trophoblasts at the feto-maternal interface. however, ip -cxcr is the most relevant to early implantation as only cxcr is expressed consistently by trophectoderm. the endometrial proteome: changes from proliferative to secretory phase. lois a salamonsen, jenny i-c chen, xian mak, peter j stanton, david m robertson, andrew n stephens. prince henry's institute of medical research, melbourne, vic, australia. global gene analyses have demonstrated major changes across the menstrual cycle, but which of these are reflected in the proteome is not known. this study aimed to globally assess proteins differentially expressed in the endometrium between the proliferative and. secretory phases. d page analysis with dige minimal dye labelling was conducted across the pi range - on endometrial tissue from either the mid-proliferative or midsecretory phases (n= /group). profiles were assessed using samespots software. differentially expressed proteins were identified using maldi-tof ms and the interrelationship of proteins examined using ingenuity software. a total of spots were detected: were differentially expressed (p < . ) with spots having an overall false discovery rate < % (q < . ). hierarchical clustering analysis revealed that these proteins lay within three main branches in the protein dendrogram. one cluster had proteins upregulated in the proliferative phase and two contained proteins up-regulated in the secretory phase. the unique protein profiles were also revealed using principle component analysis (pca): proteins clustered into two main groups, according to cycle phase. pca thus indicated similar unique protein signatures as suggested by hierarchical clustering. thirty one of the differentially expressed proteins were identified using maldi-tof ms. these proteins could be grouped into seven categories, which included structural ( ), transport ( ), regulatory ( ), membrane ( ), enzyme ( ), motor ( ) and others ( ). proteins involved in matrix assembly and those needed for subsequent establishment of secretory endometrium were up-regulated in proliferative endometrium. proteins important for cellular organisation and communication as well as products responding to environmental stress and the immune system were highly up-regulated during the secretory phase. biological pathways were constructed based on the proteins identified. the top network for secretory endometrium clustered around tgf-b. others related to inhibition of cell death / cell viability and leukocyte extravasation. these studies provide a global approach to the cyclic changes of the endometrium and highlight the complex dynamics of protein expression in human endometrium. hormonal regulation of prokineticins in the human fallopian tube: potential regulators of embryo transport. aw horne, hn jabbour, p lourenco, s wright, s battersby, arw williams, hod critchley. reproductive and developmental sciences, university of edinburgh, edinburgh, united kingdom; mrc human reproductive sciences unit, queen's medical research institute, edinburgh, united kingdom. background: understanding the factors regulating embryo transport in the fallopian tube (ft) has important clinical implications. embryo retention in the tube due to ft dysfunction is thought to lead to tubal pregnancy, a considerable cause of morbidity and occasional mortality. transport of the embryo through the ft is, for the greater part, accomplished by smooth muscle contraction. a group of multi-functional proteins and their receptors, called prokineticins, have been shown to affect smooth muscle function in other tissues, such as the intestine. the expression pattern of prokineticins, and their receptors, was examined in normal human ft obtained throughout the menstrual cycle, and the effect of the sex steroids on prokineticin expression was examined in an in-vitro model of the ft. methods: ft biopsies (n= ) and sera (for measurement of oestradiol and progesterone for endocrine staging) were collected from women undergoing gynaecological procedures for benign conditions. using a combination of quantitative taqman rt-pcr and immunohistochemistry, the mrna and protein expression pattern of prokineticins, and their receptors, were examined in the ft throughout the menstrual cycle. tubal explant culture was established using surgical tissue from the biopsies and exposed to varying concentrations, and time courses, of oestrogen and progesterone. results: prokineticin (pk ) and prokineticin receptor (pkr ) mrna are up-regulated in the progesterone-dominant mid-secretory phase. pk and pkr protein are expressed in the epithelium, smooth muscle and around the blood vessels of the ft. stimulation of tubal explant cultures with a physiological concentration of progesterone showed an up-regulation of pk and pkr . conclusions: prokineticins show temporal variation in expression in human ft and appear to be regulated by progesterone. their role in embryo transport needs to be investigated to further understanding of pregnancy complications, such as tubal pregnancy. translating mouse to human: a dynamic model of xenografted human endometrium. alex j polotsky, liyin zhu, nanette santoro, jeffrey w pollard. albert einstein college of medicine, bronx, ny, usa. in the mouse endometrium, the hormonal environment controls cellular proliferation and cell cycle activity. estradiol (e ) inhibits glycogen synthase kinase beta (gsk- ), resulting in nuclear accumulation of cyclin d and progression of the cell cycle, as well as dna replication licensing. in utero administration of the gsk- inhibitor, licl, results in epithelial cell proliferation in the absence of e (zhu, pollard. pnas. ; : ) . in this study, we derived a functional model of xenografted human endometrium to perform mechanistic studies of human endometrial proliferation. methods: human endometrial samples were obtained from volunteers aged - . immuno-compromised mice were transplanted with disaggregated/ recombined human epithelial glands and stroma under the kidney capsule. after weeks of out-growth, mice were ovariectomized, and replaced with e or licl. xenografts were harvested and processed for immunohistochemistry (ihc) and glandular labeling index (li). t test was used to compare group means. results: - % of engraftments were successful, resulting in a vascularized endometrium with characteristic architecture (a, b). hoechst staining confirmed that xenografts were made up of human cells ©. e (e) induced significantly greater proliferation compared to control (d) as assessed by ihc for ki , with li of . ± . and . ± . , respectively, p = . ). minichromosome maintenance- , a protein involved in dna replication licensing, was more sensitive for e -treated cells synthesizing dna than was ki staining (i). estrogen (g) and progesterone receptors (h) were expressed in xenotransplant tissue, the latter being up-regulated by e . licl (f) induced proliferation similar to e and greater than control (li = . ± . , p= . for e vs. licl). conclusion: xenografted human endometrium provides a dynamic model of endometrial proliferation that is well suited for translational studies. administration of licl in the absence of e induced glandular proliferation, supporting the notion that similar mechanisms are operative in human proliferation as in the mouse. gnrh analogs have been extensively used in assisted reproduction. although the main effects of gnrh analogs are via gnrh receptors on the pituitary gonadotrope, gnrh and gnrh receptors have been identified in many reproductive tissues including human endometrium, suggesting their potential action at endometrial level. in the present study, we examined the potential regulatory action of gnrha, la on sex steroid mediated gene expression in human endometrium. human endometrial surface epithelial (hes) cells and isolated endometrial stromal (esc) cells were used as in vitro models. all experiments lasted for h. the cells were treated with estradiol (e , nm, h), progesterone (p , nm, h), gnrha (la μm, h), e ( h) followed by p (last h) and gnrha plus e plus p where, gnrha added either first, second or last in order at h intervals. total rna was extracted, reversed-transcribed and subjected to real-time pcr simultaneously, measuring the expressions of il- , il- , il- , il- , il- , il- , il- , il- p , il- p , il- , ifn-and tnf . both hes and esc expressed majority of these cytokines with the exception of low to undetectable levels of il- , il- , il- and ifn-. treatment with e and p , either alone, or in combination significantly upregulated the expression of many of these cytokines at varying extend as compared to controls. however, gnrha either alone or in combination with e and p significantly diminished e , p or e plus p induced mrna expression of cytokines. the suppressive effects of gnrha on some of the cytokines varied significantly by the order which gnrha was introduced into the culture medium. we conclude that gnrha by acting directly on the endometrial cells effectively suppresses the mrna expression of several key proinflammatory cytokines upregulated by ovarian steroids. our results imply that gnrha therapy during assisted reproduction may modify endometrial receptivity via downregulation of proinflammatory cytokines induced by estradiol and progesterone. supported by nih grant hd . introduction: endometrial angiogenesis is characterized by a rapid increase during the early proliferative phase that peaks midcycle, followed by a gradual decrease in the secretory phase, while menses involves generalized endometrial inflammation, necrosis, and vascular thrombosis. vascular endothelial growth factor (vegf), whose expression is regulated by sex steroids, is a mediator of endometrial angiogenesis. recently, myoferlin, a kd transmembrane protein, was identified in endothelial cells where it mediates vegf-dependent endothelial cell proliferation, migration, and nitric oxide synthesis by affecting vegf receptor- function and stability. myoferlin also appears to play a role in vesicle trafficking and membrane repair. objective: to characterize myoferlin protein expression in endometrium. methods: western analysis of cultured human endometrial stromal cells (hesc) and human endometrial endothelial cells (heec) treated with physiologic concentrations of estradiol and progesterone was performed using a polyclonal rabbit antibody against myoferlin. subsequently, immunohistochemistry (ihc) was performed on human endometrium samples obtained from various stages of the menstrual cycle. results: myoferlin protein was expressed in hescs and heecs, but expression was not affected by sex steroids. ihc staining for myoferlin was specific, intense, and localized to the apical membrane of glandular epithelial cells and endothelial cells, with less intense staining in the stroma. h-score quantification showed that in endometrial endothelial cells, myoferlin protein expression was highest during the early proliferative and early secretory phases, while glandular and stromal myoferlin expression peaked during the late proliferative/early secretory phase. conclusion: myoferlin expression in human endometrium correlates with periods of greatest endometrial angiogenesis, and expression is not limited to endothelial cells, but also includes glandular and stromal cells. given the involvement of myoferlin in vegf signaling as well as membrane repair, understanding its role in human endometrium may further elucidate an understanding of endometrial development both under physiologic and pathologic conditions. the human embryo is the primary regulator of embryo-endometrial molecular cross talk during early implantation. gijs teklenburg, , cobi heijnen, esther baart, karima amarouchi, carolien boomsma, janet carver, helen mardon, annemieke kavelaars, nick macklon. reproductive medicine and gynaecology, and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands; nuffield department of obstetrics and gynaecology, university of oxford, women's centre, john radcliffe hospital, oxford, united kingdom. introduction: uterine receptivity and implantation are controlled by locally acting trophic factors and cytokines. in humans, the regulation of the embryo-endometrial dialogue beyond the early blastocyst stage is poorly understood. we hypothesized that the interaction between a healthy conceptus and the receptive endometrium is associated with a distinct local regulation of cytokine production favouring implantation. methods: human embryos, cryopreserved at day after fertilization and donated for research, were thawed and cultured under standard conditions until day five. forty-two embryos from donors developed to the blastocyst stage. following removal of the zona pellucida, they were placed in individual coculture on a confluent monolayer of endometrial stromal cells. on day , the developmental potential of each embryo was assessed as early arrested, late arrested or developing. culture supernatants were analysed for concentrations of il- , il- , il- , il- , il- , il- , il- , il- , tnf-, mcp- , ip- , eotaxin and hb-egf using a multiplex immunoassay. day supernatants from culture systems in which no embryo had been placed were also analysed as controls. results: out of ( %) embryos continued to develop and were able to attach and invade into the stromal cell compartment of the co-culture environment. twelve late arrested embryos showed signs of degradation on day and the totally disintegrated embryos were assigned to the early arrested group. supernatants from both early and late arrested embryo cultures contained significantly lower levels of a number of cytokines and growth factors in comparison to developing embryo cultures. moreover, the levels of these mediators in co-cultures were significantly lower than those in non-embryo control stromal cell culture supernatants. conclusion: these data suggest a pivotal role of the embryo in embryoendometrial cross talk. whether reduced mediator expression in co-cultures reflects a selective down regulation of stromal cell cytokine and growth factor production is now being investigated. epithelial cell dynamics during human implantation. hiroshi uchida, tetsuo maruyama, toru arase, masanori ono, takashi kajitani, maki kagami, hideyuku oda, sayaka nishikawa, yasunori yoshimura. department of obstetrics and gynecology, keio university school of medicine, shinjuku, tokyo, japan. epithelio-mesenchymal transition (emt) is thought to play a role in functional differentiation of endometrial epithelial cells during human implantation. molecular mechanism of epithelial sheet remodeling caused by embryo invasion remains elusive. to address this, we investigated cellular dynamics of n-cadherin and vimentin, the two representative major markers of emt, during implantation. in in vitro implantation assay using a human endometrial epithelial cell line, ishikawa, and a human choriocarcinoma cell line, jar (uchida et al., hum reprod ), we pre-treated human ishikawa cells with or without ovarian steroid hormones ( -estradiol + progesterone; ep), fa- (n-cadherin blocking ab), or suberoylanilide hydroxamic acid (saha), one of histone deacetylase inhibitors, which has a potential to improve in vitro implantation (ibid). implantation or treatment with or without ep or saha enhanced the expression of n-cadherin and vimentin but down-regulated ecadherin. furthermore, treatment with ep or saha accelerated ishikawa cell motility and increased the number and spreading area of jar spheroids. in vitro implantation assay, the most prominent staining intensity of n-cadherin was observed just around the adhered spheroid from which its intensity decreased away. functional blockade of n-cadherin by fa- resulted in the complete suppression of ishikawa cell motility, the unique distribution of n-cadherin around jar spheroids, and the spreading area of jar spheroids, while it did not affect the number of the adhered spheroids. human implantation consists of the multiple steps, including apposition, adhesion and penetration. thus, these results collectively indicate that emt may take place after the apposition and that n-cadherin may be required for the remodeling and emt of the epithelial sheet during embryo invasion. n-cadherin may enhance the recruitment of spheroid-neighboring cells, suggesting its role in the covering-up of the invading embryo through acceleration of epithelial cell motility. endocannabinoid regulation in human endometrium. jessica g scotchie, marc a fritz, steven l young. obstetrics and gynecology, university of north carolina, chapel hill, nc, usa. background: research in mouse models demonstrates two cyclically regulated endocannabinoids produced in murine endometrium, anandamide and -arachidonoyl glycerol ( -ag); both play critical roles in murine embryonic implantation. no studies of endocannabinoids in human endometrium have been performed. objectives: determine menstrual cycle expression and localization of synthetic and degradative enzymes for anandamide and ag in human endometrium. methods: human endometrium was collected from volunteers across the menstrual cycle (n= ). quantitative rt-pcr was performed analyzing the expression of: n-acylphosphatidylethanolamine (nape) and fatty acid amide hydrolase (faah), the synthetic and degradative enzymes for anandamide, respectively; sn- -diacylglycerol lipase-a (dagla), and b (daglb), the synthesis enzymes for ag; and monoacylglycerol lipase (magl) and cyclooxygenase- (cox ), the degradative enzymes for ag. the constitutive gene ppia was used for comparison. immunohistochemical localization of nape protein was performed using nape-pld polyclonal antibody (cayman chemical, # ). anova and student's t-test analysis performed on samples grouped by proliferative (pro), early, mid-, and late secretory (es, ms, ls) phases. results: mrna expression of all enzymes responsible for synthesis and degradation of anandamide and ag was detected throughout the cycle. no significant cyclic change in nape, faah, or daglb gene expression was seen. a decrease in dagla gene expression in the ms and ls phases compared to the pro and es phases (p= . ) was seen. magl gene expression was higher in the secretory phase than the pro phase (p= . ). cox gene expression was detected at low levels in the pro, es and ms phases, with marked increase in the ls phase (p< . for all comparisons). protein localization of nape showed a cytoplasmic epithelial location, with increased staining on pro and es samples compared to ms and ls samples. immunolocalization of remaining proteins is ongoing. conclusion: this is the first report documenting the presence of endocannabinoid synthetic and degradative enzymes in human endometrium. genes controlling anandamide expression do not fluctuate significantly across the cycle. however, ag's degradative enzymes increase in the secretory phase, suggesting that lower ag levels may be advantageous for embryo implantation. our findings suggest that human endometrial endocannabinoid regulation differs from murine regulation. interleukin- beta (il- ) regulates il- signaling in decidua-implication in the pathophysiology of preeclampsia (pe). sj huang, cf yen, cp chen, f schatz, cj lockwood. obstetrics, gynecology and reproductive sciences, yale university, new haven, ct, usa; ob/gyn, chang gung memorial hospital, tao-yuan, taiwan; ob/gyn, mackay memorial hospital, taipei, taiwan. objective: previously, we found much higher cytoplasmic immunoreactive il- levels in the preeclamptic decidual cells than in adjacent interstitial trophoblasts. such decidual cell-derived il- contributes to the systemic endothelial cell dysfunction that elicits the proteinuria and hypertension of the maternal syndrome. il- promotes the transition from innate to adaptive immunity. moreover, by skewing monocyte differentiation from a dendritic to a macrophage phenotype, decidual il- may promote the macrophage excess observed in the preeclamptic decidua. macrophages impair trophoblast decidual invasion to foster incomplete spiral artery remodeling that elicits placental ischemia and hypoxia. the current study: ) localized il- mrna levels in preeclamptic versus normal decidual sections; ) evaluated mechanisms regulating il- synthesis by targeting intracellular signaling pathways with specific inhibitors; ) identified potential il- targets by immunolocalizing the il- receptor (il- r) to specific cell types in placental bed biopsies. methods: in situ hybridization localized il- mrna in normal versus preeclamptic decidua. il- r was immunolocalized in placental bed biopsies. leukocyte-free first trimester decidual cells were incubated with e and mpa ± il- ( ng/ml) ± an inhibitor of p mapk (sb ) or protein kinase c (calphostin c) or nf b (activation inhibitor iii) for hrs. an elisa measured secreted il- levels. results: il- mrna was present primarily in decidual cells with increased il- mrna levels observed in pe. preferential expression of the il- r was observed on decidual cells in placental bed biopsies. compared with basal il- levels ( . ± . pg/ml/ g cell protein) by decidual cells, il- enhanced il- output by decidual cells ( . ± . pg/ml/ g cell protein). only the p mapk inhibitor significantly reduced this output to . ± . pg/ml/ g cell protein (n= , p< . ). our results indicate that inflammatory cytokine enhances il- synthesis in decidual cells of the preeclamptic decidua by a mechanism involving p mapk. such il- is likely to act as an autocrine/paracrine effector via decidual cell-expressed il- r to contribute to the macrophage excess observed in the preeclamptic decidua. heparanase is up-regulated by estrogen and during the secretory phase of the human endometrium. ronit haimov-kochman, shira natanson-yaron, caryn greenfield, achinoam lev-sagie, lichtenstein michal, haya lorberboum-galsky, israel vlodavsky, simcha yagel, arye hurwitz. ob/ gyn, jerusalem, israel; cellular biochemistry human genetics, hadassah hebrew university medical centers, jerusalem, israel; cancer and vascular biology research center, technion school of medicine, haifa, israel. introduction: heparanase is an endoglycosidase that cleaves heparan sulfate (hs) proteoglycan of the extracellular matrix. the full-length proheparanase is activated by cleavage into an active isoenzyme, resulting in the release of hsbound cell-differentiation factors, such as hb-egf. the cycling endometrium involves remarkable steroid hormone-induced tissue remodeling. in vivo, increasing exposure to unopposed estrogen may lead to endometrial malignant transformation. aim: to investigate heparanase expression and regulation in the cycling endometrium. materials and methods: heparanase mrna levels were measured by quatitative rt-pcr in naturally menstruating women and in hec a, estrogen receptor (er)-negative and ishikawa, er-positive endometrial carcinoma cell lines exposed to increasing doses of estradiol. heparanase isoenzymes were localized by immunohistochemistry using specific anitbodies in murine endometrium and human normal, hyperplastic and malignant endometrium. results: heparanase mrna level increased fold in secretory phase (d ) compared to proliferative phase (d ) endometrium. heparanase transcript levels increased fold during hr culture in er positive adenocarcinoma cell line exposed to increasing doses of estradiol, but not in hec a, er negative cell line. both heparanase isoforms were localized to murine glandular endometrium. human glandular endometrium at both proliferative and secretory phases was immunoreactive with the active isoform of heparanase. proheparanase was detected in basal membrane of endometrial glands and endometrial stroma during secretory phase. along with malignant transformation of the endometrium the presence of proheparanase increased dramatically from none in stroma of normal and hyperplastic endometrium to abundance in malignant tumors. conclusions: heparanase gene expression is higher during the window of implantation and up-regulated with estrogen in endometrial cells via er in vitro and vivo. heparanase is differentially localized in the secretory phase of the endometrium compared to the proliferative phase, suggesting a role for this molecule during the window of implantation in man. interleukin- (il- ) system mrna and protein expression in the human fallopian tube with ectopic implantation. hong-yuan huang, , tien-hung huang, chin-jung li, chyi-long lee, , hsin-shih wang, , yung-kuei soong. , obstetrics and gynecology, chang gung memorial hospital, kwei-shan, tao-yuan, taiwan; obstetrics and gynecology, chang gung university and school of medicine, kwei-shan, tao-yuan, taiwan. objective: ectopic pregnancy, an abnormal implantation of a fertilized ovum outside the uterine cavity, has been increasing in number at a staggering pace of all pregnancies. il- system is one of the major cytokines involved in human endometrium during embryo implantation and might perform a defensive role against maternal immune response. very little information is available regarding the expression and synthesis of cytokines in the pathogenesis of fallopian tube with ectopic gestation. the purpose of this study is to investigate il- system expression in human fallopian tubes with ectopic pregnancy. methods: paired segments of human fallopian tubes with ectopic implantation site and side portion close to ectopic gestation (n= ) were collected from women undergoing laparoscopic salpingectomy after informed consent and irb approval. segments of fallopian tubes from women undergoing tubal ligation (n= ) were used as control groups. total extracted rna was reverse transcribed and amplified by pcr using specific primers for gapdh ( bp), il- ( bp), il- bp ( bp) and il- r ( bp). quantitative il- and il- bp mrna expression in human fallopian tube was determined by real-time pcr. to determine the presence of il- system proteins, tissues were fixed and processed for immunohistochemical study. data analysis was done with anova and pearson's correlation. results: il- and il- bp as well as il- r mrna were all expressed in tubal ectopic implantation and normal tubes. according to real-time pcr with c t value quantification and -ct method, a significantly higher il- expression in tubal ectopic implantation and lower ratio of il- antagonist to agonist in portion close to ectopic implantation is demonstrated in comparison to normal tubes (p< . ). immunoreactive il- system at the protein levels was also present in human fallopian tubes with ectopic implantation and normal tubes. conclusions: these results suggest that fallopian tube il- system expression may play a crucial role during the process of early embryonic implantation. the expression and ratio of antagonist to agonist in fallopian tubes may indicate an earlier "dialogue " in human fallopian tubal gestation prior to uterine implantation. replacement. marcia c ferreira, ines kd cavallo, fernando m reis. gynecology, ufmg, belo horizonte, mg, brazil. activin a is a growth factor expressed in the endometrium, where it modulates tissue remodelling and enhances decidualization. the effects of activin a are counteracted by two binding proteins, namely follistatin and follistatin-like (fstl ). while the endometrial expression of activin a increases during the secretory phase of menstrual cycle, the effects of ovarian steroids on these proteins and their mrnas has not been assessed yet in postmenopausal women or in ovariectomized animals. we have evaluated the effects of estrogen alone or estrogen plus progestin on the endometrial expression of activin beta-a subunit, follistatin and fstl in ovariectomized rats. adult female wistar rats (n= ) were ovariectomized and received one week later a single dose of estradiol benzoate ( . mg/kg body weight, i.m. injection), either alone (n= ) or associated with depot medroxyprogesterone acetate ( . mg/kg body weight, i.m. injection, n= ), or oil vehicle (control group, n= ). one week after the hormone or placebo treatment, the animals were sacrificed and their uteri were removed and processed by immunohistochemistry and real-time pcr. data were normalized to the expression of ribosomal phosphoprotein p (rpp ) and analyzed with the delta-delta ct method, anova and newman-keuls test. activin beta-a subunit mrna levels increased significantly in the uteri of rats treated with estradiol alone ( . fold increase over controls, p< . ) and to the same extent in rats receiving estradiol plus medroxyprogesterone ( . fold increase over controls, p< . ). this was accompanied by increase of beta-a subunit immunostaining in estradiol and estroprogestin-treated rats, which was noted only in the surface endometrial epithelium. follistatin mrna expression, conversely, showed a significant decrease in the groups treated with estrogen alone ( . fold compared to controls, p< . ) and estrogen plus progestin ( . fold compared to controls, p< . ), while follistatin immunostaining in the glandular epithelium was weaker in estradiol and estroprotestin-treated rats compared to controls. fstl expression was similar in the groups. in conclusion, the expression of activin beta-a subunit increases and that of follistatin decreases following estrogen replacement in the endometrium of ovariectomized rats, and these effects are not further altered by the addition of progestin. endometrial nk cells are a unique inert nk subset until pregnancy. simcha yagel, irit manaster, jacob hanna, ronit haimov-kochman, miri godin, yuval bdolach, caryn greenfield, shira natanson-yaron, arye hurwitz, debra s goldman-wohl, ofer mandelboim. obstetrics and gynecology, hadassah-hebrew university medical center, jerusalem, israel; lautenberg center for tumor immunology, hadassah-hebrew university medical center, jerusalem, israel. introduction: we recently demonstrated that nk (natural killer) cells play a critical role in trophoblast migration and angiogenesis at the fetal maternal interface. nk cells populate the endometrium at the secretory phase of the menstrual cycle, the time of anticipated blastocyst implantation. peripheral blood (pb) nk cells and decidual nk (dnk) cells express a variety of activating receptors, including nkp , nkp and nkp , collectively known as natural cytotoxicity receptors (ncrs), and nkg d which regulate nk cell killing and growth factor production. to compare endometrial nk cell (enk) activating receptor expression and function to pbnk and dnk cells and endometrial ligand expression, with a focus on their roles in blastocyst implantation. patients and methods: subjects were ivf patients undergoing natural menstrual cycles. endometrium samples were collected on treatment days and . a lymphocyte profile of the endometrial cells and pb was performed. facs analysis was performed on isolated endometrial nk cells, pbnk cells and dnk cells for cd , cd , nkp , nkp , nkp and nkg d. ncr ligand expression was characterized on adherent endometrial cells using ncr-ig fusion proteins and nkgd -ig and nkg d specific ligands as well as control ccmi-ig. redirected killing assays and cytokine secretion assays of ifn , vegf, plgf, and il- with and without il- were performed. results: endometrial lymphocytes of day and in these women are mostly cd bright cd -nk cells, with a significant amount of t cells, similar to pbnk cells and in marked contrast to dnk cells. unlike pbnk and dnk cells, enk receptors do not express nkp , nkp . nkp and nkg d are the only activating enk receptor expressed. like decidual cells, adherent stromal endometrial cells expressed the ligands for nkp , nkp and nkg d, suggesting that these nk cells have potential for activation. finally enk cells could not kill or secrete cytokines. conclusions: these findings of a unique activating receptor profile on endometrial nk cells, unlike that of dnk and pbnk, suggest that enk cells are a special local population of nk cells that change dramatically and are activated at the onset of pregnancy. variation in platelet activation throughout the menstrual cycle. fiona c denison, amy o robb, imogen b smith, nicholas l mills, hilary od critchley, david e newby. centre for reproductive biology; centre for cardiovascular sciences, the university of edinburgh, united kingdom. background: platelet-monocyte aggregation (pma) is a sensitive and novel measure of platelet activation with important proinflammatory consequences including release of cytokines and chemokines. previous studies using less sensitive techniques suggest that platelet activation alters during the menstrual cycle in response to circulating concentrations of sex steroids. the effect of sex steroids on circulating (c) pmas during a single menstrual cycle is not known. objective: to determine whether cpmas, platelet surface (ps) p-selectin and plasma (p) p-selectin vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: healthy, nulliparous, pre-menopausal, non-smoking women (mean age years), with regular menses ( - days) were studied. subjects gave written informed consent and the study had ethical approval. serial venous blood samples were taken at menstrual, follicular, periovulatory and luteal phases of a single cycle (days - , - , - and - ). cpmas (monocytes positive for the platelet marker cd a) were measured by flow-cytometry. psp-selectin expression was calculated on cd a positive cells. isotype-matched controls were used. serum oestradiol (e) and progesterone (p), plasma and pp-selectin were measured by elisas. data were analysed by one-way anova with repeated measures and bonferroni's post-tests for multiple comparisons. results: luteal phase p was > nmol/l in all women. numbers of cpmas and expression of psp-selectin were both significantly higher during menstrual compared with periovulatory phase of the menstrual cycle ( . ± . vs. . ± . %, p= . and . ± . vs . ± . %, p< . , respectively). there was no significant difference in pp-selectin concentration during the menstrual cycle (p= . ). there was no correlation between levels of serum e or p and numbers of cpmas, expression of psp-selectin or pp-selectin concentration. conclusions: numbers of cpmas and expression of pspselectin are maximal at menstruation with neither numbers of cpmas nor expression of psp-selectin correlating with serum e or p levels. this study suggests that activated platelets may potentially contribute to the inflammatory response at menstruation by releasing inflammatory mediators. by angiogenic factors and peri-cellular proteases in decidual secretory endometrium (dse), decidua parietalis (dp), and basalis (db) of miscarriage patients and matched controls. comparison of these parameters between the two groups enabled hypothesizing about their correlation with the occurrence of miscarriages. methods: decidua was obtained during st trimester termination of pregnancy (control group) and vacuum aspiration of missed abortions (case group). vascularization was studied by cd -immunohistochemistry. the expression of vascular endothelial growth factor-a, placental growth factor, flt- , kdr, angiopoietin- , angiopoietin- , tie- and the membrane-type matrix metalloproteinases mt -, mt -, mt -and mt -mmp were determined at mrna and antigen level and cd -positive unk cells, cd -positive macrophages, proliferation (ki ) and apoptosis (activated caspase- ) were evaluated by immunohistochemistry in consecutive serial sections. results: the decidual vascularization pattern showed differences between cases and controls: i.e. fewer vessels with larger circumference in cases, and this correlated with the differential expression of various angiogenic factors and proteases at mrna and antigen level. moreover, the endothelial protein expression of flt , kdr, mt -and mt -mmp was increased at the implantation site of cases. ki and active caspase- showed similar levels in the two groups and also the immune cells, both unk cells and macrophages, showed no differences at the implantation site between both groups. conclusion: differences between cases and controls appeared not to be based on altered proliferation, apoptosis, and/or inflammation. the differences in vascularization pattern and in the expression of angiogenic factors and proteases between both study groups suggest a correlation between decidual vascularization and the occurrence of miscarriages. respond to adrenomedullin. yaun-lin dong, hong y wen, janice endsley, alison hogg, hui-qun wang, manubai nagamani, chandra yallampalli. background: natural killer (nk) cells are the predominant lymphocytes present in human implantation site. decidual nk cells express perforin, an essential molecule required for lysis. formation of the placenta involves cooperation between maternal nk cells and fetal trophoblast cells that remodels the blood supply; however, the interaction between trophoblasts and decidual nk cells is largely unknown. adrenomedullin (adm) has been implicated in regulating early placental function and fetal growth. objective: to determine the role of multifunctional peptide adm in the decidual nk cells and fetal trophoblast cells interactions. methods: decidual and placental tissues were obtained from normal firsttrimester pregnancies terminated for social reasons. ethical approval to use these tissues was obtained from the irb of university of texas medical branch. cell preparations containing all decidual mononuclear cells were isolated by collagenase enzymatic disaggregation. cd decidual nk cells were purified by magnetic bead isolation. results: ) immunohistochemical analysis showed that adm is expressed primarily in decidual cells and trophoblast cells at the human implantation site; ) confocal imaging analysis demonstrated that decidual nk cells, which were identified by anti-cd staining, express adm receptor components crlr/ramp /ramp and their mrna expressions were futher confirmed by rt-pcr; ) k target cell killing assay indicates that adm inhibits cytokine il- /il- -induced decidual nk cell cytotoxicity; and ) immunofluorescent labeling and flow cytometric analysis revealed that adm suppresses perforin expression by decidual nk cells. conclusion: trophoblast-derived adm inhibits decidual nk cell cytotoxicity via suppressing perforin expression, thus, our results provide evidence for a new paradigm of embryonic-maternal communication involving a adm mediated interaction between decidual nk cells and fetal trophoblasts. leandro g oliveira, gendie e lash, judith n bulmer, barbara a innes, roger f searle, stephen c robson. institute of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom. background: we have previously demonstrated that co-culture with extravillous trophoblast cells (evt) (expressing hla-g) alters cytokine secretion by uterine natural killer (unk) cells, particularly at - weeks gestation. we have also reported that unk cells can stimulate evt invasion, but only at - weeks gestation (not at - weeks gestation). in addition, unk cell cytokine profiles alter with increasing gestational age. other reports have suggested that evt or hla-g expressing cells may alter the expression of cytokines and angiogenic growth factors by unk cells. hypothesis: hla-g expressing cells alters unk secretion of cytokines. methods: cd + unk cells were isolated from early pregnancy decidua ( - and - weeks gestation, n= each group) using enzyme digestion and positive immunomagnetic bead separation. the human b lymphoblastoid . transfected with either hla-g ( g) or a mock cdna ( cdna) were obtained as a kind gift from mr r apps (university of cambridge, uk). isolated unk cells were cultured in the presence or absence of the two cell lines in either direct or indirect contact (n= each group and each gestational age) for hours. cell supernatants were analysed for cytokines using a fastquant® th /th multiplex protein assay (il- , il- , il- , il- , il- , tnf-, il- , il- , ifn-) or by standard elisa (tgf- ). the effect of direct co-culture of unk cells with g compared with co-culture with cdna at each gestational age was tested using mann whitney u test. the effect of co-culture of unk cells with g in both direct and indirect contact was also tested using mann whitney u test. results: there was no difference in the level of cytokines secreted by the g or cdna cells. cytokine secretion by unk cells was not altered after direct co-culture with either g or cdna cells at either gestational age. in addition, direct or indirect co-culture of g or cdna with unk did not alter cytokine secretion at either gestional age. conclusions: hla-g does not alter the secretion of cytokines by unk cells from either - or - weeks gestation. other evt or decidua derived factors (including hla-e) may be responsible for the alteration in secretion of cytokines by unks with increasing gestational age. introduction: natural killer (nk) lymphocytes are central to innate immunity and contribute to tissue homeostasis by eliminating altered cells. their nkg d receptor pathway plays a fundamental role in target elimination through binding nkg d ligands on the cell surface. reduction in the nkg d ligand, ulbp , expression is associated with immune resistance in neoplastic processes. we have previously shown that fibroblasts from adhesion tissue (at) are characterized by increased extracellular matrix molecules and inflammatory cytokines compared with normal peritoneal (np) fibroblasts. objective: to determine if there is a difference in nk lymphocyte-mediated elimination between np and at fibroblasts and to investigate potential role of nkg d pathway in this process. material and methods: expression of nkg d ligands; ulbp , ulbp , mica, and micb was evaluated by flow cytometry and western blot in primary cell cultures of fibroblasts from np and at, established from two patients. peripheral blood nk lymphocytes (cd +cd -) from three healthy volunteers were isolated using macs system with purity greater than % and kept in interleukin overnight. fibroblast elimination with and without ulbp blocking was investigated following -hour co-incubation with allogeneic nk lymphocytes using our established flow cytometric cell mediated cytotoxicity assay. paired t test was used in statistical analysis. results: the flow cytometry studies showed that nkg d ligands (ulbp , ulbp , mica and micb) were lower in at compared to np fibroblasts, reaching a statistical significance in ulbp expression (p = . ). western blot analysis also revealed a lower ulbp protein level in at than np fibroblasts. furthermore, nk lymphocyte-mediated elimination was % lower in at in comparison with np fibroblasts. blocking ulbp expression resulted in decreased nk lymphocyte-mediated np fibroblast elimination by %, supporting the role of nkg d receptor pathway in the process. conclusions: our results demonstrate that nkg d pathway is operational in at fibroblast resistance to immune elimination, and extends our prior observations of the potential role of immunological mechanisms in the pathogenesis of adhesion development. objective: galectin- is an anti-inflammatory lectin that has pleiotropic regulatory functions at the crossroad of innate and adaptive immunity. human galectin- is expressed in the placenta and immune privileged sites and it has been implicated in establishing immune tolerance. the aim of this study was to examine the evolution and placental expression of the lgals gene in primates. methods: seven primate nucleotide sequences were generated, aligned to vertebrate orthologs from all classes and subjected to phylogenetic analysis. deduced amino acid sequences were analyzed for functionally important substitutions. placental galectin- expression was studied by immunohistochemistry and western blot. results: ) the lgals gene had high sequence identity among all investigated species. ) phylogenetic analysis revealed that intense purifying selection had been acting on the lgals gene in placental mammals (dn/ds= . ); ) residues responsible for sugar binding or molecule stabilizing were highly conserved in primates. ) immunostaining showed a uniformly abundant and ubiquitous galectin- expression pattern in human, old and new world monkey and prosimian placentas, regardless the type of placentation. the lgals gene has conserved sequence and placental expression pattern in primates that may suggest its important function in maternal-fetal immune interactions. these results support the view that immune interactions at the maternal-fetal interface evolved in concert with invasive placentation and that these interactions have been maintained regardless of the degree of placental invasion in primates and other mammals. expression of interleukin- in human endometrium throughout the menstrual cycle and early pregnancy. yesim h uz, , william murk, umit a kayisli, aydin arici. department of obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa; department of histology and embryology, trakya university school of medicine, edirne, turkey. background: interleukin- (il- ) is a recently discovered heterodimeric cytokine, comprised by a novel p subunit and a p subunit shared by il- . it has biological activities that are similar to but distinct from il- , and is known to be involved in th /th cell class switching and the regulation of cytokines such as ifn-gamma, il- , tnf-alpha, and il- . early pregnancy is associated with alterations in the maternal immune response, such as changes in cytokine expression, and leukocyte recruitment and subtype switching. we hypothesized that expression of il- in the human endometrium is menstrual cycle-and pregnancy-dependent. materials and methods: endometrial samples from women (n= ) undergoing surgery for benign gynecologic conditions, and decidual tissues from women (n= ) with clinically normal pregnancies terminated voluntarily in the first trimester, were obtained after receiving informed consent. endometrial samples were grouped according to menstrual phase. paraffin sections were stained with il- p antibodies and evaluated semi-quantitatively with hscore. statistical analysis of the data was done using anova, with p< . considered significant. results: il- immunoreactivity was predominantly located in the cytoplasm of both endometrial stromal (esc) and glandular (egc) cells. escs showed mild il- immunoreactivity without significant changes in intensity throughout the menstrual cycle. on the other hand, first trimester decidual cells showed significantly stronger il- staining compared to escs from non-pregnant endometrium (p< . ). il- immunoreactivity in egcs was high in the late proliferative phase, as compared to other cycle phases and first trimester tissues (p< . ). moreover, egcs from the early secretory phase (p< . ) and first trimester tissues (p< . ) showed higher il- immunoreactivity compared to the early proliferative and late secretory phases. conclusions: this is the first study describing il- expression in the human endometrium and decidua. these results suggest that il- has a cycledependent expression in endometrial cells and may be involved in regulating cytokine expression and immune cell modulation during the menstrual cycle and early pregnancy. gercel-taylor, douglas d taylor. obstetrics, gynecology, and women's health, university of louisville, louisville, ky, usa. objective: estrogen appears appear to be a critical regulator of the immune system. since hypoestrogenism is present in the postmenopausal woman, our objective was to determine whether t cell activation and function, defined as il- production and signaling molecule expressions at the transcriptional and translational levels, were affected by a low estrogen environment. design: prospective study in a university research laboratory. materials and methods: jurkat . t cells, initially grown in estrogen free media, were incubated in pm (representing postmenopausal levels) or pm (premenopausal levels) of estradiol (e ) for hours. cells were either resting or activated with a phorbol ester, -phorbol -myristate -acetate (pma), and ionomycin. enzyme-linked immunosorbent spot assay (elispot) was performed to analyze production of il- . expression of signaling protein components, cd and jak, were determined by western immunoblotting. real time-polymerase chain reaction was performed to quantify cd , jak , and jak gene expression. a p value of < . was considered significant. results: jurkat cells exposed to pm e and activated exhibited significantly diminished numbers of il- producing colonies compared to t cells exposed to pm ( . ± . vs. . ± . colonies, p< . ). analysis of cellular cd and jak protein demonstrated that jurkat cells incubated in pm e expressed a . -fold decrease in cd and . -fold decrease in jak compared with cells incubated in pm e (p< . ). these diminished protein levels appeared to be the consequence of suppressed transcription, as the mrna levels of cd , jak and jak were significantly decreased in jurkat cells incubated in low levels of estrogen ( . , . , and . fold, respectively, compared to pm). conclusions: jurkat cells exposed to low postmenopausal estrogen levels produce significantly less il- following activation, which was associated with a significant decrease in signaling proteins. the diminished levels of signaling proteins appear to result from decreased cd , jak and jak gene expression in the presence of low estrogen. these findings support the observation of decreased cellular immune response in postmenopausal women and may provide a basis for the increased risk of infections and cancer proliferation associated with aging. support: dept. of ob/gyn research seed fund. the expression pattern of novel cytokines (il- and ) in human fetal membranes. judith eckardt, , stephen j fortunato, holger maul, ramkumar menon. the perinatal research center, nashville, tn, usa; womens' hospital, university of heidelberg, heidelberg, baden-wuerttemberg, germany. objective: interleukin (il) and are novel cytokines produced by various immunological cells in response to microbial antigens. the functions of these cytokines in reproductive system is unknown. this study examines the expression pattern of il- and il- in human fetal membranes from preterm and term births and in in vitro in normal term membranes in response to bacterial endotoxin (lipopolysaccharide-lps). methods: fetal membranes collected (n= ) from cesareans at term (normal, not in labor) were placed in an organ explant system for hours and were stimulated with lps for an additional hrs. fetal membranes were also collected (n= ) either at preterm or term after vaginal deliveries. in a case -control study (preterm birth vs. normal term deliveries) amniotic fluids (af) (n= ) were collected to document the role of il- and il- in ptb. tissue expressions of il- and il- were studied by rt-pcr using specific primers. elisa documented culture media and af cytokine concentrations. statistical analysis was performed using non-parametric mann-whitney u test. results: both il- and il - expressions were seen in fetal membranes in culture (in vitro) regardless of stimulation. in vivo in membranes from preterm and term deliveries and membranes at term not in labor also documented the expression of these cytokines. culture media analysis documented higher concentration of il- after lps stimulation (lps- . vs. . pg/ml; p= . ) whereas no difference was noticed in il- concentration (lps- . control- . pg/ml; p= . ) between the two groups. af analysis, regardless of the status, did not document detectable concentrations of either of the cytokines (lower limit . pg/ml for both). conclusion: this is the first study to document the expression of two novel cytokines in laboring and non laboring human fetal membranes and also in membranes from preterm deliveries. il- production was stimulated by lps whereas il- was not affected. these cytokines are not physiological components of af and their role in fetal membranes is unclear. higher il- concentration in response to lps but lack of its presence in term or preterm af is suggestive of an autocrine immune response during pregnancy in response to a microbial antigen. evidence for a selective migration of fetus specific cd + cd bright regulatory t cells from the peripheral blood to the decidua in human pregnancy. tamara tilburg, , dave l roelen, barbara j van der mast, godelieve m de groot, carin kleijburg, sicco a scherjon, frans hj claas. department of obstetrics, leiden university medical centre, leiden, netherlands; department of immunohematologie and bloodbank, leiden university medical centre, leiden, netherlands. during pregnancy the maternal immune system has to tolerate the persistence of fetal alloantigens. many mechanisms contribute to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic fetus. murine studies suggest that cd + cd + t cells provide mechanisms of specific immune tolerance to fetal alloantigens during pregnancy. previous studies by our group demonstrate that a significantly higher percentage of activated t cells and cd + cd bright t cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy ( ) . in this study we examined the phenotypic and functional properties of cd + cd bright t cells derived from maternal peripheral blood and decidual tissue. depletion of cd + cd bright t cells from maternal peripheral blood demonstrates regulation to a rd party umbilical cord blood cells comparable to non-pregnant controls, whereas the suppression capacity to umbilical cord blood cells of her own child is absent. furthermore, maternal peripheral blood shows a reduced percentage of cd + cd bright foxp + and cd + cd bright hla-dr + cells compared to peripheral blood of non-pregnant controls. in contrast, decidual lymphocyte isolates contain high percentages of cd + cd bright t cells with a regulatory phenotype that are able to down regulate fetus-specific and non-specific immune responses. these data suggest a preferential recruitment of fetus-specific regulatory t cells from maternal peripheral blood to the fetal-maternal interface where they may contribute to the local regulation of fetus specific responses. ( ) tilburgs t, roelen dl, van der mast bj, van schip jj, kleijburg c, de groot-swings gm, kanhai hh, claas fh, scherjon sa. differential distribution of cd (+) cd (bright) and cd (+) cd (-) t-cells in decidua and maternal blood during human pregnancy. placenta. apr; suppl a:s - . alicia del toro-arreola, lourdes nunez-atahualpa, juan velazquez-rodriguez, laura gonzalez-lopez, jorge i gamez-nava, adrian daneri-navarro. there is evidence that the maternal immune system is influence by changes in the hormonal levels during the menstrual cycle (mc). so far, the information related to the levels of t, treg, nk cells and receptors of activation and inhibitors is scarce. aim: to analyze the populations of t, treg, nk cells and their receptors of peripheral blood of healthy women and their correlation with hormones during mc. material and methods: we studied to women not using hormonal contraceptives in the day th of the follicular phase and st of the luteal phase of the mc. pbmc subsets and their receptors were determined by flow cytometry and hormone levels by chemiluminescence method. we found that the progesterone and prolactin were positive correlated (rho= . , p< . and rho= . , p< . , respectively) with cd /nkg in t cells and negative correlated (rho= - . , p< . and rho= - . , p< . , respectively) with nk cells. meanwhile cortisol was positive correlated (rho= . , p< . ) with the receptor nkg d expressed in nk cells. the results observed in this study in the luteal phase of mc on the expression of cd /nkg inhibitor receptor and nkg d activator receptor were related to a particular hormone (progesterone, prolactin and cortisol) might contribute to understanding the physiological role of the neuroendocrine axis on the expression of some receptors of the immune system in order to keep the homeostasis milieu of the mc. objective: sp-d, a key component of the innate immune system, is detected in amniotic fluid (af) and believed to originate in the fetal lung. however, sp-d is produced by other cells and therefore extra-pulmonary sources must be considered. the objective of this study was to determine the maternal and fetal plasma and af concentrations of sp-d to gain insight into the behavior of this natural antimicrobial peptide in pregnancy. moreover, we studied sp-d expression in maternal and fetal peripheral leukocytes. methods: maternal and fetal plasma and af samples were obtained from patients in the following groups: ) term not in labor (tnl; n= ); ) term in labor (til; n= ); ) preterm labor without histologic chorioamnionitis (ptl; n= ) and ) preterm labor with histologic chorioamnionitis (ptl-hc; n= ). sp-d concentration was measured by elisa. sp-d mrna expression in maternal and fetal leukocytes was evaluated by real-time qrt-pcr. flow cytometry and confocal microscopy were used to study the localization of sp-d in leukocytes. results: ) the af sp-d concentration increased as a function of gestational age (mean, til: , . ng/ml vs. ptl: , . ng/ml; p< . ); ) in contrast, the maternal and fetal plasma sp-d concentrations decreased with advancing gestational age (mean, til: . ng/ml vs. ptl: . ng/ml; p< . , and til: . ng/ml vs. ptl: . ng/ml; p< . , respectively); ) the maternal plasma sp-d concentration was lower than that of fetal plasma (mean: . ng/ml vs. . ng/ml; p< . ); ) however, sp-d mrna expression in maternal leukocytes was . fold higher than that of fetal leukocytes (p< . ); ) neutrophils (both maternal and fetal) expressed sp-d as demonstrated by flow cytometry and confocal microscopy. conclusion: ) the concentrations of sp-d in the maternal and fetal circulation decreased with gestational age while the af concentration increased; ) the expression of sp-d mrna is higher in maternal leukocytes than in fetal leukocytes; ) we report for the first time that maternal and fetal neutrophils are a source of sp-d and propose that this molecule plays a role in host defense against infection and in the modulation of the maternal and fetal immune response. introduction intrauterine insemination (iui) is a fertility technique that allows for couples to have intercourse after the procedure is performed. it has been postulated that intercourse after iui may increase the pregnancy rate by either endometrial stimulation or because it may represent a second spermatic flow in the periovulatory period. in the present study we evaluate the effect of intercourse on the pregnancy rate of patients undergoing iui. material and methods: from to patients were enrolled in the study. every couple undergoing iui was instructed at the moment of insemination to decide whether to have or not intercourse on the same day of the procedure. all couples were abstinent three to seven days before iui. the information regarding intercourse was recorded the day after treatment. ovulatory, insulin resistant, cervical, male, tubal and endometrial factors as well as parity and time of infertility were compared between the two groups. all these factors were analyzed based on number of follicles that ovulated in each group. our principal outcome was to determine the pregnancy rate. intercourse was practiced by . % of the couples. the global pregnancy rate was . %. the pregnancy rate for the couples who had intercourse was . % and . % for those who did not have intercourse (p< . ). even though age, parity, time of infertility and stimulation protocols were similarly distributed in both groups, the proportion of tubal and endometrial factors were higher among those who had intercourse (p< . ). when subjects with tubal and endometrial factors were excluded, the pregnancy rate between both groups (n= ) was similar ( . % vs . % for positive and negative intercourse, respectively). the average number of ovulatory follicles was . + . for the group that had intercourse and . + . for those who did not. according to our results, intercourse after iui does not improve pregnancy rate after this procedure is performed. furthermore our study indicates that iui does not interfere with sexual intimacy since almost % of the couples decided to have intercourse on the same day of the procedure. . we sought to investigate the effects of gravidity on mmc and fmc in healthy, parous women. methods: mc was assayed in dna extracted from peripheral blood mononuclear cells (pbmc). hla-genotyping was first conducted and mc quantified employing a q-pcr assay targeting a non-shared maternal-or fetalspecific hla polymorphism. gravidity was dichotomized as a history of one pregnancy compared with two or more, and the prevalence of mc was analyzed using logistic regression. possible confounders were included as appropriate, including subject age and time since last pregnancy. adjustment for possible correlation between values was also made when there were repeated measures for the same subject. results: for the mmc analysis, there were subjects with observations. for the fmc analysis, there were subjects with observations. table provides a summary of mmc and fmc by gravidity. mmc was significantly decreased with higher gravidity. fmc was not affected by gravidity. gravida gravida or more adjusted* or ( %ci) mmc / ( %) / ( %) . ( . - . ) fmc / ( %) / ( %) . ( . - . ) *adjusted for possible correlation between values within a subject, subject age, and time from last pregnancy, as appropriate. increasing gravidity is significantly associated with a decreased prevalence of mmc. despite additional sources of fmc, there does not appear to be an increase in fmc prevalence with increasing gravidity. the biology of mc is incompletely understood, and the nature of mmc and fmc are likely to be different given that acquisition of the former, but not the latter, occurs within a nascent immune system. these data raise interesting questions when considered as interactions of acquired grafts within a host, including whether emergence and persistence of one dominant source of mc may be most advantageous for an individual. anti-igd antibody treatment as a novel immunosuppressive agent for autoimmune diseases and its effects on th /th gene expression. tommie g nguyen, eileen d gallery, , jonathan m morris. elevated t-helper cell type- (th ) and type- (th ) cytokine expression have a role in autoimmune diseases, allograft rejection and pregnancy-related complications. thus, molecules that can shift the immunity away from th /th responses toward a th response represent a novel therapeutic treatment for these conditions. we have previously demonstrated that pregnancy is associated with a suppression of t-bet in peripheral t cells. in this study, we examined a novel effect of anti-igd antibody on t-bet expression, th /th gene expression in human peripheral blood mononuclear cells (pbmc) and its therapeutic effects in an animal model of collagen-induced arthritis. methods: human pbmc were isolated and then cultured in the presence of anti-igd antibody at various time points followed by stimulation with pma/ionomycin (p/i) for hrs. gene expression was examined by rt-pcr, western blotting and elisa. for in vivo study, arthritis-prone dba/j mice were induced to undergo joint inflammation by intradermal injections with bovine type-ii collagen. these mice were then given daily doses of mg/kg of intravenous injection with anti-igd antibodies as preventive or therapeutic treatments (n = per group). results: treatment with anti-igd antibodies significantly suppressed p/iinduced expression of t-bet (a master regulator of th immunity), tnf-(a classical pro-inflammatory th cytokine), and il- (a critical proinflammatory th cytokine) in human pbmc. this suppression is highly specific to these genes because anti-igd antibodies have no effects on the expression of ifn-g and il- (two classical th cytokines). in vivo experiment showed that anti-igd antibody treatment markedly reduced clinical severity of joint inflammation when comparing the clinical score of control mice group ( . ± . , mean ± s.e.m), preventive group ( . ± . ) and therapeutic group ( . ± . ) . conclusions: our study has demonstrated that suppression of t-bet by anti-igd antibodies, similar to the changes seen in human pregnancy is a novel in vivo anti-inflammatory effect. given the essential role of t-bet, tnf-and il- in the pathogenesis of human autoimmune diseases, anti-igd antibodies may represent a novel immunosuppressive treatment that needs further studies and evaluation. objective: women with circulating anti-phospholipid antibodies (apl) are at risk for recurrent miscarriage, preeclampsia and preterm labor. apl antibodies directly target the placenta by binding to phospholipids or phospholipid-binding proteins expressed on the surface of viable trophoblasts. the objective of this study was to determine the effects of apl antibodies on first trimester trophoblast cells. methods: two mouse igg anti-human beta -glycoprotein i monoclonal antibodies (mabs), designated id and iic , were used in these studies. the first trimester trophoblast cell line, htr , was incubated with either medium, a mouse igg control, id or iic ( - g/ml), in the presence or absence of unfractionated heparin ( ng/ml). trophoblast cell death and apoptosis was determined using a viability assay, hoechst staining and a caspase activity assay. cytokine production was evaluated by multiplex analysis. results: following a hour incubation, significant trophoblast cell death was induced by iic ( . ± . %) and id ( . ± . %) at the high dose of g/ml, when compared to the medium and mouse igg controls (p< . ). hoechst staining showed that id -and iic -induced trophoblast cell death was a result of apoptosis. moreover, id and iic induced a significant increase in caspase- , - and - activity (p< . ). treatment of trophoblasts with heparin significantly inhibited the effects of iic and id on cell death by . ± . % and . ± . % %, respectively (p< . ). following a hour incubation at lower concentrations ( g/ml), treatment of trophoblast cells with id or iic resulted in a significant upregulation of il- , mcp- , gro production (p< . ), and a significant reduction in il- secretion (p< . ). conclusion: this study demonstrates that at low levels apl antibodies can modulate trophoblast cytokine production, while at higher levels, the same antibodies induce trophoblast apoptosis in a caspase-dependent manner. these findings shed new light on the mechanisms by which apl antibodies may impact placental survival and function. antigenic targets for the diagnosis of premature ovarian failure. hc bohler, c gercel-taylor, lt ku, st nakajima, dd taylor. obstetrics, gynecology, women's health, university of louisville, louisville, ky, usa. objective: premature ovarian failure (pof) is a premature depletion of ovarian follicles before the age of , affecting approximately % of women < years. the involvement of autoimmune mechanisms in pof has been suggested and similar mechanisms have been postulated for other ovarian pathologies, including idiopathic infertility, polycystic ovary syndrome (pcos), or endometriosis. while the association of autoantibodies has been demonstrated for these ovarian pathologies, variation in specificity and frequency of false positivity have limited the diagnostic use of autoantibodies. the objective of this study was to develop an antigen array to differentiate antibody recognition patterns of pof from other infertility pathologies. design: prospective study in a university research laboratory. materials and methods: patients diagnosed with infertility were included in this pilot study: endometriosis (n= ), pcos (n= ) and pof (n= ). autoantibodies were assayed by dot immunoblotting using an antigen array derived from endometrial and ovarian cells. for the cellular antigen preparations, solubilized total proteins were separated by reverse phase-hplcquid chromatography and the individual proteins were blotted onto nitrocellulose membranes and reactivity visualized by peroxidase-labeled antihuman igg. results: patients with pof, endometriosis, and pcos all exhibited autoantibodies reactive with these cellular proteins. while some antigenic reactivities were shared by all infertility patients, the pattern of antigen recognition was distinct for patients with pof. patients with pof all recognized a common antigenic proteins (row , antigens a,b,d-g). conclusions: alterations in autoreactivity are observed in patients with the diagnosis of infertility; however, distinct patterns of autoantibody recognition can be demonstrated for patients with different pathologies. while this study needs to be expanded to reliably establish the specificity, sensitivity and positive and negative predictive values, patients with pof clearly exhibit a shared recognition pattern that may be useful a diagnostic marker. cicek gercel-taylor. ob/gyn, university of louisville, louisville, ky, usa. objective: americans' consumption of nutraceuticals is one of the most rapidly expanding health markets, growing at a rate of % annually. multiple nutraceuticals containing phytoestrogens have been marketed as "immune boosters" despite suboptimal evidence-based medicine to support such statements. as immunomodulatory therapies should affect downstream cytokine expression, the relative effects of estradiol and genistein in regulating expression of cd -and jak were tested. these markers were chosen since they are central to t cell signaling. cd -is a critical transducer of tcr activation and regulates t cell proliferation and cytokine production. jak upregulation is a specific marker for hematopoietic cell stimulation. methods: to test the immunomodulatory effects of phytoestrogens and estrogen, jurkat . (t cell leukemia) cells were grown in estrogen-free, phenol red-free media for hours. these cells were then exposed for hours to pm, pm (postmenopausal), or pm (premenopausual) of estradiol in the presence of increasing concentrations of genistein ( , . , . , . , , and m). cells were then solubilized and cellular protein quantitated. protein concentrations were standardized and western blots for each set of culturing conditions were run in triplicate. cd -and jak expression were quantitated following visualization with chemiluminescence by digital pixel quantification. results: our findings show that in the absence of estradiol and at postmenopausal levels of estradiol, genistein induced a dose dependent increase in cd -reaching a maximum of fold. although cultivation of t cells in pm of estradiol significantly increased the levels of cd -and jak relative to hypoestrogenic conditions, the genistein mediated dose response was not observed. conclusions: these in vitro results indicate that genistein can at least partially reverse suppression of signaling molecules observed in postmenopausal estrogen environments. clinically, this suggests that phytoestrogens may have greater immunomodulatory properties for postmenopausal females than those that are premenopausal. maternal serum il- as a biomarker of acute immunologic rejection of pregnancy. joaquin santolaya-forgas, juan deleon-luis, isabel galan. obstetrics and gynecology, brigham and women's hospital, boston, ma, usa; amarillo women's health research institute, texas tech university health science center, amarillo, tx, usa. objective: markers of acute rejection of pregnancy are very scarce. in this study we aimed at determining if rapid changes in maternal serum concetration of a variety of biomarkers could be used for this purpose. we used an established baboon model for in utero stem cell therapy to introduce at - days from conception and via ultrasound-guided celocentesis, human hematopoietic stem cells with different proportions of natural killer t-cells (nk). maternal blood was collected before and hours after celocentesis for quantification of hormones and il- using solid phase, enzyme labeled, chemiluminescent sequential immunometric assays. pearson correlation analysis was used for determination of significant changes from baseline (p< . ). results: all animals survived their pregnancies. seven animals receiving < % concentration of nk delivered at term ( days gestation) while animal receiving more than % concentration of nk had dead fetuses on ultrasound evaluation hours after celocentesis. table depicts mean maternal serum concentration of the biomarkers investigated (all n.s.). figure shows mean il- changes from baseline in continuing (n.s.) and rejected pregnancies (p< . ). conclusions: we have described a model in which in utero graft vs host disease can be studied. these preliminary results suggest that of all the biomarkers investigated, il- might be the most sensitive for detection of an acute rejection of pregnancy. biomarkers of acute immunologic rejection of pregnancy biomarker unit pre-celocentesis ( ) the activity of cytotoxic cd + t cells during pregnancy protects the mother and fetus from infection. however, pregnancy's effect on the proliferation and apoptosis of cd + t cells has not been clearly defined. objective: to determine if normal pregnancy changes the number of proliferating or apoptotic splenic cd +t cells. methods: female c bl/ mice were used unmated (um) or underwent timed mating. one day prior to harvest, mice were i.p. injected with bromodeoxyuridine (brdu), which is incorporated into replicating dna. harvested spleens were homogenized, enumerated, and stained for cell surface expression of cd and t cell receptor beta chain (tcr ). apoptotic cells were detected by treatment with terminal transferase and fitc-dutp (tunel). the numbers of cd +tcr + cells that were brdu+ or tunel+ were calculated from the percentage of positive cells obtained by flow cytometry and the absolute number of cells counted. for each experiment, the ratio of the number of positive cells in pregnant to um mice was compared by anova with dunnett's post-test. results: at day of pregnancy (n= ), the number of brdu positive cd + t cells was two fold higher than that found in um (n= , p< . ). the number of proliferating cd + t cells continued to be non-significantly elevated at day ( . x, n= ), day ( . x, n= ), and day of pregnancy ( . x, n= ) compared to um. by day of pregnancy (n= ) the number of proliferating cd + t cells returned to the um level, however by this time the total number of splenic cd + t cells was . fold higher than um (n= p< . ). on gestational day , the number of proliferating cd + t cells declined further ( . x, n= ), and the number of splenic cd +t cells returned to the um level (n= , p> . ). compared to um mice, there was no significant difference in the number of cd + t cells undergoing apoptosis at any gestational day examined ( . - . x, p> . ). in normal murine pregnancy, the number of cd + t cells is increased in late gestation, and then returns to baseline at the end of pregnancy. this is due to an early increase then gradual decline in cd + t cell proliferation, accompanied by a steady rate of apoptosis. this argues that the maternal immune system undergoes dynamic homeostatic changes, and is not globally suppressed. supported by nihro hd - niht ai . arturo cerbulo-vazquez, cun li, , gene hubbard, natalia e schlabritz-loutsevitch, , peter w nathanielsz. objectives: early thymocyte (t) maturation occurs in the cortex while later stages occur in the medulla. thymic epithelial cells (tec) synthesize gc and t express gr. tec may influence t cell maturation by regulating apoptosisinduced gc-gr interactions. igf- (also synthesized by tec) may support thymocyte prolifetarion. human fetuses of mothers in premature labor are exposed to gc. gc administered to pregnant baboons at . , . , and . gestation (g) alters fetal lymphocyte populations at . (g) (j repro immunol, , : ) . we determined if fetal gc exposure alters thymic ) structure; ) gr and igf-i protein. methods: pregnant baboons received saline (control ctr; n= ) or betamethasone i.m. ( μg/kg daily for two days at . , . and . g; gc group; n= ), c-sectioned at at . g under general anesthesia and thymic gr and igf-i evaluated by immunohistochemistry. results: gr localized to medulla and a few cortical cells. igf-i localized to cortex with little medullary expression. medullary necrosis was greater in ctr than gc fetuses. t gr was located in cytoplasm. no gross differences were observed between ctr or gc fetuses for either igf-i or gr. conclusions: a) early thymocyte maturation may be supported by igf- , b) later differentiation involves gr, and c) after exposure to gc doses equivalent to human therapy, no gross effects were detected on gr or igf, but d) natural thymic necrosis was inhibited. lindsay s christensen, peyman bizargity, elizabeth a bonney. ob/gyn, university of vermont, burlington, vt, usa. background: the exact mechanism by which bacterial products trigger preterm delivery and the immune cellular circuits involved remain unclear. our recent data in normal c bl/ (b ) or recombinase deficient c bl/ mice (rag-ko) indicates that t and b cells are not critical for lps-induced preterm delivery and stresses the importance of related innate mechanisms. rag-ko mice are more susceptible to lps, suggesting that t or b cells may control the innate response. macrophages are vital to innate immunity and produce proinflammatory cytokines that can activate prostaglandin synthesis and myometrial contraction. we questioned whether differences in susceptibility between the strains are due to differences in the uterine macrophage response to lps and thus examined macrophages at the maternal-fetal interface early after injection. objective: to compare uterine macrophages levels at and hours after lps injection in pregnant b and rag-ko mice. methods: b and rag-ko mice were mated and on gestation day , females were injected intraperitoneally with μg lps in l saline (pbs) or l pbs alone. euthanasia and uterine harvest occurred or hours after injection. frozen uterine sections were stained with the macrophage marker f / or an isotype-matched control followed by an alexafluor -conjugated secondary and a nuclear stain (dapi). sections were visualized by fluorescence microscopy. for each mouse, f / + dapi+ and total dapi + cells were counted in areas of representative section and the percentage of f / + dapi+ cells was calculated. the mean percentage for at least representative areas per experimental group was analyzed by anova. results: two hours post-injection, macrophages levels were similar in b and rag-ko mice injected with pbs (b , n= , . ± . ; rag-ko, n= , . ± . % + per area). lps injection increased macrophages (p< . ) in both strains (b , n= , . ± . ; rag-ko, n= , . ± . ,); no difference was evident between strains. the percentage of f / + cells was similar hours post-injection (b , n= , . ± . v. rag-ko, n= , . ± . ), and not elevated relative to the hour time point. objective. decay-accelerating factor (cd ), is expressed in the plasma membranes and protects mammalian cells against the lytic action of serum complement. phosphoinositide -kinases (pi ks) are involved in the regulation of cell functions by synthesizing a second messenger molecule ptdins ( , , ) p . akt, a serine-threonine kinase acts downstream of pi k and regulates cell survival, growth and proliferation. the pi k-akt activity is controlled by tumor suppressor gene pten. in this study we assessed whether the pi k-akt activity affects the expression of cd in human endometrial and cervical cells. methods. endometrial and cervical cell lines which differ in the constitutive pi k activity were used in this study. ishikawa and rl - endometrial cell lines harbor pten mutation and have high levels of phosphorylated akt (p-akt). hec- -a and kle endometrial cell lines and hela cells express wild-type pten and have minimal or no demonstrable levels of p-akt. the expression of cd was evaluated by rt-pcr, immunoblotting and flow cytometry. the pi k activity was assessed by immunoblotting with anti-p-akt antibodies. the effect of inhibition of pi k-akt pathway on cd expression was evaluated in cells treated with wortmannin ( nm), ly ( mm), or with akt inhibitor sh ( mm). results. immunoblotting densitometry and measurements of mean fluorescence intensities showed that the level of cd expression correlates with the status of pi k-akt pathway. the cd expression was lowest in hec- -a, ishikawa and rl - cells which constitutively express p-akt. higher cd expression was found in hela cells and kle cells which express wild-type pten product and has no detectable phospho-akt. mean fluorescence intensities were . -fold higher for kle cells and -fold higher for hela cells compared to hec- -a cells. treatment of cells with akt inhibitor led to . - . -fold increase in cd expression. the . - . -fold increase following treatment with pi k inhibitor wortmannin was found in ishikawa cells, rl - and kle cells. conclusions. human endometrial cell lines with elevated pi k-akt activity express lower level of cd compared to cell lines with intact pten gene function. these findings may indicate that structural alteration at the dna level and resultant overexpression of pi k-akt pathway are involved in the downregulation of cd . endometrial and cervical cells. pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cell shape is determined by the cytoskeleton, which provides the mechanical support and is involved in cellular signaling. apoptotic cells undergo major morphological changes such as rounding and contraction, a process regulated by caspases, the cysteine proteases responsible for events controlling the cell disassembly. the motifs in certain cytokeratins make them substrates for caspase degradation. anti-apoptotic serine/threonine kinase akt provides a survival signal by phosphorylating downstream effector molecules including caspase- . while studying the akt distribution in human endometrial cell line we found that akt shows filamentous pattern of staining resembling cytoskeleton organization. in this study we evaluated whether akt staining correlates with the microfilaments (mf), microtubules (mt) or intermediate filaments. endometrial ishikawa, rl - , hec- -a, kle and cervical hela cell lines were used in the study. incubation with cytochalasin d, ( ug/ml) or nocodazole ( mg/ml) and labeling with bodipy fl phallacidin, anti -tubulin or anti-akt antibody were used to assess whether cytoskeleton disruptors affect akt distribution and mf and mt organization. for colocalization, cells were stained with anti-cytokeratin mouse antibody followed by anti-mouse alexa conjugate, and then stained with anti-akt rabbit antibody and anti-rabbit alexa conjugate. the scans collected with laser scanning confocal microscope from channels filtered for alexa and alexa were combined digitally and evaluated with imaris colocalization analysis software. filamentous pattern of akt staining was most pronounced in ishikawa and less obvious in hec- -a cells. treatment with cytochalasin d or nocodazole resulted in disruption of mf and mt but had no effect on cytokeratin organization and akt distribution. double staining with anti-cytokeratin- and anti-akt antibody showed overlapping staining for cytokeratin and akt. analysis of digitally acquired images showed highest correlation for colocalized channels in rl - cells ( . ) followed by ishikawa ( . ) and hela cells ( . ). lowest correlation was found for kle ( . ) and hec- -a cells ( . ) conclusions. this study indicates a strong colocalization pattern of serine/ threonine kinase akt with cytokeratins, and suggests a mechanism by which cytokeratins might be protected against cleavage by caspase- and caspase- in the early apoptotic stages. background: cd + cd + t regulatory cells (t-reg), express foxp ,suppress antigen-specific immune responses and are important for allograft tolerance. during pregnancy the mother tolerates an allograft expressing paternal antigens (the fetus), requiring substantial changes in immune regulation over a programmed period of time. the presence of t-reg cells (cd + cd highfoxp +) was assessed in the peripheral venous blood of non-pregnant, pregnant and seven postnatal healthy women by flow cytometry. human decidua was obtained by surgical termination of pregnancy in the first (n= ), second (n= ) and third (n= ) trimester of human pregnancy. paraffin sections were immunostained for foxp and cd . foxp +cells were quantified in x fields and results compared between first, second and third trimester samples and according to the presence of extravillous trophoblast. results: fluorometric studies of blood samples indicate an increase % of circulating cd + cd highfoxp t-cells in pregnant ( . % [range . - . %]) vs. non-pregnant controls ( . % [range . - . %]; p< . ). a progression from st, nd and rd trimesters indicated the % of cd + cd highfoxp t-cells was . %, . % and . %, respectively. low numbers of foxp + cells were detected in all decidua samples and their distribution mirrored that of cd + cells. in st trimester samples, foxp + cells were often detected in lymphoid aggregates adjacent to endometrial glands. increased numbers of foxp + cells were detected in st ( . ± . ) compared with nd trimester decidua ( . ± . ; p< . ) but there was no difference between st and rd trimester ( . ± . ), nor between nd and rd trimester decidua. in st trimester decidua, numbers of foxp + cells were increased in areas without extravillous trophoblast. conclusion: normal human pregnancy is associated with an increase in the number of circulating cd + cd highfoxp t-cells. the presence of foxp + cells in early gestation human decidua may be important in the initiation of materno-fetal tolerance at an autocrine level. (supported by mrc). aims: -defensins are small cationic peptides with antibiotic and antimicotic activity. hyaluronan and its degradation products have been described as endogenous ligands for tlr and tlr , whose involvement in -defensin expression has been reported in different epithelial tissues and cell lines. we aim to investigate weather low molecular weight hyaluronic acid induces -defensin release by keratinocytes, via tlr and tlr . methods: the induction of -defensin production following in vitro treatment of human keratinocytes with a low molecular weight hyaluronic acid solution was evaluated by pcr-analysis and elisa techniques. studies on the involvement of tlr and tlr in -defensin production have been performed using specific blocking antibodies. results: pcr and elisa revealed an intense -defensin production following hyaluronic acid treatment in human keratinocytes. the -defensin production induced by hyaluronan was abolished following block of tlr and tlr by specific antibodies, demonstrating the involvement of these receptors. the same hyaluronic acid treatment did not induce activation of inflammatory genes, such as il- , tnf-, il- and il- . conclusion: our data show that hyaluronic acid is an efficient inducer ofdefensin production in keratinocytes, via tlr and tlr . this observation might be important to open new perspectives in the development of possible topical products containing hyaluronic acid, to improve the release ofdefensins by keratinocytes, ameliorating the self-defence of the skin in case of skin infections. therefore, one of the possible applications for this kind of topical products might be the treatment of infective vulvitis, one of the most distressing gynaecological diseases for adult women. pregnancy outcome? kiera von besser, serena wu, mary d stephenson. , obstetrics and gynecology, university of chicago, chicago, il, usa; obstetrics and gynaecology, university of british columbia, vancouver, bc, canada. objective: to investigate whether gender of an ongoing pregnancy impacts the probability of a successful outcome, and, to ascertain whether the gender of prior live birth(s) impacts subsequent pregnancy outcome, among women with rm/aps. materials and method: cohort-control study. rm subjects were evaluated by mds between - (stephenson, . couples who met aps criteria (wilson et al, ) , restricted to rm only, were followed prospectively. cohort data was compared to live birth data from the vital statistics agency of british columbia from - . secondary sex ratios (ssrs) among successful pregnancy outcomes were calculated by dividing the number of male live births by female. sex ratios were calculated for all pregnancies weeks, regardless if they ended in success or demise. pearsons test with yates continuity correction was applied. results: subjects were identified. subjects had prior live births of known gender ( male/ female), giving a ssr of . . there were also prior fetal demises wks of known gender ( / ) giving a sex ratio for all prior pregnancies at wks of . . subjects delivered subsequent live births of known gender ( / ), giving a ssr of . . there were also subsequent fetal demises ( / ) giving a sex ratio for all subsequent pregnancies of . . subjects delivered both prior and subsequent live births. the ssr was . ( / ) among their prior and . ( / ) among their subsequent live births. including fetal demises, subjects had ongoing prior and subsequent pregnancies. the sex ratio was . among their prior pregnancies and . among their subsequent. as the control, a ssr of . ( , / , ) was calculated from vital statistics data. when the prior and subsequent ssrs of the cohort were compared to each other, as well as to the control, there were no statistically significant differences. conclusions: our findings, from the largest study of its kind to date, suggest that, in patients with rm/aps, the gender of an ongoing pregnancy does not significantly affect the probability of a successful outcome, to any greater degree than it does in the general population. also, the gender of a prior ongoing pregnancy does not significantly impact the likelihood of developing rm/aps. oocyte maturation. jk friend, fb bezirci, e seli. ob gyn, yale u., new haven, ct, usa. introduction: oocyte maturation is associated with repression of transcription. during oocyte maturation, fertilization, and early embryo development until the onset of zygotic gene expression, proteins are synthesized from maternallyderived mrnas. the regulation of protein expression from these maternal mrnas is post-transcriptional, and occurs mainly via poly(a)-tail elongation. the embryonic poly(a)-binding protein (epab) is the predominant poly(a)binding protein before the activation of the zygotic genome, and plays a critical role in the activation of certain maternal mrnas, those bound by cpeb and probably pumilio. we are characterizing additional functions of epab during the process of oocyte maturation. methods and results: our model system is the xenopus laevis oocyte where oocyte maturation is induced by the addition of progesterone. our preliminary findings indicate that epab is phosphorylated, and that levels of phosphorylated epab increase upon progesterone-induced oocyte maturation. moreover, glycerol gradient centrifugation revealed that nonphosphorylated and phosphorylated epab are contained in distinct complexes that change mobility upon oocyte maturation. furthermore, oligo-dt selection for poly(a)-containing mrnas strongly suggests that these mrnas are bound exclusively by phosphorylated epab. using affinity purification, we have determined that nonphosphorylated epab exists primarily in a large protein complex prior to oocyte maturation that is later disassembled after the addition of progesterone. conclusions: based on these preliminary findings, we conclude that prior to oocyte maturation, the bulk of epab is nonphosphorylated and is found in a protein-rich complex separate from poly(a)-containing mrnas. upon oocyte maturation (when certain maternally-derived mrnas are activated for translation), the majority of epab becomes phosphorylated, and this phosphorylated form of epab is likely bound to translationally-active mrnas. we are currently investigating what kinase phosphorylates epab and whether this phosphorylation plays a role in translational up-regulation of epab-bound mrnas. introduction: reactive oxygen species (ros) play important roles in all aspects of cellular fate. nadph oxidase isoforms, a family of genetically preserved enzyme complexes, have been shown to be the main sources of ros in various cell types. however, the role of nadph oxidase isoforms in human myometrium proliferation and differentiation has not been defined. in the myometrium, different smooth muscle phenotypes maybe associated with specific physiologic functions. we have shown that angiotensin ii (angii) stimulates hypertrophy but not cell proliferation in ultr cells, an in vitro model of human myometrium. ultr cells at greater than passages display a replicative senescent phenotype. by introducing human telomerase reverse transcriptase (htert) gene, we have obtained a stable cell line (ultr-ht) which has a significantly increased division rate and distinct cellular morphology than the original ultr cells. objective: to determine the relationship of expression of nadph oxidase to ultr cellular fate. methods: early and late passages (p - ) of ultr and ultr-ht (p - ) cells were grown on either plastic or collagen iv (cn )-coated surfaces. ultr-ht cells were further stimulated with angii ( . um) for hrs. expression of nadph oxidase core (nox - and duox / ) and associated subunits (p phox, p phox, noxo , p phox, noxa , p phox, and rac / ), and angii receptors at / was identified by rt-pcr from cellular total rna. fluorescent immunohistochemistry (ihc) was employed to determine protein expression and localization. results: the mrna level of house keeping gene -actin was unchanged by any cellular manipulation. the senescent phenotype of ultr cells was accompanied by an apparent down-regulation of nox , p phox, and noxa genes, and an up-regulation of at / . overexpression of htert did not reverse nox , p phox and noxa expression while cell division rate was increased. however, there was a down-regulation of nox , at / and rac . plating ultr-ht cells on cn induced nox / down-regulation and up-regulation of duox / , with no apparent change of at / . however, exposure to cn re-directed cellular response to angii such that only nox was induced by angii stimulation. conclusion: expression of nadph oxidase isoforms nox , , , and duox / are correlated with ultr cell differentiation and cell fate control. data also suggests that ang ii-induced myometrial hypertrophy involves nox mediated ros generation. fluids from reproductive women -the influence of aging. eriko y fujii, masahiro nakayama. women's health, national center for child health and development, tokyo, japan; aska reproductive clinic, nara, japan. [introduction] receptor for advanced glycation end products (rage) is a multiligand type glycoprotein, and is characterized based on its ability to bind ages, adducts formed by non-enzymatic glycation and oxidation of protein and lipids. this process occurs during normal course of aging. ages/rage interaction regulates various physiological function, such as inflammation, angiogenesis through vegf inducement. a soluble form of rage (srage) works as a decoy in the body and inhibits intracellular signaling. [objectives] the balance of these factors may contribute to reproductive dysfunction by aging. we aimed to measure the ages, srage and vegf concentrations in plasma and follicular fluids from reproductive women, and examined the differences of those factors between young group and old group. [material and methods] patients' plasma and follicular fluid were collected with consent based on regulations of the ethical committee, and we measured ages (pentosidine, cml), srage and vegf in duplicate using commercially available elisa kits (fushimi co, r d and cyelex). concentrations were calculated from each standard curve, and compared between the young group under years old, and the old group over y.o. data were evaluated for the difference in two groups by student's t test , and the significance was determined by p< . . [results] ) srage in plasma, ± pg/ml (mean±sd), n= in the young group was significantly higher than ± pg/ml, n= in the old group. there was no significant difference in plasma vegf. ) vegf in follicular fluid was ± pg /mg protein, n= in the young, and ± pg /mg protein, n= in the old was increased significantly. ) we could not see statistical difference of pentosidine nor cml concentrations between two groups in plasma and follicular fluid samples. [conclusions] it has been reported that higher concentration of vegf in follicular fluid may relate to worse pregnancy rate in art. there was a significant decrease of plasma srage in older group in our result, and because of this decrease of 'decoy', focal ages-rage-vegf signaling might be activated in older women. our results showed the possibility that ages/rage and vegf regulation may contribute to the reproductive dysfunction by aging. and leiomyosarcoma cells. qun pan, xiaoping luo, nasser chegini. ob/ gyn, university of florida, gainesville, fl, usa. as a part of a novel endogenous rna silencing machinery, a noncoding short rna strand referred to as "microrna" (mirna) has been identified to regulate the stability of the target gene expression through mrna degradation and repression. we have identified the expression of many of these mirnas in leiomyoma, myometrium, their isolated smooth muscle cells (lsmc and msmc), transformed lsmc (t-lsmc) and sklm (leiomyosarcoma cell line), including mir- which is predicted to target the expression of many genes, including tgf-b and tgf-b type ii receptor (tgf-brii). however, the biological significance of these mirnas in various cellular processes remains to be established. as such in the present study we examined the expression, regulation and function of mir- in lsmc, msmc, t-lsmc and sklm. we found that mir- is expressed and regulated by b-estridiol and medroxyprogesterone acetate ( - m) in these cells (p< . ). we further assessed the regulatory function of gain of and loss of function of mir- on the expression of tgf-brii. transfection of lsmc, msmc, t-lsmc and sklm with pre-mir-and anti-mir- oligonueclotides resulted in a significant increase and/or inhibition of mir- expression in these cells, respectively as determined by realtime pcr (p< . ). over-expression of mir- resulted in a significant reduction, while transfection with antimir- increased the expression of tgfbrii mrna and protein in these cells as compared to controls (p< . ). we concluded that mir- is expressed in leiomyoma and myometrial cells, its expression is regulated by the ovarian steroids and it functions by targeting the expression of tgfbrii and possibly other genes with key regulatory action on cell growth, angiogenesis, transcription regulation, ecm turnover and apoptosis that results in leiomyomas growth and regression. (supported by nih grant hd ). objective: placenta and a number of gestational tissues are well recognized to express corticotrophin releasing factor (crf), urocortin (ucn ), ucn , ucn and crf -r and crf-r receptor subtypes together with crfbinding proteins locally. ucn and ucn are implicated in the reversal of stress responses initiated by crf. in the present investigation, we evaluated functions of crf and ucns by quantifying their contents in venous smooth muscle layers using image pro . in human umbilical cords collected at preterm and term gestation methods: umbilical cord specimens ( - mm thickness, pieces per umbilical cord) collected at preterm and term (n= each) at delivery were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analyses with polyclonal antibodies to crf ( : ), ucn ( : ), ucn ( : ) and ucn ( : ) (peninsula laboratory, pa and sigma aldridge, ms) by standard abc technique. immunoreactive materials on the sections were identified using , '-diaminobenzidine as a chromagen. immunostaining intensities (od/area) on uc-sections were quantified by image pro . software and expressed as arbitrary units (au). all values were expressed as mean ± sem. differences between groups were evaluated by anova, followed by the post-hoc tukey test for multiple comparison. results: antibodies to crf and ucns elicited positive immunostaining of variable intensity in venous smooth muscle layers in uc-sections of preterm and term gestations. immunostaining intensity (au) of venous smooth muscle layers at preterm (pt) and term (t) are as follows: crf-pt= . ± . vs crf-t= . ± . (p< . ); ucn -pt= . ± . vs ucn -t= . ± . (p< . ); ucn -pt= . ± . vs ucn -t= . ± . (p=ns); ucn -pt= . ± . vs . ± . (p< . ). conclusion: crf content in venous smooth muscle layer markedly increased at term, while ucn and ucn contents significantly decreased and no significant change occurred in ucn content. based on the opposing changes in crf vs ucn and ucn immunostaining, we speculate that crf, but not ucn or ucn , is the major key player of vasodilation and function locally at the level of venous smooth muscle cells at term. ucn and ucn may perform cytoprotective functions at preterm. physiol. ; : - , am j physiol. ; : - ) . these studies showed that when ad libitum (al) feeding was resumed in cr females, fertility was sustained well beyond the age at which al-fed females became infertile. however, much of what is known on the effects of cr on fertility derives from models in which cr was initiated at weaning. further, there is large variation in how the experiments were conducted. objective: herein we tested if adult-onset cr could delay age-related infertility in females. methods: cr ( %, nia protocol as described in j geront. a:b -b ) was initiated in c bl/ female mice at months and continued until . months of age, at which time al feeding was resumed. matings were initiated at months of age. for mating during cr, a male mouse was housed overnight in a cage with a female and removed the next morning, so that the female mice could be fed their dietary food ration. al-fed and cr females followed the same mating regimen. the number of offspring born and that survived to weaning (day ) were recorded. results: fertility was lost in of al-fed females by months of age and continued to decline through . months of age. age-matched females maintained on cr during the same period exhibited poor fertility, with a total of pregnancies achieved out of females. although cr females showed poor fertility while on cr, their fertility improved dramatically after the reinitiation of al feeding at . months of age. while only out of , al-fed females achieved a pregnancy between . - . months of age, out of age-matched cr-then-al-fed females achieved a total of pregnancies in this -month time. notably, % of the pups born to cr-then-al-fed females between . - . months of age survived and developed to weaning without complications. conclusions: adult-onset cr allows maintenance of female fertility into advanced maternal age after the reinitiation of al feeding. how long fertility can be maintained and the minimum time needed for the beneficial effects of cr to be realized remain to be investigated. nonetheless, these observations suggest that there may be ways to safely extend fertility in females at ages when reproductive function is suboptimal (nih r -ag ). bile acids in human ovarian follicular fluid. laura p smith, , kaila deiorio-haggar, jason reindollar, alan s penzias, , anny usheva-simidjiyska. ob/gyn reproductive endocrinology infertility, bidmc, boston, ma, usa; boston ivf, boston, ma, usa; endocrinology, bidmc, boston, ma, usa. introduction: bile acids are known to serve important functions in the hepatobiliary and gastrointestinal systems. the presence of bile acids in the human ovary and relation with fertility potential have never been previously evaluated. methods: human follicular fluid (ff) from large follicles and small follicles was obtained at vaginal oocyte retrieval. human serum was obtained before and hours after human chorionic gonadotropin (h-cg). follicular fluid and serum samples were analyzed for total bile acids by spectrophotometry. bile acid concentrations were correlated with age, number of retrieved and mature oocytes, number of fertilized oocytes, and pregnancy. results: bile acid concentrations were analyzed and compared to the normal human serum bile acid concentration which ranges from to micromoles/l. bile acids are present in follicular fluid with a mean concentration of . micromoles/l in large follicles and . micromoles/l in small follicles (p= . ). pre and post h-cg serum bile acid concentrations differed significantly ( . micromoles/l vs. . micromoles/l, p= . ). there was a trend toward higher bile acid concentration in large follicles of young patients < years old compared to older patients years old ( . ± . vs. . ± . , p= . ). there was also a trend toward higher pre h-cg serum bile acid concentrations in older patients ( . ± . vs. . ± . , p= . ). there was no correlation between serum and follicular fluid bile acid concentrations and number of oocytes retrieved or number of mature oocytes, but there did appear to be a positive correlation between pre h-cg serum bile acid concentration and number of fertilized oocytes (spearman's correlation coefficient . , p= . ). conclusions: this is the first demonstration of the presence of bile acids within human ovarian follicular fluid. there may be a relationship between bile acid concentration and fertility potential. the precise function of bile acids in human ovarian follicular fluid is under investigation. objective to investigate the impact of ovarian hyperstimulation treatment on the biomarkers of the homocysteine pathway in blood and follicular fluid, and their association with the follicle diameter as a measure of follicular maturation. methods in women undergoing ivf/icsi treatment blood samples were collected on cycle day and the day of hcg administration. during oocyte retrieval in each woman the diameter of two follicles was measured and the corresponding follicular fluids were collected. in blood and follicular fluid total homocysteine (thcy), folate, cobalamin and pyridoxal' 'phosphate (plp) concentrations were determined. women with a serum folate . nmol/l were classified as folic acid supplemented. results ovarian hyperstimulation significantly decreased thcy and cobalamin blood levels (both p . ). the blood and follicular fluid concentrations of thcy, folate, cobalamin and plp were significantly correlated (all p . ). in the total group, a two-fold increase of thcy in follicular fluid resulted in a . mm decrease of the follicular diameter (p . ). in non-supplemented women this decrease was . mm (p . ). in supplemented women a twofold increase of follicular fluid folate resulted in a . mm decrease of the follicular diameter (p . ). conclusions ovarian hyperstimulation reduces thcy blood levels independent of folic acid supplementation. however, high follicular fluid thcy and folic acid supplementation may have detrimental effects on the maturation of the follicle. ), post-fixed for rain in % w/v osmium tetroxide in the same buffer, quickly dehydrated in a series of ethanol solutions, and embedded in epon. thin sections were stained with uranyl acetate followed by lead citrate and were observed at electron microscope. results: ten blastocysts from each group were collected and analyzed. compared to the in vivo embryos, blastocysts generated in vitro exhibited significant differences. the surface of their trophectoderm (te) layer had a reduced number of microvilli, the number of the apoptotic cells in the inner cell mass (icm) was higher and the presence of non functional mitochondria was elevated. in this study we have, for the first time, compared the ultrastructure of the in vivo and in vitro conceived blastocysts. taken together, these results suggest that both, the higher rate of apoptosis and the morphological alterations in the mitochondrial structure in ivf embryos, are associated with stress during in vitro embryo culture. therefore, these parameters can be used, in the future, as markers for the assessment of the embryo wellbeing in the ivf settings. deteriorating oocyte quality is a critical hurdle in the management of infertility, especially one associated with advancing age.here, we explore a newly discovered role of nitric oxide (no) in the sustenance of oocyte quality. methods: sibling oocytes from superovulated mice were subjected to intracytoplasmic sperm injection (icsi) with cauda-epididymal spermatozoa following exposure to either the no donor, s-nitroso n-acetyl penicillamine (snap, . m/min); a soluble guanylyl cyclase inhibitor, h-[ , , ] oxadiazolo [ , -a] quinoxalin- -one (odq, m) or an no synthase inhibitor, n w -nitro-l-arginine methyl ester (l-name, mm). their sibling oocytes were subjected to icsi either before (young) or after culture for the corresponding period (old). outcomes of fertilization, cleavage and development to the morula and blastocyst stages were compared. some embryos from each subgroup were also subjected to tunel assay for apoptosis. results: oocytes deteriorated in their ability to undergo normal fertilization and development to morulae/blastocysts after aging in culture, as compared to their sibling cohorts subjected to icsi immediately after ovulation (p< . ). this deterioration was prevented after oocyte exposure to snap. while, exposure to l-name or odq resulted in a significant compromise in fertilization and development to the morulae/blastocysts (p< . ) with detection of apoptosis, which was also noted in embryos derived from aged oocytes but not in those from young or snap exposed oocytes. conclusions: no is essential to sustain oocyte fertilizability and developmental ability, and to prevent blastomere apoptosis. objective to investigate the effects of the levels of the biomarkers of the homocysteine pathway on ivf outcome. methods from women undergoing an ivf or icsi procedures, two blood samples and two mono follicular fluid samples were collected for determination of folate, cobalamin, and total homocysteine (thcy). total protein was determined in follicular fluid to adjust the biomarker concentrations for follicular maturation. primary endpoint of the study was oocyte quality measured by fertilization and embryo quality (range - ; with being best quality). secondary endpoint was the occurrence of pregnancy. results % of the included women used a folic acid supplement (serum folate . nmol/l). in non-supplemented women higher cobalamin levels in follicular fluid correlated with a better embryo quality (estimate - . ; p . ) and higher thcy levels (median . mol/l, range . - . ) correlated with a worse embryo quality (estimate . ; p . ). in supplemented women higher follicular fluid thcy (median . mol/l, range . - . ) correlated with better embryo quality (estimate - . ; p . ). the follicular fluid folate level of oocytes that did not fertilize was . -fold higher than in the fluids of a fertilized oocyte ( % ci . - . ; p . ). a two-fold increase of follicular folate corresponded with a . higher chance to achieve pregnancy ( % ci . - . ). conclusions cobalamin levels in follicular fluid are correlated with embryo quality. folic acid supplementation modifies the thcy and folate levels in follicular fluid and thereby affects oocyte quality. the level of folate in follicular fluid is important in the fertilization process. we recently reported that increasing relative oocyte immaturity is associated with worsening outcome, and that cycles with many immature oocytes are more common in younger women. (moore et al, asrm annual meeting, ) to further investigate this trend, we conducted a case/control analysis of patients with repeated cycles of high-level oocyte immaturity (hloi). methods: oocyte maturity data was collected on all icsi cycles starting in . we defined a cycle with hloi as having > % immature oocytes (> sd's above the median). a case was defined as a patient with hloi on more than one cycle. control subjects were age-matched and defined as having </= % ( sd above median) immature oocytes on all cycles. results: from subjects, we identified cases of recurrent hloi (comprising icsi cycles) and control subjects. at baseline, cases had more oocytes retrieved per cycle than controls ( . vs. . , p<. ). all other baseline characteristics were similar. all case subjects were younger than . outcomes are shown in table . discussion: clinical pregnancy and live births were reduced in the case group, but more than expected based on % immaturity. during cycles, there were no live births among the case group. patients with recurrent hloi represent a previously undescribed group of young patients with a very poor prognosis during icsi treatment. this study may suggest that the problem underlying the immaturity may relate to overproduction of oocytes during stimulation, a factor which may be modifiable. however, the additional trend (though not significant) that even the mature oocytes from the case patients tend to have poor outcomes suggests the possibility of a maturation defect and warrants further consideration. data on response to gonadotropins, fert rates, and implantation rates will be presented. amongst those, were performed for an initial diagnosis of premature ovarian aging (poa), for the diagnosis of premature ovarian failure, and for repeated pregnancy loss. in women under age , b-fsh levels over miu/ml and up to < miu/ml denoted a diagnosis of poa, while levels of miu/ml were considered diagnostic for pof. all patients signed consent permitting review of medical records for clinical research purposes. results: day fsh levels ranged from . to . miu/ml (mean . ± . miu/ml). amh levels ranged from < . to . ng/ml (mean . ± . ) and allele repeats ranged from to , (mean for allele- , . ± . ; allele- , . ± . ). mixed model analysis of variance for all patients, adjusted for age, race, and allele- revealed a statistically significant correlation between b-fsh levels and number of repeats in allele- ( figure ; p < . ). we did not observe a significant linear relationship between serum amh levels and cgg repeat counts, however cgg repeat counts above were significantly associated with serum amh levels less than ng/ml (chi square = . ). conclusions: triple repeats above , a level currently considered well within normal, are associated with evidence of premature decline in ovarian function. the evaluation of triple repeats in frm gene offers a first objective assessment of ovarian function and should be predictive of autologous reproductive life span in most women. objective: the levels of mis in the serum and in the ovary have been demonstrated to decrease in response to increasing doses of cisplatin. we hypothesize that ovarian stimulation also will reveal dose related decreases in ovarian and serum mis levels after cisplatin exposure, further demonstrating the follicular damage from chemotherapy. methods: adult female sprague-dawley rats were treated with saline, . mg/kg cisplatin or . mg/kg cisplatin as two weekly injections. five days following the last cisplatin injection, the rats were injected with pregnant mare serum gonadotropin (pmsg) and euthanized hours following the pmsg injection. serum was collected and both ovaries were obtained for protein analysis. serum and ovarian levels of mis were determined using an elisa kit. western blot analysis, immunohistochemical analysis, and tunel analysis were also performed on the ovarian proteins. results: stimulated mis levels declined in a linear fashion in response to increasing cisplatin doses (p< . ). ovarian protein levels measured by elisa and western blot both indicated that stimulated ovarian mis levels also decrease in a linear fashion (p= . and p< . respectively.) immunohistochemical analysis revealed that while the total number of follicles remains the same, both the total number of mis positive follicles, and the percentage of mis positive follicles declines in a linear fashion (p= . for both). tunel analysis reveals that there is no change in the incidence of apoptosis in the stimulated ovaries from any treatment group. conclusions: the administration of pmsg after treatment with cisplatin causes a dose dependent decrease in the levels of mis in both the serum and in the ovary. serum mis levels following ovarian stimulation may be useful as a serum biomarker in this animal model to assess ovarian damage following the administration of cisplatin. correlations of micro-rnas a, b, - pp, and - p with inflammatory and steroidogenic factors in human granulosa/cumulus cells. tannaz toloubeydokhti, orhan bukulmez, kenneth c drury, r stan williams, nasser chegini. obstetrics and gynecology, university of florida college of medicine, gaineville, fl, usa. as part of the novel endogenous rna silencing machinery, noncoding short rna strands, referred to as micrornas (mirnas), have been identified to regulate the stability of the target gene expression through degradation and repression. the expression of many of these mirnas has been identified in human tissues however, their biological significance in various cellular processes remains to be elucidated. in this cross-sectional study of human granulosa cells, we aimed to study the potential correlations between five selected mirnas, mir- a, mir- b, mir- - p, mir- and mir- - p, and mrna expressions of some of their predicted target genes namely, cyclooxygenase (cox)- , il- , steroidogenic acute regulatory protein (star), aromatase and estrogen receptor (er) . granulosa/cumulus cells were obtained from women (age range - ; mean age . ± . ) undergoing oocyte retrieval for assisted reproduction. total rna was extracted from these cells and reversed-transcribed and real-time pcr was performed to measure steady-state levels of mirnas and mrna transcripts. we observed that the expression of mir- a, mir- - p, mir- and mir- - p displayed a significant and positive correlation with the expression of cox- . moreover, mir- b significantly correlated with star mrna expression, but not with other genes tested (p< . ). with advancing age, the expression of mir- - p and cox- was significantly decreased, with cox- expression displaying a positive correlation with aromatase, star and er mrna expressions (p< . ). none of the factors tested showed any significant correlations with peak estradiol levels and number of oocytes retrieved. receiver operating characteristic curve analysis revealed that female age cut-off value of . y best predicted whether mir- - p was below its mean arbitrary value. in conclusion, given the importance of mirnas' regulatory function in gene expression, our results suggest that mir- b may play an important role in gene expression related to granulosa cell luteinization, while variation in mir- - p expression with female age may suggest its potential role as a predictor of oocyte quality and granulosa cell function. given the importance of mirnas in gene regulation, the role of mirnas in ovarian physiology and aging requires further investigation. support: nih grant . introduction: a symbiotic relationship between ovarian granulosa cells (gc) and the enclosed developing oocyte is critical to reproductive efficiency. genetic modulations in gc's can lead to reproductive insufficiency, highlighting the critical role of gc's in reproductive competence. defects in the oocyte-gc repertoire in women with dor include lower e levels, luteal deficiency, poor fertilization rates, higher incidence of aneuploidy, lower pregnancy and higher miscarriage rates. utilizing global gene expression analyses in cumulus gc's, we attempt to enhance our understanding of biological mechanisms that may contribute to poor reproductive capacity in young women with dor. methods: infertile women (< years old) undergoing ivf were prospectively enrolled into two groups based on ovarian reserve (normal reserve, fsh< dor, fsh > miu/ml). at egg retrieval, cumulus gc's were isolated, rna extracted transcribed into cdna. microarray targets were generated cdna hybridized to affymetrix human genome u plus . genechips. microarray data were analyzed (array assist) and normalized (robust multichip analysis). a difference in gene expression of > fold was considered biologically relevant. results: of the , genes identified to be differentially expressed in young women with dor compared to normal reserve, genes demonstrated consistency of expression across five different normalization schema; were down regulated and up-regulated. expression of gremlin, a member of the dan family of genes known for its highly regulated expression pattern during folliculogenesis, was noted to be down-regulated -fold over two probe sets (- . ) in women with dor versus normal reserve; this down-regulation was confirmed by real-time pcr (- . ) . conclusions: this is the first demonstration linking differential expression of gremlin with etiology of infertility in women. gremlin is a downstream effector of oocyte-derived gdf- which facilitates cumulus cell expansion, a critical event in reproductive physiology. our finding of a significant downregulation of gremlin expression in cumulus gc's associated with dor may partly explain the physiology of poor reproductive performance. nih k cd . nih k rr . ferring pharmaceuticals. the objective of the present study was to investigate the effects of the gnrh antagonist ca on expression of mis and aromatase (cyp ) using luteinized human granulosa cells obtained during in-vitro fertilization cycles and an immortalized human granulosa cell line (hgl ). the granulosa cells were cultured +/-dibutyryl camp ( mm) and incubated +/-the gnrh antagonist, ca, for - h. rna was isolated, reversed-transcribed and real-time pcr was performed to measure mrna transcripts for ovary-specific cypiia and mis. we observed that camp markedly induced aromatase mrna in both the primary and immortalized human granulosa cells. interestingly, camp treatment of these cells also caused an upregulation of mis mrna. objectives: bisphenol a (bpa), a known endocrine disruptor, is a chemical used as a plasticizer in the manufacture of polycarbonate plastics and epoxy resins and is present in multitude products, including the interior coating of food cans, milk containers, and baby formula bottles. bpa can leach into foods during heat processing and is known to exert a variety of endocrine-like effects on different cell types acting as an estrogen because it contains two hydroxyl groups in its diphenyl structure. in this study we focused on the effects of bpa on aromatase expression and estradiol production in the human granulosa kgn cell line. we also evaluated its effects on several transcription factors crucial in cyp expression. materials and methods: kgn cells were cultured in f- dmem and were starved for h before experiments. subsequently they were treated for h with vehicle (control), fsh ( ng/ml), and/or bpa ( , , , , um) . messenger rna expression was quantified by real time pcr and estradiol secrection was measured in supernatants by elisa. results: fsh induced a -fold increase in aromatase expression. bpa induced a dose-dependent decrease in cyp production, with the greatest effect at um (p< . ), resulting in +/- % (mean+/-sem) inhibition, compared to aromatase expression induced by fsh alone. bpa also reduced levels of estradiol secretion in a dose-dependent manner, with the greatest inhibition at um (p< . ) resulting in +/- % decrease. we also evaluated expression of transcription factors known to be involved in regulating the activity of the ovary-specific aromatase proximal pii promoter. interestingly factors known for induction of aromatase such us steroidogenic factor- and gata- , mimic the pattern of cyp expression after bpa treatment, whereas, other receptors previously reported to act as aromatase inhibitor, such as ppar gamma were up-regulated by the addition of bpa. moreover, expression of creb remained virtually intact, suggesting that most likely mechanisms governing endocrine disruption by bpa are highly selective. conclusions: bpa inhibited fsh stimulated aromatase expression and downstream estradiol secretion. we propose that constant exposure to this chemical may result in endocrine disruption which may contribute to reduced fertility and early ovarian senescence. oocyte maturation occurs during folliculogenesis as a result of complex cell-tocell communications between the granulosa cells and the oocyte. maintaining the granulosa cells' spherical structure and network of gap junctions surrounding the oocyte is critical. this project tests the ability of a novel substrate-free threedimensional culture system to form viable granulosa cell spheroids. methods: after irb approval, freshly obtained follicular fluid from in vitro fertilization was obtained and granulosa cells were purified by percol gradient. nonadhesive agarose hydrogels, containing cylindrical round bottom recesses m in diameter, were cast from micro-molds designed using computer-assisted rapid prototyping. granulosa cells seeded at a density of , cells per gel were incubated for up to days. cellular viability was assessed with live:dead assay. results: after three days in culture, granulosa cells formed spheroids of densely packed cells that were difficult to disperse with multiple pipettings. the cells remained viable for at least days. conclusions: granulosa cells can be cultured in a novel substrate-free threedimensional culture system. the cells form tightly adherent spheroids that remain viable for extended culture. the cohesiveness of the cells suggests the formation of gap junctions. this is under investigation with immunohistochemistry and electron microscopy. these experiments suggest that a substrate-free threedimensional hydrogel culture system may be ideal to reassemble follicular structure important for future in vitro evidence testing and oocyte maturation. known to function as responders to pathogens, inflammation, and tissue injury. previous studies in our laboratory demonstrated that saa was produced in mouse granulosa and production was regulated by cytokines. ovulation has long been considered an inflammatory reaction and patients with chronic inflammatory conditions often experience infertility. the present study was undertaken to explore the role of saa in human ovarian function. methods: ovarian granulosa and luteal cells were obtained from surgically removed specimens and mural and cumulus granulosa-luteal cells were obtained from ivf aspirates. rna was extracted from fresh or cultured cells. some cells were treated in vitro with tnf or other cytokines for h. expression of saa was assessed by quantitative rt-pcr. in addition, serum levels of saa were determined using a commercial elisa in women undergoing ovulation induction (oi) and art. saa levels were measured at baseline, during oi, on the day of hcg administration and at the time of the pregnancy test. results: saa mrna was expressed in theca, granulosa, and granulosa-luteal cells. in granulosa-luteal cells both saa and saa mrnas were expressed at higher levels in cumulus compared to mural granulosa. expression of saa and saa in theca was increased following treatment with tnf in vitro. serum levels indicated that patients with ovulatory dysfunction had increased levels of saa at the time of hcg injection while patients without ovulatory dysfunction had lower saa levels as compared to the baseline level. in addition, patients undergoing oi who achieved pregnancy exhibited increased levels of saa at the time of the pregnancy test compared to baseline levels, whereas patients who did not become pregnant had lower post-cycle levels of saa. interestingly, saa levels did not change in art patients that became pregnant without undergoing oi (donor egg or frozen embryo transfer) conclusions: human ovarian cells express saa mrnas which can be altered in vitro. serum levels of saa may correlate with the responsiveness of the ovary to gonadotropins as evidenced by altered levels in women with ovulatory dysfunction, and by an increase in pregnant patients following ovarian stimulation. yeh, beom su kim, felicia hercules, jennifer peresie, armando arroyo. gynecology-obstetrics, university at buffalo, buffalo, ny, usa. objective: cisplatin is a common chemotherapeutic agent given to women for treatment for a wide variety of cancers. we hypothesize that one mechanism by which cisplatin may cause damage to ovarian structures is by decreasing the amount of anti-oxidant activity in the ovary. we examined super-oxide dismutase (sod ), a critical anti-oxidant enzyme that has been shown to be affected by cisplatin in other tissues, in the ovaries of cisplatin treated animals. methods: adult female sprague dawley rats were injected with saline, cisplatin . mg/kg or cisplatin . mg/kg as weekly doses. five days following the last injection, the rats were euthanized and both ovaries were excised. one ovary was processed for immunohistochemistry and the other was processed for protein analysis using western blot techniques for sod . the anti-sod antibody was purchased from santa cruz. the immunohistochemical sections were scored using a semiquantitative h scoring method. results: immunohistochemistry analysis of the expression pattern of sod following cisplatin administration revealed that there was a significant linear decrease in a dose response pattern in the expression of sod in antral follicles and in corpora lutea (p< . for both). no change was found in the h score of sod in other ovarian structures. western blot analysis of sod in the ovaries following increasing doses of cisplatin revealed no changes in the overall protein levels of sod in the ovary. conclusions: this is the first report that administration of cisplatin causes changes in the expression pattern of sod in antral follicles and in copora lutea. cisplatin decreases the amount of sod available in these structures, possibly leading to increased oxidative stress and free radical damage, thereby leading to ovarian damage found after cisplatin administration. is there evidence for aromatase activity in the stroma of postmenopausal ovaries? mf landay, rh fogle, rb allen, s patel, fz stanczyk, rj paulson. ob/gyn, usc, keck school of medicine, los angeles, ca, usa. background: following menopause, the ovaries continue to secrete androgens and estrogens. we have recently confirmed the production of androstenedione, testosterone and estradiol (e ) up to ten years after menopause by measuring gradients from ovarian venous effluent and peripheral blood. anti-müllerian hormone (amh) and inhibin b have been shown to be markers of follicular activity. peripheral levels of these hormones have previously been found to be undetectable in menopause, suggesting the absence of follicular activity in the postmenopausal ovary. objective: to investigate if the postmenopausal ovary continues to demonstrate evidence of follicular activity as the source of steroid production. design: observational study materials and methods: eight subjects aged ± . yr (range - ) were enrolled. postmenopausal status was confirmed by preoperative fsh levels of more than u/l and/or amenorrhea greater than months. serum was collected from the ovarian veins during total abdominal hysterectomy and bilateral oophorectomy. peripheral blood was also collected pre-operatively, intraoperatively and postoperatively. all samples were analyzed for amh and inhibin b using elisas with sensitivities of . ng/ml and pg/ml, respectively. androgen and estrogen levels in these samples have previously been reported, and documented a gradient between ovarian venous effluent and peripheral serum in all cases. results: ) six patients had no detectable follicular activity by amh and inhibin b levels. ) one patient demonstrated detectable inhibin b levels with an -fold gradient between ovarian venous effluent ( pg/ml) and peripheral blood ( pg/ml), however no amh was detected. ) in one patient, aged and months postmenopause, both amh and inhibin b were detected. peripheral inhibin b levels were high at pg/ml. amh was detectable at levels of . ng/ml. conclusions: ) in the majority of patients, continued e and androgen production in the ovary occurs in the absence of follicular activity as detected by amh and inhibin b production. ) some patients have evidence of follicular function up to ten years after menopause. ) since e is produced in post-menopausal ovaries in the absence of follicular activity, these data provide evidence for aromatase activity in the stroma of post-menopausal ovaries. to elucidate the process by which prostaglandin f (pgf ) mediates luteal regression, this study examined the role that the transcription factor yin yang (yy ) and histone deacetylase (hdac ) play in altering luteal cholesterol uptake by the scavenger receptor class b type i (sr-bi), intracellular cholesterol transport by steroidogenic acute regulatory protein (star), and cholesterol processing by p side chain cleavage enzyme (p scc) expression. rat luteal cells ( days post-ovulation) were treated with pgf ( hr) and trichostatin a (tsa; hr), a potent hdac inhibitor. protein expression was measured post-treatment by western blot and cholesterol was localized via filipin staining. star and sr-bi promoter activation was also assessed in hek t cells treated with yy , myy , a deletion mutant lacking an essential region required for transcriptional repression, and tsa. pgf caused a significant -fold decline in star (p< . ), and smaller declines in sr-bi and p scc which occurred concomitantly with an increase in yy ( -fold, p< . ) and intracellular lipid staining ( % increase). in contrast, nm tsa treatment caused a dose dependent increase in sr-bi, star, and p scc protein levels, . -fold (p< . ), . -fold (p< . ), . -fold (p< . ), respectively, and a . -fold decline (p< . ) in intracellular lipid levels. tsa prevented the pgf -induced decline in sr-bi, star, and p scc expression. promoter analysis demonstrated that wildtype yy , but not myy , repressed sr-bi and star activation while the addition of tsa overcame the repressive effects. this study shows the critical role that yy plays in pgf induced luteal regression by recruiting a histone deacetylase to block three essential processes in steroid production. in luteal cells yy -mediated global repressive action prevents cholesterol metabolism by inhibiting cholesterol uptake, intracellular transport, and processing thus leading to functional and structural luteal demise. supported by nih hd and nih hl . regulation of intermedin expression in cycling rat ovary. madhu chauhan, rebekah elkins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd) is a ct/cgrp family peptide involved in a variety of physiological functions, including vasodilatation and fetoplacental growth. this peptide is expressed in a variety of tissues such as stomach, placenta, uterus, pituitary and ovary suggesting its different functions including in reproduction. imd gene has an estrogen response element and we have shown that the plasma concentration of immuno-reactive imd is elevated in rats with pregnancy. however, expression of imd in the ovary and its regulation during estrous cycle is not known. we hypothesize that imd is expressed in the ovary and its expression is hormonally regulated throughout the estrous cycle in rat. objective: ) to assess expression of imd mrna and its receptors components calcitonin receptor like receptor (crlr), and receptor activity modifying proteins, ramp and ramp mrna in rat ovary; on diestrus, proestrus and estrus stages of rat estrus cycle and ; ) to demonstrate immunohistolocalization of imd, crlr, ramp , ramp and ramp in rat ovary. methods: groups of sprague-dawley non-pregnant and pregnant rats on day of gestation were used in this study. non-pregnant rats were sacrificed on diestrus, proestrus and estrus stage. ovaries were collected and total rna was extracted using trizol method. rna was treated with dnase before performing the reverse transcriptase polymerase chain reaction (rt-pcr). immunohistolocalization of imd, ramp , ramp and ramp proteins were assessed in tissue sections of ovaries from pregnant rats sacrificed on day . results: ) imd mrna is regulated in rat estrus cycle and its expression is significantly downregulated in estrus stage compared to the diestrus and proestrus; )expression of imd receptor crlr is highest in the diestrus stage, followed by a decline in proestrus which further declined during estrus; ) expression ramp mrna is higher in proestrus compared to diestrus and estrus (p< . ) but there is no significant change in the ramp expression during the estrus cycle and ; ) imd, ramp , ramp and ramp are immunolocalized in rat ovary in granulose cells of all follicles and granulosa cells of the corpus luteum. conclusion: imd mrna and protein are expressed in rat ovary suggesting a possible role for imd in ovarian function. defining and defying deterioration in oocyte quality with advancing chronological age: role of nitric oxide. pt goud, ap goud, mp diamond, b gonik, hm abu-soud. div reprod endocrinology, dept ob/gyn, wayne state university, detroit, mi, usa; div maternal and fetal medicine, sinai grace hospital, wayne state university, detroit, mi, usa. nitric oxide (no) is a ubiquitous free radical essential for oocyte maturation, function and sustenance of oocyte quality post-ovulation. the current study investigates the role of no insufficiency in the causation of oocyte quality deterioration with advancing chronological age. methods: in set , oocytes were retrieved from the following superovulated b d f mice: (a) young breeders (yb, n= ); (b) retired breeders (rb, n= ), and © old animals (oa, n= ), aged - , - , and - weeks respectively; and studied for zona pellucida dissolution time (zpdt), spindle ( -tubulin fluorescence immunocytochemistry, sigma) and chromosome morphology (dapi, vector), ooplasmic microtubule (mt) dynamics (omd) in response to taxol [ ], and cortical granule (cg) status (rhodamine conjugated lectin, vector). in set (n= ), oocytes from retired breeders were studied after exposure in vitro to an no-donor, s-nitroso acetyl penicillamine (snap in m- , . m no/min , n= , h, °c, % co ), while their sibling control oocytes were cultured for corresponding period under identical conditions without snap. [ ]. zpdt, spindle and chromosome morphology, omd and cg status were assessed with a confocal microscope and compared between the subgroups using anova, chi square and/or fisher's exacts test and appropriate post-hoc tests. results: in set , a significantly fewer oocytes were retrieved per animal (mean) in rb ( ) and oa ( ) compared to yb ( . , p< . ). advancing age was also associated with a significant increase in zpdt, omd and cg loss in rb compared to yb (p< . ). furthermore, significantly fewer oocytes from rb than yb had normal spindle and chromosome morphology (p< . ). oocytes from oa had significant spindle and chromosome disarray, a near total cg loss and significantly harder zp (p< . ). these oocytes also exhibited diminished omd in response to taxol although, metaphases were exquisitely sensitive to disruption with taxol. in set , exposure to snap in oocytes from rb resulted in a significantly lowerzpdt, omd and cg loss, and significantly higher incidence of normal spindles ( the gamma-aminobutyric acid (gaba), its biosnthetic enzyme gad and gaba-a receptors have been found in the oviduct and ovary. objective: this study examined the expression of gaba-a receptor subunit genes and gad and whether the gaba-a receptor could alter cytosolic ca + in gc. methods: for qrt-pcr, both human cumulus and mural gc were obtained at the time of oocyte retrieval for ivf and cultured separately. total rna was isolated separately from each gc type and from human brain ( positive control). the total rna from patients was pooled for each gc type and all rna was treated with dnase i. two-step qrt -pcr was performed using gene-specific lux™ primers for all gaba-a receptor subunits, gad and gad plus gapdh. single and specific qrt-pcr products were verified by melting curve analysis, gel electrophoresis, and dna sequencing. for ca + study, gc were cultured on coverslips. gc were loaded with fura- -am and changes in ca + concentration of gc were studied using a dynamic digital ca + imaging system. gaba-a agonist, muscimol, was used to study any dose-dependent effects on gc. gaba-a antagonist, m bicuculline, were perfused min. prior to and during application of muscimol. the responses were represented as changes in the / nm fluorescence ratio over time. m atp was used as positive control. results: the qrt-pcr results indicated that all gaba-a receptor subunits were expressed to various degrees in both types of gc, with the expressed highest in both cell types. gaba-a receptor subunits showing the next highest expression in both cell types were , and . gad isoenzyme was more abundantly expressed in cumulus and mural gc than gad . ca + imaging showed that muscimol, had the ability to increase ca ++ in gc, about % gc (n= ) cells responded to muscimol. muscimol increased intracellular ca + in a dose-dependent manner. the muscimol responsive cells was reduced by bicuculline, from % to % (n= , p< . ). conclusion: qrt-pcr indicates that gaba-a receptor subunit gene and gad expression occurs in both cumulus and mural gc. the ability of bicuculline to inhibit the ca + response to muscimol suggests the activation of gaba-a receptor. the current study confirms the presence of functional gaba-a in gc for the first time, and suggests that gaba may exert trophic effects in the ovary via gaba-a receptor. the present study test the hypothesis that administration of l-nitro-l-arginine methyl ester (l-name), a nos inhibitor, prior to hypoxia prevents the hypoxiainduced activation of p mapk, erk and jnk in the cerebral cortex of the guinea pig fetus during gestation. to test this hypothesis guinea pig fetuses at and days gestation were assigned to normoxic (nx, n= ), hypoxic (hx, n= ) and hypoxic pretreated with nos inhibitor (hx+l-name, mg/kg i.p., n= ) groups. hypoxia in the fetuses was induced by exposing the pregnant guinea pigs at both gestational ages to an fio of . for min. cerebral tissue hypoxia was documented biochemically by determining the tissue levels of atp and phosphocreatine (pcr). neuronal nuclei were isolated, purified and proteins separated by sds-page, and then probed with specific phosporylated erk, jnk and p antibodies. in the days gestation group: expression of p-p was . ± . (nx), . ± . (hx) . ± . (hx+l-name). p-erk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-jnk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). in the days gestation group: expression of p-p was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-erk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-jnk expression was . ± . (nx), . ± . (hx) . ± . (hx+l-name). the data show that administration of l-name prior to hypoxia decreased the expression of phosporylated p , erk and jnk at both gestation ages however expression of phosporylation was higher at term as compared to preterm. since a nos inhibitor prevented the hypoxia-induced phosphorylation of p , erk and jnk in both gestational ages, we conclude that the hypoxia-induced activation of p , erk and jnk in the cerebral cortical nuclei of preterm and term guinea pig fetus is no-mediated. we speculate that no-mediated modification of cysteine residue leading to inhibition of map kinase phosphatases results in increased activation of p , erk and jnk in the guinea pig fetus. (nih-hd , nih-hd and st. christophers foundation for children). the the endocannabinoid, anandamide (aea), is involved in the hormone-cytokine network that regulates implantation and early pregnancy maintenance with levels at the endometrial level considered a major checkpoint . high levels ( nm) in culture are associated with embryo death while plasma levels (> nm) at weeks in women undergoing ivf-et are associated with failed pregnancy . what is uncertain is whether systemic aea levels after spontaneous conception in women presenting with threatened miscarriage are predictive of pregnancy outcome. our aim was therefore to measure plasma aea levels in women presenting with threatened miscarriage and to relate these to outcomes. methods: plasma aea levels were measured using a sensitive and previously validated hplc-ms method at - weeks gestation in women (nonsmokers, bmi < kg/m ) presenting with threatened miscarriage and in whom a viable pregnancy was confirmed by ultrasound scan. results: nine of the women subsequently had a miscarriage. the plasma aea levels in those women who had live births was . -fold lower than that in those who subsequently miscarried ( . ± . nm versus . ± . nm, mean ± sem; respectively; p< . unpaired student's t-test). the roc analysis revealed an area under the curve of . ± . with a sensitivity of % ( %ci of . % to . %) and a specificity of . % ( % ci of . % to . %). using an aea level of . nm as the optimal cut-off point, a single plasma aea measurement provided a sensitivity of % and a specificity of % with a negative predictive value of % and a positive predictive value of % for subsequent miscarriage. conclusion: these findings suggest a possible predictive role for plasma aea in women presenting with threatened miscarriage. the data also indicate that systemic aea levels may reflect local endometrial levels and therefore the local hormonal milieu in the early stages of normal pregnancies and in those complicated by threatened miscarriage. introduction: follicle stimulating hormone (fsh) mediates cyclic follicle growth and development and is widely used for controlled ovarian stimulation. the ovarian response of different women to fsh is variable, ranging from poor response to ovarian hyperstimulation and has been partly attributed to two common variants of the fsh receptor (fshr). we have previously identified four abnormal fshr splicing products ( exon deletions and intron insertion) in women with low and high response to fsh. two of the splice variants, deletion of exon and deletion of exon , showed a correlation with low and high response to fsh, respectively. in the current study, we evaluated the functional competence of the mutant fshrs in vitro. methods: we established stable hek cell lines expressing wild-type (wt) and splice-variant (del) fshr under the control of the inducible tet on/off system. the cells were transfected with a camp-responsive luciferase reporter plasmid and stimulated with fsh. results: the subcellular distribution of all splicing variants was the same as the controls and the protein localized mainly on the cell surface. all four splicing variants showed markedly decreased camp activation compared to controls when stimulated with fsh. however, all variants were able to form functional heterodimers with the wt receptor when co-expressed. interestingly, the heterodimer containing the form of fshr lacking exon , found in patients with decreased response to fsh, resulted in lower intracellular camp compared with the wild-type homodimer. conclusion: our findings suggest that fshr variants can contribute to abnormal response to stimulation in certain women undergoing ivf treatment. the variants require the presence of wild-type receptor in order to initiate signaling in response to fsh. further analysis of this signaling cascade in granulosa cells is underway to estimate the final production of estrogen from these heterodimeric receptors. objective: to compare the proteome of human endometrium in the prereceptive versus the receptive phases of the menstrual cycle. m m: endometrial biopsies were collected and days after urinary lh surge in the same menstrual cycle from three fertile women (n= ). proteins were extracted using sample grinding kit (ge healthcare) and interfering substances removed using -d clean-up kit (ge healthcare). labelling was performed with cydye dige fluors (ge healthcare) and proteins were separated using difference gel electrophoresis (dige). for the isoelectric focusing, cm ipg-strips in the nonlinear range of ph - were used. the second dimension was carried out using sds-page. differentially expressed proteins were detected by image analysis using decyder v . and the statistical module eda (ge healthcare). the spots of interest were subject to protein identification based on in-gel digestion, maldi-tof/tof mass spectrometry and database searching. western blot analysis were performed in the same biopsies in order to validate some candidate proteins. results: table displays the differentially protein abundance between days lh+ and lh+ (> -fold change). of these proteins, were increased at lh+ in comparison with lh+ , whereas only proteins were decreased. stathmin was found more than -fold decrease in lh+ compared to lh+ in the three patients studied in the validation studies performed by western blot. conclusions: this study shows that the human endometrium has a differential protein repertoire during the window of implantation compared to the prereceptive phase. the role of these proteins in the molecular events directed to embryo implantation is under research. differential protein abundance in human endometrium ( the extracellular signal-regulated kinases and (erk / ) are a mitogen-activated protein kinase (mapk) subfamily that act as key links in eukaryotic intracellular signaling transduction. activated by phosphorylation in response to specific stimuli, erk / is known to play a role in the regulation of cellular proliferation and survival. the human myometrium is a tissue known to undergo cycle-dependent proliferative and apoptotic changes in response to sex steroids. we hypothesized that erk / activity is involved in mediating menstrual cycle-dependent changes in the myometrium. materials and methods: immunostaining for phospho-erk and total-erk was performed on myometrial tissues obtained from normal women (n= ) at different phases of the menstrual cycle. staining intensities were evaluated by hscore. myometrial smooth muscle cells were isolated and cultured from normal women and treated with vehicle, estrogen ( - m), and progesterone ( - m) for and minutes, and then subjected to western blot analysis for p-and t-erk. statistical analysis was performed using one-way anova. results: tissue staining revealed that p-erk was mostly nuclear in all tissues, while t-erk was cytoplasmic and nuclear. p-erk staining was significantly stronger in the secretory phase and strongest at the early secretory phase, compared to other phases (p< . ). t-erk staining intensity was moderatehigh without variation across the menstrual cycle. in cultured myometrial cells, progesterone significantly increased p-erk levels within and minutes (p< . ) when compared to control. our results suggest that erk activity in the human myometrium is regulated in a menstrual cycle-dependent manner. the increased phosphorylation in the secretory phase suggests the involvement of progesterone in erk activation, as supported by our in vitro results. this increased erk activity may play a role in regulating myometrial proliferation. measures of insulin resistance (ir) including: fasting serum insulin, gir and homa. measures of plasma levels of endothelin- (et- ), soluble intercellular adhesion molecule- (sicam- ), soluble vascular cell adhesion molecule- (svcam- ) and high sensitivity c-reactive proteins (hscrp). results: women with pcos exhibited significantly higher levels of et- (p < . ), sicamp- (p < . ), svcam- (p < . ) and hscrp (p < . ) as compared with age-matched controls, respectively. positive correlations were evident between et- and fai (r = . ; p < . ) but et- negatively correlated with shbg (r = - . ; p < . ). svcam- positively correlated with total t (r = . ; . ), hscrp correlated with: bmi (r = . ; p < . ), and homa (r = - . ; p < . ), respectively. conclusions: women with pcos exhibited abnormal levels of endothelial and inflammatory markers, which appear to be inter-related to hyperandrogenaemia. ( ), and a higher expression of fatty acid amide hydrolase (faah) in peripheral lymphocytes post-ovulation ( ), suggest an involvement of the endocannabinoid system in menstrual cycle control. our aims were to investigate changes in plasma aea levels and in endometrial faah expression throughout the menstrual cycle. methods: plasma aea levels were measured using a hplc ms/ms method from women, median age yrs (range - ) and bmi kg/m (range - ), with regular menstrual cycles, with no medical problem and not on any medication for the preceding months. the menstrual cycle was divided to early follicular d - (n= ), late follicular d - (n= ), ovulatory d - (n= ), early luteal d - (n- ) and late luteal phases d - (n= ). uterine biopsies were taken from hysterectomy specimens taken for benign conditions and subjected to immunohistochemistry for faah with polyclonal antibodies. results: aea levels were significantly higher around ovulation in comparison to the pre-ovulatory or post-ovulatory phases as shown in the figure. faah expression in the endometrial stroma was unchanged throughout the follicular phase but increased during the mid to late luteal phase reaching a maximum in the late luteal phase. a high aea level in the early follicular phase and during ovulation suggests a role for aea in ovulation. the lower levels of aea in the luteal phase, essential for successful implantation, may be regulated by increased faah expression at the uterine level. the modulation of plasma aea levels during the menstrual cycle strongly suggests that it is regulated by gonadal steroid hormones. ( , , , , , , and , as well as at pregnancy. binding activities of igfbp and their protein levels (igfbp , , , , , ) were assessed by western ligand blot (wlb) and western blot (wb), respectively. the glyscosylation and phosphorylation status of igfbps were examined by deglycosylation treatment. our results showed that both glycosylated and unglycosylated igfbp elevate in fetal and newborn rat and graduately decline to a lower level at day and kept lower constant level since then. interestingly, biding activity of glycosylated igfbp was not detected by wlb assay. the igfbp- cleaved products were observed after rat day age day , which associated with a decrease in full length of igfbp- , suggesting endoproteolytic processing may involved in decreasing igfbp content. igfbp- , with heterogeneous glycosylation, were appeared after age day and disappeared during pregnancy, recurrence again postpartum. glycosylation of igfbp has no effect on its binding activity. unglycosylated and glycosylated igfbp were constant in life time. physiologically constant igfbp were detected by wb in protein, but not by wlb in binging activity. conclusion: highly elevated circulation igfbp suggest physiological role in new born and early postnatal development. its binding activity are well regulated by its posttranslation modification, such as glycosylation of igfbp in inactivating binding activity and possible involvement of active endoproteolytic processing in maintaining binding active igfbp- at low background: cigarette smoking affects hormone biosynthesis, storage, release, binding, transport and clearance, resulting in changes in circulating hormone levels. we used metabolomics to analyze the effects of cigarette smoking and second hand smoke (shs) on changes in hormone levels in women of childbearing age. methods: this is a three arm study; women aged - years who are active smokers, exposed to shs (passive smokers), or non-exposed were recruited from the washington d.c. area. all women completed a detailed staffadministered questionnaire probing their medical history, occupational, lifestyle factors and diet. blood and urine samples were collected at the follicular phase. hormone profiles were determined using metabolomics for serum thyroxine and triiodothyronine and steroid hormones, as well as for cotinine using isotopedilution tandem mass spectrometry (lc/ms/ms-api ). in addition, all samples were analyzed for serum lh, fsh, tsh and creatinine. results: the relationships of cigarette smoking, age, relative weight, and dietary intake to serum thyroxine, triiodothyronine, estradiol (e ), estrone (e ), progesterone (p), -hydroxyprogesterone ( ohp),dehydroepiandrosterone (dhea), dehydroepiandrosterone sulfate (dheas), androstenedione, cortisol, -deoxycortisol, corticosterone, testosterone, aldosterone and vitamin d were analyzed using lc/ms/ms. mean tsh in non-exposed, shs-exposed and smokers: . , . , and . miu/ml respectively. similarly, mean t . , . ( % increase) (p= . ), . μg/dl (- %) in active smokers; (active vs. exposed p= . ). shs increased dhea levels ( % higher, p = . ), dheas ( % higher, p = . ), cortisol ( % lower, p = . ), aldosterone ( % lower, p = . ) and androstenedione ( % higher, p = . ). these data suggest that active smoking and shs can have a profound effect on serum t , adrenal steroid and sex hormone concentrations in women of childbearing age. objective: to determine the prevalence and predictors of the metabolic syndrome (mbs) among in saudi women with polycystic ovary syndrome (pcos) in comparison to women without pcos and to assess the role of androgens and insulin resistance (ir) in mbs development. design: a prospective case control study. setting: tertiary referral university hospital. subjects: six hundreds saudi women living in the jeddah area were classified as follows: with pcos and age-matched women without pcos. interventions: blood samples were collected from all women with or without pcos between : - : , after an overnight fast. main outcome measures: measures of abdominal obesity, blood pressure and that of serum levels of lh, fsh, tsh, ft , -ohp, -a, dheas, total t, free t, shbg, insulin, hdl-c, triglycerides and plasma levels of glucose. measures ir including: fasting serum insulin, gir and homa. results: age-adjusted prevalence of mbs was higher in women with pcos ( . %, % ci: . - . %) as compared with women without pcos ( . %, % ci: . - . %) (p< . ). in the same age group, the risk of mbs in women with pcos was greater than that for women without pcos (p< . ). markers of ir were significantly abnormal in women with both pcos and mbs in comparison to those without mbs (p< . ). the most common abnormal components of mbs in women with both pcos and mbs (after adjustment for age) were: decreased hdl-c ( . ± . %); increased triglycerides ( . ± . %); and increased bmi ( . ± . %), respectively. the prevalence of mbs from lowest to highest tertile of free t level was . , . and . %, respectively; in women with both pcos and mbs. in women with pcos, % exhibited all components of mbs; . % had components, and . % had components. conclusions: women with pcos exhibited significantly higher prevalence of mbs ( . -fold) as compared with age-matched control without pcos. ir is a possible common pathogenetic factor for both mbs and the pcos. it is suggested that more intensive screening and/or therapy monitoring of mbs among women with pcos should be part of the therapeutic modalities of the condition. case of sisters with complete androgen insensitivity syndrome and discordant mullerian remnants. jennifer l nichols, jennifer j gell, eric j bieber. reproductive endocrinology and infertility, geisinger medical center, danville, pa, usa. complete androgen insensitivity is an x-linked recessive disorder resulting in the abnormal expression of the androgen receptor. affected individuals are most commonly phenotypically female but genotypically male. the prevalence of this disorder is in , live male births. we present a case of complete androgen insensitivity in members of the same family with differing residual mullerian tissue. sister a presented at age for evaluation of primary amenorrhea. a karyotype revealed ,xy. an mri of the pelvis showed a hypoplastic uterus but no ovaries. this patient underwent laparoscopic bilateral gonadectomy and hemihysterectomy. on examination under anesthesia, she was noted to have a normal vagina with no cervix noted. at laparoscopic evaluation, she was noted to have bilateral elongated gonads and what appeared to be a remnant of uterine tissue. pathology revealed portions of immature testicles and fragments of smooth muscle in what grossly appeared to be the uterine remnant. the patient's total testosterone following surgery was noted to be elevated at . ng/ml. other laboratory evaluation showed fsh . miu/ml, lh . miu/ml, free testosterone . pg/ml, and estradiol . pg/ml. approximately two years later sister b presented at age for evaluation of primary amenorrhea. no uterus or ovaries were visualized on pelvic ultrasound. again a karyotype revealed ,xy. laboratory evaluation demonstrated fsh . miu/ml, lh . miu/ml, estradiol . pg/ml, total testosterone . ng/ml, and free testosterone . pg/ml. she underwent a laparoscopic bilateral gonadectomy. no uterus, cervix or pelvic masses were identified on exam under anesthesia. at laparoscopy, both gonads were visualized and removed without difficulty but no uterus was visualized. pathology reported two testicular and epididymal-like structures. this case demonstrates the presentation and laparoscopic results of complete androgen insensitivity syndrome discovered in two siblings. although both girls are genotypically male, they differ in the presence of vestigial mullerian tissue. the case shows with complete androgen insensitivity, as an x-linked defect, one should consider apparent sisters of affected individuals, as well as offspring of unaffected individuals with a family member diagnosed. background: anandamide (n-arachidonoylethanolamine, aea) is an endocannabinoid that binds to and activates the cannabinoid receptors, cb and cb and may have important roles in the regulation of human reproduction. the traditional lipid extraction methods used for aea are cumbersome, slow and of low efficiency. the aim of this study was to determine whether the use of a solid phase (spe) method of aea extraction from human plasma would offer any advantages over the traditional liquid phase (lpe) method. methods: pooled human plasma was obtained from the local blood transfusion unit and aliquots stored at - °c prior to extraction. an internal standard of . pmol of deuterated aea (aea-d ) was added to each plasma sample and aea extracted from aliquots on each occasion over three days using the lpe and spe methods. spe was performed with waters oasis hlb cc/ mg cartridges on a waters vacuum manifold. cartridges were activated with methanol and water, the samples applied and washed with % methanol at ml/min. aea was eluted with ml acetonitrile and the eluents dried under a stream of nitrogen before reconstitution in ml acetonitrile. aea levels were measured using uplc-ms/ms against authentic standards. results: these are shown in the table. conclusion: the spe method was -fold more efficient at extracting aea compared to the traditional lpe method. the intra-day and inter-day assay variability were similar for both techniques, although the spe method was quicker, cheaper and required less plasma to generate data similar to that from the traditional lpe method, suggesting that the spe method may be more efficient than the lpe method, and thus making it more suitable for routine analysis of multiple plasma aea samples. background: the precise role of the endocannabinoid, anandamide (narachidonoylethanolamine, aea) in reproduction has been hampered by difficulties in its accurate measurement. aea levels have previously been measured by tlc, gc-ms and hplc-ms but these are laborious. our aim was to improve the analysis of aea using uplc and tandem ms/ms with a standard isotope-dilution method , . methods: authentic non-labelled and deuterium-labelled aea (aea-d ) diluted in acetonitrile were maintained at °c before analysis and separation by uplc on a c ( . x mm) column maintained at °c using a gradient of % a, . min: % a, . min: % a, . min: % a, . min: % a with a flow rate of . ml/min. the mobile phases were a ( mm ammonium acetate, . % formic acid) and b (acetonitrile, . % formic acid). the analytes were quantified using multiple-reaction monitoring in positive ion mode with a quattro premier mass spectrometer. entry, collision and exit energies were - , and - ev, respectively. calibration curves were performed in triplicate with . pmol aea-d as the internal standard. transitions employed were . > . and . > . for aea and aea-d , respectively. results: calibration curves ( . to fmol aea on column; n= ) were linear, producing a mean (±sd) gradient of y = . ± . x, crossing the y-axis very close to the origin ( . ± . units). variability was limited, with an r = . . measurements were precise, fmol aea produced a cv of only . %, and the retention time was consistent at . ± . min (n= ). the limit of quantification (signal/noise> ) was . fmol on column and the limit of detection (lod) was . fmol on column (signal/noise= ). accuracy for . fmol, . fmol and fmol aea was . ± . %, . ± . % and . ± . %, respectively. conclusions: the method described is an improvement over other lc-ms/ms methods , with a lower lod [ . fmol v. fmol or fmol ] and shorter run time [ min v. min or . min ]. thus, an improvement in terms of speed, increased sensitivity and better reproducibility will allow for a more accurate assessment of aea concentrations in a number of biological samples. objectives: the gata family of transcription factors consists of six zinc-finger proteins with a critical role in tissue-specific and developmentally-regulated gene expression. gata factors exert their effects alone and through interactions with cofactors, such as friend of gata (fog), as well as with nuclear hormone receptors, including steroidogenic factor- (sf- ), a well-described activator of gonadotropin gene expression. the objective of these studies was to define the role of gata family members in the gonadotrope. methods: ) total rna was extracted from the gonadotrope cell line, l t , and analyzed by standard rt-pcr analysis. ) the cv- fibroblast cell line was transiently transfected by the calcium phosphate precipitation method with a rat - /+ lh promoterreporter vector as well as cmv-driven expression vectors for gata- , gata- , dngata- , fog- and/or sf- . results: gonadotrope l t cells were found to express transcripts encoding gata- , gata- , and gata- as well as fog- and fog- . gata- and gata- stimulated lh gene promoter activity by approximately -fold (p< . ) and synergistically increased the sf- response ( -fold versus -fold for sf- alone; p< . ). the gata-mediated increase in lh gene expression was nearly eliminated with co-transfection of fog- . similarly, co-transfection with a gata- dominant negative construct blunted wild-type gata- effects in a dose-dependent fashion. conclusions: these data demonstrate expression of both gata and the functionally related fog proteins in a well-characterized gonadotrope cell line. furthermore, they demonstrate a functional role for these factors in regulation of gonadotrope function, specifically expression of the lh gene. low-dose dexamethasone (dex) therapy early in pregnancy is used in fetuses with suspected risk of congenital adrenal hyperplasia. several adverse neurological events in prenatally treated children have been reported and the fetal hypothalamic-pituitary-adrenal (hpa) axis may be involved. aim: to investigate the immediate and long-term effects of early maternal dex administration on fetal growth and pituitary-adrenal activity in sheep. method: pregnant ewes carrying singleton fetuses (total n= ) were randomized to control ( ml saline/ewe) or dex treatment ( . mg/kg ewe weight) consisting of four intramuscular injections at -hourly intervals over hours on - days of gestation (dg). at , , , and dg fetal weights were recorded. i -ria, qrt-pcr and in-situ hybridisation were used to measure fetal plasma cortisol and acth levels and to analyse adrenal and pituitary mrna expression. results: dex-exposed fetuses were lighter than control animals at dg*, but not at other times; in general fetal organ weights were similar between treatment groups. fetal plasma acth was unaffected by dex and did not differ between genders. similarly, pomc and pc- mrna in pars distalis were unaltered after dex. however, fetal plasma cortisol was reduced after dex in both male and females at dg*, was similar at and dg, then elevated at dg*. plasma cortisol in female fetuses in control and after dex was significantly higher than in males. the increases in cortisol after dex at dg* were associated with increased fetal adrenal expression of p c and bhsd mrna in females, reduced expression of mc r in males, but no difference in star mrna. conclusion: we conclude that in sheep, early dex programs the developmental trajectory of the fetal hpa with increased activation directly of the adrenal, but not pars distalis function. in females this effect may be attributed to increased fetal adrenal steroidogenic activity. the effect of dex in increasing cortisol in males, albeit at a significantly lower level than in females, appears to be independent of the enzymes that we have measured.*p< . . synaptophysin and gonadotropin-releasing hormone (gnrh) are colocalized in rat hypothalamus. armando arroyo, beom su kim, amanda biehl, blenna cl bett, , john yeh. gynecology-obstetrics, university of buffalo, buffalo, ny, usa; physiology and biophysics, university at buffalo, buffalo, ny, usa. the cellular and molecular mechanisms that control gonadotropin-releasing hormone (gnrh) release are not completely understood. gnrh is stored in synaptic vesicles and released by exocytosis at gnrh nerve terminals. there are currently nine families of synaptic vesicle proteins that are involved in neurotransmitter release by exocytosis. synaptophysin is one of the most common synaptic vesicle proteins present in synaptic vesicles in neurons. the hypothesis of this study is that synaptophysin is expressed in gnrh neurons. we obtained sections from the hypothalamus of female sprague dawley rats. double-label fluorescence immunohistochemistry was performed on free-floating sections. sections were incubated with a mixture of mouse monoclonal antibody against gnrh ( : -chemicon international) and with a rabbit polyclonal antibody against synaptophysin ( : -santa cruz biotechnology) for h. after incubation the sections were washed and incubated with a mixture of alexa conjugated goat antimouse and alexa conjugated goat antirabbit ( : ; molecular probes) for h. slices were visualized with confocal microscopy (zeiss lsm- ). fifteen out of a total of fifteen gnrh cell bodies in the medial preoptic area and most gnrh neuron terminals in the median eminence showed intense immunostaining for synaptophysin. this is the first study to demonstrate that synaptophysin is expressed in rat gnrh neuron terminals. this suggests that synaptophysin is present in gnrh neuron vesicles. thus, gnrh release by exocytosis may be mediated by synaptic vesicle proteins. objective: our aim was to identify the effects of early gestation gc exposure on fetal and postnatal hpa axis development and function in postnatal life. method: pregnant ewes carrying singleton fetuses were randomized to control ( ml saline/ewe) or dex groups ( . mg/kg), consisting of four at h intervals on days - of pregnancy. at months postnatal age, catheters were implanted; a bolus injection of crh ( . mg/body weight) and avp ( . mg/body weight) were administered and arterial blood samples were taken at - , - , , , , , , , and min. levels of hepatic cbg mrna were determined by qpcr, expressed relative to s rrna. plasma cortisol and cbg levels were measured by radioimmunoassay. results: both total and free cortisol levels in the dex females (n= ) were lower than in dex males (n= ) from to min (p . ) and lower than in control females (n= ) at minutes (p . ), also in the dex-m group were higher than in control males (n= ) at min (p . ). plasma cbg levels and cbg mrna expression were not altered by dexamethasone exposure or sex. conclusions: these findings suggest that prenatal glucocorticoid exposure alters the development of the hpa axis differentially according to the sex of the exposed fetus. to determine maternal injections of synthetic glucocorticoids in early gestation can alter expression of hippocampal corticosteroid receptors at months postnatal age. method: pregnant ewes carrying singleton fetuses were randomized to control ( ml saline/ewe) or dexamethasone treatment ( . mg/kg) consisting of four injections at h intervals on days - . hippocampal was collected from the offspring at months postnatal age. levels of mrna of gr and mr were determined using qpcr and levels relatived to s rrna. results: dexamethasone-treated male animals (n= ) had significantly higher levels of mr gene expression than both control males (n= ; p= . ) and females (n= ; p= . ). gr gene expression levels were higher in treated vs. control males (p= . ), but in females levels in treated and control animals were similar. total body, brain and hippocampus weights were similar. conclusions: maternal dexamethasone administration in early pregnancy resulted in gender-dependent changes in hippocampal gene expression when measured in the offspring seven months after birth. objective: diminished ovarian reserve (dor) affects many younger women. we analyzed superovulation with intrauterine insemination (so) cycles in vitro fertilization (ivf) cycles of women with dor to determine if women < with dor differ from their older counterparts. method: irb approved retrospective review of so ivf cycles from / - / with follicle stimulating hormone (fsh) levels based on age < or . results: so cycles were performed in women. no differences were noted in clinical pregnancy. women < were more likely to have inseminates with a lower mean tms/ins, median tms/ins was . among pregnancy cycles compared to for unsuccessful cycles. for ivf, mean total gonadotropin dosage was significantly lower in women < , mean number of follicles mm peak e were significantly higher in women < . so ivf measures are in tables , respectively. conclusions: similar clinical pregnancy outcomes were seen despite age. in ivf, women < required less gonadotropins and generated more follicles but with no significant difference in number of mature oocytes or clinical pregnancies. of note, pregnancy rates for so in women < with dor are substantially lower than expected in our clinical practice. as ivf yielded a substantially higher chance of pregnancy, consideration should be made to expedite progression to ivf. to assess the effects of intraovarian injection of ad-fshr on the reproductive system of fshr (-/-) mice. methods: about l containing x pfu of ad-hfshr were injected directly into each ovary of treated group and same amount of ad-lacz were injected into the ovaries of control animals. vaginal smears were collected and body weight was measured daily. four weeks after the injection animals were sacrificed and all organs were weighted and evaluated by h e. fsh, e and p measured before and after treatment. results: ad-hfshrtreated mice showed obvious estrogenic changes in vaginal smear while in control animals vaginal smear remained at diestrus stage. significant increase in total body weight and estrogen dependent organs weight (uterus, ovary, vagina) was observed in treated animals compare to control group (p< . ). no significant weight changes were observed in other organs. h e evaluation of the ovaries showed significant increases in both the total number of follicles and the collective diameter of the follicles in treated animals compare to controls. on average follicles/ovary were observed in ad-hfshr-treated group of which follicles were at the antral stage while only follicles observed in ad-lacz control group, with zero follicles at antral stage. a . to folds increase in e and about % decrease of fsh observed in treated animals compared to control mice. there was no significant change in serum progesterone level between treated and control groups. conclusion: intraovarian injection of an adenovirus expressing human fshr gene is able to restore folliculogenesis and resume estrogen hormone production in female forko mice. objective: the sentinel issue surrounding multiple gestations following ivf is the inability to precisely estimate the reproductive potential of individual embryos with the currently used embryo grading systems based on embryo cleavage rate and morphology. recently, metabolomic profiling of spent culture media using raman and near-infrared spectroscopy have been reported to predict reproductive potential of embryos. in this study we applied proton nuclear magnetic resonance (¹h nmr) spectroscopy to analyze metabolomic profile of embryo culture media and to identify components of the media that correlate with reproductive potential. methods: eighteen spent media samples from embryos that failed to implant, and samples from embryos that resulted in pregnancy and delivery were individually collected after embryo transfer on day , and evaluated using ¹h nmr. the spectra obtained were analyzed using a selective genetic algorithm (ga) to determine regions predictive of pregnancy outcome as determined by logistic regression. to avoid random correlations, a leave-one out crossvalidation was used. sensitivity and specificity of predicting pregnancy (described as implantation and delivery) were calculated. results: using the ga, two areas in the ¹h nmr spectral region were identified as most discriminatory between the two groups. viability indices calculated by ¹h nmr using these regions were significantly higher in culture media of embryos with proven reproductive potential ( . ± . ) compared to those that failed to implant ( . ± . ) (p< . ). compiled outcomes from the leave-one-out cross-validation of the logistic regression using the ¹h nmr measurements resulted in a sensitivity of % and a specificity of . %. quantification by integration showed significantly decreased glutamate levels (p= . ) and a trend toward an increase in pyruvate levels (p= . ) in culture media of embryos that did not cause pregnancy. conclusion: metabolomic profile of spent embryo culture media using ¹h nmr correlates with the reproductive potential of embryos. the lower glutamate levels detected in culture media of embryos that failed to implant could potentially be due to the toxicity associated with increased embryonic glutamate uptake. additional studies using complementary approaches are needed to further delineate molecular components associated with reproductive potential. objective: beta-carotene and other carotenoids are known antioxidants previously identified in human follicular fluid (ff). in addition to their inherent antioxidant properties, carotenoids have been identified as precursors of the antioxidant retinol in bovine ff. high retinol levels in bovine ff are associated with non-atretic follicles. this would suggest a possible role for retinol and its carotenoid precursors in follicular heath and the general oxidative state of the follicle. we sought to measure carotenoids and retinol in the ff of women undergoing ivf and correlate these levels with normal fertilization as a marker of follicular/oocyte health. design: prospective cohort study materials and methods: ff from a single - mm follicle was obtained from women (age - ) undergoing ivf and intracytoplasmic sperm injection (icsi). serum was also obtained at the time of oocyte retrieval. retinol, vitamin e ( , , and tocopherol) and carotenoids ( -carotene,cryptoxanthin, lycopene and lutein/zeaxanthin) were measured using hplc. we correlated ff carotenoid and retinol levels with oocyte fertilization status following icsi. results: as previously reported, retinol, vitamin e and carotenoids were all identified in the ff. each fat-soluble vitamin level was significantly lower in ff compared to serum (p< . for all analytes) and the levels were strongly correlated (r > . , p< . for all analytes). mean levels of ff -carotene were significantly higher in those follicles that resulted in a fertilized oocyte ( . +/- . g/ml vs. . +/- . g/ml, p= . ). other carotenoids, retinol, and vitamin e levels did not correlate with fertilization outcomes. conclusions: our finding of a strong association between ff -carotene concentration and subsequent normal fertilization of the oocytes suggests an important antioxidant role for -carotene in the health of the human ovarian follicle/oocyte. the lack of correlation with other carotenoids, retinol and vitamin e suggests that the antioxidant properties of -carotene act by preventing singlet oxygen and scavenging the peroxyl radical and may directly influence oocyte competence. mature oocytes obtained during egg retrieval have been shown to undergo spindle depolymerization as a result of cooling. for this reason, ivf programs strive to maintain constant temperature during follicle aspiration. we sought to elucidate if there was a difference in temperature of fluid aspirated into a hand held (hh) or heating block (hb) encased collection tube. the experiment was performed in an ambient temperature of °c. thermistors, pinhead sized sensors with an accuracy of %, were used to monitor temperature differences between control fluid (cf) (sterile water ºc) and aspirated fluid. data was recorded using an -channel data acquisition system from dataq instruments. one thermistor was placed into the cf, the other was placed into an empty collection tube set in the heating block or held in a gloved hand. a cm, gauge, single lumen needle connected to cm of tubing was used to aspirate into the empty collection tube. temperature was recorded x/second, each experiment was repeated times. baseline empty collection tube temperature was significantly cooler in the hh vs the hb group ( . ± . °c vs . ±. °c, p=. ). two seconds after aspiration, the lowest aspirate temperature was observed, (hh . ±. °c, hb . ±. °c, p>. ) no difference between groups. in both groups, temperature quickly increased as aspiration progressed (hh . ±. °c, hb . ±. °c, p>. ). the hb group took an average of . min to return to baseline ( °c), the hh group never returned to baseline. substantial cooling of aspirated fluid occurs during oocyte retrieval, with a mean temperature decrease of . ±. °c corresponding to . °c. considering this dramatic decrease, the difference between temperatures in the hh vs. the hb group is negligible. current aspiration systems poorly regulate temperature, thus the choice of aspirating into a test tube warmed by hand or by heating block is inconsequential. clinical management of twin gestations with recurrent preterm labor symptoms. lesley de la torre, luis roca, niki b istwan, debbie j rhea, gary j stanziano, victor hugo gonzalez-quintero. obstetrics and gynecology, university of miami medical center, miami, fl, usa; clinical research, matria healthcare, marietta, ga, usa. objective to examine pregnancy outcomes in women with twin pregnancies receiving nifedipine tocolysis (nt) who experienced recurrent preterm labor symptoms (rptlsx). study design twin pregnancies enrolled for outpatient preterm labor (ptl) surveillance services prescribed nt following an initial episode of ptl were identified from a database (n= ). eligible for inclusion were patients later hospitalized with acute rptlsx (n= ). included were those < weeks' gestational age (ga), with intact membranes, remaining undelivered for > hours after rptlsx . pregnancy outcomes of women resuming nt (rnt group, n= ) following hospitalization were compared to those having an alteration in treatment (alttx group, n= ) to continuous subcutaneous terbutaline. per normality assumptions, either independent student´s t or mann-whitney u test statistics were used for continuous variables; pearson´s chi-square for categoric. all p-values presented as two-sided, significant at < . . results overall, ( . %) of twin pregnancies prescribed nt experienced rptlsx; ( . %) were not eligible for continued tocolysis. pregnancy outcomes are presented in table. conclusion rptlsx are common. in twin pregnancies receiving nt, alteration of tocolytic treatment following rptlsx had a positive impact on pregnancy prolongation and neonatal outcomes. routine cervical dilatation during elective cesarean section -is it really necessary? arie koifman, avi harlev, eyal sheiner, fernanda press, arnon wiznitzer. obstetrics and gynecology, soroka university medical center, ben-gurion university of the negev, beer-sheva, israel. objective: the purpose of this study was to examine the necessity of routine cervical dilatation during elective cesarean section. material and methods a retrospective cohort study was performed, including all cases of elective cesarean sections performed at a tertiary medical center during . stratified analysis, using the mantel-haenszel technique, was done to control for confounders. results out of a total of elective cesarean deliveries (cd), underwent routine cervical dilatation. the overall rate of febrile morbidity was . %. no significant differences in postpartum febrile morbidity were noted between the groups ( . and . %; p= . ). in addition, hospitalization duration did not differ between the groups ( . ± . and . ± . days p= . ). about % of all elective operations were repeated cd. there was no difference in febrile morbidity between the groups even in that subgroup of the elective cd. likewise, there was no difference in anemia rate between the two groups (hemoglobin . ± . mg and . ± . mg p= . ). controlling for a previous vaginal delivery, using the mantel-haenszel technique, no significant association was noted between cervical dilatation and fever (weighted or= . ; % ci . - . ; p= . ). nevertheless, in a subgroup of patients following a previous vaginal delivery, cervical dilatation was significantly associated with post-operative fever (or= . the majority of women with an unfavorable cervix undergoing iol at term deliver vaginally, even with a prolonged first stage of labor. this is important information to discuss with women prior to iol when establishing labor expectations. providers should consider the ongoing success of labor induction when contemplating a diagnosis of "failed induction". objective: this study was performed to investigate and compare changes of the lipid peroxide levels and the protein carbonyls formation in the maternal venous plasma of preterm premature rupture of membrane (pprom) during antibiotics administration. materials and methods: thirty-six pregnant women with pprom between and weeks of gestation were chosen for this study. eighteen patients (group ) were treated with amoxicillin and erythromycin for day period while the other patients (group ) were treated with rd generation cephalosporin and erythromycin for the same period. samples of maternal blood were obtained from the two groups at before the antibiotics administration, day , and day after the antibiotics administration. lipid peroxide levels were measured by thiobarbituric acid reaction and protein carbonyl contents were determined by the , -dinitrophenylhydrazine method. results: . the lipid peroxide levels and protein carbonyls formation in the maternal venous plasma of pprom was significantly higher than that of normal pregnancy ( . ± . vs . ± . nmol/mg protein, p< . ), ( . ± . vs . ± . nmol/mg protein, p< . ). . there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the maternal venous plasma with pprom mixed and incubated by amoxicillin, cefodizime, and erythromycin (in vitro). . there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the venous plasma of group among before the antibiotics administration, day , and day after the antibiotics administration. . the protein carbonyls formation in the venous plasma of group was significantly decreased at day and day after the antibiotics administration than that of before the antibiotics administration ( . ± . , . ± . , . ± . nmol/mg protein, p< . ). conclusion: in the maternal venous plasma of pprom, the lipid peroxide levels and protein carbonyls formation were increased. the results suggest that reactive oxygen species formation by inflammatory reaction is suppressed by combined treatment of rd generation cephalosporin and erythromycin. objective: the aim of the present prospective cohort study was to evaluate the correlation between blood flow pulsatility index in fetal middle cerebral artery (mca) and the onset of spontaneous labor. study design: doppler evaluation of fetal mca were performed between and weeks gestation in consecutive pregnant women with a singleton pregnancy and known gestational age. the study population was divided according to the mca pi (< . or >/= . mom) and, using survival analysis and cox regression, the two subgroups obtained were compared. in these analyses, the event was delivery (following spontaneous labor) and the time variable was time elapsed since doppler exam. results: the median time elapsed between doppler evaluation and spontaneous labor was significantly shorter in the women with mca pi lower than . mom ( . days, interquartile range - ) in comparison with the women with mca pi higher or equal than . mom ( . days, interquartile - . days (p< . , mann-whitney test). after correction for birth weight and umbilical artery pi, survival analysis and cox regression confirmed that mca pi was independently associated with the number of days elapsed from doppler to spontaneous labor and delivery (p< . , exp(b) . , ci % . - . ). conclusions: the present data show that, at term of pregnancy, fetal cerebral resistance reduction anticipates the onset of spontaneous labor. novel calcium sensitizers and human uterine contractility. mark p hehir, audrey t moynihan, terry j smith, john j morrison. department of obstetrics and gynaecology, national university of ireland, galway, ireland. objective: the factors regulating contractility of uterine smooth muscle are central to the occurrence of preterm labour and delivery. calcium sensitizers are a novel class of drugs with unique molecular actions. levosimendan, the best characterized of these compounds, is used in the treatment of acute and chronic heart failure and is a compound which exerts a number of effects on smooth muscle. it can exert an inotropic effect via sensitization of myofilaments to calcium and also exerts a relaxant effect by opening atp-dependent potassium channels. for these reasons we hypothesized that levosimendan may have an effect on myometrial contractility and investigated its action on both spontaneous and agonist induced contractions. method: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of levosimendan in the concentration range of nmol/l to mmol/l. two sets of control experiments were performed simultaneously as follows: . strips exposed to either physiological salt solution (pss) only, for spontaneous contractions, or . nmol/l oxytocin; . strips exposed to pss/oxytocin and vehicle for levosimendan. results: levosimendan exerted an inhibitory effect on spontaneous and agonist induced contractions, compared to control strips. the mean maximal inhibition values were as follows: . ± . % for spontaneous contractions (n= ; p< . ) and . ± . % for oxytocin-induced contractions (n= ; p< . ). no significant difference was found between control and control for both spontaneous and oxytocin induced contractions. conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on uterine contractility in vitro. this action was seen in both spontaneous and agonist induced contractions. the results from this study raise the possibility of calcium sensitizers holding potential tocolytic properties in vivo and further studies are required to investigate the potential benefits of this novel class of drugs. to determine to what degree extensive patient movement affects uterine emg signals and toco signals. study design: pregnant term labor patients were recorded using both uterine emg and toco simultaneously. baseline recordings were obtained when patients were still, and these were used as control records. test recordings for both devices were obtained from all patients by asking the patients to perform movements. area under the rectified-voltage amplitude curve of the uterine emg signals and area under the curve for the toco signals were found. mean %-increase was calculated for the uterine emg and toco devices (each device %-increase values were averaged over all patients). mann-whitney rank-sum test was used to look for any statistical differences in %-increase in area for uterine emg vs. toco methods (p < . considered significant). results: there was a large increase in activity (artifact) on both devices' signals during patient movement (figure ) . both devices' traces eventually returned to baseline after the patient movement stopped. toco movement artifact was significantly higher than emg movement artifact ( table ) . conclusions: both uterine emg and toco signals experience artifact when patients move the uterine emg electrodes and toco pressure transducer, respectively. toco seems to be more adversely affected by such movements. uterine emg may be a preferred method for monitoring contractions of laboring patients in the clinic. supported by grant nih r -hd . results: % of obstetricians completed the questionnaire. none would counsel patients against labour unless there were contraindications. the majority would recommend labour for all indications for previous caesarean section investigated although in all instances, personal preferences were lower (p< . ); after a failed instrumental delivery, % obstetricians would recommend labour but only % would choose that option for themselves (p< . ). overall, female obstetricians would contemplate and recommend labour more readily than male obstetricians. labour augmentation and induction were recommended to patients more frequently ( % and % respectively) than chosen for personal care ( % and %). reluctance for labour augmentation and induction was greatest among younger consultants. conclusion: consultants have responded to consumerism and aim to meet the requests of their patients. they more readily recommend labour than they would choose for personal care, and a majority would recommend labour induction when necessary. informed patient choice is paramount rather than attaining a target figure for women attempting to labour, and the views of those requesting delivery by caesarean section should be respected. (table) . cd increased in nullipara singleton, cephalic, term pregnancy in spontaneous labor, mostly due to dystocia; nullipara singleton, cephalic term pregnancy with induced labor, mostly due to non-reassuring fetal status and dystocia; singleton, cephalic term pregnancy with previous cd, mostly due to elective cd; singleton cephalic preterm. no changes in neonatal outcome were observed. conclusion: clinical audit was useful to keep under control cd rate in our institution in comparison with italian reality, but it was not sufficient to maintain stable cd rate suggesting the need of other, multifaceted strategies. elective delivery at weeks' gestation is a common obstetric intervention. the purpose of this study is to estimate the maternal mortality rates (mmr) associated with this acog -approved intervention. there are no reliable us data describing mmr by mode of delivery. therefore, we base our estimates on british data indicating a procedure related mmr of . / , , . / , and . / , for vaginal, elective cesarean section (c/s) and emergency-unplanned c/s deliveries, respectively. a decision tree model was constructed assuming that all eligible patients (approx. , , /annually in the u.s.) would be delivered at weeks by either an elective c/s or an induction of labor. we further assumed that % of inductions would result in a vaginal delivery. our estimates show that the annual, delivery-related maternal mortality associated with an elective delivery of all patients at weeks would be and for elective c/s and induction of labor, respectively. because vaginal delivery results in the lowest mmr, we performed a oneway sensitivity analysis to identify the impact of changing the success rate of induction on the estimated mmr. the results indicate that once the success rate exceeds %, this intervention would be associated with a lower mmr as compared to elective c/s. our estimates indicate that although the overall mmr associated with elective delivery at weeks is relatively low (approximately . / , deliveries), it is certainly not negligible. in addition, the mmr is highly dependent on the likelihood of a successful vaginal delivery following induction of labor. when the success rate for induction of labor falls below %, an elective c/s appears to be the safer delivery method. background: the first obstetric visit is an opportunity to provide the pregnant patient information regarding substances that can cause potential harm to the pregnancy. little is known about how obstetric care providers handle these topics. objective: to examine patient-provider communication about substance use during the first prenatal visit. methods: we audiotaped first prenatal visits and qualitatively analyzed those tapes in which patients disclosed substance use. we invited patient participants to return for a semi structured interview during which they reviewed their audiotaped conversations and described their reactions to the providers' communication styles. results: providers ( residents, midwives, nurse practitioners) and patients participated. providers asked about smoking, alcohol and drug use in all ( %) visits. patients reported being smokers, reported alcohol use, and reported drug use. provider responses to smoking disclosures included brief discussions of smoking effects on pregnancy, encouragement to quit/cut down, and referral to smoking cessation programs. provider responses to alcohol or drug disclosures included only general statements regarding effects on pregnancy (e.g., "we find that this is bad for babies.") and referral to ultrasound/genetics for reassurance. few alcohol or drug discussions assessed whether the patient had intentions or concerns regarding continued use during the pregnancy. few discussions addressed strategies for behavioral change. none included assessment for motivations, readiness, or barriers to change. in follow up interviews, patient participants said they expected to be asked about substance use but advised providers to ask non-judgmentally. those who used alcohol/drugs wanted more information on potential effects of these substances on the pregnancy/fetus and appreciated the reassurance from referrals to ultrasound/genetics. discussion: counseling for risky behaviors in the first obstetric visits contained only limited discussion of the effects of the risky behaviors and primarily focused on referral-which may be a proxy for avoiding a difficult and time consuming conversations. background. there are conflicting results regarding the association of maternal psychiatric disorders or psychological distress due to stressful life events with pre-term birth and low birth weight. aims. to investigate the association between maternal psychiatric disorders and/or stressful life events and intrauterine growth abnormality, low birth weight or preterm birth. method. three mutually exclusive and homogeneous groups of pregnant women ( with actual psychiatric disorder, with stressful life events, and healthy comparisons) underwent serial fetal ultrasound examinations and uterine and umbilical artery doppler velocimetry at (+ ), (+ ) and (+ ) weeks of gestational age. subjects were recruited from all consecutive women attending two antenatal clinics. the presence of any maternal medical illness, drug treatments, fetal chromosomal and/or structural malformations, were exclusion criteria. all women recruited underwent a structured interview at - weeks for the psychiatric diagnosis (mini international neuropsychiatric interview -mini) (sheehan et al, ) ; moreover, the -item hamilton rating scale for depression and the hamilton rating scale for anxiety were included in the assessment. the person who obtained obstetrical clinical data was blind to the results of psychological evaluations. results. the three groups were comparable for: age, parity, socioeconomic status, smoking, alcohol consumption, body mass index. gestational age at birth was not different in the three groups. infants of women with psychiatric disorders had significantly lower birthweight ( + g) and higher percentage of birth weight below the th centile for gestational age ( %) than infants of healthy mothers ( + g and %, respectively). there was also a trend towards lower mean birth weight ( + g) and higher incidence of birth weight below the th centile ( %) in the stressful life event group. there was no significant difference among groups in the percentage of abnormal uterine or umbilical doppler results. conclusions. maternal psychiatric disorders are associated with a lower birth-weight, but the effect is unlikely to be due to abnormal utero-placental or feto-placental vascularisation. objective: the purpose of this study was to evaluate antenatal ultrasound as a tool for the detection of intrauterine growth restriction (iugr) and small for gestational age (sga) infants among subjects with elevated human chorionic gonadotropin (hcg) levels on second trimester serum screening. although iugr has been linked to elevated hcg levels, the optimal screening regimen for antenatal sonographic surveillance has not been previously established. methods: a retrospective cohort study was performed at saddleback memorial medical center where serial ultrasounds from weeks-delivery are generally recommended for patients with hcg levels > . mom. all pregnancies were dated by second trimester ultrasound ± last menstrual period. subjects with an hcg > . mom, who had at least one antenatal ultrasound evaluation for iugr, were identified from an electronic ultrasound database used for clinical report generation. ultrasound data were then linked to an obstetrical birth outcomes database for relevant demographic/delivery information using unique identifiers. iugr was defined as a sonographic estimated fetal weight (efw) < %ile for the estimated gestational age (ega). sga was defined as an actual birthweight < %ile for the ega at the time of delivery. results: from - , there were subjects with elevated hcg levels who underwent antenatal ultrasound surveillance for iugr and who had known delivery information. a total of ultrasound examinations were performed. the median number of examinations per subject was with a range from - examinations per subject. the incidence of iugr and of sga were . % (n= / ) and . % (n= / ), respectively. no fetus with iugr demonstrated absent or reverse end diastolic umbilical artery doppler flow. antenatal ultrasound examinations only identified . % (n= / ) of sga infants. however, the sensitivity for the detection of sga was % when an efw cut-off of % was used. conclusions: although the majority of sga infants did not demonstrate growth restriction on antenatal ultrasound, a sonographic efw > %ile appears to be a safe cut-off to rule out fetuses at risk for sga. neonatal dry lung syndrome after pprom: reason to change intrauterine referral practice in the netherlands? ellen s hoogakker, christiaan v hulzebos, gerda g zeeman. obstetrics and gynecology, university medical center groningen, groningen, netherlands; pediatrics, division of neonatology, university medical center groningen, groningen, netherlands. background: neonatal dry lung syndrome (dls) is a distinct clinical entity following pprom, mimicking pulmonary hypoplasia but with dramatic respiratory improvement during the first - h . its pathogenesis implies complete collapse of small airways to a degree that capillary forces impede distension by ordinary ventilatory pressures. in the netherlands, women with pprom remain admitted at a level iii nicu perinatal center until they reach wks. when they are referred back to their community hospital, unless they live in the neighborhood of the tertiary center. we question this referral pattern because we still see severe respiratory problems occur > wks. we sought to determine the prevalence of such morbidity, in particular dls, in women with pprom who deliver > wks. methods: retrospective descriptive study of neonatal outcome data of singleton pregnancies complicated by pprom between - wks, who deliver > wks (latency > h) , during the -yr period of - at a single academic center. data were extracted from medical records and electronic department databases. results: pprom pregnancies were identified. all but received at least full course of steroids. ( %) delivered > wks. newborns born at the community hospital needed emergency transportation to a level iii nicu for respiratory morbidity. neonatal outcome data (means ± sd) are listed in the table. there were no cases of late onset sepsis, nec or perinatal mortality. conclusions: respiratory morbidity still occurs after pprom with delivery > wks. further investigation of pregnancy-related characteristics, such as the presence of anhydramnios and the latency period, with regards to dls is needed. modification of current referral practice depends upon complete data derived from all dutch level iii perinatal care centers (n = ). tertiary care center (n = ) to determine if an association exists between macrosomia (birthweight > g) and perinatal outcomes in women with and without gestational diabetes mellitus (gdm). study design: this is a retrospective cohort study of , singleton pregnancies. the study cohort was stratified by the diagnosis of gdm, with the presence or absence of macrosomia as the dependent variable. perinatal outcomes examined included neonatal hyperbilirubinemia, hypoglycemia, respiratory distress syndrome (rds), shoulder dystocia and erb's palsy. chisquare tests were performed as well as multivariable analyses controlling for confounders, using p< . to indicate statistical significance. results: in women diagnosed with gdm, macrosomia is associated with a higher frequency of hypoglycemia, respiratory distress syndrome, shoulder dystocia and erb's palsy. though the prevalence of these outcomes is relatively decreased in patients without gdm, they are still more prevalent in macrosomic patients. macrosomia is associated with a higher prevalence of adverse perinatal outcomes in women with and without gdm. therefore, it is important to evaluate neonates with birthweights greater than grams for hypoglycemia and unrecognized erb's palsy. conclusion a significant proportion of our obstetrical patients are obese and many do not perceive themselves to be obese. while our finding of an inverse correlation between level of education and bmi may be confounded by socioeconomic status, our results suggest that in order to address the problems of obesity in our population an important first step will be improving the education of our reproductive age women regarding normal weight gain and nutrition in pregnancy. this may have a significant impact on improving pregnancy outcomes of today's obstetrical population. objective: the aim of the study was to evaluate the impact of a "flat" oral gct upon the outcome of pregnancy. the gct was considered flat when the difference between the basal value and the after load value was %. methods: we prospectively analyzed the outcomes of pregnancies who delivered at our department. inclusion criteria were singleton pregnancy; bmi < kg/m , absence of major risk factors for diabetes and of pregestational diabetes. g -hour oral gct was performed at - weeks of gestation. women were subdivided into groups according to the result of the gct: group = negative (load glucose > % and < mg/dl) women ( . %); group =flat (load glucose % than basal and < mg/dl) women ( . %); group =positive gct/negative ogtt, women ( . %). data are mean ± sd. differences were calculated with the student t test for unpaired samples and test. regression analysis were performed by the least squared method. p-values were considered significant at p< . . results: the characteristics and obstetric outcomes in the three groups are presented in the table . in all patients there was a significant linear relationship between the load and basal glucose values (load value= . + . basal value; r= . ; p< . ) and between birthweight and load values (birthweight= . + . load value; r= . ; p< . ). the relationships were significant also in group and separately but not in group . in this preliminary study we found no major outcome differences in women with flat gct compared to women with normal and gct positive/ ogtt negative results. it seems important, however, to futher investigate the meaning of a flat curve in a bigger population and/or by means of metabolic studies with the use of stable isotopes. results characteristic of the study population and obstetric outcomes are presented in table . the probability to be primiparous and to deliver babies < ° centile decreased significantly with bmi whereas the risk of cesarean section, of post partum hemorrhage at vaginal delivery and to deliver babies > ° centile increased significantly. also the risk to develop preeclampsia and gestational diabetes was increased, although not significantly, with bmi. compared to lean and normal, obese were more likely to be hypertensive and diabetic before pregnancy (p< . ) and to start pregnancy without any medical and obstetrical risk but obesity ( . % vs . and . %; p< . ). african women exhibited the highest bmi and the highest rate of obesity (table ). conclusions we confirm that obese women have an increased risk of prepregnancy and pregnancy complications: less than % have an uncomplicated pregnancy. at delivery there is an increased risk of cesarean section and post partum haemorrhage. objective: overweight and obese women often give birth to larger babies, which is associated with traumatic birth injuries and an increased risk to develop obesity, diabetes and hypertension in childhood and later in life. the mechanisms underlying fetal overgrowth in obese pregnant women are largely unknown. the aim of this study was to establish a mouse model of obesity/high fat diet in pregnancy. we hypothesized that a moderately high fat diet prior to and during pregnancy would result in increased maternal adiposity, fetal overgrowth and a metabolic profile similar to that of obese newborn offspring with persistent pulmonary hypertension, despite enhanced pregnant women. method: c bl/ j female mice were fed control (c, % of energy from fat) or isocaloric high fat (hf, % of energy from fat) diets ad libitum for weeks prior to mating and during gestation. at gestational day . maternal blood samples were obtained to measure adiponectin, leptin and cytokine levels and maternal fat pads were isolated and weighed. in a sub set of animals measurements of transplacental transport of neutral amino acids and glucose were performed in vivo under ketamine anesthesia using h-meaib, and c-glucose. results: no significant differences were observed in maternal pre-pregnancy bodyweight, total caloric intake, weight of the dam at e . or litter size between treatment groups. fetal weight was increased in the hf group by % (p< . ). maternal adiponectin levels were significantly (p< . , n= ) decreased (hf ± , c ± g/ml) and leptin levels increased by % in animals fed a high fat diet, but this difference did not reach statistical significance (n= ). adiposity (fat pad weight) was increased by % (p< . , n= in the dams fed high fat diet, however no difference was observed in maternal il- levels and neither group had measurable levels of tnf-. maternal red blood cell lipid profiles were altered in high fat animals with an increase in stearic and linoleic acids but decreased oleic acid levels. preliminary data showed that placental uptake and transfer to the fetus of glucose and meaib were increased by at least % in dams fed high fat diet. conclusion: this murine high fat diet model has several features consistent with human obesity in pregnancy and the maternal metabolic environment is similar to that seen in the human. the increase in placental uptake and transfer of nutrients constitute a key mechanism underlying fetal overgrowth in overweight/obesity in pregnancy. objective: epidemiological studies have demonstrated a positive relationship between maternal overnutrition and the development of the metabolic syndrome in the offspring. we previously reported that lambs of well fed ewes have increased plasma glucose levels in early life, this may be a consequence of altered hepatic glucose production. increased hsd expression is associated with an increase in intracellular cortisol, promotion of hepatic insulin resistance and a consequential increase in gluconeogenic activity. hypothesis: we hypothesised that maternal overnutrition in late gestation in the sheep results in increased hepatic expression of hsd in the lamb before and after birth. methods: ewes were provided with either % (control,c) or % (well fed, wf) of maintenance energy requirements from d gestation until delivery. post-mortem was performed on either ± d gestation (c= ,wf= ) or and postnatal day (c= ,wf= ). plasma glucose and leptin concentrations in the fetuses and lambs were determined. the relative hepatic mrna expression of hsd and reference gene rpp were determined by qrt-pcr. results: relative liver weight was significantly higher in lambs of wf ewes compared to c at d (p< . ). the expression of hsd mrna was significantly higher in the postnatal compared to the fetal lamb (p< . ) independent of maternal nutritional treatment. however, there was no effect of maternal overnutrition on the hepatic expression of hsd mrna before or after birth. there was no effect of prenatal nutrition on fetal or postnatal plasma cortisol concentrations. the expression of hsd in the liver of lambs of wf, but not c ewes, was inversely related to plasma glucose concentrations in the first hrs after birth (r =- . , p= . ). we have therefore demonstrated that exposure to prenatal overnutrition results in an inverse relationship between hsd mrna expression in the liver at d and plasma glucose concentrations on the first day of life. this suggests that exposure to high glucose levels before and immediately after birth results in a reduced expression of hepatic hsd . we would expect a reduction, rather than promotion of intra-hepatic cortisol production, and therefore it is unlikely to explain the hyperglycemia present in lambs of wf ewes in early life. ninety-seven ( %) of mothers were classified as obese (bmi> ) based on their first pregnancy weight. glycemic control at weeks was superior in the non-obese group (table ) . there were no differences in glycemic control during the last week of pregnancy. obesity was significantly associated with increased maternal weight gain during pregnancy. mean birth weights, ponderal indices and rates of macrosomia were significantly higher in infants born to obese women when compared to non-obese (table ) . primary cesarean deliveries rates were comparable. the rate of neonatal hypoglycemia, hyperbilirubinemia, phototherapy and neonatal icu admissions did not differ between obese and non-obese diabetic women (table ) . although not statistically significant, there was a trend towards an increased rate of birth injuries in the obese group. a similar comparison between obese and non-obese women treated with medication (glyburide or insulin) demonstrated a higher mean birth weight ( g+ vs. g+ , p=. ) and higher rate of macrosomia ( vs. %, p= . ). there were no differences in glycemic control, cesarean delivery rates and other neonatal outcomes between the obese and non-obese treated with medication. conclusion: obese women with type ii and gdm give birth to larger infants than their non-obese counterparts and have a higher incidence of fetal macrosomia. there was a trend towards increased rate of birth injuries in the obese group. despite these differences other maternal and neonatal outcomes were similar which may be a reflection of glycemic control. introduction: tlrs are key components of the innate immune system which recognize conserved sequences on the surface of pathogens and trigger effector cell functions. the placenta, and more specifically the trophoblast, may play an important role in the response to infection. previously, we described the expression of tlr- by human trophoblast and their ability to respond to polyinosinic-polycytidylic acid (polyi:c), a synthetic double strand rna which mimics viral rna. in the present study we evaluate the effect of polyi:c in mouse pregnancy and characterize the local and systemic response. material and methods: human first trimester trophoblast cell line, htr , was treated with polyi:c. c b/ wild type and tlr- knock out mice were injected intraperitoneally with polyi:c at . gestation day. cytokine and chemokine level were determined in supernatant and lysates using the bio-rad multiplex assay and analysis was done using the bio-plex is. results: polyi:c induced cytokine (il , il and il ) and chemokine (il , rantes, gro , mcp and mip ) secretion and production by human cultured trophoblast in a time ( - h) and a dose ( - g/ml) dependent manner. injection of polyi:c to c b/ wild type mice induced preterm delivery within h at a dose of mg/kg body weight. no effect was observed in tlr- knock out mice. a robust systemic (spleen and serum) and local (placenta and amniotic fluid) inflammatory response was observed and h following polyi:c treatment. trophoblast cell cultures from tlr- ko mice confirmed that the response to polyi:c is tlr- dependent. conclusion: we demonstrate that viral infection may trigger an immune response leading to preterm labor. furthermore we show that the trophoblast is able to recognize and respond to viruses through the expression of tlr- . our findings provide a novel mechanism of pathogenesis of preterm labor associated with tlr- mediated inflammatory response. introduction: adverse neurological outcome is a major cause of neonatal morbidity after a preterm birth (ptb). a growing body of evidence demonstrates the involvement of inflammatory pathways in ptb and implicates these pathways as a causative factor in fetal brain injury. however, activations of cytokine pathways in normal labor (whether iatrogenic preterm or at term) has been observed. what remains understudied is the effect of labor on the preterm fetal brain and whether an inflammatory stimulus is essential for fetal brain injury. methods: mouse models were utilized: ( ) a model of intrauterine inflammation (lps into uterine horn) (n= ); controls received intrauterine saline (n= ) and ( ) autism is a neurodevelopmental disorder with a strong genetic component and several known environmental risk factors, such as infection. in addition, its onset of etiology is likely to occur during prenatal development. we propose that subjecting fetal sheep via amniocentesis to the bacterial endotoxin lipopolysaccharide (lps) injected to the amniotic fluid at gestational day (gd) will result in morphological alterations in the offsprings' cerebellum resembling alterations found in the cerebellum of patients with autism. using high precision design-based stereology, we investigated mean total-and layer-specific volume and mean total granule and purkinje cell (pc) number in the cerebellum of lps infected animals and controls. the results of the present study showed preserved volumes of the total cerebellum as well as of the molecular layer, outer and inner granular cell layers and white matter. interestingly, compared to controls, the lps infected brains showed a statistically significant increase (+ . %) in the mean total number of granule cells, whereas the pcs did not show any difference between the groups. these seemingly paradoxical results might be explained by ( ) the so-called time of origin of these neurons, i.e. the pcs develop prenatally whereas the granule cells develop postnatally or ( ) the direct correlation between pcs and granule cell number in the cerebellum. these results might contribute, as an animal model, to our understanding of the biological basis for interindividual differences in morphological alterations found in the brains of patients with autism. the previous studies have shown that there is a higher incidence of spontaneous preterm birth and poorer neonatal outcome in pregnancies with a male fetus. in vitro studies also report a sex dependent pattern in different placental enzymatic systems. we have shown previously that lactobacillus rhamnosus gr- supernatant is able to antagonize the actions of lps on cytokines and ptgs in placental trophoblasts. we hypothesize that fetal sex will influence the production of cytokines and prostaglandin regulating enzymes in lps and lactobacilli treated placental trophoblast cells. methods: term placentae were collected from women undergoing elective caesarean section. placental trophoblasts were isolated using established primary culture protocols. cells were pretreated with lactobacilli supernatant and subsequently treated with lps. pgdh and ptgs expression levels were measured by western blot analysis and tnf-, and il- concentrations measured by elisa. results: lps stimulation caused a marked increase in production of tnf-( . pg/ml to . pg/ml, n= , p< . ), an effect that was greater in placentae of the male fetuses ( . pg/ml, n= ) compared to female fetuses ( . pg/ml, n= ). lactobacilli supernatant abolished this response in both sexes. lps-activated trophoblasts from placentae of the male fetuses showed an increase in il- production (n= , p< . ) and ptgs expression (n= , p< . ). however, there was no response to lps in placentae of the female fetuses. lactobacilli supernatant up-regulated pgdh (n= ) by %, and this effect was greater in placentae of the female fetuses (n= ). conclusion: we conclude that human placentae from pregnancies carrying male fetuses are more responsive to lps by producing more pro-and anti-inflammatory cytokines, as well as ptgs . conversely, placentae of the female fetuses upregulate pgdh with lactobacilli treatment. these findings may explain the underlying mechanism for the higher incidence of preterm birth and adverse pregnancy outcomes seen with male fetuses in the clinical setting. , ) . this study investigates whether iai is associated also with altered expression of neuropilin- . methods: (i) immunohistochemistry (ihc) was performed on tissue sections of term decidua with or without clinical / histologic evidence of iai (n= for each). neuropilin- expression was scored by an investigator blinded to the identity of the samples. (ii) cultured term dscs were retrieved from elective cesarean (n= ), purified, and depleted of leukocytes. after treatment with - m estradiol (e ), - m medroxyprogesterone acetate (mpa), both, or vehicle for days, dscs were stimulated with il- b ( - ng/ml), tnfa ( ng/ ml), or thrombin ( . iu/ml) for h. since no elisa exists for neuropilin- , protein expression was determined by immunocytochemistry (icc). (iii) total rna was extracted and the effect of il- b on neuropilin- mrna expression measured by real-time rt-qpcr and corrected for b-actin mrna. results: neuropilin- expression in term decidua was increased in tissues with iai vs controls (p< . ), and localized primarily to dscs. using icc, an increase in neuropilin- was noted after stimulation with il- b and tnfa, but not thrombin. il- b increased neuropilin- mrna expression in dscs by . ± . -fold (from . ± . to . ± . neuropilin- mrna/b-actin mrna; p= . ). conclusions: iai is associated with increased expression of the vegf receptor, neuropilin- , in term decidual tissues. il- b and tnfa (but not thrombin) stimulated neuropilin- expression in term dscs, and this effect appears to be mediated at the level of gene transcription. since aberrant vegf function alters vascular permeability, these data provide a mechanism by which iai can promote 'decidual activation' and preterm labor. inflammation, university of glasgow, glasgow, united kingdom. objective: asthma is associated with inappropriate activation of airway smooth muscle, chemokine expression and accumulation of mast cells which drive smooth muscle reactivity. labour is similarly associated with smooth muscle activation and expression of cxcr and cxcr ligands. the role of mast cells in human parturition is unknown; however, mast cell products can stimulate myometrial contractions and preterm labour in animal models. we have quantified mast cells in association with human labour and determined whether they express cxcr and cxcr . methods: lower segment myometrial and cervical biopsies were taken at term caesarean section from women not in labour (nil) (myometrium n= ; cervix n= ) and in labour (il) (myometrium n= ; cervix n= ). mast cells were localised in myometrial and cervical sections by icc with a primary antibody against c-kit. the number of cell transects in randomly selected high-powered fields ( x) was quantified blindly by two observers for each specimen, with median density and interquartile range (iqr) calculated. backto-back icc was performed to determine whether c-kit co-localised with the chemokine receptors cxcr and cxcr . results: mast cells were in close association with myometrial smooth muscle in non-labouring lower segment myometrium. labour was associated with a significant influx and increase in mast cells numbers (nil median . , iqr . - . ; il median . , iqr . - . , p= . ). in contrast no significant increase in mast cells was observed in cervical tissue in association with labour (nil median . , iqr . - . ; il median . iqr . - . , p= . ). analysis of chemokine receptor expression demonstrated co-localisation of cxcr to c-kit positive cells present within the myometrium. conclusions: human labour at term is associated with an increase in mast cells within the myometrium, with close approximation to smooth muscle bundles. these mast cells express the chemokine receptor cxcr , the ligands of which we have previously shown to be up-regulated in labouring myometrium. mast cells are not accumulated in cervix in association with labour suggesting a less critical role in cervical ripening. further analysis of the role of mast cells in modulating myometrial smooth muscle physiology is warranted. progestins and the glucocorticoid receptor in human myometrial and amnion-derived wish cells. alison j tyson-capper, stephen c robson. reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. background and objectives: progesterone (p) can reduce the risk of preterm birth in some high risk women. in this context there is accumulating evidence that this, at least in part, may be due to anti-inflammatory and immunoregulatory properties of p. target tissue responsiveness to p is considered to be determined by the progesterone receptors (pr) and nuclear co-factors that directly interact with pr. pr and glucocorticoid receptors (gr) share several structural and functional characteristics, including similarities in dna sequence recognition by binding to the same hormone response elements. pr and gr interact with similar chaperones in the absence of ligand and with a similar group of co-activators in the presence of hormones; both can display comparable anti-inflammatory activities under specific physiological conditions. in this study we aimed to investigate whether the anti-inflammatory properties of progestins may be mediated by pr and gr signalling. methods: primary cultures of non-pregnant and term pregnant human myometrial (passage - ) and wish cells were serum starved for hrs and treated with -hydroxyprogesterone ( -hp), progesterone (p), dexamethasone (dex) and immunofluorescent staining and immunoblotting analyses performed. in some experiments cells were pre-treated with ru (a pr/gr antagonist) or org (a pure pr antagonist). results: in the absence of hormone gr appeared to be predominantly cytoplasmic, whereas, upon treatment with -hp and p ( and m) and dex ( nm) gr was abundant within the nuclei of myometrial cells. immunoblotting analyses demonstrated that levels of gr progressively increased within the nuclear fractions of both pregnant myometrial and wish cells in response to increasing concentrations of p ( nm to m), and decreased sequentially within cytoplasmic fractions. in the presence of org gr protein levels remained constant within the cytoplasm. there also appeared to be a slight increase in gr expression, though not statistically significant (p> . ) within both cells types in response to p. conclusion: in this study we show that gr is activated by -hp and p and translocates to the nuclei of human myometrial and amnion-derived cells. in addition, levels of gr increase in response to p. whether p and -hp act as agonists or antagonists for gr in the regulation of hormone response genes associated with the onset of term and preterm labour remains to be elucidated. objective: using a mouse model of inflammation-induced preterm birth (ptb), we have demonstrated dramatic cytokine elevations in the uterus and placenta with concomitant, though less dramatic, cytokine elevations in the fetal liver and brain, associated with neuronal injury. because precise mechanisms of fetal injury in ptb remain unclear, we sought to examine inflammatory cell trafficking, and target organ damage by histopathologic assessment of the placenta, fetus, and fetal brain. study design: hours after intrauterine infusion of saline or lps into the right lower uterine horn of cd- mice, the left upper horn, with the gestational sacs(gs) in situ, was removed en bloc(n= per group) each with - gs with fetuses/treatment group. specimens were fixed, bisected and processed for histology and ihc. inflammatory and hematopoietic cells were quantified using pas, gata- (erythroid precursors), cd , and bm (macrophage-mp) within the placenta, liver, extremity mesenchyme, brain and leptomeninges. the presence of hemorrhage, necrosis, and apoptosis (h ax stain) was assessed. erythopoietin (epo) levels were measured in brain and liver by elisa. results: more neutrophils were present in maternal decidual vessels in lps compared to saline (p= . ). in lps-exposed, fetal mp were increased in the placenta (p= . ), fetal extremity mesenchyme (p= . ), fetal liver (p= . ) and leptomeninges (p= . ) but not in the brain or spinal cord compared to saline. no necrosis, hemorrhage or increased apoptosis was noted in the fetal brains. % of lps-exposed fetuses and % of saline-exposed had liver hemorrhages (p< . ). increases in nucleated erythrocytes and erythroid precursors were found in fetal vasculature of the placenta in lps-exposed (p= . ). epo levels were not elevated in either group. conclusion: intrauterine lps infusion induces acute inflammation predominantly in the maternal circulation of the placenta. in the fetus, there is widespread mp activation, liver hemorrhage and increased erythroid precursors seen in the fetal circulation of the placenta. although histologic evidence of cns damage was not evident, the increased mps present in the leptomeninges may play an important role in inflammatory-mediated cns damage. non-toll-like innate immune proteins: do they change during pregnancy? juan m gonzalez, hua xu, ella ofori, michal a elovitz. obgyn; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: trem- (trigger receptors expressed on myeloid cells) is an important regulator of innate immunity. the natural ligand for trem has not been identified. activation of trem- in the presence of toll-like receptors results in substantial amplification of the host inflammatory response (klesney-tait et al nature immunology ). since inflammatory pathways are implicated in adverse pregnancy outcomes, this novel mediator of inflammation may play a critical role in preterm birth (ptb). therefore, we sought to determine trem- expression in the uterus, cervix, and placenta across gestation and to determine if trem- levels are altered by intrauterine inflammation. methods: in cd- mice, trem- was investigated in non-pregnant (np) and throughout gestation e , e (n= - per group). uterine, cervical, and placental tissues were harvested. using an established mouse model of inflammation-induced ptb, uterine tissue was collected hours after intrauterine infusion of saline (n= ) or lipopolysaccharide (lps) (n= ). for a non-pregnant model, using cd- mice, lps (n= ) or saline (n= ) was injected into the uterine horn following same procedures as with pregnant mice. uteri were harvested hrs later. quantikine ® mouse trem- immunoassay was utilized for these studies. statistical analysis was performed using oneway anova followed by pair-wise comparison if statistical significance was reached (p< . ) results: trem levels are significantly different between np and pregnant uterine tissues (p= . ). e and e trem expression is significantly increased . and . -fold compared to np (p= . and . respectively). trem- levels in the placenta and cervix were not significantly different between e and e . trem levels increased about -fold in the uterus after intrauterine infusion of saline or lps compared to e controls. in nonpregnant, trem levels were significantly different (p= . ) with a -fold increase in trem expression in uteri exposed to lps or saline compared to controls. conclusions: non-toll-like innate immune proteins are differentially regulated during pregnancy compared to the non-pregnant state. the role of trem- in inflammation-induced ptb requires further study. research is warranted to determine if uterine up-regulation of trem in gestation is associated with an increased likelihood of responding to pathogens or severe as a protective mechanism. reduced plasma levels of vitamin d in caucasian women at term are associated with increased rate of infection. chander p arora, adegoke adeniji, susan e jackman, babak forooghi, isaac mostadim, phillip yadegari, calvin j hobel. og-gyn, cedars-siani medical center, los angeles, ca, usa; university of california los angeles, los angeles, ca, usa. background: vitamin d plays an important role in human pregnancy by acting as a regulator of immunity at the fetal-maternal interface. inflammatory changes associated with pro-inflammatory cytokines were reduced by vitamin d while anti-inflammatory cytokines were increased in t lymphocytes. vitamin d status has been defined as deficiency (< . nmol/l), insufficiency ( . to nmol/l) and sufficiency (> nmol/l) . objective: to assess the involvement of vitamin d in the occurrence of maternal infection during pregnacy in women with term deliveries. hypothesis: vitamin d metabolism could affect the rate of infection during pregnancy. study design plasma levels of (oh)d were determined by elisa and the rate of infection was recorded in a behavior in pregnancy study. in this study, ethnically diverse women were evaluated at - weeks (t ), - weeks (t ) and - weeks (t ). maternal infections were documented at each stage as well as at baseline visit with history of infection in current pregnancy. of these subjects who delivered at term two groups ( with no infection during pregnancy, with infection or history of infection) were matched further for non-smoking status, non-diabetics, ethnicity and maternal age. plasma from these were assayed for (oh)d and analyzed for the rate of maternal infection using fisher´s exact test or chi-square test. results although the women delivered at term, the levels of (oh)d in caucasians were significantly lower in the subjects with infection than the ones without (p<. ). women with vitamin d insufficiecy in the first trimester were more likely to develop infection during pregnancy ( . nmol/l ± . at t , . nmol/l ± . at t and . nmol/l ± . at t ; all p<. ) but not subjects with sufficient vitamin d at t ( . nmol/l ± . at t , . nmol/l ± . at t and . nmol/l ± . at t ; all p<. ). conclusion the results reveal a positive association between (oh) d concentrations and greater risk of infection. vitamin d deficiency or even insufficiency may, therefore be involved in the pathogenesis of maternal infection during pregnancy. it is probable that vitamin d deficiency or even insufficiency could modulate the maternal susceptibility to infection during pregnancy by a proinflammatory mechanism. in vitro and in vivo observational data suggest that infection leads to caspase activation and apoptosis in the placenta and membranes, however currently there are no data on the role of apoptosis in the pathogenesis of infection associated preterm delivery. here we used group b streptococcus (gbs) as a model pathogen and examined the role of caspase dependent and independent apoptosis in preterm delivery. methods: we injected ( . x ) heat killed group b streptococcus organisms (hk-gbs) intraperitoneally (i.p.) in . day pregnant c b/l mice. the mice were euthanized at hr (n= ) and hr (n= ), the placentas and membranes were removed and assessed for apoptosis by tunel staining. caspase activation and expression were determined by immuno-histochemistry (ih) and western blotting. the effect of apoptosis on preterm delivery was assessed by i.p. treating the pregnant mice with pbs (n= ), dmso (n= ) or pancaspase inhibitor z-vad-fmk (n= ) prior to hk-gbs and observing the animal for delivery for hrs. results: there was a time dependent, gbs-induced increase in apoptosis by tunel assay and caspase activation in the placenta and membranes. in addition hk-gbs-induced the expression of caspase and caspase independent m-calpain in the placenta. z-vad-fmk ( mg/kg), at the maximum concentration that did not induce maternal illness, did not prevent hk-gbsinduced preterm delivery. conclusions: caspase dependent and independent mechanisms are activated in the placenta upon exposure to gbs. systemic adminstration of a pan-caspase inhibitor did not impact upon the occurance or timing of bacterially induced preterm delivery. further studies are needed to assess the role of apoptosis in the pathogenesis of infection associated preterm delivery. early and ( . %) had a late sptb. the mean + sd gestational age at blood draw was + weeks. the median level of bb was higher in women with early as compared with late sptb or term births (p= . for trend). women with bb in the top quartile were . times more likely to have an early sptb as compared with women who had lower levels of bb ( % ci . to , p = . ). there was no association between bb and late sptb (rr= . , % ci = . to ). the adjusted or of an elevated bb for early sptb was . ( % ci = . to , p= . ). when the analysis was restricted to the women with sptb the rr of an elevated bb for early sptb was . ( % ci . to . , p = . ). conclusions: a significant relationship was found between an elevated bb in early pregnancy and early sptb suggesting inflammatory events in early pregnancy, perhaps infection-related, are part of the pathogenic mechanisms. objective: genital tract infection and/or inflammation appears to contribute to the majority of ptds preceding weeks of gestation. ptd in humans has been associated with colonization and/or infection with a variety of different organisms including gram positive and negative bacteria, mycoplasma, ureaplasma, trichomonads, malaria parasites and viruses. the innate immune response to these pathogens is produced by a family of pattern-recognition cell membrane receptors known as the toll-like receptors (tlrs). these studies sought to characterize the tlr isoforms expressed in the preterm mouse uterus, and their modulation during lipopolysaccharide (lps)-induced ptd. methods: using sterile surgical technique, day- pregnant cd- mice underwent intrauterine injection of g lps. subsequently, the mice were euthanized at , , , , and hours after lps injection. uterine tissue was harvested and placed in rnalater; subsequently total rna was isolated using the trizol reagent and genomic dna was removed using turbo dnafree. cdna was made using iscript cdna synthesis kit. pcr primers were designed using published mouse tlr gene sequences. real-time quantitative rt-pcr was performed using the power sybr green master mix and an abi prism multicycler. to confirm tlr amplicon sizes, the rt-pcr products were visualized on a % agarose/tbe gel stained with gelred. results: these studies have confirmed mrna expression of all of the reported mouse tlr isoforms. these tlr amplicons range in size from to bp as expected; amplicon sequences are pending. quantitative rt-pcr studies performed using uterine tissues from five mice at each time point demonstrated that at hours after lps injection, tlr increased -fold and tlr increased -fold (both p< . ). in contrast, the expression of tlr (the ligand for lps) remained stable during the hours after lps; the expression of tlr , , , , , and also remained stable. tlr expression trended upward and tlr trended downward, although neither was statistically significant. conclusions: these studies have confirmed expression of all tlrs within the preterm pregnant mouse uterus, along with characterization of their modulation during lps-induced ptd. these observations are important because they contribute to our understanding of the immunologic signaling events leading to ptd. (funded by nih hd ). udp-glucose and its receptor p y as a new innate immune system in the female reproductive tract. toru arase, tetsuo maruyama, hiroshi uchida, takashi kajitani, masanori ono, maki kagami, hironori asada, yasunori yoshimura. obstetrics and gynecology, keio university, shinjukuku, tokyo, japan. objective: innate immune system involving toll-like receptors has recently emerged in the female reproductive tract (frt). we hypothesize that there may exist new other mucosal immunity in frt. recently, it has been reported that p y , a g protein-coupled p y receptor for udp-glucose (udp-g), is involved in the lung epithelial immune system. the aim of this study is to investigate whether udp-g and p y have a potential as the defense immune system in frt, in particular endometrium. materials and methods: we obtained human endometrial tissues from consenting reproductive-aged patients. the spatiotemporal expression of p y in human and mouse endometrial tissues was analyzed using rt-pcr and ihc. isolated human endometrial cells and a human endometrial epithelial cell line, ishikawa, were cultured, treated with udp-g, and subjected to rt-pcr analysis for il- mrna expression. we also measured the il- secretion using elisa. small interfering rna was used to knock down p y expression. the chemotactic activity of udp-g on neutrophils was tested using transwell assay with ishikawa cells. lastly, mouse uterine tissues were incubated with udp-g and subjected to rt-pcr analysis for mrna expressions of kc and mip- , the il- homologues in mice. results: p y was exclusively expressed in the glandular and luminal epithelium both in human and mouse uteri. treatment with udp-g induced the secretion of il- in ishikawa and human endometrial glandular cells, but not stromal cells, in a dose-and a time-dependent manner. p y knockdown abrogated udp-g-induced il- production. treatment with udp-g also significantly increased the chemotaxis of neutrophils, which was attenuated by co-addition of anti-human il- neutralizing antibody. in the mouse uterus stimulation of udp-g significantly up-regulated the expressions of kc and mip- mrna. conclusions: udp-g is an endogenous molecule and released into the extracellular environment in a lytic manner after cell damage. taken together, our results collectively substantiate a model in which udp-g released from endometrial cells damaged by infection stimulates il- production via p y in endometrial glandular epithelium, which, in turn, recruits neutrophils thereby preventing the progression of infection. thus, udp-g and its receptor p y may be involved in the trans-species mucosal immune system in frt. (jsgi ; , (suppl) , abst # ) but in utero effects on fetal lung remain to be established. we have examined the relationship between duration of iai and subsequent azi treatment on the severity of fetal lung histopathology. we hypothesized that early treatment would prevent the development of advanced lesions, while late treatment may reduce the severity of lung damage. study design. thirteen chronically instrumented rhesus monkeys received intraamniotic inoculation of u. parvum (serovar ; - x cfu) at ± . days gest. age (dga, mean ± sem, term= d). six of these animals received maternal i.v. azi ( . mg/kg q h or q h for d) either alone (n= ) or in combination (n= ) with dexamethasone (dex; mg/kg/d i.v. for d) and indomethacin (indo; mg/d p.o for d). tissues were obtained at cesarean section for histopathologic assessment. leukocytic infiltration of aveolar spaces and septal walls, type ii pneumocyte hyperplasia and peribronchiolar lymphocytic aggregates were scored as absent ( ), minimal ( ), moderate ( ) and severe ( ). results. inoculation-to-delivery interval was - d for combined treatment groups and was similar to long duration infection without treatment ( - d). treatment effects were tabulated as mean scores and compared as follows: control (n= ), score ; short duration infection ( - d; n= ), score ; long duration infection ( - d; n= ), score ; short duration infection + treatment (n= ), score ; long duration infection + treatment (n= ), score . conclusions. histopathologic findings of fetal pneumonia progressively worsen with duration of u. parvum iai. early maternal azi treatment prevents development of advanced lung lesions, while later treatment may reduce the severity of fetal lung damage. our results suggest that in utero treatment of iai may lower the risk of neonatal bronchopulmonary dysplasia. support: nih hd , rr . brain associated with adverse neurodevelopmental outcome. lps triggers proinflammatory responses through toll-like receptor- (tlr ). mitogen activated protein kinases including c-jun-n-terminal kinase (jnk) have been reported to be implicated in tlr signalling pathways and play important role in both onset of labor and brain injury. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of specific inhibitor of jnk signaling, d-jnk inhibitory peptide (d-jnki) can (i) inhibit jnk activity in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr- specific lps to cd pregnant mice at day of pregnancy caused preterm delivery after to h with % pup mortality. in vitro kinase assay demonstrated the activation of jnk in response to lps in the maternal uterus and fetal brain. furthermore, the brain specific jnk was found to be the major jnk isoform activated by lps in the fetal brain. co-administration of d-jnki with lps to pregnant mice delayed significantly (p< . ) lps-induced preterm delivery and reduced pup mortality up to %. this was associated with inhibition of jnk activity in both maternal uterus and fetal brain. in addition, we have found that treatment with lps significantly up-regulated cox- , cxcl (il- equivalent) and ccl in myometrium and this is significantly suppressed after co-administration of d-jnki. we conclude that specific inhibition of jnk signaling may have a potential of controlling preterm labor and preventing fetal brain damage as a result of infection/inflammation. the prostaglandin -deoxy- , -prostaglandin j delays lps-induced preterm delivery and reduces mortality in the mouse. intrauterine infection is a common trigger for preterm birth, and is also a risk factor for the development of neurodevelopmental abnormalities in the neonate. bacterial lipopolysaccharide (lps) binds to toll-like receptor- (tlr ) to activate pro-inflammatory signaling pathways. the transcription factor nuclear factor kappa b (nf-kb) is a key player in the orchestration of the inflammatory response and has a central role in parturition. we have previously shown that exposure to the anti-inflammatory cyclopentenone prostaglandin -deoxy- , -prostaglandin j ( d-pgj ) inhibits il- b-induced nf-kb activity and cox- expression in human myometrial and amnion epithelial cells in vitro. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of d-pgj can (i) inhibit nf-kb in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr specific lps to cd pregnant mice at day of pregnancy caused preterm delivery after to h with % pup mortality. co-administration of d-pgj with lps to pregnant mice delayed significantly (p< . ) lps-induced preterm delivery and conferred protection from lps-induced fetal mortality up to %. we have looked at the expression profile of several labor associated genes in myometrium hours after lps administration. (otr, connexin and , cox- , cox- , cxcl (il- equivalent) and ccl ). we have found that treatment with lps significantly up-regulated cox- , cxcl and ccl and this is significantly suppressed after with co-administration of d-pgj . western analysis for ser -phosphorylated p and ikkb in-vitro kinase assay has demonstrated that lps induced activation of nf-kb at both h and h. co-administration of d-pgj was associated with inhibition of nf-kb activation in both the maternal uterus and the fetal brain. conclusion d-pgj may have potential as a therapeutic agent in the management of preterm labor and, by targeting the player nf-kb, may have the added advantage of preventing detrimental effects to the fetus that may result from infection/inflammation. synergistic macrophage response to co-activation of tlr- and tlr- : mechanisms and implications for bacterial/viral co-infection. vladimir ilievski, emmet hirsch. , department of ob/gyn, evanston northwestern healthcare, evanston, il; department of ob/gyn, feinberg school of medicine, northwestern university, chicago, il. background: toll-like receptors (tlrs) recognize structural components of pathogens and initiate host defenses. tlr- responds to gram-positive organisms and peptidoglycan (pgn), a gram-positive cell wall constituent. tlr- is activated by viral infection in response to double-stranded rna. polyinosinic-cytidylic acid (poly(i:c)) is a tlr- ligand. we have shown that pgn and poly(i:c) have a synergistic effect on the expression of downstream genes for both tlr- and tlr- . here we identify mechanisms underlying this synergy. methods: mouse peritoneal macrophages or a mouse macrophage cell line (raw . ) were treated in tissue culture with either pgn ( g/ml), poly(i: c) ( g/ml) or both pgn and poly(i:c) either simultaneously or sequentially for - hours. total rna was extracted and duplex rt-pcr was performed for inducible nitric oxide synthase (inos), interleukin (il- ), tumor necrosis factor (tnf), the chemokine rantes and tlr- , normalized to the housekeeping gene gapdh. results: compared to stimulation with either pgn or poly(i:c) alone, costimulation of raw . cells with both pgn and poly(i:c) resulted in synergistic expression of inos, il- , tnf and rantes (p< . for all) at and hours . sequential stimulation with either pgn or poly(i:c) for h followed by incubation for an additional h with the alternate ligand also induced synergistic expression of the same rnas, albeit at lower levels than were elicited by simultaneous stimulation. in contrast, incubation with either pgn or poly(i:c) for h followed by medium for h induced minimal to no gene expression. both pgn and poly(i:c) induced tlr- mrna after h but not h. tlr- mrna was not detectable by rt-pcr. in primary peritoneal macrophages, similar synergy due to pgn and poly(i:c) was seen. conclusions: simultaneous or sequential exposure to pgn and poly(i:c) exerts a synergistic effect on the expression of inflammatory mediators in macrophages. interestingly, either one of these two tlr pathways can prime cells for activation of the other pathway. a possible mechanism for this effect may be induction of tlr- by either tlr- or tlr- activation. these observations have implications for bacterial/viral co-infection. this is a secondary analysis of a prospective cohort study. after irb approval, daily blood samples were obtained from pprom subjects and analyzed for il- by elisa. paired maternal serum il- levels from subjects were divided into groups: il- levels obtained - hours prior to completion of antibiotics and those obtained - hours after completion of antibiotics. the wilcoxon signed rank test was used to perform the data comparison on the analyze-it statistical software program. statistical significance was defined as p< . . of the pprom subjects, the maternal age was . yrs; gestational age at admission was . weeks; latency was . days; gestational age at delivery was weeks; and infant birth weight was grams. median maternal serum il- levels obtained off antibiotics were significantly higher when compared to those on antibiotics ( . vs. . pg/ml, p< . ). the results of this investigation suggest that maternal serum il- levels rise after discontinuation of antibiotics. the optimal duration of antibiotics administration in the setting of pprom is unknown. this data suggests a role for continuation of antibiotics in women with pprom in order to prolong the latency period and potentially decrease neonatal morbidity. to identify clinical and pathologic factors associated with fetal inflammatory responses in the placenta from term parturients. methods: retrospective cohort study of consecutive term parturients with submitted placentas in . placentas with histologic chorioamnionitis were divided into two cohorts: group -maternal inflammatory responses only, and group -maternal and fetal inflammatory responses. maternal and fetal inflammatory responses in the placenta were classified according to guidelines established by the amniotic fluid infection nosology committee of the perinatal section of the society of pediatric pathologists. selected demographic, intrapartum, newborn and placental characteristics were analyzed with t-tests and chi-square tests as appropriate. multiple logistic regression was used to identify independent predictors of fetal inflammatory responses in the placenta. results: of consecutively submitted placentas, had histologic chorioamnionitis: with maternal inflammatory responses only (group ) and with both maternal and fetal inflammatory responses (group ). fetal inflammatory responses observed in group were associated with higher stages of maternal inflammatory responses (p<. ). % of fetal inflammatory responses were stage i (acute chorionic vasculitis or phlebitis).group patients were more likely to have had amnioinfusion ( % v %, p=. ) and less likely induction of labor ( % v %, p=. ). group was more likely to have had intrapartum fever ( % v %, p=. ) and maternal tachycardia ( % v %, p=. ). newborns from group were more likely to have been observed for sepsis ( % v %, p <. ) and have an apgar score at minutes ( % v %, p=. ). a logistic regression model showed that stage ii or greater maternal inflammatory responses (or . ) and amnioinfusion (or . ) were independent predictors of fetal inflammatory responses. conclusion: higher stages of maternal inflammatory responses in the placenta and amnioinfusion were independent predictors of fetal inflammatory responses in term parturients with histologic chorioamnionitis. objective: previous studies have shown that sulfasalazaine (sasp) inhibits lipopolysaccharide (lps)-induced nf-b activation and decreases lpsstimulated interleukin- (il- ) production by cultured explants of placenta, amnion and choriodecidua with no effect on cell viability. bacteria-induced il- production in the cervix is a potential mechanism for premature cervical ripening that may lead to preterm birth. it is unclear, however, whether sasp can inhibit il- production by endocervical cells. therefore, we performed a series of in vitro studies to determine if sasp inhibits il- production by endocervical epithelial cells stimulated with bacterial pathogens associated with preterm birth. methods: human endocervical epithelial cells were incubated with - . mm sasp overnight and then stimulated with ccu/ml ureaplasma parvum, cfu/ml escherichia coli, or x cfu/ml gardnerella vaginalis for another overnight incubation in -well cultures. conditioned medium was then harvested and production of il- was quantified by elisa. viability of the cells was ascertained at the end of the experiment with the mtt-assay. each treatment was applied in quadruplicate wells and experiments were repeated times using different batches of cells for each pathogen. results were evaluated using the general linear models procedure of sas for a randomized block design. results: sasp had no detectible effect on il- production by endocervical cells not treated with bacteria. at the highest concentration tested ( . mm), sasp significantly inhibited il- production by cultures stimulated with e. coli (p< . ), u. parvum (p< . ), and g. vaginalis (p< . ). viability of the cells, however, was significantly reduced by sasp at . and . mm in the both the presence and absence of bacteria for all experiments. conclusion: although high concentrations of sasp inhibit il- production by cultures of endocervical cells stimulated with pathogens associated with preterm birth, this effect may be due to toxicity of the antibiotic on the cells. the effect of -hydroxyprogesterone caproate on lipopolysaccharide stimulated peripheral blood mononuclear cells from pregnant women. richard h lee, aimin li, frank z stanczyk, jinwen i lin, t murphy goodwin. obstetrics and gynecology, university of southern california, los angeles, ca, usa. introduction: -hydroxyprogesterone caproate ( -ohpc) reduces the rate of recurrent preterm birth in high-risk women. however, there are lines of evidence to suggest that -ohpc alters inflammatory response in the setting of gram-negative infection. in a mouse model, -ohpc decreased the rate of preterm birth but was associated with significantly increased maternal morbidity when mice were exposed to lipopolysaccharide (lps). in non-pregnant women, -ohpc pre-treatment of whole blood exposed to lps significantly increased the production of tnf-. objective: to determine if -ohpc has an effect on the production of proinflammatory cytokines from peripheral blood mononuclear cells (pbmc) in pregnant women. methods: pbmc were isolated from fresh whole blood samples of pregnant women between and weeks. pregnant women had no prior history of preterm birth. cells were treated with -ohpc ( m) and escherichia coli lipopolysaccharide (lps, g/ml) alone or in combination. after and hours of culture, supernatants were collected and tested for tnf-and il- levels by enzyme-linked immunosorbent assay (elisa). statistical analysis was performed using non-parametric tests. p < . was considered significant. results: pbmc exposed to lps significantly increased tnf-and il- secretion compared to untreated controls (p= . and p= . ). tnf-concentrations were not significantly different between lps and lps+ -ohpc treated cells at both and hours (p= . and p= . ). il- production was significantly increased after -hour treatment with lps+ -ohpc compared to lps alone ( , [ , - , ] background. recent clinical trials indicate that progestins reduce the incidence of preterm birth in some high risk pregnancies. it has been proposed that progesterone promotes uterine quiescence, in part, via its antiinflammatory properties with inhibition of pro-inflammatory gene expression. it is intriguing that progestins are clinically effective given the considerably increased background circulating levels of the hormone during pregnancy. we hypothesised that non-classical progesterone signalling pathways contribute towards mediation of the anti inflammatory effects of progestins. to the endotoxin lipopolysaccharide (lps) by activation of inflammatory pathways. myometrial cell cultures were treated with lps ( g/ml)+/progestin ( nm). two progestin compounds were investigated. natural progesterone (p) is known to have a strong affinity with progesterone receptor (pr) analogues in contrast to -hydroxyprogesterone ( -hp) which, in the absence of esterification with caproate or acetate, has been reported to have no progestogenic activity at pr. the effect of p and -hp on the activation of two inflammatory genes known to be associated with labour (cycloxygenase [cox- ], toll-like receptor [tlr- ]) was evaluated. cox- and tlr- were detected at the protein and mrna levels using sds-page and rt-pcr. results. lps-induced expression of cox- and tlr- proteins were significantly inhibited by both p (p< . and p< . , respectively) and -hp (p< . and p< . , respectively). furthermore, preliminary results indicate that co-incubation with the anti-progesterone mifepristone, fail to abrogate the anti inflammatory effect associated with progestin treatment. conclusion. non-classical progesterone signalling pathways have a role in mediating the anti-inflammatory properties of progestins. further elucidation of the molecular actions of progestins may allow novel approaches to ameliorate the inflammatory response associated with preterm labour. detection and transcriptional expression of tlr- , tlr- and tlr- at the maternal-fetal interface. jacqueline p tilak, karen zakharian, alexandra tungol, gabriel o schubiner, david m svinarich. patient care research, providence hospital, southfield, mi, usa. preterm labor and delivery remains a leading cause of neonatal morbidity and mortality and bacterial infection is considered to be the most common etiology. toll-like receptors (tlr's) and the associated components of the innate immune system may represent a first line of defense against these pathogens. tlr's belong to a family of pattern-recognition receptors that bind to highly conserved pathogen-associated molecular patterns (pamps) including lipopolysaccharide (lps), lipotechoic acid (lta) and cpg dna, and are a key component of the innate immune system. this study was undertaken to characterize the transcriptional expression and regulation of tlr- , tlr- and tlr- within gynecologic and gestational tissues. human first trimester trophoblasts, endometrial mesoderm, ectocervix, choriocarcinoma and neonatal epithelium, were cultured in media alone or in the presence of either lps ( mg/ml) or lta ( mg/ml) for , , , , and hours. total rna was isolated and semi-quantitative rt-pcr was performed using intron-spanning amplimers to tlr- , tlr- , tlr- and gapdh. following gel electrophoresis, the integrated optical densities were determined for the corresponding pcr products and normalized with respect to gapdh levels. inducible transcription of tlr- with lta was observed in choriocarcinoma cells ( -fold, h), and endometrial mesoderm cells ( -fold, h). tlr- induction with lps was observed in ectocervical cells ( -fold, h), choriocarcinoma cells ( -fold, h) and endometrial mesoderm cells ( -fold, h). tlr- induction with lps was observed in choriocarcinoma cells ( fold, h) and neonatal epithelial cells ( -fold, h). all cell lines showed at least constitutive levels of transcription for tlr- , tlr- and tlr- . these data demonstrate that tlr- , tlr- and tlr- are transcriptionally expressed either constitutively or inducibly in both gynecologic (endometrial mesoderm, ectocervix) and gestational (chorion, trophoblast), tissues. translation of these receptors suggests that the innate immune system may function at the maternal-fetal interface to help protect the fetus against infection and prevent or diminish the likelihood of prematurity. objective: further to our development of a sheep model of intrauterine ureaplasma spp infection, we aimed to examine the capability of ureaplasma colonization in the amniotic fluid to infect the fetus and alter lung and brain development. methods: at days of gestation (d, term= d) ewes bearing single fetuses were given a single ultrasound-guided intra-amniotic injection of (a) x colony forming units (cfu) of u parvum (serovar , n= ; serovar , n= ), (b) x cfu of u parvum (serovar , n= ; serovar , n= ) or (c) media control (n= ). at d, fetuses were delivered by cesarean section. amniotic fluid and umbilical arterial blood samples were collected. fetal body weight was recorded, fetal cerebrospinal fluid (csf) collected and a descending pressurevolume curve constructed after inflation of the lungs to cmh o. fetal membranes were immersion fixed and the fetal brain was perfusion fixed with % paraformaldehyde. the fetal brain and membranes were blocked, paraffin embedded, stained and viewed under the light microscope. results: chronic intra-amniotic colonisation with u parvum serovar or (ureaplasmas) did not result in fetal abortion or death. amniotic fluid ureaplasma titers at delivery were not dose-dependent. chronic ureaplasma exposure did not affect fetal body or brain weights, or result in a fetal systemic inflammatory response. umbilical arterial blood gases at delivery were similar between ureaplasma-and media-exposed fetuses. chronic intra-amniotic exposure to ureaplasmas resulted in higher inflammatory cell scores in the fetal membranes compared to media controls (p< . ). lung compliance was increased in ureaplasma-exposed fetuses compared to controls (p< . ). there were no gross anatomical changes observed in the white or grey matter in the cerebral hemispheres of fetuses that had been exposed to ureaplasmas; even in animals (n= ) that had csf ureaplasma colonisation. conclusion: chronic ureaplasma colonisation enhances fetal lung compliance without gross anatomical changes in the fetal brain. background: an important source of vitamin d is its synthesis by skin from uv solar radiations. the skin pigment melanin absorbs uv photons thus reducing the vitamin d synthesis by more than % making african americans at high risk for vitamin d deficiency. low maternal vitamin d status during pregnancy has been linked to molecular pathways for adverse outcomes. objective: the nf b transcription factor regulates genes involved in inflammation and immune processes, and is proposed to play a major role in the successful outcome of pregnancy and labor. prior immunohistochemical (ihc) and biochemical studies have been conflicting regarding nf b activation in human intrauterine tissues. the aim of this study was to quantify nuclear localization of p , the major tranactivating nf b subunit, in full-thickness fetal membranes (fm) and myometrium using ihc. methods: a retrospective analysis of paired fm, decidua, and myometrial samples was conducted using tissues collected from women in cohorts: preterm no labor (pnl, n= ), preterm labor (ptl, n= ), spontaneous term labor (stl, n= ), and term no labor (tnl, n= ). subjects were delivered between gestational ages - weeks (preterm) and - weeks (term) by cesarean. paraffin sections were stained with polyclonal (rabbit) anti-human p and microscopically examined. myometrial samples were categorized in a blinded fashion to groups of staining: no nuclear p (-), rare nuclear p (+), and > % nuclear p (++). a p nuclear labeling index (nli; % nuclear p /total cells) was calculated for each histological layer in full-thickness fm specimens using a blinded targeted randomization scheme consisting of non-overlapping low-magnification fields. results: nuclear p labeling was rare in amnion and inconsistently present in chorion. in decidua, p nuclear labeling was observed in all cases; however, decidual nli did not vary significantly across cohorts [pnl- % ( - %); ptl- % ( - %); tnl- % ( - %); stl- % ( - %); all values are median (iqr)]. in myometrium, ++ p nuclear labeling was significantly associated with labor, but present only in a portion of cases (ptl- %; stl- %). despite a trend, decidual nli was not significantly correlated with myometrial nuclear p labeling: myometrial specimens classified as -, +, and ++ corresponded with decidual nli of % ( - %) [median (iqr)], % ( - %), and % ( - %), respectively. conclusions: in a comprehensive ihc analysis, significant nuclear p immunolabeling was observed in myometrial cells following labor. nuclear p labeling in decidua was present in all cases, and did not vary significantly among the cohorts. the inflammatory cytokines interleukin- and tnf-increase g-csf expression in term decidual cells. objectives: chorioamnionitis (cam) elicits premature rupture of the membranes and pre-term delivery. previously, we found that the decidua from cam patients contains much higher neutrophil numbers than control decidua, but macrophage numbers are similar in both groups. granulocyte colony-stimulating factor (g-csf) enhances granulocyte colony formation chemoattraction and activation. the amniotic fluid during cam contains elevated tnf-and il- levels. to account for the marked influx of neutrophils infiltration in cam-complicated decidua, tnf-and il-effects were assessed on g-csf expression in term decidual cell (dc) monolayers. methods: confluent leukocyte-free term dcs from normal term deliveries were primed with - m estradiol (e ) + - m medroxyprogesterone acetate (mpa) for days, then switched to serum-free defined medium (dm) with steroids ± tnf-or il- . after h, aliquots of conditioned dm supernatants, cell lysates and extracted rna from h parallel incubations were frozen. secreted g-csf was measured by elisa in conditioned dm and normalized to cell protein and mrna was assessed by quantitative real time rt-pcr and normalized to -actin mrna. results: in cultured first trimester dcs, basal g-csf levels in conditioned dm were . ± . pg/ml/ug cell protein [mean ± sem, n= ] and did not differ from e +mpa basal levels. the addition of ng/ml of tnf-or il- significantly elevated g-csf output to . ± . and ± respectively (p< . ), representing more than a -fold and , -fold change; respectively. western blotting confirmed the elisa results. quantitative rt-pcr demonstrated corresponding changes in g-csf mrna levels as found for the elisa measurements. concentration-dependent effects for both tnfand il- from . to . ng/ml were observed. conclusions: when extrapolated to the placental milieu of cam, the marked increase in g-csf elicited in term dcs by tnf-and il- may provide a mechanism to account for the selective increase in decidual neutrophils versus macrophages. background. the immunological mechanisms of the female reproductive tract are unclear. toll-like receptors (tlrs), innate immune receptors that combat microbial infections and establish adaptive immunity, may play a role. infection in pregnancy has been associated with preterm birth and tlrs may modulate the host response to such infections. we hypothesised that the distribution of tlr and tlr in cervical epithelial cells may differ with pregnancy, and that oestrogen may contribute to the modulation of these receptors. aims and objectives. . to compare tlr and tlr protein expression, using immunocytochemistry, in the cervical epithelium of pre-menopausal non-pregnant women with pregnant women at term. methods. fresh non-pregnant (n= ) and pregnant (n= ) human cervical cells were obtained by smear and tlr and tlr expression investigated by immunocytochemistry. cervical epithelial cells from nonpregnant women obtained by smears were then coincubated with varying concentrations of estradiol, and tlr and tlr protein expression quantified by immunocytochemistry. results. using pixel-based image analysis software, we demonstrated a reduction in tlr expression in late pregnant compared with non-pregnant cervical epithelium (p= . ), whilst tlr did not appear to differ. there was an apparent up-regulation of tlr protein expression in response to beta-oestradiol (n= ) objective: to evaluate in vitro antimicrobial activity of cranberry-exposed urine against common urinary pathogens. subjects were pregnant women enrolled in a clinical trial evaluating the effect of daily cranberry juice cocktail or matching placebo on asymptomatic bacteriuria and other urinary tract infections. methods: -hour urine samples from pregnant women who were randomized to cranberry or placebo in three treatment arms: a. cranberry two times daily with meals (c, c; n= ), b: cranberry in the am, then placebo at dinner (c, p; n= ), c: placebo two times daily with meals (p, p; n= ). we identified non-pregnant, reproductive-aged controls, randomized them to the same treatment groups and collected -hour urine specimens from them. the ph of all urine specimens was adjusted to ph= and filtered. aliquots of each were independently inoculated with overnight culture of - cell/ml each of single strains of e. coli with fimbriae type i and type ii, k. pneumoniae, and c. albicans. after hours of incubation for e. coli and k. pneumoniae, and hours for c. albicans cfu/ml of each specimen were enumerated by subculture with quantitative plate counts in duplicate. results: there were no differences for any of the antimicrobial activities between pregnant and non-pregnant groups, or based on treatment allocation. we were able to perform antimicrobial assays on the urine of women exposed to cranberry juice or placebo, but unable to demonstrate differences based on treatment allocation or pregnancy. this may be due to beta-error. further studies are planned. supported by r dk - ; clinical trials registration nct . ...,.. e. coli . x .:!: . x . x .:!: . x group a (c, c) group b ( c, p) . x + . x . x + . x . . . group c (p, p) . x o"::!>s x o" . x ~ .ox: o• k. ~neumoniae group a ( c, c) . x + . x . x + . x . . . group b (c, p) . x + . x . x + . x group c (p, pl . x ~ . x . x ~ . x c. al bicans group a (c, c) . x '+ . x . x + . x group b (c, p) . x o•+ . x . x + . x . . . group c (p, p) . x ~ . x . x ~ . x • objective: to measure compliance, we sought to evaluate the use of a bioassay for cranberry in the urine of women enrolled in a clinical trial designed to determine the effect of daily dosing of cranberry juice cocktail or matching placebo on the incidence of asymptomatic bacteriuria (asb) and other urinary tract infections (uti) during pregnancy. methods: we collected -hour urine specimens from pregnant women who were randomized to ingest cranberry or placebo in three treatment arms: a: cranberry two times daily with meals (n= ), b: cranberry in the am, then placebo at dinner (n= ), c. placebo two times daily with meals (n= ). we identified non-pregnant, reproductive-aged controls, randomized them to the same treatment regimens (group a: n= , group b: n= , group c: n= ), and collected -hour urine specimens from them. bacterial anti-adhesion effects of the cranberry-exposed urine were evaluated utilizing a human red blood cell hemagglutination assay specific for uropathogenic p-fimbriated e. coli. activity was quantified as , , and %. results: when combining all subjects and dosing regimens, the sensitivity of the assay was %, range % in the pregnant group assigned single daily dose of cranberry to % in the non-pregnant group assigned to multiple daily doses. the specificity ranged from % to %. conclusions: these data indicate that the bioassay can be applied to the pregnant patient population, although the sensitivity of the assay is variable. higher daily dosing appears to confer greater sensitivity. further studies are needed to evaluate the utility of this bioassay for compliance. supported by r dk - . clinical trials registration nct . objective: increasing evidence suggests that inflammation plays a crucial role in initiation of labour both at term and preterm. we have previously shown upregulation of pro-inflammatory cytokines in myometrium, cervix and membranes at term labour. we have also shown that myometrium and cervix is invaded by leukocytes at the time of labour, and these leukocytes express pro-inflammatory cytokines. in this study, we aimed to test the hypothesis that pro-inflammatory cytokines and toll-like receptor and (tlr and ) mrna expression are up-regulated in circulating leukocytes during term and preterm labour. methods: peripheral blood samples were taken from pregnant women: - weeks gestation (w) preterm not in labour (ptnl) n= ; - w preterm in labour (ptl), n= ; - w term not in labour (tnl) n= ; and - w term in labour (tl) n= . leukocytes were isolated using dextran sedimentation. total rna was isolated and reverse transcribed using high capacity cdna archive kit, and mrna expression determined by real-time pcr (abi, taqman). student's t-test was used to compare outcomes between groups. results: messenger rna expression of il- , icam- , mcp- , tlr and tlr was significantly increased in tl compared to gestation matched tnl. the expression levels of il- b, il- and tlr- were significantly greater in ptl compared with gestation matched ptnl. (table i) . conclusions: we show up-regulation of pro-inflammatory cytokine production in circulating leukocytes in both term and preterm labour. thus, the pathophysiology of labour seems to involve the upregulation of proinflammatory cytokine production in peripheral blood leukocytes, followed by their invasion into the myometrium and cervix. this work further contributes to our understanding of the pathophysiology of parturition, and suggests that peripheral blood leukocytes may be potential targets for therapeutic agents aimed at modifying the time course of parturition. introduction. the mechanisms of innate immunity and tolerance in the female reproductive tract (frt) are ill-understood but involve the pattern recognition toll-like receptors (tlrs). we have previously demonstrated high expression levels of tlr gene and protein in fresh human cervical epithelium. aims. in order to gain insight into the immunological role of cervical epithelial cells (cecs), we sought to determine cec responses to the following tlr- agonists: macrophage-activating lipopeptide (malp- ), pam csk , and peptidoglycan. methods. cecs were isolated by smears and compared between pregnant ( rd trimester) and nonpregnant women. following cell preparation, flow cytometry was performed using a direct staining procedure with mouse anti-human tlr primary antibody and its igg , isotype control, to determine tlr- protein expression. a further nonpregnant smear samples were each prepared, and seeded at a concentration of , cervical epithelial cells/ml into -well cell plates. cells were incubated at °c in % co overnight with the tlr agonists malp- and pam csk (at concentrations of and ng/ml), peptidoglycan( ng/ml), il- ( ng/ml, positive control) and rpmi + -glutamine media only (vehicle). following centrifugation, all resulting supernatants were stored at - °c until the concentration of il- was determined by elisa and an array of cytokines by bead assays. results. both pregnant and nonpregnant cecs expressed tlr (specific mean fluorescence . vs . respectively) but did not differ (p = . ). unstimulated cells incubated with buffer alone demonstrated high il- levels ( pg/ml), which did not differ following stimulation with malp- ( pg/ml), pam csk ( pg/ml) or peptidoglycan ( pg/ml). results of an array of pro-and anti-inflammatory cytokines following incubation of cells stimulated with tlr agonists are pending. conclusion. high basal il- levels suggest constitutive expression by cecs but the physiological relevance of this intriguing observation is unclear. that cec stimulation with tlr- agonists did not lead to further release of il- may epitomise the resistance of these cells to antigenic challenge by the vaginal commensal flora. cecs may play a pivotal role in modulating the immunological tolerance necessary to minimise inappropriate inflammation in the cervix. there are several epidemiological and clinical studies that omega- fatty acids and related fish oils can decrease the premature labor and birth. however, the scientific mechanisms are not well elucidated. this study was carried out to investigate the effects of omega- fatty acids on producing pge and il- , in human umbilical vascular endothelial cell(huvec) media with artificial inflammation as an infection-induced premature labor tissue model. also, if there are some significant effects with omega- fatty acids, we want to investigate the possible mechanisms. materials and methods; human umbilical vascular vascular cell primary culture was done. in the adequate cell confluence in each cell dish, pretreatment with various concentrations of epa, dha and troglitazone (another ppar-ligand) and incubation were done for hours in °c, co incubator. after that, g/ml conc. of lipopolysaccharide(lps) was treated to the each cell dishes and incubated for hours. the cell media were collected, and pge and il- concentration were checked by elisa. the each cell lysates were collected and western blot anaysis for cyclooxygenase(cox)- were done. on the other hand, nuclear factor kappa b (nf b) luciferase vector was transfected and then did the same pretreatment with epa, dha and troglitazone and lps treatment to each cell dishes. after that, nf b luciferase activity was checked with luminometer. statistical analysis was done by student t-test. results: epa, dha and troglitazone decreased the pge (p< . ) and il- (p< . ) significantly in elisa. cox- expression revealed the significant reduction with pretreatment of epa, dha and troglitazone in higher concentration ( , m) in the western blotting (p< . ). nf b luciferase activity were also significantly decresed with pretreatment of epa, dha and troglitazone in higher concentration (p< . ). conclusion; this study offers some scientific mechanisms, by which omega- fatty acids (epa, dha) and troglitazone may be one kind of the preventive medicine for infection-induced preterm labor and delivery. also, these effects may come from the common mechanism of epa, dha and troglitazone, ppar-ligands. the next study would be how the cox- , nf b and pparare related. ( mg/ml). cytokine expression was measured in medium using the bio-plex suspension array system (bio-rad). mean expression was compared between the two groups using t test. results: -aminopurine significantly inhibited lps stimulated cytokine production by human myometrium. in contrast, there were no significant differences in expression after mpa treatment, although a trend towards inhibition of proinflammatory cytokine and an increase in il- production was noted. introduction: intrauterine infection is now recognised as a major antecedent of white matter injury (wmi) in the preterm infant brain, which can manifest later as cerebral palsy or as varying degrees of cognitive dysfunction. wmi in these infants is characterized by damage to immature oligodendrocytes, and has been linked both to microglial activation and to elevated levels of tnfa, il- b and il- . we have developed a fetal sheep model for diffuse and focal wmi, based on repeated administration of e. coli lipopolysaccharide (lps) as the stimulus for an inflammatory response. methods: surgery to implant fetal vascular catheters was performed on pregnant ewes carrying single fetuses at d - of gestation. fetuses received repeated iv injections of lps ( ng/kg estimated fetal weight/day) between d and d . plasma levels of pro-inflammatory cytokines were determined in fetal arterial blood samples taken between d and d . at d ewes and fetuses were euthanized and fetal brain tissue samples collected for histological analysis. results: five days after final administration of lps to fetuses we observed a pattern of wmi similar to that seen clinically, ranging from focal to diffuse injury within the periventricular region, impairment of white matter (cnpase; marker for immature/mature oligodendrocytes) tracts, and thinning of the corpus callosum, characterised by oligodendrocyte disruption and microglial proliferation in the surrounding and sub-cortical white matter. we also found a progressive rise in fetal plasma tnf levels between days and (day two of treatment to third day following final dose of lps). background: plasma fatty acid (fa) levels are associated with altered host inflammatory responses, blood pressure regulation, and insulin resistance, characteristic features of pregnancy-induced hypertension (pih). most studies compare the n- and n- polyunsaturated fatty acids (pufas). in addition, recent data demonstrate that saturated and trans-fas promote inflammation. based on the immunomodulatory activity of various fas, we explored their effects on placenta inflammatory responses ex vivo. methods: placenta cells were isolated from fresh human (anonymous), term placentas and treated with/without lipopolysaccharide (lps) with various fas, including saturated fas, trans-fas, and n- and n- pufas (at physiological concentrations). after an overnight treatment, tnf-alpha (tnf) production was determined. data were analyzed by analysis of variance (anova) followed by the dunnett's test. results: oleic acid ( : n- ), a cis-monosaturated fa common in the mediterranean diet, did not induce significant placenta tnf production (fig. a) . by contrast, elaidic acid ( : n- ), a trans-monosaturated fa, induced tnf production by -fold compared to vehicle (fig. a) . likewise, palmitic acid ( : ), a saturated fa, induced placenta tnf production by -fold (fig. a) . to mimic inflammation, placenta cells were treated with lps ex vivo in the presence/absence of fas. docosahexanoic acid ( : n- , dha) reduced placenta tnf production by up to % following stimulation (fig. b) . similarly, treatment of placenta cells with linoleic acid ( : n- , la) or arachidonic acid (n : n- , aa) suppressed tnf production by up to % and %, respectively (fig. b) . conclusions: both saturated fas and trans-fas, which positively correlate with hypertension, induce placental tnf production. the n- fa precursors to prostaglandins, aa and linoleic acid, reduce placental tnf production following stimulation. likewise, dha, a product of n- fa metabolism commonly consumed in fish oil and associated with improved blood pressure, suppresses tnf production by stimulated placenta cells. vegf and flk mediated glomerulogenesis in offspring exposed to maternal hypernatremia. roy z mansano, mina desai, ahmed abdel-hakeem, thomas r magee, tazmia q henry, cynthia nast, john s torday, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: growth restricted human and animal offspring, resulting from maternal nutrient restriction or uteroplacental insufficiency, consistently demonstrate reduced glomerular number and a predisposition to adult systemic hypertension and renal compromise. in contrast, maternal water restriction (wr) produces newborns with a unique programmed phenotype of increased glomerular number. glomerulogenesis is dependent, in part, on appropriately timed and quantified vascular development. as vegf and its receptor flk have crucial stimulatory effects on renal vasculogenesis, we hypothesized that maternal wr-induced morphologic renal changes are secondary to vegfmediated vasculogenic effects. methods: from day to term gestation, pregnant rats received either ad libitum food and water (control, n= ), or wr to produce an increment of meq/l in plasma sodium (n= ). following delivery, all dams received ad libitum food and water. at d after birth, offspring kidneys were extracted for mrna. vegf and its receptor, flk , mrna levels were determined using real-time rt-pcr (presented as fold difference normalized to s). at d after birth, offspring glomerular number were determined in formalin fixed m sections using histomorphometric analysis. values are means±se. results: wr offspring (day ) had higher glomerular number than control (wr ± , control ± per mm , p< . ). flk mrna expression was increased in wr offspring kidneys (wr . ± . , control . ± . , p< . ) as compared to controls. in contrast, vegf mrna expression in wr offspring kidney was comparable to control (wr . ± . , control . ± . , p= . ). conclusion:prenatally wr offspring demonstrate significantly increased glomerular number. as vegf recruits angioblasts to the developing glomerulus via its receptor flk , increased receptors (flk ) with the same level of the ligand (vegf) suggest that enhanced vasculogenesis may represent a putative mechanism for increased nephrogenesis in wr offspring. modulation of newborn vasculogenesis via vegf and flk expression may represent a therapeutic option for growth restricted offspring with decreased glomerular number. objective: maternal food restriction (mfr) during gestation (embryonic day to birth) reduces rat kidney glomerular number by % at weeks of age. undernutrition during human gestation leads to similar impaired nephrogenesis and increased hypertension in adults. renal development may be delineated into stages including ureteric bud branching, mesenchymal to epithelial transformation and glomerulogenesis. previously, we detected dysregulation of genes controlling nephrogenesis at gestational ages, e -e , suggesting that mfr has significant effects on ureteric bud branching. in the present study we evaluated the effect of this dysregulation on early nephron development in e fetal kidney explants from dams with mfr. methods: e fetal kidneys were collected from % mfr pregnant rats and ad libitum control rats (n= per group and time point), incubated in dmem/f k medium for , , , , and days, and fixed with % paraformaldehyde. kidneys were stained with fluorescein-labeled dolichos biflorus agglutinin (dba), images captured and terminal ureteric buds quantitated. all values are presented as mean ± sem. differences were considered significant at p< . . results: mfr ex-vivo kidneys at day demonstrated an initial moderate reduction in terminal branches versus control (c) samples (mfr: ± vs c: ± terminal branch ends per kidney). both mfr and control (c) kidneys demonstrated significant (p< . ) increases in branching in explant culture to maximum values at days (mfr: ± vs c: ± ). with increasing culture days, the percent reduction in mfr branching increased with terminal branch point decreases of % at day , % at day , % at day , % at day , and % at day (p< . , mfr vs c at day ). conclusion: kidney explant cultures from mfr treated fetuses display basal and culture-based decreased branching compared to controls. this decrease in terminal ureteric buds, in combination with our previous findings of dysregulation of branching-associated kidney transcription and growth factors, suggests mfr induces early (e ) dysregulation of branching as a major mechanism of the associated nephropenia. these results indicate that early programming events in kidney development induce nephropenia and renal disease in adults. intrauterine growth restriction (iugr) has important implications for the neonate not only at the time of birth, but also as an adult. in humans and animals with iugr, the elevated risk of developing hypertension is thought to involve an upregulation of the renin-angiotensin system (ras). however, the link between the intrauterine insult and enhanced ras is not known. a novel mechanism leading to hypertension has emerged recently involving two orphan g-protein coupled receptors (gcr and gcr ) and their endogenous ligands, succinate and -ketoglutarate. infusion of succinate into adult mice induces hypertension, an effect which was eliminated in gcr knockout mice or by pretreatment with ace inhibitors. interestingly, succinate levels have been shown to increase in the circulation under conditions of oxidative stress, one of the hypothesized mediators of developmental programming of hypertension. thus, we hypothesized that gcr and gcr are upregulated in a rat model of iugr and may contribute to in utero programming of hypertension. timedpregnant rats were fed either control (c, % casein) or low protein (lp, % casein) diet throughout gestation. kidneys were collected on embryonic day (e ), post-natal day (d ) or (d ), n . real-time pcr was used to compare gcr , gcr and renin expression. data were standardized to a housekeeping gene (s ) and are expressed as fold increase/ decrease compared to control. a student's t-test was used to determine significance between groups (p< . ). offspring from lp dams were significantly smaller at birth and did not display catch-up growth. in kidneys from lp fetuses, both gcr and gcr were significantly elevated compared with controls ( . fold and . fold respectively; p= . ), whereas renin was . fold lower. on post-natal d , there was a trend towards increased expression of both gcr ( . fold; p= . ) and renin ( . fold; p= . ) in offspring from lp dams. at d , there were no significant differences between groups. in conclusion, these preliminary data suggest that in iugr offspring from lp rats, the gcr / pathway may be enhanced, indicating a novel mechanism for the programming of hypertension. objective: in addition to excess adipose tissue, obesity is accompanied by increased fat storage in organs such as the liver. peroxisome proliferatoractivated receptors (ppars) are ligand-activated transcription factors involved in the regulation of lipid metabolism, and lipid-associated inflammatory response. obesity represents a state of chronic low-level inflammation, with ppar and ppar implicated in this process. we have previously shown that nutrient restriction in pregnancy results in intrauterine growth restricted (iugr) newborns which develop adult obesity with elevated c-reactive protein (crp) levels. as crp is derived from the liver, we hypothesized that iugr-induced obesity inhibition of hepatic ppar and ppar is associated with an increased inflammatory response. methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to to produce iugr newborns. all pups were nursed by control dams and weaned at weeks to ad libitum feed. at day and months of age, male offspring were analyzed for hepatic ppar , ppar and crp mrna (real time rt-pcr) and protein (western blot) expression. data was normalized to -actin and presented as fold change for protein levels. at months, hepatic triglyceride content was determined enzymatically. results: at d of age, iugr pups showed significant downregulation of ppar ( . -fold) and ppar ( . -fold) expression, though crp expression was significantly upregulated ( -fold). findings persisted at months of age, with continued downregulation of ppar and ppar ( . -fold) and upregulation of crp expression ( -fold). furthermore, iugr adults had significantly increased hepatic triglyceride content ( ± vs. ± mg/g liver, p< . ). conclusions: reduced expression of hepatic ppar and ppar in iugr offspring may contribute to elevated hepatic crp levels and triglyceride content. thus, developmental hepatic dysregulation leads to programmed obesity-induced inflammation in iugr offspring. offspring. mina desai, ederlen casillas, guang han, darran n tosh, , michael g ross. dept. of ob/gyn, labiomed at harbor-ucla med. ctr., torrance, ca, usa; dept. of physiology, univ. of adelaide, australia. objective: leptin and insulin mediate central anorexigenic signaling responses via different receptor molecules: leptin binds to the obrb receptor activating jak-stat and pi k pathways, whereas insulin activates pi k pathway by binding to its receptor (ir) and substrate (irs ). maternal food restriction in pregnancy results in iugr newborns that develop hyperphagia and adult obesity. the iugr newborns have significantly decreased plasma leptin levels with increased hypothalamic expression of basal obrb, stat and decreased expression of ir and irs , suggesting altered anorexigenic pathways. we studied the response of hypothalamic leptin/insulin signal molecules to peripheral leptin in iugr newborns. methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to . day old male offspring were given saline or leptin ( g/g, i.p). hypothalamus was dissected at , and minutes and protein expression of total stat , phosphorylated stat (p-stat ), inhibitor of leptin signal (socs ), ir, irs , total akt and phosphorylated akt was determined by western blot (normalized to -actin). data is compared between leptin and saline treatments in iugr and controls. results: in response to peripheral leptin, iugr newborns showed marked dysfunction in stimulated hypothalamic leptin and insulin signaling responses. ( ) jak-stat : leptin-treated controls show progressively increased pstat ( -fold) with initial suppression of socs ( . -fold) as compared to salinetreated controls. conversely in leptin-treated iugr, the pattern is reversed such that there is sustained decline in pstat expression ( . -fold) with failure to downregulate socs . ( ) pi k pathway: leptin-treated controls showed a significant reduction in irs ( . -fold) and pakt ( . -fold) as compared to saline-treated controls. however, leptin-treated iugr newborns exhibited a paradoxical increase expression of irs ( -fold) and pakt ( -fold). conclusion:the iugr offspring demonstrate persistent upregulation of leptin receptor, a reduced phosphorylated stat (p-stat ) response in conjunction with an enhanced socs- response. the persistent increase in insulin responses indicates a dysfunction in dynamic signaling, leading to altered anorexigenic response and development of programmed obesity. we have previously demonstrated that maternal food restriction (mfr) in rats induces a marked increase in the expression of vegf protein in the aorta and mesenteric arterioles, accompanied by an increase in tgf-and collagen in both vessel types in adult rat offspring (am j physiol, ) . the aim of this study was to determine if this in vivo finding could be reproduced in an in vitro preparation. methods: two types of preparations were used in this study. we isolated endothelial cells from week old male control rat aortas. these cells were used after the third passage. staining with von willerbrand factor demonstrated that these cells were pure endothelial cells. the second type of preparation was aortic explants from week old male control rats. endothelial cells and aortic explants were transfected with a vegf adenovirus ( - viral infective particles) or a -galactosidase-adenovirus as a control. after hours of culture protein was isolated from cells and explants for western blot analysis using rat specific antibodies. culture media was assayed for vegf by elisa. results: transfection of vegf adenovirus induced a dose-dependant increase in the expression of vegf protein in primary endothelial cells and aortic explants. the transfection of vegf into the endothelial cells showed a bell shaped curve, and was accompanied by an increase in media levels of vegf protein. maximal secretion of vegf was found with viral infective particles. vegf adenovirus transfection induced a dose-dependant increase in c-reactive protein (crp) (inflammatory marker), and tgf-protein in both aortic explants and primary endothelial cells. these results indicate that upregulation of vegf in blood vessels induces inflammation and tgf-expression which in turn can induce collagen synthesis. thus the increased collagen expression and reduced compliance previously reported by us in vessels of mfr offspring can be explained by the over-expression of vegf which we reported. therapeutic intervention aimed at prevention of the increase in vascular vegf expression in mfr offspring could potentially prevent programmed hypertension. maternal regulation of high fat nourishment during lactation period reduce a hypertension of male offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med.univ., fukushima, fukushima, japan. objective: exposure to undernutrition or high fat nourishment during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of maternal feeding regulation during lactation period on blood pressure of offspring is unclear. our objective was to investigate the effects of either high-fat diet (hfd) during gestation to lactation period or restrictive fed a hfd during lactation period on blood pressure in male rat offspring. we use types pregnant wistar rats as fed with normal nutrition (group a), with a high fat diet (hfd) during gestation to lactation period (group b) and with hfd nutritionally restricted by feeding with % of the normal lactationmatched dietary intake from the day of delivery to the end of lactation period (group c). the male offspring was measured blood pressure at , and weeks by using indirect tail-cuff method. statically analysis was performed using oneway annova. results: body weight was significantly reduced in c offspring compared to a and b in male offspring at day after delivery (p< . ). at weeks old, the body weight of c offspring was no difference to catch up compared to a and b offspring. systolic and diastolic blood pressures were significantly elevated at all , and weeks in offspring of b > c >a. (p< . , vs. a) conclusions: under high-fat nutrition during gestation to lactation period induced hypertension in male rat offspring. maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. background: uterine artery (uta) doppler velocimetry has been validated in populations of heterogeneous parity in the second trimester for prediction of obstetric outcomes requiring preterm delivery to include: fetal growth restriction, fetal demise, hypertensive disorders of pregnancy, abruption, and indicated preterm delivery. understanding that parity may affect uta doppler indices in subsequent pregnancies, we sought to validate these predictive values in the first trimester in a homogeneous population of multiparous women. study design: multiparous women undergoing first trimester screening of singleton pregnancies were enrolled and followed prospectively until delivery (n= ). these women were divided into controls, ri < . (n= ) and cases, ri . (n= ), based on prior studies. demographic, clinical, and sonographic data (including uta indices and assessment of notching) were obtained. statistical analysis included student's t test and chi square. results: cases were not significantly different from controls in terms of maternal age, ethnicity, bmi, or medical history. uta doppler indices were significantly different between the two cohorts in terms of the presence of unilateral or bilateral notching ( % vs, % p< . and % vs. %, p< . , respectively). in contrast to that observed in patients of heterogenous parity previously, ri . was not associated with adverse pregnancy outcomes despite an average ri of . , significantly above this threshold. conclusions: in this multiparous cohort ri was not predictive of adverse obstetrical outcome, in contrast to that observed in cohorts including nulliparous patient. parity may affect uta vasculature and obscure the ability of doppler velocimetry to predict adverse obstetric outcome in multiparous women. presence of uta doppler notching in the first trimester remained a robust predictor of adverse obstetric outcomes in multiparous patients. objective: epidemiological studies have shown that offspring exposed to preeclampsia during fetal development are more susceptible to airway disease later in life. we have shown previously that gender, but not sflt- over-expression during pregnancy determines higher reactivity in the offspring airways at months of age. the objective of this study was to examine the effect of preeclampsia on the trachea from female and male offspring in our model of sflt- induced preeclampsia at year of age and compare responses between the two age groups. methods: cd- mice at day of gestation were injected via the tail vein with adenovirus carrying sflt (adsflt , pfu/ l) or mfc (admfc, pfu/ l). mice were allowed to deliver. tracheas were isolated from female and male offspring at months and year of age, and rings were mounted in organ chambers for isometric tension recording. responses to potassium chloride (kcl, mm), the mast cell degranulating agent compound / ( / , g/ml), and concentration-responses curve to acetylcholine ( - - - m) were obtained. results: there was no significant difference in responses to acetylcholine, kcl, or compound / between year old offspring born to the sflt and mfc groups. when comparing offspring within the same pregnancy exposure groups, responses to acetylcholine in adsflt -treated group were significantly higher in year old females than males. comparison between age groups by pregnancy exposure revealed that in the mfc group, year old male offspring had higher responses to compound / and acetylcholine than months old males. responses to kcl were significantly higher in months old males than year olds independent of maternal treatment during pregnancy. in females, the only difference between age groups was observed in the mfc group, where months old offspring demonstrated significantly higher responses to acetylcholine compared to year old offspring. conclusions: our findings did not show that airways of year old offspring born to mice with a preeclampsia-like syndrome induced by sflt- over-expression have airway hyperreactivity. however, sex and age differences in airway responses dependent on maternal exposure during pregnancy were observed, and needs to be explored further to elucidate underlying mechanisms. objective: maternal food restriction (fr) results in iugr newborns that when normally nursed exhibit rapid catch-up growth and adult obesity. continued fr during nursing delays catch-up growth and prevents adult obesity. igf- , which modulates growth and is secreted by the liver, may contribute to these morbidities. igf- is epigenetically regulated involving two promoters, alternative exon splicing and multiple transcription termination sites. we determined if hepatic igf- mrna levels correlate with obesity, and whether these changes are due to programmed epigenetic modification. methods: control pregnant rats received ad libitum food from gestation day to and lactation, whereas study dams were % fr. fr pups were nursed by either control (fr/adlib) or fr dams (fr/fr) and weaned to ad libitum feed. at day and months, male livers were analyzed for igf- mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h k trimethyl, and associated levels of each igf- species were measured by pcr. results: at months, obese fr/adlib males showed increased mrna levels of igf- a, igf- b, igf- exon , and igf- exon as compared to controls ( ± , ± , ± , and ± %). comparing fr/adlib month to newborn offspring, h /k was increased at igf -promoter , promoter , exon , utr# and utr# ( ± , ± , ± , ± , and ± %), though there was no differences between control month and newborns. in contrast, month fr/fr males had comparable mrna levels to the controls except for igf- b (% of control: ± ). further, fr/fr month h /k was only different from newborns at utr# (% of newborn: ± ). conclusion: iugr newborns with rapid catch-up growth and adult obesity have increased postnatal hepatic igf- mrna levels, likely a result of igf- histone and chromatin structure modifications to h k trimethylation. conversely, iugr with delayed catch-up growth and absence of adult obesity have levels similar to that of controls. thus, modulation of the rate of iugr newborn catch-up growth may protect against igf- epigenetic modifications. introduction: igf-ii is synthesized as a pro-hormone (proigf-ii; -amino acid peptide) which is then processed into its active forms: "big" igf-ii ( - ) and mature igf-ii ( - ). these active forms are essential for placental and fetal development and have also been shown to persist into postnatal life. since maternal smoking is known to adversely affect feto-placental growth and postnatal development, we postulated that these effects might be mediated through nicotine-induced alterations in igf-ii processing. methods: in the present study, nulliparous female wistar rats ( - g) were given nicotine ( mg/kg/day) or saline for days prior to mating, during pregnancy, and throughout lactation. at gestational day , and , dams were euthanized and we collected serum (fetal and maternal), amniotic fluid and recorded fetal body weight. a subset of dams were allowed to deliver at term. following parturition, serum samples from the offspring were collected at birth (pnd ) and weaning (pnd ). body weight was recorded weekly from birth to weaning. pro, "big" and mature igf-ii levels were determined by western blot analysis. results: maternal nicotine exposure during pregnancy resulted in a significant reduction in fetal body weight by gestational day . however, there was no effect of nicotine on fetal serum or amniotic fluid igf-ii levels at any gestational age examined. in maternal serum, mature igf-ii in control animals decreased with advancing gestational age such that igf-ii levels were lowest at gestational day . nicotine administration prevented this decline, which resulted in significantly higher mature igf-ii levels in nicotine-exposed mothers at gestational day . in postnatal life, nicotine exposed offspring had significantly lower levels of "big" igf-ii expression at weaning (pnd ). conclusions: these data demonstrate that nicotine can alter the amount of the active forms of igf-ii in the mother and the newborn. dysregulation of maternal igf-ii occurs concomitantly with suboptimal fetal growth. results from this study suggest a mechanism by which maternal smoking causes impaired fetal growth and adverse postnatal health outcomes. objective: maternal food restriction in pregnancy results in iugr newborns which develop adult metabolic syndrome. programming of both increased appetite-mediated hyperphagia and enhanced adipogenesis contribute to the development of obesity. transcription factors, peroxisome-proliferatoractivated-receptor (ppar ), ccaat/enhancer binding-protein (c/ebp ), and sterol regulatory element binding-protein (srebp c) regulate adipogenesis and lipogenesis. although iugr offspring exhibit acute upregulation of the adipogenesis signaling cascade prior to the development of obesity, we determined if this increased adipogenic potential was an intrinsic cellular response, and thus maintained in cell culture. we further examined the responses to adipocyte stimulators (ppar activator-ligand rosiglitazone) and inhibitors (ppar repressor-ligand badge). methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to term. adipocytes from day old iugr and controls were isolated and cell proliferation rate was determined (mtt). primary adipocyte cell cultures were established and following % confluence, iugr and control adipocytes were treated to two doses ( and m) of either rosiglitazone or badge for h. mrna and protein was extracted for expression of ppar , c/ebp , srebp c. data was normalized to -actin and compared to the respective untreated cells. results: iugr adipocytes had significantly increased protein expression of ppar ( . -fold) and c/ebp ( -fold) as compared to control adipocytes, though srebp c levels were unchanged. mrna levels showed similar changes in iugr newborns. importantly, iugr adipocytes exhibited increased cell proliferation ( % of control, p< . ) and showed greater response to rosiglitazone ( . -fold), though similar response to badge, as the control adipocytes conclusion: iugr primary adipocytes cell culture exhibit basal phenotypic characteristic of programmed upregulation of adipogenic transcription factors which promote adipose cell proliferation. the enhanced response to the adipogenic stimulant is further evidence of the predisposition to obesity. in contrast, the normal suppressive response to the inhibitor suggests that iugr adipocytes may respond to pharmacologic approaches to prevent obesity during this period. objective: maternal nutrient restriction results in intrauterine growth restricted (iugr) newborns which develop programmed obesity despite a normal post-weaning diet. the epidemic of obesity has been attributed in part to programmed "thrifty phenotype" and exposure to "western" diets. hepatic igf- is epigenetically regulated involving two promoters, alternative exon splicing, and multiple transcription termination sites. iugr offspring with normal post-weaning diet have increased postnatal hepatic igf- mrna levels, likely a result of igf- histone and chromatin structure modifications to h k trimethylation. we hypothesized that iugr newborns that develop programmed obesity would demonstrate discernable hepatic igf- changes which are distinct from diet-induced obesity. we determined igf- hepatic mrna levels and epigenetic characteristics in programmed (iugr) and dietinduced (dio) offspring. methods:: control pregnant rats received ad libitum food whereas study dams were % maternal food restricted from day to . all pups were nursed on ad libitum fed dams. controls were weaned to high-fat (fat, %) diet whereas iugr were weaned to normal ad libitum diet (fat, %) to produce diet-induced (dio) and programmed obese groups, respectively. at months, male hepatic igf- were analyzed for igf- mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h k dimethyl and h k trimethyl, and associated levels of each igf- species were measured by pcr. result: relative to dio control males, iugr had increased mrna of igf- a, exon and exon ( ± , ± , ± %). chip with h k dimethyl showed increased igf- exon ( ± %) and with h k trimethyl, increased igf- promoter and promoter ( ± , ± %) as compared to dio controls. conclusion: adult obese iugr males exposed to normal postweaning diet have increased hepatic igf- a mrna and h k dimethylation and trimethylation of igf- than dio controls. changes in igf- in adulthood from a prenatal insult thus suggest that igf- is programmed during the fetal period and may be associated with programmed adult obesity. rebekah elkins, pandu gangula, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa; internal medicine, university of texas medical branch, galveston, tx, usa. objectives: we previously reported that the offspring of rats fed % protein during gestation develop hypertension at two to four months and that the hypertension is exacerbated in males. this study is to evaluate: ) changes in estrogen receptor (er) angiotensin ii subtype receptor (at -r) and endothelial nitric oxide synthase (enos) in the mesenteric artery and aorta of offspring and assess if these changes, if any, are gender specific. methods: pregnant sprague dawley rats were fed either % protein (ctrl) or % protein (lpd) from day of gestation. the offspring were evaluated for hypertension by means of systolic blood pressure measurements. at four months for the males and nine months for the females, mesenteric artery and aorta were collected in rnalater. expression of estrogen receptor a (er-a) and b (er-b), at -r, and enos were analyzed by western immunoblotting and rt-pcr and expressed relative to b-actin or s. results: mesenteric artery shows no differences between ctrl and lpd female offspring in at -r, enos, er-a or er-b. similarly mesenteric artery shows no diet exposure related changes in at -r, er-a or er-b in male offspring. however, enos expression was lower in mesenteric artery of lpd male offspring. on the other hand, in the aorta both er-a and er-b levels are lower in lpd female offspring while there were no changes in at -r or enos. no changes in at -r, er-a or er-b were observed in male offspring aorta of ctrl and lpd rats. conclusion: the in utero exposure to lpd results in adult hypertension in both male and female offspring. some mechanisms for hypertension include the decrease in er-a and er-b but not at -r or enos in females, and the decrease in enos but not at -r or er in males indicating gender related differences. offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. our objective was to investigate the effects of either severe undernutrition during late gestation or lactation period on blood pressure and the development of vascular function in male rat offspring. we use normal pregnant wistar rats (group a), nutritionally restricted by feeding with % of the normal gestation-matched dietary intake from day of gestation to delivery (group b) and % restricted after delivery to the end of lactation period (group c). the offspring was measured blood pressure at and weeks by using indirect tail-cuff method. rings of thoracic aorta with intact endothelium from the male offspring of a and b at weeks, were equilibrated at g passive tension in organ chambers filled with krebs-henseleit solution continuously bubbled with %co in air ( °, ph . ) for isometric tension recording. concentration-response relationships to norepinephrine (ne) and angiotensinii(atii) were obtained in the absence or presence of n(omega)-nitro-l-arginine methyl ester (l-name) or a selective atii type- receptor blocker (valsartan). responses to cumulative concentrations of sodium nitroprusside (snp) and to - m oxyhemoglobin (hb, nitric oxide scavenger) were also determined. contractions were expressed as a percent of the reference contraction induced by potassium chloride ( mm). statically analysis was performed using one-way annova. results: body weight was significantly reduced in b offspring compared to a and c in male offspring at day (p< . ). at weeks the body weight of offspring of b was no difference to catch up compared to a and c offspring. systolic and diastolic blood pressures were significantly elevated at both and weeks in offspring of b > c >a. ne concentration-dependently stimulated tension of aortic rings from in a and b offspring, which was not significantly (n= ). maximal contractions to ne were significantly stimulated by l-name in a (p< . ), but not b offspring. valsartan significantly inhibited aortic contractions by ne in r (p< . ), but not a offspring. there was no significant difference on responses of aortic rings by atii, snp and hb in a and b offspring. conclusions: severe under nutrition during not only late gestation but also lactation period induced hypertension in male rat offspring in adulthood. fetal origin of adult hypertension might be vascular endothelial dysfunction. fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. objective: exposure to undernutrition during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of either high fat nourishment or undernutrition during lactation period on blood pressure is unclear. our objective was to investigate the most effective maternal nourishment and feeding period for offspring induced adulthood hypertension in using high-fat diet (hfd). study design: we use types pregnant wistar rats as fed with normal nutrition (group a), nutritionally restricted by feeding with % of the normal gestation-matched dietary intake from day of gestation to delivery (group b), % restricted after delivery to the end of lactation period (group c), with a high fat diet (hfd) during gestation to lactation period (group d) and with hfd nutritionally % restricted from the day of delivery to the end of lactation period (group e). the offspring was measured body weight (bw) and measured blood pressure at , and weeks by using indirect tail-cuff method. statically analysis was performed using one-way annova. results: bw was significantly reduced in b offspring compared to another (a, c, d, e) male offspring at day (p< . ). at day after delivery, bw was significantly reduced in c, e offspring compared to a, d in male offspring (p< . ). at weeks old, bw of all type offspring was no difference. systolic and diastolic blood pressures were significantly elevated at weeks in offspring of d> b > e > c >a. (p< . , vs. a). at weeks, hypertensive offspring as b>d>e>c>a (p< . , vs. a). at weeks, d> e > b > c >a (p< . , vs. a). conclusions: maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. in case with normal food, restrictive feeding during late gestation is more effective than lactation period for inducing hypertensive male offspring. regulation of maternal feeding not only during late gestation but also lactation period may control adulthood hypertension. the strongest epigenetical factor of maternal nutrition is high fat feeding during pregnancy to lactation period for f blood pressure, respectively. or residence at high altitude, impacts fetal growth and development. in a preliminary study, we observed a significant decrease in birth weight, subsequent compensatory postnatal growth, and an increase in relative right ventricular (rv) weight at postnatal (pn) day in female offspring of rats exposed to hypoxia ( , ft; . % po ) from days thru of gestation (dga). thus, our objective was to further elucidate the impact of prenatal hypoxia on fetal growth and postnatal development. methods: pregnant dams (hx, n= ) were hypoxic from - dga with additional control dams either fed ad libitum (al, n= ), pair-fed with the hx dams throughout gestation (pg, n= ), or only pair-fed during the window of hypoxia (ph, n= ). female offspring from hx, pg, and ph dams were cross-fostered onto additional al dams (n= /litter) by h after birth. results: at birth, there was no difference in litter size; however, body weight (bw) of the hx, pg, and ph pups was lower (p< . ) than that of al pups, and hx pups were lighter (p< . ) than ph pups. weight of hx offspring remained lower (p< . ) than al pups until the termination of the study at pn , while the pg and ph pups reached weights comparable to the al offspring by pn . relative to bw, heart weight and left ventricular/septal (lvs) weight was not different among groups; however, right ventricular weight (rv/bw) was greater (p< . ) in the hx offspring at pn as was rv/lvs (p< . ). cardiac function was evaluated by echocardiography at pn . rv wall thickness was % greater (p< . ) in hx pups as compared to al pups, confirming the significantly higher relative rv weight observed at necropsy. pep, pep/at, and pep/et were %, %, and % higher respectively in the hx offspring relative to the al offspring. lv end diastolic and end systolic diameters were smaller (p< . ) in hx and ph offspring relative to the al group. myosin heavy chain (mhc) and mrna concentrations in the rv were evaluated by qrt-pcr, and the mhc / mrna ratio was greater (p< . ) in the hx pups. conclusion: prenatal hypoxia from - dga impacted both fetal and postnatal growth, altered postnatal heart development and function, with the primary impact being on the rv. supported by nih hd . reproductive sciences; pharmacology experimental therapeutics, university of maryland, baltimore, md, usa. background: exposure to nicotine (nic) is a significant risk to normal fetal development. fetal nic, which readily crosses the placenta, can be acquired from pregnant mothers by smoking or nicotine replacement therapy. the impact of nic on fetal organs may be mediated directly and/or via intrauterine hypoxia (hpx) via constriction of the uterine circulation. in adult hearts, both nic and hypoxia stimulate gene expression of matrix metalloproteinases (mmp), although the study of nic and hypoxia on gene expression in fetal organs remains incomplete. because mmps are involved in the regulation of extracellular matrix turnover and cardiac remodeling, we tested the hypothesis that prenatal nic and intrauterine hpx upregulate protein expression and activity of mmp in the fetal guinea pig heart. methods: pregnant guinea pigs were placed in either normoxia (nmx) or hpx ( . %o in chamber) for d prior to term ( d). in two separate groups, nic was also added to the drinking water ( mg/kg/d) for d at a dose that generates fetal nic levels ( ng/ml cotinine) equivalent to a moderate smoker. anesthetized near-term fetuses ( d) were excised and weighed. left ventricles of hearts were obtained and frozen at - c for storage. mmp protein levels and enzymatic activity were measured by western analysis and gel zymography, respectively. results: nic alone (nmx+nic) decreased (p< . ) fetal body wt by %, increased (p< . ) the relative fetal brain wt (brain wt/fetal body wt ratio) by . % and had no effect on relative placental or fetal heart wts. hpx alone decreased (p< . ) fetal body wt by . %, increased the relative fetal brain wt by % but, in contrast to nic alone, increased relative placental wt by . %. both mmp protein levels (mmp /a-actin density values) and activity (clear band density) were increased (p< . ) by nic alone (by . and . fold, respectively) and hpx alone (by . and . fold, respectively). in addition, both protein and activity levels of hpx hearts were further increased by nic (by . and . fold) compared to hpx alone. conclusion: prenatal nic upregulates mmp expression in nmx fetal hearts and is potentiated by hpx. this suggests that under conditions of intrauterine stress cardiac remodeling by mmp activation may be an important mechanism by which nic and hpx affect fetal heart function. objective: in addition to peripheral hypoglycemic effects, insulin induces central anorexigenic responses via stimulation of the phosphoinositide- kinase (pi k) pathway and cellular growth by mitogen activated protein kinase (mapk) pathway. the pi k signaling cascade is activated by insulin binding to its receptor (ir), recruiting ir substrate (irs- ), and phosphorylating pi k. activated pi k in turn causes phosphorylation of protein kinase b/akt which subsequently modulates hypothalamic anorexigenic responses. in contrast, the mapk (erk /erk ) signaling pathway likely involves irs- . further, insulin signaling is inhibited by the lipid phosphatase pten. we have previously shown that maternal food restriction (mfr) during pregnancy results in iugr newborns that develop hyperphagia, obesity and insulin resistance as adults. we sought to determine if altered hypothalamic basal insulin signaling expression of pi k and mapk pathways contribute to reduced satiety responses and thus enhanced growth in iugr newborns methods: pregnant control dams received ad libitum (n= ) food, whereas study dams were % mfr (n= ) from pregnancy day to to produce iugr newborns. at day , hypothalamic region was dissected and analyzed for mrna levels (real time rt-pcr) of insulin signaling components via pi k (ir, irs , pi k and akt) and mapk (irs , erk , erk ) pathways, and pten. data is presented as fold difference normalized to beta- -microglobulin. results: at d of age, iugr pups exhibited downregulation of the entire pi k pathway with significantly decreased (p< . ) mrna levels of ir ( . -fold), irs- ( . -fold), pi k ( . -fold) and akt ( . -fold). further, iugr pups showed similar decreased mrna expression of erk ( . -fold) and erk ( . -fold). however pten expression was similar to the controls. conclusion: reduced insulin-mediated pi k signaling likely contributes to the suppressed anorexigenic responses and development of obesity in iugr offspring. reduction of central mapk signaling suggests a potential maldevelopment of additional neuronal pathways in iugr offspring. objectives: in the rat, uteroplacental insufficiency restricts fetal growth and impairs mammary development further compromising postnatal growth. both male and female growth-restricted offspring have a reduced nephron endowment but only males develop hypertension with glomerular hypertrophy, which can be reversed by improving the lactational environment. this study used cross-fostering to assess the influence of the prenatal and postnatal environments on renal development and nephrogenesis. methods: bilateral uterine vessel ligation (restricted, r) or sham surgery (control, c) was performed on day of gestation in wky rats. control and restricted pups were cross-fostered onto c or r mothers on postnatal day (pn ). post mortem was carried out on pn (c and r) and pn (c-on-c, c-on-r, r-on-c, r-on-r). results: body and kidney weights were decreased in r and r-on-r pups on pn and pn (p< . ). there was some evidence of accelerated pup growth for r-on-c relative to r-on-r on pn . male, but not female, relative bmp mrna expression on pn was higher in r than c (p< . ) while gdnf, tgf and at receptor were not different. on pn , wnt (but not at r, vegf-a) mrna expression (males only) was relatively higher in r-on-r (p< . ) when compared to c-on-c (p< . ). this and the histological analyses suggests an up-regulation of nephrogenic activity with more immature nephrons (males and females) in r-on-r (p< . ) when compared to c-on-c, while r-on-c remained intermediate. intrauterine growth-restricted pups were born lighter and with smaller kidneys. this was partially rescued by improving lactational nutrition (r-on-c) at pn . higher bmp mrna expression indicates impaired branching morphogenesis in pn r male, but not female kidneys, suggesting the timing and/or molecular mechanisms underlying the nephron deficit may be sex specific. at pn there was evidence of extended and increased nephrogenic activity in r-on-r, however, this was unable to restore the later nephron deficit. improved lactation for r-on-c, which prevented the adult nephron deficit and hypertension, increased and extended nephrogenesis to a lesser degree than r-on-r suggesting that the restoration of nephron endowment was likely to have occurred prior to pn . amy m tetrault, sarah b lieber, marya shanabrough, tamas l horvath, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa. objective: classically recognized for its role in energy balance, body weight and appetite, ghrelin has also been implicated in reproduction. ghrelin (-/-) mice are infertile while administration of ghrelin to wt mice results in decreased litter size and constrained embryonic growth. here we investigate the effect of maternal ghrelin deficiency on in utero developmental programming of the female reproductive tract. hox genes determine developmental identity of the paramesonephric duct. we determined that hoxa is regulated by ghrelin in vitro and that in utero ghrelin deficency alters f hoxa gene expression and reproductive success. methods: wild-type females mice parented by ghrelin +/-b d f (ghrellin deficient) mice were analyzed for litter size, oocyte, and corpus luteum number. rna was extracted from the uterus of mice exposed to ghrelin deficiency in utero. ishikawa cells were treated with ghrelin with/without receptor (ghsr) antagonist, or saline and rna extracted. in both hoxa expression was analysed by real time rt-pcr normalized to -actin and also determined by ihc. experiments were repeated in triplicate and mrna expression compared by student's t-test. results: wild-type female offspring of ghrelin deficient dams had smaller litter sizes than controls (n= , . ± . pups; n= , . ± . pups, respectively; p< . ). no differences were seen in oocyte or corpus luteum number suggesting a uterine defect. hoxa mrna and protein expression were decreased in the uterus of the f females. ghsr was expressed in uterine endometrium. treatment of ishikawa cells with nm to nm ghrelin resulted in a to % increase (p< . ) in hoxa expression. treatment with ghrelin and ghsr antagonist resulted in similar increases in hoxa expression indicating a non-receptor mediated mechanism. conclusion: ghrelin contributes to reproductive tract developmental programming; in utero ghrelin deficiency compromises reproduction in female offspring. the developmental effects of ghrelin were mediated by alteration in hox gene expression and not through the classic ghsr receptor. obesity and decreased ghrelin may lead to defects in developmental programming of the reproductive tract. these findings demonstrate the importance of nutrition, energy utilization and appropriate ghrelin levels on normal uterine development. we have previously studied the deleterious effects of lack of the endothelial nitric oxid synthase (nos ) in mouse dams and their offspring. our laboratory demonstrated that adaptive responses in subsequent pregnancies may offset the harmful effects of the genetic deficiency of nos . in this study we aimed to determine hepatic and renal histopathologic damage in nos deficient pregnant mice comparing animals carrying their first versus their second pregnancy. study design: gravid nos +/+wt and nos -/-ko mice during their first (p ) or second (p ) pregnancy were sacrificed at day of gestation. livers and kidneys were stained and analyzed for the presence and extent of histopathologic lesions. results: nos +/+wt dams displayed a low incidence of significant renal or hepatic lesions in either the first or the second pregnancy. in nos -/-ko mice the incidence of liver necrosis and inflammation during the first pregnancy was % and %, respectively. in nos -/-ko dams sacrificed during the second gestation the incidence rates for the same lesions were % and %, respectively (p< . ). this correlation persisted when we analyzed the relative severity of hepatic lesions between p and p animals. although a similar trend was observed, the difference between p and p animals with regards to kidney lesions did not reach the level of statistical significance in our study. conclusions: a second pregnancy in this animal model of hypertension was associated with a significantly improved hepatic histopathology compared with the first pregnancy. this observation is consistent with our previous studies showing a decrease in systemic vascular resistance in p versus p nos -/-ko mice. the beneficial effects of a prior pregnancy may partially underlie the phenomenon of a decreased risk of preeclampsia in multiparous versus nulliparous women. further studies are required to delineate the counterregulatory mechanisms leading to improved cardiovascular function in subsequent pregnancies in these genetically modified animals. maternal hypomethylation is associated with congenital heart defects in down syndrome. lmjw van driel, , r de jonge, wa helbing, bd van zelst, j lindemans, eap steegers, rpm steegers-theunissen. , , , obstetrics gynecology; pediatrics; clinical chemistry; epidemiolog y biostatistics; clinical genetics; erasmus university mc, rotterdam, netherlands. background: maternal age and hyperhomocysteinemia are risk factors for having a child with down syndrome (ds) and congenital heart defects (chds), respectively. evidence is rising that ageing is associated with a state of hypomethylation. objectives: to investigate whether the risk of a child with ds and chd is associated with maternal hypomethylation. methods: we conducted a case-control triad study at months after the index-pregnancy. case-children (n= ) were included if they had ds and chd. children (n= ) without a major congenital malformation served as controls. the concentrations of s-adenosyl methionine (sam), s-adenosyl homocysteine (sah), sam/sah ratio, and homocysteine in maternal blood were measured as biomarkers for methylation. the data were analyzed using the mann-whitney u test and a logistic regression model. results: maternal age was included in the model as potential confounder. the levels and the crude and adjusted or( %ci) of the biomarkers are shown in table . an increase of the sam/sah ratio with unit decreases the risk of a child with ds and chd with percent. moreover, every increase of mol/l of homocysteine . fold increases this risk. conclusions: maternal hypomethylation is significantly associated with an increased risk of having a child with ds and chd. since, the effects are confounded by maternal age, hypomethylation can be considered as feature of ageing. the developmental origins hypothesis postulates that during critical ontogenetic periods, transient environmental stimuli perturb developmental pathways and induce permanent changes in gene expression, metabolism, and chronic disease susceptibility. one likely mechanism is via early nutritional influences on epigenetic gene modification consisting of the presence of a methyl group on the carbon of a cytosine residue. this modification is responsible for an important form of gene regulation in eukaryotes. in the present study, we have tested the hypothesis that maternal low-protein diet altered epigenetic regulation of specific gene of the offspring. c bl/ female mice were mated and on the day the plug was detected, these females were then randomly allocated to be fed isocaloric diets consisting % protein or % protein. at delivery, offspring were killed and the livers were removed immediately, frozen in liquid nitrogen and stored at - c. genomic sequencing after bisulfite modification is used to study site-specific dna methylation. dna methylation status of oct- and sphk- gene upstream regions in the mouse liver was analyzed. hepatic oct- or sphk- promoter methylation was not significantly different between both groups. however, dna methylation pattern of the genomic dna is specific in low-protein diet group. aberrant oct- and sphk- gene expression may cause perturbations in cell differentiation. we suggest that the epigenetic mechanism consisting of dna methylation underlies the fetal programming theory. primary human cytomegalovirus (hcmv) infection during pregnancy can have devastating consequences for both the mother and fetus. hcmv infection has been implicated in the development of pre-eclampsia and intrauterine growth retardation (iugr), as well as congenital cmv syndrome in newborns exposed in utero. previously, we have shown that hcmv infection of placental cytotrophoblasts inhibits their normal invasion, proliferation, and migration. however, the mechanisms occurring during early establishment of placental infection are largely unknown. we assessed the impact of hcmv infection on cytotrophoblasts by performing immuno-based assays for various cytokines and cellular growth factors. we detected significant cytokine dysregulation at both and hours after in vitro hcmv infection of cytotrophoblast cells. soluble cytokines involved in recruitment of monocytes and macrophages (gro-a, mcp- ) were downregulated at both and hours after infection. sdf- , which is chemotactic for lymphocytes during early inflammation, was also decreased. these results suggest that recruitment of cells involved in the anti-viral immune response is being interrupted early in the course of infection by hcmv. additionally, a large decrease in the amount of soluble hgf was seen. hgf normally induces migration of cytotrophoblasts along the invasive pathway, and downregulation of this factor could severely affect these processes. finally, we saw increased amounts of soluble icam- , contrasted by decreased amounts of vcam- , indicating dysregulation of adhesion molecules that are necessary for successful placental invasion to occur. all together, these data indicate significant alterations in cytokine profiles as early as hours after hcmv infection, which could provide important clues to the pathogenesis of hcmv in placental invasion and inflammation. additional studies will further elucidate this dysregulation, and determine whether these effects are due to alterations in pre-existing cellular factors or if transcriptional alterations are involved. method: studies were performed in pregnant ewes (n= in each group) with twin fetuses at - days (very preterm) and - days (near-term). neither mother nor fetuses were instrumented prior to the time of blood collection. maternal blood was collected from the jugular vein before sedation. anesthesia was then induced with isofluorane, the abdomen was opened, fetuses exteriorized and blood collected from umbilical cords. blood samples were transferred to plastic tubes containing ethylene-diamino tetraacetic acid and reduced glutathione, plasma separated and stored at - c. commercial radioimmunoassay kits for ovine-crf (phoenix pharmaceuticals, b : . ± . pg/ml) and cortisol (diagnostic laboratory, b : . ± . ng/ml) were used according to the manufacturer's instructions. results: in very-preterm gestations, maternal and fetal plasma crf levels were undetectable. maternal, but not fetal, plasma cortisol levels were measurable ( ± ng/ml). in near-term gestations, both cortisol and crf were measurable in maternal (crf: ± pg/ml; cortisol: ± ng/m) and fetal plasma (crf: ± pg/ml; cortisol: ± ng/ml). plasma crf levels were higher in nearterm fetuses than in their maternal ewes (p < . ). conclusion: ovine plasma crf levels are measurable in maternal and fetal plasma in near-term but not very-preterm gestations. the absence of crf in preterm plasma, perhaps due to reduced placental expression and/or placental crf release, may contribute to the rarity of in utero meconium passage in preterm gestations. interleukin - objectives: interleukin- (il- ) is a pro-inflammatory cytokine produced in adipose cells. recent studies suggest il- may be a marker of maternal obesity and in utero fetal programming. our hypotheses were ) il- correlates with maternal obesity and ) il- mediates the effect of maternal obesity on infant birth weight. methods: the parity, inflammation, and diabetes (pid) study is a longitudinal study of adipokine levels in a diverse sample of pregnant women. we present a cross-sectional analysis of first trimester il- levels from non-diabetic women who underwent a live birth. the independent variable was il- (pg/ml), measured with monoclonal antibody elisa assays. the dependent variable was infant birth weight (gms). maternal bmi categories were: normal/underweight (< kg/m ); overweight ( - kg/m ); and obese (> kg/m ). data on demographic and clinical factors, nutrition and physical activity were collected at baseline. average il- levels were compared across bmi categories using anova. the association of il- levels with infant birth weight was estimated using multiple linear regression, adjusting for covariates. results: average il- levels were significantly higher in obese women ( . ± . ) compared to overweight ( . ± . ) and normal/underweight women ( . ± . ) [p< . ]. after adjustment, il- levels was positively correlated with pre-pregnancy bmi [regression coefficient (rc) . ; % ci: ( . , . )]. as shown in the table below, elevated levels of il- were statistically significantly associated with a . gm higher infant birth weight after full adjustment. each unit increase in pre-pregnancy bmi was associated with a gm higher infant birth weight. table . association of il- with infant birth weight characteristic regression coefficient % confidence interval interleukin- . . , . pre-pregnancy bmi . , gestational weight gain - - , bmi = body mass index; coefficients adjusted for demographics, clinical factors, pre-pregnancy bmi, gestational weight gain, non-fasting first trimester glucose levels, gestational age, nutritional intake and physical activity conclusion: il- and pre-pregnancy bmi were associated with infant birth weight after adjustment for covariates. our findings suggest that the effect of maternal obesity on infant birth weight may be mediated through il- or an alternative independent pathway. we have demonstrated that -tetrahydrocannabinol (thc), in physiologically relevant concentrations, inhibits the growth and tight transcriptional control of the bewo trophoblast cell line . the mechanism involved the decreased expression of the transcriptional regulator histone deacetylase (hdac ). in these experiments we sought to answer the question, 'does anandamide (aea) work in the same manner as thc?' methods: the first trimester human trophoblast cell, bewo were plated at or x cells /well to -well and -well plates for growth and rna experiments, respectively. after growing to - % confluence, cultures were treated with varying concentrations of aea up to a maximum of m for hr. cell numbers were determined using the xtt apoptosis/proliferation assay. total cellular rna was prepared and the relative levels of hdac and gapdh determined by end-point rt-pcr. aea exhibited an inhibitory effect on the bewo cell cultures, but only at concentrations in excess of m where confluency was significantly reduced from % at m to - % at m and m (*p< . ; one-way anova with tukey's hsd test; n= ). cultures treated with m and m aea did not exhibit increased cell death or failure to attach to the substratum, as evidenced by the lack of increase in the shedding of cells into the spent medium. bewo cells treated with aea showed a dose-dependent decrease in hdac mrna expression with a significant effect at . m (*p< . ; oneway anova with tukey's hsd test; n= ). at this dose, the effect of aea had reached an effective maximum decrease in hdac mrna levels ( %) because hdac mrna levels were not decreased further by either m aea ( %) or m aea ( %). the alteration of hdac gene expression by aea in bewo cells with its associated decrease in cell number suggest that the trophoblast cell may be an important target for circulating endocannabinoids during the st trimester of pregnancy. the data indicates that although exocannabinoids and endocannabinoids both inhibit bewo cell growth, they do so using different transcriptional mechanisms. further understanding of the mechanism(s) by which aea alters placental physiology may lead to new strategies for the prevention of pregnancy complications such as st trimester miscarriages. ( ) taylor background: anandamide (aea) exerts its effects by acting on two cannabinoid receptors, cb and cb with the main regulator for aea levels being the metabolising enzyme, fatty acid amide hydrolase (faah). aea, cb , cb and faah constitute the endocannabinoid system and previous studies have shown that faah and cb are expressed in term human placenta( ) suggesting that the endocannabinoid system might be present earlier in gestation. this study aimed to document changes in faah, cb and cb expression in the placenta during the first trimester of pregnancy. methods: first trimester samples ( to weeks gestation) were fixed in % neutral-buffered formalin for days before embedding into paraffin wax or frozen in liquid nitrogen for rna analysis. for immunohistochemistry, faah polyclonal antibodies were used at an optimal dilution of : in pbs, cb at : and cb at : . transcripts were measured using q-pcr with gene-specific primers. results: immunoreactive faah, cb and cb were detected in all samples. faah immunoreactivity in the syncytiotrophoblast increased between the th and th gestational week and by week faah was barely detectable within large parts of the placenta. simultaneously, faah immunoreactivity increased in the mesenchymal core of the developing villi. immunoreactive cb and cb localised to the syncytiotrophoblast, cytotrophoblast and mesenchymal core with cb immunoreactivity showing diminished intensity after week , although this did not reach significance at the transcript level. cb immunoreactivity was absent from fetal blood cells and infiltrating maternal plasma cells, whereas cb and cb immunoreactivity was detected in endothelial cells but not in the vascular smooth muscle cells of blood vessels. the intensity of cb immunoreactivity in the syncytiotrophoblast differed from that of cb and faah in that it remained constant throughout. conclusion: the data suggest that placental faah and cb levels do not alter significantly during the first trimester, but alter their cellular distribution from the syncytiotrophoblast to the mesenchymal core. the significant loss of cb expression from the syncytiotrophoblast after the th week of gestation, a point of critical alteration in the developing placenta, suggests that its retention may be detrimental to normal placental development. reference: park, b. et al., ( ) hankins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: numerous angiogenic proteins synthesized in the placenta are thought to be involved in placental vascularization and development; however, the molecular mechanism modeling the angiogenic process in early pregnancy remains elusive. calatonin gene-related peptide (cgrp) is a multifunctional peptide expressed at the human implantation site; but its influence on in vitro angiogenesis by human micro vascular endothelial cells is not known. objective: the present study was designed to determine the influence of cgrp on angiogenesis by human dermal microvascular endothelial cell (hdmvec) in vitro. methods: hdmvecs (vec technologies) were cultured in mcdb- complete solution containing cgrp ( - m), cgrp plus its antagonist cgrp - ( - m), in well plates with x cells per well. the existence of cgrp receptor components calcitonin receptor-like receptor (crlr) and receptor activity modifying protein (ramp ) was determined using immunofluorescent staining. cell proliferation was examined using methylthiazoltetrazolium (mtt) assay. the pro-angiogenic bioactivity of cgrp was evaluated using cell migration and capillary like tube formation on the matrigel. results: ) immunofluorescent staining showed that cgrp receptor components crlr and ramp are abundantly expressed by hdmvecs. replacement of the primary antibodies with preimmune serum resulted in a negative staining; ) cgrp dose-dependently ( - to - m) stimulated hdmvec proliferation, and this effect was totally blocked by cgrp antagonist, cgrp - ; ) quantitative analysis for cell migration revealed that cgrp increases hdmvec migration in a dose and time-dependant manner; and ) cgrp promotes hdmvec capillary like tube formation, and the length of capillary tube induced by cgrp ( - m) was significantly increased over that of the untreated controls. this increase was observed at hours of treatment and further increase was noted at hours of culture. conclusion: cgrp induces in vitro angiogenesis by promoting microvascular endothelial cell proliferation, migration and capillary like tube formation. therefore, trophoblast derived cgrp at the implantation site may play a role in placental angiogenesis and fetal growth. over-expression of socs- gene promotes il- production by jeg- trophoblast cells. qin dong, , ruping fan, yang gu, david f lewis, yuping wang. biochemistry, harbin medical university, harbin, heilongjiang, china; obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: suppressor of cytokine signaling- (socs- ) plays an important role in negative regulation of inflammatory response in biological cells. evidence has shown anti-inflammatory cytokine il- expression was significantly reduced in trophoblasts of preeclamptic (pe) placentas. we sought to determine if over-expression of socs- in placental trophoblasts could promote il- production. methods: full-length socs- open reading frame (socs- cdna) was generated by rt-pcr from total rna samples isolated from human leukocytes and cloned into a pzsgreen -n vector, which encodes a green fluorescent protein zsgreen . successful socs- cloning was confirmed by sequencing. for transfection, jeg- cells were placed into well/cluster plates at a concentration of . x /well. the tranfection was carried out with . g of socs- /zsgreen plasmid (psocs- /zsgreen ) for hours when cell reached - % confluence. siport lipid were used. jeg- cells transfected with zsgreen plasmid (pzsgreen ) only was used as control. after approximately hours of transfection, cells were treated with il- at and ng/ml. medium was then collected and measured for il- by elisa. il- production was calculated as the percentage of increase by psocs- /zsgreen transfected cells compared to the cells transfected with pzsgreen only. result: il- production was increased by psocs- /zsgreen transfected jeg- cells compared to the cells transfected with pzsgreen only when stimulated by il- , control: . % increase; ng/ml il- : . % increase and ng/ml il- : % increase, p< . , respectively. data are means from three independent experiments. conclusion: over-expression of socs- gene could promote il- production by placental trophoblast cells. reduced sil- r release and increased ratio of sgp /sil- r production by placental tissues from women with preeclampsia. shuang zhao, ruping fan, jingxia sun, yang gu, david f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa; obstetrics and gynecology, first hospital, harbin medical university, harbin, heilongjiang, china. objective: placental tissue/trophoblasts release more inflammatory cytokines (il- , il- and tnf ) in preeclampsia (pe) than in normal pregnancies. however, the reason for increased inflammatory cytokines released by pe placentas is not clear. soluble il- receptor (sil- r) and membrane receptor il- /gp complex play an important role in the negative regulation of cytokine signaling in suppressor of cytokine signaling (socs) pathway. in contrast, soluble gp (sgp ) is an antagonist for il- /il- r trans-signaling. this study was undertaken to determine sil- r and sgp production by villous tissues from normal and pe placentas. methods: placentas delivered by normal and pe pregnant women were used in this study. placental explants were incubated with dmem for h. the culture medium was collected. placental villous tissue productions of sil- r and sgp were measured by elisa. all samples were assayed in duplicate. data are expressed as mean ± se and analyzed by mann whitney test. a p level < . was considered statistically different. results: placental tissues from pe produced significantly less sil- r than tissues from normal pregnancies, . ± . vs. . ± . pg/mg of wet tissue, p< . . soluble gp production was relatively compatible between pe and normal placental tissues: . ± . vs. . ± . pg/mg of wet tissue. the ratio of sgp /sil- r release was significantly higher in pe than in normal placentas, . ± . vs. . ± . , p< . . conclusion: reduced sil- r production and/or increased ratio of sgp /sil- r production by pe placentas suggest less cytokine inhibitory activity in pe placentas, which may contribute to the increased toxic cytokine production in placentas from pe. using chemiluminescence immunoassays. peter s uzelac, jing dai, frank z stanczyk, daniel r mishell, jr. obstetrics and gynecology, university of louisville, louisville, ky, usa; obstetrics and gynecology, university of southern california, los angeles, ca, usa. objective: characterizing human chorionic gonadotropin (hcg) levels throughout normal pregnancy is critical to its use as a bio-marker for abnormal gestations. there is a paucity of data describing hcg trends during pregnancy and most of the relevant studies use older, less specific assays. our first objective was to characterize hcg levels throughout normal gestation using two different contemporary chemiluminescence immunoassays. our second objective was to compare hcg patterns in healthy gestations with pregnancies affected by diabetes, a common obstetrical complication. methods: a single blood sample was collected from healthy pregnant women and diabetic pregnancies. gestational age was confirmed by ultrasound. serum hcg levels were quantified by chemiluminescence immunoassays using the acs- and immulite systems. data was grouped in -week intervals until weeks of gestation and -week intervals thereafter, with to samples in each interval for healthy women and to samples in each interval for diabetic women. paired t-test and wilcoxon rank sum test were used for statistical analysis. results: using the acs- system, mean hcg levels (miu/ml) for healthy pregnant women were , at - weeks, , at - weeks, , at - weeks, , at - weeks, , at - weeks, and , at - weeks. mean hcg levels obtained from the immulite system were similar. for diabetic pregnancies, mean hcg levels (miu/ml) were , , , , , , , , , and , , respectively. between - weeks of gestation, the hcg levels were significantly lower in diabetic pregnancies (p= . ) compared to healthy controls. ) compared to healthy pregnancies, diabetic gestations have significantly lower peak hcg levels. the cause of this difference is an area deserving further investigation. background: mouse and human placentae share several cellular and molecular features, including a haemochorial interface, allowing the murine labyrinth to be compared with the human fetal placenta. there is an autonomous embryonic ras from at least the time of implantation. ace converts angiotensin i to the active angiotensin ii (angii). angii's actions as a pro-inflammatory agent, in promoting cell migration, angiogenesis and cellular growth and apoptosis, strongly suggest a rôle in placentation. hypothesis: there would be counterregulation of the placental ras if the effect of ace were removed. this study investigated for the first time whether the knockout of somatic ace affected the expression and localisation of various components of the ras in the placentae of wild-type (wt/wt), heterozygous (wt/ ) and ace knockout ( / ) mice. methods: immunohistochemistry (dako envision plus) was used to localise and semiquantify ang type receptor (at r), ang type receptor (at r), ace, and ace- in the three genotypes (n= /group). placental sections were blinded to genotype; a score range of - was used. the test was used (spss version . ) to analyse the difference in staining score by genotype. results: immunoreactivity of all antigens increased in the placental labyrinth of / mice compared to wt/wt and wt/ mice (at r p< . ; at r p<< . ; ace p<< . ; ace- p<< . ). at r and ace- displayed increased staining in the fetal vascular endothelium of / mice (at r p<< . ; ace- p= . ), and in the cells lining the maternal central artery (at r p<< . ; ace- p<< . ). ace- expression was very high in cytotrophoblast lining the maternal blood space in all genotypes. no gross structural differences were seen. comment: the antibody used did not differentiate between ace and the membrane-bound "testicular"-ace (tace). immunohistochemically-identified ace expression was upregulated in / placentae despite the loss of somatic ace, suggesting that t-ace can be expressed placentally. we believe this is the first demonstration of such expression. ace and t-ace are catalyticallysimilar in converting angi to angii. furthermore, angii acting via the at rs is vasodilatory and ace- catalyses production of the vasodilatory ang ( - ); placental blood flow was presumably well-maintained and pregnancy outcome is normal in / mice. it is generally accepted that prostaglandin production plays a crucial role in both term and preterm parturition, and recently the administration of alpha hydroxyprogesterone acetate has been shown useful in preventing preterm labor. what is not clear is the biochemical pathway of prostaglandin production during labor and what, if any, effect alpha hydroxyprogesterone acetate has on this pathway. in order to address this question, we used immunohistochemical staining techniques to evaluate the effect of treating human placental amnion and chorion decidua with alpha hydroxyprogesterone acetate. membranes from unlabored patients were obtained at cesarean section and immediately seperated into control and drug treated specimens. controls were from the same placenta and were subjected to all experimental procedures except for the addition of alpha hydroxyprogesterone acetate to the culture media. specimens were compared at zero, six, and twenty four hour intervals. at the appropriate time, each specimen was formalin fixed and then paraffin blocked. tissue sections were then mounted on slides which were immunohistochemically stained using appropriate primary and secondary antibodies and standard techniques. the slides were then analyzed via light microscopy for changes in staining of three enzymes involved in prostaglandin production--cyclooxygenase (cox- ), cyclooxygenase (cox- ), and -hydroxy prostaglandin dehydrogenase (pgdh). compared to control, the slides treated with alpha hydroxyprogesterone acetate had differing amounts of enzyme expression. cox- was relatively unchanged and pgdh was only slightly increased, but cox- was noticeably decreased in the treated slides. these results were time dependent. this data suggests that alpha hydroxyprogesterone acetate decreases prostaglandin production in fetal membranes primarily by downregulation of the cyclooxygenase enzyme. objectives: in the human placenta, proliferation, differentiation and fusion of cytotrophoblasts (ct) are essential events in the formation of the multinucleated syncytiotrophoblast, however the regulation of these processes is poorly understood. using an explant model of human first trimester placenta we have established that both igf-i and -ii enhance ct proliferation, differentiation and survival mediated via igf r signalling. we have also shown that non-specific inhibition of protein tyrosine phosphatases inhibits igf-mediated signalling in trophoblast; therefore, we have now used sirna-mediated knockdown to investigate the role of the tyrosine phosphatase shp- in this pathway. methods: amaxa nucleofector technology was used to deliver shp- or scrambled sirna ( nm) to bewo cells or first trimester villous tissue fragments. knockdown was confirmed by q-pcr and western blotting. transfected cells and tissue were maintained in culture for hours, then treated with igf-i or igf-ii ( nm) for a further hours before immunohistochemical (ihc) analysis for cell proliferation (ki , brdu) or apoptosis (m ). results: sirna-mediated knockdown of shp- in bewo cells ( % reduction on western blot) demonstrated that igf-induced proliferation was reduced from . ± . % to . ± . % (p< . , n= ). ihc analysis of tissue demonstrated that shp- is localised to ct. following knockdown ( % decrease by q-pcr), igf-i-and igf-ii-induced ct proliferation was decreased by . ± . % and . ± . % respectively (p< . , n= ). furthermore, the ability of igf-i-and igf-ii to prevent ct apoptosis (m staining) was reduced by . ± . % and . ± . % respectively (p< . , n= ) after shp- knockdown. conclusions: igf stimulation of cytotrophoblast proliferation is mediated by shp- . exogenous igf rescues cytotrophoblast from apoptosis, and this pathway is also shp- -dependent. villous endothelial cells. emily j su, zhi-hong lin, ping yin, scott reierstad, joy innes, serdar e bulun. obstetrics and gynecology, northwestern university feinberg school of medicine, chicago, il, usa. background: within the human vascular system, estrogens have been shown to enhance vasodilatation in both normal and abnormal endothelium. estrogenic function occurs by activation of one or both of two estrogen receptors, estrogen receptor-alpha (esr ) and estrogen receptor-beta (esr ). these estrogen receptors are expressed in a wide variety of tissues. within the vasculature, estrogen receptors regulate the expression of multiple vasodilator and vasoconstrictor proteins. specifically, esr has been shown to be critical in maintaining normal vascular physiology in a murine model, where esr knock-out mice demonstrate significant systolic and diastolic hypertension. we hypothesize that within placental endothelium, estrogen plays an important role in maintaining normal vascular function that is critical for normal fetal growth and development. methods: term placentas from uncomplicated pregnancies were obtained, and the decidua was removed. an iv cannula was inserted into the umbilical vein, which was perfused with a collagenase/dispase solution. the perfusate was collected and subjected to further purification. these cells were cultured in complete medium, and after the initial passage of these cells, purity was confirmed via immunofluorescence and flow cytometric studies. estrogen receptor expression was determined in these cells via western blotting. additionally, these endothelial cells underwent treatment with varying doses of estradiol ( - m to - m), and quantitative real-time pcr was performed thereafter for mrna levels of various genes important in prostanoid production. results: western blotting demonstrated that esr is the only estrogen receptor expressed within villous placental endothelial cells. estradiol induced cyclooxygenase- (cox- ) mrna levels -to -fold, as quantified by real-time pcr (p< . ). conversely, there was no effect of estradiol on cyclooxgenase- (cox- ). conclusion: these results suggest that estradiol and esr are important in mediating the balance of prostanoid production that is essential in maintaining placental vascular health. future studies will further delineate estrogenic effects on prostanoid production within placental endothelial cells in health and disease. supported by the smfm/aaogf scholarship award and the nih grant u -hd . previously we established that proteins secreted by the decidua promote the differentiation of extravillous trophoblasts (evt) from a proliferative phenotype (characterized by cx , her- and alpha integrin protein expression) to an invasive phenotype (characterized by her- and alpha protein expression). the ability of decidua-conditioned media (dcm) to induce trophoblast differentiation was inhibited in the presence of the her- receptor antagonist, ag . furthermore, dcm-induced jar cell migration was also attenuated in the presence of ag . thus, the purpose of this study is to define the role of her signaling in evt differentiation and invasion. methods: evt differentiation was assessed in placental villous explant outgrowths and jar cells using antibodies against markers of the proliferative and invasive phenotypes. trophoblast migration was assessed using jar cells in transwell migration assays. results: treatment of placental villous explants with egf, a her- ligand, resulted in the downregulation of her- and an upregulation of her- expression, as well as an induction of alpha integrin expression. pre-treatment of placental villous explants with ag blocked this effect. in the jar cell line, egf treatment mimicked the differentiation-promoting effects of dcm by downregulating her- and upregulating her- expression, effects that were both blocked when jar cells were pre-incubated with ag . in contrast to dcm however, egf stimulation did not induce jar migration. stimulation of jar cells with hb-egf, a her- /her- heterodimer ligand, induced jar migration in a dose-dependent manner. analysis of dcm using antibody arrays confirmed the presence of many members of the egf family including hb-egf. immunohistochemical assessments of placental villous explants verified the expression of her- in evt outgrowths and in jar cells; her- expression was not affected by stimulation with either egf or hb-egf. conclusions: her signaling is an important and necessary component of the invasive evt differentiation cascade. our data supports a role for her- signaling in the induction of the invasive evt phenotype. chandrasekhar thota, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: parathyroid hormone related peptide (pthrp) is expressed in trophoblast cells and may play a role in placental growth and function. studies conducted in pregnant rats using pthrp antagonist showed decreases in fetoplacental growth during mid gestation. objective: to assess the effects of pthrp silencing on the expression of growth factors in immortalized first trimester trophoblast cells (htr- /svneo cells). methods: htr- /svneo cells cultured at º c and %co in rpmi- medium supplemented with % fbs were transiently transfected with nm of three different sirna sequences of pthrp, si , auaccuaacucaggaaacuuu; s i , g a g c u g u g u c u g a a c a u c a u u ; a n d s i , caagauuuacggcgacgauuu. for control, a scrambled sirna sequence was used. at % confluency, cells from each well were split and transfected again in triplicates with respective sirna sequences. total rna was isolated using trizol reagent h after transfection, and protein was extracted using lysis buffer h after transfection. the isolated rna and protein were subjected to reverse transcription and polymerase chain reaction (rt-pcr) and western analysis, respectively, using primers and antibodies specific for pthrp, plgf, vegf and lif. the results are expressed relative to either s for changes in mrna expression or -actin for changes in protein expression. results: rt-pcr of total rna obtained from htr cells subjected to double transfection with sirnas for pthrp showed a significant decrease in pthrp expression. expression of growth factors plgf, vegf and lif showed decreases with all the three sirna sequences used compared to the scrambled sequence. western analysis of cell lysates obtained from htr cells subjected to transfection with sirnas for pthrp showed a significant decrease in protein expression of pthrp and vegf. however the protein expression for plgf, and lif decreased in cells transfected with only si sequence of pthrp. conclusions: our studies showed that transient transfection of htr cells with sirna for pthrp caused decreases in both mrna and protein expression of pthrp. our results further suggests that decrease in pthrp peptide in transfected cells resulted in a decrease in vegf, plgf and lif suggesting that pthrp may play role in regulating trophoblast cell functions. objective. maternal obesity poses an increased risk to the fetus during pregnancy, and has long term consequences for the progeny. we tested the hypothesis that maternal caloric excess effects growth-related gene expression changes in the murine placenta. methods. female c bl/ mice were fed a hypercaloric diet ( % fat, % sugar) or standard chow for six weeks prior to mating and throughout pregnancy. near-term (day gestation) the dams ( controls, overfed) were sacrificed. following placental rna extraction, we used the affymetrix mouse a_ . array to measure gene expression changes. we performed pathway analysis on regulated genes. results. maternal overfeeding was associated with a two-fold increase in body fat mass. probe sets, corresponding to genes showed differential expression (p < . ); twenty-seven of which were up-regulated, and ten down-regulated, as compared to the placenta of control fed dams. of note, several genes related to obesity, diabetes, dna methylation, and the tgf beta pathway were differentially expressed. conclusions. diet-induced obesity in mice was associated with altered placental gene expression, including genes involved in tgf beta signaling and dna methylation pathways. these findings may have important implications for placental growth and epigenetic regulation. (supported by usphs hd- , earnest eu framework , tommy's the baby charity, uk). objective. maternal dietary protein restriction has been shown to have deleterious effects on placental development, and has long-term consequences for the progeny. to comprehend more completely stress responses to maternal protein restriction, we measured gene expression changes in the mouse placenta. methods. pregnant fvb/nj mice were fed an isocaloric diet containing % less protein than normal chow ( % vs. % protein content) from embryonic day . (e . ) to e . . following placental rna extraction, we used the affymetrix mouse a_ . array to measure gene expression changes. we performed pathway analysis on the regulated genes, and used both qrt-pcr and immunohistochemistry to verify the results. results. the weights of the e . pups were decreased % (p< . ). probe sets, corresponding to genes, were regulated by protein restriction (p< . ); ninety-one being up-regulated and down-regulated. of particular note, several genes related to the p pathway were up-regulated. along with p itself, positive regulators of p (zmiz , jmy, hipk ) and genes activated by p (inpp d, cebpa) were induced. for selected genes we confirmed these results using qrt-pcr and immunohistochemistry. conclusions. by microarray analysis, we have described the genetic response to maternal protein deprivation in the mouse placenta. we observed that pups were growth restricted, and genes related to the p pathway were regulated. we propose a model through which intrauterine growth restriction is triggered, in part, by activation of the p pathway. (supported by usphs hd- and the sgi medical student grant to cpg). purpose: human placental villous tissue cultures have been underused in the study of placental drug disposition. thus we assessed the utility of this model by studying the effect of time in culture on the viability and integrity of the tissue and the, expression and function of proteins involved in the formation and efflux of -chloro- , -dinitrobenzene (cdnb) conjugate , -dinitrophenyl-s-glutathione (dnp-sg) as a model system for phase ii metabolism and cellular efflux. methods: placental tissue samples were obtained within minutes of cesarean deliveries following normal pregnancies in three patients. villous tissue was cultured in m medium to hr. at , , , , , and hr post culture, villous tissue was preincubated without or with atpase inhibitor sodium orthovanadate, exposed to μm cdnb, rinsed and incubated in buffer at °c to determine formation and efflux of dnp-sg, which was assayed by hplc. changes in expression of gstp - , abc transporter isoforms b , c and g (abcb , abcc , and abcg , resp.) were assessed by immunoblotting. lactate dehydrogenase (ldh) release, methyl tetrazolium thiazolyl blue (mtt) incorporation, and total tissue glutathione content were monitored up to hr. villous tissue morphology was assessed by immunohistochemistry. results: villous tissue structure and protein expression of glutathione-stransferase isoform p - (gstp - ) and abcg remained unchanged over hr in culture. expression of abcb and abcc , and total tissue glutathione decreased with culture time. ldh release was unchanged up to hr and increased at hr, while mtt incorporation remained constant to hr and decreased at and hr suggesting a decline in tissue integrity and viability at hr. however, dnp-sg formation, dnp-sg buffer/tissue ratio, and the extent of inhibition of dnp-sg efflux by sodium orthovanadate remained unchanged through hr. sodium orthovanadate decreased the dnp-sg buffer/tissue ratio by . ± . % (p< . ), consistent with inhibition of apical abc transporters. conclusions: these results support the use of this model to study the coordinated function of metabolizing enzyme gstp - and apical abc transporters in the formation and efflux of the model substrate dnp-sg. the model may be useful to study metabolism and transport of other compounds. syncytiotrophoblast. shauna f williams, ewa fik-rymarkiewicz, stacy zamudio, nicholas p illsley. obstetrics, gynecology and women's health, umd-new jersey medical school, newark, nj, usa. introduction: multiple inputs influence placental protein synthesis. nutritional, endocrine and metabolic factors have been implicated but its regulation has not been investigated. one of the factors shown to be associated with the inhibition of protein synthesis is hypoxia. the goal of this study was to determine the effects of hypoxia on a marker of placental protein synthesis. eukaryotic initiation factor (eif ) is a subunit of eif which is required for initiation of translation however when phosphorylated, eif is unable to participate in the assembly of the initiation complex. hypoxia has been shown previously to cause increased phosphorylation of eif . we hypothesized that hypoxia would increase the levels of phosphorylated eif in term syncytiotrophoblast, thus inhibiting protein synthesis. methods: primary syncytiotrophoblast cultured from term cytotrophoblast were incubated for hr in atmospheres of , , or % o or in the presence of the hypoxia-mimetic, dimethyloxalylglycine (dmog, . - . mm) in % o . cell extracts were analyzed by western blotting to determine the degree of eif phosphorylation. results: incubation in , , or % o did not increase eif phosphorylation relative to the % o control (n= , separate placental preparations). incubation in dmog concentrations up to . mm did not affect eif phosphorylation however incubation in . mm dmog increased eif phosphorylation by ± % (p < . , n= ). conclusions: contrary to our expectations, inhibition of protein synthesis via the eif regulatory pathway was not apparent except when induced by the highest concentration of dmog, consistent with severe hypoxia. thus while eif phosphorylation does occur, we did not observe changes at dissolved oxygen levels of %. these data suggest that a reduction in syncytial protein synthesis via the eif pathway takes place only under severe hypoxic stress. (supported by nih hd ). the increasing prevalence of overweight and obese women of childbearing age is a growing public health concern. the impact of maternal obesity on placental aa transport, which is essential for normal fetal development, remains poorly defined. there are three sub-types of the placental na-dependent system a transporter, snat , and which mediate neutral aa transport. snat is ubiquitously expressed in mammalian tissues and is likely responsible for the majority of placental system a activity. objective: to examine the impact of maternal obesity and over-nutrition on the fetal: maternal (f:m) aa ratio and placental protein abundance for snat . methods: nonpregnant ewes were randomly assigned to a control (c, % of nrc recommendations) or obesogenic (ob, % of nrc) diet from - to days of gestation (dg). under isofluorane anesthesia, maternal and fetal blood samples were collected for aa analysis by hplc from five twin bearing ewes in each dietary group. after euthanasia, placental cotyledonary (cot) tissue was separated from caruncular tissue, frozen in liquid nitrogen and stored at - c for western blot analysis. results: fetuses from ob ewes were % heavier (p< . ) than those from c ewes at dg ( ± vs. ± g). blood concentrations of asn, thr, cit, arg, tau, tyr, trp, val, phe, leu and orn were higher (p< . ), or tended to be higher (met and lys, p< . ) in ob than c ewes. in contrast, f:m ratios, for asn, ser, gln, his, gly, thr, cit, arg, b-ala, tau, ala, tyr, trp, met, val, phe, leu, orn and lys were reduced (p< . ) in ob compared to c ewes. snat content in cot tissue was reduced in ob when compared to c ewes ( . ± . vs. . ± . arbitrary units; p< . ). conclusions: maternal obesity in pregnancy reduced expression of placental snat protein and efficiency of placental aa transport in ewes, providing a mechanism whereby fetuses may mitigate excessive delivery of aa under conditions of maternal obesity and over-nutrition. decreased aa transport to the fetus may play a role in altered cellular structure and function. nih inbre p rr . early gestation utero-placental hemodynamics in an ovine model of fetal growth restriction. lucia dohnal, james s barry, henry l galan, randall b wilkening, russell v anthony. perinatal research center, university of colorado health sciences center, aurora, co. objective: fetal growth restricted (fgr) pregnancies, during late gestation, exhibit altered placental hemodynamics, and reduced capacity for o and nutrient transfer. it was our objective to examine utero-placental hemodynamics and o uptake during early gestation in an ovine model of fgr. methods: singleton-bearing ewes were instrumented with uterine artery flow probes, uterine venous and femoral artery catheters before being placed into a highambient temperature (fgr; n= ) or normothermic (con; n= ) environment at days of gestation (dga). maternal arterial and venous blood, uterine artery flow, heart rate, arterial pressure and respiration rate was collected until dga, at which time umbilical venous blood, fetal weight, placental weight and tissue were harvested. data reported here were analyzed by students t-test. results: maternal respiration rate ( . ± . vs . ± . breaths/min) and arterial po ( . ± . vs . ± . mmhg) were increased (p . ), whereas maternal heart rate ( . ± . vs . ± . beats/min), blood pressure ( . ± . vs . ± . mmhg) and arterial pco ( . ± . vs . ± . mmhg) were reduced (p . ) in fgr pregnancies. at dga, fetal weight was not different (p . ), but placental (total placentome) weight ( . ± . vs . ± . g) was reduced (p . ) in fgr pregnancies. while uterine artery (pregnant horn) flow ( . ± . vs . ± . ml/min) tended (p= . ) to be reduced in fgr pregnancies, relative uterine artery flow ( . ± . vs . ± . ml/min/g fetus; . ± . vs . ± . ml/min/ g placenta) was not different (p . ). uterine o uptake (mmol/min), relative uterine o uptake (ml/min/g fetus or ml/min/ g placenta) and uterine o extraction (%) were not different (p . ) between fgr and con pregnancies. at dga, umbilical vein po (mmhg), o content (mm) and o capacity (mm) were also not different between fgr and con pregnancies. conclusions: reduction in absolute uterine artery flow (ml/min) did not impact utero-placental o uptake or transfer to the umbilical vein, and may have resulted from reductions in maternal cardiac output. relative uterine artery flow was not reduced, suggesting that uterine blood flow and delivery of o to the conceptus does not mediate the ongoing placental growth restriction initiated during early gestation. supported by nih hd . barton c staat, anna maria marconi, cinzia paolini, alex cheung, henry l galan, frederick c battaglia. obstetrics gynecology and pediatrics, univ of colorado at denver health sciences center, aurora, co, usa; dept of obstetrics and gynecology, san paolo institute of biomedical sciences, university of milano, milano, italy. objective: to determine relative contributions of transplacental flux vs fetal production for myo-inositol and mannose in normal term pregnancies using stable isotopic methodolgy. background: myo-inositol and mannose are important in biologic functions. an external supply of mannose may be required for glycoprotein synthesis. low maternal myo-inositol is associated with spina bifida. mannose concentrations are known to be higher in the mother than the fetus. in contrast, myo-inositol concentrations are higher in the fetus than the mother. what remains unknown is whether fetal levels of these polyols are a result of direct maternal transport or from conversion of glucose. design: four term uncomplicated pregnancies undergoing an elective ceasaran section were infused with c labeled isotopes of glucose, myo-inositol and mannose over hours prior to delivery. maternal samples were obtained prior to infusate being administered, and at hour (h), . h and h. fetal concentrations were measured from umbilical artery and vein plasma. the concentrations of labeled and unlabeled glucose, mannose and myo-inositol were measured using high pressure anion exchange chromatogrpahy permitting detection of polyols and sugars at concentrations in the μm range. the feto-maternal molar percent enrichment (mpe) ratio was calculated for each glucose, mannose, and myo-inositol as the ratio between fetal plasma enrichment and the maternal plasma enrichment at steady state. steady state was calculated as the mean of the three maternal samples taken during infusion. results: the feto-maternal mpe ratios of mannose ( . ± . , p= . ) and glucose ( . ± . , p= . ) were not significantly different from . , consistent with transplacental supply. the feto-maternal ratio for myo-inositol ( . ± . , p= . ) indicates little transplacental flux ( % of fetal inositol derived from maternal plasma). conclusion: in normal term pregnancies, fetal mannose and glucose concentrations are dependent upon maternal transplacental supply. in contrast, fetal myo-inositol concentration is not dependent upon transplacental supply, but fetal demands are met by placental conversion, likely from glucose. christian wadsack, manuela augsten, christian guelly, ursula hiden, ingrid lang, manfred moertl, uwe lang, gernot desoye. clinic ob/gyn; center of med res; inst cell biol, histol embryol, med univ graz, austria. background placenta and fetus need lipids for growth and synthesis functions. part of the lipids is supplied from maternal sources by transplacental transfer. recently, we identified in human placenta the high density lipoprotein (hdl) receptor scavenger receptor class b type i (sr-bi). among other functions it mediates hdl-induced ser -phosphorylation of endothelial nitric oxide synthase (enos) resulting in enos activation. this mechanism allows hdl to contribute to regulation of vasotonus in arteries. we hypothesized that term placental endothelial cells (ec) express sr-bi at levels different between arteries and veins. methods sr-bi was localized by ihc and quantified by qrt-pcr in rna isolated from arterial and venous vessels. arterial (eca) and venous (ecv) placental ec were rigorously characterized. sr-bi levels were measured by qrt-pcr and immunoblotting. hdl binding and uptake was measured in eca and ecv with i-labelled hdl. hdl from human donors was used to stimulate ser enos phosphorylation. epigenetic regulation was studied by methylationspecific pcrs for cpg-rich promoter regions of sr-bi. pdzk a key adaptor for sr-bi mediated enos activation was measured by sqrt-pcr. in situ analyses (ihc, qrt-pcr) showed more sr-bi in arteries than in the vein. the differential expression persisted in vitro in isolated eca and ecv even after passages and culture under same conditions suggesting epigenetic mechanisms regulating sr-bi. however, no methylation was found in eca or ecv. sr-bi was functional since hdl cell association was -fold higher in eca than in ecv ( ± . vs ± . ng hdl/mg prot). hdl did not induce ser enos phosporylation in eca or ecv, which was stimulated by ionomycin about -fold in both cell types. pdzk was undetectable in eca and ecv, whereas it was expressed in placental tissue. conclusion more sr-bi is expressed in ec from arteries than from veins in situ and in vitro. this is not the result of different methylation of sr-bi promoter and, hence, unlikely an epigenetic phenomenon. mechanism of differential expression and its functional consequences for vasotonus regulation is yet unknown. the lack of pdzk may account for the failure of hdl to activate enos. (grants , , oenb). cells. juan a arroyo, brad ziebell, mi-hye park, henry l galan. obstetrics and gynecology, university of colorado andgealth sciences center, aurora, co, usa. introduction: mtor is a protein that regulates cell growth in response to nutrients and growth factors. downstream effectors of the mtor pathway include the p and the ebp proteins. activation by phosphorylation of these proteins increases protein synthesis. given that various signaling pathways are regulated by hypoxia in human trophoblast and that mtor is expressed in human trophoblast, our objective was to determine the effects of hypoxia in the activation of mtor, p and ebp in cultured human trophoblast. study designs: trophoblast cell were isolated from term uncomplicated placentas using a trypsin, dnase and disapase solution. cytokeratin immunocytochemistry confirmed trophoblast cells culture purity. trophoblast cells were treated with hypoxia ( % o ) or normoxia ( % o ) for and hours. western blot for p-mtor, mtor, p-p , p , p- ebp , and ebp were done for each time studied. results: trophoblast cells demonstrated: ) positive staining for cytokeratin, ) non significant differences for mtor at either ( . -fold; p= . ) or hours ( . -fold, p= . ), ) no differences in p protein at ( . fold; p= . ) or hours ( . -fold, p= . . ), ) no differences for ebp at either or hour. conclusion: we conclude that the mtor pathway is not regulated under hypoxic conditions in cultured trophoblast, which suggests that hypoxia does not affect protein synthesis in cultured human trophoblast. however, this may not reflect what happens in vivo in iugr. (supported by nih grant r hl - a ). increased expression of phospho-mtor, phospho-p , phospho-akt and phospho-erk in an ovine model of fetal growth restriction. juan a arroyo, brad ziebell, henry l galan. obstetrics and gynecology, university of colorado and health sciences center, aurora, co, usa. objective: both phosphorylated (p) mtor and p are known to be involved in protein synthesis and are regulated by physiological conditions such as fetal growth restriction (fgr). in a hyperthermic (ht) ovine model of fgr we hypothesize that mtor, p , ebp , erk and akt will be phosphorylated (activated) in the placentae of age (dga) animals. study design: ewes were exposed to ht conditions for days to induce iugr and were placed in ambient conditions. at necropsy ( dga), placentomes were separated into the maternal (caruncle) and fetal (cotyledon) components and frozen for western blot analysis with antibodies against (p) mtor, mtor, (p) p , p , (p) ebp , ebp , (p) erk, erk, (p)akt and akt. results: compared to control animals, fgr animals had smaller fetuses ( ± g v. ± g; p= . ) and smaller placentae ( ± g v. ± g; p= . ) at dga. fgr cotyledon showed an increase in p-mtor ( . -fold; p= . ), p-p ( . -fold; p< . ), p-erk ( . -fold; p< . ) and p-akt ( . -fold; p< . ). in contrast, caruncle (maternal) did not show any changes for the mtor pathway. conclusion: in fgr ovine pregnancies, the fetal placental tissues (cotyledons) showed upregulation of the mtor pathway for protein synthesis via phosphorylation of the p but not ebp while this was not seen in the maternal (caruncle) tissues. in addition neither the cotyledon or caruncle tissues at mid-gestation ( dga) showed changes in these endpoints, which is prior to the exponential fetal growth that starts at mid-gestation. (supported by nih grant r hl - a ). hyperuricemia has long been recognized as a common clinical finding in preeclamptic (pe) women. to date, elevated uric acid concentrations in these women have been considered a marker of disease severity. however preeclamptic pregnancies with hyperuricemia, are associated with an increased frequency of preterm birth and fetal growth restriction. over the past decade several pathogenic roles for uric acid have become evident, raising the possibility of a role(s) for uric acid in the altered vascular and placental functions associated with pe. objective: examine the effects of syncytial uric acid uptake on system a amino acid transport across the human placenta using a primary placental villous explant model. methods: placental villous explants from placentae of healthy, term pregnancies were incubated for hours with uric acid ( . mg/dl), corresponding to concentrations of uric acid observed in pe women. these experiments were conducted in the presence or absence of probenecid ( m), a uric acid cellular uptake inhibitor. system a amino acid transport was subsequently assayed using a radiochemical assay in which na+-dependant uptake of radio-labeled system a substrate, [ c] methyl-amino-isobutyric acid, was measured over minutes. data were analyzed using a paired student's t-test and presented as mean ± sem. results: uric acid attenuated system a amino acid placental transport by % (± . %, p< . ). this inhibitory effect of uric acid on system a activity was prevented by probenecid. conclusions: uric acid reduces placental amino acid transport at concentrations observed in pe women. this inhibitory effect of uric acid is dependant upon syncytial uptake of uric acid, being inhibited by the uric acid transporter inhibitor probenecid. these results may be relevant to the increased frequency of fetal growth restriction observed in hyperuricemic pe. additionally the results of this study, indicating a detrimental effect of hyperuricemia on placental function, also suggest a role for uric acid in the pathophysiology of pe. hyperuricemia, a well-documented clinical finding in preeclamptic women, is associated with pre-term birth and intrauterine growth restriction. uric acid is higher in women destined to develop preeclampsia as early as weeks of gestation at a time when cytotrophoblast are invading decidua and myometrium and remodeling uterine spiral arterioles. we propose that elevated concentrations of uric acid may have detrimental effects on placental development in part through inhibition of trophoblast invasion through the decidua. objective: examine the effects of increasing concentrations of uric acid on trophoblast invasion through a reconstituted extracellular matrix. methods: using the in-vitro matrigel invasion assay, the effects of increasing concentrations of dissolved uric acid ( . mg/dl, . mg/dl and . mg/dl) on the ability of immortalized first trimester extravillous trophoblast cells (htr -svneo) to invade through a reconstituted extracellular membrane were assessed. the concentrations of uric acid used were comparable to those measured in healthy pregnant women and preeclamptic women with an increase in uric acid of two or four standard deviations above normal. cells that successfully invaded through the matrigel membrane within hours were fixed with methanol, stained with hematoxylin and counted. data were analyzed using a one-way analysis of variance with fisher's post-hoc analysis. results: uric acid attenuated trophoblast invasion in a dose-dependent fashion (p< . ), with decreases of % (± . %), % (± . %) and % (± . %) respectively compared to untreated controls. conclusions: exogenous uric acid, at physiological and pathological concentrations, is capable of attenuating trophoblast invasion through a reconstituted extracellular membrane in a dose dependent fashion. these results suggest uric acid is a potential contributor to the pathophysiology of altered placental perfusion in preeclamptic pregnancies. previous studies have shown that transforming growth factor (tgf)-is a key inhibitory factors in the invasion of early trophoblast cells, suggesting therefore that overcoming tgf-beta signaling may be necessary for successful implantation. smad ubiquitin regulatory factor (smurf ), a hect type e ubiquitin ligase, is a key regulator of tgf-signaling pathway, targeting tgf-receptors and various smads for proteasome-mediated degradation. in this context, smurf has been shown to play important roles in embryonic development, cell senescence and tumor formation. as a key regulator of tgfbeta signaling, we wished to determine whether smurf has a physiological role during embryo implantation, especially in trophoblast invasion. we have examined the spatio-temporal expression of smurf in human placental villi during pregnancy. we have also investigated the possible function of smurf in trophoblast cell migration and invasion in a model system involving a human extravillous trophoblast cell line, htr /svneo. our results showed that expression of smurf in placental villi was the highest during the first trimester and the expression decreased in the nd trimester. expression of smurf was lowest in placental villi at parturition. overexpression of smurf in htr /svneo cells reduced tgf beta type i receptor levels and attenuated the inhibitory effect of tgf-on cell migration and invasion. conversely, rnai-mediated down-regulation of smurf resulted in significant increase of tgf-type i receptor protein levels. in contrast, the levels of smad , another potential target of smurf , was unchanged. in conclusion, the present study suggests that smurf participates in trophoblast cell migration and invasion by down-regulating the expression of tgf-type i receptor. our previous data demonstrated the extensive expression of mmp- in various kinds of trophoblast cells in human placenta at the early pregnancy. however, the modulation of the enzyme in trophoblasts is largely unclear. in the present study, the effects of the two types of gonadotropin releasing hormone (gnrh) on mmp- expression were examined in an immortalized human cytotrophoblast cell line, b tert- that has been established in this lab. real-time quantitative pcr and western blot analysis revealed that both types of gnrh (gnrh i and gnrh ii) could increase mmp- mrna and protein levels in b tert- cells in time-dependent manners. in particular, regulatory effect of gnrh i on mmp- expression was concentration-independent, whereas that of gnrh ii was dose-dependent. moreover, both gnrh i and gnrh ii could evidently activate jnk kinase, and sp , an inhibitor of a jnk kinase, reversed the up-regulation of mmp- induced by either gnrh i or gnrh ii. on the other hand, it is not likely that erk / pathway participates in the signaling of gnrh i or gnrh ii. collectively, our observations suggest that gnrh i and gnrh ii elicit their modulation effects in human trophoblastic cells through jnk pathway leading to up-regulation of mmp- . during the first trimester of pregnancy, the oxygen tension of the developing trophoblast cells is less than %. however, the majority of studies on primary trophoblast cell development have been performed at % oxygen. primary third-trimester trophoblast cells are believed to be nonproliferative syncytiotrophoblast cells. we have previously demonstrated that low oxygen tension dramatically affects the differentiation pathway of these cells. we now hypothesize that cell culture in low oxygen tension will improve cell growth and restore proliferation. methods: primary trophoblast cells were purified from third-trimester placenta by enzymatic dispersion and cd- negative selection and cultured at %, % or . % oxygen tension for up to days. the number of cells in culture was assessed by cell counting and by measuring genomic dna. live:dead and mtt assays were used to determine viability. proliferation was assayed with brdu and immunohistochemistry for proliferating cell nuclear antigen. to assess cellular activity, radioactivity of protein precipitated from cells cultured in the presence of tritiated leucine was measured. results: there were no obvious morphologic changes in the cells cultured in different oxygen tensions. the amount of cell loss was directly proportional to oxygen tension: at % oxygen % of the cells remain in culture; at . % oxygen tension % of the cells remained. the cells at . % oxygen tension were proliferating and had a five-fold increased metabolic activity. conclusions: it was previously believed that third-trimester trophoblast cells are non-proliferative. we have demonstrated that low oxygen tension increases the survival of primary third-trimester trophoblast cells. this may reflect the change in the differentiation pathway of these cells. however, the cells also begin to proliferate and increase their metabolic activity. trophoblast cells in vivo form a three dimensional structure which promotes critical complex cell-to-cell interactions that cannot be studied with traditional monolayer cell culture. we developed a substrate-free three-dimensional trophoblast culture system capable of studying cellular interactions without a confounding artificial matrix. methods: nonadhesive agarose hydrogels containing cylindrical recesses m in diameter were cast from molds designed using computer-assisted prototyping. tcl trophoblast cells were seeded into the gels ( , cells per) for up to days. viability and cellular stress were assessed and the threedimensional structures of the spheroids were analyzed. results: tcl trophoblast cells formed uniform spheroids within three days of seeding. the spheroids remained intact after being removed from the mold. when placed in traditional cell culture dishes the cells adhered to the plate within one hour and rapidly proliferated into a monolayer. repetitive reseeding allowed easy transition between monolayer and spheroid without affecting cellular morphology. serial sectioning on days , and revealed central vacuolization forming a trophoblast vesicle with an outer rim . m (+/- m) thick. this rim size remained constant for at least days. live:dead assay demonstrated that the outer cells remained viable and staining against proliferating cell nuclear antigen demonstrated that the cells were proliferating. the inner cells undergo apoptosis as demonstrated by caspase- staining. there is an abundance of vegf staining in the cells remaining in the on the inside of the sphere suggesting a gradient of nutrient or oxidative stress. the formation of a vesicle has been confirmed with confocal imaging. em imaging revealed the structure of the rim. conclusions: trophoblast cells cultured in a novel substrate-free three dimensional system form trophoblast vesicles within days of seeding. these vesicles remain viable after long-term culture and can be repeatedly reformed with repetitive seeding. this new cell culture technique allows us to better study placental cell-cell interactions with the potential of forming microtissues. the transcription factor glial cell missing- (gcm ) mediates cell cycle arrest and differentiation of human trophoblast progenitors into villous syncytiotrophoblast and invasive extravillous cytotrophoblast (evt). micro-array analysis of total rna extracted from cultured bewo cells, in which gcm mrna and protein were repressed using sirna, identified tissue inhibitor of metalloproteinase- (timp- ) in the highest ( -fold) upregulated group of genes. confirmatory rtpcr demonstrated a -fold mrna induction. in placental villi, gcm acts as a transcription factor promoting expression of the fusogenic protein syncytin that mediates syncytial fusion into the overlying syncytiotrophoblast. by contrast, syncytial fusion is uncommon in evt. rather these cells invade several millimeters into the distal myometrium where they transform spiral arterioles. to investigate the role of timp and gcm in the trophoblast we assessed its mrna by qrt-pcr and protein by western blot in cellular extracts from both bewo cells grown under standard cultivation conditions (synchronized by prior thymidine exposure) and floating cultured first trimester villous explants cultured in % oxygen with prior exposure to either gcm sirna or anti-sense oligo-nucleotides to gcm . gcm inhibition in the bewo system was associated with a - % increase in timp- protein expression and alteration of cell proliferation and differentiation in both models. we are presently utilizing the explant model of evt invasion (explant tips cultured on matrigel in % oxygen) to test the hypothesis that gcm mediates metallo-proteinase expression and evt invasion via timp- . presently we conclude that gcm -mediated evt differentiation involves more than an arrest of mitosis and may include promotion of invasion via repression of timp- . funding: cihr. scott h purcell, jeremy d cantlon, virginia d winn, russell v anthony. , colorado state university, fort collins, co; university of colorado health sciences center, aurora, co. background: periattachment factor (pf) is a nuclear protein first described in the bovine conceptus. our research in sheep has shown pf mrna concentration peaks when the conceptus is undergoing elongation and initial apposition to the endometrium, and that pf is a nuclear protein localized to the trophoblast. in silico analysis identified a human homolog, hprr . objective: the objective of this experiment was to determine if pf was expressed in the human placenta, and to develop short-hairpin (sh) rnas for hpf to begin investigating its function. materials and methods: immunohistochemistry was performed on paraffin embedded first and second trimester human placental samples. placental sections were immuno-stained using rabbit polyclonal antiovine pf or anti-human cytokeratin- . cytotrophoblasts from first trimester pregnancies (n= ) were subjected to an in vitro invasion assay and rna was harvested following , , and h. quantitative rt-pcr was performed on these samples with intron-spanning primers for hpf, and normalized on hs mrna concentrations. based on the human pf sequence, four putative shrna constructs were generated and cloned into a lentiviral expression vector. bewo human choriocarcinoma cells were treated with one of four shrna contructs or an empty vector for h and then rna was harvested from cells for analysis by quantitative rt-pcr. results: periattachment factor was present in the nuclei of both first and second trimester cytotrophoblasts. hpf mrna concentration increased as invasion occurred from , , to h in all samples; while hypoxia decreased expression at h of invasion compared to h under normoxic conditions. the four lentiviral vectors expressing shrna against hpf resulted in hpf mrna concentrations at , , and % of hpf mrna concentration with the control vector. conclusion: the presence of pf in the human placenta and the increase in pf mrna during cytotrophoblast invasion may indicate this gene plays a role during implantation. we have developed shrnas against pf that result in greater than % mrna knockdown and will be using these to begin to elucidate the function of pf in the human placenta, specifically during the invasion process. recently we demonstrated that infusion of imd antagonist (imd - ) in rat caused distorted labyrinth indicative of a deficient vasculature in placenta. we hypothesize that imd has a role in migration of first trimester trophoblast cell (htr- /svneo) via regulating human leukocyte antigen (hla-g) and stimulating mek / / erk / phosphorylation. objectives: ) to asses the effect of imd on migrating capacity of htr- sv/neo cells using scratch assay in presence or absence of mek and ras/raf inhibitor, u and manumycin a, respectively ; ) to assess the effect of imd peptide on phosphorylation of mek / and erk / in first trimester htr cells ) to analyze the effects of imd on the expression of human leukocyte antigen, hla-g, a critical factor involved in the invasion and vascular remodeling of spiral uterine arteries and subsequent pregnancy in human. methods: htr- sv/neo cells were used to assess the effect of imd ( - m) on the expression of hla-g mrna and phosphorylation of erk / and mek / protein by reverse transcriptase polymeration chain reaction (rt-pcr) and western blot analysis respectively. scratch wound assay was used to determine the migration capacity of htr cells. total rna was isolated from cells using trizol reagent and processed for rt-pcr and results are expressed relative to s mrna. trichloroacetic acid was used for the extraction of total protein for western blot analyses. results: our data demonstrates that, ) imd enhances the migrating capacity of htr cells (compared to the untreated cells) and these effects are inhibited by mek and ras/raf inhibitors, u and manumycin a, respectively; ) imd ( - m ) stimulates phosphorylation of erk / and mek / proteins in htr cells, ) imd increases the expression of hla-g mrna in htr cells. conclusion: imd promotes migration of first trimester htr cells through mek/ erk signaling pathway and modulates the expression of immunoregulatory molecule, hla-g in these cells. chymotrypsin-like protease promotes the placenta tissue release of sflt- . yang gu, shuang zhao, david f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: the placenta is a major source of soluble vegf receptor- (sflt- ) in the maternal circulation during pregnancy. increased placental release of sflt- is believed to play an important role in the pathophysiology and pathogenesis in pe. however, the mechanism of increased placental sflt- release in pe is unknown. we recently reported increased chymotrypsin-like protease (clp) activity and expression in placental trophoblasts from pe. in this study, we tested if proteolytic effects of chymotrypsin may play a role in promoting sflt- release by placental trophoblasts. methods: placentas delivered by normal pregnant women (n = ) were used. we tested if chymotrypsin could promote sflt- release by placental tissue, in which villous explants were cultured with dmem containing chymotrypsin at . , . , and . g/ml for hours. the culture medium was then collected for measuring sflt- . we then determined the specificity of chymotrypsin induced sflt- release. villous tissues were cultured with or without chymotrypsin inhibitor (ci) in culture and then the medium was collected and measured for sflt- . soluble flt- was measured by elisa. all samples were assayed in duplicate. data are presented as mean ± se and analyzed by anova. a p level < . is considered as statistically different. results: ) sflt- concentrations in the medium were increased when chymotrypsin was present in culture and the increased sflt- release induced by chymotrypsin was in a concentration-dependent manner: control: . ± . ; . g/ml: . ± . ; . g/ml: . ± . ; and . g/ml: . ± . (p< . ) pg/mg tissue/hour. ) ci could attenuate sflt- release. this inhibitory effect was also revealed in a concentration-dependent manner: control: . ± . ; ci . g/ml: . ± . ; and ci . g/ml: . ± . (p< . ) pg/mg tissue/hour. conclusions: increased placental sflt- release stimulated by chymotrypsin and decreased placental sflt- release inhibited by chymotrypsin inhibitor suggest that the proteolytic effect of clp may play a role in sflt- generation. therefore, increased clp activity in placental trophoblasts may contribute to the increased placental sflt- production in pe. (supported nih grants hl and hd ). the change of autophagy-related proteins, lc and beclin- , by tnfa stimulation in cultured primary trophoblasts. soo-young oh, kyung hee kim, suk-joo choi, jong-hwa kim, cheong-rae roh. department of obstetrics and gynecology, samsung medical center, sungkyunkwan university school of medicine, seoul, korea. objective: our previous work have demonstrated that the expression of lc , but not beclin- , was increased in placentas from pregnancies complicated by severe preeclampsia (sgi abstract # ) . to understand the regulatory mechanism of these autophagy-related proteins in trophoblast cells, we investigated the changes in these proteins in response to cytokine or hypoxic stimulation in cultured primary trophoblast. material and methods: primary human cytotrophoblasts obtained from normal term placenta were cultured with stimulation of tnf-a or cocl for a given time and the changes of beclin- and lc were assessed using immunoblot analysis. paired t test was used for statistic analysis. results: tnf-a stimulation induced a significant increase of the expression of lc -ii in cultured primary trophoblasts while decreasing the expression of beclin- (p< . for each). however, cocl stimulation did not induce a significant change of both lc -ii and beclin- . conclusions: our data suggests that tnf-a stimulation in cultured primary trophoblasts is associated with increased autophagic activity. background: thyroid hormones play vital roles in the development of the fetal brain. mutations in mct , recently recognised as a specific thyroid hormone transporter, define a novel syndrome of severe x-linked psychomotor retardation accompanied by elevated serum t . we previously reported that mct expression in n-tera- (nt ) cells (a human embryonal cell line with characteristics of cns precursors), as well as mct -null jeg- choriocarcinoma cells, resulted in markedly reduced cell proliferation. further, the s x mct mutation, as reported in males affected by severe psychomotor impairment, resulted in a similar repression of proliferation to wild type, whereas the l p mutant failed to influence cell turnover compared with control. methods: we now examine the effect of "knocking down" mct via sirna and evaluate the effects of cell proliferation (mtt and [ h]-thymidine assays) and tri-iodothyronine (t ) uptake. results: repression of endogenous mct expression in nt cells by % caused a significant increase in proliferation compared to matched-dose nonspecific sirna treatment, independent of t concentration ( . %, . % and . % induction at , and nm t , n= , p< . ). we also sought to examine the role of mct in t uptake. in jeg- cells, wild type mct induced a . -fold increase in the uptake of i-labelled t . by contrast, mutants s x and l p failed to significantly augment t uptake, though r h caused a mild but significant . fold induction in uptake, hence retaining approximately % of wt activity. in parallel experiments, co-transfection of mu-crystallin, a t binding protein, resulted in a similar increase in t uptake compared with control ( . -fold; n= ; p< . ), implying that mct plays only a minor role in thyroid hormone efflux in jeg- cells. mutants s x, l p and r h showed analogous responses to those in the absence of mu-crystallin. conclusion: these results further extend the evidence of a potential role for mct in the modulation of cell proliferation, independent of t transport. to determine predictors of failure for labor induction in women with preeclampsia. we conducted a retrospective cohort study to examine cesarean delivery rates in all the preeclamptic women at a single institution undergoing labor induction between - with a singleton pregnancy >= weeks gestational age (ga). bivariate analyses informed the creation of multivariable logistic models to predict the risk of cesarean delivery using multiple predictors (maternal age, race/ethnicity, unfavorable cervix, gestational diabetes, diabetes, and gestational age). analyses were stratified by parity. our study population included , preeclamptic women undergoing labor induction. in the bivariate analyses, the risk of cesarean delivery ranged from as low as . % (p= . ) among multiparous women - weeks ga to as high as . % (p< . ) among nulliparous women with diabetes. a total of , women had adequate data to be included in the multivariable analyses. odds ratios of the predictors are presented in the objective: preeclampsia and cardiovascular disease share many risk factors, and women with preeclampsia are at increased risk of cardiovascular mortality later in life. we investigated whether risk factors associated with cardiovascular disease and preeclampsia remain elevated months postpartum. methods: we measured plasma sflt , endoglin, plgf, cellular fibronectin (cfn), uric acid, homocysteine, and asymmetric dimethylarginine (adma) in women with uncomplicated normotensive pregnancies compared to women with preeclampsia in samples collected at pre-delivery and again several months postpartum (average . ± . months). data are mean±sd or median (interquartile range). statistical analysis was by wilcoxin rank-sum or students unpaired t-tests with statistical significance accepted at p< . . results: the mean concentration of sflt , endoglin, plgf, homocysteine, adma, cfn, and uric acid were all significantly different in samples collected pre-delivery in subjects with preeclampsia compared to controls (table). adma, cfn and uric acid remained significantly higher postpartum in subjects with previous preeclampsia compared to postpartum controls (table) . conclusions: biological markers associated with altered vascular function or cardiovascular risk are elevated in women with preeclampsia, and some remain significantly higher in postpartum preeclamptic women. these data suggest that vascular dysfunction persists in women with previous preeclampsia, and may contribute to the increased risk of future cardiovascular disease. funded in part by national institutes of health nih- mo -rr and nih- po -hd . (ajp, ) . as leptin is a known potent angiogenic factor we hypothesized that leptin deficiency and/or resistance to leptin-induced vegf expression might be a mechanism for reduced angiogenesis in mfr offspring. methods: pregnant sprague-dawley rats had % mfr from day of gestation until delivery. mfr and control offspring were sacrificed on day of life (p ). some tissues were used to determine the expression of leptin by western blot analysis. for culture experiments, thoracic aortas were dissected, cut into - mm explants and incubated with leptin ( - ng/ml) in dmem ( % fbs). after hours of culture, rna was extracted from the tissues and subjected to real time rt-pcr using specific rat primers for vegf, vegfr and r , and ob-ra, stat and socs ( s mrna as control). culture media was analyzed for vegf protein by elisa. results: expression of leptin mrna and protein in p mfr aortas was significantly reduced. in culture, leptin significantly increased expression of vegf, vegfr and vegfr mrna in explants of aortas obtained from the control but not mfr tissues. as expected, control but not mfr aortic explants secreted significantly more vegf in vitro. to determine the mechanism for resistance to leptin-induced vegf in mfr offspring, we assessed expression of leptin receptor (ob-ra) in explants treated with leptin. leptin was found to induce the expression of ob-ra in aortas from both dietary groups. this upregulation of leptin receptor was accompanied by significant upregulation of stat and socs mrna in the control tissues. in contrast, in mfr explants only the ng/ml concentration of leptin induced an increase in stat mrna, and the magnitude of socs mrna increase by both concentrations of leptin was significantly less in the mfr explants. conclusion: these results indicate that reduced angiogenesis in mfr vessels is in part due to reduced leptin expression and ability of leptin to stimulate vegf expression. although in vitro leptin induced the expression of its receptor in both groups, it was only in the mfr group in which leptin up-regulated vegf and its receptors. our results suggest that this defect in leptin receptor function in mfr vessels is due, in part, to defects in jak/stat signaling. objective: women with a history of early-onset preeclampsia are at increased risk of developing major cardiovascular disease (cvd) related events, that have a detrimental effect on their long-term health and life expectancy. in this follow-up study, we measured established risk factors predictive of first cvd events after early-onset preeclampsia. study design: over a -year interval, primiparous women with a history of early-onset preeclampsia (delivery < weeks gestation) were included and tested for major cardiovascular risk factors at least six months after delivery, in addition to a population-based control group of healthy non-pregnant women. women with chronic hypertension were excluded. results: mean age was . years for cases compared to . years for controls (p<. ). after adjustment for age, we observed significantly increased mean values for weight (p=. ), body-mass index (p<. ), systolic blood pressure (p<. ), diastolic blood pressure (p<. ), total cholesterol (p=. ), ldl cholesterol (p<. ), triglycerides (p=. ), fasting blood glucose (p<. ), and lower hdl cholesterol (p<. ) in women with previous early-onset preeclampsia. no difference was found for height, smoking, diabetes, and ethnicity. estimated -year risk of first cvd events by framingham risk scores remained < % for all women (low-risk). nonetheless, at mean (sd) . ( . ) years after early-onset preeclampsia, % of women met the criteria for metabolic syndrome, % of women exhibited >= , % of women >= and % of women >= major cvd risk factors. conclusion: the majority of women with a history of early-onset preeclampsia exhibit at least one modifiable risk factor for future cvd. although most of these women are classified as low-risk according to the current aha guidelines, this is mainly due to their young age masking other, mostly modifiable, major risk factors. our data thus support life-style intervention programs aimed at primary prevention of cvd in women with a history of early-onset preeclampsia. it has recently been shown that antihypertensive drugs can stimulate cytokine release in normal and hypertensive pregnancy. there is evidence that these cytokines alter the secretion of inhibin a. inhibin a and activin a levels are increased in pre-eclampsia (pe), but it is not known if antihypertensive therapy can affect their secretion. patients and methods we recruited women with hypertensive disorders in pregnancy ( pe and non-proteinuric hypertension [ht]) and matched normotensive controls. inhibin a and activin a levels, before and - hours after initiating antihypertensives, were measured in serum and urine, using an elisa. the same markers were measured using validated assays in placentas delivered at cesarean section at similar gestational age ( pe, ht and controls). analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. marker levels before and after antihypertensive therapy were compared using paired t-test. we compared placental concentrations between the group which received antihypertensive therapy and the group which did not, using an independent t-test. data were analysed using spss®. in pe, both serum and urine levels of inibin a and activin a were increased at all gestations (p< . ). activin a (but not inhibin a) level was also increased at all gestations in ht (p< . ). after weeks' gestation (but not before), antihypertensive treatment was associated with a significant fall in both inhibin a and activin a serum levels, and urinary inhibin a, in both pe and ht. the placental concentration of inhibin a, but not activin a, was significantly higher in women with pe compared with controls (p< . ). there was no significant difference in either marker between controls and women with ht. the fall in serum levels of inhibin a and activin a following antihypertensive treatment after weeks' gestation may indicate that these drugs have an effect on the pathophysiology of pe other than their known antihypertensive action. pre-eclampsia (pe) is a placental disease of unknown etiology. anti-angiogenic factors, such as soluble fms-like tyrosine kinase (sflt- ) and soluble endoglin (seng), and pro-angiogenic factors, such as vascular endothelial growth factor (vegf) and placental growth factor (plgf), are believed to play an important role in its pathophysiology. maternal plasma concentrations of these markers are altered in pe, even weeks before the clinical manifestations. the aim of this study was to compare the concentration of these markers in placental extracts of normotensive pregnant women, and women with pe and non-proteinuric hypertension (ht). patients and methods placental samples were collected at cesarean section from women with pe (n = ), ht (n = ) and normotensive pregnancies of similar gestational age (n = ). these samples were stored at - ºc. the frozen tissue samples were homogenised and these four markers measured by specific, validated enzymelinked immunosorbent assays. analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. data were analysed using spss®. the concentrations of both sflt- and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p= . ). there was no significant difference in plgf concentration between controls and women with pe or ht. placental vegf concentrations in both pe and ht were higher than in controls (p< . ); there was no significant difference between the levels in pe and ht (p= . ). the fact that placental concentrations of sflt- and seng mirrored the known rise in serum levels in pe suggests that the placenta is the main source of these circulating factors. although sflt- was significantly raised in pe, plgf was not reduced. this suggests that the lower levels of free plgf found in the serum of women with pe are not the result of impaired placental production or secretion, but are due to increased binding by (the increased levels of) sflt- in the serum. objective. the purpose of this study was to evaluate whether systematic screening with uterine artery doppler (utad) and serum biochemical markers of oxidative stress, endothelial dysfunction and vasculogenesis performed during the first trimester predict efficiently pre-eclampsia (pet), specifically early-onset pet, in an unselected chilean population. methods. this nested case-control study involved asymptomatic pregnancies scanned at + - + week of gestation. the subjects for biochemical testing were women who were delivered due to pet (n= ) and normotensive controls (n= ) that were enrolled during the first trimester scan. mean pulsatility index (pi) of the utad was calculated. blood samples were obtained and stored at - o c until biochemical analysis of oxidative stress, endothelial dysfunction and vasculogenesis were performed. normally distributed data were analysed by the unpaired t test, and non-normally distributed data by the mann-whitney rank sum test. chi-square tests were used for the comparison of categorical variables. a probability level of p< . was considered significant. multiple logistic regressions were used to develop a combined predictive index. results. there was % and % significantly increased of the mean pi utad in women who later developed pet or early-onset pet compared to control pregnancies during the first trimester scan. although oxidative stress and endothelial dysfunction biochemical markers were not different between all pet pregnancies and control groups, plasma levels of sflt ( . ± . vs. . ± . pg/ml, p< . ) and placenta growth factor ( . ± . vs. . ± . pg/ml, p< . ) were significantly higher in women who subsequently developed early-onset pet compared to controls. multivariate logistic regression showed that a combination between abnormal utad and both biochemical markers of abnormal vasculogenesis were the best predictor test for early-onset pet, being its detection rate % with % false positive rate. conclusion. this study has shown early and selective changes in markers of impaired placentation and angiogenic state in women who later developed earlyonset pet, without alteration in oxidative stress and endothelial dysfunction. supported by fondecyt . currently it is unknown whether maternal inflammatory changes are specific to pregnancy or reflect an innate susceptibility to inflammation. c-reactive protein (crp) and interleukin- (il- ) are markers of the acute-phase inflammatory response and predictive of future cardiovascular events. we compared crp and il- levels after influenza vaccination, as an in vivo model for lowgrade inflammation, in non-pregnant women with a history of early-onset preeclampsia and controls with only uneventful pregnancies. methods: forty-four women with a history of early-onset preeclampsia (delivery < weeks' gestation) and twenty-nine controls with at least one uneventful pregnancy received an influenza vaccination. we then compared plasma levels of crp and il- at baseline, . days and . days after vaccination. results: median baseline crp and il- levels of women with a history of early-onset preeclampsia were comparable to controls ( . versus . mg/l; p= . and . versus . pg/l; p= . , respectively). however, high crp and il- responses to vaccination were more common in cases (ors for response > th, > th, > th, > th and > th percentile based on the distribution of control values of . , . , . , . and for crp [p for trend . ] and of . , . , . , . and . for il- [p for trend . ], respectively). the relationship between high il- responses and early-onset preeclampsia persisted after adjustment for body-mass index (p for trend . ). conclusion: women with a history of early-onset preeclampsia more frequently exhibit an innate pro-inflammatory phenotype not specific to pregnancy. background altered maternal inflammatory responses play a role in the development of preeclampsia and the hemolysis, elevated liver enzymes and low platelets (hellp) syndrome. we examined whether allelic variants of the innate immune receptors toll-like receptor (tlr ) and nucleotide-binding oligomerization domain (nod ), that impair the inflammatory response to endotoxin, are related to preeclampsia and hellp syndrome. we determined five common mutations in tlr (d g and t i) and nod (r w, g r and l fs) in primiparous women with a history of early-onset preeclampsia, of whom women developed hellp syndrome and in women with a history of only uneventful pregnancies as controls. in addition, we assessed plasma levels of pro-inflammatory biomarkers c-rp, il- , sicam- , fibrinogen and von willebrand factor in a subset of women included at least six months after delivery. after adjustment for maternal age and chronic hypertension, attenuating allelic variants of tlr were more common in women with a history of early-onset preeclampsia than in controls (or . [ % ci . - . ]). highest frequencies for tlr variants were observed in women who developed hellp syndrome (adjusted or . [ % ci . - . ]). in addition, high levels of il- and fibrinogen were associated with a history of early-onset preeclampsia. combined positivity for any of the tlr and nod allelic variants and high levels of il- was . -fold more common in women with a history of early-onset preeclampsia ( % ci . - . ) compared to controls. we observed an association of common tlr and nod gene variants, and pro-inflammatory phenotype with a history of early-onset preeclampsia and hellp syndrome, that suggests involvement of the maternal innate immune system in severe hypertensive disorders of pregnancy. thus, we sought changes in pbef in the serum of patients with mild and severe pe, compared with matched controls. immunodistribution of pbef in fetal membranes and placentas from similar patients was also studied. methods: serum samples ( ) were collected with clinical data including; gestational age, medications, ethnicity and recognized complications. patients in labor or infection were excluded. the standard bp and proteinuria criteria was utilized to classify cases for pe grouping; no pe (n= ), mild pe (n= bp; / - / , proteinuria; trace to + or - mg/ hr urine) and severe pe (n= bp; > / , proteinuria; > + or > g/ hr, or other associated symptoms). pbef concentration was determined by eia (phoenix pharmaceuticals) in accordance with the manufacturers instructions. fetal membranes and placentas of additional patients, no pe (n= ) and with pe (n= ) were fixed and embedded in paraffin. sections ( um) were immunostained with pbef antibody / (pheonix) and treated with abc reagent (vector labs) followed by dab ( . % mg/ml), washed, counterstained, mounted and viewed under brightfield microscopy. results: the concentrations of pbef in serum were between - ng/ml and were significantly higher in those patients with mild pe (p= . ) and further significantly elevated in those with severe pe (p= . ) compared with the matched controls. pbef was detected by immunocytochemistry in the placental syncytiotrophoblast and in the amnion and choriodecidua of the fetal membranes. conclusions: pbef was elevated in the serum of patients according to the degree of pe severity and may be derived from the placenta and/or fetal membranes. (supported by nih #u rr - to the pacific research center for early human development, university of hawaii). background: platelet-monocyte aggregation (pma) is a novel sensitive measure of platelet activation and indicates a proinflammatory state (cytokine release). less sensitive techniques demonstrate platelet activation during pregnancy and pre-eclampsia (pe) but platelet activation has not been assessed by pma.objective: longitudinal study of pma in normal pregnancy and pe. methods: healthy, non-smoking primigravida with an uncomplicated pregnancy and primigravida women with pe were studied. pe was defined by standard definitions. informed consent was obtained and the study had ethical approval. serial venous blood collected at , , , wks in controls, at time of diagnosis in pe cases and wks post-natal (pn) in all. pma, platelet surface p-selectin (psp-sel) and monocyte cd expression (mcd ) were analysed by flow-cytometry and plasma (p) p-sel by elisa. results: groups were matched for mean age and bmi. in controls, pmas, psp-sel and mcd expression and pp-sel increased with gestation and decreased post-natally (table ) . for pe analysis, data was divided into pre-term (sampled at mean wks), and term (mean wks). pp-sel was lower in pre-term pe than control (normal pregnancy wks; p= . ) there was no significant difference in other measures between pe and control ( objective: much effort has been put into the evaluation of novel markers to identify pregnant women at risk for the development of pre-eclampsia. soluble endoglin (seng) and soluble fms-like tyrosine kinase (sflt ), two antiangiogenic agents, appear to be involved in the pathogenesis of pre-eclampsia. despite several studies describing higher midtrimester serum concentrations of these markers in women with subsequent pre-eclampsia, information on first trimester serum levels is scarce. the aim of this study was to assess seng and sflt as first trimester serum markers for the prediction of pre-eclampsia. methods: sera were obtained between + and + weeks of gestation from women who later developed late-onset pre-eclampsia and from controls matched for gestational age, maternal age, maternal pre-pregnancy weight, and storage. using commercially available microplate enzyme immunoanalytical methods, seng and sflt were determined and the results analyzed using non-parametric statistical tests. results: the serum concentration of seng was found to be increased in women with subsequent pre-eclampsia when compared to controls (mean ± sd, . ± . ng/ml versus . ± . ng/ml, p = . , unpaired mann-whitney test). similarly, the serum levels of sflt were higher in women later developing late-onset pre-eclampsia ( ± ng/ml) when compared to controls ( ± ng/ml, p = . ). sensitivities and specificities for predicting pre-eclampsia were % and % for seng and % and % for sflt- , respectively. the combination of the two markers by multiplication yielded a sensitivity of % and a specificity of %. conclusion: seng and sflt , showing increased first trimester serum levels in women with subsequent pre-eclampsia, might both fulfill the characteristics of first trimester markers to predict pre-eclampsia. the combination of the two, however, did not improve the sensitivity nor the specificity compared to their individual determinations. moderate sensitivities and specifities, however, limit the clinical use of these molecules as single markers. pregnancy is associated with increased monocyte/platelet aggregate formation in whole blood. beth a bouchard, adrienne schonberg, gary j badger, ira m bernstein. biochemistry; obstetrics and gynecology; medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is associated with increased rates of platelet clearance, changes in platelet function and platelet activation. the goal of the current study was to examine basal levels of platelet activation through pregnancy beginning prior to conception, and to examine the association of platelet activation with the development of hypertensive complications during pregnancy. methods two indices of platelet activation, platelet cd expression (%cd ) and monocyte/platelet aggregate (%mp) formation, were measured in whole blood by flow cytometry using specific, fluorescentlylabeled monoclonal antibodies in healthy, nonsmoking women during the follicular phase of their menstrual cycle (pp, cycle day . + . ). all women subsequently conceived singleton pregnancies and were re-examined in early (ep, - wks) and late pregnancy (lp, - wks). five of these women were diagnosed with hypertensive complications ( hypertension, preeclampsia) at term although hypertension was not observed at any study time point. data are expressed as mean±sem. p< . was accepted for significance. results subjects were . + . years old with a bmi of . + . kg/m at the time of prepregnancy studies. a significant increase in the %mp formed over time of pregnancy was observed (p= . ). there was little change in the %mp formed between pp and ep (pp, . ± . % and ep, . ± . %, p= . ). however, the %mp increased significantly in lp ( . ± . %) as compared to pp (p= . ) and ep (p= . ). this increase occurred independent of the development of hypertensive complications (p= . ) and independent of pp platelet activation status. although statistically significant increases in cd expression were not observed, the change in cd expression over pregnancy correlated with the change in %mp over pregnancy (r= . , p= . ) and cd expression correlated with %mp in lp (r= . , p= . ). conclusion these combined observations suggest that pregnancy is associated with increases in levels of unstimulated platelet activation and that these increases occur in the presence or absence of subsequent hypertensive complications. furthermore, we observed a correlation between the changes in two distinct platelet activation events, %mp and cd expression, during pregnancy. supported by nih hl . backgound sildenafil citrate (sc) has been proposed as a therapy to improve uterine perfusion in pregnancies complicated by iugr or preeclampsia. we sought to determine the effects of sc on uterine vascular resistance, uterine blood flow and cardiac output in young healthy nulliparous women. methods eleven young healthy nulligravid women were studied during the luteal phase (cycle day + ) of the menstrual cycle. women were randomized in a double-blind fashion to receive placebo (pl), or sc at a dose of or mg. uterine artery vascular resistance, uterine artery volumetric flow, brachial artery volumetric flow and cardiac output were measured at baseline and at and hours post dosing employing color doppler ultrasound. comparisons were made by anova between those randomized to pl (n= ) versus sc (n= ). p< . was accepted for significance. data are expressed as mean + s.e.m. results there were no significant differences in subject age, cycle day, body mass index, uterine blood flow, brachial blood flow or cardiac output at baseline comparing the two groups. there was a tendency towards increased uterine blood flow in subjects randomized to receive sc ( % increase) compared to pl (no change), changes in uterine blood flow, brachial blood flow and cardiac output are outlined in the objective reduced maternal plasma volume in the third trimester has been associated with both fetal growth restriction and preeclampisa. we sought to determine the degree to which third trimester plasma volume is dependent on plasma volume prior to pregnancy. methods sixteen young ( . + . years) healthy nulligravid women had their plasma volume measured during the follicular phase (cycle day . + . ) of the menstrual cycle and subsequently conceived. subjects were predominantly caucasian ( / ) with a mean prepregnancy bmi of . + . kg/m . plasma volume was re-estimated at - weeks gestation. all patients were placed on sodium and total calorie balanced diets for days prior to each plasma volume determination. plasma volume was determined employing evans blue dilution with multiple post injection sampling time points. data are expressed as mean + s.d. results baseline prepregnant plasma volume was , + ml or + ml/unit bmi. third trimester plasma volume was , + ml representing a % increase (p< . ). the range of plasma volume expansion was - % dependent upon prepregnant plasma volume. plasma volume in the third trimester of pregnancy was strongly correlated to prepregnant plasma volume r= . (p= . ). plasma volume expansion was consistent across the range of prepregnancy plasma volume. conclusions pre-pregnancy plasma volume contributes approximately % of the variance in third trimester plasma volume. the observed increase is plasma volume is independent of prepregnancy volume resulting in a greater percentage increase for those starting at the lower end of the plasma volume range. as third trimester plasma volume is strongly associated with pregnancy outcome the correlation of prepregnancy plasma volume to third trimester plasma volume suggests that prepregnancy status contributes to these adverse reproductive events.this work was supported by nih hl . background: hydatidiform mole is a rare disorder of pregnancy and may predispose the mother to severe morbidity. molar pregnancies are known to be associated with high risk for the development of early onset preeclampsia. in recent years, the expression of sflt- (soluble vegfr- ) was found to be increased in preeclampsia, and contributes to the pathogenesis of the maternal systemic disease. the objective of the present study was to examine the expression of sflt- in placentae from molar pregnancies. methods: placental samples from unique cases of twin pregnancies with complete molar pregnancy in one sac and developing fetus in the other sac were prospectively collected (n= ). the first set delivered at weeks due to excessive bleeding. the second set delivered at weeks due to severe iugr and elevated blood pressure. mrna level of sflt- was measured by quantitative real-time pcr using specific taqman primers and probe. protein expression of sflt- in placental tissue lysates were measured by western blot analysis using a polyclonal antibody against flt- . immunohistochemistry of paraffin embedded samples was performed using specific antibody for sftl- . results: mrna level of sflt- was increased by . fold in the molar placentae compared to matched controls. the placentae of the developing fetuses which were growth restricted exhibited . fold increase compared to controls. sflt- protein expression in the molar placentae was increased by . fold compared to controls, while the co-twin placentae exhibited a . fold increase compared to controls. immunohistochemistry revealed strong positive immunoreactivity for sflt- in the trophoblast layer of both molar pregnancies and iugr co-twin relative to controls. conclusion: our data suggest that sflt- expression is increased in placentae from molar pregnancies and thus may explain the increased risk for developing early onset preeclampsia. the expression of sflt- in the growth restricted twin placenta is also increased compared to controls and support our previous observation (supported by cihr and owh/igh). (n= ); ) neonates of patients with pe (n= ); and ) sga neonates (n= ). cord blood was collected immediately after delivery and pz plasma concentrations were measured by elisa. pz deficiency was defined as a cord plasma concentration < th percentile of the normal pregnancy group. non-parametric statistics were used for analysis. results: ) cord plasma pz concentration differed significantly among the study groups (kruskal wallis, p< . ); ) neonates of patients with pe and sga neonates had a significantly lower median cord plasma pz concentration than those delivered after normal pregnancy (pe: median . g/ml, range . - . , p< . ; sga: median . g/ml, range . - . , p= . ; normal pregnancy: median . g/ml, range . - . ); ) there were no differences in the rate of pz deficiency among the groups; and ) there was no relationship between placental histologic findings and median cord plasma pz concentrations between and among the sga and pe groups. conclusions: ) at the time of delivery, the median cord plasma pz concentration was lower in sga neonates and those born to women with pe than in neonates born to normal pregnancies; ) there was no difference in the rate of pz deficiency among the study groups, suggesting that the lower median pz cord blood concentrations in pe and sga groups may result from activation of the coagulation cascade rather than an inherited pz deficiency. objective: periconceptional multivitamin (mv) use may be related preeclampsia risk. we examined the relation between timing and frequency of periconceptional multivitamin use and the risk of preeclampsia. methods: women in the danish national birth cohort who delivered singleton liveborn infants (n= , ) reported upon enrollment at . weeks (sd . ) the number of weeks of regular multivitamin use during a week periconceptional period (lmp- to lmp+ ). preeclampsia cases were identified using icd- codes (n= , . %). logistic regression was used to estimate the effect of frequency (number of weeks of use) and timing of use to lmp+ ] and post-conception [lmp+ to lmp+ ]). results were stratified a priori by overweight status. results: overall, , women ( %) reported mv use in the periconceptional period. after adjustment for bmi, smoking, parity and chronic hypertension, infrequent mv use (< weeks of use) had no relation to risk (or . ; % ci . , . ) but regular use (>= weeks) was associated with modestly reduced risk ( . ( . , . ). similarly, when mv use was modeled as a continuous variable, each additional week of use was related to reduced risk for preeclampsia (or . , % ci . - . ). this potential dose effect of periconceptional mv use appeared to be limited to normal weight women (bmi < kg/m², or . ; % ci . - . ), with no apparent effect among overweight women (bmi kg/m², or . ; % ci . - . ). a total of , women reported regular mv use in both the preconception and post-conception periods, and , women reported regular use only in the post-conception period. among normal weight women, regular use in the preconception period had no effect on preeclampsia risk (or . , % ci . - . ). in contrast, use in the post conception period was associated with reduced risk for preeclampsia (or . , % ci . - . ). conclusions: regular periconceptional mv use was associated with a modestly reduced risk for preeclampsia among normal weight women. if causal, mv use immediately after conception appeared to be the critical exposure window. background: pre-eclampsia (pe), a disorder of pregnancy characterized by maternal inflammation, results in immune, cardiovascular and metabolic dysfunction. in non-pregnant persons, inflammatory disorders are treated with and prevented by pharmaceuticals and lifestyle methods such as physical activity (pa). while most pharmaceuticals are contraindicated for pregnant women, pa during pregnancy has been found safe, healthy and beneficial for both mother and baby. clinical evidence has found pa can beneficially affect pregnancy outcome, decrease excessive inflammation and decrease the risk of pe. epidemiological studies indicate that pa may be useful in preventing pe. unfortunately, previous studies have quantified pa based on recall of postpartum women and have not controlled for differences in women's interpretations of amount, type or intensity of pa. however, investigating pa utilizing a laboratory-based exercise intervention to control these variables inflicts difficulties translating the intervention into a community-based program that attracts and retains pregnant women in order to enhance public health. method: a retrospective study was performed to determine the rates of pe among the , women who gave birth at yale-new haven hospital (ynhh) during and , and a person subset of this group who performed prenatal pa in a community-based program that is evidence-based and standardized, thereby controlling for type and intensity of pa. additionally, the program is established in the community, has been offered to the public for years and is internationally known. results: during during - , the pe rate for the general population at ynhh was . %. for the pa group, the rate was . %. two women in the pa group were diagnosed with pe in the last month of pregnancy and delivered normal infants at term. no pe was observed in this group (pa group) during the second or early third trimesters nor was there any prematurity in this group. significance: these findings support the hypothesis that adequate physical activity provided in a standardized community-based group setting may provide a non-pharmacological approach for preventing pe. growth restriction. margreet plaisier, esther streefland, pieter koolwijk, frans m helmerhorst, jan jaap hm erwich. department of gynecology and reproductive medicine, leiden university medical center, leiden, netherlands; department of obstetrics and gynecology, university medical center groningen, groningen, netherlands; department of physiology, vu univerity medical center, amsterdam, netherlands. objective: disturbances in decidual and placental vascular development may play a role in the pathogenesis of pregnancy complications, like pre-eclampsia (pe) or fetal growth restriction (fgr). whether the regulation of decidual vascular adaptation to implantation is altered in these illnesses, is not elucidated yet. the present study focused on the role of first-trimester angiogenic factors in the pathogenesis of pe and or fgr. methods: first-trimester decidua samples were obtained during routine chorionic villous sampling. the expression of vascular endothelial growth factor (vegf-a), placental growth factor (plgf), flt- , kdr, angiopoietin- (ang- ), angiopoietin- (ang- ) and tie- mrna was determined by rt-pcr. the expression of the angiogenic factors was related to the pregnancy outcome, i.e. uncomplicated, pe or fgr. results: the first-trimester decidual tissues expressed all angiogenic factors. mrna levels of vegf-a, plgf, kdr, ang- , ang- and tie- appeared increased in fgr cases compared to matched controls. in addition, plgf, ang- and tie- mrna appeared increased in pe cases compared to matched controls. the differential expression of angiogenic factors was more pronounced in cases with fgr than pe. the large inter-individual variation disallowed a significant outcome. conclusions: various angiogenic factors showed differential mrna expression in st trimester decidua of patients developing pe or fgr in later pregnancy compared to their matched controls. the first-trimester decidual samples provided a unique opportunity to obtain information regarding the onset of pe and fgr. early st trimester changes in angiogenic factor expression may well occur as a compensatory mechanism. in turn, this may set the stage for increased non-branching angiogenesis and altered decidual and placental vascular adaptation, which may be part of the pathogenesis of pe and/or fgr. complications. beth a bouchard, adrienne schonberg, gary j badger, ira m bernstein. biochemistry; obstetrics and gynecology; medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is characterized by endothelial dysfunction. the goal of the current study was to prospectively measure plasma levels of the soluble endothelial cell adhesion molecules, sicam- and svcam- , beginning prior to pregnancy and determine if subjects destined to develop hypertension complicating pregnancy had differences in the concentrations of these molecules. methods serum levels of sicam- and svcam- were measured in healthy, nonsmoking women (cycle day . + . , prepregnancy) by elisa. all women subsequently conceived singleton pregnancies and were re-examined in early (ep, - weeks) and late pregnancy (lp, - weeks). five of these women developed hypertensive complications ( gestational hypertension, pre-eclampsia) near term. all subjects were normotensive at all study time points. data are expressed as mean ± sem. p< . was accepted as significant. results subjects were . + . years old with a mean bmi of . + . kg/m at the time of prepregnancy studies. significant differences in sicam- levels as a function of pregnancy were observed (p= . ) and are outlined in table p= . differences were dependent upon the stage of pregnancy in those women who were not diagnosed with hypertensive complications with a decrease in sicam- levels in ep (p= . ) followed by an increase in sicam- levels in lp (p= . ). in women with hypertension in pregnancy, these differences in sicam- levels were not evident (p= . ). there were no differences in sicam- levels comparing women with or women without hypertensive complications prior to pregnancy (p= . ). in contrast to sicam- , we observed no significant differences in svcam- levels over pregnancy or between those with and without hypertension. conclusions these combined observations suggest that levels of the soluble adhesion molecule sicam- change significantly over time in normal pregnancies. subjects destined to develop hypertension did not demonstrate the early pregnancy reduction in sicam- . supported by nih hl . yuval bdolah, uriel elchalal, shira natanson-yaron, hadas caspi, tali bdolah-abram, angelika bord, caryn greenfield, debra goldman-wohl, ariel milwidsky, franklin h epstein, s ananth karumanchi, simcha yagel, drorith hochner-celnikier. ob/ gyn, jerusalem, israel; ob/gyn medicine, beth israel deaconess medical center, harvard medical school, boston, ma, usa. objectives: nulliparity is a risk factor for preeclampsia (pe) with a reported incidence of up to - times higher than multiparous pregnancies. soluble fms-like tyrosine kinase- (sflt ), a circulating anti-angiogenic molecule of placental origin plays a pivotal role in pe by antagonizing placental growth factor (plgf). increased sflt and sflt /plgf have been shown to antedate clinical signs in pe. we therefore hypothesized, that the higher risk of pe in nulliparous pregnancies is associated with high sflt (or sflt /plgf). methods: maternal serum samples from nulliparous (n= ) and multiparous (n= ) term singleton pregnancies without pe, at the time of admission to delivery room, were used. serum samples were analyzed for levels of sflt and plgf by elisa. statistical analysis was performed applying t-test and the kruskall-wallis test and using spss software. results: for nulliparous and multiparous pregnancies, the mean serum sflt levels were , ± and , ± , (p= . ), the mean serum plgf levels were ± and ± (p= . ), and the mean ratios of sflt- /plgf were ± and ± (p= . ), accordingly. in a subgroup of multigravidous nulliparous pregnancies, sflt levels were , ± . correcting for maternal age did not alter the results. moreover, results did not differ between multiparous pregnancies with a - years interpregnancy interval compared with a - years interval. conclusions: in nulliparous pregnancies, circulating sflt levels and sflt / plgf ratios are significantly higher than in multiparous pregnancies. these findings suggest that the increased risk of pe in nulliparous pregnancies may involve anti-angiogenic imbalance. nulliparity may be more substantial than primigravity, as a risk factor for pe, suggesting that first semester abortions in primigravidas may not protect from pe in a subsequent term pregnancy. nevertheless, even - years intervals from the previous gestation do not increase the risk for pe. different normograms of angiogenesis should be used, when assessing the risk for pe in multiparous versus nulliparous pregnancies. objective: we determined whether maternal serum levels of angiogenic proteins namely soluble fms like tyrosine kinase (sflt- ), soluble endoglin (seng), and placental growth factor (plgf) -measured during the first trimester are associated with the subsequent development of placental abruption. methods: we performed a prospective, nested case-control study of women enrolled in the massachusetts general hospital obstetric maternal study (moms). first trimester serum samples from placental abruption cases and normal pregnancies were measured for angiogenic factors. cases and controls were matched by body mass index and age. placenta abruption was diagnosed by standard clinical findings and pathological examination of the placenta. women with confirmed preeclampsia or chronic hypertension were excluded. results: compared to controls, cases had more pregnancies, delivered infants at an earlier gestational age and with lower birth weight. first trimester levels of seng were significantly increased in cases compared to controls: . ± . ng/ml vs. . ± . ng/ml, p < . . there were no significant difference in serum levels of plgf, . ± . ng/ml versus . ± . ng/ml, p=ns, although sflt levels were lower in cases: . ± . ng/ml vs. . ± . ng/ml, p=ns. in logistic regression analysis adjusted for age, race, smoking, number of pregnancies, gestational age at delivery, gestational age of blood sampling, and blood pressure at first prenatal, seng levels remained independently associated with subsequent risk (odds ratio . , % ci . - . ) of placental abruption. examining this relationship by tertiles of seng, in the unadjusted model, women in the second (or . , % ci . - . ) and third (or . , % ci . - . ) tertiles were at increased risk of developing placental abruption compared with women in the lowest tertile. after adjusting for known risk factors of placental abruption, women in the second (or . , % ci . - . ) and third (or . , % ci . . ) tertiles remained at increased risk for placental abruption. conclusion: increased first trimester maternal serum levels of seng are associated with increased risk of subsequent placental abruption. ob/gyn, univ of vermont. shear stress is the most potent physiologic stimulus for elevating endothelial no production for flow-mediated vasodilatation. we measured in vivo shear stress during the proliferative and secretory phases and at and weeks of gestation hypothesizing that uterine blood flow (ubf) elevations in turn increase shear stress in early and late gestation. methods: during proliferative, secretory phases, and at and - weeks of pregnancy ua blood velocity and internal radius were measured bilaterally using color doppler ultrasound. blood viscosity was measured at shear rates in excess of /sec. results: compared to the proliferative phase viscosity was decreased at weeks and more so at weeks gestation (p< . ). . ± . . ± . nsd . ± . *** . ± . *** ua velocity (cm/sec) . ± . . ± . *** . ± . *** . ± . *** ubf (ml/min) . ± . ± *** . ± . *** . ± . *** shear stress (dynes/cm ) . ± . . ± . *** . ± . *** . ± . *** internal ua radius was not altered by the menstrual cycle, and was greater at weeks (+ %) and weeks (+ %). compared to proliferative, secretory phase showed significant rises in unilateral ubf and velocity that rose progressively during gestation. in contrast, shear stress increased in secretory ( %) and did not rise further in early pregnancy but by weeks shear stresses was further elevated %. conclusions: equivalent rises in shear stress during the secretory phase and weeks gestation demonstrate increases in radius and profound remodeling of uas that reflect the physiologic process of "normalization of shear". by late gestation, continued but modest rises in radius illustrate that further increases in shear stress occur almost solely due to rises in ubf via falls in down stream impedance. continued rises in shear stress into late gestation provide progressive stimuli for no production by ua endothelium. nih hl , hd , hl background: hypertension during pregnancy is associated with altered uterine vascular reactivity and blood flow, although its effects on arterial myogenic behavior have not been explored. the purpose of this study was to evaluate the effects of hypertension and no inhibition on myogenic tone in pregnancy, as the ability of a vessel to constrict and dilate in response to pressure plays a key role in regulating blood flow to the uterus. methods: three groups of sprague dawley late pregnant (day ) rats were used: control (n= ), hypertensive ( . g/l l-name in the drinking water, n= ), and treated with l-name and hydralazine (also in the drinking water, . g/l, n= ) to prevent the blood pressure increase, yet maintain no inhibition. resistance-sized radial arteries (< m) were mounted in a pressure arteriograph and equilibrated at mmhg (in pss containing l-nna and indomethacin) to induce a myogenic response. vessels were then subjected to pressure steps from to mmhg. tone (%) was calculated by comparing the vessel diameter at each pressure with the passive diameter at the same pressure (determined by incubation with . mm papaverine and m diltiazem). results: myogenic tone developed in controls ( ± % maximal), and was maintained over a broad range of transmural pressures . arteries from the l-name group did not develop tone at any pressure. co-treatment with hydralazine reinstated tone ( ± % maximal) over the same range of pressures as in the control group. the reduction in average placental weights in the l-name group ( vs. mg, p< . ) was restored by hydralazine ( mg, p< . vs. l-name). average fetal weights were also reduced in the l-name group ( . vs. . g, p< . ), but only partially restored by hydralazine co-treatment ( . g, p< . vs. control and l-name groups). conclusions: surprisingly, radial uterine arteries from the l-name group did not develop tone over any range of pressures, despite the fact that matched arteries from late-pregnant controls developed significant myogenic tone. this abolishment of tone was reversed by hydralazine, which also had beneficial effects on fetal and placental growth. these results implicate hypertension rather than no inhibition as the key factor in the suppression of myogenicitiy and dysregulation of uterine blood flow. charles r rosenfeld, timothy roy. division of neonatal-perinatal medicine, ut southwestern medical center at dallas, dallas, tx, usa. background: upbf b rises -fold in ovine pregnancy; but the mechanisms responsible for the rise and maintenance are unclear. we (jsgi ) reported that uterine vascular smooth muscle bk ca k + channels contribute to uterine vasodilation and upbf b maintenance; but up-stream activators are unclear. uterine vascular prostacyclin synthesis increases in pregnancy, but cyclooxygenase inhibition does not alter upbf b (ajp ) . vascular nitric oxide synthase (nos) also increases; but acute inhibition with l-name decreases upbf b only % (jci ) . it is unclear if l-name doses were insufficient or if prolonged nos inhibition has a greater effect. objective: to determine if local nos inhibition with l-name dose-dependently decreases upbf b and if prolonged inhibition exerts a greater effect on upbf b . methods: pregnant ewes were studied at - d gestation age (ga). had doseresponse studies with uterine artery l-name infusions to achieve . - . mg/ml over min. had h arterial l-name infusions to achieve and maintain levels of . mg/ml at (n= ), (n= ) and d (n= ) ga, while measuring arterial pressure (map), heart rate (hr) and upbf b before, during ( , , , , , , and h) and after ( , h) infusions. uterine arterial and venous cgmp were measured. data were analyzed by anova. results: acute nos inhibition decreased upbf b - %, but was not doserelated. h arterial l-name infusions decreased upbf b by - h at each ga (p . , anova) and values returned to baseline by h postinfusion. sensitivity did not differ between ga (p= . , anova), upbf b falling - % at each ga. contralateral upbf b was unaffected at all ga. map rose and hr fell during infusions at and d ga, p . ; but were unaffected at d ga. venous-arterial cgmp concentration differences were seen at d and absent at h of l-name infusion, p= . . conclusions: uterine vascular nos increases in ovine pregnancy, but its inhibition decreases upbf b %, suggesting study doses were insufficient to fully inhibit vascular nos activity. alternatively, nos contributes to the maintenance of upbf b , but other mediators, not yet identified, are more important in activating bk ca and regulating upbf b . notably, l-name reached the systemic circulation, and although further diluted, map rose - %, suggesting the systemic vasculature may be more sensitive to nos inhibition than upbf b . charles r rosenfeld, xiao-tie liu. division of neonatal-perinatal medicine, university of texas southwestern medical school, dallas, tx, usa. background: uteroplacental blood flow (upbf) rises -fold in ovine pregnancy, reflecting increases during pre-implantation, placentation and finally, in the last third of gestation. we reported that bk ca density in uterine vascular smooth muscle (uvsm) increases in ovine pregnancy (sgi ) and inhibition with tetraethylammonium ( . mm) dose-dependently decreases basal upbf % in the last third of pregnancy (jsgi ) . it is unclear how bk ca subunit expression changes and activity is regulated in pregnancy; since uterine vascular nitric oxide is increased, signaling via cgmp-dependent protein kinase g (pkg) is one potential pathway. objective: to determine simultaneous changes in uvsm bk ca density and regulatory -subunit expression and the cgmp signaling pathway for activation in ovine pregnancy. methods: endothelium-denuded segments of nd generation uterine arteries obtained from nonpregnant (n= ), pregnant (n= , - d gestation; term d) and postpartum (n= , - d) sheep were used to measure expression of bk ca -and regulatory -subunits and signaling proteins in uvsm by immunoblot analysis and immunohistochemistry. results: uvsm bk ca density, reflected as a change in the kda -subunit, rose % with placentation (p< . ) and was unchanged thereafter. expression of the kda regulatory -subunit paralleled the rise in bk ca density during placentation, increasing % (p< . ), but increased another -fold and exponentially in the last third of pregnancy (p< . , r = . , n= ). changes in subunit immunostaining in uvsm paralleled increases in protein. although uvsm soluble guanylyl cyclase was unchanged in pregnancy (p= . , r= . , n= ), pkg expression rose -fold (p= . , r= . , n= ) and gradually returned to nonpregnant levels by d postpartum (p= . , r= . , n= ). uvsm cgmp is being measured. conclusions: these are the st data suggesting that increases in ovine upbf during placentation involve vascular growth and bk camediated vasodilation. the rise in upbf in the last third of pregnancy reflects bk ca -mediated vasodilation due to enhanced channel activation via increases in uvsm pkg, bk ca phosphorylation and changes in subunit stoichiometry due to an exponential rise in the regulatory -subunit. it is unclear what initiates and directs these changes in bk ca expression and sensitivity. relaxin (rlx) is a hormone traditionally associated with changes in the female reproductive tract during pregnacy. recent evidence suggests that rlx may play a pivotal role in regulating cardiovascular function during gestation. analogous to pregnancy, administration of recombinant human relaxin (rhrlx) to nonpregnant female rats reduces systemic vascular resistance, as well as increases global arterial compliance. additionally, we demonstrated that, concurrent with rlx´s influence on overall cardiovascular function, small renal arteries (sra) from rhrlx-treated mice and rats are characterized by reduced passive stiffness and increased arterial wall area whereas external iliac arteries (eia) are not. we hypothesized that rlx regulates arterial passive mechanical properties by altering the cellular and biochemical composition of the arterial wall. nonpregnant female mice were administered rhrlx for days after which sra and eia were isolated. we measured arterial collagen, elastin, and total protein using the sircol collagen assay, the fastin elastin assay, and the pierce bca protein assay, respectively. additionally, we quantified arterial smooth muscle cell (smc) density using immunofluorescent techniques. sra isolated from rhrlx-treated mice were characterized by a significant reduction in collagen to total protein ratio ( . ± . vs . ± . μg collagen/μg protein; mean±sem; p< . ) as well as a significant increase in smc density ( . ± . vs . ± . cells/ μm ; p< . ) compared to control mice with no significant change in elastin content ( ± vs ± μg elastin/mg dry weight). in contrast, there were no significant changes in collagen to total protein ratio ( . ± . vs . ± . μg collagen/μg protein), smc density ( . ± . vs . ± . cells/ μm ) or elastin content ( ± vs ± μg elastin/mg dry weight) in eia from the rhrlx-treated mice compared to control mice. of note, comparable results were observed for rlx knock-out (rlx -/-) and wild-type mice with rlx -/mice exhibiting increased arterial collagen and decreased smc density. we conclude that the rlx-induced decrease in passive stiffness of sra that we previously reported is, at least in part, due to rlx-induced alterations in arterial wall cellular and biochemical composition. further, our findings suggest that these vessel wall remodeling effects of rlx are artery-type specific. relaxin is a peptide hormone that emanates from the corpus luteum of the ovary and circulates during pregnancy. this hormone plays an important role in renal vasodilation and hyperfiltration, two fundamental maternal adaptations in pregnancy. chronic administration of recombinant human relaxin (rhrlx) to both virgin rats and mice inhibits myogenic reactivity and increases compliance of small renal arteries, thus further mimicking pregnancy. we hypothesize that these arterial responses to rhrlx are mediated by the lgr , and not the lgr receptor. both lgr and lgr receptor-deficient, and wild-type virgin mice were investigated. the mice were chronically infused with rhrlx or vehicle (veh) for days. small renal arteries were isolated and mounted in a pressure arteriograph and myogenic reactivity was assessed (% change in diameter over baseline in response to a mmhg step increase in intraluminal pressure). small renal arteries from rhrlx-infused lgr wild-type mice showed inhibited myogenic reactivity with a . ± . % increase in diameter whereas the arteries from rhrlx-infused lgr knock-out mice exhibited robust myogenic reactivity with only a . ± . % change in diameter (p= . ). in contrast, myogenic reactivity of small renal arteries was inhibited in both the rhrlx-infused lgr knock-out and wild-type mice. the veh-treated lgr and lgr mice, regardless of genotype, exhibited robust myogenic reactivity. arterial compliance was also assessed for each genotype/treatment group. rhrlx infusion increased arterial compliance of small renal arteries from lgr wild-type, but not from lgr knock-out mice (p= . ). in contrast, the arteries from rhrlx-infused lgr wild-type and knock-out mice showed increased compliance relative to veh-infused animals. conclusion: relaxin-induced inhibition of myogenic reacitivity and increase in compliance of small renal arteries is mediated by the lgr , and not the lgr receptor. smoking is associated with adverse pregnancy outcomes including fetal growth restriction. pathologic effects of smoking on maternal vasculature is a potential mechanism leading to fetal growth restriction. the objective of this study was to determine whether cigarette smoke exposure during pregnancy affects the functional properties of uterine and peripheral arteries using a gravid murine model. study design: pregnant and virgin c bl/cj mice were exposed to whole body side stream smoke using an inhalational chamber for hours/day. smoke exposure was increased from day of gestation until late pregnancy (day - ) with mean total suspended particle levels of mg/m , representative of moderate to heavy smoking in humans. control animals were exposed to ambient room air. late pregnant and virgin mice were sacrificed and uterine, mesenteric, and renal arteries were isolated and studied in a pressure arteriograph system (n= - in each group). plasma cotinine was measured by elisa. means were compared using t-test or analysis of variance. results: fetal weights were significantly reduced in mice exposed to smoke compared to control fetuses ( . ± . g vs . ± . g, p= . ), while litter sizes were not different. cotinine levels in smoking mice were significantly elevated compared to control mice ( . ± . vs . ± . ng/ml for virgin mice and . ± vs . ± . ng/ml for pregnant mice). there was no significant difference in phenylephrine responses between groups. endothelial mediated relaxation responses to methacholine were significantly impaired in both the uterine and mesenteric vasculature of pregnant mice exposed to cigarette smoke during gestation. no difference in endothelial-mediated relaxation was seen in isolated renal arteries in pregnant mice exposed to cigarette smoke, however relaxation was significantly reduced in renal arteries from smokeexposed virgin mice. conclusions: passive cigarette smoke exposure is associated with impaired vascular relaxation of uterine and mesenteric arteries in a gravid murine model. functional vascular perturbations during pregnancy, specifically reduced uterine blood flow and impaired peripheral vasodilation, may be a mechanism by which smoking results in fetal growth restriction. human chorionic gonadotropin (hcg) is essential during early human gestation for "rescue" of the corpus luteum. however, its potential contribution to the widespread maternal vasodilation of pregnancy that occurs at this stage of pregnancy remains uncertain. our objective was to use the renal circulation of conscious rats as an experimental model in which to test the vasodilatory potential of hcg. in addition, we investigated both myogenic reactivity and relaxation responses in small renal and mesenteric arteries isolated from rats, as well as in small human subcutaneous arteries using a pressure arteriograph. we chronically instrumented rats for measurement of renal function. five were ovariectomized, and sham-ovariectomized. after days of surgical recovery, baseline glomerular filtration (gfr) and effective renal plasma flow (erpf) were measured on two separate days and the values averaged. then, an osmotic minipump containing hcg ( iu/min) was implanted s.c. and renal function was again assessed and days thereafter. gfr and erpf significantly increased and calculated effective renal vascular resistance decreased from baseline in the intact (p< . vs. baseline), but not ovariectomized (p=ns) rats on both days and of hcg administration. in the intact, but not ovariectomized rats, plasma osmolality declined and progesterone increased (both p< . vs baseline). plasma hcg concentrations were , miu/ml and comparable in both groups of rats. incubation of small renal arteries from rats with recombinant human relaxin (rhrlx, - ng/ml), but not hcg ( , - , miu/ml) in vitro inhibited myogenic reactivity and relaxed phenylephrine (pe)-constricted arteries. in contrast, both rhrlx and hcg inhibited myogenic reactivity and relaxed pe-constricted small mesenteric arteries from rats and small human subcutaneously arteries. in conclusion, consistent with our earlier work showing a virtually exclusive role for relaxin in mediating the renal circulatory changes of pregnancy, hcg is likely to play little or no role. in contrast, hcg and relaxin are both likely to contribute to the vasodilation of other organ circulations during pregnancy. pierre-andre scott, , michele brochu, jean st-louis. , research centre, chu ste-justine, montréal, qc, canada; pharmacology, université de montréal, montréal, qc, canada. uterine vasculature undergoes major structural and functional changes during pregnancy. estrogens have been shown to induce increased uterine blood flow in this circulation. we have reported that estradiol ( -e ) induced direct vasorelaxant response on smooth muscle of the uterine arteries that, for it major part, is not mediated through tissue nitric oxide. to investigate the cellular effectors mediating this vasorelaxant effect of -e , we set-up uterine arteries of non-pregnant rats in wire myograph systems for microvessels. -e and -e were equipotent in relaxing phe ( μmol/l)-preconstricted uterine arteries, although the later produced significant smaller relaxations. genistein, a phytoestrogen presumed to inhibit phosphatases, also produced uterine artery relaxation with significant lower potency. to try interfere with the vasorelaxant effect of -e , tissues were preincubated with different substances. cycloheximide (protein biosysthesis inhibitor), ici , (estrogen receptor modulator), and kt (pka inhibitor) did not significantly influenced the response to -e . rp- -pcpt-cgmps (pkg inhibitor) slightly displaced the concentration-relaxation curve to -e to the right. inhibitors of potassium channels, penitrem a (bkca) and glybenclamide (katp), showed opposite effects for -e concentration-response curve; the former producing a right shift and the latter a small not significant left shift. these data indicated that the direct acute effect of -e in uterine artery is the result of complex interactions within smooth muscle cells, involving potassium channels, and protein kinases and phosphatases. the renin-angiotensin-aldosterone system is paradoxically activated during pregnancy, since blood pressure decreased. earlier data showed that the high levels of aldosterone present during pregnancy may be involved in cardiovascular adaptation to pregnancy. in order to delineate this effect of aldosterone on vascular tone, potassium canrenoate, an antagonist of mineralocorticoid receptors (mr), was administered ( mg/kg/day) to pregnant rats from the day to of gestation. rats were sacrificed at day , and of gestation together with untreated and day pregnant rats. the thoracic aorta was quickly removed and set up in tissue baths as ring ( - mm) preparation. as observed previously, canrenoate enhanced responsiveness to phe for all time of treatment tested, but only at day ( days treatment) for kcl responsiveness. aortic contractile responses to tea (tetraethylammonium) gradually decreased during pregnancy to almost disappear at the end of gestation. in the present results, canrenoate treatment made the response statistically different by day of pregnancy. finally, the activity of na/k-atpase was measured by the relaxant effect of kcl added to physiological solution without potassium. the activity of the pump was decreased when approaching parturition compare to day of pregnancy. canrenoate treatment abolished this effect. the present data show that vascular changes that occurred during pregnancy are markedly modified by treatment of rats with an antagonist of mineralocorticoid receptors, suggesting that aldosterone may be involved in vascular adaptation to pregnancy. background impaired endothelium-dependent vasodilatation has previously been demonstrated in small myometrial arteries from women with gestational diabetes. this impairment may play a role in mediating the complications observed in diabetic pregnancies. it is not known which mechanisms of endothelium-dependent vasodilatation are affected in myometrial arteries by gestational diabetes. aim to investigate mechanisms of endothelium-dependent vasodilatation in uterine arteries using a mouse model of pregnancy complicated by diabetes. methods diabetes was induced in female c bl /j mice (streptozotocin; mg/kg) prior to mating. mice were culled at day of pregnancy (term) and uterine arteries dissected, mounted on a wire myograph, normalised to mmhg and equilibrated ( °c; %co /air). arteries were constricted with phenylephrine ( m) and a concentration-response curve to the endotheliumdependent vasodilator acetylcholine (ach; . nm- m) constructed in the presence and absence of a nitric oxide synthase inhibitor (l-nna; m), a cyclooxygenase inhibitor (indomethacin; m) or a combination of the two to determine the contribution of nitric oxide (no), prostacyclin and endothelium derived hyperpolarising factor (edhf) to vasodilatation. results sensitivity to ach was comparable between diabetic and vehicle treated mice (ec . ± . nm vs . ± . nm). the contribution of individual endothelium-dependent vasodilators was significantly altered in arteries from diabetic mice. at m ach, edhf-mediated relaxation was significantly reduced (p= . , one-way anova) compared with controls ( . ± . vs . ± ). in comparison, no-mediated relaxation was significantly increased (p= . , one-way anova) compared with controls ( . ± . vs . ± . %). conclusions endothelium-dependent relaxation was not reduced in uterine arteries of diabetic mice compared with controls. however, there was a profound change in the contribution of endothelium-dependent vasodilators in arteries of diabetic mice. this may alter compensatory capacity as disease progresses. supported by mfn training grant (cihr). raf- serine/threonine protein kinase has been extensively studied as the upstream kinase linking ras activation to the mek/erk module. mek/erk has been shown to play a role in the modulation of vascular contraction. however, the role of raf kinase in vascular contraction and its possible involvement in alteration of maternal vascular function during pregnancy is not known. objectives: to determine ( ) if raf kinase contributes to phenylephrine (pe)induced contractile response, ( ) the role of raf kinase inhibitor (gw ) in regulating vascular tone during pregnancy, and ( ) mechanism by which gw produces vasodilatation in rat mesenteric arteries. methods and results: conscious non pregnant (np) and pregnant (p) sprague dawley rats received increasing doses of pe in the absence or presence of gw . pe induced a dose-dependent increase in map in both np and p rats but responses were substantially depressed in pregnancy. gw shifted the pe-induced dose response to the right in both np and p rats. gw itself did not affect basal blood pressure. isometric tension studies in mesenteric arteries showed that gw did not change the kcl-evoked contraction but significantly inhibited the contraction to pe in both np and p arteries. interestingly, at a given concentration, gw produced greater inhibition of pe-induced contractile response in p than in np arteries. also, in p arteries the inhibitory effects of gw were greater as the pregnancy progressed from day to day . raf kinase expression and activity was significantly decreased in arteries of p compared to np rats. in mesenteric vascular smooth muscle cells (vsmcs), pe stimulated activation of raf kinase as indicated by phosphorylation of its immediate target, mek / in a time-and dose-dependent manner. measurement of [ca + ] i with fura- showed that gw -mediated inhibition of pe-induced contraction was not associated with decrease in [ca + ] i . vsmcs treated with pe exhibited higher levels of the contractile proteins, p-mypt and p-mlc which was inhibited by gw . conclusion: to our knowledge, for the first time we show that raf kinase plays an important role in the regulation of vascular contractility. decreased expression and/or activity of raf kinase during pregnancy may explain in part, for decreased systemic vascular reactivity during late gestation. the mechanism(s) that contribute to reduced vascular sensitivity to phenyleperine (pe) during pregnancy is not well understood. pe, in addition to activating the classical contractile pathways, also stimulates growth factor pathways that results in activation of ras/mitogen-activated protein kinases. it is not clear whether these pathways play a role in the modulation of vascular contraction. hence, the present study was designed to determine ) if ras is involved in mediating the pressor response to pe in non pregnant (np) and pregnant (p) rats, ) if so, the mechanism by which ras protein contributes to pe-induced vascular contraction, and ) any differential expression and/or activity of ras during pregnancy that might contribute to altered vascular response. methods: ) mean arterial pressure (map) was measured in conscious np and p rats. ) isometric tension was measured in mesenteric arteries of np and p rats. ) expression of contractile proteins, p-mlc and p-mypt were studied in cultured vascular smooth muscle cells (vsmcs). ) expression and activity of ras in mesenteric arteries of np and p rats were measured by western blot analysis. results: ( ) intravenous administration of pe resulted in a dose-dependent increase in map but responses were substantially depressed in pregnancy. inhibiting ras activation with manumycin, decreased pe-induced increase in map in both np and p rats. manumycin by itself had no effects on basal map. ( ) isometric contraction studies in myograph with mesenteric arteries showed that manumycin shifted pe-induced dose response curve to the right with a decrease in e max in np and p rats. higher concentration of manumycin was required to inhibit pe-induced contraction in np ( μm) compared to p ( - μm) rats. ( ) pretreatment with manumycin inhibited pe-induced increase in the extent of phosphorylation of both mlc and mypt in mvsmcs. ( ) compared to p rats, mesenteric arteries of np rats revealed increased basal and pe-induced expression and activity of ras. reduced expression of this contractile ras pathway might contribute to decreased vascular reactivity during pregnancy. conclusion: ras protein plays a role in regulation of vascular contraction. the decreased amount and activity of vascular ras may be a novel mechanism that explains the reduced vascular resistance during pregnancy. overweight affects the relaxin-induced response of mesenteric arteries. joris van drongelen, arianne van koppen, marc ea spaanderman, paul abm smits, frederik k lotgering. obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; pharmacology and toxicology, radboud university nijmegen medical centre, nijmegen, netherlands. objective: overweight affects pregnancy-induced vascular adaptation and predisposes to gestational hypertensive disease. relaxin plays a key role in normal gestational vascular adaptation by increasing endotheliumdependent vasodilation and compliance, and reducing myogenic reactivity. we hypothesized that overweight blunts the vascular response to relaxin. the vascular responses to flow and pressure of mesenteric arteries after pre-treatment to human recombinant relaxin (rlx) or placebo (plac) were examined in overweight (ow) and normal weight (nw) rats in a pressure-perfusion myograph. overweight was established by a high-fat diet. the endothelium-dependent vasodilatation was measured in response to flow: e (flow inducing % dilatation) and e max (maximal dilative effect). active contractile (myogenic reactivity) and passive dilative (compliance) vascular responses to pressure were determined. all vascular responses were calculated as the proportional change in diameter to % precontraction with u . results: in nw rats rlx decreased e and increased e max to flow and attenuated myogenic reactivity without affecting vascular compliance. in ow plac-treated rats, compared to nw rats, an upregulated vasodilative state was present (decreased e and increased e max , and lower myogenic reactivity). in ow rats rlx increased e to flow and unaffected e max . rlx increased myogenic reactivity mildly in absence of changes in vascular compliance. values are presented as mean±sem; * p< . between nw/ow, # p< . within nw/ow. whereas rlx stimulates endothelium-dependent vasodilation to flow and myogenic reactivity in nw rats, ow overturns the flow-induced response and decreases myogenic reactivity to a lesser extent than present in nw rats. we speculate that these overweight-induced adverse effects of rlx prelude to vascular maladaptation and gestational hypertensive disease. compared to the luteal (lut) phase, uterine blood flow is increased in vivo during the follicular (fol) phase and more so during pregnancy (preg). both of these are physiologic states of high estrogen and shear stress. endothelial cells express enos and produce greater amounts of nitric oxide (no) in response to elevations of shear stress. phosphorylation of enos is a signaling marker of activation. we have recently validated lut, fol and preg uaec culture models for evaluating enos phosphorylation responses to shear stress and vascular mediators. we hypothesized that uaecs derived from fol and preg sheep will show greater enos phosphorylation than lut phase uaecs, and with more robust responses in the presence of estrogen (e ). methods: uaecs were cultured until % confluence, and then subjected to (static control), or dynes/cm for hrs in the absence or presence of e ( nm). western analysis was used to compare optical densities of ser -penos (penos) normalized to total-enos (mean + sem). results: in lut uaec penos was equally increased two fold by and dynes/cm (from . + . to . + . and . + . , respectively). compared to lut uaecs, the static control fol uaecs appeared to have higher penos levels ( . + . ) and this was further increased . - . fold with dynes/cm ( . + . ) and dynes/cm ( . + . ). as seen with fol uaecs, preg uaec static penos ( . + . ) appeared higher than lut uaec, but unexpectedly, neither nor dynes/cm significantly raised these levels of penos ( . + . and . + . ). regardless of shear stress level, e replacement in the culture media only increased the penos levels in the nonpregnant, but not the pregnant derived uaecs. conclusions: increasing amounts of shear stress have a corresponding increase in the ratio of enos that is phosphorylated at serine in lut and fol uaecs. pregnant uaecs do not appear to increase the constitutive ratio of serine phosphorylation of enos at either or dynes/cm . in contrast to our hypothesis, chronic treatment with e for hrs did not augment the ratio of enos phosphorylation in pregnancy. nih hl , hd , hl . cells. honghai zhang, wu xiang liao, dong-bao chen. reproductive medicine, university of california san diego, la jolla, ca, usa. s-nitrosylation (sno) is a rapid, reversible, and nitric oxide (no) dependent post-translational protein modification critical for signal transduction. estrogen stimulates endothelial cell (ec) no production but yet to be determined is if this leads to increased formation of sno-proteins in ec. hypothesis: we hypothesize that estrogen stimulates the formation of sno via a receptor and endogenous no dependent pathway in huvec or ovine uterine artery ec. methods: cells on glass coverslips were treated with mm of no donor gsno or dea nonoate, estradiol- ( nm) in the presence of l-name ( mm) for min. the cells were fixed with methanol. in situ sno-proteins were detected by a rapid biotin derivatization method by blocking thiols by methylmethanethiosulfonate (mmts), followed by ascorbate reduction and labeling with texas red-labeled ethylmethanethiosulfonate (mtsea) and examined by fluorescence microscopy. cells ( x /group) were lysed in hen buffer, and then similarly prepared but labeled with biotin-mtsea. a portion of the samples were separated on sds-page and blotted with anti-biotin antibody for total sno-protein patterns. the biotin-labeled sno-proteins were captured by avidin, followed by immunoblotting with specific antibodies. results: basal sno-protein labeling was apparent in both ec types. exogenous no donated by gsno or dea nonoate significantly increased red fluorescence labeling of sno-proteins in the cytosol of both ec types. treatment with estradiol- also increased total sno-protein labeling in both ec types. pretreatment with l-name significantly reduced sno-protein labeling. sds-page and immunoblotting with anti-biotin antibody analyses identified multiple bands of sno-proteins. in avidin captured biotinylated samples, multiple proteins were indentified. conclusion: exogenous no donated by gsno or dea nonoate and endogenous no upon estrogen stimulation increased s-nitrosylated proteins in ec (supported by nih ro hl and ). background and objective: the renin-angiotensin system (ras) functions both systemically and locally as a primary regulator of blood pressure and fluid volume and is therefore involved in the etiopathogenesis of essential hypertension as well as preeclampsia, a pregnancy-induced hypertensive disorder in pregnant women. while much progress has been made in the development of strategies to block the systemic ras and thus treat essential hypertension, relatively less advance has been achieved in the development of effective local ras inhibitors to treat preeclampsia, primarily due to the fact the conventional systemic ras inhibitors generate potential teratogenic risks to pregnant women during critical stages of their pregnancies. ginger has been widely and effectively used in pregnant women to treat pregnancyinduced nausea and vomiting with minimal adverse effects. the present study was designed to investigate whether ginger could down-regulate renin secretion by human decidual cells (the major source of renin in the tissue-based uteroplacental ras), as an initial step toward a long-term goal to develop potential safe and effective ras inhibitors for use in obstetric patients. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for days in a serum-containing medium, the decidual cells were treated with ginger juice at various doses in a serum-free medium for hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. the ginger juice used was freshly prepared from the ginger roots purchased from a local grocery store and different batches of juice preparations were standardized based on their optical density values at a wavelength of μm on a spectrophotometer. results: a dominant band of renin at approximately kd was detected in all samples. when compared with the control (i.e. %), ginger juice decreased the renin protein contents in the culture supernatants in a dose-dependent manner. no significant difference in the cell morphology was observed between the control and treatment groups. conclusion: ginger root juice down-regulates renin secretion in human decidua, showing a great potential of a novel ras inhibitor, particularly, in obstetric patients. noninvasive quantification of the autonomic nervous system throughout pregnancy. dietmar schlembach, karoline pickel, daniela ulrich, philipp klaritsch, isa alkan, uwe lang, manfred g moertl. obstetrics and gynecology, medical university of graz, graz, styria, austria; cnsystems medizintechnik ag, graz, styria, austria. introduction: the analysis of heart rate variability (hrv), blood pressure variability (bpv), and baroreflex sensitivity (brs) has become a powerful tool for the assessment of autonomic control. although hrv analysis was initially developed for risk stratification in cardiology, the field of clinical application has broadened in recent years. the aim of our study was to investigate the adaptation of autonomic control during pregnancy based on analysis of heart rate variability and baroreceptor sensitivity. methods: patients with uncomplicated pregnancy were measured with the taskforce® monitor i made by cnsystems. all measurements were performed in supine position under standardized resting conditions. autonomic parameters were recorded at rest and within the "deep breathing method" as a "cardiovascular challenge test". results: throughout pregnancy a shift to higher sympathetic activity respectively a lower parasympathetic activity was observed. , and . ± . [> wks]) (figure ). during the deep breathing maneuver we could show an increase of the sympathetic / parasympathetic ratio (figure ). the noninvasive determination of autonomic parameters throughout pregnancy is possible. these results can be used as basic parameters for classifying and assessing autonomic changes in pathological conditions in pregnancy such as hypertensive disorders. how far the characterization and challenge of these autonomic functions can be utilized for diagnosis and prediction of preeclampsia has to be shown in future studies. hemodynamic parameters measured noninvasively throughout pregnancy. daniela ulrich, manfred g moertl, karoline pickel, philipp klaritsch, isa alkan, uwe lang, dietmar schlembach. obstetrics and gynecology, medical university of graz, graz, styria, austria; cnsystems medizintechnik ag, graz, styria, austria. background: hemodynamic changes throughout pregnancy have been measured predominantly by invasive techniques. the discussion on the valence und the low acceptance of these invasive procedures by pregnant women demands a noninvasive method for evaluating and distinguishing cardiovascular adaptative mechanisms throughout normal and especially complicated pregnancy. method: healthy patients with uncomplicated pregnancy were measured with the taskforce® monitor i (cnsystems, austria) at different time points throughout pregnancy. cardiovascular parameters were recorded in supine and left lateral position under standardized conditions. results: throughout pregnancy an increase of global parameters such as heart rate (hr), blood pressure (bp), total peripheral resistance (tpr) and total peripheral resistance index (tpri) were observed. stroke volume (sv), stroke index (si), cardiac output (co), contractility index (ci), acceleration index (aci), and left ventricular ejection time (lvet) decreased throughout pregnancy (table). discussion: the noninvasive determination of cardiovascular parameters throughout pregnancy is possible and the results of this pilot study can serve as basic parameters for classifying and assessing cardiovascular changes in pathological conditions in pregnancy such as hypertensive disorders. assigning pregnant hypertensive women into hyper-and hypodynamic groups may aid in planning individual therapeutic strategies. objective: both gnrh i and gnrh ii are expressed in humans. gnrh i is produced by the hypothalamus under the regulation of gonadal steroids and stimulates pituitary gonadotropins. during pregnancy gnrh i is also produced by the placenta and affects hcg. gnrh ii, the ancient isoform, is produced by numerous human tissues including immune and reproductive tissues and circulates in blood in quantifiable levels. we have demonstrated that gnrh ii analogs directly affect fertilization and uterine function, and propose that it acts via immune regulation. during pregnancy it is known to regulate numerous hormone productions, yet the levels of gnrh ii has not been reported. in these studies we have determined the circulating levels of gnrh ii throughout pregnancy and in early pregnancy loss. study design: thirty-three women having normal pregnancies were followed prospectively. plasma samples were drawn at , , , , , , weeks gestation and during labor. plasma was also collected from patients have spontaneous abortions (n= ). circulating gnrh ii and crh and gnrh i were measured by specific radioimmunoassays. results: circulating gnrh ii increased from non-pregnant levels ( +/- pg/ ml, mean+/-sem) to +/- pg/ml by -week lmp. gnrh ii concentrations continued to increase through weeks gestation over the -week levels, although a significant increase was only attained by weeks. gnrh ii continued to increase in late term, attaining levels of +/- pg/ml which was significantly higher than that of early gestation. during labor and delivery gnrh ii in maternal plasma was further increased to +/- pg/ml, i.e., . times that of -week gestation ( +/- pg/ml). circulating gnrh ii did not parallel gnrh i in early pregnancy but did in late gestation. gnrh ii did correlate with crh in early pregnancy but not in late gestation. patients having spontaneous abortion had increased circulating gnrh ii at -weeks lmp ( +/- pg/ml) as compared to normal pregnancies ( +/- pg/ml). conclusion: gnrh ii increased throughout pregnancy attaining highest concentrations during labor and delivery. patient with early pregnancy loss had increased gnrh ii expression. the function of gnrh ii or factors affecting this increased gnrh ii in normal or abnormal human pregnancy should be investigated. introduction: plasma levels of the endocannabinoid, anandamide (aea) decrease during the luteal phase of the menstrual cycle and early pregnancy and increase during parturition. high plasma aea levels at weeks in women undergoing ivf-et was associated with a failure to achieve an ongoing pregnancy . what is uncertain is what happens to aea levels when the pregnancy has already failed. we aimed to quantify aea levels in women presenting in early pregnancy with a diagnosed non-viable pregnancy and to compare them to those of a viable pregnancy. methods: plasma aea was measured by a sensitive isotope dilution hplc-ms/ms method from women in early pregnancy ( - weeks) of whom had a viable pregnancy and had a non-viable pregnancy. serum hcg and progesterone were measured in blood samples by standard elisa methods. results: the ages and bmi of both groups were similar ( ± . versus ± . years; mean ± sd; p= . ; student's unpaired t-test and ± . versus ± . kg/m ; p= . ) respectively. plasma aea levels in women with non-viable pregnancies were significantly higher than those in women with viable pregnancies ( . ± . versus . ± . nm; p= . ). there was no correlation found between aea levels and hcg or progesterone levels p= . and r= . ; p= . , respectively) , despite progesterone being significantly lower in the non-viable group ( . ± . versus . ± . ng/ml; p= . ). conclusion: higher plasma aea levels were associated with early pregnancy failure, and this association appeared to be independent of serum hcg or progesterone concentrations. these data suggest that plasma aea levels are linked with pregnancy failure through a mechanism that does not involve hcg or progesterone production. the precise involvement of anandamide in early pregnancy needs to be investigated further. n- ) and arachidonic (aa, : n- ) acids, the major brain long-chain polyunsaturated fatty acids (lc-pufa) are generated by elongation-desaturation of dietary essential fatty acids (efa). the maternal liver is principally responsible for efa elongation-desaturation. objetive. we determined effects of protein restriction in pregnancy on expression of d, d and elongases and (elov and ) in the maternal liver rat. methods. pregnant rats were fed control ( % casein; c) or restricted ( % casein; r) isocaloric diets. at day gestation maternal blood and livers were collected. serum triglycerides, cholesterol and glucose were determinate with the synchron cx autoanalyser and leptin and insulin by ria. liver fat determination was performed by the soxhlet method. liver ara and dha were calculated by gas chromatography based upon retention times from methyl ester standards. liver d, d, elov and mrna were measured by rt-pcr and northern blot. data are m ± sem; analysis by t-test. results. liver weights did not differ in c and r. total liver fat ( ± vs ± mg) serum leptin ( ± . vs ± . ng/ml) and insulin ( . ± . vs . ± . ng/ml) differed in c and r respectively (p< . ). serum triglycerides, cholesterol, and glucose did not differ. desaturase and elongase mrna and % of maternal liver aa and dha were lower in r (fig ) . conclusion. low liver fat content and desaturase and elongase mrna in r indicate impaired lc-pufa synthesis which may adversely impact fetal development, especially the brain. objective: to test the hypothesis that obstetrical dic results from an excessive leak of fetal material into the maternal circulation. a secondary objective was to assess maternal morbidity. methods: all cesarean hysterectomy cases for hemorrhage at our hospital from to were included. intravascular presence of fetal material was determined by two pathologists, blinded to each other and any clinical information. the percentage of those with any fetal material in the maternal circulation was calculated for each diagnosis for hemorrhage. for a given diagnosis, the percentage of intravascular fetal material in those with the given diagnosis was compared to those without that diagnosis using fisher's exact test. most patients had multiple hemorrhage diagnoses. a two sample t-test was used to evaluate the difference in mean blood loss between those with and without intravascular fetal material. results: primary outcome* % of patients received blood products and % received clotting agents. the average blood loss was cc. there were no maternal deaths and injuries to adjacent organs, all to the bladder. conclusions: there was no association between the presence of intravascular fetal material and any specific hemorrhage diagnosis or amount of blood loss. although the power to detect a relationship was low, the excessive leakage of fetal material as a specific and exclusive mechanism of obstetrical dic, as postulated, seems unlikely. while % of the population was transfused, there were few intraoperative complications and no maternal deaths. hypertension, tel-aviv university, sheba medical center, ramat-gan, israel. objective: the joint national committee on high blood pressure (jnc ) determined blood pressure of - / - in adults to be prehypertension that requires health promoting life style modifications to prevent cardiovascular disease. similarly, we hypothesize that gestational prehypertension in pregnancy is associated with increased risk of pregnancy complications and its management would improve pregnancy outcome. methods: prospective and retrospective recruitment resulted in a study group consisting of patients who were diagnosed in the first and early second trimester (< weeks) with blood pressure of - / , and a control group consisting of patients with blood pressure < / at similar gestational age. comparisons between the two groups were accomplished for demographic characteristics and outcome measures using the chi-square test statistic and two-sample t-tests for categorical and continuous variables, respectively. results: all outcome measures analyzed resulted in poorer results being associated with the study group compared with control as follows: % versus % of the study and control group pregnancies, respectively, experienced preeclampsia (p= . ); and % versus % of the study and control group, respectively, were associated with gestational hypertension (p= . ). % of the control group deliveries were associated with admission to the nicu, compared to only % in the control group (p= . ); % versus % of the study and control group deliveries, respectively, were preterm (p= . ); twenty-nine percent of the study group were cesarean deliveries, compared to % in the control group (p= . ). on admission mean sbp and dbp was and mmhg, respectively, in the study group, compared to and mmhg in the control group (p= . for sbp and p= . for dbp). conclusions: "gestational pre-hypertension" may be a real pregnancy condition that when is recognized, physician attention, patient close monitoring during prenatal care and delivery and early intervention in patients at risk, may all promote improved pregnancy outcome. larger scale study is in progress to evaluate and confirm these findings in the larger pregnant population. are contractions at - weeks gestation less painful than those at full term? kristy a ruis, karin blakemore, abimbola aina-mumuney, valerie jones. gynecology and obstetrics, johns hopkins hospital, baltimore, md, usa. objective to determine if gestational age plays a role in severity of pain associated with uterine contractions. study design self-reported pain from women in labor, on a scale of - , is assessed and recorded in an obstetrical database by nurses at regular intervals at two hospitals within our medical system and was retrospectively reviewed from - . pain at various dilatations ( - cm) of women who were laboring and then delivered at - weeks' gestation was compared to women in labor at term. maternal demographics of age, race, education level, bmi, presence of support person, request for analgesia at any point in labor, and epidural placement were abstracted from maternal records. categorical data were analyzed using chi square or fisher exact test; continuous variables using student t-test. results laboring patients between - weeks gestation and at term were identified. term and preterm patients did not differ with respect to maternal demographics. pain reported by preterm patients was significantly less at - cm dilatation ( . vs. . ‚ p= . ) and significantly greater at - cm ( . vs. . ‚ p= . ). pain reported at - cm and - cm was not statistically different between the groups. of note, fewer of the preterm patients received an epidural despite the request for analgesia. conclusion preterm laboring patients between - weeks gestation may perceive less pain at - cm dilatation compared to term laboring patients; however, their pain perception at advanced dilatation was comparable to those at term despite the fact that they were less likely to have an epidural in place at the time of pain evaluation. in light of these findings, the widely accepted etiology of labor pain as the result of cervical dilatation may need to be re-examined. also, factoring in the patient's discomfort level into the evaluation for onset of early active labor may not be valid in the preterm patient. background and objective: asthma is a risk factor for adverse pregnancy outcome. this has been attributed to the effect of allergy in pregnancy. mast cell mediators can induce uterine contractility. the objective of the study was to test the hypothesis that pregnant women with asthma have different uterine electrical signals from those generated by normal pregnant women. materials and methods: uterine electromyography (emg) recordings from gestational age matched pregnant women at term were analyzed. the cases consisted of patients with asthma and the controls, those women without asthma. emg was recorded for approximately minutes from surface electrodes placed upon the maternal abdomen, with the electrical signals filtered in the uterine-specific range of . to . hz to remove noise components. the recordings were analyzed in their entirety first by power spectrum analysis and then by lyapunov analysis. the power-spectrum-largest-peak frequency and the largest lyapunov exponent were calculated for each patient, and then the mean and standard deviation of these parameters was compared using student's t-test. results: patients with asthma had significant lower power spectrum frequency and higher lyapunov exponent than women without asthma. see ) the power spectrum frequency and the lyapunov exponent used to analyze uterine emg signals were different in women with and without asthma; ) the results suggest that uterine electrical activity is altered in women affected by asthma; ) these findings may explain the observed increased frequency of preterm birth in women with asthma. further studies are required to dissect the mechanisms. fetal microchimeric cells trafficked into the maternal circulation persist in blood and tissues for years after pregnancy. increasing data suggest that microchimerism occurs after every pregnancy, but the biological role of fetal microchimerism or the cell types involved is unclear. while persistent fetal cells were initially implicated in autoimmune disease, animal studies suggest these fetal cells play a broader role in response to tissue injury. methods: appendix specimens were acquired from women undergoing appendicectomy during pregnancy. detailed reproductive histories were obtained. fluorescence in situ hybridisation (fish) with two different probes allowed investigation of the presence of male presumed-fetal cells and nested pcr amplification of sry gene confirmed male dna in the appendix. immunostaining was used to determine the fetal cell phenotype. results: male cells were identified in appendix tissues from women with known male pregnancies (n= ) and also from a woman with no sons and a previous miscarriage of undetermined gender (n= ). no male cells were observed in the control (n= ), a woman with daughters. male cells of presumed fetal origin were evenly distributed in the muscle, mucosa and submucosa layers of the appendix. morphology and co-localisation analysis suggested the identified male cells had differentiated primarily into muscle cells or lymphocytes. combined immunostaining and y-fish demonstrated male desmin+ muscle cells and cd + and cd + lymphocytes. finally, the presence of male dna in the appendix specimens was confirmed by nested pcr. male cells of presumed fetal origin were identified in the appendix of pregnant women. microchimerism frequency varied according to the reproductive history and the degree of inflammation. microchimerism rates were higher in the appendix from women with current male pregnancies than in those with previous male pregnancies and were higher in those with histological acute inflammation, when compared to milder cases. male microchimeric cells identified were of haematopoietic and mesenchymal origin. this study suggests that fetal cells are present in sites of tissue injury and may participate in tissue repair during pregnancy. collagen i and collagen-binding integrins mrna expression in rat cervix during gestation. huiling ji, tanya l dailey, vit long, edward ks chien. obstetrics and gynecology, maternal fetal medicine, women and infants' hospital of rhode island, providence, ri, usa. objective cervical remodeling is associated with cell proliferation and extracellular matrix (ecm) remodeling. integrins are a family of multifunctional cell adhesion receptors which mediate ecm-cell interactions including binding collagen. of the integrin (i) heterodimers, , , and are primary receptors for collagen. the predominant cervical collagen is collagen type . the purpose of this study is to investigate collagen and integrin expression in the pregnant rat cervix. the cervix was harvested from timed pregnant sprague-dawley rats. non pregnant (np)and timed pregnant day , , , , and animals were euthanized using a protocol approved by the iacuc. four animals were sacrificed on each day of gestation. quantitative rt-pcr was used to evaluate mrna expression and normalized to -actin. a standard curve was generated from a single sample. data was analyzed using anova and multiple comparisons testing. collagen i mrna expression increased through mid-gestation but decreased to np levels on day . a similar pattern was seen with the integrin beta subunit. the pattern of integrin alpha subunit expression was different for each subunit during pregnancy. the changes in collagen, integrin alpha , , and integrin beta were found to be statistically significant. (figure) . the pattern of collagen binding integrin alpha subunit expression during the rat gestation appears to vary independently of each other. the expression of the subunit paralleled col expression. collagen binding integrins may play independent roles in signaling and biomechanics during gestation. funding: women infants' hospital research fund; phs nih-ncrr p rr p rr . background. the effects of chemotherapy on human fetal development are poorly studied, mainly due to the lack of adequate animal models in which placentation and embryological development resembles humans and pharmacokinetic studies, prenatal sonography as well as histological studies can be performed. aim of the study. to test the baboon as model for studying pharmacokinetics and fetal effects of chemotherapy administered during pregnancy. methods. experiments were performed in pregnant baboons at a mean gestational age of (+/- ) days. detailed ultrasound examination (biometry, doppler, screening) was performed days before, at and day after the drug administration. the administration of different schemes of chemotherapy and the fetal samplings occurred under endotracheal anaesthesia. maternal blood samplings, as well as percutaneous ultrasound guided fetal blood ( ml) and amniotic fluid ( ml) samplings were performed at least once immediately after drug administration. in case of fetal demise, the fetus underwent detailed macro-and microscopic examination. in the other animals mother and fetus were euthanized h after the experiments, with collection of blood, amniotic fluid and tissues for further analysis. results. all mothers survived the experiments, one mother developed paralitic ileus. none of the fetuses died during the acute phase of the experiment but / ( %) fetuses died within h following the experiment. none of the animals showed significant clinical or histological signs of infection or anaemia. a total of ultrasound examinations were performed in fetuses, allowing the creation of sonographic growth charts in this model. at the time of drug administration the mean maternal weight was . kg (range . - . ) and the estimated fetal weight was gr (range - ). sixteen out of cordocenteses ( %) and all amniocenteses ( %) were successful in obtaining the required samples for analysis. conclusion. the pregnant baboon can be used as a model for studies on pharmacokinetics and fetal effects of chemotherapy administered during gestation. prenatal ultrasound is comparable to the human situation, and amniocentesis and cordocentesis can be performed with a high success rate and short term survival. therapy to prevent preterm birth is limited". moreover, -hydroxyprogesterone ( ohp) caproate is a category d drug according to the fda (evidence of fetal harm). p is produced in the adrenal glands, gonads and brain. after weeks gestation p is secreted from the placenta independently to the mother and the fetus. p is the precursor of aldosterone and after conversion to ohp, of cortisol and androstenedione. baboons have similar reproductive system and development to humans and are used to study mechanisms of labor and prevention of preterm labor. currently, there are no normative data on the concentration of p, ohp, cortisol ©, estradiol (e ) or inhibin (i) throughout their pregnancy. therefore, a doseresponse or the teratogenic effects of p or ohp caproate cannot be studied in a controlled manner in this animal model. the aim of this study was to generate reference values for these hormones during baboon gestation and early postpartum life. material and methods: hormonal quantitative measurements were performed (immulite analyzer) results: the mean concentrations of these hormones are depicted in figure (maternal serum, from conception to term days) and figure (newborn serum) conclusion: this study provides reference values for hormones quantified during the baboon gestation and early postpartum life. this may be useful for future research concerning the effects of p and ohp administration during pregnancy. ( )non-pregnant women eligible for ivf treatment and ( )pregnant women receiving prenatal care. the pre- / samples were drawn in the year prior to / / ; the post- / samples were drawn within months after. the pre- / and post- / normal pregnant samples were drawn at + weeks' gestation. the non-pregnant samples were from ivf candidates with serum e levels< newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced pg/ml and fsh serum levels< . miu/ml. the samples were assayed for acth and cortisol by a commercially available immulite™ system. hsp and hsp were measured by commercially available elisa. these results were compared in the patient populations before and after / . t-tests were used for analysis. statistical significance was set at p< . . results: in the pregnant subjects, there were no statistical differences in the mean levels of acth, cortisol, and hsp pre-and post- / (table ) . only serum hsp levels were significantly increased in the pregnant women post- / .* mean serum acth, cortisol, hsp , and hsp levels were all significantly different in non-pregnant subjects pre-and post- / . conclusions: except for hsp , serum markers for stress were unchanged in pregnant subjects after the stress of / / in comparison to non-pregnant women whose serum hsp , hsp , cortisol, and acth levels were significantly different. the lack of change in serum markers of stress in pregnant women suggests that the hormonal milieu of pregnancy may buffer against acute environmental stressors. objective: in human pregnancy, maternal body composition provides an indicator of maternal nutritional status and metabolic capacity. previously, we reported that ß hsd- activity was significantly reduced in term placentas from thin women and women with a smaller mid-upper arm circumference before conception. this suggests that these fetuses may have been exposed to inappropriate levels of maternal cortisol, which is a mediator of gestation length. in this study, we hypothesize that the duration of gestation will be shorter in women who tend to be thin and have a lower mid-upper arm circumference prior to conception. methods: within the longitudinal, population-based southampton women's survey (sws), analyses were performed on women whose estimated date of conception was set using an algorithm that combined menstrual and early ultrasound scan data and who had a spontaneous onset of labour and delivered after weeks of gestation. linear regression was used to examine the relationships between maternal body composition and the duration of gestation. results: within the women surveyed, lower maternal body mass index, mid-upper arm circumference and arm muscle area before conception were all associated with shorter duration of gestation at delivery (r= . , p= . ; r= . , p= . ; and r= . , p= . , respectively). a lower subscapular skinfold thickness, sum of skinfolds and height were also associated with shorter duration of gestation (r= . , p= . ; r= . , p= . and r= . , p= . , respectively). mother's age, own birthweight and ratio of subscapular/triceps skinfold thickness were not related to the duration of gestation. conclusions: in this study, we found that thinner women with a lower midupper arm circumference and arm muscle area tended to have a shorter duration of gestation. our findings of shorter gestation length in thinner women are in keeping with our previous observation of lower ß hsd- activity in term placentas from thinner women. we conclude that metabolic capacity prior to conception could influence duration of gestation through mechanisms that include alteration in placental metabolism and fetal cortisol exposure. fecal incontinence (fi) is a debilitating condition affecting - % of the us. our prior studies found that % of women report new onset fi after childbirth. the goal of our study was to examine the impact of fi on postpartum quality of life (qol). women reporting fi on a statewide survey who agreed to participate in a -year study of qol were included in the analysis. women were considered to have fi based upon the nih definition of fi. the quality of life survey was based upon the uebersax incontinence impact questionnaire and was administered every months for years. qol in women with fi was examined using chi square, and impact of severe fi (stool incontinence) was determined by multivariate logistic regression. results women with fi were surveyed and of those, ( %) returned at least survey during the study period, completed all survey questions, and were included in the final analysis. among women with fi, % felt frustrated due to fi, % reported fi impacted their emotional health, . % reported fi impacted child-caring abilities, and . % reported a negative impact on social activities. qol was similar across survey periods. one out of three women with fi reported severe symptoms (incontinence of stool). women with severe symptoms were - times more likely to report negative impacts on qol compared to milder (e.g. flatus) fi after adjusting for age, parity and urinary incontinence. . % felt their stool was stored elsewhere before bowel movements, . % reported using digital defecation and more than half ( %) reported symptoms of urinary leakage. despite the substantial impact on postpartum quality of life, few women sought medical help with only % of women at months, . % at year, and . % at years ever reporting their symptoms to a medical provider. postpartum women report that fecal incontinence has a substantial negative impact on their qol after delivery including their emotional health and ability to care for their newborn. despite this profound impact, few women will discuss fi with medical providers. these data suggests there may be a benefit for providers to inquire about fi at postpartum visits. objective: the objective of this study was to determine what characteristics contribute to racial/ethnic differences in breastfeeding rates. study design: a retrospective cohort study of all women who delivered a viable infant (n= , ) was conducted. the primary outcome was breastfeeding upon discharge of the hospital. we first examined the association between race/ ethnicity and breastfeeding. next, we conducted stratified analyses examining a variety of predictors of breastfeeding within the racial/ethnic groups including maternal demographics, obstetric interventions, and perinatal complications. results: we found that both asians (or . , % ci . - . ) and blacks ( . , % ci . - . ) had statistically significant lower rates of breastfeeding, while latinas (or . , % ci . - . ) showed a trend towards higher rates of breastfeeding. in latinas, we found that rd and th degree lacerations were significantly associated with lower rates of breastfeeding, but were not predictive of breastfeeding in the other racial/ethnic groups. epidural use was predictive of lower rates of breastfeeding in caucasians and blacks, but was not predictive in latinos and asians. some of the other predictors which differed between the racial/ethnic groups were obesity and induction of labor (table ) . conclusion: race/ethnicity is significantly associated with breastfeeding, with blacks and asians having the lowest rates of breastfeeding at discharge. a variety of other factors are associated with breastfeeding and interestingly, their effect appears to differ between the racial/ethnic groups. future studies might elucidate the sociocultural and biomedical reasons that explain the differences. these results help focus our efforts during the peripartum period to advocate for mothers who have factors that might impede breast feeding. epidemiology, erasmus medical centre, rotterdam, netherlands; pediatrics, erasmus medical centre, rotterdam, netherlands; public health, erasmus medical centre, rotterdam, netherlands; clinical genetics, erasmus medical centre, rotterdam, netherlands. background recommendations on folic acid use to prevent neural tube defects are launched in several countries. however, the adequate use of folic acid supplements during the periconception period seems to be low. objective to assess the prevalence of adequate folic acid use, defined as the preconception start of supplements, and to identify its determinants in a multi-ethnic population. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods from all women in the cohort who delivered between april and january information on folic acid use and potential determinants was obtained by questionnaires and physical examination. logistic regression models were used to identify determinants of periconception folic acid use. results. data from , pregnant women were available. of all women % adequately used folic acid supplements. the most important risk factors for inadequate use were unplanned pregnancy (or . , p< . ), nonwestern ethnicity (or . , ci . - . , p < . ) and a low educational level (or . , ci . - . , p< . ). other risk factors were single marital status, smoking, multiparity (all p< . ) and alcohol use (p< . ). prior spontaneous abortion was associated with increased adequate folic acid use (p< . ). conclusion adequate periconceptional folic acid use is low. improved preconception care and public health education programs are necessary to improve the uptake of folic acid. although methamphetamine (ma) use has been front and center of the united states drug control policy since , little attention has been given to ma use in pregnancy, despite the fact that pregnant admissions to drug treatment for ma have been rising sharply. to determine trends in the prevalence of ma treatment admissions during pregnancy, we undertook a secondary analysis of the treatment episode data set (teds), an administrative data set that captures at least % of all known treatment admissions in the us. in particular, we investigated risk factors for ma use and how these characteristics have changed over time. demographic, geographic and substance use data were collected for the , pregnant admissions captured between and . logistic regression models were constructed by year. confounding was assessed via backwards elimination with a change-in-estimate criteria of . considered substantial. trend results were reported as adjusted proportions and represented graphically. overall ma prevalence, reported as the primary drug of use upon admission, rose from . % in to . % in . although white women had times the odds of using ma in (adjusted or ( %ci) . [ . , . ]), this had dropped to . [ . , . ] by , mostly due to an increase in latina ma use in pregnancy. pregnant women admitted for ma had fewer prior treatment admissions, were more likely to be unemployed, and less likely to use alcohol or marijuana. we found great geographic variability in use. ma was more common in the pacific region and least common in new england. there was little change in regional variation over the study time period. less is known about the perinatal effects of ma compared with other substances, yet it is the primary drug of choice for pregnant women admitted into treatment. as pregnant women occupy a unique place in drug treatment, analysis of national trend data is essential to guide both policy and research. background: epidemiologists have grouped the multiple disorders that lead to extremely preterm delivery in a variety of ways. we sought to identify characteristics that would support the combining or dividing of the disorders that lead to preterm delivery. methods: we enrolled , women who delivered a live born singleton infant between and completed weeks gestation at tertiary centers in the united states. each delivery was classified according to the complication that prompted presentation: preterm labor, preterm premature rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes (pprom), preeclampsia, placental abruption, cervical incompetence, and fetal indication/intrauterine growth restriction (iugr). we compared these entities on the frequency of characteristics identified by standardized interview, chart review, histological examination of the placenta, and culture of placenta parenchyma. results: the percents of women who presented with each antenatal complication were: preterm labor ( ), pprom ( ), preeclampsia ( ), placental abruption ( ), cervical insufficiency ( ), and fetal indication/iugr ( ). after considering antecedents and correlates of the processes leading to preterm delivery, we observed two overarching epidemiologic patterns. the first pattern, characterized by recovery of organisms from the placenta and by histologic chorioamnionitis, tended to be associated with preterm labor, pprom, placental abruption, and cervical insufficiency. the second pattern, characterized by a paucity of organisms and inflammation and the presence of histologic features of dysfunctional placentation, tended to be associated with preeclampsia and fetal indications/iugr. conclusions: disorders leading to preterm delivery can be categorized broadly into two groups: those associated with intrauterine inflammation and those with aberrations of placentation. predictors of compliance with tuberculosis screening in pregnancy. nadav schwartz, sarah wagner, sean keeler, may tam, julian mierlak, , aaron caughey. obstetrics and gynecology, nyu school of medicine, new york, ny; obstetrics and gynecology, ucsf, san francisco, ca; obstetrics and gynecology, gouverneur health care services, new york, ny. objective: poor compliance with tuberculosis screening and treatment is a major obstacle to the containment of this disease. we sought to identify predictors of ppd and cxr compliance during pregnancy. methods: a retrospective cohort study at a single institution which serves a largely immigrant population in nyc, from november , through june , . data on maternal age, ethnicity, country of origin, level of education, ppd and cxr status were collected. results: of the , pregnancies, asian and hispanic race/ethnicities accounted for . % and . % of the population, respectively, with . % being us-born. the mean age was . years and the overall ppd+ rate was . %. there was % non-compliance with ppd testing, and % of ppd+ patients were non-compliant with their chest x-rays. asian women were more likely to be ppd-compliant than hispanic or caucasian women. ppd+ asian women were also more likely to be compliant with cxr. us-born women were significantly less likely to be compliant with their ppd or with their cxr. women > years old were less likely to be compliant with their cxr, while women with an elementary school education or less were more likely to be compliant. (see table for odds ratios and %ci) conclusions: age, education, immigrant status and other cultural and ethnic factors appear to play a role in compliance with tuberculosis screening. further elucidation of these effects may help clinicians target at-risk subpopulations and improve overall compliance, working towards better control of this disease. background: in the us, differential outcomes in cervical cancer among medically underserved women are linked to multiple barriers impacting loss to follow-up and failure or delay in diagnostic resolution. prior studies have found risk factors for acquisition of hpv and for inability to obtain a pap smear. no study has explored health literacy and physician communication as a key factor. methods: this research represents the formative phase of a randomized controlled trial of african american (aa) and hispanic women aimed to improve communication and abnormal pap smear follow-up in chicago. semi-structured interviews and focus groups were conducted face to face in spanish or english. each interview lasted minutes, and each focus group lasted - minutes. we recruited patients ( hispanic, aa) and providers from a purposive sample representative of two large clinics that serve low-income women. results: all interviews were transcribed by two investigators. each interview was coded by two investigators separately and then a third investigator reviewed the transcripts and coding to achieve triangulation. codes for these themes were developed and the responses were tabulated using the coding scheme. atlas ti was utilized to analyze all qualitative data. from these data, we uncovered provider-patient challenges in cancer communication including: the providers' trade off between medical accuracy and/or literacy when communicating with their patients. for the patients, the word cancer was important to hear since they wanted the truth and needed to hear this word in order to encourage them to respond more quickly. however, many providers believed that the word cancer was too "scary" and to "extreme" of a word that may communicate too much exaggerated information. both the hispanic and aa patients did not seem to differ in their responses. conclusion: results exemplify not only the importance of health literacy and patient provider communication, but demonstrate the wealth of information gained from qualitative research-a method of data collection seldom utilized in ob/gyn investigations. funding: women's reproductive health research award: hd - ; r ca (spring). population. kristine beckers, isabelle guelinckx, greet vansant, roland devlieger. obstetrics and gynaecology, university hospital gasthuisberg, leuven, belgium; clinical nutrition, katholic university leuven, leuven, belgium. objective: to generate reference charts for weight gain during pregnancy for the different bmi-categories (underweight, normal weight, overweight, obesity), based on recent data in a homogeneous caucasian population. methods: in a retrospective study at the department of obstetrics of the leuven university hospital (belgium), weight gain and pre-pregnancy bmi were determined in belgian pregnant women with accurately dateable, uncomplicated singleton pregnancies. centile curves for the different bmicategories were constructed using of the linear mixed model, one set of charts based on the absolute weight gain, another set based on the relative (expressed as percentage) weight gain. the effect of parity on weight gain was examined. results: overall mean weight gain was . ± . kg ( . ± . lbs). mean weight gain was . ± . kg ( . ± . lbs) in the underweight population, . ± . kg ( . ± . lbs) in the population with normal weight, . ± . kg ( . ± . lbs) in the overweight population and . ± . kg ( . ± . lbs) in the obese population. weight gain (pattern and amount) of the underweight and normal weight patients differed significantly of the overweight and obese patients. parity had a statistical, but no clinical significant influence on amount and evolution of weight gain. conclusion: by using strict inclusion criteria, bmi-category-specific reference charts were generated representing the optimal gestational weight gain, rather than the mean weight gain. this enables the weight charts to be used as a clinical tool during the counselling of pregnant women. further studies are required to assess the effectiveness of this clinical tool. female fshr(-/-) mice are sterile, because of a block in folliculogenesis at the primary follicle stage, show decrease in e and elevated fsh. this animal model is an appropriate model for studying hypergonadotropic ovarian dysgenesis and infertility, caused by c t mutation in fshr gene. objective: to investigate the effects of bmt on serum hormonal levels, follicular maturation and fertility of fshr(-/-) mice. methods: fshr (-/-) mice at - weeks of age were randomized into treated versus control groups.bm from syngenic female donor was injected into the tails of recipients. control group received vehicle alone. vaginal smears were collected, body weight was measured daily. sample animals were sacrificed at , , , , and weeks post bmt. all organs were weighted and examined by h e. fsh, e , and p were measured before and after treatment. for donor cell tracking, dna was extracted from various organs. specific primer sets were designed for normal and mutant hfshr gene. pcr amplification was done and pcr products were analyzed in % agarose gel. results: total body weight significantly increased in treated animals(p< . ). significant increase in both the total number of follicles, and the collective diameter of the follicles in treated animals observed(p< . and p< . ). six out of treated animals showed estrogenic changes in daily vaginal smear. e level increased . - . times and fsh level dropped to % in treated animals. normal (donor allele) fshr gene was amplifiable in out of recipient mice, and was detected only in the ovaries and uterus but not in any other tested organs.control group did not show any changes in vaginal smear, hormonal level, and normal fshr allele. conclusion: intravenously injected syngeneic bone marrow cells were able to home to the ovaries of female fshr (-/-) mice and restore folliculogenesis and resume steroid hormone production. potential mechanisms for these observations will be discussed. conclusion: neural stem cells (nscs) give rise to progenitors, which expand by rapid proliferation until cell cycle arrest followed by differentiation along one of cns cell lineages: neurons, astrocytes and oligodendrocytes. increased differentiation of neuronal cells with ins and even more with leptin suggests that iugr-associated reduction of these growth factors may contribute to impaired hypothalamic pathway development. objective: to develop a technology of modulating hucb in order to achieve neuronal cells for future potential replacement of damaged neuronal tissue. design: we developed a two-dimensional ( d) tissue culture technology for isolation and differentiation of collagen-adherent hucb neural progenitors (hucbnps). we further used the extracellular matrix protein collagen, organized in a three-dimensional ( d) gel, supplemented with neuronal conditioning medium and nerve growth factor (ngf), to facilitate ex-vivo long term neuronal differentiation of hucbnps. we developed a stable green fluorescence protein (gfp)-pc cell model, to be used as a positive control for monitoring neuronal outgrowth for proper evaluation of the hucbnps growth in the d scaffold, mimicking a neural tissue organization. results: our experimental data indicate that d collagen environment is neuronal biocompatible, supporting attachment, long-term survival, proliferation and differentiation of both hucbnps and gfp-pc cells. conclusions: improvement of the d technology with cultures of hucbnps that is in progress in our laboratory might be the first step in validating the concept for the feasibility of generating a neuronal d tissue construct for future potential treatment of neurodegenerative disorders. piane, justin tsai, gnanaratnam giritharan, emin maltepe, paolo rinaudo. reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: ivf embryos are often used to generate stem cells. however, it is unknown if stem cell lines originated from in vitro generated embryos are different from stem cell originated from in vivo embryos. trophoblastic cells, due to their external position within the embryo, are most susceptible to environmental factors encountered in vitro. furthermore, our previous data showed that trophoblast transport functions may be impaired after culture in vitro and that the number of trophoblastic cells is reduced in the embryo after ivf. in this study, we assess the differentiation characteristics of ts cell lines obtained from in vivo and in vitro fertilized embryos. methods: oocytes were isolated from superovulated c bl/ j female mice and in vitro fertilized with sperm from male c bl/ j mice. the resulting late-cavitating blastocysts were harvested. in vivo controls were obtained by flushing the blastocysts from the uteri of superovulated pregnant mice days post hcg. ts cells from the two groups were allowed to develop and maintained in vitro in the presence of fgf and heparin, using a feeder layer of human placental fibroblasts. ts cells were then allowed to differentiate without fgf and heparin, fixed on day , stained with -tubulin and zo- antibodies and then observed under fluorescence microscopy. three ts cell lines per group were analyzed and their differentiative capacity was evaluated using morphological criteria. results: in vivo and ivf derived ts exhibit a similar differentiation pattern. in particular, the number and timing of trophoblast giant cells and spongiotrophoblasts derivation is similar in the two groups. there are no obvious abnormalities in the immunologic staining morphology of the different cell lines at different time points. conclusion: there are no apparent morphological alterations in ts cells lines derived from ivf embryos as compared to in vivo embryos. this finding in an animal model increases our confidence in the reliability of human stem cells derived from ivf. as a further investigation, markers of trophoblast giant cells, spongiotrophoblast, syncytiotrophoblast and chorionic trophoblast cells will be examined and compared in the two different groups by northern blot hybridization. genomewide high density snp-cgh reveals several new deletion copy number variants on the x chromosome in pof patients. erik ah knauff, cisca wijmenga, ruben van 't slot, lude franke, bart cjm fauser. reproduction gynecology, university medical centre, utrecht, netherlands; complex genetics group, university medical centre, utrecht, netherlands. introduction: around % of women have a post-menopausal hormonal profile before age , referred to as premature ovarian failure (pof). pof could act as a genetic model for accelerated follicle loss and may render useful information about the polygenetic background of major individual differences in menopausal age. macrodeletions on the x chromosome are associated with pof but karyotyping has a maximal resolution of mb. when using genotyping whole genome arrays it is now possible to detect submicroscopic deletions and duplications (copy number variants (cnv)) up to kb effective resolution. we have screened for microdeletions on the x chromosome in a well-phenotyped cohort of pof patients. methods: our study included caucasian, ,xx patients with spontaneous secondary amenorrhea before age , fsh > iu/l and absence of (low) x/ xx mosaicism and fmr premutations. dna analysis was performed using illumina k cnv arrays containing , probes. , probes were located on the x chromosome ( probe for every kb), of which , probes were specifically designed to detect known cnvs. ten samples with call rates < % were removed from the analysis and one sample gave inconsistent x probe intensities. we screened for deletions ( kb) using an algorithm detecting at least five consecutive probes with intensities (logr > - . ) below the mean probe intensity. we identified a total of x chromosomal microdeletions, divided in loci and varying between - kb in size. of the samples showed at least one deletion on the x chromosome. % of the identified deletions had already been recorded in the database of genomic variants. in the newly identified newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced loci, we found coding regions containing genes. eight of these are established or potential pof candidate genes, including five that are clustered on the terminal xq critical pof region. conclusions: we observed abundant variation in the cnv regions, a proof-of-principle for this specially designed cnv-chip. snp comparative genomic hybridization revealed possible new deletions specific to pof on the x chromosome. data on duplications, validation and whole genome cnv analysis, compared to a control cohort, will become available soon and will be presented at the sgi annual scientific meeting. unexplained intrauterine fetal death (iufd) is associated with long qt syndrome. irene cetin, patrizio antonazzo, sabrina cozzolino, stefania calabrese, lia crotti, francesca ferrari, roberto insolia, fabio facchinetti, peter j schwartz. dept. of obst/gynecol, univ. of milano, milano, italy; molec cardiol lab, policlinico san matteo, pavia, italy; mother-infant dept., univ. of modena/reggio emilia, modena, italy. introduction: in developed countries, nearly in every pregnancies ends in late fetal loss. many iufds can be attributed to maternal disorders, fetal pathology, placental pathology and fewer to complications of labor and delivery. however, - % of cases remain unexplained. we recently demonstrated that % of sudden infant death syndrome (sids) cases carry functionally relevant genetic variants in long qt syndrome (lqts) genes. aim of the study was to analyze whether lqts genes are associated with iufd. materials and methods: patients with iufd were enrolled in two years, as part of an italian multicentre study. iufd was defined as fetal death at weeks or more of gestation according to the definition of late fetal death of the who. out of cases were classified as "unexplained stillbirths" according to the wigglesworth and aberdeen classifications. at birth placental and/cord samples were collected and dna extracted. the main lqts genes kcnq , kcnh , scn a, kcne and kcne were screened through dhplc and sequence analysis. any amino-acid substitution identified in the samples was checked for in a control population of caucasian women with uneventful pregnancies. preliminary data on the first cases (gestational age of death: - weeks) are reported. results: a total of missense mutations were identified in of stillbirths ( %), two on scn a and one on kcnh . the two mutations on scn a (v l; p a) were observed in two iufd occurred at term; they had been previously associated to sids and shown to increase the late sodium current. the mutation on kcnh is a novel genetic variant absent in reference alleles, never described in any control populations; this mutations was present in a case of iufd diagnosed at weeks of gestation. we are currently performing the electrophysiological cellular studies to define its functional effect. conclusions: these preliminary data indicate that a potentially significant number of currently unexplained iufd might be caused by ion channel diseases such as lqts. the potential prevention of sids or iufd recurrence and the identification of other affected family members could have important implications for the affected families. alternative splicing of epab is regulated by exonic splicing enhancers. e seli, a yaba, o guzeloglu-kayisli, md lalioti. ob gyn, yale u., new haven, ct, usa. introduction: alternative splicing is an important mechanism by which the genome gives rise to the observed diversity of proteins. embryonic poly(a) binding protein (epab), expressed exclusively in oocytes and early embryos, mediates translation of maternal mrnas. we identified an alternatively spliced form of epab lacking exon (cex del), and investigated its regulation as a model for alternative splicing in early development. specifically, we evaluated: imprinting (expression from maternal or paternal allele only); rna editing (post-transcriptional single nucleotide substitution); and exonic splicing enhancers (eses, exonic sites that bind splicing proteins). methods: a single nucleotide polymorphism (snp) detected in exon (c a/g) served as a marker for the parental origin of the spliced form. snp genotyping was performed by pcr amplification of exon followed by restriction enzyme digestion. to evaluate imprinting, we characterized heterozygous mice (a/g) that inherited the snp from either the mother or the father. to test for rna-editing and exonic enhancer contribution we tested mice homozygous for the exon snp (a/a or g/g). efficiency of alternative splicing in different genetic backgrounds was tested using real-time pcr normalized to actin expression. results: in mice heterozygous (a/g) for the exon snp, cex del was only expressed from the (a) allele. however, this was independent of the parental origin of the allele, ruling out imprinting. in mice homozygous (g/g) for the exon snp, the cex del variant also contained (g). therefore, rna editing did not occur. further sequence analysis led to the identification of an additional snp in exon (c g/c) that co-segregated with the exon snp. presence of c g led to the formation of an ese that binds splicing regulatory protein srp , leading to efficient exclusion of exon . real time pcr revealed a five-fold increase in the expression of the cex del alternative splicing variant in animals carrying the enhancer (homozygous g/g) for the exon snp compared to those that did not (homozygous c/c) at the same locus (p < . ). conclusions: in this study, we found that eses mediate the alternative splicing of oocyte-specific transcripts. our findings suggest that single nucleotide polymorphisms may alter the ratio between alternative splicing variants of oocyte-specific proteins. the role that these subtle differences play in determining individual reproductive outcome remains to be identified. epigenetics and chromosomal abnormalities in human oocytes. ilse van den berg, , joop se laven, robert jan galjaard, j hikke van doorninck. , obstetrics gynaecology; clinical genetics, erasmus medical center, rotterdam, netherlands. humans have a low fertility rate compared to other mammalian species. moreover their fertility declines with increasing age. both phenomena are largely due to an increasing number of chromosomal abnormalities in human oocytes during life. identifying factors that cause aneuploidy in oocytes may offer possibilities to diminish these abnormalities in vitro or in vivo. chromosomal segregation errors can result from aberrant recombination but little is known about epigenetic factors that may cause aneuploidy. epigenetic modifications such as histone acetylation and subsequent de-acetylation in oocytes are necessary for a correct progress through meiosis. if disturbed it may lead to aberrant segregation of chromosomes or chromatids due to decreased kinetochore function and imperfect spindle figures resulting in aneuploidy. mice oocyte data have shown a correlation of abnormal histone de-acetylation and aneuploidy and a correlation between age, remaining histone acetylation and aneuploidy (akiyama et al., ) . our research focused on human oocytes and investigated whether a similar relationship between histone acetylation, age and aneuploidy is present. human oocytes were surplus from standard ivf/icsi treatments (ic+). human oocytes showed immunostaining for histone , lysine acetylation (h k ) at the germinal vesicle stage and complete deacetylation at the mii stage in % of the oocytes, while % keep high levels of h k acetylation. treatment of germinal vesicle oocytes with a histon deacetylase inhibitor (tsa) during in vitro maturation until mii stage resulted in high levels of acetylation. remaining acetylation in tsa treated oocytes was correlated significantly with abnormal spindle figures (tubulin staining), a hallmark of developing aneuploidy. similarly, % of oocytes with naturally remaining h k acetylation showed abnormal spindle figures. in contrast % of the normal de-acetylated oocytes showed normal spindles (p= . ). this suggests that defective de-acetylation of h k in human oocytes leads to abnormal spindle figures and subsequent aneuploidy. advanced maternal age is associated with a reduction in de-acetylation during in vitro maturation and an increase in spindle abnormalities. these results may stimulate the development of assays for histone modifications as biomarkers to follow oocyte quality in in vitro maturation studies or in optimizing general ivf treatments. objective: racial disparity in preterm birth (ptb) is unexplained and genetic risk factors are suspected as a major cause. this large scale candidate gene study examines differences association of single nucleotide polymorphisms [snps] in caucasians (c) and african americans (aa) to help elucidate racial disparity in preterm birth (ptb) methods: in this case (preterm birth < weeks) control study (term birth > weeks) maternal and fetal dna from ( cases and controls) c and ( ptb and term) aa were collected. a high-throughput candidate gene association study was performed examining snps in genes of selected from hypothesized ptb pathways. single locus association analyses were performed separately on maternal and fetal samples. results: snps in genes associated with ptb (p< . ) were common between races with both maternal and fetal dna analyses. however, snps in genes in c and in aa in both maternal and fetal dna differed in its association with ptb. in c maternal dna, the single strongest association between ptb and snp was in plasminogen activator tissue (plat) gene (c- t; rs ) at both allelic (p = . x - ) and genotypic (p= . x - ) level with an odds ratio (or) of . ]. the single strongest effect in c fetal dna was observed in a snp in the interleukin- receptor antagonist gene (a g; rs ) for both allele (p= . ) and genotype (p= . x - ), or . ). in aa, the strongest associations were in interluekin- (c t-rs , allele p= . x - , genotype p= . x - ) in maternal dna with an or= . [ci . - . ] and in fetal dna interleukin- receptor b (a g; rs , allele p= . x - , genotype p= . x - ) with an or . ]. conclusion: large scale high throughput analyses of snps in candidate genes of ptb pathway reveals significant differences between races at both maternal and fetal levels. we found that the strongest single locus associations differed in the two races in both maternal and fetal dna samples. these findings support the hypothesis that underlying genetic predispositions may differ between these populations, perhaps partly explaining racial disparity in preterm birth. candidate gene association study indicates differential etiologies in gdm and in t dm. johann urschitz, tarik sultan, kenneth ward. obgyn, jabsom, university of hawaii, honolulu, hi, usa. objective: recently several genome-wide association studies have associated multiple single nucleotide polymorphisms (snps) in multiple genes with increased risk of type diabetes. as risk factors are similar between t dm and gestational diabetes (gdm), we investigated a possible association of those snps with gdm. study design: blood was collected from caucasian ( cases, controls) women who met coustan-carpenter criteria for gdm and non diabetic controls. dna was extracted and a candidate gene association study was performed (taqman, abi). results: chi contingency tests were used to analyze genotype and allele frequencies in controls and gdm affected pregnancies (see table below). none of the snps showed a significant association after bonferroni correction. conclusion: several polymorphisms which displayed highly significant associations with type diabetes are not associated with gestational diabetes. these results suggest that gdm is not a simple unmasking of a t dm predisposition by the metabolic demands of pregnancy; rather it appears that different biological mechanisms are responsible for the respective diseases. introduction: histone acetylation/deacetylation plays an important role in the regulation of gene expression. histone deacetylase inhibitors (hdaci), in general, maintain gene expression, although in some cases they cause repression of specific genes. in this context we have previously shown that the hdaci tsa suppresses activation of the pro-contractile gene cox- in human myometrial and amnion-derived cells [reproductive sciences, vol , no (supplement, # )]. we have also shown that expression profiles for hdacs ( - , ) differ significantly within upper and lower regions of the myometrium during pregnancy and in labour. objectives: since changes in both acetylation and phosphoryation status can influence the activity of individual hdacs we aimed to define whether there are any changes in the levels of acetylation and phosphoryation of myometrial hdac , and during pregnancy and labour. methods: protein homogenates prepared from non-pregnant, term and labouring myometrium were treated +/-shrimp alkaline phosphatase and subjected to sds-page and western immunoblotting (wb) using antibodies to hdac , and . the acetylation status of the hdacs was assessed by a co-immunoprecipitation assay using anti-hdac and anti-acetylated lysine antibodies. results: distinct patterns of acetylation were observed for the individual hdacs; hdac and appeared to be more acetylated in non-pregnant and labouring lower tissues when compared to pregnant myometrium. in contrast, hdac appeared to be slightly more acetylated in lower uterine samples from pregnant and labouring myometrium. in experiments to evaluate changes in phosphorylation status we observed that ) myometrial hdac appeared to less phosphorylated in both upper and lower labouring samples when compared to non-pregnant and pregnant samples; ) hdac appeared to be more phosphorylated in labouring samples than non-pregnant and pregnant myometrium; ) no changes in phosphorylation levels were observed for hdac using this test system. conclusions: the difference in hdac acetylation and phosphorylation levels in the human myometrium may indicate differential regulation of the activity of the hdacs within the distinct myometrial regions, perhaps leading to the alteration of their epigenetic effect on genes related to myometrial quiescence and contraction and the subsequent onset of both term and preterm labour. objective: native hawaiians and pacific islanders experience a higher perinatal morbidity secondary to the increased incidence and earlier onset of heart failure. this increased incidence is likely multi-factorial and includes co-morbidities and genetic factors. mutations in the human adrenergic receptor gene may play a role in the determination of heart function. mutations in the b -adrenergic receptor (adrb ) located on chromosome have been identified as a cause progressive cardiomyocyte loss leading to a dilated cardiomyopathy. the minor allele frequencies for most of these polymorphisms have been determined for caucasian, japanese, african-american and chinese as part of the hapmap project. the frequencies have not been determined in the pacific islander groups. this study helped determine the hawaiian allele frequency and determine the allele frequency in the presence of cardiac or other vascular co-morbidities such as hypertension, preeclampsia, and gestational diabetes. study design: real time pcr technology (taqman genotyping assays, applied biosystems) was used to screen maternal dna (n= ) from the phenotyping sample core of the pacific center for early human development (prcehd) for the rs single nucleotide polymorphisms (snps). genotype frequencies were also determined in % complete ethnic controls as determined by a four grandparent descent. results: chi square was used to analyze allele and genotype frequencies differences between controls and affected pregnancies. the results are summarized in the following tables. conclusion: the rs snp was not significantly associated with hypertension, preeclampsia or gdm in our overall hawaiian population. the genotype frequency in the % pacific islander group was significantly different from the caucasians (p= . ) and asians (p= . ) in our overall hawaiian population. angiotensin-converting enzyme and -adducin polymorphisms in preeclamptic mothers and fetuses. c mando, p antonazzo, s tabano, f colleoni, a martinelli, s calabrese, c benedetto, l marozio, f facchinetti, m miozzo, i cetin. inst. obstetrics and gynecology, fondaz. irccs policlinico mangiagalli regina elena, univ. milan, milan, italy; dept. biology and genetics, univ. milan, italy; dept.obstetrics and gynecology, univ. torino, italy; univ. modena and reggio emilia, modena, italy. background the genes encoding angiotensin-converting enzyme (ace) and -adducin (add ) share the potential of influencing blood pressure. previous studies demonstrated in humans the association of hypertension with the combined effect of both ace insertion/deletion (i/d) polymorphism, which leads to a different activity of the enzyme, and add g w non-sense single nucleotide polymorphism (snp). ace i-i genotype has been associated with low serum ace activity. controversial studies concerning the association of ace polymorphism with preeclampsia (pe) were reported and the possible combined effect of both ace i/d and add g w polymorphisms yet remains to be investigated. population association study we genotyped polymorphisms (ace i/d and add g w) in women: with pe ( / with severe pe) and controls. moreover, we investigated a subset of their fetuses: from severe pe, from mild pe and controls, in order to identify specific maternal and/or fetal genotypes conferring a higher risk to develop pe. we both evaluated the single and the combined effects of ace and add genotypes on mother-fetus couples and singularly on mothers and fetuses. ace i/d genotype was analyzed using a pcr method; an allelic discrimination approach was performed to detect the add snp. in mother-fetus genotype couples, neither ace nor add polymorphisms are associated with pe, nor are the combination of their genotypes separately in mothers and in fetuses. nevertheless in mothers with mild pe ace i-i genotype is significantly less frequent ( % vs %; p< . ) and ace i-d is significantly more frequent ( % vs %; p< . ) compared to controls. ace i allele frequency is not significantly different in mild pe compared to controls ( % vs %). in women with mild pe the i allele seems to move from i-i to i-d genotype. it leads to the increase in mild pe women of those genotypes with a higher ace activity conferring a higher susceptibility to develop pe. this association with mild pe and not with severe pe could be due to the contribution in the latter of many other genetic and environmental factors. introduction: maternal mrnas stored in the oocyte are critical for early development as transcription ceases upon oocyte maturation, and gene expression until zygotic genome activation (zga) is mediated by translation of maternal transcripts. cytoplasmic polyadenylation element binding protein (cpeb) plays a central role in this process by stabilizing maternal mrnas and regulating their timely activation. this function has been well characterized in xenpous, and a similar role in mammals is suspected as the cpeb knockout mouse displays infertility as a result of arrested oogenesis. in order to investigate if translational regulation of maternal transcripts by cpeb is maintained in mammals, we characterized the spatial and temporal expression of cpeb- in the mouse and human. methods: ten different somatic tissues, testes, and ovaries were tested by rt-pcr for the expression of cpeb mrna in mouse and human. cpeb mrna expression in mouse was also tested in prophase i (pi) and metaphase ii (mii) oocytes, -cell, -cell, -cell, -cell embryos and blastocysts. in human, pi and mii oocytes, -cell embryos and blastocysts were evaluated. sequencing of the pcr products was performed to confirm specific amplification of cpeb. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results and discussion: highest level of cpeb mrna expression was detected in ovaries and testis of both mouse and human. in addition, differential expression of cpeb in somatic tissues was also observed. among these, prominent expression was present in brain, where a role for cpeb in facilitating gene expression through cytoplasmic polyadenylation has been proposed. these data would suggest that translational control of mrna by cpeb may be a mechanism utilized by multiple somatic tissues to regulate gene expression. in the mouse, cpeb was expressed in pi and mii oocytes and -cell and -cell embryos, and became undetectable in -cell or more advanced embryos. human cpeb was expressed in pi and mii oocytes, but not in -cell embryos or blastocysts. zga occurs at late -cell stage and -to- -cell stage, in mouse and human, respectively. the tightly controlled temporal expression of cpeb prior to zga in both mouse and human is consistent with a conserved role for cpeb in the regulation of maternal mrna expression during early development in mammals. background embryonic stem cells (esc) express specific transcription factors that are indicative of their ability to maintain pluripotency. these factors are activated during preimplantation embryo development. the interplay between the transcription factors pou f (oct / ) and caudal-homeobox domain (cdx ) is thought to regulate inner cell mass (icm) and trophectoderm (te) differentiation; however, the mechanism of cell fate determination in mammalian embryos is poorly understood. genetic ablation of oct / in mice prevents icm development, while cdx knockout embryos fail to implant. esc deficient in nanog, a second key regulator of pluripotency, fail to maintain pluripotency and undergo differentiation. expression of nanog in primate embryos has not been investigated, therefore the localization of oct / , nanog and cdx was determined in rhesus macaque blastocysts. methods oocytes were collected from rhesus macaque females, fertilized and cultured in vitro to the blastocyst stage. embryos were collected hours post insemination, then fixed prior to incubation with primary antibodies directed against oct / , nanog and cdx . following incubation with cy conjugated secondary antibodies, nuclei were counterstained with dapi and embryos visualized using an olympus bx fluorescence microscope. results nanog protein was restricted to the icm of monkey blastocysts. unlike the mouse embryo, oct / protein was detected in both the icm and te, based on two different antibodies. the expression of cdx was localized specifically to the te. conclusions the ubiquitous pattern of oct / expression is consistent with observations in human, cow and pig embryos. significantly, lack of restricted oct / protein, and icm localization of nanog in primate blastocysts, suggests that nanog more specifically determines cell fate in primate embryos. these results contrast markedly with current mechanistic hypotheses, although other factors may lie upstream of nanog. importantly, this difference may underlie observations that regulatory mechanisms in esc differ between mice and primates. further investigations will focus on determining the onset of marker expression, and the upstream regulators of nanog activation. supported by nih grants r hd r rr , and the intramural research program of nichd. in vitro-conceived mice tend to be smaller at birth and throughout their life. luisa delle piane, annemarie donjacour, francesca di sebastiano, gnanaratnam giritharan, paolo rinaudo. obstetrics, gynecology and reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: epidemiological evidence indicates that ivf is associated with an increased incidence of low birth weight. this phenomenon has not been studied in a mouse model; in addition, it is unknown if the resulting offspring show different growth pattern later in life. we therefore created an ivf mouse model to follow growth patterns till adulthood. methods: we generated experimental groups of b mice: one cohort of in vivo generated mice (in vivo group), one cohort of in vitro generated animals (ivf group) transferred to cd foster mothers and one cohort of animals fertilized in vivo and transferred to cd foster mothers (flushed blastocysts group, fb). the fb group was generated because our preliminary results showed that embryo transfer to b foster mothers was not successful. all pups were delivered at term, measured and weighed at birth and then weekly up to weeks. parametric tests (anova with bonferroni correction) were used as appropriate. a mixed model was used to compare growth curves. results: average litter size was . (in vivo), . (fb) and . (ivf). birth weight of male mice both ivf ( . mg) and fb ( . mg) was larger than male in vivo mice ( . mg) (p< . ). ivf mice tended to be smaller than fb mice in both sexes (p= . ). the bmi (body mass index) was not different among all the groups. male fb growth curves were different from in vivo mice (p< . ) and more importantly from ivf growth curves (p= . ) (fig. ) . conclusion: the method of conception and the maternal environment play a significant role in determining birth weight in this mouse model, emphasizing that the fb group is the best control for the effects of ivf in this model. these preliminary results confirmed for the first time an ivf effect on growth and interestingly the ivf males did not show a catch-up growth. the finding of smaller ivf mice when compared to fb is both noteworthy, because it confirms human data, and worrisome, because lower birth weight is associated with long term sequelae according to the barker hypothesis. the effect of betamethasone exposure in mid-gestation on renal sodium excretion in male sheep. lijun tang, luke carrey, nancy valego, philip deibel, james perrott, jorge figueroa, mark chappell, james c rose. obestetrics and gynecology, wake forest university, medical school, nc, usa; hypertension center, wake forest university, medical school, nc, usa. objective: whereas prenatal exposure of ovine fetuses to clinically relevant doses of glucocorticoids during the time of peak nephrogenesis results in a reduction in nephron number in adulthood, there is little information about its effect on sodium excretion. in the present study, we evaluated the effect of exposure to betamethasone on renal sodium excretion in adult male sheep. methods: we studied nineteen conscious adult rams at . years of age which were exposed to either vehicle or betamethasone at - days gestation. we implanted vascular and bladder catheters and then allowed the animals a - days recovery period prior to study. inulin and para-aminohippuric acid (pah) clearances were performed for estimating glomerular filtration rate (gfr) and renal plasma flow (rpf) respectively. following determination under basal conditions, an acute hypertonic sodium load was administrated intravenously by a continuous infusion of nacl ( . meq/kg/min at . ml/min) for minutes. urine was continuously collected for determination of na + excretion. results: basal gfr was decreased in steroid exposed adults ( . ± . ml/min/kg) compared with the vehicle animals ( . ± . ml/min/kg) (p= . ). rpf was similar in the vehicle and steroid exposed group ( . ± . ml/min/kg vs . ± . ml/min/kg). at basal conditions, na + excretion (u na v) was similar in vehicle and steroid exposed group ( . ± . μmol/h vs . ± . μmol/h). this similarity was also present after normalization by body weight ( . ± . μmol/h/kg vs . ± . μmol/h/kg). the vehicle group excreted . ± . % of the dose of na during the experiment while the betamethasone exposed group excreted only . ± . % (p< . ). gfr and prpf did not change during the experiments. these results suggest that prenatal exposure to glucocorticoids affects renal function in adult male sheep which results in a decreased basal gfr and an attenuated ability to excrete on acute sodium load. the elevated blood pressure previously observed associated with this prenatal steroid treatment may be related to the alteration in renal function. the study is supported by nih grants hd , hl and hd . characterisation of human fetal cardiac stem cells. marah alfakir, nicholas dawe, annette meeson, stephen c robson. school of surgical reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. objectives: for many patients with severe cardiac disease treatment is limited to surgical intervention and/or heart transplantation. the heart has a limited regenerative capacity but it is insufficient to repair extensive damage. cellular therapies might provide an alternative treatment. we have identified stem cells (sp cells) in the adult mouse and human heart and have shown that the mouse cardiac sp cells can differentiate along a cardiac lineage. sp cells can be identified using facs combined with hoechst dye efflux. this ability to efflux hoechst dye is due to expression of abcg (an abc transporter). we have hypothesized that the fetal heart would contain more cardiac stem cells, relative to the adult, and the aim of this project was to determine the spatial and temporal expression of known stem cells markers in the embryonic/fetal heart. methods: human fetal hearts were collected aged between - wk. we used rt-pcr, using primers for several stem cell (abcg , cd , cd ) and cardiac specific (mlc- v, mhc) markers, and ihc on frozen and paraffin sections, using a panel of antibodies (abcg , cd , cd , islet ). cdna was generated from the following regions of the heart at different developmental stages: right and left atrium, right and left ventricle, and outflow tract. results: abcg mrna was highly expressed in all regions of the fetal heart between - wk. expression was down regulated at - wk especially in the la and lv. a similar pattern of expression was observed for cd . cd showed low/moderate expression in all heart regions examined; however, this expression was absent in the lv at - wk. mrna and protein analysis showed similar results. whilst protein expression of abcg was robust at wk (approximately - %), it declined with increasing gestational age. cd was also strongly expressed at weeks of age. there was no co-localisation between abcg and islet . conclusion: abcg and cd are both expressed between - wk of age. based on this analysis, further studies are underway to isolate and characterise both the abcg and cd expressing fetal cardiac cells which might be a potential source of cardiac stem/progenitor cells. xanthine oxidase plays a significant role in the fetal cardiovascular defence to hypoxia. ja hansell, a kane, e herrera, da giussani. physiology, cambridge university, united kingdom. prenatal hypoxia remains a major concern in obstetrics. the fetal defence to hypoxia includes redistribution of the cardiac output, away from peripheral and towards essential ciculations, such as those perfusing the brain (cohn et al . ajog : , ) . the physiology underlying this response is well characterised and involves chemoreflex and endocrine responses (giussani et al. fet mat med rev : , ) . more recently, local factors such as nitric oxide (no) and reactive oxygen species (ros), and the interaction between them, have been shown to play a role. antioxidants, such as vitamin c, scavenge hypoxia-induced ros, maintain no high and thereby depress peripheral constriction (thakor et al., sgi ) . in this study, we address the source of hypoxia-induced ros production and investigated the effects on the in vivo fetal femoral constrictor response to hypoxia of maternal treatment with the xanthine oxidase inhibitor allopurinol in sheep. methods: under anaesthesia, sheep at . gestation, were instrumented with maternal and fetal catheters and a fetal femoral transonic probe. five days later, all animals were subjected to h normoxia, . h hypoxia (mat fio to reduce fetal pao to ca. mmhg) and h recovery, either following maternal i.v. treatment with vehicle or allopurinol in low ( mg.kg - over min) or high ( mg.kg - over min ) doses. treatments finished min prior to hypoxia. the low allopurinol dose was adopted from human studies (benders et al. arch dis child : , ) . the timing of the hypoxic challenge coincided with peak concentrations in fetal sheep plasma of oxypurinol (active metabolite). we evaluated the effect of bpa exposure on uterine gene expression in a nonhuman primate model. method: a total of nine vervet monkeys (cercopithecus aethiops sabaeus) were ovariectomized. three monkeys were treated with bpa via alzet pumps ( microgram/kg/day), two with estradiol benzoate and three received combined treatment with bpa and estradiol. the remaining monkey was untreated and served as a control. following days of treatment the monkeys were hysterectomized and immunohistochemistry performed to examine endometrial gene expression. we evaluated hoxa , proliferating cell nuclear antigen (pcna), and caspase iii gene expression as markers of differentiation, proliferation, and apoptosis respectively. differential expression was evaluated by h-score. results: exposure to a combination of estradiol and bpa resulted in increased expression of hoxa and pcna compared to control (p< . ). lower, but increased, levels of hoxa and pcna gene expression were seen in the specimens exposed solely to estradiol (p< . ). those specimens exposed to bpa alone demonstrated a small induction in hoxa expression (p< . ) with no change in caspase iii or pcna expression when compared to the control. conclusion: bpa induced the expression of hoxa in the uteri of a non-human primate. exposure in the presence of endogenous estrogen further augmented estrogenic stimulation confirming that bpa is a xenoestrogen. bpa's effect of developmental gene expression may alter uterine differentiation. bpa acts as an endocrine disruptor by altering estrogen regulation on a gene required for fertility. contractility and meconium passage. jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, ahmet karadag, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: endogenous glucocorticoids (gcs) increase with advancing fetal age suggesting that gcs function as a hormonal modulator of organ maturation. although fetal colonic motility and meconium passage primarily occur near term, the role of gc in colonic maturation is poorly understood. we sought to examine gc-nuclear receptor (gc-nr) expression in ovine fetal distal colon smooth muscle from very-preterm to term gestation to define the cascade of gc initiated biochemical changes as a prerequisite for maturation-associated meconium passage. methods: ovine fetal distal colonic segments removed at very-preterm (vpt: - d gestation), preterm (pt: - d), near term (nt: - d) and term (t: - d) (n= fetuses in each group) were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochmical analysis with gc-receptor antibody ( : , sc- , santacruz biotechnology, ca) using standard abc regimen. immunoreactive material identified by , 'diaminobenzidine as chromogen. the percentage of gc-nr staining in smooth muscle-enteric unit was analyzed by counting dark brown immunostained nuclei at different fields at x magnification. all values are expressed as mean ± sem. results: the gc-receptor antibody immunostained nuclei both in smooth muscle and enteric units and the observed pattern (expressed as percentage of total nuclei) are as follows: nuclear gc expression varies with gestational age with maximal expression occurring near-term in smooth muscle and enteric neurons, in a synchronized manner. a near total disappearance of gc-nr expression occurs at term. these results suggest that peak gastrointestinal gc-nr mediated effects may occur prior to term with secondary signaling effects resulting in distal colonic motility maturation and meconium passage. the rna-binding protein hud co-localizes with choline acetyl mechanisms of in utero meconium passage. jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, octavio balbuena, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: we have previously speculated that crf, acting through its receptor crf-r , increases gastrointestinal (gi) motility and potentiates in utero meconium passage. patients with paraneoplastic syndrome with auto-antibodies to hud, a neuronal rna binding protein, develop severe gi dysmotility, indicating a role for hud in gi motility. hud binds mrna for actylcholinesterase implying a role in the cholinergic neurotransmitter system. based on these known functions, we hypothesized that hud may regulate post-transcriptional activity in fetal colonic cholinergic neurons expressing crf-r . method: bouin's solution-fixed paraffin-embedded sections of distal colonic segments were prepared from very preterm (vpt: - days), preterm (pt: - days), near term (nt: - days) and term ( - days) ovine fetuses (n= at each age). sections were immunostained with anti-human neuronal protein huc/hud antibodies ( : to : ) with abc reagents and examined at x. neurons positive for hud staining were quantified. double immunofluorescence and laser confocal analyses evaluated co-expression of hud in neurons expressing peripheral choline acetyl transferase (pchat) (a marker for peripheral cholinergic neurons) and crf-r receptor. results: hud immunostaining was seen in either entire cytoplasm (c) or cytoplasm and nuclear regions (c+n) in both submucosal and myenteric neurons. a greater percentage of submucosal (vpt: ± , pt: ± , nt: ± , and t: ± %) than myenteric neurons (vpt: ± , pt: ± , nt: ± , t: ± %) exhibited positive staining at all ages. the percentages of neurons with c+n staining in submucosal neurons were significantly lower at very-preterm gestation. confocal studies co-localized the hud staining with pchat and crf-r receptor immunoreactivity both in submucosal and myenteric neurons. conclusion: in the fetal enteric nervous system hud may function both as a rna-binding protein and as a nuclear cytoplasmic shuttling protein. colocalizaion studies suggest that both pchat-mrna and crf-r mrna species are target transcripts for hud in myenteric neurons. we speculate upregulation of hud contributes to in utero meconium passage. meconium passage during mild stressors. jayaraman lakshmanan, guo l liu, john d richard, sharon k sugano, raina khan, octavio balbuena, kimberly chap, virender rehan, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: mr exhibit a greater affinity to glucocorticoids (gc) than do glucocorticoid receptors (gr). thus low circulating gc levels may preferentially bind mr during mild-stress periods, while the high gc levels may bind gc-receptors. mr antagonism increases gc-mediated responses, suggesting that mrs have a regulatory impact on gc-mediated stress responses. we recently documented that fetal in utero meconium passage is a neurovisceral stress response. to test our hypothesis that mrs play a primary role in mediating suppressive gc effects, we examined the expression patterns of mr in ovine fetal distal colon. methods: bouin's solution fixed paraffin sections of distal colonic segments collected from ovine fetuses (n= for each gestational ages) at very preterm (vpt: - days gestation), preterm (pt: - days), near term (nt: - days) and term (t: - days) were subjected to immunohistochemistry with polyclonal antibodies to mr. digital photos ( field per colonic ring, rings each gestational age) taken at x were used to count the number of intensely stained neurons, referred to as "mr-capped neurons". differences over time were determined with anova. results: mr antibody elicited a punctate staining pattern in all layers of ovine fetal distal colon. significant immunoreactive intensity was observed in submucosal and myenteric ganglia at all ages: submucosal ganglia: vpt= . ± . , pt= . ± . , nt= . ± . , t= . ± . ; a subpopulation of enteric neurons exhibited dense staining ("mr-capped"). advancing gestation was associated with a significant decreased in the percentage of mrcapped myenteric (vpt = ± , pt = ± , nt = ± , t = ± %; p< . ), though not submucosal ganglia neurons. results indicate a significant decrease in the percentage of mr capped myentric neurons with advancing gestation. in view of the potential inhibitory effect of mr on gc-mediated stress responses, these results suggest that the decrease in mr expression may contribute to near term and term in utero meconium passage. the ambiguity of myometrial progesterone receptor expression in pregnancy and labour. alison j tyson-capper, elizabeth a shiells, stephen c robson. surgical reproductive sciences, institute of cellular medicine, newcastle university, newcastle upon tyne, united kingdom. background and aims: in contrast to many other species, human parturition is not due to a reduction in circulating levels of progesterone (p) but appears to be related to changes in expression, ratios or signalling events of p receptors (pr-a and pr-b). the literature on pr expression in human myometrium in pregnancy and labour remains conflicting. we hypothesise this is due, at least in part, to differing specificities of the various pr antibodies employed. we carried out a comprehensive analysis of pr expression using ten 'pr-specific' antibodies that recognise different amino acid epitopes within the pr proteins. two phosphorylation-specific antibodies for pr were also included to define whether phosphorylation status of myometrial pr changes in pregnancy and in labour. methods: western immunoblotting and semi quantitative rt-pcr were undertaken using protein lysates and rna prepared from myometrial tissue from: -first (n= ) and second (n= ) trimesters, paired upper and lower segment myometrium from preterm ( - wks, n = ), term not in labour (n = ), term spontaneous labour (n = ) and non pregnant (n = ). results: using the same set of myometrial lysates for each antibody, we found that the specificity of individual pr antibodies, in particular those raised against internal and c-terminal epitopes of the pr proteins, varied considerably resulting in different patterns of expression. there also appeared to be temporal and spatial differences in levels of myometrial pr proteins ( - kda/ - kda) in pregnancy and in labour with use of the phosphorylation-specific pr antibodies, however, it remains to be resolved which pr isoforms these may be. in contrast, data using four antibodies all of which react with the amino terminal domain of pr consistently indicated that expression of pr, in particular pr-b, decreased significantly at term (p < . ) and in labour (p < . ) when compared to non-pregnant levels. a decrease in levels of pr-b protein was also observed when comparing preterm levels to non-pregnant levels. rt-pcr using primers specific to pr-b consistently indicated that levels of pr-b mrna decreased at term and in labour. conclusion: interpretation of gestation-related changes in myometrial pr expression and phosphorylation status must take into account the pr antibodies used. further studies are now underway to validate the specificity of the pr antibodies. objective to test the hypothesis that expression of fshr in mural gl cells from ivf follicles varies with the infertility diagnosis and correlates with the outcome of ovulation induction (oi). materials and methods women undergoing ivf were classified as: . "no ovarian factor", (tubal or male factor and egg donors, nof;n= ); . endometriosis (endo; n= ); . poor responders (pr; n= ); . polycystic ovary syndrome (pcos; n= ). ovulation induction was carried out using a long or microflare or antagonist protocol based on clinical parameters and gonadotrophin doses were selected based on ovarian reserve (day fsh and e and basal antral follicle count) and adjusted to the individual response. after ultrasound guided egg retrieval, mural gl cells were isolated from pooled follicular fluids from each patient using a percoll gradient and anti-cd immunobeads to eliminate wbcs, viability was assessed by trypan blue. fshr was measured by rt-pcr as relative expression compared to beta actin. statistical analysis was performed with the spss using pearson´s correlation, one way anova and student´s t-test. results fshr expression in nof ( ± . ) was statistically significantly higher than in pr ( ± . ) and lower than in pcos ( ± . ). both endo ( ± . ) and pr levels of fshr were statistically significantly lower than in pcos. analysis of all cycles showed that fshr expression correlates positively with the number of total (r= , ; p< , ) and mii (r= , ; p< , ) oocytes and negatively with the units of fsh (r=- , ; p< , ) and lh (r=- , ; p< , ) administered for oi. separate analysis of nof showed a positive correlation with the number of total and mii oocytes retrieved and with estradiol levels on day of hcg but not with the total dose of gonadotropins received during oi. fshr expression correlates negatively with day fsh levels in all patients except in pcos (r=- , ; p< , ). conclusions these results suggest that expression of fshr in the hyperstimulated ovarian follicle is "average" in normoresponders, high in high responders (pcos) and low in poor responders (pr and endo). knowledge of the level of expression of fshr might be useful to individualize gonadotrophin doses and oi protocols thus improving pregnancy rates. comparison of intracellular atp levels between non-vitrified and surviving post-vitrification/thawed human oocytes. somjate manipalviratn, zhi-bin tong, eric widra, , alan h decherney. reproductive biology and medicine branch, nichd/nih, bethesda, md, usa; department of obstetrics and gynecology, georgetown university hospital, washington, dc, usa; shady grove reproductive science center, washington, dc, usa. objective: to compare intracellular atp level in non-vitrified and surviving post-vitrification/thawed human oocytes using an atp bioanalysis assay. design: prospective match-controlled laboratory study material and methods: all oocytes were obtained from women undergoing controlled ovarian stimulation for ivf/icsi. oocytes were discarded due to nuclear immaturity at the time of planned icsi. all immature oocytes (gv and mi) were incubated overnight. oocytes from patients with or more discarded eggs which matured to mii stage were used in this study. oocytes from each women were randomly divided into groups. in group , the oocytes were placed in l of ultrapure water and kept at - o c for further atp analysis. in group , the oocytes were vitrified using % ethylene glycol, % dimethyl sulphoxide and . m sucrose as cryoprotectant. these oocytes were kept in liquid nitrogen for - days before thawing with a rapid thawing method. oocyte survival was determined by morphological assessment after minutes of incubation then oocytes were placed in l of ultrapure water and kept at - o c for further atp analysis. intracellular atp level was determined using luciferin-luciferase bioluminescent assay. result: ninety-one discarded human oocytes were obtained from women. oocytes were vitrified/thawed before measuring for atp content. the other oocytes were measured for their atp content without undergoing vitrification/thawing process. oocyte survival rate after vitrification/thawing is . % ( / ). mean oocyte atp levels in non-vitrified oocytes is significantly higher than surviving post-vitrification/thawed oocytes (table ). coefficient of variation of luciferin-luciferase bioluminescent assay is less than %. conclusion: oocyte atp level in surviving post-vitrification/thawed human oocytes is significantly lower than non-vitrified oocytes. objective to study the expression of genes involved in cell proliferation and steroidogenesis in the human follicle (kl , kl , fshr, papp, p ) and its relationship with ivf outcome. materials and methods patients with different infertility diagnosis underwent ovulation induction with either a long or microflare or antagonist protocol based on clinical parameters; the dose of gonadotrophin used for ovulation induction was selected based on the ovarian reserve (day fsh and e and basal antral follicle count) and adjusted to the individual patient response. ultrasound guided egg retrieval was performed hours after administration of . iu of hcg. mural granulosa-lutein cells (gl cells) were isolated from pooled follicular fluids (ff) from each patient using a percoll gradient and anti-cd immunbeads to eliminate wbcs, viability was assessed by trypan blue. the genes under study were measured by rt-pcr as relative expression compared to actin. statistical analysis was performed with the spss statistical software using pearson´s correlation and mann-whitney u. results kl expression correlates positively with kl levels (r= , ; p< , ) and with genes implicated in granulosa cell function such as fshr (r= , ; p< , ), papp (r= , ; p< , ) and p (r= , ; p< , ). the number of mii oocytes obtained is positively correlated with both fshr (r= , ; p< , ) and papp (r= , ; p< , ) expression, but not with the other genes. kl expression in women who became pregnant during the ivf cycle studied was higher ( , ± ) than in non pregnant women ( , ± , ).(n= , mann-whitney u p< , ). conclusions patients who become pregnant have increased expression of kl , which is associated with optimal granulosa cell proliferation and maturation. this is in accordance with the presence of lower apoptosis in granulosa cells in the same patients. syndrome during assisted reproduction treatment. k jayaprakasan, r jayaprakasan, h al-hasie, js clewes, bk campbell, ir johnson, nj raine-fenning. school of human development, university of nottingham, nottingham, united kingdom. objective: to test the hypothesis that an increased pre-treatment ovarian blood flow is associated with the development of ovarian hyperstimulation syndrome (ohss) and to evaluate ovarian vascularity as a predictor of ohss during invitro fertilization (ivf). methods: subjects undergoing first cycle of ivf had d transvaginal ultrasound in the early follicular phase of the menstrual cycle preceding ivf. of them developed ovarian hyper-response, defined as retrieval of oocytes and ohss. subjects had normal ovarian response with retrieval of to oocytes in the absence of ohss. antral follicle count (afc), ovarian volume (ov), and ovarian vascularity (vascularisation index, vi; flow index, fi and vascularisation flow index, vfi) were measured and an unpaired t-test was used to compare these parameters between the ohss and the control groups. multiple logistic regression analysis was used to assess the predictive value of these variables against age, bmi and basal fsh for the development of ohss. results: the ovarian vi ( . ± . vs. . ± . ), fi ( . ± . vs. . ± . ) and vfi ( . ± . vs. . ± . ) were similar in both the groups. afc and ov were significantly higher (p< . ) in the ohss group ( . ± . and . ± . cm respectively) than in the control group ( . ± . and . ± . cm respectively). afc was the only significant (p= . ) predictor of ohss on multiple regression analysis (table ) . conclusion: women developing ohss during ivf do not demonstrate an increased pre-treatment ovarian blood flow as measured by d ultrasound but do have a significantly higher afc, which is the only significant predictor of ohss. compared to other species, little is known about the steroid biosynthetic capacity and gene expression profiles of human cumulus cells. objective: to examine the ability of human cumulus cells in primary and long-term culture to synthesize steroids and respond to gonadotropin or camp-dependent stimulation. methods: human cumulus cells were isolated from cumulusoocyte complexes during assisted reproductive technology (art) and placed in primary culture or propagated to third passage. at subconfluence, cells were transferred into serum-free conditions in the presence of vehicle, forskolin, fsh, or hcg. at h, media was collected and progesterone (p ) and estradiol (e ) levels were determined by eia. aromatase activity was measured by the tritiated water assay. aromatase (cyp ) and cholesterol side-chain cleavage (cyp a ) mrna abundance was determined by quantitative real-time pcr (qrt-pcr). transcriptional regulation of the cyp and cyp a promoters was investigated by transient transfection of cumulus cells with promoter luciferase constructs. results: substantial amounts of p and e were synthesized by cumulus cells in culture, which were further elevated by forskolin, hcg, or fsh treatment. the presence of cyp and cyp a mrna in cumulus cells was confirmed by qrt-pcr, and the relative mrna abundance of both transcripts was induced by forskolin, hcg, or fsh treatment. changes in cyp and cyp a gene expression in response to camp and gonadotropin stimulation were associated with increased transcriptional regulation of both the cyp and cyp a gene promoters. our studies demonstrate that human cumulus cells are sites of significant p and e biosynthesis and respond to camp-and gonadotropin-stimulation in vitro. the ability to examine human cumulus cells in primary and long-term culture provides a unique model system to investigate cumulus cell function, and the paracrine role of cumulus cells in oocyte development, maturation, and subsequent fertilization. background: there is increasing evidence suggesting that events occurring during fetal development may result in increased predisposition to adult conditions, such as diabetes. in vitro fertilization (ivf) is a new environmental stressor and the long-term effects of these manipulations are currently unknown. there is some evidence that lower number of insulin-secreting cells at birth signals predisposition to diabetes later in life. in this study, we compare areas of pancreatic insulin-secreting cells in newborn mice conceived in vivo versus in vitro. methods: oocytes were collected from super ovulated cf- mice and fertilized in vitro with cauda epididymal sperm from b d f /j mice. fertilized eggs were cultured in whitten medium under % co in humidified air at °c for h. blastocysts were transferred to the uteri of pseudo-pregnant recipients. control mice were allowed to conceive in vivo. the newborn animals were sacrificed within hours of birth. pancreases were sectioned, immunostained with anti-insulin, and total areas and stained areas were compared using t-test with two-tailed distribution. p value of < . was considered significant. results: there were total of newborn animals: females ( ivf, controls) and males ( ivf, controls). percentage of total pancreatic area occupied by insulin-secreting cells was lower in female ivf ( . mm , . %) animals compared to female controls ( . mm , . %) and higher in male ivf ( . mm , . %) animals compared to controls ( . mm , . %). the average of males and females was slightly lower for ivf ( . mm , . %) than controls ( . mm , . %). none of these differences were statistically significant. there was a trend in newborn female mice towards lower amount of insulin-secreting cells in the ivf offspring, suggesting possible predisposition to diabetes later in life. this effect was not observed in newborn males. while our study failed to demonstrate consistent and significant differences in the areas of insulin-secreting cells between newborn mice conceived in vitro and in vivo, the number of animals in the study may have been too small to show significant results. larger studies are needed to further investigate this question. peter s uzelac, phyllis risch, kassi shelton, steven t nakajima. obstetrics and gynecology, university of louisville, louisville, ky, usa. objective: several fertility preservation methods currently available may not be viable options to certain patients due to their high cost and limited number of centers offering these services. for women undergoing bilateral oophorectomy, in vitro maturation (ivm) of oocytes retrieved from unstimulated whole ovary specimens may represent a more practical solution. here we describe out initial experience in offering ivm as a fertility-preserving measure to two patients undergoing total abdominal hysterectomy and bilateral salpingo-oophoectomy (tah-bso). case one was a year old nulligravid with chronic pelvic inflammatory disease unresponsive to medical management. case two was a year old nulligravid with stage ia endometrial carcinoma. surgery was planned for the mid-follicular phase in order to collect prophase i oocytes before the onset of late-follicular phase atresia. upon surgical removal, suction aspiration of all identifiable follicles was performed. ovaries were then serially sectioned and all tissue was examined for the presence of prophase i oocytes. results: total number of prophase i oocytes retrieved was and , maturation rate after hours was % ( / ) and % ( / ), fertilization rate after hours was % ( / ) and % ( / ), maturation rate after hours was % ( / ) and % ( / ) and fertilization rates after hours % ( / ) and % ( / ), for case and case respectively. ivm of oocytes retrieved from unstimulated whole ovary specimens may be a simple and inexpensive approach to fertility preservation in women undergoing bilateral oophorectomy. by requiring minimal adjustments to in vitro fertilization protocols, this treatment could easily be implemented in centers which currently have no fertility preservation program. patient age at time of retrieval may be predictive of developmental potential. continued patient enrollment and follow-up studies on the developmental potential of embryos derived from this technique are necessary to fully evaluate its potential for a role in fertility preservation. the routine use of progesterone as luteal phase support in women with a diagnosis of polycystic ovary syndrome (pcos) undergoing ovulation induction cycles with oral agents has not been fully elucidated. we hypothesize that women with pcos utilizing either clomiphene citrate or letrozole, an aromatase inhibitor, should administer intravaginal progesterone in the luteal phase to increase pregnancy rates. design: retrospective chart review. materials and methods: cycle data from women with pcos undergoing ovulation induction with clomiphene citrate or letrozole at the university of cincinnati medical center from to were evaluated. diagnosis of pcos was based on rotterdam criteria and all other infertility diagnoses were excluded. clinical pregnancy rates (presence of fetal cardiac activity on ultrasound at - weeks gestation) in women who received intravaginal micronized progesterone ( mg bid) following ovulation induction (with and without intrauterine insemination) were compared to those who did not receive progesterone. results: no significant differences were noted in demographic parameters, including patient age or bmi. a total of cycles were evaluated in women treated with clomiphene citrate. clinical pregnancies were documented in . % ( / ) of cycles in the progesterone group compared to . % ( / ) of the non-progesterone group (p < . ). forty-three cycles were evaluated in patients treated with letrozole. clinical pregnancies were documented in . % ( / ) of cycles in the progesterone group compared to none ( / ) in the non-progesterone group (p < . ). conclusions: patients with pcos who used letrozole for ovulation induction had superior clinical pregnancy rates when using intravaginal micronized progesterone compared to women who did not receive luteal phase support. there was a trend toward increased clinical pregnancy rates in pcos patients utilizing luteal support following clomiphene citrate for ovulation induction. therefore, in women with pcos undergoing ovulation induction with oral agents, luteal supplementation with progesterone should be strongly considered, especially in those using letrozole. were measured and an unpaired t-test was used to compare these parameters between poor and normal responders. multiple logistic regression analysis was used to assess the predictive value of these variables against age and basal fsh for poor ovarian response. results: the ovarian vi ( . ± . vs. . ± . ), fi ( . ± . vs. . ± . ) and vfi ( . ± . vs. . ± . ) were similar in both poor and normal responders. afc and ov were significantly lower (p< . ) in poor responders ( ± . and . ± . cm respectively) than in normal responders ( . ± . and . ± . cm respectively). afc was the best (p< . ) predictor of poor ovarian response on multiple regression analysis ( objective: gay male couples increasingly seek parenthood through in vitro fertilization using an oocyte donor and a gestational carrier, but no studies describe this unique experience. the purpose of this study was to determine medical and psychosocial issues unique to gay men using art. design: qualitative analysis of semi-structured interviews with gay male couples seeking parenthood through art. materials and methods: sixteen gay males (eight couples) entering an art program were assessed through the use of a semi-structured interview. characteristics evaluated included age, relationship status, duration of their relationship, psychological health and stability, how the decision was made concerning who would donate the sperm, the decision to use an anonymous or known oocyte donor, and whether their gestational carrier was someone previously known to them or not. results: the average age of the men in this study was years. all eight couples were in a committed relationship and had been together for an average of . years. five of the couples ( %) had been joined in a civil union which is legal in the state of connecticut. all subjects were psychologically stable and in good health. six couples ( %) were very clear about which partner would inseminate the oocytes. of those couples, two felt that the older partner should donate; two felt that the partner who cared more about a genetic connection to the child should donate; and two felt that the partner with "better genes" should donate. the remaining two couples chose to inseminate equal numbers of oocytes in order to transfer an embryo from each partner. all couples chose an anonymous oocyte donor, two couples chose relatives as their gestational carriers, while the others chose carriers recruited through an agency. conclusions: participants in this study were determined to become parents through assisted reproduction. they had given thoughtful consideration to the medical and psychosocial issues unique to this process including which partner would be the genetic parent, and why. clomiphene superovulation. rb allen, az steiner, rh fogle, mj kalan, rj paulson. ob/gyn, usc keck school of medicine, la, ca, usa; ob/gyn, unc, chapel hill, nc, usa. intro: micro-dose hcg has been demonstrated to increase ovulation and pregnancy rates following clomiphene administration in women resistant to clomiphene. since micro-dose hcg stimulates growth in follicles expressing the lh receptor, we hypothesized that its use following clomiphene in women with unexplained infertility would increase the number of follicles that ovulate and subsequently, pregnancy rates. m m: irb approval was obtained for this prospective pilot study of women with unexplained infertility. on day # a baseline ultrasound was performed and serum fsh and e levels were measured. subjects were given mg of clomiphene daily from days - and then returned for serial ultrasounds on day # . when at least follicles mm were present, iu of hcg im daily was initiated. cycles were monitored by ultrasound every days until a +lh surge occurred. all subjects had previously undergone a cycle using clomiphene alone for superovulation followed by iui and these cycles were used for comparison. results: subjects, aged . ± . yrs (mean±sem) and bmi . ± . kg/m with . ± . yrs of infertility were enrolled. the mean fsh and e levels were . ± . miu/ml and . ± . pg/ml, respectively. there was an average of . ± . days from starting clomiphene to the attainment of follicles mm. out of the subjects had follicles mm by day # . a mean of . ± . days of treatment were required until ovulation occurred. there were twice as many follicles in the micro-dose hcg cycles, although due to the small sample size this did not reach statistical significance ( . ± . follicles, measuring . ± . mm in the micro-dose hcg cycles, compared to . ± . (p= . ), measuring . ± . mm (p= . ) in the control cycles). there was no difference in the endometrial thickness at the time of ovulation between the micro-dose hcg cycles and the control cycles: . ± . mm vs. . ± . mm (p= . ), respectively. all subjects had at least ovulatory sized follicles in the study cycles. % of subjects had an iui performed, however there were no pregnancies resulting from this study. conclusions: ) the addition of micro-dose hcg to clomiphene may allow more follicles to reach an ovulatory size, which should theoretically increase pregnancy rates ) this pilot study demonstrates that adding micro-dose hcg may increase the effectiveness of clomiphene when given in a superovulatory protocol. jessica salas, donald maier, john nulsen, claudio benadiva, lawrence engmann. obstetrics gynecology, university of connecticut, farmington, ct, usa. objective: to evaluate the antepartum, intrapartum, and neonatal complications in patients undergoing ivf using a gnrh-antagonist protocol where a gnrhagonist was used to induce final oocyte maturation. materials and methods: a retrospective review of data from high responders undergoing their st or nd ivf cycle using a gnrh-antagonist protocol who were triggered with leuprolide acetate (study group) or hcg (control) and achieved at least a singleton pregnancy reaching the third trimester. patients younger than years old were included. both groups received luteal phase support with intramuscular progesterone. the study group received estrogen patch supplementation. outcomes measured were antenatal and intrapartum complications, order of gestation, gestational age at delivery, birth weight, neonatal adverse outcomes, and congenital anomalies. pearson chi square, fisher's exact test, or independent t-test were used as appropriate. results: the baseline characteristics were different (table ) . maternal antenatal and intrapartum complications were similar in both groups ( . % vs . %, p= . ). there were more singletons in the study group ( . % vs . %, p< . ). a subgroup analysis of gestational age at delivery, birth weight, neonatal complications and congenital anomalies in singletons showed no difference (table ) . conclusions: this is the first study reporting the maternal and perinatal outcomes after gnrh-agonist trigger. differences in response to ovarian stimulation may dictate use of gnrh-agonist triggering for prevention of ohss, which may explain the differences in baseline characteristics. maternal and neonatal complications remain unaffected. table lupron (n= ) objective: moderate to severe ovarian hyperstimulation syndrome (ohss) occurs in - % of assisted technology cycles and carries the potential for severe complications. traditional management consists of hospitalization with intravenous fluids (ivf), bedrest, and close monitoring. early and aggressive paracentesis is an alternative method of treatment for ohss in an outpatient setting, but the economic consequences of the two treatment regimens have not been compared. design: cost-effectiveness analysis materials and methods: two scenarios, outpatient management with transvaginal paracentesis and conservative therapy with hospitalization, were compared. potential initial outcomes were analyzed for the conservative group to include hospitalization either in a ward hospital bed or the intensive care unit (icu) for an average of seven days. costs included ivf administration, ultrasound, and daily bloodwork. initial outcomes for the outpatient management group included no further therapy beyond the initial transvaginal paracentesis, bloodwork, ivfs, and ultrasound versus admission for an average of three days to a ward bed with similar management. the probability of the negative outcome for the conservative group was set at % (icu admission) and % for the outpatient group (regular hospitalization). results: the cost of conservative therapy including first tier complications ranged from a low of $ , to a high of $ , . the cost of outpatient management with aggressive paracentesis and its first tier complications ranged from a low of $ to a high of $ , . this resulted in an estimated cost burden of $ , to $ , for conservative management with hospitalization. the main improvement factor was that patients in the outpatient management group had a much lower likelihood for prolonged hospitalization than the conservatively managed group. conclusions: aggressive outpatient treatment of moderate to severe ohss with early paracentesis appears to be a cost-effective strategy. induction. zeynep alpay, michael p diamond, michael l kruger, elizabeth e puscheck. obstetrics and gynecology, division of reproductive endocrinology and infertility, hutzel hospital, wayne state university, detroit, mi, usa. introduction: aromatase inhibitors (ai) are a new method of ovulation induction and is proposed to replace clomiphene citrate (cc) due to their reported advantages. their superior effect is thought to be due to up-regulation of the estrogen receptors and increase in the sensitivity of the endometrium, resulting in better proliferation despite low estrogen levels in the circulation. objective: to compare the effect of different ovulation induction methods, including ai, cc and gonadotropins (gt) on the endometrium in infertility patients. methods: we reviewed ovulation induction cycles performed in our institution in the last one-year period retrospectively. there were gt, cc and ai cycles. age, gravida, day (d et) and midcycle (mcet) endometrial thickness, endometrial pattern (ep) on day of hcg injection, endometrial growth during the induction cycle (d-et), which was calculated by the difference between mcet and d et, and the pregnancy rates (pr) were compared. the ep was categorized as type a (homogenous and hyperechogenic), type b (intermediate isoechogenic pattern and a poorly defined central echogenic line), and type c (multilayered triple-line). chi-square, anova, t test and pearson correlation were used for statistical analysis. results: in all cycles, ep was closely related to the d-et (p < . ). this correlation appeared to be more pronounced when observed ep was compared with d-et (r = . ; p < . ) rather than with mcet (r = . ; p = . ). there was a reverse relationship between the age of the patients and ep (r = - . ; p < . ). a negative correlation was also found between the gravida and ep (r = - . ; p < . ). pregnancy rate was significantly correlated with ep (r = . ; p < . ). pregnancy rate was % in type a ep, . % in b, % in c when all cycles were analyzed. aromatase inhibitors and gt treatments each resulted in % type c pattern in contrast to % with cc (p = . for ai vs. cc; p = . for gt vs. cc). the d-et was greater in gt cycles compared with ai or cc (p < . ). conclusion: these results show that ep has a strong correlation with pr in ovulation induction cycles. additionally, ai and gt treatments have similar effects on ep. both ep and the growth of the endometrium during the induction (d-et) are good prognostic variables for the successful pregnancy initiation. ovarian response in patients undergoing ovarian stimulation after myomectomy. hyacinth browne, , desiree mccarthy-keith, , barbara stegmann, , alicia armstrong. , reproductive biology and medicine branch, nichd, nih, bethesda, md, usa; reproductive endocrinology and infertility, walter reed army medical center, washington, dc, usa. objective: the impact of myomectomy on ovarian function has not been wellstudied. other surgical treatments of fibroids, such as hysterectomy and uterine artery embolization, have shown an increase of fsh into the peri-menopausal range. the objective of this study is to examine ovarian response in infertile women undergoing ovarian stimulation after abdominal myomectomy. design: retrospective analysis. materials and methods: a retrospective analysis of all infertile women with known fibroids who had a failed art cycle, from january to , followed by an abdominal myomectomy and a subsequent art cycle was performed. women served as their own controls. ovarian function pre and post-myomectomy was assessed by age, day and fsh levels, days of stimulation, total gonadotropins used, peak estradiol level, number of oocytes retrieved, embryos obtained, and high-grade embryos, and pregnancy outcome. quantitative results are presented as mean + sd. results: four women had a failed art cycle and underwent an abdominal myomectomy prior to a subsequent art cycle. the mean age was and pre-and post-myomectomy, respectively. all subjects had uterine factor infertility. two of these women also had tubal factor infertility, and one had endometriosis and male factor infertility. we found no difference in ovarian response pre and post-myomectomy. pre-myomectomy post-myomectomy age (years) + . + . day fsh (u/l) . + . . + . day fsh (u/l) . + . . + . objective: anti-mullerian hormone ( amh) is produced by developing primordial follicles. amh levels are stable through the menstrual cycle and therefore can be drawn randomly. the objective of this study was to assess the association of anti-mullerian hormone ( amh) and oocytes obtained at ivf retrieval. design: cross-sectional cohort study. materials and methods: the study cohort is comprised of thirty women in cycles of in vitro fertiization (ivf). serum levels antimullerian hormone (amh) were drawn before starting an ovulation induction cycle for ivf. standard ovulation induction was performed with leuprolide flare and miu/ml per day of follicle stimulating hormone. we performed linear regression of log convert d oocyte number against the log converted amh level. serum amh was measured using an enzymatically amplified two-site immunoassay dsl- - active mis/amh elisa. statistical analysis was performed using spss version . . continuous values are presented as mean std error. results: the log number of oocytes retrieved was directly related to the log value of amh (p = . ). conclusions: our data demonstrate an association between serum amh and oocytes produced in response to ovulation induction. using amh levels in addition to fsh levels in evaluating for ovarian reserve and pregnancy outcome may help improve predictions of reproductive performance. support: foundation for reproductive medicine. context: prediction of outcome after in vitro fertilization (ivf) can be difficult due multiple factors. human chorionic gonadotropin (hcg) levels correlate with pregnancy outcome, but data that can be used to easily counsel patients on their possible outcome is lacking. objective: to investigate the use of hcg levels along with other significant factors to predict the likelihood of an ivf pregnancy progressing to the point of detection of cardiac activity by ultrasound design: retrospective data analysis of ivf cycles performed from january to july resulting in pregnancies. multiple logistic regression analysis modeling was performed to determine the factors most predictive of an ongoing early pregnancy and to assess possible confounding variables. setting: an academic fertility center. patients: patients undergoing in vitro fertilization using autologous fresh embryos. intervention: none main outcome measure: pregnancy continuation to the documentation of cardiac activity by ultrasound. results: maternal age, day (post-oocyte aspiration) hcg level, and day hcg levels were significant in predicting pregnancy outcome. day and day hcg levels were highly correlated and can be considered proxies for each other. the most accurate predictive model used only a single day hcg level and maternal age. the type of fertilization method used, the cycle number for that patient, and the number of embryos transferred were not found to be significantly different. ongoing pregnancy rates were directly proportional to day hcg level, and inversely proportional to maternal age. the incidence of multiple pregnancies also increased proportionally to the initial hcg level. % of ongoing pregnancies had hcg level > miu/ml. conclusions: a single day post-oocyte aspiration hcg level and maternal age are the most predictive of an ongoing ivf pregnancy. there was no difference in outcome between fertilization methods or number of embryos transferred. there is no benefit to obtaining serial hcg levels after the initial one. erk activation with u ( m)) reduced fsh stimulated cyclin d mrna expression by %. fsh has also been shown to stimulate mtor, a regulator of growth and proliferation of many cell types. inhibiting mtor activation with nm rapamycin for min significantly reduced fsh-mediated cyclin d mrna expression. dht exposure for hours also inhibited fsh stimulated mtor signaling, as shown by a % reduction in the phosphorylation of its down stream target p s kinase. furthermore, pretreatment with dht resulted in significantly reduced fsh-mediated tsc- phosphorylation, which is an upstream regulator of mtor pathway. further studies revealed that fsh mediated tsc- phosphorylation and mtor signaling is regulated by erk, but independent of akt. based on these results we conclude that elevated reduced metabolites of androgens inhibit fsh-stimulated pka-erk pathway resulting in the inhibition of multiple mitogenic signaling pathways leading to defective follicle maturation culminating in anovulation. thus, rescuing the erk activation might serve as a potential therapeutic target to restore normal granulosa cell proliferation in hyperandrogenic states. (supported by nih grant hd- ). searching pcos-genes: results of a genome screen for pcos in a dutch founder population. olivier valkenburg, annemarie g mulders, aida bertoli-avella, ben a oostra, joop se laven. department of gynecology and obstetrics, erasmusmc, rotterdam, netherlands; department of internal medicine, erasmusmc, rotterdam, netherlands. context: although the etiology of pcos is not yet fully understood, evidence has accumulated for a complex genetic background i.e. a combination of multiple genetic and environmental factors. to reduce genetic heterogeneity, this study was conducted in a genetically isolated population in the netherlands . objective: in order to identify genomic loci that are associated with pcos, a whole genome screen using highly polymorphic microsatellite markers was performed in a founder population. subjects and methods: pcos patients ( rotterdam criteria) were identified in the founder population (rucphen, the netherlands) of ± . inhabitants. patients underwent a standardized screening procedure that included clinical, ultrasound and endocrine evaluation. all patients plus unaffected first-degree family members were genotyped using polymorphic markers on (microsatellites) from the abi prism ® linkage mapping set md- (average spacing cm). association-analysis was performed with the transmission disequilibrium test (tdt, genehunter software). linkage analysis was performed in clusters of or more closely related patients (simwalk ). results: there was an average number of . alleles per polymorphic marker. the tdt identified two non-adjacent markers on chrome (d s and d s ) that showed significant association with pcos (p , ). other markers that showed significant association were positioned on chromosomes (d s ), (d s ), (d s ) , (d s ), (d s ) and seventeen (d s ) (all p values . ). linkage analysis in sub-pedigrees revealed no significant linkage for these, or other, loci with pcos. moreover the results of a prior report of linkage of a marker on chromosome (d s ) could not be confirmed. conclusions: the lack of consistent results suggests the absence of a consistent genetic background in this select group of pcos patients. this supports the hypothesis of a complex genetic background for pcos that allows relatively small contributions of multiple risk-genes to be involved in the pathogenesis of this syndrome. in this founder-population the genetic heterogeneity was not sufficiently reduced to find risk-loci in a genome wide screen using highly polymorphic markers with an average spacing of cm. objectives: to objectively quantify uterine and endometrial blood flow in women with polycystic ovarian syndrome (pcos) and to examine if this was different in women with different phenotypic expressions of the disease. methods: transvaginal d and d ultrasound was performed in women with pcos, as defined by the rotterdam criteria, and sub-group analysis conducted based on the subjects' body mass index (bmi), ovulation status, and hirsutism score. anova was used to compare the mean values between the groups. results: pcos women with clinical hyperandrogenaemia had significantly lower endometrial and subendometrial blood flow than their anovulatory normoandrogenic counterparts (table ). there were no differences between lean and obese women or between anovulatory and ovulatory women with pcos. the pulsed wave doppler parameters were similar in all three phenotypic groups. conclusions: hirsute women with pcos have impaired endometrial perfusion compared to their normoandrogenic counterparts which is only evident with d ultrasound and not conventional pulsed wave doppler. nusayba a bagegni, jill blaine, anuja dokras. obstetrics gynecology, university of iowa hospitals clinics, iowa city, ia, usa; obstetrics gynecology, university of pennsylvania, philadelphia, pa, usa. background: there is conflicting evidence on the association between pcos and early and late obstetric complications. it is unclear if the reported risks are independent of bmi, preexisting hypertension and diabetes. we examined the risk of early and late obstetrical complications in a large group of women with pcos compared to controls. methods: we reviewed pregnancy records of women with pcos (rotterdam criteria, n= ) and controls (tubal infertility, n= ) after in vitro fertilization at university of iowa from - . the wilcoxon rank sum test and fisher's exact test were used to evaluate differences between variables and logistic regression analysis was used to determine the independent risk of pcos. results: subject demographics and medical history are shown in table. the first trimester miscarriage rate was % in women with pcos and % in controls. after logistic regression analysis pcos was not associated with miscarriage (p= . ). the prevalence of gestational dm (gdm) was similar in both groups % pcos vs % controls. pcos was not associated with gdm after adjusting for age and bmi (p= . ). however, bmi was significantly associated with gdm after adjusting for age and pcos (p= . ). risk of both pre-eclampsia and pih was % in pcos and % in controls, but not statistically significant after adjusting for age, bmi and twin gestation. preexisting htn showed a significant association with preeclampsia (p< . ). there was no significant difference in preterm delivery, cesarean section, twin gestation, intrauterine fetal death and intrauterine growth restriction in the groups. conclusion: despite adequate power, our study did not detect an increased risk of miscarriage in women with pcos. obesity was a significant contributor to late obstetric complications, namely gdm. these findings may warrant aggressive counseling of women with pcos on the potential benefits of weight loss prior to pregnancy. objective: to assess the prevalence of polycystic appearing ovaries (pcao) as defined by the rotterdam criteria; and to determine whether metabolic parameters differ between regularly cycling women with pcao and those with normal ovaries. studies have demonstrated a population frequency of pcao of - %, with - % among women with regular cycles. most were performed in a young reproductive age population; none examined the impact of age. all were performed prior to adoption of the rotterdam criteria. recent studies have shown an increased prevalence of metabolic syndrome among women diagnosed with polycystic ovarian syndrome (pcos). however studies focused on women in their s found no increase in bmi or fasting insulin with pcao but regular menses. participants: women aged - with regular cycles (every - days) enrolled in the ova study, a population-based study of ovarian aging. each subject underwent a transvaginal ultrasound for assessment of ovarian volume and antral follicle count (afc). outcomes collected included waist measurements and hdl, ldl, total cholesterol, triglycerides, fasting glucose and insulin. pcao were determined by the rotterdam criteria (afc of on one ovary or ovarian volume > cc). student's t-test and chi-square tests were used to assess differences between those with and without pcao for continuous and categorical variables, respectively. the prevalence of pcao decreased with increasing age (table ) . of the women with pcao, % met the afc criterion only, while % met the volume criterion only, and % met both. women with and without pcao did not differ in waist measurements, or in fasting lipids, insulin, or glucose. the rotterdam criteria, while less subjective than those described by adams in , have led to an increased prevalence of pcao among women with regular menses, over / of women in their s. women with pcao do not differ from those with normal ovaries in metabolic parameters associated with pcos. consideration should be given to adopting an age-adjusted criterion for afc, or a combination of afc and ovarian volume, for diagnosing pcos. background: pcos, especially accompanied by obesity, has been reported to be associated with a characteristic dyslipidemia comprising elevated triglycerides (tgs) and depressed hdl, especially the hdl- fraction. this is an atherogenic profile; in fact, studies suggest low hdl- may correlate most strongly with cardiovascular disease risk. weight loss is a mainstay of treatment and improves all manifestations of the disease, but the optimal diet to recommend remains undetermined. preliminary studies show a high protein diet may improve total hdl levels and insulin responses. however, the effect of weight loss and dietary composition on hdl- levels in pcos has not been investigated. objective: to evaluate the fasting lipid profile in newly diagnosed obese pcos patients and to determine the effects of a high-protein diet with or without metformin on weight loss, hdl- and other lipoproteins, and menstrual cyclicity. methods: in this pilot retrospective observational study, the fasting lipid profile of obese women newly diagnosed with pcos was determined. they were then placed on a high protein ( - g/day), low carbohydrate ( - g/day) diet with or without metformin ( and %, respectively) and followed monthly for an average of months (range - ). results: at diagnosis, % had low hdl and % had low hdl- ; only % had elevated tgs. on the diet, the patients demonstrated an average weight loss of . lbs ( . to . , p< . ) and decreased bmi of . kg/m ( . to . , p< . ). hdl levels increased significantly ( % increase from . to . , p< . ), especially the hdl- fraction ( % increase from . to . , p< . ). triglycerides decreased as well ( . to . , p< . ). ldl decreased but did not reach statistical significance. resumed menstrual cycles, were started on oral contraceptives, and had a hysterectomy. pregnancies occurred. no difference was seen with metformin use. conclusion: a majority demonstrated decreased hdl and hdl- at diagnosis. the high protein diet resulted in significant weight loss and improvement in hdl- levels, as well as improvements in total hdl, tgs and menstrual cyclicity. metformin produced no added benefit. prospective trials based on these data will help determine the optimal diet to reduce the significant short-and long-term morbidity in the large population of women with pcos. resistin is an adipokine that has been associated with obesity and insulin resistance in animal models. studies on the role of resistin on insulin resistance in humans have been controversial. recently resistin has been shown to exert atherosclerotic effects and elevated resistin levels have been observed in women with coronary heart disease (chd). women with polycystic ovary syndrome (pcos) are at high risk for chd. our present study investigates potential association of resistin and markers of inflammation, c-reactive protein (crp) and insulin resistance in women with pcos. methods: thirty two women with pcos participated in the study. all were hirsute and had irregular cycles. nineteen women with normal ovulatory cycles who matched the pcos patients in bmi served as controls. fasting glucose, insulin, resistin and crp levels were measured in all women. after a high carbohydrate diet for days, a standard oral glucose tolerance test (ogtt) was performed. blood samples were collected for glucose and insulin before and , and hrs after g oral glucose. the area under the curve (auc) for insulin and glucose was calculated. women with overt diabetes were excluded from the study. results: fasting insulin levels ( . + . μu [±se] /ml) and insulin response to oral glucose (auc) ( . + . μu/ml) were higher (p < . ) in women with pcos compared to controls. all were insulin resistant with homa-ir value > . mol x μu / l . resistin levels in women with pcos ( . ± . ng/ml) was significantly (p < . ) higher compared to control women ( . ± . ng/ml). crp levels in women with pcos ( . ± ng/ml) was also significantly (p < . ) higher than the controls ( . ± ng/ml). there was a significant positive correlation between resistin and crp levels (r = . , p < . ). there was no correlation between resistin levels and fasting insulin levels or insulin auc. there was also no correlation between resistin levels and fasting glucose or glucose auc. conclusions: our results indicate that ( )women with pcos and insulin resistance have increased resistin levels, ( ) there is a strong association between resistin and crp ( ) elevated resistin and crp levels may predict women who are at increased risk for chd ( ) ) it is unlikely that resistin plays a major role on insulin resistance in women with pcos. vuk p jovanovic, enrico carmina, prati vardhana, michel ferin, rogerio a lobo. department of obstetrics and gynecology, columbia university, new york, ny, usa; department of internal medicine, university of palermo, palermo, italy. current diagnostic criteria for pcos includes both ovulatory (ov) and anovulatory or "classic" (c) phenotypes. in an effort to further characterize differences and /or similarities between these phenotypes, we studied hyperandrogenic women with pcos (age . ± . , bmi . ± . ) and age matched controls (age . ± . , bmi . ± . ). women with pcos were divided into weight matched (ov) n= and (c) n= groups. fat and weight distribution were assessed by dexa (total fat, r fat, trunk fat) as well as fasting levels of lh, e , mis/amh, kisspeptin and testosterone, the adipocytokines (leptin, adiponectin, visfatin and retinol-binding-protein- rbp ) and serum glucose, insulin and crp. the hyperandrogenic pcos groups had characteristically altered hormone profiles compared to matched controls. although total fat mass was comparable, women with c-pcos had a significantly larger waist circumference ( . . vs. cm, p< . ) trunk fat, r fat and %trunk and %r fat compared to ov-pcos (p< . ). leptin, rbp and visfatin did not significantly differ among the pcos subgroups although adiponectin was lower in the c-pcos group (p< . ). quicki was significantly lower in c-pcos ( . ± . vs. . ± . , p< . ) and insulin was higher ( . ± . vs. . ± . , p< . ). serum lh was also higher ( . ± . vs. . ± . , p< . ) but kisspeptin, testosterone and estradiol were similar. mis/amh ( . ± . vs. . ± . ng/ml, not significant) and crp ( . ± . vs. . ± . , p< . ) were higher in c-pcos. significant correlations (p< . ) were noted among kisspeptin/rbp (r . ), mis/testosterone (r . ), insulin/ %trunk fat (r . , p< , ), total fat/leptin (r . , p< . ), %trunk fat/adiponectin (r - . ). in conclusion, women with c-pcos when compared to similarly hyperandrogenic women with ov-pcos with similar bmi, have increased abdominal fat, and appear to have differences in serum lh, crp and increased insulin resistance. the latter, as well as subtle differences in the adipocytokines may explain these differences in anthropometric findings. problems of normal oogenesis and folliculogenesis but also disturbed oogenesis and folliculogenesis in polycystic ovaries are not fully understood. oocyte specific genes play an essential role in oogenesis and folliculogenesis. there are suggestions about possible role of some oocyte specific genes in etiopathophysiology of pcos. zona pellucida gene (zp ) is recently identified gene, which belongs to zona pellucida genes such as zp , zp and zp . the role of zp , contrary to above-mentioned other zp genes is not well described. the aim of this study was to analyze zp coding sequence and expression in patients with polycystic ovary syndrome. material included blood received from patients (mean age , +/- , years; mean bmi , +/- , kg/m ) with polycystic ovary syndrome. all patients with pcos were diagnosed with the use of eshre/asrm criteria from . dna was isolated from blood cells( after separation of blood cells from serum) using a dna isolation kit (qiagen ). genomic dna was used for in vitro amplification by pcr with a specific set of primers complementary to the coding sequence of the zp gene. products from each pcr reaction were examined by sscp method. samples with changes detected by sscp in comparison to control probes were cloned into plasmid vector and then automatically sequenced from a total of patient samples with pcos, we identified nucleotide changes in the zp coding seguence : silent nucleotide changes in exons , , , , and nucleotide change in the exon ( position , t>g). the mutation in exon ( t>g ) results in substitution of cystein for glycin of amino acid in position of zp protein. in summary, our data demonstrate that zp nucleotide changes account for % of patients with pcos. relationship between serum mullerian inhibiting substance levels and insulin in women with polycystic ovary syndrome. rebecca a chilvers, shilla chakrabarthy, summer james, xin ma, manubai nagamani. ob/gyn, university of texas medical branch, galveston, tx, usa. müllerian-inhibiting substance (mis) is a member of the transforming growth factor-superfamily of growth factors. it is expressed exclusively in granulosa cells and is believed to play a role in the regulation of follicle selection and maturation. women with pcos have anovulation and most of them have hyperinsulinemia. the purpose of our study is to investigate possible association between insulin and mis in the dysregulation of folliculogenesis in pcos. methods: twenty one women with pcos who had anovulatory cycles and hyperandrogenism were recruited for the study. sixteen women with ovulatory cycles and matched the pcos patients in age and bmi served as controls. an oral glucose tolerance test (ogtt) was performed in all women. blood samples were obtained at fasting and , and hours after glucose ingestion for measurement of glucose and insulin. to investigate the effect of hyperinsulinemia on mis secretion, mis levels were measured in ten patients during the ogtt. fasting mis, testosterone, dheas, fsh, and lh levels were measured in all patients. results: fasting insulin levels ( . + . μu/ml) vs . + μ u/ml [+ se]) (p < . ) and area under the curve (auc) of insulin ( . + . vs . + . μu/ml) (p < . ) were significantly increased in women with pcos compared to control women, while the glucose levels were normal indicating insulin resistance. mis levels were significantly (p < . ) increased in women with pcos ( . + . ng/ml) compared to controls ( . + . ng/ml). there was no correlation between age and mis levels in pcos patients while there was a highly significant negative correlation between the age and mis levels in the control women ( r = - . , p< . ). there was significant negative correlation between mis and fasting insulin levels (r = - . , p < . ) and insulin auc during the ogtt (r = - . , p < . ). there were no changes in mis levels during ogtt. conclusions: results of our study indicate that in women with pcos, ( ) there is an increase in secretion of mis, ( ) higher insulin levels are associated with lower mis levels, ( ) acute increase in insulin levels has no effect on mis levels, ( ) lower mis levels in women with severe hyperinsulinemia could be due to associated increased follicular atresia and a decrease in the ovarian reserve. further studies are needed to investigate the role of insulin on mis secretion. hiroyuki asakura, noriko tanaka, kyoko nishio. ohgimachi ladies' clinic, osaka, japan. objective: elevation of serum anti-müllerian hormone (amh) levels among polycystic ovary syndrome (pcos) patients has been reported. however, the regulatory factors of amh in pcos remain unknown. we examined correlations between amh values and various indices of insulin resistance in pcos women. methods: infertile women compatible with rotterdam criteria for pcos were recruited under informed consent. the subjects underwent g oral glucose tolerance test ( ggtt, sampling at , , minutes), and -step elisa for amh (sensitivity . pmol/l, inter intra-assay c.v. : . %, . %, respectively). p< . was considered as statistical significance. results: average amh level of the subjects (age: . + . years, bmi: . + . kg/m , mean+s.d.) was . + . pmol/l, which was higher than normo-ovulatory women ( . + . pmol/l, n= ). amh levels had significant positive correlation with total ovarian volume by ultrasound ( . + . cc), but not with bmi, waist-hip ratio ( . + . ), and levels of serum lh ( . + . iu/l), total testosterone ( . + . ng/dl). although fasting glucose levels ( . + . mg/dl) were positively correlated with total ovarian volume (r= . ), amh levels had no significant correlation with fasting insulin ( . + . iu/l), fasting glucose/insulin ration ( . + . ), homa-ir ( . + . ), and the sum of insulin levels ( . + . iu/l)during ggtt. conclusions: increased serum amh level of pcos and its positive correlation with total ovarian volume implies that determination of serum amh level would aid in confirming diagnosis of the ovulatory disorder. absence of relationship between amh and various clinical indices of insulin resistance suggests alternative regulatory factors of amh gene expression in the follicular compartment. reproductive tissues. amisra a nikrodhanond, keeley l mui, helen h kim. ob/gyn, university of chicago, chicago, il, usa. background: although primarily a neuroendocrine hormone, gonadotropinreleasing hormone (gnrh) is also produced in reproductive tissues, where it acts locally. the study of gnrh regulation in these tissues is limited by low levels of expression. objective: to elucidate mechanisms that regulate gnrh expression in male tissues, our objective was to identify regions of the gnrh gene that target expression in vivo. methods: transgenic mice were generated with different fragments of the mouse gnrh gene promoter (- /+ and - /+ bps), fused to the luciferase reporter gene. in these mice, luciferase activity (detected as light) reflects gnrh promoter activity. using bioluminescent imaging, gnrh promoter activity was assayed in live gnrh-luc mice and in their reproductive tissues ex vivo. to confirm imaging results, luciferase activity was also measured as relative light units (rlu) in tissue homogenates. for each dna construct, male mice were examined. with whole-body imaging, bioluminescence was detected in the genital region of the gnrh-luc transgenic mice (fig.a) . in mice that incorporated the - luc transgene, examination of reproductive tissues ex vivo revealed bioluminescence in the prostate and penis, but not in the seminal vesicles, epididymis, or testes (fig.b) . in contrast, in the - luc mice, bioluminescence was detected in the testes, but not in other tissues (fig.c) . examination of luciferase activity in tissue homogenates from - luc mice confirmed gnrh promoter activity in the prostate ( ± rlu) and penis ( ± rlu) and absence in the testes ( ± rlu). in the - luc mice, however, luciferase activity was detected only in the testes ( ± rlu) and not in the prostate ( ± rlu) or penis ( ± rlu). conclusions: our studies demonstrate that gnrh is present in male reproductive tissues, but may be differentially regulated in the testes vs. prostate/penis. promoter elements, contained within the proximal - bp of the mouse gnrh gene, are sufficient to mediate testicular gnrh expression while - bp of the gene promoter are necessary to direct expression to the prostate and penis. characterization of mouse ringo/speedy homologues. z walton, s uckac, o guzeloglu-kayisli, md lalioti, d sakkas, e seli. ob gyn, yale u., new haven, ct, usa. introduction: ringo/speedy (ringo/spy) is a recently discovered cyclindependent kinase (cdk) activator that functions similar to cyclins in controlling the cell cycle. ringo/spy plays a crucial role during meiotic maturation in xenopus by inducing meiotic g /m progression and germinal vesicle breakdown (gvbd). more recently, padmanabhan and richter demonstrated that ringo/spy mrna is repressed in the xenopus oocyte cytoplasm by pumilio . upon meiotic reactivation, pumilio loses its interactions with ringo/spy permitting its translation. ringo/spy is then expressed, leading to activation of cpeb by phosphorylation, which in turn elicits polyadenylation and translation activation of the mrna for a critical oocyte maturation factor, the mos kinase. in this study, we investigated the expression of ringo/spy in mouse. methods: ten different somatic tissues, testes, and ovaries were tested by reverse transcription-polymerase chain reaction (rt-pcr) for the expression of ringo/spy homologues and their alternative splicing variants. ringo/spy mrna expression was also tested in prophase i (pi) and metaphase ii (mii) oocytes, -cell, -cell, -cell, -cell embryos and blastocysts. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results: we analyzed the two previously identified homologues of ringo/spy (a and b). the two alternative splicing variants of ringo/spy a (a and a ) were separately studied for their expression profile. we also identified an additional alternative splicing variant of ringo/spy b and evaluated similarly. ringo/spy a ( and ) were expressed in testes, ovaries, and certain somatic tissues including brain, and spleen. ringo/spy b (both variants) were only expressed in testis. ringo/spy a or b were not present in mouse oocytes or early embryos. conclusions: our findings indicate that the previopusly described ringo/spy homologues a and b are not expressed in mouse oocytes or early embryos suggesting that either an alternative cdk-activator or a yet to be identified homologue of ringo/spy may be mediating meiotic g /m progression in mouse. testis specific expression of ringo/spy b, and previously described role of ringo/spy in meiosis suggests a role for this protein in male gametogenesis in mouse. treated with glyburide. jennifer aguayo, gladys a ramos, alethea hanley, carri r warshak, thomas r moore. reproductive medicine, university of california, san diego, san diego, ca, usa. objective: to determine the risk factors that may predict inadequate response to first-line glyburide monotherapy in pregnant women with type diabetes mellitus (dm) or gdm and to assess if non-responders are at increased risk of adverse pregnancy and neonatal outcomes. study design: this was a retrospective cohort of women diagnosed with type ii or gdm initially treated with glyburide at a single institution from - . maternal characteristics, adequate glycemic control defined as more than % of fasting glucose < mg/dl and one-hour post-prandials < mg/dl, and neonatal outcomes were assessed. non-responders were defined as failure to achieve adequate glycemic control on maximum daily doses of glyburide or intolerance due to side effects, necessitating switch to insulin therapy. statistical methods included bivariate analyses. results: of the women initially treated with glyburide, ( %) failed to achieve adequate glycemic control or did not tolerate glyburide. reasons for glyburide non-response were maximum dose ( mg/d) reached ( %), maternal hypoglycemia ( %) and other side effects ( %). there were no statistically significant differences between non-responders and responders with respect to family history of diabetes ( % vs. %, p= . ), prior history of gdm ( % vs. %, p= . ) and macrosomia (> g) ( % vs. %, p= . ) or hour glucose challenge test ( + vs. + mg/dl, p= . ). non-responders had a higher rate of obesity (bmi> ) ( % vs. %, p= . ) and earlier gestational age at initiation of therapy ( + . vs. + . wks, p= . ). there were no differences between the groups in the mean -week fasting ( + vs. + mg/dl, p= . ) and post-prandial glucose values ( + vs. + mg/dl, p= . ). no significant differences were observed in incidence of pre-eclampsia, primary cesarean delivery or birth weight. neonatal outcomes including ponderal index, neonatal hypoglycemia, nicu admission, and birth injuries also did not differ between the groups. conclusion: women who are obese and who require earlier initiation of glyburide therapy are at increased risk of non-response to glyburide monotherapy. however, despite non-response, these women can be managed with subsequent insulin therapy to achieve similar glycemic control and pregnancy and neonatal outcomes. a different diagnostic strategy using the gram, -hour glucose tolerance test for the diagnosis of gestational diabetes mellitus. yvonne w cheng, ingrid block-kurbisch, jennifer lydell, aaron b caughey. obstetrics, gynecology and reproductive sciences, university of california, san francisco, san francisco, ca, usa. objective: to examine whether a simplified strategy using the gm, -hour glucose tolerance test (gtt) may be useful for the diagnosis of gestational diabetes mellitus (gdm). methods: this is a retrospective cohort study of women with singleton pregnancy who received the -gm, -hour glucose challenging test (gct) for initial screening and the gm, -hour gtt as confirmatory tests for the diagnosis of gdm between and . various combinations of the gm, -hour gtt results were examined and compared to the diagnostic criteria for gdm established by the carpenter and coustan criteria using the receiver-operator characteristic (roc) curves. perinatal outcomes of women who would have had a false-positive or false-negative test results were compared to those who did not have gdm using the carpenter and coustan criteria. potential confounding factors were controlled for using multivariable regression models. results: , women had gm, -hour gtt results available for analysis during the study period. using gdm diagnosed by the carpenter and coustan criteria as reference, various diagnostic strategies was compared using roc curves (figure ) . summation of the -hour and -hour gtt results with a diagnostic threshold of mg/dl yielded the most optimal balance between sensitivity ( . %) and specificity ( . %). when compared to women without gdm, women who were diagnosed with gdm by c c criteria but not by summation of -hour and -hour gtts had higher odds of operative vaginal delivery (aor= . , % confidence interval [ci] . - . ) and neonatal birthweight > gm (aor= . , % ci . - . ). conclusion: using only the summation of only the -hour and -hour gtt results of the gm -hour gtt offers an alternative test strategy which may be more convenient and less costly for the diagnosis of gdm. however, women who would have gdm by the carpenter and coustan criteria but not by the summation method have higher odds of having an operative vaginal delivery and neonatal birthweight > gm. outcome of induction of labor indicated for term prom among women with or without a uterine scar. yifat ochshorn, avital skornick rapaport, adi reches, joseph b lessing, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. objective: we aimed at comparing the outcome of induced term deliveries presenting with prom with or without a previous uterine scar. methods: the computerized files of women delivered following induction of labor due to term prom, were reviewed. perinatal outcome parameters such as the mode of delivery, indication for cesarean section, rate of low ' apgar scores and nicu admissions were compared between women with and without a previous cesarean. results: during the study period women delivered in our institution following prom and induction of labor , of them had a previous uterine scar. parturients of both groups (with and without a scar) were similar with regard to age, gestational age at delivery and parity. cesarean section rate was higher for the previous scar group ( % vs %, p< . ). the most common indication for cesarean was arrest of dilatation and/or descent among women with previous scar accounting for % and % (p< . ) for women with and with no uterine scar, respectively. no differences were noted in neonatal outcome parameters such as rate of low ' apgar scores and nicu admission rate between the two groups. conclusion: induction of labor due to prom culminates in higher cesarean rate in women with one previous scar compared with parturients with no scar. perinatal outcome is similar between the two groups. acid base balance and immediate perinatal outcome of vertex compared with breech presentation in elective cesarean section. avital skornick rapaport, yifat ochshorn, joseph b lessing, yuval yaron, michael kupferminc, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. backround: apgar scores, umbilical blood ph and bicarbonate are generally lower and pco levels are higher in vaginally delivered breech neonates compared to cephalic deliveries. although cesarean delivery improved apgar scores there is a debate wether it improved the acid base status. this retroprospective study compared umbilical cord blood acid-base values and perinatal outcome of elective cesarean breech-delivery with those of elective cephalic cesarean delivery and to determine whether a different metabolic status and perinatal outcome should be expected in neonates in breech presentation. study design: the study group included singleton pregnancies delivered by elective cesarean section at term between january and march . computerized files of singleton breech presentation elective cesarean sections were compared to those of singleton vertex neonates delivered by elective cesarean section. demographic data included: maternal age, pregnancy week at delivery and parity. perinatal outcome measures checked were: birth weight, apgar scores at 'and ', umbilical cord venous and arterial ph and base excess. results: during the period between january and march there were singleton elective cesarean sections, of them were breech and vertex. the mean age, gravida and parity were significantly different between groups ( . vs. . , . vs. . . and . vs. . respectively, p< . ). the birth weight was significantly different - . gram for the vertex and . gram in the breech deliveries (p< . ) there were no differences in either apgar scores or umbilical ph between the breech and vertex neonates delivered by cesarean section at term ( - + w). venous and arterial po and pco levels were significantly different, though the differences were very small and we doubt if these differences have any clinical importance. conclusion: although vaginal breech deliveries are associated with increased risk of asphyxia during delivery, elective cesarean breech deliveries are not at increased risk for lower ph levels or apgar scores. the clinical significance of bleeding during the second trimester of pregnancy. arie koifman, amalia levi, yaron zaulan, avi harlev, eyal sheiner. obstetrics gynecology, soroka university medical center, ben gurion university of the negev, beer-sheva, israel; epidemiology and health services evaluation, faculty of health sciences, ben gurion university of the negev, beer-sheva, israel. objective: this study aimed at investigating clinical importance and pregnancy outcome in women suffering from bleeding during the second half of their pregnancies. methods: a population based study including all deliveries which took place in the soroka university medical center between the years - were examined. comparison was performed between patients with and without second trimester bleeding pregnancies terminated before weeks, multiple gestations and women lacking prenatal care were excluded . stratified analysis, using the mantel-haenszel technique, and a multiple logistic regression model were performed . results: during the study period, , singleton deliveries occurred in our institute. of these, ( . %) were complicated with bleeding upon admission during the second half of pregnancy. the cases were attributed to placental abruption ( . %; n= ) and placenta previa ( . %; n= ). independent risk factors associated with bleeding, were oligohydramnios, polyhydramnions, (odds ratio [or]= . ; % confidence interval [ci] . - . ; p= and . ; . - . ;p< . respectively ), suspected intra uterine growth restriction (iugr, . ; . - . ; p<. ), gestational age, previos abortions and maternal age. these patients subsequently were more likely to deliver by cesarean section (cs, . % vs. . %, or= . ; %ci . - . ; p< . ). perinatal mortality among patients admitted due to second half bleeding was significantly higher as compared to patients without bleeding (p<. ). conclusion: bleeding upon admission during the second half of pregnancy is an independent risk factor for perinatal mortality. careful surveillance, including fetal monitoring, is suggested in these cases in order to reduce the adverse perinatal outcome. crude and adjusted odds ratios for perinatal mortality among patients with vaginal bleeding. characteristics or % ci p crude or for perinatal mortality . . - . < . or adjusted for: < . iugr . . - . < . oligohydramnios . . - . < . premature rupture of membranes . . - . < . the predictors included neonatal pi, bmi, or birthweight while the outcomes included hypoglycemia, shoulder dystocia, acidemia and hyperbilirubenemia. roc curve analyses were utilized to evaluate the relationship between sensitivity and specificity for each of the predictors and outcomes, with an area under the curve (auc) significantly greater than . indicating a screening test that is better than chance. results: neonatal pi, bmi and birthweight are poor predictors of nicu admission, acidemia and hypoglycemia with all auc values not statistically significantly different than chance alone. they are a reasonable predictor of shoulder dystocia, with birthweight functioning the best (p< . ). in general, neonatal anthropometric measurements do not appear to be good predictors of short term neonatal outcomes. although they are a reasonable predictor of shoulder dystocia, they are not useful clinically for this outcome since they cannot be calculated until after birth. in future studies, new models of neonatal anthropometrics should be created to better predict adverse neonatal outcomes. neonatal anthropometric measurements and their relationship to perinatal outcomes expressed as auc and % confidence intervals history of at least one spontaneous preterm delivery were offered protocolbased prenatal care including first-or second trimester bacterial vaginosis screening, repeated ultrasound cervical length assessment, and supportive care by specialized nurses. women with a positive test for bacterial vaginosis were treated with oral metronidazol therapy, and women with a cervical length below . cm (before weeks of gestation) underwent a vaginal cerclage. progesterone administration was not provided. data on obstetrical and medical history, pregnancy, delivery and neonatal outcome were prospectively collected and evaluated. results: in total, pregnant women were prospectively followedup. eighty-seven women had experienced a preterm delivery in their last pregnancy, of whom had delivered before weeks. three women were diagnosed previously with a bicornual or septal uterus. twelve women received metronidazol therapy and women underwent a vaginal cerclage. eighty-four women ( . %) delivered at weeks or beyond. one-hundred and three women ( . %) delivered no earlier than weeks, and only one woman delivered at weeks. two women gave birth to a twin (at and weeks, respectively). two neonatal deaths due to pulmonary hypoplasia were recorded. conclusion: women at high risk of preterm delivery who were offered protocol-based prenatal care not including progesterone therapy had a better pregnancy outcome than would be expected from previous studies. our findings may question the reported beneficial effects of prenatal progesterone administration. premature rupture of membranes. tracy a manuck, alexandra g eller, m sean esplin, robert m silver. obstetrics gynecology, university of utah health sciences, salt lake city, ut, usa. objective: historically, outcomes following expectant management of midtrimester preterm premature rupture of membranes (pprom) have been uniformly poor. thus, many patients elected pregnancy termination. however, outcomes may be improved with recent advances in neonatal medicine. our purpose was to assess outcomes in expectantly managed early pprom in an era of improved maternal and neonatal care. study design: this is a retrospective cohort of patients from tertiary healthcare systems from - experiencing pprom . weeks gestation. patients electing immediate termination of pregnancy, carrying fetus(es) with lethal anomalies, or delivering within hours of pprom were excluded. survival without major morbidity was the primary outcome. data were analyzed using student t-test and chi-square as appropriate. results: a total of women carrying fetuses ( singleton, twin, triplet, quadruplet) met inclusion criteria. only the fetus in the "ruptured sac" was studied. pprom occurred at a mean of . (+/- . , range . - ) weeks. the average latency period was . (+/- . , range . - ) days, with a mean delivery gestational age of . (+/- . , range . - ) weeks. this communication provides the first report of fundal uterine necrosis following placement of multiple uterine compression sutures. only two previous published cases report occurrence of uterine necrosis following application of a uterine compression suture --both identified as the b-lynch technique --and neither of these cases report necrosis confined to the fundus. in our case, uterine atony was refractory to pharmacologic therapy and manual compression of the uterus appeared to decrease the amount of blood loss. therefore, we applied a traditional b-lynch which effectively contracted the majority of the uterus, while the fundus remained atonic. a second horizontal, square suture was then placed between the cornua and carried over the fundus (see figure one: red reflects b-lynch suture; blue represents second fundal, square stitch). the patient subsequently experienced significant, persistent fevers despite antibiotic therapy. a repeat laparotomy was performed, at which time we found uterine necrosis confined to the uterine fundus (see figure : photograph taken at the time of hysterectomy). the placenta showed no histologic evidence of chorioamniotis --hence, the patient's main risk factor for fundal necrosis was the compression sutures. physicians should be aware of the risk of uterine necrosis, especially after placement of multiple compression sutures and, more specifically, after placement of a compression suture confined to the uterine fundus. background: migraines are far more common in women than in men. the incidence of migraines increases in girls after puberty, reaching an incidence of % in women who experience migraine at least once a year around middle age. migraine has been postulated as one of the major risk factors for stroke during pregnancy and the puerperium.triptans are a class of serotonin receptor agonists used in the treatment of migraine headaches. triptans administered in combination with other drugs have been known to precipitate serotonin syndrome, a rare but potentially life-threatening condition clinically manifested by a triad consisting of mental-status changes, autonomic hyperactivity, and neuromuscular abnormalities. objective: to determine whether triptan monotherapy is associated with serotonin syndrome methods: using data mining techniques we analyzed the entire food and drug administration's (fda) adverse event reporting system (aers) database. results: after excluding reports of concomitant serotonergic medication or other potentially confounding medication, eleven reports remained of serotonin syndrome associated with triptan use without concomitant serotonergic medication. the mean age for these eleven cases was . years; nine cases occurred in females and two occurred in males. there were no apparent instances of overdose among these eleven cases. conclusions: serotonin syndrome is a rare but serious occurrence with the use of triptan monotherapy. such cases were seen with eletriptan, rizatriptan, sumatriptan, and zolmitriptan. because of the spontaneous nature of voluntary reporting to aers, the actual number of occurrences of serotonin syndrome in patients using triptans is probably higher and cannot be assessed from aers data. users of triptan in combination with an ssri or snri should be warned of this rare but serious adverse effect. during periods of drug initiation, dose escalation, or the addition of another serotonergic agent, patients should be particularly vigilant for symptoms of concern and seek urgent medical attention if any occur. the offending agents should be withdrawn and the patients closely monitored and treated with supportive measures as required. rising maternal mortality in the midst of modern technology. oormila p kovilam, jane khoury, padmini c sekar, ralph c buncher. obstetrics gynecology, university of cincinnati, cincinnati, oh, usa; center for epidemiology and biostatistics, cincinnati children's hospital medical center, cincinnati, oh, usa; environmental health, university of cincinnatic, cincinnati, oh, usa. introduction: maternal mortality rate (mmr) is an index of overall wellbeing of the community and safe motherhood should be given utmost priority in obstetric care. as per , who estimates % of maternal mortality occurs in developed countries . this rate has established without much decline in recent years. the mmr for ohio for to was reported by cdc to be . per , live births with % confidence interval . to . . objective: our objective was to audit the trend in maternal mortality in the state of ohio for the last years. methods: information from the death certificates completed by attending physicians, medical examiners, coroners, funeral directors filed with ohio state registration offices were analyzed. we only used women who were residents of the state of ohio at the time of death, and cause of death was coded as a "complication of pregnancy, childbirth and the puerperium", icd codes to and icd codes o to o . five year maternal mortality pattern and long term trend was assessed. results: the number of maternal deaths recorded as due to pregnancy complications varied over the years from a high of in to a low of in and . in general the five-year mortality rate was decreasing over time until the last four years to , when we may be seeing an upward trend compared to to (p= . ). the rates and associated % confidence intervals (ci) are shown in the table below. years mmr % ci ci - ci . . - . ci - . . - . - . . - . - . . - . - . . - . the rate of maternal mortality is on the rise after a period of downward trend. we should develop a multidisciplinary approach to analyze and target the root causes of maternal death. introduction. women with thrombophilia are at higher risk of vte during pregnancy due to the acquired hypercoagulable state. it is likely that not only maternal veins but also placental vessels are more prone to the development of thrombosis. our objective was to compare maternal and fetal placental circulation disorders in women with intra-uterine fetal death (iufd) and thrombophilia and women without thrombophilia. methods. in a dutch multi-centre study on iufd, during the period - we studied singleton deaths > weeks of gestation for which the diagnosis of iufd was determined before labour. factor v leiden and prothrombin g a were tested at induction of labour. panel classification of cause according to the tulip classification was performed by assessors after individual investigation of structured patient information. we studied the cause of death group "maternal and fetal placental circulation disorders": placenta bed pathology with abruption or infarction as origin of mechanism and placental parenchyma pathology with fetal thrombotic vasculopathy and massive perivillous fibrin deposition as origin of mechanism. results. of the women tested for factor v leiden, ( . %) were carriers. of the deaths caused by "maternal and fetal placental circulation disorders" ( . %) mothers were carriers of factor v leiden. of the deaths with another cause of death ( . %) mothers were carriers of factor v leiden (p= . ). of the women tested for prothrombin g a, ( . %) were carriers. of the deaths caused by "maternal and fetal placental circulation disorders" ( . %) mothers were carriers of prothrombin g a mutation. of the deaths with another cause of death ( . %) mothers were carrier of prothrombin g a mutation (p= . ). in women with iufd and factor v leiden or prothrombin g a mutation "maternal and fetal placental circulation disorders" did not seem to cause iufd more often than in non-carriers. , university medical center groningen, groningen, netherlands; pathology, university medical center groningen, groningen, netherlands; trial coordinating center, dept of epidemiology, university medical center groningen, groningen, netherlands; hematology, university medical center groningen, groningen, netherlands. introduction. growing evidence suggests that women with thrombophilic defects may be at higher risk of fetal loss. although some paternal components to the predisposition of preeclampsia have been demonstrated it is not known whether paternal components contribute to intra-uterine fetal death (iufd). our objective was to investigate the relation between paternal thrombophilic defects and iufd. methods. in a dutch multi-centre study on iufd, from - we studied singleton deaths > weeks of gestation for which the diagnosis of iufd was determined before labour. we tested male partners of women with iufd for antithrombin (at), protein c, protein s type i and iii, factor v leiden, prothrombin g a (factor ii) and factor viii: ag. standard tests were performed in one laboratory. normal ranges were determined in healthy male blood donors. we compared prevalence of thrombophilic defects to reference values from the literature. . of the men tested for factor v leiden, ( . %) were carrier versus ( . %) non-carriers. prevalence of factor v leiden in the normal population is % (p= . ). men were tested for prothrombin g a, ( . %) were carriers and ( . %) non-carriers. all were heterozygous. prevalence of prothrombin g a mutation in the normal population is % (p= . ). decreased levels of antithrombin, protein c, protein s type i and iii and increased levels of factor viii: ag were observed significantly more often in male partners of women with iufd compared to the normal population. conclusion. in our iufd group the prevalence of male factor v leiden carriers was comparable to the normal population, prevalence of prothrombin g a mutation was lower. the difference in other factors imply an as yet unexplained association of male thrombophilia with fetal death in their partner. objective fetal macrosomia, the most important complication of gdm, increases the risk of shoulder dystocia, erb's palsy and intrauterine hypoxia. we compared frequencies of fetal macrosomia and erb's palsy in two gdm cohorts with different severity of glycemic disturbance and in healthy controls. methods we studied consecutive gdm women with singleton childbirth and or abnormal values in -h ogtt with g of glucose. abnormal plasma glucose values of the ogtt were: fasting . , -h . and -h . mmol/l. insulin treatment was started when values were . preprandially or . mmol/l postprandially in the -h glucose profile done within days of diagnosis. if the same woman had more than childbirth during the study period, only the last pregnancy was included. the control group consisted of women from a nearby town with singleton childbirth in the same hospital. women with diabetes were excluded from controls. macrosomia was defined as birth weight (bw) > . sd above the mean of a standard population. erb's palsy was diagnosed by pediatric surgeons. results gdm women were older, more obese, had more childbirths and more often a previous child with bw > g than controls. insulin was started in gdm women ( . %). c/s rate was . % in controls, . % in diettreated and . % in insulin-treated gdm women. macrosomia rate was . % in controls, . % in diet-treated and . % in insulin-treated gdm women (p< . compared with controls or diet-treated gdm women). the frequency of macrosomia did not differ between the diet-treated gdm and control women. erb's palsy occurred in . % of controls, in . % of diet-treated and in . % of insulin-treated gdm women. the frequency of erb's palsy was significantly higher in both gdm groups than in controls (p= . ). by regression analysis, previous child with bw > g (p< . ), previous c/s (p= . ), mother's age (p= . ), bmi (p= . ), insulin treatment (p= . ) and fasting plasma glucose of the -h profile (p= . ) were significant independent predictors of fetal macrosomia. conclusions the -h glucose profile done shortly after the diagnosis of gdm clearly distinguishes between low-risk (diet-treated) and high-risk (insulintreated) gdm pregnancies for fetal macrosomia. unfavourable fetal body composition of diet-treated gdm women is the likely explanation for the high rate of erb's palsy in this group. . '-:·:tpartq,-, '-:·:tpartq,-, '-:·:tpartq,-, % p= . ). first trimester uta doppler indices were similar in the two cohorts in terms of the resistance and pulsatility indices (ri . vs. . , p= . ; pi . vs. . , p= . ) . however, bilateral notching was much more common in the cohort of prior adverse outcomes ( % vs. % p= . ) as well as in patients destined to have a subsequent poor outcome (p= . ). conclusions: not surprisingly, prior poor obstetric outcome was strongly associated with recurrent adverse obstetric outcome. uta notching was robustly associated with prior poor obstetric history as well as a recurrent poor outcome. this clinical history had no discernable influence on ri or pi. reassurance may not be offered based on first trimester uta ri and pi. anti-retroviral therapy is associated with increased blood loss and uterine atony for patients undergoing primary cesarean section. carey eppes, alice cootauco, melissa russo, jessica bienstock. gynecology and obstetrics, johns hopkins university school of medicine, baltimore, md, usa. objective: anti-retroviral therapy has been associated with gastrointestinal smooth muscle dysfunction. it has also been hypothesized that myopathies can occur secondary to anti-retroviral therapy due to mitchondrial alterations. in our experience, we have noticed an increased incidence of uterine atony in our hiv patients on anti-retroviral therapy. we sought to examine the incidence of postpartum hemorrhage and uterine atony in patients currently on anti-retroviral therapy. study design: a retrospective case-controlled study was conducted on all hiv positive pregnant women on anti-retroviral therapy undergoing a primary low segment transverse cesarean section from through . these patients were obtained from an irb approved database containing hiv positive patients within our institution. patients' medical records were abstracted for demographic data, use of uterotonics, preoperative and postoperative hematocrits, and incidence of blood transfusions. controls were matched for age, parity, gestational age and surgical indication. data was analyzed using the t-test, fischer's exact test and chi square. results: there were no differences in demographics, incidence of chorioamnionitis or magnesium sulfate use between groups. patients on anti-retroviral therapy had a statistically greater decrease in hematocrit and estimated blood loss compared to controls. they also had an increased need for uterotonics and blood transfusions. conclusion: anti-retroviral therapy may impact uterine smooth muscle contractility in pregnancy, as evident by the increased incidence of uterine atony and change in hematocrit. additional research is needed to elucidate the mechanism. clinicians should be aware of the potential for uterine atony and excessive blood loss in patients on anti-retroviral therapy. background: adolescent pregnancy is frequently associated with adverse outcomes, especially small-for-gestational age (sga) deliveries. some studies have implicated maternal nutritional status in these poor outcomes. methods: in a prospective longitudinal study, ethnically-diverse, pregnant adolescents were studied from booking to parturition, with collection of anthropometric and nutritional variables. blood samples ( - weeks' gestation; n= subjects) were assayed for a spectrum of nutritional biomarkers. logistic regression was used to determine significant associations between studied variables and pregnancy outcomes. results: median age at recruitment was . years (iqr: . - . ). outcome data was available for subjects. ( . %) had uncomplicated pregnancies. median birthweight was , g (iqr: , - , g) and median birthweight centile was . % (iqr: . - . %). ( . %) infants were born sga and ( . %) were preterm. spontaneous vaginal deliveries occurred in . % of cases. there were ( . %) cases of pre-eclampsia and ( . %) admissions to neonatal care. . % of subjects reported smoking at booking. iron deficiency anaemia was prevalent in . % of subjects by - weeks and was strongly associated with higher infant birthweight centile (p< . ). . % had serum -hydroxy vitamin d concentrations < ng/ml, although this was not associated with any outcomes. low folate status, as indicated by low red cell folate (p= . ), low serum folate (p= . ) and high serum homocysteine (p= . ) concentrations, was associated with higher rates of sga birth. conclusion: adolescent pregnancy in inner-city populations is associated with a high risk of sga and preterm birth, increasing the likelihood of health problems in later life and perpetuating social disparities in health. the association between anaemia and higher birthweight is unexplained. impaired maternal folate status may contribute to impaired fetal growth. we suggest that sga birth in this population could be reduced by the use of antenatal supplements or the mandatory fortification of flour with folic acid. amount of cigarette use pre-pregnancy and during pregnancy were self-reported and subgrouped into nonsmokers, quitting during pregnancy, and continued smoking throughout pregnancy. categorical outcomes were compared using chi-square test. multivariable logistic regression analyses were used to control for potential confounders (continued smoking compared to quitting during pregnancy). results: women who quit smoking during pregnancy had higher rates of pregnancy-associated hypertension and cesarean delivery but lower rates of preterm delivery and neonatal birthweight < gm compared to non-smokers. compared to women who quit smoking during pregnancy, those who continue to smoke have lower odds of pregnancy-associated hypertension and cesarean delivery, but higher odds of preterm delivery, neonatal birthweight < gm, and apgar score < at minutes (see table) conclusion: quitting cigarette smoking during pregnancy appears to reduce undesirable neonatal outcomes, though an increase in pregnancy associated hypertension. these findings should be emphasized to women who are smoking during pregnancy. to achieve effective tamponade the balloon was inflated initially up to - ml with incremental increases of volume by ml until bleeding stops. to measure intrauterine balloon pressures we used a standard pressure transducer (edwards lifesciences) connected to the balloon port and to a pressure monitor (ge dash ). the pressure transducer was mounted on the bedrail at uterine level, primed with sterile saline and then connected to the balloon port of the catheter. the system was zeroed to atmospheric pressure, the stopcock of the intrauterine balloon port was opened and the pressure was recorded (p : the total pressure on the fluid). to verify the accuracy of measurements we used another balloon catheter (reference), inflated with the same amount of saline, connected to the pressure transducer and zeroed to atmospheric pressure. the reference balloon measured p (the intrinsic balloon pressure caused by distension at that volume). the system was then zeroed to the reference balloon. the pressure transducer was then reconnected with the intrauterine balloon and the actual intrauterine pressure was recorded (p ). we calculated the correlation coefficient between p and systolic, diastolic and mean blood pressure using a linear regression (statsdirect statistical software there is a growing body of evidence to suggest that peripartum assessment of fetal or neonatal lactate levels are as good as or better than standard blood gas analysis in the prediction of neonatal outcome. in this study we have evaluated the ability of umbilical cord blood gases and lactate levels in the prediction of neonatal hypoxic-ischaemic encephalopathy (hie). department of neonatal paediatrics, king edward memorial hospital, perth, western australia, australia; women and infants research foundation, king edward memorial hospital, perth, western australia, australia. objective: current evidence suggests that umbilical cord ph at delivery provides the most sensitive reflection of birth asphyxia. paired umbilical artery/vein blood gases have been routinely collected at king edward memorial hospital over the last years. the objective of this study was to determine: local reference ranges; accuracy of sampling; and the rates of metabolic acidosis. of the , births ( ) ( ) ( ) ( ) , accurate paired results were available on % of births. over the study period there was a progressive improvements in accuracy rates of paired sampling (p< . ). the median ( . th , . th centile) values for cord arterial blood gases were: ph . ( . , . ); po . mmhg ( . , . ); pco . mmhg ( . , . ); base excess - . (- . , . ); and lactate . mmol/l ( . , . ). there was a progressive improvement in all blood gas measures over the years of this study (all p< . ). moreover, there were significant reductions in all measures of metabolic acidosis (see table) . the progressive improvement in the measures of metabolic acidosis remained significant after multivariate analysis including obstetric, fetal, and demographic factors associated with metabolic acidosis. the introduction of universal umbilical cord blood gas analysis to all births is associated with significant improvements in all markers of metabolic acidosis. together with other compelling evidence in the literature, these data support the routine use of cord arterial and venous gases at all births; however, improved accuracy rates on paired sampling requires an ongoing education program umbilical artery indicators of acidosis, percentage of total ph < . ph < th centile* objective: intrapartum pcn prophylaxis aims to prevent early-onset gbs sepsis by interrupting vertical transmission from colonized mothers to their newborns. however, despite its wide clinical use, systematic pharmacokinetic evidence in support of the current pcn dosage regimen is lacking. current cdc guidelines recommend intensified surveillance and testing of infants exposed to < h of prophylaxis. our goal was to examine the relationship between maternal time of exposure to pcn and fetal serum pcn levels among maternal-fetal dyads exposed to short durations of pcn prophylaxis (< h) compared to those exposed to longer durations. study design: ninety-eight laboring gbs positive women were administered million units (mu) of intravenous pcn to be followed by . mu every hours until delivery (cdc ) . subjects with renal disease, multiple gestation, and preterm delivery (< wks) were excluded. umbilical cord blood samples were collected at delivery and pcn levels measured by high-performance liquid chromatography. intra and inter-assay coefficients of variation were < %. results: the pcn concentrations (mean±sd) by duration of prophylaxis were: < h, . ± . g/ml (n= ); - h, . ± . g/ml (n= ); - h, . ± . g/ ml (n= ); - h, . ± . g/ml (n= ); - h, . ± . g/ml (n= );> h . ± . g/ml (n= ); and for those without a second dose after h, . ± . g/ml (n= ). fetuses exposed to short duration (< h) had higher levels of pcn than those exposed to > h (p= . ). in multivariable linear regression analysis, fetal pcn levels were determined by total duration of exposure, time since last dose, dosage, and number of doses, but not maternal bmi. pcn levels in cord serum increased linearly until hour; thereafter, they decreased rapidly, but all groups were significantly above the minimum inhibitory concentration (mic) for gbs ( . g/ml)(p< . ). furthermore, every sample individually remained - fold above the mic. conclusion: in this study, even short durations of prophylaxis achieved levels above the mic, suggesting a benefit to prophylaxis even in precipitous labors. the data also suggests that the current cdc designation of infants exposed to < h of pcn prophylaxis as particularly at risk for gbs sepsis may be inaccurate from a pharmacokinetic standpoint. objective: -oh aa is a metabolite of tryptophan with pro-oxidant and proapoptotic properties and has been shown to increase in umbilical cord blood in pregnancies with intra-uterine infection. since labour-related events may also activate inflammatory pathways, we sought to determine the placental release of -oh aa into the umbilical circulation in labouring vs non-labouring patients at term. methods: twenty-six patients were studied (term labour n= , and term elective cesarean section n= ) with blood sampling from a clamped segment of umbilical cord after delivery of the fetus and from the cord at its insertion into the placenta after delivery of the placenta, with subsequent measurement of blood gases/ph and -oh aa (isocratic hplc using fluorometric detection with assay sensitivity at pmol). results: -oh aa measurements from respective umbilical and placental cord vessels were all variably higher in the labouring group vs the elective cesarean group patients (table ) . for labouring group patients, the -oh aa levels from the umbilical vein were significantly higher than those from the umbilical artery, indicating net release from the placenta into the fetal circulation. placental vein levels were also significantly higher than those from the umbilical vein, indicating continued placental release of -oh aa into the cord blood after delivery of the fetus. conclusion: labour at term is associated with changes in the placental metabolism of tryptophan resulting in the increased release of -oh aa into the fetal circulation with the potential for pro-oxidative and apoptotic effects in many tissues, including the brain. obstetrics, gynecology, and reproductive sciences, new brunswick, nj, usa; department of obstetrics and gynecology, mineola, ny, usa. objective: ischemic placental disease (preeclampsia, small for gestational age, sga and placental abruption) is a major contributor to pregnancy-related morbidity. although the placenta is considered a fetal organ, it is accepted that ischemic placental disease (ipd) can present clinically with either fetal or maternal manifestations. we hypothesized that the pattern of diagnosis (maternal versus fetal) varies by gestational age and would provide insights in to origins of indicated and spontaneous preterm birth. methods: this was a retrospective cohort study utilizing the maternallylinked reproductive history data for missouri residents , restricted to singleton live births. women who experienced spontaneous onset of labor and subsequently delivered preterm were classified as spontaneous preterm birth. medically indicated preterm birth included women who delivered preterm through a labor induction or (prelabor) cesarean delivery. ipd was classified as maternal (preeclampsia only), fetal (sga only) or both (preeclampsia with sga or abruption, and all conditions). results: among term births with ipd, . % presented as maternal disease only, . % as fetal disease, and the remainder ( . %) as both. among spontaneous preterm births with ipd, a greater proportion were of fetal presentation ( bethesda, md, usa; dept ob/gyn, wayne state univ, detroit, mi, usa; dept pathology, wayne state univ, detroit, mi, usa; dept ob/gyn, soroka univ medical center, israel. objective: hemoglobin (hg) and its catabolic products have been observed in cases of amniotic fluid (af) discoloration, which is a risk factor for intraamniotic infection/inflammation (iai). the study aimed to determine the association between af fetal hg concentration and gestational age, term and preterm labor and iai. study design: this cross-sectional study included: ) mid-trimester (n= ); ) term not in labor (tnl) (n= ); ) term in labor (tin) (n= ); ) preterm labor (ptl) who delivered at term (n= ); ) ptl without iai (n= ); ) ptl with iai (n= ); ) preterm prelabor rupture of membranes (pprom) with (n= ) and without iai (n= ). af fetal hg concentrations were determined by elisa. results: ) fetal hg was detected in . % of all af and . % of mid-trimester samples; ) women at tnl had a higher median af fetal hg concentration than patients at mid-trimester ( . ng/ml, iqr - vs . ng/ml, iqr . - . , p= . ); ) no differences were found in median af fetal hg concentration among patients with and without labor at term (til: . ng/ml, iqr - . ; p= . ); ) median af fetal hg concentration was not significantly different among the ptl subgroups [ptl with iai: . ng/ml, ptl who delivered preterm: . ng/ml, ptl without iai who delivered at term: . ng/ml, p= . (kruskal wallis) ]; ) in pprom, there were no differences among patients with and without iai ( . ng/ml, respectively; p= . ) ; ) median af fetal hg concentrations were significantly higher in ptl or pprom, with or without iai than in pregnant women at term, with and without labor (p< . for all comparisons). conclusions: ) immunoreactive af fetal hg increases with gestational age; ) among women with ptl or pprom, the median af fetal hg concentration is not associated with iai; ) the median af fetal hg concentration is higher in pregnancies complicated with ptl or pprom than in term pregnancies. the impact of latency time to delivery after preterm premature rupture of membranes (pprom) on neonatal outcome. dan nayot, deborah penava, barbra de vrijer, orlando da silva, bryan s richardson. obstetrics and gynaecology; pediatrics, university of western ontario, london, on, canada. objective: there continues to be controversy as to the management of pprom with conservative management to advance gestational age (ga) versus aggressive management with early induction to avoid chorioamnionitis. we have therefore used the perinatal and neonatal databases of a large regional patient population to determine the association of pregnancy variables with latency time to delivery after pprom and the impact of latency duration on adverse neonatal outcomes. methods: the perinatal/neonatal database of st. joseph's health care, london, ontario was used to obtain demographic and neonatal outcome information for all patients with pprom > and < weeks gestation, singleton and no major anomalies, delivering between january , and december , . patients were grouped according to ga at pprom stratified for latency time < hrs vs > hrs with incidences for those pregnancy related variables and neonatal outcomes available from the database then compared with the use of logistic regression analysis. results: there were patients who met the inclusion criteria of whom %, %, and % had pprom at - wks , at - wks, or at - wks, respectively, and with these pprom groupings showing a stepwise decrease in the percentage of patients with latency to delivery > hrs, at %, %, and %, respectively. pregnancy related variables and neonatal outcomes for these patient groupings are as shown in table . conclusion: despite a to fold increase in the incidence of chorioamnionitis with latency to delivery > hrs, a policy of conservative management to advance ga after pprom will result in decreased severe infant morbidity until weeks, and moderate infant morbidity until weeks. hospital. outcomes studied were prolongation of latency period with intact membranes and prom using magnesium sulfate (mgso ), nifedipine, terbutaline and tocolytic agents. secondary outcomes examined were maternal complications. statistical analysis performed were t-test and . comparisons were expressed as odds ratios ( % ci). results: in a -year period, twin gestations were admitted in ptl and administered tocolytic agent(s) with mean gestational age of . weeks. when tocolytic agents were used, maternal complications were: ( . %) with intact membranes and ( . %) with prom had pulmonary edema (or= . , ci . - . ); ( . %) with intact membranes and ( . %) with prom had chorioamnionitis (or= . , ci . - . ); ( . %) with intact membranes and ( . %) with prom had postpartum hemorrhage (or= . , ci . - . ). conclusion: there is no difference in prolongation of latency period achieved in twin gestations in ptl with intact membranes or prom using tocolytic agents. similarly, there is no difference between the groups with latency period hrs. there is an increased likelihood of pulmonary edema and chorioamnionitis with use of tocolytic agents. however, results are not significant due to type ii error. mean latency period and latency ≥ hrs using single or multiple tocolytic agent ( introduction: high sensitivity crp (hscrp) is a serum marker of inflammation and has proven clinical utlility in predicting cardiovascular disease (cvd). considering the hypothesized association between preeclampsia (pre) and inflammation and cvd, it is plausible that hscrp may have utility in predicting pre. prior to widespread utilization of this marker, the affect of labor on levels of crp needs to be clarified. we assessed the association between labor and hscrp levels in term deliveries and between elevated hscrp and adverse perinatal outcomes. a secondary analysis comparing hscrp in women with preeclampsia (pre) to those without was performed. methods: women presenting for term delivery or pre were prospectively identified as part of a case-control study. clinical data and serum were collected for all subjects. a standard immunoturbidimetric assay was used to measure hscrp levels. women presenting for induction of labor or planned cesarean delivery (non-labor) were compared to women presenting in labor. a secondary analysis comparing non labor women with and without pre was performed. serum was collected prior to labor induction in the non-labor group. nonparametric comparisons were made using wilcoxon rank sum tests. mvlr was used to evaluate dichotomous outcomes and control for confounders. results: women were included (non-labor group (n= ), labor group (n= ), non-labor pre (n= )). the median and mean hscrp levels were and and and in the non-labor and labor groups respectively (p= . ). elevated levels of hscrp in these term deliveries were not associated with chorioamnionitis (p= . ), maternal postpartum complications (endometritis, hemorrhage, transfusion) (p= . ), mode of delivery (p= . ), or admission to the nicu ( . ). levels of hscrp were significantly greater in the non-labor pre group compared to non labor without pre (mean . vs. . , median vs. , p< . ). conclusion: use of hscrp as a biomarker may improve clinical prediction of obstetrical complications such as pre. levels of hscrp are affected by labor and this should be taken into account when studying the utility of this biomarker. further, crp levels are elevated in women with pre even after excluding patients in labor. further investigations to determine if crp elevation in term labor is associated with adverse outcomes may be warranted. the role of hscrp as a valid and discriminating biomarker in pre should be assessed. review of the electronic labor record facilitated collection of maternal demographic, intrapartum, and newborn data. data analyzed with t-tests, chi-square tests, and calculated odds ratios with % cis as appropriate. a forward stepwise logistic regression analysis was used to identify predictors of histologic chorioamnionitis. results: of submitted placentas, had histologic chorioamnionitis (cases) and did not (controls). the groups were similar with respect to age, race, gbs status, and mode of delivery. gestational age, birthweight, duration of labor and ruptured membranes, and number of vaginal exams were greater in the cases (p . ). the cases were more likely to have had epidural anesthesia (or . ), internal monitoring (or . ), fever (or . ), maternal tachycardia (or . ) and fetal tachycardia (or . ) and less likely to have had induction of labor (or . ). newborns in the histologic chorioamnionitis group were more likely to have been observed for sepsis (or . severe sepsis defined as sepsis associated with acute respiratory distress syndrome (ards) or cardiovascular dysfunction(cvd) or with or more other organ dysfunction.all patients were resuscitated with fluids and treated with broad spectrum antibiotics and supportive care as needed. outcome data were: etiology, management, maternal complications, duration of icu stay and perinatal survival. results patients were young (mean age= . ± . years) with a mean gestational age at delivery . ± . weeks. etiologies were pyelonephritis(n= ), septic abortion(n= ), endomyometritis (n= ), chorioamnionitis (n= ), ruptured appendix(n= ), pneumonia( n= ) and one unknown. eighteen ( %) were diagnosed during antepartum and ( %) postpartum period.there were ( %) maternal deaths and high rate of major morbidities (table) . among the antepartum patients,there were, abortions, iufd, neonatal death for a perinatal survival rate of only %. conclusion pregnancies complicated with severe sepsis/septic shock are associated with substantial maternal and perinatal morbidities. the low maternal mortality rate in our study as compared to previous reports is attributed to early diagnosis and aggressive management of maternal complications. fetal loss rate, however continues to be high when septic shock develops antepartum. and tnfa levels are elevated. we developed a dynamic computer (in silico) model of pregnancy (uterine myometrial environment -infection/inflammation and endocrine crosstalk). mathematical differential equations were used to describe the interactions between molecules. infection was represented as increased levels of activated, nuclear transcription factor nf-kb. in the model ru inhibited both the glucocorticoid receptor and progesterone receptors. simulations were run adding different concentrations of ru ( . um= dose used in patients, . um, . um) at different time points during infection (before, at the time of or after nf-kb activation). ru degradation kinetics was also included. the effect of ru on nf-kb induced il- and tnfa levels was assessed. results: infection induced nf-kb activation led to increased il- and tnfa levels. there was a subsequent increase in cortisol that led to dampening of nf-kb activation, il- and tnfa levels. in the presence of ru , il- and tnfa levels continued to rise. the effect of ru on nf-kb induced il- and tnfa was dose dependent and was more prominent in slow metabolizers who had ru in the system for a longer time. addition of ru after the onset of nf-kb activation led to increased il- and tnfa levels above those observed without ru . the addition of misoprostol (prostaglandin e analogue), at the concentrations used together with ru for medical abortion, did not add to the effect of ru on nf-kb induced il- or tnf. conclusions: ru has dose dependent effects on infection induced immune activation and may contribute to the pathogenesis of c sordelii induced sepsis syndrome. the increased susceptibility of neonates to infection remains a major clinical problem. we previously found that after intraperitoneal (ip) listeria monocytogenes infection, neonatal mice have an ld that is orders of magnitude lower than adults. we also found that the inflammatory response of neonatal mouse macrophages, but not neutrophils, in the peritoneal fluid was deficient, and that this correlated with low levels of macrophage chemokines mcp- and rantes. given that the liver and spleen are important organs in listeria infection, we sought to characterize the innate immune response in these organs. a sublethal dose of listeria was injected intraperitoneally into balb/c - week old adult mice and - day old neonatal mice. liver and spleen was collected at , , and hours, sectioned serially for staining with hematoxylin-eosin and primary antibodies (rabbit anti-listeria monocytogenes igg; mhc class ii rat anti-mouse igg, a marker for activated macrophages; f / rat antimouse igg, a marker for macrophages) and secondary antibodies (cy- goat anti-rabbit igg; af goat anti-rat igg) were applied. negative controls used only secondary antibody. real time pcr was used to compare the levels of the chemokines mcp- and rantes in adult and neonatal liver. after ip infection, both adults and neonates showed similar influx of neutrophils to the sites of infection within the liver and spleen. at hours post-infection with listeria, adult liver and spleen showed increased staining for f / and class ii, which increased further and became confluent surrounding microabscesses at and hours. in contrast, the listeria infected neonatal mouse showed some increase in f / around microabscesses but no apparent increase in staining for mhc class ii. the neonates showed greater staining for listeria at each time point. mcp- and rantes levels were higher in infected neonatal liver compared to adults. in the neonatal mouse, the innate immune response in the liver and spleen was characterized by a deficiency of activated macrophages. this deficiency was not correlated with hepatic expression of macrophage chemokines. is there a seasonal pattern in the incidence of post-cesarean endometritis? tamula m patterson, alan tn tita, william w andrews. obstetrics and gynecology, the university of alabama at birmingham, birmingham, al, usa. objective: several theories, including one suggesting a peak in july coincident with resident turnover, postulate seasonality in post-cesarean infections. we assessed whether there is seasonal variation in endometritis. a retrospective cohort study of post-cesarean endometritis at our university-based institution using our obstetric computerized database to compare annual variation in monthly incidence patterns from to . prior to establishing an average aggregate seasonal pattern for all years, years were assessed separately for a recurrent pattern of peaks and nadirs in incidence. peak incidence (or nadir) was defined as any monthly incidence that differed from the mean incidence for the year by over %. results: a total of . % ( , ) of , deliveries from to were by cesarean. annual cesarean rates increased by an absolute rate of over %; while, post-cesarean endometritis rates decreased from % to %. monthly incidence of post-cesarean endometritis did not reveal a consistent recurrent pattern of peaks (figure ) or nadirs. the month of july accounted for only out of a total of peaks for all years. the adjacent months of june and august accounted for only each. the month with the highest number of peaks was april with only . these findings contraindicated the establishment of an average aggregate monthly pattern for all years. the incidence of post-cesarean endometritis did not follow a seasonal pattern. chi-square analyses were used to compare associations between race (black (bl) vs non-black (nbl)) and dichotomous characteristics. student´s t-test was used to compare continuous variables. results , patients were evaluated ( cases and controls). % and % of cases and % and % of the controls were bl and nbl respectively. the baseline prevalence of chtn in bl and nbl controls was . % and . % (p= . ). when comparing bl and nbl cases, bl women had a higher mean systolic blood pressure and screening bmi. bl women also had a trend toward being discharged on post partum blood pressure medicine when compared to nbl women. there was no difference in chtn, diabetes, severity of disease, iugr, or delivery < wks between the two groups (table) . to determine the leading causes of death in a case series of stillborn infants examined in a large hospital autopsy service, and to describe the most common post-mortem observations. study design: one hundred and sixty one stillborn infants were examined. gross pathology observations were recorded at autopsy and during the placental exam, and tissue sections were collected and examined microscopically. immediate and underlying cause of death (cod) were recorded, along with contributory cod, concomitant/significant cod, and incidental findings. statistical analysis was conducted using the software spss v. . . results: the immediate anatomic cod could be determined in . % of all infants examined. in over % of these cases, cod was attributable to placental or umbilical cord findings affecting the maternal-fetal blood supply. the most prevalent among these findings were placental lesions (maternal floor infarction, placental abruption, fetal thrombotic vasculopathy), umbilical cord lesions (entanglement, true knot, compression, excessive length/twisting), and infectious/inflammatory processes (chorioamnionitis, chronic villitis objective: advanced maternal age (ama) is associated with increased risk of intrauterine fetal demise (iufd). antenatal testing (at) is widely used in clinical practice to prevent iufd due to uteroplacental insufficiency and has been suggested to reduce the risk of iufd in ama women. we sought to assess the impact of at on obstetrical interventions and compliance with new practice recommendations. methods: retrospective cohort of ama women ( and older at their due date) who delivered at or after weeks. non-exposed women delivered from july to december (when at for ama was not routinely recommended); exposed women delivered from july to december (after at for ama was introduced at our institution). subjects were identified through the perinatal database; records were abstracted for demographics, medical history, and labor/delivery variables. outcomes included rates of at and induction of labor (iol) and mode of delivery. associations between at for ama and outcomes were tested using t test and chi square. assuming a baseline rate of iol of %, we had % power to detect an increase to % after the introduction of at. results: women met the inclusion criteria: delivered before the introduction of at (non-exposed=before at) and delivered after the introduction of at (exposed=after at). baseline clinical characteristics were similar in both groups. as anticipated, at was more common in the after at group than in the before at group ( % vs %; p< . ). women were not eligible for or declined trial of labor, thus not "at risk" for iol and not included in all analyses. ob intervention rates were increased after at compared to before at (table ) . the corrected iufd rates were similar in both groups ( / vs / ). conclusion: at an academic center, compliance with new practice recommendations was excellent. introducing at testing for a new indication seemed to increase iol and cs rates. these findings should be considered when assessing the risks:benefits ratio of antenatal testing. . we stratified indications for cesarean into the following: failed induction (cervix < cm dilated), arrest of dilation, arrest of descent, failed operative delivery, fetal intolerance of labor (fil), and "other" reasons (e.g., pre-eclampsia, abruption, chorioamnionitis, malpresentation). both fil and "other" reasons were more likely to occur among the medically indicated group (rr . , . physicians in solo practice had higher rates of elective inductions (p< . ), but there was no association between cesarean and practice type. conclusions: inductions accounted for . % of cesareans. these results suggest an increased risk for cs for patients undergoing medically indicated inductions at our institution. there was no association between cesarean and type of practice, whether solo or group, suggesting institutional clinical policies may be more important than practice type in determining delivery outcome after induction. further research is needed to understand how age, race/ethnicity, or other unmeasured patient factors may impact these findings. given rising rates of both cesarean delivery and inductions, this information may be pertinent to women considering elective induction prior to weeks. objective: fetal demise can lead to a consumptive coagulophathy ("fetal death syndrome") traditionally attributed to the release of "tissue thromboplastin", now known as "tissue factor" (tf). tf is the most potent activator of coagulation. despite the appeal and acceptance of this proposed pathophysiology, there is no evidence supporting this view. this study was undertaken to determine if fetal death prior to development of fetal death syndrome is associated with changes in maternal plasma concentration of cd l (a marker of platelet activation), tf and its soluble inhibitor (tfpi). methods: a cross-sectional study included the following groups: ) women with normal pregnancy (n= ) and ) patients with fetal demise without disseminated intravascular coagulation (n= ). plasma concentrations of scd l, tf and tfpi were measured by elisa. standard coagulation tests were performed. non-parametric statistics were used for analysis. results: ) patients with fetal demise had a higher median maternal plasma scd l concentration than women with normal pregnancy (median . pg/ml, range - vs. median . pg/ml, range . - . , p< . ); ) there was no significant difference between the groups in the median maternal plasma tf concentration and ) in contrast, the median maternal plasma tfpi concentration was significantly lower in patients with fetal demise than in women with normal pregnancy (median . ng/ml, range . - . vs. median . ng/ml, range . - . , p< . ). conclusions: ) a change in the plasma concentration of tf was not demonstrated; ) a change in the ratio of tf/tf inhibitor pathway may predispose to thrombin generation and activation of the coagulation cascade; ) however, maternal platelet activation is present in patients with a fetal demise without fetal death syndrome and ) the role of tf in fetal death syndrome remains to be proven. lipoic acid inhibits matrix metalloproteinase production, activity and prostaglandin e secretion by cultured amnion epithelial and mesenchymal cells. r moore, j novak, d kumar, j moore. case western reserve university, cleveland, oh, usa. introduction: cytokines, free radicals, matrix metalloproteinases (mmp) and prostaglandins (pg) have been implicated in processes of fetal membrane rupture and labor. dietary anti-oxidant supplementation has been suggested as a possible therapy for high risk patients, however, clincal evidence supporting the efficacy of agents such as vitamin c or n-acetylcysteine remains controversial. in fact, we have previously shown that vitamin c increases matrix metalloproteinase (mmp) activity in isolated fetal membrane fragments and fails to inhibit tumor necrosis factor (tnf) induced fetal membrane weakening in vitro. in this study, we examine the effect of the naturally occurring anti-oxidant, -lipoic acid, on tnf induced mmp activity/protein and pge secretion in isolated amnion epithelial and mesenchymal cells. methods: amnion epithelial and mesenchymal cells were pre-treated with increasing doses of -lipoic acid ( - mm/ h), then with increasing doses of tnf ( - ng/ml/ h). medium and cells were analyzed by gelatin zymography/western blotting for mmp /mmp activities/protein. pge output was determined by immunoassay. results: tnf induced a dose dependent increase in mmp production, secretion and activity in amnion epithelial cells. tnf ( ng/ml) induced an fold increase in cellular active mmp production and fold increase in secreted mmp enzyme activity by amnion epithelial cells. these increases were reduced - % following h pre-treatment with . - . mm -lipoic acid. mmp protein/activity and pge secretion by amnion epithelial cells were barely detectable and unaffected by tnf and/or -lipoic acid treatment. in striking contrast, mesenchymal cells exhibited little basal or tnf induced mmp protein/activity. mmp protein/activity in mesenchymal cells were unaffected by either tnf and/or -lipoic acid. however, tnf treated mesemchymal cells exhibited a dose dependent increase in pge production ( fold increase/ ng/ml tnf/ h) that was inhibited by %- % following . objective: nearly fifty years after the discovery of microphthalmia-associated transcription factor (mitf), its gene was identified as a specialized transcription factor that dictates cell-specific differentiation. unique mitf isoforms are generated from alternative promoter usage. an isoform of mitf (mitf-cx) is down-regulated in cervical stromal cells of the ripened cervix. further, mitf-cx inhibits il- gene expression and thereby suppresses signaling of the final pathway in cervical ripening. since mitf binds to canonical eboxes (canntg) in promoter regions of target genes, we sought to determine if mitf regulated its own promoter through ebox motifs. methods: the kb genomic dna sequence upstream of the mitf-cx transcription start site was cloned into pgl luciferase reporter vectors which were co-transfected with wild type or mutmitf-cx (impaired dna binding) into cervical stromal cells or hek cells. at h, promoter activity was determined and normalized for transfection efficiency. results: gel-shift assays conducted with oligonucleotides corresponding to eboxes in the mitf-cx promoter revealed two strong binding sites (eboxes - to - and - to - ). specific binding was established using oligonucleotides with or without mutated ebox, antibody supershift experiments, competition with cold probe, and absence of binding to mutmitf-cx. binding specificities were confirmed in nuclear extracts from cells that overexpressed mitf-cx, but not control or mutmitf-cx. reporter gene studies indicated that mitf-cx, but not mitf-m, increased mitf-cx promoter activity -to -fold. whereas mutations in ebox or resulted in significant decreases in mitf-stimulated promoter activity, mitfinduced increases in promoter activity were abolished by mutations in both eboxes. conclusions: collectively these experiments indicate that mitf-cx is a vital regulator of its own promoter activity and acts in a positive feedforward loop through two specific binding sites in its promoter. moreover, isoform-specific amino acids are important to mediate mitf-induced mitf-cx promoter activity. decreasing mitf protein or mutating its promoter would interrupt this loop resulting in rapid reduction of mitf synthesis. since mitf-cx suppresses il- gene expression in cervical stromal cells, we suggest that preservation of mitf-cx-induced mitf gene expression is an important mechanism to maintain the "brake" on cervical ripening and ensure cervical competency during pregnancy. the role of cd in cervical remodeling. denisse sanchez, brenda timmons, mala mahendroo. obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa. objective: prior to the onset of parturition, the uterine cervix undergoes a remodeling process from a closed, rigid structure, to one that is soft and dilatable. many changes occur, including increases in hyaluronan (ha), a glycosaminoglycan that facilitates loosening of the collagen matrix. in the postpartum period, the concentration of ha is reduced to that of the nonpregnant state. cd , a transmembrane glycoprotein expressed in hematopoietic and epithelial cells, is a receptor for ha. cd expression by immune cells is important in extravasation of leukocytes into tissue. cd may also be required for ha catabolism through the action of hyaluronidases and . in the cervix, cd is expressed in the endo-cervical epithelia as well as in immune cells localized in the stromal matrix. to study the importance of cd in cervical remodeling, mice with a null mutation for cd (cd -/-) were evaluated during pregnancy, parturition and postpartum. methods: changes in ha amount and size distribution were assessed using ha molecular weight gels and fluorophore assisted carbohydrate electrophoresis. to identify defects in cervical remodeling in the cd -/mice, differences in expression for genes regulated in the cervix were studied by quantitative real time pcr . to determine whether cd plays a role in the recruitment of immune cells during cervical ripening and postpartum repair, immunohistochemistry with antibodies against leukocytes was done. in the postpartum period, there is a higher ratio of high molecular weight ha relative to low molecular weight ha in the cd -/mice. this suggests that postpartum breakdown of ha in cd -/cervices is delayed. as compared to wt cervix, there was a significant increase in hyaluronidase (hyal- ) mrna in the postpartum period. the distribution and relative numbers of immune cells in the cd -/cervix was similar to wt. conclusion: these studies provide evidence that cd may play a role in remodeling of the postpartum cervix back to the nonpregnant state. our current data suggests that the catabolism of ha after birth is delayed in the mutant mice and upregulation of hyal may compensate to allow ha removal. furthermore, the activity of hyal- may be dependent on cd . little difference in the recruitment of immune cells between cd and wt animals suggest that cd expression is not required for this process. these experiments provide a greater understanding for the role of cd and ha in cervical remodeling. in background: during in vitro experiments we have shown that separation of amnion from choriodecidua occurs as an integral part of the process of fetal membrane (fm) rupture. although spontaneous amnion and choriodecidual separation is seen in fm after both svd and elective c/s deliveries, its etiology is uncertain. biochemical degradation at the amnion-choriodecidua interface may be a key contributing factor. our previous biomechanical studies have demonstrated that separated fm require less physical work to rupture than intact membranes. the purpose of this study was to determine whether fm separation was associated with clinical differences in the birth process. hypothesis: during term, normal labor, spontaneous separation of fm is associated with differences in the clinical parameters of labor and delivery. study design: fm from consecutive term deliveries were cut off the placental disk. separated areas of fm were cut from the intact areas. both were weighed and their weight ratios determined. maternal medical, pregnancy, and delivery data were collected and analyzed. results: term fm had the following characteristics: maternal age ± . yr, gravida . ± . , gestation ± . wks, elec. cs . %, duration of rom ± min, duration of contractions ± min, african american %. % of the fm had < % separation; % had more than % separation. srom fm with > % separation (vs. < %) had significantly shorter duration of rom (p= . ) and admission to birth (p= . ) times. srom fm with > % separation (vs. < %) had even shorter rom (p= . ), duration of contractions (p= . ) and admission to birth (p= . ). the > % group (vs. < %) was further along, gestationally (p= . ). srom fm (vs. arom) had shorter admission to birth (p= . ), but longer rom to birth (p< . ) times. absence of epidural (p= . ), srom mode of rupture (p= . ), svd (vs. elec. c/s) (p= . ), and the presence of meconium (p= . ), were all associated with increased fm separation. conclusion: spontaneous separation of fetal membranes is nearly universal and is associated with increased gestation, spontaneous rupture of membranes, shorter duration of contractions, and svd. speculation: we speculate that programmed biochemical changes initiate fm separation which then facilitates rom and childbirth. whole genome array and si-rna investigation of the function of nfkb in human amnion epithelial cells. sheri e lim, shirin khanjani, yun s lee, tg teoh, , philip r bennett. institute of reproductive and developmental biology, imperial college, london, united kingdom; maternal fetal medicine, st. mary's hospital, london, united kingdom. introduction: labour is associated with activation of nfkappab in the amnion. nfkappab increases prostaglandin synthesis through the upregulation of cyclooxygenase- (cox- ), which is essential to the labour process. cox- mrna expression increases with gestation in the amnion. primary amnion epithelial cells cultivated from tissue collected prior to the onset of labour display a spectrum of nfkappab activation, similar to the spectrum of cox- expression presumably relating to the nearness of labour. our aim was to investigate the full range of genes under nfkappab control in amnion epithelial cells by using whole genome arrays. methods: amnion from women undergoing elective caesarean section was collected and primary cell cultures established. total rna and protein were extracted from each culture. nuclear localization of nfkappab is required for its activation. western analysis of nuclear p was therefore performed to identify the samples displaying the lowest and highest nfkappab activity. the corresponding rna samples displaying the three lowest and three highest nuclear p protein concentrations were used for whole genome analysis using affymetrix u arrays. results and conclusions: we identified significantly regulated genes. the gene with the highest fold change was cox- (x . ) followed by oxytocin receptor (x . ), ch orf (x . ), integrina (x . ), and connective tissue growth factor (x . ). other significant genes included interleukin- (il- ) (x . ). pathway analysis revealed the majority of other nfkappab associated genes were involved in cell signaling, turnover and proliferation. we used real-time pcr (rtq-pcr) to validate cox- and il- expression. to prove that cox- is directly regulated by nfkappab, primary amnion epithelial cells were then transiently transfected with nfkappab p sigenome smart pool and sicontrol non-targeting sirna pool. western blot analysis confirmed knockdown of nfkappab p associated with inhibition of cox- demonstrating that nfkappab is essential for cox- expression. objective: premature birth is a major public problem accounting for over , deaths and , surviving infants with life-long morbidity yearly. in order to develop a rational basis for treatment and prevention of premature fetal membrane (fm) failure, we first need to understand the sub-failure fm structural and mechanical behavior at near full term. methods: we utilized planar biaxial mechanical testing, which approximates the physiologic loading state, for mechanical evaluation of the fm, and a structural constitutive model approach was used to offer insight into the structure-strength of the fm by integrating information on tissue composition and structure. small angle light scattering (sals) was used to nondestructively quantify the collagen fiber architecture of both intact and separated fm layers. results: in the stress free state, the gross collagen fiber architecture of the fm and separated layers were not homogenously align but exhibited small regions of fiber alignment. the amnion layer displayed the greatest alignment. the model fit the equi-biaxial strain data well (r = . ) and indicated that fm collagen fibers were rapidly recruited and straightened well below failure stress levels. collagen fibers were gradually recruited followed by a drastic increase in fiber recruitment. conclusion: this study provided the first data on the effective collagen fiber stiffness in the intact fm under physiologic biaxial loading, which was related to quantitative collagen fiber architectural measures. modeling results indicated that the collagen fibers became fully loaded and straighten well below physiological loading levels. failure did not occur during physiological loading, indicating that fibers do not begin to fail until all collagen fibers are fully straightened and bearing load. this result suggested modest structural reserve in the fm collagen architecture, and may be an important aspect of its failure properties. previously, we demonstrated that a physically "weak zone" exists overlying the cervix in the fm, evident of collagen remodeling and cellular apoptosis. we are currently extending the present study to include the "weak zone" tissues, allowing us to elucidate the micro-mechanical mechanisms that facilitate failure in this newly identified fm zone. supported by nih . the extracellular matrix of the cervix undergoes extensive remodeling during parturition. hyaluronan (ha) is a major constituent of the extracellular matrix of the term pregnant cervix. the onset of labor is preceded by an increase in ha and after delivery, the concentration of cervical ha gradually decreases to that of the non-pregnant state. these dramatic changes suggest that ha plays an important role during parturition. hyaluronan synthase (has ) is one of three known ha synthases and the most abundant isoform in the pregnant cervix. transcripts for has are regulated by two alternative promoters, one upstream of the first coding exon (proximal promoter) and another upstream on an untranslated exon (distal promoter). the focus of the current study is to further our understanding of the transcriptional regulation of has during cervical ripening. methods: rna blotting was carried out using transcript specific probes corresponding to the distal and proximal promoter of the mouse has gene. the regulation of has was evaluated in a cervical epithelial cancer cell line (caski cells) which we have previously shown to express endogenous has . regulation of has expression by epidermal growth factor was assessed in the caski cells by western blotting and quantitative real time pcr assessment of transcripts. results: has mrnas in the nonpregnant (np) and pregnant cervix are transcribed from the distal promoter upstream of exon . two transcripts of approximately . kb and . kb were detected that arise from use of polyadenylation sequences. as compared to np, the expression of has is increased , , and fold on gestation days , and shortly postpartum respectively. has mrna expression is increased upon treatment of caski cells with epidermal growth factor ( ng/ml) and is suppressed in cells treated with ag ( m), an inhibitor of egf receptor phosphorylation. maximal stimulation was observed at and hours of treatment. conclusion: has is the major ha synthase expressed during cervical ripening and the majority of transcripts are driven by the distal promoter in the has gene. in vitro studies using caski cells suggest has is regulated in part by the egf signaling pathway resulting in a several fold increase in has expression. these results provide an understanding of has gene regulation at the time of cervical ripening which will ultimately enhance our understanding of the molecular mechanisms important to cervical ripening. chorion and decidua cells. chad a grotegut, bernard j canzoneri, liping feng, phil heine, amy p murtha. obstetrics and gynecology, duke university, durham, nc, usa. objective: preterm premature rupture of the fetal membranes accounts for approximately % of all preterm deliveries. cigarette smoking independently carries a fourfold increase risk for pprom. our laboratory has previously demonstrated that the chorion layer undergoes apoptosis in women with pprom. this study was conducted to determine if extract of cigarette smoke causes cell death in specific cells of the fetal membrane. fetal membranes were collected at the time of elective cesarean section from women without labor and at term. the chorion and decidua layers were separated and purified on a gradient spin column and then plated near confluence. cigarette smoke extract (cse) was collected in cell media and used to treat chorion and decidua cells in culture at concentrations ranging from % to % in -well plates. cell viability was determined at , and hours following treatment with a non-radioactive cell viability assay. data were analyzed using paired t test (analyse-it, leeds, uk). chorion and decidua cells underwent cell death when exposed to cse in a dose dependent fashion. increasing concentrations of cse resulted in increased cell death at hours in both cell types (figure ). at hours, chorion exhibited greater percent cell death compared to decidua at concentrations of , and % cse (p= . , . , and . , respectively). for any given concentration of cse, the degree of cell death increased with increasing length of exposure ( , and hours) for each cell type. chorion cells routinely exhibited greater percentage of cell death following treatment with cse at concentrations ranging from - % compared to decidua cells. human chorion and decidua cells in primary cell culture exhibit a dose-response and time dependent cell death in the presence of cse. human chorion cells show greater sensitivity to cell death when compared to decidua cells. further studies are needed to determine the mechanisms through which these cell types undergo cell death and the implications for the differential sensitivity to cigarette smoke. yoon ha kim, tae-bok song, cheol hong kim, jong woon kim, moon kyoung cho, sung yeul yang, bong whan ahn. obstetrics gynecology, chonnam national university medical school, gwangju, korea; biochemistry, chonnam national university medical school, gwangju, korea. objective: to investigate the lipid peroxide levels and protein carbonyls levels in the amniotic fluid of pregnant women with preterm premature rupture of membranes (pprom). the lipid peroxide levels in the amniotic fluid of normal pregnancy (n= ) and pregnant women with pprom (n= ) were newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced measured by thiobarbituric acid reaction. the protein carbonyl contents in the amniotic fluid of normal pregnancy (n= ) and pregnant women with pprom (n= ) were determined by the , -dinitrophenylhydrazine method. after amniotic fluid of them were mixed and incubated up to hours with . ml of mm moxalactam, cefodizime, amoxacillin, erythromycin, the lipid peroxide levels and protein carbonyl contents in them were measured. results: . the lipid peroxide levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy ( . ± . vs. . ± . nmol/mg protein, p< . ). . the protein carbonyl levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy ( . ± . vs. . ± . nmol/mg protein p< . ). . the lipid peroxide levels and protein carbonyls formation by moxalactam in the amniotic fluid of pregnant women with pprom was significantly higher than basal level ( . ± . vs. . ± . nmol/mg protein, . ± . vs. . ± . nmol/mg protein, p< . ). . the lipid peroxide levels and protein carbonyls formation by cefodizime in the amniotic fluid of pregnant women with pprom was significantly lower than basal level ( . ± . vs. . ± . nmol/mg protein, . ± . vs. . ± . nmol/mg protein, p< . ). . there were no significant differences in the levels of lipid peroxide and protein carbonyls by amoxacillin and erythromycin in the amniotic fluid of pregnant women with pprom between antibiotics-induced and basal levels. background: chorioamnionitis (cam) is a major antecedent of preterm delivery (ptd) associated with elevated amniotic fluid tnf and il . we hypothesized that these cytokines enhance the term decidual cell (dc) expression of the matrix metalloproteinases (mmp) and , which can then promote ptd by degrading the extracellular matrix of the decidua, fetal membranes, and cervix. methods: immunostaining for mmp- , mmp- , and vimentin (a dc marker) was performed on cam-complicated (n= ) and gestational age-matched control decidua (n= ), and staining intensities were evaluated by hscore. confluent, leukocyte-free term dcs were primed with - m estradiol (e ) or e + - m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e +/-mpa with or without ng/ml of il or tnf . secreted mmp- and mmp- levels were measured by elisa (n= ), and quantitative rt-pcr assessed mmp- and mmp- mrna levels (n= ). results: tissue staining revealed that mmp- and mmp- levels in cam-complicated decidua (hscore mean±sem: ± and ± , respectively) were significantly higher than in control decidua ( ± , and ± respectively; p< . ). in cultured term dcs incubated with e , tnf and il significantly increased secreted levels of mmp- compared to e alone (pg/ml/ g protein: . ± . and . ± . , respectively, vs. . ± . ; p< . ). in parallel incubations with e +mpa, basal mmp- output was lowered by %, and tnf -and il -elicited mmp levels were blunted by % and %, respectively. rt-pcr confirmed that tnf and il increased mmp mrna levels (p< . ), although mrna levels in e +mpa incubations were not different from those of e alone. mmp levels in all treatments were similar. conclusions: mmp- and mmp- are elevated in cam decidua compared to controls. our in vitro results suggest that mmp- expression is enhanced by the high levels of il and tnf associated with cam, and that mpa may be able to blunt this effect. we have previously found a similar regulatory mechanism of mmp- and mmp- and their over-expression in cam-complicated tissues. synergy among these mmps may represent a potent pathogenic mechanism of cam that can be targeted through the therapeutic use of progestins in preventing cam-induced ptd. domerudee preechapornprasert, patama promsonthi, wasun chantratita, mana rochanawutanon, patcharee karnsombut, chutatip srichunrusami, stephen j lye, boonsri chanrachakul. obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; pathology, ramathibodi hospital, mahidol university, bangkok, thailand; obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: cervical ripening is an inflammatory process involving chemokines, cytokines and various mediators. recent study has shown that the level of monocyte chemotactic protein (mcp) , a chemokine, increases in amniotic fluid during spontaneous labor. the aim of this study was to examine the localization and expression of mcp in human cervix before pregnancy, during pregnancy and after the onset of labor. methods: this study was approved by the local ethics committee and written informed consent was obtained from each participant. cervical biopsies were taken from groups of women; nonpregnant women, first trimester pregnant women, term pregnant women with and without labor. tissue samples were fixed in % formal saline for paraffin section. immunohistochemistry (n = each) was performed by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp . the mcp messenger(m) rna was identified by reverse transcription-polymerase chain reaction using gene specific primer against mcp and mcp receptor (n = each). results: immunohistochemistry demonstrated mcp in cervical tissues from all four groups of women. mcp was localized on plasma membrane and cytoplasm of both squamous epithelial and columnar cell lining of endocervical gland. mcp and mcp receptor mrna were identified in nonpregnant, first trimester and term with and without labor human cervix. conclusion: mcp and mcp receptor were located in cervical tissues of nonpregnant and pregnant women at different gestation both before and after the onset of labor.ongoing studies are investigating the role of this chemokine during pregnancy and labor. whether preterm cervical ripening is just an aberrant regulation in timing or whether divergent mechanisms and pathways are involved in preterm versus term cervical ripening remains to be elucidated. methods: cervical tissue was collected from groups of cd- mice. group : mouse model of ptb that utilizes intrauterine infusion of lipopolysaccharide (lps). group : e dams representing preterm controls. group : e . - dams selected from a timed pregnant batch where half of the dams had delivered representing term cervical ripening. n= dams/treatment group. separate rna samples were used for microarray analysis (ma). significance analysis for ma and partek software was used for biostatistical analysis. pathway analysis was performed using david. quantitative pcr was performed to confirm the most differentially regulated genes. results: using a cut-off of -fold change with p value of < . , genes in the cervix were differently regulated between the groups. principal component analysis revealed three distinct groups (see graph). functional annotation clustering demonstrated the following pathways: ) in preterm cervical ripening (e lps vs e ): immune and inflammation response, defense response ) in term cervical ripening compared to preterm controls: negative regulation of cellular process and biological process, ecm, cell-cell communication. qprc confirmed the highly significant differences found in ma (see table) . conclusions: the molecular mechanisms and pathways governing preterm and term cervical ripening are distinctly different. elucidating these unique pathways can lead to improved therapeutics for prevention of ptb as well as for postdate pregnancies. cervical ripening at term involves activation of apoptotic enzymes. maria kb sennstrom, valentina ciani, gunvor e ekman. obstetrics and gynecology, women and child health, karolinska institute, stockholm, sweden; obstetric and gynecology, university of siena, siena, italy. aim: to investigate if the human cervical ripening at term involves programmed cell death. during the final cervical ripening the extracellular matrix dominated cervix undergoes an extensive remodelling of the tissue. inflammatory mediators such as the cytokines increase in ripening cervical tissue at term. programmed cell death, apoptosis, has been suggested as important in this process. apoptosis can be induced by inflammatory mediators such as cytokines. we also looked upon the distribution of inflammatory cells in cervical tissue. materials and methods: cervical biopsies from pregnant women at term and post partum women with fully ripened cervix were studied. biopsies from non-pregnant women served as controls. immunohistochemical analysis of the apoptotic enzyme caspase- and the inflammatory cell marker cd was performed on paraffin embedded sections of cervical tissue. double staining was performed. mann-whitney u-test was used for statistical analysis. results: there was a significantly higher frequency of caspase- staining in the post partal sections from ripened cervical tissue compared to tissue from term pregnant (p= . ) with unripe cervix and from non-pregnant patients (p= . ). the inflammatory cells staining for cd increased in post partal and term pregnant sections compared to non-pregnant (p= . ). there was a higher frequency of caspase- positive cells in the post partal tissue than of cd positive cells (p= . ). the localization of cd- positive staining was highest in the epithelia and basal lamina while caspase- staining was most pronounced in stromal tissue and around vessels. conclusion: our data show apoptotic activity in stromal tissue in fully ripened human cervix at term of pregnancy, suggesting that apoptotic mechanisms are involved in the extracellular matrix remodelling at term. the apoptotic activity is not co-localized with inflammatory cells suggesting non-infectous inflammation with apoptosis as important for cervical ripening at term. objective: cervical biomechanical responses are important for accommodating the increased stress induced by an enlarging uterus. a mechanical testing system was modified to evaluate the stress relaxation response in the pregnant cervix. methods: tissue harvested from the non pregnant and timed pregnant (days , , , , and ) sprague-dawley rats underwent tensile testing using an instron material testing system. the testing regimen consisted of tissue extension to near maximal strain over seconds followed by a period of constant strain for minutes, then return to rest over seconds. this cycle was repeated additional times with a minute rest period between cycles. strain and force measurements were recorded at second intervals. - animals were used for each time point. in addition, stress and strain at the yield point was also determined for each gestational time point. results: the pregnant and non pregnant samples exhibit marked differences in response in both stress and strain. the timed pregnant tissue demonstrated progressively increased compliance and lower nominal stress compared to non pregnant tissue. peak nominal stress declined with each successive cycle. this was demonstrated in the peak nominal stress and strain values at the yield point. conclusion: the cervix becomes more distensible (compliant) but less resistant to force with increasing gestational age. ...,.. e. coli . x .:!: . x . x .:!: . x group a (c, c) group b ( c, p) . x + . x . x + . x . . . group c (p, p) . x o"::!>s x o" . x ~ .ox: o• k. ~neumoniae group a ( c, c) . x + . x . x + . x . . . group b (c, p) . x + . x . x + . x group c (p, pl . x ~ . x . x ~ . x c. al bicans group a (c, c) . x '+ . x . x + . x group b (c, p) . x o•+ . x . x + . x . . . group c (p, p) . x ~ . x . x ~ . x • introduction: a growing body of evidence supports that inflammatory processes are implicated in spontaneous preterm birth (ptb). using an inflammatory and non-inflammatory mouse model of ptb, we sought to determine if activation of these inflammatory pathways are essential for ptb and/or cervical ripening to occur. methods: timed pregnant cd- mice were used in these two models of ptb: ) a model of intrauterine inflammation where lipopolysaccharide (lps) is injected into the uterine horn (n= ); controls for this model received intrauterine saline (n= ) and ) a non-infectious model of ptb using ru sq ( ugrams/dam) (n= ); controls for this model received no intervention (n= ) were used for these studies. for both models, hours later uterine and cervical tissues were harvested. the tissues were processed for protein and rna studies. elisas were performed to assess il- and tnf-alpha in the uterine tissue. mrna expression of ifn , il- , il- , il- , tnf-alpha were assessed in cervical tissue from both models by quantitative pcr. results: in uterine tissues, both il- and tnf were significantly elevated in the lps-induced ptb when compared to control, saline, and non-infectiousinduced preterm birth (p< . ) (figure ). in cervical tissue, an increase in il- , il- beta and tnf mrnawas observed in both models, while ifn-gamma and il- were only increased in the lps model. (figure ). conclusions: up-regulation of pro-inflammatory cytokines in the uterus do not appear to be essential for ptb. cytokine expression in the cervix is greater in an inflammatory model of ptb but is also present in a non-infectious model. these studies suggest that targeting a cytokine response in the cervix may hold the most promise in prevention of ptb. the initiation of labor at term and preterm is associated with an inflammatory response, with increased interleukins in amniotic fluid (af) and infiltration of the myometrium by neutrophils and macrophages (m ). whereas, in preterm labor, intra-amniotic infection may provide the stimulus for increased af interleukins and inflammatory cell migration, the stimulus for these events at term has remained uncertain. in studies using pregnant mice, we observed that the m that invade the maternal uterus near term arise from the fetus. furthermore, we obtained compelling evidence that surfactant protein-a (sp-a), a developmentally regulated c-type lectin secreted by the fetal lung into af near term, activates af m , which migrate to the uterus where they promote an inflammatory response culminating in labor. we propose that interactions of m surface receptors with sp-a, at term, or bacterial lipopolysaccharide at preterm, initiate changes in m phenotypic properties, resulting in the enhanced expression of genes that promote their migration to the uterus. the objectives of the present study were to analyze the numbers and phenotypic properties of mouse af m during late gestation and to identify their putative tissue source(s) of origin. to assess changes in the number of m in af during late gestation, af cells were isolated and stained for the m marker f / . the density of adherent f / + cells greatly increased in equivalent volumes of af between . and . days postcoitum (dpc) ( . dpc = term). interestingly, the f / + cells at . dpc were highly similar in morphology to those present in . dpc fetal lung, but distinctly different from those in fetal liver, suggesting their pulmonary origin. the af m were foam cell-like, suggesting the presence of lipid inclusions, a property shared by adult alveolar m . to further analyze gestational changes in the m population(s) in mouse af, we used flow cytometric analysis. in our initial studies, cells isolated from af were stained for f / and for cd , a pan-leukocyte marker. we observed that af from . and . dpc mice contained two sub-populations of cd + f / + cells. studies are in progress to analyze these af m populations for expression of cell surface antigens indicative of their maturity, activation state and chemotactic properties in association with the developmental induction of sp-a synthesis and secretion by the fetal lung. april bleich, patrick keller, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. the role of prs, proinflammatory cytokines, cell adhesion molecules, cox- , and toll-like receptors in mediating cervical ripening prior to labor is not clear. the objective of this study was to quantify pr isoforms and determine the relative expression of certain inflammation-related genes in cervical stroma from nonpregnant and pregnant women in early gestation (eg), term before and after cervical ripening, and during labor. methods: standard curves of pr-b, -a, pra+b and qpcr were used to quantify total and pr-b in cervical stroma from nonpregnant (proliferative, n = ; progestin treatment, n = ) and pregnant women undergoing hysterectomy (eg, n = ; term before ripening, n = ; after ripening, n = ; in labor, n = ). cervical status was determined by modified bishop scoring. results: total pr expression was maximal in nonpregnant women in the proliferative phase ( . ± . pg/ug cdna) and decreased % by progestins ( . ± . pg/ug cdna). this level was maintained in stroma from pregnant women before labor ( . ± . , eg.; . ± . before ripening; . ± . after ripening pg/ug cdna). in contrast, total pr was decreased significantly in the dilated cervix ( . ± . pg/ug, p < . ). interestingly, whereas pr-b was ± % that of total pr in the nonpregnant cervix, pr-b mrna levels were ± % to ± % in cervical tissues from all pregnant women and did not vary with labor status. using immunoblot analysis and pr-specific antibodies (pgr ), pr-b immunoreactivity was % that of total pr in all samples from pregnant women, and both pr-a and -b were downregulated significantly in the dilated cervix (from ± to ± units/atub). decreased expression of pr in the dilated cervix was accompanied by significant increases in il- , mcp- , tlr- , cd l, cox- , and s a mrna (all p < . , anova) but not cd b or tlr- . pgdh mrna was decreased significantly in the dilated cervix. with the exception of cox- , expression of these genes was similar before labor regardless of cervical ripening. conclusions: both pr and pr-b are decreased proportionately in the dilated cervix, but not during cervical ripening. further, a number of inflammatory gene products are increased dramatically in the cervix during labor, but not before. taken together, the results suggest that cervical ripening is distinct from cervical dilation and involves upregulation of cox- but not il- , mcp- , or toll-like receptors. ifn-y il- il- , il- tnf- "p value < . lps/saline ru /controls . "" . " . " . * " " . * . . " association between interleukin- (il- ) and il- to examine association of amniotic fluid interleukin (il- ) concentration with il- and its receptor il -r haplotypes in term and preterm caucasians (c) and african americans (aa) samples. methods: in this study case (preterm birth -ptb [< weeks]) and control (term [> weeks]) amniotic fluid (af) il- concentrations were analyzed for association with haplotypes of the il- and il r genes in aa and c separately. in il- , eight, and in il- r, single nucleotide polymorphisms (snps) were examined. aa and c maternal and fetal genotypes were assessed (aa: maternal:cases- controls- fetal: cases- fetal controls-; c: maternal cases- , controls- , fetal:cases- ; controls= ). haplotype associations were performed by using a sliding window with outcome il- concentration. analyses were performed separately on maternal and fetal dna. results: the strongest haplotype associations were observed in il- r rather than in il- . in c fetal dna il- r haplotypes defined by markers - bp from the transcription start site associated most strongly with af il- concentrations (global p= . x - ) and in aa maternal il- r haplotype markers at - - - (global p= . x - associated with il- concentrations. in the c fetal cases the - haplotype with the highest concentration was a-g (log(cytokine) = . pg/ml). in aa maternal samples the highest concentration was observed for haplotype t-t-g-c at - - - .this was seen in both cases and controls at (case log (cytokine) = . ; control log(cytokine) = . pg/ml). significant associations from haplotype analyses converged on three regions of the il- r in both races. no strong differences were observed between the haplotypes of cases with and without microbial invasion of the amniotic cavity (miac), with the exception of aa fetal samples that showed two overlapping haplotypes in il- that associated in cases with miac but not in cases without miac (- and - ; - and - )(both with p < x - ) conclusion: differences in the af il- concentration in ptb do not result from single snp effect on il- but are a result of complex relationships between il- and il- r haplotypes. these associations exhibit racial disparity. the role of tnf-in parturition. helen alexander, amanda tattersall, mark tattersall, suren sooranna, peta grigsby, leslie myatt, mark johnson. obstetrics gynaecology, imperial college, london, united kingdom; obstetrics gynaecology, university of cincinnati college of medcine, cincinnati, oh, usa. introduction: tumour necrosis factor-alpha (tnf-) is thought to play a role in inflammation-induced preterm labour since the decidua produces tnfin response to bacterial products and amniotic fluid tnf-concentrations are increased in the presence of intra-amniotic infection. the aims of this study were to (i) investigate the expression of myometrial tnf-and its receptors in relation to the onset of preterm and term labour; (ii) to identify which intracellular pathways are activated by tnf-; and to investigate the effect of tnf-alone and in combination with il- or il- on gene expression in uterine myocytes. methods: biopsies of human myometrium were taken at caesarean section from women before and after the onset of preterm and term labour and analysed for tnf-and its receptor mrna expression. a further samples were obtained before the onset of labour (n= ) from which myocytes were isolated and cultured in -well plates. when cells were - % confluent either tnf-at a concentration of ng/ml was added to the cells for , , , and min and the cells analysed by western blotting or tnf-at concentrations of , . and ng/ml was added either alone or in combination with similar concentrations of il- or il- to cells for hours and mrna was extracted and converted to cdna to determine il- and gapdh gene expression by qpcr. results: there was no change in tnf-mrna expression in relation to the onset of labour, but the expression of tnfr and tnfr mrna levels were significantly increased with gestation and further increased with the onset of labour. incubation of uterine myocytes with tnf-( ng/ml) activated all three mapk substypes: erk, jnk, and p , activation peaked between - minutes. preliminary data suggest that tnf-induces il- mrna expression but exposure to il- or il- itself did not enhance this response. conclusions: although there is no significant increase in tnf-concentration from baseline during labour we have shown tnf receptor mrna levels do increase with labour at term. exposure of isolated uterine myocytes to tnfcauses activation of all mapk subtypes and an increase in il- mrna expression. this enhanced mapk-dependent il- expression at term may be mediated via increased myometrial sensitivity to tnf-through increased tnf receptor expression. remodeling. brenda c timmons, anna-marie fairhurst, mala s mahendroo. obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa; immunology, ut southwestern medical center, dallas, tx, usa. objective: the molecular mechanisms involved in cervical ripening are not well understood. immunohistochemical studies from our lab report a recruitment of inflammatory cells to the cervical stroma one day before birth in the mouse using a neutrophil/monocyte marker (neutrophil / ). in this study, we sought to identify and quantitate inflammatory cells migrating into the mouse cervix and to determine if this recruitment was affected by changes in progesterone levels. peripheral blood was also evaluated to see if changes in the cervix was paralleled in blood. methods: flow cytometric analysis was performed using cervical cells and peripheral blood obtained before and during cervical ripening along with - h postpartum. dispersion of cervical cells was optimized. these cells were stained with a panel of fluorescent conjugated antibodies directed against leukocyte antigens and analyzed on an lsrii flow cytometer. cells were also sorted and stained to visualize cell morphologies. to determine the effect of progesterone on the migration of leukocytes, gestation d mice were treated for h with a progesterone receptor (pr) antagonist prior to tissue collection. results: neutrophils do not appear to increase in the cervix until after birth. monocyte (mo) numbers do increase during cervical ripening (late day , d . ) and remain high through postpartum (pp). macrophages (mØ) are present prior to cervical ripening and steady state levels are maintained during labor and pp. pr antagonist treatment on d resulted in a premature increase in mo but not neutrophils or mØ. in contrast to the cervix, mo and neutrophil numbers do not significantly increase in the peripheral blood until pp. results from lymphocyte studies suggest a low level of b and t cells in the cervix. in the peripheral blood, b cells remain consistent through parturition and the t cells decrease by d . and continue to decrease pp. conclusion: tissue mo are increased in the cervix during ripening. this recruitment is dependant on loss of pr function. in contrast, neutrophils are increased in the pp cervix while mØ numbers appear constant. timing of changes in mo and neutrophil numbers in the peripheral blood differed from that observed in the cervix suggesting quantitation of these cell types in blood is not reflective of what is occurring in the cervix during ripening, dilation and pp repair. novel interactions between nf-b and other labour-associated transcription factors identified by a tf-tf array. shirin khanjani, yun s lee, suren r sooranna, mark r johnson, phillip r bennett. irdb, imperial college, london, united kingdom. introduction: external stimuli lead to changes in cellular gene expression through activation of inducible transcription factors. nf-b is a ubiquitous transcription factor classically associated with inflammation, which is activated in response to infection and proinflammatory cytokines such as those prevalent during the labour. the tf-tf interaction array uses a novel technology for detecting interactions between transcription factors based on binding of tfs to their own consensus dna binding sequence. materials and methods: primary myometrial cells were grown until - % confluent and stimulated with ng/ml il- prior to nuclear protein extraction. the nuclear extracts were incubated with the provided set of biotin-labeled, double-stranded oligonucleotide probes, which represent a known library of cis-elements. during the incubation step, these tf probes bind to their specific tfs in the nuclear extract. next, immunoprecipitation was performed using an antibody against nf-bp , which pulled out nf-bp and any tfs bound to it, bound to corresponding cis-elements. normal igg was used in a parallel experiment to represent a negative control. free cis-elements and nonspecific binding proteins were washed away and the cis-elements were finally eluted and hybridized to the array membrane, which is spotted with different tf consensus sequences. results: table shows the different tfs interacting with nf-bp based on the degree of binding stimulated by il- compared to no-il- control. conclusion: these data show that il- stimulation causes nf-b to bind to a wide variety of other treansciption factors. of particular interest in the area of parturition is binding to the other pro-inlammatory tfs such as c/ebp and ap- , which are known to regulate labour-associated genes in synergy with nf-b. the lack of association between nf-b and pr without il- stimulation supports the concept that with the onset of labour inflammation leads to functional progesterone withdrawal, rather than pr acting to inhibit inflammation. table high ap- , c/ebp, cbf, creb , c-myb, e f- , ets, ets- /pea ,fast- , gas/isre, hse, mef- , mef- , myc-max, nf- , nfatc, nf-e , nf-e , pax- , objective: pre-partum cervical ripening involves remodeling of collagen structure and inflammatory immune cell activity (jsgi : , ; reprod biol endo : , ) . although parturition is associated with systemic or local progesterone withdrawal (ajog : , ) , effects of a decline in progesterone on cervical ripening is not known. the present study tested the hypothesis that progesterone withdrawal promotes collagen degradation, innervation, and immune cell trafficking in the cervix of nonpregnant mice. methods: adult virgin female c bl mice received capsules (sc) with oil vehicle (v) or estradiol (e) and progesterone (p) to simulate concentrations in pregnancy (hum reprod : , ) . after days, mice in the v and e+p groups were euthanized. the p capsule was removed from some mice on day (e-p) and groups killed on days and . cervix sections were stained for collagen, nerve fibers, macrophages, or neutrophils (n= /group/day; sections/ cervix; biol reprod : , ; jsgi : , ) . stained macrophages and neutrophils were counted (image pro-plus , media cybernetics). results: e+p treatment for days promoted hypertrophy of the cervix compared to v controls, i.e., collagen content and structure diminished, cell nuclei density declined, and nerve fibers increased. removal of p did not affect these endpoints. for immune cells, e+p for , , or days decreased immune cell numbers. by contrast, p removal increased macrophages and neutrophils in the cervix on days and (p< . , e-p vs e+p groups, respectively). the census of resident immune cells in e-p groups at and h after p removal equaled that in the v group. conclusions: mimicking gonadal steroid concentrations in circulation during pregnancy promotes hypertrophy and suppresses immigration of immune cells in the cervix. in this non-pregnant murine model for parturition, progesterone withdrawal recruits immune cells, but fails to promote further remodeling or hyperplasia of nerve fibers in the cervix. the findings raise the possibility that ripening of the cervix requires not only recruitment, but also activation of immune cells. whether proinflammatory activities by specific immune cells affect nerve fiber hypertrophy or neural activity, as part of the mechanism for ripening of the cervix, remains to be determined. we have previously identified and characterized a truncated pr (pr-m), that localizes to the mitochondrion by multiple experimental techniques, including confocal imaging of a recombinant gfp fusion protein, western blot analysis after cellular fractionation of nuclear pr negative t d-y breast cancer cells and western blot analysis of purified human heart mitochondrial proteins. initial studies with nuclear pr negative mcf- a breast epithelial cells shown to express pr-m demonstrated an increase in mitochondrial membrane potential (mmp) with progesterone/progestin treatment. these studies led to the hypothesis that progesterone modulates cellular respiration via the mitochondrial receptor, pr-m. objectives: the present studies sought to further localize pr-m to the outer, inner or matrix portion of the mitochondrion and to correlate the increase in mmp with total cellular atp production. additionally, the potency of progesterone and synthetic progestins on the change in mmp was evaluated. methods: the location of pr-m was determined by western blot analysis after fractionation of human heart mitochondria with digitonin treatment and differential centrifugation. mmp was determined in mcf- a breast epithelial cells and a rhabdomyosarcoma cells by the change in fluorescent emission of jc- . total atp was determined by a bioluminescent assay. results: western blot analysis after mitochondrial fractionation showed pr-m localization exclusively in the outer membrane. a dose-dependent increase in mmp was seen in cell lines with - min treatment with progesterone and r which were inhibited by a specific pr antagonist, rti- - b, and not affected by the translational inhibitor, cycloheximide. similar changes in mmp were seen with the same concentration of progesterone, mpa and r . progesterone/progestin treatment for min led to an increase in total cellular atp without a change in cell number. conclusions: progesterone/progestin treatment results in an increase in cellular respiration in cells expressing an outer mitochondrial membrane pr and known to lack nuclear pr expression. this may represent a mechanism whereby progesterone enhances cellular energy production to meet the demands of pregnancy. introduction pge is a major product of the fetal membranes, decidua and myometrium and plays an important role in cervical ripening and myometrial contractions. there are four pge receptors, ep- and ep- mediate contractions whilst ep- and ep- mediate quiescence. ep- contains multiple consensus sequences for transcription factors known to be of importance in labour, in particular nfkappab. we therefore performed experiments to determine the effect of activation and inhibition of nfkappab upon ep- expression. myocytes plated in well plates were treated with il- ( ng/ml), to activate nfkappab. myometrial cells were also transiently transfected with nfkappab p sigenome smart pool and sicontrol non-targeting sirna to knock down nfkappab p . rna was extracted for amplifying ep- using quantitative rt-pcr with amplification of l as a control to normalise data. il- caused an increase in expression of ep- . knock down of nfkappab using si-rna resulted in a further increase in ep- . this data suggests that although il- stimulates expression of ep- it does so through an nfkappab independent mechanism. the upregulation of ep- with sirna knock down of p suggests that activation of nfkappab would inhibit ep- expression consistent with the concept that activation of nfkappab at term causes the myometrium to adopt a more contractile phenotype. introduction: a role for the pro-inflammatory cytokine il- is suggested in preterm and term birth, independent of the presence of infection. we previously showed that mice with a null mutation in the il- gene (ko) delivered one day later than wild type (wt) mice due at least in part to altered timing of uterine expression of prostaglandin (pg) f receptor, ptgfr, mrna. we also observed differences in mrna expression of other uterine activation proteins (uaps), pg h synthase (pghs)- , oxytocin receptor (otr) and connexin- (cx- ), suggesting multiple physiological effects for il- in term delivery. objective: to examine the effect of il- deficiency on the peri-partal uterine mrna expression of the pge receptors (ep) , and ; the post-partum changes of all uaps; and the relationship of these to serum progesterone (p ) concentrations. methods: gestational length was observed in pregnant c bl/ wt (n= ) and ko (n= ) mice. uap mrna levels were measured by real time rt-pcr in wt and ko dams sacrificed from d through delivery and up to h postdelivery. serum p was determined by ria. data were analyzed by one-way and two-way anova using the holm-sidak test to differentiate treatment effects at p< . . results: birth was delayed in the il- ko mice ( . ± . d vs. . ± . d), and this affected the timing of peri-partal changes for all uaps similarly. both ep and ep (relaxatory receptors) were elevated at d in ko dams, but returned to low levels before delivery and were elevated at delivery (ep ) or afterwards (ep ). ep levels did not change. otr increased several hours before delivery in all dams. cx- increased at delivery, then fell, while pghs- increased at delivery and remained elevated afterwards. ptgfr mrna increased - -fold at delivery and a further - -fold after delivery, suggesting loss of pgf permitted enhanced ptgfr expression. p serum concentrations fell pre-partum in both groups. conclusions: il- ko alters the expression pattern of several pregnancy and parturition-related genes and may delay the pre-partum p fall, suggesting a potential ovarian effect. a uterine role for il- in regulating the timing of normal term parturition cannot be ruled out. objective: recent studies have highlighted the prevalence of vitamin d deficiency in pregnant women, particularly in those from ethnic groups with darker skin who require higher levels of uv light to make parental vitamin d. as the active form of vitamin d, , -dihydroxyvitamin d ( , (oh) d ) is a potent immunomodulator, we postulated that vitamin d deficiency may lead to dysregulated placental immunity. both trophoblast and decidua express the enzyme -hydroxylase (cyp b ) which catalyzes synthesis of , (oh) d from the inactive pro-hormone -hydroxyvitamin d ( ohd ). in view of the role of trophoblast as a barrier site protecting the fetus against infection, we investigated the impact of cyp b , ohd and , (oh) d on innate immune responses in trophoblastic cells. methods: a trophoblast cell line was used to assess the effect of vitamin d on innate immune responses. the cells were treated for hrs with various concentrations of , (oh) d ( - nm) and antimicrobial cathelicidin expression was assessed by real time pcr. to assess whether expression of cyp b was affected by pathogenic stimuli, a cells were treated with ligands for toll-like receptor (tlr) - , cyp b expression (real time pcr) and ohd utilization were assessed. results: a trophoblast cell line; which shows temperature-sensitive differentiation, revealed expression of cyp b with higher levels of enzyme activity under conditions of syncytiotrophoblast development. cells treated for hrs with , (oh) d showed dose-dependent induction of the antimicrobial defensin cathelicidin expression ( . - fold induction), whilst cells treated pro-hormone ohd ( nm) showed . -fold induction of cathelicidin expression. activation of tlr (poly i:c) and tlr (lipopolysaccharide) enhanced the expression of cyp b ( -and . -fold) and increased the sensitivity to ohd as a consequence. conclusion: these data show that autocrine synthesis of , (oh) d from ohd can stimulate trophoblast immune responses in a similar fashion to macrophages. as ohd is the major circulating form of vitamin d, we hypothesize that trophoblast innate immunity may be significantly compromised under conditions of vitamin d deficiency. introduction: secretory leukocyte protease inhibitor (slpi) is a potent -kda protein inhibitor of neutrophil elastase and it is a mediator of mucosal immunity and an inhibitor of nfkb regulated inflammatory responses. however, its source, function and regulation within the uterus during pregnancy and at parturition are not well defined. it has previously been shown to be present in fetal membranes and cervical mucus and in amniotic fluid, where its levels is increased from second trimester to term and with a further increase at parturition. slpi has also been shown to be responsive to progesterone in human epithelial cells. our aim was to determine the effects of il- on slpi gene expression in human myometrium. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in well plates and when cells were - % confluent they were serum starved overnight and incubated with m methyl- -hydroxy-progesterone acetate for h and with or without ng/ml il- for a further hours. at the end of incubations rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n= for ptnl, ptl, tnl and l). copy numbers of slpi, gapdh and beta-actin were measured by qpcr. results: h incubation of uterine myocytes with ng/ml il- caused a marked increase in slpi by % (n= ; p< . ). incubation of uterine myocytes with m progesterone for h also increased slpi by % (n= ; p< . ). incubation with il- for h in the presence of h with progesterone increased slpi: gapdh mrna ratio from . ± . to . ± . (mean ± sem; p< . ). slpi expression was similar in the upper and lower segment myometrium in preterm patients. in term myometrium the slpi: beta-actin mrna ratio was increased by -and -fold in term labour versus term non labour samples in the lower and upper segment respectively (mean ± sem; p< . ). these data show slpi is present in human myometrium and that it is increased by il- . progesterone also increases its expression. its expression is increased in term labour where its anti-bacterial, anti-fungal and anti-viral properties could allow it to act as an endogenous block to infections. objective: pre-b cell colony-enhancing factor (pbef) downstream mapk signaling and transcription factor activation. introduction: pbef is expressed in all layers of the human fetal membranes and the myometrium and is upregulated by labor, infection, nf-kb, ap- and stretching. pbef has the ability to protect a variety of cells from apoptosis, however it also appears to act as an insulin mimetic via the insulin receptor (fukuhara et al. science : ; - ) . its levels are elevated in obese patients, those with type diabetes and in gestational diabetes. because pbef has poorly understood biological activities an investigation of mapk signaling and transcription factor activation induced by pbef has been undertaken in order to gain insights into its mechanisms of action. methods: primary aec were isolated from fetal membranes and treated with rhpbef ( ng/ml) for h, lysed and the resultant proteins labeled with cy or cy (amersham). there were used on a protein microarray containing constituents of the mapk signaling pathways (sigma). isolated aec were transfected with luciferase constructs for nf-kb, ap- , cre, hse and gre response elements using the exgen reagent. the cells were treated with rhpbef ( . , . , , ng/ml) hrs, lysed and ul of sample was analyzed for luciferase activity with dual-luciferase reporter assay system (promega). co-transfection with gfp and sv luciferase constructs to control for transfection efficiency and cell number (respectively) was also performed for each experiment. results: pbef significantly upregulated mapk signaling components including functioning as part of the erk pathways and belonging to p signaling. it also activated nf-kb and camp response elements. however, it significantly down regulated signaling molecules belonging to the jnk pathway that resulted in decreased c-jun phosphorylation. conclusions: pbef signaling in aec is consistent with its anti-apoptotic ability and its upregulation of the pro-inflammatory cytokines. although some mapk components responsive to pbef could be associated with insulin receptor signaling, some may not. therefore, it appears likely that pbef interacts with another potentially unique, but currently unidentified receptor. was the first effector to be characterised, but camp is now known to have other effectors, including camp receptor protein, cyclic nucleotide-gated channels and camp-guanine nucleotide exchange factor/exchange protein directly activated by camp (camp-gef/epac). two epac's have been identified and they consist of functional domains: camp-binding domains, a dep domain, a ras exchange motif and a gef domain. our aim was to determine the presence of epac's in human myometrium and to study the effect of camp responses on prolabour genes such as il- , pghs- , otr and fp. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in well plates until - % confluent. cells were serum starved overnight and incubated with μm methyl progesterone, . mm sodium -bromo-camp, . mm forskolin, μm kt , ng/ml brefeldin a and . mm -pmeopt- 'o-me-camp either alone or in combination for h. rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n= for ptnl, ptl, tnl and l). copy numbers of epac , epac , il- , pghs- , otr, fp, gapdh and -actin were measured by qpcr. results: epac and epac were present in the upper and lower segment of myometrium with epac levels being some -fold higher than those of epac . there was no change with the onset of labour at or before term. treatment with il- for h significantly increased epac (n= ; p< . ) but had no effect on epac . when conditions mimicked pregnancy, (the presence of forskolin and progesterone), brefeldin a, an epac antagonist, increased basal pghs- and fp mrna expression (n= ; p< . ), but had no effect on il- and tended to reduce otr. the il- -induced increase in pghs- and il- , but not fp, was greater with the epac antagonist. conclusions: these data show epac's are present in human myometrium and that camp acts via epacs to reduce pghs- and fp expression during pregnancy. background: inflammatory events have been implicated in the process of labour. glucocorticoids mediate strong anti-inflammatory effects through binding to the glucocorticoid receptor (gr), which on activation translocates to the nucleus and either increases or decreases the expression of responsive genes thereby suppressing inflammation. objective: to characterise the expression profile for gr protein and mrna in human myometrium during fetal maturation and parturition. methods: western immunoblotting (wb) was employed to characterise gr protein expression in first (n= ) and second trimester myometrium (n= ), and in paired upper and lower segment pregnant (non-labouring, p, n= ) and labouring (l, n= ) myometrium; as compared to non-pregnant (np, n= ) control samples. immunofluorescence staining with confocal microscopy and rt-pcr were also undertaken. results: detection of gr protein by wb revealed two bands at - kda, representing the alternatively spliced isoforms, gr-and gr-. densitometric analysis showed that gr levels decreased significantly during pregnancy and remained at very low levels at term and in labour when compared with np samples (p< . ); this decrease was seen for gr-and gr-. no significant temporal variations were observed in gr protein levels at term or during labour. gr protein was localised by immunofluorescence staining to the nuclei and cytoplasm of cells. less intense staining was apparent in p compared to np tissues; consistent with wb data. rt-pcr showed a consistent predominance of gr-to gr-mrna in all the tissues used. the observed decrease in protein expression was also mirrored at the mrna level: gr-mrna levels appeared to gradually decrease throughout gestation (p< . ). differences between the upper and lower myometrial regions were only observed in the labouring samples, where gr-mrna levels were significantly decreased in the lower segment (p< . ). nested pcr was additionally used to amplify gr-, but no consistent pattern of mrna expression was obtained. conclusions: these data are the first to characterise gr expression in human myometrium. spatial and temporal variations have been found with expression evolving through the three trimesters of pregnancy and in labour. further studies are now underway to evaluate whether gr contributes to the sequence of inflammatory events implicated in triggering term and preterm labour. these studies sought to determine the mechanism by which pas modulate the immune response. methods: ) an in vitro co-culture model mixed with human cervical epithelial hela cells and pma-induced human macrophage u cells at epithelial/macrophage cell ratio of : was employed. ) the co-culture was pre-treated with medroxyprogesterone acetate (mpa), progesterone (prog) and dexamethasone (dex) at concentrations of , , and nm for hrs followed by day stimulation of g/ml lipopolysaccharide (lps). these experiments were repeated using hela cells transfected with sirna for glucocorticoid receptor (gr). the production of cytokines il- , il- and il- , il- and tnf were determined using elisas. ) the co-culture was pre-treated with nm of each pas for hours prior to lps stimulation at g/ml for . , . , , , and hours. the phosphorylation of p mapk and gr and the expression of mapk phosphatase (mkp ) were determined by western-blotting. results: il- , il- and il- , il- and tnf were elevated by . fold, . fold, fold, . fold and . fold in the co-culture model in response to lps(p< . for all). pretreatment of mpa and dex, but not prog, inhibited the lps-induced cytokine production. the anti-inflammatory effect of mpa occurs in a dose-dependent manner. in the absence of gr, the inhibitory effect of mpa and dex on il- , but not on il- , was lost. mpa and dex, but not prog, induced the phosphorylation of gr and expression of mkp . p mapk was activated by lps for up to hrs and pretreatment of mpa and dex attenuated this response. the ability of mpa and dex to suppress the inflammatory response in cervical tissues appears to be mediated through a gr-dependent pathway, specifically though p mapk. our studies suggest that prog is not a significant immunomodulator in these tissues. background: progesterone (p ) has been shown to play a critical role in maintaining pregnancy and preventing preterm birth. these effects are likely related to its immunomodulatory properties. we investigated whether camp plays a role in the immunosuppressive action of progesterone on fetal mononuclear cells. methods: umbilical cord blood mononuclear cells were isolated using density gradient centrifugation. to establish a p effect and optimize concentrations, cells were pretreated with p ( - m- - m), dexamethasone ( um) or vehicle for hour prior to overnight lps ( ng/ml) stimulation. supernatants were assayed for tnf-using elisa. ldh assays confirmed the absence of a cytotoxic effect. next, cells were pre-incubated with forskolin (adenylate cyclase activator) or db-camp (camp agonist) prior to lps to determine the effects of camp on tnf production. finally, cells were pretreated with rp-camp (camp antagonist) prior to p incubation and lps stimulation to determine whether the p effect was reversed by a camp antagonist. results: p significantly inhibited lps-induced tnf production in a dose dependent manner, with maximum suppression observed at - m. both forskolin and db-camp suppressed lps-induced tnf production in a doserelated manner (fig ) . finally, a dose-dependent partial reversal of tnf production was observed when cells were pretreated with rp-camp. (fig ) conclusions: progesterone suppresses the inflammatory response in fetal mononuclear cells as measured by tnf expression. this effect is mimicked by adenylate cyclase activators and camp agonists and partially reversed by camp antagonists. this implicates the camp pathway in mediating the immunomodulatory actions of p . objectives estrogen improves endothelial function after vascular injury via largely unknown mechanisms. endothelial progenitor cells (epcs) are known to be implicated in various vascular events requiring endothelialization. we hypothesized that estrogen and progesterone could influence the function and the change of epcs in menstrual cycle. material and methods peripheral blood mononuclear cells (pbmcs) were isolated from peripheral blood of healthy young women in each menstrual period by density gradient centrifugation with ficoll separating solution. pbmcs were seeded in endothelial basal medium with or without estrogen or progesterone. on the th day in culture, nonadherent cells were removed. on the th day in culture, adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. the number of ldl-and lectin-positive cells was measured as epcs using flowcytometry. the expression of estrogen and progesterone receptor mrna in epcs were measured by real time pcr in menstrual and luteal period. the number of epcs was significantly increased in the menstrual and luteal period compared with the follicular phase. estrogen and progesterone significantly increased the number of adherent epcs dose dependently in menstrual period, but not in luteal period. the expression of estrogen-alpha receptor in menstrual period was higher than luteal period. also, estrogen-beta receptor in luteal period was strongly expressed compared with menstrual period. these results suggest the expression of estrogen receptor and progesterone receptor play important roles to regulate epcs' proliferation during menstrual cycle. objective: elevated levels of fetal plasma avp are associated with the development of oligohydramnios, in part a result of avp-mediated reduction in fetal urine production. as avp urinary concentration effects are mediated via upregulation of renal tubular aqp water channels, we propose that avp modulates placental aqp channels, influencing bidirectional maternal-fetal water flow. we sought to study the effect of avp on trophoblast aqp gene expression using the first trimester-derived extravillous htr- /svneo cells and the term placenta-like trophoblast carcinoma cells jeg- . methods: cultures of both cell lines were treated with a physiological concentration of avp ( . nm) to determine aqp mrna and protein expression. negative controls consisted of cells incubated in medium supplemented with % fbs without avp. to determine whether avp regulation of aqp occurs by a camp signaling pathway, jeg- cells were preincubated with μm -(tetrahydro- '-furyl) adenine (sq ), a cell-permeable camp inhibitor, before being treated with avp ( . nm). cells were incubated for hrs for aqp mrna expression and for hrs for protein extraction at °c. after harvest, real time pcr and western blotting analysis were used to detect the aqp mrna and protein expression levels, respectively. results: avp increased aqp mrna expression in both cell lines by . and . fold after hrs. aqp protein expression paralleled the increase seen in the mrna (p< . ). pretreatment of jeg- cells with sq inhibitor completely blocked the stimulatory effect of avp. conclusion: aqp gene expression is up-regulated by avp in first trimester and term trophoblast cells, with a higher induction in the later. avp activation of aqp gene expression occurs via a camp mediated pathway, as the adenyl cyclase inhibitor blocked avp effects on aqp gene expression. these results suggest that increased fetal plasma avp may contribute to oligohydramnios by an increase in aqp-mediated fetal to maternal water flow. objective: changes in expression, ratio and activity of progesterone receptors within human myometrium have been proposed as potential contributory mechanisms for the functional progesterone withdrawal effect which precedes labour. progesterone receptor c (pr-c) is an n terminally truncated isoform of the full length progesterone receptor; pr-c has been shown to be abundant in fetal membranes, placenta and upper segment of laboring myometrium ( , ). the function and regulation of pr-c is at present unclear. in this study we aimed to investigate the regulation of pr-c in cultured human myometrial and amnion-derived wish cells. methods: myometrial primary cell cultures were prepared from non pregnant and term pregnant uteri. both myometrial and wish cell cultures were treated with natural progesterone ( , nm, mm, mm, mm) for , and hrs. western immunoblotting was employed using nuclear and cytoplasmic extracts prepared from treated cells to observe the effect of progesterone on a) expression and b) subcellular localisation of pr-c within both cell types. immunofluorescent staining and confocal microscopy were also employed. experiments were also undertaken whereby nuclear and cytoplasmic extracts were subjected to shrimp alkaline phosphatase treatment to evaluate the phosphorylation status of pr-c in response to hormone. results: data indicates that a kda cytoplasmic protein, representing pr-c is abundant in myometrial and amnion cell cultures. we show that expression of pr-c increases in both cytoplasmic and nuclear fractions within both cell types in response to progesterone. evidence also suggests that pr-c is phosphorylated in a progesterone-independent manner. concurrent treatment with progesterone and the pr-antagonists ru and organon in amnion-derived cells still led to an increased abundance of pr-c but seemed to alter the phosphorylation status of pr-c. conclusion: pr-c expression and subcellular localisation within myometrial and wish cells alters in response to progesterone. speculation is generated about potential role of pr-c in reproductive tissues and its contribution to functional progesterone withdrawal. . taylor smooth muscle responses have been studied, data on regional blood flow (bf)distribution are scarce. we have studied the effect of a single dex course on relative bf distribution within the brain, skeletal muscle, heart, omental fat, placenta and adrenal in fetal sheep at . g, the equivalent of weeks. methods: fetal sheep received amniotic, jugular, carotid and femoral artery and vein catheters at days g (dg). experiments started at dg in dex ( mg) and saline treated controls (ctr), each receiving injections h apart. fetal blood pressure was recorded continuously (windaq pro+). microspheres ( . x total; sterispheres, biopal) were injected at : am into fetal jugular and femoral veins before maternal i.m. injection (t ) and h after the rd injection (t ) in both groups. tissues were collected hours after the th injection for assessment of bf (biopal). results: fetal bp increased above baseline after dex ( . + . mmhg; p= . ) with no change in ctr (- . + . mmhg). regional bf distribution is presented as a percent of the total counts per g tissue (table ) and after normalization within each animal to placentomal flow, shown previously to be unchanged by dex, in table . no significant differences in flow were found. discussion: although dex altered bp, regional distribution of bf among key organs was unaffected. whether gc does not affect regional bf, or the rise in bp is the result of a non-specific, system-wide vasoconstriction, or the impact of gc on bf is similar in all organs, will be subject of further investigation that will include determination of regional vascular resistance and absolute flow. stimulator while crf-r is an inhibitor of gi motility at times of stress. we recently hypothesized that stress-induced in utero meconium passage in fetuses is analogous to stress-induced defecation in adult rats and likely mediated by crf pathway. in support, we demonstrated hypoxia-induced meconium passage in conjunction with marked increases in fetal plasma crf levels. as the mouse is a common experimental model, with known genome, we sought to determine the presence of crf-r and crf-r receptors in mouse fetuses. methods: time-dated cd- pregnant mouse were anaesthetized and fetuses (n= ) were collected at e (term = e ). whole gi tracts were dissected, fixed in bouin's solution, paraffin embedded and sectioned at five micron thickness. sections were immunostained with rabbit polyclonal antibodies to crf-r and crf-r by abc technique with vector abc reagents. immunoreactivity was identified as brown staining using ', diaminobenzamidine as chromagen. slides were counter stained with mayer's hematoxylin and examined under the microscope. immunoreactive signals in the smooth muscle layers of gi tracts were quantified by image analysis using image pro . plus software. results: immunostaining for both crf-r and crf-r was seen in the smooth muscle layers of all lumens examined ( lumens per section). the immunostaining intensity expressed as intensity over density (iod) in arbitrary units for crf-r (maximal iod: . ± . au and minimal iod: . ± . au) and for crf-r (maximal iod: . ± . au and minimal iod: . ± . au) greatly varied between lumens. the mouse fetal gi tract expressed both crf-r and crf-r receptors, suggesting that the mouse is an appropriate model for fetal-stress induced neurovisceral motor responses. the non-uniform distribution of crf receptors in the fetal gi tract suggests the existence of regional differences in the expression patterns of crf-receptors of both types. we speculate that mouse devoid of crf and crf-r or r receptors will be useful to confirm the participation of crf pathway in stress-induced in utero meconium passage. antenatal exposure to glucocorticoids (gc) is associated with a reduction in nephron number and hypertension in adult life. one of the mechanisms for the development of hypertension is thought to be the long term effect of single nephron hyperfiltration. however, in most studies there is only a - % reduction in nephron number with no change in gfr. thus, although a reduction in nephron number may be a contributing factor it is unlikely the only variable. the aim of the present study was to evaluate nephron mass, renal function and blood pressure in a cohort of adult sheep exposed antenatally to betamethasone. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (ctr) -hs apart at days gestational age and allowed to deliver at term. at . yr of age in female (f) and male (m) offspring, glomerular filtration rate (gfr) was measured as inulin clearance and effective renal plasma flow (erpf) as pah clearance. blood pressure was measured though an indwelling catheter in the femoral artery over a -hour period. nephron number was measured by the acid maceration technique. data mean±sem were analyzed by anova and/or two sample t test. results: antenatal bm was associated with an elevation in arterial blood pressure and a reduction in nephron number in both f and m offspring. in contrast, a reduction in gfr and erpf was present only in males. plasma and urinary electrolytes as well as urinary protein excretion were not different from ctr. conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects on blood pressure regulation. interestingly, while the decrease in nephron number was of similar magnitude in m and f, evidence of alterations in renal function was present only in males. these suggest that the decrease in renal function observed in males is not solely a consequence of the decrease in nephron number. furthermore, it is possible that different mechanisms are responsible for the elevation in arterial blood pressure observed in m and f. hl ; hd p hd . clr l>t-x to evaluate the effects on blood pressure, urine output, sodium excretion and gfr of the intrarenal infusion of angiotensin - in the male sheep with or without prenatal exposure to b. we studied male sheep which had received either b (n= ) or vehicle (n= ) at - days gestation and were born at term. catheters were placed in the femoral artery, femoral vein, left renal artery and the bladder. the right kidney was removed. after days recovery the sheep received an infusion of ang - ( ng/kg/min) with or without its antagonist, [d-ala ]-ang-( - )(d-ala, ng/kg/min), into the renal artery for hours. blood pressure, gfr, urine output and sodium excretion were measured before and after the infusion. two-way analysis of variance (anova) was used to test mean values between the groups. with or without d-ala, map and urine output didn't change significantly during ang - infusion. however, the infusion of ang - resulted in a decrease (change from baseline) (p= . ) in na + excretion of . ± . meq/kg/ h in the vehicle animals and . ± . meq/kg/ h in the b animals. there was no significant change in gfr during ang - infusion. gfr was lower in the b group during the combined ang - and d-ala infusion ( . ± . ml/min vs . ± in vehicle group, p= . ). intrarenal infusion of ang - at ng/kg/min is followed by a significant decrease in sodium excretion but no significant change in urine output and gfr. the decrease tended to be greater in the vehicle animals and was not markedly attenuated by the antagonist d-ala. this suggests the effect of ang - may be mediated by a receptor other than the mas receptor for ang - and may be of lesser magnitude in animals exposed to b before birth. and norepinephrine ( . - . g/kg/min) and to an l-name ( mg/kg) bolus were studied. vascular reactivity was analyzed by curve fitting of either the blood pressure or hr responses to obtain the maximal response. data are expressed as mean±sem of change from baseline. statistical significance was established using t test. results: bm-exposed animals displayed a higher blood pressure response to at-ii (atii max ± vs ± % of baseline, p< . , figure left panel). no differences in blood pressure or heart rate responses to norepinephrine infusion were detected in bm when compared to control animals (figure right panel) . maximal response to l-name was also elevated in bm-exposed animals but did not reach statistical significance with the current sample size. conclusion: we have shown that antenatal gc treatment significantly increases blood pressure in sheep. here we show an enhanced in vivo response to at-ii. this response may contribute to the increased blood pressure observed. the greater response to l-name most likely represent an increased no production as a compensatory mechanism to the higher blood pressure observed. hl . women at risk of delivering preterm are frequently treated with glucocorticoids to facilitate fetal lung maturation. animal studies have revealed that prenatal exposure to glucocorticoids may alter aspects of hypothalamic-pituitary adrenal functioning in later life. in this study we examined the effects of clinically relevant prenatal betamethasone treatment on the responsiveness of pituitary cells (in terms of adrenocorticotropin, acth, secretion) isolated from adult sheep. time-dated pregnant sheep were injected with betamethasone ( . mg/kg) or vehicle on days and of gestation. thereafter pregnancy was allowed to continue unimpeded and offspring were born. pituitaries were isolated from adult sheep aged between and months and cells were dispersed and plated at a density × cells per well in well plates. after h, cells were stimulated with normal medium, nm arginine vasopressin (avp), nm avp, nm corticotropin releasing factor (crf) or nm crf for h. medium was collected and analyzed for acth using a commercially available kit. acth secretion (given as % increase from untreated cells) was similar between the control and betamethasone groups following nm avp ( . ± . vs. . ± . ) and nm crf ( . ± . vs. . ± . ). at higher stimulatory concentrations of both avp and crf, acth secretion remained similar in control cells ( . ± . and . ± . respectively), but was significantly decreased in betamethasone cells ( . ± . and . ± . respectively). these findings suggest that prenatal exposure to clinically relevant doses of betamethasone can alter pituitary responsiveness to both avp and crf in adulthood. this research was supported by nih grants hd and hd . lcc is supported by an rjr-leon golberg fellowship in pharmacology and toxicology. objective: newborn meconium (mec) passage normally occurs within the first - h after birth. in contrast, more than half a million infants born annually in the us pass mec in utero. neither the physiological mechanism(s) nor causes for in utero mec passage are well understood, though both fetal maturation and stress are associated. we recently reported in utero mec passage and marked increases in plasma crf levels in term fetal rats exposed to maternal hypoxic stress. we hypothesize that stress-induced in utero mec passage is mediated by the crf pathway in a manner analogous to stress-induced defecation in adult rats. in the present investigation we examined placental crf content prior to and following maternal hypoxia, to determine the source of increased plasma crf. methods: time-dated pregnant rats (n= ) were exposed to a paradigm of graded, stepwise hypoxia on day (term= ) (pediatr. res. : - , ) . control pregnant rats were exposed to % oxygen for a similar duration as experimental animals. at the end of the study, placentas were harvested, fixed in % paraformaldehyde and paraffin embedded. sections (n= per placenta) were subjected to immunohistochemical analyses with rat/human crf antibody (peninsula laboratory) by abc technique. immunoreactivities on placental sections were quantified using the image pro . software and intensity expressed as arbitrary unit (au). all values are mean± sem. results: in control maternal rats exposed to normoxia, positive staining for crf was seen in decidua ( . ± . au) and in all three major placental cells types (giant trophoblast cells: . ± . au, spongiotrophoblast cells: . ± . au and labyrinth cells: . ± . au.) in contrast, in animals exposed to hypoxia, there was a total absence of crf staining in the placental cells, with only positive crf staining seen only in the decidua ( . ± . au). conclusion: the total absence of crf staining in both the basal and labyrinth zones in placentas of pregnant rats subjected to hypoxic stress suggests that placental cells rapidly release crf into the maternal and fetal circulation in response to hypoxic-stress. our findings support our hypothesis that the peripheral crf pathway mediates in utero mec passage. offspring. angela g massmann, jie zhang, jorge p figueroa. department of obstetrics and gynecology, wake forest university school of medicine, winston-salem, nc, usa. objective: in rats and sheep, exposure to glucocorticoids (gc) in the perinatal period induces hypertension in adult life. recent evidence suggests that endothelin b (etb) receptor plays an important role in the regulation of sodium balance and blood pressure. renal medulla is an important site of expression and action of etb receptor. furthermore, in kidney it is the medulla (km) where the highest concentrations of immunoreactive et- and etb receptor are found. the aim of this study was to measure eta and etb expression in adult sheep kidney exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (v) hours apart at days of gestational age and allowed to deliver at term. at . yr of age, male and female offspring exposed to either vehicle or bm were euthanized and the kidneys harvested. kidney medulla (km) was obtained by sharp dissection. eta and etb protein and mrna expression were evaluated by western blot and rnaase protection assay. data are expressed as mean±sem and were analyzed by two way anova. results: a significant decrease in etb (f= . , p= . ) but not eta protein was observed in km of bm exposed male and female adult sheep. at the mrna level, bm exposure also affected etb, but not, eta expression. however, bm treated animals had higher mrna levels than control sheep (f= . ; p= . ). conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects. the activation of etb receptor by et- inhibits sodium transport function in the collecting duct and the selective gene deletion of the etb in medulla results in hypertension.therefore, our finding of a significant reduction in etb protein suggests that there may be an impairment in the kidney's ability to regulate sodium reabsorption. the functional relevance of the alterations in etb protein expression as well as the discrepancies in mrna regulation need to be established. hl ; hd p hd . in the placenta, -hydroxysteroid dehydrogenase type ( hsd ) regulates the transfer of cortisol. maternal glucocorticoid (gc) administration has been shown to affect the ovine fetal growth trajectory and regulate hsd expression in mid and late gestation. however, little is know about the effects of gc administration in early gestation. we hypothesized that maternally administered low-dose dexamethasone (dex) in early gestation would affect hsd expression, which will subsequently alter fetal birth weight. pregnant ewes were randomized and received intramuscular injections consisting of either saline ( ml saline/ewe) or dex ( . mg/kg) given hours apart starting on days of gestation (dg). the animals were sacrificed at , , , and dg and fetal weights were recorded and placental tissue was collected. qrt-pcr was used to measure hsd placental mrna expression. statistical analysis was done by anova with student's t-test posthoc. overall, there was no significant effect of dex treatment on placental hsd mrna expression across gestation. however at dg, there was a significant increase in hsd expression after dex (p= . ). there was an increase in placental hsd mrna progressively from dg (mean = . ) to dg (mean = . ), independent of treatment. overall, a positive correlation between hsd and fetal weight (r = . ) was apparent at dg and at dg (p= . ) in both control (p= . ) and dex (p= . ). significant positive correlations between placental hsd and fetal weight (r = . ) were found in females (p= . ) and a similar trend was found in male fetuses (p= . ). we conclude that in the sheep placenta, hsd expression increases throughout gestation and may respond, at least transiently in early gestation, to elevations in maternal gc. the positive correlation between hsd and fetal weight is consistent with the thesis that the fetal growth trajectory may be influenced by maternal cortisol levels and that placental hsd is an important mediator of these effects. antenatal gc exposure induces hypertension in the young adult rat (neuroendocrinol; ; : ) and increases femoral vascular resistance in the lamb (jphysiol; ; : ) . unpublished own examinations in preparation of this study have shown age dependency of the vascular reactivity. vascular contractility of the middle cerebral artery (mca) decreased in response to k + and noradrenaline (na) between mo and , y of age (p< . ). vascular relaxation to acetylcholine (ach) but not to pge decreased during the same time (p< . ). vascular contractility of the renal artery (ra) increased in response to k + (p< . ). aims: to examine if antenatal gc alter vascular reactivity in the aged individual when cardiovascular diseases are predominant. we studied the ra because its vascular tone is involved in modulation of arterial pressure control (amjphysiol; ; :r ) and the mca because depressive disorders that are associated with dysregulation of the hpa axis (jcem; ; : ) increase stroke mortality for unknown reasons (amjpsychiatry; ; : ) . to be clinically relevant, we administered dexamethasone at the dose used to enhance lung maturation in babies threaten premature labor. methods: pregnant dams received saline (n= ) or μg/kg dexamethasone (n= ) i.p. at day e / equivalent to x mg dexamethasone administered to a kg pregnant woman. at . years of age, vascular response of the ra and mca to endothelium-dependent and independent mediators was measured using wire myography. vessels were inspected histologically for intact endothelium. results: basal vasoconstrictory and dilatory responses to all mediators were less pronounced in the mca than in the ra probably reflecting autoregulatory properties of cerebral vessels (p< . ). after prenatal dexamethasone exposure, contractility but not sensitivity to k + and na was enhanced in the mca and even more pronounced in the ra (p< . ). relaxation to ach and pge was similarly diminished in both vessels after precontraction with na (p< . ). conclusions: prenatal dexamethasone exposure at the dose used clinically increases renal and cerebral vascular tone in the aged rat by endotheliumdependent and independent mechanisms. this effect may be a potential mechanism of fetal programming of cardiovascular diseases in later life. betamethasone (bmz) therapy during pregnancy to improve fetal lung maturity may result in long term side effects, including hypertension, during adulthood. the kidney is an important organ which regulates systemic blood pressure via the local renin angiotensin system (ras). the major enzymes of the intrarenal ras include angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin. the hypothesis of this study is that prenatal bmz exposure will result in alterations of the enzymes regulating the intrarenal ras. specifically, sheep that are treated with bmz in utero will have higher amounts of ace activity in kidney cortex compared to control animals. pregnant sheep of known mating date were either treated with vehicle (n= ) or with two doses of bmz (n= ), . mg/kg, hours apart at days of gestation. renal cortex from the male offspring was harvested at - months of age. cortical membranes were then solubilized and incubated at °c with either i-ang i or i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln to inhibit ace activity, or sch to inhibit neprilysin activity. the metabolic products were separated by reversephase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p< . was considered significant. results: bmz treatment resulted in a significant increase in ace activity compared to control animals (p= . ). ace and neprilysin levels were not significantly different between treatment groups. when expressed as a ratio of ace/ace activity, bmz treated animals exhibited a . fold greater proportion of ace activity versus control animals (p= . ). this study demonstrates that bmz exposure during fetal life results in a significant increase in renal ace activity during adult life. this increase in enzyme activity would be expected to be associated with increased levels of ang ii in the kidney and na + retention, possibly underlying the development of hypertension. hd , hd . we recently hypothesized that stress-induced in utero meconium passage in term fetuses utilizes peripheral and/or central crf pathways in a manner analogous to stress-induced defecation in adult rats. as participation of central crf pathway requires the crf circuitry system in brain-gut axis, we exploited immunohistochemical techniques to address whether term fetal colon is wired by crf-nerve fibers. methods: fetal distal colon segments (n= ) were dissected from term ovine fetuses ( - d gestation). in addition, lumbosacral spinal cord with spinal roots and dorsal root ganglia (drg) (n= ) were dissected from ovine fetuses, and vagal nerve trunk with nodose ganglia (n= ) were dissected from mouse fetuses. paraffin sections fixed either in bouin's solution or zamboni's were subjected to immunohistochemistry with rabbit polyclonal antibodies specific to ovine-crf (ocrf). immunoreactivity on the sections was identified by standard abc technique, immunostaining quantified by image-pro plus software and expressed (intensity over area) as arbitrary units (au). results: ocrf antibody elicited strong positive staining on the serosal surface (port of entry for extrinsic nerve fibers) in distal colonic segments objective: we hypothesize that stress-induced in utero meconium passage is mediated by the crf pathway. ucn-i, similar to crf, is a potent contractility inhibitor of preterm ovine fetal distal colon, which has abundant crf-r receptors. both ucn- and crf thus may inhibit colonic motility and prevent preterm in utero meconium passage via crf-r receptors. however, ucn-i is reported to stimulate contractility of adult rat colonic smooth muscle strips more strongly than crf. we sought to study the pattern of ucn-i innervation in distal colon of ovine fetuses to delineate whether ucn- functions as a facilitator or inhibitor of colonic propulsive motility. methods: bouin's solution fixed, paraffin sections of very preterm (vpt: - days gestation), preterm (pt: - days), near term (nt: - ) and term (t: - days) ovine fetal distal colon rings were subjected to immunohistochemistry with polyclonal antibodies to human ucn- ( : sigma) by abc system. intensity of ucn- immunostaining in smooth muscle layers and myenteric neurons were quantified by image-pro plus software and expressed (intensity/area) in arbitrary units (au). differences over time were assessed with anova. in all groups very preterm was significantly greater than term (p< . ). conclusion: ucn- immunoreactivity in all three muscle layers, as well as myenteric and submucosal neurons, is highest at very preterm as compared to more mature gestations. these results suggest that the reduction of ucn- inhibitory function may facilitate stress-induced meconium passage at term. to evaluate the effect on blood pressure (bp), urinary output (uop), sodium excretion (nae) and gfr of the intrarenal infusion of ang ii in the male sheep after prenatal exposure to b. methods: we studied male sheep which had received either b (n= ) or vehicle (n= ) at - days gestation. catheters were placed in the femoral artery and vein, left renal artery and in the bladder. a right side nephrectomy was performed. after days of recovery, the sheep were infused with ang ii ( ng/kg/min) with or without candesartan ( ng/kg/day) or pd (pd g loading dose and ng/kg/min infusion) into the renal artery over hours. bp, gfr, uop and nae were measured before and after the infusion. two-way analysis of variance was used to test mean values between the groups. results: bp and uop did not change with ang ii infusion with or without candesartan. the infusion of pd did not affect uop but did increase bp in the b sheep (p= . ). ang ii decreased the nae in both groups(p= . ). candesartan increased nae among controls(controls . and b . meq/kg/h, p= . ). pd infusion decreased nae among controls but this was not significant. baseline gfr was higher among vehicle compared to b animals (p= . ). during ang ii infusion there was a decrease in gfr among control compared to b sheep (- . vs. - . ml/min, p= . ). this difference was not seen during candesartan or pd infusion. conclusion: ang ii infusion led to a decrease in nae in both groups and gfr among controls but not b animals. infusion of ang ii with candesartan increased nae while pd decreased nae among controls but not b animals. this suggests that prenatal steroid exposure can alter at receptor mediated response in renal function by decreasing the effect of ang ii on renal sodium excretion. the differences in gfr suggest that this may be due to an effect on tubular sodium reabsorption rather than glomerular perfusion. antalarmin antagonism of acth-induced cortisol secretion by ovine adrenal cells probably not at acth receptor. nancy k valego, james c rose. center of research for ob/gyn, wake forest u. school of medicine, winston-salem, nc, usa. in addition to regulating the hpa axis, crh and its type receptor (crhr- ) occur peripherally and in the female reproductive system. late in human pregnancy, a surge in placental crh probably stimulates adrenal secretion of cortisol and dheas requisite to parturition. we previously reported that, in dispersed fetal or adult ovine adrenal cortical cells, hour incubation with the specific crh-r antagonist, antalarmin (ant), significantly reduced both cyclicamp and cortisol responses to acth, suggesting an interaction with acth-r (like crh-r , a g-protein-coupled membrane receptor). however, forskolin (fsk; direct stimulant of adenylyl cyclase)-stimulated cortisol secretion was also attenuated by hour co-incubation with ant. our objective was to clarify the effect of ant on binding of acth to its receptor after a short ( . hours) treatment. method: acth binding: the adrenals from adult sheep were obtained at necropsy and the cortex cells dispersed and plated @ cells/well. after hours in culture, wells were rinsed x and refilled with serum-free dmem/ f containing vehicle (dmso) with or without ant. after . hours, wells were washed very gently and the binding assay (adapted from rainey et al; j biol chem, : , ) completed. triplicate vehicle or ant wells were treated with i -tyrosine human acth ( - ) with or without - m acth (for non-specific binding). after hour, wells were chilled, washed, and the cells lysed and counted on a gamma counter. fsk treatment: cells were treated as above except that fsk ( - m) was added to the wells. after . hours, medium was removed and stored @ - ºc for camp eia and cortisol ria. results (mean±se) of . hour incubation on acth binding and fskinduced secretion. vehicle antalarmin acth binding (net cpm) n= ± ± cortisol (ng/ml/ . hr) n= ± ± * (p=. ) camp (pmol/ml/ . hr) n= . ± . . ± . * (p=. ) * indicates significant difference from vehicle alone. conclusion: the crh-r antagonist, antalarmin, attenuates acthstimulated cortisol secretion but not by preventing acth receptor binding. however, its effect is early in the secretory process and is associated with a reduced camp response. objective glucocorticoids are often administered to pregnant women to prevent neonatal respiratory distress syndrome. prenatal steroid treatment increases blood pressure in adult sheep. exposure to excess corticosteroids before birth is hypothesized to be a key mechanism underlying the fetal origins of adult disease hypothesis and effects on the renin-angiotension system (ras) may modulate the steroid-induced increases in blood pressure. we therefore sought to determine if renin processing and secretion were altered in adult female sheep exposed antenatally to betamethasone ( ) and to compare them with data from males studied previously. methods pregnant sheep were randomized to receive doses of . mg/kg of or vehicle, at and days of gestation; the offspring were studied at and months of age. in female offspring, active renin concentration (arc) and total renin concentration in plasma were measured by ria of angiotensin i generated by incubation with excess substrate. prorenin concentration (prc) is the difference between total and active renin. nine or control exposed female animals born at term ( - days of gestation) were brought from the farm at - months of age, and had vascular and bladder catheters placed. five days after surgery, a sodium load of hypertonic nacl ( . meq/kg/min at . ml/min) was given for min. blood samples were obtained. data are expressed as mean sem and were analyzed by t test. the prc was significantly higher in the females than in the males ( . ± . vs . ± . p< . ) but there was no effect of prenatal steroid treatment. arc was similar in both genders. however, arc was a significantly greater percent of the total plasma renin concentration in the males ( . ± . vs . ± . p< . ) during the na infusion experiment, the exposed females had lower arc than did the control females ( . ± . vs . ± . p< . ). conclusion the data suggest that prenatal exposure to didn't alter the processing and secretion of renin in adult female sheep. prenatal steroid treatment does not appear to alter the effect of gender on plasma renin levels in adult sheep.it seems unlikely that the elevated blood pressure seen in adult ewes after prenatal exposure is the result of increased secretion or processing of renin. supported by hd and hd . background: mrap is a recently discovered protein with alpha and beta isoforms, a common amino terminal region and divergent c-terminal sequences generated by alternative splicing. in human and mouse mrap plays an essential role in the generation of a functional, g-protein coupled acth receptor (melanocortin- receptor; mc r) but has not been described for ruminants. we previously reported that lth fetal sheep exhibited elevated basal acth - yet decreased expression of key steroidogenic enzymes and the mc r in the adrenal cortex while basal cortisol levels were not different from control. we hypothesized that mrap could play a key role in mediating the effect of lth as well as developmental regulation of fetal adrenal cortisol synthesis in fetal sheep. the goals of the present study were to determine the ontogenic pattern of mrap expression in the sheep fetus and to determine if lth alters mrap expression. methods: we searched the bovine genomic sequence database (www.ncbi.nlm. nih.gov/genome/guide/cow/) and found a sequence with > % homology to the human mrap n-terminal residues, with partial homology to the beta isoform in the carboxyl region ( %). using primers based on the bovine sequence, we confirmed the presence of mrap in the ovine fetal adrenal cortex. adrenal cortical tissue was collected from sheep fetuses from - (n= ) and near term ( - ; n= ) days of gestation (dg) as well as from lth (n= ) fetuses (exposed to high altitude hypoxia from to - dg) and age-matched normoxic controls (n= ). cyclophilin was used as a housekeeping mrna. data are expressed in fg mrna/ ng total rna. results: the expression of mrap, based on quantitative rt-pcr, was low between - dg ( . ± . ) and increased (p< . ) approx. -fold near term ( - dg; . ± . ). levels of mrna for mrap were highly correlated to cyp mrna in individual samples. mrap expression in control ( . ± . ) and lth ( . ± . ) did not differ between groups. gender differences in hypertension are well described and there is growing evidence that the regulation of the renin angiotensin system (ras) is influenced by sex hormones. the major enzymes of the ras include angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin. the peptide products of these enzymes have opposing actions. angiotensin ii (ang ii), the product of ace, is a potent vasoconstrictor. on the other hand, the peptide products of ace and neprilysin, ang ( - ) and ang ( - ), exhibit vasodilatory properties. thus, modifications in the relative proportions of these enzymes and their peptide products can result in alterations of systemic blood pressure. the purpose of this study was to describe the gender differences in angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin (nep) enzyme activities in adult sheep kidney cortex. renal cortex from male (n= ) and female (n= ) sheep were harvested at - months of age. the tissue membranes were solubilized and incubated at °c with either i-ang i or i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln to inhibit ace activity, or sch to inhibit neprilysin activity. the metabolic products were then separated by reverse-phase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was then quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p< . was considered significant. results ace activity was over times greater ( ± . vs. . ± . fmol/mg/min, p< . ), ace activity was . times greater ( ± . vs. . ± . fmol/mg/min, p= . ) and nep activity was nearly times greater ( . ± . vs. . ± . fmol/mg/min, p= . ) in female kidney cortex. in adult sheep, key enzymes of the intrarenal ras have signficantly greater activity in female kidney cortex. these findings suggest that there are fundamental, gender specific, differences in the regulation of the enzymes of the intrarenal ras. the physiologic significance of these findings remain to be elucidated. hd , hd . in rats and sheep exposure to glucocorticoids (gc) in the perinatal period is associated with a reduction in nephron number and hypertension in adult life. furthermore, antenatal exposure to gc alters glucose tolerance in animals and in people. the aim of the present study was to determine ) if insulin resistance is a contributing factor for the development of hypertension in adult sheep exposed antenatally to gc and ) if diet-induced obesity has a more pronounced effect in sheep exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (ctr) -hours apart at days gestational age and allowed to deliver at term. at mo of age, female sheep were randomly allocated to be fed at either % of recommended nutritional allowance or ad libitum for three months. sheep were chronically instrumented under general anesthesia to place intravascular catheters. insulin sensitivity was evaluated both by iv glucose tolerance test (ivgtt) and euglycemic clamp (hec)techniques. for the ivgtt a . g/kg glucose bolus was used and for the hec mu/kg human insulin was used. data mean±sem were analyzed by anova and/or two sample t test. results: ad lib fed sheep gain > % of the original weight. antenatal bm was associated with an elevation in basal and ivgtt plasma insulin values. as shown on the figure, diet-induced obesity significantly increased insulin plasma levels during ivgtt in bm-exposed adult female sheep (f= . ;p< . ). insulin sensitivity derived from hec was significantly decreased by obesity in bm-exposed adult female sheep. conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects on glucose metabolism regulation. bm-exposed sheep exhibit alterations in glucose tolerance, hyperinsulinemia and insulin resistance. these abnormalities are exagertated by diet-induced obesity. hl . syngergistic induction of -hydroxysteroid deyhdrogenase type expression by cortisol and interleukin- in human fetal lung fibroblasts. z yang, p zhu, cm guo, l myatt, k sun. school of life sciences, fudan university, shanghai, china; obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. objectives: glucocorticoids acting via glucocorticoid receptor (gr), serve as crucial hormones in fetal lung maturation. glucocorticoids and proinflammatory cytokines to induce b-hydroxysteroid dehydrogenase type ( b-hsd ) which converts inactive cortisone to active cortisol, but their effect on b-hsd expression has not been addressed in human fetal lung. we examined the interactions and mechanism of cortisol and interleukin- b (il- b) effect on b-hsd in human fetal lung fibroblasts (hfl- cells). methods: the expression of b-hsd in hfl- was examined with immunocytochemistry and pcr. b-hsd , prostaglandin h synthase- (pghs- ) and cytosolic phospholipase a a (cpla ) mrna levels in cultured human fetal lung fibroblasts treated with cortisol and il- b were measured with real time pcr. the roles of gr and c/ebps in the effect of cortisol and il- b were studied using a gr antagonist (ru ) and transfection of plasmid carrying c/ebp-specific dominant-negative gene (cmv -a/cebp). results: hfl- cells expressed a high level of b-hsd . both cortisol ( - - - m) and il- b ( . - ng/ml) induced b-hsd mrna expression in a concentration-dependent manner, an effect blocked by the mrna transcription inhibitor , -dichlorobenzimidazole riboside ( μm). ru ( - m) blocked the induction of b-hsd by cortisol. induction of b-hsd mrna expression by cortisol ( - m) was synergistically increased by co-treatment with il- b ( . - ng/ml) in a concentration-dependent manner. in contrast, the induction of cpla and pghs- expression by il- b was inhibited by cortisol, suggesting a different mechanism of interaction. transfection of the cells with c/ebp-specific dominant-negative plasmid attenuated induction of b-hsd mrna expression by either cortisol or il- b. these data suggest induction of b-hsd expression by cortisol is a gr dependent process involving c/ebps, which also mediate induction of b-hsd expression by il- b. conclusions: cortisol and il- b synergistically induce b-hsd expression in human fetal lung fibroblasts. this would lead to greater local cortisol production, perhaps providing either a self-attenuating mechanism for control of inflammation or a mechanism for enhancing fetal lung maturation when the fetus is exposed to cytokines e.g. with infection-induced preterm labor. do alterations in placental -hydroxysteroid dehydrogenase ( hsd) activities explain differences in fetal hypothalamic pituitary adrenal function following periconceptional undernutrition or twinning in sheep? kl connor, pl van zijl, cw rumball, , al jaquiery, , je harding, , mh oliver, , fh bloomfield, , jrg challis. medicine, university of toronto. periconceptional undernutrition (pcun) leads to activation of fetal hypothalamic pituitary adrenal (hpa) function, whereas twinning results in delayed fetal hpa activation. we hypothesized that these differences in fetal hpa activity were the result of altered patterns of expression of placental of hsd isozymes and hence of the maternal glucocorticoid (gc) effect on the fetus. we developed a mass spectrometric assay for the measurement of hsd- and - activities and validated this method against a widely used thin layer chromatography method. sheep were randomly assigned to ad libitum (n control) concentrates throughout gestation or were undernourished (un) from days before until days after mating to reduce maternal body weight by %, with ad libitum feeding thereafter. placentomes were collected on days , , , and of gestation (term, d) and hsd- and - activities were determined by measuring the rate of interconversion between cortisone to cortisol. with un, there was a trend towards lower hsd- activity at d (p= . ), a significant reduction at d (p< . ), but no difference at d or d . hsd- activity was not different between n and un animals at any time. there was no effect of twinning on hsd- or - at d . however, with twins both hsd- (p= . ) and - (p< . ) activities increased at d . hsd- activity was reduced in twins (p= . ) at d but was higher than any singleton pregnancies at d (p= . ). hsd- was not different between singletons and twins at either d or d . overall, hsd- was lower in placentae of male compared to female fetuses in late gestation; hsd- was higher in male than female fetuses. there was no interaction with un in either sex. we conclude that pcun and twinning result in alterations of placental hsd activities in sheep. modifications in these enzymes during critical periods of fetal development may affect transplacental transfer or placental generation of gcs reaching the fetus potentially influencing the timing of activation of the fetal hpa axis, fetal maturation and later life development. methods: pregnant sprague-dawley rats had % mfr from day of gestation until delivery. control animals had ad libitum food. offspring were sacrificed on day of life (p ) and at months (n= - per group). adrenals were dissected and snap frozen in liquid nitrogen for later extraction of rna. real time rt-pcr using specific rat primers was used to quantify mrna levels ( s as control). we evaluated the expression of -beta hydroxylase (cyp b ), aldosterone synthase (cyp b ), acth receptor (mcr ), p side chain cleavage enzyme (cyp a ), star protein, -hydroxysteroid dehydrogenase type (hsd ) and type (hsd ), glucocorticoid receptor (gr), and mineralocorticoid receptor (nr c ). fold changes in mrna expression in controls and mfr offspring was compared by student's t-test. results: there was a marked downregulation in expression of cyp b (p=. ), cyp b (p=. ), hsd (p=. ), p (p=. ), acth receptor (p=. ), star (p=. ) and nr c (p=. ) mrna in p mfr offspring, with no changes in hsd and gr. gender specific differences were found in the adult mfr offspring. in the male mfr offspring the expression of hsd and gr were significantly upregulated with a trend towards an increase in acth receptor (p=. ) whereas in the female mfr the expression of acth receptor (p=. ) was increased and nr c (p=. ) and cyp b were decreased (p=. ). in combined data for adult male and female offspring the expression of acth receptor was significantly (p=. ) increased. conclusion: these results indicate that mfr has a suppressive effect on steroidogenic enzymes of the newborn offspring regardless of gender. this may be an adaptive mechanism in the fetus/newborn to offset the high circulating maternal glucocorticoids in response to undernutrition. in adult male mfr offspring, increased hsd indicates an increase in gc synthesis whereas in the females there were no changes in gc synthesizing enzymes with a suppression of mc synthesizing pathway. the common finding of an increased acth receptor expression in both genders would suggest an increased sensitivity of adult mfr offspring adrenals to the effects of stress. objective: during gestation the fetal adrenal undergoes phases of active growth ( - d), quiescence ( - d) followed by reactivation (> d) before birth. insulin like growth factors play an important role in stimulating adrenal growth throughout late gestation. interestingly the prepartum activation of the adrenal is delayed in the female compared with the male fetus, but the mechanisms underlying this delay are unknown. hypothesis: we hypothesize that there are gender specific differences in the gestational profile of adrenal igf mrna expression between male and female fetuses. methods: a total of twin fetuses were used in this study. post mortem was performed at either - d, - d or - d gestation. adrenal mrna expression of igf , igf , igf r, igf r and cyp was determined by qrt-pcr. results: fetal weight was not different between males and females, and increased (p< . ) with increasing gestational age. the relative adrenal weight was lower however after d when compared to the earlier gestation age group (p< . ). adrenal igf mrna expression was lower (p< . ) at - d when compared to the earlier gestation age groups. interestingly, igf r expression was highest at - d (p< . ). there was an interaction between the effects of age and gender on adrenal igf and igf r mrna expression such that the expression was higher in males compared to females at - d (p< . ), but was not different to either the earlier or later gestational ages. there was no effect of either gender or gestational age on adrenal cyp mrna expression. conclusions: it has been speculated that a delay in prepartum activation of the adrenal in female fetuses may be due to gender specific differences in the intra-adrenal bioavailability of igf . in this study we have demonstrated there is an increase in both igf and igf r mrna expression in males compared to female fetuses. this may be evidence that adrenal igf expression plays a role in the earlier activation of the adrenal gland in male fetuses. we have previously shown that in response to a secondary stressor, in vivo cortisol secretion is elevated in long term hypoxic (lth) ovine fetus despite lower acth receptor mrna expression, and no differences in plasma adrenocorticotropic hormone (acth) levels when compared to normoxic controls. the present study was designed to determine the potential mechanism(s) of this enhanced cortisol secretion. specifically we tested the hypothesis that post receptor signaling events including camp production and expression of steroidogenic acute regulatory protein (star) are enhanced following lth. methods: for the lth group, pregnant sheep were maintained at high altitude ( , m) from day to near term. on days - (term = days), fetal adrenal glands were collected from lth (n= ) and age-matched, normoxic signal transduction pathways that control embryogenesis, cell differentiation, cell proliferation and cell death. the roles of extracellular signal-regulated kinase / (erk / ) and p map kinase in the differentiation and invasion of human trophoblasts have been studied. however, the in vivo expression and activation of erk / and p at the placental bed has not been elucidated. the study group consisted of placental bed biopsy tissues obtained from the pregnancies without preeclampsia (n= ) and with preeclampsia (n= ) between and weeks of gestation. we evaluated the expressions and phosphorylations of erk / and p map kinase in the invasive trophoblasts in the placental bed tissues using immunohistochemistry. results: p and phospho-p map kinase were not detected in invasive trophoblasts in cases or controls. erk / and phospho-erk / were positive in invasive trophoblasts albeit with variable staining. phosphorylation of erk / was significantly less frequent in invasive trophoblasts in placental bed biopsies from women with preeclampsia compared with normotensive controls. conclusion: these findings suggest that preeclampsia is associated with decreased activation of erk / in invasive trophoblasts in vivo. objective: placentae from preeclamptic pregnancies are characterized by an excess of immature hyperproliferative trophoblast cells, however the molecular mechanisms regulating cell cycle progression in this pathology are unclear. our aim was to examine the expression of g phase cell cycle regulators in normal and preeclamptic placentae and to establish whether the hyperproliferative state of trophoblast cells in preeclampsia may result from a developmental delay. methods: human placental samples were collected from normal pregnancies throughout gestation (n= ) and from severe early onset preeclamptic (n= ), and age-matched control placentae (n= ). protein expression of cyclin e , cyclin d , cyclin d and cell cycle inhibitors, p , p , p , and p was assessed by western blot analysis. spatial and temporal localization of cyclins e , d , d , p and p was determined by fluorescence immunohistochemistry. expression of cyclin e and cyclin d mrna was evaluated by qpcr analysis. results: immunohistochemical analysis showed cyclin e and cyclin d to be localized to cytotrophoblast (ct) cells of the chorionic villi; additionally cyclin e was also expressed in the extravillous trophoblast cells of the anchoring columns. in contrast, cyclin d expression was predominantly restricted to the villous stroma. cell cycle inhibitor p was expressed in both ct and syncytiotrophoblast (st) cells whereas p was restricted to the st. during normal placentation, levels of both cyclin e and cyclin d were high in the first trimester and decreased with advancing gestation. the expression of cyclin d , p and p showed an inverse correlation to cyclins e and d whereby their expression increased towards term. levels of p and p remained constant throughout pregnancy. preeclamptic placentae showed a significant increase in both cyclin e and p and a decrease cyclin d and p expression, as compared to age-matched control tissues. interestingly, preeclampsia was associated with an increased number of cyclin e positive progenitor ct cells in the floating villi and an increased expression of p in the endothelial cells lining the villous vessels. conclusion: preeclampsia displays an altered expression of g phase cell cycle regulatory molecules portraying an expression profile that closely resembles that of first trimester placentae. (supported by cihr, owh/igh). we have also demonstrated elevated, hif- -mediated placental and circulating sflt- at high altitude. we sought to correlate circulating levels of plgf, free vegf and sflt- with maternal and fetal oxygen tensions. we hypothesized that circulating sflt- and free vegf would be increased at m, and that plgf, known to be up-regulated by higher oxygen tension, would be decreased. methods: we collected both serum and plasma samples, the latter treated with inhibitors of platelet activation. maternal and umbilical samples were obtained from and healthy mother-infant pairs living at m and m respectively. plgf, free vegf and sflt- were measured in both serum and plasma using commercially available elisa kits (r d). data were log-transformed and analyzed by unpaired student's t test or anova as appropriate. results: plgf did not differ between altitudes. free vegf ( ± pg/ml vs. ± pg/ml, p<. ) and sflt- ( . ± . vs. . ± . ng/ml) were increased at m. however concentrations of all the angiogenic growth factors were reduced when platelet activation was prevented (- ± % plgf; - ± % free vegf; - ± % sflt- , p<. ). cord blood free vegf was -fold greater than in the mothers, but inhibition of peripheral cell activation abolished detectable levels in % of the babies. similar results were obtained for sflt- . thus, while free and total maternal circulating vegf and sflt- are elevated at high altitude, these increases are due to activation of peripheral blood cells. moreover, free vegf does not exist in detectable amounts in human pregnancy. there was no relationship between variation in the angiogenic growth factors and maternal or fetal oxygen tensions. the objective of this study is to identify candidate genes responsible for the variance between severe preeclampsia (spe) and hellp syndrome (hs). placental biopsies from spe (n= ) and hs (n= ) were collected. diagnosis of spe was confirmed by blood pressure and protein criteria. hs was diagnosed in preeclamptic patients who developed characteristic laboratory abnormalities. placental tissues were embedded in oct for sectioning, h e staining and rna isolation (invitrogen, carlsbad). gene expression data was obtained by hybridizing fluorescently labeled reverse transcription products to spotted cdna microarrays. the microarrays were produced by the laboratory of microarray technology at the van andel institute (grand rapids, mi) using a custom microarrayer. microarrays were scanned using an agilent g b scanner. images were analyzed using genepix . (axon). limmagui was used to generate lists of discriminating genes for these data, while david provided functional annotations. there were differentially expressed genes between spe and hs (p<. ). among these candidate genes, were up-regulated and were downregulated. the most up-regulated genes are ( )-deoxyribonucleotidase (dnt- ), superoxide dismutase , hydroxy-delta- -steroid dehydrogenase, caspase , and general transcription factor iih. further analysis of functional groups revealed the most enriched gene categories are related to cellular energy (mitochondria), cell cycle regulation, and protein metabolism. the underlying role of the placenta in the development of hs is currently unknown. this study shows that there is a relatively mdest number of genes that are differentially expressed between hs versus spe placentals. thes data provide the molecular context for placental changes seen in this variant of preeclampsia and provides an opportunity to study the role of the effected genes in disease variance. ( ), severe preeclampsia ( ), hellp syndrome ( ) and eclampsia ( ) and control group ( patients) uncomplicated pregnancies. total rna was isolated and reversely transcribed to c-dna. the mrna level of tert was detected with a probe-specific real-time quantitative pcr assay using ß-actin as the reference gene. crossing point (cp) values were obtained during the pcr amplification and the relative expression level of htert equals to (cp htert -cp ß-actin) . statistical analysis was performed using the student's t test. immunohistochemistry (ihc) staining was employed to localize htert protein on placenta tissue sections using abc method, incubation with rabbit anti-htert antibody followed by application of a goat anti-rabbit antibody with results evaluation using microscope. objective tgf s are involved in the regulation of trophoblast differentiation and invasion, and we have previously reported that tgf- is over expressed in pre-eclamptic placentae. tgf s signal via a receptor complex composed of type ii (t r-ii) and type i (t r-i) receptor. to date, seven type i receptors, designated as activin receptor-like kinase (alk - ) have been identified. our aim was to investigate the expression pattern of alk and alk receptors, known to exert tgf signaling, in preeclamptic and iugr placentae. methods human placental tissue throughout gestation was used in order to determine the development profile of the receptors. in addition, placental tissue from preeclamptic and iugr pregnancies and from age matched controls was collected. all iugr pregnancies were characterized by absence of end diastolic velocity in the umbilical artery and had no evidence of preeclampsia. expression of alk and alk mrna was measured by real-time pcr analysis, and protein by western blot analysis using alk and alk antibodies. immunoblot analysis demonstrated a unique developmental profile whereby alk expression increased with advancing gestation. in preeclamptic placentae alk expression was significantly increased compared to preterm and term controls, whereas alk expression was significantly decreased. preeclamptic placentae also exhibited decreased phosphorylation of smad , a tgf signaling molecule, which is activated by alk and increased phosphorylation of smad , which is triggered by alk . the expression of alk and alk in iugr placentae differed from that of preeclampsia as both alk and alk mrna levels were significantly increased in iugr compared to preterm and term controls. however, only alk expression was significantly increased in iugr placentae at the protein level, while no differences in alk protein levels were noted between iugr and controls. conclusions imbalance between alk and alk signaling pathways might play a role in the pathogenesis of preeclampsia and iugr. as tgf signaling via either alk or alk has been found to differentially regulate vasculogenesis, changes in these signaling pathways may contribute to the altered vasculogenesis found in these pregnancy-related disorders. (supported by cihr and owh/igh). ( ), severe preeclampsia ( ), hellp syndrome ( ) and eclampsia ( ) and patients, the control group who had uncomplicated pregnancies. total protein was isolated from placental tissue. gadd a and its downstream signal proteins--phospo-p , phospho-mkk /mkk were assessed by western blot. immunohistochemistry (ihc) staining was employed to localize the expression of gadd a and sflt- proteins in placenta tissue sections using abc method. huvec cells were cultured to - % confluence and were divided into groups: control, stress induction (sorbitol, . m, h), p inhibition (sb- , ug/ml, ug/ml and ug/ml, hour) + sorbitol ( . m, h). total protein was isolated from cells and the supernatant of huvec was collected. western blot was processed to detect the induction of gadd a and phospho-p . supernatant sflt- was measured with an eia kit and the results were read at nm wavelength. results: gadd a protein was elevated in the preeclamptic placentas with its downstream proteins (mkk and p ) activation compared with control. overexpression of gadd a and sflt- in preeclamptic placentas was observed with ihc staining. in huvec cells, gadd a was induced by sorbitol, triggering the activation of the downstream p- pathway and the accumulation of sflt- in the supernatant. the up-regulation of sflt- secretion by inducing gadd a was depleted when treated with p- inhibitor. conclusions: our study reveals that gadd a and its down stream p- pathway were activated in preeclamptic placentas and this stress inducible signal pathway regulates the secretion of sflt- , which is a key player in preeclampsia. it provides novel evidence that links placental stress to sflt- secretion via the gadd . trophoblast adipose triglyceride lipase (atgl) expression is upregulated in preeclampsia. beth a plunkett, jennifer a doll, emily j su, serdar e bulun, mona cornwell, susan e crawford. obstetrics and gynecology, northwestern university, chicago, il, usa; pathology, northwestern university, chicago, il, usa. objective: preeclampsia is characterized by placental endothelial cell dysfunction and elevated maternal triglyceridemia (tg). tg traverse the placenta in a process of uptake followed by lipolysis with subsequent release of fatty acids to the fetus. although three placental lipases (hormone sensitive lipase, endothelial lipase and lipoprotein lipase) have been identified, they do not account for all lipolytic activity. here, we introduce a new lipase, adipose triglyceride lipase (atgl), which is responsible for the hydrolysis of triglycerides to diglycerides in adipocytes and was recently identified as a receptor for endothelial cell modulator pigment epithelium-derived factor. the purpose of this study is to determine if expression of atgl, a potential modulator of both lipid metabolism and vasculature, is upregulated in preeclampsia. methods: immunohistochemical studies were performed on placental tissues from normal pregnancies (n= ) and those complicated by severe preeclampsia (n= ) with anti-atgl antibodies. the degree of positivity in the trophoblasts and endothelial cell was scored ( =none, =spotty, light, =consistent, dark). to determine if atgl is a product of placental endothelial cells (plec), microvascular cells were isolated from normal placental tissue. purity of the sample was confirmed using flowcytometry (> / positivity for factor antigen). atgl was detected immunohistochemically and via western blot using anti-atgl antibodies. results: mean atgl expression in preeclamptic trophoblast was significantly higher than normal placentas ( . + standard deviation versus . + . , p = . ). endothelial expression was not significantly different in preeclamptic ( . + . ) versus normal placentas ( . + . , p> . ). atgl stained intensely and demonstrated a beaded pattern in the endothelial cells, suggestive of a lipid droplet pattern. immunohistochemistry of plec and western blot analysis of cell lysates revealed strong immunopositivity for atgl, although at a smaller size than anticipated. conclusion: these findings demonstrate that a novel lipase, atgl, is produced by trophoblasts and is upregulated in severe preeclampsia. plec express high levels of atgl, suggesting that atgl could prove to be important in the vascular dysfunction and lipid abnormalities characteristic of preeclampsia. increased endothelial chymotrypsin-like protease (chymase) expression is responsible for endothelial activation in preeclampsia. yuping wang, yang gu, yanping zhang, david f lewis. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: endothelial (ec) activation is an important component of inflammatory phenotypic changes in preeclampsia (pe). our previous study showed enhanced chymotrypsin-like protease (clp)/chymase expression in the maternal vessel endothelium in women with pe. in this study, specific effect of placental-derived clp on ec activation was examined. methods: human uterine microvascular endothelial cells (utmvecs) were used. placental conditioned medium (cm) was prepared by culturing villous explants from normal and pe placentas. confluent utmvecs were treated with placental cm with or without depletion of chymotrypsin. ec adhesion molecule expressions for icam, vcam, p-selectin and e-selectin were determined by a colorimetric assay at od nm. depletion of chymotrypsin from cm was performed by immunoprecipitation. to further determine if activation of endogenous clp/chymase in ecs is responsible for up-regulation of p-selectin and e-selectin expression, chymase sirna was applied to ec culture before the cells were treated with normal or pe cm and then ec adhesion molecule expressions were examined. data was expressed as mean ± se and analyzed by anova. a p level < . was set for statistically different. results: ) expressions of vcam, p-selectin and e-selectin, but not icam, were significantly increased in pe-cm treated utmvecs compared to those of normal-cm treated cells and untreated controls, p< . ; ) there was no difference for adhesion molecule expression in utmvecs between normal-cm treated with untreated controls; ) utmvecs transfected with chymase sirna significantly reduced p-selectin and e-selectin expressions when exposed to pe-cm, p< . . conclusion: placental-derived clp/chymase is responsible for activating ecs and inducing ec adhesion molecule expression. activation of ec chymase may be directly related to the inflammatory phenotypic changes that occur in ecs in pe. (supported nih grants hd and hl ). corin is a transmembrane serine protease that is important in processing natriuretic peptides (nps) and maintaining normal blood pressure. genetically modified mice without corin function develop a syndrome during pregnancy similar to preeclampsia. corin is present in the pregnant uterus and a deficiency in the enzyme may lead to hypertension and preeclampsia. we tested the hypothesis that corin expression was increased in human myometrium from women with preeclampsia. methods: myometrium was obtained from groups of women at the time of primary cesarean section: (i) preterm no labor with preeclampsia (n= , ptspe, . weeks); (ii) preterm labor (n= , ptl, . weeks); (iv) term no labor (n= , tnl, . weeks); (v) term labor (n= , tl, . weeks). microarray gene profiling was performed using affymetrix human genome u plus . arrays. women who were not in active labor at the time of delivery (tnl) served as the control group. conventional and real time pcr was performed to verify corin expression in the arrays was directionally accurate. corin protein expression was examined by western blot. results: compared to tnl control, corin levels decreased nearly -fold in myometrium from women in term labor (tl). there was a small increase in corin gene expression of preeclamptic women (ptspe) and little-to-no change in myometrium from women in preterm labor (ptl). these results were confirmed by pcr analysis. conclusion: blood volume surges during pregnancy, increasing the potential for hypertension and preeclampsia in the mother. the corin-np control system could contribute to this condition. however, there are no reports on corin message or protein levels in women with preeclampsia. our preliminary results suggest that preeclampsia is not a consequence of a corin deficiency (decreased corin preeclampsia). acknowlegement: supported by grants from the phs (r hl - , cpw) and cdc grant (u dp - , cpw). the expression of gp at implantation site: implication for the pathogenesis of preeclampsia. objective: gp is a common shard signal transducing subunit of the il- cytokine family which is critical for implantation, and viewed as marker of endometrial blastocyst receptivity. preeclampsia (pe) is highly related to the restricted trophoblast invasion, which leads to impaired spiral artery remodeling. our hypothesis was that the poor placentation of pe is associated with the altered expression of gp at the implantation site. methods: human decidua from patients with pe and uncomplicated term deliveries (n = , respectively) were immunostained for gp . the intensity and distribution of immunostaining on decidual cells and extravillous traphoblasts were evaluated with hscore. statistical analysis of the data was performed using student's t-test and kruskal-wallis one way anova on ranks followed by post hoc test. results: immunostaining of gp was significantly higher in decidual cells of patients with pe compared with normal specimens, with hscore (median, [interquartile range]) value [ . - . ] and , respectively (p< . ). in pe specimen, the hscore of decidual cells was also significantly higher than that of trophoblast ( [ - ]; p< . ). there were no difference in the comparison of hscore of trophoblasts between the pe and normal specimens, and in normal specimens between decidal cells and trophoblasts. conclusions: the increase of gp expression in the decidual cells of preeclamptic placenta may be implied to the pathogenesis of poor placentation in preeclampsia. we have previously demonstrated that decidual cells are the major source of renin at the human uteroplacental interface, but little is known regarding the human decidual renin expression in hypoxic conditions. therefore, the present study was undertaken to determine the effect of hypoxia on renin secretion by human decidual cells in vitro. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for days in a serum-containing medium, the decidual cells were exposed to normoxia or hypoxia ( % or % oxygen) in a serum-free medium for hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. a dominant band of renin at approximately kd was detected in all samples. when compared with the cells cultured in the normoxic condition, the cells cultured in both hypoxic conditions (i.e. % and % oxygen) had significantly lower renin protein contents in their culture supernatants. conclusion: our data for the first time show that hypoxia down-regulates renin secretion in human decidua, suggesting a link between the uteroplacental ras and oxygen tension during human gestation. the effect of nucleated fetal red blood cells derived from preeclamptic patients on endothelial progenitor cell proliferation. keiichi matsubara, emiko abe, yuko matsubara, shinji hyodo, masaharu ito. obstetrics and gynecology, ehime university school of medicine, toon, ehime, japan. objectives inadequate uteroplacental circulation results in placental ischemia and the development of preeclampsia (pe). endothelial progenitor cells (epcs) are thought to be a key player in the fetal angiogenesis. vascular endothelial growth factor (vegf), which is up regulated in pe, is involved in epcs proliferation. recently, it was reported that fetal nucleated red blood cells (nrbcs) have the capability to generate vegf. we hypothesized that nrbcs could influence epcs proliferation in the placenta and may be involved in the pathogenesis of pe. material and methods mononuclear cells (mncs) were isolated from the umbilical venous blood of normal pregnant women and preeclamptic patients by density gradient centrifugation. mncs were incubated with anti-cd antibody conjugated with microbeads. nrbcs were collected using a mini macs separator. nrbcs were incubated for hours with or without angiotensin ii (ang ii) and erythropoietin (epo). vegf and placental growth factor (plgf) concentrations in the supernatant were measured using elisa. also, pbmcs without nrbcs were seeded in endothelial basal medium with or without nrbcs using a boyden chamber. these samples were incubated for days with or without ang ii and epo. the adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. di-ldl and lectin positive cells was considered to represent epcs and the number was measured using flowcytometry. the number of nrbcs derived from umbilical venous blood was significantly increased in pe. both ang ii and epo significantly increased vegf concentration in the supernatant of nrbcs derived from normal pregnant women. however, ang ii and epo did not influence the nrbcs' vegf production in pe patients. plgf was not detectable in the supernatant. the number of epcs in the umbilical venous blood was significantly decreased in pe and the number was not changed by nrbcs. on the other hand, the number of epcs was significantly decreased in the culture with nrbcs. epo significantly increased the number of epcs in pe at the lower concentration of epo compared with normal pregnant women. conclusions it appears that fetal nrbcs may inhibit fetal epcs proliferation in pe. since epo reduced the inhibitory reaction of nrbcs without vegf production epo may affect epcs proliferation independently of nrbcs. relationship between the hypoxia-inducible factor- (hif- ) and the receptor svegf-r /sflt- : implication for phatophysiology of preeclampsia. julio e valdivia-silva, , juan c gonzalez-altamirano, keisy lopez- the trophoblast invasion is critical for the establishment of the uteroplacental circulation. at early phases of this process local oxygen pressure in the placenta is lower, that pathologically in preeclampsia remain constant. because of this, is important to understand the response of placental cells against these stimuli. in the present work, we use primary cultures of trophoblast cells, fibroblasts of the villous, and human umbilical endothelial cells, isolated of preterm and term placentas (with and without preeclampsia), to explore the effect of the oxygen pressure in the expression and synthesis of vegf, svegfr- /sflt- , hif- and- . our results show that the low pressure of oxygen resulted in a significant increase of the mrna and the protein of the receptor svegf-r selectively in the cts. the vegf's expression and synthesis was raised in three cellular types, but the free protein (not bounded to svegf-r ) of the cts was diminished. on the other hand, the expression of the arnm of hif- or - in cells was comparable in all the types of placentas, nevertheless, the protein hif- was more increased in the cts of preeclamptic placentas. to evaluate the relation of hif- and the increase of the receptor svegfr- , we used sirna-hif . in response to the inhibition, the expression of the receptor svegf-r diminished dramatically. the blockade of hif- did not alter vegf's expression. our data are the first that propose that the protein of the factor of transcription hif- is one of the molecules involved in the selective expression of the receptor svegf-r in trophoblast cells during hypoxia. figure: reduction of the expression to soluble receptor flt- /svegfr- after transfection with sirna-hif in trophoblast cells. sirna= interference rna, lu=luciferase. luc-sirna was used as control. effects of estradiol on synthesis, secretion, and activation of von willebrand factor in endometrial endothelial cell. shumei zhao, chainarong choksuchat, michael s scholfield, todd d deutch, thomas d kimble, david f archer. obstetrics and gynecology, eastern virginia medical school, norfolk, va, usa. objectives: heavy menstrual bleeding (hmb) is a serious clinical condition affecting % of women. due to inefficient and ineffective medical interventions, women with hmb often elect endometrial ablation or hysterectomy to eliminate hmb symptoms. morbidity and loss of fertility linked to these surgical treatments support the search for more effective medical remedies. von willebrand factor (vwf), a principle initiator of blood clotting produced by endothelial cells. women with von willebrand disease (vwd), have a high incidence of hmb indicating poor clotting. these findings suggest vwf heavily impacts the amount of blood loss during menstruation. estrogen stops/reduces hmb and has been used to treat hmb in women with vwd, although the mechanism(s) is unknown. this proposal addresses the hypothesis that estradiol (e ) increases the synthesis and activation of vwf, promoting clotting. materials and methods: immortalized human endometrial endothelia cells (heecs) were used for the studies. to determine if e increases synthesis of vwf, we treated heecs with e at . μm, . μm and . μm for hours. vwf protein and mrna levels were determined by western blotting and real time pcr, respectively. to establish if e can convert vwf from an inactive to an active conformation, the release of activated vwf by cells into culture medium will be assessed by elisa. decreased adamts (a disintegrin and metalloproteinase with thrombospondin type motif) activity results in increased amounts of active vwf. rrelease of activated adamts will be determined by fret. to ascertain if e regulated vwf secretion is by genomic pathway, heecs will be exposed to the estrogen receptor antagonist ici , . elisa and fret will assess the release of active vwf and adamts , respectively. results: western blotting demonstrated e increases vwf mrna and protein levels in a dose-dependent manner in heecs.conclusion: the project will determine if e acts at the heecs to increase synthesis, secretion and activation of vwf. preliminary results show e increases intracellular vwf protein and mrna levels in heecs, supporting our hypothesis that e increases synthesis of vwf in heecs in vitro. if e increases activated vwf, it could reduce/stop hmb by increasing activated vwf, providing justification for a clinical trial of e to treat hmb. over-expression of vegf-d does not induce lymphangiogenesis in the mouse endometrium. peter a rogers, jacqui f donoghue, marc g achen, steven a stacker, jane e girling. obstetrics gynaecology, monash university, melbourne, victoria, australia; ludwig institute for cancer research, melbourne, victoria, australia. background: the human endometrial functionalis has reduced lymphatics compared to the basalis and myometrium . this study examines the distribution of lymphatics in mouse uterus and investigates if over-expression of the lymphangiogenic growth factor vascular endothelial growth factor-d (vegf-d) stimulates growth of new endometrial lymphatic vessels. methods: the distribution of uterine lymphatics was examined in c bl/ jxcba mice collected during the oestrus cycle, early pregnancy and following oestrogen and progesterone treatment. human ebna cells with/without stable transfection of vegf-d were injected into the uterine horn of nod/scid mice. uteri were collected after weeks. serial sections were immunostained with lyve- and/or vegfr (lymphatic endothelial cell markers), cd (blood endothelial cell marker), mab (human vegf-d), mab (human mitochondria) and pcna (proliferative cell nuclear antigen). results: lymphatic vessel profiles were mostly found in the connective tissue between the longitudinal and circular muscle layers of the myometrium. they were rare in the endometrium and only observed in % of the sections. when present in endometrium, lymphatic vessel profiles were usually situated adjacent to the endometrial/myometrial border. ebna tumours formed inside and outside the uterine horn of both the control (n= of ) and vegf-d group (n= of ). localization of ebna cells within the mouse uterus was confirmed by anti-human mitochondrial expression. vegf-d immunostaining confirmed that transfected ebna cells expressed vegf-d in vivo. over-expression of vegf-d did not stimulate endometrial lymphangiogenesis, although there was an increase in vessel diameter of lymphatics in the myometrium adjacent to tumours. initial analysis shows no significant effect of vegf-d ebna cells on endometrial blood vascular density or endothelial cell proliferation. conclusions: minimal lymphatics are present in the mouse endometrium, as is the case for lymphatic vessels in the human endometrial functionalis. the lack of endometrial lymphangiogenesis in response to vegf-d suggests the presence of an inhibitory factor limiting lymphatic growth in this tissue. in early pregnancy, decidual-derived vegf mediates angiogenesis and is required for implantation and placentation. the role of decidual vegf in later pregnancy is poorly understood. decidual hemorrhage (placental abruption) generates excess thrombin and is a major risk factor for pprom and preterm birth. this study compares immunohistochemical (ihc) localization of vegf in decidual tissue sections from term pregnancies complicated by abruption and gestational age-matched controls, and investigates the effect of thrombin on vegf expression by cultured human term decidual stromal cells (dscs). study design: ihc was performed on serial sections of term placental tissues (no labor) with (n= ) and without (n= ) abruption. purified term dscs were passaged until > % free of cd + cells by facs. confluent dscs were primed with - m estradiol (e ), - m medroxyprogesterone acetate (mpa), both, or vehicle for days. after h incubation in defined medium with corresponding steroids ± thrombin ( . - . iu/ml), conditioned supernatants were analyzed for vegf by elisa. extracted total rna was used to assess vegf mrna levels by quantitative rt-pcr using established primers. results: vegf expression was localized by ihc primarily to dscs in placental tissue sections, and was increased in tissues from placental abruption vs controls. in term dscs, thrombin increased vegf secretion in a dosedependent fashion irrespective of the hormonal milieu (eg, . -fold stimulation by . iu/ml thrombin from . ± . to . ± . pg/ml per mcg protein for e +mpa; p= . ). this effect was abrogated by the thrombin inactivator, hirudin. vegf mrna were similarly increased by thrombin with or without steroid hormones (eg, . -fold for e +mpa; p< . ). conclusions: placental abruption is associated with increased vegf expression in term decidual tissues in vivo with thrombin enhancing vegf mrna and protein expression in term dscs in vitro. excess thrombinmediated vegf expression in term decidua aberrantly increases endothelial cell permeability to further generate thrombin by continuous exposure of tissue factor-expressing decidual cells to circulating factor vii. thrombin-enhanced matrix metalloproteinase expression in term dscs would degrade decidual and fetal membrane extracellular matrix to induce pprom and preterm birth. basal directional release of angiotensin ii by endothelial cells stimulated by chymotrypsin-like protease (clp)/chymase. yuping wang, david f lewis, yang gu. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: chymotrypsin-like protease (clp)/chymase is a serine protease which plays a major role in angiotensin ii (ang ii) generation in the human heart. our previous study showed a higher clp activity in the maternal plasma in women with pe than in normal pregnancies. we also found enhanced chymase expression in the maternal vessel endothelium in women with pe. in this study, we determined if clp could promote endothelial cell (ec) generation of ang ii. methods: we specifically examined basal directional release of ang ii by cultured ecs. ecs were grown on cell culture insert ( well/plate, micron pore size). when ecs reached confluence, chymotrypsin (chy) at concentrations of . , . , . , . , and . g/ml were added to the upper chamber of the cell insert. after hours of culture, medium in the lower chamber was collected. medium concentrations of ang ii were measured by enzymelinked immunoassay (eia). all samples were measured in duplicate. data are expressed as mean ± se and analyzed by anova. a p level < . was considered statistically different. results: chymotrypsin produced a concentration-dependent increase in basal directional release of ang ii by cultured ecs, control: . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml (p< . ); chy . : . ± . g/ml (p< . ), respectively. data are means from independent experiments. conclusion: apical exposure of ecs with chymotrypsin-like protease could promote basal directional release of ang ii. our result implicates that in pe, elevated clp levels in the maternal circulation are very likely to affect ec generation of ang ii. basal directional released ang ii may bind to its receptor on underlying vascular smooth muscle cells and contribute to the increased vasoconstriction in pe. (supported nih grants hd and hl ). vegf stimulates angiogenesis and vasodilation critical for dramatic rises in materno-feto interface blood flows directly linked to fetal growth/survival. extracellular signal-regulated kinase (erk / ) pathway mediates partially vegf-induced angiogenic and vasodilatory responses in placental endothelial cells (ec). it is, however, unknown how this vegf-induced signaling is organized in placental ec. objectives: ovine fetoplacental artery ec (ofpaec) and its transformed counterpart, sv -of to test whether: ) vegf-activated erk / signaling is compartmentalized in the caveolae and disruption of caveolae interferes vegf-induced erk / activation and; ) caveolin- , the structure protein of caveolae, regulates vegf-stimulated erk / phosphorylation. methods: ofpaec or sv -of cells were cultured in mcdb- / % fbs/antibiotics. serum-starved subconfluent ( %) cells were treated with rhvegf ( to ng/ml) for various times. caveolae were disrupted by -cyclodextrin ( -cd, mm, min) or caveolin- scaffolding domain (cav-sd, m, hr). sv -of cells were used for fractionation of caveolae membranes by discontinuous sucrose gradient ( %/ %/ %) ultracentrifugation. activation of erk / signaling pathway were analyzed by western-blotting with specific antibodies. results: in total cell extracts, vegf stimulates erk / phosphorylation in a time-and dose-dependent manner. erk / phosphorylation maximized by vegf ( ng/ml) at - min, which was abrogated by -cd or cav-sd. all the molecules for compromising the erk / signaling module, plc , pkc , src, ras, raf- , mek / and erk / , were detectable in purified caveolae membranes positive for various markers including caveolin- , enos, flotillin- , and -adaptin. in caveolae, vegf dramatically increased phosphorylated erk / without altering total erk / in a time-dependent manner similar to that in total cell extracts, which also maximized at - min. pretreatment with -cd or cav-sd blocked vegf stimulation of erk / phosphorylation in caveolae. conclusion: vegf activates the erk / signaling pathway in caveolae and caveolae integrity is essential for vegf-activated erk / signaling pathway. we conclude that caveolae/caveolin- serves as a platform for compartmentalizing the vegf-induced erk / signaling pathway in placental ec (hl and hl ). hypoxia upregulates gcm in human placenta. david mccaig, fiona lyall. institute of medical genetics, university of glasgow, glasgow, united kingdom. introduction: studies in transgenic mice have shown that a variety of genes regulate the differentiation of trophoblast cells. these genes include gcm . gcm is also expressed in the human placenta. placental gcm- protein has been reported to be reduced in pre-eclampsia, in view of the close link between hypoxia, hypoxia-reoxygneation, pre-eclampsia, placental development and the reported reduction in gcm we hypothesised that gcm expression would be affected by hypoxia. aim: the aim of this study was to determine the effects of hypoxia on gcm expression in the human placenta. two model systems were used; ( ) free floating villous explants and ( ) cultured primary cytotrophoblast and syncytiotrophoblast cells as described previously*. methods: explants or cell cultures were exposed to either hypoxia or hypoxia followed by re-oxygenation. western blot analysis was used to assess gcm protein levels. bands on the gels were quantified using scanning densitometry. statistical differences (n= experiments for both models used) were calulated by anova and turkey's post-hoc test. results: gcm protein was detectable at a low level in villous explants maintained for h in % o . a striking increase in gcm protein was observed when villous explants were incubated for h in % o (p< . ). incubation of villous explants for h in % o followed by re-oxygenation for h in % o resulted in a marked decline in gcm protein (p< . ). expression of gcm was also analysed in primary cytotrophoblast and syncytiotrophoblast cultured in % o or reduced oxygen ( % o ) conditions. gcm protein was not detected in any of the experimental conditions used. discussion: the present study has shown that acute hypoxia increases gcm- protein in villous explants. the experiments with purified trophoblast do not support a role for hypoxia increasing gcm- in these cells under the experimental conditions used. the present findings are in keeping with the complex effects of oxygen depending on the conditions used. the observed hypoxic effects on gcm warrant further investigation. the effect of acute alcohol exposure on histone -lys modification in the mid-gestation embryonic lung. xiangyuan wang, debra wolgemuth, , , laxmi baxi. ob/gyn; genetics development; human nutrition, columbia university medical center, new york, ny, usa. objective maternal alcohol abuse during pregnancy produces an array of birth defects comprising fetal alcohol syndrome. lung development depends on a balance between cell proliferation and apoptosis. we have previously shown that the acute alcohol exposure in the mid-gestation embryo can delay lung development and induce apoptosis. acute exposure to ethanol of selected tissues in mouse embryos has been reported by others to initiate apoptosis within hours after exposure and result in histone modifications. specifically, histone acetylation and deacetylation are involved in transcriptional activation and repression, respectively, but can also involve apoptosis. in the present study, we have investigated the effect of alcohol on acetylation of histone at lysine (ach lys ) in the mid-gestation embryonic lung. pregnant c bl/ j mice at day . of gestation (e . ) were injected intraperitoneally with doses of % ethanol ( . g/kg ), hr apart (alcoholexposed: ae) or with ringers solution (controls: c). ae and five c fetuses were retrieved and hr later and the lungs were fixed and processed for morphological evaluation and staining with rabbit polyclonal anti-ach ly antibody. the entire lung tissue field was evaluated for the levels of ach lys staining and scored as (-) to (++++). three areas were selected randomly from each sample and the total number of cells and staining positive cells were counted in the bronchial epithelium and in the mesenchyme. twelve hr after alcohol exposure at e . , the morphology of ae embryonic lungs was normal. however, high levels of ach lys were detected in % of the bronchial epithelial cells and % of the mesenchymal cells. the expression level in both lineages decreased hr after alcohol exposure. in the controls, the expression of ach lys was virtually undetectable in both the bronchial epithelium and the mesenchymal cells. our previous study showed that ae e . lungs significantly increased apoptotic cell in both bronchial epithelium and mesenchyme hours after alcohol treatment. we now observe that elevated expression of ach lys in the embryonic lung preceded the observation of apoptosis, suggesting that alteration in the acetylation of h could be one of the molecular mechanisms involved in the induction of apoptosis following acute alcohol exposure. objective: our purpose was to evaluate the effect of vitamin c and e supplementation on lipid peroxide levels, total antioxidant ability, and antioxidant levels in the umbilical venous plasma. materials and methods: women at risk for preeclampsia (nullipara, previous preeclampsia, chronic hypertension) were recruited at to weeksgestation and randomly assigned to receive either mg of vitamin c and iu of vitamin e (study group, n= ) or placebo (control group, n= ) daily until delivery. umbilical venous blood were collected after full term delivery. lipid peroxide levels, oxygen-radical absorbance capacity (orac) values, antioxidant levels were measured by each method (thiobarbituric acid reaction, cao's method, and high performance liquid chromatography). results: . the lipid peroxide levels in the umbilical venous plasma of study group were significantly lower than that of control group ( . ± . vs. . ± . nmol/mg protein, p< . ). . the orac values in the umbilical venous plasma of study group were significantly higher than that of control group ( . ± . vs. . ± . u/ml, p< . ). . the -tocopherol levels in the umbilical venous plasma of study group were significantly higher than that of control group ( . ± . vs. . ± . nmol/ml, p< . ). . there were no significant differences in the ascorbic acid, uric acid, -carotene, retinol, and -tocopherol levels in the umbilical venous plasma between control and study group. conclusion: this suggested that maternal vitamin c and e supplementation may affect the oxidant-antioxidants balance of the utero-placenta unit and fetus. placental tnf-related apoptosis-inducing ligand (trail) in normal pregnancy and pre-eclampsia. xilian bai, jenny e myers, philip n baker, john d aplin, ian p crocker. maternal and fetal health research group, the university of manchester. introduction: enhanced placental trophoblast apoptosis is well known to occur in pre-eclampsia. however, the potential role of membrane associated or soluble trail, an apoptosis-inducing ligand, and its death receptor, dr , is not known. we tested the hypotheses that the trail/trail-receptor system is compromised in pre-eclampsia and that soluble trail is a circulating factor which triggers the vascular complications of pre-eclampsia. method: this study was conducted on placental samples and plasma (edta) from women with uncomplicated pregnancies (n= ) and with pre-eclampsia (n= ) at - weeks gestation. protein expression levels and tissue localisation of trail and dr were defined in villous tissue by western blotting and immunohistochemistry. soluble trail (strail) was measured in maternal plasma from both groups using a commercial elisa (diaclone). results: placental villous trail and dr protein was unaltered in preeclampsia compared to normal pregnancy. whilst there was differential distribution of trail and dr within the component cells, this also was unaffected in pre-eclampsia. within the villi, trail was mainly confined to the cytoplasm and perinuclear regions of cytotrophoblast, syncytiotrophoblast, stromal cells and the fetal capillary endothelium. conversely, dr was restricted to trophoblast only, distributed evenly between cytoplasm, plasma membranes and nucleus. strail was present in plasma from non-pregnant women of childbearing age [ . ( . - . ) , median (interquartile range), pg/ml, n= ]. however, there was no significant increase either in pregnancy [ . ( . - . ) pg/ml, n= ] or pre-eclampsia [ . ( . - . ) pg/ ml, n= ] (kruskal-wallis test, p> . ). conclusions: these results suggest that placental villous trail is not adversely regulated in pre-eclampsia. the absence of dr in stromal and endothelial cells may increase resistance to apoptotic stimuli in cells of the villous core. the co-localisation of trail and dr in trophoblast suggests a role in autocrine regulation of cell turnover in this cell type. introduction: preeclampsia is associated with apoptosis of the syncytial layer of placenta and the release of particulate and soluble factors which are deleterious to maternal endothelial function. the goal of the current study was to determine whether laser capture microdissection (lcmd) and western blotting could be used to assess levels of syncytial fas ligand (fasl), a key protein in the apoptotic cascade. methods: frozen sections of term placenta delivered from uncomplicated pregnancies at term were used for study (n= ). following staining with mayer's hematoxylin (n= ), lcmd (leica instruments) of an intact terminal villus was carried out using a focused laser pulse directed to the area of interest using a microscope. in the first round of microdissection the placental villus core consisting of fibroblast, macrophages, fetal vessels, and connective tissue, were removed. in the second round of lcmd, the syncytial layer of that same villus was removed and collected in lysis buffer containing detergent and protease inhibitors, and the number of nuclei per sample was recorded. this procedure was repeated until syncytial tissue from - villi were collected. electrophoretic separation of lysate proteins was then carried out, and western blotting and immunodetection using an anti-fasl antibody was performed. results: we observed that fasl was detected in microdissected syncytial specimens at a molecular weight of approximately kda ( figure) , consistent with our previous reports. it is of note, that western blotting of samples containing approximately (lane ), (lane ), (lane ), and (lane ) nuclei revealed that fasl could be reliably detected in specimens containing as few as nuclei. conclusions: since fasl is a cytokine of low to moderate abundance in placenta, this suggests that lcmd coupled with western blotting will be a valuable methodology to elucidate pathways of syncytial apoptosis and pathophysiology in pregnancies complicated by preeclampsia. supported in part by nih grant hd . the placenta of preeclamptic pregnancies shows oxidative and nitrative stress. we have shown low protein abundance but paradoxically high activity of inducible nitric oxide synthase (inos) in the placenta with severe preeclampsia compared with normal pregnancies. protein nitration and phosphorylation are post-translational modifications possibly involved in nos activity. objectives: examine inos localization and tyrosine phosphorylation in the preeclamptic placenta and the effect of a peroxynitrite generator (sin- ) on inos expression in primary human placental microvascular endothelial cell (hpmec) cultures. methods: placental lysates from normal (n= ), mild (n= ) and severe (n= ) preeclamptic pregnancies were western blotted for inos and what are the roles played by peroxynitrite in preeclamptic women? yoshikatsu suzuki, tamao yamamoto, yoshimasa watanabe, takeo itoh, hidetaka izumi. obstetrics and gynecology, nagoya city university, nagoya, japan; pharmacology, nagoya city university, nagoya, japan; obstetrics and gynecology, izumi women's hospital, fukuoka, japan. aim preeclampsia is characterized by hypertension plus proteinuria. it is hypothesized that the endothelial cell function might be activated by placental faculty in early pregnancy and the activation might cause the vascular disease in preeclampsia. peroxynitrite, which is produced by combination with superoxide (o -) and nitric oxide (no), is strong oxidant as well as o -.we investigated whether or not the localization of peroxynitrite in both placenta and resistance artery might play important roles of developing preeclampsia. omental arteries or placentas were obtained from severe preeclamptic and term-normotensive pregnant women at cesarean section. they were stained by ant-nitrotyrosine (nt, a marker of peroxynitrite) antibody. furthermore, the concentration of nt was measured in omental arteries. in formed consent was obtained from all patients in written. preeclampsia was diagnosed according to the criteria of japan society of obstetrics and gynecology. the localization of nt was seen in placentas obtained from sever preeclamptic women ( / ), although it was not seen from normotensive pregnant women ( / ). the localization was also seen in placentas from of severe preeclamptic women with intrauterine fetal restriction. in omental arteries from both groups, the localization of nt was seen to same degree, and the concentration of nt was similar ( . ± . for sever preeclampsia and . ± . for normotensive pregnant women). from these results, it was suggested that an increase in o production, a decrease in no as well as peroxynitrite production in the placentas might cause the vascular dysfunction and subsequently damage uteroplacental blood circulation in preeclampsia. however, the role played by peroxynitrite in resistance artery might be different and more complicated. background: preeclampsia (pe) is associated with shallow cytotrophoblast (ct) invasion of the decidua, leading to impaired vascular transformation and poor uteroplacental perfusion. ct invasion requires the selective proteolysis of the peri-decidual cell (dc) extracellular matrix. we hypothesized that il and tnf , cytokines that have been linked to pe, may induce aberrant expression of the matrix metalloproteinases (mmp) and in dcs, thereby preferentially degrading the decidual ecm and interfering with sequential ct invasion. methods: immunostaining for mmp- , mmp- , and vimentin (a dc marker) was performed on decidua from normal (n= ) and preeclamptic (n= ) women, and staining intensities were evaluated by hscore. confluent, leukocyte-free first trimester dcs were primed with - m estradiol (e ) or e + - m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e +/-mpa with or without ng/ml of il or tnf . secreted mmp- and mmp- levels were measured by elisa (n= ) and confirmed by western blotting. quantitative rt-pcr assessed mmp- and mmp- mrna levels (n= ). results: tissue staining revealed that mmp- and mmp- levels in preeclamptic decidua (hscore mean±sem: ± and ± , respectively) were significantly higher than in normal decidua ( ± and ± , respectively; p< . ). in cultured first trimester dcs incubated with e , tnf increased secreted mmp- and mmp- levels by ± and ± -fold, respectively, while il increased them by ± and ± fold, respectively (p< . ). in parallel incubations with e +mpa, basal mmp- and mmp- output were lowered by approximately % and %, respectively, while tnf -and il elicited mmp- and mmp- levels were blunted by - %. western blotting confirmed the elisa results, and mrna levels corresponded to changes in mmp- and mmp- secreted protein levels. conclusions: over-expression of mmp- and mmp- in decidual cells may promote pe by disrupting decidual ecm and impairing normal ct invasion. the high levels of il and tnf associated with pe may be contributing to this over-expression. our in vitro observations that mpa blunts tnf -and il -elicited mmp expression suggests that exogenous progestin may offer a novel therapeutic approach in preventing pe. to produce -methoxyestradiol ( -me), a compound with diverse biological activities including inhibition of hif- , a transcription factor that mediates cellular response to hypoxia. circulating levels of -me and placental comt activity are significantly reduced in preeclampsia, raising the possibility that reduced production of -me contributes to the pathophysiology of preeclampsia by altering placental response to hypoxia. genetic variation in the comt gene is linked to comt activity and has been associated with intrauterine fetal growth restriction. we determined if a snp in exon of the comt gene (rs ), which does not change amino acid sequence ( leu ), but reduces comt mrna translation, was associated with preeclampsia. we analyzed comt genotypes in paired dna samples extracted from maternal and cord blood from normal pregnancies and pregnancies complicated by preeclampsia by allele discrimination. the study population was predominantly (> %) african-american. the frequency of the minor rs "g" allele, which is associated with low comt activity, was similar in maternal cases and controls (cases: %; controls: %, p= . ), but was significantly greater in fetal dna from pregnancies complicated by preeclampsia compared to control pregnancies (g allele frequency cases: %; control: %; p< . ). likewise, fetal carriage of the rs "g" allele conferred a significantly greater risk of preeclampsia (odds ratio: . ; % c.i.: . , . , p< . ). there was also a significant discordance between paired maternal and fetal rs genotypes with significantly greater discordance for the "g" allele in fetuses hosted in preeclamptic pregnancies (odds ratio: . ; % c.i.: . , . ). we conclude that genetic variation in the comt gene is associated with risk of preeclampsia, possibly through a mechanism involving reduced production of -me. supported by nih p md . smoking is associated with elevated adma in preeclampsia. michael p frank, robert w powers. , magee-womens research institute, pittsburgh, pa, usa; obstetrics gynecology, university of pittsburgh, pittsburgh, pa, usa. objective: cigarette smoke exposure paradoxically reduces the risk of preeclampsia. asymmetric dimethylarginine (adma) is an endogenous competitive inhibitor of nitric oxide synthase (nos), an independent risk factor for cardiovascular mortality, adma is elevated in women who develop preeclampsia, and adma has been reported to be both higher and lower in smokers. the objective of this study was to investigate the concentration of adma in pregnant smokers and nonsmokers with and without preeclampsia. study design: case-control study of women with uncomplicated pregnancy (controls), and women with preeclampsia matched for gestational age at sample collection. adma was measured by hplc. cigarette smoke exposure was determined by questionnaire and confirmed by plasma cotinine. data are mean±sd. analysis was by two factor anova with fishers post-hoc testing, significance accepted at p< . results: as previously reported, maternal plasma adma concentrations were higher in women with preeclampsia compared to controls (p< . ). in addition, the concentration of adma was significantly higher in preeclampsia smokers compared to controls and preeclampsia nonsmokers (p< . ). in contrast, there was no difference in adma concentration between control smokers and nonsmokers. conclusion: these data may suggest a differential effect of cigarette smoke exposure on circulating adma concentrations between women who do and do not develop preeclampsia. previous data has suggested that cigarette smoking is associated with lower adma in low risk elderly patients, and higher adma in high-risk subjects with diabetes. therefore, the data in preeclamptic and non-preeclamptic subjects may be consistent with these studies, however, the underlying biological explanation for this differential effect has yet to be determined. objective: eclampsia is similar to hypertensive encephalopathy in which an acute elevation in blood pressure causes autoregulatory breakthrough, hyperperfusion and edema formation. we previously reported that the pressure of breakthrough was similar between nonpregnant (np) and late-pregnant (lp) rats, but only lp animals developed edema. this study tested the hypothesis that lp animals have decreased in cerebrovascular resistance (cvr) and hyperperfusion in response to breakthrough vs. np. we further hypothesized that acute hypertension would cause greater blood-brain barrier (bbb) permeability in lp rats due to elevated hydrostatic pressure. methods: in vivo models of bbb permeability and cerebral blood flow (cbf) were used in np (n= ) and lp (d - ; n= ) rats that were either normotensive or hypertensive (np-htn, lp-htn) by infusion of phenylephrine to raise mean arterial pressure. permeability was determined in anterior and posterior brain regions by calculating the flux of kd dextran into the brain tissue, measured by a fluorescent spectrophotometer after flushing the vasculature with saline. cbf and cvr were measured by infusion of m fluorescent microspheres and determined based on the flow rate and fluorescence intensity of a reference sample for each animal. animals were ventilated to maintain blood gases within normal ranges (po > mmhg, pco = - mmhg). results: although the pressure change was similar between np and lp ( and mm hg), lp animals responded to acute hypertension with hyperperfusion. cbf increased from ± to ± ml/ g/min in np ( %) and ± to ± ml/ g/min in lp ( %; p< . vs. np). hyperperfusion in lp animals was associate with decreased cvr vs. np ( . ± . vs. . ± . mm hg/(ml/ g/min); p< . ). bbb permeability was significantly increased in lp animals at breakthrough vs. np in both anterior and posterior brain regions. the flux of dextran in anterior and posterior brain regions for np vs. lp animals was: ± vs. ± for anterior (p< . ) and ± vs. ± for posterior (p< . ). conclusions: these data demonstrate that pregnancy decreases cvr and causes hyperperfusion of the brain during acute hypertension. because increases in cvr is a protective function in the brain, impairment of these mechanisms during pregnancy may predispose the brain to edema when blood pressure is elevated, as in eclampsia. introduction: this study used the in vitro dually perfused human placental lobule to test the hypothesese that placental release of vegf and the fetoplacental vasodilatory response to exogenous vegf- are altered by tissue oxygenations that mimic healthy and preeclamptic pregancies. methods: lobules were dually perfused for six hours under one of two oxygenation conditions, representing 'normoxia' and 'hypoxia' (n = each): delivering maternal side inflow perfusate at oxygen concentrations of . % and %, respectively, distributed via cannulae; and fetal side inflow perfusate oxygen concentration of - % in both systems. venous perfusates were sampled and assayed, appropriate to side of release, for erythropoietin (epo), macrophage inflammatory protein- alpha (mip- alpha) (reference oxygen sensitive hormones), free vegf, svegfr- and plgf. in separate perfusions, fetoplacental vasodilation in response to pm vegf was investigated, following preconstriction of the fetoplacental vasculature to steady state fetal-side inflow hydrostatic pressure (fihp) with the thromboxane mimetic, u , (n = each). results: maternal-side mip- alpha release was higher in the 'hypoxic' than the 'normoxic' system ( ± and ± pg/ml, respectively, at hours, mean ± se; -way anova: p< . ). maternal-side epo and fetal-side soluble free vegf and svegfr- release were not different between groups. fetal-side release of plgf was higher in the 'hypoxic' group than the 'normoxic' group ( . ± . and . ± . pm, respectively, at hours, mean ± se; -way anova: p < . ). there was no difference in the vasodilatory response to vegf- in the fetoplacental vasculature between the groups ( . ± . and . ± . % change in fihp, mean ± se). discussion: differences in mip- alpha and plgf release provide evidence for metabolic separation of the adapted systems, caused by a changed oxygen environment. our failure to observe differences in epo, vegf and svegfr- release may be explained by longer lag-times for up regulation of their gene expression. vegf associated endothelial signalling appears to be unaffected by 'hypoxia' in the placenta over the time course studied here. background: there is a placental renin-angiotensin system (ras) from very early pregnancy. ang iv mediates various effects by binding to its specific receptor, the at r, the active site of which is an insulin-regulated aminopeptidase (irap). there is at r expression in both endothelial and smooth muscle cells. ang iv at low concentrations is vasodilatory, increasing blood flow via the at rs; it also stimulates nf-kappa beta and modulates glut- . to date at r expression has not been investigated in the placenta. we propose that at r plays a part in the placental vascular development necessary for successful pregnancy, and that reduced at r expression may be associated with inadequate vascular adaptation contributing to pre-eclampsia (pe). aim: to identify and locate at r expression in both np and pe placentae. methods: the study had hospital ethical approval; written informed consent was obtained from all women. placental samples were obtained from np and pe at delivery (gestational ages: and weeks respectively). samples were taken from areas, near cord, middle and outer edge of the placenta. paraffin embedded sections were immunostained for irap reactivity using a rabbit polyclonal antibody (gift from professor david james, garvan institute, australia). immunoreactivity of trophoblast and uterine cell populations was assessed using a semi-quantitative grading system. grade = no positive labelling, = - %, = - %, = - % and = - % of cells positively labelled. median (max, min) are shown. results: ) at r immunostaining was prominent in the syncytiotrophoblast and hofbauer cells of all placental villi examined, with no differences in expression between sampling sites. ) at r positivity was reduced in near cord pe samples ( . ( . , . )) compared to np ( . ( . , . ) p= . ). conclusion: we have shown for the first time, dense at rs in syncytiotrophoblast and hofbauer cells in np placentae, and their down-regulation in pe. reduced ang iv/at r binding may contribute to increased placental vasoconstriction resulting in increased ischemia/reperfusion. this in turn may stimulate xanthine oxidase, which is itself stimulated by angii, leading to increased superoxide production. further work is needed to clearly define the role of this newly identified component of the renin-angiotensin system in normal pregnancy and pe. pre-eclampsia is associated with lower percentages of regulatory t cells in maternal blood. jr prins, hm boelens, jj hm erwich, j heimweg, s van der heide, ajm van oosterhout, aej dubois, jg aarnoudse. obstetrics, university medical center groningen, groningen, netherlands; laboratory of allergology and pulmonary disease, university medical center groningen; pediatric allergology, beatrix children's clinic, university medical center groningen. pre-eclampsia is a serious disease of human pregnancy and immunological mechanisms play a role in its pathophysiology. normal pregnancy is associated with an increase in regulatory t (treg) cells and with a predominant th immune profile. treg cells are a subpopulation of cd + lymphocytes and are specifically characterized by the lineage specific transcription factor foxp . treg cells seem to induce immunological changes that have a protective role in maintaining normal pregnancy. we hypothesised that percentages of treg cells are decreased in pregnancies complicated by pre-eclampsia. methods in total, women with pregnancies complicated by pre-eclampsia and healthy pregnant controls were enrolled. to obtain control umbilical cord blood as well, control group i consisted of eighteen healthy pregnant women at term. in addition, since women with pre-eclampsia delivered preterm, control group ii (peripheral blood only) consisted of women during normal pregnancy with a gestational age matched for the preterm pre-eclamptic group. treg cells were measured from whole blood using four-color flow-cytometry. women with a pregnancy complicated by pre-eclampsia had a significantly lower percentage of cd + foxp + treg cells ( . vs . %; p< . ). in the pre-term group the pregnancies complicated by pre-eclampsia showed a significantly lower percentage of cd + foxp + cells in the peripheral blood as compared to the healthy pregnant controls. at term this percentage was also lower but not significantly so. between pre-term and term pregnancies both complicated by pre-eclampsia no significant difference was found in the percentage of cd + foxp + treg cells. no difference was found in umbilical cord blood ( . vs . %). conclusions our data suggest that pre-eclampsia is associated with a diminished percentage of treg cells in peripheral blood. we conclude that a deficiency of regulatory t cells may play a role in the pathophysiology of pre-eclampsia. background: preeclampsia and eclampsia are significant causes of maternal and fetal death. however, the pathophysiology of these conditions is unclear. we and others have reported that inhibition of endogenous nitric oxide (no) synthesis produces symptoms similar to preeclampsia in pregnant rats. several studies demonstrate that fetoplacental weights are altered in pregnancies of spontaneous hypertensive (shr) rats. in addition, impaired synthesis of tetrahydrobiopterin (bh ), a major co-factor for endothelial nitric oxide synthase (enos) activity and enhanced expression of enos has been observed in the pathogenesis of hypertension. in the current study, we examined whether supplementation of bh and sepiapterin (sep, a precursor for bh biosynthesis in the salvage pathway) reduces increased blood pressure and improves fetoplacental weights in shr pregnant rats. methods: groups ( - ) of shr pregnant rats were either treated with bh , sep ( mg/k.g body weight/day/rat, oral tablets) or vehicle (normal diet) beginning from day of pregnancy until day of gestation. animals were sacrificed on day of gestation and fetoplacental weights were recorded immediately. western blot analysis was performed to determine vascular enos expression (enos/gapdh). results: significant (p< . ) elevations in blood pressure (bp, mmhg) were observed in shr (shr, ± . vs. wky, ± . mmhg) compared to wistar-kyoto (wky) group. supplementation of either bh or sep ( ± . ), significantly (p< . ) reduced elevated bp beginning from day of pregnancy. fetal but not placental weights were significantly (p< . ) reduced in shr ( . ± . grams) compared to wky ( . ± . ) rats. bh ( . ± . ) treatment partially (p< . ) increased fetal weights compared to the shr group. vascular enos expression is significantly (p< . ) elevated in shr ( . ± . ) compared to wky rats ( . ± . ). further, treatment with bh ( . ± . ) but not sep ( . ± . ) significantly (p< . ) reduced elevated enos protein expression in shr rats. conclusions: bh may be beneficial treatment of preeclampsia to reduce blood pressure and improve fetal perfusion to increase fetal weights. genetic risk factor for severe preeclampsia: significance of endothelial nitric oxide synthase gene t- ->c and missense glu asp variants. toshihiro yoshimura, michihiro yoshimura, masafumi nakayama. obstetrics and gynecology, kumamoto university school of medicine, kumamoto, japan; cardiovascular medicine, jikei university school of medicine, tokyo, japan; cardiovascular medicine, kumamoto university school of medicine, kumamoto, japan. introduction: we recently identified two endothelial nitric oxide synthase (enos) gene polymorphisms, a glu asp missense variant in exon and a t- -->c variant in the '-franking region, which are associated with coronary spasm and myocardial infarction in japanese population. and we also identified a missense glu asp variant is associated with severe preeclampsia and placental abruption. our objective was to analyze the association between the t- -->c and severe preeclampsia. materials and methods: the study participants included patients with histories of severe preeclampsia. this is a preliminary study, therefore, the comparisons were made with the general normal population. results: the analyses revealed that the frequency of the missense glu asp variant (n= / , %) was significantly higher than the general population (n= / , %), as we previously published. however, the frequency of the t- -->c variant (n= / , %) was not different from the general population (n= / , %). interestingly, only one patient had both t- -->c and missense glu asp variants, and she developed placental abruption. conclusion: although our sample size is small, it is very unlikely that the t- -->c variant is associated with severe preeclampsia. the t- -->c variant may not be a genetic susceptibility factor to severe preeclampsia. the t- -->c may have some reproductive significance in combination with missense glu asp variant, however huge number of patients would be needed to analyze such rare ( . %) combination of the variance. the association between the development of preeclampsia and methylenetetrahydrofolate reductase, angiotensinogen, vascular endothelial growth factor single nucleotide polymorphism genotype combinations. hyun soo park, jong kwan jun, chan-wook park, joong shin park, bo hyun yoon, hee chul syn. obstetrics and gynecology, dongguk university international hospital, goyang-si, gyeonggi-do, republic of korea; obstetrics and gynecology, seoul national university college of medicine, seoul, republic of korea. objective this study was conducted to investigate if there exists any genotype combination of multiple single nucleotide polymorphism (snp)s which is frequently found in preeclampsia patients. study design one hundred sixty two preeclampsia patients and normotensive pregnant women were included in this study between jan and jul . diagnosis of preeclampsia and assignment of severity were made according to the criteria by national high blood pressure education working group and american college of obstetricians and gynecologists. the patients were reclassified as early ( weeks or before) and late-onset ( weeks or beyond) disease. genotypes were measured with pcr-rflp for methylenetetrahydrofolate reductase (mthfr) c t, angiotensinogen (agt) m t, vascular endothelial growth factor (vegf) c t with the dna extracted from maternal blood. case-control study for each snp was done and the frequencies of genotype combination were compared. anova, t-test, chi-square test, fisher's exact test and logistic regression analysis were used for statistical analysis. a p value of < . was considered statistically significant. results genotypes of mthfr polymorphism showed significant difference between late onset preeclampsia and control (cc+ct/tt, or: . , p< . ) but agt and vegf polymorphism did not show statistical difference between any case-control combination. only out of possible genotype combinations were found and there was no statistical difference in the frequencies of genotype combination between case and control group. conclusion mthfr polymorphism might be associated with the development of preeclampsia, but there was no combination of mthfr, agt and vegf polymorphisms which is associated with the development of preeclampsia. diffuse staining and vascular smooth muscle (vsm) staining. resistance-sized vessels ( - μm) were evaluated. results: for mpo, the intensity of staining (fig) and the % vessels with neutrophil, diffuse and vsm staining was significantly greater for obese than for overweight or normal weight patients: % diffuse staining ( . ± . vs. . ± . vs. . ± . , p< . ); % vsm staining ( . ± . vs. . ± . vs. . ± . , p< . ). for mmp , obese and overweight patients had a greater (p< . ) % vessels with neutrophil, diffuse and vsm staining than normal weight patients: % diffuse staining ( . ± . vs. . ± . vs. . ± . ); % vsm staining ( . ± . vs. . ± . vs. . ± . ). conclusions: neutrophils infiltrate the systemic vasculature of obese women and release mpo and mmp . speculation: obesity may put women at risk for pe because their vasculature may already be dysfunctional due to neutrophil infiltration and release of mpo and mmp . hl . collagen is an important protein that maintains the structural integrity of tissues. disruption of vascular smooth muscle collagen could result in vascular dysfunction in women with preeclampsia. recently, neutrophil infiltration of the systemic vasculature was demonstrated in preeclamptic women. neutrophils produce inflammatory mediators, such as reactive oxygen species (ros) and tnf-. we hypothesized that neutrophils, ros and tnf-would alter expression of collagen regulating genes. methods: primary cultures of human vascular smooth muscle cells (vsmc) were seeded into t- flasks ( , cells/flask) and grown for days to % confluence. the cells were treated for hours with medium control, ros (hx, . mm + xo . u/ml), tnf-( ng/ml); and neutrophils ( , ) activated with arachidonic acid, μm, ( : ratio of neutrophils to vsmc). rna was extracted from cell homogenates and analyzed for gene expression with an rt profiler pcr array system for human extracellular matrix genes (superarray). to determine the fold-change of gene expression, the results were first normalized to a housekeeping gene and then ct was calculated across two rt-pcr arrays where group was the control and group was the experimental treatment. results: table . conclusions: neutrophils, ros and tnf increased mmp expression. interestingly, genes involved in collagen synthesis (col a ) or inhibition of mmp- activity (timp ) were either not affected or down-regulated. these data suggest that neutrophil infiltration in preeclamptic women could cause vascular dysfunction by creating an imbalance between collagen synthesis and collagen breakdown favoring breakdown. hl , fogarty d tw , p md . objective: hypoxia increases membrane attack complex (mac) binding to cultured human trophoblasts, and mac enhances apoptosis in trophoblasts exposed to low compared to normal fio . trophoblast microparticles and cellular fragments released into the maternal circulation in vivo may contribute to the systemic pathophysiology of preeclampsia. we tested the hypothesis that hypoxia induced mac deposition on cultured human trophoblasts yields microparticles and fragments coated with mac. study design: primary cytotrophoblasts from term human placentas (n= ) were cultured h in % and % oxygen in dmem with % human serum with active mac or heat inactivated serum (control). media were centrifuged to obtain pellets of microparticles and cell debris which were immunostained for mac or exposed - h to confluent, phorbol myristate acetate differentiated u macrophages. the percentage of macrophages that ingested trophoblast debris was quantified by counting the number of macrophages with immunofluorescence for trophoblast cytokeratin filament staining, as assessed by confocal microscopy. results: cultures exposed to normal human serum, but not heat inactivated control serum, showed mac immunofluorescence on microparticles and fragments in medium, with the highest level of mac in cultures exposed to extreme hypoxia. the maximal percentage of macrophages that ingested the trophoblast debris coated with mac from cultures with % oxygen was . %, not different from the . % from cultures exposed a % fio and control serum. conclusion: trophoblasts exposed to hypoxia and active complement release microparticles and cellular fragments coated with mac into the extracellular environment. mac coating does not influence phagocytic removal of the debris by macrophages suggesting that placental derived, membrane bound mac could circulate to yield systemic affects on maternal endothelium. supported by nih hd and hd . is there a role for fatty acids in the pathogenesis of pre-eclampsia? nicola j robinson, laura j minchell, jenny e myers, philip n baker, carl a hubel, ian p crocker. maternal and fetal health research group, the university of manchester; obstetrics, gynecology and reproductive sciences, university of pittsburgh. objectives: women with pre-eclampsia (pe) display altered lipid metabolism as characterized by elevated circulating triglycerides and non-esterified fatty acids (nefa) and these changes are evident before the disease is clinically apparent. we have tested the hypothesis that the increased circulating levels of nefa contribute to endothelial dysfunction in pe. methods: human umbilical vein endothelial cells were incubated for h with pooled plasma ( %) from normal or pe pregnancies, or with palmitic, oleic and linoleic acid in culture media at the concentrations and molar ratios to albumin identified in normal ( , , m, ratio . ) and pe pregnancies ( , , m, ratio . ) , . lipid droplet accumulation was determined using an oil red o absorbance assay. endothelial metabolism was measured using the mtt test and mitochondrial membrane potential determined by jc- assay as a marker of early apoptosis. results: plasma from pe pregnancies increased endothelial cell lipid droplet accumulation compared to normal plasma (p< . , wilcoxon signed ranks, n= ). this change was replicated following exposure to nefa at the combined concentrations found in pe compared to normal pregnant controls (p< . , n= ). plasma from women with pe caused a significant decrease in mitochondrial dehydrogenase activity (mtt test; p< . , n= ) and a reduction in jc- fluorescence (p< . , n= ), compared to normal plasma, suggestive of mitochondrial membrane depolarization and increased cellular apoptosis. again these effects were replicated using nefa in culture medium at the levels found in pe compared to normal pregnancies (mtt test: p< . , n= ; jc- assay: p< . , n= ). conclusions: in endothelial cell cultures, plasma from women with pe caused increased lipid droplet accumulation, decreased cellular metabolism and increased apoptosis. these changes to cellular function were mirrored using nefa in culture medium at the concentrations and molar ratios to albumin previously reported in pe. these findings provide evidence that the changes in endothelial cell function induced by plasma from women with pe may be due to the increased nefa circulating levels and that increased palmitic, oleic and linoleic acid, in combination, could play a role in pathogenesis of pe. lorentzen ( ) bjog; endresen ( ) ajog. background: skewing of the maternal endothelial phenotype in pre-eclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. we hypothesise that factors secreted from pe placental tissue will impair endothelial cell function. we have tested the effect of soluble factors by menopausal status, there was a significant increase in bmi in pre-but not in postmenopausal women. in postmenopausal women there was insufficient power to note a statistically significant change in bmi. results are summarized with the follow-up for each group represented by "n" in the graph below. premenopausal patients were divided into hysterectomy with oophorectomy (pbso) versus hysterectomy alone (ph). the ph group showed an increase in bmi that plateaus at time . in the pbso group the bmi continued to increase over time. subgroup analysis comparing ph to pbso demonstrates initial weight loss in pbso but a significant increase in bmi from baseline at time compared to ph. conclusions: hysterectomy appears to be associated with an increase in bmi over time. subgroup analysis suggests that, in premenopausal women, oophorectomy is more strongly associated with continuing weight gain than hysterectomy alone. purpose: obesity is implicated as a key risk factor in chronic disease, but no studies have associated central obesity to the presence of chronic abdominal and/or pelvic pain. we set out to identify the prevalence of chronic abdominal/ pelvic pain in an underserved, primarily latina population by a cross-sectional study in the olive view-ucla outpatient gynecology clinic. we sought to identify an association between the presence and severity of abdominal/pelvic pain and central obesity. methods: nonpregnant women presenting to the gynecology clinic were prospectively evaluated and grouped according to the presence of abdominal/ pelvic pain ('none-mild' or 'moderate-severe' pain). body mass index (bmi) and abdominal circumference (ac) were measured. patients with 'moderate-severe' pain completed standardized questionnaires for pelvic pain and global health scores. results: / ( %) of patients has 'none-mild' pain, and ( %) had 'moderate-severe' pain. pain prevalence was not significantly associated with bmi (mean: 'none-mild' . + . kg/m ; 'moderate-severe' . + . kg/m , p= . ), nor was pain severity (p= . ). pain prevalence was significantly associated with ac (mean: 'none-mild' . + . inches; 'moderate-severe' . + . , p= . ). a borderline positive association exists between ac and pain severity (p= . ). conclusions: we demonstrate an association between both the presence and severity of chronic abdominal/pelvic pain and central obesity, independent of bmi. ac appears to be a more relevant factor than other traditional measures of habitus in patients with this chronic malady. to improve preventative care in women's health management, further evaluation of the role of central obesity in the pathogenesis of chronic pain is necessary. aims: vulvitis is one of the most frequently diagnosed gynaecological infections. we aim to assess the efficacy against infective vulvitis of a new topical medical device containing low molecular weight hyaluronic acid (lmw-ha). the ability of this molecule to stimulate -defensin release in keratinocytes has been recently shown. methods: we report preliminary data regarding women suffering from infective vulvitis, as assessed by a gyneacologist: patients were randomly selected to receive sphg ( patients), a cream containing low-molecular weigh hyaluronic acid, or vehicle ( patients). patients were asked to apply the cream to the vulva twice-daily for days. at the end of treatment, evaluation of efficacy, tolerability and acceptability of the cream was assessed by a specific questionnaire. results: preliminary results show that patients receiving sphg report a significative improvement of vulvitis symptoms, in terms of itch, redness of the skin and burning, in comparison to vehicle. sphg also showed a good tolerability, cosmetic acceptability and symptomatic relief perceived by patients. conclusion: sphg seems to be efficacious in ameliorating the symptoms of infective vulvitis. this activity may be probably related to the lmw-ha presents in this formulation: in fact, low molecular weight hyaluronic acid has been recently shown to induce -defensin production by human keratinocytes. since this peptide exerts antimicrobial and antimicotic activity, the improvement of symptoms assessed in patients receiving sphg might be linked to a reduced infective charge, attributable to the activity of -defensin . background: allogeneic hematopoietic stem cell transplant (hsct) is a treatment used for many malignant and nonmalignant diseases of the bone marrow and immune system. hsct may be complicated by chronic graft-versus-hostdisease (cgvhd) in to % from matched unrelated donors. genital cgvhd complicates about % of hsct and may uncommonly result in labial fusion. case: year old woman with a history of ewing's sarcoma and acute myelogenous leukemia, had received chemotherapy and total body irradiation (tbi) followed by a matched unrelated donor hsct. menarche occurred at years of age after normal pubertal development. she menstruated regularly until cancer diagnosis. premature ovarian failure resulted after chemotherapy and tbi and oral contraceptive pills were used for hormone replacement. after transplant, she developed chronic gvhd involving the skin, eyes, mouth and joints, and concomitantly complained of vulvar pruritus. she was presumed to have a yeast infection which was treated with fluconazole without a pelvic exam. she was evaluated by a gynecologist when she was unable to insert a tampon. pelvic exam revealed dense labia minora adhesions from the clitoris to urethral meatus and posteriorly leaving a cm opening at the urethra. pelvic mri revealed a normal uterus and ovaries. after weeks of topical estrogen cream, the adhesions remained dense and were lysed under general anesthesia. vaginal examination revealed pale, minimally rugated, normal mucosa. cervical cytology was normal. post-operatively she used daily topical estrogen and hydrocortisone creams. at months after surgery, her urinary stream was stronger. on pelvic exam, the labial opening was cm, but a small posterior forchette adhesion elicited severe pain. after using dilators coated with topical steroids and estrogen, she was able to insert a tampon. conclusion: genital gvhd should be considered in women with genital tract complaints after hsct. labial fusion secondary to chronic gvhd may be treated successfully with surgery and medical therapy. support: rbmb/nichd/nih. dana r ambler, mazen e abdallah, rahi victory, michael p diamond, elizabeth e puscheck, jay m berman. obstetrics and gynecology, wayne state university/detroit medical center, detroit, mi, usa. objective: to determine which factors are predictive of a ruptured ectopic pregnancy, and whether endometrial stripe thickness can be used as an alternative to such criteria in the diagnosis of an ectopic pregnancy. design: retrospectively collected ectopic pregnancy database. setting: detroit medical center, detroit, michigan. patients or participants: women with a diagnosis of an ectopic pregnancy were studied, with surgically confirmed tubal rupture cases. interventions: abstracted data included hcg(iu), gestational age (days), presenting symptoms of pain and/or bleeding, hemoglobin (hgb), hematocrit (hct), historical risk factors, ultrasound-determined ectopic size, endometrial stripe thickness (mm), amount of cul de sac fluid (cds), tubal rupture at time of surgery, and estimated blood loss (ebl)(ml). covariates included demographics. results were significant when p< . . results: chi square analysis revealed that there is a relationship between endometrial stripe thickness and hcg levels. logistic regression models demonstrated that endometrial stripe thickness was not predictive of ectopic rupture, (or . , p= . ) . however, logistic regression, both forced and forward stepwise analysis, demonstrated that hcg (or= . , p< . ), a large volume of cul-de-sac fluid (or= . , p< . ), and increasing pain (or= . , p= . ) were associated with increased risks of rupture. gestational age, and ectopic related risk factors were also not predictive of rupture. conclusions: endometrial stripe thickness is not a useful predictor for the diagnosis of a ruptured ectopic pregnancy. serum hcg measurement, cds fluid volume and the presence of pain are much stronger diagnostic indicators of ectopic pregnancies. clinical profile of migraineurs in a university hospital gynecology department in japan. [objectives] the changes in hormonal milieu associated with menarche, pregnancy, menopause, and post-menopause are frequently accompanied by changes in the patterns and frequency of migraine. little is known on the relationship of women's issues of migraine though the balance between estrogen and progesterone is critical in the elimination of migraine. our aim was to investigate the relations among the prevalence of migraine, the reproductive stage, and gynecologic diseases. [materials and methods] female patients (average age: . years old) who consulted a physician and agreed to answer about the questionnaires during september, -june, . migraineurs were diagnosed with the migraine screener by the japanese headache medical treatment promotion committee in . and the patients answered questionnaires that screened about menopausal disorder and abnormal menstruation at once. they were conducted a survey in the form of a questionnaire and sometimes were taken blood samples. [results] in patients had migraine ( . % average age: . years old). prevalence were . % in one's twenties, higher than another ages. most patients with migraine was complicated by menstruation disorders (premenstrual syndrome %, dysmenorrhea . %), sterility ( . %), and severe menopausal disorder ( %). when trh and the lh-rh test were examined for the sterility patient, migraineurs had higher prolactin basal level and lh level after lord but lower fsh level before and after lord than nonmigraineurs. on the other hand, the prevalence of migraine for postmenopausal women and women who had gynecology cancer treatment was low, and there was no relation between migraine and pregnancy history. [conclusions] this study provides migraine headache is influenced by reproductive stage and that women with migraine are frequently complicated by menstruation disorder. it is thought that migraine account for abnormality of hormone milieu including abnormality of the hypothalamus-pituitary system. objective: this study evaluated the efficacy of doses of estradiol (e ) gel . % (divigel ® ), a novel formulation of e consisting of mg e per g transdermal gel, to reduce frequency and severity of vasomotor symptoms and signs of vulvar and vaginal atrophy (vva) associated with menopause. design: postmenopausal women were evaluated in a -week study comparing placebo to e gel . % at doses of . g/day, . g/day, and . g/day with estimated nominal daily deliveries of . mg, . mg, and . mg of e respectively. endpoints included mean change from baseline in daily frequency and severity of moderate to severe hot flashes (msvs). vaginal ph and % superficial cells were collected at baseline and end of study. results: e gel . % showed statistically significant improvements from placebo gel as early as week (table) that were maintained throughout treatment. signs of vva (vaginal ph and % of superficial cells) showed statistically significant improvements from baseline with all doses of e gel . % compared to placebo. conclusion: e gel . % significantly decreased the frequency and severity of msvs at all doses evaluated in this trial. e gel . % offers multiple dosing options to individualize patient therapy, including the lowest effective dose that was studied ( . mg e , delivering . mg e /day), to treat vasomotor symptoms associated with menopause. estradiol (e ) gel . % (divigel ® ) is a novel formulation of e consisting of mg e per g transdermal gel for the treatment of vasomotor symptoms associated with menopause. safety and tolerability of e gel . % were evaluated in a large placebo-controlled trial. design: postmenopausal women participated in a -week study comparing placebo to . g/day, . g/day, and . g/day of e gel . %. circulating e and estrone (e ) concentrations were measured. safety analyses included the incidence of adverse events (aes) and clinical laboratory evaluations, including plasma levels of sex hormone binding globulin (shbg). application site tolerability was assessed using the draize scale. results: all doses of e gel . % produced physiologic e :e ratios similar to those seen in premenopausal women. e :e increased from a baseline mean of . to . , . , and . with, respectively, the . , . , and . g/day doses of e gel . %. the most frequently reported aes were breast tenderness and postmenopausal bleeding that appeared to be dose-related and would be expected with increased circulating estrogen concentrations. there were no remarkable changes in hematology, blood chemistry, urinalysis, lipid, coagulation, and carbohydrate values following treatment with e gel . %. shbg levels remained unchanged after weeks of treatment at all doses. the vast majority of patients had no evidence of skin irritation throughout the treatment period. mean draise scale scores after , , and weeks of treatment were . for all treatment groups except for a mean value of . ± . for the . g dose group after weeks of treatment. conclusion: e gel . % is a safe and well-tolerated therapy for the treatment of menopausal symptoms. design: pharmacokinetic parameters, dosing, efficacy, and safety information for divigel ® , elestrin ™ , evamist ™ , estrogel ® , and estrasorb ® were obtained from current prescribing information (obtained from manufacturer websites) and the data were compared. results: together, these new transdermal therapies offer multiple dosing/ delivery options and contain a wide range of e ( . mg to . mg), with the lowest systemic daily delivery of e attained by the divigel ® . g dose. following weeks of treatment, across the dosing options, each treatment significantly reduced both the frequency and severity of moderate to severe vasomotor symptoms (msvms) compared to placebo. change in frequency of msvms ranged from - . (divigel ® . g) to - . (estrasorb ® ); change in severity scores for msvms ranged from - . (divigel ® . g) to - . (divigel ® . g). all treatments are safe and well tolerated. breast tenderness was the only adverse event reported in % of subjects, occurring with all therapies. conclusions: current treatment guidelines recommend using the lowest effective dose of estrogen for the treatment of menopausal symptoms. this side-by-side comparison of the recently available e non-patch transdermal options to the standard transdermal therapies is meant to assist physicians in individualizing treatment for their patients. introduction: cdb- (cdb) is a relatively new progesterone receptor modulator being clinically evaluated for contraception and treatment of fibromas. its use in an intrauterine device/system (ius) has not been reported. in this study we prepared cdb-filled intrauterine devices (cdb-ius) and evaluated their effects on endometrial growth and bleeding patterns in rhesus macaques. methods: short ( . - . cm) lengths of silastic tubing (od . mm), either empty (n= ),or filled with silicone rubber matrix containing % of micronized cdb (n= ), were inserted into the uterine lumens of ovariectomized rhesus macaques. animals were induced to cycle by sequential treatment with systemic estradiol and progesterone (p) as reported (brenner et al, ann ny acad, ) . after . cycles, at the end of the follicular phase, the uterus was removed and processed. results: when systemic p treatment was withdrawn at the end of each cycle, animals with empty ius menstruated normally, while animals with cdb-ius bled little or not at all. over the whole . cycles, animals bearing a blank ius bled for an average of . ± . days while cdb treated animals bled for an average of only ± . days. at the end of treatment, animals exposed to blank ius had mean endometrial wet weights of ± mg while the cdb-treated endometria weighed only ± mg. the proliferation markers ki- and phospho-h were substantially lower in the cdb-ius treated than in the blank treated animals. histologically, the cdb exposed endometria were atrophied with evidence of glandular degeneration while the blank controls were proliferative and normal. summary: in cycling rhesus macaques, a cdb-ius prevented progestational development, blocked menstruation after p withdrawal, and suppressed endometrial proliferation. if these effects are confirmed in women, the cdb-ius could provide estrogen-free and bleed-free contraception, and could help control heavy menstrual bleeding. supported by rr , hd and the population council. introduction: irregular uterine bleeding is a major side effect and cause for discontinuation of ltpoc use. while endometria of ltpoc-exposed women display abnormally enlarged, fragile blood vessels (bv), decreased blood flow and evidence of oxidative stress, the mechanisms by which structural and vasomotor endometrial dysfunction occurs remains unknown, in part by the difficulty of manipulating hormone levels in women. the aims of this study were ) to validate the guinea pig (gp) as a model to study uterine effects of ltpoc and ) to investigate ltpoc-effects on endometrial histology and oxidative stress markers. methods: oophorectomized gps were implanted s.c. with time release pellets containing either placebo (crl,n= ); estradiol (e ,n= ); medroxyprogesterone acetate (mpa,n= ) or e +mpa (n= ). after days, uterine horns were weighed and frozen or paraffin embedded. angiogenesis was assessed by quantitative image analysis of vonwillebrand factor staining and included bv density and size. oxidative stress was detected by isoprostane and -oh-deoxyguanosine ( oxog). apoptosis was investigated by the tunel method. statistical analysis was by -way and -way anova. results: gp uteri were enlarged by both e (p< . ) and mpa (p= . ). effects of mpa on uterine weight differed significantly depending on e levels (p< . ), where mpa opposed the e effect in combined treatments. angiogenesis parameters were similarly impacted upon. thus, mpa alone increased bv density (p= . ) and bv average area (p= . ). the presence of e significantly decreased these parameters (bv density mean± sem: crl: . ± . %, e : . ± . %, mpa: . ± . %, e +mpa: . ± . %, p= . ). these changes were associated with highly elevated -isoprostane content in e +mpa-treated uteri compared to all other groups (p< . ). abnormalities in the e +mpa group were consistent with chromatin redistribution, nuclear pyknosis, karyolysis and increased nuclear oxog staining and a marked increase in tunel labeling. conclusions: ltpoc exposure alters endometrial vascular and tissue morphology consistent with oxidative stress and apoptosis in a complex interplay with endogenous estrogens. the gp is an excellent model for the study of ltpoc effects on the uterus. physiologic and psychological symptoms associated with injectable and oral contraceptive use. abbey b berenson, susan odom, carmen r breitkopf, mahbubur rahman. ob/gyn, utmb, galveston, tx, usa. objective: to compare physiologic and psychological symptoms over mo among users of depomedroxyprogesterone acetate (dmpa), an oral contraceptive with micrograms ethinyl estradiol (oc), and non-hormonal (nh) contraception. methods: a total of women reported the presence of symptoms prior to initiating contraception ( dmpa, oc, nh) and every mo thereafter for mo. longitudinal relationships between symptoms and contraceptives (reference: nh), as well as race/ethnicity (non-hispanic black, non-hispanic white, and hispanic) were assessed by gee-analysis after adjusting for age, visits and baseline status of symptoms. persistence, resolution, and new development of symptoms were noted by method in mo increments for mo and compared with that of nh controls. the gee analyses showed that oc was protective against mastalgia (or= . ), cramping (or= . ), hair loss (or= . ), acne (or= . ), nervousness (or= . ) and mood swings (or= . ). when race was considered, oc was protective for all women against acne, for whites against mastalgia and cramping, and for non-whites against nervousness. for whites, it was a risk factor for bleeding between menses. oc use resulted in resolution of acne within mo in nearly % of those with it at baseline, but no significant resolution after mo. also mastalgia ( % at baseline) was less likely to persist at and mo. bleeding between menses was reported for the first time at mo by % of oc users. dmpa users of all races had an increased risk of missed periods (or= . ), bleeding between menses (or= . ), bleeding > d (or= . ) and loss of libido (or= . ) relative to nh users. it reduced cramping (after mo) and bloating (all mo). racial differences were observed with dmpa protective against mastalgia and mood swings in whites, cramping in whites and hispanics, and bloating in non-whites. it was a risk factor for loss of energy in whites only. neither method affected depressive symptoms. conclusion: side effects of these two methods are mostly related to abnormal bleeding. very low dose pills can be protective against symptoms (mastalgia, cramping, hair loss, acne, nervousness and mood swings) commonly associated with pills while dmpa protects against cramping and bloating. knowledge about racial differences will allow physicians to individualize therapy. counseling should include that some symptoms can develop after mo while resolution often occurs within mo. methods: twenty-four women were enrolled in irb approved prospective, randomized, cross-over trial comparing months of oc (ortho cyclen) vs. tc ortho evra). the daily oc administers micrograms of ee; the weekly tc contains . milligrams of ee. each treatment was followed by months of washout and months of the alternative contraceptive. blood was drawn at baseline and final week of treatment for each arm of the study. ee was quantified by ria, with preceding organic solvent extraction and celite column partition chromatography. data were analyzed by t test with boferroni's correction, p < . . results: after two months of treatment the mean (+/-sem) ee levels for tc = . pg/ml (+/- . ) and oc = . pg/ml (+/- . ). the ee level is not significant different for the two medication (p = . ). conclusions: there is no difference in ee levels with the use of these oral and transdermal contraceptives. this suggests the transdermal contraceptive, despite the lack of the hepatic first pass effect, has similar levels of ethinyl estradiol compared to oc. the continuous elevation in ee seen with the tc route of administration, versus the episodic increases seen with the oc route, raises concerns of constant exposure to ee. this may explain the increased risk of estrogen-induced thrombotic events with this tc route of administration. impact of paracervical block, in combination with general anesthesia, on post-abortion pain. gweneth b lazenby, tod aeby. obstetrics and gynecology, university of hawaii, honolulu, hi, usa. objective to evaluate the impact of paracervical block with a long-acting local anesthetic, in conjunction with general anesthesia, on post-operative pain. methods a power analysis determined patients per arm were needed to demonstrate a significant difference of in mean pain scores. seventy-two patients were allocated to one of two arms using urn randomization. all patients received standardized anesthesia; intravenous sedation for gestational age under weeks and general anesthesia for over weeks. thirty-nine patients were randomized to receive a paracervical block with . % bupivacaine and thirtythree were randomized to no local anesthesia prior to surgical abortion. patients completed visual analog scales for pain and anxiety prior to the procedure, upon awaking, and minutes post-operatively, and prior to discharge. data were analyzed using an anova and students t-test the experimental and control groups were equivelent in age, ethnicity, gravidity, parity, prior abortions, prior vaginal deliveries, prior c-sections, gestational age, number of laminaria, pre-operative and intra-operative dilation, operative time, estimated blood loss, and reported complications. pain and anxiety were not significantly affected by placement of a paracervical block. these data do not support the hypothesized benefit of local anesthesia, prior to surgical abortion under general anesthesia, to reduce post-operative pain. we do not recommend the routine use of a paracervical block to decrease post-operative pain. age, parity, history of abortion and contraceptive choices affect the risk of repeated abortion. oskari heikinheimo, mika gissler, satu suhonen. ob gyn, university of helsinki, helsinki, finland; national research and development centre for welfare and health, helsinki, finland. objective the rate of repeat abortion varies from to % in northern europe. however, risk factors for repeat abortion are poorly understood. we characterized risk factors (demographic, as well as those related to abortion and postabortal contraception) of repeat abortion. design a prospective cohort study of women undergoing medical abortion between august and december . the subjects were followed by means of finnish registry on induced abortions until december ; the follow-up time (mean ± sd) was . ± . months. results altogether ( . %) of the subjects requested repeat abortion within the follow-up time. in univariate analysis previous abortion, parity, young age, smoking and failure to attend the follow-up visit were associated with increased risk of repeat abortion. immediate -in contrast to postponed -initiation of any contraceptive method was linked to lower risk of repeat abortion. in comparison to combined oral contraceptives, use of intrauterine contraception was most efficacious in reducing the risk of another pregnancy termination. in multivariate analysis the effects of young age, parity, previous abortion and type of contraception on the risk of another abortion persisted. conclusions increased focus on young, parous and those with the history of an abortion may be efficacious in decreasing repeat abortion. contraceptive choices made at the time of abortion have an important effect on the rate of reabortion. postabortal use of intrauterine contraception, specifically that of lng-ius, might decrease the rate repeat abortion. objective. to determine the rate of failure and to analyze factors associated with failure for the essure permanent birth control device at the detroit medical center (dmc). methods. a chart review was conducted on patients who underwent essure placement at the dmc from january through june . patient demographics, past medical and surgical history, anesthesia type, procedure time, intraoperative complications, and procedure failures were noted. data were analyzed for statistical significance using spss. results. there were essure procedures attempted at the dmc from january through june . of the attempted procedures, there were failures ( . %). of the failures were attributed to difficulty visualizing the ostia ( %). other causes of failure included expulsion of the device ( ), tubal spasm ( ), uterine perforation ( ), and tubal ostia too large for the device ( ). there were cases of failed placement for undocumented reasons, one case requiring a laparoscopic tubal ligation secondary to postprocedure tubal patency, and post-procedure pregnancies. age, race, body mass index, gavidity, parity, history of sexually transmitted infections, medical history, history of cesarean section, tobacco or illicit drug use, anesthesia type, and physician experience with the procedure were not significantly associated with placement failure or difficulty visualizing the ostia. a longer procedure time was significantly associated with failure ( . vs . min, p = . ), and history of ectopic pregnancy was significantly associated with difficulty visualizing the ostia ( . % vs . %, p = . ). conclusion. the failure rate for placement of the essure permanent birth control device at the dmc is . % with a pregnancy rate of . %. the majority of failures may be attributed to difficulty visualizing the ostia. a history of ectopic gestation was significantly associated with difficulty visualizing the ostia; thus, it may be reasonable to advise these women that success in essure placement may be reduced. introduction. the essure permanent birth control device is a relatively new form of minimally invasive sterilization for women. under hysteroscopic guidance, a dynamically expanding micro-insert is introduced into the proximal portion of the fallopian tube. the micro-insert induces local fibrosis and ultimately occlusion of the tubal lumen. a hysterosalpingogram (hsg) is performed three months after the procedure to confirm bilateral tubal occlusion. objective. to determine the follow-up rate for the post-essure hsg for a clinic population. methods. a retrospective chart review was conducted on university health center (uhc) patients who underwent placement of the essure permanent birth control device at the detroit medical center from january through june . follow-up for the post-essure hsg as well as the result of the hsg were noted for each patient. results. placement of the essure permanent birth control device was attempted in uhc patients of which were successfully completed. of the patients, ten underwent a post-essure hsg ( . %). the hsg was performed three to six months after placement of the essure permanent birth control device. bilateral tubal occlusion was documented in all ten patients. conclusion. despite counseling patients prior to their procedure that a hsg is needed and providing an information sheet, the follow-up rate for the post-essure hsg for this clinic population is only . %. for those in whom a hsg was performed three to six months after essure placement, bilateral tubal occlusion was confirmed in all. steps and or approaches to improve compliance with post-procedure confirmation of tubal occlusion should be employed to increase follow-up in the future. towards fibroids gene therapy: adenovirus mediated delivery of herpes simplex virus thymidine kinase gene/ganciclovir shrinks uterine leiomyoma in the eker rat model. memy hassan, dong zhang, salama salama, cheryl walker, hala el-mazar, ayman al-hendy. ob/gyn, utmb; md anderson; ob/gyn, meharry medical college, nashville, usa. aim: assessment of the efficacy of gene therapy of uterine leiomyoma in the immune-competent eker rat model using adenovirus mediated delivery of herpes simplex- -thymidine kinase gene followed by ganciclivir treatment (ad-tk/gcv). method: female eker rats with mri-confirmed uterine fibroid lesions were randomized to a single treatment with direct intratumor injection of ad-tk/gcv, ad-lacz/gcv, or medium.the tumor volume was evaluated by serial mri scanning and confirmed with caliper measurement at time of euthanasia. sample rats were selected randomly and killed at the following time points; , and days post treatment. samples were collected from tumors, other body organs and blood to assess the safety and efficacy of the treatment. results: ad-tk/gcv treatment produced dramatic shrinkage of the total uterine fibroid volume by % ± , % ± and %± of pretreatment volume at days , and respectively. the tumor size in negative control animals receiving ad-lacz/gcv continued to grow by + %± , + %± , + % ± while receiving media continue to grow by + % ± , + % ± and + % ± at same time points. ad-tk/gcv induced significant increase in caspase activity, bax expression, decrease in bcl and parp proteins expression and increased tunnel apoptosis index. additionally ad-tk/gcv treatment decreased cyclin d and pcna expressions. ad-tk/gcv did not produce any significant change in liver function tests or relative uterine horns weight to total body weight. the adenovirus transfection did not disseminated significantly to other distal organs except to liver and myometrium in limited number of animals. h e staining of non targeted organs did not revealed any sign of tissue damage. ad-transfection increased local cd and cd expressions as well as serum anti-ad antibodies. conclusion: ad-mediated delivery of hsv tk gene by direct intra-tumor inoculation followed by sc treatment of gcv for ten days effectively shrinks uterine leiomyoma lesions in eker rats. this effect is mediated via induction of apoptosis and decreasing the proliferation. the treatment regimen is well tolerated. these studies provide essential preclinical data for the development of gene therapy as an alternative non-surgical treatment option for women with symptomatic uterine fibroids. inhibitors of catechol o-methyl transferase shrinks uterine fibroids in the eker rat model. memy hassan, dong zhang, hala el-mazar, cheryl walker, ayman al-hendy. ob/gyn, utmb; md anderson; ob/gyn, meharry medical college, nashville, usa. background :the sex hormone dependent pattern of uterine leiomyomas and their high content of catechole -o-methy transferase (comt) raise the possibility for the development of novel treatment option using comt inhibitors.aim : to assess the potential therapeutic utility of a synthetic comt inhibitor (ro - ) in the eker rat model of uterine leiomyoma. methods female eker rat were evaluated by mri to confirm the prescence uterine fibroid lesion, then randomized for sc treatment with ro - mg /kg ,twice/ day for days versus vehicle injection.fibroid tumor burden was evaluated by serial mri measurement and confirmed by direct caliper measurement at time of euthanasia. sample animals were euthanized at and weeks. at that time tumor tissue, blood and most of animal organs including long bones were collected and subjected for further evaluations. hours urine samples were collected for evaluation of estrogen (e ) metabolites and bone resorption marker. results:animals treated with ro - exhibited significantly lower uterine fibroid tumor burden ( % and %) of pretreatment volume at and weeks post treatment respectively. conversely, the tumor size in control animals continued to grow and reached %, and % of pretreatment size at the same time points. ro - treatment resulted in an increase in urinary / hydroxy e metabolite ratio. in addition ro - increased bax expression and decreased parp , pcna and cyclind experssions . all ro - treated animals tolerated the treatment protocol with no signs of toxicity. h e staining of different body organs did not reveal any signs of tissue damage. furthermore, there was no significant change in both liver function tests (alt, ast, billirubin) and bone resorption marker , deoxypyridinoline croslinks, between treated and control rats. conclusion ro - , a synthetic selective comt inhibitor, caused immediate arrest of the growth of eker rat uterine leiomyoma. this effect might be in part due to modulation of various estrogen dependent genes regulating leiomyoma apoptosis (parp, bax) and proliferation (pcna, cyclin d ). this anti-estrogenic effect is due to the accumulation of the the antiestrogenic metabolite hydroxy estrogen secondary to comt inhibition.comt inhibitors might present an alternative non-surgical option for the treatment of women with symptomatic uterine fibroids. background development of uterine leiomyomas (fibroids) is the most common pathological feature in the female reproductive tract. they negatively impact patients of virtually every gynecologist. despite such morbidity, leiomyoma development is poorly understood. we have recently demonstrated that leiomyomas have a genomic expression pattern that limits retinoic acid (ra) exposure. our group and others have demonstrated that tgf-beta regulation is altered as well. these two pathways likely play central roles in leiomyoma development. the central feature of uterine leiomyomas, the extracellular matrix (ecm), is regulated by both all-trans retinoic acid and tgf- , focusing on versican as a critical ecm component. human uterine leiomyoma and patient-matched myometrium were obtained from surgical specimens under an irb-approved protocol. these tissues were immortalized and treated with either all-trans retinoic acid, tgf- , or anti-tgf- antibody. rna was isolated for qrt-pcr. human immortalized leiomyoma cells demonstrated the same increased template expression of tgf- ( . ± . fold), retinoic acid metabolizing protein (cyp a ; . ± . ), and versican variant v ( . ± . fold) as was found in the progenitor tissue. when treated with all-trans ra, expression of versican variant v decreased to levels found in myometrial cells ( . ± . fold). conversely, when treated with tgf- , expression of versican variant v increased . ± . fold. to confirm that tgf- was central to the overexpression of v , we treated leiomyoma cells with anti-tgf- antibody, and found that baseline over-expression of v template was decreased to expression levels similar to untreated myometrial cells. finally, we elucidated a link between the ra and tgf-pathways by assessing the impact of ra treatment of tgf- expression, demonstrating that tgf- template decreased to levels comparable to myometrial cell expression ( . ± . fold). the disrupted leiomyoma ecm, of which versican is a central component, defines the leiomyoma phenotype. in this study, the leiomyoma fibrotic phenotype regressed when treated with ra and increased when treated with tgf- , providing the basis for novel therapeutic interventions directed at cell differentiation and ecm formation. objectives: uterine fibroids are the leading cause for hysterectomies in the us. the lack of an appropriate in vitro cell model for the initiation of fibroid growth has hindered advancement in understanding the cellular and molecular basis for the development and progression of uterine fibroids. fibrosis is the underlying mechanism of uterine fibroid formation and myofibroblasts cells are the principal fibrogenic cell type in the uterus. we sought to develop a myofibroblast in vitro cell model for analyzing the initiating molecular events of uterine fibrosis. methods: smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and cultured in the presence of % serum until % confluent. for the next h cells were cultured in serum-free media followed by replacement with serum containing media. cells were fixed at , , , , m later. cell fine structure and cytoskeletal organization were evaluated by transmission electron microscopy. smooth muscle specific alpha-actin ( -sma) and progesterone receptors (pr) were detected by western blot. results: we observed smc differentiation into myofibroblasts, marked by the presence of notched nuclei ( figure) and the increased expression of -sma m after serum replacement. pr-a and pr-b were detectable at , and m. conclusions: the development of myofibroblasts is important in wound healing and fibrosis. we show for the first time that uterine myofibroblasts can be derived in culture from myometrial smcs. thus, these cells will be utilized as a model for developing "in vitro fibroids". this model will enable the study of myofibroblast activation, cytokine signaling, intracellular regulation of uterine fibrogenesis, production of extracellular matrix proteins and development of antifibrotic drugs. the presence of prs in our model enables us to evaluate pr mediated events in fibroid pathogenesis and treatment. this model will be more useful in determining the molecular biology of fibroid initiation than cell models derived from established fibroids that are already well past their initial stages of development. novel approach to genome-wide expression profiling analysis. liping feng, morgan walls, insuk sohn, millie behera, sin-ho jung, phyllis leppert. obgyn; biostatistics and bioinformatics, duke university medical center, durham, nc, usa. background: microarray studies have examined the differential gene expression between uterine fibroid and normal myometrium. all previous studies considered the fibroid as a whole and analyzed only fold changes. we have developed a novel statistical approach to genome-wide expression analysis comparing two fibroid tissue sites to myometrium. methods: using affymetrix tm u a genechip, we have compared the gene expression between c and e and matched adjacent m. data has been analyzed by considering the specimens per subject and subjects as individuals. we used a block one-way anova method to test if each gene was differentially expressed among the three sites. the p-value is calculated using a permutation method accounting for possible dependency among three lesions. the multiple testing issue was addressed by controlling the false discovery rate. expression values were calculated using the robust multichip average (rma) method. rma estimates are based upon a robust average of background corrected perfect/mismatch (pm) intensities. normalization was done using quantile normalization. expression values were then transformed by taking logarithm base . confirmatory rt-pcr was performed. results: we applied a hierarchical clustering analysis to all raw data sets and then displayed a dendrogram, where the height of each branch point indicates the similarity level at each generated cluster. identical gene expression among sites clusters together. due to a strong site effect, m tissues clustered separately from e and c combined. genes were differentially expressed when we used a . q-value cut off. expression data revealed concordant changes in genes regulating cholesterol biosynthesis, gene transcription, estrogen and extracellular matrix formation when both e and c were examined. cyp was detected and we report for the first time that scc- (a cell cycle-regulated molecule) folliculin and l-selectin are differentially expressed suggesting that they may be involved in the regulation of cell growth and proliferation of uterine fibroids. conclusions: our novel robust analysis of gene expression provides new clues to the relevant pathways of fibroid development. this new statistical approach that can be used in clinical and/or translational studies to identify differentially expressed genes comparing treatment regimens, cells or tissues. objectives: thrombospondin- (tsp- ) is a large matricellular glycoprotein secreted by many cell types. matricellular proteins modulate interactions between cells and their environment, regulate cell adhesion and are expressed during tissue formative processes. they are especially important in fibrosis. tsp- plays an important role in angiogenesis and is an activator of tgf - . in a previous study, we found that differential expression of tsp- in uterine fibroids may contribute to an altered healing process leading to fibrosis. this alteration in tissue response to injury initiates the development of abundant, nonaligned collagen fibrils and changes in other components of the ecm. methods: we measureed the pattern of mrna and protein expression of tsp- by rt-pcr and western blot in an in vitro serum-deprived differentiated myometrial cell (myofibroblast) model. specifically, smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and grown in primary culture to % confluence. then smc were serum deprived for h and treated back with % serum for , , , and m. cells were collected for rna and protein, and tsp- expression was evaluated. in addition, cells were stained using the combination of anti-cd and anti-smooth muscle -actin or combination of anti-cd d and anti-smooth muscle -actin as well as the appropriate single and double negative controls. stained sections were analyzed using zeiss axio imager widefield fluorescence confocal microscopy. results: tsp- mrna and protein was present in cells in this serumstarvation model after the addition of serum and the expression level remained elevated for m following the addition of serum. fluorescence staining analysis indicated that these cells were positive for human smooth muscle -actin, but negative for leukocyte antigen cd and platelet marker cd d suggesting that the myofibroblasts cells themselves were the source of the tsp- . conclusions: unlike skin wounds, where tsp- is derived from the blood macrophages, monocytes and platelets, differentiated myometrial cells appear to produce tsp- . elucidating the roles of tsp- in myometrium physiology and pathobiology will increase our understanding of the etiology of uterine fibroids and may lead to improved therapies. further studies utilizing this cell model to determine the role of tsp- in the activation of tgf - are indicated. phospholipid s p via gi, rac and rho pathways. yoel smicun, armando wu pimentel, jennifer gilman, david a fishman. obstetrics gynecology, new york university school of medicine, new york, ny, usa. objectives: sphingosine- -phosphate (s p) levels are elevated in serum and ascites of ovarian cancer patients. we have demonstrated that low concentration s p enhances while high dose s p inhibits invasion of epithelial ovarian carcinoma (eoc) cells in a dose and attachment mode dependent manner. we sought to further dissect the pathways by which s p affects invasion, using specific inhibitors. methods: dov eoc cells were pretreated for -hrs with vehicle, . m or m s p and with inhibitors for gi, p -mapk, rac and rock, thereafter cells were detached and tested for invasion towards m lpa chemoattractant in matrigel-coated chambers. conditioned media from pretreated cells and invading cells were quantified for upa activity using colorimetric assays. the significance of results was calculated by student's t-test. results: inhibition of gi mildly increased invasion of both control ( p= . ) and m s p treated cells ( p= . ). inhibition of both p -mapk and rac did not affect m s p treated cells, in contrast invasion of control cells was mildly increased (p= . , . ). inhibition of rock, a protein effector downstream of rho, highly elevated invasion of both control and m s p treated cells ( fold, p= . , fold, p= . ). both upa and gelatinase activities were higher in conditioned media of invading cells than of attached cells. gelatinase activity was enhanced by both concentrations of s p (p= . , . ). ptx fully inhibited gelatinase activity of control and . m s p treated cells, and partially of the m s p treated cells (p< . ). . m s p significantly increased upa activity of attached (p= . ) but not of invading cells. this increase was sensitive to ptx and rac inhibitor. m s p inhibited upa in both attached and invading cells (p= . ), this inhibition was rock dependent. conclusions: these findings suggest a strong inhibition of invasion and upa by the rho pathway, of both control and m s p treated cells. this inhibition is induced partially by upstream gi protein. increased invasion by . m s p is associated with elevation of gelatinase activity through gi, rac and rock pathways. this suggests that attached cells and invading cells affect upa activity through different pathways. objectives: sphingosine- -phosphate (s p) levels are elevated in serum and ascites of epithelial ovarian cancer (eoc) patients. we have demonstrated that invasion of attached eoc cells differentially react to s p as compared to invading cells. we examined the impact of the inhibitors for gi and rac on attached and invading eoc. methods: dov eoc cells were pretreated for -hrs with vehicle, . m or m s p and with inhibitors for gi (pertussis toxin (ptx)), and rac (nsc , (rac-i)), and cells were detached and assayed for invasion towards m lpa in matrigel-coated chambers. to distinguish the response of attached from invading cells, inhibition of cells pretreated with inhibitors was either continued or not in the invasion chambers. conditioned media (cm) of invading cells were quantified for upa and gelatinase activity by fluorometric and colorimetric assays. significance of results was calculated by student's t-test. results: the significant (p= . ) increase of invasion by . m s p was inhibited by both ptx and rac-i, either by pretreatment alone or by continuous treatment (p= . - . ). however, the invasion was higher in cells inhibited continuously than cells inhibited only in dishes. both inhibitors did not affect cells treated with m s p. control cells invasion was increased ( . fold, p= . ) by continuous rac-i treatment. . m s p increased gelatinolysis in cm of invading cells. this and control cells activity was inhibited by ptx pretreatment. continuous treatment with ptx or rac-i elevated and fold gelatinase activity of control and m s p treated cells. in contrast upa activity was inhibited by both . m and m s p. activity of control cells was inhibited by both pretreatment and continuous treatment with both ptx and raci. upa activity of cells treated with . m s p was increased only by pretreatment with ptx and raci. in contrast, in cells treated with m s p upa was inhibited only by continuous inhibition with ptx and raci. conclusions: invasion of s p induced eoc cells correlated with the gelatinase and upa activities in their microenvironment. ptx and rac-i affect attached and invading cells in different manner, inhibition of invasion and gelatinolysis of attached and increased invasion of invading cells. this suggests a dual effect of the gi-rac pathway, inhibiting attached and stimulating invasion via gelatinolysis of invading cells. lysophosphatidic acid (lpa) levels are elevated in the ascites and plasma of early-and late-stage ovarian cancer patients, underscoring the unique role this bioactive lipid plays in the pathobiology of epithelial ovarian cancer (eoc). lpa binding to its cognate g-protein-coupled receptor can transactivate the receptor tyrosine kinase, egfr, which is often overexpressed in ovarian tumors. in the current study, we investigated the role that lpa activation of egfr plays in the processing of the metalloproteinase, mmp- , and eoc dissemination. lpa stimulates tyrosine phosphorylation of egfr in ovarian cancer cells, and egfr kinase activity is required for optimal lpa induction of pro-mmp- activation. lpa-dependent pro-mmp- activation does not require the egfr ligands, amphiregulin, b-cellulin, egf, hb-egf, and tgf-a. we previously reported that when cells are cultured at high density, lpa represses rhoa activity to induce loss of stress fibers, and these changes in actin microfilament organization contribute to pro-mmp- activation. in the current study, inhibition of rho-kinase/rock with y- reversed the repression of lpa-stimulated pro-mmp- processing observed with treatment of the egfr kinase inhibitor, ag . correspondingly, lpa induced the loss of stress fibers, while inhibition of egfr kinase restored stress fiber formation in lpa-treated cells. this suggests that lpa acts through egfr to modulate microfilament organization and pro-mmp- processing. finally, lpa-induced cellular haptotactic migration and invasion are abrogated when egfr kinase activity is blocked. taken together, these results suggest that egfr signaling plays a critical role in lpa regulation of metastatic pathways by mediating changes in the cytoskeleton which impact protease activity. objectives: membrane-derived vesicles are active modulators of tumor dissemination; they promote and contribute to extracellular matrix degradation and tumor cell invasion. we have isolated vesicles from ascites of ovarian cancer patients and from ovarian cancer cells in vitro. here we analyzed the functional consequences of exposure of cancer cells to vesicles derived from a different type of malignancy in order to evaluate their proinvasive properties. methods: ovarian cancer (dov ), breast cancer (mda mb ) and mesothelioma (hp- ) cells were grown in media supplemented with % fbs. after serum deprivation, % vesicle-free fbs was added for hr to induce vesicle release. vesicles were isolated from media by differential centrifugation and quantified with a bradford assay. fusion to cells was followed by fluorescence of the lipophilic tracer dii. each cell line was stimulated with and g/ml of each type of vesicles and tested in a matrigel invasion assay. changes in proliferation were evaluated in an mts assay. gelatin zymography was used to assess matrix metalloproteinase activity of vesicles. results: zymographic analysis showed that vesicles contained mmp- and . fusion experiments showed that all vesicles fused to all cell types. all vesicles induced the invasion of their respective cell types in a dose-dependent manner. when vesicles were used at g/ml they induced significantly high levels of invasion in all cell types tested. at g/ml they significantly inhibited invasion in different cell types. both concentrations of vesicles stimulated cell proliferation in all cell types tested. conclusions: membrane derived vesicles are potent mediators of invasion for mesothelioma, breast and ovarian cancer cells in vitro. this effect occurs in a dose-dependent manner when vesicles induce invasion of the same cell type from which they were isolated. when vesicles are used to induce a different cell type, low concentrations induce invasion while high concentrations inhibit invasion, this effect was independent of cellular proliferation. these results suggest that a novel mechanism may be at play where activation of recognition of self and non-self specific pathways may determine the invasive potential of the tumor cell upon fusion to microvesicles. the identification of signaling pathways responsible for this heterotypic signaling is a current effort. vegfr- as a potential target to inhibit lpa-induced epithelial ovarian cancer (eoc) invasion. sonia dutta, fengqiang wang, elaine barfield, david a fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objective: vegf and vegf receptors (vegfrs) play important roles in ovarian cancer metastasis. in this study, we examined the expression profiles of vegf and vegfrs (vegfr , vegfr , co-receptor nrp and nrp ) in established eoc cells lines (dov , r , ovca , skov ) and an immortalized normal ovarian epithelium (iose- ). the effect of lysophosphatidic acid (lpa) on vegf and vegfrs expression and the effect of vegfr- silencing by rnai on lpa-induced invasion were also evaluated. methods: vegf and vegfrs expression in ovarian cancer cell lines and normal ovarian epithelium was quantified by real time pcr. cell invasion differentiate into either a pro-tumor or anti-tumor phenotype depending on the specific tumor microenvironment. previously, we described a subgroup of epithelial ovarian cancer (eoc) cells with a functional tlr- -myd -nf-b pathway (type i eoc cells). these cells have constitutive nf-b activity and constitutively secrete il- , il- , mcp- , and gro . we hypothesize that type i eoc cells, but not type ii, can promote macrophage differentiation into a tumor-supporting immune cell. methods: eoc cell conditioned media (cm) was prepared by incubating eoc cells in log-phase growth in optimem for h. migration assay was done using an in vitro cell culture insert with m-size pore and pkh red fluorescentlabeled thp- . cytokine profile of thp- cells co-cultured with eoc cell cm was determined using xmap technology. modulation of response to apoptotic bodies was determined by "pre-educating" thp- cells with eoc cell cm for h prior to exposure to apoptotic bodies ( : ratio with thp- cells). results: monocytes migrate toward eoc cells. however, migration is significantly higher towards type i eoc cells. type i, but not type ii, eoc cells alter monocytes' cytokine profile by inducing the secretion of high levels of progrowth and angiogenic cytokines il- , il- , mcp- , and gro . furthermore, type i eoc cells modify monocytes response to apoptotic bodies by inducing a significant increase in the secretion of il- , il- , mip- , mip- , and gro ( -fold, . -fold, -fold, -fold, and -fold respectively). conclusion: we demonstrate for the first time a differential interaction between two subtypes of eoc cells and monocytes. we showed that the microenvironment created by type i eoc cells is able to modify the function and differentiation of immune cells towards a tumor supporting phenotype. understanding the molecular mechanisms mediating this tumor-immune interaction will help to design appropriate immune therapies that will take into consideration the tumor microenvironment. objectives: the standard of treatment for patients with ovarian cancer (oc) is intravenous combination chemotherapy (ct) after primary cytoreductive surgery. although initial response is above %, most of these patients experience recurrence. the only approach for these patients is salvage ct which may prolong their lives for months. early detection of patients who are not responding to current therapy or are at risk of experiencing an early relapse of disease might improve response rates and survival if alternative therapy is possible. no single predictive marker has yet been proven sufficiently sensitive or specific to find a place in the daily clinic. in the present study we use a newly described biomarker test for the detection of oc (visintin et al clinical cancer research) and evaluated the ability of the test to monitor ct response methods: patients with oc, figo stage i-iv, were included in this study. all patients recieved postsurgery first line combination ct (paclitaxel/carboplatin). samples were evaluated at ) baseline (mean days after surgery), prior to the first cycle of ct, and ) after cycles of ct. μl serum samples were analyzed by multiplex assay (beadlyte® cancer biomarker panel kit) for six markers. changes during ct and differences in markers between patients with short time to progression (ttp) and patients with long ttp were determined. results: positive test for oc was observed in % of the patients evaluated at baseline. all patients had residual disease after surgery. from patients with long ttp, / patients (specificity %) had a negative test after cycles. from the patients with short ttp, / had a positive test (sensitivity %) p= . ( ²). the risk of experiencing a ttp shorter than months when having a positive test after cycles of ct was %. conclusion: we describe for the first time the use of a panel of biomarkers for oc that might have an application for monitoring ct response and risk of relapse. the test detects the presence of residual disease following debulking surgery, and differentiates between long term and short term progression. a longitudinal study is performed to determine how early during ct the test can identify responder versus non responder. ksp inhibitor (arry- ) as an alternative for paclitaxel in myd -positive epithelial ovarian cancer cells. ki h kim, ayesha b alvero, yanhua xie, david trollinger, gil mor. obstetrics, gynecology, and reproductive sciences, yale university school of medicine, new haven, ct, usa; array biopharma inc., boulder, co, usa. background: we previously described that epithelial ovarian cancer (eoc) cells ubiquitously express tlr- , but not the adaptor protein myd . when treated with paclitaxel, a known tlr- ligand, myd -positive eoc cells exhibited nf-b activation, increased secretion of il- , il- , mcp- , and gro , and increased p-erk levels . since majority of eoc patients are given paclitaxel in combination with a platinum drug, it is not only important to distinguish those patients that should not be given paclitaxel; it is also important to identify alternative chemotherapy agents that would benefit this sub-group of patients. the objective of this study is to determine if the ksp inhibitor, arry- , can be an alternative for paclitaxel in myd -positive ovarian cancer patients. methods: myd -positive and myd -negative eoc cell lines isolated from either ascites or tumor tissue were treated with increasing concentrations of arry- ( to nm) or paclitaxel ( . to m) for , , and hours and cell viability was determined using the celltiter aqueous one solution cell proliferation assay. cytokine profiling was performed from supernatants using xmap technology. nf-b activity was determined using a luciferase reporter system. p-erk levels were measured by western blot analysis. results: arry- and paclitaxel exhibited the same cytotoxic effect on myd -negative and positive eoc cells. the ic at h for myd -negative eoc cells was . m and . um for arry- and paclitaxel, respectively. for myd -positive eoc cells, the ic at h was m and m for arry- and paclitaxel, respectively. however, unlike paclitaxel, arry- did not induce nf-b activation or enhance the secretion of il- , il- , mcp- , and gro , and did not induce erk phosphorylation on myd positive cells. conclusions: administration of paclitaxel to patients with a myd -positive tumor could have detrimental effects due to the paclitaxel-induced enhancement of cytokine production which promotes chemoresistance and tumor growth. arry- has similar anti-tumor activity in eoc cells as that of paclitaxel. however, unlike paclitaxel, it does not induce cytokine production in myd positive eoc cells, and therefore, the ksp inhibitor arry- may represent an alternative to paclitaxel in this subgroup of eoc patients. introduction: nf-b activation has been associated with cell proliferation, angiogenesis, metastasis and suppression of apoptosis in ovarian cancer. in addition, nf-b activity induces the production of pro-inflammatory cytokines which may contribute to chemoresistance. inhibition of nf-b may represent a new approach to prevent tumor growth and reverse chemoresistance. in the present study we described the characterization of a novel nf-b inhibitor, eriocalyxin b (erib) that is able to re-establish the apoptotic cascade in chemoresistant ovarian cancer cells by suppressing pro-inflammatory cytokines and antiapoptotic proteins. materials and methods: eoc cell lines were isolated from malignant ovarian cancer ascites. caspase activity was determined by caspase-glotm assay. cytokine production and secretion were determined using multiplex assay. erib effect on cancer cells was evaluated in a time and dose dependent manner using celltiter cell proliferation assay. protein expression was determined by western blot analysis. combination studies were done with paclitaxel and carboplatin in addition to erib treatment. nf-b activity was determined by monitoring the expression of a nf-b luciferase reporter. results: erib decreased cell viability of ovarian cancer cells with an ic of . - μm in hours and was associated to increasing levels of caspases , and activity. intracellular changes induced by erib included: ) inhibition of nf-b activity; ) decrease in cytokine production; ) down regulation of anti-apoptotic proteins xiap and flip, and reversal of resistance to tnf-and fasl-mediated cell death; and ) chemosensitization to carboplatin and paclitaxel. at present, evidence is accumulating regarding the existence of unique populations of specialized tumor-initiating, stem-like cells within various tumor types of distinct origins. these cancer stem cells (csc), with characteristics reminiscent of normal stem cells, are thought to be responsible for driving tumor growth. we propose that ovarian cancers arise from csc, and are using microarray-based technology to identify specific genes/cell surface markers associated with ovarian csc that will permit distinction of these rare cells from the remaining tumor bulk. to identify unique gene signatures associated with an ovarian tumor-initiating cell population, we have utilized an in vivo serial transplantation model. this model selects for primary human ovarian tumor cells with increased tumorigenic capacity, given that time to tumor formation decreases with successive serial transplant despite fewer cells injected. our initial studies used cells derived from a human ovarian clear cell carcinoma, serially transplanted for three passages in nod/scid mice. rna derived from these transplanted tumors was analyzed on human genome microarrays. from these analyses, several differentially expressed genes were identified. the differential expression noted for potential genes of interest is currently being validated by rt-pcr. of particular interest, expression of the transmembrane glycoprotein muc was found to increase both at the rna and protein level with successive transplant of this clear cell carcinoma. further studies are ongoing to determine the functional significance of muc and other identified differentially expressed genes in ovarian clear cell cancer. in addition, we are carrying out parallel microarray analyses in other ovarian tumor subtypes. background recent observations suggest a decreased incidence of neoplastic lesions in hiv infected individuals treated with highly active anti-retroviral therapy comprised of protease inhibitors, such as ritonavir. the polycomb group protein ezh is associated with aggressive human malignancies via transcriptional suppression of dna repair proteins. objective objective of the present study was to assess the antineoplastic impact of ritonavir on epithelial ovarian cancer (eoc) cell lines. methods eoc cell lines (mdah and skov- ) were treated with serial dilutions of ritonavir ( - mm) dissolved in dmso. normal diploid human fibroblasts served as controls. growth curves, apoptosis and cell cycle analysis were performed with cell counting kit- , annexin v and flow cytometry. signal transduction was studied with western blotting. dna double strand breaks (dsb) were induced with . mm etoposide treatment for hours. homologous recombination (hr) repair of dna damage was measured by assessment of rad foci formation in the nucleus of the cells as visualized by fluorescence microscopy according to previously published criteria. untreated eoc cells expressed higher levels of ezh and lower levels of rad and xrcc as compared to controls and in turn, had lower prevalence of rad repair foci formation in response to dsb. serial treatments of eoc cells with ritonavir resulted in a decrease in expression of ezh and an increase in expression of rad and xrcc when compared to untreated eoc cells. after induction of dsb, the rad repair foci formation was significantly more prevalent in eoc cells treated with ritonavir as compared to untreated eoc cells. in addition, ritonavir induced apoptosis in ovarian cancer cell lines by down-regulation of akt pathway and caused g cell cycle arrest mediated by down modulating levels of prb phosphorylation and depleting the cyclindependent kinase and . ritonavir effectively induces apoptosis, cell cycle arrest and improves repair of dna damage by hr in ovarian cancer cell lines. as impaired hr is a key event in causation and progression of neoplastic lesions, ritonavir may have a role in chemoprophylaxis and treatment of human malignancies. a rare case of ovarian cystic lymphangioma. tomer singer, tamer seckin, noa feldman, susan jormark, michael divon. department of obstetrics and gynecology, lenox hill hospital, n.y., ny, usa; department of pathology, lenox hill hospital, n.y., ny, usa. cystic lymphangioma (cl) is a rare, benign malformation of the lymphatic system whose exact etiology remains uncertain. cl may arise in different sites: the spleen, the mediastinum, the axillary region, the retroperitoneum, and the mesentery. retroperitoneal cl is extremely rare and its true incidence is unknown. the majority of cases are symptomatic during childhood. clinical presentation of adult cl is variable and may be misleading. typically, this is a slow-growing tumor and it remains asymptomatic for a long period of time. it is most often found incidentally during abdominal or pelvic imaging studies, surgeries or autopsies. total surgical removal of the lesions with microscopically clear margins is the best approach when it is possible. we report, for the first time, a case of cystic lymphangioma arising from the ovary in a post-menopausal woman and present the feasibility and the advantages of laparoscopic surgery, allowing accurate diagnosis, optimal treatment and minimal risk for the patient. lgsc) is a chemoresistant ovarian neoplasm that has been molecularly linked to low malignant potential tumor (lmp), which often behaves in a non-invasive fashion. micropapillary features within lmp (lmp-mp) are associated with increased invasive behavior. the aim of this study was to determine the differential gene expression of lmp, lmp-mp, and lgsc to identify genes involved in malignant transformation and carcinogenesis. methods: laser capture microdissection was used to isolate epithelial cells from snap-frozen lmp (n= ), lmp-mp (n= ) and lgsc (n= ). rna was extracted, amplified, reverse transcribed to cdna and hybridized to affymetrix u plus arrays. data was analyzed by significance analysis methods: medical records were reviewed for patients who underwent hysterectomy for all indications at wsu hospitals from / / - / / . pathology reports were reviewed to identify the incidence of adenomyosis. data obtained from medial records included: age, race, insurance, bmi, social history, medical history, and presenting symptoms. the correlation between adenomyosis and all the above factors was tested using the appropriate statistical methods. results: patients were included. adenomyosis was confirmed by pathology in patients, an incidence of . %. incidence was not significantly different among races after controlling for parity. . % in african americans, . % in caucasians, and . % in others. incidence was not statistically different between uninsured( . %), privately insured ( . %), and medicaid ( . %) patients. p=. . incidence of adenomyosis was not different between smokers ( . %) and nonsmokers ( . %). p= . . table i shows the correlation between adenomyosis and different risk factors. conclusion: adenomyosis was diagnosed in . % of hysterectomy specimens. race, socioeconomic status or social habits didn't affect its incidence. diabetes and endometrial cancer were negative risk factors for adenomyosis, whereas htn, hypothyroid, breast cancer, fibroids, polyps and endometriosis didn't affect its incidence. menorrhagia, dysmenorrhea, dyspareunia, and chronic pelvic pain but not metrorrhagia had a positive correlation with adenomyosis. there is a protective adipocytokine profile. we hypothesize that this finding may precede some ill-defined threshold of fat mass and/or insulin resistance after which adiponectin decreases. the objective: to correlate reproductive hormone production with menstrual bleeding patterns among women in the menopause transition. methods: a sub-cohort of the swan study consisting of women age - was studied. each woman collected daily first void urine samples for one complete menstrual cycle or days (whichever came first) once a year for years. urine was assayed for excreted levels of fsh, lh, estrogen metabolites and progesterone metabolites which were normalized for creatinine concentration. ovulation was detected by a validated algorithm. menstrual bleeding parameters were derived from daily calendars. correlations between bleeding characteristics, hormone concentrations, and other potential clinical predictors were analyzed using multivariate logistic regression models. results: the cohort was ethnically diverse with a median age of and with % in the early perimenopause at the start of the study. % of all cycles were anovulatory. short cycle intervals (< days) were associated with the early perimenopause (or . , ci . , . ) and with anovulation (or . , ci . , . ). long cycle intervals ( + days) were associated with late perimenopause (or . , ci . , . )and with anovulatory cycles (or . , ci . , . ). both short ( - days) and long ( + days) duration of menstrual bleeding were significantly associated with anovulation (or . and . , respectively). women with anovulatory cycles were less likely to report heavy menstrual bleeding than women with ovulatory cycles. menorrhagia was not associated with steroid hormone concentrations but was associated with obesity (or . , ci . , . ) and with the self-reported presence of fibroids (or . , ci . , . ). conclusions: among women in the menopause transition, abnormalities in timing of menstrual bleeding (cycle intervals or bleeding duration) have a hormonal basis and are frequently associated with anovulation. in contrast, abnormally heavy periods do not have a hormonal basis and are less likely following anovulatory cycles. heavy periods are associated with obesity and fibroids. to undetectable levels. the use is vaginal e is contraindicated, although these women have even higher rates of av. topical testosterone (tt) has successfully treated vulvar atrophy; testosterone receptors are also present in the vaginal epithelium. objective: assess the effect of tt on vaginal maturation index (mi) and relief of av symptoms. methods: postmenopausal women on aromatase inhibitor with symptomatic atrophic vaginitis were enrolled in prospective, irb approved study. estradiol (e) and testosterone (t) levels, av questionnaires (score - ; none to most severe symptoms of dryness, irritation, and dyspareunia), gynecologic exam, and vaginal smears (for ph and vaginal maturation index, mi, by meisels criteria) were performed at baseline and after month of daily treatment with mcg of tt. data was assessed by t test and fischer's exact test; significant p < . . results: e levels were undetectable at baseline and following treatment. t levels increased (mean +/-sem) from baseline ( +/- . ) to treatment ( +/- . ); the difference was not significant; p = . , although one patient had an appreciable rise in serum t level. two av symptoms improved significantly with tt use; comparing baseline to treatment scores: vaginal dryness ( . vs. . ; p = . ) and dyspareunia ( . vs. ; p = . saliva analysis is a convenient, non-invasive and rapid method for assessing estradiol (e ) levels. however, particularly in postmenopausal women, the low salivary e levels are often near or below the sensitivity of available assays, compromising both accuracy and precision. we present results using an extraction step prior to e assay, which concentrates the sample to increase sensitivity and removes potentially interfering substances. morning saliva samples were obtained from premenopausal (mid-luteal phase, n= , ) and postmenopausal women (n= , ) not taking hormones, and from postmenopausal women receiving oral conjugated equine estrogens (cenestin, n= ; premarin, n= ), oral micronized e (estrace, n= ; compounded e , n= ), transdermal e patches (climara, n= ; vivelle, n= ); or topical e cream (compounded e , n= ). e levels were determined by an automated enzyme immunoassay (eia) after solid phase extraction. the functional sensitivity of the assay was determined to be . pg/ml, compared with > pg/ml without extraction. . . . . . oral conjugated equine estrogens; oral e ; e patch; topical cream salivary e levels corresponded with the hormone dosage, suggesting a reliable assessment of unbound e levels with each formulation, dosage and type of estrogen therapy. extraction prior to eia in an automated assay dramatically increased precision and accuracy at low concentrations. omitting the extraction step may have contributed to poor serum versus saliva correlations in other studies. this method may therefore allow reliable monitoring of estrogen therapy without the need for expensive and inconvenient blood tests. the role of fibulin- in pelvic organ prolapse. david d rahn, jesus f acevedo, patrick w keller, lihua marmorstein, r ann word. ob-gyn, ut southwestern, dallas, tx, usa; visual sciences, univ arizona, tuscon, az, usa. objectives: fibulin- (fib- ) is crucial for normal elastic fibers in the protein showed a statistically significant reduction in its expression (p= . ). lox, loxl , and proteins were detected by immunohistochemistry in all three layers of vaginal skin biopsies: ( ) stratified squamous epithelium; ( ) the lamina propria and ( ) the muscularis layer from both patients with pop and controls. significantly, in both groups we detected a numerous macrophages scattered throughout the vaginal thickness which displayed a very strong immunostaining to loxl . patients with severe pop showed reduced expression of proteins regulating collagen and elastin biogenesis. our finding raises the possibility that failure of ecm homeostasis could underlie the etiology of pop in women. fibroblast proliferation is regulated by hoxa : molecular implications for pelvic organ prolapse. kathleen a connell, marsha k guess, richard bercik, hugh s taylor. obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa. objectives: the integrity of the extracellular matrix (ecm) is maintained by a delicate balance between collagen synthesis and degradation. previously, we have demonstrated that hoxa is essential for the development of the uterosacral ligament in mice and regulates the expression of collagen type iii and mmp . we have also shown that hoxa is deficient in the uterosacral ligament of women with pelvic organ prolapse (pop) compared to women with normal support. the exact mechanisms by which hoxa regulates pelvic organ integrity and repair remains to be elucidated. the aim of this study was to determine the effect of hoxa expression on fibroblast proliferation, and its potential role in pop. methods: nih t cells, a murine fibroblast cell line, were seeded onto a six well plate ( x cells/well) and transfected with either a vector carrying a hoxa cdna or empty vector alone as a control. immunohistochemistry using bromodeoxyuridine (brdu) was performed to evaluate cell proliferation. cell division in the uterosacral ligament (usl) was also compared in women with and without pop. usl specimens were obtained at the time of hysterectomy for benign disease. immunohistochemistry was performed on paraffin embedded sections of the usl to evaluate expression of two mitotic markers, cyclin b and phospho-histone . results: constitutive expression of hoxa in murine fibroblasts resulted in significantly higher proliferation. cells transfected with hoxa had a mean brdu incorporation of . + . cells/ cells compared with . + . cells/ cells in controls (p= . ). in the usl obtained from women with and without pop, cell proliferation as determined by cyclin b and phospho-histone expression was not significantly different. cyclin b and phosphor-histone were expressed in comparable numbers of cells in both groups. conclusion: hoxa is necessary for usl development and promotes proliferation of adult fibroblasts. hoxa may have a similar function in vivo in usl, and may regulate fibroblast proliferation during growth and the acute phase response following trauma when fibroblasts are activated to proliferate and remodel the ecm. it is likely that hoxa mediated proliferation of usl fibroblasts contributes to the tensile strength and resilience of these structures and thereby prevents pop. supported by nichd wrhr: k hd - . connective tissue composition, in term of collagen i, iii and proteoglycans content, in support and nonsupport structures of women with uterine prolapse. elisabetta trabucco, gennaro acone, sara d'avino, marco torella, gennaro trezza, annamaria marenna, nicola colacurci, luigi cobellis. department of obstetrics and gynecology, second university of naples, naples, italy; loreto mare hospital, naples, italy. objective. connective tissue consists mainly of collagen and elastic fibers, glycoproteins and proteoglycans (pgs) and is considered an important factor of the support and nonsupport structures of the genitourinary region. it has been already demonstrated altered morphologic features in the pelvic support connective tissue in women with genital prolapse. however, analysis of nonsupport tissue may provide a more accurate reflection of body collagen. the objective of our study was compare the expression of collagen i, iii and four pgs (decorin, fibromodulin, lumican, biglycan), essential for synthesis and regular assembly and diameter of the collagen fibrils, in uterosacral ligaments and in a nonsupport tissue, the uterine cervix, between premenopausal women with uterine prolapse respect to age matched controls. we characterized uterosacral ligaments and uterine cervix of premenopausal women with uterine prolapse and controls. this immunohistochemical study was performed on paraffin-embedded sections. results. uterosacral ligaments of the prolapsed uteri are characterized by a higher expression of collagen iii, decorin, fibromodulin and byglican and a lower quantity of collagen i. no differences in the immunoreactivity of lumican between the two patients groups. the abnormalities of support connective tissue composition are not observed in the uterine cervix of patients with prolapse. conclusions. our results suggest an altered remodeling of connective tissue in the ligaments of premenopausal patients with prolapse, with a significant decrease in collagen i content and an increase in collagen iii and pgs expression . in the prolapse patients this abnormal collagen metabolism and organization, mainly related to the observed change in pgs expression, might affect significantly the tensile strength of the connective tissue and consequently the support that is provided by the suspensory apparatus to the uterus. the alterd remodeling of support tissues, not detectable in nonsupport tissues, may suggest a predominant role of local biomechanical stresses (childbirth, chronic straining) in the pathogenesis of prolapse respect to systemic connective tissue deficiency. objective: to achieve vaginal delivery with minimal maternal injury, vaginal smooth muscle (sm) contractility likely decreases. the objective of this study was to characterize changes in rat vaginal sm contractility in pregnancy that affords circumferential vaginal distension. methods: a tubular segment of the vagina of virgin, late pregnant , immediate and late post vaginal delivery rats were mounted in an organ bath between a force transducer and an adjustable support block. dose response curves to norepinephrine (ne) and potassium (k+) were used to assess contractility. relaxation capacity was determined in ne preconstricted vagina, using cumulative doses of sodium nitroprusside (snp). differences in active vs. passive length-tension curves were used to measure sm contractility relative to the vaginal wall forces. sm -actin levels were measured using quantitative confocal immunofluorescence. results: cumulative doses of ne induced a maximum constriction force of . ± mn/g in virgin vagina while no measurable force was generated in response to ne in late pregnant or postpartum vaginas. virgin vagina relaxed with cumulative doses of snp; no measurable relaxation response was observed from pregnant or postpartum animals. in the presence of the high dose k+ ( mm), virgin vagina generated the greatest contractile force ( . ± mn/g) vs. late pregnant vagina ( . ± . mn/g, p< . ) or immediate post partum vagina ( . ± mn/g, p= . ). four week postpartum k+ induced forces were similar to virgin levels ( . ± mn/g). sm force generation (difference in active vs. passive length tension curves at mm of displacement) was decreased in late pregnancy ( . ± mn/g) compared with virgin ( ± mn/g, p < . ) and week postpartum vagina ( ± mn/g, p < . ). the expression of sm -actin was lowest in late pregnancy ( ± . , p < . ) and immediate postpartum vagina ( ± . , p < . ) relative to virgin ( ± ) with a return to virgin levels week postpartum ( ± ). conclusions: vaginal sm contractility diminishes in pregnancy. the altered contractility is mirrored by a decrease in sm -actin protein expression. near complete recovery to pre-pregnancy levels occurs by weeks post partum. these functional and biochemical changes likely represent maternal adaptations designed to minimize trauma during passage of the fetus(es). background: diagnosis or exclusion of endometriosis usually requires laparoscopy and peritoneal biopsy. we have described a novel and consistent observation of small nerve fibers in the functional layer of eutopic endometrium in women with endometriosis (tokushige et al, ) . these nerve fibers are not present in women without endometriosis. this finding allows the possibility endometriosis in the nude-mouse and inhibited mmp- expression in human endometrial stroma. the present findings may lead to the development of novel treatments of endometriosis involving statins. supported by nih u hd . k-ras actived immunocompetent retrograde menstruation model of endometriosis. ching-wen cheng, , di licence, , cristin g print, , d stephen charnock-jones. , pathology, university of cambridge, cambridge, united kingdom; obstetric gynaecology, university of cambridge, cambridge; department of molecular medicine pathology, university of auckland, auckland, new zealand. objective: to establish a new murine model of endometriosis that mimics the retrograde menstruation theory using immuno-competent mice. method: ovariectomised donor female k-ras v +/-/cre +/+ /rosa r-lacz +/+ transgenic mice were treated sequentially with steroid hormones. to induce decidualization and activation and k-ras transgene, mg/kg b-nf dissolved in maize oil was injected into the uterine lumen. tissue degeneration mimicking menstruation was induced by hormone withdrawal. menstruating endometrial tissue was collected from donor mice hours after last hormone treatment, re-suspended in matrigel, and implanted subcutaneously in female c bl/ recipients. immunohistochemical and morphometric methods were used to characterize the endometriosis-like lesions. microarray analysis (illumina, n= in each group), was used to study the molecular changes in "menstruating" uteri following k-ras activation. result: simple transplantation of decidualised endometrial tissue into immunocompetent animals does not lead to endometriotic lesion development but using tissue with the genetic modification described here overcomes this. viable endometriosis-like lesions are visible days after implantation. these lesions are histologically similar to those seen in man with intact glands, functional blood vessels, leukocyte infiltration and collagen deposition. statistical analysis revealed that transcripts were differentially regulated in the ras activated and control tissue. gene ontology (go) analysis indicated that transcripts associated with epithelial cell function and differentiation were over represented within this gene set (chloride transport and epidermis development) as were those associated with the acute inflammatory response and neutrophil chemotaxis. we developed a new model of endometriosis using immunocompetent mice to mimic retrograde menstruation induced endometriosis. this permits for the first time, the ready use of transgenic and knock-out tools to investigate the cellular and molecular mechanisms underlying endometriosis. since the animals have an intact immune system it also allows the testing of therapeutic agents that modify the inflammatory response. stra is expressed and induced by progesterone in the endometrium and is absent in endometriosis. mary ellen pavone, serdar e bulun, scott s reierstad, joy innes, magdy p milad, you-hong cheng. obstetrics and gynecology, northwestern univeristy, chicago, il, usa. objective: retinoic acid (ra) plays important roles in development, growth and differentiation by regulating the expression of target genes. in the mouse endometrium, ra deficiency leads to hyperkeratinization, while high concentrations of retinoids promote secretory characteristics. this leads many to believe that ra mediates important actions of progesterone in the endometrium and may account for progesterone resistance in endometriosis. the mechanism for regulation of ra production by progesterone is unknown. moreover, the conversion from retinol to retinoic acid is not different between normal endometrium and endometriotic tissue. we hypothesize that retinol intake into cells rather than conversion of retinol to ra is the critical step that determines ra activity in the endometrium and endometriosis. stra , a widely expressed multitransmembrane domain protein and member of a group of "stimulated by retinoic acid" genes, has been identified as the retinol binding protein receptor. it is strongly expressed in adult mice in several tissues including the female genital tract. we hypothesize that ra activity in the endometrium may be regulated by stra . here we investigate the differential expression of stra in the endometrium and endometriosis. design: we studied primary stromal cells isolated from the eutopic endometrium of disease free women and walls of ovarian endometriomas from women with endometriosis with respect to stra expression and regulation by progesterone. materials and methods primary culture of eutopic endometrial and endometriotic stromal cells were treated with - m estradiol (e ), - m r , a progesterone agonist, or vehicle for hours. real-time pcr was employed to quantify stra mrna levels. we treated cells for hours and quantified protein levels by immunoblotting. results basal mrna of stra levels in endometrial cells (n= ), were > , fold higher than those in vehicle-treated endometriotic cells (n= , p<. ). in endometrial stromal cells (n= ), r stimulated stra mrna levels by . - fold. r induced stra protein levels in endometrial stromal cells (n= ). conclusions stra is highly expressed and regulated by progesterone in endometrial stromal cells, whereas it is practically undetectable in endometriotic cells. stra may mediate important actions of progesterone in endometrium. upregulation of aromatase expression in endometrial cells disseminated into the peritoneal cavity (pc) may influence their survival and persistence via local estrogen synthesis. the inducing factors for the upregulation of aromatase in endometriosis are not well defined, but increased expression of sf- has been suggested to play an important role. given that the aromatase substrate androstenedione (a ) is the predominant steroid in peritoneal fluid, we aimed to determine whether a has a role in the regulation of aromatase expression in human endometrium. we found that culture of primary endometrial stromal cells and explants with a ( - nm) for h significantly upregulated expression of aromatase mrna transcripts containing exon iia at their '-ends. moreover, in human endometrial surface epithelial cells (hes), dose-response studies with a ( - nm) revealed that nm a maximally upregulated expression of both aromatase and sf- . when tissue samples were evaluated from women with endometriosis and control endometrium, expression of aromatase mrna mirrored sf- . treatment of hes cells ( h) with tritiated a demonstrated its metabolism to estradiol (e ), testosterone (t), dihydrotestosterone (dht) and androstanediol (a-diol). although equimolar concentrations of a , t and e upregulated aromatase and sf- mrna expression in hes cells, the nonaromatizable androgens, dht and a-diol, had no effect. a positive feedback role of estrogen in aromatase upregulation was suggested by the finding that the estrogen receptor antagonist, ici , , markedly diminished aromatase and sf- mrna expression induced by a . finally, chromatin immunoprecipitation revealed that a significantly enhanced recruitment of sf- to its response element (- bp) upstream of cyp exon iia. collectively, our findings strongly suggest that exposure of endometrial cells within the pc to c -steroids may cause an acute upregulation of cyp gene expression through their aromatization to estrogens. thus, estrogen may play a critical positive feedback role in the pathogenesis of endometriosis. supported by nih r -dk . the hpa axis and depression/anxiety scores in chronic pelvic pain patients. b stegmann, b frank, j gemmill, g chrousos, m ballweg, p stratton. rbmb, nichd/nih, bethesda, md; som, wake forest university, winston-salem, nc; endometriosis association, milwaukee, wi. stress, pain, anxiety and depression adversely affect the hypothalamic-pituitaryadrenal (hpa) axis. we examined the influence of depression and anxiety on the hpa axis response to corticotropin-releasing hormone (crh) testing in women with and without chronic pelvic pain (cpp). methods: healthy women, aged - , with without cpp, with regular menses and off hormonal contraception were studied. none had pelvic infections, untreated depression, manic depression, fibromyalgia, or chronic fatigue syndrome. after ovine crh injection ( mcg/kg), serial blood samples (- , , + , + , + minutes) were obtained for acth and cortisol measurements. depression and anxiety were scored using the duke quality of life questionnaire. hpa response was abnormal if peak acth levels were > pg/ml without a rise in cortisol levels. subject groups were: no pain and normal acth response (npnr), pain and normal acth response (pnr), no pain with an abnormal acth response (npar) and pain with an abnormal acth response (par). student t-test was used for comparisons. results: women ( cpp, controls) had a mean age of . ± . years (range: - ). women responded normally ( pnr, npnr) and women responded abnormally ( par, npar). peak and absolute rise in acth were significantly higher in abnormal (a) vs normal (n) responders; (peak: . a vs . pg/ml n, p< . ; absolute: . a vs . pg/ml n, p= . ). there was a significant difference within groups: peak acth: . par vs . pg/ml pnr, p= . ; . npar vs . pg/ml npnr, p< . ; absolute acth: . par vs . pg/ml pnr, p= . , . npar vs . pg/ml npnr, p< . ). however, total cortisol level was not different between or within the groups ( . a vs . mcg/dl n, p= . ). mean depression score did not differ among cpp patients ( . par vs . pnr, p= . ), but differed among controls ( npar vs . npnr, p= . ). anxiety scores did not differ between or within groups ( . npar vs . npnr p = . ; par vs . pnr, p= . ). conclusions: chronic pelvic pain is associated with an abnormal hpa response, regardless of anxiety or depression symptoms. abnormal hpa responses in control women, however, appear to be influenced by depression. the mechanism and clinical significance of these findings should be explored. support: rbmb/nichd/nih and endometriosis association. objective: the eutopic endometrium in women with endometriosis demonstrates altered endometrial receptivity and altered gene expression. it is unknow if the endometrium is defective giving rise to a predisposition toward endometriosis or alternativly if the endometriosis causes the altered endometrial receptivity. here we created experimental endometriosis in a mouse model through allotransplantation of the uterus to the peritoneal cavity in immunocompetent mice and examined the expression of several markers of endometrial receptivity in the eutopic endometrium. materials and methods: the uterus of -week-old cd female mice was transected at cervicovaginal junction and each horn divided and transplantated into the abdomidnal cavity of eight cd mice. seven controls received sham surgery only. after weeks the uterus was removed, snap-frozen in trizol. total rna was extracted and cdna generated. quantitative real time rt-pcr using sybrgreen was performed and normalized to -actin. fold changes in normalized hoxa , hoxa , bteb , emx , igfbp , integrin - , total progesterone receptor (pr-ab) and progesterone receptor-b (pr-b) were assessed. all experiments were conducted in duplicate, repeated at least three times and compared using mann-whitney rank sum test. results: hoxa , hoxa , bteb , emx , and igfbp mrna expression showed . -fold (p= . ), . -fold (p< . ), . -fold (p= . ), . fold (p= . ), and . -fold (p< . ) decrease in the uterus of mice with experimental endometriosis compared with controls. pr-ab and pr-b mrna showed . -fold (p= . ) and . -fold (p= . ) increase in endometriosis group compared to controls, respectively, however the ratio of pr-b to pr-ab (b/ab) showed no significant change in both groups ( . vs. . , endometriosis vs. control, p> . ). there was no significant change in integrin - mrna expression ( . -fold, p> . ). conclusion: we demonstrate significant changes in multiple markers of endometrial receptivity in eutopic endometrium after induction of endometriosis. these findings suggest that origionally normal endometrium can develop defects with the creation of endometriosis; an abnormal endometrium is not a prerequisite for the development of endometriosis or associated abnormalities. this data also suggest the existance of a signal conduction pathway from endometriosis that alteres endometrial gene expression. endometrial expression patterns of relaxin and relaxin receptor mrna suggest involvement of relaxin in endometriosis. sara s morelli, felice petraglia, gerson weiss, stefano luisi, pasquale florio, jeff gardner, andrea wojtczuk, laura t goldsmith. obstetrics, gynecology and women's health, new jersey medical school, newark, nj, usa; pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. objective: in normal endometrium, relaxin is a potent inhibitor of matrix metalloproteinases, which have been implicated in the invasive process of endometriosis. we tested the hypothesis that relaxin plays a role in endometriosis by comparing expression of relaxin and its lgr receptor in normal human endometrium to levels in samples from patients with endometriosis. materials and methods: total rnas, extracted from ectopic (n= ) and eutopic (n= ) endometrium of patients with endometriosis and from endometrium of normal controls (n= ), were reverse transcribed into cdnas. real-time pcr was performed using primers previously shown to identify human lgr relaxin receptor mrna, h relaxin mrna, and beta-actin mrna, with sybr-green detection of double stranded dna products. the comparative c t method ( -ct ) determined relative lgr and relaxin mrna expression (normalized to beta-actin mrna expression). relaxin mrna was detectable in normal endometrium from of ( %) control patients. in contrast, relaxin mrna was detectable in a lower proportion of samples [ of ( . %)] from patients with endometriosis, among whom relaxin mrna was detectable in a lower proportion of ectopic samples [ of ( %)] than in eutopic samples [ of ( . %)]. lgr relaxin receptor mrna was detectable in all samples, with lower expression in endometriosis samples than in endometrium from normal controls. relaxin receptor lgr mrna levels vary with cycle phase, with greater expression in the secretory phase (sp) than in proliferative phase (pp): in normal controls . -fold higher levels in the sp than pp; and in endometriosis patients . -fold higher levels in the sp than pp. in both phases, lgr mrna levels were lower in ectopic samples than in either eutopic samples ( . -fold lower in pp and . -fold lower in sp) or endometrium from normal controls ( . -fold lower in pp and . -fold lower in sp). eutopic endometrium had similar lgr mrna expression to controls throughout the cycle. decreased local expression of relaxin and relaxin receptor mrna in ectopic endometrium from patients with endometriosis throughout the menstrual cycle suggests that relaxin may be protective against endometriosis. ovarian reserve after laparoscopic cystectomy of endometriotic ovarian cysts. shinichi hayasaka, takashi murakami. obsterics and gynecology, tohoku university, sendai, japan. objective laparoscopic ovarian cystectomy is generally recommended for endometriotic ovarian cysts because it has been associated with a higher pregnancy rate and a lower recurrent rate. however, residual ovarian function after laparoscopic cystectomy of endometriotic ovarian cysts has been questioned. in this study, we retrospectively evaluated ovarian response to hyperstimulation in women selected for ivf and icsi cycles who previously underwent laparoscopic cystectomy of a monolateral endometriotic ovarian cyst. methods a retrospective review of the patients between january and september was performed. the operated ovary and contralateral intact ovary were compared in terms of number of oocytes retrieved, number of follicles with a mean diameter > mm at the time of hcg administration. the patients who had recurrent endometriotic ovarian cysts were excluded. in total, ten patients were identified. the mean(±sd)number of oocytes retrieved was . ± . in the control ovary and . ± . in the previously operated ovary(p= . ). the mean(±sd)number of follicles with a mean diameter > mm was . ± . in the control ovary and . ± . in the previously operated ovary(p= . ). the age of patients and diameter of the operated ovary had little influence on the difference between the response of the control ovary and one of the previously operated ovary. laparoscopic cystectomy of endometriotic ovarian cysts is associated with a significant reduction in ovarian reserve. background pre-eclampsia (pe) is associated with systemic maternal endothelial dysfunction, which is thought to result from the presence of circulating factors released following placental damage. it is hypothesised that this occurs as a result of reduced oxygen (o ) delivery. some features of placental pathology seen in pe, such as increased apoptosis, can be reproduced by culture of placental explants in % o . metabolomics operates to study 'all' metabolites within a biological system. this strategy has previously identified differences in maternal plasma between normal pregnancies and those complicated by pe. in these experiments we used metabolomics to investigate differences in the metabolic profile of explants cultured in different o tensions. hypothesis the metabolic profile of placental villous explants is altered by culture in different o tensions. methods placental villous explants were cultured with either % serum (n= ) or in serum-free conditions (n= ) for h in %, % and % o . after h, conditioned culture medium and tissue were collected and snap frozen. tissue was homogenised in % ice cold methanol prior to analysis. samples were analysed using gas chromatography-mass spectroscopy (gc-ms). samples cultured at %, % and % o were compared to identify concentration differences in metabolites in conditioned cultured medium and tissue lysate. kruskal-wallis test was used for statistical analysis. due to the large number of metabolites identified, a p value of . was considered significant. results the mean intra-assay variability was . %. there were no differences in the metabolic profile of conditioned culture medium from % and % o . several metabolites differed in culture medium from % compared to % and % o including: deoxyribose, glycerol and threionic acid. these changes were present in both serum and serum-free conditions. using these metabolites alone, culture in % o could be completely discriminated from % and % o . deoxyribose was also elevated in tissue lysate from % o compared to % o . conclusion this metabolomic strategy can identify differences in metabolic profile in placental tissue cultured in % o . these novel compounds may provide further insight into pathophysiology of pe. objective a strategy of metabolic footprinting (the study of extracellular metabolites, which are related to intracellular metabolism) was used to detect a wide array of low molecular weight metabolites. metabolic profiles were employed to differentiate between placental explants cultured at different oxygen tensions. two separate studies were combined to validate initial observations. methods metabolic footprints of placental villous explants, obtained from uncomplicated pregnancies, (n= ) were cultured for h in %, % and % oxygen. conditioned culture medium was then collected and frozen, prior to analysis by uplc/ltq-orbitrap mass spectrometry in both negative and positive ion mode. samples cultured at %, % and % were compared to identify differences in the metabolic profiles. this procedure was repeated for different explants and the results compared across the studies in order to validate initial findings. approximately unique peaks were detected in both sample sets in negative ion mode and peaks in positive ion mode. a number of metabolites were identified as being significantly different using kruskal-wallis (pval< . ) under different oxygen tensions. some differences were seen between the results obtained from both runs due to separate batch preparation of the medium but good reproducibility was obtained for many metabolites between batches. metabolites of biological interest included amongst others -deoxyribose, valine, tyrosine and aspartic acid. the largest differences were those seen between o % and o %. comprehensive metabolic profiles were detected and employed to identify differences in the metabolic footprints of explants cultured under differing oxygen conditions. a number of biologically interesting metabolites were characterized. objective: brain weight and dna content were reduced in leptin deficient (lep ob /lep ob ) mice postnatally. leptin is detected in the sera and its receptors are expressed in the brain of the mouse fetus. we examined the role of leptin in the proliferation and differentiation of neural lineage cells in the mouse fetus. methods: the number of total cells in the cerebral cortex was compared between (lep ob /lep ob ) and wild type fetuses from embryonic day (e) to . the number of brdu positive cells was also compared on e and e . brdu uptake, the ratio of viable colony number to plated cell number, the proportion of multipotent, neuronal or glial progenitor colonies, and the expression levels of hes mrna were compared between leptin-and non-treated neurosphere cells. moreover, we examined stat phosphorylation by leptin stimulation with elisa to investigate whether or not jak/stat pathway transduces the leptin signal in the prenatal period as in the adult. results: lep ob /lep ob fetuses had reduced total cell numbers in the ventricular zone (vz) on e and e , and in the cortical plate on e . the number of brdu-positive cells was reduced in vz of e and e lep ob /lep ob fetuses. brdu uptake and the number of viable colonies were increased by days-leptin treatment in the neurosphere culture. the proportions of glial-restricted and multipotent precursor colonies were increased by leptin, whereas that of oligodendrocyte precursor colonies were decreased. hes mrna expression was enhanced in neurosphere cells by leptin. neither the amount of phosphorylated stat nor that of stat was increased in neurosphere cells by leptin stimulation. conclusion: our study suggests that leptin maintains the neural stem and glial-restricted precursor cells through upregulation of hes expression and increases the number of cerebrocortical cells in the mouse fetus, however, jak/stat pathway does not mediate the leptin signal in undifferentiated neural lineage cells. a vaginal wall, and > % of fib- knockout (ko) mice develop pelvic organ prolapse by weeks of age. in contrast, fib- and - deficiencies do not result in prolapse, and fib- kos die shortly after birth. herein, we evaluated the role of fib- in pelvic organ support. methods: two observers serially measured the degree of vaginal, perineal, and anal prolapse in fib- ko (fib -/-) and in fib -/-mice severity of prolapse was significantly related to age, but not parity, and the average age at diagnosis was wks. fib -/-animals with advanced prolapse had attenuated uterosacral ligaments and descent of the bladder and uterus caudal to the symphysis. pro-mmp ( -fold, p= . ), active mmp ( -fold, p= . ), and prommp ( -fold, p= . ) were increased in vaginal tissues from mice with gross prolapse compared with age-, strain-, and cycle-matched controls. this increase in protease activity, however, was also accompanied by increased expression of fib- and tropoelastin ( . -fold, p< . ). regardless of prolapse maternal morbidtties tf ards-n(%) ( ) acute ren~m f lure-n (%) ( ) car~tovascular ~ysfunrtion-n ("/o) ( ) hepatc fa..lure-n (%) dt sst'dlm atl:d inlfavascul..-coagul tipalhy-n(%) ( ) durallon of !cu suy (days. mean+sd) :t . hospital my (days.. me +sd) . condon, j. c., hardy, d. b., kovaric, k., and mendelson, c. r. ( ) mol endocrinol ( ), - . objective: innervation of the uterine cervix is important for the process of parturition (physiol beh : , ; j histochem cytochem : , jsgi : , ) . the hypogastric nerve is a major pathway that innervates the uterine cervix, yet its contribution to processes associated with cervical ripening and parturition is not known. the objective of this study was to determine the effect of hypogastric nerve transection on processes associated with cervical remodeling and parturition. methods: time-dated pregnant rats were sham-operated or the hypogastric nerve bilaterally transected just anterior to the inferior mesenteric ganglion on day post-breeding. live pups were born spontaneously by all rats at term on days - post-breeding. on the day of birth, the cervix was excised, postfixed overnight, sectioned, and processed to evaluate collagen content and structure (nih image j). sections were also processed by immunohistochemistry to assess cell nuclei density, the census of resident macrophages, and area of tissue that contained nerve fibers. results: hypogastric neurectomy did not affect cell nuclei density, the number of macrophages, or density of nerve fibers in the cervix. the failure of hypogastric nerve transection to affect indices of cervical remodeling is consistent with the finding that duration of pregnancy and timing of birth were not different in sham-operated and hypogastrectomized rats. conclusions: nerve fibers in the hypogastric nerve that innervate the lower uterus and cervix, including sympathetic and neuropeptidergic projections, are thus not essential for birth. these novel findings provide support for the contention that innervation of the uterine cervix other than through the hypogastric nerve contributes to processes associated with cervical ripening and parturition. receptor system in prostaglandin synthesis in pregnancy. eugenie r lumbers, , della m yates, carolyn m mitchell, jonathan j hirst, , tamas zakar. , school of health sciences, university of newcastle, newcastle, nsw, australia; mothers and babies research centre, hunter medical research institute, newcastle, nsw, australia; obstetrics and gynaecology, john hunter hospital, school of medical practice and public health, university of newcastle, newcastle, nsw, australia. the fetal membranes and decidua contain prorenin, which requires proteolytic activation. ngyuen (j clin invest. : ) described a renin/prorenin receptor that conformationally activates prorenin, so that it can form ang i from aogen. in addition, receptor-bound prorenin can stimulate intracellular pathways directly. prostaglandins (pgs) participate in the control of term and preterm labour, and renin can stimulate pg production by amnion and decidua when angiotensin's action is blocked mitchell placenta : ) . the prorenin receptor may mediate these effects. our aim was to find out if the prorenin receptor gene is expressed and colocalised with prorenin and prostaglandin h synthase- (pghs- ) in human placenta, amnion, chorion and decidua. we have also determined if there is any change in the expression of the prorenin and prorenin receptor genes with labor. placentae with attached membranes were collected after term birth either by elective caesarian section or by spontaneous labor, and prorenin, prorenin receptor and pghs- mrna levels were quantitated relative to beta-actin mrna by real-time rt-pcr in amnion, chorion, decidua and placenta. before labor, mean prorenin mrna levels were . times higher in decidua and . times higher in chorion and placenta than in amnion (p< . ). there were no significant changes with labor. proenin receptor mrna was expressed in all tissues. proenin receptor mrna levels in prelabor amnion, chorion and placenta were similar, while levels in decidua were % of those in amnion (p= . ). this low level of prorenin receptor mrna in decidua persisted after labor (p< . ). pghs- mrna expression was highest in amnion; in decidua and placenta it was only and % of amnion. similar differences were present after labor. we conclude that the decidua is the principal source of prorenin within the pregnant uterus, and all gestational tissues are targets for prorenin. decidual prorenin may affect pghs- expression in amnion through the prorenin receptor forming a maternal-fetal paracrine system that stimulates pg production at labor. the immuno-suppressive effects of progesterone on umbilical cord blood mononuclear cells and the effect of culture media estradiol content. nadav schwartz, xiangying xue, christine n metz. obstetrics and gynecology, nyu school of medicine, new york, ny, usa; feinstein institute for medical research, manhasset, ny, usa. objective: progesterone (p ) is known to supress the maternal immune response and similar effects have been seen with fetal cells. we sought to ascertain whether p action was modulated by the phenol red and/or estrogen content of the culture media. methods: umbilical cord blood mononuclear cells were isolated using a density gradient centrifugation. the cells were incubated with p ( - m) hour prior to overnight stimulation with lps. supernatants were assayed for tnf-using elisa. the following culture media were used: a)'red' media: rpmi+ % fbs, b)'yellow' media: phenol red-free rpmi+ % fbs, and c)'clear' media: phenol-free rpmi+ % dextran/charcoal treated fbs. finally, the 'clear' media experiments were repeated with estradiol add-back. results: p suppressed tnf production in all medias. tnf levels were suppressed to . % (p< . ) of controls using 'red' media and to . % (p< . ) in 'yellow' media. the degree of suppression was not as substantial using 'clear' media where tnf levels were . % (p< . ) of control. (fig ) adding estradiol to the 'clear' media had little or no effect on the degree of tnf suppression. (fig ) conclusions: progesterone exerts an immuno-suppressive effect on lpsstimulated fetal mononuclear cells which is more pronounced when using media containing untreated fetal bovine serum. the phenol-red/estradiol content of the culture media did not modulated the p effect. dextran/charcoal treatment of fbs appears to deplete the media of factors other than estradiol that are necessary for p to exert its full effects. culture conditions should be optimized when studying progesterone's effects on immune response. ovine fetal regional blood flow following prenatal dexamethasone (dex) at . gestation (g). antonine d van heesewijk, jan g nijhuis, susan l jenkins, peter w nathanielsz, mark j nijland. ob/gyn, academisch ziekenhuis maastricht, maastricht, netherlands; ob/gyn, cpnr, uthscsa, san antonio, tx, usa. background: pregnant women at risk for premature labor receive antenatal glucocorticoids (gc) to reduce neonatal morbidity and mortality. while fetal blood pressure (bp), heart rate and vascular endothelial and control fetuses (n= ). dispersed adrenal cortical cells ( . x cells/tube; in duplicate) were challenged with - m acth. samples were collected at time (baseline), , , , and min after acth treatment, and tissue and media samples were frozen for determination of cortisol, camp, and star. results: cortisol output (ng/ . x cells) was higher in the lth group compared to the control, (p< . ) at min ( . ± . vs. . ± . ), min ( . ± . vs. . ± . ), and min ( . ± . vs. . ± . ). peak camp levels (fmol/ . x cells) were observed at min but did not differ between control ( . ± . ) and lth ( . ± . ) adrenal cells. western analysis demonstrated that star protein expression was higher in the lth adrenal cortex compared to control (p< . ) at min ( . ± . vs. . ± . ), and at min ( . ± . vs. . ± . ), relative optical density units. conclusions: results from the present study taken together with those of previous in vivo studies suggest that the enhanced cortisol output in lth fetuses is the result of increased adrenal acth sensitivity. this enhanced sensitivity is not due to differences in camp generation. star, which is a key element involved in cholesterol transfer at the level of the mitochondrial membrane appears to play a key role in the enhanced cortisol response to acth in the lth group. (nih grants hd , hd and llu-nih imsd r gm - ). expression. hiroaki itoh, makoto kawamura, shigeo yura, haruta mogami, tsuyoshhi fujii, norimasa sagawa. obstetrics and gynecology, national hospital organization osaka national hospital, osaka, japan; gynecology and obstetrics, kyoto university graduate school of medicine, kyoto, japan; obstetrics and gynecology, mie university school of medicine, tsu, japan. objective: epidemiological evidences suggested that undernutrition in utero develops risk factors for adult cardiovascular disorders, such as blood pressure increase. the present study was designed to prove the hypothesis that maternal iso-caloric high protein diet alleviates blood pressure increase in the adult offspring by affecting placental beta-hydroxysteroid dehydrogenase type ( beta-hsd ) expression. methods: the % calorie restriction was applied to pregnant mice by using either standard protein diet (spd; % casein protein; spd-un) or high protein diet (hpd; % casein protein; spd-un). in some groups, these pregnant mice were sacrificed at . d.p.c.; then maternal and fetal plasma as well as placental tissues were corrected. while in other groups, systolic blood pressure was measured in the offspring at wks. fetal and maternal corticosterone levels were measured by elisa. the placental beta-hsd gene expression was measured by quantitative taqman pcr. results: systolic blood pressure in the spd-un offspring at weeks ( mmhg) was higher than that in spd-nn offspring ( . mmhg). by contrast, systolic blood pressure in the hpd-un offspring ( . mmhg) was similar to that in spd-nn offspring. maternal calorie restriction with spd and hpd caused similar elevation of maternal plasma corticosterone concentrations. by contrast, fetal plasma corticosterone levels in hpd-un offspring ( ng/ml) was lower than those in spd-un offspring ( ng/ml), indicating the amelioration of fetal exposure to high corticosterone by maternal hpd. the placental beta-hsd gene expression in the hpd-un was % higher than that in spd-un, suggesting that maternal hpd augmented placental beta-hsd gene expression in the placenta and probably facilitated the inactivation of maternal cortocsterone in the process of placental transfer to fetal circulation. conclusion: the preset study suggests that maternal high protein ingestion may elevate placental beta-hsd gene expression and ameliorate blood pressure increase in the adult offspring via modification of fetal exposure to maternal corticosterone. elevated cortisol levels at birth exert critical maturational effects through action at glucocorticoid receptors, gr. in contrast, the low cortisol concentrations in the preterm fetus, before the time of increased fetal cortisol secretion, are near to the kd for cortisol at the higher affinity mineralocorticoid receptor, mr. we hypothesized that endogenous cortisol in the fetus exerts physiologic actions at mr in tissues without appreciable cortisol-inactivating enzyme, hsd , including the fetal lung and brain. we therefore tested the effect of infusion of a mr-antagonist, ru (mra, . mg/h over h) into - day fetal sheep. we tested for effects on lung liquid production rate, lung liquid and plasma electrolytes, plasma acth and cortisol, and hematocrit at - h after start of the infusion. although there was no significant difference in the liquid production rate in fetuses infused with ru , the ratio of na + to k + in lung liquid was significantly greater in mra-treated fetuses than in the control fetuses ( . ± . vs . ± . ). plasma acth was significantly greater in the mra-treated fetuses than in control fetuses at h ( ± vs ± pg/ml); in the mra-treated fetuses, acth concentrations at h were greater than in the same fetuses at h ( ± pg/ml), wheras there was no increase in plasma acth in the control fetuses ( h: ± pg/ml). similarly, plasma cortisol concentrations at and h were greater in the mra infused fetuses than in control fetuses ( h: . ± . vs . ± . ng/ml), and cortisol increased significantly over time in the fetuses infused with mra, but not in control fetuses ( h, mra: . ± . , control: . ± . ng/ml). infusion of mra did not alter fetal plasma electrolyte, fetal blood pressure or fetal heart rate. however, fetal packed cell volume was significantly increased at - h of infusion of mra ( h: ± vs ± %) and fetal pco was significantly greater at h of infusion of mra as compared to control fetuses ( . ± . vs . ± . ). these results suggest that endogenous cortisol concentrations in the preterm fetus exert negative feedback control of acth via action at mr in the fetal brain, and play a role in regulation of fetal lung liquid composition and in fetal fluid balance. preparturient increases in fetal acth secretion are cyclooxygenase- (cox- ) dependent. charles e wood. dept. physiology and functional genomics, university of florida college of medicine, gainesville, fl, usa. maturation of the fetal hypothalamus-pituitary-adrenal axis is critical for the timely somatic development of the fetus and readiness for birth. in sheep, increased preparturient activity of the fetal hpa axis appears to be responsible for triggering parturition itself. this study was designed to test the hypothesis that prostaglandin generation, mediated by cox- in the fetal brain, is critically important for stimulation of the preparturient increase in fetal acth secretion. singleton fetal sheep were chronically catheterized with vascular, amniotic, and lateral cerebral ventricular catheters. nimesulide, a selective cox- inhibitor, was infused ( mg/day, n= ), and vehicle ( % dimethylsulfoxide, n= ) was infused. arterial blood samples were drawn from fetuses at hour intervals for measurement of plasma hormone concentrations and arterial blood gases. in the vehicle-treated fetuses, fetal plasma acth, pomc, and cortisol concentrations increased exponentially (p< . by anova) before spontaneous parturition at ± . days. in the nimesulide-treated fetuses, fetal plasma acth and pomc were constant and did not increase prior to parturition (p=ns by anova). in contrast, fetal plasma cortisol increased independent of acth (p< . by anova) and the fetuses were born spontaneously on ± . days gestation. the slight delay in spontaneous parturition in the nimesulide-treated fetuses was statistically significant (p< . by mantel-cox test). intracerebroventricular nimesulide infusion did not decrease fetal plasma concentrations of prostaglandin e , demonstrating that the action of the nimesulide was restricted to the fetal brain. fetal blood gases were normal in all of the fetuses, and there were no differences in blood gases between groups. we conclude that the spontaneous increase in fetal acth and pomc prior to parturition is cox- dependent. however, we also conclude that the increase in fetal plasma cortisol concentration can occur independent of increases in fetal acth, suggesting that the increase in cortisol can result from increases in adrenal sensitivity to acth. the results of this study demonstrate that the timing of parturition in the sheep is not dependent upon increased acth secretion, and the results suggest that parturition is regulated primarily by changes in adrenal sensitivity. expression of extracellular signal-regulated kinase / and p mitogen-antiphosphotyrosine. immunolocalization of inos was performed in the same tissues. hpmec were used to determine effect of sin- ( mm) on inos protein expression ( min, and h). results: inos protein was detected in microvascular endothelium, smooth muscle and syncytiotrophoblast of the placenta. inducible nos levels in placentas from severe preeclampsia were significantly decreased compared to normal (p< . ) but were not different from mild preeclampsia. aligning western blots of anti-phosphotyrosine and inos revealed a phosphorylated band corresponding to the molecular weight of inos. the proportion of tyrosine phosphorylated inos was reduced by % in severe preeclampsia compared with normotensive. preliminary data with ip for phosphotyrosine and crossblotting with inos confirmed this finding. sin- treatment decreased inos protein abundance at and h in hpmec. conclusions: our results demonstrate decreased tyrosine phosphorylation of inos in preeclamptic placenta. phosphorylation of tyrosine in inos has been reported to negatively regulate its activity. we therefore postulate that decreased phosphorylation of inos may be responsible for the increased catalytic activity of the enzyme that we have previously observed. the decreased levels of inos observed on sin- treatment may be due to nitration, an effect analogous to preeclampsia, where the presence of peroxynitrite has been well established. it remains to be seen if protein nitration has an effect on enzyme stability. objective: -isoprostane ( -iso), a prostaglandin-like compound formed in vivo, is a reliable measure of oxidative stress which occurs in response to many different stimuli, including infection. periodontal disease is a chronic oral infection associated with fetal growth restriction and preeclampsia. our objective was to determine if maternal periodontal disease is associated with oxidative stress as measured by -iso. methods: a secondary analysis was conducted using prospective data from the oral conditions and pregnancy study. a cohort of healthy women enrolled < weeks underwent oral health examination and serum sampling. maternal periodontal disease was categorized as healthy, mild, or moderate-severe by clinical criteria. maternal serum was analyzed for -iso by ultra-sensitive elisa. elevated -iso was defined by a value th %tile. maternal factors associated with elevated -iso were determined using chi-square or t-tests as appropriate. a logistic regression model was created to determine adjusted odds ratios ( th %ci) for elevated -iso. results: women had complete data for analysis. the median -iso level (iqr) was , ( - , pg/dl). using bivariate analysis, moderate-severe periodontal disease, non-white race, use of wic/food stamps, unmarried status, obesity, and lack of insurance were associated with elevated -iso. the logistic regression model for elevated -iso is shown below. adjusted or ( th ci)* mild periodontal disease . maternal periodontal disease and utilization of public assistance are associated with oxidative stress in pregnancy. the relationship between periodontal disease, social hardship, oxidative stress, and adverse pregnancy outcome remains to be determined. antioxidant therapy could represent novel therapy for the prevention of adverse pregnancy outcome. severe transient immunological thrombocytopenia in a preeclampsia patient whose bone marrow production of platelets had been restricted. toshihiro yoshimura. obstetrics and gynecology, ntt-west kyushu general hospital, kumamoto-city, kumamoto, japan. introduction: idiopathic myelofibrosis is a chronic myeloproliferative disorder in which clonal haemopoetic stem cell proliferation is accompanied by reactive fibrosis. case report: a -year-old primiparous woman was referred to our hospital for prenatal care after weeks of gestation. her medical history was significant for idiopathic myelofibrosis, and congenital agenesis of the spleen. the laboratory workup showed the patient's white blood cell count to be , / l; hemoglobin, . gm/dl; and platelet count, , / l. her bleeding time was normal ( min). until the th week, the patient's pregnancy was uneventful. during the th week, however, the patient's platelet count declined to , / l, when proteinuria became evident. her bleeding time was prolonged to min. the coagulation system was normal. by the th week, the patient's blood pressure was elevated to / mmhg, and her urinary protein excretion exceeded g/day. our initial diagnosis was thrombocytopenia due to bone marrow suppression. the patient had no history of platelet transfusion, but we expected it would increase the platelet count, particularly since in the absence of destruction by the spleen, the lifespan of the platelets would be prolonged. this was not the case, however. elective induction of labor was carried out after weeks. at the same time, the patient was transfused with units of platelets, but her platelet count remained unchanged ( , / l). it was then that we realized that immunological thrombocytopenia may be involved, and massive doses ( mg/kg) of gamma globulin were given intravenously for one day. thereafter, platelets ( units) were again transfused, this time raising the platelet count to , / l. cesarean section was promptly carried out under general anesthesia. we later found that the patient's serum platelet associated immunoglobulin (paigg) level was positive, though antiplatelet and antinuclear antibodies were negative. the postoperative course was uneventful. the maternal platelet count declined to the pretransfusion level within days after delivery, but gradually increased to the prepregnancy level by weeks. comment: this idiopathic myelofibrosis patient in whom bone marrow production of platelets had been severely restricted demonstrates that immunological destruction may play a major role in the development of thrombocytopenia in preeclampsia. early l-arginine therapy improves notably the fetal growth in preeclamptic women. a randomized controlled trial. keisy lopez-molina, juan c gonzalez-altamirano, julio e valdivia-silva. , oncoinmunología y biología vascular, facultad de medicina, universidad nacional san agustín, arequipa, peru; cardiología y cirugía de tórax, hospital nacional case essalud, arequipa, peru; inmunología, instituto de investigaciones biomédicas, méxico, distrito federal, mexico. objective: to assess the benefit of early administration to l-arginine, the precursor to nitric oxide, on fetal growth. methods: one hundred women with preeclampsia were randomized to receive either l-arginine or placebo until day postpartum. live singleton infants were born after preeclamptic pregnancies and compared those with other control infants. birth size was expressed as the ratio between observed and expected birth weights, and infants smaller than two standard deviations from expected birth weights were classified as small for gestational age (sga). all the women had neither previous precedents of the preeclampsia nor other factors that they cause sga. results: no significant differences existed between the groups with preeclampsia before randomization. preeclampsia was associated with a % ( % confidence interval [ci] %, %) reduction in birth weight. women with hellp syndrome had to leave the study. the risk of sga was three times higher (relative risk [rr] = . ; % ci . , . ) in infants born after preeclampsia without l-arginine therapy than in control pregnancies, and two times higher (relative risk [rr] = . ; % ci . , . ). in infants born after preeclampsia with l-arginine therapy. conclusion: fetal growth improve markedly with l-arginine therapy in women with preeclampsia. introduction: although the pathogenesis of severe preeclampsia (pe) and intrauterine growth restriction (iugr) is poorly defined, inadequate remodeling of uterine spiral arties may promote reperfusion injury leading to focal infarction and reduced nutrient flow between mother and fetus. our goal was to determine whether an intravillous inflammatory cytokine cascade was associated with pe and iugr. methods: immunohistochemistry (ihc) examined il- and il- expression in preterm placentas with severe pe+iugr, and idiopathic preterm controls (ptc) with no evidence of clinical or histological chorioamnionitis. cultures of fibroblasts (fibs) and syncytiotrophoblasts (scts) from term placentas (n= ) were treated with il- and cytokine levels in conditioned media were determined using elisa or multiplex array. results were analyzed by student's t test or anova. results: the gestational age at delivery was not significantly different in pe+iugr and ptc groups ( . ± . vs . ± . wks). ihc of pe+iugr samples revealed intense villus core staining of il- adjacent to infarcts. villi distal to infarcts stained with a lower intensity. in contrast, staining was virtually undetectable in ptc. the pattern of il- staining was nearly identical in pe+iugr and ptc groups, was localized to the syncytium, and did not differ with respect to distance from infarct. to identify cellular targets of il- action, primary cultures of fibs and scts were incubated for h in serum-free medium ± ng/ml il- . by elisa we observed that il- treatment of fibs increased il- levels from ± to ± pg/ g protein (p< . ). similarly, multiplex array revealed that levels of il- , il- , and il- were induced -, -, and -fold, respectively in fibs. the presence of ng/ml il- receptor antagonist reduced the il- -mediated increase in il- levels ± % (p< . ), indicating that this response was mediated through il- receptor. in marked contrast, il- treatment did not significantly affect levels of il- , il- , il- or il- in scts, indicating that this response was cell type-specific. conclusions: these results indicate that increased expression of il- in the placental villus core in pe and iugr may promote an inflammatory cascade in fibs at this site leading to focal infarction and reduced flow of nutrients to the fetus. introduction: in human pregnancy, decreased responsiveness to angiotensin ii (angii) starts in week , promoting an expanded vascular bed. at the same time levels of the vasodilatory hemopexin increases . during pre-eclampsia the vascular bed is contracted and the responsiveness upon angii persists. this may be due to the increased levels of extra cellular atp, a natural inhibitor of hemopexin . in the present study we tested whether extra cellular atp is toxic in the pregnant condition. methods: pregnant rats were infused with either atp ( g/kg bw; n= ) or saline (n= ) on day of pregnancy (permanent jugular vein cannula/ hr infusion). non-pregnant rats (n= ) were infused with atp identically. one day before and , , days after infusion, hr urine and blood samples (wbc count and hemopexin activity) were collected. seven days after the infusion, rats were sacrificed and kidney tissue processed for immunohistology. results: urinary albumin excretion was increased in pregnant atp infused rats only (fig ) . wbc were also increased only in pregnant rats infused with atp (days and vs pre-infusion value). in pregnant atp infused rats intraglomerular influx of monocytes was increased, which correlated with urinary albumin excretion on the same day (r = . ). staining of glomeruli for the angii receptor (at- r) showed decreased at- r expression in control pregnant rats as compared to non-pregnant rats, while at- r expression in atp infused pregnant rats was increased as compared to control pregnant rats. hemopexin activity was increased on days and in control pregnant rats as compared to all other groups. discussion: these data support the notion that atp is toxic exclusively in the pregnant condition. it may be suggested that atp induced an inflammatory response, although the exact mechanism by which atp induced its effects needs further investigation. background: hepatocyte growth factor (hgf) is a multifunctional cytokine that is known to promote division, motility, invasion and morphogenesis of a wide range of cell types and to inhibit apoptosis. the effects of hgf are mediated through its interaction with the tyrosine kinase receptor c-met. in pregnancy hgf/met system is involved in the physiologic growth and development of the feto-placental unit. hgf/met effects are counteracted by transforming growth factor- (tgf- ), that is known to promote progressive fibrosis in human tissues. moreover tgf- plays a major role in trophoblast growth and differentiation. tgf- inhibits hgf expression as well as hgf inhibits tgf- expression. an understanding of the mechanisms regulating placental balance of these growth factors may provide insights into the processes that occur in complications of pregnancy, such as preeclampsia (pe) and fetal growth restriction (fgr). objective: to verify the hypothesis that hgf, c-met and tgf- are differently expressed in tissues of normal and pe placentas. methods: we studied placentas from pregnancies complicated by pe ( with normal fetal growth and with fgr) and placentas from normal pregnancies. from each placenta random samples were excised and rna was extracted; quantitative real-time rt-pcr analysis was used to investigate mrna expression of hgf, c-met and tgf- in pe (with or without fgr) and in normal placentas. results: gestational age, neonatal weight and placental weight were, as expected, lower in pe groups. the mrna expressions of the three molecules are higher in pe than in normal placentas, but the difference is statistically significant only for c-met. the higher values are mainly due to the increase in the pe group without fgr for c-met and tgf- and the increase in the pe-fgr group for hgf. conclusion: increased levels of expression of hgf/met system in pathological placentas could be explained as an attempt to repair placental damages; nevertheless placental regeneration could be inhibited by the increase of tgf- , that promote fibrosis. placental expression of hgf, c-met and tgf- is increased in all pe placentas, but particularly in placentas of pe with normal fetal growth, where placental tissue is probably less compromised. obesity is a risk factor for preeclampsia (pe), but the reason for this risk is unknown. previously, we found that neutrophils infiltrated into the vasculature of pe women released myeloperoxidase (mpo) and matrix metalloproteinase (mmp ), products that can cause oxidative stress and vascular dysfunction. if neutrophils infiltrate the vasculature of obese women and release mpo and/or mmp , this may help explain why they are at risk of developing pe. hypothesis: systemic vascular tissue of obese women will have a significant presence of mpo and mmp as a result of neutrophil infiltration. methods: subcutaneous fat, which is highly vascularized, was obtained at abdominal surgery from normal weight, overweight and obese women. formalin fixed, paraffin embedded μm sections of fat biopsies were stained using immunohistochemistry with specific antibodies for mpo and mmp . data were evaluated for intensity of vessel staining by visual score ( - ), density of staining using image analysis software, and % vessels with neutrophil staining, liberated from serum-free placental villous explant cultures from normal and pe pregnancies on human endothelial cells, and identified candidate mediators of their differential effects on metabolism and behaviour. methods: term placental villous tissue from normal (n= ) or pe (n= ) pregnancies were explanted for days at % oxygen. conditioned h medium (day - ) was applied to human umbilical vein endothelial cells (huvecs). an angiogenesis assay was conducted in which tubule length and number were measured by morphometric imaging following seeding on % matrigel. the effect of explant conditioned medium on endothelial cell metabolism was determined by mtt and bioluminescent atp assay. the release of vasoactive metabolites (nitrite, endothelin- , prostacyclin) from huvecs exposed to this medium was also measured. finally, a luminex bead array was used to screen the explant media for a panel of chemokines/cytokines. results: in the angiogenesis assay, tubule length (p< . , mann-whitney u test) and number (p< . ) were significantly decreased in pe compared to normal pregnancy medium. there was no change in mtt reduction, but endothelial cellular atp levels were also significantly reduced following exposure to pe explant medium (p< . ). both normal and pe-derived medium stimulated huvecs to produce vasoactive metabolites. the following cytokines were detectable in the explant media: interleukin (il)- , il- , gro-, monocyte chemotactic protein- and macrophage inflammatory protein- (mip- ). higher levels of both il- and gro-were present in the normal medium compared to pe (both p< . ). mip- was present in pe conditioned media but undetectable in media generated from normal placental explants. conclusions: these results suggest that pre-eclampsia stimulates the release of soluble factors from the placenta which have adverse effects on endothelial cell angiogenic potential and metabolism. altered levels of several cytokines were detected in the explant medium, and the effects of these on endothelial cell function are currently being addressed. yang gu, davis f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: placentas from women with preeclampsia (pe) release more chymotrypsin-like protease (clp). the purpose of this study was to determine if placenta-derived clp was responsible for altering endothelial (ec) barrier function in pe. approaches: endothelial junctional protein complex ( -catenin/ve-cadherin/ p ) expression and junctional protein ve-cadherin distribution were examined in ecs treated with pe placental conditioned medium. methods: ) confluent ecs were treated with pe placental conditioned medium (cm). the association of ec junctional protein complex ve-cadherin/catenin/p was examined by a combined immuno-precipitation (ip) and immuno-blotting (ib) assay, in which total cellular protein was immunoprecipitated with monoclonal antibody against -catenin and then immunoblotted with antibodies against ve-cadherin or p- . ) confluent ecs grown on cover slips were exposed to pe placental cm with or without depletion of chymotrypsin. ec junction protein ve-cadherin distribution was examined by fluorescent microscopy. results: ) ve-cadherin and p are expressed in control ecs but not in ecs exposed to pe cm, which indicate that the junctional protein complex vecadherin/ -catenin/ p is lost in ecs exposed to pe cm. ) ecs exposed to pe placental cm showed a discontinuous distribution and reduced expression for ve-cadherin at cell contact areas. the zipper like structure was lost and cleft was formed at cell-cell contacts. these observations indicate that the homotypic cell-cell adhesion and junction protein intracellular partner complex are disrupted. these disruptive phenomena in cells treated with pe conditioned medium were not present in control cells and in cells treated with cm after depletion of chymotrypsin. conclusion: clp released by the placenta could be a candidate agent that is responsible for disrupting ec integrity and inducing endothelial permeability in pe. (supported by nih grants hl and hd ). introduction: chronic pelvic pain (cpp) syndromes such as endometriosis, irritable bowel syndrome, and interstitial cystitis are associated with visceral hyperalgesia, and often coexist in the same patient. one possible explanation for this phenomenon is viscero-visceral cross-sensitization in which increased nociceptive input from an inflamed pelvic organ sensitizes neurons that receive convergent input from an unaffected organ to the same dorsal root ganglion (drg). nociception induces upregulation of perk and substance p. the purpose of this study was to determine, in a rodent model, whether uterine inflammation increased the number of perk-and sp-positive neurons in sensory ganglia innervating both uterus and colon. methods: colonic and uterine drgs were retrogradely labeled with fluorescent tracer dyes micro-injected into the colon and uterus. ganglia were harvested, cryoprotected and cut for fluorescent microscopy. results:approximately % neurons were colon-specific and % were uterus-specific. among these uterus-or colon-specific neurons, up to % of labeled drg neurons in the l -s levels innervated both visceral organs. uterine inflammation increased the number of perk and sp-immunoreactive neurons in drg neurons innervating colon, uterus, and those innervating both organs. furthermore, this effect was specific as non-retrograde labeled drg neurons did not manifest a significant increase in perk or sp immunoreactive cells. conclusions: localized uterine inflammation leads to increased expression of sp and perk in uterine afferents as well as dichotomizing afferents innervating both uterus and colon, suggesting that viscero-visceral convergence is present at the level of drg primary afferent cell bodies. this visceral sensory integration may underlie the co-morbidity of female pelvic pain disorders and may provide basic information regarding the etiology of cpp syndromes. purpose: success rates of medical and surgical modalities for the treatment of severe fecal incontinence due to obstetric trauma are modest. we tested whether the intrasphincteric injection of autologous myoblasts is clinically safe and feasible for postobstetric fecal incontinence. methods: using ultrasound guidance a suspension of autologous myoblasts was injected in the anal sphincter muscle complex in three women with severe postobstetric fecal incontinence. main outcome measures were safety, feasibility and the wexner grading score. secondary outcome measures were incontinence episodes, rockwood fecal incontinenece quality of life scale, external anal sphincter morphology and anal pressure values. results: all procurement procedures and injections were performed without complications; there were no clinically or laboratory signs of infection or inflammation. three months post myoblast injection, wexner continence grading scores and rockwood incontinence scales were markedly improved and incontinence episodes reduced in all three patients. no change could be observed in sonographic anal sphincter morphology. mean anal squeeze pressure improved. conclusion: autologous myoblast injection for fecal incontinence is clinically feasible and safe; further studies will evaluate the efficacy of this approach. objective: to evaluate whether hysterectomy is associated with a change in postoperative bmi. methods: a retrospective cohort study was conducted of hysterectomies performed for benign indications from june to june . institutional review board approval was obtained. the data were collected by a medical student blinded to the hypothesis. basic demographic data were recorded. body mass index (bmi) was determined at time points: time (immediately postoperatively), time ( weeks to months postoperatively), time ( months to year postoperatively) and time ( to . years postoperatively). one-way anova was performed using ncss statistical software. a bonferroni multiple comparison test (p< . ) was used to compare median changes in bmi from baseline. the starting bmi served as the control group. results: there was a statistically significant increase in bmi at time compared to baseline in all women ( . to . ). when sorted was measured by matrigel invasion assay. small interference rna targeting vegfr- were predesigned by ambion inc. and cells were transfected using ambion siport neofx according to the optimized protocol. results: of the four receptors (vegfr- , vegfr- , nrp- and nrp- ), vegfr- was the predominant receptor that expressed in the more invasive cell lines (dov , skov and r ). lpa, at - m, significantly induced vegfr- expression in dov and skov cells (p< . ), without significantly affecting vegfr- expression. lpa ( - m) also significantly induced the expression of vegf and vegf (p< . ) in dov and skov cells. by small interference rna (sirna) transfection, we demonstrated that inhibition of vegfr- expression could significantly decrease dov cells' invasiveness (p< . ) and moderately decrease skov cells' invasiveness. moreover, silencing of vegfr- by sirna significantly suppressed lpa-induced dov and skov invasion. conclusion: these results suggest that knocking down vegfr- expression by rnai may be an effective strategy to inhibit lpa-induced ovarian cancer metastasis. cancer. fengqiang wang, david fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objectives: expression of matrix metalloproteinases, such as mmp- and mmp- , has implicated in epithelial ovarian cancer (eoc) invasion and metastasis. osteopontin (opn) was also expressed in various human cancers and associated with tumor progression, invasion and metastasis. in the present study, we examined the correlation of mmp- , mmp- and osteopontin expression with tumor stage in ovarian tumor tissues and normal ovaries using a commercial tissue scan array. methods: complimentary dna (cdna) from normal ovaries (n = ) and ovarian tumors (n = ) of different stages (stage i, n = ; stage ii, n = ; stage iii, n = ; stage iv, n = ) was amplified by real time pcr using gene specific primer pairs for mmp- , mmp- , and osteopontin. gapdh was also amplified as a reference control. the expression level of mmp- , mmp- and osteopontin was calculated as relative expression normalized to that of gapdh in each tissue sample, and the expression in sample that has the lowest target gene expression was arbitrarily set as . the average expression of mmp- in ovarian tumor tissues ( ± ) is fold of that in normal ovaries ( ± , p = . ). using an arbitrarily set standard, of normal ovaries ( . %) has elevated mmp- expression; in ovarian tumor tissues, the percentage is . % ( of ), significantly higher than in normal tissues (p = . ). in early stage tumors (stage i/ii, n = ), of them have elevated mmp- expression ( . %, p = . vs. normal ovaries), whereh the average expression of mmp- is fold of that in normal ovaries (p = . ) and . fold of that in late stage tumors (stage iii/iv, n = ). the mmp- expression in ovarian tumors (n = , ± ) is fold of that in normal ovaries (n = ), however, the difference is not significant (p > . ) due to the extremely high expression of mmp- in two tumor tissues. osteopontin expression in ovarian tumor tissues is . fold of that in normal ovarian tissues ( ± . , n= vs. . ± . , n = , p< . ). in early stage (i/ii) ovarian tumors, osteopontin expression is . fold of that in normal ovaries, while in late stage (iii/iv) ovarian tumors, its expression is . fold of that in normal ovaries (p< . ), suggesting an increase trend associated with disease stages. conclusions: our results suggest that mmp- , mmp- and osteopontin alone or combined may have clinical value for ovarian cancer detection. objectives: -tubulin acetylation has been proposed to control the dynamic nature of microtubule stability. acetylated -tubulin plays a role in regulating microtubule functions in mitosis and cell migration. here, we sought to identify the relationship between post-translational -tubulin acetylation and the expression of epithelial cell adhesion molecules (ep-cam) after exposure to microtubule interacting agents in ovarian cancer cells. methods: epithelial ovarian cancer cell line, hey was treated with microtubule stabilizing agents (taxol, epothilone b and discodermolide), microtubule-destabilizing agent (vinblastine), hdac inhibitor trichostatin a (tsa), anti-metabolite fluorouracil ( fu), or alkalyting agents (cisplatin and carboplatin). cells were separately treated with either ic or -fold ic of each agent, and incubated at °c for h, h and h. acetylation oftubulin and pan--tubulin were evaluated by western blot analysis followed by protein quantification. cell surface ep-cam expression was examined by flow cytometry. results: increased acetylation of -tubulin was seen with taxol, epothilone b, discodermolide, vinblastine and tsa. -tubulin acetylation was time and dose dependent. the highest level of -tubulin acetylation ( . -fold) was observed with vinblastine at -fold ic after h. exposure to microtubule interacting agents and tsa resulted in increased cell surface expression of ep-cam in a time and dose dependent manner. the highest level of cell surface ep-cam expression ( . fold) was observed with -fold ic of vinblastine at h. the increase in acetylated -tubulin and ep-cam expression was clearly detectable after h treatment. this data reveals a similar dose and time dependent increases between the acetylation of -tubulin and the increase of ep-cam expression. conclusions: these data demonstrate the promotion of -tubulin acetylation and cell surface ep-cam expression by a microtubule destabilizing agent and by microtubule stabilizing agents. interestingly, vinblastine induces the highest -tubulin acetylation and ep-cam expression. acetylation of alpha-tubulin may be associated with redistribution of cell surface antigens in ovarian cancer cells. objective: epothilone b (epob) is similar to taxol in its ability to enhance tubulin polymerization and to stabilize microtubules. epob is currently being evaluated as an antitumor agent and is in phase iii clinical trials. the tumor suppressor gene, p plays an important role in the induction of apoptosis by a variety of anticancer drugs. p mitogen-activated protein kinase is activated by a wide array of stress stimuli including chemotherapeutic agents and promotes apoptosis. since both p and p activation induces apoptosis, we hoped to evaluate the relationship between p , p-p and parp cleavage, an early indicator of apoptosis, in ovarian cancer cells after treatment with epob. methods: hey cells were treated with epob ( to nm) for h. parp cleavage product p as well as p-p , p , phospho-p (ser- ), p and survivin were determined by western blot analysis. a wild type ovarian cancer cell line hey, was treated with or without m sb , a specific inhibitor of p-p , followed by treatment with epob ( or nm) for h. lysates were prepared and western blot analysis was performed with polyclonal phospho-p and anti-phospho-p antibodies. results: epob induces p activation and apoptosis demonstrated by increased parp cleavage product. time course studies indicated that phosphorylation of p precedes phosphorylation of p in hey cells. the expression of p targeted gene, p (survivin) were differentially expressed depending on the dose of epob and the duration of drug exposure. pretreatment with specific inhibitor of p markedly inhibited the p phosphorylation at serine . conclusions: nm epob (a concentration that triggered mitotic arrest) causes a decrease in p expression and an increase in survivin expression. epob induces parp cleavage. p inhibitor sb inhibits epo-b -induced p phosphorylation. these results suggest that phosphorylation of p may lead to p activation and these signaling events may be related to epo-b induced cell death in ovarian cells. conclusions: nf-b has been shown to control the expression and function of anti-apoptotic proteins and pro-inflammatory cytokines. in the present study we demonstrated that specific inhibition of nf-b by erib reversed the anti-apoptotic state of chemoresistant ovarian cancer cells, therefore may provide a new potential venue for the treatment of ovarian cancer patients. expression in cervical cancer. madhu chauhan, chandra yallampalli. gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd)/adrenomedullin is a ct/cgrp family peptide. imd is expressed in a variety of tissues such as pituitary, stomach, placenta, uterus, and ovary .we have shown that imd plays a role in a variety of physiological functions, including vasodilatation and fetoplacental growth regulation. further we have data indicating an angiogenic activity of imd in first trimester trophoblast cells. we hypothesize that imd is expressed in cervix and may have a role in the pathology of cervical cancer objective: ) to analyze expression of imd mrna in human cervix, rat cervix from non-pregnant and pregnant rats; ) to assess the differences in the expression of imd in normal human cervix and cervical carcinoma tissues and ) to analyze the effect of imd on the expression of tnf-alpha and il- beta in human epithelial cervical carcinoma cells (hela). methods: groups of sprague-dawley, non-pregnant and pregnant rats on day of gestation were used in this study. cervical tissues were collected from the non-pregnant and pregnant rats, women undergoing hysterectomy and from women diagnosed with cervical carcinoma. total rna was extracted using trizol method. hela cells were grown to % confluency in rpmi medium supplemented with % fbs. the cells were starved in %fbs for hrs followed by treatment with imd ( - m) in presence or absence of imd antagonist, imda ( - m). cells were further incubated for hrs and total rna was extracted using trizol reagent. rna was treated with dnase before performing the reverse transcriptase polymerase chain reaction (rt-pcr). the rt-pcr data was normalized to that of s. results: ) imd mrna is expressed in rat and human cervix and in hela cells. ) expression of imd is elevated in pregnant rat cervix as compared to the non-pregnant. ) imd has no effect on tnf-alpha expression in hela cells treated with imd but caused a decline in the expression of il- beta mrna and, ) expression of imd mrna is significantly elevated (p< . ) in cervical carcinoma as compared to the normal cervix. conclusion: imd is expressed in both rat and human cervix and is elevated in pregnant rat suggesting that it may have a role in cervical function during pregnancy in rat. in addition we demonstrate that imd is involved in the pathology of cervical carcinoma and thus may have a clinical significance in the pathology of cervical cancer. objective: there is minimal information about the impact of treatment delays or dose reductions on chemotherapeutic treatment responses and overall outcomes in ovarian cancer patients. the primary objective of this study was to quantify the impact of treatment delays and dose reductions in an in vivo xenograft ovarian cancer model and to evaluate if the growth factors could improve overall survival. methods: heya- and skov -ip xenograft mouse models to determine the effect of treatment delays or dose reductions on tumor response. results: the results indicated that full dose of paclitaxel ( mg/kg) and carboplatin ( mg/kg) delivered on timeevery daysachieved better tumor response in both aggressive (heya- ) and metastatic (skov -ip ) human ovarian tumors compared to the non-treatment and vehicle groups. in heya- mice, paclitaxel/carboplatin alone and paclitaxel/carboplatin followed by growth factor support agents including pegfilgrastim ( μg/kg) and darbepoetin ( μg/kg) improved overall survival rate. growth factors also improved the tolerability in heya- model. for skov -ip mice, the treatment delays resulted in a significant reduce in overall survival time compared to full dose, on time treatment (p< . ). for the dose reduction group, there was a significant different survival comparing to full dose, on time treatment (p< . ). conclusion: treatment delays had a negative impact on tumor response and overall survival compare with treatment controls. in addition, use of growth factor agents also improved treatment response and tolerability of chemotherapy and ultimately overall survival. - ) . median age at recurrence was ( - ), and median interval to recurrence . months ( - ). ( %) patients died of recurrent disease and ( %) of other causes. site of recurrence and was local in , groin in and distant in . of the local recurrences were managed with wide local excision (wle) radical surgery ( radical excisions with skin flaps, posterior exenterations, anterior exenteration, total exenteration), wle and radiotherapy (rt)/chemort, chemo/chemort/rt, and palliation. groin recurrences were managed surgically ( adjuvant rt), rt alone, chemotherapy alone, and palliation. patients with distant metastases were managed surgically, with rt/chemo/chemort, and palliation. overall median survival after recurrence was months. median survival (months) by site of recurrence was: local , groin , distant , groin or distant . there was a significant difference in survival in local vs groin node recurrence (p< . ), local vs groin and distant (p< . ), and groin node vs distant (p . ). and years after recurrence survival rates were: local % and %, groin % and % distant , . conclusion patients with vscc remain at risk of recurrent disease therefore long term follow up these patients is essential. patients with local recurrence often have a good prognosis. outcomes for groin and distant recurrences are poor, but a proportion of those with groin recurrences can be salvaged. therefore early identification of local and groin recurrences is essential for improving outcomes in recurrent vscc particularly in cases with more conservative management of primary disease. introduction: endocrine disrupting chemicals (edcs) are environmental agents possessing hormone-like activity. exposure to edcs during differentiation can interfere with hormonal signaling, resulting in predisposition to some cancers. it has been suggested that some of these effects may be epigenetic, mediated by changes in dna and histone methylation. we hypothesized that edcs, diethylstilbestrol (des), and bisphenol-a (bpa), may act by altering the expression of dna and histone methyltransferases (dnmts and hmts). methods: ishikawa (endometrial) and mcf (breast) cells were cultured in steroid-free, phenol-free media with bpa ( m), des ( x - m) or vehicle for hours. pregnant cd- mice were treated with intraperitoneal injections of des ( mg/kg) or vehicle on days - of gestation. weeks after birth, offspring were sacrificed and tissue obtained. mrna was extracted, cdna was generated and quantitative real time rt-pcr was performed and normalized to -actin. all experiments were conducted in triplicate, repeated three times and compared using anova. results: dnmts (dnmt , a, and b) and hmts (mll and ezh ) were screened after in vitro exposure to edcs (table ) . ezh mrna expression was significantly increased in ishikawa cells after treatment with des or bpa. a similar response in ezh expression was seen in mcf cells treated with des or bpa. due to the consistent induction of ezh in all cells with either treatment, we examined the effect of des exposure on ezh expression in vivo. in adult mice exposed to des in utero, ezh expression was persistently increased ( . ± . fold, p< . ). conclusions: breast and uterine cell lines show increased expression of ezh in response to bpa or des exposure. this differential expression persists in the adult offspring of mice exposed to des in utero. ezh has been identified as a risk for neoplastic progression in the breast and for increased proliferation in uterine cancers. des induced ezh expression may be a mechanism for the increased incidence of breast and reproductive tract cancers seen in des exposed women. defects in cellular programs that control apoptosis lead to an imbalance of cell proliferation and cell death in ovarian cancer. recent evidence suggests that the use of some anti-inflammatory drugs decreases risk of ovarian cancer. natural curcumin-based anti-inflammatory therapies were shown to be beneficial in preclinical models of ovarian cancer (lin et al., ) . in this study, we examined the effects of plant derived curcumin on cell proliferation, apoptosis, and vegf expression in cultivated ovarian cancer cells. ovarian cancer igrov cells were cultured under standard conditions to study the effects of curcumin on cell kinetics and on vegf expression. cell proliferation was measured by srb and mtt assays. apoptosis was determined by measuring cytoplasmic histone-associated-dna-fragments (cell death detection elisa, roche, germany). vegf gene promoter-reporter activation and real-time quantitative reverse transcription polymerase chain reaction were used. conditioned media concentrations of vegf were measured with a commercially available enzyme-linked immunosorbent assay (quantikine, r d systems, minneapolis, mn). we observed that curcumin dose-dependently suppressed cell growth in igrov cancer cells (ic = um). treated cells showed a - fold increase in dna fragmentation compared to controls. curcumin also resulted in a significant decrease of vegfmrna expression and vegf protein secretion into the conditioned media in a dose-dependent manner. in this study, we have demonstrated that curcumin induces apoptosis, suppresses growth, and inhibits vegf gene and protein expression in an ovarian cancer cell line. experiments are underway to identify specific mechanism of curcumin action. curcumin may act as a chemosensitizing drug by potentiating the antitumor effects of standard treatments including taxols and platins in ovarian cancer. supported by the caldwell family foundation and the vesa w. and william j. hardman, jr. charitable foundation inc. daniel r christie, faheem m shaikh, john a lucas iii, susan l bellis. obsterics and gynecology, university of alabama at birmingham, birmingham, al, usa; physiology and biophysics, university of alabama at birmingham, birmingham, al, usa. introduction: previous work has shown that an upregulation of the enzyme st gal i, which is responsible for the , sialylation of integrins, confers a more tumorigenic/metastatic phenotype to colon carcinoma cell lines. it is not known, however, if this is unique to colon cancer, or if it is more broadly applicable to other forms of cancers. quantitatively increased expression of st gal i has been reported in ovarian cancers versus controls, but no biochemical or functional assays have been described to date. objective: because integrins are involved in cell adhesion and migration, and because metastasis of epithelial ovarian cancers is largely an intraperitoneal dissemination, we hypothesized that upregulation in the expression of st gal i in an ovarian cancer cell line would enhance binding with the extracellular matrix, increase invasiveness, and alter migration. methods: in the present study we forced a stable transduction of st gal i into the ov ovarian tumor cell line, which we found to be lacking the st gal i enzyme, establishing a parental (par), an empty lentiviral vector (ev), and an st gal i expressing (st ) cell line. a collagen i cell adhesion assay was performed and quantified by staining adherent cells and measuring absorbance. a haptotactic collagen i cell migration assay was performed by seeding cells in boyden chambers lined with collagen i concentration gradient, and quantified with cell staining and absorbance measurement. cell invasion through a reconstituted basement membrane (matrigel) was quantified as previously described for the cell migration assay. results: we were able to demonstrate successful creation of the ov -st gal i cell line by western blot analysis. functional assays demonstrated increased adhesion to collagen i (p < . ), increased haptotactic collagen i cell migration (p < . ), and increased invasiveness (p < . ) in the st cell line as compared to par and ev when analyzed by one-way anova. conclusion: this initial study into st gal i in ovarian cancer may have future therapeutic implications, and, in addition, lend greater insight into the intraperitoneal dissemination of disease.of microarrays (sam), arrayassist and binary tree structured vector quantization. differentially expressed genes were integrated with a database of known predicted protein-protein interactions (ophid) and key genes are being validated with real-time pcr and immunohistochemistry. results: unsupervised hierarchical clustering revealed collective clustering of all tumors, irrespective of their pathological classification. sam analysis has highlighted probe sets as differentially expressed between lmp and lmp-mp, probes between lmp and lgsc, and probes between lmp and lmp-mp+lgsc. no differential gene expression was detected between lmp-mp and lgsc. ophid analysis demonstrated gene members of the egfr and mapk / pathways to be differentially regulated between the non-invasive and the invasive tumors. to date, we have successfully validated members of the mapk / pathway-poly adp ribose polymerase (ppol) and traf family associated nf kappa b activator (tank)-using real-time pcr. conclusion: our data demonstrate that although the tumors have related genetic profiles, lmp-mp and lgsc are similar to each other and different from lmp, in keeping with their clinical behavior. members of the mapk / and egfr pathways appear to play a key role in low grade serous cancer. identification of novel genes associated with malignant transformation, may lead to development of more effective targeted therapy for lgsc. background: approximately % of pap smears with the ambiguous diagnosis of atypical squamous cells, cannot exclude high grade (asc-h), are negative for dysplasia in follow up colposcopic examination and biopsy. although hpv testing provides excellent negative predictive value (npv) ( %), the prevalence of high risk hpv infection is high in young women and the positive predictive value (ppv) in asc-h pap smears is no better than cytologic diagnosis alone ( %). recent studies have shown that immunostaining for p ink a , or proex tm c, supports a diagnosis of dysplasia in surgical biopsies. our objective was to determine whether staining for p or proexc provides sufficient predictive value to reliably distinguish high grade dysplasia in asc-h pap smears. design: we retrospectively collected samples from liquid based pap smears diagnosed as asc-h at either ohsu or portland oregon kaiser permanente ( - ). known hsil and negative pap cases were included as immunostaining controls. asc-h cases with followup cervical biopsies (n= ) were included for subsequent immunostaining according to manufacture's instructions. samples were also sequestered for hpv testing (pending). immunostained slides were scored as positive or negative by two independent cytopathologists (as and tm) while blinded to pap diagnoses and surgical biopsy outcomes. results: we observed excellent agreement between pathologists' scores (pairwise kappa statistic . ). the correlation between p and proexc scores was moderate (kappa . ). chi-square analysis comparing staining to biopsy outcome revealed a significant association between proex c positivity and cervical dysplasia (*** p< . jing lin, zhenmin lei, ch v rao. ob/gyn women health, university of louisville health sciences center, louisville, ky, usa. introduction: matrix metalloproteinases (mmps) are a family of highly homologous zinc-dependent endopeptidases, which degrade all kinds of extracellular matrix proteins. the degradation process is required for tissue growth and remodelling. these enzymes are probably involved in uterine growth and devolopment and its differentiated functions. ovarian steroid hormones, estradiol (e ) and progesterone (p ), are known to regulate some of the uterine mmps. since it is now known that lh/hcg can directly regulate uterine growth and devolopment and its functions, we questioned whether these gonadotropins could also regulate mmps in the uterus. methods: sixty day old wild type (wt) and lh receptor knockout (lhrko) mice and -day e and p treated -day old animals were used. in addition, primary cultures of uterine epithelial cells from wt animals were treated for hrs either with no hormone or with a single or a combination of pg/ml of e or p , ng/ml of hcg. then mmp- , - and - mrna levels were quantified by the semiquantitative rt-pcr. results: while the uterine mmp- mrna levels were unaffected, mmp- mrna levels significantly decreased and mmp- mrna levels significantly increased in lhrko animals as compared with wt siblings. e and p treatment reversed mmp- and mmp- changes in lhrko animals. treatment of wt type primary endometrial epithelial cell cultures with hcg had no effect on mmp- mrna levels, but it did increase mmp- mrna levels. this increase was synergistic with both e or p . conclusion: while lh/hcg do not regulate uterine mmp- and mmp- , they seem to co-regulate mmp- with ovarian steroid hormones. cancer. radhika gogoi, christine ardalan, dorota popiolek, leslie gold, john curtin, stephanie v blank, bhavana pothuri. new york university, new york, ny, usa. objective: megesterol, a synthetic progestin with strong androgenic properties, is used in the medical management of patients (pts) with atypical endometrial hyperplasia (aeh) or endometrial carcinoma (emca). our hypothesis was that androgen receptor (ar) expression in the endometrium of aeh and emca pts would correlate with degree of response to treatment. methods: pre-and post-treatment endometrial biopsy specimens were obtained from pts treated with megesterol for aeh or emca. ar expression was investigated by immunohistochemical (ihc) analysis with appropriate positive and negative controls. ihc staining was scored for intensity ( - ) and percentage of positive cells ( - ) in the glandular and stromal compartments by a pathologist, blinded to clinical response data. a composite score utilizing both intensity and percentage of positive cells was calculated. we evaluated pre-and post-treatment ar expression in responders and nonresponders as well as ar expression in emca, aeh and normal endometrium. the mann whitney u and the wilxocon signed rank tests were utilized for statistical analysis. results: eight pts' pre-and post-treatment samples were obtained; with emca and with aeh. three pts had no response, , a partial response and , complete responses. ar expression in emca samples when compared to the normal endometrium was significantly lower in both the glands (mean . vs. ; p< . ) and the stroma (mean . vs. ; p< . ). although there was no statistically significant difference in glandular ar expression in the pretreatment biopsies of responders compared to nonresponders (mean . vs. . ; p= . ), there was a significantly higher level of glandular ar expression in the post treatment biopsies of responders compared to non-responders (mean . vs. ; p< . ). furthermore, we noted a trend towards higher levels of glandular ar expression in the post-treatment versus the pre-treatment biopsies in the responders (mean . vs. . ; p= . ). we noted a significant decrease in ar expression in emca compared to the normal endometrium. increased ar expression after treatment in responders suggests an important role of the ar in pts treated conservatively with progestational therapy, and needs further prospective validation as a means to predict treatment response in these pts. objectives: study the correlation between adenomyosis and some potential non-surgical risk factors. objective: o -methylguanine-dna methyltransferase (mgmt) acts to repair dna damaged by alkylation of guanine residues. mgmt promoter methylation and gene silencing is seen in a variety of cancers and pre-cancerous changes. the loss of mgmt activity is associated with increased sensitivity to alkylating agents and is a favorable prognostic indicator in gliomas. we sought to determine if mgmt promoter methylation plays a role in endometrial cancer. methods: one hundred and twenty primary endometrial cancers were analyzed for mgmt promoter methylation by combined bisulfite restriction analysis (cobra). the cohort included endometrioid endometrial cancers, endometrial tumors of adverse histologic type, and endometrial cancer cell lines. twenty one endometrioid and mixed endometrioid ovarian cancers were also analyzed. a subset of the primary tumors was analyzed for mgmt expression by immunohistochemistry. results: no mgmt promoter methylation was seen in the endometrial cancers evaluated or the endometrial cancer cell lines. one of the ovarian cancers showed methylation. immunohistochemistry for mgmt expression is ongoing. conclusion: mgmt promoter methylation is an infrequent event in endometrial cancer. mgmt expression in tumor cells and repair of alkylguanine residues could explain in part the limited response of endometrial tumors to alkylating chemotherapy. objective: uterine sarcomas ( % of gynecological malignancies) originate from uterine mesenchyma. patients with high-risk disease (i.e. high grade) or at advanced stages have poor -years overall survival (< - %). pelvic radiation and/or chemotherapy have not demonstrated to improve survival. identification of new therapies for this malignancy is a major goal. few in vitro models have been established to test therapeutic agents for uterine sarcoma. here we sought to establish a new human uterine sarcoma cell line and to test the effects of chemotherapeutic drugs: tnf-related apoptosis inducing ligand (trail) used alone or in combination. methods: tissue sample was obtained from a woman with uterine sarcoma undergoing hysterectomy. sarcoma cell lines were established using a published protocol for endometrial cancer. phenotypic characterization was made through the different passages ( to ) by western blot. levels of estrogen (er), progesterone (pr) and trail receptors were also studied (rt-pcr, w-b). cells were incubated with chemotherapy agents (cisplatin, paclitaxel and doxorubicin and trail) and cytotoxicity (mts assays) and apoptosis (flow cytometry, parp cleavage by w-b, and dna laddering) measured. results: human uterine sarcoma cell line was established from a highgrade uterine sarcoma. through the different passages the cell line remains expressing cytokeratin, vimentin, tissue factor, caveolin- and -actin. this cell line expresses low er and pr levels. trail receptors (r and r ) were also detectable (rt-pcr, w-b). cisplatin, paclitaxel and doxorubicin ( um for h) produced low cell cytotoxicity (< %). trail ( ng/ml for h) induced about % cell cytotoxicity. apoptosis was confirmed by parp cleavage. doxorubicin significantly enhances trail mediated cytotoxicity (up to %), this was demonstrated by a significant increase in the sub g /g region in the dna histogram. conclusions: we establish a human uterine sarcoma cell lines using protocols for endometrial cancer. more importantly, we demonstrated that doxorubicin enhances trail effect on this uterine sarcomas cell line. thus, this combination might be considered as a treatment for high-risk uterine sarcomas. (financial support fondecyt ). defining the tumor initiating capacity of cd + human endometrial cancer cells. anne m friel, petra a sergent, christine l cummings, rosemary foster, current data suggest rare subpopulations of cells with tumor initiating capabilities are a common feature of solid tumors. several investigators have recently identified cd , a cell surface marker expressed on primitive cells of neural, hematopoietic, endothelial and epithelial lineages, as a potential tumor initiating cell (tic) marker in solid tumors of the brain, colon and pancreas. in our efforts to investigate such a population in human endometrial cancers (enca), we developed an in vivo model that is based on serial transplants of tics from endometrial tumor explants. serial transplant experiments were initiated in nod/scid mice that were injected with primary human enca cells. a subset of cells derived from the generated tumor was subsequently transplanted into new recipients. tumors formed following serial transplantation retained the original histological phenotype of the primary enca, and the number of cells required to initiate tumor formation was reduced at each successive transplant stage suggesting an increase in the ratio of tics to non-tics. we exploited this model in our initial efforts to investigate the tumor initiating potential of cd -expressing enca cells. to evaluate the tumorigenic potential of cd + cells, the tumor initiation capacity of xenograft derived cd + and cd cells were compared following subcutaneous injection of each population into nod/scid mice. only the cd + fraction was tumorigenic consistent with the hypothesis that tumors are generated and maintained by a subpopulation of cells with phenotypically distinct profiles. we are further investigating the functional significance and characteristics of this fraction in vitro and in vivo. objectives. central issues in tumor biology are the understanding of factors that control tumor cell proliferation and the identification of extracellular matrix cues controlling the signaling transducing repertoire that make cancer cells proliferate and invade the host tissues. among these factors, small leucine-rich proteoglycans (srlps):decorin, fibromodulin, lumican, biglycan, are emerging as powerful modulators of angiogenesis and cell growth, by affecting several key elements including matrix assembly, growth factor binding, and receptor tyrosine kinase activity. recently has been demonstrated that srlps can act as a pan-erbb ligand and, in doing so, down-regulate the activity of one of the most potent oncogenic proteins, erbb , whose overexpression is linked to poor prognosis and increased cancer mortality in breast, ovary, and prostate. since srlps have not been previously investigated in the endometrial cancer biology, we investigated their role in the benign and malignant endometrial neoplasia. method. the sampling has been obtained in women (n= ; mean age ± . ) with endometrial hyperplasia (n= ), polyps(n= ), and cancer(n= ), during therapeutic and diagnostic procedures (hysterectomy, colpohysterectomy, hysteroscopy). physiological endometrial samples (n= ) were obtained from menopausal women (n= ; mean age ± . ), during procedures for others gynaecologic indications. immunohistochemestry was the biology technique used for the detection of srlps.results. in the physiological endometrial samples immunoreactivity for decorin, fibromodulin and biglycan was intense (+++), while it was weak in polyps and hyperplasia (+) and absent (-) in cancer. no significant difference in the staining of lumican between the physiological and pathological samples. conclusions. these results could provide a mechanism by which naturally occurring proteins normally synthesized by fibroblasts and smooth muscle cells, the two key components of the tumor stroma, may play a protective role in the genesis and progression of endometrial neoplasia counteracting the growth of neoplastic cells and suppressing tumor cell-mediated angiogenesis. although further studies are necessary to understand mechanisms whereby srlps might influence endometrial cell growth and survival, these molecules may represent potential target for pharmacological cancer therapy. myostatin is a member of the tgf-beta superfamily of protein and a wellknown inhibitor of skeletal muscle proliferation. the muscular component of the uterus is the myometrium, a tissue that regulates its mass in response to different physiologic conditions under the influence of steroids. we determined the expression of myostatin mrna in immortalized pregnant human myometrial (phm ) cells and we verified its biological activity. functional assays showed myostatin induced phosphorylation of smad- and reduction of proliferation of phm cells in a time and dose-dependent manner. to investigate the physiological relevance of our in vitro findings, the expression of myostatin in rat uterus was examined at various phases of the estrous cycle. for the first time we report that myostatin is expressed in rat uterus and that levels of myostatin mrna change during distinct phases of the estrus cycle. uterine levels of myostatin peaked during late estrous and were the lowest at pro-estrous. to further examine the role of steroids in myostatin regulation, we examined the effects of gonadal steroid administration in ovariectomized (ovx) rats. ovaryectomy increased myostatin expression compared to normal cycling controls. estrogen treatment strong decreased myostatin levels while progesterone produced less robust effects on myostatin expression. these findings taken together suggest an important role for myostatin in the regulation of myometrial functionality. her neu over-expression and pi kinase/akt pathway activation in paget's disease of the vulva. amy stenson, bita behjatnia, jaime shamonki, jianyu rao, amer karam, jonathan berek, oliver dorigo. ob/gyn and pathology, ucla, los angeles, ca, usa; ob/gyn, stanford university, palo alto, ca, usa. background: paget's disease of the vulva is rare with high recurrence rates. treatment of recurrent disease is challenging due to its extent and location. non-surgical approaches have limited clinical efficacy, obviating the need for novel therapies. in contrast to paget's of the breast with well-described overexpression of her neu, molecular features of vulvar paget's are poorly characterized. our objective was to study therapeutic targets in vulvar paget's, including the her neu protein and the phosphorylated oncoprotein akt (pakt). in addition, detailed clinical characteristics were correlated with molecular expression. methods: specimens with vulvar paget's disease were retrieved from ucla's department of pathology. protein expression was evaluated by immunohistochemistry on slides from paraffin embedded tissue using the hercep test (dako) for her neu expression and a standard protocol to assess expression of activated pakt. slides were scored by two independent pathologists based on a nominal scale of (negative) to (strongly positive). clinical data was retrieved via chart review. results: between and , patients with vulvar paget's were identified. median age was yrs ( - yrs). a family history of cancer was found in / ( %), / ( %) were smokers and / ( %) had a history of hormone use. intraepithelial lesions accounted for the majority ( / , %), while / ( %) demonstrated invasion and / ( %) were associated with underlying gi malignancy. / ( %) had at least one recurrence, with median time to recurrence months. so far, specimens were stained for her neu and pakt. overexpression of her neu was found in / ( %.) positive staining for pakt was evident in / ( %.) statistical analysis suggested a correlation between her neu and pakt expression. conclusions: our study demonstrates that overexpression of her neu and activation of the pi kinase/pakt pathway are important features of vulvar paget's disease. to the best of our knowledge, this is the largest series evaluating these molecular pathways in vulvar paget's. our data suggest that her neu and pakt may be important molecular targets for novel therapies using for example the monoclonal antibody trastuzumab directed against her neu, or a pi kinase pathway inhibitor like rapamycin. introduction: uterine leiomyomas are the most frequent tumor of the female reproductive tract and are the primary indication for hysterectomy in women worldwide. these tumors occur in up to % of adult women, and their prevalence is especially high in africa-american women. currently there is no effective and safe medical treatment for uterine fibroids and surgery is the main stay. epigallocatechin gallate (egcg) constitutes the main solid extract of green tea and is believed to contributes most of the antioxidant and antiinflammation capacity of green tea. egcg has been shown to have beneficial effects on prostate cancer and breast cancer by inducing apoptosis and inhibiting proliferation of cancer cells. in this study, we investigated the ability of egcg to inhibit proliferation of human leiomyoma cells (hlm) in vitro. methods: human immortalized leiomyoma cells were cultured at ºc in a humidified atmosphere of % co - % air in smbm medium supplied with %fbs, . % insulin, . % hfgf-b, . % ga- and . % hegf(lonza). the cells were plated at density of × cells/well in -well plates and grown under the same conditions above. the monolayer cultures at approximately % confluence were treated with various concentrations ( , . , . , , , and μm) of egcg (sigma) for days. a fluorometric assay using hoechst dye (sigma) for dna quantitation was conducted at the following designed time points, day , , , and post egcg treatment. the cells were lysed and dna content was determined using hoechst dye solution ( μg/ml). fluorescence was measured after excitation at nm and emission at nm. results: egcg exhibited marked anti-proliferation effect on the growth of hlm cells. compared with untreated control, the inhibitory effect of egcg on hlm cells was observed at μm and peaked at μm concentration. the difference was statistically significant (p = . ). evaluation of the mechanism of action of egcg is cuurently under investigation in the lab.conclusion: egcg significantly inhibited the proliferation of human leiomyoma cells in a dose-depended pattern. egcg may present a potential effective and safe medicinal treatment for uterine fibroids. objective: choriocarcinoma is an aggressive form of germ cell tumor that exhibits rapid growth with early metastases. choriocarcinomas autonomously secrete hcg which acts as an autocrine/paracrine growth factor in these cancers. we hypothesize that a novel hcg antagonist (hcg-ant) can limit tumor expansion by blocking hcg-induced expression of pro-invasive genes. we investigated if hcg-ant could alter rna expression of matrix metalloproteinases (mmp- and mmp- ), which facilitate basement membrane degradation and hence invasion, and metastin (kisspeptin), a strong suppressant of metastasis, in the choriocarcinoma cell line jeg- . design: in vitro experiments using the jeg- cell line. materials and methods: after plating jeg- cells in a well tray overnight in rpmi media containing % fbs, cells were washed twice with pbs and then cultured in ul of serum-free rpmi media containing one of four treatments: ) saline; ) hcg ( iu); ) hcg ( iu) plus hcg-ant ( iu); or ) hcg-ant ( iu). rna was extracted from each well using trizol and reverse transcribed using sensiscript (qiagen). the relative expression of mmp- , mmp- , and metastin mrna was quantified using sybr-green based real time pcr. the expression of the housekeeping gene hprt was used to normalize expression data. results: treatment of jeg- cells with hcg-ant vs. hcg alone reduced expression of mmp- ( . ± . vs. . ± . ) and . hcg-ant reduced mmp- and mmp- expression by % and %, respectively (p< . ). treatment with hcg-ant vs. hcg increased metastin expression ( . ± . vs. . ± . ). metastin expression was increased by % following hcg-ant treatment. conclusion: treatment of the jeg- choriocarcinoma cell line with hcg-ant reversed the increased expression of mmp- and mmp- following treatment with hcg. metastin expression was increased by hcg-ant. this data suggests that hcg antagonism is capable of altering gene expression thereby inhibiting invasion in a choriocarcinoma cell line. the role of hcg-ant as an adjuvant therapy in hcg sensitive tumors offers promise. retinoids, but not progesterone, directly induce differentiation and apoptosis of endometrial cancer cells. you-hong cheng, serdar e bulun. ob/gyn, northwestern university feinberg school of medicine, chicago, il, usa. objectives: the opposing actions of estrogen and progesterone during the menstrual cycle regulate endometrial proliferation, differentiation and secretion. the unopposed action of estrogen contributes to the development of type i endometrial cancer. however, the mechanisms for progesterone protection of estrogen-induced carcinogenesis in endometrium remain unclear. methods and results: in the current study, we demonstrated that retinoids ( -cis retinoic acid (ra) or all-trans ra (atra)) significantly inhibited basal and hormone-stimulated ishikawa cell proliferation by over % using mtt assay. the same experiment indicated that estrogen had no significant effect, whereas progesterone slightly induced, on cell proliferation. cell cycle analysis indicated that atra significantly increased the g /g cell population by % and decreased s phase cells by %, suggesting that ra induces cell cycle arrest at the s phase. knock-down of rar alone or both rar and rxr significantly increased proliferating cell nuclear antigen (pcna) levels in epithelial ishikawa cells, suggesting that ra signaling via rar/rxr activation is critical for normal endometrial growth and differentiation. to determine whether retinoids are naturally secreted by the endometrial stromal cells, we cultured primary stromal cells and analyzed the culture media using hplc. we found that retinol is the predominant retinoid form secreted by stromal cells. the average concentration of retinol in the cultured media of eutopic endometrial stromal cells was approximately to ng/ml/ cells (n= ). although there was less than . ng/ml/ cells of all-trans retinal or atra in the cultured media, we did find a small peak for all-trans retinal and atra in the media using hplc analysis. furthermore, progesterone significantly increased secreted retinol levels from eutopic endometrial stromal cells, but decreased retinol levels secreted from endometriotic stromal cells. retinol is a precursor for ra that is converted to retinal and then to ra. conclusions: we demonstrated that progesterone signaling via pr induces endometrial stromal cells to secrete paracrine retionids that in turn control the phenotype of adjacent epithelial cells. conversely, this interaction is disrupted in diseased endometrial tissues, such as endometrial cancer and endometriosis. the effects of hormonal contraceptives on antimullerian hormone by obesity status. anne z steiner, frank z stanczyk, stan patel, alison edelman. ob/gyn, university of north carolina, chapel hill, nc, usa; ob/gyn, usc keck school of medicine, los angeles, ca, usa; ob/gyn, oregon health and sciences university, portland, or, usa. background: antimullerian hormone (amh) is emerging as a predictor of reproductive potential. serum levels of fsh, a commonly used measure of ovarian reserve, are suppressed with the use of oral contraceptives (ocs) thereby limiting its use. the impact of ocs and on serum amh levels in normal and obese women is unknown. objective: to examine the impact of ocs on serum amh levels by obesity status in reproductive-age women. materials and methods: ovulatory women, ages - years, of normal (< kg/m ; n = ) and obese (> kg/m ; n = ) body mass index (bmi) received a low dose oc ( mcg ethinyl estradiol/ mcg levonorgestrel) for two cycles. serum was obtained at three time points: after days of active pills (cycle , day ), at the end of the -day hormone-free interval (cycle , day ), and during the first week of active pills in cycle (cycle , day , , or ). amh levels were quantified by elisa; fsh and lh levels were determined by chemiluminescent immunoassay. amh at the three time points was compared using repeated measures anova. models were used to assess the relationship between amh and cycle day by obesity status. amh and gonadotropin levels were compared using spearman's correlation. results: amh levels did not differ by oc cycle day (p anova = . ) or by active versus placebo pill use ( . ng/ml ± . vs. . ng/ml ± . , p= . ) among normal bmi women. among obese women, amh levels differed by oc cycle day (p anova = . ), but not by use of active or placebo pill ( . ng/ml ± . vs. . ng/ml ± . , p = . ). across the cycle, cv(standard deviation/mean) averaged . %± . in the obese and . %± . in the normal bmi women ( p= . ). modeling to determine differences in amh throughout the cycles based on obesity status showed a significant interaction (p = . ) and lower amh levels in the obese group (p< . ). among both bmi groups, serum amh and fsh levels did not correlate during active pills or after days of placebo pills. conclusions: in young, normal bmi women serum amh levels do not appear to fluctuate during oc use. among obese women, amh levels are lower and fluctuate significantly. these fluctuations do not appear to mirror changes in gonadotropins and may complicate clinical interpretation of amh. background: menopausal hormone therapy (ht) is a confusing topic for many clinicians and patients. objective: to assess comprehension of basic clinical trial features among prospective participants for the keeps trial, designed to study the effects of ht initiated within years of menopause on chd markers. methods: screening materials were provided giving an overview of study purpose, duration, medications, likelihood of receiving drug vs. placebo, ht related risks and side effects. at a subsequent interview, a -item questionnaire assessed the participant's level of comprehension. a score of % was adequate to complete the informed consent process. those scoring < % were re-counseled and retested. demographic variables determining the likelihood of an initial score > % were evaluated by univariate and multivariable analyses. results: % ( / ) scored > % on initial testing. all women scored > % after re-counseling and retesting. likelihood of an initial response > % correct was unrelated to age or time since menopause. ability to correctly respond was influenced by highest educational level attained. none of women whose furthest educational level was high school scored > % on initial testing, significantly less than those with a college education (p= . ). a higher proportion of college graduates ( / ) scored > % compared to those attaining further education ( / ) (p= . ). comprehension was greatest for study purpose and duration ( / and / correct responses respectively) and least for questions related to results of the whi hormone trial breast cancer and chd. that e alone was not associated with an increased risk of chd ( %) or breast cancer ( %) was poorly understood. conclusion: comprehension of the risks and benefits of ht by potential research subjects is low despite provision of reading materials prior to the informed consent process. (supported by the montefiore medical center/albert einstein college of medicine site for the kronos longevity research institute and k -hd to ns). variation in menopausal symptoms within a sample of hispanic women: swan, the study of women's health across the nation. robin r green, alex j polotsky, aileen p mcginn, carol a derby, rachel p wildman, lhasa ray, kavitha t ram, gerson weiss, nanette f santoro. albert einstein coll of med, bronx, ny; univ of med and dent of new jersey, newark, nj. background: menopausal symptoms are experienced by over % of women. purpose: to describe symptom frequency in a sample of midlife hispanic women from different countries of origin. methods: the study of women's health across the nation (swan) recruited women at baseline who self-identified as hispanic. their baseline responses to validated questionnaires regarding common menopausal symptomatology were examined. symptoms were reported over the previous two weeks and scored on a frequency scale ranging from (not at all) to (every day). for all analyses, symptoms were dichotomized into "absent" vs. "present" variables. responses were correlated with acculturation ( -item scale: marin, hisp j behav sci : , ) and analyzed by sub-ethnicities: central/south american (c/s am), puerto rican (pr), dominican (dr), and cuban (cu). associations between symptoms and sub-ethnicity were tested by chi-square. logistic regression was used to test for associations with acculturation and being us-born. there was significant variation in several menopausal symptoms. while puerto-rican women had the highest likelihood of reporting trouble sleeping (or= . , %ci: . - . ) and headaches (or= . , %ci: . - . ), dominican women were most likely to report vaginal dryness (or= . , %ci: . - . ) acculturation and being us-born did not explain the variation between subethnicities in any of the models tested conclusion: there appear to be significant differences among hispanic women with respect to menopausal symptomatology. these differences were not readily explained by measures of acculturation. (supported by the study of women's health across the nation (swan) has grant support national institutes of health, dhhs, through the national institute on aging, from the national institute of nursing research and the nih office of research on women's health (grants nr ; ag , ag , ag , ag , ag , ag , ag , ag and cd ). purpose: to compare the relative effects of conjugated equine estrogen, raloxifene, and tamoxifen therapies on cognition among women aged years or older. participants and methods: annual modified mini mental state ( ms) examinations were used to assess global cognitive function among the , women enrolled in the two randomized placebo-controlled clinical trials of the women s health initiative memory study (whims) and women enrolled in co-star, the cognitive substudy of the nsabp's study of tamoxifen and raloxifen (star) trial who had baseline ms testing. associations between baseline cognitive risk factors common to both trials and baseline ms scores were assessed and interactions used to examine whether risk factor relationships differed between cohorts. factors for which relationships were similar were used as covariates in analyses comparing ontrial ms scores. factors for which relationships did not appear to be similar were used to stratify analyses. results: compared to placebo, each of the active therapies was associated with a small mean relative deficit in ms scores of . units or less, which was fairly consistent between women with and without prior hysterectomy. overall, relative deficits appeared to be most marked for tamoxifen (unadjusted p= . ) but were also evident for raloxifene (p= . ) and cee (p= . ). conclusions: while unmeasured differences between trials may have confounded our analysis, these findings suggest that both tamoxifen and raloxifene may adversely affect cognitive function in older women. weight gain and increased abdominal fat have been found in women after menopause and is associated with higher levels of leptin, and decreased levels of the cardioprotective adipocytokine adiponectin. at the same time, bmd characteristically decreases. in an effort to determine the evolution and correlates of these changes, we studied postmenopausal women (pm) within years of menopause (age . ± yrs) of normal weight (bmi . ± ) and compared them to weight matched (bmi . ± ) premenopausal (pre) controls (age . ± yrs.) all subjects had bmd and body composition studies by dexa and measurements of leptin, adiponectin, visfatin and retinol-binding protein (rbp .) while total fat was similar in the groups, pm had more trunk and abdominal fat ( ± ; ± g) compared to pre ( ± g p< . ; ± g p< . ) pm also had greater %trunk fat and %central abdominal fat compared to pre, p< . .serum leptin ( . ± . vs ± pg/ml) and visfatin ( . ± vs. . ± . ng/ml) were similar but adiponectin ( ± vs. . ± g/ml) and rbp ( . ± vs. . ± ng/ml) were higher, p< . in pm. while in pre, abdominal fat correlated negatively with adiponectin (p< . ) in pm only leptin correlated with various parameters of fat mass, p< . , and adiponectin did not correlate but correlated positively with age (r . , p< . .) as expected, pm had reduced bmd at the lumbar spine and hip ( . ± . vs. . ± . g/cm ; . ± . vs. . ± . g/cm , p< . ) but there was a correlation between total and trunk fat in pm and lumbar bmd (r . , . , p< . ) but not with hip bmd; or any correlations in pre. there was a correlation between leptin and lumbar bmd in pm (r . , p< . ) but not in pre. in summary, in normal weight early pm, abdominal fat is increased, but only adiponectin and rbp are altered with an increase in the former correlating with age. lumbar bmd correlated with fat mass in pm and is partially explained by increases in leptin. it is suggested that in spite of increasing abdominal fat in normal weight early pm, (which correlates with a higher lumbar bmd) david d rahn, jesus f acevedo, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. objectives: matrix metalloprotease (mmp) activity is increased in the postpartum vagina of wild type (wt) animals, and this activity is accompanied by a burst in elastic fiber synthesis. the mechanisms that precipitate these changes are unclear. the goals of this study were to determine how vaginal distention (such as in parturition) affects elastic fiber homeostasis in the vaginal wall and the potential significance of these changes in the pathogenesis of pelvic organ prolapse. methods: nonpregnant ( ) and pregnant (d ) wt mice underwent either vaginal distention with a μl balloon x min (to simulate parturition) or sham procedure. tissues were obtained at and h for immunoblot analysis, zymography, and histology. distention was also performed in young fbln -/-, fbln +/-, and wts, and prolapse was quantified for weeks. results: distention resulted in marked increases in mmp activity in nonpregnant animals (prommp , . -fold; active mmp , . -fold; prommp , . -fold; active mmp , . -fold; all p < . compared with sham) which was accompanied by fragmented and disrupted elastic fibers in the vaginal wall. abundant amounts of tropoelastin and fibulin- in the nonpregnant vagina were not increased further by distention. in pregnant animals (which normally have suppressed vaginal wall fibulin- and tropoelastin), however, distention resulted in -fold upregulation of both proteins (p< . ). distention also induced increased mmps in pregnant animals (mmp- , . -fold; prommp- , . -fold; active mmp- , . -fold; all p < . ). thirteen young fbln -/-( - wks prior to age of prolapse development), het siblings, and age-matched wts underwent serial exams after ballooning. distention induced rapid increases in perineal bulge and vaginal diameter (within d) in fbln -/mice which never recovered. conclusions: in pregnant mice, vaginal distention results in increased protease activity in the vaginal wall but also increased synthesis of proteins important for elastic fiber assembly. distention may thereby contribute to the burst of elastic fiber synthesis in the postpartum vagina. the finding that distention results in accelerated pelvic organ prolapse in fbln -/animals, but not wt, indicates that distention results in loss of pelvic organ support if elastic fiber synthesis is compromised. further, the data suggest that elastic fiber synthesis is crucial for recovery of the vaginal wall from parturition/distention-induced increases in vaginal protease activity. the molecular etiology of pelvic organ prolapse (pop) is complex and not yet well understood. defects in the connective tissue, such as a decrease in extracellular matrix (ecm) protein content may predispose women to pop. our objective was to study the expression and the enzymatic activity of matrix metalloproteinases (mmps) and their tissue inhibitor (timps) in vaginal tissue of patients with advanced pop and controls. after informed consent, pre-menopausal caucasian patients affected by pop ( grade by pop-q), and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer. western immunoblot analysis was performed (patients: n= , controls: n= ) to study the protein expression of mmp- , - , timp- , - , - . gelatin zymography was used to quantify the activity of mmp- and - . immunohistochemical analysis for timp- and - was performed on pfa-fixed vaginal biopsy tissue (n= ) in each group. both patient and control vaginal biopsy samples expressed latent and active forms of mmp- , and mmp- . the protein expression of the kda active form of mmp- was significantly increased in patients with pop compared to controls (p= . ). zymography detected the enzymatic activity of the proform and active form of both mmp- and mmp- . we found a significant increase in gelatinolytic activity of both kda pro-form and kda active forms of mmp- (p= . and p< . respectively) as well as kda active form of mmp- (p< . ) in patients with pop compared to controls. timp- protein expression in vaginal tissue showed a significant reduction in pop patients compared to controls (p= . ). timp- and - immunostaining were mainly localized in the smooth muscle cells at the muscularis layer of the vaginal biopsies. in vaginal tissue of patients with pop, we have shown a decrease in timp protein expression paralleled by an increase in mmp protein expression and activity. these findings reflect an active ecm remodelling that may compromise the structural integrity of the pelvic floor leading to pop. aberrant elastin and collagen synthesis may play a role in the pathogenesis of pelvic organ prolapse (pop). the lysyl oxidase (lox) family of enzymes direct cross linking of collagen and elastin polymers, however to-date no information is available on the expression and localization of these proteins in human vaginal tissue. our objectives were to study the expression and in situ localization of lox, loxl , loxl and loxl proteins in the vaginal tissue of patients with advanced pop and asymptomatic controls. pre-menopausal caucasian patients affected by pop ( grade by pop-q) and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer and western immunoblot analysis was performed (patients: n= , controls: n= ). pfa-fixed vaginal biopsy tissues (n= for each group) were used for immunohistochemical study. vaginal biopsy samples from both patient and control groups expressed all four members of lox family proteins: lox ( kda pro-form and kda active form), loxl ( kda pro-form and kda active form), loxl ( kda) and loxl ( kda). the expression of all lox family proteins was reduced in patients with pop compared to controls; however only the pro-form of lox of making the diagnosis of endometriosis from an endometrial biopsy. objective: to evaluate the diagnostic value of examining endometrial biopsy specimens for small nerve fibers in women with pelvic pain or infertility in a double-blinded prospective comparison with laparoscopy. methodology: we undertook to compare the detection of endometrial nerve fibers with laparoscopy and peritoneal biopsy for the diagnosis of endometriosis in women (aged . years; range - years) who presented with chronic pelvic pain or infertility. small nerve fibers were detected in the functional layer of endometrium using immuno-histochemical staining with the highly specific pan-neuronal marker pgp . , using a carefully validated technique and blinded assessment of nerve fiber density. laparoscopic assessment of the presence of endometriosis and peritoneal biopsies was undertaken by three experienced gynecologic endoscopic surgeons. data from these assessments were recorded independently of endometrial findings and maintained under blinded coding until the codes were broken. results: small nerve fibers were detected in all of the women in whom endometriosis was surgically diagnosed and in none of the women in whom endometriosis was excluded at laparoscopy, giving the specificity and sensitivity of %. the density of nerve fibers in the endometriosis cases was . per mm²± . (mean ± sd). conclusions: endometrial biopsy provided a reliability of detection or exclusion of endometriosis which was equal to that of diagnostic laparoscopy carried out by experienced gynecologic laparoscopists. background: erk / are mapk intracellular signaling proteins involved in cell survival and differentiation. endometriosis requires angiogenesis for ectopic implant growth. we hypothesized that the endometriotic peritoneal environment, known to be high in estrogen (e ), vegf, and cytokines such as il- and mcp- , stimulates angiogenesis in human endometrial endothelial cells (heec) through erk signaling. methods: serial sections from normal (n= ) and ectopic (n= ) endometrium were immunostained for total-(t-) and phospho-(p-) erk / and cd (an endothelial progenitor cell marker); results were quantified by computer densitometry and grouped by menstrual phase. cultured normal heec were treated with control, e ( - m), il- , mcp- , and vegf (all ng/ml) for min, and western blotted for p-/t-erk (n= ). heec were treated with peritoneal fluid (pf; diluted : in basal media) from normal (n= ) and endometriotic (n= ) women, with or without pd erk / inhibitor ( m) for h, and cell viability was analyzed by mtt assay. statistical analysis was done with one-way anova. results: tissue staining revealed that ectopic cd + endothelial progenitor cells undergoing angiogenesis (vessel sprouting and/or angiogenic cell cord formation) exhibited the strongest levels of p-erk. heec of ectopic foci showed moderate-high p-erk staining, while surrounding mesothelial capillaries were weak for p-erk. in normal endometrium, p-erk was cycledependent, with low levels in the late secretory phase vs. other phases (p< . ). p-erk of ectopic heec showed no cycle dependence, with moderate-high levels in all phases. t-erk in all tissues was high and invariable. in cultured heec, treatment with pf from endometriotic women significantly increased heec viability after h compared to normal pf (p< . ). this effect was abrogated by erk / inhibitor. among factors known to be high in endometriotic pf, vegf increased p-erk in cultured heec in and min (p< . ), while e , il- , and mcp- had no effect. conclusions: high p-erk found specifically in sprouting endothelial progenitor cells and focal ectopic capillaries suggests that erk / is involved in endometriotic angiogenesis. the peritoneal microenvironment of endometriosis may be persistently stimulating erk-mediated endometrial angiogenesis through vegf. further investigation into erk / inhibitors may offer a novel treatment of endometriosis. the events leading to the development of post operative adhesions are unknown, though recent observations emphasize the crucial role played by the superoxide ion generated by hypoxia. exposure of normal peritoneal fibroblasts to hypoxia caused the development of the adhesion phenotype, which is characterized by increased extracellular matrix molecules and inflammatory cytokines as well as high protein nitration and low nitric oxide. superoxide dismutases (sod) are a family of metalloenzymes that eliminates superoxide by converting it to hydrogen peroxide, protecting mammalian organisms against superoxide. objective: to determine whether sod is differentially expressed between normal peritoneal and adhesion fibroblasts and whether its expression is modulated by hypoxia. methods: fibroblasts from normal peritoneal and adhesion tissues were cultured under normal ( % o ) and hypoxia ( % o ) conditions for and hours. real time rt/pcr was utilized to measure the absolute mrna levels for sod in both cell lines before and after hypoxia exposure. results: normal peritoneal fibroblasts exhibited significantly higher basal mrna levels for sod ( . pg/ grna, p< . ) as compared to adhesion fibroblasts ( . pg/ grna, p< . ). short exposure to hypoxia resulted in a significant increase in sod mrna levels to . and pg/ grna in normal and adhesion fibroblasts respectively, p< . . in contrast, long exposure to hypoxia resulted in a significant decrease in sod mrna levels to . and . pg/ grna in normal peritoneal and adhesion fibroblasts respectively, p< . . conclusion: sod mrna levels are lower in adhesion as compared to normal fibroblasts, both basally and following short and longer exposure to hypoxia. reduced sod expression creates an milieu with greater free radical levels, which leads to the development of the adhesion phenotype. enhancement of sod levels and/or function are likely to be of benefit for reduction of postoperative adhesion development. objectives: development of endometriosis requires ectopic attachment and proliferation of endometrial tissue. the invasive process required to establish endometriosis may involve matrix metalloproteinases (mmps), including mmp- . recently, we have demonstrated that statins inhibit proliferation of endometrial stroma. this study evaluated the effects of simvastatin on a nude mouse model of endometriosis and on modulation of mmp- . methods: proliferative phase human endometrial biopsies were established as organ cultures or utilized for stromal cell isolation. to establish endometriosis in the nude mouse, endometrial tissues were maintained in nm estradiol (e) for hrs and subsequently injected intraperitoneally into ovariectomized nude mice. mice were treated with e ( μg, silastic capsule implants) and simvastatin ( - mg/kg/day) by gavage for days beginning day after tissue injection. control mice received e+vehicle. subsequently, animals were sacrificed and endometrial implants were evaluated. studies of endometrial stroma involved plating the cells in the presence of e ( nm) or e+medroxyprogesterone acetate (mpa; pm) with and without simvastatin ( - μm). some cultures additionally received interleukin- (il- , ng/ml). conditioned media was subjected to western analysis for expression of mmp- . results: in vivo studies demonstrated a dose-dependent inhibitory effect of simvastatin on number and volume of endometrial implants in mice. at the highest dose of simvastatin ( mg/kg/day), the number of endometrial implants was decreased by % and the volume by % in comparison to the control group. isolated stromal cells expressed abundant levels of mmp- following treatment with e, but minimal levels in cultures additionally receiving mpa or simvastatin. although il- induced a dramatic increase in mmp- secretion from cells pretreated with e alone, treatment with either mpa or simvastatin prevented this induction. cultures receiving e+mpa+simvastatin were the most resistant to mmp- induction by il- . objective: to increase awareness of potential presentations of adenomyosis and the differential of a uterine mass. material and method: in a tertiary medical center, a patient was being evaluated for a uterine cystic mass and cyclic dysmenorrhea. the patient is a year old nulliparous woman who has been complaining of cyclic dysmenorrhea for several years. the pain starts with the onset of menses and lasts around weeks. the patient did not improve on contraceptives. the patient had prior laparoscopy and imaging studies which misdiagnosed the mass as either a leiomyoma or an adnexal hemorrhagic cyst. the patient underwent exploratory laparotomy and resection of the mass. results: on ultrasound, the mass appeared as an echodense cyst within the uterus. intraoperatively, a cm thick-walled well circumscribed uterine chocolate cyst was identified. the resection of the cyst was performed in similarly to an ovarian cystectomy. tissue examination confirmed the diagnosis of cystic adenomyosis. following surgical intervention, the patient reported significant improvement of symptoms. conclusion: this case highlights an unusual presentation and treatment of adenomyosis and cyclic dysmenorrhea. cystic adenomyosis should be on the differential of uterine cystic mass, particularly in young women with cyclic dysmenorrhea and menorrhagia. the earlier misdiagnosis probably resulted from the unfamiliarity of physicians with this condition. similar clinical presentations may occur with congenital uterine anomalies (with an obstructed uterine segment) and cystic degeneration of a leiomyoma. the incidence and pathogenesis of cystic adenomyosis are unknown. oral contraceptives may be helpful as a first line therapy. however, resection of the adenomyotic cyst appears to be more effective, particularly in women seeking fertility. objective: it is hypothesized that abnormalities within the eutopic endometrium of females with endometriosis can cause the ectopic growth of the endometrial tissue at extrauterine sites. previous studies have shown that gene expression in eutopic endometrium of women with endometriosis is markedly different from disease-free females. inflammatory cytokines and receptor-dependent kinase pathways are widely recognized as therapeutic targets for immune disorders, which is believed to be the underlying pathogenesis of endometriosis. in this study we asked whether responses of primary eutopic endometrial cell cultures are dysregulated between females with or without endometriosis. methods: a total of biopsies of endometriotic eutopic endometrium (eee) and disease-free endometrium (dfe) were obtained from proliferative phase females. the primary cell cultures established from these biopsies were treated with tnfa to induce expression of inflammatory mediators. parallel cultures were also treated with kinase inhibitors of p , mek, pi k and ikk. after a period of or hours, concentrations of il- , mcp- , and gm-csf in cell culture supernatants were analyzed by elisa. results: in eee, basal concentrations of mcp- and gm-csf were - times higher, while il- was times higher compared to dfe. as expected, tnfa treatment stimulated higher levels of cytokine secretion in dfe mimicking disease state, however, the same treatment had almost no effect on eee. kinase inhibitors were very effective in lowering the cytokine levels of dfe, however, their effect on eee were less dramatic. conclusion: eee expresses higher levels of inflammatory cytokines under basal conditions, which is in concert with the current literature. our results validate that high il- levels in endometrium are diagnostic for females with disease. the increase of gm-csf, il- and mcp- following tnfa treatment was expected in dfe however; tnfa's effect was blunted in eee, which implies that eee are already highly activated. the effect of kinase inhibitors on cytokine production from eee was unaltered relative to dfe, which implies that tnfa stimulated kinase pathways are modified even in eee. endometrial cancer is the fourth leading cause of cancer in women, and hyperplasia and adenocarcinoma are more commonly seen in women with polycystic ovary syndrome (pcos). mig- , a negative inhibitor of egf signaling, is expressed in normal secretory phase endometrium and associated with hyperplasia in mig- knockout mice. objective: we examined and compared endometrium from normal and pcos women for mig- expression and characterized its regulation using in vivo and in vitro models. methods: immunohistochemistry (ihc) and real-time pcr were performed in endometrium from normal (n= ) women and pcos (n= ) women. regulation of mig- was studied in ishikawa and ecc- endometrial cell lines, treated with sex steroids or egf. endometrial xenografts from normal and pcos women were implanted in ovxd rag -(c) immunocompromised mice, treated with e or e + p pellets to mimic a natural cycle. results: mig- protein expression was low in the functionalis of the proliferative phase and high in the secretory phase; this pattern was reversed in the basalis layer. pcos secretory phase endometrium had significantly less mig- protein and mrna than normal endometrium (p < . ). xenografts using pcos samples had paradoxically elevated expression of mig- compared to normal, and appeared unresponsive to steroid treatments. mig- expression in endometrial cell lines was regulated by egf but not by ovarian steroids, e or e + p. conclusions: mig- expression is low in proliferating endometrium and regulated by egf. risk of hyperplasia or cancer in pcos women can be explained by altered expression of mig- . reduced expression mig- provides insight into endometrial function and may lead to better treatment options for disorders of endometrial proliferation including endometriosis, adenomyosis, endometrial hyperplasia and cancer. supported in part by nih-u -hd (sly), u hd (lcg) and u hd (fjd). could androgens influence human luteal cells function? anna tropea, mariateresa orlando, maria francesca gangale, federica romani, isamaria loiudice, federica tiberi, stefania catino, antonio lanzone, rosanna apa. cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. in pcos patients reproductive dysfunctions are frequently observed even if ovulation occurs. an impaired luteal function could partially explain this subfertility, since luteal steroidogenesis deficiency and premature luteal degeneration have been described in pcos patients. based on the frequent observation of high plasmatic levels of androgens in pcos, we investigated whether hyperandrogenism could negatively affect luteal function.to this aim, we tested the in vitro effects of androgens on progesterone (p) and on vascular endothelial growth factor (vegf) production by human luteal cells. indeed, vegf is essential for normal luteal development and function being an important regulator of angiogenesis and vascular permeability. since prostaglandins (pgs) play a central role in modulating luteal function, the influence of androgens on pge and pgf a secretion was also investigated. in order to investigate whether testosterone and androstenedione act directly or after local aromatisation, we tested the in vitro effects of estriol, estrone and --estradiol on p, vegf, pge and pgf secretion by human luteal cells. moreover, we tested the effects of testosterone and androstenedione in presence of exemestane -an aromatase inactivator. highly purified human luteal cells were cultured for h with medium alone (control) or in presence of increasing concentrations of testosterone or dihydrotestosterone or androstenedione (from - to - m) or in presence of increasing concentrations of estriol, estrone and --estradiol (from . to ng/ml). moreover, testosterone and androstenedione - m were tested in presence of exemestane (from to nm). in the culture medium vegf, p, pge and pgf secretion was assayed by commercially available elisa kits. in order to evaluate the influence of androgens and estrogens on vegf mrna expression on luteal cells real-time rt-pcr has been performed. our results demonstrated that testosterone, androstenedione and dihydrotestosterone were all able to significantly reduce vegf secretion in human luteal cells, while no effect was seen on vegf mrna expression. androgens were also able to significantly decrease p and pgf secretion, while an increase was observed on pge production. moreover our preliminary results demonstrated that in human isolated luteal cells estriol, estrone and -estradiol at all tested doses are able to mimic androgens effects on p and pge production. on the contrary estrogens were able to increased vegf release. estrogens seemed to have no effect on pgf  released. data regarding exemestane inhibition of testosterone and androstenedione are still in progress. increased levels of androgens were able to decrease luteal vegf, p and pgf release and might be involved in pcos-luteal phase defect. nevertheless, the observed effects could probably be attributed -at least in part -to estrogens locally produced via the aromatase enzyme, rather than be directly mediated by testosterone and androstenedione. the in vitro effect of proghrelin-derived peptides on purified human luteal cells. anna tropea, mariateresa orlando, maria francesca gangale, federica romani, isamaria loiudice, federica tiberi, stefania catino, antonio lanzone, rosanna apa. cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. ghrelin, well known mediator of neuroendocrine effects, has been demonstrated to have a role as signal for energy insufficiency. several evidences suggested that ghrelin might also operate as regulator of reproductive function. indeed, we recently demonstrated that both p and vegf released were significantly decreased by ghrelin in isolated human luteal cells. moreover ghrelin was also able to reduce prostaglandin (pg) e and increase pgf luteal cells release. in the present work we investigated whether two ghrelin-related peptides derived by the same ghrelin precursor (unacylated ghrelin and obestatin) might affect human isolated luteal cells function. conventionally regarded as an inert form of ghrelin, unacylated ghrelin has been recently proven as biologically active in specific cellular contexts. obestatin has been suggested to directly control porcine ovarian cell functions. in these assumptions, we investigated whether unacylated ghrelin and obestatin could directly affect luteal progesterone (p) release. moreover, since vascular endothelial growth factor (vegf) is known to play a critical role in luteal development and function, the influence of unacylated ghrelin and obestatin on luteal vegf, pgf and pge production was also analysed. highly purified human luteal cells were incubated for h with medium alone (control) or with hcg ( ng/ml) or with cocl ( μm) or in presence of increasing concentrations of unacylated ghrelin ( - nm) and obestatin ( - nm). in the culture medium vegf, pgf , pge and p secretion was assayed by commercially available elisa kits. moreover real-time rt-pcr has been performed in order to evaluate whether unacylated ghrelin and obestatin could affect vegf mrna in human luteal cells. our preliminary results demonstrated that either unacylated ghrelin or obestatin are able to negatively affect luteal steroidogenesis. moreover, both peptides seemed to increase the release of two luteotropic factors -vegf and pge and to reduce pgf release -a luteolytic prostanoid -from isolated human luteal cells. finally data regarding the expression of vegf mrna are still in progress. our results suggest that unacylated ghrelin and obestatin -two ghrelin-related peptides -could play a role in regulating luteal function affecting both luteal steroidogenesis and luteotropic/luteolytic imbalance. the mechanisms responsible for labor progression have yet to be fully elucidated. in a previous study, over-expression of small conductance calcium-activated k + channel isoform (sk ) in transgenic mice compromised parturition, suggesting a role for sk channels in this process. based on these findings, we hypothesized that sk channel expression is reduced late in pregnancy to enable the uterus to produce the forceful contractions required for parturition. we investigated the effects of sk channel expression on gestation and parturition by comparing transgenic mice over-expressing mice sk (sk t/t ) mice to wild-type (wt). in wt mice, sk transcript and protein are significantly reduced during pregnancy. the force produced by uterine strips from sk t/t mice on the day of delivery was significantly less than wt or sk knockout control (sk dox ), and the contractile force reached wt levels by application of the sk channel inhibitor, apamin. moreover, lipopolysaccharide and ru , which induce pre-term labor in wt mice, failed to result in completion of delivery in sk t/t mice. thus, stimuli that initiate parturition under normal circumstances are insufficient to coordinate the uterine contractions needed for the completion of delivery when sk channel activity is in excess. the mechanism(s) down-regulating this channel in the uterus during pregnancy is unknown. the sk gene promoter region contains two specificity protein (sp) binding sites; ) sp , is a transcription factor known to enhance the transcription of genes in response to estrogen, and ) sp , which competes for the same binding motif as sp , reduces gene expression. sp protein expression in mice uteri dramatically decreases in late pregnancy, while sp protein level remains consistent. our data indicate that sk channels must be downregulated for the gravid uterus to generate labor contractions sufficient for delivery in both term and preterm mice. the changes in sk channel expression may be transcriptionally regulated by sp and sp . key: cord- -atbjwpo authors: nan title: poster sessions date: - - journal: febs j doi: . /febs. sha: doc_id: cord_uid: atbjwpo nan ** each poster has been given a unique number beginning with the letter p; the next part relates to the session in which the poster will be presented. moreover, klf is also acting on cellular processes such as cell migration, apoptosis, inflammation, angiogenesis and differentiation. previous studies showed a novel role for klf as a regulator of proliferation and differentiation in skeletal muscle stem cells. detecting klf at the protein level harbored technical obstacles. commercially available antibodies exhibited low affinity, low specificity and failed to recognize post-translationally modified forms that are directly relevant to the function. thus, these obstacles prevent further functional protein studies such as western blots, protein co-immunoprecipitation and chromatin immunoprecipitation (chip) assays. therefore, we used crispr/cas system to establish a stable cell line which carry v epitope tag into the n-terminal of klf gene. insertion into the target side of klf gene via crispr-cas system provided an opportunity to overwhelm the above mentioned obstacles. v epitope tag would not interfere with the function of the klf and also enable us to recognize endogenous klf via anti-v antibody in the mouse myoblast cell lines (c c ). we confirmed the targeted insertion into the exon of the klf gene both at the dna and protein levels. the conformational dynamics of structural domains plays an important role in functioning of many proteins. the reca proteins from e. coli are known to be the central catalyst of homologous recombination and repair in bacteria. it forms a helical filament on ssdna capable to bind homologous dsdna and catalysis of the exchange of the complementary strand. significant mobility if its c-terminal domain has been observed experimentally by cryo-electron microscopy. however its potential significance for reca protein activities still remains unclear. in this work we investigated this question by construction of a mutant reca protein with artificial disulfide bridge between central and c-terminal domains. the wild type protein has no disulfide bonds, and therefore its native mobility can be restored in vitro, by addition of b-mercaptoethanol. our data suggest that the s-s bridge decreases both the rate of atp hydrolysis in vitro and the e. coli resistance to uv in vivo. thus, our experimental results indicate that the flexibility of the c-terminal domain significantly affects recombination activity of reca protein in vivo and in vitro. hydroxiurea (hu) is an inhibitor of ribonucleotide reductasethe enzyme that catalyzes the process of free dntps synthesis in living cells. treating cells with hu causes diminishment of the nucleotide pool. as a result, single-stranded dna regions are generated, which leads to s-phase checkpoint activation. the progression of replication forks is blocked and the completion of dna replication is prevented. this results in s-phase cell arrest. nevertheless, our results demonstrate that after prolonged hu treatment, the saccharomyces cerevisiae cells seam to escape the arrest and continue the progression of their cell cycle. we show that when cells re-enter the cell cycle, mrc , but not ctf is detached from chromatin. our data also shows that meanwhile, rad checkpoint activity is diminished in order to allow s-phase checkpoint escape and completion of the cell cycle. moreover, cells not only continue the cell cycle, but steadily surmount in the presence of hu. all this data indicates that cells have made the decision to compromise s-phase checkpoint and to adapt to the novel environmental conditions in order to survive. as both mrc and ctf are known to be responsible for polymerase and helicase harmonization during replicative arrest, our data indicates that mrc has a more specific role in the process of adaptation. our data demonstrates that mrc is a leading protein to regulate the stability of s-phase checkpoint arrested replication forks. zinc finger domain is the most common dna binding domain in metazoa. almost drosophila proteins with c h zinc fingers also have zinc finger associated domain (zad). several proteins with zad (zw , pita and zipic) were found to interact with cp and act as insulator proteins. for some of the zad-containing proteins (for example, weekle and grauzone) it was shown that their zad domains can form dimers with each other. the ability of these proteins to dimerize appears to be especially important in the light of the model suggesting that dna-binding insulator proteins can support genome looping and organization of chromatin structure via interaction with each other. in this work we aimed to understand the role that zadmediated protein-protein interactions play in maintenance of dna loops, focusing on proteins: zw , pita, zipic and cg . first, we performed co-precipitation and yeast two hybrid assays to confirm dimerization of isolated zads in vitro. we observed that only zads from the same protein can specifically interact with each other (homodimerization) and they are unable to interact with zads from different proteins (heterodimerization). we confirmed homo-but not heterodimerization of zads in vivo with coimmunoprecipitation experiments in s cells. furthermore, we found that zad proteins can support longdistance interactions in transgenic constructs in flies. using model system with cg protein, we demonstrated that zad is essential for these interactions. proteins without zad cannot maintain loop formation. finally, analysis of chip-seq experiments for zw , pita, zipic and cg revealed that binding sites of zad proteins often overlap with regions of inter-chromosomal contacts known from hi-c experiments. we conclude that zad-containing proteins can support longdistance genomic interactions and dimerization of zads is necessary for these interactions. this study was supported by the russian science foundation (project № - - ). over the years a large body of clinical knowledge has accumulated on pharmacological effects of drugs on thyroid function. antipsychotics are administered over long periods in humans; therefore their possible adverse side effects should be taken into consideration. the aim of this study is to evaluate the effects of haloperidol and clozapine on plasma t and t concentrations in adult male wistar rats. fifty rats aged between and weeks ( ae g) were divided into five groups (n = in each group), and drugs were administered each day intraperitoneally (ip) for days. the first group was a sham group. the other four groups were considered as low and high treatment doses of the drugs. after a one-week habituation period, animals was administered haloperidol ( . mg/kg, n = and mg/kg, n = ) and clozapine ( . mg/kg, n = and mg/kg, n = ). the rats were anesthetized with ether, and bloods were collected by direct cardiac puncture hours after the last injection. the t and t plasma concentration levels were analyzed with chemiluminescent immunoassay. statistical analysis was performed with ibm spss v . . kruskal-wallis and bonferroni tests were used. t plasma concentration levels significantly differ between sham (median= . mg/kg) and haloperidol ( mg/kg) (median= . mg/kg), haloperidol ( . mg/kg) (me-dian= . mg/kg) and clozapine ( mg/kg) (median= . mg/ kg), haloperidol ( mg/kg) (median= . mg/kg) and clozapine ( mg/kg) (median= . mg/kg) groups (p < . ). however, no significant differences between the groups regarding to t plasma levels were observed. in conclusion, haloperidol and clozapine increased the t plasma concentrations, but didn't have any significant effect on t plasma concentrations. p- . . - isolation of lipase producing strains of bacillus obtained from olive wastewater and screening for substrate specificity in this work, wastewater samples of an olive factory from yusufeli (artvin, turkey) were collected carefully. after a centrifugation period of samples, supernatants were applied to a . lm filter and incubated on lb agar medium for hours. based on differencies of colony morphologies, isolates were selected and purified for identification. s lipase activity assay was carried out by rhodamine b. all of the strains exhibited lipase activity. for determining the substrat specificity of isolates, different substrates were used; nitrophenyl-butyrate, -nitrophenyl-caprylate, -nitrophenyl-laurate, -nitrophenyl-myristate, -nitrophenyl-palmitate. results were measured spectrophotometrically at nm. all of the strains hydrolyzed -nitrophenyl-butirat, while there was no activity with -nitrophenyl-palmitate. bacillus sp. l was the most efficient strain that hydrolyzed all of the substrates. the gene encoding for lipase of bacillus sp. l will be cloned and expressed for more analyses and industrial applications. p- . . - some quantitative aspects of hair follicle layers differentiation e. vsevolodov , , v. golichenkov , a. mussayeva , , i. latypov llc "kazcytogen", almaty, kazakhstan, "institute of general genetics and cytology" sc mes, almaty, kazakhstan, lomonosov's moscow state university, moscow, russia in the course of stable hair growth the differentiation of hair bulb cambium cells to several layers with dissimilar cytochemistry and morphology takes place. this means the activation of different genes in the cells of different layers. depending upon the hair diameter some layers may be absent (medulla in the thin hairs). the hair diameters of the carpet sheep breeds vary widely even within the same square mm of the skin. we compared the different layers thicknesses proportions for the follicles with varying hair diameters. the follicle layers were measured on microphotos of transverse histological sections of the follicles made under the standard magnification. all follicles belonged to the same skin biopsy. the measurements were made at the levels just below the fissure separating the hair and inner root sheath appeared. the empirical regressions of the layers thicknesses and of ratios of different layers against hair diameters were counted. the computer model was made on the basis of these regressions which allowed to obtain the absolute parameters of the layers as well as ratios of these parameters for every chosen hair diameter. using this model we found an essential trend in changing the proportions in relative layers dimensions as we choose the follicles with more and more thick hair. when we change the follicles with mcm hair diameter for those with the hair diameter mcm the ratio of hair medulla diameter to hair diameter increases from . to . . the ratio of hair diameter to the diameter of inner root sheath increased from . to . . it means that the thicker is the hair the higher proportion of cells produced by cambium are spent to build innermost layers (medulla layer within the hair or hair within the complexinner root sheath + hair). these data may throw some light on positional information mechanism of layers differentiation. lignin is a heterogeneous polymer that constitutes % of woody plant cell walls. microorganisms that degrade lignin are fungi, actinomycetes and to a lesser extent, bacteria. in case of industrial applications, the use of fungi is not feasible due to the structural hindrance caused by fungal filaments, requirement of particular culture conditions such as humidity, aeration which are not compatible with industrial processing environments. bacteria are worthy of being studied for their ligninolytic potential due to their immense environmental adaptability. environmental concerns and increasingly stringent emissions standards have led the pulp and paper industry to devise ways to decrease the level of chlorinated lignin residues in its effluents through both production process changes and improved treatment technologies. bleaching with the enzymes is the most promising because the enzymes may be very efficient, and can be used under industrial conditions. the main objective of this study was to investigate the adequency of klebsiella pnuemonia gst (glutathione-s-transferase) pretreatment for bleaching of calabrian pine kraft pulp. for this purpose the following conditions were investigated: enzyme loadings from to u/g pulp basis and the consistecy of the pulp was between and %. enzyme at the desired concentration was added to the pulp and the mixture was incubated at °c for hours. after the enzymatic pretreatment to determine the optimum conditions the kappa number of all reactions were analyzed according to tappi standarts. as a result of this study we determine the optimum conditons as % pulp consistecy, u/g enzyme for pulp treatment. after the enzymatic treatment carried out under optimum conditions we are planning to submit a short bleaching sequence and analyze for physical properties such as viscosity and brightness. owing to this bleacing sequence we are going to able to compare the enzymatic and chemical treatments of pulp in bleaching prosess. p- . . - biochemical characterization of lipase from bacillus subtilis strain a from olive waste water f. ay sal, m. kac ßagan, s. c ß anakc ßi, a. o. beld€ uz karadeniz technical university, trabzon, turkey lipases (triacylglycerol acyl hydrolases, ec . . . ) are regarded as mild and environment-friendly biocatalysts for triacylglycerols hydrolysis. in addition to this hydrolytic reaction, they also catalyze reverse reactions of esterification, transesterification, and interesterification in non-aqueous environments. substrate, stereo-, regio-and enantio-specificities, and chiral selectivity are certain unique attributes of lipases that make them industrially attractive. these properties are often exploited in the manufacturing of detergent formulations, synthesis of fine chemicals, useful esters and peptides, food processing, paper manufacturing, degreasing of leather as well as in bioremediation. in this study, lipase from bacillus subtilis strain a is partially purified and characterized. bacillus subtilis strain a is isolated from olive factory from soke (aydin, turkey) and identified with s rrna analysis. lipase activity is screened on petri supplemented with rhodamine b. bacteria was grown in lb medium supplemented with % olive oil (vol/vol) for hour at °c. after incubation, cells were harvested by centrifugation at , rpm for minutes, resuspended in mm tris-hcl (ph . ) buffer, followed by sonication with sartorius labsonic m to release intracellular proteins. q-sepharose is used as ionexchange column chromatography for lipase purification. effects of temperature on activity and stability were determined spectrophotometrically using p-nitrophenyl laurate as the substrate. effects of ph on activity and stability were also determined. the effects of various metal ions and other reagents on the hydrolytic activity were assayed at °c. the enzyme was active and stable in the broad ph range of . - . and temperature range of - °c. bacillus subtilis strain a have high lipolytic activity. after cloning this enzyme to an expression vector and detailed characterization, this may suggests its usefulness in industrial applications. p- . . - investigation of pin as a nuclear factor one binding partner s. saritas, a. e. yilmaz, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey the nuclear factor one (nfi) proteins are important regulators of gene expression in the developing embryo and in adult stem cell niches. this transcription factor family has four members: nfia, nfib, nfic, and nfix. nfi proteins bind a consensus sequence on gene regulatory regions as homo or heterodimers. each member of nfi family has a highly conserved n-terminal dna binding and dimerization domain and a diverse proline rich c-terminal transcriptional activation/repression domain. as knockouts of nfi genes display distinct developmental phenotypes, we hypothesized that specificity of nfi protein function may arise from their interactions with binding partners. a yeast-two hybrid screen identified protein interacting with never in mitosis a (pin ) as a potential nfib interactor. pin is a ubiquitously expressed protein that specifically recognizes and binds to a phospho-serine or a phospho-threonine followed by a proline (ps/pt-p motif), and catalyzes isomerization of peptidyl-prolyl bonds. interestingly, both n-terminal and c-terminal domains of four nfi isoforms contain several conserved putative ps/pt-p motifs and some of these are reportedly phosphorylated. we looked for nfi pin interactions in vitro by gst-pulldown and co-immunoprecipitation assays. while gst-pin fusion protein interacts with all of four nfi isoforms, it binds nfib most strongly, nfia and nfic moderately, nfix most weakly. moreover, deletion of the cterminal domain leads to loss of nfi affinity for pin implicating this domain in nfi-pin interactions. co-immunoprecipitation assays where we co-expressed various epitope tagged nfi and pin proteins in hek t cells showed that pin precipitates nfib, as well as other nfi isoforms and nfib can, in turn, precipitate pin . we are currently carrying out site-directed mutagenesis on nfib to identify the specific residues that pin recognizes. we will further explore if this interaction regulates nfi function during embryonic development. that pre-adapt migrating fish to the life in seawater. among others, smoltification induces intense growth of fish that enter the ocean at a size where risk of predation is significantly reduced. skeletal muscle growth depends on a tightly controlled balance between protein synthesis and degradation. protein synthesis driven by hormone regulation is well studied in smoltified atlantic salmon; while less is known on protein degradation occurring via a number of pathways including cytosolic ubiquitin-proteasome system and calcium dependent calpains. the aim of this study was to compare calpain and proteasome enzymatic activities in the skeletal muscles of s. salar parr, pre-smolts and smolts. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from indera river (kola peninsula, russia). our results demonstrated the significant differences in studied protease activity levels between parr and smolts. calpain and proteasome activities in s. salar smolt muscles showed a significant drop compared with that of parr. the negative correlation between proteases activity levels in the muscle tissue and overall fish growth rate was shown. so, our data indicated life stage specificity in skeletal muscle protein degradation capacity in migrating fish. we suppose that intense muscle growth in s. salar pre-smolts is supported by various mechanisms including accelerated muscle protein accretion through the reduction of protease activities. obtained results enhance our knowledge in the mechanisms of atlantic salmon smoltification. the work was supported by the russian scientific foundation, project no. - - . p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the sociodemographic characteristics of the pregnant women who double and triple prenatal screening test h. d€ ulger, s. yabanci€ un meram medical faculty, n.e.university, konya, turkey double and triple prenatal screening tests which are applicable during first and second trimesters of pregnancy predict existent abnormalities at early stage. the aim of this study is to investigate the relationship between positive results of double and triple tests, further confirmatory tests during prenatal phase, postnatal status of babies and maternal age. in this study, double and triple test results of pregnant women who were admitted to meram faculty of medicine during - period were scanned from archive and test results indicating risk were detected. from these results, those which were above cut-off values for down syndrome, trisomy , open spina bifida were determined. a questionnaire was carried out with voluntary participants by reaching to these individuals. positive-negative result ratio of all double and triple test results and sociodemographic features such as age, occupation, presence of consanguineous marriage were investigated. all data from archive and answers from survey questions were assessed statistically. participants of the study were to years old and their average age was . ae . . ofthem ( . %) were under years of age whereas of them ( . %) were above years of age. number of pregnancies were scaling between to with an average of . ae . . of mothers ( . %) were not undergone amniocentesis, whereas babies with chromosomal abnormalities were detected among mothers who were undergone amniocentesis. in conclusion, there may be regional, sociological and such that reasons for those who were not undergone amniocentesis despite positive double and triple test results. ( . %) chromosomal abnormalities were detected among pregnancies with increased risk assessment with positive double and triple results. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the effects of oil on the growth and development of amphibians l. sutuyeva, t. shalakhmetova, a. ondassynova al-farabi kazakh national university, almaty, kazakhstan currently, the pollution of ecosystems by oil and oil products is increasing everywhere. the oil gets into water and ground during oil production, transportation and accidents. as a result, terrestrial animals and hydrobionts are exposed to oil contamination. thus, populations of animals decline. it can be assumed that the most sensitive to the effects of pollutants are animals in early stages of development. amphibians have established themselves as the most convenient bioindicator species. since lake frog (rana ridibunda) and green toad (bufo viridis) are the bioindicator species in kazakhstan, the study of the effects of oil on their larvae was carried out. we used water-soluble fraction of the oil from zhanazhol field (aktobe region) in our test. the larvae of control group were kept in pure water, and larvae of test groupsin aquariums with . , . and % concentrations of the oil fractions. the concentrations were chosen in accordance with the level of pollution of kazakhstan's water bodies with oil. mortality of larvae, morphometric parameters and morphogenesis were studied. it was found that high mortality of larvae is the most visible reaction when exposed to oil. this indicator rose noticeably depending on the doses ( . , . % and %) in both species with percentages %, % and % in r. ridibunda and %, % and % in b. viridis, respectively, while in the control group it was about %. furthermore, delayed larval development was detected. thus, the larvae from the control and . % oil group reached gosner stage (gs) , tadpoles from . % and % groups were at gs- and gs- , respectively, by the th day of life. moreover, behavioral abnormalities (sluggish movements) and decreased sensitivity to mechanical stress (touch) were observed under the influence of high concentrations of oil fractions. thus, oil in low concentrations alters the growth and development of tadpoles of anurans, and causes their increased mortality in high concentrations. p- . . - effect of catechin loaded plga nanoparticles on glioma cell line histone h t is a linker histone which binds to dna and contribute in chromatin condensation as well as regulation of specific genes through spermatogenesis. replacement of this histone h subtype and hyperacetylation of histone h tail, facilitate the replacement of histones with sperm chromatin condensing proteins of tnps and prms. ethical approval and informed patient consent was gained from infertile men referred to royan institute. testicular biopsies were collected from patients through assisted reproductive techniques (art) procedure. based on pathological results samples were classified into the following three subgroups: obstructive azoospermia (as positive control), complete maturation arrest and sertoli cell only syndrome (negative control). chromatin of tissues evaluated for presence/absence of histone h t protein in regulatory regions of tnps and prms genes using chip-real time pcr. results showed lower incorporation of h t protein on regulatory regions of tnps and prms genes in two spermatogenic failure group versus positive control. in this study, it can be concluded that the decreased levels of h t histone variant in testis tissues and failure in chromatin condensation have significant association with male infertility. p- . . - serum dickkopf- levels in obese children and adolescents that obesity is detrimental to bone health despite potential positive effects of mechanical loading conferred by increased body mass on bones. the wnt/b-catenin pathway is essential for normal osteogenesis. serum dickkopf- (dkk- ) is one of the most important inhibitors of the wnt//b-catenin pathway. the aim of this study was to investigate the serum dkk- levels in obese and non-obese children and adolescents. materials and methods: the study included obese children and adolescents ( males and females) aged from to years and healthy normal-weight controls ( males and females) aged from to years. serum dkk- levels were measured by elisa method using commercially available kit. results: body mass index of the obese children was significantly higher than that of non-obese children (p = . ). however, there was no significant difference between dkk- levels of the groups. (these results are preliminary and the study is continuing). discussion and conclusion: our result showed that serum dkk- levels were not changed in obese children and adolescents. p- . . - transcriptional regulation of cdo by nuclear factor one proteins b. kutay, c. lektemur, v. g€ uler, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey nuclear factor one (nfi) transcription factors play important roles in regulation of central nervous system development. three of the four members of nfi family, nfia, nfib, and nfix are expressed in neural progenitors, as well as neurons and glia in the embryo. inactivation of these genes in mice show that they function in development of neocortex and hippocampus in the forebrain, cerebellum, spinal cord and precerebellar nuclei of the hindbrain, regulating neurogenesis, gliogenesis, as well as neuronal migration, axonal outgrowth and guidance. all three neural specific nfis are expressed in precerebellar neuroprogenitors, however, only deletion of nfib leads to a delay in development of precerebellar neurons. investigation of misregulated genes in nfib À/À precerebellar neuroprogenitors identified cell adhesion associated, oncogene regulated (cdo) as a potential downstream target of nfib. interestingly, this gene has been reported to be upregulated in nfia À/À hippocampus as well. cdo, a cell surface glycoprotein of the ig superfamily, has been found to regulate neurogenesis in vivo, is highly expressed in the developing brain and can induce neural differentiation by promoting heterodimerization of basic helix loop helix transcription factors with e proteins. bioinformatic analysis of the kb human cdo promoter region identified five nfi binding sites: one cluster in the first kb region, another in the . kb upstream region. electrophoretic mobility shift and supershift assays showed that nfib binds to all five sites. furthermore, nfib, along with the other neural nfis, inhibits the proximal cdo promoter driven luciferase activity by up to % in hek t cells. preliminary data indicate that nfis bind to sites in both clusters in human neural stem cells (hnscs) suggesting that these sites are functional in vivo. we are currently investigating this possibility through nfi overexpression and silencing experiments that will examine regulation of cdo in hnscs. differentiation. the aim of this study is to investigate bdnf and drd /ankk gene variants in eos development. in this study, eos patients and healthy controls were used. genomic dna extraction was performed from peripheral blood leukocytes. drd /ankk taq a (rs ) and bdnf val met (rs ) polymorphisms were determined by real-time polymerase chain reaction (rt-pcr). positive and negative syndrome scale (panss) was used to determine eos severity. for drd /ankk rs polymorphism, there was a significant difference in the genotype frequencies between patients and controls for the co-dominant model (p = . , or = . ; % ci: . - . ). however, no significant relationship was observed in the genotype frequencies of bdnf val met polymorphism between eos patients and controls (p = . ). these results indicate that, drd /ankk rs genotypes may affect eos development. however, bdnf val met polymorphism may not be associated with eos. lack of association of bdnf val met polymorphism may be due to limited number of patients. our findings need to be confirmed by further studies. various dyes used in the textile industry are discharged in large quantities to the receiving environment in the manufacturing process. this is the beginning of a process that is difficult to compensate for environmental and human health. therefore, contaminated areas should be cleaned. in addition, technologies with high polluting potential should be integrated with biological approach and thereby the impurities consisting of dyes should be reduced. in this experiment; burdirect black meta konz (c.i. direct black ) was intended to decolorization using laccase. firstly, enzymatic decolorization of the dye was determined using spectrophotometry. the wavelengths of maximum absorption of burdirect black meta konz (c.i. direct black ) was determined between and nm. then, optimization studies have been done. for optimization studies; dye concentration, laccase activity, ph, buffer concentration, temperature, mediator effect, mediator concentration and time parameters were determined. lastly, in optimal conditions, atr-ftir and gc-ms analyzes of ensuring decolorization of dye were analyzed. decolorization of burdirect black meta konz (c.i. direct black ) was performed successfully and the absence of any metobolite in the decolorization medium has been provided by atr-ftir and gc-ms analyzes. assessing in terms of application, it can be easily applied by provided the reaction conditions in textile factories. laccase is a tool of decolorization of dyes in environmental friendly process. thus for the development of spermatids into mature sperm able to fertilize the oocyte.one of the causes of male infertility is in fact impaired sperm fertilization capacity due to sperm chromatin abnormalities and aberrant protamine replacement.recent research has focused on protamine biology,including protamine gene and protein structure,mechanisms of protamine expression regulation and involvement of the protamines in male fertility.various studies reported abnormal expressions of protamine (prm) genes in sperm of infertile men.the aim of the study is to investigate the gene expression of prm , prm and their relationship with defective spermatogenesis. materials and methods: this study has been performed on infertile and fertile turkish men.total rna was extracted from the sperm pellet using trizol reagent.after rna extraction and cdna synthesis,real-time quantitative polymerase chain reaction (rt-qpcr) was used to determine the expression of prm and prm . results: distinct levels of spermatozoal prm and prm mrna were found in infertile patients compared to fertile control groups.we found that the mrna levels of prm was reduced in (% ), and the mrna levels of prm was reduced in (% ) out of infertile patients.in the current study,we found statistical significant association between the prm expression and infertility (p < . ).although prm gene expression was decreased in most of infertile patients compared to fertile control groups,the differences between the groups were statistically insignificant (p > . ). discussion: the results of the study suggested that, the protamine expressions which were associated with spermatogenesis may be important in infertility treatment. further studies are required in a large series of different populations to clarify the role both prm and prm themselves and their mrna expression on male fertility. the study was conducted to characterize the processes of muscle growth in atlantic salmon (salmo salar l.) of different ages inhabited rivers indera and varzuga (kola peninsula, russia) in summer and autumn. the expression levels of genes myosin heavy chain myhc, myostatin (mstn), and myogenic regulatory factors myf , myogenin) in white muscle were studied in salmon parr of age groups +, +, + in june and october. the changes in expression levels of mrfs, myhc and mstn indicating the extent of hyperplasia, hypertrophy, and restriction of muscle growth at different ages of parr were revealed. the pattern of age-related changes differed between seasons. especially, the expression of genes myod, myogenin and myhc peaked in yearling parr ( +) in summer, that indicated the high rate of hyperplastic and hypertrophic muscle growth in yearlings ( +). at the same time, the mstn expression level, the negative regulator of muscle growth, was highest in parr at age +. possibly, it is the necessary regulation mechanism to attenuate hyperplasia and hypertrophy and control muscle growth. in autumn, the expression level of myhc and myogenin were higher in salmon of age + and + then in +, indicating the higher intensity of hypertrophy in parr at both first ages in comparison to +. there was no differences in expression level of myod, myf and mstn between age groups in autumn. moreover, the expression levels of genes studied were lower in autumn than in summer. thus, it indicated the decrease of muscle protein synthesis and muscle growth rate in autumn. these findings expand knowledge on age-and season-related features of muscle development in young atlantic salmon in their natural habitat. the study was supported by the grant of the russian science foundation no. - - . p- . . - lmp and lmp gene polymorphisms in the southeastern anatolia population of turkey d. mihc ßioglu , f. ozbas gerceker sanko university, gaziantep, turkey, gaziantep € universitesi, gaziantep, turkey introduction: the low molecular weight polypeptide (lmp ) and low molecular weight polypeptide (lmp ) genes are located in the class ii region of the major histocompatability complex (mhc) locus on chromosome . these genes encode peptides forming the large components of the proteosome complex which degrades short-lived cytoplasmic proteins. due to the significant role of lmp products antigen presentation, these genes can be accepted as strong candidates of susceptibility factors for different diseases. population genetic studies can also contribute to understanding of the possible role of lmp gene polymorphisms. the aim of this study was to determine the allele and genotype frequencies of the lmp and lmp gene polymorphisms in southeastern anatolia population and to compare these with the frequencies in other populations previously reported. material and methods: a total of healthy and unrelated individuals participated in this study. polymorphism analyses were done by polymerase chain reaction (pcr)-restriction fragment length polymorphism (rflp) method and allele/ genotype frequencies of lmp and lmp genes were determined. results: a deviation from the hardy-weinberg equilibrium (v = . ,p < . ) was found for the genotype distribution of lmp gene polymorphism, while the lmp genotypes found to be distributed (v = . ,p > . ). discussion: available allele frequency data for different populations were used to calculate genetic distances and to construct a neighbor-joining tree. among the included populations, nahuas (mexico) population was found to have the lowest genetic distance from the southeastern anatolia-turkey population. conclusion: it can be concluded that, more studies using different types of genetic markers are needed to clarify the filogenetic relationships of southeastern anatolia population with other populations and also the number of population studies on lmp and lmp genes should be increased to understand their effects as a genetic marker. p- . . - investigation of in vitro antioxidant activity of quercetin loaded plga nanoparticles pharmacological effects. but its usage is restricted because of low aqueous solubility, poor bioavailability, poor permeability and instability in physiological medium. these problems can be overcome with encapsulation of quercetin into nanocarriers such as biodegradable plga based nanoparticles. polymeric nanoparticles which have - nm particle size and providing controlled released of biological active agent are prepared by using biodegradable and biocompatible polymers. in this study, encapsulation of quercetin molecules into plga nanoparticles was carried with using the single emulsion (w/o) solvent evaporation method. size measurements of the obtained nanoparticles were performed by zetasizer and their size were found . ; . ; . nm respectively. the morphological features were examined by sem images. antioxidant activities of q , q ve q nanoformulations have been investigated by dpph and no (nitric oxide) methods. it is thought that the nanoparticular formulations that is developed in this study can be useful model for the other antioxidant molecules and will provide a significant contribution to the food and pharmaceutical industry. "this research has been supported by yıldız technical university scientific research projects coordination department. project number: - - -gep ". p- . . the effect of environmental enrichment on spatial memory and certain nmdars, and ht a expressions in rat pups introduction: the aim of the study was to investigate the effect of environmental enrichment exposed during whole childhood on spatial learning and memory and certain nmdars, and ht a in the hippocampi of pups. materials and methods: four-weeks old, male, weaning rats were randomised into groups as enviromental enrichment (ee, n = ) and standard cage control (scc,n = ) groups. eeg housed in an enriched environment and sccg were kept in standard cages for weeks. following the experiment the rats were trained and tested in the morris water maze (mwm) , open field test (oft) and forced swim test (fst) in order to assess the neurobehavioural effects of ee. nr a, nr b, ht a protein levels were analyzed by western blotting from hippocampi of rats. results: the positive effect of ee was seen at the learning phase in the mwm as 'latency to locate the hidden platform' between groups thoughout the training days showed that eeg located the hidden platform significantly earlier than sccg on days , (p = . , p < . ). also eeg significantly spent lower time in the outer zone of the maze on days , which was the sign of low anxiety level (p = . , p = . ). the parameters of oft which indicated increased locomotion, exploration and low anxiety were significantly higher in eeg (p < . ), in fst comparison of groups showed no difference (p > . ). the levels of nr b and ht a were significantly increased as compared to sccg as well (p < . , p = . ). discussion & conclusion: these findings showed that exposure to ee throughout the whole childhood causes several neurobehavioural effects like increased exploration and low anxiety. these effects may lead to improvement in speed of learning. increase in the nr b and ht a concentrations which are the receptors that are related to learning and memory in the hippocampi accompanied these changes which may be basis of the neurobehavioural improvements or may provide contribution to positive neurobehavioural effects. p- . . - effects of monosodium glutamate exposure during prepubertal term on several biochemical parameters in rats h. i. b€ uy€ ukbayram, d. kumbul doguc ß, i. ilhan, a. y. ismail s€ uleyman demirel university, isparta, turkey monosodium glutamate, which is commonly used in processed foods as flavor enhancer, is considered 'generally recognised as safe' by fda; however many studies have revealed the negative effects of msg.we aimed to evaluate the effects of msg in childhood on several serum parameters. sixty-six rats, ( weeks old) were divided into groups as control (cg, n = ; + , male+female) , experiment (msg-low dose, e g, n = ; + , male+female) and experiment (msg-high dose, e g, n = ; + ) groups. msg was administered at mg/kg/d to e g, . g/kg/d to e g for weeks by oral gavage. the rats were sacrified and blood samples were collected from aorta. the blood samples were centrifuged, the serum samples were separated and glucose, alt, total protein, albumin, creatinine, cholesterole and triglyceride levels were analysed by beckmann au autoanalyser. level of total protein was significantly increased in e g and e g groups when compared to cg (p < . ). level of alb€ umine was also increased in both egs but significant difference was seen in e g as compared to cg. creatinine levels were significantly increased in egs when compared to cg (p < . ). although the glucose levels in both egs were increased, the increase in e g was statistically significant (p < . ). the alt levels of in egs were also increased but the significant increase was seen in e g (p < . ). the effect of msg seem to be dose dependent and especially effect on carbonhydrate metabolism. increasing doses caused increase in glucose level, and tendency to glucose intolerance. increasing doses of msg also caused increase in creatinine and urea. another apparent effect of msg was detected on alt activity. in conclusion the negative effect of msg on glucose level, liver and kidney functions depends on daily dose intake. consumption of msg seem to be inevitable it has to be restrained in children otherwise early metabolic problems may be future problems for these children. ( mg/kg) + tartrazine ( mg/kg) + brilliant blue fcf ( mg/kg) + ponceau r ( mg/kg) + azorubine ( mg/kg) + indigotine ( mg/kg) + erythrosine ( mg/kg). artificial food color mixture were administered to g and g and drinking water was applied simultaneously to g by oral gavage per day for weeks. after application all rats were sacrificed, the total oxidant (tos)/antioxidant (tas) capacity were analyzed in rats' brain, liver, kidney homogenate and serum with rel tos-tas diagnostics assay kit.the statistical analysis was carried out by using kruskal wallis test. tas and tos levels in liver homogenate were not found significantly different between all groups (p > . ). in serum and kidney and brain homogenate, tas levels were not significantly different between all groups. tos levels in g were higher than g and g in serum and kidney and brain homogenate (p < . ). exposure to synthetic food colors may increase oxidative stres in vitale organs such as brain, kidney in female rats. these alterations differ according to organ and dose. parallel with increasing trends on healthy eating habits, consumption of prebiotics and probiotic microorganisms have been popular due to their benefits on human health. functional dairy foods such as probiotic yoghurt and cheese are the most common foods including probiotic microorganisms. due to some considerations such as standardization and quality in bulk production, starter cultures are used in industrialised fermentative food production to start fermentation. starter culture basically refers the microorganisms which induce and maintain fermentation of the fermentative foods and starter cultures including probiotic microorganisms are called as probiotic starter cultures. in this study, probiotic cheese starter cultures as a microbial community were investigated using computational systems biology tools. a metabolic network model of probiotic cheese starter culture was reconstructed using microbial community network modeling approach. literature-based genome-scale metabolic models of commonly used lactic acid bacteria were used for the microbial community metabolic model. the microbial community metabolic model simulated metabolic interactions of the microorganisms in the probiotic starter culture. metabolic flux values computed by the metabolic network model also predicted the metabolic pattern of the glycolysis (conversion of lactose), lipolysis (conversion of fat) and especially amino acid catabolism which are associated cheese flavor metabolism. simulations obtained by metabolic network-based analysis of cheese starter cultures can also be used for other fields like genetic engineering, upstream processing of the functional cheese production. p- . . - er quality control protein network in cf to modulate f del-cftr rescued phase ii xenobiotic metabolizing enzymes convert parent compounds into more hydrophilic metabolite by catalyzing conjugation reactions including glutathione and amino acid conjugation, glucuronidation, sulfation and acetylation. this study was aimed to describe the best cell line model for studying phase ii xenobiotic metabolizing nqo and gst-pi enzymes. for this purpose, mrna and protein expression of nqo and gst-pi enzymes were analyzed in ht and sw (colon); hepg and huh (liver); pnt a and pc (prostate) cell lines by qrt-pcr and western blotting techniques, respectively. protein expression analysis revealed that nqo protein was expressed in all cell lines and relative protein expression is highest in the hepg ( %) and pnt a ( %) while huh ( . %) showed relatively low expression of nqo . in addition, nqo mrna expression was relatively high in ht ( . fold) and pnt a ( . fold) when compared with liver cell line hepg ( . fold). gst-pi protein expression was found very high in huh ( %) while there was no expression in hepg . gst-pi mrna expression was relatively higher in pnt a ( . fold) and ht ( . fold) when compared with huh ( . fold). according to these results, choosing the best cell line as model depends on the purpose of the research. for studying metabolism of a chemical by nqo and gst-pi or effect of a chemical on translational regulation of these enzymes, it is better to consider protein expression of the cell lines for choosing best model. however, if the aim is to study effect of a chemical on transcriptional regulation of these enzymes, it is better to choose a cell line that expressing highest mrna of gene of interest. in conclusion, considering both mrna and protein expression levels together, the best model cell lines for studying phase ii xenobiotic metabolizing nqo and gst-pi are ht and huh , respectively. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the studies on the pancreatic cells' surface glycoconjugates profiles in rats fed with high fat with lectin labelling methods by flouresans microscopy y. mater, s. beyhan ozdas gebze technical university, kocaeli, turkey in this study, the backbone of the cellular adhesion-recognition mechanism, located in the cell membrane. the study material selected pancreas tissue, has a privileged structures. the pancreas is one of the main organs to aid in digestion. the pancreas functions as an exocrine gland and role in digestion. in addition, the pancreas also functions as an endocrine gland, secreting several hormones into the blood that control the blood levels of glucose and other nutrients. due to the pancreas have been selected for this unique feature. thus, different types of cells in the same sample will be able to study the structure of the surface glycoconjugates. generally researches about determination of carbohydrates in the cell, glycoproteins or/and glycolipids are cut with enzymes. next step, the oligosaccharide mixture obtained, than establishing the complete structure of oligosaccharides and polysaccharides requires determination of branching positions, the sequence in each branch, the configuration of each monosaccharide unit, and the positions of the glycosidic links. this is a more complex problem than protein and nucleic acid analysis. these processes are indispensable for the understanding of the chemical structure of the sugar. whereas in cells using labeled lectins specific sugars, it is possible to accurately determine. in this study, was used triticum vulgaris (wga) labeled with fluorescein (fitc). thus, the cells located on the cell surface and neu ac (sialic acid) for wga sugar residues were investigated. according to preliminary results of this study, wga labeled with fitc is specifically binding of these sugars. when this study is completed, the differences of sugar on the surface of different type of cells in the pancreas can be distinguished in micrographs. thus, in the cells of the pancreas, the sugar units involved in adhesion-recognition can be possible to determine specifically. large scale gene networks could be topologically analyzed in order to obtain possible global system-level structure cancer gene co-expression networks can have lower connectivity as compared to normal samples. using colorectal tissue gene expression datasets, we observed that tumor specific networks are less connected than normal networks. functional enrichment analysis suggested that cell cycle genes and methylation-associated cell adhesion genes can specifically play a role in the connectivity loss of carcinoma samples. literature confirmation provided a gene network including significant genes playing roles in the intersections between cell cycle, cell adhesion, and cell skeleton dynamics. this network can provide novel insight to our understanding of the molecular mechanisms of colorectal cancer. p- . . - tf-mirna circuits specific to epithelial cancers y. oztemur, a. aydos, b. gur dedeoglu ankara university biotechnology institute, ankara, turkey cancer is the most common cause of death in the world but there are still a lot of uncertainties about the exact mechanism taking roles in regulation of it. cancers can be classified according to cell type; in which they start. carcinomas are the most prevalent types of cancer and start in epithelial tissues. they are also named as epithelial cancers (ecs) and make up about out of every cancers. over the past few years, many studies are concentrated on mirnas, which have emerged as important regulators of gene expression like transcription factors (tfs). tfs are regulators at transcriptional level while mirnas are post-transcriptional regulatory key-elements. otherwise the transcription of mrnas and mirnas are known to be regulated by tfs and tfs are the targets of mirnas. therefore, it is crucial to characterize the relation of tfs, mirnas and their targets by building circuits in diseases such as in ecs. for this study, mirna and mrna expression studies including epithelial tumors and normal samples searched in geo and array express microarray databases. mrna studies and mirna study, which were designed for different ecs (breast, lung, ovary and colorectal) were selected to be analyzed. differentially expressed (de) mrnas and mirnas between epithelial tumors and normal samples were extracted (p ≤ . , fold change). among de genes, transcription factors and mirnas were identified and listed for epithelial tumor vs. normal comparison. circuit analysis resulted with remarkable circuit, which was common for all the types of ecs that includes klf transcription factor and hsa-mir- . in the literature hsa-mir- and klf are known as important regulators in different types of cancer, which indicated that the motifs involving tfs and mirnas might be useful for understanding the regulation of ecs. as a conclusion finding out new and common circuits may aid us in predicting new or alternative diagnostic and/or prognostic biomarkers for ecs. mesenchymal stem cells (mscs) are multipotent stromal cells that can differentiate into a variety of cell types which are used in cell therapy. although they are the most attractive cell type for cell therapy studies, primary mscs lose their differentiation potential with increasing time in culture and passage so they are of limited use. due to this disadvantage, msc lines are more suitable for in vitro researches owing to their immortality. in this study we compared primary bone marrow-derived msc (bm-msc) with bone marrow derived msc line (rcb ) in terms of cell characteristics and gene expression profiles to determine the functional differences among mscs types. firstly, mscs were identified by using cd , cd , cd and cd as positive markers and cd as a negative marker. gene expression profilling was investigated using affymetrix hg-u -plus arrays. the significant go biological process terms and kegg pathway enrichment analyses of the identified degs were performed using david (p < . , fold change≥ ). the analysis showed similar pathway clustering in both cell types. the resulting quantitave transcriptome of genes were identified that differentially expressed in msc line versus primer mscs ( upregulated and down-regulated). functional classification of changed genes was mainly clustered in cell cycle, cell death and mismatch repair. kegg pathway analysis revealed that the genes were significantly enriched in pathways including "cell cycle, dna replication and focal adhesion" pathways. in conclusion, our results indicate that msc lines can be used instead of primary mscs. these quatitative results provide an important basis to adapt cell lines to more closely resemble physiological conditions as oppossed to animal experimentation. this could help to minimize the use of animals in research. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] association between loss of q , gain of q . and progression in sporadic colorectal cancer n. belder , b. savas , m. a. kuzu , i. pak , h. s€ umer c ß elebi , a. ensari , h. € ozdag biotechnology institute, ankara university, ankara, turkey, school of medicine, ankara university, ankara, turkey, ankara oncology training and research hospital, ankara, turkey colorectal cancer (crc) is one of the most diagnosed cancer and the third leading cause of cancer deaths throughout the world. identifying of copy number variation (cnv) profiles between early and late stage cancers can be useful to understand the progression and aggressiveness of cancer. the main goal of this study was to construct a comprehensive insight of association between cnv and sporadic crc stages in order to identify novel candidate targets which may contribute to tumor progression. affymetrix . genechip snp arrays were used for characterization of cnvs in tumor and matched normal formalin-fixed, paraffin-embedded (ffpe) tissues from stage i, stage ii and stage iii samples. paired cnv analyses were performed using partek genomic suite . and genomic segmentation algorithm was performed using a minimum of markers per segment, a signal-to-noise ratio of . and the cut-off value for the gain and loss was set of ae . . the adjusted p-value ≤ . were considered to be significant. whole genome cnv analysis revealed that amplification of q . with genes was found the most frequent ( . %) in stage ii tumors. the most frequent ( %) amplifications were q . and p . in stage iii tumors. while deletion of chromosome q . in stage iii with a frequency of % was found the most frequent loss, deletion of q . was seen the most frequent ( . %) in stage ii tumors. two tumor suppressor genes smad and smad which are found in these deletion regions were common genes between stage ii and stage iii. our results showed that gain of q . might have a significant role in the progression of cancer. loss of q comprising two tumor suppressor genes is also another important finding. q loss can be a significant prognostic value in colorectal cancer even though validation of target genes requires additional study and larger sample size. this work was supported by tubi-tak project no: s . p- . . - meta-analysis based mirna signature discriminates cervical cancer from normal samples a. yucel polat, y. oztemur, a. aydos, b . gur dedeoglu biotechnology institute, ankara university, ankara, turkey gynaecological cancers are common problems in female health. squamous cell carcinoma (scc) is a type of these malignancies. this tumor type is derived from pre-cancerous lesions, which is called cervical intraepithelial neoplasia (cin). cin is classified as cin , cin and cin according to their dysplasia grade in the cervical tissues. mirnas are small non-coding rnas that were shown to have important roles in the development and progression of various cancers. the aim of this study is identifying mir-nas, which are playing a part in progression of cervical lesions by a ranking based meta-analysis approach. two mrna and three mirna expression studies, which include normal, cin , cin and scc samples were selected from arrayexpress and gene expression omnibus (geo) databases. three mirna studies were combined with anova dependent ranking based meta-analysis program which was developed in our laboratory to find out a mirna signature that can discriminate cin , cin and scc samples from normal samples. the top five mirnas with the highest ranks in meta-list were selected for further analysis. predicted targets of these mirnas were identified by mirdb target prediction tool. additionally two mrna datasets were selected for mirna-target validation studies. common genes, which were obtained from meta-mirna targets and differentially expressed genes between normal and cin , cin and scc groups from two independent studies, were identified and they were subjected to pathway enrichment analysis. pathway enrichment analysis that was performed with common genes showed that these targets were significantly enriched (p < . ) in especially cell proliferation, cell survival and cell cycle pathways, which are the key players of cancer development and progression. the meta-analysis results together with validation analysis of their targets may point out the potential roles of mirnas as biomarkers for the diagnosis and the treatment of cervical cancer. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the hypoglycaemic and regenerative activity of thymbra spicata in alloxanized-diabetic rats thymbra spicata (labiatae), a carvacrol and thymol containing plant, is one of the medicinal herbs used by diabetic individuals to reduce blood glucose in turkey. we investigated the hypoglycaemic and anti-lipemic effects of the aqueous extract prepared from dried leaves and flowers of this plant in alloxanized-diabetic rat model. rats were divided as: diabetic control (group ), dia-betic+glibenclamide (group ), diabetic+plant extract (group ), untreated control (group ) and control+plant extract (group ) groups (n = for each group). serum glucose, lipid levels and body weight changes were mesasured and pancreas and liver histology of the rats were examined. each rat in all groups were administered the plant extract ( mg), and the reference drug glibenclamide ( mg/kg) by gastric gavage every day for weeks. in group , blood glucose, serum alt, ast, triglyceride, cholesterol and ldl cholesterol levels increased while body weights decreased. in group , serum glucose, alt, ast, triglyseride and hba c levels decreased compared to group while cholesterol and ldl levels were high. in group , serum alt, ast, trigliserit, cholesterol, ldl levels decreased significantly but serum glucose and hba c were higher compared to group . body weights increased except group and hdl levels were not altered. histologically degenerative changes observed on pancreas of group were decreased in groups and . there was no difference on liver histology of the groups. in conclusion, thymbra spicata showed a protective and regenerative effect on diabetic pancreas. the hypo-lipidemic effect of the plant extract was also more effective than glibenclamide possibly due to the flavonoids, saponins and triterpenoids contents in the extract. its hypoglycaemic and protective activity should be tested for different doses and extract preparations and for longer periods. our study suggests that thymbra spicata is an excellent candidate for future studies on diabetes mellitus. with three different transcriptome data sets from the public gene expression omnibus database: time dependent data of dphop mutant, dargr mutant and wild type strain. the dynamic data spanned both primary and secondary phases of the metabolism. statistical results of transcriptome data were used for reporter metabolite analysis and reporter pathway analysis, which identify the metabolites (or pathways) with a significant coordinated transcriptional change in response to gene deletion perturbation in phosphate and nitrogen metabolisms. further, the production of actinorhodin, a pharmaceutically important compound, was modeled in the two deletion strains by calculating the metabolic fluxes subject to transcriptional level constraints on enzyme-coding genes. the metabolic switch from primary to secondary metabolism was highlighted in terms of the activity of pathways and fluxes as a result of the computational analyses in this work, leading to a better understanding of the role of phosphate and nitrogen metabolisms in increasing production levels. introduction: as a member of legume family licorice (glycyrrhiza glabra l.) has been widely used by human kind for many years as food constituent. especially by folks in rural sites licorice consumed widely. beside food constituent licorice has been used for medical purposes as well. licorice found effective with scientific datas on peptic skin infections, ulcers, inflammation, eczema, alzheimer disease, liver disease, and cancers. it also has been used as natural sweetener and food additive for preparing candies, chewing gum and beverage since ancient times. like all other medicine it has not been free of adverse event or toxicological effects. material and methods: alcoholic extracts of plant obtained by maceration process. for in vitro examination of anti-oxidant profile of licorice dpph free radical scavenging, abts cation radical scavenging and cupric ion reducing antioxidant capacity assay applied. application of extract made by oral route to rats for a week. anti-oxidant profile has been evaluated by myeloperoxidase (mpo), arylesterase (ares), total oxidative stress (tos) and total antioxidant status (tas) of serum levels. determination of toxicological effects alt, ast, ldh and alp values studied. histological investigation applied on liver and kidney tissues. results and discussion: results compared with control and standarts. antioxidant potential of licorice has been observed by in-vitro assays. serum mpo and ares values also compared with in-vitro results and correlation between them has evaluated. toxicological investigations made after evaluation of ast, alt, ldh and alp values. conclusion: in vitro assays has showed that licorice has potential anti-oxidant effect. investigation revealed that a mild toxic effect of licorice by biochemical tests. toxicological profile compared with control group and alt, ast values found slightly decreased and a mild elevation has been seen in ldh and alp values. for further and detailed investigation is needed. p- . . - on the applications of a metabolic network model of mesenchymal stem cells h. fouladiha, s. a. marashi, m. a. shokrgozar, m. farokhi mesenchymal stem cells (mscs) have several applications in tissue engineering and regenerative medicine. mscs can be very useful in stem cell therapy, because they can be isolated bone marrow or adipose of an adult. these cells have also been used as gene or protein carriers. therefore, maintaining them in a desire metabolic state has been the subject of several studies. here, we have used a genome scale metabolic network model of bone marrow derived mscs for exploring the metabolism of these cells. then, we try to validate the computational results by experimenal tests. we analyzed metabolic fluxes in order to increase stem cell proliferation using the metabolic model. consequently, the experimental results were in consistency with computational results. in the present work, the applicability of the metabolic model was successfully approved. therefore, this metabolic model can be useful in biomedical researches of stem cells. p- . . - qtl analysis for body weight and fatness in bxd recombinant inbred mouse strains a. dogan , c. neuschl , r. alberts , g. a. brockmann school of medicine, istanbul kemerburgaz university, istanbul, turkey, department for crop and animal sciences, humboldt-universit€ at zu berlin, berlin, germany, helmholtz-zentrum f€ ur infektionsforschung, braunschweig, germany genetic variation in body weight and composition is under the influence of many genes and have different genetic architectures. in the present study, the genetic factors contributing to body weight and fatness were examined under energy rich feeding conditions. growth traits, lean and fat weight, fat mass gain were analyzed to map qtls in a set of bxd ri strains. genome-wide analyses were revealed several genomic loci that control body weight and associated bodily changes in a sex and age-specific manner. the genetic data provided evidence for significant qtls on chromosome (chr) , , , and . most likely candidate genes within or near the regions with the highest significance levels were identified. the genes f rik, gbe , a n , and four genes cenpc , stap , uba , gnrhr for example, are suggested as most likely positional candidates accounting for the qtl effects on chr for fat mass, on chr for fat mass gain and on chr for lean weight, and chr for body weight, respectively. our results showed that body composition and fatness are highly complex that many genetic factors regulating and suggested candidate genes, which may help for studies of human fatness. related to serotonergic and gaba systems in response to hormonal changes. the nutrients involved in neurotransmitter synthesis may be the cause of relationship between diet and premenstrual syndrome. therefore, the aim of this study was to investigate the effect of various nutrients and premenstrual syndrome. this study was conducted to healthy women aged - years. participants were asked to fill in premenstrual assessment form. dietary intakes (three days in each phases) were recorded during premenstrual, menstrual and postmenstrual phases. energy, protein, amino acids, iron, calcium, and magnesium intakes were estimated. statistical analyses were performed using the spss software. friedman tests were conducted and differences were considered to be statistically significant for p-values lower than . . . % of the participants reported premenstrual symptoms and premenstrual symptoms related nutrient intake were increased in these women. it was determined that energy (p = . ) and protein (p = . ) intakes were higher in the premenstrual phase. during premenstrual phase; tyrosine (p = . ), isoleucine (p = . ), leucine (p = . ), lysine (p = . ), methionine (p = . ), cysteine (p = . ), tryptophan (p = . ), and glutamic acid (p = . ) intakes were higher than other phases. likewise, iron intake was higher on premenstrual phase (p = . ). on the other hand, intake of other potential premenstrual syndrome related nutrients like fat, cholesterol, calcium, magnesium, and vitamin b were not significantly different within the menstrual phases. amino acids including tyrosine, tryptophan, glutamine, and vitamin b are involved in neurotransmitter synthesis and might be related to premenstrual symptoms. consequently, elevated intakes of dietary protein and some amino acids during premenstrual phase may be related to premenstrual syndrome symptoms. until now far uv cd spectra of only two potexviruses were published. the papaya mosaic virus (papmv) spectrum, measured by leclerc and co-authors contained no obvious anomalies and was similar to the spectrum of isolated papmv coat protein (cp). but measured years earlier by homer and goodmanfar uv cd spectrum of potato virus x (pvx) itself had anomalous character and differed strongly from the spectrum of isolated pvx cp. in the present work we measured far uv cd spectra for two more members of potexvirusgenus: alternanthera mosaic virus (altmv) and potato aucuba mosaic virus (pamv) and their free cps. the altmv virion and altmv cp spectra were similar to each other and to the spectra of papmv and its cp. the pamv spectrum resembled the pvx spectrum in anomalously low ellipticity of the negative band at nm, but in contrast to pvx, did not have additional peak at nm. homologous modeling showed that cp of the three viruses is very similar in the core structure, and the observed difference may be explained by differences in disordered parts of proteins. possible reasons of potexvirus structural variability are discussed and it is suggested that the intravirus potexvirus cps may assume different conformations in different virions of the same preparation or even along the length of one virus particle. this work was supported by the russian science foundation (grant - - ). the antimicrobial potential of different phenolics was tested on pectobacterium in search of possible mode of action. in this respect, biofilm formation, exoenzyme activity, gene expression and virulence on its natural host (potato, cabbage, calla lily) were performed. also computational approach to show interaction between phenolic compounds and target protein was carried out using docking tools. the virulence determinants of pectobacterium were significantly impaired, at compound concentrations that did not affect bacterial cell growth. these observations suggested a mechanism which specifically interferes with bacterial virulence. since, these virulence determinants in pectobacterium are controlled by quorum sensing (qs), we focused on the effect of phenolics on the qs system in pectobacteria. the study revealed an inhibiting effect of the tested compounds on the expression level of central qs system and controlled genes, using qrt-pcr. also, there was a prominent reduction in the level of qs signal molecules n-acyl-homoserine lactone (ahl) accumulation. in addition infection capability was also practically blocked, which was completely recovered by application of exogenous-ahl. these results were supported by a potential interaction of plant phenolics with qs targets, as shown by molecular docking tool. collectively, results suggest the potential interference of phenolic compounds with qs central components (expi/expr proteins). moreover, it holds potential for future development of control measures against pectobacterium, and possibly other pathogens with similar mode of virulence. saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including double-stranded rna (dsrna) viruses. the l-a dsrna virus family of s. cerevisiae is widely distributed in nature. several versions of l-a virus are described and new ones continue to be discovered. some s. cerevisiae strains along with l-a dsrna possess smaller dsrnas, called m satellites. these dsrnas encode a sole secretable protein, known as k , k , k and k-lus toxin. l-a genome encodes the gag major structural protein and gag-pol fusion protein, formed by ribosomal frameshifting. gag-pol has transcriptase and replicase activities are necessary for maintenance of both l-a and m satellite dsrnas. so far, it's not known whether certain l-a virus has evolved to maintain a distinct type of satellite dsrna or this phenomenon lacks inherent specificity. we developed universal strategy to obtain full length l-a and m dsrna genomes from s. cerevisiae. complete viral dsrna genomes can now be cloned, as evidenced by l-a- dsrna, analyzed and sequenced directly from any yeast strain by means of enzymatic manipulations on total or fractioned rna content. we have identified previously undescribed l-a variant from different yeast strains specifically associated with certain type of m satellites. moreover, we identified for the first time full -utr and -utr sequences of m satellite. highly conserved sequence regions along with variable fragments were discovered at protein level, revealing clear trend to form clusters among different l-a gag-pol proteins. the obtained data suggest that each l-a virus variant can specifically maintain a distinct type of satellite dsrna. p- . . - physic-chemical characterization of plga adjuvants for immunization per os t. chudina, d. kolybo palladin institute of biochemisry of the national academy of sciences of ukraine (nasu), kyiv, ukraine antibodies against diphtheria toxin play the most important role in the immunity against corynebacterium diphtheriae. all current diphtheria vaccines have parenteral route of administration. undoubtedly, oral administration of antigens would be the most patient-friendly way of immunization. however, the efficacy of free antigens oral administration is limited by their degradation in the gastrointestinal tract and poor absorption by m-cells. biodegradable and biocompatible polymers, like poly (d,l lactide-co-glycolide) (plga), are widely used for the design of mucosal immunizing agents. importantly, that the way of particle preparation plays an important role in plga biodegradation and antigen release. the aim of this work was to characterize the main physic-chemical properties of two types of plga particles: with immobilized antigen (plga ) and with encapsulated antigen (plga ) . we have prepared two types of plga particles containing egfp-sbb proteins (non-toxic recombinant fragment b of dt fused with egfp). the antigen loading efficiency of particles was determined based on the ratio of protein concentration in solution before and after loading and shown better results for plga particles (plga - . %, plga - . %). the flow cytometry results demonstrated that % of plga particles conjugated with egfp-sbb, and only . % of plga particles conjugated with protein.the particle sizes had the slight difference by the results of two different techniques (ntanumber based, the software tracks individual particles; dls -scattering intensity weighted), however demonstrate similar patterns. dls data showed that the mean plga particles size was . nm and plga - . nm. nta data also showed that mean plga particles size a little smaller than plga ( . nm and . nm respectively) . demonstrated differences in the properties of synthesized particles may have an influence on the immunogenicity of the used for oral immunization antigen. p- . . - a suitable system for studying the functionality of a plasmodial protein in mammalian cell lines cho-mt , a mutant cell line was proved to be an appropriate tool for investigating intracellular function of cct. in this cell line, the endogenous cct activity decreases dramatically at °c, blocking membrane synthesis and ultimately leading to apoptosis. we have studied the rescuing potential of pfcct in cho-mt cells with the isogenic cho-k cells as a control. cells after transient transfection were incubated at °c and then analysed by facs using the fluorescence of egfp fused to pfcct. the proportion of cells undergoing apoptosis was determined by propidium-iodide staining. we have demonstrated for the first time that heterologously expressed pfcct is able to complement endogenous cct activity in mammalian cells. thus, a suitable system has been established for functional investigation of structural elements of pfcct. in order to reveal the role of different protein sequences in enzymatic function, we redesigned the structural gene of pfcct obtaining a modular system where different domains are easy to be removed or exchanged. here we designed a series of different truncation and deletion constructs to reveal the role of plasmodium specific sequences. in parallel, heterologous expression experiments of different constructs in the mutant cho-mt and the wild type control cell lines are performed to validate the reported model system. p- . . - host-pathogen interactions: is there a relationship between tlr polymorphisms and tuberculosis in a group of turkish patients? introduction: tuberculosis (tb) is a global health problem and according to world health organization (who) each year more than million individuals die from tb and each year , cases of tb are notified in turkey. malatya is the third largest city in east anatolian region of turkey and tb incidence rate is higher ( . / , ) comparing to the general population of the country. for this reason it is important to determine the factors that lead to tb in this population. disease agent can stay in the latent phase for long periods of time after infecting the individuals. while some infected individuals show the symptoms some others never do and even % of these never develop clinical disease. various mechanisms take place during the host response to infectious agents. toll-like receptor (tlr) genes are shown to be candidate genes in these responses. materials and methods: in this study tb patients and healthy controls were included. tlr genotyping for rs , rs was performed by using a commercial taqman snp genotyping assay kit. data were summarized by count and percent. hardy-weinberg equilibrium was tested by chi-square distribution with df. differences between groups due to allelic and genotypic distributions were analyzed by pearson's exact or fisher's exact tests. in all comparisons significance level was considered to be . . results: the single nucleotide polymorphisms (snps) which were subject of this study haven't been screened in turkish population earlier. no significant association was found between tb and the snps we screened in our group of patients. discussion and conclusion: unlike other populations results we couldn't find a significant association between the disease and the genotypes of our patients. the study should be performed in bigger populations in order to confirm the results. p- . . - lytic action of bacteriophages as a tool for the obtaining of images p. boltovets , r. radutny , t. shevchenko institute of semiconductor physics nas of ukraine, kyiv, ukraine, scientific and technical center of advanced technologies nas of ukraine, kyiv, ukraine, taras shevchenko national univercity of kyiv, kyiv, ukraine obtaining of images by different types of bacteria now became a very special branch of skill at the interface between science and art. however the authors did not found any scientific article, where bacterial lawn was used as the background and the image was formed by the lytic action of the virus (bacteriophage). whereas the mentioned approach could be used not only with artistic aims but for the practical use. the aim of this work was to demonstrate a possibility to obtain the image on the bacterial lawn by the lytic action of the bacteriophage. the bacterial lawn was obtained by the standard metod using the . % agar with the nutrient medium and the . % agar containing escherichia coli culture. stencils with the preparation of the bacteriophage t were applied. samples were incubated during the twenty-four hours at + °c. after that stencils were removed and the samples were stained by coomassie blue r- or fuchsine (with further fixation by the % acetic acid). several approaches to obtain the image by the lytic action of the virus were applied. first of all stencils made from printing paper and filter paper were compared. it was demonstrated, that the use of filter paper stensil allows to obtain more accurate and controllable images, than the use of the printing paper stensil. in the next series of the experiment the possibility of the reversed stencil use (where the image is formed not by the lytic zone but by the zone of bacterial growth) was demonstrated. also the possibility of the partial staining of the obtained image was explored. it gives an opportunity to obtain polychrome images using available colorants. summarizing the above it should be noted, that it was the first time when the graphical image was obtained by the lytic action of the virus on bacteria. this approach could be used not only for the artistic aims but as well for the practical use, for example, for the restriction of the action of microorganisms in out-of-theway places. burgdorferi the identification and characterization of possible antigens is essential for the improvement of current laboratory diagnostics for lyme disease and vaccine development. in this study, several recombinant b. burgdorferi outer surface proteins have been obtained and their antigenic properties have been evaluated in an effort to characterize novel immunodominant antigens. because b. afzelii and b. garinii are the most prevalent species in latvian ticks, proteins with conserved domains were included in this study. a panel of serum samples of lyme disease patients with early and disseminate disease stage was used. the controls were matched by age and sex to the patients and represented the same geographic area. the results show that proteins of several b. burgdorferi gene families have properties with respect to their candidacy as a subunit assay for a novel lyme disease immunodiagnostic. especially, the difference in their size in a range on the western blot assay may provide good discrimination between protein bands. however, they have potential for diagnosis if used in combination with other antigens but not as a "stand alone" test. in conclusions, this study showed the existing challenges in serological testing of early lyme disease. the conservation of the sequence of antigen between species of b. burgdorferi complex is essential for the most successful serodiagnostic marker candidate. the presence of homologous proteins in treponema species could lead to the cross reactivity in syphilis patients, and should be carefully evaluated. antimicrobial resistance is one of the greatest challenges in modern medicine. there is a pressing need for better understanding of the specific mechanisms that contribute to resistance to optimize existing therapies. in in georgia extended-spectrum beta-lactamase (esbl)-producing e. coli strain was isolated from the post-surgical sample obtained from gallbladder of the patients with chronic calculous cholecystitis which belongs to the sequence type (st ) complexes with ctx-m gene. is this strain characterized by other differences on a proteome level? are antibiotics against which the strain is resistant inducing the changes in bacterial proteome? the present work was aimed (i) to study the differences on a proteome level (i) between e. coli - / -g and attc e. coli-reference strain and (ii) to compare the proteomes of strain at two conditions: with and without antibiotics. strain was grown in the presence of three antibiotics: rocephin (ceftriaxone), fortum (ceftazydym) and claforan (cefotaxime sodium) together. proteomic expression was analyzed using two-dimensional gel electrophoresis and mass spectrometry. significant differences were found for several proteins, including putative abc trnsporter arginine protein , cystine-binding periplasmic protein, fkbp-type peptidyl-prolyl cis-trans isomerase, outer membrane protein a, d-galactose binding periplasmic protein and some others. the importance of these differences for anti-microbial resistance will be discussed. p- . . - molecular characterization of resistance and virulence features in staphylococcus aureus clinical strains isolated from cutanaeus lesions in patients with drug adverse reactions i. lupu , i. gheorghe , , m. popa , , a. ion , m. mihai , v. lazar , , m. c. chifiriuc , carol davila" university of medicine and pharmacy, bucharest, romania, research institute of the university of bucharest-icub, bucharest, romania, faculty of biology, university of bucharest, bucharest, romania patients treated with epidermal growth factor inhibitors often experience cutaneous adverse reactions. however, the infectious complications of these toxic effects and the contribution of specific pathogens, such as the community emergent methicillin resistant staphylococcus aureus strains. the present study was aimed to identify the types of sccmec and virulence genes profile in clinical s. aureus isolated from cutaneous lesions of different severity degrees in patients with dermatologic toxic effects. this study was conducted on a total of s. aureus clinical strains isolated in from acneiform reactions pustulae and periungual lesions in patients with drug cutaneous adverse reactions. multiplex pcr was performed on genomic dna from isolates in order to identify the sccmeccentral elements and the virulence genes: bbp (bone bound sialoprotein), ebps (elastinbinding protein), fnbb, fnba (fibronectin-binding proteins), fib, clfa, clfb (clumping factors a and b), cna (collagen-binding protein), luk-pv (panton-valentine leucocidin), hlg (haemolysin), tst (toxic shock toxin). the mrsa phenotype was genetically confirmed by the presence of meci gene in case of . %, meca in . %, sscmec type ivd element in . %, ccrb in . % and sccmec types i, iii, iv in . % of the studied s. aureus strains. regarding the virulence genes encountered in s. aureus strains, the most frequent was clfa ( . % of the isolates), followed by clfb ( . %), fib ( . %), hlg ( . %) and bbp ( . %). these results confirm the high prevalence of mec i and sscmec type iv elements, usually encountered in communityacquired mrsa strains, in cutaneous isolates from patients with dermatologic toxic effects. more data on the virulence and genetic background of these local strains are needed to appropriately assess the risk of such infections and avoid the inappropriate administration of beta-lactams. p- . . - analysis of toxicogenic properties of staphylococcus aureus strains isolated from cows with subclinical form of mastitis in the central area of russian federation. pore-forming toxins and enterotoxins), which are present in s. aureus strains isolated from clinically healthy cows. staphylococcus strains were isolated from cow's milk. disk diffusion method was used to determine the sensitivity to antibiotics. pcr analysis was used for detection of meca, mecc genes and genes of toxins. investigated strains were resistant to oxacillin ( %), vancomycin ( %) . it was found that all strains, which contain meca and mecc genes, showed resistant to more than antibiotics. it was determined that among the investigated strains % contained meca, % -mecc, % contained both meca and mecc. some strains contained genes of panton-valentine leukocidin (pvl) or alpha-hemolysin and several strains contained both types of genes. enterotoxin a (sea) gene was detected in . % of cases, sed - %, seg - %, sei - %. genes of staphylococcal toxins b, c, e, h were not found. the presence of phenol soluble modulin biosynthesis genes was determined: genes of alpha peptide synthesis were found in % of strains, beta peptide toxin genes in %, delta toxin gene in %. it was determined, that clinically healthy animals are carriers of s. aureus strains that cause mastitis. high level of antibiotic resistance was found in strains containing meca and mecc genes. the major part of the strains carried genes of phenol soluble modulin biosynthesis. the role of phenol soluble modulins as well as of pvl and alpha-hemolyzin in the development of mastitis is not completely clear. we conclude that pore-forming toxins have dominant role in the latent form of mastitis. p- . . - impact of lactoferrin on the hydrophobicity and adherence to the inert substratum of staphylococcus aureus strains isolated from patients with cutaneous drug reaction skin healing is a complex biological process that requires the involvement of different cell types and humoral effectors. one of the main factors are aggravating and delaying the healing process is represented by the supra-infection with pathogenic or opportunistic microorganisms that grow in specialized consortia embedded in a self-produced extracellular polymeric matrix, called biofilms, which are extremely resistant to any antimicrobials and host immune response. lactoferrin (lf) is an ironbinding glycoprotein which promotes skin healing by enhancing the initial inflammatory phase, but also by inducing an overabundant immune response. the aim of this study was to investigate the influence of lf, one of the main components of innate, humoral anti-infectious immunity on some microbial features, involved in the first steps of the infectious process, such as hydrophobicity and adherence of staphylococcus aureus strains isolated from maculo-pustular lesions in patients with adverse reactions to epidermal growth factor inhibitors. for hydrophobicity measurement the bacterial suspensions were grown in the presence or absence of lf, and then, the "microbial adherence to hydrocarbons test" (math) was performed. the capacity to develop biofilms on inert substrata and the influence of lf on this feature was spectrophotometrically quantified using an adapted microtiter method, after crystal violet staining. our results showed that lf decreased the hydrophobicity and limited the biofilm development of all s. aureus tested strains, in a dose and time dependent manner. the decreasing effect on the microbial hydrophobicity was accompanied by a lowering effect on the adhesion of microbial strains to the inert substratum. in conclusion these observations indicate that lf exhibits a wound pro-healing effect, by limiting the microbial colonization and biofilm formation and thus, the occurrence of infectious complications of skin lesion. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] host-specificity determinants of bacteriophage vb_ecom_fv considered vehicles of s.aureus intoxication in humans throughout the world. the objective of the present study was to assess the presence of enterotoxigenic and methicillin-resistant s. aureus in water buffalo milk and dairy products. a total of water buffalo milk and dairy products ( water buffalo cream and water buffalo cheese) were collected from different dairy farms, smallholders and local bazaars in samsun, turkey. all samples were analyzed using the standard procedure en iso - and isolates were confirmed for the presence of the s rrna and nuc gene by polymerase chain reaction. s. aureus was identified in of water buffalo milk ( %), of water buffalo cream ( %), and of water buffalo cheese ( %). a total of isolates were confirmed as s. aureus by pcr. genotypic methicillin resistance was evaluated using pcr for the meca gene. out of isolates, ( %) were found to be methicillin resistant (meca gene positive) by pcr. the enterotoxigenic s. aureus was identified in out of ( %) isolates by the mpcr technique. five isolates produced staphylococcal enterotoxins sea ( / ; . %), two isolates produced sec ( / ; . %), one isolate produced ( / ; . %) sed, one isolate produced ( / ; . %) see and three isolates produced sec+sed ( / ; %) . none of samples were positive for seb. in conclusion, the presence of enterotoxigenic and methicillin-resistant s. aureus in milk and dairy products is of significant for public health concern and also these enterotoxin genes sea and sed are predominant toxins that can cause staphylococcus intoxication in humans. this study was funded by ondokuz mayıs university, samsun, turkey, scientific research project programs (project no: pyo. vet - . . ) and this article was part of a phd thesis. p- . . - identification and biochemical characterization of an immune modulating protein from helicobacter pylori b. kaplan t€ urk€ oz faculty of engineering, department of food engineering, ege university, izmir, turkey helicobacter pylori is able to achieve persistent infection with minimal immune response. the first line of defence during h.pylori infection is through gastric epithelial cells which present toll like receptors (tlr). a family of bacterial proteins which share homology with the toll/il- receptor (tir) domain were identified. the structure of btpa from brucella showed that bacterial tir proteins (btp) mimick human tir domain proteins and act on myd signaling pathways to suppress tlr signaling. h.pylori might also produce a similar protein. a putative h. pylori tir protein was found based on sequence homology and the corresponding gene; hp ; was cloned in fusion with an n terminal cleavable his-tag. the recombinant protein, his- was purified using nickel affinity chromatography. was subjected to limited proteolysis and the bands were analyzed by peptide mass fingerprinting (pmf). oligomerization of was investigated by in vitro pull-down and size-exclusion chromatography. , a amino acid protein, has a predicted c terminal tir domain similar to other btps and sequence alignments verified the presence of tir domain signature regions. recombinant his- was produced with a yield of mg/l culture. a structurally stable kda fragment was obtained from limited proteolysis which contained the tir domain as verified by pmf. in vitro pull down assays showed interacts with itself forming dimers as shown by size-exclusion chromatography. tir domain proteins function by interacting with themselves and other tir domains. our results showed that also form dimers, supporting that it is a btp. current research is focused on solving the structure of and investigating its interaction with myd . might play a direct role in reduced immune response against h.pylori by binding to myd analogous to other btps. further characterization of will provide the first solid evidence of presence of a tir domain protein in h.pylori. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] lipopolysaccharides with different lipid a acylation status from vibrio cholerae and campylobacter jejuni contribute differently to il production by bone marrow-derived macrophages k. korneev , , e. sviriaeva , lipid a is a biologically active part of lipopolysaccharide (lps) from gram-negative bacteria that is responsible for the activation of the innate immunity through interaction with toll-like receptor (tlr ) and subsequent production of proinflammatory cytokines. bacteria frequently transform their lipid a so that its recognition by tlr is not sufficient for induction of effective antibacterial immune response. we compared biological activity of various lps from pathogenic bacteria vibrio cholerae and campylobacter jejuni. we purified r-form lps for each strain by hydrophobic chromatography. the biological activity of lps preparations was evaluated by their ability to activate production of proinflammatory cytokine il by bone marrow-derived macrophages from c bl/ mice, using tlr -deficient macrophages to control for specificity of tlr signaling. lps from e. coli and inactive lps from f. tularensis were used as positive and negative controls. lps from v. cholerae demonstrated biological activity similar to that of lps from e. coli, consistent with the presence of highly acylated lipid a in both strains. however, the former was a slightly weaker activator than the latter, because lipid a from v. cholerae had on average shorter acyl chains. lipid a from c. jejuni had on average longer acyl groups than in e. coli, while degree of acylation was lower, and as a result its lipid a displayed significantly lower biological activity. our study demonstrates importance of functional groups of lipid a in the ability of lps to activate production of il by macrophages. in line with our previous reports, we confirmed a direct correlation between biological activity of various lps species with their lipid a acylation status: the biological activity increases with increase in the length and in the number of the acyl chains. excess proinflammatory cytokine production through tlr activation can cause sepsis, while inefficient activation may result in the failure to clear bacteria. clostridium perfringens phospholipase c (cpplc) is the most toxic extracellular enzyme produced by this bacterium and it is an essential virulence factor in the pathogenesis of gas gangrene. cpplc may lead to cell lysis at concentrations that causes extensive degradation of plasma membrane phospholipids. however, at sublytic concentrations it induces cytotoxicity without causing evident membrane damage. the results of this work demonstrated that the cytotoxic effect of cpplc requires its internalization and the activation of the mek-erk pathway. cpplc internalizartion occurs through a dynamin-dependent mechanism and in a time progressive process: first, cpplc colocalizes with caveolin both at the plasma membrane and in vesicles, and later it colocalizes with early and late endosomes and lysosomes. the results also showed that cpplc requires endocytosis in order to activate mek-erk, because treatment with the dynamin inhibitor, dynasore, prevents cpplc endocytosis, erk / activation and cytotoxcity. cholesterol sequestration as well as inhibition of actin polymerization also prevents cpplc internalization and cytotoxocity, involving endocytosis in the signaling events required for cpplc cytotoxic effect. once internalized, cpplc induces reactive oxygen species production through the activation of pkc, mek/erk and nfjb dependent pathways. inhibition of either of these signaling pathways prevents cpplc's cytotoxic effect. in addition, it was demonstrated that nfjb inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of cpplc in mice. these data provide new insights about the mode of action of this bacterial phospholipase c, previously considered to act only locally on cell membrane. understanding the role of these signaling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridial myonecrosis. p- . . - apoptosis induced by clostridium perfringens phospholipase c is mediated by reactive oxygen species m. flores-d ıaz , l. monturiol-gross , m. j. pineda padilla , c. araya-castillo , a. alape-gir on bacterial phospholipases are lipolytic esterases surface associated or secreted by a wide variety of bacterial pathogens. clostridium perfringens, the most broadly distributed pathogen in nature, secretes a prototype phospholipase c (plc), also called a-toxin, which plays a key role in the pathogenesis of gas gangrene. this toxin causes death to cultured cells and extensive myonecrosis when injected intramuscularly in experimental animals. the results of the present study showed that c. perfringens plc ( - ng/ml) induces morphological and biochemical changes characteristic of apoptosis in cultured cells, as determined by scanning electron microscopy. nuclei condensation and fragmentation were observed by fluorescence microscopy and a typical ladder fragmentation pattern of genomic dna was detected by dna in agarose gels. cell death was prevented by the caspases inhibitors z-devd-fmk and z-vad-fmk. c. perfringens plc induces oxidative stress in cultured cells as determined by fluorescence microscopy and flow cytometry using the membrane permeable probe dcfda. different antioxidants including the gluthation precursor nac, several iron chelators and the free radical scavengers tiron and edaravone prevent cell death induced by c. perfringens plc in cultured cells or in mice challenged intramuscularly with . lg of that toxin. thus, this work provides compelling evidence that superoxide, hydrogen peroxide, and the hydroxyl radical are involved in the cytotoxic and myotoxic effects of c. perfringens plc. furthermore, the data demonstrated that edaravone, a clinically used hydroxyl radical trap, reduced the myonecrosis and the mortality caused by c. perfringens in a murine model of gas gangrene, induced by intramuscular bacterial injection of bacteria. this knowledge provides new insights for the development of novel therapies to reduce tissue damage during clostridial myonecrosis. lectins are ubiquitous proteins able to recognize mono-and oligosaccharides with high specificity and low affinity. lectins do not have any catalytic activity, unlike enzymes, and they are not products of the immune system in contrast to antibodies. lectins play a crucial role in cell interactions on molecular level showing their importance in various physiological and pathophysiological processes as well as both mutualistic and parasitic interactions between microorganism and hosts. photorhabdus luminescens is a gram-negative bacterium from the family enterobacteriaceae. the bacteria have a complex life cycle that involves mutualistic and pathogenic interaction with two different invertebrate hosts. it is highly pathogenic towards insect larvae. in addition, p. luminescens lives in the intestine of infective juveniles of nematode heterorhabditis bacteriophora, together forming an effective entomopathogenic complex. we have identified several soluble lectins produced by p.luminescens. in this study, we focus on proteins from p. luminescens, which show a high sequence homology with each other. a wide range of methods was used for structural and functional studies of photorhabdus lectins, e.g. surface plasmon resonance, isothermal titration calorimetry, analytical ultracentrifugation and x-ray crystallography. all lectins from p.luminescens recognize l-fucose and d-mannose. despite being closely related, they differ in fine binding specificities. to determine their biological function, knock-out mutants of p. luminescens are being prepared to study its interaction with axenic nematodes and insect larvae. breast cancer is the major disease of women in developed countries occuring predominantly after the age of . triple negative breast cancer (tnbc) is a typical subtype of epithelial breast cancer which lacks estrogen receptor (er), progesterone receptor (pr) and human epidermal growth factor receptor (her ) all together. although various researches have been focused on characterizing tnbc and enlightening different molecular markers with the aim of improving the overall outcome, currently the sole affective therapy action for tnbc is chemotherapy. thus chemoresistance is the main clinical challange and accounts for % of failures in terms of treating the disease. multidrug resistance (mdr) is defined as simultaneous resistance towards the drugs which do or do not demonstrate structural resemblance and have different effects on their molecular targets. p-glycoprotein (p-gp) is a membrane protein coded by abcb (mdr- ) gene. p-gp is an atp-dependent pump which pumps a wide range of drugs out of the cells including chemotherapeutic agents such as doxorubicin (dox) and pactilaxel. in the present study, tnbc cell line mda-mb- was treated with increasing doses of dox, cell viability was examined with srb assay and development of mdr was investigated through mdr assay and rt-pcr. results demonstrated that cell viabiliy decreased significantly with the treatment of higher doses. mdr was shown to be increased when cells were treated with , and nm of the drug respectively along with lm of p-gp inhibitor verapamil. rt-pcr results were obtained to be consistent with mdr assay results and indicated increased mdr- gene expression with the treatment of dox. especially after nm of dox treatment, mdr- was overexpressed to be fold when compared to control. in conclusion, it was demonstrated that mda-mb- cells have shown to display elevated resistance to higher doses of dox. p- . . - targeting dna damage response pathway in cancer cells under heat stress and the mechanical effect of ultrasound y. furusawa , t. kondo toyama prefectural university, imizu-shi, japan, university of toyama, toyama, japan ultrasound (us) has been widely utilized for diagnosis and therapy in many medical fields. the biophysical modes of us are divided into three classes, thermal, cavitation and non-thermal non-cavitation effects. in clinical use for cancer therapy, the thermal effect was utilized for hyperthermia therapy with focusing us on cancer to rise the temperature from °c to °c, or further which could induce thermal ablation of cancers. cavitation leads to a variety of mechanical stress such as shear stress, shock wave, high pressure, and chemical stress such as free radical formation, both of which have been inferred to act simultaneously on all biological materials. it has been indicated that us induces cell killing, cell lysis, loss of viability, and loss of clonogenicity. recently, we found that heat stress as well as us without thermal effect induce not only dna single-strand breaks but also dna double-strand breaks, a most cytotoxic region of dna, in chromatin dna detected by both gammah ax staining and neutral comet assay. in response to the stresses which induce dna damage, the dna damage sensor protein kinase, ataxia telangiectasia mutated (atm), atm and rad related (atr), and dna-dependent protein kinase (dna-pk) become activated form to initiate signal transduction pathways activating cell-cycle checkpoints, dna repair, and apoptosis. the molecules consisting of dna damage response pathway were expected as therapeutic targets because defects in the response to dna damage agents can be lethal. this work was designed to explore the possible therapeutic targets of the molecules in dna damage response pathways for future us-aided therapy. finally, several kinases (e.g., checkpoint kinase) on dna damage response pathway seems to be the targets for hyperthermia and us therapy. (ural branch) , ekaterinburg, russia, shemyakin and ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia based on the recently synthesized (s)-( -aminopurin- -yl) amino acids (gly, ala, val, phe, pro), we obtained a series of novel modified nucleosides using the transglycosylation reaction. for the first time, it has been demonstrated that the corresponding nucleobases are good substrates for the genetically engineered recombinant e. coli purine nucleoside phosphorylase (conversion to nucleosides reached - %). nucleosides, such as ribosides, -deoxyribosides, and arabinosides were obtained in high yields ( - %). it has been found that yield in the transglycosylation reaction does not depend on the structure of the amino acid fragment. the nucleosides synthesized are considered as potential inhibitors of intracellular adenosine deaminase (ad), the increasing activity of which is observed in hepatitis, cirrhosis, hemochromatosis, obstructive jaundice, prostate and bladder cancer, hemolytic anemia, rheumatic and typhoid fever, gout, and cooley's anemia. cytotoxicity of the synthesized nucleosides was tested in the jurkat (model of human t-lymphoblastic leukemia) and el- (model of mice t-lymphoblastic leukemia) cell lines. the compounds studied did not exhibit cytotoxic activity compared to the activity of the known antitumor agent nelarabin. the work was financially supported by the russian science foundation (grant - - ). p- . . - dna binding, dna cleavage, antimicrobial activities, antimutagenic and anticancer studies of a schiff base and its complexes n. yildirim , n. demir , m. yildiz health services vocational school, c ß anakkale onsekiz mart university, c ß anakkale, turkey, department of biology, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, department of chemistry, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. the rational design and synthesis of new schiff bases and their metal complexes have been drawing great interest because of their diverse biological and pharmaceutical activities. so, exploring and designing novel molecules that have biological activities and capable of interacting with nucleic acids has a great significance for disease defence and to discover new dna-targeted anticancer drugs for chemotherapy. in this study, we report the synthesis and characterization of a novel schiff base and its ni(ii) and cu(ii) complexes. the minimal inhibitory concentration (mic) of the compounds was screened in vitro against bacteria and yeast cultures using broth micro dilution test. dna binding and dna cleavage activity of the compounds were investigated by uv-vis spectroscopy and agarose gel electrophoresis. antimutagenic activity of compounds were tested in the absence of microsomal enzymes (s -). also, cytotoxicity of the compounds against hepg cell lines was assayed by the mtt ( -( , -dimethylthyazolyl- )- , -diphenyltetrazolium bromide) method. consequently, uv-vis spectroscopy studies indicated that the compounds interact with calf thymus dna (ct-dna) via intercalative binding mode. dna cleavage activity studies showed that the cu(ii) complex can effectively cleave pbr plasmid dna. compounds inhibited the base pair mutation with high inhibition rate in the absence of s . also, schiff base complex had cytotoxic activity towards hepg cell line, that it was found to be more potent than the control cisplatin. p- . . - single particle electron tomography of rnap elongation complex, stalled at position + genome in vivo is constantly exposed to the damaging effects of the environment. single-strand breaks (ssbs) are the most frequently occurring dna lesions. accumulation of unrepaired ssbs can interfere with the cells metabolism and increase genomic instability. in vivo, ssbs are repaired in specific pathway, but, in eukaryotic nuclei, dna is organized in chromatin that could affect the accessibility of lesions to sensor proteins. breaks in a template strand induce arrest of rna polymerase ii (polii) in vitro and in vivo and can be revealed in a transcription-dependent manner. our recent biochemical studies identified two key intermediates formed during transcription through a nucleosome by rnap that are nearly homogeneous, active and stable by biochemical criteria (complexes stalled after entering or bp into the nucleosome; ec+ or ec+ , respectively). hear we produced two complexes, both stalled in the + position, one without break in the dna, and the other with introduced ssb at position + of a non-template dna strand. complexes were purified using affinity chromatography and applied to a carbon-coated, glow-discharged em grid. tomographic studies were performed at ae °in a jeol microscope at kv accelerated voltage. images were recorded using a gatan ccd camera. image analysis was performed using the imod software. the resulting structure of the ec+ complex with no break in dna consist of two domains, connected by a single dna string. the complex with a break introduced into the dna has a more compact appearance and its two domains were connected by two dna strings, thus forming an intranucleosomal dna loop. our data suggest that ssbs in a non-template strand can induce the formation of stable non-productive transcription intermediate. the inhibitory effect of ssbs onto transcription may suggest a possible mechanism for their recognition in vivo with a transcription-dependent pathway. this work has been supported by the rsf grant # - - . colorectal cancer (crc) is one of the leading causes of cancerrelated deaths in the developed countries. according to who report new incidence rate of crc in turkey is . % among other cancer types. owing to difficulty of the low allele frequency variations detection, genetic association profiles of crc have not been entirely identified. low allele frequency variations mlh À g>a (rs ) promotor substitution, mlh g>c (rs ) exonic substitution, mthfr c t (rs ) and apc t>a (rs ) were investigated in this study. these snps "rs , rs , rs , rs " are located on p . , q , p respectively. colonoscopic investigations were performed on both cancer and control group. the snps were genotyped using kompetitive allele specific pcr technology in cases and healthy controls. statistical analysis was carried out with cochran-armitage chi-square test. in this study these of the snps in mlh , mthfr genes were examined for the first time in turkish sporadic crc cases. statistical analysis showed no significant association within our turkish sporadic crc population. percentage of mlh À aa genotype in group aged ≥ was found to be . % in cancer versus % in control group. moreover apc a, mlh c alleles were detected only and allele respectively. previously, apc a allele was determined in . % of a turkish cohort. however in the present study apc a allele was detected on allele only. studies showed mlh À promoter variation as a risk factor for microsatellite instabile crc but for the current study this data is not available. in spite of literature mthfr c t and mlh g>c snps were not found to be associated with sporadic crc in turkish population. this research demonstrates that importance of population based studies in multifactorial disease. p- . . - excision of damaged bases from transcription intermediates by fpg/nei superfamily dna glycosylases k. makasheva, d. zharkov sb ras institute of chemical biology and fundamental medicine, novosibirsk, russia oxidative lesions are abundant due to constant presence of reactive oxygen species in living cells. repair of oxidative base lesions is initiated by dna glycosylases. for example, bacterial fpg and nei dna glycosylases excise oxidized purines and pyrimidines, respectively, from dna. their human homologs, neil and neil , have been reported to show preference towards oxidized lesions in dna bubbles. from these observations, it had been hypothesized that neil proteins may be involved in the repair of lesions in dna bubbles generated during transcription. however, it is not presently clear how neils would behave on bubbles more closely resembling transcription intermediates (e. g., containing the rna strand), and bacterial homologs fpg and nei had never been investigated with bubble substrates. we have studied excision of either -oxoguanine ( -oxog) or , -dihydrouracil (dhu) by e. coli fpg and nei and human neil and neil from single-strand oligonucleotides, perfect duplexes, bubbles with different number of unpaired bases ( to ), d-loops with dna or rna and from complexes with rna polymerase. fpg, neil and neil efficiently excised dhu located inside a bubble. fpg and neil was generally more active than neil in excision of -oxog from ssdna and bubbles. nei, on the other hand, was active only on dhu located in dsdna (either perfect duplex or dna/dna d-loop). fpg and neil also have shown activity in d-loops with rna. the presence of an additional unpaired -tail of the third strand of d-loops didn't affect the glycosylases activity. the activity of fpg was observed in pre-assembled transcriptional complexes with e. coli rna polymerase and depended on the position of the lesion in the transcription bubble, possibly reflecting local accessibility of the lesion within the elongation complex. this work was supported by rsf ( - - ). nucleotide excision repair (ner) is a multistep process that eliminates a wide range of lesions in dna, including uv photoproducts and base modifications by many carcinogenic and chemotherapeutic agents. one of the advanced approaches to ner process investigation is based on reproducing the repair reaction by mixing protein extracts from mammalian cells with model linear dnas, bearing lesions. long linear dnas ( bp) containing efficiently recognized and processed by ner system lesions (fluoro-azidobenzoyl photoactive lesion fab-dc, nonnucleoside lesions nflu and nant) in both strands have been synthesized. we have demonstrated that dnas containing closely positioned lesions in the both strands represent difficult-to-repair (fab-dc/nflu(+ ), fab-dc/nflu(À )) or unrepairable (nflu/nflu (+ ), nflu/nflu(À ), nant/nflu(+ ), nant/nflu(À )) structures. besides, it has been shown that model dnas bearing bulky lesions in opposite positions (fab-dc/nflu( ), nflu/nflu( )) represent unrepairable structure as well. the model substrates with increasing distance between lesions in the duplex demonstrated the full recovery of substrate properties in ner process (fab-dc/nflu(+ ), fab-dc/nflu(À ), fab-dc/nflu(À ), nflu/nflu (+ ), and nant/nflu(+ )), whereas the level of specific excision from nflu/nflu(À ), nflu/nflu(À ) and nant/nflu(À ), nant/nflu(À ) was approximately % of the nflu/dg or nant/dg dna respectively. it has been shown that modified dna-duplex ( bp) with fab-dc has decreased structurally dependent affinity for xpc-hr b compared to duplexes containing lesions in both strands being analyzed (fab-dc/dg, fab-dc/nflu(+ ), fab-dc/nflu (À ), fab-dc/nflu(+ ), fab-dc/nflu(À ), fab-dc/nflu(- )) and increased compared to umdna. the data provide an argument that the ner system of higher eukaryotes recognizes and eliminates injured dna fragments on a multi-criteria basis. it is well known that dna plays crucial role in the biological system because of including all the genetic information for cellular function. therefore, the interaction of molecules with dna has gained interest in the medicinal chemistry to explore new anticancer agent. photodynamic therapy which is alternative cancer treatment method depends on free radicals and singlet oxygen to destroy tumor tissue via necrosis and apoptosis. phthalocyanines (pcs) are used for photodynamic therapy because of their absorption of high wavelength light ability and they have high triplet quantum state yields and long lifetimes in triplet states. also they do not have any toxic effect without light. in this study the novel synthesized - [ -( morpholin- -ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanines were used. the potential properties of phthalocyanine compounds for photodynamic therapy were purposed to reveal by the preliminary work. for this aim, the mode of dna binding, photocleavage and topoisomerase i inhibition of these compounds were investigated. - [ -( -morpholin- -ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanine compounds have been synthesized. the interaction of novel pcs compounds with calf thymus (ct) dna was investigated by using uv-vis spectroscopy, thermal denaturation studies and viscosity measurements. additionally, dna photocleavage and topoisomerase i inhibition studies were performed to pbr dna by using agarose gel electrophoresis. the interaction studies indicated that pcs compounds powerfully bound via an intercalation mechanism with ct-dna. these compounds showed efficiently dna photocleavage under irradiation at nm. the all of pcs inhibited topoisomerase i in a dose-dependent manner. all the experimental studies showed that pc compounds might be used agents for photodynamic therapy. p- . . - target search by base excision repair dna glycosylases e. dyatlova, g. mechetin, d. zharkov institute of chemical biology and fundamental medicine, novosibirsk, russia the problem of rapid target search in dna is faced by transcription factors, restriction endonucleases, dna repair enzymes and other sequence-or structure-specific dna-binding proteins. theoretically, the fastest target search in dna can be achieved by combining one-dimensional diffusion along the dna contour (processive search) and three-dimensional diffusion (distributive search). the balance between these search modes depends on many factors affecting dna-protein interactions, such as the presence of mono-and divalent cations, competing proteins, crowding effect, etc. presently, the mechanisms of target search are understood only for a handful of enzymes. we have recently developed an assay to study target search by dna repair enzymes, based on cleavage of oligonucleotide substrate containing two targets. thus, the distance between the targets can be precisely controlled, and any modification can be introduced into dna. subsequently, the probability of correlated cleavage (p cc ) is estimated, reflecting the efficiency of enzyme transfer between the specific sites. in this work, we have investigated five repair enzymes: e. coli endonuclease viii (nei), its human homologs neil and neil , and uracil-dna-glycosylases (ung) from e. coli and vaccinia virus. as expected, p cc of all enzymes depended on the ionic strength of the solution and the presence of mg + . ung from vaccinia virus was the most sensitive to these factors, raising questions about its proficiency as a suggested processivity factor of viral dna polymerase. nei, neil and neil showed a peak of p cc at low but non-zero ionic strength indicating that nonpolar interactions contribute to binding of these proteins to nonspecific dna. this conclusion was also supported by analyzing amino acid conservation in the catalytic core of nei. introduction of bulky fluorescent group between two specific sites greatly reduced the ability of glycosylases to slide along dna. this work was supported by rsf ( - - ). p- . . - does causes mhz magnetic field application kras and p mutations in colon?: occurences histopatologically and microbiologically changes in colon determination of kirsten rat sarcoma (kras) and p gene mutations in colon. materials and methods: in this study, three groups were prepared as control,sham and electromagnetic field (emf) group. mhz radiofrequency (rf) radiation was produced by using an electromagnetic energy generator.the emf group rats were exposed to electromagnetic field for weeks as minutes per day.at the end of experiments, rats were sacrificed under ethyl ether anesthesia and the rat colons were dissected.fecal speciments were collected.fecal dna (for detection of fusobacterium and bacteroides) and colonic dna (for detection of kras and p mutations) were isolated.rt-pcr tchnique was used for detection of bacterias and mutations. results: no any differences was observed histopathologically between control and sham groups.erosions and partial losses were observed at mucosal epitelium in the emf group.the corrupted gland structure, the mucosal edema and the inflammatory cell infiltration were observed.the amout of collagen was increased and fibrosis was detected in emf group.goblet cell number decreased statistically significant when compared to control and sham groups (p < . ).the amount of fusobacterium increased significantly in emf group compared to controls.the difference was not detected between groups in the amount of bacteroides.all the samples analysed for kras and tp mutations in the colon tissue were found to be wild type.no significant difference was observed between the control group and the emf applied group. discussion and conclusion: in conclusions,for weeks minute/day exposure to mhz emf caused histopatological damage in rat colon.the amount of fusobacterium is increased.emf exposure did not caused to kras and p mutations in colon tissue. p- . . - synthesis, antimicrobial activity, genotoxicity, dna binding and dna cleavage studies of new glycine methyl ester derivative schiff base there has been an increasing focus on the binding study of small molecules to dna during the last decades, since dna is an important genetic substance in organisms. therefore, the current growing interest in small molecules that are capable of binding and cleaving dna is related to their utility in the design and development of synthetic restriction enzymes, new drugs, dna agents, and also to their ability to probe the structure of dna itself. in recent years, schiff bases have found increased application in pharmaceutical research, organic synthesis, and bio-processes. schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. in this study, we report the synthesis and characterization of a novel glycine methyl ester derivative schiff base. the minimal inhibitory concentration (mic) of the compound was screened in vitro against bacteria and yeast cultures using broth micro dilution tests. antimutagenic activity of compound was tested in the absence of metabolic activation. also, dna binding and dna cleavage were investigated of compound by uv-vis spectroscopy and agarose gel electrophoresis respectively. consequently, this compound differs significantly in its activity against tested microorganisms. this difference may be attributed to the fact that the cell wall in gram-positive bacteria is a single layer, whereas the gram-negative bacteria cell wall is a multilayered structure, and the yeast cell wall is quite complex. the compound inhibited the base pair mutation in the absence of s with high inhibition rate. uv-vis spectroscopy studies of the interactions between the compound and calf thymus dna (ct-dna) showed that the compound interacts with dna via intercalative binding. to date a large number of the sequences in the human genome (g motifs) with the potential to form a spatial structure, gquadruplexes is known. g motifs were found in the promoter regions of most of the known oncogenes. recent experimental studies have shown that genome instability directly related to the non-canonical dna structures, including g-quadruplexes. in this work we study the distribution of somatic snvs within the g motifs in tumor samples with the aim to identify involvement of the motifs in the process of mutagenesis in pancreatic cancer. using the access kindly provided by the international icgc consortium to the database, we analyzed samples of pancreatic ductal adenocarcinoma and samples of pancreatic endocrine neoplasms. we considered only the promoter regions as the richest with g-quadruplex motifs. we found that quadruplex sequences have the ability to focus somatic snvs. this could be explained by the errors of polymerase during replication through secondary dna structures. furthermore, the snvs occur much more often in loops of g motifs than in g blocks, without changing the motive. in addition, t>g(a>c) and t>c(a>g) substitutions occur significantly more likely in loops which in turn stabilize the g-quadruplex structure. the cancer-related mutations tend to increasing the length of g blocks. the conservation of g motifs may indicate an important functional significance of g-quadruplex structures in human genome. supported by project no. - - of the russian science foundation. background: multiple myeloma (mm) is a rare, leading to bone destruction and marrow failure, largely incurable malignant disease of plasma cells. anemia (mostly normocytic normochromic) is seen in most patients. mean platelet volume (mpv) is a laboratory marker of platelet function and activity, the most accurate measure of platelet size. the aim of this study was to investigate the mean platelet volume (mpv) values in this disease. materials and methods: whole blood samples were collected from healthy controls and patients with mm. the mean age for controls and patients were . ae . and . ae . years, respectively. mpv levels were calculated with cancer is a chronic disease in the world which is the second leading cause of death, after cardiovascular diseases. benzimidazoles have been known to act as antiproliferative or anticancer agents in chemotherapeutic drug research area. in this regard we aimed to investigate the cytotoxic and apoptotic properties of novel benzimidazole derivatives bearing pyridyl/pyrimidinyl piperazine moiety against a lung adenocarcinoma cells. a lung adenocarcinoma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by mtt assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic values of the compounds were determined for a cell line. compounds , and which were including -chlorophenyl, -nitrophenyl on pyridine ring; -fluorophenyl on pyrimidine moiety, had significant cytotoxic activity with ic values lower than . ae . lg/ml. compound showed the highest cytotoxic activity with a ic value of . ae . lg/ml, whereas cisplatin ic values were . ae . lg/ml lg/ml against a cells. cytotoxic activity of compound and with a ic value were . ae . and . ae . lg/ml, respectively. also, compound showed the highest population of early apoptotic cells ( . %) of the tested compounds which was . -fold higher than for cisplatin. compound produced a comparable population of apoptotic cells with a percentage of . %, respectively according to cisplatin's percentage of . %. it was determined that synthesized compounds , and had considerable anticancer activity against a cell lines compared to cisplatin. compound including -florophenyl on pyrimidine ring was the most cytotoxic compound against the a cell line. our study results demonstrated that compound , also induced apopototic pathway on a cells. p- . . in vitro/in vivo antimitotic activity and structure-activity relationships of new glaziovianin a isoflavone series glaziovianin a (gva), isolated from the leaves of the astelia glazioviana, demonstrated cytotoxicity, disrupting microtubule structure and dynamics of hl- cells. the aim of the present work was to devise a concise synthetic route toward gva and its derivatives in order to expand structure-activity relationship studies and to investigate their anti-mitotic effect. a concise six-step protocol for the synthesis of gva and its alkoxyphenyl derivatives starting with readily available plant metabolites from dill and parsley seeds was developed. the sea urchin embryo tests confirmed that gva directly affects tubulin/ microtubule dynamics and structure. the b-ring substitution pattern of gva derivatives exhibited strong effects on activity. according to the assay results, the anti-mitotic activity decreased in the following order: gva > myristicin ≥ , , -trimethoxyphenyl = -methoxyphenyl > dillapiol > -methoxyphenyl> , dimethoxyphenyl > , , , -tetramethoxyphenyl derivatives. a methylenedioxy moiety was essential for the activity of compounds substituted with four b-ring alkoxy groups. the mts assay of the limited panel of cancer cell lines shows that gva displayed the highest inhibitory activity, with ic values ranging from . (a cells) to . lm (mda-mb- cells). compounds, containing , , -trimethoxy and apiol-derived b-rings, respectively, were less active. other isoflavones did not affect cancer cell growth up to lm. anti-proliferative effects of isoflavones observed in both the sea urchin embryo model and human cancer cell lines correlated well. importantly, none of the synthesized isoflavones demonstrated cytotoxicity in human pbmcs, up to lm. in summary, gva and its analogues were synthesized via a scalable six-step reaction sequence. the gva and its analogues containing , , -trimethoxy and apiol-derived b-rings were found to be promising anti-mitotic microtubule destabilizing agents with low toxicity against human pbmcs. bag- is a multifunctional protein which has interactions with a number of cellular proteins; nuclear hormone receptors, bcl- , hsp /hsc family, growth hormone receptors, raf- , ubiquitin machinery and dna to regulate cell survival. for this reason, bag- is a critical molecular player in the regulation of cell survival signaling and apoptosis mechanism. elevated expression levels of bag- are associated with progression of cancer. in the treatment of breast cancer, silencing tools as a promising combined therapy strategies in the presence of classical chemotherapeutics gain importance to investigate interaction networks of cell death and survival signaling pathways. therefore, we aim to understand potential role of bag- silencing in the treatment of breast cancer cells with apoptotic agents; cisplatin or paclitaxel. our results showed that, silencing of bag- enhanced cisplatin or paclitaxel-induced apoptosis in mcf- cells by down-regulating antiapoptotic and upregulating proapoptotic bcl- family proteins, changes on cell cycle, upregulation on subg phase, activating caspases and cleavage of parp. in addition, knockdown of antiapoptotic bag- has a suppressive role in pi k and akt signaling pathway in mcf- breast cancer cells through inhibition of akt phosphorylation and downregulation on pi k. investigation targets of akt pathway showed that mtor cell survival pathway also affected through bag- silencing. bag- silencing inhibited mtor signaling via downregulating both rictor and raptor proteins which are the members of rapamycininsensitive mtorc and rapamycin-sensitive mtorc complexes, respectively. knockdown strategies of bag- is important to enlighten the network interactions of bag- and clarify its interaction partners in the cells. therefore utilization of bag- targeted strategies might further increase therapeutic efficiency of drugs through inhibiting cell survival machinery in the treatment of metastatic breast cancer. p- . . - biological activity evaluation of new , , trisubstituted triazine derivatives bearing different heterocyclic rings against lung cancer cell lines l. yurttas, g. akalin c ß iftc ßi, h. e. temel, b. demir anadolu university, eskisehir, turkey cancer is one of the major death causing disease worldwide. among the various cell types occurs on different organs, lung cancer is one leading cause of cancer death accounting for approximately % of all female and % of all male cancer deaths in . the resistance development, cytotoxicity and inadequacy are the main encountered problems by the treatment with existing chemotherapeutic agents. therefore, there is continuous need to discover new active and non-toxic molecules. -[ -( , -bis( -substituted phenyl)- , , -triazin- -yl)piperazin- -yl]- -[benzimidazole/benzoxazole/benzothiazole- -yl)thio]ethanone ( - ) derivatives were synthesized with a four-step synthetic procedure using toluil, anisil and -chlorobenzil as starting materials. the anticancer activity of the compounds was evaluated using the methods mtt ( -( , -dimethylthiazol- -yl )- , -diphenyltetrazolium bromide), brdu (bromodeoxyuridine) assays and flow cytometric analysis against lung cancer cell lines. the lipoxygenase enzyme inhibition activity of the compounds were also investigated using the method described by baylac and racine. compounds was found to have (inhibition concentration) ic values between - lg/ml. the early and late apoptotic cell percentage was determined as . for compound by flow cytometric analysis. the lox inhibition activity was found . ae . for compound . compound bearing -chlorobenzil and benzoxazole moieties was found as the most active compound when we evaluate anticancer potential of all compounds. the lox enzyme inhibition was indicated for the compound including methyl substituent on phenyl rings. the dna synthesis inhibition of the compounds has been still studied at the concentrations ic / , ic and ic x . p- . . - single amino acid substitutions and deletions modulate the drp-lyase activity of human dna polymerase iota n. miropolskaya, i. petushkov, a. kulbachinskiy, a. makarova institute of molecular genetics, moscow, russia dna polymerase iota (pol ι) is a y-family dna polymerase that possesses an unusual combination of properties. due to the special organization of the active site pol ι has a very low accuracy of dna synthesis but possesses an ability to bypass a variety of dna lesions. in addition to the dna polymerization activity, human pol ι also possesses an intrinsic -deoxyribose phosphate (drp)-lyase activity. removal of the drp group is a pivotal step in base excision repair (ber) in vivo. although pol b plays a key role in the drp group cleavage and dna synthesis during ber, pol ι was shown to complement the in vitro single-nucleotide ber deficiency of pol b null cell extracts and was suggested to be involved in ber under oxidative stress. the drp-lyase active site in pol ι is still not known. to address the mechanism of the drp-lyase activity of pol ι we obtained a series of pol ι mutant variants including point mutations of conserved lysine residues and deletions in different locations. we purified human pol ι variants from yeast saccharomyces cerevisiae and tested the effect of mutations on the cleavage of an internal -drp group in oligonucleotide dna substrates in the presence or absence for me + ions. the experiments revealed several point amino acids substitutions that significantly affected the drp-lyase activity of pol ι, thus suggesting a possible location of the drp-lyase active site. furthermore, we showed that deletions in the n-terminus of pol ι and metal ions modulate its drp-lyase activity, which may play an important role in the regulation of pol ι activities in vivo. this work was supported by russian foundation for basic research grants - - -a and - - -mol-a-mos and by the russian academy of sciences presidium program in molecular and cellular biology. rosmarinus officinalis, commonly known as rosemary, is an aromatic plant belongs to lamiaceae family. from past to now, rosemary have been used as a traditional medicine to cure for various illnesses such as diabetes, rheumatism and cancer. recent studies have shown that rosemary is effective for various cancer types. in this study we aimed to investigate the effect of rosemary in glioblastoma cells (gbm) by comparison with etoposide and the effect of rosemary by concurrent application with the etoposide. gbm cells (u mg) were seeded into the well plates and cultured with dmem supplemented with % fetal bovine serum. rosmarinus officinalis tea was prepared just as traditional usage and filter sterilized. at the second day of the culture rosemary in / (v/v) dilution ratio was given to first group, lm etoposide was given to second group, / (v/v) diluted rosemary and lm etoposide together were given to third group. after one day incubation cell viability was measured by neutral red assay. it was observed that rosemary reduced the viability of gbm cells by nearly % , etoposide reduced the viability by nearly % and rosemary with the etoposide reduced the viability by nearly % . the results showed that rosemary was able to reduce the viability of gbm cells but hadn't got an increasing or inhibiting potential over the etoposide's cytotoxic effect. from our previous studies we know that rosemary increases the proliferation of mouse embryonic fibroblasts. it is considered that rosemary might have a protection potential from dna damages and when rosemary is used with etoposide during the cancer treatment, it might reduce the side effects on healthy cells. in conclusion rosemary promises hope for developing new cancer treatment strategies and reducing the side effects of chemotherapeutics. for further studies it is aimed to examine the effects of rosemary with other chemotherapeutics and if rosemary has got a protection potential from the genotoxic stress. morpholine moiety has been found to be an excellent pharmacophore in medicinal chemistry and a number of molecules possessing morpholine skeleton are the clinically approved drugs. in this present study, we aimed to investigate the possible underlying apoptotic mechanism for the cytotoxicity of new morpholine dithiocarbamate derivatives bearing -( -aryl- -oxoethyl)- -substituted benzimidazole moiety on c glioma. c glioma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by cell proliferation analysis using standard ( -( , -dimethylthiazol- -yl)- , diphenyltetrazolium bromide (mtt) assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic values of the compounds were determined for c cell line. compounds , , , , and , which were including hydrogen, -methyl, -methoxy, -chloro and -floro substituents on phenyl acetyl moiety, had significant cytotoxic activity with ic values lower than lg/ml. compound showed the highest cytotoxic activity with a ic value of lg/ml, whereas cisplatin ic values were lg/ml against c cells. cytotoxic activity of compound , , , and with a ic value were , , and lg/ml, respectively. compound , and showed the highest population of early apoptotic cells as . , . , and . % respectively compared to cisplatin ( . %). also, compounds caused dna synthesis inhibition depend on their ic values by brdu assay. conclusions: it was concluded that synthesized compounds had considerable anticancer activity against c cell lines. however, compound , and including -methyl, -chloro and -floro substituents were the most active compounds against the c cell line. also our study results showed that compound , , induced apoptosis in c glioma cells. rutin is a glycosided flavonoid and known to have antioxidant and anti-inflammatory properties.trail induces the apoptosis of tumor cells and has no significant toxic effect on normal cells. although trail is a promising anticancer agent, trail resistance is a major barrier to effective cancer therapy. this study was conducted to examine the utility of the combined use of rutin and trail in prostate cancer cells. pc- and du prostate cancer cells were treated with rutin ( - um) and/or trail ( ng/ml), cell viability and migration were examined. cell viability was determined by trypan blue exclusion and mtt assay. cell migration was determined by wound healing assay. furthermore, lactate dehydrogenases (ldh) levels of medium were determined as biochemical markers of cell viability. pc- and du- prostate cancer cells were treated with rutin for and hours incubation and ic doses for hours incubation were determined um and um respectively. treatment with rutin, pc- cells is more sensitive than du cells. rutin and rutin plus trail inhibit prostate cancer cell growth in a dose-dependent manner. treatment with trail has no effect at inhibiting growth of pc- and du prostate cancer cells. the combination of rutin and trail elicit a synergistic antitumor effect on pc- and du prostate cancer cells. there is a significant increased in rutin and rutin+trail treatments group of ldh activities with respect to control and trail group. conclusion: present data show that rutin efficiently enhanced trail effects in prostate cancer cells. combined treatment with rutin and trail is more effective than the individual treatments of trail at inhibiting growth of prostate cancer cells. p- . . - determination of antigenotoxic, proliferative and cytotoxic properties of ellagic acid since ancient time, people use plant for traditional treatment. plants or fruits are produced different type of secondary metabolites. particularly phenolic phytochemicals from plants play an important role in the prevention and treatment of radical damage by inactivating the reactive oxygen compounds due to their antioxidant properties. however, the structure and the activities of many herbal products are not fully elucidated yet and there are several studies about the toxicity of herbal antioxidants and their possible risks to human health. ellagic acid, phenolic compounds, is an important substance. ellagic acid is a naturally occurring plant phenol found in numerous fruits, including blackberries, raspberries, strawberries, cranberries, walnuts, pecans, pomegranates and wolfberries. different researchers give some information about the biological activities of ellagic acid. in this study, we aimed to determine the cytotoxic, proliferative and antigenotoxic effects of ellagic acid, which is phenolic compounds found in natural products. cytotoxic effects of ellagic acid on huvec is investigated by lactate dehydrogenase (ldh) and cell proliferation (wst- ) methods; and antigenotoxic effects against ccl on human lymphocytes is investigated by single cell gel electrophoresis (comet) methods. the rusults showed that high concentration ( and lm) of ellagic acid has cytotoxic and mutagenic effects, but showed antiproliferative effects. on the contrary, low concentrations ( , , . lm) of ellagic acid has anticytotoxic and antimutagenic effects. as a conclusion, low concentrations of ellagic acid might be use treatment of some disease. but high concentrations of ellagic acid constitute a risk factors for people. keywords: cytotoxicity, antiproliferation, wst- , ldh, rtca-sp the constitutive nuclear factor kappa b (nf-kb) activation is widely found in diverse types of hematologic malignancies such as acute myeloid leukemia (aml) and chronic myeloid leukemia (cml) as well as solid tumors. inhibition of nf-kb signaling via proteasome inhibitors such as bortezomib can induce apoptosis in myeloid leukemia cell lines. however it is not clear whether the cytotoxic effects of bortezomib on myeloid leukemia cell lines is due to direct inhibition of nf-kb or another pathway, such as dna damage. in this study, cml cell line k and aml cell line hl- were treated with bortezomib (bor) , etoposide (eto) and camptothecin (cpt) alone or in dual combination with these drugs, following by measuring the effects on cell viability, apoptosis and signal pathways. the effect on cell viability was determined using the mtt assay. the data were used in combination index and isobologram analysis. the expression levels of apoptototic genes (bcl , bax and caspase ), the related dna damage genes (atm and atr) and the involved genes in nf-kb signaling (rela and p ) were determined by real time rt-pcr. we showed that combinations of bor with topoisomerase inhibitors (cpt and eto) exhibited synergistic cytotoxic effect in k cell line but not in hl- cell line. the combination treatment increased apoptosis and dna damage response. dnadamage-sensing kinases were detected in k and hl- cells following treatment with bor as similar as topoisomerase inhibitors. bor increased the mrna levels of atm and atr dramatically, which indicated active dna damage in the myeloid cell lines. furthermore, bor induced apoptotic cell death by decreasing bcl and increasing bax and caspase levels. these effects of bor were observed to correlated with increasing the p expression levels. this study on the mechanism of action of bor indicates that this compound affects several pathways involved in the control of cell cycle progression, apoptosis and dna damage. p- . . - analysis of molecular cytogenetic alterations in gastric and colon carcinoma by array-based comparative genomic hybridization (array cgh) introduction: genomic dna regions are frequently lost or gained during tumor progression. we aimed to evaluate tumor samples of patients with gastric cancer and colorectal carcinoma to show these genetic alterations by array-based comparative genomic hybridization (array cgh) method. materials and methods: dna isolation was performed from the tumor samples obtained from sixteen patients with primary gastric adenocarcinoma and twelve patients with colon adenocarcinoma. then, agarose gel electrophoresis was performed in those dna samples. following electrophoresis of dna, array cgh procedure was performed to four patients with gastric adenocarcinoma and three patients with colon adenocarcinoma who had dna breaks with - kb. results: after array-cgh study, many common genetic changes in gastric and colon cancer genome were determined. in gastric cancer dna samples, common losses were detected in chromosome p . , p . , q , q , p . , q . , q . , q . , q . , p , q . , q . , q . , q . , q . , q . , and q . , and also common gains were detected in chromosome p . , q , q . , q . and xq . in colon cancer dna samples, common losses were detected in chromosome p . , q , p . , p . , q . , q . , q . , q . , p . , p . , q . , and q . , and also common gains were detected.in chromosome q . , xp . , xp . , xp . , xp . and xq . both in gastric and colon cancer dna samples, common losses were detected in chromosome q . , q . , and p . , and common gains were detected in xq . discussion and conclusion: we think that these common changes, generally in dna loss areas harboring tumor suppressor genes and dna gain areas harboring oncogenes, may important in gastrointestinal tumorigenesis. the dna of every cell is under a constant attack by various mutagenic factors which damage the dna and can cause cell cycle arrest and even cell death. accumulation of dna damage is the basis for cancer development and one of the reasons for aging of the organisms. in order to preserve the integrity of its dna cells have evolved an impressive array of dna repair pathways, which are precisely coordinated with the progression of the cell cycle. one of the first events at the site of dna damage is poly(adp-ribose) polymerase (parp ) recruitment which is a sensor for single strand breaks in dna. parp catalyzes the synthesis of poly(adp-ribose) or par which is needed for the recruitment of many other dna repair proteins by means of par-binding domains. we used high speed confocal spinning-disk microscopy of living cells to obtain precise kinetics of recruitment of par-dependent proteins to the sites of laser induced dna damage. our results show that the investigated par-dependent proteins are recruited to dna damage sites in the matter of seconds, they reach peak intensities for to seconds after damage infliction and start dissociating. the recruitment of the proteins is entirely dependent on par because addition of parp inhibitor abbrogated their recruitment. the use of spinning-disk microscopy of living cells allowed us to obtain the kinetics of recruitment of the studied proteins to the sites of dna damage. the results are consistent with the fact that parp and par-dependent proteins are quickly recruited to damage sites and generation of par is essential for other dna repair protein recruitment. the precise kinetic curves may serve as a basis for investigating how they will change or if they will change at all when cells are put in different conditions or treated with various chemical substances affecting dna metabolism and repair. introduction: chronic myeloid leukemia (cml) is a myeloproliferative disease associated with reciprocal translocation between chromosomes and . bcr-abl fusion gene which exhibits constitutively active tyrosine kinase activity has a main role in cml. the tyrosine kinase inhibitor imatinib is used as a first line treatment in cml patients, but imatinib resistance leads to failure in therapy. the application of imatinib in combination with other anticancer agents may be a strategy to increase the antileukemic effect of imatinib. in this study, we have investigated the antiproliferative effect two novel agents: a benzamide derivative xt and a benzoxazole derivative xt b in combination with imatinib. these molecules were investigated in imatinib-sensitive (k s) and imatinib-resistant (k r) cml cell lines. materials and methods: antiproliferative and apoptotic effects were assessed by mtt assays and flow-cytometry, respectively. we also evaluated the effects of these compounds on the expression of apoptosis-related genes bax, bcl- , bad, bim, bcl-xl and mcl by real-time quantitative pcr. results: treatment of k cells with xt increased the expression levels of the pro-apoptotic genes bax, bad and bim in both sensitive and resistant cells. however, xt b was not found to have similar effects on k r and k s cells. combined application of xt increased cell death in the mtt assay. mtt assay demonstrated that ic for xt treated cells in k r with imatinib (ic = . ) is lower than k r without imatinib (ic = . ). discussion and conclusion: our results showed that combining xt with imatinib has more antiproliferative and apoptotic effect on a cml cell line. as a result combination of xt with imatinib can be an alternative approach to overcome imatinib resistance. introduction: the mmr(mismatche repair) system recognizes base-base mismatches and insertion or deletion loops in doublestranded dna, and it degrades the error-containing region of the newly synthesized strand, allowing the polymerase to correctly resynthesize the second strand according to the template sequence. the human mmr system includes the mlh and msh . alteration in expression or a defect in mlh or msh can cause resistance to anti-cancer drugs used in chemotherapy. the attempt of the mmr system to detect drug induced dna damage, triggers the activation of apoptosis, a mechanism which may enhance the cytotoxicity of chemotherapy. loss of the mmr system would make the neoplastic cell less able to initiate apoptosis. inability to initiate apoptosis could be a mechanism of resistance to drugs. chronic myeloid leukemia (cml) is a clonal disease originating from aberrations in hematopoietic stem cell. imatinib, a tyrosine kinase inhibitor has significantly improved clinical outcome for cml patients. however, patients develop resistance when the disease progresses to the blast phase (bp) and there are several mechanisms involved in imatinib resistance. in this study we investigated the role of mmr system in imatinib resistance. materials and methods: k s (sensitive) and k r (resistance) were grown in rpmi- . k r cells were maintained in rpmi- medium supplemented with lm imatinib rna isolation, cdna synthesis, rt-pcr was performed respectively. results: the results demonstrated that expression of mlh in k r cells is dramatically lower than equal amount of imatinib treated k s cells, whereas msh expression level did not change in both cell lines. conclusion: it can be suggested that alteration and down-regulation of mlh genes leads to imatinib resistance. p- . . - characterization of interaction between rad inhibitor dids and human serum albumin d. velic, s. henry, c. charlier, m. popova, p. weigel, j. masson, i. nabiev, f. fleury cnrs/university of nantes, nantes, france -diisothiocyanostilbene- , -disulfonic acid (dids) has been largely used during the last years for its inhibitory effect on anion transporters and channels. more recently, ishida and colleagues have described a possible mechanism by which dids inhibits rad -mediated homologous pairing and strand exchange, key processes in dna repair by homologous recombination. thus, dids could act as a potential revertant of radioand chemo-resistance in cancer cells, which is the major cause of failure during therapeutic protocols. new drugs targeting rad protein have since been developed with potential use for medical applications. in this context, we attempted to determine the behaviour of dids towards blood and plasma proteins such as serum albumins. firstly, we analysed the effects of several environmental factors such as solvent polarity, which may affect the stability of the molecule. secondly, we analysed the spectroscopic properties of dids in the presence of human or bovine serum albumin proteins. uv-visible absorption, circular dichroism, fluorescence spectroscopy and isothermal calorimetry were used. here we show for the first time that dids can interact with both serum albumins. we have also determined the characteristics of these interactions. the comparison of several dids derivatives led us to identify the essential chemical moiety of this compound involved in the interaction. moreover, by using site competition approaches we show that the main binding site for this molecule is in subdomain ib of the protein. these findings show that the binding of dids to serum albumin proteins may change the equilibrium between the free and bound dids forms, thereby affecting its bioavailability and efficiency against the rad recombinase protein. p- . . - mechanism of tap beta-mdm autoregulation p is a transcription factor which is the member of a p family. it regulates many cellular processes, such as apoptosis, cell cycle, and senescence. in contrast to p , p is rarely mutated in tumors and elevated p expression is observed in many types of cancers including hepatocellular carcinoma, neuroblastoma, and lung. defining regulatory mechanisms which control p protein abundance and activity will be crucial for the development of new therapeutic strategies for cancers. mdm is known as the key player in regulation stability and activity of p . in addition, p induces mdm transcriptional activity, and caspase- , activations which cleave mdm n-terminal at asp . cleaved form of mdm binds p and promotes its stabilization. mdm suggested as a candidate to modulate p activity and stability too. however, an interaction between p and mdm has not defined well. in this study, we aimed to analyze the role of mdm in p stability. to define this relationship, firstly, we overexpressed the tap beta isoform using trex system in hep b. tap beta and mdm protein levels were determined by western blot. to examine whether mdm mediate tap beta protein degradation by the proteasomes, cells were treated with proteasome inhibitor, mg for hours prior to analysis. previous studies showed that p -induced caspase- and caspase- activation cleaves mdm . considering this, we firstly examined caspase- activation by western blot in hep b tap beta cells. then we analyzed expression of cleaved mdm and tap beta levels following caspase inhibitor, z-vad-fmk treatment. as a conclusion, tap beta-induced full-length mdm- expression. furthermore, tap beta enhanced cleavage of mdm via increased caspase- activation. in addition, inhibition of caspase- activation caused a decrease in cleaved-mdm levels in parallel with tap beta expression repression. our results suggested positive regulation between mdm -tap beta. hepatocellular carcinoma (hcc) is one of the most common type of liver cancer and third leading cause of cancer related deaths in worldwide. discovery of new targets is important in survival of hcc patients. p is a transcription factor which is the member of p family. it has two promoters; while p promoter expresses apoptotic ta isoforms, p promoter expresses anti-apoptotic dn isoforms. in addition, alternative splicing in c terminal creates many isoforms of ta and dn p . it has been shown that both tap and dnp isoforms are expressed in hcc patient tissue and cell lines. the ratio between tap and dnp affects the apoptotic response, drug response and prognosis. accordingly, identification of the role of p and its targets are important in discovery of new treatment strategies in hcc. to understand the role of p isoforms in hcc, firstly we performed mtt assays following dna-damaging drugs and multikinase inhibitor, sorafenib treatment to categorize hcc cell lines as resistant or sensitive. after that, we analyzed the expression levels of tap isoforms via western blot in all hcc cell lines. then we overexpressed the tap beta isoform using trex system in hep b and snu cells. these two clones were analyzed for dna damaging drug response by mtt, cell cycle and apoptosis by flow cytometry, and tumor formation by in vitro and in vivo experiments. in scope of our study; . only tap alpha isoform is expressed in a few hcc cell lines. . there is no correlation between basal expression of p isoforms and drug responses in hcc cell lines. . there is no change in expression of p isoforms after treatment of drugs. . we showed that the ectopic expression of tap beta in hep b arrested the cell cycle in g / s and decreased the colony formation. therefore, the capacity of tumor formation of the cells dramatically decreased in scid mice. as a result, we revealed that tap beta play role in tumor formation, cell cycle arrest, dna damage responses in hcc. p- . . - biochemical characterization of exonuclease iii-family ap endonuclease point mutants reveals role of conserved amino acid residues in the nir-specific enzymes a. mursalimov, z. koshenov, t. yeleussizov, m. redrejo-rodriguez, a. ishchenko, b. t. matkarimov, m. saparbaev national laboratory astana, astana, kazakhstan oxidative dna damage caused by reactive oxygen species is believed to be a major type of endogenous cellular damage. oxidatively damaged dna bases are substrates for two overlapping repair pathways: dna glycosylase-initiated base excision (ber) and apurinic/apyrimidinic (ap) endonuclease-initiated nucleotide incision repair (nir). in the ber pathway, an ap endonuclease cleaves dna at ap sites and -blocking moieties generated by dna glycosylases, whereas in the nir pathway, the same ap endonuclease incises dna to a number of oxidized bases. majority of characterized ap endonucleases possess classic ber activities and about half of them are able to catalyze nir activity. at present, the molecular basis of dna substrate specificities of various ap endonucleases remains unclear. here, we examined amino-acid sequence requirement of the nir activity of human major ap endonuclease (ape ). amino acid sequence alignment of various ap endonucleases including e coli exonuclease iii (xth), human ape and archaeal mth revealed conserved amino acid residues in the nir-specific ap endonucleases ape , mth and exoa that are absent in xth. based on these data, we constructed four ape point mutants y h, n q, g s and t d and examined their dna substrate specificities. results obtained from biochemical characterization of ape mutants are discussed in the light of the evolutionary conserved dna repair functions of ap endonucleases and whether these functions can be mutationally separated from. since its discovery some years ago, cisplatin has evolved for its efficacy in one of the most used drugs in treatment of various cancer types. huge effort was invested in understanding the action of cisplatin and development of more potent drugs. they target mainly neighboring purine bases of nuclear dna forming covalent intra-or inter-strand cross-links that affect inhibition of replication and transcription, cell cycle arrest, and attempted repair of the damaged nucleotides. if such damage cannot be removed the cell dies. we have studied the details of the binding site of the short oligonucleotide modified by a platinum compound using complementary solution techniques used in modern structural biology, including raman spectroscopy with dft calculations aided interpretation of the obtained vibrational spectra. moreover, the calculated structure of the dna duplex was verified using saxs (small angle x-ray scattering) curve. in our contribution, we will present an nmr structure of a dna cross-linked with a cisplatin derivative containing a cyclohexane ring. at this atomic level resolution, structural features probably influencing cytostatic effects are described and compared with previously published structures. common structural features of previously determined structures are: a significant roll ( - °) of the guanine bases involved in the cross-link, bending and unwinding of the double helix at the site of cross-link and orientation towards the major groove. also, the platinum-guanine plane angle varies between and °. although the experimental structures were often used as the starting models for molecular dynamics (md) simulations, results of these md still leave many questions unresolved. the results of this research have been acquired within ceitec (lq ) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. p- . . - ercc /xpd polymorphisms and colorectal cancer risk: a case control study in a north eastern iranian population j. mehrzad islamic azad university, neyshabur, iran excision repair cross-complimentary group (ercc ) is one of the important dna repair genes.ercc codon and polymorphisms has been shown to modulate cancer risk. we therefore assessed the relationship between the ercc polymorphisms and the susceptibility to colorectal cancer in a case-control study. there were lung cancer cases and matched healthy controls in this study. information concerning demographic and risk factors was obtained, each person donated ml blood for biomarker testing. ercc genotypes were determined by t-arms-pcr method. all of the statistical analyses were performed with spss (v . ). there was significant difference between the frequencies of ercc polymorphism in cancer cases and controls (p < . ). the frequencies of ercc gln allele were . % in controls and . % in cancer cases. the individuals with lys/gln+gln/gln combined genotype were at an increased risk for lung cancer as compared with those carrying the lys/lys genotype (adjusted or= . , %=ci . À . ). the above findings indicate that the genetic polymorphism in the ercc codon is associated with the risk of colorectal cancer in an iranian population (neyshabur citizenship). peptide pore blockers are potent tools to study structure and function of potassium voltage-gated channels (kv). kcsa-kv .x chimeras, in which a ligand-binding site of eukaryotic kv-channel is inserted into bacterial kcsa channel, mimic properly the pore domain of kv-channels. a fluorescence-based approach to study the binding of peptide blockers with kcsa-kv . -kcsa-kv . chimeras was developed by us. this approach rested on high-level expression of kcsa-kv .x chimeras in e.coli inner membrane, binding of fluorescently-labeled toxin at the surface of the spheroplast and analysis of competitive binding of studied ligands by laser scanning confocal microscopy (lscm). here we report on a new analytical system for search and study of kv . -channel blockers that combines bl (de ) cells expressing kcsa-kv . and rhodamine-labelled agitoxin (rh-agtx ) as a fluorescent probe. by tuning cultivation conditions, the high-level of membrane expression of kcsa-kv . was achieved. it was found that lowering both the growth temperature and the concentration of inducer resulted in significant increase in membrane-embedded kcsa-kv . . for system validation, wellknown kv channel blockers were studied by the method of competitive binding, and equilibrium dissociation constants were estimated for agtx , osk , and kaliotoxin. a new system was applied to study molecular determinants of peptide-kv . channel binding using a number of agtx mutants constructed by us, whose affinities to kcsakv . were measured. a new bioengineering fluorescent system is a robust and sensitive assay for assessing the binding activity of kv . channel blockers. it can be used to study interaction interfaces of toxinchannel complexes, to search for novel peptide blockers and to develop new potent and selective kv . -blockers for scientific and medical purposes. the work was supported by the grant - - from russian science foundation. asparagus racemosus root extracts (ar) have been exhibited to show a wide range of pharmacological benefits. in this study, liposomes of ar were developed and assessed their physicochemical properties and anti-inflammatory activity in monocytic leukemia cell line (thp- ). liposomes containing ratios of ar to lipid and phosphatidylcholine to cholesterol ratio were synthesized by thin-film hydration (tf), reverse-phase evaporation (rev), and polyol dilution (pd). the in vitro anti-inflammatory activity was assessed in terms of inhibition of tumor necrosis factor alpha (tnf-a) in lipopolysaccharide activated thp- by elisa. the size of ar liposomes prepared by tf were larger, whereas those prepared by rev and pd were smaller. ar to lipid ratio was shown to have no influence on particle size, whereas zeta potential enhanced with increasing ar to lipid ratio. ar liposomes with lipid ratio of : achieved the highest value of entrapment efficiency and were at the highest with polyol dilution method. ar was found to have no toxic effects on thp- cells. the anti-inflammatory activities of ar and ar liposomes in terms of tnf-a in thp- cells were was exhibited to possess the highest values of around % at ar concentration of lg/ml and % tnf-a inhibition tended to decline with the increasing amount of ar. this result may be attributed to the increased amount of liposomal particles being uptaken into the cells as a result of the increasing ar concentrations. it can be suggested that ar liposomes could be an alternative choice of topical/transdermal drug delivery for anti-inflammatory activity. p-mis- inhibition of ire signaling enzyme increases the expression of tumor suppressor genes and modifies their hypoxic regulation in u glioma cells d. tsymbal, o. minchenko palladin institute of biochemistry of the national academy of sciences of ukraine (nasu), kyiv, ukraine gliomas constitute one of the most aggressive groups of malignant neoplasms with poor survival prognosis and scarce therapeutic options. plentiful studies have proven the connection between endoplasmic reticulum stress and malignant growth. we have studied the effect of inhibition of ire (inositol requiring enzyme ), which is a central mediator of endoplasmic reticulum stress and controls cell proliferation and tumor growth, on hypoxic regulation of the expression of different proliferation related genes in u glioma cells. it was shown that inhibition of ire leads to up-regulation of the expression of krt , cd , mest, cenpu, myl , ing , ing , mybl , and mybl genes at the mrna level in u glioma cells, with more profound changes for mest, mybl , and cd genes. hypoxia leads to up-regulation of the expression of cd , ing , and ing genes and to down-regulationof krt gene in glioma cells. at the same time, inhibition of ire modifies the effect of hypoxia on the expression of all studied genes: suppresses effect of hypoxia on ing gene, eliminates hypoxic regulation of krt , cd , and ing genes in glioma cells. the present study demonstrates that inhibition of ire enhances the expression of all studied genes and modifies the hypoxic regulation of these gene expressions in gene specific manner and thus possibly contributes to slower glioma cell proliferation, but several aspects of this regulation remain to be further clarified. amplification and clonig of dna polymerase (pol ) of thermus scotoductus k isolated from an armenian goethermal spring a. saghatelyan, h. panosyan, a. trchounian, n. birkeland yerevan state university, yerevan, armenia the most important enzyme ''mined'' from thermophilic microorganisms is dna polymerase, which widely used in molecular biological studies. although dna polymerase produced by thermus aquaticus (taq polymerase) was launched into the market long back, isolation of more processive, reliable and stable dna polymerases from other species is a demand. the purpose of this work was to amplify and clone the pol gene of t. scotoductus strain k recently isolated from an armenian geothermal spring. the draft genome sequence of strain k was deposited under accession number ljjr . . genomic dna was isolated using genelute bacterial genomic dna kit. primers for the pol gene were designed manually. the gene was amplified using pfu polymerase, and amplicons (~ . kb) were ligated into the pet- b(+) vector (novagen) and transformed into chemically competent top escherichia coli. inserts were sequenced with t prom and t term primers, which showed that the gene sequence was correct and in the right reading frame and could be expressed in mesophilic e.coli. dna polymerases patented form different species of thermus are mostly comparable, suggesting that only limited natural variations in taq-like dna polymerase may be discovered. the pol gene from k shares % and % similarity with pol of t. scotoductus sa- ( . kb) and t. aquaticus, respectively. although the difference is not huge at sequence level, possible functional differences (e.g. stability, proofreading activity, resistance to different pcr inhibitors etc.) may occur. therefore, it is important to express and purify dna polymerase from strain k for further investigations. peptide ligands of the immunoglobulin g fc region identified by screening phage libraries and site-directed mutagenesis n. kruljec, p. molek, t. bratkovic young researcher, ljubljana, slovenia affinity chromatography based on immunoglobulin (ig)-binding proteins, such as staphylococcal protein a and streptococcal protein g, typically represents the initial step in therapeutic antibody purification process. however, this approach suffers from high cost, poor ligand stability and the requirement for relatively harsh elution conditions that can negatively impact activity and immunogenicity of antibodies. compared to protein ligands, peptides represent an interesting alternative due to higher stability and less expensive production. furthermore, the expected lower affinity for immunoglobulins should allow for elution under milder conditions. the aim of our research was to identify short peptide ligands for the fc region of human iggs. we have screened three commercially available phage display libraries of random cyclic and linear peptides for binding to human fc region in solution using an optimized biopanning approach. five non-homologous linear peptides were shown to specifically interact with the fc portion of immunoglobulins as verified by a set of phage elisa assays. individual phage-displayed peptides were able to recognize specific subclasses of igg. the highest-affinity peptide ( l- fc), which competed for fc binding with protein a, was subjected to mutagenesis studies. we displayed on phage several variants of l- fc with individual amino acid residues exchanged for alanine as well fragments of the parent peptide of different lengths and evaluated binding to fc with phage elisa to identify the minimal binding motif. binding characteristics of the minimized peptide were further analyzed using spr biosensor. the details will be disclosed at the meeting. diverse effects of ganoderma lucidum in combination with tamoxifen citrate and doxorubicin in mcf- breast cancer cells ganoderma lucidum, an edible medicinal fungus, has been known with its anti-metastatic, anti-carcinogenic bioactivities and widely used in asian countries in complementary and alternative medicine. however, there is no information regarding its combined usage with tamoxifen and doxorubicin in breast cancer treatment. we investigated the interactions between ganoderma lucidum and tamoxifen or doxorubicin in mcf- human estrogen receptor positive breast cancer cell line. anti-proliferative properties of six extracts were assessed by wst- method. the most effective extract in inhibition of mcf- cell viability was then evaluated in terms of its anti-metastatic activity by boyden chamber assay. apoptosis and cell cycle assays were performed by flow cytometry. ganoderma lucidum ether extract (g.ether) was the most effective extract on inhibition of cell viability among others with ic ( ) values of lg/ml and . lg/ml at h. and h. respectively. we found that g.ether is capable of inducing apoptosis and changing cell cycle dynamics. however, incubation with g.ether did not affect mcf- cell motility significantly. we then assessed the interactions between g.ether and tamoxifen or doxorubicin in mcf- cells. the interactions between g.ether and cancer therapeutics were examined by combination index analysis and macsynergy ii software. interestingly, g.ether increased the anti-proliferative effect of tamoxifen although exhibited strong antagonism with doxorubicin in mcf- cell line. testing the best matrix/analyte combination for maldi tof mass spectrometric detection of steroid hormones, amino acids, vitamins and carbohydrates in spite of numerous advantages, there are serious drawbacks of the application of matrix assisted laser desorption/ionization time-of-flight mass spectrometry (maldi tof ms) for smallmolecule analyses (below da) and quantification. the main problem is the background interference from commonly used maldi matrix materials. the aim of this work is to evaluate maldi tof mass spectra of physiologically relevant small molecules: steroid hormones, vitamins, amino acids and carbohydrates, acquired with several organic, traditional matrices. small volume, . ll, of each sample solution (testosterone, progesterone, estradiol, l-cysteine, l-alanine, dl-methionine, glutathione, d-(+)-glucose, d-(+)-maltose, vitamin a, vitamin e) was mixed on the sample plate with the same volume of organic matrix solutions (dhb, thap, chca, -aa). for each molecule/matrix pair, we determined quantitative and qualitative parameters of ms analysis. to calculate within day and day-today variation we used excel tools (anova tests). in addition, homogeneity of the sample/matrix distribution on the target was also calculated and expressed as the coefficient of variation of a series of measurements. our results show selectivity of the detection of individual molecules related with the matrix applied. the statistical analysis of certain molecule/matrix pairs gave within and day-to-day variations less than %. additionally, homogeneity of the sample/ matrix mixture distribution on the target plate was with some matrices, also less than %. some of the used matrices have a great potential for the analysis of small molecules with good analytical parameters, with low variations and high homogeneity of samples on the maldi target plate. these results hold potential for quantification of metabolically-significant small molecules and are very promising for future applications of maldi tof ms analyses. stress causes different expression of mitochondrial biogenesis markers in rat steroid-producing cells of adrenal gland and testes i. starovlah, s. radovic, t. kostic, s. andric faculty of science univeristy of novi sad, novi sad, serbia functional mitochondria of steroid producing cells of adrenal cortex and leydig cells of testes are essential for steroid hormones biosynthesis and regulation. the aim of this study was to determine transcriptional profile of mitochondrial biogenesis markers in adrenal cortex and leydig cells by applying in vivo and in vitro studies. immobilization stress (imo), was performed for hours daily for one ( ximo), two ( ximo) or ten ( ximo) consecutive days. in in vitro studies, primary cultures of purified leydig cells from undisturbed rats were stimulated with stress hormone adrenaline, propranolol (nonselective b-adrs-blocker) and prazosin (the selective a -adrs antagonist). rq-pcr results showed that the transcription of the main regulator of mitochondrial biogenesis, ppargc a and ppargc b, significantly decreased in adrenal cortex of ximo rats. oppositely, the significant increase of the same transcript was registered in leydig cells from the same rats. in parallel, transcription of ucp , the mediator of regulated proton leak, decreased in adrenal cortex, but increased in leydig cells of the same group of rats. incubation of leydig cells with adrenaline, increased transcription of the main markers of mitochondrial biogenesis (ppargc a, ppargc b, nrf and nrf a). nonselective b-adrsblocker attenuated this effect. the selective a -adrs antagonist did not change adrenaline-induced stimulation of ppargc a, ppargc b, nrf and nrf a transcription in leydig cells, indicating that the most of the effects are probably mediated by b-adrenergic receptors, not by a -adrs of leydig cells. in summary, the results suggest that reduction of transcription of mitochondrial biogenesis markers could be a possible mechanism that protects body from excessive glucocorticoid production from adrenal glands in stress conditions, while at the same time stimulation of mitochondrial biogenesis markers transcription in leydig cells could serve as mechanism to preserve testosterone production. p-mis- generation of new mitochondria is possible protection mechanism of basal steroidogenesis in leydig cells s. radovic, i. gak, t. kostic, s. andric faculty of science, university of novi sad, novi sad, serbia mitochondria are the most important component of stress response in all cells and for steroid-hormones-producing cells they are the starting point for steroid biosynthesis. here we investigated the parameters of mitochondrial biogenesis in these cells from rats exposed to the psychophysical stress by immobilization (imo). imo stress was applied for hours daily for one ( ximo), two ( ximo) or ten ( ximo) days.hormone levels were measured employing eia, elisa kit or ria. mitochondrial membrane potential (Δwm) was measured by tmre fluorescence, mitochondrial mass was detected by quantitative analysis of mitotracker-green fluorescence as well as relative intensity of fluorescence, since number of mitochondria and mitochondrial architecture were defined using transmission electron microscopy. relative gene expression and proteins analyses were performed by rq-pcr and western blot. there was positive correlation between Δw m of leydig cells and androgens production of leydig cells. both of them were reduced in all stressed rats but partially recovered in ximo group. the mitochondrial mass in leydig cells from ximo group was increased. transmission electron microscopy analyses showed that acute and two times repeated stress altered architecture of mitochondrial cristae, while ximo increased number of mitochondria and recovered mitochondrial architecture. there was significant increase in the expression of the all markers of mitochondrial biogenesis in leydig cells from ximo rats compared with other groups. accordingly, stress-triggered mitochondrial biogenesis represents an adaptive mechanism and does not only correlate with but also is an essential for testosterone production, being both events depend on the same regulators. supporting the evidence that stress, a constant factor in life of humans, induces mitochondrial biogenesis in leydig cells, our results indicate this mechanism probably protects the basal steroid production in stress conditions. targeting survival pathways in leukemic cells through synergism of metformin and thymoquinone u. glamoclija, m. suljagic international university of sarajevo, sarajevo, bosnia and herzegovina generation of resistance to current treatment options is common problem in the therapy of many hematological malignancies. combined therapies utilizing compounds with low toxicity that act synergistically, are proposed to overcome this problem. metformin and thymoquinone (tq) are two molecules which have proven safety profile and represent potential candidates for treatment of hematological malignancies. there are more than clinical trials, at different stages, exploring metformin anticancer activity. metformin activates amp activated protein kinase (ampk) leading to inhibition of the mammalian target of rapamycin (mtor) and induction of apoptosis in different cancers. however, human leukemic cells with increased basal protein kinase b (akt) phosphorylation were shown to be resistant to metformin-induced apoptosis. it was found that activity of metformin can be enhanced by combination with akt and/or nuclear factor 'kappa-lightchain-enhancer' of activated b-cells (nf-jb) inhibitors. tq is phytochemical compound that has shown inhibitory capacity on both of these targets. wst- assay was used to evaluate the effects of metformin and tq in dhl (b cell lymphoma) and k (chronic myelogenous leukemia) cell lines. compusyn software was used in order to calculate the combination index (ci). the ci value indicates whether two drugs have synergistic (ci< ), additive (ci= ) or antagonistic effects (ci> ). we have shown that separately, metformin and tq, exhibit dose dependent inhibition of dhl and k cells. in combinatorial study with fixed constant ratio and simultaneous drug exposure, in dhl and k cell lines, ci values were . and . , respectively. to our knowledge, this is the first report showing synergistic effects of metformin and tq in lymphoma and chronic myelogenous leukemia derived cell lines. these promising data are currently being investigated in order to obtain the insight into their molecular mechanisms. for the last decade many methods of calculating and analysing the physical characteristics of dna has been developed. these methods allow to estimate distributions of free energy, propensity to bend, stress-induced duplex destabilization (sidd), electrostatic potential (ep) etc. and most of them have been used for prediction of genomic regulatory site positions. the main idea of such approach is that proteins recognize genome regulatory sites by these physical and chemical properties, so the physical characteristics are used to predict the location of regulatory sites. most of the characteristics mentioned above describe properties of dna at equilibrium or steady state, but we propose to use characteristics of internal dna dynamics. in this work we used the coarse-grained model of dna, developed recently, to simulate dynamics of the dna open states. with this model we were able to calculate trajectories of the open states moving along the molecule and their dynamical characteristics, such as: open state activation energy, size, half decay time and sound velocity in dna. we use distribution of four dynamical characteristics around transcription start site of experimentally found e.coli promoters taken from regulon db to organise them in stable clusters. clusterization was made with ward method and consensus clustering technique was applied to clusterization results for analysis of its consistency. the same procedure was applied to equilibrium dna characteristics for comparison. distribution of go functions among clusters was also analysed. stable promoter clusters obtained with different physical properties share some similarity. it was not surprise that clusters obtained with dynamical characteristics of dna more similar to sidd clusters then to ep clusters. the data highlights the possible role of dna dynamical properties in transcription initiation and its applicability to promoter identification together with other physical and textual properties of dna. chromium complex with -hydroxyflavone acts on metabolic pathways the development of novel therapeutic strategies for obesity treatment are urgently required as obesity is currently the main leading cause in type ii diabetes and insulin resistance. among natural compounds, flavonoids have recently gained interest due to their positive role in maintaining blood glucose levels and insulin secretion. their association with trace elements, wellknown for their capacity in increasing the efficiency of insulin, might potentiate flavonoids biological effects. in this context, the aim of our study was to investigate the in vitro changes in energetic metabolism related genes expression profile in the presence of a chromium complex with -hydroxyflavone. dna microarray technology was used for a large scale screening of differentially expressed genes in human adipose stem cells (hascs) after weeks of adipogenic induction in the presence of the chromium complex with -hydroxyflavone. moreover, perilipin expression was assessed by flowcytometry. the chromium complex with primuletin negatively regulates the expression of key genes involved in adipogenesis and also modulates the expression of the genes associated with triglyceride synthesis and subsequent fat storage in mature adipocytes. consequently, the chromium complex with -hydroxyflavone can be further employed in studies on animal models to investigate the possible improvement of metabolic disorders. deinococcus radiodurans is a highly radioresistant and stress-resistant bacterium. despite extensive studies, the mechanisms of transcription regulation that contribute to the stress-resistance are still poorly understood. d. radiodurans encodes multipe stress-related proteins including three members of the gre-family of transcription factors: grea, gfh and gfh . while grea is a universal bacterial factor that stimulates rna cleavage by rna polymerase (rnap), the functions of lineage-specific gfh proteins remain unknown. we cloned, expressed and purified d. radiodurans rnap and gfh factors and their mutant variants and analyzed their properties using various in vitro transcription approaches. we tested gfh effects on rnap activity in promoter, elongation and termination complexes assembled on natural and synthetic dna templates under different conditions. we found that the gfh factors strongly enhance site-specific pausing and intrinsic transcription termination by d. radiodurans rnap but do not act on active transcription complexes and do not compete with the grea factor. uniquely, the pause-stimulatory activity of gfh is greatly enhanced by manganese ions, which are accumulated in d. radiodurans cells under stress conditions, and is modulated by the secondary rna structure. we revealed functionally important regions in the gfh factors and the rnap active site involved in transcriptional pausing. we propose that gfh factors inhibit rna extension in paused complexes through binding within the secondary rnap channel, coordinating metal ions in the rnap active site and stabilizing an inactive enzyme conformation. this may serve as a sensitive mechanism to regulate transcription under stress conditions and coordinate it with dna repair and replication. our data suggest that gre and gfh proteins target different structural states of the transcription elongation complex and reveal functional diversity of the factors that bind within the secondary channel of rnap. from planktonic to biofilm state of growth, flagella formation is turned off, and the production of fimbriae and extracellular polysaccharides is activated. bola protein is widespread in nature and has been associated with several cellular processes. using high-troughput techniques we showed that bola protein is a new bacterial transcription factor, which regulates the switch between motile and sessile lifestyle. it negatively modulates flagellar biosynthesis and swimming capacity in escherichia coli. moreover, bola overexpression favors biofilm development, involving fimbriae-like adhesins and curli production. our recent results show that bola action in these pathways is related with cdi-gmp a relevant intracellular signaling molecule involved in biofilm formation. we demonstrate that bola contributes to a fine-tuned expression of different diguanylate cyclases and phosphodiesterases and c-di-gmp has a negative influence in the bola mrna transcription. herein we propose that bola is a key player in motile/adhesive transcriptional switch, contributing to a fine-tuned regulation of these important pathways. background: deep venous thrombosis (dvt) is an important health problem worldwide. its pathophysiology is multicausal and involves environmental, genetic and acquired factors. factor v leiden (fvl), prothrombin g a (pt g a), and methylenetetrahydrofolate reductase (mthfr) gene mutations are to predispose to venous thrombosis. the aim of this study was to compare the frequency of fvl, pt g a and mthfr polymorphisms between patients with dvt and healthy controls. methods: this study was conducted at the bozok university hospital. total participants were included in this study, patients with dvt and healthy blood donors. in order to identify fvl, pt g a, mthfr c t and mthfr a c, the polymerase chain reaction (pcr) method was utilized combined with the amplification refractory mutation system. results: in patients fvl was present in ( . %) patients while in controls fvl was present in only ( . %). frequency of fvl was significantly higher in cases as compared to controls (p < . ). pt g a mutation was present in patients ( . %) and in healthy participants ( . %). mthfr c t and mthfr a c polymorphisms were almost equally distributed among patients and healthy participants. however, the concomitant presence of fvl and double heterozygous polymorphisms of mthfr c t/a c was found in patients ( . %) and in healthy controls ( . %), showing significant association with deep venous thrombosis. conclusion: in this study, the frequencies of fvl and pt g a polymorphisms were found significantly higher in patients with dvt than those in healthy participants. thus, fvl and pt g a polymorphisms have a contributory role on the development of dvt in contrast, mthfr c t and mthfr a c genotypes were not associated with a predisposition to development of dvt. but, a combination of double heterozygous polymorphisms of mthfr c t/a c with fvl may be associated with increased risk of dvt. p-mis- self-assembling micellar clusters comprising drugs, nanoparticles and fluorescent compounds for bilogical applications when designing drug carriers, the drug-carrier ratio is an important consideration, because the use of wrong drug-carriers relation can result in toxicity as a consequence of poor metabolism and elimination of the carriers. solubility problem of various substances also plays an important role in many aspects of fundamental science and practical field. specifically, it is an important parameter as well as bioavailability, which determines the required concentration of drug in the body needed to achieve a pharmacological response. among the variety of solubilization methods micellar solubilization is widely used as an alternative to the dissolution of poorly soluble drugs. here, we show a specific approach based on sequential selfassembly of nonionoc detergent micelles (t , tx ) followed by enacpsulation of various nanoparticles (noble metals, magnet etc.), drugs, fluorescent compounds leads to the formation of stable micellar nano-amd microcomplexes. we propose ways of micellar clusterisation. in the first one micelles are modified by semi-hydrophobic chelator followed by addition of metal ion to make cross-linking. the second way is similar to the first one and suggests application of the metal complex with incresed denticity instead of naked metal ion, and the third one involves micelles clusterisation by semi-hydrophobyc metal complex directly. therefore, one can stabilize micellar network by means of 'interactions on interface': semi-hydrophobyc metal complexes are embedded inside micelle due to hydrophobyc interactions. hydrophobic fluorescent compounds-loaded micellar complexes demonstrates better optical response in aqueous media without crystallization. such obtaining clusters are also very flexible and can be modified by nanoparticles to obtain various nanocomposites, such as fluoromagnetic clusters. this work was supported by russian foundation of basic research grants no. - - r_center_a ( no. - - r_center_a ( - no. - - r_center_a ( ) and - - mol_a_ved ( no. - - r_center_a ( - . lamellipodia and membrane blebs utilize different signalling pathways to induce directional movement of walker carcinosarcoma wc cells in a physiological electric field clear if those reactions are mediated by similar mechanisms. to establish that, we performed proteomic analysis and subsequent investigation of the role of differential signalling pathways in electrotaxis of cells representing various strategies of movement. cells were exposed to ef in galvanotaxis apparatus and their reaction was recorded. in some experiments cells were pre-incubated with erk / or btk- inhibitors. the phosphorylation of erk / and btk- was determined by western blot analysis. proteomic analysis was performed by ultimate rs lc nanosystem coupled with a q-exactive mass spectrometer. both blebbing (bc) and lamellipodial (lc) cells show cathodal migration in a physiological ef ( v/cm). comparative analysis of bc and lc cells proteomes revealed about differential proteins. functional analysis in ingenuity analysis pathway allowed to determine the statistically significant signalling pathways in which these proteins are engaged. among the most distinctively regulated pathways are tec kinase and erk/ mapk signalling activated in lc but not bc. it was found that btk- is required for directional movement of lc but not for bc cells. moreover, ef induced stronger and faster btk- phosphorylation in lc than bc cells. in contrast erk / activity was not necessary for electrotaxis of lc cells and ef did not induce erk / phosphorylation. our results reveal that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of wc cells but it is mediated by different signalling pathways. this work was supported by a grant from the national science centre / /b/nz / , poland. newborn screening for congenital hypothyroidism in turkey: a regional evaluation € o. demirelce , n. y. saral , f. b. aksungar , , a. coskun , , m. serteser , , i. unsal , acibadem labmed, istanbul, turkey, acibadem university, istanbul, turkey congenital hypothroidism (ch) is the most common congenital endocrine disorder and the most important cause of preventable mental retardation. it is important to begin the treatment within weeks before the development of brain damage. tsh based newborn screening programs are shown to be useful for implementing early treatment of ch. in this study, regional results of ch screening program in turkey between and were assessed retrospectively. we have evaluated the results of marmara, central anatolia, aegean and mediterranean regions in which our laboratories are located. screening was based on tsh determination in dried blood spot specimens. tsh limits determined to be lu/ml for cut off point and lu/ml for clinical decision point. tsh was measured using enzyme immune assay (eia). blood spot tsh data for newborns during this time period were evaluated. permanent or transient ch was determined according to the results of thyroid function tests. confirmed ch cases were based on local endocrinologists' report and initiation of thyroxine treatment. the frequency of neonatal tsh levels were found to be under the cut off level of lu/ml in ( . %), between and lu/ml in ( . %) and above the level of lu/ml in ( . %) babies, respectively. recall rate was . %. ch cases of neonatal tsh levels greater than lu/ml were . the incidence of ch of this group was : . there were no significant differences in the number of congenital hypothyroidism between males and females (p > . ). the preliminary results of our study indicate that the incidence of ch in our region is higher than the worldwide reports as has been proved by preceding studies. iodine deficiency, dyshormonogenesis, highly consanguineous population, may contribute to the high incidence of ch in turkey. newborn screening of ch must be developed for detecting true cases and tsh cut off point must be reviewed for decreasing redundant recall rate. in silico analysis of the first complete genome sequence of lactobacillus acidipiscis species k. papadimitriou , m. kazou , v. alexandraki , b. pot , e. tsakalidou agricultural university of athens, athens, greece, institut pasteur de lille, lille, france introduction: lactic acid bacteria (lab) constitute a significant group of microorganisms for the food industry, as they play a key role in food fermentation and consequently in human health. lactobacillus acidipiscis aca-dc is a gram-positive, motile, rod-shaped lab isolated from traditional greek kopanisti cheese. here we present the in silico analysis of the first complete genome sequence of l. acidipiscis in order to explore the biology of the species. materials and methods: sequencing of l. acidipiscis genome was performed using the hiseq and pacbio rsii sequencing platform technologies and the genome assembly was validated against an nhei optical map of the l. acidipiscis genome. protein-coding sequences were predicted by glimmer, rrna genes by rnammer and trna genes by the trnascan-se server. potential genomic islands were detected using the island-viewer software tool, prophage regions by phast and the subsystem-based annotation by rast server. finally, the circular representation of l. acidipiscis genome keyed to the cog groups was constructed by cgview server. results: the sequencing analysis resulted in one continuous genomic scaffold of , , bp with a g+c content of . %. the genome contains , protein-coding genes on the chromosome covering up to . % of the genome sequence, trna and rrna. according to the subsystem-based annotation, , protein-coding genes were assigned to metabolic subsystems. the most abundant of the subsystems are related to carbohydrates (n = , . % of total protein-coding genes) and protein metabolism (n = , . % of total protein-coding genes). furthermore, three prophage regions were detected; one intact ( . kb), one incomplete ( . kb) and one questionable ( . kb). discussion and conclusion: the whole genome analysis of l. acidipiscis aca-dc provided interesting information about a not well-studied species. investigation of serum irisin levels of patients with metformin taking new onset type diabetes mellitus increases glucose tolerance and energy expenditure and improves carbohydrate homeostasis. metformin is a biguanidine class antidiabetic drug which inhibits liver gluconeogenesis and decreases insulin resistance and is frequently recommended in treatment of new onset type diabetes mellitus (t dm). irisin has a role in the regulation of energy metabolism pathways and its level in blood of persons with t dm has been reported to decrease. regarding this relationship, it was aimed to reveal the effect of metformin on serum irisin levels. patients with impaired oral glucose tolerance test were included to this investigation. they were recommended to take metformin and to change their life style, such as exercise and diet. their blood were taken at the beginning and after month. also, a healthy control group (n = ) was formed from persons with similar age and sexual distribution as the patient group. irisin levels of their sera were measured by enzyme-linked immunosorbent assay (elisa) method. statistical evaluation of the measurements showed no significant difference (p = . ) between the irisin levels of the patients at the beginning and after month treatment. a similar result was found between the control and the treated groups (p = . ), while a significant difference (p = . ) was observed between the control and untreated patients groups. the results obtained from this study do not show a clear and significant change in the blood irisin levels of the patients with new onset t dm taking metformin together with life style change. a longer period of treatment and a higher number of patients may be needed for more reliable results. thermodynamics of dna ligands binding at specific sites of telomeric g-quadruplex dna g-quadruplexes are a perspective target for anticancer therapy. for example stabilization of the telomeric g-quadruplex dna formed by single-stranded ends of the chromosomes leads to inhibition of telomerase, which is active in % of cancer cells. similarly, small molecules targeted to a specific g-quadruplex would inhibit various cellular processes. stoichiometry and affinity of interaction of these compounds to dna is determined by specific structural motifs within a g-quadruplex. rational design of novel chemical compounds requires an in depth knowledge of interactions between known ligands and g-quadruplex structures. experimental methods that are used for determination of thermodynamic binding parameters, such as isothermal titration calorimetry, differential scanning calorimetry, ultraviolet absorption and circular dichroism spectroscopy provide a collective characteristic for all of the ligand molecules bound to dna, while the information on ligand affinity to individual dna binding sites is lost. we propose a complimentary method for detailed analysis of thermodynamic parameters of ligand binding based on the introduction of fluorescent probes in the structure of g-quadruplex. monitoring fluorescence quenching of the fluorescent labels allows to derive binding constants of the dna-ligand interaction at a specific binding site. temperature dependence of the fluorescence quenching determines the thermodynamics of the dnaligand complex formation. since only a proximal ligand is able to quench the fluorescence, this method allows characterization of the ligand binding to a particular site the g-quadruplex structure. the study was supported by project no. - - of the russian science foundation. the correlation between biochemical and dynamic surface tension parameters of calves blood serum during the animal ontogenesis, as well as by various pathologies or poor diet, the imbalance of protein, mineral, lipid components is observed (the changes in all parameters of biological liquids are accompanied of these metabolism peculiarities). the dynamic surface tension (dst) of serum essentially depends on these factors and (in combination with the biochemical parameters) can provide the valuable information for evaluation of the physiological and biochemical status of the organism (can be used as an express test for animal diagnostics in future). the aim of the work was to study dst and biochemical parameters of calve serum, as well as their correlations, as the main indicators of the animals. ) of calve serum were in the range of the normal values for healthy animals and can be considered as reference data for animal science and practice. the obtained results enable us to establish correlations between the dst and biochemical parameters of calves serum. this work was supported by the russian scientific foundation (grant - - ). the middle strong correlations of dst values of calves serum with the level of total protein, albumin, billirubin, some enzymes and cholesterol, whereas only weak correlations with the other biochemical parameters (urea, calcium, magnesium, phosphorus, etc.) were found. in the veterinary science and practice such correlations are important for the estimation of the organism physiologicaland biochemical status, for general inspections of cattle before vaccination (immunization) or slaughter, for "quick separation" of healthy and ill animals in the case of infection, etc. role of protein kinase c in the regulation of astrocytic glutamine transporter sn in ammonia-exposed mouse cortical astrocytes (bisi; lm). total pkc activity was analyzed by a direct pkc assay and phosphoserine detection by western blot (wb) analysis. protein level of sn and sn , second astrocytic gln transporter belonging to system n, in a membrane fraction was also analyzed. the total uptake and system n-mediated (l-ala and l-leu-inhibitable) gln uptake was tested. treatment of astrocytes with ammonia resulted in a decrease of pkc activity, whereas pma treatment increased pkc activity in ammonia-independent way. bisi treatment reversed fully, and ammonia partially, the pma-induced pkc activity. pma treatment resulted in only a slight decrease in sn protein level in both control and ammonia-treated astrocytes, while a decrease of total and system n-mediated gln uptake were noted in control astrocytes, an effect not exacerbated by ammonia. in turn, cotreatment with pma and bisi reversed the decrease of total gln uptake and showed tendency towards increase in system nmediated gln transport. the results suggest that: a) ammonia changes the dominating direction of system n transport from release to uptake, which may be related to decreased phosphorylation or to alterations in relative phosphorylation by different pkc isoforms. this inference remains to be verified in further studies; b) changes in system n transporter function induced by ammonia appear to involve mechanisms other than changes in transporter expression. evidence for human ghrelin ghs-r a and orexin ox heteroreceptor complex formation in a heterologous system ghrelin and orexin are two peptides implicated in the regulation of energy balance and modulation of food-related motivation at the level of the midbrain dopamine reward system. their function in the hypothalamic arcuate nucleus and the ventral tegmental area (vta) has already been described, but the modulation at the level of receptors remains unclear. the action of these peptides is mediated by g-protein-coupled receptors (gpcrs): ghrelin a and b (ghs-r a , ghs-r b ) for ghrelin, and orexin and (ox , ox ) for orexin. traditional approaches to know the mechanism of neurotransmission of dopaminergic neurons in the mesolimbic system have focused on targeting neuronal receptors as single entities. from the discovery that gpcrs for neuromodulators may form heteroreceptor complexes, our hypothesis is that ghrelin and orexin receptors may interact and form novel functional units that may specifically participate in the central regulation of food intake and energy balance. as a proof of concept we have investigated the potential of human ghs-r a and ox receptors to form heterocomplexes. formation of ghs-r a -ox receptor heteromers in transfected hek t cells was detected by bioluminescence resonance energy transfer (bret) and proximity ligation (pla) assays. furthermore, a negative crosstalk was identified in cells co-expressing both receptors by assessing mitogen-activated protein kinase (mapk) and adenylyl cyclase (camp) pathways, and by a label-free dynamic mass redistribution assay. experiments in sources endogenously expressing ghs-r a and ox receptors are needed to know the functional relevance of the heteromer. from the negative crosstalk here identified, it is tempting to speculate that ghs-r a -ox receptor heteromers are important players in mediating the response to the combination of different orexigenic signals. lysosomal storage diseases which are related to deficiency of specific lysosomal hydrolases resulted to clinical aspects due to accumulation of substrates in different tissues. since dried blood spot (dbs) is non-invasive, low-cost, easy transportable, acceptable enzyme stability compared to leucocyte and/or fibroblast culture, it's recommended as a first screening test. however the false positive rate with dbs sample is higher compared to other samples. we aimed to investigate any possible effect of leucocyte number on enzyme activity in dried blood samples in a retrospective study. we re-evaluated the lysosomal enzyme activity results in regard to leucocyte number among data within last year. enzyme activities had measured by using fluorometric and lc msms method. we determined the correlations between the lysosomal enzyme activities of alpha glycosidase, glycocerebrosidase, alpha galactosidase, sphingomyelinase, galactocerebrosidase and alpha-l-iduronidase in healthy population (n = ). while glycocerebrosidase and galactocerebrosidase positively correlated with the number of neutrophils, alpha galactosidase, sphingomyelinase and alpha-l-iduronidase positively correlated with the number of lymphocytes. alpha glycosidase activity showed a correlation both lymphocytes and neutrophils. the patients having the glycocerebrosidase enzyme activity which was lower than . nmol/ml/hour (which is accepted as the cut off value to recall the patients) existed significantly lower number of leukocyte, lymphocyte and neutrophil compared to those of patients having higher enzyme activity than . . our data indicated that the enzyme activity in dried blood samples including low leucocyte number might be found lower than reference intervals resulting in false positive diagnosis. therefore we suggest that the laboratory scientists should evaluate the number of leucocyte levels while they were interpreting data. using dna-markers for estimation of genetical variability of two kazakh sheep breeds a. mussayeva , , b. bekmanov , , a. amirgalieva , k. dosybaev , , z. orazymbetova , , r. zhapbasov , a. zhomartov , n. zumadillaev , n. zumadillaev llp "kazcytogen", almaty, kazakhstan, "institute of general genetics and cytology" sc mes, almaty, kazakhstan, branch "scientific research institute of sheep" llp "kazakh research institute of animal husbandry and feed", almaty, kazakhstan to compare the frequencies of different microsatellite loci in sheep breeds subpopulations genomic structure of edilbay and kazakh archaromerinos was investigated. different methods for homogeneity testing of two breeds were elaborated. inter simple sequence repeats (issr) pcr analysis of the breeds studied displayed species and breed specific fragments with different frequencies (population frequency more than . ) there were found rarely met fragments (frequency lower than . ). the combinations of these fragments present the specific issr-spectra which arrange genofond profiles of breeds. using panels of microsatellits (recommended by isag) breeds ( populations) were characterized. informative value and resolving capacity of the sum of str-loci were estimated. wide polymorphism of alleles length was demonstrated both when the breeds were compared and within the breeds. informative markers were chosen for both two breeds, markers being used for both breeds, while other markers were informative for one of the breed only. when the animals of one breed were compared unique alleles which were met only within one of populations were of much interest. for example the allele of bm was met in birlik population of edilbay breed as often as in % of animals while in two other populations there were no this allele. in kumtekey population one can meet % animals having particular locus (dyms ), while in the other population (cf ablay) this locus was not met at all. basing on genetical distances obtained using fragment analysis phylogenetic relationships between populations were estimated. so for example edilbay population of cf ajar has the larger distance from two other populations (birlik and bayserke-agro) than each of them from one another. two subpopulations of kazakh arkharomerinos breed (cf kumtekei and cf ablay) also have the genetical difference. how preeclampsia affects oxidant status and antiinflammatory potential of breast milk? preeclampsia is a pregnancy syndrome associated with hypertension, proteinuria and edema, leading to maternal morbidity/mortality and preterm delivery. in this study we aimed to investigate if the breast milk of preeclamptic mothers is effected in oxidative status and anti-inflammatory activity in comparison to the breast milk of mothers with healthy pregnancies. for the aim of the study, hyaluronidase and myeloperoxidase activities (mpo), total oxidant status (tos), total antioxidant status (tas), oxidative stress index (osi) and tbars levels were measured in breast milks of preeclamptic mothers and mothers with healthy pregnancies as control group. when the control group and preeclamptic group were compared, hyaluronidase activity, tas, tos and osi levels showed statistically significant differences in the preeclamptic group. hyaluronidase activity was significantly higher in the preeclamptic mothers' breast milk ( vs u/ml, p = . ). while tos levels were significantly higher in the preeclampsia group ( . vs . lmol/l, p = . ), the tas levels were significantly higher in the control breast milks ( . vs . mmol/l, p = . ). as expected osi levels (tos/tas ratio) were significantly higher in the preeclampsia group. even though the mean levels were higher in preeclamptic group, the difference in mpo activities and tbars levels did not show statistic significance. oxidant status parameters also suggest that preeclampsia effects in both ways by increasing oxidant status and also decreasing antioxidant capacity shifting the balance to the increased oxidant stress side. as the results showed that the preeclampsia group had higher hyaluronidase activity, this can be interpreted as preeclamptic mothers' milk have higher inflammatory potential as this enzyme enhances inflammation by catalyzing the depolymerization of certain acidic glycosaminoglcans. p-mis- investigation of relationship between postprandial lipemia and erythrocyte membrane cholesterol level postprandial lipemia is a metabolic condition related to an increase in plasma triglycerides. remnant-like lipoprotein particles are predominant in postprandial phase and they play an important role in development of atherosclerosis. cholesterol is a prominent component of erythrocyte membranes and regulates the membrane functions such as viscosity and permeability. free cholesterol derived from erythrocytes is thought to participate in the atherosclerotic plaque formation. in the current study, it was aimed to investigate the relationship between postprandial lipemia and erythrocyte membrane cholesterol level in healthy subjects. study group included subjects ( female and male with age range of - years). then these individuals were divided into three groups according to the values of area under curve (auc) calculated by using triglyceride levels at the fasting state and at nd, th and th hours after the high fat diet (ottt). lipid and erythrocyte membrane cholesterol (emc) values were compared between groups with low and high ottt response. while tc, tg, ldl-c and emc were significantly higher, hdl-c was significantly lower in high ottt response group than low ottt response group. it was not observed any statistically significant difference when compared emc values between women and men study groups. on the other part, it was seen positive correlation between emc and auc (r = . , p = . ), tg (r = . , p = . ), tc (r = . , p = . ), ldl-c (r = . , p = . ) in the total study group. it was concluded that, postprandial lipemia may show atherosclerotic tendency not only with atherogenic lipid profile but also with increasing emc. p-mis- eu-openscreen: the european infrastructure for chemical biology b. stechmann, p. gribbon eu-openscreen, fmp leibniz institute for molecular pharmacology, berlin, germany small molecules that can be applied as chemical 'tool' compounds (or 'probes') have become indispensable in basic research for the elucidation of fundamental biological mechanisms. they act directly with the protein-of-interest and often allow for the interrogation of biological processes that cannot be properly studied with traditional genetic or rna interference approaches. eu-openscreen (www.eu-openscreen.eu) is the largest emerging academic chemical biology research infrastructure initiative in europe and will provide access for molecular and cell biologists to screening infrastructure, well-characterized highquality chemical libraries, and facilities for medicinal chemistry services for compound optimization. molecular biologists who have a robust and suitable biological assay and are interested in collaboratively developing chemical tool compounds to validate their targets-of-interest are welcome to work with eu-openscreen. selected assays are screened against a collection of more than , compounds, incl. confirmatory and counter screening, ic/ec determination, sar (structure-activity relationships) and qc of confirmed hit compounds. eu-openscreen will start operations in , but it can already look back on a growing number of transnational activities: joint screening projects, exchange of local compound libraries, development of new design principles for its compound collection; exchange of experimental data through its pilot database etc. steps towards an arthrobacter nicotinovorans based biotechnology for production of hidroxy-nicotine as the archetypal agonist of nachr, nicotine stands up as a powerful scaffold for developing new alzheimer disease therapeutic agents in form of nicotine derivatives. in this context, arthrobacter nicotinovorans pao and its wide range of nicotinederivatives produced when grown on nicotine have a huge biotechnological potential. indeed, the metabolic intermediate -hydroxy-nicotine ( hnic) produced by a. nicotinovorans pao was shown to bind to the nachrs, and by modulating their function, to sustain spatial memory formation in a rat model of ad. the current work presents the first attempts to produce and isolate hnic by using a genetically engineered a. nicotinovorans strain. the growth and the hnic accumulation were compared for two strains: . a. nicotinovorans pao wild type strain and . a genetically engineered a. nicotinovorans pao strain (part ndh) containing the nicotine-dehydrogenase (ndh) genes cloned in the nicotine inducible part vector. the growth curves were followed spectrophotometrically. the consumption of nicotine and accumulation of hnic were monitored by hplc using a mn nucleodur - c ec column and . m sulfuric acid at a flow rate of ml/minute. the growth curve of the part ndh strain shows that the bacteria grow slower when compared with the wt. as a result, in the wt strain, the nicotine is quickly depleted from the medium and only low amounts of hnic are observed. although the sds-page analysis of the total protein extracts from the part ndh strain did not show clear signs of ndh overexpression, the enzyme is produced and is active, allowing a fold accumulation of hnic in the growth medium. the first attempts to purify ndh from the part ndh strain using imac were nevertheless unsuccessful. in conclusion, using the part ndh strain for hnic production is feasible. further improvements of the growth condition and strain are envisioned (i.e. knocking the ndh downstream genes; adding inhibitors for the downstream enzymes). studies on the impact of butyrylcholinesterase (bche) on the symptoms and progression of cognitive impairments like alzheimer's disease (ad) or other neurodegenerative disruptions speak in favour of selective bche inhibitors as a new approach in future ad pharmacotherapy. some derivatives of quinine and quinidine, present in the cinchona species bark, have already been identified as selective bche inhibitors with respect to acetylcholinesterase (ache); therefore, further investigation of these compounds might result in promising leads for enhanced anti-ad drugs. we synthesised ten quaternary derivatives of cinchonines and their corresponding pseudo-enantiomeric cinchonidines. quaternization of quinuclidine moiety was carried out with groups diverse in size: methyl and differently meta and para substituted benzyl groups. all of the compounds were prepared in good yields, characterized by standard analytical spectroscopy methods, and were tested for their bche and ache inhibition potency. the inhibition potency of the compounds was defined by the dissociation constants of the enzymeÀinhibitor complex (ki). all of the tested compounds reversibly inhibited both human bche and ache. the compounds inhibited bche with ki constants in the range of . - lm, and ache in the range . - lm. five cinchonidines displayed a - times higher inhibition selectivity to bche over ache, and four of them were potent bche inhibitors with ki constants up to nm. bche affinity toward the studied compounds depended on the size of the substituent on the nitrogen of the quinuclidinium part of the molecule and on the resonance stabilization of the substituent at the quaternized nitrogen. based on the presented results, cinchonidine cd-(pbr) can be pointed out as a potent and selective bche inhibitor that could be considered for further research in alzheimer disease pharmacotherapy. exposure to nmda ( lm) for h increased the expression of kir . mrna and decreased that aqp -and gs mrna. the expression of kir . was decreased by h exposure to glu ( mm) and tnfa ( ng/ml). at h incubation, nmda induced a decrease of kir . expression in the presence but not in the absence of calcium in the medium. nmda did not alter the expression of nmda receptor subunits. tnfa increased the expression of the nr subunit, and decreased that of nr b mrna. glu decreased the expression of out of subunits. the study demonstrates, to our knowledge for the first time, that prolonged exposure of astrocytes to nmda alters the expression of mrna coding for critical astrocytic proteins. the dependence of the decrease of kir . mrna expression on extracellular calcium suggests the ionotropic nature of nmda receptor stimulation. the effects of nmda receptor stimulation occurred by a mechanism bypassing changes in subunit composition of the nmda receptor. experiments are under way to establish whether the tnfa-induced changes in the expression of nmda receptor subunits contribute to modulation of nmda receptor stimulation by inflammation. the importance of education in reducing preanalytical errors the preanalytical phase includes the request of test, the preparation of patient, the obtaining of sample from the patient, the transport of the sample to the laboratory, and the pretreatment of sample. the preparation of patient and the obtaining of sample are considered as the most common error sources. in order to reduce preanalytical errors, we aimed to provide training for phlebotomists and to also determine their knowledge level about the preanalytical phase before and after these training. it was given the training related with preanalytic phases to pediatric nurses and adult nurses, other phlebotomists in march. the surveys which are made before and after the training were consisted of questions that are related with demographyic features and preanalytic phases. in order to determine the effects of training to the preanalytic phase, the preanalytic error rates before (in february) and after (in april) traning was calculated with the formula of: (the number of rejected samples/the number of total samples)x . the average age of participants was ae years. it was not found significant difference between their correct answers rate before the training and the education degree of the participants. the correct answer rate before the training was % and after the training it was %, which showed an increase of %. the preanalytic error rates which were . % in february were decreased to . % in april. in our study, the positive results were obtained through the training aimed to reduce the preanalytical errors. by providing regular training to the phlebotomists and also providing pretraining to the beginners, the updating of their information about preanalytic phase can be achieved. in this way, the loss of labor and economic related to preanalytical errors can be avoided and the accurate results can be obtained in short time. curcumin, the active compound of turmeric (curcuma longa) has antiinflammatory, antioxidative and antitumour effects. unfortunately, curcumin has a poor absorption and low stability. both can be solved by encapsulation of curcumin using a proper technique like electrospray. it was reported that piperin, the active compound of black pepper, enhances the intestinal absorption of curcumin and thus its bioavailability. due to these facts it was aimed in this study to nanoencapsulate turmeric extract in order to enhance its absorption and stability. for that purpose, it was encapsulated with the maize protein zein, chitosane and black pepper extract by varying the voltage and flow rate of electrospray and the concentration of the compounds. the nanocapsules were characterised by measuring their particle size and with help of sem photographs. the particle size of the final nanocapsule formulation was nm and had a sufficient stability over a period of months, visually determined. by encapsulating turmeric extract into double layer nanocapsules with help of black pepper extract, zein and chitosane, the turmeric extract could be protected from degradation, which was observed for the pure turmeric extract in form of clearing its yellow colour. analysis of the human genome reveals that potential g-quadruplex sequences are enriched in promoters of the oncogenes. growing body of evidence suggests that g-quadruplexes (g ) may play putative roles in various biological processes, such as the regulation of gene expression. consequently, targeting the oncogenic g-quadruplexes using small molecules is an alternative strategy for the potential treatment of cancers. porphyrin derivatives are promising class of drug in this respect, being nucleic acids binders and generators of reactive oxygen species under visual light irradiation. interaction between porphyrin derivatives and g dna from oncogene promoter region has been studied in vitro. we applied chemical probing, circular dichroism spectroscopy and uv melting techniques in order to map the oxidized bases, monitor structural rearrangements and evaluate stability of the resulting dna structures. specifically, we observed that g dna is considerably more susceptible to lightinduced modification than duplex dna; -terminal tetrads of the g dna are preferably oxidized; structural changes induced by oxidation result in decrease of the thermodynamic stability of the g dna. irreversibility of these effects on dna make porphyrin derivatives perspective lead compounds for rational design of ligands targeting human oncogenes. the study was financially supported by project no. - - from the russian science foundation. resistin levels in denervated obese rats n. saglam , t. ahmedi rendi , c. kahraman , a. alver department of medical biochemistry, faculty of medicine, karadeniz technical university, trabzon, turkey, school of health, d€ uzce university, d€ uzce, turkey the sympathetic nervous system is an important factor affecting the metabolic and secretory function of the white adipose tissue. resistin is mainly expressed by mononuclear cells, also it is expressed by adipocytes, pancreatic cells, and muscle. resistin induces insulin resistance and glucose intolerance in mice. resistin plasma levels depend on fat depots size and sex. resistin levels decrease in short-term fasting in mice, then it increase refeeding. also, it increase as a response to fed with the high fat diet. in our study we aimed to determination of the effect of high-fat diet and denervation on serum resistin levels in rats. in this study experimental groups were formed each consisted of rats. during weeks, first two groups are fed with high-fat diet and other two groups are fed with standart diet which they purchased from research diets company. at the beginning of the feeding periods, retroperitoneal fat tissiues of animals assigned to the first and the third groups were denervated. second and fourth groups were not denervated. at the end of the week feeding periods, blood collected from rats and blood resistin concentration was determinated by elisa. in denervated and fed with high fat diet groups serum resistin levels higher than control groups (p < . ). according to our literature research, there are no studies demonstrating the relationship between resistin and the sympathetic nervous system. also, denervation may lead to increase in serum resistin levels. the amount of resistin is possibly reduced by b -adrenergic activation. in conclusion, it was concluded that there is differences on serum resistin levels depending on diet in bilateral denervation of retroperitoneal fat tissues of rats. stress activated protein kinases regulates the ribosomal frameshift rate in est gene, encoding subunit of telomerase s. t€ urkel, s. sarica uludag university, bursa, turkey est gene (ever shorter telomere) of s. cerevisiae encodes one of the essential subunits of telomerase enzyme. expression of est gene is regulated at the translation level by + programmed ribosomal frameshift (prf). it is known that the physiological stresses affect telomere length. in this study, we have investigated the effects of stress activated protein kinases snf p (ampk) and gcn p (eif kinase) on the prf rate in est gene. prf rate of est gene was quantified in plasmid based expression system. expression vectors were transformed in to the wild type and mutant yeast strains that deleted for snf , or gcn genes. yeast cells were grown in normal conditions or subjected to acid stress, osmotic stress, or glucose limitations to activate protein kinases gcn p, and snf p, respectively. prf rate of est gene was measured as % in the normal growth conditions in the wild type cells. but, the prf rate of the wild type strain grown in glucose limited conditions decreased more than -fold, giving less than % prf rate. contrary to glucose limitation, osmotic or acid stress activated frameshift rate by -fold in the wild type cells and prf rate increased to %. when the prf rate was analyzed in gcn and snf mutants, frame shift rate of est was - % in normal growth conditions. when these mutants were subjected to acidic or osmotic stress, prf rate activated slightly. we have also shown that gcn p and gcn p, positive regulator of gcn p, is also essential for the regulation of prf in est in response to stress conditions. it is clear that the basal level expression of est is highly dependent on the gcn p kinase complex. gcn p is also associates with ribosomes, indicating that gcn p might have a significant function in connecting the stress signals to biosynthesis of the full length est peptide. this regulation might also link the biosynthesis of functional telomerase and telomere replications to cell physiology through protein kinases such as snf p and gcn p. inflammation might have a role in erosive esophagitis but not in non-erosive reflux disease the relationship between inflammatory activation mechanisms and acid-peptic injured esophageal tissue is not clear. we evaluated whether there are differences between inflammation and tight junctional proteins such as e-cadherine among subtypes of gastroesophageal reflux disease. the aim of this study was to investigate any possible role of inflammation in pathologic mechanism of reflux disease by determining the inflammatory markers in injured esophageal tissue as well as serum of patients. three groups (erosive-ee, n = ; nonerosive-nerd, n = ; healthy controls-hc, n = ) were evaluated with upper gastrointestinal endoscopy. the esophageal biopsies and blood samples were collected. serum e-cadherine levels, nfkb, chitotirosidase (chit), myeloperoxidase (mpo) activities in serum and homogenized tissues were determined. nkfb levels in tissue was significantly higher in subjects with ee ( . ae . ng/mg.prt) versus hc ( . ae . ng/mg.prt, p = . ). mpo tissue activities in ee group were significantly lower ( . ae . u/mg.prt) than hc ( . ae . u/mg.prt, p = . ) while mpo serum levels were higher in ee ( . ae . ul) versus hc ( . ae . ul, p = . ). tissue chit levels were three fold increased in ee versus hc (p = . ). none of these measurements showed any differences in nerd group. nfkb and mpo levels had a negative correlation (r=À . , p = . ) in tissue. nfkb and ecad levels had a positive correlation in serum (r = . , p < . ). inflammatory process might play a pivotal role in injured mechanism only in erosive esophagitis but not in nerd. noninflammatory mechanisms might be responsible such as hypersensitivity in patients with non-erosive reflux disease. d-dimer (a fibrin degradation product) test is used to aid in the diagnosis of intravascular coagulation. the aim of this study is to investigate the correlation between d-dimer levels and other inflammatory markers including procalcitonin. anonymized data on d-dimer, fibrinogen, hscrp, wbc, neutrophil% (neut%) and procalcitonin levels from , patients (mean age ae sd, . ae . ) were used for the correlation (excel analyze-it v . . ) and linear regression (pasw statistics v . ) analysis between the measured parameters. there was a significant (p < . ) age-dependent increase in d-dimer levels between different age groups. patients with the highest d-dimer levels were also found to have an increased frequency of hscrp levels. d-dimer levels showed a significant correlation with hscrp, wbc and neut%. a model describing the positive association between these parameters were built. the resulting equation is as follows: d-dimer = (hscrp* . ) + ( . *age) + ( . *wbc) + ( . *neut%)À . . correlation analysis between procalcitonin and d-dimer levels gave pearson's correlation coefficient of . . our results suggest that the age-dependent variations should be taken into account while interpreting d-dimer test results. in addition, neut% ratio was found to be the most important parameter for estimating d-dimer levels. our equation can be used when the d-dimer test is not available or for control purposes only. in the field of cancer research great hope lies in finding more powerful and selective way for the direct elimination of cancer cells. this task can be solved by means of nanobiotechnology. recent progress in this field has arisen interest in a carbon nanostructurefullerene c . fullerene exhibits not only unique physico-chemical properties and biological activity but also a significant potential to serve as a nanocarrier for selective drug delivery into cancer cells. the aim of this study is to analyze a unique tool for cancer therapy. the main idea is realized by the non-covalent conjugation of c with the well-known anticancer drug -doxorubicin (dox). two types of conjugate with different c -dox ratio ( : and : ) were studied. conjugates absorbance and fluorescence, size distribution as well as a mass data were recorded utilizing optical and analytical equipment (microplate reader, zetasizer, lc-ms/ms and maldi-tof). in vitro studies were performed including evaluation of c -dox conjugate effects on human leukemic cells (jurkat, ccrf-cem, thp ad molt- ) viability. conjugates accumulation and distribution within cancer cells was monitored using fluorescent microscopy accompanied with fluorescence-activated cell sorting. it was evidently proven that both c -dox conjugates were stable and could be used as reliable candidates for biological application. cellular accumulation and distribution studies showed that conjugation of dox with fullerene promoted its entry into leukemic cells. accumulation of dox in the form of conjugates within cancer cells was intensified compared to the free drug. the results show that conjugated dox is more cytotoxic and the value of its ic are lower compared with the free dox. obtained results confirm nanocarrier function of fullerene c and the perspective of its application for optimization of doxorubicin efficiency against leukemic cells. comperative investigation of protective effects of tea and tea-related wastes on reducing potaential of h o -induced erythrocytes tea processing waste (tpw) formed during the tea production process in tea factories is up to , tones/year in turkey. tpw is one of the abundant available phenolic biomass among plantal wastes. in this study, black and green teas and their wastes were used. the aim of the study is to determinate the phenolic content and the radical scavenging activities of the samples, and to measure their effects on hydrogen peroxide-induced erythrocyte damage due to analyzing the reducing potential of erythrocyte involving glutathione reductase (gr), glutathione peroxidase (gpx) activities and reduced glutathione (gsh) content. total polyphenol content of samples was determined as mg catechine per dry mass by using folin-ciocalteau reactive and dpph radical scavenging activity was estimated by cuendet method as equivalent catechine standard. in erythrocyte, gsh level was measured by method of sedlak and lindsay while gr and gpx activities were assayed by the methods of bergmeyer and beutler, respectively. the highest phenolic content was observed in green tea and its wastes (p < . ) whereas black fiber waste had the lowest phenolic content. therefore, the highest radical scavenging activity and gsh level were detected in green tea and its wastes (p < . ). erythrocyte with the extracts of the teas and their wastes had the similar enzyme activities for both gpx and gr. in sum, the teas and wastes have antioxidant activity but, green tea and its leaf waste hade higher antioxidant activity than other samples. the tea wastes might be evaluated as many of protective health products, particularly in cosmetic fields thus, these by-products no application for any area is expected to become an economical value. fluorouracil ( -fu) is a chemotherapeutic drug classified as an "antimetabolite". it works through irreversible inhibition of thymidylate synthase. chemical derivatization of -fu with carbohydrtates is being investigated widely in order to enhance its bioavailability, therapeutic efficiency and to reduce its toxicity. however, water solubility of the newly derived compounds is usually very low. so, in order to obtain a pharmaceutically relevant formulation they need to be formulated appropriately. in this study, we prepared micellar delivery system for the new tetra-o-acetylglycose derivative of -fu synthesized via "click reaction", namely f -[{ -( ″, ″, ″, ″-tetra-o-acetyl-b-dglycopyronosyl)- h- , , -triazole- -yl}methyl] -fluorouracil. since the water solubility of this compound is very limited, we tested its solubility in several pharmaceutically relevant solvents by visual estimation after stiring increasing amount of the compound in ml of solvent for h. to estimate the carcinogenic potential of this compound, salmonella/microsome mutagenicity assay (ames test) was performed in four histidine-requiring strains of s. typhimurium, tester strains ta , ta (for the detection of frameshift mutations) ta and ta (for detection of base pair substitutions) according to the oecd guideline . the drug was solubilized ( lg/ml) with no precipitation in lutrol Ò -f /ethanol/water ( . : . : . , wt/wt) micelles ( . ae . nm). the results of ames test were negative so the compound neither produced frame shift mutations nor base pair mutations in s. typhimurium strains. the results imply that the new compound can be dissolved in aqueous micellar delivery system in order to be used for further studies, and that it was not mutagenic in the tested s. typhimurium strains. in conclusion, the formulation of the newly synthesized compound is not carcinogenic, and can be evaluated for anticancer activity in vitro and in vivo. integral metabolism parameters of dairy goats during reproductive cycle periods d. solovyeva, e. zarudnaya, s. zaitsev moscow savmb, moscow, russia study of the goat metabolism at different periods of the reproductive cycle allows to correct feeding ration, to increase the age of the productive use of animals and to receive high-quality products. the aim of the work was to determine the metabolic parameters of blood serum of goats, expressed in terms of biochemical parameters and interfacial tensiometry and study their relationship to metabolic processes in the body goats depending on the age and the period of the reproductive cycle. the healthy goats were divided into groups. the dynamic surface tension (dst) parameters were obtained from dependences of a surface tension (r) vs. time (t): at t? (r ), at t = . s (r ), t = s (r ) and t?∞ (r ). this work was supported by the russian scientific foundation ( - - ). all animals had - % fat content. the contents of total protein ( . %), albumin ( . %) and urea ( . %) are higher for the lactating animals as compared to the normal goat values. the levels of total cholesterol ( . %) and creatinine ( . %) are higher for the lactating animals. in lactating animals have the highest level of, which along with high phosphorus level talks about the intensification of energy processes during lactation. the correlations were found between the biochemical and dst parameters of the goat blood: lipids or cholesterol levels with r (r = À . ), r (r = À . ), r (r = À . ); total protein or albumin levels with r (r = À . ), r (r = À . ), r (r = À . ); aminotransferase activity with r (r = À . ), r (r = À . ). the correlations were found between the total protein and albumin levels with k (r = . ), k (r = . ); glucose levels and r (r = . ), r (r = . ). thus, the dst and biochemical parameters of goats have strong correlation relationships that are important for biomedical and veterinary applications. the relation of the severity of atherosclerotic disease with oxidative stress in patients with stable coronary artery disease h. sezen harran university, sanliurfa, turkey introduction: because, to the best of our knowledge, the relationship of total oxidant status (tos) and total antioxidant status (tas) with the severity of stable coronary artery disease (cad) has not been investigated in the literature so far, the present study was conducted to address this issue. materials and methods: this study consisted of consecutive patients and controls who underwent coronary angiography. for each patient, the total gensiniscore (gs) was calculated andthose with a gs of > were classified as the high gs group (hgg), and those with a gs less than were defined as the low gs group (lgg). the total oxidant status (tos) and total antioxidant status (tas) levels were measured using the erelmethod. the osi, which is an indicator of the oxidative balance, was calculated as the percentage ratio of tos to tas. results: the tas was lower in the hgg than lgg. the tos and osi were higher in the hgg than lgg. the correlation analysis showed that gs was negatively associated with the tas and positively with the tos and the osi. the multivariate analysis showed that age, tos, and hdl-c were independent variables for a high gs. the cut-off level of . lmol h o equiv./ l for serum tos levels predicted high gs with a sensitivity of % and a specificity of %. discussion: information on the severity of atherosclerosis is requiredtopredicttheprognosis of an individualpatientandtodetermine the proper treatment modality. the gs system has beenproventodemonstratethe severity of atherosclerotic disease. inthepresentstudy, thepatientswith a high gs had increasedlevels of oxidants. inaddition, tos was an independentindicator of theseverity of atherosclerosis. the optimal cut-offvaluefor tos topredict high-gens score was . (sensitivity % and specificity %). conclusions: the results suggest that the severity of atherosclerosis in stable cad is associated with increased oxidative status. evaluation of roemerine as a multidrug resistance pump inhibitor f. g. avci , c. velioglu , e. recber , c. unsal , g. gulsoy , b. sariyar akbulut marmara university, istanbul, turkey, istanbul university, istanbul, turkey efflux by multidrug resistance (mdr) pumps is a common defense mechanism used against antimicrobials. by pumping the drugs out, these pumps significantly reduce the efficacy of drugs. one approach to overcome this limitation is offered by the combinatorial therapies where drugs are co-administered with together with pump inhibitors. by simply preventing the efflux of the drug, the presence of inhibitors enhance drug efficacy. (-)-roemerine is an aporphine type alkaloid with significant antibacterial (against bacillus cereus, escherichia coli) and antifungal (against candida strains) activities. interestingly, (-)-roemerine was also found to enhance the cytotoxic response mediated by vinblastine in multidrug-resistant kb-v cells. in the same study, this finding was linked to its possible interaction with p-glycoprotein, a eukaryotic mdr pump. taking this finding as the starting point, the current study investigates the potential of roemerine as an inhibitor of the p-glycoprotein homologue pump, bmra, in bacillus subtilis . the antimicrobial agent berberine was used as the model agent since its efficacy is reduced by efflux through mdr pumps. to this end, bacillus subtilis cells were subjected to lg/ml berberine, a value well below the mic. this concentration only slightly retarded growth for hours but then cells resumed their regular growth. upon addition of lg/ml (-)-roemerine to the bacillus subtilis cells treated with berberine, growth pattern changed, indicating possible interaction with bmra. further investigation for the change in the expression of bmra was achieved with real time pcr analysis. glucose oxidase is an enzyme that catalyzes the oxidation of glucose to d-glucono- , -lactone and hydrogen peroxide. we hypothesized that enzyme would cause a double negative effect on cancer cells, by reducing the presence of glucose in cancer microenvironment and producing reactive oxygen species. to increase enzyme stability and enhance cellular uptake we encapsulated the enzyme with a thin acrylamide layer. the purpose of this work was to optimize the synthesis of these glucose oxidase nanoparticles and investigate their effect on cancer cells. nanoparticles containing single glucose oxidase were synthesized in two steps; first by introducing the vinyl groups onto the surface of enzyme by acyloylation followed by polymerization step with acrylamide monomers. encapsulated enzymes are approximately nm in size and retain most of their activity. after the optimization of nanoparticles, the anticancer potency of these nanoparticles was in vitro tested in mcf- breast cancer cell line. according to results, both nanoparticles and free enzyme are capable of inhibiting viability of cancer cells in a similar manner at very low concentrations. currently we are investigating mechanisms involved in this viability inhibition. initial results demonstrated that glucose supplement does not rescue cells from death induced by the activity of glucose oxidase, suggesting an oxidative stress related cause of inhibition. further studies are required to elucidate the exact mechanism. until now there is no determined advantage of glucose oxidase encapsulation against proteolysis. however, encapsulation may induce the accumulation of enzyme in cancer microenvironment. furthermore results suggest that glucose oxidase has a high effect on the viability of mcf- breast cancer cells indicating that this enzyme may have a potential use in cancer treatment. studies on the interaction of human phospholipid scramblase with c-terminal domain of topoisomerase iia u. sivagnanam, s. n. gummadi applied and industrial microbiology lab, bhupat and jyoti mehta school of biosciences, indian institute of technology madras, chennai, india human phospholipid scramblase (hplscr ) is a multifunctional protein that plays key roles in several cellular processes including apoptosis, tumorigenesis, anti-viral defense, cell signalling and several protein-protein interactions. it has been shown that hplscr interacts with the c-terminal domain of topoisomerase iia (topo iia) and enhances its decatenation activity in vitro. the interacting region in topo iia was identified but till date, no reports exist on the binding region in hplscr . this study aims to identify the region of hplscr that interacts with topo iia. to identify the topo iia interacting sites in hplscr , nterminal deletion constructs of hplscr viz Δ -hplscr , Δ -hplscr , Δ -hplscr , Δ -hplscr and Δ -hplscr were generated by pcr, cloned, overexpressed and purified to homogeneity using ni + -nta purification. the cterminal domain (ctd) of topo iia was cloned in pgex p- and was expressed as a gst fusion protein. gst pull down assays will be performed with the deletion constructs of hplscr and the gst-ctd-topo iia. the binding region in hplscr will be confirmed by peptide competition assays. our initial results show that the decatenation activity of topo iia was enhanced when the topo ii was pretreated with hplscr . Δ -hplscr did not show any enhancement of the decatenation activity compared to full length hplscr . hence, the binding region could be in the - region of hplscr . further deletions were done in the - region of hplscr as described earlier. gst-pull down assays and decatenation assays will be performed for the deletion constructs to narrow down the region of hplscr that binds to topo iia. we conclude that hplscr interacts with and enhances the activity of topo iia and the - region of hplscr is critical for enhancement of decatenation activity. further work is under progress to identify the exact topo iia binding region of hplscr and the physiological relevance of this interaction in the cell. a. ugur kurtoglu , v. aslan , e. kurtoglu department of biochemistry, antalya education and research hospital, antalya, turkey, department of hematology, antalya education and research hospital, antalya, turkey beta-thalassemia is a common autosomal recessive disorder resulting from over different mutations of the beta-globin genes. our aim was to creat a mutation map of beta thalassemia in province of antalya, turkey. in this study, mutation analysis of a total of beta-thalassemia patients followed up at the thalassemia center of the antalya education and research hospital, antalya, turkey, were included. according to our results, the ivs . is the most frequent mutation type in our province same as other geographical regions of turkey. the most frequent mutations in heterozygous or homozygous patients are ivs . , ivs . , ivs . and ivs . . our results indicate the importance of micromaping and epidemiology studies of thalassemia, which will assist in establishing the national prevention and control program in turkey. keywords: beta-thalassemia, beta-globin gene, mutation p-mis- investigation of the in vitro effects of some antibiotics on the purified beta-glucosidases from the rat liver n. t€ urkmen , h. kara karadeniz technical university medical biochemistry department, trabzon, turkey, balikesir university veterinary faculty biochemistry department, balikesir, turkey beta-glucosidases catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in b-d-glucosides and oligosaccharides.b-glucosidases are widely distributed in the living world.b-glucosidases which in mammals, primarily found in the liver and kidneys;lysosomal b-glucosidase (gba ),non-lysosomal b-glucosidase (gba ), cytosolic b-glucosidase (gba ),intestinal lactase-phloriz the hydrolase (lph). liver tissues of wistar-albino rats were homogenized with homogenizer in the extraction buffer and crude extract was obtained after centrifugation.ammoniumsulfate precipitation range designated crude extract was purified by sepharose b-ltyrosine- -naphthylamine hydrophobic gel.commercially availabled antibiotics were prepared with substrate buffer.it was investigated inhbition effects of cefuroximesodium, ampicillin-sulbactam, amoxicillin trihydrate/potassium clavulanate, cefazolin sodium, gentamicin sulfate and ceftriaxone disodium antibiotics onto gba .inhibition types and k i values of related antibiotics were determined with p-npg substrate.lineweaver-burk plot was used for that purpose. rat liver gba was purified at . -fold with . % yield.gba was illustrated and kda at sds-page.ic value of ampicillin/sulbactam antibiotic for gba was found . mg/ml with competitive type inhibition and other antibiotics didn't inhibit. purfication methods are being used in the literature for the purified b-glucosidase from different sources.purified gba was illustrated and kda at sds-page.about molecular weight of bglucosidases is presented different information in the literat€ ure. this has been reported because of acid beta glucosidases are abnormal migration at the acrylamide or agarose gels.it was investigated inhbition effects of various antibiotics onto purified gba .ampicillin/sulbactam antibiotic inhibited to purfied gba at the competitive type.similiar antibiotics studies have been made in the literature for different enzymes. effect of glutamine on insulin resistance and endoplasmic reticulum stress g. aydogdu , p. b. sermikli , a. abbasi taghidizaj , e. yilmaz ankara university, institute of science, ankara, turkey, ankara university, biotechnology institute, ankara, turkey obesity and diseases are one of the most important public health problems of the world.excess fat storage in adipocytes leads to the release of increased amounts of non-esterified fatty acids, glycerol, hormones, cytokines, which are factors involved in the development of insulin resistance that cause type diabetes. one of the major differences between obese and lean individuals is the amino acid concentration in the circulation. although there are many studies about the amino acid metabolism associated with insulin resistance in obese individuals, the effect of glutamine metabolism in insulin resistance mechanisms are not well understood yet. glutamine can be used as fuel and its levels in tissues and circulation can regulate cell responsiveness to insulin and cellular metabolism. therefore, glutamine is a potentially important factor that might help us better understand insulin resistance and type diabetes. to determine whether glutamine effect on insulin resistance and endoplasmic reticulum stress, t -l cell is treated with different concentration of glutamine and analyzed by western blot for er stress markers. our results indicated that glutamine reduced endoplasmic reticulum stress and related with that attenuated insulin resistance. in case of transport of amino acids, insulin resistance, how it is affected when we have the information about the important tips on energy requirements and metabolism reach insulin resistance and type- diabetes treatment is likely to reveal a possible new targets. how does different lead levels affect tsh, ft , ft , vitamin b and folate? e. ozkan ankara occupational disease hospital, ankara, turkey exposure to heavy metals is increasing with the industrialization of society. one of the most intense exposure to heavy metals is pb on this issue. this study was aimed to determine the relationship between different blood pb levels and serum thyroid hormones (th), vit b , folate. the cases were - years old, male individuals who admitted to our hospital between april -march for periodic inspections because of occupational exposure to pb. the parameters of the cases were retrospectively retrieved. according to their pb levels, exposed workers (n: ) were divided into four subgroups; group (g) : - . lg/dl, g : - . , g : - . , g : ≥ . from these, the number of cases whose th levels were measured (n: ) given respectively; g : , g : , g : , g : cases. also the number of cases whose vit b and folate levels were measured (n: ) given respectively; g : , g : , g : ,g : cases. levels of pb were determined by icp-ms. th, vit b , folate were determined by cmia. between the groups formed according to pb levels, there was no significant difference in terms of average t , tsh and vitamin b (p > . ). on the other hand there was statistically significant difference between t and group , , (p < . ) but there was no difference between group (p > . ). the average folate belongs to the first group was about % higher than the other groups, and found that the difference was statistically significant (p < . ). there are many publications which have various results between pb levels and t ,t , tsh. but this study is important to compare the effect of different levels of pb. up to day there was no publication about the relation between different pb levels and vit b , folate. it was seen that there was no significant clinical relation between different pb levels and thyroid parameters, vit b . but the low levels of folate in the high pb levels groups shows us that we need further studies about this relationship. fluorescent study of in meso crystallization of membrane proteins with the introduction of membrane protein in meso crystallization years ago by landau and rosenbusch, a new era of membrane protein structural research has emerged ( ). since that time this method became associated with a number of major breakthroughs in the field ( ) including exceptional successes in structural studies of microbial rhodopsins and g-protein coupled receptors ( ) . here we used fluorescence microscopy to study in meso crystallization process of bacteriorhodopsin. several observations provide new insights into the in meso crystallization process. the crystallization starts with formation of microcrystals, followed by growth of a dominating crystal at the expense of smaller ones and formation of a depletion zone around it. these observations demonstrate an ostwald ripening mechanism of the in meso crystal growth. the depletion zone formed around the growing crystal is consistent with the previously proposed analogy relating in meso crystallization with the crystallization in a microgravity convection-free environment. this work is supported by rsf - - . ( ) landau, e. m.; rosenbusch, j. p. proc. natl. acad. sci. u. s. a. , , À . ( ) caffrey, m. acta crystallogr., sect. f: struct. biol. commun. , , - . ( ) katritch v., cherezov v., stevens r.c. ( ) . annu rev pharmacol toxicol , - . p-mis- stamp is critical for both ar and mtor signaling in prostate cancer cells x. sheng, y. jin, f. saatcioglu university of oslo, oslo, norway androgen receptor (ar) signaling plays a central role in the initiation and progression of prostate cancer (pca), including when the disease progresses to castration-resistant pca (crpc). the second central signaling pathway in pca, similar to various other cancers, is the pi k-akt-mtor signaling. importantly, these two oncogenic pathways cross-regulate each other in pca cells by reciprocal feedback, thereby maintaining tumor cell survival even when one is suppressed. we have previously identified that the six transmembrane protein of prostate (stamp ) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk mapk signaling. human pca cell lines lncap and vcap were used in the study. colony formation, soft-agar growth, prostatosphere formation assays were performed. for in vivo xenograft experiment, the cells were implanted subcutaneously into the flanks of nude mice. here, we show that stamp knockdown caused defects in colony formation, anchorage-independent growth and prostatosphere formation in lncap and vcap cells both in vitro, as well as tumor formation and growth in vivo. this may be due to the impaired ar and mtor signaling in these cells upon stamp knockdown. interestingly, in the crpc cell line rv , where-stamp knockdown did not affect mtor signaling, there was a remarkable repression of tumor take rate and growth. these results clearly indicate that stamp is essential for both ar and mtor signaling, and is crucial for pca growth in vitro and in vivo. however, the detailed molecular mechanism requires further investigation. taken together, these data unveil a critical role for stamp in coordinating the ar and mtor signaling pathways in pca cells, solidifying the basis for its pro-survival effects in pca, including in advanced disease. quantification of d thin layer chromatograms using d gel analysis software and gel documentation system o. kaynar, m. ileriturk, d. kaynar, s. kurt ataturk university, erzurum, turkey introduction: thin layer chromatography (tlc) is an important chromatographic technique that is widely used as a cost-effective method for rapid-sensitive analysis of compounds in plants, animals, and humans. however, one dimentional ( d) tlc is not sufficient for the separation of complex compounds. therefore, two-dimentional ( d) tlc was developed. the quantitative evaluation of plates are performed with tlc scanners or documentation systems. however, these systems specific for d plates, and cannot be adapted to quantitative evaluations of d plates. in this study, the applicability of the gel documentation systems and d analysis software for the analysis of d tlc plates were examined. material and method: d tlc of lipids: st dimension: methyl acetate/n-propanol/chloroform/methanol/ . % kcl ( / / / / v/v); nd dimension: chloroform/methanol/acetic acid/ water ( / / / v/v); detection: charring. d tlc of aminoacids: st dimension: . % (v/v) formic acid; nd dimension: toluene/glacial acetic acid ( : v/v); detection: uv. phospholipid and aminoacid standards, each include different classes were developed by d tlc. plates visualized with biorad geldoc xr, and band volumes on plates were calculated with biorad pdquest d gel analysis software. for the method validationa) plates containing same lipid classes were developed in the same day, and results were used for the calculation of intra-assay cv;cv% = average of each sample standard deviation/mean of sample b) plates containing same lipid classes were developed in different days, and results were used for the calculation of interassay cv; cv% = standard deviation of each sample average/mean of the plates results: volume of each phospholipid and aminoacid had less than % intra and inter-assay cv. conclusion: gel documentation system with d gel analysis software can be used for the quantitative analysis of the d tl plates both at uv and visible light. the role of na + k + atpase activity in the vasodilatory effect of n-acetylcysteine introduction: spasm occurred at the stage of and after the preparation of arterial grafts used in coronary artery bypass surgery (cabg) is effective on morbidity and mortality in the first hours of postoperative patients. n-acetyl cysteine (nac) that vasodilatory effect is known,may be considered as a suppressor agent for vasospasm developing during cabg. however, for the prevention of complications that may arise during or after cabg mechanism of these vasodilatory effects should be described. this study was aimed to investigate the role of atpase enzyme on the vasodilatory effect of nac. materials and methods: in this study, adult male wistar albino rats were used. rats were separated into four groups as control rats (g ), mm nac (g ), mm nac (g ) and mm nac (g ). a portion of the thoracic aorta isolated from rats was used for the relaxation response recording, and the other portion was used for measurement of nakatpase activity. isolated smooth muscle rings are suspended in the ml organ bath containing krebs solution for relaxation responses. in all groups, level of smooth muscle contraction were allowed to reach a plateau by adding mm kcl to the organ bath. then, in the first minutes of application relaxation responses which created by adding nac to the medium were recorded and the maximum relaxation responses were measured. nak atpase activity was determined using the mazzanti method. groups means were compared by one-way analysis of variance (anova). the threshold for statistical significance was set at . . results: the contraction force decreased in all nac dose groups compared to control group and this reduction was statistically significant (p < . ). similarly, nak atpase activity is also decreased in a dose dependent manner (p < . ). the findings obtained in this study suggest that vasodilator effect of nac formed in thoracic aortic smooth muscle was associated with the activity of the enzyme na k atpase. in the presented study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic activity. a bacillus uk , which was isolated from the soil samples taken from farmland on kahramanmaras sutcu imam university campus, showed high keratinolytic activity when cultured on feather meal medium. the enzyme activity was studied in the ph range of . - . . the optimum temperature for keratinase activity was investigated by varying the incubation temperature between °c and °c. optimum keratinolytic activity was observed at °c and ph . . the enzyme was stable at °c. the activity was investigated in the presence of some chemicals, including sds, tween , dmso, triton x- , edta, nacl, zncl , cacl , glucose. the keratinolytic activity was inhibited by all chemicals tested to some degree. the molecular weight of keratinase was determined by polyacrylamide gel ( %) using standard molecular weight marker and estimated about kda by sds-page. the keratinase isolated from bacillus uk could be used in biotechnological processes i.e. feather degradation, wastewater treatment and in industrial processes, such as detergent, food and leather industries. introduction: hemoglobinopathies, including thalassemia, abnormal hemoglobins, constitutes a major group of inherited disorders of hemoglobin synthesis. the reduced or absent of the beta (b) or alpha (a) globin chains of the adult human hemoglobin molecule results beta or alpha thalassemias, leading to imbalanced a-globin/non a-globin chains. the aim of this study was to give a quik desicion with a/b-globin mrna ratio for sequencing of a or b gene, when the anemia is not detectable. materials and methods: mrna and cdna extraction of bthalassemia and a-(including two of . kb del./hbs) thalassemia subjects and normal controls were accomplished using the high pure rna isolation kit and transcriptor first strand cdna synthesis kit, respectively, following the manufacturer's instructions. we used cdna as a template in the real-time pcr amplification using primers specific for a, b globin genes. amplification was performed in a lightcycler Ò instrument. the a/b-globin mrna ratio of each sample was calculated based on the Àddct method. results: a/b-globin mrna ratios calculated in a-thalassemia subjects relative to normal control as a result of numbers of defective a-globin genes. the a/b-globin mrna ratio was found higher in b-thalassemia subjects. coinheritance of a-thalassemia in hb s subjects concluded a stabile a/b-globin mrna ratio as per a-thalassemia or b-thalassemia subjects. discussion and conclusion: instability in a/b-globin chains is a significant factor of thalassemia disease severity and can be used before deciding type of gene sequencing when the anemia is not detectable. this study indicates that imbalance in globin gene expression could be demonstrated by measuring a/b-globin mrna ratio, which was conveniently and accurately determined by qrt-pcr and give an information about globin gene function which gene should be correct to investigate an individual for globin gene mutation. p-mis- self-assembling peptides mimic supramolecular biochirality r. garifullin , , m. o. guler bilkent university unam, institute of materials science and nanotechnology, ankara, turkey, kazan federal university, institute of fundamental medicine and biology, kazan, russia supramolecular chirality is rooted in asymmetric spatial arrangement of structural elements (e.g. molecules or units with higher hierarchy). self-assembled systems giving rise to this kind of chirality are of great importance because they closely resemble natural biological systems and potentially can lead to new advanced functional materials. in the process of self-assembly, both molecularly chiral and achiral structural units can organize into chiral nanostructures. chiral arrangement of chromophore molecules in space is known to result in emergence of chiroptical properties of a chromophore. organization of pigment-protein complexes into macrodomains in green plants gives rise to biochirality emanating from long-range chiral order of complexes. owing to this order, macrodomains start to absorb circularly polarized light intensively and thus exhibit huge circular dichroism (cd) signal. in our study, a simple approach which was aimed at mimicking the biochirality phenomenon makes use of self-assembling peptide amphiphiles and their interactions with pyrene chromophore. designed peptide amphiphiles are capable of self-assembly into nanofibers with chiral interior, which in principle gives an opportunity to achieve long-range chiral order. two modes of interactioncovalent and noncovalentwere utilized in order to induce supramolecular chirality. covalent interaction mode included direct covalent attachment of pyrene to peptide sequence. upon self-assembly of peptide amphiphile into nanofibers intense circular dichroism phenomenon was observed. noncovalent interaction mode envisioned encapsulation of pyrene molecules in the hydrophobic core of nanofibers of another peptide amphiphile. co-assembly of peptide amphiphile and pyrene molecules led to chiral order and intense cd signal. in addition, it was possible to control the sign of cd signals by using either of peptide isomers, l or d. p-mis- pon activity in hdl subgroups of obese, overweight and normal weight subjects objective: the aims of this study were isolation of hdl-c subgroups by using precipitation method, determination of pon- activity in both total and hdl subgroups, and evaluation of performance characteristics of pon- activity measurement method in newly diagnosed obese, overweight and normal subjects. material and methods: the study population consists of newly diagnosed obese, overweight and normal subjects. fasting morning blood samples were taken from all study groups. hdl subgroup was obtained by heparin-mn-dextran sulphate precipitation method and cholesterol was measured with direct (homegenous) hdl-c method. hdl -c concentrations were calculated with the subtraction of hdl -c from total hdl-c. hdl -c and total pon- activity were determined by using eckerson method. non-hdl pon- activitiy was calculated with subtraction of hdl pon- activity from total pon- activity. results: total hdl-c, hdl -c and hdl -c concentrations and the activity of total pon- and hdl pon- were found lower in obesity according to overweight and normal subjects (p < . ). negative correlations were found between body mass index and hdl -c, total pon- and hdl pon- (r = À . , p < . ; r = À . , p < . ; r = À . , p < . , respectively). conclusion: our findings indicated that hdl-c metabolism and lipoprotein associated antioxidant defense mechanisms were adversely affected with obesity. in conclusion we think that precipitation method using for separating hdl subgroup, is simple and cost effective for routine applications in clinical laboratories. besides hdl -c measurements, pon activity, measurement of total and hdl -c subgroup might be helpful to evaluate the atherosclerotic process in obese subjects. keywords: obesity, body mass index, paraoxonase, hdl subgroup, cholesterol p-mis- hepatitis e virus antibody prevelance among persons who work with animals in north cyprus introductions: hepatitis e infection is a major cause of viral hepatitis in many developing countries. the objectives of the present study was to determine the seroprevelance of hev infection in peoples who work with animals in northern cyprus. materials and methods: prevelance of hev infection were determined in study group population: persons without occupational exposure to animals; persons who work with animals; veterinarian and butcher. a total of blood samples were collected. all serum samples were tested elisa using a commercially available kit according to the manufacturer's instructions. ti-test were used for istatistical analyses. p > . was accepted as significant value. results: in a study of blood donors ( male, female), the overall prevelance of anti-hev igg antibodies were . %. the blood samples were collected different areas. the prevelance of anti-hev igm antibodies was . % and he was years and acting a butcher during years. the prevelance of anti-hev igg of women were approximately two fold higher than men. no significant difference in anti-hev prevelance was observed between the age of the blood donors. according to the anti-hev igg prevelance, the without occupation expose to animal animal were %, the animal husbandry were % and the veterians and the butcher were % were found. discussion: the prevelance of anti-hev in the north cyprus ( %) was found low such as the prevelance of the turkey ( %). the prevelance of anti-hev igg in animal husbandry were higher that the other groups because of they may be more spend of time and contact with animals. the prevelance of igm results suggested that the possibility of outbreaks may be low in north cyprus. conclusion: this study was the first seroprevelance analysis of north cyprus according to the population number.the further studies could be included the seroprevelance of anti-hev from the animals. most errors in the clinical laboratory occur in the preanalytical phase the aim of this study was to investigate the causes and rates of rejected samples, regarding to certain test groups in our laboratory. this study was designed on the rejected samples between january and january . clinical chemistry, coagulation, hormone, cardiac markers, total urine evaluation and other (ethanol level, hba c, hb electrophoresis, neonatal bilirubin, drug level, blood gas, fecal occult blood) test groups were included. the total number of specimen and rejected samples was obtained from the hospital information system retrospectively. types of inappropriateness were evaluated as follows: erroneous coding, clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen. it was determined that blood samples were sent to our laboratory in one-year period. . % of them were rejected because of preanalytical errors. erroneous coding was found as the most common rejection cause ( %). rejection rates of clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen were found to be %, %, %, %, % and % respectively. in our study, erroneous coding was the most common cause of preanalytical errors. education of medical secretaries is relevant and important as can be seen in the decrease of sample errors and the resulting quality improvement. glycosylated hemoglobin test (hba c) is important for screening, diagnosing, and monitoring diabetes and prediabetes. however, hba c levels may dependent on patient ethnicity suggesting that the diagnostic cut-offs should be evaluated for specific populations. therefore, our aim in this study was to evaluate the efficiency of hba c for predicting diabetes in comparison to oral glucose tolerance test (ogtt) results for turkish population. the study included anonymous lab results (acibadem labmed laboratories in turkey) of patients ( female, male) aging . ae . years ( - ) who had an initial diagnosis of diabetes. glucose and insulin levels during ogtt were measured after the initial administration of g sugar ( hour), -hour and -hour. these parameters were statistically analyzed in comparison to simultaneous hba c results. glucose measurements at hour had better distinction power (p < . ) between these individual groups than initial and -hour glucose measurements. the average hba c (%) levels for healthy, pre-diabetic and diabetic individuals were . ae . , . ae . and . ae . , respectively. roc curve analysis showed . % sensitivity and . % specificity for the clinically accepted hba c cut-off value of . %. hba c cut-off value of . % had a higher sensitivity of . % and comparable specificity of . %. the highest discrimination power between healthy, pre-diabetic and diabetic individuals was observed at glucose concentration at -hour after sugar administration in ogtt test as opposed -hours generally used for diagnosis. low sensitivity was observed for the clinically adapted . % cut-off value of hba c. the cut-off value of . % for hba c was found to be more sensitive with comparable specificity than the . % cut-off values for diabetes screening in our population. our results suggest that . % for hba c should be considered for diabetes cutoff value for turkish population. induction of the glutathione-dependent detoxification capacity is involved in the hepatoprotective effect of silymarin against acetaminophen-induced hepatotoxicity y. kim, d. kwon, c. ahn seoul national university, seoul, south korea recent findings in this laboratory showed that silymarin was capable of promoting hepatic glutathione (gsh) synthesis via a modification of the transsulfuration reactions in the liver. to investigate its pharmacological significance, we examined the hepatoprotective effect of silymarin against liver injury induced by acetaminophen (apap). adult male mice were treated with silymarin ( mg/kg, po) every hours for a total of doses prior to an apap challenge ( mg/kg, ip). the apap-induced liver injury was assessed by histopathological examination and measurement of changes in plasma enzyme activities, lipid peroxidation and formation of nitrotyrosine protein adducts in the liver. plasma levels of apap and its major metabolites were monitored for hours to estimate the metabolic transformation of apap. also protein and activity of the major cyp subtypes involved in the metabolic activation of apap into a toxic metabolite were determined in liver of the mice treated with silymarin only. silymarin pretreatment attenuated the apap-induced liver injury significantly when determined hours later. plasma concentrations of apap, apap-glucuronide or apap-sulfate in plasma were not changed, but thiol conjugates of apap, such as apap-glutathione, apap-cysteine and apap-n-acetylcysteine, were elevated significantly in the mice pretreated with silymarin. however, silymarin treatment did not affect protein expression of cyp e , cyp a , or cyp a in the liver. also hepatic microsomal enzyme activities measured using p-nitrophenol, ethoxyresorufin and erythromycin as substrates, were not increased by silymarin, indicating that the elevation of apap-thiol conjugates should be attributed to an augmentation of the gsh conjugation capacity. it is suggested that silymarin may protect the liver against an electrophilic substance-induced toxicity by increasing gsh availability which would enhance the detoxifying capacity of liver cells. prostate cancer (pca) is the second leading cause of death among men in western countries. we have previously found that the six transmembrane protein of prostate (stamp ) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk/mapk signaling. we also found that stamp is highly mobile in pca cells and shuttles between the plasma membrane and the golgi, often found in vesiculotubular structures in the cytosol. using advanced imaging techniques, we have now characterized the trafficking of stamp from the plasma membrane to early endosomes in lncap cells, by analysing its dynamic targeting to the three main endocytosis pathways: clathrin-mediated endocytosis, caveolae/lipid rafts, and the arf -dependent pathway. we found that stamp fused to cyan fluorescent protein (cfp-stamp ) is present at the plasma membrane where it accumulates in punctate structures. live cell confocal imaging showed that these puncta were dynamic over time indicating that stamp may be constitutively delivered to the plasma membrane and removed from it by endocytosis. co-expression of cfp-stamp with various fluorescent protein markers revealed that cfp-stamp puncta corresponded to lipid rafts that were labelled with caveolin- -rfp or antibodies against flotillin. live cell imaging showed that cfp-stamp and caveolin- -rfp disappeared at the same time from the same region of the plasma membrane suggesting that lipid rafts are likely to be responsible for stamp internalization. notably, stamp was absent from other endocytosis structures such as clathrin-coated pits/vesicles. further work is needed to determine whether stamp internalization is required for its function, such as its link to erk signaling, and whether interference with lipid rafts influences stamp effects on pca cell proliferation and survival. antithrombin-iii, mpv and plasma total homocysteine levels in behcet's disease introduction: behcet's disease is a multi-systemic and chronic inflammatory vasculitis of unknown etiology characterized by recurrent oral and genital ulcers, uveitis, arthritis, arterial aneurysms, venous thrombosis and skin lesions. platelet indices such as mean platelet volume (mpv) is a standart indicator of platelet function in disease pathophysiology. antithrombin, a glycoprotein synthesized in the liver, is the major plasma inhibitor of thrombin thus modulating blood coagulation. antithrombi-iii (at-iii) is a enzyme even moderate deficiency significantly increases the risk of thrombosis. homocysteine (hcy), that is formed during the metabolism of methionine. several clinical studies have clarified that elevated blood hcy levels are related to atherosclerotic disease. in our study, we investigated ovocystatin is one of the best characterized members of cystatin superfamily of protease inhibitors, and it has been frequently used for pathophysiological studies as the model protein, representative for this superfamily. its application has been supported by high structural similarity to human cystatin c as well as several common biological activities. as regard to biological activity, cystatins, including ovocystatin, are best characterized as inhibitors of cysteine proteases of papain family (c ), such as cathepsins b, h, l and s. these inhibitors participate in intra-and extracellular control of proteolytic events, both in physiological and pathological states. in the recent decade also new activities of cystatins, not assigned to inhibition of papain-like cysteine cathepsins, were found. these activities are associated with an alternative active center for legumain-type proteases in the molecule. here we report a chemical modification of ovocystatin that disables the anti-papain activity of the inhibitor but does not affect its anti-legumain activity. the chemical knockout has been obtained by reaction with -hydroxy- -nitrobenzyl bromide (hnbb) that covalently modifies the trp residue in the molecule. the reaction has been monitored by uv-vis and fluorescence spectroscopy. the anti-papain activity of the inhibitor has been measured colorimetrically against bana as a substrate. the anti-legumain activity was assessed fluorometrically using z-ala-ala-asn-amc. the reacted inhibitor exhibited an additional, characteristic for hnbb, band at nm in uv-vis scan. accordingly, an ablation of trp fluorescence was also observed. the molecule fully retained the anti-legumain activity, while only residual antipapain activity ( %) was observed. the modified ovocystatin can be a useful molecular tool for studying the physiological and pathological processes specifically associated with legumain activity. departments of medicine (hematology/oncology) and biochemistry and molecular biology, university of louisville, james graham brown cancer center, louisville, ky, united states -phosphofructo- -kinase/fructose- , -bisphospatase (pfkfb) family of enzymes are responsible for the conversion of fructose- -phosphate (f p) to fructose- , -bisphosphate (f , bp) and vice versa, and f , bp is an allosteric activator of phosphofructokinase- (pfk ), a rate-limiting enzyme of glycolysis. among the four identified pfkfb isozymes (pfkfb - ), pfkfb is the least studied isozyme in human cancers. there exists two different splice variants of pfkfb , variant- and variant- , coding two different isoforms, isoform a and b, respectively. in this study, we first analyzed the effect of k-ras(g d)induced oncogenic transformation on pfkfb expression in pancreatic duct cells. we found that oncogenic k-ras induction in immortalized pancreatic duct cells (ipde) was associated with decreases in total pfkfb mrna and protein expressions (mrna; ipde: ae . ; ipde+kras: . ae . and protein; ipde: ipde+kras: . ). we then, checked individual expressions of splice variants and observed that while pfkfb splice variant- (p -v ) expression was reduced by k-ras induction (ipde: ; ipde+kras: . ), pfkfb splice variant- (p -v ) expression was increased (ipde: ; ipde+kras: . ). then, we checked effects of p -v and p -v on glycolytic phenotype of ipde and ipde+kras cells. over-expression of pfkfb variants increased f , bp concentration (p -v : . ; p -v : . fold; compared to empty vec), glucose uptake (p -v : %; p -v : %) and glycolysis (p -v : %; p -v : %) in ipde+k-ras cells. we next analyzed the subcellular localizations of pfkfb isoforms and observed that both pfkfb isozymes localize to the nucleus, with more prominent nuclear localization of p -v compared to p -v . also, nuclear localization ratio of p -v increases after oncogenic transformation with mutant k-ras. taken together, these results suggest that pfkfb may have a role in the glycolytic phenotype of pancreatic cancers characterized with hyperactive k-ras signaling. effects of p map kinase inhibitors on mda-mb- cell line introduction: p mapk phosphorylates serine and/or threonine residues of the target proteins. the activation of p mapk leads to cell growth, differentiation, survival or apoptosis. in this study, we tested the effect p mapk sb and sb on mda-mb- cells to further elucidate the controversial role of p mapk on cell proliferation or cell migration. materials and methods: mda-mb- cancer line was cultured in rpmi- supplemented with % fbs. the cytotoxic and cell migration effects of sb and sb inhibitors were tested by mtt assay and wound assay, respectively. the effects of both inhibitors on proliferation and adhesion of md-mb- cells were determined by icelligence system. results: it was found that sb p map kinase inhibitor was more effective than sb . however, no significant effects of low doses of lm and lm of both inhibitors were seen on cell proliferation as compared to the dmso-treated control cells for up to hours as determined by icelligence system. on the other hand, both sb and sb significantly prevented cell proliferation at a concentration of lm. both sb and sb significantly reduced cell migration in a time-dependent manner at a concentration of lm. then, we tested whether each p mapk inhibitors have any effect on cell adhesion during a treatment period of hours using icelligence system. only lm concentration of sb reduced cell adhesion for about . hour (p < . ). conclusion: p mapk inhibitors sb and sb differentially affect cell proliferation, survival and migration. acknowledgements: this study is financially supported by dumlupınar university, scientific research project no - . mutagenicity of a series efficacious benzoxazine derivativesa new approach to evaluate ames test data e. foto , f. zilifdar , s. yilmaz , t. sarac ßbasi , i. yalc ßin , n. diril hacettepe university, ankara, turkey, ankara university, ankara, turkey testing safety of drug candidates is as crucial as evaluating their efficacy in early drug development. we previously synthesized a series of , -benzoxazine- -one derivatives showing significant antimicrobial, in vitro anticancer, topoisomerase i inhibitory activities and studied their several mechanisms of action. in this present study, we have evaluated mutagenic activities of these compounds and their potential metabolites. moreover, we aimed to develop a new statistical algorithm available for structureactivity relationship analysis to identify the regions responsible for the activity. to evaluate mutagenicity of the compounds, ames salmonella/microsome test was used. salmonella typhimurium ta and ta strains were used to detect for frameshift and basepair substitution mutagens, respectively. additionally, mutagenicity of potential metabolites of them were evaluated by adding metabolic activation system (s ) which was prepared from a pool of male sprague dawley rats. results were evaluated with student's-t test. following regression model estimation analysis, we detected minimum mutagenic doses of all tested compounds for generating a d-common features pharmacophore model with hiphop method. according to the results, only bs , bs , bs and bs exhibited strong mutagenic effects on both strains in the presence and absence of s . additionally bs , bs , bs and bs (in the absence of the s ), bs , bs and bs (in the presence of the s ) showed weak mutagenic effects on ta . hiphop analysis results revealed that mutagenicity was increased in the presence of aromatic desactivating groups which might form hydrogen bonds at the position of r and hydrophobic groups at the position of r of the benzene ring in the structure of benzoxazine. the new statistical approach developed in this study can be useful for assessing the ames test data available for structure activity relationship analyses. background: recently more than thirty different diseases can screen simultaneously with expanded newborn screening (nbs) programs by tandem ms.expanded nbs with tandem ms is performed routinely at akdeniz university hospital central laboratory since .the aim of this study was to evaluate our nbs results with some second-tier and confirmatory tests. materials and methods: nbs results (n = ) were evaluated in dried blood samples which sent to our laboratory for the study between august and august . electrospray ionisation (esi)triple quadrupole mass spectrometer (shimadzu lc-ms/ms ,japan) was used for nbs analysis,acylcarnitine and amino acid profile were screened with mrm (multiple reaction monitoring) spectrum within minutes.second-tier tests were performed as urine organic acid analysis by gas chromatography-mass spectrometry (gc-ms),plasma and urine quantitative amino acid analysis by high pressure liquid chromatography (hplc).pathological nbs results were assessed in three separate groups as amino acid metabolism disorders, fatty acid oxidation defects and organic acidemias. results: metabolic diseases were found in ( . %) patients by the second-tier tests performed.there were detected amino acid metabolism disorders in ,organic acidemia in ,fatty acid oxidation defects in patients. conclusions: the reason of high positive results in our laboratory could explain that our study includes both screening and monitoring of previously diagnosed metabolic patients.nbs is performed in only a few centers in turkey although there were the national screening programs included nbs in many foreign countries.more expanded nbs programmes in our country is required to start treatment of patients before irreversible damage is not occured. although many reports indicate the involvement of calpain in several human pathologies, it is not yet clarified how the protease can recognize the substrates to digest and how can escape to its natural inhibitor calpastatin. answers to these questions have been obtained by identifying specific intracellular localizations of calpain and its substrates and analyzing the interactions of the protease with calpastatin. these studies were carried out using human sknbe neuroblastoma cells. protein-protein interactions and intracellular localization of calpain and the related proteins were determined by immunoprecipitation and isolation of membrane microdomains. we have observed that small amounts of calpain- are localized in lipid rafts microdomains together with n-methyl-d-aspartate receptor (nmdar) containing nr /nr b subunits. immunoprecipitation experiments have demonstrated that nmdar containing nr /nr b subunits, calpain- , hsp and neuronal nitric oxide synthase (nnos) but not calpastatin and calpain- are present in specific protein complexes. thus, in this localization calpain activity is regulated by hsp that reduces the affinity for ca + of the protease. cell stimulation with nmdar agonists induces calpain activation that specifically cleaves the subunits nr b of the receptor promoting changes in lipid rafts organization and internalization of nmdar without affecting cell viability. moreover, in these conditions, also nnos is digested and converted in the active form by calpain- . our data suggest a physiological role of calpain- at specific cell sites. the protease inserted in lipid rafts microdomains is in strict contact with its targets and escapes to calpastatin which is not inserted in these structures. following an increase in ca + influx, the activated protease regulated by hsp , promotes the removal of nmdar from the plasma membranes, decreasing ca + entrance through this receptor-channel and protecting cells from ca + overloading. tissue transglutaminase (tg ) is a multifunctional protein complex that can act as a crosslinking enzyme, gtpase/atpase, protein kinase and protein disulfide isomerase. at the cell surface, tg was shown to be involved in adhesion, migration, invasion, growth, epithelial mesenchymal transition and hence implied in the metastatic development of many different tumor types. renal cell cancer (rcc) is one of the most common type of cancer in adult males that generally grows as a single tumor within a kidney. our previous findings indicate that the increased expression of tg in rcc results in tumor metastasis with a significant decrease in disease-and cancer-specific survival outcome. herewith, the role of tg in cell migration of rcc was investigated in this study by transducing the model rcc mouse cell line renca with a series of tg mutant constructs. renca cells were transduced by lentiviral particles encoding wttg , transaminase-defective tg -c s form with low gtpbinding affinity, gtp-binding deficient form tg -r a, and transaminase-inactive tg -w a. in order to investigate the role of tg transamidating and gtpase activity in cell migration, scattering assay was used where colonies for each mutant clone was followed for a time interval of hours. our results showed that non-transduced control and tg -c s mutant renca cells demonstrated a similar migration pattern with a % of scatter activity. on the other hand, % colonies formed by renca cells overexpressing wttg and tg -w a mutant scattered away from each other. a small insignificant increase in scattering was seen in % of the total number of colonies for renca cells overexpressing tg-r a construct. data from this study supports that gtp-binding activity of tg is the drive force in migration driven scattering of renca cells, suggesting that inhibitors targeting the gtp-binding activity of tg may serve as a new therapeutic approach in the treatment of rcc. background: in this study, we aimed to investigate the relationship between level of vitamin d with subclinical hypothyroidism and subclinical hyperthyroidism. material and metod: study groups planned as three groups such as euthyroid (n = ), subclinical hypothyroid (n = ), subclinical hyperthyroid (n = ). serum tsh, free t (ft ) and free t (ft ) levels were determined by chemiluminescence immunoassay and serum -hydroxy (oh) vitamin d (oh) d level were determined by liquid chromatography-tandem mass spectrometry. euthyroidism was defined as a normal level of tsh (range, . to . miu/l), ft (range, . to . ng/dl) and ft (range, . to . ng/dl). subclinical hypothyroidism is defined as an elevated serum tsh level associated with normal total or free t and t levels. subclinical hyperthyroidism is defined as low serum tsh levels associated with normal free t and free t levels. results: subclinical hyperthyroid group had significantly higher (oh) vitamin d levels compared to the euthyroid and subclinical hypothyroid groups (p < . ). (oh) vitamin d levels in subclinical hypothyroid group was not statistically significant when compared with the euthyroid group. food processing wastes provide carbon sources in high amounts for fermentative microorganisms to produce energy. converting carbon-rich biomass into bioethanol through fermentation by microorganisms both provides energy requirement for humankind and also decrease pollutant gases like co , no x and so x (ghorbani et al., ) . fermentation processes for bio-ethanol production could be achieved by saccharomyces cerevisiae, zymomonas mobilis, and escherichia coli. bacterial hemoglobin (vitreoscilla hemoglobin, vhb) is the first and best characterized prokaryotic hemoglobin molecule. the function of vhb is supporting the cellular respiration through binding to oxygen at microaerobic environment, transferring it to the terminal respiration oxidases (geckil et al., ) and thus improving growth and productivity of the microorganisms. in this study, e.coli strains fbr , ts and ts were used as ethanologenic microorganisms. expression of vhb in ts is lower than in ts strain. for the efficient ethanol production effect of different inoculum sizes, sugar species and sugar concentrations in the growth medium were investigated. vhb expression increased effectively the viability of ts strain by up to . x cfu per ml of fructose ( %, w/v) supplemented lb medium starting with small inoculum for fermentation. this indicates that vgb expression should be at the certain level to maintain sufficient the cell growth for ethanol production. geckil h, gencer s ( ) . production of l-asparaginase in enterobacter aerogenes expressing vitreoscilla hemoglobin for efficient oxygen uptake. applied microbiology and biotechnology : - . ghorbani, f., younesi, h., sari, a. e., najafpour, g. ( ) . cane molasses fermentation for continuous ethanol production in an immobilized cells reactor by saccharomyces cerevisiae. ethanol production from dairy industry by product using bacterial hemoglobin t. sar, g. seker, a. g. erman, m. yesilcimen akbas gebze technical university, depertment of molecular biology and genetics, kocaeli, turkey bioethanol production from biomass has a great potential to reduce greenhouse gases emissions. ethanol has several applications in industries (chemical, medical, pharmaceutical, food etc.) in the form of raw material, solvent and fuel. one of the most abundant liquid wastes is cheese whey generated from dairy industries. whey powder is concentrated form of whey and contains lactose and also protein, lipid, minerals and vitamins. vitreoscilla hemoglobin (vhb) is the first bacterial hemoglobin. the main function of this molecule is to improve oxygen transfer to cellular oxidases and thus supporting cellular growth and productivity at low oxygen levels (kallio et al. ) . in this work, e. coli strains fbr , ts (low level vhb expressing) and ts (high level vhb expressing) were used as ethanol producing microorganisms. fermentation medium containing whey powder supplemented with lb material was inoculated with these strains and incubated for hours at °c and rpm in a ml erlenmayer flask. the ethanol production was improved over % by using lower vhb expressing strain. the ethanol levels (v/v, %) were determined as . , . and . for fbr , ts and ts strains respectively. it is shown that the certain levels of vhb could be useful tool to increase the growth and productivity of ethanol from dairy industry wastes. kallio p.t., kim d.j., tsai p.s. and bailey j.e. ( ) . bioethanol is usually produced from cellulose, hemicellulose and lignin. the lignocellulosic wastes should be hydrolysed into fermentable sugars by using enzymes or dilute acids before microbial fermentation. acidic hydrolysis methodology is cheaper than enzymatic hydrolysis but it can cause production of some inhibitors like aliphatic acids, which affect the growth of microorganisms. vitreoscilla hemoglobin (vhb) is the first described prokaryotic hemoglobin. the recombinant strains carrying vgb gene (e. coli, p. aureginosa) which encodes vhb showed increased bacterial growth, productivity of metabolites compared to untransformant counterparts under low oxygen concentrations [nasr et al., ; geckil et al., ] . in this study, ethanologic e. coli strains fbr , its derivative strains ts (vgb+) and ts (vgb+) were used. ts was constructed in such that it could express more vhb than ts . bioethanol production by these strains in presence of lignocellulosic hydrolysates derived inhibitors was investigated. different acetic acid concentrations ( . - mm) were used as inhibitors from lignocellulose hydrolysate. . mm acetic acid was used as an inhibitor. the growth of vhb expressing ts and ts strains was inhibited about % after hours fermentation time. strain fbr was inhibited as high as % by using the same inhibitor including growth medium. it was shown that the expression of vhb could improve growth and productivity in presence of lignocellulosic inhibitors. differentiation of preadipocyte, also called adipogenesis, leads to the phenotype of mature adipocyte. however, excessive adipogenesis is closely linked to the development of obesity. thus, any drug or chemical that can inhibit adipogenesis may have preventive and/or therapeutic potential against obesity and related diseases. azd , an inhibitor of the family of pim kinases, is known for anti-cancer activity. here we investigated the effect of azd on adipogenesis in t -l preadipocytes. notably, azd treatment led to a concentration-dependent inhibition of both lipid accumulation and triglyceride (tg) synthesis during the differentiation of t -l preadipocytes into adipocytes with no cytotoxicity. on mechanistic levels, azd strongly reduced not only the expression levels of ccaat/enhancer-binding protein-a (c/ebp-a), peroxisome proliferator-activated receptor-c (ppar-c), fatty acid synthase (fas), and perilipin a but also the phosphorylation levels of signal transducer and activator of transcription- (stat- ) during adipocyte differentiation. furthermore, azd largely decreased leptin, but not adiponectin, mrna expressions during adipocyte differentiation. collectively, these results demonstrate that azd inhibits adipogenesis in t -l preadipocytes and the inhibition is largely attributable to the reduced expression and/or phosphorylation levels of c/ebp-a, ppar-c, fas, perilipin a, and stat- . effect of intrauterin exposure to artificial food colourings on dna damage in rats in many research genotoxic potential of food additives has been investigated. however there are few findings about the effect of artificial food colourings (afc) on dna. in this experimental study, we aimed to analyze whether in utero exposed artificial food colourings would have effect on dna and cause damage.thirteen female rats were included to the study which were equally divided into two groups as control (cg, n = ) and food colouring (fcg, n = ) groups. a mixture of nine food colours were given daily to fcg by oral gavage from preconception to birth. no adverse effect level (noael) of artificial food colourings for each colouring was administered to fcg. three months after the birth, offspring from each group were selected randomly as control (cg) and experiment (eg) groups. then they were sacrified under anesthesia. for performing the alkaline comet comet assay leukocytes were seperated from whole blood samples. the alkaline comet assay was performed. the extent of dna damage was assessed from the length of dna migration derived by subtracting the diameter of the nucleus from the total length of the image and graded into categories and these grades were converted into arbitrary unit (au). differences between the means of data were compared by independent samples t test. the results were given as the meanaesd, p values of less than . were considered as statistically significant. although the extent of dna damage was higher in eg, the comparison of experiment ( . ae . ) and control ( . ae . ) groups showed no statistical difference (p = . ). relationship between glucocorticoid receptor gene polymorphisms and recurrent depression l. aydogan, i. benli, z. c. ozmen, i. butun gaziosmanpasa university medical faculty, department of biochemistry, tokat, turkey objective: sensitivity to glucocorticoids varies between individuals and these differences have been implicated in the etiology of psychiatric diseases such as depression. recent studies have found relationship between common glucocorticoid receptor (gr) gene (nr c ) polymorphisms and unipolar or bipolar depression. the nr c gene is a candidate gene affecting depressive disorder risk and management. the aim of the present study was to evaluate the relative distribution of specific polymorphisms of nr c (bcl and rs ) in recurrent depressive disorder (rdd) patients. methods: our study included volunteers with recurrent depressive disorder and healthy individuals without any mental illness. depression was assessed by hamilton and madrs depression scale. nr c gene polymorphisms were detected by real-time pcr, with hybridization probe method. allele and genotype frequencies at two loci (bcli and rs ) were investigated in rdd patients and controls. results: genotype distribution among rdd patients and the control group for bcl- (g/c) were as follows: cc % and %, gc % and %, gg % and %, respectively. there was not a significant difference when the frequency of the allele (p = . ) and genotype frequency (p = . ) were compared between the patients and the control. genotype distribution in the rs region (a/t) of the patients and controls were tt % and %, ta % and %, aa % and %, respectively. allele frequency (p = . ) and the genotype frequencies (p = . ) were not significantly different among the groups. conclusion: numerous nr c gene polymorphisms were previously reported in association with modification of depressive disorders. the results of our study showed no association between gr genotype and recurrent depressive disorder. nr c polymorphism does not play a role in the development of recurrent depressive disorder. thymoquinone (tq) has been shown to supress the proliferation of various tumor cells, while it is minimally toxic to normal cells. the aim of this study is to investigate the potential therapeutic effects of tq on cell proliferation, apoptosis, invasion, migration, colony formation and wound-healing in sh-sy y human neuroblastoma cell line. sh-sy y cell line treated with - lm tq by solving medium for , and h considering a time-and dose-dependent manner. the cytotoxic effect of tq was determined by mtt method. total rna was isolated by trizol reagent. cdna synthesis was performed by using commercial kit. mdm , p , p , akt, pten, cdk , cyclin d , caspase- , - , - , - , bcl- , bax, parp, bcl-xl, bid, dr , dr , puma, noxa, mmp- , - , timp- , - and gapdh gene expression profiles were analysed by real-time pcr method. effects of tq in sh-sy y cells on invasion, colony formation and cell migration were detected by matrigel-chamber, colony formation assay and woundhealing assay, respectively. statistical analysis were performed with rt profiles array data analysis by using student's t test. ic value of tq in sh-sy y cells was detected as lm at th hours. by rt-pcr results, it was determined that tq caused a decrease in the expression of mdm , akt, cdk , cyclin d , bcl- and mmp- . it is also observed that tq caused a significant increase in the expression of p , pten, caspase- , - , bid, dr , puma, noxa and timp- . it was also found that tq in sh-sy y cells suppressed invasion, migration and colony formation by using matrigel invasion chamber, wound healing and colony formation assay, respectively. in conclusion, we demonstrate that tq significantly effect cell cycle, apoptosis, invasion, migration and colony formation of sh-sy y cells. tq may be a potential candidate as chemotherapeutic agent for the treatment of neuroblastoma. more studies have to be performed to profile the mechanisms and genome wide effects of tq to prove its therapeutic potential. dna aptamers can achieve a very high affinity to the target due to the potential of developing broad target-binding interface. however, classic strategy selection of aptamer binders is a challenging task requiring multiple rounds of panning and post-selection optimization. we have developed fast and convenient technique for the selection of dna aptamers based on the offrate selection and tandem affinity purification (tap). we constructed and produced in e.coli recombinant chimeric protein, comprising two affinity tags (his and gst) separated from each other and from the target protein (anthrax protective antigen domain iv, padiv) by sumo protease recognition polypeptide and synthetic cleavage site for the anthrax lethal factor (lf). the protein bound to aptamer library is first captured by imac resin, cleaved by sumo protease, captured by gst resin and eluted by lf following the lines of the tap method. the gst-captured aptamer-target complexes were subjected to the off-rate selection using soluble padiv as the competitor. multiple selection rounds are cumbersome and can result in carryover. high abundance of moderate affinity aptamers in the resulting pools obtained by classic selection approaches suggests that the procedure to counter-select them at the beginning of panning is needed. reduction of the contact duration between the aptamer library and the target was crucial for efficient selection of high-affinity binders. on the other hand, tap prevents contamination, and bundled with the off-rate selection, allows for clean isolation of high-affinity binders with affinity in the low nanomolar range. the developed technique is applicable for efficient selection of high affinity dna binders to soluble recombinant proteins and their fragments. dna aptamers obtained will be further used for the development of diagnostic and therapeutic tools for the detection and treatment of anthrax. the work was supported by russian science foundation research grant no. - - . the role of macab efflux pump in protection of serratia marcescens against antibiotics and oxidative stress the emergence of bacterial multi-drug resistance is a growing problem of public health worldwide. bacterial drug efflux pumps are membrane protein complexes that function to expulse drugs from the cell. they play a crucial role in the rising rates of antibiotic therapy failures. the homolog of macrolide-specific pump macab was identified in opportunistic pathogen serratia marcescens and was used in this study to characterize its role in protection against antimicrobials and other processes beyond the active efflux of antibiotics. here we used method of serial dilutions to determine minimum inhibitory concentration (mic) for s. marcescens sm wild type (wt) and its isogenic Δmacab mutant strains. we also used h o survival assay to evaluate the ability of wt and the mutant strain to withstand an oxidative stress. finally, we used b-galactosidase assay to evaluate macab promotor activation in the reporter strain and followed macab expression by western blotting analysis using macab- xhis strain. we show that in contrary to its e. coli homolog, macab pump in s. marcescens is not involved in the protection against macrolides but instead it is required for protection against aminoglycosides. we further show that similar to its salmonella typhimurium homolog, s. marcescens macab is essential for protection of bacteria against h o . transcriptional analyses demonstrate that while low level of macab promotor activity can be detected after hours of growth in lb-broth there is at least -fold increase in expression in response to the presence of h o . on the protein level macab can be detected starting from hours of growth in lb-broth and it reaches maximum expression on hour of growth. our data suggest that macab pump in s. marcescens is involved in protection of bacteria against aminoglycoside antibiotics and is crucial for protection against reactive oxygen species. we are currently working on identification of macab substrate with anti-h o properties. antiproliferative and apoptotic effects of noscapine on mcf- and mda-mb- human breast cancer cell lines approximately - % of breast cancers are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor . these are most aggressive tumor and a clinical problem because of lack of targeted therapies. noscapine is an alkaloid from opium. noscapine is a microtubule-interfering agent. it causes mitotic arrest, induces apoptosis. in this study, we aimed to investigate the effects of noscapine in mcf- and mda-mb- human breast cancer cell lines. the cytotoxic effects of docetaxel, tamoxifen, and noscapine on the mcf- and mda-mb- cell lines were analyzed by roche xcelligence system. the cells were cultured in % fetal bovine serum containing dulbecco's modified eagle medium at °c in a humidified atmosphere containing % co . h after seeding, the cells were treated with different doses of docetaxel ( . to nm), tamoxifen ( . to lm), and noscapine ( . to lm). cultured cells were harvested, fixed with % formalin, and centrifuged. pellet was blocked, fixed, and embedded in paraffin. paraffin-embedded cells blocks were sectioned at lm thickness and stained with h&e, ki- , bcl- , cyclin-d , and bax. sides were assessed under a light microscope. quantification of the analyzed proteins were evaluated by the percentage of positive cells. all drugs showed cytotoxic effects on both cell lines. all drugs inhibited the proliferation of breast cancer cells, but effects were dependent on time and dose. all drugs were especially more effective on mcf- cells. immunohistochemical examinations revealed that tamoxifen was more effective on mcf- cells, hovewer docetaxel and noscapine were more effective on mda-mb- cells. tamoxifen has more apoptotic and antiproliferative effects on mcf- cells. docetaxel and noscapine showed more apoptotic and antiproliferative effects on mda-mb- cells. noscapine may be an effective anticancer agent due to antiproliferative and apoptotic effects on breast cancer cells. negative selection of dna aptamers to reduce non-specific binding in solid-phase-based selection procedures carryover by binders specific to the components of the selection system can be a serious issue in hampering the aptamer selection campaign. solution-or "mass"-based techniques still cannot substitute classic phase-separation strategies. one approach to prevent selection of "passenger" phase-specific (plastic, beads) or blocking agent specific aptamer species is their depletion from the initial library pool. our aim was to develop the universal technique for removal of such aptamers exemplified by bsa-and casein-specific binders, while preserving the initial library complexity. the dna aptamer library was subjected to three rounds of depletion using magnetic beads with covalently attached casein and bsa. to ensure high depletion efficiency, beads were pelleted in a -ml centrifuge tube by a neodymium magnet through a -cm cushion of % sucrose, thus preventing weakly bound aptamers from re-populating the library. high complexity of the input library helped to avoid pcr amplification after depletion rounds preventing the library bias introduced by dna amplification. the depletion effciency was confirmed by real-time pcr. resulting oligonucleotide sub-library was analyzed for binding to the targets using solid-phase real-time pcr assay. we have shown that three rounds of panning under the conditions employed provided full depletion of the initial dna pool from nucleic acid structures capable of binding to protein competitors and hampering the process of aptamer selection. we compared selection efficiency of aptamers specific to type a botulinum neurotoxin light chain in depleted vs undepleted library. the yield of the target-specific aptamers was -fold higher in the library subjected to the depletion procedure. removal of undesired binders from aptamer libraries appears an important step of solid-phase selex procedure. it can become a useful approach in optimizing solid-phase selex. the work was supported by russian science foundation research grant no. - - . epithelial mesenchymal transition (emt) is a critical trans-differentiation program driving cancer metastasis. patients showing signs of emt or presence of distant metastasis have poor prognosis. another well-known feature of decreased cancer-associated survival is the lack of anti-cancer immune responses. thus we hypothesized that the emt and anti-tumor response should be linked via altered secretion of soluble factors by metastatic cells. all cell lines were grown in dmem. emt status of crc cell lines were assessed by investigating canonical markers of emt. cytokine/chemokine expression of crc cells was performed using r&d systems antibody arrays and validated using ccl sandwich elisa and rt-pcr. the mechanism of action of zeb / on ccl promoter has been studied by luciferace assay and chip. ccl coding region was cloned into pcdna . and stably transfected into dld- cells. ccl deficient ct cells were generated using lentivirual shrna transduction. cells overexpressing or knock/down ccl were injected orthotopically into mice. t lymphocyte (til) infiltration in respect to ccl and sip expression was studied using ihc or flow cytometry. emt status catagorised crc cell lines into epithelial, intermediate epithelial, intermediate mesechymal and mesenchymal. cytokine/chemokine antibody arrays showed a significant increase in ccl in induced dld-sip cells. elisa, multiplex assays and rt-pcr confirmed a significant increase of secreted ccl in the induced dld-sip cells as well as mesenchymal crc cells as compared to epithelial ones (p = . ). promoter studies showed that zeb / bind to ccl promoter and and activate ccl gene expression. no metastasis was observed for dld- cells overexpressing ccl but significant alterations of tumour associated lymphocytes were identified in syngeneic orthotopic crc models. our data shows that ccl is up-regulated by emt inducing transcription factor sip , and mesenchymal (metastatic) crc cells secrete significantly more ccl compared to epithelial (non-metastatic) ones. ccl did not induce emt per se but abundant secretion of ccl by metastatic crc cells was a crucial regulator of immune infiltrate in crc. inhibiting ccl in metastatic crc may have a therapeutic potential. barley (hordeum vulgare l.) belongs to the grass family, poaceae (gramineae). it is the fourth most important cereal crop after wheat, maize and rice and is among the top ten crop plants in the world. talbina was used to be recommended for the sick and for one who is grieving over a dead person. talbina is made by adding one or two tablespoon of barley flour (must be percent wholegrain barley flour) to one-and-a-half cups of water and placed on low heat for - minutes (optional: add milk or yoghurt and sweeten with honey). the main objectives of this investigation were determine the a-tocopherol contents and antimutagenicity activity of talbina (hordeum vulgare l.). our results showed that the total tocopherol content was in the range of . to . lmol/g fw. talbina extract was shown to have greater antimutagenic activity observed in the lg/plate concentration s. typhimurium ta . at all the doses antimutagenic response was significant at (p < . ) against both the strains with a percent mutagenicity decrease from to for ta followed by ta with percent antimutagenicity from to . the results of the study concluded that talbina is a better antimutagenic agent than vitamin e and combination of vitamins did not produce any synergistic activity. the compounds containing thiadiazoles have diverse applications as antifungals, anticancer agents, antibacterial, antiinflammatory drugs, antidepressants and carbonic anhydrase inhibitors according to literature. in this study some novel thiadiazole compounds [( , , , )-tetrathia[ . ] ( , )- , , -thiadiazolophane; ( , )dioxo- , , , )-tetrathia[ . ]( , )- , , -thiadiazolophane; ( , , , )-tetraoxo- ( , , , )-tetrathia[ . ]( , )- , , -thiadiazolophane and ( , , , , , )-hexaoxo- ( , , , )-tetrathia [ . ] ( , )- , , -thiadiazolophane] were used to evaluate the cytotoxicity on healthy human lymphocytes and the antibacterial activities. cytotoxicity tests were perfomed using mts assay and the trypan blue test. cells were incubated with the compounds for hours. at the end of the each hour, cell vitality was assessed by measuring the absorbance ( nm) of each well using a microplate reader for mts assay. in addition, viability percents of the cells were determined after trypan blue test. as a result, the compounds showed cytotoxicity in a dose dependent manner. for the concentrations of : of . mg/ml, the cytotoxic effect was eliminated. also, antioxidant capacity was determined using , -diphenyl- -picrylhydrazyl (dpph) reagent. moreover, the antibacterial activities of the compounds were analyzed using a microdilution test against e. coli and s.aureus. compounds having various concentrations showed different antibacterial effects against these two bacteria. arabidopsis thaliana ecotypes vary in their ability to utilize organic p substrates insufficient quantity of inorganic phosphorus in soil is an evergrowing problem that affects many fields of agriculture. unlike inorganic phosphates, organic phosphorus compounds are very common in many soil types, but plants are often unable to efficiently utilize them. to better characterize the extent of natural variation in the ability of the model plant arabidopsis thaliana to grow on organic phosphorus compounds, we grew arabidopsis ecotypes on several organic and inorganic sources of phosphorus. plants were grown in liquid or solid media containing naphosphate, phytate and atp as the sole supply of phosphorus or in absence of any phosphorus source. after several weeks of growth, plants were assayed for changes in their morphological and physiological characteristics. phytate was shown to be the least preferred source of phosphorus compared to inorganic phosphate and atp. the rate of biomass accumulation in all ecotypes decreased in the following order from inorganic phosphate to atp to phytate. lateral root formation was markedly reduced in the absence of any phosphorus source or in the presence of phytate. we also showed that phosphomonoesterase activity in intact roots increased when plants were grown on atp and phytate. overall phosphorus content in leaves and roots was similar when plants were grown on atp or inorganic phosphate, but it was markedly reduced on phytate. substantial differences between ecotypes were also observed in root length, p content in ash and phosphomonoesterase activity in intact roots. our analysis of the ability of arabidopsis ecotypes to grow on several different phosphorus sources provides a unique opportunity to investigate the degree of natural variation in this plant's ability to adapt to different nutritional environments. analysis of many important morphological and physiological changes observed in these plants can further extend our understanding of the full range of plant responses to phosphorus availability. laboratory tests are important in terms of confirmation of diagnosis given by clinics and implementation of appropriate treatment protocols for patients. laboratory tests used by the clinics have been increased in parallel with time.there are many reasons for increased use of the test such as increase of elderly population, increase in standard of care, lack of information and shortening of turn around time. unnecessary laboratory testing also constitute one of the reasons for increased use of laboratory tests. in our study we aimed to investigate the unnecessary laboratory testing for fpsa test. fpsa tests which are ordered with total psa tests that values of less than ng/ml or greater than ng/ml were accepted as inappropriate initial testing. fpsa tests were evaluated as unnecessary laboratory testing. the clinic which ordered the maximum unnecessary laboratory testing with was urology within all the clinics. although to the restrictions about the ordering of total psa and fpsa tests there were no decrease in the number of unnecessary laboratory testing. unnecessary usage of laboratory testing may cause increase of false positive results, increase in the use of invasive testing, unnecessary drug consumption and increase of healtcare costs. some precautions may be effective in reducing unnecessary tests such as to inform clinicians about the cost of laboratory tests, to increase the clinician education programs and to develop usage of disease specific diagnostic algorithms about test ordering. local clinical validation of blood collection tubes although the tubes with gel and clot activator are widely used due to the advantages, there are ongoing discussions about the effects of the blood collection tube on clinical outcomes in the analysis of biochemical parameters. therefore, we aimed to prove the local clinical validation of the new produced blood collection tubes with low-volume. the blood samples of patients who referred to the hospital phlebotomy unit were collected using holder into the different tubes. first tube was ml glass tube and with no additive, second was ml tube with gel separator, third was ml tube with gel separator. serum was separated and immediadiately analysed for biochemical parameters. the difference between the analyte amounts in the different tubes was evaluated using paired t-test. the clinical significance was evaluated using significant change limit. bias (%) between the other tubes with the reference tube was also evaluated according to the ''allowable total error". when we compared the other test tubes to a glass tube which was assumed reference tube, total protein, albumin, amylase, calcium, triglyceride, cholesterol, hdl-cholesterol, total and direct bilirubin, iron, gamma glutamyl transferase, magnesium, phosphorus results were statistically significant. but the results of all the analytes were within the significant changes limit and the allowable total error was not significant. while a biochemical parameters have analysed, it may be absorbed into the gel and this may caused from factors such as the chemical structure of the gel, analyte itself, the residence time in the gel, storage temperature and volume of the sample e.g. as well as the leaking of gel material to the sample was reported to be another factor for affecting the analysis. despite these factors, we observed that neither gel-clot activator tube with low nor high volume affect the clinical results. the research of the frequency of interference in thyroid function tests interference is defined as the effect of substance in the sample which changes the correct value of laboratory results. the frequency of interference in immune techniques is varied. the frequency of interference depends on population of the study, technique for detecting the reaction and researcher's method. unexpected or inconsistent results with clinical findings should suggest the possibility of interference. in this study it is aimed to investigate the frequency of interference in thyroid function tests (tsh, ft , ft ) which are the most common requested laboratory tests. thyroid function tests of patients are analyzed in ankara numune education and research hospital in october -may . five samples which had the incompatible results with clinical findings are re-evaluated just because of the suspicion of interference. the detection of interference included; repetition of test via different immune techniques, serial dilution, polyethylene glycol (peg) precipitation and incubation with heterophilic blocking tubes (hbt). the results of two different immune techniques and before/ after incubation with hbt showed no significant difference. linear curves had observed in serial dilution. after peg precipitation; below % of recovery had obtained in one sample, therefore it is interpreted as macro-tsh. the frequency of interference in thyroid function tests for -month study period was . %. no information is found about the best test for defining the cross reaction. it is also aforethought that interference should not be excluded by using any single procedure. p-mis- development of polyclonal and monoclonal antibodies against fatty acid binding protein (fabp /ap ) a. abbasi taghidizaj, g. aydogdu, b. p. sermikli, e. yilmaz ankara university, ankara, turkey recombinant proteins and antibodies can be use for therapeutic or diagnostic purposes which produced in many different host organisms. the technique for the production of immortal cell making single antibody, fusing target antibody-forming b lymphocyte precursor with a suitable myeloma cells. the fused hybrid cells (called hybridomas), as a cancer cell will reproduce rapidly and will produce large amounts of the desired antibodies. fatty acid binding protein (fabp ) is a well characterized intracellular lipid transport protein and plays a key role in the intracellular fatty acid transport and adipose tissue metabolism. fabp as a adipokine that regulates glucose homeostasis and has various features for metabolic syndrome associated with obesity. in this study, production of monoclonal antibodies against immunogenic fabp protein made by recombinant dna technology. recombinant his-fabp was expressed in e.coli and purified. balb/c mice used for immunization and serum anti-fabp antibodies determined by enzyme-linked immunosorbent assay (elisa). hybridoma cells created by fusion of splenocytes and myeloma partner cells. after selection of antibody producing cell clones, injecting hybridomas into the peritoneal cavity in balb/c mice ascites fluids was obtained. we have selected fifteen hybridoma clones that produced antibodies specific for fabp , as shown by western blotting and immunocytochemistry. as a result we produced mabs that will be useful for the scientific community working on fatty acid binding proteins and lipid metabolism. in near future, therapeutic approach for this antibody maybe a possibility in metabolic syndrome. thioridazine, an anti-psychotic drug, inhibits migration, invasion and epithelial mesenchymal transition in breast cancer cell lines thioridazine (thz), an antipsychotic drug, exhibits anti-angiogenic effects on breast cancer cell lines. however the mechanistic insight in exerting antiangiogenic effect is not clearly understood. the objective was to investigate the role of thz in epithelialmesenchymal transition (emt) by using cell migration assay, scratch assay, western blot (wb) and immunocytochemistry. thz treatment reduced cell viability on mda-mb- , mcf- and cd + /cd -cells and ic values of thz were found to be lm, . lm and lm respectively, at hours. invasion potency of mcf- , cd + /cd -and mda-mb- cells were determined as %, %, . % when compared to relevant treatment controls. migration potency of mcf- , cd + /cd -and mda-mb- cells was determined as . %, . %, % respectively. among the three cell lines mda-mb- cells display enhanced invasive and migration ability when compared to other cell lines. western blotting results demonstrate that thz significantly increases e-cadherin, cytokeratin- , b-catenin, while inhibiting n-cadherin, vimentin, fibronectin. immunocytochemistry studies revealed decrease in e cadherin and a concomitant increase in vimentin level for all three cell lines upon treatment with thz. moreover thz significantly inhibited the cell migration, invasion and emt in mda-mb- , mcf- and cd + /cd cell lines by suppressing mesenchymal markers. in conclusion, these data suggest that thz might be a novel anti-proliferative and anti-metastatic agent for treatment of breast cancer. effect of seasonal temperature and humidity on urine density in children environmental heat and humidity are important factors affecting hydration status in childhood. hereby, we aimed to investigate the effects of seasonal climate changes on urine density of children living in mediterranean climate, cyprus. healthy - year children's ( girls, boys) age, sex and urine density results were collected retrospectively for three consecutive years. the correlation of urine density with each seasonal and months' average temperature and humidity has been analysed. the urine density results had a positive correlation with temperature (r = . , p = . ) and a negative correlation with humidity (r= À . , p = . ). mean urine density in spring was higher than that of autumn (p = . ) and winter (p = . ). mean value of summer was higher than autumn (p = . ) and winter (p = . ). - months age group had lower urine density. evaluation of urine density based on gender and puberty revealed no statistically significant difference. seasonal mediterranean climate changes have an impact on urine density in children which may affect hydration status especially in infants < yrs of age. during high temperature seasons ensuring adequate water intake is essential in this age group in mediterranean climate. p-mis- implementation related to the use of antibiotics and data sources by community pharmacists in north cyprus as the resistance to antibiotics is gaining importance in today's world;the solution to this problem is possible through a common consciousness of the doctor who prescribes antibiotics,the pharmacist who sells and the patient who consumes antibiotics. irrational use of drugs is an economic and medical problem in many developed and developing countries around the world.the aim of this study is to determine the sales ratio of non-prescription antibiotics in pharmacies which is the biggest category of the antibiotic group sold as well as the indications that lead to its' prescription. eighty-four pharmacies out of pharmacies located in north cyprus were involved in the study with %stratified systematic sampling, questionnaires were filled and a consent form was signed by the participating pharmacists. the pharmacists involved in the study stated that non-prescribed antibiotics were demanded from the pharmacists and all except two ( . %),responded positively to this demand. it has also been identified in the study that . % of the daily sale of antibiotics in the first half of the year was non-prescribed. the most purchased antibiotics either with or without prescription was found to be the penicillin and its derivatives with . % and upper respiratory tract with . %. when the level of selfawareness of the pharmacists was examined, the rate is found in north cyprus to be ( . %),compared with the studies conducted in greece,italy,malta and spain % and egypt . %that designated the non prescribed antibiotics purchased from the public pharmacies. the rate of sale of non-prescribed antibiotics in north cyprus has been found to be at a higher level compared to the rates in many developed and developing countries. furthermore, the upper respiratory tract infections are amongst the most common viral causes which lead to a high consumption of both prescribed and non-prescribed antibiotics. this study was supported by turkish viral hepatitis prevention society. acrylamide has cytotoxic, antiproliferative and apoptotic effects on human lung adeno carcinoma cell line a acrylamide (aa), a widespread substance in many fields, forms in foods during high temperature processing such as baking, roasting, frying. aa is a potent neurotoxic, genotoxic and clastogenic agent being a strong electrophile and forming adduct with biological molecules or potent nucleophiles. up to now, several studies confirmed the toxicity of acrylamide to several organs. on the other hand, aa is reported to have inhibition effects both on proliferation and differentiation of different cancer cells in a time and dose-dependent manner. in addition, natural and synthetic acrylamide derivatives are also used as potent anti-cancer agents. moreover, inhibition concentration (ic ) values of aa against these cancer cells have not been investigated in detail yet. thus, the goal of this study is to investigate the cytotoxicity of aa on a cells including with ultrastructural and morphological effects. ic value of aa on a cells for h was detected with mtt ( -( , -dimethyl- -thiazolyl)- , -diphenyl- h-tetrazolium bromide) colorimetric assay. we evaluated morphological changes under confocal microscopy and ultrastructural changes under transmission electron microscopy (tem). our results demonstrate that aa inhibits the proliferation of a cells in dose-dependent manner and ic on a cells was found to be . mm for hours. confocal microscopy evaluations showed that aa caused nuclear condensations, fragmentations, cytoskeleton lacerations and membrane blebbing. tem results revealed membrane blebbing, chromatin condensations and cell shrinkage. although aa is a probable carcinogen substance, it drastically inhibited cell viability in dose-dependent manner. from microscopic assessments, aa is suggested to induce apoptosis in a cells. in conclusion, the present study confirms the high potential of aa for cytotoxic, antiproliferative and apoptotic activity on a cells. however, appropriate aa dose is critical to prevent its possible adverse effects. effect of hemolysis and lipemia on some immunochemical tests in beckman coulter unicell dxi immunoassay analyzer c. yilmaz, s. yildiz, m. senes, v. fidanci, d. y€ ucel ankara training and research hospital, ankara, turkey the aim of the study was to investigate the effects of in vitro hemolysis and lipemia on immunoassays studied by the beckman coulter unicell dxi immunoassay analyzer. we prepared a serum pool without hemolysis, lipemia and icterus. baseline serum pool concentrations of tests were measured by the beckman coulter unicell dxi . d _ ifferent serum pools, six for hemolysis and five for lipemia, were spiked with increasing concentrations of hemoglobin ( . , . , . , . , . and . g/l hemoglobin) and intralipid ( . , . , . , and g/l intralipid). the hemolysate was prepared by osmotic shock method. intralipid ( %, baxter, deerfield, il) was used to mimic the effect of lipemia. the hemolysis (h), lipemia (l) and icterus (i) indices were measured on beckman coulter au . after spiking the pools, the tests were measured again in duplicate on beckman-coulter dxi analyzer. a change of % from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. we observed a positive interference due to hemolysis for folat, vitamin b , testosterone and by lipemia for cortisol. there was a negative interference of hemolysis for ca . , ca , ca . , insulin, pth and e , and of lipemia for progesterone, ca . , vitamin b and pth. we found clinically significant effect (>total analytical error) of hemolysis on folate and insulin, and lipemia on cortisol. investigation of the effect of two different p mapk inhibitors in rats subjected to isoproterenol-induced acute myocardial injury: an experimental study objective: acute myocardial infarction is a serious acute condition. in the current study, we aimed to investigate the possible effect of two different mitogen-activated protein kinase (p mapk) inhibitors in rats subjected to isoproterenol (iso)induced myocardial injury. materials and methods: a total of male wistar-albino rats were equally and randomly seperated into four groups as follows: control, iso, iso plus sb andiso plus tak- . treatment agents were orally administered and myocardial injury was induced by subcutaneous injection of iso. serum cardiac troponin-i (ctni), ischemia modified albumin (ima), heart fatty acid binding protein (hfabp) levels and paraoxonase- (pon- ) activity, tissue tos (total oxidant status), tas (total antioxidant status), tt (total thiol), tumor necrosis factor-a (tnf-a) levels, superoxide dismutase (sod) and glutathione peroxidase (gsh-px) activity levels were measured. tissue mrna levels of nf-jb, p mapk and nuclear factor erythroid -related factor (nrf ) were analyzed. heart tissues were also immunohistochemically and histopathologically evaluated. results: both compounds have led to a decrement in myocardial damage, apoptosis, ctni, ima, hfabp, tos, and tnf-a levels, nf-jb, p mapk, phosphorylated c-jun n-terminal protein kinase (pjnk / ) expressions. on the other hand, the applied treatment increased sod, gsh-px, tas and tt levels, as well as phosphorylated extracellular signal-regulated kinase (perk / ) and nrf expressions. conclusion: data established from the current study suggest that administered agents have protective effect against cardiac injury induced by iso, which was more prominent in rats received sb treatment. p mapk inhibitors may constitute a useful choice as cardioprotective agents due to their antiinflammatory, antioxidant and anti-apoptotic effects. keywords: _ isoproterenol, myocardial infarction, myocardial ischemia, p mitogen-activated protein kinases, sb , tak- . silicosis composes the vast majority of occupational lung diseases. silicosis, caused by inhalation of crystalline silica, is a chronic lung disease characterized by parenchymal nodules and pulmonary fibrosis. the susceptibility of patients with silicosis to infection is thought to be due to toxic effects of silica on pulmonary macrophages. ada activity is considered as a nonspecific marker of t cell activation and cellular immunity. this study aimed to compare the serum ada activity in silicosis patients with spirometric values. in this study there were males in each groups which contained patients with silicosis (group ), individuals having similar symptoms with silicosis from same occupational area (group ) and healthy subjects (group ). routine hematological and biochemical parameters were also measured. the serum ada activity and spirometric values (fev , fev %, fev /fvc, fev / fvc%, fef - and fef - %) were compared. the average age of group , and are . ae . , . ae and ae . years, respectively. there was a significant difference between group and in terms of the ada level (p < . ). there was a negative correlation between ada activity and fev , fev %, fev /fvc, fev /fvc%, fef - values. elevated serum ada activity has been shown in many diseases with induced cellular immunity. despite initially toxic effects were lead to a little immunological reaction in patients with silicosis, continuation of this immunological response is important in some chronic manifestations of silicosis. the release of chemotactic factors and inflammatory mediators cause the migration of polymorphonuclear leukocytes, t lymphocytes and macrophages. in this study, the ada activity was significantly higher in patients with silicosis than others. increased immunity in patients with silicosis is being considered, increasing ada activity might be help of earlier recognition of these patients and to take better quality of life. atlantic salmon (salmo salar l.) is an important model system in evolutionary and conservation biology that provides fundamental knowledge into population persistence, adaptive response and the effects of anthropogenic change. the role of behavioral and body size variation in environmental adaptation of atlantic salmon is well known, by contrast, the underlying biochemical mechanisms are largely unknown. intracellular proteases, such as cathepsins b and d in lysosomes and calpains and proteasome in cytosol, due to their metabolic and regulatory role may contribute to phenotyping speciation of salmon young. we examined the activity of intracellular proteolytic enzymes in skeletal muscles of atlantic salmon parr from two local habitats of the varzuga river (the main channel and small tributaries) differing in hydrological and feeding parameters. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from varzuga river (kola peninsula, russia). it is known that salmon parr originated from a common hatch became phenotypically divergent during the settle in the biotopes. reliable difference in studied enzyme activities in the salmon parr from two local habitats was found; furthermore, calpain and cathepsin b proteolytic activities were found to negatively correlate with parr body size. muscle proteolytic activity data support an idea on protease contribution to environmentally-driven adaptation and speciation process in fish. the work was supported by the russian scientific foundation, project no. - - . the phylogenetic analyses of anthriscus (apiacea) species from turkey based on non-coding "trn" regions of chloroplast genome p. yilmaz sancar , m. tekin , s. civelek firat university, elazig, turkey, cumhuriyet university, sivas, turkey anthriscus pers. (apiaceae) species belongs to apiaceae family and is represented by genus on the world and by genus in turkey. anhriscus species are used extensively for treatment various disease such as asthma, alzheimer and show anti-tumoral, anti-microbial, antioxidant features. for determining exact species which treat disease it is necessary sorting species correctly with molecular markers to support morphological features. anthriscus species were defined by examining insufficient quantity of samples in turkey flora. besides, no detailed study was found in our country after flora study. for this reason a revision study was made with the aim of solving some systematical problems in by tekin. the result of the study provided important contribution to the systematic of the species in turkey. however a molecular study was also required for building the obtained results on a more solid ground. in this study, the aim to reveal systematic and phylogenetic relationship among species of anthriscus in turkey, by using trnl-f region in chloroplast genome. dna was isolated by ctab method and amplified in pcr by using e-f primaries. the obtained data was evaluated by mega . program and phylogenetic tree was prepared by using maximum likelihood method. according to the phylogenetic tree that we prepared by using the sequence line up of trnl-f section, it was observed that a. cerefolium, a. caucalis and a. tenerrima species completed their speciation and an isolation with other species in terms of speciation was provided. it was also observed that a. kotchi, a. sylvestris subs. sylvestris, a. sylvestris subs. nemarosa and a. lamprocarpa'nın provided hybridization among themselves but they did not complete their speciation. it was determined that a.lamprocarpa var. chelikhii which is one of the two different varieties of a. lamprocarpa is actually a new sub-species. this fact was supported by molecular data obtained from the study we made after morphologic data. introduction: excessive production of androstenedione can becaused by defects of adrenal steroid biosynthesis, tumors of ovarian and adrenal origin, polycystic ovarian syndrome, increased peripheral sensitivity to androgens, and increased peripheral production of androgens. most epidemiologic studies use enzyme-linked immunosorbentassay (elisa) to measure sex steroid hormones because they have acceptable turnaround times and arerelatively inexpensive. mass spectrometry-based methods are currently the most specific quantitative analytical methods for steroid determination. mass spectrometry methods are independent of matrix effects or cross-reactivity. in this study, a new liquid chromatography-tandem mass spectrometry (lc-ms/ms) method was developed. materials and methods: for serum androstenedione measurement, ll of internal standard (d - deoxycortizol) in methanol was added to ll standart or serum and centrifuged at . rpm for minutes to remove the precipitated proteins. supernatant was transferred to clean tubes and this procedure was performed twice. the supernatant was collected and dried under a nitrogen gas flow at • c and dissolved in mobile phase. ll was injected in to the ultra performance liquid chromatography analytical column for chromatography. elisa study was conducted with drg (lot. no. k ) brand kit. results: method comporison between lc-ms/ms and elisa was found slope value , , intercept value À . and r² value . . the regressione quation was elisa= À . + . lc-ms/ms. discussion and conclusion: method comparison study presented higher results in elisa compared to lc-ms/ms. in our opinion, this might due to the interference in elisa systems. our lc-ms/ms method allows rapid, sensitive and specific determination of androgens in plasma and serum.the specificity of liquid chromatography-tandem mass spectrometry (lc-ms/ ms) offers advantages over immunoassays. heparins play an important role in cell growth, differentiation, migration and invasion. however, the molecular mechanisms of heparin mediated cellular behaviors are not well defined. to determine the effect of heparin on gene expression, we performed a cdna microarray in a hepatocellular carcinoma cell line and found that heparin regulates transcription of genes involved in glucose metabolism. in this study, we showed a new role of heparin in the regulation of thioredoxin interacting protein, which is a major regulator of glucose metabolism, in hepatocellular carcinoma cell lines. we determined the importance of a unique carbohydrate response element located on its promoter for the heparin-induced activation of thioredoxin-interacting protein and the modulatory role of heparin on nuclear accumulation of carbohydrate response element associated proteins. we showed the importance of heparin mediated histone modifications and downregulation of enhancer of zeste polycomb repressive complex expression for heparin mediated overexpression of thioredoxininteracting protein. when we tested biological significance of these data; we observed that cells overexpressing thioredoxininteracting protein are less adhesive and proliferative, however they have a higher migration and invasion ability. interestingly, heparin treatment increased thioredoxin-interacting protein expression in liver of diabetic rats. in conclusion, our results show that heparin activates thioredoxin-interacting protein expression in liver and hepatocellular carcinoma cells and provide the first evidences of regulatory roles of heparin on carbohydrate response element associated factors. this study will contribute future understanding of the effect of heparin on glucose metabolism and glucose independent overexpression of thioredoxin-interacting protein during hepatocarcinogenesis. prolidase activity in chronic obstructive pulmonary disease and asthma t. g€ uc ßl€ u , h. s€ urer , g. bilgin , d. y€ ucel ankara training and research hospital, medical biochemistry department, ankara, turkey, ankara training and research hospital, chest diseases department, ankara, turkey chronic obstructive pulmonary disease (copd) is a consequence of an underlying chronic inflammatory disorder of the airways that is usually progressive and causes dysregulation in the metabolism of collagen. and asthma is a disease where there is an accumulation of collagen in the reticular basal membrane of the airway leading to chronic inflammation. prolidase has an important role in the recycling of proline for collagen synthesis and cell growth. we measured and compared prolidase activity in healthy individuals with copd and asthma patients to find out that whether its activity might reflect disturbances of collagen metabolism in the patients. patients with copd, patients with asthma and healthy control subjects with similar age range and sex were included in our study. the patient and control groups do not have any other chronic disease. serum prolidase activity was measured in the patient and control groups. ferritin and alpha- antitrypsin concentrations were also compared. there was no significant difference between serum prolidaz activities of asthma and copd patients. serum prolidase activities of both copd and asthma patients were significantly lower than those of the control subjects (p < . ). there was no significant difference for ferritine and alpha- antityripsin levels between the groups. the prolidase activity is significantly lower in asthma and copd patients comparing with control subjects. the collagen metabolism may be undergone to a change in these patients. hence, there may be an effect on the accumulation of collagen in the reticular basal membrane. the results suggest that collagen turnover are altered by the development of copd and asthma in human lungs, and prolidase activity may reflect disturbances of collagen metabolism in these pulmonary diseases. monitoring serum prolidase activity may be useful in evaluating fibrotic processes and in the chronic inflammatory lung diseases in human. acyclovir molecule in the active site of e. coli purine nucleoside phosphorylase (on the basis of x-ray study) i. kuranova , , v. timofeev , , n. zhukhlistova , y. abramchik , t. muravieva , r. esipov shubnikov institute of crystallography of fsrc "crystallography and photonics" ras, moscow, russia, national research centre "kurchatov institute", moscow, russia, shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia e. coli purine nucleoside phosphorylase (pnp), which catalyzes the reversible phosphorolysis of purine ribonucleosides, belongs to the family i of hexameric pnps. due to key role in the purine sulvage pathway pnps are attractive targets for drug design against some pathogens. they also used widely in biotechnology for the synthesis of nucleoside analogues as well as for the activation of the prodrugs in anti-cancer gene therapies. the acyclovir (acv), acyclic derivative of guanosine, is antiviral drug for the treatment of some human viral infections. the crystalline complex of e. coli pnp with acyclovir was prepared by co-crystallization using counter diffusion in capillary through the gel layer. the set of x-ray data at k from single crystal grown in space (sp. group p ) was collected on the spring- synchrotron-radiation facility (japan) and the structure was solved at . a resolution, using the molecular replacement method (pdb id i c). acv molecule was located in the nucleoside binding pocket of the enzyme in two conformations. the phosphate binding site was occupied by so ion. the hydrogen bonds network and hydrophobic interactions stabilising acv molecule in the active site as well as the conformational changes upon ligand binding were described. the comparison of e. coli pnp/acyclovir complex and the similar complexes of bacillus subtilis pnp (pdb id da ) and human pnp (pdb id pwy) allowed to establish the peculiarities of acv binding of in the e. coli enzyme. gonadotropins are glycoprotein hormones that regulate normal growth, sexual development, and reproductive function. these are large, up to kda proteins, which are synthesized and secreted by the gonadotropic cells of the anterior pituitary gland. these hormones may vary in the level of glycosylation depending on the tissue and the metabolism cycles. follicle-stimulating hormone (fsh) and upon binding to fsh receptor, a g-protein coupled receptor (gpcr), regulates the development, growth, pubertal maturation, and reproductive processes of the body. human chorionic gonadotropin (hcg) and luteinizing hormone (lh) act via a shared gpcr (lh receptor) and regulate mechanisms essential for ovulation, early pregnancy and placental function in females as well as spermatogenesis and testosterone production in males. activation of gpcrs by these hormones can be measured by monitoring formation of cellular cyclic adenosine monophosphate (camp). the level on camp was measured using a f€ orster resonance energy transfer (fret)-based biosensor tepacvv (h ) kindly provided by dr, kees jalink. the biosensor was expressed using the developed bacmam gene delivery system (recombinant baculoviruses carrying the transgene under a strong mammalian promoter). kgn cells expressing the fsh receptor and cos cells expressing the lh receptor served as study objects. monitoring of specific gpcr activation in living cells, allows detection of only the biologically active agonists, which has real impact in quantification of large hormones. differences in levels of hormone glycosylation may affect their biological function. investigation of this phenomena is planned for near future. detection of biological activity of gonadotropins is of importance for pharmaceutical industry, where today the concentration of recombinant proteins is mostly estimated using immunological assays only. development of a colorimetric aptasensor for the detection of peanut allergen protein ara h in food samples b. bora ege university, izmir, turkey food allergy, especially peanut allergy is a life-threatening problem, and severe reactions against these foods can be observed. since unintnded consumption of non-labeled foods is the most dangerous risk, any residual allergen protein should be tested and labeled by the manufacturers. an aptamer based colorimetric test is a powerful alternative to commercially available rt-pcr and elisa test kits. the main objective of this study is to develop an aptamer based colorimetric test fort he detection of major peanut allergen protein ara h . ara h aptamer was used to recognize any residual peanut major allergen protein ara h in food samples. recombinant ara h protein was produced and puirifed to be used as a target. ara h aptamer was used in combination with a blocking sequence, to prevent non-specific binding event, a biotinylated complementary strand to the blocking sequence, and finally strp-hrp interaction in order to facilitate colorimetric reaction. optimal blocking sequence length was optimized and introduced to the site of aptamer sequence to construct an aptamer-hairpin structure. liberation of the blocking sequence allows biotinylated complementary strand to bind to the blocking sequence and consequently str-hrp conjugate to achieve color development that is proportional to the target concentration. since, the aptasensor will be used for the detection of ara h in food samples, total protein extraction from chocolate samples was also optimized. in order to lower the detection limit of aptasensor, aptamer coupled magnetic bead based pre-enrichment assay was aslo optimized for the total protein extraction. as a result, a sensitive, fast and reliable aptamer based colorimetric assay was developed for the detection of peanut allergen protein from food samples. moreover, the assay has the advantages like ease of application and low cost which makes the assay a promising and a powerful alternative to commercially available rt-pcr and elisa tests. the association between lipid parameters and waist circumference in female university students in turkey s. ozen, a. cort sanko university, department of nutrition and dietetics, gaziantep, turkey a high waist circumference is associated with an increased risk for type diabetes, dyslipidemia, hypertension, and cvd in patients with a bmi in a range between and . kg/m . monitoring changes in waist circumference may be helpful, in addition to measuring bmi, since it can provide an estimate of increased abdominal fat even in the absence of a change in bmi. objective of the study was to find an association between plasma lipid profile and anthropometric parameters (waist circumference percentage of body fat and body mass index (bmi)) in abdominal obesity in turkish university students. lipid profile and anthropometric parameters of obesity were studied in a sample of women. students with high bmi (> ) had higher values of low-density lipoprotein (ldl), triglycerides (tg) and cholesterol (c) than students with low bmi (< ) but these differences were not significant. high-density lipoprotein (hdl) levels were non-significantly higher in low bmi (< ) student group. waist circumference, percentage of body fat was higher in high bmi (> ) group than low bmi (< ) group. waist circumference, percentage of body fat was positively correlated with bmi in both samples (bmi (> ) and bmi (< )). students were grouped depend on their waist circumference. healty individuals who had lower than cm waist circumference had decreased tg levels compared to cardiovascular risk group who had higher waist circumference than cm. this study shows an association between waist circumference, percentage of body fat, body mass index and lipid parameters in young female university students. with regard to the relationship, the screening females for central obesity to prevention of cardiovascular disease are recommended. a new biotechnological product from propolis with low allergen: anti-inflammatory effect propolis is extensively used in food industry due to its special medical properies (antioxidant, antimicrobial, antiseptic, antibacterial, anti-inflammatory and antimutagenic effects). even these positive properties it may cause some allergic reactions in consumers with allergic predispositons. previously, we demonstrated that biotransformation of propolis by some special strains of lactobacillus plantarum ( , , aatc strains) might decrease the allergenic molecules in propolis. in this study, we aimed to investigate the effect of biotransformation of popolis on it's antiinflammatory activities. before biotransformation, propolis samples were treated with different solutions ( % ethanol and polyethylene glycol -peg %) and different method (ultrasonic treatment w/ o c/ minutes) in order to facilitate solvation of solid samples which are very dense and not suitable for fermentation. fermantations were performed at o c/ hours under constant agitation conditions. the anti-inflammatory activity was determined in-vitro conditions using hyaluronidase's analysis and the xanthine oxidase activity. the highest inhibition (%) of radicals produced by xanthine oxidase was determined in solid samples treated by peg prior to biotransformation and using of l.plantarum strain during fermentation ( . %), followed by liquid samples treated by ultrasonic method prior to transformation ( . %). concernig the results of hyaluronidase activity (%) inhibitions, the best value were determined in the solid sample treated by peg prior to biotransformation and using of l.plantarum strain during fermentation ( . %). results indicated that the anti-inflammatory activities of analysed samples are quite high and depending of used extraction methods prior the biotransformation and used specific strain of l.plantarum could be optimized in terms of other required parameters. faceanti-mullerian hormone is not predictive for poor neonatal outcome aim: anti-mullerian hormone (amh) is a growth factor specific to ovaries. it is commonly used to predict ovarian reserve and outcomes of fertility treatments. recently, low levels of amh have been shown to be related to hypertensive diseases of the pregnancy and the risk of preterm labor. the aim of this study was to investigate the diagnostic performance of amh levels of mothers to predict poor neonatal outcome in term pregnancies and the relationship between amh and birthweights of the newborns. materials and methods: patients, having delivery beyond weeks, and who did not have any other medical problems were included in the study. the patients had normal g. oral glucose tolerance test results. they were divided as groups, based on their newborns' birthweight as " g. and g.". level of amh was determined by elisa method. results: there was not any relation with the amh of the mothers and the poor neonatal outcome of the newborns, in all groups. also no siginificant difference was observed in amh levels of the patients having delivery in early term and late term periods. when the patients of the same group were evaluated; amh levels were irrelevant to age, gravidy, delivery week, body mass index, the weight gain during pregnancy, and poor neonatal outcome. conclusion: amh is not a predictive factor for poor neonatal outcome and it is not a determinant of the weight of the newborn. objectives: the aim of the study was to investigate the effects of differing amounts of hemolysis on serum high sensitvity troponin i (hs-tni), ck-mb mass and myoglobin measurements. materials and methods: we prepared serum pools having troponin i, ck-mb and myoglobulin concentrations at low ( . ng/l, . ng/ml and . ng/ml respectively), normal ( . ng/l, ng/ml, . ng/l respectively) and high ( ng/l, ng/ml, g/ml respectively) values. the osmotic shock method was utilized to prepare a hemolysate. hemolysate was added into serum pools increasing concentrations of hemoglobin ( . , . , . , , . and . g/l hemoglobin). troponin i, ck-mb (mass) and myoglobin concentrations were measured in duplicate by beckman coulter access analyzer. the hemolysis indices were measured on beckman coulter au . a change of % from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. results: we found a positive interference due to hemolysis for ck-mb (mass) at low concentrations ( . ng/ml), and a negative interference for myoglobin at low concentrations ( . ng/ml) and high concentrations ( ng/ml). conclusions: ck-mb increase and myoglobin decrease in hemolyzed samples with hemoglobin ≥ . g/l, but the bias might not be clinically significant (< total analytical error) in samples. a retrospective study to determine a reliable marker for selective screening of pompe disease lysosomal storage diseases (lsd) are rare inherited metabolic disorders caused as consequence of a deficiency in a specific enzyme required for lysosomal function. pompe disease is one of these disorders with deficiency of a- , glycosidase enzyme with an incidence of : , - : , . as enzyme replacement therapies are available nowadays, early diagnosis is crucial and selective screening is a rational method to reach pompe patients among people who administer to healthcare with lsd suspected symptoms. this study aims to examine the relationship between basic biochemistry parameters and a- , -glycosidase activities retrospectively, in order to find a key parameter for selective screening of pompe disease. for this reason a- , glycosidase, creatine kinase (ck), creatine kinase-mb (ck-mb) activities calcium, phosphate levels of those who had been suspected to be lsd patients and administered to our laboratory for analysis are examined retrospectively. out of patients's examined, of them were diagnosed with pompe disease depending on clinical findings & low a- , glycosidase activity. enzyme activities of pompe patients were . nmol/ml/hour as lsd suspected patients'activities had a mean of . nmol/ml/hour (p = . ).comparison of ck activity was compared results showed significant difference between pompe patients and lsd suspected patients. even though ck activity levels of the lsd suspected patients were much higher ( vs - u/l) than reference interval, the levels of the pompe disease patients' were still more than twice of the lsd suspected group ( vs u/l, p = . ). ck-mb, ca, p levels didn't show a significant difference. a strong (-) correlation (p = . r=À . ) was observed between a- , -glycosidase and ck activities (n: ). selective screening is a rational way to diagnose rare diseases. this study's results show that ck activity can be used as a key parameter to determine patients for selective screening of pompe disease within lsd suspected population. the functional effect of stem cells on the reproductive organs infertitility is considered as a major health problem of recent century. importance of stem cell is increasing so it is searched new features and supposed to be involved in the infertitility treatment where oxidative stress and apoptosis play importany role. we aimed to investigate the beneficial effect of the stem cells related to free radicals and cell death on testis and ovary. biopcy model of wound healing was created in the rat testis and ovary with ppd syringe where stem cells were delivered by injection. rats were divided into four groups including controls, sham, wound healing and wound healing with stem cell. after the creation of the wound, bone marrow-derived mesenchymal stem cells from the tibia of the mature rats and medium were administered to ovaries and testes. following the applications, ovary and testis samples were investigated for oxidative stress and apoptosis by immunohistochemistry. in comparison with the medium and stem cell applications without a medium support, it was meaningfully determined that healing effect in testicles and ovaries were spotted specifically on the seven day. tissues were analysed for these staining by h-score and h-score results were determined using one-way anova test statistically. our results show the positive effects which clinic applications can bring by displaying the great contribution of the stem cell application in the treatment of testicle and ovary damage. these findings suggest that transplantation of the mesenchymal stem cells may help to promote better enviroment for the reproductive organs by the effect on oxidative stress and apoptosis. the further studies of these results in the molecular level can lead the way to solve the problem of infertility, to increase the percentage of success in the ivf and icsi techniques and more importantly to perform a differentiation from a somatic cell to a germ cell. the antimicrobial activity of ( h)-furanone derivative on staphylococcus aureus nosocomial infections caused by methicillin-resistant staphylococcus aureus strains are known to be a reason of many infectious diseases like osteomyelitis, endocarditis, sepsis etc. being organized in biofilms these bacteria become extremely resistant to antimicrobials and host immune system leading to difficulties in treatments. here we report the effect of ( h)-furanone derivative possessing sulfonyl group and l-menthol moiety (f ) on biofilms formed by s. aureus atcc and mrsa cells. while exhibiting relatively high minimal inhibiting concentration -mic ( mg/l), clear synergy with a number of antibiotics was found in the checkerboard assay. thus, in the presence of mg/l of f the mic of kanamycin was decreased -fold, and the mics of both erythromycin and ampicillin were lowered -fold. at the concentration of mg/l f also completely inhibited the biofilm formation by s. aureus; the cell growth was suppressed by two orders of magnitude as judged by differential fluorescent staining with syto and propidium iodide. the addition of f to preformed h-old biofilms increased the fraction of red-stained (dead) cells of both s. aureus atcc and mrsa strains uniformly throughout the whole profile of the biofilm. the quantitative analysis of clsm microphotographs revealed that f at concentration of mg/l led to death of up to % of biofilm-embedded cells. this fact suggests that f efficiently penetrates into the biofilm matrix and kills the cells without visible damage of biofilm structure. in summary, furanone f seems to be a promising compound for drugs design to treat biofilm-embedded s. aureus. this work is supported by the russian science foundation, project № - - and the german academic exchange service (№ ). pneumonia is an inflammatory lung disease which can be associated with inadequacy of host defense system and the proliferation of various pathogenic microorganisms into the lower respiratory tract. community acquired pneumonia (cap) is one of the leading causes of death in elderly. the incidence of pneumonia in people aged and over is - times more than young adults. creactive protein (crp) is an acute-phase protein of hepatic origin that increases following interleukin- secretion by t cells and macrophages. procalcitonin (pct) is a peptide precursor of the hormone calcitonin, the latter being involved with calcium homeostasis. it is composed of amino acids and is produced by parafollicular cells (c cells) of the thyroid and by the neuroendocrine cells of the lung and the intestine. the level of pct rises in a response to a proinflammatory stimulus, especially of bacterial origin. the aim of this study was to compare crp and pct levels in young and elderly patients with pneumonia. recently diagnosed young and elderly patients with pneumonia and their respective aged matched controls (n = , n = ) were enrolled this study. crp and pct levels were by immunoturbidometric and by elisa methods respectively. crp and pct levels for young control and patients and elderly control and patients respectively are . ae . mg/l, . ae . ng/ml, . ae . mg/l, . ae . ng/ml, . ae . mg/l, . ae . ng/ml and . ae . mg/l, . ae . ng/ml. young patients with pneumonia have significantly higher crp and pct levels than their controls (p < . and p < . ). elderly patients with pneumonia have significantly higher crp levels than their controls (p < . ). crp and pct are important markers in the diagnosis of pneumonia. effect of serum albumin concentration on total and ionized calcium z. adiyaman, c. yilmaz, s. a. peker, d. y€ ucel ankara training and research hospital, ankara, turkey objective: the aim of the study is to investigate in vitro effect of albumin concentration on total and ionized calcium concentrations. materials and methods: a serum pool with low albumin ( . g/dl) and normal calcium ( . mg/dl) concentrations was prepared from leftover sera. from this serum pool, two parts, each of ml were aliquoted. purified albumin, . g, was added to one of these pools and albumin concentration was determined as . g/dl. the low and high albumin pools were mixed at different ratios and pools with . , . , and . g/dl albumin concentrations. total calcium and albumin concentrations of these pools were measured at a beckman-coulter au analyzer and ionized calcium was measured at a radiometer abl blood gas analyzer in triplicate. total and ionized calcium concentrations were evaluated as compared to those of the original pool with an albumin concentration of . g/dl. results: total calcium concentrations are increased with the increasing albumin concentrations: . %, . %, . %, and . %, respectively. whereas, ionized calcium concentrations were decreased with increasing albumin: . %, . %, . %, and . %, respectively. conclusions: when total allowable error limits based on biological variation were considered, total calcium concentrations are significantly increased at > g/dl albumin concentrations. ionized calcium is significantly affected by . g/dl and over albumin concentrations. a regression equation based on albumin concentration may be useful for corrected ionized calcium concentrations. relationship between lipoprotein (a) and hba c in patients with type ii diabetes , is a complex lipoprotein consisting of ldl and apolipoprotein(a). lp(a) is a risk factor for coronary artery disease and stroke. the relationship between lp(a) and diabetes mellitus is not clear. in this study, the relationship between lp(a) and glycemic parameters such as hba c and fasting glucose concentration was investigated. lp(a), hba c, fasting glucose, triglyceride, total cholesterol, ldl-and hdl-cholesterol concentrations were screened retrospectively from july to july . there were patients with these test results at the same time. the patients were grouped according to hba c values: group i < . % (n = ), group ii . - . % (n = ), and group iii > . % (n = ). the relationship between these parameters were statistically within each group and all groups. there was not a statistically significant difference between the lp(a) concentrations of group i and group ii. lp(a) concentrations of group i and ii were significantly higher than those of group iii.. _ in total, lp (a) was negatively correlated with hba c (r = . ; p < . ), but there was not a significant correlation with fasting blood glucose. _ in groups, there was a significant and negative correlation between lp(a) and fasting glucose in only group i. the negative correlation between lp(a) and glycemic parameters is interesting in patients with diabetes. despite lp(a) is an independent risk factor for cardiovascular diseases, on the contrary to expectations, lp(a) concentrations are decreased in diabetes. effect of blood collection through intravenous lines on hemolysis erroneous results are one of the most important causes of medical errors and may lead to unnecessary investigations or inappropriate interventions. total testing process consists of preanalytical, analytical and postanalytical phases. hemolyzed specimens that one of the most common source of preanalytical errors are frequently observed in laboratory practice and associated with incorrect laboratory results. blood collection through intravenous lines frequently results in hemolysis especially at eds and icus. in this study, we aimed to compare the effect of blood drawing by using bd luer-lock adapters and injector on the hemolysis rates at the ed. patients who has been admitted to the ed were included in this study. all samples were drawn from newly inserted iv lines. the first blood sample was drawn with injector and the second one was drawn with luer-lock adapters to vacuum tubes. after the centrifugation routine chemistry tests and hemolysis indices were analysed on a beckman coulter au analyzer for each serum tube. the statistical significance of differences between two tubes was calculated with paired samples t test and statistical significance was accepted as p < . . there were statistically significant differences between the two groups of tubes for the following parameters: ldh, ck, ast, k + , total bilirubin, protein, albumin, alp, calcium and hemolysis index (p < . ). the use of luer-lock adapters instead of injector could reduce the hemolysis rate. because of it reduces false results and unnecessary investigations, this approach will be more appropriate and cost-effective in ed. hemolysis and test rejection: are we following a reliable process? introduction: in laboratories, some blood samples are rejected due to hemolysis. we usually cancel only some of the tests that are affected by hemolysis. however, the frequency of the test cancellation may be relative. each test is affected in different degrees of hemolysis; some of them are not even affected at all. in this study, we aim to investigate unnecessary cancellations and explain the relationship between hemolysis and test results according to their kit inserts. materials and methods: we measured hemoglobin levels of hemolyzed serum using drabkin method (abbott). interference studies are conducted using clsi protocol nccls ep -p is written in kit inserts. target values ( %) and their change due to different degree of hemolysis have been defined. results: hb concentration ranges of hemolyzed sera were found from to mg/dl. according to kit inserts, aspartate aminotransferase (ast) test results deviate . % from the target when the degrees of hb are mg/dl. when the degree of hemoglobin is mg/dl, the test strays about . %. potassium levels increase ( %) at mg/dl hb while this increase reaches to . % at mg/dl hb. sodium, calcium, ck, crea, total bil, lipase are not significantly affected even at mg/dl. in lactate dehydrogenase (ldh) tests, test reporting is not allowed at any hemolysis level. alt increases %, at the mg/dl hb. ast and potassium results were excluded from patients' reports even though those samples had low hb. some of them were reported despite of excess hemolysis. some tests are even blocked without ever being studied. discussion: prior to the approval of the lab specialist, technicians decide whether to cancel the tests affected by the hemolysis according to the visible hemolysis based on their personal knowledge. conclusion: we should use the hemolysis index, in which standards would be defined via guidelines. this way, all technicians and specialists could know which results are false. the dna-binding hu-proteins are present in all bacteria and belong to the family of nucleoid-associated proteins. these proteins can be considered precursors to eukaryotic histones. gene knockout of hu-proteins partially inhibits the growth of bacteria, their ability to resist various stressing factors and in some cases leads to their death. since the spatial structure of hu-proteins is highly conserved it is possible to create inhibitors that will affect them in a broad spectrum of pathogenic bacteria. in the present work the preparation of the recombinant hu protein from mycoplasma gallisepticum, crystallization of this protein, and x-ray diffraction study of this protein has been reported. the crystallization conditions for studying protein were found by the hanging-drop vapor-diffusion method. found conditions have been adapted to the counther-diffusion method in the capillary. the x-ray diffaction dataset from grown crystals have been collected using synchrotron radiation. d-structure of the hu protein from mycoplasma gallisepticum have been determined with a resolution. structural features of the investigated protein are described. this work is supported by russian scientific fund ( - - ). a novel sensitive disposable indium tin oxide (ito)-based electrochemical immunosensor was developed for simple, rapid and sensitive biomonitoring of sox . sox is a cancer biomarker and used for detecting of small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, skin cancer, prostate cancer, and breast cancer. in this study, indium thin oxide (ito) thin film was used as working electrode. carboxyethylsilanetriol was also used for electrode modifying so as to obtain self-assembled monolayers. the formed self-assembled monolayers were activated with -ethyl- -( -dimethylaminopropyl) carbodiimide (edc)/n-hydroxysuccinimide (nhs) chemistry. edc was used as a heterobifunctional crosslinker. nhs was used in conjunction with the crosslinker edc. anti-sox antibody was used as a biorecognition element and it was covalently immobilized onto the ito electrode modified with carboxyethylsilanetriol. immobiliztion steps were characterized by cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis), and scanning electron microscopy (sem). the optimal immobilization conditions for the best sensitivity of the new immunosensor were investigated. under optimal conditions, this immunosensor demonstrated a wide linear range ( . - pg/ml) with a detection limit as low as . ng/ml sox . furthermore, the developed sox immunosensor had good storage stability, repeatability and reproducibility. in this work, we successfully fabricated disposable ito thin film based electrodes for sensing the interaction between sox antigen and anti-sox antibody by electrochemical impedance spectroscopy and cyclic voltammetry. and our developed immunosensor has an acceptable performances for the detection of sox antigen, exhibits low detection limit, has selective and reproducible results in immunoreaction analysis. we are thankful for the support from t € ub _ itak (the scientific and technological research council of turkey, project number: z ). applying multiple linear regression model to determine the relationship between anti mullerian hormone with age, luteinizing hormone, follicle stimulating hormone and estradiol: a data mining study introduction: anti mullerian hormone (amh) has a widely used in our life because it is a good indicator of reproductive age to estimate the time of menopause. the purpose of this retrospective data mining study is the estimate of ovarian reserve by using amh and determines relationship between other indicators which are luteinizing hormone (lh), follicle stimulating hormone (fsh), estradiol and age. materials and methods: . women members were included this retrospective data mining study who were applying to acıbadem labmed laboratory. multiple regression analysis of age related changes of amh ( - ) and lh, fsh and estradiol were investigated. beckman gen ii elisa kit was used for amh and the technique of electrochemiluminescence and roche elecsys cobas analyzer were used for the measure of other hormones. results: amh shows meaningful correlation between lh, fsh, estradiol and age but also seen there is no correlation between progesterone. after the multiple linear regression analysiz amh= . -( . age)À( . fsh) + ( . lh)À( . estradiol) is detected and the model's r = . is also detected. conclusion: nowadays there are lots of methodology were developed the estimate the function of ovary and biological age of ovarian. age, fsh, lh and estradiol show ovarian reserve by indirectly. this study shows the mathematical relationship between amh and the other indicators and results are thought to lead to future developments. antioxidant and anticancer effect of artemisia absinthium extract on colon and endometrium adenocarcinoma cells plants have always been among the common sources of medicines that have many phytochemicals with various bioactivities, including antioxidant and anticancer activities artemisia absinthium (ar) has been used as an antipyretic, antiseptic, anthelmintic, tonic, diuretic, and for the treatment of stomachaches in turkish folk medicine. this study aimed to investigate antioxidant, cytotoxic, genotoxic and apoptotic effect of methanol extracts of ar activities on the human colon (dld- ) and endometrium (ecc- ) adenocarcinoma cell line. total phenolic, flavonoid content, and antioxidant activities were determined using suitable methods (abts, cuprac i.e). cytotoxic effects of ar on cells were determined by mtt and neutral red uptake assays. genotoxicity was evaluated by comet assay and, apoptosis induction were detected by apoptosis elisa and acridine orange staining methods at the half maximal inhibitory concentrations (ic ) levels. it was determined that extract have shown antioxidant activity in all tests and that they could be considered as a source of natural antioxidants. cytotoxic effects were concentration-time dependent. specifically, apoptotic and genotoxic effect increased at and lg/ml concentrations by hours. we found that ar extract had antiproliferative, genotoxic and apoptotic effects on the human cancer cell lines dld- and ecc- . however, further studies at molecular level are required to support our findings and to elucidate chemopreventive and chemotherapeutic effects of ar on colon and endometrium cancers. keywords: artemisia absinthium, antioxidant, anticancer, apoptosis, genotoxicity introduction: colorectal cancer is considered as a major gastrointestinal. this cancer is the second cancer related cause of death after lung cancer in worldwide. we designed a vaccine chimeric including cea and ca - against colorectal cancer (ce-ca). materials and methods: the construct were analyzed by bioinformatics softwares. in this study, the ce-ca gene was optimized using the codon bias of e.coli and synthesized by biomatik company. then construct (ce-ca) was cloned into an expression vector and recombinant constructs transferred to e.coli bl de bacterium and desired recombinant protein was expressed. recombinant protein was purified using ni-nta affinity chromatography. the content of secondary structures was obtained by circular dichroism (cd) spectrum. then recombinant protein was confirmed using western blot analysis and indirect elisa method. results: sds-page analysis showed that the recombinant protein was highly expressed and purified. western blot analysis confirmed recombinant protein. also cd spectrum confirmed predicted structures by bioinformatics tools. the elisa results showed significantly high affinity toward recombinant ce-ca protein. discussion: based on many studies, cea as potential immunogenic candidate could be considered in vaccine studies. also ca - is a cell-surface antigen that has significant increase of expression in colorectal cancer, thus as marker of colorectal cancer. based in available data, these two antigens, in combination can provide specificity for production of colorectal cancer vaccine. conclusion: these findings suggest that ce-ca as potential immunogenic candidate which could be considered in future vaccine studies and detection of colorectal cancer. flow cytometric cell cycle and apoptosis analyses of some wild animal species a. tas, e. koban bostanlar tubitak, marmara research center (mrc), genetic engineering and biotechnology institute (gebi), animal genetic and reproductive biology laboratory, kocaeli, turkey cell biobanking; more specifically cryopreservation of biological diversity, is promising as a tool to preserve wild animals as well as domestic ones via nuclear transfer. in this study, we investigated the viability and cell cycle characteristics of wild animal species (fallow deer, red deer, wild sheep, wolf, wild goat). auricular tissue samples were maintained in pbs+ %psa. tissues were seeded on mm petri dishes containing dmem/high glucose supplemented with % (v/v) fcs and incubated %co in air at % relative humidity and at °c. after seeding, the medium was unchanged for days and then it was changed in every days for days at maximum. once the cells were obtained; flow cytometric cell cycle and apoptosis analyses were done. in terms of apoptosis, all the groups showed high viability rates (over %) in culture when compared with the negative control ( %). the cell cycle comparisons were made between serum-starved cells and roscovitine treated cells, both for which untreated cells were used as control, which revealed different results for different species. there was no difference found between serum-starved cells and roscovitine treated cells for red deer and wolf. the serum-starved cells resulted in higher g /g phase for fallow deer and wild goat. on the contrary, roscovitine treated cells resulted in higher g /g phase for wild sheep. as a result; the cells obtained from wild animals had high viability and g /g phase rates. therefore, they may serve as a donor cell source for nuclear transfer studies.(grant: tubitak kamag, turkey, g ). the interaction of different types of antibiotics with endothelial cells in the presence of nanoparticles the interaction of nanomaterials with cells and lipid bilayers is critical in many applications such as phototherapy, imaging, and drug/gene delivery. the aim of this study was to investigate the interaction of nanoparticles (fe o ) or nanoparticles fused with different antibiotics with cell membranes in order to reveal changes in the membrane organization. endothelial cells were used to determine the effect of different antibiotics (gentamicin, kanamycin, amikacin, penicillin, polymyxin, neomycin, cefotaxime, bacitracin, moxicillin, erythromycin, streptomycin and vancomycin) on the membrane organization. for recording the anisotropy of cell suspensions treated with antibiotics or nanoparticles fused with antibiotics we used - -trimethyl- -phenyl , , hexatrien p-toluenesulfonate (tma-dph). we decided to use nanoparticles fused with antibiotics because they contain small amounts of antibiotics which makes them less toxic than simple antibiotics,which is very important in patients with genetic diseases such as cystic fibrosis, that should be treated with antibiotics for a long time. our results showed that at temperatures between and °c simple nanoparticles decreased the membrane fluidity. at physiological temperatures ( - °c) nanoparticles fused with antibiotics (gentamicin, vancomicin, cefotaxim, bacitracin, amoxicillin) increase more the membrane rigidity compare with simple antibiotics or nanoparticles.erythromycin, polymyxin and penicillin increase the membrane rigidity at °c, and at °c the same effect was obtained in the presence of nanoparticles fused with these antibiotics,suggesting that the nanoparticles are dependent to temperature for penetrating the membrane. in conclusion the membrane fluidity does not depend on antibiotics types, the modification are present in many antibiotics irrespective of class type.the presence of nanoparticles fused with antibiotics is very important for long term treatment. objectives: hypertension is an important cardiovascular risk factor for the development of atrial fibrillation (af). increased atrial electromechanical coupling time interval measured by tissue doppler is accepted as an important factor for prediction of af development in hypertensive patients. monoamine oxidases (maos), are enzymes which catalyze the oxidation of monoamines. -isoprostane is considered as an indicator of oxidative stress. mao activity and -isoprostane levels were measured in some diseases. however, there are no information on -isoprostane levels and mao activity in newly diagnosed patients with stage hypertension has not been observed in a study of literature. aim: this is the first study, we aimed to evaluate the levels of mao and -isoprostane in newly diagnosed patients with stage hypertension. the study included newly diagnosed stage hypertensive patients with no other systemic disease. patients were selected as randomized ( women, men; range of age - years) and healthy individuals as control ( women, men; range of age - years). all the underwent tissue doppler echocardiographic examination. blood samples were taken from patients and controls and, the levels of mao and -isoprostane in serum samples were measured by elisa. results: baseline blood pressures, electrocardiographic and echocardiographic findings, and atrial electromechanical coupling were similar in both groups (p > . ). compared to the control group, the activity of mao and -isoprostane levels were found significantly higher in patients (p < . ). conclusion: increased -isoprostane level indicate that there is oxidative stress in newly diagnosed patients with stage hypertension. also, increased mao activity may be biochemical biomarkers for the diagnosis of hypertension. keywords: hypertension, monoamine oxidase, -isoprostane p-mis- determining the indirect reference intervals for complete blood count parameters in bursa, turkey reference intervals (ris) for laboratory test results are defined as the most commonly used diagnostic tool in medicine. therefore, careful determination of ris by the laboratory for use is a very important task. although c -a guideline recommends the direct ris (dris) calculated from healthy subjects, ris can be calculated from laboratory data which are called as indirect ris (iris). the study was carried out at the central laboratory for clinical chemistry, teaching and research (uludag university, bursa, turkey) . the results of the laboratory analyses from , males, , females, stored for approximately one year, were used for statistical analysis. data for hospitalized patients and for ambulatory patients from the intensive care unit were eliminated. furthermore, we used evidence based criteria to enrich the health-related values. a modified bhattacharya procedure was used to estimate the iris from hospital patient data. the nested anova was used to evaluate variations among genders and ages. cell dyn analyzer (abbott diagnostics, il, us) was used for the measurements of complete blood count. the obtained iris were also compared the dris determined in our previous ri study and the ris suggested by the manufacturer. we found that the ris of rbc, hb and hct required strong gender partition and calculated the ris of rbc, hb and hct separately. the observed iris for wbc, sub-fractions of wbc and plt in both genders are in good accordance with the dris reported in previous study. age-related changes were noted for rbc, hb, and hct. the calculated iris for rbc, mcv and rdw are different from the ris suggested by the manufacturer. we believe that, using this relatively easy technique, every laboratory can produce its own iris, divided, where possible, according to sex and age and according to local conditions. these ranges can be complementary to dris obtained for reference individuals according to the ifcc recommendations. the principal sigma subunit, involved in transcription of most house-keeping genes in escherichia coli, was also shown to induce rnap pausing during transcription elongation, by interacting with promoter-like motifs in the transcribed dna. such pauses were proposed to play important roles in the regulation of phage and cellular genes. e. coli contains six alternative s subunits but little is known about their ability to induce transcriptional pausing. we expressed and purified alternative s subunits of the sigma family and tested their effects on transcription elongation in vitro on natural and synthetic dna templates containing consensus promoter motifs. the structure of the paused complexes was analyzed by dna footprinting methods. in vivo analysis of transcription was performed using reporter genes placed under the control of corresponding promoters. we demonstrated that the stationary phase sigma subunit induced efficient rnap pausing on both synthetic and natural dna templates containing promoter-like motifs in initially transcribed regions. in contrast, the sigma and sigma subunits did not affect rna elongation. we showed that the sigma -induced pausing depends on sigma contacts with both nontemplate dna strand and rnap core. the pausing results in formation of backtracked transcription elongation complexes which can be reactivated by gre factors that stimulate rna cleavage by rnap. our results for the first time reveal transcriptional pausing induced by an alternative s subunit. analysis of sigma -dependent promoters shows that a substantial fraction of them contains potential pause-inducing motifs suggesting that such pausing may be a widespread phenomenon. we propose that sigma -dependent pauses may play important roles in genetic regulation and modulate the binding of transcription repressors or activators to promoter regions. the crosstalk between streptococcus pneumoniae rnase r, ribosomes and translation c. b arria, s. domingues, c. arraiano instituto de tecnologia qu ımica e biol ogica, lisbon, portugal ribonucleases (rnases) are enzymes that ensure maturation, degradation and quality control of rna thus, contributing to the maintenance of the optimal amount of each transcript in the cells. escherichia coli rnb family of enzymes is present in all domains of life and includes rnase r, rnase ii and the eukaryotic rrp /dis , dis l and dis l proteins. in streptococcus pneumoniae only rnase r was identified. rnase r, encoded by the rnr gene, hydrolyzes rnas starting from the end. rnase r level is increased in several stress conditions such as heat shock, stationary phase or cold shock, conditions in which most of the proteins translation is blocked. moreover, rnase r is the only exoribonuclease able to degrade highly structured rnas without the help of a helicase which is critical at low temperatures. here, we investigated the role of this enzyme by comparing the wild type strain with an rnr mutant strain. for that purpose we performed northern blots analysis of transcripts involved in translation. also, we investigated rnase r connection to the ribosome and polysome fractions using sucrose gradient polysome separation and western blots. in this study, we highlight the importance of s. pneumoniae rnase r in translation. we show that this enzyme interacts with ribosomes mostly with the s subunit at °c. moreover, in the absence of this enzyme we have observed a decrease in the amount of the s ribosomal subunit, concomitantly with the increase of s and s subunits. rnase r seems also to modulate the amount of the elongation factors ef-tu and ef-g transcripts. nevertheless, preliminary results further suggest other roles of rnase r in translation. modified nucleotides are present in many rna species in all domains of life. the biosynthetic pathways of such nucleotides are well studied. however, much less is known about the degradation of rnas and the salvage of modified nucleotides, their respective nucleosides or heterocyclic bases. using an e. coli uracil auxotrophic strain, we screened the metagenomic libraries for genes, which would allow the conversion of -thiouracil to uracil and thereby lead to the growth on a defined synthetic medium. we show that a novel gene encoding previously uncharacterized domain of unknown function (duf) is responsible for such phenotype. we have purified this recombinant protein and demonstrated that it contains a fe-s cluster. the substitution of cysteines, which have been predicted to bind such clusters, with alanines abolished the growth phenotype. we conclude that this domain is required for conversion of -thiouracil into uracil in vivo. this work is supported by the research council of lithuania (lmt, mip- / modified nucleotides are present in almost all classes of rna. they have great chemical diversity and are critical for rna folding, stability, interaction with cellular proteins and thereby for various cellular processes such as translation, stress response, and signaling pathways. biosynthesis of pyrimidine nucleotides and their modified derivatives in rna is well studied. nonetheless, not much is known about the cellular degradation of these compounds and the enzymes catalyzing such processes. using an e. coli uracil auxotrophic strain, we screened metagenomic libraries for genes encoding isocytosine deaminases. three novel genes were obtained, one of which encodes a protein similar to oxoguanine deaminases. the other two encode proteins resembling hydroxydechloroatrazine ethylaminohydrolases. we confirmed that these proteins are functional in vivo, allowing growth of e.coli on minimal medium with isocytosine. we also demonstrated that such purified recombinant enzymes catalyze the conversion of isocytosine, but not cytosine, into uracil in vitro. natural products display special attributes in the treatment and prevention of various human diseases, including cancer. a significant number of organic compounds from plants exhibit anticancer properties as attested by in vitro and in vivo studies. emerging evidence supporting the antineoplastic activity of natural compounds has rendered them promising agents in the fight against cancer. in this study, skin from limnio grape, a red greek grape variety that is indigenous to the greek island of lemnos, was extracted using mixtures of methanol, water and acetone; the apoptosis-inducing properties of these extracts were studied in the human ovarian malignant adenocarcinoma cell lines tov- g and tov- d. for this purpose, tov- g and tov- d cells were treated with limnio grape skin extracts at a range of concentrations, at °c, for , and hours. untreated cells incubated for the same time intervals served as controls. cell viability was determined by measuring metabolic activity (colorimetric mtt assay) and observing cell membrane integrity (cell staining with trypan blue). after the determination of the optimal concentration of the extract, total rna was extracted from treated and untreated (control) tov- g and tov- d cells. after determination of rna concentration and subsequent first-strand cdna synthesis, mrna expression analysis of apoptosis-related genes was performed with rt-pcr using gene-specific primers. an increasing percentage of non-viable cells was observed by increasing cell exposure time and extract concentration. distinct modulations of the expression of apoptosis-related genes at the mrna level were also observed, mainly concerning bcl , bclx, bax, bak and bcl l , along apoptosis induction. in conclusion, the cytotoxic properties of limnio grape skin extracts against ovarian malignant adenocarcinoma cells merit further investigation. the intrinsic apoptotic pathway seems to be the major mechanism of action induced by these plant extracts. almost all eukaryotic mrnas are polyadenylated by a complex machinery that recognizes the poly (a) signal, cleaves the mrna and adds the poly (a) tail. % of human genes harbor multiple poly (a) signals. alternative polyadenylation (apa) generates transcript isoforms with different utr (untranslated region) lengths due to the use of proximal or distal poly (a) signals. hence, tightly regulated apa has been observed in normal physiological settings as well as in diseases. considering that utr shortening cases have been linked to increased protein levels, we hypothesized deregulated apa to be one of the potential cancer related mechanisms. we investigated the utr alterations in er(+) breast cancer patients and cell models compared to normal breast tissue, using gene expression data and a probe-based quantification tool, apadetect. based on means of proximal to distal probe sets, slr (short-long ratio) were calculated as an indication apa. significance analysis of microarrays (sam) determined significant genes. the gse numbers of the datasets are gse , gse and gse . we analyzed two datasets of er(+) breast cancer patient samples (n = , n = ) compared to normal breast tissue (n = ) using apadetect and sam. a total of utr shortening and utr lengthening events were detected in breast cancer samples compared to normal breast tissue. ontology analysis suggested almost all the utr shortening genes were proliferation related and were indeed reported to be upregulated in breast cancer. to further investigate the connection between apa and era status, we used data from a cell line model; wild type or era transfected mda-mb- cells that are otherwise of triple negative nature. our results suggested that most of the genes are utr shortened or lengthened via direct binding of era to dna. our results suggest involvement of apa mechanisms in era action mechanisms. possible link between era regulated transcription and apa remains to be elucidated. contamination of nucleic acids (na) as a result of na extraction protocols may result an inaccurate measurement of dna copy number. agarose gel electrophoresis and spectrophotometric methods are commonly used to check dna purity. however, the resolution of these methods may not be good enough for special applications such as determination of dna copy number and separation of base pairs (bp) that are close in their bp number. in this study, we have developed a new method for separating na's ranging between - bp also detecting the impurities in dna solution in %, % and % ratios to the dna of interest. the developed method was validated using the in-house dna fragments of , and bp. the dna mixture analyzed using analytical hitachi elite lachrom hplc using the guard and analytical columns tskgel dna-npr, . lm, . mm id . cm and tskgel dna-npr, . lm, . mm id . cm, respectively. the validation of the analysis was performed by running each sample five times on three different days. the linearity of the detector response was established by plotting a graph to quantity versus area of bp dna. the lod and loq were then measured by calculating the minimum level at which analyte can be readily detected and quantified. the ratios calculated with hplc were compared to the ratios calculated by quant-it kit. recovery values were calculated for each measurement and the uncertainty were calculated for each ratio. the method was found linear for bp in the range of . ng to ng dna with the regression coefficient of r = . . lod and loq for the bp dna was found to be . ng and . ng, respectively. the recovery values for the %, % and % impurity ratios were found . . . and . , respectively. the purity of the synthetic dna was determined by hplc and related uncertainty was calculated. the developed method is a simple alternative to electrophoresis and spectrophotometric methods with higher resolution and separation range. physical and chemical factors can disturb the conformation of proteins maturing within the cellular secretory pathway. in response to unfolded proteins the cell activates several stress signaling and adaptive response mechanisms. the aim of our study was to investigate small non-coding rnas as the potential regulators of cellular response to unfolded proteins (upr). for this, we conduct the next generation sequencing of small rna and transcriptome analysis of mrna from jurkat cells exposed to dithiothreitol (dtt), which reduces protein disulfide bounds. analysis of mirnas reveals the differential expression of mirnas. we observe a decrease in the normalized amount of reads aligned to mirna loci in stressed cells. affymetrix analysis with subsequent gsea reveals downregulation of reactome mirna biogenesis pathway (fdr = . ). the length distribution of small rnas revealed nt-peak corresponding to trna-derived fragments, amount of which was increased by . -fold under dtt treatment. the trna isotypes that gave rise to almost % and % of all nt rna fragments in stressed and control cells, respectively, include glycine, glutamic acid, aspartic acid and valine. the vast majority of nt fragments produced from these trnas are precisely phased halves with the characteristic cleavage patterns generated by rnase a angiogenin (ang). observed upregulation of tirna in stressed cells is accompanied with upregulation of ang mrna and down-regulation of angiogenin inhibitor (rnh ). we speculate that translational repression, associated with observed tirna, is an additional mechanism of reducing global protein synthesis in response to dtt-induced stress. collectively, our findings reveal the increase in tirna, the differential regulation of mirna expression together with the global mirna downregulation as the most prominent small rnome reprogramming events and possible fine-tuned levels of post-transcriptional regulation upon dtt-induced cellular stress response. global gene expression changes after spinal cord injury j. k. hyun , , , j. kim , , j. y. hong dankook university, cheonan, south korea, institute of tissue regeneration engineering (itren), cheonan, south korea, the neuronal regeneration is hardly achieved spontaneously after spinal cord injury (sci), and the restoration of somatic and autonomic functions after sci is also challenging in the clinical field. the pathophysiology of sci is extremely complex and many in vitro and in vivo studies continued to report opposite results each other in spite of the same treatments, therefore a fundamental analysis such as an extensive assay of global gene expression is required to find a way for spinal cord regeneration. in this study, we aimed to detect the changes of global gene expression after spinal cord contusion in rats according to the time sequence. the spinal cord tissues at contusion site were sequenced after spinal cord contusion in rats using rna-sequencing technology. for time sequence analysis, five time points was determined; hour, day, week, month and months after spinal cord contusion, and sham operated rats at each time point were used as controls. quantitative rt-pcr analysis was also performed to validate expression changes of candidate genes in each category. we found that the pattern of changes in gene expression at acute and subacute stages was quite different from that at chronic stage, especially genes associated with with neurotrophin signaling and apoptosis pathways. most of gene expression levels of inflammatory cell markers were increased and peak during acute stage ( hour to week) and maintained until chronic stage. some of regeneration-associated genes (rags) including brain derived neurotrophic factor, glial cell derived neurotrophic factor and ciliary neurotrophic factor were increased at hour or day after sci. we concluded that the information of gene expression level according to the time sequence after sci might be useful to determine treatment strategies for spinal cord regeneration especially in chronic stage. p- . . - utr length isoform generation profile in a differentiation model alternative polyadenylation (apa) is the regulated selection of a specific poly(a) signal among other proximal and/or distal signals on the utrs (untranslated region) for the endolytic cleavage and addition of a poly(a) tail to form the mature mrna. consequently, position of the poly(a) site determines the length of the utr which is known to harbor microrna and rna binding protein sites. such apa isoforms have already been linked to altered protein levels and even functions. therefore we hypothesized apa to be one of the mechanisms to generate isoform diversity in proliferating and differentiated cells to better understand the molecular basis of cancer. we used a combinatorial in silico and in vitro approach to analyze a well known enterocyte differentiation model; caco- cells. initially we analyzed gene expression datasets for the proliferative and differentiated caco- cells using a probe based apa detection tool. to better understand the significance and to validate these results, we used proliferating and differentiated (day ) caco- cells and tested sample apa events by rt-qpcr. utr isoforms were identified by using race pcr. we identified genes ( % of all apa events) to undergo utr shortening in differentiated cells compared to proliferating cells. on the contrary genes ( % of all apa events) went through utr lengthening events. several genes have been validated to follow the pattern that was seen in apa detection tool so far. to begin understanding the mechanism behind these observations, we are investigating potential inducers of apa during the complex events of differentiation. our next aim will be to further validate and investigate the consequence of such isoform generation events both in the context of differentiation in colon cancer cells. recognition of phosphorylated threonine- of rna polymerase ii c-terminal domain by end processing apparatus o. jasnovidova, m. krejcikova, k. kubicek, r. stefl central european institute of technology, masaryk university, brno, czech republic rna polymerase ii has evolved an array of heptad repeats with the consensus sequence y -s -p -t -s -p -s at the c-terminal domain (ctd) of its largest subunit, rpb . phosphorylation of serines (s , s , and s ) and tyrosine- orchestrate the binding of rna processing and transcription factors in the site of transcription. several recent studies showed that also threonine- site can be phosphorylated which has a number of functional consequences. to reveal the structural basis for the recognition of threonine- phosphorylated ctd, we set out to investigate several proteins factors that were implicated with a high levels of threonine- ctd phosphomarks using integrative structural biology. one of them, a factor involved in the -end processing and transcription termination, showed a high affinity to the phosphothreonine ctd. using nuclear magnetic resonance spectroscopy (nmr), we determined its structure bound to the ctd phosphorylated at threonine- that reveals a direct read-out of the phosphothreonine. altogether, our data provides the first insights into the recognition of this poorly understood ctd mark that plays important role in the ctd code of rna polymerase ii. the results of this research have been acquired within ceitec (lq ) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. introduction: the treatment of brain tumor glioblastoma (gbm) is still one of the greatest challenge. anti-inflammatory drug indomethacin (ind) mainly acting through the inhibition of cyclooxygenase (cox) has also anti-cancer activity including brain tumors. the aim was to investigate how ind effects an immortality enzyme telomerases' activity. materials and methods: monolayer and spheroid cultures of t g human gbm cell line were used to evaluate the effects of ind ( lm) on cell proliferation, viability, apoptosis, cell cycle, camp levels, the levels of apoptotic and anti-apoptotic proteins, morphology (sem) and ultrastructure (tem) for hours. results were analyzed using the student's t-test. results: ind decreased cell proliferation (p < . ), cell viability (p < . ), cell rate at s phase (p < . ) and g + m phase (p < . ), camp levels (p < . ), the levels of pdgfr-a (p < . ), mrp- (p < . ), nf-jb (p < . ) and cox- (p < . ) in comparison to control group. ind mildly increased apoptosis (p < . ) and caspase- levels (p < . ). interestingly, ind increased htert levels ( %, p < . ; %, p < . ). sem evaluation showed that ind led to decreased and shortened microvilli, the lost of cell interactions and the conversion of many cell shapes from spindle to oval. many cell remnants in the intercellular area, intact cell membranes, many dense lipid droplets and few autophagic vacuols in the cytoplasm were observed under tem. discussion and conclusions: the effect of ind on telomerase activity can only be found in publications at pubmed research that they only showed its' inhibitory effect in colon, gastric, head and neck cancers. in contrast to previous studies, it was shown for the first time that ind increased telomerase activity in gbm cells and this increase was independent from cox- and other tested factors. p- . . - interaction between fibrinogen and insulin-like growth factor binding protein- under physiologic conditions and influence of diabetes mellitus type on this interaction n. gligorijevic, o. nedic institute for the application of nuclear energy, university of belgrade, belgrade, serbia fibrinogen is plasma glycoprotein and principle participant in blood coagulation. it interacts with many proteins, including insulin-like growth factor binding proteins (igfbps). one of them, igfbp- , is controlled by insulin. metabolic changes due to diabetes mellitus (dm) affect igfbp- . besides glucose regulation, igfbp- stimulates wound healing. we have investigated complexes formed between fibrinogen and igfbp- , their change in dm type (dm ) patients and involvement in fibrin clot. samples from adult healthy persons and dm patients were studied: plasma, isolated fibrinogen and fibrin. the amount of igfbp- /fibrinogen complexes was determined using immunoblotting. immunoprecipitation and lectin affinity chromatography were used to confirm interaction between fibrinogen and igfbp- . in vitro incubation of fibrinogen with excess glucose or methylgyoxal (mgo) was employed to demonstrate influence of glyco-oxidation on complexes. results have shown that igfbp- /fibrinogen complexes can be differentiated from igfbp- oligomers and igfbp- /alpha- macroglobulin complexes. the amount of igfbp- /fibrinogen complexes was lower in patients with dm . complexes participated in fibrin clot formation, the amount being significantly lower in patients' samples. the quantity of igfbp- monomer in fibrin clot was greater in patients' samples. in vitro experiments revealed that complexes undergo glyco-oxidative modifications leading to their reduced formation, cross-linking and increased acidity (faster electrophoretic movement). isolated fibrinogen from patients with dm was additionally able to bind exogenous igfbp- . since igfbp- stimulates wound healing, directly and by delivering igfs, igfbp- /fibrinogen complexes may be seen as igfbp- storage instrument, ready to participate in fibrin formation and to assist in damage repair. reduction of complexes due to glyco-oxidative stress in patients with dm may be part of the mechanism responsible for impaired coagulation process. human interferon gamma (hifnc) is a proinflammatory cytokine involved in the regulation of nearly all phases of immune and inflammatory responses. its abnormal expression is associated with the aetiology of many inflammatory and autoimmune diseases. recently we have been exploring the idea to counteract the over-expression of the endogenous hifnc by competitive inhibition with inactive hifnc mutants. they are designed to have preserved affinity to the hifnc receptor, but to be deprived in their capability to trigger the intracellular signal transduction. to this end a library of mutants was created and two potential hifnc antagonists were selected for further investigations: a single point mutant k q (q substitution for k in position ) and a double mutant with additional substitution in the n-terminus. both mutants and the wild type hifnc were expressed in e. coli employing the established by us methodology for large scale production of aggregation-prone proteins in soluble native form. the purified mutants were screened for interferon activity (antiproliferative assay), binding affinity (isothermal titration calorimetry) and ability to compete with the wild type for the hifnc receptor (competition assay on wish cells). the selected mutants demonstrated (single mutant) and (double mutant) times lower antiproliferative activity than the wild type. measuring the binding thermodynamic parameters, we proved that the receptor binding affinity of both mutants was preserved, which is an indication for their potential to compete with the wild type hifnc for its receptor. finally, the biological assay performed on wish cells showed a distinct dose-dependent competition between the wild type hifnc and the mutants. based on the results presented in this study we conclude that the two hifnc mutants are potential candidates for autoimmune therapy based on selective suppression of the endogenous hifnc activity. mesencephalic astrocyte-derived neurotrophic factor (manf) is an er (endoplasmic reticulum) stress-inducible protein and widely expressed in mammalian tissues. it has been identified as a secretory protein that protects cells against er stress-induced damage. er-stress is one of the main mechanisms that play a role in ischemia/reperfusion (i/r)-induced renal injury. recent studies demonstrated that manf can protect cardiac myocytes and cortical neurons against i/r-induced injury. moreover, it has been suggested that it has a restorative effect in ischemic injury. nevertheless, the function of manf in i/r-induced renal injury is still not known. in the present study, we investigated the function of manf by manipulation its expression level in ischemic acute renal failure model established in proximal tubular kidney cells (hk- cells). for this purpose, the cells were transfected with either manf sirna or manf encoding plasmids for silencing or over-expression of manf, respectively. then, the cells were exposed to hypoxia-reperfusion (h/r) induction for indicated times. evaluations of cell viability were determined with wst- reagent. the changes in protein levels of h/r-induced stress markers were analyzed byimmunoblotting. the results showed that the overexpression of manf has provided a significant resistance to h/r-induced cell death, whereas silencing of manf has rendered the cells more susceptible to death. it was also determined that the pretreatment of cells with manf conditioned medium caused a decrease in cell death. additionally, oxidative/nitrosative stress (os/ns) and er stress levels were decreased with over-expression of manf and increased by silencing of manf in hk- cells. taken together, our study suggests that manf may have a protective role against h/r-induced renal cell injury, possibly through the reducing effects on os/ns and er stress. p- . . - his-flag tag as a fusion partner in insect expression systemgain or loss? e. krachmarova , m. tileva , k. maskos , i. ivanov , g. nacheva institute of molecular biology "roumen tsanev", sofia, bulgaria, proteros biostructures, martinsried, germany human interferon gamma (hifnc) is a glycoprotein playing major role in the regulation of innate and adaptive immunity. glycosylation is not essential for hifnc activity but is important for its stability, half-life and protease resistance in blood. the commonly used hifnc in therapy and research is produced in e. coli and therefore is not glycosylated. bearing in mind the above mentioned shortcomings of the non-glycosylated hifnc we expressed it in mammalian cells and transgenic mice, however very low yields were achieved. to obtain glycosylated hifnc, here we employed a secretory expression of n-terminal his-flag fusion protein in baculovirus-infected insect high five Ò cells. this small hydrophilic tag is designed to not affect the proper folding of the target protein and to facilitate the detection and purification procedures. in parallel the same fusion was expressed in e. coli cells. the fusion proteins were purified to high degree of purity by affinity and size-exclusion chromatography. bioassay carried out on wish cells showed that the antiproliferative activity of both fusion proteins was times lower than that of the native hifnc. this result shows that, in contrast to the generally hold view, the n-terminal his-flag tag interferes with the biological activity of hifnc despite of the protein glycosylation. in order to restore the biological activity we attempted to remove the his-flag tag enzymatically. surprisingly, we found that the fusion protein obtained from insect cells was resistant to enterokinase, independently of the enzyme source and experimental conditions, whereas the protein isolated from e. coli was susceptible and the tag-free protein showed fully restored biological activity. we are prone to explain the enterokinase resistance of the fusion protein from insect cells with either the specific conformation of the glycosylated protein or with the interaction of the carbohydrate residues with the enzymatic activity of the enterokinase. p- . . - development of fluorescence assay for highthroughput screening system based on flow cytometry for directed evolution of cellobiose dehydrogenase cellobiose dehydrogenase (cdh) is an enzyme produced by phanerochaete chrysosporium and it has been already successfully cloned in other organisms. one of the most important roles of cdh is removing products of cellulose degradation. cdh is very important for biofuel and biosensor industry. for improvements of enzyme properties we have used directed evolution. the most important step is to develop screening system that reflects properties of interest. screening in microtiter plates (mtp) is expensive, time-consuming and has low throughput with a small number of variants detected ( - in months). the aim of this work was the development of screening system for mutant libraries of cdh expressed on surface of yeast cells based on fluorescent enzymatic assay and flow cytometry. the screening method should be capable of screening cellobiose dehydrogenase variants mutated for higher activity and higher thermostability by error prone pcr. the fluorescent assay was beta-galactosidase (ec . . . ) also known as lactase is the enzyme that typically catalyzes hydrolysis of beta- , -d-galactosidic linkages in beta-d-galactosides, including disaccharide lactose, with glucose and galactose as end reaction products. this enzyme is able to catalyze synthesis of oligosaccharides, in particular galactooligosaccharides via galactosyl transfer reaction. arthrobacter sulfonivorans beta-galactosidase of unique for prokaryotes extracellular localization may find application in food industry for manufacturing lactose-free dairy products and in pharmacology as bioactive principle of medicines prescribed for patients suffering from lactase deficiency. the study was aimed at cloning of the gene encoding a. sulfonivorans beta-galactosidase, purification and characterization of the enzyme. a novel extracellular beta-galactosidase from a. sulfonivorans was recovered with an overall -fold purification, a . % yield and specific activity uÁmg À protein. the subunit molecular mass of the enzyme determined by sds-page analysis equalled kda. it was found that the enzyme displays pi . , prefers ortho-nitrophenyl-beta-galactoside as substrate (km mm) and shows maximum activity at °c and at ph . - . . the beta-galactosidase gene was isolated from the genomic dna library of a. sulfonivorans, sequenced, cloned and deposited in the genbank database under accession number km . . it was established that the gene carries an open reading frame consisting of bp ( amino acids) and encodes beta-galactosidase referred to glycosyl hydrolase family (cazy database). p- . . - different splice-forms of tdrd protein mutated in cataract's and glaucoma's interacts with s k / o. skorokhod, v. filonenko department of cellular signalling, institute of molecular biology and genetics nas of ukraine, kyiv, ukraine ribosomal s kinases (s k) are important players in cellular pi k/mtor signalling network, deregulation of which has been associated with methabolic disorders, inflammation and cancer. previously we had identified a novel binding partner of s k -tdrd (trap). tdrd is a scaffold protein detected in complexes involved in the regulation of cytoskeleton dynamics, mrna transport, protein translation, non-coding pirnas processing, transposons silensing. it was reported recently that mutations in human tdrd result in cataract and glaucoma formation, defined by elevated intraocular pressure (iop) and optic nerve damage. the aim of our study was to confirm s k-tdrd interaplay and study its role in cells. bioinformatical analysis of tdrd sequence revealed the presence of potential phosphorylation sites of s k . using in vitro kinase assay, we have demonstrated that recombinant s k phosphorylate from fragments of tdrd . formation of s k -tdrd complexes in vivo was further confirmed by coimmunoprecipitation using anti-s k and anti-tdrd antibodies generated previously in rat brain lysates. this interaction was further confirmed by confocal microscopy, oleksandr had shown that tdrd co-localize with s k in hepg cells, predominantly in perinuclear region, enreached for one of the tdrd isoforms identified previously. moreover, we have detected that c-terminal synthetic peptides of s k with methylated arg interfere with tdrd from hepg lysates. the physiological characteristics of s k -tdrd interaction and the role of this complex formation in neuropathology's development need further investigation. many biological function of placenta are performed not just a set of individual proteins, but also different oligomeric structures and complexes. herewith, activities of complexes may considerably differ from activities of individual proteins. therefore, identification and characterization of placental multi-protein complexes is an important step to understanding the placenta function. the aim of the present work was to investigate a composition and biological functions of the very stable high molecular mass multi-protein complexes (spc) from placenta of healthy mother. we isolated spcs (~ kda) from the soluble fraction of three human placentas. light scattering measurements and gel filtration showed that the spc is stable in presence salts, acetonitrile and triton x- in high concentrations, but efficiently dissociates in the presence of m urea and mm edta. such a stable complex is unlikely to be a random associate of different proteins. it was shown the spc includes a number of proteins with molecular weights of to kda. several protein components of the spc were identified, including serum albumin, transferrin, iggs, annexin a and other proteins. serum albumin, transferrin and protein with molecular weight , kda are the main proteins of the complex. it was shown high the spcs from three placentas possesses dnsase and catalase activities. an addition, investigation of cytotoxic effect on human cancerous cell lines has shown that the spcs reveal high cytotoxicity. antibody-cytochrome b fusion protein, characterization and applications for antibody development process antibodies have recently become an essential tool being a part of immunodiagnostics, therapeutics and as a valuable instrument in life science research. an enormous number of options utilizing a various tags were used to create a universal antigen-binding domain, which can be easily detectable, highly soluble and might be produced in high yields with low costs, but no multipurpose solution exists yet. we addressed the question whether a single tag could be found for enhancing solubility of recombinant fab antibody fragment and providing its detection and accurate quantification by rather simple method. a new application for hemeproteincytochrome b as the antibodies fusion partner were proposed. we have constructed of recombinant fab antibody fragment cytochrome b fusion protein. we have shown that cytochrome b enhance expression of fab antibodies fragments in bacterial system, and could be a versatile tool for recombinant proteins folding, redox (oxidation) state studies and for their precise concentration determination in the turbid solutions. fusion fab-b protein has a stable red color and characteristic absorbance spectrum with the maximum absorbance at nm in oxidized environment. cytochrome b change its spectrum maximum depending on environmental redox potential and its folded state, so one can track these events in real time spectrophotometrically. binding activities of fab-b fusion protein and hybridoma secreted immunoglobulin were measured by biolayer interferometry and elisa. no significant difference between them was revealed. due to this feature we can distinguish the chimeric protein of interest in complex mixtures and control the process of recombinant proteins expression and purification in real-time. besides, cytochrome b fusion tags multiples recombinant antibody yield (from to times) and doesn't affect antigen-binding properties. the bb - site of fibrin molecule is the site of fibrin protofibrils lateral association l. urvant palladin institute of biochemistry nas of ukraine, kyiv, ukraine previously we showed that fibrin-specific monoclonal antibody i- c (monab i- c) inhibited the fibrin protofibrils lateral association. we suggested that the epitope of monab i- c in bb - of coiled-coil region of fibrin molecule coincides with the site involved in fibrin protofibrils lateral association. the aim of this study was to localize the site of protofibrils lateral association in fibrin molecule using the synthetic peptides bb - , bb - and both their scrambled version, and bb - peptide. monab i- c was isolated from hybridoma culture medium by affinity chromatography on fibrin-sepharose. turbidity analysis was used to study the effect of synthetic peptides on fibrin polymerization. the interaction between peptides and monab i- c was investigated by spr method using plasmon- device. we investigated the effect of synthetic peptides which corresponded to amino acide sequences of fibrin molecule bb - , bb - , bb - , and the scrambled versions of bb - and bb - peptides on a binding to monab i- c and on the fibrin polymerization process. in spr analysis was showed that bb - and bb - peptides, but not their scrambled version, binds to monab i- c, immobilized to a chip. turbidity data showed that only bb - and bb - peptides caused the -fold decrease of the rate of the lateral association of protofibryls at the concentration . À m and . À m, respectively. both of them decreased the final clot turbidity. our data let us to suggest that the bb - site is the site that involved in protofibryls lateral association. it has been recently shown that irisin immunoreactivity was altered in gastrointestinal cancers. as known hematological malignancies was one of the most common malignancies through world, but no study was present how irisin was changed in this type of cancers. therefore, purpose of this was to investigate how immunoreactivity to irisin was altered in hematological malignancies (blood cancers). we used an antibody from phoenix to demonstrate how a kda band after deglycosylation of irisin altered in hematological malignancies. here we first time showed that irisin tissue immunoreactivity from acute lymphoblastic leukemia (all) and acute myelogenous leukemia (aml) patients was increased when compared with unaffected biological tissue parts. from the immune-histochemical (ihc) investigations it is concluded that hematological tissue and blood cells may be another source of irisin and increased with cancer, thus this finding might help to enlighten pathophysiology of hematological malignancies. the value of urine neutrophil gelatinaseassociated lipocalin (ngal) in acute heart failure n. serdarevic clinical centre, sarajevo, bosnia and herzegovina introduction: renal dysfunction is very common in heart failure (hf) and neutrophil gelatinase-associated lipocalin (ngal) is used as an early marker of acute renal tubular injury. recent studies have been reporting that ngal is inhibitor of inactivation of matrix metalloproteinases (mmp- ) which results in enhanced proteolytic activity with prolonged effects on collagen degradation. due to its relation to extracellular matrix degradation in myocardium and infammation, we hypothesized possible increased ngal expression in hf besides it renal dysfunction etiology. patients and methods: in study were included patients hospitalised with signs and symtoms of ahf. urine samples for ngal analysis were collected at admission and analysed by the chemiluminescent microparticle immunoassay (cmia) for the quantitative determination of neutrophil gelatinase-associated lipocalin in human urine (abbott, architect analyzer). refferent range for urine ngal is - ng/ml. on admission blood samples for bnp (brain natriuretic peptide) analysis were drawn and tested by architect bnp chemiluminescent microparticle immunoassay (cmia), abbott laboratory. results: the mean age of the patients (male= , female= ) was . years (sd . years). among them ( %) patients was diagnosed as a hf-pef (hf with preserved ejection fraction) while ( %) as a hf-ref (hf with reduced ejection fraction). mean bnp values was . pg/ml (sd . pg/ml) and mean lvef was . % (sd . %). mean urine ngal was . ng/ml (sd . ng/ml). we found significantly positive, but weak correlation among ngal and bnp only by pearson correlation test (r = . , p = . , wilcoxon signed rank test z = À . p < . ). conclusion: bnp levels are elevated in hf with reduced and preserved ejection fraction. urine ngal is not elevated in acute heart failure, but it is slightly positively correlated with serum bnp values. converging evidence implicates the intermediate and medial mesopallium (imm) of the domestic chick forebrain in memory for a visual imprinting stimulus. a number of learning-related changes have been found in plasma membrane and mitochondrial proteins of imm. for broader analysis of these changes we employed two-dimensional gel electrophoresis/mass spectrometry approach and identified differentially expressed proteins in membrane-mitochondrial fraction of the imm across chicks with different estimated levels of imprinting h after training. we further inquired whether the amounts of those proteins in the imm and a control region (posterior pole of the nidopallium, ppn) are correlated with memory for the imprinting stimulus. learning-related increase in the amounts of the following proteins was demonstrated in the left imm, but not in the right imm or left and right ppn: (i) membrane cognin;(ii) a protein resembling the p subunit of splicing factor sf ;(iii) voltage dependent anionic channel- ;(iv) dynamin- ; (v) heterogeneous nuclear ribonucleoprotein a /b . obtained results indicate that the molecular processes involved in learning and memory of imprinting cover a wide range of cellular activities, including stabilization of protein structures, increased mrna trafficking, synaptic vesicle recycling and specific changes in the mitochondrial proteome. the aim of this work is to study the substrate and inhibitory properties of uridine derivatives in the reactions catalyzed by e.coli up in order to shed some light on the substrate's conformation in the productive complex with the enzyme. we studied the e.coli up-catalyzed phosphorolysis of uridine and its derivatives modified in the heterocyclic base and the sugar moiety. the kinetic constants (km, ki, kcat ) of the phosphorolysis reaction of near uridine derivatives were determined. the combined kinetic (nnna, , ) and structural data (acta crystallogr., d , , ) provide clear evidence that up binds uridine in the most energetically unfavorable conformation, which, to the best of our knowledge, has no precedents in the enzymes of nucleic acid metabolism. this is possible due to multiple interactions between the substrate and the protein environment (active site residues) mainly through hydrogen bonds. these results are important for understanding the mechanism of action of this class of enzymes. an analysis of the conformations of nucleosides in solution and rotational barriers suggests that the energy difference between the ground state of uridine and uridine complexed with up may be high as - kj/mol. the binding in a high-energy conformation results in the weakening of the glycosidic bond. the observed conformation of uridine complexed with sulfate (mimetic of phosphate) may be very similar to its conformation in the transient state. until now, foxp (forkhead box p ) has been identified as a tumor suppressor in several correlation studies in breast cancer. although, foxp is defined as a transcriptional repressor that interacts with other transcription factors in various mechanistic studies, there is no study that explains its repressor functions in breast cancer biology. here we demonstrate the repressor function of foxp on nfat (nuclear factor of activated t cells) and the migratory effect of this repression in mda mb breast cancer cells. we performed co-immunoprecipitation experiments for the investigation of protein-protein interaction between two transcription factors. protein-protein interaction on dna was investigated with emsa and transcriptional effects of foxp on nfat, lusiferase reporter assay was performed. wound healing assay was used to analyse the effects of overexpression of foxp on tumor cell migration. our results showed that foxp has protein-protein interaction with nfat on dna and enhances breast cancer cell migration by repressing nfat transcriptional activity and foxp shows oncogenic function by regulating breast cancer cell motility. introduction: phosphodiesterase (pde ) is one of phosphodiesterase lead to hydrolyzing cgmp.the cgmp signaling pathway has an important role in proliferation of cells. previous studies showed pde was increased in cell lines cancers thus pde inhibitors can used as efficacious therapeutic option for treatment of cancers. the current study was to investigation the effect of hydroalcoholic achillea.wilhelmsii extract (hawe) on the pde gene expression and cgmp signaling in the mcf- er + and mda-mb- er À . methods and materials: the ed of the hawe on both cell lines were examined by using mtt viability test then the expression of pde and cgmp concentration were measured in timedependent manner (in the ed ) by real-time rt-pcr and colorimetric assay respectively. results: treatment with the hawe showed, lg/ml is ed for both cell lines and the hawe lead to reduction in pde mrna expression and evaluation of intracellular cgmp showed an increase pattern in the time-dependent manner. conclusion: our results showed that the hawe has anti-proliferative property in the mcf- and mda-mb- , cell lines of breast cancer through the cgmp pathway, these data suggested that the hawe can be potential source for the isolation of effective anti-proliferative molecules. keywords: achillea.wilhelmsii, breast cancer, anti-proliferative, phosphodiesterase, cgmp signaling pathway. outer membrane protein g (ompg) is a stable monomeric porin having -stranded beta barrel form from e.coli. its exact function is not fully understood; however, it allows the passage of molecules up to da in neutral ph but the pore is closed by going through a conformational change under ph . . as being monomeric and having ph-dependent gating characters, it is suitable for biosensor and targeted drug delivery applications. an attempt on ompg is to create a larger pore while its stability is undisturbed. ompg- s is obtained by adding amino acids to the primary chain in order to have a -stranded beta barrel porin. ompg- sl is formed by further adding amino acids to loop l and by replacing lysines with arginines. ompg- s and ompg- sl mutants are investigated by fourier transform infrared spectroscopy (ftir) and compared with ompg-wild type (wt) in terms of ph-dependent conformational changes and thermal stability. each mutant is prepared in na-phosphate buffer pd . / . and infrared spectra are recorded. further, temperature profiling are recorded for the range between to °c. results show that both mutants are responsive to ph changes. while turning the ph from acidic to neutral, beta sheet signals shift to lower wavenumbers showing difference in secondary structure, implying the existence of closed and open states. on the other hand, mutant proteins show structural differences compared with the wt protein. porins are known for their remarkable thermal stability. the mutans retain this character by having transition temperature of $ °c, although this is less than the wt transition at $ °c. in conclusion, two mutants show signs of open and closed states as ompg-wt and even if the mutants are less stable than ompg-wt. this study shows that the attempted alterations in ompg structure are successful in terms of ph-response but it needs improvement in terms of stability when necessary. nad is a key factor in the regulation of mitochondrial metabolism. besides its vital role as redox carrier, nad serves as substrate for protein adp-ribosylation and deacetylation, modifications which modulate enzyme activities in mitochondria. these functions depend on how nad levels are maintained in this organelle. in human cells, mitochondrial nad is segregated from the cytosolic pool and can be synthesized from nmn, which is probably imported into the matrix. here, we tested whether the nudix pyrophosphatase nudt participates in the regulation of the mitochondrial nad pool. this enzyme has a predicted nadh pyrophosphatase zinc ribbon domain and a mitochondrial targeting sequence at its n-terminus. however, it has not yet been functionally characterized. we overexpressed nudt endowed with a c-terminal flag-epitope in human cells. to evaluate changes in the mitochondrial nad concentration, we used a reporter system which includes the overexpression of the catalytic domain of poly(adpribose) polymerase (parp ) within the organelles (mitoparp). thereby mitochondrial nad is converted into protein-bound poly(adp-ribose) (par). the extent of par formation correlates with the mitochondrial nad availability and is detected by western blotting. our results established that nudt is indeed a mitochondrial protein, as it was localized exclusively to these organelles. moreover, when nudt was overexpressed along with the mitoparp detector system, a dramatic decrease of par was observed. the obtained results indicate that nudt is enzymatically active upon overexpression in the mitochondrial matrix and that it might cleave nad, thereby modulating its organellar level. however, at this point we cannot exclude the possibility of direct par cleavage by nudt . further characterization of nudt will define its substrate specificity and clarify its role in mitochondrial metabolism. the incidence of increase in colorectal cancer (crc) worldwide has become a major health problem. early diagnosis and treatment of crcs are of importance for improving survival. in the present study, it was aimed to investigate chemopreventive effect of rosmarinic acid and evaluate the angiogenesis process in azoxymethane (aom)-induced crc model. male sprague-dawley rats were randomly divided into a control group, aom-induced rat colorectal cancer group ( mg/kg body weight aom; ip, weekly for four weeks), and rosmarinic acid ( mg/kg body weight; oral, daily for four weeks)-treated group. in addition to the standart diet of the all groups . % peanut oil was added throughout the experiment. the all rats were sacrificed at the end of weeks. biochemical examinations were performed in rat plasma. histopathological adenocarcinoma rates were observed in . % of aom group. the incidence of adenocarcinoma was showed a reduction in the treatment group. significant increases in plasma tos and mcp- levels were found in the aom group compared to controls. these increases were reduced in the treatment groups but no significant. a significant increase was detected in tas levels in the treatment group when compared to the aom group. significant decreases in plasma adiponectin levels were found in the aom and the treatment groups compared to controls. in conclusion, treatment with rosmarinic acid reduced the occurrence of inflammation and was helped to maintain the oxidant-antioxidant balance in the model of aom-induced rat colon cancer. mitochondrial genome, while being strongly reduced in course of evolution, still codes for several proteins. the vast majority of them are components of the respiratory chain complexes. to produce these proteins, the system of mitochondrial translation is presented in the organelles, which is in common close to that in bacteria. translation initiation in bacterial cells is orchestrated by three protein factors called if , if and if . the orthologs of the two latter proteins are commonly found in mitochondria. however, mitochondrial if could not been identified in several groups of organisms, including s.cerevisiae, for a long time. recently we have shown that baker's yeast protein aim p possesses a function of mtif . however, the mitochondrial translation has not been stopped in the yeast strain without aim p which is surprising taking into account the fact that if is obligatory for the translation in bacterial systems. instead of blocking of mitochondrial protein synthesis in absence of aim p, we observed the translational imbalance: the synthesis rate of the complex v subunits was increased while the synthesis rate of the complex iv subunits was repressed. thus, in addition to its general role in translation initiation, aim p might specifically affect the biosynthesis of individual mitochondrial-encoded protein species. our genetic experiments have revealed that, indeed, aim p is almost indispensable for cox p synthesis, and that it affects the translation of cox mrna through its -utr, like classical mitochondrial translational activators. this is in accordance with our measurements of complex iv activity which is several times less in yeast lacking aim gene than in the wild-type. taken together, our results point on the multiple role of aim p in mitochondrial translation: in addition to its function as mitochondrial if , it specifically regulates the amount of complex iv subunits and its activity. p- . . - the circulating betatrophin and irisin levels in polycystic ovary syndrome patients with and without insulin resistance introduction: polycystic ovary syndrome (pcos) is the most common endocrine/metabolic disease in women around the world, characterized by oligo-or anovulation, polycystic ovary, and/or hyper-androgenism. insulin resistance (ir) and obesity are common findings in patients with pcos. irisin is a recently identified myokine secreted from skeletal muscle in response to physical activity. irisin has been postulated to induce the differentiation of white fat tissue into brown fat tissue. betatrophin is a currently discovered new hormone proposed to stimulate b-cell proliferation. in this study we investigated the levels of irisin and betatrophin in pcos patients. materials and methods: our study group was consisted of patients with pcos and healthy volunteers. patients group was divided into two subgroups according to presence of ir. (pcos+ir and pcos-ir). the oral glucose tolerance test (ogtt) and the homeostatic model assessment (homa-ir) were performed to assess glucose tolerance and insulin sensitivity. irisin and betatrophin levels were measured by elisa method. results: circulating irisin was significantly higher in the pco-s+ir subgroup than the control group (p < . ). circulating betatrophin was significantly lower in both patients subgroups than the control group (p < . ). there was no negative or positive correlation between irisin and betatrophin levels. discussion: these data suggest that irisin and betatrophin may act a role together in the ir mechanism in pcos patients. butyrylcholinesterase (bche) synthesized in liver has long been associated with hyperlipidemia, type diabetes and obesity. there are also reports on bche knockout mice becoming obese. the exact involvement of how bche interacts with lipids is still not clear. previously we displayed a correlation between leptin, waist circumference, fat mass and bche levels. recently, we have also shown that bche overexpression in hepg cells is regulated by alpha linoleic acid. as the next approach on the analysis of lipid metabolism and bche interaction, we considered the capability of bche to hydrolyze lipids. human serum bche was purified by subsequent deae-tris-acryl m and procainamide chromatography. the purified bche was utilized in a modified acid lipase assay with the acid lipase substrate -methylumbelliferyl palmitate ( -mu-palmitate). as the second alternative substrate trioleic acid was utilized. the triolein hydrolysis was measured by the nefa kit. verification that bche hydrolysis of these lipid substrates was not due to another esterase was done by iso-ompa inhibition studies. also, lectin binding studies with bche and rca were carried out to rule out non-specific esterase activity. using purified human serum bche and hepatic lipase as control enzyme we found that bche is able to hydrolyze the acid lipase substrate -methylumbelliferyl palmitate ( -mu-palmitate). we found that bche hydrolyses this molecule at ph rather better than at ph . . at ph values, purified human bche has a km value that was times bigger than that of human pancreatic lipase. with the bigger molecule the triolein, the difference between the km values of bche and pancreatic lipase was smaller. bche seems to hydrolyze triolein with an efficacy comparable to approximately % that of human pancreatic lipase. our results display that another function of bche may be its lipid hydrolyzing activity. p- . . - determination of regional reference ranges for erythropoietin with laboratory data mining serum erythropoietin (epo) levels are the main regulator factor of erythrocyte production and increase in response to hypoxia. our region is a location dominated by hypoxic conditions due to the high attitude. in this study we aimed to investigate the mean serum epo levels in the living conditions of our region. two hundred and eighty epo results from our laboratory data whose hemoglobin levels were normal were evaluated in the study. mean serum epo levels were analyzed via chemiluminescence method in beckman coulter dxi auto analyzer. the epo levels of samples was . ae . mul/ml (ranged between . and . ) mul/ml. when we performed ae sd for the studies population we determined normal serum epo levels were as . - . mul/ml. the upper limit determined by our results was % higher than that of determined by the manufacturer as . mul/ml and the lower limit determined by our results was % higher than that of determined by the manufacturer as . mul/ml. normal serum epo levels were considerable for our region and the upper and lower limits were higher than those of determined by the manufacturer. more detailed studies considering the physical properties of participants including a higher number participants are necessary. subclinical hypothyroidism is the precursor to hypothyroidism because it has a tendency to transform into hypothyroidism. subclinical hypothyroidism is considered one of the risk factors causing metabolic syndrome. metabolic syndrome can be characterized by plasma level of apelin released from adipocytes. in the present study, we aimed to measure serum apelin level of patients with subclinical hypothyroidism and compare them with serum apelin level from healthy individuals. our study group included patients diagnosed with subclinical hypothyroidism and healthy volunteers. serum samples were obtained from each participant for the measurement of apelin. these were then stored at À •c until the time of analysis. serum apelin concentrations were determined using an enzymelinked immunosorbent assay. the mean serum apelin levels of subclinical hypothyroidism and control groups were ng/l, control group ng/l respectively. there was no statistically significant difference in terms of the mean apelin levels between the groups (p > . ). apelin levels didn't show significant correlation with bmi (p > . ). in the present study, no significant difference of serum apelin level was observed between patients with subclinical hypothyroidism and healthy control subjects. however, the apelin levels were higher in the patients with subclinical hypothyroidism than in the control group. the possible relationship between thyroid hormones and apelins is critical to understanding the etiopathogenesis of metabolic disorders. the mitochondrial erv /mia import system does not impact cytosolic fe-s cluster protein maturation and iron regulation erv is a sulfhydryl oxidase that partners with the import receptor mia to import small cysteine-rich proteins into the mitochondrial intermembrane space. it has also been suggested that erv has an additional role in maturation of cytosolic fe-s cluster proteins and regulation of iron homeostasis in s. cerevisiae. however, these studies were performed on one particular erv mutant strain (erv - ) that we discovered has additional defects in glutathione (gsh) metabolism. since gsh is required for iron regulation and cytosolic fe-s cluster assembly, this complicates our understanding of erv s role in these processes. we discovered that the erv - strain originally tested for fe-s cluster defects was the only strain to exhibit defects in the cytosolic fe-s enzymes. mitochondrial and cytosolic fe-s protein activities in the other erv and mia mutants tested were similar to the wt control. in addition, while all the erv and mia mutants tested exhibit temperature-dependent defects in mia oxidation, only the erv - strain has significantly reduced gsh levels and more oxidized gsh: gssg redox state. we determined that the cause of gsh depletion in the erv - strain is an additional mutation in the gene encoding the glutathione biosynthesis enzyme (gsh ) that compromises gsh protein folding and/or stability. to address whether gsh deficiency in the erv - mutant is the underlying cause for the cytosolic fe-s cluster defects and iron misregulation for this strain, we measured fe-s protein activity, iron-regulated gene expression, and iron accumulation in erv and mia mutant strains. only the erv - strain exhibited iron misregulation and accumulation of mitochondrial iron, while exogenous gsh rescued these defects. these results demonstrate that the defects in cytosolic fe-s enzymes and iron homeostasis in erv - are due to gsh depletion and neither erv nor mia play significant roles in cytosolic fe-s cluster assembly and iron homeostasis. human c-peptide is a amino acid polypeptide, which is secreted into blood from b-cells in the pancreas where pro-insulin undergoes a post translational modification and cleaved into insulin and c-peptide. human c-peptide concentrations in blood plasma and urine reflect the level of insulin resistance associated b-cell function and can point out insulin secretory failure. the reference intervals in blood plasma and urine are . - . ng/ml and - ng/ml respectively. c-peptide measurement in urine and plasma provides a guide for therapy in diabetes. this study describes a method for the development and validation of picaa (peptide impurity corrected amino acid analysis) method for the determination of the purity of the human c-peptide which could be used as a reference material to measure cpeptide concentrations in plasma. two different methods were performed for the picaa; aaa-id-ms/ms for quantification of constituent amino acids following hydrolysis of the material and rp-hplc-esi-tof ms for determination of the peptide related impurities. the result of the aaa id ms/ms method was corrected for the amino acids originating from the impurities. id ms/ms-aaa was performed with zivak Ò hplc and zivak Ò tandem gold triple quadrupole ms equipped with a phenomenex ez:faast l aaa column ( mm i.d). the mobile phase was composed of, a: mm ammonium formate (af) in water, b: mm af in acetonitrile (acn). the intact peptide analysis was performed by a hitachi lachrome elite hplc and bruker microtof-q mass spectrometer equipped with a capcell pak mg-ii c column ( mm i.d., mm particle size). the purity of the synthetic c-peptide was determined by picaa analysis and related uncertainty was calculated. traceability to si was established using the amino acid standards of which the purity was determined by tub _ itak ume using qnmr analysis. picaa is a simpler alternative to the full mass balance approach which requires large quantities of the peptide material. p- . . - heat shock proteins: complementary therapies in brain tumors with viscum album e. onay-ucar, s. n. biltekin istanbul university, faculty of science, department of molecular biology and genetics, vezneciler, istanbul, turkey cancer is one of the lethal diseases in the world. different cancer types possess overexpressed hsps levels. viscum album extracts with their anticancer and antioxidant properties are being used in cancer therapies. biochemical composition of this plant is known to vary its features depending on the host trees and time of harvest. in our previous study, it has been found that v.album inhibited hsp expression and induced caspase-dependent apoptosis in c rat gliomas. the aim of the current study is to find out whether different v.album extracts have different effects on hsps expression level and apoptosis in c glioma cell line or not. in this study, three different extracts of v.album were compared for their potential inhibition effects on hsps. the cytotoxic effects of extracts have been determined via mtt test. different experiment groups were set up subjected to heat shock and/or incubated without any heat shock application. overexpression of hsps was induced by heat shock at °c for h in c cells. expression levels of hsps were determined by western blot analysis. the apoptosis inducing effect was also evaluated via caspase- activation in c glioma cells. pretreatment of the cells with non-toxic dose ( lg/ml) of v.album extracts prior to heat shock, reduced significantly the expression levels of hsps. similarly, pretreatment with the extracts prior to heat shock increased apoptosis via caspase- activation in c glioma cells. these results will be utilized in the determination of the relation between extract composition and stress protein expressions. these results suggest that different extracts of v.album are able to down regulate expression of hsps, and induce apoptosis. this warrants further exploration as a potential resource of bioactive compounds that can be used in cancer therapy. future studies targeting hsps for the development of chemosensitizers may help improve the treatment of cancer in combinational therapy. biological drugs (biologics) are the fastest-growing category of therapeutics among those approved by the agencies for drugs regulation. most biologics are proteins designed for parenteral use. however, proteins are characterized by poor pharmacokinetic and safety profiles. peg-coating (poly-ethylene glycol coating) of biologics provides several benefits, including an increased half-life related to reduced renal clearance, an increased stability to degradation, and a reduced immunogenic/antigenic response. preservation of the three-dimensional structure and activity of the pegylated form is a strict requirement for human use. the recombinant proteins used for this studies (as-sod, superoxide dismutase; mmp , matrix metalloproteases ; ansii, l-asparaginase ii) were cloned and then over-expressed in escherichia coli. pegylation reactions were performed using commercial reagents. all the protein samples were purified and analyzed by solution and solid-state nmr (fields from mhz to mhz). we developed new protocols to prepare samples of pegylated proteins, demonstrating that solid-state nmr spectra of exceptionally good quality can be obtained for pegylated proteins in the sedimented state (obtained by either ultracentrifugation or rehydration of freeze-dried samples); surprisingly, sedimentation of pegylated proteins to this end has never been attempted. the spectral quality is comparable toor better thanthat of the corresponding crystalline samples. the excellent quality of the solid-state nmr spectra would make it possible to perform extensive resonance assignment and even a conventional full structure determination of biologics. the proposed method is based on the comparison of a standard twodimensional solid-state nmr spectrum of the sedimented pegylated protein with that of the crystalline state of the native proteinfor which the x-ray structure is available. all eukaryotic creatures hereditarily have natural defense mechanisms and are protected from the infections with this defense mechanism. antimicrobial peptides (amp) contain - amino acid content, are positively charged with amphipathic feature. the antimicrobial activities of amps are thought to be depended on the microbiocidal effects by binding to the surface of microorganisms and creating pores in their membranes. defensins are both effector and mediator small antimicrobial peptides of the immune system. these peptides in cationic and amphipathic structure have broad spectrum antibacterial, antifungal and antiviral features. defensins regulate the innate and acquired immune systems by suppressing proinflammatory responses during infection. mammals have three structural subfamilies of defensins. these show differences according to the trisulfide arrays in their structure and are classified as a,b, h defensins. human beings have tissue-specific six functional a defensins. human hnp- and hnp- encoded by defa , defa and defa genes are firstly expressed in neutrophils. human hdp and hdp encoded by defa and defa are firstly expressed in paneth cells in the intestines and play important role in the defense and homeostasis. human beings have many pseudogenes such as defap and deftp in addition to these functional genes. according to literature data, defensins play an important role in defense against microbial placements on mucosal surfaces. in addition, the antimicrobial spectra of defensins include gram negative and gram positive bacteria, fungi and viruses. in addition to their antimicrobial efficiency, they can accelerate the wound healing due to their mitogenic effects on epithelium cells and fibroblasts. bile salt hydrolase (bsh) enzyme catalyzes the hydrolysis of glycine and/or taurine-conjugated bile salts into amino acid residues and the free bile acids that reduce cholesterol. however, some intestinal bacteria have an excessive deconjugation of tauro-conjugated bile salts and production of secondary bile acid having potential harmful side effects to the host. the catalytic mechanism and substrate preference of such bsh enzyme is not clear. in this study, bsh gene from lactobacillus plantarum gd strain was cloned, expressed, characterized in escherichia coli blr(de ) strain, and then val- and phe- amino acids, supposed to be responsible for substrate preference, were substituted for met- and ile- amino acids respectively by site directed mutagenesis. the hydrolysis activities and stability of the mutant recombinant bsh (mrbsh) enzymes were examined along with six different bile acids by ninhydrin assay and sds-page respectively. ninhydrin test results indicated that wild-type recombinant bsh (wrbsh) hydrolyzed six major human bile salts with an apparent preference towards glycine-conjugated to tauro-conjugated bile salts. however, the activities of mrbsh/phe ile enzyme are %, %, %, %, % and % of the activity of wrbsh against to glycocholic acid (gca), glycodeoxycholic acid (gdca), glycochenodeoxycholic acid (gcdca), taurocholic acid (tca), taurodeoxycholic acid (tdca) and taurochenodexycholic acid (tcdca) respectively. the activities of val met mrbsh enzyme are %, %, %, %, % and % of wrbsh against to gca, gdca, gcdca, tca, tdca and tcdca respectively. our findings support the suggestion that bsh enzymes recognize their substrates predominantly at the amino acid moieties and not at the cholate moieties. however, further pcr-based site-directed mutagenesis and structure-driven computational and theoretical approaches are required for the precise determination of their substrate specificities and the selection of probiotic bacteria. we deposited bacteriorhodopsin in purple membranes under applied electrical field onto ito (indium tin oxide) support. purple membranes film, highly oriented in one direction, was placed between two ito electrodes. we studied dependence of electrical properties of these films on light illumination. we argue that this setup can be used for functional studies of microbial rhodopsins. in opposite to already published results where this system was used as a photocondensor for studying functional properties of bacteriorhodopsin, we studied electric properties of such systems and we found strong light dependence of resistivity of bacteriorhodopsin in purple membranes films. optogenetics is already used in study of neuronal cells cooperation in vitro and in vivo by means of microbial rhodopsinsion pumps and channels incorporated in membranes of neurons changing their electrical potential while receiving a light quantum by laser or led source. best perspectives optogenetics will give after successful transfer to medical applications, such as the treatment of blindness, treatment of disorders like parkinson's disease etc. but to achieve these we need a broad set of tools, optogenetics tools, highly specialized to solve specific problems of neurophysiology. to the creation of such tools our work is dedicated. new optogenetic tools can be made by mutations in existing ones altering their properties (mainly spectral characteristics, selectivity and conductivity) or some promising mutations in conserved residues can be found in existing organisms. a halophilic archaeon halosimplex carlsbadens is a host of protein of our interest. according to the theoretical data based on the alignment with br and the d structure model of this novel protein, we suppose this protein functions like the light-driven h+ pump: all the key residues are the same or at worst have the similar properties, except one in the position leucine instead of the aspartic acid. a gram-positive bacteria deinococcus-thermus phylum syntheses rhodopsin with substitution of this aspartic acid to alanine. sphingomonas paucimobilis has rhodopsin where aspardic acid in position is changed to serine residue. and one yet uncharacterised guillardia theta rhodopsin even has the same as br motif (d , t , and d ) but according to alignment is closer to chr even the last one motif is e , t , n . it is expected that all of them will show us new properties. though the further experimental data are essential. the work is supported by rsf - - . evaluation of some thymus proteins in patients with crimean congo hemorrhagic fever i. b€ ut€ un , s. sahin , f. duygu university of gaziosmanpasa, department of biochemistry, tokat, turkey, oncology education and reasearch center, ankara, turkey crimean congo hemorrhagic fever (cchf) is a tick-borne viral zoonotic disease. it has a high fatal rate (% - ). tokat is one of the cities having the most reported cchf cases, in turkey. clinical presentation of the disease varies widely among patients. thymic peptides are small molecules synthesized by thymic epithelial cells. they play role in the immune response, as well as anti-inflammatory process. fourty patients referring to the hospital with tick-contact history and/or presenting clinical manifestations consistent withcchf and with positive pcr results for cchf virus in blood samples were included to the study. the wbc and platelet values at application and before the patients were discharged were recorded. the healthy control group consisted of age and gender matched healthy volunteer adults free of any chronic disease. thymosin alpha (ta ), thymuline and thymosin beta (tb ) were studied by the elisa method in this study. biochemical parameters were also analysed. ast and alt values were significantly higher (p < . ) and plt and wbc levels were significantly lower in the cchf group (p < . ). levels of tf, ta and tb were found to be significantly higher in cchf (p < . ). there was no mortality during the study period. duration of hospitalization was . ae . days. levels of tb were significantly correlated with duration of hospitalization (r= À . , p = . ). alt levels were significantly correlated with tf levels (r = . , p = . ). patients received ffp and apheresis for the supportive treatment, while patients received only ffp and patients got only apheresis. patients did not get any of these blood products. there was not a statistically significant differences in thymus peptides among these treatment groups (p > . ). we report survived cchf patients with elevated thymic peptides. pathogenesis of cchf has many points to be highlighted. thymic peptides may play role in the clinical situation of the patients with the disease. the effect of methocarbamol on the peroxiadse activity of human erythrocyte hemoglobin hemoglobin is released to blood circulation, after red blood cells lysis. it is carried in circulation by binding to haptoglobin. in normal persons, no free hemoglobin is observed in the blood, because most of hemoglobin is in the form of haptoglobin complex. in some diseases that are accompanied by hemolysis, the amount of released hemoglobin is higher than its complementary haptoglobin. as a result, free hemoglobin appears in the blood, which is a toxic compound for these patients. free hemoglobin has been showed to have peroxidase activity and considered a pseudoenzyme. in this research, the effect of methocarbamol on the peroxidase activity of human hemoglobin was studied. our results showed that the drug inhibited the pseudoenzyme by un-competitive inhibition. both k m and v max decreased by increasing the drug concentration. k i and ic values were determined as and mm, respectively. molecular docking results showed that methocarbamol did not attach to heme group directly. a hydrogen bond connected nh of carbamate group of methocarbamol to the carboxyl group of asp side chain. two other hydrogen bonds could be also observed between hydroxyl group of the drug and ser and ser residues of the pseudoenzyme. p- . . - dca reduces viability and down regulates mapk protein activations in human malignant mesothelioma cells and pericardium. microarray analyze results performed in mm patients revealed that one of the most prominent changes is upregulation of many genes involved in glycolysis and the krebs cycle. dichloroacetate (dca) is an inhibitor of pyruvate dehydrogenase kinase (pdk) that enhances the oxidative activity of cells by activating pyruvate dehydrogenase (pdh) in mitochondria. dca has shown as a promising anti-neoplastic agent that re-sensitizes cancer cells to apoptosis. the aim of this study is to elucidate the coupling between pdk inhibition and mm cell proliferation and cell cycle. human malignant mesothelioma (spc ) cell line was used as a model for dca treatments. cell viability was measured by mts assay; mapk protein activations and expressions were assessed by western blotting; cell cycle profile was analyzed by flow cytometry. statistical analysis was performed by utilizing one-way anova test. results showed that dca reduced viability of spc cells in a concentration and time dependent manner. protein analysis indicated that mapk pathway was down regulated at concentrations greater than mm. moreover, primary cell cycle analysis has indicated arrestment at g /m phase in hours. our findings corroborate with recent reports where dca treatments resulted in reduction of viability and g /g and g / m arrest in other cell lines. abnormalities in mapk signalling play a critical role in the progression of cancer. here, we showed for the first time that dca decreased mapk activation in h. our results suggest that dca is an anti-prolifertive agents for mm cells in vitro. however, it requres extra analysis with other mesothelial cells. future study will focus on investigating relation between mapk and mitochondrial apoptosis. adrenomedullin (adm) is a vasodilator peptide consisting of amino acids. adm is synthesized in many tissues. and is a biologically active peptide that has various effects including vasodilatation, the regulation of vascular endothelial function and adjusting adipogenesis. hypoxia inducible factor alpha (hif a) is a subunit of a heterodimeric transcription factor hypoxia inducible factor . it is the master transcriptional regulator of cellular and developmental response to hypoxia. the dysregulation and overexpression of hif a by either hypoxia or genetic alternations have been heavily implicated in cancer biology as well as a number of other pathophysiologies. in our research, the adm and hif -a levels in heart, kidney and lung tissues of rats were investigated in control, hypoxia, control+adm and hypoxia+adm groups. rats in hypoxia groups were provided hypoxic environment containing of - % oxygen and - % nitrogen for week. rats in adm groups were injected intraperitoneally in a dose of . nmol/kg for four days before the collection of the tissues. the control group was oxygenated with normal air. the control and treatment groups were formed from - animals and adm, hif -a levels were measured in taken tissues with immunoassay method. the aim of this study was to investigate the reaction of the organism when exposed to hypoxic conditions and the effect of adm over hif -a level. adm levels and hif -a in heart tissue were found decreased in hypoxia group, and adm levels increased in hipoxia+adm group. hif -a levels decreased in hypoxi+adm group. adm levels in liver tissue were found decreased in hipoxia and control+adm groups than control group. hif -a levels were higher in control+adm group. adm has a role in angiogenic process, and our experiment showed that adm reacts earlier than hif -a, and affects its synthesis. organism increases its vascularization as a reaction to hypoxic condition, and adm treatment may provide a rapid adjustment. p- . . - covalent conjugation and characterization of immunogenic protein of toxoplasma gondii and polyacryclic acid as vaccine candidate r. c ß akir koc ß yildiz technical university, department of bioengineering, istanbul, turkey toxoplasmosis is a major medical and veterinary disease caused by toxoplasma gondii which infect approximately half of the world's population. this infectious disease especially gains importance in pregnant women and immunodeficient individuals. also t. gondii infection has economic importance. however, there are only one attenuated-live t. gondii vaccine for veterinary uses and no vaccine against t. gondii is available for humans. therefore development of an effective vaccine would be extremely valuable for preventing disease in human and veterinary medicine. subunit vaccines are very attractive vaccine candidates but there is low antigenicity problem when they are used alone. polymers themselves don't stimulate immune response while they used with antigenic structure of various infective agents enhance immune response because when proteins are covalently conjugated with hydrophilic polymers, ( ) their circulatory-lives and stability (in different ph and temperature values) enhance ( ) binding to proteases and clearance by the reticuloendothelial-system decreases. in this study, immunogenic protein of t.gondii and polyacrylic acid with immune stimulant properties was covalently conjugated and conjugation was demonstrated by size-exclusion chromatography (sec) and fluorescence spectroscopy. it is significant to detect time of death in case of a sudden death for medical and legal concerns. there is no known method that can be used for post mortem time detection. based on this deficiency pmi detection in narrow time frame is a big problem. in this study, we aimed to investigate and determine timedependent expressional changes of apoptotic markers by western blot technique. postmortem skeletal muscle were analyzed hour periods in first -hour after death. nd and rd -hour periods were statistically significant (p < . ). keywords: post mortem interval, time of death, apoptosis. hyaluronidases are excessively found in nature and involved in numerous biological functions. hyaluronidases primarily degrade hyaluronic acid (ha) and have significant role in fertilization during acrosomal reactions. therefore, the measurement of hyaluronidase enzyme activity may provide valuable information about acrosomal function and the fertilizing ability of the sperm. the aim of this study was to investigate the semen hyaluronidase enzyme activity changes among four different sheep breeds (akkaraman, suffolk, merino, and kıvırcık). in this research, ten ram testis tissues from each sheep breed, a total of , were cut and collected on ice. ovine testicular hyaluronidase of four different sheep breeds was purified from a crude ammonium sulfate-precipitated fraction of an extract of ram testis. the semen hyaluronidase enzyme activity differences between the sheep breeds were examined by spectrophotometrically monitoring the appearance of ha at nm. analysis of variance test was used to examine the possible mean differences among the four different sheep breeds. the observed mean differences in enzyme units for kıvırcık, suffolk, akkaraman, and merino were as follows . , . , . , and . , respectively. the observed mean differences in absorbance values for kıvırcık, suffolk, akkaraman, and merino were as follows . , . , . , and . , respectively. the results showed that the observed mean differences in enzyme units and absorbance values among the four different sheep breeds were not statistically significant. despite that, in average kıvırcık had higher values for the activity of each sample and yet it had the smallest values for standard deviation. therefore, in order to achieve higher enzyme activity and more homogenous samples kıvırcık breed should be preferred. what is extra to learn from protein drying measurements? hydrations of soluble proteins are crucial for their functionality. therefore elucidating the details of protein hydration is still of interest in the proteins' action mechanisms. this is the motivation of the present study. in order to study protein hydration, changing concentrations of the well-studied serum albumin protein was measured with the spectroscopic techniques like uv-vis and ft-ir spectroscopy. spectral data is analysed and calculations were performed on the data to extract the relevant changes in the protein. experimental parameters' variation in association with the spectral changes implies the involvement of protein structure and hydrogen bonding in the drying process. the protein's reactions may not be merely a feature of the protein structure in the common sense but it could be related directly to the protein hydration states as well. this is understandable since it is already known that enzymatic proteins lose their functionality when they are dried while this drying may or may not involve dramatic structural changes. on the other hand, here it is claimed that the role of water in gaining the functionality that was lost in the dried state is not just about enhanced diffusion processes and the dynamicity but could be related to the functionality of water in the energy transfer processes as well. investigating the cellular effects of the aldoketo reductases akr b and akr b in hct- colon cancer cells b. taskoparan, e. g. seza, m. s. ceyhan, s. banerjee middle east technical university, ankara, turkey aldo-keto reductases (akr) are nad(p)h dependent oxidoreductases are best characterized as glucose reducing agents, and have been implicated in diabetic pathophysiology. increased expression of akr has been associated with tumors of lung, breast, prostate, cervix, ovarian and colon. two members of the akr superfamily that have been associated with different cancer types are akr b ; aldo-keto reductase family , member b , and akr b ; aldo-keto reductase family , member b . both are -kda cytosolic reductases that are similar in both amino acid sequence identity ( %) and tertiary structure with the (a/b) barrel topology. while hct- , a colorectal cancer cell line, cells expresses akr b robustly, there is no expression of akr b . in this study, we have stably knocked down akr b through shrna technology and overexpressed akr b in hct- cells. comparisons were made with a known akr inhibitor sorbinil. with the knock down of akr b , we have observed reduced cellular proliferation, enhanced apoptosis, delay in cell cycle progression, reduced expression of mitogenic proteins and a decrease in activation of the inflammatory transcription factor nuclear factor kappa b (nf-kappab). interestingly, although akr b overexpression did not affect cell proliferation, apoptosis or cell cycle, some effect was observed with nf-kb signaling. our data indicate that, although closely related, akr b and akr b have very different contributions towards signaling pathways in colorectal cancer. comparison of different nisin quantification methods and optimization of nisin production by lactococcus lactis z. girgin ersoy, g. demir, m. f. cesur, s. tunca gedik gebze technical university, kocaeli, turkey nisin, which is produced by certain strains of lactococcus lactis, is the only bacteriocin approved by world health organization (who) as a food additive. it prevents the growth of foodborne bacteria which cause food spoilage. nisin research and applications necessitates developing an accurate and reproducible method for its quantification. the agar diffusion bioassay is the most widely used method for quantifying nisin, although it has limitations especially diffusion-related difficulties of the active substance. in the present study, "agar diffusion bioassay", "enumeration of colony forming units", "colorimetric assay" and "flow cytometry" methods were compared with each other to determine antibacterial activity of nisin on micrococcus luteus. moreover, this study also covers the results about the effect of different cultural conditions to optimize nisin production by l. lactis. galactose, lactose and their combination in m medium (ph ) boosted nisin production at °c, as the addition of . lg/ml hemin into the fermentation broth. to our knowledge, this is the first study showing the usage of "flow cytometry" method to determine nisin activity of fermentation broth filtrates. p- . . - coronaviral nucleocapsid protein is an antiviral target for drug development institute of genomics and bioinformatics, national chung hsing university, taichung, taiwan between and , the severe acute respiratory syndrome (sars)-cov caused a worldwide epidemic and had a significant economic impact in the countries affected by the outbreak. recently, the middle east respiratory syndrome human coronavirus (mers-cov) was found in patients with severe acute respiratory tract infections in the middle east and south korea. as is true for all coronavirus infections, there are no efficacious therapies currently available against coronaviral diseases, making the development of anti-coronavirus compounds a priority. the cov genome consists of positive-sense, single-stranded rna approximately kb, and it contains several genes encoding several structural and non-structural proteins that are required for progeny virion production with a conserved order. the n proteins exist in the center of the viral particle and represent a helical structure complex. nucleocapsid protein is most abundant structural protein of covs, binds the viral rna genome to form the virion core, leading to the formation of a ribonucleoprotein (rnp) complex or to a long helical nucleocapsid structure, that is important for maintaining the rna in an ordered conformation for replication and transcription. the cov n protein is also involved in the regulation of cellular processes, such as gene transcription, interferon inhibition, actin reorganization, host cell cycle progression, and apoptosis. two strategies to inhibit oligomeric n protein function have been reported. the first strategy is to discover antiviral agents that target the rna-binding site. the second one is to impair normal n protein function by interfering with monomer-oligomer equilibrium. our recent studies suggest that n proteins in infections caused by coronaviruses will be useful antiviral drug targets because they serve many critical functions during the viral life cycle. post-translational modification of vascular endothelial growth factor (vegf) in colon cancer cells s. tunc ßer, e. solel, s. banerjee middle east technical university, ankara, turkey vascular endothelial growth factor a (vegf-a), commonly referred as vegf, is a potent secreted mitogen crucial for tumor initiation and progression. the gene for vegf is translated into a number of splice isoforms that lead to , , and amino acid proteins, with different receptor-binding and matrixbinding properties. in the present study, we discuss the functional significance of post-translational modification/processing of vegf isoform in hct- colon cancer cells. we also focus on the role of calcium in the post-translational modification of vegf . we show that vegf undergoes n-linked glycosylation in hct- cells. perturbation of cellular calcium may affect vegf driven malignant phenotypes. p- . . - novel methods for modulating the activity of bcl family proteins in apoptosis p. rowell, j. miles, a. wilson, t. edwards apoptosis, also known as programmed cell death, is an essential cellular process, but is implicated in several human diseases, including diabetes and cancer, when it is up-or down-regulated respectively. bcl- family proteins are major players in the control of intrinsic, or mitochondrial apoptosis; they respond to intracellular stress signals, function through protein-protein interactions and converge on the mitochondrial outer membrane to cause membrane permeabilisation, release of cytotoxic molecules, and initiation of an apoptotic cascade that leads to cellular demise. our work aims to identify molecules able to bind and modulate the activity of several key players in the bcl- family, including the pro-survival members bcl- , bcl xl and mcl , and the death promoting family member bax. adhirons, novel non-antibody peptide display scaffolds developed at the university of leeds, have been used to construct a phage display library containing over clones, and form a key part of the strategy to identify such molecular modulators. adhirons able to selectively bind individual bcl- family members have been identified, in vitro assays carried out to test for modulatory activity, and xray crystallography used to elucidate details of how they interact with their target proteins. more recently, studies have been carried out to identify adhirons able to target multiple bcl- family members, with the aim of selectively inhibiting defined groups of proteins in cells. this work provides opportunities to differentiate the activities carried out by different bcl- family proteins in apoptosis, enabling us to better understand how their dysregulation contributes to human disease. biophysical and evolutionary study of the structural flexibility of adp-dependent sugar kinases from mesophilic and psychrophilic archaea r. zamora , v. castro-fern andez , c. a. ramirez-sarmiento , e. a. komives , v. guixe facultad de ciencias, universidad de chile, santiago, chile, department of chemistry and biochemistry, university of california, san diego, united states of america the capability of extremophiles microorganisms to live at low temperatures is mainly attributed to the high structural flexibility of its enzymes. several sequence and structure features have been associated to a high structural flexibility that enables metabolic processes to occur at low temperatures. during evolution, the general mechanism adopted by these enzymes has been to reduce the free energy of the transition state rather than the michaelis constant, k m . increased structural flexibility and decreased affinity for its substrates in psychrophilic enzymes is compensated by an increase in the catalytic rate, k cat . few psychrophilic enzymes have been reported to performance the optimization of their catalytic efficiency (k cat /k m ) by decreasing k m values. we use the adp-dependent kinase sugar family of archaea as a model, to identify particular structure and sequence features of a psychrophilic enzyme that would make this enzyme more flexible than their thermostable homologues. we characterize the bifunctional psychrophilic enzyme phosphofructokinase/glucokinase from methanococcoides burtonii (mbpfk-gk) and the bifunctional mesophilic enzyme phosphofructokinase/glucokinase from methanococcus maripaludis (mmpfk-gk) by spectroscopic, biophysical and computational techniques. the comparison showed that the absence of two ion pairs is primarily responsible for the increased structural flexibility accounted in the psychrophilic model. this increase in structural flexibility is reflected in the exponential increase in the k m values with temperature. additionally, we reconstruct the sequences of all ancestral enzymes between the current enzymes and their last common ancestor, which was used to trace the occurrence of these electrostatic interactions during evolution in the adp-dependent sugar kinase family. our results suggest that electrostatic interactions are a dominant feature in the transition from psychrophilic to thermophilic environments. fondecyt . elucidating the domain swapping mechanism of the forkhead domain of human foxp fox transcription factors control gene transcription during key processes, such as embryogenesis and immunity, and feature a conserved dna-binding domain known as forkhead. while most forkhead domains are monomeric, solved structures of members from the p subfamily (foxp) show that they can oligomerize by domain swapping (ds), a mechanism where protein segments are exchanged between subunits leading to an intertwined dimer. the biological relevance of ds in foxp has been stressed by disease-causing mutations that impair this process. however, for many proteins ds takes days to occur and requires global protein unfolding. thus, the current mechanism impedes a conciliation of the occurrence of ds of foxp in a biological context. here, we elucidate the biological feasibility of this process by biophysically characterizing the ds mechanism of the forkhead domain of foxp using size exclusion chromatography (sec), circular dichroism, and hydrogen-deuterium exchange mass spectrometry (hdxms). our results show that ds of foxp occurs at micromolar protein concentrations, within hours and is energetically favored. remarkably, dimeric foxp follows a threestate n « i « u folding mechanism, where dimer dissociation into a monomeric intermediate (i) precedes protein unfolding, in contrast to other ds proteins. using sec and hdxms, we show that the i state of foxp largely resembles the native state, but has a larger hydrodynamic radius and a higher deuterium uptake in regions that maintain the compact monomer, suggesting that the i state is an 'open' conformation en route of ds. finally, we compared the local flexibility of the dimer and monomer of foxp , showing that only the hinge region connecting ds segments exhibits different deuteration rates. our results show that ds in foxp follows an unusual threestate folding mechanism that proceeds through transient structural changes rather than needing protein unfolding as in most ds proteins. (fondecyt , ). p- . . - the sustained release of growth factor proteins following their implantation in tissue engineering j. jang bone tissue engineering has become a promising approach for bone regeneration. however, insufficient vascularization during bone regeneration, particularly with large bone defects, results in poor and unsustainable bone formation due to central necrosis. therefore, vascularization following implantation in vivo is essential to the successful formation of new bone tissue. we evaluated the release profile of vegf from bgs using a novel fluorescence-based retention assay, which revealed that vegf loaded on bgs can be released in a sustained manner without an initial burst (near zero-order cumulative release) with a controlled release rate of . % per week for up to weeks. in contrast, an elisa-based release assay showed vegf to have an early burst-release profile for the first week. however, the biological activity of vegf released from the bgs was preserved over the -week release period, which is consistent with the sustainedrelease profile observed in the fluorescence-based retention assay. we developed a novel fluorescence-based retention assay to evaluate the release of vegf from bgs. this fluorescence-based retention assay, which detects the vegf that remains on bgs, reveals that vegf loaded on bgs can be released in a sustained manner, with a minimal initial burst, for up to weeks. these results indicate that the sustained biological activity of the vegf released from bgs over the full -week period promotes bone regeneration, and suggest its potential use for bone tissue engineering. irisin is a recently discovered myokine which regulates energy metabolism and is associated overweight. we aimed to evaluate serum irisin levels in the patients with morbid obesity. a total of sixty patients with morbidly obese and thirty healthy control subjects were included in this study. all participants were measured body weight and height, the lipid profile, and plasma glucose, hba c, insulin and irisin levels. irisin levels were measured by elisa method. serum irisin levels were significantly lower in morbidly obese patients than healthy controls (p < . ). there was no statistically significant difference in terms of age or gender. serum irisin was negatively correlated with bmi, insulin levels, and homa-ir (p = . , p = . , p = . , respectively). our study revealed lower irisin levels in morbidly obese patients with respect to control subjects. the lower irisin levels observed in morbidly obese patients might suggest a loss of browning of subcutaneous adipose tissues. p- . . - pka inhibition restores adenosine uptake in renal tubular epithelial cells under high d-glucose conditions w. garrido, j. catal an, s. alarc on, r. san mart ın universidad austral de chile, valdivia, chile introduction: diabetic nephropathy (dn) is the leading a cause of end-stage renal failure whose pathogenesis must to be elucidated. the progression of dn has been associated with elevated levels of adenosine. extracellular adenosine availability is regulated by the activity of the equilibrative nucleoside transporters (ents). due to the ents have putative sites of phosphorylation our objective was to evaluate the role of pka and ck kinases on ents activity. methods: adenosine uptake ( lm adenosine, s, °c) was assayed in hk cells preincubated with mm or mm d-glucose for h and exposed to lm -br camp, lmh or lm tbb for h. plasma membrane and intracellular proteins were fractionated by the biotinylation method and ent and ent contents were quantified by western blot. results: high d-glucose in hk cells inhibited the uptake of adenosine. this effect was reversed using a pka inhibitor (h ) through an increased ent uptake activity. we noticed this pka inhibitor did not regulate the plasma membrane localization of ent or ent under normal d-glucose ( mm) or high d-glucose conditions ( mm). also, we saw that tbb (ck inhibitor) decreased the activity of ent and ent under normal glucose conditions, decreasing the localization of ent at cell surface, while the membrane localization of ent decreases under the effect of tbb and high d-glucose conditions. conclusions: pka inhibition reversed the effect of high d-glucose, increasing the uptake of adenosine mediated by ent . this could be a new target for the restoration of adenosine levels in dn. relation between serum lipo (a), plasma fibrinogen, red cell distribution width and mean platelet volume in healthy adult men the aim of this study was to investigate the relationship between the serum lipo (a) and plasma fibrinogen, red cell distribution width (rdw) and mean platelet volume (mpv) in healthy adult men. for this purpose, healthy adult men have normal physical examination and laboratory findings and not use any drug were included to the study. serum lipo (a) levels and hematologic parameters (fibrinogen, rdw-sd and mpv) were measured by autoanalyzer and commercial kits. the mean of the age of the persons was . ; body mass index was . ; serum lipo (a) level was . and plasma fibrinogen level was . . there was significant positive correlation between the serum lipo (a) and plasma fibrinogen levels (r = . ; p = . ), significant positive correlation between the serum lipo (a) and rdw-sd (r = . ; p = . ) and significant negative correlation between lipo (a) and mpv (r= À . ; p = . ). the plasma fibrinogen and the serum lipo (a) levels have been known as the risk factors for cad (coronary artery disease) increase together in healthy adult men. similar findings have been reported in cad patients. it has reported that elevated rdw is associated with intracoronary thrombotic burden and may be associated with the severity and instability of acute myocardial infarction. in addition, mpv is predictor of severe atherosclerosis and may be used for the prediction and identification of cardiac risks in cad patients. our findings show that elevated rdw and decreased mpv may predict to increased risk of cad in the future, in healthy adult men. follicular fluid is rich in peptides, which significantly influence the growing oocyte. due to existence of a link between kisspeptin (metastin) cells and gonadal steroids kisspeptin might manipulate the gonadotropin axis and folliculogenesis. in this context, the study was planned to investigate for the first time that the follicular fluid (ff) and serum concentration of kisspeptin in high and poor responders undergoing in vitro fertilization (ivf)/intracytoplasmic sperm injection (icsi). biological samples were collected from twenty infertile women with polycystic ovary syndrome (pcos) and poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone (gnrh) antagonist protocol for ivf/icsi treatment. kisspeptin concentrations were measured in serum and follicular fluid by using elisa, whereas fsh and lh levels were detected by routine laboratory methods. it was found that kisspeptin levels were significantly lower in serum and follicular fluid of infertile women with pcos. kisspeptin levels were correlated with fsh and lh level in infertile women with pcos. it can be concluded that low level of kisspeptin might inhibit gnrh release that might cause to the inhibition of fsh and lh release and might disrupt folliculogenesis. decline in serum and ff levels of kisspeptin might be possible cause of anovulation and subfertility in pcos subjects. cryptococcus albidus d is a newly identified yeast isolates from petroleum area in _ izmir as a lipase producer. the molecular weight of the enzyme is . kda as found. optimum temperature was °c and half-life times were , , and . min at , , and °c, respectively. optimum ph value was . . however, it shows significant ph stability at ph values . and lower. the existence of acetone in the solution as a solvent enhanced lipase activity. cryptococcus albidus d lipase was able to catalyze the esterification reaction between fructose and palmitic acid to produce fructose palmitate using acetone as the solvent. due to its stability in organic solvents, we propose that in order to increase the yield of fructose palmitate, we could immobilize d lipase. therefore, the effect of immobilization on kinetic parameters of d lipase was investigated. different immobilization materials and methods were used to find efficient support materials for d lipase immobilization. additionally, fructose palmitate production processes will be optimized with immobilized lipase. introduction: the diagnosis of osteoarthritis (oa) is based on clinical symptoms and radiographic findings. it is known that the pathologic changes at the molecular level in the joint cartilage tissue start before symptoms appear in oa. c q tumor necrosis factor-related protein (ctrp- ), c q tumor necrosis factor-related protein (ctrp- ) and kallistatin are related to many different cellular processes including bone and cartilage tissue metabolism. the aim of this study was to investigate any probable association between the serum ctrp- , ctrp- and kallistatin levels and diagnosis and radiologic staging of knee oa patients. materials and methods: this study included patients with knee oa and healthy individuals for control purposes. the patient group was divided into four stages radiologically. ctrp- , ctrp- and kallistatin levels were measured in serum samples of patient and control groups with elisa method, and the differences between the groups were analyzed with statistical methods. results: the levels of serum ctrp- in the patient group were significantly higher than in the control group (p = . ), serum ctrp- and kallistatin levels were not statistically different (in order of p = . , p = . ). in the patient group, there was not a significant difference between serum ctrp- , ctrp- and kallistatin levels and radiologic stages (respectively p = . , p = . , p = . ). there was a significant positive correlation between the radiologic stage and patient's age, body mass index, western ontario and mcmaster universities arthritis index and visual analogue scale values (respectively p = . , r = . ; p = . , r = . ; p = . , r = . ; p = . , r = ). discussion and conclusion: serum ctrp- levels were detected significantly increased in patients with knee oa, but there was no significant difference in ctrp- and kallistatin levels. there was not a significant association between the radiologic stage and levels of ctrp- , ctrp- and kallistatin. enzymes in microorganisms, especially termophilic bacteria are more attractive in biotechnology and molecular biology due to the higher catalytic activity. turkey is rich in geothermal resources and it is important to determine unknown microbial content of geothermal sources. in this study, water and sludge samples were taken from ayder, kizilcahamam and gonen hotsprings. firstly, ph, temperature, salt concentration, gram reaction, mobility, endospore formation, oxidase and catalase tests were carried out as conventional characterization. molecular characterizations of isolates were achieved by fames, rep-pcr and s rrna sequencing. finally, test isolates were evaluated according to enzyme production capability by petri dish. as result of conventional tests, isolates were determined as gram positive, mobile-rod shaped, aerobic, oxidase, catalase and endospore positive. the growth range for ph and temperature of the isolates were determined as - and - °c. in consequence of the salt test, the test isolates were grown at - % nacl. of thermophilic isolates were selected by rep-pcr and according to s rrna sequencing analysis test isolates were belonging to bacillus, geobacillus, anoxybacillus and brevibacillus genus at a range of - %. enzyme tests showed that, some of the isolates were able to produce protease (f , f , f , f , f and f ), amylase (f , f , f , f and f ), cellulase (f , f , f , f , f and f ), xylanase (f , f , f , f and f ) and lipase (f , f , f , f and f ). it can be concluded that, geothermal regions are rich in bacillus and related genera. fame analysis was particularly insufficient for diagnosis of thermophilic microorganisms, but rep-pcr was successful in separation of organisms at species and even subspecies levels. most of our bacterial isolates have industrially important enzyme production capacities. it is a pioneer result to use bacteria for industrial applications which need higher temperatures. p- . . - warburg effect was investigated by studying various metabolic molecules and assays in mammalian cell lines z. i. kanbagli, b. kiratli, h. cimen yeditepe university, istanbul, turkey majority of the different cancer cells switch their metabolism from oxidative phosphorylation pathway to glycolytic pathway; in order to meet excessive energy requirement, which is also called warburg effect. acetylation is one of the most crucial post-translational modifications playing key roles in metabolic reprograming. in this study, the relationship between acetylation dependent changes in energy metabolism and apoptotic pathways were investigated in pnt a, du , hela, hep b, hek t, shsy y. immunoblotting experiments were applied by using antibodies against acetylated-lysine to examine the changes in overall acetylome. candidate proteins displaying elevated acetylation was identified with mass spectrometry based-proteomic analysis. glucose transporter (glut ) was used to detect insulin-stimulated glucose transport, total oxphos rodent antibody cocktail to identify variations in complexes which are responsible for most of the atp production. caspases (casp- , - ) to unreveal different activation levels of apoptotic pathway among the cell lines. mitochondrial membrane potential was measured by using rho-damine by employing confocal microscopy. the expression level of respiratory chain complex iv subunit mtco and casp- was higher in hek t compared to other cell lines. casp- was upregulated in cancer cell lines, mostly in hep b. glut- levels were downregulated in cancer cell lines in contrast to healthy cell lines. findings imply that these proteins might have significant roles leading to variable metabolic and apoptotic activity of each cell line during energy production. due to the results, mtco might be important in adaptation of different cell lines to regulate the overall respiratory chain complex activity. reduction in glut level demonstrates insulin desensitization in cancer cell lines, which might lead to metabolic defects in these cells. besides, since p has a repressive effect on glut , it also can lead us to study about p levels. the effect of inhibition of pi k/akt/mtor signaling pathway on receptor tyrosine kinase expression in breast cancer cells g. tunali, a. l. dogan department of basic oncology, hacettepe university cancer institute, ankara, turkey the increased expression and activation of receptor tyrosine kinases (rtk) (egfr, her , her ) play important roles in breast cancer pathogenesis. her /her interaction is the most potent heterodimer and it causes oncogenic pi k/akt activation. inhibitors of pi k/akt pathway (akt inhibitor and pi k/mtor dual inhibitors) lead to increase in rtk levels and activities while blocking signaling pathway. in this study, the time dependent effect of dual pi k/akt inhibitor pi- on receptor tyrosine kinase expressions' in breast cancer cells was investigated. two breast cancer cell lines, mda-mb- cells (which has egfr overexpression and pten deficiency) and skbr- cells (which has her overexpression) were evaluated for the effect of dual inhibitor. these cells were treated with dual pi k/akt inhibitor pi- for different time periods ( - h) . egfr, her her total rtks expression and pi k/akt pathway inhibition (p-akt and p-p s k expression) were evaluated by western blot. in mda-mb- cells, there were significant decrease in p-akt and p-p s k proteins' expression during the first h. this inhibition was followed by reactivation of the signaling pathway after h. in skbr- cells, p-akt and p-p s k proteins' expression were significantly decreased during the first h. the pi k/ akt signaling pathway in these cells were reactivated after h. basal expression of egfr and her in mda-mb- cells and basal expression of her and her in skbr- cells were found to be very high. transient inhibition of akt and mtor protein kinase activation in tumor cells followed by reactivation of signaling pathway did not result in a time-dependent difference on egfr, her and her expression levels. these results suggest that dual pi k/mtor inhibiton by pi- may trigger receptor tyrosine kinase reactivation due to the signal distruption without affecting total protein expression level. site-specific bioorthogonal reactions are one of the significant tools for discovering different aspects of biological systems in live cells. the reactions should be highly stable and rapid in physiological conditions. various chemical tools can be used in bioorthogonal reactions to monitor biological systems, therapeutics, microscopy and diagnostic applications in live cells. synthetic covalent chemistry in the study of biological systems has been used to label biomolecules selectively in their native environment. for example, small synthetic fluorophores can be added to biomolecules without disturbing other molecular biological pathways. aldehydes or ketone-based functional handles can be attached onto protein at specific sites via chemoenzymatic reactions. labeling of carboxy terminus of a-tubulin has been successfully studied in our previous studies by replacement of wild type tyrosine with unnatural amino acid -formyltyrosine in the presence of tubulin tyrosine ligase enzyme (ttl)-as its role can suggest whether certain cancer cells might grow more aggressively than others. in this work, we highlight the synthesis and spectroscopic properties of azacoumarin chemical probes to study tubulin-tubulin tyrosine ligase (ttl) system in live cells. significant increase in fluorescence quantum yield or a red shift on absorption and emission maxima is observed when the conjugated product is formed. bioorthogonal fluorescent labeling is such a favorable reaction to perform rapid kinetics, localization and high site-specificity in cell environment. newly synthesized azacoumarin fluorophores should therefore not only be useful for studying ttlbased biological systems, but also would enable broad range of high-yielding and fast diagnostics for future biolabeling applications in biochemistry, cell biology and beyond. binase is an extracellular ribonuclease from bacillus pumilus which shows antiviral and antitumor activities in cell cultures. however, the action of binase on intracellular functions and processes has not yet been identified. here, for the first time we report the whole transcriptome analysis of binase-treated human lung adenocarcinoma epithelial a cells. a plasmid-based reverse genetics system and colorimetric cell viability assay were used to identify the binase internalization and binase cytotoxicity towards a cell line, respectively. for the whole cell transcriptome analysis a cells were treated with lg/ml of binase for h followed by mrna extraction and library preparation. sequencing was performed on solid xl wildfire next-generation sequencer. we found that binase internalized into a cells after h of incubation. the binase at lg/ml was absolutely non-cytotoxic towards a cell line and was active in the cell culture medium during h incubation. the analysis of rna-seq data showed that among thousands of protein coding transcripts transcripts were up and down regulated by binase, among them transcripts were induced and repressed. binase repressed the production of s a and tnxb which act cancer biomarkers, scn a and drd which play a crucial role in cancer metastasis and responsible for pediatric tumors, respectively. the induction of transmembrane protein transcript abcb by binase can help binase to internalize into the cell as abc transporters are often account for transporting drugs across the cellular membrane. binase induced the production of nlrp , rasgrp and alpk transcripts which can activate apoptosis, cytokine or t cell activation in cancer cells. thus, binase exerts different effects in cancer cells. the rnaseq data obtained will help to understand the mechanism of binase anticancer action. . ) is a specific group of phosphatases capable of hydrolyzing myo-inositol , , , , , -hexakisphosphate (phytate) with the formation of less phosphorylated inositol derivatives (from mono-to pentaphosphate). three major types of phytases are recognized on the basis of the first phosphate group hydrolyzed by the enzymes: -phytase, / -phytase, and -phytase. due to the stereospecific way of phosphate release from the phytate molecule by the action of phytases, these enzymes by themselves and their composition may serve as a potential alternative for production of myo-inositol phosphate isomers with therapeutic properties. chemical synthesis of these compounds is inefficient and costly. pantoea sp. strain . . showing high phytase activity was isolated from the forest soil sample of the republic of tatarstan, russia. in this study the main objective was the cloning and expression of pantoea sp. . . phytase gene in e. coli. first, we amplified the phytase gene (agpp) from the genomic dna of the bacteria using specific primers phmh_dir and phmh_rev. size of phytase gene corresponded to base pairs. during the optimization of amplification conditions it was found that the optimum temperature for primer annealing was °c. this temperature increases the specificity and efficiency of annealing. then the pcr-product of agpp gene was cloned into the pbad myc/ his vector first. on the next step we carried out subcloning of the agpp into a pet a expression vector. multicopy plasmid pet a/agpp contained the sequence of the phytase gene of pantoea sp. . . under t promoter. the corresponding recombinant protein was expressed in e. coli as a fusion with a his-tag and was detected by western blotting. recombinant phytase was purified via affine chromotography on the ni-nta column and displayed high phosphomonoesterase and phytase activities. bag- (bcl- associated athanogene) is a multifunctional protein that interacts with diverse array of cellular targets and modulates a wide range of cellular processes, including proliferation, cell survival, transcription, apoptosis, metastasis and motility. in human cells bag- exists as three major isoforms (bag- s, bag- m and bag- l) derived by alternative translation initiation from a single mrna, which allows interactions with various molecular targets such as hsp /hsc molecular chaperones, components of the ubiquitylation/proteasome machinery, bcl- , raf- kinase, nuclear hormone receptors and dna. our work aims to investigate how altered bag- expression levels affect cell survival in mda-mb- (er, pr and her /neu negative) breast cancer cell lines. we first cloned bag- l gene to a cloning vector, later we transfected mda-mb- cells for overexpression of bag- . we also used bag- sirna to silence bag- gene. western blot analysis was applied to demonstrate relative expression levels of bag- , its interacting partners and certain proteins which are important for apoptosis pathway. we performed xtt cell viability assay for bag- overexpressed cells to checkbag- 's impact on cell survival, and observed enhanced survival rates on cells compared to that of the untreated cells with bag- overexpression. in addition, our study revealed that once bag- forms a complex with c-raf/b-raf/hsp /akt/bcl- , modulation of cell survival was observed. we believe that once the exact localization and involved molecular mechanisms of bag- and its isoforms are found, the role of each bag- isoform in cell survival can be understood better. this can further provide routes to study tumor development. the aim of this study is testing the recombinant glp encapsulation into a biocompatible material. we also tested if it can be a therapeutic candidate drug for type diabetes. the incretin hormones, which are also named as endogenic peptide hormones have become a more attractive therapy for type diabetes because of different physiological effects. in circulation, glp is cleaved by ddp in a very short time. if glp can be protected from cleaving, the effective time of glp would be increased and by this way it can replace the therapy of insulin. chemical synthesis methods of peptides are limited because of low efficiency and high cost. the production of peptides by recombinant e. coli is an alternative way because of effective production, simplicity and low cost. however, the major disadvantage derived from the recombinant e. coli is the frequent formation of inclusion body. for that reason, extra methods are needed for obtaining soluble recombinant peptides. glutathione s-transferase (gst) tag is commonly used as affinity and solubility tag to improve the solubility of recombinant peptides. in this study, we cloned and heterologously produced glp using the gst fusion system in e. coli. affinity purification of recombinant protein was achieved by using glutathione immobilized columns. characterization of the gst-tagged glp was performed by sds-page. the purity of fusion protein was found to be %, as confirmed by glp elisa kit. then, the fusion protein was encapsulated in a chitosan coated polygalacturonic acid. the different ph stability and in vitro release tests also in different phs was studied. morganella morganii is an opportunistic pathogen capable of causing a wide range of clinical infections. it is known that microbial metalloproteases play an important role in the development of bacterial infections. thus, investigation of m. morganii metalloproteases has a particular interest. bacteria were grown in lb medium at °c. as a bioinformatics tool blast was used. for molecular biological experiments, thermo scientific kits and sibenzyme enzymes were used. pbad/myc-his plasmid was used as an expression vector. bacterial transformation was carried out by heat shock method. bacterial cells were disrupted by sonication. gene expression products were analyzed by western blotting. to analyze the actinolytic activity of bacterial extracts sds-page electrophoresis was used. the putative metalloprotease gene (an cp . ) has been found in the genome of annotated strain of m. morganii kt. its amino acid sequence has partial homology ( %) with actin specific metalloproteases grimelysin from s. grimesii and protealysin from s. proteamaculans. using specific primers the gene with % homology was identified in the genome of clinical isolate of m. morganii . rt-pcr analysis showed that this gene had the maximum expression at h of growth. in addition, the cellular extract of m. morganii had small actinolytic activity. cloning of the gene into e. coli dh a cells led to the synthesis of the kda protein. it is known that the highest expression of serratia proteases is observed at h of growth, and the molecular weight of the mature proteins is kda. it was shown that metalloprotease gene of m. morganii expressed at the same time of growth. moreover, the recombinant e. coli cells synthesized protein with the similar weight ( kda) which perhaps is a mature form of the metalloprotease from m. morganii. as a result, in the genome of m. morganii the metalloprotease with similar properties to grimelysin and protealysin proteases was identified. the preliminary characterization of p-ii like protein glnk from lactobacillus brevis z. iskhakova , d. zhuravleva , a. laykov , k. forchhammer , a. kayumov kazan federal university, kazan, russia, eberhard-karls university tuebingen, tuebingen, germany the p-ii proteins in bacteria, archaea and plants regulate the activity of a variety of proteins in response to specific metabolic signals which affect their structural state and interaction ability. among various bacteria belonging to lactobacillus only some species have genes encoding pii protein in the genome. here we report the preliminary characterization of pii-like protein lbrglnk from lactobacillus brevis. while the amino acid sequence alignment revealed only - % homology of lbrglnk with other well studied pii proteins, lbrglnk also has the atp binding motive gdgk. trimeric structure of lbrglnk was confirmed in vitro by size exclusion chromatography, suggesting possible similarities of lbrglnk properties with pii proteins. the isothermal titration calorimetry revealed a preferential binding of adp (kd = lm) over atp (kd = lm) suggesting that they compete for binding to lbrglnk. neither -oxoglutaric acid nor other nucleotides were interacting with lbrglnk in itc measurements. the mutation gly ala in the atp binding motive completely abolished the interaction with both adp and atp. the pull down of lbrglnk with l. brevis cell extract allowed identification of chaperonin grol, transketolase and glnr-like transcriptional regulator from merr family as most probable partner proteins for interactions with lbrglnk. this work was supported by the russian foundation for basic research (project no. - - a background: hemophilia is a bleeding disorder due to the deficiency in coagulation factors viii (hemophilia a) or ix (hemophilia b). hemophilia patients are essentially treated with intravenous replacement of the missing or dysfunctional factors fviii or fix by recombinant proteins. these therapies often induce the generation of acquired antibodies, and thus, novel approaches are needed. most recent hemophilia strategies target the tissue factor pathway inhibitor (tfpi), which is the major inhibitor of the coagulation cascade, particularly of the extrinsic tenase complex. anti-tfpi agents have been empirically developed such as aptamers, peptides, monoclonal antibodies. we have followed a structure-based strategy, to design a mutated fxa that would show more affinity for tfpi, and thus trap this inhibitor. tfpi exists as two isoforms are folded as multi-kunitz domains related by linkers. the second kunitz type domain of tfpi (tfpi-k ) is known to bind the catalytic site of fxa. methods: the molecular complex of tfpi-k -fxa was modeled and submitted to molecular dynamics (md), allowing the identification of low-spots interaction. modified fxa with theoretically stronger interaction with tfpi-k were predicted using md. the mutants and wild type proteins were expressed in hek cells, and their processing status was checked. they were tested by western blotting, by chromogenic activity using a specific substrate of fxa, by thrombin generation assay in fviii depleted plasma. finally, their binding to a tfpi-k peptide array was compared. results: the mutants showed better efficiency to restore thrombin generation in plasmas from hemophiliacs and displayed stronger binding to tfpi-k than the wild type fxa. conclusions: the proof of concept of the synergistic approaches of md and mutagenesis was obtained and an efficient tfpi trap was designed. the mutated fxa is a candidate for a new hemophilia therapy. organophosphorus acid anhydride (op) nerve agents are potent inhibitors which disrupt the mechanisms of neural transmission. organophosphorus acid anhydrolase (opaa; e.c. . . . ) is a class of enzyme that potentially acts on phosphorus anhydride bonds, reported intracellularly in diverse organisms, albeit notably the enzyme belongs to alteromonas species are more extensively studied. whereas mass-transfer problem is a major issue in native whole cell biocatalysis, new anchor system derived from the n-terminal domain of ice-nucleation protein from pseudomonas syringe inav (inav-n) was used for the first time to display opaa onto the cell surface. tracing of the recombinant protein and its activity assay showed a successful presentation of opaa and its significant ability for biodegradation of organophosphorus compounds. further studies on bacterial fractions confirmed that opaa is remarkably located on the outer membrane. the specific activity of recombinant bacteria to degrade diisopropylfluorophosphate (dfp) was measured at u/mg of cell wet weight, which almost all was observed in the outer membrane fraction. recombinant cells could also degrade chlorpyrifos (cp) compound in . u/mg activity. it can be concluded that inav-n anchor is efficient for targeting opaa on the cell surface and can effectively eliminate the masstransfer problem in native whole cell bioconversion system. proper spatial and temporal organization of proteins involved in cell signal transduction is crucial for the specific and efficient information transfer. scaffold proteins coordinate the action of signaling molecules by their physical binding and organization in multiprotein complex assemblies. multiple protein binding is often mediated through intrinsically disordered regions of the scaffold, where the interaction epitopes are defined by linear peptide motifs. using a hub scaffolding protein axin as a paradigm, we have employed peptide microarray technique to identify the binding epitopes for axin interaction partners at high resolution. this enabled us to design axin-derived peptides corresponding to the respective binding epitopes that compete for the interaction in vitro. by transfection of chemically stabilized competitive peptides directly into the cells, we have shown the effect of specific interaction blocking on axin-mediated signaling in vivo. our data demonstrate a proof of concept for a rational design of inhibitors of protein-protein interactions that allow specific intervention with single function of the targeted protein (i.e. recruitment to the axin complex). contrary to the inhibitors that completely disrupt the protein function (e.g. inhibition of a kinase catalytic site), this approach provides a tool for investigating specific action within the axin complex, while the other cellular functions of the targeted protein remain preserved. the results of this research have been acquired within cei-tec (lq ) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. de novo design of an artificial biocatalyst using immunoglobulin template became rather routine procedure due to the achievements of molecular biology and crystallography. recently the 'reactibody' approach was developed based on the chemical selection of catalytic repertoires from immunoglobulin library followed by expression of these biocatalysts in eukaryotic system. in this study we structurally characterize the a antibody, its kappa and lambda variants, in order to understand the difference on the active site between a and a which although there are two antibodies sharing very high homology and sequence identity their active residues are located in a different region of the antibody. the structures of the a antibody kappa and lambda variants have been already determined, there was no structural information though about the a antibody. the structural analysis revealed dramatically different angle in position of nucleophilic residue tyr and area of solvent accessible surface. the structural difference of active center reflects on kinetics of the a organophosphate modification. both variants of antibody bind with organophosphate throw induce-fit mechanism, but rate of the step of induce fitting is different (k obs are s À and s À for a kappa and a lambda respectively). this observation may hint at novel means of regulation of velocity and specificity of artificial biocatalysts. this study was supported by grant #rfmefi x . translation elongation factor ba (eef ba) is a component of a heavy form of translation elongation factor (eef h). it functions as a catalyst of gdp/gtp exchange in translation elongation factor a (eef a) restoring its active conformation appropriate for aminoacylated trna binding. eef ba forms a tight complex with translation elongation factor bc (eef bc) via the n-terminal domain, while its c-terminal domain executes the catalytic activity. eef bc has been shown to enhance the attributed to the c-terminal domain catalytic activity of eef ba. this suggests that the eef ba n-terminal domain may influence the guanine nucleotide exchange process. to test this hypothesis we prepared a set of n-terminal truncated forms of human eef ba and checked their activity in the guanine nucleotide exchange assay on both isoforms of eef a, eef a and eef a . we showed that recombinant eef ba is a non-globular monomeric protein in solution with an elongated shape by analytical ultracentrifugation approach. the truncation of the dispensable for the catalytic activity n-terminal domain of eef ba resulted in significant acceleration of the rate of guanine nucleotide exchange in eef a comparing to full-length eef ba. similar effect on the catalytic activity of eef ba was observed after its interaction with eef bc. in contrast, the effect of full-length eef ba and its truncated forms on the rate of guanine nucleotide exchange in eef a was similar but relatively modest compared to eef a . this can be explained by higher rate of spontaneous gdp dissociation from eef a comparing to eef a and lower affinity of eef a to eef ba. thus, we propose that the n-terminal domain of eef ba via flexible linker region may interfere with eef a binding to the cterminal catalytic domain that results in a decrease of the overall rate of guanine nucleotide exchange reaction. the formation of a tight complex between eef bc and eef ba n-terminal domains abolishes this inhibitory effect. p- . . - assessment of quantitative proteomics results in large-scale data-independent with fragmentation spectra reproducibility measure reduces variation and allows to use lowintensity signals organisms with reduced genomes that lack the vast majority of transcriptional or translational regulation systems tend to adapt to changing environment with a variety of subtle changes in protein abundances. as soon as relative changes for most proteins fall below %, the power of traditional label-free proteomic analysis rapidly becomes insufficient for robust profiling of hundreds of samples. intoduction of frament-by-fragment and sample-by-sample signal quality assesment in mrm and dia experiments helps to increase accuracy of methods and at the same time reintroduce cases which could have been excluded during bulk quality assessment due to lower signal-to-noise ratio for several fragments. samples of mycoplasma gallisepticum were acquired in data-independent manner on sciex tripletof + mass-spectrometer (swath acquisition) during the year. the samples were produced from mycoplasma gallisepticum culture cultivated at different temperatures. the signals for each fragment were extracted with vendor-supplied software with the theoretical fragment ions for each peptides instead of spectral library. the results were used for relative protein quantitation in two manners the first conventional method included direct use of peptides with top most intense signals. the second included selection of peptides and ions for quantification for each pair of samples based on the reproducibility of fragmentation patterns after computing the areas of chromatographic peaks for each ion. spectral angle was used as a distance measure for fragmentation patterns for clustering. further, a base set of detected ions was selected for each peptide and a subset for comparison of each pair of runs. the first method resulted in quantitation of proteins across all samples with variation across lc-ms replicates was % on average, and the second approach led to quantitation of proteins in total, of them across % of samples, all with the variation about % on average. p- . . - interaction of plasminogen fragments k - and k with fibrin fragment dd t. yatsenko, v. rybachuk, l. kapustianenko, s. kharchenko, o. yusova, t. grinenko palladin institute of biochemistry of nas of ukraine, kyiv, ukraine introduction: plasminogen interaction with specific binding sites in c-terminal d-domains of fibrin molecule initiates the activation process of proenzyme and subsequent fibrin clot lysis. the sites are exposed under fibrin polymerization. plasminogen kringle domains ensure the proenzyme interactions with fibrin clot. in this study, we investigated the binding of human plasminogen kringle fragments k - and k with human fibrin fragment dd and their effect on glu-plasminogen interaction with dd. results: kringle-containing fragments k - and k reduce plasminogen activation by tissue-type activator on fibrin fragment dd. the level of glu-plasminogen binding to dd is decreased by - % in the presence of k - and k . fragments k - and k have high affinity to fibrin fragment dd (dissociation constant is . lm for k - and . lm for k ). analysis of k - and k binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with c-c-chains of fragment dd. k - interacts with complex of fragment dd-immobilized k as well as k with complex of fragment dd-immobilized k - . the plasminogen fragments do not displace each other from binding sites located in fibrin fragment dd, but can compete for the interaction. analysis of k - and k binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with aand c-chains of fragment dd. conclusions: widely known specific plasminogen-binding site located in aa - region of fibrin molecule is not a single binding sequence of fibrin peripheral domains or plasminogenbinding site is not linear and contains amino acid residues from other polypeptide chains of fibrin d-domains. fibrin fragment dd contains different binding sites for plasminogen kringle fragments k - and k , which can be located close to each other. possible plasminogen kringle-binding sites are located in aand c-chains. p- . . - implementation of budded baculovirus particles for characterization of ligand binding to g protein-coupled receptors a. allikalt, a. rinken university of tartu, tartu, estonia g protein-coupled receptors (gpcrs) constitute the largest class of membrane receptors involved in regulation of signal transduction into the cell in response to various extracellular stimuli. for that reason, gpcrs have become important targets for variety of drugs. as these receptors are present in native tissues at very low concentrations, efficient recombinant expression systems are needed to produce sufficient amounts of protein. we have shown that budded baculovirus particles, which display gpcrs on their surfaces can be used as a source of receptors for the investigation of ligand-receptor interactions. this expression system can be used for radioligand binding assay as well as for fluorescence anisotropy-based assay (fa). we have validated the system with budded baculovirus particles produced in spodoptera frugiperda (sf ) cells expressing human dopamine d receptors using [ h]sch- and bodipy-fl-skf- as reporter ligands for corresponding assays. this system has many advantages, for example good signal to noise ratio, homogeneity of the receptor, high expression levels and long-term stability of the receptor preparation. fa method allowed real-time monitoring of reporter ligand binding in the absence and presence of different dopaminergic ligands, giving information about their kinetic properties. association, as well as dissociation of the bodipy-fl-skf- itself were rapid with an apparent half-life of t / = . ae . s for association ( nm) and t / = . ae . s for dissociation. we determined the pharmacological profiles of different dopaminergic ligands in displacement binding assays with membranes of sf cells or budded baculovirus particles. the data were in good agreement for both membrane preparations tested in radioligand binding as well as in fa assay. obtained results indicate that budded baculovirus particles can be proposed as a source of gpcrs for performing fluorescence anisotropy as well as radioligand binding assays. gastrointestinal (gi) cancer includes a variety of cancer types affecting the structures and functions of the gi system, encompassing the gi tract and the accessory organs of digestion, from the esophagus, stomach, biliary system and pancreas to the intestine, rectum and anus. despite the significant advances however, much remains to be learned in the spectrum of gi cancer. several investigators have shown that both gas and its receptors, axl, sky, and mer are expressed in various types of cancers. however, the expression level of gas in gi cancer remains unclear. the aim of the study was to determine and compare plasma gas levels in gi cancer patients. female and male patients were included in the study (n = ): colorectal, gastric, pancreatic, liver, ampullary, gall bladder and esophageal. from all gi cancer patients, ml venous blood was collected in citrate tubes before surgery. blood samples were centrifuged at g for min, and plasma samples were carefully removed and stored in À °c prior to use. the level of plasma gas was measured using a commercial developmental elisa kit (r&d systems, minneapolis, mn) which is validated by our laboratory. plasma gas levels in cancer patients were determined as follows: . ae . ng/ml in colorectal; . ae . ng/ml in gastric; . ae . ng/ml in pancreatic; . ae . ng/ml in liver; . ae . ng/ml in ampullary; . ae . ng/ml in gall bladder and . ae . ng/ml in esophageal cancer. preliminary findings indicate that there is a relation between gi cancers and plasma gas levels. taken together, these results suggest that gas may be a candidate biomarker for diagnostic use in gi cancer. inhibition of gas would be an attractive therapeutic target for slow down the progression of gi cancer. monday september : - : computational biology p- . . - computational approaches as an assay for blactam hydrolysis in class a b-lactamases c. tooke university of bristol, bristol, united kingdom b-lactam hydrolysing enzymes, in particular carbapenem-hydrolysing enzymes, are an increasing clinical threat. herein we show that molecular dynamics (md) and combined quantum mechanics/molecular mechanics (qm/mm) approaches are a predictive tool of carbepenemase activity in class a b-lactamases. b-lactam drugs are the most prescribed class of antibiotics worldwide, especially in the treatment of gram-negative pathogens such as klebsiella pneumoniae and escherichia coli. these organisms produce b-lactamases, enzymes which hydrolyse the b-lactam ring, a key resistance mechanism. class a b-lactamases have the ability to hydrolyse carbapenems, termed 'last resort' antibiotics. in particular, the kpc (klebsiella pneumoniae carbapenemase) family are the most clinically important, and recently identified natural kpc variants show increased hydrolytic activity against ceftazidime, a third generation cephalosporin. here we use computational simulations of b-lactam hydrolysis by b-lactamases. in particular, molecular dynamics (md) combined with qm/mm approaches have been used to model the deacylation of the carbapenem meropenem across class a b-lactamases. this method has been extended to model cephalosporin hydrolysis across class a b-lactamases, including kpc variants. these approaches calculated the free energy barriers and correctly distinguished carbapenemases from carbapenem-inhibited enzymes. preliminary results suggest this protocol is also a predictive tool for ceftazidime hydrolysis. further, md simulations of kpc variants (single and double amino acid changes) were analysed to identify structural changes in the active site, highlighting that variants differ in the size of the active site opening, corresponding with experimentally derived kcat values. these computational assays provide a predictive tool of b-lactam hydrolysis and has potential to provide insights into important mechanistic differences both across class a b-lactamases and within the same families. p- . . - computational design of a novel polyglutamic dendrimer-based platform as an anticancer therapeutic approach poly (glutamic acid) (pg)-dendrimer as potential nanocarriers for cancer therapies, to specifically deliver tumor associated antigens (taa)mannosamine and melanato target cells and to modulate cancer antigen intracellular trafficking within the cytoplasm to promote an efficient and selective antitumor immunotherapeutic effect. the theoretical structures were obtained using x-plor software. the molecular dynamics simulation of pg-g -dendrimer and taas was performed using desmond. the electronic properties of the structures were determined by semi-empirical methods using mopac. docking studies of taa to pg-g -dendrimer to mannose receptor (mr ) were performed using hex . . software. taa lumo atoms were conjugated to homo atoms of pg-g dendrimer using maestro software. results showed that pg-g -dendrimer displays carboxylic end groups available for covalent interaction with taas. the homo molecular orbitals of the dendrimer was located on the a-carbon of the carboxylic acid groups from backbone chain and it preferentially interacts with lumo molecular orbitals of amine group from taas. no differences in the gap energy of homo/lumo of all pg-g -conjugates. taas bind preferentially to a-carbon of cooh of backbone chain instead of cooh from side chain. docking results showed that majority of taa conjugated pg-g -dendrimer binds to the core of the mr receptor. increasing of the number of mannosamine conjugated to pg-g -dendrimer more close and stable is the conjugated to the receptor. this system shows promising results as a novel functionalized pg-dendrimers for cancer therapy. theileria parva is one of the the economically important protozoan of the theileria genus belong to apicomplexa phylum which include plasmodium spp. and toxoplasma gondii, causative agents of malaria and toxoplasmosis respectively. this parasite is the disease agent of tick-borne east coast fever (ecf) ranks first among the tick-borne diseases of cattle in sub-saharan africa. the disease caused by the parasite affects a large proportion of domestic and wild animals and leads serious economic losses in the world. major problems in dealing with this illness are the high cost of drugs, development of resistance, and absence of effective vaccines. thus, it is important to develop an efficient and affordable antitheilerial agent. for this aim, -deoxy-d-xylulose- phosphate reductoisomerase (dxr) which subjected to identify novel drug aganist malaria and toxoplasmosis, of theileria parva was selected as potential target for improving novel inhibitors aganist ecf. a computational molecular modeling approach was conducted to determine the d structure of tpdxr by phyre . energy minimisation and root mean square deviation (rmsd) was performed by drefine and superpose servers. to ensure the quality of modelling, stereochemistry, energy profile and residue environment of modelled structure were checked by different servers and possible ligand binding pockets were identified by metapocket . server a reliable d model for dxr from t. parva was modeled by using au as a template. the ca rmsd and the backbone rmsd deviations for the model and the template crystal structure were found to be . and . a, respectively. the ramachandran plot for the predicted model by rampage reveals that model shows an acceptable stereochemistry. top three considered possible binding pockets have been identified. these results have important implications for future screens aimed at finding new and safe molecular entities active against tpdxr through docking studies. p- . . - molecular binding profile of protoberberine alkoloids on amyloid precursor proteincleaving enzyme (bace ) as a drug candidate for alzheimer's diseases g. yalcin , i. yildiz biotechnology institute, ankara university, ankara, turkey, department of pharmaceutical chemistry, faculty of pharmacy, ankara university, ankara, turkey alzheimer's disease (ad) is the most prevalent neurodegenerative disorder that leads to dementia and nowadays over million people live with dementia worldwide. because of the prevalence and economic burden of the disease, drug development studies have picked up speed and scientists especially focused on natural products. ad is basically characterized with tau hyperphosphorylation and accumulation of amyloid b (ab) proteins. ab proteins are generated from sequential cleavages of amyloid precursor protein (app) by b and c secretases, and b-site app cleaving enzyme (bace ) is a b secretase essential for ab production. the alkaloids represent a very extensive group of secondary metabolites, with diverse structures, distribution in nature and important pharmacological activities. protoberberine alkaloids, which belongs to isoquinoline alkaloid class, are widely arranged in many species of the berberidaceae, annonaceae, fumariaceae, papaveraceae, and other plant families. recent searches showed that some of the protoberberine alkaloids such as berberine, palmatine, jatrorrhizine, columbamine, magnoflorine prevents the progress of neurodegenerative disorder. however, the mechanisms of them are not absolutely clear. therefore, we have aimed to elucidate the binding and affect mechanism of these alkaloids on the bace open and closed forms in here. for this purpose, molecular docking studies were applied for these natural products to the both forms of bace by using autodock vina and it was subjected to explicit solvent simulations by amber molecular dynamic package. our preliminary studies indicate that gly , thr , gln , phe , tyr , lys , thr , arg , thr residues of binding pocket have affiliations with all of the mentioned alkaloids and the binding of them generates alterations on closed form of bace . the complexity of animal milk needs to apply numerous approaches and methods for its investigations. an understanding of the processes occurring in the milk can be used, for example, for quality control of the products. fat and protein are main components of milk which have a significant influence at its colloid properties, such as dynamic surface tension (dst). the application of regression-correlation analysis to milk data enables to develop a reliable quantitative model. the aim of our investigation was to perform the regression analysis to establish the relationship between above-mentioned parameters. for this purpose, we used a statistical software packages r version . . . dst was determined by bpa - p tensiometer. milk fat (f) and protein (p) contents were measured by analyzer bentley . this work was supported by the russian scientific foundation (grant - - ). obtained formulas characterized the degree of influence of fat and protein contents of a milk sample for each of the dst parameters (r , r , r , r , k , k ): r = . + . * p À . * f r = . + . * p À . * f r = . + . * p À . * f r = . + . * p + . * f k = . + . * p + . * f k = . À . * p À . * f these formulas show that the maximum total effect of fat and protein contents influences at r and r . a significant coefficient (> ) before the fat is observed in the formula, which describes the value of the tilt of final part of the tensiogram (k ). the resulting regression equations have fundamental importance. with their help it is possible to calculate the dst parameters without their experimental determination, positioning fat and protein contents data. obtained dst parameters promote more complete characterization of the properties of the milk that may be used for dairy products. p- . . - molecular studies of scorpion toxin and its mutants interactions with voltage-gated potassium channels the voltage-gated potassium kv . channel is mostly expressed in neurons and immune cells. its blockage has a high therapeutic potential, for example, specific inhibitor shk toxin is undergoing clinical trials on psoriasis. goal of the current study was an interface analysis in complexes of hybrid channel kcsa-kv . with peptide blockers agitoxin and its mutant forms. d structure was generated by homology modeling method using complex of mutated kcsa channel with charybdotoxin (pdb-code a h) as a template and equilibrated by molecular dynamic simulation in gromacs software. analysis of hydrophobic and stacking interactions, hydrogen and ionic bonds of the toxin and potassium channels was performed for representative frames with optimal toxin orientations using program platinum and apbs software package. we performed contacts energy characteristics estimation to predict key toxin residues for binding process and possible mutation sites for changing selectivity against kv .x channels. the results of investigation are in good agreement with the experimental values of binding constants, obtained by competitive binding assays. results of the conducted investigation may find an application in fundamental science and drug design. the research was supported by the russian science foundation grant no. - - . simulations were performed using the supercomputing center of lomonosov moscow state university. p- . . - homology modeling and molecular docking study of the paraoxonase- and its polymorphic variants q/r and m/l for non-statin lipid lowering drugs paraxonase- (pon ) enzyme is an hdl associated ester hydrolase exhibiting paraoxonase, arylesterase and lactonase activity, and reduces the formation of atherosclerosis blocking the ldl oxidation and reducing levels of oxidized lipids. in this study, molecular docking approach and molecular dynamics simulation were applied for finding the affinity of non-statin lipid-lowering drugs to pon and its polymorphic structures pon q/r and m/l . fibrates (bezafibrate, ciprofibrate, clofibrate, fenofibrate, gemfibrozil), phytosterols (beta-sitosterol, brassicasterol, campesterol, stigmasterol) and other lipid lowering drugs (ezetimibe, niacin, orlistat, probucol, and sibutramine) was obtained from pubchem database. x-ray crystallographic structure of human pon and its polymorphic variants pon q/r and m/l was generated via 'modeller', homology modelling software, from human-rabbit hybrid x-ray crystal structure of pon (pdb code: sre). ns molecular dynamic simulation analysis was performed using gromacs . . . affinity of lipid lowering drugs to pon and its polymorphic variants was predicted by molecular docking approach using autodock . suite. unlike other lipid lowering drugs that they have negative Δg values for affinity, probucol, orlistat and betasterol was calculated by positive Δg values ( . , . and . kcal/mol). these values suggest that they may have no affinity to pon q/r polymorphic structure. in all drug groups, brassicasterol and stigmasterol to pon -m/l and sibutramine to pon q/r were calculated as the highest affinity. in generally, phytosterols predicted by high affinity to pon and m/l polymorphic structures in comparison to other lipid lowering drugs. our study demonstrated that phytosterols predicted as high affinity compounds on pon structures may reduce the activity of antioxidant pon enzyme. this study need to be supported by in vitro and in vivo detailed studies. prolactin and its cognate receptor, prolactin receptor (prlr), are involved in over distinct functions in mammalians. the mammalian prlr gene consists of - exons and several and regulatory sequences. in this study, gaps and annotation errors in the rat prlr gene were corrected by comparing the genomes of mammals and rodents and new putative exons were identified. the rat prlr gene sequences from two different sources (rnor_ . , nc_ . and rn_celera, ac_ . ) were used and primary analysis showed that both sequences contain several gaps (varying from . to kbp), corresponding to about . % ( - kbp) of the gene. using the rat known prlr mrna exon sequences, it was found that the rnor_ . prlr gene has two exon- (one is about kbp long and the other immediately after this). comparisons of mammalian and rodent prlr gene structures showed that the kbp stretch is an assembly artifact. by comparing both gene sequences (and also other available rodent prlr genes), the gaps in the rat prlr gene were reduced from . % to . % (from kbp to kbp). functional annotation of the gene revealed that r. norvegicus prlr gene could have two more additional exons, exon- and - , similar to mus musculus prlr gene. in mammals, prlr mrnas contain non-protein coding exons in the utr (exon- and - ). in rats, there are exon- variants, resulting from alternative promotor usages. studies on the rat and mouse prlr genes revealed that both rodents share common non-protein coding exon- variants. in conclusion, it is found that the rnor_ . version of the prlr gene has the highest number of unidentified base pairs (corresponding to . % of the gene) and the second exon- is the assembly artifact. the rat prlr genes in both databases have several gaps and our corrected version is the best available and characterized form of the rat prlr gene. in silico affinities of some statins to paraoxonase- enzyme the structure of the statins (atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin) was obtained from pub chem database, and x-ray crystal structure of pon (pdb id: sre) from protein data bank. modeller software was used for homology modeling of pon and its polymorphic variants that's called as pon q/ r and m/l . amino acid sequence of human serum pon (uniprot: p ) was used as the modeller template. all molecular dynamics simulations were carried out with gro-macs . . software. molecular docking calculations on each of the polymorphic structure of the pon was performed with auto dock . . suite. for each substrate, y residue showed open conformation in pon and m/l polymorphic structures while q/r polymorphic structure showed closed conformation. in comparison between structures of pon variants, in most cases statins had lower affinity to q/r polymorphic structure than to the other variant. in this study, among statins, atorvastatin showed lowest but simvastatin highest affinities to pon . by considering that the high affinity drugs can have reducing effect of pon activities, it may be more appropriate to use the low affinity statins in hyperlipidemia treatment. however, these findings need to be supported with in vivo and in vitro studies. p- . . - self-assembly of lipidoids for sirna uptake and release mechanisms studied by molecular dynamics simulations o. acar , d. alpay , a. r. atilgan , c. atilgan sabanci university, istanbul, turkey, northwestern university, evanston, united states small interference rna (sirna) has the ability to bind a specific mrna which provides silencing of selected genes. nanocarriers made out of self-assembled lipidoids encapsulate sirna and deliver them into target cells effectively. in this study, a library of lipidoid structures is constructed and studied by molecular dynamics (md) simulations in different solvents, including sodium acetate, to ferret out their self-assembling mechanisms. the effect of the protonation state of the head group of lipidoids on the final shape of the self-assembled carrier is also studied. we further examine the role of the size of hydrocarbon tails in the packing. we study the final topology and the geometry of the self-assembled lipidoids both in the presence and in the absence of sirna. we find that stable clusters form with as few as chains. for lipidoids having neutral head groups, clusters are in the form of dense bundles, while those with charged head groups form spherical capsids which are depleted of the salt on the inside and having a salt rich phase on the outside. in the self-assembled structure, lipidoids are arranged so as to expose the nitrogen and oxygen atoms to the solvent. while partial capsids with these properties also form at lower lipidoid numbers, chains are necessary to form a fully closed capsid. in the presence of the sirna, the capsid assembles around the nucleotide. the free energy to remove the sirna from the assembly is calculated via repeated steered md calculations utilizing jarzynski's equality relating it to the irreversible work along and ensemble of trajectories. we therefore determine an optimal tail length for the most stable nanostructure, paving the way for designing nanocarriers with high efficacy. milk is one of the most valuable products for humans and attracts a lot of interest of researchers in various fields such as biochemistry, biology, food science and technology. the methods of milk study are quite varied. we chose the combination of the ultrasonic and dynamic surface tension (dst) measurements with the possible correlations among the obtained parameters. the aim of this work is to study the correlation between the parameters of milk, such as a content of fat, protein, lactose, minerals, dry milk solids and dst parameters. for this purpose we used milk analyzer 'klever- m' and tensiometer 'bpa- p'. three groups of animals were formed from clinically healthy holstein cows at the age of - years according to the fat content in the milk sample. group i - cows (milk fat content . ae . %), group ii - cows (milk fat content . ae . %), group iii - cows (milk fat content . ae . %). this work was supported by the russian scientific foundation (grant - - ). the biochemical parameters of the milk samples of all three groups are in the range of the 'normal' values for healthy holstein cows: protein content varies from . % to . %, lactose and mineral content varies from . % to . %, respectively. the dst parameters (r , r and k ) for the group i have strong positive correlations with the fat content in the studied milk samples. at the same time for the groups ii and iii the fat content in the milk indicates only medium positive and weak positive correlations with the r , r and k . obtained absolute values of the dst parameters of the milk samples showed some differences between all three groups. thus, the dst parameters are changing in direct proportion to fat content in the milk sample that can be explain by the primarily role of the milk lipids in the formation of the water/fat surfaces (such as fat globules, lipid-protein particles, etc.). p- . . - exploration of allosteric paths in caspase molecules using energy dissipation e. n. bingol, o. sercinoglu, p. ozbek sarica marmara university, istanbul, turkey caspases are highly regulated aspartate-specific cysteine proteases that have major roles in programmed cell death; apoptosis. effector caspases are at the terminal step of the pathway, hence they are considered as death switches. with the discovery of the presence of allosteric sites, these molecules attracted the attention of the pharmaceutical studies and became drug targets. as a result of the binding of small molecules to the dimeric interface, active site loops are shifted to an unfavorable position. this is associated with a network between distal allosteric sites and the active site loop. an energy dissipation model was applied in order to analyze this matter in further detail. perturbation of specific residues enable us to determine a possible signaling network in proteins using external energy as an input, while focusing on the dispersion of this energy between residues throughout the structure. molecular dynamics simulations were performed with and without energy perturbation using namd software with charmm force field. energy perturbation was applied by increasing the velocity of a chosen residue at the desired time step of the initial md simulation. energy change of each residue was calculated upon the application of perturbation. as a result, residue response times, corresponding to the time of the response of a residue after the perturbation of another chosen residue, are obtained. combining reponse time data with a residue interaction network, it is possible to construct a final network that shows the communication started by perturbation within the molecule. it is shown that perturbation of allosteric sites result in the disruption of the catalytic sites given in literature. our findings support this and also gives a little detail of the possible communication between distal allosteric site and the active site loops. this finding enables the usage of this methodology for similar structures where the exact allosteric mechanism is yet not known. p- . . - effect of complex mammalian membrane models with multiple membrane components on ras protein nanoclustering a. farcas , , l. buimaga-iarinca , c. floare , l. janosi faculty of physics, babes-bolyai university, cluj-napoca, romania, national institute for research and development of isotopic and molecular technologies, cluj-napoca, romania ras proteins are essential for the cellular signal transduction that regulates cell proliferation and differentiation and act as binary switches between gdp and gtp forms. a wide range of human tumors are associated with defective ras protein signaling. the production of permanently activated ras proteins is correlated with mutations in ras genes. experiments and computer simulations have shown that membrane-bound ras proteins form nonoverlapping dynamic nanosized subdomains (nanoclusters) in activation state-/isoform-dependent manner. we performed coarse-grained molecular dynamics simulations to investigate the effect of complex mammalian membrane models on formation and evolution of ras nanoclusters. a fundamental part of the plasma membrane is the phospholipids bilayer, which contains phosphatidyl-choline (pc), phosphatidylethanolamine (pe), phosphatidyl-serine (ps), sphingomyelin (sm) and cholesterol (chol). the nature of lipid-lipid and protein-lipid interactions was studied in binary (pc:chol) and quinary mixtures (pc:pe:ps:sm:chol). because the polar lipids are not uniformly distributed between the two leaflets of the membrane, the construction of the plasma membrane with five-component lipid mixtures took into account the asymmetry between the outer and inner mono-layers. the phospholipids chain saturation (combined with the presence of cholesterol) constitute the dominant factor in phase separation and was, therefore, modeled in different lipid tail combinations for various headgroups. using microsecond timescale simulations of membraneembedded ras proteins, we have shown that the nanoclusters are spontaneously forming dynamic structures whose behavioral characteristics is modulated not only by the ras isoform, but also by the complexity of the membrane model. furthermore, we showed that variations in the plasma membrane lipid composition have important implications in the localization of ras protein nanoclusters. optogenetics comprises biological methods to achieve gain or loss of function of well-defined events in specific cells of living tissue by means of targetable control tools that respond to light and deliver the effector function. microbial rhodopsins (mrs) have been established as powerful light-sensitive tools for optogenetics. acting as ion pumps or channels, mrs are used to induce cell (de)polarization to control neuronal activity in a wide range of living organisms. mrs are membrane proteins found in a large clade of organisms, including eukaryotes, bacteria, and archaea. they share a common architecture of transmembrane a-helices and a covalently linked retinal, which is employed to absorb photons for energy conversion or the initiation of cellular signaling. major efforts are put into screening of natural and generating of synthetic mrs with desirable properties for optogenetics, e.g. ion selectivity. however, experimental study of mrs is difficult and resource consuming owing to, among other factors, low expression levels and protein stability. thus, there is a need in developing of computational tools for identification of mrs with desirable properties. we used non-redundant atomic structures of mrs taken from protein data bank to develop a set of numerical descriptors that reflects functional properties of mrs. then, we calculated the descriptors for non-redundant sequences of mrs with known function taken from the uniprot database, resulting in the feature matrix. we applied the support vector machine and the fold cross-validation procedure, using the feature matrix as the training set. as a result, we obtained the classifier that discriminates mrs in terms of the ion selectivity, e.g. na + , h + , or cl À pumps, with high precision. finally, we used the derived classifier on a test set of proteins and identified mrs for the further experiment in vivo. rational design of peptides with required stability and functional activity properties becomes a real instrument for the new generation drug development. the reca bacterial protein (and human rad homolog) is considered to be the central catalyst of homologous recombination, a mechanism essential for the accurate repair of double-strand dna breaks. dna repair via homologous recombination requires reca nucleoprotein filaments assembly. using seqopt (http://mml.spbstu.ru/seqopt/), a novel method for a-helix sequence optimization we present the successful design of peptide sequences capable to maintain a very stable a-helix structure and to inhibit reca activity. novel a-helical amino acids peptide is constructed based on reca-dna complex structure. we observed in vitro inhibition of reca atp hydrolysis, dna strand exchange reaction and reca filament formation. also, we observed lower e. coli resistance to uv and sos-response suppression in vivo. computational identification of promiscous enzyme activity for the morita-baylis-hillman reaction k. ozturk, s. sayin, n. celebi olcum yeditepe university, istanbul, turkey enzyme promiscuity attracts considerable attention in terms of enzyme evolution, protein engineering and biocatalysis. especially, development of highly efficient novel biocatalysts starting from promiscuous enzymes that have the catalytic machinery to perform desired chemistry is an intense area of research in recent years. in this work, we computationally explored the catalytic promiscuity of natural enzymes for the synthesis of morita-baylis-hillman (mbh) adducts, which display antitumoral activity against human cervical cancer cells, by mining structural protein databases using quantum mechanically optimized theoretical active site models (theozymes). catalytic interactions in the active sites of selected hit proteins with potential mbh activity were evaluated in solvated dynamic environment using molecular dynamics simulations. computational screening of the protein data bank for the quantum mechanically determined optimal arrangement of catalytic functional groups for the target mbh reaction successfully identified an enzyme with experimentally determined promiscuous mbh activity. ras proteins mediate a wide variety of signal transduction pathways that regulate cell growth, proliferation and differentiation. these proteins are small gtpases that act as binary switches between gdp-bound 'off' and gtp-bound 'on' states. oncogenic point mutations of ras are associated with~ % of all cancers and up to % in specific tumors and many developmental disorders. both experimental and in silico results showed that the membrane-bound ras proteins form non-overlapping dynamic nanosized subdomains called nanoclusters in an activation state-/ isoform-dependent manner. we performed coarse-grained molecular dynamics simulations in order to investigate the formation and evolution of ras nanoclusters in mammalian model membranes. ras proteins were inserted into the cytoplasmic side of the plasma membrane model (di-c : -phosphatidyl-choline: di- : -phosphatidyl-choline: cholesterol : : ) where they formed highly dynamic nanoclusters, both in size and in composition. furhermore, we found that the presence of ras protein nanoclusters has a significant impact on the model membrane behavior. properties such as phase behavior, diffusion coefficient, surface tension and lipid tails order parameter are also influenced by the temperature variation of the model membrane. we have investigated dynamics in three different crystal forms of ubiquitin, as well as ubiquitin in solution, with particular emphasis on (i) conformational exchange between b turn type i and ii in the region - and (ii) rocking dynamics where protein molecules as a whole undergo subtle reorientational motion within the confines of the crystal lattice. experimentally, both motional processes have been probed using relaxation dispersion techniques, including recently developed near-rotary-resonance dipolar relaxation dispersion experiments. thereby it has been determined that rocking motion in one of the crystal forms (pdb id n ) occurs on the time scale of tens of microseconds, whereas the conformational exchange has characteristic time constant of ca. ls. using molecular dynamics simulations, we have shown that the similarity of motional time scales is not accidental: bi↔bii exchange and rocking motion appear to be coupled. we have investigated the mechanisms of this coupling and predicted a number of point mutations that are expected to abrogate (or enhance) rocking. the crystals of ubiquitin containing these mutations have been modeled in silico. we have also investigated the interactions (in particular, crystal contacts) that control the balance between bi and bii conformations in different crystal forms. finally, we have used md simulations as a basis for chemical shift calculations and illustrated how relaxation dispersion effects can emerge as a function of bi↔bii exchange in conjunction with the rocking motion. the md simulation study was supported by rsf grant - - . serine/threonine kinases are attractive targets in targeted cancer therapy due to their overexpression in several forms of cancer. flavonoids are highly bioactive plant secondary metabolites that are important in human health due to their antioxidant property. quercetin, a natural flavonoid derivative, has been shown to regulate several signal transduction pathways and is in phase i clinical trial as an anticancer drug. this study explored the inhibitory potential of quercetin and its derivatives using in silico methods like molecular docking and molecular dynamics simulations. quercetin and its derivatives were observed to bind to several serine/threonine kinase family proteins with binding energy significantly better than other known inhibitors and commercially available drugs. this study thus sheds light on the atomic level interactions that define the polypharmacological nature of quercetin and its ability to interfere with a number of cancer pathways. introduction: noninvasive prenatal diagnosis (nipd) of the fetal rhd status by rhd genotyping of the maternal plasma was initially applied in alloimmunized pregnant women. fetal rhesus d status detection for management of rhd incompatibility using circulating cell-free fetal dna from maternal plasma or serum is now accepted by many obstetricians in europe as reliable and useful. the aim of the study was to detect fetal rhd specific antibodies in maternal plasma using a nanopolimer based electrochemical biosensor. materials and methods: a three-electrode system, consisting of a gold electrode, an ag/agcl reference electrode and a pt counter electrode, was accommodated in a -ml electrochemical cell. anilin and jelatin were used for immobilization of rhd antibody. the polimerization was occured at nm uv light. antibodies of rhd antigen were detected using differential pulse method at between . and . v potentials by observing the differentiations in the current values. results: the rhd status of the fetus was predicted in rhdnegative pregnant women ( - th week of pregnancy). rhd antibody were detected in maternal bood using biosensor in of the fetuses. the results were confirmed with real-time pcr. the fetuses found rhd (+) for exon and of rhd gene by multiplex real-time pcr. discussion and conclusion: biosensors based studies might be useful, because they allow to monitor the molecular interactions in real-time providing qualitative and quantitative information, through kinetics, affinity and concentration analyses. we found that more advantages in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, specific, economic, practical and less time-consuming. fetal rhd detection at low concentrations and in the early week of pregnancy is possible with this method. p- . . - investigation of phylogeography of cricotopus sylvestris (diptera: chironomidae) using mitochondrial and nuclear molecular markers the family chironomidae is one of the most widely distributed insect families of diptera, and this family is distributed in all continents and all habitats from the tropics to the arctic in lakes, streams and puddles. in this study, we aimed to determine the dispersal of c. sylvestris using molecular phylogenetic markers not only in turkey but in the world and to reveal from where this species may have entered to turkey in the past. c. sylvestris larvae were collected from lakes across turkey. after total genomic dna extraction from body of larvae, fragments of two mitochondrial genes, cytochrome c oxidase subunit i (coi) and cytochrome b (cytb), and one nuclear gene, carbomyl phosphate synthase domain (cps) of cad, were amplified and sequenced. in addition, several sequences of these three genes of c. sylvestris from different countries of different continents such as south corea, japan, canada, denmark, and sweden were obtained from genbank. all sequences were aligned using mega and bioedit version . . . and were used for phylogenetic analyses. neighbour-joining (nj) tree was created in mega and paup . b with bootstrap replicates. maximum likelihood (ml) analysis was performed in raxmlgui . using gtrgamma model with bootstrap replicates. beast v . . was used for bayesian analysis. our phylogenetic analyses indicated that the japanese, south corean and american c. sylvestris were different from european and turkish members. turkish members of c. sylvestris were closely related to european ones according to our bayesian, nj and ml analyses. in turkish members, c. sylvestris collected from hazar and c ß ıldır lake was more ancient than those from marmara, sapanca, c ß ıldır, aygır, beys ßehir, e girdir and sıhke lakes. in conclusion, our results clearly suggest that several transoceanic dispersal events among the continents may have occurred and that the entrance of turkish c. sylvestris to turkey may have been from southeast and northeast of the country. metagenomics is providing great help to explore world of unculturable microorganisms in the natural samples to enhance our knowledge about microbial diversity. here, we have performed metagenomic analysis of fresh water lake bacterial community using pyrosequencing techniques. we have observed a wide array of bacteria from phylum proteobacteria and family enterobacteriaceae as well as very few viruses from podoviridae, siphoviridae and unclassified phages. we have conducted a metagenomics analysis with the primary focus on the examination of the community of bacteria in a fresh water lake ecosystem. roche gs flx software gave us total reads (with an average read length of . bp). there were contigs having > bp sequence length whereas contigs with > bp sequence length. for further analysis we have taken contigs with > bp only. further, we have analyzed the microbial community composition using blastn/blastx against nt/nr databases with e-value cutoff of À . ≥ % of total contigs were mapped to the reference with ≥ % contig match coverage. the community analysis revealed that domain bacteria is predominantly present ( . %) in the water sample, followed by eukaryota ( . %), viruses ( . %) and other sequences ( . %). most abundant phyla was proteobacteria ( . %) and the most dominant family was enterobacteriaceae ( %) followed by xanthomonadaceae ( %), vibrionaceae ( . %), pasteurellaceae ( . %), shewanellaceae ( . %). we performed functional analysis of all contigs using rapid annotation using subsystems technology (rast) which detected coding sequences and rnas in subsystems. among the classified cds from rast showed major cds hits for enzymes involved in the subsystems amino acids and derivatives and the carbohydrate metabolism. the great diversity of microorganisms present in the lake may reflect the human activity in the area. maldi-tof mass spectrometry is a ubiquitous and widespread tool for protein identification. once the protein sequence is unavailable, unambiguous identification cannot be performed, and predictability is limited by the presence of sequenced homologous proteins. we present a statistical approach to predict a number of structural, localization and functional properties of unknown proteins by direct analysis of mass distribution shapes of their post-cleavage fragments obtained from maldi-tof mass spectrometry data. secondary structure of proteins is best predicted by their specific cleavage at the inertial hydropathy group amino acid residues (filmv), with thermolysin (afilmv) being the closest commercially available reagent, leading to distinguishing between proteins with presumably ahelixes or b-sheets with % accuracy. cellular localization of proteins is best predicted by their specific cleavage at the external hydropathy group amino acid residues (dehknqr), exemplified by gluc(phosphate)+lysc(dek) cleavage. protein location in the cell membrane and its localization character (monotopic/ transmembrane, single-pass/multi-pass transmembrane) are predictable with~ % accuracy by this single cleavage, with optimal combination of - cleavages improving the accuracy tõ %. functional prediction of proteins is the best among membrane-associated proteins with characteristic structural conformations. we attribute the differences in the mass distribution shapes to the characteristic clustering of amino acids residues with respective hydropathy properties that are involved in the formation of d structural conformations of proteins. the suggested approach allows for a non-parametric statistical prediction of uncharacterized proteins from their maldi-tof mass spectrometry data without knowledge or reconstruction of their primary sequence. potential applications include proteomic studies of organisms with unavailable genomic sequences and highly variable proteins analysis. the cancer genome atlas (tcga) represents a comprehensive database of genomic, transcriptomic and epigenetic alterations across more than tumor types. earlier we developed cross-hub tool aimed at multi-way analysis of tcga data in the context of gene expression regulation. in the present work, the software was updated with new features that are described below. crosshub is a python-based application. one of the features of crosshub is the combining tcga rna-seq co-expression analysis to encode chip-seq data in order to reveal most possible transcription factor (tf) targets and coupling mirna-mrna co-expression to several algorithms of mirna target prediction in order to enhance its efficacy. the key feature of the updated crosshub version is the analysis of the associations between expression ratio of tf to its targets and tf mutation status. this allows identification of tfs that are functionally (in)activated with driver mutations in a particular cancer type. the second novel feature of crosshub is the analysis of associations between 'tf-to-targets' expression ratio and tumor characteristics (tnm classification, pathological stage), patient follow-up, etc. in turn, this analysis may result in the identification of 'tf-targets' functional relations that are important for disease progression, tumor invasion, response to chemotherapy. thus, crosshub was supplemented with new features that can be useful for comprehensive tcga data analysis. the updated version of crosshub is freely available at http://sourceforge.net/ projects/crosshub/. this work was financially supported by the russian foundation for basic research (grants - - , - - and - - ) and ras presidium program 'molecular and cellular biology'. p- . . - mutations leading to increased rnase production and streptomycin resistance in bacillus pumilus bacillus pumilus strain - which was derived from soil-isolated b. pumilus p using chemical mutagenesis is characterized by resistance to streptomycin (str, up to lg/ml) and ability to produce extracellular enzymes in quantities almost -fold higher than the parent strain. these features make the - strain suitable for industrial production of rnase (binase) which is known for its antitumour and antiviral properties and can be used as an rna-degrading tool in molecular biology. the whole genomes of both mutant and wild-type b. pumilus strains were sequenced recently. to reveal the exact genetic features responsible for rnase overproduction and str resistance we have fulfilled comparative genome analysis of b. pumilus p and - strains. facilities of rast server, edgar platform and additional bioinformatics tools (plasmid finder, prophinder, bl seq) were used. it is found that both b. pumilus genomes under study contain an intact prophage, while only wild-type strain bears a kb cryptic plasmid. none of the systems is inactivated in mutant strain according to the results of metabolic reconstruction. . % of total cdss differ in - strain in comparison to p one, % of them are hypothetical. the altered genes are involved in membrane transport, cell wall composition, chemotaxis, spore formation, carbohydrate metabolism, dna metabolism, translation and transcription regulation. mutation (k n) leading to str resistance is identified in s ribosomal protein s p. regulatory and coding regions of binase gene have no modifications. candidate genes which can account for binase overproduction are selected. mutation k n is classical in str resistance and leads to enhancement of decoding accuracy while decreasing elongation speed. rnase overproduction is brought about by non-specialized mechanism since other hydrolases are also overproduced in mutant strain. genes encoding extracellular serine protease, sporulation initiation phosphotransferase f, gnat-family acetyltransferase and cell wall modifying enzymes are reported previously to increase production of degradative enzymes. the action of encoded by them proteins lead to increase of stability and release of secreted proteins to environment and to derepression of their transcription from negative regulators bacillus pumilus strain p has been identified on its ability to produce ribonuclease and different extracellular proteases. in order to increase inherent biosynthesis of proteases the p strain was screened on culture medium supplemented by streptomycin. derivative b. pumilus strain - gains the resistance to streptomycin and also shows the increased ribonuclease activity. we used genomes of both these strains to explore streptomycin susceptibility and increased activity of hydrolytic enzymes. whole-genome shotgun sequencing was performed using a combination of pyrosequencing and ion semiconductor sequencing, which provided x ( p) and x ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) overall genome coverage. assembled genome sequences of p and - strains included and scaffolds > bp with a calculated genome size of bp and bp, respectively. the gc content was %. both draft genomes have been deposited at genbank (jojx . for p and jhud . for [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . detailed comparative genomic analyses of strains have been performed. we calculated average nucleotide identity (ani) values between the genomes of our strains and completed b. pumilus genomes deposited in ncbi database. two b. pumilus strains (sh-b and safr- ) revealed the max. ani value ( . % and . %, respectively). b. pumilus sh-b strain has been used as a reference for snp calling in strains p and - . snps for the p strain and for the - strain were classified as nonsynonymous variants. radical nucleotide substitutions from the - genome were not found in p genome. among them, the mutation in the codon of rpsl gene (coding s ribosomal protein s ) is probably associated with resistance to streptomycin. also, two mutations in rpob and nusa genes (coding rna polymerase and transcription termination factor rho, respectively) may be related to increased enzymes activity. both our strains contain protease-coding genes. twelve of them are encoding extracellular proteases. here we propose an algorithm that can predict an antibodies mutant forms with desired specificity. this algorithm allows to determine the position and type of amino acid residue for mutagenesis. approach is based on a hybrid method of quantum and molecular mechanics (qm/mm) that allows to understand the reaction mechanism and the role of active center amino acids. catalytic antibody a , that is able to hydrolyze pesticide paraoxon, was selected as a model. however, the hydrolysis efficiency of paraoxon by a antibody is only m À min À , that is insufficient for using this antibody as antidote. the main fundamental goal of our study is to determine the necessary conditions for improving the binding reaction of paraoxon by catalytic antibody a . the hybrid qm/mm method allows to study the reaction mechanism of interaction of a with paraoxon. it was shown that the reaction proceeds via the classical s n mechanism. the key step of the reaction is the proton transfer from the catalytic residue tyr- to paraoxon. qm/mm approach determines position for mutagenesis -leu- in light chain. for one of the mutant in this position -leu argwere predicted (i) increased probability of formation of a hydrogen bond between the catalytic moiety and paraoxon compared to the wild type antibody and (ii) smaller value of the diffusion coefficient, which reflects the best positioning of paraoxon in the active center. steady-state kinetic analysis shows that leu arg exhibits a -fold increase in k /k d compared to a ( m À min À vs. . m À min À ). double mutant leu arg/ser ala also has improved constants of interaction with paraoxon in comparison with the wild type antibody, however, a single mutant leu arg still binds paraoxon three times better, that may be due to the fact that the serine in position increase the nucleophilicity of tyr . thus, our results are in line with our computed predictions. this work was supported by rfmefi x . due to high prices of meat and meat products, low quality raw materials like offal tissues are commonly used in turkey. in the retrospect of the studies for evaluating and detection of unwanted tissues in the sample is basic histological examination. the light microscopy techniques are very strong method if a researcher qualification is enough. a new researcher-independent method must be developed. therefore, different tissues and organs constitute of unique mrna and protein. our method is based on this event, so the antigenic sites of the tissues can be detected by selected antibodies. the first set of the antibodies are for detecting muscle and adipose, consist on muscle actin and adipose triglyceride lipase. this set is used for calibration on standard meat sample. the second set of the antibodies are detecting of offal tissues, consist on trrap and casein. anti-casein antibody is selected because the mammary gland usage in grinded-meat is very common. immuno-staining started with hier (heat mediated epitope retrieval), then classical ihc method applied to slides with dab-chromogen. after all process completed the slides were photographed by las (leica application suite) on microscope. the capture settings were remained same on both sets. image capture size is x pixels and field of view (fov) is lm. all the image files were converted to binary for threshold operation. the threshold values of first set and second set were calculated and their ratios were compared. the formula is based on the distribution (dst) of pixel intensity (int) over threshold (thrs) values on all fov (axis: the results are good enough to detect the unwanted micro-structures on % raw meat and % offal tissue. calculations proofed with imagejÒ. future application of this method and opencv-based software algorithm is to port the source code to a single board computer (sbc) with a digital microscope connected. monday september : - : mechanisms of pro-inflammatory diseases p- . . - the effects of raas inhibition in rate limiting step by aliskiren on testicular torsion injury in rats testis torsion is a urological emergency condition that results in necrosis of the testis if the condition is not treated. unfortunately treatment of testis torsion is not fully understood, therefore clinical and experimental studies are performed continuously. reninangiotensin-aldosterone system (raas) contributed to pathophysiology of several diseases. aliskiren (als) inhibits the renin on the first step of this system. our aim is to investigate the protective effect of aliskiren on unilateral testis damage caused by experimental testis torsion and detorsion. the forty-eight rats were separated into eight groups of six animals: sham, sham+als mg/kg (oral) group, torsion group (tor), torsion/detorsion group (tor/det), tor+als mg/kg (oral) group, tor+als mg/kg (oral) group, tor/det+als mg/kg (oral) group, tor/det+als mg/kg (oral) group. in the tor and tor/det groups, the left testes were rotated °clockwise together with the spermatic cord and tunica vaginalis in the scrotal space. the left testes of the rats were subjected to torsion and detorsion during h. after experimental procedures, testicular tissues were examined by histopathologic and molecular analyses. the il- b and inos mrna expressions were increased in tor and tor/det groups when compared with sham group. both doses of aliskiren administration decreased these expressions in tor/det groups. the stereological results revealed that aliskiren administration promote the numerical density of mature spermatids in tor and tor/det groups. the numerical densities of tor/det+als and tor/det+als groups were similar and these two groups have significant difference when compared to the tor and tor/det groups. the administration of als may be useful for preventing ischemic damage on unilateral testes injury in rats. this study supported by a tubitak project, coded s . ) has recently been recognized as a potent immunomodulator which acts through regulation of gene expression involved in immunity response thus affecting various inflammatory and autoimmune diseases. the study was aimed at investigating hepatoprotective role of d in vdr-mediated regulation of pro-inflammatory factors in diabetic liver. materials and methods: type diabetes was induced in male c bl/j mice by i.p. injection of high-dose stz ( mg/kg b.w.). after weeks of stz-induced diabetes animals were treated with/without d ( iu/mouse per os) for weeks. blood serum ohd was assessed by elisa. rel-a, vpf, inos and vdr expression was measured by qrt-pcr and/or western-blot. results and discussion: diabetes caused two-fold reduction of serum ohd level, indicative of d deficiency. significant alterations in d -endocrine system were found as is evident from reduced expression of cyp a , cyp r , vdbp and vdr at transcriptional and translational levels. these changes were accompanied by a marked increase in mrna and protein levels of inflammation markers rel-a, vpf and inos in hepatic tissue of diabetic mice. diabetes also led to structural lesions in liver tissue. complete restoration of ohd content and partial normalization of liver tissue structure were achieved after d treatment. d administration partially normalized expression of cytochromes involved in d metabolism and hepatic pro-inflammatory factors. d treatment prevented overexpression of rel-a and phosphorylated p /rel-a translocation to hepatocellular nuclei that is most likely mediated through , (oh) d and vdr. conclusion: study confirmed that diabetes-induced liver abnormalities are associated with chronic inflammation that can be linked to impaired d metabolism and deficiency. our findings demonstrate protective vdr-mediated effect of vitamin d against diabetes-induced liver injury. p- . . - lavandula stoechas extract increased glucose uptake and protein levels of key signaling molecules in insulin resistant c c muscle cells s. savranoglu , h. ipek , s. arslan , h. delig€ oz , a. r. t€ ufekc ßi , i. demirtas , t. boyunegmez t€ umer graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, deparment of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, department of chemical engineering, faculty of engineering, pamukkale university, denizli, turkey, department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, turkey, department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: the aim of this is to identify remedial effects of lavandula stoechas, anatolian traditional medicine, against metabolic disorders developed on the ground of insulin resistance. ethyl acetate extract (eae) of l. stoechas was investigated in c c myotubes which were made insulin resistant by free fatty acid (ffa) treatment, for its effects on glucose uptake and as well as on the activation of akt- (by pakt/ akt ratio) molecule which plays a central role in insulin signaling through serine ( ) phosphorylation. in addition, the protein level of lipoprotein lipase (lpl) enzyme was also evaluated. material and methods: c c cells were made insulin resistant by palmitic acid (ffa) and effects of eae on p-akt (ser )/akt ratio and lpl level were determined by sds-page/western blot. the effect of eae on glucose uptake in insulin resistant cells were determined by the -deoxyglucose uptake assay. results: eae at and lg/ml significantly increased the glucose uptake and % compared to insulin resistant control cells. metformin at mm increased this parameter up to %. eae increased pakt ser /akt level - % and lpl expression - % for and lg/ml, in insulin resistant myotubes, respectively (p < . ). discussion: eae of l. stoechas improved impaired insulin sensitivity through both enhancing glucose uptake and activation of akt molecule through ser phosphorylation. in addition, it also considerably increased lpl level which has very important function in lipid metabolism. conclusion: overall, results demonstrated that l. stoechas contain phytochemicals which can be effective for the prevention and also treatment of insulin resistance and associated conditions. our research group is on the way for the identification of these 'bioactive' molecules with bioassay guided fractionation studies. tubitak (projectid: t ) supports this study. achievement of complete pain control is very difficult task, which requires a search for new molecular targets during the analgesic substances development. considering the importance of glial cells and their signaling molecules, development of new gliotropic therapeutic methods is a promising direction in pain treatment. polyunsaturated fatty acids, including docosahexaenoic acid demonstrating anti-inflammatory and antioxidant activity are of considerable interest. docosahexaenoic acid (dha, : n À ) analgesic activity was studied using a chronic constriction injury (cci) rat model. animals were subcutaneously injected with dha emulsion at a dose of . mg/kg ( mm/kg) daily during weeks after surgery. collection of material for subsequent immunohistochemistry investigation was performed on day . we clearly demonstrated that the activation of neurokinin neurotransmission and nnos synthesis are coincided with the astroglial activation in the spinal cord dorsal horn (scdh) superficial lamina during neuropathic pain development. however, the detailed mechanisms of interaction between substance p (sp)-, no-ergic systems and astrocytes in the spinal cord remain to be elucidated. systemic administration of dha to cci animals reduced neurogenic pain intensity and duration, leading to an earlier stabilization of paw weight distribution and preventing the development of degenerative changes in denervated limb. this drug treatment reduced the level of the sp-and no-ergic neurotransmission and decreased astrocytosis in the scdh superficial lamina. thus, the ability of dha to affect nociception is a promising and safe alternative to current pharmaceutical therapeutics. immunohistochemistry studies carried out with the russian science foundation financial support (agreement no. - - ), obtaining dha and all manipulations with animals of the material was funded by rfbr according to the research project no. - - mol_a. p- . . - circulating endothelial-derived apoptotic microparticles and aopps are related to highsensitive troponin t in patients with chronic hepatitis c infection the aim of this study was to evaluate non-standard risk factors for cardiovascular events, such as endothelial dysfunction assessed by endothelial-derived microparticles (emps) (cd +/ cd + ), advanced oxidation protein products (aopps), and low-grade inflammation, that are potentially associated with elevated levels of high-sensitivity troponin t (hs-tnt) and n-terminal pro-brain natriuretic peptide (nt-probnp) in patients with chronic hepatitis c (chc). methods and results: eighty-six chc patients and healthy control subjects were enrolled in the study. circulating levels of hs-tnt, nt-probnp, aopps-albumin (the ratio of aopps to albumin content), emps (cd +/ cd + ), hs-crp, and tnf-a were assessed. compared with chc patients, the chc patients with diabetes mellitus (dm) had higher levels of emps (cd +/ cd + ) and aopps-alb, which were associated with elevated hs-tnt levels (≥ . pg/ml). nt-probnp positively correlated with tnf-a level in all chc patients and this correlation was stronger in diabetic patients. in multivariate logistic regression analysis, the independent factors associated with the presence of elevated hs-tnt levels were the presence of dm (p < . ) as well as high levels of aopps-alb, apoptotic emps (cd + /cd + /an-v + ), and nt-probnp (p = . , p = . , p = . respectively). conclusion: the prevalence of elevated hs-tnt were increased significantly in the diabetic patients with chronic hepatitis c. hs-tnt was related to non-standard risk factors for cardiovascular events, and circulating endothelial-derived apoptotic microparticles (cd + /cd + /an-v + ) level was an independent predictor for elevated hs-tnt levels, potentially indicating some abnormalities in the myocardium. apnea; and healthy individuals; and assessing the connection between the pain and the dimension of the sleep disorder. material and methods: patients who were diagnosed with obstructive sleep apnea and healthy individuals who were similar in terms of age and gender were included in this study. the patients, who were diagnosed with obstructive sleep apnea with the examination and sleep tests, were assessed according to the american college of rheumatology (acr) criteria in terms of fms. serum d vitamin level was measured by using the ultra performance liquid chromatography method. findings: when the fibromyalgia syndrome and obstructive sleep apnea and pure obstructive sleep apnea patient groups are compared with the control group, the vitamin d level was found to be low at a significant level (p = . , p = . , respectively). no significant difference was found between the vitamin d levels in fibromyalgia syndrome, obstructive sleep apnea and pure obstructive sleep apnea patient groups. a negative correlation was found between the number of the sensitive points and vitamin d levels in fibromyalgia syndrome patients (p = . ). results: it has been concluded that the obstructive sleep apnea and fibromyalgia syndrome patients have low vitamin d levels, and this situation must be considered in treatment modalities. on the other hand, the results obtained in the study make us consider that vitamin d metabolism is not influential in the pathogenesis of the fibromyalgia syndrome and obstructive sleep apnea togetherness. p- . . - decreased chitotriosidase activity and levels in familial mediterranean fever discussion: familial mediterranean fever is an inflammatory disease. several cytokines and inflammatory mediators are playing role on pathogenesis of the disease. although ıt has been demonstrated that the increased concentrations of cht in patients with fmf. we found lower cht activity and concentrations in patients with fmf. conclusion: serum cht enzyme activity and concentrations may not be considered as a biomarker in fmf patients taking colchicine. new studies are needed to evaluate the changes of the enzyme activity, concentration and the role of cht in patients with colchicines negative patients. chronic hyperglycemic state leads to an increase in subclinical systemic inflammatory response in diabetes mellitus type (dmt ) patients. inflammation-based scores, neutrophil to lymphocyte ratio (nlr), platelet to lymphocyte ratio (plr) and red blood cell distribution width to platelet ratio (rpr) are biomarkers able to quantify systemic inflammation. the aim of the study was to investigate association of the inflammation-based scores with short-and long-term glycemic control markers, and whether they could be used as indicators of glucoregulation in dmt patients. the cross-sectional study included dmt patients, treated at the primary health care centre zenica from december to april , distributed into groups according to glycated hemoglobin (hba c) values: a (n = , hba c ≤ . %) and b (n = , hba c > . %). complete blood cell count, fasting blood glucose (fbg) and hba c measurements were determined at the primary health care centre zenica and at the department of laboratory diagnostics, cantonal hospital zenica by standard laboratory methods. all statistical tests were performed using spss . . p values fasting blood glucose and hba c were significantly higher in the group b compared to the group a (p < . ). there was no significant difference of nlr, plr and rpr between the groups (p = . ; p = . ; p = . , respectively). significant correlation of inflammation-based scores with fbg and hba c was found only between plr and hba c in the group a of dmt patients (r = . , p = . ). inflammation-based scores could gather meaningful clinical information, either diagnostic or prognostic, on a variety of hyperglycemic, inflammatory, cardiovascular and thrombotic disorders. since there was no statistically significant difference of nlr, plr and rpr between dmt patients with good and poor glycemic control, we conclude that these scores could not be used as indicators of glucoregulation in dmt patients. chronic inflamation plays a central role in the development and progression of diabetes and in the pathogenesis of its comlications. the neutrophil-lymphocyte ratio (nlr) and platelet-lymphocyte ratio (plr) are indicators of subclinical inflamation. mean platelet volume (mpv) is one of the platelet function indices that reflects the platelet production rate and stimulation. we investigated the association of nlr, plr and mpv with prediabetes and type diabetes mellitus (t dm) and determine whether or not these are reliable markers for diagnosis. we evalueted people's results who were carried out oral glucose tolerance test (ogtt). acording to -h values of plasma glucose in the ogtt; . group (normal glucose tolerance: ngt): under mg/dl (n = ), . group (prediabetic: impared glucose tolerance (igt)): ranging from mg/dl to mg/dl (n = ), . group (firstly diagnosed diabetic by ogtt): above mg/dl (n = ). . group is clear diabetic without complication (taking treatment) group (n = ). we compered nlr, plr, mpv and some biochemical markers between four groups. there are significantly differences between all groups in nlr (p = . ) and plr (p = . ) values. nlr values are significantly higher in prediabetic ( . it is recognized that a chronic low-grade inflammation and an activation of the immune system are involved in the pathogenesis of insulin resistance and type diabetes mellitus (t d). this study aimed to analyze the long-term impact of altered metabolism in female t d patients at the level of mediators of inflammatory response. this study included femalet d patients and control subjects, which were recruited at the clinical center university of sarajevo and the general hospital tesanj. in this study the effects of glycemic control on markers of the inflammatory response crp, fibrinogen, leukocytes, sedimentation, and cytokine il- , were analyzed. all subjects included in this study were free of evidence infections, surgery, thyroid disease, polycystic ovarian syndrome, active liver and kidney damage. all biochemical analyses were performed by employing standard ifcc protocols. results from this study demonstrated significant increase of fibrinogen (p = . ), crp (p = . ), il- (p = . ), leukocytes (p = . ) and sedimentation rate (p = . ) in female t d population compared to control subjects. interestingly, a significant correlation was shown between crp and hba c (p = . ), il- and glucose ( . ), il- and bmi ( . ). in our study, female t d compared to the healthy population had significantly higher levels of fibrinogen, leukocytes, il , crp and sedimentation. other studies conducted in female population associated elevated levels of il- and crp with t d independent of other risk factors for diabetes. crp being most robust predictor of diabetes. studies have shown that crp is an important predictor of t d for the female but not the male population. thus, our data suggest that inflammation play an important role in the pathogenesis in female diabetic population. a more detailed study on a far larger number of subjects should point out fact if they can effectively be used as biomarkers in the primary prevention of t d in this population. objectives: bone and mineral metabolism disorders hold an important place among the complications after renal transplantation. the purpose of this study was to demonstrate the relationship between vitamin d, calcium, phosphorus metabolism with graft function and to measure , (oh) d levels with lc-ms/ ms in renal transplant recipients. design and methods: this study included renal transplant recipients ( female, male; mean age: . ae . ) from living related donors were transplanted. blood samples were collected immediately before and after transplantation at month . serum creatinine, bun, calcium, phosphorus, alkaline phosphatase, glucose, albumin, pth, (oh)d and , (oh) d levels were measured. gfr values were estimated by ckd-epi. plasma , (oh) d levels were determined in a lcms- triple quadrupole tandem mass spectrometer (shimadzu corporation, japan) by mrm. spss . software was used for statistical analysis. results: although plasma , (oh) d levels significantly increased (p = . ), we did not find any significant differences for serum (oh)d levels after transplantation. when posttransplant levels of serum phosphorus, pth, creatinin, bun and alp levels were found to be significantly decreased (p = . , p = . for alp), we observed significantly higher calcium and gfr values (p = . ). vitamin d insufficiency was present . %, deficiency . %, severe deficiency % before transplantation, insufficiency was also seen . %, deficiency %, severe deficiency . % after transplantation at month . conclusions: in our study, all patients were found to vitamin d deficiency and insufficiency. determination of vitamin d deficiency and consequently treatment with vitamin d supplements could lead to better graft surveys. free fatty acids (ffa) represent important link between obesity, insulin resistance, type diabetes (t d), and dyslipidemia. increased adiposity, as approximated by body mass index (bmi), correlates well with increased serum levels of leptin-adipocyte derived hormone implicated in the regulation of adipose mass and alterations in insulin action and secretion. the main objective of the present study was to investigate the potential association of serum ffas with leptin levels in healthy and newly diagnosed type diabetic subjects. this study involved newly diagnosed type diabetics and healthy subjects. all participants in the study were free of evidence of hepatitis, viral infection or active liver and kidney injury. for biochemical analyses of glucose, glycosylated hemoglobin (hba c), and lipid profile, standard ifcc protocols were used. analysis of free fatty acids (ffas) was done by gas chromatography, while serum leptin levels were determined by the elisa kit. in addition to the expected differences in glucose, hba c, and bmi, our results also showed significant differences in leptin, myristoleic, palmitic, linolenic, arachidic, and arachidonic acids between t d and control subjects. in healthy subjects, a significant correlation was demonstrated between glucose and lauric, arachidic, arachidonic acid levels, body weight, and bmi. newly diagnosed diabetics showed significant association between glucose and lauric, myristoleic and linolenic acid levels; with leptin being associated with myristic and palmitoleic acid levels. interestingly, in all participants, significant association was found between glucose and hba c, glucose and leptin, myristoleic, arachidic, and bmi as well as between leptin, arachidic acid, and bmi. thus, our data point out association of different types of ffas with leptin levels in newly diagnosed type diabetics. however, further studies should be done in larger number of patients to confirm our results. rheumatoid arthritis (ra) and ankylosing spondylitis (as) are chronic inflammatory diseases with distinct clinical manifestations in many ways. the aim of this study is to evaluate the serum levels of molecules which may be used as markers for angiogenesis and vascular leakage in the processes of two clinically different pictures, ra and as. ra patients, as patients and healthy volunteers with mean age of - were included in the study. serum levels of vegf, angiopoetin- and tie- were measured by enzymelinked immuno-sorbentassay (elisa) using a commercially available kit. serum nitricoxide levels were evaluated by the griessreaction. serum vegf, ang- and no levels were significantly higher in the as group ml, p < . ; p < . ; p < . ). no differences were found between as and ra for tie- (p > . ). vegf, ang- , tie- and no levels were positively correlated in both as and ra patients (p < . ), but no correlation was detected between clinically activation index das- , basda _ i scores and laboratory measurements such as sedimentation, crp and anti-ccp (p > . ). when the diagnostic performance of the parameters were evaluated with the roc analysisonly the performance of the ang- in as patients was sufficient (auc ( % cl): . , p < . ). elevation of angiogenic factors in the serums of as and ra patients supports the role of angiogenesis in the etiopathogenesis of these diseases. however, lack of relationship between disease activity leads to not to use these factors as a marker for clinical follow-up. only ang- measurements may be useful for the differantial diagnosis. the evaluation of ischemia modified albumin as an early biomarker of acute myocardial infarction introduction: acute myocardial infarction (ami), remains a leading cause of morbidity and mortality worldwide. early diagnosis of ami is very important because early treatment may reduce the extent of injury to the myocardium. currently, biomarkers of myocardial necrosis such as myoglobin, ck-mb and troponins are highly sensitive and exhibit good specificity. however, these biomarkers increase after tissue injury, approximately - h after the cardiac event and detect only the consequences of prolonged ischemia. recently, ischemia modified albumin (ima) has been assessed and found to be very useful for the diagnosis of myocardial ischemia and it is considered as a serum biomarker. the aim of the present study was to evaluate the serum level of ima and determine the relation between patients with ami and control group, in order to verify its potential as a novel marker for early detection of mi. materials and methods: the study was performed with patients and healthy controls. blood samples from all subjects were collected by venipuncture in plain tubes, and immediately centrifuged at g for min at °c. the serum samples were stored at À °c until analysis. the serum levels of ima were determined using the cusabio biotech human ischemia modified albumin, elisa kit according to manufacturer's instructions. the results are given as international units/milliliter (iu/ml). results: our findings revealed that ima showed no significant difference between the groups. conclusion: our results suggest that ima assay is not a sensitive marker for early detection of ischemic hearth disease and cannot be used alone for the diagnosis of ami. prospective studies are needed to identify ima's potential as a biomarker for ami. p- . . - neutrophil-to-lymphocyte ratio and platelet-tolymphocyte ratio in polycystic ovary syndrome polycystic ovary syndrome is a complex and multifactorial disease with metabolic dysfunction and the etiopathogenesis is not well established. emerging data suggest that adiposity and chronic low-grade inflammation are involved in the development of the metabolic dysfunction. neutrophil-to-lymphocyte ratio (nlr) and platelet-to-lymphocyte ratio (plr) have recently been investigated as two new inflammatory markers used in the assessment of systemic inflammation in many diseases. the purpose of the study was to investigate their relation with pcos patients. the study population consisted of patients with polycystic ovary syndrome and healthy women controls. nlr and plr obtained by dividing absolute neutrophil to absolute lymphocyte count and absolute platelet count to absolute lymphocyte count, respectively. the neutrophil count ( . ae . vs. . ae . , p < . ) and platelet count ( . ae . vs. . ae . , p < . ) were higher in patients with pcos compared to the control group. lymphocyte count was . ae . in pcos patient and . ae . in control group. the nlr and plr of pcos patients were significantly higher compared to those of the controls ( . ae . , . ae . p < . , . ae . , . ae . p < . , respectively). in this study we found nlr and plr were significantly increased in patients with pcos compared to healty control. nlr and plr were two useful inflammatory markers for assessment of patients with pcos. imbalance in neurotransmission in conjunction with neuroinflammation contribute to neurological dysfunction observed during acute liver failure (alf). own observations indicate that alf in a mouse model is associated with altered expression and/or intracellular distribution of synaptic proteins. since neutralization of tgf-b appears to improve the neurological score in alf mice, we examined the possibility that increased levels of tgf-b , caused by liver damage, may affect the expression of selected synaptic proteins. expression and/or cytoplasmic vs. membrane distribution of a number of functionally critical synaptic proteins in cerebral cortex and blood tgf-b were measured in c bl mice with alf induced by single i.p. injection of aom ( mg/kg of b.w.) and after neutralization of tgf-b induced by single i.p. injection of ab-tgf-b ( mg/kg) h before aom injection. in alf mice, blood tgf-b was increased, and the expression of presynaptic proteins: synaptophysin and synaptotagmin was increased in the cytosolic (s ) fraction by~ % and %, respectively, but was slightly depressed in the membrane (p ) fraction by~ % and~ %. aom induced an increase of postsynaptic proteins: psd- and nnos by~ % in p fraction. tgf-b neutralization resulted in a reduction in the expression of presynaptic proteins by~ % in s fraction and~ % in p fraction, in control animals and normalized their amount in the cytosolic fraction after aom injection, but was ineffective with regard to psd- and nnos. the results indicate that in alf mouse, neutralization of cytokine tgf-b normalizes synaptophysin and synaptotagmin expression in the synaptoplasm, without affecting their synaptic membrane content. effect of tgf-b neutralization appear to be confined to the presynapse. % of acute pancreatitis (ap) patients develop severe acute pancreatitis (sap), which is is resulted in multiple organ dysfunction syndrome. an extensive inflammatory response occurs due to inflammatory mediators synthesized and secreted during sap. since preventing the inflammation in sap is important in the prognosis of the disease, new drug candidates having strong antiinflammatory effects will provide a new concept for therapeutic strategies against acute pancreatitis. non-steroidal anti-inflammatory drugs (nsaids) show their effects by inhibiting cyclooxygenases (cox- and cox- ) and they play an important role in the pathogenesis of acute pancreatitis. since conventional nsaids inhibit both cox- and cox- , they have serious side effects on gastrointestinal system. therefore, new highly selective cox- inhibitors having fewer side effects are needed. in the present study, selective cox- inhibitory activities and cytotoxic effects of new series of -benzoxazolinone and thiazolo [ , -b ]- , , -triazole derivatives previously synthesized as specific cox- inhibitors with no side effects on gastrointestinal system were investigated. permeability of the compounds was tested by pampa using caco- cells. compounds were found highly selective, non-toxic and permeable. ap was induced in rats via retrograde injection of stc into the pancreatic duct system. rats were pre-treated with saline or celecoxib or the new compounds before stc injection and sacrificed h later. the severity of ap was evaluated using biochemical and histopathological analyses. edema, inflammation, hemorrhage and acinar cell necrosis were detected in the pancreatic tissue of sap group. sap was remarkably increased serum lactate dehydrogenase, ast, alt, lipase and amylase activities and serum tnf-a, il- b, il- , il- and il- levels. tissue myeloperoxidse activity was also increased. pretreatment with the novel compounds reserved all these biochemical and histopathological parameters. alopecia areata (aa) is an inflammatory disease which is affects hair follicles, and sometimes nails. it is suggested that cytokinemediated immunity plays an important role in etiopathogenesis of aa. this study was planned to evaluate the serum ykl- and tgf-b levels of patients with aa. patients with aa and healthy volunteers were recruited into the study. fasting venous blood samples were collected from the participants and serum was obtained by centrifugation. serum ykl- and tgf-b levels were measured by enzyme linked immunosorbent assay (elisa). serum tgf-b levels in the patient group were significantly lower compared to the control group whereas serum ykl- levels were significantly higher in patient group. tgf-b levels of men and women with aa were found to be significantly lower than that of controls. while serum ykl- level of male control group is higher than the male patients, there were no significant differences between women groups. the increased serum ykl- levels in aa patients suggests that ykl- plays a crucial role in the pathogenesis of aa. arterial immune mediated inflammation participates centrally in all stages of the development of atherosclerosis, from the initial lesion to the end-stage thrombotic complications. although emerging evidence supports augmented cardiovascular morbidity and mortality in cutaneous psoriasis (psc) and psoriatic arthritis (psa) as compared to the general population its underlying mechanism is poorly understood. here we analyzed the inflammatory burden in recent onset of psa patients without traditional cardiovascular risk factors (cvrf) in a transversal study measuring carotid intima media thickness (cimt) (measured with ecodoppler), and proatherogenic inflammatory molecular markers like c-reactive protein (crp), interleukin (il- ), and soluble intercellular adhesion molecule- (sicam- ) in comparison with control patients. cimt values are similar in psa ( , ae . *) and psc ( , ae . ) patients. however, both of them were significant increase compared with control ( . ae , ). regarding inflammatory markers il- serum levels in patients with aps was higher than pcs ( ae . ) and healthy controls ( , ae . ) but the difference did not achieve statistical significance (*p > . ). on other hand mean of sicam- , value from patients with recent onset of psa is significant higher than controls. psc remain without significant changes compared to control (*p > . ). in addition mean value from patients with recent onset of psa is significantly higher than in controls (*p < . ) and psc group. overall, preliminary findings suggest for the first time that patients with early psa, without evident traditional cvrf have significant increased values of cimt, sicam- crp against the general population control group. this data strongly supports that early cv molecular markers are increased after the first symptoms and signs of this disease even in the absence of traditional cardiovascular risk factors. furthermore, this give new windows for a proper treatment. p- . . - protective effect of trail against proinflammatory cytokines on pancreatic beta cells correlated with decrease in dr and increase in dcr expressions universitesi, antalya, turkey introduction: proinflammatory cytokines are known to have destructive effects on beta cells, which contribute to type diabetes (t d) development. the combinatory effects of three of these cytokines in particular, namely tnf-alpha (tnf-a), ifngamma (ifn-c), and il- beta (il- b), are claimed to render beta cells prone to t cell-mediated destruction. the recently identified anti-inflammatory feature of tnf-related apoptosis-inducing ligand (trail), its possible protective role in this process. in this study, the effects of applications of trail with tnf-a, ifn-ᵞ, and il- b on beta cell viability and correlation of these effects with trail receptor expression patterns were investigated. methods: glucose-responsive insulin-secreting nit- mouse beta cell lines were treated with tnf-a, ifn-ᵞ, il- b, and soluble trail (strail) individually and in various combinations. cell viabilities were determined at and h by mtt assay. trail ligand and receptor expression profiles on nit- cells, and alterations in receptor expression levels following cytokine applications were determined by western blotting analysis. results: trail treatment did not have any cytotoxic effects on nit- beta cells at h, while increasing cell viability following il- /ifn/trail and il- /tnf/trail combined applications. substantial levels of death receptor (dr ) expression were detected on nit- cells before applications, yet it displayed decreased levels at h of trail treatment. lower levels of decoy receptor (dcr ) expression detected prior to treatments increased significantly in contrast. discussion: the fact that trail co-treatment with tnf-a, ifn-ᵞ and il- b increased cell viability in nit- beta cell lines along with reduction in dr death receptor expression and an increase in the decoy receptor dcr expression, points out to a possible protective effect of trail in insulitis, and strengthens its potential as a putative therapeutic molecule in prevention of beta cell loss. behc ßet's syndrome (bs) is a multisystemic inflammatory disorder with a strong and complex genetic background. being a prevalent disorder both in turkey and also in the ancient trade road 'silk road' countries, bs is an important cause of impairment and disability owing to its chronic and relapsing nature. besides, bs is reported to be an important cause of mortality among the young male patients. while the epidemiology of bs is substantially well documented, currently, the etiology, the molecular mechanisms underlying its pathogenesis, and the classification of the disorder remain to be elucidated. our aim was to disclose the disease mechanisms at molecular level in turkish bs patients by obtaining, comparing, and analysing the transcriptome data of bs patients with age and gender matched healthy controls. for this purpose, by using the affymetrix hg u plus . microarrays, peripheral blood cell mrna profiles of bs patients (b) and matched healthy controls (c) were obtained. following bioinformatics, gene ontology, and pathway analysis, validation experiments of the identified prominent mrnas were performed by qrt-pcr methodology. the comparison of b vs. c yielded differentially expressed gene numbers of and for the chosen fold changes of . and . respectively (p ≤ . for both). during gene ontology and pathway analysis, immune system process, immune system diseases, systemic lupus erythematosus, arthritis, and intestinal immune network for iga production categories/pathways were significantly enriched. clustering analysis revealed a molecular signature which accurately distinguished b and c samples, while the qrt-pcr analysis successfully validated the chosen mrna transcripts. this study documented differential expression of a large number of immune system and immune disease related genes in bs patients. the uncovering of the molecular disease mechanisms of bs will point to novel candidate molecules to be targeted for the treatment of the disorder. obesity is a public health problem in developed countries and worldwide with increasing prevalence through a relationship primarily with atherosclerotic cardiovascular diseases as well as several metabolic disturbances such as increased insulin resistance and diabetes. although several studies identified obesity as an independent risk factor for atherosclerotic cardiovascular diseases, the mechanism underlying the increased cardiovascular risk in obese patients has not been clearly delineated. adma, no, endothelin- and homocysteine are an indicator of endothel disfunction that plays an important role in the pathophysiology of atherosclerosis. in our study, obese children and the control group were compared in terms of adma, no, endothelin- and homocysteine, we also investigated whether there is a correlation between these parameters. obese and healthy children, participated in the study. when the obese group was compared to the healthy controls, the adma level of the obese group were significantly higher than those of the control group but there was no statistically significant difference in no, endothelin- and homocysteine. increased adma level might trigger the pathogenesis of atherosclerosis starting from the childhood years onward. that is why controlling obesity in this age group with diet and other treatment modalities will prevent the mortality and morbidities that will be seen in adult years. inh deficiency leads to the formation of bradykinin causing to dilation of blood vessels. furthermore, the study conducted by shagdarsuren, on the damage done by c -esterase, demonsrates that the complement system and triglyceride levels are affected. we investigated lipid oxidation and fetuin a levels in patients with c _ inh deficiency. materials and methods: people with c _ inh and people without any illnesses were taken into the study. fetuin a was studied using an el _ isa kit from raybio (usa). ferrous ion oxidation-xylenol orange test was used to find looh serum levels. sh (free thiol groups) test was studied with regards to ellmans method modified by hu. _ ibm spss . was used for statistical results. results: in assessments made between the healthy and the illness groups, there was significant differences in the levels of fetuin (p = . ), looh (p = . ) and sh (p = . ). when pearson correlation analysis was performed, we detected a significant positive correlation between fetuin a and looh levels (r: + . ) discussion and conclusion: in these patients, lipids is secreted from the adipose tissue. in response, anti-atherosclerotic fetuin a levels were risen. patients also possessed increased lipid peroxidation, this increase shows positive correlation with fetuin a levels. in conclusion, we identified that sh with antioxidant properties have increased levels. aim: high fructose corn syrups are found in soft drinks, juice beverages, breakfast cereals, most of the processed foods. it has been shown that high dose of fructose intake may lead to a reduction in the number of hepatocytes, deterioration of liver function, increasing reactive oxygen species and liver steatosis. the aim of this study was to explore whether caffeic acid phenethyl ester (cape) has any potential protective effect on high fructose diet-induced fatty liver model. materials and method: totally fifty rats were divided into five groups. control group, % fructose administered group, cape group, % fructose + cape administered group and ethanol group. after weeks, liver oxidant and antioxidant status, and blood tnf alpha, il- , and il- , tissue nfkb levels were quantified. protein levels were investigated against, nfkb and p-nfkb antibodies and normalized and analyzed against b-actin antibody by western blotting. results: serum tnf-alpha, il- , il- levels were found to be increased in fructose group compared with the control group (p < . ). in liver tissue of % fructose administered group, mda, protein carbonyls and no levels were higher than control group. however sod activity did not show any difference among the groups. in the fructose administered group, caspase showed liver apoptosis and was considered as positive. acquired data revealed that nfkb protein level was decreased in the presence of cape while increment in nfkb protein level was observed in the fructose administered group compared with control group. in case of pnfkb antibody, increment observed in fructose only and both cape and fructose administered groups, respectively. in cape only administered group, there was a decrement in the level of pnfkb protein. conclusion: depending on further analysis, experimental findings are expected to implicate the role of cape as a protective agent on high fructose diet-induced fatty liver model in relation of inos, nfkb and p-nfkb pathways. the investigating association of hepcidin levels with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction a. g. agg€ ul , n. uzun , e. c ß inar tanriverdi , h. € un agri ibrahim cecen university, agri, turkey, nenehatun maternity hospital, erzurum, turkey this study was designed to investigate hepcidin levels and their associations with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction (iugr). a total of pregnant women were included in this study. pregnant volunteers were divided into two groups ( healthy pregnant women and pregnant women with iugr). serum hepcidin, total free iron, ferritin, transferrin, transferrin receptor and interleukin- (il- ) levels were measured by elisa. also, hemoglobin (hb) and c-reactive protein (crp) levels were determined in serum samples from the healthy pregnant women and the pregnant women with iugr. there were significant differences in hepcidin, ferritin, transferrin receptor, crp and il- levels between healthy pregnant women and pregnant women with iugr. hepcidin, ferritin, crp and il- levels in pregnant women with iugr were significantly higher than healthy pregnant women (p). the mediators of systemic inflammation in lipopolysaccharide-induced neonatal sepsis rat model sepsis is an excessive inflammatory response that causes shock, multi-organ failure and high mortality. foreign bacterias and lipopolysaccharides lead to stimulation of endothelial cells to produce biologically active mediators such as proinflammatory cytokines and chemokines, cell adhesion molecules, and growth factors. then these mediators could be act on targets, which were involved in the initiation of systemic inflammation in neonatal sepsis. our aim was to indicate a protective role of thalidomide and etanercept, which have anti-tnf-a activity on systemic inflammatory response in lipopolysaccharide (lps)-induced neonatal sepsis rat samples. thirty -day-old wistar rats were randomly divided into five groups: a control group that received normal saline, a sepsis group that received lps, thalidomide, etanercept and both thalidomide and etanercept treatment group that were administered with therapeutic agents hrs after lps injection. the rats were sacrificed at hrs after lps or normal saline injection (n = ). hepatic tissue tnf-a, il- , icam- and pdgf levels were determined by enzyme-linked immuno sorbent assay (elisa) method in all groups. in sepsis group, tissue tnf-a, il- , icam- and pdgf levels were statistically significantly higher than in controls (p < . ). at same time, pretreatment with both thalidomide and etanercept were found statistically dramatically decreases the levels of tnf-a, il- , and pdgf when compared to sepsis group (p < . ). there were no significant differences in the icam- levels between the all treatment groups and the sepsis group. higher liver tissue tnf-a, il- , icam- and pdgf levels are associated with severe bacterial infection. these proinflammatory cytokines and angiogenic factors may be important in the endothelial dysregulation seen in sepsis. therapeutic agents used in the present study can be help to avoid devastating effects of neonatal sepsis. n-stearoylethanolamine (nse)is saturated minor compound of natural origin that represents the large family of signaling lipids n-acylethanolamines, which belong to endocannabinoid system. considering the crosstalk between obesity-induced inflammatory response and its key role in synaptic dysfunction and neurodegeneration, our current study aimed to investigate the biological effect of nse on brain tissue under high fat diet-induced insulin resistance. previously we found that nse administration to insulin resistant rats caused normalization of liver and pancreas lipid composition followed by the improvement of glucose tolerance and insulin sensitivity (decline in serum insulin level and homa-ir value). moreover, this effect of nse correlated with inhibition of nf-kb translocation into the nucleus of peritoneal macrophages and decreased pool of serum tnfa level in obesity-induced insulin resistant rats. further experiments showed that fat overload triggered significant reduction in the level of main phospholipids (phosphatidylethanolamine, phosphatidylcholine and sphingomyelin), while there were no changes in cholesterol content. nse at a dose of mg/kg during weeks of administration to insulin resistant rats showed a tendency to restore the phospholipid level that was accompanied by increased neural cell survival ( %) compared to rats without treatment ( %). neuroinflammation accompanied by intensive reactive oxygen species (ros) production impairs neurotransmission in a wide range of neurodegenerative pathologies. flow cytometry is used for quantitative analysis of global dna methylation, but fluorescence microscopy is mostly preferred to qualitatively reveal intranuclear localisation of dna methylation and its copattern with other markers. both methods use a similar immunostaining protocol. in this study, we aimed to compare these methods concerning the detection of the global amount of dna methylation. for this, mouse embryonic fibroblasts were cultured either with or without phenol red and then stained for dna methylation or positive controls (histone, betaactin, phosphoakt) by specific antibodies, or nonspecific control antibodies. some cells were incubated with trypan blue before or after the addition of antibodies. fluorescence intensities were measured by the green fluorescence channel ( / nm). autofluorescence spectrum of cells was analysed, and fluorescence channel used for dna methylation detection was changed to red ( nm lp). a poor discrimination between signal and noise was detected due to cellular autofluorescence interfering with specific detection of dna methylation by flow cytometry but not by microscopy. it was also the case for the other markers examined. conventional advances to reduce autofluorescence such using phenol red free culture media or trypan blue quenching were not effective, but using the red channel regarding autofluorescence spectra allows detecting specific staining of dna methylation by flow cytometry. but, green channel did work well for microscopy analysis. findings show that flow cytometry detection of dna methylation requires much attention to quench cellular autofluorescence compared to detection by fluorescence microscopy. one reason could be that flow cytometry detects all cellular content, but manual image-based analysis can exclude cytosolic components. these results suggest the usability of flow cytometry and microscopy as complimentary methods for dna methylation detection, but optimisation to reduce autofluorescence is crucial for flow cytometry. objectives: lung cancers are divided in two main groups as small cell lung cancer (sclc) and non-small cell lung cancer (nsclc) . docetaxel (dtx) and cisplatine are chemotherapeutic that has an anti-tumor activity against various solid tumors. the growing resistance against dtx and cisplatine (cis) still continues to be the biggest obstacle for the treatment success of nsclc patients. deguelin (deg.) is a natural plant derivative and has an encouraging activity against a lot of human cancers. the comparison of the treatment activity of the separate and combined usage of deg., which is a potential chemotherapeutic agent, and dtx, cis which are used in standard treatment, is aimed in this study. material-method: the ic doses of dtx, cis and deg. on the a and h nsclccell lines were determined via the cell vitality tests in our study. the active concentrations determined were applied to nsclc cell lines as deg., dtx, cis and their combinations. the impacts of the medicine are studied by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, colony formation, migration and angiogenesis analyzes on the treated cells and measuring the oxidative stress index (osi). statistical analyse program, rstudio (v. . . ) and the r-script language were used to examine the differences between the agents. the states in which the pvalue was lower than . were accepted as statistically meaningful. results: we found that deg. has pro-apoptotic, anti-migratory and cytotoxic potential on lung cancer cells. deg. amplified cis and dtx-related anti-cancer efficacy (increased apoptotic cell content and cytotoxicity, reduced migration). also, deguelin pretreatment sensitized the cells dtx-treatment (reduced ic values). these effects were remarkable in p -mutant cells. conclusion: deguelin, solely, has anti-cancer potential on nsclccells. both deguelin pre-treatment and combinantion with standart chemotherapeutics result in enhanced anticancer efficacy. the % of the lung cancers are non-small cell lung cancers (nsclc). despite docetaxel (dtx) and cisplatine (cddp) are agents used in the standard treatments of these patients and the recent improvements in the treatments, the response and remission rates observed on the patients are relatively nominal. selenium (se) is an essential diet component and is introduced to have a preventive impact on different levels of cancer. the aim of our study is to investigate the impacts of selenium addition on anticancer feature and tumor prevention before or/and during nsclc standard treatment. the ic doses of dtx, cddp and selenium on the a and h (p mutant) nsclc cell lines were determined via the cell vitality tests in our study. the active concentrations determined and the stipulated available concentrations were applied to cell lines as dtx, cddp, se combinations. the impacts were compared by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, western blot analyzes on the treated cells and measuring the oxidative stress index (osi) and thioredoksin reductase activity. selenium pre-treatments reduced dtx-related ic concentrations at lower doses in both nsclc cells. however, cddprelated ic concentrations reduced dose-dependent manner. selenium supplementation also altered cell-cycle charactheristics at several concentrations and combination regimens. the remarkably higher osi values were observed after dtx treatment and osi levels were found to be lower in selenium pre-treated nsclc cells. selenium sensitizes nsclc cells to dtx treatment at lower concentrations. however, this effect is obtained dose-dependent fashion for cddp regimen. breast cancer is the most common female malignancy worldwide. human epidermal growth factor receptor (her ) is overexpressed in % of breast cancers in association with aggressive phenotypes. the prognosis of metastatic breast cancer remains poor in spite of advances in therapy. as such, her has long been studied as a potential target for anticancer drugs. the modulation of intracellular signaling pathways leads to altered cell metabolism that triggers tumorigenesis and adapts cells to cancer cell metabolism. this characteristic hallmark of cancer metabolism is known as warburg effect meaning energy production via enhanced glycolysis. despite of several studies in breast cancer metabolism, little detail exists on the link between her overexpression and warburg effect. we have committed examining the nature of aerobic glycolysis in her overexpression. in breast cancer cell line mcf , her overexpression (mcf-her ) results in mitochondrial dysfunction with low mitochondrial membrane potential (Δᴪm) and ros accumulation. intracellular iron levels are also higher in mcf -her cells than vector control (mcf -vec). additionally, mcf -her cells show enhanced levels of atp and lactate in association with increase in glucose levels. we have found that complex i activity increases in mcf -her and decreases in knockdown of her in hcc cells that is her positive breast tumor cell line. based on these results, we conclude that there is a link between her overexpression and metabolic indicators of warburg effect. expression and methylation analysis revealed microrna genes deregulated by methylation and new potential target genes of mir- and mir- - p in breast cancer micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to assess the contribution of methylation to expression level alterations of mirna genes and to search for novel potential targets of these mirnas. to analyze alterations in expression we used qpcr technique with references (rnu , rnu ) and paired (tumor/normal) breast cancer (bc) samples. for methylation analysis a methylation specific pcr and the same set of bc samples were used. significant downregulation was shown for mir- b- p, - - p, - - p, - a- p, - b- p, - - p, - - p, and - - p (p ≤ . , fisher's exact test) in bc. we observed mirna genes to be hypermethylated and mir- hypomethylated. hypermethylation for of these mirna genes was shown for the first time: mir- , - , and - ( - % of bc cases). a significant correlation between methylation and expression alterations was revealed for mirnas with downregulation: mir- b- p, - - p, - - p, - a- p, and - b- p (spearman's correlation coefficient (rs) was in the range À . to À . , p ≤ . ), and for mirnas with both scene (down-and upregulation) as well: mir- a- p, - a, and - (rs = À . to À . , p ≤ . ). comparative analysis of the data on expression alterations of mirna genes and protein-coding genes, which were predicted as targets by mirwalk . , revealed the negative correlation between expression levels for some potential mirna-mrna interaction pairs. for example, for pairs mir- /rhoa, mir- /rassf (a), mir- - p/dapk (rs = À to À . , p ≤ . ). thus, both mirnas and methylation affect regulatory networks in bc. novel potential mirna-mrna interaction pairs could be useful in the development of bc therapy approach. this work was financially supported by grant - - from the russian science foundation. the authors thank the n.n. blokhin cancer research center for tissue samples. clear cell renal cell cancer (ccrcc) with metastases has pour prognosis: -year survival is about %. micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to find out mirnas which methylation contributed to ccrcc progression, including metastasis, and to reveal potential target genes of these mirnas. to analyze methylation status, we used a methylation specific pcr as a method and a representative set of paired (tumor/ normal) ccrcc samples. we also used post-mortal renal tissues from individuals without cancer history as additional control. for expression analysis we used qpcr method and paired ccrcc samples. we observed mirna genes (mir- a- /- /- , - - , - - , - b/c, - - , - a, - ) to be hypermethylated, (p ≤ . , fisher's exact test), mirna genes to be hypomethylated and mir- a with both scene (hyper-and hypomethylation was detected). methylation of mirna genes (mir- a- /- , - b/c, - - , - , - a, - a) correlated with advanced stage and/or tumor size and/or dedifferentiation. hypermethylation of mir- - , mir- a, and mir- significantly correlated with metastasis presence (p < . , fisher's exact test). besides, preliminary data revealed the positive correlation between hypermethylation of mir- - and up-regulation of p protein-coding genes: rarb( ), rhoa, nkiras , and chl , which were predicted as targets by mirwalk . (spearman's correlation coefficients (r s ) was in the range . - . , p ≤ . ). in conclusion, novel supposed interactions of mir- - with target genes could be useful as missing chains in signaling pathways. tests for hypermethylation of mir- - , mir- a, and mir- could be suggested as markers of metastasis and pour prognosis of ccrcc. because of difficulty in diagnosis and treatment hc is a clinical problem: early symptoms of hc are often non-specific and surgical resection is the only curative treatment for hc. it is well known that epigenetic alterations are linked to cancer development. the purpose of this study was to determine potential mechanisms of epigenetic regulation of genes related to energy metabolism in hc. we have performed bioinformatics analysis of the cancer genome atlas (tcga) project rna-seq data with crosshub software and found a number of genes involved in glycolysis and differentially expressed in cholangiocarcinoma. qpcr analysis revealed significantly decreased expression of pgm and eno genes in a majority of hc samples which were known as up-regulated in other human cancers according to the literature date. on the basis of tcga methylation dataset ( k illumina microarrays) we supposed that cpg methylation of pgm and eno promoters may play a role in their inactivation. using bisulfite sequencing study we identified several regions within the gene promoters (pgm :~ bp and bp upstream tss; eno :~ bp downstream tss) that are frequently methylated in hc samples (up to %, / ) with down-regulated pgm and eno expression. thus, we demonstrated frequent and significant pgm and eno down-regulation associated with hypermethylation of the specific regions within the gene promoters in hc. the pattern of pgm and eno gene promoter methylation suggests a possibility of ones to be used for the hc diagnosis and development new strategies for therapy. this work was financially supported by grant mК- . . from the president of the russian federation. the work was performed using the equipment of eimb ras 'genome' center. introduction: the development of stomach cancer is a multifactorial and complex process and includes multiple epigenetic, genetic alterations and dietary/non-dietary factors. iodine as an antioxidant may play a protective role against gastric cancer. the aim of this study was to investigate the changes in iodine level in rat with stomach cancer induced by n-methyl-n -nitro-n-nitrosoguanidine (mnng). materials and methods: a total of sprague dawley rats were randomly divided into six groups. rats were administered with mnng ( lg/ml) by oral gavage on days , and to initiate stomach cancer. during the experiment, rats died and those surviving were sacrificed in the rd, th, th, th and th months of the experimental period (group i, ii, iii, iv, v, respectively). the control group (group vi) contains rat which are given only food and water for months. the stomach tissue was examined histopathologically. and also, iodine levels in stomach tissue was determined using the foss method. results: a decrease in iodine level was determined in stomach cancer tissue of rats in group i-v compared with normal healthy stomach tissue in group vi. when the control (group vi) iodine level was taken as % baseline, the % iodine levels of all groups were determined as follows . , . , . , . and . for groups i-v, respectively. the pathological diagnosis of gastric cancer was adenocarcinoma. discussion and conclusion: the iodine levels of group i were higher than those of group ii (p < . ) and of groups iii, iv and v (p < . ) and also were lower than in the control group (p < . ). iodine deficiency as one of the risk factors of stomach cancer strongly supports the necessity for the application of effective iodine prophylaxis in the areas with iodine deficiency. iodine supplementation might be useful in stomach cancer therapy and therefore, further research is warranted. this study was supported by ataturk university (project number: / ). effect of water extract of turkish propolis on mitochondrial membrane potential in human laryngeal epidermoid carcinoma cell lines propolis is the generic name for the resinous substance collected by honeybees from the buds of various plant sources and it is used by bees to seal holes in their honeycombs, smooth out the internal walls, and protect the entrance of bee hive against intruders. the aim of this study is to investigate what kind of changes the turkish propolis cause on mitochondrial membrane potential (mmp) of human laryngeal epidermoid cell lines (hep- ), by considering its anticancer features. water extract of turkish propolis (wep, - mg/ml) and ethanolic extract of turkish propolis (eep, . - mg/ml) were prepared and incubated with hep- cell lines ( , , and h). mmp was investigated with a flourometric method by using dioc ( , -dihexyloxacarbocyanine iodide). the most significant mmp decrease was seen on rd hour. both wep and eep extracts at all concentrations decrease mmp according to that of control. the recent studies have shown that propolis extracts have induced apoptotic cell death by decreasing mitochondrial membrane potential in various cancer cells. it was concluded that both wep and eep decreased mitochondrial membrane potentials on hep- cell series according to control ( concentration) depending concentration and time. there are numerous transcription factors involved in the regulation of the inducible gene expression. thus, transcription of proinflammatory genes, steroid hormone receptors, etc. is controlled by the group of factors triggering gene expression which includes nf-kb. another group of factors is involved in the formation of the structure of the chromatin of the inducible genes regulatory regions, providing competence for gene expression. it is expected that this group of factors includes the proteins of nf (nuclear factor ) family. there are few data suggesting that the nf factors maintain potentially active state of the chromatin of the hormone-dependent gene promoter regions. these findings initiated studies of the correlation between presence of the nf transcription factors on the chromatin of a gene regulatory region and the functional state of the gene in vivo. as a model we chose the rat tryptophan dioxygenase (tdo) gene which is expressed tissue-specifically in the liver under control of glucocorticoid hormones. three constitutive dnase i-hypersensitive regions are identified in the regulatory region of this gene. to conduct the study we used rat liver and kidney. the basic methods were electrophoretic mobility shift assay (emsa), immunoblotting assay and chromatin immunoprecipitation combined with real-time pcr (chip-qpcr). using emsa we found that the proteins of nf family interact with the constitutive dnase i-hypersensitive regions in vitro. immunoblotting assay of the protein fraction from rat liver used in emsa experiments showed the presence of the nf -b isoform. chip-qpcr revealed statistically significant differences in the level of the factor nf enrichment of the tdo gene regulatory region between the rat liver and kidneys at p < . . these data suggest the involvement of the nf proteins in the formation of the chromatin structure of the rat tdo gene promoter region. reciprocal ( ; ) translocation and bcr-abl fusion protein that is responsible for developing leukemia are observed in more than % of chronic myeloid leukemia (cml) cases. epigallocathecin- -gallate (egcg) is a green-tea flavonoid and egcg is proposed as a natural anti-cancer agent. histone modifications which contain histone deacetylases (hdac) and histone acetyltransferases (hat) are parts of epigenetic regulations. hdacs play important roles in different human malignancies including leukemia via activation of abnormal signaling pathways. hdac inhibitors have become remarkable therapeutic molecules for malignancies. the aim of this study is to determine the expression changes of leukemia-related hdacs with the treatment of egcg in k- cells. the cytotoxic effect of egcg on k- cells was determined in time and dose dependent manner by wst- analysis. total rna was isolated from k- cells. reverse transcription procedure was performed for cdna synthesis and gene expressions were detected by rt-qpcr. the expression level of hdac , hdac , hdac gene that supports cell proliferation was down-regulated . , . , . folds in k- cells treated with ic dose of egcg, according to control, respectively. our current findings suggest that is a polyphenol egcg may be a hopeful agent in treatment of cml by hdac inhibitory effect. chronic lymphocytic leukemia (cll) is a disorder of morphologically mature but immunologically less mature lymphocytes and is manifested by progressive accumulation of these cells in the blood, bone marrow, and lymphatic tissues. carbonic anhydrase (ca) is a metalloenzyme which is widely distributed in the living world, and it is essential for the regulation of acid-base balance. anti-ca antibodies have been reported in many disorders, such as systemic lupus erythematosus, sj€ ogren's syndrome, rheumatoid arthritis, endometriosis, idiopathic chronic pancreatitis, type diabetes and graves' disease. the goal of this study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in cll. patients with cll and healthy controls were included in the study and ca i and ii autoantibody levels were investigated by elisa. the ca i autoantibody levels of cll group were significantly higher than the healthy group (p = . ) while there was no statistical difference between serum ca ii autoantibody levels of the groups (p = . ). we found a significant positive correlation between hemoglobin and hemotocrit levels in patients with cll (r = . , p = . ). cut-off value of . absu for anti-ca i was associated with % sensitivity and % specificity and a cut-off value of . absu for anti-ca ii was associated with % sensitivity and % specificity for predicting cll. the ca i autoantibody levels in patients with cll were found higher compared to control group and the results suggest that ca i autoantibody may be involved in the pathogenesis of cll. genetic and epigenetic aberrations can lead to the activation of oncogenes and inactivation of tumor-suppressor genes (tsgs) followed by the development of malignant tumors. in the present work we evaluated the frequency of alterations of cpg island methylation and dna copy number in paired (tumor/normal) breast cancer (bc) samples using comparative dna hybridization on noti-microarrays and original niman software. the microarrays contained noti-clones associated with chromosome genes. expression alterations were assessed with the use of qpcr technique, ddct method and original atg software. in total, noti-sites with high ( - % of cases) hypermethylation/deletion (hm/d) frequency were revealed in bc. among genes associated with these sites, there are both known tsgs and tsg-candidates (aldh l , vhl, ctdspl, etc.) as well as genes, which involvement in breast oncogenesis was shown for the first time (lrrn , foxp , prickle , etc.). noti-microarray data were verified selectively using bisulfite sequencing for vhl, nkiras , itga , lrrc b, and ctdspl genes. several genes with high hm/d frequency (aldh l , ephb , itga , and ropn ) were tested for expression alterations using qpcr. frequent ( - % of cases) and significant (> -fold) down-regulation was shown for all of them in bc. the most significant expression loss was observed for aldh l geneon the average -fold mrna level decrease in % of samples. the involvement of the majority of genes with high hm/d frequency in breast oncogenesis was shown for the first time. these genes are novel tsg-candidates in bc. functional hypermethylation associated with expression loss was shown for aldh l , ephb , itga , and ropn genes thereby strengthening the speculation on tumor suppressor abilities of these genes. methylation and expression analyses of genes, that were revealed by noti-microarrays, were financially supported by grant - - from the russian science foundation. functional hypermethylation of a number of chromosome genes was revealed in colon cancer using noti-microarrays cancer is a disease of genome caused by genetic and epigenetic aberrations. noti-microarrays, that were developed by prof. e.r. zabarovsky, is a unique tool that allows us to simultaneously detect hypermethylation of cpg islands and dna deletionstwo major reasons of inactivation of tumor suppressor genes (tsgs). in the present work, the frequency of chromosome genetic and epigenetic alterations in colon cancer (cc) was evaluated. noti-microarrays, that contained noti-clones associated with chromosome genes, were used for comparative (tumor/normal) hybridization of dna from paired cc samples. data analysis was performed using original niman software. expression alterations were evaluated using qpcr technique and original atg software. in total, noti-sites with % and above hypermethylation/ deletion (hm/d) frequency were revealed in cc. among genes associated with these sites, there are several known tsgs and tsg-candidates (for example, vhl, ctdspl, and itga ), but for the majority of genes, involvement in colon oncogenesis was shown for the first time (for example, lrrn , nbeal , and ube e ). the highest hm/d frequency was observed for ankrd , nkiras /rpl , itga , cmtm , and gor-asp /ttc a genes - - %. expression alterations were evaluated for genes with high hm/d frequency (plcl , prickle , and ppp r a) and significant mrna level decrease (> -fold) associated with hypermethylation was shown for all of them in the majority of samples. a number of novel potential tsg-candidates was revealed in cc. functional hypermethylation associated with expression decrease was shown for plcl , prickle , and ppp r a genes thereby enhancing the suggestion on tumor suppressor function of these genes. this work was financially supported by in many countries, radon is the second leading cause of lung cancer, which accounts from % to % of cases. it is obvious that the population of all the developed and industrial countries in the world spend most f their time, almost %. therefore it is necessary to explore the obtained radiation dose, because of the presence of radon in a room due to the radon emanation from the soil and exhalation from a variety of building materials. the developed countries solve this problem of radon pollution as well as create a special monitoring services. the paper presents some data of genes molecular-genetic analysis from patients with lung cancer who live in almaty located in a foothill area of tectonic faults. the object of research were blood samples obtained from patients diagnosed with lung cancer who are receiving a treatment at the almaty oncology center and living in the city of almaty, where the level of radon activity exceeds the norm approved by the international commission on radiation safety. as a control group relatively people living in the plains, characterized by a lower radon emanation have been considered. to determine mutations in the genes polymerase chain reaction with a subsequent analysis of restriction fragment length polymorphism has been conducted. the pcr products were subjected to hydrolysis by bstni restriction endonucleases haeiii, ras i. disturbances in the genes under consideration to variour types of cancer development. the analysis showed that examinees do not have mutations in the kras gene codons - , which corresponds to a control group consisting of people living in the city of balkhash. on the whole, molecular genetic studies have shown that examined patients do not have mutations in the kras gene. one mutation was been found in the egfr gene. aim: polyps are abnormal growths of tissue that can be found in gastro intestinal system. they are most often found in the colon and rectum. most polyps are noncancerous (benign) however, because of abnormal cell growth, they can eventually become cancerous. the aim of this study is to determine the concentrations of trace element contents in colon and rectum polyp tissues and whether there is any relationship between polyp tissue element levels and the disease. material and method: the present study was conducted on total of individuals including patients and healthy subjects. while receiving normal intestinal tissue from healthy control group; from the patient group both normal tissue and polyp samples were taken during colonoscopy procedure. the concentrations of the elements (al, cr, mn, fe, co, ni, cu, zn, as, se, ag, cd, hg and pb) were determined with induced coupled plasma-mass spectrometer. results: the mean concentrations of cr, mn, ni, se and ag in colorectal polyp tissues of patients were significantly higher than in colorectal tissues of control subjects (p is less than . ). on the other hand the mean concentration of cd and pb in colorectal polyp tissues of patients were significantly lower than in control colorectal tissues of control subjects (p is less than . ). there was no any significant difference between the groups in terms of concentrations of al, fe, co, zn, as and hg (p is more than . ). conclusion: the differences found in some elements between polyps and a control tissues may provide an indication about the role of trace elements in the early stage (polyps) in the colon carcinogenic process and encourages further studies to confirm the involvement of such elements in neoplastic processes. the use of herbal medicines is steadily growing, with approximately % of the population use herbs to treat various illnesses in the western world. vitex agnus-castus has been used since ancient times as a remedy. the aim of this study was to investigate the in vitro anticancer activities of vitexagnus-castus oil. for this purpose, the cytotoxicity of vitexagnus-castus oil in sh-sy y cells was investigated by crystal violet staining. ec was found to be . %(w/w) vitexagnus-castus oil for this cell line. this dose was applied to the cell for h, and the cells were harvested for further studies. vitex agnus-castus oil treatment increased bax and p mrna levels. on the other hand, bcl- , bcl l , erk- , jnk, caspase and mrna expression levels were reduced significantly withvitexagnus-castus oil treatment while p and pten remained unchanged. these results indicate that another effector caspase such as caspase or may be involved apoptosis process which remains to be elucidated. moreover, mapk pathways, p and erk, may be involved in vitexagnus-castus oil induced apoptosis in sh-sy y cells. these initial observations suggest that this agent might not be useful in treating cancers. further detailed studies should be carried out to elucidate the exact mechanism of vitexagnus-castus oil in neuroblastoma cell lines. melanoma is a skin cancer with a melanocyte origin that can occur in any part of the body that contain melanocytes. while melanoma is less common than other skin cancers, it causes the majority of deaths related to skin cancer. several gene expression databases have shown that interferon regulatory factor (irf ) is upregulated in melanomas, and genome wide association studies linked variation at irf locus with skin cancers. irf was first identified to have roles in lymphocyte development and function. studies have identified a 'non-oncogene addiction' of malignant cells to irf in various hematopoietic cancer types. the aim of this study is to investigate the role of irf in melanoma cell lines. lentiviral vectors were used to reduce irf levels in melanoma cell lines. a gfp competition assay was performed to study the competitive fitness of melanoma cells with irf knockdown (gfp positive cells) over melanoma cells with normal irf levels (gfp negative cells). cell cycle profiles were investigated in melanoma cells with irf knockdown by propidium iodide staining. migration potential was assessed as well by wound healing assay. our preliminary data showed a decreased competitive fitness for cells with decreased irf levels. cell cycle profiling showed increased g /g and decreased g /m levels in irf knockdown cells compared to controls. wound healing assay results showed no difference between controls for cells with reduced irf levels. taken together, these results indicate that irf knockdown affects the melanoma cell lines' survival and cell cycle profile, suggesting a non-oncogene addiction of melanomas to irf . these observations are largely similar to previous observations in hematopoietic cancers. unravelling the role of irf in melanoma will increase our knowledge about melanoma development and progression and thereby may lead to targeted therapy in melanoma treatment. humans are exposed to various chemicals having beneficial or toxic effects at a time in their daily lives. , -dimethylbenz[a] anthracene (dmba) is a carcinogenic compound produced during the incomplete combustion of carbon-containing compounds. endosulfan is an organochlorine pesticide used against insects on food. morin is an antioxidant, antiinflammatory and chemoprotective flavonoid. this study is aimed to determine the effect of morin in the presence of dmba and endosulfan. for this purpose, adult wistar male rats weighing - g were randomly selected and divided into eight groups. mg/kg body weight (b.wt.) morin and . mg/kg b.wt. endosulfan were given to morin and endosulfan treated groups three times in a week. the rats in dmba treated groups were gavaged with . mg/kg b.wt. dmba three times during the administration period ( days). cytochrome p a (cyp a) associated -ethoxyresorufin o-deethylase (erod) and glutathione s-transferase (gst) activities were measured in rat liver cytosols and microsomes. in addition, liver tissues were evaluated by histopathological analysis. erod activities of control, morin, endosulfan, dmba, morin+endosulfan, morin+dmba, dmba+endosulfan and morin+dmba+endosulfan groups were ae , ae , ae , ae , ae , ae , ae and ae pmol/min/mg protein, respectively. all treatments increased erod activities. gst activities of these groups were ae , ae , ae , ae , ae , ae , ae and ae nmol/min/mg protein, respectively. histopathological studies showed that endosulfan and dmba induced inflammation in the liver tissues and morin reduced their effects. in conclusion, morin treatment increased the metabolism of dmba and endosulfan by inducing cyp a activity. gst activities of morin+dmba+endosulfan group were not significantly different from those of dmba group. histopathological studies indicated that morin administration reduced the toxic effect of endosulfan and dmba in the liver cells. hepatocellular carcinoma (hcc) is the sixth most common cancer and third most frequent cause of cancer-related death worldwide. molecular mechanisms of hepatocarcinogenesis is still unclear. the impairment of epigenetic mechanisms is implicated in the development of multiple cancers, including hcc. transforming growth factor-beta has been shown to play both tumorsuppressive and tumor promoting roles. transforming growth factor-beta signaling pathway involves activation of smad and smad by the type i receptor and formation of smad / / heteromeric complexes that enter the nucleus to regulate transcription. -deazaneplanocin a is an inhibitor of the histone methyltransferase ezh . we aimed to reveal the effect of -deazaneplanocin a on transforming growth factor-beta /smad pathway in hepg cell line. hepg , a human liver cancer cell line cultured in dulbecco's minimal essential medium supplemented with % fbs. the cells were seeded the day before -deazaneplanocin a administration and then the cells were treated with lm -deazaneplanocin a for days. expression levels of genes were analyzed by roche lightcyclerÒ . gapdh was used as housekeeping gene. apoptosis assay was performed by the muse annexin v and dead cell assay kit. the unpaired t-test was used to compare variables and p < . was accepted as statistically significant. -deazaneplanocin treatment was significantly reduced transforming growth factor-beta, smads - in hepg cells (p < . ). we also found that -deazaneplanocin induces apoptosis in treated cell line (p < . ). as a result, -deazaneplanocin a may take place in treatment of hepatocellular cancer by its inhibitory effect on transforming growth factor-beta /smad pathway and inducing apoptosis in liver cancer cells. brefeldin a (bfa) is a lactone antibiotic first isolated from the fungus eupenicillium brefeldianium. bfa inhibits the transport of secreted proteins from endoplasmic reticulum (er) to golgi apparatus, leading to disruption of golgi function, accumulation of unfolded and not fully incompletely processed proteins in er. bfa also inhibits cell proliferation, phosphorylation and migration of cancer cells. therefore in this study, we investigated the effects of bfa on breast cancer cell proliferation of various phenotypes. in we observed that bfa inhibited the proliferation of all three phenotypes of breast cancer cells, but the effects of bfa were seen at different times and doses. according to time and dose, bfa was observed more effective to mcf- compared to other cell lines. physiological, pathological and physical factors. moreover, nlr may represent the two opposing inflammatory and immune pathways that exist together in cancer patients. we aimed to investigate nlr in breast cancer in our population. methods: using data retrieved from the medical records, women diagnosed primary breast cancer met our study inclusion criteria as they had a complete blood count with leukocyte differential performed before any anti-cancer therapy. and women with benign mammary neoplasm/disease, followed up in the outpatient clinics of mammary disease and confirmed with sonographical/histopathological examination, made up our controls. exclusion criteria included laboratory evidence of white blood cells count (wbc) > . /l. differential leukocyte counts were obtained by bc (mindray medical international ltd., china), we examined wbc, neutrophil, lymphocyte, platelet counts, and hematocrite, nlr, mean platelet volume values. results: although there is lack of evaluation of tumor-associated neutrophils and lymphocytes, higher nlr median values and lower lymphocyte mean counts (lymphopenia) were shown in women with breast cancer (p < . ). there was a weak negative correlation in breast cancer between nlr values and platelet counts (r s = À . ; p = . ). holmboe] is distributed throughout southern mediterranean europe from spain to the eastern mediterranean on anatolian peninsula of turkey. present study was designed to investigate the in vitro anti-cancer activities of turkish black pine essential oil. the essential oil was extracted by steam-hydrodistillation and its chemical composition analyzed by gc-ms. the major components of the essential oil were a-pinene, b-pinene and trans-b-caryophyllene, respectively. the crystal violet staining method was used to investigate the cytotoxicity of essential oil in sh-sy y cells. ec was found to be . % (w/w) essential oil for sh-sy y cells. neuroblastoma cells were incubated at °c for h. after h, cells were harvested for further studies. bax and p mrna levels were significantly elevated in essential oiltreated cells. on the other hand, bcl- , bcl l , casp- , casp- , erk- and jnk expression were significantly downregulated. unlike these proteins, p and pten mrnas were not changed. in this study, apoptosis was enhanced by turkish black pine essential oil treatment which was activated by the involvement of another effector caspase subfamily, like casp- and casp- . additionally, erk and p mapks may be associated with upregulation of the level of bax. based on these results, we suggest that p. nigra subsp. pallasiana essential oil might not be well-suited in cancer treatment. however, further detailed research is necessary to establish the exact role of p. nigra subsp. pallasiana essential oil in sh-sy y cells. p- . . - the protective effect of newly derivatized compound naringenin-oxime and relative to naringenin against cisplatin-induced nephrotoxicity and genotoxicity in rat background: the aim of this study was to evaluate the possible protective effect potentials of newly derivatized compound naringenin-oxime (ng-ox) relative to efficacy of free naringenin (ng) on cisplatin (cis) induced nephrotoxicity and genotoxiticity in rat. methods: totally, fifty six male wistar albino rats were equally divided into eight groups as follows: control; cis treatment ( mg/kg b.w., i.p.), ng and ng-ox ( mg/kg b.w., i.p daily for days) alone treatment; cis + ng ( or mg/kg b.w., i.p daily for days) and cis+ng-ox ( or mg/kg b.w., i.p daily for days) combination treatment. at the end of the study total antioxidant capacity (tac) levels, total oxidant status (tos), lipid peroxidation (lpo), total thiol, catalase (cat) were studied in homogenate kidney. peripheral lymphocyte cell dna damage was investigated with comet assay results: the results suggest that cis induces oxidative stress resulting in increased tos and lpo reduction thiol, tac and cat in kidney and increased peripheral lymphocyte cell dna damage. the treatment with naringenin and naringenin oxime alone or with cis treatment showed a protective effect against the toxic influence of cp on peroxidation of the membrane lipids and an altering of the total thiol status in the kidney of rats. from our results we conclude that naringenin and naringenin oxime functions as a potent antioxidant and suggest that it can control cp-induced nephrotoxicity and genototoxicity and ng-ox was found more protective than that of ng on cisplatin induced toxicity in rats. keywords: naringenin, naringenin-oxime, antinephrotoxic, antigenotoxic, comet assay. introduction: oxidative damage is considered to play a pivotal role in ageing, several degenerative diseases, and carcinogenesis. lung cancer is the most common type of cancer, resulting in over . million deaths each year worldwide. accurate and reliable determination of superoxide radicals has been widely investigated using spectrophotometric, electrochemical, amperometric, polarimetric, piezoelectric technologies. among these methods, electrochemical detection is a most promising approach to achieve accurate, separate and rapid superoxide radicals monitoring with using biosensor system. materials and methods: we used a new technic for detecting superoxide radicals in samples. superoxide dismutase (sod) enzyme immobilized on the surface of gold electrode with the help of gelatin, bovin serum albumin (bsa) and glutaraldehyde (ga) crosslinker. for the biosensor preparing benzoquinone selected as a mediator in working buffer and measurements were carried out at À . v. result: for the optimization studies, effect of the bsa, gelatin, glutaraldehyde, ph, buffer concentration on biosensor response. characterization of the biosensor commitment to the work process and answer reproducibility were evaluated. the analytical characteristic of the biosensor were evaluated by measuring the steady state current response to superoxide radical concentrations. the electrochemical response of the enzyme electrode was linearity gradually leveled of at higher concentration. we found that crosslinking of the sod (e.c. . . . ) with glutaraldehyde could be achieved over a wide range of relative mole ratios in mm phosphate buffer at ph . , glutaraldehyde concentration of % . . discuss and conclusion: in this study, a new technique for developed sod biosensors has been developed, which features effective combination of sod/gelatin/bsa/ga modified electrode, trapping of sod and glutaraldehyde cross-linking. this technique is reliable and cost effective. the effect of astaxanthin on apoptosis and cell arrest in u brain cancer cell line f. s€ og€ utl€ u, b. € ozmen yelken, c ß . kayabasi, a. asik, s. gonca, r. gasimli, s. yilmaz s€ usl€ uer, c ß . biray avci, c. g€ und€ uz department of medical biology, izmir, turkey a brain tumor is a collection, or mass, of abnormal cells in your brain. brain tumors can be cancerous (malignant) or non-cancerous (benign). the brain is one of the least accessible organ that active pharmacological compounds cannot be delivered. the two physiological barriers control and block the entry and exit of endogenous, exogenous compounds. one of these is the bloodbrain barrier and the other is the blood-cerebrospinal fluid barrier. this structures maintain protection of the brain. when there is a cancer case, it can lead to problem. astaxanthin with potent antioxidant properties can cross blood-brain barrier. in our study, we aimed to evaluate the effects of astaxanthin on apoptosis, cell cycle and also migration in brain cancer cell line. in present study, xcelligence real-time cell analyzer was used so as to determine cytotoxic effect of astaxanthin in u cell line. changes of apoptosis and cell cycle in u cell line exposured to ic dose of astaxanthin ( . nm- lm) are detected with annexin v-egfp apoptosis detection kit and cycle test plus dna reagent kit with facs, respectively. the result of apoptosis and cell cycle test was analysed in flow cytometry. the group to which active substance was not treated was used as controlled. the wound healing assay performed in order to measure migration ability of u cell line to which astaxanthin was treated or not. ic dose of astaxanthin was calculated as . lm at h by xcelligence rtca sp based on time and dose. astaxanthin decreased the migration ability at rate of % in u cells treated by ic dose of astaxanthin. astaxanthin had no apoptotic effect on viability in u cell line and astaxanthin caused an increase of g /m phase arrest ( . fold) and s phase arrest ( . fold). astaxanthin has cytotoxic effects in brain cancer. it determined that astaxantin decreases cell cycle potential at g /m even a little. the effect of anticancer of astaxanthin should be researched further. interferon regulatory factor (irf ) is a critical transcription factor in development and survival of different cell types including immune cells and melanocytes. furthermore, it has been demonstrated that irf expression levels are elevated in several lymphoid cancers, and irf is one of the key transcription factors for the survival of these cancers. several genome-wide association studies identified irf -linked genetic variants to increased melanoma incidence. in addition results from our lab and elsewhere have shown high levels of irf expression in melanoma cell lines. furthermore our preliminary results suggest melanoma cells are sensitive to irf expression levels. however, there are no published studies about irf target genes in melanoma cells. in this study, we are investigating the genome-wide target genes of irf in melanoma cell lines via high-throughput sequencing of immunoprecipitated chromatin (chip-seq). we have identified possible irf binding regions in loci with known key roles in development of melanocytes from neural-crest cells. one such key factor is mitf, which is the master regulator in melanocyte development and also plays critical roles in melanoma. integrating chip-seq and rna-seq data suggests irf as a transcriptional regulator of genes related to progression of melanoma. objectives: aim of this study was to evaluate prognostic importance of selected laboratory parameters (c-reactive protein (crp), gama glutamiltransferaz, ferritin (fer), potassium, chloride, calcium, phosphorus, magnesium, total protein, aspartat aminotransferaz, alanin aminotransferaz (alt), ifn-c, il- , tnf-a) in non-small cell lung cancer (nsclc). material and methods: patients with nsclc who were treated with chemoradiotherapy (crt) prospectively evaluated. all patients were newly diagnosed tumour. heparinized blood samples were taken from the patients before and after the completion of crt. fer analyzed by chemiluminescence method on beckman coulter dxi ; ifn-c, il- , tnf-a were analyzed with elisa kits (boster biological technology) and other biochemical parameters analyzed on abbott architect c . post-crt and pre-crt levels compared with survival. results: the lr cox regression analysis revealed that pre-crt ferritin was significantly associated with survival of patients with nsclc (hazard ratio (hr) = . , p = . , %ci; . - . ). it was also demonstrated by lr cox regression analysis, high levels of pre-crt crp was associated with worse outcome of patients (hr = . , %ci; . - . , p = . ). after crt, mean alt level was determined as . . there was survival difference in nsclc patients with high post-crt alt (hr = . , %ci; . - . , p = . ). conclusions: there exists a clinically relevant relationship between pre-crt fer concentration and the prognosis of survival in patients with nsclc. elevated fer is the result of inflammation rather than body iron overload. ferritin showed negative correlation with survival so it could be a useful biomarker to indicate bad prognosis of the patients with nsclc. additionally, crp which is easy to detect and feasible for the use in the routine clinical practice should be considered in the prognosis of nsclc patients. keywords: ferritin, nonsmall cell lung cancer, survival, c-reactive protein. epigenetic therapy tries to reverse the aberrations followed to the disruption of the balance of the epigenetic signaling ways through the use of natural and synthetic compounds, active on specific targets, such as dna methyltransferases (dnmts). we previously synthesized some benzoxazole and benzamide derivatives which might have anticancer activities on account of their heterocyclic structure. our studies showed that not only these compounds caused selective cytotoxicity towards cancer cells (hela) with little or no toxicity on normal cells (l ) but also were not genotoxic. in this study, we aimed to test whether these compounds changed global demethylation profile of normal and cancer cells. we used methylation specific comet assay (msc assay) to determine global methylation levels of cells. cells were treated with the tested compounds at ic concentrations for h. slides were prepared as did in alkaline comet assay, then they were incubated with methylation specific restriction enzymes (mspi, hpaii) before electrophoresis. differences in global methylation levels between nontreated control cells and cells treated with compounds were compared by using tail moment data. -aza-c, a demethylating agent, was used as reference drug. msc assay results revealed that none of the tested compounds caused hypermethylation on both cell lines. however, global methylation levels decreased statistically (p < . ) through both cells treated with c- and c- . only c- decreased methylation level on l but not on hela. consequently, c- and c- caused demethylation on hela cells similarly with -aza-c at low concentrations. for the reason that dna methylation is regulated mainly dnmt enzymes in the cell, c- and c- might cause global demethylation in the cell by inhibiting dnmt activity. further studies will be done to support this prediction. overall, macrophages and some subtypes of lymphoid cells are found in tumour stroma. these cells secrete a variety of growth factors, proinflammatory cytokines and chemokines, esp. tnf-a, il- b and il- , causing the formation of inflammatory microenvironment around tumour cells. tnf-a and il- b signaling increases activity of nf-kb pathway. at the same time, il- , triggers jak-stat signaling pathway, which effector is stat . nf-kb and stat activity facilitates hyperexpression of mir-nas mir- , mir- and mir- as well as down-regulates expression of mirnas mir- / , mir- and let- . this investigation aims to identify in what way these shifts in mirnaome can lead to epigenome reorganization supporting the cell transformation. mirna targets within gene transcripts were predicted in silico using targetscan software. transcripts of hdac / / / and sirt / genes encoding histone deacetylases carry targets for at least one of up-regulated mirnas mir- , mir- or mir- . also, these mirnas can silence ezh , mll, mll , nsd , setd / / , smyd , suv h genes encoding histone methyltransferases. mirna mir- suppresses gene encoding de novo dna methyltransferase dnmt b. at the same time, down-regulation of mirna mir- / can allow hyperexpression of gene encoding acetyltransferase elp . these shifts impair dna and histone methylation, cause the increase of overall level of chromatin acetylation and expression and, therefore, create epigenetic background for reactivation of silent transposons, oncogenes as well as other genes important for cell transformation. immune system can paradoxically facilitate the tumour growth instead of healing. cancer-related inflammation leads to the mir-naome and epigenome shifts contributing to the tumour promotion and progression. lysine acetylation is one of the key mechanisms to regulate chromatin structure and transcriptional activation. acetyl-lysine modifications are recognized by bromodomains, which are small interaction modules found on diverse proteins including histones. among these acetyl-lysine reader proteins is the family of the bet (bromodomain and extra-terminal) proteins which contain tandem bromodomains (bd and bd ). the recent discovery of potent and specific inhibitors for the bet family proteins has stimulated intensive research activity in diverse therapeutic areas, especially in oncology, where bet proteins regulate the expression of key oncogenes and anti-apoptootic proteins. several bet inhibitors are currently in clinical trials and reported to exhibit promising clinical activities. however, pleiotropic nature of bet proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. here, we report the recent progress in the development of bet inhibitors in korea research institute of chemical technology (krict). we have identified the bet inhibitors with a novel scaffold different from the previously reported diazepine and azepine scaffolds and specific for first bromodomains (bd s). a medicinal chemistry effort is currently made to optimize the pharmacokinetic properties of these lead compounds for further drug development. the experimental data from the biochemical and cell-based assays for these bd -selective bet inhibitors will be presented. family of small c-terminal domain serine phosphatases (scp), which includes ctdspl, ctdsp , and ctdsp , plays a regulatory role in a number of vital processes. in particular, it is shown that ctdspl is capable to activate the retinoblastoma protein (rb) which is well-known tumor suppressor and one of the key cell cycle regulators. although the question on whether ctdsp and ctdsp dephosphorylate rb is open, high similarity of sequences and three-dimensional structures of phosphatases may indicate the similar function of these enzymes. in the current study expression of scp genes was evaluated by quantitative pcr in non-small cell lung cancer (nsclc) samples. using original crosshub software, that combines an analysis of high-throughput sequencing data of the cancer genome atlas project (tcga) and databases of mrna-mirna interactions (targetscan, mirtarbase, etc), the involvement of mir- - - microrna cluster in co-regulation of ctdspl/ / genes in nsclc was predicted. the significant ( -fold on the average) and simultaneous decrease of mrna levels of ctdspl/ / genes was revealed in the majority of nsclc samples ( %, / ). such unidirectional expression change and strong positive correlation between phosphatase expression levels (r s = . - . , p ≤ . ) allowed us to suggest a common mechanism of their inactivation. we evaluated the expression of predicted co-regulators of scp gene expression, mir- - - family, in examined nsclc samples. as a result, the simultaneously increased levels of all three mir-nas in most nsclc samples ( %, / ) and negative correlation with phosphatase gene expression was shown. the results suggest the ability of investigated phosphatases to exhibit tumor-suppressive activity and the involvement of mir- - - micrornas in the regulation of rb protein activity via inactivation of ctdspl/ / in nsclc. cancer is one of the leading causes of death in all around the world. cancer is defined as a disease involving abnormal cell growth with the potential to invade or spread to other parts of the body. tumor markers are substances that are produced either directly by the tumor or as an effect of the tumor on healthy tissue. tumor markers can be used for screening, determining prognosis and monitoring effectiveness of therapy and disease recurrence. the aim of this study is to investigate the frequency of tumor markers orders and the appropriateness of these requests. laboratory information systems data for were reviewed. for , a total of patients and tumor marker requests were included. carbohydrate antigen - , cancer antigen , cancer antigen - , prostate specific antigen, alphafetoprotein and carcinoembryonic antigen were measured by chemiluminescence method. according to the data from the year of , both positive tumor markertest resultsratio and the positive patient ratio were %. in the patients group with increased marker levels, % of the patients had no history of cancer. in the patients group with tumor marker levels in referenceranges, % patients with diagnosed cancer history in remission. the ratios of positive tumor markers were % forca - , % for ca , . % for psa, %for ca - , . %for afp, and . % for cea. in conclusion; unnecessary test requests increase laboratory work load and health expenses. laboratory and clinical staff collaboration is crucial to increase the appropriate use of tumor markers. dna methylation is an epigenetic modification that is involved in both normal biological and disease states. hypermethylation of promoter regions of tumor suppressor genes have a role in tumor development. therefore, the measurement of promoter methylation of genes can be used for diagnosis and prognosis purposes of cancer. to detect dna methylation alterations in a sample (biopsy, blood, saliva, etc.), sensitive detection systems and optimization of the methods are needed. as a part of a collaboration project between national metrology institute of korea (kriss) and national metrology institute of turkiye (tubitak ume). dna methylation status of apc and gstp genes were studied. dna methylation measurements were performed using stepone real-time pcr system and results were analyzed using hrm (high resolution melting) software. the parameters effecting the quantification of dna methylation were found as primers, annealing temperature, pcr cycle number, fluorescence dye and the commercial dna methylation standards used for quantification of dna methylation. since, the accurate measurement of dna methylation is very critical in early diagnosis of cancer and choosing the right therapy, optimization of the method is required. cancer is a disease that includes heterogenic and complex molecular changes. anti-carcinogenic effects of resveratrol, a natural polyphenol, have been proved in a variety of cancer cells. considering the effects of resveratrol, the influence of the signal transduction pathways in the presence or absence of p of colon cancer cells is gaining importance. our aim was to investigate the effects of resveratrol in the presence or absence of p on cell viability, apoptotic cell death ratio and fold changes of proliferative or anti-proliferative gene expressions, which may have important effects on colon cancer, in hct colon carcinoma cells. ic doses of resveratrol were determined by wst- assay. the apoptotic cell death ratios in treatments of resveratrol were determined by annexin-v-fitc/pi assay for flow cytomety . the changes of ccnd , fra , ppard, egfr, birc , pcna, mcl , stat , fos, jun, p , atf , trail, puma, gadd a, rb , faslg, tnf, socs , stat gene expressions were evaluated by real time pcr. all data were statistically analyzed by student's t test. our research has revealed that resveratrol ( lm) causes decrease in cell viability and increase in apoptotic cell death in hct p (+/+) and hct p (À/À) cells significantly (p < . ). the fold changes of the gene expressions have shown that resveratrol has significant (p < . ) and different effects on the expressions of the genes related with the existence of p in hct cell lines. therefore we proposed that resveratrol might show proliferative or apoptotic effects related with p mutation of colon cancer cells and we predicted that unconscious consumption of resveratrol in colon cancer patient might cause adverse effects. introduction: colorectal cancer (crc) is the third most common cancer worldwide. alterations in methylation profiles of tumor suppressor genes (tsgs) have been recognized as a key mechanism in colorectal cancers. in the current study, we investigated the hypermethylation status of tsgs in colorectal cancer tissues. materials and methods: formalin-fixed paraffin-embedded (ffpe) tissue samples obtained from patients with crc. methylation specific-multiplex ligation dependent probe amplification (ms-mlpa) technique was used to assess the methylation status of tsgs. the findings were evaluated in terms of age, mortality, survival, positive lymph node status, lymphovascular invasion, and perineural invasion. results: hypermethylation-detected patients and hypermethylation-undetected patients were called as group and group , respectively. hypermethylation was detected in atm, cdkn a, and gata genes. mortality rate was ( . %) in group and group (p > . ). mean -years survival rate in group was ae months and mean -years survival in group was ae months (p > . ). positive median lymph node count was ae for group and ae for group and the difference was statistically significant (p < . ). frequencies of perineural invasion and lymphovascular invasion rate in two groups were % (p > . ). discussion and conclusion: our findings suggest that tsg hypermethylation found in crc patients may increase the lymph node metastasis. further investigations with larger sample size are required to support our results. boron (b) is known to be important for cell replication and development, but the underlying mechanism remains obscure. recently b has also become important in some specific anticancer processes. some recent reports advise using of some boron compounds for the treatment of specific forms of cancer. for instance, boron-based drugs (bortezomib) are now being developed for use as therapeutic agents with anticancer activities and several other boron-based compounds are in various phases of clinical trials. it has been shown that bortezomib disrupts the regulation of cell cycle and induces apoptosis in both hematologic and solid tumor malignancies except for colon carcinoma. colorectal cancer (crc) is the third most common cancer in men and the second in women, accounting for % of all tumour types worldwide. cytotoxic effects of boron compounds on crc cells and changing of its effects related with p mutation, which is mutated % of cancer cases, have not take part in literature yet. for this purpose; the aim of the study was designed to investigate the effects of borax pentahydrate and disodium pentaborate decahydrate compounds on cell viability, apoptotic cell death ratio and parp protein expressions in p (+/+) and p (À/À) hct colon carcinoma cells lines. the effects of the boron compounds on cell viability were assessed by xtt assay and apoptotic effects and parp protein expression of the compounds were evaluated by flow cytometry and western blot analysis respectively. our results showed that borax pentahydrate ( mm) and disodium pentaborate decahydrate ( mm) significantly causes nearly % reduction of cell viability at h (p < . ). apoptotic cell death ratios and parp expressions revealed that both of the compounds might have a potential for a candidate of anticancer agent. epithelial-mesenchymal transition (emt) is a significant event for metastasis, and could be mediated by several pathways such as pi k/akt, map kinases and many epigenetic regulators. satb is an epigenetic regulator involved in emt and osteoblastic differentiation. since preliminary results indicate that there is a crosstalk between p and akt pathways in nsclc cells, we aimed to determine whether this crosstalk has a regulatory effects on emt and satb expression in nsclc cells. we used a and h cells as a model to evaluate the effects of the crosstalk between p and akt on emt of nsclc cells. therefore, cell culture, inhibition of p activation via sb , transient expression assay for (ca-akt), western blot analysis, sirna transfection for satb , wound healing and invasion assay were performed in this study. firstly, the expression statues of e-cadherin, satb , p-p , p , p-akt and akt was examined in a and h cells by western blot analysis. we observed that e-cadherin and satb are downregulated in a cells (highly active p , lowly active akt) compared to h cells (lowly active p , highly active akt), suggesting that e-cadherin and satb are associated with the crosstalk between p and akt pathways. our results demonstrated that p inhibition in a cells leads to decreased pten expression and subsequently increased akt activation. then, we found that p inhibition upregulated satb expression, and reversed emt in a cells. furthermore, alone satb knockdown is sufficient to induce emt, and prevented the effects of p inhibition on emt. all these results strongly indicate that the crosstalk between p and akt pathways might determine satb expression and epithelial characters of nsclc cells, and satb is a critical epigenetic regulator for emt in nsclc cells. therefore, it is also need to explore how p and akt signalling pathways could regulate satb expression. this work was supported by tubitak ( s , z ). introduction: lung cancer is a disease characterized by uncontrolled cell growth in the lung tissues. the most common causes of lung cancer are tobacco smoke, radon gas, asbestos, air pollution, and genetic factors. nitric oxide (no) has potential mutagenic and carcinogenic activity and may play important roles in lung cancer. endothelial no, synthesized from l-arginine by endothelial no synthase (enos), inhibits apoptosis and promotes angiogenesis and tumor cell proliferation. the aim of the present study was to examine the possible relationship between enos gene intron vntr and exon -g t (glu asp) the stressful ecosystems exert strong adaptive pressure and proteins that facilitate these adaptation processes are candidate drug targets. nucleotides are the core of biochemical pathway required for cancer cell growth and replication and genetic changes will lead in oscillation in their pools. although it is questionable whether the warburg effect actually causes cancer, impairing dglucose uptake and metabolism induces oxidative metabolism. lproline (lproline) homeostasis is critical in a constellation of human diseases, in parametabolic linkage between cancer, epigenetics (phang et al. ) and bioenergetics (pallotta ) where degradation and biosynthesis are robustly affected by oncogenes or suppressor genes that can modulate intermediates involved in epigenetic regulation. lproline-fueled mitochondrial metabolism involves the oxidative conversion to l-glutamate by a flavin dependent lproline dehydrogenase/oxidase and a nad +dependent l-d -pyrroline- -carboxylate dehydrogenase. in saccharomyces cerevisiae an important test tube, put p and put p respectively help cells to respond to changes in the nutritional microenvironment by initiating lproline breakdown after mitochondrial uptake (pallotta ) . in this preclinical study, low molecular weight compounds were tested for inhibiting lproline mitochondrial transport and put p/put p catalytic activities. thus, in seeking for natural bioactive compounds targeting lproline pathway and its substrate channeling (becker's group ), we report data using in silico screening and in vitro researches in saccharomyces cerevisiae with genetic background atcc but different phenotypic landscape induced by nutritional stress/ ph changes. cells vitality, dΨ measurements, nad(p) + /nad(p) h pool and flavine turnover were determined in spectrofluorimeter microplater reader and via hplc (pallotta et al. (pallotta et al. , (pallotta et al. , pallotta ; di martino pallotta ) thus in supporting of future cancer therapies with decreasing side effects. evaluation of lymphocyte to monocyte ratio (lmr) in patients with colorectal cancer introduction: inflammation may play an important role in cancer progression and a high neutrophil to lymphocyte ratio (nlr) has been reported to be a poor prognostic indicator in several malignancies. the aim of this retrospective study was to evaluate the prognostic value of nlr, lymphocyte to monocyte ratio (lmr) and platelet to lymphocyte ratio (plr) in patients with colorectal cancer (crc). : patients who were diagnosed with colorectal cancer between january and january ; were evaluated retrospectively. the cutoff value was determined using receiver operating characteristics curve analysis. survival analysis was performed using the kaplan-meier method and log-rank test. the cox proportional hazard model was used to identify the influence of factors related to survival. (tnm stage, tumor differentiation, age, tumour size and lmr) results: receiver operating characteristic curves showed that lmr was superior to plr and nlr as a predictive factor in patients with colorectal cancer. the cutoff value for lmr was . . cancer-specific survival was not significantly different between the high-and low-lmr groups (p = . ). age was identified as independent prognostic factor in colorectal cancer (hazard ratio: . ; % confidence interval: . - . ; p = . ). discussion and conclusion: our preliminary study showed that the lmr was not an independent prognostic factor in crc patients, but additional large sample sized prospective studies will be needed to confirm these findings. the aim of this study is to investigate the effects of luteolin treatment on enzymatic activity of arginase, and ornithine and polyamine levels (putrescine, spermidine spermine) in serum and cancer tissues of ehrlich ascites breast cancer model. balb/c female mice were divided randomly into following groups: healthy control, healthy treatment, cancer control, treatment and treatment . . ml ehrlich ascites tumor cells was inoculated subcutaneously to medial part of left hind leg. healthy treatment and treatment groups, and the treatment group were given mg/kg and mg/kg dose of luteolin, intraperitoneally, for a days period, respectively. luteolin has a hydroxylated flavonoid structure and shows potent antioxidant, anti-inflammatory, and anticarcinogenic properties. luteolin not only leads to cell death in various tumors by suppressing cell survival pathways and stimulating apoptotic pathways, but also sensitize them to cytotoxic therapy. supporting various previous studies, tumor implantation to healthy mice resulted in statistically significant elevation of serum arginase and polyamine levels (p < . ) indicating the tumor cells as the main source of this production. furthermore, luteolin treatment abolished this increase in serum arginase and polyamine levels (p < . ). tissue measurements of arginase and polyamine levels indicated that luteolin treatment resulted with an increase in these parameters of tumor tissue while the serum levels of them showed a significant decrease. our results revealed that increased tissue arginase and polyamine levels might be related with estrogenic agonistic effect of luteolin on utilized tumor model in this experiment; and decreased serum levels of these parameters while there is a significant increase of them in tissue levels might be a result of a suppression of polyamine efflux from the tumor tissue by inhibitory effect of luteolin on plasma membrane polyamine transporters. hepatocellular carcinoma (hcc) is the third most common cause of cancer-related deaths. around - % of hcc patients are diagnosed at an early stage of the disease. hepatic resection, liver transplantation are common strategies in hcc treatment. even if, most of the patients present advanced-stage tumors and have a restricted survival rate. for the reason, resistance against existing tumor stress conditions have been demonstrated in hcc. hypoxia, hyperglycemia are general stress sources in hcc and result in aggressive cell phenotype, resistance to apoptosis and therapeutic drugs. thioredoxin interacting protein (txnip) regulates cellular responses under stress conditions. over-expression of txnip results activation of oxidative stress and apoptosis. in cancer models txnip is considered as a tumor suppressor gene. however, its role in the development, progression of hcc and mechanisms behind it warrant further investigation. in this study expression levels of txnip were examined in hcc cell lines by rt-pcr and western blotting. txnip expression was significantly high in poorly-differentiated snu- , snu- and snu- than the well-differentiated hcc cell lines such as huh- , hepg and plc/prf/ . besides, expression of txnip was examined in non-hcc and hcc tissue samples by immunohistochemical staining. txnip positivity was observed in % of well and % of poor differantiated hcc tissues. however, no txnip positivity was observed in non-hcc tissues. to investigate whether txnip might be involved in biological responses such as cell proliferation, motility and invasion, we used overexpression and silencing strategies. overexpression of txnip minimally inhibited adhesion and proliferation, whereas boyden-chamber motility and invasion assay showed that invasiveness of cells were increased. our findings suggest that txnip expression is increased in hcc and txnip over-expression is important for invasive phenotype during hepatocarcinogenesis. cardiovascular diseases are the leading cause of morbidity and mortality in the western world. it was shown that ischemic tolerance of the heart can be enhanced not only by ischemic or pharmacological conditioning (pre-and postconditioning), but also by adaptation to chronic hypoxia. different studies have indicated that these cardioprotective phenomena may at least partly share the same signaling pathways. the jak/stat signaling pathway has been demonstrated to participate in the development of cardioprotection by conditiong apparently through the inhibition of gsk- b. the aim of our present study was to determine whether this pathway also takes part in cardioprotection induced by adaptation to chronic hypoxia. we investigated the effect of inhibitor of jak kinase (ag- ) on myocardial infarct size and the jak /stat signalling pathway and other effector molecules that may participate in cardioprotection conferred by adaptation to hypoxia. adult male rats were adapted to intermittent normobaric hypoxia ( % o , weeks, h/day) and part of them recieved ag- ( mg/kg) min before ischemia. control rats were kept under normoxia. infarct size was assessed in isolated perfused hearts. relative expression of the key components of the jak /stat signalling system and other proteins was detected using western blotting. preliminary data indicate that administration of the jak inhibitor ag- caused a significant increase in infarct size in hypoxic rats. western blot analysis revealed changes in phosphorylation of jak , stat and some other proteins involved in cardioprotection (akt, erk / , gsk b). these results suggest that the jak/stat signaling pathway could participate in the development of a cardioprotective phenotype in rats exposed to chronic hypoxia. however, further research will be needed to clarify in more detail the role of this signalling pathway in the cardioprotective mechanism. p- . . - detrimental effect of hypertension on myocardium was reversed by liver x receptor agonist gw hypertension is a cardiovascular disease that causes functional and structural changes in the heart. nuclear liver x receptors (lxrs) are involved in the control of cholesterol and lipid metabolism. however, effect of lxr activation on the hypertensive heart is not well characterized. in this study, the effects of lxr agonist gw on hypertension-induced damage of myocardium were investigated. hypertension was induced by deoxycorticosterone acetate (doca) injection ( mg/kg, twice a week) following the unilateral nephrectomy in male -week-old wistar albino rats for weeks. blood pressure was measured by using tail-cuff method. gw ( mg/kg/day, i.p.) was administered last week. expression of various markers (grp , perk, p-perk, ikb-a, nf-kb p , tnf-a, bax, bcl- , mmp- ) in the ventricular tissue were examined by western blotting. inflammation and fibrosis were evaluated in histopathological examination. gw treatment reduced systolic blood pressure of hypertensive animals. expressions of endoplasmic reticulum stress markers grp and p-perk were increased by hypertension and gw treatment reversed them. hypertension-induced increase in nuclear nf-kb p expression and decrease in ikb-a expression were reversed by gw treatment. while bcl- expression was lower, bax level was higher than control in the hypertensive animals. in hypertensive group, fibrosis marker mmp- expression was augmented and gw treatment reversed this elevation. hypertension-induced increase in interstitial and perivascular collagen deposition and inflammatory cell infiltration in left ventricle were prevented by gw treatment. these results suggest that lxr activation by gw restored the hypertension-induced structural changes of heart in the doca-salt hypertension. methylphenidate (mph) is a psychostimulant prescribed for the treatment of attention deficit hyperactivity disorder (adhd), one of the most common neurobehavioral disorders of childhood and adolescence. in fact, despite the widespread use of mph the full comprehension of its cellular/molecular mechanisms is still elusive, including its effect on blood-brain barrier (bbb). this barrier is a key structure in the central nervous system since it protects the brain and its dysfunction has been described as a critical event in several brain diseases. thus, the aim of the present study was to clarify the effects of mph on the bbb function in both physiological and adhd conditions. for that, we used a rat model of adhd, the spontaneously hypertensive (shr) rats, and wistar kyoto (wky) animals as inbred comparator strain. also, to mimic a clinical dosing schedule for adhd treatment, rats were administered for monday-friday with vehicle or mph ( . mg/kg/day or mg/ kg/day, per os) from p -p . chronic mph treatment ( mg/kg/day) promoted cortical bbb permeability in both wky and shr animals; however, more prominent in wky rats. this effect can be explained by the downregulation of claudin- and collagen-iv, tight junction and basal lamina protein, respectively. noteworthy, wky animals also showed an increase in the expression of caveolin- and in both vascular cell and intercelular adhesion molecules. these bbb alterations led to subsequent infiltration of peripheral immune cells, including cd + macrophages. furthermore, hippocampal bbb disruption was only observed in wky rats with mg/kg of mph. here, mph decreased collagen iv expression and upregulated caveolin- , with no alterations in claudin- . overall, our results show that chronic exposure to mph can led to brain vascular alterations particularly under physiological conditions. this highlights the importance of an appropriate mph dose regimen for adhd, and also that mph misuse can have a negative effect. regulators of g proteins signaling (rgs) serve several cellular functions varying from tolerance, dependence, neuroprotection, transcription and tumorgenesis. despite their initial role as gtpase activating proteins, evidence suggests that rgs proteins are localized in the nucleus, interact with transcription factors thus regulating transcriptional responses. it was shown that rgs directly interacts with and interferes in opioid receptor (or) signaling. rgs is mostly expressed in brain and is implicated with brain structural alterations; however, the molecular mechanisms of how rgs could be involved in cellular differential functions remains unclear. based on these observations we examined whether rgs can regulate transcriptional responses mediated by the stat b transcription factor. isolated neural stem cells from rgs À/À mice were immunostained for the mitosis marker ph and the mrna levels of antiapoptotic genes were determined. proliferation assays were performed with brdu staining in neuro a cells stably expressing rgs . the functional assays of stat b transcriptional activation were performed in hek expressing either the erythropoietin receptor (epo-r) or the delta opioid receptor (d-or). the present data demonstrate that rgs blocks stat b phosphorylation and transcriptional activation by interfering in stat b heterodimerization upon epo-r or d-or activation triggered by cytokines or opioids administration. lack of functional rgs results in increased mrna levels of stat b target genes such as the members of the bcl anti-apoptotic family bcl- , bcl- and bcl-xl. this upregulation of stat b inducible gene transcription results in an increased proliferation rate of neural stem cells. this study demonstrates for the first time a non-canonical function of rgs in stat b mediated transcriptional responses and a novel selective role of rgs in transcription. role of the pre-molten globule structure in amyloid fibril formation a. eshaghi department of biology, faculty of science, islamic azad university, mashhad branch, mashhad, iran the major factor that caused extensive research on the protein fibrillation is their crucial roles in important diseases known as the amyloidosis diseases. neurodegenerative diseases, including alzheimer's, parkinson's, diabetes and huntington are the most important types of this disease. understanding the mechanisms of fibril formation and ways of treatment can be useful in reducing this type of disorder. in this project, the fibrillation of carbonic anhydrase protein was investigated as a model. carbonic anhydrase creates two stable intermediated known as pre-molten and molten globule, in different ph solution. this protein at ph between ph - molten globule structures was formed while the pre-molten form took place under ph . in our tests at ph . when the protein in molten globule structures only the amorphous aggregates were formed. instead, at ph . in pre-molten globule structure amyloid fibrils formed in the protein. there some reports, indicated the protein from pre-molten globule structure go toward amyloid assembly. even intrinsically unstructured proteins such as alpha-synuclein first took a structure similar to pre-molten globule and then made amyloid fibrils. it seems pre-molten globule structure have the major role in promoting to amyloid fibrils. perhaps drugs that prevent the formation of premolten globule structure have an important role in inhibiting amyloid fibrils. identification of compounds preventing the biochemical changes that underlie the epileptogenesis process and understanding the mechanism of their action is of great importance. we have previously shown that myo-inositol (mi) daily treatment for days prevents certain biochemical changes that are triggered by kainic acid (ka)-induced status epilepticus (se), [ , ] . however in these studies we have not detected any effects of mi on the first day after se. in the presented study we broadened our research and focused on ka induced other early molecular and morphological changes and influence of mi treatment on these changes. the increase in the amount of voltage-dependent anionic channel- (vdac- ), mitochondrial-plasma membrane cofilin and caspase- activity was observed in the hippocampus of ka treated rats. administration of mi h later after ka treatment abolishes these changes, whereas under the same time schedule diazepam treatment has no significant influence. the number of neuronal cells in ca and ca subfields of hippocampus is decreased after ka induced se and mi post-treatment significantly lessens this reduction. no significant changes are observed in the neocortex. obtained results indicate that mi post-treatment after ka induced se could successfully target the biochemical processes involved in apoptosis, reduces cell loss and can be successfully used in the future for translational research. references . r. . neuroscience letters, vol. , no. , pp. - . . r. solomonia, et al; . cell. mol. neurobiol. vol. , no extracellular deposits of amyloid-b peptide (ab) in brain parenchyma via proteolytic processing of amyloid precursor protein (app) are one of the typical characteristics of alzheimer's disease (ad). these aggregates mainly occur as a result of an increase in ab production or a decrease in its degradation. it was found that the neurotoxicity of ab aggregates is accelerated by acetylcholinesterase (ache). besides, ab-ache complex has a prominent neurodegenerative effect in brain. thus, cholinesterase inhibition and preventing ab production are current treatment strategies for ad. recent studies have shown that methylene blue (metb), a cholinesterase inhibitor with phenothiazine structure, inhibits the formation of amyloid plaques and neurofibrillary tangles. azure b, the major metabolite of metb, has been shown to inhibit ache and bche with ic values of . lm and . lm, respectively. in the present study, we tested whether azure b, may effectively lower the levels of ab / . we treated chinese hamster ovary cells, which stably express human wild type app and presenilin- (ps ) with - mm azure b or vehicle for h. to determine the effect of azure b treatment on ab / levels, we used separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. azure b treated ps cells were also assessed by propidium iodide in flow cytometry for cellular toxicity. we observed a significant decrease in both extracellular ab and ab levels with a dose range treatment of azure b in ps cells. ab levels were reduced by . % in lm and . % in lm azure b-treated cells when compared to control. additionally, ab levels were decreased by % in lm and . % in lm azure b-treated cells when compared to control. overall, these preliminary results suggest that azure b may have beneficial effects for the treatment of ad. the effect of green silver nanoparticles (agnps) on the amyloid formation in alphalactalbumin and chaperone action of alphacasein a. ghahghaei, m. dehvari, j. valizadeh formation and deposition of protein fibrillar aggregates in the tissues is associated with several neurodegenerative diseases such as alzheimer's and parkinson's disorders. molecular chaperones are a family of proteins that are believed to have ability for inhibiting protein aggregation. in the present study the effect of different concentrations of green synthesis silver nanoparticles (agnps) from pulicaria undulate l. on the amyloid formation of a-lactalbumin (a-la) and chaperone action of a-casein have been investigated. the effects of the agnps were determined using light scattering absorption, tht binding assay, intrinsic fluorescence assay, ans binding assay, cd spectroscopy and sds-page. light scattering and tht assay results showed that agnps have the ability in preventing aggregation of a-la in a concentration-dependent manner. consistent with these results, sds-page results represented that by increasing the concentration of agnps the adsorption and interaction between agnps and protein have increased. light scattering and tht assay results, also, revealed that the amyloid fibrilation decreased in the presence of both agnps and a s -casein compared to presence of a s -casein alone. fluorescence results, however, show that agnps have no effect on the chaperone ability of a-casein and in fact they perform their protection of protein aggregation action independently. consistent with the above experiments, cd spectroscopy also revealed that agnps have decreased structural changes in reduced a-la in absence and presence of a-casein, both the tertiary and the secondary structure of the proteins. our finding represented that agnps have preventing effects on protein aggregation and have no effect on the chaperone ability of a s -casein. in the main, results of this study show that biosynthesized agnps mediated by >pulicaria undulate l. maybe could be affective as a therapeutic agent for inhibiting aggregation in treatment of amyloidosis disorders. pink is a mitochondrial kinase with multiple cellular functions. while loss-of-function mutations of pink gene lead to early onset parkinson's disease, its over-expresion is associated with cancer development. parkinson is a multifactorial neurogenerative disease, with a complex aethiology including various cellular stressors. it is now known that genotoxic stress also triggers the release of soluble factors able to induce changes in neighboring cells enhancing the initial lesions, process known as bystander phenomena. althrough the mechanisms are still unclear, recent studies point towards a role for mitochondria in this process. our work investigates pink role in intracellular and intercellular stress response, comparatively in various models: fibroblasts (mefs) and neuroblastoma (sh-sy y) used as a tumoral model or differentiated to a neuronal phenotype. pink role in this process was analyzed using genetically engineered pink deficient cells exposed to a genotoxic agent, bleomycin. the modified cell lines showed a reduced level of basal atp production. pink proved to be involved in cellular vulnerability to stress. despite differences in cellular sensibility between our models, genotoxic treatment of pink deficient cells induced consistently higher lesions compared to corresponding wild type variant. pink deficient cells showed altered intercellular signaling of stress, impairing bystander phenomena induction, by suppression of signal formation in treated cells, but also by altering the capacity to respond to the signals in neighboring cells. our hypothesis is that pink contributes to the management of cellular stress being involved in bystander transmission of detrimental effects through intercellular communication. this is determined mainly by its role in maintaining mitochondrial homeostasy and atp levels, pink deficient cells lacking the amount of energy required for rapid dna repair and stress signaling transmission. p- . . - intranasal administration of synthetic fragments from receptor for advanced glycation end products prevents memory loss in olfactory bulbectomized mice the receptor for advanced glycation end products (rage) is a member of the immunoglobulin protein superfamily. activation of rage causes brain inflammation, oxidative stress and secretion of beta-amyloid that has been recognized as an essential phase in the development of alzheimer's disease. it is known that the receptor soluble isoform (srage) which lacks the transmembrane and cytosolic domains binds to ligands and prevents negative effects of the receptor activation in in vivo and in vitro experiments. we proposed that potential ligand-binding peptide fragments from srage would demonstrate the same biological activity. we have selected and synthesized peptide fragments from unstructured surface-exposed regions of rage. synthetic peptides were intranasally administrated into olfactory bulbectomized (obe) mice with neurodegeneration of alzheimer's type. we have found that only administration of rage fragment ( - ) effectively prevents the obe murine memory from impairment, leads to decrease of beta-amyloid level and blocks the development of neuronal pathology in the brain of experimental mice. six overlapping fragments of rage ( - ) peptide were synthesized in order to find a site, responding for the therapeutic effect. tests in obe mice carried out with these fragments showed that only the n-terminal part of the molecule is responsible for preventing obe mice memory from impairment. all fragments which do not include n-terminal - dipeptide have been fully inactive in these experiments. we have proposed that active peptides can interact with beta-amyloid or s b protein preventing these ligands from binding with rage. this interaction can inhibit the development of neurodegeneration. the aim of this study was to examine effects of social isolation, enriched environment and exercise on learning in rats. the study included female day old wistar rats. the rats were randomly divided into four different groups; control, exercise, social isolation and the enriched environment groups. the social isolation group and the enriched environment group were housed under their specific conditions and the exercise group and the control group were housed in standard conditions during weeks. the rats in the exercise group swam for weeks. after weeks, the rats were evaluated in the morris water maze. brain and blood samples were taken and the hippocampus tissue was dissected. bdnf and ngf levels were measured in these samples. in conlusion, while enriched environment was a positive effect on spatial learning, social isolation was a negative effect on spatial learning and increase thigmotactic behaviors. according to the analysis results ngf and bdnf levels in the hippocampus and plasma did not change with environmental conditions and exercise. time of exposure to social isolation, procedures of the enriched environment, time of exposure to the environment, type and duration of exercise and gender may affect the results. alzheimer's disease (ad) was characterized by dementia that typically begins with subtle recognition failure and poor memory. it slowly becomes more severe and, eventually, incapacitating. the cholinergic system seemed particularly susceptible to synapse loss, especially in cortical regions associated with memory and executive function ( ) . recent studies showed that the main cause of the loss of cognitive functions in ad patients was a continuous decline of the cholinergic neurotransmission in cortical and other regions of the human brain ( ) . acetylcholinesterase (ache) and butyrylcholinesterase (bche) are hydrolytic enzymes that act on acetylcholine (ach) to terminate its actions in the synaptic cleft by cleaving the neurotransmitter to choline and acetate. both enzymes are present in the brain and detected in neurofibrillary tangles and neuritic plaques. it was suggested that ache predominates in the healthy brain, with bche considered to play aminor role in regulating brain ach levels. however, bche activity progressively increases in patients with ad, while ache activity remains unchanged or declines. both enzymes therefore represent legitimate therapeutic targets for ameliorating the cholinergic deficit considered to be responsible for the declines in cognitive, behavioral, and global functioning characteristics of ad ( ). we initiated a study to screen their acetylcholinesterase (ache, ec . . . ) inhibitory activities, which are the key enzymes taking place in pathogenesis of ad. newly synthesized chiral benzimidazole derivatives with thioure structure showed ic values in the range of . - . nm for ache. this study was financed by turkish research council-tubi-tak (kbag z ). p- . . - f -a h, novel fingolimod derivative, activates camp-dependent signalling pathway in sk-n-sh cell line g. celik turgut , a. sen , d. doyduk , y. yildirir department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, department of chemistry, faculty of sciences, gazi university, ankara, turkey fty , a sphingosine -phosphate (s p) receptor modulator, is the first oral disease-modifying therapy to be approved for the treatment of relapsing-remitting multiple sclerosis. in this study, we have synthesized and characterized novel derivative of fty , namely f -a h, and have determined its underlying camp regulation in sk-n-sh cell lines. for this purpose, we first determined the regulation of the camp response element (cre) activity and camp concentration by f -a h along with fty using pgl . luciferase reporter assay and camp immunoassay, respectively. then, we have determined their effect on camp/pka-related gene expression profiles using custom arrays along with fty treatment at non-toxic doses. it was found that f -a h significantly activate cre and increase camp concentration in the sk-n-sh cell line, indicating that it activates camp pathway through cell surface receptors as fty does. furthermore, f -a h modulates the expression of the pathway related genes that are important in camp signaling pathway. in summary, our data demonstrate that the novel fty derivative act as a modulator of camp ultimately by influencing the gene expression via the camp and downstream transcription factor cre pathway. in conclusion, f -a h might contribute future therapies for multiple sclerosis. alzheimer disease (ad) results in memory impairment and accompanied by neuroinflammation, cholinergic deficit and amyloid-beta (ab - ) accumulation in brain. we found that bacterial lipopolysaccharide (lps) injections or mice immunizations with extracellular a nicotinic acetylcholine receptor (a - nachr) domain resulted in astrogliosis, decrease of a nachr density, accumulation of ab - in brain and episodic memory impairment. the aim was to reveal main event triggering ad-like symptoms development. c bl/ mice were injected with lps, immunized with recombinant a - or a - endoglycosidase treated to remove carbohydrates. two immunizations with week interval were performed. control mice obtained complete freund's adjuvant injections. mice were tested for memory performance, blood sera were examined for presence and fine specificity of a - -specific antibodies and brain preparations were studied for a nachr, ab - and il- levels. the original a - ('glyc') was more immunogenic than 'deglyc', and their epitopes were recognized with different efficiency. in contrast to lps and 'glyc' a - immunization with 'deglyc' a - did not stimulate il- elevation in brain and had no proinflammatory effect. immunizations with 'glyc' or 'deglyc' a resulted in similar a nachrs decrease and ab - accumulation in brain and significant episodic memory decline comparable to those after lps injections. a nachr interacts directly with amyloid-beta precursor protein and facilitates its proper processing and metabolism. our data indicate that decrease of a nachr density caused by a - -specific antibody is critical for ab - accumulation and episodic memory impairment while pro-inflammatory capacity of a - -specific antibody plays secondary role in ad-like symptoms development. in vitro antioxidant and antiacetylcholinesterase activity of achillea millefolium alzheimer diseases (ad) is a neurodegenerative condition without a current effective treatment. increase in reactive oxygen species and lipid peroxidation or decrease in total antioxidant capacity causes oxidative stress-induced tissue damage. it has been suggested that decrease in oxidative stress and inhibition of acetylcholinesterase (ache) activity play a major role in the prevention and slowing of cognitive symptoms of ad. recently, studies have been directed for the discovery of medicinal plants and natural substances that are known to have natural antioxidants. achillea millefolium (a. millefolium) is a traditional herbal medicine that contains natural compounds with antioxidant activity and has been used as a carminative, diuretic, menstrual regulator and wound healer, however the mechanism of its actions are unclear. the aim of our study was to investigate the effects of a. millefolium extracts on free radical production, acetylcholinesterase (ache) activity and lipid peroxidation in vitro. methanol (me) and ethanol (ee) extracts of a. millefolium were prepared to determine (a) in vitro antioxidant capacity (by using , -diphenyl- -picrylhydrazyl assay, radical scavenging activity, phosphomolibdenum-reducing antioxidant power, ferricreducing antioxidant power, and total phenolic-total flavonoid contents), (b) effects on ache kinetics (by using a colorimetric assay) and (c) effects on sodium nitroprusside-induced lipid peroxidation in mice brain homogenates. me showed a higher antioxidant activity compared with ee in the biochemical assays tested. similarly, me demonstrated significant inhibition of ache activity that was potent than ee. both extracts dose-dependently decreased malondialdehyde content in mice brain homogenates suggesting a strong inhibition of lipid peroxidation. these results showed that a. millefolium has a high antioxidant capacity and antiache activity, indicating a potential use as an adjuvant therapy in ad. b-cells are known to play a key role in multiple sclerosis (ms) progression and autoimmune response. cxcr is the main b-cell chemokine receptor that under normal conditions directs their migration to specific areas of secondary lymphoid organs. in ms, areas of demyelinating lesions have been reported to attract bcells due to overexpression of cxcl , the cxcr ligand. we aimed to determine whether snp rs located in the promoter of cxcr gene and associated with high risk of multiple sclerosis could have a direct effect on of cxcr promoter activity. mef c binding to dna was assessed using pull-down assay. b-cell stimulation was performed using lps, pma and ionomycin. activities of variants of cxcr promoters containing different rs alleles were estimated using luciferase reporter assay. we determined that minor rs allele creates functional mef c-binding site within one of the regions required for the basal activity of the cxcr promoter. cxcr promoter containing minor rs variant that is statistically associated with low risk of ms showed significantly decreased activity in stimulated human b-lymphoblastoid cell lines. mef c has been reported to play an essential role in b-cell survival and b-cell responses. we determined mef c as the main regulator of rs -dependent modulation of cxcr promoter activity in b-lymphoblastoid cell lines. this link may be directly related to pathogenic b-cell activities in multiple sclerosis. introduction: parkinson's disease (pd) is the second most common neurodegenerative progressive brain disease with increasing prevalence in aging population. the etiopathogenesis involves many cellular procesess, but is not fully elucidated yet. treatment of pd is based on levodopa and dopamine agonists, but mao-b inhibitors, comt inhibitors, amantadine or anticholinergics may be used as initial monotherapy or as adjuvant therapy. treatment related adverse drug reactions (adrs) are frequent, but cannot be predicted and/or prevented. non-motor adrs, such as nausea, somnolence, hallucinations and hypotension are frequent in dopamine agonist therapy, while dyskinesias along with motor fluctuations are the most common late adrs with levodopa. the aim of our study is to combine clinical data with genetic and epigenetic biomarkers in the algorithm for personalized approach to pd management. materials and methods: we are planning a clinical study to assess the combined impact of selected clinical, genetic and epigenetic factors on the progression of pd, adrs and treatment response. our study will have a retrospective and prospective arm. we will collect peripheral blood samples of pd patients and clinical data. single nucleotide polymorphisms (snps) in the genes involved in dopamine, neurotransmitter and drug metabolism and transport, receptors and signalling pathways will be genotyped. snps within inflammatory, neurodevelopmental, antioxidative defense, synaptic transmission and immune response pathways will also be analysed. in the prospective arm we will isolate the exosomes and check their mirna content at the time of diagnosis and after the treatment initiation. the combined effects of clinical, genetic and epigenetic factors will be analyzed using lasso penalized regression analysis. conclusions: we hope to identify genetic and/or epigenetic biomarkers that may predict progression of pd, adrs and treatment response and may support personalized tratment of pd. most evidence indicates that g protein-coupled receptors form heteromers between them and with other receptors. by allosteric mechanisms, them acquire a multiplicity of unique pharmacological and functional properties. recently, we discovered that dopamine d receptors (d r) and histamine h receptors (h r) form heteromers through which h r ligands can inhibit d r function. d rs also physically interact and modulate ionotropic glutamate nmda receptors (nmdar). in the present work, we investigated if nmdar, d r and h r form a heterotrimeric complex in brain. the heteromer expression was studied in slices from both rat and mouse brain cortex by co-immunoprecipitation (co-ip) and proximity ligation assays (pla). the ability of d r and h r to interact with nmdar in transfected hek cells was analyzed by bioluminescence resonance energy transfer (bret) with bimolecular fluorescence complementation (bifc) experiments. heteromer properties were studied by analyzing erk / phosphorylation and cell death in cortical slices. endogenous d r-h r heteromers were detected in rat and mouse cortical slices, where h r ligands decreased d r signaling (erk / pathway) and were also able to block the cell death induced by overstimulation of either d r or nmdar. by bret experiments in transfected hek cells, we demonstrated that both d r and h r form heteromers with nmdar subunit a in the presence of subunit b. d r-h r-nmdar heteromers were detected by bret with bifc. endogenous d r-h r-nmdar heteromers were observed in rat and mouse cortex by pla. many systems, including the glutamatergic and dopaminergic, are involved in neurodegeneration. our innovative finding is that d r, h r and nmdar form heteromers that may be a point of intervention for cognitive disorders in neurodegeneration. d r-h r-nmdar heteromers are expressed in brain cortex and a complex interaction exists between protomers in the heteromer, where h r ligands act as a 'molecular brake' for d r and nmdar signaling. studies conducted on obesity and hfd (high fat diet) revealed hypothalamus have crucial roles on development of metabolic diseases. after chronic over nutrition or high fat diet, as a neurodegenerative condition, premise inflammation, neural stress and development of functional impairments are observed. these studies generally focused on changes in neurons, but it's effects on brain vessels are still unknown. in this study, as a neuronal damage infrastructure, changes in hypothalamic vascularity investigated. experiment initiated with weeks old total male wistar rats. in order to acquire obese phenotype, the rats were fed either cafeteria diet as hfd, or normal/chow (standard diet, sd) for months. intravenous glucose tolerance tests performed before sacrification. animals were exposed for s to co and then decapitation was performed with guillotine. isolated brains were directly immersed into liquid nitrogen and stored at À °c. the hypothalamic sections were acquired with the cryostat instrument at different. immunofluorescence was performed on serial sections through the hypothalamus using the antibody smi- and cd . changes in tight junction (tj) proteins (occluding and zone occludin- ) are evaluated via western blot (wb) analysis. the hfd-treated consumed significantly more food than did control animals, when examining average food consumption per day and rats that received the hfd diet weighted significantly more at the end of month diet treatment. there were no differences acquired for glucose tolerance tests. however, after hfd treatment, wb analysis have shown that tj proteins decreased even if hypothalamic micro vessel number increased and smi- staining have shown that increased. our primary results have shown that hfd diet can affect hypothalamic vascularity and such changes might initiate neurodegenerations and functional impairments as observed in neuroretinal degeneration in relation to vasculopathy in diabetic patients. defects of mitochondrial trafficking are common problem in many neurodegenerative diseases. its dysregulation can contribute to changes in bioenergetics profile of the cell and can lead to cell death. in our study we investigate distribution of mitochondria and their transport in primary fibroblasts derived from patients with sporadic form of alzheimer's (ad) and parkinson's (pd) diseases. our data revealed that in the most cases the velocity of mitochondrial movement is lower in ad and pd cells in comparison to the control. the most intense differences between ad, pd patients and control group are observed in the case of movement of large mitochondria. owing to the fact that mitochondrial trafficking depends on mitochondrial state, we investigated the 'age' of mitochondria. we observed a diminished mitochondrial turnover in ad and pd fibroblast. evaluation of the mitochondrial distribution within the cell in all groups (ad, pd and control) showed that in the perinuclear area are accumulated 'old' and 'worn out' mitochondria, probably dedicated to remove from the cell. because mitochondrial biogenesis, shape and size depends on fusion/fission proteins we assessed their level within the cell. to summarise, our results revealed alterations in mitochondrial trafficking in fibroblasts derived from patients with alzheimer's and parkinson's diseases in comparison to the healthy control cells. carbonic anhydrases (cas, ec . . . ) is a zinc metalloenzyme that catalyzes the reversible reactions of co and water. carbonic anhydrases (cas, ec . . . ) form a family of metalloenzymes that play an important function in various physiological and pathological processes. therefore, many researchers work in this field in order to design and synthesize new drugs. carbonic anhydrase activators are important as much as inhibitors. caas have polar groups to make hydrogen bond in the main body and the activation property of enzyme increaase in this way. caas are have polar groups to make hydrogen bond in the main body and the activation property increaase in this way. furthermore, recent studies suggest that ca activation may provide a novel therapy for alzheimer's disease. in this study ca activators are determined. human carbonic anhydrase isozymes ca i and ca ii are isolated from human blood erythrocyte. hca-i and hca-ii isoenzymes were purified using sepharose- b-l tyrosine-sulfanilamide affinity colum. finally, hca-i and hca-ii isoenzymes were eluted with appropriate elution buffers. enzyme purity was checked by sds-page. the enzyme activity system contained . m tris-so ph . , r-nitro phenol in ml total volume. effects of some macrocyclic thiacrown ethers derivates were investigated. enzyme activities were measured at constant substrate and different activator concentrations to find ac value. these compounds are thought to be useful for treating alzheimer's disease. introduction: gender differences in stress models are not studied in detail. we compared different stress conditions on brain bdnf levels, in social isolation (sit) and predator scent tests (pst) in rats. bdnf levels in cortex, hippocampus and amygdala were compared, effects of chronic fluoxetine (flu) treatment were evaluated. methods: rats were used. for sit, animals were kept individually for month and for pst, rats were exposed to dirty cat litter for min at the first day of month stress. flu was given ( mg/kg, ip) through stress. controls, stress and treated groups were evaluated in elevated plus maze (epm), anxiety scores were calculated. brain bdnf levels were determined in cortex, hippocampus and amygdala by western blot. p < . were considered significant. results: sit and pst induced anxiety in both male and female rats, females having greater anxiety scores than males (p < . ). flu restored anxiety scores in both sexes (p < . ) in two settings. male and female rats exhibited reduced cortical bdnf levels in sit (p < . ). pst reduced cortical bdnf in females, but increased in males. hippocampal bdnf expression was lowered in sit (p < . ) and pst (p < . ) in both sexes. female rats had % lower bdnf expression than males in amygdala in sit. flu did not restore cortical bdnf in females in both tests, but reduced incresed bdnf levels in males (p < . ). flu did not restore reduced brain bdnf in males in hippocampus and amygdala, but restored in hippocampus, in females. discussion: our findings indicate that sex differences must be considered in studies related to mood disorders of animal models, and suggest that bdnf expression in different brain regions are altered differentially in a gender-dependent manner in rats. antianxiety effect of flu is not mediated through increasing bdnf activity in cortex in both genders. increased bdnf in hippocampus and amygdala may reflect its antidepressant effect in female rats, but not in males. perineuronal nets (pnns) are special forms of neural extracellular matrix found around neuron bodies and neurites. hyaluronan and proteoglycan link protein (hapln ) is one of the major elements of pnns. hapln interacts with tenascins and aggrecan which are other essential pnns components. in most of neurodegenerative disorders caused by neuritogenesis defects, disrupted pnns structure and decreased expression of hapln were observed. however, the role of hapln in neural differentiation is unknown. the aim of this study is to determine mrna and protein levels of hapln during differentiation using pc cell line as a neural differentation model, derived from rat pheochromocytoma. after pc cells were stimulated to differentiate into neurons by nerve growth factor on days , and ; cells were collected, qrt-pcr and western blot were performed. also, in order to find out whether there is a physical interaction between hapln and proteins related to neuritogenesis defects, spinal muscular atrophy (sma) was used as a neurodegenerative disease model. therefore, a detailed hapln and survival motor protein (smn ) network analysis were performed in-silico. as a result, we analyzed fold increase in hapln mrna level compared to undifferentiated state. on the other hand, a decrease in protein level was detected. this decrease in cellular hapln level suggests that, hapln is required for formation of pnns structure, thus secreted to extracellular environment at early stage of differentiation. in addition, according to in-silico analysis, an indirect path between hapln and smn through fibulin (fbln ) was detected. fbln was also found to be an interaction partner between different matrix molecules such as aggrecan and hapln which form a macromolecular meshwork. the results of this study will pave the way for investigating the role of hapln and fbln in neurodegenerative disease models. also it will help us to understand the mechanism of neuritogenesis defects. determination of properties of bone marrow and tissue-derived mesenchymal stromal/stem cell population in neurofibromatosis type patients neurofibromas, complex tumors deriving from schwann cells and containing fibroblasts, vascular structures and mast cells, are part of the clinical picture in nf . the risk of malignancy is increased in nf , wound healing is delayed and keloid formation is frequent. because multiple tissues are involved in malignant and non-malignant manifestations of nf , we considered the mesenchymal stem/stromal cells (msc) carrying the nf mutation might play a role in the microenvironment. mscs affect the biological behaviour of other cells: they alter their proliferation, apoptosis and migration through various secreted growth factors, cytokines, chemokines, or by direct contact. we examined the msc of nf patients. methods: the adipogenic and osteogenic differentiation potential of mscs from nf and healthy subjects was examined in vitro and by rt-pcr. msc's migration potential was measured in the scracth assay. mscs' interaction with schwann cells and their effect on tumorigenesis was examined in co-culture by apoptosis markers on flow cytometry. results: nf -mscs' adipogenic and osteogenic differentiation potential was lower than healthy controls as assessed by staining aizerin red s and oil red o and rt-pcr for osteopontin and collagen . mscs cultured from dermal neurofibroma showed faster closing of the scratch compared to the same patient's normal and caf e au lait skin. on the other hand, mscs obtained from plexiform neurofibroma healed late, while mscs derived from the same patient's caf e au lait skin showed the fastest healing. hepatic encephalopathy with ammonium ions accumulation is accompanied by some disorder in the brain due to toxic material concentration being usually detoxified in the liver. one of the reasons for hyperammoniemia could be some imbalance in brain glutamine metabolisation induced by the key enzymes glutamine transferases (gts), which catalyze the reaction of glutamine transamination resulting in neurotoxic product of a-ketoglutaramate (akgm). akgm is hydrolyzed to a-ketoglutarate and ammonia by x-amidases. in the study, the dynamics of the enzymes activity in the tissues and biological liquids of experimental animals with hepatic dysfunction induced by thioacetamide (taa) was under investigation. white laboratory rats of wistar line (female, weight of g) chronically intoxicated with hepatotrophic toxine of taa. every weeks, some biological samples were collected to assess gt-k and x-amidase activities. x-amidase activity was the highest in the kidney tissue in the control and decreased by % in the experimental group. in the experimental hepatic x-amidase activity decreased by % compared to those in the control. the average x-amidase activity in the blood serum ( . nmols/ mg/min) and in the brain ( . nmols/mg/min) differed faintly. maximal gt-k activities were revealed in the kidneys; in the controls, it was about % higher than those in the experimental animals. the difference between average enzyme activities in the liver of the control and experimental animals reached %. the average gt-k activities in the blood serum and brain of the control and experimental animals were rather similar. the decrease in x-amidase and gt-k activities obtained in the study during hepatic pathology development could testify to imbalance of glutamine metabolism, possibly aimed at declining the level of akgm neurotoxicant under the hepatic dysfunction. acknowledgments: supported by the russian federation ministry of science and education (grant no rfme-fi x ). wilson disease is an autosomal recessive disorder of copper metabolism characterized as neurodegeneration and liver abnormalities. it is caused by defects in the atp b gene. atp b is responsible for the sequestration of cu into secretory vesicles, and this function is exhibited by the orthologous ccc p in the yeast. we aimed to characterize clinically-relevant novel mutations of p.t i, p.v i and p.r g-fsx in yeast lacking the ccc gene. the patients with these mutations have copper storage abnormalities in different parts of their bodies; p.t i mutation mainly affects the liver and the nervous system, p.v i mutation affects the nervous system, and p.r g-fsx mutation causes damages to the liver. to better understand the effects of these mutations on normal functions of atp b, we cloned human atp b gene onto a yeast expression vector and created the same mutations by site directed mutagenesis. then, wild type and mutated forms of atp b genes were transformed into yeast cells lacking the homologous ccc gene for functional comparison. first, we analyzed the expression of atp b and its variants in yeast cells by a real time pcr approach and western blot to make sure that transformed cells express the plasmids. expression of human wild type atp b gene in ccc d mutant yeast restored the growth deficiency and copper transport activity, however, expression of the mutant forms did not restore the copper transport functions and only partially supported the cell growth. our data support that p.t i, p.v i and p.r g-fsx mutations cause functional deficiency in atp b functions and suggest that these residues are important for normal atp b function. in recent years, attempts were made to develop miniaturized potentiometric biosensors which is particularly important to reduce the amount of enzyme and reagents needed. the miniaturization of a biosensor is possible by using an all solid-state polymer membrane ion selective electrode which is cheap, easy to prepare and allow micro-sized construction. the use of all solidstate polymer membrane ion selective electrode as the basic sensing element also has the advantage of providing biosensors that are easy to fabricate, exhibit rapid response and have long life-times. they are also mechanically stable and allow flow-through configuration. genetical and chemical modifications for the alteration of enzyme molecule characteristics are gained considerable importance. enzymes can be modified chemically by using water-soluble polymers or some chemicals. conjugates of natural and synthetic macromolecules with enzymes provides wide usage in medicine and in many fields of biotechnology. in this study, enzyme-polymer conjugates with different molar ratios were synthesized using urease enzyme. in this study micro sized potentiometric urea sensitive biosensor has been developed in which urease-polymer conjugates were immobilized on polymeric membrane ammonium ion selective electrodes whether pvc or derivatized-pvc via glutaraldehyde cross linking reaction. biosensor is not include inner reference electrode and inner reference solution. potentiometric performance of biosensor will be examined with a computer-controlled measurement system designated. the most important features of the obtained micro sized urea biosensor by using enzyme-polymer conjugations were being highly sensitive, having long life-time, easily built at a low cost, and having short response time when compared with conventional potentiometric urea biosensor. also, these biosensors were easily built at micro-construction. this study was supported by grant from the tub _ itak research fund (project number: z ). creative drama technique as a new tool to increase enthusiasm and to achieve learning objective for medical students e. y. sozmen , , e. erem faculty of medicine, ege university, izmir, turkey, center for continuing education, ege university, izmir, turkey, recently drama in education techniques have been implemented successfully in education program of primary and secondary high school and positive effects of these techniques on learning ability and attitude of students have been shown. the aim of this study was to organize an education program based on drama in education techniques in a special module of ege university medical faculty and to test any effect of this technique on achievement of learning objectives and student's perspectives on drama. the special module program was on the oxidative stress and antioxidants. the program covered the drama in education sessions (improvisation, role play, game) linked with learning objectives (understanding of free radical generation and free radical reactions in body, evaluation of the effect of free radical reactions in diseases as well as increase the ability usage of scientific information), laboratory work (antioxidant activity determination) and searching a special scientific topic on literature. students (in rd year of faculty) who had taken theoretical lecture on this subject a year ago, participated in this special module. the opinions of the students on the program were obtained through a questionnaire form and the increase in knowledge was evaluated via pre/posttest. the mean of pretest point was . / , that increased to . / in the posttest evaluation. % of students pointed that they enjoyed participating in drama activities in the pre-questionnaire, this rate was % in the final questionnaire. they all remarked that implementation of drama in education was beneficial for their communication skill, helping them to learn more about science and increased their enthusiasm to learn and discuss the scientific information. although the preparation process might take more time and need to hard work for teachers, we concluded that the drama methods as a new tool to increase of participant's interest might be proposed for students in higher education. laboratory-based performance assessment in medical education: an opportunity for connection between scientists and medical students h. tuncel cerrahpasa medical faculty, istanbul university, istanbul, turkey number of medical students who interested in basic medical sciences is declining and medical sciences literacy is falling, it is crucial to develop ways for students and scientists to connect. students need to know that science is an intensely human endeavor, and scientists need mechanisms to bring that truth to the community at large. based on continued interest and experience on the part of faculty, and on student feedback, the development of a more effective and stimulating interactive learning tool was undertaken. an in-depth knowledge of laboratory medicine principles is vital to all practicing physicians. great variation exists in the ways that medical students learn the principles of laboratory medicine. there are a number of programs for electronic media that emphasize laboratory-related skills. some of these are appropriate for medical students in the clinical years. programs that teach skills in common laboratory procedures, such as interpretation of peripheral blood smears and microscopic examination of urine sediment, have been shown to improve student performance. to ensure that important principles are addressed, medical schools should establish goals and objectives specifically related to laboratory medicine and experiment with optimal teaching and assessment methods. we also hope that this study will inspire dialogue among primary care and specialist physicians as to the proper degree of education in this area. ideally, it will encourage scientific studies that address evidence-based possibilities for improving critical laboratory medicine educational outcomes, that is, the training of physicians who optimally use laboratory diagnostics and therapeutics. engaging medical students in scholarly scientific activities and producing clinically competent and research-oriented medical workforces are essential demands, particularly in developing countries. an experimental special study module for medical undergraduate students: learning western blot analysis and detection of b-actin protein expression in tissue and cell culture samples learning, introduce basic principles of laboratory research and to present the results.b-actin is one of six actin isoforms which is mainly expressed in all eukaryotic cells. western blotting is a widely used laboratory technique to determine specific proteins and to evaluate protein expression in tissues and cells. in our study, different concentrations of rat spleen homogenates ( , , lg/well) and lg protein/well of human lung and ovary cancer cell lysates were used. the proteins were seperated by % sds-page, transferred to pvdf membrane, incubated with specific b-actin antibody and then with hrp-conjugated antibody. protein bands were detected with ecl and densitometric analysis of proteins was quantified by imagej software. differences in protein band intensities were compared using one way anova.a value of p < . was considered statistically significant. we detected b-actin expression in rat spleen homogenates, human lung and ovary cancer cell lysates, as a kd protein. the protein band intensities were in correlation with protein concentrations. the highest concentration resulted in the highest signal intensity in rat spleen homogenates.b-actin level was higher in ovary cancer cells than in lung cancer cells, although both proteins were loaded equally. the feedback showed that students were very satisfied with this laboratory ssm. they developed their independent study skills, planned a research, worked in a laboratory, learned and performed a technique, western blotting. the hands-on experience is very important for medical undergraduate students for their future scientific career. three-dimensional structure of truncated human kv . voltage-gated potassium channel kv . belongs to the ether-ago-go family. it has been proved that its mutants are involved in development of neurological diseases and some types of tumor. directed drug design needs knowledge of details of the threedimensional channel structure. the members of the kv - subfamilies are characterized by extremely long n-and c-terminal intracellular tails, which possess a number of structural domains. the n-terminal pas domain in kv plays an important role in activation, and is thought to alter the rate of deactivation, possibly by binding at or near the s -s linker at the inner mouth of the pore. here we present an improved d structure of the truncated human kv . channel, obtained by single particle em. this channel lacks its cytoplasmic pas domain but it still forms tetrameric particles. earlier we showed that the full length kv . channel is build according to the 'hanging gondola' type, and its cytoplasmic and transmembrane parts are connected by linkers. the cytoplasmic part includes the interconnecting pas and cnbd domains. deletion of the pas domain leads to the conformational change in the cytoplasmic part of the channel. for interpretation of the d structures we used homology modeling and molecular dynamics simulation. there are several templates available to the moment including eag domain-cnbhd complex of the mouse eag channel, full-length shaker potassium channel kv . , c-linker/cnbhd of zelk channels and others. but there are still no templates for many fragments that led to necessity of partial de novo modeling. analysis of molecular trajectory allowed estimating dynamical characteristics of channel, supposing interdomain interactions. results of the conducted investigation have a great interest at both the academic and the industrial levels. the objective of this task program is to enable students to gain scientific attitude and skills for them to be able to deal with the problems they'll confront in future research careers. it's been emphasized in various studies that medical students are keen on conducting scientific research, and it's been stated that they need to be supported in this respect, as they lack the adequate fund of knowledge. this study aims to share information throughout a -year performance of the research training program (rtp) conducted at ege university, faculty of medicine. rtp is an educational program applied in addition/parallel to the bachelor's degrees for establishing scientific thought, attitude, proceeding and knowledge of the willing medical students, and enabling them to adopt study skills. the dynamic program produced by the rtp committee in has been developing each and every year via feedback received from the students. an operating principles, a manual for advisor and an introductory booklet have been laid out. applications are being accepted at the end of first academic year, making announcement to the freshmen in advance. students are being admitted to the program, taking the assessment criterias into consideration. second and third year medical students attend the didactic part of the program during the terms devoted to special study modules. thereafter, they go through the project management phase, and accomplish their projects under supervision of a faculty member until their graduation. first graduates of the program, accomplishing their projects, received their certificates at the graduation ceremony of graduation. currently, there are students in total from all classes who perpetuate their studies within the program. an inventive training pattern of ege university faculty of medicine, rtp experience is being maintained as a dynamic process and successfully keeps the students advised of conducting scientific researches, cultivating scientific awareness. objectives: objectives selection and verification of blood collection tubes is an important preanalytical issue in clinical laboratories. in this study comparison with the reference glass tube of ayset plastic tubes containing separator gel and assessment for routine clinical chemistry laboratory testing in samples were aimed. materials and methods: thirty-four volunteers were included in the study. samples were taken into two different tubes by two experienced technologists according to the clsi protocol [tube : z (becton dickinson and company, franklin lakes, nj, usa); tube : ayset (lot , turkey)]. glucose, urea, creatinine, ast, alt, total-cholesterol, triglycerides and high density lipoprotein-cholesterol were analyzed subsequently (olympus au ) and randomised samples stored at - °c for and h. th hour sample was analyzed immediately after collection and accepted as the reference for the comparison of the other samples. a paired t-test and wilcoxon signed rank sum test were used to test the significance of differences between the reference tube and test tubes. results: the difference between the results of reference tube and test tubes for glukose, urea, creatinine, ast, alt, total cholesterol, tg and hdl-cholesterol at , and h were statistically no significant (p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , respectively). conclusions: no significant difference was observed between ayset tubes' results and the reference tube's results. e. akin c ß akir d€ uzen laboratuvarlar grubu, istanbul, turkey insulin resistance underlies the development of obesity which is a global health problem. obesity is a main concern of scientists because it's associated with type diabetes, hypertension and some cancers. recently, inflammation centered mechanisms are deeply investigated as well as the effects of anti-inflammatory diets which are highly rich in vitamins, minerals, fibers and healthy oils. these diets are proposed to inhibit or supress the secretion of the inflammatory mediators and also improve the intestinal microflora. the aim in this study is to highlight the increasing trend of publications in regard to insulin resistance and inflammation based obesity along with the effects of antiinflammatory diets used for its treatment in the last decade. we performed a pubmed search with key words of 'obesity and insulin resistance and inflammation' ( / / - / / ) (search # ). besides, we performed another search with key words of '(anti-inflammatory diet or dietary supplement) and (obesity or insulin resistance)' (search # ) to highlight the value of anti-inflammatory diets and dietary supplements in combating inflammation based obesity and insulin resistance. search # revealed articles; of these were clinical trials, were observational studies. human studies were while animal studies were . overall, there were review articles and meta-analysis in the field. search # revealed articles of which were clinical trials, were review articles, were meta-analysis. human studies were and other animal studies were . the relationship of metabolic diseases with a low grade inflammatory state has opened a new area of research to understand the consequent causes of inflammation in the human body. the increasing scientific data in the field indicates that antiinflammatory diets may serve as powerful tools to solve the inflammation and the consequent insulin resistance and obesity. medical and biological illustrations for life sciences education: is 'a picture worth a thousand words' in visualizing medicine and science? medical and biological illustrations (mbi) convey complex ideas with just an image and they are powerful intersections of science and art. the clarification of complex pathways via illustrations can be effective means in education as they help the student to visualize the biomolecular world and understand the mechanisms. our aim is to illustrate how a mbi is developed over the example of mechanism of insulin action, via the phosphoinositide (pi) kinase-protein kinase b (akt) pathway. organising one's thoughts and clarifying relationships and then using the optimal complexity level to illustrate the pathway most clearly are the basics of mbi. thus, we made a thorough investigation of insulin mechanism on glucose uptake in skeletal muscle and adipose tissue; a biochemical process that includes insulin receptor (ir), ir substrate, pi kinase, pi-dependent kinase and akt. then, we found the d structures of molecules via protein databanks and accordingly created drawings and diagrams of each component in both molecular and macrolevels by adobe photoshopÒ software. graphics tablets and a compatible pc were also used in the production phase. the use of computer hardware/software enables unlimited detail in images and provides the flexibility that classical drawing techniques can not. thus, the final diagram clarifies the underlying molecular mechanisms of a biochemical pathway along with the physiologic actions. recent improvements of computer technology have resulted in the creation, and reproduction of high-quality lower cost medical art. mbi's can be used in education to explain concepts/pathways to students to enhance learning. similarly, mbi's are great tools to show mechanism/procedures to enhance patients' understanding of their medical condition. considering their unquestionable contribution to education, research and patient care, the creation of mbi's should be promoted as a graduate level course and the discipline should be represented at academic level. biochemistry is a compelling field with broad applications in many disciplines like medicine, dentistry, pharmacy and bioengineering. biochemical research increasingly combines ideas from genetics, molecular biology, etc. and collaborates with many disciplines. our objective is to conduct a scientometric analysis of the last decade's postgraduate theses in the field of biochemistry (ptb) in regard to number, collaborations, subject area distribution, etc. to discover the characteristics and trends in turkey. we searched the turkish higher education council's theses database ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) which includes master of science (msc.), doctorate (ph.d.) and specialization (s) theses in all disciplines. an electronic search with the keyword of 'biochemistry' (in the thesis subject area) was conducted, thus it brought all theses either in the biochemistry discipline or theses in other disciplines but have a biochemistry component. we performed data cleaning and further quantitative analyses in excel. we also executed word count analysis on the titles of theses to derive the main subject areas in ptb. of the total of ptb ( s, msc, phd theses) . % was in natural sciences while . % was in health sciences. the theses output-growth measured by the compund annual growth rate was % over the -years. the top clinical disciplines in collaboration with biochemistry were pediatrics, surgery and cardiology, and the top science disciplines were biotechnology, bioengineering and biology. oxidants-stress and antioxidants ( ), endocrine-metabolism ( ) and enzymology ( ) were the top research areas in ptb, followed by genetics ( ) and cancer ( ). scientometrics is a powerful tool to understand the direction of science and research. our ptb analysis indicated that prominent areas like stem cell, biosensors, geriatrics are somewhat lagging in turkish biochemistry research while postgraduate education and research in total is growing fast with sound collaborations. the st turkish in vitro diagnostic symposium evaluation objective: in vitro diagnostic (ivd) medical laboratory tests and the equipment are closely related the public health, patient safety and the safety of all who utilize these tests. it depends on auditing of the process from the production to the consumption of these materials, that they do not pose a risk to individuals and society. upon these basic requirements; 'turkish in vitro diagnostic symposium: medical laboratory tests' was held in february , organized by the cooperation of turkish biochemical society branch of izmir, and dokuz eylul university health sciences institute. it was intended to shed light on some questions such as, what is the place and importance of ivd in turkey? what are the responsibilities of educational institutions?, what is the role of ministry of health? to put across the conditions of preparing a substructure that may provide achieving success in producing ivd medical devices and in this sector, in our country. material-method: invited speakers attended the symposium, along with the participation of both as lecturers and attendees; ministry of health, turkish standards institution; representatives of manufacturer enterprises; representatives of enterprises manufacturing in turkey; scientists conducting considerable researches on health technology; students and representatives from some of the non-governmental organizations. in addition to the presentations, gathering up the written opinions of the participants, a report was prepared. results: the symposium that lasted for days was realized with participants in total, of which from universities; representatives of their companies; from chamber-institute-public establishments and of which from public hospitals. of the participants were from izmir, of them were coming from out of izmir. conclusion: at the end of the symposium, % of the participants gave feedback. among the feedback selected; . % of the participants evaluated the symposium overall, as successful. . % of them found the symposium successful with regard to its scientific content. their feedback were . % positive in terms of the symposium's contribution to the situation assessment on ivd in turkey, and . % of them stated they would consider participating in the second of the ivd symposium if it is to be organized. perceptions of molecular life science master's students on their scientific and academic competencies and prospective plans for professional development the master's education (me) in molecular life sciences (mls) is aimed at strengthening the knowledge and skills base of the young scientist, preparing him for the competitive academia/industry positions. the rapidly developing pace of science and research forces the master's student (ms) to play a central role in monitoring and guiding his scientific education and professional development (pd). thus, the aim of our study was to examine the perceptions of ms of mls, regarding their scientific and academic competencies. with this data, we planned to analyse if this awareness channels ms to take action and/or matches with their prospective plans. we developed a item online survey with sections (demographic data, current data-contributions of me, competencies and prospective data-action for pd, future plans) and distributed it via e-mail to various postgraduate institutes. at the end of the -day period, ms students (in the thesis phase) answered the questionnaire (female: . %, male: . %). the most highly rated activities that contributed to their scientific knowledge and skills gained in the me were laboratory work ( %), visits with their mentors ( %) and theoretical lessons ( %). ms expressed low levels of sufficiency both in theoretical scientific knowledge and laboratory skills (only % and % sufficiency, respectively). communication skills ( %) and team work ( %) were rated as the highest professional competencies followed by literature search and research planning (both %). it was striking that ms perceived themselves as quite insufficient in scientific writing ( %), data analysis ( %) and project writing ( %) while proficiency in english ( %) was the first area they wanted to take action. despite their perceptions of insufficiencies in many areas, a majority ( %) wanted to continue to phd education. these and similar surveys may lead to an increase in selfawareness in ms and the data may contribute to the revision of me. the report of the st turkey in vitro diagnostic symposium results the following aspects and suggestions took place in the results report prepared after the symposium: about the national ivd-tc r&d, production, forming quality assurance and innovation strategy and policy the cooperation of the university and the industry is not sufficient most of the industrialists cannot take enough advantage of the support provided by the institutions like tubitak, the ministry of science, technology and industry and the ministry of development the statistics on the ivd-tc in turkey should be carried out as soon as possible national standards should be determined in parallel with the international standards the vat rate of the exported raw materials that would be used in ivd production should be decreased. about the education and training, the job titles and positions the related graduate programs, which would focus on all steps of the whole life cycle related to the ivd-tcs one by one, are not widespread there is no 'postdoc' application in turkey. 'postdoc' staff is needed for insufficient component human resources the lecturers should not be restricted to one discipline only graduate programs on laboratory medicine are needed to be established and spread, in order to train component labor specified on the ivd-tc about research and development there are almost no researches related to product development. this should be associated with the education and training institutions about the research centers currently, there is a real infrastructure on health technology in turkey, but there are difficulties in its instituonalism the insufficient cooperation of the university and the industry does not allow the inventions to turn into products the cooperation supports of r&d, being restricted to tech-nopark and r&d centers, are open fields for improvement p-edu- phd training in medical education: career profiles and satisfaction levels of graduates from dokuz eylul university graduate school of health sciences this survey was carried out within the scope of special study modules that is entitled 'phd training in medical sciences' by a group of medical students in deu. the purpose of the survey to investigate the members of health sciences that have successfully completed their phd training in terms of the levels of satisfaction and the status of their career. from this scope we generated two hypothesis: we expected that graduate phd graduates are mostly involved in academy and find their satisfaction levels at moderate level as to phd education. the study was designed as cross-sectional. we reached phd graduates who had graduated from deu graduate school of health sciences between and from different departments via e-mail. the survey was included questions, which were prepared in the light of the existing literature. among the phd graduates, ( %) participated in the study. through this survey, perception of phd students on supervisors' scientific and educational abilities, opinions on phd training, productivity of phd training, number of articles published, their position and related satisfaction levels after graduation were investigated. according to the results, more than half of the graduates (% . ) are well satisficed from the education they had taken. beside this, interestingly we found that % . of graduates prefers staying in the academic positions and % . of them sustains their communication with their supervisors. in conclusion, most of phd graduates were contented with phd training and their career profiles. as a result of this survey, we produced a novel and precise contribution to the literature. in a further study, this survey may extend to other parts of turkey and compile the results in order to get more accurate and inclusive data. phd is an international degree that is reached by conducting an original research after finishing bachelor or master's degree. doctoral degree (phd) can open the door of academic career; on the other hand, a person with a doctoral degree is equipped to carry out important work in research, industry, or public sector. today, gradually increasing number of phd students have brought some problems in phd training. the purpose of this study is to investigate and review activities that have been done by the following international organizations: orpheus: (organization for phd education in biomedicine and health sciences) eua-cde: european university association-council on doctoral education. febs education committee: federation of european biochemical societies these three organizations have done workshops on phd training to pay attention to the following points: *a phd student must take some courses and trainings outside her/his institute, should not be limited to the institute. *the phd training programme should include transferable skills courses. *clinicians, if involved in phd training, should be given free time from their clinic duties. *with regards to potential problems with the supervisor, the institute should provide the student an advisory system. *students should be encouraged to participate in the management of doctoral programmes in the institute. *the students should be given be opportunity to select their own supervisor (thus their thesis area). the phd training has gained quality thanks to these organizations. it is advised that graduate school of health sciences should follow the recommendations and report from these organizations. itsn and itsn are genes encoded adaptor proteins with multiple isoforms participating in clathrin-mediated endocytosis (cme), mapk signaling and reorganization of actin cytoskeleton. changes in itsns expression can lead to different neurodegenerative disorders and cancers. to date little is known about regulation of itsn genes on posttranscriptional level. the aim of our work was to predict and experimentally confirm target sites for micrornas that could potentially regulate itsns expression. using web servers we analyzed utrs of short and long isoforms of human itsn mrnas and found conservative target sites for mir- , mir- , mir- , mir- / , mir- , mir- and mir- in utr of itsn -s, predicted by servers, mir- predicted by servers for itsn -l, and mir- , mir- / , mir- , mir- and mir- predicted by - servers for itsn -l. to elucidate potential impact on cme, mapk signaling and actin cytoskeleton regulation by these mirnas we performed enrichment analysis by diana-mirpath server and found that mir- , mir- , mir- / , mir- , mir- and mir- were highly enriched for all analyzed pathways. using regrna . and scan for motifs services we predicted types of different regulatory elements in utr of itsn and itsn : k-box and brd-box, musashi binding element for rbps musashi and musashi , gu-rich element (gre) and au-rich elements (are) that regulate mrna decay. to confirm itsn -s regulation by micrornas we cloned utr of itsn -s into luciferase reporter vector and transfect hek cells by this construction and mir- a, mir- a and mir- a. for mir- transfected cells, we found - % decrease of expression of itsn -s utr-bearing construction. for other mirs we did not obtain strong reproducible effect in luciferase assay. these data may confirm mir- target site in utr of itsn -s mrna but needed additional research. objective: stroke is an acute neurological disorder that is mostly caused by ischemia in central neural system. % of stroked patients lose their life in year, and remaining / of the living patients continue to their lives as dependent to others. nihss and mrs are two scales which are used in prognosis studies because they can show stroke intensity and after stroke functional recovery. microrna's which have effects on transcription and posttranscription gene regulations are small rna molecules. their roles have been investigated on pathophysiology and treatment of diseases. in this study, it was aimed to detect changes in blood serum levels of mir- a, - , - , b, - , , - a and let- f of ischemic stroke patients and to investigate role in predicting prognosis methods: patients diagnosed by acute ischemic stroke admitted to neurology service of g€ oztepe hospital and healthy individual were included in the study. after stroke patients' blood samples were taken periodically in st day, st week, and rd month, and at the same time nihss and mrs scores were determined. set mirna blood serum levels were measured by rt pcr results: when compared to the control group, we found that after stroke st day peripheric blood levels of mir- a,- ,- a and let- f were significantly low; when st week and st day serum records were compared there was a significant increase in mir- level; and when st week and rd month records were compared we noted that there was a significant increase in mir- a,- and let- f levels. from prognosis point of view; after ischemic stroke measurements showed that mir- b in the st day, mir- a and mir- in the st week showed positive significant correlation with rd month mrs scores (p = . , p = . , p = . , respectively) conclusion: according to the outcomes of this study, after stroke st day mir- b, st week mir- a and st week mir- levels can be stipulated to use in predicting patients' rd month prognosis p- . . - inhibitory rna aptamer against lambda ci repressor showed the transcriptional activator activity s. ohuchi, b. suess tu darmstadt, darmstadt, germany because of the variety of functionalities on gene regulation and the easiness of molecular engineering, functional rnas are promising parts for the construction of genetic circuits. artificial affinity rna, or rna aptamer, is one of such genetic parts. in the previous study, an inhibitory rna aptamer against a repressor protein, tetr, was developed as a transcriptional activator [ ] . the expression of this aptamer abolishes the repressor activity of tetr, resulting in the elevated gene expression under the control of tetr. because of simplicity of the mechanism, similar transcriptional activators can be generated by using rna aptamers against other repressor proteins. here, we examined the generation of an activator based on an rna aptamer against one of the most frequently applied repressor proteins, lambda phage ci. in vitro selection (selex) was performed targeting a recombinant ci protein employing an rna pool containing -nucleotides of a random region. after rounds of selex, the pool rna showed the affinity, as well as the inhibitory activity, against ci in vitro. then, rna aptamers with the transcriptional activator activity were screened from the enriched pool in vivo employing a reporter plasmid on which the expression of a reporter gene, lacz, is repressed by ci. when the variants of the rna pool were transformed to e. coli cells harboring the reporter plasmid, about half of the transformants showed the elevated reporter expression. interestingly, all of these desired rna clones shared the same sequence. quantitative analysis indicated that -fold induction of the reporter expression was achieved upon the aptamer expression. our results suggested that diversity of artificial transcriptional activators can be extended by employing rna aptamers against repressor proteins to broaden parts for the construction of genetic circuits. [ ] hunsicker, et al. ( ) an rna aptamer that induces transcription. chem. biol., : - . p- . . - microrna expression signatures between non-atherosclerotic plaque and atherosclerotic plaque in cad with humans, and parallels whole blood rnas and represent a new important class of gene regulators. the present study was designed (i) to investigate the mirna expression profile in human atherosclerotic plaques from peripheral arteries aorta as compared to non-atherosclerotic left internal mamary artery (lima); (ii) to examine the expression levels of mirna in whole with correlation mirnas of aorta tissue. material and methods: thirty-one patients with cad were enrolled in study. lima and aorta tissue samples were obtained during coronary artery bypass surgery. whole blood samples were collected before cabg surgery. each patient with cad was obtained from whole blood, aorta and limas tissues. these tissue samples were immediately soaked in rnalater solution and homogenized using a magnalyser. the rna was extracted using the trizol reagent and the mirneasyÒ mini-kit. the expression profiles of mirnas were evaluated using highthroughput qrt-pcr. results: we found that mir- - p was expressed only in aorta. mir- - p and were expressed both aorta and whole blood. mirnas were significantly up-regulated in aorta when compared to lima tissue (fc > ). mirnas were significantly down-regulated in aorta compared to lima. conclusion: in conclusion, our study suggests that mir- - p, mir- - p and might be a potential for cardiovascular disease development. also mir- - p and might serve as novel non-invasive biomarkers for cad p- . . - mir b regulates cell proliferation and colony formation in pancreatic ductal adenocarcinoma n. gurbuz, e. isildar due to the strong metastatic potential, pancreatic cancer has the highest mortality rate of all major cancers. therefore, the investigators are in urgent need of developing the new alternative therapeutic approaches for the prevention of pancreatic cancer. mirnas, small noncoding rnas, regulates as an inducer or inhibitor in expression of key mediators related molecular mechanisms in cancer promotion. to investigate the effect of mir b on pancreatic ductal adenocarcinoma cells, we performed the cell viability and clonogenic assays by mts and crystal violet dye, respectively, in panc- and miapaca- cells transfected with mir b mimic. our data revealed that mir b led to decrease the cell viability depending on enhanced mir b doses, which are , , and nm, as the ratio of % , , and , respectively, in miapaca- cells and as the ratio of % , , and , respectively, in panc- cells compared with control condition of each cell. this inhibition mediated by mir b was also obtained in colony formation both of pancreatic cancer cells. when the induced effect of mir b on the death of pancreatic cancer cells was compared with gemcitabine, which is currently used as a clinical drug for pancreatic cancer patients, we determined that mir b was more effective than gemcitabine. based on our findings, it is clearly shown that mir b serves as a tumor suppressor in pancreatic ductal adenocarcinoma cells. we strongly believed that mir b gene therapy might be more effective and targeted approach than classical gemcitabine therapy for pancreatic cancer patients. *this work was supported by tubitak (the scientific and technological research council of turkey) grant s . breast cancer is the most common cause of cancer death in women. trastuzumab is a therapeutic agent frequently used against her + breast cancers, which has role in approximately % of invasive breast cancers. with the discovery of their activity in cancerogenesis, micrornas (mirnas) have become potential candidates to mediate therapeutic actions by targeting genes that are effective in drug response. recent studies have showed that mirnas are induced by targeted therapies. in this study, we aim to find out mirna-mediated mechanism, which is driven by common trastuzumab responsive micro-rnas in her + breast cancer. for this purpose, the common trastuzumab responsive mirnas were determined in treated bt and skbr cells by microarray profiling. two datasets were intersected to find out common mirnas for both cell lines. the overlapping predicted targets of common mirnas were provided by two different mirna-target prediction databases and then a mirna-gene network was built in cytoscape by using networkanalyzer plugin. the most shared target genes were chosen to be analyzed in the ebi-embl gene expression atlas for their expression patterns in breast cancer. common mirnas were found to have overlapped targets in two target prediction algorithms that were used to build the mirna-gene regulatory network. overlapped targets were determined as the most shared genes in the mirna-gene network. expression pattern of each shared gene in the gene expression atlas showed that out of the most shared target genes were strongly dysregulated in several breast cancer types. our results suggest that mirnas might show a common response to the targeted therapies and network analysis can be profitable to have a better explanation of the regulatory mechanisms between drug responsive mirnas and their target genes. revealing the mirna-potential target interactions might provide novel key players that mediate the mechanisms of action in drug treatment. chronic myeloid leukemia (cml) is a clonal disease of primitive pluripotent stem cells that identified with a specific t( ; ) reciprocal translocation that encoding bcr-abl oncoprotein. resveratrol (res) is a natural phytoalexin found in grapes and induces apoptosis, erythroid differentiation and autophagy in leukemic cells. micrornas are small, single strand, non-coding rna molecules that regulate post-transcriptional gene expression via disrupting the stabilization of target transcripts or inhibiting protein translation. in our study we aimed to determine cytotoxic effect of res in k human cml cell line and to evaluate the expressions of mirnas that are associated with genetics of leukemia after treatment with res; to investigate target genes of mirnas which show significant expression alterations and molecular mechanisms of res treatment. k cells were treated with lm (ic dose) res during h and cytotoxicity was evaluated by using wst- assay. the rt-qpcr is used for mirna and gene expression analysis. results showed that; res up-regulated tumor suppressor mir- - p level . fold and significantly downregulated hdac gene expression (p = . ) and upregulated p / sqsmt gene expression (p = . ), according to the control cells.p /sqstm interacts with lc and plays role as a critical player in the autophagic degradation of the bcr-abl fusion protein. our findings showed that resveratrol acts as a hdac inhibitor targeting hdac and p /sqsmt gene expression level. treatment with hdac inhibitors results apoptosis, cellcycle arrest, cell differentiation, anti-angiogenesis and autophagy. downregulation of hdac provides post-translational modification for expression of tumor supressor genes and leads to cell cycle arrest and increases apoptosis. these results could be linked to hdac dependent induction of gene associated with autophagy like p /sqsmt and resveratrol could be a therapeutic candidate as a hdac inhibitor for cml treatment. the mirna used in this study are hsa-mir- , hsa-let- a, hsa-mir- b and hsa-mir- . thereafter, we bought pre-mirnas and their mirnas commercially. we apply them to the a cell line in different combination and different concentrations. these mirnas applied solely onto cells or in combination as; four of them, let + , let + b, let + , + b, + , + let + b, + let + b. the cell viability was detected by wst- kit in a well plate elisa reader. cells were seeded as per well, mirnas incubated with cells for h in an appropriate atmosphere. according to our results some combinations and mirnas didn't alter viability, however + b and + combination increased the cell viability dramatically. on the other hand let + b and only applications decreased the cell viability. the other applications' viability results are not different from the control cells significantly. in this study, we used a cell line also called non-small lung cancer (nsclc) cell line and possibly effective mirnas on lung cancer. it is important to exhibit the mirna combinations should be effective on cancer cells' viability. the prospect combinations were determined which is crucial to develop new strategies for cancer treatment. competing endogenous rnas (cernas) act as molecular sponges for the same pool of micrornas through their mirna response elements (mre), titrate mirna levels and thereby regulate gene expression post-transcriptionally. smad interacting protein (sip ), a member of the zeb family is a regulator of epithelial-to-mesenchymal transition (emt) program, which is active during embryogenesis and tumor invasion and metastasis. hence, we investigated the regulation of sip by cernas in hepatocellular carcinoma (hcc) cells. among hundreds of sip cernas listed at competive endogenous mrna database (cerdb), transcripts (pten, zeb , ptch , creb , acvr b, enah, robo , erbb , e f , foxo , rictor, klf , ets , cdk ) sharing at least common mre sites with sip were selected and their expression in hcc cell lines were determined by qrt-pcr. ets was found to be highly correlated with sip in hcc. furthermore, repressing sip expression by shrna in hcc cells resulted in decreased expression of ets , pten and zeb . our results suggest a possible bidirectional and post-transcriptional regulation of sip and its cer-nas in hcc. a meta-analysis for the identification of common microrna signatures in colorectal cancer n. sahar , , n. belder, h. ozdag biotechnology institute, ankara university, ankara, turkey, comsats institute of information technology, islamabad, pakistan colorectal carcinogenesis (crc) has quite frequent incidence and mortality rates worldwide, despite being studied for decades now. new biomarkers are needed to be identified in addition to the existing ones, due to heterogeneous nature of this disease. the regulatory molecular machinery of a cell, including micrornas (mirnas), contributes to this heterogeneity upon aberrant expression. herein, for a mechanistic understanding of differential gene expression in crc tissue we analyzed mirna expression profiles of crc tumors against normal colorectal mucosa samples, using raw data from e-mtab- and e-geod- (affymetrix microarrays), and gse and e-mtab- (agilent microarrays) datasets obtained from gene expression omnibus and arrayexpress. raw samples were normalized (different platforms separately) using quartile normalization in brb-array-tools. differential expression of mirnas was identified using cut-off values of p ≤ . , fold change ≥ . and stringent false discovery rates. mirtarbase and mirwalk . databases were explored to identify validated targets. we found thirty (including mir- and mir- ) and thirteen (mir- , mir- , mir- , etc.) mirnas commonly upregulated and downregulated respectively, in both affymetrix and agilent microarray results. predicted targets of these mirnas frequently belong to pathways related to cancer like b-catenin, phosphoinositol- kinase, and transforming growth factor-b, to name few. moreover, the target genes were significantly enriched in clusters related to cell cycle, cell differentiation and regulation of apoptosis. these promising results will further be compared with differentially expressed gene profiles from a cohort of turkish crc patients. hence the integrated study will refine the panel of diagnostic and prognostic crc markers. hsa-mir-x modulates motility and invasion in triple breast cancer cell line s. noyan , h. g€ urdal , b. g€ ur dedeoglu biotechnology institute, ankara university, ankara, turkey, department of pharmacology, ankara university, ankara, turkey breast cancer is a heterogeneous disease and expression levels of certain receptors have demonstrated subtypes which characterize clinically distinct breast tumors. a triple-negative phenotype lacks expression of er, her and pr and is known as basallike carcinoma. micrornas are a class of small non-coding rnas that participate in the gene expression in many biological processes. e-cadherin is an important mediator of adhesion in epithelial tissues, and loss of e-cadherin can play a critical role in tumor invasive behavior. another key player of cell integrity pip ( , , ) triphosphate is generated at the leading edge of the cell and leads to cell polarization. pip is generated by hydrolysis of pip ( , ) bisphosphate, which is synthesized by pip k . any dysregulation in these molecules may support the invasive behavior of the cells. the aim of this study is to find out the role of mirna precursor (hsa-mir-x) in invasion and motility in triple negative breast cancer cells. in this study a triple-negative breast cancer cell line bt- was transfected with hsa-mir-x or scrambled control sirna. to check its role in motility and invasion, wound healing and invasion assays were performed respectively. cell invasion was monitored over a period of h by xcelligence real-time cell analyzer using a double-plate and measuring impedance-based signals. additionally emt markers were analyzed by qrt-pcr to explain the molecular mechanisms beneath motility and invasion. we observed that cell motility and cell invasion diminished after transfection of bt- cells with mimic for hsa-mir-x. furthermore, qrt-pcr experiments indicated that transfection of hsa-mir-x decreased the expression level of pip k c while increasing the e-cadherin expression level. wound healing and invasion assays together with qrt-pcr results support the role of hsa-mir-x in cell motility and invasion. this process might be explained via e-cadherin mediated met or gsk- -beta related inhibition of invasion. expression level of five micrornas as diagnostic markers in glioblastoma situated in the main brain lobes, but can also be found in other brain regions. while microrna (mirna) as non-coding rnas, play a crucial function in the post-transcriptional regulation of gene expression by mrna degradation or translational repression. in the present study, we aimed to investigate the contribution of gene expression of the five mirnas and to unravel their role in brain tumor cell lines, the mirnas to the risk of gbm tumor. the five gbm cell lines (crl- , crl- , crl- , crl- and htb- ) were evaluated with non-malignant (normal) brain cell line (hcn- ) . determinations of expression level of five mirnas (mir- , mir- , mir- , mir- , and mir- ) were evaluated by monitoring quantitative rt-pcr (qrt-pcr) technique. the expression levels of four mirnas (mir- , mir- , mir- , and mir- ) were significantly decreased while the expression level of mir- was increased in gbm cell lines according to hcn- cell line. consequently, these five mirnas can potentially be used as biomarkers for gbm tumor; further studies are mandatory to a better understand and confirm our preliminary findings. background: noncoding rna are known to be crucial molecules with diverse regulatory roles in neural oncology and neurodegenerative disease. the recent study suggested that lncrna anril play role in the development of neuroblastoma and alzheimer disease via binding disease-specific micrornas. material and methods: we used lncrnadisease, hmdd v . , mir disease to predict lncrna-and mir-associated disease in our study. in addition, we utilize tardetscan to search lncrna-mirna interaction. results: disruptions of lncrna anril expression (also named as cdkn b-as, locus cdkn a/b (ink /arf), chromosome p ) have been associated with the development of neuroblastoma and alzheimer disease. here, we predicted interactions between noncoding transcripts encoded by locus cdkn a/b and their molecular partners -microrna. anril can act as decoy while containing sequences that mimic mirna target sites to titer these mirs away from their primary targets thereby act as molecular sponge. using targetscan . , we predicted target sites for hsa-mir- -p/ -p/ -p/ -p/ -p/ -p and hsa-mir- - p/ in anril utr. then, we used hmdd v . and mir disease databases to define if any of these mirs participate in alzheimer disease and neuroblastoma. according to both databases, mir- is implicated to alzheimer disease and mir- to neuroblastoma. as soon as anril participate in the development of both abovementioned disorders and can have microrna sponge activity, it could potentially positively regulate mir- and mir- targets by competing with them for micro-rna binding sites thus restoring the expression of target genes. in our further research we plan to experimentally validate predicted microrna sites in anril utr. conclusions: we predicted sites for mir- and mir- in utr of anril lncrna that could uncover its possible sponge activity in the development of neuroblastoma and alzheimer disease. aim: matrine excracted from saphora flavescens root and demonsrated that indicates pro-apoptotic and anti-proliferative effect in many types of cancer. acute lymphoblastic leukemia (all) is an acute form of leukemia, or cancer of the white blood cells which characterized by the overproduction and accumulation of lymphoblasts. mirnas play important roles in deregulated cell death mechanisms. we aimed to investigate the effects of critical mirnas during the development of matrine resistance on all cell line ccrf-cem. material-method: ccrf-cem cells were treated with different ( . - mg/ml) concentrations for h and cell viability measurements were carried out with xcelligence device to determine the cytotoxic effects of matrine. mirnas were extracted from treated and untreated ccrf-cem cells using the mirna isolation kit. cdna was synthesised using allin-one first strand cdna synthesis kit. expressions of selected mirnas were analysed with miprofiletm custom mirna qpcr array using the applied biosystem fast real-time pcr system. results: according to the cytotoxicity assay, it was determined that 'treatment with increasing concentrations of matrine, decreased the viability of the ccrf-cem cell line. expression levels of different mirnas were studied for indicated passages in two replicates. our results showed that hsa-mir- b- p (- , fold, p = . ), hsamir- - p (- , fold, p = . ), hsa-mir- a- p (- , fold p = . ), hsa-mir- a- p (- , fold, p = . ), hsa-mir- - p (- , fold, p = . ), hsamir- b- p (- , fold, p = . ), hsa-mir- b- p (- , fold, p = . ), hsamir- b- p (- , fold, p = . ), hsa-mir- - p (- , fold, p = . ), hsamir- a- p (- , fold, p = . ) expression were decreased during the development of matrine resistance. conclusion: these data suggested that especially hsa-mir- b- p plays a critical role in the matrine response. ericd (e f -regulated inhibitor of cell death) is a newly found lncrna. it is located at q . . it has two exons and its transcript size is bp. ericd is regulated by e f (transcription factor ) and modulates the cellular response to dna damage. arid a is a family member of the at rich interaction domain (arid) dna-binding proteins that participate in diverse biological processes like development, cell cycle control, chromatin remodeling and epigenetic regulation. both, ericd and arid a have just opposite roles in apoptosis in case of dna damage indicating a probability of reciprocal interaction between each other. till now, there is no work related to the interaction between lncrna and arid a in cancers. herein we try to find a probable interactive role between these in cancers. in this study, different cancer cell lines, osteoblast cell line and different types of normal human tissues rnas were selected for expression analysis of ericd and arid a. after rna isolation, cdna was converted from their rnas. expression profile analysis of ericd and arid a in different cancer cell lines and normal tissues was done using imagej program for semiquantitative and (-ddct) method for quantitative rt-pcr. among used cancer cell lines, ericd was highly expressed and arid a had lower expression in u- os (osteosarcoma), a- (glioblastoma) and a (lung cancer). at the same time, ericd expression was lower and arid a had high expression in hfob . (osteoblast cell line) and normal tissues like brain and lung . both ericd and arid a are cell cycle regulated and are commonly regulated by e f. they have just opposite roles in apoptosis during dna damage. these two genes have a probability of reciprocal interaction between each other in cancer. our results indicate that both ericd and arid a might have opposite roles in lung cancer, glioblastoma and osteosarcoma. ericd and arid a might act as cancer promoting and tumor suppressor genes respectively in these cancers. the importance of mirna expressions in infertilty implantation process is controlled with endometrium, factors secreted by the embryos and in accordance with these factors embryo and/or endometrium via receptors on. more than human microrna (mirnas) that are small noncoding rnas were shown to play an important role in intracelluler cycle regulation in both normal and pathological conditions. in this study we aim to identify mirnas and controlling molecules expressions in different time period of endometrium in fertile and infertile cases. the endometrial samples were taken from fertile and infertile patients in proliferation and early secretion periods. the samples are fixed and stained either with hematoxylen-eosin for morphological analysis or with immunohistochemistry for distributions of anti-dicer, anti-drosha, anti-eif a and anti-eif c. mir- - p, mir- a, mir- b, mir- - p, mir- , mir- a*, mir- , mir- a, mir- a, mir- b, mir- a/b/c were analyzed with qrt-pcr. while dicer immunoreactivity was detected weakly only proliferation phase of fertile group, this immunoreactivity were detected strongly in both proliferation and early secretory phases of infertile group. drosha immunoreactivitiy was also weakly detected in the proliferation phase of fertile group, it was moderately detected in both proliferation and early secretory phases of infertile group. eif a immunoreactivitiy was similar in each groups but there were a few differences between fertile and infertile group. eif c immunoreactivitiy was negative in all groups. mir- , mir- a* and mir- a were highly expressed in proliferation phase of fertile group, mir- a and mir- b were highly expressed in early secretion phase of infertile group. in conclusion, dicer and drosha immunoreactivities and different expression of mirna's were detected in all groups. implantation problems may be reason for different mirna expression which controlling with dicer and drosha in the infertile endometrium in both proliferation and early secretory phases. wheat is an important agricultural crop with an over . million metric tons harvesting capacity annually. drought and salinity are environmental stress factors that affect yield and quality of wheat, dramatically. there are different defense mechanisms against these stress conditions in plants. altering gene expression profiles by micrornas at post-transcriptional level is one of the most conserved mechanisms among plants. micrornas are an extensive class of noncoding rnas, approximately nucleotide length which regulates the expression of genes by binding to the -untranslated regions of specific mrnas. micrornas implicated under salt and drought stress have widely been reported in numerous plant species and wheat genomes in the last years; however, studies on einkorn wheat (triticum monococcum spp. monococcum) are not yet available. the goal of this study is identification of conserved micrornas from einkorn wheat using next generation sequencing technology and bioinformatic analysis. in this study, small rna molecules were extracted from pooled plant samples grown under normal, drought and salinity conditions. sequencing analysis revealed unique small rna sequences obtained from raw reads. after bioinformatic analysis based on comparative genomics approaches, we identified putative mature microrna sequences belonging to distinct microrna families. since chromosomal sequence data is not available for triticum monococcum spp. monococcum, we used available sequences from triticum urartu, a close relative, as template to extract precursor microrna sequences. of precursor sequences showing % homology to triticum urartu genome were analyzed for secondary structure prediction using mfold software. data provided in this study is critical to investigate transcriptional regulation of genes involved in stress metabolism in einkorn wheat. the role of vim-as , a natural antisense transcript, in cancer coding or non-coding transcript. in this regard, vim-as is nats located antisense to vimentin gene. in the present study, we aimed to determine tissue and cell line distribution of vim-as . materials and method: for the tissue expressions analysis, human total rna master panel ii (containing different human tissue samples) was used. total number cancer cell lines and normal cell lines included in the study. for the expression analysis rt-pcr and qpcr methods were used. results: as a result, expression levels of vim-as were found to be tissue specific. particularly, while vim-as was highly expressed in lung and thyroid gland tissues, its expression was not observed brain, stomach and adrenal gland tissues. also, vim-as was also found to be differentially expressed in cancer cell lines. vim-as expression was found to be lost in cal , pc , and hct and highly diminished a cancer cells. also, it is found to be highly expressed in bcpap, panc and beas b cells. discussion: results of the current study suggest that vim-as may have significant role in the regulation of vim gene in thyroid gland tissues, as it is highly expressed in both thyroid gland tissues and bcpap thyroid cancer cells. moreover, vim-as and vim axis can be involved in the formation of lung tumors because vim-as was highly expressed in normal lung tissues and beas b cells and expressed very low levels in a lung cancer cells. lung cancer is the leading cause of cancer related deaths in the world and approximately % patients with lung cancer ultimately die from metastatic disease. metastasis is the most dangerous step of cancer. in our recently published work showed that akt/nf-kb pathway is continuously active and induces cellular invasion and pten suppresses cellular invasion via inhibition of akt/nf-kb pathway. in this study we aimed to show nf-kb mediated induction of mirna expression can responsible for inducing nsclc invasion. we used chromatin immunoprecipitation (chip) assay kit for detection of tnf-a induced nf-kb mediated mirnas. therefore, h and pc cells treated by tnf-a ( ng/ml) for chip assay. chromatin regions, reading with chip-seq, were analyzed using bioinformatics tools. we also performed additional bioinformatics search to find nf-kb related mirnas which potentially take a role in nsclc invasion. we investigated the effects of mirna which determined at the bioinformatics analysis results on invasion using invasion chamber method. we found mirnas which potentially induced by nf-kb and related with nsclc invasion. our invasion results indicate that mir- a- p, mir- as- p, mir- , mir- , mir- - p, mir- q mimics can induce cellular invasion on h , mir- v, mir- h- p, mir- - p, mir- a- p, mir- as- p, mir- mimics can induce cellular invasion on pc . we also verified our results by qrt-pcr, because we want to sure that mirnas which can induce invasion, can also transcriptionally regulated by nf-kb or not. we found that mir- q, mir- a- p, mir- as- p, mir- , mir- in h , mir- - p, mir- a- p, mir- as- p, mir- in pc can induce cellular invasion by nf-kb. as a conclusion, our investigation indicate that nf-kb can induce nsclc invasion via mir- a- p, mir- as- p and mir- . (this study is supported by tub _ itak grand number s ). to understand of the molecular mechanisms of cellular invasion is very important for fight against cancer mechanisms and first step of invasion is emt. cells change phenotypical and genotypical in emt progress. in this study, we focussed on the explore molecular mechanism of tnf-alpha induced cellular invasion of nsclc. we use western blot, qrt pcr and mirna transfect methods for this purpose. we find that tnf-alfa treatment reduce the expression of pten and induce e cadherin expression in a cells. when we inhibit nf-kb activity by p targeted sirna tnf-alpha can not reduce pten expression means that tnf-alpha inhibits pten expression through on nf-kb. because tnf/nf-kb pathway change the transcriptional level of mir- as and pten untranslated region has recognition site for mir- as. therefore; we transfected a cells by mir- as. mir- as transfection leads to inhibition of pten expression. our results indicate that tnf-alpha mediated activation of nf-kb can inhibit pten expression on induction of emt through on mir- as. (this study is supported by tubitak grand nummber s ). introduction: the corpus luteum (cl) is an ephemeral tissue whose regulated secretion of progesterone is essential for maintenance and/or timely termination of pregnancy in rodents. our previous finding that cl of pregnant rats does not possess fas/ fasl system suggests that this tissue may undergo autophagic, but not apoptotic, cell death during regression. we here investigated the presence of autophagic system in cl and its any implications in rodent pregnancy and parturition. materials and methods: lc (-i and -ii) expression in cl was estimated by western blot analysis in comparison with progesterone secretion and luteal mass throughout pregnancy. lc was also tested by immunocytochemistry. functional implication of autophagy in this tissue was examined by local treatment with bafilomycin a (autophagy and v-atpase inhibitor, . pg/ . ml/ovary) on day of pregnancy. reproductive, biochemical, and morphological outcomes were evaluated. results: while the expression of cytosol-associated lc -i was not significantly altered throughout pregnancy, that of autophagosome-associated lc -ii increased significantly from day , showed a peak on day , and decreased on day (day of normal parturition). lc -ii / i ratio had positive correlations with steroidogenic activity and cell size. immunoreactive lc was found to be present in the cytosol of steroidogenic cells and showed marked aggregation in cells on day . in vivo treatment with bafilomycin a resulted in unaltered delivery, but in significant reductions in steroidogenic cell size and neutrophil infiltration compared to vehicle-treated control groups. discussion and conclusion: the ratio of lc -ii / i in cl was markedly up-regulated in the late phase of pregnancy. manifestation of this autophagy parameter was temporally matched with further structural and functional activation of cl. autophagy may contribute to activation, but not regression, of rodent cl and thus their female reproduction. apoptotic and necrotic effects of low dose bisphenol a in shsy y neuroblastoma cells b. ayazg€ ok, t. t€ uyl€ u k€ uc ߀ ukkilinc ß faculty of pharmacy, hacettepe university, ankara, turkey bisphenol a (bpa) is a commonly used chemical in industry to make plastics. 'low-dose' term has been expressed for the first time in studies with bpa in . the value of low dose bpa was received as < lm. in this study, matrix metalloproteinases (mmps) together with their tissue inhibitors (timps), involved in tissue remodeling after i- therapy, have been examined in patients ( m/ f) with ptc and ( m/ f) with ptc+ht. peripheral blood samples were collected just before and, subsequently, at days after i- administration ( . gbq). ptc+ht patients had positive titers of anti-thyroglobulin autoantibodies (tgab). the serum levels of tgab, mmp- , mmp- , timp- and timp- were measured by elisa. there were no significant changes in serum concentrations of mmp- , timp- and mmp- /timp- ratio after i- in the two groups. in ptc patients, i- administration resulted in an increase with % in timp- level (p = . ) and a reduction with % in mmp- /timp- ratio (p = . ). in ptc+ht patients it has been observed an increase with % in tgab level (p = . ), % in mmp- /timp- ratio (p = . ) and unchanged timp- serum concentration. tgab titers were positively correlated with mmp- /timp- ratio (r = . , p < . ). our data suggest that radioiodine therapy for ptc patients, but not for ptc+ht, modulates the balance of mmp- /timp- for anti-invasion and anti-migration by augmenting timp- . in criminal cases, the determination of the time of death of the bodies step in the analysis of events is making a big contribution. today, forensic and molecular methods utilized time of death rather than provide clear information offers potential death time interval. principally, 'time of death' is a term that should be avoided. in forensic science, the postmortem 'interval' is the term to be considered. nowadays, there is no precise molecular method that can be used alone in the determination of pmi. aim of this study is to analyze the usability of ecm components, adamts , and and mmp , and to determine pmi. for this purpose, with iliopsoas muscle tissues provided by ethical board of the ankara institute of forensic medicine, cases have been included in this study, divided into groups according to their pmis: ' - h', ' - h' and ' - h'. from these tissues, western blotting technique is used to analyse the expression of adamts , and and mmp , and . it is determined that when pmi increases; adamts- and amounts decrease. on the other hand adamts- amounts are examined to increased related to the interval., especially on the ' - h' dataset. additionally, considering metalloprotease levels, mmp- and amounts decrease as pmi increases. unlike mmp- and ; mmp- levels increase proportional to the interval, especially on the ' - h' dataset. these results are the first data on pmi determination from iliopsoas muscle tissue. in this study, it is suggested that adamts- and mmp- , proteases responsible for ecm degradation, can be used to determine pmi as markers. here we present the first observations of postmortem variation of mmp and adamts activities in skeletal muscle. in recently, popular mmps and adamts s pathways of the relationship between time-dependent changes to investigate the post mortem time interval determination to shed light on the future. the role of functional polymorphisms of matrix metalloproteinases and in spontaneous abortion samples capillaries, which are responsible for maintenance of implantation and placental nutrition, have major effects on mechanisms underlying abortion. matrix metalloproteinases (mmps) function in various cellular pathways. they play a role in patholohical conditions, metastasis; as well as normal physiological functions like tissue and bone regeneration, physiologic function of uterus, ovulation, embryogenesis and embryo implantation. mmp and mmp (gelatinases) have key roles at organisation of extracellular matrix and affect endometrial implantation. previous studies have shown that mmp - c>t and - c>t polymorphisms cause loss of gene function, and mmp - c>t polymorphism causes a decrease in gene expression, and also gene expression levels are lower in abortion specimens, compared to control specimens. the goal of this study was to investigate the potential effects of functional mmp - c>t and - c>t polymorphisms, and mmp - c>t polymorphism on etiology of abortion. restriction fragment length polymorphism (rflp) method was used to analyze the polymorphisms those evaluated in the study. study group consisted of samples collected from spontaneous abortion specimens, and control group consisted of peripheral blood blood samples collected from healthy subjects. the risk of abortion was . -fold higher in patients with heterozygous - c>t polymorphism (p = . ). combined genotype analysis showed that the risk of abortion was . -fold higher for patients with normal - c>t polymorphism, and heterozygous - c>t polymorphism (p = . ). functional polymorphisms of mmp and mmp may have a role in etiology of abortion. p- . . - changes in the specific extracellular matrix proteins and expression of adamts proteinases and effects of hypoxia in the adriamycin-induced renal fibrosis model renal fibrosis is a common cause of renal dysfunction with chronic kidney diseases. this process is characterized by excessive production of extracellular matrix (ecm) or inhibition of ecm degradation. adamts proteinases, which are widely presented in mammals, have very critical roles in ecm remodeling. we aimed to study the role of adamts proteinases in the pathogenesis of renal fibrosis and the effets of hypoxia by studying adamts expressions in rat kidney after adriamycin (adr) administration. adr was administered intravenously in consequtive two doses ( . and mg/kg) to the rats. in addition to control and adr groups, another rats were assigned into three groups as sham, min and min ischemia-reperfusion. after months following the first dose, the expression of adamtss were determined in the renal tissues using western blot analysis. additionally, renal nephropathy and fibrosis were assessed pathological and immuno-histochemical staining methods. in the adr group rats developed renal dysfuntion and tissue damage in favor of renal fibrosis pathologically. it is observed that occurred remarkable changes in the expression of adamtss. it is showed that hypoxia and hypoxia time enhanced tubulointerstitial fibrosis in the rat kidney tissues. expression differences were absorved also in the hypoxia groups, and especially the expression of adamts- and - genes were showed an increase in various rates. the restricted and different expression pattern of adamts protein profiles in the adr-induced renal fibrosis suggest that adamts family members are related with development and progression of fibrosis. moreover, our findings raise the possibility that the hypoxia promotes renal fibrogenesis. the monitoring of adamts proteinases might contribute to the early diagnosis of renal fibrosis and chronic kidney disease. adams (a disintegrin and metalloproteas) are transmembrane proteins that contain a pro-domain, a metalloprotease, a disintegrin, a cysteine-rich, an epidermal-growth factor like and a transmembrane domain, as well as a c-terminal cytoplasmic tail. they are involved in cell adhesion and they have protease activities. previous studies showed that some adam proteins act in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis, embryonic tgf-a release and the inflammatory response. although there are more researches about adam proteins, still the function of all adam proteins remain unclear. we aimed to investigate the potential link of infertilty with adam , - , and - . in this study twenty four patients were included. the patients were classified as normozoospermia (ns; n = ), oligozoospermia (os; n = ), azoospermia (as; n = ) groups. adam , - and - protein levels in infertility indviduals were analysed by western blot. adam protein level was . fold lower in the os and as groups than in the ns group. adam protein level was . fold higher in the as group than in the ns group. adam protein level was fold lower in the as group accourding to ns group. we observed no change between protein level of adam and adam of os and ns groups . in conclusion, adam proteins may have a potential role in male infertility. our study is a preliminary and first study on this issue. keywords: adam, infertility. the role of tissue metalloproteinase inhibitors thymus chemokine and thrombospondin- on prognosis of crimean-congo hemorrhagic fever s. bakir, m. bakir, s. ersan, a. engin cumhuriyet university, sivas, turkey crimean-congo hemorrhagic fever (cchf) is a disease which is caused by an arbovirus carried by ticks and characterized by the sudden onset of high fever, severe headache, dizziness, back and abdominal pain. cchf pathogenesis is still not resolved today to fully open. therefore, in this study, to determine the level of tck- , timp- and tsp serum samples obtained from cchf patients and the control group is intended to be examined for the pathogenesis and prognosis of the disease. the study sample was created patients with diagnosis of cchf. healthy volunteers were chosen control group matched for gender and similar to in terms of age. tsp, tpc- and timp- levels of patients and a control group were analyzed using the human elisa kits. serum timp- tck- and tsp levels in cchf patients were measured significantly higher than the in the control group. cchf pathogenesis of today still have not provided fully open. therefore, it reveals the importance of this work. in our study, presence of high timp- , tck- and tsp levels indicate important direction for pathogenesis and prognosis of cchf disease. p- . . - activation of calpain and protein kinase ca promotes a constitutive expression and release of matrix metalloproteinase in peripheral blood mononuclear cells from cystic fibrosis patients matrix metalloproteinase (mmp ) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies, including cystic fibrosis. we have studied if the alteration in intracellular ca + homeostasis, observed in peripheral blood mononuclear cells (pbmcs), isolated from cystic fibrosis (cf) patients homozygous for deletion of phenylalanine in gene of cystic fibrosis transmembrane conductance regulator (f del-cftr), could be involved in the abnormal presence of mmp in the extracellular fluids of cf patients. pbmcs were isolated from healthy donors and cf patients homozygous for f del-cftr. mmp levels were evaluated following h of cell incubation. our investigations show that all cf pbmcs analyzed constitutively express and release high levels of mmp ; conversely, in pbmcs from healthy donors, expression and secretion of mmp are undetectable but both events can be evoked, after h of cell culture, by a possible paracrine stimulation. we have demonstrated that in cf and h-cultured control pbmcs mmp secretion is prevented by chelation of intracellular ca + and mediated by the concomitant activation of calpain and protein kinase ca (pkca) and also that mmp expression is mediated by the sequential activation of pkc and extracellular signal-regulated protein kinases and (erk / ). moreover, the rescue of active f del-cftr reduces the extent of mmp secretion, correlating the channel defect to the [ca + ] i dysregulation of cf pbmcs. our results indicate that the high level of intracellular ca + concentration in cf pbmcs, promoting the aberrant activation of both calpain and pkca, induces a constitutive release of mmp . these data characterize new alterations in mononuclear leukocytes of cf patients that may be of primary importance in the progression of the disease and indicate that pbmcs may contribute to the accumulation of mmp in fluids of cf patients. p- . . - aebp /aclp is upregulated in differentiation, injury repair and fibrotic degeneration of skeletal muscle c. € ozdemir , , u. akpulat , i. onbasilar , c ß . kocaefe department of medical biology, faculty of medicine, hacettepe university, ankara, turkey, department of stem cell, institute of health, hacettepe university, ankara, turkey, laboratory animal breeding and research unit, faculty of medicine, hacettepe university, ankara, turkey aebp /aclp is an ambiguous gene with several attributed functions and cellular events, adipogenic differentiation, cell adhesion, pattern development and fibrosis are the well-understood. aebp is the short isoform that acts as a transcriptional repressor by targeting the ap promoter and aclp, which is the long isoform that harbors a leader sequence that directs the peptide to the extracellular compartment. the latter is known to be associated with development of the connective tissue, injury repair and fibrosis in certain pathological conditions. aebp /aclp displays a moderate expression in skeletal muscle where the role is not known. we have investigated the spatial and temporal expression of aebp /aclp in defined models of skeletal muscle differentiation, injury repair and fibrosis. aebp /aclp expression is present in steady state dividing myoblasts. this basal expression is upregulated folds upon the induction of differentiation in c c cells. considering that differentiation and post-natal injury repair share several common aspects, we also investigated the expression of aebp /aclp in acute injury-repair model. in the course of cardiotoxin induced injury, aebp /aclp expression peaks up to folds in the th day of regeneration. this time point concomitantly corresponds to tissue remodelling. since aebp /aclp is also known to be associated with fibrotic events in chronic pathological conditions, we also have investigated its expression in tenotomy induced skeletal muscle degeneration which mimics endomysial and perimysial fibrosis. aebp /aclp expression is upregulated up to folds in early time-point samples. these results depict a novel role for aebp /aclp in extracellular remodeling of the skeletal muscle during injury repair as well as fibrotic degeneration. the source of this expression might come from fibroadipogenic precursers which reside in endomisial area of muscle. our current efforts are focused on presenting of this endomysial expression. the mprbp gene from b. pumilus - encoding a novel secreted metalloproteinase was identified. based on the primary structure the enzyme has been classified as metzincin metalloproteinase that combines the features of two families: astacin and adamalysin. representatives of the adamalysin family previously have not been described for bacilli. the aim of the work was to elucidate the mechanisms of the gene expression regulation of a new bacillus pumilus - extracellular metalloendopeptidase. promoter region analysis revealed the presence of potential binding sites for transcription factors spo a (sporulation) and degu (biodegradation). study of mprbp expression in strain defective in regulatory proteins degs and degu shows that the productivity of metalloproteinase biosynthesis decline in average % compared with the strain with a complete degs-degu system. we also studied mprbp expression in strains with a mutation in the gene degu, leading to stabilization of degu~p protein. it is known, that this mutation leads to a multiple increase in the gene expression level, positively regulated by degs-degu system. our data shows a -fold increase in metalloproteinase productivity in the recombinant strain. thus, deg-system participates in control of the proteinase synthesis but not only in the regulation of mprbp gene expression. the mprbp expression in the strain deficient in regulatory protein spo a remained at the level with expression in the strain with the complete spo a. a similar pattern we observed in the study of mprbp gene expression in strains defective in other spore-specific regulatory proteins (spo b, spo f, spo k, spo j, sigf, sigh, sigk). these data indicate that mprbp gene expression is free of spo-regulatory proteins. on this basis, we concluded that the expression of metalloproteinase gene is not correlated with the sporulation. p- . . - paricalcitol attenuate activity and expression of matrix metalloproteinases in a rat model of renal ischemia-reperfusion injury matrix metalloproteinases (mmps) are endopeptidases involved in the degradation of extracellular matrix. they have been postulated to have a role in the pathogenesis of ischemia-reperfusion injury (iri). in the present study, we investigated the effect of paricalcitol, a synthetic vitamin d analog, on mmps in renal iri. wistar albino rats were divided into three groups: sham operated, ischemia-reperfusion, and paricalcitol-pretreated. iri model was induced by bilateral clamping of renal arteries for min followed by h of reperfusion. the analysis of serum creatinine levels and activities/expressions of mmp- and - were performed after h of iri. the effects of paricalcitol on activities and expressions of mmp- and mmp- levels were investigated by gelatin zymography and immunohistochemistry, respectively. the pathological examinations were performed to score tubular damage by light microscopy. creatinine levels increased significantly in the iri group. rats in the paricalcitolpretreated group showed significant decrease in expressions and activities of mmp- and mmp- during iri. moreover, pathological examinations displayed significantly lower score of tubular damage in paricalcitol-pretreated group. in conclusion, paricalcitol attenuated iri by downregulating the expressions and activities of mmp- and - . p- . . - the changes of matrix metalloproteinase , activity and hyaluronic acid level in rat's heart and serum under cadmium influence o. fomenko , o. shaulska , y. kot , g. ushakova , a. shevtsova dnipropetrovsk medical academy, dnipropetrovsk, ukraine, kharkiv national university, kharkiv, ukraine, dnipropetrovsk national university, dnipropetrovsk, ukraine the changes in the molecular mechanisms of the extracellular matrix degradation under toxic factors are not well known. the main goal of work was the investigation of the mmp and mmp activity and hyaluronic acid level in the heart and blood serum under cadmium influence at different doses. the wister rats divided to groups were used for the experiment. cdcl x . h o in doses . lg/kg and lg/kg was given to rats intragastrically in drinking water during days. the rats were decapitated under isoflurane anesthesia according to ethical rules; the heart was quickly removed. the relative activity [in arbitrary units (au)] of pro-and active forms of mmp and mmp , total protein (tp) and hyaluronic acid levels were calculated. it was shown that low doses of exogenous cadmium ( . lg/ kg) lead to reduced activity of pro-and active forms of mmp in myocardium ( . ae . au/mgtp and . ae . au/mgtp compare to the . ae . au/mgtp and . ae . au/mgtp in the control rats accordingly) and in serum ( . ae . au/mgtp and . ae . au/mgtp compare to the . ae . au/mgtp and . ae . au/mgtp in the control rats accordingly), but pro-mmp activity in heart was increased ( . ae . au/mgtp compare to the . ae . au/mgtp in the control rats); level of ha was decreased in both tissues ( . ae . lg/ml and . ae . lg/ml compare to the . ae . lg/ml and . ae . lg/ml in the control rats accordingly). high doses of cadmium ( lg/kg) caused a reliable increase of both gelatinase activity in the myocardium: mmp increased from . ae . au/mgtp to . ae . au/mgtp, prommp to . ae . au/mgtp, mmp to . ae . au/mgtp. ha level was increased in serum ( . ae . lg/ml) and decreased in heart ( . ae . lg/ml). the results indicate the dose-dependent and tissue-specific effect of cadmium on mmp-depended protein degradation and level of hyaluronic acid. a disintegrin-like and metalloproteinase domain with thrompospondin- repeats (adamts) are a large family of proteoglycanase that show proteolytic activity towards proteoglycans like aggrecan, brevican, neurocan, and versican. interleukin- (il- ) is an il- cytokine family member that uniquely plays a role as a cytokine and nuclear factor. it is released by necrotic epithelial cells and activated innate immune cells as an alarming danger signal. adamts and il- implicated in brain cancer pathogenesis. we aimed to seek the amount of adamts in u proteolytic enzymes are able to speed up wound healing by removal of the necrotic tissues and fibrin. several investigations have reported that proteases damage also the microbial biofilms formed by opportunistic bacteria including staphylococci on surfaces of chronic and acute dermal wounds. therefore, proteases are seemingly perspective enzymes for biofilm eradication by hydrolysis of both matrix proteins and adhesins, proteins providing cells attachment onto solid surface and other bacteria, as well as by the cleavage of signalling peptides of intercellular communication of gram-positive bacteria. here we report that ficin, a plant protease, efficiently degrades the structural components of biofilm matrix formed by s. aureus and s. epidermidis at concentrations of lg/ml while trypsin and chimotrypsin are used as - mg/ml solution. the spatial structure of the biofilm was analyzed by atomic force microscopy. after ficin treatment, the biofilm structure became porous, with reduced viscosity. the congo red staining of the treated biofilms confirmed the hydrolysis of the protein component of the matrix. moreover, the biofilm treatment with ficin increased the antimicrobial efficiency of ciprofloxacin against biofilm-embedded cells of s. aureus and s. epidermidis. while h antibiotic treatment did not lead to the increase of dead cells of neither s. aureus nor s. epidermidis embedded into the biofilm matrix, in the presence of ficin the fraction of viable cells decreased significantly. accordingly, soluble ficin appears beneficial for outer wound treatment biofilm eradication and reduces the reinfection risk. the wound-healing activity of ficin requires further investigations. this work was supported by the russian science foundation (project no - - ). resveratrol (resv) is an antioxidant that belongs to the group of plant compounds, called polyphenols. resv is defined as an antimicrobial substance that is produced by several plants (red grape skin, peanuts and berries) to protect them from rough environments like excessive ultraviolet light, infections and climate changes. as an antioxidant, this polyphenol protects the body against cardio-vascular and cancer diseases. besides, resv has anti-inflammatory, neuro-protective, anti-diabetic and other pharmacological effects. although the positive pleiotropic effects of this polyphenol are well documented, there is a huge need to understand its influence on the biophysical properties of lipid bilayer. in the present work, the interaction of resv with membranes composed of palmitoyl-docosahexaenoyl phosphatidylcholine (pdpc) or palmitoyl-oleoyl phosphatidylcholine (popc), sphingomyelin (sm) and cholesterol (ch) was investigated by means of fluorescence spectroscopy. generalized polarization of the fluorescent probe laurdan (gp) as a function of temperature was used to probe the changes in the fluidity of lipid bilayer induced by different resv concentration. the obtained results showed decreased lipid ordering from to lmol resv and opposite effect from to lmol in pdpc/sm/ch mixtures as compared to the control without resv. the interaction of resv with popc/sm/ch mixtures caused only an increase in the lipid ordering as a function of resv amount. popc and pdpc have the same head group but different fatty acid chains at sn- . since resv changes the physicochemical properties of lipid bilayer by different ways one might suggest that the interaction of polyphenol with the membrane depends on the level of fatty acid unsaturation. this specific effect of resv on lipid organization could be related to its unique properties to prevent the cell from oxidative stress. neurodegeneration is the umbrella term for the deseases including progressive loss of structure or function up to death of neurons. beta-amyliod peptide is proteolytic fragment of the amyloid protein. the spontaneously formation of selective, voltage-dependent, ion-permeable channels in the lipid bilayers was reported as one of amyliod peptide toxicity mechanisms. the aim of our study was the investigation of the influence of the flavonoids (phloretin, phlorizin, quercetin and genistein) on the membrane activity of amyliod peptides. virtually solvent-free bilayer lipid membranes were prepared from mixtures of phospholipids in . m kcl (ph . ) using monolayer-opposition technique. using spectrofluorimetry we estimated prepared from phospholipids by extrusion the liposomal membrane permeability for calcein. we found that the addition of phloretin into membrane bathing solution led to an signicant increase in the channel forming activity of fragments - of amyloid peptide, fragment - of [gly ]-amyloid peptide and - of human prion protein. addition of other flavonoids did not cause any changes in the steady-state amyloid-induced current. it was found that the effect was caused by electrostatic interaction with the peptide. we found that time course of amyloid induced leakage calcein from liposome's is characterized by two components: the fast one is related to sorption of b-amyloid peptide on the membrane and the slow one is related to the oligomerization of the peptides on the surface of the lipid bilayer. addition of the phloretin simultaneously with b-amyloid peptide to the suspension of liposomes caused significant reduction in time parameters characterizing fast and slow components. from this results we can proposed that phloretin compensates the positive charge of the b-amyloid peptides and leads to the changes in their oligomerization status. the study was supported in part by rfs ( - - ) and sp- . . . ferritin nanocarriers: a focus on a metal-based drug encapsulated inside the protein cavity s. ciambellotti , c. bernacchioni , f. scaletti , p. turano cerm (magnetic resonance centre), florence, italy, department of biochemical sciences, university of florence, florence, italy, chemistry department 'ugo schiff', florence, italy ferritin is one of the main player involved in the iron metabolism. the biological role of ferritin is the storage of iron atoms inside the cavity preventing the uncontrolled accumulation of toxic species inside cells. ferritins are polymers constituted by subunits that self-assemble giving rise to an almost spherical nanocage. in vertebrates, ferritins are formed by two distinct subunits, the heavy chain (h), with oxidoreductase activity, and the light chain (l) without catalytic activity. ferritins are promising nanocarriers for the delivery of contrast agents for diagnosis and of drugs for therapeutic purpose. their endogenous origin and the possibility to stabilize and protect the enclosed cargo inside the cavity, make ferritin a biocompatible vehicle. moreover, there are specific receptors on cells that recognize and incorporate ferritin by endocytosis, prospecting a kind of targeted-delivery. following the increasing interest in nanotechnology, we studied the interaction between different isoforms of ferritin with an antimetastatic drug, called nami-a, which is the first ruthenium derived anti-cancer drug to have entered clinical evaluation. this molecule is a metal-based prodrug that can release the metal ion ligands. here, we describe nami-a hydrolysis in the presence of recombinant homopolymers of ferritin followed spectrophotometrically. thanks to characteristic time dependent changes of spectral profile in the uv-visible region, we could detect differences in the hydrolysis process. the formation of a ru-adduct with hferritin was established by uv-visible and circular dichroism spectroscopies, as well as by kinetics measurements that showed inhibition of the ferroxidase activity of h-ferritin. crystallization trials are in progress to identify the binding site of ru by solving the x-ray structure of the complex. finally, we planned to test the cytotoxicity of ferritins pre-incubated with nami-a, using different cancer cell lines. a. cort , t. ozben , a. sansone , s. barata-vallejo , c. chatgilialoglu , c. ferreri sanko university, gaziantep, turkey, akdeniz university, antalya, turkey, cnr, bologna, italy, universidad de buenos aires, buenos aires, argentina, national center for scientific research 'demokritos', athens, greece liposomes as biomimetic models of cell membranes were used for examining some novel aspects of drug-metal induced reactivity with unsaturated lipids under oxidative conditions. the chemical basis of cis to trans transformation was ascertained by liposome experiments, using bleomycin-iron complexes in the presence of thiol as a reducing agent that by incubation at °c gave rise to the thiyl radical-catalysed double bond isomerisation of membrane phospholipids. the effect of oxygen and reagent concentrations on the reaction outcome was studied. as a chemical biology model for antitumoral strategies, liposomes highlight the role of cell membranes, which are not spectators but important targets of the drug effect, with synergic roles for chemotherapeutic effects. indeed, fatty acid recruitment and membrane formation are attracting a lot of interest in cancer, and in this context the loss of the natural cis geometry and the oxidationinduced lipid remodelling are worthy of deeper studies in antitumoral strategies. furthermore, the interaction between drugs and lipids can be suggestive of novel aspects of chemical reactivity for liposome carriers when circulating in vivo. gpr is a g-protein coupled receptor (gpcr), expressed in cells of brain, heart and kidney. it is related to the leukotriene and purinergic subfamilies of the rhodopsin-like class a gpcrs. gpr plays controversial role in the brain and spinal cord cells recovery after injuries. it is assumed that gpr is one of the cell death regulators immediately following an injury but at later stages it also takes part in tissue regeneration. there are also data implying some role of gpr in glucose homeostasis. drugs targeting gpr may help with treatments of multiple sclerosis and ischemia. the damage of rat's brain in artificially induced ischemia disease decreased after gpr inhibition. in addition, gpr takes part in myelin sheath formation, the lack of which is known to be the reason of multiple sclerosis. to better understand functional role of gpr and enable design of more efficient ligands we initiated structural studies of this receptor. to improve receptor stability and facilitate crystallization we created a series of chimeric constructs using different fusion partnerssmall soluble proteins inserted into the native amino acid sequence. preliminary experiments were carried out to evaluate the influence of different ligands on the receptor stability and cell surface expression in insect sf cells. this work was supported by russian federal target sterols are significant for the structural and dynamical features of cell membranes. among them, cholesterol is the major sterol in mammalian cell membranes whereas stigmasterol is the predominant sterol in plant membranes. stigmasterol varies structurally from cholesterol in having both an ethyl group at carbon and an additional trans double bond between carbons and . dimyristoylphosphatidylcholine (dmpc) is a widely studied synthetic lipid, which has a neutral (zwitterionic) pc headgroup and two symmetrical -carbon fatty acyl chains. the studies on the interactions of cholesterol and stigmasterol with dmpc membranes at molecular level are very limited. in the present study, a calorimetric comparison of the effects of the animal sterol cholesterol and the plant sterol stigmasterol on zwitterionic dimyristoylphosphatidylcholine (dmpc) multilamellar vesicles (mlvs) was investigated for the first time by using differential scanning calorimetry (dsc). our dsc studies indicate that with the inclusion of increasing cholesterol and stigmasterol concentrations from to mol% into pure dmpc mlvs, the pretransition disappears, the main phase transition shifts to lower temperatures and then disappears at cholesterol and stigmasterol concentration above mol%. the main phase transition enthalpy (dh) is progressively reduced whereas full width at half maximum (dt ½ ) gradually increases with increasing cholesterol and stigmasterol concentrations. moreover, the main phase transition peak consists of overlapping sharp and broad components, indicating the hydrocarbon chain melting of sterol-poor and sterol-rich dmpc domains, respectively. in conclusion, this study shows clearly that both cholesterol and stigmasterol interact effectively with dmpc membranes and cause changes in their structural and functional properties. p- . . - trh receptor mobility in the plasma membrane is affected by its interaction with its cognate signaling molecules and by cholesterol depletion r. moravcova, b. melkes, j. novotny department of physiology, faculty of science, charles university in prague, prague, czech republic g protein-coupled receptors (gpcrs) play a fundamental role in transferring information from extracellular environment to the cell interior. some gpcrs are supposed to form signaling complexes with their cognate g proteins and possibly other accessory proteins, which may facilitate the activation of g proteins and thus accelerate signal transmission. here we investigated the role of some components of thyrotropin-releasing hormone (trh) receptor signaling in hek cells stably expressing yfp-tagged trh receptor using fluorescence recovery after photobleaching (frap). we observed significant changes in values of the diffusion coefficients if expression of b -arrestin or gb subunit were suppressed by sirna. results of our frap experiments indicated significant difference between control and trh-treated cells as the diffusion coefficient markedly decreased after agonist stimulation. on the other hand, the same decline of the diffusion coefficient value was found after silencing with sirna and subsequent treatment with trh for most of the screened proteins. treatment of cells with À m trh led to fast internalization of trh receptor, which was revealed by real-time confocal microscopy. it is known that cholesterol is an essential component of the cell membranes and it exerts modulatory effects on the functioning of various membrane proteins. disruption of plasma membrane integrity by cholesterol depletion using b-cyclodextrin significantly reduced the apparent diffusion coefficient values. interestingly, addition of trh to cells treated with b-cyclodextrin did not further reduced trh receptor mobility. it can be concluded that stimulation with agonist and/or sirna silencing of some components of the trh receptor signaling cascade significantly affects the mobility of trh receptor in the plasma membrane. p- . . - l-opioid receptor mobility in the plasma membrane is diversely affected by biased ligands b. melkes, l. hejnova, j. novotny opioid receptors belong to the large family of g protein-coupled receptors (gpcrs), which are currently considered among the most important targets for pharmacological manipulations. during the past few years, a great deal of attention has been devoted to biased agonism. this phenomenon reflects the ability of different ligands to selectively affect the functioning of some gpcrs so they can display marked differences in their efficacies for g protein-or b-arrestin-mediated signaling. here we decided to investigate the effect of different agonists of the l-opioid receptor (l-or) on the lateral mobility of this receptor in the plasma membrane of hek cells which were stably transfected with l-or tagged with yellow fluorescent protein (l-or-yfp). it has been found previously that damgo stimulates g-protein-dependent signaling, endomorphine stimulates arrestin-dependent signaling and morphine does not show any significant bias towards these two signaling pathways. in our experiments, we used the fluorescence recovery after photobleaching (frap) method to estimate the diffusion coefficients of l-or-yfp in the resting state and after addition of the above mentioned specific agonists. we observed that addition of damgo increased the value of the diffusion coefficient and addition of endomorphin decreased the value of diffusion coefficient of l-or-yfp. addition of morphine or the l-or antagonist naloxone did not change the value of the diffusion coefficient compared to the resting state. these results indicate that different biased agonists of l-or may differently affect the mobility of this receptor in the plasma membrane. these findings provide new insights into the dynamics of l-or in the plasma membrane and contribute to better understanding of the mechanism of biased agonism at gpcrs, which is of central importance for receptor homeostasis and fine regulation of receptor activity. color tuning and adding potassium selectivity to a light-driven sodium pump k. kovalev , , v. polovinkin , , , v. shevchenko , , v. gordeliy , , moscow institute of physics and technology, dolgoprudniy, russia, research centre j€ ulich, j€ ulich, germany, institut de biologie structurale, universit e grenoble alpes, cnrs, and cea, grenoble, france recently a light-driven sodium pump has been discovered, characterized and tested as an inhibitory optogenetic tool. sodium pumping rhodopsin from dokdonia eikasta kr has an absorption maximum at nm at ph . and to create more redshifted variants we analyzed available structures of the kr (pdb codes: xtl, xtn) and did the rational mutagenesis of residues in the retinal proximity region (i.e. m , g and s ). the mutants of kr under investigation were: m a, g v, m a/g v, s a, s f, s g, s l, s m, s n, s t, s v, s y. the protein mutants were expressed in escherichia coli c strain, expression was induced by the addition of mm isopropyl b-d- -thiogalactopyranoside. the cells were then washed trice with unbuffered mm nacl or kcl solution. finally, the ph changes in cell suspensions (od = . ) were monitored. ph changes upon the addition of lm of protonophore carbonylcyanide m-chlorophenylhydrazone were also measured. the following mutants completely abolished the protein function and were not used for further characterization: s f, s l, s m, s n, s v. the remaining mutants have shown sodium pumping activity and s a mutant has gained an additional potassium pumping activity. all functionally active mutants were purified using ni-affinity chromatography and the absorption spectra were collected for them at ph . ( mm na/na-pi, mm nacl). m a mutant absorption maximum is blue-shifted to nm. g v and m a/g vblue-shift to nm. s a, a potassium pumping variant,red-shift to nm. s g, s yred-shift to nm. s tno change in absorption maximum position. based on structural analysis of kr we discovered another potassium pumping variant and provided the variants with absorption maximum blue-shift up to nm and red-shift up to nm. human endothelin receptors belong to the g-protein coupled receptors (gpcrs) superfamily. this class is pharmacologically important, with more than % drugs targeting it. the human endothelin system, which includes endothelin receptors types a and b (etb and eta), plays a highly important role in the blood pressure regulation. endothelium cells produce peptides, known as endothelins - , which activate endothelin receptors and launch cascades of reactions that lead to vasoconstriction or vasodilatation depending on the receptor subtype and the tissue. additionally, endothelin receptors take part in such processes as transmission of nerve impulses, development of neural crest, and regulation of acid-base-salt balance in kidneys. in order to stabilize etb receptor for crystallization trials we introduced a compact soluble protein, apocytochrome b ril (bril), is the third extracellular loop of the receptor. bril is known to be an effective crystallization driver for gpcrs. the engineered protein was expressed using baculovirus system in sf insect cells, purified and subject to variety of pre-crystallization assays. localization of the overexpressed protein in insect cells was visualized via the confocal microscopy. thermal stability of the protein in the presence and absence of ligands was measured by the thermal shift assay. finally, the mobility of the receptor in lipidic cubic phase (lcp) at many different conditions was probed by the lcp-frap (fluorescence recovery after photobleaching) assay. these tests showed that the obtained protein is thermally stable, functionally active and diffuses well in lcp at certain conditions, making it a suitable candidate for proceeding to in meso crystallization trials. this work was supported by the russian science foundation (project no. - - ). mitochondrial oxidative phosphorylation is the key metabolic pathway for the production of atp. mitochondrial respiratory chain (rc) defects are some of the most commonly diagnosed inborn errors of metabolism with a diverse spectrum of clinical phenotypes. the aim of the study is to evaluate the rc enzyme activities and histopathological findings in muscle biopsies of patients with suspected mitochondrial disease. muscle biopsy samples were collected from pediatric patients. the samples were homogenized in seth buffer using a glass/glass homogenizer. the activities of citrate synthase (cs) and rc enzymes (complex i, ii-iii, and iv) were measured in tissue homogenates by kinetic spectrophotometric assays by schimadzu uv spectrophotometer (uv- ). non-collagen protein was determined by the modified lowry assay. activities of complex (c) i, ii-iii and iv (cox) were normalized by cs. histopathological investigations included h&e, modified gomori trichrome, periodic acid schiff, oil-red-o, nadh, sdh, cox and atpase stains. deficiency of rc complexes was detected in biopsies ( %). c iv deficiency was most common ( %), followed by c i ( %) and c ii-iii ( %). multiple complex deficiency was present in % and isolated deficiency was present in % of the biopsies ( % c i, % c ii-iii, % c iv). cs activity was elevated in % of the biopsies. decreased c i/cs, c ii-iii/cs and c iv/cs ratio was observed in %, % and %, respectively. comparing with histological data, biochemical analysis revealed additional findings in % of biopsies. complex iv deficiency, either isolated or accompanied by other complex deficiencies, is most common in our cohort. rc analysis may reveal additional findings to histopathological results and careful clinical investigation with correlation of clinical, biochemical and histopathological data is mandatory for the challenging diagnosis of mitochondrial disorders in childhood. investigation of adipocytokines, activity of glut and na + /k + -atpase in rats fed glucose, fructose, starch-based sugars objective: all over the world, shows a significant increase in obesity and diabetes. intake of foods that contain fructose, glucose and starch-based sugar is a potential risk for metabolic syndrome. obesity and diabetes are important effects of high fructose corn syrup (hfcs). we aimed to research, activity of na + /k + -atpase in addition to glucose transporter (glut) , resistin, adiponectin and other biochemical markers in rats fed glucose, fructose and starch-based sugars. materials and methods: study was performed on rats and groups were included in the study. rats were fed with chows that were given either normal diet for control group ( % carbohydrate, % protein and % fat), high fructose ( % carbohydrate ( % fructose and % starch), % protein and % fat), or high sucrose ( % carbohydrate ( % sucrose and % starch), % protein and % fat). rats were fed with chows for weeks. in this process, the weight of the rats were followed. at the end of the experiment, blood is taken in all groups. level of hba c, glucose, resistin and adiponectin were studied. glut and na + /k + -atpase activity were studied in the liver tissue. results: a significant increase in adiponectin levels were determined in rats fed both hfcs and sucrose (p < . ). a significant decrease in level of na + /k + -atpase activity were determined in rats fed both hfcs and sucrose (p < . ). there was no significant differance level of hba c, glucose, resistin and glut in rats fed sucrose or hfcs (p > . ). conclusions: fructose-rich diet has an effect on changes in the atpase activity and is a major risk factor for obesity. keywords: adiponectin, fructose, glucose, high-fructose corn syrup, na + /k + -atpase, resistin. p- . . - nucleic acid-biomembrane lipid selfassemblies: from primordial soup to novel genome organization model and cellular nonviral nanotherapies f. k. demirsoy , n. eruygur , e. s€ uleymanoglu biotechnology central laboratory, biotechnology institute, ankara university, ankara, turkey, department of pharmacognosy, faculty of pharmacy, cumhuriyet university, sivas, turkey, department of pharmaceutical chemistry, faculty of pharmacy, gazi university, ankara, turkey turkey nucleic acid-cell membrane complexes has attracted research interest as models in gene regulation, cell cycle and division, as biosensors designs, as well as in molecular evolution. zwitterionic phospholipids, complexed with polyribonucleotides by divalent metal cations (mg +) are considered as genosome vehicles. their formations are studied by spectroscopic, thermodynamic, interfacial and microscopic approaches to build thermodynamic and kinetic models of their structural transitions. dna forms a mg +-driven ternary complexes with neutral liposomes both at the airwater interfaces and at vesicle surfaces. the described self-assemblies form relevant models for nuclear pore complexes and their further implications in gene expression and functions. such membrane contacts could be considered also in prokaryotic nucleoids important in bacterial segregation, whereas in eukaryotes these complexes can be regarded as focal points for transcription-translationtranslocation processes. the effects of ozone/oxygen mixture on citrate synthase and mitochondrial complex activities of striated muscle tissue of healthy mice we investigated the effects of ozone/oxygen mixture and oxygen on citrate synthase (cs) and succinate dehydrogenase (sdh) activities of striated muscle tissue of healthy mice. thirty-six mice were included the study. firstly muscle samples were taken from all mice's left thigh muscle in under anesthesia (group ). secondly mice were randomly divided in four groups as: group : oxygen was given once at days for days, group : ozone/oxygen was given once at days for days, group : oxygen was given once at days for days, group : ozone/oxygen was given once at days for days. ozone/oxygen mixture and oxygen were given at a dose of mg/ kg groups ( ) ( ) ( ) ( ) . after treatment animals were sacrificed, and muscle samples were taken and stored in À °c for until the analyses. muscle tissues were homogenized in . m tris-hci and . m kci. cs and sdh activities were measured with spectrophotometer. cs and sdh activities were expressed as lmol/min/g tissue. cs and sdh activity results were given as mean ae sd. cs activity has been found in group ( . ae . ), group ( . ae . ), group ( . ae . ), group ( . ae . ) and group ( . ae . ). sdh activity has been found in group ( . ae . ), group ( . ae . ), group ( . ae . ), group ( . ae . ) and group ( . ae . ). there was no statistically significant difference among all groups in terms of cs (p > . ) and sdh activities (p > . ). there was no difference between all groups in terms of inflammation, muscle fiber size, regeneration or necrosis. vascular structures, connective tissues, lipid and glycogen content of fibers were normal. cytochrome oxidase activity was also normal. ratio of ragged blue fibers of all groups were less than . %, so they were scored as . there was no difference among groups for ragged red fiber content. we have not found a significant effect of ozone/oxygen mixture and oxygen on cs and sdh activities of striated muscle tissue of healthy mice. lipidic cubic phases (lcps) consist of bicontinuous lipid bilayers and water channels. lpcs are widely used for membrane proteins crystallization and further determination of their spatial structures by means of x-ray crystallography. this approach was successfully used for studying g-protein coupled receptors structures. usually crystallization initiates by adding the precipitants (buffers with high salt concentrations). here we propose to use photo-switchable analogs of -monoolein to change lattice parameter of lpc. we synthetized a number of novel diazo-analogs of -monoolein. their structures were confirmed by nmr-spectroscopy and mass-spectrometry. being incorporated in phospholipid vesicles or detergent micelles they subjected to trans-to cis-isomerization under the light exposure at nm. also we characterized the lcp's structures and properties by small-angle x-ray scattering on the rigaku instrument. one of the synthetized compounds, -( -{-[ -(octyloxy) phenyl] diazenyl} phenoxy) propane- , -diol ( % mol), was incorporated into the -monoolein cubic phase. according to small-angle x-ray scattering data the structure of the monoolein cubic-pn m phase with lattice parameter . angstrem was not affected by insertion of this photo-switchable monoolein's analog. after the light exposure at nm we observed trans-cis-isomerization. in the same time the cubic-pn m phase was not changed but the lattice parameter reduced to . angstrem. this effect on monoolein lpc is similar to the addition of a precipitant to initiate protein crystallization process. the spontaneous return to the initial lattice parameter was completed after days in dark. thus, we demonstrated the possible controlling of the monoolein cubic phase lattice parameters by adding the photo-switchable diazo-derivatives of monoglyceride analogs, which can be used for crystallization of membrane proteins. evaluation of certain protein and phosphoprotein expression levels by using western blot technique in head and neck squamous cell carcinoma a. kalayci yigin , t. cora cerrahpasa faculty of medicine, istanbul university, istanbul, turkey, faculty of medicine, selcuk university, konya, turkey introduction: head and neck squamous cell carcinoma (hnscc) is a primer tumor type in head and neck cancers, characterized by aggressiveness, early recurrence and metastasis. while alcohol and smoking play an important role at pathogenesis of disease, deregulation of some signaling pathways, genes and protein levels related to these pathways are considered as important at contribution of development of hnscc. materials and methods: in this study, protein and phosphoprotein expression levels of the frequently phosphorylated sites (egfr, pegfr, igf-ir, pigf-ir, pdgfrb, ppgdfrb, pten, ppten, akt and pakt) were investigated by using a western blot to confirm the expression of mrna in the context of protein levels at normal-tumor tissues of hnscc and sccl-mt that is a hnscc tumor cell line and hek- that is a normal cell line. results: as a result of western blot analysis egfr, pdgfrb and igf- r were detected as highly overexpressed cell surface receptors in tumor tissues of hnscc. discussion and conclusion: the findings of this study revealed the overexpression of other cell surface receptors as well as egfr in hnscc. in conclution, potential pathways were identified by determining the cell surface receptors overexpressed in hnscc, these data support each other and may be important in pathogenesis of hnscc. introduction: the investigation of final products of lipid peroxidation is considered as the main mechanism involved in development of pathogenesis, diagnostics and prognosis of various parasitic illnesses. materials and methods: the concentration of lp-ap in the blood was determined in the study group considered of women ( %), and men ( %). results: before antiparasitic treatment, women infected with g. intestinalis showed a statistically significant . times increase of gpx activity levels; and . times ada level increase compared to the control group. after the treatment, the cat activity showed a sharp increase, whereas the ada activity decreased by . times, compared to the average level before the treatment. the results of the blood samples of the infected men with giardiasis, show the statistically significant increase in the level of all the studied parameters of lipid peroxidation, except the total primary production (tpp). the exception was the mda level, remaining significantly increased, in contrast to the control group and to the condition after antiparasitic treatment. in infected men, the level of cat activity was more than . times higher than that noted in control group. after treatment the levels of ada activity and gpx returned to the values of the control group, while the level of cat activity remained elevated. conclusion: an accumulation of primary and secondary metabolites in the course of giardiasis seems to confirm its involvement in the induction of oxidative-antioxidative homeostasis. antiparasitic treatment in giardiasis leads to normalization of the ap parameters studied in women and men, except the mda content in the blood of men. therefore, additional antioxidant treatment is advised for the antiparasitic therapy of men. in vitro effects of ethanol on rat brain synaptosome and dose-dependent antioxidative role of boric acid ethanol is a psychoactive drug that is very large and used frequently today. it has suppressive effects brain's comminication pathway. depending on its acute or chronic use and the dose, ethanol increase membrane fluidity and it causes oxidative stress. this study deals with the in vitro effects of ethanol toxicity ( mm) and potential protective effect of different doses of boric acid (ba) ( , and mm) on rat brain synaptosomes. with this aim, five male spraque dawley rats are killed by decapitation under anesthesia. after the frontal cortexes of the rats are taken out, each of them is divided into four pieces. these pieces were used as a sample in five groups (control, ethanol, ethanol+ mm ba, ethanol+ mm ba, ethanol+ mm ba) which include six samples. the synaptosomal fractions are prepared by the homogenization of the frontal cortex pieces and centrifugation for each samples. as markers of ethanol-induced oxidative stress in the synaptosome of the rats, malondialdehyde (mda), nitric oxide (no) and catalase (cat) levels were measured. mda levels in the ethahol group were a quantity increased compared with those in the control group but it unchanged significantly as statistically (p < . ). however the mda level in the ethanol+ boric acid ( mm) group was shown to be significantly decreased compared with that in the ethanol group (p < . ). the cat activity of the ethanol group was significantly higher than that in the control group and cat activity of the ba ( mm, mm) groups were close compared with control groups (p < . ). no levels in ethanol groups were decreased but unchanged compared with control groups as statistically. neverthless, no levels in ethanol+ boric acid ( mm) groups were increased (p < . ). these results demonstrate that ethanol ( mm) is capable of triggering damage to rat brain synaptosome and ba could be influential in antioxidant mechanisms against oxidative stress resulting from ethanol exposure. acute myeloid leukemia (aml) is the most common form of acute leukemia in adults and its incidence increases with age. carbonic anhydrases (cas) are zinc-containing enzymes. these enzymes catalyze a very simple physiological reaction, the inter conversion between carbon dioxide and the bicarbonate ion, and are thus involved in crucial physiological processes connected with respiration and transport of co /bicarbonate between metabolizing tissues and lungs, ph and co homeostasis, electrolyte secretion in a variety of tissues/organs, and biosynthetic reactions and many other physiologic or pathologic processes including reproductive tract. investigation of autoantibodies in aml patients have been popular research area in recent years. the aim of the current study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in the serum of subjects with aml based on the information and considerations of autoimmune relation of acute myeloid leukemia. anti-ca i and ii antibody levels were investigated by enzyme-linked immunosorbent assay method (elisa) in serum samples of thirty patients with aml and thirty healthy peers. anti-ca i and ii antibody titers of aml group were significantly higher compared with the control group (p = . ), (p = . ), respectively. we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in patients with aml (r = . , p = . ). we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in women and the men (r = . , p = . ), (r = . , p = . ), respectively. at an anti-ca i cut-off point of . absu, sensitivity was % and specificity %. at an anti-ca ii cut-off point of . absu, sensitivity was % and specificity %. the ca i and ca ii autoantibody levels in patients with aml were found higher compared to control group and the results suggest that ca i and ca ii autoantibodies may be involved in the pathogenesis of aml. aim: behc ßet's disease (bd) is a systemic autoimmune disease. recurrent oral and genital mucosal ulcers, uveitis, and skin lesions are characteristic findings for bd. platelet-lymphocyte ratio reflects a novel marker for romatological diseases. the aim of this study was to investigate the platelet/lymphocyte ratio in behc ßet's disease. methods: whole blood samples were collected from healthy control and patients with behc ßet's disease. the mean age for controls and patients were ae . and ae , respectively (p = . ). patients with chronic disease and inflammatory disorders were excluded. thrombocyte and lymphocyte counts were analyzed with abbott cell dyne heamotolgy analyzer. statistical analysis was performed with ibm spss v . results: platelet counts were higher but not statistically significant in patients with behc ßet's disease compared to control group ( ae vs. ae ) (p = . ). lymphocyte counts were lower in patients with behc ßet's disease compared to control group ( . ae . vs. . ae . ) (p = . ). platelet/lymphocyte ratio was higher but not statistically elevated in patients with behc ßet's disease compared to control group ( ae vs. ae ) (p = . ) conclusions: platelet/lymphocyte ratio (nlr) and mean platelet volume (mpv) as inflammatory markers recently became popular because of their simplicity, cost effectivity and their advantages to predict clinical prognosis of specific diseases. according to this study's results, platelet/lymphocyte ratio must be analyzed in vast scale patient populations to identify the disease. objectives: systemic lupus erythematosus (sle) is a chronic relapsing autoimmune disease characterized by production of autoantibodies against a series of nuclear antigens and by chronic inflammation. in recent years, neutrophil-lymphocyte ratio (nlr) was determined to be a good indicator of inflammatory status. nlr can be easily calculated from a whole blood count. introduction: neuroblastoma, an embryonal malignancy, is the most common extracranial solid tumor of childhood. untreated neuroblastoma tumors and cell lines are reported to have reduced hla class i expression, rendering them potentially susceptible to natural killer cell killing due to lack of engagement of hla class i-specific inhibitory killer cell immunoglobulin-like receptors (kirs). the aim of this study was to investigate whether kir genes could influence the risk of neuroblastoma and prognosis of the patients. materials and methods: study group consisted of neuroblastoma patients ( male, female, median age: months) followed at the pediatric oncology clinic of c ß ukurova university medical faculty. control group consisted of healthy children. patients had stage , , or s disease, patients had stage disease. different kir genes were analysed by sequence specific oligonucleotide probe (ssop) analyses. statistical analysis were done using fisher's exact test. results: compared to the control group, neuroblastoma patients had lower expression of activating kir gene, kir ds (p = . ), and higher expression of inhibitory kir gene dl (p = . ). additionally kir ds genes were more common in patients with early stages (stage , , or s) (p = . ) and kir dl genes were more common in patients with stage (p = . ). furthermore, there were no statistically significant differences between the rate of aa and bx genotypes and their centromeric/ telomeric regions of patients and controls. discussion: kir dl gene can have a triggering effect in neuroblastoma pathogenesis; whereas kir ds can have a protective role. investigating nk cell infiltration and kir receptors in neuroblastoma tissue samples will give more insight to the pathogenesis p- . . - neuroprotective and immunomodulatory effects of urtica urens s. arslan , g. terzioglu , b. kabalay , a. r. tufekci , a. sen , i. demirtas department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, deparment of chemistry, faculty of arts and sciences, c ß ankiri karatekin university, c ß ankiri, turkey urtica urens (small stinging nettle) has been used for medical purposes in turkey as an alternative therapy. it has been used in the treatment of inflammation that is early, non-specific immune reaction to tissue damage or pathogen invasion, plays an important role in the initiation of neurodegenerative disorders such as multiple sclerosis. however, there are limited studies that investigate anti-inflammatory activity of urtica. therefore, aim of this study is to find out theanti-inflammatory effect of chloroform extract in caco- cell line. for this purpose, firstly, chloroform extract of urtica leaves was prepared. chemical composition of extract was determined by lc-ms. the effect of chloroform extract on selected pro-inflammatory and inflammatory proteins such as tumor necrosis factor-a (tnfa), nuclear factor kappa b (nf-jb), c-x-c motif chemokine (cxcl ), and (cxcl ) were determined. whole genome transcriptome analysis was performed by using human ht- v beadchip. extract treatment caused % and % increases in protein and mrna levels of nf-jb, respectively. on the other hand, tnf-a protein and mrna levels decreased significantly ( % and %, respectively). similarly, cxcl and cxcl mrna levels decreased % and %. transcriptome analysis showed that probes were significantly changed (p < . ). pathway analysis revealed that the extract altered a group of genes involved in immune response, calcium ion homeostasis and transport, potassium channel complex, g-protein coupled receptor protein signalling pathway, etc. it is well established that calcium is very critical for brain cell death and formation of many brain disease including multiple sclerosis. these observations suggests that urtica maybe used in neurodegenerative diseases. in order to further test this hypothesis experimental autoimmune encephalomyelitis experimentsand activity guided fractionations have been still continuing. this work is supported by tubitak t . p- . . - linear low molecular weight a- , -glucan from bifidobacterium bifidum bim b- d is implicated in pathogenesis of celiac disease the bifidobacteria are recognized as human commensals and widely used as probiotics. earlier, we have found (kiseleva et al., benef. microbes, , ( ) : - ) that bifidobacterium bifidum bim b- d contains low molecular mass ( - kda) a- , glucans (g anti-tpo and g anti-tg ) that interact selectively with human autoantibodies to thyroid peroxidase (anti-tpo) and thyroglobulin (anti-tg), recognized markers of autoimmune thyroid disease (atd). the aim of the study was isolation and identification of b. bifidum bim b- d biopolymers (bps) interacting selectively with autoantibodies to tissue transglutaminase (anti-ttg) and antibodies to gliadins (anti-gl), recognized markers of celiac disease (cd). we used affinity chromatography with anti-gl, size exclusion chromatography, h and c nmr spectroscopy, elisa with immobilized bps, tissue transglutaminase (ttg) and gliadins (gl) as positive controls. the bp isolated by affinity chromatography with anti-gl (as more available marker of cd) and size exclusion chromatography was identified by two-dimensional nmr spectroscopy as - kda linear a- , -glucan identical to g anti-tpo and g anti-tg . the functional activity of the bp named g anti-gl , viz., ability to interact selectively with anti-ttg and/or anti-gl was proven by elisa with (i) serum samples of cd patients containing either both anti-ttg and anti-gl without anti-tpo and anti-tg or anti-gl without anti-ttg, anti-tpo and anti-tg vs. serum samples of healthy donors without four types of antibodies and (ii) pure anti-gl vs. pure total igg (without anti-ttg, anti-gl, anti-tpo, anti-tg). since (i) serum samples of cd patients do not contain anti-ttg without anti-gl and (ii) pure anti-gl isolated by affinity chromatography with gliadins (gl) cross reacts with tissue transglutaminase (ttg), additional studies with pure anti-ttg are necessary to find out which of the two antibodies, anti-ttg and anti-gl, bind g anti-gl . in conclusion, we proved that b. bifidum bim b- d cells contain linear low molecular mass a- , -glucan, g anti-gl , that interacts selectively with anti-ttg and/or anti-gl. since g anti-gl is identical to earlier found g anti-tpo and g anti-tg , we hypothesize that the a- , -glucan is implicated in pathogenesis of both autoimmune diseases, cd and atd. influences of elevated serum ferritin levels on insulin resistance and non-insulin-dependent diabetes mellitus (niddm) have predicted either because of increased body iron stores or influenced by several inflammatory diseases. low serum hydroxyvitamin d is known to perturb cellular function in many tissues, including the endocrine pancreas, which are involved in obesity and niddm. we planned to investigate the association between hydroxyvitamin d with hematologic parameteres and iron status in obesity vs. diabetic patients. study groups consist of control, non-diabetic obese, obese-diabetic and lean-diabetic groups. serum triglycerides, total cholesterol, ldl-c, hdl-c, fasting glucose, hba c, uric acid, creatinine, ggt, -hydroxyvitamin d, insulin, crp, esr, total blood count and iron status. apart from the three parameters, there were no significant difference (p > . ) between groups. serum ferritin and mchc levels were significantly higher in lean-diabetic patients (p < . ). on the other hand, rdw are determined to be significantly lower (p < . ) in the non-diabetic obese group. no difference was detected in -hydroxyvitamin d levels between the control and the study groups (p > . ). non-diabetic obese patients had significantly (p < . ) higher levels of tg and lower levels of hdl compared to obese-diabetics. insulin levels were higher in nondiabetic obese and obese-diabetics than lean-diabetics (p < . ). this study provides evidence that lean diabetic patients show higher ferritin and mchc levels than obese patients. the increase in serum ferritin and mchc levels is related with altered iron metabolism at cellular level. at late mitosis, the mother cell divides, leaving two daughter cells connected by a thin intercellular bridge (icb). during abscission of the icb, the ingression of the cleavage furrow is formed, and the central spindle microtubules are compacted into the structure known as midbody (mb). the mb is situated within the icb, with the abscission usually occurring at one side of the mb. as a result, only one daughter cell inherits the post-mitotic mb. these mbs can then either accumulate in the cytoplasm or be degraded. recent studies have identified mbs as novel signaling platforms regulating stem cell fate and proliferation. indeed, stem cells as well as cancer cells were shown to accumulate post-mitotic mbs, resulting in reprogramming of the cell fate and conversion to highly-proliferative, stem cell-like phenotypes. it has been proposed that regulated macroautophagy may be playing a key role in mediating pots-mitotic mb degradation. therefore, the experimental approach involved studying the dynamics and function of a protein known as fyco , which associates with postmitotic mbs and may regulate their degradation. in this study we identified fyco as a protein, which associates with post-mitotic mbs and may regulate their degradation. interestingly, fyco is also known to be present on autophagosomes, and overexpression of fyco can induce the formation of enlarged lc -containing autophagocytic structures. here we demonstrate that fyco knock-down leads to defects in autophagic mb degradation, and that fyco functions by targeting endocytic membranes to the autophagic phagophore during early stages of mb degradation. additionally, we showed that fyco depletion leads to increased proliferation and cell growth in soft agar. based on all these data, we hypothesize that fyco mediates selective mbs degradation via endosome-dependent extension of the phagophore around the post-mitotic mbs, and that mbs may be the regulators of cancer proliferation and progression. p- . . - proliferative effect of hypericine on human skin fibroblast cells and identification of the mechanism of action in molecular level to drawbacks associated with efficiency and viral genome integration. in order to improve reprogramming efficiency and compensate for viral transduction, new chemicals have been explored through ipsc research. the aim of this study was to investigate the proliferative effect of hypericine on human skin fibroblast cells (sf) in-vitro, and to identify the mechanism of action in molecular level. the proliferation was measured using the clonogenic and dimethylthiazol diphenyltetrazolium bromide (mtt) assays. real-time quantitative polymerase chain reaction (qrt-pcr) was performed to detect the mrna levels of cyclins (d and b ) and cell cycle controller genes (p and p ). sf cells were treated with different doses ( nm- lm) of hypericine for h and h. a significant cell proliferation was observed in moderate concentrations ( . - lm; % -% ), but at high concentrations ( - lm) cytotoxic effects emerged in sf cells (ic = . m, r = . ). qrt-pcr results revealed that the most proliferative dose of hypericine ( lm) stimulates cyclin d . the anti-proliferative activity of hypericine was accompanied by inhibition of cyclin b mrna, whereas it induced expression of p and p genes, and thus apoptosis was observed by dna laddering at the same dose ( lm). overall results suggested that hypericine can compensate for viral transduction and improve reprogramming efficiency of ipscs by enforcing them in g phase. hence we report that hypericine can be a good candidate component for cocktails produced to trigger ipsc proliferation. glioblastoma (gbm) is the deadliest brain tumor. the mean survival time of gbm patients is approximately months, increasing to months after treatment with temozolomide, which is the gold standard chemotherapy. the resistance of gbm to chemotherapy seems to be associated with the blood-brain barrier (bbb) that limits the delivery of chemotherapeutics, and the presence of a population of cells that expresses stem cell-like properties, which are known to be chemo-and radioresistant, the glioblastoma stem cells (gscs). the difficulties imposed by these two factors could be reduced by the use of a targeted drugdelivery liposome-based strategy that allows bbb passage and reduces the side effects of chemotherapeutics. the present study evaluated the ability of the f peptide-targeted ph-sensitive lipid-based nanoparticle containing doxorubicin (dxr) to target gscs and non-gscs. we evaluated the expression of cell-surface nucleolin by flow cytometry, as well as of stem cell-like markers, in two gbm cell lines. we also determined the ability of gbm cell lines to specifically uptake the f peptide-targeted ph-sensitive lipid-based nanoparticles, by flow cytometry, and correlated it with the expression of stem cell-like markers. moreover, to ascertain the impact of intracellular delivery of chemotherapeutic drugs into gbm cell lines, cytotoxicity was further assessed by the mtt assay. our results showed that the f peptide-targeted ph-sensitive lipid-based nanoparticles successfully targeted glioblastoma cells and particularly gscs. in addition, the results also provided evidence of the nucleolin overexpression-dependency of this strategy, emphasizing the need to adapt the therapeutic strategy to the individual patient. this study showed that f -targeted ph-sensitive liposomes may constitute an appropriate strategy to overcome the chemoresistance associated with glioblastoma cells. p- . . - leukemic cell plasticiy as a resistance mechanism towards tyrosine kinase inhibitors chronic myelogenous leukemia (cml) is a hematopoietic stem cell disease characterized by the t( ; )(q ;q ) translocation, which encodes the chimeric tyrosine kinase onco-protein, bcr-abl. the tyrosine kinase inhibitor (tki) imatinib is the first-line treatment for patients with cml. unfortunately drug resistance is one of the main problems observed. while secondary resistance is associated with bcr-abl kinase domain mutations, oncogene amplification and mechanisms interfering with intra-cellular drug concentrations; primary resistance mechanisms haven't been elucidated. we generated high dose imatinib-resistant k subclones (k -ir) by clonal selection to study primary resistance mechanisms in vitro. drug resistance was shown by caspase and annexin v/pi assays. we also showed cellular uptake and function of imatinib with western blot technics. k -ir cells are not only resistant to imatinib but also to nd, rd generation tyrosine kinase inhibitors. we demonstrated that k -ir cells have a highly adherent character, proliferate slowly and are resistant to drug-induced senescence. microarray analysis revealed that k -ir cells differentially express tissue/organ developement and differentiation genes at high levels. we showed that k -ir cells forms intact tumor spheroids in d cell culture conditions which is a marker of tumor initiating potential. cell surface maker analyses and protein analyses of k -ir cell population, points towards an epithelial-mesenchymal plastic cell capable of adopting different morphologies. we hypotizied that imatinib and other tyrosine kinase inhibitors may cause the gain of phenotypic plasticity potential in leukemic cells, by interfering with signalling pathways; which in itself may lead to therapy resistance. hypoxia has multiple effects on cancer cells, which are critically involved in tumor progression. hypoxia leads to changes in tumor cell metabolism and can promote cancer cell survival, invasion and metastasis by its critically important role on maintenance of cancer stem cell (csc) phenotype. in this research, human cd + cscs isolated from human osteosarcoma cell line saos- using macs magnetic separation technique were characterized, and their stemness properties under hypoxic ( % o ) and normoxic ( % o ) conditions were compared in two and three dimensional culture conditions. two different d culture techniques (nanofibrous bacterial cellulose scaffolds and scaffold free microtissues) were used to evaluate effects of hypoxia on csc behavior, and the results were compared with the cell behavior in classical d culture systems. the morphologies of cells were examined by scanning electron microscopy (sem); rt-pcr and immunocytochemistry staining were used to examine the cancer stem cell phenotype maintenance under hypoxic and normoxic conditions. it is shown that hypoxia supports the expression of stemness markers such as oct / , nanog and sox compared to normoxic conditions in d cultures. although similar effects of hypoxia were observed in d cultured cscs, the expression levels of stem cell phenotypeindicative markers were significantly lower on d compared to d culture systems. this study is seen as an introduction to develop a more relevant d hypoxic cancer stem cell based tumor model to study csc behavior and tumor genesis in vitro for testing of novel cancer stem cell therapeutics and to understand signal transduction in cancer stem cells. prostate cancer (pca) is the second most frequent cause of cancer-specific mortality in the world. cancer stem cells (cscs) are a subpopulation of cells that involved in drug resistance, metastasis and recurrence of cancers. the efficacy of natural flavanone apigenin on cell survival, apoptosis and migration of cscs were evaluated. cd + cscs were isolated from human pca pc cells using a magnetic-activated cell sorting system. pc and cscs were treated with different concentrations of apigenin, docetaxel and combinations of the two agents for h. apigenin dose dependently inhibited cscs and pc cell viability, and this was accompanied with a significant increase of the cell cycle inhibitors p and p (kip ). the flavonoid significantly induced apoptosis via an extrinsic caspase-dependent pathway by upregulating the mrna expressions of caspases- , - and tnfa, but failed to regulate the intrinsic pathway as determined by the bax, cytochrome c and apaf- in cscs. in contrast to cscs, apigenin induced intrinsic apoptosis pathway as evidenced by the induction of bax, cytochrome c and caspase- while caspase- , tnf-a and bcl- levels remained unchanged in pc cells. the ability of apigenin to inhibit the proliferation of cscs through apoptosis was confirmed by tali image-based cytometer. the flavanone strongly suppressed the migration rate of cscs compared to untreated cells. significant downregulation of mmp- and - exhibits the ability of apigenin treatment to suppress invasion. the expressions of pi k/akt and nf-kb p / p were significantly decreased after h apigenin treatment. taken together, these data demonstrated that flavonoid apigenin is an invaluable chemopreventive compound that inhibits proliferation, invasion and the stemness properties of cscs. this study was funded by the scientific and technological research council of turkey (tubitak, grant no. s ). (pi k), are frequently found in patients with severe early-onset segmental overgrowth. whilst differences in timing and location of the founder mutation are likely to explain part of the observed disease heterogeneity, it is less clear whether and how quantitative differences in the strength and timing of pi k activity contribute to phenotypic variability. our aim is to characterise pik ca mutant-specific signalling as well as to explore the effects of varying the strength and/or temporal pattern of pi k activation on downstream output specificity in the cell. we are currently employing crispr/cas mediated gene editing in human induced pluripotent stem cells to generate isogenic disease models of three such activating pik ca mutations. these cells will be used for signalome profiling by reverse-phase protein arrays (rppa) to compare and contrast mutant-dependent alterations to candidate signalling networks. in parallel, ongoing efforts focus on developing an endogenously expressed optogenetic p a, allowing precise spatiotemporal control over pi k signaling to unravel the extent to which pi kdependent phenotypes are determined by strength of activation and/or dynamic encoding. ultimately, the outcome of this research will yield novel insight into fundamental aspects of pi k signalling and potentially aid the development of targeted therapies for human diseases of pi k hyperactivation. e. gov, n. kaya, k. y. arga cancer stem cells (csc) have been proposed to be the cancer initiating cells. because of their highly tumorigenic and drug resistant properties, cscs offer significant potential for developing novel anticancer drugs and therapeutic strategies. in the present study we analysed eight gene expression datasets for breast, ovarian, lung cancer and glioblastoma by comparing gene expression levels between stem cells and tumor cells and integrating them with genome scale biological networks. consequently, mutual molecular signatures (i.e: differential expressed genes, transcription factor, mirna) and biological characteristics were determined via integrative analyses, which might be feasible to uncover the mutual biological mechanism insights behind the cscs. it was identified twenty mutual differential expressed genes in four cancer types; jun and klf as transcription factors, egfr and cdk as receptors come into prominence as mutual signatures. molecules and pathways that were related to mapk, wnt, p signaling and pathways in cancer were the common indicators in csc types. our results provided similarities in gene expression profiles of various cscs and gave clues about the seed of tumorigenesis. this study proposed signatures and pathways that could be considered as effective therapeutic approaches in further experimental and clinical applications to eliminate subpopulation of csc. colorectal cancer (crc) is one of the leading causes of mortality worldwide. metastasis is associated with the presence of circulating tumor cells (ctcs) in the peripheral blood of cancer patients. ctc cut-off values have been shown to predict for poorer overall survival in metastatic breast (≥ ), prostate (≥ ), and colorectal (≥ ) cancer based on assessment of . ml of blood. in our study, ctcs were detected in blood samples of colorectal cancer patients, using with our modified convenient method for the strategies of ctc enrichment and detection. . ml peripheral blood samples were firstly collected and peripheral blood mononuclear cells (pbmcs) were isolated from the fresh blood samples by ficoll gradient separation. next, the leukocytes in pbmcs were removed by magnetic microbeads conjugated with cd for a negative selection. finally, the retained cells were labeled with anti-epithelial cell adhesion molecule (anti-epcam), cytokeratins (ck , ck ) and the leukocyte-specific marker as anti-cd . all samples were analyzed by bd facs aria iii flow cytometry. in total, patients and healthy people were included in this study. the results showed that ctcs were not detected in the blood samples of healhty volunteers, but - ctcs were detected with ck , , , -based gating strategy in the blood samples of colorectal cancer patients. it is accepted that the cut off value is ctcs for colorectal cancer and ctc is negative if it is below this value or ctc is considered as a positive, if it is equal to or above this value, which might be an indication for poor prognosis. thus ctc's detection may serve a representative surrogate tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. cells were grown in culture flasks in a humidified incubator at °c with % co and were used at the proliferation and confluent stages. cultured cells were exposed to the pemf and prfe. the proliferations of the cells are measured by mtt assay for the effect of emf on the cancer cells. on the other hand the wound healing was investigated by closure of the wound by the cell proliferation with cell morphology using inverted microscope images. the proliferation decreased significantly by the effect of pemf on the semi confluent mcf- and mda-mb- cells. this effect was observed more prominent on mcf- . considering prfe therapy this effect is much more pronounced especially for mda-mb- comparing with pemf. the phase contrast observations of these results were consistent with mtt analyses. similarly, this effect was seen less for pemf but the proliferation was more suppressed with prfe on the wound models. it was considered that the emf applications could be effective in cancer cells, but this effect has not been studied how it occurs in invasive cancers. in our cell culture study, the appropriate emf applications were found to be effective though the inhibition of proliferation of cancer cells even in invasive cancer but with lower effect. this means that emf applications may support the existing treatment methods of cancer patients and even people who suffer from invasive cancer. metastasis is the one of the most known causes of death in patients diagnosed with cancer. circulating tumor cells (ctcs) are shed from primary tumors and circulating in the bloodstream, and thought to play a key role in metastasis. a hypothesis that ctcs may contribute to metastasis was first introduced in the mid th century by thomas ashworth, an austrilian pathologist. in today's research, identification and molecular characterization of ctcs are thought to be a novel target for treatment of cancer and a key factor to understand the metastatic process. existing methods of ctc capture based on the cell search system, flow cytometers, laser scanning cytometers instruments, fiber-optic array scanning technology (fast), isolation by size of epithelial tumor cells (iset), and definition fluorescence scanning microscopy. ctcs are increasingly considered as a 'liquid biopsy' and when liquid biopsy is compared to tumor tissue biopsy, liquid biopsy for ctcs detection can be carried out routinely in patients due to accessibility and ease of blood collection. also, primary tumor sampling may not reflect the actual metastatic conditions, ctcs are thought to be a novel tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. with futher works, ctcs may be used as liquid biopsies and it might provide better understanding metastatic process, new approaches in cancer diagnostics and treatment. mesenchymal stem cells (mscs) are distributed all over the organism as a source of tissue formation and regeneration. glucose is vital for the proliferation and differentiation of mscs. glucose uptake is mediated by specific glucose transporters of two families, the na-coupled glucose transporters (sglt) and glucose transporter facilitators (glut). the presence and function of glut proteins in human placental amnion derived mscs (hamscs) is unknown. we aimed to investigate the presence of glut , glut , glut proteins and genes in hamscs isolated from term placentas. mscs were isolated from human term placenta amniotic membrane, the characterization of cells were provided by flow cytometry. mscs were used to assess their chondrogenic, osteogenic and adipogenic differentiation potential. the expression of glut , glut and glut proteins was detected in hamscs by immunofluorescence. glut , glut , glut protein and gene expression in these cells were investigated by western blot and real-time pcr, respectively. flow cytometry analysis results of isolated cells showed that they were positive for cd , cd , cd , cd (mesenchymal stem cell markers) and hematopoietic markers cd , cd b, cd , cd and hla-dr were negative. the presence of glut , glut , glut proteins and genes were identified in hamscs. in this study, for the first time in literature, glut , glut and glut gene and protein presence was determined in hamscs. therefore, gluts could mediate glucose transport in human amniotic membrane mscs. proliferation and differentiation of mscs in vitro are still not optimized. further studies are required to clarify the complex mechanisms regulating the relationship between glucose and mesenchymal stem cells. disclosure of this relationship may provide a better understanding of glucose-related pathologies such as diabetes. tumors have hierarchically organized heterogeneous cell populations and a small subpopulation of cells, termed cancer stem cells (cscs), is responsible for tumor initiation, maintenance as well as drug resistance. therefore, killing the cscs along with the other cancer cells is gaining an importance. in the present study, it was aimed to evaluate the cytotoxic and apoptotic activity of a novel platin (pt) (ii) complex [pt(hepy) cl ] on mammospheres obtained from mcf- human breast cancer line. elevated expression of stemness markers were determined by western blotting. cytotoxicity was assessed using the atp viability assay. effect of the pt (ii) complex on the formation and development of mammospheres was analyzed with sphere formation (sfa) assay. apoptosis was determined via cytofluorimetric analysis (caspase / activity, annexin-v-fitc and bcl- activity) as well as gene expression analysis. cytotoxicity was confirmed with the atp viability assay after the treatment with zvad-fmk (an apoptosis inhibitor) and necrostatin (a necroptosis inhibitor). in addition, alterations in mitochondrial membrane potential were evaluated by jc- staining. mammospheres exhibited increased oct- and sox (stemness markers) expressions compared to parental mcf- cells. cytotoxicity by pt (ii) complex was evident in a dose-dependent fashion ( . - lm) . pt (ii) complex significantly prevented mammosphere formation and disrupted mammosphere structure in a dose-dependent manner. pt (ii)-induced apoptosis was determined based on the presence of caspase / activity, annexin-v-fitc positivity and bcl- inactivation. apoptosis was also confirmed with increased tnfrsf a and hrk gene expressions. in addition to apoptosis, necroptosis was also present as evidenced with increased mlkl expression. mitochondrial membrane was depolarized. in conclusion, the pt (ii) complex seems to be a powerful apoptosis-inducing compound on cancer stem cells, thereby warrants further in vivo experiments. cancer is a disease which arises from destruction of growth and proliferation mechanisms in cells and is the second leading cause of death worldwide [ ] . in the development of primary cancers, the head and neck cancer is accounting for approximately . new cases annually around the world [ ] . laryngeal cancer is a type of head and neck cancer in which malignant cells arise from the mucosal tissues of the larynx [ ] . cancer might spread from primary tumor by getting into the lymph and blood vessel system and forms secondary tumor. greater than % of deaths in cancer patients are attributed to metastasis [ ] . circulating tumor cells (ctc's) provide an opportunity to understand the metastatic process of cancer patients. identification and molecular characterization of ctc's in the peripheral blood of cancer patients is a promising research area in the field of biomarker development and novel treatment targeting in today's cancer research [ ] . the detection of ctc methods include cell search system, flow cytometry, high-definition fluorescence scanning microscopy, fiber-optic array scanning technology, isolation by size of epithelial tumor cells, and laser scanning cytometers [ ] . in our study, . ml of peripheral blood samples were collected from larynx cancer patients and healthy volunteers and the samples were analyzed by bd facs aria iii flow cytometry via biomarkers epcam, ck , ck for positive selection and cd for negative selection [ ] . according to the results of our study; ctcs were detected in larynx cancer patients by our newly modified method whereas there was no ctc's detection in the samples of controls. thus, this study may provide us monitoring of the treatment process of larynx cancer and this method might be used as diagnostic, prognostic, and predictive biomarkers in cancer therapy as a liquid biopsy. prostate cancer is the second most common cancer and the fifth leading cause of death from cancer in men . circulating tumor cells (ctcs) present in the peripheral circulation of cancer patients with different solid malignancies including prostate cancer and have a potential as a liquid biopsy to monitor disease progression and response to therapies at cell and molecular level . one of the general methods in ctc detection is flow cytometry . radical prostatectomy is the most frequently applied procedure in the surgical management of localized prostate cancer. in this surgical operation, the surgeon removes the entire prostate gland with the seminal vesicles. a radical prostatectomy procedure can be done using the da vinci robotic system (intuitive surgical, sunnyvale, ca, usa) . robotic surgery has been suggested to have fewer complications, lower risk of infections and shorter recovery period following robotic radical prostatectomy , . in this study, our aim was to detect ctcs before and after robotic radical prostatectomy in clinical localized prostate cancer patients. the ctc detection study was performed with our modified method in which . ml of peripheral blood samples were collected from each prostate cancer patient and healthy individual; the samples, using with biomarkers epcam, ck , ck for positive selection and cd for negative selection, were analyzed by bd facs aria iii flow cytometry . according to our results, we detected ctcs in the peripheral blood samples of prostate cancer patients before robotic radical prostatectomy. however, following this surgical procedure no ctc or decreased number of ctss was detected. our study might contribute to understand disease progression after robotic radical prostatectomy in clinically localized prostate cancer patients that warrants further research. keywords: circulating tumor cells, prostate cancer, flow cytometry, robotic radical prostatectomy. p- . . - determination of effect cytotoxic, apoptotic, caspace- activity and mrna expression levels of apoptototic related genes of vulpinic acid on breast cancer cell lines n. kilic ß, s. aras, d. cansaran-duman ankara university, ankara, turkey breast cancer is the most common cancer types in women. several drugs used to treat breast cancer patients are developing resistance to the treatment for this reason success rate falls. therefore the discovery of alternative therapeutic agent and molecular detection of anticarcinogen effect because of treatment for cancer patients may be a source of hope for the contributions. in this study, different concentrations ( . , . , . , . , , , lm) vulpinic acid (va) lichen seconder metabolite was determined to cytotoxic, apoptotic effect and caspase- activity in breast cancer cells (mda-mb- , mcf , bt- , sk-br ) and normal cell (mcf a). in addition to the quantitative real-time pcr (qrt-pcr) using apoptose specific primers (tp- , bcl- , bax, birc- , gapdh, caspase- , caspase- , caspase- , caspase- ) and sybr green dye were performed to determine expression patterns of transcript level in cancer cell lines, using gapdh as a reference gene. the antiproliferative characterization of va effects identification of the gene set at molecular level and we determination role of va on apoptotic pathway. according to our study, va is demonstrated significantly (p < . ) effect cytotoxic, apoptotic, caspase- activity. beside this, dose dependent expression patterns decreased apoptose spesific genes (except of bcl- ) mrna levels from six to eleven fold change more than untreated va cell lines. va will be used as candidate molecule for effective treatment on breast cancer in the future. glycosylation largely determines the variety and functions of proteins. paucimannose, a mannosidic n-glycoepitope has long been thought to be specific for plants and invertebrates. recently, it has also been detected in mammalsin physiological conditions (stem cells) and in pathophysiological conditions (inflammation and cancer). in glioblastoma cells, paucimannose also seems to play a role in cell proliferation. glioblastoma is the most frequent brain tumor in adults with poor prognosis due to a lack of suitable treatments. we hypothesize that paucimannose could be a promising new biomarker as it is otherwise rarely found in mammals. therefore, paucimannose levels were investigated in different glioblastoma cell lines differing in their proliferation rate and tumorigenicity. the highest paucimannose levels were detected in low proliferating, nontumorigenic cells. furthermore, we found that modulation of paucimannose function by application of a specific antibody regulated cell proliferation and the capability of cells to form colonies in soft agar. these data support a functional role of paucimannosidic epitopes in tumorigenic processes. glioblastoma multiforme (gbm) is the most lethal type of malignant brain tumors. recently, gbm stem cells (gscs) have been studied in great deal and accepted that they have a legitimate role in tumor formation, development, chemo-resistance and recurrence. in this study, it is aimed to investigate new therapeutic targets within apoptosis related molecules to select and eliminate cd + gscs effectively. ten primary gbm cells were isolated from gbm tissue samples and they were cultured among with the gbm cell lines (u , u , u and t ). cd + and cd À cells were seperated by macs method via anti-cd (ac ) antibody from cultured cells and cell lines. rna isolation from cd + and (À) cells, cdna synthesis was performed. finally, by performing pcr array, mrna expression levels of genes were detected. proper results were collected and analysed statistically. according to the results of pcr array; it has been found that cd + cells express approximately fold tnfrsf and fold tnfsf when they are compared with control cells. tnfsf binds to cd that is expressed on the surface of tcells. cd does not have a death domain, instead it has a cytoplasmic tail which binds to trafs. trafs act as adaptor molecules that are related with jnk and nf-jb signalling pathways. tnfrsf (dr ) is a death receptor which are known for transmitting the pro-apoptotic signals from outside to the inside of the cell. it negatively regulates t-cell activation and the release of few cytokines. as a conclusion, tnfsf and tnfrsf both are found on immune system cells, mostly on t-cells, which may mean that gbm stem cells act as a immune system cells to avoid the elimination by the immune system. to conclude, acting as an immune system cell and promoting survival via tnfsf and tnfrsf , these molecules may be essential markers to target cd + gbm stem cells. the effect of docetaxel on p , sin a and mdm gene expression in mcf- breast cell line docetaxel is a cytotoxin effective in treating breast cancer. it stabilizes microtubules and causes catastrophic cell cycle arrest in g /m. it also initiate signaling through cell death pathways that result in programmed cell death. in this study, it was aimed to investigate apoptotic and cytotoxic effects of docetaxel has on the mcf- breast cancer cells line. in this study, mcf- breast cell line was applied different doses docetaxel ( nm, nm, lm, lm, lm) as h and h. mtt analysis was performed to the mcf- breast cancer line in control group and groups of docetaxel. afterwards, evaluation of apoptosis by tunel and levels of p , sin a and mdm gene expression by real-time pcr were determined in an order. it was observed cell variable was significant lower in docetaxel groups compared to control group (p < . ) in h as mtt analysis. the lowest cell viabilty was determinated in group applied lm docetaxel. while the lowest positive cell density was determinated in control group, it was observed apoptotic cell density gradually increased with increasing docetaxel concentration in groups treated docetaxel (p < . ). the highest p , si a and mdm expressions were apperared in nm docetaxel group compared to control group. human alpha-fetoprotein (afp) and afp receptor binding domain (afprbd) are able to bind and internalize effectively by wide range of human tumor cells and tissues. as other vector molecules afprbd has insufficient quantity of chemical groups which can be conjugated with drugs or diagnostic agents. conjugation of vector molecules with macromolecular polymer carriers like dendrimers aims to solve this problem. our study describes influence of afprbd-dendrimer-doxorubicin conjugate surface charge on intracellular trafficking routes and toxicity. the amineterminated (g ) and acetyl-terminated (g ) nd generation pamam dendrimers carrying doxorubicin (dox) were used to synthesize conjugates with afprbd. unmodified by afprbd g and g dendrimer derivates labeled with dox were absorbed by the cells at °c with different efficiency. g -dox derivate characterized much slower internalization rate than nonacetylated g -dox. only g -dox shown partial colocalization with lysosomal marker lamp after h of incubation. internalization of afprbd-g -dox and afprbd-g -dox did not show significant difference. at the same time, both conjugates contained afprbd wyкy almost fully associated with lamp already after min of incubation. cytotoxicity results revealed that ic levels of g -dox and afprbd-g -dox coincided and demonstrated a bit higher activity against sensitive to dox skov and resistant skvlb cells than afprbd-g -dox conjugate after h of incubation. at the same time, after h of incubation afprbd-g -dox and afprbd-g -dox were much more than g -dox and g -dox. we may conclude that there is significant difference in ways of dendrimers internalization by tumor cells depending on nature of surface chemical groups. on the other hand, chemical modification of dendrimer conjugated with does not afprbd influence dramatically on the protein trafficking and resulting cytotoxic effect. russian scientific foundation supported this study (no. - - . ) , a key enzyme in glycolysis, catalyzes conversion of phosphoenolpyruvate (pep) into pyruvate with regeneration of adenosine triphosphate (atp). the key regulator of the metabolic alterations found in tumor cells is the glycolytic isoenzyme pyruvate kinase type m that is generally expressed in all proliferating cells and overexpressed in all tumor cells investigated to date. during carcinogenesis a shift in the pyruvate kinase isoenzyme equipment always takes place, such that the tissue-specific isoenzymes disappear, and m -pk is expressed. breast carcinoma, the third most common cancer worldwide, accounts for the highest morbidity and mortality. breast cancer tissue analysis confirmed the upregulation of m -pk in breast cancer, and high m -pk levels were associated with poor prognosis of breast cancer patients. materials and methods: poly hema (mac) nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. pyruvate immobilization was actualysed with cross linking reagent glutaraldehyde. biosensor was developed by preparing pottasiumferrociyanide, selected as a mediator. results: cyclic voltammograms have been carried out at between~ . and . v potentials vs. ag/agci. m -pk activty was detected by using differential pulse method at between . and À . v potentials by observing the differentiations in the current values. in the optimization studies, some parameters such as optimum ph, temperature, concentration of glutaraldehyde and p-hema-mac, were investigated. discussion and conclusion: the method developed for the measurement of the tumor m -pk activity by using biosensor. we found that more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. piruvat kinase tumor m -pk activity determination at low concentrations is possible with this method. p- . . - tie /tek: a potential biomarker for targeting glioblastoma stem cells role in angiogenesis, endothelial cell survival, proliferation, migration and adhesion. therefore, tie /tek could be a potential target for therapeutic strategies directed against glioblastoma stem cells and their microenvironment. in this study, we investigated the gene expression levels of tie /tek in both cd + gscs and cd À gbm cells. gbm primary cells were freshly isolated from glioblastoma tissue samples and cultured in dmem supplemented with % fetal calf serum and % penicillin-streptomycin at °c in % co -humidified incubator. we isolated cd + and cd À cells from gbm primary cells using macs system. following rna isolation from healthy brain tissues, cd À and cd + cells, cdna synthesis was performed. finally, according to microarray protocol, cell surface marker panel array was applied. expression levels were analyzed using the delta delta ct method. statistical analysis was performed using spss software for windows version . . tie /tek gene expression was determined as . fold higher in cd + gscs than normal brain tissue (p < . ). morever it was determined . fold higher compared to normal brain tissue in cd À (p < . ). according to our results tie /tek expression was higher in gscs, indicating that tie / tek may be a potential marker for targeting cancer stem cells in gbm. this research has been supported by the scientific and technological research council of turkey (no: s ). adenosine inhibited the breast cancer stemlike cell population through erk / pathway s. m. jafari, m. aghaie cancer stem cells (cscs) are immortal tumor-initiating cells that can self-renew and drive tumorigenesis in various cancers, including breast cancer and others solid cancers. in a study indicated that extracellular atp reduces tumor sphere growth and cancer stem cell population. but at present, there are no reports available in literature on the effect of adenosine on breast cancer stem cells. in this study we evaluated the effect of adenosine inhibition and its mechanism of action in breast cancer stem cells isolated from breast cancer cell lines. our result showed that adenosine significant reduces breast cancer stem cell population. reduction of erk / protein levels was also observed after treatment cancer cells with adenosine. in conclusion, our results indicate that adenosine decreases the breast cancer stem-like cell population through erk / pathway. taxanes are commonly used for the treatment of many cancers as chemotherapeutic drugs that resistance to these agents has become a major clinical obstacle. taxane based chemotherapy drugs such as paclitaxel, docetaxel and cabazitaxel bind microtubules and inhibit to microtubule polymerization appear to stimulate programmed cell death. taxane-resistance to cancer has not been clearly in progression and development of drug resistance. multiple mechanisms are involved in the drug efflux proteins as multidrug resistance protein, differences in amino acid sequences among the b-tubulin isotypes. we investigated taxane resistance with different doses of paclitaxel, docetaxel and cabazitaxel in prostate cancer stem cells. we compared the expression level of apoptotic proteins, and its functional role in resistance mechanisms in cd + /cd + prostate cancer cell lines. taxane drugs were categorized as concentration-dependent or time-dependent. cabazitaxel caused a time-dependent and dose-dependent reduction in cell viability in all tested cell lines. resistance activity was consistently higher in docetaxel in prostate cancer cells compared with the other drugs. there are many different response of clonogenic formation cd + /cd + cells with resistance to docetaxel, paclitaxel and cabazitaxel in prostate cancer stem cells. the innate of prostate cancer resistances are important characterization steps and critically limits treatment outcomes therefore novel drugs must be focus on antiresistance and molecular based combinations. mesenchymal stem cells (mscs) are self-renewing cells with ability to differentiate into organized, functional network of cells. mscs isolated from various tissues including adipose tissues, bone marrow, umbilical cord, placenta and pancreas have different differentiation and proliferation potential. good knowledge of the metabolism and proliferation mechanisms of stem cells is required for stem cell therapies. glucose is an important molecule in the culture of stem cells. glucose concentration affects the differentiation and proliferation potential of stem cells. the aim of the study was to investigate the proliferation status by identifying the proliferating cell nuclear antigen (pcna) expression under normoglycemic and hyperglycemic conditions in mscs. mscs were isolated from human term placenta amniotic membrane. characterization of the isolated cells was performed using flow cytometry. chondrogenic, osteogenic and adipogenic differentiation potential of these cells were investigated. characterized cells were cultured in normoglycemic and hyperglycemic conditions for and h and the expression of pcna protein expression in these cells were investigated by western blot. flow cytometry analysis showed that isolated cells were positive with mesenchymal stem cell markers cd , cd , cd , cd and negative with hematopoietic markers cd , cd b, cd , cd and hla-dr. western blot result of pcna protein expression statistically significantly increased in human amniotic membrane mscs under hyperglycemic conditions for and h culture. the glucose content of stem cell medium is important because glucose is an effective molecule of the proliferation of stem cells. proliferation of mscs in vitro are still not optimized. when the relationship between glucose and stem cells be understood, it will provide a better understanding for the glucose-related pathologies such as diabetes during pregnancy. prostate cancer (pca) is the second most common type of cancer among men in the world. it is revealed that some gene, protein and metabolite sets control the pca, however the whole metabolomics changes are not completely understood yet. pca is common among older men, and this is an important health problem in developed countries. sarcosine is the n-methyl derivative of the glycine amino acid. glycine n-methyl transferase produces sarcosine from glycine. besides, it is metabolized to glycine by sarcosine dehydrogenase. in , high level of sarcosine in urine was associated with pca by sreekumar et al. they identified sarcosine as a pca biomarker that was significantly increased in urine during prostate cancer progression to metastasis. following this study, several studies have been published indicating sarcosine as a pca biomarker. in our study, a preliminary biosensor system was fabricated for determination of sarcosine in urine by using sarcosine oxidase. sarcosine oxidase was immobilized on au electrode surface using gelatin as an immobilization matrix. glutaraldehyde was used as a cross-linking agent to avoid the loss of the enzymegelatin mixture. optimization and characterization studies were carried out. sarcosine concentrations were detected carefully with the developed biosensor system. the fabricated preliminary biosensor is a promising system that can allow lower detection limits after surface modifications. activation of the epithelial-mesenchymal transition (emt) program in tumor cells is associated with invasiveness and stemness. recent studies implicate emt-inducing molecules in reprogramming energy metabolism. the -phosphofructo- -kinase/fructose- , -bisphosphatase- (pfkfb ) regulates glycolysis by producing fructose , -bisphosphate (f , bp). given that pfkfb is induced by several established emt-inducers in tumor cells, e.g. hif- a and ras, we hypothesized that pfkfb may be involved in regulation of the emt in tumor cells. silencing of pfkfb in pancreatic adenocarcinoma cell lines panc and s vp was achieved using specific sirna molecules. mrna and protein expressions of the cdh gene (encoding e-cadherin, an established epithelial marker), as well as zeb and snai genes, by real-time quantitative (q)-pcr and western blot, respectively. immunfluorescence analysis was performed to visualize e-cadherin protein expression on plasma membrane. in order to test the effect of pfkfb on the invasive ability of the cells, a matrigel invasion assay was performed. ectopic expression of zeb was achived by transfecting cells with a plasmid carrying zeb cdna. cells that were depleted of pfkfb exhibited markedly increased cdh mrna and e-cadherin protein expressions and reduced snai and zeb mrna expressions. immunfluorescence analysis confirmed the upregulation of the e-cadherin protein on plasma membrane. silencing of pfkfb caused approximately % reduction in matrigel invasion, compared to non-targeting sirna. inhibition of the matrigel invasion caused by pfkfb depletion does not appear to be associated with reduced zeb expression, as ectopic expression of zeb did not reverse the effect of pfkfb silencing on invasion. taken together, these data suggest that pfkfb may be required for the maintenance of the mesenchymal phenotype and associated traits in pancreatic adenocarcinoma cell lines. introduction: leukemias are neoplasms that arise from hematopoietic cells initially proliferate in the bone marrow, and then disseminated in the peripheral blood, spleen, lymph nodes and eventually to other tissues. lymphomas occur primarily in the lymph nodes, but can be extended in peripheral blood and bone marrow infiltrate. aim: to determine the values of haematological parameters the control and test groups. to determine the prevalence of types of chronic leukemia in relation to the experimental group. compare haematological parameters in relation to the type of chronic leukemia. materials and methods: a prospective-retrospective study included subjects who were made laboratory hematology in oj clinical chemistry and biochemistry ukcs. blood tests conducted on the hematology analyzer siemens advia hematology system and abbott cell dyn and microscopic analysis of the peripheral blood smear. results and discussion: according to the age of respondents test group was established mild form of anemia, a red blood cell count is totaled . ae . x , which is signifycantly lower compared to the control group. the average number of leukocytes was significantly higher in subjects studied groups and amounted to . x , with a maximum value of x . in the peripheral blood of patients with chronic leucosis has established a significantly higher number of cells compared to the control group (p = . ), while the number of monocytes was a significantly smaller. in the group of patients with chronic leukosis largest number had chronic lymphocytic leukemia ( %), and chronic myeloid leukemia had % of respondents. conclusion: subjects with cll were statistically older than patients with cml, and as regards the gender structure, men have dominated in cll and cml in women. white bloodline was found that the number of leukocytes in both forms of chronic leukemia high above the reference value. p- . . - effect of enzymatic and non-enzymatic isolation methods of endometrial stem cells on their cell proliferative potential and mesenchymal stem cell characteristics human endometrial stem cells (hescs) are responsible for the monthly renewal of the basal layer of the human endometrium by facilitating stromal and vascular regeneration. in this study, hescs were isolated with three different isolation methods including non-enzymatic and enzymatic digestion using trypsin and collagenase type . the effect of these three isolation methods on the acquisition of mesenchymal stem cells (msc) and on hesc proliferative potential was evaluated through flow cytometric analysis of cd surface markers and wst- tetrazolium salt assay. our findings indicate that hescs isolated with these three methods have statistically similar cell proliferation rate at h time point. however, at h time point, hescs isolated with the non-enzymatic and collagenase type method displayed a higher expansion in cell number when compared to the hescs isolated with trypsin. the late passage of hescs isolated with non-enzymatic and trypsin methods showed the highest proliferation rate in comparison to the hescs obtained via collagenase type isolation method at h, h and h. the three isolation methods for the early passages of hescs had a resemblance in their msc profile with no significant difference. on the other hand, late passage hescs isolated using trypsin non-enzymatic method showed a higher cd and lower cd profile. moreover, late passage of hescs isolated with non-enzymatic method displayed a significant reduction in their cell surface cd , cd , and cd surface expression levels. only hescs isolated with collagenase type did not present a significant shift in their mesenchymal cd marker profile from early to late passages, taken together results from this study suggest that the longterm maintenance of mesenchymal markers can only be achieved in cell isolation with collagenase type , while non-enzymatic method is more suitable to obtain higher msc cell yield for immediate use. hepatocellular carcinoma (hcc) abundantly arises on the viral and/or chemical-induced cirrhosis in liver. cirrhosis is defined as one of the premalignant stage hcc in which microenvironmental changes occurred such as uncontrolled production of collagen type i and activation of hepatocyte growth factor (hgf)/c-met signaling. it has been shown that epcam+/cd + subpopulation of cells isolated from hcc tissue can initiate tumor at very low concentration in xenograft model and behaves as hepatic cancer stem cells. however, the molecular mechanisms supporting hepatic stem cell activation are not well understood and knowledge about the role of hgf/c-met pathway in this process is not clear. in this study, we aimed to define effect of collagen type i and hgf induction on the cell behaviours of epcam+/ cd . epcam+/cd + cells were sorted by magnetic separation from huh- cells. then proliferation and invasion of cells were analyzed under the hgf induction as well as branching morphogenesis in vitro. after hgf stimulation, phosphorylation level of c-met increased in epcam+/cd + subpopulation. moreover, presence of collagen type i enhanced significantly effect of hgf stimulation in the invasion of epcam+/cd + cells. we also have showed that hgf stimulation increased branching tubulogenesis capacity of epcam+/cd + subpopulation while it did not effect proliferation of cells. these effects of hgf reverted by c-met inhibitor, su , in vitro. all these findings showed that hgf and collagen type i regulates aggressive phenotype as microenvironmental changes via induction of invasiveness of epcam+/cd + subpopulation of huh- . in conclusion, we showed that hgf/c-met signaling causes to get more metastatic phenotype based on invasion and tubulogenesis in epcam+/cd + hepatic cancer stem cells in hcc and it might be possible to use c-met inhibitors to target hepatic cancer stem cells during hepatocarcinogenesis. endometriosis is defined by the migration of endometrial mesenchymal stem cells (emscs) into the peritoneal cavity or other site of body rather than uterus in a retrograde fashion. its previously known intracellular crosslinking enzyme called tissue transglutaminase (tg ) was shown to play important roles in the extracellular matrix (ecm) modelling, fibrosis, cell adhesion and migration. we have hypothesized that tg might be expressed in emscs and take part in the formation of endometriosis. the difference in the proliferation capacity of emsc isolated from endometrial tissue with/without endometriosis was determined using wst- assay and tg activity and expression levels were analysed by btc assay and rt-pcr. the biosynthesis and activity for mmp- and - were investigated with zymography and rt-pcr, respectively. although tg activity was found to be % less in emscs isolated from endometriotic tissue, these cells showed times higher tg protein expression than those isolated from the control tissue without endometriosis. emscs from endometriotic tissue have . times higher tg and . fold higher itgb mrna levels when compared to the cells of healthy group. similar results were observed in sdc- gene expression with a . fold increase. endometriotic emscs demonstrated an average of . -fold increase in the mmp- activity while a onefold increase was evident in mmp- activity when compared to the healthy emscs. emscs from patient group possessed a higher proliferative ability in comparison to that of healthy subjects within h. the fact that emscs from the control tissue showed lower tg protein levels with a high enzyme activity suggested that tg might be important in the development of endometriosis not only by destabilizing ecm but also enhancing the cell migration. in this context, the upregulation of tg along with itgb and sdc was evident in emscs of endometriosis which was possibly associated with the increase in the activity of mmp- and - . recent studies have indicated that pluripotent stem cells and some stromal stem cells such as mesenchymal stem cells (msc) are metabolically different from their differentiated counterparts. in this study, the cellular mechanisms controlling metabolic changes in stem cells was investigated using wharton jelly mesenchymal/stromal stem cells (wj-mssc). wj-msscs were isolated by the explant method and cultured in dmem-f with % fbs. endothelial differentiation was induced by the addition of vegf, egf, insulin and hydrocortisone for days. neuronal differentiation was achieved by using commercial neuronal differentiation medium (millipore) for days. in parallel experiments, cellular metabolic activity such as lactate production was measured. the msc characterization was performed by flow cytometry using antibodies against cd , cd , cd and cd (bd human msc analysis kit). the differentiation process was followed by measuring the expression of cd , cd for endothelial and gfap, neu and tyrozine hydroxylase proteins for neuronal cells by immunofluorescence. for gene expression, nanog, cd and gapdh genes were analyzed by rt-pcr. differentiation stimuli to endothelial or neuronal cells resulted in a significant decrease in msc marker proteins. expression of stem cell markers other than cd were decreased to - %. differentiation induced the expression of cd , cd for endothelial and gfap and neu proteins for neuronal cells. in vitro lactate production was decreased following differentiation in both lineages. neuronal differentiation increased glucose consumption by~ % and the extracellular calcium concentration of these cells was significantly lower than synchronous undifferentiated cells. glycolytic activity is decreased during in vitro differentiation of wj-msscs. metabolic reprogramming and glucose uptake of cells may be an early indicator of the differentiation process in wj-msscs, supporting the view on their metabolic plasticity. store-operated ca + entry (soce) activated by depletion of intracellular ca + stores has been shown to control intracellular ca + homeostasis in many physiological and pathological events. stromal interactive protein, stim , as endoplasmic reticulum (er) ca + sensor and orai protein as pore-forming subunit of soc channels play crucial roles in the activation of soce channels. stim and orai were reported to have pathophysiological roles especially in hepatocellular carcinoma (hcc). anticancer chemotherapy frequently falls back because of these tumor-initiating subpopulations, tentatively called 'cancer stem cells'. the purpose of this study was to investigate the roles of stim and orai on soce in differentiation of huh- hccs expressing epcam and cd surface adhesion molecules (epcam + cd + ). epcam + cd + subpopulations in huh- cells were separated via flow cytometry and transfected with stim and orai- over-expressing (oe) plasmids. expression levels were confirmed by rt-pcr. changes in intracellular ca + concentration were monitored via dual wavelength spectrofluorimeter in fura -loaded cells. in epcam + cd + cells, er ca + release increased without any change in soce compared to that of epcam À cd À cells. similar results were observed in stim -oe epcam + cd + cells. on the other hand, increase in orai has no effect on either parameter. cancer is globally one of the most death causes. recently, huge improvements occurred in the cancer diagnosis and treatment due to advanced technology, however recurrence occurs almost - % of patients and their survival times decreases. in this study, we aimed to investigate of relationship between the cancer stem cells which are strongly associated with chemotherapy and radiotherapy resistance and recurrence with the non-classical mhc i antigens which have immunosuppressive properties. for this purpose, we immunohistochemically evaluated the expression patterns of cd , cd , nanog, oct / , hla-g and hla-e in the advanced stage colorectal, gastric and breast cancer and also non malign biopsy samples. we detected that the cancer stem cell markers cd , cd , nanog and oct / significantly increased in the advanced stage cancer tissues. however, the immunosuppressive hla-g and hla-e expressions increased only in the colorectal and gastric tumor tissue. in addition to the presence of cancer stem cell like cells in the tumor tissues, increased expressions of hla-g and hla-e may indicate an immune evasive adaptation of tumor cells. according to our findings, the hla-g and hla-e may be potential therapy targets to elimination of cancer stem cells of colorectal and gastric cancers. however, more detailed studies are needed to support our findings and also to determinate of clinical values of these markers. endocannabinoids increase sdf- release from human mesenchymal stem cells s. k€ ose , f. aerts kaya , d. uc ßkan c ß etinkaya , p. korkusuz stem cell research and application center, hacettepe university, ankara, turkey, department of histology and embryology, hacettepe university, ankara, turkey lipid-structured endocannabinoids are endogenous morphine ligands and present widespread receptor-mediated effects at physiological and pathological levels on the nervous system as well as many other systems. these effects are partially realized through mechanisms affecting cell growth, differentiation, apoptosis and migration at the molecular level. the hematopoietic progenitor cells (hpcs) and mesenchymal stem cells (mscs) form a distinct niche in bone marrow where they interact with each other in harmony. the stromal cell-derived factor (sdf- /cxcl ) is a chemotactic factor in bone marrow and is released from mscs and their receptor cxcr is found in hpcs. with these rationale in mind, we asked if hpcs and msc interaction mediates sdf- release via endocannabinoidal system. bone marrow mscs obtained from healthy donors and passage mscs were induced with ng/ml lipopolysaccaride (lps) for h. antagonists for cb (am ) and cb (am ) receptors were added to cultures for days. after incubation with antagonists msc culture supernatants collected and processed with human sdf- beta in elisa medium. analyses demonstrated direct decreasing effect of endocannabinoid receptor antagonists on sdf- beta release from bone marrow mscs. in conclusion, endocannabinoidal system regulates sdf- release on mscs and directly act on hpcs mobilization in bone marrow microenvironment (niche). this may have a clinical implication on therapeutic mobilization strategies for hscs in hematology clinical applications. implantation is invasion of the embryo into the endometrium and occurs in three stages apposition, adhesion and invasion, via the complex cellular and molecular mechanisms. during these stages, both of maternal endometrium and embryo should be appropriate for the implantation which is the beginning of pregnancy. receptivity of uterine consists in the existence of growth factors such as tgfbeta- , igfr , vegf. it is indicated that damages of factors relesead from endometrium and blastocyst prevent implantation. recently, stem cells can be obtained from many sources to use for therapeutic purposes and mesenchymal stem cells derived from bone marrow are the most studied. in our study, it was aimed to investigate molecules play a role in blastocyst implantation after bone marrow derived mesenchymal stem cell application into the rat endometriyum. female rats were divided into three groups which were saline (sf, n: ), media (m, n: ), stem cell in media (m+bmsc, n: ). after vaginal smear technique, female rats in estrous cylcle were injected into the uterine and periton ll saline, ll culture media and cell/ ll culture media. the pregnant female rats on the day were sacrified and uterine samples removed and were stained with heamatoxylin-eosin histochemically and anti-tgfbeta- , anti-igfr , anti-vegf and anti-pcna immunohistochemically and obseved under light microscope. h-score results were determined using one-way anova test statistically. it was found that intraperitoneal administration of stem cells with media, was increased tgfbeta- , igfr , vegf and pcna parameters when compared with the intrauterine administration of stem cells. in this study, it was revealed that distribution of molecules play role in implantation were changed due to stem cell application. it is supposed that stem cell treatments can be cured the molecules caused infertility. many unconventional biochemical factors remain to be investigated for their potential effects on stem cells. among others, endogenous gasotransmitter h s, generated from l-cysteine and organosulfur-compounds (oscs) metabolisms, plays very important roles in the central nervous, respiratory and cardiovascular system. slow-releasing h s donors are viewed as powerful tools for basic studies and innovative pharmaco-therapeutic agents for cardiovascular and neurodegenerative diseases. exogenous h s administration is able to promptly scavenge ros, activate myocardial k atp channels and increase pro-cell survival signaling, very likely activating erk and phosphatidylinositol -kinase (pi k)/akt pathways. the effects of h s-releasing agents on the growth of stem cells are not yet widely investigated. therefore, stem cell therapy combined with h s may have great clinical relevance in cell-based therapy for regenerative medicine. the effects of slow-releasing h s agents on the in vitro cell growth and differentiation of human lin À sca + cardiac progenitor cells (hcpc) were here studied. in particular, the effects of h s-releasing agents, such as na s, gyy and water-soluble gsh-garlic conjugates (gsgaws), on the cell viability and differentiation of hcpc were here investigated by colorimetric assay, immune-fluorescence microscopy and western-blotting analysis. the treatment with slow-releasing h s donors increased the cell proliferation in a concentration dependent manner respect to the control. moreover, the treatment with gsgaws led to an up-regulation of the expression of proteins involved in the cell adhesion and differentiation processes. these preliminary results highlight on the effects of this gasotransmitter on the stem/progenitor cells and on the possibility to develop functional d-systems for cardiac tissue repair, that take into account the relevant biological role of h s in the cardiovascular system. p- . . - investigation of the protective effect of boric acid and omega- fatty acid in model of acute myocardial infarction changes in myocardial rats ischemic heart disease being the most common cause of the mortality and morbidity in worldwide commonly results from the occlusion or narrowing of the coronary arteries by atheromatous plaque and thus is named as coronary artery disease. male sprague dawley rats were used in the present study. rats were divided into groups with rats in each: control, mi, mi+boric acid, mi+omega- and mi+boric acid+omega- groups. control rats were treated with ml/day saline, boric acid-treated rats received mg/kg/day boric acid and omega- -treated rats received mg/kg/day for days by oral gavage. for the experimental mi model, mg/kg izoproterenol-hcl (iso) was administered subcutaneously two times with a -h interval in the last days of the boric acid and/or omega- treatments. twelve hours after the second dose of iso, general anesthesia was induced. under general anesthesia and spontaneous respiration, ecg recordings were obtained by using a computerized data recording and analysis system (mp , biopar) and d-ii recordings were used in the analysis. compared to the control group, serum ck-mb, bnp and tnf-a levels were higher in mi group (p < . , p < . and p < . respectively). in the heart tissue homogenate, biochemically measured calpain activation and mda were increased (p < . and p < . , respectively) and pon levels were decreased (p < . ). according to the ecg recordings, st wave and heart rate were found to be decreased (p < . and p < . , respectively). on the other hand, all above mentioned parameters were found to be improved in rats treated with boric acid and/or omega- after induction of mi. moreover, histological analysis including light microscopy and tem revealed a significant histological improvement in rats treated with boric acid and/or omega- after induction of mi. results of the present study suggest that omega- and/or boric acid treatment significantly decreases the cellular damage in mi. this is study is aimed at measuring the level of serum heart-type fatty acid binding protein (h-fabp) in patients presenting with diabetic ketoacidosis (dka) and diabetic ketosis (dk) and to determine its role in identifying early period cardiac ischemia by comparing this level with the level of a control group at a comparable age this study was planned to be a prospective study and it included patients diagnosed with dka, patients diagnosed with dk and voluntary pediatric and adolescent healthy control subjects. the h-fabp, creatine kinase-mb (ck-mb) and troponin-i levels were studied in patients with dka and dk as well as in the control group at the time of presentation. for dka patients, their h-fabp values were measured once again after acidosis correction and compared with the values they had at the time of presentation there were no differences among groups in terms of sex, age, height and weight. no statistically significant differences were found among groups with respect to troponin-i values ( . ae . , . ae . . ae . ; p = . ). no statistically significant differences were found among groups with respect to ck-mb values ( . ae . , . ae . , . ae . ; p = . ). the h-fabp values of dka patients at the time of presentation were found to be statistically significantly higher than those of dk patients and control group ( . ae . ; . ae . . ae . ; p = . ). the h-fabp value of the dka group at the time of presentation was found to be statistically significantly higher than the value at hour after acidosis correction ( . ae . ; . ae . ; p = . ) the fact that h-fabp levels were found to be high in pediatric patients diagnosed with dka at the time of presentation suggested that myocardial ischemia had been triggered. in diabetic patients, every ketoacidosis attack may lead to cardiac ischemia, thereby accelerating progress to necrosis. in conclusion, we would like to propose h-fabp as a potential marker for indicating myocardial ischemia. p- . . - genome-wide analysis of hypoxic stress response in human cardiomyocytes stress in human cardiomyocytes on a genome-wide scale remains poorly understood. this study aimed to identify the gene expression patterns of adaptive response of the human cardiomyocytes (hcm) to hypoxic stress. in vitro experimental models of hypoxia mimicking in-vivo coronary ischemia, are useful tools to identify molecular pathways involved in myocardial ischemia. in the current study, we cultured ac -hcms in dmem/f with %fbs. to simulate hypoxia model, cardiomyocytes were plated in hypoxia chamber ( %o , %co , %n ) for , , , h and the control group was incubated in normal conditions ( %co , %o ). cell viability was determined using mttassay. annexin-v assay was used to monitor apoptosis. gene expression profiling was analysed with affymetrix-hg-u -plus- arrays. following bioinformatic and statistical analyses differentially expressed genes (deg) were classified according to gene ontology using david and kegg pathway analysis tools. according to mtt, annexin-v and hif gene expression results, hypoxia time was determined as h. we identified genes ( down-regulated and up-regulated) (p < . , fold change ≥ . ) were differentially expressed in hypoxic-ac vs. ac . degs were mainly clustered in cell proliferation, regulation of cell death, cell adhesion and response to stress. furthermore, transcriptome analyses revealed that 'metabolic, cytokinecytokine receptor interaction, hif- signaling, tgf-beta, cell cycle and apoptosis' pathways were involved in the hypoxic stress response of human cardiomyocytes. this study provides molecular information regarding gene expression reprogramming of human myocardial hypoxia. the pathways identified in this study may pave the road for translational medicine. this study was supported by tub _ itak project number s . autologous ips cells after reprogrammed into endothelial progenitor cells (epcs) may offer several advantages in the treatment of cardiovascular disorders because of their cardiogenic and vasculogenic differentiation potential. to reach that purpose, we differentiated and characterized mouse ips cells into flk + , a well-recognized epc marker. further maturation of epc was characterized by the expression of cd and cd markers. purified ips cells were differentiated into flk + cells with the use of differentiation medium on type iv collagen-coated dishes in the absence of lif. we then analyzed flk gene expression and protein levels with qrt-pcr, western blot and immunocytochemical methods on days . to . . flk + cells isolated with macs system and then recultured these cells in differentiation medium with vegf to induce epc cells. following induction, cd and cd gene expression and protein levels were analyzed with genomic and proteomic methods. after isolating these cells and aggregate overnight, we cultured cells in three-dimensional condition in collagen type i and used differentiated medium including vegf and egf. we found that flk expressing cell number reached to a peak level ( %) on day . followed by a progressive decline subsequently. in the second step, cd and cd positive cells were generated and enriched during day of induction. we showed optimal time for harvesting flk + cells is day . of initial differentiation. following isolation of flk + progenitor cells they were further matured into functional epcs by vegf within days of induction. additionally for evaluation of angiogenic potantial differentiated cells, we monitored epcs behavior along vascular formation in d culture. our work demonstrates that epcs could be successfully derived from ips cells and these cells have vascular formation and angiogenic potential in d culture. epc drived ips cells play important role in the treatment of cardiovascular disease. p- . . - electrophysiological, biochemical and genotoxic effects of luna experience on heart tissue in rat model pesticides are widely used for the control of agricultural, industrial and domestic pests. however, the uncontrolled use of pesticides has diverse effects on ecological system and public health. fungicides are one of the pesticide type used to kill fungi or fungal spores. in this study, the effect of different doses of luna experience, a fungicide, on the cardiac electrophysiology and genotoxicity in rats were investigated. among five groups ( mg, mg, mg, control and positive control for comet assay) treatment groups received by gavage doses of luna experience for days. electrical activity of heart were recorded using electrophysiological recording techniques. tissue activities of paraoxanase (pon) and arylesterase (are) and level of malondialdehyde (mda) were measured using biochemical methods. comet assay was performed on heart tissue. we calculated genetic damage index (gdi) and damaged cell percent (dcp) from comet assay. it was observed that there is a significant decrease in heart rate in all treated groups as compared with control group (p < . ). amplitude of p wave and qrs complex did not change (p > . ). in all treated groups, statistically significant differences were found for values of pon, are, mda, gdi and dcp when compared to control group (p < . ). according to our results, exposure to different doses luna experience have a probable hazard potential for the cardiac system. the macrocyclic cage complexes iron (ii) clathrochelates are of the interest due to their bioactivity; they are able to inhibit t- rna polymerase, possess toxicity to leukemia cells hl- and suppress amyloid fibril formation. their binding to serum albumins was reported; the extreme binding affinity to albumins is observed for the compounds bearing carboxy groups. upon this interaction, clathrochelates quench protein intrinsic fluorescence and gain optical activity inducing circular dichroism (cd) signal in - nm region. here we examine the effect of spatial arrangement (isomery of substituents) of clathrochelates on their binding to globular proteins. we study bis-substituted clathrochelates bearing two same or different isomers (ortho-/meta-/para-) of carboxyphenylsulfid groups. their interaction with bovine (bsa) and human serum albumins, b-lactoglobulin and lysozyme are explored by cd and protein fluorescence quenching method. the binding of compounds to albumins evoked the cd bands of the same shape, but their intensities vary up to times depending on substituents isomery. in the presence of b-lactoglobulin, the intensities, shape, and positions of the induced cdbands differ for the compounds with different isomer groups. the cd bands induced by the lysozyme in the case of di-para substituted clathrochelate are shifted relatively to the bands of other isomeric compounds. the pronounced quenching of protein fluorescence by clathrochelates was observed only in the case of bsa, its intensity depends on the geometry of substituents ( - times). the different spatial arrangement (isomery) of carboxyphenylsulfid substituents in clathrochelates causes the distinctions in both their cd-signal induced by interaction with proteins and their effect on the protein fluorescence. the geometry of ribbed substituents is important for their binding to biomolecules (particularly proteins) and is suggested to determine the structure of the formed guest-host complex. d bioprinting is a new technology that revolutionized the field of tissue engineering and regenerative medicine, allowing reconstruction of living tissue and organs preferably using the patient's own cells. using a d printer we can design biological structures by controlling exact deposition of cells, growth factors and extracellular molecules in a spatially-controlled manner. the aim of this study was to evaluate the differentiation of human amniotic fluid stem cells (afsc) into endothelial progenitor cells using a bioinkÒ hydrogel photopolymerized in a d network resembling vascular tissue. characterization of afsc was performed by flow cytometry, followed by sorting of the cd + stem cell subpopulation. cd + stem cells were stained with cell tracker red cmtpx and then mixed with bioinkÒ hydrogel. printing was done using a lm diameter needle, under bar pressure, and mm/min speed. the network models with define distance apart were printed and analyzed by fluorescent microscopy. mtt test was used to evaluate the viability of the cd + stem cells. our results showed that afsc remained viable as shown by mtt assay. the fluorescent microscopy images confirm the viability biochemical test showing that the cd + cells viability is maintained after days of cultured in bioinkÒ hydrogel. furthermore, histological section of hydrogel showed that cells have a relatively uniform distribution forming network interactions between cells. flow cytometry assay showed that cd + cells expressed endothelial markers such as cd , cd , cd , cd and vegfr . in conclusion d printers are useful tools for creating three-dimensional scaffolds that mimics the cell microenvironment where different types of cells could proliferate, differentiate and crosslink with each other forming tissue-like structures. this study aims to reveal the biocompatibility, biodistribution and immunomodulatory impact on the production of inflammatory citokines of magnetite (fe o ) nanoparticles functionalized with natural compounds with proved antimicrobial and immunomodulatory effects. co-precipitation synthesized fe o were functionalized with plant-derived compounds: eucalyptol, carvone, limonene and b-pinene. characterization was done by ir, sem and hr tem, while in vitro biocompatibility was tested using endothelial human cells (fluorescence microscopy and proliferation assay). in vivo biodistribution was tested in a balbc mouse model at and days post-intraperitoneal injection, followed by experimental organ removal. tissue sections obtained from vital organs were stained with hematoxylin-eosin. production of inflammatory cytokines was assessed by elisa. results demonstrated that, at concentrations of lg/ml, all prepared nanosystems have a good biocompatibility in vitro and in vivo, allowing the development of cultured cells and also not affecting any visible behavior and organ morphology of the mice. microscopy evaluation of the organs sections revealed that nanoparticles are not present in vital organs such as brain, heart, kidney and liver, but aggregates were visible in the lungs and spleen. at days post-injection no visible aggregated were found in the lungs, few dark-brown nanoparticles clusters being visible in the red pulp of spleen. elisa results revealed that fe o functionalized with carvone and limonene significantly stimulated the production of il- , il- and il- , while reducing the production of tnfa. other nanosystems din not impact significantly on the cytokine production. functional fe o nanoparticles are efficient drug delivery shuttles, able to stabilize pharmacological compounds, such as plant-derived bioactives, and their biocompatibility, specific biodistribution and limited immunomodulatory effects recommend their use in pharmacological formulations. p- . . - new approach for cell imaging with fluorescent carbon nanoparticles m. dekaliuk, k. pyrshev, o. demchenko palladin institute of biochemistry, kiev, ukraine in the nanotechnology field, much interest was focused on the new carbon nanomaterials for cell imaging. recently discovered inorganic carbon nanoparticles ('c-dots') due to their excellent fluorescence characteristics and biocompatibility have ample opportunities for their use in imaging and functional transformations in living cells. their distinctive features, such as high brightness, small sizes, high biocompatibility, small negative charge on the surface and very easy methods of their preparation present a good alternative to other nanoscale materials. the focus of our research was to determine the possibility of using c-dots as the easily available probes for apoptotic cells detection. the carbon nanoparticles were prepared from alanine, citric acid, urea, etc by hydrothermal treatment at c. the studies were performed with adherent epithelial vero and hela cell lines (atcc). with these tools we demonstrate that both native and apoptotic cells can be easily visualized. the cdots uptake occurs probably by endocytosis, which allows for much larger their number to accumulate in apoptotic cells. using the different methods of sample preparation, they show the ability for labeling various structural compartments of the cell. for living cells there are the intracellular vesicles and lysosomes. in contrast, in fixed cells the nucleus is labeled preferentially. the fact that apoptotic cells accumulate strongly increased amount of cdots can be efficiently used in flow cytometry for characterizing the cell populations regarding the relative amount of apoptotic cells in different experimental conditions. the application of such cheap and easily accessible nanoparticles provides more opportunities to simplify the popular methods of cell labeling and detection. previously, our studies showed the possibility of using these nanoscale fluorophores for super resolution method sofi. a new electrochemical microbial biosensor for the fast detecting of dopamine and epinephrine based on candida tropicalis immobilized in a carbon paste electrode (cpe) modified with single wall carbon nanotube (swcnt) was described in this paper. the immobilized cells were used as a source of polyphenol oxidase (ppo) to develop voltammetric epinephrine and dopamine biosensor. voltammetric determination of phenolic compounds like epinephrine and dopamine is a simple technique available. direct oxidation of phenols can be used, but the oxidation potentials of this compounds are similar and they can not be detected distinctively. another possibility is the use of biosensors based on the polyphenol oxidase (tyrosinase) enzyme that oxidises the phenolic compounds into their corresponding quinones. by this way phenolic compounds that epinephrine and dopamine that used in this study were detected at the different potential. the effect of varying the amounts of swcnt and microorganism on the response to epinephrine was investigated to find the optimum composition of the sensor. the effects of ph and temperature were also examined. increases in biosensor responses were linearly related to dopamine concentrations between . and . mm and epinephrine concentrations between . and . mm. limits of detection of the biosensor for dopamine and epinephrine were calculated to be . and . mm, respectively. finally, proposed systems were applied to epinephrine and dopamine analysis in pharmaceutical drugs. objective: it has started a long time ago to search for a material that can replace blood. this material does not require special storage conditions, independently of the recipient's blood group and can be applied to all individual. milk, casein derivatives, starch, saline and ringer were used for this aim in the past. the determination of toxic effect of natural hemoglobin (hb) on human, researchers have focused on development modified blood. in this work, the development of an artificial biomaterial alternative of blood for using as preoperative and operative aims was aimed. material and methods: in our study, ultrapure hb molecules are immobilized on triethanolamine coated magnetic nanoparticles using various techniques. prepared nanoparticles were characterized by ft-ir, ctem, xrd and cyclic voltammetry (cv). the cytotoxic effects of artificial blood were tried on mtt cell proliferation. results: the characteristic peaks of hemoglobin were obtained from ft-ir spectra differently from support. particles size is concluded by using debye-scherrer equation as > nm from xrd spectra. sem and ctem images supported xrd result. cv results showed that hb molecule has À . v cathodic potential against ag / agcl standard electrode. significant differences were not observed in the mtt results (p < . ). conclusion: the nanoparticles were obtained in accordance with the intended desired method. it is determined that the hemoglobin molecules give the same potential with natural blood even after weeks of immobilization and carrying oxygen as natural blood. there are statistical differences between results of mtt tests due to used concentration. but, it is considered that decantation advantage of the artificial blood minimized cytotoxic effects. proteoglycans are among the most abundant molecules of the inter-cellular structure and they are present in extracellular matrices of connective tissues. these glycosylated proteins contain one or more (gag) chains that are covalently attached to the core protein and their hydrodynamic function is mainly due to the physicochemical characteristics of this gag component which provides hydration and swelling pressure to the tissue. gag levels excreted via urine are used as a marker to monitor different diseases (chronic renal disease, renal fibrosis, glomerular filtration abnormalities, bladder stones, breast and lung cancers, hypertension and diabetes, etc.) besides the well known mucopolysaccharidoses. however, their detection by using chromatographic methods is hard, because of the high polarity of negative charges and different functional groups such as acetyl sulfates that generate microheterogenity. in this study, we developed molecularly imprinted chromatographic hplc columns for specific heparan sulfate (hsa), chondroitin sulfate (cs) and dermatan sulfate (ds) detection in urine. positively charged acrylamide monomers were first polymerized by precipitation polymerization, to produce polymers which will show specific recognition for gag's via electrostatic interactions and hydrogen bond formation. these gag selective polymers were then filled in the steel hplc columns and columns eluents were chemically degraded. degradation products of gag's were examined offline column coupled with tandem mass spectrometry. the results showed that our imprinted columns separated gag's specifically and sensitively. thus, urine gag's can be specifically determined by using a gag specific molecularly imprinted column. in this study internal standart weren't used because the matrix effect was lower than % for each urine samples. %cv of ds, cs and hsa was calculated as; supported lipid bilayers (slb) were started to be used for cell culture studies to focus on cell adhesion, cell signaling etc. testing the stability of slbs is essential to utilize them as cell culture platforms. in this study, the stability of phosphatidylcholine (pc) lipid bilayers on glass was investigated under milli-q water, phosphate buffer saline (pbs) and dulbecco's modified eagle medium culture (dme) medium supplemented with/without serum. the stability was also checked by enriching slb with different lipids. pc-liposomes were prepared by hydrating the dried thin lipid film with pbs and then by extruding the suspension through a polycarbonate membrane. a negatively charged phospholipid, phosphatidylserine (ps, %); a positively charged phospholipid, dotap ( %) and cholesterol ( %) were also used for liposome preparation. liposomes were fluorescently labelled and series of slb imaging were taken for a week. in all experiments in milli-q water and pbs, the stability was conserved for days. pc bilayers in medium supplemented with serum showed hole formations on the second day and their number and size increased rapidly in time. when the bilayers were prepared in medium without serum, disruption was lowered but not completely removed as a result of other factors in medium. cholesterol providing an increased rigidity to the membrane caused higher stability. positively charged bilayer structures also showed increased stability. this can be explained by decreased mobility of bilayer as a result of electrostatic interaction between positively charged molecules and negatively charged glass surfaces. decreased mobility decreases the interactions within the medium. lastly, negatively charged bilayers did not show high stability. strong repulsive forces between the negatively charged surface and bilayer probably prevented the integrity of the bilayer and increased the deformation. in recent years the use of biopolymers has gained priority in tissue engineering and biotechnology, both as dressing material and for enhancing treatment efficiency. there is a demand for new biopolymers designed with protease inhibitors and antimicrobials. ll- is an important antimicrobial peptide in human skin and exhibits a broad spectrum of antimicrobial activity against bacteria, fungi, and viral pathogens. using lignin which is an abundant carbohydrate polymer in nature and a polyacrylic acid, we prepared a polymer film by plastifying caprolactone and polyacyrlic acid. films were actified to immobilize ll- . the structure was elucidated in terms of its functional groups by fourier transform infrared spectroscopy (ftir), and the morphology of the film was characterized by scanning electron microscopy (sem) before and after the immobilization process. the amount of ll- immobilized was determined by elisa method. . % of ll- peptide was successfully immobilized onto the films. antimicrobial activity was determined in the film samples by quantitative antimicrobial activity method. according to the results, ll- immobilized film samples were effective on test organisms; gram-positive staphylococcus aureus and gram-negative escherichia coli. in bio-compatibility assays, the ability to support tissue cell integration was detected by using t mouse fibroblasts. samples were examined under transverse microscope, non-immobilized sample showed a huge cellular death, whereas ll- immobilized film had identical cellular growth with the control group. this dual functional film with enhanced antibacterial properties and increased tissue cell compatibility may be used to design new materials for various types of biological applications. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in vitro modulation of the cross-talk between macrophages and osteoblasts by titania nanotube-modified ti surfaces p. neacsu, a. mazare, p. schmuki, a. cimpean bone remodeling is a dynamic process that maintains a fine balance between bone formation and resorption, and is highly influenced by the inflammatory state of the local microenvironment. therefore, a proper modulation in the cellular interactions and cytokine expression is a promising approach to achieve enhanced bone healing. as the biomaterials surface has a major impact on cellular behavior, the goal of the current study was to investigate the influence of tio nanotube-modified ti surfaces (ti/tio ) on the cross-talk between raw . macrophages (mf) and mc t -e preosteoblasts (ob) in mono-and co-culture systems in comparison with flat ti (cpti). raw . and mc t -e cells were seeded on the test surfaces and grown in standard culture conditions for various periods of time. for co-culture studies, the cells were cultivated using a transwell system. inflammatory mediators released by raw . cells were measured using elisa technique, while the ob capacity to produce calcified bone matrix was evaluated by alizarin red staining. in co-cultures, lps-stimulated tnf-a, il- and mcp- release was significantly increased at h, while after days only il- exhibited higher amounts when compared with mf cultures alone. moreover, the secretion of these mediators by cells exposed to ti/tio was diminished, especially in lps evoked conditions. also, alizarin red staining demonstrated the presence of calcium deposits when ob were co-cultured with mf for h and days, whereas the presence of the mf for weeks significantly inhibited mineralization. on ti/tio surface elevated calcified matrix was observed, as compared with cpti. this study reveals that the overall effect of inflammation suppression induced by ti/tio may contribute to the enhanced mineralization. also, chronic inflammation may inhibit or delay the regeneration process. therefore, an adequate modulation of mf and ob interactions is vital for the biomaterials success in stimulating bone regeneration. p- . . - synthesis and characterization of the branched magnetic polymer for drug delivery systems t. tarhan , , b. tural , s. tural mardin artuklu university, mardin, turkey, dicle university, diyarbakir, turkey magnetic nanoparticles (mnp) have gained a lot of attention in biomedical and industrial applications due to their biocompatibility, easy of surface modification and magnetic properties. magnetic nanoparticles can be utilized in versatile ways, very similar to those of nanoparticles in general. however, the magnetic properties of these particles add a new dimension where they can be manipulated upon application of an external magnetic field. this property opens up new applications where drugs that are attached to a magnetic particle to be targeted in the body using a magnetic field. often, targeting is achieved by attaching a molecule that recognizes another molecule that is specific to the desired target area. in recent years, the development of the systems in which drug is delivered magnetically to the target is drawing considerable attention since it is a current issue. it is possible to eliminate the most of the problems caused by high doses of chemotherapy by using the magnetic drug delivery systems. therefore, it is important to design delivery systems with high drug loading capacity. it is necessary to increase the number of reactive groups on the surface of nanoparticles in order to increase drug loading capacity. in this study, we synthesized a novel magnetic surface for drug delivery systems. magnetic dextran-nta (md-nta) was synthesized by using magnetic o-carboxymethyl dextran (ocmd) and nana-bis (carboxymethyl) -l-lysine hydrate (nta) in order to increase the number of reactive carboxyl groups on the surface of biocompatible and biodegradable magnetic dextran. magnetic material (md-nta) which was prepared and characterized by the analysis of transmission electron microscopy (tem), scanning electron microscope (sem), vibrating sample magnetometer (vsm), fourier transform infrared spectroscopy (ftir) and x-ray photoelectron spectroscopy (xps). there are three subtypes of the tgf-b protein that has been reported to be involved in tissue repair process; scar tissue formation has been reported on tissues that has been affected by tgf-b and due to high collagen synthesis. on the other hand the other isoform tgf-b , suppresses the dense collagen production caused by tgf-b and prevents the scar formation. to be able to use these growth factors local or iv route, new drug transport systems are needed to protect the bioactivity during the treatment and controlled release. for this purpose poly(lactic-co-glycolic) acid polymer which is widely used in controlled release systems was chosen as the matrix material. aim of the project was to design, formulate, prepare and optimize tgf-b loaded plga nanoparticular and/or plga polymeric film drug delivery systems and to test their effect on cell proliferation. tgf-b loaded nanoparticles was prepared with emulsion-solvent evaporation method; whereas polymeric film systems was prepared with film castingsolvent evaporation method. following the preparation tgf-b loaded drug delivery systems was characterized. quantification and in vitro release of the growth factor tgf-b was studied with elisa. hepg cell line was used on mtt cell proliferation assay for both tgf-b loaded nanoparticles and films on a time course study. nanoparticles and films were prepared and loading efficiency of the nanoparticles were found to be . %. particle size, zeta index and polydispersity index for this formulation were determined as . ae . nm, . mw and . , respectively. thickness of the prepared films were ae . nm. additionaly prepared nanoparticles and films were found non-toxic. tgf-b nanoparticles and films which were prepared in this study are planned to be used as an effective treatment strategy for wound healing after injury. this project was supported by grand s from the scientific and technological research council of turkey (tubitak). polyvinylpyrrolidone (pvp) is a biodegradable material and natural polymeric biomaterial in such studies. ganoderma lucidum is a natural material containing triterpenes, polysaccharides, adenosine, polypeptides, and amino acids. these constituents have been shown to exhibit anti-cancer properties, enhance and regulate immunity, resist oxidation and ageing, and promote metabolism and cell proliferation. composites of polyvinylpyrrolidone (pvp) have been prepared by solution intercalation method using ganoderma lucidum at different loading amounts. the characterization of pvp/ ganoderma lucidum composites was made by x-ray diffraction (xrd) and scanning electron microscopy (sem); the interactions between ganoderma lucidum and pvp was determined by ftir-atr; the thermal stability was determined by simultaneous tg/dta. hemocompatibility of the prepared composite samples were investigated by a -well plate spectrophotometer. in addition, contact angles and antimicrobial activity of biomaterials were also determined. ftir-atr confirms interactions formed between ganoderma lucidum and pvp. xrd and sem results give evidence that ganoderma lucidum was well dispersed and homogenously in the pvp matrix. thermogravimetric analysis indicated that introduction of clay to the polymer network resulted in an increase in thermal stability. the results of in vitro hemocompatibility test were showed that pvp/ ganoderma lucidum composites are used as biomaterial. the development of synthetic materials, textured polymers and metals and their increasing use in medicine make research of biomaterials' hemocompatibility very relevant. composite material is a multi-phase system consisted of matrix material and reinforcing material. matrix material is a continuous phase and reinforcing material is a dispersed phase. the main two bioactive components of ganoderma lucidum can be broadly grouped into triterpenes and polysaccharides. despite triterpenes and polysaccharides being widely known as the major active ingredients at anti-cancer effect. this study describes the synthesis and characterisation of biocomposites of different molecular weight of peg (polyethylene glycol) as matrix with ganoderma lucidum as a filling material at different loading (% , % . , % wt). the composites have been prepared by solution intercalation method using ground and sieved ganoderma lucidum at micron scale. the characterization of composites was made by x-ray diffraction (xrd), scanning electron microscopy (sem) and fourier transform infrared attenuated total reflectance (ftir-atr) also in this study the hemocompatibility and antibactarial properties of composite investigated. when xrd and ftir-atr results discussed, all of the composites using the different loading amunt of ganoderma lucidum (% , % . and % wt) were shown a homogen distribution in the matrix (peg). and an interaction have occured between matrix and filling material. the sem photos have confirmed these results. peg and composites have been detected as hemocompatible. these results showed that they can be used as biomaterials. p- . . - evaluation of the genotoxic potential of some nanocomposites by comet assay b. yilmaz , s. dogan , s. celikler kasimogullari department of molecular biology and genetics, balikesir university, balikesir, turkey, department of biology, uludag university, bursa, turkey due to its similar nature to the bone, nanohydroxyapatite is a biocompatible particle and poly(methyl methacrylate) (pmma) is a polymer that has been used in dentistry and orthopedic applications for years. in this study, genotoxic potential of pmma/nanohydroxyapatite nanocomposite films composed of polymers having different molecular weights and nanohydroxyapatite fillers in different concentrations ( , . and %) were investigated by comet assay which is a kind of gel electrophoresis that can be used to measure dna damage in individual cells. if the dna is damaged we expect broken ends to migrate apart from the head. at the end of the assay performed after incubation with lymphocytes of healthy humans, we measured the dna damage index (ddi) and percentage of damaged cells (pdc). in addition, to prove the morphological properties of the nanocomposites scanning electron microscope was used and an interaction between the matrix and nanoparticles with a homogeneous dispersion was observed. protein adsorption on stimuli-responsive mixed pdmaema/peo polymer brushes a. bratek-skicki , , c. dupont-gillain universit e catholique de louvain, louvain-la-neuve, belgium, j. haber institute of catalysis and surface chemistry, polish academy of sciences, krakow, poland smart polymer brushes are made of macromolecules that are sensitive to stimuli from the external environment, including ph, ionic strength, temperature, etc. when stimuli-responsive polymer brushes are introduced onto material surfaces, their properties can be adjusted by tuning the environmental stimuli. these brushes can find promising applications across many areas of research, including surface science, nanotechnology, and biotechnology. in our work, the adsorption of human serum albumin (hsa, molecular weight of . kda, isoelectric point ip at ph . ) and lysozyme (lys, molecular weight of . kda, ip~ ) was studied on polymer brushes composed of poly(ethylene oxide) (peo) and poly ( -(dimethylamine) ethyl methacrylate) (pdmaema). peo is a protein-repellent polymer and pdmaema is a polyelectrolyte bearing a variable density of positive charges depending on ph. a gold substrate was modified by these thiolated polymers according to the 'grafting to' method. the obtained polymer brushes were characterized by xray photoelectron spectroscopy, static contact angle measurements and atomic force microscopy. polymer brush formation and protein adsorption were monitored by quartz crystal microbalance. surface characterization of the mixed brushes revealed the presence of both polymers at the surface. conformational changes of pdmaema/peo brushes were experimentally evidenced, and the results indicated that the brushes collapse at ph . (pdmaema is neutral in such conditions) and were swollen at ph . (pdmaema is positively charged). protein adsorption was performed at different ph values ( . - . ) and salt concentrations ( . - . m). it was shown that pdmaema has a high affinity to hsa at ph above its isoelectric point. however, the adsorption of positively charged lysozyme in a wide range of ph was not observed. these results indicate that pdmaema/peo brushes are promising candidates for selective adsorption from a mixture of proteins. clay-polymer nanocomposites (cpn) developed in recent years as a new type of inorganic-organic hybrid materials that were conceived for medical uses such as tissue engineering or drug delivery [ ] , [ ] . the understanding of the structure and physico-chemical properties of cpn is a first step in the investigation of biomaterials, but their potential in this respect is determined by their interaction with living tissue components. in this study, pure kaolinite was intercalated with dimethyl sulfoxide (dmso) and then intercalated kaolinite was modified pyridine, -amino pyridine and , -diamino pyridine to expand the interlayer basal spacing. modified kaolinite samples as filler and poly(vinyl chloride) (pvc) polymer as matrix were used in the nanocomposite synthesis. nanocomposites of pvc have been prepared by solvent blending method using thf as a solvent. the material characterizations were carried out by xrd, afm, ftir-atr, dta/tg and dsc. the xrd results reveal the formation of intercalation/exfoliation of modified kaolinite in the pvc matrix. ftir and afm results confirm the presence of nanomaterial in kaolinite/pvc nanocomposites. tga data show that the modified kaolinit/pvc nanocomposites have significant enhanced thermal stability. the glass transition temperature (tg) of pvc nanocomposites is higher than that of pure pvc. in addition, the antimicrobial activity of clay-polymer composites were also determined. introduction: polyhydroxyalkanoates (phas) are biocompatible and biodegradable materials obtained from microorganisms. they are produced in the cytoplasm of several bacteria as energy reserve. the physical properties of poly( -hydroxybutyrate) (phb), which is from the group of phas, make it a competitive source to petrochemical plastics. phb has potential in order to be used in a variety of application fields such as packaging industry, printing materials, agriculture and food industry. furthermore, phb meets expectations for tissue engineering applications, since it is biocompatible, biodegradable, non-toxic and has good mechanical properties. although its many advantages, blending approach could be needed in order to fulfill all expectations of a material. due to its flexibility, polycaprolactone (pcl) is a promising candidate to be blended with phb. the aim of this study is to construct a scaffold by using phb produced by extreme alkaliphilic b. marmarensis gmbe t (dsm ) and commercial pcl as components and investigate its properties. materials and methods: electrospinning method was used in order to construct scaffolds from blend polymer solution containing phb from b. marmarensis and commercial pcl. results: nanofiber structures were observed on scanning electron microscope (sem) images and fourier transform infrared resonance (ftir) analyses have shown characteristic peaks for both phb and pcl. discussion and conclusion: phb could be blended with other polymers in order to enrich its properties. in addition, nanofiber structure of electrospun phb-pcl blend makes it a rewarding material as scaffold for several tissue engineering applications. q fever is a zoonotic disease that is encountered widely around the world, the most common acute form of q fever shows the following symptoms; a sudden fever, shivering, lassitude, headache, anorexia. because this disease does not show specific symptoms its diagnosis is possible with laboratory tests. current diagnostic kits lack effectiveness; this is why the main goal of our studies is to come up with a new diagnostic kit that does not have disadvantages that current diagnostic kits show. with this goal, nine mile i strain (rsa ), s serologic virulent phase i, were obtained from slovak science academy, virology institute for rickettsia reference and research from who co-operation centre. these cells were purified and lipopolysaccharide (lps) isolation from coxiella burnetii was performed. the polymeric carrier, poly (n-vinyl- -pyrrolidone-co-acrylic acid) [p(vp-co-aa)] was synthesized and characterized. physical complexes of obtained lps and p(vp-co-aa) with varying ratios. ternary complexes of lps-cu + -p(vp-co-aa) were also synthesized with copper metal mediation. structure and interaction of lipopolysaccharide-p(vp-co-aa) complexes were investigated with zeta-sizer device using zeta potential analysis and ftir spectrophotometry according to the ratios of components, reaction environment conditions and chemical structure of the polymer. the best complex ratio according to analysis results will be used in the future studies for obtaining monoclonal antibodies which will be an important step for obtaining more effective and stable diagnostic kits that can be used for q fever. this in this study; new types of water soluble polymer-biomolecule conjugates were synthesized using covalent bonding techniques between polymers and co-polymers (varying monomers of polyacrylic acid and acrylic acid) with peptides. different compositions of polymers, varying ratios of biomolecule/polymer and different molecular weight of polymer has yielded new types of bioconjugates. conjugation mechanism, composition and structure were investigated with various physicochemical methods (uv, ftir, hplc, gpc, etc.). the peptide used in this study was the antigenic peptide epitope of sheep-goat pox disease (eakssiakhfslwksyadadiknsenk). whether this peptide series was bound to polymers or whether it was bound to polymer-protein carrier; peptide-specific immunogens that were capable of producing antibodies were synthesised. it is thought that using polymer-peptide bioconjugates that contain just peptide will yield a higher peptide-specific immunogenicity compared to traditional adjuvants. in vitro and in vivo investigations of bioconjugates effectivity is planned to be done in the future studies. p- . . - bioinert fluorinated ethylene-propylene copolymer modified for keratinocyte adhesion surface properties are crucial when adhesion of a cell to a polymeric material is required for a biomedical application. one of the methods for polymer surface tailoring is argon plasma treatment. this simple and reproducible method alters the surface properties such as roughness and wettability without affecting the bulk properties of the material. for the modification of the bioinert fluorinated ethylene-propylene (fep), related to teflonÒ, we employed argon plasma treatment with the powers of and w for - s. the human keratinocytes of the hacat cell line served as a model cell line for biocompatibility testing. we studied adhesion, proliferation and morphology of the cells on modified fep matrices as well as controls (pristine fep and standard polystyrene tissue culture dish) by means of fluorescence microscopy. further morphological details were acquired by scanning electron microscopy. furthermore, fluorescence microscopy with immunochemical labelling was used to determine the size and distribution of focal adhesions in cells grown on the modified matrices. the overall effect of the matrices on metabolic activity of cultured hacat cells was also evaluated using the wst- reagent. the ar plasma treatment of fep matrices improved significantly cell adhesion and proliferation and promoted spreading of the hacat cells compared to the pristine fep, on which cells were not able to spread properly. also, increased metabolic activity rates for cells cultured on modified matrices were found in comparison to the pristine fep. altogether, we found that ar plasma treatment improved the surface properties of fep to such extent, that it allows cultivation of adherent cells on its surface. we therefore propose that ar plasma treatment is a useful method for fep surface modification. p- . . - graphene oxide enriched biomaterials display potential for tissue engineering applications tissue engineering (te) requires more efficient systems that favor local tissue regeneration with minimum cytotoxicity. materials based on natural compounds ensure biocompatibility and have better effects for regeneration. graphene oxide (go) has been shown to enhance cell adhesion and to improve the rate of cell differentiation. in this context, the aim of this study was to evaluate if the addition of different concentrations ( . - wt.%) of graphene oxide (go) improves the properties of cellulose acetate (ca) materials for biocompatibility and cell differentiation, in the prospective of using these films for te applications. go-containing ca films were tested for cytocompatibility by quantitative and fluorescence microscopy assays, and compared to the ca control. cell cytoskeleton architecture in contact with biomaterials was revealed by confocal microscopy. furthermore, bioconstructs were exposed to in vitro osteogenic and adipogenic induction and monitored for days. histological stainings were performed to validate differentiation. osteogenic and adipogenic markers gene expressions were assessed via qpcr. ca/go wt.% revealed best biocompatibility among all tested scaffolds. adhesion proved to be dependent on the percentage of go in material's composition. cells cultivated on ca/ go wt.% expressed adipogenic and osteogenic markers earlier than cells cultivated on materials with lower go content. differentiation markers displayed an increasing profile of gene expression from to days post induction, with higher levels registered for materials with high go content as compared to films with low go content and to the control. go added to ca materials positively influenced cell survival, proliferation and cell differentiation. ca/go films represent potential candidates for te applications. the design of appropriate scaffolds remains one of the most important challenges for te. current idea is that the cell-scaffold interaction could drive cell differentiation and be linked to gene expression and protein organization. therefore, their quality is essential and should favour cell attachment, growth, migration, in situ vascularization and release of biochemical and physical factors able to address the cell fate. moreover, for an ideal scaffolding material an adequate and interconnected porosity is relevant for facilitating cell spreading and colonization of the inner layers. a combination of optimal mechanical and biochemical properties were here utilized to design a d composite hydrogel scaffold ( d-chs) in order to favour cell-scaffold interactions and promote a functional differentiation of human lin À sca + cardiac progenitor cells (hcpc). the biocompatible peg-diacrylate (pegda) was used to prepare hybrid protein-pegda hydrogels with embedded albumin-microspheres (ms) as protein component. ms were able to modulate the mechanical and biological behavior of the scaffold acting as air-reservoir, porogen agents and potential carriers of biomolecules. an increase of the hcpc viability in the ms-concentration dependent manner was observed. moreover, the microarchitecture of the d scaffold also plays a key role in the stability and functionality of cellularcomposite systems. therefore, pegda-honeycomb structures were fabricated using microstereolithography process and the hcpc viability and adhesion to the microstructures were assessed. d-chss were synthesized embedding honeycombstructures into ms-pegda hydrogel and the effects on cell proliferation, cell-cell interactions and cellular alignment were investigated. these results could be of relevant interest for expanding the knowledge on cell-scaffold interaction processes and to promote the development and the application of d-chs for tissue regeneration using the emerging bioprinting technologies p- . . - gene expressions of mesenchymal stem cells after osteogenic induction on ceraform bone substitute a. kilic s€ uloglu, e. karacaoglu, h. akel, c ß . karaaslan hacettepe university, ankara, turkey ceraformÒ, is a synthetic calcium phosphate ceramic with the chemical composition of hydroxyapatite % and tricalcium phosphate %, with - % pore volume and - lm pore diameter. in this study adipose tissue derived mesenchymal stem cells were differentiated into osteoblast cells and loaded on cer-aformÒ. in order to improve cell adherence, ceraformÒ was covered with fibronectin. the cells were cultivated for -day period by osteogenic induction medium. days , , , and were selected as specific intervals for incubations. total rna was isolated and cdna was synthesized. differences in the expression of runt-related transcription factor (runx ), bone morphogenetic protein- (bmp- ), and osteocalcin (ocn) were determined by qpcr. the peptidylprolyl isomerase a (ppia) gene was used as an internal control. according to the qpcr results, ocn gene expression was observed on the day th, continued to increase in day . bmp- gene expression was increased in , , day compared to day. on the other hand, runx gene expression was increased only on days and . these findings pointed out that the osteogenic induction was successfully activated on fn coated bone material. therefore, these results can be used in bone injury treatment and related disorders. p- . . - on the in vitro cytotoxicity of graphene oxide nanomaterials v. grumezescu , i. negut , f. sima , e. axente , l. e. sima national institute for lasers, plasma and radiation physics, magurele, romania, institute of biochemistry, romanian academy, bucharest, romania during the last decade, graphene and its derivates have proven unique physicochemical properties, several applications being continuously developed. among them, electronic, catalytic, mechanical, optical, and magnetic properties have attracted huge interests. however, the merging of graphene and graphene oxide (go) with biotechnology is still in its infancy, many challenges remaining unexplored. potential applications are related to biosensors, drug delivery or gene therapy and cells imaging. in order to use gos as drug release matrices for cancer cells targeting, it is necessary to ensure that these molecules do not affect normal cells within tissues. it was shown that the cytotoxicity of graphene nanomaterials is highly dependent on surface functionalization. studies suggest that pristine and reduced go with fewer surface functional groups tend to be more toxic than go. in striking contrast, it has been reported that functionalized graphenes, can significantly reduce the cytotoxicity even at relatively high concentrations. in this study, we report on the comparison between go and protein functionalized go when submitted to in vitro cytotoxicity tests. bovine serum albumin (bsa) was used for the noncovalent go surface conjugation. three case-studies were investigated: aqueous nano-colloids consisting of serial dilutions of both go and go-bsa conjugates, dropcasted thin films and laser-assembled thin films on glass substrates. safe concentration windows were identified by live/dead staining and mts assays for different human melanoma cell lines, while melanocytes and human dermal fibroblasts were used as normality controls. the predominant melanoma subtype is represented by cells bearing braf (v e) activating mutation. with a view to target this specific melanoma subpopulation, we embedded braf inhibitors into go laser-deposited scaffolds and tested their anti-tumoral effect. our results evidence the high potential of these nanomaterials for biomedical applications. osteoporosis is a skeletal disorder associated with low bone mass and increase in bone fragility due to increased osteoclastic activity. binding of rank ligand to its receptor on osteoclast precursor cells results in the osteoclast differentiation. sirna is a dsrna, used to inhibit the translation of the target gene. the aim of the study is to develop an injectable sirna-delivery system targeted to the bone for osteoporosis treatment. pei (polyethyleneimine) and rank complex was loaded into poly(lactic acid-co-glycolic acid) (plga) nanocapsules which are bound to hydroxyapatite (hap)-specific elastin-like protein (elp) for targeting to bone tissue. plga nanocapsules were prepared by w/o/w double emulsion technique. affinity of elp to hap was determined by ftir. elp was coated on the nanocapsules by using the transition temperature of elp. elp on plga nanocapsules were crosslinked by genipin and binding of elp on plga nanocapsules were studied by xps and tem. the optimum ratio of n (pei) to p (sirna) in the complexes to be loaded into plga nanocapsules were studied by etbr staining and zeta potential measurements with varying n/p ratios and finally pei-dnaoligo encapsulation efficiency of the capsules was determined by picogreen reagent. xps results of elp treated plga (elp-plga) nanoparticles indicated the presence of nitrogen atom ( . %) in the sample which appeared as a fuzzy halo in tem micrographs. n/p ratios up to show negatively charged particles. when n/p was , the zeta potential of complex was neutralized which also resulted in larger particles compared to the others. zeta potential moved to positive values when n/p was higher than . the migration of polyplexes with different n/p ratios ( - ) was analyzed by gel electrophoresis. dnaoligo complexes show the same patterns of complexation wih that of sirna and thus were used in the encapsulation efficiency studies instead of sirna. the encapsulation efficiency of pei-dnaoligo in plga nanocapsules was %. tuesday september : - : aging p- . . - novel benzenesulfonamides exhibit low toxicity on zebrafish embryonic development and selectively inhibit carbonic anhydrase ix with nanomolar affinity in xenopus oocytes introduction: the toxic effects of two recently discovered inhibitors (vd - and vd - - ) that selectively and with extraordinary strong, picomolar affinity bind to human carbonic anhydrase (ca) ix, an anticancer target, were investigated on zebrafish embryonic development and in xenopus laevis oocytes. zebrafish has emerged as a promising animal model to evaluate the toxicity of the drug candidates. xenopus oocytes do not natively possess any ca activity and thus became a convenient in vivo model system to study the ph effects and the selectivity of synthetic ca inhibitors. materials and methods: morphological changes in zebrafish treated with the compounds were studied by light-field microscopy and histological analysis. ca activity in xenopus oocytes was monitored by measuring ph in the cytosol and at the outer membrane surface and confirmed by mass spectrometry of lysed oocytes. the toxicity studies showed lc values to be lm for vd - , lm for vd - - and lm for ethoxzolamide (eza), a non-selective ca inhibitor commonly used in clinic. the zebrafish exposed to lc doses of vd - and vd - - showed fewer phenotypic abnormalities and less morphological changes compared to the zebrafish treated with the corresponding dose of eza. vd - - exhibited - nm ic for both intracellularly and extracellularly expressed ca ix in xenopus oocytes while exhibiting strong selectivity over ca ii, ca iv and ca xii. discussion: interestingly, the compounds exhibited -fold lower toxicity, induced fewer side effects in zebrafish than eza and the amount of vd - - needed to cause complete inhibition of ca ix enzymatic activity in xenopus oocytes was -fold lower than eza. conclusions: vd compounds did not lead to deleterious effects on the zebrafish embryonic development and reached the ic of nm for ca ix in xenopus oocytes. the compounds could be further developed as anticancer drugs. cacybp/sip is present in various cells and tissues, both normal and pathological. in normal tissues, e.g. stomach or colon, cacybp/sip is weakly or barely detected whereas in gastric or colon cancer this protein is expressed at a high level. there are also data indicating that the level of cacybp/sip expression correlates with tumor metastatic potency and multidrug resistance. taking into consideration data that suggest association of cacybp/sip with many vital cellular processes, in this work we decided to investigate the possible mechanism involved in regulation of cacybp/sip gene expression, mainly by transcription factors and, on the other hand, the influence of cacybp/sip on the expression of other genes. we have shown that nfat (nuclear factor of activated t cells) influences the cacybp/sip gene expression and that overexpression of cacybp/sip has an effect on the level of ap- and on the activity of nfat and ap- transcription factors. by analyzing the cacybp/sip gene promoter sequence we also found potential binding sites for transcription factors from the stat family, which are involved in interferon signaling. microarray data indicate that indeed overexpression of cacybp/sip affects levels of the stat proteins as well as of some interferons and interleukins. based on functional analysis we have found many genes the products of which are involved in immune response. to analyze in more detail the influence of an altered level of cacybp/sip on interferon signaling pathways as well as on factors involved in expression of interleukins, including nfkappab, we plan to apply methods such as luciferase assay, real-time pcr or immunocytochemistry. one of the pathological hallmarks of alzheimer's disease (ad) is the neuritic plaques occurred as a result of the extracellular accumulation of aß peptides formed from amyloid precursor protein (app) via the ß-amyloidogenic pathway. aß is more prone to aggregation to form plaques and more toxic to neurons than aß . in addition to change in app metabolism, the decline in levels of neurotransmitter acetylcholine and cholinergic dysfunction are also observed in ad. thus, current strategies for ad treatment focus on compounds with inhibitory effect on cholinesterases as well as preventive effect on aß aggregation. in our earlier studies, toluidine blue o (tbo), a phenothiazine dye, was shown to be a highly effective inhibitor of cholinesterases with k i values in nm range. we also found that intracellular app and aß levels are reduced in human neuroblastoma cells after treatment with tbo. additionally, an earlier study revealed that tbo has a selective inhibitory effect on tau aggregation, the other pathological characteristic of ad. the aim of this study was to investigate whether tbo may effectively lower the level of extracellular aß / in an ad-like cellular model. chinese hamster ovary cells that express human wild type app and presenilin , namely ps , were treated with a dose range of tbo ( - lm) or vehicle control for h. after treatment, aß / levels in cell culture media were assayed by separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. besides, biocompatibility of tbo was evaluated in the ps cells using cell viability assay for flow cytometry. strikingly, all dose ranges of tbo inhibited both aß and aß secreted into the cell culture media. significant reduction for both aß species was evident at lm (p < . ), lm (p < . ), and lm (p < . ) of tbo vs. vehicle control. in conclusion, these results support the idea that tbo may be used as a therapeutic in ad. monitoring the changes of key molecules participating in the osmo-regulatory response of nucleus pulposus intervertebral disc cells during stress-induced senescence e. mavrogonatou, d. kletsas ncsr 'demokritos', athens, greece introduction: intervertebral disc cells are faced with a harsh extracellular milieu characterized by hyperosmotic conditions, nutrient and oxygen deficiency because of the absence of vascularization and oxidative stress due to the accumulation of their metabolism's by-products. we have previously shown that high osmolality is anti-proliferative for disc cells through the activation of the g and g cell cycle checkpoints by p and p mapk, respectively. in addition, we have shown the participation of nine solute transporters, with the a subunit of na + /k + -atpase being central in this response. here we assessed the changes in the expression of these key osmo-regulatory molecules during in vitro stress-induced senescence. materials and methods: changes in cell cycle progression were assessed using flow cytometry; overall transcriptional alterations were assessed by whole-genome arrays; differences in expression at the mrna and protein level were revealed by quantitative rt-pcr and western blotting, respectively; knocking-down of selected proteins was performed by sirna. results: high osmolality led to the differential expression of > genes, including nine genes encoding transporters. p mapk and p were demonstrated to differently participate in the regulation of the aforementioned transporters, while knocking-down of three selected transporters had a distinct outcome on the overall cellular response towards hyperosmotic stress. these molecules were found to show differences in their expression in senescent cells. discussion: given that the presence of senescent cells has been demonstrated in the intervertebral disc in vivo and could most probably attributed to the prevailing stressful conditions, here we showed differences in the expression profile of known key molecules for osmo-adaptation during senescence. conclusion: understanding disc cells' physiology is of outmost importance when designing cell-based therapies for disc degenerative disorders. p- . . - smad specific e ubiquitin protein ligase (smurf ) and its potential effects on inhibitory transmission in aging adams , , , interdisciplinary program in neuroscience, bilkent university, ankara, turkey, national nanotechnology research center (unam), bilkent university, ankara, turkey, department of molecular biology and genetics, bilkent university, ankara, turkey, molecular biology and genetics zebrafish facility, bilkent university, ankara, turkey, department of psychology, bilkent university, ankara, turkey smad specific e ubiquitin protein ligase (smurf ) is part of the tgf-b signaling pathway associated with cellular proliferation, differentiation, genomic stability and senescence. moreover, smurf , via its downstream partners, may regulate inhibitory synaptic transmission. our research group previously found that the smurf transcript is significantly higher in old zebrafish brains. thus, smurf may alter inhibitory synaptic transmission in aged animals. the focus of this study was to examine age-related changes in smurf protein levels and related key inhibitory synaptic proteins; gephyrin (gep), a scaffolding protein for gaba receptors, and gaba a , an ionotropic gaba receptor subtype. additionally, the levels of those proteins were studied in a mutant zebrafish line, which lacks acetylcholinesterase (ache) and is suggested to be a delayed aging model. whole brain tissues were isolated from young, middle-aged and old male and female zebrafish brains (ab/wildtype strain), as well as from old male and female ache mutant zebrafish (ache sb /+ ). animals were maintained and raised in standard conditions. the extracted brain tissue was homogenized in ripa buffer and subjected to western blot analysis to determine differences in the relative protein expression levels. our preliminary data indicated that smurf and gep levels remain stable in the aging brain (p = . , p = . ), and in the ache mutants gep levels are increased compared to the wildtype controls (p = . ). further analysis of the relationships between smurf and gaba a levels and brain aging is ongoing. we predicted that alterations in smurf levels would parallel changes in key synaptic inhibitory proteins during the aging process, which was the case for the gep levels. while smurf may regulate inhibitory synaptic transmission, the exact roles of those synaptic proteins in the context of normal and delayed brain aging are not known well-understood and the subject of continuing study. in recent years, express the hypothesis that aged individuals are vulnerable to infectious and other inflammatory agents and they become more prone to develop majority of severe age pathologies, including cardiovascular and oncology diseases, neurodegenerative diseases, type diabetes mellitus and inflammatory diseases, etc. one of the central components of immune response is the family of toll like receptors (tlr). there are several opinions that single nucleotide polymorphisms (snp) leading to a loss of function of the respective tlrs can be associated with age and increase the risk of age related diseases, especially cardiovascular diseases (cvd). however, many available studies focusing on tlr snps and cvd are with conflicting results. the aim of this study was to assess the potential interaction between genetic variants of tlr and tlr and ischemic heart disease (ihd) in kazakhstan population over years old. we evaluated patients with ihd and healthy subjects aged years and over (ethnical kazakhs and russians living in republic of kazakhstan). polymorphic loci of the genes tlr rs and tlr rs were genotyped by pcr with subsequent restriction analysis. our results indicated that the genotype and allele frequencies of tlr (arg gln) and tlr (asp gly) were not significantly different between the groups (p ≥ . ). statistical analysis didn't elicit any association between studied gene polymorphisms and predisposition to ihd in individuals over years old (p ≥ . ). for these polymorphisms, age, fasting blood sugar and serum lipid levels were not also significantly different among different genotypes in the ihd and control groups. in conclusion, the data shows that there is no interaction between tlr and tlr and ischemic heart disease (ihd) in kazakhstan population over years old. we plan to include other types of polymorphisms in tlr and tlr genes and increase the volume of patient cohort in our future studies. p- . . - evaluation of prognosis with total oxidant/ antioxidant status and some oxidative stress parameters in patients with acute ischemic stroke stroke is the third most common cause of death after coronary heart disease and cancer. strokes are classified into two groups according to their pathology: ischemic stroke and hemorrhagic stroke. ischemic strokes make up % and hemorrhagic strokes % of all strokes. during ischemic stroke, oxidative stress has been shown to play a major role in the occurrence and progression, formed oxidants also affect cell membranes and genetic material such asdna, rna, and various enzymatic events, and they lead to cell damage. some studies have shown oxidant-antioxidant status but have not shown the relationship with prognosis. this study has investigated the relationship between prognosis and total oxidant/antioxidant status and biochemical parameters in patients with acute ischemic stroke patients, with acute ischemic stroke and healthy controls we reenrolled in the study. blood samples were taken within st and th days, and after rd months in the patient group for analysing serum total oxidant status (tos), antioxidant status (tas), catalase, arylesterase, and thiol. prognosis was evaluated with national institutes of health stroke scale (nihss) and-modifiedrankinscale (mrs) scores. there was no significantly difference between groups by means of serum tas, tos and catalase levels. but arylesterase (p: . ) and thiol (p: . ) levels were significantly higher in first h blood samplingthancontrolgroup. statistically significant negative correlation was observed between the rd month values of tos and nihss score (r = . , p = . ). but there was no correlation between mrs scores and serum tas, tos, catalase, thiol and arylesterase. similarly, our findings suggested some serum oxidant levels were increased in acute ischemic stroke patients and total oxidant status might be used in evaluation of prognosis but larger studies are needed. p- . . - amylin and preptin regulate glucose homeostasis in infertile women with polycystic ovary syndrome and poor responders undergoing ivf/icsi disrupted glucose homeostasis leads not only metabolic disturbance such as polycystic ovary syndrome (pcos), but also influences oocyte growing. this study was designed to evaluate follicular fluid (ff) and serum levels of glucoregulatory hormones, amylin and preptin, in infertile women with pcos and poor responders undergoing ivf/icsi. human follicular and serum were obtained from infertile women with pcos and poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone antagonist protocol for ivf/icsi treatment. ff and serum amylin and preptin levels were measured by elisa. it was found that ff and serum amylin and preptin were lower in infertile women with pcos when compared with poor responder participants. ff amylin and preptin concentrations were lower than that of the serum amylin and preptin concentrations. decreased follicular fluid amylin and preptin levels suggest that amylin and preptin may have a physiological role in follicular maturation via controlling local glucose homeostasis. despite high serum levels of amylin and preptin in pcos their low concentration within the follicle may be main culprit of defective folliculogenesis seen in pcos subjects. similar to insulin resistance in pcos subjects existence of amylin and preptin resistance support the critical role of both peptides in follicular maturation in pcos. keywords: follicular fluid; amylin; preptin; polycystic ovary syndrome; infertility. the transcription initiation on p promoter of xp bacteriophage in presence of p protein, a modulator of rna-polymerase activity a. shadrin g.k. skryabin institute of biochemistry and physiology of microorganisms, ras pushchino, russia many bacteriophages are able to manage the transcription system of their bacterial host for their own needs. for example, bacteriophage xp , in the early stages of infection of xanthomonas oryzae inhibits transcriptional activity of bacterial rna-polymerase on majority of promoters via p protein, except of bacteriophage p promoter responsible for expression of the bacteriophage 'middle' class genes. the focus of this work is to study the mechanism of action of p protein in the transcription initiation and identification of the role of the individual elements of p promoter of xp bacteriophage, enabling x. oryzae rna-polymerase escapes inhibition by p protein. we have designed a set of promoter probes representing the combination of sequences of p -resistant p promoter and p sensitive t n promoter. using fret-based assay it was shown that the truncated probes corresponding to promoter dna downstream À position, relative to the transcription initiation start site, did not lead to dissociation of the sigma-factor. longer probes, containing À promoter element, induce dissociation sigma-factor. the in vitro transcription experiments show that the deletion of region , a sigma-factor domain responsible for interaction with À promoter element during the transcription initiation, is not critical for inhibition of rna-polymerase by p protein. promoter probe with up-element of p promoter had affinity to x. oryzae rna-polymerase a several times higher than a probe containing the consensus up-element for e. coli rna-polymerase . summing up the results, it seems like the transcription initiation on p promoter of bacteriophage xp can escape inhibition by p protein through a high affinity interaction between the up-element and c-terminal domains of the alpha subunit of rna-polymerase x. oryzae. p- . . - distribution of soluble form of glial fibrillar acidic protein in the different areas of gerbils brain during development and aging y. kovalchuk, g. ushakova oles honchar dniepropetrovsk national university, dniepropetrovsk, ukraine astrocytes are the most abundant cell type within the cns and play an important role in cns homeostasis and function. glial fibrillary acidic protein (gfap) forms the main astrocytic intermediate filament (if). the overall level gfap in different parts of the brain uneven and depends on the number of astrocyte cells. gfap is very sensitive to any kind of neurodegenerative diseases and aging. during aging, a glial reaction is observed in the human brain, as well as in rat and mouse brains. the aim of our study was to investigate the quantitative astrocytes-specific protein gfap in different areas of the gerbils brain at the first stages of postnatal development and aging. for the study gerbils brains were used and divided into groups (n = ): : newborn animals ( day), - : , and days respectively, : animals aged years. the animals were decapitated under mild anesthesia (thiopental), with isolated brain three divisions: the cerebellum, thalamus and hippocampus, which are then used to produce cytosolic protein fractions. the level of gfap in the obtained fractions were determined according to the method of competitive elisa. newborn gerbils found no significant content of soluble form of glial fibrillar acidic protein in all investigated parts of the brain, and a sharp increase of amount within days (in cerebellumamounted to . ae . lg/ mg tissue; to - days increased to . ae . lg/ mg tissue, and began to grow again in older individuals aged years). unlike the cerebellum, the level of sgfap in hippocampus and thalamus reached the maximum at days p.d. ( . - . lg/ mg tissue), and unchanged for days. these results revealed that the most intensive development of astrocytes in the cerebellum to p.d. of gerbils, and in the thalamus and hippocampus are formed within the first month of life. the plastid-nucleus located protein whiry acts as an upstream regulator of leaf senescence binding to the promoter of senescence associated genes (sags) like senescence marker gene hvs . in order to investigate the impact of whirly on drought stress-induced senescence, transgenic barley plants with a knockdown of whirly (hvwhy kd) were grown under untreated and drought stress conditions. the leaf senescence evolution was monitored by physiological parameters and gene expression studies of senescence and drought stress related genes. to reveal the epigenetic indexing at hvs at onset of drought-induced senescence in wild type (wt) and hvwhy kd lines, stress-responsive loading with histone modifications at gene regions of hvs ( regions in the promoter, one region around translation start site and regions located in the gene body) was analysed by chip and quantified by rtq-pcr. in barley, drought treatment caused acceleration of leaf senescence in wildtype (wt) plants, whereas why kd lines showed a staygreen phenotype. expression of senescence-associated and drought stress responsive genes expression was delayed in hvwhy kd indicating that whirly protein acts as an upstream regulator of drought stress-induced senescence. the chip results showed that drought treatment is causing in wt a significant increase in the levels of h k ac all over the analyzed gene regions, correlating with a massive induction of hvs expression, while drought stress caused no substantial increase of h k ac in why kd plants. the results suggest that drought induced expression of hvs is under epigenetic control, and furthermore that why is involved in this epigenetic control level. oncolytic viral therapy is based on the capabilities of selective lysis of tumor cells and is a prospective trend in cancer disease treatment. in vitro experiments showed that plant rhabdoviruses does not have any direct cytotoxic effect upon sarcoma cells, causes induction of apoptosis in these cells and does not pose any threat to somatic cells of warm-blooded animals, which makes it possible to use this virus for therapy of malignant neoplasms. buckwheat burn virus (bbv), the prototypic member of the family rhabdoviridae, contains surface glycoprotein and which is lectin-active. its carbohydrate branch can aid adhesion of lymphocytes to tumor cells. the present study has addressed the effect of bbv on cancer cell viability. all studies were carried out after week of inoculated with erlich cancernome ( cells/animal, i. p.) in months male balb/c mice treated at once with or without plant extract with bbv ( mg/kg, i. p.). by fluorescent microscopy and using two due staining by acredine orange and propidium iodide it was found that in the rd day of administration of bbv lead to increasing of necrotic and apoptotic cells on % and % respectively versus to untreated group. at the same time the viability of investigated cells was impaired too and according to flow cytometry analysis using propidium iodide the amount of dead cells was elevated by fivefold ( . % versus . % in untreated group). also as was shown in previously reports bbv decreased activity of macrophages in the early stages after injection and it may have a positive effect when using this drug in tumor therapy. when using this drug appears to slow down the possibility of a sharp activation of macrophages, and as a consequence of the development of cytotoxic effect will be prolonged. key words: rhabdoviruses, buckwheat burn virus, cancer, cell viability. plants are considered as one of most promising sources for new antimicrobials, based on the evidence of their use in folk medicine to treat various infectious diseases since ancient times. despite relatively small area size, armenia has large diversity of flora with many endemic species. the main goal of this study was the screening of various parts of herbs (widely being used in armenian folk medicine) for their antimicrobial activities in order to select most prospective plants for further comprehensive studies. plant crude extracts were obtained with maceration technique using five solvents: water, methanol, chloroform, acetone and hexane. agar well diffusion assay was used to evaluate antimicrobial properties of plant crude extracts at lg/ml concentration against escherichia coli vkpm-m , pseudomonas aeruginosa grp , bacillus subtilis wt-a , salmonella typhimurium mdc and staphylococcus aureus mdc , candida albicans wt- and candida guilliermondii hp- . statistical analysis was done using graphpad prism . . crude extracts of all tested plant materials expressed antimicrobial activity against at least one test strain. most of the tested extracts inhibited growth of both gram-negative and gram-positive bacteria. in contrast, only some plant materials exhibited inhibitory activity against yeast strains. according to obtained data sanguisorba officinalis, rumex confertus, hypericum alpestre, lilium armenum and agrimonia eupatoria possessed the highest and broadest antimicrobial activity. moreover, the results showed that acetone was the most effective solvent for solubilizing antimicrobial compounds from plant materials followed by methanol, chloroform, hexane and water. the results demonstrated high antimicrobial activity of medicinal plants used in armenian traditional medicine. five plant species were selected for further comprehensive studies. besides, acetone was proposed as efficient solvent in antimicrobial screening protocols. p- . . - effects of aluminum stress on photosystem-i apoprotein a gene (psab) transcription level in lichen xanthoria parietina (l.) th. fr. unal € ozakc ßa ege university, izmir, turkey in this study the effects of shortrerm aluminium (al) toxicity on the lichen xanthoria parietina (l.) th.fr. were investigated at physiological and transcriptional level. lichen thalli were treated with alcl in different doses ( . , . , and mm). lipid peroxidation and chlorophyll integrity were determined by spectrophotometer. expression level of psab gene was also investigated. chlorophyll a content was significantly (p ˂ . ) decreased after hours treatment with mm and mm of al, while chlorophyll b content was increased significantly due to treatment with increased concentration of aluminum. also treatment with . and . mm al for hours increased the gene expression level of psab by . % and . % respectively. our results indicated that aluminum treatment has decreased the chlorophyll biosynthesis and increased the lipid peroxidation depending on time and concentration. this study also demonstrates that the psi can be readily photo-inhibited by aluminum stress. in conclusion, mm al exposure for hours could damage the electron transport in photosystem i. p- . . - nigella sativa reduces paracetamol-induced nephrotoxicity and oxidative stress in rats: biochemical evaluation background: nigella sativa l. (ranunculaceae) (ns) is traditionally used to treat many conditions such as inflammation. this study evaluates the effects of ns seeds ethanol extract in paracetamol-induced acute nephrotoxicity in rats. material method: forty-eight female wistar albino rats were divided into eight groups: i = sham; ii = sham + mg/kg ns; iii = sham + mg/kg (n-acetyl cysteine) nac; iv = g/ kg paracetamol; v = g/kg paracetamol + mg/kg nac; vi, vii and viii = g/kg paracetamol + , and mg/kg ns, respectively. paracetamol administration (oral) was carried out h after ns and nac administrations (oral), and all animals were sacrificed h later. result: urea and creatinine levels were determined in serum, while glutathione, malondialdehyde levels and superoxide dismutase activity were determined in the kidney tissues. there were significant increases in the serum levels of urea and creatinine in the paracetamol-administered group. serum levels of urea and creatinine were decreased in all groups administered ns with paracetamol. ns administration dramatically restored sod, gsh, and mda levels in the kidneys. conclusion: the results suggest ns has a significant nephroprotective activity on paracetamol-induced nephrotoxicity. it may be suggested that the antiinflammatory and antioxidant effects of ns ethanolic extract originated from different compounds of its black seeds. p- . . - the study of problems of preservation of the birches e. shadenova , , e. zhumabekov , m. sembekov , m. burchaeva institute of general genetic and cytology, almaty, laboratory of genetics and reproduction of forest culture, institute of general genetics and cytology, almaty, kazakhstan nature of deciduous trees have a whole range of various medicinal properties. instead of synthetic hormone substitutes, you can use medicinal infusions and decoctions of natural phytohormones are widely used in both folk and professional medicine. one of these plants is birch, its young leaves and buds. however, they also must be used with caution because overdose of these compounds is very dangerous, not only can you not get the desired effect, but also face the opposite of his action. in our research to mass replication of plants (different types of birches (betula ajanensis, yarmolenko, jacguemontii, maximowiczii, ulmifolia, middendorffii, kelleriana, tianschanica)) we use nutritional medium excluding the application of phyto promoters in order to prevent mutation. the object of research serve as the old, the sick, being on the verge of extinction, mature trees as explant meristema. since from the moment of calling experience and most cultivation occurs at nutritional medium without hormones. as a result of molecular analysis we get without virus, genetically identical plants. molecular certification of different types of birches of interest, both in terms of organizing, and in terms of selection and genetic improvement of valuable forms, identification of lines selected from natural populations and clones obtained in vitro. relationship between clones and installed parent form by comparing profiles amplific pcr products using issr-marking. according to the results of carried out works really recovered clones obtained from one source tree, indicating the potential for certification of clones studied forms of birches pcr. a study performed in the framework of the state grant project "conservation of breeding valuable species of birches". p- . . - fractionated triterpenoid glycosides from sea cucumber inhibit invasion and metastasis in human cancer cells sea cucumbers are slow-motioned invertebrates. holothuria polii delle chiaje, is widely distributed sea cucumber in _ izmir coastline (turkey). it secretes saponins i.e. triterpenoid glycosides (ttg) as secondary metabolites. the aim of this study is to evaluate anti-invasive and anti-migrative effects of fractionated ttgs obtained from h. polii on ht- , t and upci-scc- cancer cell lines. the semi-purified ttgs was extracted from h. polii collected from coast of _ izmir-dikili. the four different fractions (fraction a-d) were collected by using hplc (high-performance liquid chromatography) and characterizated with maldi-ms/ ms. the fractions obtained from h. polii extract include holothurin a ( . m/z) and -dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer ( . m/z). anti-invasive and anti-migrative effects of the fractions on the cancer cell lines were detected with xcelligence rtca dp system. the results showed that fraction a-d inhibited migration and invasion of human cancer cell lines at th and th hours compared to control group. this study shows that holothurin a, dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer could be evaluated as promising anti-cancer agents for human cancers. acknowledgement: the authors acknowledged the scientific and technological research council of turkey (t € ub _ itak) for financial support ( z ). p- . . - alternative splicing regulation of sr proteins in response to environmental stress in chinese cabbage serine/arginine-rich protein (sr protein) family, which acts as rna-binding protein, plays a major role in post-transcriptional regulation of pre-mrna, such as alternative splicing (as). these proteins cause pleiotropic effect by regulating as of pre-mrna in a tissue and developmental stage-specific and stress-responsive manner in arabidopsis. here, we identified genes encoding sr proteins in chinese cabbage (brassica rapa chiifu- ) from brassica database and analyzed their phylogenetic relationship. b. rapa has types of sr protein that are classified into common (sr, rsz and sc) and plant specific (scl, rs z, rs and sr-like) subfamily similar with arabidopsis. interestingly, the as pattern of most sr genes changed at the late stage ( and days after germination). to verify the correlation between sr genes and environmental stress, we screened the as pattern of sr genes to various abiotic stress using rt-pcr and a microarray analysis. in particular, the expression level and the as pattern of bra and bra were affected significantly by heat stress. these results suggest that the as regulation by sr protein correlates with adaptation mechanism to the environmental stress in chinese cabbage. p- . . - characterization of recombinant prolyl oligopeptidase from myxococcus xanthus and potential use in gluten hydrolysis e. k. kocaazorbaz, f. zihnioglu faculty of science, biochemistry department, ege university, izmir, turkey a recombinant prolyl oligopeptidase from myxococcus xanthus was purified with a specific activity of u mg(- ) by using nickel-metal-chelate affinity chromatography and gel permeation chromatography. the recombinant enzyme had a monomeric molecular weight of kda. its isoelectric point, determined by two dimension polyacryl-amide gel electrophoresis, was close to . . the optimum ph and temperature was estimated as . and °c, respectively. the purified enzyme was stable from ph . - . and able to thermal stability up to °c. the k(m) and v(max) values were . mm and . micromol/ min per mg. the enzyme exhibited hydrolytic activity for suc-gly-ala-pna, suc-gly-pro-pna, z-gly-pro-pna, igf- , substance p, whereas no activity for h-gly-pro-pna, h-val-ala-pna, h-arg-pro-pna, h-ala-pro-pna, glu-ala-pna, pro-pna, leu-pna. its proteolytic activity was inhibited by activesite inhibitors of serine protease, z-pro-prolinal pmsf, and metal ions, cd + and hg +. the potential use of the enzyme was tested by the hydrolysis of the wheat gluten. the resulting gluten hydrolysate were characterized by means of their antioxidant, antibacterial, trypsin inhibition and prolyl oligopeptidase inhibition activities. keywords: serine protease, prolyl oligopeptidase, bioactive peptides, , , -trinitrobenzene sulfonic acid. proteomic analysis is probably the best approach to analyze seed germination. however, it is difficult to analyze complex samples and there are many obstacles that must be faced in order to achieve a reasonable proteome coverage. for example, the barley (hordeum vulgare) genome was fully sequenced in , but the uniprot database contains less than reviewed sequences, which is approximately -fold less than for arabidopsis thaliana. here, to improve the barley proteome coverage, we employed several fractionation methods including polyethylene glycol precipitation, strong cation exchange chromatography, off-gel separation, sds-page and acetonitrile elution gradient. proteomic analyses were performed using an lc-ms-based analyses and an uhr q-tof mass spectrometer. the candidate peptides were targeted via selected reaction monitoring (srm) and triplestage quadrupole (tsq) mass spectrometer. in total, proteins were identified, which represents a three-to four-fold increase compared with the standard shotgun analysis of the same sample. out of these, were only accessible by one of the techniques and, besides, the detection limits were not similar. we hypothesized that an srm-based targeted analysis will allow detection and quantitation of most of these proteins, even without the application of proteome fractionation. we can conclude that all peptides from the library with ms/ms spectra of the total intensity above , are easily detectable in the total protein extracts. p- . . - transcriptome sequencing based identification of alternative oxidase genes in white waterlily, nymphaea alba alternative oxidases (aoxs) are the terminal oxidases in the respiratory electron transport chain of plants. they reduce molecular oxygen to water with low proton translocation across the inner mitochondrial membrane. in plants, aoxs increase local tissue temperature to release volatile compounds thereby attracting pollinator insects and regulation of mitochondrial retrograde signaling pathway. regulation of retrograde signaling pathway is currently under investigation to improve cultivation studies in many plants. water lilies are aquatic ornamental and economically valuable plants classified under nymphaea family. nymphaea alba, white water-lily, has a special focus since its applications in landscaping of parks and gardens, farming as vegetable and medical applications. however, cultivation of n. alba is a challenging process. we hypothesized that by controlling alternative oxidases, success rate can be increased for n. alba cultivation. to identify alternative oxidase encoding genes in n. alba, we performed transcriptome analysis. by using transcriptome analysis data, aox gene sequences, subcellular localization of aox proteins and structural modelling of aox proteins were predicted. in transcripts, database search with trinotate tool revealed transcripts with aox domains characterized in known alternative oxidases. blast analysis of these sequences with known aox proteins revealed three distinct aox genes (nalba-aox , nalba-aox and nalba-aox ). after subcellular localization analysis of three identified aox proteins by using targetp server tool, nalba-aox , nalba-aox are predicted as mitochondrial while nalba-aox is localized in chloroplasts. template based structural modelling results showed that all identifed proteins are statistically similar to known structure models of corresponding aoxs. most environmental contaminants have toxic and mutagenic effects on living organisms as a result of the activation of free radical formation and inhibition of reparation activity. it is becoming relevant to search for protectors of natural origin from the effects of xenobiotics. many biologically active substances (bas) of inartificial origin are found to be antioxidants and can increase the body's resistance to the toxic and mutagenic effects of a wide range of pollutants. the aim of the study was to investigate the antioxidant and antimutagenic properties of bas from medicinal plants limonium gmelinii (plumbaginaceae) and inula britannica (compositae). the antioxidant potential of plant extracts was determined by the activity of superoxide dismutase (sod), catalase, and the content of malonic dialdehyde. mutagenic and anti-mutagenic properties of the extracts were determined in the test by counting chromosomal aberrations in root meristem of barley seeds. barley seeds were treated with an aqueous solution of unsymmetrical dimethyl hydrazine (udmh), which is highly toxic i class hazardous material, well known pro-oxidant. the results showed that udmh enhanced the process of lipid peroxidation and decreased the mitotic activity. treatment of barley seeds with extracts from i. britannica and l. gmelinii and their germination in the presence of stress factors stimulated antioxidant defenses in the primary roots of barley seeds. increase of the activity of sod and catalase, and reduction of peroxidation level of lipids were observed. cytogenetic study showed no mutagenic activity in plant extracts. when effects of plant extracts and udmh were combined there was a significant reduction in the frequency of structural mutations, induced by the toxicant. conclusion about the presence of the antioxidant and antimutagenic activity in the studied plant extracts is made. the work done within the framework of the mes project (no. gr rk ). p- . . - comparative analysis of cytokinin dehydrogenase inhibition and trans-zeatin treatment in arabidopsis seedlings j. nov ak , v. koukalov a , z. medvedov a , c. martin , j. hradilov a , l. sp ıchal , b. brzobohat y mendel university in brno, brno, palack y university in olomouc and centre of the region han a, olomouc, czech republic cytokinins are plant hormones regulating many processes during plant life ranging from germination to senescence. manipulation of cytokinin levels and their impact on plant vitality, production and ability to defend against stresses is in great interest of agriculture. in this work we focused on comparison of inhibitor of the cytokinin degradation incyde ( -chloro- -( -methoxyphenyl)aminopurine) and exogenous application of trans-zeatin on arabidopsis thaliana seedlings. transcripts of genes regulating cytokinin metabolism were analysed by rt-qpcr analysis. classical cytokinin root essay revealed that incyde effect is comparable to that of trans-zeatin in a similar concentration-dependent manner. besides a negative effect on the primary root length, both substances induce flavonoid accumulation and an increase in the root hairs formation. histochemical staining of transgenic plants expressing glucuronidase (gus) under cytokinin-responsive promoter of arr gene revealed increased gus activity in cotyledons following incyde treatment suggesting diverse localization of cytokinin modulation upon trans-zeatin and incyde treatment, respectively. possible molecular differences originating in different cytokinin population and distribution following trans-zeatin or incyde treatments were monitored on the level of gene expression and via an lc-ms proteome analysis in roots and shoots of -day-old plantlets. rt-qpcr analysis revealed an alteration in cytokinin metabolism that could explain observed differences on the proteome level between incyde and trans-zeatin treated seedlings. pharmacologically inhibited cytokinin degradation could be very efficient tool for modulation of cytokinin levels. interestingly, the application of incyde and trans-zeatin shows a contrasting spatial and temporal pattern on molecular levels. incyde represents potent growth regulator with interesting properties useful for agriculture. p- . . - the expression yield of prokaryotic alphaamylase is significantly magnified by molecular cloning techniques randomly hydrolyzing glycosidic bond alpha-amylase has been traditionally employed in bread and similar industries. in that regard, increasing the overall expression level of the enzyme is a crucial concern in biotechnology. to reach the goal, appropriate alpha-amylase producing species and expression vector were carefully selected. therefore, genome of bacillus subtilis was extracted and amplified by polymerase chain reaction (pcr) using specifically designed primers. subsequently, the extracted gene was inserted in expression vector pht and transferred to e. coli as intermediate host followed by bacillus subtilis host replacement. the recombinant vector was expressed in bacillus subtilis and the expression was evaluated by agarose gel electrophoresis. relative purification of the recombinant enzyme was performed by kda filtration to remove impurities. to identify the biochemical characteristics, starch was used as specific substrate to measure enzyme activity and the enzyme was exposed to various ph and temperatures. the extra-cellular expression of alpha-amylase enzyme was successfully elevated by folds in comparison to the native enzyme. the optimum temperature and ph for the enzyme was carefully determined as °c and , respectively. the enzyme was stable at °c, but thermal stability was dramatically decreased at higher temperatures up to °c. kinetic parameters were also measured; vmax was . u/ml min and km was . mg/ml. it is concluded that the elevated expression extent of recombinant alpha-amylase together with appropriate qualifications could make the clone a good choice for various industrial applications. flax seedlings of cultivars tmp , lira and lines g- / _o, g- / _k were treated for and hours with lm alcl solution or distilled water (control). twelve small rna libraries were constructed and sequenced using illumina gaiix. to identify known mirnas, obtained sequences were aligned with mirnas from mirbase (http://www.mirbase.org/). fold change value was calculated to identify up-and down-regulated mirnas under al stress. in total, about million raw reads were obtained and conserved mirnas from families were identified. significant expression alterations in flax plants under al treatment were shown for mir and mir . expression level of mir was varied in similar way in resistant and susceptible to al genotypes: mir was up-regulated after hours of alcl exposure and down-regulated after hours. mir expression was increased after hours of alcl exposure and decreased after hours in susceptible to al flax genotypes (lira, g- / _o), while in resistant genotypes (tmp , g- / _k) mir level was decreased after both and hours of al treatment. in other plant species, mir and mir were identified as al-responsive. mir targets mrna of tcp (teosinte branched/cycloidea/pcf) transcription factors, which control plant growth. mir targets mrna of tas protein, which regulates lateral root growth via degradation of arfs (auxin response factors). in flax, the involvement of mir and mir in response to al stress was shown for the first time. moreover, we revealed diverse expression alterations of mir in susceptible and resistant to al genotypes. this work was financially supported by grant - - from the russian science foundation. p- . . - association genetics of phenylalanine ammonia lyase (pal) and cinnamyl alcohol dehydrogenase (cad) enzymes involved in lignin biosynthesis of european black poplar (populus nigra) b. taskiran, z. kaya middle east technical university, ankara, turkey populus nigra l. are considered as one of the most economically significant forest trees with respect to production of wood, biomass, and other wood-based products. while wood quality and biomass are directly associated with high cellulose content, lignin emerges as an undesirable polymer for both pulp and biofuel manufacturing industries. the aim of the study is by choosing the superior and eliminating the inferior clones to make a contribution to woody feedstock development and to improve wood quality of populus nigra. to estimate association genetics of pal and cad enzymes which have important functions in lignin biosynthesis, the important germplasms of populus nigra has been sampled from year old poplar trees ( clones x replicates x ramets) which were grown in behic ßbey plantation clone bank in ankara. additionally, five commercially registered clones and six foreign clones were included to the study to make comparison. the average mean values of cellulose, lignin and glucose content were calculated as . ae . lg/ml, ae . lg/ml, and ae . lg/ml, respectively. even though for pal and cad enzymes, data gathering process have been still resuming, particular clones have been separated from all in terms of pal and cad activities as expected. key words: populus, poplar, lignin, pal, cad, genetic variation, feedstock p- . . - proteomic analysis of the molecular mechanisms of the response of plant seeds to pre-sowing treatment by stressors seed treatment with non-ionizing low-level radiation (nr), such as cold plasma (cp) or electromagnetic field (ef), is a modern eco-agricultural technology for stimulation of plant germination and performance. the molecular determinants of seed response to these treatments are not established and no genomic studies of plant seed response to nr have been reported. we studied the effects of pre-sowing seed treatment, using vacuum ( min), radio-frequency ef ( - min) and cp ( - min), on germination and growth of non-oilseed helianthus annuus. to gain an insight into the molecular mechanisms underlying effect of nr on sunflower seed germination and dormancy, we estimated changes induced in the balance of plant hormones and differential protein expression. the results of the germination tests and estimation of seedling morphology showed that response develops in time and is stronger when sowing is performed in days in comparison to days after seed treatment. the d dige analysis revealed differentially expressed proteoforms in kernels of seeds treated with cp or ef. proteins involved in biological processes of seed maturation, response to stress, response to abscisic acid stimulus, processes of organonitrogen compound metabolism and glucose catabolism were identified. while expression patterns for majority of the proteins were highly specific to cp and ef treated seed kernels, accumulation of several proteoforms of seed storage proteins (ssp), including vincilin-like, miraculin-like protein and albumin- were common for both experimental groups. this suggested that response to nr treatment could be at least partially associated to function of ssps in response to oxidative stress that protects proteins required for seed germination and seedling formation. variation of abundance of distinct proteoforms of helianthinin, vicilin-like and s globulin-like ssps suggested that post-translational modifications are involved in regulation of the function of ssps. p- . . - suppression of lipopolysaccharide-induced inflammatory responses in raw . macrophages by tuber extract of cyclamen l. turkey is a prominent centre of plant diversity, being the meeting point of three main floristic zones. geophytes which have underground storage organs such as, tubers, bulbs and rhizomes. cyclamen l. is a tuberous geophyte traditionally used by some people for treating whooping cough, headaches or sinusitis, and confirmed to have antioxidant, analgesic and anti-inflammatory properties by several reports. a prolonged inflammatory response is often associated with chronic diseases such as cancer, arthritis and autoimmune disorders. recently, plant based products are used as an alternative and complementary treatment of these diseases. in this respect, the present study was aimed to determine the effects of three cyclamen tuber extracts on lps-induced inflammatory responses of murine raw . macrophages. firstly, c. cilicium (endemic), c. pseudibericum (endemic) and c. graecum subsp. anatolicum were collected from different localities of turkey. the tubers of plants were air-dried and grounded to fine powder and then extracted with ethanol. cell viability assay was performed to evaluate the nontoxic concentration in cell line by mtt assay. several measurements were performed including tnf-a, no and il- concentration assay by elisa after treatment compared to non treated cells to determine the anti-inflammatory activity. also, tnf-a and inos mrna levels were evaluated by quantitative rt-pcr. the cytotoxic activity which is considered safe on raw. . cell were found as . - lg/ml. studied cyclamen taxa inhibited tnf-a and il- release on lps stimulated-raw. . in a concentration-dependent manner. among the three cyclamen tuber extracts evaluated, the highest nitrite-associated no inhibitory activity was obtained from c. pseudibericum compared to other two cyclamen l. taxa. collectively, these results suggest that cyclamen tuber extracts possess anti-inflammatory properties. p- . . - in vitro hypoglycemic activity of ziziphus jujuba recent reports have indicated that continuous treatment with nutritional jujuba (ziziphus jujuba miller) fruit extracts in diabetic rats improved glucose utilization and produced a significant decrease in the blood glucose. in the present study, hypoglycemic activity of z. jujuba was investigated using various in vitro techniques. the hypoglycemic effect of z. jujuba in phosphate buffered saline which grown in balıkesir was studied by measuring glucose adsorption, glucose diffusion and glucose uptake by yeast cells. the glucose content in the solution measured by spectrophotometrically with commercially kits. the adsorption capacity of the z. jujuba was found to be directly proportional to the molar concentration of glucose. the glucose binding capacity of extract increased in higher glucose concentratrations. there was significant differences were observed between the adsorption capacities of z. jujuba and control samples (p < . ). the rate of glucose diffusion was directly proportional to the time. diffusion rate was significantly lower in the solution containing z. jujuba compared to control (p < . ). the extract demonstrated significant inhibitory effects on movement of glucose into external solution across dialysis membrane compared to control. the rate of glucose transport across cell membrane in yeast cells was observed to be inversely proportional to the molar glucose concentration. z. jujuba inhibited glucose transport across the yeast cells. the results showed that z. jujuba reduced glucose levels at least by three mechanisms. first by increasing glucose adsorption capacity during postprandial hyperglycemia; second by retarding glucose diffusion rate and third, at the cellular level by inhibiting glucose transport across the cell membrane. all of these decreased the absorption of glucose in the intestinal cells and the concentration of postprandial serum glucose. p- . . - cucurbitacin b increased the anticancer effect of imatinib mesylate through inhibiton of matrix metalloproteinase- expression in colorectal cancer cells f. bakar ankara university, ankara, turkey several natural products have been investigated for their anticancer effects. among these, cucurbitacin b (cub) has been reported as its inhibitory effects on cancer cell proliferation. matrix metalloproteinases (mmps) belong to endopeptidase family and they are received as potential biomarkers for several types of cancer. the aim of this study is to investigate the effect of cub in combination with imatinib mesylate (im) on mmp- mrna expression of human sw colorectal carcinoma cells. the cytotoxicity analysis was performed via mtt assay. muse cytofluorimetric analysis system was performed to evaluate apoptotic cell population. the mmp- mrna expression was determined by quantitative real-time pcr. this study was supported by scientific and technological research council of turkey grant, sbag- s . data obtained from the cell culture experiments were expressed as mean ae sd and one-way anova test was applied for multiple comparisons. cub alone significantly inhibited cell growth at lm and higher concentrations. the most potent effect was observed in cub-im combination treatment with . lm ic value. in cub-im treated group, the apoptotic effect was higher than cub and im treated groups. cub-im induced apoptosis significantly at lm concentration when compared to control and lm (p < . ). cub alone showed inhibitory effects on mmp- mrna expression at lm and higher doses significantly (p < . ). the results showed that the combination treatment of cub with imatinib synergistically inhibited human sw cell growth and induced apoptosis by increasing the anti-histone antibodybound nucleosom levels and annexin v binding. although cub could inhibit mmp- expression alone at higher treatment doses, it enhanced the inhibitory effect of im on mmp- synergistically in a dose dependent manner. in conclusion, this study suggests that cub combined with imatinib mesylate may enhance the effects of chemotherapy in patients with colorectal cancer. plants are most important parts of natural resources that alternatively referred to synthetic drugs for reasons such as being less side effects and lower costs. ziziphus jujuba miller (z. jujuba), a plant used in traditional medicine, is one of the most important ziziphus species belonging to rhamnacea family. the fruit and seeds of this plant are used different purposes such as antiinflammatory, antioxidant, immune-stimulant and wound healer. in this study, we investigated the antibacterial effects of z. jujuba. the aims of this study were to screen the antibacterial activity of z. jujuba. the extract was obtained from z. jujuba fruits pulverized with the aid of ball mill using % aqueous-ethanol solution. extracts were screened for antimicrobial activity against six different standard strains of bacteria by determining minimum inhibitory concentration (mic) according to clsi criteria. serial dilutions are made between mg/ml and . mg/ml concentration range. the lowest concentration of wells that no visible growth has been accepted as mic value. materials in the mic and lower concentrated wells were transferred to % sheep blood agar petri dishes for calculation of minimal bactericidal concentration (mbc). the lowest concentration that no colony formation has been accepted as mbc value. jujuba showed the most potent effect on strain of s. aureus atcc is gram-positive cocci (mic: mg/ml). the mic values of other gram-positive bacteria s. aureus atcc , e. faecalis atcc , l. monocytogenes f and m. smegmatis cmm were detected as , , and mg/ml respectively. mic values of gram-negative bacilli were detected as > mg/ml. consequently, z. jujuba was found to be effective on grampositive cocci bacteria (s. aureus atcc , s. aureus atcc and e. faecalis atcc ). the strongest effect was observed on s. aureus atcc strain. in contrast, extract showed less effect on gram-negative bacilli. p- . . - selective cytotoxic effect of morus rubra extract in human lung cancer cells through enhancing apoptosis and cell cycle arrest cancer is a disease that develops as a result of unlimited proliferation of abnormal cells that occurs due to loss of control over the mechanisms of normal growth and differentiation of cells. morus rubra, known as "red mulberry" belongs to family of moraceae. for many years, the fruits of morus species have been used to treat many diseases in traditional medicine. biological effects of morus species is predominantly attributed to its content of polyphenolic compounds. many studies have evaluated the cytotoxic effects of different morus species, but there is no study about cytotoxic effect of m. rubra. in this study, we aimed to evaluate the cytotoxic effect of m. rubra extract in human lung cancer cells (a ) with regard to apoptosis, cell cycle and mitochondrial membrane potential. cytotoxic effect of m. rubra extract on human lung cancer cells was determined using mtt assay. then, mechanisms of cytotoxic activity of m. rubra extract on a cells were examined in terms of cell cycle, apoptosis and mitochondrial membrane potential using flow cytometric methods. m. rubra extract exhibited selective toxicity against a cells compared to normal foreskin fibroblast cells. we determined that m. rubra extract increased cell cycle arrest at g phase, the level of apoptotic cells and decreased mitochondrial membrane potential in a cells. our results showed that m. rubra extract has pro-apoptotic and antiproliferative effect in a cells. further studies are also needed to fully mechanisms underlying this effect of m. rubra extract. p- . . - dipeptidyl peptidase iv inhibitory activity of arctium tomentosum l. a. zeytunluoglu , f. zihnioglu denizli vocational school of technical sciences, pamukkale university, denizli, department of biochemistry, faculty of science, aegean university, izmir, turkey type diabetes mellitus (t dm) is rapidly growing metabolic syndrome of multiple aetiologies causing hyperglycaemia with insulin resistance at cellular level. a novel approach in the treatment of t dm is based on preventing of rapid inactivation of the incretin hormone glucagon-like peptide- (glp- ) and glucose-dependent insulinotropic polypeptide (gip) by dipeptidyl peptidase-iv enzyme. in this study; dipeptidyl peptidase iv (dpp-iv; ec . . . ) inhibitory activity of the aqueous and methanolic extracts of arctium tomentosum leaves were successfully tested in vitro conditions. our study revealed that both aqueous and methanolic extracts obtained from test material had a significant dppiv enzyme inhibitory activity in changing ratio. the ic values were also determined by nonlinear regression curve fit using graph pad prism . with appropriately diluted of lyophilized arctium tomentosum. diprotein-a (ile-pro-ile) was used as reference inhibitor. a. tomentosum aqueous extracts showed ic . lg/ml while the standard (positive control) diprotin a displayed the ic value of . lg/ml. this study demonstrates that a. tomentosum aqueous extracts could be a good lead for further development as a new antidiabetic agent. p- . . - dna recognition determinants of arabidopsis thaliana b transcription factors g. sasnauskas, k. kauneckaite, k. lapenas, v. siksnys institute of biotechnology, vilnius university, vilnius, lithuania transcription, one of the most important cellular processes, is regulated by transcription factors (tf), proteins that often directly interact with gene promoter sequences. tf binding to dna is mediated by various dna binding domains. the b tfs constitute a large, plant-specific protein family (approx. % of all tf proteins in the flowering plants), which is characterized by the presence of one or several small (approx. amino acids) b dna binding domains. currently the b tfs are divided into four groups (lec -abi /val, arf, rav and rem). the preferred recognition sites were identified for representatives of all groups except the rem family. currently, only a single structure of a dna-bound b domain (arf , arf family) is available, thus the mechanism of site-specific dna recognition by the lec -abi /val and rav b domains remains poorly understood. based on the arf -dna structure (pdb ldx) we have built homology models of dna-bound b domains from a. thaliana abi (lec -abi /val family) and nga (rav family) transcription factors, mutated putative dna-interacting amino acid residues and characterized the dna binding ability of the purified mutants using electrophoretic mobility shift assay and a set of radiolabelled dna substrates carrying various variants of the optimal recognition site. we confirm the importance of several positively charged amino acid residues, which are conserved between the abi / nga b domains and structurally related dna-binding domains of bacterial restriction endonucleases ecorii, bfii and ngoavii; furthermore, we identify residues in the 'n-arm' and 'c-arm' loops that may be involved in specific interactions with the dna bases. our results therefore help us refine the homology models of the dna-bound b domains and in the future may help us predict the dna binding properties of currently uncharacterized b domains. p- . . - immunohistochemical analysis of inhibitory effects of origanum hypericifolium oil on dipeptidyl peptidase iv in streptozotocininduced diabetic rats p. ili , a. zeytunluoglu denizli health services vocational high school, pamukkale university, denizli, denizli vocational school of technical sciences, pamukkale university, denizli, turkey diabetes mellitus (dm) is a serious metabolic disorder with micro-and macro-vascular complications that result in a significant morbidity and mortality. glp- and gip have significant role in pancreatic beta cells and prevention of inactivation of them by dipeptidyl peptidase iv (dpp iv) inhibition is a novel approach to treatment of dm. origanum hypericifolium (lamiaceae) is an endemic turkish plant and its essential oil is mainly composed of monoterpenes including carvacrol and thymol. streptozotocin (stz) is used to induce diabetes in rats. the aim of this study is to investigate the inhibitory effects of o. hypericifolium essential oil on the dpp iv in stz-induced diabetic rats. the animals (female spraque-dawley rats) were assigned to four groups (group : control, group : stzinduced diabetic, group : o. hypericifolium injected, group : stz-induced diabetic and o. hypericifolium injected). dm was experimentally induced in groups and by a single intraperitoneal injection of stz at a dose of mg/kg body weight. in groups and , rats were intraperitoneally injected with o. hypericifolium oil at a daily dose of ml/kg body weight for consecutive days. at the end of the experimental period, all animals were sacrificed by cervical dislocation under ether anesthesia and liver and kidney tissues of each animal were rapidly excised. tissues were fixed in sainte-marie fixative. after routine histological processes, samples were embedded in paraffin, immunohistochemical staining for dpp iv was performed on sections and then they were photographed. the immunohistochemical reaction intensity differences were observed between the groups. in conclusion, the immunohistochemical distribution of dpp iv in the tissues that the test oil was applied in the diabetic rats may be important for the investigation of the inhibitory effects of oil on the enzyme. moreover, our findings suggest that o. hypericifolium oil may be used for prevention of diabetic diseases. introduction: all eukaryotic cells need microtubules for purposes of nuclear and cell division, organization of intracellular structure, and intracellular transportation, as well as ciliary and flagellar motility. microtubules are made of polymerized a/btubulin subunits. mec is important for microtubules, because it encodes the enzyme that adds acetyl groups to lysine (k ) of tubulin. k is largely conserved in a-tubulins of many eukaryotes, and acetylation is thought to stabilize microtubule structure. in algae, the effect of acetylation by mec on flagellar motility and phototaxis has not been tested previously. materials and methods: in this study, mec mutant chlamydomonas reinhardtii cells were compared to wild-type cells to see the effect on flagellar motility and phototaxis. we tested phototaxis, eyespot size and quantity under the microscopy. in addition to this, we fixed cells and examined them by immunofluorescence microscopy using antibodies to tubulin, acetylated tubulin, and photoreceptor. results: we observed that some mec mutant cells contain more than one eyespot. we detected no acetylated-tubulin (ac-tub) by immunofluorescence. the cells still phototax and have normal motility discussion and conclusion: interestingly, mec cells still have the ability to phototax and they have normal flagellar motility, even though they contain occasional additional eyespots and no ac-tub. chlorella vulgaris as a model system for screening of plant growth modulators p. volynchuk , e. marusich , r. chuprov-netochin , j. neskorodov , y. mishutkina , s. leonov life sciences center, moscow institute of physics and technology, dolgoprudny, center "bioengineering", russian academy of sciences, moscow, russia the discovery of new plant growth modulators became extremely important task as an alternative approach to overcome plants resistance to herbicides and pesticides, which leads to harmful action on plants and land rising, environmental and ecological problems. small molecules provide agricultural biotechnology with valuable tools, which help to circumvent the need for genetic engineering and offer unique benefits to modulate plant growth and development. we developed a system to explore molecular modes of action of plant growth modulators using chlorella vulgaris model. our model allows applying high content screening approach in well plate format for fast and robust effect assessment of large number of tested modulators. chlorella v. was grown in climate chamber under optimized constant temperature ( °c) and light conditions ( : hours/light:dark). modulating effect of tested compounds was estimated by spectrophotometric measurement of microalgae density at the beginning of the experiment (start point- . od) and hours later. to validate our system we used known cytokinins and auxins ( mm) as positive controls of growth stimulation. we showed that in presence of each compound the density of chlorella v. was increased in - times range, compared with only times increase in control group. eight new chemicals ( lm), which demonstrated modulation effect on nicotianatabacum l. pollen and arabidopsis thaliana models, were tested on developed chlorella v. model. positive controls showed no stimulating effect at this concentration, while tested compounds were confirmed as hits and increased the density up to %. we demonstrated that developed model system, based on chlorella v., is an effective system for primary screening of plant growth modulators. the main advantages of this system are short time of assay, simplicity of performance, possibility of automation and low cost. selected hits can be recommended as perspective candidates for future test on crop field. sunflower is under a big threat of downy mildew which is a fungal disease caused by plasmopara halstedii. the disease can cause up to an % yield loss in sunflower production. downy mildew resistance genes (r) denoted as pl has been discovered to date in sunflower. in recent years, single nucleotide polymorphism (snp) markers have become widely used in plant breeding programs. in this study snp markers have been currently developing for pl , pl , pl , pl arg genes by competitive allele specific pcr (kasp) assay which enables bi-allelic scoring of snps and insertions/deletions (indels) at specific loci. in total sequence tagged site (sts) sequences from ncbi were aligned for pl , pl , pl , pl arg genes to identify conserved regions for each gene. based on the conserved regions, specific pcr primers were designed in order to make sequencing of these genes in five crosses and their f progenies. sequence data will be used to design an allele specific primer maches the target snp and amplifies the target region with the common reverse primer provided by kasp genotyping assay. snp markers linked to pl genes which are being developed in this study, have the potential to be used in marker assisted selection (mas) for sunflower breeding programs. p- . . - investigation of the antidiabetic effects of hibiscus sabdariffa, teucrium polium and myrtus communis in hepg cells line s. altundag , pamukkale university, denizli, istanbul medeniyet university, istanbul, turkey some antidiabetic plants currently are used in alternative treatment of type ii diabetes. hibiscus sabdariffa, myrtus communis and teucirum polium plants are also known for their antidiabetic properties. hibiscus sabdariffa, myrtus communis and teucrium polium; depending on the impact on hepg - cells to investigate the possible mechanisms of type ii diabetes with researches on glucolysis and glucogenesis pathways gene expressions (pk m , glut- ,pepck). plants were obtained in dried state from reliable herbalists in denizli and mersin. plants treated with the extractor device. plants obtained aqueous extract was subjected to lyophilization process. human cancer cells have been used throughout the study. the cytotoxicity of the cells was measured by elisa plate reader . total rna was isolated using trızol Ò solution was carried out according to the instructions of the manufacturer's (thermo scientific) recommended procedures were performed, but we have to optimize our own laboratory conditions. pk m , glut- ,pepck genes were synthesized by b _ ioneer. during our study the activation of certain genes (pk m , glut- ,pepck) were examined by real time pcr. in our study hibiscus sabdariffa, mrytus communis and teucrium polium plant to extract applied hepg - cell line in the gluconeogenesis and glycolysis pathways in involved in some important genes (pepck, pk m , glut- ) analyzed the expression levels . teucrum polium plant extract is applied in hepg - cells the glycolysis pathway genes (pk m glut- ) an increased expression also genes of gluconeogenesis pathway (pepck) were not decreased. however hibiscus sabdariffa and the expression of genes involved in glycolysis and gluconeogenesis pathway mrytus communis plants were observed to have a full effect as diabetic or hypoglycemic . in this context, it is considered that the plant teucrum polium on the line hepg - cells showed antidiabetic effect. p- . . - purification of b-glucosidase from malatya apricot (prunus armeniaca l.) seeds and some of its biochemical properties h. kara , s. sinan , z. ekmekci , y. turan university of balikesir faculty of veterinary, balikesir, university of balikesir faculty of arts and sciences, balikesir, turkey introduction: b-glucosidases are one of the key enzymes in carbohydrate metabolism and located in glycosyl hydrolases (ec . . ) family. plant b-glucosidases have biotechnological significance as they are effective on glycosidic bonds of flavor and aroma precursors in plants. b-glucosidases that located in fruit seeds are important because they affect the amygdalin. aim of this study is purification and partially characterization of b-glucosidase from malatya apricot seeds. materials and methods: apricot seeds were homogenized with extraction buffer to prepare of crude extract. the enzyme protein was precipitated with % ammonium sulfate then purified by hydrophobic interaction chromatography using sepharose b-ltyrosine- -naptylamine gel. para-nitrophenyl b-d-glucopyranoside (p-npglc) was used as substrate to determine biochemical properties of the enzyme. the optimum ph was determined using buffers between ph - and thermal optima was determined using - °c temperature range. inhibitory effects was determined with mm substances. results: the enzyme was . -fold purified with yield of %. purified b-glucosidase from apricot seed was visualized about kda molecular weight on sds-page. the kinetic parameters were determined against p-npglc substrate as km and vmax values of . mm and . eu, respectively. the optimum ph and temperature were determined . and °c respectively. effects of cacl , kcl, nacl, mgcl , k so , na so , cuso , fecl , pb(ii) acetate, agno , zncl and glucose on purified enzyme activity were investigated. kcl, na so , pb(ii) acetat and cuso reduced the enzyme activity. discussion and conclusion: in this study, b-glucosidase was purified from malatya apricot seed and some of its biochemical properties were determined. because this enzyme has pharmaceutical importance hydrolyzing amygdalin. the results showed that immobilized almond b-glucosidase was used to break amygdalin and release -cn compound that effective to shrink cancer mass. p- . . - a new affinity gel for purifing polyphenol oxidase enzyme a. erg€ un , , o. arslan balikesir university, science and technology application and research center, balikesir, department of medical chemistry, faculty of science, balikesir university, balikesir, turkey polyphenol oxidase (ppo) enzyme, sometimes called as phenol oxidase, catecholase, phenolase, catechol oxidase or tyrosinase, is considered to be an o-dipenol. ppo (ec . . . ), a multifunctional copper containing metalloenzyme, is widely distributed in nature. ppo exists in many kinds of plants and fungi, such as banana, mushroom, butter lettuce, napoleon grape, potato, coffee, marula fruit, artichoke heads, longan fruit, tobacco, wheat flour. in this study, a novel affinity chromatography gel was synthesized for purifing ppo enzyme. the affinity chromatography gel was synthesized by coupling aniline as a specer arm to cnbr activeted sepharose- b. then, p-amino benzoic acid was coupled to aniline as a ligand. ppo was purified from musa sapientum var. cavendishii (banana) by using sepharose- b-aniline p-amino benzoic acid affinity chromatography gel. % . yield and . fold purification were achieved. sds-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent mw of kda. the v max and k m of the purified enzyme were determined , u/ml min and . mm, respectively. p- . . - phenolic content and antioxidant capacity of gamma irradiated olive leaves m. e. diken , b. kocat€ urk , s. dogan , h. tuner balikesir university, balikesir, kavram vocational school, istanbul, turkey in this study, dried in diffirent ways (such as microwave, infrared, convection heaters and under normal atmospheric conditions) olive leaves has been used as experimental material. radiation has been applied to dried olive leaves in three diffirent dosages in room temparature. the amount of radiation to be implemented to the samples have , , kgy/min. in this study, how gamma rays (radiation) effects phenolic components, total phenolic content and antioxidant capacity of dried olive leaves has been determined. the phenolic components, total phenolic content and antioxidant capacity were analysed with hplc, folin ciocalteu and dpph radical scavenging method, respectivelty. gamma rays is well known as a decontamination method for many foodstuffs and plant materials, being an environment friendly and effective technology to resolve technical problems in trade and commercialization. for this reason, nowadays it is utilized as an alternative method of sterilization. gamma rays are of ionizing radiation. when ionizing radiation interacts with matter, generally it causes a break in the molecular bonds and/or breaks the bonds between the molecules. these intermediates have unpaired electron and called free radical. gamma radiation or the radiation-induced free radicals would break or cause damage to the dna molecules of living organisms. gamma irradiation is predict to change phenolic content and antioxidant capacity in living tissues. phenolic compounds are secondary plant metabolites naturally present in fruits and vegetables. in recent years there has been a growing interest in food phenolics because of their potential health benefits mainly due to their antioxidant and free radical scavenging activity. despite the controversy about potential risks posed by genetically modified plants on human health, environment and microorganisms, cultivation area of these crops increases day by day. this increment has revealed concerns especially related to hgt. hgt studies indicated that antibiotic resistance genes in gm plants have a potential to transfer to soil microorganisms. in this study, hgt of widely used genetic elements such as regulatory sequences, from transgenic plants to bacteria was investigated. three soybean feed and four seed examples from turkish feed manufacturers' association were used for genetic analysis based on foreign gene determination. gmo analysis were conducted by camv s promoter-specific pcr in genomic dnas. in gm positive samples, genomic dnas sheared into appropriate fragment size by ultrasonication for the purpose of bacterial transformation. presence of s promotor region in fragmented dnas was proved by pcr. escherichia coli dh a strain was transformed by fragmented dna samples according to cacl method. s promotor sequence screened by using pcr in bacterial genomic dnas. as a result of gm screening, all feed and three of the seed samples were found to be transgenic. ultrasonication conditions were optimized for shearing dna's to - bp for bacterial transformation. fragmented dnas confirmed for carrying intact s promotor sequence. none of bacterial genomic dnas were found to be s-positive. according to the transformation results, absence of s promotor sequence in all tested bacterial genomic dnas, proved the dna samples belonging to gm plants can not transfer into compotent e. coli dh under laboratory conditions. for verification of this finding, transformation studies will continue with acinetobacter baylyi bd strain which is naturally competent soil bacterium for natural transformation. we believe that all of our findings will contribute to constitute transformation system which can be used as model in hgt studies. p- . . - preliminary studies on differential methylation in a and d sub-genomes of upland cotton (gossypium hirsutum l.) four species of cotton (gossypium l.) provide raw materials for the textile industry. among the four species, two have diploid genome and another two have tetraploid genome. tetraploid genome consists of a and d sub-genomes. a sub-genome belongs to asian cotton while d-sub genome belongs to american cotton. previous studies revealed that d sub-genome of gossypium species contributes to the superior yield and quality of tetraploid gossypium l. species (atdt). dna cytosine methylation of four regions of dna sequences located on a and d sub-genomes of gossypium hirsutum l. texas marker (tm- ) was investigated using bisulfite sequencing technique. among the regions studied two could not be located on subgenomes due to sequence identity match between a and d subgenomes. on the other hand two dna regions could be located on a and d sub-genomes using the blast searches. some of the dna sequences located on different sub-genomes showed polymorphic nucleotides including c/t and g/a polymorphisms. in silico analysis indicated that some alleles located on different sub-genomes of cotton have c/t and g/a polymorphisms. c/t polymorphisms between the sub-genomes could be thought as unmethylated cytosine using the bisulfite sequencing technique. this indicated that an extra attention needs to be paid in dna total cytosine methylation studies in polyploid species such as cotton using bisulfite sequencing, methylation sensitive amplification polymorphism (msap), whole-genome bisulfite sequencing (wgbs). otherwise, t in c/t polymorphism between the subgenomes could be thought as unmethylated cytosine. based on the two genomic regions we could conclude that a sub-genome may be more methylated than d sub-genome. differential methylation of genes located on different sub-genomes may provide another mechanism responsible for differential gene expression of genes located on different sub-genomes. p- . . - cleaved minisatellite locus (cml) markers for fingerprinting of cotton cultivars grown in turkey e. u. gocer, m. karaca akdeniz university, antalya, turkey after their discovery by alec jeffreys in , minisatellites have been used in genetic studies of many organisms. minisatellites, also called variable number tandem repeats (vntrs), are composed of arrays of longer repeats mostly dispersed throughout heterochromatin (centromeres and telomeres). direct amplification of minisatellite regions of dna using a single core primer is a powerful method to amplify minisatellites (damd-pcr). although the damd-pcr technique has been applied to many plant species, the level of polymorphisms in cotton (gossypium l.) is very low due to very narrow turkish cotton genetic base. the objective of this study was to improve the level of polymorphisms by cleaving minisatellite loci by restriction enzyme digestion. genomic dna samples of twenty-one turkish cultivars, pima - , tm- and ps- were extracted. twenty-one minisatellite primers were screened using the damd-pcr technique. monomorphic amplicons were digested using several restriction enzymes. three to five micro liters of amplified products were digested with various restriction enzymes. digested products of minisatellite loci were separated in % high resolution agarose gels. comparison studies of digested and undigested markers revealed that cleaved minisatellite markers showed polymorphisms in those cotton lines that could not be differentiated by microsatellite and minisatellite markers. this approach was called cleaved minisatellite locus markers (cml). the amplification reactions of minisatellites used touch-down (td) cycling conditions. the use of td offered a simple and rapid means of optimizing polymerase chain reaction (pcr), increased specificity, sensitivity, and efficiency without the need for lengthy optimizations of minisatellite primers. the cml markers were obtained at a °c annealing temperature, which is a temperature higher than those used in random amplified polymorphic dna (rapd) inter-simple sequence repeat (issr) markers. p- . . - association between cytosine methylation and tissue specific expression of microsatellites a. g. ince, m. karaca akdeniz university, antalya, turkey heritable covalent modification of dna, rna or protein without altering their primary sequences is defined epigenetics. because all biological events are influenced by epigenetics, it is one of the most important fields in science. dna methylation is one of the most important epigenetic mechanisms. dna cytosine methylation is a process by which methyl groups are added to cytosine bases of dna. microsatellites, also knows simple sequence repeats are dna sequences consisting of - nucleotide repeats. there is a large body of information regarding the relationship between microsatellite instability and abnormal gene expression, and between dna methylation and altered gene expression. however, there is limited information on cytosine methylation of microsatellites. in the present study, we investigated whether there is any association between cytosine methylation and tissue specific expression of microsatellites. genomic dna samples of various tissues and developmental stages of pepper line demre sivrisi (capsicum annuum l.) and cotton line tm- (gossypium hirsutum l.) were extracted. cdna samples were synthesized using mrna expressed in pepper tissues. cytosine methylation levels were investigated using bisulfite sequencing methods. screening studies of microsatellites revealed that some genes containing microsatellites were differentially expressed in tissues and developmental stages of pepper. microsatellite containing genes that expressed differently among tissues had also showed different methylation levels in cg, chg and chh (where h refers to a, c or t) contexts. methylation level differences between microsatellites were also observed. as far as our knowledge, it is the first report on differential expression of genes containing methylated microsatellites. p- . . - pcr-lg: an alternative way to assign the chromosome location of genes/markers in cotton a. aydin, m. karaca akdeniz university, antalya, turkey assignment of genes and dna markers on chromosomes is very important in life sciences, especially for plant breeding and medicine. there are several methods for the assignment of a gene or dna sequence to a specific location on a chromosome. for example, the most widely used technique is the assignment of fluorescently-labeled gene or dna sequences (markers) on chromosomes using the fluorescently-labeled gene (for instance, fish technique). another example is the construction of a genetic map (linkage map) which orders the targeted genes along the dna strand based on recombination frequency. sequencing is the most precise technique in which coding (gene-containing) and noncoding dna region of genes could be located on a chromosome molecule. aneuploid lines could also be used to locate genes in a specific chromosome, but maintenance of these lines is difficult. here we report the use of chromosome substitution lines to indirectly locate genes/markers on chromosomes. we used chromosome substitution lines (csls) that carry a chromosome pair or chromosome arms from gossypium barbadense l. while the rest of chromosomes belong to g. hirsutum l. a total of chls, a homozygous cotton line tm- and a double haploid line pima - were used as plant materials. twenty microsatellite primer pairs were utilized in touch-down polymerase chain reactions. we developed a method, called polymerase chain reaction to locate gene (pcr-lg), to assign genes/markers on chromosome or chromosome arm. with the use of pcr-lg approach any polymorphic genes/markers between tm- and pima - (g. hirsutum and g. barbadense) could be assigned to a chromosome or chromosome arm. results indicated that if csls were used any polymorphic markers (genes) with known primer pairs could be assigned to cotton chromosomes. although we used cotton chromosome substitution lines to validate the proposed technique, it could be applicable all the species that have chromosome substitution lines. p- . . - pecularities of genome variability of antarctic hairgrass deschampsia antarctica desv. from the maritime antarctic deschampsia antarctica desv. (poaceae) is the only grass species native to the antarctic region, adapted to harsh environmental conditions. however, reasons for its unique success remain unexplored. stressful environmental factors can influence a plant genome and cause changes in the chromosome number and morphology and increase genetic variation. therefore, the purpose of our research was to explore alterations in the d. antarctica genome both at the chromosomal and molecular levels and to investigate species genome stability using in vitro tissue cultures. plants used for the study were grown in vitro from seeds collected in the argentine islands region during - . chromosome number was determined in plant root apical meristems and specimens prepared from tissue cultures. rrna genes localization were determined using the fish technique. moleculargenetic analysis was performed using pcr with polymorphic issr-primers. new forms of chromosome polymorphism, associated with aneuploidy ( n = - ), polyploidy ( n = - ) and the occurrence of additional b-chromosomes ( n = + - b), were observed. fish analysis also confirmed that genotypes with a different chromosome numbers varied in the number of s rdna and s rdna sites. assessment of genetic variation demonstrated a low level of diversity: differences between the plants with different chromosome numbers do not exceed the level of within-population variation. cytology analysis of d. antarctica cultured tissues revealed aneuploidy (up to . %) with predominance cells with diploid and near-diploid chromosome number irrespective of plant's initial karyotype (diploid, mixoploid or polyploid). we assume that discovered intraspecies chromosomal polymorphism is a manifestation of quick genome reaction to harsh antarctic conditions. whereas the results of molecular-genetic analysis and study of cell cultures of this species suggests on the relative stability of d. antarctica genome. p- . . - angiotensin converting enzyme inhibitory activity of morchella esculenta (l.) pers a. zeytunluoglu , i. arslan biomedical equipment technology, pamukkale university, denizli, turkey, denizli, biomedical engineering, pamukkale university, denizli, denizli, turkey hypertension is a multi-aetiological, chronic pathophysiology that leads to multi-organ dysfunctions like cardiovascular diseases, strokes, and renal complications. natural extracts play an important role in traditional medicines for the treatment of hypertension and are also an essential resource for new drug discovery. mushrooms are use as therapeutics in alternative and complementary medicine as functional food because of contain a large number of biologically active components that offer health benefits and protection against many degenerative diseases. morchella esculenta is one of the most highly priced edible mushrooms worldwide. it contains a wide range of active constituents which include tocopherols, carotenoids, organic acids, polysaccarides and phenolic compounds which exhibit a wide range of medicinal and pharmacological properties including anti-microbial, anti-inflammatory, immunustimulatory, antitumor and antioxidant. in this study; the in vitro angiotensin converting enzyme-i (ace-i) inhibitory activity of m. esculenta peptides were generated by alcalase hydrolysis were studied. the kda < peptides < kda in the ultrafiltration fractions displayed highest ace inhibition ( . ae . % at lg/ml). the results indicate that m. esculenta derived peptides may have potential as functional food ingredients in the prevention and management of hypertension. p- . . - modulations of antioxidant enzymes, gsts and catalase, by salvia absconditiflora in hepg cell line d. irtem kartal , a. altay yuzuncuyil university, van, erzincan € university, erzincan, turkey oxidative stress is considered to play a important role in the pathogenesis of aging and several degenerative diseases, such as cancer. in order to cope with an excess of free radicals produced upon oxidative stress, humans have developed several mechanisms for maintain redox homeostasis. these protective mechanisms either scavenge or detoxify ros, block their production, and include enzymatic and nonenzymatic antioxidant defenses. in enzymatic defenses include glutathione s-transferases and catalase enzymes. many epidemiological studies have revealed that there is a strong correlation between consumption of polyphenol-rich foods and the prevention of certain diseases like cancer, cardiovascular diseases and aging. phenolic compounds are abundant in all plants. so, they form an integral part of the human diet. salvia species, commonly known as sage, have been used since ancient times for more than different ailments ranging from aches to epilepsy. there are around species of salvia, of which are represented in turkey including salvia absconditiflora. in this study, s. absconditiflora collected from metu campus (ankara, turkey) is extracted with methanol and water. effects of the water and methanol extracts on the mrna expressions of antioxidant enzymes gstm and catalase in hepg cells were investigated by q-rt-pcr technique. it was also monitored the effects of the extracts on the enzyme activities of gsts and catalase by spectrophotometrically. water and methanol extracts decreased gsts mrna expression in hepg cells for hours and hours incubation and methanol extract decrease catalase mrna expression for only hours incubation. on the other hand, extracts highly increased the gsts and catalase activities in both hour incubation. overall, these results indicate that s. absconditiflora and/or its components have regulatory activities on antioxidant enzymes and they may have a potential as a therapeutic agent in the treatment of cancer. p- . . - transcriptomics and proteomics approach to drought stress mechanism in wheat b. cevher keskin tubitak marmara research center genetic engineering & biotech. institute, kocaeli, turkey birsen cevher keskin, yasemin yıldızhan, oktay kulen, bayram y€ uksel, selma onarıcı, _ ismail t€ urkan, as ßkım hediye sekmen, u gur sezerman, bu gra € ozer identification of novel stress-responsive genes and their role in drought response is an important area for the improvement of the crops. drought-related genes were investigated in leaves and roots of three wheat genotypes after different drought stress treatments by rna sequencing (rna seq) technology and de novo assembly was performed before comparative transcriptome analysis. analyzing gigabases of bp paired end illumina reads from a hexaploid wheat poly(a) rna library, we identified common and new differentially expressed transcripts. selected differentially expressed genes were confirmed by qrt-pcr. we also performed root proteome analysis with nano electrospray ionization source coupled to a high-performance liquid chromatography system (nanouplcÀesiÀqtofÀms) to identify drought-related proteins. totally proteins were differentially expressed in root tissues of tolerant and non-tolerant wheat genotypes. responses of antioxidative defense system to drought stress were comparatively studied in the same wheat cultivars. similarities between protein and rna levels help increase our confidence in novel biomarkers, differences may also reveal other post-transcriptional regulatory junctures. all these analyses will allow us to get a better idea about the possible role of these genes in the drought-response mechanism. the drought-related genes that are functionally characterized could be introduced into agronomically important wheat cultivars. this work offers a resource for accelerating drought-related gene discovery and improving this important crop. p- . . - isolation and characterization of a hexose converter from olive s. altunok , e. d€ undar department of biology, university of balikesir, balikesir, department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey introduction: hexose sugars are key components of glycolysis and photosynthesis. the genes regulating their conversion into one another, is therefore, of great importance for the control of carbon metabolism. in this study, we report isolation and characterization of a cdna associated with conversion of hexoses in olive. the cdna putatively named aldolase based on bioinformatic and experimental analyses. material-methods: characterization based on nucleotide and amino acids were conducted using bioinformatic tools such as nucleotide and protein blast, bioedit, primer , finchtv, clc genomic workbench, expasy, targetp, sosui and web promoter scan. comparison of the genomic and cdna sequence of the gene and detailed bioinformatic analyses including cellular location, hydropathy analysis, amino acid-nucleotide composition and predicted d structure were also conducted using the bioinformatics tools mentioned above. temporal expression pattern of the putative aldolase were conducted using real-time pcr experiments. sds and western blot analyses were completed while biochemical analyses are ongoing. oleocanthal is an important secondary metabolite that has been reported to be useful against important human diseases including cancer. the aim of this study was to identify and characterize the key biochemical and genetic components of oleocanthal biosynthesis. to determine the biochemical components of the pathway, multiple olive cultivars along with their spatial and temporal points were determined. the expression levels of multiple candidate genes were also aimed via real-time pcr. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes with that of other organisms) were conducted on ncbi web page. phylogenetic tree construction, amino acid composition analysis, nucleotide composition analysis, hydropathy analysis and translations through expasy were conducted. primer was used to design forward and reverse primers to amplify the target genes from different olive tissues at different times. analysis of the first candiate gene with bioedit program revealed that a+t ratio was more than g+c according to the nucleotide composition analysis. according to amino acid composition analysis isoleucine, lysine and leucine were more than other amino acids while kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. abundance of hydrophobic amino acids (leucine and isolecine) along with an abundant hydrophilic amino acid (lysine) suggest the existance of hydrophobic pockets in the protein which may mean a membrane bound protein or a sitoplasmic protein with a strong hydrophobic core. the molecular weight of the protein was kda with a pi of . . the protein was found to have a signal peptide. according to the sosui gramn , intracellular localization was found to be in the inner membrane. analysis of other candidate genes contiunes. acknowledgements: this study was supported by tubitak with grant number o . key words: olive, olea europaea l., secologanin, polymorphism, allele diversity p- . . - antioxidant potentials of propolis and its bioactive components, and their effects on cyp e gene expression in ht- adenocarcinoma cell line a. altay , d. irtem kartal erzincan € university, erzincan, y€ uz€ unc€ u yil university, van, turkey propolis is a resinous mixture that is collected by honeybees from plants, and is combined with beeswax and secretions from the bee's salivary glands plus some pollen. it is a rich mixture of polyphenols, flavonoid aglycones, phenolic acids and their esters. it has been used in traditional medicine for thousands of years because of these ingredients. propolis, just like honey, has been the subject of many studies due to its antimicrobial, antifungal, antiviral and hepatoprotective activities. the cytochrome p enzymes are involved in phase i xenobiotic and drug metabolism. cyp e is present in high levels in human tumors demonstrated by qrt-pcr and immunohistochemical studies. in this study, propolis collected from datc ßa (mugla, turkey) is extracted with % ethanol. phenolic contents of the propolis were measured by lc-ms/ms. cytotoxic effects of the propolis on ht- human colon adenocarcinoma cell lines were examined via xtt colorimetric cell proliferation assay. effects of propolis extract and its main bioactive component caffeic acid on the expression of phase i detoxification enzyme cyp e in ht- cells were investigated bu using q-rt-pcr technique. ic values for dpph radicals scavenging activities of the extract was calculated as . mg/ml. tpc and tfc were determined as . mg gae/g extract and . mg qe/g extract, respectively. caffeic acid content of the extract was measured as . lg/g extract. ic values for xtt assay in hours incubation with the extract and caffeic acid were evaluated as . mg/ ml and . mg/ml, respectively. propolis extract and its main phenolic content caffeic acid significantly dicreased cyp e mrna expression with . and . fold, respectively in ht- human colorectal adenocarcinoma cells for hours incubation. overall, these results indicate that propolis and/or its components have regulatory activities on cyp e expression and they may have a potential as a therapeutic agent in the treatment of cancer. p- . . - effects of histone h lysine inhibition on gene expression profile in tobacco (nicotiana tabacum l.) differentiation is the characteristic of multicellular organism. cellular dedifferentiation underlies topical issues in biology such as reprogramming in stem cell research, regeneration and nuclear cloning, and has common features in plants and animals. the state of dedifferentiation is evidenced by changes in cell morphology, genome organization and the pattern of gene expression as well as by the capability of plant tissues to differentiate into multiple types of cells depending on the type of stimulus applied. chromatin reorganization appears to be a fundamental theme in cellular dedifferentiation and reentry into the cell cycle both in plants and animals. the chemical modifications of histone proteins which are structural units of the nucleosome, generate 'codes' for the recruitment of proteins or protein complexes that affect chromatin structure and gene expression according to 'histone code' hypothesis. methylation of histone h at lysine residue by specific methyltransferases suv h in humans and kyp/suvh in arabidopsis generates a'code' for the recruitment of hp proteins. in this study, to enhance the dedifferentiation efficiency, chaetocin which inhibits suv h has been used at the germination stage of tobacco seeds in in vitro conditions. our results showed that chaetocin induced callus formation from leaf discs of tobacco in the early stage of the inhibitor application. chaetocin can enhance reprogramming of plant cells in seed development treatment as callus induction. it is known that the formation of callus which is the non-differentiated cell community in plants, is a consequence of the changes in the gene expression profile. it has been found that epigenetic modifications play a crucial role in some of these changes. the definition of the genes related to callus formation by the inhibitation of an epigenetic modification -h k methylation-in tobacco might be used to bear on various dedifferentiation-driven cellular processes. induction of tumor cell death by the use of some phytochemicals that consumed through diet, and derived from medicinal plants opens up new horizons for cancer treatment researches. rheum ribes species, which is studied in this research, is one of the commonly used herbs in pharmacological researches. the high content of phenolic compounds in r. ribes extracts were known to be responsible for the high antioxidant and antibacterial activities. our aim in this study is to assess cytotoxic and apoptotic changes by way of implementing methanol extract of the rheum ribes (root) to the mcf- breast cancer cell line. cytotoxic effect of rheum ribes extract was evaluated by using the xtt ( , -bis( -metoksi- -nitro- -sulfofenil)- h-tetrazolyum) test. in order to determine the dose of ic , plant extracts were applied as time and dose dependent in the range of - ug. in nd hour, the ic value is determined as ug. to examine the apoptotic effects of the extract, total rnas were isolated from dose group and the control cells firstly, then cdnas were synthesized. expression profile of the target genes(caspase- , caspase- , caspase- , caspase- , bax, bcl- , fas) are determined by qpcr. according to the results, when the control group compared with the cells, it was determined that, while . and . -fold respectively increase in the gene expressions of caspase- and caspase- of dose group cells, . -fold decrease in the gene expressions of bcl- . no significant difference was observed in the other genes examined. based on the obtained data, we believe that methanol extract of the rheum ribes induces apoptosis by activating intrinsic pathway. as a result, this plant species can be a new and effective therapeutic candidate for the medical world in search of alternative anti-cancer approaches, and could shed light on the work to be done in this area. p- . . - the effect of ferulic acid and -fluorouracil combination on apoptosis in pc- human prostate cancer cells prostate cancer is quite often seen in industrialized countries and has the second most common death rate due to cancer after lung cancer in men. -fluorouracil ( -fu) is a pyrimidine analog and cell cycle-targeting drug by inhibiting dna synthesis. it has been widely used for treatment of several cancers such as gastric, colorectal, and breast cancers. phenolic compounds found in foods are potential antioxidants of harmful oxidative processes related to cancer and also important due to induction of different mechanism such as apoptosis. ferulic acid (fa; -hydroxy- -methoxycinnamic acid), a phenolic compound, is abundant in fruits and vegetables. the purpose of this study was to investigate combination effect of fa and -fu on apoptosis in pc- human prostate cancer cell line. the effects of -fu, fa, and combination of both of them on cell viability were determined by xtt method. total rna isolation was conducted using trizol reagent. expressions of important genes in apoptosis including casp , casp , casp , casp , bcl , bax, fas and cycs were investigated in four groups by qpcr. the ic doses of fa and -fu were found to be lm and lm for hours in pc- cells, respectively. in order to determine combination effect, pc- cells were treated with <ic doses ( lm fa and lm -fu). according to qpcr results, a significant increase in the expressions of bax and cycs genes and a decrease in the expression of bcl were observed in the fa group, compared with the control group cells. after the treatment with -fu, the expression of casp was significantly increased compared with the control group. furthermore, combination of fa and -fu significantly increased expression of casp , casp , fas and cycs genes. in conclusion, comparing with the single treatments, it was observed that combination of fa and -fu affected expression of different genes in apoptosis in pc- cell line. p- . . - combination of ferulic acid and gemcitabine affects expression of some apoptotic genes in pc- human prostate cancer cells prostate cancer, the second causing of cancer-related death in men, is an important cancer type due to the late age of onset, slow the progression, high incidence. gemcitabine is an effective agent in several cancer types including non-small cell lung cancer, pancreatic, bladder and breast cancer. it is known that gemcitabine is used single agent or as a part of combination treatment in prostate cancer therapy. nowadays, new treatment methods have been tested for cancer therapy including natural compounds with low toxicity. ferulic acid (fa) one of these agents, an abundant phenolic compound found in various fruit and vegetables. in this study, objective was to investigate combination effect of ferulic acid and gemcitabine on apoptosis in pc- human prostate cancer cell line. cell viability was determined by using xtt method after the treatment with gemcitabine, fa and combination of both of them. total rna was isolated with trizol reagent. expressions of casp , casp , casp , casp , bcl , bax, fas and cycs genes are important in apoptosis, were evaluated in four groups by qpcr. the ic doses of fa and gemcitabine were found to be lm and lm for hours in pc- cells, respectively. for determination of combination effect, pc- cells were treated with <ic doses ( lm fa and lm gemcitabine). when compared with the control group, qpcr results illustrated, a significant increase in the expressions of bax and cycs genes whereas a decrease in the expression of bcl gene in the fa treatment group. after the treatment with gemcitabine, the expression of casp and fas genes were significantly increased compared with the control group. furthermore, combination of fa and gemcitabine significantly increased expression of casp , casp , casp , fas and cycs genes. in conclusion, it was observed that combination of fa and gemcitabine affected different genes in apoptosis compared with the single treatments in pc- human prostate cancer cell line. pistacia lentiscus linn. is widely distributed in mediterranean europe, morocco and turkey. the resin part of its known as mastic gum and plant called as mastic tree. it has been referred to over centuries as having medicinal properties to treat a variety of diseases. the aim of the present study are to evaluate antioxidant, cytotoxic and antimicrobial activities of heptane, chloroform and methanol extracts from p. lentiscus leaves and mastic gum. the antioxidant activities of chloroform and methanol extracts were assayed with various methods, including tpc, dpph free radical and abts radical cation scavenging activity. also, cytotoxicity of extracts was evaluated and screened against human mcf- , hela, a , and ht- cancer cell lines and nih- t cells. the viability of cells in lg/ml concentration of extracts was evaluated using mtt assay. antimicrobial activity of extracts were investigated against s aureus, s epidermidis, e coli, p aeruginosa, p vulgaris, k pneumoniae, c albicans, c glabrata, c guilliermondii, c tropicalis, c parapsilosis test organims by the agar well diffusion and broth microdilution methods . according to our results, the methanol extract of p. lentiscus leaves showed the highest dpph free radical scavenging activity (ic = . ae . mg/ml) and abts radical cation scavenging activity ( . ae . mg/ml). this study also exhibited that methanol extract of p. lentiscus leaves had the highest tpc ( . ae . mg gallic acid equivalents/g extract). the hexane extract of mastic gum showed antifungal activity against all of the tested candida species ( - mm / < . - mg/ml). the methanol extracts of p. lentiscus leaves showed the highest antimicrobial activity against all of the tested bacteria except k pneumoniae strain ( - mm/> mg/ml). on the other hand, p. lentiscus showed no cytotoxicity against cancer and normal cells. the results suggested that p. lentiscus may be natural source of antioxidant and antimicrobial activities. p- . . - antibacterial activity of and chemical composition of alcoholic extract of marjoram against some human pathogens m. ahanjan, m. rahbar mazandaran university of medical sciences, sari, iran herbs enjoy a unique value and importance in sustaining healthy communities in terms of disease prevention ( ) . in this regard, marjoram is a plant of the mint family which has antibacterial properties on microorganisms ( ) . the current study aims to investigate the anti-microbial activity of the alcohol extracts (i.e., methanol or ethanol) of marjoram plants on the bacteria of staphylococcus (atcc: ), aureus e. coli (atcc: ), and salmonella enterica (atcc: ) and p. aeroginosa through utilizing disk diffusion method. also, the minimum inhibitory concentration and the minimum bactericidal concentrationwere measured through tube. the measurement of minimum inhibitory concentration and minimum bactericidal concentration of ethanol and methanol extracts on e.coli were equal with and milligrams per milliliter, subsequently. moreover, the measurement of the minimum inhibitory concentration and of the minimum inhibitory concentration of marjoram ethanol extraction on staphylococcus aurous was reported to be milligrams and milligrams per milliliter, subsequently. in addition, the amount of ethanol and methanol extracts on salmonella enteric and p. aeroginosa was equal with and milligrams per milliliter, subsequently. the results showed that marjoram alcoholic extract enjoy antibacterial properties. also, among the alcoholic extracts, the ethanol extract has demonstrated to be the most effective extract on salmonella enterica and e. coli and p. aeroginosa. p- . . - molecular analysis of serine/arginine rich sc splicing factor from olive b. bas , e. d€ undar depertmant of biology, university of balikesir, balikesir, department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey olive (olea europaea l.) is an evergreen fruit tree adapted to mediterranean climate and rich in tannins, essential oils and organic acids. serine/arginine rich splicing factors are essential in seqeunce specific splicing of pre-mrnas. in this study we report molecular characterization of an serine/arginine rich sc splicing factor (oesarsc sf) that was isolated from a cdna library constructed from olive pedicels. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes from other organisms) were conducted on ncbi web page. amino acid composition analysis, nucleotide composition analysis, hydropathy analysis, open reading frame determiantion, through, molecular weight and the isoelectric points calculations were conducted using online expasy software. primer was used to design forward and reverse primers to amplify the target gene from different olive tissues at different times. analysis with bioedit program revealed that a+t ratio was more than that of g+c. oesarsc sf was a protein consisting of amino acids. as expected, amino acid composition analysis revealed that serines and arginines were more than other amino acids. kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. the molecular weight of the protein was kda with an isoelectric point (pi) of . . the protein was found to have a signal peptide. according to the predotar analysis results, intracellular localization was found to be in the mitochondrial. the combined results suggest oesarsc sf might function as splicing factor as its homologs from the other plants. confirming this hypothesis with futher experimental characterization including biochemical function analysis continues. acknowledgements: this study was supported by tub _ itak with grant number o . keywords: olea europaea l., oesarsc sf, alternative splcing, pre-mrna splicing, bioedit, pedicel specific cdnas. p- . . - application of three-phase partitioning for the purification of peroxidase from kiwano (cucumis metuliferus) z. denli , g. arabaci kto karatay university faculty of medicine, konya, faculty of arts and sciences, sakarya university, sakarya, turkey peroxidases are enzymes able to catalyze reduction of h o and oxidize various substrates. the kiwano is an oval shaped fruit which has an orange skin with lots of tiny horns. in this study, peroxidase isolated from kiwano is purified with three-phase partitioning (tpp). kiwano fruit was homogenized and obtained crude enzyme extract. the extract was saturated with % (w/v) ammonium sulfate ((nh ) so ) and added t-butanol with the ratio of : . (v/v). the lower and interfacial layer was collected. the influence of percent saturations of (nh ) so ( , , , , %) and tbutanol ratios ( : . , : , : . , : ) to the partitioning behaviour of peroxidase were analyzed. after dialyzed, the interfacial and lower phases were measured for peroxidase activity and protein content. the protein pattern of the peroxidases was evaluated by using gel electrophoresis. peroxidase activity recovery and the purification fold of interfacial and lower phases were . , . % and . , . . therefore, other experiments were continued with interfacial phase. at constant t-butanol with the ratio of : . , the enzyme activity recovery and purification fold of interfacial phase for saturations of (nh ) so ( , , , %) were . , . , . , % and . , . , . , . . the interfacial phase was not dissolved in % (nh ) so . at constant % (nh ) so , the enzyme activity recovery and purification fold of interfacial phase for t-butanol ratio ( : . , : , : . , : ) were . , . , . , . % and . , . , . , . . finally, at optimum conditions (% (nh ) so , t-butanol : . ) after dialyzed interfacial phase, the enzyme activity recovery and the purification fold were . % and . . the results of gel electrophoresis showed that the molecular weight of enzyme was between - kda. the applications of tpp gave the maximum recovery of . % and . -fold purification. as a result, for purfication of peroxidases, tpp is a rapid, simple and economical technique. accumulating the most robust genes and proteins in elite genotypes without any harmful effect on the potential plant yield is an urgent need to enhance productivity under various stressors. among the stressors, drought is a major challenge for agricultural productivity. brachypodium distachyon, with its close relationship to agriculturally and economically important crops, is an important model plant species. although ongoing transcriptomic analyses in brachypodium distachyon available, proteomic analyses are required to obtain an integrated picture of drought response. in the current study, a comprehensive proteome analysis was conducted on brachypodium leaves under increasing levels of drought stress. to screen gradual changes upon drought stress, brachypodium leaves subjected to drought treatment for , and days were collected for each treatment day. the cellular responses were investigated through a proteomic approach involving two dimensional difference gel electrophoresis and subsequent combined tandem mass spectrometry. for the validation of transcriptional expression, the genes encoding selected proteins were examined through quantitative real-time pcr. spot detection on cye-dyed gels revealed a total of distinct spots in brachypodium protein repertoire. a total of differentially expressed proteins (deps), with at least -fold changes in abundance, were identified by mass spectrometry and classified according to their functions. the biological functions of deps included roles in photosynthesis, protein folding, antioxidant mechanism and metabolic processes, highlighting the significant degree of overlapping between metabolic alterations induced by drought stress. identified proteins in this study and understanding the molecular mechanisms of drought response and defense mechanisms in plants will contribute to the researches on development of drought tolerant crop species. p- . . - immunohistochemical and electron microscopy investigation of tmv-based chimeric virus particles carrying conserved influenza antigen in nicotiana benthamiana recently we obtained and partially characterized set of viral vectors based on tobacco mosaic virus (tmv) genome displaying conserved influenza a m e epitope from matrix m protein. purified chimeric virus particles (cvp) conferred protection of mice against lethal homologous and heterologous influenza virus challenge. we revealed significant difference in symptoms of infections caused by tmv-m e recombinant viruses containing cysteine (cys) to serine (ser) or alanine (ala) substitutions in the human consensus m e sequence. accumulation level of m e-ala recombinant coat protein was significantly higher than m e-cys/ ser (ratio : ). tmv-m e-ser infection, in contrast to ala mutant, suspended growth and development of nicotiana benthamiana. non-inoculated leaves ( d.p.i.) were fixed with ethanol and histological sections were incubated with mouse serum to m e and secondary antibodies conjugated with either hrpo or fitc. cvps of all three mutants were detected in epidermal and stomata cells as well as in sieve elements and minor veins. electron microscopy analysis of mesophyll cells revealed typical rigid helical particles. cys/ser mutants mostly accumulated within ground cytoplasm as aggregates of discrete tubules in parallel arrangement, which were not delimited by lipid membranes. we discovered huge amount of cvps in the cytoplasm and lesser amount diffused in the central vacuole. essential part of ala particles was located in the cytoplasm, but mentioned aggregates were not found and only insignificant number of virions was revealed in vacuole. unlike wild-type tmv, none of the mutants was revealed in chloroplasts. diameters of cvps were as follows: sersingle particles in cytoplasm ae . cyanidin is a natural anthocyanidin found in a variety of fruits (grapes, blackberry, blueberry, cherry and cranberry etc.) and vegetables (red cabbage, red onion). this polyphenolic compound is a flavonoid with significant antioxidant activity. cyanidin and its glycosides have vasoprotective effects and can interfere with inflammation, carcinogenesis, obesity, and diabetes. one important role of the macrophages is the release of pro-inflammatory mediators, such as nitric oxide, various cytokines, in response to activation signals, including chemical mediators, cytokines, and bacterial lipopolysaccharide (lps). in this study, we investigated the role of cyanidin chloride in inflammation. anti-inflammatory effects of cyanidin chloride were examined in lps-stimulated murine raw . macrophages. we observed the level of various inflammation markers such as nitric oxide (no), inducible no synthase (inos), cyclooxygenase- (cox- ), tumor necrosis factor-a (tnf-a) and interleukin- (il- ) under lps treatment with or without cyanidin chloride. cyanidin chloride inhibited not only no production but also the expression of cox- and inos, without any cytotoxicity. cyanidin chloride also attenuated pro-inflammatory cytokines and other inflammation-related markers such as il- in a dosedependent manner. in conclusion, cyanidin chloride may be beneficial for the prevention and treatment of anti-inflammatory diseases. p- . . - the investigation of centranthus longiflorus plant extacts effects on cell proliferation and apoptosis activity in the cell lines of mcf- breast cancer h. askin ataturk university, erzurum, turkey introduction: in the u.s., breast cancer is the second most common cancer in women after skin cancer. current treatment of cancer can be done by surgery, chemotherapy, and radiation therapy. in addition, there is widespread use of complementary and alternative medicine in developed countries. plants and plant extracts play a critical role in the research into new anticancerogenic agents. centranthus longiflorus (cl) is used in alternative medicine for sedative and antispasmodic purposes. a plant of turkish origin, centranthus longiflorus used as traditional turkish medicine have remained uninvestigated for familial hypercholesterolemia, diabetes, coronary artery disease and cancer for their in vitro biological activity despite their use for sleep disorders. in this study, growth-inhibiting and pro-apoptotic effects of hexane, ethyl acetate and ethanol extracts of cl in mcf- breast cancer cell line were investigated. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from cl was analyzed using a gc-ms system. dose-and time-dependent cytotoxic and apoptotic effects of cl were evaluated by mtt cell proliferation kit and cell death detection elisa kit, respectively. manufecturer's protocol was followed for analyses. then, apoptotic genes; caspase , bax and p and antiapoptotic genes; bcl- and pi expression levels were determined by rt pcr. results: according to our results, cytotoxic effect on mcf- cell was only observed in and lg/ml doses of cl. however, any of the application doses showed an apoptotic effect on mcf- cells. they exhibited a necrotic effect rather than the apoptotic effect. although alterations in expression levels of these genes were determined, this alterations was statistically insignificant. discussion and conclusion: consequently, we can say that cl have a cytotoxic effect on mcf- breast cancer cell lines. p- . . - reduction of the chloroplast genome and the loss of photosynthetic pathways in the mycoheterotrophic plant monotropa hypopitys, as revealed by genome and transcriptome sequencing e. gruzdev, a. mardanov, a. beletsky, v. kadnikov, e. kochieva, n. ravin, k. skryabin institute of bioengineering, research center of biotechnology of the russian academy of sciences, moscow, russia genomes of parasitic plants represent interesting model systems to study effects of relaxed selective pressure on photosynthetic function. previous genomic studies of nonphotosynthetic plants revealed reduction of their chloroplast genomes, but the corresponding changes in their nuclear genomes are less known. here we present the data on the transcriptome and the chloroplast genome of the non-photosynthetic mycoheterotrophic plant monotropa hypopitys. the chloroplast genomes were sequenced for two specimens of m. hypopitys, collected in different regions of russia. the cpdnas are , bp (mon- kalr) and , bp (mon- volr) long and rearranged with respect to each other. both genomes contains genes encoding ribosomal proteins, infa, matk, and ribosomal rna genes. and trna genes were predicted in two cpdna. genes encoding nadh dehydrogenase, plastid rna polymerase, all genes related to photosynthetic apparatus, clpp, ycf , ycf , accd, and some genes for ribosomal proteins are missing or became pseudogenes. the reduction of gene content is associated with extensive gene order rearrangement and the lack of inverted repeats. overall, the size and gene content of m. hypopitys cpdna indicates that it is close to the end of plastid genome degradation process. in order to get insights into the changes in the nuclear genome associated with the transition to nonphotosynthetic lifestyle, we sequenced and assembled the transcriptome of m. hypopitys. as expected for holoparasites, we did not found transcripts for the nuclear genes encoding the components of photosynthetic machinery, including photosystem i and ii, cytochrome b f complex, and ribulose bisphosphate carboxylase. contrary to the holoparasitic plant phelipanche aegyptiaca, almost all genes of chlorophyll biosynthesis pathway from protoporphyrin ix were not found in the m. hypopitys transcriptome. this work was supported by the rsf grant - - and rfbr grant - - (mon- volr cpdna sequencing). introduction: beta-sitosterol is a substance found in plants. chemists call it a plant sterol ester. it is found in fruits, vegetables, nuts, and seeds. it is used to make medicine. beta-sitosterol is used for heart disease and high cholesterol. the federal food and drug administration (fda) allows manufacturers to claim that foods containing plant sterol esters such as beta-sitosterol are for reducing the risk of coronary heart disease (chd). centranthus longiflorus (cl) is used in alternative medicine for sleep disorders. a plant of turkish origin, cl used as folk medicine have remained uninvestigated for familial hypercholesterolemia, coronary artery disease and preventing colon cancer for their in vitro biological activity despite their use for sleep disorders. we investigated of the chemical constituents from dried aerial parts of centranthus longiflorus. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from the aerial parts of cl was analyzed using a perkin-elmer gc-ms system. results: ten compounds were obtained and identified as butanoic acid, hexadecanoic acid (palmitic acid), -methyl-z-tetradecen- -ol acetate, octadecanoic acid (stearic acid), diisobutyl phthalate, -octadecenamide, octacosane, nonacosane, alfa amyrin and beta sitosterol. the latter two were obtained in all extraction (hexane, ethyl acetate and ethanol). discussion and conclusion: all of these compounds are isolated from centranthus longiflorus for the first time. these findings may shed light on the design of new drugs, the cholesterollowering effect. p- . . - role of lutein for the high light-induced inhibition of photosystem ii related reactions in thylakoid membranes of arabidopsis thaliana, wt and lut k. dobrev, d. stanoeva, m. velichkova, a. v. popova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthetic reactions taking place in thylakoid membranes of higher plants are extremely sensitive towards different environmental stress conditions such as high and low temperature, high light intensity, uv radiation etc. carotenoids are intrinsic component of photosynthetic pigment-protein complexes and are involved in performing multiple important functions. their role of accessory pigments in absorbing sun light, participation in photoprotection via dissipation of excess absorbed light, deactivating of stress-induced reactive oxygen species and structural role are well documented and recognized. the role of lack of lutein in high light-induced alterations in structural organization and functional activity of the main pigment-protein complexes was evaluated using isolated thylakoid membranes of arabidopsis thaliana, wt and mutant lut , deficient in lutein, subjected to photoinhibitory treatment for different periods of time. alterations in photochemical activity of photosystem i and photosystem ii were determined by a clark-type electrode in the presence of exogenous electron donors and acceptors. activity of oxygenevolving complex and of the grana and stroma situated photosystem ii reaction centers was evaluated by determination of flash oxygen yields and initial oxygen burst under constant light without donors and acceptors. low-temperature ( k) fluorescence was applied for unraveling of light-induced alterations in energy transfer and interaction between the main pigment-protein complexes. maximal quantum efficiency of psii was registered by pulse amplitude modulated fluorescence method. results obtained are discussed in respect to the importance of lutein for the organization and sensitivity of photosynthetic apparatus towards high light intensity treatment. modern agriculture relies heavily on phosphate rock fertilizer to improve phosphorus availability in many soils, but this approach is not sustainable long-term. phytate (myo-inositol hexakisphosphate) is an organic phosphorus compound often present in many soils. however, phytate can not be utilized by most plants, and its accumulation in soil leads to substantial ecological problems. phytases are enzymes that hydrolyze phytate and release inorganic phosphate. many microorganisms such as bacteria and fungi synthesize highly diverse phytases which are suitable for plant biotechnology. generation of transgenic plants expressing phytases of bacterial origin has been proposed as one option to improve plant phosphorus nutrition. in this study, we generated and characterized transgenic arabidopsis thaliana plants expressing a modified phytase gene paphyc from pantoea sp. under strong camv s promoter. three individual transgenic a. thaliana lines expressing the bacterial phytase gene, as well as negative control plants harboring the camv s promoter alone were identified. expression of phytase in plants was verified at both transcription and translation levels. phytase-expressing plants grown on media with phytate as the sole source of phosphorus demonstrated better than wild-type growth rates, shoot dry mass, shoot phosphorus content, as well as higher phytase activity in cell-wall extracts. overall, we show that plants expressing bacterial phytase are capable of better growth on phytate as the only source of phosphorus in laboratory conditions. further research investigating the applicability of using bacterial phytase expression to improve plant growth in soil is necessary to evaluate the different routes of solving the phosphorus deficiency problem in agriculture. p- . . - the role of elevated temperature in photoinhibition and recovery of photosystem ii in thylakoid membranes from arabidopsis thaliana a. faik, m. gerganova, m. velitchkova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthesis of higher plants is the principle process to transform light energy into biochemical usable energy. in nature, plants are exposed to the environment where light, temperature, uv-b radiation varied and very often their extreme values that are unfavorable for effective performance of photosynthetic reactions. plant are developed various strategies to cope with stress including radical scavenging enzyme system, accumulation of protective compounds, etc. pigment-protein complexes of photosystem i and photosystem ii and their light harvesting antenna, situated within thylakoid membranes, are involved in the primary reactions of photosynthesis -absorption of light, charge separation and electron transport. photosynthetic process is sensitive towards higher than optimal temperatures, the photosystem ii and oxygen evolving complexes being extremely sensitive to elevation of temperature. in present work pam fluorescence was applied to evaluate the effect of long term action of elevated temperature ( / °c) on the quantum yield of photosystem ii, non-photochemical quenching and rdf, the latter quantifying the photosynthetic process. in addition, the activity of oxygen evolving complex was determined polarographically in the presence of exogenous electron acceptor , -benzoquinone. sds-page electrophoresis and western blot were applied to determine the damage of d -reaction center protein of photosystem ii. alterations of mutual organization within photosystem ii complex and its antenna and of energy interaction between them were followed by analysis of k steady state chlorophyll fluorescence spectra. the simultaneous application of high temperature and high light intensity resulted in a well pronounced reduction of non-photochemical quenching that restore to the initial values after recovery for days at optimal conditions. d was also restored while quantum efficiency of photosystem ii did not recuperate to initial values. p- . . - reorganization of the main pigment-protein complexes in thylakoid membranes from tomato (solanum lycopersicum) during long term exposure to elevated temperature the changes of earth climate resulted in unfavorable environment for plants. depending on the duration of influence of stress factors, the response of plants includes short and long term acclimation. the population, structure and organization of pigmentprotein complexes within thylakoid membranes are dynamic and flexible, thus providing for the acclimation of the photosynthetic apparatus to the changed environment. the main pigment-protein complexes, involved in energy transduction, are photosystem i, photosystem ii and light harvesting complexes. they are separated in grana and stroma regions of thylakoid membranes but it is well established that they can rearrange as a result of alterations of light intensity, temperature increase and decrease in order to balance the perception and utilization of excitation energy. in present work the effect of long term action of elevated temperature on organization and stoichiometry of main pigmentprotein complexes in the thylakoid membranes from tomato plants (solanum lycopersicum cv. m ) was investigated. three weeks old tomato grown at optimal conditions ( / °c day/ night temperature and light intensity lmol/m /s) plants were exposed for and days to elevated temperature at / °c. by means of blue-native electrophoresis the effect elevated temperature on the populations of psii (dimmer and monomers) and lhcii (monomers and trimers) was estimated and compared with the same parameters for control plants. the ability of plants to recover from this treatment was checked after days under optimal conditions. the changes of content of chlorophylls and carotenoids were determined at every stage of treatment. based on the results obtained it can be concluded that one of the mechanisms for regulating the energy balance and maintenance of efficient photosynthetic process involves a change in the organization and stoichiometry of the photosystem ii and oligomer state of light harvesting complex ii. aim of the study, was to evaluate the anti-tumor effects of silymarin, curcumin and propolis on leptin-induced mcf- cells. mcf- cells were incubated various concentrations leptin (physiological, obesity and pharmacologically doses; respectively , and ng/ml) . then different doses of silymarin ( , , , lm), curcumin ( , , , lm) and propolis ( . , . , . , . mg/ml) were added. after , , and hours incubation periods different area images were taken digital camera. then using dye release reagent we determined the intensity of apoptosis via colorimetric determination by elisa reader. absorbance was directly proportional the number of apoptotic cells (biocolor cell-apo percentageapoptosisassay). also, we examined the effect of these natural products on proliferation rate of leptin-induced mcf- cells for , , and hours (biovision cell proliferation kit) all experiments were carried out different days, at least times. all of three compounds were stimulate the apoptosis at all time points and all different doses of leptin. the differences was statistically significant at the level of p < . between and hours. it was found that there were not seen much cells at and hours time points. we thought that most of the cells were gone necrosis instead of apoptosis. the best effective doses on apoptosis of propolis was . mg/ml, silymarin and curcumin were lm. also, we evaluated the effects of on proliferation rate the mcf- cells, we found that only propolis was effective of inhibiting proliferation at all doses of leptin induced mcf- cells in hour. we hope this study will be a guide for the further studies in anti-cancer agent development field and show that the natural origin substances cause cancer cells apoptosis and provide targeted treatment for cancer therapy. p- . . - investigation of some lichen-derived substances' cosmetic potential for skin protection against ultraviolet b due to the depletion of the stratospheric ozone layer and chronic exposure, the occurrence of various skin diseases have been increased in recent decades. thence, people and cosmetic companies have progressively given more importance natural sunscreen products for the protection from harmful sun rays, especially ultraviolet b rays. we, therefore, isolated some lichen-derived substances; -hydroxyphysodic acid and protolichesterinic acid from hypogymnia tubulosa and cetraria aculeate, respectively. chemical characterization and identification of the isolated lichen substances were accomplished by using ftir, h-nmr and melting point analyses. the theoretical uv-vis spectra and d conformations of the isolated compounds were determined by using the gaussian software with hf theory at the b lyp/ - g level. the dark toxicities and ultraviolet b protection capacities of the substances were lighted up as previously described [ , ] on hacat human keratinocyte cell line by using mtt cell viability and ldh cellular membrane degradation assays. the obtained results from the assays showed that protolichesterinic acid has a more dark toxic activity on keratinocyte cells than hydroxyphysodic acid, and the toxic activities were found sufficient as much as % at the highest doses of the substances; lm. however, it was observed that the cytotoxicity of the substances were reduced at the rate of approximately % by the irradiation. consequently, we think that the substances block the ultraviolet b rays but their cytotoxic feature is an important limitation to their usage in cosmetic industry. references [ ] varol, m., t€ urk, a., candan, m., tay, t., koparal, a. t. ( ) photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes, phytotheraphy research : - . [ ] varol, m., tay, t., candan, m., t€ urk, a., koparal, a. t. ( ) a great effort and financial supports have been consumed to explore and design novel sun protection factors due to the unhindered increase of malignant and non-malignant skin diseases caused by the chronic exposure and depletion of the stratospheric ozone layer. the testing of naturally produced compounds seems to be the best and inexpensive way to search for potentially photoprotective substances. on the other hand, as photo-resistant species, lichens are still poorly exploited. norstictic acid was, therefore, isolated from the acetone extract of pleurosticta acetabulum. ftir, h-nmr and melting point analyses were performed to identify the chemical features of norstictic acid. gaussian software with hf theory at the b lyp/ - g level was also performed to determine the theoretical uv-vis spectrum and d conformation of the isolated compound. the dark cytotoxicity and ultraviolet b-protection capacity of norstictic acid were comparatively tested as previously described [ , ] by using mtt cell viability and ldh cellular membrane degradation assays. as a result of the experiments, it is observed that norstictic acid has a dark-cytotoxicity as less as % at the highest dose of the substance; lm. however, ultraviolet b-induced damage on human keratinocytes was blocked by the lower concentrations of norstictic acid as , and -lm, and % of cells were protected according to the control experiments of irradiated cells. consequently, we think that norstictic acid might be employed as a sun protection factor at the low concentrations, and further studies should be performed. years, researchers have focused on the lichen acids because of their biological activities. it is also suggested that lichens can be used as anticancer agents. vulpinic acid, an important lichen seconder metabolite, has antimicrobial activity and strong antimutagenic, anticancer and antioxidant capacity. nanotechnology has the potential to offer solutions to current obstacles in cancer therapies, because of its unique size ( - nm) and large surfaceto-volume ratios. so, in this study we aimed to determine the cytotoxic and proliferative effects of vulpinic acid and magnetic nanoparticles loaded with vulpinic acid (fe there are four species of wild rice known around the world. zizania aquatica l., zizania palustris l. and zizania texana hitche are found in north america, whereas zizania latifolia (griseb) turcz) is native to east asia. cwr mainly grows in the areas along the yangzi river and the huai river in china without any cultivation and domestication. cwr was an ancient grain that has been used in chinese herbal medicine to treat a variety of ailments associated with nutrition, including gastrointestinal disorders and diabetes. our previous studies have demonstrated that consuming chinese wild rice can significantly improve blood lipid profiles and ameliorate high-fat/cholesterol diet-induced insulin resistance. however, compared to the well studied common dietary white rice, active composition and the associated proteomic information of chinese wild rice have yet to be investigated. in this study, we compared and analysed the different proteins between chinese wild rice and white rice by proteomics method. our study provides insights and experimental evidence for further exploration of this ancient medical food in disease prevention and therapy. the homology between cwr and n is %, but significant differences also exist between the two. we gained new insight by analyzing the biological function of the high reliability (credibility score or higher, p < . ) peptide mass fingerprint of cwr -de electrophoresis revealed differences in protein composition between cwr and n . information obtained from the pmf indicates that glutelin precursor, caffeoyl coenzyme a (coa) omethyltransferase and putative bithoraxoid-like protein can provide gene evidences for its biological function. p- . . - mir and growth-regulating factors interaction during maize leaf growth under low-temperature stress g. aktug, f. aydinoglu gebze technical university, kocaeli, turkey microrna (mirna) genes are a class of non-coding small rnas about nucleotide-long which are revealed as regulators of plant growth and stress responses. the mirna mir targets and regulates growth-regulating factors (grfs) which are plant specific transcription factors family and this regulation machinery is conserved among plant species. plant growth is a result of cell division and expansion which took place as spatial gradient zones throughout maize leaf which are meristem, elongation and mature zones. cells proliferate in meristem, migrate to elongation zone and finally reach to mature zone to get its final size. it has been shown that mir affects cell division by regulating grfs and changes leaf size which are determined by cell number and cell size of leaf. this study aims to investigate the role of mir and grfs interaction during maize leaf growth under low-temperature stress. maize seedlings were grown under low-night temperature for stress treatment to generate growth retardation and control conditions as well to make comparative analysis. length of the third and fourth leaves of seedlings was measured every day and leaf elongation rate was calculated to observe stress effects on the leaves. growth zones of fourth leaves were harvested during steady-state growth phase for determining expression level of mir s and their target by q-rt-pcr. we mined mir genes sharing sequence similarity and grf targets. the expression analyses of mir s and grf are proceeding for different growth zones. in conclusion, this is the first study investigating the regulation network between mir s and grf in different developmental stages of maize leaf under low-temperature stress. oeigpd cdna was isolated from a cdna library we constructed from olive pedicels. homology searches for nucleotide, amino acids and alternative open reading frames were conducted utilizing blastn, blastp, and blastx, respectively. nucleotide sequences of homologous genes from other plants were aligned using bioedit and the number of snps were detected. the alignment was then used to generate a phylogenetic tree using mega program. another alignment with amino acid sequences of the homologues proteins was also generated to construct a phylogenetic tree displaying oeigpd's position among other plants. various aspects of oeigpd including amino acid composition, hydropathy analysis, isoelectric point (pi) and three dimentional structure of the protein were determined using online software at expasy. multiple primer pairs to amplify the full length open reading frame of the gene, to clone the gene into the expressing vector plate , and to detect expression through real-time pcr were designed using primer . amino acid composition analysis revealed that oeigpd contained serine, arginine and isoleucine predominantly while hydropathy analysis suggested it was an hydrophilic protein. isoelectric point (pi) of the protein was calculated as . . the molecular weight of the protein was calculated as kda. analyses continue to determine the polymorphism of oeigpd among olive cultivars, and biochemical function of the gene in olive. p- . . - cytotoxic effect of fractionated triterpenoid glycosides from holothuria polii in human cancer cells sea cucumbers are the members of class holothuroidea and they have more than described living species all around the world. sea cucumbers secrete special secondary metabolites from their body walls and they are called triterpene glycosides (tggs). in this study, cytotoxic activity of fractionated ttgs from h. polii on different cancer cell lines were carried out. h. polii delle chiaje, samples were collected from dikili-_ izmir. the semipurified extracts were fractioned by using hplc. four different fractions (fraction a-d) were obtained. in order to characterize the fractions, maldi-tof/ms was used. the cytotoxic activity of the fraction a-d were tested on ht- , t and upci-scc- cell lines by using xcelligence rtca sp system. the cells were treated with three different concentrations of the fractions for hours. the cell index data were compared with the control group. ic values of the fractions for three cell lines were calculated. according to the results, the fractions have holothurin a ( . m/z), -dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer ( . m/z). the fraction d was the most effective on all cell lines with ic value of . ae . mg/l, . ae . mg/l and . ae . mg/l for ht- , upci-scc- and t , respectively. in conclusion, sea cucumber ttgs are promising agents for colon adenocarcinoma, oral squamous cell carcinoma and colorectal carcinoma (metastatic) treatment. p- . . - effect of horse-chestnut (aesculus hippocastanum) seed extract on matrix metalloproteinases during diabetic wound healing impaired wound-healing in diabetics is a major public health problem. the expression and activation of matrix metalloproteinases (mmps) are also impaired in diabetic wounds according to previous studies. their main function is to degrade the various components of the extracellular matrix. also, they participate physiological processes such as inflammation, angiogenesis, tissue remodeling. horse-chestnut seeds (hc) are rich in saponins and flavonoids. it has been shown that hc has antiinflammatory, antioedema, vessel protective, and free radical scavenging properties. the aim of this study is to determine with molecular signs on cutaneous wound healing effects of the ethanol ( %) extract of hc (hce) seed in rats by excision wound model. this study was conducted on diabetic wistar albino rats, which were injected by a single dose ( mg/kg i.p.) streptozotocin. diabetic treatment rats were applied topically % (w/w) ointment with hce and control rats were applied topically simple ointment, once a day during the experimental period. the gene expression levels of mmp- , mmp- by qpcr and levels of nitric oxide (no), hydroxyproline and malondialdehyde in wound tissue investigated at the end of rd, th, and th days. wound closure was also measured. the hydroxyproline and no levels were significantly increased in the hce treated group versus control after the rd and th days. the malondialdehyde levels were significantly lower in the treatment group. mmp gene (associated with collagen processing and reepithelialization) expression levels in hce treated rats were increased in the th day while it was reduced in th day. mmp gene (associated with inflammation and gelatinase) expression levels in hce treated rats were decreased in th, and th days compared to the control. these findings indicate that hce accelerated the cutaneous wound healing process in diabetic rats via mmp and mmp regulation. p- . . - isolation and molecular molecular characterization of vps /vam from olive b. celikkaya, e. dundar molecular biology and genetic at balikesir university, balikesir, turkey vps /vam promotes aggregating and fusing of endosomes and lysosomes. it is a component of a protein complex that is found in vacuole membranes. this gene has been studied from various organisms including humans, drosophila, arabidopsis and rice. no studies on olive vps /vam , however, have been reported. the aim of this study is to report information of olive vps /vam including expression pattern and biochemical characterization. vps /vam was isolated from a cdna library we constructed from fruited olive leaves in july. to determine the putative name of the cdna, blast analyses were conducted for nucleotide, open reading frame and amino acid sequence comparisons. bioedit program was used to determine the nucleotide and amino acid composition along with its molecular weight and isoelectric point (pi). hydropathy analysis was conducted using kyte and doolittle program. phylogenetic analysis was done using mega . cellular localization of the product was predicted using sosui gramn. the three dimentional structure of the protein was calculated using i-tasser and compared to previously known structures using cn d. the blast and bioedit analyses revealed vps /vam had base pairs coding amino acids with a molecular weight of . kda, and pi of . . the at/gc ratio was very high ( . ) comparing to its homologs from other plants suggesting to expect significant differences of this gene's function from the others. amino acid composition analysis revealed high rates of serine, leusine and isoleucine indicating a hydrophobic property of the protein. the hydrophobic feature was confirmed by kyte and doolittle analysis while the cellular location was revealed to be extracellular. the hydrophobic nature despite extracellular location suggests it is a membrane associated protein which was confirmed by transmembrane domain analysis. as expected no signal peptide was detected. the d structure of the protein was similar to its previously reported homologs. p- . . - isolation and characterization of the ribosomal l protein from olive s. cinarli, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey despite as a ribosomal protein, l is known as the inhibitor of the cellular aging gene and it has been reported to have roles in apoptosis. the ribosomal l protein is larger than the lsu of ribosome and contains domains as -layered alpha/beta domain and -layered alpha/beta domain. in ribosomes, it functions in translocation and orientation of trnas. although the ribosomal l (rl ) gene has been studied in many plants, reports on olive rl (oerl ) are very rare. this study presents molecular characterization of rl gene from olive. oerl was isolated from a cdna library we constructed from unfruited olive leaves in july. homology analyses were conducted using blast programs. nucleotide and amino acid compositions, molecular weight, isoelectric point (pi) and at/gc ratio were determined using bioedit and expasy programs. cellular location of the l protein was determined using sosui-gramn program. signal peptide detection, transmembrane domain detection, three dimensional ( d) structure analysis, and phylogenetic analysis were conducted using signalp . , thhmm, i-tasser/cn d and mega , respectively. oel was found to have an open reading frame of base pairs coding amino acids that constitutes a molecular weight of . kda and a high pi of . . lysine, leucine and valine had higher rates. the hydrophilic nature suggested by kyte and doolittle analysis despite high rates of leucine and valine suggests an amphipathic nature of the protein that can bind to both hydrophilic and hydrophobic proteins and / or function in both media. a . at/gc rate is significant comparing to that of its homologs from other plants. sitoplasmic location predicted by sosui-grann is in agreement with the hypothesis suggesting an amphyphatic nature for oerl . likewise, no signal peptide was detected and it was predicted to have at least one transmembrane domain. further characterization of oerl with respect to expression pattern and biochemical function continues. p- . . - isolation and characterization of an olive splicing factor b subunit m. nurcin, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey splicing factor b subunit (sf b ) functions in the regulation of translation and gene expression. sf b forms u small nuclear ribonucleoprotein complex (u snrnp). splicing factors a and b binds pre-mrna at the site of the intron branching point. this binding joins u snrnp to pre-mrna. although sf b from various plants have been widely studied, no studies on olive sf b (oesf b ) have been reported. this study reports information on various aspects of oesf b . oesf b was obtained from a cdna library we constructed from fruited olive leaves in december. it was putatively identified as a splicing factor using blastn, blastp and blastx. to determine wheter oesfb was a sitoplasmic protein, sosui gramn was used. tmhmm was used to detect any transmembrane domains while signal peptide analysis was conducted by signalp. i-tasser and cn d were used to generate the calculated d structure and to compare it with experimentally generated models, respectively. nucleotide and amino acid compositions along with the calculated molecular weight and isoelectric point (pi) were analyzed using bioedit and online expasy software. the phylogenetic trees revealed genetic relationship of olive among other plants based on oesfb . the orf contained nucleotides coding amino acids that produce a . kda peptide with a pi of . . alanine, valine and leucine were found at high ratios suggesting a hydrophobicity which was also predicted by kyte and doolittle analysis. the at rich property of oesf b is not unusual comparing to most plant genes. cellular localization of the gene was suggested to be in mitochondria with no signal peptide indicating oesf b could be synthesizing in mitochondria. the predicted d structure of oesf b was similar to experimentally produced structures while some hydrophobic pockets were predicted. further characterization of the gene with respect to temporal and spatial expression pattern and biochemical function continues. p- . . - kafirin profile of turkish originated sorghum populations r. temizg€ ul, s. yilmaz, m. kaplan, t. akar department of biology, faculty of science, erciyes university, kayseri, turkey sorghum bicolor l. is the fifth important crop in the world with its high photosynthetic activity and resistance to unfavourable conditions as high temperature, drought, salt, and ph changes. sorghum has attracted great interest due to its intensive usage both as human and animal nutrition, and contribution to resistance against many diseases. some proteins of sorghum exerts reducing effect on nutrient digestion through making connetions with other proteins and/or carbohydrates. kafirin proteins have the highest proportion in grain with a range of - %. they are grouped into a ( - kda), b ( kda), c ( kda) and d ( kda) subunits depending on molecular weight, solubility and structure. in the current study, kafirin proteins from turkey originated sorghum populations were acquired through sequential extraction; first, non-prolamines were removed through application of % naci concentration, and second, kafirins were obtained using tertiary butanol ( %) and reducing agents. sds-page was conducted for seperating and visualising the subunits of kafirins. the a, b, c, and d subunits of populations were respectively estimated as , , , and %. of the total proteins, % was identified as a, % b, % c, % d, and % non-prolamines. non-prolamin group of proteins were visualised as different bands ranging from . to kda. c and b group of proteins were only viewed when treated with reducing agents as -me and dtt suggesting that they are connected with complex cross-links. however, a group of proteins visualized without using these agents due to not having intra molecular disulphide bridges and inter molecular cross-links. non prolamins, except for . , . , . , . and . kda, were able visualised in the presence of reducing agents. transcriptomic analysis of the genes encoding analysed proteins needs to be elucidated for better understanding of the genetic diversity and biochemical characteristics of sorghum. p- . . - untargeted metabolomic profiling of romanian and uk tomatoes varieties by high performance liquid chromatography coupled with mass spectrometry c. socaciu , university of agricultural sciences and veterinary medicine, cluj-napoca, center for applied biotechnology ccd-biodiatech at proplanta ltd, cluj-napoca, romania tomato flesh is a rich source of many phytochemicals of high nutritional value, including a large variety of carotenoid derivatives with health promoting properties. metabolomics became the most adequate technology for an accurate chemotaxonomic classification and discriminations between different varieties, based on untargeted profiling or targeted, quantitative analysis. different varieties of tomatoes (b-carotene-rich, lycopene-rich, ketocarotenoid-rich) cultivated in romania and uk were comparatively studied using enriched fractions obtained by a preliminary fractionation of the whole pulp homogenate. two methods were applied for carotenoid extraction: a mixture of hexane/ethanol ( ) and chloroform/methanol ( ) . the dried extracts were dissolved in ethyl acetate and analyzed by uv-vis spectrometry and hplc-esi(+)qtof-ms (bruker gmbh). the base-peak chromatograms were processed by specific biostatistics software (data analysis and profile analysis) and the molecular identification were determined by comparison with the data base lipidomics gateway (www.lipidmaps.org). the content of carotenoids were significantly higher using extraction ( ) , ranging from . to . mg/ g. the major carotenoid derivatives, were represented by lycopene, hidroxy-lycopene, all-trans or cis-beta-carotene, echinenone, all-trans retinyl palmitate, but also sterols, phospholipids, di/tri glycerides and ceramides. the romanian varieties were more rich in polar carotenoids and lipids, in general, while the uk tomatoes proved to be enriched in non-polar derivatives, especially esterified carotenoids, keto-carotenoids and glycerides. new molecules were identified, as good discriminatory markers of each tomato variety. acknowledgements. this work was supported by a grant of the romanian national authority for scientific research and innovation, cccdi -uefiscdi, project nr. / , pncdi . glycogen is a multi-branched polysaccharide that serves as the main form of glucose storage in the body, where the main reserves are in the liver and muscle. it has been observed that glycogen metabolism is altered in many tumor types, and that glycogen content is inversely correlated with proliferation rate. in addition, it has recently been described that when glycogen accumulation is forced in glioblastoma u cells in hypoxia, senescence is induced and tumor growth is inhibited in vivo. our laboratory has various animal models with different parts of the glycogen metabolism pathway affected. most notably, we have two animal models lacking glycogen: muscle glycogen synthase (gys ko) and liver glycogen synthase (gys ko) knockout animals. we isolated mouse embryonic fibroblasts (mefs) from gys ko to perform replicative senescence assays. in addition, we induced hepatocellular carcinomas in gys ko animals via n-nitrosodiethylamine (den) injections in order to track tumorigenesis in animals lacking hepatic glycogen. lastly, we performed partial hepatectomies (phx), which involves the resection of two thirds of the liver, on gys kos to evaluate the effect of the lack of glycogen on hepatocyte proliferation. interestingly, we have observed that glycogen levels are increased in human and mouse fibroblasts under replicative senescence, and that mefs depleted of glycogen bypass senescence and immortalize faster than wts. we have also demonstrated that senescence pathways are down regulated in mefs lacking glycogen. furthermore, gys kos treated with den show higher tumor burden and mortality than controls. we also evaluated the effect of glycogen on hepatocyte proliferation after phx. gys ko mice present faster proliferation and liver regeneration rates, when compared to wt counterparts. collectively, our preliminary data suggest that glycogen metabolism plays a crucial role in the regulation of cell cycle in both physiological and pathological states. it is established that pineal is involved in circadian regulation of testosterone secretion from leydig cells. however, the precise routes of this regulatory involvement are still unknown. as cgmp has been also regarded as modulator of steroidogenesis we sought to study the effects of pineal removal on the circadian pattern of cgmp variations and expression of the genes that encode elements of no-cgmp signaling pathway in adult rat leydig cells. the analysis was performed on testicular leydig cells obtained from pinealectomised and shame pinealectomized rats, in six time point during hours. the pinealectomy was confirmed by serum melatonin eia measurement. the androgen levels were measured by ria; cgmp by eia and gene expression was quantified by rq-pcr. all results were analyzed by cosinor method. data revealed circadian transcriptional pattern of nos , nos (genes encoded no producers) and pde a (gene for cgmp remover) in leydig cells from adult rats. pinealectomy significantly increased expression of nos which lost rhythm and increased and delayed amplitude of nos expression. further, pinealectomy initiated cyclic transcription of gucy b and noncyclic transcription of gucy a (genes encoded cgmp producers) and increased mesor and amplitude of pde transcription. the transcription of prkg , the main effector in this signaling pathway was not affected with pineal abolition. additionally, pinealectomy did not influence the circadian transcription profile of coxi or other investigated genes (coxi , nrf , nrf a, pgc a) related to mitochondrial function and biogenesis. finally pinealectomy reversed phase of circadian cgmp oscillation in leydig cells, increased amplitude and slightly advanced peak of serum testosterone oscillation. results suggested pineal influence on circadian rhythm of no-cgmp signaling in leydig cells. further studies based on these data are needed to better understand the relationship between pineal and circadian rhythm of testosterone production. influenza is a contagious respiratory infection caused by a variety of influenza viruses. neuraminidase inhibitors is a new class of antiviral drugs that inhibit influenza viruses. the most popular antiviral agents is oseltamivir, having a commercial name of tamiflu, within anti-influenza antivirals. as well as tamiflu is a member of neuraminidase inhibitor group drug. therefore, this study was performed to determine the effect of tamiflu on cultured human peripheral blood lymphocytes. material and methods: for examining the presence of the indirect mutagenic effect of oseltamivir in iver s fraction mix was used. cells were treated with . , and lg/ml oseltamivir, the tamiflu capsule ingradient, for or hours in the absence or presence of an exogenous metabolic activation system. the test chemical did not demonstrate any genotoxic effect dose-dependently but it showed a weak cytotoxicity on cells in this study. on the other hand, some concentrations of tamiflu induced sce and also decreased significantly the proliferation index (p ˂ . ) in the absence of s mix. result: tamiflu did not induce significant increases of ca or micronucleated cells in vitro in cultured peripheral blood lymphocytes under the treatment conditions used but week sce induction was observed. on the other hand, the weak cytotoxic effects observed disappeared in the cultures treated in presence of the s mix. discuss and conclusion: tamiflu weakly induced sce at the highest concentration with/without added s mix in cultured human peripheral lympocytes. it could be assumed to be a sce inducer. sces can be increased by several agents that attack dna. tamiflu decreased the proliferation index and nuclear division index at some concentrations thus interferring it as being weakly cytotoxic, though this effect disappeared in the presence of s mix applications. this finding is important for showing the inefficiency of tamiflu metabolites on the cell cycle. introduction: chronic renal failure as a result of the progression of diabetic nephropathy is the main cause of mortality in patients with type diabetes. chronic hemodialysis is a life-saving therapy for patients with strong renal disorders. the main goal of hemodialysis is toxins removal from the patient. the monitoring of hemodialysis is the best way for biomedical evaluation of correctness and efficiency of this clinical treatment. according to the published data, the markers of development of diabetes complicated with renal failure are increased levels of glucose, urea and creatinine in the patient blood. today colorimetric and spectrometric methods are most commonly used for determination of the above metabolites in biological samples. however, these methods are complex in application, have low selectivity, and require pretreatment of samples. materials and methods: we propose for levels of glucose, urea and creatinine detection the potentiometric multibiosensor based on ph-sensitive field-effect transistors and immobilized enzymes developed in our laboratory. results: we developed a potentiometric multibiosensor and studied its main analytical characteristics. linear dynamic ranges of determination of substrates were following: . - mm of glucose, . - mm of urea, and . - mm of creatinine. it was shown that the potentiometric multibiosensor had good reproducibility, and its bioselective elements were working independently from each other, because test of substrates cross-selectivity was negative. discussion and conclusion: very sensitive, fast and selective multibiosensor for simultaneous measurement of three metabolites in a single cycle based on ph-sensitive field-effect transistors and immobilized enzymes is developed. the developed potentiometric multibiosensor was verified by quantitative analysis of glucose, urea and creatinine in blood serum of patients with diabetic nephropathy. p- . . - ph-dependent interaction of asymmetrically charged peptides with a protein nanopore over the past two decades, the ability to use natural or artificial nanopores to probe at uni-molecular level the structural and kinetic features of various bio-molecules (peptides, dna, rna) was successfully achieved. the operating principles of the nanopore-based single-molecule technique are simple: the single macromolecule capture, entry and subsequent translocations through a free-standing, voltage-biased nanopore, depend upon the physico-chemical and topological features of the analyte. the concentration, identity volume and charge of the analyte are then deduced from the analysis of the stochastic current blockade events caused by the trafficked analyte across the nanopore. herein, we used the a-hemolysin (a-hl) nanopore and set up an experimental model providing efficient control of a-hl-peptide interactions, in the presence of a ph gradient across the nanopore. for this, we engineered a amino acids long peptide containing a neutral asparagines-containing sequence, flanked by oppositely charged aminoacid patches at the n-(glutamic acids) and c-termini (arginines), whose length was set as to span a single a-hl protein. when the ph of the solution in contact to the a-hl's b-barrel opening is changed from neutral to acidic values, the electrostatic interactions between the protein's mouth and either the n-or c-terminus end of the peptide occurs, and this influences strongly the dynamics of a peptide translocating the nanopore. we further proved that during the same experiment, peptide entry into the nanopore can be set to occur with either n-or c-terminus end head on, by simply changing the sign of the transmembrane potential across the nanopore. nanopores are emerging as a powerful and broadly applicable tool in biophysics, which allows one to study the features of charged macromolecules under confinement. a few noteworthy examples are: determining the electrophoretic mobility, effective charge and diffusion coefficients of charged molecules; exploring the folding and unfolding of peptides and proteins; analyzing biopolymers trafficking, protein transport, dna translocation, rna and dna sensing and sequencing. herein, we employ single molecule analysis techniques using a wild-type ahemolysin (a-hl) protein nanopore to study the capture and translocation behavior of a short cationic peptide ( amino acids in length) at an extremely low ph value. our experiments revealed that an effective absorbing field is created by the electroosmotic flow, against the electrophoretic force, which enables the peptide capture inside the nanopore. furthermore, our findings show that the trajectory of a single peptide can be experimentally visualized and the main steps determined: the peptide capture, reversible translocation across the pore's vestibule and lumen regions, and the peptide release from the nanopore. also, the kinetic analysis of the main steps observed allowed us to describe the free energy profile of the peptide interactions with the protein nanopore. the presented work provides evidence for the ability of controlling the dynamics of a single-peptide, its capture and passage inside a a-hl nanopore, that underlie the processes naturally occurring in cells, thus proving a powerful approach for probing single molecule biophysics phenomena, in general. changes in the physical conditions of the cancer microenvironment driven by elevated tissue growth and angiogenesis, may introduce exposure of laminar fluid flow, which effect the key factors of cancer, such as progression, immune-escaping and metastasis. conventional experimental models fail to mimic the physical cues on tumor microenvironment. microfluidic culture techniques allow precise control of fluids, simultaneous manipulation and analysis of cultured cancer cells. here, we present a platform that can be used for the investigation of the role of flow mediated mechanical stimuli on cancer cells. microfluidic cell culture platform was fabricated using polymethyl methacrylate and double-sided adhesive films with mm dimensions. ovary adenocarcinoma cells (efo- and onco-dg- ) were used for the optimization of the platform. to understand the fluid and gas distribution patterns, specific modeling was performed. dynamic microfluidic cell culture and static conventional cell culture conditions were compared for the differences of cancer cell phenotype, such as proliferation, viability, epithelial-mesenchymal transition. we confirmed that, the proliferation and viability of cancer cells are increasing under dynamic fluid flow. the proliferation rate of ovary adenocarcinoma cells was correlated with the increase of fluid flow rate. immunocytochemical analysis showed that fluid flow causes decrease in e-cadherin expression, and increase in n-cadherin and vimentin expressions, which indicate mesenchymal phenotype of cancer cells. our results showed that, cancer cells present different characteristics due to fluid flow of tumor microenvironment. to understand the role of physical dynamics by using microfluidic culture techniques, is a key to elucidate the mechanisms underlying disease progression, and may lead to new diagnostics and therapeutic approaches. (this study was funded by turkish scientific and technical research council (tubitak- s ). high-sensitive detection of low-affinity antibodies by immuno-pcr with supramolecular olygonucleotide-streptavidin complex detection of low affinity antibodies in blood sera and cell surface outwashes is important both in the study of molecules that bind to cellular receptors (circulating tumor cell masking antibody, for example) and medicine (diagnosis of allergy). low affinity igm and ige antibodies can not sometimes be determined by conventional methods. we using supramolecular oligonucleotide-streptavidin complex formed from single-stranded synthetic oligonucleotide ( n) contains biotin on '-and '-ends, and sterptavidin in molar ratio : . this complex represents a structure with equivalent electrophoretic mobility of bp dna and preferred "valency" of streptavidin is . this universal immuno-pcr approach make it possible to increase a signal by using several oligonucleotides per one antibody. after the method optimization we achieved - times highter sensitivity than elisa. to reduce the matrix effect we used - fold dilutions of sera samples. this approach achieved a significant advantage, because it allows working with small-volume samples (need only mkl of serum sample). antibodies to the disaccharide galb - glcnac (le c ) are typical of the natural antibodies. the igm anti-le c antibodies are found in almost healthy people without the epitope specificity variation. we have shown that the concentration of igm anti-le c antibodies was higher (p ≤ . ) for health donor sera (n = ; . ae . pg/ml) compared with sera from patients with breast cancer (n = ; . ae . pg/ml). sensitivity of igm anti-le c antibodies detection was pg/sample ( mkl) ie . molecules. thus for the immuno-pcr detection of antibodies the - tumor cells are sufficient. such amount of cells seems to be a realistic one for detection of antibodies masking circulating tumor cells. this study was supported by a grant from russian science foundation (# - - ) and by russian federation president scholarships donated to d. yu. riazantsev (# sp . . bacterial pathogen detection and identification is of crucial importance for disease diagnosis, bacterial contamination surveys and water quality assessment. we propose herein a novel method for bacterial detection based on the interaction of single gram-negative bacterial cells (i.e.: escherichia coli and pseudomonas aeruginosa) with an ahemolysin (a-hl) protein nanopore embedded in a reconstituted lipid bilayer, at neutral ph. as a consequence of an applied voltage, the negatively charged bacteria suspended in saline buffer solution are electrophoretically driven towards the pore opening, inducing reversible blockages in the ionic current through a-hl. experiments were also performed in the presence of an antimicrobial peptide, cma , as well as in acidic environment. statistical analysis of the frequency and duration of blockage events allowed us to discriminate between the two types of bacteria. the frequency of interactions was higher for escherichia coli with respect to pseudomonas aeruginosa. adsorption of cma peptides on the membrane of bacteria increased the frequency of interactions with the pore, contrary to the expected effect induced by lowering the net surface charge of the cells. in experiments performed at ph = , the frequency of blockage events was found to be two orders of magnitude higher, with longer interaction life-times. the net negative charge ( uncompensated aspartate residues) localized at the entrance of the pore contributes an additional electrostatic repulsion interaction between negatively charged bacterial cells and a-hl. thus, adsorption of cationic peptides at the interface will reduce this repulsive interaction. the same effect was recorded at ph = , when the aspartate residues are partially protonated, confirming our understanding of the previously observed results. this method could be further developed and integrated with other techniques, making nanopore-based systems a fast and reliable bacterial detection and identification tool. this study was performed to analyze the effects of tunicamycin (tm) and taurohyodeoxycholic acid (tudca) on thle- cells. cells were treated with tm to induce endoplasmic reticulum (er) stress and tudca was administered as an er stress inhibitor. cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations of either tudca, tm or both. thle cells were cultured in fibronectin, bovine collagen i and bovine serum albumin coated plates. cell lines were grown in begm media supplemented with epidermal growth factor, phosphoethanolamine, fetal bovine serum, u of penicillinstreptomycin and maintained in a humidified incubator at °c and a % co atmosphere. cell viability was measured using the colorimetric -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) assay kit. cells were grown to confluence in well plates and incubated with ll/ml dmso, - lg/ml tm, . - mm tudca, or lg/ml tm + . - mm tudca for - hours. control cells were prepared in plates containing only medium. at the end of the incubation period, mtt was added to each well and incubation was carried out for hours at °c. formazan production was expressed as a percentage of the values obtained from control cells. at all hours of incubation neither dmso or mm tudca was cytotoxic. at and hours incubations mm tudca and lg/ml tm + mm tudca were significantly cytotoxic compared to control, dmso and mm tudca groups. treatment of cells with . mm tudca hours before administrating ug/ml tm significantly decreased the cytotoxic effect of tm. we conclude that tudca may show cytotoxic effects at mm concentration when treated with tm. therefore . mm of tudca, administered hours before tm treatment should be applied to protect against er stress. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; s ). recent studies reveals that history of preeclampsia is an independent risk factor for cardiac events and stroke. lipoprotein-associated phospholipase a (lp-pla ) is a vascular inflammatory marker associated with cardiovascular diseases (cvd). we hypothesize that vascular inflammation (lp-pla mass, activity, index) related genetic variations (pla g ) increase the risk for developing future cardiovascular disease in women with pe. a group of preeclamptic patients and normal pregnant women were recruited from university of istanbul, cerrahpasa medical school, department of gynecology and obstetrics included into the study. the control group was matched for maternal and gestational age at time point of sampling. preeclamptic patients were starified into two groups; early-onset and late-onset according to the gestational weeks. enzyme-linked immunosorbent assay procedure was used to determine the serum lp-pla mass level. lp-pla activity were determined by kinetic method. plag snp genotyping performed by using the sequenom massarray iplex. the rs tt genotype had a higher lp-pla index (p = . ) for early onset preeclampsia, cc genotype had a higher lp-pla mass and lp-pla index for late onset preeclampsia. no difference were found for control. the rs gg genotype had higher lp-pla mass and index for late onset preeclampsia (p = . , p = . respectively). stepwise logistic regression analysis performed to identify cardiovascular disease related variables that independently and significantly contributed to the presence of alleles of rs and rs snps in early, late onset preeclampsia and control group. only lp-pla mass was independently and significantly associated with both snps in early onset preeclampsia. the association between lp-pla mass, index and rs , rs snps might be useful genetic markers to adress future cvd risk in patients with preeclampsia. introduction: b-thalassemia is one of the most monogenic autosomal recessive disorder characterized by defective production of the b-chain of hemoglobin. definition of the b-globin genotype is necessary for genetic counselling in the carriers, and for predicting prognosis and management options in the patients with thalassemia. dna-based diagnosis of b-thalassemias routinely relies on polymerase chain reaction (pcr) and gel electrophoresis. the aim of this study is to develop a new procedure, a dna-based piezoelectric biosensor, for the detection of b-thalassemia ivsi- mutation, the most common b-thalassemia mutation in turkey. materials and methods: b-globin gene of genomic dna isolated from whole blood, was amplified by pcr. bioactive layer was constituted by binding -hidroxymetacrilate metacriloamidoscystein (hema-mac) nanoploymers on the gold electrode's surface. single oligonucleotide probes specific for ivsi- mutation of b-thalassemia were attached to the nanopolymer via reactive cross-linker glutaraldehyde. the measurements were executed by piezoelectric resonance frequency which is caused by binding of pcr products in media with single oligonucleotide probe on the electrode surface. the results were confirmed by the conventional molecular method as arms. results: the piezoelectric resonance frequencies obtained by hybridization of the pcr products on bioactive layer were found ae , ae , and ae hz for the samples of normal b-globin, heterozygote, and homozygote of ivsi- mutation, respectively. discuss and conclusion: the developed biosensor serves as a specific result to ivsi- mutation. it could accurately discriminate between normal and ivsi- mutation samples. because of low costs, fast results, specificity and high detection/information effectiveness as compared with conventional methods, we can be offered this techique as an alternative to conventional molecular methods. the increasing use of nano-sized materials in the last several years has compelled the scientific community to investigate the potential hazards of these unique and useful materials. one of the most widely used nanoparticles is titanium dioxide. the objective of the research is to investigate the alterations in molecular and cellular responses in culture of primary lymphocytes to tio nps. human lymphocytes isolated from heparinized blood of healthy individuals were exposed to tio nanoparticles. viability, ros generation, the changes in the expression of genes encoding proinflammatory mediators tnf-a, il- b and il- and dna damage were assessed. human lymphocytes were incubated with nanoparticles of different concentrations and viability was determined in and hours after treatment, respectively. cell viability was decreased by a treatment with nanoparticles in both a time-and concentration-dependent manners. the ability of tio to induce ros formation in lymphocytes was evaluated using dcf fluorescence as a reporter of oxidant production. the fluorescence intensity of oxidized dcf was increased in cells treated with nps. this means that ros generation occurred in response to the treatment with tio . to investigate the expression level of mrna related to the inflammation responses in human lymphocytes real-time pcr was performed. the expression of il- b, il- and tnf-a genes were increased by the exposure to nanoparticles of , and lg/ml for - hours. tio nanoparticles were shown to induce the dose-dependent fragmentation of dna strands. much evidence of hazardous health effects of nps has been reported. in this study, viability was reduced under the exposure to tio . oxidative stress was elevated by the treatment with tio nps. oxidative stress can also trigger inflammation signals. induced by exposure to nanoparticles they may cause the translocation to the nucleus of transcription factors, which regulate proinflammatory genes, such as tnf-a, il- b, il- . background: endothelial cells (ec) represent one of the primary targets of the major pro-inflammatory cytokinetumor necrosis factor (tnf). development of the new approaches for the treatment of acute and chronic inflammatory conditions, including the strategies aimed to tnf neutralization, requires the usage of the adequate cellular models closely resembling the properties of the endothelium. the endothelium-derived ea.hy cell line expresses several inflammation and neoangiogenesis markers in response to activation factors however their expression can differ from the patterns demonstrated by primary ec. the aim of the current study was to compare the expression of the known endothelial cellular markers including receptor of vascular endothelial growth factor- (vegfr ) and a v b -integrin on d and d cultures (spheroids) of ea.hy . methods: the ea.hy cell line was used with permission from dr. edgell. the cells were cultivated in the presence of tnf ( ng/ml) or vegf a ( ng/ml) for hours. mrna was isolated using rneasy kit from qiagen and reverse-transcribed with revertaid kit (fermentas). rt-pcr was performed with specific primers. expression of vegfr and a v b -integrin was visualized by confocal microscopy using specific monoclonal antibodies and previously developed fluorescent hybrid proteins. results: the expression of a v b -integrin and vegfr- increased on the d culture compared to d according to confocal microscopy and rt-pcr. the aforecited methods revealed elevated expression of a v b -integrin in the d culture of the ea.hy cell line activated with tnf. also increased expression of vegfr in the d culture activated with vegf a. then by confocal microscopy, we analyzed our fluorescent hybrid proteins that bind a v b -integrin and vegfr on the surface of d and d cultures as well as antibodies with fluorescent label. conclusions: d cultures of the ea.hy cell line represent a promising model for the inflammation studies. tumor necrosis factor (tnf) is a trimeric cytokine associated with the inflammatory response to tissue injury and found to possess a key role in rheumatoid arthritis pathogenesis. spd is a highly toxic recently discovered tnf inhibitor that promotes trimer dissociation and lead to the inactivation of the protein. according to the traditional anti-tnf treatment of ra, we aim at extracellular inhibition of this pro inflammatory cytokine as an effective therapy. the project plan comprises design, synthesis and validation of candidate inhibitors (measurement of dissociation constant and aqueous solubility). because of the elevated percentage of insoluble compounds a solubility enhancement protocol has been developed. the experimental procedure was the following:: a. drug design. identification of novel drug compounds are based on two approaches: i) structure based drug design using the d structure of tnf and ii) design of more potent and less toxic spd analogues. b. drug synthesis. a series of spd analogues were in house synthesized while novel candidates discovered by in silico approaches were commercially available. the purity of the majority of the compounds exceeded %. c. solubility measurement and enhancement. samples were incubated under specific conditions that can enhance aqueous solubility and solubility measurement with a direct uv method pursued. d. measurement of the dissociation constant. a fluorescence binding assay was used in order to evaluate the inhibitory activity of the compounds. from our results it can be concluded that dmso, peg and b-cyclodextrin can be used for solubility enhancement without interfering with fluorescence assay. however peg -in contrast to dmso-is not suitable for isothermic titration calorimetry measurements. dissolution procedure also plays a crucial role in the levels of solubility reached. finally, it has been shown that some of the studied spd derivatives have better dissociation constants than spd . the effect of exercises on serum bmp- levels of knee osteoarthritis cytokines. more complicated approaches are expected to focus on molecular proteins as bone morphogenetic proteins (bmps) of the transforming growth factor (tgf)-beta superfamily. bmps associated with many cellular functions, such as proliferation, differentiation, and apoptosis. bmp- is significantly important for the endochondral bone formation. inflamation can induced serum bmp levels in oa patients. the aim of this study is to evaluate the clinical findings of oa patients after the isokinetic exercise together with the serum levels of bmp- to sustain the molecular approaches for treatments. a total of patients were included in this study. the groups are formed as follows: group , oa patients before the exercise; group , oa patients after the exercise; group , oa patients before the isokinetic exercise; group , oa patients after the isokinetic exercise clinical and biochemical findings were evaluated before and after weeks of the exercise programme. self reported severity of pain was measured using the mm visual analog scale (vas), womac scores were calculated and isokinetic knee muscle strength testing was measured using cybex dynamometer that a standardized protocol previously described was applied in a subject-specific range of motion. serum bmp- levels of all patients were studied by elisa method. results represented a better vas and womac scores for all exercise groups after treatment. the serum bmp- levels were significantly decreased in group compared to group ( . ae . ; . ae . respectively, p < . ) and in group compared to group ( . ae . ; . ae . respectively, p < . ). there is not any statistically differences between group and group (p > . ). as a conclusion, the decreased serum levels of bmp- may be suggested as a biochemical marker for oa patients during exercise programmes. p- . . - tnf-a blokade efficiently reduced severe intestinal damage in necrotizing enterocolitis c. tayman, s. aydemir, i. yakut, u. serkant, a. c ß iftc ßi g€ olbasi devlet hospital, ankara, turkey objectives: to ascertain the beneficial effects of infliximab an inhibitor of tumor necrosis factor alpha (tnf-a) on the development of nec in an experimental nec rat model. material and methods: thirty newborn sprague-dawley rats were randomly divided into three groups as nec, nec+ infliximab, and control. nec was induced by enteral formula feeding, exposure to hypoxia-hyperoxia and cold stress. pups in the nec+ infliximab group were administered infliximab at a dose of mg/kg daily by intraperitoneal route from the first day until the end of the study. all pups were sacrificed on the th day. proximal colon and ileum were excised for histopathologic, immunohistochemical (tunel and caspase- ), and biochemical evaluation, including, total antioxidant status (tas), total oxidant status (tos), malonaldehyde (mda), and myeloperoxdase (mpo) and tnf-a activities. results: we observed better clinical sickness scores, weight gain, and survival rate in the nec+ infliximab group compared to the nec group (p < . ). histopathological and apoptosis examination (tunel and immunohistochemical evaluation for caspase- ) revealed lower damage in the nec+ infliximab group compared to the damage in the nec group (p < . ). tissue mda, mpo, tnf-a levels, and tos were significantly decreased in the nec+infliximab group, whereas tas was significantly increased in the nec + infliximab group (p < . ). conclusion: tnf-a blockade with infliximab efficiently reduced the intestinal injury and preserve the intestinal tissues from severe intestinal damage by its complex mechanisms on nec. therefore, it may be an alternative option for the treatment of nec.keywords: tnf-a; infliximab; necrotizing enterocolitis; newborn; protection; rat; treatment p- . . - short-term diabetes causes cardiovascular inflammation: anti-inflammatory effect of resveratrol introduction: diabetes is a metabolic dysfunction and has been associated with various disorders including inflammation, cardiomyopathy and coronary artery disease. inflammation is a protective mechanism elicited by the host in response to infection, injury, and tissue damage. the aim of this study was to investigate the effect of intraperitoneally resveratrol administration on cardiac and vascular function in diabetic rats. materials and methods: diabetes was induced in sprague-dawley rats by using injection of streptozotocin ( mg/kg, i.p.). rats were divided into group i: control, ii: control/ mg/kg resveratrol; iii: diabetic/vehicle; and iv: diabetic/ mg/kg resveratrol. histopathological examinations with masson's trichrome and verhoeff-van gieson staining were carried out to reveal cardiac and vascular tissue damage and inflammation. in addition to plasma glucose and cardiac & vascular mda levels were measured by standard enzymatic kits while tnf-a, il- b, il- (mbl) were analyzed by elisa kit. results: final body weight decreased in all groups compared to control. in the diabetic rats, plasma glucose and vascular mda levels were enhanced while cardiac mda was unchanged compared to control. vascular tnf-a, il- b and mbl and cardiac mbl were increased in the diabetic groups compared to control. discussion and conclusion: it has been found that resveratrol has greatly normalized altered parameters. taken together, resveratrol partly improved cardiac and vascular inflammation induced by diabetes. this may be due to the healing activity of resveratrol on pro-inflammatory markers. p- . . - cytokine network is critical in growth hormone-induced resistance mechanism against curcumin which modulates jak/stat/ socs pathway in mda-mb- and mcf- breast cancer cells m. c ß elik, a. c ß oker g€ urkan, e. damla arisan, p. obakan yerlikaya, n. palavan unsal t.c. istanbul k€ ult€ ur university, istanbul, turkey curcumin (diferuloylmethane), a polyphenolic compound that triggers apoptotic cell death in various cancer cells such as prostate, colon, melanoma and breast cancer. a pituitary-derived hormone, growth hormone (gh) play role in elongation and differentiation of ductal epithelia into the breast terminal and buds. in this study, our aim is to determine the role of inflammation in curcumin induced apoptotic cell death via acting on jak/stat/socs pathway in wt and gh+ mda-mb- and mcf- breast cancer cell lines. according to mtt cell viability assay curcumin triggers cell viability loss in time and dose dependent manner in mda-mb- wt and mda-mb- gh+ breast cancer cell lines, respectively. selected concentrations of curcumin as lm (for mcf- ) and lm (for mda-mb- ) decreased cell proliferation and induced apoptosis through causing jak dephoshorylation, stat , , dimerization and acting on socs proteins expression in each cell lines. in addition, activated jak/stat/socs pathway, via forced gh expression has been suppressed following curcumin treatment for hours. lm curcumin-induced apoptotic cell death via dephosphorylating jak at tyr / residues and decreased phospho-stat , level in both breast cancer cell lines. although curcumin dephosphorylated stat in both mda-mb- and mcf- wt cells, no significant effect has been observed in mda-mb- gh+ and mcf- gh+ cell lines. in consequence, although forced gh expression induced cell proliferation in mcf- and mda-mb- breast cancer cells, curcumin overcame gh-mediated resistance mechanism via acting on jak/ stat/socs signaling, which is related to pparg-induced inflammation. acknowledgment breast cancer is one of the highest cancer type among women worldwide. various enviromental and genetic factors such as age, gender, family history, metabolic diseases and gene mutations are involved in the breast cancer pathogenesis. growth hormone (gh), a pituitary derived hormone, has essential role on postnatal growth and development. it is also established that signalling route of gh and its receptor (ghr) activity is increased in different cancer types. curcumin, a nutraceutical deriatives from rhizomes of turmeric (curcuma longa), has potential therapeutic activity against cancer cells, including breast cancer. curcumin inhibits proliferation of cancer cells such as prostate, colon, melanoma, cervical and breast cancer via induction of apoptosis and inflammation. stat , a major downstream target of gh/ghr signalling, is related to survival, proliferation and differentiation. in this study, our aim was to investigate curcumin-induced apoptotic cell death in gh overexpressed mda-mb- breast cancer cells via jak-stat/socs signalling and inflammatory response profile. according to mtt cell viability assay, curcumin decreased cell viability in time and dose dependent manner in wt and gh+ mda-mb- breast cancer cell lines. we found that lm curcumin-decreased in apoptotic cell death through inactivity at jak which led to dimerization of stat , stat , stat . concomitantly, curcumin affected stat regulating socs proteins in mda-mb- breast cancer cell line. in addition, we demonstrated that lm curcumin induced pparg expression and altered inflammatory cytokine signalling cascade. consequently, although gh overexpression led to agressive profile in mda-mb- breast cancer cells, curcumin overcame this resistance. inflammation is involved in many systemic disturbances, including osteoarthicular or skin diseases, coordinating the signaling network that contributes to tissue injuries. the aim of our study is to reveal pro-inflammatory messengers at the cutaneous barrier (keratinocytes, fibroblasts, endothelial cells), simulating the dermal impact of active principles, especially polyphenols and flavones from vegetal sources: salvia officinalis, asculum hippocastanum and calendula officinalis. we focused on il and il cytokines as main mediators of inflammation progression, correlated in keratinocytes with il a as skin irritation indicator and vegf as pro-angiogenic factor, as well as in endothelial cells with icam- and vcam- adhesion molecules expression. in order to in vitro mimic the inflammatory conditions, we used targeted stimuli for each type of cells: for fibroblasts and endothelial cells -tnfa, a systemic stimulus, single or combined with pma that activates protein kinase c and up regulates nadph oxidase, which lead to superoxide anion production; for keratinocytescontrolled uv-a and uv-b radiation, simulating the solar damages or potential uv interactions with active principles in light exposed skin. the main analysis technique was flow cytometry: beads bases assay for soluble factors and fluorescent antibodies staining. our results prove the different involvement of polyphenols and flavones in the anti-inflammatory mechanisms, depending of the vegetal source: active principles from salvia officinalis induce a strong inhibition of il and il in tnfa stimulated keratinocytes, fibroblasts and endothelial cells, reduce the icam- over-expression but have no effects on irradiated keratinocytes; biocomplexes from asculum hippocastanum inhibit only il release in stimulated fibroblasts, but protect keratinocytes from uv-a and uv-b radiation; compounds from calendula officinalis are active on il signaling in fibroblasts and counteracts only uv-b inflammation. ischemia and/or reperfusion injury is one of the most common causes of acute renal failure. ischemia-reperfusion associated with thrombolytic therapy, organ transplantation, coronary angioplasty, aortic cross-clamping, or cardiopulmonary bypass results in local and systemic inflammation. within the endothelium, ischemia produces expression of proinflammatory gene products (e.g. cytokines) and bioactive agents (e.g., endothelin), while preventing other "protective" gene products (e.g., thrombomodulin) and bioactive agents (e.g. nitric oxide). therefore, ischemia induces a proinflammatory state that increases tissue vulnerability to further injury on reperfusion. this experimental study was designed to investigate the protective effect of salvia l. extracts on kidneys from i/r injury. salvia lamiaceae have been used for treatment of some illnesses in turkish folk medicine. forty spraque dawley rats were divided into groups (n = ). right nephrectomy was performed to all groups. group i: control group; group ii: i/r group; group iii: i/r + mg/kg salvia l.group; group iv: i/r + mg/kg salvia l. group; group v: i/r + mg/kg rosmarinic acid. group. salvia l. and rosmarinic acid for days was given single dose as a gavage. minutes ischemia, minutes reperfusion were applied to groups except control. intracardiac blood samples were taken, high sensitive crp (hscrp), tumor necrosis factor-a (tnf-a), interleukin (il)- and interleukin ib (il- b) levels were detected. serum hscrp levels were also determined in our clinical laboratory using routine standard methods. serum tnf-a, il- and il-ib levels were evaluated using an enzyme-linked immunosorbent assay technique. mean values were evaluated by statistical analysis. serum hscrp, tnf-a, il- , and il- b concentrations were significantly increased after renal i/r as compared to the control group. our treatment group mg/kg salvia l. and mg/kg rosmarinic acid especially mg/kg of salvia l. were found to show a protective effect against renal structure and function. we concluded that salvia l. extracts could be beneficial in the treatment of renal ischemic injury. but mg/kg salvia l. extract were more effective than mg/kg salvia l. extract and used as synthetic mg/kg rosmarinic acid. acne vulgaris is a common chronic inflammatory skin disease of unknown etiology. excess levels of secretory phospholipase a (spla ) contributes to inflammatory diseases and studies indicate that lipoprotein lipase (lpl) has differential effects on several inflammatory pathways. the aim of the present study was to assess serum activity of spla , lpl and evaluate changes in circulating protein levels of angiopoietin-like protein (angptl ), angptl , cyclooxygenase (cox) and prostaglandin e (pge ). serum from control subjects and acne vulgaris patients with moderate and severe disease was evaluated for levels of spla , cox, pge , lpl, angptl and angptl . disease activity was determined according to the national health service (nhs) lambeth and southwark clinical commissioning group guidelines for the management of acne. lipid profile, routine biochemical and hormone parameters were assayed by standard kit methods using autoanalyzers (beckman coulter au clinical chemistry and unicel dxi immunoassay systems). serum levels of spla and lpl were significantly increased in acne vulgaris patients compared to age and gender matched controls. no significant differences were found for cox, pge , angptl and angptl levels between acne vulgaris patients and controls. the results of this study reveal the presence of a proinflammatory state in acne vulgaris as shown by significantly increased serum spla activity. increased lpl activity in serum of acne vulgaris can be protective in patients through its anti-dyslipidemic actions. to our best knowledge, this is the first study investigating spla , lpl, angptl and angptl levels in acne vulgaris. future studies are aimed to understand the regulation of spla and lpl expression in acne vulgaris patients. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; # s ). p- . . - -ohdg and hogg levels are as an oxidative dna damage markers in acne vulgaris treated with isotretinoin h. ecevit, m. izmirli, b. gogebakan, e. rifaioglu, d. sonmez, b. bulbul sen, t. sen, h. m. okuyan mustafa kemal university, hatay, turkey acne vulgaris is a skin disease that characterized by comedones, papules, pustules, nodules and cysts at face, back and body skin. isotretinoin is one of the treatment agents in acne vulgaris. about weeks after drug treatment, the amount of sebum which is produced by sebaceous gland reduces keratinization disorder and the number of propionibacterium acnes normalizes. however, isotretinoin is known that has a wide range of side effects. in recent studies, isotretinoin treatment has been shown to increase the oxidative stress. -hydroxy- -deoxyguanosine ( -ohdg), an important indicator of oxidative dna damage, hydroxyl ion is bound at the th carbon of guanine. this structure is repaired through a base excision repair mechanism and the human -oxoguanine dna glycosylase (hogg ) plays a key role in this processes. in this study we aimed to evaluate the dna damage and it's repair in acne vulgaris before and after months of isotretinoin treatment by measuring -ohdg and hogg levels. the current study includes acne vulgaris patients who are diagnosed in mustafa kemal university, department of dermatology. -ohdg and hogg levels were measured by enzymelinked immunosorbent assay (elisa) method for before and after months of isotretinoin treatment. the commercial elisa kits (cloud-clone corp; usa and cell biolabs; usa) were used for the assessment of hogg and -ohdg, respectively.both -ohdg (p as a conclusion, isotretinoin increases dna damage and high serum -ohdg and hogg levels as a result of isotretinoin treatment may effect on the amount of reactive oxygen species. the pineal gland is a circumventricular organ which serves as a major neuroendocrine gland in the brain. its primary function is the production of melatonin which is controlled by signals from the suprachiasmatic nucleus. melatonin codes the length of the night and it is well recognized for its anti-inflammatory effects. lipopolysaccharide (lps) is the essential component in the outer surface membrane of gram-negative bacteria and act as a strong stimulator of natural and innate immunity in all eukaryotic species. furthermore, lps reduces melatonin synthesis and induces the expression of the serine protease inhibitor (spi- ) in the stat -mediated manner in pinealocytes. however, the precise function of stat in the cell signaling in the pineal gland is not yet known. here we investigated the effect of inhibition of stat on lps-induced changes in melatonin levels, expression of arylalkylamine n-acetyltransferase (aa-nat) and spi- in the pineal gland. experiments were performed in vitro using organotypic and primary cultures prepared from the rat pineal glands. levels of melatonin and spi- were determined from tissue homogenate by enzyme-linked immunosorbent assay (elisa). the pinealocytes were used to carry out sirna stat transfection. the successful transfection and subsequent decline in stat expression levels were proved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the changes in synthesis of aa-nat and spi- were studied by rt-pcr. in conclusion, lipopolysaccharide can affect the immunomodulators secreted by the pineal gland. the clarification of the effect of inhibition of stat on those immunomodulators is important from the clinical point of view because inhibitors of stat are nowadays used as tumour suppressors. silica nanoparticles have a great potential for a variety of industrial, diagnostic and therapeutic applications. in this study, we have evaluated the in vitro effects of amorphous silica nanoparticles ( nm) using human lung mrc- fibroblast as model. cells were exposed to . lg/ml silica nanoparticles for , and hours. the cytotoxic and inflammatory response, and matrix metalloproteinase expression were examined. the pro-inflammatory cytokine il- b, il- , il- , tumor necrosis factor (tnf-a), matrix metalloproteinases (mmp- , mmp- , mmp- ) and tissue inhibitor of metalloproteinase- (timp- ) were analyzed by western blot method. cytotoxicity was evaluated by lactate dehydrogenase (ldh) released into the culture medium by damaged cells. the level of ldh activity was increased after exposure to silica nanoparticles, in a time-dependent manner compared to control. the protein expression of il- , il- , il- and tnf-a as well as of mmp- and timp- , was up-regulated whereas those of mmp- , mmp- was down-regulated after and hours respectively. in conclusion, our data indicate that amorphous silica nanoparticles generate a cytotoxic and inflammatory response, as well as an imbalance in extracellular matrix due to the differential regulation of mmps and tissue inhibitor of metalloproteinase- in mrc-cells after and hours. p- . . - association of fto gene variant (rs ) with markers of t dm and obesity in population from bosnia and herzegovina and kosovo fto (fat mass and obesity-associated gene), recently discovered in a genome-wide association study for type diabetes (t d) encodes a -oxoglutarate-dependent nucleic acid demethylase and is mainly expressed in the hypothalamus. this gene may play important role in the management of energy homeostasis, nucleic acid demethylation, and regulation of body fat masse by lipolysis. the aim of this study was to analyze the association of this single nucleotide polymorphisms (snps) with clinical and biochemical parameters of obesity, t d, prediabetes and at the level of healthy population from bosnia and herzegovina (bh). the study included patients with t d and prediabetes and healthy controls both sexes, aged from up to years. patients were recruited at the clinical centre university of sarajevo, university hospital of clinical centre in banja luka, general hospital in te sanj and health centre in prizren. genotyping of analyzed polymorphism was performed by rt-pcr method in cooperation with the department of clinical chemistry, faculty of pharmacy, university of ljubljana (ljubljana, slovenia) and university hospital of charles university (hradec kralove, czech republic). our results did not show significant differences in genotype frequencies of the analyzed polymorphisms between patients with t d, pre-diabetes and healthy population also, results of logistic regression analyses did not show significant association of risk a allele of fto gene polymorphism -rs with increased risk of t d (or = . , % ci . - . , p = . ). a allele was significantly associated with higher values of hba c, insulin, homa ir index, diastolic blood pressure and higher levels of inflammatory markers (fibrinogen and leukocytes). interestingly, a tendency of association of a allele with higher values of obesity markers (bmi, waist and hip circumference) was noted. further studies are needed on a larger population in order to confirm these results. the water extract of capparis ovata (cowe) has been shown to be used as an alternative medicine for the treatment of multiple sclerosis (ms). cowe was further fractionated and studied for additional anti-neuroinflammatory effects in sh-sy y cells. for this purpose, the dichloromethane sub-fraction of the cowe extract was tested for its anti-inflammatory effects on selected anti-inflammatory genes believed to be important in ms pathophysiology using sh-sy y cells. cell viability was assessed using lactate dehydrogenase (ldh) activity in the media conditioned by the crystal violet cell staining. in these cells, levels of the tumor necrosis factor-a (tnfa), nuclear factor kappa-lightchain-enhancer of activated b cells (nf-jb ), glial fibrillary acidic protein (gfap), c-x-c motif chemokine and (cxcl , cxcl ), matrix metalloproteinase (mmp ), chemokine (c-c) motif (ccl ) and tyrosine-protein phosphatase non-receptor type (ptpn ) were determined by quantitative reverse transcriptase-pcr assay (qrt-pcr). we have found out that the dichloromethane sub-fraction of cowe effectively inhibited the expression of all of the genes given above in sh-sy y cells. thus, phytochemicals present in the dichloromethane sub-fraction of the cowe extract could be beneficial in preventing/treating neurodegenerative diseases in which neuroinflammation is part of the pathophysiology. studies are underway to identify the individual compound(s) in this subextract of the cowe extract contributing to these effects. this work is supported by tubitak s and pamukkale university paubap fbe . p- . . - apigenin and luteoline were identified as active anti-inflammatory constitutents of lavandula stoeachas by bioassay guided fractionation h. ipek , s. savranoglu , a. r. t€ ufekc ßi , f. g€ ul , i. demirtas , t. boyunegmez t€ umer graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: lavandula stoechas, in the genus of lavender, has distinct therapeutic uses among anatolian people. rather than worldwide use of its essential oil in aromatherapy, specifically the aqeous portion as decoction has been traditionally used in anatolia against the components of metabolic syndrome, all of which share a state of chronic inflammation as an underlying cause. the anti-inflammatory constiutents of l. stoechas were isolated using a bioassay guided fractionation in lipopolysaccharide (lps) inflammed raw . macrophages. materials and methods: an aqeous extract was partitioned into ethyl acetate (eae) and n-butanol fractions. the eae, determined as bioactive extract was seperated into subfractions by column chromatography. e was identified as active subfraction subjected to sephadex column to get pure compounds which were then applied to nmr, ir, and uv analyses for structure determination. in raw . cells, the effects of extracts/fractions/subfractions/compounds on lps induced no production was determined by using griess method. the potential inhibitory effects of each compound on lps induced inos expression were determined by qpcr and western blot. results: p-coumaric acid, apigenin and luteoline were found in the e , and the first two compounds appeared to be primarily responsible for the anti-inflammatory activity. apigenin and luteoline at lm decreased no production and %-ic : and lm-by inhibiting inos gene expression and % as well as protein expression and %, respectively (p < . ). conclusion: this is the first time that luteoline and apigenin have been found in eae of l. stoechas, and the anti-inflammatory properties of the eae can be attributed, at least in part, to the presence of these two compounds. we are on the way to gain further insight for the action mechanism of these two active principles as anti-inflammatory agent. tubitak (project id: t ) support this work. the role of tip in the inflammation process immun response generates the first line of host defense during inflammation and plays an important role inducing pro-inflammatory response by generating early response against pathogens. il- (interleukin ) is one of the pro-inflamatory cytokines and its expression increases during the infection to activate the jak/ stat pathway. jak/stat pathway is regulated by hamp (hepcidin antimicrobial peptide). our previous study, we reported that hamp gene expression was decreased in liver-specific tip conditional knockout mice, so we thought that tip may have a direct or indirect role on inflammation mechanism. tip (tat interacting protein, kda) is a member of the myst enzyme family of histone acetyltransferases (hats) and plays an important role in multiple function including cellular signaling, dna repair, cell cycle and apoptosis. in this study, the quantitative gene and protein expression of il- were investigated by using taqman real time pcr, western blot and immunohistochemistry analysis in control group, lpsinduced inflammation group and liver-specific tip conditional knockout group mouse liver. according to our preliminary results, the gene and protein expression of il- was increased in lps-induced inflammation group (p < . , p < . ) and liver-specific tip conditional knockout group mouse liver (p < . , p < . ). our initial data suggest that tip may be essential for the inflammation process. this work was funded by grants from the scientific and technological research council of turkey (tubi-tak) (grant number: z ). although intracellular reactive oxygen species (ros) level is necessary to maintain cellular homeostasis, elevated intracellular ros level with the impact of unfavorable environmental conditions leads to oxidative stress that may cause damage to dna, proteins and lipids. in case of inflammation, organism seeks to provide cellular homeostatis by increasing ros levels via antioxidant molecules and enzymes. therefore, it was thought that there can be a direct or indirect relation between inflammation and oxidative stress. in this study, inflammation was performed by intraperitoneal injection of lipopolysaccharide (lps). the gene expression and activity of antioxidant enzyme including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glutathione s-transferase (gst), glutathione reductase (gr) and glucose -phosphate dehydrogenase (g pd). additionally, any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h o ) level are accepted as an indication for the accumulation of ros, the relative levels of them were also studied. to show our inflammation model was performed in mouse kidney with lps treatment or not, the expression of interleukin (il ), which is accepted as a inflammation marker, was investigated by real time pcr. the expression of il was significantly increased in lps treated group. while the level of mda and h o was elevated in lps treated group, gssg was decreased. no changes was seen for gsh level. the correlation was observed between enzymatic and molecular levels. while the gen expression and the enzyme activity of sod, cat, gst, gr, and g pd were decreased, gpx was increased with inflammation. in conclusion, increasing ros level was observed in the inflammation process and, the antioxidant system was affected at the molecular and protein level. this work was funded by grants from the scientific and technological research council of turkey (tubitak) (grant number: z ). the aim of our study is to evaluate effect of vitamin d levels on hemogram parameters including neutrophil %, lymphocyte %, neutrophil % / lymphocyte % ratio (nlr) and mean platelet volume (mpv) in behcet's patients. fifty eight patients with diagnosis of behcet that applied to selcuk university faculty of medicine department of dermatology are recruited to the study. clinical and laboratory characteristics of the patients were obtained from hospital automation. t test was used to examine the differences between the parameters. p < . was taken to be statistically significant. there was a statistically significant difference between vitamin d values and age (p = . ) whereas difference was not significant between vitamin d and neutrophil %, lymphocyte %, nlr, mpv values. according to the literature, there are a lot of studies that show the relationship between vitamin d and hemogram parameters. however, contrary to the previous studies, we were unable to find any significant relationship between vitamin d and these hemogram parameters. these results serve the idea that the effects of vitamin d on the hematopoietic system should be further investigated experimentally and clinically. crimean-congo hemorrhagic fever is a tick-borne disease caused by the arbovirus and characterized by a sudden onset of high fever, severe headache, dizziness, back and abdominal pains. the exact pathogenesis of cchf has not been clarified yet. the aim of this study, clinical cases of cchf in cu, se and zn is to examine the relationship between the concentration of trace elements. the study sample consisted of patients which have been diagnosed with cchf. matched for gender, healthy volunteers were similar to the control group according to age. the patients and control groups, serum cu, zn and se levels were analyzed using atomic absorption spectrophotometer. cchf patients in the group, cu zn and se serum levels were significantly lower compared with the control group. in our study, the cofactor of the antioxidant enzyme cu, zn and se elements were lower. this shows us in cchf disease, a decrease in antioxidant enzyme activity, and suggest that they contribute to the immune system's degradation. p- . . - inhibitors of mdm ubiquitin ligase as prospective modulators of autoimmunity e. bulatov, a. valiullina, r. sayarova, a. rizvanov kazan federal university, kazan, russia ubiquitin-proteasome system is seen as a pool of promising protein targets for therapeutic impact in many human diseases. mdm is an e ubiquitin ligase widely studied due to its wellknown role in cancerit negatively regulates p oncosuppressor that mediates apoptosis in tumour cells. inhibitors of p / mdm interaction have long been known as potential anticancer therapeutics. however, recent advances in the field suggest that both mdm and p might be playing a substantial role in autoimmune processes. we used a small molecule p /mdm inhibitor nutlin- a to test the effect of p activation on peripheral blood mononuclear cells (pbmcs) from both healthy volunteers and patients diagnosed with multiple sclerosis. in our study we employed a variety of molecular biology methods, such as immunoblotting, real-time pcr, mts cell proliferation assay, fluorescence flow cytometry and confocal microscopy. we demonstrated that disruption of p /mdm interaction by nutlin- a alters the p levels and also affects the lymphocyte subpopulations within pbmcs. our findings suggest that p /mdm interaction inhibitors can potentially be used as prospective modulators of immune response in autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus and other. the study was funded by rfbr research grant - - mol_a_dk. can ykl- be an inflammatory biomarker in vitamin d deficiency? object: the tumor necrosis factor (tnf) was found to be cytotoxic to tumor cells and to induce tumor regression in mice. except for one member, all receptors to the tnf superfamily bind tnf-related ligands and act mostly on inflammatory system. there are currently tnf superfamily ligands. tnf superfamily ligands share several common features. tnf ligands are generally type ii transmembrane proteins whose extracellular domains are be divided by enzyme to create cytokines. the tnf superfamily currently consists of receptors. tnf family receptors are type i or type iii transmembrane proteins that contain multiple extracellular domains. in this study, we investigated to presence, differences and effects of tnf superfamily and receptors genes in human and mice by using bioinformatics techniques. methods: the nucleotide and amino acid sequence of each protein in human and mice was determined using t-blast-n for homologous sequences. homologous sequences of human tnf family genes found an automated procedure by using psi-blast. the secondary structure of and three-dimensional of the protein were analyzed by psipred and ffas server. netcglyc . and netphos . program were used for post-translocation modifications. the web apoptosis database was also used for the lists of domains, proteins containing these domains and their associated homologs. results and conclusion: humans tnf ligands have genes encoding proteins that contain a conserved carboxy-terminal domain. this family of proteins is highly conserved between humans and mice. humans contain genes encoding tnffamily receptors. sequence data from the ncbi databases demonstrated the presence of mouse tnf-family receptors with orthologs in humans and one additional receptor found only in mice. the differences and similarities in the tnfs genes in humans and mice will provide information for understanding the utility and limitations of the mouse models of disease and comparing of immunology outcomes. the development of left ventricular remodeling after acute myocardial infarction is a predictor of shock. the genetic influence on cardiac remodeling, and shock in the early period after acute myocardial infarction are unclear. the aim of the present study was to investigate the relationship between angiotensin converting enzyme (ace) gene polymorphism and modified shock index (msi) in the early period in patients with acute anterior myocardial infarction. overall patients with a first acute ami were included in this study. dna was isolated from peripheral leukocytes. the id status was determined by pcr. based on the polymorphisms of the ace gene, they were classified into groups: deletion/deletion (dd) genotype (group , n = ), insertion/deletion (id), insertion/insertion (ii) genotypes (group , n = ). blood pressure and pulse measurements were performed in all patients within minutes admitted to coronary care unit. msi was defined as heart rate (hr) divided by mean arterial pressure (map). echocardiographic examinations were performed in accordance with the recommendations of the american echocardiography committee. one-way analysis of variance (anova) and chi-square analyses were used to compare differences among subjects with different genotypes. the study was approved by the local ethics committee, and each patient gave a written consent. there were no significant differences among clinical parameters of patients. msi was significantly higher in patients who have ace dd genotype than in patients who have ace id / ii genotypes ( . ae . and, . ae . , p < . ). presentation time hypotension or developing hypotension during admission was reported to be an important predictor of intensive care unit admission besides other vital sign measurements. our results suggested that, ace gene i/d polymorphisms d allele may affect modified shock index in patients with a first acute anterior mi. glucocorticoids (gcs) are widely used in medicine, despite their side effects, e.g. osteoporosis. however, precise molecular mechanisms of gc action, especially on bone marrow (bm) cells, remain controversial. given the osteoprotective role of vitamin d , the aim of our study was to examine prednisolone-induced changes in the rank (receptor activator of nuclear factor kappa-b)/rankl (rank ligand)/opg (osteoprotegerin) pathway of rat bm depending on the state of vitamin d endocrine system. female wistar rats received prednisolone ( mg/kg b.w.) with and without iu of d (for days). the levels of rank, rankl, opg, a-hydroxylase (cyp b ) in bm were determined by western blotting. vitamin d receptor (vdr) and rankl mrnas were measured by quantitative rt-pcr. ohd content in the serum was assayed by elisa. rankand vdr-positive bm cells were quantified using flow cytometry and visualized by confocal microscopy. prednisolone induced a marked increase in rankl and rank levels, while opg level was shown to decrease. this reflects disturbances in cytokine-mediated regulation of bm progenitor cell function. data from flow cytometry indicated a significant growth in the number of rank-positive cells (hematopoietic osteoclast precursors) compared to control. these changes were accompanied by a decrease in the levels of vdr and cyp b , which is responsible for , (oh) d synthesis, in bm and ohd content in serum. co-localization of vdr and rank in mono-and multinuclear bm cells was observed, indicating a close relation between vitamin d and rank/ rankl/opg pathway. vitamin d co-administration prevented prednisolone-induced changes in bm cells through restoration of vitamin d bioavailability and vdr signaling that resulted in a reduction of the osteoclast progenitor pool in bm. thus, prednisolone-induced imbalance in rank/rankl/ opg system components is associated with impairments of vitamin d endocrine system in bm and can be ameliorated by vitamin d treatment. p- . . - heat shock pathway in response to different stress factors the heat shock response is an emergency pathway of the cell, which mediates repair and protection from cellular stress and therefore guarantees the survival of the cell. this stress can range from heat or hypoxia to chemicals and heavy metals. it is highly conserved in all eukaryotic cells and plays an important role during atypical conditions. due to its high complexity, the pathway is not yet completely understood. most important, after activation of the pathway, is the refolding of proteins or, in case of severe misfolding, the depletion of proteins to maintain proteostasis. heat shock factor encoded by the hsf gene is known as the main switch point in heat shock regulation. after activation it trimerizes and binds to heat shock elements in target gene promoters. one of these promoters is the hspa a promoter (hsp promoter). the promoter was analyzed by dismantling it to its functional parts. especially three elements, the heat shock elements, were in the focus of this work. in first place parts of the promoter were multimerized and combined with different reporters, like luciferase, by cloning. also mutations in the natural promoter were designed by cloning. the focus now is on the heat shock elements, where hsf can bind as a trimer. the idea is that these different elements have various effects on different stressors like heat, chemicals (geldanamycine as hsp inhibitor, mg as proteasome inhibitor) or heavy metals (cadmium, arsenic, zinc) . this was tested on cells transiently transfected with those promoter variants. for promising variants stable cell lines were created. in these stable cell lines further experiments on mrna level can be conducted. in the last months experiments with the crispr/cas system were started. furthermore, experiments on transcriptional (qpcr) and translational (dual-luciferase assay) levels were done as well. in the end we hope to get a clear picture on the regulation of the hspa a promoter by different stress factors. invasive cancer cells form membrane protrusions, invadopodia, that facilitate cell invasion and metastasis. key players invadopodia include the adaptor proteins tks and tks , the actin regulators cortactin, wip and n-wasp, the kinase src and others. in spite that in the last two decades significant advances in our knowledge of the structure and development of invadopodia have been made, detailed mechanisms they are functioning is not yet available. we have identified a series of new tks binding partners including adaptor proteins itsn , itsn , crk and grb , kinase src, amph , bin , plcg and also another member of the tks family -tks . it may indicate the possible role of tks in transport and sorting of cell vesicles. current data are supported by interaction with the proteins of amph and bin , as their main functions are membrane trafficking and remodeling. adaptor proteins crk, grb and itsns are important for the actin cytoskeleton rearrangements, endocytosis and signal transduction. moreover, we have identified and characterized new tks isoform -tks -beta. we suggested that an active state of tks is regulated via intramolecular interactions between its proline-rich motifs and own sh -domains. we have shown the interaction between itsns and other prominent component of invadopodia wip. data from immunofluorescent analysis revealed co-localization of itsn and wip at the sites of invadopodia formation and in clathrin-coated pits. we have also demonstrated that the key protein itsn and wip and n-wasp can form a complex in cells. together, these findings provide insights into the molecular mechanisms of invadopodia formation and identify itsns as scaffold proteins involved in this process. we have shown the interaction between itsns and other verprolin family members cr and wire which play an important role in the reorganization of the actin cytoskeleton. we have demonstrated that cr and wire interact with sh domains of itsns in complex with actin. p- . . - correlation between proteomic and phenazine profile of pseudomonas sp. phenazines are widely known compounds with huge variativity of biological activities which are produced by pseudomonas sp. and some other bacteria species. the results of our work shows the correlation between the changes of proteomic profile of pseudomonas aeruginosa caused by a mutagenesis and the secondary metabolism of antibiotics (phenazine) profile. different strains of pseudomonas aeruginosa were obtained using mutagenesis, after that bacterial cells were destroyed by ultrasound. protein-containing fractions were isolated using methanol-chloroform method as well as phenazines compounds were extracted from culture media using liquid phase extraction. obtained proteome was analysed by shotgun-proteomics technique. as the result of the liquid phase extraction phenazine compounds were mainly extracted to the organic phase. this phase was evaporated and re-dissolved in % methanol. after sample preparation obtained solutions were analyzed by hplc-agilent with quadrupole tof mass-detector. results of the analysis were compared with the library of known phenazine compounds mass-spectras generated by cfm-id online resource. obtained phenazine profiles were compared with each other and correlation with the changes in proteome was analyzed. received results promote better understanding of mechanisms of phenazine production. this data opens possibilities for targeted changes in the methabolic pathway in order to obtain phenazine compound with required biological activity. insulators are genomic elements which block enhancer-promoter interaction and prevent spreading of heterochromatin. cp protein is an integral component of most known drosophila insulators, it interacts directly with ctcf and pita dna-binding insulator proteins using dimeric btb-domain, but function of cp within insulators still remains to be elucidated. recently we described an interaction between cp btb-domain and cterminal domain of ctcf insulator dna-binding protein, subsequent deletion analysis allowed us to isolate aa fragment within ctcf c-terminal domain sufficient for interaction with cp btb, but deletions of flanking regions also lead to the loss of interaction with cp in vivo. at the same time crosslinking experiments suggest that a dimer of btb interacts with one molecule of ctcf, presuming that it could recognize two peptide fragments within ctcf c-terminal domain. we solved crystal structure of btb-domain from cp insulator protein at . a resolution. overall structure is similar to other btb-domains. cp btb-domain has peptide-binding groove similar to that previously found in bcl btb domain. inspection of btb-domain surface revealed several possible binding sites for polypeptide fragments from ctcf protein. based on these observations a set of point mutations within peptide-binding groove of btb-domain has been designed and we tested ctcf-interaction abilities of these mutants using gst pull-down assay and yeast two-hybrid assay. the most significant impact was found with alanine-substitutions of hydrophobic residues whereas substitutions of hydrophilic amino acids were less effective. therefore our results support that cp btb-domain recognizes ctcf protein using peptide-binding groove. this study was supported by the russian science foundation (project № - - ). p- . . - comparative study of the fatty acid composition of lipids in the raw meat samples obtained from hybrid sheep one of the most important tasks in the animal biology and husbandry is to clarify the role of animal genetic diversity in providing nutrients to the diversity of animal products. the objective of our work was to study the chemical compositions of raw meat samples obtained from domestic (group i -purebred romanov sheep) and hybrid sheep (group ii -f hybrids of romanov sheep with . % of argali blood). the significant changes in fatty acid composition of the lipid fraction from the fat and muscle tissue of the hybrid sheep as compared to the control were found. the content of saturated fatty acids (sfas) in the fat samples of the hybrid animals was by . % lower ( . ae . %, p < . ), but polyunsaturated (pufas) or monounsaturated fatty acids (mufas) contents were by . % and . % higher ( . ae . and . ae . (p < . ), respectively) as compared to purebred romanov sheep. the most pronounced changes were found for palmitic acid (decreased from . % to . %) and for oleic, linoleic, arachidonic acids (increased from . %, . %, . % to . %, . %, . %, respectively). the last two acids together with the linolenic acids belong to the so-called essential acids and very important for the animal metabolism. a similar trend was observed on the composition of the lipid fraction of muscle tissue. sfas, pufas and mufas content in muscle tissue of hybrid sheep was . ae . , . ae . and . ae . %, that was . % lower (p < . ), and . % and . % higher (p < . ) compared to purebred romanov sheep. these results emphasized the difference of the pufas/sfas ratios in fat and muscle tissues, respectively) and characterized the biological value of the lipid fraction of fat and muscle tissue. the obtained data gave evidence of the positive changes in the fatty acid compositions of the lipid fractions for the hybrid animals as compared to the purebred sheep. supported by the russian scientific foundation, no. - - . foodborne illnesses resulting from the consumption of agricultural commodities contaminated with enteric pathogens are an increasing problem around the world. while various possibilities of produce contamination with pathogens exist, the global warming combined with a widespread use of animal manure in agriculture will likely contribute to an increased number of such outbreaks. thus, phages isolated from different agroecosystems may prove to be useful in detection/biocontrol of enterobacteria in produce. during the investigation of the impact of global warming on the diversity and co-evolutionary dynamics between microorganisms and viruses in lithuanian agroecosystems, a novel enterobacteria phage vb_ecos_nbd (nbd ) was isolated from agricultural soil using e. coli novablue for phage propagation. nbd genomic dna was isolated from cscl-purified phage particles, and was subjected to illumina dna sequencing. nbd is a virulent siphovirus that has a low-temperature plating profile (fails to form plaques at a temperature > °c). the genome of nbd is $ kb long, and has a total of probable protein-encoding genes as well as gene for trna ser . the genome analysis revealed that nbd orfs encode unique proteins that have no reliable identity to database entries. among the orfs that encode proteins with matches to those in other sequenced genomes, are similar to proteins from phages that infect different members of enterobacteriaceae, while nbd orfs are most similar to those from bacteria. based on the similarity to biologically defined proteins, nbd orfs were given a putative functional annotation, including genes coding for morphogenesis-related proteins, as well as associated with dna replication, recombination, and repair. phylogenetic analysis revealed that enterobacteria phage nbd is distantly related to phages belonging to the subfamily tunavirinae. this research was funded by a grant (no. sit- / ) from the research council of lithuania. p- . . - the antibiotic novobiocin affects the composition of the escherichia coli proteome n. e. arenas , j. williamson , v. schw€ ammle , s. douthwaite universidad de cundinamarca, cundinamarca, colombia, university of southern denmark, odense, denmark novobiocin (nov) is an aminocoumarin which competitively inhibits the atp binding site in the gyrase-b subunit of prokaryotic topoisomerase ii. nov remains a therapeutic choice for treating infections with bacterial pathogens that are resistant to more commonly used drugs. the aim of this study is assess the proteomic response of e. coli strain upon nov treatment. minimum inhibitory concentrations of nov were measured by standard assays. three different e. coli strains (as , as -rlma::aph and b) were grown aerobically in nutrient rich lb media at °c during one hour. the whole cell proteome (five biological replicates in each sample,) was assessed by lc-ms by using tmt labelling protocol. raw files were imported to proteome discoverer (thermo fisher scientific) and searched together with mascot against the uniprot e. coli reference proteome. mics for nov were determined to be > -fold higher the wild-type b-strain of e. coli than for the hypersusceptible as strains ( lg/ml). whole genome comparison of the b and as strains were characterized by an increase in proteasome components ( proteins), chaperones ( ), error-prone dna polymerase components ( ), ribosomal hibernation factors ( ), heat shock response ( ), electron transport coupled proton transport ( ), pentose phosphate pathway ( ), flagellar assembly ( ), oxidative phosphorylation ( ) and tca cycle ( ). whereas ribosomal proteins ( ), aminoacyl-trna synthetases ( ), rnases ( ), abc transporters ( ), mismatch repair ( ) and sec secretion pathway ( ) were significantly down-regulated upon nov treatment. the three e. coli strains respond similarly upon nov treatment and their proteomes showed upregulation of heat shock response with changes in the components of translation and transcription, the proteasome and atp biosynthesis. the changes observed can be used to define the processes that are required for antibiotic tolerance and survival of e. coli against aminocoumarin antibiotics. postnatal growth is under control of pituitary derived hormone, growth hormone (gh) that triggers bone, fat tissue growth and development via acting on protein, carbohydrate and fat metabolism. gh functions on postnatal development by jak /stat signaling following gh:gh receptor (ghr) dimerization. isolated growth hormone deficiency (ighd) is a medical condition of insufficient production of growth hormone (gh) that is caused by mutations on gh-n gene in different ethnic origin children. various mutations within gh has been determined in different populations so far, and glutamic acid to glycine (e g), asparagine to aspartic acid (n d), threonine to alanin (t- a) missense mutations, alanine to serine (a s) substitution, tryptophan to stop codon (w- x), gaaa insertion in intron of gh-n gene and both intron (+ c) and deletion of . amino acid of gh protein phenylalanine (f del) mutations were detected in turkish ighd children. the potential role of these mutations on cell growth, proliferation, emt via acting on gh signaling pathway has not been observed yet. all these mutations were performed on wild type gh-n gene inserted pc . vector by site-direct mutagenesis and stable cell line of each gh gene mutations were generated by neomycin selection. although w- x, e g, f del, a s and n d mutations suppresses gh signaling via acting on either jak dephosphorylation or stat downregulation, t- a, gaaa insertion and deletion of + c mutations have no significant effect on gh signaling. in addition, each mutation lead different growth suppression effect and colony formation potential and intracellular polyamine levels and odc expression profiles were essential role in emt potential of hek cell lines. as a result, w- x, e g, f del, a s and n d mutations prevented gh signaling and cell growth and differentiation via polyamine metabolism. pulmonary embolism (pe) is a common cardiovascular emergency and affects a large number of patients. acute pe-induced oxidative stress can lead to the accumulation of specific nitroproteins that may play a role in disease progression. the impact of nitration of a single tyrosine residue often has broad implications on the activity of biologically critical proteins, which has become increasingly related to pathological conditions. in this study, we used a proteomic approach to analyze nitrated serum proteins in patients diagnosed with acute pe and healthy controls. nitrotyrosine (no tyr)-containing proteins were immunoprecipitated from serum with a no tyr affinity sorbent. precipitated proteins were separated by sds-page and visualized by coomassie blue staining and western blotting with mouse monoclonal anti-no tyr antibody. among the numerous immunoreactive bands observed in disease patients, the kda protein band was in-gel digested and analyzed by maldi-tof mass spectrometry (ms). mass fingerprint data sets obtained from the peptide fragment ions matched human collagen alpha- (iii) chain (co a _hu-man) with mascot algorithm analysis giving a score of (p < . ). collagen alpha- (iii) chain is a fibrillar collagen that is found in extensible connective tissues such as skin, lung, and the vascular system. altered metabolism of collagen and its excessive deposition in the matrix of the connective tissue is a hallmark of chronic interstitial lung diseases. collagen can be measured in serum and bronchoalveolar lavage fluid from patients with numerous chronic interstitial lung diseases. given these considerations, future studies are aimed understand the relevance of no tyr modifications in co a relating to changes in protein structure and function. recent studies have shown that the genes involved in dislipidemia represent potential loci to be associated with diabetes as a disease. recent genome wide association (gwa) studies have associated rs in gckr gene and rs in galnt gene with parameters of t d and diabetic dyslipidemia. in this study, the association of these single nucleotide polymorphisms (snps) with t d and dyslipidemia was tested in the population from bosnia and herzegovina (bh). our study involved patients with t d and healthy subjects. biochemical and anthropometric parameters were measured in all participants. after dna extraction, sequenom iplex platform was used for the analysis of galnt polymorphism (rs ), while polymorphism in gckr (rs ) gene was analyzed by using real time pcr. our results demonstrated significant association of gckr rs variant with waist circumference (p = . ) and fasting glucose levels (p = . ) in the control group. no such association was demonstrated for rs galnt gene. in the group of diabetic patients, significant association of gckr rs variant with levels of bilirubin (p = . ) and rs galnt variant with hba c (p = . ) and triglyceride levels (p = . ) was also demonstrated. our results suggest an association of variations of gckr and galnt genes with specific markers of t d and dyslipidemia. further studies would be needed in order to confirm these genetic effects in other ethnic groups as well. osteoporosis is the most common metabolic bone disorder affecting the normal bone turnover with low bone mineral density (bmd) and risk of fragility fractures. polymorphisms at the sp binding site of the collagen type a (col a ) gene is associated with low bmd. we examined the distribution of col a gene polymorphism in young osteoporotic women and in control group in turkish population. patients had low bmd with t score ≤ . sd and controls was healthy women ( - years). mean age ( . ae . ) and ( . ae . ) respectively. the bmd, as g/cm , was measured in the hip and the lumbar spine (l -l ) with (dexa). dna was isolated from blood. col a gene was analysed with genomica clinical array system. the x test was used to compare allele and genotype frequences between patients and controls. mean of t score in patients was À . ae . . mean bmd (as g/cm ) was . ae . , and ( . ae . ) genotype distribution were ( %) ss, (% )ss, (% )ss for patients, and ( )ss, ( )ss, ( )ss for control . patients had (% )s allele, ( %) s allele, controls had ( %)s allele, ( %)s allele. when genotypes and bmd were compared in patients, there was no significant correlation between osteoporosis and genotypes. the allelic distribution was not significant between patients and controls p > . . genotypic distribution in patients were significantly different. patients had a higher frequency of the ss(% ) than controls (ss % ) p < . . this study shows that high prevalences of the ss genotype at the col a locus, in osteoporosis . _ it is possible that the presence of the s allele causes variation col a and col a mrna's producing abnormal collagene protein. since collagen protein is major protein of bone, it is to be expected that a defect in this protein will produce bone fragility. col a gene should be detected early to initiate preventative therapy for bone health. the biological activity of nigella sativa seeds is mainly attributed to its essential oil component which is pre-dominantly ( - %) thymoquinone (tq). therapeutic effect of tq was exhibited in many diseases including inflammation, cancer, sepsis, atherosclerosis and diabetes. tq has been reported to exhibit antiproliferative effects on cell lines derived from breast, colon, ovary, larynx, lung, myeloblastic leukemia, and osteosarcoma and inhibited hormone refractory prostate cancer. tq induces apoptosis in tumor cells by suppressing nf-jb, akt activation, and extracellular signal-regulated kinase signaling pathways and also inhibits tumor angiogenesis. the aim of this study was to evaluate the anti tumor effects of tq on hepatoma cells. these antitumor assays include cell viability assay, clonogenic assay, scratch assay and molecular expression studies of death related genes. cells were treated with different concentration of tq in hep b for cell proliferation by mtt and clonogenic assay. in addition, the metastatic character of tq was investigated by scratch assay in hep b at - and hours. the effect of tq was also evaluated at mrna level by real-time-pcr. tq was treated on the hep b cells in three different concentration, namely - and . lm. tq showed the cell cytotoxicity in concentration and time dependent manner. the scratch assay revealed no healing in the scratched area due to the decreased cell viability. maximum permissible dose was lm. proapoptotic genes, bax and bad, and autophagy genes, beclin- and lc , were upregulated in hep b cells after hours treatment in contrast, antiapoptotic gene, bcl- , expression level was decreased for hep b cells after hours. p- . . - association of irs genetic variation with type diabetes and insulin resistance in patients from bosnia and herzegovina insulin receptor substrate- (irs ) encodes the irs protein, a substrate for the insulin receptor tyrosine kinase and has a critical role in insulin-stimulated signaling pathways. previous studies showed that irs single nucleotide polymorphisms (snps) were associated with type diabetes mellitus (t d). this is the first study performed in a population from bosnia and herzegovina (bh) in which we examined the association of rs (g>a), rs (t>c) and rs (a>t) with t d risk and related traits. our study involved t d patients and healthy subjects. biochemical parameters, including but not limited to insulin, homa-ir, hba c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was done by spss . our results demonstrated a significant difference in frequency of rs (p < . ) and rs (p = . ) snps between t d patients and control subjects. interestingly, here we showed a significant association of irs rs risk t allele with increased insulin levels (p < . ) and homa-ir (p < . ) in t d patients. similarly, rs variant was also associated with the same markers of insulin resistance in diabetic patients, i.e. insulin levels (p = . ) and homa-ir (p = . ). no such association was demonstrated for rs . however, this irs variant was associated with changes in lipoprotein levels, where risk c allele increased vldl (p = . ) and decreased hdl levels. our results suggest that irs variants are associated with t d susceptibility in bh population, thus confirming similar findings in other population cohorts. furthermore, the associations of these variants with markers of insulin resistance and dyslipidemic metabolic changes point to their role as potential t d biomarkers. the adra a gene encodes alpha- a adrenergic receptor which mediates adrenergic suppression of insulin. a genetic variant in adra a was recently associated with defective b-cell function. the objective of this study was to analyze association of two adra a polymorphisms (rs a>g and rs g>t) with type diabetes (t d) and its related traits. in this study we have included t d patients and healthy subjects from bosnia and herzegovina (bh). biochemical parameters, including but not limited to insulin, homa-ir, hba c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was performed by ibm spss statistics software. our data showed that frequencies of both, rs and rs , variants were not significantly different between t d and control subjects. however, rs risk a allele appear to increase insulin levels (p = . ) and homa-ir index (p = . ). furthermore, this variant also seems to affect vldl levels (p = . ) and waist circumference (p = . ) in diabetic patients. the genotype analysis of rs variant demonstrated that risk g allele decreased hdl (p = . ) and increased ldl levels (p = . ), as well as affected the waist circumference (p = . ) in diabetic patients. interestingly, haplotype analysis demonstrated the association of rs a / rs g with higher homa-ir index. here we demonstrated that although both, rs and rs , adra a polymorphisms were not associated with t d risk in our cohort, they were associated with markers of dyslipidemic perturbations and insulin resistance in diabetic patients. further studies in larger cohorts are needed in order to explore these possible interactions and confirm our findings. smad-interacting protein (sip ), also known as zeb is a member of zeb family transcription factors and was shown to regulate epithelial-to-mesenchymal transition, cell cycle, cellular senescence and cancer stemness. bipartite zing finger motifs at amino and carboxyl termini of the sip mediate its binding to ebox sequences in the genome. however, there are only limited data about sip target genes. by using a home-made anti-sip monoclonal antibody, clone e , we conducted chip-seq in three hepatocellular carcinoma cell lines, namely snu , plc/prf/ and sk-hep- and found receptor tyrosine kinase-like orphan receptor (ror ) as one of the targets. sip dnabinding consensus motif cacctg was found at + kb, + kb and + kb from ror transcription start site. chip experiments validated sip binding to all consensus motifs in the ror gene region. interestingly, the strongest enrichment was at + kb suggesting that long-range interactions play an important role in the regulation of ror by sip . sip knockdown by shrna in high-sip expressing snu cells resulted in the repression of ror expression. ror is expressed in embryogenesis and fetal life, and is absent within most of adult normal tissues. however, overexpression of ror was observed in many human cancers, from hematological malignancies to solid epithelial tumors. ror -positive cancer cells have enhanced proliferation, invasion and metastasis capacities, show resistance to apoptotic stimuli and display cancer stem cell characteristics. therefore, sip and ror act in similar pathophysiological processes. our finding that ror is regulated by sip at least in hepatocellular carcinoma cells adds another level of complexity to the molecular mechanisms of proliferation, invasion and stemness of cancer cells. hepatocellular carcinoma (hcc) is the most prevalent primary liver cancer and is one of the leading causes of cancer related deaths. smad interacting protein (sip ), a member of the zeb family of emt inducers, is involved in cellular proliferation, senescence, invasion and metastasis in human tumors. however, genes regulated by sip in hcc are yet to be identified. we conducted a chip-seq study in high-sip expressing hcc cell line snu by using a home-made anti-sip antibody, clone e . among annotated genes, we selected six for further studies because of its increased expression in multiple cancers and its association with poor prognosis. sip dna-binding motif cacctg was found at - kb from transcription start site of six gene. chip qpcr experiment validated sip binding to this region with , fold enrichment. compared to healthy liver, six transcripts were upregulated in of hcc cell lines included in this study. knockdown of sip by shrna in snu cells caused upregulation of six . immunohistochemistry studies in hcc tissue arrays showed increased expression of six in tumors and inverse association with sip expression in a tumor grade dependent manner. therefore, our results strongly suggest an inverse correlation of sip and six in hcc bone mineral density (bmd) and bone turnover are under genetic control and variations in the vitamin d receptor (vdr) are related to bmd. bmd is known to be affected by -hydroxy vitamin d ( (oh)d) and intact parathyroid hormon (ipth) levels. we aimed to determine correlation blood levels of vitamin d (vitd), ipth, and vdr gene effect in healthy turkish women. the subjects were healthy women in age - years. the bmd was measured as a t score in the lumbar spine (l -l ) with dexa. all subjects had normal t score between (- . to . ) sd. vitd was measured by lc- -at shimadzu. ipth was measured by chemiluminescence method, dna was isolated from blood. the fok i (vdrf-foki) and bsmi (vdrb-bsmi) polymorphisms of vdr gene was analysed with genomica clinical array system. the mean vitd level was ( . ae . ) lg/l, mean plasma ipth level was ( . ae . ) pg/ml. pearson correlation test showed no relation of vit d with bmd. there was moderately negative correlation between ipth and bmd (r = À . ). genotype distribution and allele frequency of subjects were as follows: ( %) ff), ( %) ff, ( %) ff genotype in vdrf -fok gene, ( %) bb, ( %) bb, ( %) bb in vdrb-bsmi gene. allele frequencies were f: %, f: %; b: %, b: %. when fok and bsmi were combined, %(ff-bb) and % (ff-bb) were found as the most frequent genotypes. bsmi frequency was in hardy weinberg equilibrium (p > . ). but foki was not (p = ). it was found that vit d, ipth levels and bmd were in normal levels in all carriers of ff genotype and in combined (ff-bb) type carrying healthy women ( %). the association vdr genotype and bmd may be different in various ethnic and geographical groups. therefore it is worthwhile to assess vdr polymorphism among turkish population. these type of distribution studies of vdr in healthy and in osteoporotic women may enlighten to earlier diagnosis and treatment planning. p- . . - determination of hb a c values in beta thalassemia f. g€ uzelg€ ul , g. s. seydel , a. e. yalin , e. s€ onmez , k. aksoy c ß ukurova university, adana, nigde university, nigde, mersin university, mersin, turkey introduction: hemoglobinopathies are most commonly seen hereditary blood diseases worldwide. our aim was to compare the hba c values measured on cation-exchange high performance liquid chromatography (hplc) in beta thalassemia cases. materials and methods: we collected ml of whole blood k edta containing tubes from forty-nine cases. arms, rflp and dna sequence analysis methodologies were carried out for determination of beta thalassemia mutations. hb a c values were measured using the agilent hplc system. results: forty-nine diabetic and non-diabetic patients were diagnosed with beta thalassemias: twenty-one ivs - /ivs - , one ivs - /ivs - , one ivs - /ivs - , two ivs - /ivs - , two ivs - /ivs - , one fsc /fsc , one fsc /fsc , two - /- , two cd /cd , one cd - /cd - , one cd - /cd - , two cd /cd -g/ cd /cd -g, two ivs - / ivs - , one ivs - / ivs - , one ivs - /fsc , one ivs - /cd , one ivs - /cd , one ivs - /cd - , one ivs - /ivs - , one ivs - / ivs - , one ivs - / ivs - , one ivs - /cd and one fsc /cd . cases were classified as diabetic ( ), prediabetic ( ) and non-diabetic ( ) introduction: members of aurora kinase family aurora a, b and c are conservative kinases of cell cycle which are encoded by genes aura, aurb and aurc respectively. overexpression of aura and aurb was found in human cancers, especially in prostate cancer. moreover, there is the evidence that aurb interacts with one of the major oncogenic kinases -braf. little is known about implication of aurc in cancer, but it was demonstrated, that it can overlap aurb function and shares its location. we studied expression of genes of these kinases in urine of prostate cancer patients aiming to evaluate their involvement in this disease and their potential as tumor markers. materials and methods: urine samples from patients with prostate cancer were gathered after prostate massage before surgical invasion. we used urine samples from healthy men as control. we obtained cells from each urine sample by centrifugation and isolated rna using standard approach with phenol and guanidine thiocyanate. cdna was synthesized and taken to qpcr reactions. data was statistically analysed. results: expression of all studied genes was detected in urine of patients with prostate cancer and of healthy men. expression of aurb and aurc in cancer samples each was higher than expression of aura. the cumulative expression aurb and aurc was higher than expression of aura in samples from . we observed positive correlation between expression of aurc and braf (rs = . , p = . ). discussion and conclusion: previous investigation showed, that for normal prostate tissue % of aurora family expression was presented by aura. we suppose that presence of aurb and aurc cumulative overexpression means presence of cell cycle deviations in prostate tissue of these patients and might be further studied as prognostic marker. in this study we first showed the correlation between aurc and other carcinogenic kinase braf expression, which opens the perspective for investigation of role of aurc in carcinogenesis. bacillus marmarensis sp. nov. is an extreme obligate alkaliphile isolated from mushroom compost near marmara region of turkey. it can survive at extreme ph values up to . . based on its genome sequence, metabolic pathways for proteases, amylases, cellulases, lipase, n-butanol and a biodegradable plastic poly-b-hydroxybutyrate were annotated. in addition to being a potential extracellular hydrolase producer, its ability to survive in the high ph range of . to . makes it an attractive microorganism for different industrial applications. in the current study, the adaptation strategy of b. marmarensis sp. nov. to alkaline conditions was investigated using proteomic tools. the organism was grown at two different ph values, . and ph . . for extraction of whole cell proteins, cells were disrupted with mp bio fast prep device. protein extracts were treated with protease inhibitors and a nuclease mix. salts were removed using a cleanup kit. obtained proteins were separated based of their isoelectric points in the first dimension and then based on their molecular weights in the second dimension. proteins maps of cells grown at these two extreme ph values showed significant differences in protein expression for alkaline adaptation. p- . . - biochemical and proteomic analyses of normal human astrocytes and glioblastoma exposed to dichloroacetate treatment f. c. atilgan, h. cimen yeditepe university, istanbul, turkey glioblastoma (gbm) is an aggressive malignant tumor composed of astrocytes in brain tissue. gbm cells utilize glycolysis rather than oxidative phosphorylation to support rapid growth rate which is called warburg effect. dichloroacetate (dca) is an antiglycolytic agent that inhibits pyruvate dehydrogenase kinase (pdk) activity and induces apoptosis via normalizing the mitochondrial activity. this study aimed to demonstrate the metabolic alterations between the normal human astrocytes (nha) and gbm cell lines which are exposed to dca, and to identify the differentially expressed proteins by ms-based proteomic analyses. nha cell line, u mg and u as gbm human cell lines were examined through analyzing the alterations in the glycolysis metabolism upon dca treatment by measuring the variations in the pyruvate levels, lactate dehydrogenase a, pdk . mts was performed to investigate the effect of dca treatment on cell viability. immunoblotting of pgc -a, oxphos complexes, and mitotracker green staining was employed to reveal the mitochondrial differences between normal and the cancer cells, and upon dca treatment of these cells. proteomic analyses were utilized for the identification of candidate proteins depending on the acetylation status. in this study, compared to nha, the pyruvate and ldha levels were elevated and pdk levels in u mg were reduced by %. due to mts results, ≤ mm dca treatment showed significant decrease in gbm cells compared to nha cells. immunoblotting and mitotracker green staining results showed increase in mitochondrial mass. elevation in the pyruvate and ldha levels and reduction in pdk level in u mg and u cells indicates glycolysis dependent metabolic switch in energy metabolism. proteomic analyses demonstrate that most of the differentially expressed proteins comprised of metabolic enzymes. this study provides novel information about metabolic alterations existing between nha and gbm, which can inspire further studies for therapeutic applications. kidney stone is a complex disease resulting from environmental as well as hereditary factors and principally composes of approximately % calcium oxalate (caox) crystals, which are formed through a multi-step process. vitamin d receptor (vdr) gene encodes the nuclear hormone receptor for vitamin d ; downstream targets of this gene are chiefly contributed in mineral metabolism though the receptor regulates a variety of other metabolic pathways. calcium sensing receptor casr plays an important role in sustaining mineral ion homeostasis. the aim of this study is to profile the expression level of vdr and calcium sensing receptor (casr) genes and to unravel their role in rat kidney stone induced by ethylene glycol, in order to explain the underlying molecular mechanisms. total rna were extracted from paired sample before and after ethylene glycol treated of rats. the mrna expression level of vdr and casr gene were measured employing quantitative rt-pcr (qrt-pcr). the mrna expression levels of both genes were significantly down-regulated according to before treated. in conclusion, our data suggest reduced mrna expression in vdr and casr genes might be a risk factor for kidney stone formation. further studies are necessary to verify these findings in different ethnic groups. p- . . - apj receptor a c gene polymorphism in turkish patients with coronary artery disease against different models of expected frequencies/counts to understand the evolutionary dynamics of saars in proteins. we obtained from ensembl the assemblies of genomes/proteomes of human and nonhuman primates (chimpanzee, gorilla, and rhesus monkey), rodents (mouse and rat), and birds (chicken and zebrafinch). the expected probabilities for the occurrence of saars based on their nucleotide frequencies in coding regions and amino acid frequencies in individual protein sequences or across the whole proteome were compared with the observed repeat occurrences. we found that with all three methods and in all eight species the correlation between observed and expected repeat counts decreased above a saar length threshold. the percentage of saar proteins for each amino acid also exhibited variability among species when both the repeat length and counts were taken into account. however, clustering based on saar characteristics generally reflected the known phylogenetic relationships between species. our comprehensive bioinformatics analyses reveal that saars show amino acid-specific occurrence patterns with respect to species as well as saar length. tissue proteins play important roles in biological metabolic processes. the qualitative and quantitative analysis of tissue proteins facilitates the understanding of molecular mechanisms that differentiate between physiologic and pathologic states. health and research institutions routinely prepare formalin-fixed paraffinembedded (ffpe) tissue blocks for histopathology. proteomics on ffpe tissue still requires standardization of tissue solubilization processes to overcome variability in protein extraction results. our aim is to compare the proteomic studies of fresh frozen and ffpe rat renal tissues. fresh frozen and ffpe preparations from renal tissues were included in this study. an adult rat was sacrificed and the dissected kidneys were divided two equal section. one immediately frozen in phosphate buffer, and the other tissue specimen not thicker than mm to allow rapid penetration of the fixative put in % buffered formalin for hours. the fresh frozen tissue was dissolved and homogenised in the cold phosphate buffer solution containing protease inhibitors. paraffin blocks were performed from formalin fixed tissue specimens. we have extracted the protein from the ffpe tissues using our previously verified method. we have utilized electrophoresis three times to compare protein yield, number, intracellular and intercellular of homogenised samples obtained from ffpe and fresh frozen kidney samples. the number of proteins identified from fresh frozen kidney tissue has generally been shown to be increased compared with ffpe tissue. decrease of the qualitative results in electrophoretic bands was found similar in all replicative studies. ffpe tissues undergo extensive cross linking between protein/ dna/rna molecules during formalin fixation, which creates inter-molecular crosslinks. on the other hand, ffpe tissues represent a valuable resource to carry out retrospective studies aimed to biomarker discovery in kidney cancer as well as other kidney diseases. background: development of atrial fibrillation (af) during the course of chronic primary mitral regurgitation (mr) is common and represents complex molecular mechanisms. however, the gene expression profile of human atrial fibrillation (af) in the setting of chronic primary mr remains uncharacterized. in the current study, we aimed to compare the gene expression profiles of patients with severe degenerative mr in sinus rhythm (sr) and af. methods: left and right atrial tissue samples were obtained from patients with chronic primary severe mr in permanent af (n = ) and sinus rhythm (n = ). we performed a novel micro-dissection technique for thin sections of atrial tissue samples and immediately fresh froze intra-operatively. transcriptomes of left and right atrial appendages of degenerative mitral regurgitation patients with sr versus af were compared by microarray analysis on affymetrix hgu- plus platform. bioinformatics, data mining and pathway analyses were conducted on partek gs and webgestalt. genome-wide gene expression profiles were compared between af and sr groups among . transcripts representing . well-characterized human genes. differentially regulated genes were evaluated according to fold change (fc ≥ . ) with a p-value ≤ . . results: most remarkable pathways altered in af atrial tissues compared to sr group, were extracellular matrix-receptor interaction; mapk, adipocytokine, and calcium signaling; apoptosis and cardiac muscle contraction pathways. conclusions: this is the first human study of comparative transcriptomics in left and right atrial tissues of patients with af versus sr associated with severe degenerative mr. the main findings of this multidisciplinary translational research provide novel candidate targets for the treatment and prevention of af. in order to acquire iron under iron-limiting growth conditions, bacteria employ specific mechanisms such as production and secretion of siderophores. siderophores are low molecular metalchelating compounds that contribute not only to iron scavenging, but also participate in other important processes including oxidative stress response and cell signaling. serratia marcescens, gramnegative bacterium, could be found in various environments, including wastewater, plant rhizosphere and hospital setting where s. marcescens can cause serious life-threatening infections. in this study, we performed a detailed characterization of the siderophores of the clinically important pigment-free s. marcescens strain sr - and environmental pigment-producing s. marcescens strain sm . bioinformatic analysis of these genomes by antismash software revealed the presence of several clusters involved in non-ribosomal peptides synthesis (nrps). we found four nrps clusters in genome of s. marcescens sm . cluster has a low level of identity to enterobactin gene cluster typical for bacteria producing catechol-like siderophores. second cluster has only % of identity to xantholipin biosynthetic gene cluster. clusters and of nrps genes of s. marcescens sm did not show any homology to known nrps clusters. in contrast, the genome of s. marcescens sr - contains only one genetic cluster of nrps genes. this cluster does not have similarity to any of the known bacterial nrps genes. thus, genetic analysis of two isolates of s. marcescens allowed us to identify nrps genetic clusters and showed that the repertoire of these genes is different between strains. we hypothesized that the strain isolated from environment has competitive advantage over clinical isolate due to genetic diversity of siderophores. on the other side, clinical isolate has specific genetic cluster of siderophores which may promote s. marcescens growth and adaptation to the extreme niches present in medical facilities. p- . . - the first glance on the genome's structure and activity in hibernator edible dormouse hibernation is a unique adaptive way of survival in extreme environmental conditions where mammals decrease their metabolic rate and demonstrate physical inactivity for prolonged periods of time (up to - months). remarkably, some hibernating animals have a long average lifespan and the ability to avoid muscle atrophy caused by disuse or immobilization. to identify main molecular pathways behind the protective musculoskeletal adaptation and genome structure in hibernator edible dormouse (glis glis), whole-genome analysis of mrna expression in muscles (m. soleus and m. edl) and lumbar spinal cord samples was conducted. three groups of the dormice: ) active animals ) hibernated animals and ) animals immobilized for weeks in laboratory, were examined. rna libraries have been sequenced using hiseq illumina platform. coupled with genome dna sequencing provided x coverage of the estimated genome, we have assembled de novo transcriptome of the dormice. differentially expressed genes in response to immobilization and hibernation were determined. transcriptional program of these phenotypes was similar. pathways enriched by differentially expressed genes were identified. gene expression of the key muscle proteins and muscle atrophy markers was analyzed. muscle-specific e -ubiquitin ligases murf and mafbx revealed no changes in mrna expression. our study represents the first attempt to elucidate changes in transcription profiles of skeletal muscles and spinal cord during hibernation and hypokinesia in edible dormice. in corroboration to the gene expression data, they demonstrated minimal morphological evidence for muscle disuse atrophy during physical inactivity. edible dormice, thus, can be considered as a novel model organisms in investigation of the genetic mechanisms of hibernation and prevention of muscle atrophy. the work is performed according to the russian government program of competitive growth of kfu and supported by rfbr jsps_a no. - - . in response to diverse environmental cues bacteria form complex structured communities called biofilms. the metabolic pathways activated by these cues are remarkably different depending on the species studied. however, they all lead to the formation of an extracellular matrix that holds the cells together. non-pathogenic gram-positive spore-forming soil b. subtilis strain is recognized as a model system for the study of biofilms. to discover the pathways regulating biofilm formation in b. subtilis, we studied the natural isolate of b. subtilis strain , and constructed the recombinant strains with knocked out genes of following regulatory proteins: abrb (global transcriptional regulator), degu (two-component response regulator of signal transduction system degs-degu), ccpa (regulator of carbon catabolism) and spooa (regulator of sporulation). in the minimal medium broth b. subtilis wild-type strain forms biofilm with its maximum on th hour of culture growth. ph-optimum for biofilms formation by the wild-type strain is in the range of . - . . the temperature optimum is in the range from °c to °c. this corresponds to the natural conditions of the b. subtilis habitat in rhizosphere. the level of biofilm formation by regulatory mutant strains with deleted abrb, degu, ccpa, spooa genes is on average % lower than by the wild-type strain. this indicates that global regulatory system controlls biofilm formation process. statistically significant differences in the levels of biofilm formation between regulatory mutants haven't been identified. ph and temperature optima of mutant strains are the same as for the wildtype strain - , - and °c - °c respectively. the crataegus genus which is a member of rosaceae family, has approximately species worldwide and species in turkey. all plant species in this genus have the common name "hawthorn". crataegus microphylla (c. microphylla) c. koch which is characterised by having erect sepals in fruit and smaller leaves in comparison with the other species, is one of the wild edible fruits in turkey. crataegus species have been used as food and also in folk medicine for the treatment of various diseases. for this purpose, the potential biological properties of crataegus microphylla were aimed to reveal by the preliminary work. in this study, prevention of oxidative dna damage using supercoiled pbr plasmid dna, acetylcholinesterase, tyrosinase, a-glucosidase inhibition and antioxidant effects: , diphenyl- -picrylhydrazyl radical scavenging effect, phosphomolibdenum-reducing antioxidant power, ferric-reducing antioxidant power with total phenolic and total flavonoid contents of the c. microphylla leaves, stem barks and fruits that extracted with ethanol, methanol and water were investigated. the experiments of oxidative dna damage studies and antioxidant activities of c. microphylla extracts showed that methanol and ethanol extracts possessed a strong ability to prevent dna damage and significantly antioxidant activities. methanol extracts of stem barks from c. microphylla exhibited the highest acetylcholinesterase and tyrosinase activities ( . ae . % and . ae . %, respectively), at lg/ml. in addition, ethanol extract of leaves from c. microphylla inhibited the a-glucosidase activity significantly when compared to acarbose. this study explained significant antioxidant, enzyme inhibitory, hypoglycemic, and neuroprotective activities of methanolic or ethanolic extracts prepared with stem bark and leaf from c. microphylla and also strong ability to prevent dna damage that corresponded to antioxidant potential of methanol extracts of leaf and stem bark. the yarrowia lipolytica species (yl) is nonconventional yeast widely used for recombinant protein expression due to its system of post-translation protein modification, which is the most similar to that of higher eukaryotes. yl appears the promising producer of recombinant proteins with much more complicated molecules compared to those of prokaryotic producers. however, an important feature for a producer strain of recombinant proteins is the genes, the expression of which undertakes under controlled conditions, and consequently, search of new effective inducible promoters in the yl genome is of great interest. proteome analysis of the yl cells grown at different ph values ( . , . , . ) showed that under alkaline conditions the amount of mitochondrial porine vdac (voltage dependent anion channel), one of the most abundant protein of the mitochondrial outer membrane, increased significantly. vdac is supposed to let reactive oxygen species out of mitochondria protecting the cell against oxidative stress. therefore, the por gene expression, encoding vdac should increase in the stress conditions. the promoter of the por gene was used to construct some new expression systems based on yl w . a new genetic construct bearing a reporter bgalactosidase gene under control of the por promoter. b-galactosidase activity was assayed in the cells grown in various ph conditions and exposed to exogenous oxidants such as hydrogen peroxide, menadione, and methyl viologen. it was shown, that in h o and methyl viologen treated cells b-galactosidase activity increased . - . -fold reaching its maximum in the cells, grown at ph of . . thus, we demonstrated high inducibility of the por promoter, which is essential for effective action of the expression system based on it and potency of application for transformed lines of producers. acknowledgments: supported by the russian foundation for basic research (grant no - - mol_a). aspergillus nidulans is able to detoxify and catabolize the toxic proline analogue, lazetidine- -carboxylic acid in nature, azc serves as a plant protectant against infections and consumption. we have obtained evidence that azc is not only non-toxic for the model ascomycete aspergillus nidulans, but it can be utilized as a poor nitrogen source. in order to elucidate the molecular mechanism underlying azc detoxification, we have constructed and studied a. nidulans strains deleted in the cognate genes involved in azc detoxification in pseudomonas and saccharomyces cerevisiae. these genes, found by in silico analysis, encode a putative hydrolase, acha, and an azc acetyltransferase, ngn , respectively. gene deletion was accomplished through double crossover. a cassette containing the~ bp ' and ' flanking sequences of each gene, with the afpyrg gene as a selection marker, was contructed. crossing the achad and the ngn d strains isolated the achad ngn d double mutant strain. rt-pcr was used for gene expression analysis in the wild type strain, area-loss of function and crea-derepressed mutant strains. our results clearly show that azc can be used as a poor nitrogen source by a. nidulans. this utilization requires a) acha, a putative azc hydrolase, and b) a fully active gaba catabolic pathway, as lack of either amdr or gata abolishes azc utilization. most importantly, the double mutant, achad ngn d, shows azc toxicity, suggesting that ngn is a true orthologue of mpr , able to detoxify azc, a phenotype that can be observed only in the absence of acha. as a final point, ngn was shown to be induced by the presence of azc and and to be under nitro the spatial genome organization plays a great role in the maintenance of the nuclear architecture and regulation of all processes occurring in the nucleus. this system is controlled by a set of special proteins having an architectural function. however, the mechanisms of their action remain unknown. among these proteins are, in particular, zad-domain-containing proteins. zinc finger-associated domain (zad) is a ubiquitous motif of c h zinc finger proteins of drosophila. genes that encode zad proteins are specific for and expanded in the genomes of insects. only a few zad-encoding genes have known functions, and the role of zad is being discussed. up to date there was only one known crystal structure of zad-domain from drosophila transcription factor grauzone (grauzad). here, we present for the first time the crystal structure of the zad-domain of serendipity-d transcriptional activator of the egg-polarity gene bicoid. zad-domain was cloned, overexpressed in e. coli, purified and the structure was solved at . a by mad technique. detailed analysis of the structure proved that the protein exists in dimeric form and revealed unique spatial organization of the protein, different from those for grauzad. this work is supported in part by russian ministry of education and science grant ( . . . ). mycoplasma gallisepticum is a convenient model object for studying the regulation of transcription because it has a reduced genome, lack of cell wall and many metabolic pathways and also easy to culture and non-pathogenic to humans. due to the nature of the genomic organization and the loss of many of the known regulators, the effect of disrupting the function of some proteins may be a useful tool for studying the regulation of transcription. the gene expression study was performed on agilent onecolor microarray with custom design and random-t polymerase primer for cdna synthesis. microarray represents probes for orf including genes and ncrna. in this work, we have investigated the effect of changes in the level of gene expression of m. gallisepticum for two different types of conditions: a genetic knock-out mutants and the cell response to treatment with sub-lethal concentrations of antibiotics. we characterized transcription of m. gallisepticum when the cell responses to dysfunction of proteins with metabolic potential, possible regulators of expression, in violation of permeability of membrane by cccp, inhibition of ribosomal synthesis by tetracycline, dna gyrase by novobiocin and atp synthase by oligomycin. the data obtained allow to characterize the transcriptional response under different conditions and to identify groups of genes that change expression together. major transcriptional changes were observed in the response of cells under cccp treatment due to uncoupling of the proton gradient and further reducing the membrane potential, as well as under novobiocin treatment due to changing the topology of dna. global problem of oil pollution forces scientists to search for a new safe remediation technologies constantly. careful attention is paid to bacteria, some of which possess additional biotechnologically valuable properties, such as utilization of hydrocarbons and production of biofurfactants. in this regard, we carried out proteogenomic characterization of tsukamurella tyrosinosolvens strain ps , which was isolated from chemical sludge and capable for alkane degradation and biosurfactant production. whole genome of the strain was sequenced on the miseq (illumina) platform, assembled and annotated. proteome on mineral medium with glucose, sucrose and hexadecane as a sole carbon and energy source was studied. shotgun proteomics approach was performed on hybrid chromatography-mass spectrometry machine (maxis impact). alkane oxidation genes (alkane- -monooxygenase, rubredoxin and rubredoxin-reductase) under genome sequence, as well as two pathways of trehalose synthesis and genes for mycolic acids production were found. emulsification activity of cell-free culture liquid was about four times higher on hexadecane in comparison with sugars. proteomic profile was different at various culture conditions. all glycolysis genes, beginning with glucose- -phosphate isomerase to pyruvate kinase, were found on the media with sugar. the medium with hexadecane helped to reveal enzymes involved in the beta-oxidation of fatty acids, for example , -dienoyl-coa reductase, -ketoacyl-coa thiolase and enzymes of the initial mycolic acid synthesis pathways. thus we have established that the strain t. tyrosinosolvens ps utilizes sugar by glycolysis. also, the bacterium is capable for alkane oxidation followed by beta-oxidation of fatty acids. based on the proteogenomic data, we assume that the bacterium is able to synthesize trehalose lipids, namely, trehalose mycolates. obtained results could be useful to create conditions for increased biosurfactants production. gestational diabetes mellitus (gdm) is a glucose intolerance firstly diagnosed during pregnancy. in this study, we aimed to investigate the association between serum adiponectin, resistin levels and insulin resistance in gestational diabetic patients. a total of patients; healthy pregnant women (control group) and pregnant women diagnosed with gdm (gdm group) were included in this study. serum adiponectin, resistin, glucose, insulin, hba c levels and lipid parameters were measured. insulin resistance index homa-ir values were calculated. in this study, serum glucose, insulin, hba c levels and homa-ir were significantly higher in gdm group compared to the control group (p = . , p = . , p = . , p = . , respectively). serum adiponectin levels were significantly lower (p < . ); whereas serum resistin levels were significantly higher (p = . ) in gdm group than in the control group. it can be concluded that resistin contributes to the formation of insulin resistance, adiponectin plays an important role in the regulation of this resistance and they also have effects on gdm pathophysiology. hematological cancers including acute myeloblastic leukemia (aml) and acute lymphoblastic leukemia (all) in terms of incidence and mortality, are the second most important cancer type in turkey. numerous studies show that cancer patients respond differently to treatment thus supporting the idea of personalized therapy need for individuals. renin angiotensin system (ras) have key roles in aml and all progression and it has been shown by many studies suggests that these system's genes might be good biomarkers for aml and all personalized therapy. we aimed to identify ras gene based homogeneous subgroups of acute leukemia and determine the most effective chemotherapoetic agent for each subgroup. after validation and verification of the results, more effective drugs can be recommended for the use in clinics for chemotherapy of aml and all. results of our preliminary studies showed that we are able to identify subgroups of aml and all as well as correlating each existing subgroup with fda approved drugs. considering the long and highly cost process of developing new drugs for cancer treatment makes the present study all the more valuable. in addition, there is a serious need for change in aml and all therapy since there is no highly effective chemotherapy protocol available for their treatment. welcome trust sanger (wts) and cancer cell line encyclopedia (ccle) databases will be used to determine subgroups of aml and all based on ras genes or whole genome expression using standard deviation and hierarchical clustering analysis. the most effective drugs for each subgroup will be identified using pearson's r correlation analysis with drug sensitivity data (ic , ic , amax, aare, etc.) available in same databases. further validation tests will be performed by in vitro validation using aml and all cell lines: drug sensitivity profiles will be determined and gene expression will be shown by q-rt-pcr. p- . . - functional polymorphisms of ephx in a turkish population h. pinarbasi, i. sari cumhuriyet university, sivas, turkey soluble epoxide hydrolase (seh; ec . . . ) is encoded by ephx and catalyses the degradation of endogenous fatty acid epoxides generated by cyp epoxygenases. these fatty acid epoxides such as epoxyeicosatrienoic acids (eets) have been shown to posses vasodilator, anti-inflammatory, anti-platelet, anti-hypertensive, anti-apoptotic, anti-thrombotic and natriuretic effects. it has been reported that eet levels are associated with hypertension, stroke and cardiovascular diseases. individual differences in the ephx gene that affect the seh activity may alter the circulating levels of eets. k r and r q polymorphisms have been known to cause increased and decreased seh activity, respectively. therefore we aimed to determine the genotype frequencies of these two polymorphisms in a turkish population. k r and r q polymorphisms were determined by the real time pcr using double-dye hydrolysis probes or pcr-rflp method. the observed genotype frequencies for k r polymorphism were . % wild type (aa) and . % polymorphic genotype (ag+gg) and for r q polymorphism . % wild type and . % polymorphic genotype (ga+aa). the genotype distributions for both polymorphisms were in hardy-weinberg equilibrium. pregnancy is one of manifestations for thrombophilia factors, which in its turn leads to various complications of its course. one of the markers of hereditary thrombophilia is mutations in the folate cycle mtr, mtrr and mthfr genes. insufficient intake of folate during pregnancy disrupts the functioning of the genome, leading to miscarriage, violation of embryogenesis and various fetal malformations. however, results of studies on the role of hereditary thrombophilia in the occurrence of complications during pregnancy are rather contradictory. aim of this study was to determine the frequency of alleles and polymorphic variants of folate cycle genes mtr a g, mtrr a g and mthfr c t in women of kazakh ethnic group with pregnancy complications. we used real-time pcr. blood samples for dna isolation were obtained from pregnant women. the main group consisted of women (n = ) which had a history of two or more pregnancy complications in the form of pre-eclampsia, eclampsia, missed abortion, miscarriage, and etc. control group consisted of women (n = ) with two or more normal pregnancy outcomes, and had no complications during pregnancy in history. average age of women in experimental group was . ae . years compared with control of the age . ae . . the analysis of the frequency distribution of alleles of genes in experimental group of women with complications of pregnancy revealed no significant differences relative to the control group. analysis of the distribution of polymorphic variants of folate cycle genes showed significant difference between the study and control groups in the occurrence frequency of heterozygotes for the mutant allele g in the gene mtrr a g (or = . , ci % = . - . ; v = . , p < , ). no significant differences in alleles between homozygous wild-type and homozygous mutant alleles were observed. this work was funded by the mes kazakhstan (gr rk project number). p- . . - a study on the association between rs polymorphism in connective tissue growth factor gene and pseudoexfoliation syndrome pseudoexfoliation syndrome (pes) is a disorder of the extracellular matrix characterized by the production and progressive accumulation of an abnormal fibrillary material in many ocular tissues. pes prevalence is . % above the age in turkey. since pes is characterized by excessive synthesis of elastic microfibrillar components throughout the body, growth factors can have important roles in the pathophysiology of pes. human connective tissue growth factor (ctgf) is a protein expressed in a variety of tissues, including the anterior chamber of the eye. ctgf coding gene has several genetic polymorphisms. rs g/c single nucleotide polymorphism (snp) is found at position À , in promoter region. the presence of a c allele for rs is critical for transcriptional suppression of the ctgf gene which would reduce ctgf production. aim of this study was to investigate if there is any association between pes and rs polymorphism of the ctgf gene. study population consisted of patients with pes and controls. blood samples were collected by g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. genomic dnas were isolated from whole blood samples using manual dna isolation. the frequency of ctgf rs polymorphic allele g was . in patients, and . in controls ( . , p = . ). distribution of genotypes was gg: . %, gc: . % and cc: . % among patients, while gg: %, gc: . % and cc: . % (or = . , p = . ) in controls. statistical analysis showed that there is no significant relationship between ctgf rs snp and pes. these are the preliminary findings of a research project which is the first study analyzing the relationship between ctgf rs snp and pes. this work did not point out a role for ctgf rs in the risk for pes. a significant relationship might be found when the study population is enlarged. p- . . - evaluation of rs single nucleotide polymorphism of clusterin gene in pseuodoexfoliation syndrome risk pseuodoexfoliation syndrome (pes), an age-related systemic disorder, is characterized by production and accumulation of abnormal fibrillar extracellular material in anterior structures of the eye. clusterin (clu) is a multifunctional glycoprotein produced and secreted by almost all cell types and is found in all body fluids and in accumulated pes material. under cellular stress conditions, clu provides inhibition of stress-induced precipitation and aggregation of misfolded proteins. clu expression level in pes patients is unexpectedly low and this could be due to single nucleotide polymorphisms (snp) on the gene coding for clu. rs c/t polymorphism has been found to be associated with alzheimer's disease and pathophysiology of alzheimer and pes are similar. this study aimed to determine whether rs snp of clu gene have a role in the development of pes. study population consisted of patients with pes and controls. blood samples were obtained from g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genomic dnas were isolated from whole blood of subjects using manual dna isolation. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. t allele frequency of pes patients was . and that of controls was . ( . , p = . ). the distribution of genotypes was cc: . %, tc: . % and tt: . % among patients while cc: . %, tc: . % and tt: . % ( . , p = . ) in controls. there was no statistically significant difference between pes patients and controls in terms of tt genotype and t allele frequency. these are the preliminary findings of a research project which is the first study analyzing the relationship between clu rs snp and pes in turkish population. this work did not point out a relation for polymorphic genotype in the risk for pes. however, a relationship between clu rs polymorphism and pes can be found when we enlarge the study population. the tumor suppressor tp is the most frequently mutated gene in head neck squamous cell carcinoma cancer and represents a known transcription factor and tumor suppressor gene that regulates different microrna and target genes. the aim of our work is to construct the transcriptional and post-transcriptional network regulated by tp and to evaluate the difference at mrna and protein expression levels of the tp target genes in hpv negative head and neck squamous cell carcinoma (hnscc) patients with distinct tp mutation states and to elucidate the molecular mechanism that underlie the poor prognosis of tp mutation. to show the tp mutation landscape and its prognostic relevance for survival, we used cbioportal for cancer genomic analysis. we downloaded mutational profiles of hpv negative hnscc patients. employing different databases we constructed the tp regulatory network. and then, to evaluate the effect on mrna, protein and microrna regulated by tp we used the mrna and protein expression profiles of patients from tcga. our results show that hotspot, truncating and missense mutations have statistical significance in the univariate analysis. the tp regulatory network show the involvement of important target involved in the progression of hnscc and the deregulation of protein expression of an important key epigenetic modifier ezh was significantly associated with tp mutational state. ezh is a member of the polycomb group protein enhancer zeste homolog which is known to be directly repressed by tp and indirectly by the activation of mir- a and mir- b. we found a significant up-regulation of ezh that depend from tp mutation. it is important to understand the difference in mrna and protein expression of tp regulatory network that could depend from its mutational state. this finding suggest that ezh might be a potential therapeutic target for hnscc. p- . . - next generation sequencing based approach for monitoring of minimal residual disease in acute lymphoblastic leukemia minimal residual disease (mrd) monitoring is widely used to evaluate efficiency of chemotherapy and to choose a strategy for further treatment in acute lymphoblastic leukemia (all). the most commonly used approaches for mrd detection are based on flow cytometry and qpcr. these methods have several important limitations including insufficient sensitivity, complicated experimental setup and false positivity. the newly developed next generation sequencing (ngs) approaches could overcome the existing limitations in mrd monitoring. here we describe a new mrd monitoring approach based on targeted deep sequencing of malignant rearrangements. first, we identified bcr/tcr rearrangements specific for the leukemic clones in initial bone marrow samples of all patients. for this, we used sanger sequencing of the products of multiplex pcr, performed with bcr/tcr specific primers combined according to the optimal frequency distribution of v/j-genes in healthy donors. second, we analyzed concentration of malignant clone rearrangements, identified at the first step, in dna samples obtained from bone marrow after days of treatment. for this purpose, we performed ngs of libraries for each identified leukemic rearrangement. four libraries were amplicons of bcr or tcr gene rearrangements generated using characteristic v and j segment specific primer combination. six additional libraries were amplicons of the same primer combination from the same dna sample which contained initial leukemic dna spike-in (in concentrations corresponding to per , and , cells) for a calibration curve generation. using this approach, we analyzed all clone specific rearrangements for three patients and calculated concentration of the leukemic clones by using the calibration curve. for one patient we didn't find any leukemic cells and for two patients we found leukemic cell per , analyzed cells. znf is considered as a transcriptional target for p and plays an important role in the homologous recombination, mitosis, centrosome dynamics. as was shown by gwas some snps in znf have strong association with the risk of breast cancer (bc). however, it was unclear whether the same snps are associated with risk of bc in kazakhstan. therefore two polymorphisms (rs , rs ) of znf were investigated in kazakh population in this study. the present case-control study was carried out with participation of kazakh females with bc and cancer-free donors. additionally, subtypes of bc, stratified by estrogen receptor (er+/À), progesterone receptor (pr+/À) and human epidermal growth factor receptor (her +/À) status were estimated. pearson p-value, odds ratio, % confidence interval tests were applied to data analysis. significant differences were found in allele frequency and genotype distribution at rs locus in znf between the patients and control groups (p = . for allele; p = . for genotype). moreover, significant association with bc was revealed for rs after dividing patients according to er+/ À, pr+/À and her +/À status of the tumor. the g allele was associated with er+ (p = . , or = . , %ci: . - . ), pr+ (p = . , or = . , %ci: . - . ) and gg genotype with her -bc carriers (p = . , or = . , %ci: . - . ). also, g allele can be considered as a risk factors in er+/ pr+/her -luminal type of tumor (p = . , or = . , % ci: . - . ). our findings correlate with the data of several gwas where the association of the rs polymorphism with higher mammographic density and the risk of breast cancer have been shown. the obtained results allow us to consider g allele and gg genotype of rs as a marker of bc risk with predictive value, restricted to er, pr and her status of the tumor in the kazakh population. breast cancer (bc) is the most common cancer among women in most of countries. alternative variants of low-penetrance genes such as fgfr (rs ), tox (rs ), map k (rs ), lsp (rs ) are shown to have high frequency in north america, south-east asia, australia, europe populations and a multiplicative effect on the development of bc. in this study was investigated assosiasion between alleles/genotypes combinations of these genes with increase/decrease of bc risk. the case-control study included bc patients and healthy women from kazakh and russian populations. genotyping was performed by pcr-rflp methods. combined effect of allele and genotype variations in four different genes on bc risk was assessed by apsampler algorithm. the fisher exact test, odds ratio (or) with % confidence intervals ( % ci) were applied to data analysis. according to obtained results combinations of allele c of tox rs and a of map k rs (p = . , or = . ), also allele c of tox rs and c of lsp rs (p = . , or = . ) associated with increased bc risk in the russian population. consequently, combinations with c allele of tox rs contribute significantly to bc risk with p-value = . , or = . . on the contrary, tt genotype of tox rs with p = . , or = . and its combination with allele t of lsp rs with p = . , or = . determine a bc risk reduction in russian population. in addition, a risk combination of allele c of lsp rs and a of map k rs was found in kazakh population (p = . , or = . ). studies have shown that a genetic predisposition to bc can be determined by the cumulative effect of individual alleles and genotypes and possible epistatic interactions of studied genes. obtained combinations of alleles and genotypes can be considered as complex genetic markers of bc and may be used as predictive. cancer that is caused by excessive proliferation of cells and reduced apoptosis is a pathological condition. currently, studies that are comitted with breast cancer are great important early detection and diagnosis of breast cancer. after the discovery of cisplatin as chemoterapy drug, new transition metal based complexes have been developed for treatment of cancer. in this study, anti-cancer activity of azo-azomethide ligand and its mononuclear metal complexes is studied on human cancer cell lines (mcf- ) and mouse fibroblast (l ) cell lines. cells were studied four different concentrations ( , ; ; ; lm). xtt ( , -bis-( -methoxy- -nitro- -sulfophenyl)- h-tetrazolium- -carboxanilide) protocol was applied after and hours. in our study % fetal bovine serum (fbs), % l-glutamine, iu/ml penicillin and mg/ml streptomycin in dmem (low glucose) were used. cancer cell lines in dmem medium is produced in % humidity and % co incubator at °c. anti-cancer activity of synthesized complexes were determined on mcf- and l . in the biological activity studies, synthesized compounds showed higher anticancer activity than positive control ( -fu). finally, our new synthesized complexes can be suggested that potent ajan for anti-tumuour for breast cancer drugs. large interindividual differences in response to chemotherapy present an important issue in cancer treatment. malignant mesothelioma (mm) is an aggressive tumor with poor prognosis, treated mostly with gemcitabine/cisplatin (gem/cis) or pemetrexed/cisplatin (pmx/cis) chemotherapy. as both clinical characteristics and genetic variability may affect treatment outcome, our aim was to construct and validate clinical-pharmacogenetic prediction models of treatment outcome in mm for both chemotherapy regimens and to develop an algorithm for genotype-based treatment recommendations. clinical-pharmacogenetic models were built on gem/cistreated and pmx/cis-treated mm patients. pharmacogenetic scores were assigned by rounding the regression coefficients. gem/cis model was validated on independent mm patients. model predicting outcome of gem/cis chemotherapy included crp level, histological type, performance status, rrm rs , ercc rs , ercc rs , and xrcc rs . values ranged between and . ; cutoff value of . had sensitivity of . and specificity of . . patients with higher score had shorter progression-free and overall survival (p < . ). in the validation group, positive predictive value was . and negative predictive value was . . model predicting outcome of pmx/cis chemotherapy included crp level, mthfd rs , and abcc rs with scores ranging between and . . cutoff value of . had sensitivity of . and specificity of . . patients with higher score had lower probability of good response and shorter progression-free survival (p < . ). clinical-pharmacogenetic models could enable stratification of mm patients based on their probability of response to gem/cis or pmx/cis and improve treatment outcome. this approach could be used for translation of pharmacogenetic testing to clinical practice as it would facilitate the selection of the best treatment option for each patient. p- . . - evaluation of anti-diabetic potential of circiliol and circilineol using caco cell line diabetes mellitus is a metabolic disorder, and many people are suffering from this disease in the worldwide. oral hypoglycemic agents such as sulfonylureas and biguanides are currently used for the treatment. however, studies searching for a more effective anti-diabetic agents are being carried out continıously. based on that we aim to investigate the potential anti-diabetic effects of circilineol and circiliol isolated from teucrium alyssifolium extract, using in vitro cell culture models. for this purpose, the anti-diabetic actions were investigated by applying model caco (colorectal adenocarcinoma) cell line. we determined the level of ag (alpha-glucosidase), sglt (sodium-glucose transporter- ) and glut - and glucose transport. neither ag activity nor sglt activity was increased with either circiliol or circilineol treatment in caco cells compared to positive control. similarly, neither the activity nor the expression level of glut *- was increased in caco cell line with either circiliol or circilineol treatment relative to control. in conclusion, these results strongly suggest that circiliol and circilineol do not possess any anti-diabetic potentials.supported by tubitak z and paubap fbe the purpose of this study was to characterize and assess the impact of a novel magnetite (fe o ) nanosystem functionalized with the natural origin compound eugenol (e) on the pseudomonas aeruginosa virulence, biofilm formation and qs signaling in order to advance research aimed to find alternative and personalized therapeutic approaches for severe infections produced by this opportunistic pathogen. fe o nanoparticles were obtained by a co-precipitation method and functionalized with analytical purity e. functionalized nanoparticles (fe o @e) were characterized by ir, sem, tga and hr tem. one laboratory and p. aeruginosa clinical isolates were utilized in the study. growth and biofilm formation were assessed by an adapted microdilution method followed by absorbance reads and viable count analysis in dynamics ( , , and hours of treatment). soluble virulence factors production was assessed by enzyme activity evaluation of bacteria grown on specific media. the expression of qs core genes was analyzed by qrt-pcr and a luminescence assay. results demonstrated that the average size of the obtained nanosystem ranges - nm, particles are relatively homogenous and have a low tendency to form aggregates. subinhibitory concentrations of fe o @e limited biofilms formation in a time and strain dependent manner, and significantly inhibited the production of toxin pore forming enzymes (haemolysins and lipases) in most strains. the expression of lasi and lasr genes was three fold downregulated, while the expression of pqsr was upregulated in planktonic cultures suggesting that pqs signaling may be involved in virulence modulation after nanoparticle stimulation. the modulation of bacterial virulence and molecular signaling by functional nanoparticles utilized in subinhibitory amounts offer valuable perspectives to develop personalized antimicrobial approaches based on molecular communication control that clearly modulate pathogenicity and progression of the infectious process. p- . . - specimen processing and handling for plasma ammonia measurement b. sarac ßligil , , s. abus ßo glu , f. aky€ urek , b. € ozt€ urk selc ßuk medical school, konya, biochemistry department, selcuk university faculty of medicine, konya, turkey objectives: ammonia requires special processing and handling conditions due to its' unstability. in this study, our aim to investigate a preanalytical factor (delayed analyze) affecting plasma ammonia measurement. design and methods: blood samples were obtained from healthy volunteers. for determining different handling and storage conditions, the following protocols were applied: first protocol (a) transportation on ice and separation (centrifugation at °c) within minutes of collection and analyze immediately. second protocol (b) transportation on ice and separation (centrifugation at °c) within minutes and analyse refrigerated - °c hours (a , b ) and hours (a , b ). all plasma ammonia levels was analyzed enzimatic glutamate dehiydrogenase methods by abbott architect c clinical chemistry analyzer. results: there were statistically alterations in all protocols compared to first protocol. prolonged centrifugation time for plasma ammonia lead to have higher results ( . versus . lg/dl, p < . ). in all protocols including a , a , b , b also cause an elevation in plasma ammonia results ( . , . , . and . ; p = . , p < . , p < . , p < . , respectively). conclusions: ammonia concentration in the blood sample increases over time due to high concentrations in cells as erythrocyte or platelets (three fold). blood samples collected for ammonia determination should be stored on ice, and measured immediately. the wnt/b-catenin signaling pathway has been considered to be a factor in the development and progression of colorectal cancer. many studies have demonstrated that the presence of mutations or polymorphisms in ctnnb (gene encoding b-catenin; mostly mutations in exon ) can lead to aberrant activation of wnt/bcatenin signaling at the onset of various types of malignancies, including colorectal cancer. the aim of our study was to assess ctnnb alteration in the patients with colorectal cancer and compared their tumor and normal tissues. a total of paraffin-embedded colorectal tumor specimens were obtained from department of pathology in cerrahpasa medical faculty. also a total paraffin-embedded normal tissue was used from same cases as a control group. ten-micrometer-thick tissue sections were placed on a glass slide and stained with he. then the tissue sections were dehydrated in graded ethanol solutions and dried without a cover glass. dna was extracted from the tissues with ll of extraction buffer at °c over night. the tubes were boiled for min to inactivate the proteinase k. ctnnb exon was amplified by pcr. sscp is used to observe any difference between the groups. genomic dna was isolated as described above. ll of each pcr products mixed with ll denaturating buffer and were denatured by heating at °c for minutes in, and then were rapidly cooled on ice. the denatured pcr samples are run on % acrylamide/ bis gel in . tbe buffer for . hours at v at room temperature with water cooling system. the gel was stained with silver staining method. migration and adhesion involve continuous modulation of cell motility, beta-catenin play major roles. beta-catenin gene alterations frequency range between and % in colorectal cancer according to the different published studies. in our study no significant differences were found in the ctnnb exon between the tumor group and normal groups. p- . . - theranostic approach in agressive recurrent meningiomafirst experience in turkey m. o. demirkol , b. uc ßar koc ß university, istanbul, american hospital, istanbul, turkey meningiomas arise from the meningothelial cells of the arachnoid membranes of the leptomeninx, which are attached to the inner layer of the dura mater. meningiomas can be classified into three grades (i-iii): grade i meningiomas which are benign, exhibit slow growth; grade ii (atypical) and grade iii (anaplastic) meningiomas, which have a much more aggressive clinical behaviour. meningiomas express non-steroid hormones, including somatostatin. in the brain, somatostatin-a cyclic tetradecapeptide neuropeptide-is believed to act as a neurotransmitter and neuromodulator. somatostatin performs its physiological functions by binding to specific receptors (sstri-sstrv). sstr exhibit high affinity for octreotate (tate). tate is a polar, watersoluble peptide that does not penetrate the intact bbb (brain-blood barrier). pet and scintigraphic imagings can only demonstrate somatostatin receptor positive intracranial lesions if the bbb is disrupted. in this aim, dotatate (dota-dphe , tyr -octreotate, tate) has been labelled with the positron emitter ga and the beta and gama emitter lu. in this case, we conducted a study to evaluate peptide receptor radionuclide therapy (prrt) planning based on pet/ct imaging of meningioma in the department of nuclear medicine and molecular imaging at the amerikan hospital. [ ga] dota -tyr -oc-pet/ct has been established as the imaging modality of choice for the diagnosis and management of patient with skull-base malign meningioma (rapid progress -to mm. from mm. in d.-after fifth operation). due to its high sstr selectivity, [ lu]-tate showed significantly lower uptake/dose delivered to normal tissues, gatate-pet represents the imaging strategy of choice for an accurate assessment of sstr expression levels. although some studies have not shown a clear advantage over pet/ct, there is some evidence that it will have an advantage in selected body sites such as the head and neck, liver, and the pelvis. p- . . - cardiovascular diseases can be treated by using 'tetr-odd-vp ' and 'hre' hypoxia inducible systems a. celik , t. kaya , s. cigdem , m. g€ und€ uz department of medical genetic, turgut ozal university, ankara, faculty of medicine, turgut ozal university, ankara, turkey ischemia is an insufficient supply of blood to a tissue or organ, usually due to a blocked artery by a blood clot. up to now, the number one cause of death worldwide is caused by ischemia and related conditions such as heart attack or stroke. hif- a is a transcription activator that functions as a master regulator of oxygen homeostasis. hif- a protein levels increase under hypoxic conditions as a result of decreased o -dependent prolyl-hydroxylation, ubiqutination and degradation. we aimed to break up clots in blood vessels and to prevent damage caused by ischemia by using hypoxia inducible systems. we added oxygen dependent degredation domain (odd) of hif a between and in front of tetr dna binding domain and vp transactivation domain, so that tetr-odd-vp or odd-tetr-vp could activate transcription of tissue plasminogen activator (tpa) controlled by tetracycline response element (tre), in a hif a independent manner. in addition, we also designed tissue plasminogen activator (tpa) under control of hypoxia response element (hre) of hif a target genes. western blotting and immunofluorescence assay results showed the expression and nuclear localization of tetr-odd-vp and odd-tetr-vp constructs under hypoxic conditions, but not normoxic. in addition, using fluorometric reporter systems and tpa enzymatic assay we proved functionality of these constructs under hypoxic conditions. final apporoach to our project is predicting kinetic enzymatic activity of tpa during break up blood clots by using matlab. the results of the present investigation showed, the developed hypoxia responsible systems that can be engineered into endothelial cells to prevent ischemia related cardiovascular diseases. p- . . - osteogenic potential assessment of some original scaffolds with magnetic properties new advances in bone tissue engineering demand the development of materials that can not only replace bone, but also regenerate the damaged tissue based on external or even internal stimulus. magnetic materials inside bone scaffolds are known to be a promoting factor for bone healing especially when the therapy is accompanied by application of external magnetic stimulation. based on a recent report, the presence of iron oxide in hydroxyapatite can improve the radio opacity and osteoblast proliferation. in this view, this study focuses on the development of silk fibroin-magnetite biocomposites for potential uses in bone tissue bioenegineering. such novel composites possess good mechanical properties, biocompatibility and biomineralization potential by in vitro tests and could become smart arhiectures, able to stimulate bone regeneration. a new culture model was developed by exposing a d cell/ scaffold bioconstruct to a continuous magnetic field during weeks of osteogenic induction. in this view, mc t -e murine osteoblastes progenitor cells were seeded inside the novel silk fibroin-magnetite biocomposites and subjected to osteogenesys in a magnetic field during days. osteogenic specific markers were evaluated every week in the presence and absence of the field. our results showed that the osteogenic marker's expression started earlier when mc t -e cells were exposed to the magnetic field. consequently, in our experimental model, the magnetic field had a benefic effect on the osteogenic differentiation process as mc t -e cells differentiated more efficiently in its presence. these results suggest that the bone healing process could be improved in the presence of a magnetic field. nevertheless, further in vivo studies on animal model should be employed for validation. p- . . - impact of physical activity performed on different times of day on cardiac and skeletal muscle damage in trained and untrained male subjects s. algul, m. kara, o. ozcelik y€ uz€ unc€ u yil university, van, turkey introduction: physical activity elevates creatine kinase (ck) and creatine kinase myocardial band (ck-mb) levels which have been considered to be an indirect marker of skeletal and cardiac muscles damage. purpose: impact of physical activity performed on different times of day on serum levels of ck and ck-mb were investigated in trained and untrained male subjects. materials and methods: trained (n = , . ae . yr, . ae . kg) and untrained (n = , . ae . yr, . ae . kg) subjects performed three soccer matches ( min) in field ( m versus m) in morning (m), afternoon (a) and at night (n) on separate days. the study protocol was approved by the local ethics committee. venous blood samples were taken at onset and at end of match. serum ck and ck-mb levels are measured using autoanalyser. data are expressed as mean ae s.e., compared by wilcoxon-signed rank and mann-whitney u-tests. p < . was accepted as statistically significant. results: ck and ck-mb levels increased in three matches in both groups (p < . ). importantly, there were significant increases in ck-mb levels in a and n exercises compared to m exercise (p < . ) in trained ( . ae . u/l versus . ae . u/l, % (m) . ae . u/l versus ae . u/l, % (a) . ae . u/l versus . ae . u/l, % (n) and also untrained groups ( . ae . u/l versus . ae . u/l, % (m), . ae . u/l versus . ae . u/l, % (a) . ae . u/l versus . ae . u/l, % (n). discussion: increased metabolic stress or muscle damage during physical exercise elevate serum ck and ck-mb levels. however, higher percentage of increase in ck-mb levels in a and n exercise may reflects additional increases in cardiac muscle stress despite the similar skeletal muscle stress as indicated by ck levels. conclusion: considering the observation of higher percentage increase in ck-mb levels in untrained and also trained subjects, the caution should be taken while performing an exercise in a and n time especially in subjects who has cardiac weakness. p- . . - regional assessment of hematological and discrimination indices of complete blood count for beta-thalassemia screening beta-thalassemia is one of the most common genetic abnormality causing health problems worldwide. blood count and film of beta thalassemia trait and iron deficiency anemia have similar features. therefore, several simple screening indices have been developed for differentiating between these diseases. it was to asaimed to assess the hematological parameters and discrimination indices in patients with betathalassemia trait who admitted to our hospital. the parameters of complete blood count (cbc) in subjects ( males and females) diagnosed by mutational analysis (pcr, gene amplification, dna sequencing) between and , were retrospectively screened and the thalassemia status of patients was assessed in terms of discrimination indices (eng-land&fraser (ef), green&king (gk), mentzer (m), ricerca (r), shine&lal (s-l), srivastava (s), ehsani and sirdah). the percentages of being above the cut off value were detected by ef . %, gk . %, m . %, r . %, s-l . %, s . %, ehsani . % and sirdah . %. the percentages of falsely negatives for the indices of ricerca and shine&lal were lower than others. morever, when the first three common mutations of our study were considered, out of , out of and out of patients were up to the cut off values in terms of e&f, g&k, m, s, ehsani and sirdah indices for ivs-i- (g>a), ivsii- (g>a) and heterozygous codon deletion (-aa), respectively. the molecular diagnosis and prenatal detection for families at risk is important because of the difficulties of treatment in this disease. however, the use of discrimination indices may be valuable for distinguishing of thalassemia trait from iron defiency anemia when the equipment of molecular diagnosis are limited. in our study, ricerca and shine&lal had lower falsely negative results than others. nevertheless, further studies to detect diagnostic perfomance of discriminant indices should be conducted. p- . . - novel therapeutic agents in the development of effective drug combinations to treat glioma s. avdieiev , l. gera , r. hodges institute of molecular biology and genetics, kyiv, ukraine, university of colorado, aurora, co, united states glial tumors are driven by multiple molecular aberrations that cannot be controlled by a single targeted agent. so, it is possible to expect that the combined multitarget anti-cancer therapy aimed simultaneously at different elements of tumor formation mechanisms will be more effective and will promote the extension of patients' life. to find out which drug combinations will enable the development of therapeutic regimens with improved effectiveness and decreased toxicity, the cytotoxic effects of several bradykinin antagonists (ba) were analyzed for different glioblastoma (gb) cell lines. among all the ba under investigation, bkm- appeared to be the most effective, with ic values of lm and . lm in rat glioma c and human glioblastoma u cell lines, respectively. bkm- suppressed erk / and akt phosphorylation in u cells. temozolomide (tmz), the firstline anti-gliomic drug used in clinics, has only a temporary positive effect and severe side effects in gb patients. we showed that the combination of bkm- and tmz led to significant potentiation of tmz cytotoxicity at sub-therapeutic concentrations. recombinant proteins with cytotoxic properties are promising agents for complex therapeutic applications. we revealed that the glioma-associated protein chi l inhibited the viability of u cells more effectively than tmz. furthermore, the combination of chi l and bkm- resulted in an additive cytotoxic effect. chi l -mediated decrease of cell viability was associated with a g /s transition arrest. chi l provoked the dramatic reduction of prb phosphorylation and a significant decrease of cyclin d expression, as well as a substantial increase in p level. in addition to the accumulation of p , we observed the upregulation of cdk inhibitor p . therefore, g /s arrest in chi l -treated cells could be realized via activation of prb, downregulation of cyclin d, and activation of p . harmful hereditary mutations in brca and brca are one of the most important risk factors of breast cancer. the aim of this study is to determine the mutations which are associated with breast cancer in people which is diagnosed breast cancer and/or have breast cancer-diagnosed family members by sanger sequencing and thus provide predictive and prognostic utility. our ongoing study is present the genetic variations in brca and brca genes in breast cancer-diagnosed patients, that one of them is male, and person yet healthy whose brca was sequenced by sanger sequencing. the data were analyzed by using seqscape software . and detected variations were compared with literature. in brca , we determined different benign genetic variations and variation with unknown significance and variation which has not in literature. in brca gene of patient and healthy person, benign variations, variations with unknown significance, variations which has not in literature and mutation were determined. this mutation is c. - delgtca and is located in occr. c. - a>c variation in brca gene, was determined in only male patient.c. t>a variation in exon of brca , was observed in only the youngest patient who has no family member with breast cancer and healthy person. while this variation takes place in literature as variation with 'uncertain clinical significance', an in silico program mutation taster speculated as 'disease causing' for this variation. also, almost all of variations with 'unknown significance' literature knowledge were determined in only one and different cases. this situation increases the possibility of being pathogenic of this variations. the our findings until now can contribute to variations with uncertain clinical significance in the literature. also the variations that have not in the literature but we suggest the possible relation with breast cancer as an estimate may be added to literature by expanding the study. p- . . - inhibition of the recombinant human butyrylcholinesterase with paraoxon and coumarin analog of soman organophosphorus compounds (op) represent a class of extremely toxic chemicals that are used as warfare agents. uncontrolled utilization of op is highly dangerous due to their high potential to be efficient poisons in terrorist attacks. current therapy of op poisoning include intravenous administration of atropine and acetylcholine reactivators however, it does not completely eliminate brain damage effects. alternative experimental therapy against op poisoning is utilization of bioscavengers that irreversibly react with op and rapidly inactivate them. recombinant human butyrylcholinesterase (rhbche) is one of the most promising candidates as bioscavenger due to its pharmacokinetic characteristics and broad spectrum of op neutralizing activity. here we investigated in vitro inhibition of rhbche with two model oppesticide paraoxon (pox) and coumarin analog of soman (gd c ). both op lead to rapid and irreversible inactivation of rhbche that was monitored using ellman assay and fluorescence measurements. bimolecular inhibition rate constants dramatically differ between pox and gd c that could be explained by steric hindrance in soman analog. the next steps forward creation of catalytic bioscavengers based on rhbche should be done based on mechanisms of op-rhbche interactions. this work was performed in frame of grant rfme-fi x . non-hodgkin lymphomas (nhls) represent a heterogeneous group of malignancies that arise from the lymphoid system. at the present time exist a lot of drugs for the nhls therapy, but mostly all of them are unsafe and there is no consensus regarding the best treatment protocol. to increase the efficacy and safety of therapeutic b-lymphocyte depletion in lymphomas and leukemia's it would be preferable to induce the death of pathological b cells without affecting normal b cells to prevent side effects. similar to other types of cancer, nhls arise by a multistep accumulation of genetic aberrations that induce a selective growth advantage of the malignant clone. all b-cells of organism have a unique cell surface markerantigen b-cell receptor (bcr). we generate novel approach for personalized non-hodgkin lymphomas therapy based on peptide specific to malignant cells surface receptor. for this purpose we designed new lentiviral peptide library screening technique based on fluorescent reporter cells system. herein aa peptide library was used for screening of nhl's malignant receptor agonist. patients' lymph nodes biopsy samples mrna was used as a source of malignant bcr nucleotide sequence. variable domain of the lymphoma bcr was used for chimeric receptor generation, where bcr vh/vl part responsible for agonist recognition and bottom part of receptor was retranslate signal to the reporter gene. in this embodiment of the method, very large numbers of candidate aa peptides expressing lentivirus and eukaryotic reporter cells are packaged together in a format where each is capable of replication, thereby forging a direct link between genotype and phenotype. after four rounds of screening we discover peptides specifically interacted with malignant bcr's. selected peptide ligands were fused with chimeric antigen receptor for expression on t-cells. modified tcells selectively eliminate nhl's malignant cells ex vivo. this work was supported by grant rfmefi x . introduction: pesticides are used to prevent damage of unwanted insects, rodents, plant, moss and other pests. excessive use of pesticides may cause adverse effects in animals and humans. chlorpyrifosethylene (cpe) is an organophosphate pesticide, used in many agricultural products such as figs, cherries, olives worldwide; caused acute poisoning and chronically oxidative stress. rosadamascena mill (rosaceae) is a rose, used for production of rosewater (rw) and rose oil worldwide. rosaceae products are consumed in food and cosmetic industries. materials and methods: in this study, we investigated that cpe and rw effects on kidney tissues of rats. this study was included adult male rats, divided into groups. each group included rats. i.group: control (regular feed), ii.group: cpe ( . mg/kg/day), iii.group: rw ( mg/kg/day) and iv.group: cpe ( . mg/kg/day) + rw ( mg/kg/day). following days, kidneys were taken after sacrificed. analyzes were performed that malondialdehyde (mda), nitric oxide (no) as oxidant; superoxide dismutase (sod), glutathione reductase (gr) as antioxidant parameters. results: as compared with control, mda and no levels in cpe were a significant increase was determined (p conclusion: cpe is shown that significant increase on oxidant parameters, but significant decrease on antioxidant parameters. rw occurs opposite situation. similarly results of cpe, rw + cpe increased oxidant parameters, but decreased antioxidant parameters. these changes are lower than only cpe. these results showed that positive effects both rw and rw + cpe increasing on antioxidant parameters, also decreasing on oxidant parameters. we provide the comparative analysis of the reduced glutathione (gsh), reactive oxygen species (ros), a-tocopherol levels, an intracellular labile zn + pool and esterase activity of red blood cells of patients with diagnosed components of the metabolic syndrome (ms)arterial hypertension and diabetes mellitus type ii (ah + dm + ). as the comparison groups were selected patients with one diagnosed component of msarterial hypertension (ah + dm À ) or without any diagnosed component of ms (ah -dm -). patients of all investigated groups were at the hospital treatment with a diagnosis coronary heart disease (chd) ii degree. human blood was obtained from normal donors and patients with chd ii stage. cytosolic esterase activity was assessed using calcein-am test. ros level was evaluated using cm-h dcf-da. gsh level was estimated using lowry method. an intracellular labile zn + pool was assessed using fluozin- -am. investigations were performed on the specord m , hplc system lc- prominence (shimadzu) and facscantoii (bd). a significant decrease of the intracellular level of labile zn + in erythrocytes of patients with ah + dm + compare with ah + dm À and ah À dm À was shown. this fact confirms our assumption concerning the important role of zinc homeostasis in the etiopathogenesis of diabetes mellitus type ii. a direct relationship between the intracellular zn + level modification and erythrocytes esterase activity of patients with chd ii degree was observed. moreover, in ah + dm + group of patients this relation was more marked. the unidirectional alteration in the erythrocytes redox state (gsh and a-tocopherol levels reduction, ros formation activation) was revealed at the whole of investigated chd patients groups (ah + dm + , ah + dm À , ah À dm À ). however, the pathological erythrocytes response on in vitro action of the different antioxidants (n-acetylcysteine, ascorbic acid, a-tocopherol, quercetin) had a diverse character that can be a significant test under antioxidant therapy prescription. it is known that long-term social isolation and the disorder of natural circadian rhythm is considered an important stress factors, which cause a variety of metabolic and mental disorders. it should be noted that the impact of the stresses takes up a larger area, according to, review of the action of their mechanism is one of the topical issues of modern science. it is estimated that as a result of stress the metabolic processes change in the organizm. because of this, we've studied the functionality of the antioxidant system in laboratory rat heart muscle cells and blood under psycho-emotional stress. it was found that quantitative changes of nitric oxide (no) was initiated the process of lpo, which caused oxidative stress in the cells and decreased antioxidant enzymes activity, such as catalase,sod, gpx and gr. the results suggested that psycho-emotional stress was accompanied by oxidative stress, causing a reduction in the intensity of energy metabolism in cardiac muscle cells, which was further strengthened by the fact that the activity of the enzymes involved in atp synthesis in mitochondria was reduced. also, we've studied exogenous creatine positive and negative affects on energy metabolism and blood lipid spectrum. based on this, we proposed that psychological stress is one of the factors contributing to the development of various cardiac diseases. the importance of free radical lipid transformations, which differ from the peroxidation processes, was pointed out for the first time in our laboratory studies. we have found that ros can induce free radical destruction processes of sphingolipids with c-c-bond cleavage [lipids, , : - ; lipid insights, , - ] . in case of acylated sphingolipids, they can undergo decomposition with c-c-bond cleavage upon direct uv irradiation [photochem. photobiol., , : - ] . it was of interest to establish the possibility of photosensitized decomposition reactions of not acylated sphingolipids, which do not absorb an ultraviolet. in this work we studied photosensitized reactions of sphingosines containing a free amino group, and low molecular compounds, which simulate their structure, such as aminoalcohols (serinol). as photosensitizers, the salts of transition metals, hydrogen peroxide, and acetone were used. oxygen was removed by bubbling with argon to reduce the probability of side reactions during photolysis of sphingolipids, such as oxidation processes (including oxygen reactions with alkyl radicals). we have shown that the action of uv-radiation on aminoalcohols and sphingosines in aqueous solutions in the presence of photosensitizers induces their destruction with c-c bond rupture. the main carbonyl product of sphingosines free radical destruction was an unsaturated aldehyde - -hexadecanal. it was found, that -hexadecenal possesses a wide spectrum of biological activity: it promotes reorganization of the cell cytoskeleton and modifies the redox state of the cells [febs journal (suppl. ), abstracts: mem. biol. s , lipid signaling & dynamics, p. .] . the results of this study can expand the frontier of research regarding free radical lipid damage, which could contribute to a better understanding of the origins of diseases associated with the activation of free radical processes in living organisms. structural basis for the - - proteindependent inhibition of apoptosis signalregulating kinase protein kinase ask (apoptosis signal-regulating kinase ) is a member of the mitogen-activated protein kinase kinase kinase (map k) family that plays a crucial role in immune and stress responses. the activity of ask is regulated through homo-oligomerization and interaction with other proteins including the - - protein which binds to the phosphorylated motif located at the c-terminus of the kinase domain of ask and suppresses its catalytic activity through unknown mechanism. under stress conditions, ask is dephosphorylated at ser and the - - protein dissociates. this dissociation is then one of the factors that lead to the activation of ask . we performed low-resolution structural analysis of the kinase domain of ask (ask -cd) bound to - - using chemical cross-linking, analytical ultracentrifugation and small angle x-ray scattering. the low-resolution structural analysis shows that ask -cd binds to the - - protein in two to two stoichiometry through a small binding interface involving surface of - - outside its central channel and several regions from the c-lobe of ask -cd. the complex is dynamic and conformationally heterogeneous. phosphorus nmr and time-resolved fluorescence measurements, together with low-resolution structural analysis, indicate that binding of ask -cd to - - modulates conformation of ask 's activation segment. these results suggest that the - - binding suppresses the catalytic activity of ask through direct structural modulation of its activation segment. our study provides new insight into the interaction between the kinase domain of ask and - - and offers a plausible structural explanation for the - - protein-dependent inhibition of ask kinase activity. introduction: thymoquinone ( -methyl- -isopropyl- , -benzoquinone, tq) exerts a great antitumor activity against different types of cancer cells. a growing body of evidence indicates that reactive oxygen species (ros) generation followed by modulation of the akt and mapk pathways is a general mechanisms underlying the tq antitumor action. however, the data of tq effects on the mapk pathway are conflicting. to date, the activation or inhibition of the mapk protein family seems to depend on the cell type and on the tq concentration used. in order to elucidate the antitumor potential of tq against gliomas and the underlying molecular mechanism, tq influence on c rat glioma cells functioning was studied. results: it has been shown that the cultivation of c cells with tq in concentrations of - lm during hours strongly inhibits cell proliferation and induces cell death with id of lm. at the same time, tq induces ros generation and intracellular gsh depletion in a dose-dependent manner, that is followed by the mitochondrial potential decrease. interestingly, ros production has only cytoplasmic, but not mitochondrial origin in cells challenged with tq at the concentrations up to lm. two-electron reduction of tq by dt-diaphorase attenuates tq anticancer efficiency whereby inhibition of dt-diaphorase by dicumarol increases tq-induced c cell death by %. we analyzed mapk pathways involvement in c cells growth inhibition at tq treatment. it has been shown that inhibition of the erk pathway by pd and jnk pathway by sp does not influence on tq-induced effects. on the contrary, the specific phosphoinositide- -kinase (pi k) inhibitor (ly ) abrogates tq-induced growth arrest. conclusion: antitumor effects of thymoquinone on c glioma cells is a result of ros generation and intracellular gsh depletion, that is followed by mitochondria disfunction, and growth arrest via pi k pathway. assessment of oxidative stress and antioxidant defense activity parameters in patients with hiv-infection is of great importance, especially for hiv-positive women of reproductive age planning to have children. data of women of reproductive age with hiv-infection analyzed: patients with hiv-monoinfection (n = ) and patients with co-infection (hiv and hepatitis b and / or c) (n = ). as a control we used the data of healthy women (n = ). serum and hemolysate used as material for the study. lpo-aod products were determined by spectrophotometric and fluorometric methods. average value of initial lpo products -diene conjugates was significantly increased in the group with hiv-co-infected compared to control ( . times; p = . ) and group with hivmonoinfection ( . -fold; p = . ). the level of secondary products -ketodienes and conjugated trienes increased in patients of both groups compared to control ( . times (p = . ) and . -fold (p < . ), respectively). at the same time isolated double bonds and tba-active products content showed no significant changes (p > , ). total antioxidant activity parameter decreased . fold (p = . ) in the group with hiv-monoinfection compared to control. decrease in activity of the primary antioxidant enzyme -superoxide dismutase (p = . , compared to the control and p = . , compared with the group with hiv-monoinfection) and the level of a-tocopherol ( . -fold to control and . fold to hiv-monoinfection) was detected in the group with hiv-coinfection. . -fold higher content of retinol in hiv-coinfection group (compared to the control) revealed. in women with hiv-coinfection the oxidative stress was significantly higher than in women with hiv-monoinfection. suggested to include antioxidant supplements in the complex pathogenetic therapy in women with hiv-coinfection (hiv and hepatitis b and / or c), which will contribute to women's ability to bear children. p- . . - acute different doses of malathion induce cholinesterase inhibition, glucogenic enzymes and histopathological change in rat liver malathion, which is an organophosphorus compound, is a widely used pesticide all over the world. despite its benefits, malathion has many toxic effects on many tissues including liver. we designed to evaluate the acute different doses of malathion on cholinesterase (che) inhibition, gluconeogenic enzymes and histopathological change of rat liver. for this purpose groups were formed. rats in group served as control group animals which were only given corn oil. group , group and group were administered , , mg/kg of malathion, respectively, dissolved in corn oil by oral gavage. the rats were sacrificed after hours following administration and the livers of rats were removed. the liver che, alanine aminotransferase (alt), aspartate aminotransferase (ast) and lactate dehydrogenase (ldh) were studied using autoanalyzer. histopathological investigation was performed using microscope. the liver che activities of group , group and group were inhibition percentage of %, %, and % respectively, comparison of group 's che activity. the liver alt, ast and ldh increased in group and group compare to group and group (p < . ). we also observed that there was vacuolar and hydropic degeneration in liver of group . according to our result, acute administrations of malathion result in hepatotoxic effects with increasing doses. background and aims: an imbalance between free radicals and antioxidants is closely linked to the onset of an acute myocardial infarction (ami). the aim of this study was to investigate the antioxidant status and the lipid peroxidation in patients admitted to hospital immediately after ami. methods: the study population comprised patients with ami and healthy subjects. patients that had an acute myocardial infarction (ami) less than hours since onset were selected for this study. antioxidant status was assessed by lactonase activity of paraoxonase (pon dhc), trolox equivalent antioxidant capacity (teac) and plasma uric acid level. malondialdehyde was used as marker of lipid peroxidation. results: compare with the control group, the levels of mda and pon dhc were significantly higher in group with ami (p < . , respectively p < . ). elevated levels of mda (p < . ) were found in patients with ami compare with the control subjects. ami patients had also statistically significant reduced levels of teac (p < . ) comparative with healthy subjects. no statistically significance was found for plasma uric acid level in subjects from our study. conclusion: a high lipid peroxidation correlated with a decreased teac activity suggest an exacerbated oxidative stress in subjects admitted to hospital immediately after an ami. p- . . - dealing with copper toxicity: new insights into copper detoxification in yeast copper (cu), an essential metal, is a double-edged sword, as its essential nature is counterbalanced by the toxic effect that it can exert on cells when not properly controlled. as such, organisms have evolved defence mechanisms against cu toxicity, and in the yeast saccharomyces cerevisiae, the transcription factor ace orchestrates several of those mechanisms, by activating cu-detoxification genes. in s. cerevisiae iron (fe) uptake is partially dependent on cu, as the membrane multicopper-oxidase fet , which is part of the high-affinity iron uptake system, requires cu as a cofactor. aft , the low iron-sensing transcription factor in yeast, is known to regulate the expression of fet gene. however, we found that a strain lacking fet is more sensitive to cu surplus conditions than a strain carrying the aft gene disrupted. this finding suggests that under such conditions another regulator came into play and controls fet gene expression. we next evaluated whether ace could be the alternative regulator of fet under cu excess. to test this hypothesis we first constructed the double mutant aft ace and assayed its fitness under cu surplus. as expected, the double mutant is much more sensitive to cu than the single aft or ace mutants. we next monitored the expression of fet gene in cells lacking ace , using yeast-one hybrid and qrt-pcr approaches. our data clearly indicates that fet expression is dependent on ace when cu is overly abundant. altogether our data unveil a novel mechanism of cu detoxification relying on the activation of fet by ace in an aft independent way. experiments to understand the consequences of this regulation in terms of cu detoxification are currently being undertaken. in joint degeneracy, reactive oxygen species manifest their toxicity both through intrinsic reactivity and the inflammatory process activation, leading to cartilage dysfunctions, injuries of matrix proteins and cytokines stimulation. the study is focused on the identification of oxidative balance modulation (enzymes and oxygen free radicals) by a bioactive extract obtained from small sea fish. the cellular support was represented by the chondrocyte line chon- derived from human long bones (atcc Ò crl- tm ), that assure the reproducibility of a standardised biological system. the oxidative stress was induced through stimulation with il b, a cytokine-factor that promotes the protein catabolism and also with tnfa, the initiator of pro-inflammatory activation, combined with pma, the activator of protein-kinase c, triggering of oxygen reactive species generating cascades. the antioxidant effect was compared with known antioxidants: vitamin c, x fatty acid, n-acetyl -cysteine. the acellular antioxidant/antiradical screening was done using two complementary techniques for total antioxidant status evaluation and completed by measuring cellular catalase (cat) and superoxidedismutase (sod) activity, correlated with intracellular hydrogen-peroxide and superoxide anion monitoring through flow cytometry. the antioxidant properties of the bioactive extract proved in acellular systems are confirmed at cellular level by the involvement of the product in the enzymatic cascade cat -sod, potentiating the catalytic action of the enzymes, and by the decline of both intracellular reactive oxygen species (the hydrogen peroxide decrease with %, the superoxide anion is reduced with % compared with the stimulated control). the demonstrated antioxidant synergy assures a complete cellular protection induced by the small sea fish extract in human condrocytes. introduction: alzheimer's disease (ad) is a progressive neurodegenerative disorder characterized by memory loss, cognitive impairment. oxidative stress is a contributory factor in ad pathogenesis. glutathione (gsh) is the main antioxidant cellular defence. the ratio of gsh/gssg shows the redox status of gsh, and plays important role in maintaining intracellular redox homeostasis. the current study was carried out to determine oxidative dna damage and ratio of gsh/gssg which plays an important role in protection of target molecules from oxidation in the patients with ad. methods: the study subjects were consisted of patients with ad (n = ) and age matched control group (n = ) who were treated and followed in the cerrahpasa medical faculty hospital, department of neurology and department of internal medicine/ geriatrics. dna strand breaks and h o -induced dna damage were determined in lymphocyte dna with comet assay. the gsh and gssg levels in the erythrocyte lysates were measured by using a commercial spectrophotometric kit. the ratio of gsh/gssg were calculated. statistical analysis was performed with spss software package. results: dna strand breaks and h o -induced dna damage were found to be higher (p = . for all), the ratio of gsh/gssg was found to be lower (p = . ) in the ad group than control group. there was no significant difference between male and female for dna strand breaks and h o -induced dna damage in the ad group, but ratio of gsh/gssg were higher in male when compared with female (p = . ). no significant difference was found between the men of ad group and men of the control group for gsh/gssg ratio whereas women of the ad have a lower gsh/gssg ration than those in the women of the control group (p = . ). conclusion: increased systemic oxidative dna damage and dna susceptibility to oxidation may be resulted from diminished gsh/gssg ratio in ad patients. this finding shows the importance of antioxidant support in ad management. p- . . - validation of a liquid chromatography-tandem mass spectrometry method for the measurement of lipid peroxidation product iso-prostaglandin f a in urine m. kant , m. akıs ß , h. _ is ßlekel , department of medical biochemistry, school of medicine, dokuz eylul university, izmir, department of molecular medicine, school of medicine, dokuz eylul university, izmir turkey -iso-prostaglandin f a ( -iso-pgf a ) is a reliable indicator of lipid peroxidation resulting from oxidative lipid damage and is postulated as a gold standard biomarker for the evaluation of oxidative stress. the aim of this study was to validate non-invasive and highly accurate stable isotope dilution-multiple reaction monitoring liquid chromatography-tandem mass spectrometry (sid-mrm lc-ms/ms) method for identification and quantification of -iso-pgf a . twenty four hour urine samples were collected from healthy volunteers at medical school of dokuz eylul university for analytical performance studies. lc-ms/ms analyses were performed on conventional hplc coupled to a triple quadrupole ion trap mass spectrometer equipped with a turboionspray tm source. analyst software version . were used for data analyses. mrm transitions used were m/z? / for -iso-pgf a and m/z? / for -iso-pgf a-d . analytical performance of the method was evaluated by linearity, selectivity, sensitivity, precision and accuracy studies using pure standards and samples extracted from urine of healthy volunteers. the linear calibration range for -iso-pgf a was determined as ng/ml by using standards ranging from . - ng/ml. analytical sensitivity of the method was determined by lod with s/n of and loq with s/n of . lod and loq for iso-pgf a were . À and À ng/ml, respectively. the assay stability and reproducibility were assessed by the precision and accuracy of intra-and interday measurements (n = ). the intra-and interday cvs for -iso-pgf a were . % and . %, respectively. sid-mrm lc-ms/ms method for absolute quantification of -iso-pgf a was optimized and validated in our laboratory and therefore this highly accurate method can successfully be applied to clinical patient samples. p- . . - synergistic anticancer effects of sulforaphane and cisplatin through the induction of apoptosis and autophagy following oxidative stress in malignant mesothelioma cells malignant mesothelioma is characterized by poor responsiveness to current chemotherapeutic drugs, usually owing to high resistance to apoptosis. here, we investigated chemosensitizing effects of phytochemical resveratrol, in combination with cisplatin, on malignant meothelioma cells. cell viability was evaluated using mtt assay. cell apoptosis was detected with dapi staining, caspase / activity assay and flow cytometry. cell cycle distributions, ros levels and mitochondrial membrane potential were determined using flow cytometry. the expression of cell cycle-, apoptosis-, and autophage-related proteins was measured with western blotting. the combination treatment of cisplatin and resveratrol (cis/res) synergistically induced apoptosis, as evidenced by typical cell morphological changes, the appearance of a sub-g /g peak, an increase in the annexin v(+) cells and the cleavage of caspase- and parp. cis/res increased ros production and depolarization of mitochondrial membrane potential with an increase in the bax/bcl- ratio. these changes were attenuated by pre-treatment with n-acetylcysteine, suggesting that cis/res induced apoptosis through oxidative mitochondrial damage. compared with msto- h cells, cis/res was less efficient in killing h- cells. h- cells exhibited an enhanced autophagy to cis/res, as observed by an increase in viable cells exhibiting intense lysotracker red staining and up-regulation of beclin- and lc a. inhibition of autophagy by bafilomycin a rendered cells more sensitive to cis/res-induced cytotoxicity and this was associated with induction of apoptosis. these data indicate that the increased resistance of h- cells to cis/res is closely related to the activation of self-defensive autophagy, and provide the rationale for targeting the autophagy regulation in the treatment of malignant meothelioma. stress oxidative induced by chemotherapy with cyclophosphamide (cp) causes vulnerability in sperm and decline of fertility. this study was aimed to evaluate the role of ethyl pyruvate (ep) in the amelioration of fertility and growth of primitive embryo in animals that received cp. adult male mice ( - weeks) were randomly divided into groups: control group received normal saline ( . ml/day, ip), cp group received cp ( mg/kg/week,¬ ip), the cp+ep group received ep ( mg/kg/day, ip) along with cp, were treated for days. mice from each group were arranged for evaluation of sperm quality and in vitro fertilization (ivf) too. afterward, the separated oocytes from ovulation stimulated mice were conducted to evaluation of ivf and embryo development. the results revealed that cp caused notable decrease in percentage of fertilization in cp group, but administration of ep along with cp caused an increase in the percentage of fertilization in comparison to cp group. the percentage of the two cell zygotes in cp+ep group, and the percentage of blastocysts in control and cp+ep groups were higher than that in cp group (p < . ). results showed that the total number of arrested embryos in control and cp+ep groups was decreased in comparison to cp group (p < . ). the percentage of arrested embryos type i, ii, and iii in cp+ep group was decreased in comparison to cp group, but that decrease was significant only in types i and ii (p < . ). the average motility, viability, nucleus maturity and sperm morphology were decreased significantly in cp group in comparison with control and ep groups, whereas ep caused significant increase of these parameters (p < . ). also, the percentage of dna damage was increased significantly in cp group in comparison with control and ep groups (p < . ). the results of this study indicated that the ethyl pyruvate was able to suppress free radicals and enhance the ivf and quality of sperm in cp treated animals. malathion, which is a member of organophosphate chemical family, is used to control pests and is a widely used pesticide all over the world. however it is also known to be highly toxic on many tissues including pancreas. to test this we set groups to administer a single dose of malathion dissolved in corn oil via oral gavage at the doses of mg/kg (group ), mg/kg (group ) and mg/kg (group ). only plain corn oil was given to control group (group ). the rats were sacrificed hours after administration of the chemical and the pancreases of rats were removed. in an attempt to evaluate the dose dependent response, we measured amylase and lipase activities, insulin, malondialdehyde (mda), total oxidant status (tos) levels in rat pancreases. all of the parameters were measured spectrophotometrically. we found that pancreas insulin levels significantly increased in group compare to group , besides the insulin levels of group and group were significantly higher than group (p < . ). pancreas amylase and lipase activities significantly decreased in group and group compare to group and group (p < . ). however, there was no significant change in pancreas mda and tos levels (p > . ). according to correlation analysis, when pancreas amylase levels declined, lipase levels were decreased simultaneously and there was a strong positive correlation between them (p < . ). in addition, when the comparison was evaluated as a binary, while pancreas amylase and lipase levels diminished, pancreas insulin levels increased and a strong negative correlation between them was found (p < . ). according to our result, acute administrations of malathion leads to alterations of insulin, amylase, and lipase levels with a dose dependent manner, while it does not to change oxidant status. the aim of this study is to evaluate the potential toxic effects of mancozeb on the stress biomarkers such as catalase (cat) activity, malondialdehyde (mda) level and protein levels in the brain tissue of zebrafish (danio rerio). mancozeb, is a synthetic fungicide contaminating aquatic environments as a potential toxic pollutants, was investigated in the present study for acute toxicity. zebrafish groups (m-low and m-high) were exposed to different doses of mancozeb ( mg l- and . mg l- ) for hours except the control group. catalase (cat) activity, malondialdehyde (mda) level and total protein levels were determined by spectrophotometer. the results showed that cat activity and mda levels were decreased in all experiment groups. protein levels were increased in experiment groups when compared to the control group. in conclusion, the changes in the cat activity and mda levels were time and as well as mancozeb dose-dependent. furthermore, the biochemical parameters of mancozeb exposure on zebrafish, showed that mancozeb has significant effect on the zebrafish and/or aquatic organisims. paracetamol (para), which is antipyretic and analgesic, is widely used around the world. paracetamol can be recommended for moderate or mild pains especially in pregnancy as an analgesic. it is known that, paracetamol can cause hepatotoxicity or nephrotoxicity. we were aimed that in this study to show potential hepatoprotective and nephroprotective effect of betaine against long term paracetamol using at therapeutic doses. it has been prepared groups, control, para and para+-betain groups. paracetamol and betaine were administered by gavage to pregnant rats, from first day to the last day of pregnancy (aprox. day). ml physiological saline (% . nacl solutions), mg/kg/day paracetamol, mg/kg/day paracetamol and mg/kg/day betain was given by orally to control, para and para+betain groups respectively. the day of the birth, newborn rats anesthetized by ether and after decapitated. newborn rat's liver and kidney tissues used for biochemical analysis [malondialdehyde (mda), reduced glutathione (gsh), nitric oxide (no) and paraoxonase-arylesterase (pon-are)] and rat's liver and kidney tissues used for histological studies. we showed that, mda and no levels was significantly increased, while pon activities decreased. on the other hand gsh levels and are activities was decreased but these decline wasn't significant in the liver and kidney para group. these biochemical results showed hepatotoxicity and nephrotoxicity in neonates which can be formed in long term maternal paracetamol using at therapeutic doses. also our histological findings was support these biochemical results. we used betaine against potential hepatotoxic and nephrotoxic effect of long term maternal paracetamol using at therapeutic doses for neonates. betaine has antioxidant properties and also used as a methyl donor for transsulfuration reactions in the cell. biochemical and histological examinations showed that betaine protected the tissue injury relatively. p- . . - lipid rafts are involved in modulation of ca + responses induced by glutoxim and molixan in macrophages pharmacological analogues of oxidized glutathione (gssg) disulfide-containing drugs glutoxim Ò (gssg disodium salt with dmetal nanoaddition, «pharma-vam», st. petersburg) and molixan Ò (complex of glutoxim with inosine nucleoside) have found clinical application as a wide range immunomodulators and hemostimulators. however, the cellular and molecular mechanisms of these drugs action are still unclear. previously we showed for the first time that glutoxim and molixan cause biphasic intracellular ca + concentration ([ca + ] i ) increase due to ca + mobilization from thapsigargin-sensitive ca + stores and subsequent store-dependent ca + entry in rat peritoneal macrophages. it is known that key proteins involved in ca + signaling are localized in discrete plasma membrane lipid rafts domains. lipid rafts are cholesterol and sphingolipids enriched microdomains that function as unique signal transduction platforms. thus, the aim of the present work was to elucidate the possible involvement of lipid rafts in glutoxim and molixan effects on [ca + ] i in macrophages. [ca + ] i measurements were performed with fura- am microfluorimetry. to examine the involvement of lipid rafts in the effect of gssg-based drugs on [ca + ] i we used methyl-bcyclodextrin (mbcd), widely used to remove cholesterol from membranes, thus disrupting the lipid raft domains. we have shown for the first time that macrophage preincubation with mm mbcd for hours before molixan addition causes significant inhibition of both ca + mobilization (on average, by . ae . %) and subsequent ca + entry (on average, by . ae . %), induced by molixan. similar results were obtained in experiments with glutoxim. thus, we have demonstrated for the first time that mbcd significantly decreases both phases of glutoxim-or molixan-induced ca + responses in macrophages. the results suggest that intact rafts are required to initiate complex signaling cascade activated by glutoxim or molixan and leading to [ca + ] i increase in macrophages. plant-derived natural substances (phytochemicals) with potent pro-apoptotic activity towards cancer cells in vitro are considered as promising nutraceuticals in anticancer therapy. nevertheless, due to their relatively low bioavailability, administration of high doses of nutraceuticals that are not achievable in vivo seems to exert potentially negligible physiological effects in clinical trials. thus, there is a need for revealing novel cytophysiological effects of low doses of phytochemicals towards cancer cells. in the present study, we have considered thirty one nutraceuticals and selected four phytochemicals acting at low micromolar range ( to lm) against phenotypically different mcf- , mda-mb- and sk-br- breast cancer cells, namely diosmin, sulforaphane, ursolic acid and betulinic acid. nutraceuticals inhibited cell proliferation and caused changes in the cell cycle that was accompanied by elevated levels of p , p , p and/or p . apoptosis (annexin v staining, multicaspase and mitopotential assays) was observed exclusively when nutraceuticals were used at the concentration of lm, whereas at the concentration of lm, stress-induced premature senescence was noticed (sa-b-gal activity). nutraceuticals diminished the levels of glut- and selected glycolytic enzymes. nutraceuticals promoted oxidative and nitrosative stress as judged by increased production of total reactive oxygen species, total and mitochondrial superoxide, nitric oxide and protein carbonylation. nutraceuticals also stimulated dna single and double strand breaks that was accompanied by atm phosphorylation and to a lesser extent by histone h ax phosphorylation and bp foci formation. taken together, several responses to nutraceutical treatment were observed in breast cancer cells that may reflect the heterogeneous nature of cancer cell populations. this study was supported by grant from the national science center, / /d/nz / . nucleolus is thought to be a stress sensor and oxidative and ribotoxic stimuli may cause the inhibition of rrna synthesis by the inactivation of transcription factor tif-ia/rrn that is accompanied by the relocation of nucleolar proteins and p -based cell cycle arrest and/or apoptosis. as nutraceutical-mediated modulation of nucleolar activity may be considered an attractive anticancer strategy, in the present study, we have investigated the effects of three selected nutraceuticals, namely sulforaphane, ursolic acid and betulinic acid on nucleolus state in three breast cancer cell lines (mcf- , mda-mb- and sk-br- ). we found that low dose nutraceutical treatment resulted in p -mediated inhibition of cell proliferation, a decrease in rrna synthesis, shifts in lamin a/c and b pools, changes in the nucleolar protein levels and their carbonylation, and changes in nucleolus size and number. breast cancer cells differed in erk activity that resulted in different patterns of erk activation/inhibition, phosphorylation status of s and autophagy induction upon nutraceutical stimulation. nutraceuticals also affected dna methylation parameters, namely the levels of dnmt , dnmt a and dnmt b that resulted in both global dna hypo-and hypermethylation. taken together, after nutraceutical treatment, nucleolus-centered cellular response was revealed in breast cancer cells of different phenotypic characteristic that may be considered a potential target of anticancer therapy. this study was supported by grant from the national science center, / /d/nz / . p- . . - rate of apoptosis in human macrophages infected with leishmania tropica promastigotes infection of the cells with parasites or exposing cells to heat stress induces a cellular stress. in the present study human macrophages are infected with leishmania tropica promastigotes and exposed to heat stress. the measurement of cytoplasmic histone-associated dna-fragments was carried out using elisa technique. visualization of apoptotic cells was performed by the terminal deoxynucleotidyl transferase dutp nick end labeling staining method (tunel). degree of oxidative stress on cell is evaluated by measuring nitric oxide (no), malondialdehyde (mda), reducte glutathion (gsh) levels and superoxide dismutase (sod) activities. results of the elisa technique showed that infection of macrophages with promastigotes induced apoptosis rate significantly (p < . ), heat stress however decreased the rate of apoptosis in infected macrophages remarkably (p < . ). high levels of apoptosis rate in infected macrophages and drastic decdrease in apoptosis in heat subjected macrophages infected with promastigotes are confirmed by visualisation of apoptotic cells using tunel method. levels of glutathion (gsh) in infected macrophages decreased significantly (p < . ), while malondialdehyde (mda) levels increased notably (p < . ). however, no statistical significant alterations were detected in the nitric oxide (no) values and superoxide dismutase (sod) activities. results of the present study indicates that infection of human macrophages with leishmania tropica induces a cellular stress response, characterized by decreased values of gsh and increased levels of mda. increased rate of apoptosis in infected macrophages may be due to the increased cellular stress caused by leishmania tropica amastigotes. decreased rate of apoptosis measured in heat exposed macrophages infected with promastigotes indicates an extention in life span of macrophages. measurements of the parameters characterizing the redox and inflammatory processes in blood are essential for diagnosis and prognosis of type diabetes mellitus (t dm), but also for monitoring the effectiveness of medical treatments. along with other biochemical effects, hormonal imbalance leads to modified transport function of erythrocytes due to changes in enzyme systems involved in upholding of cellular homeostasis through a rapid degradation of altered proteins, being the second line of defense against the free radicals, which degrade and eliminate the damaged molecules. some of these enzymes are hemoglobin peroxidase (pa) and esterase (ea). the aim of this research study was to identify new parameters with a potential role of biochemical markers in t dm like hemoglobin peroxidase and esterase activity from erythrocyte. our data showed that pathological processes involved in t dm imply an increased enzymatic activity of pa ( . %), which correlates with increased levels of ea ( . %) and glycated hemoglobin (hba c) ( . %), compared with control group. the variables hba c, pa and ea are correlated: the identified pearson correlation coefficients r = . and r = . respectively, have an associated probability of p < . and p < . a value that indicates a strong positive correlation between the dependent variables pa and ea and independent variable hba c. based on these results we concluded that together with glycosylated hemoglobin assay, all the studied parameters can be successfully used as extra test for diabetes associated with oxidative stress and disorders in erythrocyte functions or blood rheology. the radioprotective effects of propolis and caffeic acid phenethyl ester on radiationinduced oxidative/nitrosative stress in brain tissue s. taysi , e. demir , k. cinar gaziantep university, school of medicine, gaziantep, haran university, sanliurfa, department of neurosurgery, medical school, gaziantep, turkey head and neck cancer patients treated with radiotherapy suffer severe side effects during and following their treatment. efforts to decrease toxicity of irradiation to normal tissue, organs and cells have led to searching for cytoprotective agent. investigations for effective and non-toxic compounds with radioprotective capability led to increasing interest in antioxidant such as propolis and caffeic acid phenethyl ester (cape). the aim of this study was to evaluate the antioxidant and radioprotective effects of propolis and cape on radiation-induced oxidative/nitrosative stress in the brain tissue. fourty sprague-dawley rats were randomly divided into five groups. group (irradiation (ir) + propolis) received total cranium irradiation and propolis was given orally through an orogastric tube daily. group (ir+cape) received total cranium irradiation plus cape, was dissolved in dimethyl sulfoxide (dmso) just before giving to the rats, intraperitoneally (ip) every day. group (ir) received gy of gamma irradiation as a single dose to total cranium plus ml saline daily. group received daily plain dmso. group received daily plain saline. at the end of the day time period, xanthine oxidase (xo), nitric oxide synthase (nos) activities, nitric oxide (no•) and peroxynitrite (onoo -) levels were significantly higher in ir group compared to all other groups. in conclusion, the results suggest the radioprotective ability of propolis and cape involving prevention of radiation-induced oxidative/ nitrosative damage. p- . . - role of leptin and adiponectin in obesityassociated oxidative stress e. becer , a. c ß irakoglu near east university, nicosia, cyprus, istanbul university, istanbul, turkey objective: increased oxidative stress is one of the major characteristics of obesity and obesity-related complications. adipokines also induce the production of reactive oxygen species and generating oxidative stress in physiological and pathological conditions of obesity. the aim of this study was to determine the association of leptin and adiponectin levels with body mass index, lipid parameters and oxidative stress biomarkers in obesity. methods: the study included obese and non-obese subjects. plasma leptin and adiponectin levels (ng/ml) were measured using commercially available enzyme-linked immunosorbent assay kits. serum lipid, superoxide dismutase, malondialdehyde and antropometric parameters were measured. results: obese and non-obese subjects did not differ in age, while plasma glucose, total cholesterol, triglycerides, ldl cholesterol and leptin levels were significantly higher and mean hdl cholesterol and adiponectin levels were significantly lower in obese than non-obese subjects. the plasma leptin (p < . ) and adiponectin (p = . ) levels were significantly correlated with bmi in both obese and non-obese subjects. in obese subjects, leptin levels were significantly correlated with superoxide dismutase (p < . ) and malondialdehyde (p < . ). strikingly, adiponectin was significantly correlated with superoxide dismutase (p = . ) and malondialdehyde (p = . ) levels in obese group. conclusion: our results suggest that leptin and adiponectin levels are associated with defective antioxidant status and increased lipid peroxidation which may have implications in the development of obesity related health problems. clinical trials of biologically active plant substances show a significant preventive effect in cancer, cardiovascular diseases and peptic ulcer disease in the form of both nutritional supplements and therapeutic intervention. anthocyanins contained in dark berries show great antioxidant potential, with the most important including a reduction in oxidative degradation of lipids or tyrosine oxidation by peroxynitrite. our previous studies of the antioxidant properties of extracts from vaccinium corymbosum, aronia melanocarpa and sambucus nigra, however, indicated that their activities largely depend on the method of extraction. while quantitative determination of anthocyanins pointed to a disproportionately larger content of anthocyanins in isolates from lyophylized berries, their scavenging activities against hydroxyl radicals was surprisingly the lowest. inflammatory processes, vascular damage, atherosclerosis and others are caused by oxidativenitrosative stress, so we tested their efficiency to scavenge no degradation products. we found that only purified extracts of lyophilized berries showed the most significant effects against no degradation products, with efficacy of around %. an extract from aronia showed greater than % efficiency, and a net ethanol extract from all the berries showed a % effect. cleaned ethanol extracts showed the lowest effects, while aronia initiated production at a concentration of mg/l. conversely, all acetone extracts consistently initiated no degradation products. these findings are in complete contrast to their determined action against reactive oxygen species. in summary, it follows that a particularly adjusted lyophilized extract of the berries could be responsible for the increased biological activity of no and the observed biological and pharmacological activities of anthocyanins in circulatory disorders. the study was supported by grant vega / / and / / . p- . . - attenuation of dysfunction, oxidative stress and apoptosis by resveratrol in benzo(a)pyrene exposed ins -e / pancreatic beta cell line s. c ß elik, b. baysal faculty of medicine, afyon kocatepe university, afyonkarahisar, turkey diabetes is one of the most important problems in the world. this disease is a very important health problem due to affects many different organs and systems. it has been well established that, environmental pollutants had deleterious effects on glucose metabolism, and caused insulin resistance and type diabetes. with this investigation, it was aimed to investigate the effects of benzo(a)pyrene on pancreatic beta cells and treatment affects of resveratrol. in this study, rat ins- e beta cell line was used. after reaching the appropriate number of cells culture operations by cell culture, benzo(a)pyrene ( lm, hours) application have been made after resveratrol ( lm, hours) application. after incubations oxidative stress, insulin secretion (in cell and in medium), cell proliferation and apoptosis were analysed in cells by biochemical and molecular techniques. b(a)p application resulted in no increase and resveratrol also increased this level of no. resveratrol increased the tas levels decrased by b(a)p, and tos levels were also increased by resveratrol interestingly. osi levels determined with tas and tos levels, has no significant change between groups. gsh levels were decreased by b(a)p while resveratrol increased its' level to control level. mrna expression levels of beta cell functions related genes ins- , ins- and sirt- were increased by resveratrol treatment. insulin levels in cell and in medium were increased after resveratrol treatment. mrna expression level of foxo- gene was increased while pdx- was decreased by resveratrol treatmeant. b(a)p suppressed the mrna expression of p gene, but resveratrol increased. the effects of b(a)p on pancreatic beta cells and the protective effects of resveratrol on this cells were investigated in vitro with this research firstly. the results obtained from this research showed that oxidative changes, functional impairment and carcinogenetic effects of b(a) p in pancreatic beta cells could be blocked by resveratrol. the protective effect of vitamin e (alphatocopherol) on ischemia-reperfusion injury in rat liver ischemia-reperfusion (i/r) process is usually used during transplantation and resection of the liver but liver dysfunction and cellular death due to lack of oxygen in tissues, changes in the balance of oxidant/antioxidant in favor of oxidants, and inflammatory response are inevitable during this process. in the present study, it was aimed to investigate whether vitamin e(alpha-tocopherol), an antioxidant agent, has a protective effect against liver ischemia reperfusion injury in rats by using morphometric methods. for this purpose, adult sprague-dawley male rats were divided into groups as; control, ischemia / reperfusion (i/r), and vitamin e+ischemia/reperfusion (evit +i/r). in experimental groups, the total hepatic ischemia was applied for minutes followed by a hour of reperfusion. in the treatment group, days before ischemia mg / kg dose of vitamin e was administered intraperitoneally once a day. after the termination of the reperfusion, the rats were perfused by cardiac way and liver tissues were dissected. following volume and weight calculations, the livers were subjected to the standard histological preparation methods and embedded in paraffin. serial sections at lm thickness were obtained from these blocks, stained with hematoxylineosin, and analyzed with morphometric methods. in light microscopic examinations of the i/r group, irregularity in lobules, dilatation in central veins and sinusoids, extensive areas of necrosis and pycnotic nuclei were seen in hepatocytes. the volume density of sinusoids to liver parenchyma was estimated as % in the control group, whereas it was % in the i/r group. this value was decreased to % in the evit + i/ r group. however, no significant difference was found among the groups in the lobule area calculated by the point counting method. these results show that intraperitoneal vitamin e administration for days prior to ischemia partially inhibits damage caused by ischemia-reperfusion injury in the liver. the leaves, fruit and bark of annona muricata, a member of the annonaceae family, are commonly used in the traditional medicine of tropical and subtropical regions. in recent years, many studies have shown their anti-cancer, anti-convulsant, antiarthritic, anti-parasitic, anti-malarial, and anti-diabetic activities. it should be noted that these characteristics have been described using different types of extracts from different parts of the plant. our studies have focused on the systematic characterization of activities most easily accessible from an aqueous extract of leaves, with hitherto documented antihypertensive and hepatoprotective effects. we found that the extract shows almost % efficiency against hydroxyl radicals. with increasing concentrations, the effectiveness weakened, reaching a second peak ( %) at a concentration of mg/l. the scavenging activity against no degradation products maintained a continuously increasing trend with a maximum at a concentration of mg/l. surprisingly, the extract initiated peroxynitrite production in a similar trend, except at mg/l, where it scavenged peroxynitrites with relatively high efficiency, up to %. these findings are consistent with the elevated levels of reduced glutathione detected after incubation of liver mitochondria with extract to a maximum concentration of mg/l, with subsequent sharp decline. the activity of glutathione s-transferase was decreased, although not significantly. this indicates a reduction of metabolic processes of compounds, allowing their action over a longer period of time. in a live system, even antihypertensive effects can be observed. however, a significant outflow of gsh to create the gsh adducts of active substances, and particularly s-nitrosoglutathione from increased production of peroxynitrites, can cause liver toxicity. the study was supported by grant vega / / and / / . the role of polyamine metabolism in curcumin induced apoptosis via reactive oxygene species (ros) generation in growth hormone (gh) overexpressed t d breast cancer cells r. genc ß, a. coker gurkan, e. d. arisan, p. obakan yerlikaya, n. palavan unsal, n. palavan unsal t.c istanbul k€ ult€ ur € universitesi, istanbul, turkey autocrine growth hormone (gh) signaling triggers cell proliferation, growth, metastasis and drug resistance in cancer cells. polyamines (pas) play an essential role in cell cycle, proliferation, growth and carcinogenesis of various cancer types such as prostate, colon and breast cancer. odc (ornithine decarboxylase) is the key enzyme in the pa biosynthetic pathway that is under control of antizyme (az) and antizyme inhibitor (azi) activity. pa catabolic enzymes polyamine oxidase (pao) and spermine/spermidine acetyle transferase (ssat) by-products triggers reactive oxygene species (ros) generation and apoptotic cell death. curcumin decreased cell viability in dose and time dependent manner in t d wt and gh+ cells. although forced gh expression induced cell proliferation, lm curcumin inhibited cell invasion. curcumin ( lm) induced apoptosis by acting on intrinsic and extrinsic pathway in both cell lines. moreover, curcumin supressed odc, azi expression in wt and gh+ t d cancer cells. although curcumin decreased az expression in t d wt cancer cell, increased az expression was determined in t d gh cancer cell. pao and ssat expressions were upregulated in t d gh+ cells. concomitantly, putrescine levels were increased in t d gh+ cancer cell compared to wt cells and curcumin depleted spermidine and spermine levels in wt and gh + t d cells. curcumin induced-apoptotic cell death via ros generation and co-treatment of n-acetyl cysteine (nac) overcame curcumin effect. conclusion, forced gh expression triggers cell proliferation and growth via acting on polyamine metabolism and curcumin-triggered ros generation was prevented by nac treatment in t d wt and gh+ breast cancer cells. acknowledgment: this study was supported by tubitak research project (project no: z ). the radio-protective effects of propolis and nigella sativa oil on oxidative/nitrosative stress in liver tissue of rats exposed to total head irradiation s. taysi our purpose is to investigate propolis and nigella sativa oil (nso) for their antioxidant effects on the liver tissue of rats exposed to ionizing radiation. a total of sprague-dawley rats were divided into five groups to test the radioprotective effectiveness of of propolis and nso administered by orogastric tube. appropriate control group was also studied. biochemical parameters in liver tissue of rats were determined by spectrophotometer. xanthine oxidase (xo), nitric oxide synthase (nos), superoxide dismutase (sod) activities, nitric oxide (no•) and malondialdehyde (mda) levels were higher in ir group while glutathione-s-transferase (gst), glutathione peroxidase (gsh-px) level in the ir group were lower in this group when compared to the other groups. gst activity in ir plus propolis group was statistically higher than in all other groups. propolis and nso clearly protect liver tissue from radiationinduced oxidative stress. the effects of royal jelly on the antioxidant parameters in the breast tissues of the rats with breast carcinoma treated with paclitaxel or not effects of royal jelly on the breast tissue antioxidant parameters in rats with breast carcinoma treated with paclitaxel or not. - weeks old female sprague-dawley rats (n = ) included in current study were divided into groups: control group (n = ) with healthy rats; breast cancer group (n = ); breast cancer group (n = ) treated with mg/kg paclitaxel injection (once a week for weeks); breast cancer group (n = ) treated with mg/kg royal jelly (by oral gavage for days); and finally breast cancer group (n = ) treated mg/kg royal jelly in addition to mg/kg paclitaxel injection. rats with breast carcinoma was obtained at th days after a single dose injection of mg/kg n-methyl-n-nitrosourea (mnu). all cancer groups were followed by days with treatment of paclitaxel and/or royal jelly. the antioxidant parameters in rat breast tissues, superoxide dismutase (sod) and catalase (cat) activities were determined by spectrophotometric colorimetric methods and glutathione (gsh) by high performance liquid chromatography (hplc). all the antioxidant parameters decreased in breast cancer group without any treatment (p < . ). but, statistically non significant increases were observed by paclitaxel and royal jelly treatment (p > . ). this study indicated that royal jelly supplementation can not be sufficient to increase the antioxidant parameters in breast cancer. we are going to continue to identify the effects of royal jelly on breast cancer detail. the effects of n-acetylcysteine on microsomal and serum paraoxonase activities at high fat diet induced obese rats obesity is a chronic disease that develops from the interaction between genotype and environmental factors and increase in the accumulation of body fat. it is related with glucose and lipid metabolism disorders, cardiovascular diseases and increased oxidative stress. paraoxanase (pon ) is an enzyme which plays a protective role in oxidative stress, inflammation and liver diseases. it has been suggested that pon has protective effects against high fat diet induced obesity and obesity related disorders. n-asetylcysteine (nac) is a potent antioxidant due to its ability to stimulate glutathione synthesis. the aim of this study was to evaluate the microsomal and serum pon enzyme activities (paraoxonase, arylesterase and lactonase) at high fat diet induced obese rats in the presence of nac. this study consisted of control, obese and nac-supplemented obese ( g/l nac) groups. eighteen sprague-dawley rats were randomized into three groups (n = ). control rats were fed by standart food and obese and nac groups were fed by high fat diet. the microsomal and serum paraoxonase, arylesterase and lactonase activities were determined by colorimetric methods. serum paraoxonase, arylesterase and lactonase activities decreased in obese and nac groups when compared to control groups. on the other hand microsomal paraoxonase, arylesterase and lactonase activities increased in nac group when compared to control and obese groups. however there was no statistically significant difference between the groups. it has been concluded that the microsomal and serum paraoxonase enzyme activities did not change at high fat diet induced obese rats in the presence of n-asetylcysteine. reactive oxygen species, playing an active role in the early and late course of acute pancreatitis, lead to dysfunction of cell membrane and releasing of lysosomal enzymes, and thereby to the injury in pancreatic cells. gallic acid, found in vegetables such as green tea, is an active component which has antioxidant, antiinflammatory, antiviral, anticancer activities. the aim of this study was to investigate the effects of gallic acid in experimental acute necrotizing pancreatitis (anp) model in rats. eighteen male sprague-dawley rats were divided into three groups ( rats in each group). group : sham + saline; group : anp induced by intraductal glycodeoxycholic acid and intravenous cerulein; and group : anp + gallic acid ( mg/kg/day, intraperitoneal). at the end of th hours, pancreas histopathology was examined. the levels of serum amylase as a diagnostic marker of pancreatitis, interleukin- (il- ), total antioxidant status (tas), nitrite + nitrate, total thiols as antioxidant marker and thiobarbituric acid reactive substances (tbars) to measure malondialdehyde (lipid peroxidation product) were determined by spectrophotometric methods. serum amylase, il- , plasma tbars levels were significantly higher but total thiols levels were lower than sham group in anp group without treatment (p < . ). however; tas and nitrite + nitrate levels did not show any significant difference (p > . ). on the other hand, while serum amylase, il- and tbars levels were lower, total thiols levels higher in gallic acid treatment group than in the untreated anp group, but statistically insignificant (p > . ). in conclusion, gallic acid treatment is beneficial but not sufficient to treat the acute necrotizing pancreatitis in rats. p- . . - evaluation of oxidant/antioxidant system parameters, il- and il- levels in amniotic fluid of pregnancies with down sydrome b. _ imge erg€ uder , s. bahsi , t. bahsi , v. topc ßu , a. bakir ankara universty faculty of medicinel, ankara, zekai tahir burak research and training, hospital genetic center, ankara, turkey introduction and aim: down sydrome (ds) can be diagnosed at high-risk of down syndrome pregnancies by invasive prenatal testing. in this study we aimed to demonstrate antioxidant/oxidant system markers, il and il levels in amniotic fluid samples of pregnancies affected by ds. materials and methods: for this purpose we collected amniotic fluid samples from pregnancies affected by down sydrome and normal healthy pregnancies who applied to zekai tahir burak research and training hospital genetic center and were proceeded with amniocenthesis. in the amniotic fluid samples; malondialdehyde (mda), superoxide dismutase (sod), glutathion peroksidase (gsh-px) xhantine oksidase (xo), catalase (cat), adenozine deaminase (ada), nitric oxide (no), nitric oxide senthase (nos) enzymatic activities were evaluated by spectrophotometric methods, il and il levels are evaluated by elisa. for statistical analysis student's t-test and spearman corralation analusis are used. results: it was found that sod levels are significantly elevated in study group compared to control group (p < . ). besides this, in study group, cat and il- levels are found singnificantly lower than control group (p < . ). we couldn't find any significant difference between two groups in terms of mda, gsh-px, xo, no, nos, ada ve il- levels (p > . ). discussion and conclusion: there is an important decrease in inflammation compared to normal pregnancie in the amniotic fluid of pregnancies having ds. based on these results, sod enzyme may be used as a marker for prenatal diagnosis of ds. for this purpose these experiments should be tried on larger sample groups. the aim of our work was to compare prooxidant and antioxidant properties of linalool, which is the oxygenated monoterpene compound reported to be a major volatile component of the essential oil of several aromatic species, in hep g cells. cytotoxicity of linalool was assayed by celltiter-blue Ò cell viability assay. malondialdehyde levels result in membrane damage in hep g cells were assayed by using fluorometric method. hep g cells were incubated with linalool at , and hours. the viability of hep g cells decreased in a manner dependent upon concentration and incubation time. the ic values were calculated . lg/ml ( hours), . lg/ml ( hours) and . lg/ml ( hours). but, cell viability of hep g cells increased when the cells preincubated with linalool at lower concentrations (˂ic ) against h o cytotoxicity. membrane-damaging effects of linalool were increased with accelerating concentrations. on the other hand, membrane damaging effect of h o was decreased when the cells preincubated with linalool before h o incubation. oxygenated monoterpene linalool had both prooxidant and antioxidant properties showing membrane damaging and protective effects on hep g cells depend on concentration. postprandial lipemia is primarily characterized by increasing triglyceride levels after the lipid rich meal. postprandial lipemia may cause oxidative stress by increasing free radical production and increasing oxidative stress could be responsible for the development of many diseases. plasma oxidant-antioxidant status was evaluated in healthy individuals with postprandial hypertriglyceridemia generated by performing oral triglyceride tolerence test (ottt). the study group included subjects ( female and male). ferric reducing ability of plasma (frap), total thiol and thiobarbituric acid reactive substances (tbars) levels were determined by colorimetric methods at fasting and nd, th and th hours following ottt. the levels of frap and thiol were significantly higher in males than females (p = . and . , respectively). thiol levels decreased significantly in both gender at postprandial nd, th and th hours as compared with fasting condition (p = . ). while tbars levels increased at postprandial nd hour, that was only significant for male individuals (p = . ). it has been concluded that postprandial lipemia may change oxidant-antioxidant balance in favor of oxidants and gender is an important criteria while assessing the oxidant-antioxidant status in postprandial lipemia p- . . - ischemia modified albumin and c-reactive protein levels in prediabetes prediabetes is a state of abnormal glucose homeostasis characterized by the presence of impaired fasting glucose, impaired glucose tolerance, or both. the aim of this study was assess serum ischemia modified albumin (ima) in prediabetes and determine its correlation with other risk factors for chronic complications such as inflammation and hyperglycemia. glucose, insulin, total cholesterol, hdl cholesterol, triglycerides, albumin, c-reactive protein (crp) and ima were measured in patients with prediabetes and controls. prediabetes patients had higher levels of ima and crp in comparison with control subjects but there was no significant difference between groups for ima. significant positive correlation was observed between crp and fasting glucose, insulin. there was no significant correlation between ima levels and the parameters tested. we have shown higher level of crp in prediabetes. these results support the hypothesis that chronic inflammation may be involved in development of hyperglycaemia. p- . . - the effects of s _ io nanoparticles of rat uterine smooth muscle specially used in textile field sio nanoparticles on uterus smooth muscle was aimed to be researched. in this study wistar albino female rats were used. rats were separated in groups as control, dose ( lg/ml), dose ( lg/ml) and dose ( lg/ml). nanoparticle's size was chosen as nm. preparations of four groups were evaluated for biochemical and histological examinations. all isolated uterine smooth muscle stripts except the controls were treated with sio for two hours. in biochemical examinations in order to evaluate oxidative stress level of malondialdehit (mda), activity of superoxide dysmutase (sod) and glutathione peroxidase (gsh-px) were measured. in histological examinations via electron microscope ultrastructure of uterus was examined as well as apoptotic cells detected with immunofluorescent labeling method. intergroups differences were defined by statistical analysis. while mda level increased depending on the dosage, sod level was decreased depending on the dosage. gsh-px rate was decreased for each dosage with respect to control. however, no significant difference is detected between groups. in electron microscopic examination no changes were observed in uterus ultrastructure with compare to control. however, in immunofluorescent labeling it was detected that apoptosis increased in dosage groups with compare to control group. as a result, it was thought that application of sio nanoparticles, in nm size and in , and lg/ml dosages caused of oxidative stress and apoptosis. this results suggested that sio has toxic effects on uterine smooth muscle. uterine myomas are the most common benign pelvic tumors arising from myometrium. they are rarely associated with mortality but responsible for significant morbidity and have adverse effects on quality of life especially in reproductive age women. reactive oxygen species and superoxide dismutases, as well as sex steroids play important roles in the reproductive physiology processes. in addition, oxidative stress and impaired antioxidant defense system have been linked to pathophysiology of various diseases including malignant gynecological disorders. clinical investigations indicate that women with myoma may have increased risk of developing malignant tumors particularly sarcomas. the present study aimed to investigate the possible role of oxidant and antioxidant status in myomas. blood and urine samples of myoma patients were collected. activities of erythrocyte antioxidant enzymes [copper-zinc superoxide dismutase (cuzn-sod), catalase (cat), glutathione peroxidase (gpx )] and levels of lipid peroxidation biomarkers [plasma malondialdehyde (mda) and urine -epi-prostaglandin f a ( -epi-pgf a)] were determined. the results were compared with those of controls. the groups were matched in terms of age, body mass index, smoking habit, coexisting chronic diseases, menopausal status and sex steroid hormone levels. all antioxidant enzyme activities were higher ( % for cuzn-sod, p = . ; % for cat, p . ) and the levels of lipid peroxidation biomarkers were lower (% for mda, p = . and % for -epi-pgf a, p > . ) in myoma patients compared to controls. correlation analyses showed a significant negative correlation between erythrocyte cuznsod activity and plasma mda levels (r = - . , p = . ). the decreased lipid peroxidation may be the consequence of elevated antioxidant enzyme activities and the data suggests a protective role of activated antioxidants especially cuznsod and cat in patients with myoma. p- . . - investigation of ischemia-modified albumin levels in patient with acute limb ischemia introduction: acute limb ischemia commonly occurs as a result of embolus caused by cardiac origin and which may end up with limb loss or even death if left untreated. thrombosis are usually seen where the arteries give branches and tendency to atherosclerosis is more serious at these sites. involvement of several arteries in either embolus or in situ thrombosis limits the adequacy of collateral circulation. restriction of blood flow due to arterial stenosis or occlusion often leads patients to complain of muscle pain on walking. any further reduction in blood flow causes ischemic pain at rest, which affects the foot. early recognition may prevent limb loss or death. ischemia can alter the capacity of the amino terminus of the albumin to bind free metal atoms such as cobalt, copper and nickel. this new, chemically changed albumin is called ischemia modified albumin (ima). ima is a sensitive marker of myocardial ischemia, skeletal muscle ischemia, pulmonary embolism, mesenteric ischemia and stroke. therefore, in this study it was aimed to investigate the ima level in acute limb ischemia. materials and methods: in this study, patients with acute limb ischemia (li group; mean age years) and healthy individuals (control group; mean age years) were included. ima levels were detected in control and li group by elisa (organo teknika, avusturya) using ima el _ isa kit. results: ima values were compared with nonparametric methods mann whitney u test, and significantly decreased ima level was statistically significantly different between li group and control group (p < . ). conclusion: there is a significant increase in serum ima in limb ischemia, so that alterations also might be clinically useful in the diagnosis of limb ischemia, but should be supported with further studies. object: polycystic ovary syndrome (pcos) is a multifaceted disorder with a pathogenetic pathway that is not fully understood yet. apart from hormonal derangements, insulin signaling defects and adipose tissue dysfunction, oxidative stress, defined as an imbalance derived from excessive formation of oxidants in the presence of limited antioxidants defenses, has been actively implicated in the etiology of the syndrome. the aim of this study was to determine of serum myeloperoxidase activity (mpo), paraoxonase activity (pon ) and arylesterase activity (are) in patients with pcos. material and methods: the study was carried out on women consisted of patients with pcos and healthy ones as control. serum pon activities were measured spectrophotometrically using diethyl-p-nitrophenylphosphate as substrate. phenylacetate was used as substrate for are measurement, and are activity was determined by measuring absorbance of the resulting phenol at nm. molar absorptivity coefficients were used in the calculation of pon and are activities as nmol phenol/ml serum/min. result: the mpo and are activities were significantly lower in the patient groups when compared with the control group ( . ae . - . ae . u/ml p < . , . ae . - . ae . u/ml p < . , recpectively). the pon activities are higher in the patient group ( . ae . u/ml) compared to the control group ( . ae . u/ml) are found, but are not statistically significant. conclusion: lower serum mpo and are activities might contribute to the increased susceptibility for the development of diseases risk in women with pcos. because free oxygen radicals are thought to contribute to the complication of many chronic diseases, the pcos may be related to oxidative stress. subclinical hypothyroidism, defined as an elevated serum thyroid stimulating hormone level associated with serum thyroid hormone concentrations within the reference range. free radicalmediated oxidative stress has been implicated in the pathogenesis of thyroid disorders. the ischemia-modified albumin (ima) has been proposed as a marker of protein oxidative damage, which has been found to reflect hypoxic stress. this study aimed to investigate the influence of subclinical hypothyroidism on serum ima levels. thirty-one subclinical hypothyroidism patients and control subjects were enrolled in the study. albumin, ima were measured and ima/albumin ratio was calculated. to determine the ima levels the measurement method based on albumin cobalt binding assay was used. serum ima levels of patients with subclinical hypothyroidism were . ae . absu, ima levels of control subjects were . ae . absu. ima levels were significantly higher in patients with subclinical hypothyroidism patients than in control subjects (p < . ). when ima values were normalized for albumin concentrations, the ima/albumin ratio was also significantly elevated in patient group compared to control group (p < . ). ima levels are increased in patients with thyroid dysfunction. elevated levels of ima can be a clinically useful marker of protein oxidative damage in subclinical hypothyroidism. p- . . - the effects on endothelial dysfunction of quercetin in streptozocin-induced diabetic rats excessively produced in pathologic conditions. ultimately, imbalance between oxidants and antioxidants results with oxidative stress (os). in this study, we investigated some os parameters in standard ( % protein, % ( % sucrose) carbohydrate, % lipid) and sucrose ( % protein, % ( % sucrose) carbohydrate, % lipid) diet fed bdnf heterozygous mice liver tissues. the male c bl strain wild type (+/+) and bdnf heterozygous (+/À) mice ( weeks) were obtained. the animals were fed ad libitum by special standard and sucrose diets. twenty four mice were divided into four groups and each group consist six mice. all mice were fed for weeks. first group involved in c bl wild type mice and fed by standard diet. second group contained c bl bdnf heterozygous mice and fed by standard diet. third group consisted c bl wild type mice and fed by sucrose diet. fourth group involved in c bl heterozygous mice and fed by sucrose diet. in first group, mda levels, sod and cat activities were higher than other groups. in second group, cat activities were lower than other groups. but, we could not find any statistically significant differences between all groups about mda, sod, cat levels in bdnf heterozygous mice liver tissues. in conclusion, standard and sucrose diet feeding may not affect mda, sod and cat levels in bdnf heterozygous mice liver tissues. brain-derived neurotrophic factor (bdnf) is member of neurotrophin family which plays critical roles in the development, differention, survival, maintenance of the central and peripheral nervous systems. bdnf also contributes to food intake and body weight control. bdnf heterozygous mice display increased body weight and mild hyperphagia. expression of bdnf is not limited to the brain, it also express some peripheral tissues like adipose tissue, liver, kidney, skeletal muscle, heart. even though roles of bdnf are well known relatively in central nervous systems, effects of this protein is not clear in peripheral tissues. as mentioned before, it is expressed in organs involved in energy, lipid and glucose homeostasis, including the liver, adipose and muscle tissues, but its role there remains unclear. in this study, we aimed to investigate role of bdnf on liver oxidative stress parameters in heterozygous mice model fed fat diet induced obese mice. in this study, we used c bl/ mice inbred strain wild type and bdnf heterozygous (+/À) mice. animals were divided to two groups: wild type (n = ) and bdnf heterozygote mice (n = ). the animals were fed ad libitum by high-fat diet during month. weight gain was recorded every th days. in liver tissues were measured malondialdehyde (mda), superoxide dismutase (sod) and catalase (cat) by spectrophotometric methods. liver mda levels decreased in obese bdnf heterozygous mice compared to obese wild type group and statistically significant difference between groups. bdnf heterozygous mice cat activities were higher than the other group and this difference was statistically significant. there was no statistically significant difference between the groups in terms of sod activities. it has been concluded that the mda levels and sod enzymes activities changed at high-fat diet induced obese bdnf heterozygous mice compared to wild type mice liver tissues. p- . . - disturbances of microelements profile in serum of overweight/obese adult females with acute and persistent pro-inflammatory chlamydia pneumoniae infection p- . . - determination of reactive oxygen species induced dna damage using modified cupric reducing antioxidant capacity (cuprac) colorimetric method s. uzunboy, s. demirci c ß ekic ß, r. apak department of chemistry, istanbul university, faculty of engineering, istanbul, turkey reactive oxygen species (ros) term is a common name of a group of species. hydroxyl radical and singlet oxygen can be taken into account as ros samples. ros may be formed as a result of endogenous or exogenous reasons. although ros have some beneficial functions, they should be balanced by antioxidants (aox). excessive amounts of ros can attack biological macromolecules including dna. dna damage is usually related with mutagenic and carcinogenic changes. that's why determination of dna damage is so important and there are a great many studies in literature comprising different techniques. one of the most common of them is the 'comet assay'. but application of the method and interpretation of the results is not easy. investigation of certain dna damage products is also very common. these methods usually need expensive instrumentation such as using tandem mass spectrometry. on the other hand, depicting total dna damage on a certain product may cause misinterpretations. in the presented study, dna was decomposed by hydroxyl radicals produced by fenton method. in the study while dna is not cuprac reactive the oxidation products can react with the cuprac reagent. the effect of aox was also investigated. for this purpose, selected aox compounds were added to the reaction medium. because of their radical scavenging effect, the cuprac absorbance decreased in the presence of aox. in the presence and absence of aox, absorbance differences were calculated. the calibration graphs between final concentration and absorbance differences were drawn for each aox. gallic acid was determined as the most effective one among the tested aox samples. for statistical comparison with the presented study, tbars was used as reference method. direct use of dna as a probe material to determine oxidative damage may be an advantage to understand dna hazard in biological systems. the proposed method can be applied in all laboratories having a spectrometer as a cost-effective and simple procedure. p- . . - effects of alpha- antagonists on oxidative system of rat heart tissue benign prostate hyperplasia is a progressive process occurring in the stromal and epithelial components of the prostate. alpha- receptor blocking agents are used for relaxation of the smooth muscles in the prostatic stroma. our aim was to investigate the effects of alpha- antagonists on oxidative system of rat heart tissue. male wistar albino rats were divided into groups randomly. ) tamsulosin ( mg/kd/day), ) terazosin ( mg/kg/day), ) doksazosin ( mg/kg/day), ) alfuzosin ( mg/kg/day), ) control. all drugs were administered every other day single doze via oral. rats were sacrificed after days. heart tissue was taken for biochemical analysis. malondialdehyde (mda), nitric oxide (no), protein carbonyl (pc) levels and superoxide dismutase (sod), glutathione peroxidase (gsh-px) enzyme activities were determined in supernatant samples. there was not an significant difference between terazosin, doxazosin, alfuzosin, tamsulosin groups in means of sod, mda and gsh-px levels. no levels were significantly different between tamsulosin group and the control group (p = . ). in addition, tamsulosin group and terazosin group were also significantly different (p = . ). according to these results we can say that tamsulosin group had higher no levels than control and terazosin group. tamsulosin's enhancer effect on no levels leads to relaxation of the heart muscle and vascular relaxation, and so fewer side effects than other alpha antagonists. the effect of rat liver tissue radical metabolism and the protective role of hippophae rhamnoides l. on cold and immobilization stress model cold and immobilization stress is a widely used model for study the changes that occur on oxidant-antioxidant balance. hippophae rhamnoides l. (seabuckthorn; sbt) a unique and valuable plant has recently gained worldwide attention, mainly for its medicinal and nutritional potential. this study was aimed to investigate the protective role of sbt which is a natural herbal product with high antioxidant content on oxidative and nitrosative stress induced by cold and immobilization stress in rats. wistar albino rats were divided into groups randomly. control (i.p. physiological saline), sbt (i.p. mg/kg/ hours sbt), stress (i.p. physiological saline; hours cold + immobilization stress) and sbt + stress (i.p. mg/kg/ hours sbt; hours cold + immobilization stress) groups were formed. nitrotyrosine levels were determined by elisa whereas total antioxidant capacity, total thiol, total glutathione, nitrite-nitrate levels and superoxide dismutase and glutathione peroxidase activities were measured by colorimetric methods. sbt + stress group nitrite-nitrat (p = . ), total glutathione (p = . ) levels and glutathione peroxidase activities (p = . ) were found to be significantly higher whereas superoxide dismutase activity was found to be lower (p = . ) when compared to stress group. there was no significant differences between stress group total thiol and total antioxidant capacity levels compared with stress + sbt group. stress + sbt group oxidative and nitrosative stress marker -nitrotyrosine level was found to be significantly higher when compared with control group (p = . ) whereas there was no significant differences between stress and stress + sbt groups. all this data show that sbt has antioxidant properties on cold and immobilization induced oxidative and nitrosative stress in rat liver tissue. obesity is a major health problem with growing incidence and accompanying complications. its relation with diminished cognitive functions was reported. this study aims to evaluate the effects of obesity induced oxidative stress and metabolic alterations on the cognitive functions of children and adults. children and adolescents with obesity (age: - ); and age and gender matched healthy subjects were enrolled. all subjects completed the battery tests of cnsvs via computer. the scores were compared by using commercial software (ibm spss statistics ). biochemical parameters, malondialdehyde (mda) and protein carbonyl (pc) levels were estimated. mda and pc levels were significantly higher in subjects with obesity ( . ae . lmol/l; . ae . nmol/ml) than the controls ( . ae . lmol/l; . ae . nmol/ml) (< . ). there was statistically significant difference between study and control groups on all cognitive performance domains. significant correlation was detected between mda, pc levels and the cognition indexes. children with obesity should be evaluated for the cognitive functions, together with the metabolic follow-up. obesity induced oxidative stress may be the reason of the diminished cognition in children as well as the changes in the lipid profile and inflammation, but we need larger study groups to lighten these complex process. p- . . - relative contribution of nitric oxide synthase (nos) isoforms to oxidative/nitrosative stress in the cerebral cortex of rat with acute liver failure (alf) acute liver failure (alf) is associated with deregulation of nmda/cgmp/no signaling and oxidative/nitrosative stress in the brain. however, the relative roles of the different nos isoforms and the mechanisms underlying alterations in their activities during alf are not fully clear. here we investigated gene and protein expression of nos isoforms, nos activity, enos uncoupling and total no production in cerebral cortex of rats with thioacetamide (taa)-induced alf. sprague dawley rats ( - g) received three i.p. injections of taa ( mg/kg) at hours intervals. the brain cortex expression nos isoforms (enos/inos/nnos) was measured by real-time pcr and western blot, nos activity was tested by monitoring the conversion of radiolabeled arginine to citrulline. reactive oxygen species (ros) were quantified in the presence of nos substrate l-arginine, using the carboxy-h dcfda probe. no was measured with the griess procedure. the enos expression was decreased, whereas the enos dimmer/monomer ratio and nnos/inos expression were elevated in taa treated rats. while the total nos activity was decreased, the inos activity was elevated and no concentration tended to increase. ros production was elevated by taa. unspecific nos inhibitors l-name and l-nna attenuated ros production in both control and taa rats, but with higher efficiency in the latter case. ca + chelation had almost the same effect as pharmacological nos inhibition suggesting that ca + -independent inos activity is not the main source of ros. incubation with high dose of tetrahydrobiopterin (bh ) with which is critical for enos dimerization and subsequent no production also reduced ros production indicating the enos uncoupling phenomenon in taa cortex. the study points to enos downregulation due to lowered protein expression and uncoupling as a novel mechanism contributing to enhanced superoxide o anion formation, and confirms the role of inos/nnos in enhancing no synthesis in alf-affected brain. introduction: diabetes mellitus (dm) is an endocrine disorder of world which is characterized by altered blood glucose levels and related complications including hepatic injury. myrtus communis l. (mc) is widely used by diabetic patients in the folk medicine of turkey as well as they are used worlwide. it is known that of leaves, oil and fruit of myrtus communis l. (mc) have therapeutic effects on diabetes mellitus (dm). this study was aimed to analyze the possible antidiabetic and hepatoprotective effects of mc berries in streptozotocin (stz) induced diabetic rats. materials and methods: a total of thirty rats composed of six groups as each included five rats were used. mg/kg stz was injected once to animals to induce dm. after stz injection, rats were exposed to three different ethanol extracts of mc berries ( , and mg/kg) by oral gavage during days. alanine aminotransferase (alt) and aspartate aminotransferase (ast) levels were determined in serum and glutathione (gsh), malondialdehyde (mda) levels and superoxide dismutase (sod) activity were determined in liver tissue. results: mc administration provided significant reducement in the altered serum glucose, ast and alt levels in all diabetic groups. mc extract showed significant antioxidant activity by altering sod activity and gsh level and reducing mda levels in diabetic rats compared to controls (p < . ). serum ast and alt levels were reduced by mc administration in all diabetic groups. mc administration provided significance increment in sod activity and gsh level, and significant reduction in mda levels compared to controls (p < . ). the maximum hypoglycemic and antioxidant effects were observed at mg/kg dose of mc. background: human serum paraoxonase (pon ) is a calcium dependent esterase that hydrolyzes organophosphates and also arylesters such as phenyl acetate. pon prevents ldl oxidation by hydrolyzing lipid peroxides. pon is inhibited by various chelating agents, heavy metal ions, and sulfhydryl reagents. in our study we investigated the effect of calcium on ldl oxidation of purified pon q r isoenzymes. methods: pon q r isoenzymes were partially purified from human serum. both allozymic forms were treated by preincubation with mm edta for minutes. ldl oxidation was induced by copper ions. formation of thiobarbituric acid-reacting substances (tbars) was used as a measure of lipid peroxidation. homocysteine thiolactonase (htlase activity) and arylesterase activities were measured spectrophotometrically by using homocysteine thiolactone and phenylacetate as the substrates. results: addition of mm edta to partially purified hdl-pon q r isoenzymes inhibited % of htlase and arylesterase activities. inactivation of pon for arylesterase/htlase activity by the addition of edta did not reduce the abilities of both allozymic forms in protecting ldl from oxidation. conclusion: ca + -dependent inhibition of pon q r arylesterase/htlase by using the metal chelator edta, did not alter pon 's ability to inhibit ldl oxidation. pon 's ability to protect ldl from oxidation may not require calcium. p- . . - evaluation of cholinesterase inhibitory effect, anti-radical and anti-lipid peroxidation activities of mentha pulegium i. hamad , college of applied medical sciences, aljouf university, aljouf, saudi arabia, faculty of medicine, bahri university, khartoum, sudan introduction: many studies indicated that intake of dietary and medicinal plants is effective in preventing or suppressing many diseases, therefore, there is a growing interest in plant'sbioactive compounds. mentha pulegium, is widely used in gulf countries in herbal teas or as additives in commercial spice mixtures for many foods to offer aroma and flavor. the aim of this study is to investigate the in vitro radical activity, the total phenol and flavonoid content, anti-lipid peroxidation and the cholinesterase inhibitory effects of mentha pulegium methanol extract. methods: the acetylcholinesterase and butyrylcholinesterase inhibitory potentials of extracts, were evaluated by colorimetric assay. the in vitro antioxidant activity was measured by dpph assay, the total phenols content was measured by folin-ciocalteau assay, the flavonoids content by the alcl colorimetric method, and the protective effect of menthe mentha pulegium extracts against lipid peroxidation was evaluated using a liposome oxidation system. results: the methanol extract showed a scavenging activity nearly equivalent to vitamin c which is attributed to its high phenolic and flavonoid contents. the extract possessed protective effect against lipid peroxidation in a dose dependent manner. the methanol extract shows very little anticholinesterase activity as compared to the standard compound, physostigmine. conclusion: results presented here indicate that mentha pulegiumpossess strong antioxidant activity and protective effects and they can therefore be used as a natural additive in food, cosmetic and pharmaceutical industries. type diabetes mellitus is a long term metabolic disorder that is characterized by hyperglycemia and insulin resistance. because of the hyperglycemia and free radicals, diabetes can cause cellular instability. micronuclei is a sensitive indicator of genetic damage and a marker of dna damage. micronuclei is also a morphological marker of chromosomal instability. in this study, we aimed to evaluate the frequencies of micronuclei in papanicolaou stained buccal cells of type diabetic patients. a total of type diabetic patients and healthy individuals were involved into our study. buccal smear samples that belong to these individuals were stained by using papanicolaou method for cytologic examination and the stained slides were evaluated by light microscopy (olympus bx- ). cells with micronuclei in each papanicolaou stained buccal smear sample were counted under light microscopy. the frequency of micronucleated epithelial cells were seen as significantly higher in type diabetic patients than the control group (p < . ). one of the boron compounds is sodium perborate tetrahydrate (nabo . h o), which the most widely used solid peroxygen compounds. it is used in safety bleach formulations, detergents and tooth powders. as known these products are commonly used in daily life. however, the actions on blood antioxidant defenses of sodium tetraborate against reactive oxygen species are not identified yet. it is reported that oxidative stress caused by ros damages. in this study, we searched enzyme activities of superoxide dismutase (sod), catalase (cat), glutathione-s-transferase (gst), glutathione reductase (gr), glutathione peroxidase (gsh-px) and glucose- -phosphate dehydrogenase (g pd) also the effect sodium perborate tetra hydrate on activities of these enzymes from human erythrocyte under in vitro conditions. according to our findings sodium perborate tetrahydrate caused significant (p < . ) increasing in the cat activity from red blood cell. the other antioxidant enzyme activities (sod, gst, gr, gsh-px and g pd) did not show any changing by influence of sodium perborate tetrahydrate. metabolism of obese individuals could be exposed to risk of chronic low-grade pro-inflammatory effect and oxidative stress. some inflammatory and oxidative markers have been studied recently. plasma total antioxidant status (tas) and total oxidant status (tos) parameters can be non-invasive markers of diseases such as fatty liver disease, laparoscopic procedures (pneumoperitoneum), accompanying inflammatory condition like urinary tract infection, diabetic neuropathy, chronic hepatitis. the study groups have been comprised of two groups with normal to over-weight children. tas and tos levels were detected and the oxidative stress index (osi) was computed as a marker of the grade of oxidative stress. the over-weight group displayed higher levels of fasting glucose, insulin resistance, the body mass index. also, we know that insulin resistance leads to increased lipolysis and free fatty acid output. higher tos as well as crp is related to the group, also lower tas than other group is shown. crp levels in plasma were positively correlated with insulin and glucose levels. in addition, there was a significant relationship between osi and insulin resistance in the over-weight group. tas and tos are together more accurate sings of oxidative and antioxidative status of people. as well as a raise over weight-related subclinical inflammation and a fall antioxidant capacity is significant even in children. this condition may eventually develop the risk of long-term vascular damage. the effects of hydrogen peroxide pretreatment on antioxidant enzyme activities in calli tissues of two eggplant genotypes under salinity o. yasarkan , e. aky€ uz , g. baysal furtana , s. s. ellialtioglu , r. tipirdamaz nezahat g€ okyigit botanic garden, istanbul, department of biology, faculty of sciences, gazi university, ankara, department of horticulture, faculty of agriculture, ankara university, ankara, department of biology, faculty of sciences, hacettepe university, ankara, turkey the effects of hydrogen peroxide (h o ) pre-treatment on catalase (cat) and superoxide dismutase (sod) were investigated and lipid peroxidation measured as malondialdehyde (mda) content of the calli from salt-sensitive (artvin) and salt-tolerant (mardin) eggplant genotypes under salinity stress. the seeds from each genotypes were germinated on ms medium for weeks and hypocotyl tissues from these plantlets were used as explants for calli induction on ms medium including mg/l , -d and . mg/l kinetin. as for the pre-treatment, the subcultured calli tissues were transplanted on the mediums containing and lm h o for hours and then transplanted on the mediums including mm nacl for hours. antioxidant enzyme analysis and mda measurement was carried out for the control, nacl-only, h o -only and h o pre-treated tissues. pre-treatment with h o decreased the deleterious effects of salt stress on mda contents. in comparison with salt stressed groups, h o pre-treatment with or without nacl reduced mda content especially in artvin. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in lm h o + nacl groups on each genotypes. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in lm h o + nacl groups on each genotypes. the result showed pre-treatment of lm h o induced acclimation of the plants to salinity. in addition, lm h o pre-treatment, as a stress signal, could trigger the activation of antioxidant enzymes in calli and in this way alleviated the oxidative damage in calli growth under salinity. the investigation of effects of ghrelin and cannabinoid cb receptor agonist and antagonist on oxidant and antioxidant mechanisms on brain tissues of penicillininduced epileptic rats the aim of this study is to investigate the individual effects of ghrelin and cannabinoid type (cb ) receptor agonist acea, the antagonist am- and the interaction of these two different systems on oxidant and antioxidant systems in the brain, cerebellum and brain stem tissues of penicillin-induced epileptic rats. in this study male wistar albino rats were used weighing - g. each group was consisted of rats. study groups: : control, :penicilin( iu), :penicillin( iu) + ghrelin( lg), :penicillin( iu) + am- ( . lg), :penicilin ( iu) + acea( . lg), :penicillin( iu) + am- ( . lg) + ghrelin ( lg), :penicillin( iu) + acea( . lg) + ghrelin( lg). than the levels of mda, gpx and sod are measured in plasma and tissue samples of these rats. penicillin was found to be induced lipid peroxidation in the brain, cerebellum and brain stem tissues in our study. ghrelin and acea, which both have anticonvulsant effects, were shown to be effective in reversing the oxidative damage caused by penicillin and proconvulsant am was found to further increase the oxidative stress caused by penicillin in these tissues. ghrelin also was found to suppress the oxidative stress caused by am in the cerebellum tissue but it did not contribute to antioxidant effects produced by acea. since the role of oxidative stress in epilepsy has been established, it may be suggested that ghrelin and acea may have anticonvulsant effects via their antioxidant features. the discovery of inhibitors for enzymes that metabolize endogenous ghrelin and cannabinoids through new studies may contribute to the improvement of seizure resistance in epilepsy. accelerated atherosclerosis in patients with ankylosing spondylitis (as) give rise to increased cardiovascular morbidity and mortality. endothelial dysfunction could be the initial process in the development of atherosclerosis. human endothelial cell-specific molecule- (endocan) is a novel human endothelial cell-specific molecule. therefore, we assessed serum endocan levels and carotid intimamedia thickness (cimt) as a surrogate marker of atherosclerosis in patients with as. a total of patients with a diagnosis of as according to newyork ctriteria and control subjects were included in our study. serum endocan, interleukin- (il- ), tumor necrozis factor-a (tnf-a), c reactive protein (crp) and cimt were measured in all participants. serum endocan, il- , tnf-a levels were measured with elisa. the other parameters were done by routine biochemical methods. as patients exhibited increased serum endocan levels and cimt compared to matched controls (p < . ). whereas, serum il- , tnf-a were similar between grous. in patient with as, there were no significant differences between active and inactive patients by means of il- , tnf-a, endocan and cimt. in as group, cimt correlated with disease duration and age (r = . , p = . ; r = . , p = . ). we could not find any significant correlation between serum endocan levels and parameters studied. our study shows increased cimt in as patients without traditional risk factors such as increased bmi, lipid profile compared to controls. although we found increased circulating endocan levels in patients with as, the other factors could affect increased atherosclerosis in this population because of lack of correlation between endocan and cimt. probable biomarkers could be related to increased cimt in patients with as should be investigated in larger study groups. keywords: ankylosing spondylitis, atherosclerosis, carotid intima media thickness, endocan p- . . - investigation of pentose phosphate pathway and oxidative stress in erthrocyte infected babesia ovis a. bildik, t. karagenc ß, p. a. ulutas, n. aysul, h. aksit adnan menderes university, aydin, turkey introduction: babesia infections occur in cattle, sheep, goat, horse, dog, cat pig and rodents. in this study, the effects of babesia ovis living and present in the erythrocytes to glucose metabolism was researched. at the same time, biochemical parameters were also associated with parasitemia. materials and methods: babesia ovis (israel) cell culture was provided from dr. abel martin gonz alez oliva (portugal). culture passaged or hours according to parasitemia state ( - %). biochemical analyses were performed in erythrocyte culture in which parasitemia between % and %. cell counts and hemoglobin concentration of erythrocytes culture suspension were measured at cell counter instrument and than it was washed times with physiological saline, erythrocyte suspensions were stored at- oc analysis. gssg (oxidize glutathione), gsh (reducte glutathione), nadph, glukoz p dehydrogenaz, gshpx (glutathione peroxidase), gshrx (glutathione reductase) were determined by commercial kits. all experiments were done in duplicate, the results were calculated by the number of erythrocytes. results: parasitemia was positively correlated with gsh, nadph and gshrx (p < . ). a correlation between other biochemical parameters was not observed. discussion: the pentose phosphate pathway in erythrocytes has an important role such as to provide pentose sugar required for the synthesis of nucleic acid, to reduce glutathione, to produce nadph and to protect from methemoglobin accumulation. in studie sthat naturally infected erythrocytes with babesia parasites, it was seen to be caused to oxidative stress, however gsh results in these investigation were obtained differently . conclusion: according to the results of this study that performed in vitro, it can be suggest that their glutathione metabolism and pentose phosphate pathway of parasites may active.key words: babesiosis, gsh, gssg, nadph, g pdh, gshpx, gshrx p- . . - in vitro protective effect of betaine on peroxidative injury caused by ethanol and aspirin exposure on rat brain synaptosomes i. sogut , g. kanbak istanbul bilim university vocational school of health services, istanbul, eskisehir osmangazi university medical school department of biochemistry, eskisehir, turkey aspirin intake of specific daily doses are advised by doctors to postmenapausal women and men above years of age to prevent heart attack and even cancer in recent times. in this study, the aim is to investigate the in vitro cytototoxic effects of different doses of ethanol ( mm, mm ve mm) alone and together with lg/ml aspirin, and possible protective role of mm betaine on rat brain synaptosomes. male sprague dawley rat forebrains were divided into equal pieces and pooled to form study groups. synaptosomal fractions extracted from pooled rat brains were incubated with different doses of ethanol, aspirin and betaine, and malondialdehyde (mda) levels, an important indicator of cellular damage, were measured. a significant increase (p < . ) was observed in mda level of mm ethanol group compared to control group. different doses of ethanol ( mm, mm ve mm) + aspirin exposure significantly increased (p < . ) mda levels compared to controls, whereas betaine administration significantly decreased (p < . ) lipid peroxidation caused by ethanol + aspirin treatment. we conclude that ethanol and ethanol + aspirin administration increases lipid peroxidation in rat brain synaptosomes while betaine helps prevent this peroxidative membrane injury.keywords: aspirin, betaine, ethanol, malondialdehyde (mda) p- . . - analyses of mitochondrial biogenesis in hepatocellular carcinoma treated with berberine f. aygenli, h. c ß imen yeditepe proteomics and mass spectrometry group (yediprot), genetics and bioengineering, yeditepe university, istanbul, turkey objective: berberine (bbr) has been demonstrated to have anticancer activities against various cancer types, particularly hepatoma. in this project, we aimed to reveal the effect of bbr treatment on mitochondrial biogenesis through sirtuins and hif- a in hepatocellular carcinoma cell line, hep b under hypoxia. method: hep b cells were subjected to normoxia ( % o ) and hypoxia ( % o ) in the presence or absence of bbr treatment. the amount of bbr was optimized via cell viability (mts) assay under normoxia. then, immunoblotting experiments were performed to identify the effect of bbr on hif- a, pgc- a, and sirtuins involved in mitochondrial stress. the variation in the oxphos complexes and the level of reactive oxygen species (ros) were also measured to investigate the effect of bbr on mitochondrial energy stress state. results: here, we present that cell viability was significantly decreased at lm. bbr treatment has shown significant reduction in hif- a and sirt which responsible for up-regulation of glycolysis. also, succinate dehydrogenase (cii) and cytochrome c oxidoreductase (ciii) of the oxphos complexes were downregulated without any change in nadh dehydrogenase (ci) or atp synthase (cv). bbr significantly abolished to oxidative stress under hypoxia, which was demonstrated as a reduction in the level of reactive oxygen species by decreasing on sirt expression. bbr induces the overexpression of sirt and its deacetylated-pgc- a, which might be an indicator of being a potent protective agent against hypoxia by normalizing mitochondrial function and inducing mitophagy in impaired mitochondria caused by deficiency of glycolysis and oxphos. conclusion: detailed information about the communication between hif- a and sirtuins and their relation to mitochondrial energy production was provided with the alteration of their activity by bbr treatment. it is highly expected that bbr and its derivatives might become important during the development of supplemental therapies. introduction: reactive oxygen species are involved in a variety of biological phenomena, such as carcinogenesis, inflammation and aging. among the targets of ros, dna appears most important in tumor biology since it is firmly established that cancer is a genetic disease. ros induce several kinds of dna damage, including strand breakage and dna-protein cross-linkage. fruit of zizyphus jujuba, a traditional chinese herb widely consumed in asian countries, has been reported to possess several vital biological activities. this study intends to evaluate their antioxidant activity on glioblastoma cells. materials methods: cell survival was quantified by colorimetric mtt assay. human gliblastoma cells were pretreated with lm h o after minutes lm ziziphus jujuba essential oil was added to the cells for three hours. then, the cell homogenates were taken and glutathione, total oxidant and total antioxidant capacity and nitric oxide levels were estimated using spectrophotometric methods. results: ziziphus jujuba treated cells could prevent intracellular glutathione from being depleted following an exposure of h o . also our data suggest that ziziphus jujuba is effective in preventing h o induced oxidative stress and nitric oxide levels. discussion: some research showed that h o was over produced in the pathological process of acute and chronic neuronal toxicity, the toxicity effect of b-amyloid on the cultured neuron and neuronal cell line was mediated by h o . the traditional medicine recommend several medicinal plants for providing relief from various inflammatory diseases. many research has been reported that the essential oil from seeds of helping to prevent the oxidative stress and neuronal diseases in brain. introduction: toxicity by oxygen radicals has been recommended as a major cause of cancer, heart disease, and aging. oxygen radicals and other oxidants appear to be toxic in large part because they start the chain reaction of lipid peroxidation. most of the analytical techniques for peroxide determination are generally time consuming and not very suitable for routine or on line analysis. we aimed to design a new biosensor for rapid determining of oxidant agent hydrogen peroxide. materials and methods: all reagents were of analytical grade unless stated otherwise, and were purchased from sigma aldrich. firstly, the -hidroxymetacrilate metacriloamidoscystein nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. free nh groups of catalase enzyme make schiff bases between nanopolymer's carbonyl groups, then immobilization was actualysed with cross linking reagent glutaraldehyde. we developed a biosensor system preparing ferrociyanide, selected as a mediator, in the buffer solution. results: polyhemamac nanopolymer and catalase complex were immobilized by glutaraldehite to construct a hydrogen peroxide biosensor. the responses of the biosensor are therefore proportional to the oxidation peaks of the complex at + . v potential. the cyclic voltammograms obtained from the experiments showed that, pottasiumferrociyanide mediator complex positively affected the biosensor responses for hydrogen peroxide determination. discussion and conclusion: as a result of this study, the method developed by the catalase enzyme electrode was found to be more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. since biosensor technology provides economical, practical, specific and sensitive results for the determination of hydrogen peroxide, it was improved very efficiently. p- . . - impact of amoxicillin, gentamicin and cefazolin sodium antibiotics on antioxidant gene expression and enzymatic activities in mouse liver p. g€ uller , h. budak , m. sisecioglu , m. c ß iftci department of chemistry, faculty of science, atat€ urk university, erzurum, department of molecular biology and genetics, faculty of science, atat€ urk university, erzurum, department of chemistry, faculty of arts and sciences, bing€ ol university, bing€ ol, turkey reactive oxygen species (ros) are highly reactive molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. living organisms have the antioxidant defence systems to block harmful effects of ros. the imbalance between oxidants and antioxidants is termed oxidative stress. the antioxidant defence mechanisms are divided into two groups as enzymatic and nonenzymatic defences. enzymatic defence mechanisms consist of enzymes like superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glucose- -phosphate dehydrogenase (g pd) and glutathione s-transferase (gst). the present study was designed to determine the effects of gentamicin, amoxicillin and cefazolin sodium antibiotics on the hepatic antioxidant system and to determine any possible correlation between enzymatic and molecular levels. for this reason, effects of these antibiotics on the transcription of the antioxidant system has been investigated by real time pcr, and then the enzyme activity of these enzymes have been measured in whole liver homogenate obtained from control group and the drug administered groups mice. our results demonstrate that administering antibiotics led to crucial inhibition of all antioxidant enzyme activity. while significant transcriptional activation for sod and cat was seen in the gentamicin treated group, the transcription of gst and g pd was decreased. however transcriptional activation was seen for sod and cat in amoxicillin administered group, the transcription of gst was decreased as compared with the control group. in the cefazolin sodium treated group, while cat and gst transcription were elevated significantly, the expression of sod and g pd were decreased. in conclusion, gentamicin, amoxicillin and cefazolin sodium affect the hepatic antioxidant system at the molecular and protein level. this work was supported by scientific research project of ataturk university of turkey (grant no: / ). p- . . - protective effects of curcumin supplementation on oxidant/antioxidant system changes created by organic phosphorus pesticide poisoning organic phosphorus pesticides (opp), widely used in agriculture or as insecticides in home, cause adverse health effects. chlorpyrifos is one of the most commonly used opp. we aimed to investigate the possible protective effects of curcumin (cur) supplementation, the principal curcuminoid of turmeric, on poisoning symptoms and oxidant/antioxidant system changes caused by chlorpyrifos. adult sprague-dawley rats were used. cur ( , and mg/kg) were administered orally for days. on the sixth day, chlorpyrifos ( mg/kg, s.c.) was administered. twenty four hours after chlorpyrifos administration, body weights, locomotor activities and body temperatures of rats were measured. following the measurements, rats were decapitated and the blood, brain and liver tissue samples were taken and prepared for the biochemical and histopathological measurements. chlorpyrifos administration increased the malondialdehyde (mda) levels but decreased catalase (cat), superoxide dismutase (sod), glutathione reductase (grx) concentrations and reduced/oxidized glutathione (gsh/gssg) ratio in the blood samples, brain and liver tissues compared with the control group (p < . - . ). the concentration of advanced oxidation protein products (aopp) were increased only in the brain tissue after chlorpyrifos administration (p < . ). cur administration reduced all of these changes (p < . - . ). similarly, cur at the doses of mg/kg reduced the decreases in body weight, body temperature and locomotor activity with chlorpyrifos (p < . ). additionally, the histopathological damage scores induced by chlorpyrifos (p < . - . ) were decreased by the administration of cur (p < . - . ). our findings suggest that cur supplementation can ameliorate the poisoning effects of chlorpyrifos via supporting the antioxidant mechanisms and cur could be used for protective purposes against oxidative stress and tissue damage caused by chlorpyrifos. the effect of ogtt applied for screening in pregnancy on adenosine deaminase and xanthine oxidase activity in normal and prediabetic pregnant women z. c. ozmen, k. deveci, i. benli department of biochemistry, gaziosmanpasa university medical faculty, tokat, turkey objective: it was reported that the activities of adenosine deaminase (ada) were different in normal pregnant women and pregnant women with gestational diabetes mellitus (gdm). it was also stated that the activity of xanthine oxidase (xo) was increased in pregnant women with gdm. the objective of this study was to evaluate if glucose have effects on oxidative stress in prediabetic women by affecting ada and ox after g ogtt which was applied in pregnant women for screening. methods: the serum specimens of pregnant women who applied to the outpatient clinic of the obstetrics and gynecology department and had g ogtt, were used in this study. ada and xo activities were analyzed in the serum specimens taken from the normal (n = ) and prediabetic pregnant women (n = ) in the th and th minutes of ogtt. ada and xo activities were measured with the spectrophotometric method and the u/l enzyme activity was calculated. findings: there was no significant difference between the th and th minutes regarding the ada activities in the normal and prediabetic pregnant woman groups. however, we observed a significant difference between th and th minutes regarding the xo enzyme activity in normal pregnant women (p = . ). in normal pregnant women, the median xo enzyme activity in the th minute was . ( . - . ) u/l and it was . ( . - . ) u/l in the th minute. nevertheless, there was no correlation between the xo activity and glucose level. as to the prediabetic pregnant women, there was no significant difference between the xo enzyme activities in th and th minutes. the results of our study showed that the xo activity increased as a response to ogtt in the normal pregnant women compared with the prediabetic pregnant women. this finding made us think that the oxidative stress caused by ogtt did not affect the xo response in prediabetic pregnant women and that there would be some adaptive mechanisms against the chronic exposure to high level glucose. rainbow trout (oncorhynchus mykiss) aquaculture continuously increases in turkey. the objective of the present study is to increase the productivity in fish farming of rainbow trout just via intervention in physical cases without the effects of any chemicals and investigate whether this conditions cause oxidative stress. in this experiment eight tanks were used and rainbow trout larvae were placed in each tank and these tanks were illuminated with light in different wavelengths; natural sunlight, and incandescent long-wave (red light), medium-wave (green light) and shortwave (blue light) led lights. the experiment took days. biochemical changes in rainbow trout exposed to light in different wavelengths (red, green, blue) were analysed via the variations in gr, gst, g pd, gpx, sod and cat enzyme activities, which are significant for enzymatic antioxidant defence system and in ache activity, which plays an important role on central nervous system. maximum activity change in liver tissue was observed for gst and g pd enzymes in fish grown under green light and for sod enzyme in fish grown under blue light. in gill tissue, sod and g pd activities were affected the most, and in brain tissue, these were gst and sod activities. it was observed that the average weight of the fish increased . times under red and blue lights and . times under green light. the highest weight increase was observed under green light, however, antioxidant enzyme activities increased in the liver and gills and decreased in the brain tissue under this light condition. in conclusion, it was observed that productivity was . times under red light when compared with control group and it was determined that the fish grown under red light can tolerate oxidative stress more than other wavelength. p- . . - effect of nigella sativa on biliary obstructioninduced oxidative stress and apoptosis in rats human safety concerns, since that these agents may not only cause acute toxicity via inhibition of acetylcholinesterase but they can also induce delayed toxicity in the nervous system. a key interest to the current work is the potential correlation between gene expression and cytoskeletal protein changes in differentiating neural cells exposed to sub-lethal neurite outgrowth inhibitory concentrations of specific ops, which was addressed by analysing the underlying changes in the levels of cytoskeletal gene expression and protein levels. to assess the molecular effects of op exposure, phenyl saligenin phosphate (psp), chlorpyrifos (cpf) and its metabolite chlorpyrifos oxon (cpfo) were applied at the point of induction of differentiation of rat c glioma and mouse n a neuroblastoma cells and incubated for hours. at sub-lethal concentrations ( , , lm) all three ops used in this study were able to inhibit the development of neurites with no significant effect on cell viability, as determined by neurite outgrowth and mtt reduction assays. to understand the possible effects of ops on cytoskeletal gene expression, primers for genes encoding glial fibrillary acidic protein (gfap), biii tubulin, growth associated protein (gap ) and neurofilament heavy chain (nefh) were optimized for qpcr analysis and the levels of the corresponding proteins were detected by western blot analysis. exposure to ops caused in most cases a reduction in the levels of cytoskeletal proteins, and the results from qrt-pcr analysis also indicated reductions in the gene expression of gfap in c cells, and of nefh and biii tubulin in n a cells, in a dose dependent manner. thus, the observed changes in protein levels are at least partly due to altered gene expression. curcumin is extracted from a perennial herbaceous plant known as curcuma longa. in recent years, considerable interest has been focused on curcumin due to its use to treat a wide variety of disorders without any side effects. earlier studies have shown that curcumin has anti-apoptotic, anti-inflammatory, antiproliferative, antiangiogenic, anticancer and antiplatelet activities. the goal of the present study was to investigate the effects of curcumin on peroxy radical-induced oxidative changes in human platelets. healthy volunteers were enrolled in the study. none of the study participants were on anticoagulation therapy. citrated venous blood samples were centrifuged at g for minute to obtain platelet-rich plasma (prp). the platelet pellet was washed and suspended with tris-nacl buffer. then, platelets were incubated with h o absence and presence of curcumin ( - lg/ ml) for hours at °c. to determine the preventive effects of curcumin on the oxidative stress and apoptosis induced by peroxy radicals in human platelets were determined by measuring levels of of lipid peroxidation, total antioxidant capacity, caspase , and activities, and mitochondrial membrane potential. additionally, we also studied the effects curcumin on platelet aggregation induced by adp. pre-treatment of platelets with curcumin caused a marked reduction in oxidative stress, activation and apoptotic markers in a dose-dependent manner. on the other hand, pre-treatment of platelets with increasing doses of curcumin resulted in inhibition of platelet aggregation induced by adp. in the light of our findings, we suggest that curcumin may have a therapeutic potential to prevent platelet activation related disorders. people have been using mushrooms in the treatment of diseases as well as food, for centuries. most of the edible and inedible mushroom species were used in important medical studies and their effects were begun to be used in the treatment of diseases. this study focuses on the hepatoprotective effects of tricholoma anatolicum, which is endemic specie in turkey, against oxidative stress based on hydrogen peroxide (h o ) on hepg liver cancer cell line. t. anatolicum used in this study was extracted with the help of ultrasonication and fraction methods. then the cytostatic effects of extracts on hepg cells were explored and their hepatoprotective effects were determined. moreover, various concentrations of aqueous extract (ehta) of t. anatolicum were determined by hepg cells's - - hours effect analysis on their cellular morphology, xtt and real-time cell analysis in of xcelligence device. ehta extract's cell pathway (apoptosis and necrosis) effects on hepg cells were determined with flow cytometry method with the help of annexin v-apc and aad fluorescent dye. finally, the phenolic compounds found in ehta extracts were determined with the help of hplc methods. according to xtt cytotoxicity analysis, the ehta extract values were determined as follows: hours ic > lg/ml, hours ic . furthermore, according to the real-time cell analysis made with xcelligence, ehta extracts were found to be; hours ic = . lg/ml, hours ic = . lg/ml, hours ic = . lg/ml. increasing concentrations of ehta extracts were determined to direct hepg cells to apoptosis. moreover, considering the hplc analysis -according to the reference point of mg in g sample-within ehta extracts, catechins and vanillic acid peaked. the final results revealed that t. anatolicum's effect on hepg was cytostatic at low doses, and cytotoxic at high doses. p- . . - relationship between serum ceruloplasmin levels and coronary blood flow background: there is growing evidence that oxidative stres plays an important role in the development of the slow coronary flow (scf) phenomenon. ceruloplasmin (cp) is a copper containing metalloenyzme which has antioxidant functionthrough its ferroxidese activity and is associated with cardiovascular diseases. we aimed to investigate the relationship between scf and serum cp level. methods: patients who underwent elective coronary angiography and had no significant epicardial coronary disease were included in the study. patients who had thrombolysis in myocardial infarction frame counts (tfcs) above the normal cutoffs were considered to have scf and those within normal limits were considered to have normal coronary flow (ncf). a total of patients ( subject as scf and subjects as ncf) were analyzed. ml blood samples were taken from the groups to study ceruloplasmin activity. serum ceruloplasmin levels were determined spectrophotometrically. results: the serum cp levels were statistically lower in scf group than in the ncf group ( ae versus ae ng/ml, p = . ). also there was a significant correlation between serum cp levels and tfcs (r = À . , p = . ). conclusion: the findings of this study suggests that patients with scf had lower serum cp levels correlated with tfcs. we concluded that reduced serum cp levels might represent a biochemical marker of scf. introduction: sleeve gastrectomy (sg) has been used for the surgical treatment of morbid obesity, as a first step or definitive treatment. alterations of thyroid hormones in gastrointestinal surgery were previously studied. the aim of the present study was to determine the effects of triiodothyronine (t ) supplementation on oxidative stress parameters in anastomotic tissue level. materials and methods: twenty-four male wistar albino rats were divided into control (n ), and experimental (n ) groups. rats were underwent a sg, with a hand-sewn suture. experimental group rats received a single dose of t ( mg/ g) in postoperative day. rats were sacrificed on postoperative day . serum thyroid stimulating hormone (tsh), free t (ft ), and free thyroxine (ft ) were analysed using elisa. each tissue was homogenized in ice-cold pbs (ph: . ) and centrifuged at rpm for minutes ( °c) to avoid contamination with cellular debris. the supernatants were used to measure total oxidant status (tos), total antioxidant status (tas), nitric oxide (no) and malondialdehyde (mda) levels. all tissue parameters were analysed by spectrophotometric methods. oxidative stress index (osi) values were calculated. results: rats given t hormone had not decline in ft levels compared with the control groups. a significant decrease in ft levels was found in t given rats on postoperative day . whereas tissue tos levels did not alter by thyroid hormone treatment, tas levels significantly decreased. osi values were not statistically different in tissues. tissue no levels were also similar in both groups. mda levels increased in t given rats compared with the control group. discussion and conclusion: this study showed that anastomosis after sleeve gastrectomy is associated with decreased ft level. although tos levels and osi values were similar in both groups, t supplementation induced lipid peroxidation by increasing tissue mda levels that might deplete tissue antioxidant level. reactive oxygen species (ros) are reactive chemical molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. although intracellular ros level is essential molecules for the signal transduction pathways, elevated intracellular level of ros leads to oxidative stress that causes damage to dna, proteins and lipids. therefore, excessive ros levels have to be eliminated by antioxidant defence systems. tip (tat interacting protein, kda) is a histone acetyltransferases (hats) that catalyses multiple functions in metabolism such as dna repair, apoptosis, etc. we thought that if tip has a role in signal transduction and apoptosis, it might have direct or indirect relationship with the antioxidant system. the present study was designed to determine the impact of tip gene on the hepatic antioxidant enzymes including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), and glutathione reductase (gr) both gene and protein level. for this reason, quantitative gene expression analysis on the antioxidant system has been investigated by real time pcr, quantitative protein expression has been investigated by western blot analysis, and then the activity of these enzymes have been measured in whole liver homogenate collected from control and liver-specific tip conditional knockout mice. additionally, since any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h o ) level in the cell might be an indication for the accumulation of ros, the relative levels of them were also studied. our data showed that the absence of tip affects the antioxidan system both gene and protein level. in conclusion, our initial data suggest that tip may be essential for the (ros) homeostasis and redox regulation. curcumin is a major chemical component produced from the rhizome of the plant curcuma longa. lt has been demonstrated that curcumin has an antioxidant, anti-inflammatory, and antiproliferative effects and, protects tissues against ischemia/reperfusion (i/ r) injury. i/r has detrimental effects on transplanted organs including uteri. the major consequence of l/r injury is oxidative stress leading to the generation of ros. uterine transplantation (ut) has been gaining popularity around the worid in the past few years. the aim of our study was to examine the antiapoptotic effects of curcumin on uterine l/r injury. the rats were randomized into three groups of seven rats each, group i consisted of rats that did not receive any treatment, group ll exposed to . hour of lschemia and hour of reperfusion, group iii of rats that received intraperitonealy curcumin ( mg/kg) . hour before the induction of i/r. then, the rat uterine tissue levels of mda, tac, and activities of caspase , and were measured. furthermore, the apoptotic index was determined immunohistochemically by the tunel method using light microscopy. biochemical analysis results showed that curcumin decreased the mda and caspase- and ieveis, and increased the uterine tissue levels of tac but, caspase activity did not changed by curcumin suggesting that curcumin induces apoptosis via intrinsic apoptotic pathway. on the other hand, an high apoptotic index was observed in i/r group ( . ae . %) and decreased after treatment with curcumin ( . ae . %). in conclusion, we demonstrated the protective effect of curcumin on apoptosis immediately after reperfusion induction in uteri and we can say that curcumin could improve ir injury and decrease apoptotic index. we propose that curcumin may be a novel approach for improvement of uteri i/r injury. glutathione and the related enzymes belong to the defence system of the tissues against chemical and oxidative stress. these enzymes especially glutathione s-transferase are often overexpressed in tumor cells and are regarded as a contributor to their drug resistance and are thought to play an important role in cancer progression. the purpose of this study is to evaluate the protective effects of chlorophylline as an antioxidant molecule which has inhibitory effects on gst p - on chemically-induced breast cancer model. in our previous work, we had observed that this molecule led to proliferation in breast cancer cells. in this study, n-methyl-n-nitrosourea (mnu) used for inducing carcinogenesis in eighteen, -day-old female sprague-dawley rats. chlorophylline and mnu solutions were injected intraperitoneally when the rats were , , and days old. their weight and tumor diameters were measured throughout the months study period. at the end of the study, all animals were sacrificed and determined both glutathione levels and related enzymes activities (gluathione s transferase, glutathione reductase and glutathione peroxidase) in tumor and tissues such as liver, kidney, heart and spleen were studied and analyzed. as a result, in breast cancer model, glutathione and related enzyme activities were protected by chlorophylline treatment whereas mnu made them decreased compared to the control group. in conclusion, chlorophylline with antioxidant features decreased the toxic effect of mnu by regeneration of glutathione and enhancement of its related enzyme activities. the use of antioxidant molecules, because of proliferative effects and defence-oriented behaviours, should be discussed in cancer therapy. p- . . - effect of overexpression of bacillus catalase on lactococcus lactis nisin production z. girgin ersoy, s. tunca gedik gebze technical university, kocaeli, turkey nisin, has been used commercially (e ) in food preservation for approximately years. it's the only bacteriocin which is approved by world health organization as a food additive. the fundamental problem that limits nisin usage in food preservation is low product yield by producer strains. because of high commercial potential of nisin, studies about increasing the production efficiency of nisin is kept in the forefront in recent years. since nisin biosynthesis and bacterial growth are occuring in parallel to each other, conditions that promote growth are also expected to encourage nisin production. it is known that, when supplied with exogenous heme, lactococcus lactis cells can respire under aerobic conditions and produce higher energy which in turn cause higher biomass. however, aerobic conditions also cause oxidative stress since catalase enzyme, which detoxify hydrogen peroxide, is absent in l. lactis. in this study, to complete the missing component of the defence mechanism of l. lactis, catalase (kate) gene of aerobic bacterium bacillus subtilis was overexpressed in facultative anaerobe l. lactis cells. for this, kate gene of b. subtilis was amplified by polymerase chain reaction (pcr). plasmid constructions were established in e. coli by using an e. coli-l. lactis shuttle vector and then the recombinant plasmid was transferred to l. lactis cells by electroporation. the presence of catalase activity in the recombinant strain grown on the solid medium was first detected by dropping hydrogen peroxide directly on the cells, then with enzyme assays. fermentation studies are going on to determine nisin production of the recombinant strain. to the best of our knowledge, this study presents the first preliminary results that shows the effect of overexpression of catalase gene on nisin production. cancer is among the leading causes of morbidity and mortality worldwide. chemotherapy is one of the major cancer treatment strategies. remarkably, natural products have garnered increased attention in the chemotherapy drug discovery field because they are biologically friendly and have high therapeutic effects. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of humic acid. the present study investigated anticancer effects of ha in several human cancer cell lines. ha was purchased from sigma-aldrich. in this study, we used several human cancer cell lines: the human breast cancer cell line, mcf- , colon cancer cell line, ht- , lung adenocarcinoma cell line, a , and servical cancer cell line, hela. the cells were maintained in dmem medium supplemented with % heatinactivated fbs and % penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at •c. five different concentrations ( ug/ml, ug/ml, ug/ ml, ug/ml, ug/ml) were prepared using a stock solution of ha. the cell proliferation and migration was measured. on the other hand, the apoptotic mechanisms induced by ha in cancer cells were investigated using "apoptosis antibody array kit". the effects of ha on cancer cell lines were evaluated over hours. according to our results, ha induced a decrease in ht- , a and hela cell numbers in a dose-dependent and time-dependent manner. contrary to this, ha induced proliferation of mcf- cells in dose dependent manner. ha inhibited cell migration in a dose dependent manner except mcf- cell line. it was also determined apoptotic pathways in cancer cells induced by ha. it was concluded that ha has an inhibitory effect on certain some cancers. since the effect of ha on tumor progression is unknown, further studies are needed to clarify the rol of ha on cancer activity. p- . . - chronic immobilization stress in rats: fluoxetine and amisulpride protects against chronic immobilization stress-induced biochemical alterations in the present study, the effects of amisulpride and fluoxetine on serum total sialic acid (tsa) and lipid bound sialic acid (lsa) levels was investigated in the rats exposed to chronic immobilization stress. the study was administered using male wistar albino rats weighing - g. rats were divided into five groups (n = / group). group i comprised the control group, group ii was exposed with saline + immobilization stress ( minutes daily immobilization stress for days and . ml saline was administered perorally minutes before immobilization), group iii was exposed amisulpride ( mg/kg/day) + immobilization stress, group iv was exposed fluoxetine ( mg/kg/day) + immobilization stress and v. group was exposed amisulpride ( mg/kg/ day) + fluoxetine ( mg/kg/day) + immobilization stress. statistical analysis showed that the saline + stress, amisulpride + stress and amisulpride + fluoxetine + stress groups was significantly higher than the control group with regards to tsa levels (p < . ). whereas, the fluoxetine group was significantly lower than the group regarding tsa levels (p < . ). on the other hand, saline group was significantly higher than the control group with regards to lsa level (p < . ). whereas, no significant differences in lsa levels were observed in the amisulpride, fluoxetine and amisulpride + fluoxetine groups, as compared to the control group (p > . ). the present study demonstrated beneficial effect of fluoxetine and amisulpride on the concentration levels of lsa and tsa in stress. p- . . - protective effect of borax and boric acid on total sialic acid and lipid-bound sialic acid levels against -methylcholanthrene and benzo(a)pyrene induced oxidative stress in rats s. ekin , g. oto, f. g€ ok, y. karakus, d. yildiz y€ uz€ unc€ u yil university, van, turkey the present study was performed to investigate total sialic acid (tsa) and lipid bound sialic acid (lsa) levels as possible in vivo chemoprotective effect of borax (bx) and boric acid (ba) again-st -methylcholanthrene ( -mc) and benzo(a)pyrene (b(a)p) induced oxidative stress in rats. the rats were divided into nine groups of six rats each. group i: control, untreated animals were given % . nacl, group ii: the b(a)p were administered mg/kg via ip. four times. group iii: the -mc-treated animals were administered mg/kg via ip. four times, group iv: ba was given mg/l/day with water. group v: bx was given mg/l/day with water. group vi: b(a)p mg/kg via ip four times + ba mg/l/day dosage with water. group vii: -mc mg/kg via ip four times + ba mg/l/day with water. group viii: b(a)p mg/kg via ip four times + bx mg/l/ day dosage with water. group ix: -mc mg/kg via ip four times + bx mg/l/day with water. the experimental period was continued for days. statistical analysis showed that the -mc + ba group was significantly higher than the control group with regards to tsa and lsa levels p < . , p < . ,p p- . . - effects of aluminum exposure on trace elements in rat tissues b. ozturk kurt, s. ozdemir department of biophysics, cerrahpasa medical faculty, istanbul university, istanbul, turkey aluminum (al) is the most abundant metal and the third most abundant element in the earth's crust. people are constantly exposed to al which is found in most rocks, soils, waters, air and foods, due to a result of an increase in industrialization and improving technology practices. the study was designed to examine the possible effects of aluminum exposure in different durations on trace elements in rat tissues. twenty-four healthy male wistar rats weighed - g were randomly divided into three groups: control group (gc) received only drinking water, short-term group (gs) and long-term group (gl). the study groups were orally exposed to mg/kg body weight alcl in drinking water for and weeks, respectively. at the end of the treatment period, rats were sacrificed and the kidney, liver, brain and cerebellum tissues were removed to analyse the levels of al, ar, b, ni, si, cr, cu, fe, mg, mn, se, cu and zn by icp-oes. the statistically significant increase were determined in cerebellum al, cu, as, b and cr levels in gl according to the gc. while as levels were statistically increased, ni levels were decreased in gl in the kidney and liver. while cu, mg and cr levels were higher, se and b levels were lower in the gs than gc in the brain. there were no significant difference in si and mn levels. as a result of our study, it may be concluded that al accumulation may lead to changes in tissue trace element levels. tacal, o., tacal, Ö., take, g., takic, m.m., taldykbayev, z., talim, b., tamashevski, a., tamer, f., tamer, l., , , taneva, s., taniyan, g., , tanrisev, m., tanriverdi, e.c., tanriverdi, k., tarhan, m., tarhan, t., tartar, s., tas, a., , taskin, a., taskin, t., taskiran, e., taskiran, b., taskoparan, b., taspinar, r., , tastan, Ö., tatli, Ö., tauraite, d., tay, t., tayman, c., taysi, s., , teker, h.t., tekes, s., tekin neijman, s., tekin, m.h., , tekin, g., , tekin, m., tekin, n., tekin, g., telci, d., , telefoncu, a., temel, h.e., , , temelie, m., temizgül, r., temlyakova, e., teplova, v., terashima, r., tercan avci, s., terekhov, s., tereshenko, o., terzi gulel, g., terzi, e., , terzi, e., terzioglu, g., terzioglu, o., testoni, g., tetik vardarli, a., tevdoradze, t., tevzadze, l., tezcan, Ö., tezcanli kaymaz, b., thielens, n., thomaidou, d., thomas, a., thornton, j.d., ticea, a.c., tikhonova, a., tileva, m., , timofeev, v., , timofeev, v., timofeeva, e., tipirdamaz, r., tiryaki, m., , tok, m., tokay, e., toker, a., , tokgun, o., toksoy Öner, e., tokyol, Ç., toman, r., tomasi, a., tombul, n., , tooke, c., topaloglu, h., topbas, m., topcu, b., topcu, c., topcu, g., , , topçu, v., topcu, c., topçuoglu, c., , topel, h., , toprak, b., toprak, m.s., torac, e., tosner, z., tosun, m., , toy, h., toymentseva, a., tozkoparan, b., trabulus, d.c., trantirek, l., trantirkova, s., trchounian, a., , trizna, e., , troshagina, d., tro t, m., truncaite, l., , tsakalidou, e., tsarkov, d., tsarkova, m., , tsigara, e.g., tsigara, m.g., tsverava, l., tsvetkova, e., tsyba, l., tsyganov, d., tsymbal, d., tüfekçi, a.r., , tufekci, a.r., tufenkci, h., tuglu, m.i., , tuglu, i., tuli, a., , , , tuli, a., tulubas, f., tunali, g., tunca gedik, s., , tunca gedik, s., tunçdemir, m., tuncel, h., tuncel, h., tunçer, s., tuncer, e., , tuncer, z.s., tuncer, b., tuncer, e., tuncez akyurek, f., tunçez akyürek, f., tuner, h., tüney kizilkaya, i., tural, b., tural, s., turan, i., turan, m., , turan, v., turan, y., turan, c., turano, p., , türel, s., , turhan, t., , turhan, y., , turk, s., turkan, a., türkcan kayhan, c., turkekul, k., türkel, s., turkeri, o.n., turkeri, n., turkkan, b., turkmen, s., türkmen, n., turkon, h., tutar, y., tüten, a., tutkun, e., , tüylü küçükkilinç, t., tuz, m.u., twardovska, m., tyapkina, o., tzartos, s., photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes evaluation of the sunscreen lichen substances usnic acid and atranorin pleiotropic anticancer activity of selected nutraceuticals against mcf- bucharest, national institute for marine research and development "grigore-antipa uk in some adult and elderly populations the acute and/or persistent infection with the common intracellular respiratory pathogen chlamydia pneumoniae (chl) may be associated with increased risk of developing obesity or cardiovascular disorders. thus, microelements modifying oxidative stress status were determined by icp-ms/ms in the hno diluted serum samples collected from chl-positive adult females (n = ) living in urbanized area in poland. chl infection was confirmed by igg+ antibody elisa and real-time pcr assay. all females were classified under their body-mass index values to the normal-weight (nw), over-weight (ow) and obese group (ob) although there are many drugs currently used in the treatment of peptic ulcer, such a drug providing radical treatment without side effects is not available. since oxidative stress is involved in peptic ulceration, this study was designed to investigate antiulcerogenic and antioxidant effects of hippophae rhamnoides l. ether extract on indomethacine-induced stomach ulcer in rats. materials and methods: thirty-five sprague dawley male rats (weights ranging - g) were randomly divided into groups, as each composed of rats. after hippophae rhamnoides l. leaf ether extracts of mg/kg, mg/kg and mg/kg doses and mg/kg doses of famotidin orally administered, mg/kg doses of indomethacine were orally applied to rats in order to make ulcer. on the sixth hour of indomethacin administration all rats were sacrificed using thiopental ( mg/kg). the stomachs were removed, and ulcer areas were evaluated macroscopically. superoxide dismutase activity (sod), glutathione (gsh) and malondialdehyde (mda) levels in stomach tissues of rats were determined by elisa method with respective kits conclusions: we can conclude that the ether extract of hippophae rhamnoides l. leaves reduces free radical formation and has antiulcerogenic effects on stomach tissue control group; hours torsion/ hours detorsion group (t/d); all other groups were saturated for four days egcg, cape and egcg+cape ( lml/kg). sections were taken from bouine's-fixed and paraffin-embedded testicular tissue blocks and stained with h&e. immunohistochemistry was applied for the detection of pi k, akt and mtorc. intensities were evaluated as mild ( ), moderate ( ) or strong ( ). serum ohdg, plasma mda levels were analyzed using elisa method. results were analyzed by anova statistical test. testis samples in control group exhibited normal histological morphology. disorganization and separation of seminiferous tubule cells and accompanying interstitial edema and vessel dilation were while mda level decreased significantly in cape+egcg group, ohdg level showed significant increase in cape group. in conclusion, cape and egcg exerted protective effects on tt. effects may be achieved through pi k/ akt/mtorc pathway involved in cell proliferation, angiogenesis, apoptosis. prophylactic use of egcg prior to tt surgery improved testicular morphology, therefore could prevent destructive effects of tt lmol/l), c ( . ae . lmol/l)), respectively. conclusions: this study is important for konya region, mitochondrial fatty acid b-oxidation disorders studies subject areas. this study is the first study to assess acylcarnitine levels of patients living in our region. we believe that our results will be useful for future studies. key words: acylcarnitine, mass spectrometry, dried blood spot p- . . - binding of fas and cu(ii) ions to hsa changes its cys thiol group antioxidant capacity and carbonylation pattern with methylglyoxal binding of cu(ii) ion ( . mol/mol hsa) led to increase of k' value if fish oil extract was present, but for other fas k' value decreased. the content of free hsacys -sh decreased for % after cu(ii) ion binding, and during hours incubation at °c, it was further decreased for % (stearic acid, mixfas) and % (myristic, fish oil extract, oleic acid). carbonylation of fa-hsa-cu(ii) complexes with mg ( mol/ mol hsa), lead to decrease in cys -sh content depending on fa present: %- % for myristic and stearic acid, % for oleic acid and mixfas and % for fish oil extract. carbonylation of fa-hsa-cu complexes could contribute to further enhancement of oxidative and carbonyl stress in diabetes as well as other diseases pirto ek p- . . - anti-proliferative and inducing apoptosis of the hydro alcholic achelia. wilhelmsii extract on human breast adenocarcinoma cell lines mcf- and mda-mb- background: vitamin d deficiency is associated with several conditions and/or diseases like inflammation, atherosclerosis, cardiovascular disease and mortality. several studies showed that lower vitamin d levels were associated with high serum levels of inflammatory biomarkers. ykl- is a glycoprotein, secreted by macrophages, neutrophils and different cell types. it is also associated with inflammation and pathological tissue remodeling. in this study, we aimed to evaluate relationship between the vitamin d deficiency and ykl- levels. methods: our study group includes subjects with vitamin d deficiency (group ) and age and sex-matched healthy subjects with normal serum levels of vitamin d (group ). plasma (oh) vitamin d levels were measured with liquid chromatography-tandem mass spectrometry (lc-ms/ms) method. plasma ykl- analysis was performed by elisa. serum hs-crp levels were measured with nephelometric method. results: plasma vitamin d levels below ng/ml were accepted as vitamin d deficiency. although we could not find any significant differences by means of serum hs-crp levels between groups (p > . ), plasma ykl- levels were significantly higher in group than group (p < . ). conclusions: in literature, vitamin d deficiency is associated with inflammation. in our study, we found similar hs-crp levels between groups and higher ykl- levels in group . vitamin d deficiency may be related to increased ykl- levels in terms of causing chronic inflammation.keywords: vitamin d deficiency, ykl- , inflammation. evaluation and comparison of tnf-family ligands and receptors genes in mice and humans by bioinformatics techniques stance called plaque builds up inside the coronary arteries. apelin is a novel endogenous peptide with inotropic and vasodilatory properties and is the ligand for the angiotensin receptor-like (apj) receptor. we aimed to determine genotype and allele frequencies of apj receptor a c gene polymorphism in turkish patients with cad and healthy controls by rflp-pcr. this study was performed on unrelated cad patients and healthy controls. we obtained aa, ac and cc genotype frequencies in cad patients as . %, . % and . %, respectively. in the control group, frequencies of genotypes were found as . % for aa, . % for ac and . % for cc. we did not observe difference in apj receptor a c polymorphism between cad patients and healthy controls (v = . ; df = ; p = . ). the a allele was encountered in % ( ) of the cad and . % ( ) of the controls. the c allele was seen in % ( ) of the cad and . % ( ) of the controls. allele frequencies were not significantly different between groups (v = . ; df = ; p = . ). the frequencies of apj receptor a c genotype were not significantly different between control and patients. individuals with cc genotypes had significantly higher weight, systolic and diastolic blood pressure levels and systolic blood pressure than other genotypes, p ≤ . . in addition, hdl-c level was found decreased, but this reduction was not statistically significant. contrarily, the low levels of weight, sbp, dbp and tc were statistically significant in the subjects with aa genotype in cad. in conclusion, cc genotype carriers may have more risk than other genotypes in the development of hypertension in cad. we suggest that this polymorphism may not be a marker of cad, but it may cause useful in function of the apelin/apj system and may be a genetic predisposing factor for diagnostic processes and can be helpfull in finding new treatment strategies. p- . . - comparative genomics/proteomics analyses of single amino acid repeat containing proteins across different vertebrate taxa a. g. keskus, o. konu department of molecular biology and genetics, bilkent university, ankara, turkeyconsecutive runs of single amino acids lead to overrepresentation of certain physicochemical properties in protein sequences. researchers also demonstrated a link between single amino acid repeat (saar) containing proteins and neurodegenerative diseases as well as biological functions. moreover, saar frequencies were shown to vary across species based on selected orthologous proteomes and/or proteins. hence, analysis of whole proteomes across multiple vertebrate taxa may provide additional species-and sequence-specific trends for saars. in addition, there is a need for testing the observed saar occurrencesthe aim of this study is to evaluate the effect of quercetin (q) on liver injury secondary to cerulein induced-acute pancreatitis (ap). for this reason, rats were randomly divided into four groups ( rats for each group) control group received physiological saline, four time and dimethyl sulfoxide, two time, at hours intervals, intraperitoneally (i.p.). cerulein group received cerulein ( lg/ kg-rat weight, in physiological saline), four times, and dimethyl sulfoxide ( %), two times, at hour intervals, i.p. quercetin pretreatment (q+cer) group received quercetin ( mg/kg-rat weight, in dimethyl sulfoxide) one time, one hour before cerulein treatment and physiological saline, one time, six hour after cerulein treatment. quercetin post-treatment (cer+q) group received dimethyl sulfoxide, one time, one hour before cerulein treatment and quercetin, one time, six hour after cerulein treatment. cerulein treatment increased significantly vascular congestion in hepatic cells. quercetin treatment also decreased significantly vascular congestion. the liver mda and carbonyl levels in cerulein group were significantly higher than the control group (p < . , p < . , respectively). the mda and carbonyl levels in q+cer group decreased significantly compared to the cer group (p < . ). the mda, carbonyl, mpo levels in cer+q group were significantly lower than the cer group (p < . ). the gssg/gsh ratio of q+cer and cer+q groups were significantly lower than the cer group (p < . , p < . , respectively). the sod activity in cer group was significantly lower than the control group, but the sod activity in q+cer and cer+q groups was significantly higher than the cer group (p < . ).this study shows that quercetin treatment was reduced the severity of liver injury secondary to cerulein induced-ap as reflected by changes in the parameters of hepatic oxidant and antioxidant. p- . . - identification of water extract of propolis components by using different columns in gas chromatography-mass spectrometry propolis is a natural material obtained by honey bees from various plants. protective effect of propolis against damages of free radicals is due to different compounds within propolis. the aim of this study is to identify qualitatively and quantitatively the chemical composition of water extract of propolis (wep) provided by erzurum region using rtx- and rtx- ms column of gas chromatography-mass spectrometry (gc-ms) and to compare with two columns.in this study, wep of mg/ml was prepared, cleared by membrane filter of . lm and freezed at À °c. then, it was lyophilized up to dry form and derivatized to apply for gaseous form. mg of dry extract was reacted with ll pyridin + ll bis-trimethylsilyl trifluoroacetamide (bstfa) mixture including % trimethylchlorosilane (tmcs) and incubated for minutes at °c. all analyses were performed with shimadzu gcms-qp ultra by using a flame ionisation detector (fid). rtx- and rtx- ms capillary columns and helium for carrier gas at a flow time of ml/minute were used. injection was applied on split mode at °c. derivatized propolis sample was injected as ll, initial column temperature was adjusted as °c, then increased to °c with increments of °c. total analysis time was determined as minutes. relative percent amount of separated compounds was calculated from total ion chromatogram with computerised integrator. all components were defined by using nist and wiley libraries.peaks obtained from rtx- column were much more than those of rtx- ms. on the other hand, analyses performing with both two columns have similar carbohydrate, aromatic acid and other acid contents.consequently chemical constituents of wep were determined qualitatively and quantitatively with gc-ms. it was concluded that rtx- column among both columns differentiating for polarities may produce more compounds in the propolis analysis. introduction: aquatic environment can be mostly contaminated by mixtures of metals. biochemical parameters have gain importance to characterize the effects of metals on aquatic organisms. glutathione s-transferase (gst) and its substrate glutathione (gsh) are important parameters of antioxidant defense system of fish metabolism due to their vital role in xenobiotic conjugation. objective: the goal of the current study to evaluate the changes in gst and gsh levels in response to cd, zn and cd+zn effects after and exposure days. materials and methods: fish were obtained from cukurova university fish culturing pools (turkey). fish were exposed to . lg/ml of cd (cdcl .h o) and zn (zncl ), and their mixture, for , and days. at the end of the exposure period, liver tissues were dissected and homogenized in a phosphate buffer (ph . ). homogenates were centrifuged at , g ( min, + °c). supernatants were stored at À °c until the analysis. one-way anova was used to compare data (mean ae se) followed by duncan's test (p < . ). results: gst and gsh changes were recorded as decreases after all metal exposures at day . although day exposure was found as less effective, combined effects caused significant decreases in gst and gsh levels. also longer exposure durations were appeared to be more effective in that situation. conclusions: significant decreases in gst and gsh levels could be occurred due to increased reactive oxygen species caused by metals particularly their combined effects. metal type, their single and combined effects and also exposure duration should be also taken into account when considering the antioxidant system response. gst and gsh might be considered as sensitive biomarker in toxicity assessment of metal mixtures.financial support: this study was supported by a grant from c ß ukurova university (turkey).background/aims: the activation of lectin-like oxidized low density lipoprotein receptor- (lox- ) on endothelial cells leads to intracellular oxidative stress and inflammation and a feed-forward cycle of injury in diabetes, since both oxidized low density lipoprotein (oxldls) and glucose increase lox- expression. quercetin (qr) is part of a subclass of flavonoids called flavonols. polyphenolic compounds affect the development of atherosclerosis not only through antioxidant properties but also by modulation of serum lipids, thereby influencing the immune and inflammatory processes associated with the development of atherogenic diseases. we investigated the effects of dietary qr on endothelial dysfunction mediated by oxidized low density lipoprotein (oxldl)/lectin-like oxidized low density lipoprotein receptor- (lox- ) in animal model of type diabetes mellitus. methods: we compared groups of male adult wistar albino rats: a control group, an untreated diabetic group, diabetic groups treated with qr, and qr group. diabetes was induced by a single injection of stz ( mg/kg). animals were kept in standard condition. in day after inducing diabetic, serum was collected for biochemical parameters. glucose, lipid profiles, microalbuminuria, oxldl and lox- levels were determined. results: serum triglyceride, ldl, vldl levels in diabetic control group (without treatment) was significantly higher than control group (normoglycemic untreated group). supplementation with quercetin decreased serum total cholesterol and increased hdl-cholesterol compared with the control group. serum oxldl and lox- levels in diabetic control group (without treatment) were significantly higher than control group (normoglycemic untreated group). conclusions: consumption of quercetin reduced oxldl and lox- levels. thus, quercetin could be effective in improving hyperglycemia, dyslipidemia, and endothelial damage in type diabetes. p- . . - investigation of some oxidative stress parameters in bdnf heterozygous mice liver tissue a. bodur , i. ince , i. abidin , a. alver objectives: the aim of this study was to evaluate the possible protective effects of nigella sativa (ns) against cholestatic oxidative stress and liver damage in the common bile duct ligated rats. methods: a total of male wistar albino rats were divided into three groups:sham control, bile duct ligation (bdl) and bdl+received ns; each group contain animals. the rats in ns treated groups were given ns ( . ml/kg) once a day orally for weeks starting days prior to bdl operation. results: the changes demonstrating the bile duct proliferation and fibrosis in expanded portal tracts include the extension of proliferated bile ducts into lobules, mononuclear cells, and neutrophil infiltration into the widened portal areas were observed in bdl group. treatment of bdl with ns attenuated alterations in liver histology. the alpha smooth muscle actin (a-sma), transforming growth factor beta (tgf-b ) and the activity of tunel in the bdl were observed to be reduced with the ns treatment. bdl significantly increased the tissue hydroxyproline (hp) content, malondialdehyde (mda) levels, and decreased the antioxidant enzyme (superoxide dismutase (sod) and glutathione peroxidase (gpx)) activities. ns treatment significantly decreased the elevated tissue hp content and mda levels and prevented the inhibition of sod and gpx enzymes in the tissues. conclusion: the data indicate that ns attenuates bdl-induced cholestatic liver injury, bile duct proliferation, fibrosis, apoptosis and oxidative stress. the hepatoprotective effect of ns is associated with antioxidative potential. organophosphorous compounds (ops) are used widely as pesticides for agricultural purposes, as oil additives or as flame retardants. however, due to their widespread use, there are major introduction: in this study, the antiulcerogenic effect of a water extract (cawe) obtained from a spices sample, cinnamomum aromaticum, was investigated using indomethacin-induced ulcer models in rats. materials and methods: experimental groups consisted of six rats. antiulcerogenic activities of , , and mg/kg body wt. doses of the cawe were determined by comparing the negative (treated only with indomethacin) and positive (famotidine) control groups. results and discussion: although all doses of the cawe showed significant antiulcerogenic activity as compared to negative control groups, the highest activity was observed with mg/kg body wt. doses ( %). the cawe showed similarly antioxidant activity when compared with trolox and ascorbic acids used as positive antioxidants. in addition, the activities of catalase (cat) and myeloperoxidase (mpo) enzymes were determined in the stomach tissues of rats and compared with those of the negative and positive control groups to expose the effects of these enzymes on antiulcerogenic activity. the enzymatic activities of cat and mpo and lipid peroxidation (lpo) level in indomethacin-administrated tissues were increased significantly by indomethacin in comparison to control groups. these enzymes and lpo level were decreased, however, by the cawe. in contrast to lpo level, cat and mpo activities, glutathione (gsh) level was decreased by indomethacin and increased by all doses of cawe and famotidine. the present results indicate that the cawe has a protective effect in indomethacin-induced ulcers, which can be attributed to its antioxidant potential. introduction: thiol groups (-sh) are important anti-oxidants and essential molecules protecting organism against the harmful effects of oxidative stress. the aim of our study is to evalute thiol-disulphide homeostasis with a novel and automated method in patients with prostate cancer (pc) before and after radical prostatectomy (rp). material and methods: patients with prostate cancer and healty control subjects were enrolled into the study. plasma samples were collected from patients before rp and months after the rp operation. thiol-disulphide homeostasis was determined with a recently developed novel method. prostate specific antigen, albumin, total thiol, native thiol, disulphide and total antioxidant status (tas) were evaulated and compared between the groups. results: native thiol levels were . ae . lmol/l in the control group, . ae . lmol/l in the patients before rp, and . ae . lmol/l in patients after rp. native thiol, total thiol and tas levels were significantly higher in the control group than the patients before rp (p values < . ). native thiol, total thiol and tas levels were higher months after rp compared to before rp in patients, but these changes were not significant statictically (p values . , . and . respectively). discussion and conclusion: our study demonstrated that antioxidant defense mechanism was weakened as indicated by the decreased thiol levels in the patients with pc. increased oxidative stress in prostate cancer patients may cause metabolic disturbance and have a role in the pathogenesis of prostate cancer. p- . . - is there any relation between g oral glucose challenge test and serum total oxidant-antioxidant status in pregnant woman? the purpose of this study was to test the hypothesis that any degree of antepartum screening for gestational diabetes mellitus with oral g glucose challenge test (gct) should be associated with oxidant-antioxidant status.in this prospectif study, oral glucose challenge test was applied to pregnant women aged - years and at - weeks of gestation. plasma glucose concentrations were measured initial, hour and in addition to test hours after ingestion of g glucose. at the same time serum insulin, cortisol, total antioxidant status (tas), total oxidant status (tos) levels were measured and the oxidative stress index (osi) was calculated.ten pregnant women (forty percent) had a positive glucose challenge test (gct). a positive moderate relation with initial and hour serum total antioxidant status (tas) levels (r = . ) and the oxidative stress index (osi) (r = . ) was found. there was a positive weak correlation with initial and hours total oxidant status (tos) levels (r = . ) but statistically significance difference was not found (p > . ).in this study after ingestion of g glucose serum total antioxidant status (tas), serum oxidant status levels (tos) and serum oxidative stress index (osi) levels were higher than the initial levels.the results of this study suggest that antepartum screening for gestational diabetes mellitus with g oral glucose challenge test (gct) weakly associated with oxidant-antioxidant status and to confirm this results the longer follow-up studies with more participants are necessary.wheat (triticum ssp.), cultivated for centuries in the middle-east, central asia, europe, and north-africa, is one leading staple crops around the world, and its marginally grown ancestor einkorn (triticum monococcum ssp. monococcum), possesses rich gene resources for wheat improvement and have bioactive compounds reducing and preventing chronic diseases such as diabetes, cancer, alzheimer, and cardio vascular diseases, beside their nutritional properties. however, as more attention has been given to wheat cultivars with strong gluten, protein content, starch composition, and resistance to biotic and abiotic stresses in bread wheat and yellow-colored pasta product in durum wheat health compounds such as fibers, phytochemicals, and bioactives have been underestimated so far. the aim of this study was, then, to examine the total phenolics and flavonoids, quantify their phenolic acids, a-tocopherol by high performance liquid chromatography (hplc), and their , -diphenyl- -picrylhydrazyl (dpph) scavenging activity of bread (triticum aestivum l.), durum (triticum turgidum ssp. durum desf.) wheat cultivars and einkorn (triticum monococcum ssp. monococcum) wheat populations collected from different provinces (bolu and kastamonu) of turkey. ferulic acid ( . - . -lg/g), p-coumaric ( . - . -lg/g), and total phenolic content (ranged . - . -lmol gae/g) of einkorn populations were significantly higher than bread and durum wheat cultivars. results suggested the possibility of production of einkorn wheat populations, and hopefully cultivars rich in particular health beneficial component(s) may provide benefit to the consumers. in addition, higher phenolic content of einkorn may offer novel wheat genetic resources for the improvement of new wheat cultivars and the development of wheat-based functional foods. oxidative damage due to ischemia and acute kidney injury (aki) after coronary artery bypass graft (cabg) surgery are the leading complication during this process. in the kidney, ischemia/ reperfusion injury contributes to aki that is a clinical syndrome with rapid kidney dysfunction and high mortality rates. some animal and clinical studies have demonstrated an increase in serum and urinary neutrophil gelatinase-associated lipocalin (ngal) expression after renal ischemic injury.in this study, our aim was to investigate the relationship betwwen ngal and oxidative stress parameters due to ischemia caused by total perfusion time (tpt) in patients who have undergone on-pump cabg. materials and methods: the study was conducted in patients who received on-pump cabg at university of istanbul, cerrahpasa medical faculty, department of cardiovascular surgery. blood samples were collected prior to surgery and after hours following the termination of cardiac pulmonary bypass (cpb) . following centrifugation, serum samples were separeted and stored at - c until analysis. serum ngal, ima (ischemia modified alb€ umin), pco (protein carbonyl), nt (nitrotyrosine), lpd (lipid peroxide) levels were determined by elisa procedure. results and discussion: serum ngal, pco, nt levels in after hours following cpb were significantly higher than the before surgery (p < . , p < . , p serum ngal levels in after hours following cpb was found to have positive correlation with ima, pco, nt, and lpo levels (r = . , p < . ; r = . , p < . ; r = . , p < . ; r = . , p < . respectively). ngal levels were positively correlated with total perfusion time after cbp (r = . , p < . ).the results of our study show that, increased ngal levels hours after cpb were positively correlated with oxidative stress parameters and total perfusion time. investigation of the effects of thymoquinone against indomethacine induced gastric damage in rats introduction: incidence and high cost of acute stomach mucosa damages make this issue a very interesting issue for study. for this reason, it is aimed to investigate the effects of different dosage of thymoquinone (tq) against indomethacine induced gastric damage in rats. material and method: in our study, six groups of wistrar male rats were used. groups are named as healthy, ind control, famotidine ( mg/kg) and three different doses of tq ( . , and mg/kg). while any treatment or drug administration will done on healthy group, model was generated to other groups by giving fam or tq with tap water via oral gavage. min later, mg/kg ind administrated to each rat. animals were sacrified about hour later and stomach samples of each groups were collected for macroscopic study and gsh levels measurements. results: lower doses of tq is more effective and all tq groups exhibit reduced ulcer region with respect to the ind control group. gsh level of ind control group is lower than healthy group. the gsh level of tq, especially in lower doses, and fam groups statistically exhibit an increase in gsh level. conclusion: it is observed that ind induced gastric damage cause ulcer and increase in free radical. it is determined that lower doses of tq ( . , and mg/kg) is also exhibit a protective effect on ind induced model. it is tought that quinone in tq structure have a strong redox feature and this feature clean up the free radicals caused by ind, it reduces the oxidative stress and protect the stomach from ulcer. anzer honey is the most famous honey in turkey with many endemic species flowers. the anzer plateau is located rize province of eastern black sea region. in this study, antioxidant and anti-hyaluronidase and anti-urease activities were investigated of the plateau bee pollen. the antioxidant capacity was determined by total phenolic content (tpc), total flavonoids contents (tfc), ferric reducing antioxidant power (frap) and , diphenyl- -picrylhydrazyl (dpph) radical scavenging activity. atherosclerosis is the leading cause of mortality worldwide, and as a chronic inflammatory disease, caused by a complex interplay between inflammatory and oxidative events.quercetin, a plant derived flavonoid and a well-known antioxidant, has shown great promises with regards to its protective effects against oxidative stress.however due to its physicochemical properties, the optimum pharmacokinetic behavior is a challenging issue.herein, we aimed to fabricate quercetin loaded solid lipid nanoparticle (quer-sln) to improve the bioavailability and therapeutics efficiency. furthermore the in-vitro capacity of quer-sln for ameliorating tnf-a induced oxidative stress in human endothelial vein cell (huvec) was evaluated. quer-slns were prepared by simple hot homogenizing method and characterized by means of drug loading (dl), encapsulation efficiency (ee), cytotoxicity, size, zeta potential and morphology. antioxidant activity of plain quercetin and quer-slns were then investigated using intracellular reactive oxygen species (ros) detection method (dcfh-da assay) by facs flowcytometryin conclusion, the results here showed superior control of oxidative stress by quercetin nanosystem as compared to plain quercetin. precirol based slns as a biocompatible/biodegradable lipid, may provide a novel drug delivery system for quercetin with improved beneficiary impact in atherosclerosis. objective: zinc is known as an antioxidant essential trace element. we aimed to evaluate the dose-dependent effects of zinc on the oxidant-antioxidant system in liver, kidney and brain tissues of rats and the histological alterations in the absence of oxidative stress (os). material and methods: thirty-nine female weighing about gr wistar albino rats were divided into four experimental groups as ad libitum (al) diet (control), al diet + mg/kg zn sulfate (low dose; group ), al diet + mg/kg zn sulfate (middle dose; group ) and al diet + mg/kg zn sulfate (high dose; group ). zn sulfate solutions were administered . ml/day orally for days and in day rats were sacrificed and tissues were excised for detecting malondialdehyde (mda), advanced oxidation protein products (aopp), superoxide dismutase (sod), glutathione peroxidase (gsh-px), glutathione reductase (gr), and glutathione-s-transferase (gst) activities. histological evaluation was also performed to confirm the effects of zinc. results: in liver tissues aopp levels decreased in all groups receiving zinc as compared to the control group. liver mda levels were increased in group and ; sod and gsh-px levels were both increased while gst levels were decreased in all groups compared to control. gr levels were increased only in group . in kidney; aopp level was decreased only in group and sod level was only decreased in group as compared to control while gr levels were increased in all doses of zinc. in brain; aopp, gsh-px and gr levels were decreased in all groups receiving zinc as compared to control group. sod activity in brain tissues was increased by the administration of middle dose of zinc (group ). gst level was decreased in only group conclusions: the biochemical and histological findings of this study suggest that zinc has various effects on liver, kidney and brain tissues in the absence of os.key words: zinc, liver, kidney, brain introduction: this study aimed to investigate the effect of diabetes mellitus (dm) on oxidative stress and antioxidant capacity in humour aqueous (ha) and venous serum using total antioxidant capacity (tac) and total oxidative stress (tos) levels in serum and ha in cataract patients. materials and methods: in this study patients were divided into two groups. group was composed of patients with type dm and cataract and group was composed of patients with cataracts who are not accompanied by dm and cataract patients who are not accompanied by systemic diseases. each group consisted of patients, totally patients were included in the study. the ha which was collected from the eyes at the beginning of the cataract surgery and venous blood serum collected from the same patients were analyzed. in both groups, ha and serum tac and tos levels were measured with elisa. results: : serum tac levels in the dm group were significantly lower than in the control group (p < . ). tos serum levels in dm group was statistically higher than the control group (p < . ). differences between tac and tos levels were not statistically significant when compared the two groups' ha results (p > . ). group , divided into two subgroups according to their hba c levels, there was no statistically significant difference between the subgroups when hba c levels were compared with the relationship between serum and ha's tac and tos levels (p > . ). there was not an association between the gender, age and the levels of tac-tos in both groups (p > . ). discussion and conclusion: presences of dm is the only risk factor for increase of oxidative stress and decrease of anti-oxidant capacity in patients without a systemic complication of dm and diabetic retinopathy. in our study, diabetic patients without retinopathy showed similar ha tos and tac levels to healthy individuals, this finding indicates that blood-aqueous barrier is protected in these patients. the effect of ferulic acid against testicular ischemia/reperfusion injury in rats u. sac ßik , g. erbil , z. c ß avdar , c. ural department of histology and embriyology, school of medicine, dokuz eyl€ ul university, izmir, department of molecular medicine, health science of institute, dokuz eyl€ ul university, izmir, turkeytestis torsion is one of the urologic emergencies occurring frequently in neonatal and adolescent period. testis is sensitive to ischemia/reperfusion (i/r) injury and, therefore, ischemia and consecutive reperfusion cause an enhanced formation of reactive oxygen species (ros) that result in testicular cell damage and apoptosis. ferulic acid, known as an antioxsidant, is a phenolic acid found in seeds and leaves of the plants. we aimed to investigate potential protective effect of ferulic acid against testis i/r injury.thirty five wistar rats were randomly divided into groups; control, ethyl alcohol, ischemia, i/r, i/r-ferulic acid groups. animals were exposed to hours of ischemia followed by hours of reperfusion. ferulic acid was administered ( mg/ kg) before reperfusion intravenously. testicular cell damage was examined by h-e staining and pas. tunnel, active caspase- , inos and mpo were evaluated by immunostaining. malondialdehyde (mda), glutathione (gsh) levels, glutathione peroxidase (gpx) and superoxide dismutase (sod) activities were assessed by biochemical methods.histological evaluation showed that ferulic acid pretreatment reduced significantly testicular cell damage and decreased tun-nel, caspase positive cells; inos and also mpo expression. in addition, ferulic acid administration decreased significantly the mda levels increased by i/r. morever, ferulic acid increased significantly the sod activity levels, which was decreased by i/r. there were no statistically significant differences in the levels of gsh and gpx activity in all groups.the present results suggest that ferulic acid is a potentially beneficial agent in protecting testicular i/r. background: in heart failure (hf), angiotensin antagonists (aa), beta-blockers (bb), spironolactone, diuretics and acetylsalicylic acid are often used. top pharmaceutical groups reduce mortality. on the increased oxidative stress (os) in patients with hf, it is known to have beneficial effects of certain groups of drugs. however, the net effect of these drugs in os is unknown. the aim of this study was to investigate the effects of drugs used in hf on os. materials and methods: patients were included in the study. all of the patients had systolic heart failure and all of them were under treatment. drugs used by the patients were recorded. the levels of total antioxidant status (tos), total oxidant status (tas), the enzymatic activity of ceruloplasmin, paraoxonase- and arylestherase were measured according to erel's method. serum total thiol levels were measured with sh modified hu method and the lipid hydroperoxide levels were measured with the ferrous ion oxidation xylenol orange assay. the percentage tos / tas was determined as osi. results: in patients treated with acetylsalicylic acid (asa), spironolactone, beta blocker and furosemide, there were increased tos, decreased tas and osi (p < . ). in patients treated with angiotensin blockers, increased tas and looh, and decreased sh were found (p < . ). in patients treated with nitrates and ccb, tos and osi were found decreased. correlation analysis showed that increased tas correlated with the use of angiotensin blockers, asa, furosemide and beta blocker positively; and with the tos and osi level correlated with the use of spironolactone, asa spironolactone and furosemide (p < . ). conclusion: current medical agents that are being used in hf are effective in reducing os in hf patients. one of the effective mechanisms to reduce the mortality of some of these drugs may decrease os.key words: heart failure, drug use, oxidative stress p- . . - across adjacent ring formed titanium phthalocyanine-mediated photodynamic therapy alters and degrades filamentous actin cytoskeleton and internal membranes photodynamic therapy (pdt) is widely accepted as a promising and minimally invasive treatment strategy due to its applicability on a wide range of cancer diseases. this clinically approved treatment method relies on the dramatic production of singlet oxygen and reactive oxygen species (ros) in target tissue to evoke apoptotic cell death [ ] . we, therefore, focused on the intracellular ros accumulation, internal membrane degradation, filamentous actin cytoskeleton alteration and nucleus morphology changes induced by pdt-mediated across adjacent ring formed titanium phthalocyanine which was previously synthesized bis(ethane- , p-phenol- , -p-phenoxy) phthalocyaninatotitanium (iv).characterization of the synthesized metallophthalocyanine was accomplished by using uv-vis, ir, h-nmr and maldi-tofmass spectroscopies. the dark and pdt-mediated activities of bare and phosphonolipids (max. %) charged titanium phthalocyanine ( . , . , . , and lm) were determined on a human lung carcinoma and hacat human keratinocyte cell lines by using intracellular ros assay, dioc( ), tritc-phalloidin and dapi staining protocols. waltmann pdt l was used as the non-toxic light source at j/cm fluence and mw/ cm fluence rate. the experiments showed that pdt-mediated titanium phthalocyanine leads to significant and concentration dependent reactive oxygen species accumulation. moreover, internal membrane degradation, apoptotic bodies on nucleus and filamentous actin cytoskeleton alteration were observed. consequently, the activity mechanism of pdt-mediated titanium phthalocyanine seems to be in a tight relationship with ros accumulation-mediated internal membrane degradation, filamentous actin cytoskeleton alteration and apoptotic pathways activation. introduction: retinal vein occlusion (rvo) is a common retinal vascular disorder that can affect visual acuity and cause blindness in elder population. sulphur containing aminoacids such as cysteine (cys), cysteinylglycine, glutathione, homocysteine and c-glutamylcysteine are reported to be associated with the pathogenesis of rvo. thiols are organosulfur compounds that are formed of a carbon-bonded sulfhydryl group. sulphur containing aminoacids slightly contribute the composition of plasma thiol pool. thiols can undergo oxidation reaction via oxidants and form disulphide bonds our purpose is to research the relationship between a novel oxidative stress marker serum dynamic thioldisulphide homeostasis and retinal vein occlusion. materials and methods: rvo patients and controls were included in the study. native thiol, total thiol, disulphide levels are measured in the serum samples of rvo and control group by using an automated method described by erel et al. also disulphide/native thiol and disulphide/total thiol ratios were calculated. results: there were no significant difference between the rvo and control group in native thiol, total thiol, disulphide disulphide/native thiol and disulphide/total thiol ratios.(p > . for all) conclusion: our study is the first report evaluating the dynamic thiol-disulphide homeostasis in rvo patients by a newly developed method by erel et al. further large sample sized studies investigating the levels of sulphur containing aminoacids may additionally be planned to verify this study. purpose: the purpose of this study was to evaluate markers of systemic oxidative stress and antioxidant capacity in subjects with severity of osas. methods: a total of osa patients were included in the study ( controls, with mild, with moderate, and with severe osa). patients were grouped according to apnea-hypopnea index (ahi) as mild, moderate and severe osa. patients with ahi< served as control group. known risk factors for oxidative stress, such as age, sex, obesity, smoking, hypelipidemia, and hypertension, were investigated as possible confounding factors. plasma arylesterase, total oxidative stress (tos), total antioxidant capacity (tac), total thiol, catalase (cat) levels were measured for all patients. results: the mean age was . ae . years and . % ( / ) of the study population was female. plasma arylesterase, tos, tac, total thiol, and cat plasma values were not different between mild, moderate, severe osa groups and controls (p > . ). catalase levels were significantly lower in women patients with severe osa compared to healthy women controls (p < . ). there was a negative correlation between ahi and serum total thiol levels (r = À . , p < . ) in severe osa groups. conclusionthe present prospective study provides evidence that osa might be associated with decreased antioxidant burden possibly via catalase way. results: the sera -ohdg, mda and il- were significantly higher in diabetic group than control group (p < . ). although there was a notable positive correlation between mda and -ohdg, there was no a relationship between -ohdg or mda with il- . discussion: in agreement with previous studies our data illustrated that high levels of oxidative stress is associated with increased production of oxidized lipids and nucleobases in diabetic patients compared to control group. also enhanced proinflammatory cytokine, il- , induced inflammmation in these patients.conclusion: oxidative stress and inflammation play pivotal roles in the development of diabetes and can cause major complications in dm. so we suggest that early detection of these measurable indicatores can help to diagnosis the severity or presence of some complication in diabet. the effect of quercetin on erythrocyte glucose- -phosphate dehydrogenase enzyme activity in ethanol treated rats in this study, we aimed to evaluate the effects of ethanol on erythrocyte (g- -p-d) enzyme activity and the effects of quercetin on erythrocyte g- -p-d activity in the recovery of the effects of ethanol.rats were randomly divided into four groups. the control group (n = ) received physiological saline. the quercetin group (n = ) received quercetin ( mg/kg/ day) via i.g. route the alcohol group (n = ) received ethanol ( % v/v, ml/day) via i.g. route. the alcohol + quercetin group (n = ) received ml of ethanol ( % v/v) hours after quercetin treatment ( mg/ kg/day). experimental procedures were peformed for days. erythrocyte g- -p-d activity was found to be higher in the quercetin group than those in the alcohol group (p < . ). in the alcohol group, the erythrocyte g- -p-d activity was found to be significantly decreased than those in the control group (p < . ). statistically significant differences were observed in erythrocyte g- -p-d activity between the alcohol group and the alcohol + quercetin group (p < . ).as a conclusion, our results demonstrate that ethanol decreased erythrocyte g pd activity and quercetin was found to be beneficial in the prevention of toxic effect raised by ethanol.key words: erythrocyte, ethanol, g pd, oxidative stress, quercetin p- . . - effects of the sulphasalazine to the cerebral hypoxia reperfusion injury in rat background: cerebral ischemia/ reperfusion (i/r) injury is still a difficult process to treat and rehabilitate today. this study was designed to investigate beneficial effects of sulfasalazine in cerebral i/r injury in rat. methods: except control group (n = ), wistar albino rats were divided into four groups for acute and chronic stage investigation of i/r injury, and temporary aneurysm clips were attempted to both internal carotid arteries for duration of minutes. four hours later, except control, sham-a, sham-c groups, mg/kg once a day sulfasalazine was administered to animals, orally. animals were sacrified and then necrotic neuronal cells of hippocampal ca , ca , and ca region, and cortical necrotic neurons, perivascular edema, pyknotic neuronal cells, irregularities of intercellular organization (iio) were counted and scaled histopathologically. tissue il- b, il- , malonyldialdehyde (mda), myeloperoxidation (mpo), no, and tnf-a levels were measured by using elisa, too. results: sulfasalazine could reduce perivascular edema, iio, cortical and hippocampal neuronal cell death in both stages. it could decreased mda in acute stage, but not reduce il- b, il- , mpo, no, and tnfa levels. it could increased il- b levels in chronic stage but not affect to il- , mpo, mda, no, tnf-a levels.conclusion: sulfasalazine could improve histopathological architecture of hypoxic tissue in both stages of i/r injury. it could inhibit lipid peroxidation cascades in acute stage but not affect to tissue mpo, no, il- , and tnf-a levels in any stage in rat. these results suggested that therapeutic mechanisms of sulfasalazine should be investigated by using more specific laboratory methods in future studies.key words: antiinflamatory, cerebral hypoxia reperfusion injury, sulfasalazine, stroke. camel and horse milk xanthine oxidase (xo) was found to catalyze the reduction of nitrate and nitrite to nitric oxide (no) under aerobic condition. to date, mammalian nitrate reductase (nar) and nitrite reductase (nir) have not been identified. no, a gas, is found to control a seemingly, limitless range of functions in animals.one assay was used to determine nar and nir activities of milk xo: ( ) nitrite formation from nitrate by nar, and ( ) nitrite utilization by nir. nitrite concentrations were determined by using sulfanylamide and n-( -naphtyl)-ethylenediamine, which form red color measured at nm.these activities of the milk xo require nadh as a physiological electron donor. high xo, nar and nir activities are detected only after heat treatment ( °c, min) of the fresh milk in the presence of molybdate. in both camel and horse milk nar activity of xo was almost two times higher than its nir activity. it is well known that xo can be reversibly converted from the dehydrogenase form to the oxidase through the oxidation of sulfhydryl groups. cysteine and, to a lesser extent, glutathione increased nir activity of milk xo but not its nar activity. the mechanism of this increase of nir activity remains unclear and is currently under study. substitution of tungsten for molybdenum under above conditions gave no detectable nar and nir activity of milk xo. the molybdenum site-directed inhibitor, tungsten inhibited in a dose-dependent manner. therefore, nitrate and nitrite are clear to interact with mo center of xo.camel and horse milk are traditional drinks in central asia and kazakhstan. therefore, it is very important that xo provide a mechanism for generation of no in camel and horse milk where nitric oxide synthase, no producing enzyme, does not exist. p- . . - the influence of phytomedicine on metabolic processes of white rats undergone to ionized radiation the study of peroxide lipids oxidation (plo) process is used as one of stability parameters of organism's changes and as a key mechanism for understanding of adaptation reactions and of pathogenesis of different diseases. it's determined by high biological activity of products which are formed in the plo reactions, in this relation lipids with high contents of fat acids play important role. to investigate the influence of phytomedicine eminium regelii on the metabolic processes (peroxide lipids oxidation) of white rats' organism in conditions of ionized radiation.the animals were exposed to ionizing radiation (gamma-radiation co) on the radiotherapeutic equipment teragam in a dose of gy and received phytomedicine eminium regelii in a dose of . mg/kg orally within days following the ionizing radiation exposure. gamma-rays caused the increase of lipid peroxidation (lpo) primary (dc) and secondary products' (mda) concentrations in spleen, liver, thymus and adrenal glands.treatment by phytomedicine resulted in contents of dc decreased in times in spleen, in times in thymus, in times in adrenal glands, in liver in times, in lymph nodes of small intestine it in times. mda decreased in liver up to and times, in spleen in . times, in thymus in times, in liver in times, in adrenal glands in time, no changes in lymph nodes of small intestine.the effect of phytomedicine treatment of organisms exposed to sublethal dozes of gamma-radiation results in the lpo primary and secondary products concentrations decrease in spleen, liver, thymus and adrenal glands. p- . . - evaluation of serum levels of ischemia modified albumin (ima) in bipolar disorder patients k. € unal , c. topc ßuoglu , m. cingi clinic of biochemistry, ankara polatli duatepe public hospital, ankara, clinic of biochemistry, ankara numune training and research hospital, ankara, clinic of psychiatry, ankara numune training and research hospital, ankara, turkey introduction: bipolar disorder is one of the most debilitating psychiatric disorders characterized by disruptive episodes of mania/hypomania and depression. considering the complex role of biological and environmental factors in the etiology of affective disorders; recent studies have focused on oxidative stress, which may damage nerve cell components and take part in pathophysiology. aim of our study is to contribute these data about oxidative stress in bipolar disorder, by detecting ischemia modified albumin (ima) levels of bipolar disorder patients in remission and also by comparing these results with healthy controls. methods: study population consisted of patients meeting the diagnostic and statistical manual of mental disorders, fifth edition (dsm- ) criteria for bipolar disorder i. healthy subjects were included as control group (hc). serum ischemia modified albumin (ima) levels of all participants were determined. results: statistical analysis on serum ischemia modified albumin (ima) levels did not show any significant difference between bipolar disorder patients in remission and healthy controls.conclusion: studies on oxidative stress in bipolar disorder have reached controversial results up till now. in this study, no statistically significant difference was detected between oxidative parameters of bipolar disorder patients in remission and healthy controls. in order to evaluate oxidative stress in bipolar disorder comprehensively, further studies are needed. keywords: bipolar disorder, ischemia modified albumin (ima), oxidative stress p- . . - xanthine oxidase and adenosine deaminase activity in patients with familial mediterranean fever (fmf) objective: fmf is an autosomal recessive dissease which is characterized by recurrent fever and inflammation of serous membranes. in this study we measured serum adenosine deaminase (ada) and xanthine oxidase (xo) levels in fmf cases. method: serum ada levels were measured with a sensitive colorimetric method described by giusti and xo levels were analysed by the method of worthington in fmf patients and healthy controls. results: there was a significant difference in xo and ada levels between controls and cases. ada and xo levels were higher in patents with fmf. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of ha. the present study was undertaken in order to evaluate the anticancer properties of the ha using a prostate cancer and osteosarcome cell lines pc- , sjsa as an in vitro model system.ha was purchased from sigma-aldrich. the cells were maintained in dmem medium supplemented with % heat-inactivated fbs and % penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at •c. five different concentrations ( ug/ml, ug/ml, ug/ml, ug/ ml, ug/ml) were prepared using a stock solution of ha. we measured cell proliferation and migration to understand of progression effects of ha in pc- and sjsa cell lines in vitro.according to our results, ha treatment caused cytotoxicity and induced cell death in vitro in pc- cells with an ic value of . lg/ml. contrary to this, ha induced proliferation of sjsa cells in dose dependent manner. ha demonstrated the highest proliferatif activity against sjsa cells with an ic value of > lg/ml. on the other hand, cell migration was reduced in pc- cell line and interestingly, migration was accelerated in sjsa cell line.our study may provide new insights into the regulatory effect of ha in cancer, but further studies are needed to clarify the role of ha in cancer pathogenesis. the febs journal (suppl. ) ( )